Antitumor agents 273. Design and synthesis of N-alkyl-thiocolchicinoids as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2010.05.081

As a part of our continuing study of colchicinoids as therapeutically useful antitumor drugs, thiocolchicine derivatives, including their phosphate and other water soluble salts, were synthesized and evaluated for inhibition of tubulin polymerization and for in vitro cytotoxicity. Three compounds, 7, 10, and 11, showed potent inhibition of tubulin assembly (IC₅₀ = 0.88-1.1 μM). In addition, compound 7, a water soluble succinic acid salt of N-deacetylthiocolchicine (4), showed potent cytotoxicity against a panel of tumor cell lines, suggesting it might be a potential lead to be developed as a therapeutic antitumor agent. Compound 8, a water soluble succinic acid salt of N,N-dimethyl-N-deacetylthiocolchicine (5), showed selective activities against HCT-8 and SK-BR-3 cells. N,N-Diethyl-N-deacetylthiocolchicine (6) seemed not to be a substrate for the P-gp efflux pump, based on the similar ED₅₀ values obtained against P-gp over-expressing KBvin (0.0146 μg/mL) cells and the parent KB (0.0200 μg/mL) cell line.

Full text of DOI:10.1016/j.bmcl.2010.05.081

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4091–4094
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antitumor agents 273. Design and synthesis of N-alkyl-thiocolchicinoids
as potential antitumor agents q
Takashi Kozaka a, Kyoko Nakagawa-Goto a, Qian Shi a, Chin-Yu Lai a, Ernest Hamel b, Kenneth F. Bastow c,
Arnold Brossi a, Kuo-Hsiung Lee a,
*
a Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA
b Screening Technologies Branch, Developmental Therapeutics Program , Division of Cancer Treatment and Diagnosis, National Institutes of Health, Frederick, MD 21702, USA
c Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our continuing study of colchicinoids as therapeutically useful antitumor drugs, thiocolchi-
cine derivatives, including their phosphate and other water soluble salts, were synthesized and evaluated
for inhibition of tubulin polymerization and for in vitro cytotoxicity. Three compounds, 7, 10, and 11,
Received 5 March 2010
Revised 17 May 2010
Accepted 19 May 2010
Available online 24 May 2010
showed potent inhibition of tubulin assembly (IC50 = 0.88–1.1 lM). In addition, compound 7, a water sol-
uble succinic acid salt of N-deacetylthiocolchicine (4), showed potent cytotoxicity against a panel of
tumor cell lines, suggesting it might be a potential lead to be developed as a therapeutic antitumor agent.
Compound 8, a water soluble succinic acid salt of N,N-dimethyl-N-deacetylthiocolchicine (5), showed
selective activities against HCT-8 and SK-BR-3 cells. N,N-Diethyl-N-deacetylthiocolchicine (6) seemed
not to be a substrate for the P-gp efflux pump, based on the similar ED50 values obtained against P-gp
Keywords:
N-Alkylthiocolchinoids
Antitumor agents
Tubulin polymerization
over-expressing KBvin (0.0146
l
g/mL) cells and the parent KB (0.0200
lg/mL) cell line.
Ó 2010 Elsevier Ltd. All rights reserved.
Colchicine (1, Fig. 1), an alkaloid isolated from Colchicum autum-
nale and Gloriosa superba, is known to possess notable anti-inflam-
matory, anti-mitotic, and anti-fibrotic effects.2 Although colchicine
has significant in vitro antitumor effects, its medicinal uses, as well
as use of its derivatives, has been limited because of high toxicity,
low bioavailability, and poor water solubility.3 Thiocolchicine (2),
originally derived from colchicine by semi-synthesis, possesses
comparable or greater biological activity than colchicine (1) and
has been studied for many years.2 Although 2 is structurally more
stable than 1, its substantial toxicity and poor water solubility limit
interest in it as a drug candidate. Many efforts have been made to
develop thiocolchicine analogs with the goal of reducing toxicity
and increasing watersolubility.3,4 2-Demethylthiocolchicine (3)
was found to be slightly less active but also less toxic than 2 and
1.5 N-Deacetylthiocolchicine (4) was previously synthesized and
was similar to 2 in its inhibitory effect on tubulin assembly.6 These
findings suggested that further modifications could lead to more
active compounds with reduced toxicity or greater water solubility
and thus have greater potential as drug candidates.
