Hydroxamic acids block replication of hepatitis c virus

Article abstract of DOI:10.1021/jm501330g

Intrigued by the role of protein acetylation in hepatitis C virus (HCV) replication, we tested known histone deacetylase (HDAC) inhibitors and a focused library of structurally simple hydroxamic acids for inhibition of a HCV subgenomic replicon. While known HDAC inhibitors with varied inhibitory profiles proved to be either relatively toxic or ineffective, structure-activity relationship (SAR) studies on cinnamic hydroxamic acid and benzo[b]thiophen-2-hydroxamic acid gave rise to compounds 22 and 53, which showed potent and selective anti-HCV activity and therefore are promising starting points for further structural optimization and mechanistic studies.

Full text of DOI:10.1021/jm501330g

Subscriber access provided by UNIV MASSACHUSETTS WORCESTER
Article
Hydroxamic Acids Block Replication of Hepatitis C Virus
Teng Ai, Yanli Xu, Li Qiu, Robert J. Geraghty, and Liqiang Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501330g • Publication Date (Web): 09 Dec 2014
Downloaded from http://pubs.acs.org on December 18, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hydroxamic Acids Block Replication of Hepatitis C Virus
*
Teng Ai, Yanli Xu, Li Qiu, Robert J. Geraghty, Liqiang Chen
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street S.E., Minneapolis, MN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
5455, USA
Abstract
Intrigued by the role of protein acetylation in hepatitis C virus (HCV) replication, we tested
known histone deacetylase (HDAC) inhibitors and a focused library of structurally simple
hydroxamic acids for inhibition of a HCV subꢀgenomic replicon. While known HDAC inhibitors
with varied inhibitory profiles proved to be either relatively toxic or ineffective, structureꢀ
activity relationship (SAR) studies on cinnamic hydroxamic acid and benzo[b]thiophenꢀ2ꢀ
hydroxamic acid gave rise to compounds 22 and 53, which showed potent and selective antiꢀ
HCV activity and therefore are promising starting points for further structural optimization and
mechanistic studies.
Key words
Hepatitis C Virus, HCV, cinnamic hydroxamic acid, hydroxamic acid, histone deacetylase,
HDAC, HDAC inhibitor, benzothiophene
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Infecting 170 million people in the world and 4 million people in the United States, hepatitis C
virus (HCV) is a major cause of liver diseases including chronic hepatitis, cirrhosis and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
hepatocellular carcinoma. For many years, the standard of care (SOC) was pegylated interferon
2
plus ribavirin, which suffered from lack of efficacy and severe sideꢀeffects. Recently approved
NS3/4A protease inhibitors (1-3, Figure 1) have demonstrated enhanced efficacy, but they are
3
undermined by rapid emergence of drugꢀresistance. Sofosbuvir (4), an approved inhibitor of the
NS5B RNA dependent RNA polymerase, is less prone to resistance and is active against the
major HCV genotypes. These and new generations of directꢀacting antivirals (DAA) in various
combinations have been aggressively pursued by the pharmaceutical industry to yield allꢀoral
4
regimens, leading to the most recent approval of the first allꢀoral pill Harvoni (a combination of
ledipasvir and sofosbuvir) by the U.S. Food and Drug Administration (FDA) to treat chronic
5
HCV genotype 1 infection.
In addition to DAA, there has been a continuing interest in exploring therapeutics that target host
cellular factors, a strategy that is generally less prone to emergence of resistance and is likely to
6
be active across different HCV genotypes. Inhibitors of cyclophilins are being investigated in
advanced clinical trials, supporting the notion that host cellular factors are promising antiꢀHCV
targets.
HCV has evolved to exploit or subvert the host cellular machinery for its replication. Alterations
of host functions generally entail reprogramming of gene expression and/or virusꢀmediated
ACS Paragon Plus Environment

Page 3 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6
modification of cellular proteins. Protein acetylation has emerged as a prominent postꢀ
translational modification of histones and other cellular proteins. The acetylation levels are
controlled by histone acetyltransferases and histone deacetylases (HDACs), the latter of which
can be divided into two major groups: zincꢀdependent HDACs and NADꢀdependent HDACs
(sirtuins). HDACs are involved in the regulation of numerous genes and the postꢀtranslational
modification of a wide range of proteins. More specifically, HDACs are regulators of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
inflammation and immunity (innate and adaptive), cellular phenomena that are closely involved
in HCV infection. Recent proteomic studies have revealed extensive acetylation in mouse and
8
human liver cells. Upꢀregulation of HDAC activity induced by HCV infection has also been
9
reported. Furthermore, polymorphisms in three HDAC enzymes (HDAC2, 3 and 5) have been
1
0
shown to be independently associated with sustained virological response in chronic HCV.
These observations suggested that HDAC inhibitors could hold promise in blocking HCV
replication. Herein we report our investigation of known HDAC inhibitors and a focused library
of simple hydroxamic acids based on the cinnamic and fused aromatic core structures using
11
cultured cells containing a subꢀgenomic HCV replicon. Very recently, benzohydroxamic acids
1
2
and a panꢀHDAC inhibitor suberoylanilide hydroxamic acid (SAHA) have been reported as
potential antiꢀHCV agents. In our current work, we have systematically investigated known
HDAC inhibitors including those that are isoformꢀselective. We have also explored novel
hydroxamic acids built upon the cinnamic acid and benzo[b]thiophene core structures. More
significantly, we have performed extensive structureꢀactivity relationship (SAR) studies to
pinpoint key structural features that elicit excellent antiꢀHCV activity and selectivity.
RESULTS AND DISCUSSION
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Evaluation of commonly used HDAC inhibitors. To investigate whether HDAC inhibitors can
block HCV replication, we tested commonly used HDAC inhibitors for inhibition of a HCV subꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
genomic replicon in Huh7 cells. 2’ꢀCꢀMethyl adenosine (2mA) , a known inhibitor of HCV
NS5B polymerase, was routinely used as a positive control in our screening for antiꢀHCV agents.
SAHA (5, Figure 2), an FDAꢀapproved HDAC inhibitor, showed a subꢀmicromolar EC value
5
0
1
2
(
Table 1), which is in agreement with its antiꢀHCV activity in a recent report. However, SAHA
showed significant cytotoxicity at a low molar concentration, leading to a low therapeutic index
14
(
TI) as defined as by CC /EC . We also tested structurally similar SMAHA (6), a dual
50
50
inhibitor of HDAC and IMPDH, the latter of which has been explored as an antiꢀcancer target.
SMAHA exhibited EC and CC values similar to those of SAHA, apparently due to their
50
50
structural similarity. We proceeded to evaluate PXD101 (7, Figure 2), a HDAC inhibitor that has
recently been approved by the FDA for the treatment of relapsed or refractory peripheral Tꢀcell
1
5
16
lymphoma, and LBH589 (8), which has been advanced into oncology clinical trials.
Compound 7 (Table 1) was more active than SAHA; however, it also exhibited elevated
cytotoxicity, resulting in a TI value almost identical to that of SAHA. Compound 8 proved to be
extremely toxic to the replicon cells as indicated by its low nanomolar CC value.
5
0
Evaluation of these HDAC inhibitors suggested that HDAC inhibitors were effective in blocking
HCV replication; however, high cytotoxicity would likely limit their application in treating HCV
infection. Since compounds 5-8 are all panꢀHDAC inhibitors that target all of HDAC1ꢀ3, 6, 8
and 10, we decided to explore whether selective HDAC inhibitors are less toxic. To this end, we
17
18
19
tested MSꢀ275 (9, Figure 2), PCIꢀ34051 (10), and Tubastatin A (11), which selectively
ACS Paragon Plus Environment

