Discovery of cycloalkyl-fused N-thiazol-2-yl-benzamides as tissue non-specific glucokinase activators: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.07.051

Glucokinase (GK) activators are being developed for the treatment of type 2 diabetes mellitus (T2DM). However, existing GK activators have risks of hypoglycemia caused by over-activation of GK in islet cells and dyslipidemia caused by over-activation of intrahepatic GK. In the effort to mitigate risks of hypoglycemia and dyslipidemia while maintaining the promising efficacy of GK activator, we investigated a series of cycloalkyl-fused N-thiazol-2-yl-benzamides as tissue non-specific partial GK activators, which led to the identification of compound 72 that showed a good balance between in vitro potency and enzyme kinetic parameters, and protected β-cells from streptozotocin-induced apoptosis. Chronic treatment of compound 72 demonstrated its potent activity in regulation of glucose homeostasis and low risk of dyslipidemia with diabetic db/db mice in oral glucose tolerance test (OGTT). Moreover, acute treatment of compound 72 did not induce hypoglycemia in C57BL/6J mice even at 200 mg/kg via oral administration.

Full text of DOI:10.1016/j.ejmech.2017.07.051

Accepted Manuscript
Discovery of cycloalkyl-fused N-thiazol-2-yl-benzamides as tissue non-specific
glucokinase activators: Design, synthesis, and biological evaluation
Zhengyu Wang, Xiaofan Shi, Huan Zhang, Liang Yu, Yanhua Cheng, Hefeng Zhang,
Huibin Zhang, Jinpei Zhou, Jing Chen, Xu Shen, Wenhu Duan
PII:
S0223-5234(17)30573-1
10.1016/j.ejmech.2017.07.051
EJMECH 9614
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2017
Revised Date: 7 June 2017
Accepted Date: 22 July 2017
Please cite this article as: Z. Wang, X. Shi, H. Zhang, L. Yu, Y. Cheng, H. Zhang, H. Zhang, J. Zhou, J.
Chen, X. Shen, W. Duan, Discovery of cycloalkyl-fused N-thiazol-2-yl-benzamides as tissue non-specific
glucokinase activators: Design, synthesis, and biological evaluation, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.051.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of cycloalkyl-fused N-thiazol-2-yl-benzamides as
tissue non-specific glucokinase activators: design, synthesis,
and biological evaluation
a, 1
b, c, 1
d
b
a
Zhengyu Wang , Xiaofan Shi
, Huan Zhang , Liang Yu , Yanhua Cheng , Hefeng
c, e
d
a,
b,
b,
e,
Zhang , Huibin Zhang , Jinpei Zhou *, Jing Chen *, Xu Shen , Wenhu Duan **
a
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing
10009, P. R. China
2
b
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China
c
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
d
Center of Drug Discovery, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, 24 Tongjia Xiang, Nanjing, Jiangsu 210009, P. R. China
e
Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China
1
These authors contributed equally to this work.
*
* Corresponding author.
Corresponding authors.
Email address: jpzhou668@163.com (J.Zhou); jingchen@simm.ac.cn (J.Chen); whduan@simm.ac.cn
W.Duan).
*
(

ACCEPTED MANUSCRIPT
Abstract
Glucokinase (GK) activators are being developed for the treatment of type 2 diabetes
mellitus (T2DM). However, existing GK activators have risks of hypoglycemia caused by
over-activation of GK in islet cells and dyslipidemia caused by over-activation of
intrahepatic GK. In the effort to mitigate risks of hypoglycemia and dyslipidemia while
maintaining the promising efficacy of GK activator, we investigated a series of
cycloalkyl-fused N-thiazol-2-yl-benzamides as tissue non-specific partial GK activators,
which led to the identification of compound 72 that showed a good balance between in
vitro potency and enzyme kinetic parameters, and protected β-cells from
streptozotocin-induced apoptosis. Chronic treatment of compound 72 demonstrated its
potent activity in regulation of glucose homeostasis and low risk of dyslipidemia with
diabetic db/db mice in oral glucose tolerance test (OGTT). Moreover, acute treatment of
compound 72 did not induce hypoglycemia in C57BL/6J mice even at 200 mg/kg via oral
administration.
Keywords:
Type 2 diabetes mellitus; Hypoglycemia; Dyslipidemia; Glucokinase activators;
N-Thiazol-2-yl-benzamides.
1
. Introduction
Type 2 diabetes mellitus (T2DM) is a chronic and complex metabolic disorder
characterized by excessive hepatic glucose production, insulin resistance, pancreatic
1
β-cells dysfunction, and ultimately hyperglycemia. The population worldwide with

