Free fatty acid receptor 1 (FFA1 or GPR40) enhances glucose-stimulated insulin secretion from pancreatic β-cells and currently attracts high interest as a new target for the treatment of type 2 diabetes. We here report the discovery of a highly potent FFA1 agonist with favorable physicochemical and pharmacokinetic properties. The compound efficiently normalizes glucose tolerance in diet-induced obese mice, an effect that is fully sustained after 29 days of chronic dosing

Blad C. C.

Briscoe C. P.

Burant C. F.

Christiansen E.

Holliday N. D.

Hopkins A. L.

Itoh Y.

Kotarsky K.

Leeson P. D.

Medina J. C.

Negoro N.

Sasaki S.

Satoh T.

Shimpukade B.

English

PubMed

Elisabeth Christiansen

Steffen V. F. Hansen

Christian Urban

Brian D. Hudson

Edward T. Wargent

Manuel Grundmann

Laura Jenkins

Mohamed Zaibi

Claire J. Stocker

Susanne Ullrich

Evi Kostenis

Matthias U. Kassack

Graeme Milligan

Michael A. Cawthorne

Trond Ulven

Crossref

ACS Medicinal Chemistry Letters

Discovery of TUG-770: A Highly Potent Free Fatty Acid Receptor 1 (FFA1/GPR40) Agonist for Treatment of Type 2 Diabetes

Christiansen, Elisabeth

Hansen, Steffen V F

Urban, Christian

Hudson, Brian D

Wargent, Edward T

Grundmann, Manuel

Jenkins, Laura

Zaibi, Mohamed

Stocker, Claire J

Ullrich, Susanne

Kostenis, Evi

Kassack, Matthias U

Milligan, Graeme

Cawthorne, Michael A

Ulven, Trond

Syddansk Universitets Forskerportal

Discovery of TUG-770:A Highly Potent Free Fatty Acid Receptor 1 (FFA1/GPR40) Agonist for Treatment of Type 2 Diabetes

Christiansen, E.

Hansen, S.V.F.

Urban, C.

Hudson, B.D.

Wargent, E.T.

Grundmann, M.

Jenkins, L.

Zaibi, M.

Stocker, C.J.

Ullrich, S.

Kostenis, E.

Kassack, M.U.

Milligan, G.

Cawthorne, M.A.

Ulven, T.

Enlighten: Publications

  
 
 
 
 
 
Christiansen, E., Hansen, S.V.F., Urban, C., Hudson, B.D., Wargent, 
E.T., Grundmann, M., Jenkins, L., Zaibi, M., Stocker, C.J., Ullrich, S., 
Kostenis, E., Kassack, M.U., Milligan, G., Cawthorne, M.A., and 
Ulven, T. (2013) Discovery of TUG-770: a highly potent free fatty acid 
receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes. ACS 
Medicinal Chemistry Letters, 4 (5). pp. 441-445. ISSN 1948-5875 
 
 
Copyright © 2013 American Chemical Society 
 
 
A copy can be downloaded for personal non-commercial research or 
study, without prior permission or charge 
 
Content must not be changed in any way or reproduced in any format 
or medium without the formal permission of the copyright holder(s) 
 
 
When referring to this work, full bibliographic details must be given 
 
 
 
http://eprints.gla.ac.uk/81349 
 
 
Deposited on: 24 June 2013 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enlighten – Research publications by members of the University of Glasgow 
http://eprints.gla.ac.uk 
Discovery of TUG-770: A Highly Potent Free Fatty Acid Receptor 1
(FFA1/GPR40) Agonist for Treatment of Type 2 Diabetes
Elisabeth Christiansen,† Steffen V. F. Hansen,† Christian Urban,‡ Brian D. Hudson,§ Edward T. Wargent,∥
Manuel Grundmann,⊥ Laura Jenkins,§ Mohamed Zaibi,∥ Claire J. Stocker,∥ Susanne Ullrich,#
Evi Kostenis,⊥ Matthias U. Kassack,‡ Graeme Milligan,§ Michael A. Cawthorne,∥ and Trond Ulven*,†
†Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
‡Institute of Pharmaceutical and Medicinal Chemistry, University of Düsseldorf, Universitaẗsstrasse 1, D-40225 Düsseldorf, Germany
§Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow
G12 8QQ, Scotland, U.K.
∥Clore Laboratory, University of Buckingham, Hunter Street, Buckingham MK18 1EG, U.K.
⊥Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany
#Department of Internal Medicine IV, Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical
Chemistry, University of Tübingen, Otfried-Müller-Str. 10, 72076 Tübingen, Germany
*S Supporting Information
ABSTRACT: Free fatty acid receptor 1 (FFA1 or GPR40) enhances
glucose-stimulated insulin secretion from pancreatic β-cells and currently
attracts high interest as a new target for the treatment of type 2 diabetes.
We here report the discovery of a highly potent FFA1 agonist with
favorable physicochemical and pharmacokinetic properties. The com-
pound efficiently normalizes glucose tolerance in diet-induced obese mice,
an effect that is fully sustained after 29 days of chronic dosing.
KEYWORDS: Type 2 diabetes, free fatty acid receptor, TUG-770, insulin secretagogue, FFA1 agonist, GPR40 agonist
The free fatty acid receptor 1 (FFA1, previously known asGPR40) has, since its deorphanization in 2003, received
considerable attention as a new potential target for treatment
of type 2 diabetes (T2D).1−3 Activation of FFA1 increases
glucose-stimulated insulin secretion but does not affect insulin
secretion at low glucose levels, providing a potentially safe and
efficient strategy for enhancing insulin levels in patients
suffering from T2D. Accordingly, the interest in FFA1 as a
new drug target has been high, and several potent agonists for
the receptor have been disclosed.4−6 Of these, TAK-875 is most
advanced with highly encouraging results from phase II clinical
trials.7 Being a fatty acid receptor, FFA1 has a natural
preference for relatively lipophilic compounds. This property
has been reflected in the majority of the reported synthetic
agonists, which mostly have been at the high end of the
generally recommended lipophilicity range. We have previously
reported a series of alkyne FFA1 agonists8 and have
subsequently directed our efforts toward lowering the lip-
ophilicity of these compounds.9,10 Herein, we report the further
optimization of this compound series, leading to a highly potent
FFA1 agonist with excellent physicochemical and pharmacoki-
netic properties and sustained glucose lowering capability in
diet-induced obese (DIO) mice after acute and chronic dosing.
The alkyne ligands with either pyridine or fluoro-substituted
benzene as the central ring were synthesized from the
corresponding 4-bromoaldehydes (Scheme 1). Initially, a
Wittig reaction with the phosphonium ylide, formed in situ
from ethyl bromoacetate and triphenylphosphine, provided the
corresponding cinnamic esters. The double bond was reduced
by NaBH4 in the presence of catalytic CoCl2.
11 Subsequently,
Sonogashira coupling with phenylacetylene followed by a base
promoted hydrolysis provided the alkyne ligands.12
The 2-fluoro substituted ligands were synthesized from the
central intermediate 2, prepared from aryl bromide 1 by an initial
Sonogashira coupling with trimethylsilylacetylene and subsequent
removal of the TMS-group (Scheme 2). A second Sonogashira
coupling of 2 with various aryl halides followed by ester hydrolysis
gave the alkyne ligands in moderate to high yields.
We set out to investigate modifications in the central ring of
the alkyne ligands (Table 1). Compounds were tested on the
human FFA1 in a calcium mobilization assay and coun-
terscreened on the human FFA4 (previously GPR120)13
because of the selectivity issues frequently observed for these
receptors.14 The central benzene ring was replaced by pyridine
due to its marked lipophilicity lowering effect. The 2-pyridyl
Received: February 17, 2013
Accepted: April 8, 2013
Published: April 8, 2013
Letter
pubs.acs.org/acsmedchemlett
© 2013 American Chemical Society 441 dx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445
  
