Design, synthesis and primary activity assay of tripeptidomimetics as histone deacetylase inhibitors with linear linker and branched cap group

Article abstract of DOI:10.1016/j.ejmech.2011.08.045

A novel series of tripeptidomimetics with spiro ring containing sulfur atoms as cap group and linear carbochain as linker was designed and synthesized as HDACs inhibitors. Several compounds possessed more potent HDAC8 inhibitory activity than clinically used drug SAHA, although their HDAC1 inhibitory activities and anti-proliferative activities against human breast cancer cell lines (MCF-7, MDA-MB-231) and prostate cancer cell line (PC-3) were not satisfactory. Among them, compounds 11l and 11k showed excellent potency against HDAC8 (IC₅₀ values were 0.021 ± 0.004 μM and 0.035 ± 0.007 μM, respectively, whereas SAHA was 0.70 ± 0.12 μM), and good selectivity over HDAC1. Up to now, few hydroxamic acid derivatives with linear linker were reported to possess HDAC8 selectivity over HDAC1.

Full text of DOI:10.1016/j.ejmech.2011.08.045

European Journal of Medicinal Chemistry 46 (2011) 5387e5397
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and primary activity assay of tripeptidomimetics as histone
deacetylase inhibitors with linear linker and branched cap group
,
Yingjie Zhanga, Jinhong Fenga, Yuping Jiaa, Yingying Xua, Chunxi Liub, Hao Fanga, Wenfang Xua
*
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
b Department of Pharmaceutics, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of tripeptidomimetics with spiro ring containing sulfur atoms as cap group and linear
carbochain as linker was designed and synthesized as HDACs inhibitors. Several compounds possessed
more potent HDAC8 inhibitory activity than clinically used drug SAHA, although their HDAC1 inhibitory
activities and anti-proliferative activities against human breast cancer cell lines (MCF-7, MDA-MB-231)
and prostate cancer cell line (PC-3) were not satisfactory. Among them, compounds 11l and 11k
Received 21 June 2011
Received in revised form
8 August 2011
Accepted 31 August 2011
Available online 7 September 2011
showed excellent potency against HDAC8 (IC50 values were 0.021 ꢀ 0.004
m
M and 0.035 ꢀ 0.007
mM,
respectively, whereas SAHA was 0.70 ꢀ 0.12
m
M), and good selectivity over HDAC1. Up to now, few
Keywords:
hydroxamic acid derivatives with linear linker were reported to possess HDAC8 selectivity over HDAC1.
1,4-Dithia-7-azaspiro[4,4]nonane-8-
carboxylic acid derivatives
Histone deacetylases
Inhibitor
Ó 2011 Elsevier Masson SAS. All rights reserved.
Synthesis
1. Introduction
aliphatic acids and benzamides, results in growth arrest, differen-
tiation and apoptosis, promoting these enzymes as promising
Histone deacetylases (HDACs) are enzymes responsible for
deacetylation of lysine residues in various proteinaceous substrates
such as nucleosomal histones. The hydrolysis of the acetyl group
from the histones results in condensation of chromosomal DNA and
transcriptional repression [1e3]. There are around 18 human
HDACs divided into 4 classes [4,5] based on their homology to yeast
models: class I (HDACs 1-3 and 8) and II (HDACs 4-7, 9 and 10) are
zinc-dependent metallohydrolases [4], whereas class III HDACs,
namely sirtuins, are NADþ dependent [6]. Class IV comprises only
HDAC11, exhibiting properties of both class I and class II HDACs.
Recently, more and more non-histone proteins related to cell
growth and survival pathways have been revealed as the HDACs
substrates, such as transcription factors, cytoskeletal proteins,
molecular chaperones and nuclear import factors [7]. Aberrant
deacetylation of histone as well as other non-histone proteins will
cause many diseases, of which cancer is the most dreadful. Many
experiments revealed that overexpression of class I and II HDACs,
especially class I isozymes, is associated with many kinds of tumors
[8e15]. Treatment of tumor cells with class I and class II HDACs
inhibitors (HDACi), including hydroxamates, cyclic peptides,
targets for anti-cancer therapy [16e18]. Currently, two HDACs
inhibitors, suberoylanilide hydroxamic acid (SAHA, Vorinostat)
[19,20] and the depsipeptide FK228 (Romidepsin) [21], have been
approved by the FDA for the treatment of cutaneous T-cell
lymphoma (CTCL), whereas some others are undergoing clinical
trials [22]. The structures of SAHA, FK228 and several clinical HDACi
are shown in Fig. 1.
Despite the variety of structural characteristics, most HDAC
inhibitors, including the intracellular active form of FK228 (Fig. 1.),
can be broadly described by a common pharmacophore according
to the HDACs active site. As shown in Fig. 1, the pharmacophore
includes three parts: a cap group, which makes contacts with the
amino acid residues on the rim of the enzymatic active site, and
a zinc ion binding group, joined by a linker domain which occupies
the hydrophobic tunnel of the active site [23]. Based on the
common pharmacophore, we designed a novel series of peptido-
mimetics HDACi (Fig. 2b). In the scaffold of our target compounds,
6-aminohexanoic acid was selected as the linker domain because
its length is quite similar with that of SAHA. Expecting that its
distinct geometrical conformation and structure could form special
interaction with the rim of the enzymatic active site, the unnatural
amino acid 1,4-Dithia-7-azaspiro[4,4]nonane-8-carboxylic acid
(Fig. 2a) was introduced into the cap group of our HDACi. Another
reason why we chose this kind of unnatural amino acid was that the
* Corresponding author. Tel./fax: þ86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.045

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Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
Fig. 1. The structures of two approved drugs SAHA, FK228, and four HDACi in clinical trials. The three parts of HDACi pharmacophore are indicated in three different colors,
respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
scaffold of 1,4-Dithia-7-azaspiro [4,4]nonane-8-carboxylic acid was
used in an approved peptidomimetic angiotensin converting
enzyme inhibitor (ACEi), Spirapril (Fig. 2c.). Therefore, the in vivo
release of this ectogenic amino acid resulted from peptide bonds
hydrolysis of our compounds would not cause metabolic toxicity.
The amino acid derivatizations at R group were expected to
recognize the amino acid residues on the rim of HDACs active site,
thus enhance affinity with the enzyme.
2. Chemistry
The target compounds 6 and 7 were synthesized following the
procedures described in Scheme 1. The starting materials
compound
1
(1,4-Dithia-7-azaspiro[4,4]nonane-8(S)-carboxylic
acid hydrobromide) and compound 3 (6-aminocaproic acid) were
protected by Boc and methyl groups, respectively, to get
compounds 2 and 4, which were connected by classical DCC/HOBT
a
b
c
Fig. 2. a. The structure of 1,4-Dithia-7-azaspiro [4,4]nonane-8-carboxylic acid. b. The structure of Spirapril, an approved ACEi. c. The structure comparison between SAHA and our
target compound.

Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
5389
Scheme 1. Reagents and conditions: (a) (Boc)2O, 1N NaOH, THF; (b) SOCl2, CH3OH; (c) DCC, HOBT, anhydrous THF; (d) NH2OK, CH3OH; (e) HCl, anhydrous EtOAc.
method to yield the key intermediate 5. The methyl ester group of
compound was treated with NH2OK in methanol to get
compound 6. Finally, compound 7 was obtained by deprotecting
the Boc group of 6.
synthesized to investigate whether substituting other R functional
5
groups for Boc group could increase the inhibitory potency of
compound 6. From the results listed in Table 1, it could be found
that most of the 11 series and 12 series compounds were more
potent than their lead compound 6, among which compounds 11f
The compounds 11ae11n and 12ae12n were synthesized effi-
ciently according to the methods described in Scheme 2. The
intermediate 5 obtained in Scheme 1 was deprotected by TFA in
CH2Cl2 to get compound 8, followed by condensation with a few
kinds of Boc protected amino acids to yield compounds 9ae9n.
Unfortunately, the yield of direct conversion of 9ae9n to 11ae11n
by NH2OK in methanol was far from satisfying. Therefore,
compounds 9ae9n were hydrolyzed to 10ae10n and then con-
verted to the corresponding target hydroxamic acid 11ae11n and
12ae12n.
(IC50 ¼ 0.60 ꢀ 0.11
(IC50 ¼ 0.021 ꢀ 0.004
(IC50 ¼ 0.14 ꢀ 0.02 M) and 11n (IC50 ¼ 0.35 ꢀ 0.08
even more potent than the clinically used drug SAHA
(IC50 ¼ 0.70 ꢀ 0.12 M). From above mentioned result that 6 was
m
M), 11k (IC50 ¼ 0.035 ꢀ 0.007
m
M), 11l
M), 11m
mM) were
m
M), 12l (IC50 ¼ 0.49 ꢀ 0.07
m
m
m
more potent than 7 and the result that most of the 11 series and 12
series compounds were more potent than their lead compound 6,
we could revealed a structureeactivity relationship (SAR) that
branched cap groups could efficiently increase compounds’
inhibitory activities against HDAC8. Such SAR could also be ob-
tained from our previously reported tetrahydroisoquinoline-based
HDACi [24] and tyrosine-based HDACi [26].
3. Result and discussion
Moreover, almost all 11 series compounds were more efficient
than their corresponding hydrochlorides 12 series compounds,
except compounds 11g and 12g. Considering the cap groups of
HDACi were highly solvent-exposed, the Boc group located on the
cap group of 11 series compounds could afford more hydrophobic
interaction with HDAC8 than their corresponding Boc-cleaved 12
series compounds. Further preliminary SAR analysis revealed that
compounds with aromatic ring (11k, 11l, 11m, 12k, 12l and 12m)
were the most potent, which was in line with the fact that most
Based on the fact that all zinc ion dependent HDACs were
highly conserved in their active sites and HDAC8 is more available
in our group, firstly, we used HDAC8 as the enzyme source to
screen our target compounds [24,25,26], with Vorinostat (SAHA)
as positive control. The results were listed in Table 1. The fact that
compound 6 (IC50 ¼ 1.86 ꢀ 0.33
mM) was more potent than
M) indicated that the substituted
compound 7 (IC50 ¼ 2.59 ꢀ 0.51
m
R group of compound 6 was probably beneficial to its activity.
