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Letter
A novel series of potent glucokinase activators leading to the discovery of AM-2394. Paul J Dransfield, Vatee Pattaropong, Sujen Lai, Zice Fu, Todd Jonathan Kohn, Xiaohui Du, Alan C Cheng, Yumei Xiong, Renee Komorowski, Lixia Jin, Marion Conn, Eric Tien, Walter E. DeWolf, Ronald J Hinklin, Thomas Daniel Aicher, Christopher F. Kraser, Steven A. Boyd, Walter C. Voegtli, Kevin R. Condroski, Murielle Veniant, Julio C Medina, Jonathan B Houze, and Peter Coward ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00140 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016
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A novel series of potent glucokinase activators leading to the discovery of AM-2394. Paul J. Dransfield†, Vatee Pattaropong†, Sujen Lai†, Zice Fu†, Todd J. Kohn†, Xiaohui Du†, Alan Cheng†, Yumei Xiong†, Renee Komorowski$, Lixia Jin†, Marion Conn†, Eric Tien$, Walter E. DeWolf, Jr.#, Ronald J. Hinklin#, Thomas D. Aicher#, Christopher F. Kraser#, Steven A. Boyd#, Walter C. Voegtli#, Kevin R. Condroski#, Murielle Veniant-Ellison$, Julio C. Medina†, Jonathan Houze† and Peter Coward† †
Departments of Therapeutic Discovery, Metabolic Disorders, and Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, United States. $
Departments of Metabolic Disorders, Comparative Biology and Safety Sciences and Pharmacokinetics and Drug Metabolism, Amgen Inc, One Amgen Center Drive, Thousand Oaks, CA 91320, United States. #
Array BioPharma Inc., 3200 Walnut St., Boulder, CO 80301, United States.
KEYWORDS: Glucokinase Activator, GKA, AM-2394 Supporting Information Placeholder ABSTRACT: Glucokinase (GK) catalyzes the phosphorylation of glucose to glucose-6-phosphate. We present the structure–activity relationships leading to the discovery of AM-2394, a structurallydistinct GKA. AM-2394 activates GK with an EC50 of 60 nM, increases the affinity of GK for glucose by approximately 10-fold, exhibits moderate clearance and good oral bioavailability in multiple animal models, and lowers glucose excursion following an oral glucose tolerance test in an ob/ob mouse model of diabetes.
v e h ic le , n = 1 0
800
glucose, m g/dL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Medicinal Chemistry Letters
A M -23 94 1 m p k , n = 10 A M -23 94 3 m p k , n = 10
600
A M -23 94 1 0m p k, n = 1 0 A M -23 94 3 0m p k, n = 1 0
400
*** *** *** *** *** ***
200
*** *** *** -30
Type 2 diabetes mellitus (T2DM) is a disease characterized by elevated plasma glucose in the presence of insulin resistance and inadequate insulin secretion. Glucokinase (GK), a member of the hexokinase enzyme family, catalyzes the phosphorylation of glucose to glucose-6-phosphate in the presence of ATP.1 Expression of GK is restricted primarily to hepatocytes, pancreatic α- and β-cells, enteroendocrine cells, and the hypothalamus. GK exhibits low substrate affinity (S0.5 or Km = 7.5 mM), positive substrate cooperativity (Hill slope h = 1.7) and a lack of product inhibition, characteristics which allow it to serve as a physiologic glucose sensor and tightly regulate plasma glucose levels.2,3 Small molecule activators of GK activity (glucokinase activators or GKAs) were first described in 20034 and proposed as potential therapeutic agents for treating diabetes.5 In fact, several compounds have entered clinical trials, although the potential for hypoglycemia, elevated plasma triglycerides and blood pressure, and lack of
0
30
60
90
120
min
durable response remain as considerable obstacles to this ther,,,, apeutic class.6 7 8 9 10 Glucokinase activators are allosteric binders that can increase the enzyme’s affinity for glucose (S0.5) and/or the maximal velocity or number of glucose molecules phosphorylated per unit time, expressed as Vmax. Physiologically, these parameters impact the degree to which the activated enzyme phosphorylates glucose at a given blood glucose concentration and reduces blood glucose levels compared to the nonactivated enzyme. In this report we describe efforts toward the discovery of 4,5-substituted-2-pyridyl ureas as GKAs. Two key assays were used to characterize compounds as GKAs and establish the structure activity relationships (SAR).
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Attachment of a phenoxy substituent at position C-4, resulted in compound 1 (see Table 1). Addition of fluorine at position 2 of the phenylether resulted in a 5-fold increase in potency, hypothesized in part to result from an improved π stacking of the aryl ring and Tyr214 (vide infra). Further installation of a chlorine atom at position 3 (3) led to a significant increase in potency, but in general, resulted in increased metabolism in both rat and human liver microsomes as shown in Table 1. However, the 2,6-difluoro substitution pattern (4) improved the potency relative to 2 and reduced the clearance in human liver microsomes compared to 3. It was hypothesized that introduction of substituents at the C-5 position based upon the crystal structure would lead to improvements in potency (Table 2). Figure 1. X-ray co-crystal structure of 5 with glucokinase.
