Synthesis and biological evaluation of A-ring biaryl-carbamate analogues of rhazinilam

Article abstract of DOI:10.1016/S0968-0896(02)00270-5

An improvement of the synthesis of biphenyl-carbamate 2a, the most active analogue of rhazinilam 1 so far, was performed using the Pd-catalyzed borylation/Suzuki coupling (BSC) method developed in our laboratories. The preparation of A-ring analogues of 2a bearing electron-withdrawing or donating groups is reported according to this new synthetic scheme. The antitubulin properties as well as the cytotoxicity of these compounds toward human cancer cell lines were evaluated in comparison with rhazinilam and 2a.

Full text of DOI:10.1016/S0968-0896(02)00270-5

Bioorganic & Medicinal Chemistry 10 (2002) 3395–3400
Synthesis and Biological Evaluation of A-Ring Biaryl-Carbamate
Analogues of Rhazinilam
Olivier Baudoin,* Fabien Claveau, Sylviane Thoret, Audrey Herrbach,
Daniel Guenard and Francoise Gueritte*
Institut de Chimie des Substances Naturelles, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France
Received 14 March 2002; accepted 3 July 2002
Abstract—An improvement of the synthesis of biphenyl-carbamate 2a, the most active analogue of rhazinilam 1 so far, was per-
formed using the Pd-catalyzed borylation/Suzuki coupling (BSC) method developed in our laboratories. The preparation of A-ring
analogues of 2a bearing electron-withdrawing or donating groups is reported according to this new synthetic scheme. The anti-
tubulin properties as well as the cytotoxicity of these compounds toward human cancer cell lines were evaluated in comparison with
rhazinilam and 2a.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
of the A-ring substitution on the biological activity.
Indeed, among the numerous rhazinilam analogues tes-
ted, only two possessed one A-ring substituent (in the
position para to the nitrogen).5 We report therein an
improved synthesis of racemic 2a as well as the use of
this new synthetic pathway to obtain A-ring analogues
of 2a in a straightforward fashion. These analogues
were biologically evaluated and compared with 1 and
2a.
(ꢀ)-Rhazinilam 1 is a tetracyclic alkaloid isolated from
various Apocynaceae,1 possessing an axially chiral
phenyl-pyrrole subunit and a 9-membered median lac-
tam ring. It was found to inhibit in vitro both micro-
tubules disassembly and assembly, the latter
phenomenon being imputable to the formation of
abnormal tubulin spirals.2 As a consequence to these
unique antitubulin properties, rhazinilam showed sig-
nificant in vitro cytotoxicity toward various cancer cell
lines,2 but no activity was found in vivo. As part of a
program directed toward the semi- and total synthesis
of 1 and analogues,3 we showed that biphenyl-carba-
mate analogue (ꢀ)-2a was the most active analogue so
far, with a 2-fold activity on microtubules disassembly
compared to 1 and a similar cytotoxicity.4 In continua-
tion with this study, we wanted to analyze the influence
Results and Discussion
Chemistry
Our previous synthesis of racemic 2a was based on a
Stille coupling between Boc-protected stannane 3 and
triethylsilyl (TES)-protected iodophenyl alcohol 4a,
furnishing biphenyl 5 in 49% yield (Scheme 1). Stan-
nane 3 was obtained by directed ortho-metalation of
Boc-protected aniline followed by transmetalation with
Bu3SnCl.6 Iodide 4a was obtained in four steps, 40%
overall yield, from commercially available material.4
After removal of the protecting groups with TFA,
cyclization occured smoothly in the presence of tri-
phosgene, giving racemic 2a in 88% yield. The active
atropisomer (ꢀ)-2a was obtained after chiral column
HPLC separation. The atropisomer (+)-2a, like
(+)-rhazinilam,5 is biologically inactive.4
*Corresponding authors. Tel.: +33-1-69823038; fax: +33-1-69077247;
e-mail: baudoin@icsn.cnrs-gif.fr; Tel.: +33-1-69824580; fax: +33-1-
69077247; e-mail: gueritte@icsn.cnrs-gif.fr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00270-5

3396
O. Baudoin et al. / Bioorg. Med. Chem. 10 (2002) 3395–3400
Scheme 3. Synthesis of aryl building blocks. Reagents and conditions:
(i) MOMCl, N,N-diisopropylethylamine, CH2Cl2, 0 ꢁC to rt, 16 h; (ii)
n-Bu4NBr3, CH2Cl2, MeOH, rt, 30 min; (iii) NaHMDS, THF, rt, 30
min, then Boc2O, 30 min; (iv) t-BuLi, THF,ꢀ20 ꢁC, 2 h, then ICH2-
CH2I, ꢀ78 ꢁC to rt, 3 h; (v) TFA, CH2Cl2, 0 ꢁC, 30 min.
