[¹⁸F]AMG 580

NOTE………CAS OF AMG 580 IS 1227067-71-1, WITHOUT 18F

AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one],

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington’s disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography–tandem mass spectrometry technology. [³H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [¹⁸F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

PAPER

We report the discovery of PDE10A PET tracer AMG 580 developed to support proof of concept studies with PDE10A inhibitors in the clinic. To find a tracer with higher binding potential (BP_ND) in NHP than our previously reported tracer 1, we implemented a surface plasmon resonance assay to measure the binding off-rate to identify candidates with slower washout rate in vivo. Five candidates (2–6) from two structurally distinct scaffolds were identified that possessed both the in vitro characteristics that would favor central penetration and the structural features necessary for PET isotope radiolabeling. Two cinnolines (2, 3) and one keto-benzimidazole (5) exhibited PDE10A target specificity and brain uptake comparable to or better than 1 in the in vivo LC–MS/MS kinetics distribution study in SD rats. In NHP PET imaging study, [¹⁸F]-5 produced a significantly improved BP_ND of 3.1 and was nominated as PDE10A PET tracer clinical candidate for further studies.

Discovery of Phosphodiesterase 10A (PDE10A) PET Tracer AMG 580 to Support Clinical Studies

PATENT FOR AMG 580

WO 2010057121

https://www.google.com/patents/WO2010057121A1?cl=en

PAPER

Nuclear Medicine and Biology (2015), 42(8), 654-663.

http://www.sciencedirect.com/science/article/pii/S0969805115000724

Phosphodiesterase 10A (PDE10A) is an intracellular enzyme responsible for the breakdown of cyclic nucleotides which are important second messengers for neurotransmission. Inhibition of PDE10A has been identified as a potential target for treatment of various neuropsychiatric disorders. To assist drug development, we have identified a selective PDE10A positron emission tomography (PET) tracer, AMG 580. We describe here the radiosynthesis of [¹⁸ F]AMG 580 and in vitro and in vivo characterization results.

AMG 580 has an in vitro K_D of 71.9 pM. Autoradiography showed specific uptake in striatum. Mean activity of 121 ± 18 MBq was used in PET studies. In Rhesus, the baseline BP_ND for putamen and caudate was 3.38 and 2.34, respectively, via 2TC, and 3.16, 2.34 via Logan, and 2.92, and 2.01 via SRTM. A dose dependent decrease of BP_NDwas observed by the pre-treatment with a PDE10A inhibitor. In baboons, 0.24 mg/kg dose of AMG 580 resulted in about 70% decrease of BP_ND. The in vivo K_D of [¹⁸ F]AMG 580 was estimated to be around 0.44 nM in baboons.

Conclusion

[¹⁸ F]AMG 580 is a selective and potent PDE10A PET tracer with excellent specific striatal binding in non-human primates. It warrants further evaluation in humans.

REFERNCES

http://jpet.aspetjournals.org/content/352/2/327.full

///Phosphodiesterase, tracer,  receptor occupancy,  positron emission tomography, radiotracer,  brain penetration, AMG 580, Phosphodiesterase 10A, PDE10A, PET Tracer, [¹⁸F]AMG 580

Difelikefalin ( INN ) (Developmental Code Names CR845 , FE-202845 ), Also Known As D -Phe- D -Phe- D -Leu- D -Lys- [Ganma- (4-N-Piperidinyl) Amino Carboxylic Acid] (As The Acetate Salt ), Is An Analgesic Opioid Peptide ^[2] Acting As A Peripherally-Specific , Highly Selective Agonist Of The kappa-Opioid Receptor (KOR). ^{[1] [3] [4] [5]} It Is Under Development By Cara Therapeutics As An Intravenous Agent For The Treatment Of Postoperative Pain . ^{[1] [3] [5]} An Oral Formulation Has Also Been Developed. ^[5] Due To Its Peripheral Selectivity, Difelikefalin Lacks The Central Side Effects Like Sedation , Dysphoria , And Hallucinations Of Previous KOR-Acting Analgesics Such As Pentazocine And Phenazocine . ^{[1] [3]} In Addition To Use As An Analgesic, Difelikefalin Is Also Being Investigated For The Treatment Of Pruritus (Itching). ^{[1] [3] [4 ]} Difelikefalin Has Completed Phase II Clinical Trials For Postoperative Pain And Has Demonstrated Significant And “Robust” Clinical Efficacy, Along With Being Safe And Well-Tolerated. ^{[3] [5]} It Is Also In Phase II Clinical Trials For Uremic Pruritus In Hemodialysis Patients. ^[4]

Difelikefalin Acts As An Analgesic By Activating KORs On Peripheral Nerve Terminals And KORs Expressed By Certain Immune System Cells . ^[1] Activation Of KORs On Peripheral Nerve Terminals Results In The Inhibition Of Ion Channels Responsible For Afferent Nerve Activity , Causing Reduced Transmission Of Pain Signals , While Activation Of KORs Expressed By Immune System Cells Results In Reduced Release Of Proinflammatory , Nerve-Sensitizing Mediators (Eg, Prostaglandins ). ^[1]

PATENT

WO 2015198505

[Formula 1] Being Represented By Compounds Are Useful As Pain Therapeutics. The Preparation Of This Compound, Solid Phase Peptide Synthesis Methods In Patent Documents 1 And 2 Have Been Described.

Document 1 Patent: Kohyo 2010-510966 JP
Patent Document 2: Japanese Unexamined Patent Publication No. 2013-241447

　Compound (1) or a salt thereof and compound (A), for example as shown in the following reaction formula, 4-aminopiperidine-4-carboxylic acid, D- lysine (D-Lys), D- leucine (D-Leu) , it can be prepared by D- phenylalanine (D-Phe) and D- phenylalanine (D-Phe) sequentially solution phase peptide synthesis methods condensation.

[Of 4]

The present invention will next to examples will be described in further detail.

Example
1 (1) Synthesis of Cbz-D-Lys (Boc) -α-Boc-Pic-OMe (3)
to the four-necked flask of 2L, α-Boc-Pic- OMe · HCl [α-Boc-4 – aminopiperidine-4-carboxylic acid methyl hydrochloride] were charged (2) 43.7g (148mmol), was suspended in EtOAc 656mL (15v / w). To the suspension of 1-hydroxybenzotriazole (HOBt) 27.2g (178mmol), while cooling with Cbz-D-Lys (Boc) -OH 59.2g (156mmol) was added an ice-bath 1-ethyl -3 – (3-dimethylcarbamoyl amino propyl) was added to the carbodiimide · HCl (EDC · HCl) 34.1g (178mmol). After 20 minutes, stirring was heated 12 hours at room temperature. After completion of the reaction, it was added and the organic layer was 1 N HCl 218 mL of (5.0v / w). NaHCO to the resulting organic layer ₃ Aq. 218ML (5.0V / W), Et ₃ N 33.0 g of (326Mmol) was stirred for 30 minutes, and the mixture was separated. The organic layer HCl 218ML 1N (5.0V / W), NaHCO ₃ Aq. 218mL (5.0v / w), NaClaq . Was washed successively with 218ML (5.0V / W), Na ₂ SO ₄　dried addition of 8.74g (0.2w / w). Subjected to vacuum filtration, was concentrated under reduced pressure resulting filtrate by an evaporator, and pump up in the vacuum pump, the Cbz-D-Lys (Boc) -α-Boc-Pic-OMe (3) 88.9g as a white solid obtained (96.5% yield, HPLC purity 96.5%).

[0033]

(2) D-Lys (Boc) Synthesis Of -Arufa-Boc-Pic-OMe (4)
In An Eggplant-Shaped Flask Of 2L, Cbz-D-Lys (Boc) -Arufa-Boc-Pic-OMe (3) 88.3g (142mmol) were charged, it was added and dissolved 441mL (5.0v / w) the EtOAc. The 5% Pd / C to the reaction solution 17.7g (0.2w / w) was added, After three nitrogen substitution reduced pressure Atmosphere, Was Performed Three Times A Hydrogen Substituent. The Reaction Solution Was 18 Hours With Vigorous Stirring At Room Temperature To Remove The Pd / C And After The Completion Of The Reaction Vacuum Filtration. NaHCO The Resulting Filtrate ₃ Aq. 441ML And (5.0V / W) Were Added For Liquid Separation, And The Organic Layer Was Extracted By The Addition Of EtOAc 200ML (2.3V / W) In The Aqueous Layer. NaHCO The Combined Organic Layer ₃ Aq. 441ML And (5.0V / W) Were Added for liquid separation, and the organic layer was extracted addition of EtOAc 200mL (2.3v / w) in the aqueous layer. NaClaq the combined organic layers. 441mL and (5.0v / w) is added to liquid separation, was extracted by the addition EtOAc 200ML Of (2.3V / W) In The Aqueous Layer. The Combined Organic Layer On The Na ₂ SO ₄　Dried Addition Of 17.7 g of (0.2W / W), Then The Filtrate Was Concentrated Under Reduced Pressure Obtained Subjected To Vacuum Filtration By an evaporator, and pump up in the vacuum pump, D-Lys (Boc) -α-Boc-Pic- OMe (4) to give 62.7g (90.5% yield, HPLC purity 93.6%).

(3) Cbz-D-Leu -D-Lys (Boc) -α-Boc-Pic-OMe synthesis of (5)
in the four-necked flask of 2L, D-Lys (Boc) -α-Boc-Pic-OMe (4) was charged 57.7 g (120 mmol), was suspended in EtOAc 576mL (10v / w). HOBt 19.3g (126mmol) to this suspension, was added EDC · HCl 24.2g (126mmol) while cooling in an ice bath added Cbz-D-Leu-OH 33.4g (126mmol). After 20 minutes, after stirring the temperature was raised 5 hours at room temperature, further the EDC · HCl and stirred 1.15 g (6.00 mmol) was added 16 h. After completion of the reaction, it was added liquid separation 1N HCl 576mL (10v / w) . NaHCO to the resulting organic layer ₃ Aq. 576ML (10V / W), Et ₃ N 24.3 g of (240Mmol) was stirred for 30 minutes, and the mixture was separated. The organic layer HCl 576ML 1N (10V / W), NaHCO ₃ Aq. 576mL (10v / w), NaClaq . Was washed successively with 576ML (10V / W), Na ₂ SO ₄　dried addition of 11.5g (0.2w / w). After the filtrate was concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, and pump up in the vacuum pump, the Cbz-D-Leu-D- Lys (Boc) -α-Boc-Pic-OMe (5) 85.8g It was obtained as a white solid (98.7% yield, HPLC purity 96.9%).

(4) D-Leu-D -Lys (Boc) -α-Boc-Pic-OMe synthesis of (6)
in an eggplant-shaped flask of 1L, Cbz-D-Leu- D-Lys (Boc) -α-Boc-Pic -OMe the (5) 91.9g (125mmol) were charged, was added and dissolved 459mL (5.0v / w) the EtOAc. The 5% Pd / C to the reaction solution 18.4g (0.2w / w) was added, After three nitrogen substitution reduced pressure atmosphere, was performed three times a hydrogen substituent. The reaction solution was subjected to 8 hours with vigorous stirring at room temperature to remove the Pd / C and after the completion of the reaction vacuum filtration. NaHCO the resulting filtrate ₃ Aq. 200mL (2.2v / w) were added to separate liquid, NaHCO to the organic layer ₃ Aq. 200mL (2.2v / w), NaClaq . It was sequentially added washed 200mL (2.2v / w). To the resulting organic layer Na ₂ SO ₄　dried added 18.4g (0.2w / w), to the filtrate concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, and a pump-up with a vacuum pump. The resulting amorphous solid was dissolved adding EtOAc 200mL (2.2v / w), was crystallized by the addition of heptane 50mL (1.8v / w). Was filtered off precipitated crystals by vacuum filtration, the crystals were washed with a mixed solvent of EtOAc 120mL (1.3v / w), heptane 50mL (0.3v / w). The resulting crystal 46.1g to added to and dissolved EtOAc 480mL (5.2v / w), was crystallized added to the cyclohexane 660mL (7.2v / w). Was filtered off under reduced pressure filtered to precipitate crystals, cyclohexane 120mL (1.3v / w), and washed with a mixed solvent of EtOAc 20mL (0.2v / w), and 30 ° C. vacuum dried, D-Leu- as a white solid D-Lys (Boc) -α- Boc-Pic-OMe (6) to give 36.6 g (48.7% yield, HPLC purity 99.9%).

(5) Synthesis of Cbz-D-Phe-D- Leu-D-Lys (Boc) -α-Boc-Pic-OMe (7)
to the four-necked flask of 1L, D-Leu-D- Lys (Boc) -α-Boc-Pic-OMe with (6) 35.8g (59.6mmol) was charged, it was suspended in EtOAc 358mL (10v / w). To this suspension HOBt 9.59g (62.6mmol), Cbz- D-Phe-OH 18.7g was cooled in an ice bath is added (62.6mmol) while EDC · HCl 12.0g (62.6mmol) It was added. After 20 minutes, a further EDC · HCl After stirring the temperature was raised 16 hours was added 3.09 g (16.1 mmol) to room temperature. After completion of the reaction, it was added and the organic layer was 1N HCl 358mL of (10v / w). NaHCO to the resulting organic layer ₃ Aq. 358ML (10V / W), Et ₃ N 12.1 g of (119Mmol) was stirred for 30 minutes, and the mixture was separated. The organic layer HCl 358ML 1N (10V / W), NaHCO ₃ Aq. 358mL (10v / w), NaClaq . Was washed successively with 358ML (10V / W), Na ₂ SO ₄　dried addition of 7.16g (0.2w / w). After the filtrate was concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, and pump up in the vacuum pump, Cbz-D-Phe-D -Leu-D-Lys (Boc) -α-Boc-Pic-OMe (7) was obtained 52.5g as a white solid (yield quant, HPLC purity 97.6%).

(6) D-Phe-D -Leu-D-Lys (Boc) synthesis of -α-Boc-Pic-OMe ( 8)
in an eggplant-shaped flask of 2L, Cbz-D-Phe- D-Leu-D-Lys ( Boc) -α-Boc-Pic- OMe (7) the 46.9g (53.3mmol) were charged, the 840ML EtOAc (18V / W), H ₂ added to and dissolved O 93.8mL (2.0v / w) It was. The 5% Pd / C to the reaction mixture 9.38g (0.2w / w) was added, After three nitrogen substitution reduced pressure atmosphere, was performed three times a hydrogen substituent. The reaction solution was subjected to 10 hours with vigorous stirring at room temperature to remove the Pd / C and after the completion of the reaction vacuum filtration. NaHCO the resulting filtrate ₃ Aq. 235mL (5.0v / w) were added to separate liquid, NaHCO to the organic layer ₃ Aq. 235mL (5.0v / w), NaClaq . It was added sequentially cleaning 235mL (5.0v / w). To the resulting organic layer Na ₂ SO ₄　dried addition of 9.38g (0.2w / w), then the filtrate was concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, pump up with a vacuum pump to D-Phe -D-Leu-D-Lys ( Boc) -α-Boc-Pic-OMe (7) was obtained 39.7g (yield quant, HPLC purity 97.3%).

351mL was suspended in (10v / w). To this suspension HOBt 7.92g (51.7mmol), Boc-D-Phe-OH HCl HCl

(8) D-Phe-D -Phe-D-Leu-D-Lys-Pic-OMe Synthesis Of Hydrochloric Acid Salt (1)
In An Eggplant-Shaped Flask Of 20ML Boc-D-Phe-D -Phe-D- Leu-D- lys (Boc) -α -Boc- Pic-OMe (9) and 2.00gg, IPA 3.3mL (1.65v / w), was suspended by addition of PhMe 10mL (5v / w). It was stirred at room temperature for 19 hours by addition of 6N HCl / IPA 6.7mL (3.35v / w). The precipitated solid was filtered off by vacuum filtration and dried under reduced pressure to a white solid of D-Phe-D-Phe- D- Leu-D-Lys-Pic- OMe 1.59ghydrochloride (1) (yield: 99 .0%, HPLC purity 98.2%) was obtained.

(9) D-Phe-D -Phe-D-Leu-D-Lys-Pic-OMe Purification Of The Hydrochloric Acid Salt (1)
In An Eggplant-Shaped Flask Of 20ML-D-Phe-D- Phe D-Leu -D-Lys- pic-OMe hydrochloride crude crystals (1) were charged 200mg, EtOH: MeCN = 1: after stirring for 1 hour then heated in a mixed solvent 4.0 mL (20v / w) was added 40 ° C. of 5 , further at room temperature for 2 was time stirring slurry. Was filtered off by vacuum filtration, the resulting solid was dried under reduced pressure a white solid ((1) Purification crystals) was obtained 161 mg (80% yield, HPLC purity 99.2% ).

(10) D-Phe-D -Phe-D-Leu-D-Lys-Pic Synthesis (Using Purified
(1)) Of (A) To A Round-Bottomed Flask Of 10ML D-Phe-D-Phe-D- -D-Lys Leu-Pic-OMe Hydrochloride Salt (1) Was Charged With Purified Crystal 38.5Mg (0.0488Mmol), H ₂ Was Added And Dissolved O 0.2ML (5.2V / W). 1.5H Was Stirred Dropwise 1N NaOH 197MyuL (0.197mmol) at room temperature. After completion of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 48.8μL (0.0488mmol), to obtain a D-Phe-D-Phe- D-Leu-D-Lys- Pic (A) (yield: quant , HPLC purity 99.7%).

D-Phe-D-Phe- D-Leu-D-Lys-Pic-OMe (1) physical properties ¹ H NMR (400 MHz, 1M DCl) [delta] ppm by: 0.85-1.02 (yd,. 6 H), 1.34-1.63 ( m, 5 H), 1.65-2.12 ( m, 5 H), 2.23-2.45 (m, 2 H), 2.96-3.12 (m, 4 H), 3.19 (ddt, J = 5.0 & 5.0 & 10.0 Hz), 3.33-3.62 (m, 1 H), 3.68-3.82 (m, 1 H), 3.82-3.95 (m, 4 H), 3.95-4.18 (m, 1 H), 4.25-4.37 (m, 2 H), 4.61-4.77 (M, 2 H), 7.21-7.44 (M, 10 H) ¹³ C NMR (400MHz, 1M DCl) Deruta Ppm: 21.8, 22.5, 24.8, 27.0, 30.5, 30.8, 31.0, 31.2, 31.7, 37.2 , 37.8, 38.4, 39.0, 39.8, 40.4, 40.6, 41.8, 42.3, 49.8, 50.2, 52.2, 52.6, 54.6, 55.2, 57.7, 57.9, 127.6, 128.4, 129.2, 129.6, 129.7, 129.8 dp 209.5 ℃

Example 2
(Trifluoroacetic Acid (TFA)
Use) (1) D-Phe-D-Phe-D-Leu-D-Lys-Pic-OMe TFA Synthesis Of Salt (1)
TFA 18ML Eggplant Flask Of 50ML (18V / W) , 1- Dodecanethiol 1.6ML (1.6V / W), Triisopropylsilane 0.2ML (0.2V / W), H ₂ Sequentially Added Stirring The O 0.2ML (0.2V / W) Did. The Solution To The Boc-D-Phe- D- Phe-D-Leu-D -Lys (Boc) -α-Boc-Pic-OMe the (9) 1.00g (1.01mmol) was added in small portions with a spatula. After completion of the reaction, concentrated under reduced pressure by an evaporator, it was added dropwise the resulting residue in IPE 20mL (20v / w). The precipitated solid was filtered off, the resulting solid was obtained and dried under reduced pressure to D-Phe-D-Phe- D-Leu -D-Lys-Pic-OMe · TFA salt as a white solid (1) (Osamu rate 93.0%, HPLC purity 95.2%).

(2) D-Phe-D -Phe-D-Leu-D-Lys-Pic synthesis of (A)
to a round-bottomed flask of 10mL D-Phe-D-Phe -D-Leu-D-Lys-Pic-OMe TFA were charged salt (1) 83mg (0.0843mmol), was added and dissolved H2O 431μL (5.2v / w). Was 12h stirring dropwise 1N NaOH 345μL (0.345mmol) at room temperature. After completion of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 84.3μL (0.0843mmol), to obtain a D-Phe-D-Phe- D-Leu-D-Lys-Pic (A) ( yield: quant, HPLC purity 95.4%).

Example
3 (HCl / EtOAc
Use) (1) In An Eggplant-Shaped Flask Of 30ML Boc-D-Phe-D -Phe-D-Leu-D-Lys (Boc) -Arufa-Boc-Pic-OMe (9) 1. It was charged with 00g (1.01mmol ), was added and dissolved EtOAc7.0mL (7.0v / w). 4N HCl / EtOAc 5.0mL (5.0v / w) was added after 24h stirring at room temperature, the precipitated solid was filtered off by vacuum filtration, washed with EtOAc 2mL (2.0v / w). The resulting solid D-Phe-D-Phe- D-Leu-D-Lys-Pic-OMe hydrochloride (1) was obtained 781mg of a white solid was dried under reduced pressure (the 96.7% yield, HPLC purity 95.4%).

(2) D-Phe-D -Phe-D-Leu-D-Lys-Pic (A) Synthesis of
eggplant flask of 10mL D-Phe-D-Phe -D-Leu-D-Lys-Pic-OMe hydrochloride were charged salt (1) 90 mg (0.112 mmol), H ₂ was added and dissolved O 0.47mL (5.2v / w). Was 12h stirring dropwise 1N NaOH 459μL (0.459mmol) at room temperature. After completion of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 0.112μL (0.112mmol), was obtained D-Phe-D-Phe- D-Leu-D-Lys-Pic (A) ( yield: quant, HPLC purity 93.1%).

4 Example
Compound (1) Of The Compound By Hydrolysis Synthesis Of (The A) (Compound (1) Without
Purification) Eggplant Flask 10ML D-Phe-D-Phe -D-Leu-D-Lys-Pic-OMe (1) Charged Hydrochloride Were (Without Pre-Step Purification) 114.5Mg (0.142Mmol), H ₂ Was Added And Dissolved O 595MyuL (5.2V / W). Was 14H Stirring Dropwise 1N NaOH 586MyuL (0.586Mmol) At Room Temperature. After Completion Of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 0.15μL (0.150mmol), was obtained D-Phe-D-Phe- D-Leu-D-Lys-Pic (A) (yield: quant, HPLC purity 95.2 %).

Example 1 Comparative
Path Not Via The Compound (1) (Using Whole Guard Boc-D-Phe-D-Phe-D-Leu-D-Lys (Boc) -Alpha-Boc-Pic-OMe
(A)) (1) D–Boc Phe- D-Phe-D-Leu-D-Lys (Boc) -Arufa-Boc-Pic-OH Synthesis Of
Eggplant Flask Of 30ML Boc-D-Phe-D -Phe-D-Leu-D- Lys (Boc) -α- Boc-Pic -OMe (9) were charged 1.00g (1.00mmol), was added and dissolved MeOH 5.0mL (5.0v / w). After stirring for four days by the addition of 1N NaOH 1.1 mL (1.10mmol) at room temperature, further MeOH 5.0mL (5.0v / w), 1N NaOH 2.0mL the (2.0mmol) at 35 ℃ in addition 3h and the mixture was stirred. After completion of the reaction, 1 N HCl 6.1 mL was added, After distilling off the solvent was concentrated under reduced pressure was separated and the organic layer was added EtOAc 5.0mL (5.0mL) .NaClaq. 5.0mL (5.0v / w) Wash the organic layer was added, the organic layer as a white solid was concentrated under reduced pressure to Boc-D-Phe-D- Phe-D-Leu-D-Lys (Boc) – α-Boc-Pic-OH 975.1mg (99.3% yield, HPLC purity 80.8% )

(2) D-Phe-D -Phe-D-Leu-D-Lys-Pic synthesis of (A)
to a round-bottomed flask of 20mL Boc-D-Phe-D -Phe-D-Leu-D-Lys (Boc) It was charged -α-Boc-Pic-OH ( 10) 959mg (0.978mmol), was added and dissolved EtOAc 4.9mL (5.0v / w). And 4h stirring at room temperature was added dropwise 4N HCl / EtOAc 4.9mL (5.0mL) at room temperature. After completion of the reaction, it was filtered under reduced pressure, a white solid as to give D-Phe-D-Phe- D-Leu-D-Lys-Pic the (A) (96.4% yield, HPLC purity 79.2%) .

