Identification of a new class of glucokinase activators through structure-based design

Article abstract of DOI:10.1021/jm401116k

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. Because 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.

Full text of DOI:10.1021/jm401116k

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Article
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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Identification of a New Class of Glucokinase Activators
Through StructureꢀBased Design
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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 pyridineꢀbased hit that was
optimized using a structureꢀbased 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 highꢀfat fed Zucker
Diabetic Fatty rats.
KEYWORDS (Word Style “BG_Keywords”). Glucokinase, glucokinase activator, structureꢀaided
design, structureꢀbased design, diabetes, type II diabetes, S0.5, Vmax, OGTT, Zucker Diabetic Fatty rat.
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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 βꢀcells,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 βꢀcell 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
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Despite the availability of a variety of antiꢀhyperglycemic 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
longꢀterm, 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 glucoseꢀ6ꢀphosphate, the first step in glucose utilization. Its
expression is restricted primarily to liver, pancreatic αꢀ and βꢀcells, 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 rateꢀlimiting 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 halfꢀmaximal 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
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GKAs contribute to controlling blood glucose concentration by enhancing the ability of the remaining
pancreatic βꢀcells to sense glucose and increase insulin secretion in a glucoseꢀdependent 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,
glucoseꢀstimulated insulin secretion occurs at lower blood glucose concentrations, and hepatic
gluconeogenesis is curtailed at a lower glucose concentration threshold.32 GKAs have been identified
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which modulate the Vmax
.
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 GKAꢀglucokinase 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.
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Around 600 unique GK mutations have been identified to date.34 Heterozygous mutations that reduce
the activity of GK lead to maturityꢀonset 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 postꢀprandial and fasting
blood glucose concentrations in patients, hypoglycemia has consistently been the doseꢀlimiting 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
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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 structureꢀbased design strategy.
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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 ꢀM 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.
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Scheme 1. Synthesis of Pyridylaminoꢀ(4ꢀmethyl)thiazolesa
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aReagents 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 4ꢀposition 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 2ꢀhaloketones.
Scheme 2. Synthesis of 5ꢀBromopyridyl Aminothiazole Derivativesa
aReagents and conditions: (a) Br2, CHCl3, 0 °C; (b) benzoylisothiocyanate, THF; (c) K2CO3, EtOH,
reflux, 4 h; (d) XCH2C(O)R2, Et3N, EtOH, reflux.
Utilizing 5ꢀbromo pyridines 13 or 15, two different routes were used to obtain 5ꢀsubstituted analogs.
The first method involved formation of the dianion of 13 (R2 = CH3) using MeLi, to remove the acidic
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NH, transmetallation with nꢀBuLi at ꢀ78 ºC, and then addition of an appropriate electrophile to afford
the 5ꢀsubstituted analogs 17-21. The second approach utilized a palladiumꢀmediated 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 baseꢀcatalyzed betaꢀelimination to afford the corresponding thiolate anions, which
were reacted in situ with alkylating agents to yield thioethers 24-26.53,54
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Scheme 3. Synthesis of 5ꢀSubstituted Pyridyl Analogsa
aReagents and conditions: (a) i. MeLi, THF, ꢀ78 °C, 10 min; ii. nꢀBuLi, ꢀ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. RꢀX.
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 Cꢀ5 suggested such substitution would not only be
accommodated, but would potentially improve potency as well.55 The 5ꢀbromo, 5ꢀmethyl, and 5ꢀchloro
compounds 13, 17 and 18 were synthesized to test this concept, and the two haloꢀsubstituted analogs
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were found to be approximately 10ꢀfold more potent than 1, importantly without substantially reducing
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the S0.5.
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Figure 2. Xꢀray crystallographic structure of the complex of 1 with glucokinase, showing the key
hydrogenꢀbonding interactions with Arg63.
