Non-Acidic Free Fatty Acid Receptor 4 Agonists with Antidiabetic Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b00685

The free fatty acid receptor 4 (FFA4 or GPR120) has appeared as an interesting potential target for the treatment of metabolic disorders. At present, most FFA4 ligands are carboxylic acids that are assumed to mimic the endogenous long-chain fatty acid agonists. Here, we report preliminary structure-activity relationship studies of a previously disclosed nonacidic sulfonamide FFA4 agonist. Mutagenesis studies indicate that the compounds are orthosteric agonists despite the absence of a carboxylate function. The preferred compounds showed full agonist activity on FFA4 and complete selectivity over FFA1, although a significant fraction of these noncarboxylic acids also showed partial antagonistic activity on FFA1. Studies in normal and diet-induced obese (DIO) mice with the preferred compound 34 showed improved glucose tolerance after oral dosing in an oral glucose tolerance test. Chronic dosing of 34 in DIO mice resulted in significantly increased insulin sensitivity and a moderate but significant reduction in bodyweight, effects that were also present in mice lacking FFA1 but absent in mice lacking FFA4.

Full text of DOI:10.1021/acs.jmedchem.6b00685

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Article
Non-Acidic Free Fatty Acid Receptor 4 Agonists with Antidiabetic Activity
Carlos M. G. Azevedo, Kenneth R. Watterson, Ed T Wargent, Steffen V. F. Hansen,
Brian D. Hudson, Ma#gorzata A. K#pczy#ska, Julia Dunlop, Bharat Shimpukade,
Elisabeth Christiansen, Graeme Milligan, Claire J Stocker, and Trond Ulven
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00685 • Publication Date (Web): 29 Aug 2016
Downloaded from http://pubs.acs.org on August 30, 2016
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NonꢀAcidic Free Fatty Acid Receptor 4 Agonists
with Antidiabetic Activity
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Carlos M. G. Azevedo†, Kenneth R. Watterson‡, Ed T. Wargent§, Steffen V. F. Hansen†, Brian D.
Hudson‡, Małgorzata A. Kępczyńska§, Julia Dunlop‡, Bharat Shimpukade†, Elisabeth
Christiansen†, Graeme Milligan‡, Claire J. Stocker§, and Trond Ulven†,*
†Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej
55, DKꢀ5230 Odense M, Denmark. ‡Institute of Molecular, Cell and Systems Biology, College
of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland,
U.K. §Buckingham Institute for Translational Medicine, University of Buckingham, Hunter
Street, Buckingham MK18 1EG, U.K.
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ABSTRACT. The free fatty acid receptor 4 (FFA4 or GPR120) has appeared as an interesting
potential target for the treatment of metabolic disorders. At present, most FFA4 ligands are
carboxylic acids that are assumed to mimic the endogenous longꢀchain fatty acid agonists. Here,
we report preliminary structureꢀactivity relationship studies of a previously disclosed nonꢀacidic
sulfonamide FFA4 agonist. Mutagenesis studies indicate that the compounds are orthosteric
agonists despite the absence of a carboxylate function. The preferred compounds showed full
agonist activity on FFA4 and complete selectivity over FFA1, although a significant fraction of
these nonꢀcarboxylic acids also showed partial antagonistic activity on FFA1. Studies in normal
and dietꢀinduced obese (DIO) mice with the preferred compound 34 showed improved glucose
tolerance after oral dosing in an oral glucose tolerance test. Chronic dosing of 34 in DIO mice
resulted in significantly increased insulin sensitivity and a moderate but significant reduction in
bodyweight, effects that were also present in mice lacking FFA1 but absent in mice lacking
FFA4.
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INTRODUCTION
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The free fatty acid receptor 4 (FFA4, also known as GPR120) has in recent years appeared as a
new potential target for the treatment of metabolic diseases.1ꢀ7 The receptor was proposed as a
possible antidiabetic and antiobesity target for the first time in 2005 when it was reported to be
expressed in the intestinal tract and activated by dietary free fatty acids to stimulate incretin
secretion,8 although this property of the receptor remains controversial.9 FFA4 was subsequently
found also to be expressed in macrophages, liver and adipose tissue, and to mediate antiꢀ
inflammatory and insulin sensitizing effects.10 The report that mice lacking FFA4 develop
obesity, insulin intolerance and fatty liver when fed a highꢀfat diet and that a human population
with a dysfunctional FFA4 variant has an increased risk of obesity supported a significant role of
the receptor in metabolic diseases.11 Further support for this was recently provided by the finding
that the selective FFA4 agonist Cpd A (Chart 1) increased insulin sensitivity and reduced
inflammation and hepatic steatosis in mice fed a highꢀfat diet.12 Moreover, FFA4 is implicated in
regulation of glucagon, ghrelin and somatostatin release, representing likely contributing
mechanisms of the observed metabolic phenotype.13ꢀ16
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Chart 1. Representative FFA4 agonists
Although unsaturated and, in particular, omegaꢀ3 fatty acids were highlighted in the initial
publications,8,10 it has become clear that FFA4 is activated by longꢀchain fatty acids with a
profile that overlaps extensively with those that activate the more established antidiabetic target
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free fatty acid receptor 1 (FFA1/GPR40).17 In 2012, we disclosed TUGꢀ891 (1, Chart 1) as the
first potent and selective FFA4 agonist, optimized from a series of FFA1 agonists originally
derived from fatty acids.18,19 However, the significantly higher potency of 1 at the murine (m)
FFA1 resulted in a selectivity of 70ꢀfold in a βꢀarrestinꢀ2ꢀbased assay and of only 3ꢀfold in a
calcium assay for mFFA4 over mFFA1, essentially rendering the compound a dual agonist in
mice, at least with respect to signals and functions mediated by the induced elevation of Ca2+.20
In 2008, a patent from Banyu disclosed a series of nonꢀacidic benzosultams as FFA4 agonists
structurally distinct from other known FFA4 agonists.21 The structures were also markedly
different from known FFA1 ligands and appeared to represent an opportunity to access FFA4
agonists with complete selectivity over FFA1. More recently, Sparks and coꢀworkers reported a
series of sulfonamide FFA4 agonists with GSK1237647A (2, Chart 1) as the preferred compound
with pEC50 = 6.3 and a lack of activity on FFA1. However, this compound was also described as
unsuitable for in vivo studies due to poor solubility.22 Herein, we report structureꢀactivity
relationship studies around these compound series, leading to the identification of a full FFA4
agonist with complete selectivity over FFA1. We further demonstrate activity in vivo and report
beneficial results on glucose regulation, insulin sensitivity and bodyweight of the selected
compound in DIO mice.
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SYNTHESIS
The initial compounds were synthesized by nucleophilic aromatic substitution at 1,3ꢀdifluoroꢀ
5ꢀnitrobenzene, reduction of 3aꢀc to anilines 4aꢀc, formation of the sulfonamides 5aꢀc, reduction
(6aꢀc) and cyclization to provide sultams 7ꢀ9, essentially following the synthetic strategy
described in Banyu’s patent (Scheme 1).21 Aniline 4a was also substrate for the synthesis of
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phthalimide 12 and for the acyclic sulfonamides 13 and 14. Acyclic sulfonamides, including 15ꢀ
19, were synthesized similarly from the corresponding sulfonyl chlorides and anilines.
