Novel agonists of free fatty acid receptor 1 (GPR40) based on 3-(1,3,4-thiadiazol-2-yl)propanoic acid scaffold

Article abstract of DOI:10.3109/14756366.2016.1142984

1,3,4-Thiadiazole was explored as a more polar, heterocyclic replacement for the phenyl ring in the 3-arylpropionic acid pharmacophore present in the majority of GPR40 agonists. Out of 13 compounds synthesized using a flexible, three-step protocol (involving no chromatographic purification), four compounds were confirmed to activate the target in micromolar concentration range. While the potency of the series should be subject of further optimization, the remarkable aqueous solubility and microsomal stability observed for the lead compound (8g) apparently attests to this new scaffold’s high promise in the GPR40 agonist field.

Full text of DOI:10.3109/14756366.2016.1142984

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Novel agonists of free fatty acid receptor 1 (GPR40)
based on 3-(1,3,4-thiadiazol-2-yl)propanoic acid
scaffold
Mikhail Krasavin, Alexey Lukin, Nikolay Zhurilo, Alexey Kovalenko, Ihor
Zahanich & Sergey Zozulya
To cite this article: Mikhail Krasavin, Alexey Lukin, Nikolay Zhurilo, Alexey Kovalenko, Ihor
Zahanich & Sergey Zozulya (2016): Novel agonists of free fatty acid receptor 1 (GPR40) based
on 3-(1,3,4-thiadiazol-2-yl)propanoic acid scaffold, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2016.1142984
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1142984
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1142984
!
RESEARCH ARTICLE
Novel agonists of free fatty acid receptor 1 (GPR40) based on
3-(1,3,4-thiadiazol-2-yl)propanoic acid scaffold
Mikhail Krasavin1, Alexey Lukin2, Nikolay Zhurilo2, Alexey Kovalenko2, Ihor Zahanich3, and Sergey Zozulya3,4
1Department of Organic Chemistry, Institutes of Chemistry and Translational Biomedicine, Saint Petersburg State University, Saint Petersburg,
2
Russian Federation, Lomonosov Moscow State Institute of Fine Chemical Technologies (MITHT), Moscow, Russian Federation, Enamine Ltd,
3
4
78 Chervonotkatska, Kyiv, Ukraine, and Taras Shevchenko National University, 62 Volodymyrska, Kyiv, Ukraine
Abstract
Keywords
1,3,4-Thiadiazole was explored as a more polar, heterocyclic replacement for the phenyl ring in
the 3-arylpropionic acid pharmacophore present in the majority of GPR40 agonists. Out of 13
compounds synthesized using a flexible, three-step protocol (involving no chromatographic
purification), four compounds were confirmed to activate the target in micromolar concen-
tration range. While the potency of the series should be subject of further optimization, the
remarkable aqueous solubility and microsomal stability observed for the lead compound (8g)
apparently attests to this new scaffold’s high promise in the GPR40 agonist field.
Agonists, aqueous solubility, bioisosteric
replacement, cLogP, free fatty acid recep-
tor 1, GPR40, metabolic stability, total polar
surface area
History
Received 14 December 2015
Revised 8 January 2016
Accepted 9 January 2016
Published online 19 February 2016
Introduction
lipophilic character of the compound6. Indeed, TAK-875 and
many other advanced compounds of this class are based on
3-phenylpropanoic scaffold and aim to mimic the endogenous
ligands of GPR40 which are fatty acids. A much exploited
strategy pursued in the design on higher-TPSA GPR40 agonists
is to use more polar moieties (such as various heterocycles) to
‘‘decorate’’ compounds’ periphery7. The other, less popular
approach is to replace 3-phenylpropanoic acid core with
heterocyclic congeners. Compounds of this sort are a lot less
abundant in the literature and can be exemplified by Takeda’s
compounds 18, 29 and 310 as well as Amgen’s indole-based
compound 411 (Figure 1). In this article, we present our results
related to the design, the synthesis of a series of hitherto
unreported compounds containing 3–(1,3,4-thiadiazol-2-yl)pro-
pionic acid moiety (cLogP ¼ ꢀ0.09) in lieu of 3-phenylpropa-
noic acid moiety (cLogP ¼ 1.84)12. Herein, we also report the
results of characterization of the series as agonists of GPR40
receptor and determination of key ADMET properties of the
most promising compounds.
Activation of free fatty acid receptor 1 (FFAR1, also known as
GPR40) in pancreatic Islets of Langerhans, where it is highly
expressed in hyperglycemic state, leads to a cascade of events that
culminate in increased levels of insulin and eventual lowering of
the glucose levels1. This mechanism of achieving the latter does
not carry the danger of causing hypoglycemia (i.e. lowering of
blood glucose concentration below physiologically acceptable
levels). Therefore, agonists of GPR40 represent a therapeutically
attractive alternative to the currently available treatments for type
2 diabetes mellitus (T2DM)2. Unfortunately, the field of GPR40
agonist-based therapies was struck severely in late-2013 with
discontinuation, due to idiosyncratic liver toxicity, of phase-III
trial of Takeda’s frontrunner compound fasiglifam (TAK-875)3.
