Structure activity relationship studies on chemically non-reactive glycine sulfonamide inhibitors of diacylglycerol lipase

Article abstract of DOI:10.1016/j.bmc.2016.02.006

N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry efforts.

Full text of DOI:10.1016/j.bmc.2016.02.006

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure activity relationship studies on chemically non-reactive
glycine sulfonamide inhibitors of diacylglycerol lipase
a
a
a
a
b
Louis S. Chupak a, , Xiaofan Zheng , Shuanghua Hu , Yazhong Huang , Min Ding , Martin A. Lewis ,
⇑
Ryan S. Westphal b, Yuval Blat c, Andrea McClure d, Robert G. Gentles a
a Discovery Chemistry, Research and Development, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA
b Neuroscience Biology, Disease Sciences and Biologics, Research and Development, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA
c Lead Evaluation, Discovery, Research and Development, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA
d Discovery Chemistry Platforms, Research and Development, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are
reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-
chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without
covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool
compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry
efforts.
Received 4 December 2015
Revised 28 January 2016
Accepted 5 February 2016
Available online xxxx
Keywords:
Ó 2016 Published by Elsevier Ltd.
Diacylglycerol
Lipase inhibitor
Endocannabinoid
2-Arachidonoylglycerol
Glycine
Sulfonamide
1. Introduction
Attempts to prepare compounds that modulate the CB1 or CB2
receptors without inducing significant side effects have met with
Regulation of the endocannabinoid signaling system is an
emerging strategy for potential therapeutic intervention in a vari-
ety of diseases,1 including; anxiety,2 depression,3 pain,4 inflamma-
tion,5 hepatic steatosis and obesity.6 Here we describe efforts to
regulate the endocannabinoid signaling system by modulating
the levels of the endogenous cannabinoid agonist, 2-arachidonoyl-
glycerol (2-AG) through inhibition of the enzymes predominantly
responsible for its biosynthesis.
The endocannabinoid signaling system consists of the cannabi-
noid receptors CB1 and CB2, the endogenous agonists (endo-
cannabinoids) anandamide (ANA) and 2-AG, and the enzymes
that regulate endocannabinoid synthesis and degradation. CB1
and CB2 are G-protein coupled receptors (GPCRs) that are widely
distributed throughout the body. CB1 is one of the most abundant
GPCRs in the brain and is also located to a lesser extent in the liver,
adipose tissue, gastrointestinal tract, as well as in vagal nerves,
pancreas and skeletal muscle. In contrast, CB2 is found mainly in
cells of the immune system.7–12
limited success. For example, the synthetic cannabinoid agonist
nabilone is approved for the treatment of chemotherapy-induced
nausea and vomiting that has not responded to conventional
antiemetics. However, nabilone produces psychoactive side effects
similar to those observed with marijuana.13 The CB1 antagonist
rimonabant and the inverse agonist taranabant inhibit food intake
and reduce body weight in obese animals and humans. However,
these agents are known to induce central nervous system (CNS)
side effects including anxiety and depression.14
An emerging alternative strategy to receptor agonism or antag-
onism is to regulate tissue levels of the endocannabinoids.12,5,15
Presented here are the SAR, profiling properties and pharmacoki-
netics of the first reported chemically non-reactive inhibitors of
2-AG synthesis.16,17 Diminished 2-AG levels are expected to pro-
duce a corresponding reduction in cannabinoid receptor (CR) acti-
vation. Unlike many typical neurotransmitters, 2-AG is not stored
in vesicles; rather, it is rapidly synthesized (in an on-demand fash-
ion) in response to rising cellular calcium levels, or alternatively,
by activation of Gq/11 protein coupled receptors.12,15,18,19 2-AG is
produced by the reaction sequences depicted in Figure 1.20 Initial
cleavage of the sugar moiety from membrane associated phos-
phatidyl inositol by phosphatidyl lipase C (PLC) results in the for-
⇑
Corresponding author. Tel.: +1 203 677 7233.
E-mail address: louis.chupak@bms.com (L.S. Chupak).
http://dx.doi.org/10.1016/j.bmc.2016.02.006
0968-0896/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Chupak, L. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.006

2
L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
R
O
O
OH
O
P
O
OHOH
O
O
HO
OH
Phosphatidylinositol
OH
O
R
O
OH
O
O
OH
OH
O
PLC
PLA1
P
O
O
OHOH
O
O
HO
OH
Lysophosphatidylinositol
Diacylglycerol (DAG)
DAGL
Lyso-PI-PLC
Phosphatase
OH
OH
OH
OH
O
O
OH
O
P
O
O
O
OH
2-Arachadinoyl glycerol (2-AG)
Lysophosphatidic acid
O
MAGL
FAAH
O
OH
OH
N
H
Arachidonic acid (ARA)
Anandamide
Figure 1. Principal endocannabinoids 2-AG and anandamide. The major routes for the anabolism and catabolism of 2-AG are shown. Enzymes exist to catalyze the reverse
reactions depicted in most of the anabolic steps shown, and the transient tissue concentrations of 2-AG represent the balance between the rates of these competing reactions.
Fatty acid amide hydrolaze (FAAH), phosphatidylinositol-specific phospholipase C (Lyso-PI-PLC), Phospholipase A1 (PLA1).
mation of diacylglycerol (DAG). DAG is subsequently hydrolyzed
by diacylglycerol lipase (DAGL) resulting in the formation of 2-
AG. Once formed, 2-AG acts as a retrograde messenger by activat-
ing CRs on presynaptic neurons. Alternatively, 2-AG can be synthe-
sized by the phosphatase mediated cleavage of lysophosphatidic
acid, though this pathway is thought to be a minor contributor to
tissue levels. In relation to its catabolism, 2-AG is cleared by the
action of several enzymes, with the major pathway thought to
involve monoacylglycerol lipase (MAGL). Thus the levels of 2-AG
are tightly controlled primarily by the actions of DAGL and MAGL.
Consistent with this observation is that genetically-modified ani-
Presynaptic membrane
Synapse
CB1
DAG
2-AG
ARA
Postsynaptic membrane
MAGL
DAGL
Lipid Bilayer
Figure 2. Sites of synthesis and degradation of 2-AG and a depiction of its role as a
retrograde neurotransmitter; activation of presynaptic CB1 receptors can result in a
variety of responses dependent on the G-protein to which the receptor is coupled.
mals that lack DAGL-
a exhibit decreased tissue concentrations of
2-AG.21,22
In this manuscript, we focus on inhibiting the activity of DAGL
as a point of potential therapeutic intervention. Inhibition of DAGL
should lead to reduced levels of 2-AG and a corresponding reduc-
tion in CR activation.23 Consequently, it is anticipated that a DAGL
inhibitor will be functionally equivalent to a CR antagonist. How-
ever, the effects of a DAGL inhibitor would be localized to sites
where 2-AG is actively being synthesized, and thus it might be
anticipated to have an improved side-effect profile relative to a
CR antagonist.
the CRs.9–11,29–31 This activation can impact the functional output
of the GABAergic and glutamatergic systems, as is the case in Alz-
heimer’s or Parkinson’s diseases where the regulatory function of
2-AG is thought to be disrupted.29 2-AG also functions in the
periphery, where DAGL inhibitors are anticipated to have thera-
peutic potential in the treatment of diseases such as obesity, meta-
DAGLs are serine hydrolases that exist as membrane associated
bolic
syndrome,
liver
fibrosis
and
allergic
contact
dermatitis.5,10,11,32
proteins. There are two known isoforms of the enzyme, DAGL-
a
and DAGL-b.24 DAGL-
a
is predominantly found in the CNS where
Prior to our initial disclosure16,17 and a subsequent related pub-
lication,25 the only reported inhibitors of DAGL were chemically
reactive molecules that function by covalently modifying DAGL.
Such irreversible inhibitors are expected to display several poten-
tial liabilities related to toxicity and a lack of selectivity. For exam-
ple, tetrahydrolipstatin (1) covalently modifies both DAGL-
DAGL-b (Fig. 3).33 In addition, 1 blocks several brain serine hydro-
lases with similar potencies to those observed against DAGL- and
it is localized in post-synaptic neurons (Fig. 2), in contrast to
DAGL-b that is found mainly in the periphery.21,25,26 Postsynaptic
neurons generate and release 2-AG which acts as a retrograde mes-
senger by activating CRs on presynaptic neurons.27,28 Activation of
the presynaptic CR regulates the release of the neurotransmitters
GABA and glutamate. In certain disease states, tissue levels of 2-
AG can be altered and result in changes to the activation level of
a and
a
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L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
chose to prioritize the identification of suitable replacements for
the biphenyl amine group while simultaneously addressing the
hydrophobic deficiencies and functional group liabilities.
O
O
P
O
O
O
F
O
O
O
H
OtBu
2. Chemistry
HN
O
With the objectives outline above, one of the first approaches
explored was to insert a spacer between the sulfonamide nitrogen
atom and the biaryl ring system. This strategy would obviate the
potential for formation of electrophilic phase II metabolites that
could result from the oxidation and conjugation of any anilines
that might be released from the parent structure.39 Three general
synthetic methods to the targeted compounds (Fig. 4) were used
to independently explore the sulfonamide, benzylic amine and
biaryl substituents. The first method used an initial sulfonylation
of suitably functionalized benzylic amines A, followed by alkyla-
tion with methyl-2-bromoacetate to provide esters B. Hydrolysis
of B provided intermediate acids C that could be subsequently cou-
pled with a range of boronic acids under standard Suzuki–
Miyaura40 conditions to provide the desired compounds D, gener-
ally in good to excellent yields. The advantage of this synthetic
sequence was the ability to control the stereochemistry and substi-
tution at the benzylic position by starting with an appropriately
functionalized benzylic amine. The second synthetic method took
advantage of commercially available benzyl and biaryl bromides.
The t-butyl ester of glycine was sulfonylated to give derivatives
E, which were reacted with the desired benzylic bromides to pro-
vide the esters of type F. Deprotection with trifluoroacetic acid pro-
vided the final compounds I. For parallel synthesis it was found
that removing the acid protecting group after completing the
Suzuki–Miyaura reaction gave more consistent results. Thus, in a
third method, t-butyl esters E were alkylated with either 4- or 3-
bromo benzylbromide to provide the intermediates G. These inter-
mediates were then coupled with boronic acids to give esters H
and deprotected to give the targeted compounds I.
tetrahydrolipstatin (1)
O-5596 (2)
DAGLα IC50 (nM) =
1
1
(n=50)
O
Cell 2AG IC50 (nM) = 20 20 (n=30)
DAGLβ IC50 (nM) = 420 300 (n=2)
O
S
N
O
O
F2HC
O
OH
O
S
N
O
O
Cl
OH
O
Cl
(4)
IC50 (nM) = 18
α
(3)
DAGL
6
DAGLα IC50 (nM) = 2.4 1.4 (n=5)
Cell 2AG IC50 (nM) = 14.3 9.1 (n=5)
DAGLβ IC50 (nM) = 40.0 20.7 (n=3)
ABHD6 Ki (μM) = 0.8
Figure 3. Reported inhibitors of DAGL.
