Fenfluramine

• DEA No. 1670
• S 768

2020/12/18, FDA APPROVED, Fintepla

Fenfluramine hydrochloride

Antiobesity

(+-)-Fenfluramine chloride

(+-)-Fenfluramine hydrochloride

Racemic fenfluramine hydrochloride

Fenfluramine hydrochloride [USAN]

AHR 3002

EINECS 206-968-2

1-(m-Trifluoromethylphenyl)-2-(ethylamino)propane hydrochloride

AHR-3002

Research Code：ZX-008

MOA：Serotonin agonist

Indication：Dravet syndrome

Company：Zogenix (Originator)

Synonyms of Fenfluramine [INN:BAN]

• (+-)-Fenfluramine
• BRN 4783711
• dl-Fenfluramine
• DL-Fenfluramine
• EINECS 207-276-3
• Fenfluramina
• Fenfluramina [DCIT]
• Fenfluramine
• Fenfluraminum
• Fenfluraminum [INN-Latin]
• HSDB 3080
• Obedrex
• Pesos
• Ponderax PA
• Rotondin
• S 768
• UNII-2DS058H2CF

mp 160-161 °C, ethyl acetate US 3198834 

nmr Salsbury, Jonathon S.; Magnetic Resonance in Chemistry 2005, V43(11), P910-917 C

IR  BIORAD: Infrared spectral data from the Bio-Rad/Sadtler IR Data Collection was obtained from Bio-Rad Laboratories, Philadelphia, PA (US).FenfluramineCAS Registry Number: 458-24-2CAS Name:N-Ethyl-a-methyl-3-(trifluoromethyl)benzeneethanamineAdditional Names:N-ethyl-a-methyl-m-(trifluoromethyl)phenethylamine; 2-ethylamino-1-(3-trifluoromethylphenyl)propaneManufacturers’ Codes: S-768Molecular Formula: C12H16F3NMolecular Weight: 231.26Percent Composition: C 62.32%, H 6.97%, F 24.65%, N 6.06%Literature References: Prepn: L. G. Beregi et al.,FRM1658; eidem,US3198833 (1963, 1965 both to Sci. Union et Cie Soc. Franc. Recherche Méd.). Prepn of optical isomers: eidem,US3198834 (1965 to Sci. Union et Cie Soc. Franc. Recherche Med.). Pharmacology: Presse Med.71, 181 (1963). Pharmacology and toxicity of isomers and racemate: J. C. Le Douarec et al.,Arch. Int. Pharmacodyn. Ther.161, 206 (1966). Pharmacokinetics: S. Caccia et al.,Eur. J. Clin. Pharmacol.29, 221 (1985). Clinical trial of dextrofenfluramine in refractory obesity: N. Finer et al.,Curr. Ther. Res.38, 847 (1985). Comprehensive review: Pinder et al.,Drugs10, 241-323 (1975).Properties: bp12 108-112°. LD50 i.p. in mice: 144 mg/kg (US3198833).Boiling point: bp12 108-112°Toxicity data: LD50 i.p. in mice: 144 mg/kg 
Derivative Type: HydrochlorideCAS Registry Number: 404-82-0Trademarks: Acino (IMA); Adipomin (Streuli); Obedrex (Beta); Pesos (Valeas); Ponderal (Servier); Ponderax (Selpharm); Ponderex (Robins); Pondimin (Robins); Rotondin (Casasco)Molecular Formula: C12H16F3N.HClMolecular Weight: 267.72Percent Composition: C 53.84%, H 6.40%, F 21.29%, N 5.23%, Cl 13.24%Properties: Crystals from ethanol + ether, mp 166°.Melting point: mp 166° 
Derivative Type:d-FormCAS Registry Number: 3239-44-9Additional Names: Dexfenfluramine; dextrofenfluramineProperties: [a]D25 +9.5° (c = 8 in ethanol). LD50 orally in rats: 114.6 mg/kg (Le Douarec).Optical Rotation: [a]D25 +9.5° (c = 8 in ethanol)Toxicity data: LD50 orally in rats: 114.6 mg/kg (Le Douarec) 
Derivative Type:d-Form hydrochlorideCAS Registry Number: 3239-45-0Trademarks: Adifax (Servier); Glypolix (Stroder); Isomeride (Ardix); Redux (Wyeth-Ayerst)Properties: Crystals from ethyl acetate, mp 160-161°.Melting point: mp 160-161° 
Derivative Type:l-FormCAS Registry Number: 37577-24-5Properties: [a]D25 -9.6° (c = 8 in ethanol). LD50 orally in rats: 195 mg/kg (Le Douarec).Optical Rotation: [a]D25 -9.6° (c = 8 in ethanol)Toxicity data: LD50 orally in rats: 195 mg/kg (Le Douarec) 
Derivative Type:l-Form hydrochlorideCAS Registry Number: 3616-78-2Properties: Crystals from ethyl acetate, mp 160-161°.Melting point: mp 160-161° 
NOTE: This is a controlled substance: 21 CFR, 1308.14.Therap-Cat: Anorexic.Keywords: Anorexic.

A centrally active drug that apparently both blocks serotonin uptake and provokes transport-mediated serotonin release.

Fenfluramine Hydrochloride has been filed an IND application with the FDA in USA to initiate phase III trials by Brabant Pharma (acquired by Zogenix in 2014) for the treatment of dravets syndrome (also known as severe myoclonic epilepsy of infancy, SMEI), this compound has been granted orphan drug designation in Europe and U.S..
Fenfluramine Hydrochloride was launched in 1963 by Servier in France and in 1973 by Wyeth （now a wholly owned subsidiary of Pfizer） in US for the treatment of obesity. However, it was withdrawn from the market in 1997 due to heart disease.

Dravet syndrome is a pediatric encephalopathy that typically manifests within the first year of life following exposure to elevated temperatures. It is characterized by recurrent pharmacoresistant seizures, which increase in frequency and severity with disease progression. Concomitantly with these seizures, patients typically display delayed development and neurocognitive impairment.^6,9,10,11 Fenfluramine is a serotonergic phenethylamine originally used as an appetite suppressant until concerns regarding cardiotoxicity in obese patients lead to its withdrawal from the market in 1997.^6,12,13 Through its ability to modulate neurotransmission, fenfluramine has reemerged as an effective therapy against pharmacoresistant seizures, such as those involved in Dravet syndrome.^3,5,8

Fenfluramine was granted initial FDA approval in 1973 prior to its withdrawal; it was granted a new FDA approval on June 25, 2020, for treatment of Dravet syndrome patients through the restricted FINTEPLA REMS program. It is currently sold under the name FINTEPLA® by Zogenix INC.¹⁶

Fenfluramine, sold under the brand name Fintepla, is a medication used for the treatment of seizures associated with Dravet syndrome in people age two and older.^[2][3]

The most common adverse reactions include decreased appetite; drowsiness, sedation and lethargy; diarrhea; constipation; abnormal echocardiogram; fatigue or lack of energy; ataxia (lack of coordination), balance disorder, gait disturbance (trouble with walking); increased blood pressure; drooling, salivary hypersecretion (saliva overproduction); pyrexia (fever); upper respiratory tract infection; vomiting; decreased weight; risk of falls; and status epilepticus.^[2]

Dravet syndrome is a pediatric encephalopathy that typically manifests within the first year of life following exposure to elevated temperatures. It is characterized by recurrent pharmacoresistant seizures, which increase in frequency and severity with disease progression. Concomitantly with these seizures, patients typically display delayed development and neurocognitive impairment.^6,9,10,11 Fenfluramine is a serotonergic phenethylamine originally used as an appetite suppressant until concerns regarding cardiotoxicity in obese patients lead to its withdrawal from the market in 1997.^6,12,13 Through its ability to modulate neurotransmission, fenfluramine has reemerged as an effective therapy against pharmacoresistant seizures, such as those involved in Dravet syndrome.^3,5,8

Fenfluramine was granted initial FDA approval in 1973 prior to its withdrawal; it was granted a new FDA approval on June 25, 2020, for treatment of Dravet syndrome patients through the restricted FINTEPLA REMS program. It is currently sold under the name FINTEPLA® by Zogenix INC.¹⁶

Medical uses

Fenfluramine is indicated for the treatment of seizures associated with Dravet syndrome in people age two and older.^[2][3]

Dravet syndrome is a life-threatening, rare and chronic form of epilepsy.^[2] It is often characterized by severe and unrelenting seizures despite medical treatment.^[2]

Adverse effects

The U.S. Food and Drug Administration (FDA) fenfluramine labeling includes a boxed warning stating the drug is associated with valvular heart disease (VHD) and pulmonary arterial hypertension (PAH).^[2] Because of the risks of VHD and PAH, fenfluramine is available only through a restricted drug distribution program, under a risk evaluation and mitigation strategy (REMS).^[2] The fenfluramine REMS requires health care professionals who prescribe fenfluramine and pharmacies that dispense fenfluramine to be specially certified in the fenfluramine REMS and that patients be enrolled in the REMS.^[2] As part of the REMS requirements, prescribers and patients must adhere to the required cardiac monitoring with echocardiograms to receive fenfluramine.^[2]

At higher therapeutic doses, headache, diarrhea, dizziness, dry mouth, erectile dysfunction, anxiety, insomnia, irritability, lethargy, and CNS stimulation have been reported with fenfluramine.^[4]

There have been reports associating chronic fenfluramine treatment with emotional instability, cognitive deficits, depression, psychosis, exacerbation of pre-existing psychosis (schizophrenia), and sleep disturbances.^[4][5] It has been suggested that some of these effects may be mediated by serotonergic neurotoxicity/depletion of serotonin with chronic administration and/or activation of serotonin 5-HT_2A receptors.^[5][6][7][8]

Heart valve disease

The distinctive valvular abnormality seen with fenfluramine is a thickening of the leaflet and chordae tendineae. One mechanism used to explain this phenomenon involves heart valve serotonin receptors, which are thought to help regulate growth. Since fenfluramine and its active metabolite norfenfluramine stimulate serotonin receptors, this may have led to the valvular abnormalities found in patients using fenfluramine. In particular norfenfluramine is a potent inhibitor of the re-uptake of 5-HT into nerve terminals.^[9] Fenfluramine and its active metabolite norfenfluramine affect the 5-HT_2B receptors, which are plentiful in human cardiac valves. The suggested mechanism by which fenfluramine causes damage is through over or inappropriate stimulation of these receptors leading to inappropriate valve cell division. Supporting this idea is the fact that this valve abnormality has also occurred in patients using other drugs that act on 5-HT_2B receptors.^[10][11]

According to a study of 5,743 former users conducted by a plaintiff’s expert cardiologist, damage to the heart valve continued long after stopping the medication.^[12] Of the users tested, 20% of women, and 12% of men were affected. For all ex-users, there was a 7-fold increase of chances of needing surgery for faulty heart valves caused by the drug.^[12]

Overdose

In overdose, fenfluramine can cause serotonin syndrome and rapidly result in death.^[13][14]

Pharmacology

Pharmacodynamics

Fenfluramine acts primarily as a serotonin releasing agent.^[15][16] It increases the level of serotonin, a neurotransmitter that regulates mood, appetite and other functions.^[15][16] Fenfluramine causes the release of serotonin by disrupting vesicular storage of the neurotransmitter, and reversing serotonin transporter function.^[17] The drug also acts as a norepinephrine releasing agent to a lesser extent, particularly via its active metabolite norfenfluramine.^[15][16] At high concentrations, norfenfluramine, though not fenfluramine, also acts as a dopamine releasing agent, and so fenfluramine may do this at very high doses as well.^[15][16] In addition to monoamine release, while fenfluramine binds only very weakly to the serotonin 5-HT₂ receptors, norfenfluramine binds to and activates the serotonin 5-HT_2B and 5-HT_2C receptors with high affinity and the serotonin 5-HT_2A receptor with moderate affinity.^[18][19] The result of the increased serotonergic and noradrenergic neurotransmission is a feeling of fullness and reduced appetite.

The combination of fenfluramine with phentermine, a norepinephrine–dopamine releasing agent acting primarily on norepinephrine, results in a well-balanced serotonin–norepinephrine releasing agent with weaker effects of dopamine release.^[15][16]

Pharmacokinetics

The elimination half-life of fenfluramine has been reported as ranging from 13 to 30 hours.^[4] The mean elimination half-lives of its enantiomers have been found to be 19 hours for dexfenfluramine and 25 hours for levfenfluramine.^[13] Norfenfluramine, the major active metabolite of fenfluramine, has an elimination half-life that is about 1.5 to 2 times as long as that of fenfluramine, with mean values of 34 hours for dexnorfenfluramine and 50 hours for levnorfenfluramine.^[13]

Chemistry

Fenfluramine is a substituted amphetamine and is also known as 3-trifluoromethyl-N-ethylamphetamine.^[13] It is a racemic mixture of two enantiomers, dexfenfluramine and levofenfluramine.^[13] Some analogues of fenfluramine include norfenfluramine, benfluorex, flucetorex, and fludorex.

History

Fenfluramine was developed in the early 1960s and was introduced in France in 1963.^[13] Approximately 50 million Europeans were treated with fenfluramine for appetite suppression between 1963 and 1996.^[13] Fenfluramine was approved in the United States in 1973.^[13] The combination of fenfluramine and phentermine was proposed in 1984.^[13] Approximately 5 million people in the United States were given fenfluramine or dexfenfluramine with or without phentermine between 1996 and 1998.^[13]

In the early 1990s, French researchers reported an association of fenfluramine with primary pulmonary hypertension and dyspnea in a small sample of patients.^[13] Fenfluramine was withdrawn from the U.S. market in 1997 after reports of heart valve disease^[22][23] and continued findings of pulmonary hypertension, including a condition known as cardiac fibrosis.^[24] It was subsequently withdrawn from other markets around the world. It was banned in India in 1998.^[25]

Fenfluramine was an appetite suppressant which was used to treat obesity.^[13] It was used both on its own and, in combination with phentermine, as part of the anti-obesity medication Fen-Phen.^[13]

In June 2020, fenfluramine was approved for medical use in the United States with an indication to treat Dravet syndrome.^[2][26]

The effectiveness of fenfluramine for the treatment of seizures associated with Dravet syndrome was demonstrated in two clinical studies in 202 subjects between ages two and eighteen.^[2] The studies measured the change from baseline in the frequency of convulsive seizures.^[2] In both studies, subjects treated with fenfluramine had significantly greater reductions in the frequency of convulsive seizures during the trials than subjects who received placebo (inactive treatment).^[2] These reductions were seen within 3–4 weeks, and remained generally consistent over the 14- to 15-week treatment periods.^[2]

The U.S. Food and Drug Administration (FDA) granted the application for fenfluramine priority review and orphan drug designations.^[2][27][28] The FDA granted approval of Fintepla to Zogenix, Inc.^[2]

On 15 October 2020, the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Fintepla, intended for the treatment of seizures associated with Dravet syndrome.^[29] Fenfluramine was approved for medical use in the European Union in December 2020.^[3]

Society and culture

Recreational use

Unlike various other amphetamine derivatives, fenfluramine is reported to be dysphoric, “unpleasantly lethargic“, and non-addictive at therapeutic doses.^[30] However, it has been reported to be used recreationally at high doses ranging between 80 and 400 mg, which have been described as producing euphoria, amphetamine-like effects, sedation, and hallucinogenic effects, along with anxiety, nausea, diarrhea, and sometimes panic attacks, as well as depressive symptoms once the drug had worn off.^[30][31][32] At very high doses (e.g., 240 mg, or between 200–600 mg), fenfluramine induces a psychedelic state resembling that produced by lysergic acid diethylamide (LSD).^[32][33] Indirect (via induction of serotonin release) and/or direct activation of the 5-HT_2A receptor would be expected to be responsible for the psychedelic effects of the drug at sufficient doses.

Research

Under the development code ZX008, the pharmaceutical company Zogenix is studying fenfluramine’s potential to treat seizures.^[34] Clinical trials have studied the use of fenfluramine in patients with Dravet syndrome.^[35] Results of a Phase III clinical trial showed a 64% reduction in seizures.^[36]Route 1

Reference：1. J. Org. Chem.1979, 44, 3580-3583.Route 2

Reference：1. EP0810195A1.

2. Chem. Ind. Times 2002, 16, 33-34.Route 3

Reference：1. ACS Symp. Ser. 2009, 1003, 165-181.

ref

BE 609630

FR 1658 M 19630218

US 3198834

DE 1593595

US 3769319

NL 7215548

Ger. (East) (1974), DD 108971

EP 3170807

SYN

US20170174613

PATENT

US 20170174613

(MOL) (CDX)

PATENT

US 20180208543

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX) a ketone, 1-(3-trifluoromethyl)phenyl-propan-2-one, is first reduced to the corresponding alcohol in the presence of yeast, D-glucose, ethanol and water. Then the alcohol is converted into the tosylate in the second step:
 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

 (MOL) (CDX)

DESCRIPTION OF THE FIGURES

SUMMARY OF THE INVENTION

 (MOL) (CDX) wherein R ₁ is CF ₃, with ethylamine or with a salt thereof, and with a reducing agent chosen from the group consisting of alkaline cation or ammonium borohydride, alkaline cation or ammonium triacetoxyborohydride and alkaline cation or ammonium cyanoborohydride, in which the alkaline cation is always different from lithium cation and mixtures thereof, to yield fenfluramine, optionally followed by the transformation of the obtained fenfluramine into a pharmaceutically acceptable salt.

Example 1

Synthesis of Fenfluramine

Example 2

Purification of Fenfluramine

Example 2a

Distillation

Example 2b

Conversion into Hydrochloride Salt and Crystallization

PAPER

Journal of Organic Chemistry (1979), 44(20), 3580-3.J. Org. Chem. 1979, 44, 20, 3580–3583

Publication Date:September 1, 1979
https://doi.org/10.1021/jo01334a031https://pubs.acs.org/doi/abs/10.1021/jo01334a031

PATENT

https://patents.google.com/patent/US20170174613A1/en

• Fenfluramine is an amphetamine drug that was once widely prescribed as an appetite suppressant to treat obesity. Fenfluramine is devoid of the psychomotor stimulant and abuse potential of D-amphetamine and interacts with the 5-hydroxytryptamine (serotonin, 5-HT) receptors to release 5-HT from neurons. Fenfluramine has been investigated as having anticonvulsive activity in the treatment of Dravet Syndrome, or severe myoclonic epilepsy in infancy, a rare and malignant epileptic syndrome. This type of epilepsy has an early onset in previously healthy children.
• [0003]
  Anorectic treatment with fenfluramine has been associated with the development of cardiac valvulopathy and pulmonary hypertension, including the condition cardiac fibrosis which led to the withdrawal of fenfluramine from world-wide markets. Interaction of fenfluramine’s major metabolite norfenfluramine with the 5-HT2B receptor is associated with heart valve hypertrophy. In the treatment of epilepsy, the known cardiovascular risks of fenfluramine are weighed against beneficial anticonvulsive activity.

• [0097]
  Chemical Abstract Service (CAS) Registry Number (RN): 404-82-0 (HCl Salt), 458-24-2 (Parent Free Base)
• [0098]
  Chemical Name: N-ethyl-α-methyl-3-(trifluoromethyl)-benzeneethanamine hydrochloride (1:1). Other Names: Fenfluramine HCl, DL-Fenfluramine, (±)-Fenfluramine
• [0099]
  Structure of Hydrochloride Salt:
• [0100]
  Stereochemistry: Fenfluramine HCl has one chiral center and is being developed as the racemate and contains dexfenfluramine and levofenfluramine
• [0101]
  Molecular Formula of hydrochloride salt: C12H16F3N.HCl
• [0102]
  Molecular Mass/Weight: 267.72 g/mol

2. General Properties

• [0103]
  Table 1 summarizes the chemical and physical properties of Fenfluramine HCl.
• TABLE 1 General Properties of Fenfluramine HCl Drug Substance Property Result Appearance (color, White to off-white powder physical form) DSC (melting 170° C. (melt/sublimation) point)^a TGA Onset 147° C. 0.03% at 150° C. 91% at 220° C. (evaporation) pKa (water) 10.15-10.38 Solubility (mg/mL) Resultant pH 25° C. 37° C. Solubility pH 6.69 (water) 54.13 71.22 (Aqueous) pH 1.73 buffer 25.34 53.68 pH 3.43 buffer 29.50 61.97 pH 6.41 buffer 37.42 95.60 0.9% NaCl (water) 22.98 — Solvent Solubility 25° C. (mg/mL) Solubility (Organic Ethanol 150 Solvents) Dichloromethane 30-35 Ethyl Acetate, 1-5 mg Tetrahydrofuran, Toluene, Acetonitrile UV Absorption Maxima: 210, 265 nm Solution pH (water) 6.69 Hygroscopicity @30% RH: ~0.05% (Dynamic Vapor @60% RH: ~0.07% Sorption (DVS) @90% RH: ~0.20%^a) Polymorphism Fenfluramine HCl has been consistently isolated as a single crystalline Form 1 as determined by DSC and x-ray powder diffraction (XRPD) Solvation/Hydration Fenfluramine HCl is isolated as a nonhydrated, nonsolvated solid Solution Stability 8 weeks @ pH 6.7 phosphate buffer medium at 40° C. and 60° C. using concentrations of 0.5, 2.5 and 5.0 mg/ml. All conditions, no new impurities >0.1% by HPLC. Solid Stability 8 weeks @ 40° C., 60° C. and 80° C. 7 days at 150° C. All conditions, no new impurities >0.1% by HPLC.

3. Synthesis of Fenfluramine Drug Substance

• [0104]
  Scheme 3.1 shows a 2-step route of synthesis used to manufacture initial clinical supplies of Fenfluramine HCl from ketone (2). The batch size is 4 kg performed in laboratory glassware (kilo lab). No chromatography is required and the process steps are amenable to scale-up. In process 1 there is one isolated intermediate Fenfluramine Free Base (1) starting from commercially supplied 1-(3-(trifluoromethyl)phenyl) acetone (Ketone 2). All steps are conducted under cGMPs starting from Ketone (2).
• [0105]
  Scheme 3.2 shows a 4-step route of synthesis to Fenfluramine HCl that can be used for commercial supply. Route 2 utilizes the same 2-step process used by Route 1 to convert Ketone (2) to Fenfluramine HCl with the exception that Ketone (2) is synthesized under cGMP conditions starting from 3-(Trifluoromethyl)-phenyl acetic acid (Acid 4). Bisulfate Complex (3) is an isolatable solid and can be purified before decomplexation to Ketone (2). In-situ intermediates which are oils are shown in brackets. Batch sizes of 10 Kg are performed. Commercial batch sizes of 20 kg are performed in fixed pilot plant equipment. Steps 1-2 of Scheme 3.2 to manufacture Ketone (2) have been demonstrated on a 100 g scale to provide high purity ketone (2) of >99.8% (GC & HPLC). Conversion of Ketone (2) to Fenfluramine using either Route 1 or 2 has provided similar purity profiles.
• Starting materials are designated by enclosed boxes. Bracketed and non bracketed compounds respectively indicate proposed in-situ and isolated intermediates. NMI=N-Methyl Imidazole.

4.1. Narrative Description (Route 1)

• [0106]
  Step 1: Reductive Amination (Preparation of Fenfluramine Free Base 1)
• [0107]
  A solution of ethylamine, water, methanol, and 1-(3-(trifluoromethyl)phenyl) acetone (Ketone 2) was treated with sodium triacetoxyborohydride and stirred for 16 h at 25° C. at which time HPLC analysis (IPC-1; In Process Control No. 1) showed the reaction to be complete and sodium hydroxide solution was added until pH>10. Toluene was added and the phases separated, and the aqueous phase (IPC-2) and organic phase (IPC-3) are checked for remaining Fenfluramine and Fenfluramine alcohol and the organic phase was reduced. Purified water was added and the pH adjusted to <2 using conc. HCl and the phases were separated. The aqueous phase was washed with toluene and the toluene phase (IPC-4) and the aqueous phase (IPC-5) was checked for Fenfluramine and Fenfluramine alcohol content. The aqueous phase containing product is pH adjusted to >10 using sodium hydroxide solution. The basic aqueous phase was extracted with MTBE until removal of Fenfluramine from the aqueous phase was observed by HPLC (<0.5 mg/ml) (IPC-6). The organic phase was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give the intermediate product Fenfluramine Free Base 1 as a pale yellow oil tested per specifications described herein which showed by NMR the material to contain 2.93% toluene giving an active yield of 88.3% with a purity of 98.23% by HPLC (0.67% Fenfluramine alcohol).
• [0108]
  Step 2: Salt Formation (Preparation of Fenfluramine HCl)
• [0109]
  To a flask was charged ethanol and acetyl chloride. The solution was stirred slowly overnight before ethyl acetate was added. The HCl in ethyl acetate solution formed was polish filtered into a clean carboy and retained for later use. To a vessel was added Fenfluramine free base 1 and MTBE. The Fenfluramine solution in MTBE was collected in two carboys before the vessel was cleaned and checked for particulate residue. The Fenfluramine solution was polish filtered into a vessel and cooled and HCl in ethyl acetate solution was added giving a final pH of 6-7. The batch was stirred for 1 h and filtered. The product was dried under vacuum at 40° C. The product (96.52% yield) was tested per IPC-7 had a purity of 99.75% by HPLC and GC headspace analysis showed MTBE (800 ppm) and EtOAc (150 ppm) to be present. The product was then tested per specifications shown herein.