Table 1. Enzymatic Kinetic Data on AminopyridineꢀBased Glucokinase Activators.a
R3
N
S
R2
N
N
H
R1
O
Compound
R1
R2
Me
Me
Me
Me
Me
Me
Me
Et
R3
H
H
H
H
EC50 (nM) Vmax (%) S0.5 (mM)
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8
Ph
3-OMe Ph
2-Pyr
3-Pyr
3-OH Ph
8-Quinoline
Ph
4940
3220
12300
7780
3440
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687
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84
72
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3.3
3.9
2.6
3.0
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3.1
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3.0
3.5
3.3
2.4
3.7
1.5
0.8
0.9
H
H
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13
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26
85
96
Br
Br
Br
Br
Me
Cl
S-Ph
S-2-Py
S-Bn
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
562
240
106
91
iPr
iBu
Me
Me
Me
Me
Me
Me
Me
iPr
356
2640
796
106
95
85
208
86
231
75
235
77
Ph
Ph
Ph
S-CH2-2-Py
S-(CH2)2-Imid
S-(CH2)2-Imid
77
65
325
119
125
117
a
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 xꢀray structure also showed a long, solventꢀaccessible channel that could be accessed by
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substituents at the 4ꢀ and 5ꢀpositions 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 4ꢀalkyl
thiazoles 13-16 performed in the same general range of EC50, Vmax and S0.5.
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At this point, it became more challenging for structureꢀbased 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 xꢀray structure. For instance, the isosteric exchange of the
methyl group for chlorine resulted in a 4ꢀfold loss in potency. However, upon soaking compound 17
into crystals of GK (see Supporting Information), the resulting xꢀray 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 5ꢀposition, directed
at this pocket.
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Figure 3. Xꢀray crystallographic structure of 5ꢀmethylpyridyl 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 contraꢀ
indications for some medications which rely on renal clearance.57 Additionally, with this class of
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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 onꢀtarget 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 5ꢀbromo analog. Additionally,
substituents occupying this induced pocket provided a wide range of Vmax and S0.5 values. Compound 26
combined the potencyꢀenhancing isopropylthiazole and 5ꢀimidazoethythio substituents to provide a
compound with an attractive balance of Vmax and S0.5. Compound 26 was advanced into in vivo efficacy
studies.
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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 nearꢀmaximal response. Confirmation of oral exposure was determined through
a single time point at the end of the OGTT. The terminal (2.5 h postꢀdose) plasma drug concentrations
averaged 4.5 ꢀg/mL (n=8), which were >70ꢀfold over the enzymatic EC50. The DPPꢀIV 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 DPPꢀIV 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, insulinꢀresistant models such as
the high fatꢀfed 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 highꢀfat 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 structureꢀbased 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 heteroarylꢀ
aminopyridines 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. LCꢀMS experiments were performed on an
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Journal of Medicinal Chemistry
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.
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9
3-(Benzyloxy)-N-(4-methylthiazol-2-yl)pyridin-2-amine
(1).
A
mixture
of
1ꢀ(3ꢀ
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(benzyloxy)pyridinꢀ2ꢀyl)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 3ꢀ(benzyloxy)ꢀNꢀ(4ꢀ
1
methylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine as a yellow oil (0.200 g, 71%). H NMR (400 MHz, CDCl3) δ 8.57
(br s, 1H), 7.94 (dd, J = 5.1, 1.4 Hz, 1H), 7.42ꢀ7.37 (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). 2ꢀ(4ꢀ
Methylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀol (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. 1ꢀ(chloromethyl)ꢀ3ꢀ
methoxybenzene (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 3ꢀ(3ꢀmethoxybenzyloxy)ꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀ
1
yl)pyridinꢀ2ꢀamine hydrochloride salt (59.3 mg, 22.5% yield). H 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,
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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)+.
