Design, synthesis, and antitumor activity of 4-halocolchicines and their pro-drugs activated by cathepsin B

Article abstract of DOI:10.1021/ml100287y

Novel colchicine derivatives possessing various substituents at the C4 position were prepared. Among them, 4-halo derivatives 3-6 were found to exhibit higher activity against cancer cell lines (A549, HT29, HCT116) as well as on mice transplanted with the HCT116 human colorectal carcinoma cell line than colchicine (1). Further, utilizing the 4-substituted colchicines, we prepared pro-drugs having a dipeptide side chain and demonstrated that these pro-drugs were activated by cathepsin B, an enzyme overexpressed in tumor cells, and exhibited selective toxicity to the tumor cells.

Full text of DOI:10.1021/ml100287y

LETTER
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Antitumor Activity of 4-Halocolchicines and
Their Pro-drugs Activated by Cathepsin B
Naoko Yasobu,† Mariko Kitajima,† Noriyuki Kogure,† Yoshiyuki Shishido,‡ Takeshi Matsuzaki,‡
Masato Nagaoka,‡ and Hiromitsu Takayama*,†
†Graduate School of Pharmaceutical Sciences, Chiba University
‡Yakult Central Institute for Microbiological Research
S Supporting Information
b
ABSTRACT: Novel colchicine derivatives possessing various substituents at the C4 position were
prepared. Among them, 4-halo derivatives 3-6 were found to exhibit higher activity against
cancer cell lines (A549, HT29, HCT116) as well as on mice transplanted with the HCT116
human colorectal carcinoma cell line than colchicine (1). Further, utilizing the 4-substituted
colchicines, we prepared pro-drugs having a dipeptide side chain and demonstrated that these pro-
drugs were activated by cathepsin B, an enzyme overexpressed in tumor cells, and exhibited
selective toxicity to the tumor cells.
KEYWORDS: Alkaloid, colchicine, structure modification, bioactivity, pro-drug, cathepsin B
’ INTRODUCTION
Colchicine (1) (see Figure 1) is a well-known bioactive alkaloid
that is used for the therapy of acute gout. It also acts as an antimitotic
agent by binding to tubulin.1 However, practical anticancer medi-
cines derived from colchicines have not been developed so far
because of their toxicity to normal cells. To solve this problem,
many groups have poured much effort into the development of
colchicine derivatives having high potency and reduced toxicity.2-11
Figure 1. Structures of colchicine (1) and gloriosamine A (2).
In our previous study of the alkaloidal constituents in the aerial
parts of Gloriosa rothschildiana, we isolated a new colchicinoid,
Table 1. IC50 Values of Colchicine Derivatives against the
gloriosamine A (2), which is the first example of natural colchi-
Tumor Cell Lines A549, HT29, and HCT116a
cinoid having a substituent at the C-4 position.12 Moreover, preli-
minary biological evaluation showed that gloriosamine A (2)
IC50 value (μM)
demonstrated significant cytotoxicity to A549 human lung
compd
A549
HT29
HCT116
adenocarcinoma cell line. This finding prompted us to develop
new colchicinoids with a substituent at the C-4 position as
effective antitumor drugs.
colchicine (1)
0.054
0.033
0.012
0.014
0.032
1.007
0.225
2.185
0.073
0.008
0.007
0.010
0.006
0.006
0.128
0.484
1.550
0.031
0.011
0.008
0.009
0.007
0.007
0.054
NT
3
4
5
6
7
8
10
11
’ RESULTS AND DISCUSSION
1. 4-Substituted Colchicinoids. First, we prepared C-4
halogensubstitutedcolchicine derivatives. As shown inScheme1,
4-fluorocolchicine (3), 4-chlorocolchicine (4), 4-bromocolchicine
(5), and 4-iodocolchicine (6) were synthesized in 15%, 82%, 99%,
and 99% yields from 1by treatment with NFSi, NCS, NBS, and NIS,
respectively. Structures of 4-halo derivatives were identified by
careful NMR analysis. In the case of compound 3, an aromatic
proton on C-4 in 1 disappeared and a quaternary carbon ascribable
to C-4 appeared at 149.1 ppm with a doublet (J = 242.7 Hz) split by
the installed fluorine atom.
NT
0.038
a NT, not tested; A549, human lung adenocarcinoma; HT29, human
colon adenocarcinoma; HCT116, human colorectal carcinoma.