Scheme 1. Original synthesis of biphenyl-carbamate analogue 2a.4
In prospect of synthesizing various A-ring analogues,
we looked for a more direct and flexible synthetic
method, in particular we were concerned that the ortho-
metalation approach could not be compatible with a
great variety of substituents on the aniline ring. Besides,
we wished to replace the Stille coupling by the more
biologically-friendly tin-free Suzuki coupling. We
recently developed a one-pot palladium-catalyzed bory-
lation/Suzuki coupling (BSC) approach which enables
the straightforward and efficient synthesis of 2,20-dis-
ubstituted biaryl compounds from two aryl halides
(Scheme 2).7
yields (10–28% overall, unoptimized). 2-Halogeno-
anilines 3b–h were commercially available or prepared
in few steps from the corresponding anilines (Scheme 3).
The electron-donating (3b–d) or withdrawing (3e–h)
substituents of these building blocks were deliberately
chosen in order to study the effect of the electronic
character of ring A on the biological activity. Dime-
thoxy- and methylenedioxy-substituted bromoanilines
3c and 3d were obtained by bromination of the corre-
sponding anilines with tetrabutylammonium tri-
bromide.9 The known 2-amino-3-iodonaphthalene 3h10
was synthesized in a convenient manner from 2-amino-
naphthalene by t-Boc protection, regioselective directed
ortho-metallation followed by quenching with diiodo-
ethane (78/22 ratio of 3- and 1-iodo isomers) and clea-
vage of the t-Boc group.
Thus, unprotected 2-bromoaniline 3a (R=H) was sub-
mitted to the Pd-catalyzed borylation with pinacol-
borane, followed by in situ Suzuki coupling with phenyl
iodide 4b, giving biphenyl 5a in 78% yield. The smaller
MOM group was chosen instead of TES as a protecting
group for the primary alcohol 6 (Scheme 3) to decrease
unfavorable steric interactions during the coupling.8
Then, after deprotection of the MOM group with HCl
in refluxing methanol, the urethane group was installed
as previously with triphosgene, furnishing 2a in 80%
yield (62% from 4b). This synthetic pathway enables the
fast incorporation of A-ring substituents from sub-
stituted 2-bromoanilines in three steps, without protec-
tion of the amino group. According to this strategy,
substituted biaryl-carbamates 2b–h (Scheme 2) were
synthesized from 2-halogenoanilines 3b–h in satisfying
Biological evaluation
The cytotoxicity and antitubulin activity of the new
analogues 2b–h were evaluated and compared to those
of (ꢀ)-1 and (Æ)-2a (Table 1). It should be noted that
compounds 2a–h tested therein were racemic. It was
shown earlier that, as (+)-rhazinilam and (+)-2a are
inactive, the activity of the racemic mixtures is twice
lower than that of the levo atropisomer.4
Table 1. Cytotoxicity and antitubulin activity of (ꢀ)-rhazinilam and
racemic A-ring analogues 2a–h
Compd Cytotoxicity
Cytotoxicity
KB cell line MCF7 cell line microtubules microtubules
Inhibition of Inhibition of
IC50 (mM)a
IC50 (mM)a
assembly
IC50 (mM)b
disassembly
IC50 (mM)b
1
0.6
1.1
2.9
>20
1
4.0
6
16
18
9
6.5
9
8
3.0
6.7
6.5
160
190
20
3.7
3.4
na
—
40
2a
2b
2c
2d
2e
2f
2g
2h
d
39
11
100
12
200
6.3
19
3.7
4.0
5.5
73
—
c
c
—
aIC50 is the concentration of compound corresponding to 50% growth
inhibition after 72 h incubation.
Scheme 2. Synthesis of rhazinilam A-ring biaryl analogues 2a–h.