Currently, only one clinical investigation of a GPR40 agonist is
underway, namely that of compound P11187 of undisclosed
structure by Piramal4. However, the proof of principle of the new
approach to treat T2DM was, in principle, established in the
course of TAK-875 investigation5. Thus, current research effort
worldwide6 is aimed primarily at tackling the toxicity profile of
GPR40 agonists.
Materials and methods
One of the ways to achieve the latter is to increase the total
2
polar surface area (TPSA, A ) of GPR40 agonists. Liver toxicity,
Chemical syntheses – general
˚
of TAK-875 in particular, has been linked to the overly
All reactions were conducted in oven-dried glassware in atmos-
phere of nitrogen. Melting points were measured with a Buchi
C-520 melting point apparatus and were not corrected. Analytical
thin-layer chromatography (TLC) was carried out on Silufol UV-
254 silica gel plates using appropriate mixtures of ethyl acetate
and hexane. Compounds were visualized with short-wavelength
Address for correspondence: Mikhail Krasavin, Department of Organic
Chemistry, Saint Petersburg State University, 26 Universitetskii Prospekt,
Institute of Chemistry, PETERHOF, 196504 Russian Federation. Tel:
+79313617872. Fax: +78124286939. E-mail: m.krasavin@spbu.ru

2
M. Krasavin et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
O
O
S
O
F3C
OH
O
O
O
O
N
N
F3C
OH
N
O
OH
Fasiglifam (TAK-875)
O
O
2
1
O
OH
O
N
N
HN
OH
O
O
O
O
3
4
Figure 1. Structures of TAK-875 and of the heterocyclic analogs of 3-phenylpropanoic acid-based GPR40 agonists 1–4.
UV light. 1H NMR and 13C NMR spectra were recorded on N(1)-Phenyl-2-hydrazino-2-thiooxacetamide (7c)
Bruker MSL-300 spectrometers in DMSO-d6 using TMS as an
1
Yield 55%, yellow solid, m. p. 152–156ꢁC; H NMR (300 MHz,
internal standard. Mass spectra were recorded using Shimadzu
DMSO-d6) d 10.21 (s, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.36
LCMS-2020 system with electron impact (EI) ionization. All and
(t, J ¼ 7.9 Hz, 5H), 7.14 (t, J ¼ 7.4 Hz, 1H); 13C NMR
reagents and solvents were obtained from commercial sources and
(75 MHz, DMSO-d6) d 167.73, 158.39, 137.91, 129.31, 125.03,
used without purification.
120.54.
N(1)– (3-chlorophenyl)-2-hydrazino-2-thiooxacetamide
(7d)
General procedure for the preparation of compounds
7a–m12
1
Yield 64%, yellow solid, m. p. 162–166ꢁC; H NMR (300 MHz,
To a suspension of elementary sulfur (1024 mg, 32.0 mmol) in dry
dimethylformamide (DMF) (40 mL) was sequentially added (in
dropwise fashion) triethylamine (4.45 mL, 32.0 mmol) and mor-
pholine (2.12 mmol) and the resulting mixture was stirred for
30 min. Then, it was treated with a solution of respective
2-chloroacetamides 5 (1.0 mmol), all of which are known
compounds. The mixture was stirred overnight, poured into
water (100 mL) and the resulting precipitate was separated by
filtration and air-dried. It was then suspended in acetone (100 mL)
and the insoluble residue of excess of unreacted sulfur was filtered
off and discarded. The filtrate was evaporated to dryness and the
dry residue (predominantly consisting of 6) was dissolved in dry
DMF (30 mL), treated with hydrazine hydrate (5.0 mL) and stirred
for 12 h. The reaction mixture was poured into water, and the pH
of the aqueous medium was adjusted to 5.0 with 2 M aqueous
HCl. The resulting precipitate was filtered off, washed with water,
air-dried and crystallized from ethanol to deliver analytically pure
compounds seven in yields indicated.
DMSO-d6) d 10.38 (s, 1H), 7.94 (t, J ¼ 2.0 Hz, 1H), 7.73–7.68 (m,
1H), 7.38 (t, J ¼ 8.1 Hz, 1H), 7.20 (dd, J ¼ 7.8, 1.6 Hz, 1H); 13C
NMR (75 MHz, CDCl3) d 167.62, 158.95, 139.49, 133.54, 130.93,
124.71, 120.17, 119.24.
