Identification of a Nonbasic Melanin Hormone Receptor 1 Antagonist

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Identification of a non-basic melanin hormone receptor 1 antagonist as an anti-obesity clinical candidate William N. Washburn, Mark Manfredi, Pratik Devasthale, Guohua Zhao, Saleem Ahmad, Andres Hernandez, Jeffrey A. Robl, Wei Wang, James Mignone, Zhenghua Wang, Khehyong Ngu, Mary Ann pelleymounter, Donald Longhi, Rulin Zhao, Bei Wang, Ning Huang, Neil Flynn, Anthony Azzara, Joel C. Barrish, Kenneth Rohrbach, James Devenny, Suzanne Rooney, Michael Michael Thomas, Susan Glick, Helen Godonis, Susan Harvey, Mary Jane Cullen, Hongwei Zhang, Christian Caporuscio, Paul Stetsko, Mary F Grubb, Brad Douglas Maxwell, Hong Yang, Atsu Apedo, Brian Gemzik, Evan Janovitch, Christine Huang, Lisa Zhang, Chris Freeden, and Brian J. Murphy J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500026w • Publication Date (Web): 28 Aug 2014 Downloaded from http://pubs.acs.org on August 31, 2014

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Journal of Medicinal Chemistry

Biology Godonis, Helen; Bristol-Myers Squibb Pharm Res Inst, Metabolic Diseases Biology Harvey, Susan; Bristol-Myers Squibb Pharm Res Inst, Metabolic Diseases Biology Cullen, Mary Jane; Bristol-Myers Squibb Pharm Res Inst, Metabolic Diseases Biology Zhang, Hongwei; Bristol-Myers Squibb Pharm Res Inst, PCO Discovery Bioanalytical Research Caporuscio, Christian; Bristol-Myers Squibb Pharm Res Inst, PCO Discovery Bioanalytical Research Stetsko, Paul; Bristol-Myers Squibb Pharm Res Inst, PCO Pharmaceutics Grubb, Mary; Bristol-Myers Squibb, Maxwell, Brad; Bristol-Myers Squibb, Yang, Hong; Pharmaceutical Research Institute, Bristol-Myers Squibb Apedo, Atsu; Bristol-Myers Squibb Pharm Res Inst, PCO Discovery Bioanalytical Research Gemzik, Brian; Pharmaceutical Research Institute, Bristol-Myers Squibb Janovitch, Evan; Bristol-Myers Squibb Pharm Res Inst, PCO Discovery Toxicology Huang, Christine; Research Institute, Bristol-Myers Squibb Pharmaceutical Zhang, Lisa; Research Institute, Bristol-Myers Squibb Pharmaceutical Freeden, Chris; Research Institute, Bristol-Myers Squibb Pharmaceutical Murphy, Brian; Bristol-Myers Squibb Pharm Res Inst, Metabolic Diseases Biology

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Identification of a non-basic melanin hormone receptor 1 antagonist as an anti-obesity clinical candidate William N. Washburn#*, Mark Manfredi#, Pratik Devasthale#, Guohua Zhao#, Saleem Ahmad#, Andres Hernandez#, Jeffrey A. Robl#, Wei Wang#, James Mignone#, Zhenghua Wang#, Khehyong Ngu#, Mary Ann Pelleymounter+, Daniel Longhi+, Rulin Zhao$, Bei Wang $, Ning Huang+, Neil Flynn+, Anthony V. Azzara+, Joel C. Barrish#, Kenneth Rohrbach+, James J. Devenny+, Suzanne Rooney+, Michael Thomas+, Susan Glick+, Helen E. Godonis+, Susan J. Harvey+, Mary Jane Cullen+, Hongwei Zhangε, Christian Caporuscioε, Paul Stetsko∞, Mary Grubb↓, Brad D. Maxwell$, Hong Yang$, Atsu Apedo>, Brian Gemzik , Evan Janovitch β, Christine Huang°, Lisa Zhang°, Chris Freeden°, and Brian J. Murphy+

β

Research and Development, Bristol-Myers Squibb Co., Princeton, NJ, USA * Address correspondence to [email protected]; 609-737-7451 #

Metabolic Diseases Chemistry,

+ Metabolic Diseases Biology, °Preclinical Candidate Optimization Metabolism and Pharmacokinetics $

Discovery Chemistry Synthesis

β

Preclinical Candidate Optimization Discovery Toxicology,

ε

Preclinical Candidate Optimization Discovery Bioanalytical Research,

↓

Preclinical Candidate Optimization Biotransformation,

∞

Preclinical Candidate Optimization Pharmaceutics,

>

Preclinical Candidate Optimization DAS SPS

Abstract 1 ACS Paragon Plus Environment

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Identification of MCHR1 antagonists with a preclinical safety profile to support clinical evaluation as anti-obesity agents has been a challenge. Our finding that a basic moiety is not required for MCHR1 antagonists to achieve high-affinity allowed us to explore structures less prone to off-target activities such as hERG inhibition. We report the SAR evolution of hydroxylated thienopyrimidinone ethers culminating in the identification of 27 (BMS-819881) which entered obesity clinical trials as the phosphate ester pro-drug 35 (BMS-830216).

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Introduction The ever increasing worldwide incidence of obesity and the attendant health costs have prompted renewed efforts by the pharmaceutical industry to focus on anti-obesity agents.1 These efforts comprise three different approaches: anorectics typically requiring CNS targeting, metabolic enhancers directed toward peripheral targets, and nutrient absorption modulators associated with GI targets.2 Each approach poses its own challenges. Agents that reduce absorption of specific nutrients achieve most of their effect by encouraging diet modification to minimize the probability of gastric disturbances.3 Metabolic elevation of cardiovascularly compromised patients has proved difficult to achieve safely due to the increased cardiac output required to support the elevated cellular metabolism.4 In principle, anorectic agents represent the most direct weight loss approach; however, modulation of certain CNS pathways controlling caloric intake may also activate brain circuits resulting unacceptable side effects as was most recently shown by the cannabanoid inverse agonists.5 The melanin concentrating hormone (MCH) pathway is one of a variety of neurological circuits reported to modulate caloric consumption. The natural ligand is a 19 amino acid cyclic peptide conserved across all mammalian species. 6,7 Predominant MCH peptide expression is limited to magnocellular neurons in the lateral hypothalamus and zona incerta. These neurons project to many areas of the brain including cortex, amygdala, dorsal and ventral striatum, olfactory tubercle, nucleus accumbens, as well as nuclei located in the hindbrain. In humans and most other species, MCH binds to two Class A GPCRs termed MCHR1 and MCHR2 for which the sequence identity is 38%. MCHR1 is primarily expressed in the brain at moderate levels in many areas including cortex, olfactory tubercle, hippocampus, outer shell of the nucleus accumbens, and amygdala. MCHR1 is also expressed in the arcuate and ventromedial portion of the hypothalamus, implicating a role for the receptor in feeding. The overall expression pattern of MCHR2 in the brain is 3 ACS Paragon Plus Environment

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similar to that of MCHR1. MCHR2 is expressed in higher mammals such as dog, monkey and primates; however, it is not expressed in rodents such as mouse and rat. At present, the physiological role of MCHR2 in humans is not known. The in vivo and transgenic studies establishing that MCHR1 was a promising anti-obesity target have been extensively reviewed; selected salient studies are briefly summarized herein.6,7 The presence of upregulated MCH mRNA in the hypothalamus of the genetically obese, leptin-deficient ob/ob mouse provided the first evidence that MCH may be an orexigenic peptide exerting its effects at the level of the hypothalamus. Intracerebroventicular (i.c.v.) injection of MCH increased acute food intake of rats; furthermore, chronic infusion of MCH into the lateral ventricle of mice resulted in significantly increased food intake, body weight, and white adipose tissue mass. Nutritional status modulates MCH peptide mRNA levels; levels were up-regulated after food deprivation in lean rats and were elevated in the hypothalami of Diet-Induced Obese (DIO) rats. Transgenic animals overexpressing MCH mRNA in the lateral hypothalamus were more susceptible to obesity when fed a high fat diet; whereas, knockout mice lacking MCH peptide were hypophagic and lean compared to wild-type littermates. Since MCH knockout mice showed an increase in metabolism, weight loss observed in the MCH knockout mice is attributed to a combination of both hypophagia and increased metabolism. MCHR1 -/- mice are lean when fed either a normal chow or high fat diet when compared to wild-type littermates. In summary, all studies with MCH peptide and both the MCHR1 and MCH peptide knockout mice are consistent with MCH playing a role in energy homeostasis, thereby establishing the potential utility of a MCHR1 antagonist for use in obesity. Pharmaceutical pursuit of MCHR1 antagonists began in earnest around 2002 with the disclosure of T226296 (1).8,9 These efforts have been guided by prior agonist SAR studies that revealed Arg 11 of the MCH peptide was essential for agonist activity due to presumed formation of a salt bridge with Asp123 4 ACS Paragon Plus Environment


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of the MCHR1 receptor.10, 11 By 2005, when BMS initiated a MCHR1 antagonist program, a number of patent applications had published disclosing a variety of high affinity MCHR1-selective competitive antagonists exemplified by 1, 2, 3, and 4. 12-14 These disclosures spanning a diverse set of lipophilic amines have been summarized in a number of reviews.15-18 Perusal of these structures, best described as a set of hydrophobic modular assemblages each containing a basic site, suggests that the MCHR1 receptor possesses a very accommodative binding site(s). Since the structural features of these chemotypes are highly conducive to hERG inhibition, removal of hERG activity from these active pharmacophores proved to be a formidable medicinal chemistry challenge.19-23 Despite the disclosures of > 200 patents for potent MCHR1 antagonists, only a few candidates including entries from GSK (GW856464), Amgen (AMG-076), Neurogen (NGD-4715), AMRI-Technology (ALB-127158) have entered clinical trials; but none progressed beyond Phase 1.15 Identification of compounds that could be safely progressed to clinical trials continues to be exceedingly challenging.24-27 Herein we report a finding that enabled identification of MCHR1 antagonists with minimal hERG activity. Figure 1: Representative MCHR1 antagonists and amide counterparts


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NMe2

O N H F

1 (T-226296) MCHR1 Ki 4.7 ± 1.5 nM; n = 4 hERG EC50 = 1.4 M

Cl

O

S

O

N OMe

N

Cl

O

S

6 MCHR1 Ki 9.6 ± 1.3 nM; n = 4 hERG EC50 > 80 M

O

N

N

OMe

N

2 (GW 803430) MCHR1 Ki 3.8 ± 2.4 nM; n = 15 hERG EC50 < 1 M

CF3O

H N O

O

N

N

Cl

O N H

N

O

S

O

N OMe

N

O N

7 MCHR1 Ki 16.4 ± 2.9 nM; n = 7 hERG EC50 > 80 M CF3O

N

3 MCHR1 Ki 3.6 ± 0.2 nM; n = 7 hERG EC50 = 6 M O

O N

H N O

O

N

N

O N

8 MCHR1 Ki 6.3 ± 1.3nM; n = 3 hERG EC50 = 7 M NN

N

S

N H

S

t-Bu OH t-Bu

Cl Cl

4 MCHR1 Ki 21.2 ± 4.1 nM; n = 5

5 MCHR1 Ki 27.3 ± 3.7 nM; n = 7 hERG EC50 > 80 M

Results and Discussion Screening the BMS chemical library revealed compound 5 (MCHR1 Ki 27 nM). Surprisingly inhibition due to 5 was not irreversible as would be anticipated if conversion of 5 to a long-lived bis 2,6-di-tbutylphenolic radical had promoted oxidative inactivation of MCHR1. Rather 5 appeared to be either a competitive or non-competitive inhibitor despite the fact that 5 lacked the structural elements associated with MCHR1 antagonists. However, our interest in this lead diminished once SAR elucidation revealed that the bis-t-butyl phenol component, which was essential for in vitro activity, precluded adequate CNS exposure to achieve in vivo efficacy. 6 ACS Paragon Plus Environment


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Spurred by the finding of MCHR1 antagonist activity of 5, we explicitly tested the assumption that the basic amine was an essential element for high affinity MCHR antagonists by ablating the basic amine of selected potent MCHR antagonists while maintaining all other structural elements intact. Preparation and characterization of the amide counterparts 6 and 7 of amine 2 revealed only a 3-4 fold decrease in affinity; moreover, 8 the amide counterpart of 3 exhibited only a 2 fold decrease in affinity findings inconsistent with the consensus conception regarding formation of a critical salt bridge between the antagonist and the MCHR1 receptor. This structural change greatly reduced aqueous solubility and dramatically attenuated affinities for hERG as well as biogenic amine receptors. For example, the hERG electrophysiological IC50 of 2 was < 1 µM; whereas 6 induced only 20% inhibition at 10 µM. When screened against 30 biogenic amine receptors and ion channels, 2 exhibited > 50 % inhibition in seven assays at 1 µM whereas an amide counterpart exhibited < 50% activity at 10 µM. Encouraged by these findings we pursued MCHR antagonists lacking basic amines by replacing the pyrrolidine moiety of thienopyrimidinone 2 with a variety of non-basic functionalities exemplified by examples 9-19. As shown in Table 1, all were potent MCHR1 antagonists; however, aqueous solubility was < 1 µg/ mL. Following oral administration as a 1% methyl cellulose/0.5% Tween 80 suspension to rats, three distinct groupings emerged based on AUC and brain/plasma ratio. Very low oral exposures were obtained with the urea 9, tertiary carbamate 10 or amide 11 or a carboxamide 12. Modest peripheral exposures were obtained with the sulfonamide 13 or secondary amide 14 as a polar group; whereas incorporation of a secondary carbamate 15 increased AUC values significantly. However, in all cases, essentially no CNS exposure was achieved with 9-15. Although alkyl ethers exhibited favorable brain/plasma ratios, exposures were unacceptably low for non-hydroxylated ethers such as 16 and marginal at 10 mg/kg for hydroxylated ethers 17-19. Table1: Comparison of MCHR1 affinities, PK profile and tissue distribution


