Synthesis and cytotoxic activity of novel quinazolino-β-carboline-5- one derivatives

Article abstract of DOI:10.1016/j.bmc.2004.03.005

A novel series of quinazolino-β-carbolinone derivatives was synthesized and evaluated for their in vitro and in vivo anticancer activity. Many compounds have shown good in vitro activity in the range 1-8μM concentration. Three of the compounds were further tested in nude mice bearing HT-29 colon cancer xenografts.

Full text of DOI:10.1016/j.bmc.2004.03.005

Bioorganic & Medicinal Chemistry 12 (2004) 1991–1994
Synthesis and cytotoxic activity of novel quinazolino-b-carboline-
5-one derivativesq
Bipul Baruah,a Kavitha Dasu,a Balasubramanian Vaitilingam,a Premkumar Mamnoor,b
Prasanthi Penubaka Venkata,b Sriram Rajagopalb and Koteswar Rao Yeleswarapua,*
aDiscovery Chemistry, Discovery Research, Dr. Reddy’s Laboratories Limited, Bollaram Road, Miyapur, Hyderabad 500050, India
bMolecular Discovery, Discovery Research, Dr. Reddy’s Laboratories Limited, Bollaram Road, Miyapur, Hyderabad 500050, India
Received 10 October 2003; revised 4 March 2004; accepted 4 March 2004
Abstract—A novel series of quinazolino-b-carbolinone derivatives was synthesized and evaluated for their in vitro and in vivo
anticancer activity. Many compounds have shown good in vitro activity in the range 1–8 lM concentration. Three of the compounds
were further tested in nude mice bearing HT-29 colon cancer xenografts.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
However, little attention was directed towards its pos-
sible antitumor activity.3 In continuation of our studies
to discover small organic molecules for different thera-
peutic indications,4 we were attracted by the structural
features offered by rutaecarpine. In the present study,
intended to discover novel antitumor agents, we have
synthesized a series of 3,10-substituted and E ring
modified quinazolino-b-carbolin-5-ones, which were
evaluated for their inhibitory activity and cytotoxic
effects on several human tumor cell lines.
Chinese herbs have been widely used as important
remedies in oriental medicine. In recent past, many
biologically active constituents were isolated and their
pharmacological actions investigated. Evodia rutae-
carpa (Chinese name: Wu-Chu-Yu) is a well-known
traditional Chinese medicine and has been used for a
long time in Chinese medical practice. It is used as a
remedy for gastrointestinal disorders (abdominal pain,
dysentery), headache, amenorrhea, and postpartum
hemorrhage.1 Rutaecarpine, a quinazolino carboline
alkaloid,2 is the major component isolated from the fruit
of this plant and has been shown to possess interesting
medicinal properties.
2. Chemistry
Synthetic methods for the preparation of rutaecarpine
derivatives are summarized in Scheme 1. The main
intermediate, substituted 1,2,3,4-tetrahydro-b-carboli-
none was prepared from substituted aniline following
the procedure described earlier by Abramovitch and
Shapiro.5 The b-carbolinone was then treated with
substituted anthranilic acid derivatives in presence of
POCl3 in toluene under reflux to provide the products in
almost 80–90% yield.6 Some of quinazolino carboli-
nones were prepared from isatoic anhydride by treating
them with tryptamine derivatives, as illustrated in
Scheme 2. The isatoic anhydride I was dissolved in
pyridine containing trifluoro acetic anhydride to give
2-(trifluoromethyl)-4-oxazinone II. The quinazolino
carbolinones were then prepared according to the
Bergman7;8 procedure. Reaction of tryptamine deriva-
tives III with oxazinone II gave IV, which were then
refluxed under acidic conditions (AcOH–HCl) for
7
8
O
6
9
N
C
10
11
4
2
5
D
A
B
3
E
N
NH 13
14
12
1
RUTAECARPINE
Keywords: Cytotoxic; Quinazolino-b-carboline-5-one derivatives.
q DRL publication No.: 343.
* Corresponding author. Tel.: +91-4023045439; fax: +91-4023045438;
e-mail addresses: y_koteswarrao@yahoo.com; koteswarraoy@
drreddys.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.005

