Alkyl esters of camptothecin and 9-nitrocamptothecin: Synthesis, in vitro pharmacokinetics, toxicity, and antitumor activity

Article abstract of DOI:10.1021/jm9607562

Eleven camptothecin esters, 6a-e and 7a-f, were prepared by straightforward acylation of camptothecins with the corresponding acylating reagents such as organic anhydrides and carboxylic acid chlorides. The in vitro pharmacokinetic determination of lacton

Full text of DOI:10.1021/jm9607562

J . Med. Chem. 1998, 41, 31-37
31
Alk yl Ester s of Ca m p toth ecin a n d 9-Nitr oca m p toth ecin : Syn th esis, in Vitr o
P h a r m a cok in etics, Toxicity, a n d An titu m or Activity
Zhisong Cao,* Nick Harris, Anthony Kozielski, Dana Vardeman, J ohn S. Stehlin, and Beppino Giovanella*
The Stehlin Foundation for Cancer Research and St. J oseph Hospital Cancer Laboratory, 1918 Chenevert Street,
Houston, Texas 77003
X
Received October 31, 1996
Eleven camptothecin esters, 6a -e and 7a -f, were prepared by straightforward acylation of
camptothecins with the corresponding acylating reagents such as organic anhydrides and
carboxylic acid chlorides. The in vitro pharmacokinetic determination of lactone levels of esters
6
a and 7b showed that the biological life span of their lactone forms in human and mouse
plasma significantly increased when compared with their mother compounds, camptothecin
3) and 9-nitrocamptothecin (4). The differences of lactone levels between human plasma and
(
mouse plasma for 6a and 7b were much smaller than what was observed for their mother
compounds. The in vivo antitumor activity and toxicity studies demonstrated that some of
these esters were very active against human tumor xenografts in nude mice and had an
exceptional lack of toxicity in nude mice, even at enormous doses.
In tr od u ction
Ch a r t 1. Topotecan (1) and Irinotecan (CPT-11, 2)
Camptothecin, a cytotoxic alkaloid first isolated from
the wood and bark of Camptotheca acuminata
(Nyssaceae) by Wall and co-workers, was shown to have
antitumor activity against the mouse leukemia L1210
system.1 The compound has a pentacyclic ring system
with an asymmetrical center in ring E with 20S con-
figuration. The pentacyclic ring system includes a
pyrrolo[3,4-b]quinoline moiety (rings A, B, and C), a
conjugated pyridone (ring D), and a six-membered
lactone (ring E) with an R-hydroxyl group. Natural
camptothecin itself is insoluble in water. Therefore,
camptothecin was evaluated clinically as the water-
soluble sodium carboxylate salt in the early stages.
Unfortunately, this form of camptothecin produced
severe toxicity and seemed devoid of anticancer
2
-6
activity,
which caused the discontinuation of phase
Ch a r t 2. Camptothecin (3), 9-Nitrocamptothecin (4),
and 9-Aminocamptothecin (5)
II trials. However, the interest in camptothecin and its
derivatives was revived by the finding that campto-
thecin inhibits topoisomerase I,7
-10
an enzyme that is
required for swiveling and relaxation of DNA during
1
1
molecular events such as replication and transcription.
A number of new syntheses and modifications of the
molecule have been reported in the literature, and many
1
2-29
new derivatives have been prepared over the years.
In 1989, it was found that camptothecin and some of
its semisynthetic derivatives possessed extraordinary
anticancer activity against human cancer xenografts in
nude mice.30 Two groups of camptothecins have been
developed leading to clinical trials since then. The first
group, which can be called water-soluble camptothecins,
consists of Topotecan (1)20 and Irinotecan (2) (Chart
31
1
). Both of them are now commercially available as
aqueous solutions which are injectable intravenously for
human treatment. The second group, water-insoluble,
comprises the mother compound, camptothecin (3), and
its semisynthetic derivatives, 9-nitrocamptothecin (4),
and 9-aminocamptothecin (5) (Chart 2). Many attempts
have been made to modify the camptothecin structure
in order to reduce its toxicity and maintain or increase
its activity. While the derivatives made today have not
met these criteria, they have offered important informa-
X
Abstract published in Advance ACS Abstracts, December 15, 1997.
S0022-2623(96)00756-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/01/1998

3
2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Cao et al.
tion about critical structural features necessary for
activity. For example, Wani and Wall demonstrated
that the carboxylate sodium salt form of camptothecin
has only about one-tenth the potency of 3 in one
Sch em e 1
1
6
antitumor assay (P388 rodent leukemia), implying
that the intact lactone ring E of 3 is the most critical
structural feature with respect to antitumor activity. It
has also been shown that administration of 3 with closed
lactone ring E is far superior to the injection of the
water-soluble carboxylate salt in inhibiting growth of
3
2
human cancer xenografts in nude mice. This further
confirmed the importance of the intact lactone ring E.
Extensive studies of the camptothecins used to treat
human cancers growing as xenografts in nude mice
showed very high activity against a wide spectrum of
human neoplasms. In our laboratory,3 we were able
to obtain a response rate of 100% of all human cancers
treated as established measurable tumors in nude mice
3
(
32/32). But, a response rate of only about 15% was
observed from a recently concluded phase II trial of
3
4
9
-nitrocamptothecin against ovarian cancer. It is not
surprising that results in mice are substantially better
than in humans when comparing the plasma lactone
levels of the drug in mice and humans. We found about
5
0% of the closed lactone form present in mice versus
Sch em e 2
about 3% in humans when determining the percentage
of the area under the curve (AUC) for 9-nitrocampto-
thecin (4). This means that the human tumor is
exposed to a concentration of the active drug 15-fold
higher in mice than in humans. It thus becomes evident
that the crucial obstacle to improving camptothecin
effectiveness is the rapid opening of the lactone ring in
the human body.
As seen above, prolonging the biological life of an
active drug by protecting the lactone moiety of the
molecule is important to drug development. An at-
tempt35 made by Wall et al. was to convert some
synthetic (RS)-camptothecin derivatives into the corre-
sponding water-soluble glycinate hydrochloric salts.
