Identification of a New Class of Glucokinase Activators through

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Identification of a New Class of Glucokinase Activators Through Structure-Based Design Ronald Hinklin, Steve Boyd, Mark Joseph Chicarelli, Kevin Condroski, Walter DeWolf, Patrice Lee, Waiman Lee, Ajay Singh, Laurie Thomas, Walter Voegtli, Lance Williams, and Thomas Aicher J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401116k • Publication Date (Web): 09 Sep 2013 Downloaded from http://pubs.acs.org on September 16, 2013

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Journal of Medicinal Chemistry

Identification of a New Class of Glucokinase Activators Through Structure-Based Design Ronald J. Hinklin,* Steven A. Boyd,† Mark J. Chicarelli, Kevin R. Condroski,# Walter E. DeWolf, Jr., Patrice A. Lee, Waiman Lee,║ Ajay Singh,∆ Laurie Thomas, Walter C. Voegtli, Lance Williams, Thomas D. Aicher‡ Array BioPharma, 3200 Walnut St. Boulder, CO 80301 RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ABSTRACT: Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high glucose concentrations. Glucose flux through GK also contributes to reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, compounds that can activate GK may serve as effective treatments for type 2 diabetes. Herein we report the identification and initial optimization of a novel series of allosteric glucokinase activators (GKAs). We discovered an initial thiazolylamino pyridine-based hit that was optimized using a structure-based design strategy, and identified 26 as an early lead. Compound 26 demonstrated a good balance of in vitro potency and enzyme kinetic parameters, and demonstrated blood glucose reductions in oral glucose tolerance tests in both C57BL/6J mice and high-fat fed Zucker Diabetic Fatty rats.

KEYWORDS (Word Style “BG_Keywords”). Glucokinase, glucokinase activator, structure-aided design, structure-based design, diabetes, type II diabetes, S0.5, Vmax, OGTT, Zucker Diabetic Fatty rat. ACS Paragon Plus Environment

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Type 2 diabetes is a chronic, progressive metabolic disease, typically characterized by hyperglycemia, hyperlipidemia and insulin resistance. Type 2 diabetes affects an estimated 300 million people worldwide and is anticipated to affect more than 472 million people by 2030 due to increasing age, sedentary lifestyle and incidence of obesity in the global population.1,2 Once patients lose approximately half of their pancreatic β-cells,3 type 2 diabetes develops as a consequence of insufficient insulin release from the pancreas to compensate for insulin resistance developed in the liver4,5 and muscle.6,7 It is believed that pancreatic β-cell loss in humans is irreversible,8 although further damage may be delayed by diet, weight loss and exercise.9,10 Inasmuch as control of blood glucose concentrations, as measured by glycosylated hemoglobin (A1C), has been correlated with a decreased risk of microvascular complications,11,12 control of blood glucose concentration is a primary goal of therapy.13 Diabetes also is a prominent risk factor for serious cardiovascular events.14,15,16 Despite the availability of a variety of anti-hyperglycemic agents, there is an unmet need for more effective therapies for type 2 diabetes. According to the National Health and Nutrition Examination Survey, in 2006 only 57% of patients with type 2 diabetes achieved glycemic control with currently available therapies.17 An ideal agent to treat diabetes would satisfy the following criteria: 1) provide long-term, durable control of postprandial and fasted blood glucose, 2) delay or prevent loss of pancreatic β−cell function, 3) cause no adverse effect on body weight, 4) present no increased cardiovascular risk, 5) provide benefit for other metabolic syndrome risk factors, such as hyperlipidemia or blood pressure and 6) delay the progression towards diabetic complications.18 A drug which acts via glucokinase activation19 is a promising strategy to achieve many of these goals.20,21,22 In this report, we communicate our initial investigation of glucokinase activators as therapeutics for type 2 diabetes. Glucokinase is a member of the hexokinase family of cellular enzymes, which are responsible for the conversion of glucose to glucose-6-phosphate, the first step in glucose utilization. Its expression is restricted primarily to liver, pancreatic α- and β-cells, enteroendocrine cells and the hypothalamus.23,24,25 The activity of glucokinase is reduced significantly in obese patients with type 2

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diabetes.26 Glucokinase is the rate-limiting enzyme for glucose metabolism, which controls insulin secretion in the pancreas.27 It enhances glucose utilization and glycogen storage, while concurrently suppressing gluconeogenesis, in the liver.28 The kinetic parameters of glucokinase are ideal for acting as a “glucose sensor.” The S0.5, the glucose concentration at GK’s half-maximal phosphorylation rate, of 7.5 mM is above the normal fasting blood glucose concentration of 5.5 mM. After a meal, the positive cooperativity for glucose (Hill slope h = 1.7) affords a rapid change in phosphorylation of glucose in response to elevated blood glucose concentrations.29,30 GKAs contribute to controlling blood glucose concentration by enhancing the ability of the remaining pancreatic β-cells to sense glucose and increase insulin secretion in a glucose-dependent manner. In addition, GKAs act in the liver to increase glucose uptake (i.e., conversion to glycogen) and to concomitantly decrease hepatic glucose output.23 Glucokinase interconverts between inactive and active conformations in response to glucose concentration.31 Each GKA has the potential to induce a unique conformation of the allosteric site of the active form of glucokinase, and each of the resulting complexes can exhibit unique enzyme kinetics. Conceptually, activation of glucokinase may be induced by reducing S0.5, and/or by raising the maximal velocity, Vmax. GKAs described in the literature reduce the S0.5 to varying extents. By reducing S0.5, glucose-stimulated insulin secretion occurs at lower blood glucose concentrations, and hepatic gluconeogenesis is curtailed at a lower glucose concentration threshold.32 GKAs have been identified which modulate the Vmax.33 GKAs with a lower S0.5 and Vmax > 100% will increase glucose phosphorylation at any glucose concentration. However, while GKAs with a lower S0.5 and a Vmax < 100% will increase glucose phosphorylation at low glucose concentration, they will begin to decrease the phosphorylation rate at higher glucose concentrations. Glucokinase in complex with GKAs may also display Hill coefficients that are different from uncomplexed glucokinase, thus altering the degree of glucose cooperativity. Both the S0.5 and the Hill coefficient are indicative of the glucose dependency of a particular GKA-glucokinase complex, with lower values of either parameter increasing the potential for