N(1)– (4-methoxyphenyl)-2-hydrazino-2-thiooxacetamide
(7e)
1
Yield 77%, yellow solid, m. p. 168–172ꢁC; H NMR (300 MHz,
DMSO-d6) d 10.10 (s, 1H), 7.66 (t, J ¼ 6.2 Hz, 2H), 6.92 (t,
J ¼ 6.1 Hz, 1H), 3.73 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d
167.79, 157.96, 156.59, 131.03, 122.15, 114.39, 55.68
N(1)– (2-fluorophenyl)-2-hydrazino-2-thiooxacetamide
(7f)
1
Yield 70%, yellow solid, m. p. 172–176ꢁC; H NMR (300 MHz,
DMSO-d6) d 10.26 (s, 1H), 8.05 (ddd, J ¼ 7.8, 5.6, 2.9 Hz, 1H),
7.40–7.29 (m, 1H), 7.28–7.19 (m, 2H); 13C NMR (75 MHz,
DMSO-d6) d 165.68 (s), 157.48 (s), 153.95 (d, J ¼ 244.9 Hz),
126.59 (d, J ¼ 7.8 Hz), 125.52 (d, J ¼ 10.9 Hz), 125.25 (d,
J ¼ 3.6 Hz), 122.77 (s), 116.02 (d, J ¼ 19.0 Hz).
N(1)–(4-methylphenyl)-2-hydrazino-2-thiooxacetamide
(7a)
1
Yield 55%, yellow solid, m.p. 155–158ꢁC; H NMR (300 MHz,
N(1)–(3-methylphenyl)-2-hydrazino-2-thiooxacetamide
(7 g)
DMSO-d6) d 10.13 (s, 1H), 7.62 (d, J ¼ 8.4 Hz, 2H), 7.16 (d,
J ¼ 8.3 Hz, 2H), 2.27 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d
167.73, 158.15, 135.41, 134.16, 129.70, 120.49, 20.98.
1
Yield 59%, yellow solid, m. p. 115–119ꢁC; H NMR (300 MHz,
DMSO-d6) d 10.12 (s, 1H), 7.56 (s, 2H), 7.25 (dd, J ¼ 11.4,
4.8 Hz, 1H), 6.96 (d, J ¼ 7.3 Hz, 1H), 2.30 (s, 3H); 13C NMR
(75 MHz, DMSO-d6) d 167.76, 158.20, 138.64, 137.78, 129.18,
125.75, 120.95, 117.57, 21.59.
N(1)– (2,4-difluorophenyl)-2-hydrazino-2-thiooxaceta-
mide (7b)
1
Yield 71%, yellow solid, m. p. 174–178ꢁC; H NMR (300 MHz,
DMSO-d6) d 10.19 (s, 1H), 7.91 (tt, J ¼ 19.2, 9.6 Hz, 1H), 7.41
(ddd, J ¼ 11.5, 9.0, 2.8 Hz, 1H), 7.28–7.04 (m, 1H), 7.19–7.08 (m,
1H); 13C NMR (75 MHz, DMSO-d6) d 165.81 (s), 159.51 (dd,
J ¼ 244.7, 11.7 Hz), 157.88 (s), 154.63 (dd, J ¼ 248.4, 12.7 Hz),
125.01 (dd, J ¼ 9.7, 2.3 Hz), 122.18 (dd, J ¼ 11.5, 3.7 Hz), 111.93
(dd, J ¼ 22.1, 3.7 Hz), 104.85 (dd, J ¼ 27.1, 23.8 Hz).
N(1)– (4-fluorophenyl)-2-hydrazino-2-thiooxacetamide
(7 h)
1
Yield 62%, yellow solid, m. p. 179–183ꢁC; H NMR (300 MHz,
DMSO-d6) d 10.28 (s, 1H), 7.78 (ddd, J ¼ 8.5, 5.2, 2.9 Hz, 1H),
7.23–7.16 (m, 2H); 13C NMR (75 MHz, CDCl3) d 167.88 (s),

DOI: 10.3109/14756366.2016.1142984
Novel agonists of free fatty acid receptor 1 (GPR40)
3
159.24 (d, J ¼ 241.5 Hz), 158.53 (s), 134.35 (d, J ¼ 2.6 Hz), 3–(5-{[(2,4-Difluorophenyl)amino]carbonyl}-1,3,4-thiadia-
122.70 (d, J ¼ 8.0 Hz), 115.88 (d, J ¼ 22.4 Hz).
zol-2-yl)propanoic acid (8b)
1
Yield 87%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d ppm d 12.44 (s, 1H), 10.90 (s, 1H), 7.58 (td, J ¼ 8.8,
6.2 Hz, 1H), 7.43–7.35 (m, 1H), 7.18–7.10 (m, 1H), 3.38 (t,
J ¼ 6.9 Hz, 2H), 2.82 (t, J ¼ 6.9 Hz, 2H); 13C NMR (75 MHz,
DMSO-d6) d 174.29 (s), 173.43 (s), 165.25 (s), 160.70 (dd,
J ¼ 245.8, 11.7 Hz), 157.12 (s), 156.65 (dd, J ¼ 250.8, 13.0 Hz),
129.13 (d, J ¼ 10.0 Hz), 121.20 (dd, J ¼ 12.6, 3.7 Hz), 111.92 (dd,
J ¼ 22.3, 3.6 Hz), 105.05 (dd, J ¼ 26.9, 24.3 Hz), 33.15 (s), 25.71
(s); MS m/z 314.2 (M + H+).
