A pentanoic acid derivative targeting matrix metalloproteinase-2 (MMP-2) induces apoptosis in a chronic myeloid leukemia cell line

Article abstract of DOI:10.1016/j.ejmech.2017.09.052

Depending on our previous observations, some compounds of pentanoic acid were designed and synthesized. Characterization of the synthesized compounds was done by mass, NMR and IR spectroscopy as well as elemental analysis. Among the synthesized molecules, (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(pentylamino) pentanoic acid (Cpd 11) was found as a lead and potent inhibitor of matrix metalloproteinase-2 (MMP-2). Molecular modeling and enzyme inhibition studies were done to confirm the interaction or inhibitory potential of this compound. Thereafter, the biological screening was done through cytotoxicity, anti-invasion and apoptosis-related assays. Docking analysis revealed that Cpd 11 interacted with the target molecule MMP-2 and with MMP-9. However, enzyme inhibition assay showed 3-fold MMP-2 inhibition compared to MMP-9. Cytotoxicity assay showed the inhibitory potential of Cpd 11 against K562 cell line having IC₅₀ value of 17.9 ± 0.01 μM after 48 h of incubation. The cell death was apoptotic in nature as revealed from the annexin V and sub-G1 cell cycle arrest assay. Besides this, Cpd 11 also exhibited dose dependent anti-invasive activity into K562 cell line. On the other hand, flow cytometry and western blot data revealed Cpd 11 induced downregulation of MMP-2 in K562 cell line after 48 h of incubation that might be linked with the anti-invasive and apoptotic activity furthermore. Therefore, the overall results validated each method and make this molecule as a potent MMP-2 inhibitor that blocked the invasion and could bring apoptosis at later stages in K562 cells sparing the normal ones.

Full text of DOI:10.1016/j.ejmech.2017.09.052

Accepted Manuscript
A pentanoic acid derivative targeting matrix metalloproteinase-2 (MMP-2) induces
apoptosis in a chronic myeloid leukemia cell line
Avinaba Mukherjee, Nilanjan Adhikari, Tarun Jha
PII:
S0223-5234(17)30767-5
10.1016/j.ejmech.2017.09.052
EJMECH 9767
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 May 2017
Revised Date: 13 September 2017
Accepted Date: 25 September 2017
Please cite this article as: A. Mukherjee, N. Adhikari, T. Jha, A pentanoic acid derivative targeting matrix
metalloproteinase-2 (MMP-2) induces apoptosis in a chronic myeloid leukemia cell line, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.052.
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ACCEPTED MANUSCRIPT

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A pentanoic acid derivative targeting matrix metalloproteinase-2 (MMP-2) induces
apoptosis in a chronic myeloid leukemia cell line
Avinaba Mukherjee#, Nilanjan Adhikari, Tarun Jha*
Natural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry,
Department of Pharmaceutical Technology, P.O. Box 17020, Jadavpur University, Kolkata
700032, India.
#Present address: Department of Zoology, Charuchandra College, Kolkata 700029, India.
*Corresponding author:
Tel: 9133 2457 2495 (o), 9133 2438 3814 (r), +919433187443 (m)
e-mail: tjupharm@yahoo.com
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Abstract:
Depending on our previous observations, some compounds of pentanoic acid were designed
and synthesized. Characterization of the synthesized compounds was done by mass, NMR
and IR spectroscopy as well as elemental analysis. Among the synthesized molecules, (2S)-5-
oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(pentylamino) pentanoic acid (Cpd 11) was
found as a lead and potent inhibitor of matrix metalloproteinase-2 (MMP-2). Molecular
modeling and enzyme inhibition studies were done to confirm the interaction or inhibitory
potential of this compound. Thereafter, the biological screening was done through
cytotoxicity, anti-invasion and apoptosis-related assays. Docking analysis revealed that Cpd
11 interact with the target molecule MMP-2 and with MMP-9. However, enzyme inhibition
assay showed 3-fold MMP-2 inhibition compared to MMP-9. Cytotoxicity assay showed the
inhibitory potential of Cpd 11 against K562 cell line having IC50 value of 17.9±0.01 µM after
48 h of incubation. The cell death was apoptotic in nature as revealed from the annexin V and
sub-G1cell cycle arrest assay. Besides this, Cpd 11 also exhibited dose dependent anti-
invasive activity into K562 cell line. On the other hand, flow cytometry and western blot data
revealed Cpd 11 induced downregulation of MMP-2 in K562 cell line after 48 h of
incubation that might be linked with the anti-invasive and apoptotic activity furthermore.
Therefore, the overall results validated each method and make this molecule as a potent
MMP-2 inhibitor that blocked the invasion and could bring apoptosis at later stages in K562
cells sparing the normal ones.
Keywords: pentanoic acid, MMP-2 inhibition, enzyme assay, apoptosis, cytotoxicity,
molecular docking study
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1. Introduction
Metalloproteinases are associated with a variety of pathological and cellular functions
including matrix degradation during cancer cell proliferation and migration [1]. Till now, 26
different matrix metalloproteinases (MMPs) are identified. These are categorized into six
subgroups (namely collagenases, gelatinases, matrilysins, stromelysins, membrane type
MMPs and other MMPs). Overexpression of MMPs is related to a number of diseases such as
cardiovascular and neurological disorders as well as the formation of cancer through
uncontrolled cellular proliferation, invasion and metastasis [2-4]. Among these MMPs,
MMP-2 is found to be upregulated in most of the cancers particularly in leukemia where the
leukemic cells proliferate in an uncontrollable manner [5]. In leukemia, leukemic cell blasts
occur in an excess amount which later triggers invasion into various tissues [6]. Regarding
this, MMP-2 expression plays a role in the increased vessel density found in the bone marrow
of leukemia patients by accelerating in vitro endothelial cell migration [7]. Moreover, the cell
surface association between MMP-2 and integrins is found to be involved in the growth and
progression of acute and chronic myeloid leukemia cells [8]. Therefore, designing and
synthesizing MMP-2 inhibitors may be a valuable strategy for blocking the leukemia cell
growth. However, very few MMP-2 inhibitors have been reported to control leukemia
particularly [9-12]. Apart from this, most of the MMP-2 inhibitors either failed to induce
apoptosis against leukemia or lacks their target specificity.
