Novel amide derivatives as inhibitors of histone deacetylase: Design, synthesis and SAR

Article abstract of DOI:10.1016/j.ejmech.2008.06.020

Enzymatic inhibition of histone deacetylase (HDAC) activity is emerging as an innovative and effective approach for the treatment of cancer. A series of novel amide derivatives have been synthesized and evaluated for their ability to inhibit human HDACs. Multiple compounds were identified as potent HDAC inhibitors (HDACi), with IC₅₀ values in the low nanomolar (nM) range against enzyme activity in HeLa cell extracts and sub-μM for their in vitro anti-proliferative effect on cell lines. The introduction of an unsaturated linking group between the terminal aryl ring and the amide moiety was the key to obtain good potency. This approach yielded compounds such as (E)-N-[6-(hydroxyamino)-6-oxohexyl]-3-(7-quinolinyl)-2-propenamide (27) (HDAC IC₅₀ 8 nM) which showed potent in vivo activity in the P388 mouse leukemia syngeneic model (an increased lifespan (ILS) of 111% was obtained).

Full text of DOI:10.1016/j.ejmech.2008.06.020

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1067e1085
http://www.elsevier.com/locate/ejmech
Original article
Novel amide derivatives as inhibitors of histone
deacetylase: Design, synthesis and SAR
Victor Andrianov a, Vija Gailite a, Daina Lola a, Einars Loza a, Valentina Semenikhina a,
Ivars Kalvinsh a, Paul Finn b,1, Kamille Dumong Petersen c, James W.A. Ritchie b,
Nagma Khan b, Anthony Tumber b, Laura S. Collins b, Sree M. Vadlamudi b,
c,d
Fredrik Bjorkling c, , Maxwell Sehested
*
¨
a Latvian Institute of Organic Synthesis, Aizkraules 21, Riga, LV-1006, Latvia
b TopoTarget UK Ltd., 87A Milton Park, Abingdon, Oxfordshire OX14 4RY, United Kingdom
c TopoTarget A/S, Symbion Science Park, Fruebjergvej 3, DK-2100 Copenhagen, Denmark
d Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen, Denmark
Received 18 March 2008; received in revised form 13 June 2008; accepted 20 June 2008
Available online 27 June 2008
Abstract
Enzymatic inhibition of histone deacetylase (HDAC) activity is emerging as an innovative and effective approach for the treatment of cancer.
A series of novel amide derivatives have been synthesized and evaluated for their ability to inhibit human HDACs. Multiple compounds were
identified as potent HDAC inhibitors (HDACi), with IC50 values in the low nanomolar (nM) range against enzyme activity in HeLa cell extracts
and sub-mM for their in vitro anti-proliferative effect on cell lines. The introduction of an unsaturated linking group between the terminal aryl
ring and the amide moiety was the key to obtain good potency. This approach yielded compounds such as (E )-N-[6-(hydroxyamino)-6-oxo-
hexyl]-3-(7-quinolinyl)-2-propenamide (27) (HDAC IC50 8 nM) which showed potent in vivo activity in the P388 mouse leukemia syngeneic
model (an increased lifespan (ILS) of 111% was obtained).
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: HDAC inhibitor; SAR; Hydroxamic acid
1. Introduction
structures blocks accessibility of the transcriptional machinery
to their target genes. Transcription is regulated by multiple
post-translational modifications of the histone tails within
the chromatin, and histone acetylation has been shown to be
one of the major regulatory mechanisms for gene expression
[2]. Control of expression is dependent on the balance between
the competing activities of histone acetyl transferases (HATs)
and histone deacetylases (HDACs) [3] on the regulation of
chromatin structure by acetylation of lysines on histone tails.
Mammalian HDACs are grouped into four distinct classes.
Class I, II and IV enzymes are zinc dependent with classes I
and II being homologous to the yeast proteins RPD3 and
Hda1, respectively [4], whereas class III HDACs are structur-
ally distinct NAD-dependent enzymes [5,6]. The discovery
and study of histone deacetylase inhibitors (HDACi) confirms
Histones are small basic proteins, which occur in five main
classes (H1, H2A, H2B, H3, and H4). A pair of each of H2A,
H2B, H3, and H4 together forms a disc-shaped octomeric pro-
tein core, around which DNA (about 140 base pairs) is wound
to form a nucleosome. In turn, nucleosomes form a structure
described as ‘‘beads on a string’’ with the DNA, and coil to
form the basis of 30 nm chromatin filaments [1]. This packag-
ing of DNA in nucleosomes and higher order chromatin
* Corresponding author. Tel.: þ45 39 17 83 92; fax: þ45 39 17 94 92.
E-mail address: fbj@topotarget.com (F. Bjorkling).
¨
Present address: InhibOx Ltd., 36-37 Pembroke Street, Oxford OX1 1BP,
United Kingdom.
1
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.06.020

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V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
that these enzymes play an important role in the regulation of
a number of cellular processes, including cell proliferation, ap-
optosis, assembly of the cytoskeleton and regulation of tran-
scription [2,7e9]. In addition, the over-expression of
HDACs leads to epigenetic inactivation of apoptotic signaling
pathways, cell cycle regulators and tumor suppressor genes
[10]. As a consequence of these findings, several HDACi are
currently undergoing clinical trials as anticancer drugs
[11,12], some of which have been designed as selective inhib-
itors in addition to the ‘‘pan-HDACi’’ that inhibit all zinc de-
pendent HDACs. The natural product inhibitor Trichostatin A
(TSA, (I)), and the synthetic inhibitor suberoylanilide hy-
droxamic acid (SAHA, (II)) broadly inhibit the class I, II
and IV enzymes and have been reported to inhibit cell growth,
induce terminal differentiation, and prevent the formation of
tumors in mice [13]. Compound II (SAHA, vorinostat, Zo-
linzaÒ, Merck Inc. [14]) was recently approved for treatment
of refractory cutaneous T-cell lymphoma (CTCL [15]). Other
synthetic HDACi are currently in clinical development for use
in cancer treatment including hydroxymates such as (E )-3-
(4-(((2-(1H-indol-3-yl)ethyl)-(2-hydroxyethyl)amino)methyl)-
phenyl)-N-hydroxyacrylamide (III e panobinostat (LBH589),
phase I/II, Novartis [16]) and N-hydroxy-3-(3-(N-phenylsulfa-
moyl)phenyl)acrylamide (IV e belinostat (PXD101), phase II,
TopoTarget [17]), benzamides (4-(2-aminophenylcarbamoyl)
benzylamino)methyl nicotinate (V e SNDX-275 (MS-275),
phase II, Syndax (Bayer-Schering AG) [18]) and N-(2-amino-
phenyl)-4-((4-(pyridin-3-yl)pyrimidin-2-ylamino)methyl)ben-
zamide (VI e MGCD0103, phase II, Methylgene [19]) and
valproic acid (VII e VPA, phase II, TopoTarget [20]) (see
Fig. 1 for structures). Furthermore, there is a growing interest
in the potential of HDACi in other therapeutic areas, such as
inflammation [21], neurodegeneration [22] and malaria
[23,24].
HDACi based on an amide-alkyl-hydroxamic acid frame-
work, such as SAHA (II) are well known. These conform to
a well-described, simple HDAC inhibition pharmacophore
AeBeC, where A represents a moiety which confers potency
and specificity (usually aryl), B is a linker group, such as am-
ide-alkyl, and C is a zinc liganding group such as hydroxamic
acid (Table 1). Most of the SAR exploration studies published
to date have investigated the role of the zinc binding group,
with considerable effort being used searching for alternatives
to hydroxamic acid, and to some extent the linker. The SAR
of the aryl moiety has been much less well explored. In the
present manuscript we therefore describe the SAR for a novel
series of hydroxamic acid containing HDACi with a wide va-
riety of aryl end groups in the side chains which are shown to
favorably interact with the residues at the entrance of the bind-
ing pocket.
2. Results and discussion
2.1. Chemistry
The routes used for synthesis of the compounds for this
study are shown in Schemes 1e3.
O
O
O
H
OH
N
OH
N
H
N
H
O
SAHA (II)
N
Trichostatin A (I)
OH
O
H
N
H
N
OH
N
OH
S
H
N
O
O
O
HN
LBH589 (III)
PXD101 (IV)
O
N
O
N
H
O
N
N
H
N
NH2
H
N
HN
H2N
N
O
MGCD0103 (VI)
MS275 (V)
O
OH
VPA (VII)
Fig. 1. Natural product and synthetic inhibitors of HDAC.

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1069
Table 1
Prepared HDAC inhibitors: synthesis sequence and biological activity
O
n
H
N
R
A
OH
N
H
O
C
B
Compound
R
n
Synthesis
Biological activity
Scheme no.:
synthesis sequence
HDAC
IC50 (nM)
WST-1
IC50 (mM)
SAR comment(s)
Reverse amide
Chain length; 4 carbons are to short
II (SAHA)
5
87
42% at 500 nM
3.1
24.7
4
1: 2a, 3a, 5
6
7
6
7
1: 2a, 3b, 6
1: 2a, 3c, 7
20
28
0.4
3.6
Chain length; 5e6 carbons optimal
Chain length
8
5
5
5
1: 2b, 3d, 8
38
21
2.6
1.1
4.1
9
1: 2a, 3e, 4a, 9
1: 2c, 3f, 4b, 10
Chain length; 5e6 carbons optimal
10
110
N
Simple phenyl; less active as compared
to naphthyl
11
16
5
5
1: 2d, 4c, 11
238
29
6.3
5.1
2: 12a, 13a, 16
Unsaturation in linker; potency retained
Alkyl spacer, potency lost
17
18
19
20
21
5
5
5
5
5
2: 12b, 13b, 17
2: 12c, 13c, 18
2: 12d, 13d, 19
2: 12e, 13e, 20
2: 12f, 13f, 21
27% at 500 nM
19% at 500 nM
32
74.8
52.7
5.5
Alkyl spacer, potency lost
Unsaturation, potency retained
Extra methylene lower activity
b-substitution allowed
53% at 500 nM
24.0
4.8
23
22
3
5
2: 12g, 13g, 22
2: 12h, 13h, 23a
22,850
101
113.5
46.4
23a
Pyridyl substitution is allowed
N
N
Small difference in activity for pyridyl
isomers
24a
5
2: 12i, 13i, 24a
46
9.5
(continued on next page)

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V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
Table 1 (continued)
Compound
R
n
Synthesis
Biological activity
Scheme no.:
synthesis sequence
HDAC
IC50 (nM)
WST-1
IC50 (mM)
SAR comment(s)
N
25a
5
5
5
2: 12j, 13j, 25a
2: 12k, 13k, 26
2: 12l, 13l, 27
141
7
28.4
18.3
12.3
Unsaturation and a naphthyl gives
potent compounds
26
27
N
9
Quinoline is a preferred substituent
Point of substitution affect the activity
28
29
5
5
2: 12m, 13m, 28
2: 12n, 13n, 29
25
36
20.9
37.9
N
N
30
31
5
5
2: 12o, 13o, 30
2: 12p, 13p, 31
201
338
8.5
9.9
Diphenyl is less potent
To large groups give lower activity
32
5
2: 12r, 13r, 32
5
1.7
Some bulky aromatics are very potent
Alkyl spacer, potency lost
33
34
5
5
2: 12s, 13s, 33
2: 12t, 13t, 34
51% at 500 nM
625
70.8
20.6
35
36
5
5
2: 12u, 13u, 35
2: 12v, 13v, 36
62
10.6
52.7
Alkyne, potency retained
9% at 500 nM
Methylene spacer, low potency
N
Substituted aryl with unsaturated
linker; highly active
37
38
5
5
2: 12w, 13w, 37
8
6.3
Cl
F
2: 12x, 13x, 14, 15, 38
51% at 500 nM
35.9
Methylene spacer; low potency
41
42
a
3
3
39% at 500 nM
46% at 500 nM
44.2
13.3
Reverse amide with alkyl spacer;
low activity
Reverse amide with alkyl spacer;
low activity
Isolated as oxalate.

