Cytotoxic and antitumoral properties in a series of new, ring D modified, olivacine analogues

Article abstract of DOI:10.1016/j.bmc.2004.09.047

The present study describes the synthesis and pharmacological profiles of new olivacine related compounds, possessing a modified D ring. The impact of this modification has been evaluated with respect to the cytotoxic and in vivo antitumoral effects of these molecules and in comparison with parent S 16020-2 previously prepared and investigated in our laboratory. The D ring size and number of nitrogen atoms as well as the position of the aminoalkyl substituent have a profound impact on the cytotoxic and antitumoral profiles. Thus out of the prepared pyrazinocarbazole compounds, 2 is devoid of any substantial cytotoxic and antitumoral activities while the pyrimidocarbazole 3 has a similar profile compared to 1 (S 16020-2). L1210 and P388 in vivo antitumoral effects are lost for both imidazocarbazoles 4 and 5, but the former conserves an in vivo antitumoral effect on B16 melanoma, this effect being the largest in the series. Structural similarities and differences amongst the studied compounds could be evidenced by calculation of global properties such as molecular electrostatic potentials (MEP maps) and partition coefficients (log P), thus adding information on the impact of chemical changes on these two parameters known to influence biological behavior.

Full text of DOI:10.1016/j.bmc.2004.09.047

Bioorganic & Medicinal Chemistry 13 (2005) 175–184
Cytotoxic and antitumoral properties in a series of new, ring
D modified, olivacine analogues
a
Claude Guillonneau, Annette Nault, Eric Raimbaud, Stephane Leonce,
a
b
c
´
Laurence Kraus-Berthier, Alain Pierre and Solo Goldstein
´
c
c
a,
*
´
aServier, Division Chimie A, 11 rue des Moulineaux, 92150 Suresnes, France
b
´
´
Servier, Modelisation moleculaire, 125 chemin de Ronde, 78290 Croissy, France
´ ´
Servier, Division de Cancerologie Experimentale, 125 chemin de Ronde, 78290 Croissy, France
c
Received 2 June 2004; accepted 24 September 2004
Available online 2 November 2004
Abstract—The present study describes the synthesis and pharmacological profiles of new olivacine related compounds, possessing a
modified D ring. The impact of this modification has been evaluated with respect to the cytotoxic and in vivo antitumoral effects of
these molecules and in comparison with parent S 16020-2 previously prepared and investigated in our laboratory. The D ring size
and number of nitrogen atoms as well as the position of the aminoalkyl substituent have a profound impact on the cytotoxic and
antitumoral profiles. Thus out of the prepared pyrazinocarbazole compounds, 2 is devoid of any substantial cytotoxic and antitumo-
ral activities while the pyrimidocarbazole 3 has a similar profile compared to 1 (S 16020-2). L1210 and P388 in vivo antitumoral
effects are lost for both imidazocarbazoles 4 and 5, but the former conserves an in vivo antitumoral effect on B16 melanoma, this
effect being the largest in the series. Structural similarities and differences amongst the studied compounds could be evidenced by
calculation of global properties such as molecular electrostatic potentials (MEP maps) and partition coefficients (logP), thus adding
information on the impact of chemical changes on these two parameters known to influence biological behavior.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and pharmacologically evaluated. In these analogues,
the number of nitrogens and the position of the basic
side chain were varied. To begin with, we sought for
practical synthetic approaches using simple reagents
and no scaling up impediments.
Previous reports from our laboratory described new
cytotoxic olivacine derivatives out of which compound
1 (Fig. 1, S 16020-2) was selected for clinical evaluation.
Pursuing our goals, we initiated a research project aimed
to investigate the impact of ring D modifications on
cytotoxic and antitumoral profiles of olivacine related
compounds. Indeed, while the impact of substitution
of the tetracycle has been studied,1 no analogues
containing modified D ring were known. Moreover,
since literature reports stressed the role of D ring
nitrogen in related tricyclics compounds,2 it was decided
to also evaluate such changes in 1. Therefore, four
types of structures namely pyrazinocarbazole, pyri-
midocarbazole, imidazocarbazole and N-substituted
imidazocarbazole (2, 3, 4, 5, Fig. 1) were synthesized
2. Chemistry
Preparation of compound 2 (Scheme 1) started with carb-
azole 6 itself prepared in good yield by a literature de-
scribed method.3 Demethylation of the methoxy group
as well as deacetylation of the acetamido group was
achieved in good yield by treatment with 48% hydrobro-
mic acid.4 The amino and hydroxyl functions in com-
pound 7 were then reacetylated with acetic anhydride.
Compound 8 was then N-methylated using sodium
hydride and iodomethane in DMF.5 Selective nitration
of 9 with nitric acid (fuming 100%) occurred at the
ortho-position to the diacetylamino group to afford 10
in good yield.6 The acetyl groups were removed in
quantitative yield by treatment with sodium hydroxide
in ethanol. Ni (catalytic) hydrogenation of the aromatic
Keywords: Cytotoxic; Antitumoral; Olivacine; Modified D ring.
*
Corresponding author. Tel.: +33 1 55 72 25 99; fax: +33 1 55 72 24
30; e-mail: solo.goldstein@fr.netgrs.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.047

176
C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
CONHCH2CH2N(CH3)2
HO
N
N
HO
CONHCH2CH2N(CH3)2
N
N
N
N
CH3
CH3
CH3
CH3
2
3
CONHCH2CH2N(CH3)2
N
HO
N
CH3
CH3
1
(S 16020-2)
CH2CH2N(CH3)2
HO
HO
O
N
N
CH3
N
NHCH2CH2N(CH3)2
C
N
H
N
N
CH3 CH3
CH3 CH3
4
5
Figure 1. Ring D modified S 16020-2 analogues.
COCH3
N
NH2
CH3
NHCOCH3
CH3
CH3O
HO
CH3COO
ii
i
COCH3
N
H
N
H
CH3
N
H
7
8
6
11
12
iii
NO2
NH2
NO2
COCH3
COCH3
N
N
CH3COO
CH3COO
HO
v
iv
COCH3
COCH3
CH3
CH3
CH3
N
N
CH3
N
CH3
CH3
9
10
vi
COOC2H5
CONHCH2CH2N(CH3)2
NH2
NH2
CH3
N
N
N
HO
HO
HO
N
vii
viii
CH3
CH3
N
N
CH3
N
CH3
CH3
13a
13b
2
N
COOC2H5
N
HO
CH3
N
CH3
Scheme 1. Synthesis of pyrazocarbazole 2: (i) 48% HBr (94%); (ii) Ac2O (62%); (iii) NaH, CH3I, DMF (64%); (iv) HNO3 (53%); (v) NaOH, C2H5OH
(100%); (vi) H2–Ni, NMP (100%); (vii) BrCH2COCOOC2H5, N(C2H5)3, NMP (29%); (viii) H2N(CH2)2N(CH3)2 (44%).
nitro group6 in compound 11 afforded in good yield
the diamino carbazole 12. The pyrazo D cycle was
constructed by condensation of diamino compound 12
with ethylbromopyruvate in the presence of triethylamine7

C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
177
in NMP to afford compound 13a in moderate yield.