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ID

R

Plasma

Brain/Plasma

AUC0-24 hr

Cmax (nM)

at 24 hr

(µ µmol*hr)

3.8 ±2.4; n = 15

585

5.3

1.4

11.9; (6.7 -21.3); n = 3

16

0

0.076

Ki ± SEM (nM); n

2

(CH2)2-pyrrolidine

9

(CH2)2-NH-CO-NEt2

10

(CH2)2-NMe-CO-OMe

15.5 ± 1.2; n =3

31

1

0.17

11

CH2)2-NEt-CO-Me

9.7 ± 1.9; n = 3

6

0

0.025

12

CH2CONEt2

8.1 ± 2.7; n = 3

8

4.7

0.05

13

CH2)2-NH-SO2Me

9.3 ± 1.6; n = 3

565

0

3.4

14

CH2)2-NH-CO-Me

11.6 ± 0.96; n = 4

1879

0

9.1

15

(CH2)2-NH-CO-OMe

10.7 ± 2.6; n = 3

10,500

0.05

63.8

16

Me

25.4 ± 7.8; n = 14

431

3

0.43

17

(CH2)2OH

7.7 ± 1.4; n =3

644

1.2

2.6



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18

(R)-CH2CH(Me)OH

9.5 ± 1.4; n = 6

2240

0.7

13.1

19

CH2C(Me)2OH

7.9 ± 0.7; n = 7

2921

1.7

16.3

The challenge was to maintain the favorable CNS/plasma partitioning conferred by the hydroxylated ether moiety while increasing exposure in a dose dependent manner. Since the failure to significantly increase exposures with higher doses suggested that solubility limited absorption was the limiting factor, pro-drugs of 18 and 19 were evaluated. Figure 2 summarizes the AUC0-24hr of 18 and 19 achieved following oral administration to rats of 10 mg/kg suspensions of the parent alcohols and their corresponding amino acid (AA) esters, mono-esters of dibasic acids, dibasic phosphate esters or as a glucoside. Based on the superior exposures achieved and given the ease of preparation and purification, the amino acid esters were selected as the pro-drug for oral administration of these 3-(4-(2hydroxyethoxy)aryl)thieno[3,2-d]pyrimidin-4(3H)-ones.


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Figure 2: Variation in exposure of 18 and 19 due to pro-drug employed for oral administration to rats

90 80

18

19

70 60 AUC (µ µM*hr)

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50 40 30 20 10 0

Although stable in plasma, hydrolysis of these AA esters readily occurred during transit the GI wall and the liver. Since these AA esters were potent MCHR antagonists (Ki ~ 3-10 nM ) as well as 1-3 nM inhibitors of hERG and cytochrome P450 (CYP) 2C9 and 2C19, it was essential that there be no peripheral exposure of the pro-drug following oral administration to avoid potentially confounding results. The preferred pro-drugs were the L-valine ester for 1° and 2° alcohols and the glycine ester for 3° alcohols. PK studies established that no detectable amount of these esters survived the initial passage through the liver whereas measureable levels were observed for more sterically encumbered AA esters. When these esters were administered orally as a HCl salt as a 0.5% methyl cellulose (Methocel) / 0.1% Tween 80 aqueous suspension, dose dependent alcohol exposures were obtained following oral administration of doses of 1-100 mg/kg. For each pro-drugged alcohol, oral PK studies were run prior to efficacy evaluation to ascertain that the peripheral and CNS exposures of the alcohol were adequate and that the AA pro-drug was not detectable. Following identification of a potential development candidate 10 ACS Paragon Plus Environment


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alcohol, our strategy entailed preparation and utilization of the corresponding phosphate pro-drug for final studies prior to advancement of the phosphate pro-drug into clinical development thereby avoiding any potential off-target risks posed by these amino ester pro-drugs. These MCHR1 antagonists were initially evaluated in a young growing rat model using eight week old Sprague-Dawley (SD) rats which had been previously acclimated to a choice diet.28 A critical determinant for efficacy was whether adequate 24 hr drug coverage was maintained. If not, breakthrough food consumption would occur once plasma drug levels became too low to maintain the prerequisite receptor occupancy. This critical Cmin value was compound dependent reflecting affinity, brain to plasma ratio, free fraction in the CNS and kinetics pertaining to receptor occupancy. Since these 3-(4-(2-hydroxyethoxy-aryl)thieno[3,2-d]pyrimidin-4(3H)-ones.were uniformly highly bound to serum proteins, drug concentrations measured in plasma and brain vastly overestimated the concentrations available to bind to MCHR1. Typically the % free drug was < 1% unless the structure contained a second polar functionality as in the case for 21 or 24; whereupon, the % free fraction increased to 2-7%. Body weights of rats administered 18 or 19 qd at 3 mg/kg as the valine and glycine ester pro-drug respectively for ten days as a Methocel/Tween 80 aqueous suspension were 2.3 and 10.3 % less than their vehicle treated cohort; whereas for a 30 mg/kg dose this differential increased to 15.7 and 15.6% respectively. Weight loss produced by these MCHR1 antagonists was gradual and continuous throughout the study course. Daily food and water consumption was measured; although the daily decrease versus vehicle was rarely significant, the cumulative difference was. Separate studies established that taste aversion was not a confounding factor. Routine liver histopathology was performed to screen for overt hepatotoxicity. After four days obstructive and proliferative lesions of small caliber intra-hepatic bile ducts were observed in all lean SD rats treated with 18 at 30 mg/kg but not in those treated with 19. Subsequently a 28- day dose dependent response study using DIO rats revealed that 19 11 ACS Paragon Plus Environment

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at 30 mg/kg/day produced identical bile duct lesions in 40% of the treated rats. Serum bilirubin and bile acid levels were only increased in those rats with the most severe and widespread bile duct lesions; biomarkers of toxicity, including transcripomic and metabolomic profiles, were descriptive rather than predictive. Rather than abandoning this chemotype, we elected to conduct mechanistic studies to ascertain whether the toxic agent was the amino acid pro-drug, parent alcohol or a metabolite. Incubation with hepatocytes revealed these thienopyrimidinones to be surprisingly resistant to metabolic oxidation. The methyl ether remained intact; rather the primary if not exclusive site subject to oxidation was the hydroxylated side chain. Metabolism of 18 occurred at two sites of the hydroxylated alkyl substituent to respectively generate phenol 20 and diol 21a which was further transformed to the α-hydroxyacid 22a. (Scheme 1) An analogous transformation of 19 generated 20, 21b and 22b. Both 21a and 21b were potent brain penetrant MCHR1 antagonists (Ki = 7 and 10 nM respectively) for which the brain/plasma was ~ 1:2. In contrast, 22a and 22b were weak MCHR1 antagonists (Ki ~ 1 µM) which were not brain penetrant.

In vivo mechanistic studies utilized 18 since lesions were formed much more quickly than with 19. To resolve the role of the pro-drug, rats were treated for four days qd with the valine ester 18a (absorbed from Gi tract and hydrolyzed in liver) and phosphate ester 18b (not absorbed from GI, hydrolyzed at the 12 ACS Paragon Plus Environment


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intestinal brush border releasing 18 which was absorbed). Comparable plasma levels of 18 were generated following oral administration of either pro-drug at 30 mg/kg. The appearance of lesions in all rats administered either the phosphate or valine ester of 18 excluded the pro-drug as the causative agent. To ascertain if 18 was toxic, rats were administered 18a daily for four days at 30 mg/kg with and without the non-specific CYP inhibitor aminobenztriazole (ABT) at 10 mg/kg. The findings that 18a alone produced lesions in all rats but co-administration of ABT with 18a completely prevented lesion formation established that the toxic agent was a metabolite. The phenol 20 was excluded since no lesions were detected when it was administered as the corresponding phenolic acetate. In contrast, administration of the bis valine ester of diol 21a generated very severe lesions in all rats after 3 days whereas less severe lesions were detected in 60% of the rats if ABT was co-administered. These results are best explained by the α-hydroxyacid 22a being the toxic agent although these findings do not preclude diol 21a being a minor contributor. Although these findings precluded advancement of 18 or 19, the understanding that the toxicity arose due to formation of a toxic α-hydroxy acid dictated future target selection. We replaced the methylcarbinol moieties with functionality that would both disfavor formation of a 1,2-diol metabolite and preclude formation of an α-hydroxy acid metabolite by employing two approaches: cyclic alcohols and secondary alcohols not prone to hydroxylation. Most alcohols evaluated were prepared by alkylating nitroguiacol with an appropriate alkyl halide or epoxide. Following catalytic reduction, the resulting aniline was condensed with 33 in either ethanol at 80 °C or in a phenol melt at 130 °C. The preparation of 27 shown in Scheme 1 illustrates this route.


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Scheme 2.Reagents and conditions: (a) Br2, MeOH, 0 °C; 59% (b) DMF, 80 °C; 90% (c) NaBH4, EtOH; 96% (d) chiral chromatography; 93% (e) H2, Pd/C; 100% (f) phenol, 130 °C; 74% (g) (SEMO)2P(NiPr2); (h) H2O2; 95% (g and h combined yield) (i) TFA, 0 °C; 73% (j) EtOH, 80 °C; 88%.

Compounds exhibiting Ki < 10 nM and acceptable 24 hr PK profiles in SD rats when administered as the amino ester pro-drug were evaluated as anorectic agents.29 PK studies with the secondary alcohols 23-28 eliminated 23 due to low exposure. Histopathology examinations following initial efficacy studies with alcohols 24-28 revealed no evidence for formation of biliary lesions. We attributed the weak anorectic effect of the ethyl sulfone 24 to poor CNS exposure (brain/plasma ratio was ~ 0.05). The trifluromethyl 25 and trifluoroethyl 26 carbinols were not further pursued because the reduction in weight gain exhibited a nonlinear dose dependence and the drug accumulations of 3-5 fold by day 5 led 14 ACS Paragon Plus Environment


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to high brain and plasma levels. Encouraged by the promising Ki, AUC and CNS exposure exhibited racemic cyclopropyl carbinol, the two enantiomers 27 and 28 were prepared. Although the R and S enantiomers were equally potent MCHR1 inhibitors, the R enantiomer 27 became the focus of the program due a superior PK profile (AUC of 27 when administered as the valine pro-drug was ~ 3-fold higher than that of 28). The absolute configuration of 27 was assigned following a chiral synthesis of 28.30 Table 2: Weight loss and exposures in Sprague Dawley rats observed after oral q.d. administration of hydroxylated thienopyrimidinone ethers as amino acid (AA) pro-drug esters for four days

R

Ki (nM);

AA ester pro-

Parent

Parent plasma

% weight

n

drug

AUC0-24

(brain) concen.

reduction vs.

administered

from PK

at 20 hr post

vehicle at day 4

at 10 mg/kg

study

dose on day 4

(µ µmol*hr) 18

9.5 ± 1.4;

Valine

157

35 nM 1.6

n=6 19

7.9 ± 0.7;

(36 nM) Glycine

45

682 nM 6.1 ± 0.5

n=7 23

31.7 ± 8.8;

(1116 nM) Valine

2.8

Valine

46

n=4 24

2.5 ± 0.8;

12,440 nM 1.7

n=4

(640 nM) 15 ACS Paragon Plus Environment

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25


OH CF3

26

13.3 ± 3.1;

Valine

35.5

16,562 nM 3.9 ± 1.4

n=4 11.4 ± 2.6;

(6544 nM) Valine

26.4

20,845 nM 4.3 ± 0.6

n=3 27

10.4 ± 0.6;

(8811 nM) Valine

2143 nM 24.5

n =37 28

11.7 ± 0.7;

3.5 ± 0.9 (1780 nM)

Valine

404 nM 8

2.2 ± 0.7 (660 nM)

n = 29

Comparable weight loss was observed with young growing SD rats following oral administration of 27 either as the valine ester pro-drug 27a or phosphate pro-drug ester 35 as would be expected since both pro-drugs were equally efficient non-circulating delivery vehicles. Note neither pro-drug was detected in plasma despite both being stable in plasma. In a 10-day dose response study the reduced weight gain relative to vehicle cohort produced by daily administration of 35 at 1, 3, 10 and 30 mg/kg was 3.4, 5.5, 7.8 and 10.6% respectively. An equivalent dose response was obtained after a 28 study with DIO SD rats. In both sets of studies, 5% reduced weight gain versus vehicle correlated with trough plasma levels equal to 1.2 µM. Given that 27 is 99.8% bound to rat serum proteins and rat MCHR1 Ki is 7 nM, this finding suggests that trough free drug plasma levels must remain above 0.4 Ki to achieve 5% weight reduction. The anorectic effect appears to be solely MCHR1 mediated as administration of 35 at 10 mg/kg for twelve days to WT mice resulted in a 7.6% reduction in body weight whereas MCHR1 KO mice gained 2.5%. FLIPR-based assays established that 27 was a potent and highly selective MCHR1 functional antagonist. 27 (Kb = 32 nM) effectively blocked MCH stimulated Ca+2 mobilization in heterologous cells overexpressing MCHR1 but failed to inhibit MCH mediated Ca+2 mobilization of cells expressing MCHR2 16 ACS Paragon Plus Environment


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at 10 µM.. No activity was observed upon screening 27 at 10 µM versus a panel of 20 GPCR’s associated with feeding homeostasis.31 The percent of 27 bound to serum proteins was species dependent ranging from 99.8, 99.6 and 99.3% respectively for rat, dog and monkey; for humans the value was 99.5%. Although 27 was a weak P-glycoprotein 1 substrate, the high permeability (> 100 nm/sec) dominated; consequently CNS exposure was adequate resulting in a brain/plasma ratio of 0.66. When screened for CYP activity, EC50 for 1A2, 2C9, 2C19, 2D6 were > 40 µM; however the 3A4 EC50 was 13 µM.32 In microsomal preparations 27 was oxidized by 3A4 to the corresponding cyclopropyl ketone 36 which was subsequently reduced to generate a mixture of 27 and 28. Microsomal incubations revealed no propensity for conjugation to glutathione. 27, 28, 36 and 35 exhibited no significant offtarget activities; all four compounds exhibited < 50% inhibition when evaluated against a panel of 38 receptors, ion channels or enzymes at 10 µM. Cardiovascular safety studies supported initiating clinical studies with 27 and 35. In the absence of serum proteins, hERG inhibition of 48 and 64% were observed for 27 at 3 and 10 µM whereas 35 at 10 µM produced 12% inhibition. The inhibition of the human cardiac Na+ channel and the L-type Ca+2 channel produced by 27 at 10 µM was 20 and 15% respectively. Studies with purkinje fibers at 30 µM revealed no drug induced effect on action potential duration or resting membrane potentials. In an anesthetized rabbit model, plasma levels of 38 µM caused no drug induced effect on QT or QRS duration. For dogs, plasma levels reaching 7.6 µM of 27 produced no drug related changes in heart rate, blood pressure or QT. Table 3: PK profile of 27 in rat, dog and cynomologous monkey Species

Compound:

Route

TMax

AUC

t1/2

CL

VSS

Dose

of

(hr)

(INF)

(hr)

(mL/min/kg))

(L/kg)


F (%)

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(mg/kg)

Admin

(µ µmol*hr)

.

