Antimicrobial and antitumor activity of n-heteroimmine-1,2,3-dithiazoles and their transformation in triazolo-, imidazo-, and pyrazolopirimidines

Article abstract of DOI:10.1016/S0968-0896(01)00294-2

The reaction of Appel's salt with o-amino nitrile heterocycles 10-19 gave the corresponding 4-chloro-5-heteroimmine-1,2,3-dithiazole 20-29 which were evaluated for their antibacterial, antifungal and antitumor activity. Although all these N-heteroimines were devoid of significant antibacterial activity, they showed significant antifungal activity. Moreover, the same derivatives represent highly versatile intermediates in heterocyclic synthesis, in fact the pyrazoleimino dithiazoles 20-26 can be converted in one step into 2-cyano derivatives of the corresponding 4-methoxy-pyrazolo[3,4-d]pyrimidines 30-35 by sodium methoxide in refluxing methanol. This provides a general and attractive route to 4-methoxy-6-cyano pyrazolo[3,4-d]pyrimidines from 1-substituted 5-amino pyrazoles 10-19 in two simple steps. Finally, the isosteric replacement of the pyrazole ring atoms to give the imidazole[3,4-d]pyrimidine and triazole [4,5-d]pyrimidine ring systems were examined. Copyright

Full text of DOI:10.1016/S0968-0896(01)00294-2

Bioorganic & Medicinal Chemistry 10 (2002) 449–456
Antimicrobial and Antitumor Activity of N-Heteroimmine-1,2,3-
dithiazoles and Their Transformation in Triazolo-, Imidazo-, and
Pyrazolopirimidines
Pier Giovanni Baraldi,a,* Maria Giovanna Pavani,a Maria del Carmen Nunez,a
Patrizia Brigidi,b Beatrice Vitali,b Roberto Gambaric and Romeo Romagnolia
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy
bDipartimento di Scienze Farmaceutiche, Via Belmeloro, 40126 Bologna, Italy
`
Dipartimento di Biochimica e Biologia Molecolare, Universita di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy
c
Received 9 July 2001; accepted 15 August 2001
Abstract—The reaction of Appel’s salt with o-amino nitrile heterocycles 10–19 gave the corresponding 4-chloro-5-heteroimmine-
1,2,3-dithiazoles 20–29 which were evaluated for their antibacterial, antifungal and antitumor activity. Although all these N-het-
eroimines were devoid of significant antibacterial activity, they showed significant antifungal activity. Moreover, the same deriva-
tives represent highly versatile intermediates in heterocyclic synthesis, in fact the pyrazoleimino dithiazoles 20–26 can be converted
in one step into 2-cyano derivatives of the corresponding 4-methoxy-pyrazolo[3,4-d]pyrimidines 30–35 by sodium methoxide in
refluxing methanol. This provides a general and attractive route to 4-methoxy-6-cyano pyrazolo[3,4-d]pyrimidines from 1-sub-
stituted 5-amino pyrazoles 10–19 in two simple steps. Finally, the isosteric replacement of the pyrazole ring atoms to give the imi-
dazole[3,4-d]pyrimidine and triazole [4,5-d] pyrimidine ring systems was examined. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
4,5-Dichloro-1,2,3-dithiazolium chloride (Appel’s salt,
1), easily prepared from chloroacetonitrile and disulfur
dichloride,1 is the most studied derivative of the 1,2,3-
dithiazolium system2 and reacts with proton-active
compounds by the nucleophilic substitution of the
chlorine at carbon atom C-5. Substrates like anilines
with electron-withdrawing as well as electron-releasing
substituents in the phenyl ring react rapidily with
Appel’s salt in the presence of pyridine to give very
stable 5-(arylimino)-4-chloro-5H-1,2,3-dithiazoles with
general formula 21 in very high yield. These heterocyclic
compounds are interesting because of their usefulnesss
as acaricides, herbicides and for their antimicrobial and
antifungal activity,3 and also because they are useful
synthetic intermediates.2
In previous works, Appel1 found that anilino com-
pounds 2 with X=H, cyclised from an ortho-carbon
onto sulfur when heated to give the 2-cyanobenzothia-
zole 32,4 with liberation of the other sulfur atom and
hydrogen chloride. For the anilines bearing nucleophilic
neighbouring group in the ortho position of the aro-
matic ring, when X=OH the imine 2 was transformed
both into the 2-cyanobenzoxazole 4 and dithiazolo-
benzoxazine 5,1,2 while when X=COOH the derivative 2
was converted into the cyanobenzoxazinone 6 or in the
cyanobenzothiazinone 7.5 When X=NHR0(R0=alkyl or
aryl), the compound 2 rearranged giving the cyano-
benzimidazole with general formula 86 by heating or in
the presence of a catalytic amount of hydrogen chloride.
*Corresponding author. Tel.: +39-0532-291293; fax: +39-0532-
291296; e-mail: pgb@dns.unife.it
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00294-2

