Synthesis and antiproliferative evaluation of new aryl substituted pyrido[3′,2′:5,6]thiopyrano[4,3-c]pyrazoles

Article abstract of DOI:10.1002/jhet.5570420715

The preparation and the cytotoxic properties of new derivatives of the planar pyrido[3′,2′:5,6]thiopyrano[4,3-c]pyrazole system, carrying an arylic side group in the 1 or 2 positions, are described. The novel substituted derivatives were obtained by react

Full text of DOI:10.1002/jhet.5570420715

Nov-Dec 2005
1357
Synthesis and Antiproliferative Evaluation of New Aryl Substituted
Pyrido[3',2':5,6]thiopyrano[4,3-c]pyrazoles
*
†
†
†
†
G. Primofiore , A. M. Marini , S. Salerno , F. Da Settimo ,
§
†
D. Bertini and L. Dalla Via
†
Dipartimento di Scienze Farmaceutiche, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy
§
Dipartimento di Scienze Farmaceutiche, Università di Padova, via Marzolo 5, 35131 Padova, Italy
Received April 5, 2005
The preparation and the cytotoxic properties of new derivatives of the planar pyrido[3',2':5,6]thiopyrano-
[4,3-c]pyrazole system, carrying an arylic side group in the 1 or 2 positions, are described. The novel substi-
tuted derivatives were obtained by reaction of suitable arylhydrazines with the appropriate key intermediate
3-hydroxymethylene-2,3-dihydrothiopyrano[2,3-b]pyridin-4(4H)-ones. Moreover the preparation was
reported of the 2-carboxamidophenyl derivatives, which was accomplished from the previously obtained
pyrido[3',2':5,6]thiopyrano[4,3-c]pyrazole nucleus, by reaction with phenylisocyanate. All the new com-
pounds were evaluated for their antiproliferative ability, by an in vitro assay on human tumor cell lines (HL-
60 and HeLa).
J. Heterocyclic Chem., 42, 1357 (2005).
Introduction.
In an effort to enhance the cytotoxic activity of this class
of compounds it seemed reasonable to further investigate
how the position or the nature of the lipophilic moiety
linked to the chromophore system was able to affect the
capacity of intercalation with DNA base pairs. In this
paper, we report the synthesis of the 1-benzyl derivatives 2
and of the 2-phenyl, 2-p-methoxyphenyl, 2-benzyl and 2-
carboxamidophenyl derivatives 3, 4, 5 and 6, respectively,
in which the pendant phenyl ring was inserted at the 2-
position of the heterocyclic scaffold both directly and with
spacer groups of different length and grade of flexibility
(Figure 2).
Many effective anticancer drugs in clinical use interact
with DNA and some of them exhibit cytotoxic activity
through DNA intercalation [1-3]. The intercalation process
reflects the ability of a planar aromatic or heteroaromatic
system to become inserted between adjacent base pairs of a
DNA molecule; this intercalative reaction can affect many
biological properties of DNA, including enzymatic block-
ade and reading errors during the replication and transcrip-
tion process, thus leading to cell death [4-7]. Although the
presence of a fused heterocyclic system plays an important
role for the DNA-binding properties, the antiproliferative
activity is often strongly dependent on the presence of suit-
able substituents or of a positive charge which could be
provided both by an aminoalkyl side chain or by a proton-
able aromatic nitrogen of the chromophore [8].
In the continual search of new classes of DNA-binding
systems, our group has been interested for several years in
the synthesis of new polycyclic derivatives, some of
which, containing the purine, benzimidazole or indole
nucleus, showed a cytotoxic activity, similar to that of
Ellipticine, mainly attributable to an intercalative mode of
binding to DNA [9-11]. As part of this screening research
activity, we recently described a series of new 1-phenyl
pyridothiopyrano[4,3-c]pyrazoles 1 (Figure 1) [12] which,
evaluated for their antiproliferative activity on two human
tumor cell lines, showed only moderate cytotoxic proper-
ties, with IC values higher than 20 µM.
50
The antiproliferative activity of the new compounds was
evaluated in vitro, by means of a cell growth inhibition
assay on two human tumor cell lines, human promyelo-
cytic leukemic cells (HL-60) and human cervix adenocar-
cinoma cells (HeLa).

