Synthesis and biological evaluation of ursolic acid-triazolyl derivatives as potential anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.05.029

A series of ursolic acid-1-phenyl-1H-[1,2,3]triazol-4-ylmethylester congeners have been designed and synthesized in an attempt to develop potent antitumor agents. A regioselective approach using Huisgen 1,3-dipolar cycloaddition reaction of ursolic acid-alkyne derivative with various aromatic azides was employed to target an array of triazolyl derivatives in an efficient manner. Their structures were confirmed by using ¹H NMR, ¹³C NMR, IR and MS analysis. All the compounds were evaluated for anticancer activity against a panel of four human cancer cell lines including A-549 (lung), MCF-7 (breast), HCT-116 (colon), THP-1 (leukemia) and a normal human epithelial cell line (FR-2) using sulforhodamine-B assay. The pharmacological results showed that most of the compounds displayed high level of antitumor activities against the tested cancer cell lines compared with ursolic acid. Compounds 7b, 7g, 7p and 7r were found to be the most potent compounds in this study.

Full text of DOI:10.1016/j.ejmech.2013.05.029

European Journal of Medicinal Chemistry 66 (2013) 238e245
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of ursolic acid-triazolyl derivatives
as potential anti-cancer agents
a
a
b
b
a,
*
Showkat Rashid , Bilal Ahmad Dar , Rabiya Majeed , Abid Hamid , Bilal Ahmad Bhat
a
Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Sanatnagar-Srinagar 190005, Jammu & Kashmir, India
Division of Pharmacology, Indian Institute of Integrative Medicine, Jammu 180001, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 January 2013
Received in revised form
A series of ursolic acid-1-phenyl-1H-[1,2,3]triazol-4-ylmethylester congeners have been designed and
synthesized in an attempt to develop potent antitumor agents. A regioselective approach using Huisgen
1,3-dipolar cycloaddition reaction of ursolic acid-alkyne derivative with various aromatic azides was
27 March 2013
employed to target an array of triazolyl derivatives in an efficient manner. Their structures were
confirmed by using 1H NMR, C NMR, IR and MS analysis. All the compounds were evaluated for anti-
cancer activity against a panel of four human cancer cell lines including A-549 (lung), MCF-7 (breast),
HCT-116 (colon), THP-1 (leukemia) and a normal human epithelial cell line (FR-2) using sulforhodamine-
B assay. The pharmacological results showed that most of the compounds displayed high level of anti-
tumor activities against the tested cancer cell lines compared with ursolic acid. Compounds 7b, 7g, 7p
and 7r were found to be the most potent compounds in this study.
Accepted 23 May 2013
Available online 7 June 2013
13
Keywords:
Ursolic acid
Click chemistry
Cancer cell lines
IC50 values
Ó 2013 Elsevier Masson SAS. All rights reserved.
Aromatic azides
1. Introduction
the most promising tumor preventive medication by Japanese re-
searchers [28]. Mechanistic studies revealed that 4 blocks the cell
Triterpenoids represent a varied class of natural products which
on account of their omnipresence constitute the major components
of some medicinal plants. Among these, some triterpene members
like betulinic acid (1), oleanolic acid (2), moronic acid (3) and
ursolic acid (4) have been exploited in recent past owing to their
significant role in various biological activities [1e4] (Fig. 1). These
triterpenes and their derivatives have been reported to possess
activities such as anti-HIV [5e11], inhibition of HIV protease [12]
and cytotoxicity to tumor cell lines [13e15]. Renewed interest in
the cytotoxic activity of triterpenes has come from the findings that
betulinic acid exhibited selective cytotoxicity against melanoma
and has apoptosis induction properties [16]. Considerable struc-
tural modification has been performed on betulinic acid and
potentially important anti-cancer derivatives have emerged from it
cycle progression in the G1 phase and its treatment results in the
triggering of apoptosis as determined by DNA fragmentation assay
[29]. Despite of its in vitro biological promise, its poor bioavailability
under in vivo restricts its clinical applications [30]. According to
previous reports, it has been observed that keeping polar sub-
stituents at the C-28 position of 4 enhanced its antitumor potential
against BGC-823 cell lines [31]. Recently, an interesting report from
Liu et al. [32] suggests that an acyl piperazine motif incorporated at
C-28 position of 4 was a useful structural modification to improve
its cell proliferation inhibition against the gastric cancer and breast
cancer cell lines.
In view of the preceding discussion, medicinal chemists globally
are in fray to generate more potent derivatives of compound 4 in
order to improve its antitumor potential [22,31e34]. An article by Liu
et al. [32] along with the work of Daneshtalab’s group [22] encour-
aged us to propose and synthesize a series of ursolic acid-triazolyl
based congeners at C-28 position to develop more effective thera-
peutic agents. A library of ursolic acid-triazolyl derivatives modified
at C-28 carboxylic position has been synthesized by employing click
chemistry protocol as potential anti-cancer compounds. Click
chemistry enables a modular approach to generate these novel
pharmacophores utilizing a collection of reliable chemical reactions.
Of particular interest is the Huisgen [3 þ 2] cycloaddition between a
terminal alkyne and an azide to generate substituted 1,2,3-triazoles
[17e21]. Similarly, compound 4, a pentacyclic triterpene isolated in
abundace from the peels of Malus pumila Mill. [22] has been re-
ported to possess a wide range of pharmacological properties,
including antiinflammatory, antiallergic, antibacterial, antiviral,
antitumor and cytotoxic activity being the most intriguing [23e27].
Owing to its biological potential, compound 4 was ranked as one of
*
Corresponding author. Tel.: þ91 1942431253/55; fax: þ91 1942441331.
E-mail addresses: bilal@iiim.ac.in, bilal.bht@gmail.com (B.A. Bhat).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.029

S. Rashid et al. / European Journal of Medicinal Chemistry 66 (2013) 238e245
239
COOH
COOH
HO
HO
Betulinic acid (1)
Oleonolic acid (2)
H
COOH
COOH
O
HO
Moronic acid (3)
Ursolic acid (4)
Fig. 1. Some triterpenes with potential biological potential as anti-cancer agents.
[35]. All the triazolyl derivatives were screened for anti-cancer ac-
region as well as by other proton absorptions in aromatic region.
13
tivity against a panel of four human cancer cell lines along with a
normal epithelial cell line. To our delight, it was observed that most
of the compounds exhibited promising cytotoxic activity against all
the tested cancer cell lines contrary to normal cell line. It has been
found from the data that some compounds exhibited better anti-
cancer potential than the parent compound 4 and standard com-
pounds, 5-fluorouracil and mitomycin-C used in this study. A close
inspection of the results confirmed that the triazolyl derivatives of
compound 4 with o-bromo, o-chloro and o-methoxy substitution at
aromatic ring were found to be the most promising compounds
The structure was further supported by C NMR and DEPT, which
showed all the expected carbon signals corresponding to ursolic
acid-triazolyl derivatives. ESI-MS of all the derivatives were also in
agreement with the products. By employing the above reaction
conditions, a series of ursolic acid-triazolyl derivatives that vary at
substitutions on aromatic ring has been synthesized from a range of
aromatic azides. It was observed that all the reactions worked
smoothly under ultra-sonication conditions, the reaction time and
yields are reported in Table 1.
having IC50 values less than 0.1
leukemia) cancer cell lines.
mM against MCF-7 (Breast) and THP-1
2.2. Biology: anti-cancer activity
(
The cytotoxic activity of compounds was studied using cultured
A-549 (lung), MCF-7 (Breast), HCT-116 (colon) and THP-1 (leuke-
mia) cancer cell lines along with a normal epithelial cell line (FR-2)
by using sulforhodamine-B assay. 5-fluorouracil and mitomycin-C
along with compound 4 were taken as reference standards in this
2
. Results and discussion
2.1. Chemistry
Taking cue from the literature, we have undertaken a research
study. The cell lines were exposed to 50 mM concentration of the
program directed toward the structural modifications of 4 at C-28
position to fine tune its biological potential as anti-cancer agent.
