Synthesis and apoptosis inducing ability of new anilino substituted pyrimidine sulfonamides as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.09.039

A series of anilino substituted pyrimidine sulfonamides were prepared and evaluated for their anticancer activity. These sulfonamides showed promising activity with IC₅₀ values ranging from 5.6 to 12.3 μM. The detailed biological aspects of some of the promising compounds (3d, 3e and 3g) on the K562 cell line were studied. Interestingly, compounds induced G1 cell cycle arrest and down regulation of G1 phase cell cycle regulatory proteins such as cyclin D1, CDK4. These compounds also exhibited inhibition of NF-κB as well as its downstream target gene Akt1 and the phosphorylated form of AKt ser 474 proteins. One of the representative compound 3e could be considered as the potential lead for its development as a new anticancer agent.

Full text of DOI:10.1016/j.ejmech.2011.09.039

European Journal of Medicinal Chemistry 46 (2011) 5817e5824
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and apoptosis inducing ability of new anilino substituted pyrimidine
sulfonamides as potential anticancer agents
,
c
,
a
b
a
a
Ahmed Kamal a , D. Dastagiri , M. Janaki Ramaiah , J. Surendranadha Reddy , E. Vijaya Bharathi ,
*
M. Kashi Reddya, M. Victor Prem Sagar a, T. Lakshminarayan Reddy b, S.N.C.V.L. Pushpavalli b,
**
Manika Pal-Bhadra b
,
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
b Chemical Biology Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
c Catalytic Chemistry Chair, Chemistry Department, College of Science King Saud University, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of anilino substituted pyrimidine sulfonamides were prepared and evaluated for their anticancer
Received 1 March 2011
Received in revised form
21 September 2011
Accepted 21 September 2011
Available online 29 September 2011
activity. These sulfonamides showed promising activity with IC50 values ranging from 5.6 to 12.3 mM. The
detailed biological aspects of some of the promising compounds (3d, 3e and 3g) on the K562 cell line
were studied. Interestingly, compounds induced G1 cell cycle arrest and down regulation of G1 phase cell
cycle regulatory proteins such as cyclin D1, CDK4. These compounds also exhibited inhibition of NF-kB as
well as its downstream target gene Akt1 and the phosphorylated form of AKt ser 474 proteins. One of the
representative compound 3e could be considered as the potential lead for its development as a new
anticancer agent.
Keywords:
Anilino substituted pyrimidines
Sulfonamides
Apoptosis
Caspases
Akt
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
hypoglycemic [12], etc. A number of sulfonamides have also been
screened particularly for their antitumor activity, and they have
Cancer is one of the most dreadful disease in the world and
despite immense advances in the field of basic and clinical research,
which have resulted in higher cure rates for a number of malig-
nancies, cancer remains the second leading cause of death in
developing as well as developed countries [1,2]. Although chemo-
therapy is the mainstay of cancer therapy, the use of available
chemotherapeutics is often limited mainly due to undesirable side
effects and it clearly underscores the need of developing novel
chemotherapeutic agents for more effective cancer treatments [3].
Among the wide range of compounds tested as potential anticancer
agents, sulfonamides comprise an important class of therapeutic
agents, that are known to exhibit a broad spectrum of biological
activities like antibacterial [4], antitumor [5e10], diuretic [7,11],
a common chemical motif of an aromatic/heterocyclic sulfonamide.
There are a variety of mechanisms for their anticancer action,
such as disruption of microtubule assembly, cell cycle arrest in the
G1 phase, functional suppression of the transcriptional activator
NF-Y, angiogenesis and carbonic anhydrase inhibition [5]. Similarly,
an anilino substituted pyrimidine moiety present in the structures
of agents like STI571 (imatinib mesylate, gleevec (1)) and anilino
substituted pyrimidine (2) (Fig. 1) is considered responsible for
the potent antitumor properties [13,14] and are also reported to
possess apoptosis inducing ability [15,16].
In continuation of our efforts in search of new effective anti-
cancer agents, we synthesized a series of anilino substituted
pyrimidine sulfonamides and evaluated for their anticancer
potential. The promising activity obtained, prompted us to inves-
tigate their role regulating the balance between cell proliferation
and apoptosis by interrupting the regulatory proteins in cell cycle
* Corresponding author. Division of Organic Chemistry, Indian Institute of
Chemical Technology, Tarnaka, Hyderabad 500 607, India. Tel.: þ91 40 27193157;
fax: þ91 40 27193189.
progression and by inhibiting the NF-
kB signaling pathway and its
downstream target genes such as Akt. This new class of sulpho-
namide compounds has a unique mode of action that differs from
that of other known antitumor compounds.
** Corresponding author. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in
(M. Pal-Bhadra).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.039

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A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 5817e5824
Fig. 1. Structures of imatinib mesylate (1), anilino substituted pyrimidine (2), and new anilino substituted pyrimidine sulfonamides (3aeh).
Scheme 1. Synthesis of anilino substituted pyrimidine sulfonamides (3aeh). Reagents and conditions: a) H2NCN, HNO3, EtOH, reflux, 24 h, 58%; b) DMFeDMA, toluene, reflux, 24 h,
72e75%; c) NaOH, n-butanol, reflux, 48 h, 60e62%; d) SnCl2$2H2O, HCl, rt, 2 h, 80e82%; e) Et3N, DMAP, dry DMF, rt, 6 h, 80e85%.
2. Chemistry
3. Results and discussions
Synthesis of anilino substituted pyrimidine sulfonamides
(3aeh) was carried out from the precursors 4 and 6 that were
obtained by employing literature methods [17]. Reaction of 2-
methyl-5-nitroaniline (4) with cyanamide in HNO3 produced the
corresponding guanidine (5). Compounds 7a,b were obtained by
the reaction of substituted acetophenones with DMFeDMA. These
compounds (7a,b) were then coupled with guanidine (6) to provide
the corresponding nitro pyridines (8a,b). Further, these compounds
(8a,b) were reduced with SnCl2 to afford the amino compounds
(9a,b). These amino compounds 9a,b were coupled to the
substituted sulfonylchlorides (10aeh) to give the target
compounds 3aeh as shown in Scheme 1.
