Open Journal of Medicinal Chemistry, 2012, 2, 10-14
http://dx.doi.org/10.4236/ojmc.2012.21002 Published Online March 2012 (http://www.SciRP.org/journal/ojmc)
Sulfuric Acid Catalyzed Preparation of Alkyl and Alkenyl
Camptothecin Ester Derivatives and Antitumor Activity
against Human Xenografts Grown in Nude Mice
Zhisong Cao*, Anthony Kozielski, Nick Harris, Dana Vardeman, Beppino Giovanella
Christus Stehlin Foundation for Cancer Research, Houston, USA
Received January 19, 2012; revised February 16, 2012; accepted February 28, 2012
Camptothecin-20-propinate (CZ48) and other camptothecin ester derivatives were prepared by the esterification reac-
tions of camptothecin or 9-nitrocamptothecin with the corresponding acylating agents such as organic acid anhydride or
chloride with concentrate sulfuric acid as the catalyst. The sulfuric acid-catalyzed reactions gave high yields of camp-
tothecin ester products.Among the 11 compounds prepared by this method, camptothecin-20-O-propionate, camptothe-
cin-20-O-crotonate, and 9-nitrocamptothecin-20-O-propionate showed good anticancer activity against various types of
human tumors grown as xenografts in nude mice. The methodology developed for the preparation of camptothecin es-
ters in this article can be applied to a wide scope of other ester derivatives.
Keywords: Anti-Cancer Drugs; Activity; Esterification; Camptothecin; 9-Nitrocamptothecin
Camptothecin, a natural product, was first isolated from a
native Chinese tree Camptotheca acuminata by Wall and
his coworkers in 1966 . Because of its remarkable
anti-tumor activity in animal models, this compound was
rushed to the human clinical trials in the late 1960s and
early 1970s. Unfortunately, with this compound, the re-
sults from these early human clinical studies were disap-
pointing due to intolerable side effects and lack of
anti-tumor activity in patients. The molecule of camp-
tothecin contains a six-membered lactone moiety with an
-hydroxyl group at the C20 position. Lactone campto-
thecin is insoluble in water by itself. The sodium car-
boxylate, a water-soluble form, of the molecule was thus
prepared and used in early human clinical trials. Pa-
tients participating in the study did not receive any
therapeutic benefits from this open acid form of the drug,
and also experienced severe toxicities [2-5]. The sub-
sequent studies showed that carboxylate salt of the
molecule has only one-tenth the potency of the lactone
form . The carboxylate form and the lactone form of
the molecule co-exist in equilibrium in aqueous solution
and are pH-dependent. As shown by Equation (1), the
molecule is present in lactone form when the solution is
acidic. The physiological pH value of human plasma is 7.4.
This slightly basic environment is not favorable in the lac-
tone form of camptothecin, and even worse, human serum
albumin has the highest affinity to the carboxylate form
of the molecule , which causes the active form of the
drug to disappear very rapidly when circulating in body.
A number of attempts at improving lactone stability of
camptothecin derivatives have been made. Acylation of
20-OH of the molecule has been proven to be a success-
ful way of obtaining stable camptothecins. A number of
different reactions are reported in literature for preparing
camptothecin esters. Direct acylation of camptothecin
with organic acid anhydride as the acylating agent and
pyridine as the reaction-helper was employed for prepar-
ing alkyl and alkenyl camptothecin esters [8,9] (Equation
(2)). This reaction usually gives high yields, but the
availability of organic acid anhydrides restricts the scope
of the reaction.
Lactone CPTCarboxylate CPT
opyright © 2012 SciRes. OJMC
Z. S. CAO ET AL. 11
dine (DMAP) reagent system is frequently used for acy-
lation reactions of carboxylic acids with alcohols [10-12]
and thiols . We previously used this method to pre-
are aromatic camptothecin esters  (Equation (3)).
is procedure gives good reaction yields only when the
carboxylic acids are very eletrophilic. When the acids are
less electrophilic the reaction gives low yield or no ex-
pected product at all. For example, when using propionic
acid to prepare camptothecin propionate with this proce-
dure, we did not obtain the ester product, and the starting
camptothecin was 100% recovered. We also used non-
anoic chloride as an acylating agent to esterify campto-
thecin with pyridine as an HCl-trapping agent in methyl-
ene chloride . The reaction occurred with low yield
(6%, Equation (4)).