27: 1

iv

35: 10

po

27: 1

iv

35: 10

po

1.3 ± 0.6

65 ± 2.4

Cyno.

27: 1

iv

-

11 ± 1.9

Monkey

35: 10

po

12 ± 11

21 ± 4.4

Rat

Dog

11 3.3 ± 1.2

5.7

3.4

1.5

26 ± 0.3 17 ± 5.6

93 32 ± 8

2.3 ± 0.9

4.1 ± 1.1

44

14 ± 3

3.5 ± 0.6

4.0 ± 1.1

23

PK studies using three species – rat, dog and cynomologous monkey - were conducted with 27 administered iv at 1 mg/kg and as the phosphate ester pro-drug 35 administered po at 10 mg/kg. For all three species no detectable plasma levels of 35 were observed. Clearance of 27 was low in all three species giving rise to the same major metabolites: oxidation to generate the cyclopropyl ketone 36, alcohol 28 from subsequent non-selective reduction of 36 and the glucuronides of alcohols 27 and 28. The projected human PK profile based on these findings supported advancement of 27 into clinical development as the phosphate ester 35 (BMS-830216). Using these parameters a 400 mg dose qd of 35 was projected to generate trough human plasma levels of 1.2 µM of 27 (BMS-819881) corresponding to the trough levels required to produce 5% weight loss in the rat models. Clinical trials were begun with BMS-830216; results will be disclosed in a subsequent publication. 1. World Health Organization Global Health Observatory (GHO) website http://www.who.int/gho/ncd/risk_factors/obesity_text/en/ (accessed Oct 18, 2013).



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2. Holes-Lewis, K.A.; Malcolm, R.; O'Neil, P.M. “Pharmacotherapy of obesity: clinical treatments and considerations” Am. J. Med. Sci. 2013, 345, 284-288. 3. Schwartz, S.M.; Bansal, V.P.; Hale, C.; Rossi, M.; Engle, J.P. “Compliance, behavior change, and weight loss with orlistat in an over-the-counter setting” Obesity (Silver Spring). 2008 ,16, 623-629. 4. (a) Larsen, T.M.; Toubro, S.; van Baak, M.A.; Gottesdiener, K.M.; Larson, P.; Saris,W.H.; Astrup, A. “Effect of a 28-d treatment with L-796568, a novel beta(3)-adrenergic receptor agonist, on energy expenditure and body composition in obese men” Am. J. Clin. Nutr. 2002, 76, 780-788. (b) Redman, L.M.; de Jonge, L.; Fang, X.; Gamlin, B.; Recker, D.; Greenway, F.L.; Smith, S.R.; Ravussin, E. “Lack of an effect of a novel beta3-adrenoceptor agonist, TAK-677, on energy metabolism in obese individuals: a double-blind, placebo-controlled randomized study” J. Clin. Endocrinol. Metab. 2007, 92, 527-531. 5. Christensen, R.; Kristensen, P.K.; Bartels, E.M.; Bliddal, H.; Astrup, A. “Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials” Lancet, 2007, 370 (9600), 1706– 1713 6. Pissios, P.; Bradley, R. L.; Maratos-Flier, E “Expanding the scales: the multiple roles of MCH in regulating energy balance and other biological parameters” Endo. Rev. 2006, 27, 606-620 7. Luthin, D. R. “Anti-obesity effects of small molecule melanin-concentrating hormone receptor 1 (MCHR1) antagonists” Life Sciences 2007, 81, 421-440 8. Forray, C. The MCH receptor family: feeding brain disorders? Current Opin. in Pharmacol. 2003, 3, 85-89


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9. Takekawa, S.; Asami, A.; Ishihara, Y.; Terauchi, J.; Kato, K.; Shimomura, Y.; Mori, M.; Murakoshi, H.;, Kato, K.; Suzuki, N.; Nishimura, O.; Fujino, M. “T-226296: a novel, orally active and selective melanin-concentrating hormone receptor antagonist” Eur. J. Pharmacol. 2002, 438, 129-35 10. Clark, D. E.; Higgs, C.; Wren, S.P.; Dyke, H. J.; Wong, M.; Norman, D.; Lockey, P. M.; Roach, A. G.; “A virtual screening approach to finding novel and potent antagonists at the melanin-concentrating hormone 1 receptor” J. Med. Chem. 2004 47, 3962-3971 11. McBriar, M. D. “Recent advances in the discovery of melanin-concentrating hormone receptor antagonists” Current Opin. Drug Discov. Devel. 2006, 9, 496-508 12. Hertzog, D. L.; Al-Barazanji, K. A.; Bigham, E. C.; Bishop, M. J.; Britt, C. S.; Carlton, D. L.; Cooper, J. P.; Daniels, A. J.; Garrido, D. M.; Goetz, A. S.; Grizzle, M. K.; Guo, Y. C.; Handlon, A. L.; Ignar, D. M.; Morgan, R. O.; Peat, A. J.; Tavares, F. X.; Zhou, H. “The discovery and optimization of pyrimidinone-containing MCH R1 antagonists” Bioorg. Med. Chem. Lett. 2006, 16, 4723-4727 13. Ulven, T.; Frimurer, T. M.; Receveur, J.M.; Little, P. B.; Rist, O.; Nørregaard, P. K.; Högberg T. “6Acylamino-2-aminoquinolines as potent melanin-concentrating hormone 1 receptor antagonists. Identification, structure-activity relationship, and investigation of binding mode” J. Med. Chem. 2005, 48, 5684-5697 14. Souers, A. J.; Gao, J.; Brune, M.; Bush, E.; Wodka, D.; Vasudevan, A.; Judd, A. S.; Mulhern, M.; Brodjian, S.; Dayton, B.; Shapiro, R.; Hernandez, L. E.; Marsh, K. C.; Sham, H. L.; Collins, C. A.; Kym, P. R. “Identification of 2-(4-benzyloxyphenyl)-N- [1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-6yl]acetamide, an orally efficacious melanin-concentrating hormone receptor 1 antagonist for the treatment of obesity” J. Med. Chem. 2005, 48, 1318-1321



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15. Johansson, A. “Recent progress in the discovery of melanin-concentrating hormone 1-receptor antagonists” Current Opin. Ther. Patents 2011, 21, 905-925 16. Rokosz, L. L. “Discovery and development of melanin-concentrating hormone receptor 1 antagonists for the treatment of obesity” Expert Opin. Drug Discov. 2007, 2, 1301-1327. 17. Kowalski, T. J.; Sasikumar, T. “Melanin-concentrating hormone receptor-1 antagonists as antiobesity therapeutics” Biodrugs 2007, 21, 311-321 18. Handlon, A. L.; Zhou, H. “Melanin-concentrating hormone receptor-1 antagonists for the treatment of obesity” J. Med. Chem. 2006, 49, 4017-4022 19. Mendez-Andino, J. L.; Wos, J. A. “MCH-R1 antagonists: what is keeping most research programs away from the clinic?” Drug Discovery Today 2007, 12, 972-979. 20. Kamata, M.; Yamashita, T.; Imaeda, T., Masada, S.; Kamaura, M.; Kasai, S.; Hara, R.; Sasaki, S.; Takekawa, S.; Asami, A.; Kaisho, T.; Suzuki, N.; Ashina, S.; Ogino, H.; Nakano, Y.; Nagisa, Y.; Kato, K.; Kato, K.; Ishihara, Y. “Melanin-concentrating hormone receptor-1 antagonists. Synthesis and structure –activity relationships of novel 3-(aminomethyl)quinolines” J. Med. Chem. 2012, 55, 23532366 21. Lim, C. J.; Kim, N.; Lee, E. K., Lee, B.H.; Oh, K.S.; Yoo, S.E.; Yi, K.Y. “Synthesis and SAR investigations of novel 2-arylbenzimidazole derivatives as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists” Bioorg. Med. Chem. Lett. 2011, 21, 2309-2312 22. Berglund, S.; Egner, B. J.; Graden, H.; Graden, J.; Morgan, D. G.; Inghardt, T.; Giordanetto, F. “Discovery of 1,3-disubstituted-1H-pyrrole derivatives as potent melanin-concentrating hormone receptor 1 (MCH-R1) antagonists” Bioorg. Med. Chem. Lett. 2008, 18, 4859-4863


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23. Kasai, S.; Kamaura, M.; Kamata, M.; Aso, K.; Ogino, H.; Nakano, Y.; Watanabe, K.; Kaisho, T.; Tawada, M.; Nagisa, Y.; Takekawa, S.; Kato, K.; Suzuki, N.; Ishihara, Y. “Melanin-concentrating hormone receptor 1 antagonists: structure-activity relationship, docking studies, and biological evaluation of 2,3,4,5-tetrahydro-1H-benzaxepine derivatives” Bioorg. Med. Chem. 2011, 19, 6261-6273 24. Kasai, S.; Kamaura, M.; Masada, S.; Kunitomo, J.; Kamaura, M.; Okawa, T.; Takami, K.; Ogino, H.; Nakano, Y.; Ashina, S.; Watanabe, K.; Kaisho, T.; Imai, Y. N.; Ryu, S.; Nakayama, M.; Nagisa, Y.; Takekawa, S.; Kato, K.; Murata, T.; Suzuki, N.; Ishihara, Y. “Synthesis, structure-activity relationship, and pharmacological studies of novel melanin-concentrating hormone receptor 1 antagonists 3aminomethylquinolines: reducing human ether a-go-go-related gene (hERG) associated liabilities” J. Med. Chem. 2012, 55, 4336-4351 25. Sasmal, P. K.; Sasmal, S.; Abbineni, C.; Venkatesham, B.; Rao, P. T.; Roshaiah, M.; Khanna, I.; Sebastian, V. J.; Suresh, J.; Singh, M. P.; Talwar, R.; Shashikuma, D.; Reddy, K. H.; Frimure, T. M.; Rist, O.; Elster, L.; Högberg, T. “Synthesis and SAR studies of benzimidazole derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists: focus to detune hERG inhibition” Med. Chem. Comm. 2011, 2, 285-289 26. Hadden, M.; Deering, D. M.; Henderson, A. J.; Surman, M. D.; Luche, M.; Khmelnitsky, Y.; Vickers, S.; Viggers, J.; Cheetham, S.; Guzzo, P. R. “Synthesis and SAR of 4-aryl-(1-indazol-5yl)pyridine-2(1H)ones as MCH-1 antagonists for the treatment of obesity” Bioorg. Med. Chem. Lett. 2010, 20, 7020-7023 27. Mihalic, J. T.; Fan, P.; Chen, X.; Chen, X.; Fu, Y.; Motani, A.; Liang, L.; Lindstrom, M.; Tang, L.; Chen, J. L.; Jaen, J.; Dai, K.; Li, L. “Discovery of a novel melanin concentrating hormone receptor 1 (MCHR1) antagonist with reduced hERG inhibition” Bioorg. Med. Chem. Lett. 2012, 22, 3781-3785



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28. Devenny, J. J.; Godonis, H. E.; Harvey, S. J.; Rooney, S.; Cullen, M. J.; Pelleymounter, M. A. “Weight Loss Induced by Chronic Dapagliflozin Treatment Is Attenuated by Compensatory Hyperphagia in Diet-Induced Obese (DIO) Rats” Obesity, 2012, 20, 16-45-1652 29. Rat MCHR1 Ki values were also determined for all compounds prior to evaluation for in vivo efficacy. In all instances the rat MCHR1 Ki value was within a factor of 2 of the human MCHR1 Ki value. 30. The chirality of 28 was assigned based on the coincident retention time of 28 using a chiral column with that of the product generated by alkylation of phenol 20 with the cyclopropanated product obtained by treatment of commercial (S)-2-hydroxybut-3-enyl 4-methylbenzenesulfonate with Et2Zn/CH2I2. 31.Angiotensin, APJ, Calcitonin Gene Related Peptide, Cholecystokinin A, Cholecystokinin B, G Protein-Coupled Receptor 8, Galanin 1, Galanin 2, Histamine H3, Leptin, Melanocortin 3, Melanocortin 4, Melatonin 1, Melatonin 2, Neuromedin U1, Neuromedin U2, Neurotensin 1, Neurotensin non-selective, Somatostatin, Urotensin II. 32. Despite aqueous solubility of 27 and related thienopyrimidinones analogs being < 1 µg/mL, nephelometry studies confirmed that stable 40 uM solutions required for in vitro profiling could be prepared by addition of a DMSO stock solution to aqueous buffer. 33. Calverley, M. J. “Synthesis of MC 903, a biologically active vitamin D metabolite analogue” Tetrahedron 1987, 43, 4609-4619 34. Leff, P,; Dougall, I. G..” Further concerns over Cheng-Prusoff analysis.” Trends Pharmacol Sci 1993, 14, 110-112 Experimental Section All reactions were carried out under a static atmosphere of argon or nitrogen and stirred magnetically unless otherwise noted. All reagents used were of commercial quality and were primarily obtained from 23 ACS Paragon Plus Environment