　If not via the compound of the present invention (1), the purity of the compound obtained (A) was less than 80%.

PATENT

http://www.google.com/patents/US20110212882

References

REFERENCES

1: Cowan A, Kehner GB, Inan S. Targeting Itch With Ligands Selective For kappa Opioid
. Receptors Handb Exp Pharmacol 2015; 226:.. 291-314 Doi:
.. 10.1007 / 978-3-662-44605-8_16 Review PubMed PMID: 25861786.

Difelikefalin

Systematic (IUPAC) Name
Amino–4 1- ( D -Phenylalanyl- D -Phenylalanyl- D -Leucyl- D -Lysyl) Piperidine-4-Carboxylic Acid
Clinical data
Of Routes Administration	Intravenous
Pharmacokinetic Data
Bioavailability	Pasento 100 ( IV ) [1]
Metabolism	Metabolized Not [1]
Biological half-life	Hours 2 [1]
Excretion	As Unchanged Excreted Drug Via Bile And Urine [1]
Identifiers
CAS Number	1024828-77-0
ATC code	None
ChemSpider	44208824
Chemical data
Formula	C 36 H 53 N 7 O 6
Molar mass	679.85 g / mol

///// Difelikefalin,  CR845 , FE-202845,  Phase III, PEPTIDES

CC (C) C [C @ H] (C (= O) N [C @ H] (CCCCN) C (= O) N1CCC (CC1) (C (= O) O) N) NC (= O) [ C @@ H] (Cc2ccccc2) NC (= O) [C @@ H] (Cc3ccccc3) N

Rovatirelin is a novel synthetic agent that mimics the actions of thyrotropin-releasing hormone (TRH). Rovatirelin binds to the human TRH receptor with higher affinity (Ki=702nM) than taltirelin (Ki=3877nM). Rovatirelin increased the spontaneous firing of action potentials in the acutely isolated noradrenergic neurons of rat locus coeruleus (LC). Rovatirelin increased locomotor activity. Rovatirelin may have an orally effective therapeutic potential in patients with SCD.

Rovatirelin ([1-[-[(4S,5S)-(5-methyl-2-oxo oxazolidin-4-yl) carbonyl]-3-(thiazol-4-yl)-l-alanyl]-(2R)-2-methylpyrrolidine) is a novel synthetic agent that mimics the actions of thyrotropin-releasing hormone (TRH). The aim of this study was to investigate the electrophysiological and pharmacological effects of rovatirelin on the central noradrenergic system and to compare the results with those of another TRH mimetic agent, taltirelin, which is approved for the treatment of spinocerebellar degeneration (SCD) in Japan. Rovatirelin binds to the human TRH receptor with higher affinity (Ki=702nM) than taltirelin (Ki=3877nM). Rovatirelin increased the spontaneous firing of action potentials in the acutely isolated noradrenergic neurons of rat locus coeruleus (LC). The facilitatory action of rovatirelin on the firing rate in the LC neurons was inhibited by the TRH receptor antagonist, chlordiazepoxide. Reduction of the extracellular pH increased the spontaneous firing of LC neurons and rovatirelin failed to increase the firing frequency further, indicating an involvement of acid-sensitive K+ channels in the rovatirelin action. In in vivo studies, oral administration of rovatirelin increased both c-Fos expression in the LC and extracellular levels of noradrenaline (NA) in the medial prefrontal cortex (mPFC) of rats. Furthermore, rovatirelin increased locomotor activity. The increase in NA level and locomotor activity by rovatirelin was more potent and longer acting than those by taltirelin. These results indicate that rovatirelin exerts a central nervous system (CNS)-mediated action through the central noradrenergic system, which is more potent than taltirelin. Thus, rovatirelin may have an orally effective therapeutic potential in patients with SCD.

PATENT

WO 9808867

PATENT

WO 9945000 

PATENT

WO 2002017954

Example

Preparation of the compound represented by Example 1 set (IX)

The second step

Two

(First step)

Method described in the literature (Synth. Commun., 20, 3507 (1990)) synthesized N- in (tert- butoxide deer Lupo sulfonyl) one 3- (4 one-thiazolyl) one L Aranin (1, 21.79 g, 80 mmol) in Torifuruoro and the mixture was stirred acetic acid (80 ml) were added under ice-cooling for 2 hours and a half. Then stirred for 30 minutes at room temperature was added to the reaction mixture p- toluenesulfonic acid hydrate (15.22 g, 80 mmol). The reaction mixture was concentrated to dryness under reduced pressure. To remove excess Torifuruoro acetic acid by the obtained residue concentrated to dryness under reduced pressure by addition of water and methanol.Obtained obtained residue was collected by filtration crystals ether was added to precipitate the compound (2) 29.8 g (quantitative).

NMR (CD ₃ OD): 9.01 (1H, d-, J = 1.8 Hz), 7.70 (2H _; yd), 7.46 (lH, d-, J = 1.8 Hz), 7.23 (2H, yd), 4.38 (1H, dd , J = 4.8 from and 3.8 from Hz), 3.45 (2H _; yd), 2.37 (3H, s).

(Second step)

I 匕合 product (2) 38.85 g E evening Nord (200 ml) of (112.8 mmol) – in THF (600 ml) solution, diphenyl di § zone methane while 攪袢 at room temperature (39 g, 201 mmol) in small portions over 30 minutes were added. The reaction mixture was stirred for 1 hour at room temperature, Ziv E sulfonyl di § zone methane (10 g, 51.5 mmol) was added and stirred for one hour. To the reaction mixture

After decomposing the excess reagent by the addition of acetic acid (0.1 ml), it was concentrated to dryness under reduced pressure and distilled off the solvent. The resulting residue (92 g) with ether (1 L) was crystallized to give compound (3) 49.05 g (96.1%).

mp: 139-140 ° C

[A] _D = -34.7 ° (C = 1.006, CHC1 ₃₎ 23 ° C)

^ Cm IRCKB ” ¹ : 1753, 1602, 1512, 1496, 1260, 1224, 1171, 1124, 1036, 1012. NMR (CD ₃ 0D): 8.92 (1H, D, J = 2 Hz), 7.70 (2H _; M ), 7.2-7.4 (13H, m) , 6.91 (1H, s), 4.62 (1H, t, J = 5.8 Hz), 3.47 (2H, d, J = 5.8 Hz), 2.36 (3H, s).

Elemental analysis (C _2E H _2S N ₂ 0 ₅ S ₂ )

Calculated: C, 61.16; H, 5.13; N, 5.49; S, 12.56.

Measured value: C, 61.14; H, 5.32; N, 5.41; S, 12.46.

(Third step)

Cis-one L one 5-methyl-2-one O Kiso O Kisa ethylbenzthiazoline one 4-carboxylic acid 13.95 g (96.14 mmol), compound (3) 49.09 g (96.14 mmol ), N-hydroxybenzotriazole To Riazoru 2.6 g (19.23 mmol) and under ice-cooling in THF (1L) solution of Toryechiruamin 14.1 ml (lOlmmol), was added to the DCC (20.83g, 101 mmol). The cooling bath was removed after stirring for 10 minutes at the same temperature, and stirred for an additional 2 0 hours at room temperature. After removing the precipitated precipitate and the filtrate concentrated to dryness under reduced pressure an oily residue (82.7 _g was obtained). The residue was filtered off and dissolved by heating to insoluble matter in acetic acid Echiru (700 ml). The filtrate was successively washed with sodium carbonate aqueous solution and water.After the addition of methanol (20 ml) the organic layer was dried with sulfuric acid mug Neshiumu, was concentrated to a small volume under reduced pressure.Precipitated collected by filtration and acetic acid E Ji Le crystals – ether (2: 3) washing to compound with a mixture (4) 35.69 g (79.8% ) was obtained. After addition was concentrated to dryness under reduced pressure of the mother liquor, and crystallized from acetic acid E Chiru ether mixture compound (4) 2.62 g (5.9% ) was obtained.

mp: 176-177 ° C

[A] _D = -39.2 ° (C = 1.007, CHC1 ₃ , 24 ° C)

^ Cm IRiKB ¹ : 1739, 1681, 1508, 1453, 1386, 1237, 1193, 1089.

NMR (CDC1 ₃ ): 8.71 (1H, d-, J = 1.8 Hz), 8.18 (lH, d-‘J = 3.9 from Hz), 7.2-7.4 (10H _; yd), 6.82 (1H, s), 6.66 (1H, d-, J = 1.8 Hz), 5.79 (1H, s), 5.12 (1H, yd), 4.94 (lH, yd), 4.35 (1H _; dd, J = 1.8 and 4.5 from Hz), 3.40 (1H _; dd, J 5.7 and 15 = Hz), 3.29 (1H _; dd, J = 4.5 of and 15 Hz), 1.27 (3H, d-, J = 6.3 Hz).

Elemental analysis (C ₂₄ H ₂₃ N ₃ 0 ₅ S)

Calculated: C, 61.92; H, 4.98; N, 9.03; S, 6.89.

Measured value: C _! 61.95; H, 5.01; N, 8.94; S ₎ 6.62.

(Fourth step)

Compound (4) 41.24 under ice-cooling to g (88.59 mmol), and the mixture was stirred Anisoru (240ml) and To Rifuruoro acetic acid (120 ml) and the mixture for 15 minutes. And the mixture was stirred for 2 hours 3 0 minutes further room temperature after removal of the cooling bath. The reaction mixture was added to the E one ether (500 ml) to the oily residue obtained by concentrated to dryness under reduced pressure was collected by filtration and pulverized. The resulting powder is water (50 ml) – was removed by filtration methanol (300 ml) warming dissolved insoluble matter in a mixture. The filtrate was concentrated to small volume under reduced pressure, and allowed to stand at room temperature for 3 days adding a seed crystal and methanol. The precipitated crystals were obtained Shi preparative filtration compound (5) 14.89 g (56.1%). The mother liquor was concentrated to dryness under reduced pressure, to give again further compound was crystallized from methanol one ether mixture of the (5) 10.3 g (38%). mp: 214-215 ° C

[]. -4.2 ° = (C = 0.5, H ₂ 0, 22 ° C)

^ Cm IRCKB ¹ : 1753, 1707, 1655, 1548, 1529, 1409, 1343, 1264, 1236, 1102, 1092. NMR (DMS0-D6): 9.02 (1H, D, J = 1.8 Hz), 8.46 (1H, d- _; J = 3.9 from Hz), 7.74 (1H, s),

7.38 (1H, d, J = 1.8 Hz), 4.77 (1H, dq, J = 6.6 and 8.7 Hz), 4.66 (1H, m), 4.21 (1H, d,

J = 8.7 Hz), 3.24 (IH, dd, J = 5.1 and 15 Hz), 3.13 (1H, dd, J = 8.4 and 15 Hz),

1.13 (3H, d, J = 6.6 Hz).

Elemental analysis (C _U H ₁₃ N ₃ 0 ₅ S)

Calculated: C _; 44.14; H, 4.38; N, 14.04; S ₎ 10.71.

Measured value: C, 43.94; H, 4.478; N, 14.09; S, 10.58.

(Fifth step)

Compound (5) 12.1 g, (40.48 mmol) and N- hydroxysuccinimide (4.66 g, 40,48 mM) under ice-cooling to THF (242 ml) suspension of,: DCC (8.35 g, 40.48 mmol) was added to 3 and the mixture was stirred for 10 minutes. The cooling bath was removed, and the mixture was further stirred at room temperature for 2 hours. The resulting compound N- hydroxysuccinimide ester solution of (5) was synthesized in a way described in the literature (Tetrahedron, 27, 2599 (1971 )) (R) – (+) – 2- Mechirupiro lysine hydrochloride (5.42 g) and Toryechiruamin (8.46 ml, was added at room temperature to THF (121 ml) suspension of 60.72 mmol). The reaction mixture was stirred for an additional 1 5 hrs. The filtrate after removal of the insoluble matter that has issued analysis was concentrated to dryness under reduced pressure. Residue (24.6 _Ga) the insoluble material was removed by filtration was dissolved in water (150 ml). The filtrate was purified by gel filtration column chromatography one (MCI Gel CHP-20P, 600 ml). 4 0% aqueous methanol solution compound of the collected crude eluted cut off fractionated (IX) was obtained 8.87 g. Then after purification by silica gel column chromatography (black port Holm one methanol mixture), to give the compound was freeze-dried (IX) 5.37 g (35.7% ).

mp: 192-194 ° C

[A] _D = -1.9 ° (C = 1.005, H ₂ 0, 25 ° C)

KB Cm- IR ¹ : 1755, 1675, 1625, 1541, 1516, 1448, 1232, 1097.

NMR (CD ₃ 0D): 8.97 (1H, t, J = 2.1 Hz), 7.34 (1H, t, J = 2.1 Hz), 5.19 and 5.04 (total the IH, the each t, J = 7.5 Hz), 4.92 (1H , Dq, J = 6.6 And 8.7 Hz), 4.36 And 4.35 (1H, D, J = 8.7 Hz), 4.07 And 3.92 (Total IH, Eac M), 3.78 (1H _; M), 3.42 (1¾ M), 3.22 (2H, m), 1.5-2.0 ( 4H, m), 1.28 and 1.22 (total 3H, each d, J = 6.6 Hz), 1.21 and 1.02 (total 3H, each d, J = 6.6 Hz).

Elemental analysis (C ₁₆ H ₂₂ N ₄ 0 ₄ S H ₂ 0)

Calculated: C, 49.99; H, 6.29; N, 14.57; S, 8.34.

Measured value: C, 49.99; H, 6.29; N, 14.79; S, 8.36.

PATENT

WO 2006028277

Example

Example 1

B

Step 1 l-N-[N<tert-butoxycarbonyl)-3-(^^^

N.N-dicyclohexylcarbodiimide (10.83 g, 52.5 mmol), N-hydroxybenzotriazole (2.03 g, 15 mmol) and triethylamine (7.7 ml, 55.2 mmol) were added to a solution (130 ml) of N-(tert-butoxycarbonyl)-3-(thiazol-4-yl)-L-alanine (1) (13.62 g, 50 mmol) obtained by the method described in literatures (J. Am. Chem. Soc. 73, 2935 (1951) and Chem. Pharm. Bull. 38, 103 (1950)) and 2(R)-2-methylpyrrolidine p-toluenesulfonic acid (2) (12.79 g, 50 mmol) obtained by the method described in a literature (HeIv. Chim. Acta, 34, 2202 (1951)) in tetrahydrofuran. The mixture was stirred for 20 hours at room temperature. After the precipitates are filtered off, the obtained filtrate was concentrated under reduced pressure. Thus-obtained residue was dissolved in ethyl acetate (200 ml) and the solution were washed with an aqueous solution of sodium hydrogencarbonate and water, successively. The organic layers were dried over magnesium sulfate and concentrated under reduced pressure to give a title compound (3) (16.45 g, 100%) as oil.

NMR (CDCl₃): O_H 8.76 and 8.75 (1 H, each d, J=2.1Hz, Thia-H-2), 7.08 (1 H, d, J=2.fflz, thia-H-5), 5.45 (1 H, m, NH), 3.45-3.64 (1 H, m, AIa-CoH), 4.14 and 3.81 (1 H, each m, Pyr-CαH), 3.51 (1 H, m, PVr-NCH₂), 3.1-3.4 (3 H, m, Pyr-CH₂and AIa-CH₂), 1.39 (9 H, s, BOC), 1.3-2.0 (4 H, m, PyT-CH₂), 1.06 (3 H, d, J=6Hz, Pyr-Me)

Step 2 l-N-[3-(thiazol-4-yl)-L-alanyl]-(2R)-2-methylpyrroHdine di-p-toluenesulfcnate (4)

Compound (3) (33.77 g, 99.48 mmol) and p-toluenesulfonic acid hydrate (37.85 g, 199 mmol) were dissolved in ethyl acetate (101 ml) and the solution was cooled with ice. To the mixture, 4 mol/L solution of hydrogen chloride-ethyl acetate (125 ml) was added, and the mixture was stirred for 2 hours 45 minutes. After the mixture was concentrated under reduced pressure, methanol was added to the residue. The mixture was concentrated. Methanol-toluene (1: 1) was added to the residue and concentrated under reduced pressure to give crystalline residue. The residue was washed with acetone and filtered to give compound (4) as crystals (36 g, 62%). After the mother liquor was concentrated under reduced pressure, methanol and toluene were added to the residue and concentrated. Obtained crystalline residue was washed with acetone to give compound (4) (10.67 g, 18.4%). mp 188-189 ⁰C [α]_D ²⁴ +2.2 (c, 1.0, MeOH) IR(KBr)Cm^“1: 3431, 3125, 3080, 2963, 1667, 1598, 1537, 1497, 1451, 1364, 1229, 1198, 1170, 1123, 1035, 1011.

NMR (CD₃OD): δ_H 9.04 and 9.03 (1 H, each d, J=2.1Hz, Thia-H-2), 7.70 (2 H, m, aromaticH), 7.46 (1H, d, J=2.1Hz, thia-H-5), 7.23 (2H, m, aromaticH), 4.49and4.46 (1 H, each d, J=6.9Hz, Ala-CαH), 4.14 and 3.75 (1 H, each m, Pyr-CαH), 3.51 (1 H, m, pyr-NCH₂), 3.2-3.4 (3 H, m, PyT-CH₂ and AIa-CH₂), 2.36 (3 H, s, aromatic Me), 1.3-2.0 (4 H, m, pyr-CH₂), 1.19 and 1.07 (3 H, each d, J=6.3Hz, Pyr-Me) Anal Calcd For C₁₁H₁₇N₃OS 2C₇H₈O₃S Calculated: C, 51.44%; H₁ 5.70%; N, 7.20%; S, 16.48%. Found: C, 51.36%; H, 5.69%; N, 7.23%; S, 16.31%.

Step 3 l-[N-[(4S,5S)-(5-methyl-2-oxooxazolidin-4-yl)carbonyl]-3-(thiazol-4-yl)-L-alanyl-(2R)-2- methylpyrrolidine trihydrate (I- 1) Step 3 (1) Method A

(4S, 5S)-5-methyl-2-oxooxazolidin-4-yl carboxylic acid (5) (1.368 g, 9.43 mmol) obtained by the method described in literatures (J. Chem. Soc. 1950, 62; Tetrahedron 48; 2507 (1992) and Angew. Chem. 101, 1392 (1989)), Compound (4) (5 g, 8.56 mmol) and N-hydiOxysuccinimide (217 mg, 1.89 mmol) were dissolved in N, N-dimethylformamide (10 ml), and tetrahydrofuran (65 ml) was added. After the mixture was cooled with ice in a cool bath, triethylamine (2.63 ml, 18.86 mmol) and N, N-dicyclohexylcarbodiimide (2.04 g, 9.89 mmol) were added with stirred and the mixture was stirred for additional 30 minutes. The cooling bath was removed and the mixture was stirred for 15 hours at room temperature. The precipitated were filtered off and the filtrate was concentrated under reduced pressure. Water (100 ml) was added to thus-obtained residue (9.95 g) and the mixture was stirred for 1.5 hours at room temperature. After insoluble substance was filtered off, the filtrate was concentrated until it was reduced to about half volume under reduced pressure. The small amount of insoluble substance was filtered off and the filtrate was concentrated until it was reduced to about 2O g under reduced pressure. After the mixture was allowed to stand in a refrigerator for 3 days, the precipitated crystals (2.98 g) were collected by filtration and washed with cold water. The filtrate was extracted twice with chloroform, dried over magnesium sulfate and concentrated under reduced pressure. Ethyl acetate (5 ml) was added to oil residue (1.05 g) and the mixture was stirred to give crystals (136 mg). The obtained crystals were combined and dissolved in purified water (45 ml) with heating. After the solution was allowed to cool to room temperature, the precipitated insoluble substance was filtered off The filtrate was concentrated under reduced pressure and allowed to stand at room temperature overnight. The mixture was cooled with ice, and the crystals were collected by filtration to give Compound (1-1, 2.89 g, 80.3%). mp 194-196 ⁰C

[α]_D ²² -2.0 ± 0.4 ° (c, 1.008, H₂O), [α]₃₆₅ +33.1 ± 0.7 ° (c, 1.008, H₂O)

IR(Nujor)cm”¹: 3517, 3342, 3276, 3130, 3092, 3060, 1754, 1682, 1610, 1551, 1465, 1442,

1379, 1235, 1089. NMR(CD₃OD): δ_H 8.97 and 8.96 (total 1 H, d, J=2.1Hz, Thia-H-2), 7.34 and 7.33 (total 1

H, d, J=2.1Hz, Thia-H-5), 5.18 and 5.04 (total 1 H, each t, J=7.5Hz, Ala-CαH), 4.92 (1

H, dq, J=6.6 and 8.7Hz, Oxa-H-5), 4.36 and 4.35 (total 1 H, d, J=8.7Hz, Oxa-H-4), 4.07 and 3.92 (total 1 H, each m, Pyr-Cα-H), 3.78 (1 H, m, Pyr-NCH₂), 3.42 (1 H, m, Pyr- 5 NCH₂), 3.22 (2 H, m, AIa-CH₂), 1.5-2.0 (4 H, m, Pyr-CH₂), 1.28 and 1.22 (total 3 H, each d, J=6.6Hz, Oxa-5-Me), 1.21 and 1.02 (total 3 H, each d, J=6.6Hz, Pyr-2-Me)

Anal. Calcd For C₁₆H₂₂N₄O₄S 3H₂O

Calculated: C, 45.00%; H, 6.71%; N, 13.33%; S, 7.63%.

Found: C, 45.49%; H, 6.60%; N, 13.58%, S, 7.88%. 10

Step 3 (2)

Method B

After Compound (1-2) (410 g, 1.119 mmol) was dissolved in purified water (6.3 L) with heating, the solution was concentrated until the total weight of the mixture was 15 reduced to 1370 g under reduced pressure. The concentrated solution was allowed to stand at room temperature overnight. The solution was cooled with ice for 1 hour and filtered to give the precipitated crystals. The obtained crystals were washed with cold water to give

Compound (T- 1) (448 g, 95.2%) as colorless crystals. Mother liquor was mixed with purified water (300 mL) with heating and the solution was concentrated to 55 g under reduced pressure. 20 After the concentrated solution was allowed to stand at room temperature overnight, the solution was filtered to give the precipitated crystals (T-1, 16.3 g, 3.5%, total amount 464.3 g, 98.7%). mp 194-196 ⁰C

[α]_D ²² -0.9 ± 0.4 ° (c, 1.007, H₂O), [α]₃₆₅ + 35.4 ± 0.8 ° (c, 1.007, H₂O)

IR(NuJOr)Cm^“1: 3511, 3348, 3276, 3130, 3093, 3060, 1755, 1739, 1682, 1611, 1551, 1465, 25. 1442, 1379, 1235, 1089.

AnalCalcdFor: C₁₆H₂₂N₄O₄S 3H₂O

Calculated: C, 45.00%;H, 6.71%;N, 13.33%; S, 7.63%.

Found: C, 45.56%; H, 6.66%; N, 13.43%, S, 7.69%.

