New synthetic thiocolchicine derivatives as low-toxic anticancer agents

Article abstract of DOI:10.1002/ardp.200500148

New thiocolchicine derivatives (1-8) were designed as less toxic anticancer agents possessing the powerful anticancer activity of colchicine. The synthesis and biological evaluation of these compounds were described. As a preliminary result of biological in vitro investigation, compounds 1, 6, and 7 showed lower toxicities than that of colchicine in combination with potent anticancer activities.

Full text of DOI:10.1002/ardp.200500148

582
DOI 10.1002/ardp.200500148
Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
New Synthetic Thiocolchicine Derivatives as Low-
toxic Anticancer Agents
a
a
a
b
Sun-Hee Lee , Sun-Kyoung Park , Jeong-Mi Kim , Myung-Hwa Kim , Kwang-Hee
b
b
c
c
a
Kim , Kwang-Woo Chun , Kyung-Hea Cho , Ji-Youn Youn , Sung Keon Namgoong
a
Department of Chemistry, Seoul Women’s University, Seoul, Korea
R&D Center, Jeil Pharmaceutical Co., Ltd., Youngin-City, Kyunggi-Do, Korea
Department of Biotechnology, Seoul Women’s University, Seoul, Korea
b
c
New thiocolchicine derivatives (1Ϫ8) were designed as less toxic anticancer agents possessing the
powerful anticancer activity of colchicine. The synthesis and biological evaluation of these compounds
were described. As a preliminary result of biological in vitro investigation, compounds 1, 6, and 7
showed lower toxicities than that of colchicine in combination with potent anticancer activities.
Keywords: Colchicine; Thiocolchicine; Synthetic derivatives; Toxicity; Anticancer activity
Received: May 31, 2005; Accepted: August 25, 2005
Introduction
B with (S)-configuration is also one of the crucial factors
to decide molecule’s anticancer activity [9].
(
7S)-(Ϫ)-Colchicine (Figure 1), a naturally occurring
Colchicum alkaloid, is a powerful antimitotic agent that
interferes with microtubules both in vitro and in vivo,
thereby causing cells to accumulate in apparent mitotic
arrest during cell cycle [1, 2]. However, its effects against
tumors were only observed at toxic or near toxic levels [1,
A keystone of this work was to develop less toxic thio-
colchicine derivatives possessing the potent anticancer ac-
tivity of colchicine. The designed compounds 1Ϫ8 shown in
Figure 2 are built with two moieties in the form of an amide
bond; one is deacetylthiocolchicine (DATC) and the others
are mainly natural or synthetic carboxylic acids. DATC is
an amine that is deacetylated from thiocolchicine at C-7
position. The counterparts of compound 1Ϫ8 are mostly
the nontoxic acids, which show diverse medicinal effects.
Perillic acid is an antitumor agent inhibiting protein prenyl-
transferases [10, 11]. The porphyrins have been well known
to show selective affinity to tumor cells and applied for pho-
todynamic cancer therapy as a photo sensitizer [12, 13].
These properties lead porphyrins to work as drug carrier
molecule to tumor cells. Receptors for folic acid are fre-
quently overexpressed on epithelial cancer cells. Therefore,
drugs including a folic acid moiety might be effective for
folate-mediated tumor cell targeting [14]. Diclofenac, an
anti-inflammatory drug, has the unique structure linking
two aromatic parts to an amino group. This structural fea-
ture may affect the anticancer activity of thiocolchicine
through introduction of Diclofenac into DATC. DHA (do-
cosahexanoic acid) displays elements of apparent synergism
when it is treated with the very toxic anti-neoplastic drug,
Methotrexate (MTX), currently in use in cancer chemo-
therapy [15].
3]. The high toxicity of this alkaloid has precluded its use
in cancer chemotherapy.
Extensive structural modifications have been performed on
three fused rings of colchicine to discover more effective and
less toxic analogues of colchicine [4Ϫ6]. Among a number
of synthetic colchicine analogues, thiocolchicine (Figure 1)
that has a C-10 methylthio group of the tropolone ring C
instead of a methoxy group is highly stable in acidic hydro-
lytic condition without racemization at the C-7 position [7].
Anticancer activities for most of its analogues are compar-
able to those of colchicines [8]. The C-7 amide group of ring
Figure 1. The structures of (-)-colchicine and thiocolchicine.
This combined therapy holds a great promise as a highly
effective cytotoxic agent that can be targeted to cancer cells
without affecting the normal cell viability. -gluconic acid
derived from -glucono-1,5-lactone is a water-soluble com-
pound and has many stereocenters in its structure. Of par-
ticular interest is the relationship between the anticancer
Correspondence: Sung Keon Namgoong, Department of Chemistry,
Seoul Women’s University, 126, Kongnung 2-dong, Nowon-gu,
Seoul 139-774, Korea. Phone: ϩ82 2 970-5658, Fax: ϩ82 2 970-
5
972, e-mail: sknam@swu.ac.kr

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
Thiocolchicine derivatives as anticancer agents
583
Figure 2. The designed target molecules.
effect and the configuration of its water-soluble colchicine
derivative. Nontoxic ferulic acid (trans-4-hydroxy-3-meth-
oxycinnamic acid) and its derivatives suppress the formation
of colonic tumor marker and skin tumor promotion [16].
Phytosphigosine and its analogues induce apoptotic cell de-
ath and show various interesting antitumor effects as pro-
tein kinase C inhibitors with low toxicity [17]. In addition,
combination of cisplatin with safingol, an analogue of phy-
tosphigosine, led to a significant decrease of cisplatin dose
in most cancer cells [18].
those of colchicine. Herein, we describe the synthesis and
preliminary data for both anticancer activities and toxicities
of thiocolchicine derivatives 1Ϫ8.
Results and discussion
Synthesis of the designed target molecule was achieved as
shown in Scheme 1 and 2. The first step was the preparation
of DATC (deacetylthiocolchicine) from the starting mate-
rial, colchicines, according to the reported procedure [7].
The compounds 1Ϫ5 and 7 were prepared from the coup-
ling reactions between DATC and various carboxylic acids
in a single step as shown in Scheme 1. The coupling reagent
was water-soluble EDC·HCl (1-(3-dimethylaminopropyl)-
Synthesis of the designed target molecules coupled to
DATC with the carboxylic acid moiety as an amide bond
was carried out to investigate their biological effects. It is
also interesting to compare their biological effects with