3.1. Evaluation of biological activity
3.1.1. Inhibition of cell proliferation
Compounds 3aeh were evaluated for their cytotoxicity in
selected human cancer cell lines of leukemia (K562), breast
(MCF-7, MDA-MB-231) using MTT method. The compounds
exhibiting IC50 ꢀ 10ꢁ5 M (10
mM) are considered to be active on
the respective cell lines. Table 1 reveals that these compounds
exhibit broad-spectrum activity in all the cell lines examined
and the activity of these compounds ranged from IC50 5.6 to
12.3
m
M. Imatinib was employed as the positive control that
M [18].
demonstrated IC50 values in the range of 0.21e1.8
m

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 5817e5824
5819
Table 1
cycle [21], suchascyclinD1and theirassociated protein CDK4that are
essential for the cell cycle progression from G1 to S. Therefore K562
IC50 valuesa (in
mM) for compounds 3aeh in selected human cancer cell lines.
cells were treated with these sulfonamides (3d, 3e and 3g) at 10 mM
Compound
K562
MCF-7
MDA-MB-231
concentration for 24 h and Western blot analysis was carried out. As
shown in Fig 4, the protein levels of cyclin D1 and its associated CDK4
level decreased in comparison to the control. These results indicated
that compound 3e induced cell cycle arrest at G1 phase.
Further, induction of apoptotic cell death is mediated by cas-
pases, such as caspase-3 and -7 are associated with the mitochon-
drial function [22]. Interestingly caspases-3 and 7 are the effector
caspases in case of intrinsic pathway and to understand their effect,
K562 cells were treated with these compounds (3d, 3e and 3g) at
1
0.21 ꢂ 0.03
10.16 ꢂ 0.02
9.49 ꢂ 0.014
9.89 ꢂ 0.011
5.88 ꢂ 0.018
5.60 ꢂ 0.005
9.12 ꢂ 0.01
8.57 ꢂ 0.03
9.81 ꢂ 0.017
0.83 ꢂ 0.0176
11.8 ꢂ 0.025
11.86 ꢂ 0.023
12.0 ꢂ 0.0276
9.96 ꢂ 0.044
9.33 ꢂ 0.044
12.3 ꢂ 0.011
11.6 ꢂ 0.0039
11.56 ꢂ 0.0069
1.8 ꢂ 0.18
3a
3b
3c
3d
3e
3f
7.79 ꢂ 0.057
8.0 ꢂ 0.0048
8.31 ꢂ 0.014
7.31 ꢂ 0.009
6.93 ꢂ 0.004
6.86 ꢂ 0.014
7.48 ꢂ 0.00074
7.55 ꢂ 0.006
3g
3h
Determined by MTT assay, 24 h incubation with test compound. Values are mean of
at least three separate determinations. Imitanib (1) was used as a control.
10 mM concentration respectively for 24 h and Western blot analysis
a
IC50 values (in lM), which the concentration required to inhibit 50% of cell
was carried out. The level of both the caspases was up regulated by
this treatment, however the levels of TNFR1 did not change (data
not shown). The data obtained is indicative for the existence of an
intrinsic pathway for these new sulfonamides (Fig. 3).
viability by the test compounds after exposure to cells.
Overall, considerable cytotoxicity was observed for these new
sulfonamide derivatives.
3.1.6. Effect on NF-kB dependent proteins
3.1.2. Cell cycle effects
NF- B is a key transcription factor that is known to have
implications in the processes such as tumor progression [23],
promotion, invasion, angiogenesis and metastasis [24] Moreover,
k
To investigate the effect of these sulphonamides on the cell cycle
progression of human cancerous cell line K562, the DNA content of
the cell nuclei was measured by flow cytometric (FACS) analysis.
Drug induced hypodiploid DNA (i.e., sub-G1 phase) is the charac-
teristic feature of apoptosis [19,20]. Treatment of K562 cells with
NF-
a crucial role in cancer cell proliferation. Therefore to study the
effect of these sulphonamides (3d, 3e and 3g) on NF- B and its
associated proteins, K562 cells were treated with these sulphona-
mides (3d, 3e and 3g) at 10 M concentration for 24 h and Western
blot analysis was carried out. It was observed that NF- B (p65)
kB and associated proteins such as Akt [25] and STAT3 [26] play
k
the most promising compounds 3d, 3e and 3g at 10 mM concen-
tration for 24 h induced apoptosis up to 29.86%, 29.74% and 21.57%
respectively, whereas imatinib (1) showed 29% of sub-G1 DNA peak.
Simultaneously a reduction in the S phase population was observed,
indicating G1 cell cycle arrest as seen from Table 2 and Fig 2a.
m
k
protein level decreased in case of compound 3e and there was not
much effect on 3d. It is also observed that the Akt (Akt1, phos-
phorylated form of Akt1 i.e., AktS473) and STAT3 proteins were
3.1.3. BrdU incorporation assay
down regulated. This suggests that NF-kB has a possible role
involving Akt and STAT proteins. It is interestingly to note that
compound 3e suppresses cancer cell growth by inhibiting Akt, STAT
and NF-kB activity as shown in Fig. 4.
To further confirm the G1 cell cycle arresting nature of these
compounds (3d, 3e and 3g) 5-bromo-2-deoxyuridine (BrdU)
incorporation assay was carried out. This assay assesses the
compounds ability to block in G1eS transition. Treatment of cells
by these compounds at 10 mM concentration results in the reduc-
3.1.7. Effect on oncogenes and survival proteins
tion of incorporation of BrdU into cellular DNA. This level of inhi-
bition of BrdU incorporation is indicative of a G1 cell cycle arrest by
these sulphonamides (3d, 3e and 3g) as shown in Fig 2b.
From previous studies it is well established that involvement of
oncogene such as C-myc [27] is considered vital in chronic and
acute myeloid leukemia [28e30]. To understand the effect of these
sulfonamides on c-myc, K562 cells were treated with these
3.1.4. Tunel assay
sulfonamides (3d, 3e and 3g) at 10 mM concentration for 24 h and
As these compounds (3d, 3e and 3g) were found to be cytotoxic
and cause G1 cell cycle arrest in K562 cells, it was considered of
interest to investigate the apoptotic effects such as DNA fragmen-
tation caused by these sulfonamides by employing Tunel assay
wherein imatinib (1) was used as the positive control. Results of the
Tunel assay revealed that the apoalert DNA fragmentation is
prominent in 3d and 3e compared to 3g. However control cells did
not show any cell death and proliferate actively as no staining or
background staining was observed.