N(1)–(2-methylphenyl)-2-hydrazino-2-thiooxacetamide
(7i)
1
Yield 73%, yellow solid, m. p. 151–156ꢁC; H NMR (300 MHz,
DMSO-d6) d 10.09 (s, 1H), 7.83 (d, J ¼ 7.9 Hz, 1H), 7.25 (dd,
J ¼ 13.5, 7.5 Hz, 2H), 7.11 (t, J ¼ 7.4 Hz, 1H), 2.28 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 166.77, 157.44, 135.79, 130.94,
130.04, 126.94, 125.75, 122.08, 17.84.
N(1)-{[(5-Methyl-1,3,4-thiadiazol-2-yl)amino]-carbonyl}-2-
hydrazino-2-thiooxacetamide (7j)
3-[5-(Anilinocarbonyl)-1,3,4-thiadiazol-2-yl]propanoic acid
(8c)
1
Yield 45%, yellow solid, m. p. 240–243ꢁC; H NMR (300 MHz,
1
Yield 80%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
CDCl3) d 3.75–3.07 (m, 4H), 2.64 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) d 161.02, 160.63, 159.87, 158.34, 15.37.
DMSO-d6) d ppm 12.42 (s, 1H), 11.10 (s, 1H), 7.84 (t, J ¼ 8.6 Hz,
2H), 7.38 (dd, J ¼ 16.9, 9.2 Hz, 2H), 7.23–7.00 (m, 1H), 3.38 (t,
J ¼ 7.0 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz, 2H); 13C NMR (75 MHz,
DMSO-d6) d ppm d 174.16, 173.49, 166.28, 156.76, 138.04,
129.25, 125.22, 121.29, 33.13, 25.68; MS m/z 278.2 (M + H+).
N(1)– (3-methoxyphenyl)-2-hydrazino-2-thiooxacetamide
(7k)
1
Yield 54%, yellow solid, m. p. 165–168ꢁC; H NMR (300 MHz,
3–(5-{[(3-Chlorophenyl)amino]carbonyl}-1,3,4-thiadiazol-
2-yl)propanoic acid (8d)
CDCl3) d 10.17 (s, 1H), 7.42 (t, J ¼ 2.0 Hz, 1H), 7.34 (d,
J ¼ 8.3 Hz, 1H), 7.26 (t, J ¼ 8.1 Hz, 1H), 6.72 (dd, J ¼ 7.9,
2.1 Hz, 1H), 3.74 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d
167.85, 159.98, 158.37, 139.02, 130.16, 112.67, 110.65, 106.20,
55.56.
1
Yield 76%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d 12.44 (s, 3H), 11.30 (s, 3H), 7.99 (t, J ¼ 1.9 Hz, 3H),
7.81 (dd, J ¼ 15.5, 7.3 Hz, 3H), 7.40 (t, J ¼ 8.1 Hz, 1H), 7.22 (dd,
J ¼ 7.9, 1.5 Hz, 3H), 3.38 (t, J ¼ 7.0 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz,
6H); 13C NMR (75 MHz, DMSO-d6) d 174.38, 173.44, 165.94,
157.05, 139.64, 133.48, 130.95, 124.87, 120.68, 119.66, 33.15,
25.73; MS m/z 312.8 (M + H+).
N(1)– (3,4-difluorophenyl)-2-hydrazino-2-thiooxaceta-
mide (7 l)
1
Yield 76%, yellow solid, m. p. 164–169ꢁC; H NMR (300 MHz,
3–(5-{[(4-Methoxyphenyl)amino]carbonyl}-1,3,4-thiadia-
zol-2-yl)propanoic acid (8e)
CDCl3) d 10.41 (s, 1H), 7.91 (ddd, J ¼ 13.0, 7.4, 2.4 Hz, 1H),
7.65–7.55 (m, 1H), 7.43 (dd, J ¼ 19.5, 9.2 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6) d 167.77 (s), 158.94 (s), 149.53 (dd,