Reagents and conditions: (i) 3a–h, (pin)BH, Et3N, Pd(OꢁAc)2, PCy2
bIC50 is the concentration of compound required to inhibit 50% of the
rate of microtubules assembly or disassembly (na=not active).
cUnable to determine (low solubility).
(o-biph), dioxane, 80 ꢁC, 1h, then H O, 4b, Ba(OH)2, 1 00 C, 1h; (ii)
2
concd HCl, MeOH, reflux, 1h; (iii) (Cl 3CO)2C¼O, pyridine, CH2Cl2,
ꢀ78 ꢁC, 30 min. MOM=methoxymethyl, pin=pinacol, PCy2 (o-
biph)=2-(dicyclohexylphosphino)biphenyl.
d200 times less active than 1 at 30% inhibition.

O. Baudoin et al. / Bioorg. Med. Chem. 10 (2002) 3395–3400
3397
The cytotoxicity of 1 and 2a on human breast adeno-
Experimental Protocols
carcinoma MCF7 cells as well as the IC50 values of these
compounds for the inhibition of tubulin polymerization
have not been reported earlier.
Chemistry
Reagents were commercially available and used without
further purification unless otherwise stated. Solvents
were distilled under argon over appropriate drying
agents immediately before use. NMR spectra were
recorded on Bruker AC-250 or AC-300 instruments and
calibrated using tetramethylsilane as an internal refer-
ence. The following abbreviations were used to desig-
nate the multiplicities: s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet, br=broad. IR spectra were
recorded on a Nicolet FT-IR 205 spectrometer. High
resolution mass spectra (HRMS) were recorded under
liquid secondary ion (LSIMS), Matrix Assisted Laser
Desorption Ionization (MALDI) or Chemical Ioni-
zation (CI) conditions at the Laboratoire de Spectro-
metrie de Masse, ICSN, Gif-sur-Yvette, France or at
the Laboratoire Central d’Analyse du CNRS, Vernai-
son, France. The physical data of compounds 3c,9 3h,10
6,4 and 2a4 were previously described.
The IC50 values of rhazinilam for tubulin polymeri-
zation and depolymerization are comparable and there-
fore are probably both responsible for the cytotoxicity
toward KB and MCF7 cells. It was shown earlier that
this unique property is related to the abitily of rhazini-
lam to form spirallike complexes with tubulin.2 Racemic
unsubstituted biphenyl analogue 2a was found to be as
active as rhazinilam on both microtubules formation
and dissociation and twice less cytotoxic towards KB
cells, consistently with previous reports.4 Similarly, 2a
was 1.5 times less cytotoxic than rhazinilam on MCF7
cells.
Racemic A-ring biphenyl-carbamate analogues 2b–h
were markedly less active than unsubstituted 2a on the
tubulin test, except the nitro derivative 2f which showed
two-fold IC50 values. Thus, by analogy with 2a, the (ꢀ)-
atropisomer of 2f should be as active as (ꢀ)-rhazinilam.
Consistently, the IC50 values of 2b–h on KB cells were
all smaller (3 to more than 20 times) than that of 2a, 2f
being again the most active compound. Less difference
between 2b–h and 2a was observed on MCF7 cells, with
IC50 values from 0.5 to 3 times that of 2a. Interestingly,
2h was slightly more active than 2a and rhazinilam on
this cell line. For low solubility reasons, we were unable
to determine if this cytotoxicity originated in the anti-
tubulin properties of 2h. For other analogues 2b–g, it
appears that the cytotoxicity toward both cancer cell
lines can be only partially linked to the antitubulin
effect. For instance compounds 2b and 2e had a very
weak antitubulin effect but were substantially cytotoxic,
which suggests that these cytotoxicities arise from other
factors. Further investigation is underway to explain
these observations.
[2-Ethyl-2-(2-iodophenyl)butoxy]methoxymethane (4b).
To a stirred solution of alcohol 6 (8.0 g, 26.3 mmol) in
dry dichloromethane (130 mL) under argon at 0 ꢁC was
added N,N-diisopropylethylamine (16 mL, 91.9 mmol)
and chloromethyl methyl ether (6 mL, 79.0 mmol)
dropwise. The temperature was allowed to warm to rt
for 16 h, then a saturated aqueous NaHCO3 solution
(200 mL) was added and the aqueous phase was
extracted with dichloromethane. The organic solution
was dried over MgSO4 and evaporated under vaccum.
