Synthesis of 9-O-substituted derivatives of 9-hydroxy-5,6-dimethyl-6H- pyrido[4,3-b]carbazole-1-carboxylic acid (2-(dimethylamino)ethyl)amide and their 10- and 11-methyl analogues with improved antitumor activity

Article abstract of DOI:10.1021/jm981093m

Analogues of the antitumor drug S 16020-2 modified at the 9, 10, or 11 position were synthesized and evaluated in vitro and in vivo on the P388 leukemia and B16 melanoma models. Starting from 9-methoxy-5,11-dimethyl-6H- pyrido[4,3-b]carbazole-1-carboxylic acid ethyl ester, the 11-CH₃ analogue of 9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylic (2- (dimethylamino)ethyl)amide (1), compound 4, was synthesized using a four- step sequence, whereas its 10-CH₃ analogue 5 was prepared using a two-step pathway, starting from compound 1. Finally starting from the 9-OH compounds 1, 4, and 5, a series of variously 9-O-substituted derivatives were synthesized. In these series, the most active compounds resulted from esterification of the 9-OH group with various aliphatic diacids, which led to 9-O-CO-()-COOH derivatives of 1, 4, and 5. For these compounds, the number of long-term surviving mice obtained at the optimal dose were 60-100% in the ip/iv P388 leukemia and 10-35% in the ip/ip B16 melanoma, corresponding to an improved therapeutic index with respect to 1 and 4. This high antitumor activity, with curative examples in both models, was not due to a higher cytotoxicity since these compounds were equally or slightly less potent in vitro than 1 and 4. The most active compounds were thus selected for further in vivo evaluation.

Full text of DOI:10.1021/jm981093m

J . Med. Chem. 1999, 42, 2191-2203
2191
Syn th esis of 9-O-Su bstitu ted Der iva tives of 9-Hyd r oxy-5,6-d im eth yl-6H-
p yr id o[4,3-b]ca r ba zole-1-ca r boxylic Acid (2-(Dim eth yla m in o)eth yl)a m id e
a n d Th eir 10- a n d 11-Meth yl An a logu es w ith Im p r oved An titu m or Activity
Claude Guillonneau,*,† Alain Pierre´,‡ Yves Charton,† Nicolas Guilbaud,‡ Laurence Kraus-Berthier,‡
Ste´phane Le´once,‡ Andre´ Michel,† Emile Bisagni,§ and Ghanem Atassi‡
Division Chimie A and Division de Cance´rologie Expe´rimentale, Institut de Recherches Servier, 11 rue des Moulineaux,
92150 Suresnes, France, and UMR176 CNRS-Institut Curie-Recherche, Baˆtiment 110, 15 rue Georges Cle´menceau,
91405 Orsay, France
Received October 11, 1998
Analogues of the antitumor drug S 16020-2 modified at the 9, 10, or 11 position were synthesized
and evaluated in vitro and in vivo on the P388 leukemia and B16 melanoma models. Starting
from 9-methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylic acid ethyl ester, the 11-
CH3 analogue of 9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylic (2-(dimethyl-
amino)ethyl)amide (1), compound 4, was synthesized using a four-step sequence, whereas its
10-CH3 analogue 5 was prepared using a two-step pathway, starting from compound 1. Finally
starting from the 9-OH compounds 1, 4, and 5, a series of variously 9-O-substituted derivatives
were synthesized. In these series, the most active compounds resulted from esterification of
the 9-OH group with various aliphatic diacids, which led to 9-O-CO-( )-COOH derivatives of 1,
4, and 5. For these compounds, the number of long-term surviving mice obtained at the optimal
dose were 60-100% in the ip/iv P388 leukemia and 10-35% in the ip/ip B16 melanoma,
corresponding to an improved therapeutic index with respect to 1 and 4. This high antitumor
activity, with curative examples in both models, was not due to a higher cytotoxicity since
these compounds were equally or slightly less potent in vitro than 1 and 4. The most active
compounds were thus selected for further in vivo evaluation.
Ch a r t 1
In tr od u ction
In a preceding paper, we described a new series of
cytotoxic olivacine derivatives, from which S 16020-2
(1) (Chart 1) was selected for further pharmacological
and toxicological evaluation.1 This new potent inhibitor
of DNA topoisomerase II2 was highly cytotoxic against
a panel of 20 tumor cell lines, with a mean IC50
comparable to that of adriamycin and markedly lower
than that of elliptinium acetate.3 In vivo, S 16020-2 was
very active against certain murine transplantable tu-
mors and human xenografts in nude mice.4,5 Due to its
antitumor activity in experimental models, favorable
pharmacokinetic characteristics, and acceptable toxic-
ity,6 S 16020-2 is currently studied in clinical trials.7
The aim of the present work was the synthesis of new
analogues and derivatives possessing comparatively
higher antitumor potency with an improved therapeutic
index. We thus focused on three positions of the parent
molecule that appear to be implicated in the biological
activity and therefore worthy of modification: methyl-
ation at positions 10 and 11 and substitution of the free
hydroxyl group at position 9.
Ellipticine (2), which bears a methyl group at position
11, was shown to be slightly more cytotoxic in vitro than
olivacine (3) and also more toxic in mice as indicated
* To whom all correspondence should be sent.
† Division de Chimie A, Institut de Recherches Servier.
‡ Division de Cance´rologie Expe´rimentale, Institut de Recherches
Servier.
by a lower LD0.8 Data available for a limited number of
pairs of olivacine/ellipticine derivatives which differ only
by the presence of the 11-CH3 group suggest that these
§ UMR176 CNRS-Institut Curie-Recherche.
10.1021/jm981093m CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/02/1999

2192 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Guillonneau et al.
Sch em e 1a
a
Method A: (a) dimethyl carbonate, adogene 464, DMF, 89%; (b) NaOH, H2O, ethanol, 52%; (c) H2N-CH2CH2N(CH3)2, PyBOP, NMP,
86%; (d) BBr3, CH2Cl2, 82%.
Sch em e 2a
a
(a) Acetic acid, dioxane, 86%; (b) 10% Pd/C, ethanol, H2, 50%; (c) cyclohexene, 10% Pd/C, NMP, 80%.
properties might be general.6 To have access to 11-CH3
derivatives, we developed a synthetic route to compound
4, herein described for the first time.
No data were available concerning derivatives bearing
a methyl group at position 10 in these series of com-
pounds. The possible involvement of a quinone imine,
generating an electrophilic center at the 10 position, in
either their antitumor activity or their toxicity9 led us
to synthesize the novel compound 5 and derivatives and
to study their pharmacological properties.
Hydroxylation at the 9 position was previously shown
to be a favorable modification in the ellipticine series:
it increases the affinity for DNA,10 stabilizes the DNA
topoisomerase II cleavable complex,11 and consequently
increases the cytotoxicity and antitumor activity.1,12
Moreover, it is interesting to note that many anticancer
drugs interacting with topoisomerases possess a phe-
nolic group. It can be hypothesized that this phenol
plays an important role in the mechanism of antitumor
action and also in the induction of some toxic side effects
through the generation of the quinone imine in the case
of ellipticines and olivacines.13 Hence we decided to
mask this function with hydrolyzable groups in order
to decrease the general toxicity of the compounds and
to allow gradual generation of the more active hydrox-
ylated compounds. Compounds 1, 4, and 5 were there-
fore subjected to these modifications, to obtain various
products corresponding to the general structure 6.
Ch em istr y
The general structure 6 is characterized by the
presence of a fused tetracycle 6H-pyrido[4,3-b]carbazole
substituted at position 1 by a 2-(dimethylamino)ethyl-
carbamoyl group and by methyl groups at positions 5
and 6. R2 and R3 substituents at positions 10 and 11
are either a proton or a methyl. In all cases, the variable
group R1 is bound to the 9 oxygen atom of the tetracycle
and includes an ester, ether, carbamate, sulfonate,
carbonate, or phosphate function.
The syntheses of original compounds 4 and 5 are
summarized in Schemes 1 and 2 (methods A and B),
whereas derivatives described in this publication were
prepared from compounds 1,1 4, and 5 using various
methods (Schemes 3-5, methods C-P).
The structures of all compounds corresponding to the
general formula 6 are described in Table 1.
Methylation of the previously described ester 71 using
dimethyl carbonate as methylating agent gave com-

Analogues of S 16020-2 with Improved Antitumor Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2193
Sch em e 3a
a
Method C: acid chloride, N(C2H5)3, THF, 25-65%. Method D: acid anhydride, pyridine, 28.5-75%. Method E: carboxylic acid, DCC,
HOBT, DMF, 40-67.5%. Method F: carboxylic acid, DCC, DMAP, pyridine, 91%. Method G: carboxylic acid, PyBOP, NMP, 24-88%.
Method H: cyclic anhydride, pyridine, 34-87%. Method I: alcohol, PPh3, DEAD, THF, CH2Cl2, 32%. Method J : bromoalkyl ester, Cs2CO3,
DMF, 69%. Method K: N-chloroformate, pyridine, 51-95%. Method L: isocyanate, DBU, pyridine, 42-92%. Method M: alkanesulfonyl
chloride, N(C2H5)3, THF, 63.5%. Method N: 3-(benzyloxycarbonyl)propyl chloroformate, pyridine, NMP, 39%. Method O: NaH, tetrabenzyl
pyrophosphate, THF, 63%.
Sch em e 4a
a
(a) NaH, THF, 63%; (b) cyclohexene, Pd/C, NMP (method P); yields and reaction conditions indicated in Experimental Section.
Sch em e 5a
a
(a) Room temperature (4 days); (b) cyclohexene, 10% Pd/C, NMP, 62.8%.
pound 8, which on condensation with 2-(dimethylamino)-
ethylamine failed to yield the amide 10 probably be-
cause of the steric hindrance due to the presence of the
11 methyl group. Indeed, at 120 °C no reaction occurred,
but when the condensation was performed at a higher
temperature, about 170 °C, compound 8 spontaneously
lost its 1-COOC2H5 group and gave the 1-H analogue
of 8. Therefore, we tried to carry out the amidification
from the acid 9, obtained by saponification of 8, using
several classical coupling agents such as ethyl chloro-
formate, DCC-HOBT,14 or DCC-DMAP15 which totally
failed. However, using PyBOP16 in NMP, which gener-
ally works with hindered carboxylic acids, the amide 10
was obtained in good yield, and subsequent demethyl-
ation in the presence of BBr3 gave the desired compound
4 (method A).
On the other hand, when performed with compound
1
using N,N,N′,N′-tetramethyldiaminomethane,
a

2194 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Ta ble 1. Biological Activity of Compounds
Guillonneau et al.