DAGL-b.34 More selective compounds have been reported: for
example, 2 is an irreversible inhibitor of DAGL- , but does not inhi-
a
bit MAGL or fatty acid amide hydrolase, a key enzyme involved in
the degradation of anandamide (see Fig. 1).35 Compound 2 is not an
antagonist of the CB1 or CB2 receptors, but shows effects that
mimic CR antagonism. Intraperitoneal administration of 2 reduced
mouse intake of palatable food consistent with a reduction in 2-AG
levels. Thus, this compound has been shown to be valuable as both
an in vitro and in vivo tool for studying 2-AG biosynthesis. How-
ever, the need remains for chemically non-reactive inhibitors of
DAGL that might function as starting points for drug design.
To that end, our corporate compound file was screened for inhi-
bitors of DAGL-a. A series of glycine sulfonamide derivatives was
discovered and initial hit to lead optimization exercises resulted
in the identification of 2-(N-(20,30-dichloro-[1,10-biphenyl]-3-yl)-
4-(difluoromethoxy)phenylsulfonamido)acetic acid (3), as an early
lead compound, see Figure 3 (manuscript in preparation). This
compound is an orally bioavailable, non-covalent inhibitor of both
3. Results and discussion
Table 1 illustrates the observed SAR associated with the intro-
duction of a methylene spacer between the aryl moiety and the
nitrogen of the sulfonamide. It was found that the simple benzyl
DAGL-a and DAGL-b, with IC50s of 2.4 and 40 nM, respectively.
Compound 3 was also shown to reduce 2-AG levels in a whole-cell
assay (EC50 14 nM) and was demonstrated to induce a reduction in
2-AG levels in liver tissue when dosed intraperitoneally (IP) in
mice. Additionally, compound 3 did not inhibit MAGL at the high-
amine derivative 5 showed no DAGL-
a inhibition up to the highest
concentration tested of 30 M. However, the combination of a
l
methylene spacer and a para phenyl ring, as found in derivative
6, immediately gave a compound in the 100 nM potency range.
Further substitution on the distal phenyl ring produced significant
advancements in potency, with several halogenated derivatives
exhibiting low double digit nanomolar inhibition. For example,
the 2,4-dichloro compound 7 gave a 3-fold improvement over
the unsubstituted phenyl compound 6. Typically, halogens as in
compounds 7–13, alkyls and weak hydrogen-bond acceptors as
presented in compounds 14–16 gave the lowest IC50’s, as illus-
trated in Table 1. Additionally, the distal aryl group was equally
well tolerated in both the meta- or para-position, as can be seen
by comparing analog 7 with 8 and 12 with 13. In contrast, basic
amines and hydrogen bond donors (data not shown) on the termi-
nal phenyl showed significantly poorer activity. The effects of sub-
stitution on the methylene spacer were also examined.
Introduction of a methyl group produced enantiomers 9 and 10.
Both compounds were approximately 20 nM regardless of the
absolute stereochemistry and did not represent a significant
improvement over compound 7. Di-substitution in the form of a
cyclopropyl or a cyclobutyl group showed interesting SAR. A signif-
icant loss in potency was observed with cyclopropyl relative to the
unsubstituted or mono-methyl substituted analogs, as can be
est tested concentrations of 30 lM.
Recently, the activity profiles of a related series of glycine sul-
fonamides were reported.36 These compounds were derived from
analogs of compound 317 by introduction of an oxygen spacer to
change the biaryl functional group into a biaryl ether. In selectivity
studies, compound 425 was shown to target at least three proteins
in a proteome-wide activity based profiling assay. Among the iden-
tified proteins was a/b-hydrolase domain 6 which is responsible for
2-AG hydrolysis and is implicated in obesity-related metabolic dis-
orders. Due to the similar character of the biarylether and biphenyl
moieties, it is possible that the latter may have similar selectivity
profiles, but in this current work, selectivity studies are limited
to DAGL, MAGL and porcine pancreatic lipase.
The N-aryl glycine sulfonamides reported to date possess sev-
eral potential liabilities. The compounds are very lipophilic as esti-
mated by their CLogP’s. All contain a carboxylic acid group which
would be expected to limit the compounds permeability. In addi-
tion, the most potent examples of this chemotype retain an
embedded biaryl amine of the type found in compound 3. Due to
the known carcinogenicity of [1,10-biphenyl]-4-amine,37,38 we
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4
L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Method 1
Ar1
O
R1
Ar1
O
R1
Ar1
O
R1
O
O
O
S
N
S
N
S
N
O
O
O
R1
R2
NH2
OMe
OH
OH
R2
R2
R2
a
b
c
Br
A
Br
B
Br
C
Ar2
D
Ar1
O
Ar1
O
O
O
Method 2
S
N
S
N
Ar1
O
O
O
O
O
S
d
e
f
O
O
OH
H2N
O
HN
O
R3
R3
E
F
I
e
f
Method 3
Ar1
Ar1
O
O
O
S
S
N
O
O
O
c
N
O
O
Br
Ar2
G
H
Figure 4. (a) (i) Aryl sulfonyl chloride, DIEA, DMF, 70 °C; (ii) methyl-2-bromoacetate, BEMP, 80 °C. (b) NaOH, MeOH, THF. (c) Arylboronic acid, sodium bicarbonate (aq),
palladium acetate, DMF, 120 °C. (d) Aryl sulfonyl chloride, pyridine, DMAP, DCM. (e) Benzyl bromide, BEMP, 70 °C. (f) TFA.
observed with the cyclopropyl compound 11 having an IC50 of
130 nM. A similar loss of potency was observed with cyclopropyl
derivative 15 versus the unsubstituted analog 14 with IC50s of 40
and 17 nM, respectively. However, increased ring size resulted in
an improvement as observed with cyclobutyls such as 12, 13 and
16 that have IC50s of 7, 11 and 8 nM, respectively. A possible expla-
nation for the reduced activity observed with the cyclopropyl ana-
logs could be attributed to the larger bond angle (ꢀ115°) between
geminal pendant substituents on the smaller cyclopropyl ring rel-
ative to the larger cyclobutyl (ꢀ112°) or acyclic derivatives
(ꢀ109°). While these differences are modest, the associated qua-
ternary carbon is at the center of the molecule, and even small
changes in bond angles here could result in significant differences
in the relative positions of distal portions of the analogs examined.
As discussed above, and as will be presented below, the terminal
moieties in the compounds are key determinants of the potencies
observed. However, the similar activities observed for the para-
and meta-biaryls seen in 12 and 13 suggests that small changes
in the placement of the distal substituents should be tolerated.
An alternative explanation for the observed SAR is that di-substitu-
tion at the benzylic position is suboptimal, but can be overcome by
increasing lipophilicity in the form of larger saturated rings. A full
understanding of these observations will have to await a co-crystal
structure. However, from the data presented it is apparent that the
cyclobutyl derivative 13 represented an excellent compromise.
This compound significantly increased potency with a minimum
addition of lipophilicity, and was therefore chosen as a starting
point for further optimization.
mined to have an IC50 of 80 nM. It was found that in all cases exam-
ined, ortho substitution negatively impacted activity, as typified
with analogs 18 and 23. Contrastingly, meta substitution had either
modest or no effect, as characterized by compounds 19 and 22.
More encouraging was the observation that para substitution sig-
nificantly improved activity, as observed with analogs 20, 21, 13
and 24. In general, it would appear that electron rich sulfonamides
are more active, as the potency order follows the sequence of
increasing electron donating character of the substituents, that is,
2,2-dimethylchromanyl > F2HCO- > -Cl > -CN. Consistent with this
observation were the results obtained in attempting to introduce
isosteric heterocycles at this vector (data not shown). A variety
of such heterocycles showed a significant loss in potency and were
not considered viable replacements for phenyl. It is interesting
however, that heteroatoms are tolerated when masked with adja-
cent lipophilicity, as in 2,2-dimethylchromanyl derivative 24 that
displayed sub-nanomolar (0.6 nM) potency. Given this finding,
2,2-dimethylchromanyl derivatives were used to reexamine the
SAR at the benzylic position of the chemotype.
Benzylic derivatives were prepared and typical trends can be
seen in Figure 5. The unsubstituted compounds 25 and 26 showed
a four-fold loss in potency relative to the cyclobutyl derivative, 24.
It was also observed that the cyclobutyl 24 and cyclopentyl 27
groups were approximately equipotent with IC50s of 0.6 and
0.9 nM, respectively. In an attempt to investigate the tolerance
for polarity in this region of the structure, the tetrahydrofuran
derivative 28 was prepared and a ten-fold loss in activity (IC50
8 nM) versus the cyclopentyl compound 27 was observed. At this
juncture, it was concluded that the preferred pendent moieties
consisted of cyclobutyl, 2,2-dimethylchromanyl sulfonyl and either
a meta or para 3,5-dichlorophenyl group. With these significant
advancements, our focus shifted to optimization of the glycine
core.
With the initially identified preferred substituents at both the
benzylic position and the terminal aryl of the biphenyl group, we
turned our attention to the optimization of the sulfonamide moi-
ety. Table 2 shows the SAR for substitution of the sulfonamide aryl
ring while holding the cyclobutyl spacer and the biphenyl sub-
stituents constant.
Efforts to replace the carboxylic acid moiety with esters, amides
or a variety of common acid isosteres were uniformly unsuccessful
resulting in almost complete loss of activity (data not shown). It
As a reference point, 2-(N-(1-(30,50-dichloro-[1,10-biphenyl]-3-
yl)cyclobutyl) phenyl-sulfonamido)acetic acid (17) was deter-
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L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 1
Alternatively, it is also possible that the two lipophilic side chains
undergo hydrophobic collapse and fill a larger, common binding
site. Currently, no X-ray crystal structures of human DAGLs are
reported that would help further refine this putative
pharmacophore.