1358
G. Primofiore, A. M. Marini, S. Salerno, F. Da Settimo, D. Bertini and L. Dalla Via
Vol. 42
Results.
the desired 2-substituted products 3, 4 and 5. All com-
pounds were purified by flash chromatography and charac-
terized by analytical and spectral data (Table 1). In particu-
The synthetic procedure utilized in the preparation of the
target compounds 2 and 2-aryl substituted analogues 3, 4
and 5 is illustrated in Figure 3. The 3-hydroxymethylene-
2,3-dihydrothiopyrano[2,3-b]pyridin-4(4H)-one 7a and its
7-methyl derivative 7b, which represent the starting key
compounds, have been recently described by us [12,13].
The condensation of compounds 7 with benzylhydrazine
hydrochloride, in refluxing methanol, gave the desired 1-
benzyl pyrazoles 2 as unique and pure isomers, since the
reaction of 7 with the hydrazines firstly involves the more
reactive methine group adjacent to the C=O function [14-
16]. Analytical and spectral data of compounds 2 confirm
the proposed structures [12].
1
lar the H nmr spectra of compounds 3, 4 and 5 showed, as
the most discriminating feature, a singlet at ~ 8.3 ppm
attributed to the proton in the 3-position of the pyrazole
moiety, while the same proton of the analogous 1-aryl [12]
and 1-benzyl (2) substituted compounds resonates at ~ 7.6
ppm. The proposed synthetic pathways have been con-
firmed by the isolation and characterization of the interme-
diate 4-phenylhydrazone derivative 9, which was obtained
by stirring 8b with phenylhydrazine hydrochloride for 1
1
hour at room temperature. The H nmr spectrum of 9
showed two singlet signals at δ 2.43 and δ 2.45 which were
The synthetic sequence leading to the 2-aryl substituted
2,4-dihydropyrido[3',2':5,6] thiopyrano[4,3-c]pyrazoles 3,
4 and 5 involved, as a first step, the conversion of the
3-hydroxymethylene compounds 7 into the intermediate
p-toluensulphonates 8a or 8b, in which the reactive
methine functionality has been protected [17]. The subse-
quent condensation of compounds 8 with the appropriate
arylhydrazine hydrochloride, in anhydrous dimethylfor-
mamide at room temperature, involves only the carbonylic
function in the 4-position, affording the intermediate aryl-
hydrazones, which easily cyclize in situ by heating (100
°C) the reaction mixture. The removal of the protective
tosyl group occurs under these conditions, thus affording

Nov-Dec 2005
New Aryl Substituted Pyrido[3',2':5,6]thiopyrano[4,3-c]pyrazoles
1359

1360
G. Primofiore, A. M. Marini, S. Salerno, F. Da Settimo, D. Bertini and L. Dalla Via
Vol. 42
EXPERIMENTAL
assigned to the methyl groups of the thiopyrane nucleus
and of the tosyl moiety, respectively. Moreover, the pres-
ence of the nine-proton multiplet signals at δ 7.12-7.23 and
δ 7.35-7.66, assigned to the aromatic protons either of the
phenylhydrazine and of the tosyl group, was consistent
with the structure of 9.
Melting points were determined using a Reichert Köfler hot-
stage apparatus and are uncorrected. Infrared spectra (ir) were
obtained on a NICOLET/AVATAR, 360 FT spectrophotometer as
Nujol mulls. Nuclear magnetic resonance (nmr) spectra were
recorded on a Varian Gemini 200 spectrometer, in dimethyl-d
6
The 2-carboxamidophenyl derivatives 6 were prepared
from the pyridothiopyranopyrazoles 10 [12], by reaction
with an excess of phenylisocianate (Figure 4). The assign-
ment of the position of the carboxamido side chain was
made on the basis of their proton nmr spectra, which
showed important similarities to the above described com-
sulfoxide solution, using TMS as the internal standard. Mass
spectra (ms) were obtained on a Finningan Polaris/GCQ Plus
spectrometer using an electron beam energy of 70 eV.
Magnesium sulfate was always used as the drying agent.
Evaporations were made in vacuo (rotating evaporator).
Analytical tlc were carried out on Merck 0.2 mm precoated silica
gel aluminium sheets (60 F-254). Elemental analyses were per-
formed by our Analytical Laboratory.
1
pounds. The H nmr spectra of 6 exhibited a singlet at ~
8.3 ppm, which was assigned to the proton in the 3-posi-
tion of the pyrazole ring, allowing us to infer that the car-
boxamidophenyl group is linked to the 2-position of the
chromophore system (Table 1).
The antiproliferative activity of compounds 2-6 was
evaluated by means of a cell growth inhibition assay on
two human tumor cell lines, HL-60 and HeLa, in accor-
dance with the experimental procedures previously
described [11]. Moreover, the cytotoxic ability of the pyri-
dothiopyranopyrazole nucleus 10 was also investigated in
order to elucidate if the scaffold itself is biologically active
or is a good carrier for other suitable molecular groups.