30 Step 4 l-[N-[(4S₎5S)-(5-methyl-2-oxooxazolidin-4-yl)carbonyl]-3-(thiazol-4-yl)-L-alanyl-(2R)-2- methylpyrrolidine (1-2)

Method A

After l-[N-[(4S,5S)-(5-methyl-2-oxooxazolidin-4-yl)carbonyl]-3-(thiazol-4-yl)-L- 35 alanyl-(2R)-2-methylpyrrolidine monohydrate (4.77 g) obtained by the method described in Patent Literature 8 was crushed in a mortar, it was dried under reduced pressure (66.5 Pa) at 100 ⁰C for 15 hours to give 4.54 g of Compound (1-2). mp 194.5-196.5 ⁰C [α]_D ²⁵ -2.1 +. 0.4 ° (c, 1.004, H₂O), [α]₃₆₅ +36.8 ± 0.8 ° (c, 1.004, H₂O) Water measurement (Karl Fischer method): 0.27%

IR(NuJOr)Cm”¹: 3276, 3180, 3104, 1766, 1654, 1626, 1548, 1517, 1457, 1380, 1235, 1102, 979. NMR(CD₃OD):δ_H 8.97 and 8.96 (total 1 H, d, J 2.1 Hz, Thia-H-2), 7.34 and 7.33 (total 1 H, d, J 2.1 Hz, Thia-H-5), 5.19 and 5.04 (total 1 H, each t, J 7.5 Hz, Ala- CaH), 4.92 (1 H, dq, J 6.6 and 8.7 Hz, Oxa-H-5), 4.36 and 4.35 (total 1 H, d, J 8.7 Hz, Oxa-H-4), 4.07 and 3.92 (total 1 H, each m, Pyr-Cα-H), 3.78 (1 H, m, Pyr-NCH₂), 3.42 (1 H, m, Pyr-NCH₂), 3.22 (2 H, m, AIa-CH₂), 1.5-2.0 (4 H, m, Pyr-CH₂), 1.28 and 1.22 (total 3 H, each d, J 6.6 Hz, Oxa-5-Me), 1.21 and 1.02 (total 3 H, each d, J 6.6 Hz, Pyr-2-Me). Anal Calcd For: C₁₆H₂₂N₄O₄S

Calculated: C, 52.44%; H, 6.05%; N, 15.29%; S, 8.75%. Found: C, 52.24%; H, 5.98%; N, 15.27%, S, 8.57%.

Method B

After Compound (1-1) (17.89 g, 47.3 mmol) was crushed in a mortar, it was dried under reduced pressure (66.5 Pa) at 100 °C for 14 hours to give Compound (1-2, 17.31 g). mp 193-194 ⁰C [α]_D ²⁵ -1.9 ± 0.4 ° (c, 1.002, H₂O), [α]₃₆₅ +37.2 ± 0.8 ° (c, 1.002, H₂O)

Water measurement (Karl Fischer method): 0.22%

IR(NuJOr)Cm^“1: 3273, 3180, 3111, 1765, 1685, 1653, 1626, 1549, 1516, 1456, 1346, 1331,

1277, 1240, 1097, 980.

Anal Calcd For C₁₆H₂₂N₄O₄S Calculated: C, 52.44%; H, 6.05%; N, 15.29%; S, 8.75%.

Found: C, 52.19%; H, 5.98%; N, 15.42%, S, 8.74%.

REFERENCES

1: Ijiro T, Nakamura K, Ogata M, Inada H, Kiguchi S, Maruyama K, Nabekura J,
Kobayashi M, Ishibashi H. Effect of rovatirelin, a novel thyrotropin-releasing
hormone analog, on the central noradrenergic system. Eur J Pharmacol. 2015 Aug
15;761:413-22. doi: 10.1016/j.ejphar.2015.05.047. Epub 2015 Jul 2. PubMed PMID:
26142830.

////////Rovatirelin Hydrate, S-0373, Rovatirelin, 204386-76-5, clinical, phase 3

C[C@@H]1CCCN1C(=O)[C@H](Cc2cscn2)NC(=O)[C@@H]3[C@@H](OC(=O)N3)C

[F-18](2S,4S)-4-(3-Fluoropropyl)glutamine

CAS 1196963-79-7

The early diagnosis of malignant tumors plays a very important role in the survival prognosis of cancer patients. In this non-invasive diagnosis, diagnostic imaging procedures are an important tool. In the last few years has mainly PET technology (P ositronen- E mission- Tomographie) proved to be particularly useful. The sensitivity and specificity of PET technology depends significantly on the used signal-emitting substance (tracer) and their distribution in the body from. In the search for suitable tracers one tries to take advantage of certain properties of tumors differ, the tumor tissue from healthy, surrounding tissue. The preferred commercially used isotope which finds application for PET, ¹⁸ F ¹⁸ F represents by its short half-life of less than 2 hours special requirements for the preparation of suitable tracer. Complex, long synthetic routes and purifications are with this isotope is not possible, because otherwise a significant portion of the radioactivity of the isotope has already decayed before the tracer can be used for diagnosis. It is therefore often not possible to established synthetic routes for non-radioactive fluorination to be applied to the synthesis of¹⁸ F-tracer. Furthermore, the high specific activity of ¹⁸ F (80 GBq / nmol) at very low substance amounts of ^[18 F] fluoride for the tracer synthesis, which in turn an extreme excess of precursor-related and the success of a non-radioactive fluorination based Radio synthetic strategy designed unpredictable

FDG ^([18 F] F 2 luoro d esoxy lukose g) -PET is a widely accepted and popular tool in the diagnosis and other clinical tracking of tumor diseases. Malignant tumors compete with the host organism to glucose supply to the nutrient supply (Warburg O. About the metabolism of carcinoma cell Biochem;.. Kellof G. Progress and Promise of FDG PET Imaging for Cancer Patient Management and Oncologic Drug Development Clin Cancer Res 2005;.. 11 (8): 2785-2807) where tumor cells compared to surrounding cells of normal tissue usually an increased glucose metabolism. This is used when using fluorodeoxyglucose (FDG), a glucose derivative, which is amplified transported into the cells, but there included metabolically after phosphorylation as FDG-6-phosphate (“Warburg effect”). ¹⁸ F-labeled FDG is Therefore, an effective tracer for the detection of tumors in patients using PET technology. Imaging were looking for new PET tracers in recent years increasingly amino acids for ¹⁸ F PET used (eg (review): Eur J Nucl Med Mol Imaging 2002 May; 29 (5):.. 681-90). In this case, some of the ¹⁸ F-labeled amino acids for the measurement of the speed rate of protein synthesis, the most useful derivatives but for the direct measurement of the cellular uptake in the tumor. Known ¹⁸ F-labeled amino acids are, for example, from tyrosine, phenylalanine, proline, aspartic and unnatural amino acids derived (eg J. Nucl Med 1991; 32:.. 1338-1346, J Nucl Med 1996; 37: 320-325, J Nucl Med 2001; 42: 752-754 J Nucl Med and 1999, 40: 331-338).. Glutamic acid and glutamine than ¹⁸ F-labeled derivatives not known, whereas non-radioactive fluorinated glutamine and glutamic acid derivatives are well known; Thus, for Example those which at γ-position (for Ex (review):Amino Acids (2003) April; 24 (3):… 245-61).. or at β-position (e.g. ExTetrahedron. Lett. .; 30; 14; 1989, 1799-1802, J. Org Chem .; 54; 2; 1989, 498-500, Tetrahedron: Asymmetry, 12, 9; 2001; 1303-1312) havefluorine..

Of glutamic acid having the chemical functionalities protecting groups in β and γ position or a leaving group, has already been reported in the past. So was informed of glutamate as mesylate or bromide in γ-position whose acid and amine functions were provided with ester or Z-protecting groups (J. Chem Soc Perkin Trans. 1;.. 1986, 1323-1328) or, for example, of γ-chloro-glutamic acid without protecting groups(Synthesis, (1973); 44-46). About similar derivatives, but where the leaving group is positioned in β-position has also been reported on several occasions. Z Ex. Chem. Pharm. Bull .; 17; 5; (1969); 879-885,J.Gen.Chem.USSR (Engl.Transl.); 38; (1968); 1645-1648, Tetrahedron Lett .; 27; 19; (1986); 2143-2144, Chem. Pharm. Bull .; EN; 17; 5; 1969;873-878, patent FR 1461184 , Patent JP 13142 .)

The current PET tracers, which are used for tumor diagnosis have some undisputed disadvantages: in FDG accumulates preferably in those cells with increased glucose metabolism on, but there are also other pathological and physiological conditions of increased glucose metabolism in the cells involved and tissues, eg, Ex. of infection or wound healing (summarized in J. Nucl. Med. Technol. (2005), 33, 145-155). It is still often difficult to decide whether a detected by FDG-PET lesion actually neoplastic origin or due to other physiological or pathological state of the tissue. Overall, the diagnostic activity by FDG-PET in oncology has a sensitivity of 84% and a specificity of 88% to(Gambhir et al., ” A tabulated summary of the FDG PET literature “J. Nucl. Med. 2001, 42, 1- 93S). Tumors in the brain can be represented very difficult in healthy brain tissue, for example, by the high accumulation of FDG.

The previously known ¹⁸ F-labeled amino acid derivatives are in some cases well suited to detect tumors in the brain ((review): Eur J Nucl Med Mol Imaging 2002 May; 29 (5):. 681-90), but they can in other tumors do not compete with the imaging properties of the “gold standard” ^[18 F] 2-FDG. The metabolic accumulation and retention of previously F-18 labeled amino acids in tumorous tissue is usually lower than for FDG. Moreover, the accessibility of isomerically pure F-18-labeled non-aromatic amino acids is chemically very demanding.

Similar to glucose increased metabolism in proliferating tumor cells has been described (Medina, J Nutr 1131: 2539S-2542S, 2001; Souba, Ann Surg 218:. 715-728, 1993) for glutamic acid and glutamine. The increased rate of protein and nucleic acid synthesis and energy production per se be accepted as reasons for increased Glutaminkonsum of tumor cells. The synthesis of the corresponding C-11 and C-14 labeled with the natural substrate thus identical compounds, has already been described in the literature (eg. Ex.Antoni, enzymes Catalyzed Synthesis of L- [4-C-11] Aspartate and L – [5-C-11] Glutamate J. Labelled Compd Radiopharm 44; (4) 2001: 287-294) and Buchanan, The biosynthesis of showdomycin: studies with stable isotopes and the determination of principal precursor J….. Chem. Soc. Chem. Commun .; EN; 22; 1984, 1515-1517). First indications with the C-11 labeled compound indicate no significant tumor accumulation.

Although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. In particular, tumor cells with the upregulated c-Myc gene are generally reprogrammed to utilize glutamine. We have developed new 3-fluoropropyl analogs of glutamine, namely [(18)F](2S,4R)- and [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. Optically pure isomers labeled with (18)F and (19)F (2S,4S) and (2S,4R)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (≥95%). Cell uptake studies of both isomers showed that they were taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was approximately two times higher than that of l-[(3)H]glutamine ([(3)H]Gln). These in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [(18)F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dual-isotope experiment using l-[(3)H]glutamine and the new probe showed that the uptake of [(3)H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (<10%). This suggests a different mechanism of retention. In vivo PET imaging studies demonstrated tumor-specific uptake in rats bearing 9L xenographs with an excellent tumor to muscle ratio (maximum of ∼8 at 40 min). [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.

[¹⁸F](2S,4S)-4-(3-Fluoropropyl)glutamine as a Tumor Imaging Agent

http://pubs.acs.org/doi/full/10.1021/mp500236y

Abstract

Although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. In particular, tumor cells with the upregulated c-Myc gene are generally reprogrammed to utilize glutamine. We have developed new 3-fluoropropyl analogs of glutamine, namely [¹⁸F](2S,4R)- and [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. Optically pure isomers labeled with ¹⁸F and ¹⁹F (2S,4S) and (2S,4R)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (≥95%). Cell uptake studies of both isomers showed that they were taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was approximately two times higher than that of l-[³H]glutamine ([³H]Gln). These in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [¹⁸F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dual-isotope experiment using l-[³H]glutamine and the new probe showed that the uptake of [³H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (<10%). This suggests a different mechanism of retention. In vivo PET imaging studies demonstrated tumor-specific uptake in rats bearing 9L xenographs with an excellent tumor to muscle ratio (maximum of ∼8 at 40 min). [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.

PATENT

US 20100290991

http://www.google.com/patents/US20100290991

PATENT

WO 2009141091

https://patentscope.wipo.int/search/ko/detail.jsf?docId=WO2009141091&recNum=70&maxRec=287&office=&prevFilter=%26fq%3DOF%3ACU&sortOption=Relevance&queryString=&tab=PCTDescription

PATENT

http://www.google.co.ug/patents/EP2123621A1?cl=en

REFERENCES

https://www.researchgate.net/publication/264538736_F-182S4S-4-3-Fluoropropylglutamine_as_a_Tumor_Imaging_Agent

Molecular Pharmaceutics (2014), 11(11), 3852-3866

////////

CCT 245737

CAS:1489389-18-5
M.Wt: 379.34
Formula: C16H16F3N7O

2-Pyrazinecarbonitrile, 5-[[4-[[(2R)-2-morpholinylmethyl]amino]-5-(trifluoromethyl)-2-pyridinyl]amino]-

(R)-5-(4-(Morpholin-2-ylmethylamino)-5-(trifluoromethyl)pyridin-2-ylamino)pyrazine-2-carbonitrile

(+)-5-[[4-[[(2R)-Morpholin-2-ylmethyl]amino]-5-(trifluoromethyl)pyridin-2-yl]amino]pyrazine-2-carbonitrile

Cancer Research Technology Limited   INNOVATOR

SAREUM

IND Filed, Sareum FOR CANCER

Synthesis, Exclusive by worlddrugtracker

5-[[4-[[morpholin-2-yl]methylamino]-5- (trifluoromethyl)-2-pyridyl]amino]pyrazine-2-carbonitrile compounds (referred to herein as “TFM compounds”) which, inter alia, inhibit Checkpoint Kinase 1 (CHK1) kinase function. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit CHK1 kinase function, and in the treatment of diseases and conditions that are mediated by CHK1 , that are ameliorated by the inhibition of CHK1 kinase function, etc., including proliferative conditions such as cancer, etc., optionally in combination with another agent, for example, (a) a DNA topoisomerase I or II inhibitor; (b) a DNA damaging agent; (c) an antimetabolite or a thymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent; (e) ionising radiation; (f) an inhibitor of a mitosis regulator or a mitotic checkpoint regulator; (g) an inhibitor of a DNA damage signal transducer; or (h) an inhibitor of a DNA damage repair enzyme.

Checkpoint Kinase 1 (CHK1)

Progression through the cell division cycle is a tightly regulated process and is monitored at several positions known as cell cycle checkpoints (see, e.g., Weinert and Hartwell,

1989; Bartek and Lukas, 2003). These checkpoints are found in all four stages of the cell cycle; G1 , S (DNA replication), G2 and M (Mitosis) and they ensure that key events which control the fidelity of DNA replication and cell division are completed correctly. Cell cycle checkpoints are activated by a number of stimuli, including DNA damage and DNA errors caused by defective replication. When this occurs, the cell cycle will arrest, allowing time for either DNA repair to occur or, if the damage is too severe, for activation of cellular processes leading to controlled cell death.

All cancers, by definition, have some form of aberrant cell division cycle. Frequently, the cancer cells possess one or more defective cell cycle checkpoints, or harbour defects in a particular DNA repair pathway. These cells are therefore often more dependent on the remaining cell cycle checkpoints and repair pathways, compared to non-cancerous cells (where all checkpoints and DNA repair pathways are intact). The response of cancer cells to DNA damage is frequently a critical determinant of whether they continue to proliferate or activate cell death processes and die. For example, tumour cells that contain a mutant form(s) of the tumour suppressor p53 are defective in the G1 DNA damage checkpoint. Thus inhibitors of the G2 or S-phase checkpoints are expected to further impair the ability of the tumour cell to repair damaged DNA. Many known cancer treatments cause DNA damage by either physically modifying the cell’s DNA or disrupting vital cellular processes that can affect the fidelity of DNA replication and cell division, such as DNA metabolism, DNA synthesis, DNA transcription and microtubule spindle formation. Such treatments include for example, radiotherapy, which causes DNA strand breaks, and a variety of chemotherapeutic agents including topoisomerase inhibitors, antimetabolites, DNA-alkylating agents, and platinum- containing cytotoxic drugs. A significant limitation to these genotoxic treatments is drug resistance. One of the most important mechanisms leading to this resistance is attributed to activation of cell cycle checkpoints, giving the tumour cell time to repair damaged DNA. By abrogating a particular cell cycle checkpoint, or inhibiting a particular form of DNA repair, it may therefore be possible to circumvent tumour cell resistance to the genotoxic agents and augment tumour cell death induced by DNA damage, thus increasing the therapeutic index of these cancer treatments.

CHK1 is a serine/threonine kinase involved in regulating cell cycle checkpoint signals that are activated in response to DNA damage and errors in DNA caused by defective replication (see, e.g., Bartek and Lukas, 2003). CHK1 transduces these signals through phosphorylation of substrates involved in a number of cellular activities including cell cycle arrest and DNA repair. Two key substrates of CHK1 are the Cdc25A and Cdc25C phosphatases that dephosphorylate CDK1 leading to its activation, which is a

requirement for exit from G2 into mitosis (M phase) (see, e.g., Sanchez et al., 1997). Phosphorylation of Cdc25C and the related Cdc25A by CHK1 blocks their ability to activate CDK1 , thus preventing the cell from exiting G2 into M phase. The role of CHK1 in the DNA damage-induced G2 cell cycle checkpoint has been demonstrated in a number of studies where CHK1 function has been knocked out (see, e.g., Liu et ai, 2000; Zhao et al., 2002; Zachos et al., 2003).

The reliance of the DNA damage-induced G2 checkpoint upon CHK1 provides one example of a therapeutic strategy for cancer treatment, involving targeted inhibition of CHK1. Upon DNA damage, the p53 tumour suppressor protein is stabilised and activated to give a p53-dependent G1 arrest, leading to apoptosis or DNA repair (Balaint and Vousden, 2001). Over half of all cancers are functionally defective for p53, which can make them resistant to genotoxic cancer treatments such as ionising radiation (IR) and certain forms of chemotherapy (see, e.g., Greenblatt et al., 1994; Carson and Lois, 1995). These p53 deficient cells fail to arrest at the G1 checkpoint or undergo apoptosis or DNA repair, and consequently may be more reliant on the G2 checkpoint for viability and replication fidelity. Therefore abrogation of the G2 checkpoint through inhibition of the CHK1 kinase function may selectively sensitise p53 deficient cancer cells to genotoxic cancer therapies, and this has been demonstrated (see, e.g., Wang et al., 1996; Dixon and Norbury, 2002). In addition, CHK1 has also been shown to be involved in S phase cell cycle checkpoints and DNA repair by homologous recombination. Thus, inhibition of CHK1 kinase in those cancers that are reliant on these processes after DNA damage, may provide additional therapeutic strategies for the treatment of cancers using CHK1 inhibitors (see, e.g., Sorensen et al., 2005). Furthermore, certain cancers may exhibit replicative stress due to high levels of endogenous DNA damage (see, e.g., Cavalier et al., 2009; Brooks et al., 2012) or through elevated replication driven by oncogenes, for example amplified or overexpressed MYC genes (see, e.g., Di Micco et al. 2006; Cole et al., 2011 ; Murga et al. 2011). Such cancers may exhibit elevated signalling through CHK1 kinase (see, e.g., Hoglund et al., 2011). Inhibition of CHK1 kinase in those cancers that are reliant on these processes, may provide additional therapeutic strategies for the treatment of cancers using CHK1 inhibitors (see, e.g., Cole et al., 2011 ; Davies et al., 2011 ; Ferrao et al., 2011).

Several kinase enzymes are important in the control of the cell growth and replication cycle. These enzymes may drive progression through the cell cycle, or alternatively can act as regulators at specific checkpoints that ensure the integrity of DNA replication through sensing DNA-damage and initiating repair, while halting the cell cycle. Many tumours are deficient in early phase DNA-damage checkpoints, due to mutation or deletion in the p53 pathway, and thus become dependent on the later S and G2/M checkpoints for DNA repair. This provides an opportunity to selectively target tumour cells to enhance the efficacy of ionising radiation or widely used DNA-damaging cancer chemotherapies. Inhibitors of the checkpoint kinase CHK1 are of particular interest for combination with genotoxic agents. In collaboration with Professor Michelle Garrett (University of Kent, previously at The Institute of Cancer Research) and Sareum (Cambridge) we used structure-based design to optimise the biological activities and pharmaceutical properties of hits identified through fragment-based screening against the cell cycle kinase CHK1, leading to the oral clinical candidate CCT245737. The candidate potentiates the efficacy of standard chemotherapy in models of non-small cell lung, pancreatic and colon cancer. In collaboration with colleagues at The Institute of Cancer Research (Professor Louis Chesler, Dr Simon Robinson and Professor Sue Eccles) and Newcastle University (Professor Neil Perkins), we have shown that our selective CHK1 inhibitor has efficacy as a single agent in models of tumours with high replication stress, including neuroblastoma and lymphoma.

The checkpoint kinase CHK2 has a distinct but less well characterised biological role to that of CHK1. Selective inhibitors are valuable as pharmacological tools to explore the biological consequences of CHK2 inhibition in cancer cells. In collaboration with Professor Michelle Garrett (University of Kent, previously at The Institute of Cancer Research), we have used structure-based and ligand-based approaches to discover selective inhibitors of CHK2. We showed that selective CHK2 inhibition has a very different outcome to selective CHK1 inhibition. Notably, while CHK2 inhibition did not potentiate the effect of DNA-damaging chemotherapy, it did sensitize cancer cells to the effects of PARP inhibitors that compromise DNA repair.

Synthesis 

PATENT

http://www.google.com/patents/WO2013171470A1?cl=enSynthesis 1 D

5-[[4-[[(2R)-Morpholin-2-yl]methylamino]-5-(trifluoromethyl)-2-pyridyl]amino]py

carbonitrile (Compound 1)

A solution of (S)-tert-butyl 2-((2-(5-cyanopyrazin-2-ylamino)-5-(trifluoromethyl)pyridin-4- ylamino)methyl)morpholine-4-carboxylate (1.09 g, 2.273 mmol) in dichloromethane (8 mL) was added dropwise over 10 minutes to a solution of trifluoroacetic acid (52.7 mL, 709 mmol) and tnisopropylsilane (2.61 mL, 12.73 mmol) in dry dichloromethane (227 mL) at room temperature. After stirring for 30 minutes, the mixture was concentrated in vacuo. The concentrate was resuspended in dichloromethane (200 mL) and

concentrated in vacuo, then resuspended in toluene (100 mL) and concentrated.

The above procedure was performed in triplicate (starting each time with 1.09 g (S)-tert- butyl 2-((2-(5-cyanopyrazin-2-ylamino)-5-(trifluoromethyl)pyridin-4- ylamino)methyl)morpholine-4-carboxylate) and the three portions of crude product so generated were combined for purification by ion exchange chromatography on 2 x 20 g Biotage NH2 Isolute columns, eluting with methanol. The eluant was concentrated and 10% methanol in diethyl ether (25 mL) was added. The resulting solid was filtered, washed with diethyl ether (30 mL), and dried in vacuo to give the title compound as a light straw coloured powder (2.30 g, 89%). H NMR (500 MHz, CD₃OD) δ 2.62 (1 H, J = 12, 10 Hz), 2.78-2.84 (2H, m), 2.95 (1 H, dd, J = 12, 2 Hz), 3.27-3.38 (2H, m), 3.63 (1 H, ddd, J = 14, 9.5, 3 Hz), 3.73-3.78 (1 H, m), 3.91 (1 H, ddd, J = 11 , 4, 2 Hz), 7.26 (1 H, s), 8.18 (1 H, s), 8.63 (1 H, s), 9.01 (1 H, s).

LC-MS (Agilent 4 min) R_t 1.22 min; m/z (ESI) 380 [M+H⁺]. Optical rotation [a]_D ²⁴ = +7.0 (c 1.0, DMF).