4.2. Narrative Description (Route 2)

• [0110]
  Step 1: Preparation of Ketone Bisulfite Adduct
• [0111]
  Procedure: Charge acetic anhydride, (2.8 vol, 3.0 wt, 5.0 eq.) to a vessel and commence stirring. Cool the solution to −5 to 5° C., targeting −4° C. Charge 1-methylimidazole, (0.2 vol, 0.21 wt, 0.5 eq.) to the mixture at −5 to 5° C. Caution: very exothermic. If necessary, adjust the temperature to 0 to 5° C. Charge ZX008 acid, (1.00 wt, 1.0 eq.) to the mixture at 0 to 5° C. Caution: exothermic. Stir the mixture at 0 to 5° C. until ≦2.1% area ZX008 acid by HPLC analysis, typically 7 to 9 hours. Charge 15% w/w sodium chloride solution (2.0 vol) to the mixture at 0 to 5° C., 60 to 90 minutes. Caution: very exothermic which will be slightly delayed. Warm the mixture to 18 to 23° C. over 45 to 60 minutes and continue stirring for a further 30 to 45 minutes at 18 to 23° C. Charge TBME, (5.0 vol, 3.7 wt) to the mixture and stir for 10 to 15 minutes at 18 to 23° C. Separate the aqueous layer and retain the organic layer. Back-extract the aqueous layer with TBME, (2×3.0 vol, 2×2.2 wt) at 18 to 23° C. retaining each organic layer. Adjust the pH of the combined organic layer to pH 6.5 to 9.0, targeting 7.0 by charging 20% w/w sodium hydroxide solution (5.3 to 8.3 vol) at 18 to 23° C. Caution: exothermic. Separate the aqueous layer and retain the organic layer. Wash the organic layer with 4% w/w sodium hydrogen carbonate solution (2×3.0 vol) at 18 to 23° C. Determine the residual ZX008 acid content in the organic layer by HPLC analysis, pass criterion ≦0.10% area ZX008 acid. Wash the organic layer with purified water, (2×3.0 vol) at 18 to 23° C. Concentrate the organic layer under reduced pressure to ca. 2 vol at 40 to 45° C., targeting 43° C.
• [0112]
  Determine the w/w assay of ZX008 ketone (WIP) in the mixture by 1H-NMR analysis for information only and calculate the contained yield of ZX008 ketone (WIP) in the mixture. Note: This step can be removed from the process since the process is robust and consistently delivers 80 to 90% th yield. The achieved yield was factored into the charges of the subsequent steps.
• [0113]
  Charge n-heptane, (4.0 vol, 2.7 wt) to the mixture at 40 to 45° C., targeting 43° C. Concentrate the mixture to ca. 2 vol at 40 to 45° C., targeting 43° C. Determine the TBME content in the mixture by 1H-NMR analysis, (pass criterion ≦5.0% w/w TBME vs. ZX008 ketone). Charge n-heptane, (2.4 vol, 1.6 wt) at 40 to 45° C., targeting 43° C., vessel A. To vessel B, charge sodium metabisulfite, (0.82 wt, 0.88 eq.) at 18 to 23° C. To vessel B, charge a solution of sodium hydrogen carbonate, (0.16 wt, 0.4 eq.) in purified water, code RM0120 (2.0 vol) at 18 to 23° C. followed by a line rinse with purified water, code RM0120 (0.4 vol) at 18 to 23° C. Caution: gas evolution. Heat the contents of vessel B to 40 to 45° C., targeting 43° C. Charge the contents from vessel A to vessel B followed by a line rinse with n-heptane, (0.8 vol, 0.5 wt) at 40 to 45° C., targeting 43° C. Stir the mixture for 1 to 1.5 hours at 40 to 45° C., targeting 43° C. Charge n-heptane, code RM0174 (3.2 vol, 2.2 wt) to the mixture with the temperature being allowed to cool to 18 to 45° C. at the end of the addition. Cool the mixture to 18 to 23° C. at approximately constant rate over 45 to 60 minutes. Stir the mixture at 18 to 23° C. for 1.5 to 2 hours.
• [0114]
  Sample the mixture to determine the residual ZX008 ketone content by 1H-NMR analysis, (pass criterion ≦10.0% mol, target 5.0% mol ZX008 ketone vs. ZX008 ketone bisulfite adduct). Filter the mixture and slurry wash the filter-cake with n-heptane, (2×2.0 vol, 2×1.4 wt) at 18 to 23° C. Dry the solid at up to 23° C. until the water content is <10.0% w/w water by KF analysis according to AKX reagent. At least 16 hours. Determine the w/w assay of the isolated ZX008 ketone bisulfite adduct by 1H-NMR analysis and calculate the contained yield of ZX008 ketone bisulfite adduct.
• [0115]
  Yields and Profiles: The yield for the stage 1 Demonstration batch is summarized Table below. Input: 1700.0 g uncorr., acid, 99.50% area (QC, HPLC), 2-isomer not detected, 4-isomer 0.02% area, RRT1.58 (previously not observed) 0.48% area as per the preparative method. The analytical data is summarized in Table 1A below.
• TABLE 1A Table for isolated yields for step 1 Demonstration batch Corr. % area Reference Corr. Yield % w/w (HPLC, number Input Output (% th)** (¹H-NMR)* QC) Comments Batch A1 1700.0 g 1500.1 g 89.1 45.0 —.— Crude ketone as TBME sol. Batch A2 1500.1 g 1716.1 77.8 76.0 98.15 Bisulfite adduct only 67.3 Overall product
• [0116]
  Step 2: Preparation of Ketone
• [0117]
  Procedure: Charge toluene, (5.0 vol, 4.3 wt), and purified water, (5.0 vol) to the vessel and commence stirring. If necessary, adjust the temperature to 18 to 23° C. and charge ZX008 ketone bisulfite adduct, (1.00 wt corrected for % w/w assay) to the mixture at 18 to 23° C. Charge 20% w/w sodium hydroxide solution to the mixture at 18 to 23° C. adjusting the pH of the mixture to pH 8.0 to 12.0, targeting 9.0 (0.5 to 1.0 vol).
• Separate the lower aqueous layer and retain the top organic layer. Wash the organic layer with purified water, (3.0 vol) at 18 to 23° C. Concentrate the organic layer under reduced pressure to ca. 2 vol at 45 to 50° C., targeting 48° C. Charge methanol, (5.0 vol, 4.0 wt) to the mixture at 45 to 50° C., targeting 48° C. Re-concentrate the mixture under reduced pressure to ca. 2 vol at 45 to 50° C., targeting 48° C. Repeat steps 7 and 8 once before continuing with step 9. Cool the mixture to 18 to 23° C. Clarify the mixture into a tared, suitably-sized drum followed by a methanol (1.0 vol, 0.8 wt) line rinse at 18 to 23° C. Determine the w/w assay of ZX008 ketone (WIP) in the mixture by 1H-NMR analysis and calculate the contained yield of ZX008 ketone (WIP) in the mixture. Determine the toluene content in the mixture by 1H-NMR analysis.
• [0118]
  Yields and Profiles: The yield for the step 2 Demonstration batch is summarized in Table 1B below. Input: 1200.0 g corr. Ketone bisulfite adduct, 76.0% w/w assay (NMR, using DMB as internal standard in d₆-DMSO), (1.00 eq, 1.00 wt corr. for w/w assay) for input calculation.
• TABLE 1B Table for isolated yields for step 2 Demonstration batch % w/w % area Corr. Corr. Corr. Yield (¹H- (HPLC, Input Output (% th) NMR)* QC) Comments 1200.0 g 858.15 g 108.3 25.5 99.31 Purified ketone
• [0119]
  Step 3: Preparation of Fenfluramine HCl Crude
• [0120]
  Procedure: Charge the ZX008 ketone (corr. for assay, 1.00 wt, 1.00 eq. isolated as solution in MeOH in stage 2) to a vessel. Charge methanol, code RM0036 (5.0 vol, 4.0 wt) to the mixture at 18 to 23° C. Cool the solution to 0 to 5° C. Charge 70 wt % aqueous ethylamine solution (1.3 vol, 1.6 wt, 4.0 eq) to the mixture at 0 to 10° C., over 15 to 30 minutes, followed by a line rinse with methanol (1.0 vol, 0.8 wt). Warm the mixture to 15 to 20° C. and stir the mixture for a further 60 to 70 minutes at 15 to 20° C. Adjust the mixture to 15 to 18° C. if required, targeting 15° C. Charge sodium triacetoxyborohydride (2.4 wt, 2.25 eq.) to the mixture in approximately 10 portions, keeping the mixture at 15 to 20° C., targeting 17° C. Addition time 1.5 to 2 hours. Caution: Exothermic. Stir the mixture at 15 to 20° C. until complete by HPLC analysis, pass criterion ≦3.0% area ZX008 ketone, typically 2 to 3 hours. Adjust the pH of the mixture to pH>12 by charging 20% w/w aqueous sodium hydroxide solution (5.0 to 6.0 vol) to the mixture at 15 to 40° C. Addition time 10 to 30 minutes. Caution: Exothermic. If necessary, adjust the temperature to 18 to 23° C. Extract the mixture with toluene (3×3.0 vol, 3×2.6 wt) at 18 to 23° C., retaining and combining the top organic layer after each extraction. Wash the combined organic layer with purified water, (1.0 vol) at 18 to 23° C. Heat the mixture to 40 to 50° C., targeting 48° C. Concentrate the mixture under reduced pressure at constant volume maintaining ca. 5 vol by charging the organic layer at approximately the same rate as the distillation rate at 40 to 50° C., targeting 48° C. Cool the mixture to 18 to 23° C. Charge purified water (10.0 vol) to the mixture at 18 to 23° C. Adjust the pH of the mixture to 0.1<pH<1.5 at 18 to 23° C. by charging concentrated hydrochloric acid, 0.5 vol. Do not delay from this step until neutralization.
• [0121]
  Separate the layers at 18 to 23° C. retaining the bottom aqueous layer. Wash the aqueous layer with toluene, (3.0 vol, 2.6 wt) at 18 to 23° C. retaining the aqueous layer. Adjust the pH of the aqueous layer to pH>12 by charging 20% w/w sodium hydroxide solution at 18 to 23° C. 0.8 to 0.9 vol. Caution: Exothermic. Charge TBME, code RM0002 (2.0 vol, 1.5 wt) to the basic aqueous layer. Separate the layers at 18 to 23° C. retaining the top organic layer. Back-extract the aqueous layer with TBME (2×2.0 vol, 2×1.5 wt) at 18 to 23° C. retaining the organic layers. Wash the combined organic layer with purified water, (2×1.0 vol) at 18 to 23° C. Concentrate the combined organic layers under reduced pressure at 40 to 50° C., targeting 48° C. to ca. 3 vol. Determine the residual toluene content of the mixture by 1H-NMR analysis. Sample for determination of residual water content by KF analysis, AKX reagent. Charge TBME (8.7 vol, 6.4 wt) to the mixture at 40 to 50° C. Cool the solution to 0 to 5° C., targeting 2° C. Charge concentrated hydrochloric acid (0.54 vol, 0.46 wt) maintaining the temperature <15° C. Caution: Exothermic. Line rinse with TBME (1.0 vol, 0.7 wt). If necessary, adjust the temperature to 0 to 10° C. and stir the mixture at 0 to 10° C. for a further 2 to 3 hours. Filter the mixture and wash the filter-cake with TBME (2×4.4 vol, 2×3.3 wt) at 0 to 10° C. Dry the solid at up to 40° C. until the TBME content is <0.5% w/w TBME by 1H-NMR analysis. 4 to 8 hours.
• [0122]
  Yields and Profiles: The yield for the step 3 Demonstration batch is summarized in Table 1C below. Input: 856.8 g corr. Ketone, 44.2% w/w assay (NMR, using TCNB as internal standard in CDCl₃), (1.00 eq, 1.00 wt corr. for w/w assay) for input calculation. FIG. 2 and Table 1D shows an exemplary HPLC chromatogram of a crude preparation of fenfluramine hydrochloride (210 nm UV absorbance).
• TABLE 1C Table for isolated yields for step 3 Demonstration batch Corr. % area Reference Corr. Corr. Yield % w/w (HPLC, number Input Output (% th) (¹H-NMR)* QC) Comments Batch A1 856.8 g 836.31 g 85.3 44.2 99.15 Fenfluramine free base (in situ intermediate) Batch A2 880.7 84.0 based 99.5 100.00 Fenfluramine•HCl on ketone crude (step 3 an bisulfite d 4.1) adduct (77.6 based on purified ketone)
• TABLE 1D Purity of crude fenfluramine hydrochloride by HPLC (see FIG. 2) Processed Channel Descr. DAD AU Ch 1 Sample 210, Bw 4 Peak Results USP USP USP Name RT RelRT Area Height Tailing Resolution Plate Count EP s/n % Area 1 NorFenfluramine 7.46 2 2-Fenfluramine 7.68 3 Fenfluramine 8.67 1.000 3789064 778178 1.7 70796 2549.8 99.15 4 4-Fenfluramine 8.95 5 11 34 1.308 6073 1449 1.2 23.5 215529 3 8 0.16 6 ZX008 acid 12.93 7 Fenfluramine alcohol 14.16 1.633 15266 2972 1.3 24.8 215040 8.7 0.40 8 ZX008 ketone 14.83 9 Fenfluramine acetamide 15.55 10 TOLUENE 15 75 11 15.92 1.836 4110 1122 2.7 0.11 12 16.60 1.915 6861 1630 1.5 451209 4.3 0.18 Sum 3821374 100.00
• [0123]
  Step 4.2: Crystallization of Fenfluramine Hydrochloride
• [0124]
  Procedure: Charge Fenfluramine.HCl (crude) (1.00 wt, 1.0 eq.) and TBME (10.0 vol, 7.4 wt) to the vessel and commence stirring. Heat the suspension to reflux (50 to 58° C.). Charge ethanol (5.0 vol, 3.9 wt) maintaining the temperature at 50 to 58° C. Addition time 20 minutes. Stir at 50 to 58° C. for 5 to 10 minutes and check for dissolution. Stir the solution at 50 to 58° C. for 5 to 10 minutes, targeting 54 to 58° C. Clarify the reaction mixture through a 0.1 μm in-line filter at 54 to 58° C., followed by a line rinse with TBME (1 vol, 0.7 wt). Cool the solution to 48 to 50° C. Charge Fenfluramine HCl, code FP0188 (0.01 wt). Check for crystallization. Allow the suspension to cool to 15 to 20° C., target 17° C. over 5 to 5.5 hours at an approximately constant rate. Stir the mixture at 15 to 20° C., target 17° C. for 2 to 3 hours. Filter the mixture and wash the filter-cake with clarified TBME (2×3.0 vol, 2×2.2 wt) at 5 to 15° C. Dry the solid at up to 40° C. until the TBME content is <0.5% w/w TBME and the ethanol content is <0.5% w/w EtOH by 1H-NMR analysis. 4 to 8 hours. Determine the w/w assay of the isolated Fenfluramine.HCl by 1H-NMR analysis.
• [0125]
  Yields and Profiles: The yield for the stage 4 Demonstration batch is summarized in Table 1E below. Input: 750.0 g uncorr. Fenfluramine HCl crude (1.00 eq, 1.00 wt uncorr.) for input calculation. FIG. 3 shows an exemplary HPLC chromatogram of a crystallized fenfluramine hydrochloride sample (210 nm UV absorbance).
• TABLE 1E Table for isolated yields for stage 4 Demonstration batch Uncorr. Uncorr. Uncorr. Yield HPLC (% area, Input Output (% th) QC) Comments 750.0 g 608.0 81.1 100.00* Fenfluramine•HCl

PATENT

https://patents.google.com/patent/EP3170807A1/en

• Fenfluramine, i.e., 3-trifluoromethyl-N-ethylamphetamine, has the following chemical structure:
• [0003]
  The marketing of fenfluramine as a pharmaceutical active ingredient in the United States began in 1973 and was used in a therapy in combination with phentermine to prevent and treat obesity. However, in 1997 fenfluramine was withdrawn from the market in the United States and immediately thereafter in other countries, since its ingestion was associated with the onset of cardiac fibrosis and pulmonary hypertension. As a consequence of this event, the pharmaceutical compounds containing this active ingredient were withdrawn from the market. However, fenfluramine, even after its exit from the market, has continued to attract scientific interest, as will become apparent from the discussion presented hereinafter.
• [0004]
  In the literature, over the years, numerous syntheses or processes have been reported for preparing fenfluramine or its dextrorotatory enantiomer dexfenfluramine or an analog containing a highly electron-attractor group on the aromatic ring as in the fenfluramine molecule (see for example Pentafluorosulfanyl Serotonin Analogs: Synthesis, Characterization, and Biological Activity, John T. Welch and Dongsung Lim Chapter 8, pp 165-181 DOI: 10.1021/bk-2009-1003.ch008). Many of these synthesis paths are long and foresee multiple stages or synthesis steps that can include reagents that are dangerous or scarcely environment-friendly and are therefore scarcely convenient for an industrial synthesis. Hereinafter, any reference to “fenfluramine” is understood to referto the racemic form, i.e, (RS)-N-ethyl-1-[3-(trifluoromethyl)phenyl]propan-2-amine.
• [0005]
  To the best of the knowledge of the inventors, the first method for fenfluramine synthesis reported in the literature dates back to 1962 and is referenced in patent BE609630 and in analogous patents US3198833 and FR1324220 . All the synthesis methods reported in these patents provide for numerous synthesis steps. By way of example, one of the methods provides for the transformation into oxime of a ketone, 1-(3-trifluoromethyl)phenyl-propan-2-one, as shown here:
• [0006]
  The oxime is then hydrogenated in the presence of Raney nickel catalyst so as to yield the corresponding primary amine, which is acetylated subsequently with ethanoic anhydride before being converted into fenfluramine by reduction with lithium aluminum hydride.
• [0007]
  As can be seen, the final step of this chemical process provides for the use of lithium aluminum hydride and the persons skilled in the art will acknowledge that the use of this reagent should be avoided, if possible, on an industrial level, since it is extremely flammable and is the source of accidents. Furthermore, lithium is a potentially neurotoxic metal and therefore its use should be avoided where possible. Furthermore, the Raney nickel catalyst is used in the oxime reduction step and can contaminate the final active ingredient; the use of hydroxylamine also entails problems of toxicity for workers assigned to production.
• [0008]
  A further disadvantage of this process is, as already mentioned earlier, the number of steps, not only because a large number of synthesis steps entails a reduction of the overall yield of active ingredient, but also because each synthesis step in principle can generate impurities and a larger number of steps can therefore entail a higher number of impurities in the final active ingredient. Many of these impurities, furthermore, due to their structural similarity to fenfluramine, are difficult to eliminate and remove from a fenfluramine preparation. One impurity for example that can be formed in the process described above and is difficult to eliminate is the following:
• [0009]
  This impurity, which is a primary amine, shares physical-chemical properties that are similar to fenfluramine and therefore, like fenfluramine, it can form a hydrochloride salt by treatment with hydrochloric acid and thus contaminate the active ingredient fenfluramine hydrochloride. Furthermore, this impurity – as a free base – has a boiling point that is similar to that of fenfluramine (73°C vs. 89°C at 6 mmHg respectively), and therefore its elimination by distillation also can be problematic.
• [0010]
  The process described above can in principle generate other impurities, which are listed in Figure 1 .
• [0011]
  EP 0441160 claims a synthesis in 5 steps of dexfenfluramine, dextrorotatory enantiomer of fenfluramine. This synthesis can be adapted easily to produce fenfluramine instead of its dextrorotatory enantiomer simply by performing the first reduction step with a non-chiral reducing agent. In the first step, in fact:a ketone, 1-(3-trifluoromethyl)phenyl-propan-2-one, is first reduced to the corresponding alcohol in the presence of yeast, D-glucose, ethanol and water. Then the alcohol is converted into the tosylate in the second step:
• [0012]
  This reaction occurs in the presence of triethylamine and tosyl chloride in methylene chloride as solvent. After purification, the tosylate is converted to fenfluramine by means of three successive steps:
• [0013]
  In the first of these three steps, the tosylate is converted into an azide intermediate by reaction with sodium azide in dimethylformamide. The azide intermediate is then hydrogenated in the presence of a catalyst, palladium on carbon. Finally, the resulting primary amine is converted into fenfluramine by reaction with acetaldehyde and sodium borohydride.
• [0014]
  Persons skilled in the art may see easily that this process is not desirable from an industrial standpoint due to reasons related to environmental risk, safety and costs. For example, the sodium azide used in the process is a notoriously explosive compound and its use at the industrial level is dangerous. Furthermore, palladium is an expensive material and its use in the process entails an increase in the production costs of fenfluramine. Furthermore, palladium can contaminate the finished active ingredient.
• [0015]
  In another method for the synthesis of dexfenfluramine in 3-4 steps, reported by Goument et al. in Bulletin of the Chemical Society of France (1993), 130, p. 450-458, 3-bromobenzotrifluoride is subjected to a Grignard reaction with enantiopure 1,2-propylene-epoxide to yield 1-[3-(trifluoromethyl)phenyl]propan-2-ol as shown hereafter:
• [0016]
  If this reaction is performed with racemic 1,2-propylene-epoxide, the synthesis can be adapted to the preparation of fenfluramine.
• [0017]
  The alcohol thus obtained is first transformed into trifluoromethyl sulfonate by reaction with trifluoromethanesulfonic anhydride and then treated with ethylamine to yield fenfluramine, as shown in the diagram hereinafter:
• [0018]
  In this article, the authors acknowledge that the main byproducts of the reaction are isomer alkenes having the following chemical structures:
• [0019]
  The process proposed by Goument et al. is not interesting from the industrial standpoint for a series of reasons. First of all, it is known that the use of Grignard reagents, especially on an industrial scale, is problematic, because these compounds are often pyrophoric and corrosive. Furthermore, 1,2-propylene epoxide is a suspected carcinogenic compound. Finally, the formation of the three isomer alkenes as byproducts listed above is a disadvantage of the process. In the article, Goument presents methods for activation of the intermediate alcohol which are alternative to trifluoromethylsulfonate, for example by converting it to chloride (via thionyl chloride) or to mesylate (via mesyl chloride), but these process variations share the same disadvantages as the main process analyzed above.
• [0020]
  In addition to the methods with multiple synthesis steps discussed so far in detail, the literature reports other methods or processes for producing fenfluramine or dexfenfluramine. In general, persons skilled in the art acknowledge that the syntheses in the literature for producing dexfenfluramine sometimes can be applied to the preparation of fenfluramine simply by replacing the initial materials and/or enantiopure reagents with the corresponding racemates while maintaining the reaction conditions. For example, patents that present long synthesis methods in multiple steps are the following:
  • DE1593595 and US3769319
  • NL7215548
  • EP810195 and EP882700 (dexfenfluramine)
  • EP0301925 (dexfenfluramine)
• [0021]
  Other examples of preparation of fenfluramine, taken from non-patent literature, are the following:
  • Synthesis, Nov.1987, p. 1005-1007
  • J.Org.Chem, 1991, 56, p. 6019
  • Tetrahedron, 1994, 50(1), p. 171
  • Bull. Soc. Chim. France, 1993, 130(4), p. 459-466 (dexfenfluramine)
  • Chirality, 2002, 14(4), p. 325-328 (dexfenfluramine)
• [0022]
  Without analyzing in detail the individual methods described in these patents or articles, it can be stated in summary that all these methods are not attractive and interesting from the industrial standpoint because these are processes with many synthesis steps or because the initial materials described therein are not easily available and therefore have to be prepared separately, with a further expenditure of time and with further costs, or because they provide for the use of reagents that are dangerous/explosive/toxic or because they entail the use of catalysts based on heavy metals that can contaminate the final active ingredient.
• [0023]
  One should consider that in the literature there are methods for the preparation of fenfluramine that do not provide for long syntheses and multiple steps but are shorter and consist of one or two steps. These processes, which therefore would be more interesting from the industrial standpoint, have other specific disadvantages, as will become apparent in detail hereinafter. For example, in the literature there is a first group of articles or patents that describe the reaction between 1-(3-trifluoromethyl)phenyl-propan-2-one and ethylamine in the presence of hydrogen gas and of a transition metal as catalyst:
• [0024]
  In particular, in Huagong Shikan, 2002, 16(7), p. 33, the reaction is performed with hydrogen gas (2.9 – 3.38 atm), at 65-75°C, for 9 hours, in the presence of Raney nickel. Likewise, in patent DD108971 (1973), Raney nickel and hydrogen gas and methanol are used as solvent to perform this reaction.
• [0025]
  In HU55343 , instead, a similar reaction in one step is performed with hydrogen gas in the presence of another transition metal catalyst, such as palladium on carbon.
• [0026]
  Although these three methods describe short single-step processes, they have the disadvantage of the use of hydrogen gas. As is known to persons skilled in the art, hydrogen gas is a dangerous gas due to the inherent danger of forming explosive mixtures with air and must be used by expert personnel in expensive facilities dedicated to its use and built with special precautions. Despite being used in purpose-built facilities, the use of hydrogen at the industrial level is inherently dangerous and to be avoided if possible. Another danger element that is shared by the processes described above is the fact that the reactions are performed under pressure. The third industrial disadvantage then arises from the use of heavy metal catalysts, which have a high cost and therefore increase the overall cost of the final active ingredient and -on the other hand- may contaminate the active ingredient fenfluramine even after filtration of the catalyst and purification of said active ingredient.
• [0027]
  Analysis of the background art shows, however, that an attempt has been made to devise a process for the production or synthesis of fenfluramine that is short (one or two steps) and does not entail the use of hydrogen gas or of catalysts based on nickel or palladium or the like. In particular, for example, Synthesis 1987, 11, p. 1005, and then DECHEMA Monographien (1989), 112 (Org. Elektrochem.–Angew. Elektrothermie), 367-74, present a method for the synthesis of fenfluramine which starts from 1-(3-trifluoromethyl)phenyl-propan-2-one, which is made to react with ethylamine in great excess, in an electrochemical process, which uses a mercury cathode in a water/ethanol solution with pH 10-11. One obtains fenfluramine with 87% yield. This process has some drawbacks from an industrial standpoint: it is a process of the electrochemical type and therefore requires special equipment which is scarcely widespread, dedicated cells and reactors, and it is not possible to use the classic multipurpose reactors available in the pharmaceutical industry. Furthermore, the use of mercury at the industrial level poses severe environment safety problems, requiring constant health monitoring on workers who manage the equipment and systems for the management and destruction of wastewater that are particularly onerous; finally, mercury can be transferred from the cathode to the reaction environment and therefore to the active ingredient, and this obviously is to be considered very dangerous due to the accumulation of the metal in human beings; small traces of mercury are very toxic.
• [0028]
  Another method for fenfluramine synthesis in a single step is the one presented in J.Org.Chem, 1979, 44(20), p. 3580. Here the reaction is described between an alkene derivative and ethylamine in the presence of sodium borohydride and mercury nitrate:
• [0029]
  Again, this process is not interesting from an industrial standpoint since it has the same problems, if not even greater ones, related to the use of mercury (used here as a water-soluble salt) discussed previously. The complication introduced in this process with the use of mercury nitrate together with sodium borohydride highlights the level of innovation of the synthesis path found here.
• [0030]
  In past years, therefore, it has not been possible to provide a process for synthesizing fenfluramine in a small number of steps by using modern reducing agents that are commonly and easily used. Indeed, while Gaodeng Xuexiao Huaxue Xuebao, 9(2), 1988, p. 134-139, describes and exemplifies the synthesis of 2-N-ethyl-1-phenyl propane by means of (1) the treatment of the precursor ketone with ethylamine followed by (2) sodium cyanoborohydride as reducing agent, Xuexiao Huaxue Xuebao provides no example for fenfluramine. Moreover, for the latter, Xuexiao Huaxue Xuebao indicates a melting point for the hydrochloride of 161°C, a data item that matches the value indicated in the literature initially (see BE609630 ); these facts prove that fenfluramine synthesis with cyanoborohydride was not performed, otherwise one cannot explain why the author did not transcribe, in the document, the example of a product that at the time was very important. It should be noted in fact that 1-phenyl propan-2-one and 1-(3-trifluoromethyl)phenyl-propan-2-one can have different reactivities to reductive amination due to the presence of a highly electron-attractor -trifluoromethyl group, hence the need for an example to demonstrate its feasibility. The use of cyanoborohydride shares some disadvantages with other methods discussed in the preceding paragraphs. The excellent selectivity for reductive aminations of this reagent is highly appreciated, but its application can be less advantageous with respect to other reducing systems in the synthesis of fenfluramine, where the latter is intended for therapeutic application in human beings. The reasons for this are the possible contamination of the finished pharmaceutical active ingredient with cyanide ions, the toxicity of the reagent itself and finally the danger of its use. It is known to persons skilled in the art that sodium cyanoborohydride can release hydrocyanic acid if the pH of the reaction environment is acid enough and it is known that hydrocyanic acid is a powerful poison, since it competes with oxygen for hemoglobin coordination. As a consequence of this, particular care must be taken in its use and in the disposal of the production wastewater, which can be contaminated by cyanides. Not least, one must consider that the cost of sodium cyanoborohydride is considerable.
• [0031]
  To conclude, it can be seen that more than 50 years after the publication of its first synthesis dated 1962, there are still numerous disadvantages or limitations in the synthesis paths developed in the past decades in the literature for the preparation of fenfluramine.
• [0032]
  Moreover, recently there has been renewed pharmaceutical interest in the fenfluramine molecule, since the possibility of its therapeutic use in severe disorders of infancy has appeared in the medical literature. For example, mention can made of Ceulemans et al., Epilepsia, 53(7), pages 1131 to 1139, 2012.
• [0033]
  According to a certain part of medical literature, fenfluramine might therefore be interesting as a medication in a chronic therapy for the treatment of symptoms of epilepsy and other correlated severe disorders.
• [0034]
  Based on recent medical developments, therefore, the need exists for a synthesis method that is better than the existing ones and can overcome in particular the disadvantages of the processes that are present in the literature. Particularly important, in view of use in chronic therapies for children such as epilepsy and other severe disorders, it would be fundamentally important to identify a path for synthesis of the active ingredient fenfluramine or of isomers thereof and/or analogs thereof that does not entail the use of heavy metals and/or transition metals, which in a chronic therapy might accumulate in the body of the patients over the years, with severe consequences on health.
• [0035]
  More generally, it is desirable to identify a synthesis path that uses reagents from which (or from the transformation products of which) it is then possible to easily purify fenfluramine (or isomers and/or analogs thereof).
• [0036]
  It would be equally desirable to identify a synthesis path that comprises a small number of synthesis steps and uses reagents that are widely commercially available and easy to use.
• [0037]
  At the same time, the new identified synthesis path should avoid if possible the formation of byproducts.

EXAMPLES

• [0082]
  The present invention is exemplified by, but not limited to, the following examples:

Example 1 – Synthesis of fenfluramine

• [0083]
  A suspension of sodium hydroxide (34.62 g – 0.866 mol, 3.5 eq) in 170 mL of methanol, under mechanical agitation, receives the addition, drop by drop, over the course of 30 minutes, of a solution of ethylamine hydrochloride (70.59 g – 0.866 mol, 3.5 eq) in 165 mL of methanol, followed by 1-(3-trifluoromethyl)phenyl-propan-2-one (50 g – 0.247 mol). The mixture is left under agitation at 20°C for 4.5 hours, then cooling to 0°C is performed and a solution of sodium borohydride (9.36 g – 0.247 mol) in 19 mL of sodium hydroxide 1M in water is then added drop by drop, keeping the temperature below 10°C. The reaction is then left under agitation at 20°C for another 2 hours. Once the reaction is complete, 270 mL of methanol are removed at a reduced pressure at 40°C and then 200 mL of water are added and the mixture is extracted with heptane (200 mL). The aqueous phase is eliminated and the organic phase is washed with water (200 mL x 3). The organic phase is concentrated at 50°C at reduced pressure to yield free base fenfluramine as colorless oil. Yield: 72%; purity: 77% – as listed in test 3 of table A above.

Example 2 – Purification of fenfluramine

• [0084]
  Purification of free base fenfluramine can be performed in two ways:
• distillation of the free base
• crystallization of the fenfluramine hydrochloride salt
• [0085]
  Depending on the degree of purity that is desired, both purification processes are performed in sequence (distillation first and then crystallization), or only one of the two purification processes is performed.

Example 2a – Distillation:

• [0086]
  Free base fenfluramine (10 g), prepared as in Example 1, is distilled under reduced pressure with a distillation column of the Vigreux type: the distillation heads are eliminated, the fraction that is distilled at 89-90°C at 6 mmHg, which is the active ingredient fenfluramine (8.5 g) with a high degree of purity, is collected.

Example 2b – Conversion into hydrochloride salt and crystallization:

• [0087]
  Crude fenfluramine, prepared as in Example 1, or purified fenfluramine as in Example 2a, is dissolved in 125 mL of ethyl acetate, and cooling is performed to 0°Celsius under agitation. 272 mL of a solution of 1M HCl in ethyl acetate are added drop by drop at 0°C. The precipitate that forms is filtered and washed with ethyl acetate (125 mL x 2) to yield approximately 55 g of solid fraction. The solid fraction is crystallized by 2-butanol (260 mL), keeping the solid for 22 hours at 3°C under slow agitation before filtering it. Filtering is performed and washing is performed with cold 2-butanol. The solid fraction, fenfluramine hydrochloride, is dried in a vacuum stove, yielding 51.7 g of product. A DSC of the resulting product is shown in Figure 3 .

CLIP

• Synthetic Method of Dexfenfluramine hydrochloride
• (CAS NO.: ), with its systematic name of (S)-N-Ethyl-alpha-methyl-m-(trifluoromethyl)phenethylamine hydrochloride, could be produced through many synthetic methods.Following is one of the synthesis routes:The action of d-camphoric acid on (rac)-fenfluramine (I) affords the camphorate of (+)-fenfluramine (II). After purification of this salt by crystallization, sodium hydroxide in methylene chloride is added, forming (+)-fenfluramine (III) after removal of camphoric acid. Finally, the action of hydrogen chloride in methyl cyclohexane on (+)-fenfluramine produces the corresponding salt: (+)-fenfluramine hydrochloride.

PAPER

https://www.designer-drug.com/pte/12.162.180.114/dcd/chemistry/fenfluramine.html

Fenfluramine 1 is the active ingredient of a obesity drug acting on the digestion of carbohydrates, the activity being restricted mainly to the S enantiomer [1, 2], which can be obtained by separation of the diastereoisomers [3] or by preferential crystallisation of derivates, which were identified of being conglomerates [4]. Only two syntheses of optical active fenfluramine have been described until now: one by stereoselective reduction of the imine derived from the ketone 2 and (R) or (S)-alpha-phenylethylamine [5], the other starting from (S)-alanine [6]. Two recent publications [7, 8] about the synthesis of (S)-fenfluramine via the intermediate alcohol (S)-3 (scheme 1) made us publish our previous results [9]. Through yeast reduction of the ketone 2, the authors obtain the alcohol (S)-3, the configuration of which they inverse in three steps. The alcohol (R)-3, via the intermediate tosylate (R)-4a and further the azide (S)-5 leads to the amine (S)-6 after reduction and finally to the (S)-fenfluramine (S)-1 after reductive amination in presence of acetaldehyde:

The (S)-fenfluramine is such obtained in 7 steps starting from the alcohol (S)-3 or in 4 steps from the alcohol (R)-3.

In this article we present a new way of preparing the two enantiomers of the alcohol 3, a new two step synthesis of (S)-fenfluramine starting from the alcohol (R)-3, a one step synthesis of (S)-fenfluramine starting from the azide (S)-5, which doesn’t pass over the intermediate primary amine (S)-6 and finally a much faster process (3 steps) of preparing (S)-fenfluramine starting from the alcohol (S)-3.

Results and discussion

Synthesis of 1-[3-(trifluoromethyl)phenyl]propan-2-ol (R)-3

The racemic alcohol is seldom mentioned. It’s one of the metabolites of fenfluramine in the human body, secreted in urine [10]. It can also be obtained by metabolic transformations of an oxime by different kinds of microorganisms [11]. It was used as an intermediate for the synthesis of a family of anorexics [12] and a family of antispasmodic and psychotherapeutic agents [13]. It was obtained by the reaction of methyloxirane 7 with the magnesium compound 8, with a yield of 50%. This same reaction was described earlier as being little regioselective [14], a fact we observed too [9].

The only synthesis of the optically active alcohol 3 is the reduction of the ketone 2 with yeast as described above. One abtains the S enantiomer, the R enantiomer is obtained by inversion.

On our part we used the condensation of the commercial [15] methyloxirane (R)-7, of which many syntheses are known [16], with the magnesium compound 8 and cuprous chloride [17, 18].

The yield is about 90% and the reaction very selective (purity GC: 93%). The optical purity of the methyloxirane 7 was determined by 1H-NMR in presence of the europium complex Eu(hfc)3 [19]. The optical purity of the alcohol (R)-3 was obtained by 1H-NMR and HPLC over silica of the Mosher derivate [20]. The comparison of these values show that the chiral centre is preserved. This procedure has the advantage of allowing us the preparation of the alcohol (S)-3 with the same reaction, because the methyloxirane (S)-7 is also commercially available and multiple syntheses are known [21].

Two step synthesis of the fenfluramine (S)-1 starting from the alcohol (R)-3

With the goal of obtaining the simplest procedure we have studied at first the transformation of the alcohol (R)-3 into fenfluramine (S)- 1 in two steps via the intermediate of the easily obtained sulfonates (R)-4:

The substitution of the mesitylate (R)-4b and the tosylate (R)-4a with ethylamine was realised with medium yields always between 40 and 50% in spite of the large number of conditions tested: solvents (DMSO, DMF, ethanol, ethylamine), different dilutions (in proportions from 1 to 5) and temparatures from 50 to 160°C (with different times of contact). With the triflate (R)-4c the yield of the substitution is 60% but under non comparable conditions (-20°C in acetonitrile) because of its higher reactivity. In all cases the non aminated, and thus easily separated, byproducts are mainly the alkenes 9, 10Z and 10E (10E >> 10Z > 9).