IC50’s for DAGL-
a inhibition were determined in a cell-free assay at pH 6.5 as
previously described41
O
S
CO2H
F2HCO
N
R3
O
However, a homology model for the active site of DAGL-
a has
R1 R2
been proposed. The model is based on the co-crystal structure of
oleic acid bound to Thermomyces (Humicola) lanuginosa S146A
R1/R2
R3 position
R3
DAGL-
a IC50 (nM)
mutant.42 This serine hydrolase and DAGL-
a
contain the same
5
6
H/H
H/H
para
para
H
Ph
>30,000
Ser-His-Asp catalytic triad and typical
a
/b hydrolase fold motif.43
80 50 (n = 2)
30 15 (n = 2)
The docking of compound 4 into the model has been described
to provide two complimentary, high-ranking binding poses.36 In
one pose, the carboxylic acid forms a hydrogen bond with the cat-
alytic serine (Ser472). In the second pose the carboxylic acid forms
a hydrogen bond with the catalytic histidine (His650) and a second
nearby histidine (His471). In both poses the aryl rings occupy sep-
arate, large, open hydrophobic pockets. The sulfonamide forms no
Cl
7
H/H
meta
Cl
Cl
Cl
8
H/H
para
16 10 (n = 2)
direct interactions with DAGL-a in this model, but instead acts as a
Cl
Cl
Cl
Cl
9
H/CH3
para
para
para
para
18 5 (n = 3)
19 6 (n = 3)
130 65 (n = 4)
geometrical constraint to place the aryl rings into the hydrophobic
pockets. By extension, it would be concluded that the substituents
on the benzylic methylene of our inhibitors would point into the
solvent in both poses. Thus the reported homology model does
not readily explain the SAR observed when comparing compounds
27 (0.9 nM) and 28 (8 nM). Nonetheless, even without a conclusive
binding hypothesis, the key elements required for highly-potent,
non-covalent inhibitors of DAGL-a have been successfully defined,
and our attention progressed to an assessment of the physiochem-
ical characteristics of this series of compounds.
Cl
Cl
Cl
Cl
10
11
12
CH3/H
Cyclopropyl
Cyclobutyl
7
4 (n = 5)
From the structures above, it can be clearly seen that the best
inhibitors are highly lipophilic. The requirement for lipophilicity
Cl
Cl
13
Cyclobutyl
meta
11 7 (n = 3)
Table 2
IC50’s for DAGL-
described above for Table 1
a
inhibition were determined in
a
cell-free assay at pH 6.5 as
14
15
16
H/H
para
para
para
17 6 (n = 5)
40 10 (n = 3)
F3CO
F3CO
F3CO
HO2C
Cyclopropyl
Cyclobutyl
O
N
Cl
S
Aryl
O
8
3 (n = 3)
Cl
Aryl
DAGL-a IC50 (nM)
17
18
80 50 (n = 4)
was also found that substitution is not tolerated alpha to the car-
boxylic acid, nor is homologation of the acid. For example, alanine
or b-alanine derivatives displayed no inhibitory activity. Finally,
the sulfonamide linkage was found to be required. Benzyl carba-
mate, phenyl urea and benzamide derivatives were inactive at
the highest tested concentrations. Taken collectively, these
requirements suggest a very specific, sterically constrained inter-
action between the inhibitor’s carboxylic acid head group and
the enzyme. In contrast, the SAR of the N-alkyl and N-sulfonyl
groups tolerates a wide variety of lipophilic functionality consis-
tent with these groups binding in a flexible or large lipophilic
pocket. Recalling that DAGL is a serine hydrolase and that lipophi-
lic DAG is its substrate, it is tempting to suggest that the inhibitors
operate through a four point pharmacophore model (Fig. 6). In this
model, the carboxylic acid could form a hydrogen bond or a salt
bridge interaction near the active site: perhaps interacting with
one of the catalytic residues of the proton transfer network. The
tetrahedral sulfonyl moiety could act as a transition-state mimetic,
accept critical hydrogen bonds from complimentary donors near
the active site, or act to control the conformation. Finally, the large
lipophilic groups can occupy pockets normally occupied by the
hydrocarbon chains of the endocannabinoid substrate esters.
Cl
510 120 (n = 4)
Cl
Cl
19
60 30 (n = 4)
20
21
4
1 (n = 4)
NC
45 10 (n = 4)
161 40 (n = 4)
MeO
22
23
OMe
14,000 3,100 (n = 4)
F2HCO
O
13
24
11 7 (n = 3)
0.6 0.1 (n = 5)
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6
L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
O
O
O
O
O
S
N
O
S
N
O
S
N
O
O
O
O
OH
OH
OH
O
Cl
Cl
Cl
Cl
Cl
Cl
25 m-3,5 dichloro
26 p-3,5 dichloro
27
28
DAGLα IC50 =8 2 nM (n=3)
DAGL IC50 = 3 1 nM (n=3)
α
DAGLα IC50 =0.9 0.5 nM (n=3)
Figure 5. Key analogs used to characterize SAR at the benzylic position.
Aromatic Hydrophobes
H-Bond Donor / Acceptor Array
(Ser472 or His650/His471)
In the case of the 4-(difluoromethoxy)benzenesulfonyl deriva-
tives, 32, 16, 33 and 10, there is good concordance between the
cell-based assay and the enzyme assay, with a consistently modest
5-fold difference in the IC50’s observed between the two assay for-
mats. However, for the 2,2-dimethylchroman-6-sulfonyl deriva-
tives 24 and 12, there is a larger and more significant decrease in
activity in the whole cell assay, with IC50’s that are right shifted,
74 and 15-fold respectively. In the case of 24, this compound
appears quite permeable in our PAMPA assay suggesting that it
should readily penetrate into cell membranes to access DAGL.
Thus, there is no correlation observed between PAMPA permeabil-
ity and fold shift in potency in the whole-cell assay system. The
shift in activity is not readily explained other than by invoking
some non-defined binding to a component of the medium for the
cell-based assay.
O
N
H
O
Electron Rich
S
Aromatic Hydrophobes
O
O
H-Bond Acceptor Array
Figure 6. Rudimentary 4-point pharmacophore model consistent with observed
SAR. Conserved amino acid residues in parenthesis have the potential to form
interactions based on the reported homology model.
is not surprising considering the nature of the DAGL substrate
(DAG; CLogP = 6.9), and suggests that the active site of the enzyme
has a complimentary highly hydrophobic character. In addition,
many lipases access their substrates by maintaining an intimate
contact with the cell membrane, where the substrates are
located.43 Inhibitors with high LogPs would be expected to parti-
tion into the cell membrane, thereby increasing access to the
enzyme’s active site.
To fully characterize the lipophilicity of the inhibitors described
we compared their CLogP values with the corresponding measured
LogDs at a pH of 7.4. Interestingly, the experimentally determined
values were consistently and very significantly lower than the
CLogPs for all the analogs profiled. As can be seen in Table 3, the
measured LogDs were approximately 4 log units less than the cal-
culated LogPs. This discrepancy could be explained primarily
through the measured pKas. Of the analogs characterized, all have
pKas of <3.5. This is appreciably lower than might be expected for a
typical carboxylic acid (ꢀ4.8), but is consistent with what is
expected from an amino acid. Additionally, the lower LogDs may
also indicate that the compounds undergo a hydrophobic collapse
in aqueous solution, minimizing the exposed hydrophobic surfaces
and thus reducing their overall hydrophobicity.
Regardless, the most important observation from Table 4 is that
all of the compounds presented display considerable inhibitory
activity against DAGL-a under the more physiologically relevant
conditions of a cell-based assay. Of greatest import, is the observa-
tion that the reported compounds exhibit activities in both the
enzyme and cell-based assays that are similar to those observed
for the irreversible inhibitors. Tetrahydrolipstatin (1), for example,
has an IC50 of 1 nM in the enzyme assay and 20 nM in the cell-
based assay. Thus, the glycine sulfonamide chemotype clearly
demonstrates that a chemically reactive inhibitor is not required
for potent activity against DAGL in a cellular context. Hence, it
has been established that this enzyme can be targeted by conven-
tional means, opening the door to future drug design.
As detailed in the introduction, a key objective in the identifica-
tion of chemically non-reactive inhibitors of DAGL was the hope
that they would display significant selectivity against other serine
hydrolases to avoid potential off-target effects. To that end we first
explored the selectivity of a number of our analogs against DAGL-b,
with representative data being presented in Table 5. Interestingly,
all of the compounds displayed selectivity for DAGL-a over DAGL-
Given the highly acidic nature of this inhibitor class, the perme-
ability of this series was of obvious interest and was determined in
a parallel artificial membrane permeability assay (PAMPA)44 at pHs
of 5.5 and 7.4 (to simulate pH conditions in the small intestine and
blood, respectively). All of the compounds were found to be highly
permeable at the lower pH, and not surprisingly, showed a signif-
icant reduction in permeability at pH 7.4. Importantly however,
the more active examples of the chemotype, as exemplified by
compound 29, maintained high permeability at both pHs
examined.
b, with factors ranging between 5 and 70-fold within the structural
diversity explored. However, it should be noted that the most
potent compounds do retain significant inhibitory activity against
DAGL-b, and any observed in vivo effects could potentially be
attributed to inhibition of either or a combination of both isoforms.
Additionally, all of the compounds presented above showed no
activity in assays that measure their inhibitory profile against
either MAGL or porcine pancreatic lipase (PPL) at test concentra-
tions up to 30 lM. Thus the compounds are not expected to be
indiscriminant lipase inhibitors, although a more comprehensive
With the permeability of the series characterized, we next
explored the activity of the inhibitors in a whole-cell system. In
profiling in this regard is certainly merited.36
To assess if our compounds could be useful as tools for in vivo
proof of concept studies, and to gain further perspective on their
drug-like properties, we conducted some additional profiling stud-
ies, the results of which are shown in Table 6.
the assay used, DAGL-
a was transiently expressed in HEK cells,
and 2-AG concentrations were determined directly by mass spec-
trometry.41 The results obtained with a representative set of ana-
logs are shown in Table 4.