J ¼ 210.8, 12.9 Hz), 146.30 (dd, J ¼ 210.4, 13.0 Hz), 135.06 (dd,
J ¼ 9.1, 3.0 Hz), 117.93 (d, J ¼ 17.9 Hz), 117.43 (dd, J ¼ 6.1,
3.4 Hz), 109.97 (d, J ¼ 21.7 Hz).
Yield 91%, white solid, m. p. 195–196ꢁC; 1H NMR
(300 MHz, DMSO-d6) d 12.43 (s, 1H), 10.99 (s, 1H), 7.72 (d,
J ¼ 6.2 Hz, 1H), 6.94 (d, J ¼ 6.2 Hz, 1H), 3.74 (s, 1H), 3.37 (t,
J ¼ 7.0 Hz, 1H), 2.82 (t, J ¼ 7.0 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6) d 173.93, 173.46, 166.47, 156.65, 156.32, 131.13,
122.79, 114.33, 55.66, 33.16, 25.68; MS m/z 308.2 (M + H+).
N(1)- {[(5-Ethyl-1,3,4-thiadiazol-2-yl)amino]-carbonyl}-2-
hydrazino-2-thiooxacetamide (7m)
3–(5-{[(2-Fluorophenyl)amino]carbonyl}-1,3,4-thiadiazol-
2-yl)propanoic acid (8f)
1
Yield 40%, yellow solid, m. p. 183–189ꢁC; H NMR (300 MHz,
CDCl3) d 3.65–3.19 (m, 3H), 3.07–2.95 (m, 1H), 1.29 (t,
J ¼ 7.5 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) d 167.13,
166.67, 159.90, 158.02, 23.21, 14.25.
1
Yield 81%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d ppm d 12.45 (s, 1H), 10.82 (s, 1H), 7.61 (t, J
¼ 7.8 Hz, 1H), 7.40–7.11 (m, 3H), 3.39 (t, J ¼ 6.9 Hz, 2H), 2.83 (t,
J ¼ 6.9 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d 174.28 (s),
173.45 (s), 165.40 (s), 156.22 (d, J ¼ 247.9 Hz), 128.34 (d,
J ¼ 7.8 Hz), 127.50 (s), 124.79 (dd, J ¼ 26.0, 7.9 Hz), 116.45 (d,
J ¼ 19.6 Hz), 33.14 (s), 25.72 (s). MS m/z 296.6 (M + H+).
General procedure for the preparation of compounds
8a–m12
Thiohydrazide 7 (0.1 mmol) was placed in a thick-walled crew-
capped glass tube along with succinic anhydride (1.2 mmol) and
glacial acetic acid (3.0 mL). The reaction mixture was heated at
reflux temperature on vigorous stirring over 2 h, cooled down and
poured into water (25 mL). The precipitate formed was filtered off
and air-dried to deliver analytically pure compounds 8 in yields
indicated.
3–(5-{[(3-Methylphenyl)amino]carbonyl}-1,3,4-thiadiazol-
2-yl)propanoic acid (8 g)
1
Yield 84%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d 12.43 (s, 1H), 11.01 (s, 1H), 7.67 (s, 1H), 7.60 (d,
J ¼ 8.2 Hz, 1H), 7.24 (t, J ¼ 7.8 Hz, 1H), 6.97 (d, J ¼ 7.5 Hz, 1H),
3.37 (t, J ¼ 7.0 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz, 2H), 2.30 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 174.08, 173.44, 166.36, 156.69,
138.45, 138.07, 129.06, 125.83, 121.73, 118.45, 33.16, 25.71,
21.67; MS m/z 292.3 (M + H+).
3–(5-{[(4-Methylphenyl)amino]carbonyl}-1,3,4-thiadiazol-
2-yl)propanoic acid (8a)
1
Yield 73%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d ppm d 12.44 (s, 3H), 11.02 (s, 3H), 7.70 (d,
J ¼ 8.4 Hz, 6H), 7.17 (d, J ¼ 8.4 Hz, 6H), 3.37 (t, J ¼ 7.0 Hz,
7H), 2.82 (t, J ¼ 7.0 Hz, 6H), 2.27 (s, 9H); 13C NMR (75 MHz,
3–(5-{[(4-Fluorophenyl)amino]carbonyl}-1,3,4-thiadiazol-
2-yl)propanoic acid (8 h)
1
DMSO-d6) d 174.03, 173.47, 166.40, 156.54, 135.59, 134.29, Yield 95%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
129.62, 121.20, 33.14, 25.69, 20.98; MS m/z 292.3 (M + H+). DMSO-d6) d ppm d 12.44 (s, 1H), 11.19 (s, 1H), 7.84

4
M. Krasavin et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
O
O
OH
O
O
(Het)Ar
S
OH
S
N
Lipophilic
periphery
N
H
Lipophilic
periphery
+
O
N
HN
Y
Lipophilic
periphery
NH
2
O
X
cLogP=1.89
(core)
cLogP= −0.09
(core)
Figure 2. Design of new 1,3,4-thiadiazole-containing inhibitors explored in this work.