SnCl4 in CH2Cl2 in a quantitative yield. The Baeyer-Villiger
oxidation of 7 with magnesium bis(monoperoxyphthalate)
(MMPP)14 in MeOH gave 4-hydroxycolchicine (8) in 37% yield.
In addition, five 4-substituted colchicine derivatives 7-11 were
prepared. According to the literature,13 4-formylcolchicine (7)
was obtained by reacting with Cl2CHOMe in the presence of
Received: December 3, 2010
Accepted: February 13, 2011
Published: March 04, 2011
r
2011 American Chemical Society
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Scheme 1. Syntheses of 3-6 from 1a
a Reagents: (a) for 3, NFSi, HCOOH; (b) for 4, NCS, AcOH; (c) for 5, NBS, AcOH; (d) for 6, NIS, AcOH; (e) SnCl4, Cl2CHOMe, CH2Cl2; (f) 80%
MMPP, MeOH; (g) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH/H2O; (h) DPPA, Et3N, THF/H2O; (i) CAN, TFAA, CH2Cl2. Abbreviations:
NFSi, N-fluorodisulfoneimide; NCS, N-chlorosuccinimide; NBS, N-bromosuccinimide; NIS, N-iodosuccinimide; MMPP, magnesium bis(mono-
peroxyphthalate); DPPA, diphenylphosphoryl azide; CAN, cerium(IV) ammonium nitrate; TFAA, trifluoroacetic anhydride.
Table 2. In Vivo Data of 3 and 4 in Mice Intraperitoneally Injected with HCT116 Human Colorectal Carcinoma Cell Line
schedule
on days
dose (mg/
kg/day)
total dose
(mg/kg)
tumor weight
(g, mean ( S.D.)
inhibition
rate (%)
compd
control
mortality
1-5 and 8-12
1, 3, 5
1-5 and 8-12
1-5 and 8-12
1-5 and 8-12
1.61 ( 0.34
1.08 ( 0.51
1.21 ( 0.44
0.71 ( 0.28
0.51 ( 0.08
0/5
2/5
0/5
0/5
0/5
colchicine (1)
1
0.25
1
3
32.8
25.0
55.6
68.4
3
3
4
2.5
10
50
5
Figure 2. Drug release by cathepsin B.
Further, colchicine-4-carboxylic acid (9) was obtained by the
Pinnick oxidation of 7 in 99% yield. The Curtius rearrangement
of 9 gave 4-aminocolchicine (10) in 36% yield.
Moreover, 4-nitrocolchicine (11) was obtained in 15% yield by
reacting with cerium(IV) ammonium nitrate (CAN)15 and TFAA.
The cytotoxicity of novel C-4 substituted colchicine derivatives
3-11 was evaluated using the A549 human lung adenocarcinoma
cell line, the HT29 human colon adenocarcinoma grade II cell line,
and the HCT116 human colorectal carcinoma cell line (Table 1).
Interestingly, 4-halogenated derivatives 3-6 more strongly inhibited
the examined tumor cell lines than 1, while derivative 7 with a carbon
unit and 8 and 10 with an electron donor group were less active. On
the other hand, nitro derivative 11 showed potent activity. To
understand the different activities among the 4-derivatives of colchi-
cines, we performed a docking study of the colchicines binding site of
the tubulin protein. For example, 4-fluoro derivative 3 showed a
similar binding mode to that of 1, in which interaction of the tricyclic
scaffold contributes the appearance of antitumor activity, while less
active compound 8 showed a different binding mode compared to
that of 1 (see the Supporting Information), resulting in lowering its
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activity. In this binding mode, interaction of the acetylamino group
on C-7 seems to be very important for exhibiting activity besides that
of the tricyclic scaffold.
Then, we examined the cell-growth inhibitory activities of 3
and 4 using mice transplanted with the HCT116 human color-
ectal carcinoma cell line by intraperitoneal injection (Table 2).
Colchicine (1) at the dose of 1 mg/(kg day) caused lethality in 2
of 5 mice due to its inherent toxicity, although it showed 32.8%
inhibition of tumor cell growth. Meanwhile, 4-fluorocolchicine
(3) administered at the dose of 0.25 mg/(kg day) or 1 mg/(kg
day) exhibited 25.0% or 55.6% inhibition of tumor cell growth,
respectively, without exerting a lethal effect on the mice.
4-Chlorocolchicine (4) also showed significant efficacy (68.4%
inhibition, 5 mg/(kg day), no mortality). These data demon-
strated that 4-fluoro- and 4-chlorocolchicines are capable of
reducing the intrinsic toxicity of colchicine (1) and show
potential for use as a clinical drug.