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T2DM has almost doubled to 347 million since 1980, and is anticipated to rise to 472
2
million by 2030. Although several oral antihyperglycemic agents have been marketed
over the past decades, many of these therapeutic agents show adverse effects, such as
3
hypoglycemia, peripheral edema, weight gain, and potential cardiovascular effects;
therefore searching for new oral antihyperglycemic agents with improved benefits-risk
profiles is still an unmet task. As the key glucose-phosphorylating enzyme, glucokinase
(GK) represents a promising approach for improving glycemic control as well as for
4
combination therapies in patients with T2DM. Unlike other members of hexokinase
family, GK shows low substrate affinity (K ~ 7 mM), positive cooperativity with
m
glucose, and lack of product inhibition, which enable GK to be highly responsive to
glucose levels and also to ensure that the glucose metabolic flux is closely tied to the
5
, 6
glucose concentation. As a monomeric enzyme, GK achieves substrate cooperativity
7
, 8
through equilibration between inactive and active conformations.
GK is selectively
expressed in a restricted number of glucose sensing tissues including liver hepatocytes
and pancreas. In the liver, GK represents the rate-determining steps for glucose uptake
4
and promotes glycogen storage along with reduction of gluconeogenesis in hepatocytes.
In the pancreas, GK preserves pancreatic β-cells viability and modulates
9
glucose-stimulated insulin secretion (GSIS) in β-cells. The activity of GK is reduced
1
0, 11
significantly in obese patients with T2DM.
Moreover, studies found that GK
activators can increase the activity of some currently approved antihyperglycemic
1
2
agents, which makes GK activators have potential applications in combination

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therapies.
Since the report of 1 (RO-28-1675) as a carbon-centered GK activators by
Hoffmann-La Roche in 2003, many compounds with amino acid-based and aryl-centered
scaffolds have also been identified as small molecule allosteric GK activators which have
1
3−18
been evaluated in preclinical or clinical studies (Figure 1);
however, their risks of
hepatic steatosis, low efficacy durability, and hypoglycemia are concerning. Because of
the high incidence of hypoglycemia, investigation of some early stage GK activators,
1
2, 19−20
including representative 5 (piragliatin) and 8 (MK-0941), were discontinued.
To
ameliorate the problem of hypoglycemia, several design strategies have been used in the
developments of new GK activators. One strategy is based on the hepatoselective
distribution by incorporating a carboxylate moiety for recognition and active uptake via
liver specific organic anion transporting polypeptide (OATP) transporters, for instance,
hepatoselective activator 6 (PF-04991532) was investigated and showed effective
2
1
glycemic control in animal studies. However, further clinical studies on 6 have been
2
2
terminated due to the tendency of increasing triglycerides in liver, and the relationship
between over-activation of GK by liver-specific GK activators and the adverse side effect
2
3, 24
of hepatic steatosis have been identified.
Another strategy is the design of ‘partial
activators’ of GK that avoids reducing GK’s Km for glucose to inappropriately low levels,
thereby maintaining increased dependence of enzymatic activity on physiological glucose
concentrations. To identify compounds with partial GK activation, screening cascades
that would enable correlation of an activator’s intrinsic biochemical properties (i.e.

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changes in GK’s K
m
for glucose and maximal velocity (Vmax)) against its in vivo efficacy
2
5
17, 26
and safety profiles have been developed by Pfizer and other companies;
and GK
activators with the optimized therapeutic indexes are recognized to have less
hypoglycemia risk for glycemic control in T2DM patients. The representative compound
2
7
7
(PF-04937319), a partial GK activators which is now in phase II clinical trials,
showed improved glycemic control with an acceptable risk-benefit profile.
Figure 1. Structures of representative GK activators.
Because T2DM is characterized by the excessive hepatic glucose production, insulin
resistance, and pancreatic β-cells dysfunction, GK activators ameliorating these problems
2
8
have demonstrate their promising potential for the treatment of T2DM. However,
liver-specific GK activators, such as 6, may decrease β-cell replication when they
2
9
effectively achieve euglycemia with decreasing demand for insulin; in contrast, partial
GK activators 7 displayed marginal activity in our study of β-cells apoptosis (Table 5).

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Therefore, we envisioned that the novel GK activators should be able to 1) effectively
control glycemic levels with low risk of hypoglycemia, 2) to regulate hepatic glucose
metabolism without dyslipidemia, 3) to effect on GSIS, and 4) to protect β-cells from
apoptosis.
To identify GK activators that have the effect of glycemic control with low risk of
hypoglycemia, we focused on potent GK activators (EC < 1.0 µM) that could activate
5
0
the recombinant human GK with maximum activation fold between 2.0 and 3.0 versus
control level; the threshold values were based partly on a screening strategy defined by
2
5
Pfizer. To identify activators profiles with reduced hypoglycemic risk, Pfizer used a
matrix assay wherein dose response determinations for each activator were conducted at
1
6 different glucose concentrations (ranging from 0 to 100 µM). Then an activator’s
maximum effect on reducing GK’s K for glucose and its maximum effect on the
m
enzyme’s Vmax were defined as α (α = K (min) / K (max)) and β (β = Vmax(max) /
m
m
V
max(min)), respectively. After testing various risk-benefit profiles with preclinical and
clinical candidates (including, but not limited to 1, 3, 5), GK activators with less dramatic
reductions of K (α = 0.05−0.2) combined with appropriate effects on Vmax (β = 0.8−1.2)
m
would offer activation at high glucose concentration (affording efficacy) but exert
reduced activation at low glucose concentration (avoiding hypoglycemia). When
initiating our program, we sought to evaluate the potency and activation profiles of each
activator more efficiently by determining values of EC and maximum activation fold
50
1
3
(
versus control level) at the single glucose concentration of 5.0 mM; and the partial GK