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P. G. Baraldi et al. / Bioorg. Med. Chem. 10 (2002) 449–456
The process of cyclisation which led to the synthesis of
4, 7 and 8 involved the attack of the nucleophilic ortho-
substituent (X=OH, COOH and NHR0) at the imino-
carbon to form a new heterocyclic ring with loss of both
sulfur atoms from the dithiazole ring. The treatment
with alkoxides of the imine with general formula 2
bearing a cyano group in the ortho position (X=CN) of
the N-aryl group allowed the formation of 4-alkoxy-
quinazolines 97 (Scheme 1).
by the reaction of 4,5-dichloro-1,2,3-dithiazolium chlo-
ride 1 with the o-aminonitrile heterocycles 10–19 in
methylene chloride (DCM) at room temperature, after
which pyridine (2 equiv) was added to the reaction
mixture before work-up. The reaction of pyrazolimines
20–25 with sodium methoxide (2 equiv) in methanol at
reflux to close the pyrimidine ring afforded the 1-alkyl
(aryl)-2-cyano-pyrazole[3,4-c]pyrimidines
30–35
in
acceptable yield (52–72%) as outlined in Scheme 2. The
general method of synthesis of pyrazolo[3,4-d]pyr-
imidines from a pyrazole intermediate has been further
extended. In the same way, the imidazoleimine 26 and
triazoleimine 28 were treated with sodium methoxide in
methanol to give the corresponding imidazole [3,4-
c]pyrimidine 36 and triazole [4,5-c]pyrimidine 37 in 52
and 69% yields, respectively, confirmed by analysis and
spectroscopy. The 2-cyano group can be removed by
hydrolysis and decarboxylation with hot concentrated
hydrochloric acid, converted into corresponding
amides, esters, amidines and ketones and can be dis-
placed by nucleophiles such as methoxide and ethoxide
ions.
In this article, we have studied the reaction of Appel’s
salt 1 with amino heterocycles 10–19 having a nucleo-
philic cyano group in the ortho position, to give the
expected 4-chloro-5-heteroimmine-1,2,3-dithiazoles 20–
29, which were then evaluated for their antibacterial,
antifungal and antitumor activity. Although the synth-
esis of 4-chloro-5-heteroimine-dithiazoles has been
already reported,8 only a limited series of examples have
been described, and neither the biological activity nor
the possible chemical conversion was investigated. For
these reasons, we have further explored this method
increasing the range of heterocyclic imines that react
with Appel’s salt, introducing a nucleophilic neigh-
bouring cyano group in the ortho position of the aro-
matic heterocyclic ring in the hope of enhancing the
biological activity of the products 20–29 obtained. Since
the compounds 20–29 are so readily available, we have
also explored their use in heterocyclic synthesis heating
these derivatives (with the exception of 27) in a mixture
of sodium methoxide in boiling methanol, to give the
cyclised compounds 30–37.
Results and Discussion
The in vitro antimicrobial activity of the synthesized
compounds 20–29 was investigated against several
pathogenic representative Gram-negative bacteria
(Pseudomonas aeruginosa ATCC 9027, Escherichia coli
ATCC 11105 and Proteus mirabilis MB81) and Gram-
positive bacteria (Enterococcus faecalis ATCC 8043 and
Staphylococcus aureus ATCC 29213). No antibacterial
activity was showed by any of the assayed compounds:
all MIC values for the Gram-negative bacteria tested
were >100 mg/mL, as well as high MIC values, ran-
ging from 75 to >100 mg/mL, were found for the
Chemistry
The 4-chloro-5-heteroimine-5H-1,2,3-dithiazoles 20–29
were prepared clean and in acceptable yield (38–73%)
Scheme 1.

