Structure-Based Design of N-(5-Phenylthiazol-2-yl)acrylamides as Novel and Potent Glutathione S-Transferase Omega 1 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01960

Using reported glutathione S-transferase omega 1 (GSTO1-1) cocrystal structures, we designed and synthesized acrylamide-containing compounds that covalently bind to Cys32 on the catalytic site. Starting from a thiazole derivative 10 (GSTO1-1 IC₅₀ = 0.6 μM), compound 18 was synthesized and cocrystallized with GSTO1. Modification on the amide moiety of hit compound 10 significantly increased the GSTO1-1 inhibitory potency. We solved the cocrystal structures of new derivatives, 37 and 44, bearing an amide side chain bound to GSTO1. These new structures showed a reorientation of the phenyl thiazole core of inhibitors, 37 and 44, when compared to 18. Guided by the cocrystal structure of GSTO1:44, analogue 49 was designed, resulting in the most potent GSTO1-1 inhibitor (IC₅₀ = 0.22 ± 0.02 nM) known to date. We believe that our data will form the basis for future studies of developing GSTO1-1 as a new drug target for cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.8b01960

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Article
Structure-based Design of N-(5-phenylthiazol-2-yl)acrylamides as
Novel and Potent Glutathione S-Transferase Omega 1 Inhibitors
Weiyang Dai, Soma Samanta, Ding Xue, Elyse M. Petrunak, Jeanne
A. Stuckey, Yanyan Han, Duxin Sun, Yong Wu, and Nouri Neamati
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01960 • Publication Date (Web): 08 Feb 2019
Downloaded from http://pubs.acs.org on February 10, 2019
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Structure-based Design of N-(5-phenylthiazol-2-yl)acrylamides as Novel
and Potent Glutathione S-Transferase Omega 1 Inhibitors
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Weiyang Dai,† ,¶ Soma Samanta, Ding Xue, Elyse M. Petrunak, Jeanne A. Stuckey,
†
†
‡
‡
¶
, *
†, *
§
§
Yanyan Han, Duxin Sun, Yong Wu and Nouri Neamati
†Departments of Medicinal Chemistry, §Pharmaceutical Sciences, College of Pharmacy,
North Campus Research Complex, 1600 Huron Parkway, and ‡Life Sciences Institute and
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109,
United States. ¶Key Laboratory of Drug Targeting and Drug Delivery System of Ministry
of Education, West China School of Pharmacy, Sichuan University, No.17 People's South
Road, Chengdu, 610041, P. R. of China.
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ABSTRACT
Using reported glutathione S-transferase omega 1 (GSTO1-1) co-crystal structures, we
designed and synthesized acrylamide-containing compounds that covalently bind to Cys32
on the catalytic site. Starting from a thiazole derivative 10 (GSTO1-1 IC50 = 0.6 µM),
compound 18 was synthesized and co-crystalized with GSTO1. Modification on the amide
moiety of hit compound 10 significantly increased the GSTO1-1 inhibitory potency. We
solved the co-crystal structures of new derivatives, 37 and 44, bearing amide side chain
bound to GSTO1. These new structures showed a reorientation of the phenyl thiazole core
of inhibitors, 37 and 44, when compared to 18. Guided by the co-crystal structure of
GSTO1:44, analog 49 was designed resulting in the most potent GSTO1-1 inhibitor (IC50
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0.22 ± 0.02 nM) known to date. We believe that our data will inform the future studies
of developing GSTO1-1 as a new drug target for cancer therapy.
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INTRODUCTION
Glutathione S-transferases (GSTs) are a diverse family of cytosolic, mitochondrial and
microsomal enzymes that conjugate glutathione to a variety of exogenous compounds
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including drugs, industrial intermediates, pesticides, herbicides, environmental pollutants,
and carcinogens which mainly acts as part of an integrated defense strategy.1, 2 In addition,
GSTs also play an important role in biotransformation of endogenous substrates and other
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biological functions. The mammalian GSTs are classified into seven different classes,
alpha, mu, pi, theta, sigma, zeta and omega. Among them, the omega GSTs (GSTOs)
belong to an atypical cytosolic class and two isozymes, GSTO1-1 and GSTO2-2 have been
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identified in humans that share low sequence identity with other GST classes. GSTO1-1
possesses S-phenacyl glutathione reductase and monomethylarsonate reductase activity,
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while GSTO2-2 catalyzes dehydroascorbate reduction. S-(4-Nitrophenacyl)glutathione
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4-NPG) is also reported as a good substrate for GSTO1-1. They are also unique among
GSTs in terms of their catalytic amino acid residues. Rather than a catalytic serine or
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tyrosine, GSTOs possess a reactive cysteine nucleophile in the active site. Therefore, their
enzymatic activities are sensitive to molecules that are able to conjugate with the thiol
group like N-ethylmaleimides or haloacetamides8-10. The crystal structure of GSTO1
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revealed two important active sites (PDB ID: 1EEM). One is the hydrophobic or
electrophile binding site (H-site) that can easily accommodate lipophilic electrophiles; the
other is a narrow strip consisting of a series of hydrophilic residues that can selectively
bind with glutathione (G-site). 7
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GSTO1-1 is overexpressed in select human cancers such as pancreatic cancer , head and
neck cancer , urinary bladder cancer and colorectal cancer14 which leads to an
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accelerated detoxification of drug substrates and thus is implicated in drug resistance. 15-18
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We previously reported that silencing GSTO1-1 with siRNA impairs cancer cell viability,
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suggesting that GSTO1-1 is a potential target for cancer therapy. It is also reported that
GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity.19
Only a limited number of GSTO1-1 inhibitors have been reported and there is a strong
correlation between thiol-alkylating groups in these compounds and their GSTO1-1
inhibitory activities (Figure 1). The epoxide-bearing cytokine release inhibitory drug
(
CRID) has affinity towards GSTO1-1, which may be related to their inhibition of ATP-
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induced IL-1β posttranslational processing. Previous screen of the Molecular Libraries
Small Molecule Repository (MLSMR) library uncovered a class of selective α-
chloroacetamide GSTO1-1 inhibitors. After further optimization, KT53 was selected as a
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potent GSTO1-1 inhibitor that enhanced cisplatin-induced cytotoxicity. However, KT53
showed instability in PBS within 6 h at room temperature due to its intramolecular
cyclization reaction. A GSTO1 co-crystal with ML175 was subsequently reported (PDB
22
ID: 5V3Q). Additionally, an aryl fluorosulfate compound was identified as a GSTO1-1
probe by an “inverse drug discovery” strategy and its X-ray co-crystal structure was also
solved (PDB ID:5UEH).23 Previously, we developed a series of chloroacetamide-
containing GSTO1-1 inhibitors by high-throughput screening of our in-house compound
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library. The lead compound C1-27 was discovered as a low molecular weight
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chloroacetamide derivative with good potency. The co-crystal structure of GSTO1-C1-27
complex (PDB ID: 4YQM) was also obtained, showing C1-27 binding in the H-site of
GSTO1. C1-27 exhibited cytotoxicity in several cancer cell lines, but knockdown of
GSTO1-1 did not significantly affect C1-27 cytotoxicity, suggesting that C1-27 has off
target effects at higher concentrations than those required for GSTO1-1 inhibition. All of
GSTO1-1 inhibitors discussed above display similar structural features to match GSTO1
protein structure. Their hydrophobic cores (usually electron-deficient aromatic rings) allow
them to enter the H-site easily. Most importantly, they all contain electrophilic reactive
groups (epoxide, chloroacetamide, benzyl chloride, aryl fluorosulfate) that can covalently
bind with the Cys32 thiol group of GSTO1. We noticed that α,β-Unsaturated carbonyls,
like acrylamides, acrylic esters and chalcones, can also be served as covalent modifiers.
