Synthesis and in Vitro cytotoxic evaluation of new derivatives of pyrido [1,2-a]benzimidazolic ring system: The pyrido [1′,2′: 1,2]imidazo[4,5-h]quinazolines

Article abstract of DOI:10.1248/cpb.49.1061

Access to the original series of pyrido[1′,2′:1,2]imidazo[4,5-h]quinazoline was developed from a 1,3-dicarbonyl unit with some "N-C-N" bisnucleophilic reagents and the derivatives obtained were evaluated for in vitro cytotoxic activities against HL60 and

Full text of DOI:10.1248/cpb.49.1061

September 2001
Chem. Pharm. Bull. 49(9) 1061—1065 (2001)
1061
Synthesis and in Vitro Cytotoxic Evaluation of New Derivatives of
Pyrido[1,2-a]benzimidazolic Ring System:
The Pyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazolines
Marianne DUPUY,a,b Frederic PINGUET,b Olivier CHAVIGNON,c Jean-Michel CHEZAL,c
Jean-Claude TEULADE,c Jean-Pierre CHAPAT,a and Yves BLACHE*,a
Laboratoire de Chimie organique Pharmaceutique,a E.A. 2414, 15 Avenue Charles Flahault, Faculté de Pharmacie, 34060
Montpellier, Laboratoire d’oncopharmacologie, Centre Régional de Lutte contre le Cancer,b Val d’Aurelle, 34298
Montpellier Cedex 05, and Laboratoire de Chimie Organique Pharmaceutique, UFR de Pharmacie,c 28 Place Henry
Dunant, B.P. 38, 63001 Clermont-Ferrand, France. Received January 5, 2001; accepted May 21, 2001
Access to the original series of pyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline was developed from a 1,3-dicar-
bonyl unit with some “N–C–N” bisnucleophilic reagents and the derivatives obtained were evaluated for in vitro
cytotoxic activities against HL60 and A2780 cells. All compounds exhibited cytotoxic activities on resistant cell
lines (MDR؉; HL60R and A2780R) with no resistance phenomena.
Key words pyrido[1Ј,2Ј:1,2]imidazo[4,5-h]quinazoline; anticancer agent; multidrug resistance
As a part of studies related to the pharmacochemistry of line Framework Our strategy based on a direct route to the
heterocycles with a bridgehead nitrogen,1) we initiated a pro- pyrimidinic ring requiring the condensation of a bisnucle-
gram aimed at examining the synthesis and cytotoxicity of ophilic “N–C–N” equivalent reagent with the 1,3-dicarbonyl
new tetracyclic derivatives of azacarbazoles.2) Since the units 13—15 led us to investigate first the reactivity of 13 to-
pyrido[1,2-a]benzimidazole ring system has been found to ward a variety of such reagents. Results are summarized in
exhibit anticancer properties by Badaway and Kappe,3)
number of studies have been directed toward this heterocy-
cle. These investigations showed good activities of the com- prepared by reaction of 13 with formamidinium acetate. For
a
Table 1.
The unsubstituted pyrimidine derivative 16 could be easily
pounds substituted on ring A (Chart 1, compound type 1).
As a first approach to modifications which should be inte-
grated into ring C, we previously reported the regioselective
synthesis of some pyrrolo4) and pyrazolo5) derivatives and
their antitumor activities in vitro against resistant cell lines
(MDRϩ).6) To determine the effect of the nature of the hete-
rocycle, we were interested in the pyrido[1Ј,2Ј:1,2]imi-
dazo[4,5-h]quinazoline series (compound type 2). This class
of compounds is related to the general class of tetracyclic an-
gular heterocycles which are of great interest in the field of
new potential anticancer agents. For example, a number of
angular analogs of ellipticine7) have been prepared in this
way. From these studies the most promising product ap-
peared to be intoplicine 3,8) which is actually in a phase 2
clinical trial.9) In this context, we became interested in the
synthesis of the original series of pyrido[1Ј,2Ј:1,2]imi-
dazo[4,5-h]quinazoline possessing a bridgehead nitrogen
atom with a view to designing some new potential anticancer
agents. In this paper, we describe the access to this frame-
work, and our results concerning the cytotoxicity of the syn-
thesized compounds against two human cell lines exhibiting
the MDR phenotype.10)
the synthesis of the 2-amino derivative 17, guanidinium chlo-
Chart 1
Chart 2
Chemistry Retrosynthetic analysis showed that this se-
ries of compounds 4 was accessible by dehydrogenation of
dihydro precursors 5 which can themself be obtained by re-
action of an appropriate bisnucleophilic reagent with tri-
cyclic 1,3-dicarbonyl units 6 possessing the tetrahydropy-
rido[1,2-a]benzimidazolic framework11) (Chart 2).