1
2
3
4
5
6
7
8
N-(4-Methylthiazol-2-yl)-3-(pyridin-2-ylmethoxy)pyridin-2-amine dihydrochloride (7). 2ꢀ(4ꢀ
Methylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀol (75 mg, 0.362 mmol), 2ꢀ(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 dichloromethaneꢀmethanol (2 mL)
and 1 M HCl in ether (1 mL) was added and concentrated to afford Nꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ꢀ3ꢀ(pyridinꢀ
2ꢀylmethoxy)pyridinꢀ2ꢀamine dihydrochloride (96 mg, 71.4% yield) as a tan solid. 1H NMR (400 MHz,
DMSOꢀd6) 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), 7.70ꢀ7.64 (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+Hꢀ2HCl)+.
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N-(4-Methylthiazol-2-yl)-3-(pyridin-3-ylmethoxy)pyridin-2-amine dihydrochloride (8). 2ꢀ(4ꢀ
Methylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀol (75 mg, 0.362 mmol), 3ꢀ(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 dichloromethaneꢀmethanol (2
mL) and add 1 M HCl in ether (1 mL) and concentrated to afford Nꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ꢀ3ꢀ(pyridinꢀ3ꢀ
1
ylmethoxy)pyridinꢀ2ꢀamine dihydrochloride (67 mg, 49.9% yield) as a tan solid. H NMR (400 MHz,
DMSOꢀd6) 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) 7.49ꢀ7.43 (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+Hꢀ2HCl)+.
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3-((2-(4-Methylthiazol-2-ylamino)pyridin-3-yloxy)methyl)phenol (9).
2ꢀ(4ꢀMethylthiazolꢀ2ꢀ
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2
3
4
5
6
7
8
9
ylamino)pyridinꢀ3ꢀol (5.0 g, 24 mmol), (3ꢀ(bromomethyl)phenoxy)(tertꢀbutyl)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%
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hexanes,
10%
ethyl
acetate)
to
afford
5.4
g
of
Nꢀ(3ꢀ((3ꢀ((tertꢀ
butyldimethylsilyl)oxy)benzyl)oxy)pyridinꢀ2ꢀyl)ꢀ4ꢀmethylthiazolꢀ2ꢀamine 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 3ꢀ
1
((2ꢀ(4ꢀmethylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀyloxy)methyl)phenol (1.4 g, 34% yield) as a tan solid. H
NMR (400 MHz, d6ꢀDMSO) 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), 7.01ꢀ6.96 (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+HꢀHCl)+.
N-(4-Methylthiazol-2-yl)-3-(quinolin-8-ylmethoxy)pyridin-2-amine dihydrochloride (10).
mixture of 2ꢀ(4ꢀmethylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀol (75.0 mg, 0.362 mmol),
A
8ꢀ
(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
dichloromethaneꢀmethanol (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 Nꢀ(4ꢀ
methylthiazolꢀ2ꢀyl)ꢀ3ꢀ(quinolinꢀ8ꢀylmethoxy)pyridinꢀ2ꢀamine dihydrochloride (148 mg, 97.1% yield) as
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1
a tan solid. H NMR (400 MHz, DMSOꢀd6) 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), 7.78ꢀ7.71 (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+Hꢀ2HCl)+.
1
2
3
4
5
6
7
8
3-(Benzyloxy)-5-bromo-N-(4-methylthiazol-2-yl)pyridin-2-amine (13).
1ꢀ(3ꢀ(Benzyloxy)ꢀ5ꢀ
9
bromopyridinꢀ2ꢀyl)thiourea (30.0 g, 88.7 mmol) and triethylamine (21.0 mL, 151 mmol) were dissolved
in ethanol (250 mL). 1ꢀchloropropanꢀ2ꢀone (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
3ꢀ(benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (32.7 g, 98.0% yield) as a tan solid
10
11
12
13
14
15
16
17
18
19
20
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1
after drying in vacuum oven. H 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).