Synthesis 2B

(R)-tert- Butyl 2-((2-chloro-5-(trifluoromethyl)pyridin-4-ylamino)methyl)morpholine-

To a solution of 2-chloro-5-(trifluoromethyl)pyridin-4-amine (1 g, 5.09 mmol) in

dimethylformamide (32.6 mL) was added sodium hydride (60% by wt in oil; 0.407 g, 10.18 mmol) portionwise at room temperature followed by stirring for 10 minutes at 80°C. (S)- tert-Butyl 2-(tosyloxymethyl)morpholine-4-carboxylate (2.268 g, 6.1 1 mmol) was then added portionwise and the reaction mixture was stirred at 80°C for 2.5 hours. After cooling, the mixture was partitioned between saturated aqueous sodium

hydrogencarbonate solution (30 mL), water (100 mL) and ethyl acetate (30 mL). The organic layer was separated and the aqueous layer was further extracted with ethyl acetate (2 x 30 mL). The combined organic layers were washed with brine (2 x 70 mL), dried over magnesium sulfate, filtered, concentrated and dried thoroughly in vacuo. The crude material was purified by column chromatography on a 90 g Thomson SingleStep column, eluting with an isocratic mix of 2.5% diethyl ether / 2.5% ethyl acetate in dichloromethane, to give the title compound as a clear gum that later crystallised to give a white powder (1.47 g, 73%). H NMR (500 MHz, CDCI₃) δ 1.48 (9H, s), 2.71-2.83 (1 H, m), 2.92-3.05 (1 H, m), 3.18- 3.23 (1 H, m), 3.33-3.37 (1 H, m), 3.56-3.61 (1 H, m), 3.66-3.71 (1 H, m), 3.80-4.07 (3H, m), 5.32 (1 H, broad s), 6.61 (1 H, s), 8.24 (1 H, s). LC-MS (Agilent 4 min) R_t 3.04 min; m/z (ESI) 396 [MH⁺]. Svnthesis 2C

(R)-tert-Butyl 2-((2-(5-cyanopyrazin-2-ylamino)-5-(trifluoromethyl)pyridin-4-

(R)-tert-Butyl 2-((2-chloro-5-(trifluoromethyl)pyridin-4-ylamino)methyl)morpholine-4- carboxylate (1.44 g, 3.64 mmol), 2-amino-5-cyanopyrazine (0.612 g, 5.09 mmol, 1.4 eq.), tris(dibenzylideneacetone)dipalladium(0) (0.267 g, 0.291 mmol, 0.08 eq.), rac-2,2′- bis(diphenylphosphino)-1 ,1 ‘-binaphthyl (0.362 g, 0.582 mmol, 0.16 eq.) and caesium carbonate (2.37 g, 7.28 mmol) were suspended in anhydrous dioxane (33 ml_) under argon. Argon was bubbled through the mixture for 30 minutes, after which the mixture was heated to 100°C for 22 hours. The reaction mixture was cooled and diluted with dichloromethane, then absorbed on to silica gel. The pre-absorbed silica gel was added to a 100 g KP-Sil SNAP column which was eluted with 20-50% ethyl acetate in hexanes to give the partially purified product as an orange gum. The crude product was dissolved in dichloromethane and purified by column chromatography on a 90 g SingleStep Thomson column, eluting with 20% ethyl acetate in dichloromethane, to give the title compound (1.19 g, 68%). H NMR (500 MHz, CDCI₃) δ 1.50 (9H, s), 2.71-2.88 (1 H, m), 2.93-3.08 (1 H, m), 3.27- 3.32 (1 H, m), 3.40-3.44 (1 H, m), 3.55-3.64 (1 H, m), 3.71-3.77 (1 H, m), 3.82-4.11 (3H, m), 5.33 (1 H, broad s), 7.19 (1 H, s), 8.23 (1 H, s), 8.58 (1 H, s), 8.84 (1 H, s). LC-MS (Agilent 4 min) R_t 2.93 min;m/z (ESI) 480 [MH⁺].

Paper

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic–pharmacodynamic (PK–PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.

Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737)

///////////CCT 245737, IND, PRECLINICAL, Cancer Research Technology Limited, SAREUM

N#CC(C=N1)=NC=C1NC2=NC=C(C(F)(F)F)C(NC[C@@H]3OCCNC3)=C2

• Supporting Info  SEE NMR OF COMPD 4

GSK 6853

CAS  1910124-24-1

The genomes of eukaryotic organisms are highly organised within the nucleus of the cell. The long strands of duplex DNA are wrapped around an octomer of histone proteins (most usually comprising two copies of histones H2A, H2B, H3 and H4) to form a

nucleosome. This basic unit is then further compressed by the aggregation and folding of nucleosomes to form a highly condensed chromatin structure. A range of different states of condensation are possible, and the tightness of this structure varies during the cell cycle, being most compact during the process of cell division. Chromatin structure plays a critical role in regulating gene transcription, which cannot occur efficiently from highly condensed chromatin. The chromatin structure is controlled by a series of post-translational

modifications to histone proteins, notably histones H3 and H4, and most commonly within the histone tails which extend beyond the core nucleosome structure. These modifications include acetylation, methylation, phosphorylation, ubiquitinylation, SUMOylation and numerous others. These epigenetic marks are written and erased by specific enzymes, which place the tags on specific residues within the histone tail, thereby forming an epigenetic code, which is then interpreted by the cell to allow gene specific regulation of chromatin structure and thereby transcription.

Histone acetylation is usually associated with the activation of gene transcription, as the modification loosens the interaction of the DNA and the histone octomer by changing the electrostatics. In addition to this physical change, specific proteins bind to acetylated lysine residues within histones to read the epigenetic code. Bromodomains are small (=1 10 amino acid) distinct domains within proteins that bind to acetylated lysine residues commonly but not exclusively in the context of histones. There is a family of around 50 proteins known to contain bromodomains, and they have a range of functions within the cell.

BRPF1 (also known as peregrin or Protein Br140) is a bromodomain-containing protein that has been shown to bind to acetylated lysine residues in histone tails, including H2AK5ac, H4K12ac and H3K14ac (Poplawski et al, J. Mol. Biol., 2014 426: 1661-1676). BRPF1 also contains several other domains typically found in chromatin-associated factors, including a double plant homeodomain (PHD) and zinc finger (ZnF) assembly (PZP), and a chromo/Tudor-related Pro-Trp-Trp-Pro (PWWP) domain. BRPF1 forms a tetrameric complex with monocytic leukemia zinc-finger protein (MOZ, also known as KAT6A or MYST3) inhibitor of growth 5 (ING5) and homolog of Esa1 -associated factor (hEAF6). In humans, the t(8;16)(p1 1 ;p13) translocation of MOZ (monocytic leukemia zinc-finger protein, also known as KAT6A or MYST3) is associated with a subtype of acute myeloid leukemia and

contributes to the progression of this disease (Borrow et al, Nat. Genet., 1996 14: 33-41 ). The BRPF1 bromodomain contributes to recruiting the MOZ complex to distinct sites of active chromatin and hence is considered to play a role in the function of MOZ in regulating transcription, hematopoiesis, leukemogenesis, and other developmental processes (Ullah et al, Mol. Cell. Biol., 2008 28: 6828-6843; Perez-Campo et al, Blood, 2009 1 13: 4866-4874). Demont et al, ACS Med. Chem. Lett., (2014) (dx.doi.org/10.1021/ml5002932), discloses certain 1 ,3-dimethyl benzimidazolones as potent, selective inhibitors of the BRPF1 bromodomain.

BRPF1 bromodomain inhibitors, and thus are believed to have potential utility in the treatment of diseases or conditions for which a bromodomain inhibitor is indicated. Bromodomain inhibitors are believed to be useful in the treatment of a variety of diseases or conditions related to systemic or tissue inflammation, inflammatory responses to infection or hypoxia, cellular activation and proliferation, lipid metabolism, fibrosis and in the prevention and treatment of viral infections. Bromodomain inhibitors may be useful in the treatment of a wide variety of chronic autoimmune and inflammatory conditions such as rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), asthma, chronic obstructive airways disease, pneumonitis, myocarditis, pericarditis, myositis, eczema, dermatitis (including atopic dermatitis), alopecia, vitiligo, bullous skin diseases, nephritis, vasculitis, atherosclerosis, Alzheimer’s disease, depression, Sjogren’s syndrome, sialoadenitis, central retinal vein occlusion, branched retinal vein occlusion, Irvine-Gass syndrome (post-cataract and post-surgical), retinitis pigmentosa, pars planitis, birdshot retinochoroidopathy, epiretinal membrane, cystic macular edema, parafoveal telengiectasis, tractional maculopathies, vitreomacular traction syndromes, retinal detachment,

neuroretinitis, idiopathic macular edema, retinitis, dry eye (kerartoconjunctivitis Sicca), vernal keratoconjunctivitis, atopic keratoconjunctivitis, uveitis (such as anterior uveitis, pan uveitis, posterior uveits, uveitis-associated macula edema), scleritis, diabetic retinopathy, diabetic macula edema, age-related macula dystrophy, hepatitis, pancreatitis, primary biliary cirrhosis, sclerosing cholangitis, Addison’s disease, hypophysitis, thyroiditis, type I diabetes, type 2 diabetes and acute rejection of transplanted organs. Bromodomain inhibitors may be useful in the treatment of a wide variety of acute inflammatory conditions such as acute gout, nephritis including lupus nephritis, vasculitis with organ involvement such as

glomerulonephritis, vasculitis including giant cell arteritis, Wegener’s granulomatosis, Polyarteritis nodosa, Behcet’s disease, Kawasaki disease, Takayasu’s Arteritis, pyoderma gangrenosum, vasculitis with organ involvement and acute rejection of transplanted organs. Bromodomain inhibitors may be useful in the treatment of diseases or conditions which involve inflammatory responses to infections with bacteria, viruses, fungi, parasites or their toxins, such as sepsis, sepsis syndrome, septic shock, endotoxaemia, systemic inflammatory response syndrome (SIRS), multi-organ dysfunction syndrome, toxic shock syndrome, acute

lung injury, ARDS (adult respiratory distress syndrome), acute renal failure, fulminant hepatitis, burns, acute pancreatitis, post-surgical syndromes, sarcoidosis, Herxheimer reactions, encephalitis, myelitis, meningitis, malaria and SIRS associated with viral infections such as influenza, herpes zoster, herpes simplex and coronavirus. Bromodomain inhibitors may be useful in the treatment of conditions associated with ischaemia-reperfusion injury such as myocardial infarction, cerebro-vascular ischaemia (stroke), acute coronary syndromes, renal reperfusion injury, organ transplantation, coronary artery bypass grafting, cardio-pulmonary bypass procedures, pulmonary, renal, hepatic, gastro-intestinal or peripheral limb embolism. Bromodomain inhibitors may be useful in the treatment of disorders of lipid metabolism via the regulation of APO-A1 such as hypercholesterolemia, atherosclerosis and Alzheimer’s disease. Bromodomain inhibitors may be useful in the treatment of fibrotic conditions such as idiopathic pulmonary fibrosis, renal fibrosis, postoperative stricture, keloid scar formation, scleroderma (including morphea) and cardiac fibrosis. Bromodomain inhibitors may be useful in the treatment of a variety of diseases associated with bone remodelling such as osteoporosis, osteopetrosis, pycnodysostosis, Paget’s disease of bone, familial expanile osteolysis, expansile skeletal hyperphosphatasia, hyperososis corticalis deformans Juvenilis, juvenile Paget’s disease and Camurati

Engelmann disease. Bromodomain inhibitors may be useful in the treatment of viral infections such as herpes virus, human papilloma virus, adenovirus and poxvirus and other DNA viruses. Bromodomain inhibitors may be useful in the treatment of cancer, including hematological (such as leukaemia, lymphoma and multiple myeloma), epithelial including lung, breast and colon carcinomas, midline carcinomas, mesenchymal, hepatic, renal and neurological tumours. Bromodomain inhibitors may be useful in the treatment of one or more cancers selected from brain cancer (gliomas), glioblastomas, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, colorectal cancer, Wilm’s tumor, Ewing’s sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma cancer, osteosarcoma, giant cell tumor of bone, thyroid cancer,

lymphoblastic T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblastic T-cell leukemia, acute myeloid leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma, megakaryoblastic leukemia, acute megakaryocytic leukemia, promyelocytic leukemia, mixed lineage leukaemia, erythroleukemia, malignant lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, lymphoblastic T-cell lymphoma, Burkitt’s lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor) and testicular cancer. In one embodiment the cancer is a leukaemia, for example a leukaemia selected from acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia,

acute myeloid leukemia and mixed lineage leukaemia (MLL). In another embodiment the cancer is multiple myeloma. In another embodiment the cancer is a lung cancer such as small cell lung cancer (SCLC). In another embodiment the cancer is a neuroblastoma. In another embodiment the cancer is Burkitt’s lymphoma. In another embodiment the cancer is cervical cancer. In another embodiment the cancer is esophageal cancer. In another embodiment the cancer is ovarian cancer. In another embodiment the cancer is breast cancer. In another embodiment the cancer is colarectal cancer. In one embodiment the disease or condition for which a bromodomain inhibitor is indicated is selected from diseases associated with systemic inflammatory response syndrome, such as sepsis, burns, pancreatitis, major trauma, haemorrhage and ischaemia. In this embodiment the

bromodomain inhibitor would be administered at the point of diagnosis to reduce the incidence of: SIRS, the onset of shock, multi-organ dysfunction syndrome, which includes the onset of acute lung injury, ARDS, acute renal, hepatic, cardiac or gastro-intestinal injury and mortality. In another embodiment the bromodomain inhibitor would be administered prior to surgical or other procedures associated with a high risk of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS (multiple organ dysfunction syndrome). In a particular embodiment the disease or condition for which a bromodomain inhibitor is indicated is sepsis, sepsis syndrome, septic shock and endotoxaemia. In another embodiment, the bromodomain inhibitor is indicated for the treatment of acute or chronic pancreatitis. In another embodiment the bromodomain is indicated for the treatment of burns. In one embodiment the disease or condition for which a bromodomain inhibitor is indicated is selected from herpes simplex infections and reactivations, cold sores, herpes zoster infections and reactivations, chickenpox, shingles, human papilloma virus, human immunodeficiency virus (HIV), cervical neoplasia, adenovirus infections, including acute respiratory disease, poxvirus infections such as cowpox and smallpox and African swine fever virus. In one particular embodiment a bromodomain inhibitor is indicated for the treatment of Human papilloma virus infections of skin or cervical epithelia. In one embodiment the bromodomain inhibitor is indicated for the treatment of latent HIV infection.

PATENT

WO 2016062737

http://www.google.com/patents/WO2016062737A1?cl=en

Scheme 1

Example 1

Step 1

5-fluoro-1 H-benzordlimidazol-2(3H)-one

A stirred solution of 4-fluorobenzene-1 ,2-diamine (15.1 g, 120 mmol) in THF (120 mL) under nitrogen was cooled using an ice-bath and then was treated with di(1 -/-imidazol-1 -yl)methanone (23.4 g, 144 mmol) portion-wise over 15 min. The resulting mixture was slowly warmed to room temperature then was concentrated in vacuo after 2.5 h. The residue was suspended in a mixture of water and DCM (250 mL each) and filtered off. This residue was then washed with water (50 mL) and DCM (50 mL), before being dried at 40 °C under vacuum for 16 h to give the title compound (16.0 g, 105 mmol, 88%) as a brown solid.

LCMS (high pH): Rt 0.57 min; [M-H⁺]^“ = 151.1

δ_Η NMR (400 MHz, DMSO-d₆) ppm 10.73 (br s, 1 H), 10.61 (br s, 1 H), 6.91-6.84 (m, 1 H), 6.78-6.70 (m, 2H).

Step 2

5-fluoro-1 ,3-dimethyl-1 /-/-benzo[dlimidazol-2(3/-/)-one

A solution of 5-fluoro-1 H-benzo[d]imidazol-2(3H)-one (16.0 g, 105 mmol) in DMF (400 mL) under nitrogen was cooled with an ice-bath, using a mechanical stirrer for agitation. It was then treated over 10 min with sodium hydride (60% w/w in mineral oil, 13.1 g, 327 mmol) and the resulting mixture was stirred at this temperature for 30 min before being treated with iodomethane (26.3 mL, 422 mmol) over 30 min. The resulting mixture was then allowed to warm to room temperature and after 1 h was carefully treated with water (500 mL). The aqueous phase was extracted with EtOAc (3 x 800 mL) and the combined organics were washed with brine (1 L), dried over MgS0₄ and concentrated in vacuo. Purification of the brown residue by flash chromatography on silica gel (SP4, 1.5 kg column, gradient: 0 to 25% (3: 1 EtOAc/EtOH) in cyclohexane) gave the title compound (15.4 g, 86 mmol, 81 %) as a pink solid.

LCMS (high pH): Rt 0.76 min; [M+H⁺]⁺ = 181.1

δ_Η NMR (400 MHz, CDCI₃) ppm 6.86-6.76 (m, 2H), 6.71 (dd, J = 8.3, 2.3 Hz, 1 H), 3.39 (s, 3H), 3.38 (s, 3H).

Step 3

5-fluoro-1 ,3-dimethyl-6-nitro-1 /-/-benzordlimidazol-2(3/-/)-one

A stirred solution of 5-fluoro-1 ,3-dimethyl-1 H-benzo[d]imidazol-2(3/-/)-one (4.55 g, 25.3 mmol) in acetic anhydride (75 mL) under nitrogen was cooled to -30 °C and then was slowly treated with fuming nitric acid (1 .13 mL, 25.3 mmol) making sure that the temperature was kept below -25°C. The solution turned brown once the first drop of acid was added and a thick brown precipitate formed after the addition was complete. The mixture was allowed to slowly warm up to 0 °C then was carefully treated after 1 h with ice-water (100 mL). EtOAc (15 mL) was then added and the resulting mixture was stirred for 20 min. The precipitate formed was filtered off, washed with water (10 mL) and EtOAc (10 mL), and then was dried under vacuum at 40 °C for 16 h to give the title compound (4.82 g, 21 .4mmol, 85%) as a yellow solid.

LCMS (high pH): Rt 0.76 min; [M+H⁺]⁺ not detected

δ_Η NMR (600 MHz, DMSO-d₆) ppm 7.95 (d, J = 6.4 Hz, 1 H, (H-7)), 7.48 (d, J = 1 1.7 Hz, 1 H, (H-4)), 3.38 (s, 3H, (H-10)), 3.37 (s, 3H, (H-12)).

δ₀ NMR (151 MHz, DMSO-d₆) ppm 154.3 (s, 1 C, (C-2)), 152.3 (d, J = 254.9 Hz, 1 C, (C-5)), 135.5 (d, J = 13.0 Hz, 1 C, (C-9)), 130.1 (d, J = 8.0 Hz, 1 C, (C-6)), 125.7 (s, 1 C, (C-8)), 104.4 (s, 1 C, (C-7)), 97.5 (d, J = 28.5 Hz, 1 C, (C-4)), 27.7 (s, 1 C, (C-12)), 27.4 (s, 1 C, (C-10)).

Step 4

(R)-tert-but \ 4-( 1 ,3-dimethyl-6-nitro-2-oxo-2,3-dihydro-1 H-benzordlimidazol-5-yl)-3-methylpiperazine-1-carboxylate

A stirred suspension of 5-fluoro-1 ,3-dimethyl-6-nitro-1 H-benzo[d]imidazol-2(3/-/)-one (0.924 g, 4.10 mmol), (R)-ie f-butyl 3-methylpiperazine-1 -carboxylate (1.23 g, 6.16 mmol), and DI PEA (1 .43 mL, 8.21 mmol) in DMSO (4 mL) was heated to 120 °C in a Biotage Initiator microwave reactor for 13 h, then to 130 °C for a further 10 h. The reaction mixture was concentrated in vacuo then partitioned between EtOAc and saturated aqueous sodium bicarbonate solution. The aqueous was extracted with EtOAc and the combined organics were dried (Na₂S0₄), filtered, and concentrated in vacuo to give a residue which was purified by silica chromatography (0-100% ethyl acetate in cyclohexane) to give the title compound as an orange/yellow solid (1.542 g, 3.80 mmol, 93%).

LCMS (formate): Rt 1.17 min, [M+H⁺]⁺ 406.5.

δ_Η NMR (400 MHz, CDCI₃) ppm 7.36 (s, 1 H), 6.83 (s, 1 H), 4.04-3.87 (m,1 H), 3.87-3.80 (m, 1 H), 3.43 (s, 6H), 3.35-3.25 (m, 1 H), 3.23-3.08 (m, 2H), 3.00-2.72 (m, 2H), 1.48 (s, 9H), 0.81 (d, J = 6.1 Hz, 3H)

Step 5

(RHerf-butyl 4-(6-amino-1 ,3-dimethyl-2-oxo-2,3-dihydro-1 /-/-benzordlimidazol-5-yl)-3-methylpiperazine-1-carboxylate

To (R)-iert-butyl 4-(1 ,3-dimethyl-6-nitro-2-oxo-2,3-dihydro-1 H-benzo[d]imidazol-5-yl)-3-methylpiperazine-1-carboxylate (1 .542 g) in /^‘so-propanol (40 mL) was added 5% palladium on carbon (50% paste) (1.50 g) and the mixture was hydrogenated at room temperature and pressure. After 4 h the mixture was filtered, the residue washed with ethanol and DCM, and the filtrate concentrated in vacuo to give a residue which was purified by silica chromatography (50-100% ethyl acetate in cyclohexane) to afford the title compound (1.220 g, 3.25 mmol, 85%) as a cream solid.

LCMS (high pH): Rt 1 .01 min, [M+H⁺]⁺ 376.4.

δ_Η NMR (400 MHz, CDCI₃) ppm 6.69 (s, 1 H), 6.44 (s, 1 H), 4.33-3.87 (m, 4H), 3.36 (s, 3H), 3.35 (s, 3H), 3.20-2.53 (m, 5H), 1.52 (s, 9H), 0.86 (d, J = 6.1 Hz, 3H).

Step 6

(flVferf-butyl 4-(6-(2-methoxybenzamidoV 1 ,3-dimethyl-2-oxo-2,3-dihvdro-1 H-benzordlimidazol-5-yl)-3-methylpiperazine-1 -carboxylate

A stirred solution of (R)-iert-butyl 4-(6-amino-1 ,3-dimethyl-2-oxo-2,3-dihydro-1 /-/-benzo[d]imidazol-5-yl)-3-methylpiperazine-1 -carboxylate (0.254 g, 0.675 mmol) and pyridine (0.164 ml_, 2.025 mmol) in DCM (2 mL) at room temperature was treated 2-methoxybenzoyl chloride (0.182 mL, 1.35 mmol). After 1 h at room temperature the reaction mixture was concentrated in vacuo to give a residue which was taken up in DMSO:MeOH (1 :1 ) and purified by HPLC (Method C, high pH) to give the title compound (0.302 g, 0.592 mmol, 88%) as a white solid.

LCMS (high pH): Rt 1 .27 min, [M+H⁺]⁺ 510.5.

δ_Η NMR (400 MHz, CDCI₃) ppm 10.67 (s, 1 H), 8.53 (s, 1 H), 8.24 (dd, J = 7.8, 1.7 Hz, 1 H), 7.54-7.48 (m, 1 H), 7.18-7.12 (m, 1 H), 7.07 (d, J = 8.1 Hz, 1 H), 6.82 (s, 1 H), 4.27-3.94 (m, 2H), 4.08 (s, 3H), 3.45 (s, 3H), 3.40 (s, 3H), 3.18-2.99 (m, 2H), 2.92-2.70 (m, 3H), 1.50 (s, 9H), 0.87 (d, J = 6.1 Hz, 3H).

Step 7

(R)-N-( 1 ,3-dimethyl-6-(2-methylpiperazin-1 -yl)-2-oxo-2,3-dihydro-1 H-benzordlimidazol-5-yl)-2-methoxybenzamide

A stirred solution of (R)-ie f-butyl 4-(6-(2-methoxybenzamido)-1 ,3-dimethyl-2-oxo-2,3-dihydro-1 /-/-benzo[d]imidazol-5-yl)-3-methylpiperazine-1-carboxylate (302 mg, 0.592 mmol) in DCM (4 mL) at room temperature was treated with trifluoroacetic acid (3 ml_). After 15 minutes the mixture was concentrated in vacuo to give a residue which was loaded on a solid-phase cation exchange (SCX) cartridge (5 g), washed with MeOH, and then eluted with methanolic ammonia (2 M). The appropriate fractions were combined and concentrated in vacuo to give a white solid (240 mg). Half of this material was taken up in DMSO:MeOH (1 :1 ) and purified by HPLC (Method B, high pH) to give the title compound (101 mg, 0.245 mmol, 41 %) as a white solid.