Analogues of S 16020-2 with Improved Antitumor Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2195
Ta ble 1 (Continued)

2196 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Ta ble 1 (Continued)
Guillonneau et al.
a
b
Inhibition of cell proliferation measured by the MTT assay (mean of g2 values obtained in independent experiments). Mice were
inoculated ip on day 0 with 106 P388 cells and treated iv by at least three doses of the compounds. c Dose (mg/kg) giving the best antitumor
d
activity without major toxic effects (no lethality and body weight loss <20%). Median survival time of treated/median survival time of
control animals, obtained in one or two representative experiments. e Long-term survivors scored on day 60 (P388) or 90 (B16). f 0.5 mL
of a tumor brei was injected ip on day 0, and compounds were administered ip on days 1-9.
Mannich reaction yielded compound 11 according to a
previously described procedure.17 Catalytic hydrogena-
tion of derivative 11 gave the expected compound 5 (15%
yield) besides the 1,2,3,4-tetrahydro-6H-pyrido[4,3-b]-
carbazole derivative 12 (50% yield) as the major product.
Aromatization of 12 over palladium on charcoal in
xylene totally failed. Nevertheless, our study in order
to find optimized conditions allowed us to obtain com-
pound 5 in very good yield (80%) by replacing hydrogen
gas by cyclohexene and palladium on charcoal in NMP.18
9-O-Substituted derivatives of compounds 1, 4, and
5, described in Scheme 3, were readily prepared, using
classical organic reactions from 9-OH compounds 1, 4,
and 5. Benzyl esters 20 and 21 were obtained from
compound 1 and adipic and succinic monobenzyl ester,19
respectively, using DCC-HOBT14 as coupling agent
(method E). Benzyl ester 22 was prepared from 1 and
N-tBOC-L-glutamic acid R-benzyl ester using PyBOP16
as coupling agent (method G). Debenzylation of 20 and
22 was achieved with cyclohexene and palladium on
carbon as catalyst18 in N-methylpyrrolidone (method P)
to give monoesters 32 and 33, respectively. It can be
pointed out that debenzylation of benzyl ester 21, using
the same method P, failed to give the expected succinic
acid monoester of compound 1, this compound being
probably susceptible to hydrolysis. Acidic hydrolysis of
the protected glycerol ether 35 and saponification of
ethyl ester 38 gave compounds 37 and 39, respectively.

Analogues of S 16020-2 with Improved Antitumor Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2197
Condensation of compound 1 with (R,S)-trans-2-phen-
ylcyclopropylmethanol20 using the Mitsunobu proce-
dure21 gave compound 36. Condensation of crude 3-(ben-
zyloxycarbonyl)propyl chloroformate (prepared from
4-hydroxybutyric acid benzyl ester22 and phosgene in
toluene using dimethylaniline as base) with compound
1 in pyridine gave the carbonate 54 (method N). This
compound was further debenzylated to give compound
55 (method P).
Reaction between tetrabenzyl pyrophosphate23 with
compound 1 gave unstable phosphate 56 (method O,
Scheme 4) which was further monodebenzylated or
didebenzylated, according to the reaction conditions,
using method P18 to give compounds 57 and 58, respec-
tively.
After 4 days at room temperature, the unstable
compound 56 gave the zwitterion 59 resulting from the
migration of one benzyl group from the 9-dibenzyl
phosphate moiety to the dimethylamino part of the
1-substituent of compound 56. Further debenzylation
of 59, using method P, gave compound 58.
F igu r e 1. Antitumor activity against ip P388 leukemia.
Compounds were administered by iv route on day 1 to P388
leukemia-bearing mice. Results are expressed as % T/C and
long-term survivors (LTS) as indicated in the Experimental
Section.
P h a r m a cology
In vivo evaluation was performed on two murine
experimental models, the P388 leukemia and the B16
melanoma. The compounds were tested at three doses
in the first experiment, and, if active, at five doses in a
second experiment. To estimate the therapeutic index,
the doses range includes a low, nontoxic, inactive dose
and a high, toxic dose. Table 1 lists the antitumor
activity obtained at the optimal dose, which is the dose
giving the best T/C without major toxicity estimated by
a body weight loss > 20% and/or toxic death in the
experimental group. The optimal dose is generally
immediately below the toxic dose.
Compound 1 was markedly active against the P388
leukemia following a day 1, 5, 9 schedule of administra-
tion, inducing on average 62% of long-term survivors
(LTS) at 80 mg/kg (n ) 40 experiments). It was less
active on this model when administered on day 1 (4 %
of LTS on average) and moderately active on the B16
melanoma, inducing a mean T/C of 157% and 3% of LTS
(n ) 60 experiments). Elliptinium acetate was only
moderately active on the two models, without inducing
LTS.
Hence, we selected the compounds giving a significant
number of LTS on the P388 leukemia (day 1 schedule)
and the B16 melanoma for further evaluation.They are
4, 25, 27-33, and 55 for P388 and 4, 21, 25, 27, 29, 30,
and 32 for B16. Some of these compounds have a
broader therapeutic index than 1, as illustrated on
Figures 1 and 2. On P388 leukemia, the therapeutic
index (TI) of 1 was 8 versus 16 for 32, which induced
16 LTS over 19 mice at 240-320 mg/kg. On B16 mel-
anoma, the TI of 1 was 8 versus 32 for 25 (Figure 2).
All these compounds were either equally or slightly less
cytotoxic than 1 and induced a massive accumulation
of L1210 cells in the G2+M phase of the cell cycle at
cytotoxic concentrations (not shown). This modification
of the cell cycle reflects at the cellular level the inhibi-
tion of DNA topoisomerase II, as is the case for 1.2,3
F igu r e 2. Antitumor activity against ip B16 melanoma. On
day 0, 0.5 mL of B16 tumor brei at 1 g/10 mL was inoculated
ip to B6D2F1 mice. Drugs were administered ip daily for a
total of 9 injections (days 1-9). Results are expressed as %
T/C.
stitution of the 9-hydroxy group of compound 1 and
analogues 4 and 5 on cytotoxicity and antitumor activ-
ity.
Effect of Meth yla tion a t P osition 10. Although 5
(10-CH3) was more cytotoxic than 1 (10-H) on both cell
lines, it was less active in vivo in the two models as no
LTS were scored at the optimal doses. In addition, 5
was more toxic than 1 as shown by comparison of their
iv optimal doses (day 1): 20 versus 80 mg/kg, respec-
tively. The 10 methyl group, supposed to prevent the
covalent binding of nucleophiles after formation of a
quinone imine, thus led to a more cytotoxic and toxic
compound. It is interesting to note that 1 and 5 showed
a similar antitumor efficiency at low doses, which
suggests that covalent binding to a cellular target like
DNA is not involved in the mechanism of action of this
family of compounds, at least through the 10 position
of the 6H-pyrido[4,3-b]carbazole system.
A similar trend is observed for compound 26 (10-CH3)
which is less active in vivo and more toxic than
compound 32 (10-H).
Effect of Meth yla tion a t P osition 11. The cytotox-
icity of compound 4 (11-CH3) was similar to that of
compound 1 (11-H) on both cell lines. The lack of effect
of methylation at position 11 on cytotoxicity was also
Str u ctu r e-Activity Rela tion sh ip s
The present work was undertaken to determine the
effect of methylation at positions 10 and 11 and sub-

