Sulfuric Acid Catalyzed Preparation of Alkyl and Alkenyl Camptothecin Ester Derivatives and Antitumor Activity against Human Xenografts Grown in Nude Mice

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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

Email: *[email protected]

Received January 19, 2012; revised February 16, 2012; accepted February 28, 2012

ABSTRACT

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

1. Introduction

Camptothecin, a natural product, was first isolated from a

native Chinese tree Camptotheca acuminata by Wall and

his coworkers in 1966 [1]. 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 [6]. 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 [7], 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.

N

NO

O

O

OH

OH

HN

NOH

O

OH

CO2(1)

Lactone CPTCarboxylate CPT

N

NO

O

O

OH

(RCO)2O

CPT

N

NO

O

OCOR

O

CPT es

t

e

r

s

Pyridine

(2)

N

NO

O

O

OH

ArCOOH

CPT

N

NO

O

OCOAr

O

CPT este

r

s

DCC/DMAP

(3)

*Corresponding autho

r

.

C

opyright © 2012 SciRes. OJMC

Z. S. CAO ET AL. 11

Dicyclohexylcarbodiimide (DCC)/dimethylaminopyri-

dine (DMAP) reagent system is frequently used for acy-

lation reactions of carboxylic acids with alcohols [10-12]

and thiols [10]. We previously used this method to pre-

are aromatic camptothecin esters [13] (Equation (3)).

Th

p

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 [8]. The reaction occurred with low yield

(6%, Equation (4)).

N

NO

O

O

OH

CH3(CH2)7COCl

N

NO

O

OCO(CH2)7CH3

O

Pyridine/CH2Cl2

(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

co

edia, camptothecins are

th them at room temperature or an

under N atmosphere with a few

lting point apparatus and were

un

by

as surgically

.

-

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) [16] and haloalkyl

camptothecin esters [17]. 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.

2. Methodology

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

elevated temperature2

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 [18]. 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. [8] 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 [8].

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.

12

re

m

and literature-reported procedures.

ridine procedure is fre-

compounds and usually

ptothecin-20-O-pro-

Ta

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

acylation procedure

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

C613

CH=CHCH3 H

N 45

N 73

N 56

N 14

4H9

H

0 99

H 98 99

C8H17 H 6 92

31 90

CH3 O298

C2H5 O299

C3H7 O298

i-C3H7 O292

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

ogrmice and

d gooticanity., 9-

c

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

13

(a) (b)

(c) (d)

(e) (f)

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

O-prs

sizen

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

treatment.

4. Conclusion

The H2SO4-catalyzed procedure gives us an efficient way

for preparing camptothecin esters. The reaction gives

higher yields compared to those procedures reported pre-

viously. Th

Z. S. CAO ET AL.

14

compounds, 9-nitrocamptothecin-20-O-propionate

hlin Fo

ged.

amptotheca acuminata

Journal of the American Chemical Society, Vol. 88,

16,1966, pp. 300968a057

pos-

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.

5. Acknowledgements

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

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