Drugs or compounds with α,β-unsaturated carbonyls are mostly designed to undergo an
irreversible hetero-Michael addition reaction with a specific residue (typically,
unsubstituted cysteines) of the target protein. Compared with other covalent binding groups,
α,β-unsaturated carbonyls, especially acrylamides, are considered to be mild and present
in select FDA approved drugs .24-26 Previous reports showed a series of artemisinine
derivatives containing 1,2,4-trioxane acrylates displayed GSTP1 inhibition activity,
indicating that α,β-unsaturated carbonyl modifiers might be applicable for GST inhibitors.
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Currently, there is no reported GSTO1-1 inhibitor containing α,β-unsaturated carbonyl
moiety. Herein, we describe a structure-guided approach of developing novel GSTO1-1
inhibitors based on our previously published GSTO1 crystal structures.
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RESULTS AND DISCUSSIONS
Chemistry
The synthesis of compounds 1-12 is shown in Scheme 1. Compounds 1-3 were
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C1-27
intermediate
5-amino-2-chloro-N,N-
dimethylbenzenesulfonamide 4i conjugated with acryloyl chloride, 2-chloroethylsulfonyl
chloride, and methyl 4-bromocrotonate, respectively as described in our previous study.4
Commercially available heterocycle amines were treated with acryloyl chloride in DCM to
give 5-12. Scheme 2 describes the synthesis of compounds 16-27 with different
substituents. Bromination of commercially available acetophenones 13a-13l using copper
bromide in ethyl acetate gave the 2-bromoacetophenones 14a-14l.28 Treatment of 2-
bromoacetophenones with thiourea in ethanol yielded 4-phenyl-2-aminothiazole
29
derivatives 15a-15l. The amines were then conjugated with acryloyl chloride to give
compounds 16-27.
Compounds 29-34 were synthesized as described in Scheme 3 to investigate the
influence of installing substituents on the thiazole ring and the amide nitrogen. Compounds
with 5-substituents on the thiazole ring 29 and 30 were obtained by treating commercially
available α-bromoketones 14o and 14p with thiourea followed by coupling with acryloyl
chloride. Compounds with N-substitutions on acrylamide moiety were synthesized under
two different conditions. Intermediates 28c and 28d were obtained by 4f and corresponding
bromides. N-substituted 4-phenylthiazol-2-amines 28c-28d were heated with acryloyl
chloride in toluene to give 33-34. The total yield of 33-34 was relatively low because the
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bromination process resulted in bi-substituted side products. In order to overcome this
limitation and generate more derivatives with amide side chain efficiently, we developed
another synthetic route for compounds 37-45 (Scheme 4). The commercially available
starting material 2-bromoacetophenone 14m was treated with potassium thiocyanate
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followed by primary amines gave the intermediate 28e-28h. Acid hydrolysis of methyl
ester intermediate 28e provided the free carboxylic acid 35, which was then conjugated
with amines to give amides 36a-36d. Compounds 28e-28h, 36a-36d were treated with
acryloyl chloride to give 37-45.
Compounds 47-52 were synthesized as described in Scheme 5. Hydroxyl protection of
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3p and 13q gave intermediate 13r and 13s. Bromination and cyclization processes were
similar with the protocols presented in Scheme 4 to give 28i-28l from 14e, 14r, 14s and
4l, respectively. Compound 46c was obtained by conjugating 28l with 2-chloroacetyl
1
chloride. Methyl ether deprotection of 46a-46c and 48 gave the hydroxyl derivatives 49-
52.
The synthesis of compound 53 started with tert-butyl (4-bromothiophen-2-yl)carbamate
54 conjugated with 5-(bromomethyl)-3-methylisoxazole in the presence of NaH (Scheme
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). The Boc group of intermediate 55 was cleaved before conjugating with acryloyl
chloride. A Suzuki coupling with (2-hydroxyphenyl)boronic acid offered the final product
53.
Design and SAR Studies
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GSTO1-1 is considered a relatively new target for early stage drug discovery. Various
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assays can be used to identify GSTO1 inhibitors. We used several assays to evaluate
GSTO1-1 inhibitor C1-27, including 4-NPG substrate assay and in-gel competitive
fluorescence assay. Since C1-27 and all other GSTO1-1 inhibitors bind with GSTO1-1 on
the catalytic active site residues, we tested GSTO1-1 inhibitory activity of all compounds
by a competitive fluorescence assay. Using 5-chloromethylfluorescein diacetate (CMFDA),
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which has been reported to be a potent and irreversible GSTO1-1 inhibitor (Figure 1) , we
optimized the gel-based binding assay that efficiently measures competitive inhibition of
CMFDA binding to GSTO1, a representative example of this assay is shown in
Supplementary Figure S1. Recombinant GSTO1-1 was used to reduce assay interference
caused by limitation of gel separation. It is of interest to note that for irreversible covalent
inhibitor ideal measurement is Kinact, because it considers the rate of inactivation and the
binding affinity. 26 The limitation of our assay is that it cannot measure the rate of
inactivation and the binding affinity. Thus, in this study, we determined % inhibition of the
binding of CMFDA to the GSTO1 by the compound, where compounds are pre-incubated
with GSTO1 for 30 min followed by addition for CMFDA for 30 min. Pre-incubation at
various time points did not show notable difference in the IC50 values (Supplementary
Figure S2). In this study, we determined IC50 for all compounds pre-incubated for 30 min.