2198 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Guillonneau et al.
observed with 27 versus 32 and with 28 versus 25 on
L1210 cells only, 28 being surprisingly more cytotoxic
than 25 on B16 cells.
32) were selected for further evaluation in experimental
models of solid tumors and for preliminary toxicological
studies.
In vivo, 4 was more potent and markedly more active
than 1 following the day 1 schedule against the P388
leukemia (15 LTS/18 mice versus 0/12, respectively).
Following the D1,5,9 schedule, both compounds were
highly active, but 4 was 8-fold more potent than 1
(optimal doses 10 versus 80 mg/kg, respectively). Against
the B16 melanoma, both compounds were equally ac-
tive, but 4 was 2-fold more potent than 1.
Exp er im en ta l Section
Ch em istr y. Autonom 2 software was used to name com-
pounds, used or prepared, in this publication.
Melting points (cap) were determined on a Mel-temp capil-
lary apparatus and were uncorrected. Melting points (K) were
determined on a Kofler apparatus. Fast atom bombardment
mass spectra (FAB) and chemical ionization spectra (CI) were
performed on a Nermag R10-10 C instrument. 1H NMR spectra
of compounds in solution in CDCl3 or DMSO-d6 were recorded
using a Bruker AC 200 spectrometer. Chemical shifts are
expressed in ppm downfield from internal tetramethylsilane.
Significant 1H NMR data are reported in the following order:
multiplicity (s, singlet; d, doublet, t, triplet; q, quartet; m,
multiplet; AB, AB system), number of protons. Elemental
analyses were performed on a Carlo Erba analyzer 1108.
Column chromatography was performed using Amicon silica
gel (0.035-0.07 mm) under a 1 bar nitrogen pressure (flash
chromatography). All reactions were carried out under a
nitrogen atmosphere. The methods of preparation, the yields,
and the melting points for the final step products are reported.
The formula indicates possible salification or solvation of
products.
Similarly, a more pronounced antitumor activity and
a higher potency of the 11-CH3 derivatives were ob-
served in the P388 model with the two pairs of deriva-
tives: 28 versus 25 and 27 versus 32. The two 11-CH3
derivatives 28 and 27 were highly active following both
schedules of administration, curing 90% of mice at doses
4-6-fold lower than those required for their 11-H
derivatives 25 and 32. Against the B16 melanoma, 28
and 27 were more potent than 25 and 32.
Effect of Su bstitu tion of th e 9-OH Gr ou p . Among
the 9-OH-substituted derivatives, the most active com-
pounds, in vitro and in vivo, corresponded to 9-O-CO-
( )-COOH derivatives of the heterotetracyclic system.
Derivatives of 1 (25, 29, 32) were generally more active
than 1 in vivo and presented a better therapeutic index
as shown by a higher optimal dose (Figure 2), although
derivatives 27 and 28 (11-CH3) were about as active as
4 at similar doses. It is thus clear that the substitution
of the 9-OH group by an ester carrying a free COOH
function is more favorable for 11-H than for 11-CH3
derivatives. These 9-OH-substituted compounds can be
cytotoxic by themselves or by generating the corre-
sponding free hydroxyl group after cleavage of the ester
bound by cellular esterases. Preliminary results show
that 25 is less potent than 1 at inhibiting the catalytic
activity of purified DNA topoisomerase II (not shown),
which suggests that these esters are prodrugs of the
9-OH compounds.
Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole
(HOBT), 4-(dimethylamino)pyridine (DMAP), N-methyl-2-pyr-
rolidone (NMP), triphenylphosphine (PPh3), diethyl azodicar-
boxylate (DEAD), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
and (benzotriazol-1-yloxy)trispyrrolidinophosphonium hexaflu-
orophosphate (PyBOP) were obtained from Aldrich.
9-Hyd r oxy-5,6,11-tr im eth yl-6H-p yr id o[4,3-b]ca r ba zole-
1-ca r boxylic Acid (2-(Dim eth yla m in o)eth yl)a m id e Di-
h yd r och lor id e (4) (Meth od A, Sch em e 1). A 1 M solution
of boron tribromide in dichloromethane (230 mL, 230 mmol)
was added to a stirred suspension of compound 10 (9.5 g, 23
mmol) and water (3 mL) in dichloromethane (380 mL) at -50
°C, and then the temperature was allowed to rise to 10 °C
before pouring onto crushed ice and water. The mixture was
made basic by addition of a 28% ammonia solution. The solid
was collected by filtration, washed with water, and dried under
vacuum. Column chromatography, eluting with a mixture of
1% ammonia solution (28%) and 10% methanol in dichloro-
methane, gave compound 4 as the free base (8.2 g). The product
was suspended in ethanol (200 mL) and treated with 2 M
solution of hydrogen chloride in ethanol. The solid was
collected by filtration and washed with ethanol and ether
before drying at 50 °C under vacuum to yield compound 4 (8.75
g, 82%): mp(cap) 288-290 °C; 1H NMR (DMSO-d6) δ 9.5-9.0
(m, 4H, exchangeable for D2O), 8.5 (m, 2H), 7.8 (d, 1H), 7.6 (d,
1H), 7.2 (dd, 1H), 4.2 (s, 3H), 3.9 (m, 2H), 3.5 (m, 2H), 3.1 (2s,
6H), 2.95 (s, 6H). Anal. (C23H26N4O2‚2HCl‚0.5H2O) C, H, N,
Cl.
The more lipophilic compounds containing one or two
phenyl groups (14, 15, 20, 21, 42, 44, 50) or a linear
polymethylene chain (16, 41, 47) were among the most
toxic compounds studied as shown by their low optimal
doses and were either slightly active or inactive.
Con clu sion
9-Hyd r oxy-5,6,10-tr im eth yl-6H-p yr id o[4,3-b]ca r ba zole-
1-ca r boxylic Acid (2-(Dim eth yla m in o)eth yl)a m id e (5)
(Meth od B, Sch em e 2). Cyclohexene (20 mL) and 10%
palladium on activated carbon (2 g) were added to a stirred
solution of compound 11 (3.0 g, 6.9 mmol) in N-methylpyr-
rolidone (150 mL). The mixture was stirred for 10 h at 100
°C. A further three portions of cyclohexene (3 × 20 mL) and
10% palladium on activated carbon (3 × 2 g) were added
during this period. After cooling, the mixture was filtered and
the filtrate concentrated under vacuum. Column chromatog-
raphy of the residue eluting with a mixture of 0.5% ammonia
solution (28%) and 10% methanol in dichloromethane gave
Among the modifications of the olivacine/ellipticine
derivatives studied in this work, methylation at the 11
position and aliphatic diacid monoesterification of the
9-OH position were shown to markedly improve the
antitumor activity. The 11-methylated derivatives showed
a more pronounced antitumor activity at lower doses
than their corresponding 11-H derivatives. The most
active compounds described in this study (4, 27, 28, 32)
are probably the most potently active ellipticine ana-
logues described to date, dramatically more active than
elliptinium acetate, in that they cured more than 80%
of mice bearing the P388 leukemia after one iv injection.
The monoesterification of the 9-OH of S16020-2 by an
aliphatic diacid led to compounds with a very low
toxicity, the optimal dose being in the range 160-320
mg/kg. Consequently, their therapeutic index was sig-
nificantly improved. Highly active compounds (4, 25-
1
compound 5 (2.18 g, 80%): mp(K) 170 °C; H NMR (DMSO-
d6) δ 10.0 (s, 1H), 9.1 (s, 1H), 8.85 (t, 1H), 8.5 (d, 1H), 8.2 (d,
1H), 7.35 (d, 1H), 7.2 (d, 1H), 4.15 (s, 3H), 3.55 (q, 2H), 3.15
(s, 3H), 2.75 (s, 3H), 2.55 (t, 2H), 2.3 (s, 6H). Anal. (C23H26N4O2‚
0.5H2O) C, H, N.
5,6,11-Tr im eth yl-9-m eth oxy-6H-pyr ido[4,3-b]car bazole-
1-ca r boxylic Acid Eth yl Ester (8) (Sch em e 1). A mixture
of compound 7 (5.0 g, 14.3 mmol), dimethyl carbonate (50 mL),