LCMS (high pH): Rt = 0.90 min, [M+H⁺]⁺ 410.5.

δ_Η NMR (600 MHz, DMSO-d₆) ppm 10.74 (s, 1 H), 8.39 (s, 1 H), 8.05 (dd, J = 7.7, 1.8 Hz, 1 H), 7.57 (ddd, J = 8.3, 7.2, 2.0 Hz, 1 H), 7.29 (d, J = 8.1 Hz, 1 H), 7.23 (s, 1 H), 7.17-7.1 1 (m, 1 H), 4.10 (s, 3H), 3.33 (s, 3H), 3.32 (s, 3H), 3.30 (br s, 1 H), 3.07-3.02 (m, 1 H), 3.02-2.99 (m, 1 H), 2.92-2.87 (m, 1 H), 2.80 (td, J = 1 1.3, 2.7 Hz, 1 H), 2.73 (td, J = 1 1 .0, 2.7 Hz, 1 H), 2.68-2.63 (m, 1 H), 2.55 (dd, J = 12.0, 9.8 Hz, 1 H), 0.71 (d, J = 6.1 Hz, 3H).

δ₀ NMR (151 MHz, DMSO-d₆) ppm 162.1 , 156.8, 154.1 , 134.4, 133.2, 131.5, 130.1 , 126.6, 125.7, 121.9, 121.0, 1 12.5, 103.0, 99.4, 56.8, 55.4, 55.3, 53.3, 46.3, 26.8, 26.6, 16.7.

[a_D]^{25 °c} = -50.1 (c = 0.3, MeOH).

CLIPS

PAPER

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.

GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain

Dapiprazole

CAS 72822-12-9

HCL SALT 72822-13-0

Dapiprazole (Rev-Eyes) is an alpha blocker. It is used to reverse mydriasis after eye examination.^[1]

Used in the treatment of iatrogenically induced mydriasis produced by adrenergic (phenylephrine) or parasympatholytic (tropicamide) agents used in certain eye examinations.

Dapiprazole is an alpha-adrenergic blocking agent. It produces miosis by blocking the alpha-adrenergic receptors on the dilator muscle of the iris. Dapiprazole produces no significant action on ciliary muscle contraction and thus, there are no changes in the depth of the anterior chamber of the thickness of the lens. It does not alter the IOP either in normal eyes or in eyes with elevated IOP. The rate of pupillary constriction may be slightly slower in clients with brown irises than in clients with blue or green irises.

Dapiprazole acts through blocking the alpha1-adrenergic receptors in smooth muscle. It produces miosis through an effect on the dilator muscle of the iris and does not have any significant activity on ciliary muscle contraction and, therefore does not induce a significant change in the anterior chamber depth or the thickness of the lens.

Oral LD₅₀ is 1189-2100 mg/kg in mice, rats and rabbits.

Brief background information

Application

Classes substance

Synthesis

Синтез a)

Scheme illustration:By cyclization of O-methylvalerolactam (I) with 3-(4-o-tolyl-1-piperazinyl) propionic acid hydrazide (II) in refluxing xylene, followed by a treatment with ethanolic HCl.

FR 2423221; GB 2020269; JP 54157576; NL 7902489; US 4252721

Acylation of (1-methylcyclopropyl)guanidine (IV) with 3-bromo-5-chlorothiophene-2-sulfonyl chloride (III) under Schotten-Baumann conditions afforded the sulfonyl guanidine (V). This was cyclized to the desired thienothiadiazine upon treatment with Cs2CO3 and Cu2O in boiling butanol.

In a different method, (1-methylcyclopropyl)guanidine (I) is acylated by 3-bromo-5-chlorothiophene-2-sulfonyl chloride (II) to produce the sulfonyl guanidine (III). Intramolecular cyclization of (III) in the presence of Cu2O and Cs2CO3 leads to the title thienothiadiazine derivative. Similarly, acylation of guanidine (I) with 3,5-dichlorothiophene-2-sulfonyl chloride (IV) provides sulfonyl guanidine (V), which is then cyclized in the presence of Cu2O and Cs2CO3.

In an alternative method, sulfonylation of N-isopropylguanidine (V) with 2,5-dichlorothiophene-3-sulfonyl chloride (IV) produced the sulfonyl guanidine (VI). This was then cyclized to the title compound by treatment with copper bronze and potassium carbonate in boiling DMF……..WO 0102410

Trade names

Formulations

References

Structural formula

UV- Spectrum

Conditions : Concentration – 1 mg / 100 ml
The solvent designation schedule	methanol	water	0.1М HCl	0.1M NaOH
maximum absorption	235 nm	235 nm	234 nm	There decay
	212	179	172	–
e	7650	6450	6200	–

IR – spectrum

Wavelength (μm)
Wave number (cm -1 )

 

References

Dapiprazole

Systematic (IUPAC) name
3-{2-[4-(2-methylphenyl)piperazin-1-yl]ethyl}-5,6,7,8- tetrahydro-[1,2,4]triazolo[4,5-a]pyridine
Clinical data
AHFS/Drugs.com	Consumer Drug Information
MedlinePlus	a601043
Pregnancy category	B
Routes of administration	Topical (eye drops)
Legal status
Legal status	℞ (Prescription only)
Pharmacokinetic data
Bioavailability	Negligible when administered topically
Identifiers
CAS Number	72822-12-9
ATC code	S01EX02 (WHO)
PubChem	CID 3033538
IUPHAR/BPS	7155
DrugBank	DB00298
ChemSpider	2298190
UNII	5RNZ8GJO7K
KEGG	D07775
ChEBI	CHEBI:51066
ChEMBL	CHEMBL1201216
Chemical data
Formula	C19H27N5
Molar mass	325.451 g/mol

//////Дапипразол ,  Dapiprazole, AF-2139, Remydrial, Rev-Eyes, Reversil, Glamidolo

n1nc(n2c1CCCC2)CCN4CCN(c3ccccc3C)CC4

Ladostigil, TV-3,326

(N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate

(3R)-3-(Prop-2-ynylamino)indan-5-yl ethyl(methyl)carbamate; R-CPAI

Carbamic acid, ethylmethyl-, (3R)-2,3-dihydro-3-(2-propynylamino)-1H-inden-5-yl ester

Condition(s): Mild Cognitive Impairment
U.S. FDA Status: Mild Cognitive Impairment (Phase 2)
Company: Avraham Pharmaceuticals Ltd

Target Type: Cholinergic System

CAS 209394-46-7, Ladostigil tartrate

N-Ethyl-N-methylcarbamic acid 3(R)-(2-propynylamino)-2,3-dihydro-1H-inden-5-yl ester L-tartrate

In 2010, ladostigil tartrate was licensed by Technion Research & Development Foundation and Yissum to Avraham for the treatment of Alzheimer’s disease and other neurogenerative diseases.

Ladostigil (TV-3,326) is a novel neuroprotective agent being investigated for the treatment of neurodegenerative disorders likeAlzheimer’s disease, Lewy body disease, and Parkinson’s disease.^[1] It acts as a reversible acetylcholinesterase andbutyrylcholinesterase inhibitor, and an irreversible monoamine oxidase B inhibitor, and combines the mechanisms of action of older drugs like rivastigmine and rasagiline into a single molecule.^[2][3] In addition to its neuroprotective properties, ladostigil enhances the expression of neurotrophic factors like GDNF and BDNF, and may be capable of reversing some of the damage seen in neurodegenerative diseases via the induction of neurogenesis.^[4] Ladostigil also has antidepressant effects, and may be useful for treating comorbid depression and anxiety often seen in such diseases as well.^[5][6]

Ladostigil [(N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate] is a dual acetylcholine-butyrylcholineesterase and brain selective monoamine oxidase (MAO)-A and -B inhibitor in vivo (with little or no MAO inhibitory effect in the liver and small intestine), intended for the treatment of dementia co-morbid with extrapyramidal disorders and depression (presently in a Phase IIb clinical study). This suggests that the drug should not cause a significant potentiation of the cardiovascular response to tyramine, thereby making it a potentially safer antidepressant than other irreversible MAO-A inhibitors. Ladostigil was shown to antagonize scopolamine-induced impairment in spatial memory, indicating that it can cause significant increases in rat brain cholinergic activity. Furthermore, ladostigil prevented gliosis and oxidative-nitrative stress and reduced the deficits in episodic and spatial memory induced by intracerebroventricular injection of streptozotocin in rats. Ladostigil was demonstrated to possess potent anti-apoptotic and neuroprotective activities in vitro and in various neurodegenerative rat models, (e.g. hippocampal damage induced by global ischemia in gerbils and cerebral oedema induced in mice by closed head injury). These neuroprotective activities involve regulation of amyloid precursor protein processing; activation of protein kinase C and mitogen-activated protein kinase signaling pathways; inhibition of neuronal death markers; prevention of the fall in mitochondrial membrane potential and upregulation of neurotrophic factors and antioxidative activity. Recent findings demonstrated that the major metabolite of ladostigil, hydroxy-1-(R)-aminoindan has also a neuroprotective activity and thus, may contribute to the overt activity of its parent compound. This review will discuss the scientific evidence for the therapeutic potential use of ladostigil in Alzheimer’s and Lewy Body diseases and the molecular signaling pathways that are considered to be involved in the biological activities of the drug

LADOSTIGIL

Recently, Yissum Research Development Company associated with the Hebrew University of Jerusalem announced that the University will use a portion of a $9 million dollar grant awarded to Avraham Pharmaceuticals, Pontifax, Clal Biotechnology Industries and Professor Marta Weinstock-Rosin to complete the Phase II efficacy trial in patients afflicted with Alzheimer’s disease.

The trial will be conducted over the course of 52 weeks and will involve the novel drug, Ladostigil. Ladostigil is a new comprehensive drug used to combat symptoms of Alzheimer’s, Parkinson’s, depression, and anxiety. The drug is deemed multi-functional because it addresses a host of neurodegenerative problems. Ladostigil is a brain-selective monoamine oxidase inhibitor (MAOI) that protects the neurons.

PATENT

US 20140243544 http://www.google.co.in/patents/US20140243544

PATENT

WO 2008074816

http://www.google.com/patents/WO2008074816A1?cl=en

PATENT

US 20060199974

Example 3Ethylmethyl-carbamic acid 3-oxo-indan-5-yl ester

Ethylmethyl carbamyl chloride (15.5 g, 127.57 mmol) was added to a stirred suspension of 6-hydroxy-1-indanone (17.2 g, 116.1 mmol) and potassium carbonate (31.8 g, 188 mmol) in acetonitrile (800 mL) at room temperature over a period of 15 minutes. The reaction mixture was heated to reflux and refluxed for 18 hours. The reaction mixture was cooled to ambient temperature, the solvent evaporated and the residue was diluted with water (250 mL) and extracted three times with toluene (250 mL). The combined organic phase was dried on MgSO and toluene was evaporated in a rotary evaporator. The crude crystalline product was purified by crystallization from 2-propanol (200 mL), collected by filtration, and dried under vacuum at 50° C. to afford the title compound (22 g, 81.5%).

1H NMR (300 MHz, CDCl₃) δ ppm 7.47-7.44 (2H, m, Ar), 7.36 (1H, dd, J 8.4 and 2.1, Ar), 3.52-3.37 (2H, m, NCH₂CH₃), 3.14-3.108 [2H, m, OCCH₂CH₂ and incl. NCH₃ (two rotamers), at 3.08 and 2.99 (3H, s, Me)], 2.74-2.71 (2H, m, OCCH₂CH₂) and 1.25 and 1.19 (two rotamers) (3H,two triplets, J 6.9). Mass Spectrum (FAB+) [MH⁺=234

Example 6(S)-Dimethyl-carbamic acid 3-prop-2-ynylamino-indan-5-yl ester

Methanesulfonyl anhydride (296 mg, 1.7 mmol) as a solution in dichloromethane (1.5 mL+0.5 mL) was added to a stirred solution of (R)-dimethyl-methyl-carbamic acid 3-hydroxy-indan-5-yl ester (188 mg, 0.8 mmol, product of example 1a) and triethylamine (0.47 mL, 3.4 mmol) in dichloromethane (2 mL) at −78° C. (external) over 10 minutes. The reaction was maintained at this temperature for 1 hour before propargylamine (1.20 mL, 17.0 mmol) was added. The reaction was allowed to warm slowly to room temperature overnight before being partitioned between ethyl acetate (20 mL) and ice-water (20 mL). The organic material was concentrated under reduced pressure to afford a brown oil which was partitioned between methyl tert-butyl ether (10 mL) and aqueous hydrochloric acid (1M, 10 mL). The aqueous layer was basified by addition of aqueous sodium hydroxide solution (2M, 16 mL) before being extracted with ethyl acetate (10 mL). This final organic extract was dried (MgSO₄), filtered and concentrated under reduced pressure to afford the title compound (175 mg, 80%). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.19 (1H, d, J 8, Ar), 7.09 (1H, d, J 2, Ar), 6.94 (1H, dd, J 8 and 2, Ar), 4.39 (1H, dd, J 6 and 6, CHNH), 3.54 (1H, Dd, J 17 and 3, NCHH), 3.49 (1H, Dd, J 16 and 3, HNCHH), 3.09 (3H, s, Me), 3.03-2.96 [4H, m, NCHCHH and Me incl. at 3.00 (3H, s, Me)], 2.83-2.75 (1H, m, NCHCHH), 2.48-2.39 (1H, m, NCHCH₂CHH), 2.25 (1H, t, J 2, ≡CH) and 1.92-1.83 (1H, m, NCHCH₂CHH). Analysis of this material by chiral LC indicated it to be 70% e.e.

Example 7b(R)-Ethyl-methyl-carbamic acid 3-prop-2-ynylamino-indan-5-yl ester (ladostigil)

The procedure of example 7a is repeated with (S)-ethyl-methyl-carbamic acid 3-hydroxy-indan-5-yl ester instead of (R)-ethyl-methyl-carbamic acid 3-hydroxy-indan-5-yl ester. The R-enantiomer is produced.

PAPER

Tetrahedron: Asymmetry (2012), 23(5), 333-338

http://www.sciencedirect.com/science/article/pii/S0957416612001334

(R)-3-(Prop-2-ynylamino)-2,3-dihydro-1H-inden-5-yl ethyl(methyl)carbamate C16H20N2O2

ee: 89% (c 1.46, CHCl3) Source of chirality: the precursor Absolute configuration: (R)

(R)-3-(Prop-2-ynylamino)-2,3-dihydro-1H-inden-5-yl ethyl(methyl)carbamate hemi((2R,3R)-2,3-dihydroxysuccinate) C36H46N4O10

ee: 99% (c1.95, CHCl3) Source of chirality: the precursor Absolute configuration: (R,R,R)

Avraham Pharmaceuticals Announces Commencement of a Phase 2 Study of Ladostigil for the Treatment of MCI

Posted on May 17, 2012

Yaacov Michlin appointed Chairman of the Board and Dr. Yona Geffen appointed Chief Executive Officer

Yavne, Israel, May 17, 2012 — Avraham Pharmaceuticals Ltd. announced today the commencement of a Phase 2 clinical trial to evaluate the safety and efficacy of ladostigil in patients diagnosed with mild cognitive impairment (MCI). This 36-month, multi-centre, randomized, double-blind, placebo-controlled trial will include at least 200 patients in 16 centers in Europe and Israel.

In parallel, Avraham Pharmaceuticals has also completed the enrollment of 200 patients in a Phase 2 trial of ladostigil, a novel molecule for the treatment of mild to moderate Alzheimer’s disease. The Phase 2 study is a double-blind, closed-label, placebo-controlled trial taking place at 20 sites in five countries across Europe. In January 2012, the Company performed an interim analysis of this Phase 2 trial, which indicated that the drug is safe and well tolerated, as well as shows a positive trend toward efficacy. Final results of the 26-week trial are expected in the fourth quarter of 2012.

The Company also announced today that it has appointed Yaacov Michlin, CEO of Yissum Research Development Company of the Hebrew University of Jerusalem Ltd., the technology transfer arm of the University, as Chairman of the Board and Yona Geffen, Ph.D., as Chief Executive Officer.

“We strongly believe in ladostigil and are confident that Yona’s background and extensive experience in developing therapies for neurological disorders and neurodegenerative diseases renders her the perfect choice to lead Avraham,” said Yaacov Michlin, Chairman of Avraham Pharmaceuticals and Chief Executive Officer of Yissum. “In further researches performed by Prof. Weinstock-Rosin, ladostigil has showed promise also for the treatment of MCI in addition to Alzheimer’s disease. We are pleased that another Phase 2 clinical trial in patients with MCI has begun in parallel, and look forward to the final results of the Phase 2 study for the treatment of Alzheimer’s disease expected at the end of this year.”

“I am delighted to lead Avraham in these exciting times for the company, as we advance ladostigil in 2 Phase 2 clinical trials simultaneously. We believe that this unique drug candidate has the potential to transform the treatment of various neurodegenerative diseases,” said Dr. Yona Geffen, Avraham Pharmaceuticals Chief Executive Officer.

Dr. Yona Geffen joined Avraham Pharmaceuticals in January 2011 as Senior Vice President of Clinical Affairs and Chief Operating Officer. Dr. Geffen has more than 12 years of experience in the field of drug development in biopharmaceutical companies. Prior to Avraham, she was Executive Drug Development Director at BiolineRx (NASDAQ: BLRX). Before that, Dr. Geffen was a project manager at Proneuron Biotechnologies. Dr. Geffen received her Ph.D. from Ben Gurion University in Beer Sheva, Israel. She also holds an M.Sc. in business management.

Yaacov Michlin has been CEO of Yissum since 2009. Prior to Yissum, Mr. Michlin spent over a decade in leading and assisting pharmaceutical, hi-tech and biomedical companies in various technology commercialization deals, licensing agreements, capital raising activities, partnerships, mergers and acquisitions. Michlin holds a Bachelor of Law and Economics cum laude, and a Master of Law all from Bar-Ilan University, Ramat Gan, Israel. In addition, he has an MBA cum laude from the Technion Israel Institute of Technology, Haifa, Israel.

About Ladostigil

Ladostigil is a novel cholinesterase and brain-selective monoamine oxidase inhibitor, and neuroprotective agent for the treatment of Alzheimer’s disease, mild cognitive impairment and other neurodegenerative diseases. The drug, which was exclusively licensed to Avraham Pharmaceuticals by Yissum Research Development Company Ltd., and by the Technion Research and Development Foundation Ltd. (TRDF), has proven to be safe and well tolerated in Phase 1 and Phase 2 clinical trials. Like other cholinesterase inhibitors currently on the market, ladostigil targets symptomatic relief in Alzheimer’s disease patients. But unlike these drugs, ladostigil, which also causes brain selective inhibition of monoamine oxidase (MAO) provides the potential to improve the behavioral and psychological symptoms of dementia such as depression and anxiety. Moreover, ladostigil has the potential to slow progression of clinical symptoms of Alzheimer’s disease for sustained periods of time and to modify the pathology associated with the disease. In addition, the neuroprotective activity of ladostigil provides a drug candidate that may have the potential to slow progression to Alzheimer’s disease in patients diagnosed with MCI. This potential has been amply demonstrated in animal models, especially in studies of ageing rats.

Ladostigil was designed by Professor Marta Weinstock-Rosin of the Hebrew University of Jerusalem, inventor of Exelon® and Professor Moussa B.H. Youdim of the Technion Israel Institute of Technology, inventor of Azilect®. The drug substance was first synthesized by Professor Michael Chorev of the Hebrew University, who is now based at Harvard University. All three distinguished scientists act as scientific advisors to Avraham Pharmaceuticals.

About Alzheimer’s Disease

Alzheimer’s disease is the most common cause of dementia worldwide, affecting about one in 20 people 65 years of age or older, accounting for 60-80% of dementia cases. In 2010, 5.4 million people were affected by Alzheimer’s disease in the U.S., where it is the 6th leading cause of death. In Europe, more than 6 million are living with the disease. Approximately half of Alzheimer’s patients also suffer from depression, and up to 40% also exhibit Parkinson-like symptoms.

About Mild Cognitive Impairment

Mild cognitive impairment (MCI) is a syndrome defined as an intermediate stage between the expected cognitive decline of normal aging and the more pronounced decline of dementia. It involves problems with memory, language, thinking and judgment that are greater than typical age-related changes. Although MCI can present with a variety of symptoms, when memory loss is the predominant symptom it is termed “amnestic MCI” and is frequently seen as a prodromal stage of Alzheimer’s disease. Prevalence in population-based epidemiological studies ranges from 3% to 19% in adults older than 65 years. There is no proven treatment or therapy for MCI.

About Avraham Pharmaceutical

Founded in 2010, Avraham Pharmaceuticals has raised more than $12 million to advance the development of its unique, multi-functional drug substance, ladostigil, currently undergoing two Phase 2 clinical trials for the treatment of Alzheimer’s disease and mild cognitive impairment. The Company has been capitalized by Clal Biotechnology Industries Ltd., the Pontifax Fund and Yissum Research Development Company Ltd., the technology transfer arm of the Hebrew University.

References

1. Weinstock M, Bejar C, Wang RH, et al. (2000). “TV3326, a novel neuroprotective drug with cholinesterase and monoamine oxidase inhibitory activities for the treatment of Alzheimer’s disease”. Journal of Neural Transmission. Supplementum (60): 157–69.PMID 11205137.
2. Weinreb O, Mandel S, Bar-Am O, et al. (January 2009). “Multifunctional neuroprotective derivatives of rasagiline as anti-Alzheimer’s disease drugs”. Neurotherapeutics : the Journal of the American Society for Experimental NeuroTherapeutics 6 (1): 163–74.doi:10.1016/j.nurt.2008.10.030. PMID 19110207.
3. Weinstock M, Luques L, Bejar C, Shoham S (2006). “Ladostigil, a novel multifunctional drug for the treatment of dementia co-morbid with depression”. Journal of Neural Transmission. Supplementum (70): 443–6. PMID 17017566.
4. Weinreb O, Amit T, Bar-Am O, Youdim MB (December 2007). “Induction of neurotrophic factors GDNF and BDNF associated with the mechanism of neurorescue action of rasagiline and ladostigil: new insights and implications for therapy”. Annals of the New York Academy of Sciences 1122: 155–68.doi:10.1196/annals.1403.011. PMID 18077571.
5. Weinstock M, Poltyrev T, Bejar C, Youdim MB (March 2002). “Effect of TV3326, a novel monoamine-oxidase cholinesterase inhibitor, in rat models of anxiety and depression”.Psychopharmacology 160 (3): 318–24. doi:10.1007/s00213-001-0978-x. PMID 11889501.
6. Weinstock M, Gorodetsky E, Poltyrev T, Gross A, Sagi Y, Youdim M (June 2003). “A novel cholinesterase and brain-selective monoamine oxidase inhibitor for the treatment of dementia comorbid with depression and Parkinson’s disease”. Progress in Neuro-psychopharmacology & Biological Psychiatry 27 (4): 555–61. doi:10.1016/S0278-5846(03)00053-8.PMID 12787840.

Ladostigil

Systematic (IUPAC) name
[(3R)-3-(prop-2-ynylamino)indan-5-yl]-N-propylcarbamate
Clinical data
Routes of administration	Oral
Legal status
Legal status	Uncontrolled
Identifiers
CAS Number	209349-27-4
ATC code	none
PubChem	CID 208907
ChemSpider	181005
UNII	SW3H1USR4Q
Synonyms	[N-propargyl-(3R)-aminoindan-5yl]-N-propylcarbamate
Chemical data
Formula	C16H20N2O2
Molar mass	272.34 g/mol

///////////Ladostigil, TV-3,326

c1c(cc2c(c1)CC[C@H]2NCC#C)OC(=O)N(CC)C

MESSAGE FROM VIJAY KIRPALANI

A  2-day FLOW CHEMISTRY Symposium + Workshop has been organized on 16-17 June 2016 at

IICT Hyderabad, India   by Flow Chemistry Society – India Chapter (in collaboration with IICT-Hyderabad & IIT-B)

with speakers from India, UK, Netherlands and Hungary.

