(N-Hydroxycarbonylbenylamino)quinolines as Selective Histone Deacetylase 6 Inhibitors Suppress Growth of Multiple Myeloma in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.7b01404

A series of bicyclic arylamino/heteroarylamino hydroxamic acids (7-31) have been examined as novel histone deacetylase 6 (HDAC6) inhibitors. One compound (13) exhibits remarkable inhibitory activity of HDAC6 with an IC₅₀ value of 0.29 nM, which is 4,000-43,000 times more selective over other HDAC isoforms. Compound 13 was shown to have antiproliferative activity against human multiple myeloma RPMI 8226, U266, and NCI-H929 cells with no effect on normal bone marrow cells. Compound 13, as a single drug, suppresses the growth of tumors by a %TGI factor of 60.4% in human multiple myeloma RPMI 8226 xenograft models and, in combination with bortezomib, shows significant in vivo antitumor activity (%TGI = 86.2%). Compound 13 also demonstrates good human hepatocytic stability and high permeability, without any effect on mutagenicity and cytotoxicity. Thus, compound 13 is a potent HDAC6 inhibitor that could be developed for the treatment of multiple myeloma in the future.

Full text of DOI:10.1021/acs.jmedchem.7b01404

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
(N-Hydroxycarbonylbenylamino)quinolines as Selective Histone Deacetylase
6 Inhibitors Suppress Growth of Multiple Myeloma in vitro and in Vivo
Hsueh-Yun Lee, Kunal Nepali, Fang-I Huang, CHIH-YI CHANG, Mei-Jung
Lai, Yu-Hsuan Li, Hsiang-Ling Huang, Chia-Ron Yang, and Jing-Ping Liou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01404 • Publication Date (Web): 05 Jan 2018
Downloaded from http://pubs.acs.org on January 5, 2018
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 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
(N-Hydroxycarbonylbenylamino)quinolines as Selective Histone Deacetylase 6
Inhibitors Suppress Growth of Multiple Myeloma in vitro and in Vivo
9
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
＆
＆
＆
Hsueh-Yun Lee,†, Kunal Nepali,†, Fang-I Huang,§, Chih-Yi Chang,† Mei-Jung Lai,‡ Yu-Hsuan Li,†
Hsiang-Ling Huang,† Chia-Ron Yang,§,* Jing-Ping Liou*,†
†School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei
11031, Taiwan. Center for Translational Medicine, Taipei Medical University. School of Pharmacy,
College of Medicine, §National Taiwan University, Taipei, Taiwan.
ABSTRACT. A series of bicyclic arylamino/heteroarylamino hydroxamic acids (7-31) have been
examined as novel histone deacetylase 6 (HDAC6) inhibitors. One compound (13) exhibits remarkable
inhibitory activity of HDAC6 with an IC50 value of 0.29 nM, which is 4000-43000 times more selective
over other HDAC isoforms. Compound 13 was shown to have antiproliferative activity against human
multiple myeloma RPMI 8226, U266, and NCI-H929 cells with no effect on normal bone marrow cells.
Compound 13, as a single drug, suppresses the growth of tumors by a %TGI factor of 60.4% in human
† School of Pharmacy, College of Pharmacy, Taipei Medical University.
§ School of Pharmacy, College of Medicine, National Taiwan University.
‡ Center for Translational Medicine, Taipei Medical University.
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 49
1
2
3
4
5
6
7
8
multiple myeloma RPMI 8226 xenograft models, and in combination with bortezomib, shows
significant in vivo antitumor activity (%TGI = 86.2%). Compound 13 also demonstrates good human
hepatocytic stability and high permeability, without any effect on mutagenicity and cytotoxicity. Thus
compound 13 is a potent HDAC6 inhibitor that could be developed for the treatment of multiple
myeloma in the future.
9
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
Introduction
Targeting proteins related to epigenetic regulation has emerged as an attractive therapeutic strategy
for various diseases. Among these epigenetic enzymes, histone deacetylase (HDAC) is the most studied
protein. HDAC is able to remove the acetyl groups from N-terminal lysines of histones, which frees the
genetic component and triggers the transcriptional process. In addition, it also deacetylates various non-
histone protein substrates such as tubulin1 and p532,3. These crucial roles of HDAC make it a promising
target for drugs to treat various diseases. The HDAC family has eighteen members which are grouped
into four classes (I, II, III, and IV) according to their homologies. The development of HDAC inhibitors
started with the discovery of pan-HDAC inhibitors and has brought various compounds into the clinic,
for instance, vorinostat, romidepsin, belinostat, and panobinostat.4-7 A survey of the results of clinical
trials indicates that the use of pan-HDAC inhibitors results in some side effects, such as fatigue, diarrhea,
nausea,8 QTc-interval prolongation9-11, and thrombocytopenia.12 Consequently, in the past decade
scientific attention was drawn to the discovery of selective inhibitors of HDAC isoforms, specifically
ACS Paragon Plus Environment
2

Page 3 of 49
1
Journal of Medicinal Chemistry
HDAC6 inhibitors. Compound 1 (tubacin) was discovered by high-throughput screening and was
recognized as the first selective HDAC6 inhibitor,13 which opened an avenue to the subsequent
development of other HDAC6 inhibitors. For instance, Compound 2 (tubastatin A) shows remarkably
selective HDAC6 inhibition and neuroprotective activity14 and compound 3 (ACY-1215) currently in
phase II clinical trials, is able to suppress the growth of multiple myeloma in combination therapy.15 The
correlation of HDAC6 with diverse diseases has encouraged us to develop potent selective HDAC6
inhibitors with effective biological activity.
2
3
4
5
6
7
8
9
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
Figure 1. Structures of HDAC6 inhibitors.
In addition to the HDAC6 inhibitors cited above, some additional examples of HDAC6 inhibitors (4-
6) are also shown in Figure 1.16-18 HDAC6 is a zinc-dependent enzyme; therefore, the structure of
HDAC6 inhibitors comprises three parts: zinc binding domain, linker, and surface recognition region.
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 49
1
2
3
4
5
6
7
8
Upon inspection of their structures, compounds 2, 4, 5, and 6 can be seen to share a common moiety, the
4-(N-hydroxyaminocarbonyl)benzylamino group (-NHCH2PhCONHOH, shown in blue in Figure 1).
This moiety is considered to be a combination of a zinc ion binding domain and a linker section, and it
is connected with various aromatic rings acting as a surface recognition region. This led to a series of
bicyclic arylamino/heteroarylamino hydroxamic acids (7-31, Figure 2). The current study is also aimed
at assays of their activity against both HDAC6 and the growth of multiple myeloma cells.
9
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
Figure 2. Synthetic bicyclic arylamino/heteroarylamino hydroxamic acids (7-31).
Results and Discussion
ACS Paragon Plus Environment
4