The enantiomeric purity of the amine (S)-1 is analysed by HPLC chromatography through silica of the camphanylated derivate [22] and compared to the previously analysed alcohol (R)-3: we have thus shown that the optical centre is conserved during the nucleophilic substitution. One had indeed to fear that due to the participation of the aromatic ring as neighbour group there could be partial or complete racemisation with an phenonium ion as intermediate. With the results obtained, which match with the literature [23-26], one can suppose that the trifluoromethyl group in meta position is sufficiently deactivating the aromatic ring in order to prevent participation in the substitution. We probably have thus in our case a pure nucleophilic SN2 substitution in competition with an elimination reaction. We believe that this elimination reaction is due to the simultaneous nucleophilic and basic properties of the ethylamine.

Although the yields are medium, this method has the advantage of being relatively fast because it permits to prepare fenfluramine (S)-1 starting from the alcohol (R)-2 in two steps instead of four [7, 8]. As far as we know it was never mentioned in literature.

Synthesis of fenfluramine (S)-1 from 2-azido-1-[3-(trifluoromethyl)phenyl]propane (S)-5

The substitution of the mesylate (R)-4b by sodium azide (scheme 4), an only slightly basic nucleophile compared to ethylamine, forms no elimination side products. One obtains the optically pure azide (S)-5 with a yield of 95%.

The enantiomeric purity couldn’t be directly analysed on the azide (S)-5. Only for analytical purposes did we reduce it into the amine (S)- 6. Among the numerous methods for the reductions of azides to amines mentioned in the literature [27] we chose the catalytic hydrogenation with 5% Pd on calcium carbonate at standard temperature and pressure [27f]. The HPLC analysis through silica column of the champhanyl derivate [22] of the amine (S)-6 such obtained shows that the enantiomeric centre was totally inverted during the substitution when compared to the enantiomeric purity of the alcohol (R)-3.

The reductive amination of the amine 6 in presence of acetaldehyde is known for a long time [28]. It was used recently in the works listed in the introduction [7, 8]. On our part, we propose another synthetic route for fenfluramine (S)-1 starting from the azide (S)-5 which does not go via the primary amine (S)-6 (Schema 5).

The reaction of Staudinger, reacting a stoichiometric quantity of triethylphosphite on the azide (S)-5 in THF at room temperature [29], gives quantitative yields of the phosphorimide 11 in 48 hours. It’s total conversion into the phosphoramide 13, by reacting with ethyl iodide [30] could not be realised [9]. We always obtained different mixtures of the phosphoramides 12 and 13 (referential compounds prepared from the amines 6 and 1). We also noted that the phosphorimide 11 can’t be isolated. When the solvent is evaporated, a partly transformation into the phosphoramide 12 takes place. This transformation is completed in less then 2h by simple heating to 100°C under argon after evaporation of the solvent. Because the phosphorimides are strongly basic compounds, we believe that an intramolecular arrangement of the phosphorimide, pictured in Schema 6, takes place.

Having the phosphoramide 12, we investigated the alkylation into the phosphoramide 13 in DMF at room temperature [31, 32]: one deprotonates with sodium hydride then alkylates with diethyl sulfate. After treatment with hydrogen bromide [33], one obtains fenfluramine 1 with a yield of 85% and a purity of 97% (GC).

With the goal of simplifying the reaction scheme by avoiding the isolation of the intermediates we have again studied the transformation 5 -> 11 -> 12 in DMF (Schema 7). First, we noted that the reaction of Staudinger can be directly realised in this solvent. Thereafter we pinned down the transformation of the phosphorimide 11 into the phosphoramide 12 by reaction with water [34]. One then proceeds as described above. The transformation is thus performed without isolation of a single intermediate with a yield of 83%.

HPLC analysis on silica column of the camphanyl derivate of the amine (S)-1 [22] shows that the optical centre is conserved during the whole transformation.

Synthesis via the intermediate 2-chloro-1-[3-(trifluoromethyl)phenyl]propane 14

The yeast reduction of the ketone 2 gives the alcohol (S)-3, of which the authors have inverted the configuration to get the pharmacological active S enantiomer of fenfluramine [7, 8]. Independent research, using the epoxidation method of Sharpless [9, 35] lead us too to the alcohol (S)- 3 which we tried to convert into fenfluramine (S)-1 using a different method. The reaction scheme we kept uses the chloride 14 and proceeds via two inversions of the optical centre (scheme 8). Not owning enough alcohol (S)-3 during the studies, we tested the principle starting with the alcohol (R)-3, produced earlier, and studied the transformation into the azide (R)-5 (scheme 8), the latter being able to lead to (R)-fenfluramine using different methods, like the one outlined above:

It is well known that the action of thionylchloride on an optically active alcohol gives the corresponding chlorine derivate, with inversion of the configuration in presence of bases and with retention of the configuration in the other case. We have performed the reaction with a catalytical amount of pyridine. One thus obtains the chloride (S)-14 with 91% yield and a purity of 91% (GC): it contains 9% of the elimination products 9, 10Z and 10E which are not separable by chromatography on silica.

The direct substitution of the chloride 14 with ethylamine with similar conditions to those used for the mesylate (R)-4b (EtNH2, DMSO, 110°C, 5h30 or EtNH2 (solvent and reactant), 140°C, 5h), gives mainly the elimination products. The yield of fenfluramine is below 10%.

By action of sodium azide in DMSO, on the other hand, one obtains the azide (R)-5 with a yield of 78%, the elimination products formed here or in the last step can be removed by chromatography on silica. HPLC analysis on silica of the camphanyl derivate of the amine (R)-6 [22] obtained by catalytic reduction of the azide (R)-5 has confirmed the double inversion without racemisation after comparison with the starting alcohol (R)-3. Then the fenfluramine (R)-1 is prepared without racemisation with a 83% yield starting from the azide (R)-5 like detailed above.

This procedure with two inversions allows to transform the alcohol 3 in the azide 5 with the same configuration in two steps with a global yield (non optimised) of 70% and without racemisation. It’s thus preferred over the recently published one [7, 8], which needs 5 steps for a lower global yield (55%) and in addition features an epimerisation of 10% [8]. It’s a promising way to fenfluramine (S)-1 starting from the alcohol (S)- 3.

References

1. ^ “Fintepla- fenfluramine solution”. DailyMed. Retrieved 8 January 2021.
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12. ^ Jump up to:^a b Dahl, C. F.; Allen, M. R.; Urie, P. M.; Hopkins, P. N. (2008). “Valvular Regurgitation and Surgery Associated with Fenfluramine Use: An Analysis of 5743 Individuals”. BMC Medicine. 6: 34. doi:10.1186/1741-7015-6-34. PMC 2585088. PMID 18990200.
13. ^ Jump up to:^{a b c d e f g h i j k l m} Donald G. Barceloux (3 February 2012). Medical Toxicology of Drug Abuse: Synthesized Chemicals and Psychoactive Plants. John Wiley & Sons. pp. 255–262. ISBN 978-1-118-10605-1. Archived from the original on 9 May 2018.
14. ^ Mann SC, Caroff SN, Keck PE, Lazarus A (20 May 2008). Neuroleptic Malignant Syndrome and Related Conditions. American Psychiatric Pub. pp. 111–. ISBN 978-1-58562-744-8.
15. ^ Jump up to:^{a b c d e f g h i} Rothman RB, Clark RD, Partilla JS, Baumann MH (2003). “(+)-Fenfluramine and its major metabolite, (+)-norfenfluramine, are potent substrates for norepinephrine transporters”. J. Pharmacol. Exp. Ther. 305 (3): 1191–9. doi:10.1124/jpet.103.049684. PMID 12649307. S2CID 21164342.
16. ^ Jump up to:^{a b c d e f g} Setola V, Hufeisen SJ, Grande-Allen KJ, Vesely I, Glennon RA, Blough B, Rothman RB, Roth BL (2003). “3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) induces fenfluramine-like proliferative actions on human cardiac valvular interstitial cells in vitro”. Mol. Pharmacol. 63 (6): 1223–9. doi:10.1124/mol.63.6.1223. PMID 12761331. S2CID 839426.
17. ^ Nestler, E. J. (2001). Molecular Neuropharmacology: A Foundation for Clinical Neuroscience. McGraw-Hill.
18. ^ Giuseppe Di Giovanni; Vincenzo Di Matteo; Ennio Esposito (2008). Serotonin-dopamine Interaction: Experimental Evidence and Therapeutic Relevance. Elsevier. pp. 393–. ISBN 978-0-444-53235-0.
19. ^ Fitzgerald LW, Burn TC, Brown BS, Patterson JP, Corjay MH, Valentine PA, Sun JH, Link JR, Abbaszade I, Hollis JM, Largent BL, Hartig PR, Hollis GF, Meunier PC, Robichaud AJ, Robertson DW (2000). “Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine”. Mol. Pharmacol. 57 (1): 75–81. PMID 10617681.
20. ^ Jump up to:^a b c Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, Partilla JS (January 2001). “Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin”. Synapse. 39 (1): 32–41. doi:10.1002/1098-2396(20010101)39:1<32::AID-SYN5>3.0.CO;2-3. PMID 11071707.
21. ^ Rothman RB, Baumann MH (2002). “Therapeutic and adverse actions of serotonin transporter substrates”. Pharmacol. Ther. 95 (1): 73–88. doi:10.1016/s0163-7258(02)00234-6. PMID 12163129.
22. ^ Connolly, H. M.; Crary, J. L.; McGoon, M. D.; Hensrud, D. D.; Edwards, B. S.; Edwards, W. D.; Schaff, H. V. (1997). “Valvular Heart Disease Associated with Fenfluramine-Phentermine”. New England Journal of Medicine. 337 (9): 581–588. doi:10.1056/NEJM199708283370901. PMID 9271479.
23. ^ Weissman, N. J. (2001). “Appetite Suppressants and Valvular Heart Disease”. The American Journal of the Medical Sciences. 321 (4): 285–291. doi:10.1097/00000441-200104000-00008. PMID 11307869. S2CID 46466276.
24. ^ FDA September 15, 1997. FDA Announces Withdrawal Fenfluramine and Dexfenfluramine (Fen-Phen) Archived 2013-07-19 at the Wayback Machine
25. ^ “Drugs banned in India”. Central Drugs Standard Control Organization, Dte.GHS, Ministry of Health and Family Welfare, Government of India. Archived from the original on 2015-02-21. Retrieved 2013-09-17.
26. ^ “FDA Approves FINTEPLA (fenfluramine) for the Treatment of Seizures Associated with Dravet Syndrome” (Press release). Zogenix Inc. 25 June 2020. Retrieved 25 June 2020 – via GlobeNewswire.
27. ^ “Fenfluramine Orphan Drug Designations and Approvals”. U.S. Food and Drug Administration (FDA). 24 December 1999. Retrieved 25 June 2020.
28. ^ “Fenfluramine Orphan Drug Designations and Approvals”. U.S. Food and Drug Administration (FDA). 24 December 1999. Retrieved 25 June 2020.
29. ^ “Fintepla: Pending EC decision”. European Medicines Agency (EMA). 16 October 2020. Retrieved 16 October 2020. Text was copied from this source which is © European Medicines Agency. Reproduction is authorized provided the source is acknowledged.
30. ^ Jump up to:^a b John Calvin M. Brust (2004). Neurological Aspects of Substance Abuse. Butterworth-Heinemann. pp. 117–. ISBN 978-0-7506-7313-6.
31. ^ United States. Congress. Senate. Select Committee on Small Business. Subcommittee on Monopoly and Anticompetitive Activities (1976). Competitive problems in the drug industry: hearings before Subcommittee on Monopoly and Anticompetitive Activities of the Select Committee on Small Business, United States Senate, Ninetieth Congress, first session. U.S. Government Printing Office. pp. 2–.
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33. ^ Drug Addiction II: Amphetamine, Psychotogen, and Marihuana Dependence. Springer Science & Business Media. 27 November 2013. pp. 249–. ISBN 978-3-642-66709-1.
34. ^ “Zogenix ends enrolment in second Phase lll trial of ZX008”. Drug Development Technology. Kable. 1 February 2018. Archived from the original on 2018-02-04. Retrieved 3 February 2018.
35. ^ “A Two-Part Study to Investigate the Dose-Ranging Safety and Pharmacokinetics, Followed by the Efficacy and Safety of ZX008 (Fenfluramine Hydrochloride) Oral Solution as an Adjunctive Therapy in Children ≥2 Years Old and Young Adults With Dravet Syndrome”. ClinicalTrials.gov. Archived from the original on 29 September 2017. Retrieved 9 May 2018.
36. ^ “Zogenix drug cuts seizure rate in patients with severe epilepsy”. statnews.com. 29 September 2017. Archived from the original on 30 April 2018. Retrieved 9 May 2018.

Further reading

• Gustafsson BI, Tømmerås K, Nordrum I, Loennechen JP, Brunsvik A, Solligård E, Fossmark R, Bakke I, Syversen U, Waldum H (March 2005). “Long-term serotonin administration induces heart valve disease in rats”. Circulation. 111 (12): 1517–22. doi:10.1161/01.CIR.0000159356.42064.48. PMID 15781732.
• Welch, J. T.; Lim, D. S. (2007). “The Synthesis and Biological Activity of Pentafluorosulfanyl Analogs of Fluoxetine, Fenfluramine, and Norfenfluramine”. Bioorganic & Medicinal Chemistry. 15 (21): 6659–6666. doi:10.1016/j.bmc.2007.08.012. PMID 17765553.

External links

• “Fenfluramine”. Drug Information Portal. U.S. National Library of Medicine.
• “Fenfluramine hydrochloride”. Drug Information Portal. U.S. National Library of Medicine.
• Inchem.org – Fenfluramine hydrochloride

CLIP

http://www.inchem.org/documents/pims/pharm/pim938.htm

////////////Fenfluramine, 塩酸フェンフルラミン , dravet, AHR-3002, ZX-008, Fintepla

CCNC(C)CC1=CC(=CC=C1)C(F)(F)F.Cl

PATENT

https://patents.google.com/patent/US20170174613A1/en

• [0093]
  Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).
• [0094]
  Compounds as described herein can be purified by any purification protocol known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969.
• [0095]
  During any of the processes for preparation of the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie”, Houben-Weyl, 4^th edition, Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Protein”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
• [0096]
  The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. A variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.

Example 11. Fenfluramine Nomenclature & Structure

• [0097]
  Chemical Abstract Service (CAS) Registry Number (RN): 404-82-0 (HCl Salt), 458-24-2 (Parent Free Base)
• [0098]
  Chemical Name: N-ethyl-α-methyl-3-(trifluoromethyl)-benzeneethanamine hydrochloride (1:1). Other Names: Fenfluramine HCl, DL-Fenfluramine, (±)-Fenfluramine
• [0099]
  Structure of Hydrochloride Salt:
• [0100]
  Stereochemistry: Fenfluramine HCl has one chiral center and is being developed as the racemate and contains dexfenfluramine and levofenfluramine
• [0101]
  Molecular Formula of hydrochloride salt: C12H16F3N.HCl
• [0102]
  Molecular Mass/Weight: 267.72 g/mol

2. General Properties

• [0103]
  Table 1 summarizes the chemical and physical properties of Fenfluramine HCl.
• TABLE 1 General Properties of Fenfluramine HCl Drug Substance Property Result Appearance (color, White to off-white powder physical form) DSC (melting 170° C. (melt/sublimation) point)^a TGA Onset 147° C. 0.03% at 150° C. 91% at 220° C. (evaporation) pKa (water) 10.15-10.38 Solubility (mg/mL) Resultant pH 25° C. 37° C. Solubility pH 6.69 (water) 54.13 71.22 (Aqueous) pH 1.73 buffer 25.34 53.68 pH 3.43 buffer 29.50 61.97 pH 6.41 buffer 37.42 95.60 0.9% NaCl (water) 22.98 — Solvent Solubility 25° C. (mg/mL) Solubility (Organic Ethanol 150 Solvents) Dichloromethane 30-35 Ethyl Acetate, 1-5 mg Tetrahydrofuran, Toluene, Acetonitrile UV Absorption Maxima: 210, 265 nm Solution pH (water) 6.69 Hygroscopicity @30% RH: ~0.05% (Dynamic Vapor @60% RH: ~0.07% Sorption (DVS) @90% RH: ~0.20%^a) Polymorphism Fenfluramine HCl has been consistently isolated as a single crystalline Form 1 as determined by DSC and x-ray powder diffraction (XRPD) Solvation/Hydration Fenfluramine HCl is isolated as a nonhydrated, nonsolvated solid Solution Stability 8 weeks @ pH 6.7 phosphate buffer medium at 40° C. and 60° C. using concentrations of 0.5, 2.5 and 5.0 mg/ml. All conditions, no new impurities >0.1% by HPLC. Solid Stability 8 weeks @ 40° C., 60° C. and 80° C. 7 days at 150° C. All conditions, no new impurities >0.1% by HPLC.

3. Synthesis of Fenfluramine Drug Substance

• [0104]
  Scheme 3.1 shows a 2-step route of synthesis used to manufacture initial clinical supplies of Fenfluramine HCl from ketone (2). The batch size is 4 kg performed in laboratory glassware (kilo lab). No chromatography is required and the process steps are amenable to scale-up. In process 1 there is one isolated intermediate Fenfluramine Free Base (1) starting from commercially supplied 1-(3-(trifluoromethyl)phenyl) acetone (Ketone 2). All steps are conducted under cGMPs starting from Ketone (2).
• [0105]
  Scheme 3.2 shows a 4-step route of synthesis to Fenfluramine HCl that can be used for commercial supply. Route 2 utilizes the same 2-step process used by Route 1 to convert Ketone (2) to Fenfluramine HCl with the exception that Ketone (2) is synthesized under cGMP conditions starting from 3-(Trifluoromethyl)-phenyl acetic acid (Acid 4). Bisulfate Complex (3) is an isolatable solid and can be purified before decomplexation to Ketone (2). In-situ intermediates which are oils are shown in brackets. Batch sizes of 10 Kg are performed. Commercial batch sizes of 20 kg are performed in fixed pilot plant equipment. Steps 1-2 of Scheme 3.2 to manufacture Ketone (2) have been demonstrated on a 100 g scale to provide high purity ketone (2) of >99.8% (GC & HPLC). Conversion of Ketone (2) to Fenfluramine using either Route 1 or 2 has provided similar purity profiles.
• Starting materials are designated by enclosed boxes. Bracketed and non bracketed compounds respectively indicate proposed in-situ and isolated intermediates. NMI=N-Methyl Imidazole.

4.1. Narrative Description (Route 1)

• [0106]
  Step 1: Reductive Amination (Preparation of Fenfluramine Free Base 1)
• [0107]
  A solution of ethylamine, water, methanol, and 1-(3-(trifluoromethyl)phenyl) acetone (Ketone 2) was treated with sodium triacetoxyborohydride and stirred for 16 h at 25° C. at which time HPLC analysis (IPC-1; In Process Control No. 1) showed the reaction to be complete and sodium hydroxide solution was added until pH>10. Toluene was added and the phases separated, and the aqueous phase (IPC-2) and organic phase (IPC-3) are checked for remaining Fenfluramine and Fenfluramine alcohol and the organic phase was reduced. Purified water was added and the pH adjusted to <2 using conc. HCl and the phases were separated. The aqueous phase was washed with toluene and the toluene phase (IPC-4) and the aqueous phase (IPC-5) was checked for Fenfluramine and Fenfluramine alcohol content. The aqueous phase containing product is pH adjusted to >10 using sodium hydroxide solution. The basic aqueous phase was extracted with MTBE until removal of Fenfluramine from the aqueous phase was observed by HPLC (<0.5 mg/ml) (IPC-6). The organic phase was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo to give the intermediate product Fenfluramine Free Base 1 as a pale yellow oil tested per specifications described herein which showed by NMR the material to contain 2.93% toluene giving an active yield of 88.3% with a purity of 98.23% by HPLC (0.67% Fenfluramine alcohol).
• [0108]
  Step 2: Salt Formation (Preparation of Fenfluramine HCl)
• [0109]
  To a flask was charged ethanol and acetyl chloride. The solution was stirred slowly overnight before ethyl acetate was added. The HCl in ethyl acetate solution formed was polish filtered into a clean carboy and retained for later use. To a vessel was added Fenfluramine free base 1 and MTBE. The Fenfluramine solution in MTBE was collected in two carboys before the vessel was cleaned and checked for particulate residue. The Fenfluramine solution was polish filtered into a vessel and cooled and HCl in ethyl acetate solution was added giving a final pH of 6-7. The batch was stirred for 1 h and filtered. The product was dried under vacuum at 40° C. The product (96.52% yield) was tested per IPC-7 had a purity of 99.75% by HPLC and GC headspace analysis showed MTBE (800 ppm) and EtOAc (150 ppm) to be present. The product was then tested per specifications shown herein.

4.2. Narrative Description (Route 2)

• [0110]
  Step 1: Preparation of Ketone Bisulfite Adduct
• [0111]
  Procedure: Charge acetic anhydride, (2.8 vol, 3.0 wt, 5.0 eq.) to a vessel and commence stirring. Cool the solution to −5 to 5° C., targeting −4° C. Charge 1-methylimidazole, (0.2 vol, 0.21 wt, 0.5 eq.) to the mixture at −5 to 5° C. Caution: very exothermic. If necessary, adjust the temperature to 0 to 5° C. Charge ZX008 acid, (1.00 wt, 1.0 eq.) to the mixture at 0 to 5° C. Caution: exothermic. Stir the mixture at 0 to 5° C. until ≦2.1% area ZX008 acid by HPLC analysis, typically 7 to 9 hours. Charge 15% w/w sodium chloride solution (2.0 vol) to the mixture at 0 to 5° C., 60 to 90 minutes. Caution: very exothermic which will be slightly delayed. Warm the mixture to 18 to 23° C. over 45 to 60 minutes and continue stirring for a further 30 to 45 minutes at 18 to 23° C. Charge TBME, (5.0 vol, 3.7 wt) to the mixture and stir for 10 to 15 minutes at 18 to 23° C. Separate the aqueous layer and retain the organic layer. Back-extract the aqueous layer with TBME, (2×3.0 vol, 2×2.2 wt) at 18 to 23° C. retaining each organic layer. Adjust the pH of the combined organic layer to pH 6.5 to 9.0, targeting 7.0 by charging 20% w/w sodium hydroxide solution (5.3 to 8.3 vol) at 18 to 23° C. Caution: exothermic. Separate the aqueous layer and retain the organic layer. Wash the organic layer with 4% w/w sodium hydrogen carbonate solution (2×3.0 vol) at 18 to 23° C. Determine the residual ZX008 acid content in the organic layer by HPLC analysis, pass criterion ≦0.10% area ZX008 acid. Wash the organic layer with purified water, (2×3.0 vol) at 18 to 23° C. Concentrate the organic layer under reduced pressure to ca. 2 vol at 40 to 45° C., targeting 43° C.
• [0112]
  Determine the w/w assay of ZX008 ketone (WIP) in the mixture by 1H-NMR analysis for information only and calculate the contained yield of ZX008 ketone (WIP) in the mixture. Note: This step can be removed from the process since the process is robust and consistently delivers 80 to 90% th yield. The achieved yield was factored into the charges of the subsequent steps.
• [0113]
  Charge n-heptane, (4.0 vol, 2.7 wt) to the mixture at 40 to 45° C., targeting 43° C. Concentrate the mixture to ca. 2 vol at 40 to 45° C., targeting 43° C. Determine the TBME content in the mixture by 1H-NMR analysis, (pass criterion ≦5.0% w/w TBME vs. ZX008 ketone). Charge n-heptane, (2.4 vol, 1.6 wt) at 40 to 45° C., targeting 43° C., vessel A. To vessel B, charge sodium metabisulfite, (0.82 wt, 0.88 eq.) at 18 to 23° C. To vessel B, charge a solution of sodium hydrogen carbonate, (0.16 wt, 0.4 eq.) in purified water, code RM0120 (2.0 vol) at 18 to 23° C. followed by a line rinse with purified water, code RM0120 (0.4 vol) at 18 to 23° C. Caution: gas evolution. Heat the contents of vessel B to 40 to 45° C., targeting 43° C. Charge the contents from vessel A to vessel B followed by a line rinse with n-heptane, (0.8 vol, 0.5 wt) at 40 to 45° C., targeting 43° C. Stir the mixture for 1 to 1.5 hours at 40 to 45° C., targeting 43° C. Charge n-heptane, code RM0174 (3.2 vol, 2.2 wt) to the mixture with the temperature being allowed to cool to 18 to 45° C. at the end of the addition. Cool the mixture to 18 to 23° C. at approximately constant rate over 45 to 60 minutes. Stir the mixture at 18 to 23° C. for 1.5 to 2 hours.
• [0114]
  Sample the mixture to determine the residual ZX008 ketone content by 1H-NMR analysis, (pass criterion ≦10.0% mol, target 5.0% mol ZX008 ketone vs. ZX008 ketone bisulfite adduct). Filter the mixture and slurry wash the filter-cake with n-heptane, (2×2.0 vol, 2×1.4 wt) at 18 to 23° C. Dry the solid at up to 23° C. until the water content is <10.0% w/w water by KF analysis according to AKX reagent. At least 16 hours. Determine the w/w assay of the isolated ZX008 ketone bisulfite adduct by 1H-NMR analysis and calculate the contained yield of ZX008 ketone bisulfite adduct.
• [0115]
  Yields and Profiles: The yield for the stage 1 Demonstration batch is summarized Table below. Input: 1700.0 g uncorr., acid, 99.50% area (QC, HPLC), 2-isomer not detected, 4-isomer 0.02% area, RRT1.58 (previously not observed) 0.48% area as per the preparative method. The analytical data is summarized in Table 1A below.
• TABLE 1A Table for isolated yields for step 1 Demonstration batch Corr. % area Reference Corr. Yield % w/w (HPLC, number Input Output (% th)** (¹H-NMR)* QC) Comments Batch A1 1700.0 g 1500.1 g 89.1 45.0 —.— Crude ketone as TBME sol. Batch A2 1500.1 g 1716.1 77.8 76.0 98.15 Bisulfite adduct only 67.3 Overall product
• [0116]
  Step 2: Preparation of Ketone
• [0117]
  Procedure: Charge toluene, (5.0 vol, 4.3 wt), and purified water, (5.0 vol) to the vessel and commence stirring. If necessary, adjust the temperature to 18 to 23° C. and charge ZX008 ketone bisulfite adduct, (1.00 wt corrected for % w/w assay) to the mixture at 18 to 23° C. Charge 20% w/w sodium hydroxide solution to the mixture at 18 to 23° C. adjusting the pH of the mixture to pH 8.0 to 12.0, targeting 9.0 (0.5 to 1.0 vol).
• Separate the lower aqueous layer and retain the top organic layer. Wash the organic layer with purified water, (3.0 vol) at 18 to 23° C. Concentrate the organic layer under reduced pressure to ca. 2 vol at 45 to 50° C., targeting 48° C. Charge methanol, (5.0 vol, 4.0 wt) to the mixture at 45 to 50° C., targeting 48° C. Re-concentrate the mixture under reduced pressure to ca. 2 vol at 45 to 50° C., targeting 48° C. Repeat steps 7 and 8 once before continuing with step 9. Cool the mixture to 18 to 23° C. Clarify the mixture into a tared, suitably-sized drum followed by a methanol (1.0 vol, 0.8 wt) line rinse at 18 to 23° C. Determine the w/w assay of ZX008 ketone (WIP) in the mixture by 1H-NMR analysis and calculate the contained yield of ZX008 ketone (WIP) in the mixture. Determine the toluene content in the mixture by 1H-NMR analysis.
• [0118]
  Yields and Profiles: The yield for the step 2 Demonstration batch is summarized in Table 1B below. Input: 1200.0 g corr. Ketone bisulfite adduct, 76.0% w/w assay (NMR, using DMB as internal standard in d₆-DMSO), (1.00 eq, 1.00 wt corr. for w/w assay) for input calculation.
• TABLE 1B Table for isolated yields for step 2 Demonstration batch % w/w % area Corr. Corr. Corr. Yield (¹H- (HPLC, Input Output (% th) NMR)* QC) Comments 1200.0 g 858.15 g 108.3 25.5 99.31 Purified ketone
• [0119]
  Step 3: Preparation of Fenfluramine HCl Crude
• [0120]
  Procedure: Charge the ZX008 ketone (corr. for assay, 1.00 wt, 1.00 eq. isolated as solution in MeOH in stage 2) to a vessel. Charge methanol, code RM0036 (5.0 vol, 4.0 wt) to the mixture at 18 to 23° C. Cool the solution to 0 to 5° C. Charge 70 wt % aqueous ethylamine solution (1.3 vol, 1.6 wt, 4.0 eq) to the mixture at 0 to 10° C., over 15 to 30 minutes, followed by a line rinse with methanol (1.0 vol, 0.8 wt). Warm the mixture to 15 to 20° C. and stir the mixture for a further 60 to 70 minutes at 15 to 20° C. Adjust the mixture to 15 to 18° C. if required, targeting 15° C. Charge sodium triacetoxyborohydride (2.4 wt, 2.25 eq.) to the mixture in approximately 10 portions, keeping the mixture at 15 to 20° C., targeting 17° C. Addition time 1.5 to 2 hours. Caution: Exothermic. Stir the mixture at 15 to 20° C. until complete by HPLC analysis, pass criterion ≦3.0% area ZX008 ketone, typically 2 to 3 hours. Adjust the pH of the mixture to pH>12 by charging 20% w/w aqueous sodium hydroxide solution (5.0 to 6.0 vol) to the mixture at 15 to 40° C. Addition time 10 to 30 minutes. Caution: Exothermic. If necessary, adjust the temperature to 18 to 23° C. Extract the mixture with toluene (3×3.0 vol, 3×2.6 wt) at 18 to 23° C., retaining and combining the top organic layer after each extraction. Wash the combined organic layer with purified water, (1.0 vol) at 18 to 23° C. Heat the mixture to 40 to 50° C., targeting 48° C. Concentrate the mixture under reduced pressure at constant volume maintaining ca. 5 vol by charging the organic layer at approximately the same rate as the distillation rate at 40 to 50° C., targeting 48° C. Cool the mixture to 18 to 23° C. Charge purified water (10.0 vol) to the mixture at 18 to 23° C. Adjust the pH of the mixture to 0.1<pH<1.5 at 18 to 23° C. by charging concentrated hydrochloric acid, 0.5 vol. Do not delay from this step until neutralization.
• [0121]
  Separate the layers at 18 to 23° C. retaining the bottom aqueous layer. Wash the aqueous layer with toluene, (3.0 vol, 2.6 wt) at 18 to 23° C. retaining the aqueous layer. Adjust the pH of the aqueous layer to pH>12 by charging 20% w/w sodium hydroxide solution at 18 to 23° C. 0.8 to 0.9 vol. Caution: Exothermic. Charge TBME, code RM0002 (2.0 vol, 1.5 wt) to the basic aqueous layer. Separate the layers at 18 to 23° C. retaining the top organic layer. Back-extract the aqueous layer with TBME (2×2.0 vol, 2×1.5 wt) at 18 to 23° C. retaining the organic layers. Wash the combined organic layer with purified water, (2×1.0 vol) at 18 to 23° C. Concentrate the combined organic layers under reduced pressure at 40 to 50° C., targeting 48° C. to ca. 3 vol. Determine the residual toluene content of the mixture by 1H-NMR analysis. Sample for determination of residual water content by KF analysis, AKX reagent. Charge TBME (8.7 vol, 6.4 wt) to the mixture at 40 to 50° C. Cool the solution to 0 to 5° C., targeting 2° C. Charge concentrated hydrochloric acid (0.54 vol, 0.46 wt) maintaining the temperature <15° C. Caution: Exothermic. Line rinse with TBME (1.0 vol, 0.7 wt). If necessary, adjust the temperature to 0 to 10° C. and stir the mixture at 0 to 10° C. for a further 2 to 3 hours. Filter the mixture and wash the filter-cake with TBME (2×4.4 vol, 2×3.3 wt) at 0 to 10° C. Dry the solid at up to 40° C. until the TBME content is <0.5% w/w TBME by 1H-NMR analysis. 4 to 8 hours.
• [0122]
  Yields and Profiles: The yield for the step 3 Demonstration batch is summarized in Table 1C below. Input: 856.8 g corr. Ketone, 44.2% w/w assay (NMR, using TCNB as internal standard in CDCl₃), (1.00 eq, 1.00 wt corr. for w/w assay) for input calculation. FIG. 2 and Table 1D shows an exemplary HPLC chromatogram of a crude preparation of fenfluramine hydrochloride (210 nm UV absorbance).
• TABLE 1C Table for isolated yields for step 3 Demonstration batch Corr. % area Reference Corr. Corr. Yield % w/w (HPLC, number Input Output (% th) (¹H-NMR)* QC) Comments Batch A1 856.8 g 836.31 g 85.3 44.2 99.15 Fenfluramine free base (in situ intermediate) Batch A2 880.7 84.0 based 99.5 100.00 Fenfluramine•HCl on ketone crude (step 3 an bisulfite d 4.1) adduct (77.6 based on purified ketone)
• TABLE 1D Purity of crude fenfluramine hydrochloride by HPLC (see FIG. 2) Processed Channel Descr. DAD AU Ch 1 Sample 210, Bw 4 Peak Results USP USP USP Name RT RelRT Area Height Tailing Resolution Plate Count EP s/n % Area 1 NorFenfluramine 7.46 2 2-Fenfluramine 7.68 3 Fenfluramine 8.67 1.000 3789064 778178 1.7 70796 2549.8 99.15 4 4-Fenfluramine 8.95 5 11 34 1.308 6073 1449 1.2 23.5 215529 3 8 0.16 6 ZX008 acid 12.93 7 Fenfluramine alcohol 14.16 1.633 15266 2972 1.3 24.8 215040 8.7 0.40 8 ZX008 ketone 14.83 9 Fenfluramine acetamide 15.55 10 TOLUENE 15 75 11 15.92 1.836 4110 1122 2.7 0.11 12 16.60 1.915 6861 1630 1.5 451209 4.3 0.18 Sum 3821374 100.00
• [0123]
  Step 4.2: Crystallization of Fenfluramine Hydrochloride
• [0124]
  Procedure: Charge Fenfluramine.HCl (crude) (1.00 wt, 1.0 eq.) and TBME (10.0 vol, 7.4 wt) to the vessel and commence stirring. Heat the suspension to reflux (50 to 58° C.). Charge ethanol (5.0 vol, 3.9 wt) maintaining the temperature at 50 to 58° C. Addition time 20 minutes. Stir at 50 to 58° C. for 5 to 10 minutes and check for dissolution. Stir the solution at 50 to 58° C. for 5 to 10 minutes, targeting 54 to 58° C. Clarify the reaction mixture through a 0.1 μm in-line filter at 54 to 58° C., followed by a line rinse with TBME (1 vol, 0.7 wt). Cool the solution to 48 to 50° C. Charge Fenfluramine HCl, code FP0188 (0.01 wt). Check for crystallization. Allow the suspension to cool to 15 to 20° C., target 17° C. over 5 to 5.5 hours at an approximately constant rate. Stir the mixture at 15 to 20° C., target 17° C. for 2 to 3 hours. Filter the mixture and wash the filter-cake with clarified TBME (2×3.0 vol, 2×2.2 wt) at 5 to 15° C. Dry the solid at up to 40° C. until the TBME content is <0.5% w/w TBME and the ethanol content is <0.5% w/w EtOH by 1H-NMR analysis. 4 to 8 hours. Determine the w/w assay of the isolated Fenfluramine.HCl by 1H-NMR analysis.
• [0125]
  Yields and Profiles: The yield for the stage 4 Demonstration batch is summarized in Table 1E below. Input: 750.0 g uncorr. Fenfluramine HCl crude (1.00 eq, 1.00 wt uncorr.) for input calculation. FIG. 3 shows an exemplary HPLC chromatogram of a crystallized fenfluramine hydrochloride sample (210 nm UV absorbance).
• TABLE 1E Table for isolated yields for stage 4 Demonstration batch Uncorr. Uncorr. Uncorr. Yield HPLC (% area, Input Output (% th) QC) Comments 750.0 g 608.0 81.1 100.00* Fenfluramine•HCl

