Synthesis and cytotoxicity evaluation of (tetrahydro-β-carboline)-1,3,5-triazine hybrids as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2010.02.001

A series of tetrahydro-β-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts (NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic towards KB (oral cancer) cell line with IC₅₀ values of 105.8, 664.7 and 122.2?nM, respectively; while their enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC₅₀ value of 740?nM, also arrests cell cycle in G₁ phase and induces apoptosis in MCF7 and MDA MB231cell lines.

Full text of DOI:10.1016/j.ejmech.2010.02.001

European Journal of Medicinal Chemistry 45 (2010) 2265–2276
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity evaluation of (tetrahydro-
hybrids as anticancer agents
b-carboline)-1,3,5-triazine
Ravi Kumar a, Leena Gupta a, Pooja Pal b, Shahnawaz Khan a, Neetu Singh b, Sanjay Babu Katiyar a,
Sanjeev Meena b, Jayanta Sarkar b, Sudhir Sinha b, Jitendra Kumar Kanaujiya b, Savita Lochab b,
,
Arun Kumar Trivedi b, Prem M.S. Chauhan a
*
a Division of Medicinal & Process Chemistry, Central Drug Research Institute, CSIR, Lucknow 226001, India
b Drug Target Discovery & Development Division, Central Drug Research Institute, CSIR, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetrahydro-b-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for
Received 9 November 2009
Received in revised form
28 January 2010
Accepted 1 February 2010
Available online 10 February 2010
their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts
(NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic
towards KB (oral cancer) cell line with IC50 values of 105.8, 664.7 and 122.2 nM, respectively; while their
enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more
selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC50 value of 740 nM, also
arrests cell cycle in G1 phase and induces apoptosis in MCF7 and MDA MB231cell lines.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
(Tetrahydro-b-carboline)-1,3,5-triazine
hybrid
Oral cancer
Breast cancer
Anticancer
1. Introduction
The b-carboline nucleus that possess a common tricyclic pyr-
ido[3,4-b]indole ring structure, is a recurring motif in both natural
and synthetic cytotoxic compounds which can act through multiple
mechanisms [9]. In addition to simple, substituted harman and
norharman derivatives, [10] more complex structures such as
manzamine(1) [11], eudistomine K (2) [12], azatoxin (3) [13],
Cancer is a major health problem worldwide and is the leading
cause of human mortality exceeded only by cardiovascular
diseases [1]. Therefore, development of new anticancer drugs and
more effective treatment strategies for cancer are of utmost
importance as traditionally prescribed chemotherapeutic agents
have problems with toxicity and drug resistance [2]. Although,
numerous kinase inhibitors have been discovered recently and
several have been successfully developed for treatment of cancer
including Gleevec, [3] Iressa, [4] Tarceva, [5] Tykerb, [6] and
Sutent, [7] still there is strong demand for discovery of improved
cytotoxic agents. As most of the solid human cancer tumor are
multi causal in nature and their treatment with ‘‘mechanism-
based’’ agents alone is unlikely to be successful, so a combination
of these inhibitors with a better cytotoxic drug is likely to be
a good strategy [8].
fascaplysine (4) [14], and picrasidine
L (5) [15], display potent
cytotoxic activities against various cancer cell lines (Fig. 1).
Cytotoxicity of 1,3,5-triazine derivatives is well known as hex-
amethylmelamine (HMM) (6) was discovered as an effective agent
against breast, lung and ovarian cancer but it causes many adverse
effects such as nausea, vomiting, anorexia and abdominal cramps
[16]. Irsogladine (7) has been shown to have anti-tumor activity in
murine xenograft models of epidermoid cancer and glioma [17].
More recently, the effect of compound (7) was also investigated in
a human breast cancer athymic nude mouse system and the results
suggested that irsogladine can be useful in the breast cancer adju-
vant setting [18]. Moon et al. reported compound (8) as a microtu-
bule destabilizing agent with potent growth inhibition against U936
cells (GI50 ¼ 1
mM) [19]. p38 MAP kinase inhibitory activity [20] of
* Corresponding author. Tel.: þ91 522 2262411; fax: þ91 522 2623405. CDRI
comm., No. 7880.
E-mail addresses: premsc58@hotmail.com, prem_chauhan_2000@yahoo.com
(P.M.S. Chauhan).
(9), inhibitory potency of (10) against various cyclin dependent
kinases, [21] and VEGF-R2 (KDR) tyrosine kinase inhibitory activity
[22] of (11) has also been reported recently (Fig. 2).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.001

2266
R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
O
N
O
N
N
N
S
N
H
OH
O
N
H
N
H
N
Br
H
H2N
O
2
O
OMe
Cl
+N
N
MeO
N
OH
5
3
N
H
O
1
4
Fig. 1. Cytotoxic natural products containing b-carboline nucleus.
Now a days, there is an increased interest in use of hybrid
molecules for drug discovery against multitude of disease indica-
tions [23]. Lesser degree of conformational flexibility and less likely
3. Pharmacology
1–50 concentrations of (tetrahydro-b-carboline)-1,3,5-
mM
interaction of tetrahydro-
reduced toxicity and increased anticancer activity [24]. These
observations and our previous experience [25,26] led us to
hypothesize that (tetrahydro-b-carboline)-1,3,5-triazine hybrid
molecules will be more efficacious and selective cytotoxic agents.
Herein, the synthesis and cytotoxic activities of a series of (tetra-
b
-carbolines with DNA may also lead to
triazine hybrids were tested for in vitro cytotoxic activity on human
cancer cell lines representing breast cancer (MCF7), colon (SW620),
prostate (DU145), oral (KB), ovary (PA1), leukemia (K562), pancreas
(MiaPaCa-2), lung (A549) and normal fibroblasts (NIH3T3) by MTT
assay [28].
hydro-b-carboline)-1,3,5-triazine hybrid derivatives against a panel
of human cancer cell lines are described.
4. Results and discussion
Initially, anticancer activity of all the previously reported (tet-
2. Chemistry
rahydro-b-carboline)-1,3,5-triazine [24] hybrids by our group and
synthesized analogues (33–40) was assessed in vitro against
The synthetic strategy followed for synthesis of (tetrahydro-
b
-
a panel of 8 human cancer cell lines and the results are summarized
carboline)-1,3,5-triazine hybrids is depicted in Scheme 1. The tet-
rahydro- -carbolines (12–32) were obtained via Pictet–Spengler
cyclization [26] of methyl ester of tryptophan with various
substituted benzaldehydes under acidic conditions. Two diaste-
reomers so obtained were separated by flash column chromatog-
in Table 2. Previously reported (tetrahydro-b-carboline)-1,3,5-
b
triazine hybrids (general structure 11a) [24] were found to be
inactive. But we were pleased to find that compound 34 obtained
from DL-tryptophan, having trans stereochemistry, 4-methoxy
group as R and N-methylpiperazino group as R1 showed IC50 value
raphy. Spectroscopic data of the obtained tetrahydro-
b
-carbolines
of 1.87 and 4.78 mM against KB and NIH3T3 cell lines, respectively. It
were identical to that of reported earlier [24,27]. The (tetrahydro-
b
-
was 2.5 times more selectively toxic towards KB cells than NIH3T3
cell line, while its cis isomer 33 was moderately cytotoxic against all
the tested cell lines. Butylamino group as R1 as in compounds 35
and 36, was found to be detrimental to cytotoxic activity. Also the
combination of 4-methyl group as R and aminoethanol as R1 has
resulted in complete loss of activity in compounds 37 and 38.
Cytotoxicity profiles of 39 and 40 also indicated that trans isomer
was preferred over cis for cytotoxicity. Excited by these findings,
and to rapidly develop SAR around 1,3,5-triazine moiety, enantio-
carboline)-1,3,5-triazine hybrids (33–75) enlisted in Table 1 were
synthesized in a two step sequence [26] – (i) a nucleophilic
substitution of one chloro group of cyanuric chloride with N-2 of
different tetrahydro-b-carbolines, (ii) displacement of remaining
two chloro groups with various amines (Scheme 1). Compound 76
was also synthesized according the same procedure except that
tryptamine was used instead of methyl ester of tryptophan in step
(i) of above mentioned method (Scheme 2). Treatment of cyanuric
chloride with excess of N-methyl piperazine resulted in formation
of compound 77 (Scheme 2).
pure b-carboline unit having 4-methoxy group as R, obtained from
methyl ester of
L-tryptophan was kept intact while R1 was varied
MeO
NMe2
Cl
NH2
N
MeO
NH
N
N
N
N
N
N
Me2N
N
NMe2
HN
Cl
NH
N
2
MeO
HN
8
6
7
N
H
N
S
NMe
N
Cl
H
N
N
N
O
R
1
R2
MeO
N
N
N
N
NH
N
N
N
N
N
N
N
N
N
N
H
OH
R1
N
H
N
Me
R
N
H
OH
10
9
11
11a
Fig. 2. Previously reported anticancer 1,3,5-triazine derivatives.

R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
2267
COOCH3
COOH
COOCH3
R1
N
NH
NH2
(i)
N
(ii)
N
N
H
N
H
N
H
N
(iii)
R1
R
R
D/L/DL-Tryptophan
(12-32)
(33-75)
Scheme 1. Preparation of (tetrahydro-
b
-carboline)-1,3,5-triazine hybrids (33–75). For (12) R ¼ 4-methyl, Isomer ¼ DL, Trans; (13) R ¼ 4-methyl, Isomer ¼ DL, Cis; (14) R ¼ 4-methyl,
Isomer ¼ L, Trans; (15) R ¼ 4-methyl, Isomer ¼ L, Cis; (16) R ¼ 4-methyl, Isomer ¼ D, Trans; (17) R ¼ 4-methyl, Isomer ¼ D, Cis; (18) R ¼ 4-methoxy, Isomer ¼ DL, Trans; (19) R ¼ 4-
methoxy, Isomer ¼ DL, Cis; (20) R ¼ 4-methoxy, Isomer ¼ L, Trans; (21) R ¼ 4-methoxy, Isomer ¼ L, Cis; (22) R ¼ 4-methoxy, Isomer ¼ D, Trans; (23) R ¼ 4-methoxy, Isomer ¼ D, Cis;
(24) R ¼ 3,4,5-trimethoxy, Isomer ¼ DL, Trans; (25) R ¼ 3,4,5-trimethoxy, Isomer ¼ DL, Cis; (26) R ¼ 3,4,5-trimethoxy, Isomer ¼ L, Trans; (27) R ¼ 3,4,5-trimethoxy, Isomer ¼ L, Cis;
(28) R ¼ 4-chloro, Isomer ¼ DL, Trans; (29) R ¼ 4-isopropyl, Isomer ¼ L, Trans; (30) R ¼ 4-isopropyl, Isomer ¼ L, Cis; (31) R ¼ 4-isopropyl, Isomer ¼ D, Trans; (32) R ¼ 4-isopropyl,
Isomer ¼ D, Cis. Reagents and Conditions: (i) Thionyl chloride, MeOH, various benzaldehydes, (ii) Cyanuric chloride, K2CO3, THF, 0 ꢀC – r.t. (iii) Amines, K2CO3, THF, Reflux.
using various amine nucleophiles. Cytotoxic evaluation of (41–49)
demonstrated that every substituent except N-methylpiperazino
group as R1 had detrimental effect on cytotoxicity against human
cancer cell lines. To our surprise compound 42, which is enantio-
pure form of 34, was not selective towards KB cells but was 2.6
times selectively toxic towards MCF7 cell line in comparison to
NIH3T3 cells. On the other hand 41, the cis isomer of 42, was
generally cytotoxic to all cell lines and has similar cytotoxicity
profile as 33. Subsequently, we tried few other combinations,
hybrids (50–61) having R ¼ 4-isopropyl, 3,4,5-trimethoxy, 4-methyl,
4-methoxy, 3-hydroxy and R1 ¼ various cyclic amines, were
synthesized starting from L-tryptophan and evaluated for cytotox-
icity. But again, only compounds having N-methylpiperazino group
as R1 were found to be cytotoxic. Interestingly, cytotoxic activity was
also found to be decreasing with increase in carbon chain length at
N-4 of piperazine unit as evidenced by the cytotoxicity profile of 60
and 61. Compound 60 with N-ethylpiperazino group as R1 exhibited
better cytotoxicity than N-propylpiperazine derivative 61, whereas
both were less active than 34. In the meantime, compound 42 was
studied for its effect on cell cycle in MCF7 and MDA MB231 cell lines
and found to arrest mitotic cycle in G1 phase. DNA fragmentation
analysis and Hoechst staining was also carried out in MCF7 and MDA
MB231 cell lines to confirm apoptosis by 42.
