Urea-mediated regioselective nitration of (20s)-camptothecin

Article abstract of DOI:10.1081/SCC-200036276

A facile and efficient procedure for the regioselective nitration of (20S) camptothecin, using urea mediated reagent system under relatively mild experimental conditions, yielding promising anticancer drug 9-nitro-(20S)- camptothecin in 40% yield with purity of 94.5% (on HPLC) is being reported.

Full text of DOI:10.1081/SCC-200036276

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Urea‐Mediated
Regioselective Nitration of
(
20S)‐Camptothecin
a a a
S. C. Puri , G. Handa , O. P. Suri & G. N. Qazi
a
a
Natural Products of Chemistry Division , Regional
Research Laboratory, (C.S.I.R) , Jammu, 180004,
India
Published online: 10 Jan 2011.
To cite this article: S. C. Puri , G. Handa , O. P. Suri & G. N. Qazi (2004)
Urea‐Mediated Regioselective Nitration of (20S)‐Camptothecin, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 34:18, 3443-3448, DOI: 10.1081/SCC-200036276
To link to this article: http://dx.doi.org/10.1081/SCC-200036276
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SYNTHETIC COMMUNICATIONS
Vol. 34, No. 18, pp. 3443–3448, 2004
Urea-Mediated Regioselective
Nitration of (20S)-Camptothecin
#
*
S. C. Puri, G. Handa, O. P. Suri,
and G. N. Qazi
Natural Products of Chemistry Division, Regional Research
Laboratory, (C.S.I.R), Jammu, India
ABSTRACT
A facile and efficient procedure for the regioselective nitration of (20S)
camptothecin, using urea mediated reagent system under relatively mild
experimental conditions, yielding promising anticancer drug 9-nitro-
(20S)-camptothecin in 40% yield with purity of 94.5% (on HPLC) is
being reported.
Key Words: Anticancer drugs; Camptothecin; Nitration; 9-Nitro-
(
20S)-camptothecin.
#
Indian Patent 1559/DEL December 21, 1999.
Correspondence: S. C. Puri, Natural Products of Chemistry Division, Regional
*
Research Laboratory, (C.S.I.R), Jammu 180004, India; E-mail: purisc2004@yahoo.
com; scpuri2003@rediffmail.com.
3
443
DOI: 10.1081/SCC-200036276
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

3
444
Puri et al.
The present work relates to the preparation of a semisynthetic derivative
-nitro camptothecin from the natural (20S)-camptothecin isolated from
9
[
1,2]
Nothapodytes foetida (wight) Sleumer.
anti-tumor activity and results in complete tumor regression followed by no
It is reported to exhibit strong
[
3]
further growth of tumor.
Metabolic studies on 9-nitrocamptothecin (9-NC) indicated that it gets
converted into 9-amino-(20S)-camptothecin (9-AC). Preclinical studies have
revealed that administration of 9-NC, which being more stable and less
toxic than 9-AC, produces the desired effect, which being qualitatively and
[
4]
quantitatively superior to 9-AC.
[5,6]
Conventional nitration (mixture of conc. HNO3 and H SO ),
2
when
4
employed for nitration of camptothecin yielded 12-nitrocamptothecin (60%)
and the desired product 9-nitrocamptothecin was obtained in extremely poor
yield along with undesired polynitrated products. In order to select a suitable
reaction medium for the nitration of camptothecin to get better yields of
[
7]
9
-nitrocamptothecin, a mild mediated-nitrating method was used. It is
well known that a molecule like crystalline tetragonal urea (or thiourea)
does not remain rigid and acts as a host molecule and forms hexagonal
0
lattice containing the guest in longitudinal channels (5 A dia.). Since position
1
2 in the aromatic ring of camptothecin is on relatively hindered side, it was
visualized to use urea as the “host” molecule for the “guest” nitronium ion so
that the total volume of the reagent becomes larger to prefer position 9 (which
lies on least hindered side of the ring) to 12. The controlled nitration of camp-
þ
tothecin has been achieved by sustained release of NO2 trapped in flexible
urea channels due to interaction of Vander-Waal forces between the host
[8]
and guest. It is reported that using organic media reaction kinetics shifts
þ
from 2nd order to 1st order with respect to NO2 ion concentration. To the
best of our knowledge zero order nitration with respect to camptothecin has
not been reported till date.
Urea=HNO3=AcOH
CPT þexcessðCH COÞO ꢀꢀ ꢀꢀꢀ ꢀꢀꢀ ꢀꢀ ꢀ! 9ꢀnitroCPT þ12 ꢀnitroCPT
3
2
þCH3CO2NO2 þCH3COOH