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1071
O
n
O
n
O
b
a
H
N
H
N
R
R
R
OH
OMe
N
H
Cl
2a-d
O
O
3a-f
5 - 11
e
c
d
O
H
R
N
OH
5
O
4a-c
Scheme 1. Reagents and conditions: (a) H2N(CH2)nCOOMe/HCl (1, n ¼ 4, 5, 6, or 7), DIPEA, DMF; (b) H2NOH/HCl, aq. NaOH/MeOH; (c) aq. LiOH/THF;
(d) CDI/THF, then H2NOH/DMF; (e) H2N(CH2)5COOH, aq. NaOH, dioxane.
Thus, as a starting material for synthesis of hydroxamic
acids 5e11 (Table 1) served commercially available acid chlo-
rides 2aed (Scheme 1).
ester 13x was hydrolyzed by LiOH into carboxylic acid 14
and converted into O-benzyl hydroxamate 15. Removal of
O-benzyl protection of 15 by hydrogenation in the presence
of 5% Pd(C) catalyst finished the preparation of hydroxamic
acid 38.
Synthesis of hydroxamic acids 41 and 42 containing a ‘‘re-
versed amide’’ functional group in the central part of the mol-
ecule is shown in Scheme 3. Benzylamine 39a or
2-phenylethylamine 39b was acylated with imidazolide pre-
pared from 8-methoxy-8-oxooctanoic acid, and the obtained
amidoesters 40a,b were treated with hydroxylamine in the
presence of NaOH in aqueous methanol affording target hy-
droxamic acids 41 and 42 (Table 1).
Acid chlorides 2aec were condensed with appropriate
methyl u-aminoalkanoates 1 (n ¼ 4e7) in presence of diiso-
propylethyl amine in DMF to give the corresponding ami-
doesters 3aef. Amidoesters 3aed were transformed into
hydroxamic acids 5e8 by treating the former with four-fold
excess of hydroxylamine in the presence of NaOH in aqueous
methanol medium. Amidoesters 3e,f were hydrolyzed into
acids 4a,b by LiOH in aqueous THF. Benzoyl chloride 2d
was directly transformed into carboxylic acid 4c by the reac-
tion with 6-aminohexanoic acid in the presence of NaOH in
water/dioxane (1:1) mixture. The carboxylic acids 4aec
were subsequently converted into the corresponding hydroxa-
mic acids 9e11 via imidazolide intermediates followed by hy-
droxylamine treatment.
2.2. SAR studies
For synthesis of the majority of studied hydroxamic acids,
i.e. 16e38, (Table 1) appropriate starting carboxylic acids
12aex were utilized which in turn were provided from com-
mercial suppliers or prepared according to published proce-
dures (see Section 4) (Scheme 2). Syntheses of 16e37 were
quite straightforward. Thus, amidation of appropriate starting
acids 12aex with methyl 6-aminohexanoate (or methyl 4-ami-
nobutanoate in the case of 12g) in the presence of suitable con-
densing agent afforded the expected amidoesters 13aex. The
esters 13aew were treated with hydroxylamine in the pres-
ence of base (NaOH or MeONa) to give the expected hydroxa-
mic acids 16e37. In the case of amidoester 13x direct
treatment with hydroxylamine/base failed. The intermediate
2.2.1. Chain length
Synthesis of a series of homologues quickly identified that
the linking section between the hydroxamic acid and amide
must be at least five carbon atoms in length (Table 1), with
lengths of five and six being optimal. Predicted binding modes
from the results of docking experiments suggested that an ex-
tra methyl group in the spacer directs the terminal aryl group
into the surface pocket in the correct orientation to pick up fa-
vorable interactions (Fig. 2). In addition, the Zn binding dis-
tances of compounds with chain lengths of five or six
carbon atoms were found to be very similar to reference com-
pounds I and II. This is consistent with the SAR reported pre-
viously by others. Selecting a chain length of five as a basis for
O
n
O
O
a or b or c
d or e
H
N
H
N
R
R
R
OH
OMe
3a-x
N
H
n
OH
12a-x1
O
O
16 - 38
13x
h
f
O
O
5
H
H
N
g
N
OBn
N
H
OH
5
O
O
14
15
Scheme 2. Reagents and conditions: (a) CDI, 1 (n ¼ 3 or 5), TEA, THF; (b) CDI, H2N(CH2)5COOMe/HCl, TEA, DMF; (c) ClCOO-iso-Bu, TEA/THF, then 1
(n ¼ 5); (d) NaOMe, NH2OH/HCl/MeOH; (e) aq. NaOH, NH2OH/HCl/MeOH; (f) aq. LiOH, THF; (g) CDI, NH2OBn/HCl, TEA, DMF; (h) H2/Pd(C)/MeOH.

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V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
O
O
6
O
O
6
b
a
OH
Ph
Ph
Ph
n
NH2
n
N
H
OMe
n
N
H
N
H
39a, n = 1
39b, n = 2
41, n = 1
40a, n = 1
40b, n = 2
42, n = 2
Scheme 3. Reagents and conditions: (a) CDI, HOOC(CH2)6COOMe/DMF; (b) aq. NaOH, NH2OH/HCl/MeOH.
further work, the effect of introducing a spacer between the
amide and the terminal aromatic moiety was examined.
lipophilic and electrostatic binding contributions. Terminal
aryl groups in compounds with unsaturated carbon next to
the amide linker (Fig. 4) were oriented in the correct direction
to enable favorable interactions with the residues on the en-
trance of the narrow lipophilic pocket. This guided the identi-
fication of compounds with improved biological activity. In
general, compounds possessing an alkenyl linker were more
potent than their directly linked or methylene linked homo-
logues. Further substitution of the alkyl spacer was allowed
in 21, however, introduction of the alkyl spacer did not com-
pensate for reduction in the length of the linking group be-
tween the amide and hydroxamic acid 22.
2.2.2. Linker-extension
The simple phenyl analogue 11 had moderate inhibitory ac-
tivity. Introducing an alkyl spacer length of 2, 3, or 4 reduced
both enzyme inhibitory and anti-proliferative potency in 17,
18, and 33. It was also found that a methylene spacer was del-
eterious to activity, as illustrated by the homologous pairs 8/38
and 10/36 (Table 1). Introduction of 2, 3 or 4 carbon atom
spacers significantly altered the orientation of the terminal
aryl group of the inhibitors (17, 18 and 33) as opposed to
the simple phenyl analogue 11 (Fig. 3). Due to this, com-
pounds with extra carbon atoms demonstrated severe vdW
clashes with the residues on the top of the narrow aliphatic
channel and the surface aryl pocket, which lead to a significant
reduction in the biological activity.
Whenever a saturated carbon was adjacent to the amide
e.g., in 20 and 34, potency remained poor, but when unsatura-
tion was introduced, the compounds showed excellent potency,
as in 19 and 16. Strong potency was also maintained using an
alkynyl analogue 35. The nature of the SP3 or SP2 hybridized
carbon atom next to the amide linker determined the orienta-
tion of the terminal aryl group. Differences in the angles con-
necting the amide linker and terminal aryl group were
governed by the character of SP3 or SP2 in the carbon atom
next to the amide linker. The entrance of the narrow tunnel
leading to the surface pocket is predominantly lipophilic,
and small variations in the contact distances governed by the
nature of the hybridized carbon atom may change the
2.2.3. SAR of linker-extended reverse amides
In comparison with SAHA (II), two compounds (41 and
42) were made in which the directionality of the amide was
inverted. Interestingly, the SAR appears to mirror that ob-
served above in that the compounds with a methylene (41)
or ethylene (42) spacer were found to be less active than the
parent molecule.
2.2.4. Headgroup SAR
Simple aryl systems were generally well tolerated, with the
SAR being rather flat (Table 1). Activity of halogen substituted
molecules was good. In addition, the activity of pyridyl ana-
logues was less sensitive to the position of substitution in
23e25, with the 3- and 4-pyridyl compounds being only
slightly weaker inhibitors. Interestingly, quinoline substitution
yielded potent compounds 27e29, especially with regard to
enzyme inhibitory activity.
Fig. 2. Superposition of the docked conformations of compound 5 with four
carbon chain length (blue), and of compound 6 with six carbon chain length
(red) in the HDAC active site. Protein represented in the form of molecular
surface, and the active site Zn atom shown in magenta sphere throughout
the figures.
Fig. 3. Superposition of the docked conformations of the compound 11 (red
capped sticks), and compounds 17, 18 and 33 e with two (blue wire frame),
three (orange wire frame) and four carbon atom spacer (yellow wire frame) in
the HDAC active site.

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1073
Amide direction important
but not critical
Hydroxamic acid essential
for good zinc
binding and activity
O
H
OH
R
N
N
H
n
O
Aromatic substituents preferred
particularly with unsaturated linker
Optimal chain lenght 5-6 carbons
Fig. 6. SAR summary.
recently published data where the HDAC selectivity for 3 hy-
droxamic acid HDACi was studied [25]. In this study, it is con-
cluded that bulkier head groups interacting with the entrance
of the binding pocket may give some selectivity between
HDAC class I (HDAC1) and class II (HDAC6) enzymes. How-
ever, a more pronounced difference is observed between the
HDAC8 (class I) enzyme and the others, which is in agreement
with what we found. The disfavored binding of long chain am-
ide HDACi to HDAC8 as compared to the potent inhibition of
other HDACs is suggested to be due to a possible change of
divalent ion in the active site from Zn(II) to Fe(II) in this iso-
form [26].
Fig. 4. Superposition of the docked conformations of the compound 11 (red
capped sticks), compound 16 (orange wire frame) and compound 19 (blue
wire frame) in the HDAC active site.
However, bulkier head groups revealed that there were
some steric constraints to activity. Compared to the simple
phenyl analogue 19, the di-phenyl 30 lost potency. Linking
the two aromatic rings to form a tricyclic system did not im-
prove activity 31. Even so, when the tricyclic system was
made smaller and more conformationally constrained, activity
returned, with the smallest and most constrained system deliv-
ering a highly potent molecule 32. Superposition of predicted
docking orientations of compounds 32 and 19 in the HDAC
active site (Fig. 5) revealed the importance of conformation-
ally constrained head groups to achieve tight binding by occu-
pying the surface hydrophobic pocket. A summary of the SAR
is given in Fig. 6.
2.4. In vivo biology
A small number of compounds were selected for in vivo
evaluation in the syngeneic P388 mouse leukemia i.p. model.
This model was valuable as a first in vivo screening model de-
termining anti-tumor activity and general animal tolerance.
The compounds were administered i.p. thus, as an intra-tumor
treatment, which was believed to maximize exposure and limit
pharmacokinetic influences. Activity in this model was most
commonly observed using compounds with bulkier aryl
groups, or suitable substitutions on the aryl ring. Activity
was equal to, and in some cases, superior to that shown by
compounds currently under clinical investigation as HDACi.
Of the four compounds tested, none produced increased
survival when dosed at 20 mg/kg/day for 5 days. However,
when dosed at 60 mg/kg/day, two compounds showed anti-tu-
mor effect. Compound 27 (ILS 111%) was the most active fol-
lowed by compound 32 (ILS 78%). Compound 37 at 60 mg/
kg/day had a minor anti-tumor effect in vivo (ILS 22%), while
2.3. HDAC selectivity
A representative set of compounds were tested for HDAC
isoform selectivity. Using recombinant HDAC enzymes
(HDAC 1e9) inhibitory activity of the compounds was deter-
mined (Table 2). These compounds were found to be more po-
tent than SAHA, but have a similar selectivity profile. Thus,
a broad and potent (nM) inhibition of all HDAC enzymes ex-
cept HDAC8 was found for compounds 21, 27, 32 and 37.
HDAC8 was inhibited at approximately 200e900 times higher
concentration (IC50). These results correspond well with
Fig. 5. Superposition of the docked conformations of the compound 32 (red capped sticks), compound 19 (blue capped sticks) in the HDAC active site. For the sake
of clarity two orthogonal views are shown in the left and right hand panels.

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V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
Table 2
Inhibition of HDAC isoforms (IC50, nM)
Enzyme/compound
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
21
27
3.5
2.9
3.1
2.5
5.3
6.7
3.7
5.3
NDa
7.6
NDa
NDa
107.3
3.5
3.4
4.4
3.0
2116.3
2028.3
779.0
4.2
2.8
32
37
1.6
3.2
0.8
3.0
2.8
4.9
1.2
4.3
2.0
2.5
NDa
6.3
2.3
3.9
1619.7
1374.7
II (SAHA)
68.1
163.6
67.1
100.9
110.6
102.4
107.2
a
Not determined.
SAHA (II) failed to show any survival benefit at 60 mg/kg/day
using the P388 mouse leukemia model (Table 3). At higher
concentrations SAHA was found weakly active in this model
(ILS 14%, 100 mg/kg/day).
4. Experimental
4.1. Chemistry
4.1.1. General
Nuclear magnetic spectra (1H NMR) were recorded on
WH-90/DS or Mercury 200 (Varian) spectrometers at ambient
temperatures. The purity of synthesized hydroxamic acids
(>96%) was assessed by reverse phase analytical HPLC using
Symmetry C18 column (column size: 3.9 ꢀ 150 mm; mobile
phase: acetonitrilee0.1 M phosphate buffer with pH 2.5);
measurements were performed on a Varian ProStar HPLC sys-
tem equipped with a spectrophotometer. Elemental analyses
were obtained with a Carlo Erba EA 1108 instrument. Melting
3. Conclusion
A novel series of chain-extended compounds, based on the
amide linker template of HDACi, were designed and synthe-
sized using computational and medicinal chemistry. Com-
pounds within this novel series showed potent HDAC
enzymatic inhibition, as well as anti-proliferative activity su-
perior to compounds currently undergoing clinical investiga-
tion. Two of these novel compounds also demonstrated large
survival benefits when administered to an in vivo mouse can-
cer model, underlining their potential for further development.
Available structural information (from sequence compari-
son and crystallography [4]) indicates that the HDAC isoforms
are well conserved in the vicinity of the catalytic machinery,
but more variable at the cavity entrance. Interactions with
these surface regions would therefore be expected to differ be-
tween HDAC family members and potentially confer HDAC
subtype selectivity, a topic of great current interest. However,
the inhibitors described in this report did not show a selective
interaction with this region of the HDAC isoforms leading to
isoform selectivity, with the exception of a decreased activity
towards HDAC8. Using data obtained through this and other
recent investigations, we are currently exploring further com-
pounds which may exhibit improved isoform selectivity.
points were measured on a ‘‘Boetius’’ or ‘‘Fisher’’ micro melt-
¨
ing point apparatus and are uncorrected. Silica gel, 0.035e
0.070 mm, (Acros) was employed for column chromatography
(CC). All the solvents were purified before use by routine
techniques.
2-Naphthoyl chloride (2a), benzenecarbonyl chloride (2d),
[1,10-biphenyl]-4-carbonyl chloride (2b), 2-[1,10-biphenyl]-4-
ylacetic acid (12x), 4-(dimethylamino)benzenecarbonyl chlo-
ride (2c), 2-[4-(dimethylamino)phenyl]acetic acid (12v),
aniline, benzylamine (39a), methyl 3,3-diphenylacrylate, 2-
(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yliden)acetic
acid (12p), 7-aminoheptanoic acid, 8-aminooctanoic acid, and
8-methoxy-8-oxooctanoic acid were purchased from Aldrich,
3-phenylpropanoic acid (12b), 4-phenylbutanoic acid (12c), 5-
phenylpentanoic acid (12s), (E )-3-phenyl-2-propenoic acid
(12d), (E )-4-phenyl-3-butenoic acid (12e), 3-phenyl-2-propy-
noic acid (12u), 2-phenyl-1-ethanamine (39b), (E )-3-(4-
chloro-2-fluorophenyl)-2-propenoic acid (12w), 6-quinoline-
carboxylic acid (12n), 5-aminopentanoic acid, and 6-amino-
hexanoic acid were from Acros, (2E,4E )-5-phenyl-2,
4-pentadienoic acid (12a), (E )-3-(4-pyridinyl)-2-propenoic
acid (12h), (E )-3-(3-pyridinyl)-2-propenoic acid (12j),
(E )-3-(2-naphthyl)-2-propenoic acid (12k), and 9-fluorenyli-
deneacetic acid (12r) were from Alfa Aesar, (E )-3-(2-pyri-
dinyl)-2-propenoic acid (12i) was from Apollo Sci., and
methyl 4-aminobutanoate was from Fluka.
Table 3
In vivo survival test using the P388 mouse leukemia i.p. model
Treatment Dose
compound (mg/kg/day)
ID
Schedule
Median
survival
(days)
N
ILS p-Value
(%)
Vehicle
control
ꢀ1 i.p. days 3e7
9
18
32
32
37
37
20
60
20
60
ꢀ1 i.p. days 3e7 10
ꢀ1 i.p. days 3e7 16
9
9
9
9
9
9
9
9
11 NS
78 <0.05
ꢀ1 i.p. days 3e7
9
0 NS
22 <0.05
11 NS
3,3-Diphenyl-acrylic acid (12o), methyl 5-aminopenta-
noate, methyl 6-aminohexanoate, methyl 7-aminoheptanoate,
and methyl 8-aminooctanoate were prepared by routine proto-
cols from methyl 3,3-diphenylacrylate, 5-aminopentanoic
acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, and 8-
aminooctanoic acid, accordingly. SAHA was prepared as de-
scribed in Ref. [27].
ꢀ1 i.p. days 3e7 11
ꢀ1 i.p. days 3e7 10
II (SAHA) 20
II (SAHA) 60
ꢀ1 i.p. days 3e7
ꢀ1 i.p. days 3e7
9
9
0
0
NS
NS
27
27
20
60
ꢀ1 i.p. days 3e7 19
111 <0.05
N ¼ number of animal per group. Statistics: LogRank analysis, p > 0.05 is
considered ‘‘not significant’’ (NS).