In this context, some compounds of pentanoic acid have been designed and synthesized to
judge their MMP-2 inhibitory properties along with cytotoxic and apoptotic features. As far
as our earlier observations were concerned, a number of potent and selective MMP-2
inhibitors over other MMPs were reported [13, 14]. However, none of these derivatives were
able to generate cytotoxicity in cancer cells (such as A549 and MDA-MB-231 cell lines, etc)
at a lower dose (data not shown). Some of these compounds were found to be partially
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cytotoxic in chronic myeloid leukemia K562 cell line over the other MMP-2 secretive cells
like A549 (non-small cell lung cancer cell line), MDA-MB-231 (breast cancer cell line) (data
not shown). It may be assumed that poor solubility of these compounds due to the
hydrophobic aryl functions may hinder the cellular permeability and in turn, may result in a
low degree of cytotoxicity though a higher MMP-2 inhibition was achieved. In search of the
better cytotoxicity, some new compounds were designed and synthesized to achieve the
better solubility (Fig. 1). It may also be assumed that due to free carboxylic acid function
these analogs may bind to the zinc ion at the MMP-2 active site as evidenced in our earlier
observations [13, 14]. In this article, only the hydrophobic aryl substituent directed towards
the S1′ pocket was modified with the smaller substituent to enhance the solubility criteria
without altering other structural features.
Fig. 1. Molecular modification of pentanoic acids for achieving higher solubility
Among the designed and synthesized molecules, the lead compound was obtained by
analyzing the results of enzyme assay and cytotoxicity profiling against a variety of cancer
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cell lines (such as A-549, MDA-MB-231, U-937 and K562) keeping peripheral blood
mononuclear cells (PBMC) as the positive control. Moreover, MMP-2 expression analysis
and invasion assay were performed in K562 cell line by applying the best active molecule as
these molecules exhibited comparatively better cytotoxicity in K-562 cell line. Besides this,
the apoptotic efficacy of the best active MMP-2 inhibitor was also analyzed to establish the
cytotoxic property in chronic myeloid leukemia.
2. Results and discussion
2.1. Synthesis
Derivatives of pentanoic acid (Cpd 5-12) were prepared according to Scheme 1.
O
O
H2N
COOH
O2N
S
NH
a
O2N
S
Cl
+
O
COOH
O
HOOC
COOH
(1)
(2)
(3)
b
HOOC
O
S
O
S
O2N
NH
c
O2N
N
O
O
HOOC
CONHR
O
(4)
(5-12)
a: 2(N) NaOH; b: CH3COCl, Reflux, 2 hr.; c: RNH2, 6(N) HCl
Scheme 1. Synthetic route of the designed molecules
The 4-Nitrobenzenesulfonyl chloride (1) was condensed with L(+)-glutamic acid (2) in
presence of 2(N) sodium hydroxide solution to obtain 2-N-(4'-nitrobenzenesulphonyl)-L(+)-
glutamic acid (3). The diacid (3) was cyclized with acetyl chloride to get 1-(4'-
nitrobenzenesulphonyl)-5-oxopyrrolidine-2-carboxylic acid (4). The monoacid (4) was
treated with different amines to get the desired pentanoic acids (5-12). All the final products
were characterized by mass, 1H NMR, 13C NMR and FTIR spectroscopy as well as elemental
analyses. The physicochemical properties of these compounds (cpd 5-12) are shown in Table
1.
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Table 1. Physicochemical parameters of the final compounds (Cpds 5-12)
Cpda
5
R
M.P.(°C)
181-183
206-208
219-221
235-237
209-211
204-206
215-217
191-193
% Yield
63.24
81.91
78.72
92.06
89.25
76.86
86.82
88.24
Molecular Formula
C11H13N3O7S
C12H15N3O7S
C13H17N3O7S
C14H19N3O7S
C15H21N3O7S
C15H21N3O7S
C16H23N3O7S
C18H19N3O7S
MW
H
331.30
345.33
359.36
373.38
387.41
387.41
401.44
421.43
Me
6
Et
7
i-Pr
n-But
i-But
n-Pent
Benz
8
9
10
11
12
aCompound number
2.2. Enzyme inhibition study
In enzyme inhibition study, MMP kits were taken and exposed to the synthesized
compounds. Then the chromogenic substrate was applied after sufficient time of exposure.
The concentration of these substrates was detected by its absorbance value at 410 nm in a
microplate photometer. A gradual decrease in the absorbance indicated the compound
induced inhibition of the MMPs action. As far as the MMP-2 and MMP-9 inhibitory
activities of these final compounds (Cpd 5-12) were concerned, some compounds show
MMP-2 selectivity over MMP-9 enzyme (Cpds 5, 6, 8 and 12) whereas other compounds are
nonselective (Cpd 7, 9, 10 and 11) (Table 2). The Cpd5 without having any substitution at
the R position (Table 2) results in 4.24 folds MMP-2 selectivity over MMP-9 whereas
substitution with the methyl group (Cpd 6) at R position (Table 2) yields about 2 folds loss
in MMP-2 inhibition compared to Cpd 5 though it shows 2.63 folds selectivity over MMP-9.
Increasing the linear alkyl chain length (such as ethyl at R position, Table 2) results in
nonselectivity rather it (Cpd 7) shows the better affinity towards MMP-9 compared to MMP-
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2. Substitution with the branched alkyl function at R position (such as i-propyl, Cpd 8) shows
more or less similar inhibitory profile compared to the ethyl analog (Cpd 7). It also shows
2.82 folds MMP-2 selectivity over MMP-9. Increasing more linearity (chain length) with the
n-butyl function (Cpd 9) at the R position yields nonselectivity whereas branching (such as i-
butyl, Cpd 10) rather exhibits comparatively better affinity towards MMP-9. For the higher
linear alkyl substitution (such as n-butyl and n-pentyl at R position, Table 2), the MMP-2
inhibitory activity is found to be more or less similar. Moreover, the n-pentyl function (Cpd
11) at the R position (Table 2) also yields nonselectivity. Interestingly, aryl function (such as
benzyl, Cpd 12) at the R position (Table 2) yields potent MMP-2 inhibition and more than
25 folds selectivity over MMP-9. Therefore, it may be assumed that smaller alkyl groups
(methyl, ethyl) at R position favor MMP-2 inhibition and selectivity over MMP-9 whereas
the higher bulky and linear substitution (such as n-butyl, n-pentyl) disfavors MMP-2
selectivity. Moreover, the bulky branched alkyl substitution (such as i-butyl) at R position
disfavors activity. Interestingly, it is noticed that aryl substitution (such as benzyl) at R
position may be necessary for maintaining the higher MMP-2 inhibitory potency as well as
selectivity over MMP-9.