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L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Table 3
Physico-chemical and permeability analyses of select examples of DAGL inhibitors
O
S
O
CO2H
Ar2
Ar1
N
R1
Ar1
Ar2
R1
DAGL-
a
CLogP
LogD
PAMPA
pKa
IC50 (nM)
pH 7.4
pH 5.5
630
pH 7.5
430
29
H
55 60 (n = 4)
6.6
2.2
3.4
O
O
5
H
>30,000
3.6
4.6
3.9
ꢁ0.4
0.5
290
550
537
<15
75
3.4
F
F
N
30
31
H
13,500 6000 (n = 4)
2.0/5.8
O
O
CH3
16,000 (n = 1)
ꢁ0.3
32
3.4
F
F
The analogs profiled span approximately two orders of magni-
tude with respect to their potency of inhibition of DAGL- . With
also displayed a very high rate of clearance (Cl), which may reflect
sustained partitioning into membranes. However, 24 did have
excellent bioavailability when administered either IP or PO with
F values of 100% and 88%, respectively. The high bioavailability
suggests that first pass metabolism via the liver is minimal; a
result consistent with the modest in vitro clearance in liver micro-
somes. The high clearance observed in the IV study (twice hepatic
blood flow) is also consistent with non-hepatic mechanisms. In
separate studies (data not disclosed) the brain uptake of 24 was
explored, and no significant penetration into the CNS was
observed. This is consistent with other members of this series that
were also examined in brain uptake studies and were shown not to
penetrate into the brain. Thus it is expected that these compounds
would achieve sufficient concentrations to act exclusively on
peripherally located DAGLs.
a
respect to their potential for metabolic clearance, as assessed in a
liver microsome assay,45 all show potential for moderate clearance
in the rodent species examined, and somewhat lower potential for
clearance in humans. All of the compounds displayed some minor
activity in a flux assay46 used to determine blocking of the human
ether-a-go-go (HERG) channel, and would require further charac-
terization in an electrophysiology patch clamp assay to more accu-
rately determine the significance of this observation. We also
assessed the analogs ability to inhibit a panel of cytochrome
P450 (CYP) enzymes,47 with selected data reported above. While
none of the analogs displayed particularly potent inhibition of
any of the isozymes, the data suggest that the free methylene
derivatives 29 and 33, display better CYP profiles than their di-sub-
stituted congeners 24 and 16. Additionally, we noted that com-
pound 24 displayed significant activity in
a
pregnane
4. Conclusion
transactivation (PXR) assay,48 and further profiling of this com-
pound and related analogs could be warranted to fully assess the
potential for auto-induction of metabolism. However, in a general
sense as it relates to the chemotype, all of the signals observed in
the profiling assays described above appear sporadic in nature. We
concluded that the chemotype did not possess any implicit liabili-
ties in the profiling suite described above and therefore provides
an excellent lead for continued drug-design efforts.
We have described the structure activity relationships, profiling
characteristics and physico-chemical properties for the first
reported series of N-benzylic-substituted glycine sulfonamides
that reversibly inhibit diacylglycerol lipases. An effort to identify
replacements for a known toxicophore led to the identification of
2-(N-(1-(30,50-dichloro-[1,10-biphenyl]-3-yl)cyclobutyl)-2,2-
dimethylchroman-6-sulfonamido)acetic acid 24. This compound is
a highly potent, easily-prepared inhibitor of DAGL alpha and beta
that lacks the embedded biphenyl amine found in our initially
identified leads. In contrast to nearly all previously reported DAGL
inhibitors, this compound is not an irreversible inhibitor of the
DAGLs. The compound has sub-nanomolar activity versus the
alpha isoform and nanomolar activity versus the beta isoform. A
window of roughly 60-fold exists between the activities versus
the two isoforms and there is no activity versus other lipases
tested. The compound also has excellent activity in a whole-cell
Given the outstanding potency of compound 24 in both the
enzyme and whole-cell assays, its pharmacokinetic (PK) profile
was examined in mice. The results from these experiments are
shown in Table 7.
In all three dosing formats the maximal exposure (Cmax) of 24
far exceeds its measured whole-cell IC50 of 40 nM. From the IV
study the observed large volume of distribution (7.4 L/Kg) is con-
sistent with good distribution into the tissues, which is interesting
given the highly acidic nature of the compound. The compound
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8
L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 4
Comparison of enzyme and whole-cell inhibitory assay data
O
S
O
CO2H
Ar2
Ar1
N
R1 R2
IC50 (nM)
Ar1
Ar2
R1/R2
DAGL-
a
Whole cell IC50 (nM)
PAMPA pH 5.5
288
Fold loss in activity
24
Cyclobutyl
0.6 0.1 (n = 5)
37 (n = 1)
74
O
Cl
Cl
32
12
16
H/H
3
6
2 (n = 9)
14 9 (n = 5)
88 25 (n = 2)
82 30 (n = 2)
ND
ND
179
5
Cl
O
F
Cl
F
Cyclobutyl
Cyclobutyl
4 (n = 5)
15
Cl
Cl
O
O
22 7 (n = 4)
17 8 (n = 7)
19 6 (n = 3)
4
F
F
F
F3CO
F
F
F
33
10
H/H
60 10 (n = 2)
455
500
4
5
OCF3
O
O
CH3/H
100 70 (n = 2)
Cl
Cl
assay, and a promising pharmacokinetic profile. The compound
was found to have no access to the CNS and would be expected
to avoid the CNS side-effects observed with CB1 antagonist rimon-
abant and the inverse agonist taranabant while having therapeutic
potential for diet-induced obesity and metabolic syndrome. We
anticipate 24 to be a valuable tool compound for studying DAGL
inhibition, and constitute an important lead for future medicinal
chemistry efforts.
reported in Hertz (Hz). Multiplicity patterns are designated as fol-
lows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,
broad; dd, doublet of doublet; dt, doublet of triplet; dq, doublet
of quartet and most spectra were analyzed using the ACDLABS
SpecManagerTM 12.0 program software. Reactions performed under
microwave irradiation utilized either a Biotage Initiator or CEM
Discover microwave. Liquid chromatography (LC)–mass spectra
(MS) were run on a Shimadzu LC instrument coupled to a Waters
Micromass ZQ instrument. All final compounds had purity P95%,
unless otherwise noted. LC–MS and HPLC conditions are provided
in the Supplementary material.
5. Experimental section
5.1. General considerations
5.1.1. 2-(N-Benzyl-4-(difluoromethoxy)phenylsulfonamido)
acetic acid (5)
All reagents were purchased from commercial suppliers and
used without purification. All anhydrous reactions were performed
under a nitrogen or argon atmosphere using SureSealTM solvents
from Aldrich. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Varian 600 MHz spectrometer, on a Bruker
Avance III 400 MHz or 500 MHz spectrometer, each equipped with
a 5-mm TXI or BBFO probe. All spectra were determined in the sol-
vents indicated and chemical shifts are reported in parts per mil-
To
a solution of tert-butyl 2-aminoacetate hydrochloride
(0.864 g, 5.00 mmol) in dichloromethane (10 mL) was added pyri-
dine (4.04 mL, 50.0 mmol), 4-(difluoromethoxy)benzene-1-sul-
fonyl chloride (1.213 g, 5 mmol) and
dimethylaminopyridine. The reaction mixture was stirred at room
temperature overnight. The crude reaction mixture was diluted
with ethyl acetate and the organic layer was washed once each
with brine and sodium bicarbonate. The organic portion was dried
over sodium sulfate. The solvent was removed in vacuo and the
a catalytic amount of
lion (ppm)
d units downfield from the internal standard
tetramethylsilane (TMS) with inter proton coupling constants
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L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
(20 mg, 86%). 1H NMR (500 MHz, chloroform-d) d ppm 3.96 (s, 2H),
4.47(s, 2H), 6.58 (t, J = 75 Hz (C-F), 1H), 7.20–7.26 (m, 4H), 7.32 (m,
3H), 7.89 (d, J = 11.5, 2H). LC–MS 0.78 min, 372.2 (M+H), 370.2
(MꢁH).
Table 5
DAGL selectivity profiling for prototypical inhibitors
O
S
O
CO2H
Ar2
Ar1
N
5.1.2. Compounds 6, 7 and 8
Ar1
Ar2
DAGL-
IC50 (nM)
a
DAGL-b
IC50 (nM)
Fold
difference
Compounds 6, 7 and 8 were prepared as part of a larger parallel
synthesis library from t-butyl N-(4-bromobenzyl)-N-((4-(difluo-
romethoxy)phenyl) sulfonyl) glycinate.
0.6 0.1
(n = 8)
40 16
(n = 2)
24
34
67
13
5.1.2.1. t-Butyl N-(4-bromobenzyl)-N-((4-(difluoromethoxy)
O
phenyl) sulfonyl) glycinate.
This common intermediate was
prepared from 1-bromo-4-(bromomethyl)benzene and tert-butyl
((4-(difluoromethoxy)phenyl)sulfonyl)glycinate using the method
described in the preparation of 5. 1H NMR (500 MHz, methanol-
Cl
Cl
d4)
d ppm 7.96–7.90 (m, 2H), 7.53–7.46 (m, 2H), 7.34 (d,
Cl
6
3
Cl
80 (n = 1)
J = 8.9 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H), 7.19–6.86 (m, 1H), 4.47 (s,
(n = 3)
Cl
2H), 3.86 (s, 2H), 1.35 (s, 9H).). LC–MS: 2.3 min, no ion observed.
Cl
5.1.2.2. Parallel synthesis method.
t-butyl N-(4-bromoben-
zyl)-N-((4-(difluoromethoxy)phenyl) sulfonyl) glycinate. (1.2 g)
was dissolved in 23.5 mL of anhydrous NMP. Microwave vials con-
taining the appropriate boronic acid (0.062 mmol, 1.2 equiv) were
prepared. The bromide solution (0.5 mL) was added to each micro-
6
4
12
16
146 (n = 1) 24
(n = 5)
Cl
F
F
Cl
O
O
F
F
wave vial. Potassium carbonate (77.0
lL, 2 N, 3 equiv,
0.154 mmol), 3.8 mg of PdCl2(dppf) (5.13 mol, 0.1 equiv) were
l
added to each vial followed by 0.1 mL of anhydrous NMP to wash
the solids off the sides of the vial. The vials were capped, flushed
with a blanket of Argon and heated in a microwave reactor at
150 °C for 30 min, pre-stirring was 10 s. Five samples were ran-
domly chosen and all contained desired product by LC/MS analysis.
The samples were dried overnight under vacuum centrifugation.
The residue in each microwave vial (expected 1 equiv, 0.051 mmol)
was dissolved in 0.5 mL of anhydrous dichloromethane. To each of
the vials was added 0.5 mL of TFA and the solution stirred for 4 h.
LC–MS of randomly chosen samples showed complete reaction.
The samples were dried overnight under vacuum centrifugation.
To each microwave vial was added 0.6 mL DMSO and 1.0 mL of
dimethylformamide. The vials were sonicated and shaken for
10 min. The solids were removed by filtration and purified on a
22
(n = 4)
7
112 (n = 1)
5
F3CO
residue was purified with a gradient of 10:1 hexane/ethyl acetate
to 3:2 hexane/ethyl acetate on a 40 g silica gel column. The frac-
tions containing the product were collected and concentrated to
afford tert-butyl ((4-(difluoromethoxy)phenyl)sulfonyl)glycinate
as white solid (0.95 g, 54%). 1H NMR (500 MHz, chloroform-d) d
ppm 7.87 (d, J = 9.0 Hz, 2H), 7.22 (d, J = 9.0 Hz, 2H), 6.58
(t, J = 75 Hz (C-F), 1H), 5.09 (t, J = 5.5 Hz (N–H), 1H), 3.68
(d, J = 5.5 Hz, 2H), 1.34 (s, 9H). LC–MS 1.2 min, 336.0 (MꢁH).