Analogues of S 16020-2 with Improved Antitumor Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2199
potassium carbonate (5.0 g), and adogen 464 (1.0 g) in
dimethylformamide (25 mL) was stirred at reflux temperature
for 3 h and then concentrated under vacuum. The residue was
taken up with dichloromethane and the solution washed with
water and a saturated aqueous solution of lithium chloride,
dried (Na2SO4), and concentrated under vacuum. Chromatog-
raphy of the residue, eluting with 3% ethanol in toluene, gave
compound 8 (4.64 g, 89%): mp(cap) 130-133 °C; 1H NMR
(CDCl3) δ 8.4 (d, 1H), 7.8 (d, 1H), 7.7 (s, 1H), 7.2-7.0 (m, 2H),
4.5 (q, 2H), 3.8 (2s, 6H), 3.0 (s, 3H), 2.9 (s, 3H), 1.5 (t, 3H).
Anal. (C21H22N2O3) C, H, N.
(s, 3H), 2.6 (s, 3H), 2.15 (s, 6H), 3.3-3.1 (m, 3H), 2.9 (m, 1H),
2.75 (m, 2H), 2.3 (m, 2H); MS (CI) m/e 394.
(R,S)-tr a n s-2-P h en ylcyclopr opan ecar boxylic Acid 1-[(2-
(Dim eth yla m in o)eth yl)ca r ba m oyl]-5,6-d im eth yl-6H-p y-
r id o[4,3-b]ca r ba zol-9-yl Ester (14) (Meth od C). (R,S)-trans-
2-Phenylcyclopropanecarbonyl chloride24 (0.53 g, 2.9 mmol in
tetrahydrofuran (50 mL) was added at 5 °C to a stirred solution
of 9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carbox-
ylic acid (2-(dimethylamino)ethyl)amide (1) (1 g, 2.66 mmol)
and triethylamine (0.29 g, 2.87 mmol) in tetrahydrofuran (50
mL). The mixture was stirred at 20 °C for 20 h and then
concentrated under vacuum. The residue was dissolved in
dichloromethane and the solution washed with water, dried
(Na2SO4), and concentrated. Column chromatography of the
residue, eluting with 10% methanol in dichloromethane, gave
compound 14 (0.9 g, 65%): mp(cap) 176-178 °C; 1H NMR
(DMSO-d6) δ 9.65 (s, 1H), 8.75 (t, 1H), 8.4 (d, 1H), 8.2 (d, 1H),
8.1 (d, 1H), 7.65 (d, 1H), 7.3 (m, 6H), 4.15 (s, 3H), 3.55 (q, 2H),
3.1 (s, 3H), 2.8 (m, 1H), 2.6 (m, 2H), 2.3 (s+m, 7H), 1.8-1.7
(m, 2H). Anal. (C32H32N4O3‚0.5C2H5OH) C, H, N.
9-Meth oxy-5,6,11-tr im eth yl-6H-p yr id o[4,3-b]ca r ba zole-
1-ca r boxylic Acid (9) (Sch em e 1). A 2 N solution of sodium
hydroxide (36 mL) was added to a solution of compound 8 (8.7
g, 24 mmol) in ethanol (900 mL). The mixture was stirred for
16 h at reflux temperature. After cooling, an aqueous solution
of 1 N hydrochloric acid (72 mL) was added and the mixture
concentrated under vacuum. The residue was taken up with
water and the resulting solid collected by filtration and washed
with water, methanol, and dichloromethane before drying
under vacuum at 50 °C to give compound 9 (4.15 g, 52%):
mp(K) 190 °C; 1H NMR (DMSO-d6) δ 8.25 (d, 1H), 8.0-7.1 (s,
1H, exchangeable for D2O), 7.95 (d, 1H), 7.8 (d, 1H), 7.55 (d,
1H), 7.25 (dd, 1H), 4.1 (s, 3H), 3.9 (s, 3H), 3.1 (s, 3H), 3.0 (s,
3H). Anal. (C20H18N2O3‚H2O) C, H, N.
As described for 14, using suitable starting materials, the
following compounds were obtained. 15 (25%): mp(cap) 84-
1
86 °C; H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.8 (t, 1H), 8.5 (d,
1H), 8.2 (d, 1H), 8.1 (d, 1H), 7.65 (d, 1H), 7.5-7.3 (m, 6H), 4.2
(s, 3H), 4.1 (s, 2H), 3.5 (q, 2H), 3.1 (s, 3H), 2.5 (m, 2H), 2.3 (s,
6H); MS (FAB) m/e 494. Anal. (C30H30N4O3‚H2O) C, H, N. 16
(58%): mp(cap) 122-125 °C; 1H NMR (DMSO-d6) δ 9.7 (s, 1H),
8.9 (t, 1H), 8.5 (d, 1H), 8.25 (d, 1H), 8.1 (d, 1H), 7.7 (d, 1H),
7.4, (dd, 1H), 4.2 (s, 3H), 3.65 (q, 2H), 3.15 (s, 3H), 2.7 (m,
4H), 2.4 (s, 6H), 1.75 (m, 2H), 1.5-1.3 (m, 10H), 0.95 (t, 3H).
Anal. (C31H40N4O3‚0.8H2O) C, H, N. 18 (64%): mp(cap) 116-
9-Meth oxy-5,6,11-tr im eth yl-6H-p yr id o[4,3-b]ca r ba zole-
1-ca r boxylic Acid (2-(Dim eth yla m in o)eth yl)a m id e (10)
(Sch em e 1). PyBOP (25.8 g, 49.6 mmol) in N-methylpyrroli-
done (50 mL) was added over a 1-h period to a solution of
compound 9 (9.2 g, 27.5 mmol) and 2-(dimethylamino)ethyl-
amine (4 mL) in N-methylpyrrolidone (75 mL) at 65 °C.16 The
mixture was stirred at 65 °C for 30 min and concentrated
under vacuum. The residue was taken up with dichloro-
methane and the solution washed with a solution of sodium
carbonate, dried (Na2SO4), and concentrated under vacuum.
Column chromatography of the residue, eluting with a mixture
of 1% ammonia solution (28%) and 10% methanol in dichloro-
methane, gave compound 10 (9.6 g, 86%): mp(cap) 132-135
1
118 °C; H NMR (DMSO-d6) δ 9.65 (s, 1H), 8.8 (t, 1H), 8.45-
8.2 (2d, 2H), 8.05 (d, 1H), 7.65 (d, 1H), 7.35 (dd, 1H), 4.15 (s,
3H), 3.8 (t, 2H), 3.55 (q+t, 4H), 3.1 (s, 3H), 2.9 (t, 2H), 2.6 (t,
2H), 2.25 (s, 6H), 1.2 (t, 3H). Anal. (C27H32N4O4) C, H, N. 19
1
(50%): mp(cap) 120-122 °C; H NMR (DMSO-d6) δ 9.65 (s,
1H), 8.8 (t, 1H), 8.45 (d, 1H), 8.2 (d, 1H), 8.1 (d, 1H), 7.6 (d,
1H), 7.35 (dd, 1H), 4.2 (s, 3H), 3.7 (s, 3H), 3.55 (q, 2H), 3.1 (s,
3H), 2.75 (t, 2H), 2.55 (m, 4H), 2.3 (s, 6H), 2.0 (q, 2H). Anal.
(C28H32N4O5) C, H, N.
1
°C; H NMR (DMSO-d6) δ 8.6 (t, 1H, exchangeable for D2O),
8.35 (d, 1H), 8.05 (d, 1H), 7.8 (d, 1H), 7.55 (d, 1H), 7.25 (dd,
1H), 4.1 (s, 3H), 3.9 (s, 3H), 3.5 (q, 2H), 3.1 (s, 3H), 3.0 (s, 3H),
2.5 (t, 2H), 2.2 (s, 6H). Anal. (C24H28N4O2‚0.25H2O) C, H, N.
Tetr a d eca n oic Acid 1-[(2-(Dim eth yla m in o)eth yl)ca r -
ba m oyl]-5,6-d im eth yl-6H-p yr id o[4,3-b]ca r ba zol-9-yl Es-
ter (17) (Meth od D). Myristic anhydride (4.67 g, 10.64 mmol)
was added at 5 °C to a stirred solution of compound 1 (2 g,
5.31 mmol) in pyridine (60 mL). The mixture was stirred at
20 °C for 16 h and concentrated under vacuum at a temper-
ature below 40 °C. Column chromatography of the residue,
eluting with a mixture of 10% methanol in dichloromethane,
gave compound 17 (2.4 g, 28.5%): mp (K) 98 °C; 1H NMR
(CDCl3) δ 10.1 (s, 1H), 8.4 (t+d, 2H), 7.9 (s+d, 2H), 7.2 (d,
2H), 4.0 (s, 3H), 3.7 (q, 2H), 3.0 (s, 3H), 2.7 (t+t, 4H), 2.4 (s,
6H), 1.9 (m, 2H), 1.4 (m, 20H), 0.9 (t, 3H). Anal. (C36H50N4O3‚
0.7H2O) C, H, N.
Following a similar procedure as that described for 17, using
acetic anhydride and compound 1 as starting materials,
compound 13 was obtained (75%): mp(K) 166 °C; 1H NMR
(CDCl3) δ 10.1 (s, 1H), 8.4 (t, 1H), 8.3 (d, 1H), 7.9 (s, 1H), 7.8
(dd, 1H), 7.1 (m, 2H), 3.9 (s, 3H), 3.7 (q, 2H), 2.9 (s, 3H), 2.7
(t, 2H), 2.4 (s, 3H), 2.35 (s, 6H). Anal. (C24H26N4O3) C, H, N.
10-((Dim eth yla m in o)m eth yl)-9-h yd r oxy-5,6-d im eth yl-
6H-p yr id o[4,3-b]ca r ba zole-1-ca r boxylic Acid (2-(Dim eth -
yla m in o)et h yl)a m id e (11) (Sch em e 2). N,N,N′,N′-Tetra-
methyldiaminomethane17 (3.25 g, 31.8 mmol) and acetic acid
(0.3 mL) were added to a stirred solution of compound 1 (0.9
g, 2.39 mmol) in dioxane (72 mL). The mixture was then stirred
at reflux for 30 min and concentrated under vacuum. The
residue was taken up in water, the pH was made basic using
ammonia solution, and the mixture was extracted with dichlo-
romethane and ethanol. The organic layer was dried (Mg SO4)
and then concentrated under vacuum. The residue was stirred
in ether and the solid collected by filtration, washed with ether,
and dried at 30 °C under vacuum to give compound 11 (0.9 g,
86%): mp(K) 200 °C; 1H NMR (DMSO-d6) δ 9.8 (s, 1H), 8.7
(m, 1H exchangeable for D2O), 8.4 (d, 1H), 8.15 (d, 1H), 7.4 (d,
1H), 7.1 (d, 1H), 4.1 (s, 5H), 3.6 (m, 2H), 3.1 (s, 3H), 2.6 (m,
2H), 2.35 (s, 6H), 2.2 (s, 6H). Anal. (C25H31N5O2) C, H, N.
Hexan edioic Acid Ben zyl Ester 1-[(2-(Dim eth ylam in o)-
eth yl)ca r ba m oyl]-5,6-d im eth yl-6H-p yr id o[4,3-b]ca r ba zol-
9-yl Ester (20) (Meth od E). 1-Hydroxybenzotriazole (2.26 g,
16.8 mmol) and dicyclohexylcarbodiimide (4.0 g, 19.4 mmol)
were added successively to a stirred solution of adipic acid
monobenzyl ester (3.96 g, 16.8 mmol) in dimethylformamide
(230 mL) at room temperature.14 The mixture was stirred for
4 h before adding compound 1 (3.0 g, 7.97 mmol). The mixture
was stirred for 48 h at room temperature and filtered. The
filtrate was concentrated under vacuum, and the residue was
taken up in dichloromethane, washed with an aqueous solution
of sodium hydrogen carbonate, dried (Na2SO4), and concen-
trated under vacuum. Column chromatography of the residue,
eluting with a mixture of 0.5% triethylamine and 10% metha-
nol in toluene, gave compound 20 (3.2 g, 67.5%): mp(cap) 88-
9-Hyd r oxy-5,6,10-tr im eth yl-2,3,4,6-tetr a h yd r o-1H-p yr -
id o[4,3-b]ca r ba zole-1-ca r boxylic Acid (2-(Dim eth yla m i-
n o)eth yl)a m id e (12) (Sch em e 2). To a solution of compound
11 (2.6 g, 6 mmol) in ethanol (1.2 L) was added 10% palladium
on activated carbon (2.2 g). The mixture was stirred at 30 °C
under hydrogen atmosphere at ambient pressure for 30 h. The
mixture was filtered and the catalyst washed with a mixture
of dichloromethane and ethanol. The combined filtrates were
concentrated under vacuum. Column chromatography of the
residue, eluting with a mixture of 1% triethylamine and 10%
ethanol in dichloromethane, provided recovered starting mate-
rial 11 (0.26 g, 10%), compound 5 (0.35 g, 15%), and compound
12 (1.15 g, 50%): 1H NMR (DMSO-d6) δ 8.7 (s, 1H), 8.0 (2s,
2H), 7.15 (d, 1H), 6.95 (d, 1H), 4.55 (s, 1H), 3.95 (s, 3H), 2.65