The starting hydroxymethylene derivatives 13, 14 were ob-
tained according to the literature.5) Compound 15 was pre-
pared using the same procedure in two steps from 2-amino-5-
chloropyridine (Chart 3).
Chart 3
Access to the Pyrido[1,2:1؅,2؅]imidazo[4,5-h]quinazo-
To whom correspondence should be addressed. e-mail: yblache@pharma.univ-montp1.fr
© 2001 Pharmaceutical Society of Japan

1062
Vol. 49, No. 9
Table 1. Access to the Pyrimidinic Framework from Compound 13
N–C–N reagent
Products
and conditions
Chart 4
Chart 5
ride was used. Surprisingly, under the first condition used,
the expected product was not obtained, but the 6,7,8,9-
tetrahydropyrido[1,2-a]benzimidazol-9-one 10 was isolated.
Formation of 10 under these conditions was found to be the
result of a retro-claisen type reaction performed with sodium
ethylate generated from ethanol by sodium hydride. This hy-
pothesis was confirmed by the following experiments. Treat-
ment of 13 by NaH in ethanol led to the formation of 10, and
Fig. 1. 1H–13C Long-Range Correlation Observed from INEPT Spectra of
16
the same result was obtained when treating 13 with sodium In particular, in the cases of 16 and 23, assignment of the dif-
in ethanol while the treatment of 13 by NaH in THF gave no ferent signals of H-2 and H-4 (two singlets at 9.01 and
results. To prevent this reaction, compound 13 was allowed 8.48 ppm) was made on the basis of selective INEPT experi-
to react with guanidinium chloride in the presence of potas- ments as illustrated in Fig. 1. Carbon C-12b was expected to
sium carbonate in DMF, and the desired product 17 was ob- show a significant long-range coupling with both protons H-
tained. Synthesis of the thiomethyl derivative 18 was finally 2 and H-4, while carbon C-4a would exhibit a long-range
investigated and first conducted by reaction of 13 with 2- coupling only with H-4. As illustrated in Fig. 1, the unam-
(methylthio)pseudourea in refluxing ethanol. Under these bigous attribution for C-12b (154.49 ppm) and for C-4a
conditions, heterocyclization occurred to give 18 as the (125.53) were obtained by selective irradiation of H-5 caus-
minor product admixed with the acetal 19. Formation of this ing polarization transfer to C-12b and C-4a, and of H-6 (po-
acetal is probably catalyzed by sulfuric acid liberated in situ larization transfer to C-4a). Furthermore, selective irradiation
from 2-(methylthio)pseudourea sulfate. To avoid this reac- of the signal at 8.48 ppm showed the transfer of C-12b and
tion, the b-hydroxenone 13 was first converted to its benzoyl C-4a, while the selective irradiation of the signal at 9.01 ppm
ester 20, which reacted with 2-(methylthio)pseudourea in showed the transfer of C-12b only; this signal was attributed
ethanol to give the excepted product 18 with only 5% to H-2, while the signal at 8.48 ppm was attributed to H-4.
yield.12) When the reaction was performed in DMF, under Analogous correlations were observed for 23 leading to the
basic conditions (Et3N), formation of 18 occurred in 55% following attributions: H-2 at 9.38 ppm and H-4 at 9.42 ppm.
yield (Chart 4).
Pharmacology The cytotoxic properties of compounds
Compounds 14 and 15 were also allowed to react with 16, 21, 23 and 24 were evaluated by a cell growth inhibition
guanidinium acetate leading to the dihydropyrido[1Ј,2Ј:1,2]- assay against two human cell lines: HL60 (leukemia) and
imidazo[4,5-h]quinazolines 21, 22 with 61% and 46% yield A2780 (ovarian). The resistant sublines HL60R and A2780R
respectively showing that yields are increased by the methyl were established by the continuous spasm of cells to gradu-
group. Finally dehydrogenations of 16—18, 21, 22 were in- ally increasing concentrations of daunorubicin and doxoru-
vestigated by treating these derivatives with palladium on bicin. All results are expressed as IC50 and reported in Tables
charcoal. The corresponding pyridoimidazoquinazolines 2 and 3.
23—27 were obtained. The best yield was observed with the
2-amino derivative 24 while the 10-methyl derivative was ob- Results and Discussion
tained in only 7% yield (Chart 5).