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activators 7 was chosen as the benchmark activators. As shown in Table 1, compound 7
showed moderate activity with the EC50 value of 0.93 µM and maximum activation fold
of 2.11, which was the desired enzymatic activity of GK activators at this fixed glucose
2
5
concentration. Considering the hypoglycemia risk of reported GK activators, the criteria
that activators displayed EC value below 1.0 µM and maximum activation fold ranged
50
from 2.0-fold to 3.0-fold was used to include as many reasonable compounds as possible.
Compounds that met the criteria should offer substantial activation of GK without
1
3, 30
over-activation of enzyme.
In the end, optimized compounds that met the criteria
2
5
would be re-evaluated in a matrix assay using Pfizer’s criteria.
Having established the strategy for identifying GK activators with low risk of
hypoglycemia, we embarked on the selection of center scaffold for tissue non-specific
GK activators. Common structure of GK activators contains two functional blocks
(
Figure 1): (i) an amino acid-based or aryl-centered fragment with two hydrophobic
moieties attached (blue), and (ii) a conserved H-bond donor-acceptor moiety (red),
9
heterocyclic amine in most cases. Functionally, the H-bond donor-acceptor moiety can
be responsible for the H-bond interactions with the Arg63. GK activators can stabilize the
7
GK enzyme in active conformations by forming the key H-bond interactions with Arg63,
m
which leads to the reduction of GK’s K for glucose to varying extents and/or effect on
1
3
enzyme’s V max; two hydrophobic moieties, with at least one of them being an aromatic
ring, may increase binding affinity by having hydrophobic interactions with two
2
6
hydrophobic pockets. Considering the effects of some aryl-centered GK activators on

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boosting β-cell replication and apoptosis reduction in glucotoxic β-cells, such as
11
31
compounds 9 (GKA71) and 10 (YH-GKA), we chose the 1,3,5-substituted benzamide
scaffold as part of the center; the 1-methoxy-2-propanol and 4-methylsulfonylphenol
groups were selected as the initial hydrophobic moieties, to direct into specific
hydrophobic cavities in the allosteric binding pocket.
Figure 2. Drug design approaches for overcoming the toxic oxidative metabolism of
2
-aminothiazoles.
Next, we explored H-bond donor-acceptor moieties. As shown in Figure 1, the
N-aminothiazole-2-yl amide moiety is favorably used as the bidentate anchor points for
allosteric GK activators, here 2-aminothiazole also served as the critical moiety
responsible for the H-bond interactions with Arg63. However, 2-aminothiazole moiety as
32
33
in derivatives 1 and 2 is recognized as structural alerts for bioactivation-related
toxicities, which result from the generation of toxic thioureas (III) in vivo through
oxidative ring-opening at the C-5 of the thiazole and further oxidation process in liver

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3
4, 35
microsomes (Figure 2).
To prevent formation of the metabolic toxic thiourea,
approaches were employed by introducing a single substituent at the oxidation-sensitive
3
6
position (C-5) of the thiazole core (e.g. 3, PSN-GK1), or by introducing substituents at
3
7
the neighboring C-4 to exert indirect influence to the C-5 (compound 4). These
3
6
37
approaches have been proven effective by the discovery of 3 and 4 , which show
improved pharmacokinetics and better safety profiles, as well as significant reduction of
3
6, 38
blood glucose levels in rodents.
Moreover, structural biology studies have shown that
the H-bond donor-acceptor containing moiety of amino acid-based can locate at a
8
, 39
solvent-exposed channel lined with the side chain of Arg63,
indicating that this vector
might provide an opportunity to incorporate tail regions for further optimization. In our
pursuit of novel tissue non-specific GK activators, an idea was to fuse five or
six-membered rings at C-4 and C-5 on 2-aminothiazole to block the toxic metabolic
process while maintaining the H-bond network; and hydrophilic or hydrophobic
substituents were introduced as tail regions on five or six-membered rings to probe the
effects on biochemical kinetics, liver hepatocytes, and pancreas. With the
above-mentioned reasons, a class of cycloalkane-fused 2-aminothiazole-containing
benzamides were designed to investigate their effects on enzyme and cells activity.
2
. Chemistry
As described in Scheme 1, three methods were developed for the synthesis of the
-aminothiazole derivatives. Cycloalkyl-fused 2-aminothiazole derivatives 12a−b were
2
obtained by one-pot reactions of commercially available cyclopentanone or