Table 1
(Tetrahydro-b-carboline)-1,3,5-triazine hybrid analogues (33–75).
Entry
Compound
no.
Isomer
R
R1
1
2
3
4
5
6
7
8
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
DL, Cis
DL, Trans
DL, Cis
DL, Trans
DL, Cis
DL, Trans
DL, Trans
DL, Cis
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-Methyl
N-methylpiperazine
N-methylpiperazine
butylamine
4.1. Arrest of MCF7 and MDA MB231 cells in G1 and inhibition of
mitosis
butylamine
2-aminoethanol
2-aminoethanol
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-phenylpiperazine
N-phenylpiperazine
morpholine
4-Methyl
To understand the mechanism of cell death, compound 42 was
evaluated for its effect on cell cycle. Breast cancer cells MCF7 and
MDA MB231 were exposed to two concentrations of 0.50 and
0.74 mM for 24 h, which were then stained with propidium iodide
and analyzed by flow cytometry. DNA contents of the live pop-
ulation of MCF7 cells were 54.35, 0.01, and 45.64 for untreated cells
3,4,5-Trimethoxy
3,4,5-Trimethoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-methoxy
4-Isopropyl
4-Isopropyl
4-Isopropyl
4-Isopropyl
4-Isopropyl
4-Isopropyl
4-Isopropyl
4-Isopropyl
3,4,5-Trimethoxy
3,4,5-Trimethoxy
4-Methoxy
4-Methoxy
4-Methoxy
4-methoxy
-H
9
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
, Cis
, Trans
, Cis
, Trans
, Cis
, Trans
, Trans
, Cis
, Trans
, Cis
, Trans
, Cis
, Trans
, Cis
, Trans
, Trans
, Cis
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
morpholine
cyclohexylamine
o-toluidine
to 76.68, 0.12, 23.20% for the cells treated with 0.74
compound 42 after 24 h in G1, S and G2 phase, respectively (Fig. 3).
While, at a concentration of 0.50 M, 64.4% accumulation of MCF7
mM of
m
o-toluidine
cells was observed in comparison to that of 54.4% of untreated
control cells. Similar results were obtained with MDA MB231 cells
but increase in percentage accumulation in G1 phase was less in
comparison to that of MCF7 cells. Hence, compound 42 at 1 mM
concentration enhances mitotic arrests in G1 phase.
N-methylpiperazine
N-methylpiperazine
N-phenylpiperazine
N-phenylpiperazine
Morpholine
Morpholine
N,N-dimethylamine
cyclohexylamine
N-methylpiperazine
N-methylpiperazine
N-ethylpiperazine
N-propylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
N-methylpiperazine
propylamine
4.2. DNA fragmentation analysis
, Trans
, Cis
, Trans
, Trans
A nuclear associated event in apoptosis is the degradation of
DNA into nucleosomal sized fragments of approximately 180–
200 bp which forms a ladder pattern when subjected to gel elec-
trophoresis and is indeed one of the important diagnostic
biomarker of apoptosis. Both MCF7 and MDA MB231 cells were
treated with varying concentrations of compound 42 ranging from
0.25 to 0.74 mM for 24 h. DNA fragmentation analysis was carried
out to evaluate apoptosis in both cell lines. DNA fragmentation was
observed in both cell lines at all the concentrations tested (Fig. 4).
D
D
D
D
D
D
, Cis
, Trans
, Cis
, Trans
, Cis
, Trans
-H
4-Methyl
4-Methyl
4-Chloro
4-Methyl
4-Methyl
4-Methyl
4-Methyl
DL, Trans
DL, Trans
DL, Cis
DL, Trans
DL, Trans
DL, Trans
DL, Cis
Propylamine
Ethylamine
Ethylamine
Propylamine
4.3. Hoechst staining for visualization of apoptosis
4-Methoxy
4-Methoxy
4-Methoxy
For visualization of apoptosis both MCF7 and MDA MB231 cells
were treated with 0.50 and 0.74 mM concentration of compound 42
DL, Trans
Propylamine

2268
R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
Me
N
Me
N
Cl
N
N
ii
( )
N
N
NH2
N
i
ii
( ), ( )
N
N
HN
N
Cl
N
Cl
N
H
N
N
N
N
N
H
MeN
NM
e
NMe
77
76
Scheme 2. Preparation of 2,4,6-triamino-1,3,5-triazines. Reagents and Conditions: (i) Cyanuric chloride, K2CO3, THF, 0 ꢀC – r.t. (ii) N-methylpiperazine, K2CO3, THF, Reflux.
for 24 h and Hoechst staining was performed. Compound 42 causes
apoptosis, was demonstrated by unevenly displayed and frag-
mented micronuclei in significant number of cells (Fig. 5).
At that time it was thought that cytotoxicity profile of other
enantiomers of 41 and 42 i.e. 62 and 63 will be interesting. So, a few
analogues (62–67) were also synthesized starting with methyl ester
the combination of 4-methoxy group as R and N-methylpiperazino
group as R1 was not selectively cytotoxic. Both compound 71 and 75
have propylamino group as R1, but differs in R group. In this case
4-methoxy and propylamino group combination as R and R1 was
proven to be more active than 4-methyl and propylamino group
combination. Replacing
in formation of 2,4,6-triamino-1,3,5-triazine 76, which was found
to be inactive, indicating that -carboline part is essential for
b-carboline with tryptamine in 69 resulted
of
D
-tryptophan. They also showed same SAR as that of compounds
-tryptophan, i.e. only compounds
obtained from methyl ester of
L
b
having N-methylpiperazino group as R1 were found to be cytotoxic
but they differed from 34 in their selectivity towards KB cells.
A little decrease in cytotoxicity was observed when R ¼ 4-methoxy
in 63 was replaced with H (65). Similar was the case with 4-methyl
derivative 67, as it also showed decreased toxicity and selectivity.
From the above data it was evident that racemates are more
selective towards KB cell lines than their enantiopure forms.
Keeping in view this fact, we synthesized a few analogues (68–75)
having 4-methyl, 4-methoxy, 4-chloro group as R at C-1 phenyl ring
cytotoxicity. Substituting all three chloro groups of cyanuric chlo-
ride with N-methyl piperazine, another inactive compound 78 was
obtained, proving our hypothesis correct. More selectivity of
racemic mixture in comparison to pure enantiomers indicates that
both the enantiomers may be acting on different targets, and their
cumulative effect leads to selectivity, while one enantiomer alone is
not selective at all. Further studies are required to delineate the
mode of action and subsequent improvement in selectivity towards
cancer cells.
of tetrahydro-b-carboline ring system and alkyl amino groups such
as N-methylpiperazino, propylamino and ethylamino groups as R1,
starting from methyl ester of DL-tryptophan. Cytotoxicity evaluation
of (68–75) led us to discovery of compounds 69, 71 and 75, which
are selectively cytotoxic towards KB cells with IC50 values of 105.8,
664.7, 122.2 nM, respectively, and in accordance to previous SAR
their cis isomers were inactive. Compound 69 having 4-methyl
group as R and N-methylpiperazino group as R1 combination is
selectively cytotoxic towards KB cells, while compound 34 having
5. Conclusion
In summary, we have described a series of (tetrahydro-b-car-
boline)-1,3,5-triazine hybrid molecules, some of them have shown
good cytotoxicity against a panel of human cancer cell lines.
Compounds 69, 71 and 75 are selectively toxic towards KB
(oral cancer) cell line with IC50 values of 105.8, 664.7 and 122.2 nM,
while their enantiopure forms are less active and not selective.
Table 2
In Vitro cytotoxicity data of (tetrahydro-
b
-carboline)-1,3,5-triazine hybrids and 2,4,6-triamino-1,3,5-triazine derivatives.a
Entry
Compound
no.
IC50 (mM)
MCF7
SW620
DU145
KB
1.46
1.87
2.43
17.88
7.54
8.06
6.88
3.73
1.22
3.54
1.82
0.99
1.83
1.98
1.86
1.98
0.1058
0.6647
0.1222
PA1
K562
Miapaca2
A549
NIH3T3
1
2
3
4
5
6
7
8
10
11
12
13
14
15
16
17
18
19
20
33
34
39
40
41
42
50
51
60
61
62
63
64
65
67
68
69
71
75
6.06
12.44
NA
3.32
4.26
3.15
ND
1.19
1.35
1.93
5.92
NA
5.35
10.47
2.01
5.22
10.31
6.75
2.87
NA
1.67
7.11
2.45
2.58
7.76
2.81
2.14
NA
2.72
2.03
2.75
5.32
2.77
1.82
0.27
2.89
NA
7.13
NA
3.00
ND
4.83
4.6
6.43
2.24
ND
7.37
6.35
7.54
ND
3.62
2.3
1.81
NA
ND
6.30
5.83
5.67
9.84
3.13
2.1
1.75
1.76
1.61
1.67
11.46
3.00
8.62
3.21
2.34
6.55
NA
2.33
4.78
2.17
6.71
1.62
1.96
1.17
2.63
1.86
2.24
5.28
3.16
11.38
6.00
2.16
2.22
NA
5.96
9.00
0.74
5.93
6.73
1.34
2.07
12.27
10.97
8.28
6.21
5.89
NA
NA
NA
ND
ND
3.27
2.15
3.24
2.84
2.65
2.73
NA
3.34
2.05
1.79
1.93
6.91
6.39
NA
4.48
6.00
6.45
5.34
3.22
5.01
NA
5.26
6.23
5.88
6.11
7.00
7.12
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
IC50 ¼ compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as mean from the dose response curves of at least two independent
experiments with three determinations in each.
a
NA ¼ not active, that is, the IC50 is greater than 25 M. ND ¼ not done.
m

R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
2269
Fig. 3. Effect on the cell cycle as determined by flow cytometry. Uninduced MCF7 and MDA MB231 cells were taken as controls. Cells were treated with 0.50 and 0.74
concentrations of compound 42 (b, c for MCF7 & e, f for MDA MB231).
mM
Compound 42, however, is not as good as 69, 71 or 75 but is 2.5
times more cytotoxic towards MCF7 cells than normal fibroblasts
NIH3T3 with an IC50 value of 740 nM. Compound 42 arrests the cell
cycle in G1 phase in MCF7 as well as MDA MB231 cell lines,
ultimately leading to cell death which was confirmed by DNA
fragmentation analysis and Hoechst staining. In vivo studies,
additional SAR, further lead optimization, and studies regarding
mode of action are progress in our laboratories and will be reported
6. Experimental
6.1. Chemistry
All non-indigenous chemicals were purchased from Sigma–
Aldrich. All reactions were monitored by thin layer chromatog-
raphy conducted on E. Merck TLC plates (Silica gel 60 F-254,
Aluminium Back) and visualized with UV light or iodine. The crude
products were purified by column chromatography using silica gel
as adsorbent. Nuclear magnetic spectra were recorded on 200 MHz
and 300 MHz spectrometers. In the case of multiplets, the signals
in due course. These (tetrahydro-b-carboline)-1,3,5-triazine
hybrids should be considered as important lead compounds for
potential application in anticancer chemotherapy.
