Design, synthesis, and evaluation of 4- and 5-substituted o-(octanesulfonamido)benzoic acids as inhibitors of glycerol-3-phosphate acyltransferase

Article abstract of DOI:10.1039/c4md00126e

Despite a rising demand for anti-obesity therapeutics, few effective pharmacological options are clinically available that target the synthesis and accumulation of body fat. Moderate inhibition of mammalian glycerol-3-phosphate acyltransferase (GPAT) with 2-(alkanesulfonamido)benzoic acids has recently been described in vitro, accompanied by promising weight loss in vivo. In silico docking studies with 2-(octanesulfonamido)benzoic acid modeled into the active site of squash GPAT revealed an unoccupied volume lined with hydrophobic residues proximal to C-4 and C-5 of the benzoic acid ring. In an effort to produce more potent GPAT inhibitors, a series of 4- and 5-substituted analogs were designed, synthesized, and evaluated for inhibitory activity. In general, compounds containing hydrophobic substituents at the 4- and 5-positions, such as biphenyl and alkylphenyl hydrocarbons, exhibited an improved inhibitory activity against GPAT in vitro. The most active compound, 4-([1,1′- biphenyl]-4-carbonyl)-2-(octanesulfonamido)benzoic acid, demonstrated an IC ₅₀ of 8.5 μM and represents the best GPAT inhibitor discovered to date. Conversely, further substitution with hydroxyl or fluoro groups, led to a 3-fold decrease in activity. These results are consistent with the presence of a hydrophobic pocket and may support the binding model as a potential tool for developing more potent inhibitors.

Full text of DOI:10.1039/c4md00126e

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CONCISE ARTICLE
Design, synthesis, and evaluation of 4- and 5-
substituted o-(octanesulfonamido)benzoic acids as
inhibitors of glycerol-3-phosphate acyltransferase†
Cite this: Med. Chem. Commun., 2014,
5, 826
Victor K. Outlaw,a Edward A. Wydysh,a Aravinda Vadlamudi,b Susan M. Medghalchib
a
*
and Craig A. Townsend
Despite a rising demand for anti-obesity therapeutics, few effective pharmacological options are clinically
available that target the synthesis and accumulation of body fat. Moderate inhibition of mammalian
glycerol-3-phosphate acyltransferase (GPAT) with 2-(alkanesulfonamido)benzoic acids has recently been
described in vitro, accompanied by promising weight loss in vivo. In silico docking studies with
2-(octanesulfonamido)benzoic acid modeled into the active site of squash GPAT revealed an unoccupied
volume lined with hydrophobic residues proximal to C-4 and C-5 of the benzoic acid ring. In an effort to
produce more potent GPAT inhibitors, a series of 4- and 5-substituted analogs were designed,
synthesized, and evaluated for inhibitory activity. In general, compounds containing hydrophobic
substituents at the 4- and 5-positions, such as biphenyl and alkylphenyl hydrocarbons, exhibited an
improved inhibitory activity against GPAT in vitro. The most active compound, 4-([1,10-biphenyl]-4-
carbonyl)-2-(octanesulfonamido)benzoic acid, demonstrated an IC50 of 8.5 mM and represents the best
GPAT inhibitor discovered to date. Conversely, further substitution with hydroxyl or fluoro groups, led to
a 3-fold decrease in activity. These results are consistent with the presence of a hydrophobic pocket and
may support the binding model as a potential tool for developing more potent inhibitors.
Received 17th March 2014
Accepted 2nd May 2014
DOI: 10.1039/c4md00126e
www.rsc.org/medchemcomm
primary hydroxyl group of glycerol-3-phosphate (G3P), the rst
committed and rate-limiting step in the biosynthesis of TAG.9 In
Introduction
mammals, mitochondrial GPAT (mtGPAT) is an integral
membrane protein that co-localizes in the mitochondrion with
Obesity, dened as having a Body Mass Index (BMI) of 30 or
higher, has been implicated in multiple diseases, including
cardiovascular disease,1 type-2 diabetes,2 hypertension, nonal-
coholic fatty liver disease,3 infertility,4 and certain types of
cancer.5 According to the World Health Organization, it affects
approximately 500 million adults and is the h leading risk of
deaths globally, accounting for at least 2.8 million deaths a year.