1ꢀ(3ꢀ(Benzyloxy)ꢀ5ꢀ
bromopyridinꢀ2ꢀyl)thiourea (8.00 g, 23.7 mmol), 1ꢀbromobutanꢀ2ꢀone (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 3ꢀ(benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀ
1
ethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (5.32 g, 57.6% yield) as a light yellow powder. H NMR (400 MHz,
DMSOꢀd6) δ 10.21 (br s, 1H), 7.97 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.61ꢀ7.57 (m, 2H),
7.44ꢀ7.33 (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).
1ꢀ(3ꢀ(Benzyloxy)ꢀ5ꢀ
bromopyridinꢀ2ꢀyl)thiourea (7.60 g, 22.5 mmol), 1ꢀbromoꢀ3ꢀmethylbutanꢀ2ꢀone (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
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Journal of Medicinal Chemistry
water, brine, dried over sodium sulfate, filtered and concentrated. The residue was purified using
chromatography (elution, 83% hexanes, 17% ethyl acetate) to afford 3ꢀ(benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀ
isopropylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (8.09 g, 89.0% yield) as a white powder after triturating with
1
2
3
4
5
6
7
8
1
hexanes. H NMR (400 MHz, DMSOꢀd6) δ 10.17 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.62ꢀ7.57 (m, 3H),
9
7.44ꢀ7.33 (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)+
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13
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15
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17
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19
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25
26
27
28
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3-(Benzyloxy)-5-bromo-N-(4-isobutylthiazol-2-yl)pyridin-2-amine (16).
1ꢀ(3ꢀ(Benzyloxy)ꢀ5ꢀ
bromopyridinꢀ2ꢀyl)thiourea (0.500 g, 1.48 mmol), 1ꢀchloroꢀ4ꢀmethylpentanꢀ2ꢀone (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 3ꢀ(benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀ
isobutylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (0.284 g, 45.9% yield) as a white powder. 1H NMR (400 MHz,
DMSOꢀd6) 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), 7.44ꢀ7.32
(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). 3ꢀ
(Benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (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 nꢀButyl 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
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concentrated to afford 3ꢀ(benzyloxy)ꢀ5ꢀmethylꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine hydrochloride
1
2
3
4
1
(0.037 g, 12.8% yield). H NMR (400 MHz, DMSOꢀd6) δ 11.10 (bs, 1H), 7.83 (s, 1H), 7.60 (d, J = 7.2
Hz, 2H), 7.54 (s, 1H), 7.45ꢀ7.33 (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)+
5
6
7
8
9
3-(Benzyloxy)-5-chloro-N-(4-methylthiazol-2-yl)pyridin-2-amine hydrochloride salt (18). 3ꢀ
(Benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (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 nꢀButyl 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 3ꢀ(benzyloxy)ꢀ5ꢀchloroꢀNꢀ
(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine hydrochloride salt as a tan solid. 1H 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)+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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3-(Benzyloxy)-N-(4-methylthiazol-2-yl)-5-(phenylthio)pyridin-2-amine (19).
3ꢀ(Benzyloxy)ꢀ5ꢀ
bromoꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (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. nꢀButyl lithium (0.465 mL, 1.16 mmol) was slowly added and stirred for 15 minutes. 1,2ꢀ
Diphenyldisulfane (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 3ꢀ(benzyloxy)ꢀ
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Nꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(phenylthio)pyridinꢀ2ꢀamine (0.182 g, 48.2% yield). H NMR (400 MHz,
CDCl3) δ 8..00 (d, J = 1.7 Hz, 1H), 7.62 (m, 1H), 7.53 (m, 2H), 7.41ꢀ7.29 (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)+.