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an inappropriate and excessively high activation of glucokinase activity at low blood glucose concentrations. Around 600 unique GK mutations have been identified to date.34 Heterozygous mutations that reduce the activity of GK lead to maturity-onset diabetes of the young type 2 (MODY2), whereas homozygous inactivating mutations lead to permanent neonatal diabetes mellitus (PNDM). Activating mutations are rarer and result in autosomal dominant persistent hyperinsulinemic hypoglycemia in infancy (PHHI). Sixteen of the seventeen known activating GK mutations are located in or around the allosteric pocket where GKAs bind. With the central role of glucokinase in the regulation of blood glucose concentrations, there has been extensive interest in finding compounds which activate the enzyme. Scientists at Hoffman la Roche were the first to describe small molecule activators of glucokinase,35 with subsequent disclosures by AstraZeneca,36,37,38 Banyu (Merck),39 and others.40,41,42 The S0.5 values for these compounds complexed to glucokinase were markedly lower than unbound glucokinase, ranging from 0.3 to 0.6 mM glucose vs. 7.0 mM. We hypothesized that compounds with such low S0.5 values could increase the potential for hypoglycemic events by inducing inappropriately high insulin secretion and hepatic glucose uptake, as well as supression of glucose production, at low glucose concentration. While these early GKAs have been demonstrated to both lower post-prandial and fasting blood glucose concentrations in patients, hypoglycemia has consistently been the dose-limiting adverse event in clinical trials of the GKAs with low S0.5 values. More recently, there have been reports that longer term dosing of GKAs in clinical trials have shown loss of efficacy over time in some cases.43,44 One approach to modulate this risk is to target the compounds to the liver.45,46 Two risks are evident in this approach. The first is that glucokinase in the liver is regulated by the insulin promoter.47 Therefore, when insulin secretion is impaired, less GK will be expressed in the liver. This, in turn, may limit the efficacy of the final drug to a suboptimal level. A second risk to this approach is that selective activation of GK in the liver may increase the risk of inappropriately high levels of lipid storage in the liver leading to hepatic steatosis.48


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Our approach was to identify GKAs with a higher degree of glucose dependency by identifying activators with intermediate S0.5 values that retain good potencies (as measured by enzymatic EC50 values).49 With the goal of identifying GKAs with unique kinetic properties, we began by designing scaffolds that varied significantly from the common structural motifs found in known activators. At the inception of our program, all of the known GKAs contained amide or urea functionalities (Figure 1). We focused on diverse alternatives that still possessed the bidentate hydrogen bonding capability and incorporated lipophilic substituents that were strategically directed into specific hydrophobic cavities in the allosteric binding pocket, using a structure-based design strategy.

Figure 1. Lead compound 1 and contemporary glucokinase activators from the literature. A large number of scaffolds were investigated to determine if they effected enzyme activation upon binding. One of the compounds synthesized was aminopyridine 1, which showed an EC50 of 5 µM and importantly had an S0.5 = 3.3 mM. This represented a promising, low molecular weight lead compound for our program. Results and Discussion Chemistry. Target aminopyridines were synthesized via the following routes (compounds 6-10 are shown in Scheme 1). Commercially available amino pyridine 2 was reacted with benzoyl isothiocyanate to form the acyl thiourea 3, and saponification of the acyl group afforded an intermediate thiourea 4.50 Thiourea 4 was condensed with chloroacetone in a Hantzsch thiazole synthesis to afford aminothiazole


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1. Removal of the benzyl group under acidic conditions afforded phenol 5, and subsequent alkylation with various benzyl halides afforded ethers 6-10. Scheme 1. Synthesis of Pyridylamino-(4-methyl)thiazolesa

a

Reagents and conditions: (a) benzoylisothiocyanate, THF; (b) K2CO3, EtOH, reflux, 4 h; (c) chloroacetone, Et3N, EtOH, reflux, 5 h; (d) 6 M HCl, reflux, 10 h; (e) R1CH2X, K2CO3, DMF. Substitution at the 4-position of the thiazole was achieved via the route shown in Scheme 2. Reaction of aminopyridine 2 with bromine afforded bromide 11. Conversion of the aminopyridine 11 into thiazoles 13-16 was accomplished in a similar manner to that shown in Scheme 1 by reaction of thiourea 12 with various 2-haloketones. Scheme 2. Synthesis of 5-Bromopyridyl Aminothiazole Derivativesa

a

Reagents and conditions: (a) Br2, CHCl3, 0 °C; (b) benzoylisothiocyanate, THF; (c) K2CO3, EtOH, reflux, 4 h; (d) XCH2C(O)R2, Et3N, EtOH, reflux. Utilizing 5-bromo pyridines 13 or 15, two different routes were used to obtain 5-substituted analogs. The first method involved formation of the dianion of 13 (R2 = CH3) using MeLi, to remove the acidic ACS Paragon Plus Environment

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NH, transmetallation with n-BuLi at -78 ºC, and then addition of an appropriate electrophile to afford the 5-substituted analogs 17-21. The second approach utilized a palladium-mediated coupling of 13 (R2 = CH3) or 15 (R2 = iPr) with methyl thiopropionate to provide thioethers 22 and 23.51,52 Thiopropionates 22 and 23 underwent base-catalyzed beta-elimination to afford the corresponding thiolate anions, which were reacted in situ with alkylating agents to yield thioethers 24-26.53,54 Scheme 3. Synthesis of 5-Substituted Pyridyl Analogsa

a

Reagents and conditions: (a) i. MeLi, THF, -78 °C, 10 min; ii. n-BuLi, -78 °C, 15 min; iii. E+, warm to RT, 15 min.; (b) HS(CH2)2CO2Me, DIEA, Xantphos, Pd2dba3, dioxane, 95 °C, 1 h; (c) i. KOtBu, THF, 5 min; ii. R-X. Structure-based design strategies and development of structure-activity relationships.