19 ꢂ 100 mm 5
l
m Waters XbridgeTM C18 column with acetonitrile
and aqueous ammonium acetate.
Benzyl bromide (10.64 lL, 0.089 mmol) was added to an ace-
tonitrile (1.0 mL) solution of tert-butyl ((4-(difluoromethoxy)
phenyl)sulfonyl)glycinate (20 mg, 0.059 mmol) followed by 2-
tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-
diazaphosphorine (BEMP) on resin (53.2 mg, 0.107 mmol). The
mixture was agitated at 70 °C overnight. The solvent was removed
and dichloromethane (2.0 mL) was added. The mixture was filtered
to remove the resin. TFA (0.7 mL) was added. After 1 h an LC–MS
showed the reaction was complete. The solvent was removed
and the crude material dissolved in methanol and purified using
preparative HPLC employing acetonitrile/water and 0.1% TFA buf-
fer with a 30 mm ꢂ 100 mm XterraTM column. The fractions con-
taining the desired product were combined. The solvent was
removed in vacuo and the title compound obtained as a white solid
5.1.2.3. 2-(N-([1,10-Biphenyl]-4-ylmethyl)-4-(difluoromethoxy)
phenylsulfonamido) acetic acid (6).
1H NMR (600 MHz,
dimethyl sulfoxide-d6 chloroform-d) d ppm 7.92 (d, J = 8.2 Hz,
2H), 7.61 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.46 (t,
J = 63 Hz (CHF2), 1H), 7.44 (t, J = 7.3 Hz, 2H), 7.36–7.28 (m, 5H),
4.47 (br s, 2H), 3.87 (br s, 2H). LC–MS: 3.5 min, 446.5 (MꢁH).
5.1.2.4.
(difluoromethoxy) phenylsulfonamido) acetic acid (7).
2-(N-((20,40-Dichloro-[1,10-biphenyl]-4-yl)methyl)-4-
1H
NMR (600 MHz, dimethyl sulfoxide-d6 chloroform-d) d ppm 7.91
(d, J = 7.6 Hz, 2H), 7.63 (br s, 1H), 7.45 (d, J = 8.8 Hz, 1H),
Table 6
General profiling of selected benzylic analogs
Compound
Metabolic stability % remaining
HERG IC50
(lM)
Cyp IC50
2C9
(
l
M)
PXR EC50 (lM)/% activation
Human
Rat
Mouse
2C19
2D6
3A4
24
16
33
29
79
78
80
77
70
77
40
15
11
20
9
3
13
4
40
40
9
6
30
40
3/140
ND
ND
100
100
67
100
ND
87
13
40
40
27
40
23
ND
Details of the assays can be found in the Supplementary material.
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10
L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 7
Pharmacokinetic profile of 24 in mice (N = 4) when dosed intravenously (IV), orally (PO), or intraperitoneally (IP)
O
O
O
S
N
O
OH
Cl
24
Cl
Route/dose (mg/kg)
t1/2 (h)
Cmax (nM)
Tmax (h)
AUCinf (nM*h)
Cl (mL/min/kg)
Vss (L/kg)
F (%)
IV/1
PO/10
IP/10
0.58 .05
0.99 .08
1.33 .25
274 40
941 68
2863 410
—
173 14
1516 123
5784 640
168.7 13.6
—
—
7.42 0.94
—
—
—
88
ꢀ100
0.5 0.1
0.63 0.25
7
7.40–7.35 (m, 3H), 7.34–7.30 (m, 4H), 7.18 (s, (CHF2), 1H), 4.50
(s, 2H), 3.90 (s, 2H). LC–MS 4.1 min, 513.8 (MꢁH).
7.97 (d, J = 8.5 Hz, 2H), 7.48 (s, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.29
(d, J = 8.2 Hz, 1H), 7.25 (d, J = 4.3 Hz, 3H), 7.24–7.20 (m, 2H),
6.82–6.39 (m, 1H), 5.15 (q, J = 6.8 Hz, 1H), 4.06 (d, J = 18.6 Hz,
1H), 3.79 (d, J = 18.3 Hz, 1H), 1.56–1.43 (m, 3H). LC–MS 1.1 min,
528.2 (MꢁH). HRMS Calcd for C23H18O5NCl2F2S: 528.0245. Found:
528.0247.
5.1.2.5.
(difluoromethoxy) phenylsulfonamido) acetic acid (8).
2-(N-((30,50-Dichloro-[1,10-biphenyl]-3-yl)methyl)-4-
1H
NMR (600 MHz, dimethyl sulfoxide-d6 chloroform-d) d ppm 7.91
(d, J = 8.2 Hz, 2H), 7.61–7.58 (m, 3H), 7.52 (br s, 1H), 7.42
(d, J = 8.2 Hz, 1H), 7.33–7.27 (m, 4H), 7.18 (s, (CHF2), 1H), 4.49
(s, 2H), 3.91 (s, 2H). LC–MS 4.4 min, 514.5 (MꢁH).
5.1.4. (R)-2-(N-(1-(20,40-Dichloro-[1,10-biphenyl]-4-yl)ethyl)-4-
(difluoromethoxy) phenylsulfonamido) acetic acid (10)
Prepared from (R)-(ꢁ)-1-(4-Bromophenyl)ethylamine in the
same manner as compound 9. 1H NMR (500 MHz, chloroform-d)
d ppm 1.50 (d, J = 7.02 Hz, 3H) 3.79 (d, J = 18.31 Hz, 1H) 4.07 (d,
J = 18.31 Hz, 1H) 5.15 (q, J = 7.02 Hz, 1H) 6.61 (t, J = 75.00 Hz, 1H)
7.20–7.25 (m, 5H) 7.29 (d, J = 8.24 Hz, 1H) 7.32 (d, J = 8.24 Hz,
2H) 7.48 (s, 1H) 7.97 (d, J = 8.55 Hz, 2H). LC–MS 1.1 min, 528.2
(MꢁH), Exact Mass: 529.0. HRMS Calcd for C23H18O5NCl2F2NaS:
552.0221. Found: 552.0208.
5.1.3. (S)-2-(N-(1-(20,40-dichloro-[1,10-biphenyl]-4-yl)ethyl)-4-
(difluoromethoxy) phenylsulfonamido) acetic acid (9)
To
a
solution of (S)-(ꢁ)-1-(4-Bromophenyl)ethylamine
(0.078 mL,
0.500 mmol) and N,N-diisopropylethylamine
(0.131 mL, 0.750 mmol) in dimethylformamide (1.0 mL), 4-(difluo-
romethoxy)benzene-1-sulfonyl chloride (121 mg, 0.500 mmol)
was added. The reaction mixture was stirred at room temperature
for 1 hour. LC–MS analysis showed the reaction was complete:
1.4 min, 404.1, 406.1 (MꢁH) Methyl bromoacetate (0.095 mL,
1.000 mmol) and BEMP (274 mg, 1.000 mmol) were added to the
reaction containing crude (S)-N-(1-(4-bromophenyl)ethyl)-4-
(difluoromethoxy)benzenesulfonamide and the reaction was
heated at 70 °C overnight. The reaction was diluted with ethyl
acetate and washed with water and brine. The organic portion
was dried over sodium sulfate. The solvent was removed, the resi-
due dissolved in methanol and purified by preparative HPLC using
a CH3CN–H2O-TFA solvent system. Obtained methyl (S)-N-(1-(4-
5.1.5. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclopropyl)-4-
(difluoromethoxy) phenylsulfonamido) acetic acid (11)
Prepared from 1-(4-bromophenyl)cyclopropanamine in the
same manner as compound 9.
1H NMR (500 MHz, chloroform-d) d ppm 1.29 (br s, 2H) 1.68 (br
s, 2H) 4.36 (s, 2H) 6.56 (t, J = 75.00 Hz, 1H) 7.10 (m, J = 8.85 Hz, 2H)
7.19–7.24 (m, 3H) 7.24–7.31 (m, 1H) 7.31–7.36 (m, 2H) 7.52 (d,
J = 1.83 Hz, 1H) 7.76 (m, J = 8.85 Hz, 2H). LC–MS 1.1 min, 539.9
(MꢁH). HRMS Calcd for
C24H18O5NCl2F2S: 540.0245. Found:
bromophenyl)ethyl)-N-((4-(difluoromethoxy)phenyl)
sulfonyl)
540.0246.
glycinate as light yellow oil.
1H NMR (500 MHz, chloroform-d) d ppm 1.49 (d, J = 6.71 Hz, 3H)
3.79 (d, J = 18.31 Hz, 1H) 4.06 (d, J = 18.62 Hz, 1H) 5.15 (q,
J = 6.82 Hz, 1H) 6.61 (t, J = 75.00 Hz, 1H) 7.20–7.27 (m, 5H) 7.27–
7.35 (m, 3H) 7.48 (s, 1H) 7.97 (d, J = 8.55 Hz, 2H). LC–MS 1.3 min
531 (M+H).
5.1.6. 2-(N-(1-(20,40-Dichlorobiphenyl-4-yl)cyclobutyl)-4-
(difluoromethoxy) phenylsulfonamido) acetic acid (12)
4-(Difluoromethoxy)benzene-1-sulfonyl chloride (170 mg,
0.700 mmol) was added to a solution of 1-(4-bromophenyl)cy-
clobutanamine HCl (184 mg, 0.7 mmol) and BEMP (384 mg,
1.400 mmol) in dimethylformamide (1.0 mL). The reaction mixture
was stirred at 70 °C overnight. Methyl bromoacetate (0.133 mL,
1.400 mmol) and additional BEMP (384 mg, 1.400 mmol) was
added and the reaction heated at 70 °C overnight. The reaction
was diluted with ethyl acetate and washed with HCl (1 N) and
brine. The organic portion was dried over sodium sulfate.The resi-
due was purified by chromatography using 4:1 hexanes/ethyl acet-
To a toluene (1.5 mL) and ethanol (1.5 mL) solution of methyl
(S)-N-(1-(4-bromophenyl)ethyl)-N-((4-(difluoromethoxy)phenyl)
sulfonyl) glycinate (60 mg, 0.125 mmol) was added 2,4-
dichlorophenylboronic acid (28.7 mg, 0.151 mmol), followed by
Na2CO3 (1 N, 0.8 mL) and tetrakis(triphenylphosphine)palladium
(0) (7.25 mg, 6.27 lmol). The mixture was degassed, placed under
argon and heated at 120 °C in microwave reactor for 30 min. LC–
MS analysis showed that the reaction was complete. The mixture
was diluted with ethyl acetate and extracted with brine. The
organic portion was dried over sodium sulfate. The solvent was
removed and residue purified using preparative HPLC employing
acetonitrile and water containing 0.1% TFA on an XterraTM
30 mm ꢂ 100 mm column. The solvent was removed from the frac-
tions containing the desired product. A colorless oil (12 mg, 17%
yield) was obtained. 1H NMR (500 MHz, chloroform-d) d ppm
ate on
a 40 g silica column to give methyl 2-(N-(1-(4-
bromophenyl)cyclobutyl)-4-(difluoromethoxy)phenylsulfon-
amido) acetate as a yellow oil (200 mg, 57% yield).