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Aldrich Chemical Co., Sigma Chemical Co., Lancaster Chemical Co., or Acros Chemical Co.; anhydrous solvents from Aldrich Chemical Co. or EM Science Chemical Co. unless otherwise noted were utilized. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a JEOL GSX400 spectrometer using Me4Si as an internal standard unless otherwise noted. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a JEOL JNM-ECP500 spectrometer. Chemical shifts are given in parts per million (ppm) downfield from internal reference Me4Si in δ-units, and coupling constants (Jvalues) are given in hertz (Hz). All flash chromatographic separations were performed using E. Merck silica gel (particle size, 0.040-0.063 mm). Reactions were monitored by TLC using 0.25 mm E. Merck silica gel plates (60 F254) and were visualized with UV light or with 5% phosphomolybdic acid in 95% EtOH. LC/MS data were recorded on a Shimadzu LC-10AT equipped with a SIL-10A injector, a SPD10AV detector, normally operating at 220 nm, and interfaced with a Micromass ZMD mass spectrometer. LC/MS separations were obtained with a Phenomenex reverse phase C18 column 4.6 x 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA, 1 min hold; 4 mL/min, UV detection at 220 nm. Analytical HPLC separations typically employed a Phenomenex Luna S5 reverse phase C18 column 4.6 x 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.2% H3PO4 to 90% MeOH/10% H2O/0.2% H3PO4, 1 min hold; 4 mL/min, UV detection at 220 nm. Preparative HPLC purifications entailed gradient elution from a Phenomenex Luna AXIA column using an appropriate mixture of 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA. On occasion, mixtures of 10% MeCN/90% H2O/0.1% TFA and 90% MeCN/10% H2O/0.1% TFA were employed. If the molecule contained an acid sensitive component, the TFA was omitted. Purity for all compounds was greater than 95% as determined by LC/MS analysis..

Preparation of 18



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Page 26 of 52

To a solution of Ph3P (23.3 g; 88.7 mmol) in 450 mL of THF cooled to 0 oC was added a solution of dit-butylazodicarboxylate (20.4 g; 88.7 mmol) in 50 ml of THF over 15 min. After stirring at 0oC for 10 min 4-nitroguaiacol (10.0 g; 59.1 mmol) was added followed by (S)-glycidol (6.3 mL; 94.6 mmol) over 10 min. The reaction was allowed to warm to rt and stir for 2 hr prior to removal of the volatiles under vacuum.. The residue was dissolved in EtOAc, washed with H2O and brine, dried over anhydrous MgSO4 and filtered. After concentration under reduced pressure, the residue was purified by flash chromatography (silica gel, Hexanes/EtOAc; 100:0 to 1:3 gradient). Impure fractions were concentrated under reduced pressure and further purified by flash chromatography (silica gel, Hexanes/EtOAc; 100:0 to 0:100 gradient). Pure fractions from both columns were combined and concentrated under reduced pressure to afford 9.58 g (72%) of (R)-2-((2-methoxy-4-nitrophenoxy)methyl)oxirane as a yellow solid. 1

H NMR (CDCl3) δ 2.78-2.80 (m, 1H), 2.94-2.96 (m, 1H), 3.40-3.43 (m, 1H), 3.96 (s, 3H), 4.06-4.11

(m, 1H), 4.41-4.44 (m, 1H), 6.98 (d, J = 8.79 Hz, 1H), 7.75 (d, J = 2.20 Hz, 1H), 7.89 (dd, J = 8.79 Hz, 2.19 Hz, 1H); 13C NMR (CDCl3) δ 28.20, 44.57, 49.75, 56.28, 70.17, 106.80, 111.71, 117.50, 141.92, 149.20, 153.38; HPLC: 2.09 min retention time; LCMS (ES): m/z 226 [M+H]+.

To 160 mL of Et2O cooled to 0 oC was added LiClO4 (80 g; 752 mmol) in portions over 20 min. The mixture was allowed to warm to rt prior to addition of (R)-2-((2-methoxy-4-nitrophenoxy)methyl)oxirane (9.55 g; 42.5 mmol). After stirring for 10 min, BH3-NHMe2 (3.40 g; 46.6 mmol) was added and the suspension was stirred at rt for 2.5 h. The suspension was diluted with CH2Cl2 and stirred in a beaker with H2O until gas evolution ceased. The organic layer was washed with H2O, dried over anhydrous MgSO4 and filtered prior to concentration under reduced pressure. The residue was triturated in CH2Cl2, filtered and purified by flash chromatography (silica gel, Hexanes/EtOAc; 100:0 to 100:0 gradient) to afford 6.83 g (70%) of (R)-1-(2-methoxy-4-nitrophenoxy)propan-2-ol as a yellow solid. 25 ACS Paragon Plus Environment

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1


H NMR (CDCl3) δ 1.31 (d, J = 6.59 Hz, 3H), 2.77 (br s, 1H), 3.90-3.94 (m, 4H), 4.05-4.08 (m, 1H),

4.24-4.32 (m, 1H), 6.92 (d, J = 9.35 Hz, 1H), 7.74 (d, J = 2.75 Hz, 1H), 7.88 (dd, J = 8.79 Hz, 2.74 Hz, 1H); 13C NMR (CDCl3) δ 18.64, 56.18, 65.87, 74.85, 106.73, 111.66, 117.60, 141.76, 149.20, 153.63; HPLC: 2.17 min retention time; LCMS (ES): m/z 228 [M+H]+. A stirred EtOH suspension (100 mL) containing (R)-1-(2-methoxy-4-nitrophenoxy)propan-2-ol (6.0g, 26.4 mmol) and 10% Pd/C (0.15 g) was hydrogenated under 60 psi H2 for 5hr. HPLC analysis revealed no starting nitro catechol ether remained. After filtration through a a pad of celite under a blanket of N2, the resultant red colored solution was immediately concentrated using a rotary evaporator under vacuum to yield 6.4 of (R)-1-(2-methoxy-4-aminophenoxy)propan-2-ol as a dark oil which was used without further purification. Following the literature procedure for preparation of the thienopyrimidinones12, (R)-1-(2-methoxy-4aminophenoxy)propan-2-ol

(26.4

mmol)

was

condensed

with

methyl

5-(4-chlorophenyl)-3-

((dimethylamino)methyleneamino)thiophene-2-carboxylate 33 (8.8g mg, 24 mmol) by heating an EtOH solution (36 mL) of the two components at reflux for 15 hr. Upon cooling and filtration, 18 (8.2 g, 77%) was isolated as a white solid after washing with Et2O and air drying, . 1H NMR (DMSO-d6) δ 1.16 (d, J = 6.05 Hz, 3H), 3.77-3.82, (m, 4H), 3.88-3.91 (m, 1H), 3.94-4.02 (m, 1H), 4.89 (d, J = 4.39 Hz, 1H), 7.03 (dd, J = 8.79 Hz, 1.64 Hz, 1H), 7.10 (d, J = 8.25 Hz, 1H), 7.18 (d, J = 1.65 Hz, 1H), 7.57 (d, J = 8.25 Hz, 2H), 7.92 (d, J = 8.25 Hz, 2H), 7.97 (s, 1H), 8.39 (s, 1H); 13C NMR (DMSO-d6) δ 20.03, 55.58, 64.21, 73.88, 111.63, 112.68, 119.43, 121.56, 121.84, 127.66, 129.10, 129.57, 131.02, 134.09, 148.27, 148.80, 149.32, 149.60, 155.91, 157.23; [a]589 = -10.1° at 23 °C for 12.3 mg in 1 mL CH2Cl2; HPLC: 3.64 min retention time, (100%); LCMS (ES): m/z 443 [M+H]+.



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Preparation of 19 A steel bomb with ~ 55 mL capacity, fitted with an internal thermocouple, pressure gauge and safety release valve rated at 3000 psi, was charged with the potassium salt of 2-methoxy-4-nitrophenol (6g, 29 mmol), NaH2PO4 (3.3g, 27.7mmol), isobutylene oxide (2.8 g, 35 mmol) and 30 mL of 15% H2O/MeCN. The sealed bomb was heated at 170 °C for 3 hr. Following cooling, HPLC revealed all starting phenol had been converted to product. The biphasic solution was concentrated using a rotary evaporator before being partitioned between CH2Cl2 and H2O. The aqueous phase was extracted 3x with CH2Cl2; the combined CH2Cl2 fractions were washed 3x with aq KHCO3/K2CO3 and once with H2O. After drying over Na2SO4, concentration under vacuum yielded 6.9g of 1-(2-methoxy-4-nitrophenoxy)-2methylpropan-2-ol as a tan solid. (Buffering with NaH2PO4 is essential to prevent reversion of the product to starting phenol as the pH will increase during the reaction if unbuffered. Note even for small scale reactions, temperatures greater than 180 °C should be avoided to minimize the probability of explosive decomposition since the potassium salt of 2-chloro-4-nitrophenol rapidly decomposes at ~210 °C producing gaseous products.) 1

H NMR (CDCl3) δ 1.42 (s, 6H), 2.5 (s, 1H), 3.91 (s, 3H), 3.94 (s, 3H), 6.90 (d, J = 9 Hz, 1H), 7.74 (d, J

= 2 Hz, 1H), 7.87 (dd, J= 2 Hz and 9 Hz, 1H); 13C NMR (CDCl3) δ 26.02, 56.18, 70.00, 77.42, 106.77, 111.74, 117.56, 141.73, 149.37, 153.95; HPLC: 3.26 min retention time (99% API); LCMS (ES): m/z 242.1 [M+H]+. A stirred EtOH suspension (320 mL) containing 1-(2-methoxy-4-nitrophenoxy)-2-methylpropan-2-ol (13.6g, 56.4 mmol) and 10% Pd/C (0.25 g) was hydrogenated under 60 psi H2 for 6hr. HPLC analysis revealed no starting nitroarene remained. After filtration through a fiberglass filter paper under a blanket of N2, the resultant clear wine colored solution was immediately concentrated using a rotary evaporator


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under vacuum to yield 12.4 of desired 1-(2-methoxy-4-aminophenoxy)-2-methylpropan-2-ol as a dark orange oil which was used without further purification. 1

H NMR (CDCl3) δ 1.29 (s, 6H), 3.40, (br s, 2H), 3.74 (s, 2H), 3.78 (s, 3H), 6.20 (dd, J = 8.35 Hz, 2.64

Hz, 1H), 6.28 (d, J = 2.64 Hz, 1H), 6.76 (d, J = 8.35 Hz, 1H); 13C NMR (CDCl3) δ 25.90, 55.67, 70.02, 80.30, 100.76, 106.81, 118.27, 141.55, 141.93, 150.99; HPLC: 0.88 min retention time; LCMS (ES): m/z 212.1 [M+H]. The above product (12g, 57 mmol) was condensed with 33 (21g, 65 mmol) by heating overnight in refluxing EtOH. as described for the preparation of 18. Upon cooling, 19 was isolated as a white solid ((20.1g, 80%) following filtration, washing with Et2O and air drying. 1

H NMR (CDCl3) δ 1.38 (s, 6H), 2.74, (s, 1H), 3.87 (s, 3H), 3.89 (s, 2H), 6.93 (dd, J = 8.25 Hz, 2.20 Hz,

1H), 6.96 (d, J = 2.20 Hz, 1H), 7.01 (d, J = 8.25 Hz, 1H), 7.44 (d, J = 8.79 Hz, 2H), 7.52 (s, 1H), 7.65 (d, J = 8.80 Hz, 2H), 8.14 (s, 1H); 13

C NMR (CDCl3) δ 26.02, 56.11, 70.08, 78.05, 111.19, 114.49, 119.20, 120.82, 123.19, 127.61, 129.43,

130.46, 131.50, 135.66, 148.16, 149.35, 150.30, 151.64, 155.77, 157.34; HPLC: 4.29 min retention time, (99% API); LCMS (ES): m/z 457 [M+H].