O
O
O
S , morpholine
8
Cl
(Het)Ar
N
(Het)Ar
N
N
Et N, DMF, r.t.
3
H
H
S
16-20h
5
6
N H
hydrate
2
4
O
O
O
HO
O
O
O
(Het)Ar
H
S
N
N
N
(Het)Ar
H
N
H N
N
glacial AcOH
reflux, 2 h
2
H
S
8, 65-95%
7, 40-77%
Scheme 1. Synthesis of 3-(1,3,4-thiadiazol-2-yl)propanoic acids 8a–m studied in this work.
(dt, J ¼ 10.4, 4.3 Hz, 2H), 7.20 (dd, J ¼ 15.1, 6.1 Hz, 2H), 3.38 (t, J ¼ 13.0, 7.5, 2.5 Hz, 1H), 7.67 (ddd, J ¼ 6.0, 3.7, 1.9 Hz, 1H),
J ¼ 6.9 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz, 2H); 13C NMR (75 MHz, 7.44 (dd, J ¼ 19.7, 9.2 Hz, 1H), 3.38 (t, J ¼ 7.0 Hz, 2H), 2.82 (t,
DMSO-d6) d 174.15 (s), 173.44 (s), 166.19 (s), 159.28 (d, J ¼ 7.0 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d 174.36 (s),
J ¼ 241.5 Hz), 156.70 (s), 134.53 (d, J ¼ 2.7 Hz), 123.17 (d, 173.42 (s), 165.88 (s), 156.93 (s), 149.53 (dd, J ¼ 203.2, 13.0 Hz),
J ¼ 8.0 Hz), 115.86 (d, J ¼ 22.4 Hz), 33.16 (s), 25.71 (s); MS m/z 146.30 (dd, J ¼ 202.8, 12.9 Hz), 135.20 (dd, J ¼ 9.0, 3.0 Hz),
296.4 (M + H+).
117.96 (d, J ¼ 17.7 Hz), 117.70 (dd, J ¼ 6.1, 3.4 Hz), 110.29 (d,
J ¼ 21.7 Hz), 33.15 (s), 25.73 (s); MS m/z 314.2 (M + H+).
3–(5-{[(2-Methylphenyl)amino]carbonyl}-1,3,4-thiadiazol-
2-yl)propanoic acid (8i)
3–(5-{[(5-Ethyl-1,3,4-thiadiazol-2-yl)amino]-carbonyl}-
1
Yield 91%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz, 1,3,4-thiadiazol-2-yl)propanoic acid (8m)
DMSO-d6) d 12.44 (s, 1H), 10.60 (s, 1H), 7.47–7.33 (m, 1H),
1
Yield 72%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d 12.94 (s, 1H), 3.37 (t, J ¼ 6.9 Hz, 2H), 3.00 (q,
J ¼ 7.5 Hz, 2H), 2.82 (t, J ¼ 6.9 Hz, 2H), 2.41 (s, 1H), 1.30 (t,
J ¼ 7.5 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) d 174.09, 173.46,
33.03, 29.20, 25.66, 23.45, 13.85; MS m/z 314.4 (M + H+).
7.33–7.08 (m, 3H), 3.38 (t, J ¼ 6.9 Hz, 2H), 2.82 (t, J ¼ 6.9 Hz,
2H), 2.24 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 174.20,
173.61, 165.92, 156.93, 135.15, 133.97, 130.97, 127.25, 126.72,
126.68, 33.05, 25.60, 18.09; MS m/z 292.3 (M + H+).
3–(5-{[(5-Methyl-1,3,4-thiadiazol-2-yl)amino]-carbonyl}-
1,3,4-thiadiazol-2-yl)propanoic acid (8j)
Determination of biological activity and ADMET
characterization of compounds 8a–m
1
Yield 65%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d 13.04 (s, 1H), 3.37 (t, J ¼ 6.9 Hz, 2H), 2.82 (t, The calcium flux assay to determine agonistic activity of
J ¼ 6.9 Hz, 2H), 2.63 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d compounds 8a–m was conducted on Chinese hamster ovary
174.02, 173.44, 160.03, 33.05, 25.67, 15.67; MS m/z 300.2 (CHO) cells stably expressing GPR40 using the protocol earlier
(M + H+).
described by us2. The experimental details of this assay and of the
procedures used to determine aqueous solubility, metabolic
stability in the presence of mouse liver microsomes and plasma
protein binding are provided in Supplementary Material.