Besides this, MMP-1, MMP-8, MMP-12 and MMP-14 inhibitory assays were also done.
The results of the other enzyme (apart from gelatinase sub group) inhibitory assays indicated
that the Cpd 5 and Cpd 12 having the inhibitory potential against the other MMPs than
gelatinase (Table 3) whereas Cpd 11 shows the lower specificity upon the inhibition of the
other MMPs than gelatinase subgroup enzymes.
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Table 2. Enzyme inhibitory activity and cytotoxic activity profile of pentanoic acids (Cpds
5-12)
Cpda
MMP-2
(IC50)b
MMP-9
(IC50)b
Cytotoxicity (IC50)c
A-549
MDA-MB- K-562
U-937
PBMC
231
1.65±0.52 7.00±0.7 >500
2.85±0.2 7.50±0.73 >500
>500
>500
>500
>500
>500
>500
>500
>1000
>1000
5
6
5.34±0.23 4.80±0.81 221.10±0.09 >500
2.12±0.33 5.97±0.43 >500 >500
4.07±0.11 6.36±0.56 276.10±0.12 >500
8.52±0.04 5.14±0.03 >500 >500
89.37±0.12 >1000
7
19.80±0.2 95.12±0.23 >1000
19.70±0.32 94.01±0.19 >1000
42.45±0.23 93.76±0.05 >1000
8
9
10
11
12
4.80±0.03 6.80±0.04 205.80±0.1 162.00±0.13 17.90±0.04 32.14±0.07 >1000
0.21±0.02 6.37±0.07 342.60±0.09 174.80±0.09 23.41±0.02 102.33±0.03 >1000
aCompound number; bActivity in µM; cValues taken after 48 h of incubation
Table 3. MMP-1, MMP-8, MMP-12, MMP-14 enzyme inhibitory activity of pentanoic acids
(Cpds 5-12)
Cpda
Enzyme Inhibition (IC50)c
MMP-1
>500
>500
>500
>500
>500
>500
>500
>500
MMP-8
MMP-12
MMP-14
76.3±0.04
>500
198.9±0.12
401.1±0.23
298.5±0.03
>500
100.1±0.92
>500
5
6
109.1±0.03
493.2±0.53
198.3±0.01
>500
87.1±0.06
301.1±0.1
111.35±0.09
309.3±0.12
435.8±0.1
109.0±0.02
7
8
345.8±0.04
483.1±0.32
>500
9
10
11
12
493.25±0.98
95.2±0.88
172.80±0.14
aCompound number
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2.3. Cytotoxicity assay
In cytotoxicity assay, cells were incubated with the synthesized compounds. After the
exposure, MTT was added which forms crystal violet coloration in live cells after addition of
DMSO. A gradual decrease in the absorbance of the crystal violet coloration indicated
compound induced cellular death. All these synthesized compounds are screened against
some cancer cell lines (A-549, MDA-MB-231, K-562 and U-937) for their possible
cytotoxicity along with normal peripheral blood mononuclear cell (PBMC) to judge whether
these analogs are cytotoxic or not. All these compounds are found to be inactive in normal
cell line PBMC (> 1000 µM).
Regarding the cytotoxic activity in A-549 cell line, most of these compounds are inactive
(Cpds 5, 6, 8 and 10) whereas some of them exhibit cytotoxicity in high doses (Cpds 7, 9, 11
and 12). It is interesting to notice that compounds with either linear alkyl (straight chain)
functions (such as ethyl, n-butyl, n-pentyl) or aryl function (such as benzyl) may retain A-549
inhibitory activity at high doses. Branching at the R position completely abolishes the activity
profile as far as the A-549 inhibition is concerned (> 500 µM). The higher linear (straight)
function (such as n-pentyl, Cpd 11) is the highest cytotoxic in A-549 cell line (IC50
=
205.8±0.1 µM) whereas incorporation of aryl function such as benzyl (Cpd 12) may reduce
the activity (IC50 = 342.6±0.09 µM). Therefore, regarding the cytotoxicity in A-549 cell line,
the higher linear (straight chain) alkyl substitution at R position (Table 2) is favored
compared to the aryl function. Regarding MDA-MB-231 cell cytotoxicity, only the highly
linear n-pentyl (Cpd 11) and the benzyl (Cpd 12) analogs retain cytotoxicity in high doses
(IC50 = 162±0.13 and 174.8±0.09 µM respectively). Therefore, it may be assumed that n-
pentyl substitution (Cpd 11) should be optimum to retain MDA-MB-231 cellular cytotoxicity
in high dose.
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Compared to A-549 and MDA-MB 231 cell lines, these compounds are found to be better
effective in K-562 and U-937 cell lines. Regarding the cytotoxic profile in K-562 cell line,
the SAR data suggests that the smaller groups such as hydrogen (Cpd 5), methyl (Cpd 6) and
ethyl (Cpd 7) at the R position (Table 2) result in inactivity. Interestingly, branching at the R
position with the i-propyl moiety (Cpd 8) result in effective inhibition against K-562 (IC50 =
19.8±0.2 µM) whereas increasing the length with the i-butyl function (cpd 10) at R position
yields more than 2 folds reduction in cytotoxicity (IC50 = 42.45±0.23 µM). The
corresponding linear alkyl function, i.e., n-butyl (Cpd 9) results in more or less similar
activity (IC50 = 19.7±0.32 µM) compared to the i-propyl analog (Cpd 8).Increasing the chain
length with the n-pentyl function (Cpd 11) at the R position is found to be similar active
(IC50 = 17.9±0.04 µM) compared to the i-propyl (Cpd 8) and n-butyl analog (Cpd 9). Apart
from linear and branched alkyl substitution at R position, interestingly, it is observed that the
aryl function such as the benzyl moiety (Cpd 12) produces comparatively effective inhibition
(IC50 = 23.41±0.02 µM). Therefore, it may be assumed that aryl function may be important at
this position to maintain the cytotoxic profile whereas long chain alkyl functions (namely n-
pentyl) impart the highest cytotoxicity as far as these glutamine analogs are concerned.