 
 
 
 
 
Christiansen, E., Hansen, S.V.F., Urban, C., Hudson, B.D., Wargent, 
E.T., Grundmann, M., Jenkins, L., Zaibi, M., Stocker, C.J., Ullrich, S., 
Kostenis, E., Kassack, M.U., Milligan, G., Cawthorne, M.A., and 
Ulven, T. (2013) Discovery of TUG-770: a highly potent free fatty acid 
receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes. ACS 
Medicinal Chemistry Letters, 4 (5). pp. 441-445. ISSN 1948-5875 
 
 
Copyright © 2013 American Chemical Society 
 
 
A copy can be downloaded for personal non-commercial research or 
study, without prior permission or charge 
 
Content must not be changed in any way or reproduced in any format 
or medium without the formal permission of the copyright holder(s) 
 
 
When referring to this work, full bibliographic details must be given 
 
 
 
http://eprints.gla.ac.uk/81349 
 
 
Deposited on: 24 June 2013 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enlighten – Research publications by members of the University of Glasgow 
http://eprints.gla.ac.uk 
(4) and 3-pyridyl (5) analogues turned out to be twice as
potent as previously reported ligands with pyridines as the
terminal ring9 but, nevertheless, resulted in >20-fold decrease in
potency compared to 3. Aromatic fluoro-substituents often
result in higher metabolic stability and have been applied with
success in the corresponding ring of other compound
series.15,16 Thus, we selected three mono- and difluoro-
substituted analogues for synthesis and testing. The 3-fluoro
analogue (6) showed maintained potency and only a small
increase in ClogP compared to 3. The 2-fluoro analogue (7)
resulted in a 5-fold increased potency and the highest ligand
efficiency (LE)17 and ligand lipophilicity efficiency (LLE)18
values and, moreover, the highest selectivity over FFA4 (>200-
fold). Introduction of a second ortho-fluoro substituent (8) led
to a reduction of potency back to the level of 6 and 3.
With 7 showing high potency and LE, we decided to focus
on the 2-fluoro scaffold in the exploration of the terminal ring
in analogy with our previous studies (Table 2). Introduction of
a corresponding 2-fluoro substituent in the lead structure
TUG-424 (9) to give 10 resulted in increased potency but
less so than for the terminally unsubstituted pair 3 and 7
(ΔpEC50 = 0.14 vs 0.78). Moving the methyl of the terminal
ring to the meta-position (11) gave a further increase in
potency. The order of potency is thus reversed relative to the
analogues lacking the 2-fluoro substituent,8 implying that
previous SAR information is not directly transferrable to the
2-fluoro series.
Introduction of a cyano-substituent on 10 to give the
2-methyl-5-cyano analogue (12) resulted in reduced ClogP
together with doubled potency and increased selectivity over
FFA4. The difluoromethyl analogue (13) was found to be more
potent than 10 but only equipotent with 12, despite its higher
lipophilicity. The 3,5-dichloro analogue (14) was synthesized to
mimic the previously published chloro-substituted pyridine
alkyne TUG-4999 but turned out only equipotent with TUG-
499, despite its high lipophilicity.
Extension of the ortho- and meta-methyl with the hydrophilic
mesyl group was explored (15 and 16) and resulted in
significantly reduced ClogP values and improved LLE but
unfortunately also markedly reduced potency. Methoxymethyl
substituents on the terminal ring have previously shown good
potency and significantly reduced lipophilicity in the alkyne
series.10 When adding larger substituents on the terminal
ring of the alkyne ligands, the meta-substituted compounds
(18 and 20) were found to be favored over the ortho analogues
(17 and 19). Although all four analogues exhibited high
selectivity over FFA4, the potency was found to be rather low
(EC50 = 0.3−0.7 μM).
We then directed our attention to the cyanomethyl alkyne
TUG-488 (21).10 The corresponding 2-fluoro analogue 22
(TUG-770) showed a pronounced increase in potency on
FFA1 (ΔpEC50 = 0.51) with EC50 = 6 nM and 150-fold selec-
tivity over FFA4. Moving the cyanomethyl to the meta-position
(23), which had been beneficial for the methyl analogue (11),
led to 12-fold erosion of potency. Finally, homologation to the
Scheme 1a
aReagents and conditions: (a) ethyl bromoacetate, PPh3, NaHCO3,
water, EtOAc, room temp, 18 h, 87−96%; (b) CoCl2·6H2O, NaBH4,
MeOH, 0 °C→ room temp, 3 h, 59−87%; (c) PhCCH, Na2PdCl4, 2-
(di-tert-butylphosphino)-1-phenylindole (PIntB), CuI, TMEDA,
water, 70 → 80 °C, 0.5−4.5 h, 56−86%; (d) LiOH, THF, water,
