Discovery of a Potent Thiazolidine Free Fatty Acid Receptor 2 Agonist with Favorable Pharmacokinetic Properties

Article abstract of DOI:10.1021/acs.jmedchem.8b00855

Free fatty acid receptor 2 (FFA2/GPR43) is a receptor for short-chain fatty acids reported to be involved in regulation of metabolism, appetite, fat accumulation, and inflammatory responses and is a potential target for treatment of various inflammatory and metabolic diseases. By bioisosteric replacement of the central pyrrolidine core of a previously disclosed FFA2 agonist with a synthetically more tractable thiazolidine, we were able to rapidly synthesize and screen analogues modified at both the 2- and 3-positions on the thiazolidine core. Herein, we report SAR exploration of thiazolidine FFA2 agonists and the identification of 31 (TUG-1375), a compound with significantly increased potency (7-fold in a cAMP assay) and reduced lipophilicity (50-fold reduced clogP) relative to the pyrrolidine lead structure. The compound has high solubility, high chemical, microsomal, and hepatocyte stability, and favorable pharmacokinetic properties and was confirmed to induce human neutrophil mobilization and to inhibit lipolysis in murine adipocytes.

Full text of DOI:10.1021/acs.jmedchem.8b00855

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Article
Discovery of a Potent Thiazolidine Free Fatty Acid Receptor
Agonist with Favorable Pharmacokinetic Properties
2
Anders Højgaard Hansen, Eugenia Sergeev, Daniele Bolognini, Richard R. Sprenger, Jeppe Hvidtfeldt
Ekberg, Christer S. Ejsing, Christine J. McKenzie, Elisabeth Rexen Ulven, Graeme Milligan, and Trond Ulven
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00855 • Publication Date (Web): 24 Sep 2018
Downloaded from http://pubs.acs.org on September 24, 2018
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Discovery of a Potent Thiazolidine Free Fatty Acid
Receptor 2 Agonist with Favorable Pharmacokinetic
Properties
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†
‡
‡
¥
Anders Højgaard Hansen , Eugenia Sergeev , Daniele Bolognini , Richard R. Sprenger , Jeppe
¶
¥
†
†
Hvidtfeldt Ekberg , Christer S. Ejsing , Christine J. McKenzie , Elisabeth Rexen Ulven , Graeme
‡
†,§,
Milligan and Trond Ulven *
†
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej
5
5, DKꢀ5230 Odense M, Denmark
‡
Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology,
College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ,
Scotland, United Kingdom
¥
Department of Biochemistry and Molecular Biology, University of Southern Denmark,
Campusvej 55, DKꢀ5230 Odense M, Denmark
¶
Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical
Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark
§
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken
2
, DKꢀ2100 Copenhagen, Denmark
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ABSTRACT
Free fatty acid receptor 2 (FFA2/GPR43) is a receptor for shortꢀchain fatty acids reported to be
involved in regulation of metabolism, appetite, fat accumulation and inflammatory responses,
and is a potential target for treatment of various inflammatory and metabolic diseases. By
bioisosteric replacement of the central pyrrolidine core of a previously disclosed FFA2 agonist
with a synthetically more tractable thiazolidine, we were able to rapidly synthesize and screen
analogues modified at both the 2ꢀ and 3ꢀpositions on the thiazolidine core. Herein, we report
SAR exploration of thiazolidine FFA2 agonists and the identification of 31 (TUGꢀ1375), a
compound with significantly increased potency (7ꢀfold in a cAMP assay) and reduced
lipophilicity (50ꢀfold reduced clogP) relative to the pyrrolidine lead structure. The compound has
high solubility, high chemical, microsomal and hepatocyte stability, favorable pharmacokinetic
properties, and was confirmed to induce human neutrophil mobilization and to inhibit lipolysis in
murine adipocytes.
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INTRODUCTION
The shortꢀchain free fatty acid receptor FFA2 (formerly GPR43) is a G proteinꢀcoupled
receptor that is activated by physiological concentrations of shortꢀchain fatty acids (SCFAs),
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primarily acetate and propionate. FFA2 is expressed in a variety of tissues including immune
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2
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cells, adipocytes, pancreatic βꢀcells and enteroendocrine Lꢀcells. Colonic fermentation of
dietary fiber produces large amounts of SCFAs and results indicate that an interplay between
FFA2 and dietary fiber via colonic fermentation to produce SCFAs is implicated in promoting a
8
healthy composition of microorganisms in the gut. Fiberꢀrich diets and SCFAs can counteract
9,10
colitis, and exacerbated inflammation in colitis models has been reported for FFA2ꢀknockout
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mice. However, others have found that FFA2ꢀknockout mice exhibited reduced inflammatory
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responses in colitis models, and it is currently unclear if antagonists or agonists are preferable
for the treatment of intestinal inflammation. So far, the antagonist GLPG0974 (1, Chart 1) is the
only FFA2 modulator that has undergone clinical trials, but unfortunately failed to meet
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endpoints against ulcerative colitis.
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Dietary fiber is also known to exert positive effects in diabetic subjects. Acetate has been
shown to potentiate glucoseꢀstimulated insulin secretion from βꢀcells in a FFA2ꢀdependent
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manner, and SCFAs were able to promote secretion of the incretin glucagon like peptideꢀ1
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(
GLPꢀ1) from colonic cultures through FFA2, and both GLPꢀ1 and the appetite regulating
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peptide YY (PYY) in vivo, indicating that activation of FFA2 could have beneficial effects on
type 2 diabetes (T2D). Interestingly and in contrast, loss of FFA2 and FFA3 was found to
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improve insulin secretion and glucose tolerance. Moreover, SCFAꢀmediated activation of
FFA2 has been found to suppress adipose insulin signaling, leading to reduced fat accumulation
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in adipose tissue, and to promote GLPꢀ1 secretion in the gut. Thus, both agonists and
antagonists of FFA2 are considered to be potential therapeutics for treatment of T2D and
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obesity.
Several FFA2 agonists have been disclosed, including an allosteric agonist series
from Amgen represented by 2 (known as AMG7703 or 4ꢀCMTB) and an orthosteric agonist
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0,26ꢀ30
series originating from Euroscreen represented by 3.
A recent study found that agonist 4,
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also originating from Euroscreen, caused only marginal GLPꢀ1 secretion but led to significant
PYY mucosal responses, inhibited insulinꢀpromoted fat accumulation and intestinal functions,
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and suppressed food intake, supporting FFA2 as a possible antiꢀobesity target.
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Chart 1. FFA2 modulators
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clogP values are calculated using ChemBioDraw v16.0.1.4. pIC for 1 and pEC for 2 and 3
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8,30,32
are previously reported.
The FFA2 agonists disclosed hitherto have relatively high lipophilicity, which is associated
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with several undesired properties,
and only moderate potency (see e.g. Chart 1).
Furthermore, the allosteric agonist 2 has solubility issues and has been reported to give results
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that differ from orthosteric FFA2 agonists, and the orthosteric agonist 3 was found to have
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stability issues. We therefore wished to explore 4, an FFA2 agonist reported to have properties
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useful for in vivo studies. Initially, we sought to explore the SAR around the pyrrolidine
scaffold of 4, however, the synthetic route to obtain this class of FFA2 agonists is rather lengthy
and precludes efficient exploration of parts of the molecule. We therefore opted for replacing the
central pyrrolidine by a thiazolidine. This replacement strategy has previously been employed for
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HIV protease inhibitors. Herein, we report SAR investigations of substituted thiazolidine FFA2
agonists and the discovery of an FFA2 agonist with improved potency, reduced lipophilicity and
favorable physicochemical and pharmacokinetic properties.