All compounds exhibited cytotoxic activities against both
All compounds were characterized by NMR experiments. HL60S and A2780S cell lines. In the case of the HL60 cell

September 2001
1063
Table 2. Cytotoxicity of Compounds against the HL 60 Cell Line
HL60S
HL60R
Compound
RF
IC50
S.D.
IC50
S.D.
Doxorubicin
2.30ϫ10Ϫ6
1.60ϫ10Ϫ4
1.78ϫ10Ϫ3
1.90ϫ10Ϫ4
4.50ϫ10Ϫ4
0.51ϫ10Ϫ6
0.23ϫ10Ϫ4
0.64ϫ10Ϫ4
0.10ϫ10Ϫ4
0.90ϫ10Ϫ4
1.76ϫ10Ϫ4
1.60ϫ10Ϫ4
1.59ϫ10Ϫ3
1.70ϫ10Ϫ4
5.9ϫ10Ϫ4
0.93ϫ10Ϫ4
0.089ϫ10Ϫ4
0.46ϫ10Ϫ3
0.11ϫ10Ϫ4
0.08ϫ10Ϫ4
76
1
1
1
1.3
16
21
23
24
S.D., standard deviation; RF, resistance factor.
Table 3. Cytotoxicity of Compounds against the A 2780 Cell Line
A2780S
A2780R
Compound
RF
IC50
S.D.
IC50
S.D.
Doxorubicin
0.34ϫ10Ϫ6
1.50ϫ10Ϫ4
1.75ϫ10Ϫ3
3.60ϫ10Ϫ5
5.10ϫ10Ϫ5
0.05ϫ10Ϫ6
0.18ϫ10Ϫ4
0.10ϫ10Ϫ3
0.12ϫ10Ϫ5
0.30ϫ10Ϫ5
0.28ϫ10Ϫ4
1.30ϫ10Ϫ4
1.34ϫ10Ϫ3
3.70ϫ10Ϫ5
5.80ϫ10Ϫ5
1.98ϫ10Ϫ6
0.32ϫ10Ϫ4
0.63ϫ10Ϫ4
0.15ϫ10Ϫ5
0.10ϫ10Ϫ5
82
1
1
1
1.1
16
21
23
24
S.D., standard deviation; RF, resistance factor.
Experimental
line, the less active compound was the 2-methyl derivative
21, while other compounds showed similar activities against
both sensitive and resistant cells with small resistance factor
(RF ca. 1 for 16, 23, 24 and 76 for doxorubicin). In addition,
Melting points were determined on a Buchi capillary melting point appa-
ratus and are not corrected. Elemental analysis was performed by Microana-
lytical Center, ENSCM, Montpellier. Spectral measurements were taken
using the following instruments: 1H-NMR spectra were taken on a Brüker
no effect of the dehydrogenation was observed on the activi- AC 100 and a EM 400 WB instrument; 13C-NMR spectra were obtained at
26 °C with proton noise decoupling at 25 MHz with a Brüker AC 100 and a
EM 400 WB instrument. Chemical shifts are expressed relative to residual
chloroform. Mass spectra were recorded on a LKB 2091 spectrometer at
15 eV [q(source)ϭ180 °C].
ties.
The most interesting results were obtained on the A2780S
and A2780R cell lines with the dehydrogenated compounds
23 and 24 which exhibited the highest activities. These activ-
ities were approximately 10 times better than their hydro-
genated homologues and close to those observed for doxoru-
bicine on the resistant cell line. In addition, in these two
cases, the enhanced cytotoxicity did not induce the resistance
phenomena (RF ca. 1).
Finally, these results showed that the pyrido[1Ј,2Ј:1,2]imi-
dazo[4,5-h]quinazoline skeleton is an interesting framework
in which to study a new class of potential cytotoxic agents.
As for the previously described pyrrolo and pyrazolo deriva-
tives,4,5) the principal interest in these compounds resides in
their activities on resistant cell lines with better activities of
the six membered heterocyclic derivatives. Also the planerity
of the molecules is necessary to enhance the cytotoxic prop-
erties.