In this Letter, we report such modifications of thiocolchicine and
its derivatives at the amino group at C7 and at the hydroxy group
at C2 of 3 and 4.
Treatment of 46 with formaldehyde and acetaldehyde in the
presence of NaBH3CN under acidic conditions yielded N,
N-dimethyl- (5) and N,N-diethyl-N-deacetylthiocolchicine (6) in
80% and 82% yields, respectively, without obtaining N-mono-al-
kyl-substituted compounds (Scheme 1). Amines 4–6 were con-
verted to corresponding succinic acid salts 7–9 in quantitative
yields.
A series of N-mono-alkylated analogs of 4 was synthesized
through ready addition and removal of the o-nitrobenzenesulfona-
mide (o-Ns) group, which is widely tolerant of basic and acidic con-
ditions (Scheme 2). According to Fukuyama’s protocol,7 4 was
MeO
MeO
MeO
RO
NHAc
O
NHAc
O
7
2
The preparation of di-alkyl-substituted amines of N-deac-
etylthiocolchicine (4) and related pharmaceutically and clinically
accepted salts and phosphate derivatives has not been described.
MeO
MeO
10
OMe
Colchicine (1)
SMe
Thiocolchicine (2; R=Me)
q
2-Demethylthiocolchicine (3; R=H)
For part 272 of this series, see Ref. 1.
* Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966 3893.
E-mail address: khlee@unc.edu (K.-H. Lee).
Figure 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.081

4092
T. Kozaka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4091–4094
MeO
MeO
MeO
NH2
NR2
O
7
a
2
MeO
MeO
MeO
O
10
SMe
SMe
5; R=Me
4
6; R=Et
b
b
MeO
MeO
MeO
MeO
+
-
+
-
NH3 C4H5O4
NHR2 C4H5O4
MeO
MeO
O
O
SMe
SMe
7
8; R=Me
9; R=Et
Scheme 1. Reagents and conditions: (a) RCHO (R = H or Me), NaBH3CN, HOAc, CH2Cl2, 5; 80% (R0 = Me); (b) succnic acid, acetone, 7, 8, 9; quant.
MeO
MeO
MeO
MeO
H
R
N
N
a
b
c
oNs
oNs
4
MeO
MeO
O
O
SMe
SMe
11; R=Me
12
10
; R=Et
13; R=Pr
MeO
HO
MeO
HO
MeO
Me
N
NHMe
NHMe
O
O
P
d
e
oNs
O
MeO
MeO
HO
MeO
OH
O
O
SMe
SMe
SMe
14
15
16
f
MeO
HO
-
NH2Me+ C4H5O4
O O
S
MeO
oNs =
O
O2N
SMe
17
Scheme 2. Reagents and conditions: (a) o-NsCl, 2,6-lutidine, CH2Cl2; (b) RI (R = Me, Et or Pr), K2CO3, MeCN 11; 85%, 12; 97%; 13; 53%; (c) BBr3, CH2Cl2, ꢀ78 °C, 75%; (d)
thioglycolic acid, Et3N, CH2Cl2, 59%; (e) POCl3, Et3N, MeCN, 0 °C to rt; then H2O, pyridine, 43%; (f) succinic acid, acetone, quant.