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RESULTS AND DISCUSSION
Synthesis
Pyrrolidine ligands were synthesized in seven linear steps from
Lꢀpyroglutamic acid (5)
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essentially as previously described by Euroscreen, with the modifications that the ring opening
by aryl lithium was allowed to reach room temperature and that the amide coupling with
pyrrolidine 6 was performed with bis(tetramethylene)fluoroformamidinium hexafluorophosphate
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(
BTFFH).
a
Scheme 1. Synthesis of pyrrolidines 4 and 37 from ꢀpyroglutamic acid
L
a
Reagents and conditions: (a) SOCl , MeOH, rt, 70%; (b) Boc O, DMAP, DCM, rt, 4 h, 98%;
2
2
(
(
(
c) (i) 1ꢀiodoꢀ2ꢀchlorobenzene, nꢀBuLi, THF, 20 min at ꢀ78 °C → rt; (ii) sat. aq. NH Cl, 67%;
d) TFA, DCM, rt, 98%; (e) NaBH CN, AcOH (cat.), MeOH, rt, 71% (cis/trans 2:1, 49%/22%);
f) carboxylic acid, BTFFH, DIPEA, DCM, 80 °C, 8 h, 75%; (g) LiOH (aq), THF, rt, 42ꢀ93%.
4
3
Thiazolidine ligands were synthesized in three steps by condensation of (R)ꢀCysꢀOMe
hydrochloride with benzaldehydes via 7aꢀj (Scheme 2). In contrast to the pyrrolidine route, this
strategy allowed rapid access also to variations on the 2ꢀposition of the thiazolidine scaffold in a
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single step from readily available starting materials and formation of the desired (2R,4R)ꢀ
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thiazolidine amide as the major product in the next step.
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Condensation of (R)ꢀCysꢀOMe hydrochloride with aldehydes produced mixtures of C(2)
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epimeric methyl (4R)ꢀ2ꢀarylꢀthiazolidineꢀ4ꢀcarboxylates (7aꢀj). To obtain the (2R,4R)ꢀ
configuration, 7aꢀj were treated with Et N and subsequently reacted with the relevant acid
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chlorides to give the desired (2R,4R)ꢀproducts. Finally, demethylation using LiI in EtOAc
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afforded the corresponding (2R,4R)ꢀNꢀacylthiazolidineꢀ4ꢀcarboxylic acids.
a
Scheme 2. Synthesis of (2R,4R)ꢀNꢀacylthiazolidineꢀ4ꢀcarboxylic acids from (R)ꢀCysꢀOMe
a
Reagents and conditions: (a) H O/EtOH 1:1, KHCO , rt, 4ꢀ24 h, 63−81%; (b) benzoyl
2
3
chlorides, Et N, ꢀ10 °C → rt, THF, 1ꢀ14 h, 21−83%; (c) LiI, EtOAc, 80 °C, 24−92%.
3
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Scheme 3. Synthesis of biaryl analogues via coupling reactions
a
Reagents and conditions: (a) ArB(OH) or ArBPin, Pd(PPh ) , MeOH, toluene, 6ꢀ27 h,
2
3 4
2
0−86%; or ArCCH, Pd(PPh ) Cl , CuI, Et N, 4 h, rt, 76%; (b) LiI, EtOAc, 80 °C, 37−78%.
3 2 2 3
A second set of analogues aimed at exploring the Nꢀbenzoyl part of the ligands was
synthesized from central intermediates 8aꢀb by SuzukiꢀMiyaura or Sonogashira crossꢀcoupling
followed by demethylation afforded the desired biarylic compounds in low to good yields
(Scheme 3).
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The crystal structure of 14 confirms unambiguously the (2R,4R)ꢀconfiguration of thiazolidine
substituents (Figure 1). A relatively short intramolecular hydrogen bond at 2.598(2) Å is found
between the carboxylic acid and pyridine group. The crystal packing (Supporting Information,
Figure S1) shows no significant intermolecular interactions stronger than van de Waals forces.
The intramolecular hydrogen bond is a likely reason for why crystals suitable for structural
information were obtained for this compound but for none of the others. At physiological pH
with the carboxylic acid ionized, the pyridyl hydrogen bond will be absent and the overall
conformation is expected to be different. It is therefore not possible to draw conclusions
regarding the configuration around the amide in the active conformation of 14. In fact, rather
than the (Z)ꢀconfiguration apparent in the crystal structure of 14, computer minimizations
indicate a general preference for the (E)ꢀconfiguration around the amide in the compound series.
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Figure 1. Perspective view of 14 drawn with 50% probability ellipsoids
Biological Testing and SAR Analysis
Compounds were tested on the human (h)FFA2 receptor using both a bioluminescence
resonance energy transfer (BRET)ꢀbased βꢀarrestinꢀ2 interaction assay and a cAMP inhibition
assay. The former assay is an upstream signaling assay measuring BRET arising from agonistꢀ
mediated recruitment of βꢀarrestinꢀ2 to FFA2, and the latter assay evaluates inhibition of
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forskolinꢀinduced production of cAMP facilitated by coupling of FFA2 to Gα proteins.
i/o
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The FFA2 agonist activity of 4 was confirmed in both assays. Compound 4 has previously
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been reported with an EC of 81 nM. We obtained somewhat lower potencies of 1.2 ꢀM in the
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βꢀarrestinꢀ2 assay and 0.53 ꢀM in the cAMP assay. We were pleased to find that the thiazolidine
analog 9 had only moderately decreased potency in both the BRET and the cAMP assay (ꢁpEC50
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=
ꢀ0.67 and ꢀ0.31, respectively) relative to pyrrolidine 4 (Table 1). The easy access to substituted
thiazolidines enabled a rapid screen of variations in the 2ꢀposition of the thiazolidine scaffold for
2
ꢀmethoxyꢀ1,1'ꢀbiphenyl derivatives (Table 1).
Compared to 2ꢀchlorophenyl (9), 3ꢀchlorophenyl (10) or 4ꢀchlorophenyl (11) led to a decrease
in potencies in the βꢀarrestinꢀ2 assay, whereas 11 retained potency in the cAMP assay. Thus, the
ꢀposition appeared to be disfavored and 4ꢀposition substitution may be a route to G protein
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biased ligands. Substituting the 2ꢀchloro by bromo (12) or hydrogen (13) retained potency in
both assays.
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Table 1. SAR investigations at the thiazolidine 2ꢀposition
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cmpd
X
R
hFFA2
βꢀarrestin pEC50
c
d
cAMP pEC50
clogP
LLE
a
b
(
Emax
)
(Emax)
4
9
CH
2
5.91 ± 0.21 (127)
5.60 ± 0.10 (116)
6.28 ± 0.19 (96)
5.61 ± 0.06 (96)
5.07
0.84/1.21
0.53/0.54
S
5.07
1
0
1
S
S
4.91 ± 0.29 (93)
4.73 ± 0.29 (136)
5.15 ± 0.19 (106) 5.07
ꢀ0.16/0.08
ꢀ0.34/0.48
1
5.55 ± 0.19 (80)
5.45 ± 0.11 (90)
6.03 ± 0.15 (88)
5.07
5.22
4.36
12
S
S
5.65 ± 0.18 (108)
5.53 ± 0.16 (116)
0.43/0.23
13
14
1.17/1.67
1.97/3.01
S
S
4.83 ± 0.09 (124)
4.74 ± 0.38 (68)
5.87 ± 0.10 (75)
5.66 ± 0.19 (70)
2.86
3.53
1
5
6
1.21/2.13
ꢀ
e
f
1
S
~4
nr
4.50
4.12
f
f
17
O
nr
nr
ꢀ
a
b
BRETꢀbased βꢀarrestinꢀ2 recruitment assay. Efficacy (E ) is relative to propionate. cAMP
max
c
FFA2 assay. Efficacy (Emax) is relative to propionate. clogP values were calculated using
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
d
e
ChemBioDraw v16.0.1.4. (BRET/cAMP) LLE values (LLE = pEC −clogP). 50% activation at
1
5
0
f
00 ꢂM. No response up to 30 ꢂM.