The

protein crystal structure of the complex of initial lead 1 with GK confirmed that the aminothiazole made dual hydrogen bonds with Arg63 (Figure 2).31 The benzyloxy group occupied a lipophilic pocket in the allosteric site. Structural analysis identified potential areas where both lipophilic and polar groups could be incorporated on the aryl ring of the benzyl group to make improved contacts with the enzyme. An initial survey of modifications to the benzyl group did not demonstrate dramatic increases in potency (compound 1 vs. 6-10 in Table 1). Docking studies of a diverse set of compounds incorporating small hydrophobic substituents at the pyridyl C-5 suggested such substitution would not only be accommodated, but would potentially improve potency as well.55 The 5-bromo, 5-methyl, and 5-chloro compounds 13, 17 and 18 were synthesized to test this concept, and the two halo-substituted analogs ACS Paragon Plus Environment

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were found to be approximately 10-fold more potent than 1, importantly without substantially reducing the S0.5.

Figure 2. X-ray crystallographic structure of the complex of 1 with glucokinase, showing the key hydrogen-bonding interactions with Arg63. Table 1. Enzymatic Kinetic Data on Aminopyridine-Based Glucokinase Activators.a R3

R1

Compound 1 6 7 8 9 10 13 14 15 16 17 18 19 20 21 24 25 26 a

R1 Ph 3-OMe Ph 2-Pyr 3-Pyr 3-OH Ph 8-Quinoline Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph

R2 Me Me Me Me Me Me Me Et iPr iBu Me Me Me Me Me Me Me iPr

N

O

S N H

N

R3 H H H H H H Br Br Br Br Me Cl S-Ph S-2-Py S-Bn S-CH2-2-Py S-(CH2)2-Imid S-(CH2)2-Imid

R2

EC50 (nM) 4940 3220 12300 7780 3440 1700 687 562 240 356 2640 796 208 231 235 77 325 119

Vmax (%) 79 84 72 67 88 85 96 106 91 106 95 85 86 75 77 65 125 117

S0.5 (mM) 3.3 3.9 2.6 3.0 1.8 3.0 4.5 2.8 3.1 4.9 3.0 3.5 3.3 2.4 3.7 1.5 0.8 0.9

The variability of each of the parameters was estimated based on the historical performance of a standard reference compound. The estimated coefficients of variation for the EC50, Vmax and S0.5 are 30% (n = 383), 4.3% (n = 329) and 21% (n = 329), respectively


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This initial x-ray structure also showed a long, solvent-accessible channel that could be accessed by substituents at the 4- and 5-positions of the thiazole. Starting from bromopyridyl thiourea 12, a set of small alkyl groups was incorporated in an effort to further increase potency. The simple 4-alkyl thiazoles 13-16 performed in the same general range of EC50, Vmax and S0.5. At this point, it became more challenging for structure-based design to provide additional potency improvements for this series of compounds. In particular, relative potencies of compounds 17 and 18 were difficult to explain given the initial x-ray structure. For instance, the isosteric exchange of the methyl group for chlorine resulted in a 4-fold loss in potency. However, upon soaking compound 17 into crystals of GK (see Supporting Information), the resulting x-ray structure revealed that Tyr215, which previously had formed the back wall with which the chlorine and methyl group were predicted to make favorable van der Waals interactions, had rotated by ~120°, creating a large, unoccupied pocket (Figure 3). Given this observation, we investigated larger substituents at the pyridyl 5-position, directed at this pocket.

Figure 3. X-ray crystallographic structure of 5-methylpyridyl analog 17 in GK. It was found that this induced pocket was tolerant of a wide variety of groups. However, the scope of discussion in this publication will be limited to the thioethers. We chose to focus on these compounds to incorporate a metabolic “soft spot” into the structure to favor hepatic over renal clearance.56 Many patients with diabetes have impaired renal function, which has resulted in reduced dosages or contraindications for some medications which rely on renal clearance.57 Additionally, with this class of ACS Paragon Plus Environment

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compounds the expected major metabolites, the sulfones and sulfoxides, are significantly less potent than the original drug agent.58 Consequently, we expected that the on-target pharmacological activity with this class of compounds would generally result from the parent compound.

The thioether

compounds in this study were more potent than the corresponding 5-bromo analog. Additionally, substituents occupying this induced pocket provided a wide range of Vmax and S0.5 values. Compound 26 combined the potency-enhancing isopropylthiazole and 5-imidazoethythio substituents to provide a compound with an attractive balance of Vmax and S0.5. Compound 26 was advanced into in vivo efficacy studies. In vivo efficacy studies. The efficacy of compound 26 was evaluated in an oral glucose tolerance test (OGTT) in normal C57BL/6J mice (Figure 4). Dosed orally at 50 mg/kg 30 min prior to glucose load, the compound significantly blunted the glucose excursion without inducing hypoglycemia during the 2 h test period. The screening paradigm used in this program was to dose intriguing compounds at 50 mg/kg orally to ensure a near-maximal response. Confirmation of oral exposure was determined through a single time point at the end of the OGTT. The terminal (2.5 h post-dose) plasma drug concentrations averaged 4.5 µg/mL (n=8), which were >70-fold over the enzymatic EC50. The DPP-IV inhibitor vildagliptin59 (50 mg/kg) was used as a positive control.


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Figure 4. OGTT of compound 26 at 50 mg/kg, tested in comparison to the DPP-IV inhibitor vildagliptin. The normal mouse OGTT study provided a convenient in vivo screening model, but did not necessarily indicate that the compounds would work in hyperglycemic, insulin-resistant models such as the high fat-fed female ZDF rat model. The ZDF model better represents type 2 diabetes60 and allowed us to demonstrate that our GKAs were efficacious in the relevant disease state. After the rats had been on a high-fat diet (HFD) for 21 days, an OGTT was conducted. Compared to the HFD control group, the group dosed with 26 (50 mg/kg, p.o.) showed a marked decrease (23%) in blood glucose AUC over the 2 h time course of the study, comparable to the 24% reduction obtained with vildagliptin (50 mg/kg, p.o.), and into the range of the lean controls (Figure 5).


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Figure 5. OGTT in Zucker fatty rats with 50 mg/kg 26 p.o. after 21 d on HFD compared to Vildagliptin and lean controls. Conclusions. We have identified a new class of GKAs utilizing structure-based design. A preliminary investigation into the SAR of this series indicated the potential to modulate EC50, S0.5 and Vmax independently with structural modifications. The aminopyridine 26 showed blood glucose control in two different animal models without inducing hypoglycemia, with exposures far in excess of its in vitro EC50. The results of this early investigation served as the basis for a broad investigation into heteroarylaminopyridines as new cores for GKAs, and additional reports on those studies will be forthcoming.