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cyclohexanone with mixture of thiourea and iodine in sealed tubes at 110 °C with yields
4
0
of 56%−60%. However, the tetrahydropyridine-fused 2-aminothiazole derivatives,
4a−b, 20a−q, and 23a−b, were obtained with extremely low yields in the same method
as described above. Thus, amine formation of 19a−m and 19o−r with pyrrolidine in
1
4
1
isopropanol at 55 °C, followed by reaction with elemental sulfur and cyanamide, were
employed as the alternative method to obtain bicyclic product 20a−q with yields of
3
8%−96%. Deprotection of compound 20q using 4 N hydrochloric acid in dioxane
provided compound 20r, which was converted to 20s upon reaction with
N,N-dimethylcarbamoyl chloride with an overall yield of 35% for the two steps. To
obtain title compounds 20a−q, N-aryl and N-(heteroaryl-substituted)-4-piperidones 19j−n
were prepared by two-step syntheses proceed with the Buchwald-Hartwig amination
using X-Phos as ligand for the Pd(0)-catalyzed coupling reactions, and followed by
subsequent hydrolysis of resultant ketals 16a−e. The method for preparing
N-alkyl-substituted piperidones 19p and 19q is the Dieckmann cyclization of amino
diesters as 18a−b, which was produced by the Michael addition of ethyl acrylate with
4
2
substituted anilines 17a and 17b. Intermediate 19o was obtained by treating 16f with
sodium hydride in 1,3-dimethyl-imidazolidin-2-one, hydrolysis of ketal 16g, and
deprotection of the benzyl group of 19n by using 10% Pd-C/H
2
. Compounds 14a and 14b
were prepared simultaneously by treating N-Boc-3-piperidone 13 with sulfur and
4
2
cyanamide in 20% and 16% yield, respectively. Meanwhile, the desired compounds
4
4
2
3a−b were achieved via the Hantzsch’s cyclocondensation of α-bromoacetones 22a−b

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with thiourea with yields 38%−92%.
Scheme 1. Synthesis of fused 2-aminothiazole derivatives 12a−b, 14a−b, 20a−s, and
a
2
3a−d
a
Reagents and conditions: (a) thiourea, I
5 °C, (ii) NH CN, S , rt; (c) 1,4-dioxa-8-azaspiro[4.5]decane, NaO-t-Bu, Pd(OAc)
X-Phos, toluene/t-BuOH (5:1), 120 °C, microwave irradiation; (d) 2 N HCl, THF, 60 °C;
e) methyl acrylate, MeOH, 40 °C, (f) (i) MeONa/MeOH 30%aq, toluene, 110 °C, (ii)
conc. HCl, 100 °C; (g) NBS, THF, 0 °C; (h) thiourea, NaHCO , EtOH, 80 °C; (i) NaH,
2
, 110 °C, sealed tube; (b) (i) pyrrolidine, i-PrOH,
5
2
8
2
,
(
3
1
,3-dimethyl-imidazolidin-2-one, 0 °C to 90 °C; (j) 10% Pd-C/H , MeOH, 45 °C; (k) 4 N
2

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2 3
HCl in dioxane, rt; (l) N,N-dimethylcarbamoyl chloride, K CO , MeCN, DIPEA, 0 °C to
rt.
The synthesis of compounds 40−53 is shown in Scheme 2. First, the
1
-methoxy-2-propanol group was introduced to one phenolic hydroxyl of commercially
available 3,5-dihydroxybenzoic acid 24 using a Mitsunobu reaction to provide racemic
ether 25. Then, an Ullmann-type reaction of ether 25 with commercially available
4
2
-bromophenyl
methyl
sulfone
using
copper(I)
iodide
with
4
5
,2,6,6-tetramethyl-3,5-heptanedione as a ligand under microwave irradiation provided
diaryl ether 26a with the yield of 71%. Hydrolysis of methyl ester 26a yielded key
benzoic acid 27a. Finally, compounds 40−53 were installed via amidation of 27a with
2
-aminothiazole derivatives.
a
Scheme 2. Synthesis of target compounds 40−53

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a
Reagents and conditions: (a) 1-methoxy-2-propanol, PPh , DIAD, THF, 0 °C to rt; (b)
3
4
-bromophenyl methyl sulfone, Cs CO , CuI, TMHD, DMF, 120 °C, microwave
2 3
irradiation; (c) (i) 2 N NaOH, MeOH, THF, 0 °C to rt, (ii) 2 N HCl; (d) 2-aminothiazoles,
EDCI, HOBt, DMF, rt.
The synthesis of compounds 54−62 is shown in Scheme 3. Compounds 54−62 were
obtained by a three-step procedure, i.e. the Ullmann reactions between 25 with
commercially available arylbromides, hydrolysis of the diaryl ethers 26b−j, and finally
amidation.
a
Scheme 3. Synthesis of target compounds 54−62

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a
2
Reagents and conditions: (a) R Br, Cs
2
CO
3
, CuI, TMHD, DMF, 120 °C, microwave
irradiation; (b) (i) 2 N NaOH, MeOH, THF, 0 °C to rt, (ii) 2 N HCl; (c) 20q, EDCI, HOBt,
DMF, rt.
The synthesis of compounds 63−68 is shown in Scheme 4. First, copper-catalyzed
Ullmann cross-coupling of 24 with 4-bromophenyl methyl sulfone provided the diaryl
ether 28 followed by the nucleophilic substitution of the alkyl iodides or a Mitsunobu
reaction with 28, affording compounds 29a−f. Hydrolysis of esters 29a−f under basic
conditions provided the corresponding acids 30a−f which were finally reacted with
compound 20q to give compounds 63−68 in 44%−75% yield.
a
Scheme 4. Synthesis of target compounds 63−68