Compound 4 was subjected to oxidation using Jones reagent at 0 C
that resulted in the formation of C-3 oxidized derivative (5) in
almost quantitative yield. Propargylation of the carboxylic group at
compound for 48 h and the percentage of dead cells was evaluated.
Compounds that exhibited significant cytotoxic activity were
further evaluated at different concentrations to get their IC50
values. Compounds 7g, 7p and 7r with o-bromo, o-methoxy and o-
chloro substitution at aromatic ring were found to be the most
promising. From the percentage inhibition data (Table 2) and IC50
values (Table 3), we conclude:
ꢀ
C-28 was tried with different bases including NaHCO
3 2 3
, Cs CO ,
pyridine, Et N and DBU. Cs CO condition in dry THF delivered the
3
2
3
well poised alkyne derivative (6) in excellent yield. On the other
hand, aromatic azides were prepared from their respective anilines
by diazotization with sodium nitrite in acidic conditions followed
by displacement with sodium azide in good to excellent yield. 1,3-
dipolar cycloaddition reaction of 6 with aromatic azides in presence
(a) Compounds 4, 6, 7a, 7b, 7de7h, 7k, 7pe7r possess better anti-
cancer activity compared to 7c, 7i and 7j.
(b) Compounds having o-substitutions at aromatic ring (7g, 7k, 7p
and 7r) are generally more active than compounds without
substitution or substitution other than ortho-position. Howev-
er, compounds with electron withdrawing groups at ortho-po-
sition (7l and 7q) and o-fluoro substitution (7k) are less active
than compounds having electron donating ortho-substitutions.
(c) Among all the active compounds, compound 7b, 7g, 7p and 7r
were the most active in this study with IC50 values less than
4 2 2
of CuSO .5H O and sodium ascorbate in t-BuOH: H O (2:1) resulted
into the formation of 1,4-substituted-triazolyl derivatives (7ae7r)
regioselectively in excellent yields (Scheme 1). All the reactions
ꢀ
were carried out at 45 C under ultra-sonication and completed
1
13
within 0.3e8 h. The products were confirmed by H NMR, C NMR,
IR and MS analysis. In H NMR cyclization of azides to form triazoles
1
was confirmed by resonance of H-5 of triazole ring in aromatic
0.1 mM against certain cell lines. However, it is to be noted that

240
S. Rashid et al. / European Journal of Medicinal Chemistry 66 (2013) 238e245
O
COOH
COOH
a
b
O
HO
O
O
4
5
6
N3
c
R
N
N
O
N
O
R
O
7a-7r
ꢀ
ꢀ
Scheme 1. (a) CrO
3 2
, H SO
4
, acetone, 0 C, 98%; (b) propargyl bromide, Cs
2 3
CO , THF, rt, 92%, (c) sodium ascorbate, CuSO
4
, t-BuOH: H
2
O (2:1), 45 C, sonication, 72e98%.
ꢁ
1
the compound 7b was not selective between normal and can-
cer cell lines. From the IC50 values, it is quite clear that the MCF-
Infrared spectra were recorded as cm absorption using KBr pellet
1
13
method on Perkin Elmer FT-IR spectrometer. H NMR and C NMR
were recorded on 500, 400 and 100 MHz Bruker Daltonics spec-
7
and THP-1 cell lines were found to be the most susceptible to
7
g, 7p and 7r.
trometers. The chemical shifts (d) are reported in ppm relative to
(
d) From the IC50 values, it is quite clear that almost all the active
tetramethylsilane (TMS) as internal standard and coupling con-
stants were measured in Hz. Mass spectra were recorded on Bruker
Daltonics electro spray ionization apparatus. Column chromatog-
raphy was carried out on silica gel (Qualigens, 60e120 mesh) and
pre-coated silica gel thin layer chromatographic (TLC) plates were
viewed with ultraviolet light at 254 nm for fluorescence quenching
spots and at 366 nm for fluorescent spots. Ceric sulfate, anisalde-
hyde, and iodine were used as visualizing agents. 4 used in this
study was isolated from apple peels in bulk and its characterization
was assured by routine spectral techniques.
compounds exhibited less cytotoxic activity toward the normal
epithelial cell line (FR-2) compared to their anti-cancer po-
tential against cancer cell lines, which justifies the role of these
triazolyl derivatives of 4 as anti-cancer agents.
(
e) Compounds 7g, 7p and 7r with o-bromo, o-methoxy and o-
chloro substitution at aromatic ring were found to be the most
potent among the whole series with IC50 value less than 0.1 mM
against two of the tested cancer cell lines, MCF-7 and THP-1. It
was observed that the IC50 values of these compounds against a
normal cell line (FR-2) were more than 50
. Conclusion
To sum up, a series of ursolic acid-1-phenyl-1H-[1,2,3]triazol-4-
mM.
4
4
.2. Synthesis
3
.2.1. Synthesis of compound 5
To a solution of 4 (400 mg, 0.88 mmol) in acetone (9 ml) at 0 C, 2
ꢀ
ylmethylester derivatives has been designed and synthesized by
employing Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction
of terminal alkyne derivatives of 4 with various aromatic azides. All the
compounds synthesized were screened for anti-cancer activityagainst
a panel of four human cancer cell lines including A-549 (lung), MCF-7
drops of Jones reagentwere added and reaction mixture was stirred at
room temperature. Reaction was monitored by TLC till its completion
in around 2 h. After quenching the reaction with cold water, crude
product was extracted with ethylacetate (3 ꢂ 20 ml). Organic layer
was dried over sodium sulfate and purified through column chro-
matography to give pure compound 5 (365 mg, 91% yield). White
(breast), HCT-116 (colon), THP-1 (leukemia) and a normal epithelial
cell line (FR-2) using sulforhodamine-B assay. From the data, it was
evident that most of the compounds exhibited better anti-cancer ac-
tivity against all the tested cancer cell lines compared to positive
controls, 4, 5-fluorouracil and mitomycin-C used in this study. Com-
pounds 7g, 7p and 7r with o-bromo, o-methoxy and o-chloro sub-
stitutions were found to be the most promising derivatives and their
detailed mechanistic studies are currently underway in our laboratory.
ꢀ
1
solid; mp: 276e278 C; H NMR (400 MHz, CDCl
1H, d, J ¼ 12.0 Hz), 2.28 (1H, t, J ¼ 8.0 Hz), 1.97 (2H, m), 1.81 (4H, m),
.52 (12H, m), 1.30 (4H, m), 1.04 (3H, s), 0.91 (3H, s), 0.85 (3H, s), 0.82
3
): d 5.29 (1H, s), 3.20
(
1
13
(
3H, s), 0.80 (3H, s), 0.75 (3H, s), 0.68 (3H, s); C NMR (100 MHz,
CDCl ): 216.6,181.5,151.9,123.2, 53.6, 52.2, 51.2, 50.6, 49.3, 47.8, 46.8,
1.7, 40.1, 39.8, 39.7, 39.4, 39.3, 39.1, 39.1, 38.9, 38.5, 38.4, 36.6, 28.3,
3
d
4
2
7
3.3, 21.2, 17.1, 17.0, 16.1, 15.3; IR (KBr): 2930, 1718, 1657, 1456, 1378,
51 cm ; ESI-MS: 455.46 calcd. for C30
ꢁ
1
þ
H O
48 3
[M þ Na] .