Western blot analysis was carried out. It was observed from Fig. 5
that the levels of c-myc down regulated in case of compound 3e
compared to untreated control, thus suggesting the possible
involvement of signaling molecules in the cell proliferation.
4. Conclusion
In conclusion, a new class of anilino substituted pyrimidine
sulfonamides were synthesized that exhibit considerable anti-
cancer activity. The FACS analysis showed more population in sub-
G1 phase indicating that these sulfonamides (3d, 3e and 3g)
possess apoptosis inducing ability. It was observed from the
detailed biological studies that there is down regulation of cyclins
and CDK4 indicating cell cycle blocking in the G1 phase in these
3.1.5. Effect on cell cycle regulatory and apoptotic proteins
On the basis of the results of cell cycle distribution, we later
investigated the cell cycle regulatory proteins related to G1 phase cell
Table 2
Cell cycle distribution of K562 cell line at 10 mM concentration of compounds 3d, 3e,
3g and imatinib (1).
compounds (3d, 3e and 3g), apart from down regulation of NF-kB,
Akt and STAT. Based on the above results, compound 3e could be
a potential candidate for undergoing detailed biological investiga-
tions for assessing its usefulness in the treatment of cancer.
Compound
Sub-G1
G1
S
G2/M
Control
1
3d
3e
3g
0.27 ꢂ 0.04
37.0 ꢂ 2.64
29.86 ꢂ 1.02
31.00 ꢂ 1.00
21.57 ꢂ 1.93
74.25 ꢂ 1.511
62.0 ꢂ 2.64
11.23 ꢂ 0.8
0.73 ꢂ 0.12
0.20 ꢂ 0.02
0.25 ꢂ 0.025
0.63 ꢂ 0.04
14.24 ꢂ 0.76
0.26 ꢂ 0.017
0.08 ꢂ 0.01
0.14 ꢂ 0.017
0.25 ꢂ 0.05
69.84 ꢂ 1.03
69.86 ꢂ 1.55
77.53 ꢂ 1.98
5. Experimental protocols
All chemicals and reagents were obtained from Aldrich (-
Values are mean of at least three separate determinations. Imitanib (1) was used as
a control.
SigmaeAldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson

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A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 5817e5824
Fig. 2. a. The histogram depicting the percentage of cells in sub-G1 phase, an indicator of percentage of apoptosis for these sulfonamides (3d, 3e and 3g). b. The BrdU cell
proliferation assay: the K562 cells were seeded in 96-well plate at a density of 15,000 cells and were grown for 24 h, BrdU was incubated for 5 h at RT followed by treatment with
these sulphonamides (1, 3d, 3e and 3g) for 24 h at a final conc of 10
mM. The intensity of blue color at 450 nm was measured and is proportional to amount of BrdU incorporated in
proliferating cells. Lesser the O.D. reading denotes the less proliferation rate. c. Tunel staining of K562 cells after exposure to compounds (1, 3d, 3e and 3g) at 10
mM concentration
after 24 h time period. Green color stained cells represent Tunel positive cells and the control cells act as control for the staining procedure. Lack of staining in control (untreated)
cells, represents that the cells are actively proliferating, i.e., without apoptotic cell death (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.).
Matthey Company, Ward Hill, MA, USA) or Spectrochem Pvt. Ltd
(Mumbai, India) and were used without further purification. Reac-
tions were monitored by TLC, performed on silica gel glass plates
containing 60 GF-254, and visualization on TLC was achieved by UV
light or iodine indicator. Column chromatography was performed
with Merck 60e120 mesh silica gel. 1H spectra were recorded on
Gemini Varian-VXR-unity (200 MHz) or Bruker UXNMR/XWIN-NMR
voltage 3.98 kV and ESI mode positive ion trap detector. High-
resolution mass spectra (HRMS) were recorded on QSTAR XL Hybrid
MS/MS mass spectrometer. Melting points were determined with an
Electro thermal melting point apparatus, and are uncorrected.
5.1. Synthesis of anilino substituted pyrimidine sulfonamides
(300 MHz) instruments. Chemical shifts (d) are reported in parts per
5.1.1. 1-(2-Methyl-5-nitrophenyl)guanidine nitrate (5)
million downfield from internal TMS standard. ESI spectra were
recorded on Micro mass, Quattro LC using ESIþ software with capillary
To a solution of 2-methyl-5-nitroaniline (1.5 g, 10 mmol) in
ethanol (25 mL) was added drop wise nitric acid (2 mL) at room

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 5817e5824
5821
Fig. 5. Effect of compounds on oncogene c-myc protein. K562 cells were treated with
10 M concentration of compounds (1, 3d, 3e and 3g) for 24 h. The cell lysates were
m
collected and observed the levels of proteins using specific antibodies. b-Actin was
used as loading control. C: control (untreated).
Fig. 3. Effect of compounds on the expression of cell cycle regulatory proteins (cyclin
D1, Cdk4) and apoptosis associated proteins (caspase-3 and 7). K562 cells were treated
5.1.3. 3-(N,N-dimethylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (7b)
with 10
mM concentration of compounds (1, 3d, 3e and 3g) for 24 h. The cell lysates
This compound was prepared according to the method
described for compound 7a, employing compound 6b (2.1 g,
10 mmol) to obtain the pure product 7b as a yellow solid (1.98 g,
were collected and observed for expression of cyclin D1, Cdk4, caspase-3 and 7 protein
levels using specific antibodies, wherein b-actin was used as loading control. C: control
(untreated).