Despite an increase in obesity and obesity-related diseases, few
effective pharmacological therapies currently exist to combat
the physiological source of obesity, the accumulation of tri-
acylglycerol (TAG). One approach involves exploiting the
mechanisms of the lipid metabolic pathway, either by
decreasing the rate of fat synthesis, as shown with the small
molecule fatty acid synthase (FAS) inhibitors C756 and cer-
ulenin,7 or increasing the rate of fatty acid oxidation.8 GPAT
catalyzes the transesterication of fatty acyl-CoAs with the
carnitine-palmitoyl transferase
I (CPT1), the rate-limiting
enzyme in b-oxidation. These enzymes are co-regulated and
compete for fatty acyl-CoA substrates.10 Inhibition of GPAT by a
small molecule inhibitor has the potential to reduce de novo
synthesis of TAG and to divert the ux of fatty acyl-CoAs to
b-oxidation mediated by CPT1, thus making GPAT an attractive
therapeutic target against obesity.
Recently, it has been demonstrated that o-(alka-
nesulfonamido)benzoic acids such as 1a–b (Fig. 1) possess
aDepartment of Chemistry, The Johns Hopkins University, Remsen Hall, 3400 N.
Charles Street, Baltimore, MD 21218, USA. E-mail: ctownsend@jhu.edu
bFASGEN, Inc., UMB Research Park, Building One, 800 W. Baltimore St., Suite 150,
Baltimore, MD 21201, USA
† Electronic supplementary information (ESI) available: Experimental details
including synthetic procedures and characterization data for all novel
compounds is available online free of charge. See DOI: 10.1039/c4md00126e
Fig. 1 Structure of o-(alkanesulfonamido)benzoic acid GPAT inhibi-
tors (1a–c) and proposed analogs.
826 | Med. Chem. Commun., 2014, 5, 826–830
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Fig. 2 (A) Compound 1c (yellow) docked into the active site of squash GPAT using the CDOCKER protocol with Accelrys Discovery Studio
(version 2.1). (B) Compound 14b manually docked into the binding site obtained for compound 1c.
moderate inhibitory activity against mammalian GPAT in vitro.11
In an attempt to improve upon the inhibitory activity of 1c, a
The 8-carbon derivative 1c has also been synthesized and series of potential inhibitors was designed and synthesized
exhibits an inhibitory activity comparable to 1b.12 The benzoic with various aryl substituents either directly-bound to the 4- or
acid moiety of these compounds, which is deprotonated at 5-positions of 1c or coupled via one of several linkers (keto-,
physiological pH, is believed to mimic the anionic phosphate vinyl-, or ethyl-). The aryl substituents were chosen to promote
group of the native substrate. The relatively acidic aryl sulfon- p-stacking with the aromatic residues in the potential hydro-
amide hydrogen was proposed to interact with the catalytic phobic channel as well as for synthetic accessibility. The linkers
histidine responsible for deprotonating the primary hydroxyl were chosen to examine the effects of pharmacophore separa-
group of G3P necessary for acylation. The long alkyl chain of the tion and conformational rigidity versus exibility. The 8-carbon
sulfonamide was designed to bind the lipophilic palmitoyl-CoA chain of 1c was chosen for its ready commercial availability and,
binding site.
within experimental error, essentially unchanged GPAT inhibi-
While structural information regarding mammalian GPAT is tory activity compared to the nonyl side chain reported previ-
limited, X-ray crystal structures of squash GPAT revealed the ously.11 The candidates were tested for GPAT inhibition in vitro
conserved catalytic HXXXXD motif present in all GPATs as well to develop a structure–activity relationship and assess the val-
as a putative G3P binding site comprised of conserved cationic idity of the docking model as a tool to direct future medicinal
residues Lys193, Arg235, and Arg237.13a–b A complex network of chemistry efforts.