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3-(Benzyloxy)-N-(4-methylthiazol-2-yl)-5-(pyridin-2-ylthio)pyridin-2-amine
dihydrochloride
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(20). 3ꢀ(Benzyloxy)ꢀ5ꢀbromoꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (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. nꢀButyl lithium (0.166 mL, 0.415 mmol) was slowly added and stirred
for 15 minutes. 1,2ꢀdi(pyridinꢀ2ꢀyl)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, 10ꢀ35% 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 3ꢀ(benzyloxy)ꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(pyridinꢀ2ꢀ
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ylthio)pyridinꢀ2ꢀamine dihydrochloride (0.058 g, 36.4% yield). H NMR (400 MHz, DMSOꢀd6) 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), 7.42ꢀ7.34 (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+Hꢀ
2HCl)+
3-(Benzyloxy)-5-(benzylthio)-N-(4-methylthiazol-2-yl)pyridin-2-amine (21).
3ꢀ(Benzyloxy)ꢀ5ꢀ
bromoꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (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. nꢀButyl lithium (0.465 mL, 1.16 mmol) was slowly added and the resulting mixture was
stirred for 15 minutes. 1,2ꢀDibenzyldisulfane (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, 10ꢀ20% ethyl acetate in hexanes
gradient) followed by reverse phase chromatography (C18 column, elution, 5 to 95% acetonitrile in
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Journal of Medicinal Chemistry
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water gradient) to afford 3ꢀ(benzyloxy)ꢀ5ꢀ(benzylthio)ꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine (0.108
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g, 27.7% yield). H 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)+.
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3-(Benzyloxy)-N-(4-methylthiazol-2-yl)-5-(pyridin-2-ylmethylthio)pyridin-2-amine
dihydrochloride
(24).
Methyl
3ꢀ(5ꢀ(benzyloxy)ꢀ6ꢀ(4ꢀmethylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀ
ylthio)propanoate (70.0 mg, 0.168 mmol) was dissolved in THF (1 mL). Potassium 2ꢀmethylpropanꢀ2ꢀ
olate (1M in THF, 0.185 mL, 0.185 mmol) was added and stirred at room temperature for 5 minutes. 2ꢀ
(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 3ꢀ(benzyloxy)ꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀ
yl)ꢀ5ꢀ(pyridinꢀ2ꢀylmethylthio)pyridinꢀ2ꢀamine dihydrochloride (51.6 mg, 62.1% yield) as a white solid.
1H NMR (400 MHz, DMSOꢀd6) 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), 7.61ꢀ7.54 (m, 4H), 7.49ꢀ7.34 (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+Hꢀ2HCl)+
5-(2-(1H-Imidazol-1-yl)ethylthio)-3-(benzyloxy)-N-(4-methylthiazol-2-yl)pyridin-2-amine
dihydrochloride
(25).
Methyl
3ꢀ(5ꢀ(benzyloxy)ꢀ6ꢀ(4ꢀmethylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀ
ylthio)propanoate (70.0 mg, 0.168 mmol) was dissolved in THF (1 mL). Potassium 2ꢀmethylpropanꢀ2ꢀ
olate (1 M in THF, 0.185 mL, 0.185 mmol) was added and stirred at room temperature for 5 minutes. 1ꢀ
(2ꢀchloroethyl)ꢀ1Hꢀimidazole 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,
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Journal of Medicinal Chemistry
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
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afford
5ꢀ(2ꢀ(1Hꢀimidazolꢀ1ꢀyl)ethylthio)ꢀ3ꢀ(benzyloxy)ꢀNꢀ(4ꢀmethylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine
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dihydrochloride (28.1 mg, 33.6% yield) as a tan solid. 1H NMR (400 MHz, DMSOꢀd6) 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), 7.64ꢀ7.59 (m, 3H),
7.45ꢀ7.33 (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+Hꢀ2HCl)+
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5-(2-(1H-Imidazol-1-yl)ethylthio)-3-(benzyloxy)-N-(4-isopropylthiazol-2-yl)pyridin-2-amine
dihydrochloride (26).