Scheme 1. Synthesis of compounds using a previously described routea,21
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o
aReagents and conditions: (a) K2CO3, DMF, 100 C, 16 h (30ꢀ68%). (b) Y = O: NH4HCO2,
Pd/C, EtOH, 90 oC (ꢀν), 10 min (quant.); Y = CH2O: SnCl2, MeCN/EtOH (1:1), refl., 1 h (86%).
(c) Pyridine, rt, 16 h (50ꢀ96%). (d) LiAlH4, THF, 0 oC, 0.5–2 h (48ꢀ95%); e) PBr3, THF, 0 oC to
rt, 0.5 h; Na2CO3 (sat. aq.), 1 h (44ꢀ83%). (f) phtalic anhydride, acetic acid, 100 oC, 16 h (68%);
(g) MeI, NaH, DMF, 0 oC to rt, 1 h (65ꢀ78%).
Since this synthetic route to the sultam is cumbersome and unsuitable for variations in the
eastern part of the structure, a more straightforward coupling procedure was developed. Thus, the
benzosultam intermediate 20, obtained from saccharin, was subject to copper(I)ꢀcatalyzed crossꢀ
coupling with 4ꢀiodophenol followed by Ullmann coupling to give 22, with 1ꢀbromoꢀ3ꢀ
phenoxybenzene to give 23, and with 3ꢀbromoꢀ5ꢀfluorophenol to give the central intermediate 25
(Scheme 2). Further Ullmann condensation of 25 with aryl bromides or iodides provided the
target compounds (26ꢀ34). The monocyclic 24 was synthesized from 3ꢀphenoxyaniline and 3ꢀ
chloropropanesulfonyl chloride (Scheme 3). Analogues with the central fluoro substituent
removed (36) or replaced by chloro (38) were synthesized by nucleophilic aromatic substitution
of 2ꢀfluoropyridine with the respective halophenols followed by copper(I)ꢀcatalyzed crossꢀ
coupling with 20 (Scheme 4).
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Scheme 2. Synthesis of Nꢀarylbenzosultams from saccharina
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aReagents and conditions: (a) LiAlH4, THF, 0 C to rt, 16 h (78%). (b) Aryl halide (4ꢀ
iodophenol, 1ꢀbromoꢀ3ꢀphenoxybenzene or 3ꢀbromoꢀ5ꢀfluorophenol), CuI, DMEDA, K2CO3,
MeCN, 70 oC, 16 h (65ꢀ96%). (c) Aryl halide (Br or I), picolinic acid, CuI, K3PO4, DMSO, 90 oC
(12ꢀ83%), 24 h.
Scheme 3. Synthesis of sultam 24a
aReagents and conditions: (a) 3ꢀChloropropanesulfonyl chloride, pyridine, CH2Cl2, rt, 16 h. (b)
K2CO3, DMF, 50 °C, 16 h (72% over two steps).
Scheme 4. Synthesis of nonꢀfluorinated analoguesa
aReagents and conditions: (a) K2CO3, DMF, 140 C (ꢀν), 4ꢀ5½ h (62ꢀ65%). b) 20, CuI,
o
DMEDA, K2CO3, MeCN, 70 oC, 16 h (61ꢀ88%).
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RESULTS AND DISCUSSION
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The compounds were screened on human (h) FFA4 in a βꢀarrestinꢀ2 recruitment assay and
compounds of particular interest were further tested in a Ca2+ mobilization assay and on
hFFA1.18 The sultam 7, disclosed as an FFA4 agonist with pEC50 of 6.74 in the Banyu patent,21
showed somewhat lower activity in our assays with a pEC50 of 6.36 in the βꢀarrestinꢀ2 assay and
6.52 in the Ca2+ assay (Table 1). Notably, the compound did not show any activity on FFA1 at
concentrations up to 100 ꢀM. The FFA1 agonist 1023 was found to also act as a full FFA4
agonist (see the Supporting Information),18 and has been used herein as a reference compound.
Table 1. Initial exploration of the pyridine ring of 7
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Code
7
R
hFFA4
hFFA1
Ca2+ pEC50
Ca2+ pIC50
βꢀArr. pEC50
b
c
a
(Emax
)
(Imax)
(Emax
)
6.36 ± 0.05 (107) 6.52 ± 0.09 (115)
< 4
6.63 ± 0.18 (101) 6.42 ± 0.13 (78)
5.41 ± 0.01 (47)
5.61 ± 0.63 (36)
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d
<4
–
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BRETꢀbased βꢀarrestinꢀ2 recruitment based FFA4 assay. Efficacy (Emax) is relative to 100 ꢀM
10 (3ꢀ(4ꢀ(oꢀtolylethynyl)phenyl)propanoic acid, TUGꢀ424).23 bCa2+ FFA4 assay. Efficacy (Emax
)
is relative to 100 ꢀM 10. Tested in the FFA1 Ca2+ assay as agonists and antagonists. Imax
denotes % reduction of the response of 300 nM 10. All compounds were inactive as FFA1
agonists. dNot tested.
c
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Studies with 7 on FFA4 mutants directed at the orthosteric binding site revealed that the
activity depends critically on Arg992.64, the residue identified as the key anchoring point for the
carboxylate group of free fatty acids and orthosteric ligands such as 1.24 This observation was
initially surprising in light of the distinctly different structure and the lack of any acidic group.
Compound 7 was also affected by other mutations that impact the activity of 1 and αꢀlinolenic
acid, strongly suggesting that 7 also binds to the orthosteric site (Table S1).24 Thus, mutations
that eliminate or significantly reduce the activity of αꢀlinolenic acid and 1 (W104A, F1153.29A,
W2075.38A, F2115.42A, W2776.48A and F3047.36A eliminate activity of both, F882.53A, I2845.66A,
F3037.35H and T3107.42A significantly reduce activity of both αꢀlinolenic acid and 1, T1193.33A,
I1263.40A, N2155.46A, I2806.51A, I2816.52F only reduce the activity of 1, see Table S1) also affect
the activity of 7 in a similar manner. Exceptions were W1002.65A and L1143.28A, which affected
1 but not 7, as well as V2125.43A, and F3117.43A, which each significantly decreased potency of
7, while only producing smaller nonꢀsignificant decreases in the potency of 1. Computational
modeling indicated favored alternative docking poses in the orthosteric site of FFA4 with 7
directly interacting with Arg992.64 by hydrogen bonds either to the sultam oxygen atoms or to the
pyridyl nitrogen. To investigate the most likely orientation, the phenyl analogue 8, lacking the
pyridyl interaction possibility, was synthesized and found more potent on FFA4 than 7,
indicating the sulfonamide as the Arg992.64 interaction point (Figure 1).
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Figure 1. Docking pose for 7 in a homology model of hFFA4. Residues where mutation
significantly affects the functional activity of 7 have been displayed.
To probe the importance of the sulfonamide moiety, phthalimide 12, also exemplified in the
Banyu patent but without activity data,21 was produced and found to have 10ꢀfold lower activity
than 7 and low partial efficacy, indicating that the sulfonamide is important for agonist activity
but that replacement by other hydrogen bond acceptors is possible. The acyclic sulfonamide 13,
containing a weakly acidic group (calculated pKa 7.7)25 exhibited further reduced activity.
Interestingly, Nꢀmethylation to give the nonꢀacidic ringꢀopened analogue 14 resulted in complete
loss of activity, possibly reflecting that planarity between the aromatic ring and the SꢀN bond is a
requirement.