Table 2.a,b,c SAR of C-5 substitution of pyridyl urea GKA’s. X 5
The first assay determined the EC50 of the GKA in the presence of 5 mM glucose. In addition, this assay was also run in the presence of 4% human serum albumin (HSA) as a surrogate measure of plasma protein binding. The second assay generated S0.5 and Vmax values.11 Our goals were to maintain the S0.5 between 0.6 and 1.0 mM to balance efficacy with risk of hypoglycemia, and Vmax close to 100%, but no less than 90%, to protect against potential of the GKA inhibiting enzyme activity at high, but still physiologic, glucose concentrations, a possibility in severely diabetic patients. We began by modeling possible structural replacements of the amido-heterocycle found in a number of GKAs 12,13,14,15 and determined that a 2-pyridyl urea could be a suitable replacement, particularly due the potential to maintain the key bidentate hydrogen bonding interaction to Arg63 which is observed in all GKAs (Figure 1). X-ray crystal structure of compound 5 confirmed the formation of the hydrogen bonds, and thus we focused our efforts on this series.
O 4 F
5
Br
0.19
50
6
Me
0.12
3.5
0.038
0.76 0.92
n/a
0.83
n/a
1.23
0.66 0.99
173
0.050
2.37
0.76 0.79
355
0.88
8.8
2.23 0.68
201
0.045
0.42
0.62 0.88
35
N
0.23
5.5
0.60 0.95
131
N
0.036
0.62
0.79 0.91
116
0.014
1.05
0.77 0.85
27
0.088
0.65
0.68 0.93
21
Ph
8
N 9 N
11 HN
O 4 2 N 3 H R1
O
RLM GKA EC50 4% HSA S 0.5 Vmax EC50 (µM) (mM) ratio (µL/(min.mg)) (µM)
Table 1.a,b,c Initial C-4 substituent SAR of 1-methyl-3-(pyridine2-yl)-urea. N
N H
X
10
5
F
Compound
7
6
HN
N
0.9
MeO 12
O
EtO Compound
R1
GKA EC50 (µM)
S0.5 (mM)
1
Ph
10.8
0.76
0.71
184 / 36
2.13
0.99
0.67
106 / 29
0.3
n/a
0.62
265 / 168
N
13
Vmax RLM/HLM ratio (µL/(min.mg))
14
O
N
OH 2 F 3
F Cl
4
a
F
F
0.83
0.99
0.53
168 / 50
n/a
n/a
n/a
0.50
0.96
0.68
0.93
0.12
1.38
0.63
0.95
0.31
0.73
0.75
0.81
0.29
0.64
0.61
1.00
0.05
0.08
0.60
1.00
0.04
0.096
0.62
0.97
0.047
0.075
0.63
1.06
0.66
1.3
0.82
0.70
N
16
N
17 N 18 Me
N
19 N 20
Me
Me N
21 Me
N
23 MeO
N
24 EtO
Efforts to improve the overall free fraction of our molecules, were investigated using a 4% HSA assay. We focused on replacements of the 2,6-difluoro phenyl ring (Table 3). Initial introduction of a 2-pyridyl or 4-pyridyl displayed a large loss in potency (15 and 16, EC50 = 6.25 and >50 µM, respectively). Although the 3-pyridyl 17 also exhibited a loss of potency, it was only 5-fold (EC50 = 0.5 µM), but more importantly, there was only a 2-fold shift in potency in the presence of 4% HSA. From the x-ray structure (Figure 2), it appeared that a slight increase in steric bulk and lipophilicity of R1 would be tolerated. Further SAR of the 3-pyridyl group led to the finding that in general, addition of a methyl group around the pyridyl ring led to only minor improvements in potency (18, 19, 20).
N
22 Et
profile, 12 had poor solubility (27 µM in SIF; pH 6.8) and moderate clearance in rat microsomes (116 µL/(min×mg)). With compounds displaying good in vitro potency and kinetics, we returned to addressing the in vitro rat metabolism. To this end, we found that replacement of the methoxy group with an ethoxy group (13) was tolerated and improved the rat microsomal clearance, however with a substantial loss in potency in our shift assay. To address this loss in potency, the cLogP was reduced through installation of the hydroxyethyl group, 14. This achieved a reduction in cLogP from 3.9 (13) to 2.5, with only a moderate loss of potency and change in kinetic profile, while maintaining low rat liver microsomal clearance (human liver microsomal clearance for compounds containing the hydroxyethyl group were in general consider low;