Initially we modified the chloroacetamide group of C1-27 with other electrophilic
covalent binding groups. Its replacement with an acrylamide or a vinyl sulfonamide moiety
caused a significant decrease in either GSTO1-1 potency or cytotoxicity (Table 1). By
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analyzing the co-crystal structure of C1-27 and GSTO1-1, we observed an important H-
bond interaction between sulfonamide moiety and Trp180 that restricts compound’s
conformation in the binding pocket (Figure 2A). Chloroacetamide moiety and acrylamide
moiety have similar molecular shapes but different binding positions. The cysteine thiol
group binds to the alpha-carbonyl carbon atom of C1-27 but with the beta-carbonyl carbon
of the acrylamide analog 1. When we superimposed these two structures, the distance
between the two active sites was measured as approximately 1.34 Ǻ (Figure 2B). Direct
replacement of chloroacetamide on C1-27 with an acrylamide moiety might result in the
loss of H-bond interaction due to the different length of the covalent-binding groups. We
further synthesized methyl crotonate derivative 3 where the distance from the sulfonamide
group to the covalent binding site was retained. We expected that the ester moiety of 3
could interact with any residues on the G-site of GSTO1. However, this modification
resulted in a loss of activity, perhaps due to the bulky methyl crotonate motif decreasing
the chance of Micheal acceptor reaction with the thiol group of Cys32. The unsatisfying
result indicated that C1-27 core was not suitable for acrylamide replacement, and new cores
or scaffolds needed to be developed.
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We previously built a chloroacetamide-containing compound library to screen potential
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GSTO1-1 inhibitors. Based on this library, a GSTO1 covalent docking study was
conducted to search for novel inhibitors. The chloroacetamide moieties of active
compounds were replaced with arylamide and the new library generated was docked on the
GSTO1 active site. Taking into consideration the difference in physical and chemical
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properties between chloroacetamide and acrylamide, we selected C4-10, a phenylpyrazole
derivative, that showed modest potency on GSTO1-1 binding assay in our previous study
as a model compound (Figure 1), and the co-crystal structure of C4-10 with GSTO1 was
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also solved (PDB ID: 4YQV). We observed that its acrylamide analog 5 exhibited a very
similar pose with the C4-10 (Figure 2C) in our docking study. The acrylamide moiety binds
Cys32, the N-phenylpyrazole moiety occupied the H-site and the benzene ring located deep
into the hydrophobic pocket. Additionally, some other phenyl heterocyclic derivatives
were docked onto GSTO1 active site and showed similar binding poses. Thus, a series of
analogs were synthesized and investigated in GSTO1-1 binding assay to verify our docking
results. The compounds with pyrazole core were inactive (5 and 6). Compounds 7 and 8
with non-aromatic piperidine core also did not possess significant GSTO1-1 binding
activity. However, compounds with thiazole core displayed potency and the 4-phenyl (10)
was more potent than 5-phenyl derivatives (11). Most of these heterocyclic compounds are
rigid molecules, their binding conformation and low energy conformation is likely to be
quite similar. Therefore, a 3D structure superimposition was conducted to analyze the
structure-activity relationship. We observed that the benzene ring of the thiazole
compounds displayed a shift compared with the pyrazole compound 5 while other motifs
were perfectly aligned (Figure 2D), which is probably due to the unique sulfur-carbon bond
33
angle in the thiazole ring (88.6 ̊) . This special angle allows the benzene ring to insert into
the narrow hydrophobic cleft to improve potency. Two other compounds with thiophene
core structure (11 and 12) also showed good potencies, suggesting that the sulfur atom in
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the heterocyclic core displays positive contribution to potency. Thiophene analog 11
showed slightly decreased potency as compared to 10, perhaps due to the conformational
restriction imposed by its intramolecular H-bond. Endogenous GSTO1-1 binding activity
was also observed on 9 and 10 in a cell-based assay. Compound 10 exhibits most desirable
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ligand efficiency (LE) and ligand-lipophilicity efficiency (LLE) among these analogs.
Therefore, we chose compound 10 as a new GSTO1-1 inhibitor template for further studies
and optimization. Compound 10 also displayed cytotoxicity on HCT116 and HT29 cell
lines, but whether the cytotoxicity was resulted from GSTO1-1 inhibition needs further
investigation.
The binding pose of compound 10 in our docking study showed that the benzene ring
does not fully occupy the H-site and additional lipophilic groups can be accommodated.
We investigated whether modifications on the phenyl group can improve potency. All
compounds were tested in GSTO1-1 binding assay and MTT cytotoxicity assay. Since the
major interaction between GSTO1 and our compounds is hydrophobic interaction, LLE of
these compounds was also investigated. Substitution of benzene ring with chlorine atoms
showed a preference at 4-position (18, IC50 = 0.17 µM) over other positions. This may be
due to the fact that 4-chloro can fill additional space in the hydrophobic pocket. The classic
bioisosteric replacement of chlorine with methyl group resulted in a slightly decreased
potency. We hypothesized that the activity change was related to the different electron
inductive effects between chlorine and methyl group on the benzene ring. When bound to
GSTO1, the benzene ring located in the hydrophobic pocket, is surrounded by Trp180,
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Phe31, Phe225, and Tyr229. These residues can form π-π interaction with the benzene ring
and the interaction intensity is significantly influenced by electron density of the aromatic
ring. In order to test the feasibility of this hypothesis, additional analogs with substitutions
at 4-position were synthesized. As anticipated, analogs with strong electron-withdrawing
groups (e.g. trifluoromethyl analog 20, IC50 = 0.30 µM) displayed improved potency, while
analogs with 2,3-dioxole, a strong electron-donating substituent, showed decreased
potencies (IC50 = 1.22 µM).
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To validate our design strategy and to guide future analogue design, we obtained the co-
crystal structure of 18 bound to GSTO1 (PDB ID: 6MHB, Figure 3A). The 2.75 Å structure
of GSTO1:18 demonstrates similarity to the co-crystal structure of GSTO1 with
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chloroacetamide inhibitor C4-10 (PDB ID: 4YQV) in both the overall structure of GSTO1
(average RMSD between two structures of 0.49 Å) as well as the orientation of the ligand.
Compound 18 occupies the same deep hydrophobic cleft of the H-site aligned by aromatic
residues including Trp180, Phe31, Phe225, and Tyr229 (Figure 3B). The 4-chlorophenyl
moiety makes van der Waals contacts with the side chains of Val127, Arg183, Met187,
Trp222, Phe225, and Leu226 as well as the backbone amide of Phe31 (Figure 3A). The
propionamide and thiazole ring make hydrophobic contacts with Met29, Pro33, Phe34, and
Tyr229. Although Tyr229 appears to interact with the thiazole, its orientation varies
between the six chains of GSTO1 modeled as part of this structure, and a lack of electron
density in some chains disallow modeling of the Tyr229 side chain altogether. In
conjunction with the poor electron density of the thiazole ring, this seems to suggest that
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binding of 18 is likely driven by interactions of the 4-chlorophenyl moiety with GSTO1.
The most significant difference between GSTO1:18 and GSTO1:C4-10 is a change in
conformation of the Leu226 side chain, which reorients to interact with the chlorine of the
4-chlorophenyl substituent of 18.