Scheme 2. Preparation of Deacetylcolchicines 15-17a
Table 3. IC50 Values of Deacetylated Colchicine Derivatives
Using Two Tumor Cell Lines, A549 and HT29. a
IC50 value (μM)
compd
A549
HT29
15
16
17
0.036
0.101
0.301
0.025
0.080
0.250
a Reagents: (a) Boc2O, DMAP, Et3N, MeCN; (b) NaOMe, MeOH; (c)
TFA. Abbreviations: Boc, t-butoxycarbonyl; DMAP, dimethylamino
pyridine; TFA, trifluoroacetic acid.
a A549, human lung adenocarcinoma; HT29, human colon adenocar-
cinoma.
Scheme 3. Synthesis of 12 or 13a
a Reagents: (a) AllocCl,1NNaOH,H2O; (b) CbzCl, 2 M NaOH; (c) HOSu, DCC, THF; (d) NaHCO3,H2O/DME (1:1.3); (e) p-aminobenzyl alcohol, HOBt,
EDCI, NMM, THF; (f) p-nitrophenylchloroformate, pyridine, THF; (g) Et3N, NMP; (h) Pd(PPh3)4, morpholine, THF; (i) Boc2O, DMAP, Et3N, MeCN; (j)
NaOMe, MeOH; (k) TFA. Abbreviations: Alloc, allyloxycarbonyl; Cbz, benzyloxycarbonyl; HOSu, N-hydroxysuccinimide; DCC, dicyclohexyl carbodiimide; DME,
1,2-dimethoxyethane; HOBt, 1-hydroxybenzotriazole; EDCI, 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride; NMM, N-methylmorpholine; THF,
tetrahydrofuran; NMP, N-methyl-2-pyrrolidinone; Boc, tert-butoxycarbonyl; DMAP, dimethylamino pyridine; TFA, trifluoroacetic acid.
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Figure 3. Cathepsin B mediated release of free drugs from pro-drug 12 (A) and fluorinated pro-drug 13 (B) at pH 5.0, 37 °C. A 20 μL mixture was
sampled at various time points and quenched with 20 μL of MeOH. The quenched reaction mixture was stored at 0 °C, and 10 μL of the mixture was
injected into the HPLC. (HPLC conditions: Inertsil ODS-2 4.6 mm  250 mm; solvent A, 0.1% (v/v) H3PO4; solvent B, CH3CN; 0 min B conc 15%; 30
min B conc 100%; column oven 40 °C; flow rate 0.5 mL/min; λ = 254 nm.)
2. Pro-drug Study. Utilizing novel 4-halocolchicinoids that
were found to show potent antitumor activities in in vitro and
in vivo experiments, we next designed safer and more effective
antitumor agents based on a tumor-activated pro-drug strategy.
Dubowchik et al. developed a novel pro-drug system16,17 that
involved the release of free drug by the action of cathepsin B,18 an
enzyme overexpressed in tumor cells.19 We applied the system to
4-halocolchicinoids incorporating the self-immolative p-aminoben-
zyloxycarbonyl (PABC) spacer20 between the active drug and the
lysosomally cleavable dipeptide Phe-Lys, as shown in Figure 2.
According to the idea above, we planned the synthesis of pro-
drugs 12-14. For that purpose, deacetylcolchicines 15-
17,10,21,22 which would be generated by the action of cathepsin
B, were initially prepared (Scheme 2) and their cytotoxicity
evaluated. Colchicines (1, 3, and 4) were respectively converted
into N-Boc derivatives 18-20 and then sequentially deprotected
to give amine derivatives 15-17.
Table 4. Biological Evaluation of Pro-drugs 12 and 13 and
Active Compounds 15 and 16 Using HT29 Human Colon
Adenocarcinoma Cell Line and Normal HUVEC
IC50 value (μM)
compd
HT29
HUVEC
selectivity (HUVEC/HT29)
12
13
15
16
0.044
0.091
0.025
0.080
0.075
0.151
0.034
0.075
1.7
1.7
1.4
0.9
to the enzyme solution (96 μL), and the concentration of the
substrate was adjusted to 0.04 mM. The mixture was stirred by
vortexing and left to stand at 37 °C, and the reaction course was
monitored by HPLC.