converted to N-[o-Ns]-N-deacetylthiocolchicine (10) with o-NsCl in
the presence of 2,6-lutidine in CH2Cl2, followed by alkylation with
RI (R = Me, Et or Pr) in the presence of K2CO3 in MeCN to afford 11,
12 and 13 in 72%, 97% and 53% overall yields, respectively. Treat-
ment of 11 with BBr3 in CH2Cl2 at ꢀ78 °C succeeded in the regiose-
lective demethylation to afford 14 in 75% yield, while the reported
method8 by heating with H2SO4 gave a lower yield of the desired
compound. The o-Ns group was removed from 14 with thioglycolic
acid and Et3N in CH2Cl2 to obtain the secondary amine 15 in 59%
yield. Compound 16, a phosphate of 15, was synthesized by using
POCl3 in MeCN and purified with reverse phase column chroma-
tography (MeOH–H2O). Treatment of 15 with succinic acid in
acetone gave 17. The water solubility at room temperature of
analogs 7–9, 16, and 17 were 180 mg/mL, 103 mg/mL, 107 mg/mL,
15 mg/mL and 95 mg/mL, respectively.
All newly synthesized compounds were evaluated for effects on
tubulin assembly and in vitro antitumor activities against several
tumor cell lines. Studies were also performed with colchicine (1),

T. Kozaka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4091–4094
4093
Table 1
hydroxy group at C2 of compound 16 was masked with a phos-
phoryl moiety, the highly polar and H-bonding properties of the
phosphate moiety may prevent the molecule from interacting opti-
mally with the colchicine-binding domain and thereby eliminating
Inhibition of tubulin assembly of 2–17
Compound Inhibition of tubulin assembly Inhibition of colchicine binding
IC50
(
lM) SD
% Inhibition SD
its anti-tubulin activity (IC50 >40 lM).
5
lM
50 lM
In vitro antitumor activities of the newly synthesized
compounds were evaluated, and the results are summarized in
Table 2. Consistent with the tubulin data discussed above, 5 and
6, N-deacetylthiocolchicine derivatives with N,N-di-alkyl substitu-
ents at C7, were on average ten times more active than the non-al-
2
4
5
6
7
0.77 0.2
0.91 0.05
2.5 0.3
5.4 0.2
0.88 0.03
3.6 0.04
74
63
42
31
64
27
17
58
43
22
ND
20
ND
ND
ND
99
2
NDa
ND
88 0.6
1
4
3
2
4
6
2
3
5
84
ND
3
a
8
9
85 0.1
65
ND
60
26
ND
34
ND
ND
ND
ND
kyl substituted compound 4. The N,N-diethyl compound
6
16
1
1
generally had antiproliferative activity similar to that of the N,N-di-
methyl compound 5 and colchicine (1). Compound 6 was the only
highly active agent tested that was equally active against the vin-
cristine resistant, MDR nasopharyngeal cancer cell line (KBvin) as
the parental non-MDR KB cell line. This result suggests that the
size of N-alkyl substituent at C7 may affect recognition of a colchi-
cinoid by P-glycoprotein and/or the mechanism of action of 6 may
be other than inhibition of tubulin activity. Although, in general,
the succinic acid salts, 7–9, showed decreased activities relative
to their parent compounds 4–6 N,N-dimethyl compound 8 was
10
11
12
13
14
15
16
17
CAb
1.1 0.06
0.92 0.03
4.2 0.7
>40
3.6 0.2
>40
2
5
a
7
6
>40
23
1
1.2 0.2
1
a
Not done.
Combretastin A-4: positive control.
