Exploring Derivatives of Quinazoline Alkaloid l-Vasicine as Cap Groups in the Design and Biological Mechanistic Evaluation of Novel Antitumor Histone Deacetylase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00322

l-Vasicine is a quinazoline alkaloid with an electron dense ring and additional functionalities in its structure. Employing target oriented synthesis (TOS) based on in silico studies, molecules with significant docking scores containing different derivatives of l-vasicine as caps were synthesized. Interestingly, one molecule, i.e., 4a, which contained 3-hyroxypyrrolidine as a cap group and a six carbon long aliphatic chain as a linker was found to inhibit HDACs. 4a showed more specificity toward class I HDAC isoforms. Also 4a was found to be less cytotoxic toward normal cell lines as compared to cancer cell lines. 4a inhibited cancer cell growth and induced cell death by various mechanisms. However, 4a was found to induce cell death independent of ROS generation, and unlike many natural product based HDAC inhibitors, 4a was found to be nontoxic under in vivo conditions. Importantly, we for the first time report the possibility of using a 3-hydroxypyrrolidine cap for the synthesis of HDAC inhibitors with good potency.

Full text of DOI:10.1021/acs.jmedchem.7b00322

Subscriber access provided by University of Newcastle, Australia
Article
Exploring derivatives of quinazoline alkaloid l-vasicine
as cap groups in the design and biological mechanistic
evaluation of novel antitumor histone deacetylase inhibitors
Mudassier Ahmad, Mushtaq A. Aga, Javeed Ahmad Bhat, Brijesh Kumar, Abdul
Rouf, Neena Capalash, Mubashir Javeed Mintoo, Ashok Kumar, Priya Mahajan, Dilip
Manikrao Mondhe, Amit Nargotra, Parduman Raj Sharma, Mohmmad Afzal Zargar,
Ram A. Vishwakarma, Bhahwal Ali Shah, Subhash Chandra Taneja, and Abid Hamid
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00322 • Publication Date (Web): 03 Apr 2017
Downloaded from http://pubs.acs.org on April 4, 2017
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 47
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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
5
6
7
8
9
Exploring derivatives of quinazoline alkaloid lꢀ
vasicine as cap groups in the design and biological
mechanistic evaluation of novel antitumor histone
deacetylase inhibitors
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
Mudassier Ahmad,1,ǂ Mushtaq A. Aga,1,† Javeed Ahmad Bhat,1,ǂ, ᶲ Brijesh Kumar,† Abdul Rouf,†
Neena Capalash, ᶲ Mubashir Javeed Mintoo,ǂ,$ Ashok Kumar, † Priya Mahajan, ¥ Dilip Manikrao
Mondhe,ǂ,$ Amit Nargotra,¥ Parduman Raj Sharma,†,$ Mohmmad Afzal Zargar, ǁ Ram A.
Vishwakarma,# Bhahwal Ali Shah, *†,$ Subhash Chandra Taneja,*,† Abid Hamid*,ǂ,$
ǂCancer Pharmacology Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road,
Jammuꢀ180001, India.
†Natural Product Microbes Division,# Medicinal Chemistry Division, CSIRꢀIndian Institute of
Integrative Medicine, Canal Road, Jammuꢀ180001, India.
¥Discovery Informatics Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road,
Jammuꢀ180001, India.
ǁDepartment of Biochemistry, University of Kashmirꢀ190006, India
ᶲDepartment of Biotechnology, Panjab University, Chandigarhꢀ160014, India
$CSIRꢀAcademy of Scientific & Innovative Research, New Delhi, Govt. of India.
1
ACS Paragon Plus Environment

Page 3 of 47
Journal of Medicinal Chemistry
1
2
3
4
Abstract
5
6
7
8
9
lꢀVasicine is a quinazoline alkaloid with electron dense ring and additional functionalities in its
structure. Employing target oriented synthesis (TOS) based on in silico studies, molecules with
significant docking scores containing different derivatives of lꢀvasicine as caps were synthesized.
Interestingly, one molecule i.e., 4a which contained 3ꢀhyroxypyrrolidine as a cap group and six
carbon long aliphatic chain as a linker was found to inhibit HDACs. 4a showed more specificity
towards class I HDAC isoforms. Also 4a was found to be less cytotoxic towards normal cell
lines as compared to cancer cell lines. 4a inhibited cancer cell growth and induced cell death by
various mechanisms. However, 4a was found to induce cell death independent of ROS
generation and unlike many natural product based HDAC inhibitors; 4a was found to be nonꢀ
toxic under in vivo conditions. Importantly, we for the first time report the possibility of using 3ꢀ
hydroxypyrrolidine a cap for the synthesis of HDAC inhibitors with good potency.
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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
Introduction
5
6
7
8
In cancer, epigenetics has been found to play an important role in the origin, development and
metastasis1,2. Last decade has also witnessed clinical applications of epigenetics in cancer.
Epigenetic writer and reader enzymes like histone deacetylases (HDACs), DNA methyl
transferases (DNMTs), histone methyl transferases (HMTs) are being increasingly used as
targets for chemotherapeutic intervention in cancer and other diseases like diabetes and neuroꢀ
degenerative disorders3,4. Acetylation of histones is the common epigenetic mechanism used by
cells for regulating the cellular processes like gene expression, cell growth, cell death etc5,6.
Dysregulation of the acetylation has been associated with diverse cellular events in the cancer
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
pathologies7,8. Global hypoacetylation of H4 is the common feature of human tumours9,10
.
Acetylation of histones and other proteins is maintained by two antagonistic families of enzymes,
histone deacetylases (HDACs) and histone acetyl transferases (HATs)6,11. Due to its reversible
nature, acetylation has been harnessed for cancer chemotherapy1,12. So far, two HDAC inhibitors
suberoylanalide hydroxamic acid (SAHA) and romidepsin have already been approved by the
FDA as drugs against Chronic T Cell Lymphoma (CTCL)13,14. Besides, there are tens of HDAC
inhibitors at various stages of clinical trials against different cancers11,15. HDAC inhibitors
increase the acetylation level of histones and have been chiefly found to reactivate the expression
of numerous silenced genes that promotes cell death1,11. HDAC inhibitors also lead to cell
growth arrest, cell differentiation and angiogenesis inhibition which are important biological
processes for suppression of cancers16,17
.
Taking into account the potential therapeutic efficacy of HDAC inhibitors, several
classes of HDAC inhibitors have been designed. However in spite of large efforts the clinical
potential of HDACs has not been realized yet. Natural products offer a good opportunity for the
3
ACS Paragon Plus Environment

