Synthesis and preliminary anticancer evaluation of 10-hydroxycamptothecin analogs

Article abstract of DOI:10.1248/bpb.b12-00145

We have synthesized new 10-hydroxycamptothecin (HCPT) analogs and evaluated their anticancer activity in cell culture and in experimental animal tumor model. Although the new analogs were less potent against L1210 leukemia cells in vitro, some of them wer

Full text of DOI:10.1248/bpb.b12-00145

August 2012
Regular Article
Biol. Pharm. Bull. 35(8) 1295–1299 (2012)
1295
Synthesis and Preliminary Anticancer Evaluation of
10-Hydroxycamptothecin Analogs
Pei Yu, Longfei Xia, Jing Zhao, Gaoxiao Zhang, Zaijun Zhang, Ming Lang, and Yuqiang Wang*
Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of
Traditional Chinese Medicine, Jinan University College of Pharmacy; Guangzhou 510632, China.
Received February 10, 2012; accepted April 17, 2012
We have synthesized new 10-hydroxycamptothecin (HCPT) analogs and evaluated their anticancer ac-
tivity in cell culture and in experimental animal tumor model. Although the new analogs were less potent
against L1210 leukemia cells in vitro, some of them were more efficacious against L1210 leukemia in vivo
compared to the parent HCPT.
Key words anticancer; 10-hydroxycamptothecin; analog
The natural product camptothecin (CPT) (Fig. 1) is a pen- cytotoxicity. This mechanism of action has significant implica-
tacyclic alkaloid, first isolated in 1966 from the extract of a tions for the use of these agents. It suggests that a prolonged
Chinese plant, Camptotheca accuminata, by Wall et al.1) CPT exposure of CPT to tumors is needed to ensure its optimal
and its synthetic analogues are among the most promising therapeutic efficacy.
new agents for the treatment of human cancers.2,3) They act
In initial clinical trials, CPT was limited by its poor solu-
by a unique mechanism inhibiting DNA topoismerase. DNA bility in physiologically compatible media. Early attempts to
topoisomerase I covalently binds to double-stranded DNA, form a water-soluble sodium salt of CPT by opening the lac-
forming a cleavable complex and producing a single-strand tone ring with sodium hydroxide led to poor antitumor activ-
break.4) This cleavable complex facilitates the relaxation of ity.9–11) Later research found that the closed lactone form is an
torsional strain of the supercoiled DNA. Once the torsional absolute requirement for antitumor activity.12) Intensive efforts
strain is relieved, the enzyme rejoins the cleaved strands of in medicinal chemistry over the past several decades have
DNA and dissociates from the relaxed double helix.5) CPT provided a large number of camptothecin analogues, of which
binds to and stabilizes the cleavable complex and inhibits topotecan and irinotecan are among those clinically approved
the religation of DNA, leading to the accumulation of DNA for the treatment of cancers (Fig. 1).
single-stranded breaks.6,7) The single-stranded breaks are not
In irinotecan, a piperidino–piperidino carbonyl moiety was
in themselves toxic to cell, because they readily religate upon introduced at the 10-hydroxy position which greatly increases
drug removal; however, collision of the DNA replication fork the compound water-solubility. Irinotecan is converted in
with the drug–enzyme–DNA complex generates an irrevers- vivo to its much higher active metabolite 7-ethyl-10-hydroxy
ible double-strand break that ultimately leads to cell death.8) camptothecin by carboxylesterases.13) Structure–activity rela-
CPT is S phase-specific, because ongoing DNA synthesis is tionship (SAR) studies suggested that the intact lactone ring
needed to induce the above sequence of events leading to E of CPT is the most critical structural feature accounting for
Fig. 1. Structures of Camptothecin, 10-Hydroxycamptothecin, Irinotecan, Topotecan, PP-HCPT and DHA-HCPT
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: yuqiangwang2001@yahoo.com
© 2012 The Pharmaceutical Society of Japan

1296
Vol. 35, No. 8
Fig. 2. Chemical Structures of New HCPT Analogs
Chart 1. Synthesis of Compounds 1–7
its antitumor activity. For these reasons, extensive efforts are solubility problems is to introduce the alkanoic acid esters
made to modify at CPT’s 10 and 20th positions to reduce its and nitrogen-based esters into the molecule of PP-HCPT at its
toxicity and increase its anticancer activity. A member of the 20-hydroxyl via esterification produced 10-O and 20-O-dou-
CPT family, 10-hydroxycamptothecin (HCPT), is more potent ble-acylated HCPT derivatives (Fig. 2). The new compounds
and less toxic than CPT.3) We herein report synthesis of a se- have enhanced water solubility and as improved stability of
ries of new derivatives of HCPT with esterifications on both the lactone ring and showed significant antitumor activity and
the 10 and 20th-hydroxy groups. Their antitumor activity was low toxicity in vivo.