3–(5-{[(3-Methoxyphenyl)amino]carbonyl}-1,3,4-thiadia-
zol-2-yl)propanoic acid (8k)
1
Yield 78%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz,
DMSO-d6) d 12.43 (s, 1H), 11.06 (s, 1H), 7.55–7.40 (m, 2H), 7.26 Results and discussion
(t, J ¼ 8.2 Hz, 1H), 6.73 (dd, J ¼ 8.2, 2.3 Hz, 1H), 3.74 (s, 3H),
In order to mimic known GPR40 ligand with a 3-(1,3,4-
3.38 (t, J ¼ 7.0 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz, 2H); 13C NMR
(75 MHz, DMSO-d6) d 174.19, 173.48, 166.26, 159.86, 156.76,
139.25, 130.08, 113.40, 110.61, 106.99, 55.50, 33.12, 25.69; MS
m/z 308.4 (M + H+).
thiadiazol-2-yl)propanoic acid, one should perhaps consider
decorating the latter with lipophilic tail periphery and thus
increasing affinity to the receptor (which has fatty acids like
eicosatrienoic acid as endogenous ligands)13. In this work, we
selected anilide periphery for exploration of the SAR around
this novel GPR40 scaffolds. We reasoned that a series of
compounds like this would be conveniently attainable via
3–(5-{[(3,4-Difluorophenyl)amino]carbonyl}-1,3,4-thiadia-
zol-2-yl)propanoic acid (8 l)
1
Yield 90%, white solid, m. p. 195–196ꢁC; H NMR (300 MHz, cyclocondensation of the respective thiohydrazides with succininc
DMSO-d6) d ppm d 12.44 (s, 1H), 11.35 (s, 1H), 7.94 (ddd, anhydride (Figure 2).

DOI: 10.3109/14756366.2016.1142984
Novel agonists of free fatty acid receptor 1 (GPR40)
5
Table 1. Compounds 7(8)a–m synthesized in this work and GPR40 activation data obtained for 8a–m.
% GPR40 activation
at 5 mM*
Compound
Ar
Yield of 7, %
Yield of 8, %
7(8)a
55
73
33.2
*
*
7(8)b
71
87
28.6
F
F
7(8)c
7(8)d
55
64
80
76
13.2
52.7
*
Cl
*
7(8)e
7(8)f
77
70
91
81
31.4
36.7
MeO
*
*
F
7(8)g
59
84
59.9
*
*
7(8)h
7(8)i
62
73
95
91
38.3
18.6
F
*
7(8)j
45
54
65
78
57.0
32.8
S
*
N
N
7(8)k
MeO
*
7(8)l
76
40
90
72
63.9
32.3
F
*
F
7(8)m
S
*
N
N
*Relative to GPR40 activation by 20 mM of GW9509, n ¼ 2.
The synthesis of the compounds was based on the previously respect to the anilde (or heteroaromatic) portion and is practical as
reported protocol12 and was realized as shown in Scheme 1. it involves no chromatographic purification.
Known 2-chloroacetamides 5 were reacted with excess elemen-
The 13 compounds 8a–m thus synthesized were evaluated for
tary sulfur and morpholine (in the presence of triethylamine). agonistic activity toward GPR40 receptor at 5 mM concentration
Without purification, the intermediate N-aryl-2-morpholino-2- using CHO cell line stably expressing GPR40 and calcium flux
thiooxacetamides 6 was reacted with hydrazine hydrate to furnish fluorescent imaging plate reader (FLIPR) platform
good yields of the stable hydrazino products 7. The latter were (Supplementary Material). Four compounds that displayed
condensed with succinic anhydride in glacial acetic acid to 450% activation of the receptor (8d, 8 g, 8j and 8 l, Table 1) at
provide the target 3-(1,3,4-thiadiazol-2-yl)propanoic acids 8 in that concentration were selected for dose-response experiments to
good yields (Table 1). The synthesis is highly flexible with determine their EC50 values. The agonistic activity was confirmed

6
M. Krasavin et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Table 2. EC50 values for compounds 8d, 8 g, 8j and 8 l.
curve fit (Figure 3). The compounds displayed fair plasma protein
binding (with410% free fraction in plasma) and truly remarkable
metabolic stability (determined on compound’s exposure to
mouse liver microsome preparation) and aqueous solubility at
physiological pH (Table 3).
Compound
Structure
EC50 SD, mM*
8d
3.32 0.54
O
OH
O
Cl
S
N
N
H
Conclusion
N
While the potency of the initial hits presented in this paper is
lower than that of many advanced compounds,2 they are full
agonists of GPR40 receptor and can therefore serve as excellent
starting points for further SAR-guided optimization. In particular,
the sensitivity of the agonistic potency to the nature of the
(hetero)anilide periphery observed in this work, provides a clue
that this could be the part of the molecule that should be further
manipulated and perhaps elaborated into a slightly more lipophilic
one, in order to counterbalance the hydrophilicity introduced by
1,3,4-thiadiazole core and increase the affinity to the receptor and
the resulting agonistic activity. The high promise of this new
scaffold is significantly boosted by its remarkable ADMET
profile, in particular, aquenous solubility and metabolic stability.