These compounds (Cpds 5-12) are also screened against U-937 cell line. Except Cpd 5 and
6, other analogs (Cpds 7-12) exhibit moderate activity against U-937 cell line though the
cytotoxicity of these molecules (Cpds 7-12) over U-937 is found better compared to the
cytotoxicity in A-549 and MDA-MB-231 cell lines. It is interesting to notice that compounds
with no substitution and smaller alkyl (methyl) substitution (Cpds 5-6) are inactive in U-937
cell line whereas increasing the chain length and bulkiness either linear or branched alkyl
functions (Cpds 7-10) at R position yield cytotoxic feature though no such difference is
observed regarding the cytotoxicity among them. Interestingly, the bulky linear group at the
R position (such as n-pentyl: Cpd 11) is found to exhibit 3 folds higher potency (IC50
=
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32.14±0.07 µM) compared to the lesser alkyl functions (Cpds 7-10). Moreover, the aryl
moiety such as the benzyl substitution (Cpd 12) at the R position (Table 2) yields more than
3 folds loss in U-937 cytotoxicity (IC50 = 102.33±0.03 µM) compared to the n-pentyl analog
(Cpd 11).
Therefore, combining results of all these cytotoxic activities found in these different cell
lines, it may be assumed that the n-pentyl analog (Cpd 11) is the best one regarding
producing cytotoxic effects in all these four cell lines though it produces nonselectivity in
MMP assay (Table 2). As a result, Cpd 11 was taken into consideration for further biological
screening.
2.4. DNA nick generation assay
The 4',6-diamidino-2-phenylindole (DAPI) was used to stain nicked DNA, if any. Nicked
DNA showed increased fluorescence of DAPI, which bind with the nicked region of DNA.
As Cpd 11 shows the best cytotoxic efficacy in K-562 cell line, it was further studied in K-
562 cells for DNA nick generation. After staining with DAPI the fixed cells either untreated
or treated with Cpd 11 were visualized under fluorescence microscope. No fluorescence was
observed in K562 cell line (Fig. 2A). Detectable increased fluorescence of DAPI was found
after 48 h in K562 cells when treated with increasing doses (2.5-10 µM) of Cpd 11 (Fig. 2B-
2D). This dose dependent increase in fluorescence of DAPI indicates that this Cpd 11 bears
the ability to bring nick in cellular DNA which is a hallmark feature of apoptosis.
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Fig. 2. Fluorescence imaging of K562 cells after DAPI staining. K562 cells was being treated
with Cpd 11 at the doses of (A) 0µM (B) 2.5µM (C) 5µM (D) 10µM and stained with DAPI
to observe DNA nick generation. Arrow indicates nicked DNA with intense fluorescence.
2.5. Apoptotic assay after annexinV/propidium iodide staining
Flow cytometry by dual staining of annexin V and propidium iodide (PI) is a technique by
which apoptotic or necrotic cell death percentage is being calculated. The increase in both
annexin-V positive and PI negative cells indicate early apoptosis and both annexin-V and PI
positive cells indicate late apoptosis when flow cytometric data are being analyzed after dual
staining with annexin V-FITC and PI. The flow cytometric analysis reveals a progressive
increase in the annexinV-FITC positive population of cells as compared to the untreated
control (Fig. 3). Cpd 11 induces almost upto 300 fold both early and late cellular apoptosis
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when compared with the untreated control. This data suggests the state of drug induced
apoptosis.
Fig. 3. AnnexinV-FITC/PI assay. Flow cytometric analysis after K562 either (A) untreated or
treated with (B) 2.5 µM (C) 5 µM and (D) 10 µM dose of Cpd 11.
2.6. Cell cycle assay
Cell cycle stages were being identified through flow cytometry after PI staining followed by
RNAse treatment. The increase in sub-G1 cell population in cell cycle indicated cellular
apoptosis and arrest. In this experiment, flow cytometric analysis indicates a significant
increase in sub diploid cell population (sub-G1) of K562 cell line after the treatment of the
Cpd 11. An increase of sub-diploid cell population (sub-G1) from 1% (at 0 µM dose) to
4.6% (in 10 µM dose) suggests that the Cpd 11 has the potentiality to block the cell
progression and survivability by arresting the sub-G1cell cycle arrest (Fig. 4).
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Fig. 4. Cell cycle analysis after PI staining. Cpd 11 at the doses of (A) 0 µM (B) 2.5 µM (C)
5 µM and (D) 10 µM was given to K562 cells and sub-G1 cell cycle arrest (M8) was in flow
cytometry after being stained with PI. G1, G1/S and G2/M stages are designated as M3, M6,
M7 respectively.
2.7. Invasion assay of K562 cell line
The anti-invasiveness of the synthesized compounds was evaluated by a fluorimetric QCM
EC Matrix Cell Invasion Assay kit (Millipore, USA) based on Boyden chamber principle.
Modulation in the invasion process of K562 cell line was performed and the percentages of
invaded cells are graphically presented in Fig. 5A. The inhibition was found to be
12.40±1.15%, 14.20±0.9%, 24.10±0.182% after induction of 2.5 µM, 5 µM and 10 µM of
Cpd 11 treatment respectively (Fig. 5A) which shows highly significant ($P<0.001) when
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compared to untreated control (0±1.35%). These results indicate that the Cpd 11 has the
potentiality to be anti-invasive against K562 cell line.
Fig. 5. Invasion assay and MMP-2 expression analysis. (A) Invasion assay of K562 cells after
.
being treated with 0, 2.5, 5 and 10 µM of Cpd 11 for 24 hours. Data are expressed as
percentage of control and presented as mean ± SD. $P<0.001 were considered as statistically
significant. (B) Western blots of MMP-2 and β-tubulin were being done. (C) Relative band
intensities of MMP-2 and β-tubulin are calculated. β-tubulin was being used for loading
correction. Data were expressed as percentage of control and presented as mean ± SD.
$P<0.001 are considered as statistically significant. (D) Flow cytometric analysis of MMP-2
expression analysis. V2-L and V2-R were being considered as MMP-2 negative and MMP-2
positive cells respectively.
2.8. MMP2 expression assay and western blot analysis
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MMP-2 expression was analyzed through western blot and flow cytometry. Western blot was
done to confirm the Cpd 11 induced downregulation of MMP-2. Results of western blot
analysis indicated Cpd 11 downregulated the expression of MMP-2 after 48 h of induction
(Fig. 5B). Band intensity was found to be lowered after treatment of Cpd 11 which is highly
significant ($P<0.001) (Fig. 5C).
Furthermore, flow cytometry was done to check Cpd 11 induced modulation of MMP-2
expression. Untreated or Cpd 11 treated cells were fixed and stained with MMP-2 antibody.