These glycinate hydrochloric salts were tested in vivo
against L1210 cells and showed less potency when
compared with camptothecin. Whether these glycinates
could prolong the biological life of the active drug was
not addressed.
Thus, we propose the intact lactone ring would be
better protected if camptothecins were transformed into
the correponding water-insoluble alkyl esters. In this
paper, synthesis and biological activity of camptothecin
and 9-nitrocamptothecin esters, 6a -e and 7a -f, are
described.
Compounds 7a -d were prepared in moderate to high
yields by the esterification of 9-nitrocamptothecin (4)
with the corresponding organic anhydrides (Scheme 1).
Thus, product 7a was obtained in 45% yield by the
reaction of 4 with acetic anhydride. The acetyl methyl
protons absorbed at 2.26 ppm. Product 7b was obtained
in 73% yield by reacting 4 with propionic anhydride.
Ch em istr y
1
The H NMR spectrum of 7b showed a characteristic
Camptothecin esters 6a -d were prepared in high
yields by the straightforward acylation of camptothecin
with the corresponding organic anhydrides (Scheme 1).
Thus, compound 6a was obtained in 94% yield by the
reaction of 3 with propionic anhydride in pyridine at
triplet with a J value of 7.46 Hz at 1.18 ppm for the
C24-methyl group and a multiplet at 2.52-2.70 ppm for
the C23-methylene protons. On reacting 4 with butyric
anhydride or isobutyric anhydride, products 7c,d were
obtained in yields of 56% and 14%, respectively. The
1
4
0 ( 5 °C. In the same manner, compounds 6b-d were
corresponding H NMR peaks for the side propyl group
obtained in yields of 92%, 90%, and 98%, respectively,
when the corresponding butyric anhydride, valeric
anhydride, and heptanoic anhydride were used as the
acylating reagents. The H and C NMR spectra of
these esters showed the corresponding characteristic
absorptions for their side ester alkyl chains.
of 7c and the side isopropyl group of 7d were observed.
The products 6e and 7e,f were prepared by using the
corresponding carboxylic acid chlorides as the acylating
reagents (Scheme 2). Product 6e was obtained in 6%
yield by acylation of 3 with nonanoyl chloride in meth-
1
13
1
ylene chloride. Its H NMR spectrum obtained from

Alkyl Esters of Camptothecin and 9-Nitrocamptothecin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 33
Ta ble 1. Comparison of Percent Lactone of Prodrug 6a and
Ta ble 3. Changes in Body Weights of Mice during the Test
Camptothecin (CPT) in Human and Mouse Plasma
Time Period
time (h)
doses
mg/kg)
time (days)/body weight (g)
(
0
1
2
4
6
control 21/31.7 27/33.5 34/34.4 41/35.2 48/35.0 56/36.7
human plasma
5
6
7
8
21/33.0 27/33.7 34/34.4 41/33.5 48/32.9 56/33.2
21/32.9 27/33.4 34/33.8 41/33.5 48/34.0 56/32.2
21/30.8 27/31.7 34/30.6 41/31.1 48/31.6 56/31.5
21/32.9 27/34.1 34/34.0 41/33.4 48/33.9 56/33.2
%
%
lactone for 6a
lactone for CPT
100.0
100.0
86.0
12.0
77.0
0.50
68.0
00.0
56.0
00.0
mouse plasma
%
%
lactone for 6a
lactone for CPT
100.0
100.0
97.5
42.0
ND
35.0
ND
18.0
85.0
18.0
Ta ble 2. Comparison of Percent Lactone of Prodrug 7b and
9
-Nitrocamptothecin (9NC) in Human and Mouse Plasma
time (h)
0
1
6
28
51
human plasma
%
%
lactone for 7b
lactone for 9NC
100.0
100.0
93.7
7.0
64.4
00.0
16.6
00.0
5.8
00.0
mouse plasma
%
%
lactone for 7b
lactone for 9NC
100.0
100.0
97.7
30.0
84.3
18.0
73.1
18.0
59.9
ND
CDCl3 showed a multiplet for those methylene protons
in the long side ester alkyl chain. On reacting 4 with
the corresponding cyclopropanecarboxylic acid chloride
or cyclohexanecarboxylic acid chloride, products 7e,f
were obtained in yields of 30% and 63%, respectively.
The corresponding characteristic proton peaks for the
side cyclopropyl group of 7e and the side cyclohexanyl
group of 7f were observed.
F igu r e 1. Activity of prodrug 6a against human breast
carcinoma.
evident that the differences of lactone levels in plasma
between human and mouse for 7b are much smaller
than what is observed for its mother compound 4.
All 11 esters were tested in vivo with nude Swiss mice
of the NIH strain as the animal models. Esters 6a and
The mass spectral data of esters 6a -e showed char-
acteristic decomposition patterns. They all gave the M
7
b showed the best results. The in vivo toxicity study
+
H peaks and the peaks at m/e 331, 317, 303, and 287.
showed that these esters had little or no toxicity in nude
mice. Overall toxicity can be judged using various
criteria. For example, loss of body weight in a subject
over 10% of the initially recorded body weight (i.e.,
before treatment) can be considered as one sign of
toxicity. In addition, loss of mobility and activity and
signs of diarrhea or cystitis in a subject can also be
interpreted as evidence of toxicity. Table 3 shows the
toxicity of prodrug 6a in nude mice with different doses.
The change of body weight of mice is recorded as a
function of time. Body weight losses in mice during the
test time period were not observed.
These ester compounds exhibit significant antitumor
activity against human cancer xenografts growing in
nude mice. Figure 1 represents the antitumor activity
of prodrug 6a in different doses against the implanted
human breast carcinoma CLO in nude mice. Figure 2
shows the antitumor activity of 6a at a dose of 100 mg/
kg of body weight against human lung carcinoma SPA
in nude mice. Figure 3 shows the anticancer activity
of prodrug 7b at a dose of 30 mg/kg of body weight
against human colon carcinoma SQU in nude mice.