Page 5 of 49
1
Journal of Medicinal Chemistry
Chemistry
2
3
4
5
6
7
8
9
Scheme 1 shows the synthesis of 4-(N-hydroxyaminocarbonyl)benzylaminoquinolines (7-13 and 23).
Amino-substituted quinolines (32b, 32d, 32e, and 32g) reacted with methyl terephthalaldehydate in the
presence of acetic acid followed by reduction with NaBH(OAc)3 to generate the corresponding
secondary amines (33b, 33d, 33e, and 33g). To afford C2-, C4-, and C7-(4-(N-
hydroxyaminocarbonyl)benzylamino)quinolines (7, 9, and 12), the synthetic route started from
halogenated quinolines (32a, 32c, and 32f) via a Buchwald-Hartwig amination reaction with methyl 4-
(aminomethyl)benzoate with the assistance of Pd(OAc)2, which provided compounds 33a, 33c, and 33f.
Under basic conditions, the methyl ester groups of 33a-g were converted into the corresponding
hydroxamic acid by NH2OH, which generated the anticipated N-hydroxy-4-(aminomethyl)benzamide
(7-13). In addition, compound 23 was prepared from reaction of 32g with methyl 3-formylbenzoate
followed by conversion of ester (33h) into hydroxamic acid.
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
Scheme 1. Synthetic Approaches to Compounds 7-13 and 23a
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 49
1
2
3
4
5
6
7
8
9
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
aReagents and conditions: (a) for 33b, 33d, 33e, and 33g: methyl terephthalaldehydate, NaBH(OAc)3, AcOH, rt; for 33a:
methyl 4-(aminomethyl)benzoate, Pd(OAc)2, BINAP, K2CO3, toluene, 100 oC; for 33c and 33f: methyl 4-
o
(aminomethyl)benzoate, Pd(OAc)2, DPEphos, K3PO4, toluene, 100 C; (b) NH2OH, NaOH, MeOH, rt; (c) methyl 3-
formylbenzoate, NaBH(OAc)3, AcOH, rt.
To understand the effect on enzymatic activity of the linker between hydroxamic acid and the
quinoline, compounds 14-17 were synthesized as shown in Scheme 2. The synthesis of compounds 14
and 15 possessing a -C=C- linker started from the treatment of 5-aminoquinoline (32d) and 8-
aminoquinoline (32g) with methyl (E)-3-(4-formylphenyl)acrylate in the presence of NaBH(OAc)3 and
acetic acid. The preparation of 16 and 17 which have an amide linkage, was carried out by the reaction
of 32f and 32g with methyl 4-(chloroformyl)benzoate and monomethyl terephthalate, respectively. All
the resultant esters (34a-b and 35a-b) were converted into the corresponding hydroxamic acid by
treating NH2OH under basic conditions, which generates the designed compounds 14-17.
ACS Paragon Plus Environment
6

Page 7 of 49
Journal of Medicinal Chemistry
Scheme 2. Synthetic Approaches to Compounds 14-17a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
aReagents and conditions: (a) (E)-methyl 3-(4-formylphenyl)acrylate, NaBH(OAc)3, AcOH, rt; (b) NH2OH, NaOH, MeOH,
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
o
rt; (c) for 35a: methyl 4-(chloroformyl)benzoate, TEA, DCM, 0 C to rt; for 35b: monomethyl terephthalate, EDC-HCl,
DMAP, DCM, rt.
In addition to the secondary amine linker, several bridging groups such as ether, sulfide, and
ethylenyl, were inserted between the quinoline and 4-(N-hydroxyaminocarbonyl)phenyl moieties. Their
synthetic route was illustrated in Scheme 3. Access to compounds 18 and 19 possessing ether and
sulfide linkage respectively, started from the SN2 reaction of 36a and 36b with ethyl 4-
(bromomethyl)benzoate. The resulting benzoate (37a and 37b) was transformed to the hydroxamic acid
(18 and 19) under conditions similar to those shown in Scheme 1. The Wittig olefination of 36c
followed by reduction of the resulting C=C bond gave compound 37c, which possesses an ethylenyl
group, and the subsequent reaction with NH2OH under basic conditions yielded 20. To prepare
compound 21 with a reverse secondary amino group, compound 36c underwent reductive amination
with methyl 4-aminobenzoate in the presence of NaBH(OAc)3, followed by reaction with NH2OH,
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 49
1
2
3
4
5
6
7
8
which leads to the designed compound 21. Compound 22 with a shortened amino linkage was prepared
by the Buchwald-Hartwig amination reaction of 36d with methyl 4-aminobenzoate to afford the
corresponding carboxylate 37e which can be converted into compound 22.
9
Scheme 3. Synthetic Approaches to Compounds 18-22a
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
aReagents and conditions: (a) for 37a-b: methyl 4-(bromomethyl)benzoate, K2CO3, acetone, reflux; for 37c: i. triphenyl-(4-
o
methoxycarbonylbenzyl)phosphonium chloride, t-BuOK, DCM, 0 C to rt; ii. Pd/C, H2, MeOH, rt; for 37d: methyl 4-
aminobenzoate, NaBH(OAc)3, AcOH, rt; for 37e: methyl 4-aminobenzoate, Pd(OAc)2, DPEphos, K3PO4, toluene, 100 oC; (b)
NH2OH, NaOH, MeOH, rt.
The replacement of quinoline by various heterocycles or bicyclic rings is beneficial to an
understanding of the influence of quinoline on the biological activity. The synthesis of bicyclic ring-
containing molecules (24-31) is shown in Scheme 4. Halogen-containing bicyclic rings (38b-e)
underwent Buchwald-Hartwig amination with methyl 4-(aminomethyl)benzoate, and the resulting
benzoate was converted into the designed hydroxamic acid (25-28). Meanwhile, the reductive amination
of amino-containing rings (38a and 38f-h) with methyl terephthalaldehydate followed by reaction with
NH2OH gave the designed compounds 24 and 29-31.
ACS Paragon Plus Environment
8