Urea-Mediated Regioselective Nitration of (20S)-Camptothecin
3445
The reaction may proceed by in-situ generation of acetylnitrate, which may
dissociate to yield nitronium ion.
ðCH COÞO þurea þHNO !CH CðOÞONO þNH þCO
3
2
3
3
2
3
2
ꢀ
þ
CH3CðOÞONO2 !CH3COO þNO2
Conc. HNO3 on ionization forms 4% nitronium ions which enter
into urea channel as tiny guest molecules thereby protecting campto-
thecin from vigorous nitration which leads to the formation of undesirable
products.
Table 1 gives a comparative account of nitration carried out under dif-
ferent temperature conditions.
A method has been developed for the nitration of natural (20S)-
camptothecin C H N O , mol. wt. 348.35 C 69.96%, H 4.63%, N 8.04%,
2
0 16 2 4
25
O 18.37%, mp 255–2778C dec. 264–2678, [a] D þ31.38 (in MeOH–CHCl )
3
isolated from woody stem of the Indian tree Nothapodytes foetida (wight)
1
Sleumer (Fam. Icacinaceae). TLC, HPLC, MP, IR, UV, H NMR and mass
spectrometry established its purity (98.5%). The stereochemistry of the
natural compound, which is an essential requirement for biological activity,
was ascertained by optical rotation.
In conclusion we have developed a mild and effective method for the
regioselective nitration of camptothecin in good yields.
Table 1. Comparative nitration of camptothecin
Yield %
Total
nitration
Isolated
9NC
S. no.
1
Temp.
9NC
31
12NC
21
CPT
23
Byproduct
25
Room
temp.
08C
52
25.7
2
3
39
43
14
35
8
19
39
3
53
78
35.8
40.0
2208C
ꢁ
ꢁ
Note: Yields were determined by HPLC before isolation of the products. For each
reaction Camptothecin (700 mg, 2 mmole), acetic anhydride (20 mL), nitrating
mixture 0.9 mL (0.009 mole) for 3 h.
ꢁ
ꢁ
HPLC done on Partisil silica column 5 m, 4 ꢂ260 mm, isocratic mobile phase
CHCl : MeOH (98 : 2) at 366 nm.
3

3
446
Puri et al.
EXPERIMENTAL
General
1
H NMR, IR spectra and HPLC systematically characterized all products.
Melting points determined are uncorrected IR spectra was recorded on a PC-16-
1
FT Perkin Elmer Spectrometer. H NMR was recorded on an INOVA-600
“eden.” HRMS was recorded on EIL Rpos-359 spectrometer. HPLC was
recorded on Gilson. The reagents were purchased from E. Merck and
Ranbaxy. The solvents used were of commercial grade and were double
distilled. The yields reported are not theoretical yield but are on the basis of
HPLC and isolated products.
Preparation of the Nitrating Solution
Stock nitrating solution was prepared by mixing conc. HNO (60% w/w
3
specific gravity 1.41, 0.5 mole, 31.6 mL) and urea (0.5 g) and diluting the sol-
ution to 50 mL with glacial acetic acid. The solution is stable and can be kept
for 3 months under refrigerated conditions.
General Procedure for Nitration
Natural camptothecin (700mg, 2 mmole) was suspended in acetic anhy-
dride (20 mL) with stirring at 2208 in a round bottom flask (100mL) equipped
with a magnetic stirrer. The mixture was stirred for 30min, to this suspension
was added slowly, above nitrating solution (0.009mole, 0.09mL). The resulting
orange red mixture was stirred for 3 h. at (18–208C). Progress of the reaction
was monitored on TLC. The reaction mixture was allowed to stand at room
temperature for 18h. and poured into crushed ice (50 mL) when a red oily sus-
pension was obtained and kept for 3 h. with occasional stirring changed into a
yellow crude solid, which on desiccation over P O for 12h gave yellow
2
5
2
3
solid (0.25 g). It was crystallized from ethanol, mp 276–2798C, [a] D þ268
1
c 0.2, MeOH–CHCl ), identified by mp, HPLC and H NMR data and
(
optical rotation as 12-nitro-(20S)-camptothecin. After separating the solid
3
1
2-nitro CPT the yellow filtrate was extracted with CH Cl (4 ꢂ100 mL).
2
2
The extract was washed with water (2 ꢂ40mL) and dried over anhydrous
sodium sulphate. Evaporation of the solvent under reduced pressure yielded a
yellow solid (0.4g), which was admixed with celite and loaded on a column
of silica gel. The elution was carried out with CHCl : MeOH (98: 3–95: 5)
3
mixture in a gradient fashion. TLC selected eluates on evaporation afforded a
yellow solid (280mg, 40%). It was crystallized from ethyl acetate and hexane.