Page 5 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
inhibit HDAC1/2, HDAC8, and HDAC6, respectively. In comparison with SAHA, compound 9
displayed a reduction in both antiꢀHCV replication and toxicity, leading to a TI value almost
identical to that of SAHA (Table 1). Inhibitor 10 exhibited an inhibitory profile very similar to
that of 9. For selective HDAC6 inhibitor Tubastatin A, it was active at 10 µM but cell viability
was severely compromised at that concentration. In order to explore the role of HDAC class IIa
enzymes (HDAC4, 5, 7 and 9) that are marginally inhibited by compounds 5-11, we tested
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
0
TMP269 (12, Figure 2), a selective class IIa inhibitor recently reported. Compound 12 turned
out to be ineffective at 10 µM (Table 1). Collectively, our evaluation revealed a very narrow TI
for known HDAC inhibitors whether they are panꢀ or selective inhibitors while a selective class
IIa inhibitor was ineffective. Therefore, it may be challenging to develop HDAC inhibitors as
potential antiꢀHCV agents.
Evaluation of compounds based on cinnamic hydroxamic acids. In parallel to our testing of
known HDAC inhibitors, we also explored cinnamic hydroxamic acid, a core structure that is
21
well represented in HDAC inhibitors and inhibitors of neural toxins. To guide our structural
optimization, we performed our SAR studies based on the inhibition of replication of a subꢀ
genomic HCV replicon (EC ) in conjunction with cellular cytotoxicity (CC ). First, we probed
50
50
the effect of substitutions on position 3 of the unsaturated linker region (Table 2). The parent
compound cinnamic hydroxamic acid (13) possessed a promising submicromolar EC against
5
0
HCV replication and a TI value of 45. Introducing a methyl group (14) reduced the antiꢀ
replication activity and a more bulky phenyl group (15) further decreased the activity, indicating
that substitution at position 3 has a detrimental effect. Next, we investigated the influence of
electronꢀwithdrawing or –donating groups at the para position. As exemplified by inhibitors 16
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 17, an electronꢀdonating group, such as methyl or methoxy, greatly diminished the antiꢀHCV
activity, indicating that those groups are not tolerated at the para position. Surprisingly, an
electronꢀwithdrawing Cl group attached at the para position (18) also reduced antiꢀreplicon
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
activity. Nevertheless, a stronger electronꢀwithdrawing NO group (19) slightly improved the
2
antiꢀreplication activity when compared with the parent compound 13. Compounds donned with
a methylsulfone (20) or cyano (21) group showed excellent activity together with low reduction
in viability at 10 µM. However, at 1 µM their activities were greatly diminished. As a result, no
EC or CC values was determined. Gratifyingly, compound 22, in which a strong electronꢀ
50
50
withdrawing CF group was introduced, exhibited an EC value of 140 nM. More importantly, it
3
50
showed limited toxicity and possessed a TI value of 178, a marked improvement over that of the
parent compound 13. Furthermore, it displayed better antiꢀHCV activity when compared with
11
recently reported benzohydroxamic acids.
With compound 22 in hand, we proceeded to probe the importance of its structural features
(Table 3). Saturation of the olefin (23) led to a drastic reduction of the antiꢀHCV activity while
removing the olefin moiety (24) resulted in nearly 4ꢀfold decrease in activity. Noticeably,
compound 24 structurally resembles benzoic hydroxamic acids that have been reported to inhibit
1
1
HCV replication. To assess the importance of the hydroxamic acid functionality, we prepared
2
2
compounds 25-27, in which the hydroxamic acid was replaced with the corresponding methyl
ester, carboxylic acid, and primary amide, respectively. Those three compounds showed minimal
inhibitory activity against HCV, highlighting the importance of the hydroxamic acid
functionality. We also attempted to replace the hydroxamic acid with a benzamide chelator as
23
seen in 9. Compound 28 showed high activity but compromised cellular viability,
ACS Paragon Plus Environment

Page 7 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
characteristics that are consistent with those of compound 9. Our SAR study of compounds 23-
28 clearly indicates that the unsaturated hydroxamic acid as seen in compound 22 played a
crucial role in its antiꢀHCV activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Having determined that α, βꢀunsaturated hydroxamic acid is a key feature for antiꢀHCV activity,
we continued to investigate substituents on the aromatic ring. (Table 4). First, a CF group was
3
attached at the meta (29) or ortho (30) position; however, both modifications turned out to be
detrimental to the antiꢀHCV activity. Second, a CF group was fixed at the para position and
3
additional groups were introduced. With an additional fluoro group at the ortho position,
compound 31 showed an antiꢀHCV activity very similar to that of compound 22 but compound
31 exhibited a higher toxicity and therefore a relatively low TI. Incorporation of one more fluoro
group as shown in compound 32 led to further attenuation of activity and a concomitant
reduction of TI. When a chloro group was appended at the ortho position (compound 33), its
antiꢀHCV activity was severely compromised, suggesting that a chloro group is not tolerated at
this position. Intriguingly, compound 34, in which a chloro group was placed at both of the para
and ortho positions, exhibited excellent antiꢀHCV activity and a TI value comparable to that of
compound 31. Interestingly, compound 34 has been previously investigated for its ability to
21b
block the action of neurotoxin. Third, we also explored whether the phenyl ring could be
replaced with a pyridine ring that contains a nitrogen atom at the ortho (35), meta (36) or para
24
(
37) position. Compounds 35 and 36 showed good activity at 1 µM, suggesting that a pyridine
ring could be used in conjunction with an unsaturated hydroxamic acid. Consequently, we
prepared compounds 38 and 39 in an attempt to combine a pyridine ring and a CF group at the
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
para position (relative to the hydroxamic acid). Unfortunately, both compounds possessed a low
TI value even though they showed excellent antiꢀHCV activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In order to find isosteric replacements for the cinnamic hydroxamic acid core, we also explored
fused aromatic rings such as furan, benzo[b]thiophene and indole (Table 5). First, furanꢀ2ꢀ
hydroxamic acid (40) and benzo[b]thiopheneꢀ2ꢀhydroxamic acid (41) showed low micromolar
antiꢀHCV activities comparable to that of cinnamic hydroxamic acid 13 even though they all
possessed low TI values. Second, we tested indoleꢀderived hydroxamic acids in which the
functionality was placed on either position 2 or 3 (42-47). With a free NH, compound 42
displayed a weak activity. Blocking the NH with phenyl (43) did not improve the activity;
however, introducing a benzyl group (44) significantly increased the antiꢀHCV activity although
it still remained relatively low. Identical structural modifications were performed on indoleꢀ3ꢀ
hydroxamic acid to give compounds 45-47. Compounds 45 and 46 exhibited negligible activities.
In compound 47 benzylation led to a marked boost in activity, unfortunately likely due to its high
cytotoxicity. Taken together, our evaluation of fused aromatic hydroxamic acids suggested that
benzo[b]thiopheneꢀ2ꢀhydroxamic acid is a viable substitute for the cinnamic hydroxamic acid
scaffold.
Consequently, we focused our efforts on benzo[b]thiophene derivatives as shown in Table 6.
Initially, we evaluated the effect of an electronꢀwithdrawing group at position 5 or 6 of
benzo[b]thiophene. A chloro group at position 5 (48) slightly improved the activity. In contrast, a
nitro group at either position 5 (49) or 6 (50) significantly decreased the antiꢀHCV activity with a
concomitant lowering of TI. We also tested the effect of a CF group, which imparted a superior
3
ACS Paragon Plus Environment