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Interestingly, we found that the benzene ring was close to the Phe31 backbone amide
(3.50 Å), which could be treated as a potential binding site. Therefore, we generated 2-
hydroxyl analog 24. The potency was improved although not that significantly. Meanwhile,
the other 2-position substitutions or 4-hydroxyl substitution led to a potency reduction. The
2-methoxyl analog 26 was inactive. We speculated that the 2-hydroxyl group probably
formed an H-bond with the backbone oxygen of Phe31 but adding ortho-position
substituents altered the binding energy and offset the potency gained from the H-bonding.
We also tested the cytotoxicity of these compounds in MTT assay. Most of these analogs
showed inhibition of cell viability but their cytotoxicities did not show an obvious
correlation with their GSTO1 binding potency.
In order to extend our structure-activity relationships study and improve potency, we
analyzed the co-crystal structure of 18 bound to GSTO1 looking for other potential
interaction sites. Two regions on the binding pocket can be identified as exploitable sites.
One is a hydrophobic area near the “T cave” surrounded by aromatic ring residues like
Tyr229, Phe31 and Phe225. The other one is Trp180 close to the thiazole ring (distance =
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.46 Å), which plays an important role as an H-bond donor when bound with C1-27.
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Therefore, we designed analogs with substituents on 5-position of thiazole ring or adding
side chain on amide nitrogen.
Inserting a methyl on the 5-position of thiazole ring lead to a potency reduction (29, 3.1
± 3.3 µM) whereas methyl 5-carboxylate analog 30 exhibited slightly increased potency.
We assumed that 5-substitutions generally affected the low energy conformation, which
accounted for the potency decrease for 29, whereas the carboxylate group in 30 improved
the binding free energy by forming another H-bond with Trp180. Compounds 33 and 34
displayed increased potencies, suggesting that more area occupied in the H-site helps
exclude “unfavorable water molecules” and contribute to potency. Therefore, additional
compounds with amide side chains were synthesized to improve potency based on this
theory. Since ethyl acetate derivative 34 showed improved potency, we further examined
if its homologs 37, 38 and 39 could be accommodated within the cavity. A bulky tert-butyl
group can be tolerated whereas the free carboxylic acid replacement resulted in a
significant activity loss, suggesting that lipophilic groups may be beneficial to improve
potency as we expected. Additional derivatives were synthesized to investigate the
tolerance of other isosteres. Replacement of the methyl ester group with more lipophilic
cyclohexyl amides led to increased potency, which also validated our hypothesis.
Considering there are still polar residues around the molecule, we sought to design amide
derivatives with potential H-bond acceptors. Compounds with a terminal oxygen, including
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3 with a bulky N-(2-morpholinoethyl) group exhibited moderate to good potencies.
Compound 42 displayed the best potency up to this stage (IC50 = 0.03 ± 0.02 µM). All
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analogs showed improved LLE, suggesting our optimization was reasonable. In addition,
we also synthesized compounds based on 33. We hypothesized that the additional benzyl
group forms edge-to-face π-stacking with Tyr229. However, the benzyl group installation
made the compound greasy (cLogP = 4.48). In order to reduce lipophilicity, pyridine
derivative was synthesized. Unfortunately compound 45 showed decreased potency
probably due to the electron-deficient pyridine ring impairing the π-interaction. Hence, we
designed a molecule with 3-methyl-isoxazole group, which was regarded as a preferred
drug-like and electron-rich heterocycle group. This modification resulted in fairly good
activity (44, IC50 = 0.051 µM) and improved cLogP (2.96). When the potency of GSTO1-
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binding activity improved, these derivatives, however, lost their cytotoxicity, indicating
that inhibiting GSTO1-1 might not influence cell viability.
The co-crystal structures of GSTO1 with 37 and 44 were solved to resolutions of 2.0
(PDB ID: 6MHC) and 2.15 Å (PDB ID: 6MHD), respectively. The two structures reveal
very little difference in the overall structure of GSTO1 (average RMSD = 0.33 Å) when
compared to one another but demonstrate more substantial differences when compared
with the structure of GSTO:18 (average RMSD = 0.78 Å for GSTO1:37 and 0.80 Å for
GSTO1:44) (Figure 4A). In particular, the structures of GSTO1 with 37 and 44 show a
rearrangement of the H-site as a result of a shift in the 9 helix when compared to the
GSTO1:18 structure (Figure 4A). In this state, the side chain of Trp222 is reoriented to
occupy the cavity that accommodates the 4-chlorophenyl ring of 18. In both structures,
electron density of inhibitors 37 and 44 suggest modeling the thiazole ring such that it
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engages in - stacking interactions with the side chain of Tyr229, which appears well
ordered in all chains of both structures, and to a lesser extent Phe225 (Figure 4B). Electron
density associated with the phenyl ring of both inhibitors is less definitive, suggesting that
there is likely a degree of free rotation about the bond connecting the two ring systems.
The phenyl substituent is poised to make weak hydrophobic contacts with 4 helix residues
Gly128, Ile131, Arg132, as well as Leu226 and Tyr229, which form a shallow pocket in
the H-site. In the GSTO1:37 structure, electron density did not support modeling the ester,
which is projected into bulk solvent and consequently disordered (Figure 4B). In contrast,
an interaction with the side chain of Phe34 orders the isoxazole ring of 44 which extends
this portion of the ligand toward the G-site (Figure 5A). This additional interaction likely
contributes to the improvement in potency of 44 when compared to 37 since it may block
glutathione to enter the G-site. This discovery led us to further optimization by adding more
flexible substituents attached to functional groups to interact with residues at the G-site
that may further improve potency.
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The reorientation of the phenyl thiazole core of inhibitors 37 and 44 when compared to
18 seem to suggest that the deep cleft in the H-site occupied by inhibitors C4-10 and 18
cannot accommodate much steric bulk associated with N-substitution of the acrylamide.
This ultimately forces N-substituted ligands to occupy a more solvent-exposed pocket of
the H-site which is driven mostly by hydrophobic packing of the thiazole, whereas the deep
hydrophobic cleft occupied by inhibitors C4-10 and 18 locks the phenyl ring into a more
ordered state.
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On the basis of above preliminary SAR results, we continued to design analogs based on
our new optimized compound 44. We first combined the 4-trifluromethylphenyl group and
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-methyl-isoxazole side chain on acrylamide nitrogen to provide hybrid compound 47.
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This modification showed a little improvement in potency as compared to 44. We then
examined isoxazole compound with 2-hydroxylphenyl group to match the potential
protein-ligand interaction requirement and this molecule exhibit a remarkable picomole
level potency (49, IC50 = 0.00022 µM). Meanwhile, its 2-methoxylphenyl analog 48 was
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00-fold less potent than 49, validating the hypothesis that the hydroxyl group did play an
important role on binding interaction. From the co-crystal structure of GSTO1: 44 and
docking studies, it was not apparent that the hydroxyl group would form H-bonds with any
protein residues. We believe that the increase in potency is due to charge interaction. The
partial negative charge of the oxygen atom would be highly attracted to the positive charge
of the drug-binding pocket. Thus, compound 49 with the 2’-hydroxyl substitution would
bind tighter than compound 44 (Figure 5B). We also noticed that additional space can be
occupied at 4-position on the benzene ring; therefore, analogs with 4-chloro or 4-fluoro
were designed. Compounds 50 also displayed excellent potency whereas decreased
potency was observed with fluorinated analog 51. High LLE values and optimal cLogP of
these compounds also illustrated that they also have good drug-like properties. These
compounds showed remarkable potency in GSTO1-1 assay but did not show significant
cytotoxicity, indicating that the selectivity of these compounds can be further improved.