4
. Experimental
4.2.2. Synthesis of compound 6
4.1. General
To a solution of 5 (365 mg, 0.8 mmol) in THF, cesium carbonate
521 mg, 1.6 mmol) and propargyl bromide (476 mg, 4 mmol) were
(
Melting points were determined on digital Buchi B-545 melting
point apparatus using glass capillaries and are uncorrected.
added and the reaction mixture was stirred at room temperature
for 4 h. Reaction was monitored by TLC and the crude product was

S. Rashid et al. / European Journal of Medicinal Chemistry 66 (2013) 238e245
241
Table 2
Percentage inhibition data of compounds 4, 6, 7ae7r against 4 cancer cell lines & a
normal cell line.
Table 1
a
Ursolic acid-triazolyl derivatives varying at aromatic ring.
Tissue
Lung
Breast
MCF-7
Colon
Leukemia Normal
cell line
N
Cell line
A-549
HCT-116 THP-1
FR-2
N
N
Entry Code
Conc.
mol)
O
R
(
m
O
1
2
3
4
4
50
61 ꢃ 0.3 57 ꢃ 0.4 53 ꢃ 0.3 84 ꢃ 0.2 66 ꢃ 0.2
28 ꢃ 0.2 36 ꢃ 0.2 55 ꢃ 0.2 31 ꢃ 0.3 31 ꢃ 0.2
78 ꢃ 0.4 42 ꢃ 0.1 96 ꢃ 0.2 88 ꢃ 0.3 86 ꢃ 0.1
50 ꢃ 0.1 22 ꢃ 0.4 74 ꢃ 0.1 42 ꢃ 0.2 33 ꢃ 0.5
64 ꢃ 0.2 73 ꢃ 0.2 91 ꢃ 0.1 86 ꢃ 0.1 78 ꢃ 0.3
57 ꢃ 0.3 17 ꢃ 0.3 69 ꢃ 0.3 82 ꢃ 0.4 2 ꢃ 0.2
93 ꢃ 0.3 99 ꢃ 0.1 99 ꢃ 0.2 94 ꢃ 0.2 85 ꢃ 0.4
74 ꢃ 0.1 69 ꢃ 0.3 91 ꢃ 0.1 82 ꢃ 0.4 68 ꢃ 0.5
10
O
6
50
10
Time Yielda Compd
Time Yielda
(h)
7a
7b
50
Compd
R
R
10
(
h)
50
10
b
F
5
6
7c
7d
50
50
30 ꢃ 0.2 03 ꢃ 0.2 16 ꢃ 0.3 27 ꢃ 0.4 ND
F
64 ꢃ 0.1 51 ꢃ 0.3 78 ꢃ 0.3 83 ꢃ 0.3 45 ꢃ 0.4
43 ꢃ 0.2 28 ꢃ 0.2 44 ꢃ 0.2 54 ꢃ 0.3 12 ꢃ 0.4
50 ꢃ 0.3 80 ꢃ 0.1 76 ꢃ 0.1 75 ꢃ 0.2 ND
27 ꢃ 0.4 40 ꢃ 0.2 02 ꢃ 0.2 47 ꢃ 0.3 ND
63 ꢃ 0.3 78 ꢃ 0.2 92 ꢃ 0.3 95 ꢃ 0.1 93 ꢃ 0.4
45 ꢃ 0.1 44 ꢃ 0.2 80 ꢃ 0.2 74 ꢃ 0.1 58ꢃ
99 ꢃ 0.3 99 ꢃ 0.1 99 ꢃ 0.3 99 ꢃ 0.3 99 ꢃ 0.3
85 ꢃ 0.2 80 ꢃ 0.2 41 ꢃ 0.2 92 ꢃ 0.5 2 ꢃ 0.2
26 ꢃ 0.3 34 ꢃ 0.3 67 ꢃ 0.2 55 ꢃ 0.4 46 ꢃ 0.1
13 ꢃ 0.1 17 ꢃ 0.2 44 ꢃ 0.1 32 ꢃ 0.3 14 ꢃ 0.3
26 ꢃ 0.2 52 ꢃ 0.3 49 ꢃ 0.2 09 ꢃ 0.3 ND
18 ꢃ 0.3 15 ꢃ 0.1 12 ꢃ 0.2 49 ꢃ 0.3 ND
67 ꢃ 0.4 76 ꢃ 0.3 78 ꢃ 0.4 79 ꢃ 0.2 56 ꢃ 0.4
43 ꢃ 0.2 54 ꢃ 0.4 44 ꢃ 0.3 56 ꢃ 0.3 36 ꢃ 0.3
46 ꢃ 0.3 04 ꢃ 0.3 23 ꢃ 0.4 73 ꢃ 0.1 ND
16 ꢃ 0.5 03 ꢃ 0.4 10 ꢃ 0.4 32 ꢃ 0.1 ND
03 ꢃ 0.3 04 ꢃ 0.1 03 ꢃ 0.3 02 ꢃ 0.3 ND
2 ꢃ 0.02 08 ꢃ 0.2 08 ꢃ 0.3 01 ꢃ 0.3 ND
94 ꢃ 0.4 91 ꢃ 0.1 94 ꢃ 0.3 93 ꢃ 0.5 28 ꢃ 0.1
79 ꢃ 0.5 87 ꢃ 0.4 77 ꢃ 0.5 82 ꢃ 0.2 5 ꢃ 0.5
39 ꢃ 0.2 42 ꢃ 0.4 65 ꢃ 0.2 54 ꢃ 0.4 12 ꢃ 0.2
3 ꢃ 0.3 4 ꢃ 0.2 45 ꢃ 0.4 24 ꢃ 0.4 9 ꢃ 0.1
91 ꢃ 0.5 95 ꢃ 0.6 93 ꢃ 0.4 96 ꢃ 0.1 35 ꢃ 0.5
77 ꢃ 0.3 81 ꢃ 0.3 54 ꢃ 0.5 80 ꢃ 0.3 9 ꢃ 0.4
7a
08
08
94
96
7j
03
92
10
7
8
9
1
7e
7f
50
10
F
50
10
7g
7h
50
10
50
I
7b
7k
0.5 98
0
10
11
12
13
7i
7j
7k
50
50
50
10
CF3
7c
05
95
7l
08
96
14
7l
50
50
50
50
50
1
1
5
6
7m
7n
7o
7p
CF3
17
18
10
19
7q
50
7
d
e
02
06
88
85
7m
7n
0.3 95
10
Br
20
7r
50
10
2
2
1
2
5-FUc
20
86 ꢃ 0.3 ND
76 ꢃ 0.2 96 ꢃ 0.5 ND
ND ND
Mito-Cd 01
70 ꢃ 0.3 82 ꢃ 0.3 ND
7
6.5 92
a
b
c
All experiments were carried out in triplicates.
ND ¼ not determined.
5
-FU ¼ 5-flourouracil.
F
I
d
Mito-C ¼ mitomycin-C.
7
7
7
f
05
02
08
96
88
72
7o
7p
7q
0.4 96
subjected to column chromatography to give pure 6 (356 mg, 98%
ꢀ
1
yield). White solid; mp: 279e280 C; H NMR (400 MHz, CDCl
3
):
5.30 (1H, m), 4.66 (1H, d, J ¼ 12.0 Hz), 4.58 (1H, d, J ¼ 12.0 Hz),
.54 (1H, m), 2.40 (2H, m), 2.26 (1H, d, J ¼ 8.0 Hz), 1.95 (4H, m), 1.75
d
Br
2
(
3H, m), 1.60 (4H, m), 1.45 (3H, m), 1.37 (3H, m), 1.30 (3H, m), 1.15
Br
OMe
NO2
g
h
08
94
92
(3H, s), 1.05 (3H, s), 1.00 (6H, s), 0.94 (3H, s), 0.92 (3H, s), 0.87 (3H,
13
s); C NMR (100 MHz, CDCl
3
):
5.3, 52.9, 51.6, 48.2, 47.4, 46.8, 42.2, 39.6, 39.1, 39.0, 38.8, 36.7, 36.4,
4.2, 32.6, 30.6, 28.0, 26.6, 24.2, 23.5, 23.4, 21.5, 21.1, 19.6, 17.2, 17.0,
d 217.9, 176.6, 137.9, 125.6, 78.1, 74.4,
5
3
1
5.3; IR (KBr): 2939, 2565, 2455, 2417, 1717, 1654, 1464, 1378, 1246,
1042, 779, 718 cm
ꢁ1
þ
; ESI-MS: 493.36 calcd. for C33
H
48
O
3
[M þ H] .