Page 9 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
antiviral ability in the cinnamic hydroxamic acid series when it was placed at the para position.
Unfortunately, while compounds 51 and 52 showed very good activities at 10 µM, their activities
waned at 1 µM. These observations suggested that the benzo[b]thiopheneꢀ2ꢀhydroxamic acid
series behave differently from the cinnamic hydroxamic acid in terms of SAR trends.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Aiming to identify a new position to append an electronꢀwithdrawing group, we attached a
phenyl sulfone at position 3 (53, Table 6), which resulted in a slightly enhanced activity when
compared with the parent compound 41. More importantly, compound 53 gained a significantly
improved TI, suggesting that it may represent a new scaffold suitable for further structural
manipulations. First, in order to probe whether the electronꢀwithdrawing ability of the phenyl
sulfone contributed to the antiꢀHCV activity, the corresponding sulfoxide 54 and sulfide 55,
which possess attenuated electronꢀwithdrawing capacities, were prepared and tested. Sulfoxide
54 was twoꢀfold less active than compound 53 while sulfide 55 lost antiviral activity,
highlighting the key role of the sulfone functionality. Next, in order to confirm the importance of
the hydroxamic acid, we tested the corresponding methyl ester (56) and carboxylic acid (57),
which as anticipated showed essentially no activity at 10 µM. In summary, in addition to the
cinnamic hydroxamic acid, we have identified benzo[b]thiopheneꢀ2ꢀhydroxamic acid as a new
scaffold with antiꢀHCV activity even though the position and the nature of electronꢀwithdrawing
groups may vary for optimal antiviral ability. Nonetheless, the structural similarity and the
requisite hydroxamic acid functionality strongly suggest that both scaffolds block HCV
replication through a common mechanism of action.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Secondary assay to confirm the antiviral activity of leads. To ensure that our identified lead
compounds inhibited HCV replicon production or maintenance, representative inhibitors were
examined for their ability to reduce replicon RNA by reverse transcription and quantitative PCR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(RTꢀqPCR). The relative HCV replicon RNA level in compoundꢀtreated cells was compared to
that in DMSOꢀtreated cells using HCVꢀspecific NS5B primers/probes and GAPDH control
primers/probes. Compounds 48 and 53 reduced replicon RNA 75ꢀ80% at 2 µM and the positive
13
control inhibitor 2mA treatment (0.5 µM) reduced the replicon RNA level approximately 75%
(Figure 3). We chose those compound concentrations because they were not toxic to cells and
were likely to show significant reduction in replicon RNA levels. We conclude that our hits
inhibit HCV replicon RNA production or maintenance as expected.
Preliminary examination of potential targets. We attempted to uncover clues about potential
targets of our newly discovered hydroxamic acids. Since the hydroxamic acid functionality in
compound 53 is crucial for its activity, compound 53 was evaluated for inhibition of zincꢀ
dependent enzymes that are expected to be sensitive to hydroxamic acidꢀbased molecules. First,
it was tested against a full panel of 11 human HDACs (Table S1, Supporting Information).
Compound 53 exhibited very weak inhibition of all HDACs except for HDAC8, which was
inhibited at a low micromolar level. Compound 53 therefore is unlikely to target any of the
HDACs tested with the possible exception of HDAC8. To explore the possibility that HDAC8
inhibition might be responsible for the antiꢀHCV replicon effect of 53, we tested a potent and
selective HDAC8 inhibitor, compound 10 (Figure 2) in the HCV replicon assay. Compound 10
failed to show any activity, therefore HDAC8 is also unlikely to be a molecular target of
compound 53. Second, compound 53 was tested against a panel of human matrix
ACS Paragon Plus Environment

Page 11 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
metalloproteinases (MMPs) (Table S1, Supporting Information) because MMP inhibitors
2
5
generally feature a hydroxamic acid functionality. Compound 53 displayed submicromolar IC
50
values against MMP9, MMP12 and MMP14, which suggests that inhibition of MMP might
contribute to compound 53’s antiꢀviral activity. Third, compound 53 exhibited low inhibition of
tumor necrosis factorꢀα converting enzyme (TACE), a zincꢀdependent enzyme that can also be
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
6
blocked by hydroxamic acids. Finally, compound 53 did not inhibit HCV NS3/4A or NS5B,
two prominent antiꢀHCV viral targets that have been aggressively explored.
Our preliminary studies suggest that compound 53 does not target common viral target NS33/4A
or NS5. Neither does it inhibit HDACs or TACE, both of which are zincꢀdependent enzymes.
Nevertheless, we cannot rule out the possibility that compound 53 exerts at least part of its antiꢀ
viral activity by blocking selected members of MMPs.
Chemistry. We used several synthetic methods to prepare hydroxamic acids. For the syntheses
of cinnamic hydroxamic acids 13-15, 19 and 22, the corresponding carboxylic acids 59a-e were
2
7
activated with either oxalyl chloride/DMF or 1,1’ꢀcarbonyldiimidazole (CDI) followed by
28
29
treatment with a hydroxylamine solution (Scheme 1). Carboxylic acids 59b and 59c, which
2
8
29
are not commercially available, were readily obtained from ethyl esters 58b and 58c,
respectively. For the synthesis of hydroxamic acids 16-18, 21 and 23, we adopted another
synthetic method, in which carboxylic acids 60a-e were first converted into methyl esters 61a-e.
Treatment of these methyl esters with hydroxylamine hydrochloride in the presence of methanoic
NaOMe afforded the desired hydroxamic acids.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The syntheses of hydroxamic acids 20, 29-33, and their pyridine analogs 38-39 began with
properly substituted bromides or iodides 62a and 62c-h (Scheme 2), which underwent Heck
reactions to give tertꢀbutyl esters 63a and 63c-h. After removal of the tertꢀbutyl groups under
acidic conditions, the resulting carboxylic acids as well as commercially available acid 63b were
converted into the THPꢀprotected hydroxamates 64a-h, which upon an acidic treatment gave
hydroxamic acids 20, 29-33 and 38-39. In addition, hydroxamic acids 34-37 were prepared
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1b, 24, 30
according to literature procedures.
The syntheses of fused aromatic hydroxamic acids 40-42 and 45 were accomplished through
activation of the corresponding acids 65a-d and subsequent treatment with a hydroxylamine
solution (Scheme 3). To prepare the benzylated hydroxamic acids 44 and 47, benzylation was
31
performed on carboxylic acids 65c-d to give compounds 66c-d, which were then converted into
hydroxamic acids 44 and 47. In contrast to the benzylated counterparts, introduction of a phenyl
3
2
group was achieved on methyl esters 67a-b via a copperꢀcatalyzed coupling reaction, resulting
33
34
in compounds 68a-b. After hydrolysis, the resulting acids 69a-b were transformed into
hydroxamic acids 43 and 46 (Scheme 4).
As shown in Scheme 5, benzo[b]thiophene hydroxamic acids 48-52, which contain a substituent
at either position 5 or 6, were prepared from methyl esters 70a-e after being treated with
hydroxylamine. Benzo[b]thiophene analogs that are substituted at position 3 were all derived
from chloride 71, which was first displaced by thiophenol to give sulfide 72. Oxidations of
sulfide 72 to the corresponding sulfoxide 73 and sulfone 56 were accomplished with
H O /TMSCl and oxone, respectively. These three methyl esters were individually converted
2
2
ACS Paragon Plus Environment