Based on this idea, compounds 11ae11n, 12ae12n were
Scheme 2. Reagents and conditions: (a) TFA, Na2CO3, anhydrous CH2Cl2; (b) Boc protected amino acids, TBTU, Et3N, anhydrous THF; (c) 2M NaOH, C2H5OH; (d) Isobutyl chlor-
ocarbonate, Et3N, NH2OH, anhydrous THF; (e) HCl, anhydrous EtOAc.

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Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
Table 1 (continued)
Table 1
The structures and inhibitory activities of compounds 11ae11j, 12ae12j and SAHA
against HDAC8.
a
Compounds
R
IC50 of HDAC8 (
m
M)
O
S
O
H
11g
1.31 ꢀ 0.20
S
N
N
OH
HN
H
Boc
O
N
R
O
12g
11h
1.07 ꢀ 0.16
0.87 ꢀ 0.12
Compounds
R
IC50a of HDAC8 (
m
M)
.
NH2 HCl
O
O
6
1.86 ꢀ 0.33
2.59 ꢀ 0.51
1.19 ꢀ 0.21
O
HO
HO
HN
7
H$Hcl
Boc
O
O
11a
12h
11i
2.52 ꢀ 0.37
0.76 ꢀ 0.10
HN
Boc
.
HCl
NH2
O
O
12a
1.30 ꢀ 0.22
.
HCl
NH2
N
Boc
O
O
11b
12b
1.90 ꢀ 0.36
1.93 ꢀ 0.41
HN
12i
11j
10.28 ꢀ 2.14
1.44 ꢀ 0.16
Boc
.
HCl
NH
O
O
H2N
.
HCl
N
O
H
11c
12c
0.96 ꢀ 0.19
4.12 ꢀ 0.54
Boc
N
Boc
O
O
H2N
.
HCl
12j
2.79 ꢀ 0.31
O
N
H
.
11d
1.15 ꢀ 0.16
HCl
HN
Boc
O
O
11k
0.035 ꢀ 0.007
12d
11e
5.90 ꢀ 0.57
0.72 ꢀ 0.21
HN
.
Boc
O
HCl
O
NH2
12k
11l
0.84 ꢀ 0.14
.
NH2 HCl
HN
Boc
O
O
0.021 ꢀ 0.004
12e
11f
1.07 ꢀ 0.23
0.60 ꢀ 0.11
HN
HO
HO
Boc
O
.
NH2 HCl
O
12l
0.49 ꢀ 0.07
0.14 ꢀ 0.02
.
HCl
NH2
HN
Boc
O
O
11m
12f
0.92 ꢀ 0.17
HN
HN
.
HCl
NH2
Boc

Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
5391
Table 1 (continued )
selective inhibitors with linear linker using the branched cap
strategy. Using them as leads, continued efforts are underway in
our lab to search more potent HDAC8 selective inhibitors.
Besides, according to the ClogP values of our compounds, we
postulated that membranes permeability was also a key factor for
compounds’ anti-proliferative activities because compounds
exhibiting low ClogP values were unable to permeate plasma-
lemma and nuclear membrane to inhibit HDACs, which mainly
located in cell nucleus. The result that compound 12l with potent
HDACs inhibitory activities (IC50 values against HDAC8 and HDAC1
a
Compounds
R
IC50 of HDAC8 (
m
M)
O
12m
0.69 ꢀ 0.10
0.35 ꢀ 0.08
.
NH2 HCl
HN
O
H
N
11n
12n
Boc
were 0.49 ꢀ 0.07
CLogP value (ꢁ0.46) exhibited poor anti-proliferative capacity
(IC50 > 100 M) supported our presumption to some degree.
mM and 0.45 ꢀ 0.09
mM, respectively) but low
HN
Boc
m
O
In order to gain insights into the possible reasons for selectivity
and activity of compounds 11l and 11k, we docked these two
compounds to HDAC8 crystal structures available in Protein Data
Bank (PDB entry: 1T64, 1T67, 1T69, 1VKG, 1W22, 2V5W, 2V5X,
3F0R, 3F07, 3RQD) using SYBYL 8.0. However, considering the
remarkable malleability of the HDAC8 protein surface, we would
not discuss our docking results here as they may not represent the
true binding mode. The malleability of the HDAC8 protein surface
has been revealed by many different available HDAC8 crystallo-
graphic structures (PDB entry: 1T64, 1T67, 1T69, 1VKG, 1W22,
2V5W, 2V5X, 3F0R, 3F07, 3RQD), the photoaffinity labeling
approach [33,34] and the molecular dynamic simulation results
[35]. The likely malleability of the protein surfaces in other class I
HDACs members (HDAC1, 2 and 3) are unknown due to the lack of
their crystallographic structures. Only one HDAC2 crystallographic
structure was reported in Protein Data Bank (PDB entry: 3MAX)
until this paper was finished. However, there are three crystallo-
graphic structures of HDLP (histone deacetylase-like protein, the
bacterial homologue of human class I HDACs) reported [23], and the
protein surface difference between HDAC8 and HDLP was discussed
by Somoza et al. [36]. Because the loop L1 in HDAC8 protein surface
is two residues shorter than that in HDLP, the opening to the active
site of HDAC8 is wider than that of HDLP, and the protein surface
around the entrance to the active site of HDAC8 is more malleable
than that of HDLP. Sequence alignments of HDAC8 with the other
class I HDACs members suggest that HDAC1, 2 and 3 all possess
longer loop L1, structurally similar to the loop in HDLP [36]. This
indicates that the protein surfaces around the entrance to the active
site of HDAC1, 2 and 3 might be more rigid relative to HDAC8.
Moreover, the molecular dynamic simulation of HDAC1 showed
that its active site and protein surface were fairly stable [35]. Based
on the above mentioned information, we could presume the
interaction between HDACi and the enzymes. In HDAC8 isoform,
when the zinc ion was coordinated by hydroxamic acid group and
the tunnel of the active site was occupied by carbochain, the
malleable protein surface at the rim of the active site could adjust
itself to accommodate complex structures, such as branched cap
groups. If the protein and the inhibitor could accommodate each
H2N
.
4.77 ꢀ 0.75
0.70 ꢀ 0.12
HCl
.
NH2 HCl
O
H
N
OH
SAHA
N
H
O
a
Data represent mean values of at least three independent experiments and
standard deviations are calculated based on the results of these experiments.
potent HDACi possessed aromatic or heteroaromatic rings in their
cap groups [27,28]. Compounds 11k and 11l exhibited double digit
nanomolar IC50 values, which were one order of magnitude lower
than that of SAHA. In a word, a hydrophobic R group containing
aromatic residues was optimal for compounds’ HDAC8 inhibitory
activity.
Encouraged by their potent anti-HDACs activities, compounds
11f, 11l, 12l, 11k, 11m and 11n were progressed to in vitro anti-
proliferative activity assay against human breast cancer cell lines
(MCF-7, MDA-MB-231) and prostate cancer cell line (PC-3). As can
be seen in Table 2, although the HDAC8 inhibitory activities of these
compounds were all superior to SAHA, their anti-proliferative
activities were not comparable with that of SAHA. Therefore, we
tested our compounds against HDAC1, which was reported to play
important roles in human breast cancer and prostate cell lines [29],
and found that the IC50 values of these derivatives
(0.45
mMe0.98
mM) were at least 10 fold larger than that of SAHA
(0.044
mM). This might be the reason why our compounds were less
potent in anti-proliferative assays. Compounds 11l and 11k showed
excellent potency against HDAC8 (IC50 values were
0.021 ꢀ 0.004
m
M and 0.035 ꢀ 0.007
mM, respectively) and good
selectivity against HDAC8 over HDAC1. So far, only a few HDAC8
selective inhibitors have been developed and all of them are ‘link-
less’ [30e32]. Although the selectivity of our compounds is
moderate relative to the reported ‘linkerless’ HDAC8 selective
inhibitor, our results indicate that it is feasible to find HDAC8
Table 2
The anti-proliferative activities, HDACs inhibitory activities and CLogP values of compounds 11f, 11l, 12l, 11k, 11m, 11n and SAHA.
a
compd
IC50
(m
M)
CLogP
MCF-7
MDA-MB-231
PC-3
HDAC8
HDAC1
11f
11l
12l
11k
11m
11n
SAHA
3.51 ꢀ 0.55
21.6 ꢀ 3.1
>100
7.78 ꢀ 1.14
9.16 ꢀ 1.92
42.5 ꢀ 7.2
1.15 ꢀ 0.36
3.50 ꢀ 0.23
30.5 ꢀ 4.2
>100
2.79 ꢀ 0.81
5.83 ꢀ 1.04
12.8 ꢀ 2.9
1.91 ꢀ 0.41
4.3 ꢀ 0.88
24.7 ꢀ 5.1
>100
12.7 ꢀ 2.5
13.7 ꢀ 43.1
34.6 ꢀ 7.2
1.98 ꢀ 0.33
0.60 ꢀ 0.11
0.021 ꢀ 0.004
0.49 ꢀ 0.07
0.035 ꢀ 0.007
0.14 ꢀ 0.02
0.35 ꢀ 0.08
0.70 ꢀ 0.12
0.68 ꢀ 0.10
0.45 ꢀ 0.08
0.45 ꢀ 0.09
0.98 ꢀ 0.18
0.75 ꢀ 0.13
0.98 ꢀ 0.20
0.044 ꢀ 0.01
2.07
1.36
ꢁ0.46
2.03
2.02
2.05
/
a
Data represent mean values of at least three independent experiments and standard deviations are calculated based on the results of these experiments.