Both days have intensive interactive sessions on the theory and industrial applications of Flow Chemistry followed by live demonstrations using 7 different Flow Reactor platforms — from microliters to 10,000 L/day industrial scale.

The Fees are Rs. 5,000 for Industry Delegates and Rs. 2,500 for Academic Delegates (+15% Service Tax) : contact : vk@pi-inc.co or msingh@cipla.com

I have attached a detailed program and look forward to meeting you at the event..

Best regards

Vijay Kirpalani
President
Flow Chemistry Society – India Chapter
email : vk@pi-inc.co
Tel: +91-9321342022 // +91-9821342022

ABOUT

IICT, Hyderabad, India

Dr. S. Chandrasekhar,
Director

CSIR-Indian Institute of Chemical Technology (IICT)

Hyderabad, India

SPEAKERS

Mr Vijay Kirpalani

President
Flow Chemistry Society – India Chapter, INDIA

Dr Charlotte Wiles , CHEMTRIX

UK &THE NETHERLANDS,UNIV OF HULL

Prof Anil Kumar( IIT-B), INDIA

Sreepriya Vedantam

IICT, INDIA

Kevin van Eeten

Flowid B.V, The Netherlands

TU EINDHOWEN

Laszlo Kocsis

Head of Research and Development at ThalesNano Inc, HUNGARY

 

GLIMPSE OF HYDERABAD INDIA

/////Flow Chemistry, Symposium,  Workshop,  16-17 June, IICT, Hyderabad, India

Obeticholic acid (abbreviated to OCA), is a semi-synthetic bile acid analogue which has the chemical structure 6α-ethyl-chenodeoxycholic acid. It has also been known as INT-747. It is undergoing development as a pharmaceutical agent for severalliver diseases and related disorders. Intercept Pharmaceuticals Inc. (NASDAQ symbol ICPT) hold the worldwide rights to develop OCA outside Japan and China, where it is licensed to Dainippon Sumitomo Pharma.^[2]

REVIEW
INT-747(Obeticholic acid; 6-ECDCA) is a potent and selective FXR agonist(EC50=99 nM) endowed with anticholestatic activity. IC50 value: 99 nM(EC50) [1] Target: FXR agonist in vitro: The exposure of rat hepatocytes to 1 microM 6-ECDCA caused a 3- to 5-fold induction of small heterodimer partner (Shp) and bile salt export pump (bsep) mRNA and 70 to 80% reduction of cholesterol 7alpha-hydroxylase (cyp7a1), oxysterol 12beta-hydroxylase (cyp8b1), and Na(+)/taurocholate cotransporting peptide (ntcp) [2]. in vivo: In vivo administration of 6-ECDCA protects against cholestasis induced by E(2)17alpha [2]. high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls [3]. Rats were gavaged with INT-747 or vehicle during 10 days after bile-duct ligation and then were assessed for changes in gut permeability, BTL, and tight-junction protein expression, immune cell recruitment, and cytokine expression in ileum, mesenteric lymph nodes, and spleen. After INT-747 treatment, natural killer cells and interferon-gamma expression markedly decreased, in association with normalized permeability selectively in ileum (up-regulated claudin-1 and occludin) and a significant reduction in BTL [4].

REFERENCES

Invention and development

The natural bile acid, chenodeoxycholic acid, was identified in 1999 as the most active physiological ligand for the farnesoid X receptor (FXR), which is involved in many physiological and pathological processes. A series of alkylated bile acid analogues were designed, studied and patented by Roberto Pellicciari and colleagues at the University of Perugia, with 6α-ethyl-chenodeoxycholic acid emerging as the most highly potent FXR agonist.^[3] FXR-dependent processes in liver and intestine were proposed as therapeutic targets in human diseases.^[4] Obeticholic acid is the first FXR agonist to be used in human drug studies.

Clinical studies

OCA is undergoing development in phase 2 and 3 studies for specific liver and gastrointestinal disorders.^[5]

Primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an auto-immune, inflammatory liver disease which produces bile duct injury, fibrosis, cholestasisand eventual cirrhosis. It is much more common in women than men and can cause jaundice, itching (pruritus) and fatigue.Ursodeoxycholic acid therapy is beneficial, but the disease often progresses and may require liver transplantation.^[6] Animal studies suggested that treatment with FXR agonists should be beneficial in cholestatic diseases such as PBC.^[7] OCA at doses between 10 mg and 50 mg was shown to provide significant biochemical benefit, but pruritus was more frequent with higher doses.^[8][9] The results of a randomized, double-blind phase 3 study of OCA, 5 mg or 10 mg, compared to placebo (POISE) were presented in April 2014, and showed that the drug met the trial’s primary endpoint of a significant reduction in serum alkaline phosphatase, abiomarker predictive of disease progression, liver transplantation or death.^[10]

Nonalcoholic steatohepatitis (NASH)

Non-alcoholic steatohepatitis is a common cause of abnormal liver function with histological features of fatty liver, inflammation andfibrosis. It may progress to cirrhosis and is becoming an increasing indication for liver transplantation. It is increasing in prevalence. OCA is proposed to treat NASH.^[11] A phase 2 trial published in 2013 showed that administration of OCA at 25 mg or 50 mg daily for 6 weeks reduced markers of liver inflammation and fibrosis and increased insulin sensitivity.^[12]

The Farnesoid X Receptor Ligand Obeticholic Acid in Nonalcoholic Steatohepatitis Treatment (FLINT) trial, sponsored by NIDDK, was halted early in January 2014, after about half of the 283 subjects had completed the study, when a planned interim analysis showed that a) the primary endpoint had been met and b) lipid abnormalities were detected and arose safety concerns. Treatment with OCA (25 mg/day for 72 weeks) resulted in a highly statistically significant improvement in the primary histological endpoint, defined as a decrease in the NAFLD Activity Score of at least two points, with no worsening of fibrosis. 45% (50 of 110) of the treated group had this improvement compared with 21% (23 of 109) of the placebo-treated controls.^[13] However concerns about longterm safety issues such as increased cholesterol and adverse cardiovascular events may warrant the concomitant use of statins in OCA-treated patients.^[14]

Portal hypertension

Animal studies suggest that OCA improves intrahepatic vascular resistance and so may be of therapeutic benefit in portal hypertension.^[15] An open label phase 2a clinical study is under way.

Bile acid diarrhea

Bile acid diarrhea (also called bile acid malabsorption) can be secondary to Crohn’s disease or be a primary condition. Reduced median levels of FGF19, an ileal hormone that regulates increased hepatic bile acid synthesis, have been found in this condition.^[16] FGF19 is potently stimulated by bile acids and especially by OCA.^[17] A proof of concept study of OCA (25 mg/d) has shown clinical and biochemical benefit.^[18]

SYNTHESIS

CN 105541953

Take 10g of austempered cholic acid 89.6% purity crude (single hetero greater than 2%), 3 times its weight of acetone and added to their 20% by weight of triethylamine was added, was heated at reflux for 2h, cooled slowly to 10 ° C, the precipitated crystals were filtered to give Obey acid organic amine salt crystals.

Acidification [0020] The organic amine salts Obey acid crystals were dissolved with purified water after 10wt% by mass percentage to the PH value of 2.0 with dilute hydrochloric acid, filtered and dried to give purified Obey acid.

[0021] The purified Obey acid ethyl acetate dissolved by heating and then cooling to 20 ° C, the precipitated crystals were filtered and dried to obtain a purity of 98.7% recrystallization Obey acid (single hetero less than 0.1%), recovery was 84.5%.

PATENT

 WO 2016045480 

The synthetic process can be achieved, although the enlarged combined, however, the compound Vb produce large amounts of byproducts under strongly alkaline conditions palladium on carbon hydrogenation process for preparing high temperature and strong alkaline compound XI during this step leading to the separation of income a lower rate (about 60%), low yield of this step may be due to compound Vb and XI in unprotected hydroxy dehydration occurs under strongly basic (30% NaOH) and high temperature (95-105 ℃) conditions side effects caused.

synthesis of bile acids Obey,

PATENT

CN 105175473

According to Obey acid 6 was prepared in the form of C Patent Document W02013192097A1 reaction of Example 1, as follows:

The 3 a – hydroxy -6 a – ethyl-7-keto -5 P – 24-oic acid (. 86g, 205 4mmol), water (688mL) and 50% (w / w) hydrogen sodium hydroxide solution (56. 4mL) and the mixture of sodium borohydride (7. 77g, 205. 4mmol) in a mixture of 50% (w / w) sodium hydroxide solution (1.5 mL of) and water (20 mL) in 90 ° in C to 105 ° C reaction. Was heated with stirring under reflux for at least 3 hours, the reaction was completed, the reaction solution was cooled to 80 ° C. Between 30 ° C at 50 ° C of citric acid (320. 2g, anhydrous), a mixture of n-butyl acetate (860 mL of) and water (491mL) to ensure an acidic pH of the aqueous phase was separated. Evaporation of the organic phase was distilled to give the residue was diluted with n-butyl acetate, slowly cooled to 15 ° C to 20 ° C, centrifugation. The crude product was crystallized from n-butyl acetate. After Obey acid isolated by n-butyl acetate (43mL, 4 times), dried samples were dried at 80 ° C under vacuum. To give 67. 34g (77. 9%) crystalline form C Obey acid.

References

External links

• “Intercept pharmaceuticals”.
• NashBiotechs Several articles on drug candidates in NASH

Obeticholic acid

Systematic (IUPAC) name
(3α,5β,6α,7α)-6-Ethyl-3,7-dihydroxycholan-24-oic acidOR (4R)-4-[(3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid
Clinical data
Routes of administration	Oral
Legal status
Legal status	Investigational New Drug
Identifiers
CAS Number	459789-99-2
ATC code	A05AA04 (WHO)
PubChem	CID 447715
IUPHAR/BPS	3435
ChemSpider	394730
UNII	0462Z4S4OZ
KEGG	C15636
ChEMBL	CHEMBL566315
Synonyms	6α-ethyl-chenodeoxycholic acid; INT-747
Chemical data
Formula	C26H44O4
Molar mass	420.62516 g/mol

/////////6-ECDCA,  DSP-1747,  INT-747, 459789-99-2, Obeticholic acid

CC[C@@H]1[C@@H]2C[C@@H](CC[C@@]2([C@H]3CC[C@]4([C@H]([C@@H]3[C@@H]1O)CC[C@@H]4[C@H](C)CCC(=O)O)C)C)O

CCC1C2CC(CCC2(C3CCC4(C(C3C1O)CCC4C(C)CCC(=O)O)C)C)O

AM 2394

1-(6′-(2-hydroxy-2-methylpropoxy)-4-((5-methylpyridin-3-yl)oxy)-[3,3′-bipyridin]-6-yl)-3-methylurea

Urea, N-[6′-(2-hydroxy-2-methylpropoxy)-4-[(5-methyl-3-pyridinyl)oxy][3,3′-bipyridin]-6-yl]-N‘-methyl-

CAS 1442684-77-6
Chemical Formula: C22H25N5O4
Exact Mass: 423.1907

Array Biopharma Inc., Amgen Inc. INNOVATORS

AM-2394 is a potent and selective Glucokinase agonist (GKA), which catalyzes the phosphorylation of glucose to glucose-6-phosphate. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes

Type 2 diabetes mellitus (T2DM) is a disease characterized by elevated plasma glucose in the presence of insulin resistance and inadequate insulin secretion. Glucokinase (GK), a member of the hexokinase enzyme family, catalyzes the phosphorylation of glucose to glucose-6-phosphate in the presence of ATP.

Glucokioase i exok ase IV or D> is a glycolytic enssyiris that plays, an importaat. role irt blood sugar regulation .related to glucose utifeattoti a»d metabolism in the liver and pancreatic beta ^•cells. Serving as a glucose sessor, gtoeokiuase controls lasma glucose, levels. Glucokinaae plays a doal rob in .reducing plasma glucose levels; glucose-mediated activation of the en¾ymc in hepatocytes facilitates hepatic giocose npiafcc aad glycogen synthesis, while that la pancreatic beta ceils ultimately induces ins lin seeretio«. Both of these effects in turn reduce plasma glucose levels.

Clinical evidence has shown that, glueokitiase variants with, decreased, and increased activities are associated with mature easel, diabetes of the y ung { O0Y2) and persistent: hyperinsul nemic hypoglycemia &( infancy (PHHI), respectively. lso, aoo n.sulin dependent diabetes rneilitos (NIDDM) patients have been reported to have inappropriately lo giueokaiase activity; Ftirtherrnare. overexpressioa of glucokiuase it* dietary or gesetie animal models of diabetes either prevents, aoKiiorafes, or reverses the progress of pathological. symptoms in the disease. For these reasons, compounds that activate gfecokiaase have been sought by the pitasaaceatjeai liidustry.

International patent application, Publication No. WO 2 7/OS3345, which was published on May 10, 200?, discloses as giocokinase act ators certain 2-an«.aopyridiiie derivatives bearing at the 3 -position a meihyieneoxy-dkrked aromatic group a d on. the ammo group a heteroaryl ring, such as dna/oly! or i A4-lmadiazoiyl

it has .now been found that pyridyl ureas are useful as glneokirtase activators. Cettain of these •compounds have been, found to have an outstanding combination of properties that especially adapts them, for oral use to control plasma glucose levels.

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394

Glucokinase (GK) catalyzes the phosphorylation of glucose to glucose-6-phosphate. We present the structure–activity relationships leading to the discovery of AM-2394, a structurally distinct GKA. AM-2394 activates GK with an EC₅₀ of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes.

PATENT

WO 2013086397

 http://www.google.com/patents/WO2013086397A1?cl=en

 COPYING ERROR

Example. 1734 t¾^Jtiyi¾rea

Step A: In 100 mL of DMA were corafeiaed 1 ^545miSO- -ll«omp ridinr2-yl)-3-i«e hir8a- (17.5 g, 70,5 ii!-!to!). 5-o:ieS:t}yI yiidlii~3- ). (9,24 g, S4.7 ΪΗΪΪΪΟ!}, sad CO · (10.1 g, 77.6 mmo!) mid heated to 90 *C for 5 days. After that time, the reaction was om lete a d to it was added water arid DCM and stirred vigorously for 3 hr. The resulting solid was isolated via vacuum .filtratiott nd the cake was wasted mill rater and DCM. The DCM in tli aqueous rime was dried vdth a stream of aidogeji aad vigorous sbrriug. Use resulting solid was then collected via vacuum filtration aad these solids were

Stirred vig rousl in f 0% MeOH irt EtOAc arid die res dtipg solid was colleeied. via vactiiars fiirfati m.

Trie two batches wen i coiiibiaed to yield I-(5-bmmo-4 5^»ie†fey pyiidin-3-yl xy)p Tidin-2- d 3~ metbySurea (I S J g, 5 3.7 om»)i, 76% yield).

S e .8: In 2 niL ofc ioxane

yI) iyridMJ-2-yios:y)pf¾ps3i-2-oI (0,098 g, 0.33 «ΜΠΟΪ), ^“ -i5-bs¾tao-4-{5-a3fidiy I py f idia-3 – ylosy)f5yridia-2-yl)-3-raethyl«rea (0.075 g, 0.22 tn ol._. t, and.2M poiass.ua» carbonate (0.33 ml, 0.67 m oi} artd tfets was s parged wi h At .for 10 mia before PdC§4dppl)*DCM (0.01 g g, 0.022 msttol) was added and dre reae!io a was sparged for aaotber 5 ma-, ir efore a was sealed and heated to 100 oversight The react! art was then loaded directly onto s ilica gel (50% acetone to PCM w4i. }%

MH40H) to afford i – (6′-(2diydioxy-2ⁱ-H5eth:ylpropCis:y) -4-{ 5″i:t re th y Ipy r i d i rt -3- io s y ) -3 ,3 ^: -bipyr id i rt -6- yl)-3-aie5¾ylt)rea φ.? 42 , 0.096 m ol, 43 % yield). ^!1 1 HMR (400 Mife, CDCij) 3 ppm 9.06 is,. !H),

S.33 is, 1H>, 8,27 (rs 2H), 8. Π (s, I H)_: K. (s, IHU 82 (dd, j-S.fi, 5.9 H HI), 1.21 (S !H), 6,«8

(d, Hz, i i i ). 6. ,4 (s_:. m>, 4.25 (s, 2H), 2,87 (dj =4,3 Hz„ 3H) 2,37 (s, 3H>. 1 .33 is, <SH). Mass speetram (apci) tar/, ^: – 423.9 (M÷H).

REFERENCES

Novel Series of Potent Glucokinase Activators Leading to the Discovery of AM-2394
Paul J. Dransfield, Vatee Pattaropong, Sujen Lai, Zice Fu, Todd J. Kohn, Xiaohui Du, Alan Cheng, Yumei Xiong, Renee Komorowski, Lixia Jin, Marion Conn, Eric Tien, Walter E. DeWolf Jr., Ronald J. Hinklin, Thomas D. Aicher, Christopher F. Kraser, Steven A. Boyd, Walter C. Voegtli, Kevin R. Condroski, Murielle Veniant-Ellison, Julio C. Medina, Jonathan Houze, and Peter Coward
Publication Date (Web): May 23, 2016 (Letter)
DOI: 10.1021/acsmedchemlett.6b00140

/////////Glucokinase activator,  GKA,  AM-2394, 1442684-77-6, AM 2394, Amgen

O=C(NC)NC1=CC(OC2=CC(C)=CN=C2)=C(C3=CC=C(OCC(C)(O)C)N=C3)C=N1

JNJ-54257099,

1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione

MW 374.28, C₁₄ H₁₉ N₂ O₈ P

CAS 1491140-67-0

2,4(1H,3H)-Pyrimidinedione, 1-[(2R,2′R,4aR,6R,7aR)-dihydro-2-(1-methylethoxy)-2-oxidospiro[4H-furo[3,2-d]-1,3,2-dioxaphosphorin-7(6H),2′-oxetan]-6-yl]-

1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphos-phinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione

Janssen R&D Ireland INNOVATOR

Ioannis Nicolaos Houpis, Tim Hugo Maria Jonckers, Pierre Jean-Marie Bernard Raboisson, Abdellah Tahri, 

Tim Hugo Maria Jonckers

Tim Jonckers was born in Antwerp in 1974. He studied Chemistry at the University of Antwerp and obtained his Ph.D. in organic chemistry in 2002. His Ph.D. work covered the synthesis of new necryptolepine derivatives which have potential antimalarial activity. Currently he works as a Senior Scientist at Tibotec, a pharmaceutical research and development company based in Mechelen, Belgium, that focuses on viral diseases mainly AIDS and hepatitis. The company was acquired by Johnson & Johnson in April 2002 and recently gained FDA approval for its HIV-protease inhibitor PREZISTA™.

DATA

Chiral SFC using the methods described(Method 1, R_t= 5.12 min, >99%; Method 2, R_t = 7.95 min, >99%).

¹H NMR (400 MHz, chloroform-d) δ ppm 1.45 (dd, J = 7.53, 6.27 Hz, 6 H), 2.65–2.84 (m, 2 H), 3.98 (td, J = 10.29, 4.77 Hz, 1 H), 4.27 (t,J = 9.66 Hz, 1 H), 4.43 (ddd, J = 8.91, 5.77, 5.65 Hz, 1 H), 4.49–4.61 (m, 1 H), 4.65 (td, J = 7.78, 5.77 Hz, 1 H), 4.73 (d, J = 7.78 Hz, 1 H), 4.87 (dq, J = 12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J = 8.03 Hz, 1 H), 7.20 (d, J = 8.03 Hz, 1 H), 8.78 (br. s., 1 H);

³¹P NMR (chloroform-d) δ ppm −7.13. LC-MS: 375 (M + H)⁺.

HCV is a single stranded, positive-sense R A virus belonging to the Flaviviridae family of viruses in the hepacivirus genus. The NS5B region of the RNA polygene encodes a RNA dependent RNA polymerase (RdRp), which is essential to viral replication. Following the initial acute infection, a majority of infected individuals develop chronic hepatitis because HCV replicates preferentially in hepatocytes but is not directly cytopathic. In particular, the lack of a vigorous T-lymphocyte response and the high propensity of the virus to mutate appear to promote a high rate of chronic infection. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, end-stage liver disease, and HCC (hepatocellular carcinoma), making it the leading cause of liver transplantations. There are six major HCV genotypes and more than 50 subtypes, which are differently distributed geographically. HCV genotype 1 is the predominant genotype in Europe and in the US. The extensive genetic heterogeneity of HCV has important diagnostic and clinical implications, perhaps explaining difficulties in vaccine development and the lack of response to current therapy.

Transmission of HCV can occur through contact with contaminated blood or blood products, for example following blood transfusion or intravenous drug use. The introduction of diagnostic tests used in blood screening has led to a downward trend in post-transfusion HCV incidence. However, given the slow progression to the end-stage liver disease, the existing infections will continue to present a serious medical and economic burden for decades.

Therapy possibilities have extended towards the combination of a HCV protease inhibitor (e.g. Telaprevir or boceprevir) and (pegylated) interferon-alpha (IFN-a) / ribavirin. This combination therapy has significant side effects and is poorly tolerated in many patients. Major side effects include influenza-like symptoms, hematologic

abnormalities, and neuropsychiatric symptoms. Hence there is a need for more effective, convenient and better-tolerated treatments.

The NS5B RdRp is essential for replication of the single-stranded, positive sense, HCV RNA genome. This enzyme has elicited significant interest among medicinal chemists. Both nucleoside and non-nucleoside inhibitors of NS5B are known. Nucleoside inhibitors can act as a chain terminator or as a competitive inhibitor, or as both. In order to be active, nucleoside inhibitors have to be taken up by the cell and converted in vivo to a triphosphate. This conversion to the triphosphate is commonly mediated by cellular kinases, which imparts additional structural requirements on a potential nucleoside polymerase inhibitor. In addition this limits the direct evaluation of nucleosides as inhibitors of HCV replication to cell-based assays capable of in situ phosphorylation.

Several attempts have been made to develop nucleosides as inhibitors of HCV RdRp, but while a handful of compounds have progressed into clinical development, none have proceeded to registration. Amongst the problems which HCV-targeted

nucleosides have encountered to date are toxicity, mutagenicity, lack of selectivity, poor efficacy, poor bioavailability, sub-optimal dosage regimes and ensuing high pill burden and cost of goods.

Spirooxetane nucleosides, in particular l-(8-hydroxy-7-(hydroxy- methyl)- 1,6-dioxaspiro[3.4]octan-5-yl)pyrimidine-2,4-dione derivatives and their use as HCV inhibitors are known from WO2010/130726, and WO2012/062869, including

CAS-1375074-52-4.

There is a need for HCV inhibitors that may overcome at least one of the disadvantages of current HCV therapy such as side effects, limited efficacy, the emerging of resistance, and compliance failures, or improve the sustained viral response.

The present invention concerns HCV-inhibiting uracyl spirooxetane derivatives with useful properties regarding one or more of the following parameters: antiviral efficacy towards at least one of the following genotypes la, lb, 2a, 2b, 3,4 and 6, favorable

profile of resistance development, lack of toxicity and genotoxicity, favorable pharmacokinetics and pharmacodynamics and ease of formulation and administration.

Such an HCV-inhibiting uracyl spirooxetane derivative is a compound with formula I

including any pharmaceutically acceptable salt or solvate thereof.

PATENT

WO 2015077966

https://www.google.com/patents/WO2015077966A1?cl=en

Synthesis of compound (I)

(5) (6a)

Synthesis of compound (6a)

A solution of isopropyl alcohol (3.86 mL,0.05mol) and triethylamine (6.983 mL, 0.05mol) in dichloromethane (50 mL) was added to a stirred solution of POCI₃ (5)

(5.0 mL, 0.055 lmol) in DCM (50 mL) dropwise over a period of 25 min at -5°C. After the mixture stirred for lh, the solvent was evaporated, and the residue was suspended in ether (100 mL). The triethylamine hydrochloride salt was filtered and washed with ether (20 mL). The filtrate was concentrated, and the residue was distilled to give the (6) as a colorless liquid (6.1g, 69 %yield).