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a
Reagents and conditions: (a) 4-bromophenyl methyl sulfone, Cs CO , CuI, TMHD,
2
3
3
3
DMF, 120 °C, microwave irradiation; (b) R I, K CO , DMF, 0 °C to 50 °C; (c) R OH,
2
3
PPh , DIAD, THF, 0 °C to rt; (d) (i) 2 N NaOH, MeOH, THF, 0 °C to rt, (ii) 2 N HCl; (e)
3
2
0q, EDCI, HOBt, DMF, rt.
The synthesis of compounds 69−70 is shown in Scheme 5. One phenolic hydroxyl
group on the 3,5-dihydroxybenzoic acid 24 was firstly protected to form tosylate 31 using
tosyl chloride in the presence of sodium bicarbonate in diethyl ether. A Chan-Lam
4
6
coupling reaction of 31 was then performed to introduce the 3,5-difluorobenzene
moiety leading to diaryl ether 32. Deprotection in basic condition of tosylate 32 produced
4
7
the phenol 33a which was then subjected to a Mitsunobu reaction
R)-1-methoxy-2-propanol to produce (S)-isomeric compound 34a. Hydrolysis of ester
4a yielded acid 35a followed by amidation to afford compound 69. Compound 70 was
prepared through Ullmann condensation between 24 with
with
(
3

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azetidin-1-yl(4-bromo-2-fluorophenyl) methanone under microwave irradiation followed
by a Mitsunobu reaction with (R)-1-methoxy-2-propanol, hydrolysis of ester 34b, and
finally amidation with compound 20q.
a
Scheme 5. Synthesis of target compounds 69 and 70
a
Reagents and
conditions: (a) TsCl, sat. NaHCO3, Et O, rt; (b)
2
3
5
,5-difluorobenzeneboronic acid, Cu(OAc) , TEA, DCM, 0 °C to rt; (c) KOH, MeOH,
2
0 °C; (d) azetidin-1-yl(4-bromo-2-fluorophenyl) methanone, Cs CO , CuI, TMHD,
2
3
DMF, 120 °C, microwave irradiation; (e) (R)-1-methoxy-2-propanol, PPh , DIAD, THF,
3
0
°C to rt; (f) (i) 2 N NaOH, MeOH, THF, 0 °C to rt, (ii) 2 N HCl; (g) 20q, EDCI, HOBt,
DMF, rt.
The synthesis of compounds 71−85 is shown in Scheme 6. Compounds 39, 71−72, and
4−84 were prepared via amidation between chiral acids 38a−b and
7

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tetrahydropyridine-fused 2-aminothiazole derivatives. Deprotection of compound 72
using trifluoroacetic acid provided compounds 73; while deprotection of compound 39
using tetrabutylammonium fluoride produced compounds 85. Benzoic acid 38a−c were
prepared by a three-step produce, i.e. the Ullmann reaction between 24 with
azetidin-1-yl(4-bromophenyl)methanone,
Mitsunobu
reactions
with
(R)-1-methoxy-2-propanol, and finally hydrolysis of the diaryl ethers 37a−c.
a
Scheme 6. Synthesis of target compounds 71−85
O
O
O
HO
R3O
R3
O
O
O
O
OH
HO
O
a
b
c
O
O
O
OH
N
N
N
a R3= (R)-CH(CH3)CH2OCH3
b R3= (S)-CH(CH3)CH2OCH3
c R3= (S)-CH(CH3)CH2O-TBS
O
O
O
24
38a-c
36
37a-c
R1
X
Boc
Y
N
O
O
W
O
S
(
R)O
O
S
(S)O
(S)
O
O
N
OH
(R)
O
OH
N
O
O
N
O
N
N
H
N
H
d
d
O
O
O
O
N
O
N
O
O
O
72 W, Y = (CH2), X = NR1, R1 = Boc
3 W, Y = (CH2), X = NR1, R1 = H
4 W, Y = (CH2), X = NR1, R1 = COOMe
5 W, Y = (CH2), X = NR1, R1 = COOEt
6 W, Y = (CH2), X = NR1, R1 = CON(CH3)2
7 W, Y = (CH2), X = NR1, R1 = 2-methoxyethyl
78 W, Y = (CH2), X = NR1, R1 = 2-hydroxyethyl
9 W, Y = (CH2), X = NR1, R1 = 2-pyridyl
0 W, Y = (CH2), X = NR1, R1 = 2-pyrimidyl
1 W, Y = (CH2), X = NR1, R1 = 4-pyrimidyl
82 W, Y = (CH2), X = NR1, R1 = 4-pyridyl-2( 1H)-one
3 X, Y = (CH2), W = NR1, R1 = Boc
38a
71
38b
f
7
7
7
7
7
Boc
Boc
N
N
O
O
S
O
S
TBS
O
TBS
O
O
O
OH
O
N
N
N
HO
N
N
N
H
H
7
8
8
d
e
O
O
O
N
8
8
O
O
O
4 W, X = (CH2), Y = NR1, R1 = Boc
38c
39
85
a
2 3
Reagents and conditions: (a) azetidin-1-yl(4-bromophenyl)methanone, Cs CO , CuI,
3
TMHD, DMF, 120 °C, microwave irradiation; (b) R OH, PPh
3
, DIAD, THF, 0 °C to rt; (c)
(i) 2 N NaOH, MeOH, THF, 0 °C to rt, (ii) 2 N HCl, rt; (d) 2-aminothiazoles, EDCI,
HOBt, DMF, rt; (e) TBAF, THF, 0 °C to rt; (f) TFA, CH Cl , 0 °C to rt.
2
2
3
. Results and discussion