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1075
4.1.3. General synthesis of amidomethyl esters 3aed from
carboxylic acid chlorides 2a,b (method A)
2H), 3.63 (s, 3H), 6.32 (br s, 1H), 7.40e7.56 (m, 2H),
7.72e8.05 (m, 4H), 8 27 (s, 1H).
To a solution of u-amino acid methyl ester hydrochloride 1
(n ¼ 4, 5, 6, or 7) (2.75 mmol) and diisopropyl ethylamine
(0.96 ml, 5.5 mmol) in anhydrous N,N-dimethylformamide
(3 ml) appropriate carboxylic acid chlorides 2a,b
(2.75 mmol) in dimethylformamide (3 ml) was added. The
mixture was stirred for 3 h at room temperature, diluted with
brine (30 ml), and extracted with ethyl acetate (3 ꢀ 25 ml).
The organic phase was washed with brine (2 ꢀ 15 ml), dried
(Na2SO4) and the solvent was evaporated. The residue was pu-
rified on silica gel (20 g) with chloroform/ethyl acetate as
eluent, affording the corresponding amidoester derivatives
3aed.
4.1.6.2.
N-[7-(Hydroxyamino)-7-oxoheptyl]-2-naphthamide
(6). Compound 6 was obtained from methyl 7-[(2-naphthyl-
carbonyl)amino]heptanoate (3b) by the method B, yield
97%. M.p. 136e137 ꢁC. H NMR (DMSO-d6, HMDSO) d:
1.48e1.62 (m, 8H), 1.95 (t, J ¼ 6.8 Hz, 2H), 3.20e3.30 (m,
2H), 7.52e7.64 (m, 2H), 7.86e8.08 (m, 4H), 8.43 (s, 1H),
8.52e8.68 (m, 2H), 10.33 (s, 1H). Anal. Calcd for
C18H22N2O3: C 68.77, H 7.05, N 8.91. Found: C 68.50, H
7.08, N 8.96.
1
4.1.7. Preparation of 7
4.1.7.1. Methyl 8-[(2-naphthylcarbonyl)amino]octanoate (3c).
Compound 3c was obtained from 2-naphthoyl chloride (2a)
and methyl 8-aminooctanoate hydrochloride by the method
4.1.4. General synthesis of hydroxamic acids 5e8 from
amidoesters 3aed (method B)
1
Appropriate amidoesters 3 (1 mmol) and hydroxylamine
hydrochloride (278 mg, 4 mmol) were dissolved in methanol
(3e5 ml), and a solution of NaOH (320 mg, 8 mmol) in water
(1 ml) was added. After stirring for 15e45 min at ambient
temperature, the resultant mixture was acidified with 1 N
HCl to pH 3 and extracted with ethyl acetate (3 ꢀ 30 ml).
The organic phase was evaporated under reduced pressure
by adding several times of benzene to remove traces of water.
The crude product was washed with a small amount of ethyl
acetate and crystallized from acetonitrile to give the corre-
sponding hydroxamic acid.
A, yield 93%. H NMR (CDCl3, HMDSO) d: 0.98e1.89 (m,
10H), 2.34 (t, J ¼ 7.0 Hz, 2H), 3.47 (q, J ¼ 6.0 Hz, 2H), 3.63
(s, 3H), 6.31 (br s, 1H), 7.40e7.52 (m, 2H), 7.72e8.00 (m,
4H), 8.27 (s, 1H).
4.1.7.2.
N-[8-(Hydroxyamino)-8-oxooctyl]-2-naphthamide
(7). Compound 7 was obtained from methyl 8-[(2-naphthyl-
carbonyl)amino]octanoate by the method B, yield 84%. M.p.
1
138e139 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.14e1.66
(m, 10H), 1.95 (t, J ¼ 7.2 Hz, 2H), 3.22e3.30 (m, 2H),
7.53e7.65 (m, 2H), 7.87e8.08 (m, 4H), 8.43 (s, 1H), 8.55e
8.71 (m, 2H), 10.33 (s, 1H). Anal. Calcd for C19H24N2O3: C
69.49, H 7.37, N 8.53. Found: C 69.20, H 7.40, N 8.52.
4.1.5. Preparation of 5
4.1.8. Preparation of 8
4.1.5.1. Methyl 5-[(2-naphthylcarbonyl)amino]pentanoate
(3a). Compound 3a was obtained from 2-naphthoyl chloride
(2a) and methyl 5-aminopentanoate hydrochloride by the
4.1.8.1. Methyl 6-[([1,10-biphenyl]-4-ylcarbonyl)amino]hexa-
noate (3d). Compound 3d was obtained from [1,10-biphe-
nyl]-4-carbonyl chloride (2b) and methyl 6-aminohexanoate
hydrochloride by the method A, yield 47%. 1H NMR
(CDCl3, HMDSO) d: 1.34e1.52 (m, 2H), 1.55e1.78 (m,
4H), 2.34 (t, J ¼ 7.3 Hz, 2H), 3.49 (q, J ¼ 6.6 Hz, 2H), 3.67
(s, 3H), 6.21 (unresolved t, J w 5.8 Hz, 1H), 7.33e7.52 (m,
3H), 7.56e7.69 (m, 4H), 7.83 (d, J ¼ 8.4 Hz, 2H).
1
method A, yield 83%. H NMR (CDCl3, HMDSO) d: 1.41e
1.89 (m, 4H), 2.23e2.52 (m, 2H), 3.32e3.60 (m, 2H), 3.67
(s, 3H), 6.60 (br s, 1H), 7.36e7.63 (m, 2H), 7.72e8.00 (m,
4H), 8.27 (s, 1H).
4.1.5.2.
N-[5-(Hydroxyamino)-5-oxopentyl]-2-naphthamide
(5). Compound 5 was obtained from methyl 5-[(2-naphthyl-
carbonyl)amino]pentanoate (3a) by the method B, yield
4.1.8.2. N-[6-(Hydroxyamino)-6-oxohexyl][1,10-biphenyl]-4-
carboxamide (8). Compound 8 was obtained from methyl 6-
[([1,10-biphenyl]-4-ylcarbonyl)amino]hexanoate (3d) by the
method B, yield 85% (cryst. from acetonitrile, 38%). M.p.
1
92%. M.p. 155e156 ꢁC. H NMR (DMSO-d6, HMDSO) d:
1.44e1.62 (m, 4H), 1.94e2.06 (m, 2H), 3.20e3.30 (m, 2H),
7.52e7.64 (m, 2H), 7.86e8.08 (m, 4H), 8.43 (s, 1H), 8.58e
8.75 (m, 2H), 10.36 (s, 1H). Anal. Calcd for C16H18N2O3: C
67.12, H 6.34, N 9.78. Found: C 67.00, H 6.33, N 9.83.
1
204e206 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.20e1.1.40
(m, 2H), 1.42e1.62 (m, 4H), 1.89e2.01 (m, 2H), 3.17e3.28
(m, 2H), 7.34e7.55 (m, 3H), 7.67e7.79 (m, 4H), 7.93 (d,
2H, J ¼ 8.2 Hz), 8.50 (t, 1H, J ¼ 5.45 Hz), 8.66 (s, 1H),
10.34 (s, 1H). Anal. Calcd for C19H22N2O3: C 69.92, H
6.79, N 8.58. Found: C 69.89, H 6.89, N 8.30.
4.1.6. Preparation of 6
4.1.6.1. Methyl 7-[(2-naphthylcarbonyl)amino]heptanoate
(3b). Compound 3b was obtained from 2-naphthoyl chloride
(2a) and methyl 7-aminoheptanoate hydrochloride by the
method A, yield 85%. H NMR (CDCl3, HMDSO) d: 1.16e
1.83 (m, 8H), 2.32 (t, J ¼ 7.0 Hz, 2H), 3.49 (q, J ¼ 6.0 Hz,
4.1.9. Preparation of 9
1
4.1.9.1. Methyl 6-[(2-naphthylcarbonyl)amino]hexanoate
(3e). To a solution of methyl 6-aminohexanoate hydrochloride