b
selectively more potent against HCT-8 (ED50 = 0.0220
lg/mL) and
SK-BR-3 (ED50 = 0.0129 g/mL) than its parent compound 5, and
l
thiocolchicine (2), and N-deacetylthiocolchicine (4). The cell line
panel included KB (human epidermoid carcinoma of the nasophar-
ynx), HCT-8 (human ileocecal carcinoma), A549 (human lung ade-
nocarcinoma), DU145 (human prostate carcinoma), SK-BR-3
(human breast cancer) and KBvin (vincristine-resistant line de-
rived from the KB cell) cells. In addition, the most active inhibitors
of tubulin assembly were also evaluated as inhibitors of the bind-
ing of [3H]colchicine to tubulin. The tubulin data are summarized
in Table 1. Relative to the activity shown by 2 and 3, N,N-dimethyl-
(5) and N,N-diethyl-N-deacetylthiocolchicine (6), derived from 4
by alkylation, exhibited reduced activity as inhibitors of both tubu-
lin assembly and colchicine binding Compounds 7, 8, and 9 are
three succinic acid salts of 4, 5, and 6. Compound 7, derived from
N-deacetylthiocolchicine (4), had activity comparable with that
of its parent compound 4, while compounds 8 and 9, salts of 5
and 6, were somewhat less potent than their parent compounds.
N-o-Ns substituted compounds 10 and 11, with no other N-alkyl
(10) or a methyl substituent (11) in addition to the o-Ns group, also
displayed potent anti-tubulin activities. However, replacing the
methyl group with an ethyl (12) or propyl (13) moiety led to re-
duced or lost activity relative to compound 11 in the tubulin as-
says. This suggests that the size of alkyl groups at the C7-amino
position of N-deacetylthiocolchicines may play a role in the inter-
action of these compounds with the binding site of the target pro-
tein, while the o-Ns group may not significantly affect anti-tubulin
activity.
8 was 10-fold more cytotoxic against other tested cell lines. The
cytotoxicity of N-o-Ns-N-deacetylthiocolchicine derivatives, 10–
13, revealed the same tendency as described above. In summary,
the smaller alkyl substituents on the amino group at the C7-posi-
tion tended to have stronger activities against all cell lines (10,
R = H > 11, R = Me > 12, R = Et > 13, R = Pr). N-Non-alkyl-substi-
tuted N-[o-Ns] compound 10 showed the highest activity among
10–13 against all tested cell lines (ED50 = 0.0100–0.0270
Although 2-demethylthiocolchicine (3) showed similar activity to
thiocolchicine (2) (ED50 = 0.01 g/mL against A549, 0.01 g/mL
lg/mL).
l
l
against KB),3 the cytotoxic potency decreased for the 2-demeth-
yl-N-[o-Ns]-N-deacethylthiocolchicine series (e.g., 11 vs 14), as oc-
curred in the tubulin assay. Differing from the tubulin assay
results, compound 15, derived from 14 by removal of the o-Ns pro-
tection group, did not show significant loss in inhibition of tumor
cell growth. In addition, the inhibitory activity toward DU145
Table 2
In vitro anticancer activity data of 1–17
Compounds
ED50 (l
g/mL) a/cell lineb
HCT-8
A549
DU145
SK-BR-3
KB
KBvin
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
0.0540
0.0080
0.0050
0.0380
0.0310
0.0291
0.0220
0.1100
0.0270
0.1200
0.5200
>1.000
>1.000
>1.000
>1.000
>1.000
0.0199
0.0019
0.0077
0.0476
0.0193
0.0256
0.1902
0.1808
0.0209
0.1288
0.2116
1.6330
1.6791
1.5474
0.5332
1.2599
0.0027
0.0012
0.0018
0.0180
0.0138
0.0185
0.1243
0.1249
0.0129
0.0312
0.1630
0.3574
1.1483
0.5054
0.1572
0.3420
0.0027
0.0015
0.0016
0.0179
0.0141
0.0115
0.0129
0.1361
0.0126
0.0285
0.1482
0.6155
1.0118
0.4874
0.1368
0.3899
0.0038
0.0008
0.0018
0.0270
0.0200
0.0080
0.1200
0.0630
0.0100
0.0350
0.1300
0.9600
0.7200
0.5500
0.2500
0.3500
0.1578
0.0147
0.0139
0.0413
0.0146
0.0330
0.1250
0.1512
0.1120
0.1526
0.1737
1.3626
1.4892
1.3798
1.7062
1.3456
2-Demethylthiocolchicine (3) was previously reported as an
active inhibitor of tubulin assembly and colchicine binding.3
However, in our recent study, a series of 2-demethyl-N-deac-
etylthiocolchicine derivatives (14–17) showed reduced activity
against tubulin assembly. Compared with the parent compound
11, compound 14, the 2-demethyl derivative of 11, was about four
times less active against tubulin assembly (IC50 = 3.6 lM) and half
as potent in inhibition of colchicine binding (20% inhibition of col-
chicine binding). Compound 15, the N-de-o-Ns analog of com-
pound 14, showed remarkably reduced activity as an anti-tubulin
agent, since it was essentially inactive as an inhibitor of tubulin
a
Mean concentration giving 50% inhibition of cell growth following two days
continual exposure (three replicates varied less than 5% between and within
experiments).