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
design of new HDAC inhibitors. One of the important classes of bioactive natural products is
quinazolines. Quinazolines show potent anticancer, antiꢀmicrobial and antiꢀinflammatory
activities18. We in our current study, explored the possibility of using derivatives of quinazoline
alkaloid lꢀvasicine in the design of natural product based HDAC inhibitors. lꢀvasicine is a unique
molecule with electron dense ring system and additional functionalities in its structure, we
hypothesized that these structural features may be used to design new HDAC inhibitors with lꢀ
vasicine derivatives as cap groups. Using in silico approach, target oriented synthesis (TOS) and
biological studies, lꢀvasicine derivative 3ꢀhyroxypyrrolidine was found to act as a suitable cap
group in the design of a novel HDAC inhibitor (S)ꢀN1ꢀhydroxyꢀN8ꢀ(2ꢀ((3ꢀhydroxypyrrolidinꢀ1ꢀ
yl) methyl ) phenyl) octanediamide (4a). Figure 1 shows the chemical structures of compounds
4aꢀ4d and 9aꢀ9d.
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: Chemical structures of compounds 4aꢀ4d and 9a-9d
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
RESULTS AND DISCUSSION
5
6
7
8
In silico screening of l-vasicine derivatives. To support our hypothesis, different modifications
of lꢀvasicine were proposed and virtual screening of proposed molecular designs was carried out.
Linkers were placed on the proposed lꢀvasicine based caps at structurally and chemically feasible
positions on the ring system. Hydroxamic acid was used as chelator. In silico studies were
carried out with Schrodinger suite 2015 molecular modeling software. 3D structures of various
HDAC isoforms (HDAC 1, 4 and 8) were used as receptors. Significant docking scores were
achieved for lꢀvasicine derivatives (R)ꢀaminoꢀ1ꢀbenzylpyrrolidine and 3ꢀhydroxypyrrolidine
when attached as cap groups to aliphatic linkers of various lengths (Table 1). The molecules
which showed significant docking scores were taken as probable HDAC inhibitors and three
schemes of Target Oriented Synthesis (TOS) were carried out to synthesise probable lꢀvasicine
based HDAC 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
Table 1. Docking scores of novel HDAC inhibitors containing various analogs of lꢀvasicine as
caps. Schrodinger suite 2015 molecular modeling software was used for in silico studies.
Molecule
name
Linker
length
Cap group
HDACꢀ1 HDACꢀ2 HDACꢀ4 HDACꢀᶲ8
6 carbons
7 carbons
3 carbons
ꢀ7.5
ꢀ6.8
ꢀ7.3
ꢀ7.98
ꢀ7.82
ꢀ6.34
ꢀ6.7
ꢀ6.4
ꢀ6.0
ꢀ8.09
ꢀ7.69
ꢀ6.13
4a
4b
4c
4d
4 carbons
ꢀ6.8
ꢀ6.89
ꢀ6.6
ꢀ7.48
3ꢀhydroxyꢀ
pyrrolidine
9a
9b
9c
9d
3 carbons
4 carbons
6 carbons
7 carbons
ꢀ6.1
ꢀ6.6
ꢀ6.3
ꢀ6.3
ꢀ6.88
ꢀ7.08
ꢀ7.77
ꢀ7.34
ꢀ7.9
ꢀ7.2
ꢀ7.2
ꢀ6.7
ꢀ6.42
ꢀ6.31
ꢀ7.82
ꢀ8.06
(R)ꢀaminoꢀ1ꢀ
benzylpyrrolidine
5
ACS Paragon Plus Environment

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
4
Chemistry
5
6
For using 3ꢀhydroxypyrrolidine moiety as a cap region, synthesis started by bringing the
7
8
9
cleavage of imidine bond of lꢀvasicine (1) using NaBH4 in methanol:water (1:1) to get
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
18, 19
tetrahydrovasicine
(2). The next step was the coupling of amine (2) in the presence of
secondary hydroxyl group with hydrophobic linker through amide bond. It would be pertinent to
mention here that various hydrophobic linkers of varying chain lengths i.e., monomethyl succinic
acid, monomethyl adipic acid, monomethyl suberic acid and monomethyl azealic acid were
selected for these analogues. Here, boric acid mediated coupling of 2 with different linkers was
found to be the best method, as it gives selectively Nꢀcoupling products i.e., 3a-3d in the
presence of a free hydroxyl group. The last step was the conversion of esters (3a-3d) into
corresponding hydroxamic acids (4a-d), using hydroxylamine hydrochloride in the presence of
KOH (Scheme 1).
Scheme 1. Synthesis of Histone deacetylase inhibitors from vasicine: (a) NaBH4, MeOH:H2O, rt; (b) B(OH)3, Toluene, reflux; (c) NH2OH.HCl,
KOH, MeOH, rt.
Besides, probable HDAC inhibitors having (R)ꢀaminoꢀ1ꢀbenzylpyrrolidine (7) as a
hydrophobic cap group were also synthesized (Scheme 2). The synthesis once again initiated
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
5
6
7
8
9
from vasicine to yield anilinic nitrogen (2), which on hydrodeamination (anilinic amine) in the
presence of amylnitrite gave (S)ꢀ1ꢀbenzylpyrrolidꢀ3ꢀol (5). The 3ꢀhydroxyl group of 5 was then
converted into amino group (7) in two steps. The treatment of 5 with tosyl chloride in the
presence of triethyl amine in DCM gave 6, followed by SN2 substitution of tosyl group in 6 with
NaN3, and thereafter in situ reduction of azido intermediate in the presence of PPh3 gave (R)ꢀ3ꢀ
aminoꢀ1ꢀbenzylpyrrolidine (7). Boric acid mediated coupling of 7 with the hydrophobic linker in
toluene under reflux gave the esters, i.e. 8a-d. Again, four different linkers as described in
scheme 1 were attached. In the final step, the esters (8a-d) were converted to the corresponding
hydroxamic acids (9a-d) by using hydroxylamine hydrochloride in the presence of KOH.
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
b
a
N
N
N
NH2
(2)
(5)
OTs
OH
OH
(6)
d
N
c
N
O
O
O
HN
n
O
NH2
N
OH
(7)
O
O
O
(8)
8a, n=3.; 8b, n=4
8c, n=6.; 8d, n=7
e
HN
OH
n
HN
O
(9)
9a, n=3.; 9b, n=4
9c, n=6.; 9d, n=7
0
Scheme 2. Synthesis of HDAC inhibitors from (R)ꢀaminoꢀ1ꢀbenzylpyrrolidine: (a) Amyl nitrite, DMF, 60 C; (b) TsCl, TEA, DCM; (c) NaN3,
Bu4NBr, DMF, PPh3, 0 °C ꢀ rt; (d) B(OH)3, Toluene, reflux; (e) NH2OH.HCl, KOH, MeOH, rt.
Furthermore, the probable HDAC inhibitors were also synthesized via stereo inversion of (S)ꢀ
1ꢀbenzylpyrrolidinꢀ3ꢀol (5) to obtain (R)ꢀ1ꢀbenzylpyrrolidinꢀ3ꢀol (12) by Mitsunobu reaction in
presence of DIED, PPh3 and acetic acid in THF. The 3ꢀhydroxyl group of 12 was converted into
7
ACS Paragon Plus Environment