The mass spectral data of esters 7a -f also showed
characteristic fragmentation patterns. They all gave
the molecular ion peaks and peaks at m/e 375, 360, 347,
3
19, and 302.
Resu lts a n d Discu ssion
The in vitro determination of lactone levels in human
and mouse plasma for 6a and 7b are shown in Tables1
and 2. As shown in Table 1 the percent lactone of
camptothecin (3) in human blood is 12% after 1 h, 0.5%
after 2 h, and undetectable after 4 h. In other words,
the active form of 3 is completely lost in a very short
time period after oral administration. This is the
opposite to what is observed in mice, in which the active
drug form (i.e., the closed lactone form) lasts for a very
long time period. For example, the closed lactone form
of 3 in mouse plasma is still detectable after 6 h (18%,
Table 1). The closed lactone form of 6a in human
plasma is much more stable than its mother compound
3
. For example, 56% of 6a is still detected as the closed
lactone form even after 6 h (Table 1). Camptothecin,
in terms of lactone level, shows a big difference between
mouse and human. This kind of difference is not
observed for prodrug 6a . Even more striking are the
results obtained with prodrug 7b. As shown in Table
Con clu sion
We have described the synthesis of 11 camptothecin
esters. The compounds selected for the in vitro deter-
mination of lactone levels showed that their biological
life span in human and mouse plasma was much longer
than that of their parent compounds. The differences
of lactone levels in plasma between human and mouse
for these tested esters, such as 6a and 7b, were much
smaller than that observed for their parent compounds.
2
, after oral administration of this compound, the
lactone form of 7b is very stable in human and mouse
plasma. While its mother compound 4 stays in human
plasma as the closed lactone form for only a very short
time period, the majority of the drug present in human
plasma is the toxic carboxylate salt form. It is also

3
4
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Cao et al.
and 20 mL of pyridine. A homogeneous solution was obtained
after shaking for 30 s. To this solution was added 2.0 g of
starting camptothecin. The mixture was stirred at 40 ( 5 °C
for 48 h. After cooling to room temperature, the reaction
mixture was poured onto 400 mL of petroleum ether while
stirring. The product precipitated from petroleum ether was
collected by filtration and washed with 150 mL of petroleum
ether (50 mL × 3). After drying under air for 1 h, a white
powder, 2.17 g, was obtained, yield 94%, purity 98% (HPLC),
1
mp 250-252 °C dec. H NMR in CDCl
3
: δ 0.98 (3H, t, J )
7
.5 Hz, C19-methyl protons), 1.17 (3H, t, J ) 7.51 Hz, C24-
methyl protons), 2.12-2.34 (2H, m, C18-methylene protons),
.48-2.58 (2H, m, C23-methylene protons), 5.29 (2H, s, C5-
2
methylene protons), 5.39-5.72 (2H, dd, J ) 17.12, 17.12 Hz,
C17-methylene protons), 7.23 (1H, s, C14-H), 7.68 (1H, t, J )
6
.96 Hz, C10-H), 7.84 (1H, t, J ) 6.96 Hz, C11-H), 7.95 (1H,
F igu r e 2. Activity of prodrug 6a against human lung
carcinoma.
d, J ) 8.43 Hz, C9-H), 8.23 (1H, d, J ) 8.06 Hz, C12-H), 8.40
1
(1H, s, C7-H). H NMR in TFA: δ 1.18 (3H, t, J ) 7.5 Hz,
C19-methyl protons), 1.32 (3H, t, J ) 7.30 Hz, C24-methyl
protons), 2.30-2.80 (4H, m, C18- and C23-methylene groups),
5
.60-6.10 (4H, s + dd, s at 5.86 for C5-methylene protons, dd
with J ) 18.96, 18.32 Hz for C17-methylene protons), 7.99 (1H,
s, C14-H), 8.19 (1H, t, J ) 8.06 Hz, C10-H), 8.20-8.46 (2H,
m, C9-H, C11-H), 8.54 (1H, d, J ) 8.79 Hz, C12-H), 9.43 (1H,
1
3
s, C7-H).
(
C NMR (TFA): δ 7.42 (C19), 8.55 (C24), 28.61
C18), 33.07 (C23), 53.14 (C5), 68.77 (C17), 78.31 (C20), 105.68,
1
1
1
13.19, 117.35, 125.87, 131.23, 131.36, 132.59, 133.40, 139.30,
39.89, 140.78, 144.62, 147.00, 149.43 (C2, C3, C6-C16, C16a),
72.50, 179.76 (C21, C22). MS m/e (relative intensity): 405
+
(
M + H, 100), 404 (M , 15), 331 (M - CH
3
CH
+ H, 10), 303 (M - C
COO - CO , 9), 273 (8), 261(9). Precise
): found, 404.137; required, 404.137.
2
COO, 17), 317
H COO - CO,
2 5
(
M - C
5), 287 (M - C
mass (C23
2
H
5
COO - CH
3
1
2
H
5
5
2
H
20
N
2
O
Ca m p toth ecin -20-O-bu tyr a te (6b). Using 20 mL of bu-
tyric anhydride, 18 mL of pyridine, and 1.61 g of camptothecin,
the reaction was carried out in the same manner as in
F igu r e 3. Activity of prodrug 7b against human colon
carcinoma.
preparing 6a , yield 92%, purity 99% (HPLC), mp 225-227 °C
1
dec. H NMR in CDCl
3
: δ 0.98 (6H, t, J ) 7.51 Hz, C19- and
The overall toxicity of these tested esters against nude
mice when compared with their parent compounds was
much lower. Meanwhile, their antitumor activity against
xenografts of human tumors growing in nude mice is
maintained.
The biological studies for these esters are very pre-
liminary. Many other aspects such as topoisomerase I
inhibition (IC50), the cleavage of supercoiled DNA by
topoisomerase I in the presence of these esters, and the
rate of hydrolysis of these esters will be further studied.