These features (which we view as a positive corollary to the
replacement of the benzene ring with 1,3,4-thiadiazole under-
taken in this work) are indispensable from drug development
perspective and certainly increase the value of the newly
identified scaffold in the GPR40 agonist field.
8g
8j
5.93 0.23
O
OH
S
N
N
H
O
N
24.2 2.34
S
O
N
OH
N
S
N
H
O
N
N
8l
9.85 0.56
F
O
OH
F
S
N
N
H
O
N
*Each value is an average of n ¼ 4.
Acknowledgements
This research was supported by the Russian Scientific Fund (project grant
1450-00069).
8d
8g
8j
100
80
60
40
20
0
8l
Declaration of interest
The authors declare no conflict of interest. The authors are solely respon-
sible for the content and results presented in this paper.
References
1. Ito Y, Kawamata Y, Harada M, et al. Free fatty acids regulate insulin
secretion from pancreatic beta cells through GPR40. Nature 2003;
422:173–6.
−8
−7
−6
−5
−4
Log[compound] (M)
2. Defossa E, Wagner M. Recent developments in the discovery
of FFA1 receptor agonists as novel oral treatment for type 2
diabetes mellitus. Bioorg Med Chem Lett 2014;24:2991–3000.
3. Watterson KR, Hudson BD, Ulven T, Milligan G. Treatment of type
2 diabetes by free fatty acid receptor agonists. Front Endocrinol
2014;5:137.
Figure 3. Dose-response curves for compounds 8d, 8 g, 8j and 8l used to
determine EC50 values.
Table 3. Further in vitro characterization of lead compound 8g.
4. Available from: https://clinicaltrials.gov.
5. Kaku K, Enya K, Nakaya R, et al. Efficacy and safety of fasiglifam
(TAK-875), a G protein-coupled receptor 40 agonist, in Japanese
patients with type 2 diabetes inadequately controlled by diet and
exercise: a randomized, double-blind, placebo-controlled, phase III
trial. Diabetes Obes Metab 2015;17:675–81.
6. Mancini AD, Poitout V. GPR40 agonists for the treatment of type 2
diabetes: life after ’TAKing’ a hit. Diabetes Obes Metab 2015;17:
622–9.
O
OH
S
N
N
H
O
N
Plasma protein binding (human)*
Aqueous solubility (PBS, pH 7.4)yz
89.9%
4800 mM
1324 min
7. Zahanich I, Kondratov I, Naumchyk V, et al. Phenoxymethyl 1,3-
oxazoles and 1,2,4-oxadiazoles as potent and selective agonists of
free fatty acid receptor 1 (GPR40). Bioorg Med Chem Lett 2015;25:
3105–11.
Microsomal stability (mouse, t1/2
)
*Each value is an average of n ¼ 4, measured at c ¼ 1 mM.
yEach value is an average of n ¼ 2.
8. Maekawa T, Hara R, Odaka H, et al. Preparation of 1,2-azole
derivatives with hypoglycemic and hypolipidemic activity. PCT
Int Appl WO 2003099793A1, 564 Pp; Chem Abstr 2003;140:
951003.
zEach value is an average of n ¼ 5, measured at c ¼ 2 mM.
9. Hamamura K, Sasaki S, Amano Y, et al. Preparation of furan
derivatives for treatment of abnormal lipid metabolism, arterioscler-
osis, and diabetes. PCT Int Appl WO 2004022551A1, 325 Pp; Chem
Abstr 2004;140:220326.
and the compounds were identified as novel single-to-double-digit
micomolar agonists of GPR40 (Table 2).
One of these potent hits (8 g) was selected for further
characterization based on its potency as well as the good EC50
10. Yasuma T, Negoro N, Sasaki S. Preparation of aminophe-
nylpropanoic acid derivatives as antidiabetic agents. PCT Int

DOI: 10.3109/14756366.2016.1142984
Novel agonists of free fatty acid receptor 1 (GPR40)
7
Appl WO 2005087710 A1, 371 Pp; Chem Abstr 2005;143: 12. Yarovenko VN, Shirokov AV, Krupinova ON, et al. Synthesis of
1021733.
11. Sharma R, Akerman M, Cardozo MG, et al. Preparation of coumarin
oxamic acids thiohydrazides and carbamoyl-1,3,4-thiadiazoles. Russ
J Org Chem 2003;39:1133–9.
and related carbocycle and heterocyclic analogs useful for treating 13. Ichimura A, Hirasawa A, Hara T, Tsujimoto G. Free fatty acid
metabolic disorders. PCT Int Appl WO 2007106469 A2, 194 Pp;
Chem Abstr 2007;147:1064386.
receptors act as nutrient sensors to regulate energy homeostasis.
Prostaglandins Other Lipid Mediat 2009;89:82–8.
Supplementary material available at online