CPT CPT nonanoate
Biological studies have demonstrated that camptothe-
cin esters are potent as anti-cancer agents against human
carcinomas grown in nude mice as xenografts, and that
the toxicities of these esters in mice are low [14,15]
Thus, it is highly possible for camptothecin esters to be
edia, camptothecins are
th them at room temperature or an
under N atmosphere with a few
lting point apparatus and were
me effective agents for the treatment of human cancers.
Although there are many methods for preparing campto-
thecin esters, each procedure has certain restrictions as
discussed above. Therefore, there is still a need to de-
velop alternative procedure(s) for preparing camptothe-
cin esters. The H2SO4-catalyzed preparation of campto-
thecin ester was previously proven to be more efficient
than anhydride/pyridine when preparing crystalline cam-
ptothecin-20-O-propionate (CZ48)  and haloalkyl
camptothecin esters . In this report, we expanded this
sulfuric acid-catalyzed reaction for preparing various
different alkyl and alkenyl camptothecin ester com-
pounds and found that this method was, indeed, more
efficient than the previously reported preparations by
giving higher reaction yields. The ester products gener-
ated by this preparation procedure had identical structural
parameters as those reported and were active against
human xenografts grown in nude mice. We now wish to
report our experimental results.
Chemistry: With excessive organic acid derivatives, such
as acid chloride (or bromide) and acid anhydrides, as
acylating agents and reaction m
allowed to react wi
drops of concentrate sulfuric acid as the catalyst. After
subsequent work-up, camptothecin ester products are
obtained in high yields.
General chemicals and equipment: Dry nitrogen was
routinely used as the reaction atmosphere in all reactions.
All glassware was baked at 70˚C ± 10˚C for a minimum
of 2 h before being used. Melting points were obtained
with a MEL-TEMP me
corrected. Camptothecin was purchased from The Peo-
ple’s Republic of China and used as purchased. 9-nitro-
camptothecin was prepared in our laboratory by using an
established procedure . The 1H NMR spectrum of
approximately 10% (w/v) solution in CDCl3 was ob-
tained at 399.93 MHZ, and 13C NMR at 100.57 MHz,
with a Varion Unity PlusNMR spectrometer (Palo Alto,
CA). Chemical shifts are reported in parts per million (δ
scale), employing tetramethylsilane as an internal stan-
dard. Silica gel (70 - 230 mesh, Aldrich) for column
chromatography was used for all product separations.
Eastman chromagram (Silica gel with fluorescent indi-
cator on polyethylene) sheets were employed in thin-
layer chromatography (TLC) operations. Methylene
chloride and THF solvents used as eluent for column
chromatography were purchased from Fisher Scientific.
A typical procedure for preparation reaction: To a 200
ml round-bottomed flask equipped with a magnetic stirrer
and a sand bath, were added 20 g camptothecin (0.05747
moles) and 100 ml propionic anhydride (97%, Aldrich
Chemical Co., Milwaukee, WI). The mixture was heated
sand bath while stirring. A few drops (8 to 10) of
concentrate sulfuric acid (95% - 98%, A.C.S. reagent,
Aldrich Chemical Co.) were added drop by drop when
the sand bath temperature reached 80˚C. The mixture
was then stirred at 110˚C 10˚C for overnight (~14 hr).
After cooling down to room temperature, the reaction
mixture was poured onto 1000 ml ice water portion by
portion while stirring. After stirring for roughly 45 min,
the mixture was filtrated. The residue obtained from fil-
tration was air-dry for 24 hr. The dried crude product was
transferred to a 500 ml round-bottomed flask equipped
with a heating mantle. To this crude product was added
200 ml absolute ethanol (99.5%, 200 proof, Aldrich
Chemical Co.). The mixture was allowed to reflux for 2
hr, and then cooled to room temperature. The pure product,
camptothecin-20-O-propionate, was obtained as white
crystals after crystallization from ethanol. Purity 99.8%
(HPLC), mp 242˚C (lit.  250˚C - 252˚C dec). TLC
showed the identical Rf values with the authentic camp-
tothecin-20-propionate prepared in this laboratory previ-
ously. The 1H and 13C NMR also showed the same spec-
trum as the product reported previously .