C25-methyl groups), 1.65-1.74 (2H, m, C24-methylene pro-
tons), 2.14-2.30 (2H, m, C18-methylene protons), 2.44-2.51
(2H, m, C23-methylene protons), 5.29 (2H, s, C5-methylene
protons), 5.38-5.71 (2H, dd, J ) 17.59, 17.59 Hz, C17-
methylene protons), 7.23 (1H, s, C14-H), 7.68 (1H, t, J ) 8.06
Hz, C10-H), 7.84 (1H, t, J ) 7.96 Hz, C11-H), 7.95 (1H, d, J )
6
.96 Hz, C9-H), 8.23 (1H, d, J ) 8.06 Hz, C12-H), 8.40 (1H, s,
1
C7-H). H NMR in TFA: δ 0.75-1.15 (6H, m, C19- and C25-
methyl groups), 1.70-1.80 (2H, m, C24-methylene protons),
2
.10-2.80 (4H, m, C18- and C23-methylene groups), 5.50-
6.00 (4H, s + dd, s at 5.73 for C5-methylene protons, dd for
C17-methylene protons), 7.86 (1H, s, C14-H), 8.05 (1H, s, C10-
H), 8.30 (2H, brs, C9-H, C11-H), 8.40 (1H, s, C12-H), 9.30 (1H,
Exp er im en ta l Section
1
3
s, C7-H). C NMR (TFA): δ 7.23 (C19), 13.20 (C25), 19.20
All glassware was baked at 80-100 °C for a minimum of 2
h before being used. Melting points were obtained with a
MEL-TEMP melting point apparatus and are uncorrected. The
(
(
C24), 32.91 (C18), 36.91 (C23), 52.96 (C5), 68.58 (C17), 78.00
C20), 105.56, 113.40, 113.50, 117.00, 117.10, 131.00, 132.40,
1
13
133.16, 139.06, 139.15, 140.00, 144.36, 146.90, 149.40 (C2, C3,
C6-C16, C16a), 172.50, 178.00 (C12, C22). MS m/e (relative
H and C NMR spectra were obtained at 270.05 MHz with a
J EOL GX-270 WB NMR spectrometer. Chemical shifts are
reported in parts per million (δ scale), employing tetramethyl-
silane as an internal standard. In reporting the NMR data,
we have used the following abbreviations: coupling constants
in hertz (J ), singlet (s), doublet (d), triplet (t), broad singlet
intensity): 419 (M + H, 100), 331 (M - C
3
H
7
COO, 17), 317
COO - CO,
(
M - C
3), 287 (M - C
22 2 5
mass (C24H N O
6
H
13COO - CH
COO - CO
): found, 418.152; required, 418.153.
3
+ H, 10), 303 (M - C
3
H
7
1
3
H
7
2
, 8), 273 (2), 261 (3). Precise
(
brs), multiplet (m), etc. Mass spectra were recorded using a
Ca m p toth ecin -20-O-va ler a te (6c). Using 15 mL of va-
leric anhydride, 14 mL of pyridine, and 1.51 g of starting
camptothecin, product 6c was obtained as a gray-white
VG ZAB-SEQ mass spectrometer (VG Analytical Co., England)
with a resolution of 10 000. The numbering system used to
depict NMR for these new camptothecin esters is shown in
Schemes 1 and 2.
powder, yield 90%, purity 99% (HPLC), mp 265-267 °C dec.
1
H NMR in CDCl : δ 0.92 (3H, t, J ) 7.33 Hz, C26-methyl
3
The starting camptothecin was purchased from The People’s
Republic of China and was purified before being used. The
starting 9-nitrocamptothecin was prepared by following the
protons), 0.98 (3H, t, J ) 7.51, C19-methyl protons), 1.37-
2.00 (4H, m, C24- and C25-methylene protons), 2.10-2.28 (2H,
m, C18-methylene protons), 2.46-2.53 (2H, m, C23-methylene
protons), 5.30 (2H, s, C5-methylene protons), 5.38-5.71 (2H,
dd, J ) 17.22, 17.21 Hz, C17-methylene protons), 7.23 (1H, s,
C14-H), 7.70 (1H, t, J ) 6.96 Hz, C10-H), 7.82 (1H, t, J ) 6.96
Hz, C11-H), 7.95 (1H, d, J ) 7.32 Hz, C9-H), 8.22 (1H, d, J )
1
7
procedure described by Wall et al.
The other organic
anhydrides, carboxylic acid chlorides, and solvents used for
the experiments were all purchased from Aldrich Chemical
Co. (Milwaukee, WI).
Cam ptoth ecin -20-O-pr opion ate (6a). In a 100-mL round-
bottomed flask were mixed 25 mL of propionic acid anhydride
1
8.42 Hz, C12-H), 8.40 (1H, s, C7-H). H NMR in TFA: δ 0.83
(3H, brs, C26-methyl protons), 0.99 (3H, brs, C19-methyl

Alkyl Esters of Camptothecin and 9-Nitrocamptothecin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 35
169.98, 176.92 (C21, C22). MS m/e (relative intensity): 489
protons), 1.32 (2H, m, C25-methylene protons), 1.60 (2H, m,
C24-methylene protons), 2.19-2.58 (4H, m, C18- and C23-
methylene protons), 5.49-5.82 (4H, s + dd, s at 5.67 for C5-
methylene protons, dd with J ) 17.58, 18.68 Hz for C17-
methylene protons), 7.80 (1H, s, C14-H), 7.99 (1H, s, C10-H),
(M + H, 100), 331 (M - C
- CH + H, 13), 303 (M - C
17COO - CO , 8), 273 (3), 216 (2). Precise mass
): found, 488.230; required, 488.231.