Conc. of 42 in µM
0.50 0.74
0
0.25
0
0.25
0.50
0.74
a
b
MDA MB231
MCF7
Fig. 4. DNA fragmentation analysis. Depiction of DNA fragmentation pattern observed with treatment of MDA MB231 and MCF7 cells with 42 at conc. of 0.5, 0.74 mM concentrations,
respectively.

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R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
Fig. 5. Hoechst staining for visualization of apoptosis. Untreated MCF7 and MDA MB231 cells were taken as control.
are reported as intervals. Signals were abbreviated as s, singlet;
d, doublet; t, triplet; m, multiplet. The electron spray mass spectra
were recorded on a triple quadrupole mass spectrometer. The
samples (dissolved in suitable solvents such as methanol/acetoni-
trile/water) were introduced into the ESI source through a syringe
times), dried over anhydrous Na2SO4, concentrated in vacuo and
used further without purification. Then 2 mmol of resulting solid,
4 mmol of required amine and K2CO3 (0.55 g, 4 mmol) in dry THF
(40 ml) were refluxed for 3–6 h. The reaction mixture was filtered
and evaporated the solvent under vacuum. The solid residue was
dissolved in CHCl3 (75 mL), washed with water (three times), dried
over anhydrous Na2SO4, purified with column chromatography
using silica gel as adsorbent.
pump at the rate of 5 mL/min. The ESI capillary was set at 3.5 kV, and
the cone voltage was 40 V. The spectra were collected in 6 s scans,
and the printouts are averaged spectra of 6–8 scans. Infrared
spectra were recorded using a Perkin–Elmer RX-1 spectrometer;
the values were reported in cm-1. The purity of all tested
compounds was ascertained on the basis of their elemental analysis
and was ꢁ95%.
6.1.3. (ꢂ)cis-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-2-
yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (33)
Yield: 67%; mp. >210 ꢀC; ESMS: 612 (M þ 1); IR (KBr): 3402,
6.1.1. General procedure for synthesis of compounds (12–32)
3182, 2936, 2848, 2802, 1737, 1542, 1436, 1362 cmꢃ1 1H NMR
.
To the stirred solution of 2.04 g (10 mmol) DL
/L/D
-tryptophan in
(CDCl3, 200 MHz): d (ppm) 8.63 (bs, 1H), 7.59–7.13 (m, 7H), 6.57
methanol at 0 ꢀC, 1.34 ml (1.1 equivalent, 11 mmol) of thionyl
chloride was added drop-wise. The reaction mixture was then
refluxed for 2 h. An appropriate aromatic aldehyde (1.1 equivalent)
was added to the reaction mixture and was then refluxed for 8 h.
The solvent was removed under vacuum and the solid residue so
resulted was dissolved in water, neutralized with saturated sodium
bicarbonate solution and extracted with ethyl acetate. The organic
layer was washed with brine solution (three times), water (three
times), and dried over sodium sulfate. The solvent was removed in
vacuo and the cis and trans isomers were separated by flash column
chromatography using silica gel as adsorbent. Spectroscopic data of
(d, 2H, J ¼ 8.7 Hz), 6.22 (dd, 1H, J ¼ 6.3, 2.4 Hz), 3.78 (t, 8H,
J ¼ 4.9 Hz), 3.75 (s, 3H), 3.61–3.54 (m, 2H), 3.06 (s, 3H), 2.43 (t, 8H,
J ¼ 5.4 Hz), 2.31 (s, 6H). 13C NMR (CDCl3, 50 MHz): 21.62, 43.44,
46.63, 50.03, 51.55, 51.97, 55.32, 55.72, 108.92, 111.08, 113.75, 118.98,
119.57, 122.12, 127.36, 130.43, 131.83, 134.03, 136.74, 159.29, 165.33,
165.95, 173.19. Anal. calcd. for C33H41N9O3: C 64.79, H 6.76, N 20.61;
Found: C 64.68, H 6.80, N 20.56%.
6.1.4. (ꢂ)trans-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (34)
the obtained tetrahydro-
reported earlier [24,31].
b-carbolines were identical to that of
Yield: 71%; mp. >210 ꢀC; ESMS: 612 (M þ 1); IR (KBr): 3368,
3181, 2936, 2849, 2798, 1739, 1544, 1428, 1354 cmꢃ1 1H NMR
.
(CDCl3, 200 MHz):
d (ppm) 8.75 (bs, 1H), 7.56–6.85 (m, 7H), 6.84
6.1.2. General procedure for synthesis of compounds (33–75)
To the stirred mixture of cyanuric chloride (0.92 g, 5 mmol) and
K2CO3 (0.56 g, 5 mmol) in dry THF (50 ml) at 0 ꢀC was added the
(d, 2H, J ¼ 8.1 Hz), 4.41 (dd, 1H, J ¼ 8.7, 4.1 Hz), 3.78 (s, 3H), 3.76
(t, 8H, J ¼ 4.5 Hz), 3.57 (s, 3H), 3.49–3.07 (m, 2H), 2.37 (t, 8H,
J ¼ 5.6 Hz), 2.29 (s, 6H). 13C NMR (CDCl3, 50 MHz):
d (ppm) 22.44,
solution of tetrahydro-
b
-carboline (5 mmol) in dry THF (30 ml)
43.53, 46.55, 52.18, 53.63, 55.32, 55.70, 56.15, 109.95, 111.23, 114.25,
118.78, 119.82, 122.22, 127.39, 129.67, 133.74, 133.97, 136.77, 159.51,
165.29, 166.67, 172.39. Anal. calcd. for C33H41N9O3: C 64.79, H 6.76,
N 20.61; Found: C 64.62, H 6.74, N 20.58%.
dropwise for 1 h. After completion of the reaction, solvent was
evaporated under vacuum and solid mass was obtained. The solid
residue was dissolved in CHCl3 (100 mL), washed with water (three

R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
2271
6.1.5. (ꢂ)cis-2-[4,6-Bis-(butylamino)-[1,3,5]-triazin-2-yl]-1-
121.95, 122.30, 126.59, 129.33, 134.90, 136.19, 142.04, 150.33, 152.98,
165.16, 166.44, 174.72. Anal. calcd. for C33H45N9O5: C 62.58, H 6.75,
N 18.76; Found: C 62.47, H 6.81, N 18.73%.
(4-methoxyphenyl)-2,3,4,9-tetra-hydro-1H-b-carboline-3-
carboxylic acid methyl ester (35)
Yield: 65%; mp. 132–134 ꢀC (dec.); FAB-MS: 558 (M þ 1); IR
(KBr): 3415, 3339, 3025, 2953, 2837, 1733, 1595, 1489, 1375 cmꢃ1. 1H
6.1.10. (ꢂ)cis-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-2-
NMR (200 MHz, CDCl3):
d
(ppm) 7.71 (bs, 1H), 7.60–7.13 (m, 7H),
yl]-1-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-
6.75 (d, 2H, J ¼ 8.6 Hz), 6.27 (dd, 1H, J ¼ 6.2, 2.1 Hz), 3.75 (s, 3H),
3-carboxylic acid methyl ester (40)
3.49 (s, 3H), 3.39–3.12 (m, 6H), 1.75–1.64 (m, 4H),1.44–1.34 (m, 4H),
Yield: 68%; mp. 142–144 ꢀC; ESMS: 672 (M þ 1); IR (KBr): 3372,
0.93 (t, 6H, J ¼ 6.1 Hz); 13C NMR (50 MHz, CDCl3):
d
(ppm) 14.31,
3062, 2936, 2847, 2799, 1741, 1593, 1435, 1357, 1279 cmꢃ1. H NMR
1
20.42, 22.85, 32.55, 40.83, 53.44, 55.47, 56.65, 57.23, 109.11,
111.34 114.21, 118.72, 119.86, 122.40, 127.29, 129.47, 134.42, 136.36,
159.82, 166.12, 166.43, 172.56. Anal. calcd. for C31H39N7O3: C 66.76,
H 7.05, N 17.58; Found: C 66.67, H 6.98, N 17.55%.
(CDCl3, 200 MHz):
d
(ppm) 8.38 (s,1H), 7.64 (d,1H, J ¼ 8.8 Hz), 7.29–7.12
(m, 4H), 6.71 (d, 2H, J ¼ 8.8 Hz), 6.23 (dd, 1H, J ¼ 6.2, 2.1 Hz), 3.95–3.77
(m, 20H), 3.62–3.53 (m, 2H), 2.42–2.25 (m, 14H). 13C NMR (CDCl3,
50 MHz): d (ppm) 21.67, 43.44, 46.62, 50.27, 51.97, 52.65, 55.30, 56.46,
109.16, 111.14, 119.06, 119.68, 119.96, 122.26, 127.32, 129.61, 131.42,
133.91, 136.79, 137.44, 153.28, 165.50, 165.97, 173.36. Anal. calcd. for
C33H45N9O5: C 62.58, H 6.75, N 18.76; Found: C 62.45; H 6.83, N 18.70%.
6.1.6. (ꢂ)trans-2-[4,6-Bis-(butylamino)-[1,3,5]-triazin-2-yl]-1-
(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
acid methyl ester (36)
Yield: 65%; mp. 238–240 ꢀC (dec.); FAB-MS: 558 (M þ 1); IR
6.1.11. (1S, 3S)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
(KBr); 3418, 3315, 3060, 2965, 2853, 1734, 1591, 1489, 1378 cmꢃ1. 1H
NMR (200 MHz, CDCl3):
d
(ppm) 7.84 (bs, 1H), 7.56–7.15 (m, 6H),
carboxylic acid methyl ester (41)
6.87–6.79 (m, 3H), 4.57 (dd, 1H, J ¼ 6.2, 1.9 Hz), 3.76 (s, 3H), 3.59
Yield: 60%; mp. 150–152 ꢀC; ESMS: 612 (M þ 1); IR (KBr): 3390,
(s, 3H), 3.52–3.44 (m, 2H), 3.39–3.21 (m, 4H), 1.50–1.27 (m, 8H),
3180, 2929, 2850, 2798, 1739, 1537, 1433, 1359 cmꢃ1 1H NMR
.
(CDCl3, 200 MHz): d (ppm) 8.38 (bs, 1H), 7.62–7.12 (m, 7H), 6.76
0.94 (t, 6H, J ¼ 6.4 Hz); 13C NMR (50 MHz, CDCl3):
d
(ppm) 14.29,
20.48, 22.91, 32.36, 40.85, 52.29, 54.27, 55.65, 56.30, 109.13, 111.43,
114.25, 118.70, 119.97, 122.32, 127.35, 129.30, 134.46, 136.90, 159.32,
166.29, 166.90, 172.68. Anal. calcd. for C31H39N7O3: C 66.76, H 7.05,
N 17.58; Found: C 66.65, H 6.94, N 17.52%.
(d, 2H, J ¼ 7.9 Hz), 6.28 (dd, 1H, J ¼ 6.1, 2.6 Hz), 3.78 (s, 3H), 3.75
(t, 8H, J ¼ 4.8 Hz), 3.61–3.54 (m, 1H), 3.18–3.09 (m, 1H), 3.01 (s, 3H),
2.41 (t, 8H, J ¼ 5.9 Hz), 2.29 (s, 6H); 13C NMR (CDCl3, 50 MHz):
d
(ppm) 21.62, 43.28, 46.60, 50.05, 51.56, 51.97, 55.31, 55.72, 108.08,
111.06, 113.74, 118.96, 119.52, 122.09, 129.53, 130.42, 131.82, 134.08,
136.75, 159.27, 165.29, 165.96, 173.19. Anal. calcd. for C33H41N9O3; C
64.79, H 6.76, N 20.61; Found: C 64.66, H 6.72, N 20.57%.