The residue was purified by flash chromatography
(silica gel, heptane/ethyl acetate 9/1) to afford 4b as a
1
colorless oil (8.2 g, 90%); H NMR (300 MHz, CDCl3)
d=0.68 (t, J=7.5 Hz, 6H), 1.93 (dq, J=15, 7.5 Hz, 2H),
2.35 (dq, J=15, 7.5 Hz, 2H), 3.38 (s, 3H), 3.97 (s, 2H),
4.69 (s, 2H), 6.85 (td, J=7.4, 1.8 Hz, 1H), 7.20 (dd,
J=8.4, 1.8 Hz, 1H), 7.29 (td, J=7.5, 1.2 Hz, 1H), 8.04
(dd, J=7.8, 1.0 Hz, 1H) ppm; 13C NMR (75 MHz,
CDCl3) d=8.5 (CH3), 25.4 (CH2), 46.8 (Cq), 55.4
(CH3), 69.4 (CH2), 94.5 (Cq), 96.8 (CH2), 127.5 (CH),
127.6 (CH), 130.4 (CH), 143.8 (CH), 144.4 (Cq) ppm;
HRMS (LSIMS) calcd for C14H21ILiO2 [(M+Li)+]:
355.0746; found: 355.0770.
Finally, the electron-withdrawing or donating character
of A-ring substituents seems to have no clear effect on
the activity of these analogues. On the other hand, the
present results suggest that the increase of the steric
hindrance on ring A induced by the addition of sub-
stituents is harmful to the antitubulin activity and to the
cytotoxicity, as particularly shown with compound 2c.
2-Bromo-4,5-methylenedioxyaniline (3d). n-Bu4NBr3 (4 g,
8.3 mmol) was added to a solution of 3,4-methylene-
dioxyaniline (1g, 7.3 mmol) in dichloromethane (27
mL) and methanol (13 mL). After 20 min stirring at rt,
the solution was diluted with diethyl ether (30 mL), the
organic solution was washed successively with a satu-
rated aqueous Na2SO3 solution (30 mL) and with brine.
After drying over MgSO4, the solvents were removed
under vacuum and the residue was purified by flash
chromatography (silica gel, heptane/ethyl acetate 4/1-7/
3), to afford 3d as an oil (708 mg, 45%); 1H NMR
(250 MHz, CDCl3) d=3.74 (br s, 2H), 5.87 (s, 2H), 6.36
(s, 1H), 6.87 (s, 2H) ppm; 13C NMR (75 MHz, CDCl3)
d=97.8, 98.8, 101.3, 112.0, 138.8, 140.9, 148.1 ppm; IR
In conclusion, racemic biaryl-carbamate analogues of
rhazinilam, bearing a variety of electron-withdrawing or
donating substituents on ring A, were synthesized in a
straightforward fashion by the BSC method. These
compounds were all less active on KB cancer cells and
tubulin than the unsubstituted analogue 2a, presumably
due to steric factors. However nitro-substituted com-
pound 2f retained an interesting activity. On MCF7
cancer cells, biphenyls 2b–g were all less active than
rhazinilam and 2a, except 2h which was slightly more
active. Further biological testing is required to explain
the discrepancies observed among the present assays.
The present work also suggests future modifications on
B and C-rings to increase the antitumor potential of
compound 2a.
(film) ꢀ=1636, 3186, 3297, 3397 cmꢀ1
(MALDI) calcd for C7H6BrNO2 [M+ ]: 214.9582;
found: 214.9609.