Thereafter, cells were stained with the secondary antibody labeled with fluorescein
isothiocyanate (FITC). The MMP-2 positive cell population was found to be inhibited after
treatment with Cpd 11. Flow cytometric data revealed that at 2.5 µM, 5 µM and 10 µM of
Cpd 11 treatment 26.30%, 18.90% and 15.60% cells were found to MMP-2 positive
respectively whereas the untreated one showed 98.4% of MMP-2 positivity (Fig. 5D).
Therefore, this data indicates that the synthesized lead compound (Cpd 11) has the
potentiality to inhibit the MMP-2 expression almost upto 80.10% at the highest dose (10 µM)
which is significant.
2.9. Molecular docking study
The ligand-enzyme interaction study of these compounds (Cpd 5-12) with both MMP-2 and
MMP-9 was done by molecular docking where the particular binding site and affinity of the
ligand for the specific enzyme was calculated and interpreted. All these compounds were
docked into the active site of MMP-2 and MMP-9 (Fig. 6).
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Fig. 6. Docked conformation of compounds (Cpd 5-12) along with the structure-based
hydrophobic contour map in A) MMP-2 enzyme (pdb: 1HOV), B) MMP-9 enzyme (pdb:
2OW1)
Regarding the docking interactions of these compounds (Cpd 5-12) with both these enzymes,
it is noticed that all these compounds coordinate with the catalytic Zn2+ ion at the active site
of these enzymes through the carboxylic acid function (Fig. 6A and Fig. 6B). The 2D view of
docking interactions for all these compounds (Cpd 5-12) with both MMP-2 and MMP-9
individually are shown in Fig. 7 and Fig. 8 respectively.
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Fig. 7. Molecular docking interaction of compounds (Cpd 5-12) with MMP-2 enzyme (pdb:
1HOV)
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Fig. 8. Molecular docking interaction of compounds (Cpd 5-12) with MMP-9 enzyme (pdb:
2OW1)
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Here, the ligand-enzyme interaction suggests that the carboxyl group of these compounds
(Cpd 5-12) is making a salt bridge with the catalytic zinc for both MMP-2 (PDB: 1HOV) and
MMP-9 (PDB: 2OW1) enzymes. In case of MMP-2, the carboxyl groups of these compounds
(Cpd 5-12) interacts with Zn166 (Fig. 7) whereas the same carboxyl group interacts with
Zn444 of MMP-9 (Fig. 8) at the enzyme active site. The p-Nitrophenylsulfonyl function
enters into the hydrophobic S1′ pocket of MMP-2 enzyme formed by Leu82, Leu83, Val17,
Tyr142, Ile141, Pro140, Ala139 and Leu116 (Fig. 6 and Fig. 7) whereas the same function
enters into the hydrophobic pocket formed by the amino acid residues namely Tyr423,
Met422, Tyr420, Leu397, Ala417, Leu418, Val398 and Leu397 (Fig. 8). Regarding the
docking interaction with the MMP-2 enzyme (Fig. 7), except Cpd 6 and 9, phenyl group
associated with the sulphonyl function of other compounds (Cpds 5, 7, 8, 10, 11 and 12)
forms a π-π interaction with His120 amino acid residue at the enzyme active site. Apart from
π-π interaction, the phenyl group is found to form π-cation interaction with catalytic Zn2+ ion
(Fig. 7). In some of these compounds (Cpd 5, 7, 11), the nitro group forms salt bridge
interaction with the negatively charged Glu121 amino acid residue. Some compounds (Cpd
6, 9, 12) forms π-cation interaction with His120 by the nitro group substituted at the para
position of the phenyl ring. Regarding the hydrogen bonding interaction, it is observed that
one of the sulphonyl oxygen atoms acts as a hydrogen bond acceptor function through
interacting with the backbone amino acid residues Leu83 and Ala84 for compounds 8, 10 and
12 (Fig. 7). Moreover, backbone amino acid Ala84 is also found to form a hydrogen bond
with the amide function adjacent to the sulfonyl group (Cpd 5-9, 11-12). The long chain
alkyl functions associated with the carboxamide group tend towards the S2′ pocket
surrounded by hydrophobic amino acid residues namely His85, Ala88, Pro89, Ala125 and
Phe87 (Fig. 6 and Fig. 7). Ala86 is another important backbone amino acid residue that is
found to form hydrogen bonding interaction with the carbonyl group of the carboxamide
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function (Cpd 5-8, 12). Due to the higher flexibility of the higher alkyl function adjacent to
the carboxamide group, the carbonyl group may form hydrogen bonding interaction with
His85 (Cpd 10) or His120 (Cpd 11).
Regarding the molecular docking study of these compounds with MMP-9, The p-nitrophenyl
group associated with the sulphonyl group enters into the hydrophobic S1′ pocket surrounded
by amino acid residues Tyr423, His401, Tyr420, Leu397, Leu418, Val398 (Fig. 6).
Moreover, the phenyl group is found to interact with His401 amino acid residue through π-π
stacking interaction (Cpd 5-6, 8-10). For Cpds 7 and 11, a π-cation interaction is noticed
between the nitro group and His401 amino acid residue (Fig. 8). One of the sulphonyl oxygen
atom forms hydrogen bonding interaction with backbone amino acids Leu188 (Cpd 7) or
Ala89 (Cpd 9, 11) (Fig. 8). Again, the amide group of the carboxamide moiety is found to
form hydrogen bonding interaction with Gly186 (Cpd 5-6, 8). The long chain alkyl function
due to higher flexibility tends towards the hydrophobic S2′ pocket formed by amino acid
residues namely Gly186, Leu187, Leu188, His190 and Gly178 (Fig. 6 and Fig. 8).
3. Conclusion
In the present work, an MMP-2 targeted pentanoic acid having good cytotoxicity against
leukemia cell line has been identified as the lead. Based on our earlier observations [13, 14],
some new pentanoic acid analogs were designed and synthesized keeping in mind the
solubility factor compared to the earlier analogs. All these analogs exhibited the gelatinase
inhibition in lower micromolar concentration as it was presumed that these analogs were
structurally related to the earlier reported compounds. The major issue is to judge whether the
cytotoxicity has been achieved or not by incorporating the nitro function in the phenyl ring at
the para position of the aryl moiety. Interestingly, these newer analogs exhibited higher
cytotoxicity compared to the earlier reported analogs [14] though these newer analogs
showed lesser gelatinase inhibition over the earlier reported compounds. Among all these
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synthesized compounds, the n-pentyl analog (Cpd 11) may be considered as the lead as this
compound produces comparatively better cytotoxic profile over other analogs in this series.