5392
Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
other through interaction, a preferable binding mode and the
subsequent effective inhibition were achieved. While in the other
class I HDACs isoforms, such as in HDAC1, the rigid protein surface
could not accept complex structures very well, thus the selectivity
occurred. This hypothetical theory not only explained why
compounds with branched cap groups (7, 11 series and 12 series
compounds) exhibited more potent HDAC8 inhibition than their
unbranched parent compound 6, and why almost all three-
branched compounds (11 series, of which the Boc group could be
recognized as the third branch) exhibited more potent HDAC8
inhibition than their corresponding two-branched compounds (12
series), but also indicated the basis for HDAC8 selectivity. According
to this hypothesis, HDAC8 selective compounds with linear linker
and branched cap are being designed and synthesized in our lab,
and such molecular probes could in turn validate our hypothesis.
Furthermore, detailed experiments, such as SAR studies, X-ray or
NMR structure resolution and photoaffinity labeling approach, are
warranted to identify what kinds of functional groups are suitable
for HDAC8 binding and what exact amino acid residues of HDAC8
are involved in enzymeeinhibitor interactions.
product was used for the following reaction without further puri-
fication. 1H NMR (DMSO-d6)
1.36 (s, 9H), 2.32e2.39 (m, 1H),
2.70e2.73 (m, 1H), 3.32e3.39 (m, 4H), 3.58e3.63 (m, 1H),
3.74e3.78 (m, 1H), 4.12e4.17 (m, 1H), 12.83 (br s, 1H). ESI-MS m/z:
306.5 [M þ H]þ.
d
5.1.2. Methyl 6-aminohexanoate hydrochloride (4)
The title compound was prepared from compound 3 according
to the method described by Gigante et al. [37].
5.1.3. (S)-Tert-butyl 8-(6-methoxy-6-oxohexylcarbamoyl)-1,4-
dithia-7-azaspiro[4.4]nonane-7-carboxylate (5)
At 0 ꢃC, to a solution of compound 2 (3.05 g, 10.0 mmol) and
HOBT (1.49 g, 11.0 mmol) in anhydrous THF (40 mL), was added
a solution of DCC (2.27 g, 11.0 mmol) in anhydrous THF (10 mL), and
after 30 min, Et3N (1.11 g, 11.0 mmol) and compound 4 (1.82 g,
11.0 mmol). After stirring the mixture at room temperature over
night, THF was evaporated with the residues being taken up in
EtOAc (40 mL) and freezed in refrigerator. Then DCU was filtered
off, the filtrate washed with saturated Na2CO3 (3 ꢂ 10 mL), 1 N HCl
(3 ꢂ 10 mL), brine (3 ꢂ 10 mL), dried over MgSO4 and evaporated
under vacuum. The residues were purified by flash column chro-
matography (Petroleum/EtOAc 3:1) to give 2.89 g of desired
compound 5 as colorless oil. Yield: 67%, 1H NMR (DMSO-d6)
4. Conclusions
In summary, a novel series of tripeptidomimetics with the
scaffold 1,4-Dithia-7-azaspiro[4,4]nonane-8-carboxylic acid were
synthesized efficiently as HDACs inhibitors. Compared with SAHA,
our scaffold exhibited more preference to HDAC8 than HDAC1, and
compounds 11l and 11k, which were over 20 fold selective against
HDAC8 over HDAC1, could be used as leads to further design HDAC8
isoform selective inhibitors. As far as we know, although several
‘linkerless’ HDAC8 selective inhibitors were reported, there are few
linear HDACs inhibitors with HDAC8 selectivity. Therefore, our
results provided anther strategy for designing HDAC8 selective
inhibitor and confirmed the idea that modification of the cap
groups could change the isoform selectivity of HDACs inhibitors.
d
1.22e1.26 (m, 2H), 1.36e1.43 (m, 2H), 1.38 (s, 9H), 1.44e1.52 (m,
2H), 2.30 (t, J ¼ 7.2 Hz, 2H), 2.32e2.44 (m, 1H), 2.53e2.76 (m, 1H),
2.98e3.09 (m, 2H), 3.36e3.40 (m, 4H), 3.73 (d, J ¼ 10.8 Hz, 1H), 3.97
(d, J ¼ 10.8 Hz, 1H), 4.26e4.28 (m, 1H), 7.88 (s, 1H), 8.67 (s, 1H),
10.34 (s, 1H). ESI-MS m/z: 433.4[M þ H]þ.
5.1.4. (S)-Tert-butyl 8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1,4-dithia-7-azaspiro[4.4]nonane-7-carboxylate (6)
To a solution of compound 5 (2.16 g, 5.0 mmol) in anhydrous
methanol (20 mL), was added a solution of NH2OK (1.06 g,
15,0 mmol) in anhydrous methanol (8.8 mL). The mixture was
stirred for 0.5 h and the solvent was evaporated under vacuum. The
residues were acidified with 2 N HCl until pH 3e4 then extracted
with EtOAc (3 ꢂ 10 mL). The combined organic layers were
combined, washed with brine (3 ꢂ 10 mL), dried over MgSO4 and
evaporated with the residues being recrystallized with EtOAc/EtOH
to give 0.89 g of title compound 6 as a white powder. Yield: 41%,
5. Experimental section
5.1. Chemistry
Unless specified otherwise, all starting materials, reagents and
solvents were commercially available. All reactions except those in
aqueous media were carried out by standard techniques for the
exclusion of moisture. All reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates (60 GF-254) and
visualized with UV light, ferric chloride or iodine vapor. Proton
NMR spectrums were determined on a Brucker DRX spectrometer
mp: 103e105 ꢃC. 1H NMR (DMSO-d6)
d 1.16e1.19 (m, 2H),
1.33 þ 1.39 (s, 9H, cis/trans), 1.35e1.46 (m, 2H), 1.47e1.49 (m, 2H),
1.92 (t, J ¼ 7.2 Hz, 2H), 2.24e2.28 (m, 1H), 2.53e2.57 (m, 1H),
2.96e2.99 (m, 1H), 3.05e3.09 (m, 1H), 3.31e3.39 (m, 4H), 3.56 (d,
J ¼ 10.8 Hz, 1H), 3.80 (d, J ¼ 10.8 Hz, 1H), 4.08e4.13 (m, 1H), 7.96 (s,
1H), 8.65 (s, 1H),10.33 (s,1H). 13C NMR (150 MHz, DMSO-d6)
d: 24.9,
(600 MHz),
d
in parts per million and J in Hertz, using TMS as an
26.2, 27.9, 28.9, 32.2, 38.4, 38.5, 46.2, 60.2, 60.9, 66.1, 79.2, 152.9,
169.0, 170.8. HRMS (AP-ESI) m/z calcd for C18H32N3O5S2 [M þ H]þ
434.1783, found 434.1795.
internal standard. Measurements were made in DMSO-d6 solutions.
ESI-MS were determined on an API 4000 spectrometer. HRMS
spectrums were conducted by Shandong Analysis and Test Center.
Melting points were determined on an electrothermal melting
point apparatus and were uncorrected.
5.1.5. (S)-N-(6-(Hydroxyamino)-6-oxohexyl)-1,4-dithia-7-azaspiro
[4.4]nonane-8-carboxamide hydrochloride (7)
To a solution of compound 6 (0.45 g, 1.04 mmol) in dry EtOAc
(15 mL), was added a solution of EtOAc (15 mL) saturated by dry HCl
gas. The resulting solution was stirred at room temperature for 5 h
when the precipitation appeared. The suspension was filtered with
the filter being washed with ether to give 0.35 g of desired
compound 7 as a white powder. Yield: 92%, mp: 114e116 ꢃC. 1H
5.1.1. (S)-7-(Tert-butoxycarbonyl)-1,4-dithia-7-azaspiro[4.4]
nonane-8-carboxylic acid (2)
To a solution of compound 1 (5.72 g, 20.0 mmol) in 44 mL of 1 N
NaOH, was added a solution of (Boc)2O (4.80 g, 22.0 mmol) in THF
(10 mL). The solution was kept between pH 9e11 by addition of 1 N
NaOH. After stirring the mixture at room temperature for 6 h, THF
was evaporated in vacuum with the residues being adjusted to pH
4e5 with 1 N aqueous citric acid. Then the mixture was extracted
with EtOAc (3 ꢂ 25 mL). The extractions were combined, washed
with brine (3 ꢂ 20 mL), dried over MgSO4 and evaporated to give
5.80 g of crude product compound 2 as a light brown solid. This
NMR (DMSO-d6)
d 1.25e1.27 (m, 2H), 1.41e1.45 (m, 2H), 1.46e1.51
(m, 2H), 1.94 (t, J ¼ 7.2 Hz, 2H), 2.41e2.45 (m, 1H), 2.83e2.86 (m,
1H), 3.07e3.17 (m, 2H), 3.38e3.46 (m, 4H), 3.59e3.65 (m, 2H),
4.28e4.32 (m, 1H), 8.69 (s, 1H), 8.88 (s, 1H), 10.32 (s, 1H), 10.41 (s,
1H), 10.58 (s, 1H). HRMS (AP-ESI) m/z calcd for C13H24N3O3S2
[M þ H]þ 334.1259, found 334.1223.

Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
5393
5.1.6. (S)-Methyl 6-(1,4-dithia-7-azaspiro[4.4]nonane-8-
carboxamido)hexanoate (8)
5.1.7.8. (S)-Tert-butyl 2-((S)-8-(6-methoxy-6-oxohexylcarbamoyl)-1,4-
dithia-7- azaspiro[4.4]nonane-7-carbonyl)pyrrolidine-1-carboxylate
(9i). Yield: 55%.