; HRMS
ꢂ

3398
O. Baudoin et al. / Bioorg. Med. Chem. 10 (2002) 3395–3400
2-Amino-3-iodonaphthalene (3h). To
a
solution of
were evaporated under vacuum and the residue was
purified by flash chromatography (silica gel, heptane/
ethyl acetate 95/5–9/1) to afford 5a as an oil (282 mg,
78% from 4b); 1H NMR (300 MHz, CDCl3) d=0.70 (2t,
J=7.5 Hz, 6H), 1.70 (m, 4H), 3.28 (s, 3H), 3.47 (br s,
2H), 3.59 (d, J=9.6 Hz, 1H), 3.63 (d, J=9.6 Hz, 1H),
4.49 (d, J=6.6 Hz, 1H), 4.53 (d, J=6.3 Hz, 1H), 6.73
(m, 2H), 7.02 (dd, J=7.7, 2.2 Hz, 1H), 7.03 (dd, J=7.5,
1.5 Hz, 1H), 7.15 (td, J=7.5, 1.8 Hz, 1H), 7.25 (td,
J=7.5, 1.5 Hz, 1H), 7.33 (td, J=7.2, 1.7 Hz, 1H), 7.42
(m, 2H) ppm; 13C NMR (75 MHz, CDCl3) d=8.8, 28.0,
47.5, 55.6, 70.0, 96.9, 115.1, 117.4, 126.2, 127.6, 128.3,
129.4, 130.3, 130.5, 133.3, 138.6, 143.3, 144.3 ppm; IR
(film) ꢀ=1615, 2962, 3369, 3469 cmꢀ1; HRMS (LSIMS)
calcd for C20H27LiNO2 [(M+Li)+]: 320.2202; found:
320.2209.
2-aminonaphthalene (1.025 g, 7.2 mmol) in dry THF
(20 mL) under argon at rt was added dropwise sodium
bis(trimethylsilyl)amide (2 M solution in THF, 7.5 mL,
15.0 mmol). After 30 min stirring, a solution of di-tert-
butyl dicarbonate (1.640 g, 7.5 mmol) in 2 mL dry THF
was added dropwise and the resulting mixture was stir-
red for 30 min. A saturated aqueous NH4Cl solution
was added, and the solution was extracted with di-
chloromethane. After drying over MgSO4, the solvents
were removed under vacuum and the residue was pur-
ified by flash chromatography (silica gel, heptane/ethyl
acetate 4/1) to afford t-Boc-protected 2-aminonaphtha-
lene as pale cristals (1.443 g, 83%); 1H NMR (250 MHz,
CDCl3) d=1.53 (s, 9H), 6.78 (br s, 1H), 7.35 (m, 3H),
7.69 (m, 3H), 7.96 (s, 1H) ppm; 13C NMR (62.5 MHz,
CDCl3) d=28.3, 80.5, 114.5, 119.1, 124.3, 126.3, 127.3,
127.4, 128.6, 129.9, 133.9, 135.8, 152.8 ppm. To a solu-
tion of this compound (500 mg, 2.1mmol) in 10 mL dry
THF under argon at ꢀ20 ꢁC was added dropwise tert-
butyl lithium (1.5 M solution in pentane, 3.43 mL, 5.1
mmol) and the resulting solution was stirred for 2 h at
ꢀ20 ꢁC. After cooling at ꢀ78 ꢁC, a solution of diio-
doethane (1.448 g, 5.1 mmol) in 5 mL dry THF was
added dropwise and the temperature was allowed to
warm to rt for 3 h. A saturated aqueous NH4Cl solution
was added, and the solution was extracted with diethyl
ether. The resulting organic solution was washed with a
saturated aqueous Na2SO3 solution and dried over
MgSO4. The solvents were evaporated under vacuum
and the residue was purified by flash chromatography
(silica gel, heptane/ethyl acetate 95/5) to afford 471mg
of a 78/22 mixture (NMR) of regioisomeric 3-iodo and
1-iodo t-Boc-protected 2-aminonaphthalene. This mix-
ture (440 mg) was dissolved in dichloromethane (5 mL)
and trifluoroacetic acid (5 mL) was added dropwise at
0 ꢁC. After 30 min stirring at 0 ꢁC, the solution was