With the same procedure and using the corresponding
organic acid anhydride or chloride as acylating agents, all
listed products in Table 1 were prepared in high yields.
Antitumor activity: A tumor xenograft growing in a
nude mouse, approximately 1 cm3 in size, w
Copyright © 2012 SciRes. OJMC
Z. S. CAO ET AL.
and literature-reported procedures.
ridine procedure is fre-
compounds and usually
moved under sterile conditions, finely minced with
iridectomy scissors, and suspended in cell culture me-
dium at the ratio 1:10, v/v. One-tenth to one quarter of 1
L of this suspension, containing about 50 mg of wet-
weight tumor mince was subcutaneously inoculated on
the upper half of the dorsal thorax of the mouse. Groups
of six animals were used. Camptothecin ester product
was finely suspended in cottonseed oil and then injected
into the stomach cavity (IS) of the mouse through the
anterior abdominal wall using a 26 gauge needle or
administered intramuscularly (IM). The weekly schedule
used for IS was five days on and two days off. The IM
procedure was always performed on Monday and Thur-
sday for each week. Treatment was initiated when the
tumor had reached a volume of about 200 mm3, i.e. ,
well-vascularized, measurable, and growing exponen-
tially. Tumors growing in animals were checked daily and
measured with a caliper two times per week. The effe-
ctive doses were established when a positive response in
mouse was reached.
3. Results and Discussion
Table 1 shows the comparison of the reaction yields of
11 camptothecin esters between the H2SO4-catalyzed
The conventional anhydrides/py
quently used in preparing ester
gives high reaction yields when the corresponding anhy-
drides are available. As shown in Table 1, campto-
thecin-20-propionate, butyrate, valerate, and heptanoate
are all obtained in high reaction yields. However, organic
anhydrides are not always available. For example, we
used nonanoyl chloride as an acylating agent rather than
the corresponding anhydride when preparing campto-
thecin-20-nonanoate. In this situation, the reaction yield
of the product was only 6%. The DCC/DMAP procedure
was actually not working for the preparations of the
Table 1-listed CPT esters. The reactions we tried for pre-
paration of camptothecin-20-O-propionate, camptothe-
cin-20-O-butyrate, and 9-nitrocamptothecin-20-O-pro-
pionat did not generate the ester products, and the start-
ing camptothecins were completely recovered. The H2SO4-
catalyzed acylation of camptothecin derivatives with the
corresponding acid anhydrides or chlorides gives high
yields for every reaction as shown in Table 1. When
nonanoyl chloride was employed as an acylating agent,
the H2SO4-catalyzed reaction gave camptothecin-20-
nonanoate in a 92% yield, while the previously reported
method in literature only gave 6%.
Of 11 listed compounds in Table 1, camptothecin-20-
O-propionate, camptothecin-20-O-crotonate, and 9- nitro-
camptothecin-20-O-propionate were very active against
various human xenografts grown in nude mice. We pre-
viously reported the results of cam
ble 1. Comparison of reaction yields of H2SO4-catalyzed
esterfication of camptothecin with preothecin with previ-
ously reported procedure.
Reaction yields (%)
R R1 Prevously reported H2SO4-catalyzed
CH3 H 58 96
C2H5 H 94 99
C3H7 H 92 98
C H 9
H 98 99
C8H17 H 6 92
pionatainst ifferent an tumors [1Other
two a comwere tested againarious
human tumors grown as xenafts in nude
found ancer activ Particularlynitro-
amptothecin-20-O-propinate showed spectacular results
e ag19 dhum6].
ctivepounds alsost v
d gooticanity., 9-
against the tumors tested. Figure 1 shows the results of
this compound against 5 different human tumors and
Figure 2 shows the toxicity of this compound at two
effective doses in nude mice. The dose-dependence of
this compound was observed at the low range. For
example, the growth inhibition of this agent against MUR-
breast carcinoma was almost twice stronger at 2 mg/kg
than 1 mg/kg (Figure 1(a)). This dependency disapp-
eared when the dose was elevated to 10 mg/kg or higher.