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Compound 49, designed based on the GSTO1:44 co-crystal structure, is the most potent
GSTO1 inhibitor (IC50 = 0.22 ± 0.02 nM) known to date and did not show off-target
induced cytotoxicity. There is a possibility that the 2-OH of this molecule may make the
overall molecule a potential chelator. We tested the compound in the presence or absence
of various metals to examine whether a chelation could give rise to assay interference. We
did not see a significant effect by varioous metals in the activity of the compound
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(Supplementary Figure S3). This compound also potently inhibited GSTO1 enzyme
activity in 4-nitrophenacyl glutathione (4NPG) reduction assay (Supplementary Figure S4).
Although not tested in this study, due to 64% sequence identity of GSTO1 and GSTO2, we
expect these inhibitors may also bind to GSTO2.
Covalent inhibitors sometimes display instability in vitro or in vivo due to their
24
reactivity. Compounds 18 and 49 were selected for stability studies. They showed good
stability in 20% acetonitrile/PBS buffer solution at room temperature overnight as detected
by LC-MS. An in vitro mouse and human liver microsome stability assay was used to
evaluate their metabolic properties (Table 7). The results showed that compounds were
rapidly metabolized in both mouse and human microsomes perhaps due to the acrylamide
moiety being the main metabolic liability.
Additional optimizations were conducted to improve the metabolic stability without
major decrease in potency. Two more analogs were synthesized (Table 8).
Chloroacetamide, the most commonly used covalent reactive group of GSTO1-1 inhibitors,
was conjugated with the scaffold of compound 49 to obtain compound 52. However, the
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replacement displayed respectable potency loss, indicating that the scaffold is more
suitable for acrylamide. Concerning thiazole nitrogen of compound 49 may be attacked by
its acrylamide structure forming an inactive cyclic product like KT53, we synthesized 53
by replacing the thiazole ring by thiophene but that resulted in a 1000-fold loss in potency.
Future studies will focus on seeking analogs with better PK properties.
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CONCLUSIONS
This study reports our structure-based design efforts towards the development of novel
GSTO1 inhibitors with better potency and drug-like properties. Our efforts towards
discovering new analogs with acrylamide structure led to a potent phenylthiazole
compound 10 (IC50 = 0.5 ± 0.2 µM) possessing desirable cLogP, LE and LLE. Compound
10 was identified as a lead and a series of analogs was synthesized based on a new co-
crystal structure with GSTO1. A co-crystal structure of 18 and GSTO1 revealed that the
compound inserted into a hydrophobic cave with GSTO1 H-site. Derivatives with amide
side chain showed improved potency as compared to those without substitutions. Co-
crystal structures of 37 and 44 with GSTO1 showed slightly different binding pose than
with 18 but the core structures still located in the electrophilic pocket. The introduction of
amide side chain caused a shift in the 9 helix and resulted in a reorientation of the H-site.
We also found that the isoxazole ring of compound 44 occupies a small portion of the G-
site and contributed to improved potency.
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The inhibitors disclosed in this study present suitable lead compounds for further
development. Future studies will focus on further optimizations of 49 to acquire potent
inhibitors with improved PK and pharmaceutical properties. Detailed in vitro and in vivo
studies will also be performed to evaluate their efficacy and safety in cancer therapy.
EXPERIMENTAL SECTION
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Chemistry
Reagents and anhydrous solvents were purchased from commercial sources and used
without further purification. Reaction progress was monitored by UV absorbance using
thin-layer chromatography (TLC) on aluminum-backed precoated silica plates from
Silicycle (SiliaPlate™, 200 µm thickness, F254). Purifications using flash chromatography
were performed using a Biotage® Isolera Chromatography system equipped with 10 g and
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5 g Ultra-SNAP Cartridge columns (25 µM spherical silica). Glassware for reactions were
oven dried in reactions performed using nitrogen or argon atmosphere using standard inert
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conditions. H NMR spectra were obtained using a Bruker NMR instrument (300 or 400
MHz) and spectral data are reported using the following abbreviations: (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets); coupling constants
are reported in Hz, followed by integration. A Shimdazu LCMS 2020 system was utilized
for generating HPLC traces and obtaining mass spectrometry data. The system was
equipped with a PDA UV detector and Kinetex® 2.6 µm, XB-C18 100 Å, 75 x 4.6 mm
column, which was used at room temperature. HPLC gradient method utilized was 10% to
95% MeCN in H O with 0.1% formic acid over 15 minutes with a 0.50 mL/min flow rate.
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Reverse-phase preparative purifications were performed on a Shimdazu LC20 modular
HPLC system utilizing a PDA detector and a Kinetex® 5 µm XB-C18 100 Å, 150 x 21.2
mm column. Purification method used was 25-minute gradient of 10% to 90% MeCN in
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H O with 0.05% trifluoroacetic acid as the additive. Purity of final compounds (≥95%)
was assessed at 254 nm using the described column and method.
N-(4-chloro-3-(N,N-dimethylsulfamoyl)phenyl)acrylamide (1). To a solution of 5-
amino-2-chloro-N,N-dimethylbenzenesulfonamide 4i (50 mg, 0.21 mmol) in DCM (10 mL)
was added DIEA (89 μL, 0.63 mmol) and stirred at 0°C for 20 min. Acryloyl chloride (16
μL, 0.60 mmol) in DCM (5 mL) was added slowly. The reaction mixture was stirred at
room temperature for 2h and then partitioned between DCM and saturated NaHCO
aqueous layer was extracted with DCM for another two times. The combined organic layer
was dried with MgSO , filtered, concentrated, and purified with flash chromatography
3
. The
4
using a gradient method of 2 to 50% EtOAc in hexane to give a white solid (48 mg, 80%
1
yield). H NMR (300 MHz, CDCl
3
) δ 7.79 (d, J = 2.6 Hz, 1H), 7.55 – 7.35 (m, 2H), 7.28
(s, 1H), 7.02 (s, 1H), 6.60 (dd, J = 16.5, 9.8 Hz, 1H), 6.35 (d, J = 16.5 Hz, 1H), 6.07 (d, J
+
= 9.8 Hz, 1H), 2.93 (s, 6H). MS (ESI) 289.0[M+H] . Purity: 95.8%.