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3ꢀamine (14), following the two step reaction sequence depicted in scheme 3. Boric acid
coupling of 14 with the hydrophobic linkers in toluene gave the esters 15a-d. The esters, i.e.
(15a-d) were converted to corresponding hydroxamic acids (16a-d) using hydroxylamine
hydrochloride in the presence of KOH.
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
o
Scheme 3. Synthesis of HDAC inhibitors from (S)ꢀaminoꢀ1ꢀbenzylpyrrolidine: (a) DIAD, PPh3, AcOH, THF, 0 C; (b) LiOH, THF, H2O; (c)
TsCl, TEA, DCM; (d) NaN3, Bu4NBr, DMF, PPh3 , 0 °C ꢀ rt; (e) B(OH)3, Toluene, reflux; (f) NH2OH.HCl, KOH, MeOH, rt.
Biological evaluation.
Screening of molecules for HDAC inhibition
The molecules with significant docking scores synthesised through above schemes of synthesis
were evaluated for HDAC inhibition. First molecules were evaluated for HDAC inhibition
against HeLa nuclear extract. TSA and SAHA were used as positive controls (supporting
information Figure 2). HDAC inhibition activity of molecules was determined at different
concentrations (0.5, 1.0, 1.5 and 2 μM concentrations) so as to generate their IC50 values. The
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 47
1
2
3
4
5
6
7
8
9
molecules which did not show any significant HDAC inhibition up to 2 μM concentration were
taken as inactive. The results are shown in Figure 2. Molecules 9a, 9b, 9c, 9d, 16a, 16b, 16c and
16d did not show any significant HDAC inhibition up to 2 μM concentrations where as
molecules 4b, 4c and 4d showed little HDAC inhibition at this concentration. Importantly,
molecule 4a showed significant HDAC inhibition. Molecule 4a showed IC50 value of 370 nM
against the HeLa nuclear extract (Figure 2b). 4a contains lꢀvasicine derivative 3ꢀ
hydroxypyrrolidine as a cap which proves our hypothesis. Furthermore, HDAC inhibition
activity of 3ꢀhydroxypyrrolidine cap based molecules (4a, 4b, 4c and 4d) varied with any change
in linker lengths. Linker length of six carbons (present in 4a) was found suitable for HDAC
inhibition and any change in linker length rendered the molecule inactive. 3ꢀhydroxypyrrolidine
cap based molecule with seven carbon (4b), three carbon (4c) and four carbon (4d) long linkers
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
were found to be inactive against HDACs.
Figure 2. (a) HDAC inhibition activities of the designed compounds at 2 μM concentration against HeLa nuclear extract. SAHA and TSA were
used as positive controls at 20 ꢁM concentrations. (b) HDAC inhibition curve of active molecule 4a. 4a showed IC50 value of 370 nM against
HeLa nuclear extract. (c) Structure of active molecule 4a.
9
ACS Paragon Plus Environment

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
4
Evaluation of HDAC isoform specificity of 4a
5
6
7
8
9
After initial screening against HeLa nuclear extract, HDAC inhibition activity of active molecule
4a was evaluated against HDAC isoforms. SAHA was used as a positive control. The results are
shown in Table 2 below. 4a exhibited interesting HDAC isoform specificity. 4a was found most
active against HDAC isoforms 1, 2, 3 and 8 with IC50 values 415, 268, 368 and 211 nM
respectively. 4a was found to be moderately active against HDAC isoforms 4 and 5 with IC50
values in micro molar range while as 4a was found to be least potent against HDAC 9, 10 and
11. These results indicate that 4a is more active towards class I HDACs as compared to other
HDACs.
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. HDAC inhibition activities (IC50) of 4a and SAHA against HDAC isoforms. Values are mean
of three experiments and the standard error of the IC50 was generally less than 10%.
IC50 (nM)
Class I
Class IIa
Class IIb Class IV
Molecule
HDAC
1
HDAC
HDAC
3
HDAC
8
HDAC
10
HDAC
11
HDAC 4 HDAC 5 HDAC 9
2
415
150
268
454
368
261
211
300
7120
5391
2180
>10000
>10000
>10000
982
>10000
>10000
4a
SAHA
>10000
Molecular modeling studies of 4a
Incorporation of linker chain and hydroxamic acid functionality to cap group (i.e. lꢀvasicine and
its derivative 3ꢀhyroxypyrrolidine) led to the identification of series of compounds with varying
affinity for the HDACꢀ8 enzyme. The optimized novel HDAC inhibitor (S)ꢀN1ꢀhydroxyꢀN8ꢀ(2ꢀ
((3ꢀhydroxypyrrolidinꢀ1ꢀyl) methyl) phenyl) octane diamide (4a), displayed inꢀvitro IC50 value
of 211 nM for HDACꢀ8 and 268 nM for HDACꢀ2. The results of molecular docking studies of
4a are in close agreement with the inꢀvitro studies where 4a display selectivity for HDACꢀ8
inhibition, in contrast to other HDACs.
10
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
5
6
7
8
9
Upon docking studies, it was observed that entire 4a molecule was involved in interaction with
the HDACꢀ8, 3ꢀhrydroxypyrrolidine ring acts as cap group for molecular recognition and
involved in Hꢀbonding with Pheꢀ152 backbone amide. Similar to the CRAAꢀA and HDACꢀ8
crystal structure, drift in Pheꢀ152 side chain creates hydrophobic subꢀpocket, which provides
additional stability to the ligandꢀreceptor interactions. At biological pH pyrrolidine nitrogen,
atom gets protonated and forms a hydrogen bond with the Tyrꢀ111 phenolic hydroxyl group. In
addition to this, protonated cation is also able to interact with the Trpꢀ141 π clouds. Similar to
wellꢀknown HDAC inhibitors like SAHA and CRAAꢀA the 6ꢀcarbon chain length linker region
is quiet flexible and well accommodated in the hydrophobic tunnel. In addition to the Hꢀbonding
interactions with the Tyrꢀ306 and Hisꢀ142; the terminal hydroxamic acid functionality is also
capable of forming coꢀordination bond with the Zn2+ metal ion.
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
Overall, the introduction of lꢀvasicine and its derivatives as cap group in the suitably designed
linker and metal chelator (such as hydroxamic acid) can regulate the deacetylation of the histoneꢀ
DNA assembly. The observed little selectivity of 4a towards the HDACꢀ8 over HDACꢀ2 is
might be due to the Hꢀbonding and πꢀcationic interactions of the protonated pyrrolidine nitrogen
with Tyrꢀ111 and Trpꢀ141, respectively. These interactions were not possible in case of HDACꢀ
2 because Tyrꢀ111 and Trp141 residues are replaced Pheꢀ103 and Leuꢀ133, respectively. 2D and
3D interactions of the 4a with HDACꢀ8 are shown in Figure 3A and 3B while 2D interactions of
4a with HDACꢀ2 is shown in Supporting information section Figure S2.
Screening of molecules for cytotoxicity
4a and other 3ꢀhydroxypyrrolidine cap based molecules (4b, 4c and 4d) were evaluated for
cytotoxicity potential against a panel of human cancer cell lines. The cells of different tissue
origins were included in the panel. The panel consisted of leukemia cell lines (THPꢀ1 and HLꢀ
11
ACS Paragon Plus Environment