6.1.7. (ꢂ)cis-2-[4,6-Bis-(2-hydroxyethylamino)-[1,3,5]-triazin-2-
yl]-1-p-tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid
methyl ester (37)
Yield: 72%; mp. 202–205 ꢀC (dec.); FAB-MS: 518 (M þ 1); IR
6.1.12. (1R, 3S)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
(KBr): 3445, 3333, 3028, 2965, 2855, 1734, 1599, 1479, 1395 cmꢃ1
;
1H NMR (200 MHz, CDCl3):
6.10 (dd, 1H, J ¼ 6.2, 2.1 Hz), 3.71–3.51 (m, 8H), 3.18–3.03 (m, 2H),
3.01 (s, 3H), 2.25 (s, 3H); 13C NMR (50 MHz, CDCl3):
(ppm) 21.44,
d
(ppm) 7.95 (bs, 1H), 7.58–7.03 (m, 9H),
carboxylic acid methyl ester (42)
Yield: 68%; mp. 110–112 ꢀC; ESMS: 612 (M þ 1); IR (KBr): 3389,
d
3180, 2937, 2854, 2798, 1739, 1542, 1442, 1357 cmꢃ1 1H NMR
.
21.77, 43.74, 51.05, 52.00, 52.63, 62.61, 108.49, 111.37, 118.83, 119.85,
122.38, 127.06, 129.11, 129.46, 131.64, 136.73, 138.07, 165.69, 166.18,
172.92. Anal. calcd. for C27H31N7O4: C 62.65, H 6.04, N 18.94; Found:
C 62.57, H 6.13, N 18.85%.
(CDCl3, 200 MHz): d (ppm) 8.16 (bs, 1H), 7.56–7.06 (m, 8H), 6.84
(d, 1H, J ¼ 8.2 Hz), 4.48 (dd, 1H, J ¼ 7.4, 3.3 Hz), 3.78 (s, 3H), 3.75
(t, 8H, J ¼ 4.2 Hz), 3.58 (s, 3H), 3.17–3.10 (m, 2H), 2.40 (t, 8H,
J ¼ 5.1 Hz), 2.31 (s, 6H). 13C NMR (CDCl3, 50 MHz):
d (ppm) 22.45,
43.44, 46.52, 52.18, 53.66, 55.26, 55.69, 56.17, 109.83, 111.23, 114.24,
118.75, 119.79, 122.20, 127.36, 129.63, 133.77, 133.97, 136.77, 159.49,
165.24, 166.67, 172.38. Anal. calcd. for C33H41N9O3: C 64.79, H 6.76,
N 20.61; Found: C 64.67, H 6.69, N 20.54%.
6.1.8. (ꢂ)trans-2-[4,6-Bis-(2-hydroxyethylamino)-[1,3,5]-triazin-2-
yl]-1-p-tolyl-2,3,4,9-tetr-ahydro-1H-b-carboline-3-carboxylic acid
methyl ester (38)
Yield: 69%; mp. 218–220 ꢀC; FAB-MS: 518 (M þ 1); IR (KBr):
3451, 3315, 3055, 2965, 2853, 1735, 1559, 1472, 1378 cmꢃ1. 1H NMR
6.1.13. (1S, 3S)-2-[4,6-Bis-(4-phenylpiperazin-1-yl)-[1,3,5]-triazin-
(200 MHz, CDCl3):
1H), 4.60 (dd, 1H, J ¼ 6.5, 2.1 Hz), 3.59 (s, 3H), 3.52–3.16 (m, 10H),
2.28 (s, 3H); 13C NMR (50 MHz, CDCl3):
(ppm) 21.25, 22.75, 43.37,
52.23, 54.33, 56.44, 62.16, 109.47, 111.30, 118.48, 119.73, 122.16,
126.84, 127.25, 129.43, 134.12, 136.65, 137.34, 138.12, 166.24, 172.63.
Anal. calcd. for C27H31N7O4: C 62.65, H 6.04, N 18.94; Found: C
62.62, H 6.12, N 18.86%.
d
(ppm) 7.91 (bs, 1H), 7.51–7.10 (m, 8H), 6.88 (s,
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (43)
d
Yield: 72%; mp. 218–220 ꢀC; FAB-MS: 736 (M þ 1); IR (KBr):
3340, 3056, 2944, 2829, 1739, 1597, 1544, 1438, 1370, 1174 cmꢃ1. 1H
NMR (CDCl3, 200 MHz):
d (ppm) 7.73 (bs, 1H), 7.36–6.76 (m, 19H),
6.24 (dd, 1H, J ¼ 6.7, 2.3 Hz), 3.99 (t, 8H, J ¼ 4.8 Hz), 3.75 (s, 3H),
3.61–3.39 (m, 2H), 3.22 (t, 8H, J ¼ 5.1 Hz), 3.04 (s, 3H); 13C NMR
(CDCl3, 50 MHz):
d (ppm) 21.82, 43.67, 49.92, 50.48, 51.93, 52.08,
6.1.9. (ꢂ)trans-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
55.74, 109.33, 111.21, 113.86, 117.01, 119.01, 119.97, 120.62, 122.47,
127.35, 129.65, 130.35, 131.78, 133.77, 136.68, 151.86, 159.36, 165.29,
165.63, 166.09, 173.20. Anal. calcd. for C43H45N9O3: C 70.18, H 6.16,
N 17.13; Found C 70.07, H 6.19, N: 16.99%.
2-yl]-1-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid methyl ester (39)
Yield: 71%; mp. 169–171 ꢀC; ESMS: 672 (M þ 1); IR (KBr): 3415,
3058, 2937, 2849, 2797, 1741, 1658, 1542, 1432, 1353 cmꢃ1. 1H NMR
(CDCl3, 200 MHz):
d
(ppm) 8.78 (bs,1H), 7.56 (d, 1H, J ¼ 8.8 Hz), 7.48
6.1.14. (1R, 3S)-2-[4,6-Bis-(4-phenylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
(d, 1H, J ¼ 6.6 Hz), 7.34–7.07 (m, 3H), 6.89 (s, 1H), 6.58 (d, 2H,
J ¼ 8.8 Hz), 5.10 (dd, 1H, J ¼ 6.2, 2.2 Hz), 4.11 (s, 3H), 3.85–3.67
(m, 17H), 3.42–3.31 (m, 1H), 3.21–3.11 (m, 1H), 2.38–2.29 (m, 14H).
carboxylic acid methyl ester (44)
Yield: 75%; mp. 168–170 ꢀC; FAB-MS: 736 (M þ 1); IR (KBr):
13C NMR (CDCl3, 50 MHz):
d
(ppm) 23.36, 43.35, 46.57, 51.44, 52.21,
3338, 3146, 2945, 2848, 1730, 1593, 1519, 1433, 1352, 1168 cmꢃ1. 1H
55.28, 55.81, 56.37, 61.25, 105.74, 107.82, 111.50, 118.35, 119.72,
NMR (CDCl3, 200 MHz):
d (ppm) 7.84 (bs, 1H), 7.35–6.83 (m, 19H),

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R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
4.53 (dd,1H, J ¼ 7.6, 4.7 Hz), 3.88 (t, 8H, J ¼ 4.5 Hz), 3.78 (s, 3H), 3.59
(s, 3H), 3.53–3.46 (m, 2H), 3.15 (t, 8H, J ¼ 5.1 Hz); 13C NMR (CDCl3,
200 MHz):
J ¼ 6.4, 2.5 Hz), 3.75 (s, 3H), 3.72 (s, 3H), 3.56–3.35 (m, 2H), 2.18
(s, 6H); 13C NMR (CDCl3, 50 MHz):
(ppm) 19.82, 21.49, 40.20,
d (ppm) 7.91 (bs, 1H), 7.53–7.06 (m, 17H), 4.88 (dd, 1H,
50 MHz):
d
(ppm) 22.67, 43.59, 49.85, 52.30, 54.01, 55.72, 56.34,
d
109.72, 119.35, 113.74, 114.36, 116.95, 118.81, 120.12, 120.56, 122.48,
127.37, 129.27, 129.61, 134.10, 136.81, 151.85, 159.48, 165.55, 166.86,
172.62. Anal. calcd. for C43H45N9O3: C 70.18, H 6.16, N 17.13; Found:
C 70.03, H 6.15, N: 17.19%.
42.71, 50.54, 56.51, 57.18, 107.83, 110.00, 111.46, 111.62, 114.25,
118.75, 120.22, 122.56, 124.98, 126.18, 126.90, 127.04, 128.73, 129.73,
131.07, 136. 27, 142.78, 159.39, 165.63, 169.42, 173.56. Anal. calcd. for
C37H35N7O3: C 71.02, H 5.64, N 15.67; Found: C 70.89, H 5.56, N
15.70%.
6.1.15. (1S, 3S)-2-[4,6-Bis-(morpholin-4yl)-[1,3,5]-triazin-2-yl]-1-
(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-
b
-carboline-3-carboxylic
6.1.20. (1S, 3S)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
acid methyl ester (45)
2-yl]-1-(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
Yield: 73%; mp. 203–205 ꢀC; FAB-MS: 586 (M þ 1); IR (KBr):
carboxylic acid methyl ester (50)
3317, 2924, 2850, 1737, 1539, 1436, 1257, cmꢃ1. 1H NMR (200 MHz,
Yield: 68%; mp 166–168 ꢀC; MS: 624 (M þ 1); IR (KBr): 3417,
CDCl3):
6.77 (d, 2H, J ¼ 8.5 Hz), 6.19 (dd, 1H, J ¼ 6.2, 2.1 Hz), 3.95–3.63 (m,
22H), 3.07–3.03 (m, 4H); 13C NMR (50 MHz, CDCl3):
(ppm) 21.77,
d
7.69 (bs, 1H), 7.60 (d, 1H, J ¼ 5.6 Hz), 7.32–7.13 (m, 6H),
3066, 2937, 2852, 1741, 1666, 1541, 1436, 1365 cmꢃ1. 1H NMR (CDCl3,
200 MHz):
d (ppm) 7.99 (bs, 1H), 7.62–7.07 (m, 9H), 6.24 (dd, 1H,
d
J ¼ 6.3, 2.5 Hz), 3.77 (s, 3H), 3.35 (t, 8H, J ¼ 4.9 Hz), 3.08–2.97
(m, 2H), 2.84–2.78 (m, 1H), 2.34 (t, 8H, J ¼ 8.3 Hz), 2.04 (s, 6H), 1.17
(d, 6H, J ¼ 6.0 Hz). 13C (CDCl3, 50 MHz): 21.65, 24.52, 34.35, 43.41,
46.05, 46.67, 49.91, 51.80, 54.62, 11.34, 108.87, 118.94, 119.52, 122.04,
126.35, 129.12, 131.75, 136.86, 139.35, 148.64, 161.23, 165.34, 165.65,
165.93, 173.20. Anal. calcd. for C35H45N9O2: C 67.39, H 7.27, N 20.21;
Found: C 67.32, H 7.30, N 20.19%.
43.92, 44.18, 50.47, 52.05, 55.71, 67.26, 107.91, 109.19, 111.22, 113.82,
114.56, 118.96, 119.95, 122.46, 127.29, 128.68, 129.20, 130.28, 131.74,
133.67, 136.69, 159.35, 165.70, 172.33. Anal. calcd. for C31H35N7O5: C
63.58, H 6.02, N 16.74; Found: C 63.46, H 6.09, N 16.71%.