75%). 1H NMR (CDCl3, 300 MHz):
3.83 (s, 3H, eOCH3), 3.91 (s, 6H, 2 ꢄ eOCH3), 5.59 (d,1H, J ¼ 12.6 Hz,
olefineH), 7.09 (s, 2H, ArH), 7.70 (d, 1H, J ¼ 12.2 Hz, olefineH); MS
(EI): m/z 265 [Mþ].
d
2.80e3.25 (br, 6H, 2 ꢄ eCH3),
temperature. After the completion of addition, the reaction mixture
was stirred for 10 min at room temperature. 50% aqueous solution
of cyanamide (3 mL, 25 mmol) was added droop wise to the reac-
tion mixture and stirred for 12 h at 80e85 ꢃC. The crude product
was collected by filtration and washed with ethyl acetate and
recrystallized from ethanol to afford the compound 5 as light
yellow solid (1.12 g, 58%). Mp 216e218; 1H NMR (DMSO-d6,
5.1.4. Synthesis of N-(5-nitro-2-methylphenyl)-4-(3-pyridinyl)-2-
pyrimidineamine (8a)
To a solution of 3-(N,N-dimethylamino)-1-(pyridin-3-yl)prop-2-
en-1-one (7a) (1.76 g, 10 mmol) in 2-propanol was added phenyl
guanidinium nitrate (5) (1.94 g, 10 mmol) and sodium hydroxide
(12 mmol). The reaction mixture was refluxed at 90 ꢃC for 24 h and
cooled at 0 ꢃC. The precipitate was collected by filtration and was
washed with water and dried in air. The crude product was recrys-
tallized from propanol to afford the compound 8a as light yellow
solid (1.87 g, 62%). Mp 194e196; 1H NMR (DMSO-d6, 300 MHz):
300 MHz):
d 2.32 (s, 3H, eCH3), 7.44 (m, 4H, ArH), 7.64 (d, 1H,
J ¼ 8.2 Hz, ArH), 8.09 (d, 1H, J ¼ 2.5 Hz, ArH), 8.15 (dd, 1H, J ¼ 8.2,
2.5 Hz, ArH), 9.51 (m, 1H, ArH); MS (EI): m/z 194 [Mþ].
5.1.2. Synthesis of 3-(N,N-dimethylamino)-1-(pyridin-3-yl)prop-2-
en-1-one (7a)
d
2.43 (s, 3H, eCH3), 7.54 (m, 3H, ArH), 7.90 (dd, 1H, J ¼ 8.2, 2.2 Hz,
N,N-DMF/DMA was added to a solution of 3-acetyl pyridine (6a)
(1.2 g, 10 mmol) in ethanol (20 ml) at room temperature, the
reaction mixture was refluxed in an oil bath for 12 h. The reaction
was monitored by TLC and the solvent was evaporated under the
vacuum. The mixture was cooled to 0 ꢃC and added ether. The crude
product was removed by filtration and recrystallized from ethanol
to afford compound 7a as light yellow solid (1.26 g, 72%). 1H NMR
pyrimidineeH), 8.48 (dt, 1H, J ¼ 7.9 Hz, pyridineeH), 8.62 (d, 1H,
J ¼ 5.4 Hz, pyridineeH), 8.71 (dd, 1H, J ¼ 8.3, 2.4 Hz, pyrimidineeH),
8.79 (d, 1H, J ¼ 2.2 Hz, pyridineeH), 9.27 (d, 1H, J ¼ 1.6 Hz,
pyridineeH), 9.32 (s, 1H, eNHe); MS (ESI): m/z 308 [M þ 1]þ.
5.1.5. N-(5-nitro-2-methylphenyl)-4-(3,4,5-trimethoxyphenyl)-2-
pyrimidineamine (8b)
This compound was prepared according to the method
described for compound 8a, employing compound 7b (2.65 g,
10 mmol) and compound 5 (1.94 g, 10 mmol) to obtain the pure
product 7b as a yellow solid (2.38 g, 60%). 1H NMR (CDCl3,
(CDCl3, 300 MHz):
d 2.96 (s, 3H, eCH3), 3.15 (s, 3H, eCH3), 5.68 (d,
1H, J ¼ 12.6 Hz, olefineH), 7.36 (dd, 1H, J ¼ 8.3, 5.2 Hz, pyridineeH),
7.85 (d, 1H, J ¼ 12.8 Hz, olefineH), 8.19 (dt, 1H, J ¼ 2.2 Hz,
pyridineeH), 8.67 (dd, 1H, J ¼ 4.5, 1.5 Hz, pyridineeH), 9.09 (d, 1H,
J ¼ 1.5 Hz, pyridineeH); MS (EI): m/z 176 [Mþ].
300 MHz):
d 2.47 (s, 3H, eCH3), 3.78 (s, 3H, eOCH3), 3.87 (s, 6H,
2 ꢄ eOCH3), 7.35 (s, 2H, ArH), 7.40 (d, 2H, J ¼ 8.3 Hz,
pyrimidineeH), 7.81 (dd, 1H, J ¼ 8.3, 2.2 Hz, ArH), 8.46 (d, 1H,
J ¼ 5.0 Hz, ArH), 8.74 (s, 1H, ArH), 8.94 (d, 1H, J ¼ 1.7 Hz,
pyrimidineeH); MS (ESI): m/z 397 [M þ 1]þ.
5.1.6. Synthesis of N-(5-amino-2-methylphenyl)-4-(3-pyridinyl)-2-
pyrimidineamine (9a)
N-(5-nitro-2-methylphenyl)-4-(3-pyridinyl)-2-pyrimidineamine
(8a) (3.08 g 10 mmol) was added to a stirred suspension of stannous
chloride (50 mmol) and con HCl, the mixture was stirred at room
temperature for 12 h. The reaction mixture was poured into crushed
ice and was basified with potassium carbonate. The aqueous layer
extracted with ethyl acetate and dried over anhydrous sodium
sulfate, evaporated in vacuum to afford the compound as yellow
solid (2.21 g, 80%). Mp 138e140; 1H NMR (CDCl3, 300 MHz):
d 2.18
Fig. 4. Effect of compounds on the expression of NF-
cells were treated with 10 M concentration of compounds (1, 3d, 3e and 3g) for 24 h.