Page 9 of 49
Journal of Medicinal Chemistry
Scheme 4. Synthetic Approaches to Compounds 24-31a
1
2
3
4
5
6
7
8
9
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
aReagents and conditions: (a) for 25-28: methyl terephthalaldehydate, NaBH(OAc)3, AcOH, rt; for 24 and 29-31: methyl 4-
(aminomethyl)benzoate, Pd(OAc)2, DPEphos, K3PO4, toluene, 100 oC; (b) NH2OH, NaOH, MeOH, rt.
Biological Evaluation.
A. HDAC6 Inhibitory Activity.
All synthesized adducts (7-31) and a reference compound, trichostatin A were examined for their
HDAC6 inhibitory activity, and the results are shown in Table 1. The activities of compounds 7-13
indicate the effective substitution position where the 4-(N-hydroxycarbonyl)benzylamino group binds,
on enzymatic activity. The result shows that the 4-(N-hydroxycarbonyl)benzylamino group favors the
C5 or C8 positions of quinoline, while 10 and 13 have better HDAC6 inhibitory activity. This result
reveals that quinoline-5-yl and quinoline-8-yl are suitable moieties as surface recognition area. The IC50
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 49
1
2
3
4
5
6
7
8
values of 10 and 13 against HDAC6 are 0.795 and 0.291 nM, respectively. The shift from C5 or C8
position to C3, C6, or C7 position led to a slight decrease of HDAC inhibitory activity, but their IC50
values remain in the single-digit nM range. The C2-N-hydroxyaminocarbonylbenzylamino group
resulted in a distinct loss of enzymatic potency. The insertion of a -C=C- linkage between N-
hydroxyaminocarbonyl and phenyl ring resulted in a decrease of activity if one compares 10 and 13 with
14 and 15, respectively. The influence of the bridging groups linking the quinoline to the hydroxamate
group and the position of the hydroxamate group on HDAC6 inhibition can be studied in compounds
18-22. The replacement of nitrogen atom with oxygen (18) or sulfur (19) as well as reversal of
alignment (21) slightly reduces activity, whereas replacement of the nitrogen with carbon (20) led to
marked loss of HDAC6 inhibitory activity. The removal of a carbon atom (22) and meta-hydroxamate
group (23) resulted in a dramatic decrease of enzymatic activity. The results from 24-31 show the effect
of replacement of quinoline with a variety of heterocycles and bicyclic rings on biological activity. It
reveals that most of nitrogen-containing [6,6]- or [6,5]-heterocycles (25-29) show HDAC6 inhibitory
ability with IC50 values in single-digit nM range, the single exception being the 2-methylquinoline (24).
The bicyclic rings which incorporate saturated rings like 1,2,3,4-tetrahydronaphthalene (30) and 2,3-
dihydro-1H-indene (31) decrease their ability to inhibit HDAC6. In summary, this study provides a
detailed study of structure-activity relationship that assists in the further development of selective
HDAC6 inhibitors.
9
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
ACS Paragon Plus Environment
10

Page 11 of 49
Journal of Medicinal Chemistry
1
2
Table 1. HDAC6 inhibitory activity (IC50) of tested compounds (7-31)
3
4
5
Cpd
HDAC6 IC50 (nM)a
6
7
8
9
4.24
403
7
8
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
21.5
0.795
4.78
3.25
0.291
25.3
41.7
525
9
10
11
12
13
14
15
16
265
17
2.83
9.48
41.7
7.40
129
18
19
20
21
22
1130
11.4
3.35
2.33
2.31
5.89
3.56
25.3
19.6
3.55
23
24
25
26
27
28
29
30
31
Trichostatin A
aThese assays were conducted by the Reaction Biology Corporation, Malvern,
PA. All compounds were dissolved in DMSO and tested in at least 10-dose IC50
mode with 3-fold serial dilution starting at 10 μM. IC50 values displayed the
result of a single experiment.
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 49
1
2
3
4
5
6
7
8
Compounds 10 and 13, which showed the most potent HDAC6 inhibition were further examined for
their inhibitory ability to other HDAC isotypes (Table 2). Compounds 10 and 13 inhibit HDAC6 with
IC50 values of 0.795 and 0.291 nM, respectively. This result shows that 10 and 13 exhibit distinct
selective HDAC6 inhibition over other isoforms. The values inside the bracket of Table 2 are selectivity
ratio of tested compounds over HDAC6. The result indicates that the selectivity potency of 10 and 13
toward HDAC6 is dramatically better than that of compound 3. Importantly, compound 13 is highly
selective for HDAC6 and more active than with HDAC1, 2, 3, 4, 5, 7, 8, 9, 10, and 11 by factors of
32817, 42955, 26632, 15250, 10694, 2436, 4089, 5258, 33646 and 1292, respectively.
9
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
Table 2. Inhibitory activity (IC50, nM) of test compounds 10, 13, and reference 3.
IC50 (nM)a
3b
10
13
HDAC subtype
HDAC1
(selectivity ratio)c
(selectivity ratio)
(selectivity ratio))
7050
(8868)
9550
(32817)
58
(12)
8610
(10830)
12500
(42955)
48
(10)
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
5480
(6893)
7750
(26632)
51
(11)
4817
(6059)
4438
(15250)
7000
(1500)
2251
(2831)
3112
(10694)
5000
(1100)
0.795
(-)
0.291
(-)
4.7
(-)
766
(964)
709
(2436)
1400
(300)
636
(800)
1190
(4089)
100
(21)
ACS Paragon Plus Environment
12