6.1.16. (1R, 3S)-2-[4,6-Bis-(morpholin-4yl)-[1,3,5]-triazin-2-yl]-1-
(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
acid methyl ester (46)
6.1.21. (1R, 3S)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
Yield: 76%; mp. 203–205 ꢀC; FAB-MS: 586 (M þ 1); IR (KBr): 3346,
2923, 2852,1726,1550,1442,1357,1259,1172 cmꢃ1;1H NMR (200 MHz,
carboxylic acid methyl ester (51)
CDCl3):
d
7.85 (bs, 1H), 7.55 (d, 1H, J ¼ 8.1 Hz), 7.31–7.10 (m, 5H), 6.88 (t,
Yield: 68%; mp 186–188 ꢀC; ESMS: 624 (M þ 1); IR (KBr): 3373,
3H, J ¼ 6.7 Hz), 4.66 (dd, 1H, J ¼ 6.8, 2.1 Hz), 3.80 (s, 3H), 3.69 (bs, 8H),
3055, 2939, 2854, 1739, 1666, 1539, 1438, 1357 cmꢃ1 1H NMR
.
3.60 (s, 3H), 3.55 (bs, 8H), 3.52–3.46 (m, 1H), 3.22–3.15 (m, 1H); 13C
(CDCl3, 200 MHz): d (ppm) 8.38 (bs, 1H), 7.55–7.30 (m, 8H), 6.90
NMR (50 MHz, CDCl3):
d
(ppm) 22.61, 44.11, 52.25, 53.93, 55.69, 56.34,
(s, 1H), 4.64 (dd, 1H, J ¼ 5.1, 3.8 Hz), 3.53 (s, 3H), 3.47 (t, 8H,
J ¼ 7.7 Hz), 3.21–3.09 (m, 2H), 2.91–2.84 (m, 1H), 2.28 (t, 8H,
J ¼ 8.8 Hz), 2.12 (s, 6H), 1.22 (d, 6H, J ¼ 6.8 Hz). 13C (CDCl3, 50 MHz):
22.82, 24.54, 34.25, 43.61, 49.92, 52.45, 54.34, 55.23, 56.58, 109.23,
111.34, 116.87, 118.81, 120.12, 120.26, 122.44, 127.12, 127.34, 127.67,
129.56, 136.58, 139.87, 148.54, 151.91, 165.33, 167.02, 172.83. Anal.
calcd. for C35H45N9O2: C 67.39, H 7.27, N 20.21; Found: C 67.35, H
7.29, N 20.24%.
67.25, 109.66, 111.33, 114.31, 118.77, 120.09, 122.48, 127.32, 129.24,
134.01, 136.85, 137.55, 159.46, 165.30, 166.72, 172.54; Anal. calcd. for
C31H35N7O5: C 63.58, H 6.02, N 16.74; Found: C 63.44, H 5.98, N 16.83%.
6.1.17. (1R, 3S)-2-[4,6-Bis-(cyclohexylamino)-1,3,5-triazin-2-yl]-1-
(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
acid methyl ester (47)
Yield: 65%; mp. 151–153 ꢀC; FAB-MS: 610 (M þ 1); IR (KBr):
3203, 3082, 2931, 2858, 1735, 1558, 1454, 1352, 1245, 1170 cmꢃ1; 1H
6.1.22. (1S, 3S)-2-[4,6-Bis-(4-phenylpiperazin-1-yl)-[1,3,5]-triazin-
NMR (200 MHz, CDCl3):
(dd, 1H, J ¼ 7.5, 2.2 Hz), 3.79 (s, 3H), 3.65 (s, 3H), 3.12–2.92 (m, 2H),
1.94–1.18 (m, 22H); 13C NMR (CDCl3, 50 MHz):
(ppm) 24.78, 30.11,
32.37, 33.63, 49.41, 50.23, 52.21, 54.44, 55.67, 111.41, 113.40, 114.10,
114.27, 118.72, 119.70, 121.70, 127.30, 127.53, 131.24, 136.71, 137.16,
158.26, 159.05, 166.45, 179.74. Anal. calcd. for C35H43N7O3: C 68.94,
H 7.11, N 16.08; Found: C 68.87, H 6.92, N 16.14%.
d
7.83 (bs, 1H), 7.57–6.75 (m, 9H), 4.74
2-yl]-1-(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (52)
d
Yield: 66%; mp 178–180 ꢀC; ESMS: 748 (M þ 1); IR (KBr): 3408,
2924, 2817, 1737, 1602, 1541, 1431, 1366 cmꢃ1 1H NMR (CDCl3,
.
200 MHz):
d (ppm) 7.95 (bs, 1H), 7.92–6.86 (m, 19H), 6.26 (dd, 1H,
J ¼ 6.4, 2.4 Hz), 3.71 (s, 3H), 3.75–3.03 (m, 16H), 2.93–2.63 (m, 3H),
1.23 (d, 6H, J ¼ 10.0 Hz). 13C (CDCl3, 50 MHz): 21.28, 24.46, 34.23,
43.56, 49.87, 50.27, 51.93, 52.20, 109.45, 11.23, 117.02, 119.04,
119.92, 120.61, 122.53, 126.62, 129.03, 129.21, 129.56, 131.16, 136.27,
139.03, 148.57, 151.88, 165.67, 166.32, 173.11. Anal. calcd. for
C45H49N9O2: C 72.26, H 6.60, N 16.85; Found: C 72.12, H 6.66, N
16.89%.
6.1.18. (1S, 3S)-2-[4,6-Bis-(o-tolylamino)-1,3,5-triazin-2-yl]-1-(4-
methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
acid methyl ester (48)
Yield: 64%; mp. 188–190 ꢀC; ESMS: 626 (M þ 1); IR (KBr): 3267,
2925, 2839, 1739, 1591, 1452, 1245 cmꢃ1. 1H NMR (CDCl3, 200 MHz):
d
(ppm) 7.85 (bs, 1H), 7.57–7.17 (m, 17H), 6.66 (dd, 1H, J ¼ 6.4,
2.5 Hz), 3.74 (s, 3H), 3.72 (s, 3H), 3.56–3.34 (m, 2H), 2.17 (s, 6H); 13C
NMR (CDCl3, 50 MHz): (ppm) 18.57, 21.66, 50.41, 52.15, 52.41,
6.1.23. (1R, 3S)-2-[4,6-Bis-(4-phenylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
d
carboxylic acid methyl ester (53)
55.67, 109.15, 111.21, 113.63, 118.99, 119.99, 122.55, 123.98, 125.03,
126.73, 127.18, 130.11, 131.48, 132.76, 136. 72, 137.17, 159.40, 165.22,
166.45, 172.75. Anal. calcd. for C37H35N7O3: C 71.02, H 5.64, N 15.67;
Found: C 70.93, H 5.67, N 15.69%.
Yield: 69%; mp. 170–172 ꢀC; ESMS: 748 (M þ 1); IR (KBr): 3406,
3361, 2922, 2854, 1735, 1542, 1436, 1369 cmꢃ1 1H NMR (CDCl3,
.
200 MHz):
d (ppm) 7.83 (bs, 1H), 7.37–6.92 (m, 18H), 6.84 (s, 1H),
4.25 (dd, 1H, J ¼ 6.4, 2.1 Hz), 3.86 (s, 3H), 3.60–3.31 (m, 16H),
3.25–3.14 (m, 3H), 1.44 (d, 6H, J ¼ 6.0 Hz); 13C (CDCl3, 50 MHz):
6.1.19. (1R, 3S)-2-[4,6-Bis-(o-tolylamino)-1,3,5-triazin-2-yl]-1-(4-
d (ppm) 22.78, 24.34, 34.12, 43.25, 49.68, 52.34, 54.32, 56.58,109.01,
methoxyphenyl)-2,3,4,9-tetrahydro-1H-
b
-carboline-3-carboxylic
111.23, 116.78, 118.82, 120.11, 120.50, 122.43, 127.04, 127.32, 127.41,
127.67, 129.56, 134.34, 136.82, 139.48, 148.56, 151.82, 165.51, 166.69,
172.82; Anal. calcd. for C45H49N9O2: C 72.26, H 6.60, N 16.85;
Found: C 72.19, H 6.65, N 16.80%.
acid methyl ester (49)
Yield: 70%; mp. 188–190 ꢀC; FAB-MS: 640 (M þ 1); IR (KBr):
3244, 2949, 2842,1741,1569,1515,1448,1244 cmꢃ1. 1H NMR (CDCl3,

R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
2273
6.1.24. (1S, 3S)-2-[4,6-Bis-(morpholin-4-yl)-[1,3,5]-triazin-2-yl]-1-
6.1.29. (1S, 3S)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-
b
-carboline-3-
2-yl]-1-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboxylic acid methyl ester (54)
carboline-3-carboxylic acid methyl ester (59)
Yield: 67%; mp 180–182 ꢀC; ESMS: 598 (M þ 1); IR (KBr): 3379,
Yield: 65%; mp. 140–142 ꢀC; ESMS: 672 (M þ 1); IR (KBr): 3373,
2927, 2854, 1720, 1539, 1434, 1361 cmꢃ1. 1H NMR (CDCl3, 200 MHz):
3061, 2936, 2848, 2798, 1739, 1593, 1435, 1357, 1278 cmꢃ1. 1H NMR
d
(ppm) 7.77 (bs,1H), 7.28–7.08 (m, 9H), 6.19 (dd,1H, J ¼ 6.8, 2.4 Hz),
(CDCl3, 200 MHz):
d
(ppm) 8.36 (s, 1H), 7.62 (d, 1H, J ¼ 8.7 Hz),
3.72 (s, 3H), 3.02–2.85 (m, 19H), 1.18 (d, 6H, J ¼ 6.0 Hz). 13C (CDCl3,
50 MHz): 21.13, 24.25, 34.31, 44.20, 50.44, 51.92, 52.25, 67.34,
109.32, 11.20, 118.87, 119.92, 122.45, 126.61, 129.03, 129.34, 131.53,
136.74, 138.91, 148.62, 165.84, 166.16, 172.97. Anal. calcd. for
C33H39N7O4: C 66.31, H 6.58, N 16.40; Found: C 66.29, H 6.60, N
16.37%.
7.29–7.13 (m, 4H), 6.70 (d, 2H, J ¼ 8.8 Hz), 6.22 (dd, 1H, J ¼ 6.2,
2.3 Hz), 3.94–3.78 (m, 20H), 3.61–3.51 (m, 2H), 2.42–2.25 (m, 14H).
13C NMR (CDCl3, 50 MHz):
d (ppm) 21.64, 43.41, 46.60, 50.25, 51.97,
52.67, 55.32, 56.45, 109.15, 111.12, 119.05, 119.66, 119.97, 122.27,
127.30, 129.62, 131.41, 133.91, 136.79, 137.45, 153.29, 165.49, 165.96,
173.38. Anal. calcd. for C33H45N9O5: C 62.58, H 6.75, N 18.75; Found:
C 62.51; H 6.82, N 18.73%.
6.1.25. (1R, 3S)-2-[4,6-Bis-(morpholin-4-yl)-[1,3,5]-triazin-2-yl]-1-
(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-
b
-carboline-3-
6.1.30. (1R, 3S)-2-[4,6-Bis-(4-ethylpiperazin-1-yl)-[1,3,5]-triazin-2-
carboxylic acid methyl ester (55)
yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
70%; mp 210–212 ꢀC; ESMS: 598 (M þ 1); IR (KBr): 3379, 2958,
carboxylic acid methyl ester (60)
2852, 1732, 1542, 1431, 1361 cmꢃ1
.