2
-Chloro-N,N-dimethyl-5-(vinylsulfonamido)benzenesulfonamide (2): To a
solution of 5-amino-2-chloro-N,N-dimethylbenzenesulfonamide 4i (50 mg, 0.21 mmol) in
DCM (10 mL) was added DIEA (89 μL, 0.63 mmol) and stirred at 0°C for 20 min. 2-
chloroethylsulfonyl chloride (34 mg, 0.21 mmol) in 5 mL DCM was added slowly. The
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reaction mixture was stirred at room temperature for 2h and then partitioned between
DCM and saturated NaHCO
3
. The aqueous layer was extracted with DCM for another
, filtered, concentrated, and
two times. The combined organic layer was dried with MgSO
4
0
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purified via flash chromatography using a gradient method of 2 to 50% EtOAc in hexane
1
to give a white solid (25 mg, 36% yield). H NMR (300 MHz, CDCl
3
) δ 7.79 (d, J = 2.6
Hz, 1H), 7.55 – 7.35 (m, 2H), 7.28 (s, 1H), 7.02 (s, 1H), 6.60 (dd, J = 16.5, 9.8 Hz, 1H),
6
.35 (d, J = 16.5 Hz, 1H), 6.07 (d, J = 9.8 Hz, 1H), 2.93 (s, 6H). MS (ESI) 325.0
+
[
M+H] . Purity: 95.7%.
Methyl (E)-4-((4-chloro-3-(N,N-dimethylsulfamoyl)phenyl)amino)but-2-enoate (3).
To a solution of 5-amino-2-chloro-N,N-dimethylbenzenesulfonamide 4i (100 mg, 0.42
mmol) in acetonitrile (10 mL) was added potassium fluoride (73 mg, 12.8 mmol) and
methyl (E)-4-bromocronate (150 mg, 0.84 mmol). The solution was heated at 100°C for 4
h using a microwave reactor and concentrated in vacuo. The residue was purified with flash
chromatography using DCM as the eluent to give 3 as a white solid (73 mg, 53% yield).
1H NMR (300 MHz, CDCl
) δ 7.36 – 7.25 (m, 2H), 6.93 (dt, J = 15.7, 4.3 Hz, 1H), 6.70
3
(dd, J = 8.8, 3.1 Hz, 1H), 5.87 (dt, J = 15.8, 2.0 Hz, 1H), 4.12 (dd, J = 4.6, 1.9 Hz, 2H),
+
3
.74 (d, J = 0.8 Hz, 3H), 2.88 (d, J = 0.8 Hz, 6H). MS (ESI) 332.9[M+H] . Purity: 96.4%.
General procedure A：preparation of acrylamides 5-12. To a solution of phenyl
heterocyclic amines (0.50 mmol) in DCM (10 mL) was added DIEA (1.50 mmol) and
stirred at 0°C for 20 min. Acryloyl chloride (0.60 mmol) in DCM (10 mL) was added
2
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slowly. The reaction mixture was stirred at room temperature for 2h and then partitioned
between DCM and saturated NaHCO
3
. The aqueous layer was extracted with DCM for two
, filtered, concentrated, and
more times. The combined organic layer was dried with MgSO
4
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purified via flash chromatography using a gradient method of 2 to 50% EtOAc in hexane.
N-(1-phenyl-1H-pyrazol-4-yl)acrylamide (5). Compound 5 was prepared from 1-
phenyl-1H-pyrazol-4-amine 4a (100 mg, 0.62 mmol), DIEA (210 μl, 1.26 mmol) and
acryloyl chloride (56 mg, 0.62 mmol) according to the general procedure A as a yellow
1
solid (100 mg, 75% yield). H NMR (300 MHz, CDCl
3
) δ 8.25 (d, J = 1.4 Hz, 1H), 7.69
(d, J = 1.7 Hz, 1H), 7.39 – 7.21 (m, 3H), 6.43 (dt, J = 16.9, 1.6 Hz, 1H), 6.34 – 6.18 (m,
+
1
H), 5.74 (ddd, J = 10.1, 2.7, 1.4 Hz, 1H). MS (ESI) 214.1 [M+H] . Purity: 98.5%.
N-(1-benzyl-1H-pyrazol-4-yl)acrylamide (6). Compound 6 was prepared from 1-
benzyl-1H-pyrazol-4-amine 4b (100 mg, 0.58 mmol), DIEA (193 μl, 1.16 mmol) and
acryloyl chloride (52 mg, 0.58 mmol) according to the general procedure A as a yellow
1
solid (107 mg, 82% yield). H NMR (300 MHz, CDCl
3
) δ 8.45 (s, 1H), 8.02 (s, 1H), 7.45
(s, 1H), 7.37 – 7.24 (m, 1H), 7.29 (s, 2H), 7.20 (dd, J = 7.4, 2.2 Hz, 2H), 6.37 (dd, J = 17.0,
1.6 Hz, 1H), 6.21 (dd, J = 16.9, 10.0 Hz, 1H), 5.67 (dd, J = 10.0, 1.6 Hz, 1H), 5.23 (s, 2H).
+
MS (ESI) 227.9 [M+H] . Purity: 98.5%.
1
-(4-phenylpiperidin-1-yl)prop-2-en-1-one (7). Compound 7 was prepared from 4-
phenylpiperidine 4c (100 mg, 0.62 mmol), DIEA (207 μl, 1.24 mmol) and acryloyl chloride
(56 mg, 0.62 mmol) according to the general procedure A as a colorless oil (117 mg, 88%
1
yield). H NMR (300 MHz, CDCl
3
) δ 7.40 – 7.17 (m, 5H), 6.65 (dd, J = 16.8, 10.5 Hz,
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1H), 6.32 (dd, J = 16.8, 2.0 Hz, 1H), 5.72 (dd, J = 10.5, 2.0 Hz, 1H), 4.86 (d, J = 13.2 Hz,
1
3
H), 4.21 – 4.10 (m, 1H), 3.20 (t, J = 13.0 Hz, 1H), 2.93 – 2.75 (m, 1H), 2.87 – 2.55 (m,
+
H), 1.69 (td, J = 16.3, 12.6, 7.5 Hz, 2H). MS (ESI) 215.9 [M+H] . Purity: 100.0%.
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-(4-benzylpiperidin-1-yl)prop-2-en-1-one (8). Compound 8 was prepared from 4-
benzylpiperidine 4d (100 mg, 0.57 mmol), DIEA (190 μl, 1.14 mmol) and acryloyl chloride
(51 mg, 0.57 mmol) according to the general procedure A as a colorless oil (117 mg, 90%
1
yield). H NMR (300 MHz, CDCl
3
) δ 7.37 – 7.19 (m, 2H), 7.16 (dd, J = 5.5, 3.3 Hz, 3H),
6.59 (dd, J = 16.8, 10.5 Hz, 1H), 6.27 (dd, J = 16.8, 2.0 Hz, 1H), 5.67 (dd, J = 10.6, 2.0 Hz,
1
H), 4.14 (q, J = 7.1 Hz, 1H), 2.67 – 2.47 (m, 4H), 2.05 (d, J = 11.3 Hz, 2H), 1.71 (s, 4H).