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Table 2. Lead structures and western part variations
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hFFA1
Code
Structure
hFFA4
7
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BRET pEC50
Ca2+ pEC50
Ca2+ pIC50
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b
c
(Emax
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(Emax
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(Imax)
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d
d
5.30 ± 0.08 (45)
4.78 ± 0.15 (39)
nre
–
–
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14
d
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nre
d
–
5.27 ± 0.36 (76)
5.45 ± 0.01 (52)
nrh
nrh
5.09 ± 0.15 (68)
4.89 ± 0.10 (95)
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nre
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nre
–
4.97 ± 0.15 (57)
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2
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(Chart 1)
–
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nre
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nre
nre
nre
5.00 ± 0.09 (100)
nre
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22
d
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6.61 ± 0.07 (95) 6.68 ± 0.06 (105)
6.76 ± 1.34 (39)
nre,f
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24
nre
nre
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aBRETꢀbased βꢀarrestinꢀ2 recruitment based FFA4 assay. Efficacy (Emax) is relative to 10.
c
bCa2+ FFA4 assay. Efficacy (Emax) is relative to 10. Tested in the FFA1 Ca2+ assay as agonists
and antagonists. All compounds were inactive as FFA1 agonists. Imax denotes % reduction of the
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d
e
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response of 300 nM 10. Not tested. No response up to 100 ꢀM. Tested as antagonist in the
FFA1 Ca2+ mobilization assay with 20 nM of 11 (3ꢀ(4ꢀ((2ꢀ(cyanomethyl)phenyl)ethynyl)ꢀ
phenyl)propanoic acid, TUGꢀ770).26
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Related to 13, the sulfonamide DC260126 (15) has been described as an FFA1 antagonist and
was, somewhat surprisingly, reported to improve insulin sensitivity and βꢀcell function in rats
and db/db mice.27,28 We confirmed its FFA1 antagonist activity (pIC50 = 5.09) but found that the
compound also acts as an FFA4 agonist in the βꢀarrestinꢀ2 recruitment assay although it was
devoid of activity in the Ca2+ assay (Table 2), and thus represents a relatively lowꢀpotency, but
completely βꢀarrestinꢀbiased, FFA4 agonist. Since FFA4 is proposed to mediate antiꢀ
inflammatory effects via the βꢀarrestin pathway, it cannot be excluded that the tendency to
insulin sensitization observed with this compound is mediated by FFA4.
Replacing the butyl of 15 by phenoxy (16) gave a compound with similar FFA1 antagonistic
and FFA4 agonistic properties. NꢀMethylation of the potentially acidic sulfonamide (17) led to
loss of FFA4 activity but preserved FFA1 antagonism. The related sulfonamide 2 (Chart 1) was
recently reported as a selective FFA4 agonist.22 We confirmed a pEC50 of 6.31 in the βꢀarrestinꢀ2
assay. Replacement of the paraꢀfluoro of 16 with methoxy (18) akin to 2 resulted in complete
loss of activity, as did paraꢀphenoxyphenyl sultam 22, whereas the moderate FFA1ꢀantagonistic
activity was maintained with a paraꢀmethyl substituent (19). Interestingly, removing the metaꢀ
fluoro of the phenyl analogue 8 resulted in a compound (23) with preserved potency in both the
βꢀarrestinꢀ2 and calcium assay on FFA4 as well as preserved partial antagonist activity on FFA1,
whereas removing the benzene ring fused to the sultam (24) resulted in a compound devoid of
activity on both FFA4 and FFA1, indicating a critical role of this ring.
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Explorations of the western part of the structure suggested that the benzosultam system was
favored. Turning attention back to the eastern site, the eastern phenyl ring of 8 was scanned with
the nonꢀpolar methyl substituent (26, 27, 28) and the polar cyano (29ꢀ31) and metaꢀcyanomethyl
(32) substituents, in all cases these resulted in decreased potency with paraꢀsubstituents being
somewhat better tolerated (Table 3).
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Table 3. Exploration of the central and eastern parts
Code
26
R
X
hFFA4
hFFA1
Ca2+ pEC50
Ca2+ pIC50
βꢀArr. pEC50
b
c
a
(Emax
)
(Imax)
(Emax
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F
5.57 ± 0.10 (42)
nrd
4.35 ± 0.23 (71)
F
F
5.58 ± 0.24 (87)
6.36 ± 0.09 (91)
5.35 ± 0.17 (14)
5.96 ± 0.20 (38)
4.60 ± 0.16 (51)
5.54 ± 0.12 (52)
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e
F
4.79 ± 0.27 (76)
–
<4
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e
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F
5.24 ± 0.22 (83)
5.78 ± 0.09 (87)
–
5.47 ± 0.94 (52)
6.21 ± 0.66 (43)
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31
5.63 ± 0.16 (29)
e
F
F
5.28 ± 0.10 (57)
5.90 ± 0.05 (97)
–
4.70 ± 0.08 (44)
<4
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33
5.77 ± 0.16 (120)
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F
6.91 ± 0.04 (98)
5.81 ± 0.11 (102)
6.63 ± 0.13 (130)
<4
<4
<4f
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H
5.99 ± 0.19 (93)
7.41 ± 0.18 (78)
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Cl 6.86 ± 0.10 (113)
aBRETꢀbased βꢀarrestinꢀ2 recruitment based FFA4 assay. Efficacy (Emax) is relative to 100 ꢀM
b
c
10. Ca2+ FFA4 assay. Efficacy (Emax) is relative to 100 ꢀM 10. Tested in the FFA1 Ca2+ assay
as agonists and antagonists. All compounds were inactive as FFA1 agonists. Imax denotes %
d
e
f
reduction of the response of 300 nM 10. No response up to 100 ꢀM. Not tested. Tested as
antagonist in the FFA1 Ca2+ mobilization assay with 20 nM 11.26
Further investigation of 8 revealed antagonistic activity on FFA1, but without the ability to
completely block the agonist response (i.e. “partial antagonism”, Table 1). A similar effect was
seen with all substituted analogues exhibiting noticeable agonistic activity (EC50 <10 ꢀM) on
FFA4. These results are surprising because they indicate that this compound series also has the
ability to interact with and modulate FFA1 despite its structural dissimilarity from other FFA1
agonists and the absence of an acidic functional group. However, the inability of the compounds
to completely block activity of 10 suggests nonꢀcompetitive binding and allosteric interaction
with FFA1.
In contrast to the phenoxy compounds, the pyridines 7, 33, 13, 14 did not show any sign of
agonistic or antagonistic activity on FFA1. These structures were therefore explored further by
altering the attachment point at the pyridine. Thus, the 4ꢀpyridyl (33) derivative resulted in a 3ꢀ
fold reduced potency, whereas the 2ꢀpyridyl (34) gratifyingly showed a 3.5ꢀfold increased
potency to achieve an EC50 of 128 nM.
Finally, the importance of the metaꢀfluoro substituent at the central ring of 34 was assessed.
Removal of the fluoro substituent in 36 resulted in a 10ꢀfold reduced potency and indicated,
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contrary to 8 vs 23, that the metaꢀfluoro contributes to the potency of 34. In contrast, substitution
of fluoro by chloro in 38 led to preserved activity in the βꢀarrestinꢀ2 recruitment assay and an
EC50 of 40 nM in the Ca2+ assay, a 6ꢀfold increase in potency relative to 34, albeit with reduced
efficacy.
