Antitumor agents. 185. Synthesis and biological evaluation of tridemethylthiocolchicine analogues as novel topoisomerase II inhibitors

Article abstract of DOI:10.1021/jm980007f

Several 1,2,3-tridemethyldeacetylthiocolchicine derivatives have been synthesized and evaluated for cytotoxic activity against, various human tumor cell lines and for their inhibitory effects on DNA topoisomerases in vitro. Exhaustive demethylation of thiocolchicine analogues completely changes their biological profiles. Instead of displaying antitubulin activity, most target compounds inhibited topoisomerase II activity. Only compounds with a larger side chain, such as 15a, 23a, and 24a, did not interfere with topoisomerase II enzymatic functions. The cytotoxicity of target compounds was reduced by 3 orders of magnitude compared to that of colchicine in most cell lines. The hydrophilicity of phenolic compounds might prevent drug passage through the cell plasma membrane and, thus, be responsible for the relatively weak cytotoxicity. To test this hypothesis, 27-30 were prepared from 16a by protecting all hydroxy groups with esters with an aim to facilitate drug transportation. In vitro cytotoxicity assays indicated that 27 was more potent than its parent compound in all tested tumor cell lines and showed tissue selective cytotoxicity with a significant inhibitory effect against KB cells (IC₅₀ = 2.7 μg/mL). Therefore, we propose that 27 acts as a prodrug, liberating 16a to exert its antitopoisomerase activity and, finally, to cause cell death.

Full text of DOI:10.1021/jm980007f

1956
J . Med. Chem. 1998, 41, 1956-1961
An titu m or Agen ts. 185.† Syn th esis a n d Biologica l Eva lu a tion of
Tr id em eth ylth iocolch icin e An a logu es a s Novel Top oisom er a se II In h ibitor s
J ian Guan,‡ Xiao-Kang Zhu,‡ Yoko Tachibana,‡ Kenneth F. Bastow,‡ Arnold Brossi,*,‡ Ernest Hamel,§ and
Kuo-Hsiung Lee*,‡
Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy,
University of North Carolina at Chapel Hill, North Carolina 27599, and Laboratory of Drug Discovery Research and
Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
Frederick Cancer Research and Development Center, Frederick, Maryland 21702
Received J anuary 5, 1998
Several 1,2,3-tridemethyldeacetylthiocolchicine derivatives have been synthesized and evaluated
for cytotoxic activity against various human tumor cell lines and for their inhibitory effects on
DNA topoisomerases in vitro. Exhaustive demethylation of thiocolchicine analogues completely
changes their biological profiles. Instead of displaying antitubulin activity, most target
compounds inhibited topoisomerase II activity. Only compounds with a larger side chain, such
as 15a , 23a , and 24a , did not interfere with topoisomerase II enzymatic functions. The
cytotoxicity of target compounds was reduced by 3 orders of magnitude compared to that of
colchicine in most cell lines. The hydrophilicity of phenolic compounds might prevent drug
passage through the cell plasma membrane and, thus, be responsible for the relatively weak
cytotoxicity. To test this hypothesis, 27-30 were prepared from 16a by protecting all hydroxy
groups with esters with an aim to facilitate drug transportation. In vitro cytotoxicity assays
indicated that 27 was more potent than its parent compound in all tested tumor cell lines and
showed tissue selective cytotoxicity with a significant inhibitory effect against KB cells (IC50
) 2.7 µg/mL). Therefore, we propose that 27 acts as a prodrug, liberating 16a to exert its
antitopoisomerase activity and, finally, to cause cell death.
In tr od u ction
Colchicine (1) (Figure 1), the major alkaloid isolated
from Colchicum autumnale in 1820, is an ancient drug
used to treat gout and familial Mediterranean fever.2
Most biological effects of 1 are probably related to its
powerful action on tubulin,3 causing inhibition of mi-
crotubule polymerization and cell arrest at metaphase.4
Although colchicine is a potent antitumor agent, it is
not used clinically due to its severe toxicity.5 Subse-
quently, much interest has focused on structural modi-
fications in the hope of improving the therapeutic
potential.6-8
F igu r e 1. Structure of colchicine (1).
Both the therapeutic and toxic effects of colchicinoids
are likely caused by interaction with tubulin, resulting
in a low therapeutic index.9 We postulate that a
conformational change in tubulin results when drugs
bind to the colchicine domain, and this change may
invariably lead to undesirable side effects. Conse-
quently, further development of colchicine analogues
may require targeting the analogues to either a different
binding site on tubulin or a completely different intra-
cellular target protein. The successful conversion of
podophyllotoxin to etoposide offers an excellent example
of the second possibility.
F igu r e 2. Structures of podophyllotoxin 2, etoposide, and
teniposide.
colchicine, its medical uses are limited due to its high
toxicity.11 However, its semisynthetic derivatives, eto-
poside (3) and teniposide (4) (Figure 2), are less toxic
and function as DNA topoisomerase II inhibitors with
broad antitumor spectra (compare data for 2 and 3 in
Table 1).12-14 Because topoisomerase II appears to be
a multidrug target in cancer chemotherapy,15 it is of
great interest to develop colchicine analogues that target
topoisomerase II but have no or reduced antitubulin-
related toxicity.
Podophyllotoxin (2) (Figure 2) is another antimitotic
agent that binds at the colchicine domain.10 Like
* To whom correspondence should be sent at the University of North
Carolina. Phone: (919)-962-0066. E-mail: KHLEE@UNC.EDU.
Fax: (919)-966-3893.
† For part 184, see ref 1.
In our continuing investigation of bioactive colchici-
noids, N-(trifluoroacetyl)-1,2,3,10-tetrademethyldeacetyl-
‡ University of North Carolina.
§ Laboratory of Drug Discovery Research and Development, NCI.