1076
V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
(0.500 g, 2.75 mmol) and diisopropyl ethylamine (0.96 ml,
5.5 mmol) in dry dimethylformamide (3 ml) 2-naphthoyl chlo-
ride (2a) (0.524 g, 2.75 mmol) in dry dimethylformamide
(3 ml) was added. The mixture was stirred for 3 h at room tem-
perature, then diluted with saturated NaCl (30 ml) and ex-
tracted with ethyl acetate (3 ꢀ 25 ml). The organic phase
was washed with saturated NaCl (2 ꢀ 15 ml) and dried
(Na2SO4). The solvent was evaporated to give crude methyl
6-[(2-naphthylcarbonyl)amino]hexanoate (0.825 g) which
was used in the next step without further purification.
J ¼ 7.0 Hz, 2H), 3.03 (s, 6H), 3.43 (q, J ¼ 6.0 Hz, 2H), 3.65
(s, 3H), 6.00 (br s, 1H), 6.69 (d, J ¼ 9.0 Hz, 2H), 7.79 (d,
J ¼ 9.0 Hz, 2H).
4.1.10.2. 6-{[4-(Dimethylamino)benzoyl]amino}hexanoic acid
(4b). Compound 4b was obtained from methyl 6-{[4-(dime-
thylamino)benzoyl]amino}hexanoate (3f) by a similar proto-
col to 4a, yield 87%. 1H NMR (CDCl3, HMDSO) d:
1.16e1.92 (m, 6H), 2.34 (t, J ¼ 7.0 Hz, 2H), 3.03 (s, 6H),
3.43 (q, J ¼ 6.0 Hz, 2H), 5.97 (br s, 1H), 6.69 (d,
J ¼ 9.0 Hz, 2H), 7.79 (d, J ¼ 9.0 Hz, 2H).
4.1.9.2.
6-[(2-Naphthylcarbonyl)amino]hexanoic
acid
(4a). The crude methyl 6-[(2-naphthylcarbonyl)amino]hexa-
noate (3e) (0.825 g) was dissolved in tetrahydrofuran (5 ml)
and 1 N LiOH (5.5 ml, 5 mmol) was added. The reaction mix-
ture was stirred for 3 h at room temperature, washed with ether
(2 ꢀ 10 ml), and acidified with 2 N HCl up to pH 3. The mix-
ture was extracted with ethyl acetate (3 ꢀ 20 ml), the organic
layer was washed with brine (3 ꢀ 10 ml), and dried (Na2SO4).
The solvent was evaporated and the residue was chromato-
graphed on silica gel with ethyl acetate as eluent to give
pure 6-[(naphthalene-2-carbonyl)amino]hexanoic acid (4a)
(0.618 g, 79%). 1H NMR (CDCl3, HMDSO) d: 1.11e1.77
(6H, m), 2.22e2.39 (2H, m), 3.47 (2H, dd, J ¼ 7.0 Hz), 6.26
(1H, br s), 7.17(1H, s), 7.33e7.57 (2H, m), 7.64e7.93 (4H,
m), 8.22 (1H, s).
4.1.10.3. 4-(Dimethylamino)-N-[6-(hydroxyamino)-6-oxohex-
yl]benzenecarboxamide (10). Compound 10 was obtained
from 6-{[4-(dimethylamino)benzoyl]amino}hexanoic acid
(4b) by a similar protocol to 9, yield 71%. M.p. 137e
138 ꢁC (from acetonitrile). 1H NMR (DMSO-d6, HMDSO)
d: 1.17e1.1.33 (m, 2H), 1.35e1.56 (m, 4H), 1.93 (t, 2H,
J ¼ 7.5 Hz), 2.95 (s, 6H), 3.11e3.25 (m, 2H), 6.68 (d, 2H,
J ¼ 8.8 Hz), 7.69 (d, 2H, J ¼ 8.8 Hz), 8.01 (t, 1H,
J ¼ 5.45 Hz), 8.65 (s, 1H), 10.32 (s, 1H). Anal. Calcd for
C15H23N3: C 61.41, H 7.90, N 14.32. Found: C 61.87, H
8.08, N 14.01.
4.1.11. Preparation of 11
4.1.11.1. 6-(Benzoylamino)hexanoic acid (4c). To a solution
of 6-aminocaproic acid (0.982 g, 7.5 mmol) in water/dioxane
mixture (1:1, 30 ml) NaOH (0.8 g, 20 mmol) in H2O (2 ml)
and benzoyl chloride (2d) (1.16 ml, 10 mmol) successively
were added. The mixture was stirred for 6 h at room tempera-
ture and diluted with brine (150 ml). The mixture was washed
with diethyl ether (2 ꢀ 25 ml), acidified with conc. HCl to pH
4, and extracted with ethyl acetate (4 ꢀ 25 ml). The organic
solution was washed with brine (3 ꢀ 25 ml), dried (Na2SO4)
and evaporated. The residue was purified by column chroma-
tography on silica gel with chloroform/ethyl acetate (1:2) as
eluent to give 6-(benzoylamino)hexanoic acid (4c) (1.589 g,
90%). 1H NMR (CDCl3, HMDSO) d: 1.18e1.85 (m, 6H),
2.34 (t, J ¼ 7.0 Hz, 2H), 3.45 (q, J ¼ 6.0 Hz, 2H), 6.27 (br s,
1H), 7.29e7.52 (m, 3H), 7.61e7.85 (m, 2H), 10.41 (br s, 1H).
4.1.9.3.
N-[6-(Hydroxyamino)-6-oxohexyl]-2-naphthamide
(9). A solution of 6-[(naphthalene-2-carbonyl)amino]hexa-
noic acid (4a) (0.618 g, 2.17 mmol) in dry tetrahydrofuran
(6 ml) was cooled in ice bath under argon atmosphere and
1,10-carbonyldiimidazole (0.422 g, 2.6 mmol) was added.
The mixture was stirred for 30 min and a solution of hydrox-
ylamine (4.35 mmol) in dry dimethylformamide (3 ml) [the
solution of hydroxylamine was made from hydroxylamine hy-
drochloride (0.302 g, 4.35 mmol) and triethylamine (0.61 ml)
in dimethylformamide (3 ml) and filtered] was added. After
stirring overnight the reaction mixture was diluted with satu-
rated NaH2PO4 (30 ml) and extracted with ethyl acetate
(3 ꢀ 30 ml). The organic phase was evaporated under reduced
pressure by adding several times of benzene to remove traces
of water. The crude product was crystallized successively from
ethyl acetate and acetonitrile to give N-[6-(hydroxyamino)-6-
oxohexyl]-2-naphthamide (9) (0.260 g, 40%). M.p. 96e
98 ꢁC. 1H NMR (DMSO-d6, HMDSO) d: 1.21e1.45 (2H,
m), 1.46e1.73 (4H, m), 1.89e2.05 (2H, m), 3.25e3.37 (2H,
m), 7.65e7.81 (2H, m), 7.86e8.04 (4H, m), 8.42 (1H, s),
8.56e8.72 (2H, m), 10.32 (1H, s). Anal. Calcd: C 67.98, H
6.71, N 9.33. Found: C 68.29, H 6.97, N 8.99.
4.1.11.2. N-[6-(Hydroxyamino)-6-oxohexyl]benzenecarboxa-
mide (11). Compound 11 was obtained from 6-(benzoylami-
no)hexanoic acid (4c) by a similar protocol to 9, yield 26%.
1
M.p. 102e104 ꢁC (from acetonitrile). H NMR (DMSO-d6,
HMDSO) d: 1.15e1.1.30 (m, 2H), 1.42e1.50 (m, 4H), 1.87
(t, J ¼ 7.0 Hz, 2H), 3.16 (q, J ¼ 6.6 Hz, 2H), 7.34e7.50 (m,
3H), 7.70e7.80 (m, 2H), 8.37 (t, J ¼ 5.8 Hz, 1H), 8.60 (s,
1H), 10.27 (s, 1H). Anal. Calcd for C13H18N2: C 62.38, H
7.25, N 11.19. Found: C 62.96, H 7.47, N 10.65.
4.1.10. Preparation of 10
4.1.10.1. Methyl 6-{[4-(dimethylamino)benzoyl]amino}hexa-
noate (3f). Compound 3f was obtained from 4-(dimethylami-
no)benzoyl chloride (2c) and methyl 6-aminohexanoate
hydrochloride by the method A, yield 67%. 1H NMR
(CDCl3, HMDSO) d: 1.16e1.92 (m, 6H), 2.34 (t,
4.1.12. Preparation of 16
4.1.12.1. Methyl 6-{[(2E,4E )-5-phenyl-2,4-pentadienoyl]ami-
no}hexanoate
(13a). 1,10-Carbonyldiimidazole
2.2 mmol) was added to a solution of 5-phenyl-penta-2E,
(0.36 g,

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1077
4E-dienoic acid (12a) (0.35 g, 2 mmol) in dry tetrahydrofuran
(10 ml) and the obtained mixture was stirred for 1 h at ambient
temperature. To the mixture triethylamine (0.30 g 3.0 mmol)
and methyl 6-aminohexanoate hydrochloride (0.40 g,
2.2 mmol) were added and the resultant suspension was stirred
for 6 h at ambient temperature. The solvent was removed un-
der reduced pressure. To the residue water (15 ml) was added
and the precipitate was filtered off, washed with water and
dried. Methyl 6-{[(2E,4E )-5-phenyl-2,4-pentadienoyl]ami-
no}hexanoate (13a) (0.36 g, 60%) was obtained as a white
Calcd for C15H22N2O3: C 64.73, H 7.97, N 10.06. Found: C
64.85, H 8.07, N 9.94.
4.1.14. Preparation of 18
4.1.14.1. Methyl 6-[(4-phenylbutanoyl)amino]hexanoate (13c).
Compound 13c from 4-phenylbutyric acid (12c) and methyl
6-aminohexanoate hydrochloride by a similar protocol to
13a was obtained, yield 76%, oil. 1H NMR (CDCl3, HMDSO)
d: 1.11e2.43 (m, 12H), 2.65 (t, J ¼ 7.0 Hz, 2H), 3.23 (q,
J ¼ 5.4 Hz, 2H), 3.64 (s, 3H), 5.56 (br s, 1H), 7.21 (s, 5H).
1
solid. M.p. 125e127 ꢁC. H NMR (DMSO-d6, HMDSO) d:
1.05e1.72 (m, 6H), 2.29 (t, J ¼ 7.3 Hz, 2H), 3.12 (q,
J ¼ 6.0 Hz, 2H), 3.58 (s, 3H), 6.12 (d, J ¼ 14.8 Hz, 1H),
6.91e7.16 (m, 2H), 7.23e7.66 (m, 6H), 8.05 (t, J ¼ 5.8 Hz,
1H).
4.1.14.2. N-Hydroxy-6-[(4-phenylbutanoyl)amino]hexanamide
(18). Compound 18 from methyl 6-[(4-phenylbutanoyl)ami-
no]hexanoate (13c) by a similar protocol to 16 was prepared,
yield 73%. M.p. 92e93 ꢁC (from ethyl acetate). 1H NMR
(DMSO-d6, HMDSO) d: 1.11e2.21 (m, 12H), 2.88e3.16
(m, 2H), 7.23 (br s, 5H), 7.74 (unresolved t, 1H), 8.63 (br s,
1H), 10.32 (br s, 1H). Anal. Calcd for C16H24N2O3: C
65.73, H 8.27, N 9.58. Found: C 65.79, H 8.34, N 9.56.
4.1.12.2. (2E,4E )-N-[6-(Hydroxyamino)-6-oxohexyl]-5-phe-
nyl-2,4-pentadienamide (16). A solution of sodium methylate
(6 mmol) in methanol (5 ml) was added to a solution of hy-
droxylamine hydrochloride (0.28 g, 4 mmol) in methanol
(8 ml). The mixture was stirred for 10 min and the precipitate
was filtered off. Methyl 6-{[(2E,4E )-5-phenyl-2,4-pentadie-
noyl]amino}hexanoate (13a) (0.30 g, 1 mmol) was added to
the filtrate and the mixture was heated to complete dissolv-
ing. The resultant mixture was stirred for 4 h at ambient tem-
perature and the solvent was removed under reduced
pressure. The product was dissolved in water (10 ml) and
acidified with 3% HCl. The precipitate was filtered off and
crystallized from methanol. (2E,4E )-N-[6-(Hydroxyamino)-
6-oxohexyl]-5-phenyl-2,4-pentadienamide (16) (0.23 g, 66%)
4.1.15. Preparation of 19
4.1.15.1. Methyl 6-{[(E )-3-phenyl-2-propenoyl]amino}hexa-
noate (13d). Compound 13d was obtained from (E )-3-phe-
nyl-2-propenoic acid (12d) and methyl 6-aminohexanoate
hydrochloride by a similar protocol to 13a as a white solid,
yield 42%. 1H NMR (CDCl3, HMDSO) d: 1.11e1.86 (m,
6H), 2.32 (t, J ¼ 6.5 Hz, 2H), 3.38 (q, J ¼ 5.8 Hz, 2H), 3.65
(s, 3H), 5.68 (br s, 1H), 6.37 (d, J ¼ 15.6 Hz, 1H), 7.13e
7.61 (m, 5H), 7.59 (d, J ¼ 15.6 Hz, 1H).
1
was obtained as a white solid. M.p. 160e161 ꢁC. H NMR
(DMSO-d6, HMDSO) d: 1.05e1.69 (m, 6H), 1.81e2.07 (m,
2H), 3.12 (q, J ¼ 5.8 Hz, 2H), 6.14 (d, J ¼ 14.8 Hz, 1H),
6.89e7.14 (m, 2H), 7.23e7.65 (m, 6H), 8.05 (t, J ¼ 6.0 Hz,
1H), 8.63 (s, 1H), 10.34 (s, 1H). Anal. Calcd for
C17H22N2O3: C 65.68, H 6.61, N 10.21. Found: C 65.63, H
6.60, N 10.17.
4.1.15.2. (E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-phenyl-2-
propenamide (19). Compound 19 was obtained from methyl
6-{[(E )-3-phenyl-2-propenoyl]amino}hexanoate (13d) by
1
a similar protocol to 16, yield 60%. M.p. 154e155 ꢁC. H
NMR (DMSO-d6, HMDSO) d: 1.05e1.74 (m, 6H), 1.94 (t,
J ¼ 6.4 Hz, 2H), 3.16 (m, 2H), 6.61 (d, J ¼ 15.9 Hz, 1H),
7.16e7.66 (m, 6H), 8.06 (t, J ¼ 5.3 Hz, 1H), 8.63 (s, 1H),
10.32 (s, 1H). Anal. Calcd for C15H22N2O3: C 65.20, H
7.30, N 10.14. Found: C 64.93, H 7.33, N 10.21.
4.1.13. Preparation of 17
4.1.13.1. Methyl 6-[(3-phenylpropanoyl)amino]hexanoate
(13b). Compound 13b from 3-phenylpropionic acid (12b)
and methyl 6-aminohexanoate hydrochloride by a similar pro-
4.1.16. Preparation of 20
1
tocol to 13a was obtained, yield 76%. M.p. 34e35 ꢁC. H
4.1.16.1. Methyl 6-{[(E )-4-phenyl-3-butenoyl]amino}hexa-
noate (13e). Compound 13e from 4-phenyl-but-3E-enoic acid
(12e) and methyl 6-aminohexanoate hydrochloride by a similar
protocol to 13a was obtained, yield 49%. M.p. 49e51 ꢁC. H
NMR (CDCl3, HMDSO) d: 1.12e1.81 (m, 6H), 2.29 (t,
J ¼ 7.0 Hz, 2H), 3.14 (d, J ¼ 6.2 Hz, 2H), 3.26 (q, J ¼ 6.5 Hz,
2H), 3.64 (s, 3H), 5.65 (br s, 1H), 6.27 (dt, J ¼ 6.2, 16.0 Hz,
1H), 6.57 (d, J ¼ 16.0 Hz, 1H), 7.21e7.52 (m, 5H).
NMR (CDCl3, HMDSO) d: 0.99e1.77 (m, 6H), 2.27 (t,
J ¼ 7.2 Hz, 2H), 2.44 (t, J ¼ 7.5 Hz, 2H), 2.95 (t, J ¼ 7.5 Hz,
2H), 3.18 (q, J ¼ 6.0 Hz, 2H), 3.64 (s, 3H), 5.47 (br s, 1H),
7.22 (s, 5H).
1
4.1.13.2. N-Hydroxy-6-[(3-phenylpropanoyl)amino]hexana-
mide (17). Compound 17 from methyl 6-[(3-phenylpropanoy-
l)amino]hexanoate (13b) by a similar protocol to 16 was
prepared, yield 95%. M.p. 107 ꢁC. 1H NMR (DMSO-d6,
HMDSO) d: 1.08e1.64 (m, 6H), 1.91 (t, J ¼ 6.6 Hz, 2H),
2.33 (m, 2H), 2.66e3.12 (m, 4H), 7.08e7.36 (m, 5H), 7.73
(unresolved t, 2H), 8.61 (br s, 1H), 10.29 (br s, 1H). Anal.
4.1.16.2. (E )-N-[6-(Hydroxyamino)-6-oxohexyl]-4-phenyl-3-
butenamide (20). Compound 20 from methyl 6-{[(E )-4-phe-
nyl-3-butenoyl]amino}hexanoate (13e) by a similar protocol
to 16 was prepared, yield 52%. M.p. 126e128 ꢁC. H NMR
1