Experimental Section

Chemistry.

Reagents were purchased from commercial sources and used without further

purification. Nuclear magnetic resonance (NMR) spectra were measured in the indicated solvent with tetramethylsilane (TMS) or the residual solvent peak as the internal standard on a Varian 400 MHz spectrometer. Chemical shifts (δ) are in parts per million. LC-MS experiments were performed on an ACS Paragon Plus Environment

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Agilent 1100 HPLC coupled with an Agilent MSD mass spectrometer using ESI as ionization source. Peaks were detected by UV absorbance at 220 and 254 nm, and MS full scan was applied to all experiments. All compounds were disclosed in this paper were confirmed to be >95% purity via this method. 3-(Benzyloxy)-N-(4-methylthiazol-2-yl)pyridin-2-amine

(1).

A

mixture

of

1-(3-

(benzyloxy)pyridin-2-yl)thiourea (0.247 g, 0.953 mmol), triethylamine (0.53 mL, 3.81 mmol) and chloroacetone (0.228 mL, 2.857 mmol) were refluxed in ethanol (7 mL) for 5 hours. The solution was cooled to room temperature and concentrated. The residue was partitioned between ethyl acetate and water. The layers were separated and the aqueous layer was extracted three times with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 80% hexane, 20% ethyl acetate) to afford 3-(benzyloxy)-N-(4methylthiazol-2-yl)pyridin-2-amine as a yellow oil (0.200 g, 71%). 1H NMR (400 MHz, CDCl3) δ 8.57 (br s, 1H), 7.94 (dd, J = 5.1, 1.4 Hz, 1H), 7.42-7.37 (m, 5H), 7.10 (dd, J = 7.8, 1.4 Hz, 1H), 6.81 (dd, J = 8.0, 5.1 Hz, 1H) 6.38 (q, J = 1.0 Hz, 1H), 5.10 (s, 2H), 2.32 (d, J = 1.0 Hz, 3H); MS (ES+) m/e 298.1 (M+H)+. 3-(3-Methoxybenzyloxy)-N-(4-methylthiazol-2-yl)pyridin-2-amine hydrochloride salt (6). 2-(4Methylthiazol-2-ylamino)pyridin-3-ol (0.150 g, 0.724 mmol) and potassium carbonate (0.225 g, 1.63 mmol) were added to DMF (3 mL) and stirred at room temperature. 1-(chloromethyl)-3methoxybenzene (0.113 g, 0.724 mmol) was added and the reaction stirred at room temperature overnight. The mixture was partitioned between water and ether, separated and the aqueous was reextracted with ether. The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified using chromatography (elution, 80% hexane, 20% ethyl acetate). The free base of the title compound was then dissolved in dichloromethane (2 mL) and 1 M HCl in ether (1 mL, 1 mmol) was added and concentrated to afford 3-(3-methoxybenzyloxy)-N-(4-methylthiazol-2yl)pyridin-2-amine hydrochloride salt (59.3 mg, 22.5% yield). 1H NMR (400 MHz, CDCl3) δ 12.35 (s, 1H), 7.93 (dd, J = 4.9, 1.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.20 (dd, J = 8.0, 1.0 Hz, 1H), 7.12 (m, ACS Paragon Plus Environment

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2H), 6.97 (dd, J = 8.0, 4.9 Hz, 1H) 6.84 (m, 1H), 6.38 (s, 1H), 5.34 (s, 2H), 3.84 (s, 3H), 2.46 (s, 3H); MS (ES+) m/e 328.1 (M+H)+. N-(4-Methylthiazol-2-yl)-3-(pyridin-2-ylmethoxy)pyridin-2-amine dihydrochloride

(7).

2-(4-

Methylthiazol-2-ylamino)pyridin-3-ol (75 mg, 0.362 mmol), 2-(bromomethyl)pyridine hydrochloride (75.4 mg, 0.362 mmol), potassium carbonate (175 mg, 1.27 mmol) and DMF (3 mL) were stirred overnight at room temperature. The mixture was poured into water (20 mL) and extracted twice with 20 mL of ether. The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified using column chromatography (elution, 20% hexane, 80% ethyl acetate). The free base of the title compound was dissolved in 1:1 dichloromethane-methanol (2 mL) and 1 M HCl in ether (1 mL) was added and concentrated to afford N-(4-methylthiazol-2-yl)-3-(pyridin2-ylmethoxy)pyridin-2-amine dihydrochloride (96 mg, 71.4% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 5.1 Hz, 1H), 8.19 (t, J = 7.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1Hz), 8.04 (d, J = 4.9 Hz, 1H), 7.70-7.64 (m, 2H), 7.16 (dd, J = 8.0, 5.0 Hz, 1H), 6.87 (s, 1H), 5.54 (s, 2H), 2.34 (s, 3H); MS (ES+) m/e 299.1 (M+H-2HCl)+. N-(4-Methylthiazol-2-yl)-3-(pyridin-3-ylmethoxy)pyridin-2-amine dihydrochloride (8).

2-(4-

Methylthiazol-2-ylamino)pyridin-3-ol (75 mg, 0.362 mmol), 3-(bromomethyl)pyridine hydrochloride (75.4 mg, 0.362 mmol), potassium carbonate (175 mg, 1.27 mmol) and DMF (3 mL) were stirred at room temperature overnight. The reaction was poured into water (20 mL) and extracted twice with 20 mL of ether. The combined extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified using chromatography (elution, 7 M NH3 in 99% ethyl acetate, 1% methanol). The free base of the title compound was dissolved in 1:1 dichloromethane-methanol (2 mL) and add 1 M HCl in ether (1 mL) and concentrated to afford N-(4-methylthiazol-2-yl)-3-(pyridin-3ylmethoxy)pyridin-2-amine dihydrochloride (67 mg, 49.9% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (br s, 1H), 8.82 (s, 1H), 8.57 (d, J = 3.9 Hz, 1H), 8.06 (dt, J = 7.8, 1.7 Hz, 1H) 7.88 (dd, J = 5.1, 1.2 Hz, 1H) 7.49-7.43 (m, 2H), 6.91 (dd, J = 7.8, 4.9 Hz, 1H), 6.58 (q, J = 1.0 Hz, 1H), 5.29 (s, 2H), 2.24 (d, J = 1.0 Hz, 3H); MS (ES+) m/e 299.1 (M+H-2HCl)+. ACS Paragon Plus Environment

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3-((2-(4-Methylthiazol-2-ylamino)pyridin-3-yloxy)methyl)phenol

(9).