Although two different isomers 13a and 13b could be
expected from this reaction, only isomer 13a was
obtained; its structure was proved at the next step by
the NMR spectral data of compound 2, itself obtained
from compound 13a on which the appropriate chain
was introduced by condensation of the ethyl ester part
with the suitable amine.8 This procedure was easily per-
formed on a multigram scale, thus allowing the prepara-
tion of 2 in fair quantities.
gen peroxide gave phenolic compound 29 in moderate
yield.13 The introduction of the amide chain was per-
formed in good yield by reaction with N,N-dimethyl-
ethylenediamine to give 3.
Preparation of compound 4 (Scheme 3) started with the
benzylation of the phenolic function in compound 11
using sodium hydride and benzyl chloride in DMF to
give carbazole 15 in good yield. Reduction of the nitro
group with hydrazine hydrate in methanol and THF
using Raney Nickel as catalyst14 gave the diamino-
carbazole 16 which was acetylated with acetyl chloride
in THF in the presence of triethylamine to give a mix-
ture of isomer 17a and 17b used without separation
for the next step. Thermic cyclization15 gave the ex-
pected imidazocarbazole 18 which reacted with 2-
(dimethylamino)ethyl chloride hydrochloride in the
presence of potassium carbonate16 in DMF to give
19a. Although two different isomers 19a and 19b could
be expected from this reaction, only isomer 19a was iso-
lated; its structure was elucidated by NMR spectral data
using NOE experiment. Debenzylation using cyclohex-
ene in the presence of palladium on activated carbon17
in N-methylpyrrolidone gave compound 4.
Preparation of compound 3 (Scheme 2) started with the
known tetracyclic anhydride 20.9 Reaction with trimeth-
ylsilylazide in DMF afforded in good yield tetracycle 21,
which could be transformed into pyrimidocarbazole 22
according to a previously described procedure.10 The cy-
ano substituted pyrimidocarbazole 24 could be easily
obtained by a two-step procedure involving quantitative
reaction of 22 with phosphorous oxychloride affording
the chloro derivative 2311 followed by chlorine displace-
ment using potassium cyanide and a catalytic amount of
sodium tosylate in DMSO.12 Transformation of the nit-
rile 24 into the corresponding amide 25 was achieved by
treatment with an excess of hydrogen chloride in a mix-
ture of dichloromethane and methanol. The amide 25
was quantitatively transformed into carboxylic acid so-
dium salt 26 using sodium hydroxide in a mixture of eth-
anol and DMSO. The corresponding ester 27, obtained
in methanol in the presence of sulfuric acid, was formyl-
ated with hexamethylenetetramine in TFA13 to give
aldehyde 28 whose oxidation in methanol using hydro-
Preparation of compound 5 (Scheme 4) started with
exhaustive acylation using ethylchlorooxoacetate of the
previously described compound 12 in the presence of tri-
ethylamine in DMF. Transamidification of compound
14 using N,N-dimethylethylenediamine and concomitant
cyclodehydration led to compound 5.
O
Cl
O
O
H
N
N
O
O
O
N
H
N
N
O
ii
i
iii
N
CH3
CH3
N
CH3
CH3
N
N
CH3
CH3
CH3
CH3
20
21
23
22
iv
O
H2NC
+ -
NaOOC
NC
CH3OOC
N
N
N
N
N
N
N
N
vi
v
vii
CH3
CH3
CH3
N
N
CH3
N
N
CH3
CH3
CH3
CH3
27
26
24
25
viii
O
CH3OOC
(CH3)2 N-CH2CH2 NH-C
CH3OOC
N
N
N
N
N
N
HO
OHC
HO
x
ix
CH3
CH3
N
N
CH3
N
CH3
CH3
CH3
3
28
29
Scheme 2. Synthesis of pyrimidocarbazole 3: (i) (CH3)3SiN3, DMF (52%); (ii) formamidine acetate, 1-methoxy-2-propanol (76%); (iii) POCl3 (96%),
(iv) KCN, NaOTos, DMSO (71%); (v) HCl, CH2Cl2, CH3OH (76%); (vi) NaOH, C2H5OH, DMSO (100%); (vii) H2SO4, CH3OH (68%); (viii)
hexamethylenetetramine, TFA (83%); (ix) KHSO4, H2O2, CH3OH (68%); (x) H2N(CH2)2N(CH3)2 (88%).

178
C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
NO2
NH2
NO2
NH2
NH2
NH2
O
O
HO
ii
i
CH3
CH3
CH3
N
N
N
CH3
CH3
CH3
11
15
16
iii
NHCOCH3
NH2
O
H
N
CH3
N
CH3
N
CH3
N
O
N
17a
O
(CH2)2N(CH2)2
iv
+
CH3
N
CH3
N
CH3
CH3
NH2
19b
18
NHCOCH3
CH3
O
N
v
CH3
17b
(CH3)2N(CH2)2
O
(CH3)2N(CH2)2
CH3
CH3
N
N
N
N
HO
vi
N
CH3
N
CH3
CH3
CH3
19a
4
Scheme 3. Synthesis of imidazocarbazole 4: (i) NaH, C6H5CH2Cl, DMF (78%); (ii) H2N–NH2, H2O, Ni Raney, THF, CH3OH (94%); (iii)
CH3COCl, N(C2H5)3, THF (96%); (iv) D (68%); (v) Cl(CH2)2N(CH3)2ÆHCl, K2CO3, DMF (79%); (vi) cyclohexene, Pd/C, NMP (74%).
O
NH2
NH2
NHCOCOOC H5
2
N
NHCH2CH2N(CH3)2
NH
NHCOCOOC H5
2
HO
HO
C2H5OOC-COO
i
ii
CH3
CH3
CH3
N
N
N
CH3
CH3
CH3
12
Scheme 4. Synthesis of imidazocarbazole 5: (i) ClCOCOOC2H5, N(C2H5)3, DMF (83%); (ii) H2N(CH2)2N(CH3)2 (59%).
14
5
3. Results and discussion
Thus compound 2 including a pyrazino D ring instead
of the pyridine ring in 1 exhibits a much lower cytotoxi-
city (IC50 = 330nM vs 13.1nM). It might be assumed
that the different lateral chain position plays a role in
this diminished cytotoxicity. The same is true for the
in vivo antitumoral activity since 2 reveals either a total
lack of effect (P388; T/C = 105%) or a lower one (B16;
T/C = 114% vs 157%, respectively).
The cytotoxic effects of the newly synthesized com-
pounds were evaluated on murine L1210 leukemia cells
while the in vivo antitumoral effect was investigated
on P388 leukemia and B16 melanoma (see experimental
part for detailed protocols). These results were compared
with those obtained for the parent compound 1 (Table 1).