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

584
Namgoong et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
3
-ethylcarbodiimide hydrochloride) in the reaction con-
because it is highly polar and very insoluble in most organic
solvents. Moreover, it decomposed under the conditions for
chromatographic separation.
ditions [19]. In general, the yields of the coupling reactions
to synthesize thiocolchicine derivatives 1Ϫ5 and 7 ranged
from 70Ϫ80%. The coupling reaction of DATC with hema-
toporphyrin IX led to compound 2 in form of a dimer. On
the other hand, its monomeric product could not be isolated
Selective introduction of the DATC moiety at the β-position
of folic acid was successful to generate compound 3 through
(
2
(
2
1) NaSCH
: DATC (2.1 eq.), hematoporphyrin IX (1.0 eq.), CH
6) For 4: Diclofenac, CH Cl , 20 h, 70%; (7) For 5: DHA (docosahexanoic acid), CH
4 h, 70%; (8) For 7: ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid), 4-dimethylaminopyridine, CH
3
, H
2
O, 4 days, 63%; (2) 2 N HCl, MeOH, 90°C, 4 days, 66%; (3) For 1: (S)-perillic acid, CH
Cl , 24 h, 73%; (5) For 3: folic acid dihydrate, pyridine, DMSO, 24 h, 69%;
Cl , 2,6-Di-tert-butyl-p-cresol, CH Cl
Cl , 2,6-Di-tert-butyl-p-
2 2
Cl , 27 h, 82%; (4) For
2
2
2
2
2
2
2
2
,
2
2
cresol, 12 h, 78%; (9) For 6: DATC (1.2 eq.), -Glucono-1,5-lactone (1.0 eq.) DMF, 24 h, 80%.
Scheme 1. Synthesis of DATC and the thiocolchicine derivatives 1Ϫ7.
(
(
1
1) Di-tert-butyl dicarbonate, K
3) Pyridinium dichromate (PDC), DMF, 24 h, 79%; (4) DATC, EDC HCl, CH
h, 70%.
2 3 3
CO , CH OH, 1 h, 95%; (2) p-Toluenesulfonic acid, Acetone/2,2-Dimethoxypropane, 1:1, 5 h, 79%;
.
2
2 2 2
Cl , 48 h, 68%; (5) p-Toluenesulfonic acid, CH Cl ,
Scheme 2. Synthesis of the thiocolchicine derivative 8.