evaluated against L1210 leukemia in vitro and in experimental
animals.
Inspired by our previous work,14) modification of 10-hy-
droxy with a piperazine moiety in HCPT was used, the
MATERIALS AND METHODS
Chemistry All reagent and solvents were purchased
tertiary nitrogen of piperazine (PP-HCPT) is expected to be from commercial sources. Further purification and drying
protonated at physiologic pH, leading to increased aqueous by standard methods were employed when necessary. Melt-
solubility. In addition, the piperazine and HCPT were linked ing points were measured using a melting point detector and
1
through a carbamoyl bond, which should provide certain sta- are uncorrected. H-NMR spectra were recorded at ambient
bility to cleavage by carboxylate esterase. However, this car- temperature on an NT-400 spectrometer. Mass spectra (elec-
bamoyl bond will be cleaved by carboxylate esterase to release trospray ionization, EI) were recorded using a Micromass
HCPT, leading to the killing of cancer cells.
Q-Tof 1 mass spectrometer. Atlantic Microlab, Inc., Norcross,
The 20S-hydroxyl group is thought to participate in the GA, U.S.A. performed the elemental analyses, and the results
enhanced rate of lactone hydrolysis of HCPT at neutral pH were within 0.4% of the theoretical values unless otherwise
by shifting the lactone-carboxylate equilibrium in favor of the noted. Analytical thin-layer chromatography was performed
carboxylate form. There is an intramolecular hydrogen bond- on silica-coated plastic plates (silica gel 60 F-254, Merck)
ing in the E-ring of HCPT molecule, it would not only activate and visualized under UV light. Preparative separations were
the lactone but also diminish the interaction with the enzyme. performed by flash chromatography on silica gel (Qinghdao
Esterification of 20S-hydroxyl group blocks this process. Haiyang, 200–300 mesh).
Although 20-O-acylated HCPTs possess no intrinsic Topo I
The general synthetic method for new HCPT analogs 1–7 is
(topoisomera- se I) inhibitory activity, they can function as shown in Chart 1.
prodrugs to release the HCPTs in vivo, and improve solubil-
10-[4-(1-Piperidino)-1-piperidino]carbonyloxycampto-
ity, pharmacokinetics character and toxicity of HCPTs.15) thecin (8) Compound 8 was synthesized by treatment of the
Therefore, the esterification of 20-hydroxyl group can either precursor carbonate of HCPT with 4-piperidinopiperidine as
eliminate the intramolecular hydrogen bonding and increase reported in our previous work.14)
the steric hindrance of carbonyl group of E-ring, so lactone
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20-acetyl-
camptothecin (1) PP-HCPT (100mg, 0.18mmol) was dis-
ring stability was improved in vivo.16)
Our approach to the above chemical stability/water solved in tetrahydrofuran (THF) (20mL), and acetic anhydride

August 2012
1297
(0.1mL) was added, followed by the addition of 4-dimeth- J=9.2Hz), 8.68 (1H, s). MS m/z: 773.5 (M++H). Anal. Calcd
ylamino pyridine (DMAP) (12mg, 0.10mmol). The reaction for C39H39BrN4O8·H2O: C, 59.32; H, 5.23; N, 7.09. Found: C,
was allowed to proceed overnight at room temperature. Sol- 59.14; H, 5.06; N, 7.31.