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Overall, 34 and 38 stood out as the most potent FFA4 agonists in both the βꢀarrestinꢀ2 and the
Ca2+ assays with essentially equal activity in the former. Although 38 appeared more potent in
the Ca2+ assay, the compound showed only partial agonist activity, whereas 34 was a full agonist
in both assays as well as less lipophilic (ꢁClogP ~0.5). Compound 34 showed no activity at the
other free fatty acid receptors FFA1, FFA2 or FFA3 at up to 30 ꢀM, indicating >300ꢀfold
selectivity. Experimental log D7.4 measurements showed identical lipophilicity for 34 and 7 (3.17
and 3.12, respectively). Solubility studies for compounds 7, 34 and 38 in PBS indicated
progressively lower solubility with 38 being virtually insoluble (11, 1.3 and 0.1 ꢀM,
respectively), however, the solubility of the two latter in fasting state simulated intestinal fluid
(FaSSIF) gave a somewhat better picture for 34 (14 ꢀM for 34 and 0.9 ꢀM for 38). Thus, 34 was
selected for in vivo studies. The low solubility of the compound did not cause any problems in
relation to these. Prior to the mouse studies, the activity and specificity of 34 and the two other
most potent and selective agonists, 7 and 38, on the mFFA4 and mFFA1 were investigated.
Completely preserved activity was confirmed for all three compounds in the Gqꢀdependent Ca2+
mobilization assay on mFFA4 (pEC50 = 7.08 ± 0.05, 6.83 ± 0.08 and 7.14 ± 0.08 for 7, 34 and
38, respectively), with no activity detected up to 100 ꢀM on mFFA1. Further, 34 was also tested
in the βꢀarrestin assay, confirming full agonistic activity with pEC50 = 6.32 ± 0.06, again with no
sign of activity on mFFA1. Compound 34 also failed to induce a response when tested on the
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FFA4 R992.64Q mutant, supporting the notion that this compound binds as indicated for 7 in
Figure 1.
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FFA4 has been reported to mediate free fatty acid promoted incretin release and to improve
glucose tolerance,8,12 and mice lacking FFA4 show impaired glucose homeostasis, an effect
believed to involve improper regulation of glucagon secretion.14 Thus, the effect of 34 as well as
7 in a glucose tolerance test was investigated by oral dosing prior to an oral glucose challenge.
This resulted in significant lowering of plasma glucose levels for both compounds compared to
vehicle (Figure 2).
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Figure 2. FFA4 agonists 34 and 7 lower plasma glucose concentrations compared to vehicle
after oral dosing at 10 mg/kg 15 min before oral glucose challenge (n = 6 mice per group,
* p≤0.05; ** p≤0.01, *** p≤0.001, twoꢀway ANOVA with Bonferoni post hoc test).
FFA4 has attracted high interest as a potential antidiabetic target in particular because of
results indicating that the receptor mediates insulin sensitization, antiꢀinflammatory effects,
protection of pancreatic islets, and that it may even counteract obesity.3ꢀ6,10,11,29 However, each of
these is difficult to assess satisfactorily in acute treatment studies. To evaluate the effects of 34
on some of these, a chronic treatment study in DIO mice was performed with daily dosing of 10
mg/kg 34 over a three week period. Mice lacking FFA4 were included to assess and confirm
receptor specific activity of the ligand. The wildꢀtype mice were littermates of the FFA4(ꢀ/ꢀ)
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animals. As observed in normal mice, a glucose challenge 15 min after dosing in DIO mice on
the first day significantly reduced plasma glucose levels in 34ꢀtreated wildꢀtype mice compared
to vehicleꢀtreated wildꢀtype mice, whereas 34 had no effect on glucose levels in the FFA4(ꢀ/ꢀ)
animals (Figure 3, top). The effect was significant in the wildꢀtype animals also when calculated
relative to t = ꢀ30 min (see the Supporting Information). A significantly increased insulin level
was observed 30 min after challenge for the wildꢀtype mice treated with 34 relative to vehicle
treated wildꢀtype mice, whereas the difference was absent in mice lacking FFA4 (Figure 3,
bottom). This confirms that the effect of 34 on glucose excursion when dosed 15 minutes before
the challenge is mediated by FFA4. Studies to investigate the mechanism of this effect are in
progress.
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Figure 3. Plasma glucose levels (top) and insulin levels at t = 30 min (bottom) after an acute oral
glucose tolerance test 15 min after dosing with 34 or vehicle in DIO wildꢀtype and DIO FFA4(ꢀ/ꢀ
) mice on day 0 (n=9 mice per group; * p=<0.05, ** p=<0.01, *** p<0.001, twoꢀway ANOVA
with Bonferroni post hoc test).
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After treatment over 21 days, the fasting insulin levels of the 34 treated wildꢀtype DIO mice
was significantly reduced compared to vehicle treated mice, whereas no difference was observed
between the FFA4(ꢀ/ꢀ) groups (Figure 4, top). These results indicate that 34 promotes insulin
sensitization in mice through activation of FFA4. An oral glucose tolerance test on day 21
(Figure 4, bottom) showed an even more robust effect than on day 0, presumably an effect of
increased insulin sensitization.
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Figure 4. Fasting insulin levels after chronic dosing with 34 for 21 days (top) and oral glucose
tolerance test at day 21 (bottom) (n=9, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001, oneꢀ
way ANOVA with Dunnett’s post hoc test).
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Previous studies have linked FFA4 to obesity by demonstrating a correlation between
expression and obesity and have indicated an increased risk of obesity with p.R270H FFA4
variant in European populations or with deletion of the receptor in mouse,5,11 although a recent
study failed to find a similar association in a Danish population.30 No significant effect was
observed on either food or water intake over the course of dosing with 34 for 21 days in either
the wildꢀtype or FFA4(ꢀ/ꢀ) animals. Treatment with 34 did, however, result in a reduction in
bodyweight in the wildꢀtype mice of 3ꢀ4 grams (7ꢀ9%) towards the end of the study, whereas no
effect, or even a weak trend towards weight gain, was observed in the FFA4(ꢀ/ꢀ) animals (Figure
5). These results support the notion that FFA4 may represent a potential antiꢀobesity target, an
effect that would be an important addꢀon to regulation of glucose homeostasis in an antidiabetic
drug.
Figure 5. Bodyweight change in DIO wildꢀtype and FFA4(ꢀ/ꢀ) mice over 21 days of once daily
dosing (10 mg/kg, po) with 34 (n=9 mice per group, * p≤0.05; twoꢀway ANOVA with
Bonferroni post hoc test).
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CONCLUSION
Most currently known FFA4 agonists are carboxylic acids assumed to mimic the endogenous
free fatty acid agonists. Most of these also exhibit some degree of activity on FFA1. In the search
for highly selective FFA4 agonists, we investigated a nonꢀacidic benzosultam ligand known from
the patent literature and conducted a preliminary structureꢀactivity relationship study around this
scaffold. Interestingly, mutagenesis studies indicated that these compounds also bind to the same
site as αꢀlinolenic acid and 1 and also require carboxylate interaction partner Arg992.64 for
activity despite the absence of a carboxylate. Given the limited overall homology between FFA1
and FFA4, even though they are both activated by overlapping sets of mediumꢀ and longꢀchain
free fatty acids, it was surprising that a subꢀseries of these compounds also interacted with FFA1
to produce partial blockade of this receptor. Compound 34, a full FFA4 agonist with comparable
potency in both Ca2+ꢀ and βꢀarrestinꢀ2ꢀbased assays, showed complete selectivity over FFA1 and
other free fatty acid receptors. Its distinctly different structure from the carboxylic acid FFA4
agonists currently used as tool compounds makes 34 a suitable orthogonal tool in combination
with these for further studies of the function of FFA4. The compound was investigated in vivo
and was found to lower plasma glucose levels after an oral glucose challenge when dosed orally
15 minutes prior to the challenge, an effect that was confirmed to be mediated by FFA4 as this
was not observed in animals lacking FFA4. Furthermore, 34 robustly increased insulin sensitivity
after chronic dosing in DIO mice and led to moderately reduced bodyweight over the course of
the study, despite no significant effects on food or water intake. Altogether, 34 is a nonꢀacidic
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full agonist of FFA4 with complete selectivity over FFA1 that shows antidiabetic and antiꢀ
obesity effects after chronic oral dosing in DIO mice, corroborating FFA4 as a promising new
antidiabetic target.