Installation of 2ꢀpyridyl (14) or 2ꢀfuryl (15) onto the thiazolidine scaffold eroded potency in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the βꢀarrestinꢀ2 assay compared to the phenyl derivatives, but mostly preserved potency in the
cAMP assay, again suggesting a route to G proteinꢀbiased agonists. Since the goal was both to
33
increase potency and decrease lipophilicity, values for ligandꢀlipophilicity efficiency (LLE)
based on calculated lipophilicity of the neutral compounds (clogP) were also used to guide the
optimization. The hydrophilic pyridyl led to a marked improvement in LLE for 14 compared to
all previous compounds including 4, but insufficient potency. The introduction of a tertꢀbutyl
group in the 2ꢀposition of the thiazolidine scaffold resulted in a compound (16) that only caused
50% receptor activation at 100 ꢂM. Replacing sulfur with oxygen on 16 led to a completely
inactive compound (17), hence, the oxazolidine scaffold was not explored further.
We next directed attention to the Nꢀacyl substituent. As evident from 18 (Table 2), the
biphenyl system is important for activity, as removing the terminal ring leads to complete loss of
activity. Removing only the 2’ꢀmethoxy (19) also resulted in a pronounced loss of potency in
both assays, as did moving the terminal phenyl to the 3ꢀposition (20). The 2’ꢀfluoroꢀ3’ꢀmethoxy
(
21) and the 4’ꢀmethoxy (22) analogues both led to decreased potency in both assays relative to
the 2’ꢀmethoxy analogue 9. Compared to 19, installation of either acetyl (23) or nitrile (24) in the
ꢀposition markedly boosted potency in both βꢀarresinꢀ2 and cAMP assays, indicating that
4
certain hydrogen bond accepting groups are favored in this region.
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Table 2. SAR investigation of the Nꢀacyl substituent
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ar
cmpd
hFFA2
(
3/4ꢀposition)
βꢀarrestinꢀ2
cAMP pEC50
c
d
pEC50
b
clogP
LLE
Emax
( )
a
(Emax)
e
e
18
(4)
(4)
(3)
nr
nr
3.82
5.71
5.71
ꢀ
1
9
0
5.09 ± 0.05 (146) 5.00 ± 0.20 (93)
4.59 ± 0.13 (141) 5.40 ± 0.04 (91)
ꢀ0.62/ꢀ0.71
ꢀ1.12/ꢀ0.31
2
2
1
2
(4)
(4)
5.32 ± 0.23 (122) 5.04 ± 0.18 (82)
4.66 ± 0.16 (174) 5.35 ± 0.15 (87)
5.57
5.63
ꢀ0.25/ꢀ0.53
ꢀ0.97/ꢀ0.31
2
2
3
(4)
6.09 ± 0.04 (103) 5.68 ± 0.07 (90)
5.15
0.94/0.53
24
(4)
(4)
5.89 ± 0.08 (95)
5.89 ± 0.13 (95)
5.15
6.18
0.74/0.74
2
5
5.14 ± 0.24 (143) 6.04 ± 0.16 (74)
ꢀ1.04/ꢀ0.14
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
6
7
(4)
(4)
5.36 ± 0.12 (146) 5.40 ± 0.19 (82)
5.91
6.07
ꢀ0.55/ꢀ0.51
ꢀ1.03/ꢀ0.75
f
2
5.04 ± 0.17 (134 ) 5.32 ± 0.15 (89)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
8
9
(4)
(4)
5.22 ± 0.12 (130) 5.68 ± 0.09 (83)
6.46
6.38
ꢀ1.24/ꢀ0.78
ꢀ1.58/ꢀ0.73
2
4.80 ± 0.35 (99)
5.65 ± 0.09 (89)
3
0
1
(4)
(4)
6.01 ± 0.04 (103) 6.67 ± 0.12 (77)
4.04
3.63
1.97/2.63
2.47/3.48
O
N
3
6.10 ± 0.07 (99)
4.68 ± 0.11 (108)
7.11 ± 0.08 (86)
32
(4)
ꢀ
5.60
ꢀ0.92/ꢀ
a
b
BRETꢀbased β arrestinꢀ2 recruitment assay. Efficacy (E ) is relative to propionate. cAMP
FFA2 assay. Efficacy (E ) is relative to propionate. clogP values were calculated using
ChemBioDraw v16.0.1.4. LLE = pEC (βꢀarrestin/cAMP)−clogP. No response up to 30 ꢂM.
Average response at highest concentration relative to propionate.
max
c
max
d
e
50
f
Replacing the 2’ꢀmethoxy (9) with 2’ꢀchloro (25) or 2’ꢀmethyl (26) resulted in a marked
potency boost in the cAMP assay for 25, whereas 2’ꢀmethoxyphenyl remained the preferred ring
system in the βꢀarrestinꢀ2 assay (9 > 25 ~ 26).
Extension of the biphenyl 19 by insertion of acetylene between the rings (28) caused a minor
loss of potency in the cAMP assay but no changes in the βꢀarrestinꢀ2 assay. In a parallel manner,
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9
alkyne extension of 2ꢀmethoxyphenyl (9) into 29 reduced potency in the βꢀarrestinꢀ2 assay, but
preserved activity in the cAMP assay.
Overall, 4ꢀbiphenyl compounds with the terminal phenyl ring carrying lipophilic (25, 26) or
moderately hydrophilic (9) substituents in the orthoꢀposition showed higher potencies than a
nonꢀsubstituted phenyl analog such as 19 or 20, indicating that a nonꢀplanar biphenyl
conformation is favored for optimal binding of this compound class.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
With this and the encouraging results from 23 and 24 in mind, we wished to explore biaryl
systems with orthoꢀsubstituted polar heterocycles as the terminal ring. Satisfyingly, installation
of 1ꢀmethylꢀ1Hꢀpyrazolyl produced a significantly improved compound (30), whereas
introduction of 3,5ꢀdimethylisoxazolyl (31) appeared as the most potent compound of the series
with a pEC of 7.11 in the cAMP assay and an LLE increased by 2.3 relative to the initial
5
0
pyrrolidine 4. Replacement of the methyl groups of 31 by phenyl and cyclopropyl (32) resulted
in a 40ꢀfold lower potency in the βꢀarrestinꢀ2 assay, indicating a limit to the acceptable steric
bulk in this region.
Having identified 4ꢀ(3,5ꢀdimethylisoxazolyl)benzoyl as a preferred Nꢀsubstituent, we directed
the attention back to the 2ꢀsubstituent of the thiazolidine scaffold to screen for improved
substituents in a second iteration (Table 3). This effort showed again that the 2ꢀchloro (31) was
clearly favored over a naked phenyl (33). The same was true for 2ꢀethynyl (34) and 2ꢀmethoxy
(35). Furthermore, increasing the polarity by installation of 2ꢀpyridyl (36) resulted in a weakly
potent agonist in the βꢀarrestinꢀ2 assay, whereas 36 was devoid of any activity when evaluated in
the cAMP assay. Since pyridyl analog 14 was a reasonably potent compound in both βꢀarrestinꢀ2
and cAMP assays, the observation that 36 lost activity is likely explained by this compound
being too polar to effectively reach the lipophilic binding site of FFA2.