2-(4-Methylthiazol-2-

ylamino)pyridin-3-ol (5.0 g, 24 mmol), (3-(bromomethyl)phenoxy)(tert-butyl)dimethylsilane (7.3 g, 24 mmol) and potassium carbonate (8.3 g, 60 mmol) were dissolved in DMF (75 mL) and stirred at room temperature for 4 hours. The reaction was poured into water (350 mL) and extracted with twice with 150 mL of ether. The combined organic layers were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 90% hexanes,

10%

ethyl

acetate)

to

afford

5.4

g

of

N-(3-((3-((tert-

butyldimethylsilyl)oxy)benzyl)oxy)pyridin-2-yl)-4-methylthiazol-2-amine as an oil. The oil was dissolved in THF (50 mL) and 1 M tetrabutyl ammonium fluoride in THF (15 mL, 15 mmol) was added and stirred at room temperature for 2 days. The reaction was poured into saturated aqueous ammonium chloride and extracted with dichloromethane (2 x 100 mL). The organic layers were dried over sodium sulfate, filtered and concentrated. The residue was triturated with dichloromethane (10 mL) to afford 3((2-(4-methylthiazol-2-ylamino)pyridin-3-yloxy)methyl)phenol (1.4 g, 34% yield) as a tan solid. 1H NMR (400 MHz, d6-DMSO) δ 9.83 (br s, 1H), 9.50 (br s, 1H), 7.89 (dd, J = 4.9, 1.2 Hz, 1H), 7.38 (dd, J = 8.0, 1.2 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.01-6.96 (m, 2H), 6.91 (dd, J = 8.0, 5.1 Hz, 1H), 6.75 (ddd, J = 8.2, 2.5, 0.8 Hz, 1H), 6.62 (q, J = 1.0 Hz, 1H), 5.22 (s, 2H), 2.28 (d, J = 1.0 Hz, 3H); MS (ES+) m/e 314.1 (M+H-HCl)+. N-(4-Methylthiazol-2-yl)-3-(quinolin-8-ylmethoxy)pyridin-2-amine dihydrochloride (10). mixture

of

2-(4-methylthiazol-2-ylamino)pyridin-3-ol

(75.0

mg,

0.362

mmol),

A 8-

(bromomethyl)quinoline (80.4 mg, 0.362 mmol), potassium carbonate (125 mg, 0.905 mmol) and DMF (3 mL) was stirred at room temperature overnight. The reaction was poured into water (15 mL) and stirred for 20 minutes. The precipitate was filtered and dried. The solids were dissolved in 1:1 dichloromethane-methanol (2 mL) and 2 M HCl in ether (0.5 mL) was added. The solution was concentrated and redissolved in a small amount of 10% methanol in dichloromethane and added dropwise to vigorously stirred ether (10 mL). The solids were filtered and dried to afford N-(4methylthiazol-2-yl)-3-(quinolin-8-ylmethoxy)pyridin-2-amine dihydrochloride (148 mg, 97.1% yield) as ACS Paragon Plus Environment

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Page 16 of 28

a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (dd, J = 4.1, 1.6 Hz, 1H), 8.63 (d, J = 7.2 Hz, 1H), 8.23 (br s, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.05 (dd, J = 4.9, 0.8 Hz, 1H), 7.78-7.71 (m, 3H), 7.19 (m, 1H), 6.89 (q, J = 1.0 Hz, 1H), 5.91 (s, 2H), 2.33 (d, J = 1.0 Hz, 3H); MS (ES+) m/e 349.2 (M+H-2HCl)+. 3-(Benzyloxy)-5-bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine

(13).

1-(3-(Benzyloxy)-5-

bromopyridin-2-yl)thiourea (30.0 g, 88.7 mmol) and triethylamine (21.0 mL, 151 mmol) were dissolved in ethanol (250 mL). 1-chloropropan-2-one (9.89 mL, 124 mmol) was added and heated to reflux overnight. The reaction was cooled to room temperature, poured into water (1 L) and filtered to afford 3-(benzyloxy)-5-bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (32.7 g, 98.0% yield) as a tan solid after drying in vacuum oven. 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.42 (m, 5H), 7.23 (d, J = 2.0 Hz, 1H), 6.41 (m, 1H), 5.09 (s, 2H), 2.32 (s, 3H); MS (ES+) m/e 297.2, 378.0 (M+H)+. 3-(Benzyloxy)-5-bromo-N-(4-ethylthiazol-2-yl)pyridin-2-amine

(14).

1-(3-(Benzyloxy)-5-

bromopyridin-2-yl)thiourea (8.00 g, 23.7 mmol), 1-bromobutan-2-one (4.29 g, 28.4 mmol), triethylamine (5.77 mL, 41.4 mmol) and ethanol (100 mL) were heated to reflux for an hour. The reaction was cooled, partitioned between ethyl acetate (500 mL) and water (100 mL), washed with water, brine, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 87% hexane, 13% ethyl acetate) to afford 3-(benzyloxy)-5-bromo-N-(4ethylthiazol-2-yl)pyridin-2-amine (5.32 g, 57.6% yield) as a light yellow powder. 1H NMR (400 MHz, DMSO-d6) δ 10.21 (br s, 1H), 7.97 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.61-7.57 (m, 2H), 7.44-7.33 (m, 3H), 6.61 (t, J = 1.0 Hz, 1H), 5.29 (s, 2H), 2.60 (qd, J = 7.4, 1.0 Hz, 2H), 1.20 (t, J = 7.4 Hz, 3H); MS (ES+) m/e 390.0, 391.9 (M+H)+ 3-(Benzyloxy)-5-bromo-N-(4-isopropylthiazol-2-yl)pyridin-2-amine (15).