The cell lysates were collected and observed for expression of NF- B (p65), Akt1,
-actin was used
kB, Akt and STAT3 proteins. K562
(s, 3H, eCH3), 4.07 (br, 2H, eNH2), 6.32 (d, 1H, J ¼ 7.9, 2.2 Hz, ArH),
6.87 (d, 1H, J ¼ 8.1 Hz, ArH), 7.15 (d, 1H, J ¼ 5.2 Hz, pyrimidineeH),
7.26 (d, 1H, J ¼ 3.5 Hz, ArH), 7.41 (dd, 1H, J ¼ 7.9, 4.9 Hz, ArH), 7.59 (d,
1H, J ¼ 7.8 Hz, ArH), 8.34 (dt, 1H, J ¼ 1.8 Hz, pyridineeH), 8.43 (d, 1H,
m
k
Akt1s473, and STAT3 protein levels using specific antibodies wherein
b
as loading control. C: control (untreated).

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A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 5817e5824
J ¼ 5.0 Hz, pyrimidineeH), 8.64 (dd, 1H, J ¼ 4.7, 1.2 Hz, pyridineeH),
9.21 (s, 1H, eNHe); MS (EI): m/z 277 [Mþ].
product 3c as a yellow solid (396 mg, 85%). Mp 138e140; 1H NMR
(300 MHz, DMSO-d6):
2.22 (s, 3H, eCH3), 6.56 (dd, 1H, J ¼ 8.1,
d
7.9 Hz, ArH), 6.89 (d, 1H, J ¼ 8.1 Hz, ArH), 6.96 (s, 1H, ArH), 7.11 (d,
1H, J ¼ 5.2 Hz, pyrimidineeH), 7.32 (dd, 2H, J ¼ 4.7, 7.6 Hz, ArH),
7.41 (d, 1H, J ¼ 7.7 Hz, ArH), 7.52 (t, 1H, J ¼ 8.8, 8.3 Hz, ArH), 7.84 (d,
2H, J ¼ 8.6 Hz, ArH), 7.88 (d, 1H, J ¼ 8.3 Hz, pyridineeH), 8.13 (d, 1H,
J ¼ 2.0 Hz, ArH), 8.20 (d, 1H, J ¼ 7.5 Hz, ArH), 8.37 (d, 1H, J ¼ 5.0 Hz,
pyrimidineeH), 8.65 (d, 1H, J ¼ 4.7 Hz, ArH), 8.77 (d, 1H, J ¼ 8.3 Hz,
pyridineeH), 9.15 (s, 1H, eNHe), 10.24 (s, 1H, eNHe); 13C NMR
5.1.7. N-(5-amino-2-methylphenyl)-4-(3,4,5-trimethoxyphenyl)-2-
pyrimidineamine (9b)
This compound was prepared according to the method
described for compound 9a, employing compound 8b (3.9 g,
10 mmol) to obtain the pure product 9b as a yellow solid (3 g, 82%).
1H NMR (CDCl3, 300 MHz):
d 2.24 (s, 3H, eCH3), 3.60 (s, 2H, eNH2),
3.91 (s, 3H, eOCH3), 3.96 (s, 6H, 2 ꢄ eOCH3), 6.39 (dd, 1H, J ¼ 8.1,
2.2 Hz, ArH), 6.91 (s, 1H, ArH), 6.99 (d, 1H, J ¼ 7.9 Hz,
pyrimidineeH), 7.02 (d, 1H, J ¼ 5.2 Hz, ArH), 7.33 (s, 2H, ArH), 7.65
(d, 1H, J ¼ 2.2 Hz, ArH), 8.43 (d, 1H, J ¼ 5.2 Hz, pyrimidineeH), 9.21
(s, 1H, eNHe); MS (ESI): m/z 367 [M þ 1]þ.
(75 MHz, DMSO-d6):
134.5, 134.1, 132.5, 130.7, 130.1, 128.9, 128.1, 126.6, 124.4, 123.9,
123.6, 115.8, 113.9, 108.2, 17.4 ppm; IR (KBr) (Umax/cmꢁ1):
3424
(br), 3051, 2924, 2856, 1661, 1580, 1530, 1479, 1447, 1412, 1324, 1199,
1159, 1131, 1011, 984, 884, 802, 770, 705, 677, 638, 584; MS (ESI): m/
z 468 [M þ 1]þ; HRMS (ESI m/z) for C26H22N5O2S calcd 468.1494,
found 468.1493 [M þ 1]þ.
d 162.2, 160.0, 158.8, 150.9, 147.9, 137.8, 135.2,
n
5.1.8. Synthesis of 4-chloro-N-(4-methyl-3-(4-(pyridin-4-yl)
pyrimidin-2-ylamino)phenyl)benzene sulfonamide (3a)
To a stirred solution of compound 9a (277 mg, 1 mmol) in DMF,
triethylamine (5.0 equiv) and catalytic amount of DMAP were
added and cooled to 0e5 ꢃC, then sulfonylchloride 10a (1 mmol)
was added drop wise. The reaction mixture was stirred for 4e5 h at
room temperature (completion of reaction was confirmed by TLC)
then the reaction mixture was washed with water, followed by
saturated NaCl solution. The aqueous layer extracted with ethyl
acetate and dried over anhydrous sodium sulfate, evaporated in
vacuum to afford the compound as yellow solid (360 mg, 80%). Mp
5.1.11. N-(4-methyl-3-(4-(pyridin-4-yl)pyrimidin-2-ylamino)
phenyl)quinoline-3-sulfonamide (3d)
This compound was prepared according to the method
described for compound 3a, employing compound 9a (277 mg,
1 mmol) and compound 10d (227 mg, 1 mmol) to obtain the pure
product 3d as a yellow solid (383 mg, 82%). Mp 131e133; 1H NMR
(300 MHz, DMSO-d6):
d
2.20 (s, 3H, eCH3), 6.48 (d, 1H, J ¼ 7.8 Hz,
ArH), 6.85 (d, 1H, J ¼ 7.8 Hz, ArH), 6.91 (s, 1H, ArH), 7.17 (d, 1H,
J ¼ 4.8 Hz, pyrimidineeH), 7.54 (d, 2H, J ¼ 6.8 Hz, pyridineeH), 7.96