Page 13 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
into hydroxamic acids 53-55. In order to confirm the importance of the hydroxamic acid
functionality, sulfone 56 was also hydrolyzed to give its carboxylic acid 57.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSIONS
Prompted by the prevalence and importance of protein acetylation in liver cells, we evaluated
known HDAC inhibitors and structurally simple hydroxamic acids in human liver cells
containing a HCV subꢀgenomic replicon. HDAC inhibitors, regardless of their selectivity profile,
failed to show antiꢀHCV activity with a desired TI value. Nevertheless, our investigation of
simple hydroxamic acids led to those based on the cinnamic acid and benzo[b]thiophene core
structures. Further SAR studies produced compounds 22 and 53, which possessed excellent antiꢀ
viral activity and selectivity. Our SAR studies also revealed that electronꢀwithdrawing CF and
3
phenylsulfone groups are responsible for the enhanced activity and selectivity of compounds 22
and 53, respectively, observations that will help guide our design of improved HCV inhibitors.
We also demonstrated that compound 53 reduced replicon RNA as judged by RTꢀqPCR,
supporting our conclusion that it is a bona fide inhibitor of HCV replication. Our preliminary
investigation showed that compound 53 did not block NS3/4A or NS5B, two of the commonly
pursued viral targets. Neither does it appear to exert its antiꢀHCV activity through inhibiting
zincꢀdependent enzymes HDACs or TACE. While the exact molecular target of compound 53
remains to be elucidated, inhibition of selected members of MMPs might contribute to its
activity. Our work represents the first systematic investigation of known HDAC inhibitors and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
novel hydroxamic acids for their potential antiꢀHCV application. We are currently performing
3
5
activityꢀbased protein profiling to identify the molecular targets of compounds 22 and 53.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemical Synthesis. All commercial reagents were used as provided unless otherwise indicated.
Belinostat (7), panobinostat (8), entinostat (9), 10, and tubastatin A HCl were obtained from
Selleckchem. Compound 12 was purchased from Cellagen Technology. An anhydrous solvent
dispensing system (J. C. Meyer) using 2 packed columns of neutral alumina was used for drying
THF, Et O, and CH Cl while 2 packed columns of molecular sieves were used to dry DMF.
2
2
2,
Solvents were dispensed under argon. Flash chromatography was performed with Ultra Pure
®
®
silica gel (Silicycle) or with RediSep R silica gel columns on a Teledyne ISCO CombiFlash
f
R system using the solvents as indicated. Nuclear magnetic resonance spectra were recorded on
f
1
a Varian 600 MHz with Me Si or signals from residual solvent as the internal standard for H.
4
Chemical shifts are reported in ppm, and signals are described as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), brs (broad singlet), and dd (double doublet). Values given for
coupling constants are first order. High resolution mass spectra were recorded on an Agilent
TOF II TOF/MS instrument equipped with either an ESI or APCI interface. Analysis of sample
purity was performed on an Agilent 1200 Infinity series HPLC system with a Phenomenex
Gemini C18 column (5 ꢁ, 4.6×250 mm). HPLC conditions were the following: solvent A =
water, solvent B = MeCN or MeOH; flow rate = 2.0 mL/min. Compounds were eluted with a
gradient of from 10% to 100% MeCN/water or from 10 to 100% MeOH/water in 15 min. Purity
was determined by the absorbance at 254 nm. All tested compounds have a purity of ≥ 95%.
ACS Paragon Plus Environment

Page 15 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
7
N-Hydroxycinnamamide (13). A mixture of cinnamic acid (59a, 148 mg, 1.0 mmol) and CDI
(
243 mg, 1.5 mmol) in THF (2 mL) was stirred at rt for 80 min. NH OH·HCl (138 mg, 2.0
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mmol) was then added and the reaction mixture was allowed to stir overnight. The solvent was
evaporated under vacuum and the resulting residue was purified by flash column
1
chromatography to give hydroxamic acid 13 as a pale solid (46 mg, 28%). H NMR (CD OD,
3
ꢀ
6
00 MHz) δ 7.52ꢀ7.57 (m, 3H), 7.34ꢀ7.36 (m, 3H), 6.47 (d, J = 9.6 Hz, 1H). HRMS (ESI ) calcd
ꢀ
for C H NO 162.0555 (MꢀH) , found 162.0556.
9
8
2
2
8
(
E)-N-Hydroxy-3-phenylbut-2-enamide (14). To a solution of carboxylic acid 59b (131 mg,
0.81 mmol) and two drops of DMF in CH Cl (1.5 mL) was added oxalyl chloride (304 mg, 2.4
2
2
mmol), and the reaction mixture was allowed to stir at 0 ºC for 1 h. After the solvent being
removed under vacuum, the residue was dissolved in Et O (1 mL) and the resulting solution was
2
added to a solution of NH OH, which was prepared from NH OH·HCl (1200 mg, 17.3 mmol)
2
2
and Na CO (1200 mg, 11.3 mmol) in Et O (8 mL) and H O (1.5 mL). The reaction mixture was
2
3
2
2
allowed to stir at rt overnight and the solvent was evaporated under vacuum. The residue was
purified by flash column chromatography to give hydroxamic acid 14 as a pale solid (18 mg,
1
2
1%). H NMR (CD OD, 600 MHz) δ 7.50 (d, J = 7.2 Hz, 2H), 7.34ꢀ7.41 (m, 3H), 6.10 (s, 1H),
3
ꢀ
ꢀ
2.50 (s, 3H). HRMS (ESI ) calcd for C H NO 176.0712 (MꢀH) , found 176.0718.
1
0
10
2
3
6
N-Hydroxy-3,3-diphenylacrylamide (15). In a manner similar to that was described for the
2
9
preparation of compound 14, carboxylic acid 59c (65 mg, 0.29 mmol) was treated with oxalyl
chloride (110 mg, 0.87 mmol) followed by a solution of NH OH to give hydroxamic acid 15 as a
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
pale solid (57 mg, 83%). H NMR (DMSOꢀd , 600 MHz) δ 7.38ꢀ7.31 (m, 6H), 7.22ꢀ7.18 (m, 2H),
6
ꢀ
ꢀ
7
.16ꢀ7.12 (m, 2H), 6.27 (s, 1H). HRMS (ESI ) calcd for C H NO 238.0868 (MꢀH) , found
15 12 2
238.0867.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1b, 37
(E)-N-Hydroxy-3-(p-tolyl)acrylamide (16).
To a solution of carboxylic acid 60a (162 mg,
1
.0 mmol) in MeOH (4 mL) was added thionyl chloride (179 mg, 1.5 mmol), and the reaction
mixture was allowed to stir at rt for 2 h. After the solvent being removed under vacuum,
NH OH·HCl (278 mg, 4.0 mmol) and MeOH (15 mL) was added. To the above suspension was
2
added 25% wt NaOMe in MeOH (1.65 mL, 7.2 mmol). The reaction mixture was allowed to stir
at rt for 20 h and concentrated. The residue was suspended in H O (20 mL) and the mixture was
2
acidified with 1N HCl to pH = 8. The solid formed was filtered, washed with H O, suctionꢀdried,
2
1
and dried in vacuo to give hydroxamic acid 16 as a pale solid (115 mg, 65%). H NMR (DMSOꢀ
d6, 600 MHz) δ 7.58ꢀ7.52 (m, 3H), 7.22 (d, J = 8.4 Hz, 2H), 6.46 (d, J = 16.2 Hz, 1H), 2.32 (s,
+
+
3
H). HRMS (ESI ) calcd for C H NO (M+H) 178.0868, found 178.0866.
10 12 2
2
1b, 36ꢀ37
(E)-N-Hydroxy-3-(4-methoxyphenyl)acrylamide (17).
In a manner similar to that was
described for the preparation of compound 16, carboxylic acid 60b (178 mg, 1.0 mmol) was
treated with thionyl chloride in MeOH followed by NH OH·HCl and 25% wt NaOMe in MeOH
2
1
to give hydroxamic acid 17 as a pale solid (108 mg, 56%). H NMR (DMSOꢀd , 600 MHz) δ
6
7
.63 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 16.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.37 (d, J = 16.2 Hz,
+
+
1
H), 3.79 (s, 3H). HRMS (ESI ) calcd for C H NO (M+H) 194.0817, found 194.0826.
10 12
3
ACS Paragon Plus Environment