2200 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Guillonneau et al.
90 °C; 1H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.8 (t, 1H), 8.5 (d,
1H), 8.2 (d, 1H), 8.1 (d, 1H), 7.7 (d, 1H), 7.3 (m, 6H), 5.15 (s,
2H), 4.2 (s, 3H), 3.55 (q, 2H), 3.1 (s, 3H), 2.7 (m, 2H), 2.5 (m,
4H), 2.3 (s, 6H), 1.75 (m, 4H). Anal. (C35H38N4O5) C, H, N.
2H), 3.2 (s, 3H), 2.95 (d, 6H), 2.7 (t, 2H), 2.45 (t, 2H), 1.95 (m,
2H). Anal. (C27H30N4O5‚2HCl‚H2O) C, H, N, Cl.
As described for 25, using suitable starting materials, the
following compounds were obtained. 26 (62%): mp(cap) 215-
1
Following a similar procedure as that described for 20, using
succinic acid monobenzyl ester and compound 1 as starting
materials, compound 21 was obtained (40%): mp(cap) 174-
225 °C; H NMR (DMSO-d6) δ 10.2 (s, 1H), 9.6 (s, 1H), 9.3 (t,
1H), 8.6 (d, 1H), 8.35 (d, 1H), 7.6 (d, 1H), 7.4 (d, 1H), 4.25 (s,
3H), 3.8 (q, 2H), 3.4 (q, 2H), 3.2 (s, 3H), 2.9 (2s, 6H), 2.8 (t+s,
5H), 2.35 (t, 2H), 1.8 (m, 4H). Anal. (C29H34N4O5‚2HCl‚2H2O)
C, H, N, Cl. 27 (36%): mp(cap) 185-195 °C; 1H NMR (DMSO-
d6) δ 10.1-9.35 (2s, 2H), 8.5 (d, 1H), 8.35 (d, 1H), 8.1 (d, 1H),
7.8 (d, 1H), 7.45 (dd, 1H), 4.3 (s, 3H), 3.8 (q, 2H), 3.4 (m, 2H),
3.15 (2s, 6H), 2.9 (s, 6H), 2.7 (t, 2H), 2.35 (t, 2H), 1.75 (m, 4H).
Anal. (C29H34N4O5‚2HCl‚H2O) C, H, N, Cl. 28 (46%): mp(K)
1
176 °C; H NMR (DMSO-d6) δ 9.70 (s, 1H), 8.70 (t, 1H), 8.50
(d, 1H), 8.20 (d, 1H), 8.0 (d, 1H), 7.70 (d, 1H), 7.40 (m, 5H),
7.30 (dd, 1H), 5.20 (AB, 2H), 4.20 (s, 3H), 3.50 (m, 2H), 3.20
(s, 3H), 2.95-2.80 (m, 4H), 2.50 (t, 2H), 2.20 (s, 6H). Anal.
(C33H34N4O5) C, H, N.
Nicotin ic Acid 1-[(2-(Dim eth yla m in o)eth yl)ca r ba m -
oyl]-5,6-dim eth yl-6H-pyr ido[4,3-b]car bazol-9-yl Ester (23)
(Meth od F ). Dicyclohexylcarbodiimide (3.7 g, 18 mmol) and
4-(dimethylamino)pyridine (1.5 g, 12.3 mmol) were added to
a stirred solution of compound 1 (3.0 g, 7.97 mmol) and
nicotinic acid (1.5 g, 12.2 mmol) in pyridine15 (100 mL). The
mixture was stirred for 16 h at room temperature. Further
amounts of dicyclohexylcarbodiimide (2.0 g, 9.7 mmol) and
nicotinic acid (0.75 g, 6.1 mmol) were added; the mixture was
stirred for a further 24 h and concentrated under vacuum. The
residue was taken up in dichloromethane, washed with an
aqueous solution of sodium hydrogen carbonate, dried (Na2SO4),
and concentrated under vacuum. Column chromatography of
the residue, eluting with a mixture of 0.5% triethylamine and
10% ethanol in toluene, gave compound 23 (3.5 g, 91%):
1
180 °C; H NMR (DMSO-d6) δ 10.3 (m, 1H exchangeable for
D2O), 9.3 (m, 1H exchangeable for D2O), 8.5 (d, 1H), 8.3 (d,
1H), 8.1 (d, 1H), 7.75 (d, 1H), 7.45 (dd, 1H), 4.2 (s, 3H), 3.7
(m, 2H), 3.4 (m, 2H), 3.1 (2s, 6H), 2.7 (t, 2H), 2.6 (s, 6H), 2.4
(t, 2H), 1.9 (q, 2H). Anal. (C28H32N4O5‚2HCl‚1.5H2O) C, H, N,
1
Cl. 29 (34%): mp(cap) 190 °C; H NMR (DMSO-d6) δ 10.5 (s,
1H exchangeable for D2O), 9.65 (t, 1H), 9.55 (s, 1H), 8.5 (2d,
2H), 8.2 (d, 1H), 7.7 (d, 1H), 7.4 (dd, 1H), 4.25 (s, 3H), 3.85 (q,
2H), 3.5 (q, 2H), 3.15 (s, 3H), 2.9 (2s, 6H), 2.8 (s, 2H), 2.45 (s,
2H), 1.3 (s, 6H). Anal. (C29H34N4O5‚2HCl‚0.5H2O) C, H, N, Cl.
1
30 (42%): mp(cap) 190-196 °C; H NMR (DMSO-d6) δ 10.0
(s, 1H exchangeable for D2O), 9.7 (s, 1H), 9.35 (t, 1H), 8.55 (d,
1H), 8.4 (d, 1H), 8.2 (d, 1H), 7.75 (d, 1H), 7.4 (dd, 1H), 4.5 (s,
1H exchangeable for D2O), 4.25 (s, 3H), 3.85 (m, 2H), 3.45 (m,
2H), 3.15 (s, 3H), 2.9 (2s, 6H), 2.8 (t, 2H), 2.4 (t, 2H), 1.7-1.5
(m, 6H). Anal. (C29H34N4O5‚2HCl‚0.5H2O) C, H, N, Cl. 31
1
mp(cap) 190-192 °C; H NMR (DMSO-d6) δ 9.7 (s, 1H), 9.35
(d, 1H), 9.0 (m, 1H), 8.8 (t, 1H), 8.6 (m, 1H), 8.5 (d, 1H), 8.35
(d, 1H), 8.25 (d, 1H), 7.75 (d, 1H), 7.7 (m, 1H); 7.6 (dd, 1H),
4.25 (s, 3H), 3.5 (q, 2H), 3.15 (s, 3H), 2.55 (t, 2H), 2.2 (s, 6H).
Anal. (C28H27N5O3) C, H, N.
1
(44%): mp(cap) 210-215 °C; H NMR (DMSO-d6) δ 10.3 (m,
1H exchangeable for D2O), 9.5 (s, 1H), 8.5 (d, 1H), 8.4 (d, 1H),
8.2 (m, 1H), 7.7 (d, 1H), 7.35 (dd, 1H), 4.25 (s, 3H), 3.8 (q, 2H),
3.4 (m, 2H), 3.1 (s, 3H), 2.9 (m, 6H), 2.65 (t, 2H), 2.25 (t, 2H),
1.8-1.2 (m, 8H). Anal. (C30H36N4O5‚2HCl‚0.6H2O) C, H, N, Cl.
(R,S)-5-[1,2]Dit h iola n -3-ylp en t a n oic Acid 1-[(2-(Di-
m eth yla m in o)eth yl)ca r ba m oyl]-5,6-d im eth yl-6H-p yr id o-
[4,3-b]ca r ba zol-9-yl Ester (24) (Meth od G). PyBOP (5.2 g,
10 mmol) was added, at room temperature, to a stirred solution
of compound 1 (3.0 g, 8 mmol), (R,S)-thioctic acid (2.1 g, 10
mmol), and triethylamine (2.0 g, 19.8 mmol) in N-methyl-
pyrrolidone (42 mL).16 The mixture was stirred for 48 h, poured