S0022-2623(98)00007-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/29/1998

Antitumor Agents. 185
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1957
Ta ble 1. Evaluation of Phenolic Thiocolchicines as Inhibitors
of Tubulin and Topoisomerase II in Vitro
chlorides. Finally, exhaustive demethylation of 7 and
13-26 with excess boron tribromide gave rise to the
corresponding demethylated 7a and 13a -26a .24 Reac-
tion of compound 14a with HCl formed its water-soluble
hydrochloride salt 14b. Esterification of compound 16a
with the appropriate acid chlorides and with methyl
chloroformate in the presence of pyridine and DMAP25
afforded the esters 27-29 and carbonate 30, respec-
tively (Scheme 2).
IC50 (µM)
compd
DNA topo II inhibitiona
tubulin polymerizationb
Biologica l Resu lts a n d Discu ssion s
1
2
3
7
8
INc
IN
38
IN
IN
IN
IN
>150
38
1.5d
1.0e
IN
0.65
4
2
2
4
4
Table 1 shows the in vitro activities of phenolic
congeners (8-13) of thiocolchicine against mammalian
DNA topoisomerase II and tubulin polymerization. All
phenolic A ring derivatives had reduced inhibitory
effects on tubulin polymerization, with removal of all
three methyl groups (7a ) being the most deleterious
(IC50 > 40 µM). In contrast, in the topoisomerase II
inhibition assay, the completely demethylated 7a dis-
played the highest activity (IC50 ) 9 µM, 4-fold more
active than etoposide). Catechol 12 was as inhibitory
as etoposide; however, monophenols 8-10 and catechol
11 did not inhibit topoisomerase II. Interestingly, as
more methyl ether groups on A ring are cleaved, tubulin
inhibition decreases and topoisomerase II inhibition
increases. Therefore, complete demethylation of the A
ring is optimal for topoisomerase II inhibitory activity
in vitro.
9
10
11
12
7a
9
>40
a
Measured as ATP-dependent unknotting of P4 DNA compared
to enzyme and DNA control reactions. Etoposide (100 µM)
completely inhibited the unknotting activity. b Concentration which
inhibits tubulin polymerization by 50% compared to the control.
c IN ) inactive. Data from ref 8. e Data from ref 10.
d
To extend these SAR studies, we prepared several
exhaustively demethylated thiocolchicine derivatives
with variable side chains at C-7. Most modifications of
the C-7 substituent resulted in compounds active toward
topoisomerase II. Complete inhibition of enzyme activ-
ity was observed at drug concentrations of 100 µM in
preliminary in vitro topo II screening (Table 2). Thus,
side chain modifications in the B ring are tolerated in
the topoisomerase II enzyme inhibition assay. A few
exceptions are compounds 15a , 23a , and 24a , which
possess a large side chain. Their inactivity suggests
that increasing the steric bulk of the nitrogen substitu-
ent decreases the inhibitory activity of these molecules
toward topoisomerase II. The target compounds were
also screened against topoisomerase I; however, only
compounds 16a and 26a interacted with both topoi-
somerases. In addition, all target compounds did not
interfere with tubulin polymerization.
Table 3 shows the in vitro cytotoxic activities of
selected target compounds against five panels of human
tumor cell lines. For comparison, colchicine was also
evaluated. Generally, the demethylthiocolchicine de-
rivatives were considerably less toxic than colchicine in
most cell lines. Compounds 7a with an unmodified side
chain and 13a with a hydrolyzed side chain were not
cytotoxic. Among the compounds containing variable
side chains, 16a was active against KB cells and 26a
was active against both KB and MCF-7 cells. Com-
pound 14b, a salt form of 14a , showed enhanced
cytotoxic effects toward three cell lines compared to its
parent compound. The increased cytotoxicity might be
due to improved water solubility.
F igu r e 3. Structures of demethylisocolchicine analogues.
isocolchicine (5) and N-(3′,4′,5′-trihydroxybenzoyl)-1,2,3,-
10-tetrademethyldeacetylisocolchicine (6) (Figure 3)
demonstrated interesting activities not shared by 1.16
These properties include the ability to induce intracel-
lular protein-linked DNA breaks, inhibit DNA topoi-
somerase II in vitro, and exert cytotoxicity that is not
affected by overexpression of p-glycoprotein.17 This
encouraging discovery brought a new vision for ad-
ditional investigation of colchicinoids.
The major goal of the present work is to determine
the structural requirements and B ring side chain
modifications for specific inhibition of DNA topoi-
somerase II activity. Therefore, regioselectively and
exhaustively demethylated thiocolchicine analogues
were synthesized and evaluated in vitro for topoi-
somerase I and II inhibitory, antitubulin, and cytotoxic
activities. Limited mechanism of action studies of
selected compounds are also reported in this paper.
Ch em istr y
Monophenols 8 and 10 were prepared according to
published methods.18,19 An improved procedure of re-
gioselective demethylation of thiocolchicine to phenol 9
and catechols 11 and 12 has been reported recently by
our laboratory.20
As shown in Scheme 1, all demethylated compounds
were synthesized from thiocolchicine (7) and deacetylth-
iocolchicine (13), which were prepared from 1 by litera-
ture procedures.21 Reaction of 13 with 4-aminobenzoic
acid/DCC afforded 14, which was then acylated with (S)-
(-)-camphanic chloride to give 15.22,23 Similarly, N-
acyldeacetylthiocolchicinoids 16-26 were obtained by
acylation of 13 with various commercially available acyl
These demethylthiocolchicine derivatives are not very
potent as cytotoxic agents (for significant activity of a
pure compound, EC50 e 4.0 µg/mL is required). Their
weak cytotoxicity may result from the multiple phenolic

1958 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Guan et al.