To a solution of compound 5 (4.33 g, 10.0 mmol) in anhydrous
CH2Cl2 (40 mL), was added 25 mL of trifluoroacetic acid. When the
reaction was finished, saturated Na2CO3 was added to the solution
until the pH reach weak basic. The mixture was separated with
separatory funnel, the organic layer was washed with distilled
water, dried over MgSO4 and evaporated under vacuum to get
2.95 g of desired compound 8 as light yellow oil. This product was
used for the following reaction without further purification. ESI-MS
m/z: 333.6 [M þ H]þ.
5.1.7.9. (S)-Tert-butyl 4-(8-(6-methoxy-6-oxohexylcarbamoyl)-1,4-
dithia-7- azaspiro[4.4]nonane-7-carbonyl)piperidine-1-carboxylate
(9j). Yield: 45%.
5.1.7.10. Methyl 6-((S)-7-((S)-2-(tert-butoxycarbonylamino)-3-phe-
nylpropanoyl)-
1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamido)
hexanoate (9k). Yield: 58%.
5.1.7. (S)-Methyl 6-(7-(2-(tert-Butoxycarbonylamino)acetyl)-1,4-
dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoate (9a)
Boc protected glycine, as well as the other Boc protected amino
acids, was synthesized according to the synthesis method of
compound 2.
5.1.7.11. Methyl
6-((S)-7-((S)-2-(tert-butoxycarbonylamino)-3-(4-
hydroxyphenyl)propanoyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-
carboxamido)hexanoate (9l). Yield: 51%.
5.1.7.12. Methyl 6-((S)-7-((S)-2-(tert-butoxycarbonylamino)-3-(1H-
indol-3-yl)propanoyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-
carboxamido)hexanoate (9m). Yield: 59%.
At room temperature, to a solution of Boc protected glycine
(0.88 g, 5.0 mmol) in anhydrous THF (20 mL), was added Et3N
(0.56 g, 5.5 mmol) followed by 2-(1H-Benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU, 1.78 g, 5.5 mmol).
After 15 min, the amine compound 8 (1.67 g, 5.0 mmol) was
added. The stirring was continued for 8 h and then THF was
evaporated. The residues were taken up in EtOAc (40 mL), washed
with 1 N HCl (3 ꢂ 10 mL), saturated Na2CO3 (3 ꢂ 10 mL), brine
(3 ꢂ 10 mL), dried over MgSO4 and evaporated under vacuum. The
crude product was purified by flash column chromatography
(Petroleum/EtOAc 2:1) to give 1.25 g of desired compound 9a as
5.1.7.13. Methyl 6-((S)-7-((S)-2,6-bis(tert-butoxycarbonylamino)hex-
anoyl)- 1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoate
(9n). Yield: 44%.
5.1.8. (S)-6-(7-(2-(Tert-butoxycarbonylamino)acetyl)-1,4-dithia-7-
azaspiro[4.4]nonane-8-carboxamido)hexanoic acid (10a)
Compound 9a (1.1 g, 2.25 mmol) was dissolved in 20 mL of
ethanol and then 4 mL of 2 N NaOH was added. The mixture was
stirred for 1 h at 75 ꢃC. Then most of the ethanol was evaporated
with the residues being adjusted to pH 3e4 with 1 N HCl. The
precipitated product was extracted with EtOAc, washed with brine,
dried over MgSO4 and evaporated to give 0.99 g of desired
compound 10a as a white solid. Yield: 93%. 1H NMR (DMSO-d6)
colorless oil. Yield: 51%. 1H NMR (DMSO-d6)
d 1.23e1.27 (m, 2H),
1.36e1.42 (m, 2H), 1.38 (s, 9H), 1.48e1.53 (m, 2H), 2.29 (t,
J ¼ 7.2 Hz, 2H), 2.30e2.44 (m, 1H), 2.53e2.76 (m, 1H), 2.98e3.09
(m, 2H), 3.37e3.43 (m, 4H), 3.58 (s, 3H), 3.61e3.64 (m, 1H), 3.73
(d, J ¼ 10.8 Hz, 1H), 3.78e3.82 (m, 1H), 3.97 (d, J ¼ 10.8 Hz, 1H),
4.26e4.28 (m, 1H), 6.88 (s, 1H), 7.87 (s, 1H). ESI-MS m/z: 476.4
[M þ H]þ.
d
1.24e1.28 (m, 2H), 1.37e1.42 (m, 2H), 1.38 (s, 9H), 1.48e1.54 (m,
2H), 2.28 (t, J ¼ 7.2 Hz, 2H), 2.30e2.44 (m, 1H), 2.53e2.74 (m, 1H),
2.98e3.08 (m, 2H), 3.37e3.45 (m, 4H), 3.60e3.64 (m, 1H), 3.73 (d,
J ¼ 10.8 Hz, 1H), 3.78e3.81 (m, 1H), 3.97 (d, J ¼ 10.8 Hz, 1H),
4.27e4.30 (m, 1H), 6.88 (s, 1H), 7.87 (s, 1H), 12.83 (s, 1H). ESI-MS m/
z: 462.5 [M þ H]þ.
Compounds 9be9n were synthesized following the procedure
described above.
5.1.7.1. (S)-Methyl 6-(7-(3-(tert-butoxycarbonylamino)propanoyl)-
1,4-dithia-7- azaspiro[4.4]nonane-8-carboxamido)hexanoate (9b).
Yield: 48%.
Compounds 10be10n were synthesized following the proce-
dure described above.
5.1.7.2. (S)-Methyl
1,4-dithia-7- azaspiro[4.4]nonane-8-carboxamido)hexanoate (9c).
Yield: 55%.
6-(7-(4-(tert-butoxycarbonylamino)butanoyl)-
5.1.8.1. (S)-6-(7-(3-(Tert-butoxycarbonylamino)propanoyl)-1,4-dithia-
7-azaspiro[4.4]nonane-8-carboxamido)hexanoic acid (10b). Yield:
91%.
5.1.7.3. Methyl 6-((S)-7-((S)-2-(tert-butoxycarbonylamino)propanoyl)-
1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoate (9d).
Yield: 58%.
5.1.8.2. (S)-6-(7-(4-(Tert-butoxycarbonylamino)butanoyl)-1,4-dithia-
7-azaspiro[4.4]nonane-8-carboxamido)hexanoic acid (10c). Yield:
90%.
5.1.7.4. Methyl 6-((S)-7-((S)-2-(tert-butoxycarbonylamino)-3-meth-
ylbutanoyl)- 1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoate
(9e). Yield: 48%.
5.1.8.3. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)propanoyl)-1,4-
dithia-7- azaspiro[4.4]nonane-8-carboxamido)hexanoic acid (10d).
Yield: 95%.
5.1.7.5. Methyl 6-((S)-7-((S)-2-(tert-butoxycarbonylamino)-4-meth-
ylpentanoyl)-1,4-Dithia-7-azaspiro[4.4]nonane-8-carboxamido)hex-
anoate (9f). Yield: 48%.
5.1.8.4. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)-3-methylbutanoyl)-
1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoic acid (10e).
Yield: 91%.
5.1.7.6. Methyl 6-((S)-7-((2S,3R)-2-(tert-butoxycarbonylamino)-3-
methylpentanoyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamido)
hexanoate (9g). Yield: 51%.
5.1.8.5. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)-4-methylpentanoyl)-
1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoic acid (10f).
Yield: 85%.
5.1.7.7. Methyl 6-((S)-7-((S)-2-(tert-butoxycarbonylamino)-3-hydro-
xypropanoyl)- 1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoate
(9h). Yield: 40%.
5.1.8.6. 6-((S)-7-((2S,3R)-2-(Tert-butoxycarbonylamino)-3-methyl-
pentanoyl)- 1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamido)hex-
anoic acid (10g). Yield: 92%.

5394
Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
5.1.8.7. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)-3-hydroxypro-
panoyl)- 1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoic
acid (10h). Yield: 89%.
5.1.9.2. (S)-Tert-butyl 4-(8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1,4-dithia-7- azaspiro[4.4]nonan-7-yl)-4-oxobutylcarbamate (11c).
Yield: 60%, mp: 84e86 ꢃC. 1H NMR (DMSO-d6)
d 1.18e1.23 (m,
2H), 1.37 (s, 9H), 1.41e1.47 (m, 2H), 1.50e1.61 (m, 2H), 1.92 (t,
J ¼ 7.2 Hz, 2H), 2.13e2.25 (m, 2H), 2.26e2.41 (m, 1H), 2.55e2.62
(m, 1H), 2.86e3.08 (m, 4H), 3.32e3.45 (m, 4H), 3.75 (d,
J ¼ 10.8 Hz, 1H), 3.93 (d, J ¼ 10.8 Hz, 1H), 4.24e4.27 (m, 1H),
6.85 (s, 1H), 7.86 (s, 1H), 8.65 (s, 1H), 10.33 (s, 1H). HRMS (AP-
ESI) m/z calcd for C22H39N4O6S2 [M þ H]þ 519.2311, found
519.2321.
5.1.8.8. 6-((S)-7-((S)-1-(Tert-butoxycarbonyl)pyrrolidine-2-carbonyl)-
1,4-dithia-7- azaspiro[4.4]nonane-8-carboxamido)hexanoic acid
(10i). Yield: 88%.
5.1.8.9. (S)-6-(7-(1-(Tert-butoxycarbonyl)piperidine-4-carbonyl)-
1,4-dithia-7- azaspiro[4.4]nonane-8-carboxamido)hexanoic acid
(10j). Yield: 93%.