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2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As the initial idea was to use the thiazolidine as a bioisostere for pyrrolidine in the
optimization of the compound series, the corresponding pyrrolidine 37 of thiazolidine 31 was
synthesized and tested (Table 3). Although pyrrolidine 37 displayed appreciable FFA2 activity,
its potency was lower than that of 31.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Variations in the 2ꢀposition and pyrrolidine analogue with the optimized acyl part
cmpd
33
X
R
hFFA2
βꢀarrestinꢀ2 pEC
cAMP pEC50
50
c
d
b
clogP
LLE
a
(Emax
)
(Emax)
S
S
5.64 ± 0.04 (79)
5.99 ± 0.21 (73)
5.09 ± 0.11 (79)
5.84 ± 0.08 (94) 2.92
5.46 ± 0.12 (49) 3.19
2.72/2.92
2.80/2.27
3
4
3
3
5
6
S
S
5.78 ± 0.04 (83) 2.84
2.25/2.94
3.03/ꢀ
e
4.45 ± 0.04 (102)
5.97 ± 0.08 (90)
nr
1.42
37
CH
2
6.82 ± 0.07 (85) 3.63
2.34/3.19
a
b
BRETꢀbased β arrestinꢀ2 recruitment assay. Efficacy (E ) is relative to propionate. cAMP
max
c
FFA2 assay. Efficacy (Emax) is relative to propionate. clogP values were calculated using
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Page 15 of 70
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7
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9
d
e
ChemBioDraw v16.0.1.4. (BRET/cAMP) LLE values (LLE = pEC −clogP). No response up
to 30 ꢂM.
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Binding affinities of compounds for FFA2
a
Cmpd
pKi
4
9
6.32 ± 0.02
6.47 ± 0.06
5.92 ± 0.08
6.01 ± 0.02
6.69 ± 0.03
6.40 ± 0.06
1
1
3
3
3
9
1
7
a
3
Binding affinities of agonists determined using [ H]ꢀ1 in radioligand binding assays.
The occasionally diverging results in the cAMP and βꢀarrestinꢀ2 assays illustrate that potencies
in the functional assays do not directly reflect receptor affinity. To better understand the
relationship between affinity and functional activity, we proceeded with further examination of
3
1 and selected analogs in an FFA2 competition binding assay using a radiolabeled version of
3
antagonist 1 (Table 4). It has previously been confirmed that 1 and radiotracer [ H]ꢀ1 are
orthosteric ligands and fully displaceable by propionate and the synthetic orthosteric agonist
3
2,41
3
.
Pyrrolidines 4 and 37 and thiazolidines 9, 13, 19 and 31 were also all confirmed to fully
3
displace [ H]ꢀ1. Parent pyrrolidine 4 exhibited a moderate binding affinity and the corresponding
thiazolidine (9) was found to be a slightly stronger binder, in contrast to the slightly lower
potency observed in both functional assays. Upon comparing 9 with the analogue lacking the 2ꢀ
chloro substituent of the 2ꢀaryl (13) or lacking the 2’ꢀmethoxy (19) substituent at the biphenyl
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Journal of Medicinal Chemistry
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
system, it becomes clear that both these features contribute significantly to the affinity (ꢁpK =
i
0
.55 and 0.46, respectively). The most potent thiazolidine (31) was indeed found to also exhibit
the highest binding affinity within this selection. The corresponding pyrrolidine (37) displayed a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
significantly weaker binding affinity when compared to 31 (ꢁpK = ꢀ0.29), which correlated well
i
with the observed differences in functional potency between these two compounds. Hence, for
isoxazoles 31 and 37, the thiazolidine central scaffold boosted both affinity and receptor
activation compared to the pyrrolidine. Replacement of the 2ꢀmethoxyphenyl (9) by the more
polar 3,5ꢀdimethylisoxazole (31) not only gave rise to the more potent thiazolidineꢀbased agonist
(31) but also reduced lipophilicity resulting in a marked improvement in LLE over 4 (ꢁLLE =
2.27), and 31 indeed exhibits the highest LLE of all compounds in the study.
Molecular modeling
To better understand the interaction of 31 with FFA2, the compound was docked in a
3
2
previously established homology model of the receptor. The lowest energy pose of 31 is
depicted in Figure 2. Compound 31 was found to effectively engage Arg180 and Arg255 via
ionicꢀ and hydrogen bonding interactions, an observation which is in agreement with previous
3
0,41
reports that both residues are known to be vital for receptor binding and activation.
Simultaneously, the carboxylate carbonyl also interacted with the conserved Tyr238 through
hydrogen bonding. The 2ꢀchlorphenyl moiety situated cis to the carboxylate was observed to
interact with Phe89 via displaced πꢀπ interactions, where the chloro substituent appears to orient
the phenyl for optimal contact. Furthermore, the dimethylisoxazole ring formed πꢀcation
interactions with Lys65 and edgeꢀtoꢀface πꢀπ interactions with Trp75 (Supporting Information,
Figure S2). The shown pose also reflects higher energy poses. No alternative lowꢀenergy pose
was identified.
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9
Overall, groups of 31 found in the SAR analysis to be crucial for binding, such as 2ꢀ
chlorophenyl and dimethylisoxazole, were confirmed by docking to contribute to the ligand
receptor interaction, further supporting the proposed binding pose of 31 in hFFA2. Notably, both
the carboxylate and the amide carbonyl of 31 were oriented towards the two arginine residues
and Arg255 interacted directly with both groups through hydrogen bonds (Figure 2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
W75
B
W75
K65
R255
R180
H242
R255
F89
R180
Y238
K65
H242
Y328
Figure 2. Lowest energy pose of 31 (grey) docked into hFFA2 (gold) with strongly interacting
residues shown (cyan) and hydrogen bonds highlighted (yellow stipples). (A) Receptor as ribbon
viewed from the angle of helix 4 and 5. (B) Same pose with receptor as surface viewed from the
angle of helix 3 and 4.
Characterization of 31
Since 31 showed the highest potency and LLE based on calculated lipophilicity in both
functional assays and the binding assay, it was decided to characterize its properties in further
detail. Thus, 31 was tested and confirmed to be active on the murine FFA2 orthologue (pEC =
5
0
6
.44 ± 0.13 in the cAMP assay). The compound was found to be inactive up to 100 ꢀM
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
concentration at the closely related SCFA receptor FFA3 (formerly GPR41), and evoked no
significant agonist or antagonist response in the longꢀchain fatty acid receptors FFA1 (GPR40)
or FFA4 (GPR120), or in nuclear receptors PPARα, PPARγ, PPARδ, LXRα or LXRβ at 10 ꢀM
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(see the Supporting Information).
Previous FFA2 ligands are generally, apart from SCFAs, lipophilic compounds. In contrast,
experimental lipophilicity of 31 (Table 5) was found to be in the lower part of the optimal
3
4
range, giving an LLE of 6.2 based on the cAMP assay and measured lipophilicity. The
compound also showed high aqueous solubility and chemical stability (Table 5). Recovery after
incubation with PBS at pH 7.4 or with fastedꢀstate simulated gastric or intestinal fluid
(FaSSGF/FaSSIF) was close to quantitative, indicating excellent chemical stability. Investigation
of metabolic stability of 31 in mouse liver microsomes also resulted in a high recovery.
Table 5. Physicochemical properties
Assay
31
log D (nꢀoctanol/PBS, pH 7.4)
aqueous solubility (PBS, pH 7.4)
chemical stability
0.94
182 ꢂM
PBS, 37 °C, 3 weeks
97.6%
95%
FaSSIF, 2 h
FaSSGF, 2 h
100%
82%
metabolic stability (MLM, remaining, 1 h)
Hepatocyte stability is a better approximation to the in vivo properties than liver microsomes
since mitochondrial metabolism and conjugation processes also take place. Incubation of 31 with
primary mouse hepatocytes gave a recovery of 72% after 2 hours and low intrinsic clearance (<8
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ꢀ
L/min/10 cells), indicating high metabolic stability in mouse hepatocytes (Supporting
Information, Figure S3 and Table S6).