1078
V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
(DMSO-d6, HMDSO) d: 1.07e1.68 (m, 6H), 1.92 (t,
J ¼ 7.0 Hz, 2H), 3.03 (d, J ¼ 5.6 Hz, 2H), 3.05 (q, J ¼ 6.2 Hz,
2H), 6.29 (dt, J ¼ 5.8, 16.0 Hz, 1H), 6.48 (d, J ¼ 16.0 Hz,
1H), 7.14e7.53 (m, 5H), 7.85 (unresolved t, 1H), 8.63 (br s,
1H), 10.32 (br s, 1H). Anal. Calcd for C16H22N2O3: C 66.19,
H 7.64, N 9.65. Found: C 66.18, H 7.74, N 9.56.
(4-pyridinyl)-2-propenoic acid (12h) and methyl 6-aminohex-
anoate hydrochloride by a similar protocol to 13a, yield 34%.
M.p. 92e94 ꢁC. 1H NMR (DMSO-d6, HMDSO) d: 1.09e1.76
(m, 6H), 2.28 (t, J ¼ 7.5 Hz, 2H), 3.16 (q, J ¼ 6.0 Hz, 2H),
3.57 (s, 3H), 6.81 (d, J ¼ 16.0 Hz, 1H), 7.41 (d, J ¼ 16.0 Hz,
1H), 7.52 (d, J ¼ 6.0 Hz, 2H), 8.23 (unresolved t, 1H), 8.61
(d, J ¼ 6.0 Hz, 2H).
4.1.17. Preparation of 21
4.1.19.2.
(E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-(4-pyri-
4.1.17.1. Methyl 6-{[(2E,4E)-3-methyl-5-phenyl-2,4-pentadie-
noyl]amino}hexanoate (13f). Compound 13f was obtained
from (2E,4E )-3-methyl-5-phenyl-2,4-pentadienoic acid [28]
(12f) and methyl 6-aminohexanoate hydrochloride by a similar
protocol to 13a, yield 90%. M.p. 83e85 ꢁC. 1H NMR
(DMSO-d6, HMDSO) d: 1.01e1.78 (m, 6H), 2.28 (t,
J ¼ 7.0 Hz, 2H), 2.29 (s, 3H), 3.09 (q, J ¼ 6.0 Hz, 2H), 3.55
(s, 3H), 5.94 (s, 1H), 6.88 (s, 2H), 7.21e7.67 (m, 5H), 7.96
(unresolved t, 1H).
dinyl)-2-propenamide (23). Compound 23 was obtained
from methyl 6-{[(E )-3-(4-pyridinyl)-2-propenoyl]amino}hex-
anoate (13h) by a similar protocol to 16 and dissolved in eth-
anol. To the obtained solution an ethanol solution of oxalic
acid (2 equiv) was added. The precipitate was filtered and
crystallized from methanol to give (E )-N-[6-(hydroxya-
mino)-6-oxohexyl]-3-(4-pyridinyl)-2-propenamide
oxalate
(23), yield 50%. M.p. 168e170 ꢁC. 1H NMR (DMSO-d6,
HMDSO) d: 1.09e1.69 (m, 6H), 1.94 (t, J ¼ 7.5 Hz, 2H),
3.16 (q, J ¼ 6.0 Hz, 2H), 6.81 (d, J ¼ 16.0 Hz, 1H), 7.38 (d,
J ¼ 16.0 Hz, 1H), 7.51 (d, J ¼ 6.0 Hz, 2H), 8.19 (unresolved
t, 1H), 8.57 (d, J ¼ 6.0 Hz, 2H), 10.21 (s, 1H). Anal. Calcd
for C14H19N3O3/(COOH)2/0.25H2O: C 51.68, H 5.83, N
11.30. Found: C 51.44, H 5.62, N 11.23.
4.1.17.2.
(2E,4E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-
methyl-5-phenyl-2,4-pentadienamide (21). Compound 21 was
prepared from methyl 6-{[(2E,4E )-3-methyl-5-phenyl-2,4-
pentadienoyl]amino}hexanoate (13f) by a similar protocol to
16, yield 60%. M.p. 147e149 ꢁC. 1H NMR (DMSO-d6,
HMDSO) d: 1.01e1.74 (m, 6H), 1.94 (t, J ¼ 6.0 Hz, 2H),
2.29 (s, 3H), 3.09 (q, J ¼ 6.0 Hz, 2H), 5.94 (s, 1H), 6.89 (s,
2H), 7.14e7.69 (m, 5H), 7.96 (unresolved t, 1H), 8.67 (s,
1H), 10.29 (s, 1H). Anal. Calcd for C18H24N2O3: C 68.33, H
7.65, N 8.85. Found: C 68.32, H 7.58, N 8.89.
4.1.20. Preparation of 24
4.1.20.1. Methyl 6-{[(E )-3-(2-pyridinyl)-2-propenoyl]amino}-
hexanoate (13i). Compound 13i was obtained from (E )-3-
(2-pyridinyl)-2-propenoic acid (12i) and methyl 6-aminohexa-
noate hydrochloride by a similar protocol to 13a as an oil,
1
4.1.18. Preparation of 22
yield 45%. H NMR (DMSO-d6, HMDSO) d:1.02e1.72 (m,
6H), 2.28 (t, J ¼ 7.0 Hz, 2H), 3.16 (q, J ¼ 6.0 Hz, 2H), 3.56
(s, 3H), 7.09 (d, J ¼ 15.6 Hz, 1H), 7.23e7.42 (m, 1H), 7.42
(d, J ¼ 15.6 Hz, 1H), 7.49 (d, J ¼ 7.5 Hz, 1H), 7.76e7.99
(m, 1H), 8.23 (t, J ¼ 5.6 Hz, 1H), 8.59 (dd, J ¼ 2.0, 4.8 Hz,
1H).
4.1.18.1. Methyl 4-[(2E,4E )-4-methyl-5-phenyl-2,4-pentadie-
noyl]aminobutanoate (13g). Compound 13g was obtained
from 4-methyl-5-phenylpenta-2,4-dienoic acid [29] (12g) and
methyl 4-aminobutyrate hydrochloride by a similar protocol
1
to 13a as white crystals, yield 53%. M.p. 111e113 ꢁC. H
NMR (DMSO-d6, HMDSO) d: 1.41e1.88 (m, 2H), 2.00 (s,
3H), 2.30 (t, J ¼ 7.5 Hz, 2H), 3.12 (q, J ¼ 6.4 Hz, 2H), 3.59
(s, 3H), 6.10 (d, J ¼ 15.0 Hz, 1H), 6.89 (s, 1H), 7.30 (d,
J ¼ 15.0 Hz, 1H), 7.48 (s, 5H), 8.10 (unresolved t, 1H).
4.1.20.2.
dinyl)-2-propenamide oxalate (24). Compound 24 was ob-
tained from methyl 6-{[(E )-3-(2-pyridinyl)-2-
(E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-(2-pyri-
propenoyl]amino}hexanoate (13i) by similar protocols to 16
1
and 23, yield 46%. M.p. 126e128 ꢁC. H NMR (DMSO-d6,
4.1.18.2. (2E,4E )-N-[4-(Hydroxyamino)-4-oxobutyl]-4-methyl-
5-phenyl-2,4-pentadienamide (22). Compound 22 from
methyl 4-[(2E,4E )-4-methyl-5-phenyl-2,4-pentadienoyl]ami-
nobutanoate (13g) by a similar protocol to 16 was prepared,
yield 70%. M.p. 127e129 ꢁC. 1H NMR (DMSO-d6, HMDSO)
d: 1.42e1.85 (m, 2H), 1.87 (t, 2H), 1.98 (s, 3H), 3.16 (q,
J ¼ 6.2 Hz, 2H), 6.14 (d, J ¼ 14.8 Hz, 1H), 6.87 (s, 1H),
7.25 (d, J ¼ 14.8 Hz, 1H), 7.38 (s, 5H), 8.07 (unresolved t,
1H), 9.56 (br s, 2H). Anal. Calcd for C16H20N2O3: C 66.65,
H 6.99, N 9.72. Found: C 66.55, H 6.98, N 9.66.
HMDSO) d: 1.15e1.65 (m, 6H), 1.94 (t, J ¼ 7.2 Hz, 2H),
3.16 (q, J ¼ 6.0 Hz, 2H), 7.08 (d, J ¼ 15.4 Hz, 1H), 7.32 (dd,
J ¼ 4.8, 6.8 Hz, 1H), 7.42 (d, J ¼ 15.4 Hz, 1H), 7.56 (d,
J ¼ 7.8 Hz, 1H), 7.83 (dd, J ¼ 1.8, 7.7 Hz, 1H), 8.28 (t,
J ¼ 5.5 Hz, 1H), 8.60 (d, J ¼ 4.8 Hz, 1H), 10.35 (br s, 1H).
Anal. Calcd for C14H19N3O3/(COOH)2/0.5H2O: C 51.06,
H 5.89, N 11.16. Found: C 50.95, H 5.76, N 11.34.
4.1.21. Preparation of 25
4.1.21.1. Methyl 6-{[(E )-3-(3-pyridinyl)-2-propenoyl]amino}-
hexanoate (13j). Compound 13j from (E )-3-(3-pyridinyl)-2-
propenoic acid (12j) and methyl 6-aminohexanoate hydrochlo-
ride by a similar protocol to 13a was obtained, yield
52%. M.p. 75e77 ꢁC. 1H NMR (DMSO-d6, HMDSO) d:
4.1.19. Preparation of 23
4.1.19.1. Methyl 6-{[(E )-3-(4-pyridinyl)-2-propenoyl]amino}-
hexanoate (13h). Compound 13h was obtained from (E )-3-