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EXPERIMENTAL SECTION
Synthesis
Commercial starting materials and solvents were used without further purification. THF was
freshly distilled from sodium/benzophenone. Water was filtered and demineralized (MilliꢀQ).
TLC was performed on TLC Silica gel 60 F254 plates and visualized at 254 nm and/or by
staining with phosphomolybdic acid, vanillin, or KMnO4 stains. Petroleum ether (PE) refers to
o
alkanes with bp 60ꢀ80 C. Microwave reactions were performed in a Biotage Initiator+
microwave reactor. Purification by flash chromatography was carried out using silica gel 60
1
(0.040ꢀ0.063 mm, Merck). H and 13C NMR spectra were recorded at 400 and 101 MHz,
respectively, on Bruker Avance III 400 at 300 K. Highꢀresolution mass spectra (HRMS) were
obtained on a Bruker micrOTOFꢀQ II (ESI). Purity was determined by HPLC and confirmed by
inspection of NMR spectra. HPLC analysis was performed using a Dionex 120 C18 or a Gemini
C18 column (5 ꢁm, 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 0.05% TFA
or 0.1% HCOOH as modifier; UV detection at 254 nm. All test compounds were of ≥95% purity
unless otherwise stated.
2ꢀ(4ꢀHydroxyphenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (21). To 20 (250 mg, 1.48
mmol), CuI (42 mg, 0.22 mmol), 4ꢀiodophenol (390 mg, 1.77 mmol) and anhydrous K2CO3 (615
mg, 4.45 mmol) under argon were added dry and freshly degassed acetonitrile (5 mL) and
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DMEDA (80 µL, 0.75 mmol). The reaction mixture was heated at 70 ºC for 16 h. The reaction
was then quenched with aqueous HCl (2N, 10 mL) and the aqueous phase was extracted with
EtOAc (3x). The combined organic phases were dried (Na2SO4) and concentrated. The crude
product was purified by flash chromatography (EtOAc/PE, 1:1) and the product obtained
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1
recrystallized from EtOAc/PE (1:1) to give 21 as a yellow solid (232 mg, 65%). H NMR (400
MHz, acetoneꢀd6): 8.51 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.78 (t, J = 7.6 Hz, 2H), 7.69 (t, J = 7.6
Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.93 (s, 2H). 13C NMR (100 MHz,
acetoneꢀd6): 156.8, 136.4, 134.5, 133.7, 130.2, 129.5, 125.94, 125.93, 121.8, 116.9, 51.9. HRMS
(ESI) calcd for C13H11NNaO3S [M+Na]+ 284.0352, found 284.0347. HPLC tR = 9.4 min, 99.6%.
2ꢀ(4ꢀPhenoxyphenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (22). To 21 (75 mg, 0.29
mmol), iodobenzene (48 µL, 0.43 mmol), copper iodide (11 mg, 0.06 mmol), picolinic acid (14.8
mg, 0.12 mmol) and K3PO4 (123 mg, 0.58 mmol) under argon was added dry DMSO (0.5 mL)
and the reaction mixture was heated at 90 ºC for 24 h. The temperature was then allowed to reach
rt and the reaction mixture was partitioned between water and EtOAc. The aqueous phase
extracted with EtOAc (2x) and the combined organic phases were washed with brine, dried
(Na2SO4) and concentrated. The crude product was purified by flash chromatography
(EtOAc/PE, 3:7) and recrystallized from EtOAc/PE (2:1) to give 22 as light yellow solid (80 mg,
83%). 1H NMR (400 MHz, CDCl3): 7.88 (d, J = 7.6 Hz, 1H), 7.66 (td, J = 7.6, 0.9 Hz, 1H), 7.59
(t, J = 7.6 Hz, 1H), 7.49 – 7.44 (m, 3H), 7.35 (t, J = 8.0 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 7.12
13
(d, J = 8.9 Hz, 2H), 7.12 (d, J = 7.7 Hz, 2H), 4.84 (s, 2H). C NMR (100 MHz, CDCl3): 157.1,
155.2, 135.0, 133.0, 132.5, 131.6, 129.9, 129.5, 124.4, 123.5, 123.5, 121.6, 120.0, 118.9, 50.8.
HRMS (ESI) calcd for C19H15NNaO3S [M+Na]+ 360.0665, found 360.0653. HPLC tR = 12.3
min, 99.7%.
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2ꢀ(3ꢀPhenoxyphenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (23). The title compound
was obtained as described for 21 using 20 (100 mg, 0.59 mmol) and 3ꢀbromodiphenylether (129
µL, 0.71 mmol) as starting materials. The crude product was purified by flash chromatography
(EtOAc/PE, 3:7) and the product recrystallized from EtOAc/PE (2:1) to give 23 as a white solid
(175 mg, 88%). 1H NMR (400 MHz, CDCl3): 7.86 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H),
7.58 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.39 – 7.31 (m, 3H), 7.27 (dd, J = 8.2, 1.3 Hz,
1H), 7.13 (t, J = 7.4 Hz, 1H), 7.09 – 7.02 (m, 3H), 6.78 (dd, J = 8.2, 1.3 Hz, 1H), 4.82 (s, 2H).
13C NMR (100 MHz, CDCl3): 158.5, 156.7, 138.5, 135.0, 133.2, 131.9, 130.7, 129.9, 129.5,
124.5, 123.7, 121.6, 119.2, 114.3, 113.5, 109.4, 49.5. HRMS (ESI) calcd for C19H15NNaO3S
[M+Na]+ 360.0665, found 360.0655. HPLC tR = 12.3 min, >99.9%.
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2ꢀ(3ꢀPhenoxyphenyl)isothiazolidine 1,1ꢀdioxide (24). Step 1: To a solution of 3ꢀ
phenoxyaniline (501 mg, 2.71 mmol) in dry CH2Cl2 (10 mL) was added pyridine (0.57 mL, 7.02
mmol) and 3ꢀchloropropanesulfonyl chloride (0.4 mL, 3.24 mmol) at room temperature. The
reaction was stirred for 16 h, then diluted with CH2Cl2 and washed successively with HCl (1N,
aq) and NaHCO3 (sat. aq.), dried (Na2SO4) and concentrated to give 3ꢀchloroꢀNꢀ(3ꢀ
phenoxyphenyl)propaneꢀ1ꢀsulfonamide as a crude yellow oil that was used directly in the next
step. Step 2: To a solution of 3ꢀchloroꢀNꢀ(3ꢀphenoxyphenyl)propaneꢀ1ꢀsulfonamide in DMF (15
mL) was added K2CO3 (963 mg, 6.97 mmol) and stirred at 50 °C for 16 h. The reaction mixture
was cooled to room temperature, diluted with water and extracted with EtOAc (3x). The
combined organic phases were washed with water and brine, dried (Na2SO4) and concentrated.