in water (400 mL), and extracted with dichloromethane. The
organic phase was washed with an aqueous solution of sodium
hydrogen carbonate, dried (Na2SO4), and concentrated under
vacuum. Column chromatography of the residue, eluting with
0.5% triethylamine and 5% methanol in dichloromethane, gave
compound 24 (1.1 g, 24%): mp(cap) 140-142 °C; 1H NMR
(DMSO-d6) δ 9.70 (s, 1H), 8.95 (t, 1H), 8.45 (d, 1H), 8.20 (d,
1H), 8.10 (d, 1H), 7.70 (d, 1H), 7.45 (dd, 1H), 4.20 (s, 3H), 3.70
(m, 2H), 3.60 (m, 2H), 3.20 (m, 2H), 3.10 (s, 3H), 2.80 (m, 2H),
2.70 (t, 2H), 2.50 (m, 8H), 1.95 (m, 1H), 1.70 (m, 2H), 1.55 (m,
2H); MS (FAB) m/e 564. Anal. (C30H36N4O3S2) C, H, N.
Hexa n ed ioic Acid Mon o[1-((2-(d im eth yla m in o)eth yl)-
ca r b a m oyl)-5,6-d im e t h yl-6H -p yr id o[4,3-b]ca r b a zol-9-
yl] Ester (32) (Meth od P ). 5% Palladium on activated carbon
(3.0 g) was added to a solution of compound 20 (9.2 g, 15.47
mmol) and cyclohexene (20 mL) in N-methylpyrrolidone (200
mL) under nitrogen atmosphere.18 The mixture was stirred
at 80 °C for 30 min and filtered and the filtrate concentrated
under vacuum. The residue was triturated in ethanol and the
precipitate collected by filtration, washed with ethanol and
ether, and dried at 50 °C under vacuum to give compound 32
(7.2 g, 92%): mp(cap) 200-202 °C; 1H NMR (DMSO-d6) δ 9.65
(s, 1H), 8.8 (t, 1H, exchangeable for D2O), 8.5 (d, 1H), 8.2 (d,
1H), 8.1 (d, 1H), 7.7 (d, 1H), 7.3 (dd, 1H), 4.2 (s, 3H), 3.5 (q,
2H), 3.1 (s, 3H), 2.7 (t, 2H), 2.6 (t, 2H), 2.3 (t, 2H), 2.3 (s, 6H),
1.7 (m, 4H). Anal. (C28H32N4O5‚0.5H2O) C, H, N.
As described for 32, using suitable starting materials, the
following compounds were obtained. 33 (32%): mp(cap) 160-
164 °C; 1H NMR (DMSO-d6) δ 10.6-9.7 (2s, 2H, exchangeable
for D2O), 9.5 (s, 1H), 8.55 (d, 1H), 8.48 (d, 1H), 8.25 (d, 1H),
7.75 (d, 1H), 7.4 (dd, 1H), 7.3 (d, 1H), 4.25 (s, 3H), 4.15 (m,
1H), 3.9 (q, 2H), 3.5 (m, 2H), 3.2 (s, 3H), 2.95 (2s, 6H), 2.75 (t,
2H), 2.2-2.0 (m, 2H), 1.45 (s, 9H). Anal. (C32H39N5O7‚2HCl‚
H2O) C, H, N, Cl. 51 (55%): mp(cap) 195-198 °C; 1H NMR
(DMSO-d6) δ 9.65 (s, 1H), 8.8 (t, 1H), 8.5 (d, 1H), 8.25 (d, 1H),
8.05 (d, 1H), 7.85 (t, 1H), 7.7 (d, 1H), 7.35 (dd, 1H), 4.2 (s, 3H),
3.55 (q, 2H), 3.5 (q, 2H), 3.15 (s, 3H), 2.6 (2t, 4H), 2.3 (s, 6H).
Anal. (C26H29N5O5‚2H2O) C, H, N. 52 (76%): mp(cap) 174-
177 °C; 1H NMR (DMSO-d6) δ 9.60 (s, 1H), 8.75-7.85 (2s, 2H
exchangeable for D2O), 8.2 (d, 1H), 8.0 (d, 1H), 7.60 (dd, 1H),
7.20 (d, 1H), 4.20 (s, 3H), 3.50 (m, 2H), 3.20 (t, 2H), 3.10 (s,
3H), 2.60 (m, 2H), 2.25 (m, 2H), 2.20 (t, 2H), 1.75 (s, 6H). Anal.
(C27H31N5O5‚H2O) C, H, N. 55 (61%): mp(cap) 135-137 °C;
1H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.8 (t, 1H), 8.5 (d, 1H), 8.25
(2m, 2H), 7.7 (d, 1H), 7.5 (dd, 1H), 4.3 (t, 2H), 4.25 (s, 3H), 3.6
(q, 2H), 3.15 (s, 3H), 2.6 (t, 2H), 2.45 (t, 2H), 2.35 (s, 6H), 1.95
(q, 2H). Anal. (C27H30N4O6‚H2O) C, H, N.
Following a similar procedure as that described for 24, using
N-tBoc-L-glutamic acid R-benzyl ester and compound 1 as
starting materials, compound 22 was obtained (88%): mp(K)
120 °C; 1H NMR (DMSO-d6) δ 9.6 (s, 1H), 8.55-8.45 (2d, 2H),
8.25 (d, 1H), 7.75 (m, 1H), 7.2 (dd, 1H), 5.2 (AB, 2H), 4.35 (m,
4H), 3.85 (m, 2H), 3.45 (m, 2H), 3.2 (s, 3H), 2.9 (m, 6H), 2.8 (t,
2H), 2.2-2.0 (m, 2H), 1.4 (s, 9H).
P en ta n ed ioic Acid Mon o[1-((2-(d im eth yla m in o)eth yl)-
ca r b a m oyl)-5,6-d im e t h yl-6H -p yr id o[4,3-b]ca r b a zol-9-
yl] Ester Dih yd r och lor id e (25) (Meth od H). Glutaric
anhydride (4.56 g, 40 mmol) was added at room temperature
to a stirred solution of compound 1 (7.52 g, 20 mmol) in
pyridine (200 mL). The mixture was stirred at 55 °C for 16 h
and concentrated under vacuum at a temperature below 40
°C. The residue was taken up in dichloromethane (150 mL)
and stirred for 10 min. The solid was collected by filtration,
washed with dichloromethane, dried at 50 °C under vacuum,
and taken up in ethanol. A small excess of a 2 M solution of
hydrogen chloride in ethanol was added and the suspension
stirred for 2 min. The solid was collected by filtration, washed
with ethanol, and dried at 50 °C under vacuum to give
compound 25 (9.8 g, 87%): mp(cap) 189-195 °C; 1H NMR
(DMSO-d6) δ 9.5 (s, 1H),, 8.55 (d, 1H), 8.45 (d, 1H), 8.2 (s, 1H),
7.8 (d, 1H), 7.45 (dd, 1H), 4.3 (s, 3H), 3.85 (m, 2H), 3.45 (m,
(R ,S )-5,6-D im e t h y l-9-(t r a n s-2-p h e n y lc y c lo p r o p y l-
m et h oxy)-6H -p yr id o[4,3-b]ca r b a zole-1-ca r b oxylic Acid
(2-(Dim eth yla m in o)eth yl)a m id e (36) (Meth od I). Diethyl
azodicarboxylate (2.44 g, 14 mmol) was added over 20 min at