2-Chloro-6,7,8,9-tetrahydropyrido[1,2-a]benzimidazol-9-one (12)
This compound was obtained in 49% yield according to the methodology
given in the literature (ref. 8) for the synthesis of 10 and 11. mp: 119—
121 °C (recrystallization solvent, methanol); MS m/z: 222 (38), 220 (100),
194 (27), 192 (73), 164 (32), 129 (38). 1H-NMR (CDCl3, 400 MHz) d: 2.12
(t, 2H, J6—7ϭJ7—8ϭ6.0 Hz, H7), 2.53 (t, 2H, H8), 2.93 (t, 2H, H6), 7.29 (dd,
1H, J3—4ϭ9.0 Hz, J1—3ϭ1.5 Hz, H3), 7.47 (dd, 1H, J1—4ϭ1.1 Hz, H4), 9.16
(s, 1H, H1). 13C-NMR (CDCl3, 100 MHz) d: 23.5 (C6), 25.2 (C7), 38.1 (C8),
116.8 (C4), 119.5 (C9a), 122.4 (C2), 126.0 (C1), 130.2 (C3), 145.9 (C5a),
160.3 (C4a), 188.2 (CϭO). Anal. Calcd for C11H9N2OCl: C, 59.88; H, 4.11;
N, 12.70. Found: C, 59.75; H, 4.21; N, 12.51.
2-Chloro-8-hydroxymethylene-6,7,8,9-tetrahydropyrido[1,2-a]benzim-
idazol-9-one (15) This compound was obtained in 74% yield according to
the methodology given in the literature (ref. 5) for the synthesis of 10 and
11. mp: 226—228 °C (recrystallization solvent, methanol). MS m/z: 250
(26), 248 (71), 221 (30), 220 (48), 219 (100), 191 (17), 155 (26), 129 (13).
1H-NMR (CDCl3, 100 MHz) d: 2.68 (t, 2H, J6—7ϭ6.0 Hz, H7), 3.07 (t, 2H,
H6), 7.27 (s, 1H, CHOH), 7.45 (dd, 1H, J3—4ϭ9.4 Hz, J1—3ϭ1.9 Hz, H3),
7.69 (d, 1H, H4), 9.35 (s, 1H, H1). 13C-NMR (CDCl3, 25 MHz) dCH: 23.8
(C6), 24.5 (C7), 98.8 (CHOH), 116.7 (C4), 126.2 (C1), 131.4 (C3), 159.7 (C2).
Anal. Calcd for C12H9N2O2Cl: C, 57.96; H, 3.65; N, 11.27. Found: C, 57.79;
H, 3.81; N, 11.11.
5,6-Dihydropyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (16) To a so-
lution of 13 (600 mg, 2.8 mmol) in dry ethanol (53 ml) was added 3.5 g
(33.6 mmol) of formamidinium acetate. The solution was refluxed for 25 h
under a nitrogen stream. After evaporation of solvent, the crude product was
chromatographed on silica gel eluting with CH2Cl2/CH3OH (97/3) to give 16
(48%). mp: 103—105 °C (recrystallization solvent, methanol). MS m/z: 222
(100), 221 (70), 194 (20), 167 (20). 1H-NMR (CDCl3, 400 MHz) d: 3.21 (m,
4H, H5, H6), 7.07 (t, 1H, J10—11ϭJ10—9ϭ7.0 Hz, H10), 7.43 (t, 1H, H9), 7.72
(d, 1H, J8—9ϭ7.0 Hz, H8), 8.48 (s, 1H, H4), 9.01 (s, 1H, H2), 9.58 (d, 1H,
H11). 13C-NMR (CDCl3, 100 MHz) d: 23.3 (C6), 25.2 (C5), 113.7 (C10),
117.1 (C8), 117.2 (C-12a), 125.5 (C-4a), 127.2 (C9), 128.2 (C-11), 148.2 (C-
7a), 152.6 (C-6a), 153.3 (C4), 154.5 (C-12b), 156.9 (C2). Anal. Calcd for
Conclusion
In this paper, we have reported the synthesis of a new
class of tetracyclic bridgehead nitrogen heterocycles: the
pyrido[1Ј,2Ј:1,2]imidazo[4,5-h]quinazoline framework. Re-
activity of 1,3-dicarbonyl unit (13) derived from 6,7,8,9-
tetrahydropyrido[1,2-a]benzimidazol-9-one was reported to-
ward a set of “N–C–N” bisnucleophilic reagents showing the
influence of the reaction conditions on the reactivity of such
systems. Further studies on the pharmacomodulation of such
compounds are now in progress and should lead to an inter-
esting class of new potential anticancer agents with no resis-
tant phenomena.

1064
Vol. 49, No. 9
C13H10N4OCl: C, 70.26; H, 4.54; N, 25.21. Found: C, 70.41; H, 4.44; N,
25.15.