neutralized with a concentrated NaOH solution and the
aqueous layer was extracted with dichloromethane. The
solution was dried over MgSO4, evaporated and the
resulting solid was washed with heptane, yielding 3h as
a white powder (184 mg, 35%), which had identical
physical properties to the previously described com-
pound.10
9,9-Diethyl-8,9-dihydro-5H-7-oxa-5-azadibenzo[a,c]cyclo-
nonen-6-one (2a). To a solution of compound 5a (280
mg, 0.89 mmol) in methanol (4 mL) at rt was added
36% aqueous HCl (1mL) and the mixture was refluxed
for 1h. After cooling to 0 ꢁC, the solution was neu-
tralized with concd. aqueous NaOH and extracted with
dichloromethane. The organic solution was dried over
MgSO4 and concentrated under vacuum. The residue
was purified by flash chromatography (silica gel, hep-
tane/ethyl acetate 7/3) to afford 2-(20-aminobiphenyl-2-
yl)-2-ethylbutan-1-ol4 as an oil (208 mg, 86%). The
conversion of this amino-alcohol to carbamate 2a using
triphosgene has been previously described (93% yield).4
Carbamate 2b. General procedure from 2-bromo-4-
1
methylaniline (3b), 10% yield for three steps; H NMR
(250 MHz, CDCl3) d=0.67 (t, J=7.7 Hz, 3H), 0.95 (t,
J=7.5 Hz, 3H), 1.50–1.91 (m, 4H), 2.35 (s, 3H), 3.82 (d,
J=10.5 Hz, 1H), 4.24 (d, J=11 Hz, 1H), 5.97 (br s,
1H), 6.66 (dd, J=7.7, 1.5 Hz, 1H), 7.00 (d, J=8.8 Hz,
1H), 7.13 (m, 2H), 7.19 (td, J=7.3, 1.0 Hz, 1H), 7.35
(td, J=8.0, 1.3 Hz, 1H), 7.49 (dd, J=8.5, 1.2 Hz, 1H)
ppm; 13C NMR (75 MHz, CDCl3) d=8.1, 8.3, 21.0,
23.6, 25.0, 48.3, 73.8, 124.9, 126.1, 128.0, 128.3, 128.4,
130.5, 132.7, 133.9, 135.2, 139.6, 141.8, 144.4, 156.9
ppm; IR (film) ꢀ=1721, 2964, 3256 cmꢀ1; HRMS (CI)
calcd for C20H24NO2 [(M+H)+]: 310.1807; found:
310.1821.
General
procedure
for
the
synthesis
of
Carbamate 2c. General procedure from 2-bromo-4,5-
1
biphenylcarbamates 2a–h from anilines 3a–h and iodide
4b
dimethoxyaniline (3c),9 11% yield for three steps; H
NMR (250 MHz, CDCl3) d=0.70 (t, J=7.2 Hz, 3H),
0.96 (t, J=7.2 Hz, 3H), 1.5–1.9 (m, 4H), 3.86 (s, 3H),
3.86 (d, J=11 Hz, 1H), 3.94 (s, 3H), 4.27 (d, J=11 Hz,
1H), 5.84 (br s, 1H), 6.71 (s, 1H), 6.83 (s, 1H), 6.91 (dd,
J=7.5, 1.5 Hz, 1H), 7.23 (td, J=7.5, 1.3 Hz, 1H), 7.35
(td, J=7.3, 1.8 Hz, 1H), 7.5 (d, J=7.5 Hz, 1H) ppm;
13C NMR (62.5 MHz, CDCl3) d=8.1, 8.5, 23.4, 25.1,
48.3, 55.9, 56.1, 74.0, 108.9, 112.7, 126.3, 128.1, 128.6,
129.4, 133.0, 136.4, 139.2, 142.1, 146.2, 148.1, 157.2
ppm; IR (film) ꢀ=1732, 2962, 3332 cmꢀ1; HRMS (CI)
calcd for C21H26NO4 [(M+H)+]: 356.1862; found:
356.1840.
2-(20-Aminobiphenyl-2-yl)-2-ethylbutoxymethoxy meth-
ane (5a). To a solution of 2-bromoaniline 3a (296 mg,
1.7 mmol) in dry dioxane (5 mL) at 25 ꢁC under argon
were successively added dry Et3N (0.96 mL, 6.9 mmol),
Pd(OAc)2 (19 mg, 0.086 mmol), PCy2(o-biph) (121 mg,
0.34 mmol), and pinacolborane (0.75 mL, 5.2 mmol)
dropwise. The solution was heated to 80 ꢁC for 1h.