+
MS (ESI) 230.1 [M+H] . Purity: 100.0%.
N-(5-phenylthiazol-2-yl)acrylamide (9). Compound 9 was prepared from 5-
phenylthiazol-2-amine 4e (100 mg, 0.57 mmol), DIEA (190 μl, 1.14 mmol) and acryloyl
chloride (51 mg, 0.57 mmol) according to the general procedure A as a white solid (58 mg,
1
4
5% yield). H NMR (300 MHz, CDCl
3
) δ 7.71 – 7.55 (m, 3H), 7.51 – 7.30 (m, 3H), 6.68
(dd, J = 17.0, 1.3 Hz, 1H), 6.50 (dd, J = 17.0, 10.1 Hz, 1H), 6.00 (dd, J = 10.1, 1.3 Hz, 1H).
+
MS (ESI) 231.0 [M+H] . Purity: 97.7%.
N-(4-phenylthiazol-2-yl)acrylamide (10). Compound 10 was prepared from 4-
phenylthiazol-2-amine 4f (100 mg, 0.57 mmol), DIEA (190 μl, 1.14 mmol) and acryloyl
chloride (51 mg, 0.57 mmol) according to the general procedure A as a white solid (52 mg,
1
40% yield). H NMR (300 MHz, CDCl
3
) δ 7.83 (d, J = 7.6 Hz, 2H), 7.41 (dt, J = 23.4, 7.1
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Hz, 3H), 7.21 (s, 1H), 6.56 – 6.44 (m, 1H), 6.06 (ddd, J = 16.8, 10.4, 6.2 Hz, 1H), 5.75 (dd,
+
J = 10.4, 5.5 Hz, 1H). MS (ESI) 231.0 [M+H] . Purity: 98.0%.
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Ethyl 2-acrylamido-4-phenylthiophene-3-carboxylate (11). Compound 11 was
prepared from ethyl 4-phenylthiophene-2-amino-3-carboxylate 4g (100 mg, 0.40 mmol),
DIEA (135 μl, 0.82 mmol) and acryloyl chloride (36 mg, 0.40 mmol) according to the
1
general procedure A as a yellow solid (93 mg, 77% yield). H NMR (300 MHz, CDCl
3
) δ
8
.91 (s, 1H), 7.49 (d, J = 1.7 Hz, 5H), 6.46 (dd, J = 17.0, 1.5 Hz, 1H), 6.34 (dd, J = 17.0,
9.8 Hz, 1H), 5.85 (dd, J = 9.9, 1.6 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.14 (t, J = 7.1 Hz,
+
3
H), 0.09 (s, 4H). MS (ESI) 302.1 [M+H] . Purity: 95.0%.
Ethyl 2-acrylamido-5-phenylthiophene-3-carboxylate (12). Compound 11 was
prepared from ethyl 5-phenylthiophene-2-amino-3-carboxylate 4h (100 mg, 0.40 mmol),
DIEA (135 μl, 0.82 mmol) and acryloyl chloride (36 mg, 0.40 mmol) according to the
1
general procedure A as a yellow solid (97 mg, 80% yield). H NMR (300 MHz, CDCl
3
) δ
1
1.23 (s, 1H), 7.63 (dd, J = 7.3, 1.7 Hz, 2H), 7.50 – 7.25 (m, 5H), 6.56 (dd, J = 17.0, 1.1
Hz, 1H), 6.39 (dd, J = 17.0, 10.1 Hz, 1H), 5.93 (dd, J = 10.2, 1.2 Hz, 1H), 4.40 (q, J = 7.1
+
Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H). MS (ESI) 302.1 [M+H] . Purity: 100.0%.
4
-Chloro-2-methoxyacetophenone (13r). To
hydroxylacetophenone 13p (1.0 g, 5.86 mmol) in acetonitrile (20 mL) was added
anhydrous K CO (1.61 g, 11.7 mmol) and stirred at room temperature for 10 min. methyl
iodine (0.98 g, 7.03 mmol) was then added and the reaction mixture was heated to reflux
a
solution of 4-chloro-2-
2
3
2
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overnight and concentrated. The residue was suspended in H O and extracted with EtOAc
2
(3x). The organic layer was dried with MgSO
4
, filtered, and concentrated to give 4-chloro-
1
2
-methoxyacetophenone 13r as a white solid (1.04 g, 97% yield). H NMR (300 MHz,
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CDCl ) δ 7.72 (d, J = 8.2 Hz, 1H), 7.05 – 6.94 (m, 2H), 3.93 (s, 3H), 2.61 (d, J = 0.6 Hz,
3
H). MS (ESI) 185.1 [M+H]+.
3
4-Fluoro-2-methoxyacetophenone (13s). To
hydroxylacetophenone 13p (1.0 g, 6.49 mmol) in acetonitrile (20 mL) was added
anhydrous K CO (1.79 g, 13.0 mmol) and stirred at room temperature for 10 min. Methyl
iodine (1.10 g, 7.78 mmol) was then added and the reaction mixture was heated to reflux
overnight and concentrated. The residue was suspended in H O and extracted with EtOAc
a
solution of 4-fluoro-2-
2
3
2
(3x). The organic layer was dried with MgSO
4
, filtered, and concentrated to give 4-chloro-
1
2
-methoxyacetophenone 13s as a white solid (1.05 g, 94% yield). H NMR (300 MHz,
CDCl ) δ 7.88 – 7.77 (m, 1H), 6.77 – 6.63 (m, 2H), 3.92 (s, 3H), 2.60 (s, 3H). MS (ESI)
3
169.1 [M+H]+.
General procedure B: preparation of compounds 14a-14t. To a solution of 2-
bromoacetophenones (3.00 mmol) in EtOAc (25 mL) was added copper bromide (6.00
mmol) and the suspension was heated to reflux under vigorous stirring for 3 h. Then the
resulting white suspension was cooled to room temperature and the solid was removed by
filtration. The filtrate was washed with water, dried with MgSO
to give compounds 14a-14t (55%~97%)
4
, filtered, and concentrated
.
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2-Bromo-2′-chloroacetophenone (14a). Intermediate 14a was prepared from 2-
chloroacetophenone 13a according to the general procedure B as a colorless oil (539 mg,
1
7
7% yield). H NMR (300 MHz, CDCl
3
) δ 7.62 – 7.54 (m, 1H), 7.46 (d, J = 4.9 Hz, 2H),
1
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9
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7.39 (dq, J = 7.7, 4.6, 3.6 Hz, 1H), 4.54 (d, J = 0.9 Hz, 2H). MS (ESI) 232.9, 234.9 [M+H]+.