1H NMR (500 MHz, CDCl3) d ppm 1.69 (d, J = 10.68 Hz, 1H),
1.83–1.92 (m, 1H), 2.67 (t, J = 8.70 Hz, 2H), 2.79–2.91 (m, 2H),
4.13 (s, 2H), 6.54 (t, J = 75.00 Hz, 1H), 7.06 (d, J = 8.55 Hz, 2H),
7.22–7.31 (m, 1H), 7.37 (d, J = 8.24 Hz, 3H), 7.47–7.58 (m, 5H).
LC/MS 1.5 min, Molecular ion not observed.
Please cite this article in press as: Chupak, L. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.006

L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
Methyl
2-(N-(1-(4-bromophenyl)cyclobutyl)-4-(difluo-
5.1.10. 2-(4-(Difluoromethoxy)-N-(1-(20-(trifluoromethoxy)
romethoxy)phenylsulfonamido) acetate (1.15 g, 2.28 mmol) was
dissolved in THF (5 mL) and MeOH (5 mL). A solution of aqueous
NaOH (1 N, 3 mL) was added. The mixture was heated at 90 °C
for 20 min. The solution was then allowed to cool, acidified with
dilute HCl (0.1 N) until acidic by pH paper and the product parti-
tioned into ethyl acetate. The organic portion was washed with
dilute HCl (0.1 N),dried over sodium sulfate,filtered and concen-
trated under vacuum to give 2-(N-(1-(4-bromophenyl)cy-
clobutyl)-4-(difluoromethoxy)phenylsulfonamido)acetic acid as a
light yellow-colored, semi-solid, (1.05 g, 94%).
biphenyl-4-yl)cyclobutyl) phenylsulfonamido)acetic acid (16)
Prepared using 1-(4-bromophenyl)cyclobutanamine and (2-
(trifluoromethoxy)phenyl)boronic acid in the same manner as
compound 9.
1H NMR (500 MHz, CDCl3) d ppm 1.66–1.75 (m, 1H) 1.84–1.92
(m, 1H) 2.58–2.72 (m, 2H) 2.87 (dt, J = 11.67, 9.88 Hz, 2H) 4.13
(s, 2H) 6.51 (t, J = 75.00 Hz, 1H) 7.04 (d, J = 8.85 Hz, 2H) 7.38–
7.45 (m, 6H) 7.51–7.55 (m, 4H). 1H NMR (500 MHz, DMSO-d6) d
ppm 1.74 (m, 1H) 1.99 (m, 1H) 2.46 (m, 2H) 2.53–2.63 (m, 2H)
3.54 (m, 2H) 6.96–7.14 (m, 1H) 7.31–7.59 (m, 9H) 7.85 (d,
J = 8.55 Hz, 2H). LC/MS 1.1 min, Mꢁ1: 570.0, Exact Mass: 571.1.
HRMS Calcd for C26H21O6NF5S: 570.1004. Found: 570.1004.
1H NMR (500 MHz, CDCl3) d ppm 1.63 (d, J = 10.68 Hz, 1H), 1.84
(dd, J = 9.31, 1.68 Hz, 1H), 2.57–2.72 (m, 2H), 2.76–2.92 (m, 2H),
4.09 (s, 2H), 6.57 (t, J = 75 Hz (C-F), 1H), 7.02 (m, J = 8.85 Hz, 2H),
7.27–7.31 (m, 2H), 7.41–7.46 (m, 2H), 7.47 (m, 2H). LC/MS
1.0 min, Mꢁ1: 489.9, Exact Mass: 491.0. HRMS Calcd for C19H17O5-
NBrF2S: 487.9973. Found: 487.9974.
5.1.11. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclobutyl)
phenylsulfonamido)acetic acid (17)
Prepared using 1-(3-bromophenyl)cyclobutanamine, benzene
sulfonylchloride and (3,5-dichlorophenyl)boronic acid in the same
manner as compound 9.
The 2-(N-(1-(4-bromophenyl)cyclobutyl)-4-(difluoromethoxy)
phenylsulfonamido)acetic acid (40 mg, 0.082 mmol) was dissolve
in 1 mL of dimethylformamide. To this solution was added 2,4-
dichlorophenylboronic acid (31.1 mg, 0.163 mmol), followed by
sodium carbonate (15.13 mg, 0.143 mmol)and palladium(II) acet-
ate (3.66 mg, 0.016 mmol). The mixture was placed under argon
and heated at 120 °C in a microwave reactor for 20 min. LC/MS
showed the reaction was ꢀ40% complete. The mixture was filtered
and purified using reverse phase HPLC employing gradient of ace-
tonitrile in water with 0.1% TFA on a XterraTM 30 mm ꢂ 100 mm
column. The fractions containing product were combined and the
solvent was removed in vacuo to obtain 12 as a light gray solid.
1H NMR (500 MHz, CDCl3) d 7.57–7.48 (m, 5H), 7.39–7.32 (m,
3H), 7.28–7.26 (m, 1H), 7.06 (d, J = 8.5 Hz, 2H), 6.71–6.34 (m,
1H), 4.13 (s, 2H), 2.87 (d, J = 10.4 Hz, 2H), 2.67 (t, J = 8.7 Hz, 2H),
1.93–1.82 (m, 1H), 1.76–1.62 (m, 1H). LC/MS 1. 2 min, Mꢁ1:
554.0, Exact Mass: 555.0.
1H NMR (500 MHz, CDCl3) d ppm 1.62–1.70 (m, J = 10.60, 10.60,
7.78, 2.75 Hz, 1H) 1.82–1.90 (m, 1H) 2.59–2.67 (m, 2H) 2.85–2.94
(m, 2H) 4.17 (s, 2H) 7.27 (d, J = 2.14 Hz, 2H) 7.31–7.38 (m, 3H)
7.39–7.50 (m, 4H) 7.52–7.59 (m, 3H). LC/MS 1.5 min, Mꢁ1:
488.2, Exact Mass: 489.1. HRMS Calcd for
540.0245. Found: 540.0246.
C24H18O5NCl2F2S:
5.1.12. 2-(2-Chloro-N-(1-(30,50-dichlorobiphenyl-3-
yl)cyclobutyl) phenylsulfonamido) acetic acid (18)
Prepared
using
1-(3-bromophenyl)cyclobutanamine,
2-chlorobenzene sulfonylchloride and (3,5-dichlorophenyl)boronic
acid in the same manner as compound 9.
1H NMR (500 MHz, CDCl3) d ppm 1.51–1.58 (m, 1H) 1.73–1.80
(m, 1H) 2.53–2.60 (m, 2H) 2.91–2.98 (m, 2H) 4.47 (s, 2H) 7.15–
7.22 (m, 1H) 7.29–7.38 (m, 7H) 7.49–7.55 (m, 2H) 7.83 (dd,
J = 8.09, 1.37 Hz, 1H). LC/MS 1.6 min, Mꢁ1: 525.0, Exact Mass:
523.0. HRMS Calcd for C24H19O4NCl3S: 522.0095. Found: 522.0098.
5.1.7. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclobutyl)-4-
(difluoromethoxy) phenylsulfonamido)acetic acid (13)
Prepared from 1-(3-bromophenyl)cyclobutanamine in the same
manner as compound 12.
5.1.13. 2-(3-Chloro-N-(1-(30,50-dichlorobiphenyl-3-
yl)cyclobutyl)phenylsulfonamido) acetic acid (19)
Prepared
using
1-(3-bromophenyl)cyclobutanamine,
1H NMR (400 MHz, CD3OD) d ppm 1.58–1.69 (m, 1H) 1.78–1.88
(m, 1H) 2.56–2.65 (m, 2H) 2.97–3.08 (m, 2H) 4.18 (s, 2H) 6.69 (t,
J = 75.00 Hz, 1H) 6.90–6.99 (m, 2H) 7.38–7.44 (m, 5H) 7.44–7.49
(m, 2H) 7.59 (t, J = 1.76 Hz, 1H) 7.64 (dd, J = 7.65, 1.38 Hz, 1H).
LC/MS 1.6 min, Mꢁ1: 554.0, Exact Mass: 555.0.
3-chlorobenzene sulfonylchloride and (3,5-dichlorophenyl)boronic
acid in the same manner as compound 9.
1H NMR (500 MHz, CDCl3) d ppm 1.64–1.72 (m, 1H) 1.89 (m,
1H) 2.63–2.69 (m, 2H) 2.84–2.92 (m, 2H) 4.25 (s, 2H) 7.23–7.28
(m, 2H) 7.28–7.31 (m, 2H) 7.34–7.38 (m, 2H) 7.39–7.47 (m, 3H)
7.48–7.50 (m, 1H) 7.53–7.56 (m, 1H). LC/MS 1.6 min, Mꢁ1:
525.0, Exact Mass: 523.0. HRMS Calcd for
522.0095. Found: 522.0098.
C24H19O4NCl3S:
5.1.8. 2-(4-(Difluoromethoxy)-N-((20-(trifluoromethoxy)-[1,10-
biphenyl]-4-yl)methyl) phenylsulfonamido)acetic acid (14)
Prepared using 4-(difluoromethoxy)benzene-1-sulfonyl chlo-
ride in the same manner as compound 26.
5.1.14. 2-(4-Chloro-N-(1-(30,50-dichlorobiphenyl-3-
yl)cyclobutyl)phenylsulfonamido) acetic acid (20)
1H NMR (500 MHz, CDCl3) d ppm 3.96 (s, 2H), 4.47(s, 2H), 6.58
(t, J = 75 Hz (–CHF2), 1H), 7.12 (m, J = 8.6 Hz, 2H) 7.20 (d, J = 7.9 Hz,
2H) 7.35 (d, J = 8.2 Hz, 2H) 7.35–7.40 (m, 4H) 7.75 (m, 2H) 9.88 (br.
s, 1H). LC/MS 1.1 min, Mꢁ1: 556.0, Exact Mass: 557.1.