Page 13 of 49
Journal of Medicinal Chemistry
1915
(2409)
1530
(5258)
>10000
(> 2100)
1
2
3
4
5
6
7
8
HDAC9
HDAC10
HDAC11
SIRT1
1256
(1580)
9791
(33646)
NA
(-)
1004
(1263)
376
(1292)
>10000
(> 2100)
9
>20000
(> 25157)
>20000
(> 68729)
>10000
(> 2100)
10
11
12
13
14
15
16
17
18
19
20
21
22
aThese assays were conducted by the Reaction Biology Corporation, Malvern, PA. All compounds were
dissolved in DMSO and tested in at least 10-dose IC50 mode with 3-fold serial dilution starting at 10 μM. IC50
values displayed the result of a single experiment. Data obtained from reference 15. cSelectivity ratio:
b
selectivity ratio of HDAC subtypes over HDAC6
The comparison of the influence of compounds 10 and 13 on the acetylation of α-tubulin in RPMI
8226, U266, and NCI-H929 cell lines is shown in Figure 3. Treatment with 10 or 13 led to upregulation
of acetylated α-tubulin in a dose-dependent manner, which corresponds to the functionality of HDAC6
inhibitors.
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
Figure 3. Compounds 10 and 13 increased acetyl-α-tubulin expression in human multiple myeloma cell lines.
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 49
1
2
3
4
5
B. In Vitro Cell Growth Inhibition.
6
7
8
9
A survey of the literature indicates that HDAC6 inhibitors are specifically able to inhibit the growth
of multiple myeloma cells, and their combination with proteasome inhibitors trigger marked apoptosis
of multiple myeloma cells.19 Consequently, compounds 10 and 13 were selected to study their influence
on the growth of three multiple myeloma cells, RPMI 8226, U266, and NCI-H929, using compound 3 as
a reference compound (Table 3). It was found that U266 and NCI-H929 cells are more sensitive to
compounds 10 and 13, as compared to the reference compound 3, and both 10 and 13 have
antiproliferative activity for RPMI 8226 cells comparable to that of 3. In addition, 10, 13, and 3 have no
influence on normal bone marrow HS-5 cells. Further studies of the apoptotic effect of compound 13
and bortezomib, a proteasome inhibitor, were evaluated by flow cytometry. As shown in Figure 4,
compound 13 (5 M) alone or bortezomib (2.5 nM) alone caused mild apoptosis in U266 and NCI-
H929 cells, but not in RPMI 8226 cells. However, the combination of the drugs exhibited significant
apoptotic effect in all three multiple myelomas. We determine the apoptotic effect by Annexin V/PI
stain in response to compound 13 and bortezomib treatment in human multiple myeloma cells. As
shown as supporting information Figure S1, compound 13 (5 M) or bortezomib (2.5 nM) alone for 24
h only caused mild apoptosis (upper right quadrant) in three multiple myeloma cells. However, the
combination of the drugs exhibited significant apoptotic effect in all three multiple myelomas.
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
ACS Paragon Plus Environment
14

Page 15 of 49
Journal of Medicinal Chemistry
Table 3. Growth inhibition activity (GI50, μM) of 10, 13, and reference 3 against multiple myeloma cell
1
2
3
lines.
4
5
Multiple myeloma cells
Normal bone marrow cells
6
7
8
9
Cmpd
10
RPMI 8226
U266
28.40±1.07
40.61±9.84
>100
NCI-H929
6.20±1.41
9.14±2.33
>100
HS-5
>100
>100
>100
10.96±2.52
7.49±3.63
6.40±2.68
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
13
3
Figure 4. A combination of compound 13 with bortezomib induces significant apoptosis in three multiple myeloma cells
(U266, NCI-H929 cells, and RPMI 8226 cells). Upper panel shows the result of flow cytometry and lower panel presents the
distribution of cell cycle phases.
C. Growth Inhibition of Human Multiple Myeloma Xenografts in vivo.
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 49
1
2
3
4
5
6
7
8
Figure 5 shows the in vivo activity of the mesylate of compound 13 in human multiple myeloma
RPMI 8226 xenograft models, using bortezomib as a reference compound. Bortezomib and 13·mesylate
were used intravenously or intraperitoneally to treat human multiple myeloma RPMI 8226 xenograft
mice, in the form either of a single-drug or a combination therapy (Figure 5). The use of 13·mesylate
alone at the concentration of 30 mg/kg led to the suppression of tumor growth by a tumor growth
inhibition factor (%TGI) of 60.4% (Figure 5A), which is comparable to the response to 1 mg/kg of
bortezomib (%TGI = 70.1%). Notably, treatment with bortezomib led to the death of a test animal. The
combination treatment of bortezomib and 13·mesylate in an in vivo study showed enhancement of tumor
suppression by a %TGI of 86.2%. Unlike use of bortezomib alone, treatment with 13·mesylate alone or
combined with bortezomib has no influence on body weight of testing animals (Figure 5B).
Investigation of the maximal tolerated dose of compound 13 is described in Figure 6. Compound 13 was
administered intraperitoneally to healthy mice at doses of 50, 100, and 200 mg/kg, and body weights
were recorded over 14 days. No significant adverse reaction with 13 was observed, suggesting that it
was well tolerated and safe within the dosage range of up to 200 mg/kg.
9
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
ACS Paragon Plus Environment
16