10-Chloro-5,6-dihydropyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (22)
This compound was obtained in 46% yield according to the procedure used
for 16 (reaction time, 24 h); chromatography: silica gel eluted with
CH2Cl2/CH3OH (95/5). mp: 185—187 °C. MS m/z 256 (97), 255 (42), 220
(22), 149 (15). 1H-NMR (CDCl3, 100 MHz) d: 3.20 (m, 4H, H5, H6), 7.33 (d,
1H, H9, J8—9ϭ1.2 Hz), 7.62 (d, 1H, H8), 8.49 (s, 1H, H4), 9.00 (s, 1H, H2),
9.61 (s, 1H, H11). Anal. Calcd for C13H9N4Cl: C, 60.83; H, 3.53; N, 21.83.
Found: C, 60.75; H, 3.36; N, 21.99.
2-Amino-5,6-dihydropyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (17)
To a stirred solution of 1.62 g (16.8 mmol) of guanidinium chloride and
potassium carbonate in dry DMF (20 ml) was added 200 mg (0.9 mmol) of
13. The solution was refluxed for 2 h. After filtration and evaporation of sol-
vent the crude product was chromatographed on silica gel eluting with
CH2Cl2/CH3OH (97/3). Yield: 53%. mp: 239—241 °C (recrystallization
solvent, ethanol). MS m/z: 238 (25), 237 (100), 236 (86), 209 (18). 1H-NMR
(CDCl3, 100 MHz) d: 3.08 (m, 4H, H5, H6), 5.35 (NH2), 6.99 (t, 1H,
Pyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (23) To a solution of 16
(300 mg, 1.35 mmol) in diphenylether (6.0 ml) was added 140 mg of palla-
dium (10%) on charcoal. The solution was refluxed for 22 h. After filtration
of the palladium, the crude product was washed with methanol and chro-
matographed on silica gel with CH2Cl2/CH3OH (99/1). Yield: 34%. mp:
186—188 °C (recrystallization solvent, ethanol). MS m/z: 220 (100), 193
J
9—10ϭJ10—11ϭ7.0 Hz, H10), 7.38 (dt, 1H, J9—8ϭ7.0 Hz, J9—11ϭ1.0 Hz, H9),
7.64 (dd, 1H, J8—10ϭ1.0 Hz, H8), 7.98 (s, 1H, H4), 9.46 (dd, 1H, H11). 13C-
NMR (CDCl3, 25 MHz) d: 23.5 (C6), 24.2 (C5), 113.6 (C10), 115.5 (C-
12a),116.7 (C8), 122.6 (C-4a), 127.3 (C9), 128.2 (C11), 147.6 (C-7a), 152.7
(C-6a), 153.2 (C4), 155.9 (C-12b), 161.3 (C-2). Anal. Calcd for C13H11N5: C,
65.81; H, 4.67; N, 29.52. Found: C, 65.89; H, 4.71; N, 29.40.
1
(12), 166 (14). H-NMR (CDCl3, 400 MHz) d: 7.15 (t, 1H, J9—10ϭJ10—11ϭ
7.0 Hz, H10), 7.63 (t, 1H, J8—9ϭ7.0 Hz, H9), 7.85 (d, 1H, J5—6ϭ8.8 Hz, H5),
7.90 (d, 1H, H8), 8.06 (d, 1H, H6), 9.38 (s, 1H, H2), 9.42 (s, 1H, H4), 9.98 (d,
1H, H11). 13C-NMR (CDCl3, 100 MHz) d: 112.8 (C10), 117.6 (C8), 120.2
(C12a), 121.2 (C4a), 122.4 (C6), 124.8 (C5), 130.0 (C9), 130.1 (C11), 142.5
(C12b), 148.3 (C6a), 149.5 (C7a), 155.4 (C2), 158.6 (C4). Anal. Calcd for
C13H8N4: C, 70.90; H, 3.66; N, 25.44. Found: C, 71.02; H, 3.58; N, 25.40.
2-Aminopyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (24) This com-
pound was obtained according to the procedure used for 23 (reaction time,
6 h); chromatography: silica gel with CH2Cl2/CH3OH (98/2). Yield: 67%.