room temp, 12 h, 79−97%.
Scheme 2a
aReagents and conditions: (a) trimethylsilylacetylene, Na2PdCl4,
PIntB, CuI, TMEDA, water, 70 → 80 °C, 10 min; (b) K2CO3,
MeOH, room temp, 2 h, 74% over two steps; (c) aryl halide,
Na2PdCl4, PIntB, CuI, TMEDA, water, 80 °C, 1−4 h, 52−70%; (d)
LiOH, THF, water, room temp, 12 h, 69−100%.
Table 1. SAR Investigations of the Central Ring
aEfficacy is given as % response relative to 10 μM TUG-20.19 bEfficacy
is given as % response relative to 9; n.a. = no activity (pEC50 < 4).
14
cCalculated by BioByte’s algorithm as implemented in ChemBioDraw
Ultra 12.0 (ClogP option). dLE = RTln KD, presuming that EC50 ≈
KD. Values are given in kcal mol
−1 per non-hydrogen atom.17 LLE =
pEC50 − ClogP.18
ACS Medicinal Chemistry Letters Letter
dx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445442
corresponding cyanoethyl (24) resulted in good potency but
the compound could not compete with 22.
With 22 being the clearly superior agonist in terms of
potency and LLE, as well as displaying significantly higher
potency (EC50 = 6 vs 14 nM), lower lipophilicity (log D7.4 =
1.41 vs 2.24) and higher ligand efficiency (LE = 0.49 vs 0.29)
compared to the most advanced compound in the field TAK-
875,20 we set out to evaluate the compound further using our
previously preferred compound 21 as reference (Table 3).
Compound 22 displayed excellent physicochemical and in vitro
ADME properties, with good aqueous solubility, good chemical
stability, low lipophilicity, and decreased plasma protein
binding (PPB). In support of the lower PPB, 21 showed
significantly decreased activity on hFFA1 in a BRET assay in
the presence of 0.1% BSA (from 7.16 ± 0.09 to 6.62 ± 0.05, p =
0.0024), whereas the corresponding reduction of activity for 22
was insignificant (from 7.64 ± 0.09 to 7.58 ± 0.06, p = 0.5635).
Compound 22 furthermore showed excellent stability toward
human liver microsomes (HLM), no inhibition of selected
CYP-enzymes implicated in drug−drug interactions, no
P-glycoprotein (P-gp) inhibition, and good permeability in
the Caco-2 cell assay. Pharmacokinetic studies in mice showed
a fast oral absorption, higher plasma concentration, a longer
half-life, lower clearance, and increased bioavailability, overall
giving a markedly improved pharmacokinetic profile compared
to 21. No cytotoxicity was observed in vitro in up to 100 μM
concentration (see the Supporting Information), and no adverse
effects were seen in mice after four weeks of daily oral treatment
of 20 mg/kg and acute treatment in doses up to 250 mg/kg.
In addition to the counterscreen on FFA4, 22 showed a high
selectivity over FFA2, FFA3, PPARγ, and 54 diverse receptors,
transporters, and enzymes (see the Supporting Information).
The compound exhibited lower potency on the rodent orthologs
(mFFA1, pEC50 = 6.83 ± 0.07 (n = 3); rFFA1, pEC50 = 6.49 ±
0.05 (n = 2)). The effect of 22 was initially evaluated in vitro in
Table 2. Structure−Activity Investigations of the 2-Fluoro Alkyne Agonists
pEC50 (efficacy, %)
compd R1 X hFFA1, calciuma hFFA4, BRETb ClogPc LEd LLEe
9 2-Me H 7.34 ± 0.07 (103) 5.84 ± 0.01 (103) 5.04 0.50 2.30
10 2-Me F 7.48 ± 0.03 (107) 5.80 ± 0.03 (98) 5.18 0.49 2.30
11 3-Me F 7.65 ± 0.03 (100) 5.41 ± 0.07 (124) 5.18 0.50 2.47
12 2-Me, 5-CN F 7.77 ± 0.03 (104) 5.02 ± 0.04 (123) 4.62 0.46 3.15
13 2-CF2H, 5-F F 7.74 ± 0.04 (97) 5.93 ± 0.04 (117) 5.02 0.44 2.72
14 3,5-Cl F 7.42 ± 0.07 (99) 5.50 ± 0.25 (90) 6.11 0.46 1.31
15 2-CH2Ms F 5.84 ± 0.02 (97) n.a. 2.67 0.33 3.17
16 3-CH2Ms F 5.71 ± 0.02 (104) 4.19 ± 0.06 (26) 2.67 0.33 3.04
17 2-CH2O(CH2)2Ms F 6.21 ± 0.03 (94) n.a. 3.33 0.31 2.88
18 3-CH2O(CH2)2Ms F 6.44 ± 0.03 (93) n.a. 3.33 0.33 3.11
19 2-CH2O(CH2)3Ms F 6.14 ± 0.04 (92) n.a. 3.59 0.30 2.55
20 3-CH2O(CH2)3Ms F 6.43 ± 0.04 (83) n.a. 3.59 0.31 2.84
21 2-CH2CN H 7.70 ± 0.04 (103) 6.11 ± 0.06 (99) 3.96 0.48 3.76
22 2-CH2CN F 8.21 ± 0.03 (102) 6.03 ± 0.06 (98) 4.11 0.49 4.10
23 3-CH2CN F 7.13 ± 0.03 (104) 5.41 ± 0.07 (115) 4.11 0.42 3.02
24 2-CH2CH2CN F 7.74 ± 0.04 (97) 5.86 ± 0.00 (114) 4.25 0.44 3.50
aEfficacy is given as % response relative to 10 μM TUG-20.19 bEfficacy is given as % response relative to 9; n.a. = no activity (pEC50 < 4).
14
cCalculated by BioByte’s algorithm as implemented in ChemBioDraw Ultra 12.0 (ClogP option). dLE = RTln KD, presuming that EC50 ≈ KD. Values
are given in kcal mol−1 per non-hydrogen atom.17 eLLE = pEC50 − ClogP.
18
Table 3. Physicochemical Properties, in Vitro ADME, and