Sch em e 1. Synthesis of Exhaustive Demethylation of Thiocolchicine Analogues 7a , 13a -26a , and 14ba
a
Reagent: (a) NaSCH3, H2O, (b) 10 equiv of BBr3/CH2Cl2, 0 °C; (c) 2 N HCl, MeOH, reflux; (d) RCOCl, CH2Cl2/pyr, room temperature;
(e) 4-aminobenzolic acid, CH2Cl2, DCC, room temperature; (f) (1S)-(-)-camphanic chloride, CH2Cl2/pyr, room temperature; (g) 4 M HCl/
dioxane.
hydroxy groups, making these compounds too hydro-
philic to pass through the cell plasma membrane. To
improve the lipid solubility, we prepared three esters
27-29 and one carbonate 30 by protecting all three
hydroxy groups of 16a . According to the prodrug
hypothesis, esters and carbonates should be hydrolyzed
intracellularly by esterases to generate the hydroxy
groups of the parent compound. As the hydroxy groups
were blocked, all prodrugs (27-30) became inactive
against either topoisomerase or tubulin (Table 2). This
lack of an antitopoisomerase effect further confirmed
that the hydroxy groups are required for enzyme inhibi-
tion. Hydrogen bonds formed between the hydroxy
groups and the residues of its binding pocket might play
an important role in drug-enzyme interactions. An in
vitro cytotoxic assay revealed that 27 was about 3 times
more potent that its parent compound 16a in KB cells,
with the EC50 value of 2.7 µg/mL (Table 3). Carbonate
30 also showed improved cytotoxic activity against three
cell lines compared to 16a . However, the prodrugs
containing larger protecting groups (28 and 29) did not
display cytotoxicity superior to that of their parent
compound. Thus, the small protecting groups might be
removed easily by esterases in comparison with the
large protecting groups.
We also examined the ability of compound 16a and
its prodrug 27 to stabilize the cleavable complex in KB
cells. For comparison, etoposide was included as a
positive control. Data obtained are presented in Figure
4. As expected, etoposide produced a significant amount
of enzyme-linked DNA complexes at a 50 µM drug
concentration. In contrast, compound 16a and 27 did
not induce cleavable complex formation compared to the
control. This result indicated that the two compounds
do not arrest the enzyme at its transition state after
DNA strand breakage, and thus, they have a different
inhibitory mechanism of action from that of etoposide.
The ability of 16a and 27 to interfere with etoposide-

Antitumor Agents. 185
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1959
Sch em e 2. Synthesis of Compounds 27-30
In KB cells, demethylthiocolchicines appear inhibit
topoisomerase II without producing cleavable protein-
linked DNA complexes. The precise molecular mecha-
nism of action merits further investigation.
Exp er im en ta l Section
Melting points were measured with a Fisher-J ohns melting
point apparatus without correction. Optical rotations were
determined with a DIP-1000 polarimeter. Proton nuclear
magnetic resonance (1H NMR) spectra were measured on a
Bruker AC-300 spectrometer with Me4Si(TMS) as the internal
reference. Elemental analyses were performed by Atlantic
Microlab Inc., Norcross, GA. MS was determined by NIH.
Thin-layer chromatography (TLC) silica gel plates were pur-
chased from Analtech, Inc. Silica gel (230-400 mesh), from
Aldrich, was used for column chromatography. MCI gel was
purchased from Supelco.
Gen er a l P r oced u r es for Exh a u stive Dem eth yla tion of
N-Acyld ea cetylth iocolch icin es (7a a n d 13a -26a ). To a
solution of starting material in anhydrous CH2Cl2 was added
dropwise a 1 M solution of boron tribromide in CH2Cl2 (molar
ratio of 1:10) under ice cooling. The reaction mixture was
stirred at room temperature for 20-24 h. The reaction
mixture was cooled in an ice bath, and MeOH (20 mol) was
added dropwise. The solution was refluxed for 2 h, and then
the solvent was removed in vacuo. The residue was purified
by MCI Gel CHP-20P column chromatography using water and
MeOH as eluents.
Ta ble 2. Evaluation of Demethylthiocolchicines as Inhibitors
of Tubulin and Topoisomerases in Vitro
% inhibition of DNA
compd Topo II activity Topo I activity tubulin polymerization
no.
(100 µM)a
(100 µM)b
IC50 (µM)c
13a
14a
14b
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27
100
100
100
0
100
100
100
100
100
100
100
0
0
0
0
0
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
Tr id em eth ylth iocolch icin e (7a ): yield 61.5% (starting
from 108.5 mg of 7); amorphous; [R]25 -282.27° (c 0.11,
D
1
MeOH); H NMR (pyridine) δ 2.0 (s, 3H, COCH3), 2.1 (s, 3H,
SCH3-10), 1.88-2.9 (m, 4H, H-5,6), 5.29 (m, 1H, H-7), 6.79 (s,
1H, H-4), 6.69 (d, J ) 10.4 Hz, 1H, H-11), 7.70 (d, J ) 10.3
Hz, 1H, H-12), 9.24 (d, J ) 7.1 Hz, 1H, NHCO); CIMS m/z
373 M+. Anal. (C19H19NSO5‚13/4H2O) C, H, N.
<100
0
0
0
0
0
0
0
0
0
Tr id em et h yld ea cet ylt h iocolch icin e h yd r ob r om id e
(13a ): yield 15.8% (starting from 150.6 mg of 13); amorphous;
[R]25 -188.9° (c 0.12, MeOH); 1H NMR (CD3OD) δ 1.9-2.3
D
(m, 4H, H-5,6), 2.49 (s, 3H, SCH3-10), 4.06 (m, 1H, H-7), 6.32
(s, 1H, H-4), 7.0 (s, 1H, H-8), 7.4 (d, J ) 10.6 Hz, 1H, H-11),
7.57 (d, J ) 10.5 Hz, 1H, H-12); CIMS m/z 366 (M + 2H)+.
