ml) was added a solution of hydroxylamine-O-sulfonic acid
(100 mg) in formic acid (12 ml). A few drops of sulfuric acid
were added and the solution was refluxed for 2 d. During this
time hydroxylamine-O-sulfonic acid (300 mg) was added in
portions. The reaction was monitored with TLC (dichloro-
methane–acetone, 9:1). The reaction mixture was poured on
ice–saturated NaHCO3 when no further reaction could be
monitored. Chloroform was added and the phases were separ-
ated and the water phase was extracted twice with chloroform.
The combined organic phases were washed once with water,
dried (MgSO4) and concentrated to a yellow solid. Flash chro-
matography (dichloromethane–acetone, 20:1) gave yellow crys-
tals which could be recrystallized from ethanol (96%) to afford
yellow crystals. Yield 35–41%; mp 226–227 ЊC; ν/cmϪ1 (film):
2940, 1605, 1574, 1491, 1465, 1343, 1315, 1105, 1016 and 932;
NMR see Table 2; MS: C20H19NO5S, calc. 385.0984; found
385.0984.
7-Oxodeacetamidothiocolchicine oximes, 4a and 4b. 7-
Oxodeacetamidothiocolchicine (267 mg, 0.72 mmol) was partly
dissolved in 92% methanol (14 ml). Hydroxylamine hydro-
chloride (56 mg, 0.81 mmol) and sodium carbonate (22 mg,
0.27 mmol) were added and the mixture was refluxed. After
24 h water and ethyl acetate were added and the aqueous and
organic phases were separated. The water phase was extracted
three times with ethyl acetate and the combined organic phases
were dried (Na2SO4) and concentrated, yielding a yellow solid
as a mixture of isomers. The isomers were separated by flash
chromatography (ethyl acetate–heptane) yielding 4b as the first
eluted isomer and 4a as the last eluted isomer. The oximes were
recrystallized from ethanol (96%) to afford yellow crystals. The
oximes had the following physical properties: 4a: Yield 22%;
mp 237–238 ЊC (decomp.); ν/cmϪ1 (film): 3400, 2938, 1601,
1563, 1488, 1345, 1322, 1135, 1028 and 750; δH (CDCl3) 7.32
(1H, d, J 10.5), 7.10 (1H, d, J 10.5), 7.0 (1H, s), 6.68 (1H, s),
6.54 (1H, s), 3.90 (3H, s), 3.89 (3H, s), 3.58 (3H, s), 2.9–2.6
(4H, m); δC(CDCl3) 181.8, 159.5, 158.6, 153.5, 151.6, 143.4,
141.5, 136.2, 135.4, 134.6, 132.1, 126.5, 126.2, 107.6, 61.9,
61.3, 56.0, 37.5, 29.7, 15.3; MS: C20H21NO5S, calc. 387.1140;
found 387.1138. 4b: Yield 63%; mp 236–238 ЊC (decomp.);
ν/cmϪ1 (film): 3350, 2934, 1596, 1524, 1489, 1456, 1346, 1137,
1097 and 989; δH (CDCl3) 9.5 (1H, s), 7.34 (1H, s), 7.23
(1H, d, J 10.5), 7.10 (1H, d, J 10.6), 6.54 (1H, s), 3.90 (1H, s),
3.89 (1H, s), 3.54 (1H, s), 3.5–3.4 (2H, m), 2.8–2.5 (2H, m);
δC(CDCl3) 182.7, 161.6, 159.2, 154.0, 152.0, 145.6, 141.7,
137.9, 136.5, 135.8, 135.2, 127.3, 126.0, 107.1, 61.7, 61.6, 56.4,
32.8, 29.9, 15.6. The X-ray analysis of 4b will be published
elsewhere.
Fig. 4 Competition fluorescence development for the binding of
MTC (2 µ) with tubulin (2 µ) and subsequent addition of 1 or 2 (4 µ)
Compound 3 does not show any sign of binding. The effect of
variation of the 10-substituent has been studied including an
unsubstituted analogue and a rather large tolerance to substi-
tution in this position was found.24 The unsubstituted deriv-
ative was active.