Page 17 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
21b, 36ꢀ37
(
E)-3-(4-Chlorophenyl)-N-hydroxyacrylamide (18).
In a manner similar to that was
described for the preparation of compound 16, carboxylic acid 60c (183 mg, 1.0 mmol) was
treated with thionyl chloride in MeOH followed by NH OH·HCl and 25% wt NaOMe in MeOH
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
to give hydroxamic acid 18 as a light brown solid (89 mg, 45%). H NMR (DMSOꢀd , 600 MHz)
6
δ 7.72 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 15.6 Hz, 2H), 7.47 (d, J = 9.0 Hz, 1H), 6.55 (d, J = 15.6
+
+
Hz, 1H). HRMS (ESI ) calcd for C H ClNO (M+H) 198.0322, found 198.0322.
9
9
2
2
1b, 36ꢀ37
(E)-N-Hydroxy-3-(4-nitrophenyl)acrylamide (19).
In a manner similar to that was
described for the preparation of compound 13, carboxylic acid 59d (193 mg, 1.0 mmol) was
treated with CDI (243 mg, 1.5 mmol) and then NH OH·HCl (138 mg, 2.0 mmol) to give
2
1
hydroxamic acid 19 as a yellow solid (146 mg, 70%). H NMR (CD OD, 600 MHz) δ 8.27 (d, J
3
=
7.2 Hz, 2H), 7.80 (d, J = 7.2 Hz, 2H), 7.66 (d, J = 15.0 Hz, 1H), 6.65 (dd, J = 16.2, 1.8 Hz,
ꢀ
ꢀ
1
H). HRMS (ESI ) calcd for C H N O 207.0406 (MꢀH) , found 207.0405.
9 7 2 4
(
E)-tert-Butyl 3-(4-(methylsulfonyl)phenyl)acrylate (63a). The reaction vessel was charged
with 4ꢀbromophenyl methyl sulfone 62a (1 mmol), tꢀbutyl acrylate (3 mmol), Pd(OAc) (5%
2
mmol), Xphos ligand (10% mmol), and NEt (2.0 mmol) in DMF (5 mL). The reaction mixture
3
was heated at 140 ºC for 20 h. The reaction mixture was cooled to rt, diluted with EtOAc (20
mL), and washed with water. The organic phase was dried over anhydrous Na SO After
2
4.
removal of the solvent, the residue was purified by silica gel column using EtOAc/hexanes to
1
afford transꢀcinnamic acid tꢀbutyl ester 63a as a white solid (500 mg, 88%). H NMR (CDCl ,
3
6
00 MHz) δ 7.95 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 16.2 Hz, 1H), 6.49 (d,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ
ꢀ
J = 16.2 Hz, 1H), 3.07 (s, 3H), 1.55 (s, 9H). HRMS (APCI ) calcd for C H O S (MꢀtꢀBu)
1
0
9
4
225.0222, found 225.0219.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38
(
E)-tert-Butyl 3-(2-(trifluoromethyl)phenyl)acrylate (63c). In a manner similar to that was
described for the preparation of compound 63a, iodide 62c (136 mg, 0.5 mmol) underwent a
1
Heck reaction to give transꢀcinnamic acid tꢀbutyl ester 63c as a white solid (120 mg, 88%). H
NMR (CDCl , 600 MHz) δ 7.97 (d, J = 15.0 Hz, 1H), 7.69 (dd, J = 6.6, 6.6 Hz, 2H), 7.54 (dd, J
3
= 7.5, 7.5 Hz, 1H), 7.45 (dd, J = 7.5, 7.5 Hz, 1H), 6.34 (d, J = 15.6 Hz, 1H), 1.54 (s, 9H). HRMS
ꢀ
ꢀ
(
APCI ) calcd for C H F O (MꢀtꢀBu) 215.0320, found 215.0322
10 6 3 2
(E)-tert-Butyl 3-(2-fluoro-4-(trifluoromethyl)phenyl)acrylate (63d). In a manner similar to
that was described for the preparation of compound 63a, bromide 62d (243 mg, 1.0 mmol)
underwent a Heck reaction to give transꢀcinnamic acid tꢀbutyl ester 63d as a white solid (170
1
mg, 59%). H NMR (CDCl , 600 MHz) δ 7.70 (d, J = 15.6 Hz, 1H), 7.64 (dd, J = 7.5, 7.5 Hz,
3
1
H), 7.42 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 10.2 Hz, 1H), 6.54 (d, J = 16.2 Hz, 1H), 1.55 (s, 9H).
ꢀ
ꢀ
HRMS (APCI ) calcd for C H F O (MꢀtꢀBu) 233.0226, found 233.0226.
10
5
4
2
(
E)-tert-Butyl 3-(2,6-difluoro-4-(trifluoromethyl)phenyl)acrylate (63e). In a manner similar to
that was described for the preparation of compound 63a, bromide 62e (261 mg, 1.0 mmol)
underwent a Heck reaction to give transꢀcinnamic acid tꢀbutyl ester 63e as a white solid (200
1
mg, 65%). H NMR (CDCl , 600 MHz) δ 7.64 (d, J = 16.2 Hz, 1H), 7.23 (s, 1H), 7.21 (s, 1H),
3
ꢀ
ꢀ
6
.74 (d, J = 16.2 Hz, 1H), 1.55 (s, 9H). HRMS (APCI ) calcd for C H F O (MꢀtꢀBu) 251.0131,
10 4 5 2
found 251.0127.
ACS Paragon Plus Environment

Page 19 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
E)-tert-Butyl 3-(2-chloro-4-(trifluoromethyl)phenyl)acrylate (63f). In a manner similar to
that was described for the preparation of compound 63a, iodide 62f (153 mg, 0.5 mmol)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
underwent a Heck reaction to give transꢀcinnamic acid tꢀbutyl ester 63f as a white solid (100 mg,
1
65%). H NMR (CDCl , 600 MHz) δ 7.97 (d, J = 16.2 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.68 (s,
3
ꢀ
1
H), 7.52 (d, J = 7.8 Hz, 1H), 6.43 (d, J = 16.2 Hz, 1H), 1.55 (s, 9H). HRMS (APCI ) calcd for
ꢀ
C H ClF O (MꢀtꢀBu) 248.9930, found 248.9931.
10
5
3
2
(E)-tert-Butyl 3-(5-(trifluoromethyl)pyridin-2-yl)acrylate (63g). In a manner similar to that
was described for the preparation of compound 63a, bromide 62g (113 mg, 0.5 mmol) underwent
1
a Heck reaction to give tꢀbutyl ester 63g as a white solid (70 mg, 50%). H NMR (CDCl , 600
3
MHz) δ 8.83 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 16.2 Hz,
+
+
1
H), 6.52 (d, J = 16.2 Hz, 1H), 1.55 (s, 9H). HRMS (ESI ) calcd for C H F NO (M+H)
13 15 3 2
274.1055, found 274.1050.
(E)-tert-Butyl 3-(6-(trifluoromethyl)pyridin-3-yl)acrylate (63h). In a manner similar to that
was described for the preparation of compound 63a, bromide 62h (261 mg, 1.0 mmol)
1
underwent a Heck reaction to give tꢀbutyl ester 63h as a white solid (150 mg, 55%). H NMR
(CDCl , 600 MHz) δ 8.83 (s, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.59 (d, J =
3
+
1
5.6 Hz, 1H), 6.52 (d, J = 16.8 Hz, 1H), 1.55 (s, 9H). HRMS (ESI ) calcd for C H F NO
1
3
15
3
2
+
(
M+H) 274.1055, found 274.1057.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
E)-N-(Tetrahydropyran-2-yloxy)-3-(4-methylsulfonyl)phenyl)acrylamide (64a). A solution
of tꢀButyl ester 63a (450 mg, 1.59 mmol) in 6 mL of TFA/DCM (2:1) was allowed to stir at rt for
h. After removal of the organic solvent, Oꢀ(tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)hydroxylamine (1.0
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mmol) and EDC (1.0 mmol) were added. The reaction mixture was allowed to stir at rt overnight
and was then washed with water and brine. After removal of the solvent, the residue was purified
1
by silica gel column using EtOAc/hexanes to afford 64a as a colorless oil (400mg, 70%). H
NMR (CDCl , 600 MHz) δ 7.90 (d, J = 6.6 Hz, 2H), 7.80ꢀ7.60 (m, 3H), 6.65 (d, J = 13.8 Hz,
3
1H), 5.10 (brs, 1H), 4.08ꢀ3.98 (m, 1H), 3.68ꢀ3.62 (m, 1H), 3.08 (s, 3H), 1.92ꢀ1.78 (m, 3H), 1.70ꢀ
ꢀ
ꢀ
1
.54 (m, 3H). HRMS (ESI ) calcd for C H NO S (MꢀH) 324.0906, found 324.0910.
15 18 5
(E)-N-(Tetrahydropyran-2-yloxy)-3-(3-(trifluoromethyl)phenyl)acrylamide
solution of acid 63b (108 mg, 0.5 mmol), Oꢀ(tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)hydroxylamine (64 mg,
.55 mmol), and EDC (105 mg, 0.55 mmol) in DCM (10 mL) was allowed to stir at rt overnight
(64b).
A
0
and the mixture was washed with water and brine. After removal of the solvent, the residue was
purified by silica gel column using EtOAc/hexanes to afford 64b as a colorless oil (100 mg,
1
63%). H NMR (CDCl , 600 MHz) δ 7.80ꢀ7.72 (m, 2H), 7.67 (d, J = 7.2 Hz, 1H), 7.62 (d, J =
3
7
.8 Hz, 1H), 7.51 (dd, J = 7.5, 7.5 Hz, 1H), 6.58 (d, J = 13.8 Hz, 1H), 5.03 (brs, 1H), 3.98 (t, J =
ꢀ
9
.3 Hz, 1H), 3.70ꢀ3.64 (m, 1H), 1.92ꢀ1.82 (m, 3H), 1.70ꢀ1.58 (m, 3H). HRMS (ESI ) calcd for
ꢀ
C H F NO 314.1004 (MꢀH) , found 314.1009.
15
15
3
3
(E)-N-(Tetrahydropyran-2-yloxy)-3-(2-(trifluoromethyl)phenyl)acrylamide (64c). In
a
manner similar to that was described for the preparation of compound 64a, tꢀbutyl ester 63c (120
mg, 0.44 mmol) was converted into the corresponding acid followed by an EDCꢀmediated
ACS Paragon Plus Environment