Urea-Mediated Regioselective Nitration of (20S)-Camptothecin
3447
1
MP, TLC, HPLC, H NMR identified it to be 9-nitro-(20S)-camptothecin mp
2
3
21
1
82–1868C, [a] D þ278 (c 0.2, MeOH–CHCl ), IR (KBr, cm ) 3430
3
(
OH), 1750 (lactone), 1660 (pyridone), 1590 (aromatic) 1530 (NO ).
2
1
H NMR: (CDCl ), d ¼ 1.05 (3H, t, J ¼ 7.40 Hz, C19-methyl protons),
3
1
.92 (2H, m, C18-methylene protons), 3.82 (1H, s, C20–OH), 5.40 (2H, s,
C5-methylene protons), 5.55 (2H, dd, J ¼ 14.21, 14.21 Hz, C17-methylene
protons), 7.70 (1H, s, C14-H), 7.92 (1H, t, J ¼ 8.40 Hz, C11-H), 8.48 (1H,
d, J ¼ 8.35 Hz, C10-H), 8.55 (1H, d, J ¼ 8.35 Hz, C12-H), 9.36 (1H, s, C7-H).
þ
MS: m/z (%) ¼ 393 (M , 100), 364 (M–C H , 35), 349 (48), 334 (25),
2
5
3
20 (25), 293 (35), 274 (8), 262 (8), 246 (15), 234 (6), 218 (20), 205 (8), 190
9), 177 (5), 164 (3), 151 (3), 137 (5), 123 (4), 109 (5), 95 (5), 75 (3), 60 (23).
(
þ
HRMS: Calcd. M for C H N 0 393.0960. Found 393.0961.
20 15 3 6
ACKNOWLEDGMENTS
We thank Dr. M. C. Wani of Research Triangle Institute Research
Triangle Park, North Carolina, 277709, USA, for providing reference stan-
dards and Prof. M. Spiteller, University of Dortmund, Dortmund, Germany,
for high resolution NMR and HRMS.
REFERENCES
1
2
3
. Puri, S.C.; Handa, G.; Gupta, R.K.; Gupta, V.K.; Srivastava, T.N.;
Somal, P.; Sharma, S.N. Quantitation of camptothecin in Nothapodytes
foetida. J. Indian Chem. Soc. 1997, 36B, 25.
. Sharma, S.N.; Puri, S.C.; Srivastava, T.N.; Handa, G.; Koul, B. Enhance-
ment of seed germination in Nothapodytes foetida. J. Med. Arom. Plant
Sci. 2000, 22, 206.
. Giovanella, B.C.; Stehlin, J.S.; Wall, M.E.; Wani, M.C.; Nicholas, A.W.;
Liu, L.F.; Silber, R.; Potmesil, M. Highly effective topoisomerase-I tar-
geted chemotherapy of human colon cancer in xenografts. Sciences
(N.Y. Acad. Sci.) 1989, 246, 1046.
4
5
6
. Pantazis, P.; Harris, N.; Mendoza, J.; Giovanella, B. Conversion of 9-nitro-
camptothecin to 9-amino-camptothecin by human blood cells in vitro. Eur.
J. Haematol. 1994, 53, 246.
. Wani, M.C.; Nicholas, A.W.; Wall, M.E. Plant antitumor agents: synthesis
and antileukemic activity of camptothecin analogues. J. Med. Chem. 1986,
2
9, 2358.
. Cao, Z.S.; Armstrong, K.; Shaw, M.; Petry, E.; Harris, N. Nitration of
camptothecin with various inorganic nitrate salts in concentrate sulfuric

3
448
Puri et al.
acid: a new preparation of anticancer drug 9-nitrocamptothecin. Synthesis
998, 1724.
1
7
. Ferrarini, P.L.; Mori, C.; Badawneh, M.; Manera, C.; Martinelli, A.; Micell, M.;
Romagnolic, F.; Saccomanni, G. Heterocyclic vinyl ether VII. Rearrangement
of 1,4, dithiadiene ring system. Eur. J. Med. Chem. 1997, 32, 955.
8
. Takemoto, K.; Sonoda, N. In Inclusion Compounds; Atwood, J.L.,
Davies, J.E., Macnicol, D.D., Eds.; Academic Press: NY, 1984; Vol. 2, 47.
Received April 5, 2004