Anal. (C17H18NSO4Br‚1/2C2H5OH) C, H, N.
0
100
100
0
0
0
100
0
0
0
0
T r i d e m e t h y l-N -(4′-a m i n o b e n z o y l)d e a c e t y lt h i o -
colch icin e (14a ): yield 40% (starting from 168.7 mg of 14);
28
29
30
amorphous; [R]25 -85.9° (c 0.14, DMSO); 1H NMR (DMSO) δ
D
0
2.0-2.1 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-10), 4.61 (m, 1H,
H-7), 5.67 (s, 2H, NH2, 6.24 (s, 1H, H-4), 6.54 (d, J ) 8.4 Hz,
2H, H-3′,5′), 7.10 (s, 1H, H-8), 7.21 (d, J ) 10.4 Hz, 1H, H-11),
7.29 (d, J ) 10.7 Hz, 1H, H-12), 7.61 (d, J ) 8.6 Hz, 2H,
H-2′,6′), 8.43 (d, J ) 6.7 Hz, 1H, NHCO), 8.44 (s, 1H, OH-1),
8.56 (s, 1H, OH-2), 9.33 (s, 1H, OH-3); CIMS m/z 449 (M +
H)+. Anal. (C24H22N2SO5‚1/2H2O) C, H, N.
a
Measured as ATP-dependent unknotting of P4 DNA compared
to enzyme and DNA control reactions. Etoposide (100 µM)
completely inhibited the unknotting activity. Measured as ATP-
independent relaxation of supercoiled plasmid DNA compared to
enzyme and DNA control reaction. Camptothecin at 100 µM served
as the positive control. c Concentration which inhibits tubulin
polymerization by 50% compared to the control.
b
Tr id em eth yl-N-(4′-ca m p h a n oylben zoyl)-d ea cetylth io-
colch icin e (15a ): yield 18% (starting from 536 mg of 15);
induced protein-linked DNA complex formation was also
examined at a 80 µM drug concentration. In comparison
with the positive control, 16a stimulated the ternary
complex adducts induced by etoposide. Therefore, 16a
did not block DNA binding with the enzyme, otherwise
DNA would not be available to be trapped by etoposide.
Surprisingly, no statistically significant stimulation of
etoposide-induced cleavage complex was observed with
compound 27. The different behavior of the two com-
pounds questioned the prodrug hypothesis.
In conclusion, complete demethylation of thiocolchi-
cine in the A ring of colchicine is required for maximal
topoisomerase II inhibitory activity in vitro. Generally,
demethylthiocolchine analogues are selective topoi-
somerase II inhibitors without antitubulin activity. The
cytotoxicities were improved by side chain modifications
and further elevated by protecting the hydroxy groups.
1
amorphous; [R]25 -104.4° (c 0.12, MeOH); H NMR (DMSO)
D
δ 0.91 (s, 3H, camphanoyl CH3), 1.05 (s, 6H, camphanoyl CH3),
1.94-2.11 (m, 8H, H-5,6 and camphanoyl H-4,5), 2.40 (s, 3H,
SCH3-10), 4.65 (m, 1H, H-7), 6.26 (s, 1H, H-4), 7.10 (s, 1H,
H-8), 7.23 (d, J ) 10.8 Hz, 1H, H-11), 7.30 (d, J ) 10.4 Hz,
1H, H-12), 8.86 (d, J ) 7.7 Hz, 1H, NHCO); CIMS m/z 630 (M
+ H)+. Anal. (C34H34N2SO8‚1/2H2O) C, H, N.
T r i d e m e t h y l -N -(4 ′-n i t r o b e n z o y l )d e a c e t y l t h i o -
colch icin e (16a ): yield 30% (starting from 102 mg of 16);
1
amorphous; [R]25 -119.9° (c 0.09 MeOH); H NMR (DMSO)
D
δ 2.08-2.21 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-10), 4.65 (m,
1H, H-7), 6.26 (s, 1H, H-4), 7.09 (s, 1H, H-8), 7.24 (d, J ) 10.8
Hz, 1H, H-11), 7.32 (d, J ) 10.5 Hz, 1H, H-12), 8.10 (d, J )
8.7 Hz, 2H, H-3′,5′), 8.35 (d, J ) 8.8 Hz, 2H, H-2′,6′), 8.48 (s,
1H, OH-1), 8.60 (s, 1H, OH-2), 9.29 (d, J ) 7.5 Hz, 1H, NHCO),
9.37 (s, 1H, OH-3); CIMS m/z 481 (M + H)+. Anal. (C24H20N2-
SO7‚13/4H2O) C, H, N.
T r i d e m e t h y l -N -(3 ′-n i t r o b e n z o y l )d e a c e t y l t h i o -
colch icin e (17a ): yield 60% (starting from 845.3 mg of 17);

1960 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Guan et al.