mp Ͼ260 °C (recrystallization solvent, methanol). MS m/z: 236 (100), 235
(70), 209 (23), 208 (23). 1H-NMR (CDCl3, 100 MHz) d: 8.00 (t, 1H,
2-(Methylthio)-5,6-dihydropyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline
(18) Method A: To a solution of 13 (500 mg, 2.3 mmol) and 0.20 ml of tri-
ethylamine in anhydrous EtOH (30 ml) was added 5.2 g of methylthio-
pseudourea sulfate (18.72 mmol). The solution was refluxed for 10 h under a
nitrogen stream. After evaporation of solvent, the crude mixture was chro-
matographed on silica gel eluted with CH2Cl2/CH3OH (98/2) to give 18 in
5% yield. mp: 92—94 °C (recrystallization solvent, ethylacetate). MS m/z:
268 (100), 267 (20), 222 (40), 221 (56), 194 (28). 1H-NMR (CDCl3, 100
MHz) d: 2.55 (s, 3H, CH3), 3.02 (m, 4H, H5, H6), 6.91 (t, 1H, J9—10ϭ
J
10—11ϭ6.0 Hz, H10), 7.29 (dt, 1H, J8—9ϭ6.0 Hz, J9—11ϭ1.3 Hz, H9), 7.55 (d,
1H, H8), 8.11 (s, 1H, H4), 9.23 (d, 1H, H11). 13C-NMR (CDCl3, 25 MHz)
CH: 13.9 (CH3), 22.7 (C6), 24.2 (C5), 114.0 (C10), 116.4 (C8), 127.8 (2C,
J
10—11ϭJ9—10ϭ7.0 Hz, H10), 8.41 (t, 1H, H9), 8.50 (m, 3H, H8, H5, H6), 9.85
(s, 1H, H4), 10.81 (d, 1H, H11). 13C-NMR (CDCl3, 25 MHz) dCH: 112.3
(C10), 115.2 (C8), 115.9 (C6), 125.7 (C5), 129.7 (C9*), 130.2 (C11*), 160.2
(C4). Anal. Calcd for C13H9N5: C, 66.37; H, 3.86; N, 29.77. Found: C, 66.45;
H, 3.87; N, 29.68.
d
C9, C11), 153.2 (C4). Anal. Calcd for C14H12N4S: C, 62.67; H, 4.51; N,
20.88. Found: C, 62.85; H, 4.31; N, 20.76. Further elution yielded 8-di-
ethoxymethyl-6,7,8,9-tetrahydropyrido[1,2-a]benzimidazol-9-one (19) with
48% yield (brown oil). MS m/z: 288 (5), 259 (44), 213 (11), 185 (25), 157
(30), 103 (100). 1H-NMR (CDCl3, 100 MHz) d: 1.22 (m, 6H, CH3–CH2),
2.35 (t, 2H, J6—7ϭJ7—8ϭ6.1 Hz, H7), 2.60—3.30 (m, 3H, H6, H8), 3.61 (m,
4H, CH3–CH2), 5.1 (s, 1H, H1Ј), 6.95 (t, 1H, J1—2ϭJ2—3ϭ7.0 Hz, H2), 7.47
(t, 1H, J3—4ϭ7.0 Hz, H3), 7.59 (d, 1H, H4), 9.21 (d, 1H, H1). 13C-NMR
(CDCl3, 25 MHz) d: 15.2 (CH3–CH2); 15.32 (CH3–CH2), 22.5 (C6), 24.3
(C7), 51.1 (C8), 63.6 (CH3–CH2), 64.9 (CH3–CH2), 102.2 (C1Ј), 114.3 (C2),
116.8 (C4), 119.8 (C9a), 128.2 (C1), 129.4 (C3), 148.1 (C5a), 160.6 (C4a), 187.
1 (CϭO). Anal. Calcd for C15H20N2O3: C, 65.20; H, 7.30; N, 10.14. Found:
C, 65.31; H, 7.23; N, 10.25.
2-(Methylthio)pyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (25) This
compound was obtained according to the procedure used for 23 (reaction
time, 10 h); chromatography: silica gel with CH2Cl2. Yield: 24%; mp: 196—
198 °C (recrystallization solvent, ethyl acetate). MS m/z: 266 (100), 221
(15), 220 (73), 219 (37), 193 (26), 166 (15). 1H-NMR (CDCl3, 100 MHz) d:
2.58 (s, CH3), 6.88 (t, 1H, J9—10ϭJ10—11ϭ6.6 Hz, H10), 7.30—7.75 (m, 4H,
H5, H6, H8, H9), 8.84 (s, 1H, H4), 9.29 (d, 1H, H11). 13C-NMR (CDCl3,
25 MHz) dCH: 14.4 (SCH3), 111.9 (C10), 116.9 (C8), 119.5 (C6), 124.2 (C5),
128.8 (C11*), 129.3 (C9*), 157.8 (C4). Anal. Calcd for C14H10N4S: C, 63.14;
H, 3.78; N, 21.04. Found: C, 63.25; H, 3.99; N, 21.13.