relatively hydrophobic pockets and tunnels emanating from the
active site was also observed. Protein homology-based model-
ling of multiple human transferases, including AGPAT1,
AGPAT2, and AGPAT9, with the squash GPAT crystal structure
showed that, despite low sequence homology, the human
Results and discussion
Synthesis of 4- and 5-substituted analogs
transferases are similar in predicted secondary structure and An efficient, divergent route was devised to access the 4- and 5-
hydrophobic residue distribution.14 These ndings suggest substituted derivatives of 1c as shown in Scheme 1. Sulfon-
that, although the overall architectures of the acyltransferase amide coupling of 4- and 5-bromoanthranilates 2 and 3,
homologs differ, structural similarity in the region of the active respectively, with octanesulfonyl chloride generated sulfon-
site may be good. An in silico docking simulation of 1c with amides 4 and 5. Suzuki–Miyaura coupling reactions with
squash GPAT, as shown in Fig. 2, displayed the inhibitor bound various aryl boronic acids were then performed either under
at the putative G3P binding pocket with the carboxylate anion argon to afford coupling products 6a–l and 7a–l or under an
positioned at the phosphate binding site and the long alkyl atmosphere of carbon monoxide to afford keto-linker precur-
chain residing in the putative palmitoyl-CoA binding site sors 8a–b and 9a–d. Alternatively, coupling of 4 and 5 with
described by Turnbull et al.13a Furthermore, the docking model various trans-arylvinyl boronic acids under an argon atmo-
showed a second hydrophobic tunnel, described previously by sphere provided vinyl-linker precursors 10a–b and 11a–b. Each
Tamada et al.,13b extending away from the G3P binding site and of the benzoates was then hydrolyzed, either under Gassman
presenting from C-4 and C-5 of the benzoic acid ring of the conditions15 or in aqueous 1 M NaOH, to afford analogs 12a–l,
docked inhibitor. This volume was lined by hydrophobic and 13a–l, 14a–b, 15a–d, 16a–b and 17a–b. Alternatively, hydroge-
aromatic residues including Phe98, Gly99, Tyr102, Ile103, nation of 10a–b and 11a–b prior to hydrolysis afforded ethyl-
Ala143, Leu176, Pro179, and Phe180.
linked analogs 18a–b and 19a–b.
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groups were not well tolerated, decreasing inhibition by nearly
3-fold.
Inhibition data
The inhibitory data for the 4-aryl-substituted analogs are pre-
sented in Table 1. Several of these compounds exhibited
marked improvement over 1c. In general, hydrophobic
substituents such as p-n-butylphenyl (12d), biphenyl (12e and f),
and methoxyphenyl (12g and h) exhibited a moderate increase
in inhibitory activity of up to 2-fold. Chlorophenyl analogs (12a–
c) also demonstrated markedly more potent inhibition, partic-
ularly the meta-chloro analog which also was about 2-fold more
active than 1c. Conversely, hydroxyl- and uoro-containing
analogs (12i–l) caused a modest decrease in inhibitory activity.
The para-hydroxyl analog, for instance, showed a nearly 3-fold
decline in inhibition.
The inhibitory data for the 5-aryl analogs is presented in
Table 2. Generally, the structure–activity relationship of this
class mirrors that of the 4-aryl class. For instance, several of the
compounds showed notable improvement over 1c, particularly
those containing chlorophenyl and hydrophobic substituents.