Preparation of amino acid ester pro-drugs of 18, 19 and analogs The following procedure, illustrated by the conversion of 18 to the valine ester pro-drug, was employed to prepare all amino ester pro-drugs of all alcohols except for the glycine ester pro-drug of tertiary alcohols. A mixture of the alcohol 18 (50 mg, 0.113 mmol), diisopropylcarbodiimide (21 uL, 0.135 mmol), 4-dimethylaminopyridine (1 mg, 0.011 mmol) and N-(t-butoxycarbonyl)-L-valine (29 mg, 0.135 mmol) in 1 mL of CH2Cl2 was stirred at rt for 1 hr before additional diisopropylcarbodiimide (5 uL, 0.032 mmol) and N-(t-butoxy-carbonyl)-L-valine (5 mg, 0.023 mmol) were added. After stirring at rt for 28 ACS Paragon Plus Environment


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3.5 h, the suspension was diluted with CH2Cl2, washed with water, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2/CH3OH, 100:0 to 98:2 gradient) to afford the desired boc’d protected valine ester (76 mg) as a white solid. 1

H NMR (CDCl3) δ 0.90-0.95 (m, 3H), 0.97-1.02 (m, 3H), 1.40 (d, 3H), 1.45 (s, 9H), 2.15-2.19 (m, 1H),

3.86 (d, 3H), 4.05-4.27 (m, 3H), 5.05-5.08 (m, 1H), 5.35-5.41 (m, 1H), 6.92-6.96 (m, 2H), 7.00-7.05 (m, 1H), 7.45 (d, J = 8.79 Hz, 2H), 7.54 (s, 1H), 7.66 (d, J = 8.80 Hz, 2H), 8.15 (d, 1H); HPLC: 4.73 min retention time, (99%); LCMS (ES): m/z 642 [M+H]. The above boc’d amine was dissolved in a 1:2 mixture of TFA/CH2Cl2 (1 mL). By HPLC analysis after 1 hr at 20 °C, the reaction was complete; whereupon, the volatiles were removed under vacuum. The residue upon dissolution in CH2Cl2 was washed 2x with aq NaHCO3/Na2CO3 followed by brine prior to drying over Na2SO4. Upon concentration, 65 mg (94%) of the desired valine pro-drug was obtained. 1

H NMR (DMSO-d6) δ 0.97-1.01 (m, 6H), 1.34-1.36 (m, 3H), 2.11-2.18 (m, 1H), 3.75 (d, 3H), 3.94-

3.97 (m, 1H), 4.09-4.21 (m, 2H), 5.29-5.37 (m, 1H), 7.04-7.07 (m, 1H), 7.12-7.16 (m, 1H), 7.20-7.23 (m, 1H), 7.58 (d, J = 8.25 Hz, 2H), 7.92 (d, J = 8.80 Hz, 2H), 7.98 (s, 1H), 8.34 (br s, 3H), 8.39 (s, 1H); HPLC: 3.07 min; MS (ES): m/z 542 [M+H]+.

Preparation of the glycine ester pro-drug of 19 To a solution of 19 (0.96 g, 2.11 mmol), 4-pyrrolidinopyridine (0.31 g, 2.11 mmol) and N-(tertbutoxycarbonyl)glycine (1.11 g, 6.33 mmol) in 20 mL of refluxing CH2Cl2 was added diisopropylcarbodiimide (0.98 mL; 6.33 mmol) over 3 hr via syringe pump. After heating the suspension at reflux for 1 h, additional N-(tert-butoxycarbonyl)glycine (0.55 g, 3.17 mmol) was added followed by diisopropylcarbodiimide (0.55 mL, 3.17 mmol) over 2 hrs via syringe pump. The suspension was heated at reflux for 1 hr prior to cooling to rt. (Note: acylation of the tertiary alcohol 29 ACS Paragon Plus Environment

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was accompanied by a second acylation to extensively convert the desired product to an imide which could be converted back to the desired pro-drug by careful treatment with hydrazine.) Hydrazine monohydrate (0.34 mL, 7.01 mmol) was added and the suspension stirred at rt for 2 h. The suspension was cooled to 0oC, filtered and the filtrate washed with cold 1N HCl, cold dilute NaHCO3 solution, dried over anhydrous MgSO4 and filtered. After concentrating the filtrate under reduced pressure, the residue was purified by flash chromatography (silica gel, EtOAc/Hexanes, 0:100 to 1:1 gradient) to afford 1.37 g (100%) of the title compound as an off-white solid. 1

H NMR (CDCl3) δ 1.24 (t, 3H), 1.45 (s, 9H), 1.64 (s, 6H), 2.68-2.73 (m, 2H), 3.85 (d, J = 4.95 Hz, 2H),

4.18 (s, 2H), 4.98 (br s, 1H), 6.92 (d, J = 9.35 Hz, 1H), 7.20-7.22 (m, 2H), 7.44 (d, J = 8.79 Hz, 2H), 7.53 (s, 1H), 7.66 (d, J = 8.25 Hz, 2H), 8.13 (s, 1H); 13C NMR (CDCl3) δ 13.68, 23.39, 23.67, 28.28, 42.87, 72.72, 79.86, 81.86, 111.48, 120.82, 123.25, 125.42, 127.62, 129.42, 129.67, 131.54, 134.25, 135.59, 148.29, 151.50, 155.58, 156.69, 156.92, 157.37, 169.47; HPLC: 4.80 min retention time; LCMS (ES): m/z 612 [M+H]+.

Following the procedure described for the generalized preparation of pro-drugs, the BOC group was cleaved and the glycine pro-drug isolated as a white solid. 1

H NMR (CDCl3) δ 1.24 (t, 3H), 1.48 (br s, 2H), 1.64 (s, 6H), 2.68-2.74 (m, 2H), 3.38 (s, 2H), 4.19 (s,

2H), 6.93 (d, J = 9.34 Hz, 1H), 7.19-7.22 (m, 2H), 7.44 (d, J = 8.79 Hz, 2H), 7.52 (s, 1H), 7.66 (d, J = 8.80 Hz, 2H), 8.12 (s, 1H); 13C NMR (CDCl3) δ 13.68, 23.42, 23.75, 44.54, 72.72, 81.02, 111.43, 120.82, 123.22, 125.45, 127.62, 129.42, 129.62, 131.54, 134.17, 135.59, 148.27, 151.50, 156.77, 156.92, 157.37, 173.56; HPLC: 3.27 min retention time, (100%); LCMS (ES): m/z 512 [M+H]+.

Preparation of half oxalate ester pro-drugs 30 ACS Paragon Plus Environment


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To oxalyl chloride (0.50 mL; 5.91 mmol) cooled to 0oC was added t-butanol (0.28 mL; 2.95 mmol) over 30 min. After warming the solution to rt and concentration under reduced pressure, the residue was dissolved in 1 mL of CH2Cl2 whereupon 19 (100 mg; 0.219 mmol) and pyridine (42 uL; 0.522 mmol) were added. After the suspension was stirred at rt for 1.5 h, the solution was diluted with CH2Cl2, washed with 1N HCl and brine, dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under reduced pressure prior to purification of the residue by flash chromatography (silica gel, Hexanes/EtOAc; 100:0 to 1:1 gradient) to afford 120 mg (94%) of the desired of tert-butyl 1-(4-(6-(4chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-2-methylpropan-2-yl oxalate as a white foam. 1

H NMR (CDCl3) δ 1.55 (s, 9H), 1.70 (s, 6H), 3.87 (s, 3H), 4.24 (s, 2H), 6.92 (dd, J = 8.25 Hz, 2.20 Hz,

1H), 6.96 (d, J = 2.20 Hz, 1H), 7.04 (d, J = 8.24 Hz, 1H), 7.44 (d, J = 8.79 Hz, 2H), 7.53 (s, 1H), 7.65 (d, J = 8.79 Hz, 2H), 8.13 (s, 1H); 13C NMR (CDCl3) δ 23.14, 27.74, 56.36, 74.39, 84.26, 84.51, 111.76, 115.30, 119.27, 120.84, 123.22, 127.65, 129.47, 130.78, 131.54, 135.67, 148.19, 149.25, 150.64, 151.63, 156.77, 157.22, 157.35, 157.60; HPLC: 4.40 min retention time, (100%); MS (ES): m/z 585 [M+H]+.

A solution of tert-butyl 1-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2methoxyphenoxy)-2-methylpropan-2-yl oxalate (124 mg; 0.212 mmol) in 0.5 mL of TFA and 1 mL of CH2Cl2 was stirred at rt for 1.5 h. The solution was concentrated under reduced pressure to afford 102 mg (91%) of the oxalate pro-drug as a white foam. 1

H NMR (CDCl3) δ 1.70 (s, 6H), 3.86 (s, 3H), 4.30 (s, 2H), 6.91-6.94 (m, 2H), 7.09 (d, J = 8.80 Hz, 1H),

7.45 (d, J = 8.79 Hz, 2H), 7.66-7.69 (m, 3H), 8.74 (s, 1H); 13C NMR (CDCl3) δ 22.76, 56.26, 74.60, 85.93, 111.25, 116.62, 119.30, 122.64, 127.93, 129.47, 129.70, 130.35, 136.86, 148.88, 151.00, 151.55, 155.17, 155.98, 157.04, 158.59, 159.55, 159.95; 31 ACS Paragon Plus Environment

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HPLC: 4.13 min retention time, (99%); MS (ES): m/z 529 [M+H]+.

Preparation of half malonate ester pro-drugs A suspension of 19 (100 mg; 0.219 mmol), 3-tert-butoxy-3-oxopropanoic acid (83 uL; 0.538 mmol), diisopropylcarbodiimide (83 uL; 0.538 mmol) and 4-N,N-dimethylaminopyridine (27 mg; 0.219 mmol) in 1 mL of CH2Cl2 was stirred at rt for 17 h. After additional 3-tert-butoxy-3-oxopropanoic acid (83 uL; 0.538 mmol) and diisopropylcarbodiimide (83 uL; 0.538 mmol) were added, the suspension was stirred at rt for 3 h. The suspension was diluted with CH2Cl2 and filtered; whereupon, the filtrate was washed with 1N HCl, saturated aq. NaHCO3 and brine, dried over anhydrous MgSO4 and filtered. The residue, obtained after concentration of the filtrate, was purified by flash chromatography (silica gel, Hexanes/EtOAc; 100:0 to 1:1 gradient) to afford 104 mg (79%) of tert-butyl 1-(4-(6-(4-chlorophenyl)-4oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-2-methylpropan-2-yl malonate as a beige foam. 1

H NMR (CDCl3) δ 1.47 (s, 9H), 1.64 (s, 6H), 3.23 (s, 2H), 3.87 (s, 3H), 4.20 (s, 2H), 6.91 (dd, J = 8.24

Hz, 2.20 Hz, 1H), 6.95 (d, J = 2.20 Hz, 1H), 7.02 (d, J = 8.24 Hz, 1H), 7.44 (d, J = 8.24 Hz, 2H), 7.53 (s, 1H), 7.66 (d, J = 8.80 Hz, 2H), 8.13 (s, 1H); 13C NMR (CDCl3) δ 23.37, 27.92, 44.04, 56.23, 74.44, 81.83, 82.01, 111.53, 114.75, 119.20, 120.87, 123.22, 127.65, 129.47, 130.51, 131.54, 135.67, 148.19, 149.30, 150.49, 151.63, 156.79, 157.40, 165.85, 166.20; HPLC: 4.46 min retention time, (97%); MS (ES): m/z 599 [M+H]+.

Treatment with TFA employing previously described conditions yielded the malonate pro-drug. 1

H NMR (CDCl3) δ 1.65 (s, 6H), 3.23 (s, 2H), 3.89 (s, 3H), 4.32 (s, 2H), 6.91-6.94 (m, 2H), 7.09 (d, J =

8.80 Hz, 1H), 7.45 (d, J = 8.24 Hz, 2H), 7.55 (s, 1H), 7.65 (d, J = 8.79 Hz, 2H), 8.22 (s, 1H); 13C NMR



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(CDCl3) δ 22.89, 42.14, 56.20, 75.00, 83.88, 111.58, 116.26, 119.60, 120.54, 122.82, 127.72, 129.52, 130.00, 131.32, 135.95, 148.09, 149.58, 150.82, 152.62, 157.40, 157.58, 166.43, 167.52; HPLC: 4.22 min retention time; MS (ES): m/z 529 [M+H]+.

Preparation of half succinate ester pro-drugs To a mixture of 19 (140 mg, 0.306 mmol) and DMAP (56 mg, 0.459 mmol.) in DMA (0.6 mL) at 140 o

C was added succinic anhydride (183 mg, 1.83 mmol) over 5 hours. The reaction mixture was stirred at

140 °C for additional 2 hr and was then cooled to room temperature. After dilution with CH2Cl2, the crude product was purified silica gel chromatography. Elution with CH2Cl2 to CH2Cl2:MeCN:HOAc (90:5:5) afforded partially purified product which was recrystallized from EtOH to yield pure succinate pro-drug, 4-(1-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-2methylpropan-2-yloxy)-4-oxobutanoic acid, (90 mg, 53% yield) as an off-white solid. 1

H NMR (CDCl3) δ ppm 2.48 - 2.57 (m, 4 H), 3.90 (s, 3 H), 4.32 (s, 2 H), 6.89 - 6.96 (m, 2 H), 7.03 (d,

J=8.14 Hz, 1 H), 7.15 - 7.21 (m, 1 H), 7.45 (d, J=8.65 Hz, 2 H), 7.54 (s, 1 H), 7.66 (d, J=8.65 Hz, 2 H), 8.16 (s, 1 H). HPLC: 4.71 min retention time; MS (ES): m/z 557 [M+H]+.

Preparation of half glutarate ester pro-drugs A solution of 18 (100 mg, 0.225 mmol) in DMF (1 mL) was added to a solution of sodium hydride (8.1 mg, 0.338 mmol) in DMF (2 mL). The reaction was stirred at room temperature for 30 minutes. Glutaric anhydride (129 mg, 1.128 mmol) was then added and stirring was continued at room temperature for 18 hours. The reaction mixture was poured into a solution of 1.0 N HCl (40 mL) and extracted with EtOAc (30 mL). The EtOAc layer was dried over sodium sulfate and concentrated. The crude product was purified by prep-HPLC (ODS, water-MeOH_TFA 90:10:0.1 to 10:90:0.1 gradient) to 33 ACS Paragon Plus Environment

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yield the glutarate pro-drug, (R)-5-(1-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2methoxyphenoxy)propan-2-yloxy)-5-oxopentanoic acid (114 mg, white solid). 1

H NMR (CDCl3) δ ppm 1.81 - 1.92 (m, 2 H), 2.31 (q, 4 H), 3.89 (s, 3 H), 4.25 (s, 2 H), 6.88 - 6.96 (m,

2 H), 7.04 (d, J=8.65 Hz, 1 H), 7.15 - 7.21 (m, 1 H), 7.45 (d, J=8.65 Hz, 2 H), 7.54 (s, 1 H), 7.66 (d, J=8.65 Hz, 2 H), 8.16 (s, 1 H); HPLC: 3.50 min retention time; MS (ES): m/z 557 [M+H]+.