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3
.1. Primary in vitro assay
The activation of GK by the synthesized compounds was assessed in in vitro and in
vivo assays. The enzymatic activity of the synthesized compounds on recombinant human
GK was first evaluated. The GK activity of each compound was evaluated in 14 different
concentrations for enzymatic assay and is presented as EC50 and maximum activation
fold (versus control level) in Tables 1, 2, and 3. Compounds that exhibited EC below
5
0
1
.0 µM and maximum activation fold between 2.0-fold and 3.0-fold were chosen for
cellular assay, where glucose uptake in mouse primary hepatocytes after a 24-h treatment
with 20 µM compound and GSIS in INS-832/13 cells after 2-h treatment with 20 µM
compounds were measured. Clinical compound PF-04937319 (7) was evaluated in our
enzymatic assay for comparison.
a
Table 1. In vitro GK activity of compounds 40−53.
Glucose
Uptake (%)
ND
GSIS
b
compd
7
EC50 (µM)
Max Act
c
d
(fold)
ND
0.93 ± 0.24 2.11 ± 0.04
0.10 ± 0.04 4.80 ± 0.20
40
31 ± 1.0
NA

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4
1
2
0.89 ± 0.41 3.42 ± 0.14
31 ± 1.1
ND
NA
ND
4
＞10
＞10
2.17 ± 0.24
2.61 ± 0.10
43
ND
ND
4
4
5
8.42 ± 2.10 3.14 ± 0.15
5.42 ± 2.86 2.08 ± 0.10
ND
ND
ND
ND
4
4
6
7
7.08 ± 2.78 2.00 ± 0.11
2.22 ± 1.09 2.50 ± 0.08
ND
ND
ND
4
ND
1
.72 ±
48
0.32 ± 0.06 2.94 ± 0.06
36 ± 1.0
0
.20
49
50
51
＞10
2.05 ± 0.07
ND
ND
ND
ND
ND
0.89 ± 0.16 2.39 ± 0.10
1.09 ± 0.25 2.70 ± 0.03
ND
1
.30 ±
5
2
3
0.21 ± 0.09 2.97 ± 0.11
NA
ND
0
.19
5
＞10
2.10 ± 0.06
ND
a
b
Values are the mean ± SEM of three independent assays. Maximum fold activation of GK over
c
control level. Compounds were tested at 20 µM in the mouse primary hepatocytes as

ACCEPTED MANUSCRIPT
d
described in Experimental Section. Compounds were tested at 20 µM in INS-832/13 cells as
described in Experimental Section. NA, not active; ND, not determined.
For initial structure−activity relationship (SAR) exploration, cyclopentane-fused
2
-aminothiazole 40 and cyclohexane-fused 2-aminothiazole 41 were prepared to
determine the size of the ring. Compounds 40 and 41 showed EC values of 0.10 µM and
5
0
0
.89 µM in enzymatic assay (Table 1), respectively, thereby confirming our initial
hypothesis that cycloalkyl-fused 2-aminothiazoles would maintain the ability of GK
activation. Although none of them showed activity in increasing GSIS, the enhancement
of glucose uptake (both by 31%) was observed in cellular assays. The maximum
activation folds for these two compounds were 4.80-fold and 3.42-fold, respectively,
which were higher than the previously set criteria (ranged from 2.0-fold to 3.0-fold).
Because enzymatic activity of 41 was closer to the criteria (EC ≤ 1.0 µM, and maximum
5
0
fold between 2.0-fold and 3.0-fold) than 40, cyclohexane derivative 41 was chosen as the
lead compound for further structure optimization.
A molecular docking simulation was performed to get a putative binding mode.
Analysis of the docking poses of 41 (Figure 3) showed that the 2-aminothiazole moiety
maintained the critical interactions similarly to that of reported X-ray structure of the
1
0, 39
allosteric binding site of GK
: the aminothiazole core was anchored to the active site
via two highly conserved H-bonds with the carbonyl (2.4 Å) and the amino group (2.9 Å)
of Arg63; an additional H-bond occurred between the amino group of Gln98 and oxygen

ACCEPTED MANUSCRIPT
atom of methylether moiety (3.1 Å); the 4-(methylsulfonyl)phenyl moiety was oriented
toward a larger pocket, which is known to be tolerant of larger hydrophobic moieties; and
a H-bond interaction with Arg250 located at the bottom may further increase binding
4
8
affinity.
Figure 3. Proposed binding mode of 41 with human GK. (3A) The atoms of 41 are