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
60), colon cancer cell lines (Coloꢀ205 and Cacoꢀ2), pancreatic cancer cell line (MIAPaCaꢀ2),
prostate cancer cell line (PCꢀ3), lung cancer cell line (A549) and breast cancer cell line (MCFꢀ7).
The cytotoxicity of molecules was initially determined at 10 µM concentration (Table 3). Out of
molecules 4a, 4b, 4c and 4d, only 4a showed significant cytotoxicity against all the cell lines in
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
A
B
Figure 3: Interactions of 4a with HDAC-8 (PDB-1VKG), A; 3D representation, B; 2D
representation.
the panel and the molecules 4b, 4c and 4d were found to be inactive against all the cell lines in
panel (Table 3). Importantly, 4a is a HDAC inhibitor thus cytotoxicity of 4a against a panel of
cell lines is induced by HDAC inhibition activity of 4a and molecules other than 4a lack
significant HDAC activity and no cell death was induced by them. Moreover, cytotoxicity of 4a
was evaluated at lower concentrations to determine IC50 values of 4a against the panel of cell
lines (Table 4). IC50 value of 4a varied from 0.6 µM to 7.0 µM against various cancer cell lines in
12
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
6
7
8
9
the panel. Some cell lines were found to be more sensitive to cytotoxicity induced by 4a. 4a
showed least IC50 value of 0.6 µM against colon cancer cell line, i.e. Coloꢀ205. 4a was also
found to be very significantly cytotoxic against leukaemia cell lines (THPꢀ1, HLꢀ60 and MOLTꢀ
4), pancreatic cancer cell line (MIAPaCaꢀ2) and breast cancer cell line MCFꢀ7, while as it was
found to be moderately active against colon cancer cell line Cacoꢀ2 and non small cell lung
cancer cell line, i.e. A549. 4a was least cytotoxic against prostate cancer cell line PCꢀ3. This
selective cytotoxic behavior of the 4a against some cell lines in the panel may be attributed to the
epigenomic and genomic profiles of these cell lines.
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 3. Evaluation of cytotoxicity of synthesized molecules at 10 ꢁM concentration by MTT assay.
Results are Mean ± S.D of three experiments carried out in triplicates separately.
Tissue
Leukaemia
Colon
Pancreatic Prostate Lung Breast
Cancer cell type
THP1 HLꢀ60 MOLTꢀ4 Colo205 Cacoꢀ2 MIAPaCaꢀ PCꢀ3 A549
2
MCFꢀ7
Test molecule
Conc.
(ꢁM)
% Growth Inhibition
10
58 ± 7
29 ± 3
4a
4b
94 ± 6 95 ±5 91 ± 3 92 ± 7
17 ± 1 6 ± 3 30 ± 2 7 ± 4
93 ± 5
84±9 93 ±6
10
10
10
5
5 ± 4 2 ± 3
12 ± 2 5 ± 6
3 ± 4
5 ± 5
26±4
89±6
4c
3 ± 4 3 ± 3 27 ± 1 26 ± 4 1 ± 1 4 ± 5
37 ± 5 29± 2 38 ± 4 22 ± 4 31 ±5 12 ± 3
2 ± 2
5 ± 6
4d
15 ± 4 19±3
88 ±1 95±4
SAHA
91± 4 93± 2 90±3
88±3
93±4 86±3
13
ACS Paragon Plus Environment

Page 15 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
Table 4. IC50 value of 4a against the panel of cell lines. Results are Mean ± S.D of three
experiments carried out in triplicates separately.
6
7
8
9
Tissue
Leukaemia
HLꢀ60
Colon
Pancreatic Prostate Lung
Breast
Cancer cell
type
THP1
1.3±0.3
1.1 ±0.2
MOLTꢀ4 coloꢀ205 Cacoꢀ2 MIAPaCaꢀ2 PCꢀ3
A549 MCFꢀ7
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
IC50 of 4a
(µM)
1.0±0.2
0.81±0.3 0.6±0.1 6.0±0.8
0.7 ±0.3 1.8 ±0.1 3 ± 0.6
1.0±0.2 19.0±3 7.0±1 1.5±0.1
2.2 ±0.1 3 ±0.8 1.5±0.2 0.9±0.3
IC50 of SAHA
1.0 ± 0.2
(µM)
Molecule 4a induced acetylation of histones
HDAC inhibitors are known to increase the acetylation level of histones20, 21. In order to further
confirm that molecule 4a caused HDAC inhibition, HLꢀ60 and MOLTꢀ4 cells were treated with
various concentrations of 4a. Histones were extracted from treated cells and level of acetylation
on H3 at lysine 18 and H4 at lysine 5 was probed. 4a was found to increase the acetylation at
both H3K18 and H4K5 residues in both the cells (Figure 4). The level of acetylation increased
with increase in concentration of 4a which further proves that 4a is a HDAC inhibitor. SAHA
was used as a positive control.
Figure 4: Increase in acetylation of core histones serves as the marker for HDAC Inhibition. (a) 4a increased the acetylation level of core
histones H3 and H4 in HLꢀ60 cells (b) 4a increased the acetylation level of core histones H3 and H4 in MOLTꢀ4 cells.
4a is least cytotoxic to non-cancerous normal cell lines
Cytotoxicity of 4a was also evaluated against nonꢀcancerous normal cell line fRꢀ2 and MCF
10a. Interestingly, 4a was found to be least cytotoxic against fRꢀ2. 4a showed IC50 value of 23.0
14
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
7
8
9
± 2 ꢁM against fRꢀ2 and 37 ± 1 ꢁM which is several fold higher than IC50 values against
different cancer cell lines. Thus 4a showed selective cytotoxic behavior against cancer cell lines
as compared to normal cell line. Importantly, this selective cytotoxicity of 4a towards cancer cell
lines as compared to normal cell lines is an essential pharmacological feature with therapeutic
implications. SAHA was used as positive control which showed IC50 of 16 ± 1 against fRꢀ2 and
28 ± 1 ꢁM against MCF 10A.
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
In vivo anti-leukemic efficacy of 4a
2.5 x 106 P388 lymphocytic cells were injected intraperitoneally in CDF1 female mice weighing
18ꢀ23 g. A group of 7 animals was treated with 4a at a dose of 50 mg/kg (i.p.) for 9 consecutive
days. Although natural product based HDAC inhibitors like TSA, trapoxin and depudecin
generally show toxic effects under in vivo conditions but no significant toxic effects were
observed in animals treated with 4a. Moreover, 4a showed the median survival time of 92.47 %
at 50mg/kg i.p. dose. Usually HDAC inhibitors derived from natural resources have limited
retention time and are unstable under in vivo conditions21. The effect of 3ꢀhydroxypyrrolidine on
pharmacokinetic and pharmacodynamic properties of 4a may need further elucidation. 5ꢀ
Fluorouracil (at 20mg/kg i.p. dose) and SAHA (at 50 mg/kg i.p dose) used as positive controls
also showed significant median survival time in P388 lymphocytic model.
4a induced cell death by inducing mitochondrial membrane potential loss
The proton gradient across the inner mitochondrial membrane is required for oxidative
phosphorylation and is measured as mitochondrial membrane potential (ѱm). Ѱm is reflective of
intactness and function of mitochondria and any deviation in ѱm is used as a marker for inner
mitochondrial membrane disruption which leads to apoptosis (Figure 5). Using Rhꢀ123 as a
fluorescent probe, 4a was found to induce loss in ѱm in HLꢀ60 and MOLTꢀ4 cells. Change in ѱm
15
ACS Paragon Plus Environment