5. In-Process Controls

• [0126]
  Table 2 summarizes the in-process controls (IPCs) by IPC number as cited in the narrative procedures above used for Process 1.
• TABLE 2 In-Process Controls Performed during Process 1 Critical IPC Synthesis Process No. Step Sample Description Method Acceptance Criteria 1 1 Reaction Reaction HPLC NMT 3.0% Ketone (1) Mixture Completion 2 1 Extraction Purity HPLC Report percent Aqueous Fenfluramine Free Base and Phase Fenfluramine Alcohol 3 1 Extraction Purity HPLC Report percent Organic Fenfluramine Free Base and Phase Fenfluramine Alcohol 4 1 Extraction Purity HPLC Report percent Organic Fenfluramine Free Base and Phase Fenfluramine Alcohol 5 1 Extraction Purity HPLC NLT 98.0% Fenfluramine Aqueous HCl Phase LT 1.0% Fenfluramine Alcohol 6 1 Extraction Purity HPLC Report percent result of Aqueous Fenfluramine HCl Phase Fenfluramine Alcohol 7 2 Reaction Purity ¹H-NMR Residual Solvents by ¹H- Mixture NMR Ethanol NMT 0.50% w/w Ethyl Acetate NMT 0.50% w/w Methanol NMT 0.50% w/w Toluene NMT 0.50% w/w MTBE NMT 0.50% w/w

6. Starting Materials

• [0127]
  This section provides information and specification controls for the starting materials used to produce clinical supplies of fenfluramine per the routes shown herein.
• TABLE 3 Starting Materials via the Route 1 Chemical Name Code [CAS. No.] Name Structure Source Step 1-(3- (Trifluoromethyl)- phenylacetone [21906-39-8] Ketone (1)Fluorochem 1 Ethyl Amine Ethyl EtNH2 Alfa Aesar 1 (70% in water) Amine [75-04-7]
• TABLE 4 Starting Materials via Route 2 Chemical Name Code [CAS. No.] Name Structure Source Step 3-(Trifluoromethyl)- phenylacetic acid [351-35-9] Acid (1a)To be determined 1 Acetic Anhydride [108-24-7] Acetic AnhydrideVarious 1 Ethyl Amine Ethyl EtNH2 Various 3 (70% in water) Amine [75-04-7]
• [0128]
  Table 5 provides a list of the intermediates for the Route 2 synthesis. Both routes share the same intermediate Fenfluramine Free Base (1). Fenfluramine Free Base (1) was treated as an isolated intermediate in the Route 1 process however the Route 2 process uses fixed equipment where both Ketone (2) and Fenfluramine Free Base 1, both non-isolatable oils, are telescoped as a solution and controlled as in-situ intermediates. The Bisulfate Complex (3) is isolated as a solid thus is amenable to treatment as an isolated intermediate and released as such. Crude Fenfluramine HCl can be isolated as an intermediate before recrystallization.
• [0129]
  A Specification and Testing Strategy for Intermediates is used. Additional tests and acceptance criteria are be added based upon review of data from the primary stability batches and process validation critical parameter studies. Analytical reference standards are used in full characterization of each intermediate. HPLC methods to determine assay and impurities are the same as the drug substance release method and are validated for Accuracy, Precision: Repeatability, Intermediate Precision, Selectivity/Specificity, Detection limit, Quantitation limit, Linearity, Range, and Robustness.
• TABLE 5 In-Situ and Isolated Intermediates Chemical Name [CAS No] Code Name Step No. Control Structure Bisulfate Complex of Ketone 1 Bisulfate Complex (3) Step 1 Isolated (Solid)1-(3- (Trifluoromethyl)- phenylacetone [21906-39-8] Ketone (2) Step 2 In-Situ (oil)Fenfluramine Free Base [458-24-2] Fenfluramine Free Base (1) Step 3 In-Situ (oil)Fenfluramine HCl [404-82-0] Crude Fenfluramine HCl Step 4 Isolated (Solid)

7. Characterization

• [0130]
  Physiochemical Characteristics of Drug Substance.
• [0131]
  Fenfluramine HCl is developed as a single polymorph Form 1. A polymorphism and pre-formulation study has been conducted. Under a wide range of solvents and conditions crystalline material is produced of the same polymorph Form 1 based on a well-defined XRPD pattern and a consistent reproducible endotherm by DSC analysis. A summary of the chemophysical properties of Fenfluramine HCl from this study is provided below. Tabulated data includes example diffractograms, DSCs, and micrographs.
• [0132]
  The input Fenfluramine HCl (from precipitative isolation) was characterized to provide reference data and also to determine if the salt was of the same form as that identified from previous salt formations. The XRPD pattern of the salt reveals a crystalline solid that visually matches the reflection patterns obtained from formal crystallization of Fenfluramine HCl and has been arbitrarily termed Form 1. Comparison of the μATR-FTIR data for the salt from various batches gave profiles that had a 99.95% match.
• [0133]
  Thermal data analysis matched previous data obtained with only one major endotherm on the DSC thermograph peaking at 172.3° C. that matches the beginning of potential decomposition shown in a TGA thermograph. This also matches the reported melting point quoted for the reference standard.
• [0134]
  Isolation of the amorphous form has been shown to be difficult, with attempts using three common methods (rapid solvent evaporation, anti-solvent precipitation and lyophilization) all yielding highly crystalline solids that very closely share the same XRPD pattern of the input Form 1.
• [0135]
  Stability analysis of the salt after one week at 40° C./0% RH, three weeks at 40° C./75% RH, and under photostability conditions revealed that the input Form 1 has been maintained with no new impurities observed at 0.1% threshold.
• [0136]
  Results from DSC heat cycling analysis of Fenfluramine HCl are comparable to results generated when the material was held at 170° C. No crystallization event was noted and the amorphous was not generated but rather Form 1 was returned.
• [0137]
  Holding Fenfluramine HCl at approximately 170° C. for several hours causes a melt and evaporation event to take place with recombination and cooling to provide a white solid. Analysis of the white solid by XRPD, DSC and ¹H NMR indicates no change in chemical or physical form, purity, or dissociation.
• [0138]
  Forced degradation studies carried out have proven Fenfluramine HCl to be stable under a range of conditions. Thermal modulation of Fenfluramine HCl repeatedly yielded the input Form 1.

8. Impurities

• [0139]
  Impurities in a drug substance can be organic impurities (process impurities or drug substance-related degradants), inorganic impurities (salt residues or metals) and residual solvents; some of these impurities must be evaluated as to whether or not they are genotoxic agents. These impurities are taken into consideration and controlled in Fenfluramine HCl preparation by using either compendia or validated analytical methods per the specifications or by separate “for information only” testing. The following sections address the actual and potential impurities in Fenfluramine HCl.
• [0140]
  Actual Impurities and the Qualification of Synthesis Batch
• [0141]
  No impurities reported in cGMP drug substance batches intended for use in humans have exceeded the ICHQ3A qualification thresholds of 0.15% (Table 8). All impurities >0.1% are identified and handled as described in ICH Q3A unless they are genotoxic impurities.
• [0142]
  Process Impurities
• [0143]
  Table 6 lists the known potential impurities arising from the route of synthesis. All of these impurities are controlled to below ICHQ3A qualification threshold of 0.15% by either process changes and/or control of starting material input purities.
• TABLE 6 Fenfluramine HCl Known Potential Process Impurities (Route 1) Observed Observed in in Development cGMP Name PLC Batches Batches [Cas. No.] Source (RRT) ≧0.10%¹⁾ ≧0.10%¹⁾ Ketone (2) Starting RRT No No [351-35-9] Material or 0.89 Intermediate Fenfluramine By-product RRT Yes No Alcohol 1.60 [621-45-4] Norfenfluramine By-product RRT Yes Yes [1886-26-6] 1.67 2-Fenfluramine Starting RRT No No [172953-70-7] Material 0.89 (isomer) 4-Fenfluramine Starting RRT Yes Yes [1683-15-4] Material 1.02 (isomer) N-(3- By-product RRT Yes Yes (trifluoromethyl)- 0.53-0.57 benzyl)ethanamine [90754-95-3] ¹⁾ICH Q3A Identification threshold. The Reporting threshold (LOQ) for the HPLC method is 0.05%.
• [0144]
  Degradation Impurities
• [0145]
  No change in impurity profile is observed upon long-term storage based on forced degradation studies under the ICH Q1A(R2) conditions of heat (solid, solution), acid, base, oxidizing, and ICH Q1B photostability conditions (solid, solution). Fenfluramine HCL is stable for 7 days as a solid at 150° C. (99.90 parent area %), as a solution in water-acetonitrile at 70° C. (99.73 parent area %), as a solution in acid, base, or photosensitizing conditions at ambient. Only oxidizing conditions (peroxide conditions) produced degradation of Fenfluramine HCl to 94.42% after 1 day producing several new related substances at −1% each consistent by LC-MS with +16 oxidation by-products
• [0146]
  Organic Volatiles/Residual Solvents
• [0147]
  Table 11 in the Batch Analysis section summarizes the solvents used in the process and the resulting amounts found in drug substance. All solvents used in the GMP steps are controlled at ICH Q3A limits using a suitably qualified Head-Space (HS) GC method.
• [0148]
  Inorganic Impurities
• [0149]
  Heavy Metals conform to either USP <231> or ICP method USP <233> as well as ICH Q3D.
• [0150]
  Genotoxic Impurities
• [0151]
  The ICH guidelines Q3A and Q3B are not sufficient to provide guidance on impurities that are DNA-reactive. The European Medicines Agency (EMA) guideline (2006) “Guideline on the Limits of Genotoxic Impurities” (EMA 2006) and the ICH Guideline M7 (2014) “Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk” (ICH Guideline M7) are taken into consideration in controlling for potential genotoxic impurities. The diazonium route to prepare ketone (2) described in FIG. 5 has a disadvantage due to the potential formation of genotoxic intermediates shown as boxed compounds (e.g., N-hydroxyaryl, N-nitrosamine and Nitro compound). Muller et al. (Regulatory Toxicology and Pharmacology 44 (2006) 198-211) list potential functional alert groups that can be genotoxic. Safety guidances and regulations indicate that analysis of a process and identification of potential genotoxic agents, and control of such impurities at sub 10 parts per million levels is critical for safety. Often removal of such impurities and/or demonstrating their absence is costly and time consuming and sometimes difficult to achieve technically. For these reasons, selecting synthetic routes that circumvent the potential for such toxic intermediates is important. Because of the potential problems with the diazo route discussed above, as well as potential safety issues using diazo (shock-sensitive) intermediates, as well as the lower purity profiles with this route, this route is less preferred than the preferred route to ketone (2) starting from Nitrile (5). This route produces no potential genotoxic agents and leads to high purity Ketone (2) after isolation by distillation or via the bisulfite salt adduct—hydrolysis sequence.
• [0152]
  Additionally, attempts to remove isomer by-products present in commercial supplies of Aniline were unsuccessful whereas crystallization the Acid (4) resulting from hydrolysis of the nitrile (5) provides crystalline Acid (4) which can be purified to remove isomers early in synthesis. Removing impurities and/or isomers early in a synthesis is preferred if it is known such impurities track to final product, as the need to crystallize a final product at the end of a synthesis is more costly in losses and impacts cost of goods more greatly than removing early in synthesis before raw materials are invested along the process.
• TABLE 7 Potential Impurities in Fenfluramine Synthesis Synthesis Route No. Compound Route 1 Route 2 CAS. No. 1No Starting Material [351-35-9] 2Starting Material Intermediate [21906-39-8] 4No Intermediate Not Available 5Potential Impurity Potential Impurity [621-45-4] 6Potential Impurity Potential Impurity [1886-26-6] 7Potential Impurity Potential Impurity [172953-70-7] 8Potential Impurity Potential Impurity [1683-15-4] 9Potential Impurity Potential Impurity [90754-95-3]
• TABLE 8 Batch Analyses of Fenfluramine HCl Drug Substance Test Batch 1 Batch 2 Batch 3 Batch 4 Appearance* White solid White solid White solid White solid Identification: FTIR* ^a) a Conforms Conforms Identification: ¹H-NMR Conforms Conforms Conforms Conforms Identification: ¹³C-NMR Conforms Conforms Conforms Conforms Identification: MS Conforms Conforms Conforms Conforms Purity (HPLC area %) 99.57 99.77 ^b) b Assay (w/w %)* 99.49 100.37 100.79 100.13 Anhydrous Basis (HPLC) Impurities 2-Fenfluramine ND ND ND ND (HPLC 4-Fenfluramine) 0.16 0.15 0.11 0.12 area %) Fenfluramine Alcohol ND ND ND ND 1-((3-trifluoromethyl)phenyl)acetone ND ND ND ND Acetamide 0.27 ND ND ND N-(3-(trifluoromethyl)- ND 0.08 0.07 0.13 benzyl)ethanamine (RRT 0.53-0.57) Total 0.43 0.23 0.19 0.25 Residual Solvents Methanol ND ND ND ND (GC): ppm Ethanol ND ND ND ND MTBE 597 844 472 800 Ethyl Acetate 115 164 79 150 Toluene 4 7 ND ND Residue on Ignition (w/w %) 0.01 0.02 0.04 ND Heavy Metals (as Pb) <10 ppm <10 ppm ^b b Heavy Metals ICP (ppm) As ^a a <0.1 <0.1 Cd ^a a 0.1 0.1 Hg ^a a <0.1 <0.1 Pb ^a a 0.2 <0.4 Co ^a a <0.1 0.1 Mo ^a a <0.1 <0.1 Se ^a a <0.1 <0.1 V ^a a <0.1 <0.1 Water Determination* 0.21 0.08 0.02 0.03 (Karl Fischer) Chloride content by titration 13.19 13.09 12.92 12.93 XRPD* Form 1 Form 1 Form 1 Form 1 Differential Scanning Onset 169.42° C. 169.23° C. 169.85° C. 168.70° C. Calorimetry (DSC)* Peak 172.82° C. 171.55° C. 172.22° C. 171.97° C. Particle Size % Volume mean (D) ^a 11 11 19 Malvern (μm) D10 ^a 1 1 1 D50 ^a 5 7 9 D90 ^a 17 26 32 Microbial Total aerobic ^a a LT 100 CFU/g LT 100 CFU/g Limits Tests* microbial Count USP <61> Total yeast and ^a a LT 100 CFU/g LT 100 CFU/g molds count USP <62> Absence of ^a a Absent Absent Pathogens ^a)These tests were added to the specifications recently thus only recent lots have been tested using this test. ^b)These tests have been dropped from the specifications thus only historical lots have been tested using this test.

Example 3Method for Hydrolysis of Nitrile (5) to Acid

• [0153]
  
• TABLE 9 Step Operation 1. Charge 3-(trifluoromethyl)phenyl acetonitrile (1.0 eq., 1.00 wt) and purified water (5.0 vol) to a vessel and commence stirring. 2. Dissolve sodium hydroxide (1.1 wt, 5.0 eq.) in purified water (4.0 vol) at up to 40° C. in a suitable make-up vessel. Caution very exothermic. 3. Charge the aqueous sodium hydroxide solution to the mixture from step 1 at up to 40° C. followed by a line rinse with purified water, code RM0120 (1.0 vol) at up to 40° C. 4. Heat the mixture to 75 to 85° C., target 80° C. over 1 to 2 hours. 5. Heat the mixture at 80° C. until ≦0.1% area nitrile by HPLC analysis, expected 4 to 6 hours. 6. Cool the mixture to 18 to 23° C. 7. Adjust the pH of the mixture to pH ≦2 by charging 6M HCl (expected 7.0 vol) to the mixture at 18 to 23° C. Caution exothermic. 8. Stir the mixture for 15 to 30 minutes at 18 to 23° C. 9. Filter and wash the filter-cake with purified water (2 × 5.0 vol) at 18 to 23° C. 10. Slurry wash the filter-cake with n-heptane, code RM (2 × vol) at 0 to 5° C. 11. Dry the isolated solid at up to 45° C. until the water content is ≦.0.2% w/w by KF analysis according to MET/AN/0163, AKX-reagent. 12. Crystallization of crude stage 1 acid (1.00 wt for input calculation) 13. Charge the crude stage 1 acid (1.00 wt), ethyl acetate (0.75 vol) and n-heptane (10.5 vol) to a vessel and commence stirring. 14. Heat the mixture to 50° C. to achieve dissolution. 15. Cool the mixture to 5° C. and age at 5° C. for at least 30 mins. 16. Filter and wash the filter-cake with n-heptane (2 × 5.0 vol). 17. Dry the isolated solid at up to 45° C. until the residual solvent content by ¹H-NMR analysis is ≦.0% w/w EtOAc and ≦.0% w/w n-heptane. Expected yield: 60 to 90% th uncorr. 68 to 103% w/w Expected purity: 93.00 to 99% area by HPLC

Example 4Evaluation of Minor Components Formed During Dakin-West Reaction in Preparation of Ketone (2)

• [0154]
  The impurities formed during the Dakin-West chemistry and their subsequent removal using the distillation or via isolation of the product ketone as the bisulfite salt are described. The two major impurities found are shown below.
• [0155]
  Table 10 shows a table of analytical data for crude Ketone (2) isolated from Dakin-West reaction before and after bisulfite purification. In entry 1 crude Ketone isolated directly from the Dakin-West step (pre-bisulfite treatment) is 61.66% purity (e.g. about 62%) and contains 1.98% (e.g., about 2%) and 4.64% (e.g., about 5%) respectively of impurities having RRTs 1.20 and 1.34, which are proposed to be the acetate and dimer impurities (e.g., depicted above), respectively. In entry 2 which is post bisulfite treatment these are other impurities are removed leading to an overall purity of 95.55% (e.g., about 96%). Other entries shown in Table 10 provide other examples of this impurity enhancement by bisulfite treatment of crude Dakin-West ketone. The last two entries use pure Fluorchem ketone as input to the salt formation step and re-isolation of ketone thus illustrating that the salt formation and re-isolation does not produce any impurities itself. Additionally use of bicarbonate extraction procedure during reaction workup provides an improvement in purity of the resulting composition as it serves to remove any unreacted acid. Crude Ketone (2) made by the Diazo route showed similar improvements in purity when treated with bisulfite and isolated.
• TABLE 10 Analytical purity data for crude Ketone (2) isolated from Dakin-West reaction before and after bisulfite purification. RRT is relative retention time (min) in chromatogram. RRT 0.93 1.00 1.009 1.06 Entry 0.85 Aniline 0.95 0.99 Ketone 1.004 Nitrile 1.02 Acid 1.10 1.15 1.34 1.38 1 1.38 1.76 0.04 0.49 61.66 nd 0.29 0.29 0.26 1.98 0.66 4.64 0.14 2 0.82 nd nd nd 95.53 0.31 0.14 nd 0.23 0.01 0.10 0.43 0.27 3 nd nd nd nd 77.82 nd nd nd nd 3.12 0.01 7.76 6.16 4 nd nd nd nd 98.82 nd 0.63 nd nd nd 0.02 0.30 0.22 5 0.08 nd nd 0.05 72.02 nd 0.02 nd nd 7.11 0.04 3.58 10.33 6 nd nd nd nd 99.49 nd 0.02 nd nd nd 0.02 0.11 0.24 7 0.15 0.23 nd nd 98.35 nd nd nd nd nd nd nd 0.24 8 nd nd nd nd 99.84 nd nd nd nd nd nd nd nd Entry 1 (Crude ketone from Route 1); Entry 2 (Ketone Route 1 post bisulfite release); Entry 3 (Crude ketone using crude acid); Entry 4 (Ketone using crude acid Post bisulfite); Entry 5 (Crude ketone using cryst. acid); Entry 6 (Crude ketone using cryst. acid post bisulfite); Entry 7 (Crude ketone using cryst. acid); Entry 8 (Fluorochem ketone); Entry 9 (Fluorochem ketone post bisulfite).

Example 5

• [0156]
  
• Additional Method for Preparation of 1-(3-trifluoromethyl)phenyl-propan-2-one
• [0157]
  35 mL of water and 45 g of 37% (w/w) aqueous hydrochloric acid are put in a flask equipped with stirrer and dropping funnel. 24.25 Grams (0.151 moles) of m-trifluoromethylaniline are added after having cooled to 10 degree C. with an ice bath and then, at 5 degree C., an aqueous solution containing 12.43 g (0.180 moles) of sodium nitrite in 150 ml of water is slowly added. The reaction mixture is stirred for 30 minutes and then is poured during 30 minutes into a mixture made by 90 ml of water, 1.35 g (0.014 moles) of cuprous chloride, 2.30 g (0.013 moles) of cupric chloride dihydrate, 50 ml of acetone, 40.8 g (0.300 moles) of sodium acetate trihydrate and 23 g (0.230 moles) of isopropenyl acetate while keeping the reaction temperature at 30 degree C. After further 30 minutes of stirring, the reaction mixture is brought to 20 degree C., 50 ml of methylene chloride are added and the two layers are separated.
• The aqueous layer is discarded while the organic layer is concentrated under vacuum until an oil is obtained which is treated with 35 g of sodium metabisulfite, 70 ml of water and 150 ml of heptane under stirring at room temperature for 12 hours. The suspension is filtered, the bisulfite complex is washed on the filter with 50 ml of heptane and then suspended in a two-phase mixture made by 100 ml of methylene chloride and 150 ml of a 10% (w/v) aqueous solution of sodium hydroxide. The layers are separated after one hour of stirring at room temperature, the aqueous phase is discarded while the organic layer is washed with water and evaporated under vacuum to give pure ketone.

NIROGACESTAT

(2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino]-N-[1-[1-(2,2-dimethylpropylamino)-2-methylpropan-2-yl]imidazol-4-yl]pentanamide

489.6 g/mol, C₂₇H₄₁F₂N₅O

CAS 1290543-63-3

PF-03084014, 1290543-63-3, PF-3084014, 865773-15-5QZ62892OFJUNII:QZ62892OFJUNII-QZ62892OFJнирогацестат [Russian] [INN]نيروغاسيستات [Arabic] [INN]尼罗司他 [Chinese] [INN]ニロガセスタット;

orphan drug designation in June 2018 for the treatment of desmoid tumors, and with a fast track designation

 Nirogacestat, also known as PF-03084014, is a potent and selective gamma secretase (GS) inhibitor with potential antitumor activity. PF-03084014 binds to GS, blocking proteolytic activation of Notch receptors. Nirogacestat enhances the Antitumor Effect of Docetaxel in Prostate Cancer. Nirogacestat enhances docetaxel-mediated tumor response and provides a rationale to explore GSIs as adjunct therapy in conjunction with docetaxel for men with CRPC (castration-resistant prostate cancer).

Nirogacestat was disclosed to be a gamma-secretase inhibitor, which can inhibit Aβ-peptide production. SpringWorks Therapeutics (a spin-out of Pfizer ) is developing nirogacestat, as hydrobromide salt, a gamma-secretase inhibitor, for treating aggressive fibromatosis. In February 2021, nirogacestat was reported to be in phase 3 clinical development.

Nirogacestat is a selective gamma secretase (GS) inhibitor with potential antitumor activity. Nirogacestat binds to GS, blocking proteolytic activation of Notch receptors; Notch signaling pathway inhibition may follow, which may result in the induction of apoptosis in tumor cells that overexpress Notch. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth and survival.

Nirogacestat has been used in trials studying the treatment of Breast Cancer, HIV Infection, Desmoid Tumors, Advanced Solid Tumors, and Aggressive Fibromatosis, among others.

Nirogacestat (Gamma Secretase Inhibitor)

Nirogacestat is an oral, selective, small molecule, gamma secretase inhibitor (GSI) in Phase 3 clinical development for patients with desmoid tumors. Gamma secretase is a protease complex that cleaves, or divides, multiple transmembrane protein complexes, including Notch, which, when dysregulated, can play a role in activating pathways that contribute to desmoid tumor growth.

Gamma secretase has also been shown to directly cleave BCMA, a therapeutic target that is highly expressed on multiple myeloma cells. By inhibiting gamma secretase with nirogacestat, membrane-bound BCMA can be preserved, thereby increasing target density while simultaneously reducing levels of soluble BCMA, which may serve as decoy receptors for BCMA-directed therapies. Together, these mechanisms combine to potentially enhance the activity of BCMA therapies and improve outcomes for multiple myeloma patients. SpringWorks is seeking to advance nirogacestat as a cornerstone of multiple myeloma combination therapy in collaboration with industry leaders who are advancing BCMA therapies.

SpringWorks Therapeutics Announces Clinical Collaboration with Pfizer

By Satish  October 05, 2020 

SpringWorks Therapeutics today announced that the company has entered into a clinical trial collaboration agreement with Pfizer to evaluate SpringWorks Therapeutics’ investigational gamma secretase inhibitor (GSI), nirogacestat, in combination with Pfizer’s anti-B-cell maturation antigen (BCMA) CD3 bispecific antibody, PF‐06863135, in patients with relapsed or refractory multiple myeloma.

Gamma secretase inhibition prevents the cleavage and shedding of BCMA from the surface of myeloma cells. In preclinical models, nirogacestat has been shown to increase the cell surface density of BCMA and reduce levels of soluble BCMA, thereby enhancing the activity of BCMA-targeted therapies, including CD3 bispecific antibodies.

Saqib Islam, Chief Executive Officer of SpringWorks Therapeutics Said: This collaboration is another important step in continuing to advance our goal of developing nirogacestat as a best-in-class BCMA potentiator, and we are pleased to work with Pfizer to study nirogacestat in combination with PF‐06863135, which has recently demonstrated promising monotherapy clinical data, We now have five collaborations with industry-leading BCMA developers to evaluate nirogacestat in combinations across modalities. We look forward to generating clinical data with our collaborators to further evaluate the ability of nirogacestat to improve outcomes for patients with multiple myeloma.

Under the terms of the agreement, Pfizer will sponsor and conduct the Phase 1b/2 study to evaluate the safety, tolerability and preliminary efficacy of the combination, and will assume all costs associated with the study, other than expenses related to the manufacturing of nirogacestat and certain expenses related to intellectual property rights. Pfizer and SpringWorks Therapeutics will also form a joint development committee to manage the clinical study, which is expected to commence in the first half of 2021.

Chris Boshoff, MD, PhD, Chief Development Officer for Pfizer Oncology at Pfizer Said: Entering into this clinical collaboration is a proud milestone in our strong relationship with SpringWorks,We believe that studying nirogacestat in combination with PF-06863135 could hold significant therapeutic promise for patients with relapsed or refractory multiple myeloma, and we look forward to working together to advance this important area of research.