1-Benzyl-1,4-dihydropyrido[3',2':5,6]thiopyrano[4,3-c]pyrazole
2a and 7-Methyl derivative 2b.
General Procedure.
Benzylhydrazine hydrochloride (0.215 g, 1.10 mmoles) was
added to a solution of 3-hydroxymethylene-2,3-dihydrothiopy-
rano[2,3-b]pyridin-4(4H)-one 7a (0.193 g, 1.00 mmole) or 7-
methyl-derivative 7b (0.207 g, 1.00 mmole) in 15 mL of
methanol, and the reaction mixture was stirred at room tempera-
ture for 24 hours, then refluxed for 15 hours. After cooling, the
yellow precipitate, if present, was collected and the filtrate was
evaporated under reduced pressure. The solid and the residue
were treated with a saturated aqueous potassium carbonate solu-
tion to give crude pyrazoles 2, which were purified by recrystal-
lization from ethanol.
The results, expressed as IC values and reported in Table
50
2, indicated that either 1-benzylderivatives 2 or those sub-
stituted in the 2-position (3, 4, 5 and 6b) do not exert any
cytotoxic effect, while compound 6a showed a detectable
antiproliferative activity in both cell lines. As regard the
pyrazole system, compound 10a showed the ability to
induce a cytotoxic effect, thus appearing an interesting
chromophore system. It is also noteworthy that the inser-
tion of a methyl group in the 7-position seemed to be detri-
mental for the biological activity.
Compound 2a was obtained as a yellow solid in 32% yield:
m.p. 135-137 °C; ir: 1580, 1550, 1310, 1090, 990, 800, 750, 700
-1
1
cm ; H nmr: δ 4.12 (s, 2H, CH S), 5.65 (s, 2H, CH Ph), 7.03
2
2
(d, 2H, 2',6'-PhH), 7.10-7.17 (dd, 1H, J =4.7 Hz, J =8 Hz, 8-
8,7
8,9
H), 7.24-7.36 (m, 3H, 3',4,'5'-PhH), 7.54 (s, 1H, 3-H), 7.80 (dd,
1H, J =8 Hz, J =1.6 Hz, 9-H), 8.23 (dd, 1H, J =4.7 Hz,
9,8
9,7
7,8
+
J
=1.6 Hz, 7-H); ms: m/z 279 (90, M ), 149 (100).
7,9
Anal. Calcd. for C
H N S: C, 68.82; H, 4.66; N, 15.05.
16 13 3
Found: C, 68.69; H, 4.84; N, 14.84
Compound 2b was obtained as an orange solid in 33% yield:
-1
1
m.p. 98-101 °C; ir: 1580, 1500, 1130, 845, 765, 710 cm ; H
nmr: δ 2.35 (s, 3H, CH ), 4.09 (s, 2H, CH S), 5.63 (s, 2H,
3
2
CH Ph), 6.97-7.05 (m, 3H, J=8 Hz, 8-H, 2', 6'-Ph), 7.27-7.32 (m,
2
Table 2
3H, 3',4',5'-PhH), 7.52 (s, 1H, 3-H), 7.71 (d, 1H, J=8 Hz, 9-H);
+
ms: m/z 293 (60, M ), 149 (100).
Antiproliferative activity of compounds 2, 3, 4, 5, 6 and 10
Anal. Calcd. for C
H N S: C, 69.0; H, 5.11; N, 14.33.
16 13 3
Found: C, 68.86; H, 4.91; N, 13.96
Compound
Cellular lines IC (µM)
50
HeLa
HL-60
3-[(p-Methylphenyl)sulhonyl]oxymethylen-2,3-dihydrothiopy-
rano[2,3-b]pyridin-4(4H)-one 8a and 7-Methyl Derivative 8b.
2a
>20
>20
2b
3a
3b
4a
4b
5a
5b
6a
>20
>20
>20
>20
>20
>20
>20
6.6 0.3
>20
8.8 0.2
>20
>20
>20
>20
>20
>20
>20
>20
13.5 0.5
>20
9.0 0.5
>20
General Procedure.
p-Toluensulphonylchloride (3.96 g, 20.8 mmoles) was added
to a solution of 7a (2.0 g, 10.4 mmoles) or 7b (2.152 g, 10.4
mmoles) in 40 mL of anhydrous tetrahydrofurane in the presence
of potassium carbonate (5.72 g, 41.6 mmoles). The reaction mix-
ture, under nitrogen atmosphere, was stirred at room temperature
for 24 hours then refluxed for 15 hours. After cooling, the sus-
pension was concentrated under reduced pressure, and the
residue obtained was treated with water and then extracted with
chloroform. The organic layers were dried and evaporated to give
6b
10a
10b

Nov-Dec 2005
New Aryl Substituted Pyrido[3',2':5,6]thiopyrano[4,3-c]pyrazoles
1361
crude products 8 which were purified by flash chromatography
on a silica gel column (60/0.040-0.063 mm) using petroleum
ether 60-80°C/ethyl acetate 7/3 as the eluting system.