Page 17 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was quantified by using flow cytometry. In both the cell lines, percentage of cells undergoing
loss in ѱm increased at higher concentrations. Moreover, loss in ѱm occurred more prominently
in MOLTꢀ4 cells as compared to HLꢀ60 cells. 4a thus disrupts the architecture of inner
mitochondrial membrane and initiates intrinsic apoptotic pathway in both the cell lines used.
SAHA was used as positive control.
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
4a induced ROS independent cell death
Reactive oxygen species (ROS) are important agents that promote cell death in cancer cells.
Moreover, many HDAC inhibitors like TSA, SAHA and Sodium butyrate have been reported to
cause accumulation of reactive oxygen species in transformed cell lines which facilitates cell
death of transformed cells22.
Figure 5: Reactive oxygen species generation was quantified by flowcytometry. H202 was used as a positive control. H2DCFDA dye was used
as probe. (a) 4a did not induce accumulation of reactive oxygen species (ROS) in HLꢀ60 cells (b) 4a did not induce accumulation of reactive
oxygen species (ROS) in MOLTꢀ4 cells
We investigated whether 4a caused generation of ROS by flow cytometry using H2DCFDA dye
as probe. Interestingly, 4a was not found to induce accumulation of significant level of ROS in
16
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
HLꢀ60 and MOLTꢀ4 cells. Thus 4a induced cell death by ROS independent mechanism (Figure
5
6
6). 0.01% H2O2 was used as positive control
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 6: Mitochondrial membrane potential loss was studied by flow cytometry. Rhꢀ123 dye was added half an hour before completion of
incubation. (a) 4a induced mitochondrial membrane potential loss in HLꢀ60 cells (b) 4a induced mitochondrial membrane potential loss in
MOLTꢀ4 cells.
4a induced DNA damage
DNA damage is one of the most important events occurring during cell death. DNA damage
serves as the marker of the cell death. Comet assay or Single Cell Gel Electrophoresis (SCGE)
assay was used to assess the DNA damage. Comet assay is based on applying electric current to
the cells embedded in agarose and lysed by alkaline buffer. The broken DNA fragments migrate
away from the nucleus. The resulting fluorescent images, named as comets for their appearance,
determine the extent of DNA lesions. Length of tails formed during comet assay depicts the
extent of DNA damage induced. DNA damage induction by 4a was studied in HLꢀ60 cells using
comet assay. 4a was found to induce significant DNA damage in HLꢀ60. Moreover, the extent of
damage induced in HLꢀ60 cells by 4a increased with increase in concentration (Figure 7). DNA
damage induced by 4a was found to be maximum at 10 µM concentration.
17
ACS Paragon Plus Environment

Page 19 of 47
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 7:. DNA damage induced by 4a in HLꢀ60 cells was studied by comet assay. Length of tails depicts the damage induced by 4a.
4a induced autophagy
Autophagy generally promotes tumor suppression. HDAC inhibitors like other cytotoxic agents
generally induce autophagy. The characteristic feature of autophagy is the formation of acidic
vacuoles. We used acridine orange dye to investigate whether 4a could induce autophagy.
Interestingly, 4a induced the accumulation of autophagic vacuoles in the cytoplasm of HLꢀ60
and MOLTꢀ4 cells (Figure 8a and 8c). To further confirm induction of autophagy by 4a,
intracellular distribution of LCꢀ3B was detected by immunofluorescent microscopy. In both HLꢀ
60 and MOLTꢀ4 cells treated with 4a punctuated distribution of LCꢀ3B was seen (Figure 8b and
8d). Thus proving that 4a induced autophagy in HLꢀ60 cells.
18
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 47
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 8. (a) HLꢀ60 cells treated with 4a were stained with acridine orange dye and examined under florescent microscope. 4a induced,
formation of acidic vacuoles occurs in cytoplasm. (b) HLꢀ60 cells treated with 4a were subject to immunofluorescent stainining and examined
under fluorescent microscope.4a caused punctuated distribution of LCꢀ3B. (c) 4a induced the formation of acidic vacuoles in the cytoplasm of
MOLTꢀ4 cells. (d) 4a induced punctuated distribution of LCꢀ3B in MOLTꢀ4 cells.
4a inhibits cell migration ability of THP-1 cells
Cell motility is a necessary feature for tumor invasion and metastasis23,24. In order to study the
effect of 4a on the migration behavior of cancer cells, cell migration assay was performed
(Figure 9). It was found that 4a prevented the migration of cells to the wounds induced in the
confluent monolayers of THPꢀ1 cells. Furthermore, the degree of inhibition of cell migration was
found to be higher at higher concentrations. At higher concentrations of 4a, cell morphology got
altered. Migration assay is reflective of potential of an anticancer agent to inhibit metastasis25.
19
ACS Paragon Plus Environment

Page 21 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Localization of THPꢀ1 cells induced by 4a indicated that 4a may prevent degradation of
extracellular matrix proteins and surrounding tissues and may thus inhibit metastasis of cancer
cells in in vivo models. However, the antiꢀmetastatic ability of 4a needs further exploration in
suitable animal models.
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 9: Wound healing assay on THP1 cells. Test molecule addition was carried out after inducing wound in confluent cells. 4a prevents
migration of THPꢀ1 cells.
Conclusion
We in our current study report the designing of a novel HDAC inhibitor (S)ꢀN1ꢀhydroxyꢀN8ꢀ(2ꢀ
((3ꢀhydroxypyrrolidinꢀ1ꢀyl) methyl ) phenyl) octanediamide (4a) containing lꢀvasicine derivative
3ꢀhydroxypyrrolidine as a cap group. This is the first time that lꢀvasicine has been used in the
synthesis of HDAC inhibitors. Our hypothesis was that lꢀvasicine has essential structural features
which may be employed in the designing of novel HDAC inhibitors. During in silico studies,
molecules containing various derivatives of lꢀvasicine as caps and aliphatic linkers of various
20
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
7
8
9
lengths showed significant docking scores. Three schemes of target oriented synthesis were
framed to synthesise these probable HDAC inhibitors. Interestingly, 3ꢀhydroxypyrrolidine cap
based molecule 4a with linker length equal to six carbons was found to significantly inhibit
enzyme activity of HDACs and induce cell death in different cancer cell lines. 4a was found to
be more specific towards class I HDAC isoforms. HDAC inhibition activity of 4a was also
confirmed by increase in acetylation level of histones in 4a treated HLꢀ60 and MOLTꢀ4 cells. 3ꢀ
hydroxypyrrolidine cap was found to be highly sensitive to linker length and any change in
linker length rendered molecule inactive. Importantly, 4a showed several fold higher IC50 value
against nonꢀcancerous normal cell line fRꢀ2 and MCF 10A as compared to most cancer cell lines,
thus indicating that 4a induced cancer specific cell death. Moreover, unlike many other natural
product based HDAC inhibitors 4a was found to be nonꢀtoxic during in vivo studies on P388
lymphocytic model. Mechanistic studies revealed that 4a facilitated cell death by several
pathways. 4a promoted cell death by inducing mitochondrial membrane potential loss and
autophagy in HLꢀ60 and MOLTꢀ4 cells. However unlike many HDAC inhibitors 4a treatment
did not lead to accumulation of ROS in HLꢀ60 cells thus inducing ROS independent cell death.
Importantly, 4a showed DNA damage which is an important event in initiating cell death in
cancer cells and serves as a key marker for cell death detection. In addition, migration assay
showed that 4a induces localization in THPꢀ1 cells and decreased their ability to migrate to the
wound induced in the THPꢀ1 monolayer, thus indicating antiꢀmetastatic potential of 4a.
Moreover, molecular modeling studies revealed that 4a that cap, linker and chelator of 4a
interact with the different amino acids in the binding pockets of HDACs 2 and 8. However, the
hydrophobic cleft formed by Met274, Cys275 and Phe207 provides an additional stability to 4a
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
ACS Paragon Plus Environment