Conclusions
Three new colchicine derivatives have been synthesized and
characterized. All three compounds are atropisomers and exist
as racemates, and rotation around the pivot bond is rapid at
room temperature, with rotational barriers in the range 60–77
kJ molϪ1. The dihedral angles between planes A and C are 61.9
and 46.4Њ, for 1 and 2, respectively; two rather extreme values
for colchicinoids possessing an intact ring skeleton. Solution
studies and molecular mechanics computations give structures
which agree with crystal structures with the exception of the
orientation of the 1- and 2-methoxy groups for 2. In vivo and
in vitro experiments indicate that the compounds 1 and 2 bind
to the colchicine binding site of tubulin, but 1 is considerably
more active than 2. This is consistent with earlier findings that
colchicine analogues with small values of the A–C dihedral
angle are less active.6 Compound 3 does not seem to bind not-
ably. A quantitative analysis of the tubulin binding properties
of these and other colchicine analogues is in progress.
10,11,12-Trimethoxy-7,8-dihydro-4H-benzo[1,2]heptaleno-
[5,6-cd]isoxazol-4-one, 3. The oxime 4a (25 mg, 0.065 mmol)
was dissolved in 92% methanol (18 ml). The mixture was heated
until a clear solution was obtained. Sodium carbonate (28 mg)
was added and the solution was refluxed for 3 h. After evapor-
ation of the methanol, 2 HCl was added and the aqueous
solution was extracted twice with chloroform. The combined
organic phases were dried and concentrated to a yellow solid.
Purification with flash chromatography (ethyl acetate–heptane)
yielded a yellow solid (17 mg, 77%), which could not be
recrystallized. ν/cmϪ1 (film): 2937, 1628, 1577, 1507, 1490, 1344,
1250, 1198, 1143 and 1116; NMR, see Table 2; MS: C19H17NO5,
calc. 339.1107; found 339.1087.
Experimental
Syntheses
7-Oxodeacetamidothiocolchicine, 1. To deacetylthiocolchi-
cine (600 mg, 1.55 mmol) in a mixture of dichloromethane and
dimethylformamide (DMF) (3:1, 48 ml) was added 4-formyl-1-
methylpyridinium toluene-p-sulfonate (680 mg, 2.3 mmol). The
resulting mixture was refluxed for 3 h. The reaction mixture was
cooled in an ice–water bath and 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) (800 mg, 5.2 mmol) was added dropwise with
stirring to afford a deep-purple solution. After 20 min oxalic
acid (60 ml, 5%) was added to the vigorously stirred solution.
The stirring was continued for 90 min at room temp. The aque-
ous and organic phases were separated and the water phase was
extracted twice with dichloromethane. The combined organic
phases were dried (MgSO4) and concentrated to a yellow solid.
Recrystallization from ethanol (96%) afforded yellow crystals
as fine needles. Yield 67%; mp 228–229 ЊC; ν/cmϪ1 (film): 2938,
1702, 1601, 1562, 1488, 1344, 1095, 1025, 954 and 891; NMR
see Table 2; MS: C20H20O5S, calc. 372.1031; found 372.1033.
3-Methylthio-10,11,12-trimethoxy-7,8-dihydro-4H-benzo-
[1,2]heptaleno[5,6-cd]isoxazol-4-one, 2. To 7-oxodeacetamido-
thiocolchicine 1 (137 mg, 0.37 mmol) in 97% formic acid (9
X-Ray crystallography
Crystals of 1 and 2 were grown from ethanol solutions. The
crystals were pale yellow (001) and (100) plates, respectively.
Crystallographic data were collected on a modified Nicolet P3
diffractometer under control of local software.25 Graphite-
monochromated Cu-Kα radiation (λ = 1.5418 Å) was used.
Table 6 summarizes the crystal data, data collection and struc-
ture refinement. Three intensity standards were checked every
60 min. They showed only negligible decay. The intensity data
1702
J. Chem. Soc., Perkin Trans. 2, 1997
Berg, Ulf