assembly (IC50 >40 lM). These findings suggest that a trimethoxy-
phenyl moiety with its lipophilic molecular feature may be re-
quired for N-deacetylthiocolchicines to fit into a hydrophobic
pocket in the binding site. The acetamido group at C7 of compound
3 may play a role in maintaining the drug molecule in a suitable
conformation to interact with the target protein. Although the
b
HCT-8 = human ileocecal carcinoma cell line, A549 = human lung adenocarci-
noma epithelial cell line, DU145 = human prostate carcinoma cell line, SK-BR-
3 = human breast cancer cell line, KB = human epidermoid carcinoma of the naso-
pharynx, KBvin = vincristine-resistant nasopharyngeal cancer cell line derived from
the KB line.

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T. Kozaka et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4091–4094
and SK-BR-3 cells slightly increased. Compound 16, a phosphate of
15, showed two-three times more potent cytotoxicity than 15
against A549, DU145, SK-BR-3, and KB cell growth, suggesting
that the phosphate group may increase selectivity to the target
tumor cells. Notably, the new compounds, N,N-dialkyl-N-deac-
etylthiocolchicines, 5 and 6, as well as the succinates, 7–9, showed
comparable or better cytotoxicity against P-gp over-expressing
KBvin cells than colchicine.
compound 6 may merit further investigation to understand its
apparent resistance to the P-gp efflux pump. Finally, the succinic
acid salt of N,N-dimethyl-N-deacetylthiocolchicine, compound 8,
showed selective activities against HCT-8 and SK-BR-3 cells.
Acknowledgment
This study was supported by NIH grant CA17625 from the
National Cancer Institute awarded to K. H. Lee.
In summary, modifications of the amino group at C7 and the
methoxy group at C2 of N-deacetylthiocolchicine (4) were
achieved. Two N,N-di-alkyl-N-deacetylthiocolchicine derivatives,
5
and 6, corresponding succinates 7–9, N-[o-Ns]-N-deac-
References and notes
etylthiocolchicines 10–13, and 2-demethyl-N-methyl-N-deac-
etylthiocolchicines 14 and 15, salt 17, and C2–phosphate 16 were
generated. We analyzed this series of compounds for their anti-
tubulin activity and in vitro cytotoxicity. Although none of the
newly synthesized compounds showed more potent activity than
their parent compound 4 or thiocolchicine (2), the succinic acid
salts, especially compound 7, have potential as promising antitu-
mor agents, because of both their excellent antiproliferative
activity and their superior water solubility. The size of the N-
substituted alkyl groups played a role in inhibition of tubulin
assembly and colchicine binding and in vitro cytotoxicity. Gener-
ally, the larger alkyl substituents showed reduced activity against
both tubulin and tumor cell growth. N,N-Diethyl analog 6,
however, showed more potent cytotoxicity than its N,N-dimethyl
counterpart, which differed somewhat from the tubulin results.
This result may indicate an additional mechanism of action for 6
versus 5. In addition, compound 6 also showed higher potency
against the KBvin cell line relative to the parental KB line. Thus,
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