1992
B. Baruah et al. / Bioorg. Med. Chem. 12 (2004) 1991–1994
NH
HOOC
i
ii
R
R
O
R
NH
N2+Cl -
NH2
NH N
O
iii
O
R
NHR2
N
N
NH
O
iv
R
+
N
R1
N
R1
COOH
H
R2
1. R, R1, R2 = H; 2. R = SCH3, R1, R2 = H.
3. R = SOCH3, R1, R2 = H; 4. R =SO2CH3,R1, R2 = H.
5. R = Br, ,R1, R2 = H; 6. R = Cl, ,R1, R2 = H.
7. R = Cl,,R1 = H, R2 = CH3
;
;
8. R = Br,,R1 = H, R = CH
.
.
9. R = H, ,R1 = H, R2 = CH3
10. R = F,,R1 = H, R2 = CH3
2
3
11. R = R2
= H, R1 = NO2; 12. R = OCH3, R1, R2 = H.
13. R = OCF3, R1, R2 = H; 14. R = CF3, R1, R2 = H.
15. R = SO2NH2, R1, R2 = H.
Scheme 1. Reagents: (i) NaNO2/HCl, 0 ꢁC; (ii) AcOH, 0 ꢁC, 8–10 h; (iii) 90% formic acid, 100 ꢁC, 1 h; (iv) POCl3/toluene, reflux, 1 h.
O
N
O
NH2
O
R
+
CF3
ii
i
O
R
O
N
H
N
H
O
II
III
I
O
R
iii
N
iv
N
F3C
N
H
HN
N
IVHF3C N
V
O
R
N
N
H
N
Scheme 2. Reagents: (i) (CF3CO)2O, pyridine, 25 ꢁC, 15 min; (ii) 115 ꢁC, 3 h; (iii) HCl, AcOH, reflux, 1 h; (iv) KOH, H2O, EtOH.
30 min to give V. Elimination of CF3H was accom-
plished by treating V with alcoholic KOH to form the
quinazolino-b-carbolinone derivatives. Scheme 3 illus-
trates synthesis of compounds having different hetero-
cyclic rings as E ring in the overall structure.
The parent compound 1, without any substituent has
been reported to possess no significant cytotoxic activity
against different cell lines.3 However, in the present in
vitro eight panel cancer cell lines,9 the compound
exhibited some cytotoxic activity. We initially started
our SAR studies on quinazolino-b-carbolinone bearing
different substituents on A ring at position C-10. Com-
pounds 3, 4, and 12–15 did not have significant anti-
cancer activity in human cancer cell lines. However,
compounds 2, 5, and 6 with SCH3, Br, and Cl
substituent at C-10 position showed better activity than
the parent compound. To optimize activity of these
compounds, different N-1 methyl substituted quinazo-
lino-b-carbolinones were synthesized (7–10) and tested
for anticancer activity. Compound 7 with Cl at C-10 was
found to be potent.
3. Discussion
The quinazolino-b-carbolin-5-ones prepared in this
study were tested for in vitro anticancer activity on eight
cancer cell lines and the active compounds were further
screened for their in vivo activity. To establish the SAR
of this series of compounds, each variable of general
structures in Schemes 1–3 was systematically modified
and the GI50 values obtained are tabulated in Table 1.