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
Thiocolchicine derivatives as anticancer agents
585
a normal coupling reaction. In case of light-sensitive reac-
tions, such as the formation reactions of compounds 5 and
rials, the compounds 2Ϫ5 and 8 were significantly less cyto-
toxic against all the tumor cells tested. Especially, the com-
pound 3 was almost inactive against the ovarian cancer cell
line SK-OV-3 as well as the melanoma cell line SK-MEL-2.
In contrast, compounds 1, 6, and 7 showed potent growth
inhibitory effects as does Colchicine with IC50 values of
5Ϫ30 nM. In general, these compounds were considerably
more active toward MCF-7 and slightly more active toward
the lung cancer cell line A549 than colchicine. On the other
hand, their cytotoxic effect against both SK-OV-3 and SK-
MEL-2 was slightly weaker than the one of colchicines, in
the range of about two- to maximum three-fold, except in
7
, use of an antioxidant, BHT (2,6-di-tert-butyl-p-cresol),
was required. Application of the above coupling-reaction
condition to the synthesis of 6 proved troublesome due to
its purification problem. Compound 6 was only obtained
from the nucleophilic substitution reaction of -glucono-
1
,5-lactone with excess DATC, instead of the carboxylic
acid.
The synthetic strategy for compound 8 is shown in Scheme
2
. The synthesis of compound 8 was accomplished in five
steps from phytosphingosine via the key intermediate 8b in
8% overall yield. Phytosphingosine was converted into the
2
key intermediate 8b in 59% overall yield via a modified syn-
thesis we previously described [20]. The synthesis of the acid
Table 1. Inhibition of in vitro tumor cell growth†,‡ by the refe-
rence materials and compounds 1Ϫ8.
8
b is based on proper protection/deprotection of amino al-
cohols at the C-2, 3, 4 positions of phytosphingosine. Our
previous method required first protection of C-1 primary
alcohol with silylating agent and deprotection here later on,
but the modified procedure eliminated this laborious syn-
thetic steps. Boc (tert-butyloxycarbonyl) protection reaction
at C-2 amine functionality and subsequent acetal formation
reaction at C-3, 4-diol of phytosphingosine generated the
protected alcohol 8a as a major product even though the
other acetal formation at C-1, 3 positions occurred. The
compound 8a underwent oxidation with PDC (pyridinium
dichromate) to give the unstable carboxylic acid 8b. Next,
the coupling reaction between DATC and the unpurified
acid 8b, followed by the selective deprotection reaction of
§
IC50 [nM]
Compounds A549‡ SK-OV-3‡ SK-MEL-2ε HCT-15ε MCF-7ε
Doxorubicin 14.7
؊)-Colchicine 21.0
34.2
18.0
17.2
2863
ND††
1500
2630
29.8
24.8
676
11.7
6.0
16.4
9.0
8.10
4838
1162
306
41.9
> 300#
5.2
3215
697
296
494
7.8
5.6
(
1
2
3
4
5
6
7
8
7.5
1684
4955
520
917
14.2
5.8
17.9
2837
ND
1050
2005
12.3
16.6
130
392
18.7
10.8
204
228
303
8
c with p-toluenesulfonic acid in non-aqueous media al-
†
‡
§
lowed the separation of the thiocolchicine derivative 8.
However, compound 8 did not undergo conversion to the
expected amino alcohols as the fully deprotected form un-
der all-aqueous acidic conditions. Instead, hydrolysis at C-7
amide functionality of 8 largely occurred rather than further
deprotection at the C-3, 4 acetal of phytoshingosine moiey.
Data obtained from in vitro disease-oriented human tumor cell
screen.
A549, lung cancer; SK-OV-3, ovarian cancer; SK-MEL-2, mali-
gnant melanoma; HCT-15, colon cancer; MCF-7, breast cancer.
IC50 is the concentration of the compound that causes a 50%
reduction in absorbance at 520 nm relative to untreated cells
using SRB assay.
#
Plateau dose response observed [3].
NDϪnot determined.
δδ
The newly synthesized thiocolchicine derivatives 1Ϫ8 were
evaluated for in vitro cytotoxicities on both five different
types of tumor cells and a normal cell (Table 1 and Table
2
). The reference materials used in this assay to compare
†
Table 2. In vitro cytotoxic effects of the thiocolchicine deriva-
tives 1Ϫ8 and (Ϫ)-colchicine in Chinese hamster ovary
the cytotoxic effects of the compounds prepared were
doxorubicin and colchicine. Table 1 summarizes the ability
of compounds 1Ϫ8 and the reference material to inhibit
tumor cell growth. After 3 days of drug treatment, the IC50
values were determined as the compound concentration that
caused a 50% reduction in absorbance at 520 nm relative to
untreated cells using an SRB assay [21].
(
CHO-K1) cell line.
IC50 (µM)‡
Compounds
Compounds
IC50 (µM)
(
؊)-Colchicine
0.044
0.078
0.092
8.6
1
2
3
4
5
6
7
8
6.4
0.9
0.82
0.70
The reference compounds, both doxorubicin and colchicine,
showed similar cytotoxic effects in nanomolar concen-
trations. Colchicine invariably produced a plateau dose re-
sponse in the breast cancer cell line MCF-7 with maximum
growth inhibition of 45% as previously reported, whereas
doxorubicin did not [3]. Compared with the reference mate-
5.7
†
‡
Data obtained from in vitro CHO cell screen.
IC50 is the concentration of the compound that causes a 50%
reduction in absorbance at 520 nm relative to untreated cells using
SRB assay.