04
02
4
.2.3. General procedure for the synthesis of compounds 7ae7r
To a solution of compound 6 (15 mg, 0.03 mmol) in t-BuOH:H
2:1, 2 ml), sodium ascorbate (1.2 mg, 0.006 mmol) and CuSO
1.2 mg, 0.0045 mmol) were added at room temperature. To this
mixture, aryl azide (0.06 mmol) was added and the reaction
mixture was sonicated at 45 C till its completion, monitored by
CN
2
O
(
(
4
Cl
7i
08
88
7r
85
ꢀ
TLC. The crude mixture was extracted with ethylacetate (3 ꢂ 20 ml)
and the combined organic layer was dried over sodium sulfate and
purified through column chromatography to give pure 7ae7r in
Cl
Yields reported are isolated yields in percentage.
a
72e98% yield.

242
S. Rashid et al. / European Journal of Medicinal Chemistry 66 (2013) 238e245
Table 3
IC50 values of compounds 4, 6, 7a, 7b, 7d, 7f, 7g, 7h, 7k and 7pe7r against various cell lines.
Tissue
Cell line
Entry
Lung
Breast
MCF-7
Colon
Leukemia
THP-1
Normal cell line
FR-2
A-549
HCT-116
Code
IC 50 values in (mM)
1
2
3
4
5
6
7
8
9
4
6
33 ꢃ 0.03
15 ꢃ 0.06
6.4 ꢃ 0.08
0.5 ꢃ 0.05
24 ꢃ 0.04
18 ꢃ 0.08
2.9 ꢃ 0.05
>100
19 ꢃ 0.07
<0.1 ꢃ 0.001
>50 ꢃ 4.0
0.15 ꢃ 0.01
37 ꢃ 0.07
42 ꢃ 0.08
9.1 ꢃ 0.07
13 ꢃ 0.09
31 ꢃ 0.08
24 ꢃ 0.06
44 ꢃ 0.03
10 ꢃ 0.04
82 ꢃ 0.09
19 ꢃ 0.02
69 ꢃ 0.05
46 ꢃ 0.06
35 ꢃ 0.08
>50 ꢃ 4.1
>50 ꢃ 6.0
>50 ꢃ 3.9
56 ꢃ 0.06
30 ꢃ 0.09
2.3 ꢃ 0.07
0.47 ꢃ 0.04
<0.1 ꢃ 0.09
14 ꢃ 0.03
1.7 ꢃ 0.06
15 ꢃ 0.06
19 ꢃ 0.09
15 ꢃ 0.04
0.3 ꢃ 0.001
14 ꢃ 1.5
7a
7b
7d
7f
7g
7h
7k
7p
7q
7r
1.1 ꢃ 0.04
0.9 ꢃ 0.02
11 ꢃ 0.09
5.5 ꢃ 0.08
39 ꢃ 0.03
15 ꢃ 0.08
1.0 ꢃ 0.06
<0.1 ꢃ 0.03
37 ꢃ 0.06
<0.1 ꢃ 0.05
79 ꢃ 0.06
13 ꢃ 0.05
9.4 ꢃ 0.09
<0.1 ꢃ 0.001
48 ꢃ 4.0
10
11
12
<0.1 ꢃ 0.09
>50 ꢃ 3.0
<0.1 ꢃ 0.001
9.1 ꢃ 1.0
<0.1 ꢃ 0.001
4
d
.2.3.1. Compound 7a. White paste: 1H NMR (400 MHz, CDCl
8.02 (1H, s), 7.28 (1H, m), 6.82 (1H, m), 6.65 (1H, m), 5.23 (2H, s),
.19 (1H, m), 2.78 (1H, d, J ¼ 12.0 Hz), 2.43 (2H, m), 2.26 (4H, m),
3
):
138.0, 136.9, 129.7, 129.6, 128.8, 125.4, 122.4, 120.4, 120.0, 57.2, 55.4,
52.9, 48.2, 47.3, 46.6, 42.1, 39.2, 39.1, 38.8, 38.9, 36.6, 36.6, 34.1, 30.4,
29.7, 29.9, 26.6, 23.3, 23.3, 21.4, 21.1, 19.5, 16.9, 16.7, 15.1, 14.3; IR
5
ꢁ1
1.77 (2H, m), 1.60 (2H, m), 1.54 (2H, m), 1.47 (2H, m), 1.42 (2H, m),
(KBr): 2926, 2858, 1707, 1697, 1455, 977 cm ; ESI-MS: 634.42
þ
1.21 (3H, s), 1.04 (2H, m), 1.01 (3H, s), 0.99 (3H, s), 0.94 (2H, m), 0.90
calcd. for C39H
53
N
3
O
3
[M þ Na] .
13
(
(
1
4
2
3H, s), 0.87 (3H, s), 0.81 (2H, m), 0.77 (3H, s), 0.45 (3H, s); C NMR
100 MHz, CDCl ): 217.4, 177.2, 168.4, 166.1, 144.4, 139.6, 137.0,
23.4, 118.8, 110.9, 110.1, 103.6, 58.4, 55.4, 54.2, 53.6, 51.0, 49.8, 48.4,
d
4.2.3.5. Compound 7e. White solid; mp: 285e287 C; 1H NMR
(400 MHz, CDCl ): 8.04 (2H, m), 7.97 (1H, s), 7.66 (5H, m), 5.33
ꢀ
3
3
d
6.3, 43.5, 41.2, 39.8, 38.4, 36.8, 36.4, 34.2, 32.6, 30.6, 28.0, 26.2,
4.2, 23.4, 21.8, 21.1, 19.6, 17.2, 17.0, 16.8; IR (KBr): 2922, 2857, 1717,
(1H, m), 5.27 (2H, s), 2. 48 (1H, m), 2.26 (2H, m) 2.01 (1H, m), 1.81
(3H, m), 1.87 (3H, m), 1.74 (3H, m), 1.54 (3H, s), 1.45 (4H, m), 1.39
ꢁ
1
1703, 1628, 1540, 1432, 1341, 986 cm ; ESI-MS: 670.48 calcd. for
(4H, m), 1.03 (3H, s), 0.97 (3H, s), 0.93 (3H, s), 0.92 (2H, m), 0.89 (3H,
þ
13
C
H
39 51
N
3
O
3
[M þ Na] .
s), 0.86 (3H, s), 0.64 (3H, s); C NMR (100 MHz, CDCl
3
):
d
217.7,
177.6, 143.2, 138.2, 134.2, 133.6, 130.5, 128.4, 128.3, 127.9, 127.1,
.2.3.2. Compound 7b. White solid, mp: 287e288 C; 1H NMR
400 MHz, CDCl ):
7.98 (1H, s), 7.50 (1H, s), 741 (1H, d, J ¼ 8.0 Hz),
ꢀ
126.6, 125.5, 124.9, 123.4, 122.4, 57.4, 55.2, 52.9, 48.3, 47.3, 46.7,
42.2, 39.5, 39.3, 39.1, 38.8, 36.7, 36.6, 34.0, 32.5, 30.6, 28.0, 26.6, 24.1,
23.4, 23.3, 21.4, 21.1, 19.6, 17.1, 17.0, 15.1; IR (KBr): 2919, 2850, 1709,
4
(
3
d
7
.25 (1H, d, J ¼ 8.0 Hz), 5.26 (2H, s), 5.21 (1H, m), 2.89 (1H, d,
ꢁ
1
J ¼ 12.0 Hz), 2.52 (2H, m), 2.37 (3H, s), 2.34 (3H, s), 2.27 (2H, m),1.99
1692, 1542, 1449, 1383, 992 cm ; ESI-MS: 684.86 calcd. for
þ
(
(
2H, m), 1.88 (4H, m), 1.57 (4H, m), 1.43 (4H, m), 1.33 (2H, m), 1.26
3H, s), 1.10 (3H, s), 1.06 (3H, s), 1.00 (3H, s), 0.95 (3H, s), 0.91 (2H,
C
43
55
H N
3
O
3
[M þ Na] .