The pharmacokinetic profile of 31 was investigated in mice to establish its properties as a tool
for in vivo studies, revealing a satisfactory halfꢀlife of 2 hours and a moderate to low clearance
of 20.3 mL/min/kg in mice (Table 6). Oral dosing of 10 mg/kg resulted in a maximal plasma
concentration of 6.5 ꢀM after 15 min and a bioavailability of 32%. Furthermore, ip
administration of 31 at 5 mg/kg in mice and analysis of plasma concentrations indicated an
AUC0ꢀ∞ of 60500 ng/mL·min a halfꢀlife of 82 min and thus a favorable profile relative to 4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
21
(AUC0ꢀ∞ ~25000 ng/mL·min, t1/2 ~50 min).
a
Table 6. Pharmacokinetic properties in mice
Parameters
31
Intravenous admin.
t1/2
138 min
ꢀ1
AUC0ꢀ∞
Vd
246000 ng min mL
ꢀ1
4050 mL kg
ꢀ1
ꢀ1
CLtotal
Oral admin.
tmax
20.3 mL min kg
15 min
ꢀ1
Cmax
2880 ng mL
ꢀ1
AUC0ꢀ∞
156000 ng min mL
32%
F
a
Administered at 10 mg/kg po and 5 mg/kg iv in Balb/c mice.
Agonist 3 has been shown to affect inflammatory responses in human neutrophils via FFA2 by
4
2
induction of intracellular calcium and superoxide production. Propionate is known to exert
9
beneficial antiꢀinflammatory effects, and the elimination of FFA2 from mice has previously
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been shown to cause increased inflammation in tissue models. Although the FFA2 antagonist 1
failed to meet the endpoint in clinical trials with ulcerative colitis patients, the compound was
4
3
found to modulate neutrophil recruitment, and the therapeutic potential of FFA2 modulation in
inflammatory disease is still not clear, especially since neutrophil recruitment may have both
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44
beneficial and detrimental effects. Hence, 31 and propionate were examined for their ability to
promote human neutrophil migration, as it is already established that antagonists, such as 1, can
4
5,46
inhibit chemotaxis in vitro, and that acetate can induce chemotaxis in mouse neutrophils.
Compound 31 at 1 ꢂM significantly induced migration of human neutrophils at a level
4
7
comparable to propionate (C3) at a physiologically relevant concentration, an effect that was
more than doubled at 10 ꢂM (Figure 3).
Figure 3. The ability of propionate (C3) and 31 to induce neutrophil migration relative to the
highly potent chemotactic factor fMLP (n = 4ꢀ6). * p < 0.05, *** p < 0.01; oneꢀway ANOVA
followed by Dunnett post hoc test.
SCFAs have been shown to inhibit lipolysis in adipocytes mediated through FFA2 via the Gαi
4
8
pathway. In this study we demonstrate that thiazolidine 31, in particular when evaluated in
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cAMP assays, is a potent agonist of hFFA2 with retained activity at mFFA2. Therefore, 31 was
evaluated for its ability to inhibit isoproterenolꢀinduced lipolysis in murine adipocytes, a process
48
known to be Gα dependent. Even though 31 is slightly less potent on murine FFA2, 31 was, as
i
1
1
1
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0
predicted, 50ꢀfold more potent than propionate in inhibiting lipolysis in isoproterenolꢀstimulated
adipocytes (Figure 4).
150
propionate
100
31
5
0
0
-
10 -9
-8
-7
-6
-5
-4
-3
-2
Log [Ligand]
Figure 4. Inhibition of isoproterenolꢀinduced lipolysis by propionate (pEC = 3.6 ± 0.4) and 31
5
0
(
pEC = 5.3 ± 0.6) in murine adipocytes (n = 3ꢀ4)
50
CONCLUSION
In conclusion, substitution of the central pyrrolidine of a published FFA2 agonist by a
synthetically more tractable thiazolidine and rapid screening of substituents at this scaffold led to
the discovery of the potent and selective FFA2 agonist 31, a compound with lipophilicity in the
lower part of the optimal range, and therefore also high LLE. The compound furthermore has
excellent solubility, high chemical, microsomal and hepatocyte stability, and favorable
pharmacokinetic properties. Compound 31 was able to induce migration of human neutrophils
and to inhibit lipolysis in murine adipocytes. Collectively, these properties render 31 an
interesting tool compound for further exploration of FFA2 as a potential therapeutic target for
treatment of inflammatoryꢀ and metabolic diseases.
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EXPERIMENTAL SECTION
Synthesis
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0
All commercial starting materials and solvents were used without further purification unless
otherwise stated. THF was freshly distilled from sodium/benzophenone. MeOH was freshly
distilled from Mg. DCM was freshly distilled and stored over 4Å sieves. TLC was performed on
TLC Silica gel 60 F254 plates (Merck) and visualized at 254 nm or by staining with ninhydrin,
KMnO or FeCl stains. Petroleum ether (PE) refers to alkanes with bp 60ꢀ80 °C. Water used in
4
3
reactions was demineralized and water used for freezeꢀdrying was filtered MilliꢀQ. Purification
by flash chromatography was carried out using silica gel 60 (0.040ꢀ0.063 mm, Merck). Test
compounds suspended in MilliꢀQ water (1 mL/10 mg, 3 drops MeCN) were lyophilized on a
1
13
Heto Drywinner Freezeꢀdryer. H and C NMR spectra were recorded at 400 and 101 MHz,
respectively, on a Bruker Avance III 400 at 300 K. Spectra were calibrated relative to residual
1
13
1
solvent peaks: H NMR (CDCl ): 7.26 ppm; C NMR (CDCl ): 77.16 ppm; H NMR (DMSOꢀ
3
3
13
1
13
d ): 2.50 ppm; C NMR (DMSOꢀd ): 39.52 ppm; H NMR (CD OD): 3.31 ppm; C NMR
6
6
3
1
13
(
CD OD): 49.00 ppm; H NMR (CD Cl ): 5.32 ppm; C NMR (CD Cl ): 53.84 ppm. Rotamer
3 2 2 2 2
1
chemical shift values in H NMR are marked by ‘*’ and have been assigned where possible.
1
1
13
Rotamer peaks in H NMR are labeled by an integral equal to that of its main partner. H and C
spectra of 31 were obtained at 600 and 151 MHz, respectively, on an Agilent NMRS 600 at the
indicated temperature (Supporting Information). Highꢀresolution mass spectra (HRMS) were
recorded on a Bruker micrOTOFꢀQ II (ESI). Specific optical rotation was recorded on Anton
Paar MCP 100 polarimeter. Purity was determined by HPLC and confirmed by inspection of
1
13
NMR spectra ( H and C NMR). HPLC analysis was performed using a Dionex 120 C18
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column (5 ꢂm, 4.6x150 mm); flow: 1 mL/min; 10% MeCN in water (0ꢀ1 min), 10ꢀ100% MeCN
in water (1ꢀ10 min), 100% MeCN (11ꢀ15 min), with both solvents containing 0.1% formic acid
as modifier; UV detection at 254 nm. All test compounds were of ≥95% purity. None of the test
4
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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0
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compounds contain substructures associated with panꢀassay interfering activities (PAINS) by
inspection or by screening for PAINS or aggregators at http://zinc15.docking.org/patterns/home.