In addition to its ongoing clinical collaborations with BCMA-directed therapies, SpringWorks is also currently conducting a global Phase 3, double-blind, randomized, placebo-controlled clinical trial (the DeFi Trial) to evaluate nirogacestat in adults with progressing desmoid tumors.

About Nirogacestat

Nirogacestat is an investigational, oral, selective, small molecule gamma secretase inhibitor in Phase 3 clinical development for desmoid tumors, which are rare and often debilitating and disfiguring soft-tissue tumors. Gamma secretase cleaves multiple transmembrane protein complexes, including Notch, which is believed to play a role in activating pathways that contribute to desmoid tumor growth.

In addition, gamma secretase has been shown to directly cleave membrane-bound BCMA, resulting in the release of the BCMA extracellular domain, or ECD, from the cell surface. By inhibiting gamma secretase, membrane-bound BCMA can be preserved, increasing target density while reducing levels of soluble BCMA ECD, which may serve as decoy receptors for BCMA-directed therapies. Nirogacestat’s ability to enhance the activity of BCMA-directed therapies has been observed in preclinical models of multiple myeloma. SpringWorks is evaluating nirogacestat as a BCMA potentiator and has five collaborations with industry-leading BCMA developers to evaluate nirogacestat in combinations across modalities, including with an antibody-drug conjugate, two CAR T cell therapies and two bispecific antibodies. In addition, SpringWorks and Fred Hutchinson Cancer Research Center have entered into a sponsored research agreement to further characterize the ability of nirogacestat to modulate BCMA and potentiate BCMA directed therapies using a variety of preclinical and patient-derived multiple myeloma models developed by researchers at Fred Hutch.

Nirogacestat has received Orphan Drug Designation from the U.S. Food and Drug Administration (FDA) for the treatment of desmoid tumors (June 2018) and from the European Commission for the treatment of soft tissue sarcoma (September 2019). The FDA also granted Fast Track and Breakthrough Therapy Designations for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis (November 2018 and August 2019).

About PF‐06863135

PF‐06863135 is an anti-B-cell maturation antigen (BCMA) CD3 bispecific antibody being investigated in a Phase 1 clinical study to treat relapsed or refractory multiple myeloma. This bispecific antibody can be administered subcutaneously and has been optimized for binding affinity to both BCMA and CD3, enabling more potent T-cell-mediated tumor cell toxicity.

Source: SpringWorks Therapeutics

FDA Grants Breakthrough Designation to Nirogacestat for Desmoid Tumors

The FDA has granted nirogacestat, an investigational gamma-secretase inhibitor, with a breakthrough therapy designation for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis.

The FDA has granted nirogacestat (PF-03084014), an investigational gamma-secretase inhibitor, with a breakthrough therapy designation for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis.¹

The breakthrough designation was granted as a result of positive findings seen in phase I and II trials of nirogacestat monotherapy in patients with desmoid tumors. A phase III trial has also been initiated investigating nirogacestat in patients with desmoid tumors or aggressive fibromatosis (NCT03785964).

“We are committed to pursuing the rapid development of nirogacestat given the important need for new therapies for patients with desmoid tumors and are pleased to receive this breakthrough therapy designation,” Saqib Islam, CEO of SpringWorks, the company developing the small molecule inhibitor, said in a statement. “We are currently enrolling adult patients in our phase III DeFi trial and will continue to work closely with the FDA with the goal of bringing nirogacestat to patients as quickly as possible.”

The open-label, single-center phase II trial of nirogacestat enrolled 17 patients with desmoid tumors who were not eligible for surgical resection or definitive radiation therapy and who had experienced disease progression after at least 1 prior treatment regimen. Patients received 150 mg twice per day of continuous, oral nirogacestat in 21-day cycles.²

The median age of patients was 34 years (range, 19-69), 82% of the patients were female, and 53% of patients had aCTNNB1T41A somatic missense mutation. The median number of prior therapies was 4 (range, 1-9), which included cytotoxic chemotherapy in 71% and a tyrosine kinase inhibitor in 59%.

Sixteen patients were evaluable for response. After a median follow-up of more than 25 months, 5 patients (29%) achieved a partial response and 11 (65%) had stable disease, for a disease control rate of 100%. Ten patients (59%) remained on treatment with nirogacestat for more than 2 years.

Grade 1/2 adverse events were observed in all patients, with diarrhea (76%) and skin disorders (71%) being the most common toxicities. The only treatment-related grade 3 event was reversible hypophosphatemia, which was reported in 8 patients (47%) and was considered to be a class effect of gamma-secretase inhibitors. Four patients met the criteria for dose reduction.

Findings from the phase I study also showed a disease control rate of 100% with nirogacestat. However, the median progression-free survival was not reached in either study due to a lack of patients progressing on treatment. Only 1 patient discontinued treatment due to an adverse event between the 2 studies.¹

The FDA had previously granted nirogacestat with an orphan drug designation in June 2018 for the treatment of desmoid tumors, and with a fast track designation in November 2018 for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis.

References

1. SpringWorks Therapeutics Receives Breakthrough Therapy Designation for Nirogacestat for the Treatment of Adult Patients with Progressive, Unresectable, Recurrent or Refractory Desmoid Tumors [press release]. Stamford, CT: SpringWorks Therapeutics, Inc; August 29, 2019. https://bit.ly/30IV0Eb. Accessed September 3, 2019.
2. Kummar S, O&rsquo;Sullivan Coyne G, Do KT, et al. Clinical Activity of the &gamma;-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis).J Clin Oncol.2017;35(14):1561-1569. doi: 10.1200/JCO.2016.71.1994.

PAPER

Bioorganic & medicinal chemistry letters (2011), 21(9), 2637-40.

https://www.sciencedirect.com/science/article/abs/pii/S0960894X10018822

PATENT

WO 2016089208

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016089208

PATENT

WO-2021029854

Novel, stable crystalline polymorphic (A to N) and amorphous forms of nirogacestat hydrobromide , useful for treating desmoid tumors such as multiple myeloma, a cancer having a mutation in a Notch pathway gene, adenoid cystic carcinoma and T-cell acute lymphoblastic leukemia.

(S)-2-(((S)-6,8-difluoro-l,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(l-(2- methyl- l-(neopentylamino) propan-2-yl)-lH-imidazol-4-yl)pentanamide (“Compound 1”) is a gamma-secretase inhibitor which can inhibit Ab-peptide production.

[0003] Not all compounds that are gamma-secretase inhibitors have characteristics affording the best potential to become useful therapeutics. Some of these characteristics include high affinity at the gamma-secretase, duration of gamma-secretase deactivation, oral bioavailability, tissue distribution, and stability (e.g., ability to formulate or crystallize, shelf life). Favorable characteristics can lead to improved safety, tolerability, efficacy, therapeutic index, patient compliance, cost efficiency, manufacturing ease, etc.

[0004] In addition, the isolation and commercial -scale preparation of a solid state form of hydrobromide salts of Compound 1 and corresponding pharmaceutical formulations having acceptable solid state properties (including chemical stability, thermal stability, solubility, hygroscopicity, and/or particle size), compound manufacturability (including yield, impurity rejection during crystallization, filtration properties, drying properties, and milling properties), and formulation feasibility (including stability with respect to pressure or compression forces during tableting) present a number of challenges.

[0005] Accordingly, there is a current need for one or more solid state forms of hydrobromide salts of Compound 1 that have an acceptable balance of these properties and can be used in the preparation of pharmaceutically acceptable solid dosage forms.

Crystalline Form A

[0147] In one aspect, the present disclosure relates to crystalline Form A of a hydrobromide salt of (S)-2-(((S)-6,8-difluoro-l,2,3,4-tetrahydronaphthalen-2-yl)amino)- N-(l -(2 -methyl- l-(neopentylamino) propan-2-yl)-lH-imidazol-4-yl)pentanamide having Formula (I),

[0148] In one embodiment, crystalline Form A is anhydrous.

[0149] In another embodiment, the melting point of crystalline Form A is about 254 °C.

[0150] In another embodiment, Form A is characterized by an XRPD pattern having peaks at 8.8 ± 0.2, 9.8 ± 0.2, and 23.3 ± 0.2 degrees two theta when measured by Cu Ka radiation. In another embodiment, Form A is characterized by an XRPD pattern having peaks at 8.8 ± 0.2, 9.8 ± 0.2, 23.3 ± 0.2, 25.4 ± 0.2, 28.0 ± 0.2, and 29.3 ± 0.2 degrees two theta when measured by Cu Ka radiation. In another embodiment, Form A is characterized by an XRPD pattern having peaks at 8.8 ± 0.2, 9.8 ± 0.2, 20.0 ± 0.2, 23.3 ± 0.2, 25.4 ± 0.2, 28.0 ± 0.2, 29.3 ± 0.2, and 32.5 ± 0.2 degrees two theta when measured by Cu Ka radiation.

Patent

Product case, WO2005092864 ,

hold protection in the EU states until March 2025, and expire in the US in February 2026 with US154 extension.

PATENT

WO2020208572 , co-assigned to GSK and SpringWorks, claiming a combination of nirogacestat with anti-BCMA antibody (eg belantamab mafodotin ), for treating cancer.

PATENT

US10590087 , for a prior filing from Pfizer, claiming crystalline forms of nirogacestat hydrobromide.

////////////NIROGACESTAT, orphan drug designation, esmoid tumors,  fast track designation, PF-03084014, PF 03084014, QZ62892OFJ , UNII:QZ62892OFJ ,UNII-QZ62892OFJ, ,нирогацестат , نيروغاسيستات , 尼罗司他 , ニロガセスタット, phase 3

CCCC(C(=O)NC1=CN(C=N1)C(C)(C)CNCC(C)(C)C)NC2CCC3=C(C2)C(=CC(=C3)F)F

SEQUENCE1

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Sequence Modifications

Tozinameran

Pfizer–BioNTech COVID-19 vaccine

トジナメラン (JAN);
コロナウイルス修飾ウリジンRNAワクチン;

RNA (recombinant 5′-​[1,​2-​[(3′-​O-​methyl)​m⁷G-​(5’→5′)​-​ppp-​Am]​]​-​capped all uridine→N¹-​methylpseudouridine-​substituted severe acute respiratory syndrome coronavirus 2 secretory signal peptide contg. spike glycoprotein S1S2-​specifying plus 5′- and 3′-​untranslated flanking region-​contg. poly(A)​-​tailed messenger BNT162b2)​, inner salt

Nucleic Acid Sequence

Sequence Length: 42841106 a 1315 c 1062 g 801 umodified

APPROVED JAPAN Comirnaty, 2021/2/14

CAS 2417899-77-3

5085ZFP6SJ

UNII-5085ZFP6SJ

Bnt-162b2

Bnt162b2

Tozinameran is mRNA encoding full length of spike protein analog of SARS-CoV-2

Target Severe acute respiratory syndrome coronavirus 2 spike glycoprotein

Coronavirus disease – COVID-19

NAME	INGREDIENTS	DOSAGE	ROUTE	LABELLER	MARKETING START	MARKETING END
Pfizer-BioNTech Covid-19 Vaccine	Pfizer-BioNTech Covid-19 Vaccine (0.23 mg/1.8mL)	Injection, suspension	Intramuscular	Pfizer Manufacturing Belgium NV	2020-12-12	Not applicable

NAME	DOSAGE	STRENGTH	ROUTE	LABELLER	MARKETING START	MARKETING END
Comirnaty		30 mcg	Intramuscular	Bio N Tech Manufacturing Gmb H	2021-01-06	Not applicable
Pfizer-BioNTech Covid-19 Vaccine	Suspension	30 mcg	Intramuscular	Biontech Manufacturing Gmbh	2020-12-14	Not applicable
Pfizer-BioNTech Covid-19 Vaccine	Injection, suspension	0.23 mg/1.8mL	Intramuscular	Pfizer Manufacturing Belgium NV	2020-12-12	Not applicable

The Pfizer–BioNTech COVID‑19 vaccine (pINN: tozinameran), sold under the brand name Comirnaty,^[13] is a COVID-19 vaccine developed by the German company BioNTech in cooperation with Pfizer. It is both the first COVID-19 vaccine to be authorized by a stringent regulatory authority for emergency use^[14][15] and the first cleared for regular use.^[16]

It is given by intramuscular injection. It is an RNA vaccine composed of nucleoside-modified mRNA (modRNA) encoding a mutated form of the spike protein of SARS-CoV-2, which is encapsulated in lipid nanoparticles.^[17] The vaccination requires two doses given three weeks apart.^[18][19][20] Its ability to prevent severe infection in children, pregnant women, or immunocompromised people is unknown, as is the duration of the immune effect it confers.^[20][21][22] As of February 2021, it is one of two RNA vaccines being deployed against COVID‑19, the other being the Moderna COVID‑19 vaccine. A third mRNA-based COVID-19 vaccine, CVnCoV, is in late-stage testing.^[23]

Trials began in April 2020; by November, the vaccine had been tested on more than 40,000 people.^[24] An interim analysis of study data showed a potential efficacy of over 90% in preventing infection within seven days of a second dose.^[19][20] The most common side effects include mild to moderate pain at the injection site, fatigue, and headache.^[25][26] As of December 2020, reports of serious side effects, such as allergic reactions, have been very rare,^[a] and no long-term complications have been reported.^[28] Phase III clinical trials are ongoing: monitoring of the primary outcomes will continue until August 2021, while monitoring of the secondary outcomes will continue until January 2023.^[18]

In December 2020, the United Kingdom was the first country to authorize the vaccine on an emergency basis,^[28] soon followed by the United States, the European Union and several other countries globally.^{[29][30][6][31][32]}

BioNTech is the initial developer of the vaccine, and partnered with Pfizer for development, clinical research, overseeing the clinical trials, logistics, finances and for manufacturing worldwide with the exception of China.^[33] The license to distribute and manufacture in China was purchased by Fosun, alongside its investment in BioNTech.^[34][35] Distribution in Germany and Turkey is by BioNTech itself.^[36] Pfizer indicated in November 2020, that 50 million doses could be available globally by the end of 2020, with about 1.3 billion doses in 2021.^[20]

Pfizer has advanced purchase agreements of about US$3 billion for providing a licensed vaccine in the United States, the European Union, the United Kingdom, Japan, Canada, Peru, Singapore, and Mexico.^[37][38] Distribution and storage of the vaccine is a logistics challenge because it needs to be stored at temperatures between −80 and −60 °C (−112 and −76 °F),^[39] until five days before vaccination^[38][39] when it can be stored at 2 to 8 °C (36 to 46 °F), and up to two hours at temperatures up to 25 °C (77 °F)^[40][11] or 30 °C (86 °F).^[41][42] In February 2021, Pfizer and BioNTech asked the U.S. Food and Drug Administration (FDA) to update the emergency use authorization (EUA) to permit the vaccine to be stored at between −25 and −15 °C (−13 and 5 °F) for up to two weeks before use.^[43]

Development and funding

Before COVID-19 vaccines, a vaccine for an infectious disease had never before been produced in less than several years, and no vaccine existed for preventing a coronavirus infection in humans.^[44] After the COVID-19 virus was detected in December 2019,^[45] the development of BNT162b2 was initiated on 10 January 2020, when the SARS-CoV-2 genetic sequences were released by the Chinese Center for Disease Control and Prevention via GISAID,^[46][47][48] triggering an urgent international response to prepare for an outbreak and hasten development of preventive vaccines.^[49][50]

In January 2020, German biotech-company BioNTech started its program ‘Project Lightspeed’ to develop a vaccine against the new COVID‑19 virus based on its already established mRNA-technology.^[24] Several variants of the vaccine were created in their laboratories in Mainz, and 20 of those were presented to experts of the Paul-Ehrlich-Institute in Langen.^[51] Phase I / II Trials were started in Germany on 23 April 2020, and in the U.S. on 4 May 2020, with four vaccine candidates entering clinical testing. The Initial Pivotal Phase II / III Trial with the lead vaccine candidate ‘BNT162b2’ began in July. The Phase III results indicating a 95% effectiveness of the developed vaccine were published on 18 November 2020.^[24]

BioNTech received a US$135 million investment from Fosun in March 2020, in exchange for 1.58 million shares in BioNTech and the future development and marketing rights of BNT162b2 in China,^[35] Hong Kong, Macau and Taiwan.^[52]

In June 2020, BioNTech received €100 million (US$119 million) in financing from the European Commission and European Investment Bank.^[53] In September 2020, the German government granted BioNTech €375 million (US$445 million) for its COVID‑19 vaccine development program.^[54]

Pfizer CEO Albert Bourla stated that he decided against taking funding from the US government’s Operation Warp Speed for the development of the vaccine “because I wanted to liberate our scientists [from] any bureaucracy that comes with having to give reports and agree how we are going to spend the money in parallel or together, etc.” Pfizer did enter into an agreement with the US for the eventual distribution of the vaccine, as with other countries.^[55]

Clinical trials

See also: COVID-19 vaccine § Clinical trials started in 2020

Preliminary results from Phase I–II clinical trials on BNT162b2, published in October 2020, indicated potential for its efficacy and safety.^[17][56] During the same month, the European Medicines Agency (EMA) began a periodic review of BNT162b2.^[57]

The study of BNT162b2 is a continuous-phase trial in Phase III as of November 2020.^[18] It is a “randomized, placebo-controlled, observer-blind, dose-finding, vaccine candidate-selection, and efficacy study in healthy individuals”.^[18] The early-stage research determined the safety and dose level for two vaccine candidates, with the trial expanding during mid-2020 to assess efficacy and safety of BNT162b2 in greater numbers of participants, reaching tens of thousands of people receiving test vaccinations in multiple countries in collaboration with Pfizer and Fosun.^[20][35]

The Phase III trial assesses the safety, efficacy, tolerability, and immunogenicity of BNT162b2 at a mid-dose level (two injections separated by 21 days) in three age groups: 12–15 years, 16–55 years or above 55 years.^[18] For approval in the EU, an overall vaccine efficacy of 95% was confirmed by the EMA.^[58] The EMA clarified that the second dose should be administered three weeks after the first dose.^[59]

The ongoing Phase III trial, which is scheduled to run from 2020 to 2022, is designed to assess the ability of BNT162b2 to prevent severe infection, as well as the duration of immune effect.^[20][21][22]

Pfizer and BioNTech started a Phase II/III randomized control trial in healthy pregnant women 18 years of age and older (NCT04754594).^[60] The study will evaluate 30 µg of BNT162b2 or placebo administered via intramuscular injection in 2 doses, 21 days apart. The Phase II portion of the study will include approximately 350 pregnant women randomized 1:1 to receive BNT162b2 or placebo at 27 to 34 weeks’ gestation. The Phase III portion of this study will assess the safety, tolerability, and immunogenicity of BNT162b2 or placebo among pregnant women enrolled at 24 to 34 weeks’ gestation. Pfizer and BioNTech announced on 18 February 2021 that the first participants received their first dose in this trial.^[61]

Vaccine technology

See also: RNA vaccine and COVID-19 vaccine § Technology platforms

The BioNTech technology for the BNT162b2 vaccine is based on use of nucleoside-modified mRNA (modRNA) which encodes part of the spike protein found on the surface of the SARS-CoV-2 coronavirus (COVID‑19), triggering an immune response against infection by the virus protein.^[62]

The vaccine candidate BNT162b2 was chosen as the most promising among three others with similar technology developed by BioNTech.^[18][62][56] Prior to choosing BNT162b2, BioNTech and Pfizer had conducted Phase I trials on BNT162b1 in Germany and the United States, while Fosun performed a Phase I trial in China.^[17][63] In these Phase I studies, BNT162b2 was shown to have a better safety profile than the other three BioNTech candidates.^[63]

Sequence

The modRNA sequence of the vaccine is 4,284 nucleotides long.^[64] It consists of a five-prime cap; a five prime untranslated region derived from the sequence of human alpha globin; a signal peptide (bases 55–102) and two proline substitutions (K986P and V987P, designated “2P”) that cause the spike to adopt a prefusion-stabilized conformation reducing the membrane fusion ability, increasing expression and stimulating neutralizing antibodies;^[17][65] a codon-optimized gene of the full-length spike protein of SARS-CoV-2 (bases 103–3879); followed by a three prime untranslated region (bases 3880–4174) combined from AES and mtRNR1 selected for increased protein expression and mRNA stability^[66] and a poly(A) tail comprising 30 adenosine residues, a 10-nucleotide linker sequence, and 70 other adenosine residues (bases 4175–4284).^[64] The sequence contains no uridine residues; they are replaced by 1-methyl-3′-pseudouridylyl.^[64]

Composition

In addition to the mRNA molecule, the vaccine contains the following inactive ingredients (excipients):^[67][68][8]

• ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)
• ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide
• 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)
• cholesterol
• dibasic sodium phosphate dihydrate
• monobasic potassium phosphate
• potassium chloride
• sodium chloride
• sucrose
• water for injection

The first four of these are lipids. The lipids and modRNA together form nanoparticles. ALC-0159 is a polyethylene glycol conjugate (that is, a PEGylated lipid).^[69]

The vaccine is supplied in a multidose vial as “a white to off-white, sterile, preservative-free, frozen suspension for intramuscular injection“.^[11][12] It must be thawed to room temperature and diluted with normal saline before administration.^[12]

Authorizations

Expedited

The United Kingdom’s Medicines and Healthcare products Regulatory Agency (MHRA) gave the vaccine “rapid temporary regulatory approval to address significant public health issues such as a pandemic” on 2 December 2020, which it is permitted to do under the Medicines Act 1968.^[70] It was the first COVID‑19 vaccine to be approved for national use after undergoing large scale trials,^[71] and the first mRNA vaccine to be authorized for use in humans.^[14][72] The United Kingdom thus became the first Western country to approve a COVID‑19 vaccine for national use,^[73] although the decision to fast-track the vaccine was criticised by some experts.^[74]

On 8 December 2020, Margaret “Maggie” Keenan, 90, from Fermanagh, became the first person to receive the vaccine.^[75] In a notable example of museums documenting the pandemic, the vial and syringe used for that first dose were saved acquired by The Science Museum in London for its permanent collection.^[76] By 20 December, 521,594 UK residents had received the vaccine as part of the national vaccination programme. 70% had been to people aged 80 or over.^[77]

After the United Kingdom, the following countries expedited processes to approve the Pfizer–BioNTech COVID‑19 vaccine for use: Argentina,^[78] Australia,^[79] Bahrain,^[80] Canada,^[7][81] Chile,^[82] Costa Rica,^[83] Ecuador,^[82] Hong Kong,^[84] Iraq,^[85] Israel,^[86] Jordan,^[87] Kuwait,^[88] Mexico,^[89] Oman,^[90] Panama,^[91] the Philippines,^[92] Qatar,^[93] Saudi Arabia,^[32][94] Singapore,^[95][96] the United Arab Emirates,^[97] and the United States.^[10]

The World Health Organization (WHO) authorized it for emergency use.^[98]

In the United States, an emergency use authorization (EUA) is “a mechanism to facilitate the availability and use of medical countermeasures, including vaccines, during public health emergencies, such as the current COVID‑19 pandemic”, according to the FDA.^[99] Following an EUA issuance, BioNTech and Pfizer are expected to continue the Phase III clinical trial to finalize safety and efficacy data, leading to application for licensure (approval) of the vaccine in the United States.^{[99][100][101]} The United States Centers for Disease Control and Prevention (CDC) Advisory Committee on Immunization Practices (ACIP) approved recommendations for vaccination of those aged 16 years or older.^[102][103]

Standard

On 19 December 2020, the Swiss Agency for Therapeutic Products (Swissmedic) approved the Pfizer–BioNTech COVID‑19 vaccine for regular use, two months after receiving the application, stating that the vaccine fully complied with the requirements of safety, efficacy and quality. This is the first authorization under a standard procedure.^[1][104] On 23 December, a Lucerne resident, a 90-year-old woman, became the first person to receive the vaccine in Switzerland.^[105] This marked the beginning of mass vaccination in continental Europe.^[106]

On 21 December 2020, the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) recommended granting conditional marketing authorization for the Pfizer–BioNTech COVID‑19 vaccine under the brand name Comirnaty.^{[2][107][108]} The recommendation was accepted by the European Commission the same day.^[107][109]

On February 23, 2021, the Brazilian Health Regulatory Agency approved the Pfizer–BioNTech COVID-19 vaccine under its standard marketing authorization procedure. It became the first COVID-19 vaccine to receive definitive registration rather than emergency use authorization in the country.^[110]

Adverse effects

The adverse effect profile of the Pfizer–BioNTech COVID‑19 vaccine is similar to that of other adult vaccines.^[20] During clinical trials, the side effects deemed very common^[a] are (in order of frequency): pain and swelling at the injection site, tiredness, headache, muscle aches, chills, joint pain, and fever.^[68] Fever is more common after the second dose.^[68] These effects are predictable and to be expected, and it is particularly important that people be aware of this to prevent vaccine hesitancy.^[111]

Severe allergic reaction has been observed in approximately 11 cases per million doses of vaccine administered.^[112][113] According to a report by the US Centers for Disease Control and Prevention 71% of those allergic reactions happened within 15 minutes of vaccination and mostly (81%) among people with a documented history of allergies or allergic reactions.^[112] The UK’s Medicines and Healthcare products Regulatory Agency (MHRA) advised on 9 December 2020, that people who have a history of “significant” allergic reaction should not receive the Pfizer–BioNTech COVID‑19 vaccine.^{[114][115][116]} On 12 December, the Canadian regulator followed suit, noting that: “Both individuals in the U.K. had a history of severe allergic reactions and carried adrenaline auto injectors. They both were treated and have recovered.”^[67]

On 28 January 2021, the European Union published a COVID-19 vaccine safety update which found that “the benefits of Comirnaty in preventing COVID‑19 continue to outweigh any risks, and there are no recommended changes regarding the use the vaccine.”^[113][117] No new side effects were identified.^[113]

Manufacturing

A doctor holding the Pfizer vaccine

Pfizer and BioNTech are manufacturing the vaccine in their own facilities in the United States and in Europe in a three-stage process. The first stage involves the molecular cloning of DNA plasmids that code for the spike protein by infusing them into Escherichia coli bacteria. In the United States, this stage is conducted at a small pilot plant in Chesterfield, Missouri^[118] (near St. Louis). After four days of growth, the bacteria are killed and broken open, and the contents of their cells are purified over a week and a half to recover the desired DNA product. The DNA is stored in tiny bottles and frozen for shipment. Safely and quickly transporting the DNA at this stage is so important that Pfizer has used its company jet and helicopter to assist.^[119]

The second stage is being conducted at plants in Andover, Massachusetts^[120] in the United States, and in Germany. The DNA is used as a template to build the desired mRNA strands. Once the mRNA has been created and purified, it is frozen in plastic bags about the size of a large shopping bag, of which each can hold up to 5 to 10 million doses. The bags are placed on special racks on trucks which take them to the next plant.^[119]

The third stage is being conducted at plants in Portage, Michigan^[121] (near Kalamazoo) in the United States, and Puurs in Belgium. This stage involves combining the mRNA with lipid nanoparticles, then filling vials, boxing vials, and freezing them.^[119] Croda International subsidiary Avanti Polar Lipids is providing the requisite lipids.^[122] As of November 2020, the major bottleneck in the manufacturing process was combining mRNA with lipid nanoparticles.^[119]

In February 2021, Pfizer revealed this entire sequence initially took about 110 days on average from start to finish, and that the company was making progress on reducing that number to 60 days.^[123] Vaccine manufacturers normally take several years to optimize the process of making a particular vaccine for speed and cost-effectiveness before attempting large-scale production.^[123] Due to the urgency presented by the COVID-19 pandemic, Pfizer began production immediately with the process by which the vaccine had been originally formulated in the laboratory, then started to identify ways to safely speed up and scale up that process.^[123]

BioNTech announced in September 2020 that it had signed an agreement to acquire from Novartis a manufacturing facility in Marburg, Germany, to expand their vaccine production capacity.^[124] Once fully operational, the facility would produce up to 750 million doses per year, or over 60 million doses per month. The site will be the third BioNTech facility in Europe which currently produces the vaccine, while Pfizer operates at least four production sites in the United States and Europe.

Advance orders and logistics

Pfizer indicated in its 9 November press release that 50 million doses could be available by the end of 2020, with about 1.3 billion doses provided globally by 2021.^[20] In February 2021, BioNTech announced it would increase production by more than 50% to manufacture two billion doses in 2021.^[125]

In July 2020, the vaccine development program Operation Warp Speed placed an advance order of US$1.95 billion with Pfizer to manufacture 100 million doses of a COVID‑19 vaccine for use in the United States if the vaccine was shown to be safe and effective.^{[34][126][127][128]} By mid-December 2020, Pfizer had agreements to supply 300 million doses to the European Union,^[129] 120 million doses to Japan,^[130] 40 million doses (10 million before 2021) to the United Kingdom,^[22] 20 million doses to Canada,^[131] an unspecified number of doses to Singapore,^[132] and 34.4 million doses to Mexico.^[133] Fosun also has agreements to supply 10 million doses to Hong Kong and Macau.^[134] The Hong Kong government said it would receive its first batch of one million doses by the first quarter of 2021.^[135]

BioNTech and Fosun agreed to supply Mainland China with a batch of 100 million doses in 2021, subject to regulatory approval. The initial supply will be delivered from BioNTech’s production facilities in Germany.^[136]

The vaccine is being delivered in vials that, once diluted, contain 2.25 ml of vaccine (0.45 ml frozen plus 1.8ml diluent).^[101] According to the vial labels, each vial contains five 0.3 ml doses, however excess vaccine may be used for one, or possibly two, additional doses.^[101][137] The use of low dead space syringes to obtain the additional doses is preferable, and partial doses within a vial should be discarded.^[101][138] The Italian Medicines Agency officially authorized the use of excess doses remaining within single vials.^[139] As of 8 January 2021, each vial contains six doses.^{[68][140][141][138]} In the United States, vials will be counted as five doses when accompanied by regular syringes and as six doses when accompanied by low dead space syringes.^[142]

Temperature the Pfizer vaccine must be kept at to ensure effectiveness, roughly between −80 and −60 °C (−112 and −76 °F)

Logistics in developing countries which have preorder agreements with Pfizer—such as Ecuador and Peru—remain unclear.^[38] Even high-income countries have limited cold chain capacity for ultracold transport and storage of a vaccine that degrades within five days when thawed, and requires two shots three weeks apart.^[38] The vaccine needs to be stored and transported at ultracold temperatures between −80 and −60 °C (−112 and −76 °F),^{[39][22][38][143][144]} much lower than for the similar Moderna vaccine. The head of Indonesia‘s Bio Farma Honesti Basyir stated that purchasing the vaccine is out of the question for the world’s fourth-most populous country, given that it did not have the necessary cold chain capability. Similarly, India’s existing cold chain network can only handle temperatures between 2 and 8 °C (36 and 46 °F), far above the requirements of the vaccine.^[145][146]

In January 2021, Pfizer and BioNTech offered to supply 50 million doses of COVID‑19 vaccine for health workers across Africa between March and the end of 2021, at a discounted price of US$10 per dose.^[147]

Name

BNT162b2 was the code name during development and testing,^[17][148] tozinameran is the proposed international nonproprietary name (pINN),^[149] and Comirnaty is the brand name.^[1][2] According to BioNTech, the name Comirnaty “represents a combination of the terms COVID‑19, mRNA, community, and immunity.”^[150][151]

The vaccine also has the common name “COVID‑19 mRNA vaccine (nucleoside-modified)”^[2] and may be distributed in packaging with the name Pfizer–BioNTech COVID‑19 Vaccine.”^[152]

How the Pfizer-BioNTech Vaccine Works

By Jonathan Corum and Carl ZimmerUpdated Jan. 21, 2021Leer en español

The German company BioNTech partnered with Pfizer to develop and test a coronavirus vaccine known as BNT162b2, the generic name tozinameran or the brand name Comirnaty. A clinical trial demonstrated that the vaccine has an efficacy rate of 95 percent in preventing Covid-19.