Prepared
using
1-(3-bromophenyl)cyclobutanamine,
4-
chlorobenzene sulfonylchloride and (3,5-dichlorophenyl)boronic
acid in the same manner as compound 9.
1H NMR (500 MHz, CDCl3) d ppm 1.63–1.71 (m, 1H) 1.87 (m,
1H) 2.60–2.66 (m, 2H) 2.83–2.91 (m, 2H) 4.22 (s, 2H) 7.23–7.27
(m, 2H) 7.27–7.30 (m, 2H) 7.38 (t, J = 1.83 Hz, 1H) 7.40–7.46 (m,
4H) 7.47–7.49 (m, 1H) 7.54–7.59 (m, 1H). LC/MS 1.6 min, Mꢁ1:
5.1.9. 2-(4-(Difluoromethoxy)-N-(1-(20-(trifluoromethoxy)
biphenyl-4-yl)cyclopropyl)phenylsulfonamido)acetic acid (15)
Prepared using 1-(4-bromophenyl)cyclopropanamine and
(2-(trifluoromethoxy)phenyl)boronic acid in the same manner as
compound 9.
525.0, Exact Mass: 523.0. HRMS Calcd for
522.0095. Found: 522.0098.
C24H19O4NCl3S:
1H NMR (500 MHz, CDCl3) d ppm 1.31 (br s, 2H) 1.71 (br s, 2H)
4.39 (s, 2H) 6.56 (t, J = 75.00 Hz (C-F), 1H) 7.13 (m, J = 8.55 Hz, 2H)
7.21 (d, J = 7.93 Hz, 2H) 7.35 (d, J = 8.24 Hz, 2H) 7.37–7.42 (m, 4H)
7.77 (m, 2H) 9.88 (br s, 1H). LC/MS 1.1 min, Mꢁ1: 556.0, Exact
Mass: 557.1.
5.1.15. 2-(4-Cyano-N-(1-(30,50-dichlorobiphenyl-3-yl)cyclobutyl)
phenylsulfonamido) acetic acid (21)
Prepared
using
1-(3-bromophenyl)cyclobutanamine,
4-
cyanobenzene sulfonylchloride and (3,5-dichlorophenyl)boronic
acid in the same manner as compound 9.
Please cite this article in press as: Chupak, L. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.006

12
L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
1H NMR (500 MHz, CDCl3) d ppm 1.67 (dd, J = 7.78, 2.90 Hz, 1H)
tert-butyl 2-aminoacetate (2.308 mL, 16.39 mmol), Hunig’s base
(3.82 mL, 21.85 mmol) and a catalytic amount of DMAP. The
orange–red colored mixture was stirred at 25 °C overnight and
slowly turned bright yellow. The crude reaction mixture was
diluted with ethyl acetate and sequentially extracted with HCl
(aq), NaHCO3 (aq) and brine. The organic portion was dried over
MgSO4 and the solvent removed to leave a white powder. After
drying under vacuum overnight, tert-butyl 2-(2,2-dimethylchro-
man-6-sulfonamido)acetate (3.6 g, 9.62 mmol, 88% yield) was iso-
lated in sufficient purity to use directly in the next step with no
further purification.
1.83–1.90 (m, 1H) 2.58–2.68 (m, 2H) 2.80–2.89 (m, 2H) 4.29 (s, 2H)
7.30 (d, J = 1.83 Hz, 2H) 7.38–7.42 (m, 2H) 7.45 (d, J = 7.93 Hz, 1H)
7.52–7.56 (m, 2H) 7.58 (m, 2H) 7.63 (m, 2H). LC/MS 2.1 min, Mꢁ1:
513.2, Exact Mass: 514.1. HRMS Calcd for
513.0437. Found: 513.0436.
C25H19O4N2Cl2S:
5.1.16. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclobutyl)-3-
methoxyphenylsulfonamido) acetic acid (22)
Prepared using 1-(3-bromophenyl)cyclobutanamine, 3-meth-
oxy benzene sulfonyl chloride and (3,5-dichlorophenyl)boronic
acid in the same manner as compound 9.
1H NMR (500 MHz, CDCl3) d 7.62–7.48 (m, 2H), 6.83 (d,
J = 8.5 Hz, 1H), 3.64 (d, J = 5.2 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H), 1.81
(t, J = 6.9 Hz, 2H), 1.35 (s, 9H), 1.34 (s, 6H)
1H NMR (500 MHz, CDCl3) d ppm 1.62–1.69 (m, 1H) 1.86 (q,
J = 9.36 Hz, 1H) 2.63 (t, J = 8.85 Hz, 2H) 2.89 (dd, J = 10.22,
2.59 Hz, 2H) 3.72 (s, 3H) 4.20 (s, 2H) 6.96 (dd, J = 7.78, 1.98 Hz,
1H) 7.02–7.04 (m, 1H) 7.17 (d, J = 8.54 Hz, 1H) 7.25 (t, J = 7.93 Hz,
1H) 7.37 (t, J = 1.83 Hz, 1H) 7.39–7.43 (m, 2H) 7.48 (s, 2H) 7.57
(d, J = 6.41 Hz, 1H). LC/MS 1.7 min, Mꢁ1: 518.2, Exact Mass:
To a solution of tert-butyl 2-(2,2-dimethylchroman-6-sulfon-
amido)acetate (500 mg, 1.407 mmol) in acetonitrile (15 mL) was
added BEMP-resin (786 mg, 1.547 mmol) followed by 1-bromo-
4-(bromomethyl)benzene (602 mg, 2.409 mmol). The mixture
was heated in microwave reactor at 90 °C for 3 h. The resin was
removed by filtration and rinsed alternately with dichloro-
methane and methanol. The combined organic portions were con-
centrated by rotary evaporation to give an orange red residue.
The residue was dissolved in hexanes and a minimum amount
519.1. HRMS Calcd for
542.0563.
C25H23O5NCl2NaS: 542.0566. Found:
5.1.17. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclobutyl)-2-
methoxyphenylsulfonamido) acetic acid (23)
Prepared using 1-(3-bromophenyl)cyclobutanamine, 2-meth-
oxy benzene sulfonyl chloride and (3,5-dichlorophenyl)boronic
acid in the same manner as compound 9.
of dichloromethane, charged to a silica-column and chro-
matographed using a gradient of 10–20% ethyl acetate in hexanes
over 3 column volumes. The fractions containing the product
were combined and the solvent was removed to obtain clear
oil. The oil solidified whiling drying under high vacuum overnight
to provide tert-butyl 2-(N-(4-bromobenzyl)-2,2-dimethylchro-
man-6-sulfonamido)acetate (632 mg, 1.205 mmol, 86% yield) as
a white solid.
1H NMR (500 MHz, CDCl3) d ppm 1.50–1.60 (m, 1H) 1.70–1.82
(m, 1H) 2.54–2.63 (m, 2H) 2.81–2.90 (m, 2H) 3.83 (s, 3H) 4.43 (s,
2H) 6.80 (d, J = 8.54 Hz, 1H) 6.83 (t, J = 7.63 Hz, 1H) 7.28–7.31 (m,
4H) 7.32–7.35 (m, 1H) 7.36 (t, J = 1.83 Hz, 1H) 7.48–7.53 (m, 2H)
7.60 (dd, J = 7.93, 1.53 Hz, 1H). LC/MS 1.7 min, Mꢁ1: 518.2, Exact
Mass: 519.1. HRMS Calcd for C25H23O5NCl2NaS: 542.0566. Found:
542.0563.
1H NMR (500 MHz, CDCl3) d 7.56 (s, 2H), 7.43 (d, J = 8.2 Hz, 2H),
7.14 (d, J = 8.5 Hz, 2H), 6.89–6.81 (m, 1H), 4.45 (s, 2H), 3.78 (s, 2H),
2.79 (s, 2H), 1.83 (t, J = 6.7 Hz, 2H), 1.39–1.28 (m, 15H).
The final compounds were obtained using parallel synthesis
techniques as follows:
5.1.18. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclobutyl)-2,2-
dimethylchroman-6-sulfonamido)acetic acid (24)
Prepared using 1-(3-bromophenyl)cyclobutanamine, 2,2-
dimethylchromane-6-sulfonyl chloride and (3,5-dichlorophenyl)
boronic acid in the same manner as compound 9.
A series of ArB(OH)2 (0.210 mmol) were individually weighed
into separate reaction vials. To each tube was added an aqueous
solution of K3PO4 (0.140 mL, 0.280 mmol). A dimethylformamide
(1.5 mL) solution of tert-butyl 2-(N-(4-bromobenzyl)-2,2-
dimethylchroman-6-sulfonamido)acetate (36.7 mg, 0.070 mmol)
was distributed to each reaction tube, followed by Pd(Ph3P)4
(8.09 mg, 7.00 lmol (amount estimated without weighing)). The
reaction vial was immediately sealed under nitrogen and heated
in an oil bath at 100–105 °C overnight. The crude reaction mix-
ture was filtered through a 0.45 lm PVDF to give a clear solution
which was purified by direct injection onto a preparative HPLC
and eluted with an 50–100% step-gradient of acetonitrile in a
water 0.1% TFA buffer system. The fractions containing the
desired product were combined and the solvent removed in
vacuo. To hydrolyze the tert-butyl ester, the residue was dissolved
in dichloromethane (2 mL) and TFA (1 mL) and then stirred at
room temperature for 1 hour. The solvent was removed under
vacuum. The crude reaction mixture was filtered through a
1H NMR (500 MHz, CDCl3) d ppm 1.26 (s, 6H) 1.67 (t, J = 6.71 Hz,
3H) 1.85–1.94 (m, 1H) 2.53 (t, J = 6.56 Hz, 2H) 2.67 (t, J = 8.85 Hz,
2H) 2.83–2.92 (m, 2H) 4.14 (s, 2H) 6.68 (d, J = 8.85 Hz, 1H) 6.89
(d, J = 1.83 Hz, 1H) 7.23–7.27 (m, 3H) 7.36 (t, J = 1.83 Hz, 1H)
7.43–7.48 (m, 2H) 7.49 (s, 1H) 7.60 (dd, J = 5.49, 2.44 Hz, 1H). 13C
NMR (101 MHz, chloroform-d) d 173.2, 157.9, 143.8, 143.4, 138.7,
135.4, 130.4, 129.2, 128.8, 127.3, 127.0, 126.9, 126.3, 126.1,
125.6, 121.0, 117.4, 75.6, 65.8, 48.3, 35.2, 32.1, 26.8, 22.2, 14.7.
LC/MS 1.7 min, Mꢁ1: 572.1, Exact Mass: 573.1. HRMS Calcd for
C29H30O5NCl2S: 574.1216. Found: 574.1206.