Methyl (4R)ꢀ2ꢀ(2ꢀchlorophenyl)thiazolidineꢀ4ꢀcarboxylate (7a).
hydrochloride (1.51 g, 8.80 mmol) was dissolved in water (6.6 mL) and potassium bicarbonate
879 mg, 8.78 mmol) was added followed by a solution of 2ꢀchlorobenzaldehyde (2.49 g, 18.1
LꢀCysteine methyl ester
(
mmol) in EtOH (6.6 mL). The reaction was stirred at rt for 12 h, whereafter the reaction mixture
was diluted with water and extracted with DCM (x3). The combined organic phases were
washed with brine, dried over Na SO , concentrated in vacuo and purified by flash
2
4
chromatography (EtOAc:PE, 1:5) to give 7a as a light yellow oil (1.68 g, 75% yield, 40% minor
1
isomer and 60% major isomer): R = 0.22 (EtOAc:PE, 1:5); H NMR for minor isomer (400
f
MHz, CDCl ) δ 7.81 (dd, J = 7.6, 1.8 Hz, 1H), 7.50 – 7.24 (m, 6H)[minor and major], 6.04 (br s,
3
1
H), 4.14 – 4.06 (m, 1H), 3.90 (s, 3H), 3.56 (dd, J = 10.3, 6.9 Hz, 1H), 3.24 – 3.14 (m,
1
2
H)[minor and major], 2.83 (br s, 1H); H NMR for major isomer (400 MHz, CDCl ) δ 7.66 (dd,
3
J = 7.7, 1.7 Hz, 1H), 7.50 – 7.23 (m, 6H)[minor and major], 6.18 (s, 1H), 4.34 (app t, J = 6.6 Hz,
H), 3.91 (s, 3H), 3.43 (dd, J = 10.6, 6.5 Hz, 1H), 3.24 – 3.16 (m, 2H)[minor and major], 3.15
1
1
3
(
br s, 1H); C NMR for mixture of diastereomers (101 MHz, CDCl ) δ 172.2, 171.7, 140.2,
3
136.0, 133.9, 133.1, 130.0, 129.9, 129.8, 128.7, 128.3, 127.5, 127.0, 126.7, 68.5, 67.4, 65.6, 65.1,
+
5
2.8, 52.7, 39.1, 37.6; HRMS (ESI) calcd for C H ClNNaO S (M + Na ): 280.0169, found:
11
12
2
2
80.0162.
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Methyl (4R)ꢀ2ꢀ(3ꢀchlorophenyl)thiazolidineꢀ4ꢀcarboxylate (7b). The title compound was
prepared as described for 7a using Lꢀcysteine methyl ester hydrochloride (119 mg, 1.16 mmol)
and 3ꢀchlorobenzaldehyde (328 mg, 2.33 mmol) and obtained after flash chromatography
0
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(EtOAc:PE, 1:5) as a light yellow oil (214 mg, 71% yield, 44% minor isomer and 56% major
1
isomer): R = 0.16 (EtOAc:PE, 1:5); H NMR for minor isomer (400 MHz, CDCl ) δ 7.50 – 7.48
f
3
(m, 1H), 7.41 – 7.28 (m, 4H)[minor and major], 7.25 – 7.20 (m, 2H)[minor and major], 5.78 (s,
1
2
7
H), 4.12 (app t, J = 6.6 Hz, 1H), 3.79 (s, 3H), 3.37 (dd, J = 10.6, 7.0 Hz, 1H), 3.18 – 3.06 (m,
1
H)[minor and major], 2.92 (br s, 1H); H NMR for major isomer (400 MHz, CDCl ) δ 7.54 –
3
.52 (m, 1H), 7.41 – 7.28 (m, 4H)[minor and major], 7.25 – 7.20 (m, 2H)[minor and major], 5.50
(br s, 1H), 4.01 – 3.93 (m, 1H), 3.80 (s, 3H), 3.45 (dd, J = 10.3, 7.1 Hz, 1H), 3.18 – 3.06 (m,
13
2
H)[minor and major], 2.62 (br s, 1H); C NMR for mixture of diastereomers (101 MHz,
CDCl ) δ 172.1, 171.5, 143.9, 140.4, 134.6, 134.4, 130.0, 129.74, 129.0, 128.0, 127.8, 127.1,
3
125.9, 125.3, 71.8, 69.9, 65.6, 64.2, 52.7, 52.7, 39.2, 38.2; HRMS (ESI) calcd for C H ClNO S
1
1
13
2
+
(
M + H ): 258.0350, found: 258.0356.
Methyl (4R)ꢀ2ꢀ(4ꢀchlorophenyl)thiazolidineꢀ4ꢀcarboxylate (7c). The title compound was
prepared as described for 7a using ꢀcysteine methyl ester hydrochloride (200 mg, 1.17 mmol)
and 4ꢀchlorobenzaldehyde (328 mg, 2.33 mmol) and obtained after flash chromatography
L
(EtOAc:PE, 1:5) as a light yellow oil (208 mg, 69% yield, 42% minor isomer and 58% major
1
isomer): R = 0.19 (EtOAc:PE, 1:5); H NMR for minor isomer (400 MHz, CDCl ) δ 7.36 – 7.25
f
3
(m, 4H), 5.77 (s, 1H), 4.13 (t, J = 6.5 Hz, 1H), 3.78 (s, 3H), 3.36 (dd, J = 10.6, 7.0 Hz, 1H), 3.16
1
(
dd, J = 10.6, 6.1 Hz, 1H), 2.67 (br s, 2H)[minor and major]; H NMR for major isomer (400
MHz, CDCl ) δ 7.48 – 7.39 (m, 4H), 5.51 (s, 1H), 3.97 (dd, J = 8.8, 7.2 Hz, 1H), 3.45 (dd, J =
3
10.3, 7.1 Hz, 1H), 3.79 (s, 3H), 3.10 (dd, J = 10.3, 9.0 Hz, 1H), 2.67 (br s, 2H)[minor and
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major]; C NMR for mixture of diastereomers (101 MHz, CDCl ) δ 172.1, 171.5, 140.1, 136.8,
3
1
34.5, 133.6, 128.9, 128.9, 128.5, 128.4, 71.8, 70.0, 65.5, 64.2, 52.7, 52.6, 39.2, 38.1; HRMS
+
(ESI) calcd for C H ClNO S (M + H ): 258.0350, found: 258.0361.
1
1
13
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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Methyl (R)ꢀ2ꢀ(pyridinꢀ2ꢀyl)thiazolidineꢀ4ꢀcarboxylate (7e). The title compound was
prepared as described for 7a using ꢀcysteine methyl ester hydrochloride (200 mg, 1.16 mmol)
L
and picolinaldehyde (187 mg, 1.75 mmol) and obtained after flash chromatography (EtOAc:PE,
1:1) as a clear oil (165 mg, 63% yield, 45% minor isomer and 55% major isomer): R = 0.18
f
1
(
EtOAc:PE, 1:1); H NMR for minor isomer (400 MHz, CDCl ) δ 8.57 (d, J = 4.8 Hz, 1H), 7.72
3
–
2
2
7.61 (m, 2H)[minor and major], 7.35 – 7.27 (m, 2H)[minor and major], 7.26 – 7.17 (m,
H)[minor and major], 5.85 (s, 1H), 4.56 (dd, J = 6.4, 4.6 Hz, 1H), 3.80 (s, 3H), 3.63 (br s,
1
H)[minor and major], 3.41 – 3.31 (m, 2H); H NMR for major isomer (400 MHz, CDCl ) δ 8.63
3
(
d, J = 4.8 Hz, 1H), 7.72 – 7.61 (m, 2H)[minor and major], 7.35 – 7.27 (m, 2H)[minor and
major], 7.26 – 7.17 (m, 2H)[minor and major], 5.66 (s, 1H), 4.04 (dd, J = 9.7, 6.7 Hz, 1H), 3.83
(s, 3H), 3.63 (br s, 2H)[minor and major], 3.45 (dd, J = 10.1, 6.7 Hz, 1H), 3.09 (t, J = 9.9 Hz,
13
1
1
H); C NMR for mixture of diastereomers (101 MHz, CDCl ) δ 172.2, 171.2, 158.7, 156.8,
3
49.9, 149.6, 136.8, 136.8, 123.4, 123.0, 122.1, 121.6, 71.6, 71.1, 66.3, 65.6, 52.6[minor and
+
major], 39.4, 38.7; HRMS (ESI) calcd for C H N NaO S (M + Na ) 247.0512, found
1
0
12
2
2
2
47.0524.