Figure 1(b) shows the results of the experiment with
three different doses (10 mg/kg, 20 mg/kg, and 30 mg/kg),
and no significant differences in inhibitory effects be-
tween them were observed. This agent completely in-
hibits the growth of various types of human tumors in
mice when the dose is 10 mg/kg or higher. Figures 1(c),
(d) and (e) show that the growths of BRO-Melanoma,
BRE-Stomach, and SPA-Lung in mice were completely
inhibited. Two administration routes were used when
treating SQU-Colon with 10 mg/kg of this compound.
The result shown in Figure 1(f) clearly indicates that the
inhibitory effect was identical when this agent was
administered intrastomachly compared to when admini-
stered intramuscularly. The toxicity of this agent in mice
at the effective dose was minimal. Figure 2 shows the
body weight changes of mice during the period on
treatment with this compound at 20 mg/kg and 30
mg/kg, respectively. At 20 mg/kg level, this agent was
Copyright © 2012 SciRes. OJMC
Z. S. CAO ET AL.
Copyright © 2012 SciRes. OJMC
Figure 1. The anticancer activity of 9-nitrocamptothecin-20-opionate against various different types of human tumor
grown as xenografts in nude mice. Each group had six sames of mice. All control groups of mice were sacrificed whe
tumors reached certain of siz
es. P ≤ 0.05.
Figure 2. Toxicity of 9-nitrocamptothecin-20-O-propionate
in mice at 20 mg/kg and 30 mg/kg, respectively with a sche-
dule of 5 days on and 2 days off. Group of 6 mice. P ≤ 0.05.
same as the initial weight. However, when dose reached
30 mg/kg, sign of toxicity appeared because animals lost
e camptothecin ester derivatives prepared by
this procedure showed great anticancer activity. Of active
completely safe for animals. The end body weight was
about 10% of their body weight. The effective dose of
this agent was found to be smaller than 20 mg/kg against
all types of the tumors tested in our laboratory. This
compound has great potential to be developed for human
The H2SO4-catalyzed procedure gives us an efficient way
for preparing camptothecin esters. The reaction gives
higher yields compared to those procedures reported pre-
Z. S. CAO ET AL.
Journal of the American Chemical Society, Vol. 88,
16,1966, pp. 300968a057
sesses great potential to be further developed for human
treatment. This procedure would also be applicable to the
acylation reactions of other alcohols or thiols.
The authors wish to thank Mr. Edward Ezell at the Uni-
versity of Texas Medical Branch for NMR data. Sup-
porting funds from the CHRISTUS Stehlin Foundation
for Cancer Research and the Friends of the Steun-
dation are greatly acknowled
 M. Wall, M. Wani, C. Cook, K. Palmer, A. McPhail and
G. Sim, “Plant Antitumor Agents. I. The Isolation and
Structure of Camptothecin, a Novel Alkaloidal Leukemia
and Tumor Inhibitor from C,”
 J. Gottlieb, A.verio and J. Block,
eclinical Studies,” Cancer Chemotherapy Reports
of Medicinal Chemis-
Guarino, J. Call, V. Oli
“Preliminary Pharmacologic and Clinical Evaluation of
Camptothecin Sodium (NSC-100880),” Cancer Chemo-
therapy Reports: Part 1, Vol. 54, No. 6, 1970, pp. 461-
 J. Gottlieb and J. Luce, “Treatment of Malignant Mela-
noma with Camptothecin (NSC-100880),” Cancer Che-
motherapy Reports, Vol. 56, No. 1, 1972, pp. 103-105.
 F. Muggia, P. Creaven, H. Hansen, M. Cohen and O.
Selawry, “Phase I Clinical Trial of Weekly and Daily
Treatment with Camptothecin (NSC-100880): Correlation
Vol. 56, No. 4, 1972, pp. 515-521.