Ta ble 3. Inhibition of in Vitro Tumor Cell Growtha,b by Selected Demethylthiocolchicines and Prodrugs 27-30
cytotoxicity EC50 (µg/mL)c
compd
KB
A549
MCF-7
CAKI-1
SK-MEL-2
1
7a
0.002
>17.8 (0)e
>13.2 (0)
>18.0 (35)
4.8
7.7
9.6
2.7
12.3
0.022
>0.4 (33-45)d
13.6
>13.2 (23)
14.8
10.7
>9.6 (38)
8.6
>0.15 (23-48)
>12.9 (40)
>27.6 (43)
13.1
0.4
0.008
>17.8 (0)
>13.2 (0)
>18.0 (0)
>19.5 (42)
>9.6 (0)
>9.6 (30)
>0.3 (26-41)
12.9
>17.8 (0)
>13.2 (0)
>18.0 (39)
9.7
>9.6 (36)
>9.6 (30)
>0.15 (18-43)
>25.9 (29)
>27.6 (19)
24.2
>17.8 (22)
>13.2 (2)
>18.0 (13)
>19.5 (37)
>9.6 (20)
>9.6 (29)
>0.3 (12-38)
>25.9 (16)
>27.6 (17)
>26.2 (38)
13a
14a
14b
16a
26a
27
28
29
30
25.5
6.5
27.6
>26.2 (44)
a
b
Data obtained from our in-house in vitro disease-oriented human tumor cell screen. KB, epidermoid carcinoma of the nasopharynx;
A549, lung carcinoma; MCF-7, breast adenocarcinoma; CAKl-1, kidney carcinoma; SK-MEL-2, malignant melanoma. c EC50 is the
d
concentration of compound that causes a 50% reduction in absorbance at 562 nm relative to untreated cells using SRB assay. Plateau
dose response observed. e Observed percent inhibition in parentheses.
7.6 Hz, 1H, NHCO); CIMS m/z 453 (M + H)+. Anal. (C24H20
NSO5F‚1/2H2O) C, H, N.
-
Tr id e m e t h yl-N -(4′-ch lor ob e n zoyl)d e a ce t ylt h iocol-
ch icin e (21a ): yield 36% (starting from 100 mg of 21);
1
amorphous; [R]25 -41.7° (c 0.57, acetone); H NMR (DMSO)
D
δ 2.0-2.2 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-10), 4.65 (m, 1H,
H-7), 6.25 (s, 1H, H-4), 7.10 (s, 1H, H-8), 7.23 (d, J ) 10.7 Hz,
1H, H-11), 7.31 (d, J ) 10.4 Hz, 1H, H-12), 7.57 (d, J ) 8.5
Hz, 2H, H-3′,5′), 7.90 (d, J ) 8.6 Hz, 2H, H-2′,6′), 8.47 (s, 1H,
OH-1), 8.50 (s, 1H, OH-2), 9.0 (d, J ) 7.6 Hz, 1H, NHCO),
9.36 (s, 1H, OH-3); CIMS m/z 470 M+. Anal. (C24H20NSO5Cl)
C, H, N.
Tr id em eth yl-N-h exa n oyld ea cetylth iocolch icin e (22a ):
yield 39.3% (starting from 330 mg of 22); amorphous; [R]25
D
-270.8° (c 0.06 MeOH); 1H NMR (CD3OD) δ 0.88 (t, 3H,
hexanoyl-CH3), 1.27-1.89 (m, 8H, hexanoyl-CH2), 2.0-2.1(m,
4H, H-5,6), 2.45 (s, 3H, SCH3-10), 4.50 (m, 1H, H-7), 6.29 (s,
1H, H-4), 7.16 (s, 1H, H-8), 7.33 (d, J ) 10.5 Hz, 1H, H-11),
7.48 (d, J ) 10.4 Hz, 1H, H-12); CIMS m/z 429 (M + H)+. Anal.
(C23H27NSO5) C, H, N.
F igu r e 4. Effects of compounds 16a and 27 on cellular
protein-DNA complexes formation in KB cells.
Tr idem eth yl-N-adam an tyldeacetylth iocolch icin e (23a):
yield 75.4% (starting from 215.8 mg of 23); amorphous; [R]25
amorphous; [R]25 -90.7° (c 0.17, DMSO); 1H NMR (DMSO) δ
D
D
-176.8° (c 0.11 DMSO); 1H NMR (DMSO) δ 1.66 (s, 6H,
adamantyl CH2), 1.77 (s, 6H, adamantyl CH2), 1.98 (s, 3H,
adamantyl CH), 1.9-2.1 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-
10), 4.41 (m, 1H, H-7), 6.22 (s, 1H, H-4), 7.02 (s, 1H, H-8), 7.20
(d, J ) 10.7 Hz, 1H, H-11), 7.26 (d, J ) 10.4 Hz, 1H, H-12);
CIMS m/z 493 (M + H)+. Anal. (C28H31NSO5‚1/2CH3CO2C2H5)
C, H, N.
2.0-2.2 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-10), 4.70 (m, 1H,
H-7), 6.27(s, 1H, H-4), 7.11 (s, 1H, H-8), 7.25 (d, J ) 10.7 Hz,
1H, H-11), 7.33 (d, J ) 10.4, 1H, H-12), 7.81 (t, 1H, H-5′), 8.31
(d, J ) 7.9 Hz, 1H, H-6′), 8.41 (d, J ) 7.9 Hz, 1H, H-4′), 8.75
(s, 1H, H-2′), 9.33 (d, J ) 7.6 Hz, 1H, NHCO); CIMS m/z 480
(M + H)+. Anal. (C24H20N2SO7‚3/4CH3CO2C2H5) C, H, N.
T r i d e m e t h y l -N -(2 ′-n i t r o b e n z o y l )d e a c e t y l t h i o -
Tr id e m e t h yl-N -ca m p h a n oyld e a ce t ylt h iocolch icin e
(24a ): yield 39.4% (starting from 60.8 mg of 24); amorphous;
colch icin e (18a ): yield 58.7% (starting from 511 mg of 18);
1
amorphous; [R]25 -293.6° (c 0.24, MeOH); H NMR (DMSO)
D
[R]25 -210.4° (c 0.11, DMSO); 1H NMR (DMSO) δ 0.8 (s, 3H,
δ 1.87-2.18 (m, 4H, H-5,6), 2.42 (s, 3H, SCH3-10), 4.61 (m,
1H, H-7), 6.28 (s, 1H, H-4), 7.17 (s, 1H, H-8), 7.25 (d, J ) 10.6
Hz, 1H, H-11), 7.31 (d, J ) 10.4 Hz, 1H, H-12), 7.60 (d, J )
7.3 Hz, 1H, H-6′), 7.72 (t, 1H, H-5′), 7.84 (t, 1H, H-4′), 8.08 (d,
J ) 8.0 Hz, 1H, H-3′), 8.49 (broad, 1H, OH-1), 8.63 (s, 1H,
OH-2), 9.31 (d, J ) 7.7 Hz, 1H, NHCO), 9.38 (s, 1H, OH-3);
CIMS m/z 480 (M + H)+. Anal. (C24H20N2SO7‚1/2H2O) C, H,
N.