After cooling, degased water (1.5 mL) was added drop-
wise, then 4b (400 mg, 1.1 mmol) in degased dioxane (1
ꢂ
mL), and Ba(OH)2 8H2O (1.63 g, 5.2 mmol) were added
and the mixture was heated to 100 ꢁC for 1h. After
cooling, the solution was filtered through Celite, brine
was added and the aqueous layer was extracted with di-
chloromethane. After drying over MgSO4, the solvents
Carbamate 2d. General procedure from 2-bromo-4,5-
methylenedioxyaniline (3d), 28% yield for three steps;

O. Baudoin et al. / Bioorg. Med. Chem. 10 (2002) 3395–3400
3399
1H NMR (250 MHz, CDCl3) d=0.70 (t, J=7.2 Hz,
3H), 0.93 (t, J=7.0 Hz, 3H), 1.55–1.90 (m, 4H), 3.84 (d,
J=11.5 Hz, 1H), 4.25 (d, J=11.0 Hz, 1H), 5.83 (br s,
1H), 6.03 (s, 2H), 6.64 (s, 1H), 6.77 (s, 1H), 6.85 (dd,
J=7.5, 1.5 Hz, 1H), 7.20 (td, J=7.3, 1.3 Hz, 1H), 7.36
(td, J=7.3, 1.8 Hz, 1H), 7.48 (d, J=8.7 Hz, 1H) ppm;
13C NMR (75 MHz, CDCl3) d=8.1, 8.4, 23.7, 24.8,
48.5, 73.9, 101.7, 106.5, 109.6, 126.4, 128.2, 128.6, 130.1,
133.0, 137.8, 139.3, 142.2, 145.2, 146.8, 157.1 ppm; IR
(film) ꢀ=1717, 2964, 3256 cmꢀ1; HRMS (LSIMS) calcd
(d, J=11.2 Hz, 1H), 4.27 (d, J=11.2 Hz, 1H), 6.67 (br
s, 1H), 6.85 (dd, J=7.5, 1.2 Hz, 1H), 7.19 (td, J=7.2,
1.0 Hz, 1H), 7.39 (td, J=7.6, 1.5 Hz, 1H), 7.50 (m, 4H),
7.79 (m, 3H) ppm; 13C NMR (62.5 MHz, CDCl3)
d=8.1, 8.3, 24.0, 25.0, 48.2, 73.7, 122.3, 126.1, 126.2,
126.2, 127.2, 127.6, 128.0, 128.4, 128.5, 131.1, 132.7,
133.4, 135.1, 139.5, 141.7, 143.2, 156.7 ppm; IR (film)
ꢀ=1720, 2963, 3247 cmꢀ1; HRMS (LSIMS) calcd for
ꢂ
C23H23NO2 [M+ ]: 345.1729; found: 345.1738.
ꢂ
for C20H22NO4 [M+ ]: 339.1471; found: 339.1485.
Biological assays2,5
Carbamate 2e. General procedure from 2-bromo-4-
1
Inhibition of microtubules disassembly. The drug, dis-
solved in DMSO at different concentrations, is added to
a solution of microtubules (tubulin concentration ca. 20
mM, freshly prepared from mammalian brain) at 37 ꢁC.
Then the solution is placed in a temperature-controlled
cell at 9 ꢁC (microtubules disassembly) and the decrease
of the optical density is monitored in a UV spectro-
photometer at 350 nm for 1min. The maximum rate of
disassembly is recorded and compared to a sample
without drug. The IC50 of the compound is calculated
from the effect of several concentrations and compared
to the IC50 of rhazinilam obtained within the same day
with the same tubulin preparation.
nitroaniline (3e), 19% yield for three steps; H NMR
(300 MHz, CDCl3) d=0.72 (t, J=7.4 Hz, 3H), 0.98
(t, J=7.5 Hz, 3H), 1.65 (m, 2H), 1.81 (m, 1H), 1.91
(m, 1H), 3.84 (d, J=10.2 Hz, 1H), 4.22 (d, J=10.2 Hz,
1H), 6.70 (br s, 1H), 6.77 (dd, J=7.8, 1.5 Hz, 1H), 7.20
(d, J=8.1Hz, 1H), 7.25 (td, J=7.3, 1.2 Hz, 1H), 7.43
(td, J=8.1, 2.1 Hz, 1H), 7.55 (d, J=8.1 Hz, 1H), 8.18 (s,
1H), 8.20 (dd, J=8.4, 3.0 Hz) ppm; 13C NMR
(62.5 MHz, CDCl3) d=8.1, 23.9, 24.7, 48.4, 73.6, 123.1,
124.9, 125.1, 126.0, 128.2, 129.3, 132.5, 137.4, 141.8,
142.4, 144.7, 145.7, 155.7 ppm; IR (film) ꢀ=1736, 2987,
3054 cmꢀ1; HRMS (LSIMS) calcd for C19H21N2O4
[(M+H)+]: 341.1501; found: 341.1510.