B. Baruah et al. / Bioorg. Med. Chem. 12 (2004) 1991–1994
1993
NH
R
R
R
HOOC
i
ii
O
NH
N2+Cl -
NH2
NH N
O
iii
O
R
R1
R2
X
R
N
N
NH
O
iv
R1
R2
COOH
+
N
N
NH2
H
X
16. X = S, R, R1, R2 = H.
17. X = S, R = SCH3, R1=CH3, R2 = COOEt.
18. X = S, R = Cl, R1 = H, R2 = H.
19. X = S, R = F, R1 = H, R2 = H.
20. X = N, R = Br, R1 = CH3, R2 = H.
21. X = O, R = Cl, R1 = t-Butyl, R2 = H.
Scheme 3. Reagents: (i) NaNO2/HCl, 0 ꢁC; (ii) AcOH, 0 ꢁC, 8–10 h; (iii) 90% formic acid, 100 ꢁC, 1 h; (iv) POCl3/toluene, reflux, 1 h.
Table 1. In vitro cytotoxic activities of quinazolino-b-carbolin-5-ones (GI50 values in lM)
Compound
Breast
MCF7/ADR
CNS U251
Colon SW620 Lung H522
Melanoma
UACC62
Ovarian
SKOV3
Prostate
DU145
Renal ACHN
1
21
23
0.02
3
25
26
37
35
>100
17
3
13
0.2
15
0.1
32
3
20
0.08
2
2
3
>100
>100
62
7
>100
>100
33
91
>100
59
>100
>100
>100
>100
7
27
>100
3
4
>100
5
1
5
>100
2
6
40
2.5
12
11
9
10
2
11
2
7
1
15
6
0.3
2
1
8
14
18
1
>100
6
3
3
0.4
11
9
4
35
5
0.9
1
11
2
80
15
32
3
10
11
12
13
14
15
16
17
18
19
20
21
2
>100
2
27
3
2
3
51
2
3
12
>100
>100
>100
19
>100
6
24
>100
18
28
>100
>100
>100
>100
7
>100
>100
>100
>100
10
17
57
>100
––
>100
2
1
>100
>100
>100
>100
>100
53
>100
>100
16
>100
>100
>100
>100
>100
44
94
>100
>100
>100
>100
54
>100
>100
74
>100
13
7
20
12
2
12
1
2
46
>100
26
11
>100
33
––
7
17
14
9
14
34
15
20
40
18
8
2.4
At this juncture, to see the effect of substituent on the E
ring, we synthesized few compounds with a heterocycle
fused to E ring (16–21). These compounds were found to
be inactive. However, introducing a NO2 group in the E
ring imparted good anticancer activity, with an average
GI50 value 2 (compound 11). It was subsequently
observed that this compound was also highly stable in
both human and mouse plasma.
fragments (ꢀ60 mm3) from established tumors in athy-
mic nude mice. Tumor fragments were implanted by s.c.
in the right flank. The s.c. tumors were measured with
calipers, and mice were weighed every alternate day.
When tumors reached a volume of ꢀ100 mm3, mice were
randomized to control and treated groups. Test com-
pounds were administered orally at specified doses, once
daily for 20 days. Tumor size was measured every
alternate day and the volume was calculated using the
equation V ¼ ðD ꢁ d2Þ=2 where V (mg) is tumor vol-
ume, D is longest diameter in mm and d is shortest
diameter in mm. The results of the xenograft studies are
tabulated in Table 2.
Our efforts to improve the cytotoxic activity of com-
pound 1 resulted in compounds 6, 7, and 11, which
showed promising in vitro cytotoxic activity with GI50
values ranging from 1 to 2 lM. These compounds were
tested for their antitumor activity against human xeno-
grafts in nude mice. In this antitumoral assay, HT-29
human tumors were initiated by implantation of tumor
The antitumor activity is expressed as the optimum
T =C% (median tumor volume of test/control · 100).