General Procedure.
Phenylisocyanate (0.3 mL, 2.8 mmoles) was added dropwise
to a stirred solution of compound 10a (0.454 g, 2.4 mmoles) or
10b (0.487 g, 2.4 mmoles) in 15 mL of anhydrous tetrahydrofu-
rane, under nitrogen atmosphere. The reaction mixture was
stirred at room temperature for 24 hours adding during this time
three additional amounts of 0.1 mL of phenylisocyanate, then the
mixture was heated at 60 °C for 1 hour. After cooling, the sus-
pension obtained was filtered and the pinkish solid was collected
to give the crude products 6 which were purified by recrystalliza-
tion from ethanol (Table 1).
Compound 8a was obtained as an orange solid in 39.4% yield:
m.p. 60-63 °C; ir: 1670, 1600, 1200, 1130, 935, 840, 740, 675
-1
1
cm ; H nmr: δ 2.44 (s, 3H, 4'-CH ), 4.02 (s, 2H, CH S), 7.32
3
2
(dd, 1H, J =4.6 Hz, J =7.9 Hz, 6-H), 7.49 (s, 1H, CHO), 7.55
6,7
6,5
(d, 2H, J=8.2 Hz, 3',5'-PhH), 7.97 (d, 2H, J=8.2 Hz, 2',6'-PhH),
8.24 (dd, 1H, J =7.9 Hz, J =1.6 Hz, 5-H), 8.56 (dd, 1H,
5,6
5,7
+
J
=4.6 Hz, J =1.6 Hz, 7-H); ms: m/z 347 (2, M ), 149 (100).
7,6
7,5
Anal. Calcd. for C
H NO S : C, 55.33; H, 3.74; N, 4.03.
16 13 4 2
Found: C, 55.42; H, 3.55; N, 4.04
Compound 8b was obtained as an orange solid in 57.7% yield:
Acknowledgments.
m.p. 135-137°C; ir: 1670, 1605, 1195, 1070, 940, 835, 760, 670
This work was supported by grants from MIUR (Research
fund Cofin 2004).
-1 1
cm ; H nmr: δ 2.43 (s, 3H, 4'-CH ), 2.45 (s, 3H, 7-CH ), 3.98 (s,
3
3
2H, CH S), 7.17 (d, 1H, J=8.0 Hz, 6-H), 7.45 (s, 1H, CHO), 7.55
2
(d, 2H, J=8.3 Hz, 3',5'-PhH), 7.97 (d, 2H, J=8.3 Hz, 2',6'-PhH),
+
8.13 (d, 1H, J=8.0 Hz, 5-H); ms: m/z 361 (1, M ), 149 (100).
REFERENCES AND NOTES
Anal. Calcd. for C
H NO S : C, 56.51; H, 4.15; N, 3.88.
17 15 4 2
Found: C, 56.09; H, 4.51; N, 3.87
*
To whom correspondence should be addressed. E-mail:
marini@farm.unipi.it; Tel: +39-050-2219555; Fax: +39-050-2219605.
[1] E. Borowski, D. Shugar, in "Molecular Aspects of
Chemotherapy", Pergamon Press, New York, (1991).
[2] S. Neidle, Anti-Cancer Drug Des., 12, 433, (1997).
[3] S. Neidle, D. E. Thurston, In New Targets for Cancer
Chemotherapy, D.J. Kerr, P. Workman, Eds.; CRC Press: Boca Raton,
FL, (1994).
[4] L.P.G. Wakelin, M.J. Waring, "DNA intercalating agents" in
Comprehensive Medicinal Chemistry, Pergamon Press, New York,
(1990).
[5] B. C. Baguley, Anti-cancer Drug Des., 6, 1 (1991).
[6] U. Pindur, G. Fisher, Curr. Med. Chem., 3, 379 (1996), and
references therein.
[7] M. F. Brana, M. Cacho, A. Gradillas, A. Ramos, Curr.
Pharmaceutical Des., 7, 1745 (2001).
[8] S. D. G. Crow, C. Bailly, C. Garbay-Jaureguiberry, B.