-Nitr oca m p toth ecin -20-O-a ceta te (7a ). Acetic anhy-
8
H
17COO, 23), 317 (M - C
8
H17COO
17COO - CO, 17), 287 (M -
3
8
H
C
8
H
2
(C29
32 2 5
H N O
8
.23 (2H, brs, C9-H, C11-H), 8.33 (1H, s, C12-H), 9.24 (1H, s,
9
1
3
C7-H). C NMR (TFA): δ 4.48 (C26), 10.37 (C19), 20.23 (C24),
dride (3 mL) and pyridine (2 mL) were mixed in a 50-mL
round-bottomed flask in which 140 mg of 9-nitrocamptothecin
was placed. The mixture was stirred at room temperature for
24 h. The mixture was then poured onto 200 mL of petroleum
ether while stirring. The crude product was precipitated and
collected by filtration. After separation by column chroma-
tography with chloroform-methanol as eluent, the pure
product 7a (70 mg) was obtained as a yellow powder by
2
7
1
4.98 (C25), 30.15 (C18), 32.03 (C23), 50.20 (C5), 65.82 (C17),
5.36 (C20), 102.84, 109.89, 110.24, 114.06, 114.39, 128.25,
28.39, 129.65, 130.41, 136.30, 137.00, 141.62, 148.57, 149.28
(
C2, C3, C6-C16, C16a), 169.00, 176.80 (C21, C22). MS m/e
relative intensity): 433 (M + H, 100), 331 (M - C COO,
7), 317 (M - C 13COO - CH + H, 10), 303 (M - C COO
CO, 13), 287 (M - C COO - CO , 7), 273 (2), 261 (4).
Precise mass (C25 ): found, 432.168; required, 432.169.
(
4
H
9
1
6
H
3
4 9
H
-
4
H
5
9
2
H
24
N
2
O
reprecipitation from petroleum ether, yield 45%, purity 99%
1
Ca m p toth ecin -20-O-h ep ta n oa te (6d ). Using 15 mL of
(HPLC), mp 195 °C (deformed at 165 °C). H NMR (CDCl
3
):
heptanoic anhydride, 13 mL of pyridine, and 1.55 g of starting
δ 1.02 (3H, t, J ) 7.51 Hz, C19-methyl protons), 2.10-2.40
(5H, s + m, s at 2.26 for C23-methyl protons, m for C18-
methylene protons), 5.40 (2H, s, C5-methylene protons), 5.41-
5.75 (2H, dd, J ) 17.59, 17.95 Hz, C17-methylene protons),
7.23 (1H, s, C14-H), 7.96 (1H, t, J ) 6.96 Hz, C11-H), 8.53
(1H, d, J ) 10.99 Hz, C10-H), 8.58 (1H, d, J ) 9.98 Hz, C12-
camptothecin, 6d was obtained as a gray-white powder, yield
1
9
8%, purity 99% (HPLC), mp 270 °C (deformed at 210 °C). H
3
NMR in CDCl : δ 0.82 (3H, t, J ) 7.51 Hz, C28-methyl
protons), 0.98 (3H, t, J ) 7.01 Hz, C19-methyl protons), 1.20-
1
2
.80 (8H, m, C24-, C25-, C26-, and C27-methylene protons),
.10-2.30 (2H, m, C18-methylene protons), 2.40-2.60 (2H, m,
+
H), 9.31 (1H, s, C7-H). MS m/e (relative intensity): 435 (M ,
C23-methylene protons), 5.29 (2H, s, C5-methylene protons),
25), 375 (M - CH
3
COOH, 100), 360 (M - CH
3
3
COOH - CH
3
,
5
.38-5.72 (2H, dd, J ) 17.69, 17.22 Hz, C17-methylene
40), 347 (M - CH
COOH - CO, 87), 332 (M - CH
3
COOH -
protons), 7.23 (1H, s, C14-H), 7.68 (1H, t, J ) 7.30 Hz, C10-
H), 7.84 (1H, t, J ) 7.42 Hz, C11-H), 7.95 (1H, d, J ) 8.06 Hz,
C9-H), 8.22 (1H, d, J ) 8.32 Hz, C12-H), 8.40 (1H, s, C7-H).
CO - CH
3
, 37), 319 (13), 302 (11), 291 (10), 286 (11), 274 (10),
): found,
258 (4), 246 (5), 216 (8). Precise mass (C22
35.107; required, 435.107.
9-Nitr oca m p toth ecin -20-O-p r op ion a te (7b). Using 6
17 3 7
H N O
4
1
H NMR in TFA: δ 0.74 (3H, s, C28-methyl protons), 0.99 (3H,
s, C19-methyl protons), 1.21 (6H, brs, C25-, C26-, and C27-
methylene protons), 1.62 (2H, s, C24-methylene protons),
mL of propionic anhydride, 5 mL of pyridine, and 600 mg of
starting 9-nitrocamptothecin, the pure compound (500 mg) was
2
6
.10-2.30 (4H, m, C18- and C23-methylene groups), 5.50-
obtained as a yellow powder, yield 73%, purity 99% (HPLC),
1
.00 (4H, s + dd, s at 5.67 for C5-methylene protons, dd for
mp 163 °C (deformed at 155 °C). H NMR (CDCl
3
): δ 0.99
C17-methylene protons), 7.80 (1H, s, C14-H), 7.99 (1H, s, C10-
(3H, t, J ) 7.51 Hz, C19-methyl protons), 1.18 (3H, t, J ) 7.46
Hz, C24-methyl protons), 2.10-2.30 (2H, m, C18-methylene
protons), 2.52-2.70 (2H, m, C23-methylene protons), 5.37 (2H,
s, C5-methylene protons), 5.39-5.73 (2H, dd, J ) 17.58, 17.58
Hz, C17-methylene protons), 7.22 (1H, s, C14-H), 7.93 (1H, t,
J ) 8.06 Hz, C11-H), 8.50 (1H, d, J ) 10.60 Hz, C10-H), 8.54
1
3
H), 8.23 (2H, s, C9-H, C11-H), 9.24 (1H, s, C7-H). C NMR
TFA): δ 8.63 (C19), 14.99 (C28), 24.66 (C27), 27.14 (C26),
(
3
6
1
1
1.07 (C24), 33.68 (C25), 34.29 (C18), 36.45 (C23), 54.34 (C5),
9.98 (C17), 79.50 (C20), 106.97, 114.39, 118.55, 127.11,
32.41, 133.79, 134.55, 140.46, 141.11, 142.00, 145.79, 148.14,
50.62, 153.00 (C2, C3, C6-C16, C16a), 180.57, 193.10 (C21,
(1H, d, J ) 8.43 Hz, C12-H), 9.28 (1H, s, C7-H). MS m/e
+
C22). MS m/e (relative intensity): 461 (M + H, 100), 331 (M
(relative intensity): 449 (M , 28), 375 (M - C
2
H
5
COOH, 100),
COOH -
, 26), 319 (9), 302
(8), 291 (7), 274 (7), 258 (2), 245 (2), 216 (2). Precise mass
): found, 449.122; required, 449.122.