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

586
Namgoong et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
the case of 1 against SK-OV-3. The inhibition data of com-
pounds 1 and 7 against colon cancer cell line HCT-15 were
comparable to those of colchicine. Consequently, a marked
difference in anticancer activity was observed between two
groups of thiocolchicine derivatives. The poor anticancer
effect of 2Ϫ5 and 8 is probably due to their big side chains
at C-7 position of the B ring that can interfere favorably
between the thiocolchicine derivative and its binding do-
main on tubulin [22]. The other compounds 1, 6, and 7
have relatively smaller C-7 side chains, possessing a specific
configurations at the α-carbon such as either a trans-double
bond or a stereo center on their structures as compared with
rest of the synthetic compounds. Their excellent anticancer
activities seem to be primarily attributed to these struc-
tural features.
than colchicine together with the potent anticancer activity
of it. They can be considered as new lead compounds in the
field of colchicinoid chemistry. Compounds 6 and 7 demon-
strated to be the most promising lead compounds among
them.
Acknowledgments
This work was supported by the Korea Research Foun-
dation Grant funded by the Korean Government
(
0
MOEHRD) R06-2002-018-01002-0 and R06-2002-018-
1004-0.
Experimental
Table 2 shows the preliminary result for in vitro cytotoxicity
Chemistry
of colchicine as the reference molecule and the compounds
General
1Ϫ8 against normal cell (CHO-K1, Chinese hamster ovary
Melting points of all the compounds were recorded on an Electro-
thermal 9100 (Electrothermal, Essex, UK) without correction.
Infrared spectra were obtained using a Midac PRS-INT FT-IR
spectrophotometer (Midac Corporation, Costa Mesa, CA, USA).
Infrared spectra of samples were normally run as solutions in
chloroform. 1H and C NMR spectra were recorded with Bruker
DPX-300 and Bruker AMX-500 NMR spectrometers (Bruker,
Rheinstetten, Germany). All Chemical shifts are reported on the δ
scale using residual solvent as internal standard. Mass spectra were
recorded on a Jeol JMS-AX 505WA mass spectrometer (Jeol,
Tokyo, Japan) using fast atomic bombardment (FAB) technique.
Flash chromatography was carried out using Merck Kiesegel 60
flash silica gel (Merck, Darmstadt, Germany). All the solvents and
chemicals were purchased from common commercial suppliers and
directly used without further purification.
cell). The IC50 values were determined in the same manner
mentioned above. All the synthetic compounds were much
less toxic than colchicine toward CHO-K1. Considering
their anticancer activities, the toxicity of 2Ϫ5 and 8 could
be expected to be low. However, it is unusual that 1, 6, and
13
7
exhibited low toxicities together with strong tumor growth
inhibitory effects. At present, there is no clear explanation
for those results. The virtual difference of cytotoxicities
ranges from two-fold to 20-fold between synthetic com-
pounds and colchicine, except the weak inhibitors for tumor
cell growth. In particular, compounds 6 and 7 are at least
10-fold less toxic than compound 1. Most importantly, most
normal cells survived to almost 100% at the concentration
of IC50 for those three compounds against five kinds of can-
cer cells. They can be used as lead compounds for de-
veloping more powerful and less toxic colchicinoids. Com-
pounds 6 and 7 are better candidates than compound 1 as
to toxicity.
General method for the synthesis of compounds 1Ϫ8
Dichloromethane was added to a mixture of DATC (1.20 eq or
2.10 eq in case of the compound 2), the carboxylic acid (1.00 eq),
and EDC·HCl (1.20 eq) under an atmosphere of nitrogen. The reac-
tion mixture was stirred at room temperature 24Ϫ48 h. The re-
sulting solution was washed with 1 M hydrochloric acid and water.
The dichloromethane was dried with magnesium sulfate, filtered,
and evaporated in vacuo. Purification of the residue by flash chro-
matography afforded the products. The solvents were DMSO and
DMF, respectively, for the syntheses of compounds 3 and 6. Com-
pound 3 was purified by recrystallization from the mixed solvents
These two kinds of compounds, 6 and 7, have several chemi-
cal and structural features. Compound 6 is water-soluble
and conformationally flexible. Its sugar moiety has four
stereo centers with proper configurations. It is valuable to
examine structure-activity relationship (SAR) for analogues
of compounds 6 according to its configurational variation
for each stereo center of the sugar moiety. Compound 7
possesses trans-double bond and a disubstituted aromatic
group on a ferulic acid backbone and it is conformationally
more rigid than compound 6. It is also interesting to investi-
gate SAR for derivatives of the compound 7 through struc-
tural modification from trans- to cis-geometry and variation
of substituents and their positions on aryl group. Their SAR
study is in progress.
(ethyl acetate:methanol, 1:1) without separation by flash chroma-
tography. To purify compound 6 the resulting residue is washed suc-
cessively with ethyl acetate and methanol after removal of the reac-
tion solvent without separation by flash chromatography.
N-[(4S)-Isopropenylcychlohexene-1-carboxyl]-(Ϫ)-deacetyl-10-
thiocolchicine 1
Reagents: DATC (124 mg, 0.348 mmol), (s)-perillic acid (50.0 mg,
0.290 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (EDC·HCl) (68.0 mg, 0.348 mmol), and CH
mL). Purification: flash column chromatography (chloro-
form:methanol, 15:1). Yellow syrup (124 mg, 82%). IR (neat): 3332
2 2
Cl
(5
Ϫ1
1
(
3
NH), 1706 (CϭO), 1675 (NHCϭO) cm . H NMR (CDCl , 300
In conclusion, new thiocolchicine derivatives 1Ϫ8 were syn-
thesized. As a preliminary result of biological investigation
in vitro, the compounds 1, 6, and 7 showed lower toxicity
MHz, δ): 7.29 (m, 2H, H-8 and H-11), 7.05 (d, 1H, J11,12 ϭ 10.3
Hz, H-12), 6.68 (m, 1H, CϭCH), 6.54 (s, 1H, H-4), 4.71 (m, 3H,
H-7 and CϭCH ), 3.94 (s, 3H, OCH ), 3.91 (s, 3H, OCH ), 3.58
2 3 3