Table 1. Cytotoxic and antitumoral profiles
Compound
LogPa
Cytotoxicity
L1210 IC50 (nM)
Antitumoral activity
P388 Leukemia
B16 Melanoma
MTDb
T/C (%)
MTDb
T/C (%)
1
2
3
4
5
2.78
2.78
2.30
2.80
1.57
13.1
330
10
80
80
184–262
105
208
30
30
60
60
20
157
114
129
185
138
160
80
300
16900
110
102
80
a LogP values were calculated using Scilog P – Ultra version 1.5 software (MDL, San Leandro, CA, USA).
b MTD: maximal tolerated dose (mg/kg).

C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
179
However if a pyrimidine ring replaces the pyridine D
ring in 1 like in compound 3, cytotoxic and antitumoral
effects of the two olivacine derivatives are quite similar
(cytotoxicity: L1210 IC50 = 10nM vs 13.1nM, respec-
tively; antitumoral activity: P388: T/C = 208% vs
184–262%, respectively and B16: T/C = 129% vs 157%,
respectively). In this case, unlike compound 2 the lateral
chain position is identical for 1 and 3.
Lipophilicity, another global parameter may be appreci-
ated from calculated logP (Table 1). Thus very close val-
ues are observed for compounds 1, 2, 3 and 4 while a less
lipophilic value was calculated for 5. This last com-
pound exhibits no cytotoxicity under the experimental
condition employed. It is clear that lipophilicity alone
cannot be invoked to explain the difference in toxicity
for the first four compounds.
Further insight on the factors influencing the pharma-
logical profile was obtained by preparing and investigat-
ing analogues 4 and 5. Thus cytotoxic effect as well as
antitumoral effect on P 388 leukemia exhibited by
compound 4 are much lower compared to reference
Further studies18 are needed in order to ascertain the
precise impact of these two global parameters on biolog-
ical behavior.
compound
1
(IC50 = 300 nM vs 13.1 nM and
4. Conclusion
T/C = 110% vs 184–262%, respectively). On the con-
trary, comparative to 1, this imidazocarbazole tetracycle
exhibited a more pronounced antitumoral effect on B16
melanoma T/C = 185% vs 157%, respectively). Unlike
In this paper, the structure–activity relationship arising
from the previously unknown ring D modification in
compound 1 (S 16020-2) has been disclosed. It was
shown that such structural variations strongly impact
on the cytotoxic and in vivo properties of the studied
molecules. Thus, while pyrazinocarbazole 2 is devoid
of any substantial cytotoxic and antitumoral activity,
pyrimidocarbazole 3 has a similar profile compared to
1. Interestingly while L1210 cytotoxicity and P388 in
vivo antitumoral effects are lost for imidazocarbazoles
4 and 5, the compound 4 conserves an in vivo antitumo-
ral effect against B16 melanoma, this effect being even
the largest in the series.
compound 4, compound
5 was devoid of both
cytotoxicity (L1210) and antitumoral effect on P388
leukemia. Antitumoral effect of B16 is lower for com-
pound 5 than for reference 1 (T/C = 138% vs 157%,
respectively).
Structural similarities and dissimilarities among struc-
tures cannot be simply based on superpositions that will
evidence presence/absence of functional groups or atoms
and interconnectivity patterns.
Indeed global properties that evidence molecular char-
acteristics as a whole should be taken into consideration
especially since it is well known that they largely impact
on biological behavior.
5. Experimental
5.1. Chemistry
Thus from simple atom by atom superposition of com-
pounds 1, 3 and 4, one might hastily conclude on very
large similarities between these molecules.
Melting points (cap) were determined on a Mel-temp
capillary apparatus and are uncorrected. Melting points
(K) were determined on a Kofler apparatus.
However when molecular electrostatic potentials (MEP
maps) are calculated (Fig. 2, color coded and repre-
sented on Connolly surfaces), it is easily observed that
while shape similarities are observed for all three com-
pounds, distribution of electrostatic potential values
are identical for 1 and 3, they are very different for com-
pound 4 (Fig. 2, 1, 3, 4 from right to left).
Elemental analysis were performed on a Carlo Erba
analyser 1108. Column chromatography was performed
using Merck silica gel 60 (0.040–0.063mm) under a 1bar
nitrogen pressure (flash chromatography).
All reactions were carried out under a nitrogen atmos-
phere except where otherwise stated.
Figure 2. Color coded molecular electrostatic potentials for compounds 1, 3 and 4.

180
C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
1H NMR spectra were recorded at 200MHz or 300MHz
(as indicated) on a Bruker AC 200 or a Bruker AM 300
spectrometer. Specific high field studies (NOESY experi-
ment on compound 19a) were performed at 500MHz on
a Bruker AMX 500 spectrometer. Solvent is indicated
for each compound.
DMF (1.1L). After 30min, methyl iodide (32.4g,
0.228mol) was added at room temperature for 2h fol-
lowed by concentration under vacuum. The residue
was taken up with dichloromethane and water, the
organic layer was washed with a 10% aqueous solution
of lithium chloride, dried (Na2SO4) and concentrated
under vacuum. Chromatography of the residue eluting
with 3% acetone in dichloromethane afforded com-
pound 9 (43.2g, 64%).
1
Significant H NMR data are reported in the following
order: chemical shifts expressed in ppm downfield from
internal tetramethyl silane, number of protons, multi-
plicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet).
1
Mp (K): 198ꢁC. H NMR (300MHz, DMSO-d6) d 8.04
(1H, d), 7.91 (1H, d), 7.62 (1H, d), 7.22 (1H, dd), 7.02
(1H, d), 4.17 (3H, s), 2.57 (3H, s), 2.32 (3H, s), 2.21
(6H, s). Anal. Calcd for C20H20N2O4: C, 68.17; H,
5.72; N, 7.95. Found: C, 68.11; H, 5.80; N, 7.94.
Electrospray (ESI) high resolution mass spectrometric
analysis was performed using a QtoF2 instrument
(Waters Micromass, Manchester, UK) operating in the
positive mode. 10À6 M solutions of compounds x to y
were introduced by flow injection in a 75/25 acetonit-
rile/water mixture. High measurements were precisely
done by locking the mass calibration with an internal
reference ion (resolving power ca 8500 FWHM at m/z
556). The low resolution mass spectra were recorded
using a Nermag R 10-10 C simple quadrupole mass
spectrometer fitted with a fast atom bombardment
(FAB) source operating in the positive mode.