5.1.19. 2-(N-((30,50-Dichloro-[1,10-biphenyl]-3-yl)methyl)-2,2-
dimethylchroman-6-sulfonamido)acetic acid (25)
Prepared from 1-bromo-3-(bromomethyl)benzene in the same
manner as compound 26.
1H NMR (500 MHz, CDCl3) d 7.61–7.57 (m, 2H), 7.44 (d,
J = 7.8 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 1.8 Hz, 2H),
7.34 (s, 1H), 7.32 (t, J = 1.8 Hz, 1H), 7.26 (d, J = 7.0 Hz, 1H), 6.87–
6.83 (m, 1H), 4.51 (s, 2H), 3.95 (s, 2H), 2.77 (t, J = 6.7 Hz, 2H),
1.79 (t, J = 6.7 Hz, 2H), 1.32 (s, 6H). LC/MS 2.5 min, Mꢁ1: 532.2,
Exact Mass: 533.1.
0.45 lm PVDF to give a clear solution which was purified by
direct injection onto a preparative HPLC and eluted with a 40–
100% step-gradient of acetonitrile in a water 0.1%TFA buffer sys-
tem. The fractions containing the desired product were combined
and the solvent removed in vacuo. The purified compounds were
dried under high vacuum overnight.
1H NMR (500 MHz, CDCl3) d 7.62–7.57 (m, 2H), 7.46 (d,
J = 8.2 Hz, 2H), 7.41 (d, J = 1.8 Hz, 2H), 7.33 (t, J = 1.8 Hz, 1H),
7.31 (d, J = 8.2 Hz, 2H), 6.85 (d, J = 8.5 Hz, 1H), 4.48 (s, 2H), 3.95
(s, 2H), 2.80 (t, J = 6.7 Hz, 2H), 1.83 (t, J = 6.7 Hz, 2H), 1.35 (s,
6H). LC/MS 2.5 min, Mꢁ1: 532.2, Exact Mass: 533.1.
5.1.20. 2-(N-((30,50-Dichloro-[1,10-biphenyl]-4-yl)methyl)-2,2-
dimethylchroman-6-sulfonamido)acetic acid (26)
To a ꢁ30 °C solution of 2,2-dimethylchroman-6-sulfonyl chlo-
ride (3.165 g, 10.92 mmol) in dichloromethane (50 mL) was added
Please cite this article in press as: Chupak, L. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.006

L. S. Chupak et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
5.1.21. 2-(N-(1-(30,50-Dichlorobiphenyl-3-yl)cyclopentyl)-2,2-
dimethylchroman-6-sulfonamido)acetic acid (27)
1H NMR (400 MHz, CDCl3) d 7.93 (d, J = 8.8 Hz, 2H), 7.52–7.46
(m, 1H), 7.45–7.38 (m, 1H), 7.38–7.33 (m, 1H), 7.32–7.30 (m,
1H), 7.27–7.21 (m, 4H), 7.20–7.15 (m, 1H), 6.60 (t, J = 72.6 Hz,
1H), 4.59 (s, 2H), 4.04 (s, 2H). LC/MS 3.9 min, Mꢁ1: 514.5, Exact
Mass: 515.0. HRMS Calcd for C22H16O5NCl2F2S: 514.0089. Found:
514.0089.
Prepared using 1-(3-bromophenyl)cyclopentanamine, 2,2-
dimethylchromane-6-sulfonyl chloride and (3,5-dichlorophenyl)
boronic acid in the same manner as compound 9.
1H NMR (500 MHz, CD3OD)
d
ppm 1.28 (s, 6H)
1.69 (t, J = 6.71 Hz, 2H) 2.35–2.42 (m, 2H) 2.50–2.65 (m, 4H)
2.74–2.80 (m, 2H) 2.88 (t, J = 6.87 Hz, 2H) 4.33 (s, 2H) 6.61 (d,
J = 8.85 Hz, 1H) 7.08 (d, J = 2.14 Hz, 1H) 7.29 (dd, J = 8.55, 2.44 Hz,
1H) 7.45 (m, 4H) 7.59 (d, J = 3.05 Hz, 1H) 7.61 (m, 2H). LC/MS
1.9 min, Mꢁ1: 586.3, Exact Mass: 587.1.
5.1.27. 2-(4-(Difluoromethoxy)-N-((20-(trifluoromethoxy)-[1,10-
biphenyl]-3-yl)methyl) phenylsulfonamido)acetic acid (33)
Prepared using 4-(Difluoromethoxy)benzene-1-sulfonyl chlo-
ride,
1-bromo-3-(bromomethyl)benzene
and
(2-trifluo-
romethoxyphenyl)boronic acid in the same manner as compound
26.
5.1.22. 2-(N-(3-(30,50-Dichlorobiphenyl-4-yl)tetrahydrofuran-3-
yl)-2,2-dimethylchroman-6-sulfonamido) acetic acid (28)
Prepared using 3-(4-bromophenyl)tetrahydrofuran-3-amine,
2,2-dimethylchromane-6-sulfonyl chloride and (3,5-dichlorophe-
nyl)boronic acid in the same manner as compound 9.
1H NMR (600 MHz, DMSO/CDCl3) d 7.91 (d, J = 8.2 Hz, 2H), 7.51–
7.35 (m, 6H), 7.32–7.24 (m, 4H), 4.51 (s, 2H), 3.89–3.79 (m, 2H) LC/
MS 4.0 min, Mꢁ1: 530.6, Exact Mass: 531.1.
1H NMR (500 MHz, CDCl3) d ppm 1.39 (s, 6H) 1.87 (t, J = 6.71 Hz,
2H) 2.31–2.45 (m, 2H) 2.82–2.90 (m, 1H) 2.90–2.97 (m, 1H) 3.51–
3.63 (m, 1H) 4.02–4.07 (m, 1H) 4.35 (q, J = 18.31 Hz, 2H) 4.46 (d,
J = 9.46 Hz, 1H) 4.64 (d, J = 9.46 Hz, 1H) 6.53 (d, J = 8.55 Hz, 1H)
6.91 (d, J = 8.55 Hz, 1H) 7.00 (s, 1H) 7.16 (dd, J = 8.70, 1.98 Hz,
1H) 7.32–7.39 (m, 2H) 7.41 (d, J = 1.83 Hz, 2H) 7.62–7.72 (m, 2H).
LC/MS 1.6 min, Mꢁ1: 588.2, Exact Mass: 589.1.
5.1.28. 2-(3,4-Dichloro-N-(1-(20,40-dichloro-[1,10-biphenyl]-3-
yl)cyclobutyl) phenylsulfonamido)acetic acid (34)
Prepared using 3,4-dichlorobenzene-1-sulfonyl chloride,
1-bromo-3-(bromomethyl)benzene and (2,4-dichlorophenyl)boro-
nic acid in the same manner as compound 26.
1H NMR (500 MHz, CDCl3) d ppm 1.65–1.73 (m, 1H) 1.88 (d,
J = 10.68 Hz, 1H) 2.66 (t, J = 8.85 Hz, 2H) 2.82–2.92 (m, 2H) 4.18
(s, 2H) 7.19 (d, J = 8.24 Hz, 1H) 7.33–7.42 (m, 5H) 7.46 (d,
J = 1.22 Hz, 1H) 7.47–7.53 (m, 3H). ESI-MS m/z 560 (MH+).
5.1.23. 2-(N-([1,10-Biphenyl]-3-ylmethyl)-2,2-
dimethylchroman-6-sulfonamido)acetic acid (29)
Prepared using 3-(bromomethyl)-1,10-biphenyl and 2,2-
dimethylchromane-6-sulfonyl chloride in the same manner as
compound 5.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.02.006.
1H NMR (500 MHz, CDCl3) d 7.61 (d, J = 6.3 Hz, 1H), 7.60 (s, 1H),
7.51 (d, 1H), 7.50 (d, J = 7.4 Hz, 2H), 7.41 (dd, J = 13.4, 5.5 Hz, 2H),
7.39 (s, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.34 (t, J = 7.3 Hz, 1H), 7.19
(d, J = 7.6 Hz, 1H), 6.87–6.83 (m, 1H), 4.52 (s, 2H), 3.97 (s, 2H),
2.77 (t, J = 6.7 Hz, 2H), 1.79 (t, J = 6.7 Hz, 2H), 1.33 (s, 6H). LC/MS
1.4 min, Mꢁ1: 464.3, Exact Mass: 465.2.
References and notes
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5.1.24. 2-(2,2-Dimethyl-N-(quinolin-8-ylmethyl)chroman-6-
sulfonamido)acetic acid (30)
Prepared
using
8-(bromomethyl)quinoline
and
2,2-
dimethylchromane-6-sulfonyl chloride in the same manner as
compound 5.
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1H NMR (600 MHz, DMSO/CDCl3) d 8.85–8.81 (m, 1H), 8.29 (d,
J = 8.2 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 7.0 Hz, 1H),
7.52 (d, J = 7.6 Hz, 1H), 7.51–7.47 (m, 1H), 7.42–7.37 (m, 2H),
6.69 (d, J = 8.2 Hz, 1H), 5.03 (s, 2H), 4.06 (s, 2H), 2.67 (t,
J = 6.7 Hz, 2H), 1.76 (t, J = 6.7 Hz, 2H), 1.29 (s, 6H). LC/MS 3.4 min,
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441.1479. Found: 441.1466.
5.1.25. (R)-2-(4-(Difluoromethoxy)-N-(1-phenylethyl)
phenylsulfonamido)acetic acid (31)
Prepared using (R)-1-phenylethan-1-amine and 4-(difluo-
romethoxy)benzene-1-sulfonyl chloride in the same manner as
compound 9.
1H NMR (500 MHz, chloroform-d) d ppm 1.46 (d, J = 7.02 Hz, 3H)
3.73 (d, J = 18.31 Hz, 1H) 4.01 (d, J = 18.62 Hz, 1H) 5.10 (q,
J = 7.02 Hz, 1H) 6.61 (t, J = 75 Hz (H-F), 1H) 7.15 (br s, 1H) 7.16
(d, J = 2.44 Hz, 1H) 7.19–7.36 (m, 5H) 7.96 (d, J = 8.85 Hz, 2H). LC/
MS 0.8 min, Mꢁ1: 384.3, Exact Mass: 385.1.
5.1.26. 2-(N-((20,30-Dichloro-[1,10-biphenyl]-3-yl)methyl)-4-
(difluoromethoxy) phenylsulfonamido)acetic acid (32)
Prepared using 4-(Difluoromethoxy)benzene-1-sulfonyl chlo-
ride, 1-bromo-4-(bromomethyl)benzene and (2,3-dichlorophenyl)
boronic acid in the same manner as compound 26.
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