vent was removed under vacuo. The product was purified by
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20-(N-
column chromatography, eluting with trichloromethane and BOC)-glycinylcamptothecin (6) N-BOC-glycine (53mg,
methanol (20:1, v/v) to afford 1 as a yellow solid (38mg, 0.3mmol) in THF (10mL) was treated with triethylamine
1
35%), mp 188–189.3°C. H-NMR (DMSO-d6) δ: 0.91 (3H, t, (0.25mL, 1.80mmol) and ethyl chloroformate (0.13mL), and
J=7.4Hz), 1.24 (2H, s), 1.50 (6H, s), 1.91 (1H, s), 2.14 (4H, the reaction mixture was stirred at room temperature for 2h.
m), 2.22 (3H, s), 3.28 (4H, s), 4.27 (2H, m), 4.43 (2H, m), 5.31 Then compound 8 (50mg, 0.09mmol) and DMAP (10mg,
(2H, s), 5.48 (2H, s), 7.06 (1H, s), 7.68 (1H, dd, J=2.5, 6.7Hz), 0.08mmol) were added. The reaction was allowed to proceed
7.91 (1H, d, J=2.6Hz), 8.18 (1H, d, J=9.2Hz), 8.66 (1H, s). at room temperature for 24h. Solvent was removed in vacuo,
MS m/z: 602.0 (M++H). Anal. Calcd for C33H36N4O7·2H2O: C, and the product was purified by column chromatography,
62.25; H, 6.33; N, 8.80. Found: C, 62.57; H, 6.51; N, 8.57.
Compounds 2–4 were synthesized by using the similar syn- 6 as a yellow solid (15mg, 23%). H-NMR (MeOD) δ: 1.01
thetic procedure as for compound 1.
eluting with chloroform and methanol (10:1, v/v) to afford
1
(3H, t, J=7.4Hz), 1.43 (2H, m), 1.53 (9H, m), 1.68 (6H, m),
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20-pro- 1.96 (4H, m), 2.70 (4H, m), 2.99 (1H, s), 3.08 (2H, s), 4.35
1
pionylcamptothecin (2) Yellow solid, yield: 21%. H-NMR (4H, m), 5.27 (2H, s), 5.37 (1H, d, J=16.3Hz), 5.56 (1H, d,
(DMSO-d6) δ: 0.92 (3H, t, J=7.4Hz), 0.99 (2H, m), 1.06 (3H, J=16.3Hz), 7.61 (2H, m), 7.75 (1H, d, J=2.4Hz), 8.13 (1H, d,
t, J=7.5Hz), 1.50 (6H, s), 1.78 (1H, m), 2.20 (4H, m), 2.53 (2H, J=9.2Hz), 8.53 (1H, s). MS m/z: 716.5 (M++H). Anal. Calcd
s), 3.17 (4H, m), 4.08 (2H, m), 4.09 (2H, m), 5.30 (2H, s), 5.48 for C38H45N5O9·2H2O: C, 60.71; H, 6.57; N, 9.32. Found: C,
(2H, s), 7.04 (1H, s), 7.67 (1H, dd, J=2.5, 6.6Hz), 7.90 (1H, d, 60.35; H, 6.81; N, 9.05.