Page 17 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
Figure 5. Anticancer activities of 13·mesylate alone and combined with bortezomib in human multiple myeloma RPMI 8226
xenograft models. Tumor growth is tracked by the mean tumor volume (mm3) ± S.E and calculated as % tumor growth
inhibition (%TGI). Tumor volume was determined using caliper measurements and was calculated as the product of 1/2 x
length x width2. *, p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the control group. TR, treatment related death.
Figure 6. Maximum tolerance dose (MTD) evaluation of compound 13. Thirty animals were randomized into three groups.
The treated animals were administered compound 13 at doses of 50, 100, and 200 mg/kg. The animals were dosed
intraperitoneally daily for 7 days and then monitored for one week. The animals were weighed daily.
D. Kinase selectivity analysis
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 49
1
2
3
4
5
6
7
8
To know if compound 13 has off target effects, the kinase selectivity profiling against a
comprehensive panel of 97 kinases was assessed using DiscoveRx’s KinomeScan technology.
Compound 13 was screened at a concentration of 1 µM. In this study, the potential of the test compound
to disrupt the complex between a high affinity ATP-mimic probe immobilized on a solid support and the
selected kinase was measured. The screening platform provides “Percent of Control” (POC) values
which are indicative of a compound’s affinity for kinases. Low POC values indicate stronger binding of
the test compound with the test kinase. The results of the preliminary screening clearly indicates that
there is no high affinity binding of compound 13 against the kinases tested (Supporting Information
Table S1).
9
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
E. In vitro hepatocyte stability
A preliminary investigation of the ADME properties of compound 13 to ascertain its potential for
further development was conducted. In vitro hepatocyte stability of compound 13 was determined using
substrate depletion methodology for the determination of half-life (t1/2)
CLint in cryopreserved
and
hepatocytes from male SD rats, male beagle dogs and human hepatocytes. As shown in Table 4,
compound 13 demonstrated good human hepatocytic stability. The stability order is human > dog > rat.
Table 4. Compound 13 stability, apparent half-life and in vitro clearance in rat, dog and human
hepatocytes spiking concentration of 10 µM.
Cryopreseved
Hepatocytes
60 min
remaining (%)
Half life
(min)
CLint
(mL/min/kg)
ACS Paragon Plus Environment
18

Page 19 of 49
Journal of Medicinal Chemistry
Rat
Dog
28.4 ± 1.5
70.6 ± 1.4
86.4 ± 5.8
55.83
118.17
239.82
150.85
39.59
32.52
1
2
3
4
5
6
7
8
9
10
Human
F. Caco-2 cell permeability study
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
An in vitro Caco-2 bi-directional permeability study was conducted to gain insights into membrane
penetration, efflux from cells and possible interaction with the P-glycoprotein (P-gp) of compound 13.
Compound 13 (10 µM) and rhodamine 123 (10 µM) were added to either the apical or basolateral
chambers of the trans well plate assembly and were incubated at 37 °C for a 2-hour period. Compounds
with an efflux ratio greater than 2 are typically considered to be a substrate of the efflux transporter,
especially the P-glycoprotein. Table 5 presents the data as apparent permeability (Papp) and efflux ratio.
The Caco-2 cell permeability coefficients for apical to basolateral (AP → BL) movement and basolateral
to apical (BL → AP) movement of compound 13 are 24.03 ± 2.87 x 10-6 cm/sec and 20.13 ± 3.13 x 10-6
cm/sec. This result shows that the (Papp,A→B) of compound 13 (24.03 ± 2.87 x 10-6 cm/sec) was higher
than the high (Papp,A→B)cutoff (10 x 10-6 cm/sec). On the basis of permeability coefficient (Papp,A→B),
compound 13 could be considered to be highly permeable. The efflux ratio ((Papp,B→A)/(Papp,A→B)) of
compound 13 is 0.84 indicating that it should not serve as a substrate of apical efflux transporters.
Table 5. Permeability, recovery and efflux ratio of compound 13 and P-gp control in both directions
across Caco-2 cell Monolayers
Papp
Recovery
(%)
Compound
Direction
Efflux ratio
(x 10-6 cm/sec)
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 49
A → B
B → A
A → B
B → A
24.03 ± 2.87
20.13 ± 3.13
1.16 ± 0.33
7.52 ± 0.48
72.21 ± 2.93
69.05 ± 2.38
89.75 ± 3.90
93.86 ± 2.93
1
2
3
4
5
6
7
0.84
6.50
13
Rhodamine 123
(P-gp control)
8
9
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
G. Antimutagenicity test
An Ames test was performed to evaluate the mutagenicity of compound 13 against S. typhimurium
TA 98 and TA 100 both with and without metabolic activation. Compound 13, at concentrations of 3000,
300, 30 or 3 μg/plate was evaluated for possible activity to induce reversion of mutations at the histidine
loci of two His-auxotrophic strains of Salmonella typhimurium (TA98 and TA100). In this assay,
positive mutagenicity (induction of reversion) was evidenced by 3-fold increase in the reversion
frequency of the treated group compared to the spontaneous reversion of the vehicle control group.
Colonies numbering ≤50% of the vehicle control indicated cytotoxicity. Results clearly indicate that
TA98 and TA100 strains failed to increase the number of revertant colonies when the bacteria was
treated with compound 13 at various concentrations either in the presence or absence of metabolic
activation enzymes (S9) (Supporting Information Table S2 and S3). The results of the antimutagenicity
test revealed the absence of any significant mutagenicity and cytotoxicity.
Conclusion
This study resulted in the synthesis of a series of bicyclic arylamino/heteroarylamino hydroxamic
acids (7-31) and testing of their HDAC6 inhibitory activity. Among them, compounds 10 and 13, with a
ACS Paragon Plus Environment
20