-
C
6
H
13COO, 20), 317 (M - C
M - C 13COO - CO, 15), 287 (M - C
73 (2), 261 (2). Presice mass (C27
6
H
13COO - CH
13COO - CO
): found, 460.199;
3
+ H, 10), 303
360 (M - C
2
H
5
COOH - CH
3
2 5
, 35), 347 (M - C H
(
6
H
6
H
2
, 8),
CO, 82), 332 (M - C
2
H
5
COOH - CO - CH
3
2
H
28
N
2
O
5
required, 460.120.
(C23
H
19
N
3
O
7
Ca m p toth ecin -20-O-n on a n oa te (6e). To 40 mL of meth-
ylene chloride in a 100-mL round-bottomed flask were added
mL of nonanoyl chloride and 2 g of starting camptothecin.
9-Nitr oca m p toth ecin -20-O-bu tyr a te (7c). Using 2 mL
of butyric anhydride, 2 mL of pyridine, and 60 mg of starting
9-nitrocamptothecin, the product 7c (40 mg) was obtained as
5
The mixture was stirred under reflux for 48 h. After the
solvent was evaporated by a rotary evaporator, the residue
was chromatographically separated with chloroform-methanol
as eluent. Product 6e (169 mg) was obtained as a pale yellow
a yellow powder, yield 56%, purity 99% (HPLC), mp 182 °C.
1
H NMR (CDCl ): δ 0.98 (6H, m, C19- and C25-methyl groups),
3
1.65-1.70 (2H, m, C24-methylene protons), 2.10-2.40 (2H, m,
C18-methylene protons), 2.41-2.60 (2H, m, C23-methylene
protons), 5.36 (2H, s, C5-methylene protons), 5.38-5.72 (2H,
dd, J ) 17.59, 17.96 Hz, C17-methylene protons), 7.22 (1H, s,
C14-H), 7.92 (1H, t, J ) 7.52 Hz, C11-H), 8.49 (1H, d, J )
10.80 Hz, C10-H), 8.53 (1H, d, J ) 9.53 Hz, C12-H), 9.27 (1H,
1
powder, yield 6%, purity 99% (HPLC), mp 180 °C. H NMR
in CDCl
3
: δ 0.84 (3H, t, J ) 6.60 Hz, C30-methyl protons),
1
.02 (3H, t, J ) 7.69 Hz, C19-methyl protons), 1.20-1.80 (12H,
m, C24-C29-methylene protons), 2.10-2.38 (2H, m, C18-
methylene protons), 2.40-2.60 (2H, m, C23-methylene pro-
tons), 5.33 (2H, s, C5-methylene protons), 5.40-5.80 (2H, dd,
J ) 17. 22, 17.22 Hz, C17-methylene protons), 7.26 (1H, s,
C14-H), 7.71 (1H, t, J ) 8.06 Hz, C10-H), 7.88 (1H, t, J ) 8.43
+
s, C7-H). MS m/e (relative intensity): 463 (M , 14), 375 (M -
C
C
3
H
H
7
COOH, 100), 360 (M - C
3
H
7
COOH - CH
3
, 32), 347 (M -
3
7
COOH - CO, 78), 332 (M - C
3
H
7
COOH - CO - CH ,
3
25), 319 (9), 302 (8), 291 (7), 274 (7), 258 (2), 245 (3), 216 (5).
Precise mass (C24 ): found, 463.137; required, 463.138.
Hz, C11-H), 7.99 (1H, d, J ) 7.33 Hz, C9-H), 8.26 (1H, d, J )
21 3 7
H N O
1
8
(
.79 Hz, C12-H), 8.44 (1H, s, C7-H). H NMR in TFA: δ 0.96
9-Nitr oca m p toth ecin -20-O-isobu tyr a te (7d ). By the
same manner as for the preparation of 7c, product 7d was
3H, s, C30-methyl protons), 1.24 (3H, s, C19-methyl protons),
1
.38 (10H, brs, C25-C29-methylene protons), 1.87 (2H, m,
obtained as a yellow powder, yield 14%, purity 99%, mp 197
1
C24-methylene protons), 2.40-2.90 (4H, m, C18- and C23-
methylene protons), 5.74-6.07 (4H, s + dd, s at 5.91 for C5-
methylene protons, dd with J ) 17.90, 18.21 Hz for C17-
methylene protons), 8.05 (1H, s, C14-H), 8.24 (1H, t, C10-H),
°C. H NMR (CDCl
3
): δ 0.96 (3H, t, J ) 7.52 Hz, C19-methyl
protons), 1.21 (6H, t (d + d), J ) 6.99, 6.99 Hz, C24- and C25-
methyl groups), 2.00-2.30 (2H, m, C18-methylene protons),
2.60-2.80 (1H, m, C23-tertiary proton), 5.32 (2H, s, C5-
methylene protons), 5.33-5.67 (2H, dd, J ) 17.35, 17.35 Hz,
C17-methylene protons), 7.20 (1H, s, C14-H), 7.88 (1H, dd, J
) 8.08, 8.28 Hz, C11-H), 8.45 (1H, d, J ) 7.51 Hz, C10-H),
8
.48 (2H, m, C9-H, C11-H), 8.57 (1H, d, C12-H), 9.48 (1H, s,
1
3
C7-H). C NMR (TFA): δ 4.99 (C30), 11.45 (C19), 21.17 (C29),
2
3
1
1
3.53 (C28), 27.82 (C24, C26-C27), 30.52 (C25), 30.63 (C18),
2.80 (C23), 50.68 (C5), 66.34 (C17), 75.82 (C20), 103.28,
10.73, 114.91, 123.47, 128.79, 128.90, 130.14, 130.93, 136.84,
37.46, 138.33, 142.17, 144.47, 146.94 (C2, C3, C6-C16, C16a),
1
3
8.50 (1H, d, J ) 8.54 Hz, C12-H), 9.23 (1H, s, C7-H). C NMR
(CDCl ): δ 7.60 (C19), 18.65 (C24, C25), 31.84 (C18), 33.60
(C23), 50.24 (C5), 67.00 (C17), 75.41 (C20), 96.80, 120.99,
3

3
6
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Cao et al.