5.1.4. 7-(Diacetylamino)-8,9-dimethyl-6-nitro-9H-carb-
azol-3-yl acetate (10). Fuming nitric acid (d = 1.52,
5.2mL, 0.12mol) was added at 15ꢁC to a stirred solu-
tion of 9 (43g, 0.12mol) in acetic acid (2L). The mixture
was stirred at room temperature for 3h and concen-
trated under vacuum. The yellow residue was taken up
with diethyl ether collected by filtration, washed with
diethyl ether and dried under vacuum. Column chroma-
tography of the residue eluting with 5% THF in toluene
afforded compound 10 (25.3g, 53%).
5.1.1. 7-Amino-8-methyl-9H-carbazol-3-ol (7). A mixture
of 6 (88.7g, 0.33mol) and 48% hydrobromic acid (2.6L)
was heated under reflux for 2h and then concentrated
under vacuum. The residue was dissolved in water, fil-
tered and the pH was brought to basic using sodium car-
bonate. The precipitate was filtered off, washed with
water and dried at 40ꢁC under vacuum to yield com-
pound 7 (66g, 94%).
1
Mp (K): 266ꢁC. H NMR (400MHz, DMSO-d6) d 9.07
(1H, s), 8.2 (1H, d), 7.8 (1H, d), 7.38 (1H, dd), 4.25 (1H,
s), 2.72 (3H, s), 2.38 (3H, s), 2.28 (6H, s). Anal. Calcd
for C20H19N3O6: C, 60.45; H, 4.82; N, 10.57. Found:
C, 60.77; H, 4.97; N, 10.56.
5.1.5. 7-Amino-8,9-dimethyl-6-nitro-9H-carbazol-3-ol (11).
Compound 10 (25.2g, 0.063mol) was dissolved in etha-
nol (350mL) and 20% aqueous sodium hydroxide solu-
tion (350mL ). The mixture was stirred for 5h at reflux
temperature. Then, the cooled mixture was neutralized
with 4N hydrochloric acid and the ethanol was distilled
off under vacuum. The red precipitate was collected by
filtration, washed with water and diethyl ether and dried
at 40ꢁC under vacuum to yield compound 11 (17.2g,
100%).
Mp (K): 239ꢁC. 1H NMR (300MHz, DMSO-d6) d 10.25
(1H, s), 8.60 (1H, s), 7.45 (1H, d), 7.15 (1H, d), 7.1 (1H,
s), 6.65 (1H, dd), 6.45 (1H, d), 4.8 (2H, bs), 2.2 (3H, s).
Anal. Calcd for C13H12N2O, O. 17 H2O: C, 72.52; H,
5.78; N, 13.01. Found: C, 72.18; H, 5.75; N, 12.82.
HRMS calcd for C13H13N2O (M + H)+: 213.1028;
found: 213.1916.
1
5.1.2. 7-(Diacetylamino)-8-methyl-9H-carbazol-3-yl ace-
tate (8). A mixture of 7 (66g, 0.31mol) and acetic anhy-
dride (3L) was heated under reflux for 2h and then
concentrated under vacuum. The residue was taken up
in ligroin and the solid was collected by filtration and
dried under vacuum. Chromatography of the residue
eluting with a mixture of 4% THF in dichloromethane
led to compound 8 (65.5g, 62%).
Mp (K): 243ꢁC. H NMR (200MHz, DMSO-d6) d 9.15
(1H, br s, exchangeable for D2O), 8.6 (1H, s), 7.37 (1H,
d), 7.25 (1H, d), 7.04 (2H, br s, exchangeable for D2O),
6.85 (1H, dd), 3.9 (3H, s), 2.52 (3H, s). Anal. Calcd for
C14H13N3O3: C, 61.99; H, 4.83; N, 15.49. Found: C,
61.50; H, 4.93; N, 15.03.
5.1.6. 6,7-Diamino-8,9-dimethyl-9H-carbazol-3-ol (12). A
solution of compound 11 (17g, 0.062mol) in NMP
(680mL) in the presence of activated nickel (4mL) was
hydrogenated under a 5bar pressure of hydrogen during
16h. the catalyst was removed by filtration and the fil-
trate was concentrated under vacuum. The residue was
taken up with diethyl ether, collected by filtration,
washed with diethyl ether and dried at 40ꢁC under vac-
uum to yield compound 12 (15.0g, 100%).
1
Mp (K): 184ꢁC. H NMR (200MHz, DMSO-d6) d 11.5
(1H, s, exchangeable for D2O), 8.05 (1H, d), 7.9 (1H, d),
7.55 (1H, d), 7.2 (1H, dd), 7.0 (1H, d), 2.3 (6H, s), 2.2
(3H, s). Anal. Calcd for C19H18N2O4: C, 67.45; H,
5.36; N, 8.28. Found: C, 67.39; H, 5.52; N, 8.16.
5.1.3. 7-(Diacetylamino)-8,9-dimethyl-9H-carbazol-3yl
acetate (9). Sodium hydride (60% dispersion in mineral
oil, 7.6g, 0.19mol) was added at room temperature to
a stirred solution of compound 8 (65.5g, 0.19mol) in
Mp (cap): dec > 250ꢁC. 1H NMR (300MHz, DMSO-d6)
d 8.66 (1H, s, exchangeable for D2O), 7.1 (1H, d), 7.02

C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
181
(1H, d), 6.99 (1H, s), 4.35 (4H, br s, exchangeable for
D2O), 3.82 (3H, s), 2.48 (3H, s). Anal. Calcd for
C14H15N3O: C, 69.69; H, 6.27; N, 17.41. Found: C,
68.80; H, 6.25; N, 16.80. (Water dosage impossible).
120ꢁC for 20h. The mixture was concentrated under
vacuum. Chromatography of the residue eluting with
1% ammoniac solution (28%) and 10% methanol in
dichloromethane yielded compound 5 (1.4g, 59%) as a
mixture of tautomers.
5.1.7. Ethyl 9-hydroxy-5,6-dimethyl-6H-pyrazino[2,3-b]-
carbazole-2-carboxylate (13). Ethyl bromopyruvate
(1.63mL, 0.013mol) was added, dropwise, to a solution
of 12 (3.0g, 0.0124mol) in NMP (120mL). The mixture
was stirred for 16h at room temperature and concen-
trated under vacuum. Chromatography of the residue
eluting with a mixture of 3% methanol in dichlorometh-
ane gave compound 13 (1.2g, 29%).
Mp (K): 273ꢁC. 1H NMR (300MHz, DMSO-d6) d 13.02
(0.5H, s, exchangeable for D2O), 12.94 (0.5H, s,
exchangeable for D2O), 8.93 (1H, s, exchangeable for
D2O), 8.6 (0.5H, t, exchangeable for D2O), 8.51 (0.5H,
t, exchangeable for D2O), 8.13 (0.5H, s), 7.84 (0.5H,
s), 7.5 (0.5H, d), 7.44 (0.5H, d), 7.3 (1H, d), 6.92 (1H,
dd), 4.06 (1.5H, s), 4.05 (1.5H, s), 3.44 (2H, t), 3.1
(1.5H, s), 3.0 (1.5H, s), 2.5 (2H,t), 2.2 (6H, s). Anal.