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1079
1
1.01e1.78 (m, 6H), 2.25 (t, J ¼ 7.2 Hz, 2H), 3.16 (q,
J ¼ 6.0 Hz, 2H), 3.57 (s, 3H), 6.75 (d, J ¼ 16.0 Hz, 1H),
7.46 (d, J ¼ 16.0 Hz, 1H), 7.46 (dd, J ¼ 4.8, 8.0 Hz, 1H),
7.98 (dt, J ¼ 1.8, 8.0 Hz, 1H), 8.14 (t, J ¼ 5.3 Hz, 1H), 8.56
(dd, J ¼ 1.8, 4.8 Hz, 1H), 8.76 (d, J ¼ 1.8 Hz, 1H).
61%. H NMR (DMSO-d6, HMDSO) d: 1.12e1.67 (m, 6H),
2.31 (t, J ¼ 7.3 Hz, 2H), 3.19 (q, J ¼ 6.4 Hz, 2H), 3.58 (s,
3H), 6.83 (d, J ¼ 15.8 Hz, 1H), 7.54 (dd, J ¼ 4.2, 8.3 Hz,
1H), 7.62 (d, J ¼ 15.8 Hz, 1H), 7.81 (dd, J ¼ 1.4, 8.6 Hz,
1H), 8.00 (d, J ¼ 8.6 Hz, 1H), 8.15 (s, 1H), 8.19 (t,
J ¼ 5.8 Hz, 1H), 8.36 (dd, J ¼ 1.5, 8.3 Hz, 1H), 8.92 (dd,
J ¼ 1.5, 4.2 Hz, 1H).
4.1.21.2.
(E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-(3-pyri-
dinyl)-2-propenamide oxalate (25). A solution of sodium
methylate (6 mmol) in methanol (5 ml) was added to a solution
of hydroxylamine hydrochloride (0.28 g, 4 mmol) in methanol
(8 ml). The mixture was stirred for 10 min and NaCl was fil-
tered off. Methyl 6-{[(E )-3-(3-pyridinyl)-2-propenoyl]ami-
no}hexanoate (13j) (0.28 g, 1 mmol) was added to the
filtrate and the resultant mixture was stirred for 4 h at ambient
temperature. The solvent was removed under reduced pres-
sure, the product was dissolved in ethanol (10 ml) and then ox-
alic acid (0.36 g, 4 mmol) was added to the solution. The
precipitate was filtered and crystallized from water. (E )-N-
[6-(hydroxyamino)-6-oxohexyl]-3-(3-pyridinyl)-2-propena-
mide oxalate (25) (0.22 g, 68%) was obtained as a white solid.
4.1.23.2. (E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-(7-quino-
linyl)-2-propenamide (27). Compound 27 from methyl 6-
{[(E )-3-(7-quinolinyl)-2-propenoyl]amino}hexanoate
by a similar protocol to 16 was prepared, yield 58%. M.p.
(13l)
1
163e165 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.19e1.38
(m, 2H), 1.38e1.61 (m, 4H), 1.95 (t, J ¼ 7.2 Hz, 2H), 3.18
(q, J ¼ 6.6 Hz, 2H), 6.83 (d, J ¼ 15.8 Hz, 1H), 7.54 (dd,
J ¼ 4.3, 8.3 Hz, 1H), 7.62 (d, J ¼ 15.8 Hz, 1H), 7.81 (dd,
J ¼ 1.4, 8.5 Hz, 1H), 8.01 (d, J ¼ 8.5 Hz, 1H), 8.14 (s, 1H),
8.18 (t, J ¼ 5.8 Hz, 1H), 8.36 (dd, J ¼ 1.6, 8.3 Hz, 1H), 8.68
(br s, 1H), 8.92 (dd, J ¼ 1.6, 4.3 Hz, 1H), 10.35 (br s, 1H).
Anal. Calcd for C18H21N3O3$H2O: C 62.59, H 6.71, N
12.17. Found: C 62.44, H 6.69, N 11.83.
1
M.p. 157e159 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.03e
1.72 (m, 6H), 1.96 (t, J ¼ 7.2 Hz, 2H), 3.18 (q, J ¼ 6.0 Hz,
2H), 6.74 (d, J ¼ 15.8 Hz, 1H), 7.44 (d, J ¼ 15.8 Hz, 1H),
7.44 (dd, J ¼ 5.0, 8.0 Hz, 1H), 7.99 (dt, J ¼ 1.8, 8.0 Hz, 1H),
8.18 (t, J ¼ 5.2 Hz, 1H), 8.56 (dd, J ¼ 1.6, 5.0 Hz, 1H), 8.74
(d, J ¼ 2.0 Hz, 1H), 10.34 (br s, 1H). Anal. Calcd for
C14H19N3O3/0.5(COOH)2/2H2O: C 50.27, H 6.75, N
11.73. Found: C 50.28, H 6.71, N 11.60.
4.1.24. Preparation of 28
4.1.24.1. Methyl 6-{[(E )-3-(8-quinolinyl)-2-propenoyl]amino}-
hexanoate (13m). Compound 13m from (E )-3-(8-quino-
linyl)-2-propenoic acid [31] (12m) and methyl 6-
aminohexanoate hydrochloride by a similar protocol to 13a
1
was obtained, yield 46%. H NMR (DMSO-d6, HMDSO) d:
4.1.22. Preparation of 26
1.10e1.68 (m, 6H), 2.24 (t, J ¼ 7.1 Hz, 2H), 2.97 (q,
J ¼ 6.0 Hz, 2H), 3.61 (s, 3H), 6.97 (d, J ¼ 15.8 Hz, 1H),
7.53e7.78 (m, 2H), 7.95e8.13 (m, 2H), 8.26 (t, J ¼ 5.2 Hz,
1H), 8.44 (dd, J ¼ 1.6, 8.0 Hz, 1H), 8.60 (d, J ¼ 15.8 Hz,
1H), 9.00e9.17 (m, 1H).
4.1.22.1. Methyl 6-{[(E )-3-(2-naphthyl)-2-propenoyl]amino}-
hexanoate (13k). Compound 13k from (E )-3-(2-naphthyl)-2-
propenoic acid (12k) and methyl 6-aminohexanoate hydro-
chloride by a similar protocol to 13a was obtained, yield
94%. M.p. 74e76 ꢁC. 1H NMR (DMSO-d6, HMDSO) d:
1.10e1.78 (m, 6H), 2.25 (t, J ¼ 6.5 Hz, 2H), 3.21 (q,
J ¼ 5.6 Hz, 2H), 3.58 (s, 3H), 6.78 (d, J ¼ 15.5 Hz, 1H),
7.46e8.23 (m, 9H).
4.1.24.2. (E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-(8-quino-
linyl)-2-propenamide (28). Compound 28 from methyl 6-
{[(E )-3-(8-quinolinyl)-2-propenoyl]amino}hexanoate (12m)
by a similar protocol to 16 was prepared, yield 52%. M.p.
1
120e122 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.19e1.39
4.1.22.2.
(E )-N-[6-(Hydroxyamino)-6-oxohexyl]-3-(2-naph-
(m, 2H), 1.39e1.66 (m, 4H), 1.97 (t, J ¼ 7.2 Hz, 2H), 3.20
(q, J ¼ 6.6 Hz, 2H), 6.93 (d, J ¼ 16.0 Hz, 1H), 7.62 (dd,
J ¼ 4.2, 8.3 Hz, 1H), 7.67 (t, J ¼ 8.0 Hz, 1H), 8.02 (d,
J w 8.0 Hz, 1H), 8.05 (d, J w 8.0 Hz, 1H), 8.21 (t,
J ¼ 5.6 Hz, 1H), 8.42 (dd, J ¼ 1.6, 8.3 Hz, 1H), 8.63 (d,
J ¼ 16.0 Hz, 1H), 8.70 (br s, 1H), 9.00 (dd, J ¼ 1.6,
4.2 Hz, 1H), 10.37 (br s, 1H). Anal. Calcd for
C18H21N3O3/H2O: C 62.59, H 6.71, N 12.17. Found: C
62.36, H 6.52, N 11.97.
thyl)-2-propenamide (26). Compound 26 from methyl 6-
{[(E )-3-(2-naphthyl)-2-propenoyl]amino}hexanoate (13k) by
a similar protocol to 16 was prepared, yield 74%. M.p.
161e163 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.07e1.74
(6H, m, CH2), 1.81e2.14 (unresolved t, 2H), 3.03e3.41 (m,
2H), 6.74 (d, J ¼ 16.0 Hz, 1H), 7.43e8.21 (m, 9H), 8.63 (br
s, 1H), 10.32 (br s, 1H). Anal. Calcd for C19H22N2O5/
H2O: C 66.26, H 7.02, N 8.13. Found: C 66.51, H 7.11, N
8.01.
1
4.1.25. Preparation of 29
4.1.23. Preparation of 27
4.1.25.1. Methyl 6-[(6-quinolinylcarbonyl)amino]hexanoate
(13n). Compound 13n from quinoline-6-carboxylic acid
(12n) and methyl 6-aminohexanoate hydrochloride by a similar
protocol to 13a was obtained, yield 48%. 1H NMR (DMSO-d6,
HMDSO) d: 1.15e1.70 (m, 6H), 2.21 (t, J ¼ 7.1 Hz, 2H), 2.88
4.1.23.1. Methyl 6-{[(E )-3-(7-quinolinyl)-2-propenoyl]amino}-
hexanoate (13l). Compound 13l from (E )-3-(7-quinolinyl)-2-
propenoic acid [30] (12l) and methyl 6-aminohexanoate
hydrochloride by a similar protocol to 13a was obtained, yield

1080
V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
(q, J ¼ 6.0 Hz, 2H), 3.64 (s, 2H), 7.65 (dd, J ¼ 4.0, 8.0 Hz,
C9HN), 8.10 (d, J ¼ 8.7 Hz, 1H), 8.10e8.21 (m, 1H), 8.42e
8.61 (m, 2H), 8.51 (t, J ¼ 5.1 Hz, 1H), 9.11 (dd, J ¼ 1.6,
4.0 Hz, 1H).
HMDSO) d: 1.00e1.19 (m, 2H), 1.19e1.33 (m, 2H), 1.42
(qui, J ¼ 7.0 Hz, 2H), 1.90 (t, J ¼ 7.4 Hz, 2H), 2.96 (q,
J ¼ 6.0 Hz, 2H), 3.32e3.37 (m, 4H), 6.21 (s, 1H), 7.05e7.15
(m, 3H), 7.15e7.25 (m, 4H), 7.15e7.33 (m, 1H), 7.80 (t,
J ¼ 5.6 Hz, 1H), 8.66 (br s, 1H), 10.33 (br s, 1H). Anal. Calcd
for C23H26N2O3: C 72.99, H 6.92, N 7.40. Found: C 72.71, H
6.93, N 7.46.
4.1.25.2. N-[6-(Hydroxyamino)-6-oxohexyl]-6-quinolinecar-
boxamide (29). Compound 29 from methyl 6-[(6-quinolinyl-
carbonyl)amino]hexanoate (13n) by a similar protocol to 16
was prepared, yield 52%. M.p. 180e182 ꢁC. 1H NMR
(DMSO-d6, HMDSO) d: 1.21e1.44 (m, 2H), 1.44e1.71 (m,
4H), 1.97 (t, J ¼ 7.3 Hz, 2H), 3.31 (q, J ¼ 6.4 Hz, 2H), 7.61
(dd, J ¼ 4.2, 8.3 Hz, 1H), 8.08 (d, J ¼ 8.8 Hz, 1H), 8.17 (dd,
J ¼ 1.8, 8.8 Hz, 1H), 8.47 (dd, J ¼ 1.6, 8.3 Hz, 1H), 8.49 (d,
J ¼ 1.8 Hz, 1H), 8.70 (br s, 1H), 8.71 (t, J ¼ 5.6 Hz, 1H),
8.98 (dd, J ¼ 1.6, 4.2 Hz, 1H), 10.36 (br s, 1H). Anal. Calcd
for C16H19N3O3/H2O: C 60.18, H 6.63, N 13.16. Found: C
59.84, H 6.50, N 13.01.
4.1.28. Preparation of 32
4.1.28.1. Methyl 6-{[2-(9H-fluoren-9-yliden)acetyl]amino}hex-
anoate (13r). Compound 13r from fluoren-9-ylidene-acetic
acid (12r) and methyl 6-aminohexanoate hydrochloride by
a similar protocol to 13a was obtained, yield 73%. M.p.
1
54e56 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.05e1.76 (m,
6H), 2.28 (t, J ¼ 7.3 Hz, 2H), 3.27 (q, J ¼ 6.1 Hz, 2H), 3.59
(s, 3H), 7.11 (s, 1H), 7.22e7.59 (m, 4H), 7.64e7.95 (m,
3H), 8.51 (t, J ¼ 5.2 Hz, 1H), 8.62e8.84 (m, 1H).
4.1.26. Preparation of 30
4.1.28.2. 6-{[2-(9H-Fluoren-9-yliden)acetyl]amino}-N-hydrox-
yhexanamide (32). Compound 32 from methyl 6-{[2-(9H-flu-
oren-9-yliden)acetyl]amino}hexanoate (13r) and methyl 6-
aminohexanoate hydrochloride by a similar protocol to 16
was obtained, yield 84%. M.p. 174e176 ꢁC. 1H NMR
(DMSO-d6, HMDSO) d: 1.20e1.41 (m, 2H), 1.41e1.63 (m,
4H), 1.96 (t, J ¼ 7.2 Hz, 2H), 3.24 (q, J ¼ 6.1 Hz, 2H), 7.09
(s, 1H), 7.25e7.49 (m, 4H), 7.73e7.88 (m, 3H), 8.53 (t,
J ¼ 5.5 Hz, 1H), 8.65e8.74 (m, 1H), 8.68 (s, 1H), 10.36 (s,
1H). Anal. Calcd for C21H22N2O3: C 71.98, H 6.33, N 7.99.
Found: C 71.91, H 6.37, N 8.03.
4.1.26.1. Methyl 6-[(3,3-diphenylacryloyl)amino]hexanoate
(13o). Compound 13o from 3,3-diphenyl-acrylic acid (12o)
and methyl 6-aminohexanoate hydrochloride by a similar pro-
1
tocol to 13a was obtained, yield 82%, as an oil. H NMR
(DMSO-d6, HMDSO) d: 0.91e1.70 (m, 6H), 2.30 (t,
J ¼ 6.9 Hz, 2H), 3.01 (q, J ¼ 5.6 Hz, 2H), 3.59 (s, 3H), 6.45
(s, 1H), 7.03e7.50 (m, 10H), 7.80 (t, J ¼ 5.0 Hz, 1H).
4.1.26.2. N-[6-(Hydroxyamino)-6-oxohexyl]-3,3-diphenylacry-
lamide (30). Compound 30 from methyl 6-[(3,3-diphenylacry-
loyl)amino]hexanoate (13o) and methyl 6-aminohexanoate
hydrochloride by a similar protocol to 16 was obtained, yield
1
4.1.29. General synthesis of amidomethyl esters 13seu
from carboxylic acids 12seu (method C)
63%. M.p. 123e125 ꢁC. H NMR (DMSO-d6, HMDSO) d:
0.90e1.63 (m, 6H), 2.01 (t, J ¼ 7.0 Hz, 2H), 2.97 (q,
J ¼ 5.5 Hz, 2H), 6.43 (s, 1H), 7.01e7.47 (m, 10H), 7.78 (t,
J ¼ 5.0 Hz, 1H), 8.63 (br s, 1H), 10.32 (br s, 1H). Anal. Calcd
for C21H24N2O3: C 71.57, H 6.86, N 7.95. Found: C 71.56, H
6.87, N 7.98.
A solution of carboxylic acids 12seu (2.75 mmol) in dry
dimethylformamide (3 ml) under argon atmosphere was
cooled in ice bath and 1,10-carbonyldiimidazole (490 mg,
3.01 mmol) was added. The mixture was stirred for 30 min
and then triethylamine (1.0 ml, 7.2 mmol) followed by a solu-
tion of methyl 6-aminohexanoate hydrochloride (2.75 mmol)
in dry dimethylformamide (3 ml) were added. The reaction
mixture was stirred at ice bath temperature for 1 h and 20 h
at room temperature, diluted with 50 ml of brine and extracted
with ethyl acetate (3 ꢀ 25 ml). The organic phase was washed
with brine, 5% NaHCO3, brine, saturated KH2PO4, and brine.
The organic layer was dried (Na2SO4) and the solvent was
evaporated. The residue was purified on silica gel (20 g)
with chloroform/ethyl acetate as eluent affording the corre-
sponding amidoester derivatives 13seu.
4.1.27. Preparation of 31
4.1.27.1. Methyl 6-{[2-(10,11-dihydro-5H-dibenzo[a,d]cyclo-
hepten-5-yliden)acetyl]amino}hexanoate
(13p). Compound
13p from 2-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-
yliden)acetic acid (12p) and methyl 6-aminohexanoate hydro-
chloride by a similar protocol to 13a was obtained, yield 75%.
1H NMR (DMSO-d6, HMDSO) d: 0.89e1.65 (m, 6H), 2.21 (t,
J ¼ 7.1 Hz, 2H), 2.99 (q, J ¼ 6.0 Hz, CH2), 3.05 (s, 4H), 3.59
(s, 3H), 6.18 (s, 1H), 6.89e7.34 (m, 8H), 7.34 (t, J ¼ 5.2 Hz,
1H).
4.1.30. Preparation of 33
4.1.27.2. 6-{[2-(10,11-Dihydro-5H-dibenzo[a,d]cyclohepten-
5-yliden)acetyl]amino}-N-hydroxyhexanamide (31). Compound
31 from methyl 6-{[2-(10,11-dihydro-5H-dibenzo[a,d]cyclo-
hepten-5-yliden)acetyl]amino}hexanoate (13p) and methyl 6-
aminohexanoate hydrochloride by a similar protocol to 16
was obtained, yield 64%. M.p. 141e143 ꢁC. (DMSO-d6,
4.1.30.1. Methyl 6-[(5-phenylpentanoyl)amino]hexanoate
(13s). Compound 13s from 5-phenylpentanoic acid (12s)
and methyl 6-aminohexanoate hydrochloride by the method
C was obtained, yield 35%. 1H NMR (CDCl3, HMDSO)
d: 1.22e1.81 (m, 10H), 2.02e2.41 (m, 4H), 2.49e2.75