Methyl 3ꢀ(5ꢀ(benzyloxy)ꢀ6ꢀ(4ꢀisopropylthiazolꢀ2ꢀylamino)pyridinꢀ3ꢀ
ylthio)propanoate (200 mg, 0.451 mmol) was dissolved in THF (3 mL). Potassium 2ꢀmethylpropanꢀ2ꢀ
olate (1 M in THF, 1.58 mL, 1.58 mmol) was added and stirred at room temperature for a minute. 1ꢀ(2ꢀ
chloroethyl)ꢀ1Hꢀimidazole 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 5ꢀ(2ꢀ(1Hꢀimidazolꢀ
1ꢀyl)ethylthio)ꢀ3ꢀ(benzyloxy)ꢀNꢀ(4ꢀisopropylthiazolꢀ2ꢀyl)pyridinꢀ2ꢀamine dihydrochloride (155 mg,
65.5% yield) as a white solid. 1H NMR (400 MHz, DMSOꢀd6) 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), 7.45ꢀ7.33 (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+Hꢀ2HCl)+
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
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PDB nos.: 4MLE for GKꢀ1; 4MLH for GKꢀ17.
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Author Information:
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Corresponding author
*rhinklin@arraybiopharma.com tel:(303)ꢀ386ꢀ1275
Present address:
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† 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,NꢀDiisopropylethylamine; DPPꢀIV, Dipeptidyl peptidaseꢀ4; GK, glucokinase; GKA,
glucokinase activator; HFD, highꢀfat diet; Imid,1ꢀimidazole; OGTT, oral glucose tolerance test;
MODY2, maturityꢀonset diabetes of the young type 2; PHHI, persistent hyperinsulinemic hypoglycemia
in infancy; PNDM, permanent neonatal diabetes mellitus; S0.5, the concentration of glucose at halfꢀ
maximal velocity; Vmax, Enzymatic rate of glucokinase at saturating glucose concentration; Xantphos,
4,5ꢀBis(diphenylphosphino)ꢀ9,9ꢀdimethylxanthene
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Journal of Medicinal Chemistry
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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 countryꢀyears
and 2.7 million participants. Lancet 2011, 378, 31ꢀ40.
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increased betaꢀcell apoptosis in humans with type 2 diabetes. Diabetes 2003, 52, 102ꢀ110.
4 Home, P. D.; Pacini, G. Hepatic dysfunction and insulin insensitivity in type 2 diabetes mellitus: a
critical target for insulinꢀsensitizing agents. Diabetes Obes. Metab. 2008, 10, 699ꢀ718.
5 Rizza, R. A. Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications
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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), 773ꢀ795.
7 Wasserman, D. H. Four grams of glucose. Am. J. Physiol. Endocrinol. Metab. 2009, 296, E11ꢀ21.
8 Perl, S.; Kushner, J. A.; Buchholz, B. A.; Meeker, A. K.; Stein, G. M.; Hsieh, M.; Kirby, M.;
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10 Zhang, X.; Gregg, E. W.; Williamson, D. F.; Barker, L. E.; Thomas, W.; Bullard, K. M.; Imperatore,
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14 Panzram, G., Mortality and survival in type 2 (nonꢀinsulinꢀdependent) diabetes mellitus. Diabetologia
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15 Kannel, W. B.; McGee, D. L. Diabetes and cardiovascular disease. The Framingham study. J. Amer.
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34 Osbak, K. K.; Colclough, K.; SaintꢀMartin, C.; Beer, N. L.; BellanneꢀChantelot, C.; Ellard, S.; Gloyn,
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37 Johnson, D.; Shepherd, R. M.; Gill, D.; Gorman, T.; Smith, D. M.; Dunne, M. J. Glucoseꢀdependent
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38 Coope, G. J.; Atkinson, A. M.; Allott, C.; McKerrecher, D.; Johnstone, C.; Pike, K. G.; Holme, P. C.;
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39 Futamura, M.; Hosaka, H.; Kadotani, A.; Shimazaki, H.; Sasaki, K.; Ohyama, S.; Nishimura, T.; Eiki,
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40 Fyfe, M. C.; White, J. R.; Taylor, A.; Chatfield, R.; Wargent, E.; Printz, R. L.; Sulpice, T.;
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