5.1.9.3. Tert-butyl (S)-1-((S)-8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-1-oxopropan-2-ylcarbamate
(11d). Yield: 49%, mp: 105e107 ꢃC. 1H NMR (DMSO-d6)
5.1.8.10. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)-3-phenylprop-
anoyl)-1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamido)hexanoic
acid (10k). Yield: 90%.
d
1.11e1.16 (m, 3H), 1.18e1.23 (m, 2H), 1.37 (s, 9H), 1.35e1.41 (m,
2H), 1.44e1.48 (m, 2H), 1.91 (t, J ¼ 7.2 Hz, 2H), 2.22e2.37 (m, 1H),
2.52e2.63 (m, 1H), 2.92e3.08 (m, 2H), 3.32e3.41 (m, 4H), 3.82 (d,
J ¼ 10.8 Hz, 1H), 3.96 (d, J ¼ 10.8 Hz, 1H), 4.14e4.19 (m, 1H),
4.27e4.31 (m, 1H), 7.20 (s, 1H), 7.82 (s,1H), 8.65 (s, 1H),10.33 (s, 1H).
HRMS (AP-ESI) m/z calcd for C21H37N4O6S2 [M þ H]þ 505.2155,
found 505.2171.
5.1.8.11. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)-3-(4-hydroxy-
phenyl)propanoyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carbox-
amido)hexanoic acid (10l). Yield: 81%.
5.1.8.12. 6-((S)-7-((S)-2-(Tert-butoxycarbonylamino)-3-(1H-indol-3-
yl)propanoyl)- ,4-dithia-7-azaspiro[4.4]nonane-8-carboxamido)hex-
anoic acid (10m). Yield: 89%.
5.1.9.4. Tert-butyl (S)-1-((S)-8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-3-methyl-1-oxobutan-2-ylcar-
5.1.8.13. 6-((S)-7-((S)-2,6-Bis(tert-butoxycarbonylamino)hexanoyl)-
1,4-dithia-7- azaspiro[4.4]nonane-8-carboxamido)hexanoic acid
(10n). Yield: 91%.
bamate (11e). Yield: 58%, mp: 91e93 ꢃC. 1H NMR (DMSO-d6)
d 0.82
(d, J ¼ 6.6 Hz, 3H), 0.86 (d, J ¼ 6.6 Hz, 3H), 1.19e1.24 (m, 2H),
1.33e1.36 (m, 2H), 1.37 (s, 9H),1.44e1.49 (m, 2H),1.85e1.90 (m, 1H),
1.91 (t, J ¼ 7.2 Hz, 2H), 2.25e2.28 (m, 1H), 2.51e2.54 (m, 1H),
2.95e3.06 (m, 2H), 3.35e3.41 (m, 4H), 3.77 (d, J ¼ 10.8 Hz, 1H),
3.90e3.92 (m, 1H), 4.28e4.29 (m, 1H), 4.31 (d, J ¼ 10.8 Hz, 1H), 6.89
(s, 1H), 7.89 (s, 1H), 8.65 (s, 1H), 10.33 (s, 1H). 13C NMR (150 MHz,
DMSO-d6) d: 18.3, 19.1, 24.9, 25.9, 28.2, 28.7, 29.5, 32.2, 38.4, 38.5,
44.3, 57.8, 60.0, 61.2, 67.1, 78.0, 155.6, 169.1, 169.9, 170.2. HRMS (AP-
ESI) m/z calcd for C23H41N4O6S2 [M þ H]þ 533.2468, found
533.2568.
5.1.9. (S)-Tert-butyl 2-(8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1,4-dithia-7-azaspiro[4.4]nonan-7-yl)-2-oxoethylcarbamate (11a)
Compound 10a (0.85 g, 1.79 mmol) was dissolved in anhydrous
THF (15 mL), then Et3N (0.20 g, 1.97 mmol) was added. The solution
was stirred at 0 ꢃC and isobutyl chlorocarbonate (0.27 g, 1.97 mmol)
was dropped into the solution. After 3e5 min, the solution of
NH2OH in anhydrous methanol (3 mL, 1.8 mmol/mL) was added.
The stirring was continued for 6 h and then THF was evaporated.
The residues were acidified with 1 N HCl, extracted with EtOAc,
washed with brine, dried over MgSO4 and evaporated. The crude
product was purified by C18 reversed-phase column chromatog-
raphy (H2O/MeOH 3:7) to give 0.55 g of desired compound 11a as
a white powder. Yield: 62%, mp: 98e100 ꢃC. 1H NMR (DMSO-d6)
5.1.9.5. Tert-butyl
(S)-1-((S)-8-(6-(hydroxyamino)-6-oxohexylcar-
bamoyl)-1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-4-methyl-1-oxopentan-
2-ylcarbamate (11f). Yield: 63%, mp: 99e101 ꢃC. 1H NMR (DMSO-d6)
d
0.86e0.87 (m, 6H),1.18e1.26 (m, 2H),1.33e1.38 (m,13H),1.44e1.47
(m, 2H), 1.58e1.63 (m, 1H), 1.91 (t, J ¼ 7.2 Hz, 2H), 2.23e2.27 (m, 1H),
2.52e2.55 (m, 1H), 2.91e3.07 (m, 2H), 3.36e3.43 (m, 4H), 3.76 (d,
J ¼ 10.8 Hz, 1H), 4.05e4.15 (m, 1H), 4.19 (d, J ¼ 10.8 Hz, 1H),
4.27e4.29 (m, 1H), 7.05 (s, 1H), 7.83 (s, 1H), 8.65 (s, 1H), 10.33 (s, 1H).
d
1.17e1.22 (m, 2H), 1.38 (s, 9H), 1.35e1.40 (m, 2H), 1.45e1.48 (m,
2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.26e2.29 (m, 1H), 2.39e2.44 (m, 1H),
2.99e3.07 (m, 2H), 3.34e3.37 (m, 4H), 3.60e3.64 (m, 1H), 3.72 (d,
J ¼ 10.8 Hz, 1H), 3.79e3.83 (m, 1H), 3.97 (d, J ¼ 10.8 Hz, 1H),
4.26e4.29 (m, 1H), 6.90 (s, 1H), 7.89 (s, 1H), 8.66 (s, 1H), 10.34 (s,
13C NMR (150 MHz, DMSO-d6)
d: 21.7, 23.0, 24.1, 24.9, 25.9, 28.2, 28.7,
32.2, 38.4, 38.7, 44.4, 50.7, 60.1, 61.0, 67.1, 78.1, 155.4, 169.1, 169.9,
170.7. HRMS (AP-ESI) m/z calcd for C24H43N4O6S2 [M þ H]þ 547.2624,
found 547.2684.
1H). 13C NMR (150 MHz, DMSO-d6)
d: 25.3, 26.4, 28.6, 29.1, 32.6,
38.9, 39.2, 43.0, 44.6, 60.4, 61.1, 67.5, 78.4, 156.2, 168.1, 170.3, 170.4.
HRMS (AP-ESI) m/z calcd for C20H34N4NaO6S2 [M þ H]þ 513.1817,
found 513.1861.
Compounds 11be11n were synthesized following the procedure
described above.
5.1.9.6. Tert-butyl (2S,3S)-1-((S)-8-(6-(hydroxyamino)-6-oxohexyl-
carbamoyl)-1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-3-methyl-1-oxopentan-
2-ylcarbamate (11g). Yield: 51%, mp: 105e107 ꢃC. 1H NMR (DMSO-
d6)
d 0.78e0.87 (m, 6H), 0.88e0.92 (m, 1H), 1.03e1.09 (m, 1H),
5.1.9.1. (S)-Tert-butyl 3-(8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1.20e1.23 (m, 2H), 1.38 (s, 9H), 1.36e1.42 (m, 2H), 1.43e1.46 (m,
2H), 1.69e1.72 (m, 1H), 1.91 (t, J ¼ 7.2 Hz, 2H), 2.25e2.29 (m, 1H),
2.49e2.53 (m, 1H), 2.94e3.05 (m, 2H), 3.27e3.38 (m, 4H), 3.76 (d,
J ¼ 10.8 Hz, 1H), 3.91e3.94 (m, 1H), 4.27e4.29 (m, 1H), 4.36 (d,
J ¼ 10.8 Hz, 1H), 6.97 (s, 1H), 7.87 (s, 1H), 8.65 (s, 1H), 10.33 (s, 1H).
1,4-dithia-7- azaspiro[4.4]nonan-7-yl)-3-oxopropylcarbamate (11b).
Yield: 66%, mp: 97e99 ꢃC. 1H NMR (DMSO-d6)
d 1.18e1.22 (m,
2H), 1.37 (s, 9H), 1.36e1.40 (m, 2H), 1.44e1.48 (m, 2H), 1.92 (t,
J ¼ 7.2 Hz, 2H), 2.26e2.32 (m, 1H), 2.37e2.43 (m, 2H), 2.54e2.57
(m, 1H), 2.96e3.17 (m, 4H), 3.32e3.47 (m, 4H), 3.60e3.64 (m, 1H),
3.75 (d, J ¼ 10.8 Hz, 1H), 3.95 (d, J ¼ 10.8 Hz, 1H), 4.24e4.27 (m,
1H), 6.69 (s, 1H), 7.89 (s, 1H), 8.65 (s, 1H), 10.33 (s, 1H). 13C NMR
13C NMR (150 MHz, DMSO-d6)
d: 10.6, 15.0, 24.1, 24.9, 25.9, 28.2,
28.7, 32.2, 35.5, 38.4, 38.5, 44.3, 56.5, 60.0, 61.1, 67.1, 78.1, 155.5,
169.1, 169.8, 170.3. HRMS (AP-ESI) m/z calcd for C24H43N4O6S2
[M þ H]þ 547.2624, found 547.2642.