Proques, B. R. Shaw, M. J. Waring, Biochemistry, 42, 8672, (2002).
[9] A. Da Settimo, A. M. Marini, G. Primofiore, F. Da Settimo,
S. Salerno, G. Viola, L. Dalla Via, S. Marciani Magno, Eur. J. Med.
Chem., 33, 685 (1998).
[10] A. Da Settimo, A. M. Marini, G. Primofiore, F. Da Settimo,
S. Salerno, L. Dalla Via, O. Gia, S. Marciani Magno, Farmaco, 56, 159
(2001).
[11] L. Dalla Via, O. Gia, S. Marciani Magno, A. Da Settimo, G.
Primofiore, F. Da Settimo, F. Simorini, A. M. Marini, Eur. J. Med.
Chem., 37, 475 (2002).
[12] G. Primofiore, A. M. Marini, F. Da Settimo, S. Salerno, D.
Bertini, L. Dalla Via, S. Marciani Magno, J. Heterocyclic Chem., 40,
783 (2003).
2-(Phenyl)- 3a, 2-(p-Methoxyphenyl)- 4a, 2-(Benzyl)-2,4-dihy-
dropyrido[3',2':5,6] thiopyrano[4,3-c]pyrazole 5a and 7-Methyl
derivatives 3b-5b.
General Procedure.
The required arylhydrazine hydrochloride (2.4 mmoles) was
added to a solution of 8a (0.694 g, 2.00 mmoles) or 8b (0.722 g,
2.00 mmoles) in 10 mL of anhydrous dimethylformamide. The
reaction mixture, under nitrogen atmosphere, was stirred at room
temperature for 2 hours, then refluxed at 100 °C for 15 hours.
After cooling, the suspension was poured into a saturated aque-
ous potassium carbonate solution and extracted with chloroform.
The organic layers were dried and evaporated under reduced
pressure to give desired crude pyrazoles 3, 4 and 5, which were
purified by flash chromatography on a silica gel column
(60/0.040-0.063 mm) using petroleum ether 60-80 °C/ethyl
acetate 3/2 as the eluting system (Table 1).
7-Methyl-3-[(4-methylphenyl)sulphonyl]oxymethylen-2,3-
Dihydrothiopyrano[2,3-b]pyridin-4(4H)-phenylhydrazone (9).
Phenylhydrazine hydrochloride (0.048 g, 0.332 mmole) was
added to a solution of 8b (0.100 g, 0.277 mmole) in 10 mL of
anhydrous dimethylformamide. The reaction mixture was stirred
at room temperature for 1 hour under nitrogen atmosphere then
poured into a saturated aqueous potassium carbonate solution.
The suspension obtained was filtered to give the desired crude
phenylhydrazone 9 as an orange solid (84 % yield), which was
analyzed without crystallization, due to its instability to warming
up. m.p. 75-77 °C; ir: 3400, 1580, 1350, 1230, 1065, 940, 830,
[13] A. Da Settimo, A. M. Marini, G. Primofiore, F. Da Settimo,
S. Salerno, F. Simorini, G. Pardi, C. La Motta, D. Bertini, J.
Heterocyclic Chem., 39, 1001 (2002).
[14] A. Da Settimo, G. Primofiore, F. Da Settimo, F. Simorini,
Drug Design and Discovery, 11, 307 (1994).
[15] L. Mosti, G. Menozzi, P. Schenone, J. Heterocyclic Chem.,
21, 361 (1984).
[16] G. Winters, A. Sala, B. Barone, E. Baldioli, J. Med. Chem.,
28, 934 (1985).
-1
1
760, 675 cm ; H nmr: δ: 2.43 (s, 3H, 4'-CH ), 2.44 (s, 3H, 7-
3
CH ), 3.99 (s, 2H, CH S), 6.82 (s, 1H, CHO), 7.12-7.23 (m, 3H,
3
2
PhH), 7.35-7.66 (m, 6H, PhH), 7.96 (d, 1H, J=8.3 Hz, 6-H), 8.21
+
(d, 1H, J=8.4 Hz, 5-H); ms: m/z 451 (2, M ), 162 (100), 225 (95).
Anal. Calcd. for C
H N O S : C, 61.19; H, 4.66; N, 9.31.
23 21 3 3 2
Found: C, 61.37; H, 4.55; N, 9.04
2-(N-Phenylcarboxamido)pyrido[2',3':5,6]thiopyrano[4,3-c]-
pyrazole 6a and 7-Methyl derivative 6b.
[17] I. K. Kostakis, P. Magiatis, N. Pouli, P. Marakos, A. Pierrè,
J. Med. Chem., 45, 2599 (2002).