A Piece of the Coronavirus

The SARS-CoV-2 virus is studded with proteins that it uses to enter human cells. These so-called spike proteins make a tempting target for potential vaccines and treatments.

Spikes

Spike

protein

gene

CORONAVIRUS

Like the Moderna vaccine, the Pfizer-BioNTech vaccine is based on the virus’s genetic instructions for building the spike protein.

mRNA Inside an Oily Shell

The vaccine uses messenger RNA, genetic material that our cells read to make proteins. The molecule — called mRNA for short — is fragile and would be chopped to pieces by our natural enzymes if it were injected directly into the body. To protect their vaccine, Pfizer and BioNTech wrap the mRNA in oily bubbles made of lipid nanoparticles.

Lipid nanoparticles

surrounding mRNA

Because of their fragility, the mRNA molecules will quickly fall apart at room temperature. Pfizer is building special containers with dry ice, thermal sensors and GPS trackers to ensure the vaccines can be transported at –94°F (–70°C) to stay viable.

Entering a Cell

After injection, the vaccine particles bump into cells and fuse to them, releasing mRNA. The cell’s molecules read its sequence and build spike proteins. The mRNA from the vaccine is eventually destroyed by the cell, leaving no permanent trace.

VACCINE

PARTICLES

VACCINATED

CELL

Spike

protein

mRNA

Translating mRNA

Three spike

proteins combine

Spike

Cell

nucleus

Spikes

and protein

fragments

Displaying

spike protein

fragments

Protruding

spikes

Some of the spike proteins form spikes that migrate to the surface of the cell and stick out their tips. The vaccinated cells also break up some of the proteins into fragments, which they present on their surface. These protruding spikes and spike protein fragments can then be recognized by the immune system.

Spotting the Intruder

When a vaccinated cell dies, the debris will contain many spike proteins and protein fragments, which can then be taken up by a type of immune cell called an antigen-presenting cell.

Debris from

a dead cell

Engulfing

a spike

ANTIGEN-

PRESENTING

CELL

Digesting

the proteins

Presenting a

spike protein

fragment

HELPER

T CELL

The cell presents fragments of the spike protein on its surface. When other cells called helper T cells detect these fragments, the helper T cells can raise the alarm and help marshal other immune cells to fight the infection.

Making Antibodies

Other immune cells, called B cells, may bump into the coronavirus spikes on the surface of vaccinated cells, or free-floating spike protein fragments. A few of the B cells may be able to lock onto the spike proteins. If these B cells are then activated by helper T cells, they will start to proliferate and pour out antibodies that target the spike protein.

HELPER

T CELL

Activating

the B cell

Matching

surface proteins

VACCINATED

CELL

B CELL

SECRETED

ANTIBODIES

Stopping the Virus

The antibodies can latch onto coronavirus spikes, mark the virus for destruction and prevent infection by blocking the spikes from attaching to other cells.

ANTIBODIES

VIRUS

Killing Infected Cells

The antigen-presenting cells can also activate another type of immune cell called a killer T cell to seek out and destroy any coronavirus-infected cells that display the spike protein fragments on their surfaces.

ANTIGEN-

PRESENTING

CELL

Presenting a

spike protein

fragment

ACTIVATED

KILLER

T CELL

INFECTED

CELL

Beginning

to kill the

infected cell

Remembering the Virus

The Pfizer-BioNTech vaccine requires two injections, given 21 days apart, to prime the immune system well enough to fight off the coronavirus. But because the vaccine is so new, researchers don’t know how long its protection might last.

First dose, 0.3ml

Second dose, 21 days later

A preliminary study found that the vaccine seems to offer strong protection about 10 days after the first dose, compared with people taking a placebo:

Cumulative incidence of Covid-19 among clinical trial participants 2.5% 2.0 People taking a placebo

1.5 1.0 Second dose First dose People taking the

Pfizer-BioNTech vaccine

0.5

0

1

2

3

4

8

12

16

Weeks after the first dose

It’s possible that in the months after vaccination, the number of antibodies and killer T cells will drop. But the immune system also contains special cells called memory B cells and memory T cells that might retain information about the coronavirus for years or even decades.

For more about the vaccine, see Pfizer’s Covid Vaccine: 11 Things You Need to Know.

Preparation and Injection

Each vial of the vaccine contains 5 doses of 0.3 milliliters. The vaccine must be thawed before injection and diluted with saline. After dilution the vial must be used within six hours.

A diluted vial of the vaccine at Royal Free Hospital in London.Jack Hill/Agence France-Presse

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108. ^ “Comirnaty”. Union Register of medicinal products. Retrieved 8 January 2021.
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120. ^ Hughes M (20 December 2020). “Andover’s piece of the vaccine: Pfizer”. The Eagle-Tribune.
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122. ^ Mullin R (25 November 2020). “Pfizer, Moderna ready vaccine manufacturing networks”. Chemical & Engineering News. Washington, D.C.: American Chemical Society. Retrieved 21 December 2020.
123. ^ Jump up to:^a b c Weise, Elizabeth (7 February 2021). “Pfizer expects to cut COVID-19 vaccine production time by close to 50% as production ramps up, efficiencies increase”. USA Today.
124. ^ “BioNTech to Acquire GMP Manufacturing Site to Expand COVID-19 Vaccine Production Capacity in First Half 2021 | BioNTech”. investors.biontech.de. Retrieved 5 February2021.
125. ^ “Statement on Manufacturing | BioNTech”. investors.biontech.de. Retrieved 5 February2021.
126. ^ Erman M, Ankur B (22 July 2020). “U.S. to pay Pfizer, BioNTech $1.95 bln for millions of COVID-19 vaccine doses”. Reuters. Archived from the original on 22 July 2020. Retrieved 22 July 2020.
127. ^ “U.S. Government Engages Pfizer to Produce Millions of Doses of COVID-19 Vaccine”. US Department of Health and Human Services. 22 July 2020. Archived from the original on 22 July 2020. Retrieved 23 July 2020.
128. ^ Nazaryan A (9 November 2020). “So is Pfizer part of Operation Warp Speed or not? Yes and no”. Yahoo!. Archived from the original on 10 November 2020. Retrieved 9 November 2020.
129. ^ Pleitgen F (11 November 2020). “EU agrees to buy 300 million doses of the Pfizer/BioNTech Covid-19 vaccine”. CNN. Archived from the original on 24 November 2020. Retrieved 26 November 2020.
130. ^ “Japan and Pfizer reach COVID-19 vaccine deal to treat 60 million people”. The Japan Times. 1 August 2020. Archived from the original on 10 November 2020. Retrieved 21 November 2020.
131. ^ Tasker JP (9 November 2020). “Trudeau says promising new Pfizer vaccine could be ‘light at the end of the tunnel'”. CBC News. Archived from the original on 9 November 2020. Retrieved 9 November 2020.
132. ^ “Pfizer and BioNTech to Supply Singapore with their BNT162b2 mRNA-based Vaccine Candidate to Combat COVID-19”. pfizer.com.sg. Pfizer Singapore. 14 December 2020. Retrieved 1 February 2021.
133. ^ de Salud S. “233. Firma secretario de Salud convenio con Pfizer para fabricación y suministro de vacuna COVID-19”. gob.mx (in Spanish). Retrieved 17 December 2020.
134. ^ Ng E (27 August 2020). “Fosun Pharma to supply Covid-19 vaccine to Hong Kong, Macau once approved”. South China Morning Post. Archived from the original on 20 November 2020. Retrieved 21 November 2020.
135. ^ Ting V, Lau C, Wong O (11 December 2020). “Hong Kong buys 15 million Covid-19 vaccine doses from Sinovac, Pfizer”. South China Morning Post. Retrieved 18 December2020.
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External links

“Tozinameran”. Drug Information Portal. U.S. National Library of Medicine.

• Global Information About Pfizer–BioNTech COVID‑19 Vaccine (also known as BNT162b2) Pfizer
• Comirnaty assessment report European Medicines Agency Committee for Medicinal Products for Human Use
• A Phase 1/2/3 Study to Evaluate the Safety, Tolerability, Immunogenicity, and Efficacy of RNA Vaccine Candidates Against COVID‑19 in Healthy Individuals Pfizer clinical protocol
• Pfizer Vaccince News, updates and tracking of Israel’s vaccinaion campaign
• “How the Pfizer-BioNTech Covid-19 Vaccine Works”. The New York Times.

/////////Tozinameran, APPROVALS 2021,   JAPAN 2021,  Comirnaty, Coronavirus disease, COVID-19, BNT162b2 , BNT162b2 SARS-CoV-2 Vaccine, RNA ingredient BNT-162B2

The Pfizer-BioNTech COVID-19 vaccine (Tozinameran, INN), also known as BNT162b2, is one of four advanced mRNA-based vaccines developed through “Project Lightspeed,” a joint program between Pfizer and BioNTech.^2,3 Tozinameran is a nucleoside modified mRNA (modRNA) vaccine encoding an optimized full-length version of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein. It is designed to induce immunity against SARS-CoV-2, the virus responsible for causing COVID-19.² The modRNA is formulated in lipid nanoparticles for administration via intramuscular injection in two doses, three weeks apart.^1,3

Tozinameran is undergoing evaluation in clinical trials in both the USA (NCT04368728) and Germany (NCT04380701).^4,5 Tozinameran received fast track designation by the U.S. FDA on July 13, 2020.⁶ On December 11, 2020, the FDA issued an Emergency Use Authorization (EUA) based on 95% efficacy in clinical trials and a similar safety profile to other viral vaccines over a span of approximately 2 months.¹ Tozinameran was granted an EUA in the UK on December 2, 2020,⁸ and in Canada on December 9, 2020⁷ for active immunization against SARS-CoV-2.¹²

Currently, sufficient data are not available to determine the longevity of protection against COVID-19, nor direct evidence that the vaccine prevents the transmission of the SARS-CoV-2 virus from one individual to another.⁹ Fact sheets for caregivers, recipients, and healthcare providers are now available.^10,11

Tozinameran has not yet been fully approved by any country. In both the UK and Canada, Tozinameran is indicated under an interim authorization for active immunization to prevent COVID-19 caused by SARS-CoV-2 in individuals aged 16 years and older.^7,8

On December 11, 2020, the U.S. Food and Drug Administration granted emergency use authorization (EUA) for Tozinameran to prevent COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients aged 16 years and above.⁹ Safety and immune response information for adolescents 12-15 years of age will follow, and studies to further explore the administration of Tozinameran in pregnant women, children under 12 years of age, and those in special risk groups will be evaluated in the future.¹

This vaccine should only be administered where appropriate medical treatment for immediate allergic reactions are immediately available in the case of an acute anaphylactic reaction after vaccine administration.¹² Tozinameran administration should be postponed in any individual suffering from an acute febrile illness. Its use should be carefully considered in immunocompromised individuals and individuals with a bleeding disorder or on anticoagulant therapy. Appropriate medical treatment should be readily available in case of an anaphylactic reaction following vaccine administration.^7,8

Tozinameran contains nucleoside modified mRNA (modRNA) encapsulated in lipid nanoparticles that deliver the modRNA into host cells. The lipid nanoparticle formulation facilitates the delivery of the RNA into human cells.¹² Once inside these cells, the modRNA is translated by host machinery to produce the SARS-CoV-2 spike (S) protein antigen, which is subsequently recognized by the host immune system. Tozinameran has been shown to elicit both neutralizing antibody and cellular immune responses to the S protein, which helps protect against subsequent SARS-CoV-2 infection.^7,8

Tozinameran is a nucleoside modified mRNA (modRNA) vaccine encoding an optimized full-length version of the SARS-CoV-2 spike (S) protein, translated and expressed in cells in vaccinated individuals to produce the S protein antigen against which an immune response is mounted. As with all vaccines, protection cannot be guaranteed in all recipients, and full protection may not occur until at least seven days following the second dose.^7,8

In U.S. clinical trials, the vaccine was 95% effective in preventing COVID-19; eight COVID-19 cases occurred in the vaccine group and 162 cases occurred in the placebo group. Of the total 170 COVID-19 cases, one case in the vaccine group and three cases in the placebo group were considered to be severe infections.^1,9

1. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez JL, Perez Marc G, Moreira ED, Zerbini C, Bailey R, Swanson KA, Roychoudhury S, Koury K, Li P, Kalina WV, Cooper D, Frenck RW Jr, Hammitt LL, Tureci O, Nell H, Schaefer A, Unal S, Tresnan DB, Mather S, Dormitzer PR, Sahin U, Jansen KU, Gruber WC: Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020 Dec 10. doi: 10.1056/NEJMoa2034577. [PubMed:33301246]
2. Gen Eng News: BNT162 vaccine candidates [Link]
3. BioNTech BNT162 Update [Link]
4. Clinical Trial NCT04368728 [Link]
5. Clinical Trial NCT04380701 [Link]
6. FDA fast track designation: BNT162b1 and BNT162b2 [Link]
7. Health Canada Interim Product Monograph: BNT162b2 SARS-CoV-2 Vaccine [Link]
8. MHRA Interim Product Monograph: BNT162b2 SARS-CoV-2 Vaccine [Link]
9. FDA News Release: FDA Takes Key Action in Fight Against COVID-19 By Issuing Emergency Use Authorization for First COVID-19 Vaccine [Link]
10. Pfizer: Fact Sheet for Healthcare Providers Administering Vaccine, Pfizer-BioNtech COVID-19 vaccine [Link]
11. Pfizer: Fact Sheet for Recipients and Caregivers, Pfizer BioNTech COVID-19 vaccine [Link]
12. FDA Emergency Use Authorization: Full EUA Prescribing information, Pfizer-BioNTech COVID-19 vaccine [Link]
13.  
    PHASESTATUSPURPOSECONDITIONSCOUNT2Active Not RecruitingPreventionCoronavirus Disease 2019 (COVID‑19)12, 3Active Not RecruitingPreventionCoronavirus Disease 2019 (COVID‑19)11, 2Active Not RecruitingPreventionCoronavirus Disease 2019 (COVID‑19)11, 2RecruitingTreatmentCoronavirus Disease 2019 (COVID‑19) / Protection Against COVID-19 and Infections With SARS CoV 2 / Respiratory Tract Infections (RTI) / RNA Virus Infections / Vaccine Adverse Reaction / Viral Infections / Virus Diseases1 

(Heavy chain)
EVQLVESGGG VIQPGGSLRL SCAASGFTFD DYAMNWVRQG PGKGLEWVSA ISGDGGSTYY
ADSVKGRFTI SRDNSKNSLY LQMNSLRAED TAFFYCAKDL RNTIFGVVIP DAFDIWGQGT
MVTVSSASTK GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP
AVLQSSGLYS LSSVVTVPSS SLGTKTYTCN VDHKPSNTKV DKRVESKYGP PCPPCPAPEF
LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE
QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVYTLPPS
QEEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSRLTVDK
SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK
(Light chain)
DIQMTQSPST LSASVGDRVT ITCRASQSIR SWLAWYQQKP GKAPKLLIYK ASSLESGVPS
RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSYSYTFGQ GTKLEIKRTV AAPSVFIFPP
SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
(Disulfide bridge: H22-H96, H140-L214, H153-H209, H232-H’232, H235-H’235, H267-H327, H373-H431, H’22-H’96, H’140-L’214, H’153-H’209, H’267-H’327, H’373-H’431, L23-L88, L134-L194, L’23-L’88, L’134-L’194)

Evinacumab

エビナクマブ (遺伝子組換え)

Immunoglobulin G4, anti-​(human protein ANGPTL3 (angiopoietin-​like 3)​) (human monoclonal REGN1500 heavy chain)​, disulfide with human monoclonal REGN1500 light chain, dimer

Protein Sequence

Sequence Length: 1334, 453, 453, 214, 214multichain; modified (modifications unspecified)

FDA APPROVED,  2021/2/11, EVKEEZA

Antihyperlipidemic, Anti-angiopietin like 3

Monoclonal antibody
Treatment of dyslipidemia

• REGN 1500
• REGN-1500
• REGN1500

Sequence:

1EVQLVESGGG VIQPGGSLRL SCAASGFTFD DYAMNWVRQG PGKGLEWVSA51ISGDGGSTYY ADSVKGRFTI SRDNSKNSLY LQMNSLRAED TAFFYCAKDL101RNTIFGVVIP DAFDIWGQGT MVTVSSASTK GPSVFPLAPC SRSTSESTAA151LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS201SLGTKTYTCN VDHKPSNTKV DKRVESKYGP PCPPCPAPEF LGGPSVFLFP251PKPKDTLMIS RTPEVTCVVV DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE301QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR351EPQVYTLPPS QEEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT401PPVLDSDGSF FLYSRLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS451LGK

Sequence:

1EVQLVESGGG VIQPGGSLRL SCAASGFTFD DYAMNWVRQG PGKGLEWVSA51ISGDGGSTYY ADSVKGRFTI SRDNSKNSLY LQMNSLRAED TAFFYCAKDL101RNTIFGVVIP DAFDIWGQGT MVTVSSASTK GPSVFPLAPC SRSTSESTAA151LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS201SLGTKTYTCN VDHKPSNTKV DKRVESKYGP PCPPCPAPEF LGGPSVFLFP251PKPKDTLMIS RTPEVTCVVV DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE301QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR351EPQVYTLPPS QEEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT401PPVLDSDGSF FLYSRLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS451LGK

Sequence:

1DIQMTQSPST LSASVGDRVT ITCRASQSIR SWLAWYQQKP GKAPKLLIYK51ASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSYSYTFGQ101GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV151DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG201LSSPVTKSFN RGEC

Sequence:

1DIQMTQSPST LSASVGDRVT ITCRASQSIR SWLAWYQQKP GKAPKLLIYK51ASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSYSYTFGQ101GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV151DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG201LSSPVTKSFN RGEC

Sequence Modifications

PATENTS

WO 2017024062

 US 20170305999 

Evinacumab, sold under the brand name Evkeeza, is a monoclonal antibody medication for the treatment of homozygous familial hypercholesterolemia (HoFH).^[1][2]

Evinacumab is a recombinant human IgG4 monoclonal antibody targeted against angiopoietin-like protein 3 (ANGPTL3) and the first drug of its kind. The ANGPTL family of proteins serve a number of physiologic functions – including involvement in the regulation of lipid metabolism – which have made them desirable therapeutic targets in recent years.² Loss-of-function mutations in ANGPTL3 have been noted to result in hypolipidemia and subsequent reductions in cardiovascular risk, whereas increases in function appear to be associated with cardiovascular risk, and it was these observations that provided a rationale for the development of a therapy targeted against ANGPTL3.³

In February 2021, evinacumab became the first-and-only inhibitor of ANGPTL3 to receive FDA approval after it was granted approval for the adjunctive treatment of homozygous familial hypercholesterolemia (HoFH) under the brand name “Evkeeza”.⁸ Evinacumab is novel in its mechanism of action compared with other lipid-lowering therapies and therefore provides a unique and synergistic therapeutic option in the treatment of HoFH.

Common side effects include nasopharyngitis (cold), influenza-like illness, dizziness, rhinorrhea (runny nose), and nausea. Serious hypersensitivity (allergic) reactions have occurred in the Evkeeza clinical trials.^[2]

Evinacumab binds to the angiopoietin-like protein 3 (ANGPTL3).^[2] ANGPTL3 slows the function of certain enzymes that break down fats in the body.^[2] Evinacumab blocks ANGPTL3, allowing faster break down of fats that lead to high cholesterol.^[2] Evinacumab was approved for medical use in the United States in February 2021.^[2][3]

NAME	DOSAGE	STRENGTH	ROUTE	LABELLER	MARKETING START	MARKETING END
Evkeeza	Injection, solution, concentrate	150 mg/1mL	Intravenous	Regeneron Pharmaceuticals, Inc.	2021-02-11	Not applicable
Evkeeza	Injection, solution, concentrate	150 mg/1mL	Intravenous	Regeneron Pharmaceuticals, Inc.	2021-02-11	Not applicable

History

The effectiveness and safety of evinacumab were evaluated in a double-blind, randomized, placebo-controlled, 24-week trial enrolling 65 participants with homozygous familial hypercholesterolemia (HoFH).^[2] In the trial, 43 participants received 15 mg/kg of evinacumab every four weeks and 22 participants received the placebo.^[2] Participants were taking other lipid-lowering therapies as well.^[2]

The primary measure of effectiveness was the percent change in low-density lipoprotein (LDL-C) from the beginning of treatment to week 24.^[2] At week 24, participants receiving evinacumab had an average 47% decrease in LDL-C while participants on the placebo had an average 2% increase.^[2]

The U.S. Food and Drug Administration (FDA) granted the application for evinacumab orphan drug, breakthrough therapy, and priority review designations.^[2] The FDA granted approval of Evkeeza to Regeneron Pharmaceuticals, Inc.^[2]

References

1. ^ Jump up to:^a b https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/761181s000lbl.pdf
2. ^ Jump up to:^{a b c d e f g h i j k l m n} “FDA approves add-on therapy for patients with genetic form of severely”. U.S. Food and Drug Administration (FDA). 11 February 2021. Retrieved 12 February 2021.  This article incorporates text from this source, which is in the public domain.
3. ^ “FDA Approves First-in-class Evkeeza (evinacumab-dgnb) for Patients with Ultra-rare Inherited Form of High Cholesterol” (Press release). Regeneron Pharmaceuticals. 11 February 2021. Retrieved 12 February 2021 – via PR Newswire.

Further reading

• Dewey FE, Gusarova V, Dunbar RL, O’Dushlaine C, Schurmann C, Gottesman O, et al. (July 2017). “Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease”. N Engl J Med. 377 (3): 211–221. doi:10.1056/NEJMoa1612790. PMC 5800308. PMID 28538136.

External links

• “Evinacumab”. Drug Information Portal. U.S. National Library of Medicine.

//////////////Evinacumab, Peptide, APPROVALS 2021, FDA 2021, Monoclonal antibody, dyslipidemia, エビナクマブ (遺伝子組換え) , REGN 1500, REGN-1500, REGN1500,

Fluvoxamine

• Molecular FormulaC₁₅H₂₁F₃N₂O₂
• Average mass318.335 Da
• 54739-18-3

(E)-5-Methoxy-1-[4-(trifluoromethyl)phenyl]-1-pentanone O-(2-Aminoethyl)oxime1-Pentanone, 5-methoxy-1-[4-(trifluoromethyl)phenyl]-, O-(2-aminoethyl)oxime, (1E)-2-[({(1E)-5-Methoxy-1-[4-(trifluoromethyl)phenyl]pentylidene}amino)oxy]ethanamine
2-{[(E)-{5-Methoxy-1-[4-(trifluoromethyl)phenyl]pentylidene}amino]oxy}ethanamine1-Pentanone, 5-methoxy-1-(4-(trifluoromethyl)phenyl)-, O-(2-aminoethyl)oxime, (E)- 
387954739-18-3[RN]5583954[Beilstein]5-Methoxy-4′-(trifluoromethyl)valerophenone (E)-O-(2-aminoethyl)oximeA selective serotonin reuptake inhibitor that is used in the treatment of DEPRESSION and a variety of ANXIETY DISORDERS.

Fluvoxamine, sold under the brand name Luvox among others, is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class^[5] which is used primarily for the treatment of obsessive–compulsive disorder (OCD).^[6] It is also used to treat depression and anxiety disorders, such as panic disorder, social anxiety disorder, and post-traumatic stress disorder.^[7][8]

FLUVOXAMINE MALEATE

C₁₉H₂₅F₃N₂O₆, 434.4 g/mol

1-Pentanone, 5-methoxy-1-(4-(trifluoromethyl)phenyl)-, O-(2-aminoethyl)oxime, (E)-, (Z)-2-butenedioate (1:1)

(Z)-but-2-enedioic acid;2-[(E)-[5-methoxy-1-[4-(trifluoromethyl)phenyl]pentylidene]amino]oxyethanamine

Luvox

61718-82-9

CAS 54739-20-7

Fevarin, Luvox CR

Synonyms

• 5-Methoxy-4′-(trifluoromethyl)valerophenone (E)-O-(2-aminoethyl)oxime, maleate (1:1)
• 5-Methoxy-4′-trifluoromethylvalerophenone (E)-O-2-aminoethyloxime monomaleate
• DU23000
• • Fevarin
  • Fluvoxamine maleate
  • Luvox
  • Luvox CR
  • SME 3110
  • UNII-5LGN83G74V

Medical uses

Fluvoxamine is approved in the United States for OCD,^[9][6] and social anxiety disorder.^[10] In other countries (e.g., Australia,^[11][12] the UK,^[13] and Russia^[14]) it also has indications for major depressive disorder. In Japan it is currently^[when?] approved to treat OCD, SAD and MDD.^[15][16] Fluvoxamine is indicated for children and adolescents with OCD.^[17] The drug works long-term, and retains its therapeutic efficacy for at least one year.^[18] It has also been found to possess some analgesic properties in line with other SSRIs and tricyclic antidepressants.^[19][20][21]

There is tentative evidence that fluvoxamine is effective for social phobia in adults.^[22] Fluvoxamine is also effective for GAD, SAD, panic disorder and separation anxiety disorder in children and adolescents.^[23] There is tentative evidence that fluvoxamine may help some people with negative symptoms of chronic schizophrenia.^[24][25]

A double-blind controlled study found that fluvoxamine may prevent clinical deterioration in outpatients with symptomatic COVID-19. The study had important limitations: it was run fully remotely; it had a small sample size (150) and short follow-up duration (15 days).^[26] The accompanying editorial noted that, although this study is important enough to choose out of more than 10,000 other COVID-19 related submissions, it “presents only preliminary information” and “the findings should be interpreted as only hypothesis generating; they should not be used as the basis for current treatment decisions.”^[27] Similarly, the study authors themselves cautioned that “the trial’s results should not be treated as a measure of fluvoxamine’s effectiveness against COVID-19 but as an encouraging indicator that the drug warrants further testing.”^[28] A prospective open-labelled cohort study showed similar results.^[29]

Adverse effects

Gastrointestinal side effects are more common in those receiving fluvoxamine than with other SSRIs.^[30] Otherwise, fluvoxamine’s side-effect profile is very similar to other SSRIs.^{[2][9][11][13][31][32]}Common (1–10% incidence) adverse effects

• Nausea
• Vomiting
• Weight loss
• Yawning
• Loss of appetite
• Agitation
• Nervousness
• Anxiety
• Insomnia
• Somnolence (drowsiness)
• Tremor
• Restlessness
• Headache
• Dizziness
• Palpitations
• Tachycardia (high heart rate)
• Abdominal pain
• Dyspepsia (indigestion)
• Diarrhea
• Constipation
• Hyperhidrosis (excess sweating)
• Asthenia (weakness)
• Malaise
• Sexual dysfunction (including delayed ejaculation, erectile dysfunction, decreased libido, etc.)
• Xerostomia (dry mouth)

Uncommon (0.1–1% incidence) adverse effects

• Arthralgia
• Hallucination
• Confusional state
• Extrapyramidal side effects (e.g. dystonia, parkinsonism, tremor, etc.)
• Orthostatic hypotension
• Cutaneous hypersensitivity reactions (e.g. oedema [buildup of fluid in the tissues], rash, pruritus)

Rare (0.01–0.1% incidence) adverse effects

• Mania
• Seizures
• Abnormal hepatic (liver) function
• Photosensitivity (being abnormally sensitive to light)
• Galactorrhoea (expulsion of breast milk unrelated to pregnancy or breastfeeding)

Unknown frequency adverse effects

• Hyperprolactinaemia (elevated plasma prolactin levels leading to galactorrhoea, amenorrhoea [cessation of menstrual cycles], etc.)
• Bone fractures
• Glaucoma
• Mydriasis
• Urinary incontinence
• Urinary retention
• Bed-wetting
• Serotonin syndrome — a potentially fatal condition characterised by abrupt onset muscle rigidity, hyperthermia (elevated body temperature), rhabdomyolysis, mental status changes (e.g. coma, hallucinations, agitation), etc.
• Neuroleptic malignant syndrome — practically identical presentation to serotonin syndrome except with a more prolonged onset
• Akathisia — a sense of inner restlessness that presents itself with the inability to stay still
• Paraesthesia
• Dysgeusia
• Haemorrhage
• Withdrawal symptoms
• Weight changes
• Suicidal ideation and behaviour
• Violence towards others^[33]
• Hyponatraemia
• Syndrome of inappropriate antidiuretic hormone secretion
• Ecchymoses

Interactions[edit]

Luvox (fluvoxamine) 100 mg film-coated scored tablets

Fluvoxamine inhibits the following cytochrome P450 enzymes:^{[34][35][36][37][38][39][40][41][42]}

• CYP1A2 (strongly) which metabolizes agomelatine, amitriptyline, caffeine, clomipramine, clozapine, duloxetine, haloperidol, imipramine, phenacetin, tacrine, tamoxifen, theophylline, olanzapine, etc.
• CYP3A4 (moderately) which metabolizes alprazolam, aripiprazole, clozapine, haloperidol, quetiapine, pimozide, ziprasidone, etc.^[43]
• CYP2D6 (weakly) which metabolizes aripiprazole, chlorpromazine, clozapine, codeine, fluoxetine, haloperidol, olanzapine, oxycodone, paroxetine, perphenazine, pethidine, risperidone, sertraline, thioridazine, zuclopenthixol, etc.^[44]
• CYP2C9 (moderately) which metabolizes nonsteroidal anti-inflammatory drugs, phenytoin, sulfonylureas, etc.
• CYP2C19 (strongly) which metabolizes clonazepam, diazepam, phenytoin, etc.
• CYP2B6 (weakly) which metabolizes bupropion, cyclophosphamide, sertraline, tamoxifen, valproate, etc.