The crude was purified by flash chromatography (EtOAc:PE, 1:1) to give 24 as a pale yellow oil
which solidified to an offꢀwhite solid after a week under high vacuum (596 mg, 72% over two
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steps): H NMR (400 MHz, CDCl3) δ 7.37 – 7.27 (m, 3H), 7.14 – 7.00 (m, 4H), 6.88 (t, J = 2.2
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Hz, 1H), 6.74 (dd, J = 8.2, 2.2 Hz, 1H), 3.74 (t, J = 6.5 Hz, 2H), 3.37 (t, J = 7.5 Hz, 2H), 2.52 (p,
J = 7.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 158.2, 156.9, 139.3, 130.5, 129.9, 123.7, 119.2,
114.3, 113.9, 109.7, 48.5, 46.8, 18.8; HRMS (ESI) m/z: calculated C15H15NNaO3S [M+Na+] =
312.0665, found 312.0663. HPLC: tR = 11.62 min, 99.5%.
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2ꢀ(3ꢀFluoroꢀ5ꢀhydroxyphenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (25). The title
compound was obtained as described for 21 using 20 (500 mg, 3 mmol) and 3ꢀbromoꢀ5ꢀ
fluorophenol (677 mg, 3.55 mmol) as starting materials. The crude product was purified by flash
chromatography (EtOAc/PE, 1:1) to give the desired compound 25 as a white solid (793 mg,
96%). 1H NMR (400 MHz, DMSOꢀd6): 10.23 (broad s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.82 (dt, J
= 7.6, 1.0 Hz, 1H), 7.71 – 7.67 (m, 2H), 6.80 (t, J = 1.9 Hz, 1H), 6.67 (dt, J = 11.1, 2.2 Hz, 1H),
13
6.39 (dt, J = 10.7, 2.2 Hz, 1H), 5.04 (s, 2H). C NMR (100 MHz, DMSOꢀd6): 163.4 (d, J =
240.6 Hz), 159.6 (d, J = 14.1 Hz), 139.2 (d, J = 13.9 Hz), 133.9, 133.7, 132.2, 129.6, 125.2,
121.0, 101.0 (d, J = 2.4 Hz), 98.0 (d, J = 23.8 Hz), 95.9 (d, J = 26.8 Hz). 49.0. HRMS (ESI)
calcd for C13H10FNNaO3S [M+Na]+ 302.0258, found 302.0256. HPLC tR = 10.3 min, 99.2%.
The spectra is in accordance with the literature.21
2ꢀ(3ꢀFluoroꢀ5ꢀ(oꢀtolyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (26). The
title compound was obtained as described for 22 using 25 (70 mg, 0.25 mmol) and 2ꢀ
bromotoluene (45 µL, 0.38 mmol) as starting materials. The crude product was purified by flash
chromatography (EtOAc/PE, 1:4) and recrystallized from EtOAc/PE (3:1) to give 26 as a white
1
solid (23 mg, 25%). H NMR (400 MHz, CDCl3): 7.86 (d, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz,
1H), 7.59 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.28 (d, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6
Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.93 (dt, J = 10.3, 1.9 Hz, 1H), 6.81
(s, 1H), 6.29 (dt, J = 10.0, 1.9 Hz, 1H), 4.80 (s, 2H), 2.22 (s, 3H). 13C NMR (100 MHz, CDCl3):
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164.1 (d, J = 245.6 Hz), 160.3 (d, J = 12.8 Hz), 153.1, 139.3 (d, J = 13.2 Hz), 134.8, 133.4,
131.8, 131.5, 130.4, 129.6, 127.5, 125.2, 124.5, 121.6, 120.6, 102.5 (d, J = 2.9 Hz), 99.6 (d, J =
25.5 Hz), 99.5 (d, J = 26.9 Hz), 49.3, 16.1. HRMS (ESI) calcd for C20H16FNNaO3S [M+Na]+
392.0727, found 392.0715. HPLC tR = 12.2 min, 99.2%.
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2ꢀ(3ꢀFluoroꢀ5ꢀ(mꢀtolyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (27). The
title compound was obtained as described for 22 using 25 (70 mg, 0.25 mmol) and 3ꢀiodotoluene
(48 µL, 0.38 mmol) as starting materials. The crude product was purified by flash
chromatography (EtOAc/PE, 3:7) and recrystallized from EtOAc/PE (3:1) to give 27 as a white
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solid (33 mg, 36%). H NMR (400 MHz, CDCl3): 7.86 (d, J = 7.7 Hz, 1H), 7.68 (t, J = 7.7 Hz,
1H), 7.59 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.26 (t, J = 7.7 Hz, 1H), 7.01 – 6.94 (m,
2H), 6.90 – 6.82 (m, 3H), 6.42 (dt, J = 9.8, 2.0 Hz, 1H), 4.80 (s, 2H), 2.36 (s, 3H). 13C NMR (100
MHz, CDCl3): 164.0 (d, J = 245.8 Hz), 160.0 (d, J = 12.8 Hz), 155.7, 140.3, 139.3 (d, J = 13.1
Hz), 134.8, 133.4, 131.5, 129.7, 129.6, 125.3, 124.5, 121.6, 120.4, 116.8, 103.8 (d, J = 2.9 Hz),
101.0 (d, J = 25.1 Hz), 100.0 (d, J = 26.9 Hz), 49.3, 21.4. HRMS (ESI) calcd for
C20H16FNNaO3S [M+Na]+ 392.0727, found 392.0716. HPLC tR = 13.1 min, 99.4%.
2ꢀ(3ꢀFluoroꢀ5ꢀ(pꢀtolyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (28). The
title compound was obtained as described for 22 using 25 (70 mg, 0.25 mmol) and 4ꢀiodotoluene
(82 mg, 0.38 mmol) as starting materials. The crude product was purified by flash
chromatography (EtOAc/PE, 3:7) and recrystallized from EtOAc/PE (3:1) to give 28 as a white
solid (24 mg, 26%). 1H NMR (400 MHz, CDCl3): 7.87 (d, J = 7.7 Hz, 1H), 7.68 (td, J = 7.7, 0.8
Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 2H), 7.00 – 6.92
13
(m, 3H), 6.84 (s, 1H), 6.41 (dt, J = 9.9, 2.0 Hz, 1H), 4.80 (s, 2H), 2.36 (s, 3H). C NMR (100
MHz, CDCl3): 164.0 (d, J = 245.7 Hz), 160.4 (d, J = 12.6 Hz), 153.2, 139.3 (d, J = 13.2 Hz),
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134.8, 134.2, 133.4, 131.5, 130.5, 129.6, 124.5, 121.6, 119.9, 103.4 (d, J = 3.0 Hz), 100.7 (d, J =
25.3 Hz), 99.9 (d, J = 27.0 Hz), 49.3, 20.8. HRMS (ESI) calcd for C20H16FNNaO3S [M+Na]+
392.0727, found 392.0714. HPLC tR = 13.1 min, 99.7%.