Page 23 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
for HDAC8 which is not seen in HDACꢀ2. From this analysis it was concluded that the molecule
4a has more potency for HDACꢀ8.
7
8
9
Thus our results indicate designing of a novel HDAC inhibitor (4a) employing 3ꢀ
hydroxypyrrolidine derivative of quinazoline alkaloid lꢀvasicine as the cap group. Moreover, the
cytotoxicity of 4a against a spectrum of cancer cells of different tissue origins and significantly
high specificity against cancer cell lines are scientifically valuable results which warrants further
exploration. 4a is thus a lead candidate with a therapeutic potential. Moreover, importance of 4a
also lies in understanding its unique mechanism of action essentially the fact that unlike other
HDAC inhibitors it does not produce ROS and is nonꢀtoxic to in vivo models unlike many
natural product based HDAC inhibitors. Furthermore, it will be interesting to evaluate the
applicability of 3ꢀhydroxypyrrolidine cap in HDAC inhibitors containing chelators other than
hydroxamic acid. Also the effect of 3ꢀhydroxypyrrolidine cap on bioavailability of 4a may be
studied.
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 Procedure:
13
Chemistry.1H and C NMR spectra were recorded on Bruker 400 and 500 MHz spectrometers
with TMS as the internal standard. Chemical shifts are expressed in ppm. ESIꢀHRMS are
recorded on a (Agilent Technology 6540 UHD, Acurrat Mass QꢀTOFꢀLcꢀMs) instrument. Silica
gel coated aluminium plates were used for TLC. Optical rotations were measured on Perkinꢀ
Elmer 241 polarimeter. Reagents and solvents used were mostly of LR grade. Dry Solvents were
procured from Merck. HPLC analysis was done on Shimadzu HPLC system (model: LCꢀ6AD)
equipped with a PDA detector (model: SPDꢀM20A) using Hypersil BDS C18 (5.0 ꢁ, 4.6 mm ×
250 mm) column. Mobile phase used was ACN: Buffer (40:60) isocratic elution at flow rate of 1
22
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 47
1
2
3
4
5
6
ml/min. The purity of final compounds was determined by HPLC. All compounds have ≥95%
purity.
7
8
9
Buffer Preparation: Dissolve 13.61 g of potassium dihydrogen phosphate in 500 ml of water.
Shake well to mix and adjust the pH to 2.8±0.05 with dilute orthophosphoric acid. Shake well to
mix and sonicate to degas.
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
Synthesis of (S)-1-(2-aminobenzyl) pyrrolidin-3-ol (2). The natural (ꢀ)ꢀvasicine (1) (10g, 53
mmol) was dissolved in 100 mL of MeOH/H2O (1:1) in a 250 mL of round bottom flask with
continuous stirring, followed by the addition of NaBH4 (5.87g , 158 mmol) in small amounts
over a period of 1 h. The mixture was stirred for an additional 1 h, and then extracted with
CH2Cl2 (3x100 mL). The solvent was dried over anhydrous Na2SO4 and removed in vacuo to
25
give the compound (2) in 99% yield (10.1 g) as a light yellow semiꢀsolid, [α]D = ꢀ199 (c 3,
CHCl3). 1H NMR (500 MHz, CDCl3) δ 1.63ꢀ1.79 ( m, 1H), 2.1ꢀ2.2 (m, 1H), 2.24ꢀ2.30 (m, 1H),
2.4ꢀ2.6 (m, 2H), 2.73ꢀ2.84 (m, 1H), 3.60 (dd, 2H), 4.22ꢀ4.38 (m,1H), 6.6ꢀ6.74 (m, 2H), 7.00
13
(d,1H), 7.07 (t, 1H). C NMR (100 MHz, CDCl3) δ 34.9, 52.4, 59.1, 62.6, 71.1, 115.6, 117.8,
123.5, 128.3, 129.8, 146.2. HRMS (+ESI): calc. for C11H16N2OH 193.1341; found 193.1343.
A: General procedure for boric acid coupling:
(S)-Methyl-8-((2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)amino)-8-oxooctanoate
(3a):
Suberic acid monomethyl ester (1 g, 5.32 mmol) and 20 mol% boric acid was weighed into
reaction vessel, followed by mounting on assembly of a microꢀSoxhlet apparatus loaded with
activated 3Å molecular sieves under nitrogen. Dry toluene (15 mL) and (S)ꢀ1ꢀ(2ꢀ
aminobenzyl)pyrrolidinꢀ3ꢀol (2) (1 g, 5.23 mmol) then added to reaction vessel and the reaction
heated to reflux. The mixture was allowed to reflux for 3 h before being concentrated in vacuo.
The residue was then redissolved in ethyl acetate (20 mL), washed with brine (15 mL), solvent
23
ACS Paragon Plus Environment