By so doing, fluvoxamine can increase serum concentration of the substrates of these enzymes.^[34]

The plasma levels of oxidatively metabolized benzodiazepines (e.g., triazolam, midazolam, alprazolam and diazepam) are likely to be increased when co-administered with fluvoxamine. However the clearance of benzodiazepines metabolized by glucuronidation (e.g., lorazepam, oxazepam, temazepam)^[45][46] is unlikely to be affected by fluvoxamine.^[47] It appears that benzodiazepines metabolized by nitro-reduction (clonazepam, nitrazepam) are unlikely to be affected by fluvoxamine.^[48] Using fluvoxamine and alprazolam together can increase alprazolam plasma concentrations.^[49] If alprazolam is coadministered with fluvoxamine, the initial alprazolam dose should be reduced to the lowest effective dose.^[50][51]

Fluvoxamine and ramelteon coadministration is not indicated.^[52][53]

Fluvoxamine has been observed to increase serum concentrations of mirtazapine, which is mainly metabolized by CYP1A2, CYP2D6, and CYP3A4, by 3- to 4-fold in humans.^[54] Caution and adjustment of dosage as necessary are warranted when combining fluvoxamine and mirtazapine.^[54]

Fluvoxamine seriously affects the pharmacokinetics of tizanidine and increases the intensity and duration of its effects. Because of the potentially hazardous consequences, the concomitant use of tizanidine with fluvoxamine, or other potent inhibitors of CYP1A2, should be avoided.^[55]

Fluvoxamine’s interaction with St John’s wort can lead to increased serotonin levels and potentially lead to serotonin syndrome.^{[citation needed]}

Pharmacology

Fluvoxamine is a potent selective serotonin reuptake inhibitor with around 100-fold affinity for the serotonin transporter over the norepinephrine transporter.^[35] It has negligible affinity for the dopamine transporter or any other site, with the sole exception of the σ₁ receptor.^[59][60] It behaves as a potent agonist at this receptor and has the highest affinity (36 nM) of any SSRI for doing so.^[59] This may contribute to its antidepressant and anxiolytic effects and may also afford it some efficacy in treating the cognitive symptoms of depression.^[61] Unlike fluoxetine, fluvoxamine’s metabolites are inactive, without a significant effect on serotonin or norepinephrine uptake.^[62]

History

Fluvoxamine was developed by Kali-Duphar,^[63] part of Solvay Pharmaceuticals, Belgium, now Abbott Laboratories, and introduced as Floxyfral in Switzerland in 1983.^[63] It was approved by the U.S. Food and Drug Administration (FDA) in 1994, and introduced as Luvox in the US.^[64] In India, it is available, among several other brands, as Uvox by Abbott.^[65] It was one of the first SSRI antidepressants to be launched, and is prescribed in many countries to patients with major depression.^[66] It was the first SSRI, a non-TCA drug, approved by the U.S. FDA specifically for the treatment of OCD.^[67] At the end of 1995, more than ten million patients worldwide had been treated with fluvoxamine.^{[68][failed verification]} Fluvoxamine was the first SSRI to be registered for the treatment of obsessive compulsive disorder in children by the FDA in 1997.^[69] In Japan, fluvoxamine was the first SSRI to be approved for the treatment of depression in 1999^[70][71] and was later in 2005 the first drug to be approved for the treatment of social anxiety disorder.^[72] Fluvoxamine was the first SSRI approved for clinical use in the United Kingdom.^[73]

Society and culture

Manufacturers include BayPharma, Synthon, and Teva, among others.^[74]

SYN

SYN

J. Zhejiang Univ. (Medical Sci.) (2003), 32 (5), 441-442

PATENT

WO 2014178064

The present invention relates to an improved and industrially applicable process for the preparation of fluvoxamine maleate of formula I,

Fluvoxamine or (E)-5-methoxy-1 -[4-(trifluoromethyl)phenyl]pentan- 1 -one-O-2-aminoethyl oxime is an antidepressant which functions as a selective serotonin reuptake inhibitor (SSRI). Fluvoxamine is used for the treatment of major depressive disorder (MDD), obsessive compulsive disorder (OCD), and anxiety disorders such as panic disorder and post-traumatic stress disorder (PTSD). Fluvoxamine CR (controlled release) is approved to treat social anxiety disorder.

Fluvoxamine maleate and compounds were first disclosed in US patent 4,085,225. According to said patent, Fluvoxamine maleate prepared by alkylation reaction of 5-methoxy-4′-trifluoromethylvalerophenone oxime, compound of formula III with 2-chloroethylamine hydrochloride in dimethylformamide in the presence of a base such as potassium hydroxide powder for two days at 25°C.

Subsequently the solvent is removed under vacuum then the residue is acidified and extracted with ether to remove the unreacted oxime followed by basification. The obtained fluvoxamine base in ether extract is washed with sodium bicarbonate solution. The fluvoxamine base is then treated with maleic acid in absolute ethanbl and the residue obtained by concentration under vacuum is recrystallized from acetonitrile to obtain fluvoxamine maleate. The process is very much tedious, time consuming as it requires two days for the reaction completion. Operations like removal of dimethylformamide, ether, ethanol makes process cumbersome at plant level. Requirement of

various solvents lead the process to be non-eco-friendly. Moreover the patent is silent about yield and purity of the product.

In an alternate route described in US patent 4,085,225, the oxime of formula III is converted to formula I in a five step process i.e. alkylation of formula III with ethylene oxide. The reaction solvent is ethanol in which lithium is already dissolved. The reaction further involves addition of acetic acid to give the hydroxyethyl compound of formula A as oil. The compound of formula A is purified chromatographically over the silica gel, which is converted to a mesylate compound of formula B by treating with methanesulfonyl chloride and triethylamine at -5 to 0°C, then aminated with ammonia in methanol at 100°C using autoclave for 16 hours followed by removal of methanol and extraction in ether to give fluvoxamine base.

The base is then converted to the maleate salt formula I, which is finally purified by recrystallization from acetonitrile.

There are lots of disadvantages involve like more unit operations, use of various solvents and handling of ethylene oxide which is also known for its carcinogen effect. More unit operations lead to long occupancy of reactors in the plant as well as man power, high energy consumption and require bigger plant. These all parameters make the process commercially unviable as wel l as environmentally non-feasible. Further, purification of the compound of formula A requires cumbersome technique i.e chromatography over silica gel as well as lengthy work-up procedure in U.S. Pat. No. 4,085,225 requires complete removal of organic solvents at various stages.

US patent 6,433,225 discloses the process for preparing fluvoxamine maleate, prepared by alkylating 5-methoxy-4′-trtfluoromethylvalerophenone oxime, compound of formula III with 2-chloroethylamine hydrochloride in toluene and PEG-400 (polyethyleneglycol-400) as facilitator in the presence of a base potassium hydroxide powder at 30-35°C to obtain fluvoxamine base in

toluene layer is then treated with maleic acid in water. The precipitated fluvoxamine maleate is filtered and washed with toluene and dried. The obtained dried cake recrystallized with water to get fluvoxamine maleate. The process disclosed in the patent is silent about actual purity of the product. As per our scientist’s observation alkylation reaction at the temperature of 30-35°C may lead to non completion of reaction and results lower yield. Additional step of purification may further lead to loss of yield.

Thus, present invention fulfills the need of the art and provides an improved and industrially applicable process for preparation of fluvoxamine maleate, which provides fluvoxamine maleate in high purity and overall good yield.

EXAMPLES:

Stage – 1 : Preparation of (1E)-N-hydroxy-5-methoxy-1-(4-trifluoromethyI pheny 1) pentan-1-imine formula III

To a stirred solution of 5-methoxy- 1 -(4-trifluoromethylphenyl) pentan-1 -one ( 150 gm) in methanol (750 ml), sodium carbonate (granule) (72 gm) and hydroxylamine hydrochloride (59.64 gm) were added at temperature 25-30°C. The reaction mass was heated 45-50°C for 10- 15 minutes followed by maintaining the reaction mass at temperature 45-50°C for 8-9 hours under stirring. The reaction mass was cooled to 25-30°C and filtered under vacuum to remove unreacted inorganic matter, then distilled out the methanol completely from the collected filtrate under vacuum at temperature below 50°C. The obtained slurry was cooled to 25-30°C and water (300 ml) was added into the residue followed by the addition of hexane (300×2 ml) and stirred for 30 minutes. The layers were separated. The collected organic layer was stirred for 5- 10 minutes at temperature 25-30°C followed by cooling the mass at temperature -5°C to – 10°C, stirred for 30-40 minutes and filtered at the same temperature. The product was suck dried at -5 to -10°C and further in vacuum at 25-30°C for 2-3 hours to give 138 – 142 gm of title compound. HPLC purity: >98.5%

Stage – 2: Preparation of crude fluvoxamine maleate formula I

To a prepared solution of dimethyl sulphoxide (575 ml), potassium hydroxide flakes ( 1 14.64 gm) and water (69 ml), stage-1 (1 15 gm) was added at temperature 40-45°C. The reaction mixture was stirred to get clear solution followed by adding 2-chloroethylamine hydrochloride (86.36 gm) drop wise into the reaction mixture at temperature 40-45°C and maintained for 1 -2 hour. Water (1 150 ml) was added in to the reaction mixture at temperature 25-30°C and stirred for 20-25 minutes. Then toluene (575 ml x 2) was added and stirred for 30 minutes and preceded for separation of layers followed by washing the toluene layer with water ( 1 1 50 x 5 ml). The solution of maleic acid (48.47 gm) dissolved in water (98 ml) was added into above obtained toluene layer and stirred at temperature 25-30°C for 2-3 hours. The reaction mixture was cooled to 0-5°C and maintained for 30-40 minutes at the same temperature. The obtained material was washed with toluene, filtered and suck dried. The wet cake was then added hexane (600 ml) and stirred for 30 minutes at temperature 25-30°C, filtered, washed with hexane and dried to get 161 gm of title compound. HPLC purity: >98.5%

Stage – 3: Preparation of pure fluvoxamine maleate formula I

In to the reaction assembly, water (600 ml) was added and heated to 40-45°C. Stage -2 ( 1 50 gm) was added into the hot water under stirring. The reaction mixture was stirred for 5- 10 minutes, filtered and cooled to 25°C. Toluene (68 ml) was added into the reaction mixture at temperature 25°C and stirred for 30 minutes. Filtered the solid, washed with 10-15°C chilled water and dried to get the pure 127.5 gm fluvoxamine maleate. HPLC purity: >99.8%

Process for isolation of 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one formula II

To a solution of cone. HCl (600 ml) and water ( 160 ml), organic residue (250 gm) of ( 1 £)+( 1 Z) of 1 -N-hydroxy-5-methoxy- 1 -[4-(trifluoromethyl) phenyl]pentan-1 -imine and traces of 5-methoxy- 1 -[4-(trifluoromethyl)phenyl]pentan- 1-one (obtained after hexane recovery from stage-1 filtrate) was added at temperature 25-30°C under stirring. The reaction mixture was heated to 67-75°C and maintained for 13-14 hours followed by cool ing the reaction mixture at temperature 25-30°C. Then after hexane (500 x 2 ml) was added into the reaction mixture and stirred for 15 minutes at 25-30°C. The organic layers were separated and sodium bicarbonate solution (25 gm sodium bicarbonate dissolved in 250 ml water) was added into the hexane layer and stirred for 15 minutes. The layers were separated and water (250ml) was added into hexane layer and stirred for 15 minutes at temperature 25-30°C. Further the layers were separated and hexane layer was added activated charcoal ( 12.5 gm) and stirred for 20-30 minutes at temperature 30-35°C. The reaction mixture was filtered and stirred for 5-10 minutes at 25-30°C followed by cooling at 0 to -5°C and stirred for 30-40 minutes at 0 to -5°C. The reaction mixture was filtered and dried to get 150 to l 75 gm of title compound. HPLC purity: >99%.

PATENT

 US 20140243544

 IN 2013MU01290/WO 2014178064

WO 2014035107

PATENT

https://patents.google.com/patent/US9783492B2/en

Fluvoxamine or (E)-5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one-O-2-aminoethyl oxime is an antidepressant which functions as a selective serotonin reuptake inhibitor (SSRI). Fluvoxamine is used for the treatment of major depressive disorder (MDD), obsessive compulsive disorder (OCD), and anxiety disorders such as panic disorder and post-traumatic stress disorder (PTSD). Fluvoxamine CR (controlled release) is approved to treat social anxiety disorder.

Fluvoxamine maleate and compounds were first disclosed in U.S. Pat. No. 4,085,225. According to said patent, Fluvoxamine maleate prepared by alkylation reaction of 5-methoxy-4′-trifluoromethylvalerophenone oxime, compound of formula III with 2-chloroethylamine hydrochloride in dimethylformamide in the presence of a base such as potassium hydroxide powder for two days at 25° C.

Subsequently the solvent is removed under vacuum then the residue is acidified and extracted with ether to remove the unreacted oxime followed by basification. The obtained fluvoxamine base in ether extract is washed with sodium bicarbonate solution. The fluvoxamine base is then treated with maleic acid in absolute ethanol and the residue obtained by concentration under vacuum is recrystallized from acetonitrile to obtain fluvoxamine maleate. The process is very much tedious, time consuming as it requires two days for the reaction completion. Operations like removal of dimethylformamide, ether, ethanol makes process cumbersome at plant level. Requirement of various solvents lead the process to be non-eco-friendly. Moreover the patent is silent about yield and purity of the product.

In an alternate route described in U.S. Pat. No. 4,085,225, the mine of formula III is converted to formula I in a five step process i.e. alkylation of formula III with ethylene oxide. The reaction solvent is ethanol in which lithium is already dissolved. The reaction further involves addition of acetic acid to give the hydroxyethyl compound of formula A as oil. The compound of formula A is purified chromatographically over the silica gel, which is converted to a mesylate compound of formula B by treating with methanesulfonyl chloride and triethylamine at −5 to 0° C., then aminated with ammonia in methanol at 100° C. using autoclave for 16 hours followed by removal of methanol and extraction in ether to give fluvoxamine base.

The base is then converted to the maleate salt formula I, which is finally purified by recrystallization from acetonitrile.

There are lots of disadvantages in like more unit operations, use of various solvents and handling of ethylene oxide which is also known for its carcinogen effect. More unit operations lead to long occupancy of reactors in the plant as well as man power, high energy consumption and require bigger plant. These all parameters make the process commercially unviable as well as environmentally non-feasible. Further, purification of the compound of formula A requires cumbersome technique i.e chromatography over silica gel as well as lengthy work-up procedure in U.S. Pat. No. 4,085,225 requires complete removal of organic solvents at various stages.

U.S. Pat. No. 6,433,225 discloses the process for preparing fluvoxamine maleate, prepared by alkylating 5-methoxy-4′-trifluoromethylvalerophenone oxime compound of formula III with 2-chloroethylamine hydrochloride in toluene and PEG-400 (polyethyleneglycol-400) as facilitator in the presence of a base potassium hydroxide powder at 30-35°C. to obtain fluvoxamine base in toluene layer is then treated with maleic acid in water. The precipitated fluvoxamine maleate is filtered and washed with toluene and dried. The obtained dried cake recrystallized with water to get fluvoxamine maleate. The process disclosed in the patent is silent about actual purity of the product. As per our scientist’s observation alkylation reaction at the temperature of 30-35° C. may lead to non completion of reaction and results lower yield. Additional step of purification may further lead to loss of yield.

EXAMPLES

Stage-1: Preparation of (1 E)-N-hydroxy-5-methoxy-1-(4-trifluoromethyl phenyl)pentan-1-imine Formula III

To a stirred solution of 5-methoxy-1-(4-trifluoromethylphenyl)pentan-1one (150 gm) in methanol (750 ml), sodium carbonate (granule) (72 gm) and hydroxylamine hydrochloride (59.64 gm) were added at temperature 25-30° C. The reaction mass was heated 45-50° C. for 10-15 minutes followed by maintaining the reaction mass at temperature 45-50° C. for 8-9 hours under stirring. The reaction mass was cooled to 25-30° C. and filtered under vacuum to remove unreacted inorganic matter, then distilled out the methanol completely from the collected filtrate under vacuum at temperature below 50° C. The obtained slurry was cooled to 25-30° C. and water (300 ml) was added into the residue followed by the addition of hexane (300×2 ml) and stirred for 30 minutes. The layers were separated. The collected organic layer was stirred for 5-10 minutes at temperature 25-30° C. followed by cooling the mass at temperature −5° C. to −10° C., stirred for 30-40 minutes and filtered at the same temperature. The product was suck dried at −5 to −10° C. and further in vacuum at 25-30° C. for 2-3 hours to give 138-142 gm of title compound. HPLC purity: >98.5%

Stage-2: Preparation of Crude Fluvoxamine Maleate Formula I

To a prepared solution of dimethyl sulphoxide (575 ml), potassium hydroxide flakes (114.64 gm) and water (69 ml), stage-1 (115 gm) was added at temperature 40-45° C. The reaction mixture was stirred to get clear solution followed by adding 2-chloroethylamine hydrochloride (8636 gm) drop wise into the reaction mixture at temperature 40-45° C. and maintained for 1-2 hour. Water (1150 ml) was added in to the reaction mixture at temperature 25-30° C. and stirred for 20-25 minutes. Then toluene (575 ml×2) was added and stirred for 30 minutes and preceded for separation of layers followed by washing the toluene layer with water (1150×5 ml). The solution of maleic acid (48.47 gm) dissolved in water (98 ml) was added into above obtained toluene layer and stirred at temperature 25-30° C. for 2-3 hours. The reaction mixture was cooled to 0-5° C. and maintained for 30-40 minutes at the same temperature. The obtained material was washed with toluene, filtered and such dried. The wet cake was then added hexane (600 ml) and stirred for 30 minutes at temperature 25-30° C., filtered, washed with hexane and dried to get 161 gm of title compound. HPLC purity: >98.5%

Stage-3: Preparation of Pure Fluvoxamine Maleate Formula I

In to the reaction assembly, water (600 ml) was added and heated to 40-45° C. Stage-2 (150 gm) was added into the hot water under stirring. The reaction mixture was stirred for 5-10 minutes, filtered and cooled to 25° C. Toluene (68 ml) was added into the reaction mixture at temperature 25° C. and stirred for 30 minutes. Filtered the solid, washed with 10-15° C. chilled water and dried to get the pure 127.5 gm fluvoxamine maleate. HPLC purity: >99.8%

Process for isolation of 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one Formula II

To a solution of conc. HCl (600 ml) and water (160 organic residue (250 gm) of (1 E)+(1 Z) of 1-N-hydroxy-5-methoxy-1-[4trifluoromethyl)phenyl]pentan-1-imine and traces of 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one (obtained after hexane recovery from stage-1 filtrate) was added at temperature 25-30° C. under stirring. The reaction mixture was heated to 67-75° C. and maintained for 13-14 hours followed by cooling the reaction mixture at temperature 25-30° C. Then after hexane (500×2 ml) was added into the reaction mixture and stirred for 15 minutes at 25-30° C. The organic layers were separated and sodium bicarbonate solution (25 gm sodium bicarbonate dissolved in 250 ml water) was added into the hexane layer and stirred for 15 minutes. The layers were separated and water (250 ml) was added into hexane layer and stirred for 15 minutes at temperature 25-30° C. Further the layers were separated and hexane layer was added activated charcoal (12.5 gm) and stirred for 20-30 minutes at temperature 30-35° C. The reaction mixture was filtered and stirred for 5-10 minutes at 25-30° C. followed by cooling at 0 to −5° C. and stirred for 30-40 minutes at 0 to −5° C. The reaction mixture was filtered and dried to get 150 to 175 gm of title compound. HPLC purity: >99%.
Claims (5)Hide Dependent 

We claim:1. An improved process for the preparation of fluvoxamine maleate of formula I,

wherein the improvements comprises the steps of:a). condensing the compound of formula II,

with hydroxylamine hydrochloride in the presence of sodium carbonate granules at temperature 45-50° C. in suitable solvent to form a compound of formula III, wherein the compound of formula III comprises a mixture of (1E)+(1Z) isomers of 1-N-hydroxy-5-methoxy-1-[4(trifluoromethyl)phenyl]pentan-1-imine, and wherein the mixture of (1E)+(1Z) isomers of 1-N-hydroxy-5-methoxy-1-[4(trifluoromethyl)phenyl]pentan-1-imine comprises 98% of E-isomer and 2% of Z-isomer;

b). isolating compound of formula III;c). treating compound of formula III with 2-chloroethylamine hydrochloride in the presence of base in suitable solvent at 40-45° C. to form compound of formula IV;

d). extracting compound of formula IV with suitable solvent to form an organic layer;e). treating organic layer of step d) with maleic acid;f). isolating crude fluvoxamine maleate of formula I; andg). optionally purifying fluvoxamine maleate of formula I.

2. The process according to claim 1, wherein in step a), said suitable solvent is selected from the group consisting of alcohol, ketone, nitrile, and hydrocarbons in any suitable proportion or mixtures thereof;in step c), said base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, triethylamine and diisopropylethyamine;in step c), said solvent is selected from the group consisting of dimethylformamide (DMF), dimethylsulphoxide (DMSO) and hexamethylphosphoramide (HMPA) in any suitable proportion or mixtures thereof; andin step d) said suitable solvent is selected from the group consisting of toluene and xylene.3. A process for the isolation of 5-methoxy-1-[4-(trifluoromethyl)phenyl]pentan-1-one of formula II from mixture of (1E)+(1Z) of 1-N -hydroxy-5-methoxy-1-[4-(trifluoromethyl) phenyl]pentan-1-imine of formula III by treating compound of formula III with aqueous hydrochloric acid, wherein the mixture of (1E)+(1Z) of 1-N-hydroxy-5-methoxy-1-[4-(trifluoromethyl) phenyl]pentan-1-imine of formula III comprises 98% of E-isomer and 2% of Z-isomer.4. The process according to claim 3, wherein the reaction is performed at temperature 65-75°C.5. The process according to claim 1, wherein in step a), said suitable solvent is methanol. 
Publication numberPriority datePublication dateAssigneeTitleUS4081551A *1975-03-201978-03-28U.S. Philips CorporationOxime ethers having anti-depressive activityUS4085225A1975-03-201978-04-18U.S. Philips CorporationOxime ethers having anti-depressive activityCN1079733A *1993-04-081993-12-22中国科学院成都有机化学研究所The synthetic method of a-benzoin oximeUS6433225B11999-11-122002-08-13Sun Pharamaceutical Industries, Ltd.Process for the preparation of fluvoxazmine maleateCN101654419A *2009-09-122010-02-24西北师范大学Preparation method of fluvoxamine maleate 
SynUS 6433225 SUN 

SYN US 4085225

SYNGB 1535226

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External links

• “Fluvoxamine”. Drug Information Portal. U.S. National Library of Medicine.

/////////DU23000, Fevarin, Fluvoxamine maleate, Luvox, Luvox CR, SME 3110, UNII-5LGN83G74V, Fluvoxamine, sme 3110, DU 23000

#DU23000, #Fevarin, #Fluvoxamine maleate, #Luvox, #Luvox CR, #SME 3110, #UNII-5LGN83G74V, #Fluvoxamine, #sme 3110, #DU 23000

DETOMIDINE1H-Imidazole, 4-[(2,3-dimethylphenyl)methyl]-
4-(2,3-Dimethylbenzyl)-1H-imidazole
507876631-46-4[RN]7N8K34P2XH

• Molecular FormulaC₁₂H₁₄N₂
• Average mass186.253 Da

UNII-7N8K34P2XHдетомидинديتوميدين地托咪定

Formal Name5-[(2,3-dimethylphenyl)methyl]-1H-imidazole, monohydrochlorideCAS Number90038-01-0Synonyms

• Domosedan
• MPV 253AII

Molecular FormulaC₁₂H₁₄N₂ • HClFormula Weight222.7DetomidineCAS Registry Number: 76631-46-4CAS Name: 4-[(2,3-Dimethylphenyl)methyl]-1H-imidazoleAdditional Names: 4-(2¢,3¢-dimethylbenzyl)imidazoleMolecular Formula: C12H14N2Molecular Weight: 186.25Percent Composition: C 77.38%, H 7.58%, N 15.04%Literature References: a2-Adrenoceptor agonist with sedative and analgesic activity. Prepn: A. J. Karjalayne, K. O. A. Kurkela, EP24829; eidem,US4443466 (1981, 1984 both to Farmos). Physical studies: E. Laine et al.,Acta Pharm. Suec.20, 451 (1983). Crystal structure: L. H. J. Lajunen et al.,ibid.21, 163 (1984). Pharmacology: R. Virtanen, L. Nyman, Eur. J. Pharmacol.108, 163 (1985); R. Virtanen, E. MacDonald, ibid.115, 277 (1985). Mechanism of action: eidem,J. Vet. Pharmacol. Ther.8, 30 (1985).Properties: Crystals from acetone, mp 114-116°. LD50 i.v. in mice: 35 mg/kg (Karjalayne, Kurkela).Melting point: mp 114-116°Toxicity data: LD50 i.v. in mice: 35 mg/kg (Karjalayne, Kurkela) Derivative Type: HydrochlorideTrademarks: Domosedan (Farmos)Molecular Formula: C12H14N2.HClMolecular Weight: 222.71Percent Composition: C 64.72%, H 6.79%, N 12.58%, Cl 15.92%Properties: Crystals, mp 160°. Converts reversibly to monohydrate at room temp, 80% humidity.Melting point: mp 160° Therap-Cat-Vet: Sedative.

Detomidine is an imidazole derivative and α₂-adrenergic agonist,used as a large animal sedative, primarily used in horses. It is usually available as the salt detomidine hydrochloride. It is a prescription medication available to veterinarians sold under the trade name Dormosedan.

Currently, detomidine is only licensed for use in horses in the US but it is also licensed for use in cattle in Europe and Australasia.^[1]

Properties

Detomidine is a sedative with analgesic properties.^[2] α2-adrenergic agonists produce dose-dependent sedative and analgesic effects, mediated by activation of α2 catecholamine receptors, thus inducing a negative feedback response, reducing production of excitatory neurotransmitters. Due to inhibition of the sympathetic nervous system, detomidine also has cardiac and respiratory effects and an antidiuretic action.^[3]

Effects

UsesA profound lethargy and characteristic lowering of the head with reduced sensitivity to environmental stimuli (sound, pain, etc.) are seen with detomidine. A short period of reduced coordination is characteristically followed by immobility and a firm stance with front legs spread. Following administration there is an initial increase in blood pressure, followed by bradycardia and second degree atrioventricular block (this is not pathologic in horses). The horse commonly sweats to excess, especially on the flanks and neck. Other side effects reported include pilo erection (hair standing erect), ataxia, salivation, slight muscle tremors, and (rarely) penile prolapse. 

Sedation and anaesthetic premedication in horses and other large animals, commonly combined with butorphanol for increased analgesia and depth of sedation. In conjunction with ketamine it may also be used for intravenous anaesthesia of short duration.

The drug is normally administered by the intravenous route, and is fastest and most efficient when given intravenously . However, in recalcitrant animals, detomidine may be administered by the intramuscular or sublingual routes. The dose range advised by the manufacturers is 20–40 µg/kg intravenous for moderate sedation, but this dose may need to be higher if given intramuscularly.

When given intravenously, detomidine usually takes effect in 2–5 minutes, and recovery is full within 30–60 minutes. However, this is highly dependent upon the dosage, environment, and the individual animal; some horses are highly resistant to sedation.

Detomidine is a poor premedication when using ketamine as an anesthetic in horses.As detomidine is an arrhythmogenic agent, extreme care should be exercised in horses with cardiac disease, and in the concurrent administration of other arrhythmogenics. The concurrent use of potentiated sulfonamide antibiotics is considered particularly dangerous.

Anesthetic recoveries in horses that have received ketamine following a detomidine premedication are often violent with the horse having multiple failures to stand resulting in trauma to itself. Xylazine is a superior premedication with ketamine resulting in safer recoveries.

PATENT

EP-03782989

Novel crystalline forms of detomidine hydrochloride monohydrate, processes for their preparation and compositions comprising them are claimed. Also claimed is their use as alpha2-adrenoreceptor agonists.

Detomidine hydrochloride (1H imidazole,4-[(2,3-dimethylphenyl)methyl]-hydrochloride (CAS Number: 90038-01-0) is a synthetic alpha ₂-adrenoreceptor agonist with sedative and analgesic properties widely used for sedation of large animals like horses and cattle. This substance displays various other pharmacologic effects related to the cardiovascular and respiratory system as well as on muscles. Detomidine hydrochloride is available as a parenteral solution with 10 mg/ml as active ingredient which is indicated for use as a sedative and analgesic to facilitate minor surgical and diagnostic procedures in mature horses and yearlings (e.g. DORMOSEDAN®). Furthermore, detomidine hydrochloride is supplied as an oromucosal (i.e. sublingual) gel (e.g. DORMOSEDAN GEL®) with 7.6 mg/ml as active ingredient which is indicated for sedation and restraint in horses.
Further details regarding the clinical pharmacology and side effects as well as contraindications for this drug substance (i.e. active pharmaceutical ingredient) can be found in: Veterinary Psychopharmacology; Sharon L. et al., 2nd edition (2019), Wiley & Sons (pages 161 – 162). According to these authors detomidine has not been used in humans to date.
Detomidini hydrochloridum ad usum veterinarium is included in the EUROPEAN PHARMACOPOEIA (Ph. Eur. 9.0) but currently not included in the United States Pharmacopoeia (USP). It has to be noted that in the absence of a statement regarding a specific hydrate form, like a degree of hydration or mono-, di-, etc., in the title of the monograph – as is the case for detomidine hydrochloride – the anhydrous form is indicated for this substance.
According to a prior version of the respective monograph, namely Ph. Eur. 8.0, the substance exists as a white or almost white, hygroscopic, crystalline powder. The substance is soluble in water, freely soluble in ethanol (96 %), very slightly soluble in methylene chloride and practically insoluble in acetone. The molecular weight (M _r) amounts to 222.7. The melting point (mp) is specified at about 160 °C. In the current monograph (Ph. Eur. 9.0) the content of detomidine hydrochloride is specified at 99.0 % to 101.0 percent (anhydrous substance).

[0003]  In the current monograph (Ph. Eur. 9.0) the content of detomidine hydrochloride is specified at 99.0 % to 101.0 % (anhydrous substance).
The current monograph includes the three following known impurities:

Impurity A: (RS)-(2,3-dimethylphenyl) (1H-imidazol-4-yl)-methanol

Impurity B: (RS)-(1-benzyl-1H-imidazol-5-yl)(2,3-dimethylphenyl)-methanol

Impurity C: 4-[(2,3-dimethylcylohexyl)methyl]-1H-imidazole

The related substances are specified at ≤ 0.20 % for any unspecified impurities and ≤ 0.5 % for total impurities with a reporting threshold of 0.10 %.
The water content of detomidine hydrochloride as determined by Karl Fischer (KF) titration is limited to ≤ 2.0 % for release as well as shelf-life testing. As detomidine hydrochloride is hygroscopic, the compound has to be stored in airtight containers.

[0004]  A synthesis method for detomidine was disclosed in US 4,584,383.
Specific details on the last two steps of a synthesis method for detomidine hydrochloride (including a reaction scheme) were published in Drugs Future 10, 17 (1985).

[0005]  Detomidine hydrochloride is known to exist in two crystalline forms, namely the anhydrous form, as described above, and the monohydrate form B (M _r: 240.7, CAS Number: 90038-00-9) which can easily interconvert, depending on ambient temperature and air humidity ( Veldre, K. et al., Eur. Journ. Pharm. 44, 273-280 (2011)). At 80 % air humidity and room temperature the monohydrate is reversibly formed. The theoretical water content of detomidine hydrochloride monohydrate amounts to 7.48 %.

[0006]  To date, all commercially available (i.e. veterinary) drug products (i.e. parenteral solutions and oromucosal gels) only contain the anhydrous form. In general, hygroscopic substances like detomidine hydrochloride tend to absorb moisture so that they have to be protected from a humid environment during production and storage of the drug substance and corresponding drug product to avoid an inacceptable uptake of water. It has to be noted that such uptake during storage will reduce the content of the drug substance so that this would have to be taken into consideration during production of the corresponding drug product, like pharmaceutical preparation.

[0007]  The problem to be solved is to provide a pure and stable active pharmaceutical ingredient (API), namely detomidine hydrochloride monohydrate, that can advantageously be used for the production of pharmaceutical compositions comprising the active pharmaceutical ingredient detomidine hydrochloride.

Example 1

Preparation of detomidine hydrochloride monohydrate (DHM)

[0053]  Detomidine hydrochloride was synthesized starting from 1-benzyl-imidazole-4-carboxyaldehyde and 2,3-dimethylphenlymagnesiumbromide according to the two-step synthesis described in Drugs Future 10, 17 (1985).