1H NMR (CDCl3, 200 MHz):
Yield: 72%; mp. 162–164 ꢀC; ESMS: 640 (M þ 1); IR (KBr): 3401,
d
(ppm) 7.81 (bs, 1H), 7.35–7.10 (m, 8H), 6.90 (s, 1H), 4.80 (dd, 1H,
3183, 2935, 2813, 1739, 1550, 1439, 1350 cmꢃ1 1H NMR (CDCl3,
.
J ¼ 7.8, 4.0 Hz), 3.66 (s, 3H), 3.58–3.47 (m, 16H), 3.44–3.21 (m, 2H),
2.90–2.83 (m, 1H), 1.21 (d, 6H, J ¼ 8.0 Hz). 13C (CDCl3, 50 MHz):
22.86, 24.44, 34.25, 44.12, 52.35, 54.42, 56.93, 67.22, 108.97, 111.34,
118.82, 120.15, 122.43, 127.02, 127.35, 127.52, 134.24, 136.81, 139.86,
148.53, 165.45, 165.91, 172.72. Anal. calcd. for C33H39N7O4: C 66.31,
H 6.58, N 16.40; Found: C 66.34, H 6.60, N 16.35%.
300 MHz):
d
(ppm) 8.57 (bs, 1H), 7.56 (d, 1H, J ¼ 8.8 Hz), 7.35–7.27
(m, 3H), 7.20–7.09 (m, 2H), 7.01 (s, 1H), 6.86 (d, 2H, J ¼ 8.8 Hz), 4.48
(dd, 1H, J ¼ 8.9, 3.0 Hz), 3.80 (s, 3H), 3.76 (t, 8H, J ¼ 4.9 Hz), 3.60
(s, 3H), 3.56–3.46 (m, 1H), 3.18–3.12 (m, 1H), 2.43 (bs, 12H), 1.11
(t, 6H, J ¼ 6.0 Hz). 13C NMR (CDCl3, 50 MHz):
d (ppm) 12.13, 22.48,
43.40, 52.15, 52.77, 53.11, 53.71, 55.69, 56.16, 109.91, 111.18, 114.25,
118.76, 119.85, 122.25, 127.39, 129.56, 133.91, 136.73, 159.51, 165.13,
166.68, 172.40. Anal. calcd. for C35H45N9O3: C 65.71, H 7.09, N 19.70;
Found: C 65.62, H 7.14, N 19.65%.
6.1.26. (1S, 3S)-2-[4,6-Bis-(dimethylamino)-1,3,5-triazin-2-yl]-1-
(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (56)
Yield: 60%; mp 176–178 ꢀC; ESMS: 514 (M þ 1); IR (KBr): 3408,
6.1.31. (1R, 3S)-2-[4,6-Bis-(4-propylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
2945, 2817, 1737, 1541, 1431, 1362 cmꢃ1. 1H NMR (CDCl3, 200 MHz):
d
(ppm) 7.80 (bs, 1H), 7.35 (d, 1H, J ¼ 8.0 Hz), 7.28–7.11 (m, 7H), 6.94
carboxylic acid methyl ester (61)
(s, 1H), 4.52 (dd,1H, J ¼ 8.0, 4.2 Hz), 3.59 (s, 3H), 3.25–2.93 (m, 15H),
1.22 (d, 6H, J ¼ 6.0 Hz). 13C (CDCl3, 50 MHz): 24.10, 24.46, 34.22,
36.53, 53.22, 55.91, 108.93, 111.21, 118.76, 119.98, 123.31, 126.72,
126.91, 127.84, 133.32, 136.45, 138.83, 148.75, 165.83, 166.92, 173.71.
Anal. calcd. for C29H35N7O2: C 67.81, H 6.87, N 19.09; Found: C 67.78,
H 6.86, N 19.12%.
Yield: 72%; mp. 158–160 ꢀC; ESMS: 668 (M þ 1); IR (KBr): 3400,
3184, 2935, 2811, 1740, 1547, 1439, 1352 cmꢃ1 1H NMR (CDCl3,
.
300 MHz):
d
(ppm) 8.50 (bs, 1H), 7.57 (d, 1H, J ¼ 6.4 Hz), 7.35–7.27
(m, 3H), 7.20–7.12 (m, 2H), 7.01 (s, 1H), 6.86 (d, 2H, J ¼ 8.8 Hz), 4.48
(dd, 1H, J ¼ 6.4, 2.1 Hz), 3.80 (s, 3H), 3.76 (t, 8H, J ¼ 4.9 Hz), 3.60
(s, 3H), 3.54–3.46 (m, 1H), 3.18–3.12 (m, 1H), 2.43 (t, 8H, J ¼ 5.7 Hz),
2.29 (t, 4H, J ¼ 5.9 Hz), 1.54 (m, 4H), 1.11 (t, 6H, J ¼ 6.0 Hz). 13C NMR
(CDCl3, 50 MHz): 12.32, 20.17, 22.48, 43.37, 52.15, 53.53, 55.68,
56.16, 61.15, 71.83, 109.94, 111.18, 114.27, 118.77, 119.84, 122.24,
127.40, 129.58, 133.91, 136.73, 159.49, 165.14, 166.67, 172.40. Anal.
calcd. for C37H49N9O3: C 66.54, H 7.40, N 18.88; Found: C 66.47, H
7.44, N 18.91%.
6.1.27. (1S, 3S)-2-[4,6-Bis-(cyclohexylamino)-1,3,5-triazin-2-yl]-1-
(4-isopropylphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (57)
Yield: 60%; mp 170–173 ꢀC; ESMS: 622 (M þ 1); IR (KBr): 3386,
2940, 2853, 1740, 1545, 1457, 1355 cmꢃ1. 1H NMR (CDCl3, 200 MHz):
d
(ppm) 7.73 (bs, 1H), 7.61 (d, 1H, J ¼ 4.0 Hz), 7.58–7.09 (m, 8H), 6.25
(dd, 1H, J ¼ 6.2, 2.2 Hz), 4.79 (bs, 2H), 3.63 (s, 3H), 2.94–2.82
(m, 5H),1.53–1.12 (m, 26H). 13C (CDCl3, 50 MHz): 21.62, 24.45, 25.31,
26.23, 33.71, 34.32, 47.78, 48.52, 49.55, 52.24, 109.41, 111.22, 118.94,
119.42, 122.40, 126.53, 127.44, 129.32, 131.67, 136.73, 138.91, 148.65,
165.87, 166.12, 173.21; Anal. calcd. for C37H47N7O2: C 71.47, H 7.62, N
15.77; Found: C 71.37, H 7.66, N 15.69%.
6.1.32. (1R, 3R)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (62)
Yield: 63%; mp. 125–127 ꢀC; ESMS: 612 (M þ 1); IR (KBr): 3392,
3180, 2928, 2850, 2796, 1739, 1537, 1432, 1359 cmꢃ1 1H NMR
.
(CDCl3, 200 MHz):
d (ppm) 8.70 (bs, 1H), 7.62–7.12 (m, 7H), 6.76
(d, 2H, J ¼ 8.5 Hz), 6.20 (dd, 1H, J ¼ 6.1, 2.6 Hz), 3.80 (s, 3H), 3.75
6.1.28. (1R, 3S)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
(t, 8H, J ¼ 4.8 Hz), 3.53–3.10 (m, 2H), 3.07 (s, 3H), 2.61 (t, 8H,
2-yl]-1-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-
b
-
J ¼ 5.4 Hz), 2.30 (s, 6H). 13C NMR (CDCl3, 50 MHz):
d (ppm) 21.64,
carboline-3-carboxylic acid methyl ester (58)
43.38, 46.59, 50.13, 51.63, 51.96, 55.31, 55.71, 108.95, 111.07, 113.75,
118.96, 119.60, 122.14, 129.43, 130.40, 131.83, 134.03, 136.73, 159.29,
165.55, 165.55, 173.19. Anal. calcd. for C33H41N9O3: C 64.79, H 6.76,
N 20.61; Found: C 64.67, H 6.84, N 20.59%.
Yield: 67%; mp. 148–150 ꢀC; ESMS: 672 (M þ 1); IR (KBr): 3364,
3060, 2937, 2851, 2799, 1739, 1658, 1549, 1439, 1352 cmꢃ1. 1H NMR
(CDCl3, 200 MHz):
d (ppm) 8.72 (s, 1H), 7.56–7.06 (m, 6H), 6.92
(s, 1H), 4.65 (dd, 1H, J ¼ 6.1, 2.2 Hz), 3.72 (bs, 17H), 3.57 (s, 3H),
3.51–3.44 (m, 1H), 3.23–3.13 (m, 1H), 2.34 (t, 8H, J ¼ 5.9 Hz), 2.21
(s, 6H). 13C NMR (CDCl3, 50 MHz): 21.64, 43.41, 46.60, 50.25, 51.97,
52.68, 55.32, 56.45, 109.15, 111.13, 119.05, 119.67, 119.97, 122.27,
127.30, 129.67, 133.82, 131.41, 136.79, 137.45, 153.29, 165.49, 165.96,
173.38. Anal. calcd. for C33H45N9O5: C 62.58, H 6.75, N 18.75; Found:
C 62.52, H 6.78, N 18.64%.
6.1.33. (1S, 3R)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylic acid methyl ester (63)
Yield: 71%; mp. 125–128 ꢀC; ESMS: 612 (M þ 1); IR (KBr): 3372,
3180, 2937, 2849, 2797, 1739, 1548, 1441, 1356 cmꢃ1 1H NMR
.
(CDCl3, 200 MHz): d (ppm) 8.40 (bs, 1H), 7.56–7.09 (m, 6H), 6.97 (s,

2274
R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
1H), 6.84 (d, 2H, J ¼ 8.4 Hz), 4.47 (dd, 1H, J ¼ 7.7, 4.1 Hz), 3.78 (s,
3H), 3.75 (t, 8H, J ¼ 4.2 Hz), 3.54 (s, 3H), 3.49–3.46 (m, 1H),
2.64–2.58 (m, 1H), 2.35 (t, 8H, J ¼ 5.8 Hz), 2.27 (s, 6H). 13C NMR
6.1.38. (ꢂ)trans-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-
triazin-2-yl]-1-(4-chlorophenyl)-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylic acid methyl ester (68)
(CDCl3, 50 MHz):
d
(ppm) 22.50, 43.43, 46.53, 53.77, 54.63, 55.31,
Yield: 68%; mp. 224–227 ꢀC (dec.); ESMS: 616 (M þ 1); IR (KBr):
55.70, 56.18, 109.82, 111.23, 114.25, 118.76, 119.89, 122.27, 127.37,
129.47, 133.94, 136.77, 159.46, 161.14, 165.26, 166.65, 172.43. Anal.
calcd. for C33H41N9O3: C 64.79, H 6.76, N 20.61; Found: C 64.73, H
6.81, N: 20.56%.