Fluorescence imaging indicated that Cpd 11 induced nicked DNA of K562 cell line which is
a hallmark of apoptosis. Flow cytometric analysis studies also reveal that the compound (Cpd
11) induced cytotoxicity was apoptotic in nature as a significant number of cells are
annexinV positive. This apoptotic efficacy is further being confirmed by cell cycle assay as
the n-pentyl analog (Cpd 11) may arrest the sub-G1 stage of cell cycle in a significant
proportion. Besides the apoptotic property, the Cpd 11 is being found to block the invasion
of K562 cell line as evidenced by the matrigel invasion assay.
Molecular docking analysis reveals a significant interaction of this compound with both
MMP-2 and MMP-9. However, the enzymatic study indicates all these compounds including
Cpd 11 to have more or less inhibitory activity only on MMP-2 over MMP-9. Besides this,
flow cytometry and western blot analysis show significant inhibition of MMP-2 expression
after Cpd 11 exposure against K562 cell line. Early studies of several workers have
suggested that MMP-2 downregulation might be linked with the anti-invasive and apoptotic
cell death property of cancer cells [15-19]. Therefore, our compound (Cpd 11) which is
found to be a nonspecific inhibitor of MMP-2 and MMP-9 might lead to generate the anti-
invasive and apoptotic nature of the compound against K562 cell line. As all these molecules
have better solubility compared to the earlier reported molecules [14], these may exhibit
better cytotoxicity against cancer cell lines such as K-562 and U-937. However, their
gelatinase inhibitory activity may be lower as gelatinase inhibition may be mainly dependent
on the bulky hydrophobic aryl group directed towards the S1′ pocket. Due to the presence of
carboxylic acid function as zinc binding group, these molecules exhibit gelatinase inhibition
but due to the presence of the p-nitrophenyl group instead of bulky higher aryl function, these
molecules show lower gelatinase inhibition. Therefore, the lower hydrophobic character is
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the main reason controlling the cytotoxicity of these molecules. Even though all these
compounds (Cpd 5-12) exhibit lower gelatinase inhibition, compounds with higher linear or
branched chain alkyl and aryl functions (Cpd 8-12) exhibit cytotoxicity against K-562 and U-
937 cell lines. Interestingly, it may be the long chain alkyl (n-pentyl) function of Cpd 11 that
may be able to bind properly with both these MMP-2 and MMP-9 enzymes and produce
nonselective gelatinase inhibition. The unique elongated linear n-pentyl function may,
therefore, be able to exhibit cytotoxic feature along with the downregulation and reduced
expression of the MMP-2 enzyme. Therefore, this study may help to validate the mechanism
of MMP-2 inhibition mediated cytotoxicity against chronic myeloid leukemia cell line
(K562). Regarding this, there is no significant compound has been reported so far which is
found to be a potent MMP-2 inhibitor and also an apoptosis inducing agents against leukemia
cells [9-12]. Therefore, this study particularly emerges the significance. Furthermore, this
analysis may be taken into consideration for advance exploration of the lead candidate to
design the better gelatinase inhibitor with the higher cytotoxic profile in future.
4. Experimental section
4.1. Chemicals and reagents
All major chemicals/culture/antibodies media were of the analytical/extra-pure grade.
Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin-
streptomycin-neomycin (PSN) antibiotics, trypsin and ethylenediamine tetraacetic acid
(EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic
wares were purchased from BD Bioscience (USA).
All organic solvents used were of HPLC grade. [3-(4, 5-dimethyl-thiazol-2-yl)-2, S-
diphenyltetrazolium bromide (MTT), propidium iodide (PI), 4', 6-diamidino-2- phenylindole
(DAPI) etc were purchased from Sigma Aldrich (USA). Annexin V-FITC apoptosis
detection kit was purchased from Calbiochem, Germany. The MMP-2 and MMP-9 enzyme
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assay kit were purchased from Enzo Life Science International, Inc. (Plymouth Meeting, PA,
USA). Anti-MMP-2 monoclonal antibodies and anti-rabbit secondary antibody were
purchased from Santa Cruz Biotechnology Inc, USA. The primary antibodies of β-tubulin
and FITC-conjugated secondary antibody were obtained from Cell Signalling Tech (USA).
The DAB substrate system was purchased from Amresco, USA.
4.2. Synthesis
The designed compounds were synthesized and characterized. Chemical reactions were
checked by analytical thin layer chromatography (TLC) using silica gel G plates (TLC silica
gel GF254, Merck, Germany). Spots were developed using iodine chamber. Melting points
were uncorrected and measured on a capillary melting point apparatus and mel-temp electro-
thermal apparatus. These were checked in CTRONICS digital melting point apparatus.
Optical rotations of compounds were seen in Parkin-Elmer type 141 polarimeter. Mass
spectroscopic analysis was done in an LC MS/MS instrument [The LC, Agilent coupled to
Electrospray ion (ESI) mass spectrometer having Mass Hunter Quantitative Analysis
software], Nuclear magnetic resonance (NMR) spectra were obtained from an AC Bruker 400
MHz FT-NMR spectrometer with tetramethylsilane (TMS) as the internal standard.
Compounds were dissolved in dimethyl sulfoxide-d6 (DMSO-d6). Splitting patterns are
mentioned as s (singlet), d (doublet), t (triplet) and m (multiplet). 13C NMR of these
compounds was performed in Bruker 400 MHz FT-NMR spectrometer. Infrared (IR) spectra
were recorded on a Bruker alpha 11960095 FT-IR instrument. Elemental analyses of these
compounds were conducted in a PerkinElmer 2400 Series II CHNS/O Elemental Analyzer.
As the diacid (3) and the monoacid (4) were reported earlier [20], general procedures for
preparations of these compounds are not discussed here.
4.2.1. General procedure for preparation of pentanoic acid analogs (Cpd 5-12):
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The monoacid (4) was subjected to amination with some amines to get the desired products
(Cpd 5-12). In a conical flask, a slurry of the monoacid compound (4) was prepared in 20 ml
distilled water. Excess amines are added individually to these suspensions. The reaction
mixture was kept overnight and heated on a water bath to remove the unreacted excess
amines. Finally, the mixture was cooled down to room temperature. 6(N) hydrochloric acid
was added dropwise to the reaction mixture in an ice bath for liberation of pentanoic acids.