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2ꢀ(3ꢀ(1,1ꢀDioxidobenzo[d]isothiazolꢀ2(3H)ꢀyl)ꢀ5ꢀfluorophenoxy)benzonitrile (29). The title
compound was obtained as described for 22 using 25 (79 mg, 0.28 mmol) and 2ꢀ
bromobenzonitrile (40 mg, 0.22 mmol) as starting materials. The crude product was purified by
flash chromatography (EtOAc/PE, 1:4) and the title compound 29 was recrystallized from
EtOAc/PE (3:1) as a white solid (10 mg, 12%). 1H NMR (400 MHz, CDCl3): 7.88 (d, J = 7.7 Hz,
1H), 7.73ꢀ7.69 (m, 2H), 7.62 (t, J = 7.7 Hz, 1H), 7.57 (td, J = 7.7, 1.6 Hz, 1H), 7.51 (d, J = 7.7
Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.09 (dt, J = 10.2, 2.0 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.96
(s, 1H), 6.55 (dt, J = 9.1, 2.0 Hz, 1H), 4.85 (s, 2H). 13C NMR (100 MHz, CDCl3): 164.0 (d, J =
247.8 Hz), 158.2, 157.3 (d, J = 12.9 Hz), 139.9 (d, J = 12.7 Hz), 134.6, 134.5, 134.1, 133.6,
131.2, 129.8, 124.5, 124.1, 121.6, 118.2, 115.5, 104.8 (d, J = 3.2 Hz), 104.7, 102.4 (d, J = 25.3
Hz), 101.8 (d, J = 26.6 Hz). 49.3. HRMS (ESI) calcd for C20H13FN2NaO3S [M+Na]+ 403.0523,
found 403.0528. HPLC tR = 11.9 min, 99.5%.
3ꢀ(3ꢀ(1,1ꢀDioxidobenzo[d]isothiazolꢀ2(3H)ꢀyl)ꢀ5ꢀfluorophenoxy)benzonitrile (30). The title
compound was obtained as described for 22 using 25 (58 mg, 0.21 mmol) and 3ꢀiodobenzonitrile
(40 mg, 0.17 mmol) as starting materials. The crude product was purified by flash
chromatography (EtOAc/PE, 3:7) and recrystallized from EtOAc/PE (3:1) to give 30 as a white
1
solid (20 mg, 30%). H NMR (400 MHz, CDCl3): 7.88 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.5 Hz,
1H), 7.62 (t, J = 7.5 Hz, 1H), 7.53 – 7.43 (m, 3H), 7.35 – 7.28 (m, 2H), 7.00 (dt, J = 10.2, 2.0
Hz, 1H), 6.95 (s, 1H), 6.48 (dt, J = 9.3, 2.0 Hz, 1H), 4.84 (s, 2H). 13C NMR (100 MHz, CDCl3):
164.1 (d, J = 247.5 Hz), 158.0 (d, J = 12.7 Hz), 156.7, 139.9 (d, J = 12.8 Hz), 134.6, 133.6,
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131.2, 131.0, 129.8, 127.7, 124.5, 123.6, 122.2, 121.6, 118.0, 114.0, 104.5 (d, J = 3.1 Hz), 101.9
(d, J = 25.0 Hz), 101.2 (d, J = 26.9 Hz), 49.2. HRMS (ESI) calcd for C20H13FN2NaO3S [M+Na]+
403.0523, found 403.0523. HPLC tR = 12.2 min, 98.6%.
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4ꢀ(3ꢀ(1,1ꢀDioxidobenzo[d]isothiazolꢀ2(3H)ꢀyl)ꢀ5ꢀfluorophenoxy)benzonitrile (31). The title
compound was obtained as described for 22 using 25 (74 mg, 0.26 mmol) and 4ꢀ
bromobenzonitrile (40 mg, 0.22 mmol) as starting materials. The crude product was purified by
flash chromatography (EtOAc/PE, 1:3) and recrystallized from EtOAc/PE (3:1) to give 31 as a
white solid (37 mg, 45%). 1H NMR (400 MHz, CDCl3): 7.88 (d, J = 7.7 Hz, 1H), 7.71 (t, J = 7.7
Hz, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.63 (t, J = 7.7 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.11 (d, J =
8.8 Hz, 2H), 7.03 (dt, J = 10.2, 2.0 Hz, 1H), 6.97 (s, 1H), 6.55 (dt, J = 9.1, 2.0 Hz, 1H), 4.84 (s,
2H). 13C NMR (100 MHz, CDCl3): 164.1 (d, J = 248.0 Hz), 160.2, 157.2 (d, J = 12.9 Hz), 140.0
(d, J = 12.8 Hz), 134.6, 134.4, 133.6, 131.2, 129.8, 124.5, 121.7, 118.8, 118.5, 107.2, 105.1 (d, J
= 3.1 Hz), 102.8 (d, J = 24.8 Hz), 101.6 (d, J = 26.7 Hz), 49.2. HRMS (ESI) calcd for
C20H13FN2NaO3S [M+Na]+ 403.0523, found 403.0503. HPLC tR = 12.2 min, 99.2%.
2ꢀ(3ꢀ(3ꢀ(1,1ꢀDioxidobenzo[d]isothiazolꢀ2(3H)ꢀyl)ꢀ5ꢀfluorophenoxy)phenyl)acetonitrile
(32). The title compound was obtained as described for 22 using 25 (70 mg, 0.25 mmol) and 3ꢀ
bromophenylacetonitrile (40 mg, 0.38 mmol) as starting materials. The crude product was
purified by flash chromatography (EtOAc/PE, 3:7) and the title compound 32 was recrystallized
from EtOAc/PE (3:1) as an orange solid (20 mg, 20%). 1H NMR (400 MHz, CDCl3): 7.86 (d, J =
7.7 Hz, 1H), 7.69 (td, J = 7.7, 1.0 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.40
(td, J = 1.6, 7.6 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.06 – 7.02 (m, 2H), 6.99 (dt, J = 10.2, 2.1 Hz,
1H), 6.89 (s, 1H), 6.46 (dt, J = 9.6, 2.1 Hz, 1H), 4.82 (s, 2H), 3.76 (s, 2H). 13C NMR (100 MHz,
CDCl3): 164.0 (d, J = 246.6), 159.0 (d, J = 12.8), 156.6, 139.5 (d, J = 13.2), 134.7, 133.5, 132.1,
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131.4, 130.8, 129.7, 124.5, 123.7, 121.6, 119.09, 119.08, 117.5, 104.0 (d, J = 3.1), 101.5 (d, J =
25.0), 100.6 (d, J = 26.8), 49.3, 23.5. HRMS (ESI) calcd for C21H15FN2NaO3S [M+Na]+
417.0680, found 417.0667. HPLC tR = 12.0 min, 99.5%.
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2ꢀ(3ꢀFluoroꢀ5ꢀ(pyridinꢀ4ꢀyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (33).
The title compound was obtained as described for 22 using 25 (79 mg, 0.28 mmol) and 4ꢀ
bromopyridine hydrochloride (46 mg, 0.22 mmol) as starting materials. The crude product was
purified by flash chromatography (EtOAc/PE, 9:1) to provide 33 as a white solid (34 mg, 40%).
1H NMR (400 MHz, CDCl3): 8.54 (d, J = 3.9 Hz, 2H), 7.88 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.5
Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 10.2 Hz, 1H), 6.97 (s,
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1H), 6.93 (d, J = 5.5 Hz, 2H), 6.61 (d, J = 9.0 Hz, 1H), 4.84 (s, 2H). C NMR (100 MHz,
CDCl3): 164.0 (d, J = 248.0), 163.6, 156.2 (d, J = 12.9), 151.7, 139.9 (d, J = 12.9), 134.6, 133.6,
131.2, 129.8, 124.6, 121.6, 112.7, 105.5 (d, J = 3.2), 103.4 (d, J = 24.5), 102.0 (d, J = 26.7),
49.3. HRMS (ESI) calcd for C18H14FN2O3S [M+H]+ 357.0704, found 357.0717. HPLC tR = 8.6
min, 98.8%.