D
camphanoyl CH3), 0.90 (s, 6H, camphanoyl CH3), 0.99 (cam-
phanoyl H-4,5), 1.54-1.95 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-
10), 4.62 (m, 1H, H-7), 6.22 (s, 1H, H-4), 7.09 (s, 1H, H-8), 7.22
(d, J ) 10.6 Hz, 1H, H-11), 7.27 (d, J ) 10.4 Hz, 1H, H-12),
8.73 (d, J ) 8.3 Hz, 1H, NHCO); CIMS m/z 512 (M + H)+.
Anal. (C27H29NSO7‚3/4H2O) C, H, N.
T r i d e m e t h y l -N -( 4 ′-b r o m o m e t h y l e n e b e n z o y l ) -
d ea cetylth iocolch icin e (25a ): yield 90.7% (starting from 353
Tr id e m e t h yl-N -(3′,5′-d in it r ob e n zoyl)d e a ce t ylt h io-
colch icin e (19a ): yield 76.3% (starting from 658.2 mg of 19);
mg of 25); amorphous; [R]25 -57.8° (c 0.1, acetone); 1H NMR
D
1
amorphous; [R]25 -38.7° (c 0.7 MeOH); H NMR (DMSO) δ
(CD3OD) δ 1.9-2.2 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-10), 4.68
(m, 1H, H-7), 4.75 (s, 2H, CH2Br-4′), 6.28 (s, 1H, H-4), 7.11 (s,
1H, H-8), 7.23 (d, J ) 10.7 Hz, 1H, H-11), 7.32 (d, J ) 10.4
Hz, 1H, H-12), 7.56 (d, J ) 8.1 Hz, 2H, H-3′,5′), 7.85 (d, J )
8.6 Hz, 2H, H-2′,6′), 8.97 (d, J ) 7.7 Hz, 1H, NHCO); CIMS
m/z 530 (M + 2H)+. Anal. (C25H22NSO5Br‚1/2H2O) C, H, N.
Tr id em eth yl-N-(3′,4′,5′-tr ih yd r oxylben zoyl)d ea cetylth -
iocolch icin e (26a ): yield 23% (starting from 165 mg of 26);
D
2.0-2.2 (m, 4H, H-5,6), 2.40 (s, 3H, SCH3-10), 4.72 (m, 1H,
H-7), 6.27 (s, 1H, H-4), 7.11 (s, 1H, H-8), 7.25 (d, J ) 10.7 Hz,
1H, H-11), 7.32 (d, J ) 10.5 Hz, 1H, H-12), 8.48 (s, 1H, OH-1),
8.63 (s, 1H, OH-2), 8.97 (s, 1H, H-4′), 9.11 (s, 2H, H-2′,6′), 9.37
(s, 1H, OH-3), 9.63 (d, J ) 7.5 Hz, 1H, NHCO); CIMS m/z 525
(M + H)+. Anal. (C24H19N3SO9‚11/2H2O) C, H, N.
Tr id e m e t h yl-N -(4′-flu or ob e n zoyl)d e a ce t ylt h iocol-
ch icin e (20a ): yield 78.5% (starting from 545.6 mg of 20);
amorphous; [R]25 -189.1° (c 0.07, MeOH); 1H NMR (CD3OD)
D
1
amorphous; [R]25 -203.7° (c 0.07 MeOH); H NMR (DMSO)
δ 2.0-2.36 (m, 4H, H-5,6), 2.45 (s, 3H, SCH3-10), 4.73 (m, 1H,
H-7), 6.33 (s, 1H, H-4), 6.89 (s, 2H, H-2′,6′), 7.28 (s, 1H, H-8),
7.37 (d, J ) 10.7 Hz, 1H, H-11), 7.53 (d, J ) 10.6 Hz, 1H, H-12);
CIMS m/z 483 (M + H)+. Anal. (C24H21N3SO8‚21/4H2O) C, H,
N.
D
δ 1.9-2.2 (m, 4H, H-5,6), 2.08 (s, 3H, SCH3-10), 4.67 (m, 1H,
H-7), 6.26 (s, 1H, H-4), 7.10 (s, 1H, H-8), 7.23 (d, J ) 10.7 Hz,
1H, H-11), 7.31 (d, J ) 11.2 Hz, 1H, H-12), 7.35 (d, J ) 8.9
Hz, 2H, H-3′,5′), 7.95 (d, J ) 8.3 Hz, 2H, H-2′,6′), 8.96 (d, J )

Antitumor Agents. 185
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1961
(3) Brossi, A.; Yeh, H. J . C.; Chrzanowska, M.; Wolff, J .; Hamel,
E.; Lin, C. M.; Quin, F.; Suffness, M.; Silverton. J . Colchicine
and Its Analogues: Recent Findings. Med. Res. Rev. 1988, 8,
77-94.
(4) Hastie, S. B. Interaction of Colchicine with Tubulin. Pharm.
Ther. 1991, 51, 377-401 and references therein.