Calcd for C20H23N5O2, 1.6 H2O: C, 60.93; H, 6.70; N,
17.76. Found: C, 60.40; H, 6.39; N, 17.29. MS calcd
for C20H24N5O2 (M + H)+: m/z 366.
1
Mp (K): 269ꢁC. H NMR (300MHz, DMSO-d6) d 9.35
(1H, s, exchangeable for D2O), 9.3 (1H, s), 8.75 (1H, s),
7.75 (1H, d), 7.50 (1H, d), 7.15 (1H, dd), 4.5 (2H, q), 4.2
(3H, s), 3.3 (3H, s), 1.45 (3H, t). This compound, which
contained NMP, was used without further purification
for the next step.
5.1.11. 6-(Benzyloxy)-1,9-dimethyl-3-nitro-9H-carbazol-
2-amine (15). Sodium hydride (60% dispersion in min-
eral oil, 0.86g, 0.021mol) was added at room tempera-
ture to a stirred solution of compound 11 (5.7g,
0.021mol) in DMF (120mL). After 1h at room temper-
ature, benzyl chloride (3.59g, 0.021mol) was added, the
mixture stirred 2 h and concentrated under vacuum. The
residue was taken up with dichloromethane and water,
the organic phase was dried (Na2SO4) and concentrated
under vacuum. Chromatography of the residue eluting
with dichloromethane yielded compound 15 (5.9g,
78%).
5.1.8. N-[2-(Dimethylamino)ethyl]-9-hydroxy-5,6-dimeth-
yl-6H-pyrazino[2,3-b]carbazole-2-carboxamide (2). Com-
pound
13
(1.2g,
0.0036mol)
and
N,N-
dimethylethylenediamine (25mL) was stirred in a auto-
clave at 120ꢁC for 20h. The mixture was concentrated
under vacuum. Chromatography of the residue eluting
with a mixture of 1% ammoniac solution (28%) and
10% methanol in dichloromethane yielded compound
2 (0.6g, 44%).
1
1
Mp (cap): 247–250ꢁC. H NMR (400MHz, DMSO-d6)
Mp (K): 193ꢁC. H NMR (300MHz, DMSO-d6) d 8.8
d 9.4 (1H, s), 9.3 (1H, s), 8.9 (1H, t), 8.7 (1H, s), 7.7
(1H, d), 7.55 (1H, d), 7.15 (1H, dd), 4.20 (3H, s), 3.55
(2H, q), 3.35 (3H, s), 2.6 (2H, t), 2.3 (6H, s). Anal. Calcd
for C21H23N5O2: C, 66.83; H, 6.14; N, 18.55. Found: C,
66.25; H, 6.08; N, 18.17.
(1H, s), 7.9 (1H, s), 7.5 (2H, d), 7.4 (3H, m), 7.4 (1H,
m), 7.1 (1H, dd), 7.10 (1H, s, exchangeable for D2O),
5.2 (2H, s), 3.95 (3H, s), 2.55 (3H, s). Anal. Calcd for
C21H19N3O3: C, 69.79; H, 5.30; N, 11.63. Found: C,
69.74; H, 5.35; N, 11.48.
5.1.9. 6,7-Bis{[ethoxy(oxo)acetyl]amino}-8,9-dimethyl-
9H-carbazol-3-yl ethyl oxalate (14). Ethyl chorooxoace-
tate (7.9g, 0.0581mol) was added to a stirred solution of
compound 12 (4.0g, 0.0166mol) in DMF (100mL) in
the presence of triethylamine (5.9g, 0.0581mol) at a
temperature below 10ꢁC. The mixture was stirred at
10ꢁC for 16h and concentrated under vacuum. The res-
idue was taken up with dichloromethane and water and
the organic phase was dried (Na2SO4) and concentrated
under vacuum. Chromatography of the residue eluting
with 4% THF in dichloromethane yielded compound
14 (7.5g, 83%).
5.1.12. 6-(Benzyloxy)-1,9-dimethyl-9H-carbazole-2,3-di-
amine (16). Compound 15 (6.5g, 0.018mol) was dis-
solved in THF (260mL) and methanol (130mL).
Activated nickel was added and the mixture was heated
at reflux temperature. Hydrazine hydrate was added
dropwise and the mixture stirred for 15min. The catalyst
was removed by filtration and the filtrate concentrated and
dried under vacuum to yield compound 16 (5.62g, 94%).
Mp (K): 164ꢁC. 1H NMR (300MHz, DMSO-d6) d
7.5(2H, d), 7.4 (3H, m), 7.35 (1H, d), 7.25 (1H, d), 7.1
(1H, s), 6.9 (1H, dd), 5.1 (2H, s), 4.4 (4H, m, exchange-
able for D2O), 3.9 (3H, s), 2.5 (3H, s). HRMS calcd for
C21H22N3O [M + H]+: 332.1763; found: 332.1765.
Mp (K): 237ꢁC. 1H NMR (300MHz, DMSO-d6) d 10.39
(1H, s, exchangeable for D2O), 10.13 (1H, s, exchange-
able for D2O), 8.25 (1H, s), 8.04 (1H, d), 7.7 (1H, d),
7.38 (1H, dd), 4.44-4.27 (6H, m), 4.17 (3H, s), 2.64
(3H, s), 1.4-1.29 (9H, m). Anal. Calcd for
C26H27N3O10: C, 57.67; H, 5.03; N, 7.76. Found: C,
57.53; H, 5.11; N, 7.56.
5.1.13. N-[2-Amino-6-(benzyloxy)-1,9-dimethyl-9H-carb-
azol-3-yl] acetamide (17). Acetyl chloride (1.2g,
0.0143mol) was added at 5ꢁC to a stirred solution of
compound 16 (4.5g, 0.0136mol) in THF (225mL) in
the presence of triethylamine (1.45g). The mixture was
stirred 1h at room temperature and concentrated under
vacuum. The residue was taken up with dichlorometh-
ane and water, the organic phase was dried (Na2SO4),
concentrated under vacuum and dried at 40ꢁC under
vacuum to yield compound 17 (4.9g, 96%).
5.1.10. N-[2-(Dimethylamino)ethyl]-8-hydroxy-4,5-dimethyl-
1,5-dihydroimidazo[4,5-b]carbazole-2-carboxamide (5).
Compound 14 (3.5g, 0.0065mol) and N,N-dimethyle-
thylenediamine (100mL) are stirred in an autoclave at

182
C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
1
Mp (cap): 198–204ꢁC. H NMR (300MHz, DMSO-d6)
d 9.1 (1H, s, exchangeable for D2O), 7.65 (1H, s), 7.5
(1H, d), 7.5–7.35 (5H, m), 7.3 (1H, d), 6.95 (1H, dd),
5.2 (2H, s), 4.75 (2H, bs), 4.0 (3H, s), 2.55 (3H, s), 2.1
(3H, s). HRMS Calcd for C23H24N3O2 [M + H]+:
374.1869; found: 374.1846.