Page 21 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
coupling to afford 64c as a white solid (80 mg, 58%). H NMR (CDCl , 600 MHz) δ 8.73 (brs,
3
1
7
1
H), 8.08 (d, J = 15.6 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.55 (dd, J =
.2, 7.2 Hz, 1H), 7.46 (dd, J = 7.5, 7.5 Hz, 1H), 6.50ꢀ6.20 (m, 1H), 5.03 (brs, 1H), 4.04ꢀ3.94 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
H), 3.70ꢀ3.63 (m, 1H), 1.90ꢀ1.80 (m, 3H), 1.70ꢀ1.56 (m, 3H). HRMS (ESI ) calcd for
ꢀ
C H F NO 314.1004 (MꢀH) , found 314.1008.
15
15
3
3
(
E)-N-(Tetrahydropyran-2-yloxy)-3-(2-fluoro-4-(trifluoromethyl)phenyl)acrylamide (64d).
In a manner similar to that was described for the preparation of compound 64a, tꢀbutyl ester 63d
(
160 mg, 0.55 mmol) was converted into the corresponding acid followed by an EDCꢀmediated
1
coupling to afford 64d as a white solid (100 mg, 55%). H NMR (CDCl , 600 MHz) δ 9.59 (brs,
3
1H), 7.79 (d, J = 13.8 Hz, 1H), 7.62 (s, 1H), 7.44ꢀ7.30 (m, 2H), 6.72ꢀ6.58 (m, 1H), 5.08 (brs,
ꢀ
1
H), 4.06ꢀ3.96 (m, 1H), 3.70ꢀ3.64 (m, 1H), 1.90ꢀ1.80 (m, 3H), 1.70ꢀ1.58 (m, 3H). HRMS (ESI )
ꢀ
calcd for C H F NO 332.0910 (MꢀH) , found 332.0916.
15 14
4
3
(
E)-N-(Tetrahydropyran-2-yloxy)-3-(2,6-difluoro-4-(trifluoromethyl)phenyl)acrylamide
(64e). In a manner similar to that was described for the preparation of compound 64a, tꢀbutyl
ester 63e (170 mg, 0.55 mmol) was converted into the corresponding acid followed by an EDCꢀ
1
mediated coupling to afford 64e as a colorless oil (110 mg, 57%). H NMR (CDCl , 600 MHz) δ
3
8.89 (brs, 1H), 7.80 (d, J = 15.6 Hz, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 6.82ꢀ6.70 (m, 1H), 5.05 (brs,
ꢀ
1
H), 4.04ꢀ3.92 (m, 1H), 3.70ꢀ3.60 (m, 1H), 1.94ꢀ1.76 (m, 3H), 1.70ꢀ1.58 (m, 3H). HRMS (ESI )
ꢀ
calcd for C H F NO 350.0816 (MꢀH) , found 350.0813.
15 13
5
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
E)-N-(Tetrahydropyran-2-yloxy)-3-(2-chloro-4-(trifluoromethyl)phenyl)acrylamide (64f).
In a manner similar to that was described for the preparation of compound 64a, tꢀbutyl ester 63f
(60 mg, 0.22 mmol) was converted into the corresponding acid followed by an EDCꢀmediated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
coupling to afford 64f as a white solid (45 mg, 65%). H NMR (CDCl , 600 MHz) δ 9.05 (brs,
3
1H), 8.07 (d, J = 14.4 Hz, 1H), 7.70ꢀ7.60 (m, 2H), 7.52ꢀ7.46 (m, 1H), 6.57ꢀ6.40 (m, 1H), 5.05
(brs, 1H), 4.4ꢀ3.95 (m, 1H), 3.70ꢀ3.64 (m, 1H), 1.92ꢀ1.79 (m, 3H), 1.70ꢀ1.58 (m, 3H). HRMS
ꢀ
ꢀ
(ESI ) calcd for C H ClF NO 348.0614 (MꢀH) , found 348.0609.
1
5
14
3
3
(E)-N-(Tetrahydropyran-2-yloxy)-3-(5-(trifluoromethyl)pyridin-2-yl)acrylamide (64g). In a
manner similar to that was described for the preparation of compound 64a, tꢀbutyl ester 63g (70
mg, 0.26 mmol) was converted into the corresponding acid followed by an EDCꢀmediated
1
coupling to afford 64g as a colorless oil (40 mg, 48%). H NMR (CDCl , 600 MHz) δ 8.86 (s,
3
1
7
1
H), 8.58 (brs, 1H), 7.05 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 14.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H),
.14ꢀ7.00 (m, 1H), 5.05 (brs, 1H), 4.02ꢀ3.94 (m, 1H), 3.72ꢀ3.65 (m, 1H), 1.90ꢀ1.81 (m, 3H),
+
+
.70ꢀ1.58 (m, 3H). HRMS (ESI ) calcd for C H F N O 317.1113 (M+H) , found 317.1106.
1
4
16
3
2
3
(E)-N-(Tetrahydropyran-2-yloxy)-3-(6-(trifluoromethyl)pyridin-3-yl)acrylamide (64h). In a
manner similar to that was described for the preparation of compound 64a, tꢀbutyl ester 63h (150
mg, 0.55 mmol) was converted into the corresponding acid followed by an EDCꢀmediated
1
coupling to afford 64h as a colorless oil (90 mg, 52%). H NMR (CDCl , 600 MHz) δ 8.86 (s,
3
1
H), 8.02ꢀ7.92 (m, 1H), 7.76 (d, J = 16.2 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 6.62ꢀ6.57 (m, 1H),
5
.04 (brs, 1H), 4.02ꢀ3.94 (m, 1H), 3.72ꢀ3.64 (m, 1H), 1.90ꢀ1.81 (m, 3H), 1.70ꢀ1.58 (m, 3H).
+
+
HRMS (ESI ) calcd for C H F N O 317.1113 (M+H) , found 317.1117.
14
16
3
2
3
ACS Paragon Plus Environment