J=2.6Hz), 8.16 (1H, d, J=9.2Hz), 8.65 (1H, s). MS m/z: 615.5
(M++H). Anal. Calcd for C34H38N4O9·2H2O: C, 62.76; H, 6.51; glycinylcamptothecin·HCl (7) Compound
N, 8.61. Found: C, 62.60; H, 6.24; N, 8.27. 0.028mmol) was dissolved in a mixture of THF and ethyl ace-
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20-
6
(20mg,
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20- tate. Then anhydrous HCl in ethyl acetate was added. The pre-
butyrylcamptothecin (3) Yellow solid, yield: 18%, mp cipitate was filtered, and the solid was dried to afford 7 as a
1
1
198.5–199.3°C. H-NMR (DMSO-d6) δ: 0.92 (6H, t, J=7.3Hz), salt (13mg, 71%). H-NMR (MeOD) δ: 1.01 (3H, t, J=7.3Hz),
1.41 (2H, t, J=2.4Hz), 1.52 (4H, m), 1.59 (2H, q), 1.82 (1H, 1.22 (1H, s), 1.91 (6H, m), 1.96 (2H, m), 2.01 (2H, s), 3.10 (4H,
m), 2.15 (4H, m), 2.47 (2H, s), 3.17 (2H, m), 3.31 (4H, m), 4.08 m), 3.26 (4H, s), 3.58 (2H, m), 4.15 (4H, m), 5.25 (2H, m), 5.52
(2H, s), 4.26 (2H, s), 5.31 (2H, s), 5.49 (2H, s), 7.04 (1H, s), (2H, m), 6.99 (1H, d, J=7.6Hz), 7.60 (1H, m), 7.79 (1H, d,
7.67 (1H, dd, J=2.3, 6.9Hz), 7.91 (1H, d, J=2.3Hz), 8.16 (1H, J=2.3Hz), 8.11 (1H, d, J=8.9Hz), 8.63 (1H, s). MS m/z: 616.5
d, J=9.1Hz), 8.66 (1H, s). MS m/z: 629.9 (M++H). Anal. Calcd (M++H). Anal. Calcd for C33H37N5O7·3H2O·HCl: C, 56.13; H,
for C35H40N4O7·2H2O: C, 63.24; H, 6.67; N, 8.43. Found: C, 6.28; N, 9.92. Found: C, 56.46; H, 6.19; N, 9.57.
63.44; H, 6.43; N, 8.65.
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20-iso-
PHARMACOLOGY
butyrylcamptothecin (4) Yellow solid, yield: 44%, mp
1
183.7–185.0°C. H-NMR (DMSO-d6) δ: 0.93 (3H, t, J=7.4Hz),
For the cytotoxicity study, drugs were dissolved in dimethyl
1.06 (2H, d, J=7.0Hz), 1.17 (6H, dd, J=2.1, 4.9Hz), 1.50 (6H, sulfoxide (DMSO) to provide a stock solution of 1mg/mL,
s), 1.80 (1H, m), 2.12 (4H, m), 2.79 (1H, m), 3.17 (4H, m), which were stored at −20°C. For each experiment, drug so-
4.11 (2H, m), 4.25 (2H, m), 5.30 (2H, s), 5.48 (2H, dd, J=2, lutions were freshly prepared from the stock solution by the
16.9Hz), 7.01 (1H, s), 7.66 (1H, dd, J=2.6, 6.6Hz), 7.91 (1H, d, addition of sterile water to afford concentrations suitable for
J=2.6Hz), 8.16 (1H, d, J=9.2Hz), 8.65 (1H, s). MS m/z: 630.1 the experiment. For animal experiments, drugs were first dis-
(M++H). Anal. Calcd for C35H40N4O7·2H2O: C, 63.24; H, 6.67; solved in DMSO and Tween 80 was then added. The solution
N, 8.43. Found: C, 63.53; H, 6.81; N, 8.12.
was then diluted with sterile water.
10-[4-(1-Piperidino)-1-piperidino]carbonyloxy-20-(4-bro-
L1210 mouse leukemia cells were cultured in RPMI-1640
mophenoxy)-acetoxycamptothecin (5) PP-HCPT (120mg, plus 10% fetal bovine serum (FBS) with the addition of 100U/
0.22mmol) in THF (30mL) was treated with 4-bromophenoxy mL penicillin and 100µg/mL streptomycin. Cytotoxic effects
acetic acid (120mg, 0.52mmol) in the presence of a coupling of drugs were measured by inhibition of DNA synthesis.