ACCEPTED MANUSCRIPT
colored as follows: carbon green, oxygen red, nitrogen blue, sulfur yellow, and hydrogen
white. The docked protein structure of GK is shown in the cartoon and binding atoms of
key residues are colored as follows: carbon pink, oxygen red, and nitrogen blue. The
H-bonds are colored in maroon. (3B) The docked protein structure of GK is shown in
surface. The cyclohexane-fused 2-aminothiazole moiety fit in a vector lined with the side
chain of Arg63. The H-bonds between 41 and residues of GK are omitted for clarity.
Having established the activity of cyclohexane derivative 41, compounds with isosteric
replacement of methenes for O, (CO) and N(CH ) were evaluated in enzymatic assay
3
(Table 1). The dihydropyran derivative 42 and cyclohexanone derivative 43 were inactive,
1
while tetrahydropyridine derivative 44 containing a methyl moiety as the tail region (R )
displayed a marginal activity of EC50 value of 8.42 µM and maximum activation by
3
.14-fold. Then we embarked on evaluating the effect of various substituents at tail
1
region (R ) on GK activity. Docking overlays with protein crystal structure of 44 showed
that there should be accessible space to accommodate the groups larger than methyl to
increase potency. Consistent with our expectations, addition of larger groups than methyl
resulted in increases in the potency (cf. 45, 46, 47 and 48 vs 44), among four derivatives,
4
8 with a Boc group as tail region was the most potent; the values of EC50 (0.32 µM) and
maximum activation fold (by 2.94-fold) of 48 met our criteria; moreover, it was observed
that 48 increased glucose uptake in mouse primary hepatocytes and GSIS in INS-832/13
cells by 36% and 1.72-fold, respectively. Further investigation of the tail region was

ACCEPTED MANUSCRIPT
pursued to determine what effects aromatic rings had on potency and maximum
activation fold. As shown in Table 1, benzene derivative 49 and benzyl derivative 53 led
to deterioration of activity, which indicated that phenyl and benzyl might not be tolerated
as the tail region; surprisingly, pyridine derivatives showed increased potencies (cf. 50,
5
1 and 52 vs 44). When considering the cellular activities of these derivatives (40−53),
the Boc group of compound 48 was selected to remain constant while investigating
2
3
structure−activity relationship (SAR) of hydrophobic moieties R and R .
a
Table 2. In vitro GK activity of compounds 54−72 and 85.
Glucose
Uptake (%)
36 ± 1.0
GSIS
2
3
b
compd
R
R
EC50 (µM)
Max Act
c
d
(fold)
4
5
8
4
0.32 ± 0.06 2.94 ± 0.06
0.20 ± 0.10 4.07 ± 0.17
1.72 ± 0.20
1.42 ± 0.17
35 ± 1.3
5
5
5
6
＞10
1.89 ± 0.11
ND
ND
0.82 ± 0.21 2.74 ± 0.05
0.14 ± 0.03 2.37 ± 0.13
25 ± 0.2
1.14 ± 0.28
5
7
34 ± 0.3
NA

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5
5
8
9
0.23 ± 0.05 3.12 ± 0.06
0.90 ± 0.21 3.26 ± 0.11
ND
NA
ND
1.37 ± 0.07
6
6
6
0
1
2
0.29 ± 0.05 2.89 ± 0.05
0.09 ± 0.04 1.91 ± 0.06
0.87 ± 0.22 2.10 ± 0.07
33 ± 0.5
2.29 ± 0.20
1.24 ± 0.04
5.40 ± 0.21
NA
NA
6
6
6
6
6
6
3
4
5
6
7
8
2.23 ± 0.74 2.30 ± 0.07
0.17 ± 0.07 3.55 ± 0.13
0.18 ± 0.09 3.17 ± 0.11
0.55 ± 0.18 1.71 ± 0.10
ND
ND
NA
NA
NA
ND
NA
34 ± 0.6
26 ±1.5
29 ± 0.7
ND
＞10
2.11 ± 0.18
7.00 ± 1.85 2.30 ± 0.06
21 ± 1.6
7
1
2.60 ± 0.76 2.96 ± 0.12
ND
ND

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7
2
0.16 ± 0.04 2.84 ± 0.06
15 ± 0.4
2.69 ± 0.14
8
6
5
9
0
1.30 ± 0.38 2.62 ± 0.07
0.20 ± 0.06 2.72 ± 0.14
ND
ND
15 ± 0.6
19 ± 1.7
1.30 ± 0.14
1.10 ± 0.21
7
0.33 ± 0.08 2.83 ± 0.08
a
b
Values are the mean ± SEM of three independent assays. Maximum fold activation of GK over
c
control level. Compounds were tested at 20 µM in the mouse primary hepatocytes as
d
described in Experimental Section. Compounds were tested at 20 µM in INS-832/13 cells as
described in Experimental Section. NA, not active; ND, not determined.
2
Then the substituent effect of the hydrophobic moiety (R ) was explored (Table 2).
2
Replacement of the phenyl of R with pyridine (54) maintained enzymatic activity.
However, maximum activation by 4.07-fold for 54 was beyond our criteria. Modification
of the methyl sulfone from para-position (48) to meta-position (56) led to an slight
decrease in potency (cf. 0.82 µM for 56 versus 0.32 µM for 48). In contrast, the
ortho-methyl sulfone derivative 55 exhibited a significant decrease in GK activation.
2
6, 49
Based on the reported X-ray structure of the allosteric binding site of GK,
a possible