 C. Moertel, A. Schutt, R. Reitemeier and R. Hahn, “Phase
II Study of Camptothecin (NSC-100880) in the Treatme
of Advanced Gastrointestinal Cancer,” Cancer Chemo-
therapy Reports , Vol. 56, No. 1, 1972, pp. 95-101.
 M. Wani, P. Ronman, L. Lindley and M. Wall, “Plant
Antitumor Agents. 18. Synthesis and Biological Activity
of Camptothecin Analogs,” Journal
try, Vol. 23, No. 5, 1980, pp. 554-560.
 T. Burke, “Chemistry of the Camptothecins in the Blood-
stream: Drug Stabilization and Optimization of Activity
eman, J. Stehlin
Esters of Camptothecin and
in the Camptothecins—From Discovery to Patients,” An-
nals of the New York Academy of Sciences, Vol. 903,
1993, pp. 29-31.
 Z. Cao, N. Harris, A. Kozielski, D. Vard
and B. Giovanella, “Alkyl
9-Nitrocamptothecin: Synthesis, in Vitro Pharmacokinet-
ics, Toxicity, and Antitumor Activity,” Journal of Me-
dicinal Chemistry, Vol. 41, No. 1, 1998, pp. 31-37.
 Z. Cao, J. Mendoza, A. DeJesus and B. Giovanella, “Syn-
thesis and Antitumor Activity of Alkenyl Camptoth
Esters,” Acta Pharmacoecin
logica Sinica, Vol. 26, No. 2,
2005, pp. 235-241.
 B. Neises and W. Steglich, “Simple Method for the
Esterification of Carboxylic Acids,” Angewandte Ch
International Edition, Vol. 17, No. 7, 19
78, pp. 522-524.
 A. Hassner and V. Alexanian, “Direct Room Temperature
Esterification of Carboxylic Acids,” Tetrahedron Letters,
Vol. 19, No. 46, 1978, pp. 4475-4478
 F. Ziegler and G. Berger, “A Mild Method for the Esteri-
fication of Fatty Acids,” Synthetic Com
9, No. 6, 1979, pp. 539-543.
 Z. Cao, J. Mendoza, A. DeJusus, D. Vardeman and B.
Giovanella, “Synthesis and Antit
matic Camptothecin Esters,” International Jo
umor Activity of Aro-
Molecular Medicine, Vol. 21, No. 4, 2008, pp. 477-487.
 Z. Cao, P. Pantazis, J. Mendoza, J. Early, A. Kozielski, N.
Harris and B. Giovanella, “Structure-Activity Relation-
ship of Alkyl 9-Nitrocamptothecin Esters,” Acta Phar
macologica Sinica, Vol. 24, No. 2, 2003, pp. 109-119.
 Z. Cao, P. Pantazis, J. Mendoza, J. Early, A. Kozielski, N.
Harris, D. Vardeman, J. Liehr, J. Stehlin and B. Gio-
vanella, “Structure-Activity Relationship of Alkyl Cam
tothecin Esters,” Annals of the New York Academy of
Sciences, Vol. 922, 2000, pp. 122-135.
 Z. Cao, A. Kozielski, X. Liu, Y. Wang, D. Vardeman and
B. Giovaniella, “Crystalline Camptothe
pionate Hydrate: A Novel Anticancer Ag
ent with Strong
Activity against 19 Human Tumor Xenograts,” Cancer
Research, Vol. 69, No. 11, 2009, pp. 4742-4749.
 Z. Cao, J. Mendoza, A. Kozielski, X. Liu, A. DeJesus, Y.
Wang, C. Zhan, D. Vardeman and B. Giovanella
cancer Activity of New Haloalkyl Ca
against Human Cancer Cell Lines and Human Tumor
Xenografts Grown in Nude Mice,” Submission.
 Z. Cao, K. Armstrong, M. Shaw, E. Petry and N. Harris,
“Nitration of Camptothecin with Various Inorganic Ni-
trate Salts in Concentrated Sulfuric Acid: A Ne
ration of Anticancer Drug 9-Nitrocamptothecin,” Synthe-
sis, Vol. 1998, No. 12, 1998, pp. 1724-1730.
Copyright © 2012 SciRes. OJMC