Page 25 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
dried over anhydrous Na2SO4 and removed in vacuo. The residue obtained after evaporation was
chromatographed over silica gel column using hexane/ethyl acetate (70:30) as eluent to give the
compound (3a) in 80% yield (1.55 g) as a light yellow semiꢀsolid. 1H NMR (400 MHz, CDCl3) δ
8.23 (d, J = 8.0 Hz, 1H), 7.27ꢀ7.22 (m, 1H), 7.02 (d, J = 6.5 Hz, 1H), 6.93 (td, J = 7.4, 1.0 Hz,
1H), 4.42 (ddt, J = 7.5, 5.1, 2.5 Hz, 1H), 3.74ꢀ3.62 (m, 6H), 3.61 (d, J = 2.9 Hz, 3H), 2.82 (dt, J =
11.0, 9.5 Hz, 1H), 2.63 (ddd, J = 10.7, 10.4, 3.6 Hz, 2H), 2.42ꢀ2.21 (m, 5H), 2.21ꢀ2.14 (m, 1H),
1.82ꢀ1.61 (m, 3H), 1.60 (dd, J = 14.5, 7.4 Hz, 2H), 1.43ꢀ1.27 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 174.3, 171.6, 138.5, 129.0, 128.2, 126.1, 123.2, 121.0, 70.7, 62.1, 59.1, 52.0, 51.4, 38.1,
34.7, 33.9, 28.8, 28.7, 25.4, 24.6. HRMS (+ESI): calc. for C20H30N2O4H 363.2284; found
363.2279.
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
(S)-Methyl-9-((2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)amino)-9-oxononanoate
(3b):
Prepared by the general procedure A and purified on silica gel (ethyl acetate/hexane: 30/70) to
obtain the semiꢀsolid product (75%). 1H NMR (400 MHz, CDCl3) δ 8.19 (t, J = 8.5 Hz, 1H), 7.28ꢀ
7.12 (m, 1H), 7.05 (d, J = 6.9 Hz, 1H), 6.93 (dt, J = 7.4, 3.7 Hz, 1H), 4.41 (bs, 1H), 3.63 (t, J = 5.5
Hz, 2H), 3.62 (s, 3H), 2.89ꢀ2.74 (m, 1H), 2.34 (ddt, J = 25.1, 15.0, 8.6 Hz, 6H), 2.20ꢀ2.04 (m,
1H), 1.81ꢀ1.70 (m, 1H), 1.70ꢀ1.61 (m, 2H), 1.58 (dd, J = 13.2, 7.4 Hz, 2H), 1.44ꢀ1.21(m, J = 6H).
13C NMR (100 MHz, CDCl3) δ 174.3, 171.7, 154.0, 138.6, 128.9, 128.2, 126.1, 123.1, 120.6, 70.8,
62.2, 59.3, 52.0, 51.4, 51.4, 48.0, 42.6, 36.0, 34.8, 34.0, 25.6, 25.1, 24.8, 23.4, 22.5, 22.5, 20.9.
HRMS (+ESI): calc. for C21H32N2O4H 377.2440; found 377.2438.
(S)-Methyl-5-((2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)amino)-5-oxopentanoate
(3c):
Prepared by the general procedure A and purified on silica gel (ethyl acetate/hexane: 30/70) to
obtain the semiꢀsolid product (80%). 1H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 8.2 Hz, 1H), 7.08
(d, J = 7.4 Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 6.79 (t, J = 7.3 Hz, 1H), 4.25 (s, 1H), 3.59 (d, J =
24
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
5
6
7
8
9
12.8 Hz, 1H), 3.48 (s, 3H), 3.41 (d, J = 12.8 Hz, 1H), 2.73 (d, J = 8.7 Hz, 1H), 2.63 (d, J = 9.9 Hz,
1H), 2.32 (dd, J = 10.1, 4.9 Hz, 1H), 2.23 (dd, J = 14.2, 7.1 Hz, 4H), 2.16ꢀ2.00 (m, 3H), 1.95ꢀ1.80
(m, 2H). 13C NMR (100 MHz, CDCl3) δ 174.1, 170.7, 138.5, 129.0, 128.3, 126.2, 123.3, 120.8,
70.9, 62.4, 59.2, 52.0, 51.7, 37.2, 34.7, 33.1, 23.4, 21.1. HRMS (+ESI): calc. for C17H24N2O4H
321.1214; found 321.1217.
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
(S)-Methyl 6-((2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)amino)-6-oxohexano-ate (3d):
Prepared by the general procedure A and purified on silica gel (ethyl acetate/hexane: 30/70) to
obtain the semiꢀsolid product (80%). 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 8.3 Hz, 1H), 7.33ꢀ
7.15 (m, 1H), 7.04 (d, J = 6.4 Hz, 1H), 6.94 (dt, J = 7.2, 6.3 Hz, 1H), 4.43 (qd, J = 4.4, 2.0 Hz,
1H), 3.74 (d, J = 21.2, 1H), 3.65 (d, J = 12.5 Hz, 4H), 2.88 (td, J = 8.7, 5.2 Hz, 1H), 2.71 (d, J =
10.3 Hz, 1H), 2.54 (dd, J = 10.1, 5.3 Hz, 1H), 2.45ꢀ2.26 (m, 5H), 2.24ꢀ2.01 (m, 2H), 1.84ꢀ1.58 (m,
13
4H). C NMR (100 MHz, CDCl3) δ 174.2, 171.2, 138.6, 129.0, 128.3, 126.0, 123.2, 120.8, 70.8,
62.3, 59.1, 51.9, 51.6, 37.9, 34.7, 33.6, 25.2, 24.4. HRMS (+ESI): calc. for C12H26N2O4H
335.1971; found 335.1973.
B: General procedure for hydroxamic acid synthesis.
(S)-N1-Hydroxy-N8-(2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)octanediamide
(4a):
Hydroxylamine hydrochloride (3.51 g, 5.1 mol) in methanol (10 mL) was mixed with KOH (2.83
gm, 0.051 mol) at 40 °C in methanol (20 mL) and filtered. The compound (3a) (1g, 2.76 mmol)
was then added to the filtrate followed by the addition (over 30 min) of KOH (231 mg, 4.1 mmol).
The reaction mixture was stirred at room temperature for 6 h. The mixture was added to stirring
cold water (90 mL), pH adjusted to 7 by adding acetic acid, followed by the extraction with ethyl
acetate. Solvent was dried over anhydrous Na2SO4 and removed in vacuo. The residue obtained
after evaporation of the ethyl acetate was chromatographed over silica gel column using
25
ACS Paragon Plus Environment