[0054]  For the second step of this synthesis (RS)-(3-Benzyl-3 H-imidazol-4-yl)-(2,3-dimethyl-phenyl)-methanol (HCl) was suspended in a mixture of water and hydrochloric acid. The catalyst (i. e. palladium on activated carbon) suspended in demineralized water was added. Hydrogenation (i.e. removal of the benzyl group and reduction of the hydroxyl group with hydrogen (H ₂/Pd-C in HCl)) was performed at elevated temperature (50 – 80 °C) and the obtained suspension was filtered after the hydrogenation was finished. Subsequently ethyl acetate and a solution of ammonium hydroxide were added under continuous stirring. After discontinuation of stirring, phase separation occured after which the aqueous phase was repeatedly extracted with ethyl acetate. The combined organic phases were washed with demineralized water and filtered.

[0055]  After addition of 5 – 6 N hydrogen chloride in 2-propanol and cooling precipitation of detomidine hydrochloride occured. After filtration the filtercake (i.e. raw product) was washed with ethyl acetate and dried.

[0056]  A fraction of the resulting raw product (i.e. 5 g batch RSO E-190604 RP) was recrystallized from 5 g demineralized water by heating (until complete dissolution was obtained) and subsequent cooling on an ice bath. The resulting crystals were separated by filtration and the resulting filter cake washed with 2-propanol. Subsequently, the washed product was dried under vacuum (10 mbar) at 23 °C. The obtained yield for the white crystalline substance amounted to 66.0 % of the theory.

[0057]  The resulting drug substance showed a water content (KF) of 7.49 %. The corresponding DSC curve was in line with the expectation (see for example Figure 1) and showed the two typical peaks routinely observed for DHM. Other than 2-propanol used for final washing none of the other solvents employed during the overall synthesis of this compound were found above the respective LOQ by GC-FID.

Example 2

Impurities after preparation of detomidine hydrochloride monohydrate (DHM)

[0058]  A larger batch of detomidine hydrochloride (i.e. 50 g NK E-190709-I A K1) was synthesized in line with Example 1. However, the final crystals obtained after recrystallization from 50 ml demineralized water were washed with 25 ml demineralized water instead of 2-propanol. Drying was performed at 21 °C and 10 mbar until constant weight. The obtained yield for the white crystalline substance amounted to 87.2 % of the theory which was markedly higher than the yield obtained in Example 1. The water content of this substance was determined at 7.54 % (KF) and the corresponding DSC curve showed two peaks with an onset at 95.7 °C and 159.3 °C.

[0059]  As shown below, recrystallization of the initial raw product from water (incl. washing) resulted in significant removal/reduction of impurities eluting before the detomidine peak (i.e. more polar compounds, e.g. Impurity A) as well as impurities eluting behind the detomidine peak (i.e. less polar compounds, e.g. Impurity C).

[0060]  The final substance showed a very high HPLC purity of 99.80 area% (Ph. Eur. test method) and only a limited number of unknown impurities in addition to those

PATENT

https://patents.google.com/patent/WO2006108910A1/en

Example 1. Preparation of 4-[(2,3-dimethylbenzyl)]imidazole hydrochloride

(detomidine HCl)

l-Benzyl-5-(2,3-dimethylphenylhydroxymethyl)imidazole (20 kg), water (225 1), 30 % HCl (20 1), ethanol (5 1) and palladium on charcoal 10 % (4.4 kg) are charged. The mixture is stirred under 2.2 bar overpressure of hydrogen at 75 ± 5 °C for 24 hours. The reaction mixture is filtered at 45 ± 3 ⁰C and the filter cake is washed with water (30 1). 170 1 of water is distilled off under reduced pressure and 30 % HCl (8 1) is added. The solution is cooled to 3 ± 3 ⁰C during 2 h. The solution is seeded with crystals of detomidine HCl at 40 ± 5 °C, 30 ± 5 ⁰C, 20 ± 5 °C and at 10 ± 5 ⁰C, until the crystallization starts. The mixture is agitated for two hours. The crystalline compound is collected by centrifugation and washed with toluene. The crude product and water (250 1) are charged. The solution is heated to about 50 °C and stirred for 1 hour. The solution is cooled to 10 °C during 1.5 hour. The solution is filtered and 180 1 of water is distilled off under vacuum. 30 % HCl (20 1) is added and the solution is warmed to 60 ⁰C, and then cooled to 3 ± 3 °C during 2 hours. The solution is seeded as above until the crystallization starts and agitated for two hours. The crystalline compound is collected by centrifogation and washed with toluene. The product is dried under vacuum at 39 ± 5 °C for 20 hours, at 61 ± 5 °C for 6 hours and at 85 ± 5 °C for 16 hours. The yield is 10.5 kg (78 %).

PATENT

https://patents.google.com/patent/US20080287685A1/en

• Detomidine which is 4-[(2,3-dimethylbenzyl)]imidazole of formula I
• is a well known pharmaceutical agent currently used as its hydrochloride salt in animal sedation.
• [0003]The synthesis of detomidine is described in U.S. Pat. Nos. 4,443,466 and 4,584,383. The preparation of detomidine hydrochloride salt is described in U.S. Pat. No. 4,584,383, wherein detomidine obtained from the hydrogenation step is first recovered from alkaline solution as a free base after which the crystalline product is converted into its hydrochloride salt by treatment with HCl-isopropanol in ethyl acetate.
• [0020]1-Benzyl-5-(2,3-dimethylphenylhydroxymethyl)imidazole (20 kg), water (225 l), 30% HCl (20 l), ethanol (5 l) and palladium on charcoal 10% (4.4 kg) are charged. The mixture is stirred under 2.2 bar overpressure of hydrogen at 75±5° C. for 24 hours. The reaction mixture is filtered at 45±3° C. and the filter cake is washed with water (30 l). 170 l of water is distilled off under reduced pressure and 30% HCl (8 l) is added. The solution is cooled to 3±3° C. during 2 h. The solution is seeded with crystals of detomidine HCl at 40±5° C., 30±5° C., 20±5° C. and at 10±5° C., until the crystallization starts. The mixture is agitated for two hours. The crystalline compound is collected by centrifugation and washed with toluene. The crude product and water (250 l) are charged. The solution is heated to about 50° C. and stirred for 1 hour. The solution is cooled to 10° C. during 1.5 hour. The solution is filtered and 180 l of water is distilled off under vacuum. 30% HCl (20 l) is added and the solution is warmed to 60° C., and then cooled to 3±3° C. during 2 hours. The solution is seeded as above until the crystallization starts and agitated for two hours. The crystalline compound is collected by centrifugation and washed with toluene. The product is dried under vacuum at 39±5° C. for 20 hours, at 61±5° C. for 6 hours and at 85±5° C. for 16 hours. The yield is 10.5 kg (78%).

PATENT

https://patents.google.com/patent/WO2020016827A1/en

Detomidine

Detomidine, 4-[(2,3-dimethylphenyl)methyl]-lH-Imidazole, is an a-2-andregenic agonist available under the brand name Equimidine® and Dormosedan® for use as a veterinary sedative. Detomidine is not currently approved for human use.

Detomidine and related compounds, including its 3,4 dimethyl isomer, iso-detomidine (4-(3,4- Dimethylbenzyl)-lH-imidazole) were first described in US4,443,466. Both the‘466 patent and the later US4, 584,383 describe the synthetic method of manufacturing detomidine as being based on coupling of an imidazole moiety with l-Bromo-2, 3-dimethyl benzene using a Grignard reaction. RU2448095 describes an alternative route of synthesis of the molecule based on coupling in presence of a Titanium catalyst. According to both the‘383 and‘095 patents, detomidine is purified by crystallization of its hydrochloride salt from water. The chemical structures of detomidine HC1 and iso-detomidine are shown below:

Detomidine HC1 Iso-detomidine

Two solid state forms of detomidine HC1 are known, the anhydrous and monohydrate forms.

Synthesis of the anhydrous form by crystallization of the monohydrate and further decomposition at elevated temperatures is described in US7,728,l47. Synthesis of the anhydrous form via decomposition of the monohydrate in reduced pressure is described in Laine et al (1983). According to Veldre et al (2011), the anhydrous and monohydrate forms of detomidine HC1 can easily interconvert depending on temperature and humidity.

The European Pharmacopeia 9.0 monograph (January 2014) describes detomidine HC1 for veterinary use. The monograph lists the established HPLC method for identification of detomidine and its impurities as using a Symmetry C8, 5 pm, 4.6 x 150 mm column, with a mobile phase of Ammonium phosphate buffer pH 7.9 – 65% and Acetonitrile – 35% at a flow rate of 1.0 mL/min and UV detection at 220 nm. That procedure is listed as recording three distinct impurities of detomidine:

Impurity A: (RS)-(2, 3 -dimethylphenyl)(l/f-imidazol-4-yl)m ethanol

– l/f-imidazol-5-yl)(2,3-dimethylphenyl)m ethanol

Impurity C: 4-| (2.3 -dimcthy ley clohcxyl)m ethyl |- 1 /7-im ida/olc

PCT/US18/012579 discloses topical formulations of detomidine and their uses in treating pain.

Purified detomidine for use in human pharmaceutical formulations is not known in the art.

EXAMPLE 5: Purification of organic impurities from detomidine HC1 monohvdrate

Two potential procedures for purification of organic impurities from sourced monohydrate were compared. The first attempted procedure was by direct re-crystallization of detomidine HC1 from 2.88 volumes of water, while the second included carbon treatment and precipitation of detomidine free base followed by the free base being reacted with HC1 and crystallized as monohydrate. Both procedures used the same non-GMP, off white anhydrous detomidine HC1 starting material which had previously been shown in Table 7 to contain 0.21% of iso-detomidine and 0.07% of Impurity A. All the re-crystallized materials were found to have practically the same purity level. The direct re-crystallization procedure was found to provide a product with a high yield and purity and at the same time provides a practical and scalable crystallization process which could be controlled by process parameters such as seeding and cooling rate.

Example 5 a: Direct recrvstallization

Anhydrous detomidine HC1 (4.5g) was introduced to a round-bottom flask with a magnetic stirrer and thermometer. Deionized water (l3ml) was then added and the mixture stirred and heated in a water bath. At 39°C, the complete dissolution of solids was observed, providing a clear yellow solution with a pH = 4.

The batch was gradually cooled by stirring. At 3 l°C, intensive crystallization was observed. The resulting slurry was cooled in an ice-water bath for 20 min and filtered. Flask and cake were then washed with 2 ml of cold deionized water and 3.97g of a white to cream colored solid was collected. 2.03g of the material was dried in a vacuum desiccator at ambient temperature and 20 mbar to a constant weight over 23 hrs producing a dry monohydrate – l .96g off-white crystalline solid (sample 1).

An additional l .9 lg of the material was dried in a vacuum oven at 90°C under house vacuum to a constant weight over about 24.5 hrs producing a dry anhydrate , l .68g off-white solid (sample 2)

The two samples were subjected to physical characterization and purity analysis by HPLC. The XRPD spectra and DSC and TGA thermograms of sample 1 are presented in Figures 8 -10 and of sample 2 are presented in Figures 11-13, respectively.

As shown in Table 11, direct re-crystallization resulted in the effective purification from all organic impurities, but was not effective for color. The content of iso-detomidine and of Impurity A was reduced to a level below the QL, but the off white color remained after re-crystallization.

Table 11 : properties following direct recrystallization (sample 1)

¹ – below the QL

² – system peak

Example 5b(i): Carbon treatment and detomidine free base isolation

Anhydrous detomidine HC1 (70.3g) and deionized water (220ml) were introduced to a 0.5 liter jacketed glass reactor equipped with a mechanical stirrer, thermocoupler and a circulating oil bath for heating and cooling.

The mixture was heated while stirring. At 40°C, complete dissolution was observed. Active carbon (CXV type, 5.2g) was added to the clear yellow solution and the batch stirred at 45°C for 50 minutes. Following this, the batch was filtered on through paper filter on Buchner funnel, reactor and filter washed with deionized water (20ml).

The slightly yellowish clear filtrate was reintroduced to the 0.5 liter reactor, stirred and 40% NaOH solution was added at 40°C. After 10ml NaOH solution was added, a pH of 7 was reached and precipitation began. An additional 13ml of NaOH was added over 1 hour at 42 – 52°C and intensive stirring (400 – 450 rpm) performed. The mixture at the end of the addition of NaOH had a pH of 13.

The batch was stirred at 33 – 35°C overnight then cooled to l6°C over 4 hours and stirred at this temperature for an additional hour. The resultant solid was filtered on Buchner filter, reactor and cake washed with two portions of deionized water (2><200ml). The wet solid (86g) was dried in a vacuum oven at 45°C to constant weight to produce a dry product (53.2g, Yield 90.7%) – white powder, m.p.=l 18.6 – 119.2

The dry detomidine base was analyzed for purity by HPLC, the results presented in Table 12. Table 12: Properties of detomidine base (intermediate in sample 2)

¹ – system peak

Example 5b(nT Monohvdrate crystallization from detomidine base

The dry detomidine free base (53.0g) from Example 5b(i) was introduced together with 37% HC1 (29.7g) and deionized water (159g) into a 0.5 liter jacketed glass reactor equipped with a mechanical stirrer, a thermocoupler and a circulating oil bath for heating and cooling. The batch was stirred and heated to 45°C, at 37°C complete dissolution of solid was observed. The clear solution had a pH of 1. The solution was cooled gradually to 37°C and seeded with detomidine HC1 monohydrate and cooled gradually to 3°C over 4 hours, and then the batch was stirred for 45 minutes at this temperature. The solid was filtered on Buchner filter, reactor and cake washed with cold deionized water (80ml). The wet solid (61.9g) was dried in vacuum oven for 16 hours at 45°C to produce a dry product (57.8g, Yield 84.3%) – white crystalline powder (sample 2)

The dry detomidine HC1 monohydrate was analyzed for water by CKF (¾0 = 7.46%) and for purity by HPLC with the results presented in Table 13. Microscopic observation for particle morphology (regular prisms) was performed and the microscopic photograph is shown in Figure

14.

Table 13 : Properties of detomidine HC1 (sample 2)

¹ – system peak

Example 5c: Re-crvstallization of detomidine HC1 to monohvdrate. bench scale experiment Anhydrous detomidine HC1 (754.6g) 37% HC1 (116. Og) and deionized water (2008g) were introduced to a 3 liter glass jacketed reactor equipped with a mechanical stirrer, two baffles, a thermocoupler and a circulating oil bath for heating and cooling. The batch was stirred and heated to 52°C, at 47°C complete dissolution was observed and the clear solution was found to have a pH of 0-0.5.

The solution was cooled gradually and at 45°C seeded with detomidine HC1 monohydrate (0.5g). Crystallization initiation was observed at 43°C and the batch was then cooled to 1.5°C during 5 hours and stirred for 12 hours at this temperature. The solid was filtered on Buchner filter and conditioned on the filter with vacuum for 40 minutes. The wet product (817g) was dried in vacuum oven to constant weight. For the first 13 hours, the material was dried at 30°C and 35-27 mbar, then for an additional 7 hours at 40°C and 30-18 mbar to produce a dry product (771.2g, Yield 94.6%) – white crystalline powder (Batch“90” in Tables 8-9; sample 3)

Dry detomidine HC1 monohydrate was analyzed for water by CKF (FhO = 7.37%) and for purity by HPLC, the results presented in Table 14. The physical characterization results are shown in Table 10 above.

The material was subjected to physical characterization and microscopic observation for particle morphology (regular prisms) microscopic photograph presented in Figure 7.

Table 14: Properties of detomidine HC1 (sample 3)

¹ – system peak

EXAMPLE 6: Synthesis of iso -detomidine

Scheme 1 outlines a process for the synthesis of iso-detomidine was developed to produce a solid iso-detomidine HC1 in high yield and substantially free of impurities.

Scheme 1 : Route of synthesis of iso-detomidine

Example 6a: Sandmever Reaction

3,4 dimethyl aniline (150g, 1.24M) was mixed with acetonitrile (0.6 liter) in a 5 liter flask, chilled to lO°C and water (1.2 liter) added dropwise over 5 minutes. The mixture was cooled to 5°C with ice-ethanol bath and concentrated H2SO4 (98% wt, 363g 3.71M) was added dropwise over 30 min at 5-l0°C. Sodium nitrite (NaNC ) aqueous solution (89.7g in 300 ml water, 1.30M) was then added dropwise over 30 min at 0-5°C to give a brown solution. The resulting solution of diazonium salt was stirred at 0-5°C for an additional 30 min.

In another 5 liter flask KI (225g, 1.36M) was dissolved in water (0.8 liter) during stirring and cooled. The diazonium salt solution was added dropwise to the KI solution at 7-l3°C during 35 min, the batch stirred at 7-l3°C for 1.25 hr to give a black solution. MTBE (2.0 liter) was then added to the reaction mixture and Na₂SC>₄ (23.4g) was introduced in small portions during 5 min.

The mixture was settled and the organic phase separated and washed with two portions of brine (2 500ml). The organic solution was concentrated under vacuum to a volume of about 250ml.

The product was purified by vacuum distillation at ca. 40Pa, BP = 52 – 60°C to give 246g of intermediate 1 as a brown oil with a product yield of 86%.

Example 6b: TRT protection reaction

lH-Imidazole-4-carbaldehyde (45.2g, 0.47M) and acetonitrile (0.8 liter) are introduced into a 2 liter flack and cooled to 8°C, then TRT-C1 (131. Og, 0.47M) was added at 8°C and TEA (57. lg, 0.56M) was added dropwise during 20 min. The reaction mixture was stirred at 8 to l8°C for 2 hrs.

The reaction mixture was poured into a stirring mixture of water (0.72 liter) and MTBE (0.72 liter) and stirred for 10 minutes. The resulting solid was isolated by filtration on Buchner funnel and dissolved with THF (3 liter). The solution was dried over Na₂SC>₄ and concentrated to remove most of the solvent.

MTBE (400 ml) and PE (200ml) was added to the residue, the mixture stirred at 8°C for 16 hrs. The precipitated solid was isolated by filtration on Buchner filter and dried in air for 16 hrs at room temperature. Then the filter cake is dried by azeotropic drying with 2-Me-THF (2×500 ml) to give l29g of intermediate 2 as white solid with a yield of 66.5%.

Example 6c: Grignard reaction

A 2M solution of i-PrMgCl in THF (0.275 liter, 0.55M) and THF (1.0 liter) was introduced to a 2 liter flask at l2°C. Intermediate 1 (121.8g, 0.525M) was added dropwise during 20 min. The mixture was stirred at l2-l5°C for 3 hrs.

Intermediate 2 (84.6g, 0.25M) was added in small portions without cooling during 30 min, with a temperature rise to 25°C, to give a light brown solution. The solution was stirred for 2.5hrs at l5°C and added to aqueous solution of NH4CI (117g in 0.7 liter water) during 10 min at 5°C. PE (1.6 liter) was added during 5 min and the mixture stirred for extra 25 min.

Precipitated solid filtered on Buchner funnel and then re-slurred with mixture of MTBE (400 ml), water (600 ml) and PE (200 ml). Then the solid was filtered on Buchner funnel and re-slurred with MeOH (700 ml) at 60°C for 10 min, cooled to 20°C with cold water bath and filtered again on Buchner funnel. The solid product was dried in an air oven at 45 °C for 2 hrs to give 112 g of intermediate 3 as a white solid with a yield of 89.9%.

Example 6d: Reductive dehvdroxylation and de-protection

Intermediate 3 (l07g, 0.240M) and DCM (1.10 liter) were introduced to a 2 liter flask at 1 l°C, TFA (214 ml) was added dropwise over 5 mins with a temperature rise to l4°C.

The mixture was stirred for about 5 mins and EhSiH (94.4g, 0.794M) added dropwise during 5 mins. After stirring at 25-30°C for 16 hrs the mixture was concentrated by rotary evaporation at 40°C to a residue.

The residue of evaporation was dissolved in DCM (600 ml) and washed with 1.5M aq. HC1 (0.241iter). Organic phase was separated and washed with aq. NaOH (11.5g in 200ml water), pH of aqueous phase 13. Two phases were separated and the organic phase washed with brine (200 ml) dried over Na2S04 and filtered. The resulting solution was concentrated by rotary evaporation.

The evaporation residue was dissolved in mixture of EtOAc (500 ml) and EtOH (30 ml) and then 4M HC1 solution in dioxane (40 ml) was added dropwise in 5 minutes, pH = 1 – 2 adjusted and a white solid precipitated out.

The solid product was filtered on Buchner funnel, the cake dried in air for 16 hrs to give 36g of white solid.

The solid product was re-crystallized from iPrOH / Acetone. The dry cake (36g) and iPrOH were introduced into a 1 liter flask and heated to dissolution. Acetone (360 ml) was added to the resulting colorless solution at reflux during 10 mins. The mixture was cooled to 8°C and stirred at this temperature for additional 4.5 hrs. The solid product was filtered on Buchner funnel and dried in air for 36 hrs. 29.2g of iso-detomidine as a white solid was obtained with a yield of 54.4%. The ¹H-NMR spectra of iso-detomidine is shown in Figure 15. EXAMPLE 7 : Re-crvstallization of detomidine HC1 spiked with 2% iso-detomidine

Detomidine HC1 monohydrate (26. Og), iso-detomidine HC1 (0.52g) and deionized water (68.7g) were introduced to a 100 ml glass jacketed reactor equipped with a mechanical stirrer, a thermocouple and a circulating oil bath for heating and cooling. The batch was stirred and heated to 51°C, at 47°C complete dissolution was observed.

The solution was cooled gradually and at 42°C seeded with detomidine HC1 monohydrate. Crystallization initiation was observed at 39°C and then the batch was cooled to 3°C for 5 hours, filtered on Buchner filter and conditioned on the filter with vacuum. The wet product (20.7 g) was dried in vacuum oven to constant weight to produce a dry product (20.13g, Yield 75.9%) – white crystalline powder

Dry detomidine HC1 monohydrate was analyzed for PSD and morphology, the results are presented in Table 8 (Sample. No. 91). The purity of re-crystallized material was analyzed using the optimized HPLC process disclosed herein, and the results are presented in Table 15.

Table 15 : Properties of detomidine HC1 following recrystallization from iso-detomidine spiked material

a area %

b Spiked amount, calculated

References

1. ^ Clarke, Kathy W.; Hall, Leslie W.; Trim, Cynthia M., eds. (2014). “Principles of sedation, anticholinergic agents, and principles of premedication”. Veterinary Anaesthesia. pp. 79–100. doi:10.1016/B978-0-7020-2793-2.00004-9. ISBN 978-0-7020-2793-2.
2. ^ England GC, Clarke KW (November 1996). “Alpha 2 adrenoceptor agonists in the horse–a review”. The British Veterinary Journal. 152 (6): 641–57. doi:10.1016/S0007-1935(96)80118-7. PMID 8979422.
3. ^ Fornai F, Blandizzi C, del Tacca M (1990). “Central alpha-2 adrenoceptors regulate central and peripheral functions”. Pharmacological Research. 22 (5): 541–54. doi:10.1016/S1043-6618(05)80046-5. PMID 2177556.

External links

• “Medication Protocols for Horses”. The Ontario Association of Equine Practitioners. 2005. Archived from the original on 2007-04-17.
• Compendium of data sheets for animal medicines. National Office of Animal Health. 2005. ISBN 978-0-9548037-0-4.

////////////// DETOMIDINE, UNII-7N8K34P2XH , детомидин ,ديتوميدين, 地托咪定 , Domosedan, Farmos, SEDATIVE

#DETOMIDINE, #UNII-7N8K34P2XH , #детомидин ,#ديتوميدين, #地托咪定 , #Domosedan, #Farmos, #SEDATIVE

https://patents.google.com/patent/WO2020016827A1/en

EXAMPLES

EXAMPLE 1 : Elemental analysis of impurities found in commercially available anhydrous detomidine HC1

Example la: Anhydrous detomidine HC1 was sourced from two commercial API suppliers. Properties of the commercial batches, GMP1, GMP2 and GMP3, are presented below.

Elemental impurity analysis was performed by inductively coupled plasma mass spectrometry (ICP-MS) on four different batches of sourced anhydrate. The results of the analysis are found in Table 1.

Table 1 : Elemental impurities in anhydrous detomidine HC1

¹¹ Elements having levels L.T. 0.5 mg/kg (Ti, As, Hg, Pb, Mo, Pt, etc) are not presented in the table

The screening of elemental impurities shows that the GMP products contained significant levels of Pd (0.9 – 5.3 mg/kg). Pd is understood to be a catalyst used in the synthesis of detomidine (e.g., in reduction/hydrogenation methods).

Example lb: Characterization of commercially sourced material

Samples of the anhydrous detomidine products described in Table 1 were analyzed for water content and characterized by microscope, XRPD and thermal analyses. The results are summarized in Table 2.

Table 2: Characterization of commercial anhydrous detomidine HC1

a Anhydrous + mono hydrate The values presented in T able 2 demonstrate that the commercial samples of detomidine HC1 labeled as anhydrous contain some amount of monohydrate and this amount varied depending on storage conditions and packaging.

EXAMPLE 2: Stability assessment of anhvdrate and monohvdrate forms of detomidine base and detomidine HC1

Pure forms of crystalline free base, and HC1 salt (both monohydrate and anhydrate) were prepared from commercially sourced anhydrous detomidine HC1 as outlined in Table 3, and characterized using XRPD and thermal analysis. The solids were crystallized from aqueous solutions and then dried under different conditions. The crystallization and drying conditions are summarized in Table 3.

Table 3: Preparation of detomidine HC1 crystalline forms

The properties of the solids crystallized according to Table 3 are described in Table 4.

Table 4: Properties of Detomidine HC1 crystalline forms

These results demonstrate that crystallization from 2.8 – 2.9 volumes of water is effective for isolation and purification of the detomidine HC1 monohydrate drug substance. Drying of the monohydrate under mild conditions (20-40 mbar and temperatures from at least ambient to about 45 °C) provided pure monohydrate without traces of the anhydrous form.

The same monohydrate dried at elevated temperature (30-40 mbar 90°C) converted completely into the anhydrous form. The vacuum dried, hermetically closed anhydrate did not absorb water from the atmosphere and did not convert into the monohydrate. After exposure to atmospheric air, however, the anhydrate absorbed water and converted to a mixture of anhydrate and

monohydrate.

Melting points (m.p.) of the intermediate detomidine free base and hydrochloride of Sample 5 measured in open capillary corresponded with the published literature and the DSC data and are presented in Table 5. In order to evaluate effect of humidity on different forms of detomidine, a hydration study was performed. Samples of detomidine free base and hydrochloride salt were subjected to DVS analysis. These observations are in accordance with the DVS results shown in Figures 5 and 6, for detomidine free base and detomidine HC1, respectively.

Table 5: Composition and properties of known solid forms of detomidine

a -literature data

The free base was found to be crystalline and insoluble in water but it reacted readily with aqueous HC1 giving soluble detomidine hydrochloride.

Crystallization from water provided effective purification of the detomidine HC1 and formation of large regular crystals. Anhydrous detomidine hydrochloride appeared as small irregular particles whereas the possibility to control particle size distribution by crystallization parameters existed for the monohydrate.

The detomidine free base was found to be non-hygroscopic, but also able to absorb more than 1% of water at relative humidity (RH) >50%. An increase of humidity from RH 70% to RH >90% did not lead to absorption of additional water to monohydrate. During the dehydration cycle, the monohydrate began to lose water at RH -10% and converted into the anhydrate at RH =0%. Anhydrate did not absorb water at RH <30% and transformed completely to into the monohydrate at RH between 30% and 50%.

Four cycles of hydration-dehydration demonstrated good reproducibility of anhydrate- monohydrate interconversion.

An anhydrous detomidine HC1 of Sample 2 was shown to absorb water to a level of cKF 7.7% which corresponds well to the theoretical amount of water in the monohydrate form (Table 5).

The hydration profile of detomidine hydrochloride showed that the monohydrate is stable in a wide range of humidity between 10% and >90% RH. At the same time, the anhydrous form is not stable in atmospheric air and absorbs water at RH = 30 – 50%.

This data demonstrates that the anhydrous form is challenging in the aspects of water content and solid form stability and that detomidine HC1 monohydrate is more suitable for pharmaceutical development.

Example 3 : Impurity analysis of commercially sourced detomidine HC1

Using the established Pharmacopeia HPLC protocol (Symmetry C8, 5 pm, 4.6 x 150 mm column, with a mobile phase of 65% Ammonium phosphate buffer pH 7.9 and 35% Acetonitrile at a flow rate of 1.0 mL/min and UV detection at 220 nm), sourced samples of detomidine HC1 were assayed for impurities. As shown in Figure 1, a previously unreported peak was identified, which partially overlapped with that of detomidine. By LC-MS/MS analysis, this impurity was shown to have the same molecular weight as detomidine.

The established Pharmacopeia HPLC protocol did not separate the detomidine from the impurity. Therefore, for further identification of the elusive impurity, new HPLC protocols for assaying detomidine HC1 were developed. One protocol (“HPLC Protocol A”) comprised using a SunFire C8 column, IOqA, 3.5 pm, 4.6 x l50mm column with an initial mobile phase of 70% Ammonium Phosphate buffer solution, pH 7.9 and 30% Acetonitrile, at a flow rate of 1.0 mL/min and UV detection at 220 nm. To remove late eluting peaks, the flush gradient shown in Table 6 was applied after each run. This HPLC protocol allowed for a resolution factor of 3.9 between detomidine and the unidentified impurity. The quantitation level (QL) for impurities and degradation products is 0.025%. The detection level (DL) for impurities and degradation products is 0.01%.

Table 6: Flush gradient for HPLC protocol

Given its molecular weight, it was hypothesized that the impurity was iso-detomidine.

A solution of 100 pg/ml detomidine HC1 and about 1 pg/mL (about 1% of the working concentration) of detomidine impurity A and iso-detomidine were prepared and assayed using the new HPLC protocol (HPLC Protocol A), disclosed hereinabove. Figure 2 is a chromatogram showing that the previously unreported peak is confirmed as being iso-detomidine.

The analysis of commercially sourced detomidine HC1 revealed a significant additional impurity. Table 7 provides levels of the various detomidine impurities in different commercial batches. In all batches, total impurities were observed at levels of > 0.1% area.

Table 7: Impurity levels (% area) in commercial batches of detomidine.

provided by commercial supplier after undergoing the reciystallization process of Example 5, provided by inventors.

Further analysis of the peak at RRT=0.38 showed that it actually consisted of 2 separate, overlapping peaks. As shown in Figure 3, LC-MS/MS analysis confirmed one of these peaks as iso-impurity A. Further analysis, as shown in Figure 4, identified the second peak as (2,3- dimcthylphcnylX 1 //-imidazol-4-yl) methanone.

EXAMPLE 4: Optimization of the crystallization method of detomidine HC1 monohvdrate from commercial batches of anhydrous detomidine HC1

Crystallization experiments on 25, 65, and 770 gram scale were performed in 100 ml, 500 ml and 3 liter jacketed glass reactors, respectively, equipped with CBT (curved blade turbine) mechanical stirrers, circulating oil bath, thermocouples, and condensers. Stirrer speed in all experiments was between 300 – 600 rpm. Variable process parameters were: amounts of HC1, solvent ratio, cooling time/rate, seeding and cake wash. The parameters and the variation ranges were chosen according to production conditions. The crystallization parameters are summarized in Table 8.

Table 8: Crystallization parameters

a Seeding with detomidine HC1 monohydrate

b Time 24 hrs

c Seeding with anhydrous detomidine HC1

d 5.5 hrs cooling and overnight stirring at 1-3° C

e Spiked with 2% iso -detomidine

The drying parameters and solid properties of batches shown in Table 8 are described in Table 9. Table 9: Drying parameters and solid properties of detomidine monohydrate crystals

microscopic observation: Rods – aspect ratio > 2; prisms – aspect ratio < 2