1-(3-(Benzyloxy)-5-

bromopyridin-2-yl)thiourea (7.60 g, 22.5 mmol), 1-bromo-3-methylbutan-2-one (5.19 g, 31.5 mmol), triethylamine (5.48 mL, 39.3 mmol) and ethanol (150 mL) were heated to reflux for an hour. The reaction was cooled, partitioned between ethyl acetate (500 mL) and water (100 mL), washed with


16

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water, brine, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 83% hexanes, 17% ethyl acetate) to afford 3-(benzyloxy)-5-bromo-N-(4isopropylthiazol-2-yl)pyridin-2-amine (8.09 g, 89.0% yield) as a white powder after triturating with hexanes. 1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.62-7.57 (m, 3H), 7.44-7.33 (m, 3H), 6.60 (s, 1H), 5.29 (s, 2H), 2.88 (sep, J = 6.8 Hz, 1H), 1.22 (d, J = 6.8 Hz, 6H); MS (ES+) m/e 404.0, 406.0 (M+H)+ 3-(Benzyloxy)-5-bromo-N-(4-isobutylthiazol-2-yl)pyridin-2-amine

1-(3-(Benzyloxy)-5-

(16).

bromopyridin-2-yl)thiourea (0.500 g, 1.48 mmol), 1-chloro-4-methylpentan-2-one (0.341 g, 1.77 mmol), triethylamine (0.361 mL, 2.59 mmol) and ethanol (15 mL) were heated to reflux for an hour. The reaction was cooled, partitioned between ethyl acetate (100 mL) and water (20 mL), washed with water, brine, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 87% hexane, 13% ethyl acetate) to afford 3-(benzyloxy)-5-bromo-N-(4isobutylthiazol-2-yl)pyridin-2-amine (0.284 g, 45.9% yield) as a white powder.

1

H NMR (400 MHz,

DMSO-d6) δ 10.18 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.58 (m, 2H), 7.44-7.32 (m, 3H), 6.61 (s, 1H), 5.28 (s, 2H), 2.43 (d, J = 7.0 Hz, 2H), 1.99 (m, 1H), 0.88 (d, J = 6.6 Hz, 6H); MS (ES+) m/e 418.0, 420.0 (M+H)+ 3-(Benzyloxy)-5-methyl-N-(4-methylthiazol-2-yl)pyridin-2-amine hydrochloride salt (17).

3-

(Benzyloxy)-5-bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (0.350 g, 0.930 mmol) was dissolved in THF (30 mL) and cooled to -78 °C. 2 M Methyl lithium (0.639 mL, 1.02 mmol) was added slowly and the resulting mixture was stirred for 10 minutes. 2.5 M n-Butyl lithium (0.409 mL, 1.02 mmol) was added slowly at -78 °C and the resulting reaction was stirred for 15 minutes. Iodomethane (0.165 g, 1.16 mmol) was added and the reaction warmed to room temperature. The reaction was stirred at room temperature for 15 minutes, quenched with saturated aqueous ammonium chloride and extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 90% hexanes, 10% ethyl acetate). The free base of the title compound was dissolved in dichloromethane and 2 M HCl in ether (0.5 mL) was added and ACS Paragon Plus Environment

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Page 18 of 28

concentrated to afford 3-(benzyloxy)-5-methyl-N-(4-methylthiazol-2-yl)pyridin-2-amine hydrochloride (0.037 g, 12.8% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.10 (bs, 1H), 7.83 (s, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.54 (s, 1H), 7.45-7.33 (m, 3H), 6.84 (s, 1H), 5.31 (s, 2H), 2.32 (s, 3H), 2.29 (s, 3H); MS (ES+) m/e 312.1 (M+H)+ 3-(Benzyloxy)-5-chloro-N-(4-methylthiazol-2-yl)pyridin-2-amine hydrochloride salt (18).

3-

(Benzyloxy)-5-bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (0.225 g, 0.598 mmol) was dissolved in THF (30 mL) and cooled to -78 ºC. 2 M Methyl lithium (0.467 mL, 0.747 mmol) was slowly added and the resulting mixture was stirred for 10 minutes. 2.5 M n-Butyl lithium (0.299 mL, 0.747 mmol) was added and the resulting mixture was stirred for 15 minutes. Perchloroethane (0.991 g, 4.19 mmol) was added and the reaction was warmed to room temperature and stirred for 15 minutes. The reaction was quenched with aqueous ammonium chloride and extracted with dichloromethane, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 40% hexanes, 60% ether). The free base of the title compound was dissolved in dichloromethane and 2 M HCl in ether (0.5 mL) was added, concentrated and dried in vacuo to afford 3-(benzyloxy)-5-chloro-N(4-methylthiazol-2-yl)pyridin-2-amine hydrochloride salt as a tan solid.

1

H NMR (400 MHz, CDCl3) δ

12.41 (s, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.59 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.0 Hz, 2H), 7.33 (m, 1H), 7.20 (d, J = 2.0 Hz, 1H), 6.41 (bs, 1H), 5.36 (s, 2H), 2.47 (s, 3H); MS (ES+) m/e 332.1 (M+H)+. 3-(Benzyloxy)-N-(4-methylthiazol-2-yl)-5-(phenylthio)pyridin-2-amine (19).

3-(Benzyloxy)-5-

bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (0.350 g, 0.930 mmol) was dissolved in THF (30 mL) and cooled to -78 ºC. Methyl lithium (0.727 mL, 1.16 mmol) was slowly added and stirred for 10 minutes. n-Butyl lithium (0.465 mL, 1.16 mmol) was slowly added and stirred for 15 minutes. 1,2Diphenyldisulfane (0.203 g, 0.93 mmol) was added and the resulting mixture was warmed to room temperature and stirred for 15 minutes. The reaction was quenched with aqueous ammonium chloride, extracted with dichloromethane, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 90% hexanes, 10 EtOAc) followed by reverse phase chromatography (C18 column, elution, 5 to 95% acetonitrile in water gradient) to afford 3-(benzyloxy)ACS Paragon Plus Environment

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N-(4-methylthiazol-2-yl)-5-(phenylthio)pyridin-2-amine (0.182 g, 48.2% yield). 1H NMR (400 MHz, CDCl3) δ 8..00 (d, J = 1.7 Hz, 1H), 7.62 (m, 1H), 7.53 (m, 2H), 7.41-7.29 (m, 5H), 7.26 (m, 1H), 7.20 (m, 2H), 6.83 (s,1H), 5.32 (s, 2H), 2.31 (s, 3H); MS (ES+) m/e 315.1, 406.0 (M+H)+. 3-(Benzyloxy)-N-(4-methylthiazol-2-yl)-5-(pyridin-2-ylthio)pyridin-2-amine (20).