Method B: To a solution of 20 (500 mg, 1.56 mmol) and 0.26 ml of tri-
ethylamine in anhydrous DMF (30 ml) was added 5.2 g of methylthio-
pseudourea sulfate (18.72 mmol). The solution was refluxed for 23 h under a
nitrogen stream. After evaporation of solvent, the crude mixture was chro-
matographed on silica gel eluted with CH2Cl2/CH3OH (98/2) to give 18 in
55% yield.
8-[(Benzoyloxy)methylene]-6,7,8,9-tetrahydropyrido[1,2-a]benzimida-
zol-9-one (20) To a solution of 13 (1 g, 4.7 mmol) and potassium carbon-
ate (0.78 g, 5.6 mmol) in dry acetone (67 ml) was added 1.62 ml (14.1 mmol)
of benzoyl chloride over 20 min. The solution was stirred at room tempera-
ture under a nitrogen stream overnight. After evaporation of solvent, the
crude product was taken up in CH2Cl2 and poured over ice. The layers were
separated and the organic layer was washed with 10% NaHCO3 and brine,
dried over Na2SO4 and concentrated. Yield: 64%; mp: 189—191 °C (recrys-
tallization solvent, ether). MS m/z: 318 (34), 213 (13), 185 (14), 105 (100).
1H-NMR (CDCl3, 100 MHz) d: 3.16 (m, 4H, H6, H7), 7.02 (t, 1H, J1—2ϭ
10-Methylpyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (26) This com-
pound was obtained according to the procedure used for 23 (reaction time,
2 h); recrystallization in ether. Yield: 7%. mp: 207—209 °C. MS m/z: 236
(8), 235 (29), 234 (100), 233 (58). 1H-NMR (CDCl3, 100 MHz) d: 2.56
(s, CH3), 7.48 (d, 1H, J8—9ϭ8.8 Hz, H9), 7.84 (d, 1H, H8), 7.86 (d, 1H,
J5—6ϭ8.8 Hz, H5), 8.10 (d, 1H, H6), 9.42 (s, 2H, H2, H4), 9.86 (s, 1H, H11).
13C-NMR (CDCl3, 25 MHz) dCH: 18.5 (CH3), 116.9 (C8), 122.5 (C6), 124.3
(C5), 127.6 (C11*), 133.0 (C9*), 155.1 (C4), 158.4 (C2). Anal. Calcd for
C14H10N4: C, 71.78; H, 4.30; N, 23.92. Found: C, 71.75; H, 4.26; N, 23.99.
10-Chloropyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (27) This com-
pound was obtained according to the procedure used for 23 (reaction time,
10 h); chromatography: silica gel with CH2Cl2/CH3OH (98/2). Yield: 52%.
mp: 216—218 °C. MS m/z 254 (25), 221 (25), 167 (57), 149 (80). 1H-NMR
(CDCl3, 100 MHz) d: 7.59 (d, 1H, H9, J8—9ϭ9.1 Hz), 7.80—8.10 (m, 3H,
H5, H6, H8), 9.44 (s, 2H, H2, H4), 10.09 (s, 1H, H11). 13C-NMR (CDCl3,
25 MHz) dCH: 117.3 (C10), 126.1 (C6), 128.2 (C5), 128.9 (C9*), 130.9 (C11*),
153.6 (C2), 157.0 (C4). Anal. Calcd for C13H7N4l: C, 61.31; H, 2.77; N,
22.00. Found: C, 61.45; H, 2.58; N, 21.91.
J
2—3ϭ7.0 Hz, H2), 7.53 (m, 5H, HAr), 8.10 (m, 2H, H3, H4), 8.52 (s, 1H, H1Ј),
9.29 (d, 1H, H1). 13C-NMR (CDCl3, 25 MHz) d: 22.7 (C7), 23.9 (C6), 114.2
(C2), 116.4 (C4), 119.8 (C9a), 120.7 (C8), 127.9 (CAr), 128.0 (CAr), 128.3 (C2,
CAr), 129.4 (C1*), 129.8 (2C, CAr), 133.7 (C3*), 140.3 (C1Ј), 147.9 (C5a),
158.9 (C4a), 176.8 (CϭO). Anal. Calcd for C19H14N2O3: C, 71.69; H, 4.43;
N, 8.80. Found: C, 71.75; H, 4.26; N, 8.99.
Biological Assay Doxorubicin hydrochloride (Pharmacia, St Quentin en
Yvelines, France); RPMI 1640 medium and fetal calf serum (Polylabo,
Paris, France) were used in this study. All other reagents were of analytical
grade and were obtained from commercial sources.