Specically, both the meta-chloro (13b) and para-n-butyl (13d)
analogs effected a 2.9-fold increase in inhibitory activity. Also
similar to the 4-aryl class, hydroxyphenyl and uorophenyl
The data for the 4- and 5-aryl analogs are consistent with the
presence of a hydrophobic binding pocket extending from C-4
and C-5 of the benzoic acid of 1. Both steric and electronic
factors may be affecting binding in this pocket. Sterically, the
pocket is large enough around the 4-position of 1c to accom-
modate the relatively voluminous and rigid ortho- and para-
biphenyl analogs 12e and 12f. The region near the 5-position,
however, appears more constrained in size. Consequently, the
para-biphenyl analog 13f shows a large decrease in activity,
possibly due to steric interactions with residues on the edge of
the binding pocket. The ortho-biphenyl analog 13e, however,
may orient the terminal phenyl substituent away from the edge
and back into the binding pocket resulting in a 2.5-fold increase
in activity. van der Waals and p-stacking interactions seem to be
favored within the pocket as phenyl-, alkylphenyl-, and alkox-
yphenyl substituents showed an increase in activity. Conversely,
the more polarized uorine-containing analogs demonstrated
moderately diminished activity possibly due to binding at a site
intolerant of the inhibitor's high charge density or disruption of
key p–p interactions within the hydrophobic channel.
Scheme 1 Reagents and conditions: (a) ClSO2C8H17, NEt3, CH2Cl2, 62–76%; (b) Pd(PPh3)4, Ar-B(OH)2, Na2CO3, PhMe/MeOH, 90 ꢀC, 45–80%; (c)
Pd(PPh3)4, Ar-B(OH)2, K2CO3, dioxane, 90 ꢀC, CO (1 atm), 28–46%; (d) Pd(PPh3)4, (E)-Ar-(CH)2B(OH)2, K2CO3, PhCH3, 105 ꢀC, 58–83%; (e) Pd/C,
H2 (1 atm), MeOH, 80–91%; (f) KOtBu, H2O, Et2O, 0 ꢀC-r.t., 71–94%; (g) 1 M NaOH, THF, 40 ꢀC, 73–92%.
828 | Med. Chem. Commun., 2014, 5, 826–830
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While the 3-fold increase in observed activity for the 4-aryl of separating the substituents from the pharmacophore and to
and 5-aryl derivatives was promising, we worried that the explore conformational rigidity versus exibility in extending
kinked nature of the hydrophobic tunnel observed in the the substituents into the hydrophobic tunnel. Inhibition data
docking model was preventing the analogs from extending fully for the 4- and 5-substituted analogs are shown in Tables 3 and 4,
into it. Therefore, we next sought to incorporate various linker respectively. In general, the analogs possessing a keto linker
regions on a subset of the more promising hydrophobic leads. were slightly to moderately less potent than the corresponding
Keto, vinyl, and ethyl linkers were chosen to examine the effect structures without a linker. For example, 5-keto-substituted
para-n-butyl analog 15a exhibited a 3.5-fold decrease in inhibi-
tion compared to compound 13d. The para-biphenyl analogs
14b and 15b, however, were notable exceptions. Compound 14b
Table 1 GPAT Inhibition by 4-substituted derivativesa
showed a 2-fold more potent inhibition compared to the linker-
free analog 13f. While the rigid linear carbon skeleton of 13f
may sterically interfere with residues on the face of the hydro-
phobic pocket, the ketone linker in 15b could potentially direct
the large biphenyl moiety back towards the cavity. Compound
14b, with an IC50 of 8.5 mM, was 3-fold more potent than 1c and,