dihydrochloride

3-(Benzyloxy)-5-bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (0.125 g, 0.332 mmol) was

dissolved in THF (30 mL) and cooled to -78 ºC. Methyl lithium (0.260 mL, 0.415 mmol) was slowly added and stirred for 10 minutes. n-Butyl lithium (0.166 mL, 0.415 mmol) was slowly added and stirred for 15 minutes. 1,2-di(pyridin-2-yl)disulfane (0.0732 g, 0.332 mmol) was added and the resulting mixture was warmed to room temperature and stirred for 15 minutes. The reaction was quenched with aqueous ammonium chloride, extracted with dichloromethane, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 10-35% ethyl acetate in hexanes gradient) to afford a residue that was dissolved in 10% MeOH in dichloromethane and excess 2M HCl in ether was added and concentrated to afford 3-(benzyloxy)-N-(4-methylthiazol-2-yl)-5-(pyridin-2ylthio)pyridin-2-amine dihydrochloride (0.058 g, 36.4% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.37 (m, 1H), 8.13 (d, J = 1.7 Hz, 1H), 7.74 (m, 1H), 7.63 (m, 1H), 7.57 (m, 2H), 7.42-7.34 (m, 3H), 7.17 (m, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.85 (s, 1H), 5.34 (s, 2H), 2.32 (s, 3H); MS (ES+) m/e 407.1 (M+H2HCl)+ 3-(Benzyloxy)-5-(benzylthio)-N-(4-methylthiazol-2-yl)pyridin-2-amine (21).

3-(Benzyloxy)-5-

bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (0.350 g, 0.930 mmol) was dissolved in THF (30 mL) and cooled to -78 ºC. Methyl lithium (0.727 mL, 1.16 mmol) was slowly added and stirred for 10 minutes. n-Butyl lithium (0.465 mL, 1.16 mmol) was slowly added and the resulting mixture was stirred for 15 minutes. 1,2-Dibenzyldisulfane (0.229 g, 0.930 mmol) was added and the reaction was warmed to room temperature and stirred for 15 minutes. The reaction was quenched with aqueous ammonium chloride, extracted with dichloromethane, dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 10-20% ethyl acetate in hexanes gradient) followed by reverse phase chromatography (C18 column, elution, 5 to 95% acetonitrile in ACS Paragon Plus Environment

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Page 20 of 28

water gradient) to afford 3-(benzyloxy)-5-(benzylthio)-N-(4-methylthiazol-2-yl)pyridin-2-amine (0.108 g, 27.7% yield). 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 1.8 Hz, 1H), 7.57 (m, 2H), 7.53 (m, 1H), 7.42 (m, 2H), 7.37 (m, 1H), 7.23 (m, 5H) 6.77 (s, 1H), 5.28 (s, 2H), 4.18 (s, 2H), 2.29 (s, 3H). MS (ES+) m/e 420.1 (M+H)+. 3-(Benzyloxy)-N-(4-methylthiazol-2-yl)-5-(pyridin-2-ylmethylthio)pyridin-2-amine dihydrochloride

(24).

Methyl

3-(5-(benzyloxy)-6-(4-methylthiazol-2-ylamino)pyridin-3-

ylthio)propanoate (70.0 mg, 0.168 mmol) was dissolved in THF (1 mL). Potassium 2-methylpropan-2olate (1M in THF, 0.185 mL, 0.185 mmol) was added and stirred at room temperature for 5 minutes. 2(bromomethyl)pyridine hydrobromide (42.6 mg, 0.168 mmol) was added and the resulting mixture was stirred at room temperature for 10 minutes. The reaction was quenched with aqueous ammonium chloride, extracted twice with 10 mL of ethyl acetate. The combined extracts were dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 85% hexanes, 15% ethyl acetate) to afford a residue that was dissolved in 10% MeOH in dichloromethane and excess 2M HCl in ether was added and concentrated to afford 3-(benzyloxy)-N-(4-methylthiazol-2yl)-5-(pyridin-2-ylmethylthio)pyridin-2-amine dihydrochloride (51.6 mg, 62.1% yield) as a white solid. 1

H NMR (400 MHz, DMSO-d6) δ 8.63 (d, J = 4.3 Hz, 1H), 8.04 (t, J = 7.0 Hz, 1H), 7.81 (d, J = 1.8 Hz,

1H), 7.61-7.54 (m, 4H), 7.49-7.34 (m, 4H), 6.77 (s, 1H), 5.30 (s, 2H), 4.42 (s, 2H), 2.28 (s, 3H); MS (ES+) m/e 421.0 (M+H-2HCl)+ 5-(2-(1H-Imidazol-1-yl)ethylthio)-3-(benzyloxy)-N-(4-methylthiazol-2-yl)pyridin-2-amine dihydrochloride

(25).

Methyl

3-(5-(benzyloxy)-6-(4-methylthiazol-2-ylamino)pyridin-3-

ylthio)propanoate (70.0 mg, 0.168 mmol) was dissolved in THF (1 mL). Potassium 2-methylpropan-2olate (1 M in THF, 0.185 mL, 0.185 mmol) was added and stirred at room temperature for 5 minutes. 1(2-chloroethyl)-1H-imidazole hydrochloride (28.1 mg, 0.168 mmol) was added and the resulting mixture was stirred at room temperature for an hour. The reaction was quenched with aqueous ammonium chloride, extracted twice with 10 mL of ethyl acetate. The combined extracts were dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, ACS Paragon Plus Environment

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50% hexanes in ethyl acetate to 10 % methanol in ethyl acetate gradient) to afford a residue that was dissolved in 10% MeOH in dichloromethane and excess 2M HCl in ether was added and concentrated to afford

5-(2-(1H-imidazol-1-yl)ethylthio)-3-(benzyloxy)-N-(4-methylthiazol-2-yl)pyridin-2-amine

dihydrochloride (28.1 mg, 33.6% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 9.16 (t, J = 1.4 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.76 (t, J = 1.6 Hz, 1H), 7.66 (t, J = 1.6 Hz, 1H), 7.64-7.59 (m, 3H), 7.45-7.33 (m, 3H), 6.77 (s, 1H), 5.34 (s, 2H), 4.35 (t, J = 6.2 Hz, 2H), 3.47 (t, J = 6.2 Hz, 2H), 2.29 (d, J = 1.0 Hz, 3H); MS (ES+) m/e 424.0 (M+H-2HCl)+ 5-(2-(1H-Imidazol-1-yl)ethylthio)-3-(benzyloxy)-N-(4-isopropylthiazol-2-yl)pyridin-2-amine dihydrochloride

(26).