Preparation of glucoside pro-drugs



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To a mixture of 18 (100 mg, 0.225 mmol), silver carbonate (311 mg, 1.12 mmol) and 4 Å molecular sieves (500 mg) in chloroform (7 mL) was slowly added a solution of acetobromo-a-D-glucose (232 mg, 0.564 mmol) in CHCl3 (3 mL). The reaction mixture was stirred at reflux for 48 hours. After removal of the precipitated material by filtration, the filtrate was concentrated and subjected to ISCO flash chromatography (silica gel/hexane-EtOAc 100:0 to 0:100 gradient) to obtain the tetraacetate (139 mg, 80% yield) as a brown gum. To a solution of the tetraacetate (60 mg, 0.077 mmol) in MeOH (1 mL) was added a NaOMe solution freshly prepared by reaction of Na metal (75 mg, 3.12 mmol) with MeOH (3 mL). After stirring at room temperature for 1.5 hour, the mixture was concentrated, dissolved in H2O (10 mL) and loaded onto an ODS column (10 g). The column was initially eluted with H2O followed by a progressively increasing MeOH/H2O gradient to elute the glucoside pro-drug, (R)-6-(4-chlorophenyl)-3-(3-methoxy-4-(2((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yloxy)propoxy)phenyl)thieno[3,2-d]pyrimidin-4(3H)-one (39 mg, 83% yield) as a white solid. 1

H NMR ( MeOD) δ ppm 1.20 - 1.32 (m, 3 H), 3.06 - 3.15 (m, 1 H), 3.24 - 3.32 (m, 1 H), 3.52 - 3.63 (m,

1 H), 3.68 - 3.82 (m, 5 H), 3.90 - 4.04 (m, 2 H), 4.11 - 4.30 (m, 2 H), 4.33 - 4.59 (m, 1 H), 6.88 - 6.95 (m, 1 H), 7.02 - 7.10 (m, 2 H), 7.39 - 7.45 (m, 2 H), 7.59 - 7.64 (m, 1 H), 7.69 - 7.76 (m, 2 H), 8.22 8.29 (m, 1 H); HPLC: 2.65 min retention time; MS (ES): m/z 605 [M+H]+.

Preparation of phosphate pro-drugs A suspension of 19 (3.00 g; 6.56 mmol), 1,2,4-triazole (1.36 g; 19.7 mmol) and dibenzyl N,Ndiisopropylphosphoramidite (6.62 mL; 19.7 mmol) in 40 mL of CH2Cl2 was heated at reflux for 16 h. The solution was cooled to rt; whereupon, 30% H2O2 in water (4.00 mL; 35.3 mmol) was added. After stirring at rt for 2.5 h, the reaction was diluted with CH2Cl2, washed with 1M sodium metabisulfite, 1N 35 ACS Paragon Plus Environment

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HCl, water and brine, dried over anhydrous MgSO4 and filtered. After concentration of the filtrate under reduced pressure, the residue was purified by flash chromatography (silica gel, Hexanes/EtOAc; 100:0 to 0:100 gradient) to afford 3.79 g (80%) of dibenzyl 1-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-2-methylpropan-2-yl phosphate as a white solid. 1

H NMR (CDCl3) δ 1.65 (s, 6H), 3.75 (s, 3H), 4.07 (s, 2H), 5.04-5.06 (m, 4H), 6.87-6.96 (m, 3H), 7.29-

7.35 (m, 10H), 7.44 (d, J = 8.24 Hz, 2H), 7.53 (s, 1H), 7.66 (d, J = 8.25 Hz, 2H), 8.10 (s, 1H); 13C NMR (CDCl3) δ 22.56, 25.04, 56.08, 68.98, 69.03, 75.36, 82.87, 111.30, 113.91, 119.15, 120.87, 127.42, 127.67, 127.88, 128.33, 128.48, 129.47, 130.33, 131.54, 135.69, 136.07, 148.19, 149.10, 150.19, 151.63, 156.79, 157.37; HPLC: 4.85 min retention time, (100%); MS (ES): m/z 717 [M+H]+.

A solution of dibenzyl 1-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2methoxyphenoxy)-2-methylpropan-2-yl phosphate (166 mg; 0.231 mmol) in 2 mL of TFA and 0.11 mL of water was stirred at rt for 2 h. The solution was diluted with methanol and concentrated under reduced pressure. The residue was purified by prep HPLC to afford 59 mg (48%) of the phosphate prodrug, 1-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-2methylpropan-2-yl dihydrogen phosphate, as a white solid. 1

H NMR (DMSO-d6) δ 1.52 (s, 6H), 3.79 (s, 3H), 4.03 (s, 2H), 7.04-7.12 (m, 2H), 7.20 (s, 1H), 7.57 (d,

J = 8.25 Hz, 2H), 7.91 (d, J = 8.80 Hz, 2H), 7.96 (s, 1H), 8.39 (s, 1H); 13C NMR (DMSO-d6) δ 25.00, 56.42, 75.75, 79.18, 112.54, 113.80, 120.13, 122.12, 122.40, 128.25, 129.66, 130.47, 131.61, 134.67, 148.84, 149.52, 149.88, 150.18, 156.48, 157.82; HPLC: 3.73 min retention time, (98%); MS (ES): m/z 537 [M+H]+.

Preparation of 27 36 ACS Paragon Plus Environment


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Following the procedure described by Calverley33, Br2 (21.72 mL, 422 mmol) was added over 5 min to a solution of 1-cyclopropylethanone (35.44 g, 421 mmol) in MeOH (250 mL) at 0°C. After removal of the ice bath, the mixture was stirred at 20 °C for another 0.5 h; whereupon, 30 ml of water was added. After stirring an additional 15 min, 90 ml water was added prior to extraction of the resulting mixture with 200 mL of Et2O (4x). The combined organic layers were sequentially washed with 1M Na2CO3 (150 ml) and brine (100 ml) and dried over anhydrous MgSO4. After filtration, the filtrate was concentrated using a rotary evaporator prior to distillation at 13 mm Hg to yield 40.9 g (59%) of 29, 2-bromo-1cyclopropylethanone as a colorless oil bp 58 – 62 °C. 1

H NMR (500 MHz, (CDCl3) δ 0.95-1.03 (m, 2H), 1.08–1.15 (m, 2H), 2.13–2.21 (m, 1H), 4.00 (s, 2H)

1-Cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 30 An orange suspension of 4-nitroguaiacol potassium salt hydrate (31.7 g, 153 mmol) and 29 (29.4 g, 180 mmol) in DMF (310 mL) was heated at 80 °C for 1h. The resulting yellow reaction mixture was diluted with H2O (932 ml) and stirred for 4 hr at 20 °C. Subsequent filtration yielded a yellow filter cake which after washing 3x with 150 mL of H2O and air drying yielded 34.6g (90%) of 1-cyclopropyl-2-(2methoxy-4-nitrophenoxy)ethanone as a light yellow solid. 1

H NMR (400 MHz, CDCl3): δ 0.95-1.03 (m, 2H), 1.13-1.18 (m, 2H), 2.15-2.23 (m, 1H), 3.95 (s, 3H),

4.86 (s, 2H), 6.73 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 2.7 Hz, 1H), 7.82 (dd, J = 8.7, 2.7 Hz, 1H). 13

C NMR (125 MHz, CDCl3) δ 205.2, 152.7, 149.1, 117.3, 111.6, 106.9, 73.5, 56.3, 17.1, 12.0.

HPLC: 2.98 min retention time; LC/MS: m/e 252.3 (M+H); 4 min gradient; 2.35 min retention.

(R)-1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanol 31 37 ACS Paragon Plus Environment

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To a yellow suspension of 30 (34.6 g, 138 mmol) in 356 mL of EtOH at 0 °C was added NaBH4 (3.1 g, 82 mmol) over 15 min. After removal of the ice bath, the temperature was not allowed to exceed 20 °C while the reaction was stirred for 35 additional min. The stirred reaction was cooled to ~10 °C prior to cautious slow addition of HOAc (12 mL, 210 mmol). After stirring for 0.5 hr once gas evolution had ceased, the yellow suspension was concentrated under vacuum to remove ~ 300mL of EtOH. The resulting precipitate was collected by filtration, washed with H2O and air dried to yield a light yellow solid (28.7g). Filtration, after further concentration of the filtrate to remove most of the EtOH, yielded an additional 4.9 g of desired product. The two fractions were combined to yield 33.6g (96%) of racemic 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanol. Racemic 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanol (45.1g, mmol) in 2/1 MeCN/i-PrOH (451 mL) was resolved by chiral chromatography resolution using a Chiralpak AD-H (3x25cm, 5µm) column under the Chiral- SFC conditions. The chromatographic conditions employed an 85/15 mixture of CO2/i-PrOH as the mobile solvent with a flow rate of 130 mL/min at 35 °C with the BPR pressure maintained at 100 bar and detector wavelength at 234 nM. Each 0.7 mL injection required a run time of 7 min. The chiral purity of the R enantiomer was determined using analytical SFC conditions to be greater than 99.9% at 234 nm based on SFC/ UV area%. Concentration of the resultant eluant under vacuum followed by subsequent dissolution in 150 ml EtOH and reconcentration yielded a yellow oil which solidified to form a light yellow solid (20.9 g, 93%) upon drying under high vacuum overnight. 1

H NMR (400 MHz, CDCl3): δ 0.30-0.37 (m, 1H), 0.42-0.50 (m, 1H), 0.55-0.69 (m, 2H), 0.97-1.08 (m,

1H), 2.40-2.70 (bs, 1H), 3.41 (ddd, J = 8.3, 8.3, 2.7 Hz, 1H), 3.93 (s, 3H), 4.10 (dd, J = 9.3, 8.0 Hz, 1H), 4.23 (dd, J = 9.3, 2.7 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.8, 2.2 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 153.7, 149.2, 141.7, 117.6, 111.5, 106.7, 74.4, 73.5, 56.2, 13.4, 2.7, 2.0. HPLC: 3.23 min retention time; LC/MS: m/e = 254.3 (M+H). 38 ACS Paragon Plus Environment


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(R)-2-(4-amino-2-methoxyphenoxy)-1-cyclopropylethanol 32 To a solution of 31 (20.90 g, 83 mmol) in EtOH (546 ml) was added 5% Pd/C (3.0 g, 0.705 mmol). The suspension was hydrogenated (1 atm. H2, balloon) at 20 °C for 2.5 h; whereupon, LC/MS analysis revealed the reaction to be complete. After filtration of the reaction mixture through Celite pad and subsequent washing of the cake with EtOH, the filtrate was concentrated under vacuum using a rotary evaporator to yield (R)-2-(4-amino-2-methoxyphenoxy)-1-cyclopropylethanol as a brownish solid. (18.34 g, 100%). 1

H NMR (400 MHz, CDCl3): δ 0.18-0.27 (m, 1H), 0.38-0.43 (m, 1H), 0.45-0.61 (m, 2H), 0.82-0.92 (m,

1H), 3.21 (ddd, J = 8.8, 8.8, 2.6 Hz, 1H), 3.80 (s, 3H), 3.86 (dd, J = 10.1, 8.8 Hz, 1H), 4.09 (dd, J = 10.1, 2.6 Hz, 1H), ), 6.21 (dd, J = 8.3, 2.7 Hz, 1H). 6.29 (d, J = 2.7 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H). 13

C NMR (125 MHz, CDCl3) δ 151.2, 142.1, 140.8, 118.7, 106.9, 100.5, 76.5, 74.4, 55.7, 12.9, 2.5, 1.6.

LC/MS: m/e 224.5 (M+H);.

(E)-Methyl 5-(4-chlorophenyl)-3-(2-(dimethylamino)vinyl)thiophene-2-carboxylate 33 To a mixture of commercially available methyl 3-amino-5-(4-chlorophenyl)thiophene-2-carboxylate (75 g, 279 mmol) in EtOH (450 mL) was added 1,1-dimethoxy-N,N-dimethylmethanamine (56 mL, 420 mmol). The stirred reaction mixture was heated to reflux; whereupon within 30 min, the suspension became a clear solution. LC/MS analysis revealed that the reaction was complete after 4 hr. The mixture was cooled to room temperature and then concentrated under vacuum using a rotary evaporator to obtain a yellow-green oil. After addition of Et2O (100 mL), the mixture was stirred as seed crystals were added. Continuation of stirring resulted in a rapid formation of a precipitate which was collected by 39 ACS Paragon Plus Environment

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filtration. After drying overnight under vacuum, 74.9g of a light yellow solid was obtained. Concentration of the filtrate yielded another 4.5g resulting in a combined yield of 79.4g (88%) of methyl 5-(4-chlorophenyl)-3-(2-(dimethylamino)vinyl)thiophene-2-carboxylate. 1

H NMR (400 MHz, CDCl3): δ 3.06 (s, 3H), 3.08 (s, 3H), 3.81 (s, 3H), 6.98 (s, 1H), 7.33-7.38 (m, 2H),

7.51-7.56 (m, 2H), 7.68 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 163.2, 159.1, 156.0, 145.7, 134.4, 132.2, 129.1, 126.9, 122.3, 112.4, 51.4, 40.2, 34.3.

LC/MS: m/e 323.3 (M+H)..