T r i d e m e t h y l-N -(4′-a m i n o b e n z o y l)d e a c e t y lt h i o -
colch icin e h yd r och lor id e (14b): A solution of 14a (50 mg)
in MeOH was allowed to react with 4 M HCl in dioxane for 1
min with TLC monitoring. The reaction mixture was concen-
trated to give a pure powder: yield 100.4%; [R]25 -176.8° (c
D
0.1, CH3OH); 1H NMR (CH3OH) spectrum was identical with
that of 14a . Anal. (C24H23N2SO5Cl‚3/4CHCl3) C, H, N.
Gen er a l P r oced u r es for Syn th esizin g 1,2,3-O-Acyl-
1,2,3 -d em et h yl-N-(4′-n it r ob en zoyl)d ea ct ylt h iocolch i-
cin es 27-30. To a solution of 16a in dry THF was added
pyridine (0.1 mL), DMAP (0.5 equiv), and appropriate acyl
chlorides or methyl chloroformate (10-12 equiv). The mixture
was allowed to stir for 4-8 h at room temperature, and the
volatiles were evaporated in vacuo. The residue was diluted
with water and extracted three times with CHCl3. The organic
layer was washed with brine, dried over anhydrous NaSO4,
and concentrated in vacuo. The residue was chromatographed
on a preparative TLC plate.
(5) Caplan, Y. H.; Orloff, K. G.; Thompson, B. C. A Fatal Overdose
with Colchicine. J . Anal. Toxicol. 1980, 4, 153-155.
(6) Staretz, M. E.; Hastie, S. B. Synthesis and Tubulin Binding of
Novel-10 Analogues of Colchicine. J . Med. Chem. 1993, 36, 758-
764.
(7) Muzaffar, A.; Brossi, A.; Lin, C. M.; Hamel, E. Antitubulin
Effects of Derivatives of 3-Demethylthiocolchicine, Methylthio
Ethers of Natural Colchicinoids, and Thioketones Derived from
Thiocolchicine. Comparison with Colchicinoids. J . Med. Chem.
1990, 33, 567-571.
(8) Pei, X. F.; Lin, C. M.; Hamel, E.; Brossi, A. N-acetoacetamides
of the Thiocolchicine Series: Synthesis and Inhibition of Tubulin
Polymerization. Med. Chem. Res. 1994, 4, 563-568.
(9) Zweig, M. H.; Chignel, C. F. Interaction of Some Colchicine
Analogs, Vinblastine and Podophyllotoxin with Rat Brain Mi-
crotubule Protein. Biochem. Pharmacol. 1973, 22, 2141-2150.
(10) Hoebeke, J .; Van Nijen, G. Quantitative Turbidimetric Assay
for Potency Evaluation of Colchicine-like Drug. Life Sci. 1975,
17, 591-595.
(11) Wilson, L.; Bryan, J . Biochemical and Pharmacological Proper-
ties of Microtubules. Adv. Cell. Mol. Biol. 1974, 3, 21-72 and
literature cited therein.
(12) Clark, P. I.; Slevin, M. L. The Clinical Pharmacology of Etoposide
and Teniposide. [Review]. Clin. Pharmacokinet. 1987, 12, 223-
252.
(13) J ardine, I. In Anticancer Agents Based on Natural Products
Model; Cassady, J . M.; Douros, J ., Eds.; Academic Press: New
York, 1980; pp 319-351 and references therein.
(14) Issell, B. F.; Muggia, F. M.; Cartes, S. K. Etoposide [VP-16]
Current Status and New Developments; Academic Press: Or-
lando, 1984; pp 1-353.
1,2,3-O-Ac e t yl-1,2,3-d e m e t h yl-N -(4′-n it r ob e n zoyl)-
d ea cetylth iocolch icin e (27): yield 46.6% (starting from 52
mg of 16a ); amorphous; [R]25 -47.6° (c 0.1, DMSO); 1H NMR
D
(DMSO) δ 2.12-2.22 (m, 4H, H-5,6), 2.9 (s, 3H, CH3CO2-1),
2.31 (s, 3H, CH3CO2-2), 2.34 (s, 3H, CH3CO2-3), 2.43 (s, 3H,
SCH3-10), 4.59 (m, 1H, H-7), 6.99 (d, J ) 10.3 Hz, 1H, H-11),
7.09 (s, 1H, H-4), 7.26 (s, 1H, H-8), 7.31 (d, J ) 10.6 Hz, 1H,
H-12), 8.11 (d, J ) 8.7 Hz, 1H, H-3′,5′), 8.37 (d, J ) 8.7 Hz,
1H, H-2′,6′), 9.30 (d, J ) 7.2 Hz, 1H, NHCO); CIMS m/z 606
M+. Anal. (C30H26N2SO10) C, H, N.
1,2,3-O-P r op ion yl-1,2,3-d em eth yl-N-(4′-n itr oben zoyl)-
d ea cetylth iocolch icin e (28): yield 35.3% (starting from 62.8
1
mg of 16a ); amorphous; [R]25 +55° (c 0.85, CHCl3); H NMR
D
(CDCl3) δ 1.17-1.39 (m, 9H, CH3CH2CO2-1,2,3), 2.14-2.27 (m,
4H, H-5,6), 2.44 (s, 3H, SCH3-10), 2.51-2.69 (m, 6H, CH3CH2-
CO2-1,2,3), 5.02 (m, 1H, H-7), 7.02 (d, J ) 10.3 Hz, 1H, H-11),
7.05 (s, 1H, H-4), 7.13 (d, J ) 10.6 Hz, 1H, H-12), 7.58 (s, 1H,
H-8), 8.08 (d, J ) 8.7 Hz, 2H, H-3′,5′), 8.16 (d, J ) 8.7 Hz, 2H,
H-2′,6′); CIMS m/z 649 (M + H)+. Anal. (C33H32N2SO10) C,
H, N.
(15) Smith, P. J .; Soue`s, S. Multilevel Therapeutic Targeting by
Topoisomerase Inhibitors. Br. J . Cancer Suppl. 1994, 23, S47-
51.