Pharmacokinetics of 21 and 22
physicochemical properties 21 22
aqueous solubility (PBS, pH 7.4)a 196 μM 197 μM
chemical stab. (PBS, 37 °C, 12 days) 99.8% 99.1%
log D (n-octanol/PBS, pH 7.4)b 1.28 (1.32) 1.35 (1.44)
in vitro ADME propertiesc
PPB (human) >99.9% 97.3%
metabolic stability (HLM) 81% 87%
CYP inhibition (10 μM)
CYP1A2 −3% −10%
CYP2C9 11% −33%
CYP2C19 −2% −5%
CYP2D6 5% −1%
CYP3A4 8% −1%
P-gp inhibition (% @ 30/100 μM) −4.0/−1.8 −4.4/−3.6
Caco-2 (A to B, TC7, pH 6.5/7.4) 91 × 10−6 cm/s 72 × 10−6 cm/s
pharmacokinetic propertiesd 21 22
Intravenous
Cmax (ng/mL) 5071 7811
tmax (min) 5 5
t1/2 (min) 17 119
AUC0‑∞ (μg/mL·min) 174 809
Vd (L/kg) 0.35 0.53
CLtotal (mL/min/kg) 14 3.1
Oral
Cmax(ng/mL) 7757 12340
tmax (min) 30 15
t1/2 (min) 50 355
AUC0‑∞ (μg/mL·min) 732 4388
F (%) 105 136
aThe maximum concentration of the assay is 200 μM. bDetermined by
shake-flask method.16 The values given in parentheses were
determined at Cerep Inc. cDetermined at Cerep Inc. dData are
mean concentrations in mouse plasma (n = 3) following a single
2.5 mg/kg intravenous dose or 10 mg/kg oral dose.
ACS Medicinal Chemistry Letters Letter
dx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445443
the rat INS-1E cell line, performed as previously reported,9
where the compound caused significantly increased insulin
secretion (10.75 ± 0.74% of total content with 10 μM 22 vs
8.74 ± 0.54 with vehicle, p < 0.05) at high glucose concen-
tration (12.4 mM) and, as expected, no effect (4.14 ± 0.15% of
total content with 10 μM 22 vs 4.02 ± 0.08 with vehicle) at low
glucose concentration (2.8 mM).
In vivo examination of 22 in an acute intraperitoneal glucose
tolerance test (IPGTT) in normal mice revealed a good dose
dependent response with maximal reduction in glucose level
reached at 50 mg/kg (Figure 1). The study was followed up by
a chronic oral glucose tolerance test (OGTT) study in DIO
mice, which showed that 22 was more effective than 21 (see the
Supporting Information) and that the effect of 22 was fully
sustained after 29 days of daily oral treatment. Additional
evaluation of 22 in rats confirmed a significant glucose lowering
effect for the high doses already after 10 min and for all doses
after 30 min (Figure 2). This was in agreement with an
observed increase in plasma insulin concentration, with
maximum concentration 15 min after glucose challenge. With
an approximately 30-fold higher potency on human than on
rodent receptors, it appears reasonable to expect that the
effective dose would be correspondingly lower in humans.
In conclusion, optimization of the FFA1 alkyne agonists has
resulted in the discovery of 22, a highly potent FFA1 agonist
with excellent physicochemical and pharmacokinetic properties.
The compound demonstrated a potent effect on glucose
tolerance in DIO mice, a situation that was sustained after 29
days of chronic dosing. The compound all together appears as a
promising candidate for development of improved T2D
therapeutics.
■ ASSOCIATED CONTENT
*S Supporting Information
Synthetic procedure, compound characterization, and biological
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*(T.U.) E-mail: ulven@sdu.dk.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank Lone Overgaard Storm for excellent technical
assistance and the Danish Council for Independent Research|
Technology and Production (grant 09-070364), the Danish
Council of Strategic Research (grant 11-116196), and the
Canadian Institutes of Health Research (fellowship to B.D.H.)
for financial support.
■ ABBREVIATIONS
BRET, bioluminescence resonance transfer; FFA1, free fatty
acid receptor 1 (GPR40); IPGTT, intraperetoneal glucose
Figure 1. In vivo evaluation of 22 in mice on glucose tolerance. (A)
Effect of 22 on acute IPGTT in normal mice. Mice were dosed ip with
22, vehicle, or control (sitagliptin, 10 mg/kg). (B) Effect of 22 on
OGTT in a chronic study in DIO mice: acute (4 weeks vehicle prior to
treatment with 22), chronic (4 weeks treatment with 22), and control
(vehicle). Means ± standard errors (n = 6) are shown (*, p < 0.05;
**, p < 0.01; ***, p < 0.001).
Figure 2. In vivo evaluation of 22 in Sprague−Dawley rats on glucose
tolerance after oral dosing. (A) Effect on plasma glucose levels. (B)
Effect on plasma insulin levels. Means ± standard errors (n = 6) are
shown (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
ACS Medicinal Chemistry Letters Letter
dx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445444