ACCEPTED MANUSCRIPT
explanation for the decrease in enzymatic activity of 55 might be the hindrance factor of
2
ortho-position of methyl sulfone for adapting of aromatic ring of R into the larger
hydrophobic pocket, and forming potential H-bond interactions. A tendency could be
observed that the increase of the size of alkyl groups led to a reduction in potency (cf.
0
.14 µM for 57 versus 0.23 µM and 0.90 µM for 58 and 59, respectively). Further
potency enhancement was realized upon replacing the sulfone of 59 with a carbonyl (60).
Surprisingly, 60 displayed favorable potency and maximum activation fold, and increased
hepatic glucose uptake in mouse primary hepatocytes by 33% and GSIS in INS-832/13
cells by 2.29-fold. Although N,N-dimethyl analogue 61 also showed a comparable
potency to that of 60, the enhancement of glucose uptake of 60 was not observed.
Replacing the phenyl of 60 with pyridine (62) could maintain the activation of GK,
however, the effect on increasing glucose uptake in mouse primary hepatocytes was not
observed. Compared to 72, fluoro-containing analogues 69 and 70 were also found to
display comparable enzymatic potency, whereas GSIS in INS-832/13 cells was slightly
increased by 1.30-fold and 1.10-fold, respectively.
3
Next, we explored the SAR of hydrophobic moiety (R ) by incorporating various
lipophilic substituents (Table 2). In these studies, compound 63 without the branched
methyl showed a loss of potency, while the truncated analogues 64 offered the enzymatic
potency with EC of 0.17 µM and 3.55-fold maximum activation. Replacement of the
5
0
isopropyl with larger lipophilic moieties (65−68), a tendency of decreasing of enzymatic
activity was accompanied with the increasing of the size of lipophilic moieties.

ACCEPTED MANUSCRIPT
Additionally, the potency of compounds 64−66 in modulating GSIS was lost, indicating
the importance of the methoxy group of 48. To quickly identify promising compounds for
in vivo studies, compounds bearing the 1-methoxy-2-propanol moiety (40−62) were
synthesized as racemates for preliminary evaluation. To confirm the effect of the chiral
center of 1-methoxy-2-propanol moiety on GK activity, chiral isomers were also assessed
by enzymatic and cellular assays. The (S)-isomer 72 showed a favorable enzymatic
potency with EC of 0.16 µM and maximum activation fold by 2.84-fold, and it had
5
0
approximately 16-fold improved potency in EC50 as compared to (R)-isomer 71.
Moreover, 72 increased hepatic glucose uptake in mouse primary hepatocytes by 15%
and GSIS in INS-832/13 cells by 2.69-fold. A similar trend was observed for the
benzene-centered GK activators with (S)-1-methoxy-2-propanol moiety for the in vitro
4
8, 50
GK activation assays, showing superior activity to corresponding (R)-isomers.
Based
on binding mode analysis of reported molecular docking studies and X-ray structures of
4
9–51
the allosteric binding site of GK,
the favorable (S)-1-methoxy-2-propanol moiety
bounds in a small hydrophobic pocket and forms hydrophobic interactions with Gly97
4
9, 52
and Leu451,
which might be conserved for (S)-isomer 72 and explain why in the
conformationally flexible 1-methoxy-2-propanol moiety the absolute chirality of the
methyl group has a significant effect on its activity. Therefore, we assumed that the
methyl group of (R)-isomer 71 might hamper the hydrophobic interactions with Gly97
and Leu451 and lead to the decrease in enzymatic activity. Additionally, in an attempt of
replacing the methoxy group of 72 with hydrophilic hydroxyl moiety (85), a significant

ACCEPTED MANUSCRIPT
loss of potency was also observed, further validating the hypothesis that
1
-methoxy-2-propanol moiety might bound in the small hydrophobic pocket and form
hydrophobic interactions.
a
Table 3. In vitro GK activity of compounds 73−84.
Glucose
GSIS
b
compd
EC50 (µM)
Max Act
c
d
Uptake (%)
(fold)
7
7
7
7
7
2
3
4
5
6
0.16 ± 0.04 2.84 ± 0.06
1.13 ± 0.40 2.20 ± 0.06
0.69 ± 0.20 2.38 ± 0.07
0.23 ± 0.06 2.22 ± 0.04
0.50 ± 0.19 2.35 ± 0.07
15 ± 0.4
ND
2.69 ± 0.14
ND
16 ± 1.8
17 ± 1.4
21 ± 1.0
5.68 ± 0.11
5.45 ± 0.43
3.52 ± 0.34
7
7
7
8
0.21 ± 0.06 1.76 ± 0.04
4.60 ± 1.87 1.97 ± 0.05
13 ± 0.3
ND
6.07 ± 0.60
ND