Synthesis of compound (4):

CAS 1255860-33-3 is dissolved in pyridine and 1,3-dichloro-l, 1,3,3-tetraisopropyldisiloxane is added. The reaction is stirred at room temperature until complete. The solvent is removed and the product redissolved in CH₂CI₂ and washed with saturated NaHC0₃ solution. Drying on MgSC^ and removal of the solvent gives compound (2). Compound (3) is prepared by reacting compound (2) with p-methoxybenzylchloride in the presence of DBU as the base in CH₃CN. Compound (4) is prepared by cleavage of the bis-silyl protecting group in compound (3) using TBAF as the fluoride source.

Synthesis of compound (7a)

To a stirred suspension of (4) (2.0 g, 5.13 mmol) in dichloromethane (50 mL) was added triethylamine (2.07 g, 20.46 mmol) at room temperature. The reaction mixture was cooled to -20°C, and then (6a) (1.2 g, 6.78mmol) was added dropwise over a period of lOmin. The mixture was stirred at this temperature for 15min and then NMI was added (0.84 g, 10.23 mmol), dropwise over a period of 15 min. The mixture was stirred at -15°C for lh and then slowly warmed to room temperature in 20 h. The solvent was evaporated, the mixture was concentrated and purified by column chromatography using petroleum ether/EtOAc (10: 1 to 5: 1 as a gradient) to give (7a) as white solid (0.8 g, 32 % yield).

Synthesis of compound (I)

To a solution of (7a) in CH₃CN (30 mL) and H₂0 (7 mL) was add CAN portion wise below 20° C. The mixture was stirred at 15-20° C for 5h under N₂. Na₂S0₃ (370 mL) was added dropwise into the reaction mixture below 15°C, and then Na₂C0₃ (370 mL) was added. The mixture was filtered and the filtrate was extracted with CH₂C1₂

(100 mL*3). The organic layer was dried and concentrated to give the residue. The residue was purified by column chromatography to give the target compound (8a) as white solid. (Yield: 55%)

1H NMR (400 MHz, CHLOROFORM- ) δ ppm 1.45 (dd, J=7.53, 6.27 Hz, 6 H), 2.65 -2.84 (m, 2 H), 3.98 (td, J=10.29, 4.77 Hz, 1 H), 4.27 (t, J=9.66 Hz, 1 H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz, 1 H), 4.49 – 4.61 (m, 1 H), 4.65 (td, J=7.78, 5.77 Hz, 1 H), 4.73 (d, J=7.78 Hz, 1 H), 4.87 (dq, J=12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J=8.03 Hz, 1 H), 7.20 (d, J=8.03 Hz, 1 H), 8.78 (br. s., 1 H); ³¹P NMR (CHLOROFORM-^) δ ppm -7.13; LC-MS: 375 (M+l)+

PATENT

https://www.google.co.in/patents/WO2013174962A1?cl=en

The starting material l-[(4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-l,6-dioxa- spiro[3.4]octan-5-yl]pyrimidine-2,4(lH,3H)-dione (1) can be prepared as exemplified in WO2010/130726. Compound (1) is converted into compounds of the present invention via a p-methoxybenzyl protected derivative (4) as exemplified in the following Scheme 1. cheme 1

Examples

Scheme 2

Synthesis of compound (8a)

Synthesis of compound (2)

Compound (2) can be prepared by dissolving compound (1) in pyridine and adding l,3-dichloro-l,l,3,3-tetraisopropyldisiloxane. The reaction is stirred at room temperature until complete. The solvent is removed and the product redissolved in CH₂CI₂and washed with saturated NaHC0₃ solution. Drying on MgSC^ and removal of the solvent gives compound (2).

Synthesis of compound (3)

Compound (3) is prepared by reacting compound (2) with p-methoxybenzylchloride in the presence of DBU as the base in CH₃CN.

Synthesis of compound (4)

Compound (4) is prepared by cleavage of the bis-silyl protecting group in compound (3) using TBAF as the fluoride source.

Synthesis of compound (6a)

A solution of isopropyl alcohol (3.86 mL,0.05mol) and triethylamine (6.983 mL, 0.05mol) in dichloromethane (50 mL) was added to a stirred solution of POCl₃ (5) (5.0 mL, 0.055 lmol) in DCM (50 mL) dropwise over a period of 25 min at -5°C. After the mixture stirred for lh, the solvent was evaporated, and the residue was suspended in ether (100 mL). The triethylamine hydrochloride salt was filtered and washed with ether (20 mL). The filtrate was concentrated, and the residue was distilled to give the (6) as a colorless liquid (6.1g, 69 %yield).

Synthesis of compound (7a)

To a stirred suspension of (4) (2.0 g, 5.13 mmol) in dichloromethane (50 mL) was added triethylamine (2.07 g, 20.46 mmol) at room temperature. The reaction mixture was cooled to -20°C, and then (6a) (1.2 g, 6.78mmol) was added dropwise over a period of lOmin. The mixture was stirred at this temperature for 15min and then NMI was added (0.84 g, 10.23 mmol), dropwise over a period of 15 min. The mixture was stirred at -15°C for lh and then slowly warmed to room temperature in 20 h. The solvent was evaporated, the mixture was concentrated and purified by column chromatography using petroleum ether/EtOAc (10:1 to 5: 1 as a gradient) to give (7a) as white solid (0.8 g, 32 % yield).

Synthesis of compound (8a)

To a solution of (7a) in CH₃CN (30 mL) and H₂0 (7 mL) was add CAN portion wise below 20°C. The mixture was stirred at 15-20°C for 5h under N₂. Na₂S0₃ (370 mL) was added dropwise into the reaction mixture below 15°C, and then Na₂C0₃ (370 mL) was added. The mixture was filtered and the filtrate was extracted with CH₂C1₂

(100 mL*3). The organic layer was dried and concentrated to give the residue. The residue was purified by column chromatography to give the target compound (8a) as white solid. (Yield: 55%)

1H NMR (400 MHz, CHLOROFORM- ) δ ppm 1.45 (dd, J=7.53, 6.27 Hz, 6 H), 2.65 – 2.84 (m, 2 H), 3.98 (td, J=10.29, 4.77 Hz, 1 H), 4.27 (t, J=9.66 Hz, 1 H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz, 1 H), 4.49 – 4.61 (m, 1 H), 4.65 (td, J=7.78, 5.77 Hz, 1 H), 4.73 (d, J=7.78 Hz, 1 H), 4.87 (dq, J=12.74, 6.30 Hz, 1 H), 5.55 (br. s., 1 H), 5.82 (d, J=8.03 Hz, 1 H), 7.20 (d, J=8.03 Hz, 1 H), 8.78 (br. s., 1 H); ³¹P NMR (CHLOROFORM-^) δ ppm -7.13; LC-MS: 375 (M+l)+ Scheme 3

Synthesis of compound (VI)

Step 1: Synthesis of compound (9)Compound (1), CAS 1255860-33-3 ( 1200 mg, 4.33 mmol ) and l,8-bis(dimethyl- amino)naphthalene (3707 mg, 17.3 mmol) were dissolved in 24.3 mL of

trimethylphosphate. The solution was cooled to 0°C. Compound (5) (1.21 mL, 12.98 mmol) was added, and the mixture was stirred well maintaining the temperature at 0°C for 5 hours. The reaction was quenched by addition of 120 mL of tetraethyl- ammonium bromide solution (1M) and extracted with CH₂CI₂ (2×80 mL). Purification was done by preparative HPLC (Stationary phase: RP XBridge Prep CI 8 ΟΒϋ-10μιη, 30x150mm, mobile phase: 0.25% NH₄HCO₃ solution in water, CH₃CN) , yielding two fractions. The purest fraction was dissolved in water (15 mL) and passed through a manually packed Dowex (H⁺) column by elution with water. The end of the elution was determined by checking UV absorbance of eluting fractions. Combined fractions were frozen at -78°C and lyophilized. Compound (9) was obtained as a white fluffy solid (303 mg, (0.86 mmol, 20%> yield), which was used immediately in the following reaction. Step 2: Preparation of compound (VI)

Compound (9) (303 mg, 0.86 mmol) was dissolved in 8 mL water and to this solution was added N . N’- D ic y c ! he y !-4- mo rph line carboxamidine (253.8 mg, 0.86 mmol) dissolved in pyridine (8.4 mi.). The mixture was kept for 5 minutes and then

evaporated to dryness, dried overnight in vacuo overnight at 37°C. The residu was dissolved in pyridine (80 mL). This solution was added dropwise to vigorously stirred DCC (892.6 mg, 4.326 mmol) in pyridine (80 mL) at reflux temperature. The solution was kept refluxing for 1.5h during which some turbidity was observed in the solution. The reaction mixture was cooled and evaporated to dryness. Diethylether (50 mL) and water (50 mL) were added to the solid residu. N’N-dicyclohexylurea was filtered off, and the aqueous fraction was purified by preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-ΙΟμιη, 30x150mm, mobile phase: 0.25% NH₄HCO₃ solution in water, CH₃CN) , yielding a white solid which was dried overnight in vacuo at 38°C. (185 mg, 0.56 mmol, 65% yield). LC-MS: (M+H)⁺: 333.

1H NMR (400 MHz, DMSO-d₆) d ppm 2.44 – 2.59 (m, 2 H) signal falls under DMSO signal, 3.51 (td, J=9.90, 5.50 Hz, 1 H), 3.95 – 4.11 (m, 2 H), 4.16 (d, J=10.34 Hz, 1 H), 4.25 – 4.40 (m, 2 H), 5.65 (d, J=8.14 Hz, 1 H), 5.93 (br. s., 1 H), 7.46 (d, J=7.92 Hz, 1 H), 2H’s not observed

Paper

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.6b00382,

Discovery of 1-((2R,4aR,6R,7R,7aR)-2-Isopropoxy-2-oxidodihydro-4H,6H-spiro[furo[3,2-d][1,3,2]dioxaphosphinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1H,3H)-dione (JNJ-54257099), a 3′-5′-Cyclic Phosphate Ester Prodrug of 2′-Deoxy-2′-Spirooxetane Uridine Triphosphate Useful for HCV Inhibition

JNJ-54257099 (9) is a novel cyclic phosphate ester derivative that belongs to the class of 2′-deoxy-2′-spirooxetane uridine nucleotide prodrugs which are known as inhibitors of the HCV NS5B RNA-dependent RNA polymerase (RdRp). In the Huh-7 HCV genotype (GT) 1b replicon-containing cell line 9 is devoid of any anti-HCV activity, an observation attributable to inefficient prodrug metabolism which was found to be CYP3A4-dependent. In contrast, in vitro incubation of 9 in primary human hepatocytes as well as pharmacokinetic evaluation thereof in different preclinical species reveals the formation of substantial levels of 2′-deoxy-2′-spirooxetane uridine triphosphate (8), a potent inhibitor of the HCV NS5B polymerase. Overall, it was found that 9 displays a superior profile compared to its phosphoramidate prodrug analogues (e.g., 4) described previously. Of particular interest is the in vivo dose dependent reduction of HCV RNA observed in HCV infected (GT1a and GT3a) human hepatocyte chimeric mice after 7 days of oral administration of 9

////////////JNJ-54257099, 1491140-67-0, JNJ54257099, JNJ 54257099

O=C(C=C1)NC(N1[C@H]2[C@]3(OCC3)[C@H](O4)[C@@H](CO[P@@]4(OC(C)C)=O)O2)=O

3,5-Dibromo-N-(4,6-difluorobenzo[d]thiazol-2-yl)thiophene-2-carboxamide

New and novel anti-norovirus agents

There is an urgent need for structurally novel anti-norovirus agents. In this study, we describe the synthesis, anti-norovirus activity, and structure–activity relationship (SAR) of a series of heterocyclic carboxamide derivatives. Heterocyclic carboxamide 1 (50% effective concentration (EC₅₀)=37  µM) was identified by our screening campaign using the cytopathic effect reduction assay. Initial SAR studies suggested the importance of halogen substituents on the heterocyclic scaffold and identified 3,5-di-boromo-thiophene derivative 2j (EC₅₀=24 µM) and 4,6-di-fluoro-benzothiazole derivative 3j (EC₅₀=5.6 µM) as more potent inhibitors than 1. Moreover, their hybrid compound, 3,5-di-bromo-thiophen-4,6-di-fluoro-benzothiazole 4b, showed the most potent anti-norovirus activity with a EC₅₀ value of 0.53 µM (70-fold more potent than 1). Further investigation suggested that 4b might inhibit intracellular viral replication or the late stage of viral infection.

3,5-Dibromo-N-(4,6-difluorobenzo[d]thiazol-2-yl)thiophene-2-carboxamide (4b)

According to the same procedure used for 2f, starting from 3,5-dibromothiophene-2-carboxylic acid (286 mg, 1.00 mmol) and 4,6-difluorobenzo[d]thiazol-2-amine (204 mg, 1.10 mmol), 4b (270 mg, 60%) was obtained as white powder. mp: 245–246°C. ¹H-NMR (DMSO-d₆) δ: 7.43 (1H, dt, J=10.2, 2.0 Hz), 7.56 (1H, s), 7.83 (1H, dd, J=8.4, 2.0 Hz). ¹³C-NMR (DMSO-d₆) δ: 102.2 (dd, J=28.0, 23.1 Hz), 104.7 (dd, J=26.4, 3.3 Hz), 114.3, 118.4, 131.4 (d, J=7.4 Hz), 134.3 (d, J=10.7 Hz), 134.9, 135.2, 152.7 (d, J=241.2, 20.7 Hz), 158.3 (dd, J=242.2, 10.7 Hz), 159.0, 159.7. HPLC purity: >99%, ESI-MS m/z 453 [M+H]⁺.

Antiviral Activity and Cytotoxicity of Tetra-halogenated Hybrid Compounds

Compound	R6	R7	R8	EC50 (µM)a)	CC50 (µM)b)
4a	Cl	H	H	2.1	>100
4b	Br	H	Br	0.53	>100
4c	Cl	H	Cl	1.1	>100
4d	Cl	Cl	H	1.4	31

Discovery and Synthesis of Heterocyclic Carboxamide Derivatives as Potent Anti-norovirus Agents

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Methyl {(2S,3R)-1-[(2S)-2-{5-[(2R,5R)-1-{3,5-difluoro-4-[4-(4-fluorophenyl)piperidin-1-yl]phenyl}-5-(6-fluoro-2-{(2S)-1-[N-(methoxycarbonyl)-O-methyl-L-threonyl]pyrrolidin-2-yl}-1H-benzimidazol-5-yl)pyrrolidin-2-yl]-6-fluoro-1H-benzimidazol-2-yl}pyrrolidin-1-yl]-3-methoxy-1-oxobutan-2-yl}carbamate

Dimethyl N,N’-(((2R,5R)-1-(3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl)pyrrolidine-2,5-diyl)bis((6-fluoro-1H-benzimidazole-5,2-diyl)((2S)-pyrrolidine-2,1-diyl)((2S,3R)-3-methoxy-1-oxobutane-1,2-diyl)))biscarbamate

Methyl ((2S,3R)-1-((2S)-2-(5-((2R,5R)-1-(3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl)-5-(6-fluoro-2-((2S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl)pyrrolidin-2-yl)-1H-benzimidazol-5-yl)pyrrolidin-2-yl)-6-fluoro-1H-benzimidazol-2-yl)pyrrolidin-1-yl)-3-methoxy-1-oxobutan-2-yl)carbamate

MW 1113.1925 MW

Pibrentasvir

• UNII-2WU922TK3L
• CLINICAL https://clinicaltrials.gov/search/intervention=A-1325912.0%20OR%20ABT-530%20OR%20Pibrentasvir

Pibrentasvir

SYNTHESIS

PATENT

WO 2012051361

http://www.google.com/patents/WO2012051361A1?cl=en

Example 3.52 methyl {(2S,3R)-l-[(2S)-2-{5-[(2R,5R)-l-{3,5-difluoro-4-[4-(4- fluorophenyl)piperidin-l-yl]phenyl}-5-(6-fluoro-2-{(2.S)-l-[A^-(methoxycarbonyl)-0-methyl-L- threonyl]pyiTolidin-2-yl}-l f-benzimidazol-5-yl)pyiTolidin-2-yl]-6-fluoro-l f-benzimidaz yl}pyrrolidin-l-yl]-3-methoxy-l-oxobutan-2-yl}carbamatelH NMR (400 MHz, DMSO) δ 12.36 – 12.06 (m, 2H), 7.41 (dd, J = 11.2, 6.3, 1H), 7.34 (dd, J = 10.4, 4.8, 1H), 7.30 – 7.20 (m, 3H), 7.17 – 6.98 (m, 5H), 5.98 – 5.82 (m, 2H), 5.65 – 5.47 (m, 2H), 5.17 – 5.06 (m, 2H), 4.25 (dd, J = 15.6, 8.1, 2H), 3.88 – 3.74 (m, 3H), 3.53 (d, J = 1.3, 6H), 3.49 – 3.38 (m, 2H), 3.31 (d, 1H), 3.25 (d, J = 3.7, 1H), 3.13 (d, J = 1.3, 3H), 3.03 (d, J = 2.3, 3H), 3.00 – 2.84 (m, 3H), 2.60 – 2.53 (m, J = 2.5, 2H), 2.26 – 1.55 (m, 14H), 1.28 – 1.13 (m, 1H), 1.10 – 0.88 (m, 6H). MS (ESI; M+H) m/z = 1113.4.

PATENT

WO 2015171993

The present invention features crystalline polymorphs of methyl {(2S,3R)-1- [(2S)-2-{5-[(2R,5R)-l-{3,5-difluoro-4 4-(4-fluorophenyl)piperidin-l-yl]phenyl}-5-(6-fluoro-2-{(2S)- 1 -[N-(methoxycarbonyl)-0-methyl-L-threonyl]pyrrolidin-2-yl} – 1 H-benzimidazol-5-yl)pyrrolidin- -yl] -6-fluoro- 1 H-benzimidazol-2-yl} pyrrolidin- 1 -yl] -3 -methoxy- 1 -oxobutan-2-

yl} carbamate
, herein “Compound I”). Compound I is a potent HCV NS5A inhibitor and is described in U.S. Patent Application Publication No. 2012/0004196, which is incorporated herein by reference in its entirety.

//////////1353900-92-1, PHASE 3, ABT-530, Pibrentasvir, ABT 530, A 1325912.0

C[C@H]([C@@H](C(=O)N1CCC[C@H]1c2[nH]c3cc(c(cc3n2)[C@H]4CC[C@@H](N4c5cc(c(c(c5)F)N6CCC(CC6)c7ccc(cc7)F)F)c8cc9c(cc8F)[nH]c(n9)[C@@H]1CCCN1C(=O)[C@H]([C@@H](C)OC)NC(=O)OC)F)NC(=O)OC)OC

C[C@H]([C@@H](C(=O)N1CCC[C@H]1c2[nH]c3cc(c(cc3n2)[C@H]4CC[C@@H](N4c5cc(c(c(c5)F)N6CCC(CC6)c7ccc(cc7)F)F)c8cc9c(cc8F)[nH]c(n9)[C@@H]1CCCN1C(=O)[C@H]([C@@H](C)OC)NC(=O)OC)F)NC(=O)OC)OC

SYNTHESIS

NMR CDCL3 FROM NET

SEE……http://www.slideserve.com/truda/discovery-of-the-novel-orally-active-s1p-1-receptor-agonist-act-128800-ponesimod

Ponesimod (INN, codenamed ACT-128800) is an experimental drug for the treatment of multiple sclerosis (MS) and psoriasis. It is being developed by Actelion.

Clinical trials

In a 2009–2011 Phase II clinical trial including 464 MS patients, ponesimod treatment resulted in fewer new active brain lesions thanplacebo, measured during the course of 24 weeks.^[3][4]

In a 2010–2012 Phase II clinical trial including 326 patients with psoriasis, 46 or 48% of patients (depending on dosage) had a reduction of at least 75% Psoriasis Area and Severity Index (PASI) score compared to placebo in 16 weeks.^[3][5]

SEE https://clinicaltrials.gov/ct2/show/NCT02425644

Adverse effects

Common adverse effects in studies were temporary bradycardia (slow heartbeat), usually at the beginning of the treatment,dyspnoea (breathing difficulties), and increased liver enzymes (without symptoms). No significant increase of infections was observed under ponesimod therapy.^[3] QT prolongation is detectable but was considered to be too low to be of clinical importance in a study.^[6]

Mechanism of action

Like fingolimod, which is already approved for the treatment of MS, ponesimod blocks the sphingosine-1-phosphate receptor. This mechanism prevents lymphocytes (a type of white blood cells) from leaving lymph nodes.^[3] Ponesimod is selective for subtype 1 of this receptor, S1P₁.^[7]

PAPER

Bolli, Martin H.; Journal of Medicinal Chemistry 2010, V53(10), P4198-4211 CAPLUS

2-Imino-thiazolidin-4-one Derivatives as Potent, Orally Active S1P₁Receptor Agonists

Martin H. Bolli*, Stefan Abele, Christoph Binkert, Roberto Bravo, Stephan Buchmann, Daniel Bur, John Gatfield,Patrick Hess, Christopher Kohl, Céline Mangold, Boris Mathys, Katalin Menyhart, Claus Müller, Oliver Nayler,Michael Scherz, Gunther Schmidt, Virginie Sippel, Beat Steiner, Daniel Strasser, Alexander Treiber and Thomas Weller

Drug Discovery Chemistry, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland

J. Med. Chem., 2010, 53 (10), pp 4198–4211

DOI: 10.1021/jm100181s

Publication Date (Web): May 06, 2010

Copyright © 2010 American Chemical Society

*To whom correspondence should be addressed. Phone: + 41 61 565 65 70. Fax: + 41 61 565 65 00. E-mail:martin.bolli@actelion.com.

Sphingosine-1-phosphate (S1P) is a widespread lysophospholipid which displays a wealth of biological effects. Extracellular S1P conveys its activity through five specific G-protein coupled receptors numbered S1P₁ through S1P₅. Agonists of the S1P₁ receptor block the egress of T-lymphocytes from thymus and lymphoid organs and hold promise for the oral treatment of autoimmune disorders. Here, we report on the discovery and detailed structure−activity relationships of a novel class of S1P₁ receptor agonists based on the 2-imino-thiazolidin-4-one scaffold. Compound 8bo (ACT-128800) emerged from this series and is a potent, selective, and orally active S1P₁ receptor agonist selected for clinical development. In the rat, maximal reduction of circulating lymphocytes was reached at a dose of 3 mg/kg. The duration of lymphocyte sequestration was dose dependent. At a dose of 100 mg/kg, the effect on lymphocyte counts was fully reversible within less than 36 h. Pharmacokinetic investigation of8bo in beagle dogs suggests that the compound is suitable for once daily dosing in humans.

(Z,Z)-5-[3-Chloro-4-((2R)-2,3-dihydroxy-propoxy)-benzylidene]-2-propylimino-3-o-tolyl-thiazolidin-4-one (8bo)

…………..DELETED…………… column chromatography on silica gel eluting with heptane:ethyl acetate 1:4 to give the title compound (1.34 g, 37%) as a pale-yellow foam.

¹H NMR (CDCl₃): δ 0.94 (t, J = 7.3 Hz, 3 H), 1.58−1.70 (m, 2 H), 2.21 (s, 3 H), 3.32−3.48 (m, 2 H), 3.82−3.95 (m, 3 H), 4.12−4.27 (m, 4 H), 7.07 (d, J = 8.8 Hz, 1 H), 7.21 (d, J = 7.0 Hz, 1 H), 7.31−7.39 (m, 3 H), 7.49 (dd, J = 8.5, 2.0 Hz, 1 H), 7.64 (d, J= 2.0 Hz, 1 H), 7.69 (s, 1 H).

¹³C NMR (CDCl₃): δ 11.83, 17.68, 23.74, 55.42, 63.46, 69.85, 70.78, 133.48, 120.75, 123.71, 127.05, 128.25, 128.60, 129.43, 130.06, 131.13, 131.50, 134.42, 136.19, 146.98, 154.75, 166.12. LC-MS (ES⁺): t_R 0.96 min. m/z: 461 (M + H).