1994
B. Baruah et al. / Bioorg. Med. Chem. 12 (2004) 1991–1994
Table 2. Result of HT-29 xenograft studies
Compound
MTD, PO in
athemic nude mice
Schedule
Dose
(mg/kg/day)
Total dose
(mg/kg)
Max. mean%
weight loss (day)
% T=C (day)
1
>1000
>1000
100
QD · 20
QD · 20
QD · 20
QD · 20
300
100
25
6000
2000
500
6 (20)
10 (20)
3 (20)
12 (20)
132 (20)10
94 (20)11
94 (20)12
75 (20)13
6
7
11
600
100
2000
P.; Misra, P.; Mullangi, R.; Casturi, S. R.; Yeleswarapu,
K. R. Ind. J. Chem. 2003, 42B, 593.
Compound showing an optimum T=C% value of <43%
was considered as active in this xenograft model.
Though compounds 6, 7, and 11 showed very good in
vitro activity, the activity did not translate in the xeno-
graft model, probably because of their poor bioavail-
ability.
5. Abramovitch, R. A.; Shapiro, D. J. Chem. Soc. 1954,
4263; Abramovitch, R. A.; Shapiro, D. J. Chem. Soc.
1956, 4589; Phillips, R. R.. In: Organic Reactions; John
Wiley & Sons: New York, 1959; Vol. X, Chapter 2, p 143.
6. Rahman, A. V.; Ghazala, M. Synthesis 1980, 372–374.
7. Bergman, J.; Bergman, S. Heterocycles 1986, 16, 347.
8. Bergman, J.; Bergman, S. J. Org. Chem. 1985, 50, 1246.
9. In vitro cell growth assay: Cells were seeded on a 96-well
cell culture plates at a concentration of 10,000 cells/well in
a volume of 100 lL of RPMI medium with 10% fetal
bovine serum and were incubated at 37 ꢁC in a CO2
incubator. After 24 h cells were treated with varied
concentrations of compounds in 100 lL of medium and
the control wells received vehicle. The plates were further
incubated at 37 ꢁC in a CO2 incubator for 48 h. The assay
was terminated by addition of 50% cold TCA to a final
concentration of 10% to the cells, followed by incubation
at 4 ꢁC for 1 h. At the time of compound addition, a
separate set of cells having a 24 h growth were terminated
for time zero growth (T0). The TCA fixed plates were
washed thrice with distilled water, air dried, and stained
with 0.4% SRB in 1% acetic acid for 30 min at room
temperature. The plates were washed with 1% acetic acid,
dried, and the protein bound dye was dissolved in 100 lL
of 10 mM Tris buffer and read at 515 nm. The percentage
growth of treated cells is calculated using the following
formulae: if T is greater than or equal to T0, percentage
growth ¼ 100½ðT ꢂ T0Þ= ðC ꢂ T0Þꢃ and if T is less than T0,
percentage growth ¼ 100½ðT ꢂ T0Þ=T0ꢃ, where C, T and T0
are optical densities of control, test, and time zero,
respectively. From the growth curves, the GI50 values
were extrapolated.
In conclusion, in this communication, we have described
the synthesis, in vitro and in vivo anticancer activity of a
new series of quinazolino-b-carbolinone derivatives.
Many compounds have shown good in vitro activity, in
the range 1–8 lM concentration. Further work to
impart the desired in vivo activity among these novel
compounds is presently under progress.
Acknowledgements
We thank Drs. R. Rajagopalan and A. Venkateswarlu
for discussions and analytical department for providing
the spectral data.
References and notes
1. Li, S. C. In Pentssu Kang Mu, 1976; Chapter 32, p 1064;
Lia, J. F.; Chen, C. F.; Chow, S. Y. J. Formosan Med.
Assoc. 1981, 79, 0–38; Chow, S. Y.; Chen, S. M.; Yang,
C. M. J. Formosan Med. Assoc. 1976, 75, 349–357; Anon.
In Encyclopedia of Chinese Medicine; People’s: Shangai,
People’s Republic of China, 1977; p 1118.
2. Asahina, Y.; Ishimasa, S. Synthese des Rutaecarpins.
Yakugaku Zasshi 1926, 534, 61–63; Bergman, J. In The
Alkaloids; Brossi, A. R., Ed.; Academic: New York, 1983;
Vol. 21, pp 29–54.
10. Compound 1: mp 160–161 ꢁC; (lit.7 159 ꢁC) IR: (KBr)
3325, 1655 cmꢂ1
;
1H NMR (CDCl3) d 3.23 (2H, t,
J ¼ 7 Hz), 4.60 (2H, t, J ¼ 7 Hz), 7.30–7.80 (8H, m) and
8.30 (1H, s); CIMS m=z 288 (MHþ, 100%).
3. Yang, L. M.; Lin, S. J.; Lin, L. C.; Kuo, Y. H. The Chin.
Pharm. J. 199, 51, 219–225; Yang, L. M.; Chen, C. F.; Lee,
K. H. Bioorg. Med. Chem. Lett. 1995, 5, 465–468.
11. Compound 6: mp 308–309 ꢁC; IR: (KBr) 3335, 1653 cmꢂ1
;
1H NMR (CDCl3) d 3.23 (2H, t, J ¼ 6:9 Hz), 4.60 (2H, t,
J ¼ 7 Hz), 7.20–7.80 (7H, m) and 8.30 (1H, s); CIMS m=z
322 (MHþ, 100%).
4. Pal, M.; Madan, M.; Padakanti, S.; Pattabiraman, V. R.;
Kalleda, S.; Vanguri, A.; Mullangi, R.; Casturi, S. R.;
Yeleswarapu, K. R. J. Med. Chem. 2003, 46, 3975; Pal,
M.; Veeramaneni, V. R.; Nagabelli, M.; Misra, P.;
Casturi, S. R.; Yeleswarapu, K. R. Biorg. Med. Chem.
Letts. 2003, 13, 1639; Barua, B.; Dasu, K.; Vaitalingam,
B.; Misra, P.; Casturi, S. R.; Yeleswarapu, K. R. Biorg.
Med. Chem. Lett. 2004, 14, 445; Pal, M.; Veeramaneni, V.
R.; Padakanti, S.; Nagabelli, M.; Vanguri, A.; Mamnoor,
12. Compound 7: mp 300–301 ꢁC; IR: (KBr) 3332, 1652 cmꢂ1
;
1H NMR (CDCl3) d 2.99 (3H, s), 3.23 (2H, t, J ¼ 7 Hz),
4.65 (2H, t, J ¼ 7 Hz), 7.40–7.84 (7H, m) and 8.40 (1H, s);
CIMS m=z 338 (MHþ, 100%).
13. Compound 11: mp 294–295 ꢁC; IR: (KBr) 3325,
1655 cmꢂ1; 1H NMR (DMSO-d6) d 3.13 (2H, t, J ¼ 7 Hz),
4.70 (2H, t, J ¼ 7 Hz), 7.30–8.50 (8H, m) and 8.90 (1H, s);
CIMS m=z 333 (MHþ, 100%).