Inhibition of tubulin assembly. The assay is conducted in
a reverse manner as above: the drug, dissolved in DMSO
at different concentrations, is added to a solution of free
tubulin at 0 ꢁC. Then the solution is placed in a tem-
perature-controlled cell at 37 ꢁC (microtubules assembly)
and the increase of the optical density is monitored in a
UV spectrophotometer at 350 nm for 1min. The max-
imum rate of assembly is recorded and compared to a
sample without drug. The IC50 of the compound is calcu-
lated from the effect of several concentrations and com-
pared to the IC50 of rhazinilam obtained within the same
day with the same tubulin preparation.
Carbamate 2f. General procedure from 2-bromo-5-
1
nitroaniline (3f), 15% yield for three steps; H NMR
(300 MHz, CDCl3) d=0.70 (t, J=7.2 Hz, 3H), 0.96 (t,
J=7.2 Hz, 3H), 1.57 (m, 2H), 1.81 (m, 1H), 1.92 (m,
1H), 3.83 (d, J=10.8 Hz, 1H), 4.24 (d, J=10.8 Hz, 1H),
6.53 (br s, 1H), 6.75 (dd, J=7.5, 1.2 Hz, 1H), 7.24 (td,
J=7.5, 1.2 Hz, 1H), 7.44 (td, J=8.1, 1.8 Hz, 1H), 7.46
(d, J=9.0 Hz, 1H), 7.55 (d, J=8.1Hz, 1H), 7.97 (d,
J=2.4 Hz, 1H), 8.10 (dd, J=8.7, 2.4 Hz) ppm; 13C
NMR (75 MHz, CDCl3) d=8.0, 8.1, 24.1, 24.7, 48.4,
73.6, 120.1, 120.4, 126.6, 128.4, 129.3, 130.6, 131.8,
137.6, 137.7, 141.6, 147.2, 152.0, 155.9 ppm; IR (film)
ꢀ=1728, 2966, 3256 cmꢀ1; HRMS (CI) calcd for
C19H21N2O4 [(M+H)+]: 341.1501; found: 341.1505.
Cytotoxicity assays. the effect of the drugs on the
growth of KB and MCF7 human cell lines was mon-
itored at the Laboratoire de Cultures Cellulaires, ICSN,
Gif-sur-Yvette, France. The IC50 value refers to the
concentration of drug corresponding to 50% growth
inhibition after 72 h incubation. Docetaxel IC50 values:
4 nM for KB cells and 3.5 nM for MCF7 cells.
Carbamate 2g. General procedure from 2-bromo-4,5,6-
trifluoroaniline (3g), 27% yield for three steps; 1H
NMR (300 MHz, CDCl3) d=0.71(t, J=7.2 Hz, 3H),
0.96 (t, J=7.2 Hz, 3H), 1.60 (m, 2H), 1.83 (m, 2H), 3.86
(d, J=10.5 Hz, 1H), 4.23 (d, J=11.4 Hz, 1H), 5.78 (br
s, 1H), 6.80 (dd, J=7.8, 2.2 Hz, 1H), 6.96 (ddd,
JHF=10.2, 7.8, 1.8 Hz, 1H), 7.24 (td, J=7.8, 0.9 Hz,
1H), 7.41 (td, J=8.1, 1.5 Hz, 1H), 7.52 (d, J=8.1Hz,
1H) ppm; 13C NMR (62.5 MHz, CD3OD) d=8.3, 8.5,
25.7, 26.6, 49.6, 74.7, 113.8 (dd, JCF=19, 3 Hz), 124.9
(dd, JCF=11, 3 Hz), 127.6, 129.5, 130.3, 133.7, 138.2–
142.2 (ddd, J1=247 Hz), 138.2, 142.4, 143.4, 145.0–
149.0 (ddd, J1=248 Hz), 146.9–150.7 (ddd, J1=246
Acknowledgements
We thank Dr. C. Thal for his constant support and C.
Gaspard for cytotoxicity assays. This work was finan-
cially supported by the Centre National de la Recherche
Scientifique (France).
Hz), 157.9 ppm; IR (film) ꢀ=1713, 2964, 3233 cmꢀ1
;
HRMS (CI) calcd for C19H19F3NO2 [(M+H)+]:
350.1368; found: 350.1378.
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