7.45 (1H, d), 7.30 (1H, d), 6.90 (1H, dd), 4.30 (2H, t),
4.05 (3H, s), 3.00 (3H, s), 2.65 (2H, t), 2.60 (3H, s),
2.25 (6H, s). MS: m/z 337 [M + H]+.
5.1.17. 10,11-Dimethyl-1,10-dihydro[1,3]oxazino[4,5-b]carb-
azole-2,4-dione (21). Trimethylsilylazide (5.1mL,
0.0383mol) was added to a stirred solution of com-
pound 20 (1.8g, 0.00678mol) in DMF (90mL) at
60ꢁC. The mixture was stirred at 80ꢁC for 2h, at
100ꢁC for 15min and at room temperature for 16h.
The solid was collected by filtration, washed with diethyl
ether and dried at 40ꢁC under vacuum to yield com-
pound 21 (1.0g, 52%).
5.1.14. 8-(Benzyloxy)-2,4,5-trimethyl-1,5-dihydroimidazo-
[4,5-b]carbazole (18). Compound 17 (4.9g, 0.013mol) in
DMF (260mL) was heated under reflux for 4h and then
concentrated under vacuum. The residue was taken up
with dichloromethane and water. The organic phase
was dried (Na2SO4) and concentrated under vacuum.
Chromatography of the residue eluting with a mixture
of 4% methanol in dichloromethane afforded compound
18 (3.12g, 68%) as a mixture of tautomers.
1
Mp (cap): 338–345ꢁC. H NMR (300MHz, DMSO-d6)
d 8.7 (1H, s), 8.25 (1H, d), 7.65 (1H, d), 7.5 (2H, t),
7.3 (2H, t), 4.1 (3H, s), 2.7 (3H, s). MS: m/z 281
[M + H]+.
Mp (K): 251ꢁC. 1H NMR (300MHz, DMSO-d6) d
(majority form) 12.00 (1H, m, exchangeable for D2O),
7.9 5 (1H, s), 7.80 (1H, d), 7.50 (2H, d), 7.35 (4H, m),
7.10 (1H, dd), 5.20 (2H, s), 4.05 (3H, s), 3.90 (3H, s),
2.55 (3H, s). MS: m/z 356 [M + H]+.
5.1.18. 10,11-Dimethyl-3,10-dihydro-4H-pyrimido[4,5-b]carb-
azol-4-one (22). Compound 21 (24.7g, 0.088mol) and
formamidine acetate (36.6g, 0.352mol) were dissolved
in 1-methoxy-2-propanol (2.47L) and stirred for 1h at
reflux temperature. Silica gel (120g) was added and
the suspension was concentrated to dryness under
vacuum. The residue was eluted with a mixture of 30%
THF in dichloromethane to yield compound 22
(17.65g, 76%).
5.1.15. {2-[8-(Benzyloxy)-2,4,5-trimethylimidazo[4,5-b]carb-
azol-1(5H)-yl]ethyl}dimethylamine (19a). A mixture of
compound 18 (0.3g, 0.0008mol), THF (20mL), DMF
(10mL), potassium carbonate (0.22g, 0.0016mol),
2-dimethylamino ethyl chloride hydrochloride and ado-
gene 464 (0.1g) was stirred at reflux temperature for 16h
and concentrated under vacuum. The residue was taken
up with water, collected by filtration, dissolved with a
mixture of methanol and dichloromethane, dried
(Na2SO4) and concentrated under vacuum. Chromato-
graphy of the residue eluting with a mixture of 5% meth-
anol in dichloromethane afforded compound 19 (0.27g,
79%).
1
Mp (cap): 307–315ꢁC. H NMR (500MHz, DMSO-d6)
d 11.9 (1H, s), 8.8 (1H, s), 8.3 (1H, d), 8.1 (1H, s),
7.65 (1H, d), 7.5 (1H, t), 7.25 (1H, t), 4.2 (3H, s), 3.15
(3H, s). MS: m/z 264 [M + H]+.
5.1.19. 4-Chloro-10,11-dimethyl-10H-pyrimido[4,5-b]carb-
azole (23). Compound 22 (17.6g, 0.0668mol) was stirred
at reflux temperature in phosphorus oxychloride
(400mL) for 3h. The suspension was concentrated
under vacuum. The residue was washed with diethyl
ether, collected by filtration and stirred with an aqueous
solution of sodium hydrogencarbonate. The yellow solid
was collected by filtration, washed with water and dried
at 40ꢁC under vacuum to yield compound 23 (18.0g,
96%).
1
Mp (K): 213ꢁC. H NMR (300MHz, DMSO-d6) d 8.00
(1H, s), 7.85 (1H, d), 7.55 (2H, d), 7.45 (1H, d), 7.40
(3H, m), 7.10 (1H, dd), 5.20 (2H, s), 4.30 (2H, t),
4.05 (3H, s), 3.00 (3H, s), 2.65 (2H,t), 2.50 (3H, s), 2.25
(6H, s).
In a NOESY experiment, specific Overhauser were ob-
served between, CH3 (d = 2.50ppm) and CH2
(d = 4.30ppm); between CH2 (d = 4.30ppm) and CH
(d = 8.00ppm); between CH (d = 8.00ppm) and CH
(d = 5.85ppm) and between CH (d = 7.85ppm) and
CH2 (d = 5.20ppm). All these observations led to a
non ambiguous determination of the position of substi-
tution. MS: m/z 427 [M + H]+.
Mp (K): 258ꢁC. 1H NMR (300MHz, CDCl3) d 9.1 (1H,
s), 8.8 (1H, s), 8.25 (1H, d), 7.65 (1H, t), 7.4 (1H, d), 7.35
(1H, t), 4.25 (3H, s), 3.3 (3H, s). MS: m/z 282 [M + H]+.
5.1.20. 10,11-Dimethyl-10H-pyrimido[4,5-b]carbazole-4-
carbonitrile (24). Compound 23 (10.0g, 0.035mol),
potassium cyanide (22.8g, 0.35mol) and sodium tosylate
(2.3g, 0.012mol) were stirred at 80ꢁC for 4h 30min
DMSO (500mL). The suspension was poured in sodium
chloride saturated aqueous solution (2L) and extracted
with dichloromethane. The organic phase was washed
with water, dried (Na2SO4) and concentrated under vac-
uum. Chromatography of the residue eluting with
dichloromethane yielded compound 24 (6.8g, 71%).
5.1.16. 1-[2-(Dimethylamino)ethyl]-2,4,5-trimethyl-1,5-di-
hydroimidazo[4,5-b]carbazol-8-ol (4). A mixture of com-
pound 19 (0.36g, 0.0008mol), cyclohexene (4mL), NMP
(12mL) and 10% palladium on activated carbon (0.1g)
was stirred at 90ꢁC for 1h. The catalyst was filtered
off and the filtrate was concentrated under vacuum.