Analogues of S 16020-2 with Improved Antitumor Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2201
room temperature to a stirred suspension of compound 1 (5.3
g, 14 mmol), triphenylphosphine (3.67 g, 14 mmol), and (R,S)-
trans-2-phenylcyclopropylmethanol20 (2.1 g, 14 mmol) in tet-
rahydrofuran (330 mL). The mixture was stirred for 2 h at
room temperature. Further aliquots of triphenylphosphine
(3.67 g, 14 mmol) and diethyl azodicarboxylate (2.44 g, 14
mmol) were added (Mitsunobu reaction21); the mixture was
stirred for 16 h and then concentrated under vacuum. Column
chromatography of the residue, eluting with 10% methanol in
dichloromethane, gave a crude product which was recrystal-
lized from ethanol to give compound 36 (2.25 g, 32%): mp(cap)
120-123 °C; 1H NMR (DMSO-d6) δ 9.65 (s, 1H), 8.8 (t, 1H,
exchangeable for D2O), 8.45 (d, 1H), 8.15 (d, 1H), 7.8 (d, 1H),
7.55 (d, 1H), 7.2 (m, 6H), 4.15 (m, 5H), 3.55 (m, 2H), 3.05 (s,
3H), 2.6 (t, 2H), 2.3 (s, 6H), 2.05 (m, 1H), 1.6 (m, 1H), 1.1 (m,
2H). Anal. (C32H34N4O2) C, H, N.
ca r ba zol-9-ylca r ba m a te (43) (Meth od K). [1,4′]Bipiperidi-
nyl-1′-carbonyl chloride (1.5 g, 7.3 mmol) was added over 5
min at room temperature to a stirred solution of compound 1
(1.6 g, 4.25 mmol) in pyridine (80 mL). The mixture was stirred
for 16 h and concentrated under vacuum. The residue was
dissolved in dichloromethane, washed with an aqueous solu-
tion of sodium hydrogen carbonate, dried (Na2SO4), and
concentrated under vacuum. The residue was taken up with
a mixture of ethyl acetate and ether. The solid was collected
by filtration, washed with ether, and dried at 50 °C under
vacuum to give compound 43 (2.3 g, 95%): mp(cap) 190-200
°C; 1H NMR (DMSO-d6) δ 9.65 (s, 1H), 8.8 (t, 1H), 8.5 (d, 1H),
8.25 (d, 1H), 8.1 (s, 1H), 7.65 (d, 1H), 7.35 (dd, 1H), 4.2 (s,
3H), 4.3-4.15 (m, 2H), 3.6 (q, 2H), 3.35-3.0 (m, 3H), 3.15 (s,
3H), 2.6 (t+m, 5H), 2.3 (s, 6H), 1.9-1.5 (m, 10H). Anal.
(C33H42N6O3‚0.7H2O) C, H, N.
Following a similar procedure as that described for 36, using
(R,S)-2,2-dimethyl-1,3-dioxolane-4-methanol and compound 1
as starting materials, compound 35 was obtained (32%):
mp(cap) 96-98 °C; 1H NMR (DMSO-d6) δ 9.70 (s, 1H), 8.80 (t,
1H), 8.45 (d, 1H), 8.20 (d, 1H), 7.80 (d, 1H), 7.55 (d, 1H), 7.25
(dd, 1H), 4.50 (m, 1H), 4.20-3.85 (m, 2H), 4.20 (m, 5H), 3.50
(m, 2H), 3.15 (s, 3H), 2.50 (t, 2H), 2.20 (s, 6H), 1.45-1.30 (2s,
6H). Anal. (C28H34N4O4‚0.5H2O) C, H, N.
As described for 43, using suitable carbamoyl chlorides
prepared according to a previously described method,25 the
following compounds were prepared. 40 (75%): mp(cap) 188-
190 °C; 1H NMR (CDCl3) δ 10.05 (s, 1H), 8.5 (t, 1H, exchange-
able for D2O), 8.3 (d, 1H), 8.0 (d, 1H), 7.9 (d, 1H), 7.3 (dd, 1H),
7.2 (d, 1H), 4.0 (s, 3H), 3.7 (q, 2H), 3.2 (s, 3H), 3.1 (s, 3H), 3.0
(s, 3H), 2.75 (t, 2H), 2.4 (s, 6H). Anal. (C25H29N5O3‚0.5H2O) C,
1
H, N. 41 (51%): mp(cap) 90-93 °C; H NMR (CDCl3) δ 10.1
(s, 1H), 8.5 (t, 1H, exchangeable for D2O), 8.4 (d, 1H), 8.0 (m,
1H), 7.95 (d, 1H), 7.2 (m, 2H), 4.1 (s, 3H), 3.8 (q, 2H), 3.5 (m,
2H), 3.1 (2s, 3H), 3.0 (s, 3H), 2.65 (t, 2H), 2.4 (s, 6H), 1.8-1.2
(m, 20H), 0.9 (m, 3H). Anal. (C36H51N5O3) C, H, N. 42 (86%):
(R,S)-9-(2,3-Dih ydr oxypr opoxy)-5,6-dim eth yl-6H-pyr ido-
[4,3-b]ca r ba zole-1-ca r boxylic Acid (2-(Dim eth yla m in o)-
eth yl)a m id e (37) (Meth od Q). Compound 35 (2.4 g, 4.89
mmol) was dissolved in acetic acid (60 mL) and water (40 mL).
The mixture was stirred for 8 h at reflux temperature and
concentrated under vacuum. The residue was dissolved in
dichloromethane; the solution was washed with an aqueous
solution of sodium hydrogen carbonate, dried (Na2SO4), and
concentrated under vacuum. Column chromatography of the
residue, eluting with a mixture of 0.75% triethylamine and
15% ethanol in toluene, gave compound 37 (0.75 g, 34%):
mp(cap) 136-138 °C; 1H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.8 (t,
1H), 8.45 (d, 1H), 8.2 (d, 1H), 7.8 (d, 1H), 7.6 (d, 1H), 7.25 (dd,
1H), 5.0 (d, 1H), 4.7 (t, 1H), 4.2 (s, 3H), 4.2-4.1 (m, 2H), 3.9
(m, 1H), 3.55 (m, 4H), 3.15 (s, 3H), 2.6 (t, 2H), 2.3 (s, 6H);
purity 98.3% (HPLC C18, 210 nm, CH3CN/H2O/CH3SO3H).
Anal. (C25H30N4O4) C, H, N.
5-[1-((2-(Dim eth ylam in o)eth yl)car bam oyl)-5,6-dim eth yl-
6H-p yr id o[4,3-b]ca r ba zol-9-yloxy]p en ta n oic Acid Eth yl
Ester (38) (Meth od J ). A suspension of compound 1 (3.0 g,
7.98 mmol), ethyl 5-bromopentanoate (3.75 g, 18.1 mmol), and
cesium carbonate (5.85 g) in dimethylformamide (100 mL) was
stirred at reflux temperature for 24 h at 50 °C and then
concentrated under vacuum. The residue was taken up in
dichloromethane, and the mixture was washed with an aque-
ous solution of sodium hydrogen carbonate. The organic layer
was dried (Na2SO4) and concentrated under vacuum. Chro-
matography of the residue eluting with a mixture of 0.5%
triethylamine and 10% ethanol in toluene gave compound 38
(2.8 g, 69%): mp(cap) 101-104 °C; 1H NMR (DMSO-d6) δ 9.7
(s, 1H), 8.8 (t, 1H), 8.45 (d, 1H), 8.2 (d, 1H), 7.8 (d, 1H), 7.6 (d,
1H), 7.2 (dd, 1H), 4.2 (s, 3H), 4.2 (t, 2H), 4.1 (q, 2H), 3.55 (q,
2H), 3.15 (s, 3H), 2.55 (t, 2H), 2.4 (t, 2H), 2.3 (s, 6H), 1.8 (m,
4H), 1.2 (t, 3H). Anal. (C29H36N4O4) C, H, N.
5-[1-((2-(Dim eth ylam in o)eth yl)car bam oyl)-5,6-dim eth yl-
6H -p yr id o[4,3-b]ca r b a zol-9-yloxy]p en t a n oic Acid (39)
(Meth od R). A solution of 38 (2.8 g, 5.55 mmol) and an
aqueous solution of 1 N sodium hydroxide (10 mL) in ethanol
(50 mL) were stirred at reflux temperature for 24 h and then
concentrated under vacuum. The residue was taken up in
water, and an aqueous solution of 1 N hydrochloric acid (10
mL) was added. The solid was collected by filtration, washed
with water and ethanol, and dried at 60 °C under vacuum to
give compound 39 (2.1 g, 80%): mp(cap) 196-200 °C; 1H NMR
(DMSO-d6) δ 9.65 (s, 1H), 8.8 (t, 1H), 8.45 (d, 1H), 8.2 (d, 1H),
7.8 (d, 1H), 7.6 (d, 1H), 7.25 (dd, 1H), 4.2 (s+t, 5H), 3.6 (q,
2H), 3.15 (s, 3H), 2.6 (t, 3H), 2.4 (t, 2H), 2.3 (s, 6H), 1.8 (m,
4H). Anal. (C27H32N4O4‚H2O) C, H, N.
1
mp(cap) 125-128 °C; H NMR (CDCl3) δ 10.1 (s, 1H), 8.5 (t,
1H, exchangeable for D2O), 8.4 (d, 1H), 8.0 (m, 1H), 7.95 (d,
1H), 7.5-7.2 (m, 7H), 4.0 (s, 3H), 3.7 (q, 2H), 3.45 (s, 3H), 3.0
(s, 3H), 2.7 (t, 2H), 2.5 (s, 6H). Anal. (C30H31N5O3) C, H, N. 44
(78%): mp(cap) 135-138 °C; 1H NMR (DMSO-d6) δ 9.7 (s, 1H),
8.8 (t, 1H), 8.45 (d, 1H), 8.2 (d, 1H), 8.05 (d, 1H), 7.6 (d, 1H),
7.55 (d, 6H), 7.4 (m, 4H), 7.3 (m, 2H), 4.45 (s, 1H), 4.15 (s,
3H), 3.8 (m, 2HJ ), 3.6 (q, 2H), 2.6-2.4 (m, 8H), 3.1 (s, 3H), 2.3
(s, 6H). Anal. (C40H42N6O3‚0.25H2O) C, H, N. 45 (87%):
1
mp(cap) 134-137 °C; H NMR (CDCl3) δ 10.0 (s, 1H), 8.5 (t,
1H, exchangeable for D2O), 8.3 (d, 1H), 8.0 (d, 1H), 7.8 (d, 1H),
7.3 (dd, 1H), 7.15 (d, 1H), 4.2-3.7 (m, 4H), 4.0 (s, 3H), 3.7 (q,
2H), 2.9 (s, 3H), 2.7 (t, 2H), 2.6 (m, 4H), 2.4 (s, 9H). Anal.
(C28H34N6O3‚0.5H2O) C, H, N. 46 (93%): mp(cap) 190-192 °C;
1H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.8 (t, 1H), 8.45 (d, 1H), 8.2
(d, 1H), 8.1 (d, 1H), 7.65 (d, 1H), 7.4 (dd, 1H), 4.2 (s, 3H), 3.6
(s, 8H), 3.5 (q, 2H), 3.1 (s, 3H), 2.5 (t, 2H), 2.25 (s, 6H). Anal.
(C27H31N5O4‚0.4H2O) C, H, N.
Dod ecylca r b a m ic Acid 1-((2-(Dim et h yla m in o)et h yl)-
ca r ba m oyl)-5,6-d im eth yl-6H-p yr id o[4,3-b]ca r ba zol-9-yl-
ca r ba m a te (47) (Meth od L). Dodecyl isocyanate (3.2 g, 15.2
mmol) in pyridine (10 mL) was added to a stirred solution of
compound 1 (3.8 g, 10 mmol) and 1,8-diazabicyclo[5.4.0]undec-
7-ene (0.05 g) in pyridine (40 mL). The mixture was stirred
for 16 h at 50 °C and concentrated under vacuum. Column
chromatography of the residue, eluting with a mixture of 1.5%
ammonia solution (28%) and 5% methanol in dichloromethane,
gave compound 47 (3.9 g, 66%): mp(cap) 162-164 °C; 1H NMR
(DMSO-d6) δ 9.3 (s, 1H), 8.8-7.8 (2t, 2H), 8.45 (d, 1H), 8.2 (d,
1H), 8.0 (d, 1H), 7.6 (d, 1H), 7.3 (dd, 1H), 4.15 (s, 3H), 3.55 (q,
2H), 3.1 (m, 5H), 2.6 (t, 2H), 2.3 (s, 6H), 1.55 (m, 2H), 1.4-1.2
(m, 18H), 0.85 (t, 3H). Anal. (C35H49N5O3) C, H, N.
As described for 47, using suitable isocyanates26 and com-
pound 1 as starting materials, the following compounds were
obtained. 48 (92%): mp(cap) 120 °C; 1H NMR (DMSO-d6) δ
9.6 (s, 1H), 8.8 (t, 1H exchangeable for D2O), 8.45 (d, 1H), 8.2
(d, 1H), 7.95 (d, 1H), 7.9 (t, 1H exchangeable for D2O), 7.6 (d,
1H), 7.3 (dd, 1H), 4.2 (s, 3H), 4.15 (q, 2H), 3.6 (q, 2H), 3.4 (q,
2H), 3.1 (s, 3H), 2.6 (m, 4H), 2.25 (s, 6H), 1.2 (t, 3H). Anal.
(C28H33N5O5), C, H, N. 49 (58%): mp(cap) 112-114 °C; 1H
NMR (DMSO-d6) δ 9.63 (s, 1H), 8.75 (t, 1H exchangeable for
D2O), 8.45 (d, 1H), 8.18 (d, 1H), 7.97 (d, 1H), 7.90 (t, 1H
exchangeable for D2O), 7.60 (d, 1H), 7.40 (m, 5H), 7.3 (dd, 1H),
5.17 (s, 2H), 4.15 (s, 3H), 3.52 (q, 2H), 3.40 (q, 2H), 3.10 (s,
3H), 2.68 (t, 2H), 2.53 (t, 2H), 2.25 (s, 6H). Anal. (C33H35N5O5)
C, H, N. 50 (42%): mp(cap) 125-128 °C; 1H NMR (DMSO-d6)
[1,4′]Bip ip er id in yl-1′-ca r boxylic Acid 1-((2-(Dim eth yl-
a m in o)eth yl)ca r ba m oyl)-5,6-d im eth yl-6H-p yr id o[4,3-b]-