(150 MHz, DMSO-d6) d: 24.8, 26.0, 28.2, 28.6, 32.2, 34.4, 36.0,
38.4, 38.7, 44.6, 60.0, 61.6, 66.9, 77.7, 155.4, 169.8, 170.0, 170.1.
HRMS (AP-ESI) m/z calcd for C21H37N4O6S2 [M þ H]þ 505.2155,
found 505.2168.
5.1.9.7. Tert-butyl
(S)-3-hydroxy-1-((S)-8-(6-(hydroxyamino)-6-
oxohexylcarbamoyl)-1,4-dithia-7-azaspiro[4.4]nonan-7-yl)-1-

Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
5395
oxopropan-2-ylcarbamate (11h). Yield: 40%, mp: 98e100 ꢃC. 1H
NMR (DMSO-d6) 1.18e1.23 (m, 2H), 1.37 (s, 9H), 1.33e1.38 (m, 2H),
2.26e2.31 (m, 1H), 2.54e2.57 (m, 1H), 2.89e3.07 (m, 4H),
3.31e3.39 (m, 4H), 3.75 (d, J ¼ 10.8 Hz, 1H), 4.21 (d, J ¼ 10.8 Hz,
1H), 4.31e4.34 (m, 1H), 4.41e4.45 (m, 1H), 6.94 (d, J ¼ 8.4 Hz, 1H),
6.95e7.10 (m, 2H), 7.22 (s, 1H), 7.32e7.33 (m, 1H), 7.52e7.53 (m,
1H), 7.69 (s, 1H), 8.63 (s, 1H), 10.32 (s, 1H), 10.90 (d, J ¼ 8.4 Hz, 1H).
d
1.43e1.48 (m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.27e2.31 (m, 1H),
2.60e2.62 (m, 1H), 2.94e3.05 (m, 2H), 3.36e3.41 (m, 4H),
3.51e3.53 (m, 2H), 3.90 (d, J ¼ 10.8 Hz, 1H), 4.19 (d, J ¼ 10.8 Hz, 1H),
4.26e4.29 (m, 1H), 4.33e4.36 (m, 1H), 5.02 (s, 1H), 6.93 (s, 1H), 7.73
(s, 1H), 8.65 (s, 1H), 10.33 (s, 1H). HRMS (AP-ESI) m/z calcd for
C21H37N4O7S2 [M þ H]þ 521.2104, found 521.2114.
13C NMR (150 MHz, DMSO-d6)
d: 25.3, 26.3, 28.6, 28.7, 29.2, 32.7,
38.9, 39.1, 44.7, 53.5, 60.3, 61.7, 67.5, 78.6, 110.1, 111.9, 118.5, 118.8,
121.3, 124.4, 127.8, 136.4, 155.6, 169.6, 170.3, 170.9. HRMS (AP-ESI)
m/z calcd for C29H42N5O6S2 [M þ H]þ 620.2577, found 620.2596.
5.1.9.8. (S)-Tert-butyl
2-((S)-8-(6-(hydroxyamino)-6-oxohexylcar-
bamoyl)-1,4- dithia-7-azaspiro[4.4]nonane-7-carbonyl)pyrrolidine-
5.1.9.13. Tert-butyl (S)-6-((S)-8-(6-(hydroxyamino)-6-oxohexylcar-
1-carboxylate (11i). Yield: 60%, mp: 115e117 ꢃC. 1H NMR (DMSO-d6)
bamoyl)-1,4-
diyldicarbamate (11n). Yield: 47%, mp: 94e96 ꢃC. 1H NMR (DMSO-
d6) 1.20e1.24 (m, 4H), 1.30e1.38 (m, 22H), 1.45e1.53 (m, 4H), 1.92
dithia-7-azaspiro[4.4]nonan-7-yl)-6-oxohexane-1,5-
d
1.20e1.23 (m, 2H), 1.33 þ 1.38 (s, 9H, cis/trans), 1.35e1.40 (m, 2H),
1.44e1.49 (m, 2H), 1.67e1.77 (m, 2H), 1.78e1.83 (m, 1H), 1.92 (t,
J ¼ 7.2 Hz, 2H), 2.05e2.16 (m, 1H), 2.19e2.25 (m, 1H), 2.50e2.56 (m,
1H), 2.90e3.09 (m, 2H), 3.23e3.31 (m, 2H), 3.36e3.43 (m, 4H), 3.73
(d, J ¼ 10.8 Hz, 1H), 4.07 (d, J ¼ 10.8 Hz, 1H), 4.26e4.31 (m, 1H),
4.43e4.46 (m, 1H), 7.87 (s, 1H), 8.66 (s, 1H), 10.33 (s, 1H). HRMS
(AP-ESI) m/z calcd for C18H31N4O4S2 [M þ H]þ 431.1787, found
431.1805.
d
(t, J ¼ 7.2 Hz, 2H), 2.23e2.27 (m, 1H), 2.51e2.55 (m, 1H), 2.86e2.89
(m, 2H), 2.93e3.07 (m, 2H), 3.37e3.41 (m, 4H), 3.77 (d, J ¼ 10.8 Hz,
1H), 4.06e4.12 (m, 1H), 4.20 (d, J ¼ 10.8 Hz, 1H), 4.27e4.29 (m, 1H),
6.77 (s, 1H), 6.95 (s, 1H), 7.86 (s, 1H), 8.65 (s, 1H), 10.33 (s, 1H). 13C
NMR (150 MHz, DMSO-d6) d: 22.8, 25.3, 26.3, 28.6, 28.7, 29.1, 29.6,
32.6, 38.8, 39.1, 39.5, 44.8, 52.8, 60.4, 61.5, 67.5, 77.8, 78.5, 155.8,
156.0,169.5,170.4,170.9. HRMS (AP-ESI) m/z calcd for C29H52N5O8S2
[M þ H]þ 662.3257, found 662.3280.
5.1.9.9. (S)-Tert-butyl 4-(8-(6-(hydroxyamino)-6-oxohexylcarbamoyl)-
1,4-dithia-7- azaspiro[4.4]nonane-7-carbonyl)piperidine-1-carbox-
ylate (11j). Yield: 55%, mp: 100e102 ꢃC. 1H NMR (DMSO-d6)
5.1.10. (S)-7-(2-Aminoacetyl)-N-(6-(hydroxyamino)-6-oxohexyl)-
1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamide hydrochloride
(12a)
To a solution of compound 11a (0.25 g, 0.51 mmol) in dry EtOAc
(8 mL), was added a solution of EtOAc (8 mL) saturated by dry HCl
gas. The reaction solution was stirred at room temperature for 3 h
when the precipitation appeared. The suspension was filtered with
the filter being washed with ether to give 0.20 g of desired
compound 12a as a white powder. Yield: 90%, mp: 147e149 ꢃC. 1H
d
1.18e1.24 (m, 2H), 1.29e1.37 (m, 4H), 1.39 (s, 9H), 1.44e1.50 (m,
2H), 1.62e1.64 (m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.23e2.27 (m, 1H),
2.52e2.56 (m,1H), 2.61e2.65 (m,1H), 2.76e2.82 (m, 2H), 2.91e3.08
(m, 2H), 3.32e3.43 (m, 4H), 3.79 (d, J ¼ 10.8 Hz, 1H), 3.90e3.93 (m,
2H), 4.06 (d, J ¼ 10.8 Hz, 1H), 4.23e4.26 (m, 1H), 7.85 (s, 1H), 8.65 (s,
1H), 10.33 (s, 1H). HRMS (AP-ESI) m/z calcd for C24H41N4O6S2
[M þ H]þ 545.2468, found 545.2506.
5.1.9.10. Tert-butyl (S)-1-((S)-8-(6-(hydroxyamino)-6-oxohexylcar-
bamoyl)-1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-1-oxo-3-phenylpropan-
NMR (DMSO-d6) d 1.17e1.21 (m, 2H), 1.36e1.40 (m, 2H), 1.46e1.49
(m, 2H), 1.93 (t, J ¼ 7.2 Hz, 2H), 2.26e2.29 (m, 1H), 2.39e2.44 (m,
1H), 2.99e3.08 (m, 2H), 3.30e3.37 (m, 4H), 3.58e3.63 (m, 1H), 3.72
(d, J ¼ 10.8 Hz, 1H), 3.75e3.81 (m, 1H), 3.97 (d, J ¼ 10.8 Hz, 1H),
4.26e4.29 (m,1H), 7.96 (s,1H), 8.11 (s, 3H), 8.68 (s,1H),10.37 (s,1H).
HRMS (AP-ESI) m/z calcd for C15H27N4O4S2 [M þ H]þ 391.1474,
found 391.1452.
1
2-ylcarbamate (11k). Yield: 49%, mp: 102e104 ꢃC. H NMR (DMSO-
d6)
d
1.19e1.25 (m, 2H), 1.32 þ 1.37 (s, 9H, cis/trans), 1.36e1.41 (m,
2H), 1.46e1.50 (m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.27e2.35 (m, 1H),
2.52e2.57 (m, 1H), 2.72e2.90 (m, 2H), 2.94e3.04 (m, 2H),
3.28e3.41 (m, 4H), 3.81 (d, J ¼ 10.8 Hz, 1H), 4.25 (d, J ¼ 10.8 Hz,
1H), 4.29e4.31 (m, 1H), 4.33e4.36 (m, 1H), 7.06 (s, 1H), 7.19e7.33
(m, 5H), 7.82 (s, 1H), 8.66 (s, 1H), 10.33 (s, 1H). 13C NMR (150 MHz,
5.1.10.1. (S)-7-(3-Aminopropanoyl)-N-(6-(hydroxyamino)-6-
oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamide hydro-
chloride (12b). Yield: 88%, mp: 142e144 ꢃC. 1H NMR (DMSO-d6)
DMSO-d6)
d: 25.3, 26.3, 28.6, 29.2, 32.7, 36.7, 38.8, 39.1, 44.4, 54.1,
60.6, 61.9, 67.5, 78.5, 126.7, 128.4, 129.7, 138.3, 155.7, 169.5, 170.2,
170.3. HRMS (AP-ESI) m/z calcd for C27H41N4O6S2 [M
581.2468, found 581.2497.