The precipitate was filtered off and washed with cold water thoroughly to obtain the desired
compounds (Cpd 5-12). These compounds were recrystallized with hot ethanol with charcoal
treatment.
All the final products were characterized through mass spectroscopy, 1H NMR, 13C NMR and
FTIR analysis as well as elemental analysis. These analytical data are given below:
4.2.2. (2S)-5-amino-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino] pentanoic acid (5)
Yield 63.24%, mp 181-183oC, MS (ESI Positive) m/z [M+H+]: 332.06. 1H NMR (DMSO-d6,
400 MHz, ppm): δ 8.40-8.37 (d, J = 8.8 Hz, 2 Ar-H), δ 8.02-8.00 (d, J = 8.8 Hz, 2 Ar-H), δ
7.26 (m, 1H, SO2NH), δ 6.75 (s, 2H, CONH2), δ 3.78 (m, 1H, CH), δ 2.09-2.05 (t, J = 7.2
13
Hz, 2H, CH2), δ 1.91-1.63 (m, 2H, CH2). C NMR (DMSO-d6, 101 MHz, ppm) δ 173.47,
172.80, 149.84, 147.18, 128.54, 124.80, 55.86, 31.08, 28.14. Anal. Calc. for C11H13N3O7S:
C, 39.88; H, 3.96; N, 12.68; S, 9.68. Found. C, 40.14; H, 3.79; N, 12.89; S, 9.86. FTIR (KBr,
cm-1): 3324 (NH str of SO2NH), 3125 (NH str Of CONH), 3041 (Ar-C-H str), 2857, 2825
(ali C-H str), 1685 (CO str of COOH), 1565, 1518 (N=O str of Ar-NO2Asym.), 1428 (ali C-H
def), 1334, 1228 (S=O str of SO2NH), 855, 834, 798, 724 (Ar C-H def)
4.2.3. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(methylamino) pentanoic acid (6)
Yield 81.91%, mp 206-208oC, MS (ESI Positive) m/z [M+H+]: 346.10, [M+H++Na+] 368.10.
1H NMR (DMSO-d6, 400 MHz, ppm): δ 8.61-8.59 (m, 1H, CONH), δ 8.40-8.38 (d, J = 8.8
Hz, 2 Ar-H), δ 8.02-8.00 (d, J = 8.8 Hz, 2 Ar-H), δ 7.70-7.69 (m, 1H, SO2NH), δ 3.80-3.79
25

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(m, 3H, CH3), δ 3.45-3.40 (m, 1H, CH), δ 2.09-2.05 (t, J = 7.6 Hz, 2H, CH2), δ 1.94-1.62 (m,
2H, CH2). 13C NMR (DMSO-d6, 101 MHz, ppm) δ 172.79, 171.70, 149.84, 147.13, 128.51,
124.79, 55.76, 31.24, 28.20, 25.88. Anal. Calc. for C12H15N3O7S: C, 41.74; H, 4.38; N,
12.17; S, 9.29. Found. C, 41.42; H, 4.69; N, 12.45; S, 9.51. FTIR (KBr, cm-1): 3372 (NH str
of SO2NH), 3254 (NH str of CONH), 3068 (Ar CH str), 2934 (ali CH strAsym), 2867 (ali
CH str), 1712 (CO str of COOH), 1582 (N=O str of Ar-NO2Asym.), 1428 (ali C-H def), 1298
(asym. S=O str of SO2NH), 1137 (sym. S=O str of SO2NH), 958, 865, 826, 756 (Ar C-H def)
4.2.4. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(ethylamino) pentanoic acid (7)
Yield 78.72%, mp 219-221oC, MS (ESI Positive) m/z [M+H+]: 360.0, [M+H++Na+] 382.0.
1H NMR (DMSO-d6, 400 MHz, ppm): δ 8.61-8.59 (m, 1H, CONH), δ 8.40-8.38 (d, J = 8.8
Hz, 2 Ar-H), δ 8.02-8.00 (d, J = 8.4 Hz, 2 Ar-H), δ 7.78-7.75 (m, 1H, SO2NH), δ 3.83-3.78
(m, 1H, CH), δ 3.05-2.95 (m, 2H, N-CH2-1), δ 2.11-2.01 (m, 2H, CH2), δ 1.94-1.62 (m, 2H,
CH2), δ 0.98-0.94 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (DMSO-d6, 101 MHz, ppm) δ 172.78,
170.90, 149.83, 147.19, 128.53, 124.79, 55.80, 33.75, 31.38, 28.26, 15.09. Anal. Calc. for
C13H17N3O7S: C, 43.45; H, 4.77; N, 11.69; S, 8.92. Found. C, 43.83; H, 5.07; N, 12.03; S,
8.76. FTIR (KBr, cm-1): 3334 (NH str of SO2NH), 3220 (NH str of CONH), 3015 (Ar CH
str), 2845, 2812 (ali CH str), 1674 (CO str of COOH), 1563 (N=O str of Ar-NO2Asym), 1462
(ali C-H def), 1266 (asym. S=O str of SO2NH), 1134 (sym. S=O str of SO2NH), 943, 862,
824, 786 (Ar C-H def)
4.2.5. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(isopropylamino) pentanoic acid
(8)
Yield 92.60%, mp 235-237oC, MS (ESI Positive) m/z [M+H+]: 374.0. 1H NMR (DMSO-d6,
400 MHz, ppm): δ 8.59 (m, 1H, CONH), δ 8.40-8.38 (d, J = 8.8 Hz, 2 Ar-H), 8.02-8.00 (d, J
= 8.8 Hz, 2 Ar-H), 7.68-7.66 (m, 1H, SO2NH), 3.81-3.78 (m, 1H, CH of i-Propyl), 3.74-3.71
26

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(m, 1H, CH), 2.08-2.04 (t, J = 7.8 Hz, 2H, CH2), 1.93-1.61 (m, 2H, CH2), 1.01-0.98 (m, 6H,
2CH3). 13C NMR (DMSO-d6, 101 MHz, ppm) δ 172.74, 170.26, 149.82, 147.19, 128.54,