3367, 2937, 2852, 2799, 1738, 1549, 1488, 1440, 1354, 1277 cmꢃ1. 1H
NMR (200 MHz, CDCl3):
d
8.35 (bs, 1H), 7.54 (d, 1H, J ¼ 6.0 Hz),
7.37–7.10 (m, 7H), 6.84 (s, 1H), 4.86 (dd, J ¼ 7.0, 2.3 Hz), 3.70 (t, 8H,
J ¼ 4.7 Hz), 3.58 (s, 3H), 3.51–3.43 (m, 1H), 3.22–3.12 (m, 1H),
2.35–2.27 (m, 14H); 13C NMR (50 MHz, CDCl3):
d (ppm) 22.67, 43.40,
6.1.34. (1R, 3R)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-phenyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid
46.54, 52.29, 54.11, 55.28, 56.29, 109.48, 111.34, 118.85, 120.02,
122.48, 127.26, 129.07, 129.35, 133.34, 133.61, 136.90, 141.06, 165.17,
166.76, 172.46. Anal. calcd. for C32H38ClN9O2: C 62.38, H 6.22, N
20.46; Found: C 62.29, H 6.19, N 20.42%.
methyl ester (64)
Yield: 73%; mp. 120–122 ꢀC; ESMS: 582 (M þ 1); IR (KBr): 3405,
3062, 2937, 2850, 2798, 1738, 1539, 1489, 1436, 1363 cmꢃ1. 1H NMR
(CDCl3, 300 MHz):
d
(ppm) 8.43 (s, 1H), 7.62 (d, 1H, J ¼ 7.8 Hz),
6.1.39. (ꢂ)trans-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-
7.45–7.14 (m, 9H), 6.22 (dd, 1H, J ¼ 7.8, 2.5 Hz), 3.84 (t, 8H,
triazin-2-yl]-1-p-tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-
J ¼ 6.1 Hz), 3.63–3.56 (m, 1H), 3.22–3.02 (m, 1H), 2.98 (s, 3H),
carboxylic acid methyl ester (69)
2.43–2.26 (m, 14H). 13C NMR (CDCl3, 50 MHz):
d
(ppm) 21.64, 43.26,
Yield: 63%; mp. 160–162 ꢀC; FAB-MS: 596 (M þ 1); IR (KBr):
46.63, 50.18, 51.82, 52.14, 55.29, 108.99, 111.05, 119.01, 119.58,
122.15, 127.33, 127.88, 128.54, 129.17, 131.47, 136.75, 141.83, 165.30,
166.04, 173.09. Anal. calcd. for C32H39N9O2: C 66.07, H 6.76, N 21.67;
Found: C 66.02, H: 6.88, N 21.62%.
3387, 3078, 2936, 2855, 1739, 1543, 1443, 1359, 1259 cmꢃ1; 1H NMR
(200 MHz, CDCl3):
d 8.45 (bs, 1H), 7.54–7.08 (m, 8H), 6.92 (s, 1H),
4.61 (dd, 1H, J ¼ 7.7, 2.6 Hz), 3.90 (t, 8H, J ¼ 5.1 Hz), 3.59 (s, 3H),
3.19–3.10 (m, 2H), 2.36 (t, 8H, J ¼ 4.9 Hz), 2.32 (s, 3H), 2.25 (s, 6H);
13C NMR (50 MHz, CDCl3):
d 21.49, 22.63, 43.34, 46.55, 53.99, 55.31,
6.1.35. (1S, 3R)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-phenyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid
56.57, 57.78, 109.53, 111.24, 118.76, 119.91, 122.28, 127.39, 128.00,
129.58, 134.09, 136.82, 137.54, 139.11, 165.23, 166.81, 172.54. Anal.
calcd. for C33H41N9O2: C 66.53, H 6.94, N 21.16; Found: C 66.47, H
6.98, N 20.97%.
methyl ester (65)
Yield: 75%; mp. 144–146 ꢀC; ESMS: 582 (M þ 1); IR (KBr): 3394,
2936, 2851, 2799, 1739, 1548, 1437, 1356 cmꢃ1 1H NMR (CDCl3,
.
300 MHz):
d
(ppm) 8.18 (s, 1H), 7.56 (d, 1H, J ¼ 8.1 Hz), 7.42–7.12
6.1.40. (ꢂ)cis-2-[4,6-Bis-(propylamino)-[1,3,5]-triazin-2-yl]-1-p-
(m, 8H), 6.91 (s, 1H), 4.76 (dd, 1H, J ¼ 6.3, 2.6 Hz), 3.73 (t, 8H,
J ¼ 5.9 Hz), 3.60 (s, 3H), 3.56–3.48 (m, 1H), 3.24–3.18 (m, 1H),
tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid methyl
ester (70)
2.44–2.29 (m, 14H). 13C NMR (CDCl3, 50 MHz):
d
(ppm) 22.60, 43.46,
Yield: 70%; mp. 138–140 ꢀC; FAB-MS: 514 (M þ 1); IR (KBr):
46.45, 52.24, 54.00, 55.33, 56.84, 109.53, 111.24, 118.80, 119.85,
122.26, 127.34, 127.95, 128.17, 128.54, 128.93, 133.80, 136.82, 141.75,
142.14, 165.17, 166.79, 172.51. Anal. calcd. for C32H39N9O2: C 66.07, H
6.76, N 21.67; Found: C 65.96, H 6.83, N 21.61%.
3408, 3311, 3060, 2968, 2855, 1736, 1570, 1449, 1378 cmꢃ1. 1H NMR
(200 MHz, CDCl3):
d (ppm) 7.72 (bs, 1H), 7.62–7.03 (m, 9H), 6.28
(dd, 1H, J ¼ 6.2, 1.9 Hz), 3.39–3.30 (m, 4H), 3.15–3.05 (m, 2H), 2.97
(s, 3H), 2.28 (s, 3H), 1.69–1.58 (m, 4H), 0.96 (t, 6H, J ¼ 7.3 Hz); 13C
NMR (50 MHz, CDCl3):
d (ppm) 11.88, 21.50, 22.95, 23.45, 42.88,
6.1.36. (1R, 3R)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-p-tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid
50.16, 51.83, 52.68, 109.06, 111.33, 118.90, 119.85, 122.35, 127.03,
129.08, 129.46, 131.65, 136.73, 137.52, 138.22, 165.78, 173.16; Anal.
calcd. for C29H35N7O2: C 67.81, H 6.87, N 19.09; Found: C 67.73, H
6.96, N 19.12%.
methyl ester (66)
Yield: 67%; mp. 150–152 ꢀC; ESMS: 596 (M þ 1); IR (KBr): 3425,
2937, 2849, 2800, 1738, 1589, 1541, 1436, 1355, 1277 cmꢃ1. 1H NMR
(200 MHz, CDCl3):
d
9.05 (bs, 1H), 7.60–6.94 (m, 9H), 6.21 (dd, 1H,
6.1.41. (ꢂ)trans-2-[4,6-Bis-(propylamino)-[1,3,5]triazin-2-yl]-1-p-
J ¼ 6.8, 1.9 Hz), 3.78 (t, 8H, J ¼ 4.6 Hz), 3.61–3.53 (m, 1H), 3.10–3.03
tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid methyl
(m, 1H), 2.82 (s, 3H), 2.81–2.01 (m, 14H); 13C NMR (50 MHz, CDCl3):
ester (71)
d
(ppm) 21.46, 21.67, 43.34, 46.61, 50.25, 51.77, 51.94, 55.31, 108.85,
Yield: 63%; mp. 180–183 ꢀC; FAB-MS: 514 (M þ 1); IR (KBr):
111.06, 118.95, 119.55, 122.08, 127.36, 128.05, 129.10, 129.58, 131.82,
133.82, 136.75, 137.44, 138.80, 165.34, 165.03, 173.16; Anal. calcd. for
C33H41N9O2: C 66.53, H 6.94, N 21.16; Found: C 66.42, H 6.95, N
20.98%.
3430, 3315, 3033, 2951, 2860, 1733, 1575, 1480, 1378 cmꢃ1; 1H NMR
(200 MHz, CDCl3):
d (ppm) 7.89 (bs, 1H), 7.48–7.02 (m, 8H), 6.87
(s, 1H), 4.60 (dd, 1H, J ¼ 6.5, 2.1 Hz), 3.56 (s, 3H), 3.41–3.32 (m, 4H),
3.29–3.15 (m, 2H), 2.31 (s, 3H), 1.67–1.55 (m, 4H), 0.97 (t, 6H,
J ¼ 7.2 Hz); 13C NMR (50 MHz, CDCl3):
d (ppm) 11.82, 21.49, 22.95,
6.1.37. (1S, 3R)-2-[4,6-Bis-(4-methylpiperazin-1-yl)-[1,3,5]-triazin-
2-yl]-1-p-tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid
23.47, 42.87, 52.27, 54.39, 56.60, 109.68, 111.33, 118.73, 119.99,
122.31, 126.74, 127.82, 129.57, 134.41, 136.85, 137.44, 138.55, 162.85,
166.35, 172.73; Anal. calcd. for C29H35N7O2: C 67.81, H 6.87, N 19.09;
Found: C 67.78, H 6.75, N 19.23%.
methyl ester (67)
Yield: 72%; mp. 138–140 ꢀC; ESMS: 596 (M þ 1); IR (KBr):
3368, 2937, 2852, 2798, 1739, 1549, 1439, 1353 cmꢃ1 1H NMR
.
(CDCl3, 200 MHz):
d
(ppm) 8.68 (bs, 1H), 7.54 (d, 1H, J ¼ 6.6 Hz),
6.1.42. (ꢂ)trans-2-[4,6-Bis-(ethylamino)-[1,3,5]-triazin-2-yl]-1-p-
7.30–7.09 (m, 7H), 6.95 (s, 1H), 4.52 (dd, 1H, J ¼ 6.3, 2.2 Hz), 3.70
tolyl-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic acid methyl
(t, 8H, J ¼ 4.9 Hz), 3.70 (s, 3H), 3.57–3.43 (m, 1H), 3.18–3.11
ester (72)
(m, 1H), 2.34 (m, 17H). 13C NMR (CDCl3, 50 MHz):
d
(ppm) 21.55,
Yield: 65%; mp. 232–234 ꢀC (dec.); FAB-MS: 486 (M þ 1); IR
22.44, 43.55, 46.57, 52.19, 53.84, 55.39, 56.53, 109.70, 111.20,
118.77, 119.78, 122.18, 127.39, 128.29, 129.59, 133.95, 136.80,
137.69, 138.81, 165.21, 166.75, 172.44. Anal. calcd. for
C33H41N9O2: C 66.53, H 6.94, N 21.16; Found: C 66.38, H 6.98, N
21.18%.
(KBr): 3439, 3315, 3035, 2975, 2865, 1735, 1580, 1483, 1385 cmꢃ1
;
1H NMR (200 MHz, CDCl3):
d (ppm) 7.86 (bs, 1H), 7.46–7.02 (m, 8H),
6.92 (s, 1H), 4.56 (dd, 1H, J ¼ 6.6, 2.1 Hz), 3.58 (s, 3H), 3.44–3.31 (m,
4H), 3.25–3.12 (m, 2H), 2.29 (s, 3H), 1.12 (t, 6H, J ¼ 7.2 Hz); 13C NMR
(50 MHz, CDCl3):
d (ppm) 15.58, 21.69, 22.60, 36.07, 52.31, 54.49,

R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
2275
56.78, 109.78, 111.63, 118.89, 119.73, 122.39, 126.84, 127.62, 129.88,
134.61, 136.87, 137.64, 138.45, 162.90, 166.75, 172.68; Anal. calcd. for
C27H31N7O2: C 66.78, H 6.43, N 20.19; Found: C 66.65, H 6.66, N
20.05%.
41.43, 43.36, 46.66, 55.39, 111.59, 113.85, 119.26, 119.61, 122.41,
123.02, 127.89, 136.82, 165.63, 166.76; Anal. calcd. for C23H33N9: C
63.42, H 7.64, N 28.94; Found: C 63.25, H 7.66, N 28.97%.
6.1.48. Typical procedure for synthesis of compound 77
To a solution of cyanuric chloride in THF was added 3.3 equiva-
lents of N-methylpiperazine and refluxed for 4 h. Solvent was
removed in vacuo and resulting solid was crystalized from ethanol
to get pure compound 77.