10-Methyl-5,6-dihydropyrido[1؅,2؅:1,2]imidazo[4,5-h]quinazoline (21)
This compound was obtained according to the procedure used for 16 (reac-
tion time, 24 h); chromatography: silica gel, CH2Cl2/CH3OH (95/5); yield:
61.5%. mp: 207—209 °C (recrystallization solvent, methanol). MS m/z: 236
Cells and Cultures: The human promyelocytic leukaemia cell line, HL60
was obtained from the American type culture collection (Rockville, MD,
U.S.A.). The human ovarian carcinoma were generously given by Dr P.
Canal (C. R. L. C. Val d’Aurelle). The doxorubicin resistant sublines HL60R
and A2780R were established by the continuous spasm of cells to gradually
increasing concentrations of daunorubicin and doxorubicine, respectively,
and were maintained in medium supplemented with daunorubicin and dox-
orubicin at 0.1 mg/ml, respectively. The MDR phenotype expression of the
HL60R and A2780R cell lines was assessed by an immunohistochemistry
method, using the two P-glycoprotein-specific murine monoclonal antibod-
ies C219 (Cantocor, Malvern, PA, U.S.A.) and JSB1 (Tebu, le Perray en
1
(100), 235 (66), 208 (7), 181 (7). H-NMR (CDCl3, 100 MHz) d: 2.39 (s,
CH3), 3.08 (m, 4H, H5, H6), 7.17 (d, 1H, J8—9ϭ9.0 Hz, H9), 7.51 (d, 1H, H8),
8.36 (s, 1H, H4), 8.89 (s, 1H, H2), 9.20 (s, 1H, H11). 13C-NMR (CDCl3,
25 MHz) d: 18.07 (CH3), 22.7 (C6), 24.7 (C5), 115.7 (C8), 116.3 (C-12a),
123.4 (C-10), 125.1 (C-4a), 125.7 (C9), 129.9 (C11), 148.6 (C-7a), 151.6 (C-
6a), 152.5 (C4), 154.1 (C-12b), 156.3 (C2). Anal. Calcd for C14H12N4: C,
71.17; H, 5.12; N, 23.71. Found: C, 71.25; H, 5.22; N, 23.53.

September 2001
1065
Yvelines, France). Cultures were grown in RPMI 1640 medium supple-
mented with 10% fetal calf serum, antibiotics and glutamine at 37 °C in a
humidified atmosphere containing 5% CO2.
Teulade J.-C., Chapat J.-P., Fauvelle F., Grassy G., Dauphin G., J. Het-
erocyclic Chem., 34, 765—771 (1997).
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J. C., Teulade J. C., Chapat J. P., J. Org. Chem., 62, 8553—8556
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(1994).
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4) Dupuy M., Pinguet F., Chavignon O., Teulade J. C., Chapat J. P, Blache
Y., Heterocyclic Chem., 7, 23—28 (2001).
5) Dupuy M., Pinguet F., Blache Y., Chavignon O., Teulade J. C., Chapat
J. P., Chem. Pharm. Bull., 46, 1820—1823 (1998).
6) Chevillard S., Vielh P., Oncologia, 5, 5—13 (1993).
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ibid., 7, 166—174 (1996).
Cytotoxicity assays: In all experiments, parental sensitive and resistant
HL60 and A2780 cells were seeded at a final density of 6000 cells/well in 96
well microtiter plates and were treated with drugs (doxorubicin and com-
pounds 16, 21, 23, 24). Ten dilutions were used for each drug. After 96 h of
incubation, 20 ml of MTT solution in PBS (5 mg/ml) was added to each well
and the wells were then exposed to 37 °C for 4 h. This colorimetric assay is
based on the ability of live and metabolically unimpaired tumor-cell targets
to reduce MTT to a blue formazan product.8) Then, 100 ml of a mixture of
isopropanol and 1M hydrochloride acid (96 : 4, v/v) was added to each well.
After a vigorous shaking, the absorbance was measured on a microculture
plate reader (Dynatech MR5000, France) at 570 nm. For each assay, at least
four experiments were performed in triplicate. The resistance factor (RF)
was calculated from the ratio between the IC50% growth-inhibitory concen-
trations (IC50 values) recorded from HL60 R, A2780 R and HL60, A2780
cells, respectively, for all drugs tested (doxorubicin; compounds 16, 21, 23,
24).
Acnowledgements We gratefully acknowledge financial support from
the Ligue Nationale de Lutte contre le Cancer (Comité de l’Hérault).
References and Notes
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