13
4.2.3.6. Compound 7f. White solid; mp: 279e281 C; 1H NMR
(400 MHz, CDCl ): 8.08 (1H, s), 7.71 (4H, m), 5.32 (1H, m), 5.28
ꢀ
m), 0.86 (3H, s), 0.48 (3H, s); C NMR (100 MHz, CDCl
77.8, 143.7, 138.3, 138.1, 137.6, 130.6, 125.5, 122.7, 122.3, 121.7, 117.8,
7.6, 56.2, 55.2, 54.4, 47.4, 46.8, 41.8, 41.0, 39.2, 39.1, 36.7, 34.1, 33.1,
1.9, 31.6, 30.7, 30.3, 29.7, 29.3, 26.5, 25.6, 23.6, 22.7, 21.4, 19.9, 19.4,
6.7, 14.9, 14.1; IR (KBr): 2925, 2856, 1714, 1700, 1620, 1539, 1444,
3
): d 217.7,
1
5
3
1
3
d
(2H, s), 2.34 (1H, m), 2.31 (1H, m), 2.19 (1H, d, J ¼ 8.0 Hz), 2.09 (1H,
m), 1.95 (3H, m), 1.77 (4H, m), 1.64 (4H, m), 1.61 (3H, m) 1.48 (3H, s),
1.33 (3H, s), 1.09 (3H, s), 1.0 (3H, s), 0.99 (3H, s), 0.96 (2H, m), 0.93
ꢁ
1
13
1391, 1325, 1230, 972 cm ; ESI-MS: 662.34 calcd. for C41
H
57
N
3
O
3
(3H, s), 0.91 (3H, s), 0.61 (3H, s); C NMR (100 MHz, CDCl
3
):
d
216.6,
þ
[M þ Na] .
176.5, 143.0, 137.1, 134.9, 131.9, 124.5, 121.5, 121.3, 120.8, 56.1, 54.2,
2.0, 47.2, 46.3, 45.7, 41.2, 38.5, 38.3, 38.1, 37.8, 35.6, 35.6, 33.1, 31.5,
28.9, 26.7, 25.6, 23.1, 22.4, 22.3, 20.4, 20.1, 18.9, 16.0, 15.8, 14.1; IR
5
.2.3.3. Compound 7c. Light yellow solid; mp: 282e284 C; 1
):
ꢀ
H
4
ꢁ1
NMR (400 MHz, CDCl
3
d
8.11 (1H, s), 7.90 (2H, d, J ¼ 4.0 Hz), 7.81
(KBr): 2919, 2854, 1721, 1705, 1588, 1490, 1457, 1394, 990 cm ; ESI-
þ
(
2
2H, d, J ¼ 8.0 Hz), 5.29 (1H, m), 5.26 (2H, s), 2.51 (1H, d, J ¼ 12.0 Hz),
MS: 713.24 calcd. for C39
H52BrN
3
O
3
[M þ Na] .
.48 (2H, m), 2.36 (1H, d, J ¼ 8.0 Hz), 2.00 (2H, m), 1.92 (3H, m), 1.81
4.2.3.7. Compound 7g. White solid, mp: 280e282 C; 1H NMR
(500 MHz, CDCl ):
ꢀ
(
(
4H, m), 1.60 (2H, m), 1.52 (2H,m), 1.40 (2H, m), 1.32 (4H, m), 1.09
3H, s),1.06 (3H, s), 0.89 (3H, s) 0.87 (3H, s), 0.85 (3H, s), 0.84 (3H, s),
3
d
8.01 (1H, d, J ¼ 10.0 Hz), 7.77 (1H, m), 7.56 (1H,
0
.46 (3H, s); 13C NMR (100 MHz, CDCl
3
):
d
217.7, 177.6, 144.2, 139.3,
m), 7.50 (1H, m), 7.41 (1H, m), 5.28 (3H, m), 2.51 (1H, m), 2.45 (2H,
m), 2.28 (1H, d, J ¼ 15.0 Hz), 2.01 (2H, m), 1.95 (3H, m), 1.74 (2H, m),
1.67 (4H, m), 1.47 (2H, m), 1.27 (2H, m), 1.25 (3H, s), 1.20 (3H, s), 1.17
138.0, 127.1, 127.1, 125.5, 122.4, 120.3, 57.0, 55.2, 53.0, 48.2, 47.3,
4
2
6.6, 42.1, 39.4, 39.2, 39.1, 38.8, 36.6, 36.6, 34.1, 32.4, 30.6, 29.7, 27.9,
6.5, 24.1, 23.7, 23.3, 22.7, 21.4, 21.1, 19.5, 17.0, 16.7, 15.0, 14.1; IR
(3H, s), 1.08 (3H, s), 0.99 (3H, s), 0.87 (4H, m), 0.84 (3H, s), 0.68 (3H,
ꢁ
1
13
(
KBr): 2926, 2862, 1731, 1701, 1610, 1512, 1455, 1389, 982 cm ; ESI-
s); C NMR (100 MHz, CDCl
3
):
d
217.8, 177.5, 142.9, 138.2, 136.4,
þ
MS: 701.94 calcd. for C40
H
52
F
3
N
3
O
3
[M þ Na] .
134.0, 131.2, 128.5, 128.1, 126.0, 125.5, 118.3, 57.2, 55.2, 52.9, 48.2,
7.4, 46.8, 46.7, 42.2, 41.2, 39.5, 39.3, 39.2, 39.1, 38.8, 36.7, 34.1, 30.6,
30.5, 28.3, 26.6, 24.4, 23.5, 22.6, 22.4, 19.6, 17.0, 15.2; IR (KBr): 3015,
4
1
4
d
.2.3.4. Compound 7d. Gummy liquid; H NMR (400 MHz, CDCl
3
):
ꢁ
1
8.03 (1H, s), 7.73 (2H, m), 7.54 (2H, m), 7.44 (1H, t, J ¼ 12.0 Hz),
2951, 1704, 1692, 1522, 1462, 1434, 1372, 1322, 1285, 982 cm ; ESI-
þ
5
.32 (1H, m), 5.25 (2H, s), 2.58 (1H, m), 2.30 (3H, m), 2.04 (2H, m),
MS: 713.44 calcd. for C39
H52BrN
3
O
3
[M þ Na] .