Page 27 of 47
Journal of Medicinal Chemistry
1
2
3
chloroform/methanol (85:15) as eluent to give the compound (4a) in 82% yield (820 mg) as a light
4
5
6
1
yellow semiꢀsolid. HPLC: tR = 25.6 min (99% purity). H NMR (400 MHz, DMSOꢀd6) δ 10.74
7
8
9
(bs, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.30ꢀ7.25 (m, 1H), 7.09 (d, J = 6.5 Hz, 1H), 6.98 (dt, J = 7.4,
1.0 Hz, 1H), 4.46 (ddt, J = 7.5, 5.1, 2.5 Hz, 1H), 3.78ꢀ3.66 (dd, J = 12.2, 8.1 Hz, 2H), 2.82 (dt, J =
12.0, 9.5 Hz, 1H), 2.66 (ddd, J = 15.7, 10.4, 3.6 Hz, 2H), 2.46ꢀ2.27 (m, 5H), 2.26ꢀ2.15 (m, 1H),
1.86ꢀ1.69 (m, 3H), 1.63 (dd, J = 14.5, 7.4 Hz, 2H), 1.46ꢀ1.30 (m, 4H). 13C NMR (100 MHz,
MeOD) δ 172.3, 171.7, 138.5, 129.1, 128.3, 126.2, 123.3, 121.0, 62.2, 59.1, 52.0, 51.5, 38.1, 34.7,
33.9, 28.8, 28.7, 25.5, 24.7. HRMS (+ESI): calc. for C19H29N3O4H 364.2236; found 364.2233.
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
(S)-N1-Hydroxy-N9-(2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)nonanediamide
(4b):
Prepared by the general procedure B and purified on silica gel (chloroform/methanol: 85/15) to
obtain the semiꢀsolid product (70%). HPLC: tR = 27.2 min (98% purity). 1H NMR (400 MHz,
DMSOꢀd6) δ 10.85 (bs, 1H), 8.21 (t, J = 8.4 Hz, 1H), 7.30ꢀ7.21 (m, 1H), 7.08 (d, J = 6.7 Hz, 1H),
6.98 (dt, J = 7.3, 3.5 Hz, 1H), 4.45 (s, 1H), 3.68 (t, J = 5.4 Hz, 2H), 2.92ꢀ2.75 (m, 1H), 2.37 (ddt, J
= 25.1, 15.0, 8.5 Hz, 6H), 2.24ꢀ2.08 (m, 1H), 1.84ꢀ1.74 (m, 1H), 1.74ꢀ1.65 (m, 2H), 1.62 (dd, J =
14.1, 7.0 Hz, 2H), 1.49ꢀ1.25 (m, J =6H). 13C NMR (100 MHz, MeOD) δ 173.2, 171.7, 154.1,
138.6, 128.9, 128.2, 126.2, 123.1, 120.7, 62.3, 59.3, 52.1, 51.5, 51.4, 48.1, 42.7, 36.1, 34.8, 34.0,
25.7, 25.2, 24.8, 23.5, 22.5, 22.5, 20.9. HRMS (+ESI): calc. for C20H32N3O4H 378.2393; found
378.2389.
(S)-N1-Hydroxy-N5-(2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)glutaramide (4c): Prepared
by the general procedure A and purified on silica gel (methanol/chloroform: 15/85) to obtain the
1
semiꢀsolid product (72%). HPLC: tR = 22.4 min (97% purity). H NMR (400 MHz, DMSOꢀd6) δ
10.72 (bs, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.81 (t,
J = 7.5 Hz, 1H), 4.28 (s, 1H), 3.61 (d, J = 13.1 Hz, 1H), 3.45 (d, J = 13.2 Hz, 1H), 2.78 (d, J = 8.6
26
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
Hz, 1H), 2.66 (d, J = 10.1 Hz, 1H), 2.36 (dd, J = 10.5, 5.2 Hz, 1H), 2.27 (dd, J = 14.4, 7.2 Hz,
5
6
13
4H), 2.12ꢀ2.02 (m, 3H), 1.97ꢀ1.83 (m, 2H). C NMR (100 MHz, MeOD) δ 173.0, 170.7, 138.5,
7
8
9
129.1, 128.3, 126.2, 123.3, 120.9, 70.9, 62.5, 59.2, 52.0, 51.7, 37.2, 34.8, 33.1, 23.4, 21.2. HRMS
(+ESI): calc. for C16H23N3O4H 322.1767; found 322.1771.
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
(S)-N1-Hydroxy-N6-(2-((3-hydroxypyrrolidin-1-yl)methyl)phenyl)adipamide (4d): Prepared
by general procedure A and purified on silica gel (methano/chloroform: 15/85) to obtain the semiꢀ
1
solid product (70%). HPLC: tR = 23.8 min (98% purity). H NMR (400 MHz, DMSOꢀd6) δ 10.89
(bs, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.35ꢀ7.12 (m, 1H), 7.08 (d, J = 6.6 Hz, 1H), 6.98 (td, J = 7.4,
1.0 Hz, 1H), 4.46 (qd, J = 4.7, 2.1 Hz, 1H), 3.79 (dd, J = 12, 11.1 Hz, 1H), 3.65 (d, J = 12.5 Hz,
4H), 2.90 (dt, J = 8.7, 5.0 Hz, 1H), 2.74 (d, J = 10.4 Hz, 1H), 2.57 (dd, J = 10.4, 5.1 Hz, 1H),
13
2.48ꢀ2.28 (m, 5H), 2.27ꢀ2.05 (m, 2H), 1.88ꢀ1.61 (m, 4H). C NMR (100 MHz, MeOD) δ 173.2,
171.3, 138.6, 129.0, 128.3, 126.1, 123.2, 120.8, 70.9, 62.3, 59.1, 51.9, 51.6, 37.9, 34.7, 33.6, 25.3,
24.48. HRMS (+ESI): calc. for C17H25N3O4H 336.1923; found 336.1912.
C: (R)-1-Benzylpyrrolidin-3-amine (7): (S)ꢀ1ꢀBenzylpyrrolidinꢀ3ꢀol (1 g, 5.68 mmol) and TEA
(170.4 mg, 1.136 mmol) were added to DCM (50 mL) at 0 °C. A solution of tosyl chloride (1.62
g, 8.52 mmol) in DCM (30 mL) was added dropwise. The reaction mixture was stirred at 0 °C
for 30 min then at room temperature for 12 h. Water (50 mL) was added and the organic phase
washed with saturated solution of NaHCO3 (2x50 mL), followed by brine. The organic layer was
collected and dried over anhydrous Na2SO4 and the solvent evaporated under reduced pressure to
obtain tosyl alcohol (6). The compound (6) (4 mmol), sodium azide (520 mg, 8.0 mmol) and
o
tetrabutylammonium bromide (5 mol %) in DMF (5 mL) are stirred for 6 h at 60 C. It was then
cooled to room temperature and then triphenylphosphine (1.153 g, 4.4 mmol) added to it and
stirred overnight at room temperature. The resultant mixture was then poured into water (100
27
ACS Paragon Plus Environment

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mL) and extracted with DCM (2x10 mL). The acid and base treatments given to the DCM layer
to partially purify the product. The organic phase was dried over anhydrous Na2SO4 and
removed in vacuo to obtain the crude compound, which was chromatographed over silica gel
column using chloroform/methanol (85:15) as eluent to give the compound (7) in 60% yield (600
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
25
1
mg) as a light yellow liquid, [α]D = ꢀ1.92 (neat). H NMR (400 MHz, CDCl3) δ 7.43ꢀ7.15 (m,
5H), 3.64 (q, J = 12.8 Hz, 2H), 3.51 (s, 1H), 2.72 (dd, J = 7.8, 5.0 Hz, 2H), 2.47 (dd, J = 14.3,
7.4 Hz, 1H), 2.30 (dd, J = 15.0, 10.1 Hz, 1H), 2.27ꢀ2.12 (m, 1H), 1.48 (d, J = 5.0 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ 138.4, 128.7, 128.3, 127.2, 58.8, 52.8, 52.2, 51.5, 34.8. HRMS
(+ESI): calc. for C11H17N2H 177.1392; found 177.1386.
(R)-Methyl 5-((1-benzylpyrrolidin-3-yl)amino)-5-oxopentanoate (8a): Prepared by the
general procedure A and purified on silica gel (ethyl acetate/hexane: 30/70) to obtain a semiꢀ
1
solid product (80%). H NMR (400 MHz, CDCl3) δ 7.28 (m, 5H), 4.44 (s, 1H), 3.68ꢀ3.50 (m,
5H), 2.88 (m, 1H), 2.64ꢀ2.48 (m, 2H), 2.25 (t, J = 7.2 Hz, 3H), 2.07 (dd, J = 16.1, 9.1 Hz, 2H),
13
1.74 (s, 4H). C NMR (100 MHz, CDCl3) δ 173.6, 172.1, 133.8, 129.9, 128.7, 128.5, 128.3,
59.0, 58.9, 52.1, 51.5, 51.4, 33.5, 33.1, 20.9, 20.8. HRMS (+ESI): calc. for C17H24N2O3H
305.1265; found 305.1264.
(R)-Methyl-6-((1-benzylpyrrolidin-3-yl)amino)-6-oxohexanoate (8b): Prepared by the
general procedure A and purified on silica gel (ethyl acetate/Hexane: 30/70) to obtain a semiꢀ
solid product (80%). 1H NMR (500 MHz, CDCl3) δ 7.32ꢀ7.15 (m, 5H), 6.03 (s, 1H), 4.41 (dd, J
= 6.6, 2.1 Hz, 1H), 3.65ꢀ3.64 (m, 4H), 3.60 (d, J = 3.8 Hz, 1H), 2.92 (dd, J = 13.7, 8.5 Hz, 1H),
2.59 (dd, J = 11.0, 2.8 Hz, 1H), 2.51 (dd, J = 11.0, 3.2 Hz, 1H), 2.37ꢀ2.24 (m, 4H), 2.14 (t, J =
13
6.1 Hz, 2H), 1.70ꢀ1.57 (m, 5H). C NMR (101 MHz, CDCl3) δ 173.9, 172.0, 136.8, 129.1,
28
ACS Paragon Plus Environment