þ
H]þ
d 1.20e1.24 (m, 2H), 1.36e1.40 (m, 2H), 1.44e1.49 (m, 2H), 1.92
(t, J ¼ 7.2 Hz, 2H), 2.29e2.32 (m, 1H), 2.58e2.62 (m, 1H),
2.62e2.69 (m, 2H), 2.96e3.08 (m, 4H), 3.35e3.41 (m, 4H), 3.81
(d, J ¼ 10.8 Hz, 1H), 3.95 (d, J ¼ 10.8 Hz, 1H), 4.28e4.31 (m, 1H),
7.94 (s, 3H), 8.01 (s, 1H), 8.70 (s, 1H), 10.38 (s, 1H). HRMS (AP-
ESI) m/z calcd for C16H28N4NaO4S2 [M þ H]þ 427.1450, found
427.1458.
5.1.9.11. Tert-butyl (S)-1e((S)-8-(6-(hydroxyamino)-6-oxohexylcar-
bamoyl)-1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-3-(4-hydroxyphenyl)-
1-oxopropan-2-ylcarbamate (11l). Yield: 51%, mp: 130e132 ꢃC. 1H
NMR (DMSO-d6)
d
1.20e1.24 (m, 2H), 1.32 þ 1.37 (s, 9H, cis/trans),
1.35e1.40 (m, 2H), 1.46e1.50 (m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H),
2.26e2.30 (m, 1H), 2.50e2.56 (m, 1H), 2.60e2.64 (m, 1H),
2.76e2.79 (m, 1H), 2.95e3.06 (m, 2H), 3.28e3.41 (m, 4H), 3.78 (d,
J ¼ 10.8 Hz, 1H), 4.22 (d, J ¼ 10.8 Hz, 1H), 4.25e4.31 (m, 2H), 6.64 (d,
J ¼ 8.4 Hz, 2H), 6.99 (s, 1H), 7.10 (d, J ¼ 8.4 Hz, 2H), 7.76 (s, 1H), 8.66
5.1.10.2. (S)-7-(4-Aminobutanoyl)-N-(6-(hydroxyamino)-6-
oxohexyl)-1,4-dithia- 7-azaspiro[4.4]nonane-8-carboxamide hydro-
chloride (12c). Yield: 91%, mp: 108e110 ꢃC. 1H NMR (DMSO-d6)
d
1.19e1.24 (m, 2H), 1.34e1.38 (m, 2H), 1.41e1.45 (m, 2H),
(s, 1H), 9.18 (s,1H), 10.34 (s, 1H). 13C NMR (150 MHz, DMSO-d6)
d:
1.75e1.79 (m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.25e2.29 (m, 1H),
2.40e2.43 (m, 2H), 2.55e2.59 (m, 1H), 2.74e2.82 (m, 2H),
2.88e3.08 (m, 2H), 3.33e3.45 (m, 4H), 3.80 (d, J ¼ 10.8 Hz, 1H),
3.96 (d, J ¼ 10.8 Hz, 1H), 4.25e4.28 (m, 1H), 7.97 (s, 4H), 8.71 (s,
1H), 10.39 (s, 1H). HRMS (AP-ESI) m/z calcd for C17H31N4O4S2
[M þ H]þ 419.1787, found 419.1810.
24.9, 25.9, 28.2, 28.8, 32.3, 35.5, 38.5, 38.8, 44.3, 54.1, 60.2, 61.2, 67.1,
78.1, 114.9, 127.8, 130.3, 155.3, 155.8, 169.1, 169.9, 170.3. HRMS (AP-
ESI) m/z calcd for C27H41N4O7S2 [M þ H]þ 597.2417, found 597.2425.
5.1.9.12. Tert-butyl (S)-1-((S)-8-(6-(hydroxyamino)-6-oxohexylcar-
bamoyl)-1,4- dithia-7-azaspiro[4.4]nonan-7-yl)-3-(1H-indol-3-yl)-1-
oxopropan-2-ylcarbamate (11m). Yield: 39%, mp: 116e118 ꢃC. 1H
5.1.10.3. (S)-7-((S)-2-Aminopropanoyl)-N-(6-(hydroxyamino)-6-
oxohexyl)- 1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamide hydro-
chloride (12d). Yield: 92%, mp: 167e169 ꢃC. 1H NMR (DMSO-d6)
NMR (DMSO-d6)
d
1.19e1.24 (m, 2H), 1.32 þ 1.37 (s, 9H, cis/trans),
1.36e1.41 (m, 2H), 1.46e1.51 (m, 2H), 1.93 (t, J ¼ 7.2 Hz, 2H),

5396
Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
d
1.19e1.24 (m, 3H), 1.31e1.34 (m, 2H), 1.35e1.39 (m, 2H), 1.44e1.49
(s, 1H), 10.26 (s, 1H). HRMS (AP-ESI) m/z calcd for C18H31N4O4S2
(m, 2H), 1.93 (t, J ¼ 7.2 Hz, 2H), 2.25e2.35 (m, 1H), 2.57e2.67 (m,
1H), 2.97e3.06 (m, 2H), 3.37e3.44 (m, 4H), 3.77e3.81 (m, 1H),
4.01e4.07 (m, 1H), 4.10e4.19 (m, 1H), 4.28e4.37 (m, 1H), 8.08 (s,
1H), 8.27 (s, 3H), 8.70 (s, 1H), 10.47 (s, 1H). HRMS (AP-ESI) m/z calcd
for C16H28N4NaO4S2 [M þ H]þ 427.1450, found 427.1462.
[M þ H]þ 431.1787, found 431.1805.
5.1.10.9. (S)-N-(6-(Hydroxyamino)-6-oxohexyl)-7-(piperidine-4-
carbonyl)-1,4- dithia-7-azaspiro[4.4]nonane-8-carboxamide hydro-
chloride (12j). Yield: 91%, mp: 171e173 ꢃC. 1H NMR (DMSO-d6)
d
1.20e1.23 (m, 2H),1.33e1.38 (m, 2H),1.44e1.49 (m, 2H),1.68e1.70
5.1.10.4. (S)-7-((S)-2-Amino-3-methylbutanoyl)-N-(6-(hydrox-
yamino)-6- oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carbox-
amide hydrochloride (12e). Yield: 89%, mp: 159e161 ꢃC. 1H NMR
(m, 2H), 1.79e1.84 (m, 2H), 1.93 (t, J ¼ 7.2 Hz, 2H), 2.23e2.27 (m,
1H), 2.54e2.58 (m, 1H), 2.76e2.79 (m, 1H), 2.92e3.06 (m, 4H),
3.24e3.30 (m, 2H), 3.35e3.43 (m, 4H), 3.80 (d, J ¼ 10.8 Hz, 1H), 4.06
(d, J ¼ 10.8 Hz, 1H), 4.25e4.27 (m, 1H), 7.87 (s, 1H), 8.20 (s, 2H), 8.66
(s, 1H), 10.34 (s, 1H). HRMS (AP-ESI) m/z calcd for C19H33N4O4S2
[M þ H]þ 445.1943, found 445.1974.
(DMSO-d6)
d
0.90 (d, J ¼ 6.6 Hz, 3H), 1.01 (d, J ¼ 6.6 Hz, 3H),
1.20e1.24 (m, 2H), 1.3e1.39 (m, 2H), 1.44e1.49 (m, 2H), 1.92 (t,
J ¼ 7.2 Hz, 2H), 2.11e2.15 (m, 1H), 2.26e2.30 (m, 1H), 2.55e2.58
(m, 1H), 2.98e3.04 (m, 2H), 3.35e3.45 (m, 4H), 3.74 (d,
J ¼ 10.8 Hz, 1H), 4.17e4.19 (m, 1H), 4.25 (d, J ¼ 10.8 Hz, 1H),
4.37e4.40 (m, 1H), 8.10 (s, 3H), 8.11 (s, 1H), 8.66 (s, 1H), 10.36 (s,
5.1.10.10. (S)-7-((S)-2-Amino-3-phenylpropanoyl)-N-(6-(hydrox-
yamino)-6- oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamide
hydrochloride (12k). Yield: 93%, mp: 161e163 ꢃC. 1H NMR (DMSO-
1H). 13C NMR (150 MHz, DMSO-d6)
d: 16.6, 18.5, 24.8, 25.9, 28.7,
28.9, 32.2, 38.4, 38.8, 44.0, 55.6, 60.2, 61.4, 67.1, 166.7, 169.1, 169.4.
HRMS (AP-ESI) m/z calcd for C18H33N4O4S2 [M þ H]þ 433.1943,
found 433.1991.
d6) d 1.22e1.26 (m, 2H),1.37e1.42 (m, 2H),1.44e1.51 (m, 2H),1.94 (t,
J ¼ 7.2 Hz, 2H), 2.25e2.31 (m,1H), 2.38e2.56 (m, 1H), 2.94e2.98 (m,
2H), 3.03e3.08 (m, 2H), 3.28e3.43 (m, 4H), 3.88 (d, J ¼ 10.8 Hz, 1H),
4.17 (d, J ¼ 10.8 Hz, 1H), 4.23e4.38 (m, 1H), 4.49e4.51 (m, 1H),
7.21e7.40 (m, 5H), 8.06 (s,1H), 8.40 (s, 3H), 8.70 (s,1H),10.42 (s,1H).
HRMS (AP-ESI) m/z calcd for C22H33N4O4S2 [M þ H]þ 481.1943,
found 481.1961.