Page 21 of 49
1
Journal of Medicinal Chemistry
quinoline moiety showed the most potent HDAC6 inhibition with IC50 values in sub-nM range. The
screening of 10 and 13 for HDAC isoforms inhibition revealed that 10 and 13 possess remarkable
HDAC6 selectivity over other isoforms, and are between ten and several thousand times more potent
than compound 3, an HDAC6 inhibitor currently in clinical trials. The result of cellular assays
demonstrated that 10 and 13 exhibit potent antiproliferative activity against multiple myeloma cells such
as RPMI 8226, U266, and NCI-H929 cells. The mesylate of 13, as a single drug or in combination with
bortezomib is able to suppress the growth of tumors in human multiple myeloma xenograft models.
Notably, combinatorial use of 13·mesylate with bortezomib avoided the death of test animals. This
study provides a series of (N-hydroxycarbonylbenzylamino)quinolines that show marked HDAC6
selectivity and could be further developed as efficient and safer therapeutic agents for the treatment of
multiple myeloma.
2
3
4
5
6
7
8
9
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
Experimental Section
(A) Chemistry. Nuclear magnetic resonance (1HNMR) spectra were obtained with a Bruker DRX-500
spectrometer operating at 300 MHz, with chemical shift reported in parts per million (ppm, δ) downfield
from TMS, an internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL
(JMS-700) electron impact (EI) mass spectrometer. Elemental analyses were performed on a Heraeus
varioIII-NCH. The purity of the final compounds was determined using an Agilent 1100 series HPLC
system using C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm × 150 mm) and was
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 49
1
2
3
4
5
6
7
8
found in all cases to be ≥ 95%. Flash column chromatography was carried out using silica gel (Merck
Kieselgel 60, no. 9385, 230-400 mesh ASTM). All reactions conducted under an atmosphere of dry N2.
N-Hydroxy-4-((quinolin-2-ylamino)methyl)benzamide (7)
9
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
NaOH powder (10 eq) was added to a mixture of hydroxylamine hydrochloride (10 eq) in MeOH at 0
oC and stirred for 30 min. The resulting mixture was filtered and the filtrate was added to a solution of
o
compound 33a (1 eq) in MeOH (0.1 M). Additional NaOH powder (1-4 eq) was added at 0 C and the
mixture was stirred at room temperature until the reaction was complete. The reaction mixture was then
quenched with H2O and the pH value was adjusted to pH 7. The precipitate was filtered and washed
with boiling MeOH to give compound 7 (yield 85%). 1HNMR (300 MHz, DMSO-d6) δ 4.67 (d, J = 5.7
Hz, 2H), 6.83 (d, J = 9.0 Hz, 1H), 7.10-7.17 (m, 1H), 7.42-7.46 (m, 4H), 7.54-7.62 (m, 2H), 7.69 (d, J =
8.1 Hz, 2H), 7.87 (d, J = 9.0 Hz, 1H), 8.98 (s, 1H), 11.14 (s, 1H). HRMS (ESI) for C17H18N3O2 [M +
H]+ calcd 294.1243, found 294.1208.
N-Hydroxy-4-((quinolin-3-ylamino)methyl)benzamide (8)
The title compound was obtained in 93% overall yield from compound 33b in a manner similar to
that described for the preparation of 7: 1HNMR (300 MHz, DMSO-d6) δ 4.43 (d, J = 5.7 Hz, 2H), 6.93-
6.98 (m, 2H), 7.27-7.38 (m, 2H), 7.67 (d, J = 8.1 Hz, 2H), 7.54-7.58 (m, 1H), 7.69-7.78 (m, 3H), 8.54 (d,
J = 2.7 Hz, 1H). HRMS (ESI) for C17H18N3O2 [M + H]+ calcd 294.1243, found 294.1209.
N-Hydroxy-4-((quinolin-4-ylamino)methyl)benzamide (9)
ACS Paragon Plus Environment
22

Page 23 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The title compound was obtained in 83% overall yield from compound 33c in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.59 (d, J = 5.1 Hz, 2H), 6.29
(d, J = 5.4 Hz, 1H), 7.42-7.48 (m, 3H), 7.59-7.65 (m, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 8.4 Hz,
1H), 7.95 (s, 1H), 8.29 (d, J = 5.4 Hz, 1H). HRMS (ESI) for C17H18N3O2 [M + H]+ calcd 294.1243,
found 294.1209.
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
N-Hydroxy-4-((quinolin-5-ylamino)methyl)benzamide (10)
The title compound was obtained in 91% overall yield from compound 33d in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.54 (d, J = 5.7 Hz, 2H), 6.37
(d, J = 7.5 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.24 (t, J = 6.0 Hz, 1H), 7.34-7.48 (m, 4H), 7.69 (d, J = 8.4
Hz, 2H), 8.69 (d, J = 8.4 Hz, 1H), 8.80 (dd, J = 1.5, 4.2 Hz, 1H), 9.00 (s, 1H), 11.14 (s, 1H). HRMS
(ESI) for C17H18N3O2 [M + H]+ calcd 294.1243, found 294.1236.
N-Hydroxy-4-((quinolin-6-ylamino)methyl)benzamide (11)
The title compound was obtained in 93% overall yield from compound 33e in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.56 (d, J = 5.7 Hz, 2H), 6.62
(d, J = 2.4 Hz, 1H), 6.88 (t, J = 5.7 Hz, 1H), 7.22-7.30 (m, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 9.0
Hz, 1H), 7.88-7.92 (m, 3H), 8.46 (dd, J = 1.5, 4.2 Hz, 1H). HRMS (ESI) for C17H18N3O2 [M + H]+ calcd
294.1243, found 294.1242.
N-Hydroxy-4-((quinolin-7-ylamino)methyl)benzamide (12)
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 49
1
2
The title compound was obtained in 90% overall yield from compound 33f in a manner similar to
3
4
5
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.45 (d, J = 5.7 Hz, 2H), 6.70
6
7
8
9
(d, J = 2.1 Hz, 1H), 6.99-7.14 (m, 3H), 7.47 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.71 (d, J =
8.4 Hz, 2H), 8.00 (dd, J = 1.2, 8.0 Hz, 1H), 8.54-8.57 (m, 1H), 9.02 (s, 1H), 11.15 (s, 1H). HRMS (ESI)
for C17H18N3O2 [M + H]+ calcd 293.1243, found 294.1222.
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
N-Hydroxy-4-((quinolin-8-ylamino)methyl)benzamide (13) and 13·mesylate
The title compound was obtained in 92% overall yield from compound 33g in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.57 (d, J = 6.3 Hz, 2H), 6.51
(dd, J = 1.2, 7.8 Hz, 1H), 7.04 (dd, J = 1.2, 8.4 Hz, 1H), 7.21-7.28 (m, 2H), 7.45 (d, J = 8.1 Hz, 2H),
7.50 (dd, J = 4.2, 8.4 Hz, 1H), 7.68 (d, J = 8.1 Hz, 2H), 8.20 (dd, J = 1.8, 8.4 Hz, 1H), 8.76 (dd, J = 1.8,
4.2 Hz, 1H), 8.96 (brs, 1H), 11.11 (brs, 1H). HRMS (ESI) for C17H18N3O2 [M + H]+ calcd 294.1243,
found 294.1217. For 13·mesylate: a solution of MsOH in dioxane (0.2M) was added to a solution of
compound 13 in dioxane (0.2M), and the resulting mixture was stirred at room temperature. The
1
reaction mixture was filtered and washed with dioxane to afford the 13·mesylate. HNMR (300 MHz,
DMSO-d6) δ 2.34 (s, 3H), 4.59 (s, 2H), 6.56 (dd, J = 0.9, 7.8 Hz, 1H), 7.08 (dd, J = 1.2, 8.1 Hz, 1H),
7.26-7.32 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.55 (dd, J = 8.4, 8.4 Hz, 1H), 7.68 (d, J = 8.1 Hz, 2H),
8.26 (dd, J = 1.8, 8.4 Hz, 1H); Anal. Calcd for C18H19N3O5S: C, 55.52; H, 4.92; N, 10.79; S, 8.23.
Found: C, 55.65; H, 4.81; N, 10.72; S, 7.91.
(E)-N-Hydroxy-3-(4-((quinolin-5-ylamino)methyl)phenyl)acrylamide (14)
ACS Paragon Plus Environment
24