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
Thiocolchicine derivatives as anticancer agents
587
(
9
s, 3H, OCH
H), 1.62 (s, 3H, CH
MHz, δ): 182.3, 168.1, 158.2, 153.7, 151.9, 151.3, 148.9, 141.8,
3
), 2.52 (m, 1H), 2.39 (s, 3H, SCH
3
), 2.30Ϫ1.68 (m,
N-(cis-4,7,10,13,16,19-Docosahexaenoyl)-(Ϫ)-deacetyl-10-thio-
colchicine 5
1
3
3
), 1.47 (m, 1H) ppm. C NMR (CDCl3, 125
Reagents: DATC (55.8 mg, 0.156 mmol), DHA (docosahexanoic
1
6
38.6, 135.4, 134.6, 133.5, 132.8, 129.0, 126.6, 125.9, 109.2, 107.5,
1.8, 61.5, 56.2, 52.2, 40.2, 36.6, 30.9, 30.2, 27.2, 24.9, 20.8, 15.2
.
acid) (49.0 mg 0.130 mmol), EDC HCl (30.5 mg, 0.156 mmol), 2,6-
ϩ
di-tert-butyl-p-cresol (5.00 mg, 0.014 mmol), and CH Cl2 (5 mL).
ppm. FAB-MS: m/z [MϩH] 522.
2
Purification: flash column chromatography (dichlorometh-
ane:methanol, 40:1). Yellow syrup (62.0 mg, 70%). IR (neat): 3310
Ϫ1
1
(
NH), 1700 (CϭO), 1680 (NHCϭO) cm . H NMR (CDCl
3
, 300
Bis(N-deacetamidocolchicinyl)-7,12-bis(1-hydroxyethyl)-3,8,13,17-
tetramethyl-21H,23H-porphine-2,18-dipropanamide 2
MHz, δ): 7.30Ϫ7.26 (m, 2H, H-8 and H-11), 7.04 (d, 1H, J11,12
ϭ
1
5
0.5 Hz, H-12), 6.53 (s, 1H, H-4), 6.39 (d, 1H, J ϭ 8.2 Hz, NH),
.34 (m, 12H, CϭCH), 4.60 (m, 1H, H-7), 3.94 and 3.90 (s ϫ 2, 6H,
ϫ 2), 3.66 (s, 3H, OCH ), 2.80 (m, 10H, ailylic CH ϫ 5),
.60Ϫ1.80 (m, 13H), 0.94 (t, 3H, J ϭ 7.5 Hz, CH
) ppm. 13C NMR
CDCl3, 125 MHz, δ): 182.6, 172.2, 158.4, 153.8, 151.5, 151.4, 141.9,
138.5, 134.8, 134.5, 132.2, 129.5, 128.8, 128.7, 128.6, 128.5, 128.4,
Reagents: DATC (125 mg, 0.350 mmol), hematoporphyrin IX (100
OCH
2
(
3
3
2
mg, 0.170 mmol), EDC·HCl (72.3 mg, 0.350 mmol), and CH
5 mL). Purification: flash column chromatography (chloroform :
methanol, 40:1). Red solid (166 mg, 73%). mp: > 400°C. IR (KBr):
2 2
Cl
3
(
-1 1
3
(
414 (OH and NH), 1720 (CϭO), 1687 (NHCϭO) cm . H NMR
DMSO, 300 MHz, δ): 10.70 (brs, 1H, porphyrin-H), 10.23 (s, 1H,
porphyrin-H), 8.60 (brs, 2H, porphyrin-H), 7.27 (d, 2H, J11,12
0.8 Hz, H-11 ϫ 2), 7.13 (s, 2H, H-8 ϫ 2), 7.10 (d, 2H, J ϭ 10.5
Hz H-12 ϫ 2), 6.51 (brs, 2H, H-4 ϫ 2), 6.41 (d, 1H, CONH), 6.19
brs, 1H, CONH), 4.40Ϫ4.08 (m, 4H, H-7 ϫ 2 and HOCH ϫ 2),
1
6
2
28.3, 128.2, 128.1, 128.0, 127.9, 127.2, 126.6, 125.9, 107.6, 61.9,
1.6, 56.3, 52.1, 40.5, 37.1, 36.3, 30.2, 25.8, 25.7, 25.4, 25.0, 23.5,
0.7, 15.3, 14.5 ppm. FAB-MS: m/z [MϩH] 684.
ϭ
ϩ
1
(
N-[d-(ϩ)-Gluconyl]-(-)-deacetyl-10-thiocolchicine 6
3
2
.79Ϫ3.35 (m, 30H, OCH
3
ϫ 6 and CH
3
ϫ 4), 3.03Ϫ1.12 (m, 16H),
ϫ 2), -3.90 (s, 2H, porphy-
Reagents: DATC (240 mg, 0.672 mmol), d-glucono-1,5-lactone (100
mg, 0.560 mmol), and N,N-dimethylformamide (DMF) (5 mL).
Purification: wash the resulting residue with ethyl acetate and meth-
.42 (s, 6H, SCH ϫ 2), 2.15 (s, 6H, CH
3
3
ϩ
rin-NH) ppm. FAB-MS: m/z [MϩH] 1309.
anol after removal of the reaction solvent. Very hygroscopic yellow
1
syrup (247 mg, 80%). H NMR (CD
3
OD, 300 MHz, δ): 7.86 (s, 1H,
N-(Pteroyl-5-L-glutamyl)-(Ϫ)-deacetyl-10-thiocolchicine 3
H-8), 7.24 (m, 2H, H-11 and H-12), 6.60 (s, 1H, H-4), 4.43 (m, 1H,
H-7), 4.24 (m, 2H), 4.06Ϫ3.73 (m, 4H), 3.73Ϫ3.49 (m, 13H), 2.80
Reagents: DATC (85.8 mg, 0.240 mmol), folic acid dihydrate (100
mg, 0.200 mmol), EDC·HCl (46.9 mg, 0.240 mmol), pyridine
(
s, 3H, SCH
3
) ppm. 13C NMR (CDCl3, 125 MHz, d): 182.6, 173.1,
1
1
3
58.4, 153.9, 153.8, 151.5, 141.7, 138.6, 136.0, 134.6, 128.8, 126.7,
26.0, 107.6, 75.0, 74.3, 71.5, 65.8, 61.6, 61.5, 59.0, 56.0, 53.6, 37.0,
0.2, 15.3 ppm. FAB-MS: m/z [MϩH] 552.
(
0.0100 mL), and dimethyl sulfoxide (DMSO) (5 mL). Purification:
recrystallization from the mixed solvents (ethyl acetate : methanol,
:1). Yellow solid (112 mg, 69%). mp: 265°C. IR (KBr): 3403 (OH
ϩ
1
-1 1
and NH), 1700 (CϭO), 1687 (NHCϭO) cm . H NMR (DMSO,