Page 23 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
E)-N-Hydroxy-3-(4-(methylsulfonyl)phenyl)acrylamide (20). To a solution of 64a (300 mg,
0
.92 mmol) in DCM (5 mL) was added HCl in Et O (3 mL, 2M). The reaction mixture was
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
stirred at rt for 2 h. The white solid precipitate was filtrated and washed with DCM to give
1
hydroxamic acid 20 as white solid (86.2 mg, 39%). H NMR (DMSOꢀd , 600 MHz) δ 7.94 (d, J
6
=
7.2 Hz, 2H), 7.81 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 15.6 Hz, 1H), 6.66 (d, J = 15.6 Hz, 1H), 3.23
+
+
(
s, 3H). HRMS (ESI ) calcd for C H NO S (M+H) 242.0487, found 242.0481.
10 12 4
21b
(E)-3-(4-Cyanophenyl)-N-hydroxyacrylamide (21).
In a manner similar to that was
described for the preparation of compound 16, carboxylic acid 60d (100 mg, 0.58 mmol) was
treated with thionyl chloride in MeOH followed by NH OH·HCl and 25% wt NaOMe in MeOH
2
1
to give hydroxamic acid 21 as a white solid (43 mg, 40%). H NMR (DMSOꢀd , 600 MHz) δ
6
7.86 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H), 7.50 (d, J = 16.2 Hz, 1H), 6.63 (d, J = 16.2 Hz,
ꢀ
ꢀ
1
H). HRMS (ESI ) calcd for C H N O (MꢀH) 187.0508, found 187.0501.
1
0
7
2
2
3
7
(E)-N-Hydroxy-3-(4-trifluoromethylphenyl)acrylamide (22). In a manner similar to that was
described for the preparation of compound 13, carboxylic acid 59e (216 mg, 1.0 mmol) was
treated with CDI (243 mg, 1.5 mmol) and then NH OH·HCl (138 mg, 2.0 mmol) to give
2
1
hydroxamic acid 22 as a white solid (68 mg, 29%). H NMR (CD OD, 600 MHz) δ 7.75 (d, J =
3
8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 15.6 Hz, 1H), 6.60 (d, J = 15.6 Hz, 1H).
ꢀ
ꢀ
HRMS (ESI ) calcd for C H F NO 230.0429 (MꢀH) , found 230.0428.
10
7
3
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N-Hydroxy-3-(4-(trifluoromethyl)phenyl)propanamide (23). In a manner similar to that was
described for the preparation of compound 16, carboxylic acid 60e (109 mg, 0.50 mmol) was
treated with thionyl chloride in MeOH followed by NH OH·HCl and 25% wt NaOMe in MeOH
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
to give hydroxamic acid 23 as a white solid (17 mg, 15%). H NMR (DMSOꢀd , 600 MHz) δ
6
7.63 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 3.31 (brs, 2H), 2.90 (t, J = 7.8 Hz, 2H), 2.57 (t,
ꢀ
ꢀ
J = 8.1 Hz, 2H). HRMS (ESI ) calcd for C H F NO (MꢀH) 232.0585, found 232.0581.
1
0
9
3
2
(E)-N-Hydroxy-3-(3-(trifluoromethyl)phenyl)acrylamide (29). In a manner similar to that was
described for the preparation of compound 20, a solution of compound 64b (50 mg, 0.16 mmol)
in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M) for 2 h to give hydroxamic acid 29 as
2
1
a white solid (23 mg, 63%). H NMR (DMSOꢀd , 600 MHz) δ 10.79 (s, 1H), 9.12 (brs, 1H), 7.92
6
(s, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.65 (dd, J = 8.1, 8.1 Hz, 1H), 7.55 (d,
+
+
J = 16.2 Hz, 1H), 6.60 (d, J = 15.6 Hz, 1H). HRMS (ESI ) calcd for C H F NO (M+H)
10
7
3
2
230.0429, found 230.0424.
(E)-N-Hydroxy-3-(2-(trifluoromethyl)phenyl)acrylamide (30). ). In a manner similar to that
was described for the preparation of compound 20, a solution of compound 64c (60 mg, 0.19
mmol) in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M) for 2 h to give hydroxamic
2
1
acid 30 as a white solid (40 mg, 91%). H NMR (DMSOꢀd , 600 MHz) δ 10.91 (s, 1H), 9.16 (s,
6
1H), 7.83ꢀ7.77 (m, 2H), 7.76ꢀ7.56 (m, 2H), 7.59 (dd, J = 7.5, 7.5 Hz, 1H), 6.52 (d, J = 15.0 Hz,
ꢀ
ꢀ
1
H). HRMS (ESI ) calcd for C H F NO (MꢀH) 230.0429, found 230.0426.
10
7
3
2
ACS Paragon Plus Environment

Page 25 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
E)-3-(2-Fluoro-4-(trifluoromethyl)phenyl)-N-hydroxyacrylamide (31). In a manner similar
to that was described for the preparation of compound 20, a solution of compound 64d (60 mg,
0
.18 mmol) in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M) for 2 h to give
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
hydroxamic acid 31 as a white solid (12 mg, 27%). H NMR (DMSOꢀd , 600 MHz) δ 7.89 (dd, J
6
= 7.8, 7.8 Hz, 1H), 7.77 (d, J = 11.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 15.6 Hz, 1H),
+
+
6
.69 (d, J = 15.6 Hz, 1H). HRMS (ESI ) calcd for C H F NO (M+H) 248.0335, found
10 6 4 2
248.0338.
(
E)-3-(2,6-Difluoro-4-(trifluoromethyl)phenyl)-N-hydroxyacrylamide (32). In a manner
similar to that was described for the preparation of compound 20, a solution of compound 64e
(60 mg, 0.17 mmol) in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M) for 2 h to give
2
1
hydroxamic acid 32 as a white solid (13 mg, 29%). H NMR (DMSOꢀd , 600 MHz) δ 11.05 (s,
6
1
H), 9.27 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 16.2 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H).
+
+
HRMS (ESI ) calcd for C H F NO (M+H) 266.0240, found 266.0246.
10
5
5
2
(E)-3-(2-Chloro-4-(trifluoromethyl)phenyl)-N-hydroxyacrylamide (33). In a manner similar
to that was described for the preparation of compound 20, a solution of compound 64f (50 mg,
0
.14 mmol) in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M) for 2 h to give
2
1
hydroxamic acid 31 as a white solid (33 mg, 90%). H NMR (DMSOꢀd , 600 MHz) δ 7.95 (s,
6
1
H), 7.92 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 15.6 Hz, 1H), 6.64 (d, J =
ꢀ
ꢀ
1
6.2 Hz, 1H). HRMS (ESI ) calcd for C H ClF NO (MꢀH) 264.0039, found 264.0032.
10
7
3
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
E)-N-Hydroxy-3-(5-(trifluoromethyl)pyridin-2-yl)acrylamide hydrochloride (38). In a
manner similar to that was described for the preparation of compound 20, a solution of
compound 64g (40 mg, 0.13 mmol) in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M)
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
for 2 h to give hydroxamic acid 38 as a white solid (6 mg, 18%). H NMR (DMSOꢀd , 600 MHz)
6
δ 8.98 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 15.0 Hz, 1H), 7.07
ꢀ
ꢀ
(d, J = 15.0 Hz, 1H). HRMS (ESI ) calcd for C H F N O (MꢀH) 231.0381, found 231.0389.
9 6 3 2 2
(E)-N-Hydroxy-3-(6-(trifluoromethyl)pyridin-3-yl)acrylamide hydrochloride (39). In a
manner similar to that was described for the preparation of compound 20, a solution of
compound 64h (45 mg, 0.14 mmol) in DCM (1 mL) was treated with HCl in Et O (1 mL, 2M)
2
1
for 2 h to give hydroxamic acid 39 as white solid (8 mg, 22%). H NMR (DMSOꢀd , 600 MHz) δ
6
1
7
2
0.93 (s, 1H), 9.19 (brs, 1H), 8.96 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H),
ꢀ
ꢀ
.58 (d, J = 16.2 Hz, 1H), 6.71 (d, J = 15.6 Hz, 1H). HRMS (ESI ) calcd for C H F N O (MꢀH)
9
6
3
2
2
31.0381, found 231.0391.
N-Hydroxybenzofuran-2-carboxamide (40). In a manner similar to that was described for the
preparation of compound 13, carboxylic acid 65a (100 mg, 0.62 mmol) was treated with CDI
(150 mg, 0.93 mmol) and then NH OH·HCl (85 mg, 1.2 mmol) to give hydroxamic acid 40 as a
2
1
pale solid (71 mg, 65%). H NMR (CD OD, 600 MHz) δ 7.71 (d, J = 7.8 Hz, 1H), 7.58 (d, J =
3
ꢀ
7
.8 Hz, 1H), 7.44ꢀ7.47 (m, 2H), 7.32 (t, J = 7.2 Hz, 1H). HRMS (ESI ) calcd for C H NO
9
6
3
ꢀ
1
76.0348 (MꢀH) , found 176.0348.
ACS Paragon Plus Environment