(d, 1H, J ¼ 7.8 Hz, ArH), 8.14 (s, 1H, ArH), 8.23 (d, 1H, J ¼ 8.7 Hz, ArH),
8.29 (s, 1H, ArH), 8.34 (d, 2H, J ¼ 6.8 Hz, ArH), 8.38 (d, 1H, J ¼ 4.8 Hz,
pyrimidineeH), 8.58 (d, 1H, J ¼ 6.8 Hz, pyridineeH), 8.74 (d, 1H,
J ¼ 2.9 Hz, ArH), 9.18 (s, 1H, eNHe), 10.29 (s, 1H, eNHe); 13C NMR
215e217; 1H NMR (DMSO-d6, 300 MHz):
d 2.42 (s, 3H, eCH3), 6.61
(d, 1H, J ¼ 7.7 Hz, ArH), 7.20 (d, 1H, J ¼ 7.7 Hz, pyrimidineeH), 7.31
(d, 1H, J ¼ 4.8 Hz, ArH), 7.37 (dd, 2H, J ¼ 8.7, 7.7 Hz, ArH), 7.46 (d, 2H,
J ¼ 8.7 Hz, ArH), 7.82 (s, 1H, ArH), 7.86 (d, 2H, J ¼ 8.7 Hz,
pyridineeH), 8.24 (s, 1H, ArH), 8.35 (d, 1H, J ¼ 7.7 Hz,
pyrimidineeH), 8.46 (d, 2H, J ¼ 5.8 Hz, pyridineeH), 9.44 (s, 1H,
(75 MHz, DMSO-d6):
d 158.7, 151.3, 151.1, 148.2, 137.7, 137.0, 135.3,
135.0, 133.4, 132.5, 131.7, 130.5, 125.6, 125.0, 123.9, 122.1, 117.1, 115.4,
eNHe), 10.45 (s, 1H, eNHe); 13C NMR (75 MHz, DMSO-d6):
d
159.8,
108.3, 29.6, 17.4 ppm; IR (KBr) (Umax/cmꢁ1):
n
3388 (br), 2923, 2856,
158.8, 151.4, 147.9, 146.4, 138.8, 138.3, 138.0, 135.2, 132.4, 130.8,
130.0, 129.1, 128.9, 128.6, 125.0, 124.0, 122.7, 108.4, 17.9 ppm; IR
1579, 1524, 1483, 1448, 1404, 1329, 1206, 1148, 1009, 877, 835, 794,
697, 591; MS (ESI): m/z 469 [M þ 1]þ; HRMS (ESI m/z) for
C25H21N6O2S calcd 469.1446, found 469.1453 [M þ 1]þ.
(KBr) (Umax/cmꢁ1):
n 3409 (br), 3090, 2923, 2855, 1577, 1529, 1476,
1446, 1381, 1280, 1175, 1089, 1015, 920, 865, 757, 707, 657, 617, 544;
MS (ESI): m/z 452 [M þ 1]þ; HRMS (ESI m/z) for C22H19ClN5O2S
calcd 452.0870, found 452.0863 [M þ 1]þ.
5.1.12. N-(4-methyl-3-(4-(3,4,5-trimethoxyphenyl)pyrimidin-2-
ylamino)phenyl)biphenyl-4-sulfonamide (3e)
This compound was prepared according to the method
described for compound 3a, employing compound 9b (366 mg,
1 mmol) and compound 10b (252 mg, 1 mmol) to obtain the pure
product 3e as a yellow solid (494 mg, 85%). Mp 194e196; 1H NMR
5.1.9. N-(4-methyl-3-(4-(pyridin-4-yl)pyrimidin-2-ylamino)
phenyl)biphenyl-4-sulfonamide (3b)
This compound was prepared according to the method
described for compound 3a, employing compound 9a (277 mg,
1 mmol) and compound 10b (252 mg, 1 mmol) to obtain the pure
product 3b as a yellow solid (409 mg, 83%). Mp 138e140; 1H NMR
(300 MHz, CDCl3): d 2.43 (s, 3H, eCH3), 3.79 (s, 3H, eOCH3), 3.86 (s,
6H, 2 ꢄ eOCH3), 6.64 (d, 1H, J ¼ 7.5 Hz, ArH), 7.02 (d, 1H, J ¼ 5.2 Hz,
pyrimidineeH), 7.10 (s, 1H, ArH), 7.16 (s, 2H, ArH), 7.19 (t, 1H,
J ¼ 3.7 Hz, ArH), 7.45 (t, 2H, J ¼ 8.3 Hz, ArH), 7.55 (d, 2H, J ¼ 6.7 Hz,
ArH), 7.62 (d, 2H, J ¼ 8.3 Hz, ArH), 7.87 (d, 1H, J ¼ 8.3 Hz, ArH), 8.05
(d, 1H, J ¼ 8.3 Hz, ArH), 8.29 (s, 1H, ArH), 8.31 (d, 1H, J ¼ 5.2 Hz,
pyrimidineeH), 9.23 (s, 1H, eNHe), 10.14 (s, 1H, eNHe); 13C NMR
(300 MHz, DMSO-d6):
d
2.42 (s, 3H, eCH3), 6.70 (d, 1H, J ¼ 8.3 Hz,
ArH), 7.22 (d, 1H, J ¼ 5.4 Hz, pyrimidineeH), 7.35e7.47 (m, 5H, ArH),
7.57 (d, 2H, J ¼ 7.3 Hz, pyridineeH), 7.69 (d, 2H, J ¼ 7.3 Hz, ArH), 7.89
(d, 1H, J ¼ 8.3 Hz, ArH), 7.95 (d, 2H, J ¼ 8.3 Hz, ArH), 8.33 (d, 2H,
J ¼ 6.2 Hz, ArH), 8.42 (d, 1H, J ¼ 4.1 Hz, pyrimidineeH); 8.61 (d, 1H,
J ¼ 7.3 Hz, pyridineeH), 9.09 (s, 1H, eNHe), 10.39 (s, 1H, eNHe);
(75 MHz, CDCl3):
132.7, 132.1, 130.7, 130.1, 129.1,128.5, 127.3, 125.2, 122.5, 109.0, 104.3,
96.1, 60.7, 56.3, 18.1 ppm; IR (KBr) (Umax/cmꢁ1):
3434 (br), 2924,
d 165.0, 160.0, 158.0, 153.4, 146.5, 139.1, 138.2,
13C NMR (75 MHz, DMSO-d6):
d
160.3, 159.2, 151.9, 148.4, 146.9,
n
139.2, 138.7, 138.4, 135.6, 132.9, 131.3, 130.5, 129.5, 129.4, 129.1,
127.7, 127.3,125.5, 125.1, 124.4,123.1, 122.7,108.9, 18.3 ppm; IR (KBr)
2853, 1735, 1593, 1556, 1526, 1501, 1448, 1406, 1376, 1336, 1230,
1165, 1124, 1005, 930, 870, 836, 807, 760, 717, 693; MS (ESI): m/z
583 [M þ 1]þ; HRMS (ESI m/z) for C32H31N4O5S calcd 583.1937,
found 583.1929 [M þ 1]þ.