1
1
(
.87 (3H, m), 1.60 (4H, m), 1.50 (4H, m), 1.48 (4H, m), 1.32 (3H, s),
.21 (3H, s), 1.10 (3H, s), 1.01 (3H, s), 0.95 (2H, m), 0.89 (3H, s), 0.85
3H, s), 0.53 (3H, s); 13C NMR (100 MHz, CDCl
): 217.7, 177.5, 143.7,
ꢀ
1
4.2.3.8. Compound 7h. Yellowish solid, mp: 284e285 C; H NMR
(400 MHz, CDCl ): 7.94 (1H, s), 7.75 (2H, m), 7.44 (2H, m), 5.27 (2H,
3
d
3
d

S. Rashid et al. / European Journal of Medicinal Chemistry 66 (2013) 238e245
243
s), 5.26 (1H, m), 4.19 (2H, s), 2.60 (1H, m), 2.41 (1H, m), 2.17 (1H, d,
J ¼ 8.0 Hz), 2.02 (2H, m),1.97 (4H, m),1.77 (4H, m),1.62 (2H, m),1.54
s), 5.26 (1H, m), 2.52 (1H, m), 2.40 (1H, m), 2.22 (1H, d, J ¼ 8.0 Hz),
2.15 (2H, m), 2.06 (4H, m), 1.96 (3H, m), 1.85 (2H, m), 1.60 (4H, m),
1.43 (3H, m), 1.26 (3H, s), 1.10 (3H, s), 1.00 (3H, s), 0.98 (3H, s), 0.90
(2H, m), 0.84 (3H, s), 0.83 (3H, s), 0.50 (3H, s); C NMR (100 MHz,
3
CDCl ): d 217.8, 177.6, 144.0, 138.0, 137.8, 131.8, 130.9, 125.5, 123.6,
(
2H, m), 1.51 (3H, s), 1.48 (4H, m), 1.25 (3H, s), 1.08 (3H, s), 1.00 (3H,
13
13
s), 0.94 (3H, s), 0.85 (2H, m), 0.72 (3H, s), 0.68 (3H, s); C NMR
100 MHz, CDCl ): 217.6, 177.6, 152.6, 144.1, 139.4, 138.3, 132.8,
27.3, 127.1, 124.9, 122.4, 120.2, 120.0, 57.3, 55.2, 52.5, 48.7, 47.3,
(
3
d
1
4
2
2
123.3, 122.6, 118.9, 57.3, 55.2, 53.0, 48.2, 47.3, 46.6, 42.1, 39.4, 39.1,
38.8, 36.6, 35.4, 33.6, 34.1, 31.3, 30.9, 27.4, 26.6, 23.3, 21.4, 21.1, 19. 2,
18.4, 17.0, 16.6, 15.2, 14.4; IR (KBr): 2922, 2851, 1728, 1705, 1592,
6.5, 45.7, 43.7, 41.3, 39.4, 39.0, 38.1, 36.8, 36.5, 35.3, 34.1, 33.6, 29.7,
6.4, 24.6, 23.8, 22.5, 21.5, 20.9, 17.8, 16.2, 15.8, 14.3; IR (KBr): 2926,
ꢁ1
ꢁ1
þ
848, 1709, 1697, 1517, 1462, 1424, 1324, 997 cm ; ESI-MS: 650.47
1456, 996 cm ; ESI-MS: 713.38 calcd. for C39
H52BrN
3
O
3
[M þ Na] .
þ
calcd. for C41
H
54
N
4
O
3
[M þ H] .
4
.2.3.14. Compound 7n. White paste: 1H NMR (400 MHz, CDCl
3
):
4
d
.2.3.9. Compound 7i. Gummy liquid: 1H NMR (400 MHz, CDCl
8.01 (1H, s), 7.68 (2H, d, J ¼ 4.0 Hz), 7.51 (2H, d, J ¼ 4.0 Hz), 5.26
3
):
d
8.09 (1H, s), 7.90 (2H, d, J ¼ 8.0 Hz), 7.80 (2H, d, J ¼ 8.0 Hz), 5.30
(1H, m), 5.29 (2H, s), 2.53 (1H, m), 2.42 (1H, m), 2.25 (1H, m), 2.11
(4H, m), 2.00 (2H, m), 1.88 (3H, m), 1.76 (3H, m), 1.57 (4H, m), 1.50
(2H, m), 1.32 (3H, s), 1.10 (3H, s), 1.05 (3H, s), 0.99 (3H, s), 0.92 (2H,
m), 0.85 (3H, s), 0.84 (3H, s), 0.53 (3H, s); C NMR (100 MHz,
CDCl ): 217.9, 177.6, 144.2, 139.3, 138.1, 127.1, 125.5, 122.4, 120.3,
(
1H, m), 5.24 (2H, s), 2.50 (1H, m), 2.37 (1H, m), 2.27 (1H, d,
J ¼ 8.0 Hz), 2.01 (2H, m),1.90 (4H, m),1.70 (4H, m), 1.55 (4H, m),1.32
4H, m), 1.10 (3H, s), 1.06 (3H, s), 0.99 (3H, s), 0.91 (3H, s), 0.89 (2H,
13
(
13
m), 0.85 (3H, s), 0.84 (3H, s), 0.53 (3H, s); C NMR (100 MHz,
CDCl ): 217.8, 177.6, 144.0, 138.0, 135.4, 134.7, 129.9, 125.5, 122.4,
21.6, 57.1, 55.2, 52.9, 48.2, 47.4, 46.7, 42.1, 42.0, 39.4, 39.3, 39.1, 38.8,
3
d
3
d
120.3, 57.0, 55.2, 53.0, 48.2, 47.3, 46.6, 42.1, 39.1, 38.8, 36.6, 35.4,
33.6, 34.1, 32.0, 30.6, 29.7, 27.4, 26.5, 24.1, 23.4, 22.0, 21.2, 19.7, 18.8,
17.0, 16.8, 15.0; IR (KBr): 2928, 2850, 1713, 1705, 1622, 1539, 1382,
1
3
6.6, 34.1, 32.4, 29.8, 28.4, 26.5, 24.1, 23.4, 23.3, 21.4, 21.1, 19.5, 17.0,
ꢁ1
þ
1
9
6.7, 15.1; IR (KBr): 2924, 2853, 1716, 1701, 1604, 1496, 1446, 1392,
1345, 978 cm ; ESI-MS: 652.17 calcd. for C39
H52FN
3
O
3
[M þ Na] .
ꢁ
1
þ
83 cm ; ESI-MS: 647.26 calcd. for C39
H52ClN
3
O
3
[M þ H] .
4
d
.2.3.15. Compound 7o. Yellow paste: 1H NMR (400 MHz, CDCl
8.00 (1H, s), 7.69 (2H, m), 7.51 (2H, m), 5.30 (2H, s), 5.26 (1H, m),
3
):
ꢀ
4
.2.3.10. Compound 7j. White solid, mp: 279e282 C; 1H NMR
): 8.12 (1H, s), 7.98 (1H, m), 7.45 (1H, m), 7.34 (2H,
(
400 MHz, CDCl
3
d
2.52 (1H, m), 2.37 (1H, m), 2.25 (1H, d, J ¼ 8.0 Hz), 2.10 (2H, m), 1.98
(4H, m), 1.76 (3H, m), 1.57 (4H, m), 1.50 (2H, m), 1.40 (3H, m), 1.30
(3H, s), 1.10 (3H,s), 1.06 (3H, s), 0.98 (3H, s), 0.88 (2H, m), 0.85 (3H,
m), 5.26 (1H, m), 5.25 (2H, s), 2.51 (1H, m), 2.40 (1H, m), 2.25 (1H, d,
J ¼ 8.0 Hz), 2.01 (2H, m), 1.94 (3H, m), 1.75 (4H, m),1.64 (2H, m),1.56
13
(
3H, m), 1.38 (4H, m), 1.20 (3H, s), 1.08 (3H, s), 1.01 (3H, s), 0.94 (3H,
3
s), 0.84 (3H, s), 0.55 (3H, s); C NMR (100 MHz, CDCl ): d 217.6,
13
s), 0.92 (2H, m), 0.86 (3H, s), 0.84 (3H, s), 0.58 (3H, s); C NMR
100 MHz, CDCl ): 217.9, 177.4, 143.5, 138.1, 130.3, 130.2, 125.5,
25.3, 125.2, 124.8, 117.2, 117.0, 57.2, 55.1, 52.9, 48.2, 47.3, 46.7, 42.1,
177.6, 144.0, 138.1, 135.4, 134.7, 129.9, 125.5, 122.3, 121.6, 57.1, 55.2,
53.4, 48.2, 47.4, 46.7, 42.2, 39.5, 39.1, 38.8, 36.6, 36.6, 34.1, 32.0, 31.0,
29.7, 28.0, 26.6, 24.2, 23.4, 22.6, 22.1, 20.0, 18.8, 17.3, 17.4, 15.4; IR
(
3
d
1
3
2
1
C
ꢁ
1
9.4, 39.3, 39.1, 38.8, 36.6, 34.1, 32.5, 31.1, 30.0, 29.7, 26.6, 24.1, 23.4,
3.2, 21.4, 21.1, 19.5, 17.0, 16.7, 15.1; IR (KBr): 2926, 2859, 1716, 1695,
(KBr): 2922, 2850, 1705, 1504, 1459, 1375, 1329, 975 cm ; ESI-MS:
þ
759.90 calcd. for C39
H52IN
3
O
3
[M þ Na] .