1
1
21.54, 125.82, 127.46, 128.57, 131.53, 136.56, 145.00, 146.16,
deviation of 2%. The percent lactone of 6a was determined
by the ratio of drug measured at each time point to drug
measured at starting time point (t ) 0 h). A Waters C-8 Sep-
Pak cartridge was preconditioned by vacuuming through 1 mL
of methanol and then 1 mL of H
2
O; 100 µL of plasma was then
added and vacuumed through. The salt was removed by
eluting with 2 mL of 20% methanol in water. The lactone was
48.91, 153.00, 157.23 (C2, C3, C6-C16, C16a), 167.23, 175.88
+
(C21, C22). MS m/e (relative intensity): 463 (M , 48), 375 (M
-
(CH
5), 347 (M - (CH
CHCOOH - CO - CH
Precise mass (C24
-Nit r oca m p t ot h ecin -20-O-cyclop r op a n eca r b oxyla t e
7e). The starting 9-nitrocamptothecin (0.5 g, 0.0013 mol) and
3
)
2
CHCOOH, 100), 360 (M - (CH
CHCOOH - CO, 100), 332 (M - (CH
, 72), 319 (23), 302 (24), 291 (17).
): found, 463.138, required, 463.138.
3
)
2
CHCOOH - CH
3
,
8
3
)
2
3
)
2
-
3
21 3 7
H N O
2
eluted with 1 mL of a solution of 50% methanol/H O/0.1%
9
acetic acid. For total drug determination, 100 µL of plasma
was added to 1000 µL of 0.35% perchloric acid. The mixture
was vortexed for 10 s and left for 5 min at room temperature.
The solution was then passed through a preconditioned C-8
Sep-Pak cartridge and washed with 2 mL of 20% methanol in
water. The total drug was then eluted with 1 mL of 50%
methanol/water/0.1% acetic acid. HPLC analysis: 500 µL of
solution obtained above was injected through a 2-mL loop onto
a C-8 Microsorb column and chromatographed with 30%
acetonitrile/water/0.1% acetic acid as mobile phase. The drug
was detected by a fluorescence detector at 347/418 nm. The
peak area was integrated, and the lactone levels of drug at
different time points were determined.
(
cyclopropanecarboxylic acid chloride (8 mL) were added to 20
mL of acetone in a 100-mL round-bottomed flask equipped
with a magnetic stirrer. To this mixture was added 7 mL of
pyridine dropwise while stirring. After stirring at room
temperature for 15 h, the mixture was poured onto 750 mL of
5
% hydrochloric acid solution in water while stirring. The
yellow suspension obtained was extracted with 400 mL of
methylene chloride (100 mL × 4). The combined extracts were
washed with 200 mL of distilled water and dried over
anhydrous sodium sulfate for 4 h. After filtration the solvent
was removed by a rotary evaporator. The residue was
chromatographically separated with methanol-chloroform as
eluent. The pure product was obtained as a yellow powder,
yield 30%, purity 99% (HPLC), mp 274 °C. H NMR (CDCl
In Vitr o Deter m in a tion of La cton e Levels in Hu m a n
a n d Mou se P la sm a for 4 a n d 7b. To 1 mL of plasma
(human or mouse) was added 500 ng of drug in methanol. The
mixture was incubated at 37 °C of the following time points:
0.5, 1, 2, 4, 6, 28, and 51 h. The percent lactone of 4 was
determined by the ratio of lactone to total drug. The total drug
at each time point was consistent with a relative average
deviation of 2%. The percent lactone of 7b was determined
by the ratio of drug measured at each time point to drug
measured at starting time point (t ) 0 h). A Waters C-8 Sep-
Pak cartridge was preconditioned by vacuuming through 1 mL
1
3
):
δ 0.62-1.07 (7H, m, C19-methyl, C24- and C25-methylene
groups), 1.70-1.80 (1H, m, C23-tertiary proton), 2.10-2.70
(2H, m, C18-methylene protons), 5.32 (2H, s, C5-methylene
protons), 5.33-5.65 (2H, dd, J ) 17.35, 17.35 Hz, C17-
methylene protons), 7.23 (1H, s, C14-H), 7.88 (1H, t (d + d), J
)
8.03, 8.28 Hz, C11-H), 8.44 (1H, d, J ) 7.51 Hz, C10-H),
.51 (1H, d, J ) 8.55 Hz, C12-H), 9.23 (1H, s, C7-H). 13C NMR
CDCl ): δ 7.60 (C19), 9.19, 9.36 (C24, C25), 12.78 (C23), 31.90
C18), 50.20 (C5), 67.00 (C17), 75.64 (C20), 96.99, 120.99,
8
(
(
3
1
1
21.59, 125.81, 127.46, 128.61, 131.55, 136.51, 144.94, 146.04,
of methanol and then 1 mL of H O; 100 µL of plasma was then
2
48.92, 153.86, 157.21 (C2, C3, C6-C16, C16a), 167.00, 173.75
added and vacuumed through. The salt was removed by
eluting with 2 mL of 20% methanol in water. The lactone was
eluted with 1 mL of a solution of acetonitrile, methanol, and
10 mM ammonium formate (1:4:5, pH 2.0) and collected. To
+
(C21, C22). MS m/e (relative intensity): 461 (M , 13), 375 (M
-
cyclopropanecarboxylic acid, 100), 360 (M - cyclopropane-
carboxylic acid - CH , 30), 347 (M - cyclopropanecarboxylic
acid - CO, 66), 332 (M - cyclopropanecarboxylic acid - CO -
CH , 27), 319 (9), 302 (8), 291 (4). Precise mass (C24 ):
found, 461.123; required, 461.122.