Methyl (4R)ꢀ2ꢀ(2ꢀmethoxyphenyl)thiazolidineꢀ4ꢀcarboxylate (7f). The title compound was
prepared as described for 7a using ꢀcysteine methyl ester hydrochloride (254 mg, 1.48 mmol)
and 2ꢀmethoxybenzaldehyde (199 mg, 1.46 mmol) and obtained after flash chromatography
L
(
EtOAc:PE, 1:4) as a clear oil (297 mg, 81% yield, 40% minor isomer and 60% major isomer):
1
R = 0.21 (EtOAc:PE, 1:4); H NMR for minor isomer (400 MHz, CDCl ) δ 7.42 (dd, J = 7.6, 1.5
f
3
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Hz, 1H), 7.25 – 7.19 (m, 1H), 7.00 – 6.92 (m, 2H), 5.99 (br s, 1H), 4.25 (app t, J = 6.6 Hz, 1H),
3
.85 (s, 3H), 3.79 (s, 6H)[minor and major], 3.29 (dd, J = 10.4, 6.6 Hz, 1H), 3.06 (br s,
1
2H)[minor and major], 3.12 – 3.00 (m, 2H)[minor and major]; H NMR for major isomer (400
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
MHz, CDCl ) δ 7.49 (dd, J = 7.6, 1.6 Hz, 1H), 7.32 – 7.26 (m, 1H), 6.92 – 6.83 (m, 2H), 5.83 (s,
3
1
1
H), 3.98 – 3.91 (m, 1H), 3.86 (s, 3H), 3.79 (s, 6H)[minor and major], 3.42 (dd, J = 10.1, 6.8 Hz,
1
3
H), 3.06 (br s, 2H)[minor and major], 3.12 – 3.00 (m, 2H)[minor and major]; C NMR for
mixture of diastereomers (101 MHz, CDCl ) δ 172.3, 171.8, 157.4, 156.8, 130.1, 129.7, 128.7,
3
1
3
27.8, 126.2, 125.8, 120.9, 120.5, 111.2, 110.7, 67.7, 66.0, 65.8, 64.9, 55.7, 55.5, 52.51, 52.49,
+
9.0, 37.6; HRMS (ESI) calcd for C H NO S (M + H ) 254.0845, found 254.0855.
12 16
3
Methyl (4R)ꢀ2ꢀphenylthiazolidineꢀ4ꢀcarboxylate (7g). The title compound was prepared as
described for 7a using ꢀcysteine methyl ester hydrochloride (327 mg, 1.91 mmol) and
benzaldehyde (288 ꢂL, 2.83 mmol) and obtained after flash chromatography (EtOAc:PE, 1:4) as
L
a clear oil (340 mg, 80% yield, 37% minor isomer and 63% major isomer): R = 0.32 (EtOAc:PE,
f
1
1:4); H NMR for minor isomer (400 MHz, CDCl ) δ 7.49 – 7.46 (m, 2H), 7.39 – 7.22 (m, 6H)
3
[
minor and major], 5.81 (s, 1H), 4.20 (app t, J = 6.1 Hz, 1H), 3.77 (s, 3H), 3.37 (dd, J = 10.6, 7.1
1
Hz, 1H), 3.19 (dd, J = 10.6, 5.8 Hz, 1H), 2.84 (br s, 1H); H NMR for major isomer (400 MHz,
CDCl ) δ 7.54 – 7.49 (m, 2H), 7.39 – 7.22 (m, 6H) [minor and major], 5.55 (s, 1H), 4.02 – 3.93
3
(m, 1H), 3.78 (s, 3H), 3.45 (dd, J = 10.3, 7.1 Hz, 1H), 3.10 (dd, J = 10.1, 9.2 Hz, 1H), 2.66 (br s,
13
1
H); C NMR for mixture of diastereomers (101 MHz, CDCl ) δ 172.2, 171.6, 141.2, 138.3,
3
1
28.73, 128.72, 128.5, 127.9, 127.5, 127.0, 72.7, 70.9, 65.6, 64.4, 52.6, 52.5, 39.3, 38.2; HRMS
+
(
ESI) calcd for C H NNaO S (M + Na ) 246.0559, found 246.0565.
1
1
13
2
Methyl (2R,4R)ꢀ2ꢀ(2ꢀchlorophenyl)ꢀ3ꢀ(4ꢀiodobenzoyl)thiazolidineꢀ4ꢀcarboxylate (8a). A
preꢀdried vial with 7a (926 mg, 3.59 mmol) in THF (7.4 mL, anhydrous) was cooled to ꢀ10 °C
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and added triethylamine (1.25 mL, 8.97 mmol) dropwise. After stirring for 15 min at rt the
solution was evaporated, the triethylammonium salt was reꢀdissolved in THF (11 mL) and the
solution was cooled to ꢀ10 °C. 4ꢀIodobenzoyl chloride (1.05 g, 3.95 mmol) dissolved in THF (15
mL, anhydrous) was added dropwise and the reaction mixture was allowed to reach rt. After
stirring at rt for 4 h the solvent was evaporated off, the crude residue was added water (18 mL)
and acidified with 1 M HCl (pH 2ꢀ3) and extracted with EtOAc (x3). The organic phases were
washed with brine, dried over Na SO , concentrated in vacuo and purified by flash
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
4
4
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4
4
4
4
4
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
chromatography (EtOAc:PE, 1:3) to give central intermediate 8a as a white foam (1.43 g, 82%
1
yield): R = 0.21 (EtOAc:PE, 1:3); H NMR (400 MHz, CDCl ) δ 8.32 (d, J = 6.4 Hz, 1H), 7.64 –
f
3
7.47 (m, 2H), 7.40 – 7.31 (m, 2H), 7.30 – 7.22 (m, 1H), 7.07 – 6.91 (m, 2H), 6.17 (br s, 1H),
13
5
1
.07 (app s, 1H), 3.86 (s, 3H), 3.45 – 3.32 (m, 1H), 3.20 (m, 1H); C NMR (101 MHz, CDCl ) δ
3
70.3, 169.9, 138.9, 137.3, 134.1, 131.4, 129.8, 129.2, 128.5, 127.5, 127.3, 97.6, 65.8, 65.5, 52.8,
+
20
31.1; HRMS (ESI) calcd for C H ClINO S (M + H ) 487.9579, found 487.9573. [α] = +45°
1
8
16
3
D
(c = 1.0, DCM).