(R)-6-(4-chlorophenyl)-3-(4-(2-cyclopropyl-2-hydroxyethoxy)-3-methoxyphenyl)-thieno[3,2d]pyrimidin-4(3H)-one 27 A mixture of 33 (85 g, 263 mmol), aniline 32 (52 g, 233 mmol) and phenol (230 g, 2444 mmol) was heated at 130 °C for 30 min. After cooling the black sticky syrup to rt, the resulting mixture was diluted with Et2O (300 mL), stirred at room temperature for 20 min, then filtered and washed with Et2O (600 mL). Dissolution of the filter cake in CH2Cl2 (200 mL) generated an orange solution which, upon being stirred after dilution with Et2O (400 mL), generated a precipitate. The resulting solid was collected by filtration and dried in oven to give the desired title compound as an off-white solid (81 g, 74.2% yield). 1

H NMR (500 MHz, DMSO-d6) δ 0.29 - 0.45 (m, 4 H), 0.91 - 1.01 (m, 1 H), 3.34 - 3.39 (m, 1 H), 3.79

(s, 3 H), 3.96 - 4.05 (m, 2 H), 7.04 (dd, 1 H), 7.13 (d, J=8.2 Hz, 1 H), 7.19 (s, 1 H), 7.58 (d, J=8.8 Hz, 2 H), 7.92 (d, J=8.2 Hz, 2 H), 7.97 (s, 1 H), 8.40 (s, 1 H); 13C NMR (125 MHz, DMSO-d6) δ 1.33, 1.66, 14.11, 55.79, 71.16, 73.18, 111.86, 112.81, 119.61, 121.71, 122.04, 127.84, 129.27, 129.68, 131.22, 134.27, 148.61, 148.99, 149.48, 149.78, 156.09, 157.40

LC/MS: m/e 469.3 (M+H);



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Preparation of 35 bis(2-(trimethylsilyl)ethyl) diisopropylphosphoramidite A solution of 2-(trimethylsilyl)ethanol (12.6 g; 106.55 mmoles) and Et3N (15.4 g; 152.19 mmoles) in Et2O (84 mL) was added dropwise over 30 minutes under N2 to a stirred 0 to -2 °C solution of diisopropylphosphoramidous dichloride (10.8 g 50.78 mmoles) in diethyl ether (53 mL).

A mild

exotherm (+1-2 °C) accompanied the formation of a thick white suspension. After stirring overnight at 20°C, the mixture was filtered. The resultant cake was washed twice with 30 mL portions of Et2O. The combined filtrates were washed 2 x 100 mL of saturated aqueous NaHCO3 followed by 40 mL of brine. After drying over MgSO4 and concentrating to dryness under vacuum at room temperature, bis-(2(trimethylsilyl)ethyl) diisopropylphosphoramidite (18.12 g; 49.6 mmoles; 97.6% yield) was obtained as a clear colorless liquid. 1

H NMR δ (CDCl3): 3.90-3.78 (m, 4H), 3.77-3.68 (m, 2H), 1.31 (d, J= 6.6 Hz, 12H), 1.17-1.12 (m, 4H),

0.15 (s, 18H); 13C NMR δ (CDCl3): 60.7 (2, d, JC-P = 19.1 Hz, 2C), 42.7 (1, d, JC-P = 12.7 Hz, 2C), 24.6 (3, d, JC-P = 7.6 Hz, 4C), 20.1 (2, d, JC-P = 7.6 Hz, 2C), -1.4 (3, 6C). 31

P NMR δ (CDCl3): 143.5 (s)

(R)-2-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-1cyclopropylethyl bis(2-(trimethylsilyl)ethyl) phosphate 34 Addition of 1H-1,2,4-triazole (1.89 g; 27.02 mmoles) to a 250 mL 3-neck round bottom flask containing a stirred solution of 27 (6.33 g; 13.50 mmoles) in anhydrous CH2Cl2 (65 mL) at 20 °C under N2 produced a thick white suspension. To the resulting thick white suspension was added bis(2(trimethylsilyl)ethyl) diisopropylphosphoramidite (9.8 g; 26.80 mmoles). 41 ACS Paragon Plus Environment

After heating the stirred

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reaction mixture at reflux for 18 hr, HPLC analysis revealed complete conversion. The reaction mixture was cooled to -3 to -4 °C prior to dropwise addition of H2O2 (8.8 mL; 100.14 mmoles) over 15 minutes. Note the exotherm was most severe during addition of the first 1.3-1.5 mL of the H2O2. After the addition was complete, the reaction was stirred for 2 hrs at 0-5 °C; whereupon, HPLC analysis revealed the oxidation to be have generated a new component (~92.9-93 AP). The reaction was quenched by dropwise addition of cold 60 mL of 1N aqueous Na2S2O5 over 12-15 minutes. Note a cooling bath was required as the first 15-20 mL of the quench produced an exotherm resulting in the temperature rising to 17-18 °C; the remainder of the addition was endothermic. The mixture was stirred for 20 minutes at 1015 °C prior to separating the phases. The organic layer was washed sequentially with 70 mL of 1N HCl, 65 mL of H2O and 50 mL of brine prior to drying over MgSO4. After filtration, the filtrate was concentrated to approximately 30 mL using a rotary evaporator. The residue was redissolved in 65 mL of methyl t-butyl ether (MTBE); reconcentration to ~ 30-35 mL produced a slightly hazy residue. Dilution with an additional 35 mL of MTBE and 45 mL of hexanes induced formation of a solid. Concentration of the residue to dryness yielded 24.5 g of a white solid which upon trituation with 40 mL of hexanes produced a suspension that after further dilution with 40 mL hexanes + 5 mL of MTBE was collected by filtration. The cake was washed twice with 21 mL each of 95:5 hexanes/MTBE and airdried for 1 hr on the filter with vacuum suction. After drying over the week-end at 20 °C under vacuum, (R)-2-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-1cyclopropylethyl bis(2-(trimethylsilyl)ethyl) phosphate (9.64 g; 12.86 mmoles; 95.30% yield) was obtained as a white crystalline product with 96.6% purity. 1

H NMR δ (500 MHz, CDCl3): 8.10 (s, 1H), 7.64 (d, J= 8.8 Hz, 2H), 7.51 (s, 1H), 7.42 (d, J= 8.8 Hz,

2H), 7.05 (d, J= 8.8 Hz, 1H), 6.94 (d, J= 2.7 Hz, 1H), 6.90 (dd, J= 8.8, 2.7 Hz, 1H), 4.31-4.20 (m, 2H), 4.21-4.80 (m, 4H), 4.80-4.00 (m, 1H), 3.85 (s, 3H), 1.30-1.18 (m, 1H), 1.13-1.04 (m, 4H), 0.70-0.60 (m, 3H), 0.47-0.38 (m, 1H), 0.02 (s, 18 H); 13C NMR δ (125 MHz, CDCl3): 157.4, 156.8, 151.7, 150.3, 42 ACS Paragon Plus Environment


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149.1, 148.2, 135.7, 131.6, 130.5, 129.5, 127.7, 123.2, 120.1, 119.2, 114.3, 111.4, 81.1 (d, JC-P = 5.1 Hz), 71.8 (d, JC-P = 5.1 Hz), 66.1 (d, JC-P = 6.4 Hz, 2C), 56.2, 19.6 (d, JC-P = 6.4 Hz), 13.1 (d, JC-P = 5.1 Hz), 3.6, 3.0, -1.5 . 31

P NMR δ ( CDCl3): -1.11 (m, JP-H = 7.4 Hz)

MS (electrospray, + ions) m/z 749, 751.

(R)-2-(4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)-1cyclopropylethyl dihydrogen phosphate 35 A mixture of 34 (35.27 g, 47.06 mmoles) and anhydrous CH2Cl2 (315 mL) in a 500 mL Chemglass jacketed reactor was stirred at 20 °C until dissolution was complete; whereupon, the internal temperature was reduced to -2 °C. Once the temperature had stabilized, TFA (30.2 mL; 399.40 mmoles) was added dropwise to the stirred solution taking care to maintain the internal reaction temperature between -0.5 °C and 1 °C since higher temperatures will promote solvolytic decomposition. Aliquots were periodically withdrawn to monitor the reaction progress by HPLC analysis. Immediately following completion of the TFA addition, HPLC analysis revealed the composition to be 9.29% starting 34, 44.78% monodeprotection, 42.2% 35, 1.21% 27 and 1.25% of the main side-product.

After 35 min, the

composition was 0.05% starting 34, 5.34% monodeprotection, 90.47% 35, 1.35% 27 and 1.99% of main side-product. After 64 min, the composition was 0.0% starting 34, 0.62% monodeprotection, 94.36% 35, 1.52% 27 and 2.69% of main side-product. After 95 minutes, the reaction was terminated by reduction of the reaction temperature to - 3 °C prior to the addition of MeOH (28.5 mL) over 5 min. After stirring for 30 min between - 1 and 0 °C, the volume was reduced under vacuum at 15 °C to ~ 134 mL. After warming to 19 °C, slow addition of 120 mL of MTBE over 12 min produced a white precipitate which was stirred for 2 hours at 19-20 °C prior to collection of the solid by filtration. The filter cake was 43 ACS Paragon Plus Environment

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washed twice with 120 mL of MTBE/CH2Cl2 2.5:1 v/v, air-dried for 15 min with vacuum suction before drying overnight in a. vacuum oven at 45 °C. The crude product was recrystallized by heating to 55-57 °C in 200 mL of THF, and 16 mL of water with stirring to achieve complete dissolution. The solution was heated at 60 °C for an additional 15 min, cooled to 45 °C over 10 min; whereupon 50 mL of acetone was added over ca 5 min while maintaining the temperature above 44 °C throughout the addition. Once seeding with 35 induced crystallization, acetone (245 mL) was added over 30 minutes while maintaining the temperature above 42.5 °C. The resultant thick slurry was cooled to 22 °C over ca 60 minutes and stirred for 90 min at 20-21 °C before collecting the solid by filtration. Both the reactor and the filter cake were washed first with 120 mL of acetone/THF 3:1 v/v and then with acetone (110 mL). After air drying for 40 min with vacuum suction, the solid was dried in a vacuum oven at 50 °C for 18 hr to yield 18.96 g of 35 (99.4% purity in 73.40% yield). 1

H NMR (500 MHz, DMSO-d6) δ 0.41 (m, 2H), 0.52 (m, 2H), 1.26 (m, 1H), 3.82 (m, 1H), 4.20 (d, 2H,

J = 4.29 Hz), 3.80 (s, 3H),7.06 (dd, 1H, J = 8,57, J = 2.34 Hz), 7.15 (d, 1H, J = 8.57Hz), 7.22 (d, 1H, J = 2.34 Hz), 7.58 (d, 2H, J = 8.57 Hz), 7.93 (2H, J = 8.57 Hz), 7.98 (s, 1H), 8.40 (s, 1H) 13

H NMR (126 MHz, DMSO-d6) δ 2.4, 3.1, 13.1, 56.0, 71.0, 77.9, 112.2, 113.1, 119.8, 121.9, 122.1,

128.0, 129.4, 130.1, 131.3, 134.4, 148.4, 149.1, 149.6, 149.9, 156.2, 157.5 31

P NMR δ (162 MHz, DMSO-d6)): -0.75

HPLC: 95.4% API; LC/MS: m/e 549.1 (M+H); High Res. Mass: C24H23O7N2ClPS calc. 549.06522; exp. 549.06531.

Spectral Characterization of Examples 6-17, 21-23, 25 and 26



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6: 1H NMR (CDCl3) δ 2.02-2.08 (m, 2H), 2.39-2.42 (m, 2H), 3.65-3.68 (m, 2H), 3.74-3.76 (m, 2H), 3.89 (s, 3H), 4.21-4.23 (m, 2H), 6.93 (dd, J = 8.25 Hz, 2.20 Hz, 1H), 6.97 (d, J = 2.20 Hz, 1H), 6.99 (d, J = 8.25 Hz, 1H), 7.44 (d, J = 8.24 Hz, 2H), 7.53 (s, 1H), 7.66 (d, J = 8.25 Hz, 2H), 8.14 (s, 1H); 13C NMR (CDCl3) δ 18.20, 30.77, 42.22, 48.99, 56.11, 67.92, 111.03, 113.22, 119.18, 120.84, 123.17, 127.63, 129.43, 130.28, 131.50, 135.66, 148.12, 148.72, 149.90, 151.66, 156.79, 157.38, 175.36; LCMS: m/z 496 [M+H]+.

7: 1H NMR (CDCl3) δ 1.84-1.91 (m, 2H), 1.96-2.03 (m, 2H), 3.52-3.59 (m, 4H), 3.91 (s, 3H), 4.76 (s, 2H), 6.91 (dd, J = 8.35 Hz, 2.63 Hz, 1H), 6.98 (d, J = 2.20 Hz, 1H), 7.08 (d, J = 8.79 Hz, 1H), 7.45 (d, J = 8.35 Hz, 2H), 7.53 (s, 1H), 7.66 (d, J = 8.79 Hz, 2H), 8.13 (s, 1H) LCMS: m/z: 496 [M+H].

9: 1H NMR (CDCl3) δ 1.15 (t, 6H, J = 7.4 Hz), 3.28 (q, 4H, J = 7.4 Hz), 3.7 (m, 2H), 3.89 (s, 3H), 4.18 (t, 2H, J = 4.8Hz), 4.97 (m, 1H), 6.93 (dd, 1H, J= 8.4 Hz, J = 2.2 Hz), 6.97 (d, 1H, J = 2.2 Hz), 7.05 (d, 1H, J = 8.4 Hz), 7.44 (d, 2H, JAB = 8.3 Hz), 7.54 (s, 1H), 7.66 (d, 2H, JAB = 8.3 Hz), 8.14 (s, 1H) LCMS: m/z: 527 [M+H].

10: 1H NMR (CDCl3) δ 3.07 (s, 3H), 3.71 (br s, 5H), 3.88 (s, 3H), 4.17-4.27 (m, 2H), 6.92-7.04 (m, 3H), 7.44 (d, J = 8.25 Hz, 2H), 7.55 (s, 1H), 7.66 (d, J = 7.70 Hz, 2H), 8.19 (s, 1H) LCMS: m/z: 500 [M+H].