As a result, it was demonstrated that both pro-drugs 12 and 13
released free drugs 15 and 16 by the action of cathepsin B. As
shown in Figure 3, the amounts of pro-drugs decreased while
those of free drugs increased with time. We confirmed that pro-
drugs 12 and 13 remained stable (for almost 24 h) in the absence
of cathepsin B. HPLC analysis manifested the generation of the
dipeptide fragment Z-Phe-Lys with the appearance of the free
drugs, demonstrating a sequence of reaction involving the
cleavage of the dipeptide residue by the action of cathepsin B
and the fragmentation of the spacer moiety to generate the free
drugs, as expected in Figure 2.
The cytotoxicity of pro-drugs 12 and 13 and deacetyl deriva-
tives 15 and 16 to the HT29 human colon adenocarcinoma grade
II cell line and normal HUVEC was evaluated (Table 4) to
estimate their selectivity for tumor cells. As a result, we found that
pro-drugs 12 and 13 have approximately 2-fold higher selectivity
for the HT29 tumor cell line compared with normal HUVEC.
Further, the data concerning the fluoro-derivatives (13 and 16)
reveals that utilization of fluoro-derivatives would be more
efficient for the creation of safer antitumor medicines based on
colchicines.
Among them, deacetylcolchicine (15) and 4-fluorodeacetyl-
colchicine (16) remained potent compared with 17, as shown in
Table 3. Therefore, we planned to prepare pro-drugs 12 and 13
for further investigation.
To prepare the dipeptide region, the Nε position in L-lysine
(24) was protected with allylchloroformate to give 25 (61%
yield). On the other hand, the amino group in phenylalanine
(26) was protected by a Cbz group to afford 27 (99% yield)23,24
and then the carboxylic function was activated with N-hydro-
xysuccinimide to give 28 (81% yield).16,25,26 Condensation of 25
with 28 in the presence of NaHCO3 in H2O/DME afforded
dipeptide 2916 in 79% yield. The coupling reaction between 29
and p-aminobenzyl alcohol by using EDCI and HOBt in THF
gave 3016 in 96% yield. Carbonate 3116 was obtained in 95% yield
by reacting 30 with p-nitrophenylchloroformate in the presence
of pyridine in THF.
The coupling of dipeptide carbonate 31 with deacetylcolchi-
cine (15) or 4-fluorodeacetylcolchicine (16) in the presence of
Et3N in NMP afforded Z-Phe-Lys(alloc)-PABC-deacetylcolchi-
cine (32) in 80% yield or Z-Phe-Lys(alloc)-PABC-4-fluorodeace-
tylcolchicine (33) in a quantitative yield, respectively. Deprotection
of the alloc group on the primary amine in 32 or 33 by using
Pd(PPh3)4 and morpholine gave Z-Phe-Lys-PABC-deacetylcolchi-
cine (12) in 59% yield or Z-Phe-Lys-PABC-4-fluorodeacetylcolchi-
cine (13) in 55% yield, respectively (Scheme 3).
After obtaining pro-drugs 12 and 13, we next examined their
enzymatic conversion. As the enzyme, we chose bovine spleen
cathepsin B (EC 3.4.22.1, Mw ca. 40000) and prepared a stock
solution by dissolving the lyophilized solid. For each assay, a
1 mM DMSO solution of pro-drug compound (4 μM) was added
’ CONCLUSION
A series of novel colchicinoids having a substituent at the C-4
position were synthesized from the natural alkaloid colchicine
(1) and evaluated for their cytotoxicity to tumor cells, A549,
HT29, and HCT116. As a result, some 4-halogenated derivatives
exhibited more potent cytotoxicity to the tumor cells than 1. On
evaluation of cell-growth inhibitory activity using mice trans-
planted with the HCT116 human colorectal carcinoma cell line,
4-fluorocolchicine (3) and 4-chlorocolchicine (4) exhibited less
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LETTER
toxicity to mice and more potent cell-growth inhibitory activity
than 1. Further, we synthesized pro-drugs 12 and 13 having a
dipeptide side chain cleavable by cathepsin B, an enzyme over-
expressed in solid tumors, and we confirmed that 12 and 13 were
cleaved by cathepsin B to release the active drugs, resulting in an
approximately 2-fold higher selectivity for tumor cells than
normal cells.
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’ ASSOCIATED CONTENT
S
Supporting Information. Procedures for the preparation
b
and spectral data of compounds 3-14, 16, 17, 19 and 20, and
22-33, procedures for in vitro and in vivo assays, and the data of
the docking study. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: (81)43-290-2901. E-mail: takayama@p.chiba-u.ac.jp.
’ ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science and
Adaptable and Seamless Technology Transfer Program through
Target-Driven R&D (A-STEP, Exploratory Research) from
Japan Science and Technology Agency.
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