Discovery of TUG-770: a highly potent free fatty acid receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes

Enlighten

       Christiansen, E., Hansen, S.V.F., Urban, C., Hudson, B.D., Wargent, E.T., Grundmann, M., Jenkins, L., Zaibi, M., Stocker, C.J., Ullrich, S., Kostenis, E., Kassack, M.U., Milligan, G., Cawthorne, M.A., and Ulven, T. (2013) Discovery of TUG-770: a highly potent free fatty acid receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes. ACS Medicinal Chemistry Letters, 4 (5). pp. 441-445. ISSN 1948-5875   Copyright © 2013 American Chemical Society   A copy can be downloaded for personal non-commercial research or study, without prior permission or charge  Content must not be changed in any way or reproduced in any format or medium without the formal permission of the copyright holder(s)   When referring to this work, full bibliographic details must be given    http://eprints.gla.ac.uk/81349   Deposited on: 24 June 2013                   Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Discovery of TUG-770: A Highly Potent Free Fatty Acid Receptor 1(FFA1/GPR40) Agonist for Treatment of Type 2 DiabetesElisabeth Christiansen,† Steﬀen V. F. Hansen,† Christian Urban,‡ Brian D. Hudson,§ Edward T. Wargent,∥Manuel Grundmann,⊥ Laura Jenkins,§ Mohamed Zaibi,∥ Claire J. Stocker,∥ Susanne Ullrich,#Evi Kostenis,⊥ Matthias U. Kassack,‡ Graeme Milligan,§ Michael A. Cawthorne,∥ and Trond Ulven*,††Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark‡Institute of Pharmaceutical and Medicinal Chemistry, University of Düsseldorf, Universitaẗsstrasse 1, D-40225 Düsseldorf, Germany§Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, GlasgowG12 8QQ, Scotland, U.K.∥Clore Laboratory, University of Buckingham, Hunter Street, Buckingham MK18 1EG, U.K.⊥Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany#Department of Internal Medicine IV, Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and ClinicalChemistry, University of Tübingen, Otfried-Müller-Str. 10, 72076 Tübingen, Germany*S Supporting InformationABSTRACT: Free fatty acid receptor 1 (FFA1 or GPR40) enhancesglucose-stimulated insulin secretion from pancreatic β-cells and currentlyattracts high interest as a new target for the treatment of type 2 diabetes.We here report the discovery of a highly potent FFA1 agonist withfavorable physicochemical and pharmacokinetic properties. The com-pound eﬃciently normalizes glucose tolerance in diet-induced obese mice,an eﬀect that is fully sustained after 29 days of chronic dosing.KEYWORDS: Type 2 diabetes, free fatty acid receptor, TUG-770, insulin secretagogue, FFA1 agonist, GPR40 agonistThe free fatty acid receptor 1 (FFA1, previously known asGPR40) has, since its deorphanization in 2003, receivedconsiderable attention as a new potential target for treatmentof type 2 diabetes (T2D).1−3 Activation of FFA1 increasesglucose-stimulated insulin secretion but does not aﬀect insulinsecretion at low glucose levels, providing a potentially safe andeﬃcient strategy for enhancing insulin levels in patientssuﬀering from T2D. Accordingly, the interest in FFA1 as anew drug target has been high, and several potent agonists forthe receptor have been disclosed.4−6 Of these, TAK-875 is mostadvanced with highly encouraging results from phase II clinicaltrials.7 Being a fatty acid receptor, FFA1 has a naturalpreference for relatively lipophilic compounds. This propertyhas been reﬂected in the majority of the reported syntheticagonists, which mostly have been at the high end of thegenerally recommended lipophilicity range. We have previouslyreported a series of alkyne FFA1 agonists8 and havesubsequently directed our eﬀorts toward lowering the lip-ophilicity of these compounds.9,10 Herein, we report the furtheroptimization of this compound series, leading to a highly potentFFA1 agonist with excellent physicochemical and pharmacoki-netic properties and sustained glucose lowering capability indiet-induced obese (DIO) mice after acute and chronic dosing.The alkyne ligands with either pyridine or ﬂuoro-substitutedbenzene as the central ring were synthesized from thecorresponding 4-bromoaldehydes (Scheme 1). Initially, aWittig reaction with the phosphonium ylide, formed in situfrom ethyl bromoacetate and triphenylphosphine, provided thecorresponding cinnamic esters. The double bond was reducedby NaBH4 in the presence of catalytic CoCl2.11 Subsequently,Sonogashira coupling with phenylacetylene followed by a basepromoted hydrolysis provided the alkyne ligands.12The 2-ﬂuoro substituted ligands were synthesized from thecentral intermediate 2, prepared from aryl bromide 1 by an initialSonogashira coupling with trimethylsilylacetylene