Page 27 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
39
N-Hydroxybenzo[b]thiophene-2-carboxamide (41). In a manner similar to that was described
for the preparation of compound 13, carboxylic acid 65b (100 mg, 0.56 mmol) was treated with
CDI (137 mg, 0.84 mmol) and then NH OH·HCl (78 mg, 1.1 mmol) to give hydroxamic acid 41
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
as a pale solid (63 mg, 58%). H NMR (CD OD, 600 MHz) δ 7.93 (d, J = 8.4 Hz, 1H), 7.89 (d, J
3
ꢀ
= 7.8 Hz, 1H), 7.85 (s, 1H), 7.42ꢀ7.47 (m, 2H). HRMS (ESI ) calcd for C H NO S 192.0119 (Mꢀ
9
6
2
ꢀ
H) , found 192.0120.
4
0
N-Hydroxy-1H-indole-2-carboxamide (42). In a manner similar to that was described for the
preparation of compound 14, carboxylic acid 65c (322 mg, 2.0 mmol) was treated with oxalyl
chloride (279 mg, 2.2 mmol) followed by a solution of NH OH to give hydroxamic acid 42 as a
2
1
tan solid (184 mg, 52%). H NMR (CD OD, 600 MHz) δ 7.57ꢀ7.61 (m, 1H), 7.43ꢀ7.46 (m, 1H),
3
ꢀ
7
.19ꢀ7.23 (m, 1H), 7.04ꢀ7.08 (m, 1H), 6.97 (s, 1H). HRMS (ESI ) calcd for C H N O 175.0508
9
7
2
2
ꢀ
(
MꢀH) , found 175.0511.
41
N-Hydroxy-1H-indole-3-carboxamide (45). In a manner similar to that was described for the
preparation of compound 13, carboxylic acid 65d (161 mg, 1.0 mmol) was treated with CDI (243
mg, 1.5 mmol) and then NH OH·HCl (138 mg, 2.0 mmol) to give hydroxamic acid 45 as a tan
2
1
solid (154 mg, 88%). H NMR (CD OD, 600 MHz) δ 8.05 (d, J = 7.8 Hz, 1H), 7.81 (s, 1H), 7.43
3
ꢀ
(d, J = 8.4 Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H). HRMS (ESI ) calcd for
ꢀ
C H N O 175.0508 (MꢀH) , found 175.0505.
9
7
2
2
3
1a
1
-Benzyl-1H-indole-2-carboxylic acid (66c). A mixture of indoleꢀ2ꢀcarboxylic acid (65c, 805
mg, 5.0 mmol) and NaH (800 mg, 60% dispersion, 8.0 mmol) in DMF (24 mL) at was stirred at
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
ºC for 1h, and benzyl bromide (940 mg, 5.5 mmol) was added. The reaction mixture was
allowed to stir at rt overnight, and a solution of 10% NH Cl (5mL) and H O (10 mL) was
4
2
carefully added. The resulting mixture was extracted with EtOAc (15×3 mL) and the combined
organic layer was washed with brine and evaporated under vacuum. The residue was purified by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
flash column chromatography to give carboxylic acid 66c as a yellowish solid (176 mg, 17%).
1
H NMR (CD OD, 600 MHz) δ 7.70 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.39 (s, 1H),
3
7
.29 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 6.6 Hz, 2H), 7.19 (q, J = 7.2 Hz, 1H), 7.14 (t, J = 7.8 Hz,
ꢀ
ꢀ
1H), 7.04 (d, J = 7.8 Hz, 2H), 5.91 (s, 2H). HRMS (ESI ) calcd for C H NO 250.0868 (MꢀH) ,
1
6
12
2
found 250.0865.
3
1b
1-Benzyl-1H-indole-3-carboxylic acid (66d). In a manner similar to that was described for
the preparation of compound 66c, indoleꢀ3ꢀcarboxylic acid (65d, 895 mg, 5.56 mmol) was
1
alkylated to give carboxylic acid 66d as a pinkish solid (1.07 g, 76%). H NMR (CD OD, 600
3
MHz) δ 8.11ꢀ8.13 (m, 1H), 8.04 (s, 1H), 7.40ꢀ7.42 (m, 1H), 7.34 (t, J = 7.2 Hz, 2H), 7.29 (t, J
ꢀ
=
7.2 Hz, 1H), 7.21ꢀ7.23 (m, 4H), 5.46 (s, 2H). HRMS (ESI ) calcd for C H NO 250.0868 (Mꢀ
1
6
12
2
ꢀ
H) , found 250.0861.
1-Benzyl-N-hydroxy-1H-indole-2-carboxamide (44). In a manner similar to that was described
for the preparation of compound 14, carboxylic acid 66c (100 mg, 0.40 mmol) was treated with
oxalyl chloride (56 mg, 0.44 mmol) followed by a solution of NH OH to give hydroxamic acid
2
1
4
4 as a pale solid (36 mg, 34%). H NMR (CD OD, 600 MHz) δ 7.64 (d, J = 7.8 Hz, 1H), 7.42
3
(d, J = 8.4 Hz, 1H), 7.21ꢀ7.25 (m, 3H), 7.17ꢀ7.19 (t, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H),
ACS Paragon Plus Environment

Page 29 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ꢀ
7
.06 (d, J = 7.8 Hz, 2H), 6.99 (s,1H), 5.82 (s, 2H). HRMS (ESI ) calcd for C H N O 265.0977
16 13 2 2
ꢀ
(MꢀH) , found 265.0975.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
-Benzyl-N-hydroxy-1H-indole-3-carboxamide (47). In a manner similar to that was described
for the preparation of compound 14, carboxylic acid 66d (261 mg, 1.0 mmol) was treated with
oxalyl chloride (150 mg, 1.1 mmol) followed by a solution of NH OH to give hydroxamic acid
2
1
4
7 as a pale solid (112 mg, 40%). H NMR (CD OD, 600 MHz) δ 9.89 (s, 1H), 8.20ꢀ8.22 (m,
3
1H), 8.15 (s, 1H), 7.43ꢀ7.44 (m, 1H), 7.33 (t, J = 7.2 Hz, 2H), 7.26ꢀ7.30 (m, 3H), 7.24 (d, J = 7.2
+
+
Hz, 2H), 5.46 (s, 2H). HRMS (ESI ) calcd for C H N O 267.1134 (M+H) , found 267.1133.
1
6
15
2
2
3
3a
Methyl 1-phenyl-1H-indole-2-carboxylate (68a). A mixture of methyl ester 67a (70 mg,
.40 mmol), bromobenzene (75 mg, 0.48 mmol), CuI (3.8 mg, 0.020 mmol), transꢀN,N’ꢀ
dimethylcyclohexaneꢀ1,2ꢀdiamine (11.2 mg, 0.08 mmol), and K PO (178 mg, 0.84 mmol) in
0
3
4
toluene (2 mL) in a sealed tube was heated at 120 °C for 22 h. The solvent was evaporated under
vacuum and the residue was purified by flash column chromatography to give compound 68a as
1
a white solid (89 mg, 89%). H NMR (CDCl , 600 MHz) δ 7.73 (d, J = 7.8 Hz, 1H), 7.51ꢀ7.53
3
(m, 2H), 7.46ꢀ7.48 (m, 1H), 7.44 (s, 1H), 7.34 (d, J = 7.8 Hz, 2H), 7.25ꢀ7.28 (m, 1H), 7.19 (t, J =
+
6.6 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 3.78 (s, 3H). HRMS (ESI ) calcd for C H NO 252.1025
16
14
2
+
(M+H) , found 252.1022.
3
3b
Methyl 1-phenyl-1H-indole-3-carboxylate (68b). In a manner similar to that was described
for the preparation of compound 68a, methyl ester 67b (700 mg, 4.0 mmol) and bromobenzene
(753 mg, 4.8 mmol) underwent a CꢀN coupling to give compound 68b as a yellowish syrup (202
ACS Paragon Plus Environment