Page 25 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The title compound was obtained in 96% overall yield from compound 34d in a manner similar to
that described for the preparation of 7: 1HNMR (300 MHz, DMSO-d6) δ 4.51 (d, J = 6.0 Hz, 2H), 6.36-
6.43 (m, 2H), 7.15-7.24 (m, 2H), 7.37-7.45 (m, 5H), 7.50 (d, J = 8.1 Hz, 2H), 8.69 (d, J = 8.4 Hz, 1H),
8.78-8.81 (m, 1H), 9.01 (s, 1H), 10.72 (s, 1H). HRMS (ESI) for C19H18N3O2 [M + H]+ calcd 320.1399,
found 320.1375.
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
(E)-N-Hydroxy-3-(4-((quinolin-8-ylamino)methyl)phenyl)acrylamide (15)
The title compound was obtained in 88% overall yield from compound 34g in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.55 (d, J = 6.6 Hz, 2H), 6.40
(d, J = 15.6 Hz, 1H), 6.53 (dd, J = 0.9, 7.5 Hz, 1H), 7.02-7.06 (m, 1H), 7.19-7.29 (m, 2H), 7.38-7.53 (m,
6H), 8.20 (dd, J = 1.8, 8.4 Hz, 1H), 8.76 (dd, J = 1.5, 4.2 Hz, 1H), 8.99 (brs, 1H), 10.72 (brs, 1H).
HRMS (ESI) for C19H18N3O2 [M + H]+ calcd 320.1399, found 320.1382.
N1-Hydroxy-N4-(quinolin-5-yl)terephthalamide (16)
The title compound was obtained in 93% overall yield from compound 35a in a manner similar to
that described for the preparation of 7: 1HNMR (300 MHz, DMSO-d6) δ 7.55 (dd, J = 4.2 Hz, 1H),7.70-
7.73 (m, 1H), 7.77-7.83 (m, 1H), 7.90-7.98 (m, 3H), 8.14 (d, J = 8.4 Hz, 2H), 8.41 (d, J = 8.1 Hz, 1H),
8.93 (dd, J = 1.5, 4.2 Hz, 1H), 9.17 (s, 1H), 10.64 (s, 1H), 11.41 (s, 1H). HRMS (ESI) for C17H14N3O3
[M + H]+ calcd 308.1035, found 308.1035.
N1-Hydroxy-N4-(quinolin-8-yl)terephthalamide (17)
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 49
1
2
The title compound was obtained in 88% overall yield from compound 35b in a manner similar to
3
4
5
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6): δ 7.61-7.77 (m, 3H), 7.95-8.16
6
7
8
(m, 4H), 8.45 (d, J = 8.4 Hz, 1H), 8.69-8.75 (m, 1H), 8.97 (d, J = 3.6 Hz, 1H), 10.68 (s, 1H). HRMS
9
(ESI) for C17H14N3O3 [M + H]+ calcd 308.1035, found 308.1035.
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
N-Hydroxy-4-((quinolin-8-yloxy)methyl)benzamide (18)
The title compound was obtained in 90% overall yield from compound 37a in a manner similar to
that described for the preparation of 7: 1HNMR (300 MHz, DMSO-d6) δ 5.37 (s, 2H), 7.25-7.29 (m, 1H),
7.46-7.57 (m, 3H), 7.61 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H), 8.30-8.34 (m, 1H), 8.85-8.88 (m,
1H), 9.03 (brs, 1H), 11.22 (brs, 1H). HRMS (ESI) for C17H15N2O3 [M + H]+ calcd 295.1083, found
295.1057.
N-Hydroxy-4-((quinolin-8-ylthio)methyl)benzamide (19)
The title compound was obtained in 92% overall yield from compound 37b in a manner similar to
that described for the preparation of 7: 1HNMR (300 MHz, DMSO-d6) δ 4.38 (s, 2H), 7.47-7.61 (m, 5H),
7.68-7.73 (m, 3H), 8.35 (dd, J = 1.2, 8.4 Hz, 1H), 8.88 (dd, J = 1.8, 4.2 Hz, 1H), 9.00 (brs, 1H), 11.17
(brs, 1H). HRMS (ESI) for C17H15N2O2S [M + H]+ calcd 311.0854, found 311.0848.
N-Hydroxy-4-(2-(quinolin-8-yl)ethyl)benzamide (20)
The title compound was obtained in 92% overall yield from compound 37c in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 3.03-3.09 (m, 2H), 3.47-3.53
ACS Paragon Plus Environment
26