ꢁ1
586, 1522, 1481, 1342, 1309, 989 cm ; ESI-MS: 652.12 calcd. for
þ
4.2.3.16. Compound 7p. Yellow paste: 1H NMR (500 MHz, CDCl
39
H
52FN
3
O
3
[M þ Na] .
3
):
d
8.16 (1H, d, J ¼ 5.0 Hz), 7.89 (1H, d, J ¼ 5.0 Hz), 7.42 (1H, t,
4
.2.3.11. Compound 7k. Yellow paste; 1H NMR (400 MHz, CDCl
3
):
J ¼ 5.0 Hz), 7.10 (2H, t, J ¼ 10.0 Hz), 5.27 (3H, m), 3.90 (3H, s), 2.50
(1H, m), 2.38 (1H, m), 2.27 (1H, d, J ¼ 15.0 Hz), 2.00 (2H, m), 1.92
(4H, m), 1.70 (6H, m), 1.58 (2H, m), 1.46 (2H, m), 1.32 (6H, s), 1.20
(2H, m), 1.12 (3H, s), 1.05 (3H, s), 0.95 (6H, s), 0.89 (2H, m), 0.62 (3H,
d
8.01 (1H, s), 7.91 (1H, d, J ¼ 4.0 Hz), 7.51 (1H,m), 7.49 (1H, m), 7.44
(
(
1H, m), 5.28 (1H, m), 5.26 (2H, s), 2.51 (1H, m), 2.42 (1H, m), 2.30
1H, d, J ¼ 8.0 Hz), 2.01 (2H, m), 1.95 (3H, m), 1.72 (4H, m), 1.60 (2H,
13
m), 1.45 (4H, s), 1.32 (3H, m), 1.08 (3H, s), 1.04 (3H, s), 1.00 (3H, s),
3
s); C NMR (100 MHz, CDCl ): d 218.0, 177.3, 143.9, 142.4, 138.1,
13
0
.95 (3H, s), 0.91 (2H, m) 0.87 (3H, s), 0.86 (3H, s), 0.72 (3H, s);
NMR (100 MHz, CDCl ): 217.8, 177.4, 143.0, 140.4, 139.9, 138.2,
31.5, 129.2, 127.8, 125.9, 125.4, 93.6, 57.3, 55.2, 52.9, 48.2, 47.4, 46.7,
C
130.1, 126.3, 125.4, 125.3, 122.2, 121.1, 112.2, 57.7, 56.4, 56.0, 53.2,
48.2, 47.4, 46.7, 42.1, 39.4, 39.2, 39.1, 38.8, 36.6, 34.1, 32.0, 31.0, 29.7,
27.2, 26.6, 25.1, 23.3, 22.4, 21.1, 19.5, 17.0, 16.7, 15.2, 14.1; IR (KBr):
3
d
1
4
2
ꢁ1
2.2, 39.5, 39.3, 39.1, 38.8, 36.7, 36.6, 34.1, 32.5, 30.6, 29.7, 28.0, 26.6,
4.1, 23.5, 23.4, 21.5, 21.1, 19.6, 17.1, 15.3; IR (KBr): 2929, 2842, 1711,
3010, 2940, 2852, 1703, 1512, 1465, 1450, 1420, 1385, 972 cm ; ESI-
þ
MS: 642.78 calcd. for C40
H
55
N
3
O
4
: [M þ H] .
ꢁ
1
1
698, 1581, 1529, 1462, 1404, 1379, 1322, 995 cm ; ESI-MS: 759.94
þ
4.2.3.17. Compound 7q. Yellow solid, mp: 282e285 C: 1H NMR
500 MHz, CDCl ):
ꢀ
calcd. for C39
H52IN
3
O
3
[M þ Na] .
(
3
d
8.10 (1H, m), 7. 91 (1H, d, J ¼ 10.0 Hz), 7.80 (1H,
ꢀ
1
4
(
.2.3.12. Compound 7l. Gray white solid, mp: 283e285 C; H NMR
400 MHz, CDCl ): 7.87 (1H, s), 7.71 (2H, m), 7.57 (2H, m), 5.27 (2H,
s), 5.26 (1H, m), 2.51 (1H, m), 2.42 (1H, m), 2.26 (1H, d, J ¼ 8.0 Hz),
.01 (2H, m), 1.94 (2H, m), 1.76 (2H, m), 1.67 (4H, m), 1.48 (4H, m),
.25 (3H, s), 1.10 (3H, s), 1.01 (3H, s), 0.98 (3H, s), 0.93 (4H, m), 0.89
m), 7.73 (1H, m), 7. 63 (1H, m), 5.29 (3H, m), 2.51 (1H, m), 2.39 (1H,
m), 2.27 (1H, d, J ¼ 15.0 Hz), 2.08 (2H, m), 1.95 (4H, m), 1.72 (4H, m),
1.65 (2H, m), 1.48 (2H, m), 1.33 (2H, m), 1.30 (3H, s), 1.22 (3H, s), 1.12
(3H, s), 1.08 (3H, s), 0.99 (3H, s), 0.92 (3H, s), 0.88 (4H, m), 0.68 (3H,
3
d
2
1
(
13
3
s); C NMR (100 MHz, CDCl ): d 217.9, 177.4, 144.3, 143.7, 138.1,
13
2H, m), 0.87 (3H, s), 0.86 (3H, s), 0.72 (3H, s); C NMR (100 MHz,
CDCl ): 217.5, 177.4, 143.2, 138.2, 135.7, 133.0, 130.5, 128.9, 128.8,
27.4, 126.5, 125.4, 112.6, 57.2, 54.4, 52.9, 48.2, 47.5, 46.7, 42.2, 39.5,
9.3, 39.1, 38.8, 36.7, 36.5, 34.2, 32.5, 30.6, 29.7, 28.0, 26.6, 24.6, 24.1,
3.4, 21.5, 21.1, 19.6, 17.0, 15.2; IR (KBr): 2926, 2854, 1728, 1701, 1611,
133.8, 130.9, 130.1, 127.8, 125.7, 125.4, 122.2, 57.5, 57.4, 55.1, 52.8,
48.2, 47.4, 46.8, 46.7, 42.2, 41.9, 39.5, 39.3, 39.0, 38.7, 36.6, 34.1, 30.7,
29.7, 26.7, 25.5, 23.3, 21.5, 21.1, 19.5, 17.0, 15.2, 15.0; IR (KBr): 3117,
3
d
1
3
2
ꢁ1
2630, 1715, 1519, 1440, 1418, 1378, 981 cm ; ESI-MS: 657.58 calcd.
þ
for C39H
52
N
4
O
5
: [M þ H] .
ꢁ
1
1
512, 1456, 1389, 993 cm ; ESI-MS: 701.94 calcd. for C40
H
52
F
3
N
3
O
3
þ
4.2.3.18. Compound 7r. White paste: 1H NMR (500 MHz, CDCl
[M þ Na] .