Methyl (2R,4R)ꢀ2ꢀ(2ꢀchlorophenyl)ꢀ3ꢀ(3ꢀiodobenzoyl)thiazolidineꢀ4ꢀcarboxylate (8b). The
title compound was prepared as described for 8a using 7a (300 mg, 0.16 mmol) and 3ꢀ
iodobenzoyl chloride (347 mg, 0.13 mmol) and obtained after flash chromatography (EtOAc:PE,
1
1
:3) as a colorless oil (237 mg, 42% yield): R = 0.26 (EtOAc:PE, 1:3); H NMR (400 MHz,
f
CDCl ) δ 8.30 (d, J = 7.1 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.57 – 7.48 (m, 1H), 7.40 (t, J = 7.2
3
Hz, 1H), 7.35 (dd, J = 7.9, 1.3 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.24 – 7.17 (m, 1H), 7.01 –
6
.91 (m, 1H), 6.15 (s, 1H), 5.13 – 4.96 (m, 1H), 3.89 (s, 3H), 3.47 – 3.32 (m, 1H), 3.32 – 3.15
13
(
m, 1H); C NMR (101 MHz, CDCl ) δ 170.5, 169.3, 139.7, 139.1, 136.6, 136.2, 131.6, 130.0,
3
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1
29.4, 127.8, 127.6, 126.1, 93.8, 65.8, 65.6, 53.0, 31.4; HRMS (ESI) calcd for
+
C H ClINNaO S (M + Na ) 509.9398, found 509.9393.
18
15
3
(2R,4R)ꢀ2ꢀ(2ꢀChlorophenyl)ꢀ3ꢀ(2'ꢀmethoxybiphenylꢀ4ꢀcarbonyl)thiazolidineꢀ4ꢀcarboxylic
acid (9). Step 1: The methyl ester of the title compound 9a was prepared as described for 8a
using 7a (100 mg, 0.39 mmol) and 2'ꢀmethoxybiphenylꢀ4ꢀcarbonyl chloride (107 mg, 0.44
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
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9
0
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9
0
mmol) and obtained after flash chromatography (EtOAc:PE, 1:3) as a clear viscous oil (141 mg,
1
7
7
3
7% yield): R = 0.20 (EtOAc:PE, 1:3); H NMR (400 MHz, CDCl ) δ 8.38 (d, J = 7.0 Hz, 1H),
f
3
.49 – 7.19 (m, 9H), 7.07 – 6.91 (m, 2H), 6.32 (br s, 1H), 5.18 – 5.08 (m, 1H), 3.89 (br s, 3H),
13
.76 (s, 3H), 3.45 – 3.34 (m, 1H), 3.27 – 3.18 (m, 1H); C NMR (101 MHz, CDCl ) δ 171.0,
3
170.8, 156.5, 141.4, 139.5, 133.0, 131.7, 130.7, 129.9, 129.6, 129.4, 129.3, 129.1, 127.9, 127.4,
+
1
26.9, 121.0, 111.5, 66.1, 65.9, 55.6, 52.8, 31.3; HRMS (ESI) calcd for C H ClNO S (M + H )
2
5
23
4
4
68.1031, found 468.1028.
Step 2: Methyl ester 9a (40 mg, 86 ꢂmol) and LiI (46 mg, 0.34 mmol) were dissolved in
degassed EtOAc (120 ꢂL), the vial was capped, flushed with argon (x3), and the reaction stirred
at 80 °C in the dark. The reaction mixture was added water and 1 M HCl (1:1) and extracted with
EtOAc (x3). The combined organic phases were washed with NaHSO , water and brine. The
3
organic layers were dried over Na SO , concentrated in vacuo, and purified by flash
2
4
chromatography (EtOAc:PE, 2:1 [1% AcOH]) to give the title compound 9 after freeze drying as
1
an amorphous solid (20 mg, 51% yield): R = 0.13 (EtOAc:PE, 2:1 [1% AcOH]); H NMR (400
f
MHz, CDCl ) δ 8.03 (d, J = 6.1 Hz, 1H), 7.45 – 7.40 (m, 2H), 7.39 – 7.29 (m, 5H), 7.29 – 7.21
3
(m, 2H), 7.05 – 6.93 (m, 2H), 6.37 (br s, 1H), 5.28 – 5.16 (m, 1H), 3.77 (s, 3H), 3.50 – 3.35 (m,
1
3
2
H); C NMR (101 MHz, CDCl ) δ 172.9, 172.6, 156.6, 141.9, 138.9, 132.4, 131.7, 130.8,
3
130.1, 129.6, 129.5, 129.5, 129.4, 127.6, 127.0, 121.1, 111.6, 66.6, 66.1, 55.7, 31.0; HRMS
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+
(
ESI) calcd for C H ClNNaO S (M + Na ) 476.0694, found 476.0680; HPLC: t = 12.28 min,
2
4
20
4
R
20
9
8.4%. [α] = +13° (c = 0.25, DCM).
D
(
2R,4R)ꢀ2ꢀ(3ꢀChlorophenyl)ꢀ3ꢀ(2'ꢀmethoxybiphenylꢀ4ꢀcarbonyl)thiazolidineꢀ4ꢀcarboxylic
1
1
1
1
1
1
1
1
1
1
2
2
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2
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2
2
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2
3
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0
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0
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0
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9
0
acid (10). Step 1: The methyl ester of the title compound 10a was prepared as described for 8a
using 7b (100 mg, 0.39 mmol) and 2'ꢀmethoxybiphenylꢀ4ꢀcarbonyl chloride (105 mg, 0.43
mmol) and obtained after flash chromatography (EtOAc:PE, 1:3) as a clear viscous oil (152 mg,
1
8
3% yield): R = 0.18 (EtOAc:PE, 1:3); H NMR (400 MHz, CDCl ) δ 7.71 – 7.64 (m, 1H), 7.57
f
3
(
d, J = 5.8 Hz, 1H), 7.48 – 7.41 (m, 4H), 7.35 – 7.22 (m, 4H), 7.06 – 6.92 (m, 2H), 6.15 (br s,
1
3
1H), 5.17 (app s, 1H), 3.85 (s, 3H), 3.79 (s, 3H), 3.47 – 3.14 (m, 2H); C NMR (400 MHz,
CDCl ) δ 171.0, 170.5, 156.5, 143.5, 141.4, 134.4, 133.5, 130.8, 129.8, 129.6, 129.5, 129.4,
3
1
28.2, 127.2, 126.9, 125.2, 121.0, 111.4, 67.8, 65.2, 55.6, 52.9, 32.0; HRMS (ESI) calcd for
+
C H ClNNaO S (M + Na ) 490.0850, found 490.0830.
25
22
4
Step 2: The title compound 10 was prepared as described for 9 using methyl ester 10a (100
mg, 0.21 mmol) and LiI (4 equiv) and obtained after flash chromatography (EtOAc:PE, 1:2 [2%
AcOH]) and subsequent freeze drying as a white amorphous powder (65 mg, 67% yield): R =
f
1
0.16 (EtOAc:PE, 1:2 [2% AcOH]); H NMR (400 MHz, CDCl ) δ 7.61 – 7.39 (m, 5H), 7.37 –
3
7
.30 (m, 2H), 7.29 – 7.22 (m, 3H), 7.05 – 6.92 (m, 2H), 6.68 (br s, 1H), 6.16 (br s, 1H), 5.23 (app
1
3
s, 1H), 3.78 (s, 3H), 3.49 – 3.35 (m, 2H); C NMR (101 MHz, CDCl ) δ 172.5, 172.1, 156.5,
3
142.8, 141.9, 134.7, 132.7, 130.9, 130.0, 129.8, 129.5, 129.4, 128.5, 127.0, 124.9, 121.1, 111.5,
+
6
4
8.3, 65.0, 55.7, 31.6; HRMS (ESI) calcd for C H ClNNaO S (M + Na ) 476.0694, found
24
20
4
2
0
76.0687; HPLC: t = 12.10 min, 97.6%. [α] = +23° (c = 1.0, DCM).
R
D
(
2R,4R)ꢀ2ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(2'ꢀmethoxybiphenylꢀ4ꢀcarbonyl)thiazolidineꢀ4ꢀcarboxylic
acid (11). Step 1: The methyl ester of the title compound 11a was prepared as described for 8a
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