2202 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Guillonneau et al.
δ 9.6 (s, 1H), 8.8 (t, 1H exchangeable for D2O), 8.4 (d, 1H), 8.2
(d, 1H), 8.0 (d, 1H), 7.7 (t, 1H, exchangeable for D2O), 7.3 (m,
6H), 5.1 (s, 2H), 4.15 (s, 3H), 3.5 (q, 2H), 3.2 (q, 2H), 3.0 (s,
3H), 2.6 (m, 4H), 2.3 (s, 6H), 1.8 (m, 2H). Anal. (C34H37N5O5)
C, H, N.
(1.5 g, 43.5%): 1H NMR (DMSO-d6) δ 10.6 (s, 1H), exchange-
able for D2O), 9.6 (s, 1H), 9.20 (t, 1H), 8.25 (d, 1H), 8.0 (d,
2H), 7.5 (dd, 1H), 7.3-7.2 (m, 6H), 4.85 (s, 2H), 4.15 (s, 3H),
3.8 (m, 2H), 3.3 (m, 2H), 2.9 (s, 3H), 2.85 (s, 6H). Anal.
(C29H31N4O5P‚2H2O) C, H, N.
P h osp h or ic Acid Mon o[1-((2-(d im eth yla m in o)eth yl)-
ca r b a m oyl)-5,6-d im e t h yl-6H -p yr id o[4,3-b]ca r b a zol-9-
yl] Ester (58) (Meth od P , Sch em e 4). 5% Palladium on
activated carbon (2.5 g) was added to a stirred solution of
compound 56 (4 g, 6.28 mmol) and cyclohexene (75 mL) in
N-methylpyrrolidone (750 mL). The mixture was stirred for 1
h at 100 °C. After cooling, the solid was collected by filtration
and extracted with water. The aqueous solution was concen-
trated under vacuum and the residue taken up with a mixture
of 20% ethanol in ether. The solid was collected by filtration
and dried at 50 °C under vacuum to give compound 58 (2.3 g,
80%): mp(cap) 220-224 °C; 1H NMR (D2O + NaOD) δ 8.8 (s,
1H), 8.1 (d, 1H), 8.0 (s, 1H), 7.7 (d, 1H), 7.5 (d, 1H), 7.15 (d,
1H), 3.9 (t, 2H), 3.6 (s, 3H), 2.9 (t, 2H), 2.6 (s, 3H), 2.5 (s, 6H).
Anal. (C22H25N4O5P‚0.7H2O) C, H, N.
58 (Sch em e 5). 10% Palladium on activated carbon (2 g)
was added to a stirred solution of compound 59 (4.0 g, 6.28
mmol) and cyclohexene (50 mL) in N-methylpyrrolidone (400
mL). The mixture was stirred at reflux temperature for 1 h. A
further 2-g portion of catalyst was added and the mixture
stirred at reflux temperature for 4 h. After cooling, the solid
was collected by filtration and extracted with water. The
aqueous phase was concentrated under vacuum and the
residue taken up with a mixture of 20% ethanol in ether. The
solid was collected by filtration and dried at 60 °C under
vacuum to give compound 58 (1.85 g, 64,5%), identified as the
same product as that obtained in method P, Scheme 4.
Meth a n esu lfon ic Acid 1-((2-(Dim eth yla m in o)eth yl)-
car bam oyl)-5,6-dim eth yl-6H-pyr ido[4,3-b]car bazol-9-yl Es-
ter (53) (Meth od M). Methanesulfonyl chloride (1.2 g, 10.5
mmol) was added to a stirred solution of compound 1 (3 g, 7.97
mmol) and triethylamine (1 g) in tetrahydrofuran (200 mL)
at 5 °C. The mixture was stirred at room temperature for 16
h and then concentrated under vacuum. The residue was
dissolved in dichloromethane, and the mixture was washed
with an aqueous solution of sodium hydrogen carbonate, dried
(Na2SO4), and concentrated under vacuum. Column chroma-
tography of the residue, eluting with a mixture of 0.5%
ammonia solution (28%) and 5% methanol in dichloromethane,
gave compound 53 (2.3 g, 63.5%): mp(cap) 192-194 °C; 1H
NMR (DMSO-d6) δ 9.75 (s, 1H), 8.8 (t, 1H), 8.5 (d, 1H), 8.3 (d,
1H), 8.25 (d, 1H), 7.75 (d, 1H), 7.6 (dd, 1H), 4.2 (s, 3H), 3.55
(q, 2H), 3.5 (s, 3H), 3.15 (s, 3H), 2.6 (t, 2H), 2.3 (s, 6H). Anal.
(C23H26N4O4S) C, H, N.
3-(Ben zyloxyca r bon yl)p r op yl Ch lor ofor m a te. A mix-
ture of 4-hydroxybutyric acid benzyl ester22 (14.0 g, 72 mmol)
and dimethylaniline (10.6 g) in toluene (47 mL) was added
over 45 min at 0 °C to a 1.93 M solution of phosgene in toluene
(41 mL). The mixture was stirred for 1 h at 5 °C, washed with
iced water, 0.1 N hydrochloric acid, and water, dried (Na2SO4),
and then concentrated to 37 mL under vacuum. This unstable
compound (not isolated) was used immediately as a solution
in toluene in the following procedure.
4-[[1-((2-(Dim e t h yla m in o)e t h yl)ca r b a m oyl)-5,6-d i-
m eth yl-6H-p yr id o[4,3-b]ca r ba zol-9-yloxy]ca r bon yloxy]-
bu tyr ic Acid Ben zyl Ester (54) (Meth od N). The above
solution of 3-(benzyloxycarbonyl)propyl chloroformate in tolu-
ene (37 mL) was added over 30 min to a solution of compound
1 (2.6 g, 6.9 mmol) and pyridine (1.86 mL) in N-methylpyr-
rolidone (70 mL) at 5 °C. The mixture was stirred for 2 h at 5
°C followed by 16 h at room temperature and then concen-
trated under vacuum. The residue was dissolved in dichloro-
methane; the solution was washed with an aqueous solution
of sodium hydrogen carbonate, dried (Na2SO4), and concen-
trated under vacuum. Chromatography of the residue, eluting
with a mixture of 0.5% ammonia solution (28%) and 5%
methanol in dichloromethane, gave compound 54 as a gum
(1.63 g, 39%): 1H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.75 (t, 1H),
8.4 (d, 1H), 8.15 (m, 2H), 7.4 (m, 7H), 5.15 (s, 2H), 4.3 (t, 2H),
4.1 (s, 3H), 3.6 (m, 2H), 3.05 (s, 3H), 2.6 (m, 4H), 2.3 (s, 6H),
1.9 (m, 2H). Anal. (C34H36N4O6) C, H, N.
P h osp h or ic Acid Diben zyl Ester 1-((2-(Dim eth yla m i-
n o)et h yl)ca r b a m oyl)-5,6-d im et h yl-6H -p yr id o[4,3-b]ca r -
ba zol-9-yl Ester (56) (Meth od O, Sch em e 4). Sodium
hydride (60% dispersion in mineral oil, 0.6 g, 15 mmol) was
added at room temperature to a stirred solution of compound
1 (3.76 g, 10 mmol) in tetrahydrofuran (200 mL). After 1 h,
tetrabenzyl pyrophosphate23 (6.5 g, 12 mmol) was added, and
the stirring was continued for 6 h. The mixture was filtered,
and the filtrate was concentrated under vacuum at a temper-
ature below 35 °C. The residue was dissolved in dichloro-
methane, washed with an aqueous solution of sodium hydrogen
carbonate, dried (Na2SO4), and concentrated under vacuum
to give unstable compound 56 (4 g, 63%) which was used
immediately in the following procedure: 1H NMR (CDCl3) δ
9.7 (s, 1H), 9.65 (t, 1H), 8.45 (d, 1H), 8.1 (d, 1H), 7.7-7.1 (m,
13H), 5.2 (m, 4H), 4.1 (s, 3H), 3.8 (m, 2H), 3.3 (s, 3H), 2.7 (m,
2H), 2.3 (s, 6H).
Ben zyl[2-(5,6-dim eth yl-9-(ben zyl ph osph ate)-6H-pyr ido-
[4,3-b]ca r b a zol-1-yl)ca r b on yla m in oet h yl]m et h yla m m o-
n iu m (59) (Sch em e 5). Unstable compound 56 (6.37 g, 10
mmol) was left for 4 days at room temperature. Column
chromatography, eluting with a mixture of 2% ammonia
solution (28%) and 20% methanol in dichloromethane, gave
compound 59 (4.0 g, 62.8%): 1H NMR (DMSO-d6) δ 9.6 (s, 1H),
9.4 (t, 1H, exchangeable for D2O), 8.3 (d, 1H), 8.1 (m, 2H), 7.7-
7.0 (m, 12H), 4.8 (d, 2H), 4.7 (s, 2H), 4.15 (s, 3H), 4.0 (m, 2H),
3.6 (m, 2H), 3.2 (s, 6H), 3.0 (s, 3H); MS (FAB) m/e 636.
Biology. 1. Cell Cu ltu r e a n d Cytotoxicity. The murine
L1210 leukemia and B16 melanoma cells were cultured in
RPMI 1640 medium (Gibco) supplemented with 10% fetal calf
serum, 2 mM l-glutamine, 100 units/mL penicillin, 100 µg/mL
streptomycin, and 10 mM HEPES buffer (pH 7.4). Cytotoxicity
was measured by the microculture tetrazolium assay as
described.3,27 Cells were exposed to graded concentrations of
drug (nine serial dilutions in triplicate) for about four doubling
times (48 h for L1210 and 96 h for B16). Results were
expressed as IC50, the concentration which reduced by 50%
the optical density of treated cells with respect to untreated
controls.
For the cell cycle analysis, L1210 cells (5 × 105 cells/mL)
were incubated for 21 h with various concentrations of drugs.
Cells were then fixed by 70% ethanol (v/v), washed, and
incubated in PBS containing 100 µg/mL RNase and 25 µg/mL
propidium iodide for 30 min at 20 °C. For each sample, 10 000
cells were analyzed on an ATC3000 flow cytometer (Brucker,
Wissembourg, France).
2. An titu m or Activity. The antitumor activity of the
compounds was evaluated on the P388 leukemia and the B16
melanoma, all provided by NCI, Frederick, MD. Groups of 6-7
mice were used. P388 cells were inoculated ip (106 cells/mouse)
into B6D2F1 mice (Iffa credo, L’Arbresle, France) on day 0.
The dihydrochloride salts of the compounds were dissolved in
water and injected iv on day 1 or days 1, 5, and 9. The results
are expressed in terms of % T/C survival (median survival time
of treated animals/median survival time of control animals)
× 100. For the ip B16 melanoma, 0.5 mL of a tumor brei (1 g
of tumor in 10 mL of 0.9% NaCl) was injected ip on day 0, and
compounds were administered ip on days 1-9. The optimal
dose was the dose which gave the higher T/C without major
P h osp h or ic Acid Ben zyl Ester 1-((2-(Dim eth yla m in o)-
eth yl)ca r ba m oyl)-5,6-d im eth yl-6H-p yr id o[4,3-b]ca r ba zol-
9-yl Ester (57) (Meth od P , Sch em e 4). 5% Palladium on
carbon (1 g) was added to a solution of compound 56 (4 g, 6.28
mmol) and cyclohexene (10 mL) in N-methylpyrrolidone (100
mL).18 The mixture was stirred for 35 min at 100 °C. After
cooling, the solid was collected by filtration and extracted with
water. Aqueous solution was lyophilized to give compound 57

Analogues of S 16020-2 with Improved Antitumor Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2203
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Ack n ow led gm en t. We thank J . P. Volland and
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