(Umax/cmꢁ1):
n 3417 (br), 3031, 2923, 2855, 1717, 1579, 1524, 1446,
1407, 1372, 1303, 1166, 1088, 1014, 924, 863, 800, 760, 672; MS (ESI):
m/z 494 [M þ 1]þ; HRMS (ESI m/z) for C28H24N5O2S calcd 494.1650,
found 494.1646 [M þ 1]þ.
5.1.13. 4-Methoxy-N-(4-methyl-3-(4-(3,4,5-trimethoxyphenyl)
pyrimidin-2-ylamino)phenyl)benzene sulfonamide (3f)
5.1.10. N-(4-methyl-3-(4-(pyridin-4-yl)pyrimidin-2-ylamino)
phenyl)naphthalene-2-sulfonamide (3c)
This compound was prepared according to the method
described for compound 3a, employing compound 9a (277 mg,
1 mmol) and compound 10c (226 mg, 1 mmol) to obtain the pure
This compound was prepared according to the method
described for compound 3a, employing compound 9b (366 mg,
1 mmol) and compound 10e (206 mg, 1 mmol) to obtain the pure
product 3f as a yellow solid (468 mg, 84%). Mp 198e200; 1H NMR
(300 MHz, CDCl3): d 2.39 (s, 3H, eCH3), 3.83 (s, 3H, eOCH3), 3.89 (s,

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 5817e5824
5823
6H, 2 ꢄ eOCH3), 3.91 (s, 3H, eOCH3), 6.52 (d, 1H, J ¼ 9.0 Hz, ArH),
6.86 (d, 1H, J ¼ 8.6 Hz, ArH), 7.08 (s, 1H, ArH), 7.16 (s, 2H, ArH), 7.27
(d, 1H, J ¼ 5.2 Hz, pyrimidineeH), 7.71 (d, 1H, J ¼ 8.6 Hz, ArH), 7.91
(d, 2H, J ¼ 8.8 Hz, ArH), 8.35 (s, 1H, ArH), 8.39 (d, 1H, J ¼ 5.2 Hz,
pyrimidineeH), 9.05 (s, 1H, eNHe), 10.33 (s, 1H, eNHe); 13C NMR
dehydrogenase. In this assay K562 cells were seeded in a 96-well
plate at a density of 10,000 cells/well. After overnight incubation
cells were treated with 1, 3aeh at 10
bated for 24 h. Then the medium was discarded and replaced with
10
L MTT dye. Plates were incubated at 37 ꢃC for 2 h. The resulting
formazan crystals were solubilized in 100 L extraction buffer. The
mM concentration and incu-
m
(75 MHz, CDCl3):
132.3, 131.2, 130.7, 129.3, 128.6, 125.0, 122.0, 113.8, 108.9, 104.2, 60.8,
55.5, 18.0 ppm; IR (KBr) (Umax/cmꢁ1):
3416 (br), 2935, 1593, 1551,
d
164.9, 163.6, 160.0, 158.1, 153.4, 138.7, 132.9,
m
optical density (O.D.) was read at 570 nm with micro plate reader.
n
1526, 1495, 1449, 1409, 1370, 1340, 1264, 1234, 1159, 1121, 1014, 930,
870, 830, 715, 666; MS (ESI): m/z 537 [M þ 1]þ; HRMS (ESI m/z) for
C27H28N4O6NaS calcd 559.1627, found 559.1638 [M þ Na]þ.
5.4. BrdU cell proliferation assay
Cell proliferation analysis by the BrdU incorporation method by
used to assess the effect of compounds (1, 3d, 3e and 3g) on K562
cell proliferation. K562 cells were seeded at a density of 15,000 per
well of 96-well plate. After 24 h time period cells were incubated
with BrdU for 5 h. Then the culture was treated with compounds (1,
5.1.14. 3,4-Dimethoxy-N-(4-methyl-3-(4-(3,4,5-trimethoxyphenyl)
pyrimidin-2-ylamino)phenyl)benzene sulfonamide (3g)
This compound was prepared according to the method
described for compound 3a, employing compound 9b (366 mg,
1 mmol) and compound 10f (236 mg, 1 mmol) to obtain the pure
product 3g as a yellow solid (464 mg, 82%). Mp 205e207; 1H NMR
3d, 3e and 3g) at 10 mM concentration and incubation carried for
24 h. Fixation of cells was done for 30 min at room temperature
followed by three washings. Anti BrdU antibody was added to the
cell suspension and incubated for 1 h allowing it to bind to incor-
(300 MHz, CDCl3): d 2.42 (s, 3H, eCH3), 3.81 (s, 3H, eOCH3), 3.87 (s,
6H, 2 ꢄ eOCH3), 3.89 (s, 3H, eOCH3), 3.91 (s, 3H, eOCH3), 6.49 (d,
1H, J ¼ 7.7 Hz, ArH), 6.80 (d, 2H, J ¼ 8.6 Hz, ArH), 7.10 (d, 1H,
J ¼ 5.0 Hz, pyrimidineeH), 7.14 (s, 1H, ArH), 7.18 (s, 2H, ArH), 7.47 (s,
1H, ArH), 7.56 (d, 1H, J ¼ 8.4 Hz, ArH), 8.34 (d, 1H, J ¼ 5.0 Hz,
pyrimidineeH), 8.39 (s, 1H, ArH), 9.81 (s, 1H, eNHe), 10.33 (s, 1H,
porated BrdU. After 1 h incubation 100 mL of anti BrdU goat anti
mouse HRP secondary antibody (1:2000) was added and allowed
for 30 min, washing procedure was repeated for 3 times, with PBS.