-Nitr ocam ptoth ecin -20-O-cycloh exan ecar boxylate (7f).
3
this 1 mL of eluent was added 200 µL of Fe/H O (100 mg of
2
3
H
19
N
3
O
7
reduced pentacarbonyliron/1 mL of H O). The mixture was
2
vortexed for 10 s, sonicated for 30 min, and centrifuged at
6000g for 30 s. HPLC analysis: 500 µL of solution was taken
from the top homogeneous layer of the above-centrifuged
mixture, injected through a 2-mL loop onto a C-8 Microsorb
column, and chromatographed with 45% methanol in water-
10 mM ammonium formate (pH 2.0) as mobile phase. The
drug was detected by a fluorescence detector at 360/455 nm.
The peak area was integrated, and the lactone levels of drug
at different time points were determined.
9
The starting 9-nitrocamptothecin (0.38 g, 0.0010 mol) and
cyclohexanecarboxylic acid chloride were added to 25 mL of
acetone in a 100-mL round-bottomed flask equipped with a
magnetic stirrer. To this mixture was added 5 mL of pyridine
dropwise. The mixture was stirred at 40 ( 5 °C for 15 h. After
workup and separation same as for product 7e, the product
7
f was obtained as a yellow powder, yield 63%, purity 99%
1
(HPLC), mp 186 °C. H NMR (CDCl
3
): 0.80-1.15 (5H, m, C19-
Gen er a l P r oced u r e for Mea su r em en ts of in Vivo
Toxicity a n d An titu m or Activity. All the animal experi-
ments were performed on nude Swiss mice of the NIH high-
methyl, C26-methylene protons), 1.20-2.40 (10H, m, C18-,
C24-, C25-, C27-, and C28-methylene groups), 2.42-2.60 (1H,
m, C23-tertiary proton), 5.36 (2H, s, C5-methylene protons),
fertility strain. They were bred and raised in our laboratory
5
.37-5.72 (2H, dd, J ) 17.34, 17.34 Hz, C17-methylene
protons), 7.23 (1H, s, C14-H), 7.92 (1H, t (d + d), J ) 8.02,
.28 Hz, C11-H), 8.48 (1H, d, J ) 7.77 Hz, C10-H), 8.56 (1H,
under strict pathogen-free conditions.36 The tumors used for
the anticancer activity determination were human xenografts
originally obtained from human biopsies and then carried in
our laboratory by serial transplantation from nude mouse to
8
1
3
d, J ) 8.54 Hz, C12-H), 9.28 (1H, s, C7-H). C NMR (CDCl
3
):
7
3
9
1
.63 (C19), 25.23 (C25, C27), 26.65 (C24, C28), 28.58 (C26),
33
nude mouse. For the in vivo toxicity and antitumor activity
1.84 (C18), 42.49 (C23), 50.20 (C5), 66.98 (C17), 75.32 (C20),
6.87, 120.99, 121.54, 125.75, 127.42, 128.51, 131.54, 136.56,
44.96, 146.02, 146.23, 148.96, 153.83, 157.21 (C2, C3, C6-
determination, a tumor xenograft growing in a nude mouse,
approximately 1 cm3 in size, was excised sterilely, minced
finely with iridectomy scissors, and suspended in MEM tissue
culture medium at the ratio 1:10 (v/v). One-half of 1 mL of
this suspension, containing about 50 mg of tumor mince wet
weight, was inoculated subcutaneously on the upper half of
the dorsal thorax of the mouse. Groups of six animals were
used. The drug (ester) was finely suspended in cottonseed oil
and then injected into the stomach cavity of a mouse through
the anterior abdominal wall by using a 26-gauge needle. The
weekly schedule previously established for 9-nitrocamptothecin
injection was once a day, 5 days on, and 2 days off. This
schedule was employed throughout all the animal experiments.
Treatment was initiated when the tumor had reached a
C16, C16a), 167.23, 174.77 (C21, C22). MS m/e (relative
intensity): 503 (M , 20), 375 (M - cyclohexanecarboxylic acid,
1
+
3
00), 360 (M - cyclohexanecarboxylic acid - CH , 33), 347 (M
-
cyclohexanecarboxylic acid - CO, 90), 332 (M - cyclohexane-
carboxylic acid - CO - CH
3
, 26), 319 (10), 302 (10), 291 (4).
Precise mass (C27
25 3 7
H N O ): found, 503.170; required, 503.169.
In Vitr o Deter m in a tion of La cton e Levels in Hu m a n
a n d Mou se P la sm a for 3 a n d 6a . To 1 mL of plasma
(
human or mouse) was added 500 ng of drug in methanol. The
mixture was incubated at 37 °C for the following time points:
.5, 1, 2, 4, and 6 h; 100-µL aliquots were taken for analysis.
0
3
The percent lactone of camptothecin was determined by the
ratio of lactone to total drug at each time point. The total drug
at each time point was consistent with a relative average
volume of about 200 mm , i.e., well-vascularized, measurable,
and growing exponentially. Tumors growing in animals were
checked and measured with a caliper once a week.

Alkyl Esters of Camptothecin and 9-Nitrocamptothecin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 37
Ack n ow led gm en t. The authors wish to thank Mr.
Edward Ezell at the University of Texas Medical Branch
for NMR data and Baylor Medical College for mass data.
Supporting funds from the Stehlin Foundation for
Cancer Research and The Friends of The Stehlin
Foundation are gratefully acknowledged.
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K. W.; Pommier, Y. Plant Antitumor Agent. 29. Synthesis and
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(
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(
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(
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(
(
24) Wang, H.-K.; Liu, S.-Y.; Hwang, K.-W.; McPhail, A. T.; Lee, K.-
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I
treatment with camptothecin (NSC-100880): Correlation with
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