The residue was washed with ether and dried under vac-
uum at 40ꢁC to yield compound 4 (0.2g, 74%).
Mp (K): 249ꢁC. 1H NMR (300MHz, CDCl3) d 9.4 (1H,
s), 8.8 (1H, s), 8.3 (1H, d), 7.7 (1H, t), 7.5 (1H, d), 7.4
(1H, t), 4.3 (3H, s), 3.3 (3H, s). MS: m/z 273 [M + H]+.
1
Mp (cap): 275–280ꢁC. H NMR (300MHz, DMSO-d6)
d 8.85 (1H, s, exchangeable for D2O), 7.85 (1H, s),

C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
183
5.1.21. 10,11-Dimethyl-10H-pyrimido[4,5-b]carbazole-4-
carboxamide (25). Anhydrous hydrogen chloride was
slowly bubbled for 5h through a solution of compound
24 (3.2g, 0.01175mol) in methanol (500mL) and dichlo-
romethane (500mL) at a temperature below 20ꢁC. The
mixture was stirred for 16h at room temperature and
concentrated under vacuum to a volume of approxi-
mately 100mL. The solid was collected by filtration,
washed with water, ethanol and diethyl ether, dried
at 40ꢁC under vacuum to yield compound 25 (2.6g,
76%).
Mp (K): 268ꢁC. 1H NMR (300MHz, DMSO-d6) d 10.10
(1H, s), 9.40 (1H, s), 9.15 (1H, s), 9.00 (1H, s), 7.85 (1H,
d), 8.15 (1H, d), 4.30 (3H, s), 4.20 (3H, s), 3.35 (3H, s).
Anal. Calcd for C19H15N3O3: C, 68.46; H, 4.54; N,
12.61. Found: C, 68.28; H, 4.53; N, 12.45.
5.1.25. Methyl 7-hydroxy-10,11-dimethyl-10H-pyrimido-
[4,5-b]carbazole-4-carboxylate (29). Compound 28
(3.09g, 0.0092mol) and methanol (300mL) were vigor-
ously stirred at room temperature. Potassium hydrogen-
sulfate (0.9g) was added and 30% hydrogen peroxide (6
mL) was added over a 15 min period. The mixture was
then heated at reflux temperature for 1h. After cooling
to 40ꢁC, the solid (mixture of starting material 28 and
mineral products) was collected by filtration and reused,
without further purification, in the conditions described
above. This process was repeated several times until
completion of the reaction. Each time, filtrates were
poured in a mixture of dichloromethane (400mL) and
sodium hydrogencarbonate aqueous solution (400mL).
Combined organic phases were dried (Na2SO4) and
concentrated under vacuum. Chromatography of the
residue, eluting with a mixture of 5% THF in dichloro-
methane, afforded compound 29 (1.78g, 68%).
1
Mp (K): 226ꢁC. H NMR (300MHz, DMSO-d6) d 9.3
(2H, 2s), 8.4 (1H, s, exchangeable for D2O), 8.35 (1H,
d), 8..0 (1H, s, exchangeable for D2O), 7.7 (1H, d),
7.65 (1H, t), 7.35 (1H, t), 4.25 (3H, s), 3.3 (3H, s). MS:
m/z 291 [M + H]+.
5.1.22. 10,11-Dimethyl-10H-pyrimido[4,5-b]carbazole-4-
carboxylic sodium salt (26). Compound 25 (4.3g,
0.0154mol) was dissolved in ethanol (200mL) and
DMSO (100mL) in the presence of 1N sodium hydrox-
ide aqueous solution (46.2mL). The mixture was stirred
at reflux temperature for 20h and concentrated under
vacuum. The residue was taken up with ethanol and
diethyl ether, collected by filtration, washed with diethyl
ether and dried at 40ꢁC under vacuum to yield com-
pound 26 (5.0g) which was used without further purifi-
cation for the next step.
1
Mp (K): 246ꢁC. H NMR (300MHz, DMSO-d6) d 9.40
(1H, s), 9.35 (1H, s), 9.30 (1H, s, exchangeable for D2O),
7.75 (1H, d), 7.55 (1H, d), 7.15 (1H, dd), 4.20(3H, s),
4.15 (3H, s), 3.25 (3H, s). HRMS calcd for
C18H16N3O3 [M + H]+: 322.1192; found: 322.1160.
1H NMR (300MHz, DMSO-d6) d 9.0 (1H, s), 8.85 (1H,
s), 8.20 (1H, d), 7.65 (1H, d), 7.60 (1H, t), 7.30 (1H, t),
4.25 (3H, s), 3.25 (3H, s). MS: m/z 314 [M + H]+.
5.1.26. N-[2-(Dimethylamino)ethyl]-7-hydroxy-10,11-di-
methyl-10H-pyrimido[4,5-b]carbazole-4-carboxamide (3).
Compound 29 (1.0g, 0.0031mol) and N,N-dimethyle-
thylenediamine (40mL) were stirred at 60ꢁC for 1h
and concentrated under vacuum. Chromatography of
the residue eluting with a mixture of 0.5% ammonia
solution (28%) and 5% methanol in dichloromethane
yielded compound 3 (1.03g, 88%).
5.1.23. Methyl 10,11-dimethyl-10H-pyrimido[4,5-b]carb-
azole-4-carboxylate (27). Compound 26 (0.8g,
0.00255mol) was dissolved in methanol (500mL) in
the presence of concentrated sulfuric acid. The mixture
was stirred at reflux temperature for 3h. After cooling,
the mixture was neutralized using sodium hydrogencar-
bonate, concentrated under vacuum, the residue taken
up with water, collected by filtration, washed with water
and dried at 40ꢁC under vacuum to yield compound 27
(0.53g, 68%).
1
Mp (cap): 235–240ꢁC. H NMR (300MHz, DMSO-d6)
d 9.35 (1H, s), 9.30 (1H, s, exchangeable for D2O),
9.25 (1H, s), 8.25 (1H, t, exchangeable for D2O),
7.60(1H, d), 7.50 (1H, d), 7.15 (1H, dd), 4.20 (3H, s),
3.55 (2H, q), 3.25 (3H, s), 2.55 (2H, t), 2.25 (6H, s).
Anal. Calcd for C21H23N5O2: C, 66.83; H, 6.14; N,
18.55. Found: C, 66.72; H, 6.06; N, 18.57. HRMS calcd
for C21H24N5O2 [M + H]+: 378.1930; found: 378.1916.
Mp (K): 222ꢁC. 1H NMR (300MHz, DMSO-d6) d
9.30 (1H, s), 9.00 (1H, s), 8.40 (1H, d), 7.70 (2H, m),
7.35 (1H, m), 4.20 (6H, 2s), 3.35 (3H, s). HRMS
calcd for C18H16N3O2 [M + H]+: 306.12.43; found:
306.1236.