00 MHz, δ): 11.5 (brs, 1H, COOH), 8.63 (m, 2H, CONH and
heterocyclic-H), 7.95 (brs, 1H, CONH), 7.58 (m, 2H, ArH), 7.24 (s,
H, H-8), 7.16 (d, 1H, Jϭ10.3 Hz, H-11), 7.13 (d, 1H, H-12), 7.01
brs, 2H, NH ), 6.78 (s, 1H, H-4), 6.60 (m, 2H, ArH), 4.50Ϫ4.10
m, 4H, NHCH , α-CH and H-7), 3.83 and 3.78 (s ϫ 2, 6H, OCH
), 2.72 (s, 3H, SCH ), 2.60Ϫ1.70 (m, 8H,
3
N-(trans-4-Hydroxy-3-methoxycinnamyl)-(Ϫ)-deacetyl-10-thio-
colchicine 7
1
(
(
Reagents: DATC (80.2 mg, 0.221 mmol), ferulic acid (trans-4-
hydroxy-3-methoxycinnamic acid) (37.0 mg, 0.187 mmol),
EDC·HCl (43.0 mg, 0.221 mmol), 4-dimethylamino-pyridine (4-
DMAP) (1.00 mg, 0.00940 mmol), 2,6-di-tert-butyl-p-cresol (6.00
2
2
3
ϫ 2), 3.58 (s, 3H, OCH
CH CH O, H-5, 5Ј, H-6 and H-6Ј) ppm. C NMR (DMSO
MHz, δ): 181.0, 173.9, 173.8, 172.0, 166.4, 157.8, 156.5, 153.8,
3
3
1
3
2
2
,
125
mg, 0.028 mmol), and CH
chromatography (chloroform : methanol, 30:1). Yellow syrup (43
mg, 78%). IR (CDCl ): 3326 (OH, NH), 1701 (CϭO), 1680 (NHCϭ
O) cm . H NMR (CDCl , 300 MHz, δ): 7.43Ϫ7.26 (m, 3H), 7.04
(d, 1H, Jϭ12.3 Hz, H-11), 6.90 (m, 3H), 6.56 (m, 2H), 6.33 (d, 1H,
2 2
Cl (5 mL). Purification: flash column
1
1
6
53.7, 153.4, 150.8, 150.7, 148.5, 142.9, 141.2, 138.1, 135.0, 134.1,
33.9, 129.4, 128.9, 127.9, 125.7, 125.4, 124.0, 121.3, 111.2, 107.0,
3
Ϫ1
1
1.5, 61.1, 56.1, 53.6, 51.7, 45.9, 40.4, 30.6, 30.4, 26.0, 15.1 ppm.
3
ϩ
FAB-MS: m/z [MϩH] 797.
J
3
trans ϭ 17.4 Hz, trans-H), 5.85 (brs, 1H), 4.89 (m, 1H, H-7),
.96Ϫ3.89 (s ϫ 3, 9H, OCH ϫ 3), 3.70 (s, 3H, OCH ), 2.68Ϫ1.86
) ppm. C NMR
(CDCl3, 125 MHz, d): 182.5, 171.2, 158.5, 158.2, 153.7, 151.8, 151.1,
3
3
13
N-[{2-(2,6-Dichloroanilino)phenyl}acetyl]-(Ϫ)-deacetyl-10-thio-
colchicine 4
(m, 4H, H-5, 5Ј, H-6 and H-6Ј), 2.50 (s, 3H, SCH
3
1
1
3
50.8, 141.5, 141.3, 139.1, 138.3, 135.1, 134.6, 129.3, 127.3, 127.0,
26.4, 125.6, 125.3, 107.5, 107.4, 61.3, 61.2, 56.2, 56.1, 53.7, 39.3,
0.4, 15.2 ppm. FAB-MS: m/z [MϩH] 550.
Reagents: DATC (60.0 mg, 0.168 mmol), Diclofenac (42.0 mg, 0.140
mmol), EDC·HCl (32.8 mg, 0.168 mmol), and CH Cl (5 mL). Puri-
2 2
ϩ
fication: flash column chromatography (dichloromethane:meth-
anol, 50:1). Yellow syrup (64.0 mg, 70%). IR (neat): 3320 (NH),
N-[(2R,3S,4R)-2-{(tert-Butoxycarbonyl)amino}-3,4-O-isopropyl-
ideneoctadecanoyl]-(Ϫ)-deacetyl-10-thiocolchicine 8c [20]
-1 1
1
710 (CϭO), 1680 (NHCϭO) cm . H NMR (CDCl
3
, 300 MHz,
δ): 7.78 (s, 1H, ArNH), 7.73 (d, 1H, J ϭ 6.9 Hz), 7.50 (s, 1H, H-8),
7
7
.33 (d, 1H, J11,12 ϭ 10.3Hz, H-11), 7.24 (m, 3H), 7.09 (d, 1H, J ϭ
.0 Hz H-12), 7.00 (dd, 1H, J ϭ 7.1 Hz), 6.91 (m, 2H), 6.51 (s, 1H,
Reagents: DATC (51.5 mg, 0.144 mmol), 8b (58.0 mg, 0.12 mmol),
EDC·HCl (28.2 mg, 0.144 mmol), and CH Cl (5 mL). Yellow syrup
2 2
H-4), 6.40 (d, 1H, J ϭ 7.7 Hz), 4.6 (m, 1H, H-7), 3.95 (s ϫ 2, 6H,
(51.8 mg, 68%). Purification: flash column chromatography (chloro-
OCH
OCH
3
ϫ 2), 3.84 (ABq, 2H J ϭ 13.8 Hz, ArCH
2
), 3.69 (s, 3H,
form:methanol, 15:1). IR (neat): 3428 (NH), 1709 (Boc CϭO and
CϭO), 1680 (NHCϭO) cm . H NMR (CDCl , 300 MHz, δ): 7.25
3
(d, 1H, J ϭ 10.5 Hz, H-11), 7.12 (s, 1H, H-8), 7.02 (d, 1H, H-12),
6.95 (d, 1H, J ϭ 6.0 Hz, CONH), 6.53 (s, 1H, H-4), 5.04 (d, 1H,
J ϭ 6.0 Hz, BocNH), 4.67 (m, 1H, H-7), 4.27 (m, 2H), 4.13 (m,
-
1 1
3
), 2.51Ϫ1.93 (m, 3H, H-5, 5Ј and H-6), 1.90 (m, 1H, H-6Ј),
1
3
2
1
1
1
3
.45 (s, 3H, SCH
3
) ppm. C NMR (CDCl3, 125 MHz, δ): 182.8,
72.4, 158.6, 153.9, 152.7, 151.4, 143.2, 142.0, 139.1, 138.1, 135.1,
34.6, 133.8, 131.0, 129.8, 129.0, 128.9, 128.7, 127.6, 127.1, 126.0,
25.2, 123.6, 121.4, 117.4, 107.8, 61.9, 61.6, 56.3, 52.7, 40.4, 36.7,
1H), 3.94 and 3.91 (s ϫ 2, 6H, OCH
3
ϫ 2), 3.66 (s, 3H, OCH
3
),
ϩ
0.2, 15.4 ppm. FAB-MS: m/z [MϩH] 651.
2.62Ϫ2.19 (m, 3H, H-5, 5Ј and H-6), 2.45 (s, 3H, SCH
3
), 1.79 (m,