2ꢀ(3ꢀFluoroꢀ5ꢀ(pyridinꢀ2ꢀyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (34).
The title compound was obtained as described for 22 using 25 (75 mg, 0.27 mmol) and 2ꢀ
iodopyridine (24 µL, 0.22 mmol) as starting materials. The crude product was purified by flash
chromatography (EtOAc/PE, 2:3) and the title compound 34 was recrystallized from acetone/PE
as a white solid (36 mg, 45%). 1H NMR (400 MHz, CDCl3): 8.23 (dd, J = 4.7, 1.4 Hz, 1H), 7.86
(d, J = 7.6 Hz, 1H), 7.73 (dd, J = 7.6, 1.9 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz,
1H), 7.49 (d, J = 7.6 Hz, 1H), 7.08 – 7.04 (m, 2H), 7.01 (s, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.69
(dt, J = 9.4, 1.8 Hz, 1H), 4.83 (s, 2H). 13C NMR (100 MHz, CDCl3): 163.7 (d, J = 246.3 Hz),
162.7, 156.2 (d, J = 13.3 Hz), 147.8, 139.8, 139.2 (d, J = 12.8 Hz), 134.8, 133.4, 131.4, 129.6,
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124.5, 121.6, 119.4, 112.1, 106.5 (d, J = 3.2 Hz), 104.3 (d, J = 24.5 Hz), 101.8 (d, J = 26.7 Hz),
49.3. HRMS (ESI) calcd for C18H14FN2O3S [M+H]+ 357.0704, found 357.0704. HPLC tR = 11.6
min, 98.1%.
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2ꢀ(3ꢀIodophenoxy)pyridine (35). A vial was charged with 3ꢀiodophenol (500 mg, 2.27
mmol), K2CO3 (345 mg, 2.5 mmol), dry DMF (2 mL) and 2ꢀfluoropyridine (196 µL, 2.27
mmol). The vial was capped and heated at 140 ºC under microwave irradiation for 6 h. The
reaction was partitioned between water and EtOAc, the aqueous phase was extracted with EtOAc
(2x). The combined organic phases were washed with brine, dried (Na2SO4) and concentrated.
The crude product was purified by flash chromatography (EtOAc/PE, 1:9) to give 35 as colorless
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oil that solidified upon standing (416 mg, 62%). H NMR (400 MHz, CDCl3): 8.20 (dd, J = 4.9,
1.3 Hz, 1H), 7.70 (td, J = 8.3, 1.9 Hz, 1H), 7.55 – 7.48 (m, 2H), 7.16 – 7.09 (m, 2H), 7.02 (dd, J
= 6.8, 5.4 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H). 13C NMR (100 MHz, CDCl3): 163.2, 154.6, 147.8,
139.6, 133.7, 130.9, 130.3, 120.7, 119.0, 111.8, 94.0. HRMS (ESI) calcd for C11H9INO [M+H]+
297.9723, found 297.9716. HPLC tR = 12.5 min, 99.8%.
2ꢀ(3ꢀ(Pyridinꢀ2ꢀyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (36). The title
compound was obtained as described for 21 using 20 (75 mg, 0.44 mmol) and 35 (158 mg, 0.53
mmol) as starting materials. The crude product was purified by flash chromatography
(EtOAc/PE, 1:1) and the title compound (36) was recrystallized from EtOAc/PE (1:1) as a white
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solid (110 mg, 73%). H NMR (400 MHz, CDCl3): 8.21 (dd, J = 5.0, 1.3 Hz, 1H), 7.86 (d, J =
7.5 Hz, 1H), 7.70 (ddd, J = 8.3, 7.4, 2.0 Hz, 1H), 7.65 (td, J = 0.8, 7.5 Hz, 1H), 7.58 (t, J = 7.5
Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H), 7.36 (dd, J = 8.2, 1.3 Hz, 1H), 7.21 (t,
J = 2.1 Hz, 1H), 7.01 (ddd, J = 6.9, 5.0, 0.6 Hz, 1H), 6.98 – 6.92 (m, 2H), 4.85 (s, 2H). 13C NMR
(100 MHz, CDCl3): 163.4, 155.2, 147.8, 139.6, 138.4, 135.0, 133.2, 131.9, 130.6, 129.5, 124.5,
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121.5, 118.8, 117.0, 115.0, 111.9, 111.8, 49.5. HRMS (ESI) calcd for C18H15N2O3S [M+H]+
339.0798, found 339.0787. HPLC tR = 11.2 min, 99.6%.
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2ꢀ(3ꢀBromoꢀ5ꢀchlorophenoxy)pyridine (37). The title compound was obtained as described
for 35 using 3ꢀbromoꢀ5ꢀchlorophenol (100 mg, 0.48 mmol) and 2ꢀfluoropyridine (41 µL, 0.48
mmol) as starting materials. The crude product was purified by flash chromatography
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(EtOAc/PE, 1:9) to give 37 as colorless oil that crystallized upon standing (89 mg, 65%). H
NMR (400 MHz, CDCl3): 8.21 (ddd, J = 5.0, 2.0, 0.7 Hz, 1H), 7.74 (ddd, J = 8.3, 7.2, 2.0 Hz,
1H), 7.34 (t, J = 1.9 Hz, 1H), 7.22 (t, J = 1.9 Hz, 1H), 7.11 (t, J = 1.9 Hz, 1H), 7.07 (ddd, J = 7.2,
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5.0, 0.7 Hz, 1H), 6.96 (dt, J = 8.3, 0.7 Hz, 1H). C NMR (100 MHz, CDCl3): 162.5, 155.2,
147.7, 139.8, 135.5, 127.6, 122.9, 122.7, 120.5, 119.5, 112.1. HRMS (ESI) calcd for
C11H8BrClNO [M+H]+ 283.9472, found 283.9483. HPLC tR = 13.4 min, 98.9%.
2ꢀ(3ꢀChloroꢀ5ꢀ(pyridinꢀ2ꢀyloxy)phenyl)ꢀ2,3ꢀdihydrobenzo[d]isothiazoleꢀ1,1ꢀdioxide (38).
The title compound was obtained as described for 21 using 20 (37 mg, 0.22 mmol) and 37 (75
mg, 0.26 mmol) as starting materials. The crude product was purified by flash chromatography
(EtOAc/PE, 3:7) to provide 38 as a colorless oil that crystallized upon standing (50 mg, 61%). 1H
NMR (400 MHz, CDCl3): 8.22 (ddd, J = 5.0, 1.9, 0.6 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.73
(ddd, J = 8.3, 7.2, 2.0 Hz, 1H), 7.69 (td, J = 7.6, 1.1 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.49 (d, J
= 7.6 Hz, 1H), 7.29 (t, J = 2.0 Hz, 1H), 7.17 (t, J = 2.0 Hz, 1H), 7.06 (ddd, J = 7.2, 5.0, 0.9 Hz,
1H), 7.00 – 6.95 (m, 2H), 4.85 (s, 2H). 13C NMR (100 MHz, CDCl3): 162.8, 155.6, 147.8, 139.8,
139.1, 135.9, 134.8, 133.4, 131.5, 129.6, 124.49, 121.6, 119.3, 117.2, 114.5, 112.1, 109.7, 49.4.
HRMS (ESI) calcd for C18H14ClNO3S [M+H]+ 373.0408, found 374.0406. HPLC tR = 12.1 min,
> 99.9%.
Computational modeling
ACS Paragon Plus Environment
29