5.1.24. Methyl 7-formyl-10,11-dimethyl-10H-pyrimido-
[4,5-b]carbazole-4-carboxylate (28). Compound 27
(3.5g, 0.0115mol) was dissolved in trifluoroacetic acid
(175mL). Hexamethylenetetramine (18.2g) was added
over a 30-min period. The mixture was stirred at reflux
temperature for 40min and concentrated under vacuum.
The residue was taken up with a sodium hydrogencarbo-
nate aqueous solution, the solid was collected by filtra-
tion, washed with water and dried at 40ꢁC under
vacuum. Chromatography of this product eluting with
3% THF in dichloromethane gave compound 28
(3.15g, 83%).
5.2. Biology
5.2.1. Cell culture and cytotoxicity. The murine L1210
leukemia cells were cultured in RPMI 1640 medium
(Gibco) supplemented with 10% fetal calf serum, 2mM
L-glutamine, 100 units/mL penicillin, 100lg/mL strepto-
mycin and 10 mM HEPES buffer (pH = 7.4). Cytotoxi-
city was measured by the microculture tetrazolium assay
as described.19,20 Cells were exposed to graded concen-
trations of drug (nine serial dilutions in triplicate)
for about four doubling times (48h). Results were ex-
pressed as IC50, the concentration that reduced by

184
C. Guillonneau et al. / Bioorg. Med. Chem. 13 (2005) 175–184
50% the optical density of treated cells with respect to
untreated controls.
References and notes
´
1. Guillonneau, C.; Pierre, A.; Charton, Y.; Guilbaud, N.;
´
Kraus-Berthier, L.; Leonce, S.; Michel, A.; Bisagni, E.;
5.2.2. Antitumor activity. The antitumor activity of
the compounds was evaluated on the P388 leukemia
and the B16 melanoma, all provided by the NCI, Fred-
erick, U.S.A. P388 cells were inoculated ip (106 cells/
mouse) into B6D2F1 mice (Iffa credo) on day 0. The
dichlorohydrate salts of the compounds were dissolved
in water and injected iv on day 1. The results are ex-
pressed in terms of percent T/C survival (median sur-
vival time of treated animals/median survival time of
control animals, · 100). For the ip B16 melanoma,
0.5mL of a tumor brei (1g of tumor in 10mL 0.9%
NaCl) were injected ip on day 0, and compounds were
administered ip on days 1, 5, 9. The optimal dose was
the dose which gave the higher T/C without major toxi-
city (no toxic death, weight loss <20%). Results are ex-
pressed as % T/C survival.
Atassi, G. J. Med. Chem. 1999, 42, 2191.
2. Wang, H.-K.; Morris-Natschke, S. L.; Lee, K.-H. In
Medicinal Research Reviews; John Wiley & Sons, 1997;
Vol. 17, pp 367–427.
3. Moron, J.; Landras, C.; Bisagni, E. J. Heterocycl. Chem.
1992, 29, 1573.
4. Bhatt, M. V.; Kulkarni, S. U. Synthesis 1983, 249.
5. Albanese, D.; Landini, D.; Penso, M.; Spano, G.;
Trebicka, A. Tetrahedron 1995, 51, 5681.
6. Lancelot, J.-C.; Gazengel, J.-M.; Robba, M. J. Heterocycl.
Chem. 1981, 18, 1281.
7. Sanna, P.; Carta, A.; Loriga, M.; Zanetti, S.; Sechi, L.
Farmaco 1999, 54, 169.
´
8. Jasztold-Howorko, R.; Landras, C.; Pierre, A.; Atassi, G.;
Guilbaud, N.; Kraus-Berthier, L.; Leonce, S.; Rolland, Y.;
Prost, J.-F.; Bisagni, E. J. Med. Chem. 1994, 37,
2445.
9. Lancelot, J.-C.; Landelle, H.; Robba, M. Chem. Pharm.
Bull. 1984, 32, 902.
10. Stevenson, T. M.; Leonard, N. J. J. Org. Chem. 1984, 49,
2158.
11. Venugopalan, B.; Desai, P. D.; de Souza, N. J. J.
Heterocycl. Chem. 1988, 25, 1633.
12. Miyashita, A.; Suzuki, Y.; Ohta, K.; Higashino, T.
Heterocycles 1994, 39, 345.
5.2.3. Molecular modelling. Structures discussed in this
study were modified using standard bond lengths as
implemented in SYBYL version 6.9.1 from Tripos21
running on an OCTANE R12000 Silicon Graphics
workstation.
13. Plug, J. P. M.; Koomen, G.-J.; Pandit, U. K. Synthesis
1992, 1221.
14. Hongbin, L.; Rong, Z.; Hui, W.; Yi, P.; Yaozeng, S.
Synth. Commun. 1997, 27, 3047.
15. DeLuca, M. R.; Kerwin, S. M. Tetrahedron 1997, 53,
457.
16. Letois, B.; Lancelot, J.-C.; Saturnino, C.; Robba, M. J.
Heterocycl. Chem. 1993, 30, 1525.
A comparison of electrostatic potentials was performed
for molecules 1, 3 and 4 (same side chain position rela-
tive to the tetracyclic skeleton. Thus the X-ray structure
of 1 (S 16020-2) was used as a model to set the basic side
chain position, followed by a short AM1 semiempirical
optimization22 to remove any unfavorable interactions.
17. Graybill, T. L.; Dolle, R. E.; Osifo, I. K.; Schmidt, S. J.;
Gregory, J. S.; Harris, A. L.; Miller, M. S. Bioorg. Med.
Chem. 1995, 5, 387.
Electrostatic potentials were then calculated using par-
tial Gasteiger–Huckel charges, and were projected on
¨
the Connolly surfaces of the molecules, visualized using
the MOLCAD software within SYBYL.
´
´
18. Le Mee, S.; Pierre, A.; Markovits, J.; Atassi, G.; Jacque-
min-Sablon, A.; Saucier, J.-M. Mol. Pharmacol. 1998, 53,
213.
´
`
19. Leonce, S.; Perez, V.; Casabianca-Pignede, M. R.; Anstett,
M.; Bisagni, E.; Atassi, G. Invest. New Drugs 1996, 14,
169.
Energy levels (29.2 to À60.5kcal/mol) are represented by
a color scheme (Fig. 2).
20. Alley, M. C.; Scudiero, O. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.;
Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1989, 48,
589.
21. Tripos Inc., 1699 South Hanley Road, Saint-Louis, MO
63144-2913.
Acknowledgements
We thank Dr. J. P. Volland and his team for analytical
data, the team of the division of experimental oncology
for performing biological assays and Miss C. Pancin for
the skillful typing of the manuscript.
22. Dewar, M. J. S.; Zoebish, E. G.; Healy, E. F.; Stewart, J.
P. J. Am. Chem. Soc. 1985, 107, 3902.