and subsequentremoval of the TMS-group (Scheme 2). A second Sonogashiracoupling of 2 with various aryl halides followed by ester hydrolysisgave the alkyne ligands in moderate to high yields.We set out to investigate modiﬁcations in the central ring ofthe alkyne ligands (Table 1). Compounds were tested on thehuman FFA1 in a calcium mobilization assay and coun-terscreened on the human FFA4 (previously GPR120)13because of the selectivity issues frequently observed for thesereceptors.14 The central benzene ring was replaced by pyridinedue to its marked lipophilicity lowering eﬀect. The 2-pyridylReceived: February 17, 2013Accepted: April 8, 2013Published: April 8, 2013Letterpubs.acs.org/acsmedchemlett© 2013 American Chemical Society 441 dx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445       Christiansen, E., Hansen, S.V.F., Urban, C., Hudson, B.D., Wargent, E.T., Grundmann, M., Jenkins, L., Zaibi, M., Stocker, C.J., Ullrich, S., Kostenis, E., Kassack, M.U., Milligan, G., Cawthorne, M.A., and Ulven, T. (2013) Discovery of TUG-770: a highly potent free fatty acid receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes. ACS Medicinal Chemistry Letters, 4 (5). pp. 441-445. ISSN 1948-5875   Copyright © 2013 American Chemical Society   A copy can be downloaded for personal non-commercial research or study, without prior permission or charge  Content must not be changed in any way or reproduced in any format or medium without the formal permission of the copyright holder(s)   When referring to this work, full bibliographic details must be given    http://eprints.gla.ac.uk/81349   Deposited on: 24 June 2013                   Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk (4) and 3-pyridyl (5) analogues turned out to be twice aspotent as previously reported ligands with pyridines as theterminal ring9 but, nevertheless, resulted in >20-fold decrease inpotency compared to 3. Aromatic ﬂuoro-substituents oftenresult in higher metabolic stability and have been applied withsuccess in the corresponding ring of other compoundseries.15,16 Thus, we selected three mono- and diﬂuoro-substituted analogues for synthesis and testing. The 3-ﬂuoroanalogue (6) showed maintained potency and only a smallincrease in ClogP compared to 3. The 2-ﬂuoro analogue (7)resulted in a 5-fold increased potency and the highest ligandeﬃciency (LE)17 and ligand lipophilicity eﬃciency (LLE)18values and, moreover, the highest selectivity over FFA4 (>200-fold). Introduction of a second ortho-ﬂuoro substituent (8) ledto a reduction of potency back to the level of 6 and 3.With 7 showing high potency and LE, we decided to focuson the 2-ﬂuoro scaﬀold in the exploration of the terminal ringin analogy with our previous studies (Table 2). Introduction ofa corresponding 2-ﬂuoro substituent in the lead structureTUG-424 (9) to give 10 resulted in increased potency butless so than for the terminally unsubstituted pair 3 and 7(ΔpEC50 = 0.14 vs 0.78). Moving the methyl of the terminalring to the meta-position (11) gave a further increase inpotency. The order of potency is thus reversed relative to theanalogues lacking the 2-ﬂuoro substituent,8 implying thatprevious SAR information is not directly transferrable to the2-ﬂuoro series.Introduction of a cyano-substituent on 10 to give the2-methyl-5-cyano analogue (12) resulted in reduced ClogPtogether with doubled potency and increased selectivity overFFA4. The diﬂuoromethyl analogue (13) was found to be morepotent than 10 but only equipotent with 12, despite its higherlipophilicity. The 3,5-dichloro analogue (14) was synthesized tomimic the previously published chloro-substituted pyridinealkyne TUG-4999 but turned out only equipotent with TUG-499, despite its high lipophilicity.Extension of the ortho- and meta-methyl with the hydrophilicmesyl group was explored (15 and 16) and resulted insigniﬁcantly reduced ClogP values and improved LLE butunfortunately also markedly reduced potency. Methoxymethylsubstituents on the terminal ring have previously shown goodpotency and signiﬁcantly reduced lipophilicity in the alkyneseries.10 When adding larger substituents on the terminalring of the alkyne ligands, the meta-substituted compounds(18 and 20) were found to be favored over the ortho analogues(17 and 19). Although all four analogues exhibited highselectivity over FFA4, the potency was found to be rather low(EC50 = 0.3−0.7 μM).We then directed our attention to the cyanomethyl alkyneTUG-488 (21).10 The corresponding 2-ﬂuoro analogue 22(TUG-770) showed a pronounced increase in potency onFFA1 (ΔpEC50 = 0.51) with EC50 = 6 nM and 150-fold selec-tivity over FFA4. Moving the cyanomethyl to the meta-position(23), which had been