Table 3 4-Substituted inhibitors with linkersa
Compound
R
IC50 (mM) ꢁ SD
12a
12b
12c
12d
12e
12f
12g
12h
12i
o-Cl
m-Cl
p-Cl
26.9 ꢁ 4.1
13.8 ꢁ 2.5
18.1 ꢁ 3.5
15.7 ꢁ 1.6
17.1 ꢁ 0.8
13.5 ꢁ 3.2
16.1 ꢁ 0.5
13.3 ꢁ 1.1
47.2 ꢁ 2.0
32.6 ꢁ 3.6
63.1 ꢁ 5.5
64.8 ꢁ 3.8
p-n-C4H9
o-C6H5
p-C6H5
o-OCH3
p-OCH3
o-F
m-F
o-OH
p-OH
Compound
Linker
R
IC50 (mM)
12j
12k
12l
14a
14b
p-n-C4H9
p-C6H5
15.8 ꢁ 0.8
8.5 ꢁ 2.1
38.0 ꢁ 9.9
38.7 ꢁ 6.3
37.5 ꢁ 4.8
43.0 ꢁ 9.3
16a
16b
18a
18b
–H
p-OCH3
–H
a
A description of assay conditions can be found in the ESI.†
p-OCH3
Table 2 GPAT Inhibition by 5-substituted derivativesa
a
A description of assay conditions can be found in the ESI.†
Table 4 5-Substituted inhibitors with linkersa
Compound
R
IC50 (mM) ꢁ SD
13a
13b
13c
13d
13e
13f
13g
13h
13i
o-Cl
m-Cl
p-Cl
14.4 ꢁ 1.3
8.9 ꢁ 0.9
Compound
Linker
R
IC50 (mM)
9.2 ꢁ 0.7
p-n-C4H9
o-C6H5
p-C6H5
o-OCH3
p-OCH3
o-F
m-F
o-OH
p-OH
9.0 ꢁ 0.4
15a
15b
15c
15d
17a
17b
19a
19b
p-n-C4H9
p-C6H5
o-OCH3
p-OCH3
–H
p-OCH3
–H
p-OCH3
31.7 ꢁ 3.2
13.0 ꢁ 0.6
59.2 ꢁ 3.8
44.3 ꢁ 9.2
16.3 ꢁ 5.3
37.1 ꢁ 8.3
37.5 ꢁ 4.8
26.3 ꢁ 2.4
10.1 ꢁ 0.7
29.1 ꢁ 6.3
27.6 ꢁ 2.0
19.5 ꢁ 5.5
34.6 ꢁ 2.0
37.6 ꢁ 9.7
61.7 ꢁ 13.8
47.3 ꢁ 6.4
13j
13k
13l
a
a
A description of assay conditions can be found in the ESI.†
A description of assay conditions can be found in the ESI.†
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´
to our knowledge, represents the most active GPAT inhibitor
discovered to date. The rigid vinyl and conformationally-exible
ethyl linkers were generally not well tolerated. For example, 16b,
a vinyl-linked para-methoxyphenyl analog, produced an activity
of 37.5 mM compared to 13.3 mM for the linker-free derivative
12h. No signicant benet was apparent from the incorporation
of the conformationally-exible ethyl linkers. The same para-
methoxyphenyl substituent with a linker region in compound
18b demonstrated an IC50 of 43.0 mM or 3.2-fold lower activity
than the corresponding compound 12h.
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Conclusions
Several 4- and 5-substituted analogs of GPAT inhibitor (octane-
sulfonamido)benzoic acid were synthesized and analyzed for in
vitro inhibitory activity. In general, hydrophobic substituents led
to an increase in inhibitory activity while more polar and
hydrogen-bonding substituents showed a decrease in inhibitory
activity, consistent with the presence of a hydrophobic pocket
identied by in silico studies. Taking advantage of the hydro-
phobic pocket, p-biphenylketone-substituted 14b improved the
inhibitory activity 3-fold to 8.5 mM, representing the most potent
GPAT inhibitor to date. Future studies will aim to determine
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Acknowledgements
We would like to thank Dr I. P. Mortimer for performing HRMS
analyses. This work was supported by the NIH 1R43DK65423 to
FASgen, Inc. Under a license agreement between FASgen, Inc.
and The Johns Hopkins University, C.A.T. is entitled to share
royalty received by the University on sales of products described
in this article. C.A.T. owns FASgen, Inc. stock, which is subject 14 A. K. Agarwal, S. Sukumaran, R. Bartz, R. I. Barnes and
to certain restrictions under university policy. The Johns Hop-
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830 | Med. Chem. Commun., 2014, 5, 826–830
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