reagent 1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide hy- L1210 leukemia cells in RPMI-1640 plus 10% FBS medium
drochloride (EDCI, 240mg, 1.25mmol) and a catalyst 4-di- were seeded at 5×104 cells/well in a 96-well plate. Drugs
methylamino pyridine (DMAP, 20mg, 0.16mmol), and the (10µL) at increasing concentrations were added to each well,
reaction mixture was stirred at room temperature for 12h. and the total volume was adjusted to 0.1mL/well using the
Solvent was removed in vacuo and the product was puri- same medium. The plate was incubated for 24h at 37°C fol-
fied by column chromatography, eluting with chloroform and lowed by the addition of drugs. The plate was incubated for
methanol (20:1, v/v) to afford 5 as a yellow solid (55mg, another 48h. The cells were harvested and radioactivity was
1
40%), mp 196.4–197.8°C. H-NMR (DMSO-d6) δ: 0.93 (3H, counted using the Packard Matrix 96 beta counter. The per-
t, J=7.4Hz), 1.08 (2H, t, J=7.2Hz), 1.64 (6H, s), 1.95 (4H, s), centage growth inhibition was calculated as follows:
2.15 (1H, m), 3.16 (4H, m), 4.13 (2H, m), 4.35 (2H, m), 5.05
(2H, m), 5.31 (2H, s), 5.52 (2H, d, J=3.2Hz), 6.86 (2H, dd,
J=2, 6.8Hz), 6.98 (2H, dd, J=2.4, 7.2Hz), 7.21 (1H, s), 7.72
(1H, dd, J=2.8, 6.4Hz), 7.94 (1H, d, J=2.4Hz), 8.23 (1H, d,
%growth inhibition
[(total cpm－experimental cpm)]
=
×100
total cpm

1298
Vol. 35, No. 8
For all experiments, tumors were implanted on day 0 to leukemia in mice, and the result was shown Table 2. When
male BDF1 mice, weighing 18–22g (6/group for the L1210 one dose was administered, PP-HCPT-PA showed the best
model), and drugs (0.1mL) were administered intraperitone- efficacy and was more efficacious than its parent HCPT.
ally (i.p.) on the dates as indicated in the experiment. Animals For example, at the optimal dose (100mg/kg), PP-HCPT-PA
were monitored and weighed daily. L1210 leukemia cells (105 had a 128% increase in life span (ILS) while HCPT (at an
cells/mouse, 0.1mL) were inoculated i.p. Antitumor activity optimal dose of 15mg/kg) had an ILS of 71%. Furthermore,
was determined by comparing the median survival time of the PP-HCPT-PA had a greatly decreased toxicity compared to
treated groups (T) with that of the control group (C), and was HCPT (bodyweight loss of 13% for PP-HCPT-PA at a dose of
expressed as a percentage of ILS [increase of life span, where 200mg/kg while 14% for HCPT at a dose of 15mg/kg). Ex-
%ILS=(T/C−1)×100]. The calculations considered dying ani- cept for PP-HCPT-ACO, all new compounds showed a higher
mals only. The median number of days in the untreated group therapeutic efficacy than HCPT. It is interesting to note that
of mice (given the vehicle only) died was 7.5.
PP-HCPT-ACO was the least potent new compound against
L1210 leukemia cells in vitro (Table 1).
After testing the compounds at the same dose levels, we
then evaluated PP-HCPT-PA, the one with the best therapeu-
RESULTS AND DISCUSSION
The new compounds were first tested in vitro against tic index, the free drug HCPT and PP-HCPT at an equimolar
L1210 leukemia cells, and the results are shown in Table 1. In dose. The result was shown in Table 3. PP-HCPT produced a
agreement with what we have reported previously that add- 79% ILS, which is much better than that of its parent HCPT
ing a 4-(1-piperidino)-1-piperidino moiety to the 10-hydroxy with an ILS of 64%. In addition, the former was much less
increased HCPT’s IC50 from 1.15µM to 134µM (PP-HCPT).14) toxic. This result is in agreement with what we had previously
This data are also in agreement with what was reported for reported,14) and once again demonstrated that modifying the
irinotecan, where the 4-(1-piperidino)-1-piperidino moiety sig- 10-hydroxyl group with a 4-(1-piperidino)-1-piperidino moiety
nificantly increased the free drug SN-38’s in vitro potency.17) improved therapeutic efficacy. Modification of the 20-hydroxyl
When the 20-hydroxy of PP-HCPT was converted to its acetic group of PP-HCPT with a propionyl moiety further improved
ester, there were little changes in their potencies (IC50 values the therapeutic index, increasing the ILS from 79% to 114%.