HPLC (ChiralPak AD-H, 4.6 mm × 250 mm, 0.8 mL/min, 70% hexane in ethanol): t_R 11.8 min. Anal. (C₂₃H₂₅N₂O₄SCl): C, H, N, O, S, Cl.

PATENT

WO 2014027330

https://www.google.com/patents/WO2014027330A1?cl=3Den

The present invention relates inter alia to a new process for the preparation of (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one (hereinafter also referred to as the “COMPOUND” or “compound (2)”), especially in crystalline form C which form is described in WO 2010/046835. The preparation of COMPOUND and its activity as immunosuppressive agent is described in WO 2005/054215. Furthermore, WO 2008/062376 describes a new process for the preparation of (2Z,5Z)-5-(3-chloro-4-hydroxy-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one which can be used as an intermediate in the preparation of COMPOUND.

Example 1 a) below describes such a process of preparing (2Z,5Z)-5-(3-chloro-4-hydroxy-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one according to WO 2008/062376. According to WO 2008/062376 the obtained (2Z,5Z)-5-(3-chloro-4-hydroxy-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one can then be transformed into COMPOUND by using standard methods for the alkylation of phenols. Such an alkylation is described in Example 1 b) below. Unfortunately, this process leads to the impurity (2Z,5Z)-5-(3-chloro-4-((1 ,3-dihydroxypropan-2-yl)oxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one which is present in about 2% w/w in the crude product (see Table 1 ) and up to 6 recrystallisations are necessary in order to get this impurity below 0.4% w/w (see Tables 1 and 2) which is the specified limit based on its toxicological qualification.

the obtained (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde (1 ) with 2-[(Z)-propylimino]-3-o-tolyl-thiazolidin-4-one to form (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one (2):

.

The reaction of (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde (1 ) with 2-[(Z)-propylimino]-3-o-tolyl-thiazolidin-4-one can be performed under conditions which are typical for a Knoevenagel condensation. Such conditions are described in the literature for example in Jones, G., Knoevenagel Condensation in Organic Reaction, Wiley: New York, 1967, Vol. 15, p 204; or Prout, F. S., Abdel-Latif, A. A., Kamal, M. R., J. Chem. Eng. Data, 2012, 57, 1881-1886.

2-[(Z)-Propylimino]-3-o-tolyl-thiazolidin-4-one can be prepared as described in WO 2008/062376, preferably without the isolation and/or purification of intermediates such as the thiourea intermediate that occurs after reacting o-tolyl-iso-thiocyanate with n-propylamine. Preferably 2-[(Z)-propylimino]-3-o-tolyl-thiazolidin-4-one obtained according to WO 2008/062376 is also not isolated and/or purified before performing the Knoevenagel condensation, i.e. before reacting 2-[(Z)-propylimino]-3-o-tolyl-thiazolidin-4-one with (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde (1 ), i.e. in a preferred embodiment compound (2) is prepared in a one-pot procedure analogous to that described in WO 2008/062376.

Example 1 : (2Z,5Z)-5-(3-Chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one

a) Preparation of (2Z,5Z)-5-(3-chloro-4-hydroxy-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one:

Acetic acid solution: To acetic acid (149.2 mL) are added sodium acetate (1 1 .1 1 g, 2.00 eq.) and 3-chloro-4-hydroxybenzaldehyde (10.60 g, 1.00 eq.) at 20 °C. The mixture is stirred at 20 °C until complete dissolution (2 to 3 h).

n-Propylamine (4.04 g, 1.00 eq.) is added to a solution of o-tolyl-iso-thiocyanate (10 g, 1.00 eq.) in dichloromethane (100 mL) at 20 °C. The resulting pale yellow solution is agitated for 40 min at 20 °C before IPC (conversion specification≥ 99.0 %). The reaction is cooled to -2 °C. Bromoacetyl bromide (13.53 g, 1.00 eq.) is added and the resulting solution is stirred for 15 min at -2 °C. Pyridine (10.92 g, 2.05 eq.) is then added slowly at -2 °C. The intensive yellow reaction mixture is stirred for 15 min at -2 °C before IPC (conversion specification≥ 93.0 %). 70 mL of dichloromethane are distilled off under atmospheric pressure and jacket temperature of 60 °C. The temperature is adjusted to 42 °C and the acetic acid solution is added to the reaction mixture. The resulting solution is heated to 58 °C and stirred at this temperature for 15 h before IPC (conversion specification≥ 95 %). 25 mL of solvents are distilled off under vacuum 900 – 500 mbars and jacket temperature of 80 °C. The temperature is adjusted to 60 °C and water (80.1 mL) is added to the reaction mixture over 1 h. The resulting yellow suspension is stirred at 60 °C for 30 min. The suspension is cooled to 20 °C over 1 h and stirred at this temperature for 30 min.

The product is filtered and washed with a mixture of acetic acid (30 mL) and water (16 mL) and with water (50 mL) at 20 °C. The product is dried under vacuum at 50 °C for 40 h to afford a pale yellow solid; yield 25.93 g (78 %).

b) Preparation of crude (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one:

To a suspension of (2Z,5Z)-5-(3-chloro-4-hydroxy-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one (10.00 g, 1.00 eq.) in ethanol (47.2 mL) is added (R)-3-chloro-1 ,2-

propanediol (3.37 g, 1.18 eq.) at 20 °C. Potassium tert-butoxide (3.39 g, 1.13 eq.) is added in portions at 20 °C. The resulting fine suspension is stirred at 20 °C for 25 min before being heated to reflux (88 °C). The reaction mixture is stirred at this temperature for 24 h before IPC (conversion specification≥ 96.0 %). After cooling down to 60 °C, acetonitrile (28.6 mL) and water (74.9 mL) are added. The resulting clear solution is cooled from 60 °C to 0 °C over 2 h. During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one seeds of crystalline form C (0.010 g, 0.001 eq.; crystalline form C can be prepared as described in WO 2010/046835) are added at 50 °C. The suspension is heated from 0 °C to 50 °C, cooled to 0 °C over 6 h and stirred at this temperature for 12 h.

The product is filtered and washed with a mixture of acetonitrile (23.4 mL) and water (23.4 mL) at 0 °C. The product is dried under vacuum at 45 °C for 24 h to afford a pale yellow solid; yield 1 1.91 g (84 %).

c) Purification of (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one:

Recrystallisation I: The crude (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one (10 g) is dissolved in acetonitrile (30 mL) at 70 °C. The reaction mixture is cooled from 70 °C to 0 °C over 2 h. During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one seeds of crystalline form C (0.0075 g, 0.00075 eq.) are added at 50 °C. The suspension is heated up to 52 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h. The product is filtered and washed with acetonitrile at -10 °C (2 x 12.8 mL).

Recrystallisation II: The wet product is dissolved in acetonitrile (27.0 mL) at 70 °C. The reaction mixture is cooled from 70 °C to 0 °C over 2 h. During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one seeds of crystalline form C (0.0075 g, 0.00075 eq.) are added at 50 °C. The suspension is heated up to 52 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h. The product is filtered and washed with acetonitrile at -10 °C (2 x 1 1.3 mL).

Recrystallisation III: The wet product is dissolved in acetonitrile (24.3 mL) at 70 °C. The reaction mixture is cooled from 70 °C to 0 °C over 2 h. During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4- one seeds of crystalline form C (0.0075 g, 0.00075 eq.) are added at 50 °C. The suspension is heated up to 52 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h. The product is filtered and washed with acetonitrile at -10 °C (2 x 10.1 mL).

Recrystallisation IV: The wet product is dissolved in acetonitrile (21.9 mL) at 70 °C. The reaction mixture is cooled from 70 °C to 0 °C over 2 h. During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one seeds of crystalline form C (0.0075 g, 0.00075 eq.) are added at 50 °C. The suspension is heated up to 52 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h. The product is filtered and washed with acetonitrile at -10 °C (2 x 9.1 mL).

Recrystallisation V: The wet product is dissolved in acetonitrile (19.7 mL) at 70 °C. The reaction mixture is cooled from 70 °C to 0 °C over 2 h. During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one seeds of crystalline form C (0.0075 g, 0.00075 eq.) are added at 50 °C. The suspension is heated up to 52 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h. The product is filtered and washed with acetonitrile at -10 °C (2 x 8.2 mL).

Recrystallisation VI: The wet product is dissolved in acetonitrile (23.9 mL) at 70 °C. Water (20 mL) is added at 70 °C. The reaction mixture is cooled from 70 °C to 0 °C over 2 h.

During the cooling ramp, (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2- (propylimino)-3-(o-tolyl)thiazolidin-4-one seeds of crystalline form C (0.0075 g, 0.00075 eq.) are added at 50 °C. The suspension is heated up to 52 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h. The product is filtered and washed twice with a mixture of acetonitrile (4.5 mL) and water (4.5 mL) at -10 °C.

The product is dried under vacuum at 45 °C for 24 h to afford a pale yellow solid; yield: 7.0 g (70 %).

Example 2: (R)-3-Chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde

Potassium tert-butoxide (1 18 g, 1.20 eq.) is added to n-propanol (963 mL) followed by 3-chloro-4-hydroxybenzaldehyde (137 g, 1.00 eq.). To the mixture is added (R)-3-chloro-1 ,2-propanediol (126 g, 1.30 eq.). The suspension is heated to 90 °C and stirred at this temperature for 17 h. Solvent (500 mL) is distilled off at 120 °C external temperature and reduced pressure. Water is added (1.1 L) and solvent (500 mL) is removed by distillation. The turbid solution is cooled to 20 °C. After stirring for one hour a white suspension is obtained. Water (500 mL) is added and the suspension is cooled to 10 °C. The suspension is filtered and the resulting filter cake is washed with water (500 mL). The product is dried at 50 °C and reduced pressure to yield 149 g of a white solid (73%), which is (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde in crystalline form A.

Example 3: (R)-3-Chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde

Potassium tert-butoxide (8.60 g, 1.20 eq.) is added to n-propanol (70 mL) below 15 °C, the temperature is allowed to rise. After the addition the temperature is corrected again to below 15 °C before addition of 3-chloro-4-hydroxybenzaldehyde (10 g, 1 .00 eq.). The suspension is heated to 40 °C and stirred for 30 min. (R)-3-Chloro-1 ,2-propanediol (9.18 g, 1.30 eq.) is added at 40 °C. The resulting suspension is heated to 60 °C and stirred at this temperature for 15 h then heated to 94 °C till meeting the IPC-specification (specification conversion≥ 90.0 %). The mixture is cooled to 30 °C and n-propanol is partially distilled off (-50 mL are distilled off) under reduced pressure and a maximum temperature of 50 °C, the jacket temperature is not allowed to raise above 60 °C.

Water (81 mL) is added and a second distillation is performed under the same conditions (24 mL are distilled off). The mixture is heated till homogeneous (maximum 54 °C) and then cooled to 24 °C. At 24 °C the mixture is seeded with crystalline (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde of form A (0.013 g, 0.00085 eq.). How to obtain the crystalline seeds is described in Examples 2 and 5. The reaction mixture is cooled to 0 °C over 7.5 h.

The product is filtered and washed with water (2 x 35 mL) and once with methyl tert-butyl ether (20 mL) at 5 °C. The product is dried under vacuum at 40 °C for 20 h to afford an off-white solid; yield: 10.6 g (72 %), which is (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde in crystalline form A.

Example 4: (2Z,5Z)-5-(3-Chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)- 3-(o-tolyl)thiazolidin-4-one

a) Preparation of crude (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one:

n-Propylamine (5.23 g, 1.32 eq.) is added to a solution of o-tolyl-iso-thiocyanate (10 g, 1.00 eq.) in dichloromethane (100 mL) at 20 °C. The resulting pale yellow solution is agitated for 15 min at 20 °C before IPC (conversion specification≥ 99.0 %). The reaction is cooled to -2 °C. Bromoacetyl bromide (14.88 g, 1.10 eq.) is added and the resulting solution is stirred for 15 min at -2 °C. Pyridine (10.92 g, 2.05 eq.) is then added slowly at -2 °C. The intensive yellow reaction mixture is stirred for 15 min at -2 °C before IPC (conversion specification≥ 93.0 %). Dichloromethane is partially distilled off (66 mL are distilled off) under atmospheric pressure and jacket temperature of 60 °C. Ethanol (1 1 1.4 mL), sodium acetate (12.75 g, 2.30 eq.) and (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde from Example 3 (14.38 g, 0.93 eq.) are added. The remaining dichloromethane and a part of ethanol are distilled off (49.50 mL are distilled off) under atmospheric pressure and jacket temperature up to 85 °C. The reaction mixture (orange suspension) is stirred for 3 – 5 h under reflux (78 °C) before IPC (conversion specification≥ 97.0 %).

Water (88.83 mL) is added and the temperature adjusted to 40 °C before seeding with micronized (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one in crystalline form C (0.075 g, 0.0024 eq.). The reaction mixture is cooled to 0 °C over 5 h, heated up to 40 °C, cooled to 0 °C over 6 h and stirred at this temperature for 2 h.

The product is filtered and washed with a 1 :1 ethanohwater mixture (2 x 48 mL) at 0 °C. The product is dried under vacuum at 45 °C for 10 h to afford a pale yellow solid; yield: 24.71 g (86 %).

b) Purification of (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one:

The crude (2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one (10 g) is dissolved in ethanol (40 mL) at 70 °C. The temperature is adjusted at 50 °C for seeding with micronised (2Z,5Z)-5-(3-chloro-4-((R)-2,3- dihydroxypropoxy)benzylidene)-2-(propylimino)-3-(o-tolyl)thiazolidin-4-one in crystalline form C (0.016 g, 0.0016 eq.). The reaction mixture is cooled from 50 °C to 0 °C over 4 h, heated up to 50 °C, cooled to 0 °C over 6 h and agitated at this temperature for 2 h.

The product is filtered and washed with ethanol at 0 °C (2 x 12.8 mL). The product is dried under vacuum at 45 °C for 10 h to afford a pale yellow solid; yield: 9.2 g (92 %).

Example 5: Preparation of crystalline seeds of (R)-3-chloro-4-(2,3-dihydroxypropoxy)- benzaldehyde

10 mg of (R)-3-chloro-4-(2,3-dihydroxypropoxy)-benzaldehyde of at least 99.5% purity by 1 H-NMR assay is dissolved in a 4 mL vial by adding 1 mL of pure ethanol (puriss p. a.). The solvent is allowed to evaporate through a small hole in the cap (approx. 2 mm of diameter) of the vial until complete dryness. The white solid residue is crystalline (R)-3-chloro-4-(2,3- dihydroxypropoxy)-benzaldehyde in crystalline form A. Alternatively, methanol or methylisobutylketone (both in puriss p. a. quality) is used. This procedure is repeated until sufficient seeds are made available.

PATENT

WO 2005054215

SEE https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2005054215

References

ABOUT PONESIMOD

CURRENT STATUS

AVAILABLE CLINICAL DATA

MILESTONES

KEY SCIENTIFIC LITERATURE

Ponesimod

Systematic (IUPAC) name
(2Z,5Z)-5-{3-Chloro-4-[(2R)-2,3-dihydroxypropoxy]benzylidene}-3-(2-methylphenyl)-2-(propylimino)-1,3-thiazolidin-4-one
Clinical data
Routes of administration	Oral
Legal status
Legal status	Investigational
Pharmacokinetic data
Metabolism	2 main metabolites
Biological half-life	31–34 hrs[1]
Excretion	Feces (57–80%, 26% unchanged), urine (10–18%)[2]
Identifiers
CAS Number	854107-55-4
ATC code	none
PubChem	CID 11363176
ChemSpider	9538103
ChEMBL	CHEMBL1096146
Synonyms	ACT-128800
Chemical data
Formula	C23H25ClN2O4S
Molar mass	460.974 g/mol

////Ponesimod, Phase III , A sphingosine-1-phosphate receptor 1, S1P1 agonist, multiple sclerosis.  ACT-128800; RG-3477; R-3477, autoimmune disease, lymphocyte migration, multiple sclerosis, psoriasis, transplantation

CCC/N=C\1/N(C(=O)/C(=C/C2=CC(=C(C=C2)OC[C@@H](CO)O)Cl)/S1)C3=CC=CC=C3C

ND 630, NDI 010976,  ND-630, NDI-010976

1,4-dihydro-1-[(2R)-2-(2-methoxyphenyl)-2-[(tetrahydro-2H-pyran-4-yl)oxy]ethyl]-α,α,5-trimethyl-6-(2-oxazolyl)-2,4-dioxo-thieno[2,3-d]pyrimidine-3(2H)-acetic acid

2-[l-[2-(2-methoxyphenyl)-2-(oxan-4-yloxy)ethyl]-5- methyl-6-(l,3-oxazol-2-yl)-2,4-dioxo-lH,2H,3H,4H-thieno[2,3-d]pyrimidin-3-yl]-2- methylpropanoic acid

2-[1-[(2R)-2-(2-methoxyphenyl)-2-(oxan-4-yloxy)ethyl]-5-methyl-6-(1,3-oxazol-2-yl)-2,4-dioxothieno[2,3-d]pyrimidin-3-yl]-2-methylpropanoic acid

CAS 1434635-54-7

Thieno[2,3-d]pyrimidine-3(2H)-acetic acid, 1,4-dihydro-1-[(2R)-2-(2-methoxyphenyl)-2-[(tetrahydro-2H-pyran-4-yl)oxy]ethyl]-α,α,5-trimethyl-6-(2-oxazolyl)-2,4-dioxo-

Molecular Formula:	C28H31N3O8S
Molecular Weight:	569.62604 g/mol

Company	Nimbus Therapeutics LLC
Description	Small molecule allosteric inhibitor of acetyl-coenzyme A carboxylase alpha (ACACA; ACC1) and acetyl-coenzyme A carboxylase beta (ACACB; ACC2)
Molecular Target	Acetyl-Coenzyme A carboxylase alpha (ACACA) (ACC1) ; Acetyl-Coenzyme A carboxylase beta (ACACB) (ACC2)
Mechanism of Action	Acetyl-coenzyme A carboxylase alpha (ACACA) (ACC1) inhibitor; Acetyl-coenzyme A carboxylase beta (ACACB) (ACC2) inhibitor
Therapeutic Modality	Small molecule

Preclinical Diabetes mellitus; Hepatocellular carcinoma; Metabolic syndrome; Non-alcoholic steatohepatitis; Non-small cell lung cancer

Acetyl CoA carboxylase 1/2 allosteric inhibitors – Nimbus Therapeutics

The Liver Meeting 2015 – American Association for the Study of Liver Diseases (AASLD) – 2015 Annual Meeting, San Francisco, CA, USA

Nimbus compounds targeting liver disease in rat models

Data were presented by Geraldine Harriman, from Nimbus Therapeutics, from rat models using acetyl-CoA carboxylase (ACC) inhibitors NDI-010976 (ND-630) and N-654, which improved metabolic syndrome endpoints, decreased liver steatosis, decreased expression of inflammatory markers and improved fibrosis. The hepatotropic ACC inhibitor NDI-010976 had IC50 values of 2 and 7 nM for ACC1 and 2, respectively, EC50 values in HepG2 serum free and 10% serum of 9 and 66 nM, respectively, and 2-fold C2C12 fatty acid oxidation (FAOxn) stimulation at 200 nM. Rat FASyn (synthase), malonyl-CoA (liver) and malonyl-COA (muscle) respective ED50 values were 0.14 mg/kg po, 0.8 and 3 mg/kg. The rat respiratory quotient (RQ) MED was 3 mg/kg po. ADME data showed low multispecies intrinsic clearance (human, mouse, rat, dog, monkey). NDI-010976 was eliminated predominantly as the parent drug. Additionally, P450 inhibition was > 50 microM. In liver and muscle, NDI-010976 modulated key metabolic parameters including a dose-dependent reduction in the formation of the enzymatic product of acetyl coA carboxyloase malonyl coA; the ED50 value was lower in muscle. The drug also decreased FASyn dose dependently and increased fatty acid oxidation in the liver (EC50 = 0.14 mg/kg). In 28-day HS DIO rats, NDI-010976 favorably modulated key plasma and liver lipids, including decreasing liver free fatty acid, plasma triglycerides and plasma cholesterol; this effect was also seen in 37-day ZDF rats

 PATENT

http://www.google.com/patents/WO2013071169A1?cl=en

Example 76: Synthesis of 2-[l-[2-(2-methoxyphenyl)-2-(oxan-4-yloxy)ethyl]-5- methyl-6-(l,3-oxazol-2-yl)-2,4-dioxo-lH,2H,3H,4H-thieno[2,3-d]pyrimidin-3-yl]-2- methylpropanoic acid (1-181).

Synthesis of compound 76.1. Into a 250-mL 3 -necked round-bottom flask, purged and maintained with an inert atmosphere of nitrogen, was placed oxan-4-ol (86 g, 842.05 mmol, 2.01 equiv) and FeCl₃ (10 g). This was followed by the addition of 57.2 (63 g, 419.51 mmol, 1.00 equiv) dropwise with stirring at 0 °C. The resulting solution was stirred for 3 h at room temperature. The resulting solution was diluted with 500 mL of H₂0. The resulting solution was extracted with 3×1000 mL of ethyl acetate and the organic layers combined. The resulting solution was extracted with 3×300 mL of sodium chloride (sat.) and the organic layers combined and dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1 : 10). This resulted in 22 g (21%) of 76.1 as a white solid.

Synthesis of compound 76.2. The enantiomers of 76.1 (22g) were resolved by chiral preparative HPLC under the following conditions (Gilson Gx 281): Column: Venusil Chiral OD-

H, 21.1 *25 cm, 5 μιη; mobile phase: hexanes (0.2% TEA) and ethanol (0.2% TEA) (hold at 10% ethanol (0.2%TEA) for 13 min); detector: UV 220/254 nm. 11.4 g (52%) of 76.2 were obtained as a white solid.

Synthesis of compound 76.3. Into a 500-mL 3-necked round-bottom flask, purged and maintained with an inert atmosphere of nitrogen, was placed 70.1 (12 g, 20.49 mmol, 1.00 equiv), tetrahydrofuran (200 mL), 76.2 (6.2 g, 24.57 mmol, 1.20 equiv) and DIAD (6.5 g, 32.18 mmol, 1.57 equiv). This was followed by the addition of a solution of triphenylphosphane (8.4 g, 32.03 mmol, 1.56 equiv) in tetrahydrofuran (100 mL) dropwise with stirring at 0 °C in 60 min. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1 :5). This resulted in 17 g (crude) of 76.3 as a white solid.

Synthesis of compound 76.4. Into a 500-mL 3-necked round-bottom flask, purged and maintained with an inert atmosphere of nitrogen, was placed 76.3 (17 g, crude), toluene (300 mL), Pd(PPh₃)₄ (1.7 g, 1.47 mmol, 0.07 equiv) and 2-(tributylstannyl)-l,3-oxazole (8.6 g, 24.02 mmol, 1.16 equiv). The resulting solution was stirred overnight at 110 °C. The resulting mixture was concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1 : 10). Purification afforded 6 g of 76.4 as a white solid.

Synthesis of compound 1-181. Into a 250-mL 3-necked round-bottom flask, was placed 76.4 (6 g, 7.43 mmol, 1.00 equiv), tetrahydrofuran (100 mL), TBAF (2.3 g, 8.80 mmol,

I .18 equiv). The resulting solution was stirred for 1 h at room temperature. The resulting mixture was concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (50: 1). This resulted in 3.4 g (80%) of Compound 1-181 as a white solid.

Purification: MS (ES): m/z 570 (M+H)⁺, 592 (M+Na)⁺.

1H NMR (300 MHz, DMSO- d₆): δ 1.22-1.36 (m, 2H), 1.62 (m, 8H), 2.75 (s, 3H), 3.20-3.39 (m, 3H), 3.48-3.58 (m, 2H), 3.80 (s, 3H), 3.85-4.20 (m, 2H), 5.30 (m, 1H), 7.03 (m, 2H), 7.33-7.50 (m, 3H), 8.2 (s, 1H).

WO-2014182951 

WO-2014182950 

/////// ND 630, NDI 010976,  ND-630, NDI-010976, NIMBUS, GILEAD, 1434635-54-7

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