TMB substrate (100 mL) was added and incubated for another
30 min at room temperature. Finally the O.D. reading was taken at
450 nm. A higher O.D. reading indicates higher BrdU incorporation
in the sample (which indirectly depicts the higher proliferation
rate).
eNHe); 13C NMR (75 MHz, CDCl3):
d
164.9,159.8, 158.1,153.3, 148.7,
138.8, 133.2, 132.0, 130.4, 127.9, 124.9, 122.8, 121.3, 110.8, 109.9,
108.9, 104.2, 96.1, 60.7, 56.3, 56.0, 18.1 ppm; IR (KBr) (Umax/cmꢁ1):
n
3427 (br), 3085, 2926, 2851, 1586, 1554, 1510, 1445, 1408, 1375,
1344, 1265, 1236, 1160, 1135, 1090, 1019, 928, 869, 823, 767, 690; MS
(ESI): m/z 567 [M þ 1]þ; HRMS (ESI m/z) for C28H31N4O7S calcd
567.1835, found 567.1828 [M þ 1]þ.
5.5. Cell cycle analysis
5 ꢄ105 K562 cells were seeded in 60 mm dish and were allowed
to grow for 24 h, 10 mM concentration of 1, 3d, 3e and 3g
5.1.15. N-(4-methyl-3-(4-(3,4,5-trimethoxyphenyl)pyrimidin-2-
ylamino)phenyl)naphthalene-2-sulfonamide (3h)
This compound was prepared according to the method described
for compound 3a, employing compound 9b (366 mg, 1 mmol) and
compound 10c (226 mg, 1 mmol) to obtain the pure product 3h as
a yellow solid (450 mg, 81%). Mp 191e193; 1H NMR (300 MHz,
compounds were added to the culture media, and the cells were
incubated for an additional 24 h. Cells were harvested with Trypsin/
EDTA, fixed with ice-cold 70% ethanol at 4 ꢃC for 30 min, washed
with PBS and incubated with 1 mg/ml RNAase solution (Sigma) at
37 ꢃC for 30 min. Cells were collected by centrifugation at 2000 rpm
for 5 min and further stained with 250 mL of DNA staining solution
CDCl3):
d
2.17 (s, 3H, eCH3), 3.83 (s, 3H, eOCH3), 3.91 (s, 6H,
[10 mg of propidium iodide (PI), 0.1 mg of trisodium citrate, and
0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ
water at room temperature for 30 min in the dark]. The DNA
contents of 20,000 events were measured by flow cytometer (DAKO
CYTOMATION, Beckman Coulter, and Brea, CA). Histograms were
analyzed using Summit Software.
2 ꢄ eOCH3), 6.61 (dd, 1H, J ¼ 8.3 Hz, ArH), 6.86 (d, 1H, J ¼ 8.3 Hz,
ArH), 7.13 (d, 1H, J ¼ 5.2 Hz, pyrimidineeH), 7.30 (s, 2H, ArH), 7.43 (t,
1H, J ¼ 7.3, 8.3 Hz, ArH), 7.55 (t, 1H, J ¼ 7.3 Hz, ArH), 7.62 (t, 1H,
J ¼ 7.3 Hz, ArH), 7.73e7.77 (m, 2H, ArH), 7.88 (d, 1H, J ¼ 8.3 Hz, ArH),
7.97 (d, 1H, J ¼ 8.3 Hz, ArH), 8.17 (d, 1H, J ¼ 7.3 Hz, ArH), 8.34 (d, 1H,
J ¼ 5.2 Hz, pyrimidineeH), 8.77 (d, 1H, J ¼ 8.3 Hz, ArH), 9.81 (s, 1H,
eNHe),10.17 (s,1H, eNHe); 13C NMR (75 MHz, CDCl3):
d
169.5,160.7,
159.1, 151.3, 142.8, 139.2, 137.0, 135.5, 134.1, 130.6, 128.4, 128.1, 127.3,
126.2, 123.4, 119.0, 106.2, 104.5, 102.6, 56.4, 56.1, 18.3; IR (KBr) (Umax
5.6. Tunel assay
/
Tunel assay (Terminal Transferase dUTP Nick End Labeling) was
conducted by using the Apoalert DNA fragmentation Assay kit
(Clone tech). Apoptosis induced nuclear DNA fragmentation was
determined using this assay. This assay was conducted according to
the manufacturer’s recommendations and is based on the principle
of terminal deoxy nucleotidyl transferase (TdT)-mediated dUTP
nick end labeling. TdT catalyzes incorporation of fluorescein-dUTP
at the free 30-hydroxyl ends of fragmented DNA. Flourescein-
labeled DNA can be detected via confocal microscope.
cmꢁ1):
n 3337 (br), 2932, 1554, 1488, 1447, 1412, 1335, 1233, 1163,
1128,1007, 915, 809, 770, 673; MS (ESI): m/z 557 [M þ 1]þ; HRMS (ESI
m/z) for C30H29N4O5S calcd 557.1853, found 557.1882 [M þ 1]þ.
5.2. Cell culture
The K562 (Chronic myeloid leukemia) were incubated by using
RPMI media, supplemented with 10% fetal calf serum, 100 mg/mL
pencillin-G and 100 mg/mL streptomycin sulfate. The cell line was
maintained at 37 ꢃC in a humidified atmosphere containing 5% CO2
5.7. Protein extraction and Western blot analysis
in the incubator.
Total cell lysates from cultured K562 cells were obtained by
5.3. MTT cell viability assay
lysing the cells in ice-cold RIPA buffer (1 ꢄ PBS, 1% NP-40, 0.5%
sodium deoxycholate and 0.1% SDS) and containing 100 mg/mL
Cell viability was assessed by the MTT assay, a mitochondrial
function assay. It is based on the ability of viable cells to reduce the
MTT to insoluble formazan crystals by mitochondrial
PMSF, 5
100 g/mL NaF. After centrifugation at 12,000 rpm for 10 min, the
protein in supernatant was quantified by Bradford method (BIO-
mg/mL aprotinin, 5 mg/mL leupeptin, 5 mg/mL pepstatin and
m

5824
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