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

588
Namgoong et al.
H, H-6Ј), 1.63Ϫ0.99 (m, 44H, alkyl protons), 0.88 (t, 3H, CH
Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
1
3
)
medium or no additions (control), and further incubated for 48 h.
After incubation, 50% TCA was added and fixation was allowed to
proceed for 1 h at 4°C. The TCA-fixed cells were stained for 1 h
with 0.4% SRB dissolved in 1% acetic acid. After staining, un-
bound dye was removed by washing five times with 1% acetic acid
and the plates were air-dried. Bound stain was dissolved with 10
mM Tris base (pH 10.5) and the optical density was measured at
1
3
ppm. C NMR (CDCl3, 125 MHz, δ): 182.4, 172.7, 157.9, 155.3,
1
1
3
2
53.7, 153.4, 149.0, 141.1, 137.9, 134.9, 134.1, 129.0, 127.8, 125.6,
25.5, 109.0, 107.1, 64.3, 61.1, 61.0, 56.1, 53.2, 52.3, 51.9, 50.4, 40.0,
3.3, 32.1, 31.7, 30.7, 29.9, 29.8, 29.7, 29.6, 29.5, 29.0, 28.7, 28.0,
7.1, 26.5, 26.2, 24.3, 22.9, 19.4, 15.0, 14.5 ppm.
5
20 nm using a microplate reader (Molecular devices, Sunnyvale,
N-[(2R,3S,4R)-2-Amino-3,4-O-isopropylideneoctadecanoyl]-(Ϫ)-
deacetyl-10-thiocolchicine 8
CA, USA). Using absorbance measurements [time zero (Tz), control
growth (C), and test growth in the presence of test compounds (T)],
the percentage growth was calculated at each concentration of the
test compound. Percentage growth inhibition was calculated accord-
ing to:
p-Toluenesulfonic acid (36 mg, 0.19 mmol) was added to a solution
of the compound 8c (60 mg, 0.074 mmol) in anhydrous dichloro-
methane (4 mL) under an atmosphere of nitrogen. The reaction mix-
ture was stirred at room temperature for 1 h. It was then neutralized
with potassium carbonate (76 mg, 0.56 mmol). The resulting solu-
tion was washed with water (4 mL ϫ 2). The dichloromethane was
dried with magnesium sulfate, filtered, and evaporated. The residue
was purified by a silica gel column chromatography (dichlorometh-
ane : methanol, 15:1) to give the product 8 (32 mg, 70%). IR (neat):
(
(
T-Tz)/(C-Tz) ϫ 100 for concentrations for which T ϭ Tz
T-Tz)/Tz ϫ 100 for concentration for which T > Tz
IC50, the concentration that can inhibitor cancer cell growth by
0%, was calculated by using a regression analysis of the lotus pro-
5
Ϫ1
1
gram based on the degree of cytotoxicity obtained by the above
mathematical formula. Each IC50 of doxorubicin and colchicines,
which were used as controls, was also calculated as described above.
3
428 (NH), 1710 (Boc CϭO and CϭO), 1680 (NHCϭO) cm . H
NMR (CDCl , 300 MHz, δ): 7.26 (m, 2H, H-8 and H-11), 7.06 (d,
H, J ϭ 10.5 Hz, H-12), 6.54 (s, 1H, H-4), 5.84 (d, 1H, J ϭ 6.0 Hz,
CONH), 4.53 (m, 1H, H-7), 4.44 (d, 1H J ϭ 6.0 Hz, BocNH), 3.94
and 3.91 (s ϫ 2, 6H, OCH ϫ 2), 3.66 (m, 5H, SCH and CH ϫ 2),
.48 (m, 2H), 3.30 (m, 1H) 2.56Ϫ2.20 (m, 3H, H-5 and H-6), 2.43
s, 3H, SCH ) 2.00Ϫ1.80 (m, 3H, CH , H-5, 5Ј, H-6 and H-6Ј), 1.62
s, 6H, acetal CH ϫ 2), 1.25 (m, 22H, alkyl protons), 0.86 (t, 3H,
) ppm. C NMR (CDCl3, 125 MHz, δ): 182.4, 172.7, 157.9,
3
1
3
3
3
(
(
References
3
2
3
[1] O. Boye, A. Brossi, Alkaloids 1992, 41, 125Ϫ176.
1
3
CH
3
[
[
2] S. B. Hastie, Pharmacol. Ther. 1991, 51, 377Ϫ401.
1
1
3
2
53.7, 153.4, 149.0, 141.1, 137.9, 134.9, 134.1, 129.0, 127.8, 125.6,
25.5, 109.0, 107.1, 64.3, 61.1, 61.0, 56.1, 53.2, 52.3, 51.9, 50.4, 40.0,
3.3, 32.1, 31.7, 29.9, 29.8, 29.7, 29.6, 29.5, 29.0, 27.1, 26.5, 26.2,
3] J. Guan, X-K. Zhu, Y. Tachibana, K. F. Bastow, A. Brossi, E.
Hamel, K-H. Lee, Bioorg. Med. Chem. 1998, 6, 2127Ϫ2131.
ϩ
2.9, 19.4, 15.0, 14.5 ppm. FAB-MS: m/z [MϩH] 727.
[4] X. Pan, W. Fang, Acta Pharmaceutica Sinica 2002, 37,
21Ϫ827.
8
[
5] R. Brecht, G. Seitz, D. Guenard, S. Thoret, Bioorg. Med.
Chem. 2000, 8, 557-562 and references cited therein.
Pharmacology
Cell culture
[
6] Q. Shi, K. Chen, A. Brossi, P. Verdier-Pinard, E. Hamel, A. T.