124.75, 55.83, 40.66, 31.51, 28.34, 22.76. Anal. Calc. for C14H19N3O7S: C, 45.03; H, 5.13;
N, 11.29; S, 8.59. Found. C, 45.23; H, 4.85; N, 10.88; S, 8.78. FTIR (KBr, cm-1): 3360 (NH
str of SO2NH), 3246 (NH str of CONH), 3025 (Ar CH str), 2886 (ali CH strAsym), 1695
(CO str of COOH), 1549, 1519 (C=C str of Ar), 1314 (S=O str of SO2NH), 1261 (CO str and
OH def of COOH), 1125 (sym. S=O str of SO2NH), 824, 733 (Ar C-H def)
4.2.6. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(butylamino) pentanoic acid (9)
Yield 89.25%, mp 209-211oC, MS (ESI Positive) m/z [M+H+]: 388.0. 1H NMR (DMSO-d6,
400 MHz, ppm): δ 8.60-8.59 (m, 1H, CONH), 8.40-8.38 (d, J = 8.4 Hz, 2 Ar-H), 8.02-8.00
(d, J = 8.8 Hz, 2 Ar-H), 7.76-7.73 (m, 1H, SO2NH), 3.81 (m, 1H, CH), 3.00-2.94 (m, 2H, N-
CH2-1), 1.93-1.61 (m, 2H, CH2), 1.34-1.21 (m, 4H, N-CH2-2 and N-CH2-3), 0.87-0.83 (t, J =
7.0 Hz, 3H, CH3). 13C NMR (DMSO-d6, 101 MHz, ppm) δ 172.78, 171.05, 149.80, 147.21,
128.54, 124.75, 55.82, 38.58, 31.61, 31.41, 28.34, 19.99, 14.05. Anal. Calc. for
C15H21N3O7S: C, 46.50; H, 5.46; N, 10.85; S, 8.28. Found. C, 46.27; H, 5.32; N, 11.18; S,
8.16. FTIR (KBr, cm-1): 3312 (NH str of SO2NH), 3120 (NH str of CONH), 2965, 2935 (Ar
CH str), 2833 (ali CH str), 1682 (CO str of COOH), 1512 (C=C str of Ar), 1298, 1185 (S=O
str of SO2NH), 842, 798, 735 (Ar C-H def)
4.2.7. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(isobutylamino) pentanoic acid
(10)
Yield 76.86%, mp 204-206oC, MS (ESI Positive) m/z [M+H+]: 388.10. 1H NMR (DMSO-d6,
400 MHz, ppm): δ 8.61-8.59 (m, 1H, CONH), 8.40-8.38 (d, J = 8.8 Hz, 2 Ar-H), 8.02-8.00
(d, J = 8.8 Hz, 2 Ar-H), 7.79-7.77 (m, 1H, SO2NH), 3.81-3.80 (m, 1H, CH), 2.84-2.77 (m,
2H, N-CH2-1), 2.13-2.09 (t, J = 7.4 Hz, 2H, CH2), 1.94-1.85 (m, 1H, CH of i-Butyl), 1.71-
27

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1.58 (m, 2H, CH2), 0.81-0.79 (m, 6H, 2CH3). 13C NMR (DMSO-d6, 101 MHz, ppm) δ
172.77, 171.18, 149.82, 147.21, 128.55, 124.78, 55.84, 46.45, 31.42, 28.46, 28.42, 20.53.
Anal. Calc. for C15H21N3O7S: C, 46.50; H, 5.46; N, 10.85; S, 8.28. Found. C, 46.79; H, 5.82;
N, 10.48; S, 8.54. FTIR (KBr, cm-1): 3325 (NH str of SO2NH), 3155 (NH str of CONH),
3036 (Ar CH str), 2871, 2826 (ali CH strAsym), 1702 (CO str of COOH), 1562 (N=O str of
Ar-NO2), 1438 (C=C str of Ar), 1316, 1137 (S=O str of SO2NH), 916, 872, 794, 712 (Ar C-H
def)
4.2.8. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(pentylamino) pentanoic acid (11)
Yield 86.82%, mp 215-217oC, MS (ESI Positive) m/z [M+H+]: 402.00, [M+H++Na+] 424.00.
1H NMR (DMSO-d6, 400 MHz, ppm): δ 8.60-8.58 (m, 1H, CONH), 8.40-8.38 (d, J = 8.8
Hz, 2 Ar-H), 8.02-8.00 (d, J = 8.8 Hz, 2 Ar-H), 7.76-7.74 (m, 1H, SO2NH), 3.81-3.80 (m,
1H, CH), 3.02-2.90 (m, 2H, N-CH2-1), 2.10-2.04 (m, 2H, CH2), 1.93-1.61 (m, 2H, CH2),
1.37-1.16 (m, 6H, N-CH2-2, N-CH2-3 and N-CH2-4), 0.87-0.83 (t, J = 7.0 Hz, 3H, CH3). 13C
NMR (DMSO-d6, 101 MHz, ppm) δ 172.78, 171.01, 149.82, 147.21, 128.54, 124.78, 55.82,
38.87, 31.40, 29.19, 29.05, 28.34, 22.28, 14.36. Anal. Calc. for C16H23N3O7S: C, 47.87; H,
5.77; N, 10.47; S, 7.99. Found. C, 46.60; H, 5.59; N, 10.08; S, 8.26. FTIR (KBr, cm-1): 3362
(NH str of SO2NH), 3192 (NH str of CONH), 3025 (Ar CH str), 2865 (ali CH str), 1683 (CO
str of COOH), 1582, 1525 (N=O str of Ar-NO2), 1437 (Ali C-H def), 1358, 1142 (S=O str of
SO2NH), 968, 878, 826, 741 (Ar C-H def)
4.2.9. (2S)-5-oxo-2-[(nitrobenzene-4-yl sulfonyl) amino]-5-(benzylamino) pentanoic acid (12)
Yield 88.24%, mp 191-193oC, MS (ESI Positive) m/z [M+H+]: 422.00. 1H NMR (DMSO-d6,
400 MHz, ppm): δ 8.61 (m, 1H, CONH), 8.39-8.37 (d, J = 8.8 Hz, 2 Ar-H), 8.34-8.32 (m,
1H, SO2NH), 8.02-7.99 (d, J = 8.4 Hz, 2 Ar-H), 7.32-7.28 (t, J = 7.0 Hz, 2 Ar-H of benzyl),
7.24-7.20 (t, J = 7.4 Hz, 3 Ar-H of benzyl), 4.22-4.20 (m, 2H, CH2 of benzyl), 3.83 (m, 1H,
28