11:

1

H NMR (CDCl3) δ 1.16 (t, 0.75H, J = 7 Hz), 1.24 (t, 2.25H, J = 7 Hz), 2.13 (s, 2.25H), 2.24(s,

0.75H), 2.25 (s, 0.75H), 3.49 (q, 2 H, J = 7 Hz), 3.76 (t, 2H, J = 5.7 Hz), 3.87 (s, 0.75H), 3.885 (s, 2.25H), 4.16 (t, 0.5 5H, J = 5.7 Hz), 4.26 (t, 1.5H, J = 5.7 Hz), 6.95 (m, 2H), 7.06 (d, 1H, 9 Hz), 7.44 (d, 45 ACS Paragon Plus Environment

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2H, JAB = 8.7 Hz), 7.53 (s, 1H), 7.66 (d, 2H, JAB = 8.7 Hz), 8.14 (s, 1H). (Rotamers coalesce upon heating); LCMS: m/z: 498 [M+H].

12: 1H NMR (CDCl3) δ 1.14 (t, 3H), 1.22 (t, 3H), 3.38-3.43 (m, 4H), 3.89 (s, 3H), 4.80 (s, 2H), 6.89 (dd, J = 8.80 Hz, 2.20 Hz, 1H), 6.97 (d, J = 2.20 Hz, 1H), 7.05 (d, J = 8.25 Hz, 1H), 7.43 (d, J = 8.24 Hz, 2H), 7.51 (s, 1H), 7.64 (d, J = 8.25 Hz, 2H), 8.12 (s, 1H) LCMS: m/z: 498 [M+H].

13: 1H NMR (CDCl3) δ 3.06 (s, 3H), 3.60 (m, 2H), 3.89 (s, 3H), 4.20 (t, 2H, J = 4.8Hz), 4.99 (m, 1H), 6.95 (dd, 1H, J= 8.8 Hz, J = 2.2 Hz), 6.98 (d, 1H, J = 2.2 Hz), 7.03 (d, 1H, J = 8.8 Hz), 7.45 (d, 2H, JAB = 8.7 Hz), 7.54 (s, 1H), 7.66 (d, 2H, JAB = 8.7 Hz), 8.14 (s, 1H) LCMS: m/z: 506 [M+H].

14: 1H NMR (CDCl3) δ 2.03 (s, 3H), 3.72 (m, 2H), 3.91 (s, 3H), 4.16 (t, 2H, J = 4.8Hz), 6.13 (m, 1H), 6.94 (dd, 1H, J= 8.4 Hz, J = 2.2 Hz), 6.98 (d, 1H, J = 2.2 Hz), 7.02 (d, 1H, J = 8.4 Hz), 7.45 (d, 2H, JAB = 8.7 Hz), 7.54 (s, 1H), 7.66 (d, 2H, JAB = 8.7 Hz), 8.14 (s, 1H); LCMS m/z: 498 [M+H].

15: 1H NMR (CDCl3) δ 3.65 (m, 2H), 3.70 (s, 3H), 3.89 (s, 3H), 4.14 (t, 2H, J = 4.8Hz), 4.97 (m, 1H), 6.93 (dd, 1H, J= 8.4 Hz, J = 2.2 Hz), 6.97 (d, 1H, J = 2.2 Hz), 7.02 (d, 1H, J = 8.4 Hz), 7.44 (d, 2H, JAB = 8.3 Hz), 7.54 (s, 1H), 7.66 (d, 2H, JAB = 8.3 Hz), 8.14 (s, 1H) LCMS: m/z: 486 [M+H].



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16: 1H NMR (CDCl3) δ 3.89 (s, 3H), 3.93 (s, 3H), 6.9 – 7.0 (m, 3H), 7.43 (d, 2H, JAB = 8.7Hz), 7.51 (s, 1H), 7.63 (d, 2H, JAB = 8.7Hz), 8.12 (s, 1H) LCMS: m/z: 399 [M+H].

17: 1H NMR (DMSO-d6) δ 3.72-3.75 (m, 2H), 3.77 (s, 3H), 4.03 (t, 2H), 4.91 (t, 1H), 7.03 (dd, J = 8.80 Hz, 2.20 Hz, 1H), 7.10 (d, J = 8.80 Hz, 1H), 7.18 (d, J = 2.75 Hz, 1H), 7.56 (d, J = 8.79 Hz, 2H), 7.91 (d, J = 8.80 Hz, 2H), 7.97 (s, 1H), 8.39 (s, 1H) LCMS m/z: 429 [M+H].

21a: 1H NMR (DMSO-d6) δ 3.43-3.50 (m, 2H), 3.77 (s, 3H), 3.77-3.85 (m, 1H), 3.90-3.94 (m, 1H), 4.02-4.05 (m, 1H), 4.68 (t, 1H), 4.98 (d, J = 4.83 Hz, 1H), 7.03 (dd, J = 8.35 Hz, 2.20 Hz, 1H), 7.10 (d, J = 8.79 Hz, 1H), 7.18 (d, J = 2.20 Hz, 1H), 7.57 (d, J = 8.35 Hz, 2H), 7.92 (d, J = 8.35 Hz, 2H), 7.97 (s, 1H), 8.39 (s, 1H) LCMS: m/z: 459 [M+H]. 21b: 1H NMR (DMSO-d6) δ 1.15 (s, 3H), 3.32-3.43 (m, 2H), 3.78-3.90 (m, 5H), 4.53 (s, 1H), 4.65 (t, 1H), 7.02 (dd, J = 8.25 Hz, 2.20 Hz, 1H), 7.10 (d, J = 8.24 Hz, 1H), 7.17 (d, J = 2.75 Hz, 1H), 7.57 (d, J = 8.80 Hz, 2H), 7.91 (d, J = 8.25 Hz, 2H), 7.96 (s, 1H), 8.38 (s, 1H) LCMS: m/z: 473 [M+H].

22a: 1H NMR (DMSO-d6) δ 3.77 (s, 3H), 3.92-3.97 (m, 2H), 4.24-4.29 (m, 1H), 6.73-6.76 (m, 1H), 7.01 (dd, J = 8.79 Hz, 2.64 Hz, 1H), 7.08-7.16 (m, 3H), 7.57 (d, J = 8.79 Hz, 2H), 7.92 (d, J = 8.79 Hz, 2H), 7.97 (s, 1H), 8.40 (s, 1H) LCMS: m/z: 473 [M+H].


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22b: 1H NMR (CD3OD) δ 1.52 (s, 3H), 3.90 (s, 3H), 4.09 (d, J = 9.67 Hz, 1H), 4.34 (d, J = 9.66 Hz, 1H), 7.00 (dd, J = 8.35 Hz, 2.19 Hz, 1H), 7.09 (d, J = 2.63 Hz, 1H), 7.13 (d, J = 8.35 Hz, 1H), 7.48 (d, J = 8.79 Hz, 2H), 7.63 (s, 1H), 7.75 (d, J = 8.35 Hz, 2H), 8.29 (s, 1H) LCMS: m/z: 487 [M+H].

23: 1H NMR (CDCl3) δ 1.46 (t, 3H), 3.16-3.38 (m, 5H), 3.89 (s, 3H), 4.14-4.15 (m, 2H), 4.63-4.69 (m, 1H), 6.95 (dd, J = 8.35 Hz, 2.63 Hz, 1H), 6.99 (d, J = 2.20 Hz, 1H), 7.06 (d, J = 8.35 Hz, 1H), 7.45 (d, J = 8.35 Hz, 2H), 7.54 (s, 1H), 7.66 (d, J = 8.35 Hz, 2H), 8.14 (s, 1H) LCMS: m/z: 535 [M+H].

25: 1H NMR (DMSO-d6) δ 3.78 (s, 3H), 4.10-4.13 (m, 1H), 4.22-4.25 (m, 1H), 4.40-4.45 (m, 1H), 6.71 (d, J = 6.59 Hz, 1H), 7.06 (dd, J = 8.25 Hz, 2.20 Hz, 1H), 7.18 (d, J = 8.80 Hz, 1H), 7.23 (d, J = 2.20 Hz, 1H), 7.57 (d, J = 8.80 Hz, 2H), 7.91 (d, J = 8.80 Hz, 2H), 7.97 (s, 1H), 8.39 (s, 1H) LCMS: m/z: 497 [M+H].

26: 1H NMR (DMSO-d6) δ 2.28-2.56 (m, 2H), 3.68 (s, 3H), 3.82-3.93 (m, 2H), 4.04-4.05 (m, 1H), 5.37 (d, J = 6.05 Hz, 1H), 6.95 (dd, J = 8.25 Hz, 2.20 Hz, 1H), 7.02 (d, J = 8.79 Hz, 1H), 7.11 (d, J = 2.20 Hz, 1H), 7.46 (d, J = 8.79 Hz, 2H), 7.81 (d, J = 8.79 Hz, 2H), 7.86 (s, 1H), 8.29 (s, 1H) LCMS m/z: 511 [M+H]. Bioological Assays Determination of binding affinity (Ki) Membranes from stably transfected HEK-293 cells expressing a mutated (E4Q, A5T) hMCHR1 receptor were prepared by dounce homogenization and differential centrifugation. Binding experiments were carried out with 0.5 -1.0 ug of membrane protein incubated in a total of 0.2 ml in 25 mM HEPES 48 ACS Paragon Plus Environment


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(pH 7.4) with 10 mM MgCl2, 2 mM EGTA, and 0.1% BSA (Binding Buffer) for 90 min. For competition binding assays, reactions were carried out in the presence of with 0.06 -0.1 nM [Phe13, [125I]Tyr19]-MCH and increasing concentrations of unlabeled test molecules. Reactions were terminated by rapid vacuum filtration over 96 well-GFC Unifilter plates pre-coated with 0.075 ml binding buffer containing 1% BSA, and washed 3 times with 0.4 ml of Phospho-buffered Saline (pH 7.4) containing 0.01% TX-100. Filters were dried, 0.05 ml microscint 20 was added to each well and radioactivity was subsequently quantified by scintillation counting on a TopCount microplate scintillation counter (Packard). Inhibitory constants were determined by nonlinear least squares analysis using a four parameter logistic equation. Determination of MCHR functional Activity Stable HEK-293 cells expressing human MCHR1 or MCHR2 receptor were plated at a density of 50,000 cells / well in 96 well poly-lysine coated plates (BD #35-6640) and cultured overnight in DMEM (high glucose (4.5 g/ml), 25 mM HEPES, pH 7.4, 10% Fetal Bovine Serum, 1 mM Na Cl) at 37°, 5% CO2 conditions. For assay, media was replaced with 90 ml per well dye solution consisting of 3.8 mM Fluo4 AM (Invitrogen #F14201), 0.04% Pluronic F-127 (Invitrogen #P3000MP), 2.5 mM Probencid (Sigma #P8761), in Base Buffer (Hank’s balanced salt solution, 25 mM HEPES, 0.1% BSA). Dye solution was allowed to ‘load’ for 1 hr at room temperature in subdued light. Dye was subsequently removed and replaced with 75 ml of Base Buffer and 75 ml diluted test compound and incubated for an additional 15 minutes. Test compound dilution plates were prepared by serial diluting test and reference compounds from 100% DMSO stocks first 1:50 in Base Buffer and then serially (1:3.26) in Base Buffer containing 2% DMSO to generate twelve half log test concentrations. Serially diluted test compounds were assessed for their ability to inhibit MCH-stimulated calcium flux initiated by a fixed concentration of MCH (20% to 90% of full agonism). Agonist source 49 ACS Paragon Plus Environment

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plates also contained a reference MCH twelve point concentration response curve (CRC). 50 ml of agonist from agonist source plate was injected into the cell plates and then measures of fluorescence over a 150 second window (Ex 488 nm, Em 515-570 nm) in a FLIPR instrument (Molecular Devices). The ∆RFU data for each concentration response curve was analyzed by nonlinear regression analysis using a four parameter logistic equation in Excel Fit (Equation 205) to determine IC50 or EC50, Hill slope, as well as the maximum, and minimum RFU for each CRC. Kb’s were calculated using the modified Cheng-Prusoff correction from the IC50 values using the EC50, Hill slope, and concentration of the agonist peptide.34 Protocol for efficacy studies using young growing rat model

Male Sprague Dawley rats were obtained from Charles River Laboratories at 8 weeks of age. The rats were maintained on a 12:12 light/dark cycle in the BMS vivarium. Animals were acclimated to the facility for 10 days prior to use. Five days prior to the start of testing, rats were acclimated to the choice diet regime which entailed ad libitum access to two different diets: Harlan rat chow (standard chow, 5% fat, 3.5kcal/g) and Research Diets D12327 (a highly palatable 40% fat diet, 4.59kcal/g). Rats typically display ~85% (by kcal) preference for the Research Diets chow.

At the start of the study rats were randomized and grouped based upon body weight, daily kcal intake, and diet preference. Rats were dosed orally with vehicle (0.5% Methocel / 0.1% Tween 80 / 99.4% distilled H2O) or compounds dissolved in vehicle, at a volume of 5ml/kg. Test compounds were administered orally one hour before the onset of the dark cycle. Body weight and food consumption were measured daily at the time of drug administration. Upon completion of the study total caloric consumption, diet preference, and change in body weight were tabulated. Drug effects on body weight were analyzed using body weights converted to percent change from baseline and evaluated across treatment groups using a one-way ANOVA. Post-hoc day by day group comparisons were evaluated 50 ACS Paragon Plus Environment


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using Fisher’s PLSD test. Drug effects on daily caloric consumption were similarly evaluated. In addition, cumulative caloric consumption for each animal over the entire treatment period was calculated and the results were analyzed using one-way ANOVA followed by Fisher’s PLSD.


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Table of Contents graphic


Identification of a Nonbasic Melanin Hormone Receptor 1 Antagonist

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