Page 27 of 49
1
2
3
4
5
6
7
Journal of Medicinal Chemistry
(m, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.45-7.58 (m, 3H), 7.68 (d, J = 8.1 Hz, 2H), 7.81(dd, J = 1.5, 8.1 Hz,
1H), 8.31-8.35 (m, 1H), 8.95 (dd, J = 1.8, 4.2 Hz, 1H), 8.98 (s, 1H), 11.15 (s, 1H). HRMS (ESI) for
C18H17N2O2 [M + H]+ calcd 293.1290, found 293.1262.
8
9
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
N-Hydroxy-4-(quinolin-8-ylmethylamino)benzamide (21)
The title compound was obtained in 87% overall yield from compound 37d in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.95 (d, J = 6.0 Hz, 2H), 6.57
(d, J = 8.7 Hz, 2H), 6.79-6.84 (m, 1H), 7.45-7.68 (m, 5H), 7.87 (d, J = 8.1 Hz, 1H), 8.37-8.41 (m, 1H),
8.64 (s, 1H), 8.96-8.99 (m, 1H), 10.71 (brs, 1H). HRMS (ESI) for C17H16N3O2 [M + H]+ calcd 294.1243,
found 294.1238.
N-Hydroxy-4-(quinolin-8-ylamino)benzamide (22)
The title compound was obtained in 75% overall yield from compound 37e in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 7.33-7.38 (m, 2H), 7.57-7.63
(m, 2H), 7.70-7.78 (m, 4H), 8.62 (dd, J = 1.5, 8.4 Hz, 1H), 8.97 (dd, J = 1.5, 4.5 Hz, 1H). HRMS (ESI)
for C16H14N3O2 [M + H]+ calcd 280.1086, found 280.1081.
N-Hydroxy-3-((quinolin-8-ylamino)methyl)benzamide (23)
The title compound was obtained in 92% overall yield from compound 33h in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.57 (d, J = 6.3 Hz, 2H), 6.54
(d, J = 7.2, 8.1 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 7.19-7.29 (m, 2H), 7.34-7.40 (m, 1H), 7.47-7.60 (m,
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 49
1
2
3
4
3H), 7.82 (s, 1H), 8.17-8.22 (m, 1H), 8.75-8.77 (m, 1H), 8.99 (s, 1H), 11.19 (s, 1H). HRMS (ESI) for
C17H16N3O2 [M + H]+ calcd 294.1243, found 294.1209.
5
6
7
8
9
N-Hydroxy-4-(((2-methylquinolin-8-yl)amino)methyl)benzamide (24)
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
The title compound was obtained in 91% overall yield from compound 39a in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 2.67 (s, 3H), 4.57 (d, J = 6.6
Hz, 2H), 6.47 (d, J = 7.5 Hz, 1H), 6.97-7.05 (m, 2H), 7.17 (t, J = 8.1 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H),
7.44 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 8.4 Hz, 1H), 8.96 (brs, 1H), 11.11 (brs,
1H). HRMS (ESI) for C18H18N3O2 [M + H]+ calcd 308.1399, found 308.1399.
N-Hydroxy-4-((isoquinolin-8-ylamino)methyl)benzamide (25)
The title compound was obtained in 90% overall yield from compound 39b in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.56 (d, J = 5.7 Hz, 2H), 6.42
(d, J = 7.8 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 7.34-7.40 (m, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.58-7.63 (m,
2H), 7.70 (d, J = 8.1 Hz, 2H), 8.40 (d, J = 5.7 Hz, 1H), 9.03 (brs, 1H), 9.64 (s, 1H), 11.14 (brs, 1H).
HRMS (ESI) for C17H16N3O2 [M + H]+ calcd 294.1243, found 294.1221.
N-Hydroxy-4-((isoquinolin-5-ylamino)methyl)benzamide (26)
The title compound was obtained in 90% overall yield from compound 39c in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.55 (d, J = 6.0 Hz, 2H), 6.51
(d, J = 7.5 Hz, 1H), 7.20-7.24 (m, 2H), 7.31 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.1
ACS Paragon Plus Environment
28

Page 29 of 49
1
Journal of Medicinal Chemistry
Hz, 1H), 8.11 (d, J = 6.0 Hz, 1H), 8.43 (d, J = 5.7 Hz, 1H), 8.97 (s, 1H), 9.11 (s, 1H), 11.11 (s, 1H).
2
3
4
5
HRMS (ESI) for C17H16N3O2 [M + H]+ calcd 294.1243, found 294.1210.
6
7
8
9
N-Hydroxy-4-((isoquinolin-4-ylamino)methyl)benzamide (27)
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
The title compound was obtained in 88% overall yield from compound 39d in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.58 (d, J = 6.0 Hz, 2H), 7.13
(d, J = 6.0 Hz, 1H), 7.46-7.54 (m, 3H), 7.59-7.65 (m, 1H), 7.67-7.74 (m, 3H), 7.88-7.96 (m, 1H), 8.29
(d, J = 8.4 Hz, 1H), 8.98 (s, 1H), 11.13 (s, 1H). HRMS (ESI) for C17H16N3O2 [M + H]+ calcd 294.1243,
found 294.1212.
N-Hydroxy-4-((quinoxalin-5-ylamino)methyl)benzamide (28)
The title compound was obtained in 78% overall yield from compound 39e in a manner similar to
1
that described for the preparation of 7: HNMR (300 MHz, DMSO-d6) δ 4.59 (d, J = 6.6 Hz, 2H), 6.56
(dd, J = 0.6, 7.8 Hz, 1H), 7.16 (dd, J = 0.9, 8.4 Hz, 1H), 7.43-7.52 (m, 4H), 7.68 (d, J = 8.4 Hz, 2H),
8.77 (d, J = 1.8 Hz, 1H), 8.89 (d, J = 1.8 Hz, 1H), 8.97 (brs, 1H), 11.12 (brs, 1H). HRMS (ESI) for
C16H15N4O2 [M + H]+ calcd 295.1195, found 295.1190.
4-((1H-Indazol-7-ylamino)methyl)-N-hydroxybenzamide (29)
The title compound was obtained in 88% overall yield from compound 39f in a manner similar to
that described for the preparation of 7: 1HNMR (300 MHz, DMSO-d6) δ 4.50 (d, J = 5.7 Hz, 2H), 6.14-
6.18 (m, 1H), 6.27 (d, J = 7.2 Hz, 1H), 6.79-6.86 (m, 1H), 6.95 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 8.4 Hz,
ACS Paragon Plus Environment
29