McPhail, K-H. Lee, Helv. Chim. Acta.1998, 81, 1023Ϫ1037
and references cited therein.
For a normal cell lines, the CHO-K1 (Chinese hamster ovary) cells
were purchased from Korea Cell Line Bank (KCLB , Seoul,
Ǟ
Korea). The cells were maintained as monolayer cultures in Dul-
becco’s modified Eagle’s medium (DMEM) (Gibco BRL, Gaithers-
burg, MA, USA) supplemented with 10% fetal bovine serum (FBS,
Gibco BRL), 100 µg/mL of streptomycin, and 100 U/mL of penicil-
lin (Gibco BRL). The cells were incubated at 37°C in a humidified
[7] Q. Shi, P. Verdier-Pinard, A. Brossi, E. Hamel, A. T. McPhail,
K-H. Lee, J. Med. Chem. 1997, 40, 961Ϫ966.
[
[
8] R. Dumount, A. Brossi, C. F. Chignell, F. R. Quinn, M. Suff-
ness, J. Med. Chem. 1987, 30, 732Ϫ735.
9] H. J. C. Yeh, M. Chrzanowska, A. Brossi, FEBS Lett. 1988,
2
atmosphere of 5% CO . The five different tumor cell lines including
229, 82Ϫ86.
A549 (human non-small cell lung cancer), SK-OV-3 (human ovarian
cancer), SK-MEL-2 (human melanoma), HCT-15 (human colon
cancer), and MCF-7 (human breast cancer) cells were provided by
Korea Research Institute of Chemical Technology, Daejeon, Korea.
The cells were maintained as monolayer cultures in RPMI 1640
medium (Gibco BRL) supplemented with 10% FBS, 100 µg/mL of
streptomycin, and 100 U/mL of penicillin. The cells were incubated
[10] M. H. Gelb, F. Tamanoi, K. Yokoyama, F. Ghomashchi, K.
Esson, M. N. Gould, Cancer Lett. 1995, 91, 169Ϫ175.
[
[
11] I. R. Hardcastle, M. G. Rowlands, A. M. Barber, Biochem.
Pharmacol. 1999, 57, 801Ϫ809.
12] J. L. Sessler, A. Gebauer, E. Vogel in The Porphyrin Handbook,
Vol. 2 (Eds.: K. M. Kadish, K. M. Smith, R. Guilard), Aca-
demic Press, San Diego, CA, 1999, chapter 8.
2
at 37°C in a humidified atmosphere of 5% CO .
[
13] D. Lash, Angew. Chem. 2000, 112, 1833Ϫ1837.
Cytotoxicity
[
14] R. J. Lee, P. S. Low, Biochimica Biophysica Acta 1995, 1233,
134Ϫ144.
The cytotoxicity of the test compounds against the cancer cell lines
and a normal cell line was evaluated by sulforhodamine B (SRB)
assay as described previously [21]. In order to assay the cytotoxicity
of test compounds against a normal cell line, CHO-K1 cells were
plated in 96-well plates (8000 cells/well) and incubated for 24 h in a
DMEM conditioned medium. The five different kinds of human
tumor cells were plated in 96-well plates at plating densities ranging
from 5000 to 20,000 cells per well depending on the doubling time
of individual cell lines and incubated for 24 h. The plates of each
cell line were treated with various concentrations ranging from 0.01
nM to 1 µM of test compounds, which were dissolved in the culture
[
[
15] M. Zerouga, W. Stillwell, L. J. Jenski, Anti-Cancer Drugs 2002,
13, 301Ϫ311.
16] A. Murakami, Y. Nakamura, K. Koshimizu, D. Takahashi,
K. Matsumoto, K. Hagihara, H. Taniguch, E. Nomura,
A. Hosoda, T. Tsuno, Y. Maruta, H-W. Kim, K. Kawabata, H.
Ohigashi, Cancer Lett. 2002, 180, 121Ϫ129.
[17] M. Macchia, S. Bertini, S. Fogli, E. Giovanetti, F. Minatolo, S.
Rapposelli, R. Danesi, Farmaco 2003, 58, 205Ϫ203 and refer-
ences cited therein.

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2005, 338, 582−589
Thiocolchicine derivatives as anticancer agents
589
[18] T. K. Hoffmann, K. Leenen, D. Hafner, V. Balz, C. Gerharz,
[20] E. O. Kim, M.S. Thesis, Seoul Women’s University, 2003.
A. Grund, H. Ballo, U. Hauser, H. Bier, Anti-Cancer Drugs
[
21] H. M. Kim, S. B. Han, M. S. Kim, J. S. Kang, G. T. Oh, D.
2
002, 13, 93Ϫ100.
19] M. C. Desai, L. M. Stephens-Stramiello, Tetrahedron Lett.
993, 34, 7685-7688.
H. Hong, J. Pharmacol. Toxicol. Methods 1996, 36, 163Ϫ169.
[
[22] L. Sun, H. Hamel, C. M. Lin, S. B. Hastie, A. Pyluck, K-H.
1
Lee, J. Med. Chem. 1993, 36, 1474Ϫ1479.

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com