Methyl

3-(5-(benzyloxy)-6-(4-isopropylthiazol-2-ylamino)pyridin-3-

ylthio)propanoate (200 mg, 0.451 mmol) was dissolved in THF (3 mL). Potassium 2-methylpropan-2olate (1 M in THF, 1.58 mL, 1.58 mmol) was added and stirred at room temperature for a minute. 1-(2chloroethyl)-1H-imidazole hydrochloride (82.8 mg, 0.496 mmol) was added and the resulting mixture was stirred at room temperature for 30 minutes. The reaction was quenched with aqueous ammonium chloride, extracted twice with 10 mL of ethyl acetate. The combined extracts were dried over sodium sulfate, filtered and concentrated. The residue was purified using chromatography (elution, 0 to 10% methanol in ethyl acetate gradient) to afford a residue that was dissolved in 10% MeOH in dichloromethane and excess 2M HCl in ether was added and concentrated to afford 5-(2-(1H-imidazol1-yl)ethylthio)-3-(benzyloxy)-N-(4-isopropylthiazol-2-yl)pyridin-2-amine dihydrochloride (155 mg, 65.5% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.80 (br s, 1H), 9.16 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.75 (m, 1H), 7.66 -7.59 (m, 4H), 7.45-7.33 (m, 3H), 6.78 (s, 1H), 5.35 (s, 2H), 4.35 (t, J = 6.2 Hz, 2H), 3.49 (t, J = 6.2 Hz, 2H), 2.96 (m, 1H), 1.25 (d, J = 6.8 Hz, 6H); MS (ES+) m/e 452.1 (M+H-2HCl)+ Supporting Information Available: Structural biology, pharmacology and enzymology protocols, along with procedures for the synthesis of all intermediates are provided in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. Accession codes ACS Paragon Plus Environment

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Page 22 of 28

PDB nos.: 4MLE for GK-1; 4MLH for GK-17. Author Information: Corresponding author *

[email protected] tel:(303)-386-1275

Present address: †

Knopp Biosciences LLC, 2100 Wharton Street, Suite 615, Pittsburgh, PA 15203

#

Celgene Corporation, 10300 Campus Point Drive Suite 100, San Diego, CA 92121

║

Deceased

∆

11448 Central Court #108 Broomfield, CO 80021

‡

Lycera Corporation, 2800 Plymouth Road, NCRC, Building 26, Ann Arbor, MI, 48109.

Abbreviations Used: DIEA, N,N-Diisopropylethylamine; DPP-IV, Dipeptidyl peptidase-4; GK, glucokinase; GKA, glucokinase activator; HFD, high-fat diet; Imid,1-imidazole; OGTT, oral glucose tolerance test; MODY2, maturity-onset diabetes of the young type 2; PHHI, persistent hyperinsulinemic hypoglycemia in infancy; PNDM, permanent neonatal diabetes mellitus; S0.5, the concentration of glucose at halfmaximal velocity; Vmax, Enzymatic rate of glucokinase at saturating glucose concentration; Xantphos, 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene


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Table of Contents Graphic:


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1

Danaei, G.; Finucane, M. M.; Lu, Y.; Singh, G. M.; Cowan, M. J.; Paciorek, C. J.; Lin, J. K.; Farzdfar, F.; Khang, Y.-H.; Stevens, G. A.; Rao, M.; Ali, M. K.; Riley, L. M.; Robinson, C. A.; Ezzati, M. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: Systemic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 2011, 378, 31-40. 2

The diabetes pandemic. Lancet 2011, 378, 99.

3

Butler, A. E.; Janson, J.; Bonner-Weir, S.; Ritzel, R.; Rizza, R. A.; Butler, P. C. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003, 52, 102-110.

4

Home, P. D.; Pacini, G. Hepatic dysfunction and insulin insensitivity in type 2 diabetes mellitus: a critical target for insulin-sensitizing agents. Diabetes Obes. Metab. 2008, 10, 699-718. 5

Rizza, R. A. Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy. Diabetes 2010, 59, 2697-2707.

6

Defronzo, R. A. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes 2009, 58 (4), 773-795. 7

Wasserman, D. H. Four grams of glucose. Am. J. Physiol. Endocrinol. Metab. 2009, 296, E11-21.

8

Perl, S.; Kushner, J. A.; Buchholz, B. A.; Meeker, A. K.; Stein, G. M.; Hsieh, M.; Kirby, M.; Pechhold, S.; Liu, E. H.; Harlan, D. M.; Tisdale, J. F. Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating. J. Clin. Endocrinol. Metab. 2010, 95, E234-239. 9

Sigal, R. J.; Kenny, G. P.; Wasserman, D. H.; Castaneda-Sceppa, C. Physical activity/exercise and type 2 diabetes. Diabetes Care 2004, 27, 2518-2539. 10

Zhang, X.; Gregg, E. W.; Williamson, D. F.; Barker, L. E.; Thomas, W.; Bullard, K. M.; Imperatore, G.; Williams, D. E.; Ann L. Albright, A. L. A1C level and future risk of diabetes: A systemic review. Diabetes Care 2010, 33, 1665-1673. 11

The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment on the development and progression of long-term complications in insulin dependent diabetes mellitus. New Engl. J. Med. 1993, 329, 977-986. 12

Statton, I. M.; Adler, A. I.; Neil, H. A.; Matthews, D. R.; Manley, S. E.; Cull, C. A.; Hadden, D.; Turner, R. C.; Holman, R. R. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observation study. Brit. Med. J. 2000, 321, 405-412.

13

Standards of medical care in diabetes—2012. Diabetes Care 2012, 35, S11-63.

14

Panzram, G., Mortality and survival in type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1987, 30, 123-131.


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Identification of a New Class of Glucokinase Activators through

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