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1081
(m, 2H), 3.22 (q, J ¼ 6.0 Hz, 2H), 3.65 (s, 3H), 5.52 (br s, 1H),
7.05e7.38 (m, 5H).
atmosphere triethylamine (0.36 ml, 2.6 mmol) was added
and the mixture was cooled in an ice bath. Then to the reaction
mixture iso-butylchloroformate (0.3 ml, 2.3 mmol) was added
and the resulting mixture was stirred for 20 min at ice bath
temperature. At the same time, a suspension of methyl 6-ami-
nohexanoate hydrochloride (364 mg, 2 mmol), anhydrous tet-
rahydrofuran (3 ml) and triethylamine (0.31 ml, 2.2 mmol)
was prepared and stirred for 20 min at room temperature.
This suspension to the above prepared reaction mixture of ac-
tivated ester was added. The resulting mixture was stirred at
ice bath temperature for 15 min and 1 h at room temperature,
then diluted with brine (50 ml) and extracted with ethyl ace-
tate (3 ꢀ 25 ml). The organic phase was washed with brine,
5% NaHCO3, brine, saturated KH2PO4 and brine. The organic
layer was dried (Na2SO4) and the solvent was evaporated. The
residue was purified on silica gel (20 g) with chloroform/ethyl
acetate as eluent affording title product 13v or 13w,
accordingly.
4.1.30.2.
N-Hydroxy-6-[(5-phenylpentanoyl)amino]hexana-
mide (33). Compound 33 was obtained from methyl 6-[(5-
phenylpentanoyl)amino]hexanoate (13s) by the method B,
1
yield 52%. M.p. 97e98 ꢁC. H NMR (DMSO-d6, HMDSO)
d: 1.09e1.61 (m, 10H), 1.91 (t, J ¼ 7.3 Hz, 2H), 2.06 (t,
J ¼ 6.7 Hz, 2H), 2.56 (t, J ¼ 7.2 Hz, 2H, overlapped with a sig-
nal of DMSO), 2.99 (q, J ¼ 6.3 Hz, 2H), 7.11e7.34 (m, 5H),
7.75 (t, J ¼ 5.4 Hz, 1H), 8.67 (s, 1H), 10.33 (s, 1H). Anal.
Calcd for C17H26N: C 66.64, H 8.55, N 9.14. Found: C
66.63, H 8.65, N 9.14.
4.1.31. Preparation of 34
4.1.31.1. Methyl 6-[(E )-5-phenyl-4-pentenoyl]aminohexanoate
(13t). Compound 13t from 5-phenyl-pent-4E-enoic acid [32]
(12t) and methyl 6-aminohexanoate hydrochloride by the
method C was obtained, yield 82%. 1H NMR (CDCl3,
HMDSO) d: 1.10e1.78 (m, 6H), 2.07e2.69 (m, 6H), 3.25
(q, J ¼ 6.0 Hz, 2H), 3.65 (s, 3H), 5.53 (br s, 1H), 6.20 (dt,
J ¼ 6.0, 16.0 Hz, 1H), 6.49 (d, J ¼ 16.0 Hz, 1H), 7.07e7.45
(m, 5H).
4.1.34. Preparation of 36
4.1.34.1. Methyl 6-({2-[4-(dimethylamino)phenyl]acetyl}ami-
no)hexanoate (13v). Compound 13v from (4-dimethylamino-
phenyl)-acetic acid (12v) and methyl 6-aminohexanoate hy-
1
drochloride by the method D was prepared, yield 73%. H
4.1.31.2. (E )-N-[6-(Hydroxyamino)-6-oxohexyl]-5-phenyl-4-
pentenamide (34). Compound 34 was obtained from methyl
6-[(E )-5-phenyl-4-pentenoyl]aminohexanoate (13t) by the
NMR (CDCl3, HMDSO) d: 1.16e1.92 (m, 6H), 2.34 (t,
J ¼ 7.0 Hz, 2H), 3.03 (s, 6H), 3.22 (s, 2H), 3.43 (q,
J ¼ 6.0 Hz, 2H), 3.65 (s, 3H), 6.00 (br s, 1H), 6.69
(d, J ¼ 9.0 Hz, 2H), 7.79 (d, J ¼ 9.0 Hz, 2H).
1
method B, yield 10%. M.p. 131e133 ꢁC. H NMR (DMSO-
d6, HMDSO) d: 1.12e1.54 (m, 8H), 1.90 (t, J ¼ 6.8 Hz,
2H), 2.10e2.40 (m, 2H), 3.00 (q, J ¼ 6.0 Hz, 2H), 6.25e
6.50 (m, 2H), 7.18e7.42 (m, 5H), 7.81 (t, J ¼ 5.2 Hz, 1H),
8.65 (s, 1H), 10.32 (s, 1H). Anal. Calcd for C17H24N: C
67.08, H 7.95, N 9.20. Found: C 66.67, H 7.94, N 9.17.
4.1.34.2. 6-({2-[4-(Dimethylamino)phenyl]acetyl}amino)-N-
hydroxyhexanamide (36). Compound 36 was obtained from
methyl 6-({2-[4-(dimethylamino)phenyl]acetyl}amino)hexa-
noate (13v) by the method B, yield 39%. M.p. 124e126 ꢁC.
1H NMR (DMSO-d6, HMDSO) d: 1.11e1.56 (m, 6H), 1.91
(t, J ¼ 7.4 Hz, 2H), 2.84 (s, 6H), 2.98 (q, J ¼ 6.4 Hz, 2H),
3.22 (s, 2H), 6.64 (d, J ¼ 8.6 Hz, 2H), 7.04 (d, J ¼ 8.6 Hz,
2H), 7.85 (t, J ¼ 5.4 Hz, 1H), 8.66 (s, 1H), 10.33 (s, 1H).
Anal. Calcd for C16H25N3O3: C 62.52, H 8.20, N 13.67.
Found: C 62.32, H 8.21, N 13.68.
4.1.32. Preparation of 35
4.1.32.1. Methyl 6-[(3-phenyl-2-propynoyl)amino]hexanoate
(13u). Compound 13u was obtained from phenylpropynoic
acid (12u) and methyl 6-aminohexanoate hydrochloride by
the method C, yield 89%. 1H NMR (CDCl3, HMDSO) d:
1.25e1.92 (m, 6H), 2.34 (t, J ¼ 7.0 Hz, 2H), 3.34 (q,
J ¼ 6.0 Hz, 2H), 3.65 (s, 3H), 7.27e7.63 (m, 5H).
4.1.35. Preparation of 37
4.1.35.1. Methyl 6-{[(E )-3-(4-chloro-2-fluorophenyl)-2-prope-
noyl]amino}hexanoate (13w). Compound 13w from 3-(4-
chloro-2-fluorophenyl)acrylic acid (12w) and methyl 6-amino-
hexanoate hydrochloride by the method D was prepared, yield
76%. 1H NMR (CDCl3, HMDSO) d: 1.16e1.85 (m, 6H), 2.32
(t, J ¼ 7.0 Hz, 2H), 3.40 (q, J ¼ 6.0 Hz, 2H), 3.65 (s, 3H), 5.80
(br s, 1H), 6.52 (d, J ¼ 16.0 Hz, 1H), 7.00e7.54 (m, 3H), 7.56
(d, J ¼ 7.0 Hz, 1H).
4.1.32.2. N-[6-(Hydroxyamino)-6-oxohexyl]-3-phenyl-2-propy-
namide (35). Compound 35 was obtained from methyl 6-[(3-
phenyl-2-propynoyl)amino]hexanoate (13u) by the method B,
yield 70%. M.p. 112e113 ꢁC. 1H NMR (DMSO-d6, HMDSO)
d: 1.15e1.55 (m, 6H), 1.94 (t, J ¼ 7.2 Hz, 2H), 3.10 (q,
J ¼ 6.2 Hz, 2H), 7.39e7.61 (m, 5H), 8.66 (s, 1H), 8.76 (t,
J ¼ 5.4 Hz, 1H), 10.33 (s, 1H). Anal. Calcd for C15H18N2O3:
C 65.68, H 6.61, N 10.21. Found: C 65.49, H 6.61, N 10.24.
4.1.35.2.
(E )-3-(4-Chloro-2-fluorophenyl)-N-[6-(hydroxya-
4.1.33. General synthesis of amidomethyl esters 13v,w from
carboxylic acids 12v,w (method D)
To a solution of appropriate carboxylic acid 12v or 12w
(2.0 mmol) in anhydrous tetrahydrofuran (5 ml) under argon
mino)-6-oxohexyl]-2-propenamide (37). Compound 37 was
obtained from methyl 6-{[(E )-3-(4-chloro-2-fluorophenyl)-2-
propenoyl]amino}hexanoate (13w) by the method B, yield
1
27%. M.p. 155e157 ꢁC. H NMR (DMSO-d6, HMDSO) d:

1082
V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1.16e1.60 (m, 6H), 1.94 (t, J ¼ 7.3 Hz, 2H), 3.15 (q,
J ¼ 6.2 Hz, 2H), 6.72 (d, J ¼ 16.0 Hz, 1H), 7.35 (dd, J ¼ 2.2,
8.4 Hz, 1H), 7.42 (d, J ¼ 16.0 Hz, 1H), 7.53 (dd, J ¼ 2.2,
11.0 Hz, 1H), 7.68 (t, J ¼ 8.4 Hz, 1H), 8.23 (t, J ¼ 5.4 Hz,
1H), 8.67 (d, J ¼ 1.8 Hz, 1H), 10.34 (s, 1H). Anal. Calcd for
C15H18ClFN2O3: C 54.80, H 5.52, N 8.52. Found: C 54.60,
H 5.55, N 8.60.
was enhanced by crystallization from acetonitrile. M.p. 226e
1
227 ꢁC. H NMR (DMSO-d6, HMDSO) d: 11.25e1.48 (m,
6H), 1.92 (t, J ¼ 7.2 Hz, 2H), 3.35 (q, J ¼ 6.0 Hz, 2H), 3.42
(s, 2H), 7.26e7.54 (m, 5H), 7.56e7.69 (m, 4H), 8.04 (t,
J ¼ 6.0 Hz, 1H), 8.65 (s, 1H), 10.33 (s, 1H). Anal. Calcd for
C20H24N2O3/0.35H2O: C 69.28, H 7.18, N 8.08. Found: C
69.33, H 7.24, N 8.15.
4.1.36. Preparation of 38
4.1.37. General synthesis of amidoesters 40a and 40b
(method E)
4.1.36.1. Methyl 6-{[(E )-3-(4-chloro-2-fluorophenyl)-2-prope-
noyl]amino}hexanoate (13x). Compound 13x from 2-[1,10-
biphenyl]-4-ylacetic acid (12x) and methyl 6-aminohexa-
noate hydrochloride by the method C was prepared, yield
79% (white solid). 1H NMR (CDCl3, HMDSO) d: 1.09e
1.89 (m, 6H), 2.27(t, J ¼ 7.0 Hz, 2H), 3.20 (q, J ¼ 6.0 Hz,
2H), 3.58 (s, 2H), 3.65 (s, 3H), 5.43 (br s, 1H), 7.18e7.74
(m, 9H).
A solution of 8-methoxy-8-oxooctanoic acid (2.75 mmol)
in anhydrous dimethylformamide (3 ml) under argon atmo-
sphere was cooled in an ice bath and to the solution carbon-
yldiimidazole (490 mg, 3.01 mmol) was added. The mixture
was stirred for 30 min at ice bath temperature and then a so-
lution of appropriate amine 39a or 39b (2.75 mmol) in dime-
thylformamide (3 ml) was added (1.0 ml triethylamine
additionally was added if amine 39a or 39b in a salt form
was utilized). The reaction mixture was stirred for 1 h at
ice bath temperature and 20 h at room temperature. Then
the mixture was supplemented with brine (50 ml) and ex-
tracted with ethyl acetate (3 ꢀ 25 ml). The organic phase
was washed with brine, 5% NaHCO3, brine, saturated
KH2PO4, and brine. The organic layer was dried (Na2SO4)
and the solvent was evaporated. The residue was purified
on silica gel (20 g) with chloroform/ethyl acetate as eluent af-
fording the corresponding reaction product 40a or 40b,
accordingly.
4.1.36.2. 6-[(2-[1,10-Biphenyl]-4-ylacetyl)amino]hexanoic acid
(14). Compound 14 was obtained from methyl 6-{[(E )-3-(4-
chloro-2-fluorophenyl)-2-propenoyl]amino}hexanoate (13x)
by a similar protocol to 4a, yield 87%. M.p. 148e150 ꢁC.
1H NMR (CDCl3, HMDSO) d: 1.09e1.69 (m, 6H), 2.32 (t,
J ¼ 7.0 Hz, 2H), 3.20 (q, J ¼ 6.0 Hz, 2H), 3.61 (s, 2H), 5.43
(br s, 1H), 7.29e7.69 (m, 9H).
4.1.36.3. N-(Benzyloxy)-6-[(2-[1,10-biphenyl]-4-ylacetyl)ami-
no]hexanamide (15). A solution of 6-[(2-[1,10-biphenyl]-4-
ylacetyl)amino]hexanoic acid (14) (0.619 g, 1.9 mmol) in an-
hydrous dimethylformamide (7.5 ml) under argon atmosphere
was cooled in an ice bath and carbonyldiimidazole (0.339 g,
2.09 mmol) was added. The resulting mixture was stirred for
30 min at ice bath temperature and then triethylamine
(1.2 ml, 8.6 mmol) followed by a solution of O-benzylhydrox-
ylamine hydrochloride (0.456 g, 2.86 mmol) in anhydrous di-
methylformamide (7.5 ml) were added. The mixture was
stirred at ice bath temperature for 1 h and for 20 h at room
temperature. The reaction mixture was diluted with brine,
the precipitate was filtered and chromatographed on silica
gel (20 g) with chloroform/methanol (9:1) as eluent to give
4.1.38. Preparation of 41
4.1.38.1. Methyl 8-(benzylamino)-8-oxooctanoate (40a). Com-
pound 40a was obtained from 8-methoxy-8-oxooctanoic acid
and benzylamine by the method E, yield 80%. 1H NMR
(CDCl3, HMDSO) d: 1.14e1.83 (m, 8H), 2.21 (t,
J ¼ 7.0 Hz, 2H), 2.27 (t, J ¼ 7.0 Hz, 2H), 3.63 (s, 3H), 4.43
(d, J ¼ 6.0 Hz, 2H), 5.69 (br s, 1H), 7.29 (s, 5H).
4.1.38.2. N1-Benzyl-N8-hydroxyoctanediamide (41). Com-
pound 41 was obtained from methyl 8-(benzylamino)-8-ox-
ooctanoate (40a) by the method B, yield 60%. M.p. 126e
1
the title compound 15 (0.550 g, 67%). M.p. 150e151 ꢁC. H
NMR (CDCl3, HMDSO) d: 1.07e1.69 (m, 6H), 1.85e2.23
(m, 2H), 3.18 (q, J ¼ 6.0 Hz, 2H), 3.58 (s, 2H), 4.87 (s, 2H),
5.56 (br s, 1H), 7.29e7.65 (m, 14H).
1
126.5 ꢁC. H NMR (DMSO-d6, HMDSO) d: 1.16e1.32 (m,
4H), 1.36e1.60 (m, 4H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.12 (t,
J ¼ 7.4 Hz, 2H), 4.24 (d, J ¼ 5.6 Hz, 2H), 7.16e7.36 (m,
5H), 8.30 (t, J ¼ 5.6 Hz, 1H), 8.66 (s, 1H), 10.33 (s, 1H).
Anal. Calcd for C15H22N2O3/0.5H2O: C 62.70, H 8.07, N
9.75. Found: C 62.84, H 7.83, N 9.73.
4.1.36.4. 6-[(2-[1,10-Biphenyl]-4-ylacetyl)amino]-N-hydroxy-
hexanamide (38). N-(Benzyloxy)-6-[(2-[1,10-biphenyl]-4-yla-
cetyl)amino]hexanamide (15) (0.550 g, 1.28 mmol) was
dissolved in chloroform/methanol mixture (1:1, 20 ml) and
250 mg of 5% Pd/C catalyst was added. The suspension was
vigorously stirred under hydrogen atmosphere until the start-
ing material disappeared (ca. 5 h). The resultant reaction mix-
ture was filtered through a small amount of silica gel, the
sorbent was washed with chloroform, and the combined fil-
trates were evaporated in vacuum to give the title compound
38 as a white solid (0.320 g, 73%). The purity of the material
4.1.39. Preparation of 42
4.1.39.1. Methyl 8-oxo-8-(phenethylamino)octanoate (40b).
Compound 40b was obtained from 8-methoxy-8-oxooctanoic
acid and phenethylamine (39b) by the method E, yield 63%.
1H NMR (CDCl3, HMDSO) d: 1.05e1.81 (m, 8H), 2.09 (t,
J ¼ 7.0 Hz, 2H), 2.27 (t, J ¼ 7.0 Hz, 2H), 2.78 (t, J ¼ 7.0 Hz,

V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
1083
2H), 3.52 (q, J ¼ 6.0 Hz, 2H), 3.65 (s, 3H), 5.56 (br s, 1H),
7.00e7.43 (m, 5H).
complimentary steric and electrostatic interactions between
˚
putative small molecule inhibitor. A 15 A grid was computed
using the centroid of co-crystallized ligand to perform dock-
ing calculations. The following hydrogen bond and metal
constraints selected from the protein structure were included
in the grid calculation: A. 1036A: His132: NE2:NE2: Hydro-
gen bond acceptor constraint. B. 1026A: His131: NE2:NE2:
Hydrogen bond acceptor constraint. C. 5200A: Tyr 297:
HH:HH: Hydrogen bond donor constraint. D. 2790A: Zinc
501: ZN:ZN: Metal constraint. The receptor grid was then
used to rank order the docked orientations. Compounds
were docked using Glide extra-precision mode with hydro-
gen bond and metal constraints with up to five poses saved
per molecule. The docked poses for each ligand were then
analyzed for steric and electrostatic complimentarity with
the receptor in order to select the best pose for further
analysis.
4.1.39.2. N1-Hydroxy-N8-phenethyloctanediamide (42). Com-
pound 42 was obtained from methyl 8-oxo-8-(phenethylami-
no)octanoate (40b) by the method B, yield 30%. M.p. 113e
114 ꢁC. 1H NMR (DMSO-d6, HMDSO) d: 1.10e1.30 (m,
4H), 1.34e1.56 (m, 4H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.01 (t,
J ¼ 7.4 Hz, 2H), 2.68 (t, J ¼ 7.6 Hz, 2H), 3.25 (q,
J ¼ 6.8 Hz, 2H), 7.12e7.34 (m, 5H), 7.85 (t, J ¼ 5.6 Hz,
1H), 8.66 (d, J ¼ 1.6 Hz, 1H), 10.33 (s, 1H). Anal. Calcd for
C16H24N2O3/0.17H2O: C 65.05, H 8.30, N 9.48. Found: C
65.04, H 8.25, N 9.44.
4.2. Computational chemistry
All molecular modeling operations were performed using
the Schrodinger suite of software tools (Maestro, ver-
¨
sion70110) running on a HP8200 Linux work station.
4.3. In vitro assays
4.2.1. Preparation of ligands for docking
4.3.1. Histone deacetylase activity
The compounds dataset was imported into Maestro and
prepared for docking runs using Ligprep module. This pro-
cess consists of a series of steps for generating 3D structure
from 2D (SD file) representation, searching for tautomers and
steric isomers, and performing a geometry minimization of
ligands.
Human cervix epithelial cancer HeLa cells (ATCC Ref. No.
CCL-2) were cultured in DMEM containing 10% foetal bo-
vine serum, 100 U/ml penicillin and 100 mg/ml streptomycin.
Subconfluent cells were harvested by trypsinisation, and
washed twice in ice cold PBS. Cells were then resuspended
in two volumes of buffer (60 mM TriseHCl, pH 7.4, 30%
glycerol, 450 mM NaCl), and lysed by three freeze and thaw
cycles (dry ice and 30 ꢁC). Cell debris was removed by centri-
fugation at w20,000g and supernatant aliquoted and stored at
ꢂ80 ꢁC.
4.2.2. Protein preparation and refinement
The X-ray crystal structure of bacterial HDACeTSA com-
plex (PDB_ID:1C3R) was obtained from the Protein Data
Bank [33]. Bacterial HDACeTSA complex is a dimer with
duplicate binding sites. Thus, after manual inspection and
cleaning of structure, we have retained the ‘A’ sub unit for
modeling calculations. The protein was prepared for docking
The activity of the compounds as HDACi was determined
using a commercially available fluorescent assay kit (Fluor
de LysÔ, BioMol Research Labs, Inc., Plymouth Meeting,
USA). HeLa extract was incubated for 1 h at 37 ꢁC in assay
buffer (25 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2, pH 8.0) with 15 mM acetylated substrate in the pres-
ence of test compound.
˚
runs as follows: amino acid residues within a 20 A radius of
the co-crystallized ligand (TSA) were used to define the bind-
ing site. The zinc metal atom was assigned with correct atom
type (87) and charge (þ2) from metal ion. Maestro atom types
and ligand (TSA) checked for correct atom types, formal
charges, bond orders. Using the pprep command, amino acid
residues that form salt bridges in the binding site of the protein
4.3.2. Cell proliferation
HeLa cells were seeded into 96-well plates at a density of
3000 cells/well in 100 mL of culture medium, allowed to set-
tle overnight, and compounds added using increasing concen-
trations the following day. Plates were then incubated at
37 ꢁC in 5% CO2 for 48 h, 10 mL/well of WST-1 reagent
added, followed by a further 1 h incubation at 37 ꢁC. Absor-
bance was then measured using a wavelength of 450 nm (ref-
erence wavelength 690 nm). Percent activity (% activity) in
reducing the number of viable cells was calculated for each
test compound as % activity ¼ {(SC ꢂ B)/(S0 ꢂ B)} ꢀ 100.
SC denotes signal measured in the presence of the compound
being tested, S0 signal measured in the absence of the com-
pound being tested and B the background signal measured
in blank wells containing medium only. IC50 values were cal-
culated using the non-linear regression (variable slope) curve
fitting option within Prism 3.0 (GraphPad Software Inc., San
Diego, CA).
˚
(except the residues within 3.5 A radius of ligand) were neu-
tralized and hydrogen bonding conflicts corrected. After add-
ing hydrogen atoms, the prepared ligand and protein entries
were subjected to a series of restrained, partial minimizations
using imprep command. The maximum rms deviation allowed
during the minimization process was 0.3. Finally, the com-
bined minimized structure was exported in .mae files for re-
ceptor grid calculations.
4.2.3. Docking and scoring
All molecular docking calculations were performed using
Glide [34] with the prepared and refined protein structure.
Generation of a receptor grid is the first step of the Glide
docking process. The shape and properties of protein are rep-
resented by different sets of fields on
a grid with

1084
V. Andrianov et al. / European Journal of Medicinal Chemistry 44 (2009) 1067e1085
M.W. Knuth, R.V. Swanson, D.E. McRee, L.W. Tari, Structure 12
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4.3.3. Recombinant human histone deacetylase (rhHDAC)
activity
Recombinant HDACs 1e9 were produced and assays per-
formed as described in Ref. [35].
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Hepes buffer (25 mM Hepes, 137 mM NaCI, 2.7 mM KCI
and 4.9 mM MgCI2, pH 8.0).
Plates were incubated at 37 ꢁC for 3 h (HDAC8 assays were
performed at room temperature) and the reaction quenched
with HDAC-FDL Developer (Biomol International: HDACs
1e7 and 9 e KI-105; HDAC8 e KI-176) 20 ꢀ stock diluted
to 1:400 in Hepes buffer and containing 2 mM TSA. Further
incubation for 25 min at room temperature allowed a fluores-
cence signal to develop. Generated fluorescence was measured
at wavelengths 355 nm (excitation) and 460 nm (emission).
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The in vivo anti-tumor activity of compounds, 32, 37, II
(SAHA) and 27 was investigated [36]. B6D2F1 female mice
were obtained from Taconic M&B (Ry, Denmark) and kept
in a light- and temperature-controlled environment with ad li-
bitum access to water and standard laboratory diet. On day 0,
mice weighing 19e21 g were inoculated with 106 P388 mouse
leukemia cells intraperitoneally (i.p.). On days 3e7 mice were
treated once daily i.p. with the different HDACi formulated as
8 and 24 mg/ml in 100% DMSO and dosed at 50 ml per mouse
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significant.
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EORTC International Conference, 14e18 November 2005, Philadel-
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Experiments were conducted according to the institutional
and national guidelines for the care and use of laboratory an-
imals, and approved by the Danish Experimental Animal In-
spectorate, Department of Justice.
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Acknowledgements
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We kindly thank CuraGen Inc. for providing us with the re-
combinant HDAC isoforms used for selectivity determina-
tions. We also thank Angela Finn and Ruth Hollinshead for
excellent technical assistance.
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