beneﬁcial for the methyl analogue (11),led to 12-fold erosion of potency. Finally, homologation to theScheme 1aaReagents and conditions: (a) ethyl bromoacetate, PPh3, NaHCO3,water, EtOAc, room temp, 18 h, 87−96%; (b) CoCl2·6H2O, NaBH4,MeOH, 0 °C→ room temp, 3 h, 59−87%; (c) PhCCH, Na2PdCl4, 2-(di-tert-butylphosphino)-1-phenylindole (PIntB), CuI, TMEDA,water, 70 → 80 °C, 0.5−4.5 h, 56−86%; (d) LiOH, THF, water,room temp, 12 h, 79−97%.Scheme 2aaReagents and conditions: (a) trimethylsilylacetylene, Na2PdCl4,PIntB, CuI, TMEDA, water, 70 → 80 °C, 10 min; (b) K2CO3,MeOH, room temp, 2 h, 74% over two steps; (c) aryl halide,Na2PdCl4, PIntB, CuI, TMEDA, water, 80 °C, 1−4 h, 52−70%; (d)LiOH, THF, water, room temp, 12 h, 69−100%.Table 1. SAR Investigations of the Central RingaEﬃcacy is given as % response relative to 10 μM TUG-20.19 bEﬃcacyis given as % response relative to 9; n.a. = no activity (pEC50 < 4).14cCalculated by BioByte’s algorithm as implemented in ChemBioDrawUltra 12.0 (ClogP option). dLE = RTln KD, presuming that EC50 ≈KD. Values are given in kcal mol−1 per non-hydrogen atom.17 LLE =pEC50 − ClogP.18ACS Medicinal Chemistry Letters Letterdx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445442corresponding cyanoethyl (24) resulted in good potency butthe compound could not compete with 22.With 22 being the clearly superior agonist in terms ofpotency and LLE, as well as displaying signiﬁcantly higherpotency (EC50 = 6 vs 14 nM), lower lipophilicity (log D7.4 =1.41 vs 2.24) and higher ligand eﬃciency (LE = 0.49 vs 0.29)compared to the most advanced compound in the ﬁeld TAK-875,20 we set out to evaluate the compound further using ourpreviously preferred compound 21 as reference (Table 3).Compound 22 displayed excellent physicochemical and in vitroADME properties, with good aqueous solubility, good chemicalstability, low lipophilicity, and decreased plasma proteinbinding (PPB). In support of the lower PPB, 21 showedsigniﬁcantly decreased activity on hFFA1 in a BRET assay inthe presence of 0.1% BSA (from 7.16 ± 0.09 to 6.62 ± 0.05, p =0.0024), whereas the corresponding reduction of activity for 22was insigniﬁcant (from 7.64 ± 0.09 to 7.58 ± 0.06, p = 0.5635).Compound 22 furthermore showed excellent stability towardhuman liver microsomes (HLM), no inhibition of selectedCYP-enzymes implicated in drug−drug interactions, noP-glycoprotein (P-gp) inhibition, and good permeability inthe Caco-2 cell assay. Pharmacokinetic studies in mice showeda fast oral absorption, higher plasma concentration, a longerhalf-life, lower clearance, and increased bioavailability, overallgiving a markedly improved pharmacokinetic proﬁle comparedto 21. No cytotoxicity was observed in vitro in up to 100 μMconcentration (see the Supporting Information), and no adverseeﬀects were seen in mice after four weeks of daily oral treatmentof 20 mg/kg and acute treatment in doses up to 250 mg/kg.In addition to the counterscreen on FFA4, 22 showed a highselectivity over FFA2, FFA3, PPARγ, and 54 diverse receptors,transporters, and enzymes (see the Supporting Information).The compound exhibited lower potency on the rodent orthologs(mFFA1, pEC50 = 6.83 ± 0.07 (n = 3); rFFA1, pEC50 = 6.49 ±0.05 (n = 2)). The eﬀect of 22 was initially evaluated in vitro inTable 2. Structure−Activity Investigations of the 2-Fluoro Alkyne AgonistspEC50 (eﬃcacy, %)compd R1 X hFFA1, calciuma hFFA4, BRETb ClogPc LEd LLEe9 2-Me H 7.34 ± 0.07 (103) 5.84 ± 0.01 (103) 5.04 0.50 2.3010 2-Me F 7.48 ± 0.03 (107) 5.80 ± 0.03 (98) 5.18 0.49 2.3011 3-Me F 7.65 ± 0.03 (100) 5.41 ± 0.07 (124) 5.18 0.50 2.4712 2-Me, 5-CN F 7.77 ± 0.03 (104) 5.02 ± 0.04 (123) 4.62 0.46 3.1513 2-CF2H, 5-F F 7.74 ± 0.04 (97) 5.93 ± 0.04 (117) 5.02 0.44 2.7214 3,5-Cl F 7.42 ± 0.07 (99) 5.50 ± 0.25 (90) 6.11 0.46 1.3115 2-CH2Ms F 5.84 ± 0.02 (97) n.a. 2.67 0.33 3.1716 3-CH2Ms F 5.71 ± 0.02 (104) 4.19 ± 0.06 (26) 2.67 0.33 3.0417 2-CH2O(CH2)2Ms F 6.21 ± 0.03 (94) n.a. 3.33 0.31 2.8818 3-CH2O(CH2)2Ms F 6.44 ± 0.03 (93) n.a. 3.33 0.33 3.1119 2-CH2O(CH2)3Ms F 6.14 ± 0.04 (92) n.a. 3.59 0.30 2.5520 3-CH2O(CH2)3Ms F 6.43 ± 0.04 (83) n.a. 3.59 0.31 2.8421 2-CH2CN H 7.70 ± 0.04 (103) 6.11 ± 0.06 (99) 3.96 0.48 3.7622 2-CH2CN F 8.21 ± 0.03 (102) 6.03 ± 0.06 (98) 4.11 0.49 4.1023 3-CH2CN F 7.13 ± 0.03 (104) 5.41 ± 0.07 (115) 4.11 0.42 3.0224 2-CH2CH2CN F 7.74 ± 0.04 (97) 5.86 ± 0.00 (114) 4.25 0.44 3.50aEﬃcacy is given as % response relative to 10 μM TUG-20.19 bEﬃcacy is given as % response relative to 9; n.a. = no activity (pEC50 < 4).14cCalculated by BioByte’s algorithm as implemented in ChemBioDraw Ultra 12.0 (ClogP option). dLE = RTln KD, presuming that EC50 ≈ KD. Valuesare given in kcal mol−1 per non-hydrogen atom.17 eLLE = pEC50 − ClogP.18Table 3. Physicochemical Properties, in Vitro ADME, andPharmacokinetics of 21 and 22physicochemical properties 21 22aqueous solubility (PBS, pH 7.4)a 196 μM 197 μMchemical stab. (PBS, 37 °C, 12 days) 99.8% 99.1%log D (n-octanol/PBS, pH 7.4)b 1.28 (1.32) 1.35 (1.44)in vitro ADME propertiescPPB (human) >99.9% 97.3%metabolic stability (HLM) 81% 87%CYP inhibition (10 μM)CYP1A2 −3% −10%CYP2C9 11% −33%CYP2C19 −2% −5%CYP2D6 5% −1%CYP3A4 8% −1%P-gp inhibition (% @ 30/100 μM) −4.0/−1.8 −4.4/−3.6Caco-2 (A to B, TC7, pH 6.5/7.4) 91 × 10−6 cm/s 72 × 10−6 cm/spharmacokinetic propertiesd 21 22IntravenousCmax (ng/mL) 5071 7811tmax (min) 5 5t1/2 (min) 17 119AUC0‑∞ (μg/mL·min) 174 809Vd (L/kg) 0.35 0.53CLtotal (mL/min/kg) 14 3.1OralCmax(ng/mL) 7757 12340tmax (min) 30 15t1/2 (min) 50 355AUC0‑∞ (μg/mL·min) 732 4388F (%) 105 136aThe maximum concentration of the assay is 200 μM. bDetermined byshake-ﬂask method.16 The values given in parentheses weredetermined at Cerep Inc. cDetermined at Cerep Inc. dData aremean concentrations in mouse plasma (n = 3) following a single2.5 mg/kg intravenous dose or 10 mg/kg oral dose.ACS Medicinal Chemistry Letters Letterdx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445443the rat INS-1E cell line, performed as previously reported,9where the compound caused signiﬁcantly increased insulinsecretion (10.75 ± 0.74% of total content with 10 μM 22 vs8.74 ± 0.54 with vehicle, p < 0.05) at high glucose concen-tration (12.4 mM) and, as expected, no eﬀect (4.14 ± 0.15% oftotal content with 10 μM 22 vs 4.02 ± 0.08 with vehicle) at lowglucose concentration (2.8 mM).In vivo examination of 22 in an acute intraperitoneal glucosetolerance test (IPGTT) in normal mice revealed a good dosedependent response with maximal reduction in glucose levelreached at 50 mg/kg (Figure 1). The study was followed up bya chronic oral glucose tolerance test (OGTT) study in DIOmice, which showed that 22 was more eﬀective than 21 (see theSupporting Information) and that the eﬀect of 22 was fullysustained after 29 days of daily oral treatment. Additionalevaluation of 22 in rats conﬁrmed a signiﬁcant glucose loweringeﬀect for the high doses already after 10 min and for all dosesafter 30 min (Figure 2). This was in agreement with anobserved increase in plasma insulin concentration, withmaximum concentration 15 min after glucose challenge. Withan approximately 30-fold higher potency on human than onrodent receptors, it appears reasonable to expect that theeﬀective dose would be correspondingly lower in humans.In conclusion, optimization of the FFA1 alkyne agonists hasresulted in the discovery of 22, a highly potent FFA1 agonistwith excellent physicochemical and pharmacokinetic properties.The compound demonstrated a potent eﬀect on glucosetolerance in DIO mice, a situation that was sustained after 29days of chronic dosing. The compound all together appears as apromising candidate for development of improved T2Dtherapeutics.■ ASSOCIATED CONTENT*S Supporting InformationSynthetic procedure, compound characterization, and biologicalassays. This material is available free of charge via the Internetat http://pubs.acs.org.■ AUTHOR INFORMATIONCorresponding Author*(T.U.) E-mail: ulven@sdu.dk.NotesThe authors declare no competing ﬁnancial interest.■ ACKNOWLEDGMENTSWe thank Lone Overgaard Storm for excellent technicalassistance and the Danish Council for Independent Research|Technology and Production (grant 09-070364), the DanishCouncil of Strategic Research (grant 11-116196), and theCanadian Institutes of Health Research (fellowship to B.D.H.)for ﬁnancial support.■ ABBREVIATIONSBRET, bioluminescence resonance transfer; FFA1, free fattyacid receptor 1 (GPR40); IPGTT, intraperetoneal glucoseFigure 1. In vivo evaluation of 22 in mice on glucose tolerance. (A)Eﬀect of 22 on acute IPGTT in normal mice. Mice were dosed ip with22, vehicle, or control (sitagliptin, 10 mg/kg). (B) Eﬀect of 22 onOGTT in a chronic study in DIO mice: acute (4 weeks vehicle prior totreatment with 22), chronic (4 weeks treatment with 22), and control(vehicle). Means ± standard errors (n = 6) are shown (*, p < 0.05;**, p < 0.01; ***, p < 0.001).Figure 2. In vivo evaluation of 22 in Sprague−Dawley rats on glucosetolerance after oral dosing. (A) Eﬀect on plasma glucose levels. (B)Eﬀect on plasma insulin levels. Means ± standard errors (n = 6) areshown (*, p < 0.05; **, p < 0.01; ***, p < 0.001).ACS Medicinal Chemistry Letters Letterdx.doi.org/10.1021/ml4000673 | ACS Med. Chem. Lett. 2013, 4, 441−445444

Discovery of TUG-770: a highly potent free fatty acid receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes

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