of 134µM for PP-HCPT and 146µM for PP-HCPT-ACO, com- These results demonstrate that di-esterification of the 10- and
pound 1). Replacing the 20-hydroxy of PP-HCPT with a bu-
tyryl (compound 3), isobutyryl (compound 4) or BOC-glycinyl
(compound 6) had no significant effects on the IC50 values
Table 2. Antitumor Activity against L1210 Leukemia in Mice*
either. What was surprised was that replacing the 20-hydroxy
%Weight
Drug
Dose (mg/kg)
%ILS
changea)
of PP-HCPT with a propionyl group significantly increased
the compound’s potency from 134µM for PP-HCPT to 25.6µM
for PP-HCPT-PA (compound 2). What was more surprised
was that replacing the 20-hydroxy of PP-HCPT with a bulky
OCOCH2OPhBr (compound 5) also significantly increased the
compound’s potency from 134µM for PP-HCPT to 35µM for
PP-HCPT-CPA. The reasons for these results were not under-
stood currently. The rationale to place a glycine moiety at the
20-hydroxy position was to further increase water-solubility,
and the resulting compound PP-HCPT-GL·HCl (compound
7) had an increased potency compared with its precursor PP-
HCPT. The data in Table 1 did not reveal any obvious SAR
when the 20-hydroxy group was replaced by different sub-
stituents.
HCPT
15
200
100
50
−14
−13
−5
−3
−8
−6
−5
−9
−6
−4
−9
−7
−4
−12
−9
−4
71
82
128
118
67
53
40
93
80
67
93
80
67
93
93
80
PP-HCPT-PA
PP-HCPT-ACO
PP-HCPT-BUA
200
100
50
200
100
50
PP-HCPT- iso-
BUA
200
100
50
Next, we tested selected new compounds against L1210
PP-HCPT-CPA
200
100
50
Table 1. Antitumor Activity against L1210 Leukemia in Vitro*
*Male C57BL mice (6/group) were injected i.p. with 105 cells on day 0. Drugs
were administered i.p. on day 1. a) Group bodyweight change was between day 0 and
the day at which time the group of mice had the lightest weight. The median number
of days of survival of the untreated mice was 7.5.
Drug
IC50 (µM)
HCPT
1.15 0.61
134 36
PP-HCPT
PP-HCPT-ACO (1)
PP-HCPT-PA (2)
146 20.1
25.6 16.7
113 12.5
93 12.3
Table 3. Antitumor Activity against L1210 Leukemia in Mice*
Drug
HCPT
Dose (mg/kg)
%Weight changea)
%ILS
PP-HCPT-BUA (3)
PP-HCPT-isoBUA (4)
PP-HCPT-CPA (5)
PP-HCPT-GL-BOC (6)
PP-HCPT-GL·HCl (7)
10
91
−10
−4
64
79
35.24 10.72
116 13
PP-HCPT
PP-HCPT-PA
100
−3
114
55.7 27.6
*Male C57BL mice (6/group) were injected i.p. with 105 cells on day 0. Drugs
were administered i.p. on day 1. a)Group bodyweight change was between day 0 and
the day at which time the group of mice had the lightest weight. The median number
of days of survival of the untreated mice was 7.5.
*Cytotoxicity was measured in a 48-h proliferation assay. The results were re-
ported as the minimal drug concentration that inhibits uptake of MTT assay by 50%
and were the mean values of at least three experiments.

August 2012
1299
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20-hydroxy positions of HCPT is a valid strategy to increase
its therapeutic efficacy. It will be very interesting to find if
this strategy can be used to improve the therapeutic efficacy
of irinotecan, the first CPT analog used to treat cancer.
In conclusion, we have found that the therapeutic efficacy
of HCPT can be first improved by modifying the 10-hydroxy
group with a 4-(1-piperidino)-1-piperidino moiety, and then
further improved by modifying its 20-hydroxy group with a
propionyl moiety. That is PP-HCPT-PA (compound 2) showed
the best efficacy and was more efficacious than its parent
HCPT. The novel PP-HCPT-PA merits further investigation as
an anticancer agent.
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