-Bromo-3′-chloroacetophenone (14b). Intermediate 14b was prepared from 3-
2
chloroacetophenone 13b according to the general procedure B as a white solid (616 mg,
1
8
7
8% yield). H NMR (300 MHz, CDCl
3
) δ 7.98 (t, J = 1.9 Hz, 1H), 7.93 – 7.83 (m, 1H),
.66 – 7.56 (m, 1H), 7.47 (t, J = 7.9 Hz, 1H), 4.44 (s, 2H). MS (ESI) 232.9, 234.9 [M+H]+.
2-Bromo-4′-chloroacetophenone (14c). Intermediate 14c was prepared from 4-
chloroacetophenone 13c according to the general procedure B as a white solid (658 mg,
1
95% yield). H NMR (300 MHz, CDCl
3
) δ 7.95 (d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.6 Hz,
2
H), 4.43 (s, 2H). MS (ESI) 232.9, 234.9 [M+H]+.
-Bromo-4′-methylacetophenone (14d). Intermediate 14d was prepared from 4-
2
chloroacetophenone 13d according to the general procedure B as a white solid (658 mg,
1
8
8% yield). H NMR (300 MHz, CDCl
3
) δ 7.91 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz,
1H), 4.46 (d, J = 0.6 Hz, 2H), 2.45 (s, 3H). MS (ESI) 212.9, 214.9 [M+H]+.
-Bromo-4′-trifluoromethylacetophenone (14e). Intermediate 14e was prepared from
2
4
-trifluoromethylacetophenone 13e according to the general procedure B as a white solid
1
(440 mg, 55% yield). H NMR (300 MHz, CDCl
3
) δ 8.13 (d, J = 8.2 Hz, 2H), 7.79 (d, J =
8
.2 Hz, 2H), 4.48 (s, 2H). MS (ESI) 266.9, 268.9 [M+H]+.
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2-Bromo-4′-methoxyacetophenone (14f). Intermediate 14f was prepared from 4-
methoxyacetophenone 13f according to the general procedure B as a grey solid (612 mg,
1
9
0% yield). H NMR (300 MHz, CDCl
3
) δ 7.99 (d, J = 9.1 Hz, 1H), 6.98 (d, J = 8.7 Hz,
0
1
2
3
4
5
6
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1H), 4.42 (s, 1H), 3.91 (s, 2H). MS (ESI) 228.2, 230.2 [M+H]+.
-Bromo-3′,4′-dioxolacetophenone (14g). Intermediate 14g was prepared from 3,4-
2
dioxolacetophenone 13g according to the general procedure B as a yellow solid (568 mg,
1
7
9% yield). H NMR (300 MHz, CDCl
3
) δ 7.66 – 7.54 (m, 2H), 7.51 – 7.34 (m, 2H), 6.90
d, J = 8.2 Hz, 1H), 6.09 (s, 2H), 4.40 (s, 2H). MS (ESI) 242.9, 244.9 [M+H]+.
(
2-Bromo-2′-acetonaphthone (14h). Intermediate 14h was prepared from 2-
acetonaphthone 13h according to the general procedure B as a white solid (720 mg, 97%
1
yield). H NMR (300 MHz, CDCl
3
) δ 8.53 (d, J = 1.6 Hz, 1H), 8.05 (dd, J = 8.6, 1.6 Hz,
1
H), 8.00 (d, J = 8.0 Hz, 1H), 7.97 – 7.88 (m, 3H), 7.72 – 7.54 (m, 3H), 4.61 (s, 2H). MS
ESI) 248.9, 250.9 [M+H]+.
(
2-Bromo-2′-hydroxylacetophenone (14i). Intermediate 14i was prepared from 2-
hydroxylacetophenone 13i according to the general procedure B as a yellow solid (460 mg,
1
72% yield). H NMR (300 MHz, CDCl
3
) δ 11.76 (s, 1H), 7.77 (dd, J = 8.1, 1.6 Hz, 1H),
7
2
.55 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 7.03 – 6.90 (m, 1H), 4.47 (s,
H). MS (ESI) 214.9, 216.9 [M+H]+.
2-Bromo-4′-hydroxylacetophenone (14j). Intermediate 14j was prepared from 4-
hydroxylacetophenone 13j according to the general procedure B as a yellow solid (496 mg,
2
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Journal of Medicinal Chemistry
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73% yield). H NMR (300 MHz, Acetone-d6) δ 9.40 (s, 1H), 7.98 (d, J = 8.6 Hz, 2H), 6.98
d, J = 8.7 Hz, 2H), 4.65 (s, 2H). MS (ESI) 214.9, 216.9 [M+H]+.
-Bromo-2′-methoxyacetophenone (14k). Intermediate 14k was prepared from 2-
(
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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2
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0
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0
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0
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0
methoxyacetophenone 13k according to the general procedure B as a yellow solid (612 mg,
1
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1% yield). H NMR (300 MHz, CDCl
3
) δ 7.85 (dd, J = 7.7, 1.9 Hz, 1H), 7.61 – 7.48 (m,
1H), 7.12 – 6.97 (m, 2H), 4.64 (s, 2H), 3.97 (s, 3H). MS (ESI) 228.2, 230.2 [M+H]+.
-Bromo-2′-fluoroacetophenone (14l). Intermediate 14l was prepared from 2-
2
fluoroacetophenone 13l according to the general procedure B as colorless oil (611 mg, 94%
1
yield). H NMR (300 MHz, CDCl
3
) δ 7.96 (td, J = 7.6, 2.1 Hz, 1H), 7.61 (q, J = 7.1 Hz,
1
H), 7.36 – 7.23 (m, 1H), 7.19 (dd, J = 11.0, 8.0 Hz, 1H), 4.55 (dt, J = 2.5, 1.3 Hz, 2H).
MS (ESI) 216.9, 218.9 [M+H]+.
-Bromo-2′-methoxy-4′-chloroacetophenone (14r). Intermediate 14r was prepared
2
from 2-methoxy-4-chloroacetophenone 13r according to the general procedure B as a
1
yellow solid (687 mg, 87% yield). H NMR (300 MHz, CDCl
3
) δ 7.73 (dd, J = 8.3, 6.2 Hz,
1
H), 7.02 – 6.97 (m, 2H), 4.56 (s, 2H), 3.95 (s, 3H). MS (ESI) 262.9, 264.9 [M+H]+.
2-Bromo-2′-methoxy-4′-fluoroacetophenone (14s). Intermediate 14s was prepared
from 2-methoxy-4-fluoroacetophenone 13s according to the general procedure B as a
1
yellow solid (666 mg, 90% yield). H NMR (300 MHz, CDCl
3
) δ 7.89 (dd, J = 8.6, 6.9 Hz,
1H), 6.85 – 6.58 (m, 2H), 4.57 (s, 2H), 3.95 (s, 3H). MS (ESI) 246.9, 248.9 [M+H]+.
2
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ACS Paragon Plus Environment