5.1.10.5. (S)-7-((S)-2-Amino-4-methylpentanoyl)-N-(6-(hydrox-
yamino)-6- oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carbox-
amide hydrochloride (12f). Yield: 94%, mp: 166e168 ꢃC. 1H NMR
(DMSO-d6)
d 0.89e0.95 (m, 6H), 1.22e1.25 (m, 2H), 1.34e1.37 (m,
2H), 1.41e1.47 (m, 2H), 1.48e1.54 (m, 2H), 1.78e1.81 (m, 1H), 1.92 (t,
J ¼ 7.2 Hz, 2H), 2.26e2.30 (m,1H), 2.56e2.59 (m,1H), 2.95e3.08 (m,
2H), 3.37e3.44 (m, 4H), 3.77 (d, J ¼ 10.8 Hz, 1H), 4.15 (d, J ¼ 10.8 Hz,
1H), 4.20e4.23 (m, 1H), 4.34e4.37 (m, 1H), 8.02 (s, 1H), 8.15(s, 3H),
5.1.10.11. (S)-7-((S)-2-Amino-3-(4-hydroxyphenyl)propanoyl)-N-(6-
(hydroxyamino)-6-oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-
carboxamide (12l). Yield: 90%, mp: 181e183 ꢃC. 1H NMR (DMSO-
d6)
d 1.21e1.26 (m, 2H), 1.37e1.41 (m, 2H), 1.46e1.51 (m, 2H), 1.93
8.63 (s, 1H), 10.35 (s, 1H). 13C NMR (150 MHz, DMSO-d6)
d: 21.4,
(t, J ¼ 7.2 Hz, 2H), 2.26e2.30 (m, 1H), 2.52e2.56 (m, 1H),
2.82e2.86 (m, 1H), 2.89e2.96 (m, 1H), 3.03e3.08 (m, 2H),
3.32e3.47 (m, 4H), 3.87 (d, J ¼ 10.8 Hz, 1H), 4.16 (d, J ¼ 10.8 Hz,
1H), 4.35e4.42 (m, 2H), 6.72 (d, J ¼ 8.4 Hz, 2H), 7.16 (d,
J ¼ 8.4 Hz, 2H), 8.06 (s, 1H), 8.12 (s, 3H), 8.33 (s, 1H), 9.37 (s,1H),
23.2, 23.3, 24.8, 25.8, 28.7, 32.2, 38.4, 38.8, 44.3, 49.5, 60.3, 60.6,
67.2, 167.6, 169.0, 169.3. HRMS (AP-ESI) m/z calcd for C19H35N4O4S2
[M þ H]þ 447.2100, found 447.2132.
5.1.10.6. (S)-7-((2S,3S)-2-Amino-3-methylpentanoyl)-N-(6-(hydrox-
yamino)- 6-oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carbox-
amide hydrochloride (12g). Yield: 86%, mp: 131e133 ꢃC. 1H NMR
10.40 (s, 1H). 13C NMR (150 MHz, DMSO-d6)
d: 24.8, 25.9, 28.7,
32.2, 35.0, 38.6, 38.7, 44.0, 52.2, 60.4, 60.8, 67.1, 115.2, 124.0, 131.2,
156.7, 166.8, 169.1, 169.3. HRMS (AP-ESI) m/z calcd for
C22H32N4NaO5S2 [M þ H]þ 519.1712, found 519.1717.
(DMSO-d6)
d
0.85 (t, J ¼ 7.2 Hz, 3H), 0.99 (d, J ¼ 6.6 Hz, 3H),
1.09e1.14 (m, 1H), 1.21e1.24 (m, 2H), 1.34e1.39 (m, 2H), 1.44e1.49
(m, 3H),1.80e1.88 (m,1H),1.94 (t, J ¼ 7.2 Hz, 2H), 2.27e2.31 (m,1H),
2.55e2.58 (m, 1H), 2.97e3.04 (m, 2H), 3.43e3.49 (m, 4H), 3.74 (d,
J ¼ 10.8 Hz, 1H), 4.14e4.20 (m, 1H), 4.25 (d, J ¼ 10.8 Hz, 1H),
4.37e4.40 (m,1H), 8.10 (s,1H), 8.16 (s, 3H), 8.68 (s,1H), 10.39 (s, 1H).
HRMS (AP-ESI) m/z calcd for C19H35N4O4S2 [M þ H]þ 447.2100,
found 447.2150.
5.1.10.12. (S)-7-((S)-2-Amino-3-(1H-indol-3-yl)propanoyl)-N-(6-
(hydroxyamino)- 6-oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-
carboxamide hydrochloride (12m). Yield: 86%, mp: 180e182 ꢃC. 1H
NMR (DMSO-d6)
d 1.22e1.27 (m, 2H), 1.37e1.42 (m, 2H), 1.44e1.50
(m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.26e2.33 (m, 1H), 2.55e2.59 (m,
1H), 2.85e3.21 (m, 4H), 3.26e3.49 (m, 4H), 3.69e3.75(m, 1H),
4.05e4.10 (m, 1H), 4.37e4.39 (m, 1H), 4.40e4.42 (m, 1H), 7.00e7.13
(m, 2H), 7.39e7.48 (m, 2H), 7.64 (s,1H), 8.05 (s,1H), 8.16 (s, 3H), 8.37
(s, 1H), 8.68 (s, 1H), 10.39 (s, 1H), 11.12 (s, 1H). 13C NMR (150 MHz,
5.1.10.7. (S)-7-((S)-2-Amino-3-hydroxypropanoyl)-N-(6-(hydrox-
yamino)-6- oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carbox-
amide hydrochloride (12h). Yield: 88%, mp: 158e160 ꢃC. 1H NMR
DMSO-d6) d: 25.3, 26.4, 26.5, 29.2, 32.6, 39.0, 39.2, 44.8, 53.4, 61.0,
(DMSO-d6)
d
1.20e1.25 (m, 2H), 1.33e1.38 (m, 2H), 1.44e1.48 (m,
61.2, 67.3, 106.6, 111.9, 119.0, 120.5, 121.5, 126.1, 127.7, 136.5, 167.7,
169.5, 169.7. HRMS (AP-ESI) m/z calcd for C24H34N5O4S2 [M þ H]þ
520.2052, found 520.2143.
2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 2.27e2.31 (m, 1H), 2.59e2.62 (m, 1H),
2.95e3.07 (m, 2H), 3.39e3.46 (m, 4H), 3.64e3.78 (m, 2H), 3.85 (d,
J ¼ 10.8 Hz, 1H), 4.22 (d, J ¼ 10.8 Hz, 1H), 4.30e4.32 (m, 1H),
4.37e4.40 (m, 1H), 5.47 (s, 1H), 7.97 (s, 1H), 8.23 (s, 3H), 8.64 (s, 1H),
10.38 (s, 1H). HRMS (AP-ESI) m/z calcd for C16H29N4O5S2 [M þ H]þ
421.1579, found 421.1585.
5.1.10.13. (S)-7-((S)-2,6-Diaminohexanoyl)-N-(6-(hydroxyamino)-6-
oxohexyl)-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamide dihydro-
chloride (12n). Yield: 89%, mp: 195.197 ꢃC. 1H NMR (DMSO-d6)
d
1.20e1.24 (m, 2H),1.35e1.49 (m, 6H),1.50e1.58 (m, 2H),1.68e1.73
5.1.10.8. (S)-N-(6-(Hydroxyamino)-6-oxohexyl)-7-((S)-pyrrolidine-
2-carbonyl)- 1,4-dithia-7-azaspiro[4.4]nonane-8-carboxamide hydro-
chloride (12i). Yield: 88%, mp: 125e127 ꢃC. 1H NMR (DMSO-d6)
(m, 2H), 1.93 (t, J ¼ 7.2 Hz, 2H), 2.25e2.29 (m, 1H), 2.57e2.62 (m,
1H), 2.70e2.80 (m, 2H), 2.95e3.07 (m, 2H), 3.38e3.44 (m, 4H), 3.85
(d, J ¼ 10.8 Hz, 1H), 4.20 (d, J ¼ 10.8 Hz, 1H), 4.29e4.31 (m, 1H),
4.37e4.40 (m, 1H), 7.91 (s, 3H), 8.17 (s, 1H), 8.33 (s, 3H), 8.68 (s, 1H),
d
1.20e1.24 (m, 2H), 1.34e1.39 (m, 2H), 1.44e1.49 (m, 2H), 1.81e1.90
(m, 2H), 1.88e1.96 (m, 3H), 2.26e2.29 (m, 1H), 2.32e2.36 (m, 1H),
2.59e2.62 (m, 1H), 2.95e3.08 (m, 2H), 3.13e3.24 (m, 2H),
3.39e3.47 (m, 4H), 3.73 (d, J ¼ 10.8 Hz, 1H), 4.18 (d, J ¼ 10.8 Hz, 1H),
4.37e4.39 (m, 1H), 4.54e4.59 (m, 1H), 8.15 (s, 1H), 8.52 (s, 2H), 8.66
10.41 (s, 1H). 13C NMR (150 MHz, DMSO-d6)
d: 20.0, 24.9, 25.9, 26.3,
28.7, 32.2, 38.2, 38.5, 38.7, 43.9, 50.7, 60.2, 61.0, 67.2, 167.0, 169.2,
169.5. HRMS (AP-ESI) m/z calcd for C19H36N5O4S2 [M þ H]þ
462.2209, found 462.2273.

Y. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5387e5397
5397
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Acknowledgements
This work was supported by National Nature Science Foundation
of China (grant no. 30772654 and no. 36072541), National High
Technology Research and Development Program of China (863
project; grant no. 2007AA02Z314), National Science and Technology
Major Project (grant no. 2009ZX09103-118), and Academic Award
for Young Doctoral Postgraduate of Ministry of Education of China.
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Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.08.045.
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