(16) Bastow, K. F.; Tatematsu, H.; Sun, L.; Fukushima, Y.; Lee, K.-
H. Synthesis and Biological Evaluation of Tetrademethyl iso-
colchicine Derivatives as Inhibitors of DNA Topoisomerase
Action In Vitro. Bioorg. Med. Chem. 1993, 3, 227-234.
(17) Bastow, K. F.; Tatematsu. H.; Bori, I. D.; Fukushima, Y.; Sun,
L.; Goz, B.; Lee, K.-H. Induction of Reversible Protein-Linked
DNA Breaks in Human Osteogenic Sarcoma Cells by Novel
Cytocidal Colchicine Derivatives Which Inhibit DNA Topoi-
somerase II In Vitro: Absence of Cross-Resistance in A Colchi-
cine-Resistance Sub-clone. Bioorg. Med. Chem. Lett. 1993, 3,
1045-1050.
(18) Muzaffar, A.; Chrzanowdka, M.; Brossi, A. Phenolic Congeners
of Colchicine: Preparation and Characterization of Phenolic and
Catecholic Analogues of Colchicine, Colchiceine and Thiocolchi-
cine. Heterocycles 1989, 28, 365-372 and reference sited therein.
(19) Sharma, P. N.; Brossi, A. Selective Ether-Cleavage of Thio-
chochicoside and Thicolchicine: Characterization of 3- and 2-
Demethylthiocolchicine and Catecholic Congeners. Heterocycles
1983, 20, 1587-1590.
(20) Guan, J .; Brossi, A.; Zhu, X. K.; Wang, H. K.; Lee, K.-H.
Oxidation Products of Phenolic Thiocolchicines: Ring A Quino-
nes and Dienones. Synth. Commun., in press.
(21) Hamel, E. Interactions of Tubulin with Small Ligands. In
Microtubule Proteins; Avila, J ., Ed.; CRC Press: Boca Raton,
FL, 1990; pp 89-191 and references therein.
(22) Sun, L.; Hamel, E.; Lin, C. M.; Hastie, S, B.; Pyluck, A.; Lee,
K.-H. Synthesis and Biological Evaluation of Novel Thiocolchi-
cine Analogs: N-acyl-, N-aroyl-, and N-(substitutedbenzyl)-
deacetylthiocolchicines as Potent Cytotoxic and Antimitotic
Compounds. J . Med. Chem. 1993, 36, 1474-1479.
(23) Sun, L.; McPhail, A. T.; Hamel, E.; Lin, C. M.; Hastie, S. B.;
Chang, J . J .; Lee, K.-H. Synthesis and Biological Evaluation of
Thiocolchicine Analogs 5,6-Dihydro-6(S)-(acyloxy)- and 5,6-Di-
hydro-6(S)-[(aroyloxy)methyl]-1,2,3-trimethoxy-9-(methylthio)-
8H-cyclohepta[a]naphthalen-8-ones as Novel Cytotoxic and An-
timitotic Agents. J . Med. Chem. 1993, 36, 544-551.
(24) McOmie, J . F. W.; Watts, M. L.; West, D. E. Demethylation of
Aryl Methyl Ethers by Boron Tribromide. Tetrahedron 1968, 24,
2289-2292.
1,2,3-O-Isobu tyr yl-1,2,3-d em eth yl-N-(4′-n itr oben zoyl)-
d ea cetylth iocolch icin e (29): yield 68.8% (starting from 58.2
mg of 16a ); amorphous; [R]25D +30.8° (c 0.42, CHCl3); 1H NMR
(CDCl3) δ 1.15-1.38 (m, 18H, (CH3)2CHCO2-1,2,3), 2.43 (s, 3H,
SCH3-10), 2.48-2.5 (m, 4H, H-5,6), 2.64-2.83 (m, 3H,
(CH3)2CHCO2-1,2,3), 5.02 (m, 1H, H-7), 7.01 (d, J ) 10.6 Hz,
1H, H-11), 7.06 (s, 1H, H-4), 7.12 (d, J ) 10.3 Hz, 1H, H-12),
7.53 (s, 1H, H-8), 8.07 (d, J ) 8.8 Hz, 2H, H-3′,5′), 8.15 (d, J
) 8.8 Hz, 2H, H-2′,6′); CIMS m/z 691 (M + H)+. Anal.
(C36H38N2SO10) C, H, N.
1,2,3-Meth oxylca r bon yl-N-(4′-n itr oben zoyl)d ea cetylth -
iocolch icin e (30): yield 11.8% (starting from 106 mg of 16a );
amorphous; [R]25 -50° (c 0.12,DMSO); 1H NMR (DMSO) δ
D
2.09-2.9 (m, 4H, H-5,6), 2.43 (s, 3H, SCH3-10), 3.81 (s, 3H,
CH3OCO-1), 3.89 (s, 3H, CH3OCO-2), 3.90 (s, 3H, CH3OCO-
3), 4.60 (m, 1H, H-7), 7.06 (d, J ) 10.2 Hz, 1H, H-11), 7.11 (s,
1H, H-4), 7.32 (d, J ) 10.6 Hz, 1H, H-12), 7.49 (s, 1H, H-8),
8.12 (d, J ) 8.7 Hz, 2H, H-3′,5′), 8.37 (d, J ) 8.8 Hz, 2H,
H-2′,6′), 9.32 (d, J ) 7.2 Hz, NHCO); CIMS m/z 655 (M + H)+.
Anal. (C30H26N2SO13‚3/4CH3CO2C2H5) C, H, N.
Ack n ow led gm en t. This work was supported by
Grants CA 54508 and CA 17625 (in part) from the
National Cancer Institute awarded to K.-H. Lee.
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J M980007F