3
):
d
8.05 (1H, d, J ¼ 10.0 Hz), 7. 60 (2H, m), 7.46 (2H, m), 5.29 (3H, m),
ꢀ
4
.2.3.13. Compound 7m. Gray white solid, mp: 280e283 C; 1
):
J ¼ 8.0 Hz), 7.58 (1H, d, J ¼ 8.0 Hz), 7.42 (1H, t, J ¼ 8.0 Hz), 5.29 (2H,
H
2.51 (1H, m), 2.40 (1H, m), 2.27 (1H, d, J ¼ 15.0 Hz), 2.00 (2H, m),
1.93 (4H, m), 1.73 (4H, m), 1.65 (2H, m), 1.45 (2H, m), 1.32 (6H, s),
1.20 (2H, m), 1.18 (3H, s), 1.10 (3H, s), 0.98 (3H, s), 0.95 (3H, s), 0.85
NMR (400 MHz, CDCl
3
d 8.01 (1H, s), 7.92 (1H, s), 7.68 (1H, d,

244
S. Rashid et al. / European Journal of Medicinal Chemistry 66 (2013) 238e245
(
4H, m), 0.65 (3H, s); 13C NMR (100 MHz, CDCl
43.7, 143.0, 138.2, 134.7, 130.9, 128.4, 127.7, 126.1, 126.0, 125.5, 57. 3,
5.2, 52.2, 48.2, 47.4, 46.8, 46.7, 42.2, 41.8, 39.5, 39.3, 39.1, 38.7, 36.7,
6.6, 34.1, 32.3, 32.2, 30.9, 29.8, 29.7, 26.7, 23.4, 21.8, 19.5, 17.0, 15.3,
3
):
d
217.8, 177.5,
[2] S.B. Mahato, S.K. Sarkar, G. Poddar, Triterpenoid saponins, Phytochemistry 27
1988) 3037e3067.
(
1
5
3
[
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S. Shibata, Inhibition of the tumor-promoting action of 12-O-tetradacanoyl-
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IR (KBr): 2994, 2708, 1705, 1512, 1462, 1421, 1405, 1380, 1367,
9
[
[
4] K.R. Price, I.K. Johnson, G.R. Fenwick, The chemistry and biological signifi-
cance of saponins in food and feeding stuffs, Crit. Rev. Food Sci. Nutr. 26
ꢁ1
þ
75 cm ; ESI-MS: 669.12 calcd. for C39
H52ClN
3
O
3
[M þ Na] .
(1987) 27e135.
5] J.F. Mayaux, A. Bousseau, R. Pauwels, T. Huet, Y. Henin, N. Dereu, M. Evers,
F. Soler, C. Poujade, E.D. Clercq, J.B. Le Pecq, Triterpene derivatives that block
entry of human immunodeficiency virus type 1 into cells, Proc. Natl. Acad. Sci.
U.S.A. 91 (1994) 3564e3568.
4
4
.3. Biological assay
.3.1. Human cancer cell lines culture
The optimum density of seeded cell suspension was introduced
[
6] M. Evers, C. Poujade, F. Soler, Y. Ribeill, C. James, Y. Lelièvre, J.C. Gueguen,
D. Reisdorf, I. Morize, R. Pauwels, E.D. Clercq, Y. Hénin, A. Bousseau,
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to each well of 96-well plates (Iwaki) and exposed to a range of
compound concentrations. Cells were cultured in humidified at-
mosphere at 37 C in 5% CO
known cytotoxic agents, 5-flourouracil and Mitomycin-C (Sigmae
Aldrich) were used as positive controls. The cells were incubated
with sample for 48 h and fixed in cold ice TCA for 1 h at 4 C.
Plates were washed with distilled water and allowed to dry in the
air. Sulforhodamine-B (SRB) solution (0.4%) was added to each
well of 96-well plates and allowed to stain at room temperature
for 30 min. The unbound SRB solution was removed by washing
the plates quickly with 1% (v/v) acetic acid. The bound SRB dye
ꢀ
2
. In the cultured RPMI-1640 medium,
[
[
[
7] Y. Kashiwada, T. Nagao, A. Hashimoto, Y. Ikeshiro, H. Okabe, L.M. Cosentino,
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(
[
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was solubilized by adding 100 ml of 10 mM unbuffered Tris base
(
pH ¼ 10.5) to each well and shaken for 5 min on shaker platform.
The plates were read in a 96-well plate reader at 540 nm
wavelength.
[
[
4
.3.2. Cytotoxicity assay
Sulforhodamine-B assay was performed against four human
cancer cell lines namely THP-1 (leukemia), A-549 (lung), MCF-7
Breast) and HCT-116 (colon) in addition to a normal epithelial
cell line (FR-2). An increase in growth inhibition was seen during
8 h incubation at all tested concentrations. DMSO control was set
(
4
[
[
15] S.L. Holz-Smith, I.C. Sun, L. Jin, T.J. Matthews, K.H. Lee, C.H. Chen, Role of
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up separately to cancel out the cell death occurred by DMSO, which
was used as a solvent for dissolving samples homogeneously.
Initially, the compounds were screened at 50 mM concentrations to
get the percentage of cell growth inhibition and compounds which
showed significant cytotoxic activity were further evaluated at 30,
(2001) 60e66.
16] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farns-worth, G.A. Cordell,
C.W.W. Beecher, H.H.S. Fong, A.D. Kinghorn, D.M. Brown, M.C. Wani,
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a selective inhibitor of human melanoma that functions by induction of
apoptosis, Nat. Med. 1 (1995) 1046e1051.
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1
0 and 5
expressed as concentration of drug inhibiting cell growth by 50%
IC50). The results showed that the inhibition of different human
mM concentration to get the IC50 values. Cytotoxicity is
(
cancer cell lines of varying tissue origin with above mentioned
sample concentrations imparted significant cellular cytotoxic ef-
fects on all the cell lines particularly on breast and leukemia cancer
cells.
[
[
19] M.N. Laszczyk, Pentacyclic triterpenes of the lupane, oleanane and ursane
group as tools in cancer therapy, Planta Med. 75 (2009) 1549e1560.
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Acknowledgments
[
We acknowledge our Director, Dr. Ram Vishwakarma for his
great support and encouragement in establishing medicinal
chemistry laboratory facilities in Srinagar Campus of IIIM. We
highly appreciate the help rendered by Drs. Aravinda and Deepika
for recording all NMR data of samples. One of the authors, B.A.D
acknowledges UGC for supporting through FIP program.
[
21] H.J. Jeong, H.B. Chai, S.Y. Park, D.S.H.L. Kim, Preparation of amino acid con-
jugates of betulinic acid with activity against human melanoma, Bioorg. Med.
Chem. Lett. 9 (1999) 1201e1204.
[22] C.M. Ma, S.Q. Cai, J.R. Cui, R.Q. Wang, P.F. Tu, M. Hattori, M. Daneshtalab, The
cytotoxic activity of ursolic acid derivatives, Eur. J. Med. Chem. 40 (2005)
582e589.
[
23] J. Li, W.J. Guo, Q.Y. Yang, Effects of ursolic acid and oleanolic acid on human
colon carcinoma cell line HCT15, World J. Gastroenterol. 8 (2002) 493e495.
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N.D. Kim, Y.H. Choi, S.H. Lee, K.W. Kim, Apoptotic activity of ursolic acid may
correlate with the inhibition of initiation of DNA replication, Int. J. Cancer 87
[
Appendix A. Supplementary data
(
2000) 629e636.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.05.029.
[
[
25] P.O. Harmand, R. Duval, C. Delage, A. Simon, Ursolic acid induces apoptosis
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apoptosis by ursolic acid through activation of caspases and down-regulation
of c-IAPs in human prostate epithelial cells, Int. J. Oncol. 17 (2000) 565e571.
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