6.1.43. (ꢂ)trans-2-[4,6-Bis-(ethylamino)-[1,3,5]-triazin-2-yl]-1-(4-
methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
acid methyl ester (73)
Yield: 61%; mp. >200 ꢀC (dec.); FAB-MS: 502 (M þ 1); IR (KBr);
3429, 3318, 3029, 2971, 2859, 1732, 1585, 1483, 1380 cmꢃ1; 1H NMR
(200 MHz, CDCl3):
d
(ppm) 7.88 (bs, 1H), 7.56–7.15 (m, 6H), 6.86–
6.1.49. 2,4,6-Tris(4-methylpiperazin-1-yl)-1,3,5-triazine (77)
Yield: 86%; mp. 160–162 ꢀC; ESMS: 376 (M þ 1); IR (KBr): 2934,
6.78 (m, 3H), 4.54 (dd, 1H, J ¼ 6.2, 2.3 Hz), 3.77 (s, 3H), 3.62 (s, 3H),
3.52–3.23 (m, 6H), 1.17 (t, 6H, J ¼ 6.8 Hz); 13C NMR (50 MHz, CDCl3):
2852, 2800, 1534, 1497, 1444, 1278 cmꢃ1 1H NMR (300 MHz,
;
d
(ppm) 15.64, 21.97, 36.11, 52.30, 54.47, 55.43, 56.79, 109.26, 111.25,
CDCl3): 3.21 (t, 8H, J ¼ 4.5 Hz), 2.46 (t, 8H, J ¼ 4.5 Hz), 2.28 (s, 6H);
118.92, 119.87, 122.39, 127.32, 129.12, 129.37, 131.69, 136.72, 137.57,
138.37, 163.14, 165.98, 173.42; Anal. calcd. for C27H31N7O3: C 64.65,
H 6.23, N 19.55; Found: C 64.57, H 6.32, N 19.45%.
13C NMR (50 MHz, DMSO-d6):
d
(ppm) 46.66, 53.56, 58.24, 179.62;
Anal. calcd. for C18H33N9: C 57.57, H 8.86, N 33.57; Found: C 57.42, H
8.89, N 33.45%.
6.2. Biological assays
6.1.44. (ꢂ)cis-2-[4,6-Bis-(propylamino)-[1,3,5]-triazin-2-yl]-1-(4-
methoxyphenyl)-2,3,4,9-tetra-hydro-1H-b-carboline-3-carboxylic
MTT, PI, DAPI, guanosine 50-triphosphate (GTP), and PIPES were
obtained from Sigma, U.S.A. DMEM (Dulbecco’s modified eagles
medium) and fetal bovine serum (FBS) were procured from Gibco
BRL, U.S.A. DMSO was from Merck, India, and antibiotic solution
(containing penicillin and streptomycin) was obtained from
Hyclone, U.S.A. Human cancer cell lines representing breast cancer
(MCF7), colon (SW620), prostate (DU145), oral (KB), ovary (PA1),
Leukemia (K562), pancreas (MiaPaCa-2), Lung (A549) and normal
fibroblasts (NIH3T3) were procured from NCCS, Pune, India.
acid methyl ester (74)
Yield: 72%; mp. 140–143 ꢀC (dec.); FAB-MS: 530 (M þ 1); IR
(KBr): 3429, 3315, 2953, 2829,1732,1599,1479,1380 cmꢃ1; 1H NMR
(200 MHz, CDCl3):
d (ppm) 7.89 (bs, 1H), 7.59–7.15 (m, 7H), 6.83
(d, 2H, J ¼ 8.6 Hz), 6.25 (dd, 1H, J ¼ 6.1, 2.1 Hz), 3.73 (s, 3H), 3.52
(s, 3H), 3.42–3.35 (m, 4H), 3.30–3.10 (m, 2H), 1.57–1.49 (m, 4H),
0.98 (t, 6H, J ¼ 6.8 Hz); 13C NMR (50 MHz, CDCl3):
d (ppm) 15.57,
21.49, 21.62, 36.08, 50.16, 51.19, 52.01, 52.06, 109.25, 111.24, 118.95,
119.86, 122.38, 127.34, 129.09, 129.36, 131.64, 136.75, 137.58, 138.36,
165.97, 166.74, 173.11; Anal. calcd. for C29H35N7O3: C 65.76, H 6.66,
N 18.51; Found: C 65.72, H 6.78, N 18.44%.
6.2.1. Cytotoxicity assay
Various concentrations of (tetrahydro-b-carboline)-1,3,5-triazine
hybrids weretested forinvitrocytotoxicactivityonhumancancercell
lines representing breast cancer (MCF7), colon (SW620), prostate
(DU145), oral (KB), ovary (PA1), leukemia (K562), pancreas (MiaPaCa-
2), lung (A549) and normal fibroblasts (NIH3T3). Cytotoxicity was
measured by MTT assay, which is based on the principle of uptake of
MTTbythemetabolicallyactivecells,whereitismetabolizedbyactive
mitochondria into a blue-colored formazan product that is read
spectrophotometrically [29]. Briefly, tumor cells were seeded (5000–
10000 cells/well) in 96-well culture plates and incubated at 37 ꢀC in
6.1.45. (ꢂ)trans-2-[4,6-Bis-(propylamino)-[1,3,5]-triazin-2-yl]-1-
(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylic
acid methyl ester (75)
Yield: 68%; mp. 220–223 ꢀC; FAB-MS: 530 (M þ 1); IR (KBr):
3419, 3340, 3060, 2959, 2870, 1734, 1562, 1459, 1353 cmꢃ1. 1H NMR
(200 MHz, CDCl3):
d (ppm) 7.89 (bs, 1H), 7.57–7.13 (m, 7H),
6.86–6.75 (m, 3H), 4.69 (dd, 1H, J ¼ 6.3, 2.1 Hz), 3.76 (s, 3H), 3.59
(s, 3H), 3.53–3.45 (m, 2H), 3.40–3.22 (m, 4H),1.52–143 (m, 4H), 0.91
(t, 6H, J ¼ 7.2 Hz); 13C NMR (50 MHz, CDCl3 þ DMSO-d6):
d (ppm)
a CO2 incubator with various concentrations of (tetrahydro-
line)-1,3,5-triazine hybrids analogues ranging from 1 to 100
with relevant controls in triplicate wells. After 72 h, the assay was
terminated by the addition of 25 L of MTTsolution (5 mg/mL) in each
b-carbo-
11.64, 22.76, 23.22, 42.52, 52.04, 54.20, 54.46, 56.07, 111.48, 113.92,
118.18, 119.28, 121.66, 126.92, 128.90, 134.69, 136.76, 136.92, 158.97,
165.85,166.51,173.16. Anal. calcd. for C29H35N7O3: C 65.76, H 6.66, N
18.51; Found: C 65.63, H 6.70, N 18.40%.
mg/mL,
m
well. Percentage cytotoxicity was calculated as given below. The IC50
valuesweredeterminedbynonlinearregressionusingPrismsoftware
v 4.01.
6.1.46. Typical procedure for synthesis of compound 76
To 1.5 equivalent of cyanuric chloride in dry THF was added
dropwise a solution of tryptamine (1 equivalent) at 0 ꢀC in dry THF
over a period of half an hour. The reaction mixture was stirred for
1 h and solvent was removed in vacuo, washed with water and
extracted with chloroform. Organic layers were combined, dried
with Na2SO4 and solvent was evaporated under reduced pressure.
Resulting solid was refluxed with 2 equivalent of N-methylpiper-
azine in THF. When TLC analysis showed completion of reaction,
solvent was evaporated and resulted solid was column chromato-
graphed to get pure compounds.
percentage cytotoxicity ¼ 100ꢄ½1 ꢃ ðX=R1Þꢅ
where
X
¼
absorbance of treated sample at 540 nm and
R1 ¼ absorbance of control sample at 540 nm.
6.2.2. Analysis of cell cycle by flow cytometry
For cell cycle analysis of MCF7 and MDA MB231 cells,
1 ꢄ105 cells/ml were plated in 6-well plate. After treatment of cells
with the compound 42 and incubation, cells were trysinized and
washed with 1 ꢄ PBS, 2 ml of cold 70% ethanol was added, and cell
suspension was fixed on ice for at least 30 min. Cells were centri-
fuged for 5 min at 1200 rpm and washed with 1 ꢄ PBS. Cell pellets
6.1.47. N-[2-(1H-indol-3-yl)ethyl]-4,6-bis-(morpholin-4-yl)-1,3,5-
triazin-2-amine (76)
Yield: 76%; mp. 160–162 ꢀC; ESMS: 436 (M þ 1); IR (KBr): 2934,
were resuspended in 500
staining solution (0.1% Triton X-100, 0.1% sodium citrate, 50
PI and 10
g/mL of RNase A in PBS (with Ca2þ and Mg2þ) [30]. Cells
ml of freshly made propidium iodide (PI)
2852, 2800,1534,1497,1444,1278 cmꢃ1. 1H NMR (300 MHz, CDCl3):
m
g/mL
d
8.01 (bs, 1H), 7.62 (d, 1H, J ¼ 7.8 Hz), 7.33 (d, 1H, J ¼ 7.8 Hz),
m
7.19–7.08 (m, 3H), 5.08 (s, 1H), 3.54 (t, 8H, J ¼ 4.5 Hz), 2.44 (t, 8H,
were analyzed in Flow Cytometer (FACS Calibur, Beckon Dickinson,
USA).
J ¼ 4.5 Hz), 2.31 (s, 6H); 13C NMR (50 MHz, CDCl3):
d (ppm) 26.18,

2276
R. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 2265–2276
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6.2.3. Analysis of apoptosis by DNA fragmentation
The DNA fragmentation assay was performed according to the
protocol [31] described previously by Nianyu et al. MCF7 and MDA
MB231 cells were treated with different concentration of
compound 42. After 24 h,1 ꢄ105 cells were trypsinized and washed
with PBS (4 ꢀC, pH 7.4) and collected by centrifugation at 1200 rpm
for 5 min. The pellet was then treated with 0.5 ml of lysis buffer
(10 mM Tris-HCL, pH 7.4, 10 mM EDTA, 0.5% sodium dodecyl
sulfate) for 10 min on ice. After treatment with RNase A (final
concentration, 100
at 50 ꢀC for 4 h in the presence of 100
precipitated by addition of 50
m
g/mL) for 1 h at 37 ꢀC, the cells were incubated
g/mL proteinase K. DNA was
l of 3 M sodium acetate (pH 5.2) and
m
m
1 ml of cold (4 ꢀC) 100% ethanol to the solution. DNA was then
collected and dissolved in TE buffer (10 mM Tris pH 8.0, EDTA
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1 mM). For analysis, 10–20
ml of DNA was loaded on a 1.5% agarose
gel containing 10 g/mL ethidium bromide. DNA was visualized
m
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under ultraviolet light and photographed.
6.2.4. Visualization of apoptotic bodies by Hoechst staining
For Hoechst staining in MCF7 and MDA MB231, 1 ꢄ 104 cells
were plated in 6-well plate on coverslip. After treatment of cells
with compound 42, they were washed with 1 ꢄ PBS and fixed with
4% paraformaldehyde for 15 min. After fixing cells were stained
with 1.0ug/ml of Hoechst stain for 15 min at 4 ꢀC. Excess stain was
washed with 1 ꢄ PBS, coverslips were mounted with fluorescent
mounting solution. Cells were visualized under fluorescent
microscope [32].
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J. Wen, J. Wityak, S.T. Wrobleski, H. Wu, J. Wu, M. Desai, K.M. Gillooly, T.H. Lin,
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Financial Support from Department of Science and Technology,
India, and Dabur Research Foundation, and CSIR network project
IAP-001 is highly acknowledged. Ravi Kumar is thankful to CSIR,
India, for senior research fellowships. L. G. and P. P. are thankful to
UGC and ICMR New Delhi for senior research fellowships respec-
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