P. G. Baraldi et al. / Bioorg. Med. Chem. 10 (2002) 449–456
451
Gram-positive bacteria (Table 1). None of the deriva-
tives 20–29 showed a better spectrum of activity than
the reference drug ampicillin.
activity against fungi. 20, 21 and 23 products appeared
to be scarcely active only against some microorganisms
such as Penicillium and C. utilis, whereas 22 and 29
products presented the lowest antifugal activities against
all the fungi tested. It is interesting to note that for the
compounds 20–25 obtained from o-amino nitriles 13–18
which possess the same heterocyclic ring, the biological
activity is affected by substituents on the N1-position of
the pyrazole ring.
The in vitro antifungal activity of compounds 20–29
was performed against yeasts (Candida albicans ATCC
10231, Candida utilis ATCC 9950, Candida lipolytica
CBS 6124, Saccharomyces cerevisiae ATCC 26785,
Pichia stipitis CBS 5776) and moulds (Aspergillus niger
L32 and Penicillium sp.) and the results are summarized
in Table 2. All the derivatives showed significant anti-
fungal activity against the yeasts tested. The compounds
24–28 exhibited the highest antifungal activity, showing
good MIC values included in the range of 10–50 mg/mL.
Particularly, the efficacy of these products against the
moulds can be compared with that showed by ampho-
tericin B, used as reference compound for inhibitory
Based on their fungicidal activity, the heteroimine 20–29
may represent interesting leads for further study and, as
previously reported,3e it seems likely that the 1,2,3-
dithiazole ring, having nucleophilic centers at the S1,
S2, C4 and C5 positions, could be implicated in inhibi-
tory activity against fungi acting as a potent inhibitor of
some enzymes like serine proteases.3c,d
Scheme 2.
Table 1. Minimum Inhibitory Concentration (MIC) against some strains of Gram–positive and negative bacteria
Compound
MIC (mg/mL)
Bacteria tested
Pseudomonas aeruginosa
ATCC 9027
Escherichia coli
ATCC 11105
Proteus mirabilis
MB
Enterococcus faecalis
ATCC 8043
Staphylococcus aureus
ATCC 29213
20
21
22
23
24
25
26
27
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.5
100
100
100
75
100
75
100
100
75
>100
0.5
28
29
Ampicillin
MIC, minimum inhibitory concentration represents the mean from dose–response curves of at least three experiments.

452
P. G. Baraldi et al. / Bioorg. Med. Chem. 10 (2002) 449–456
Table 2. Minimum Inhibitory Concentration (MIC) against some fungi
Compound
MIC (mg/mL)
Fungi tested
Candida albicans Candida utilis Candida lipolytica Saccharomyces cerevisiae Pichia stipitis Aspergillus niger Penicilliumsp
CBS 5776
ATCC 10231
ATCC 9950
CBS 6124
ATCC 26785
20
21
22
23
24
25
26
27
505025
50
>100
25
25
25
25
25
5025
100
2.5
25
25
25 0
10
50
>100
25
>100
>100
100
5025
25
25
5025
25
100
2.5
25
100
25
25
50
25
25
25
25
100
100
25
25
25
1025
25
25
5050
50
25
25
25
25
25
1025
50
25
25
25
5025
28
29
100
20102.5
100
100
100
100
Amphotericin B
20 20
MIC, minimum inhibitory concentration represents the mean from dose–response curves of at least three experiments.
Table 3. In vitro biological effects on L1210and K562 cell lines
receptor9 and the anticonvulsant activity10 of the same
compounds has already appeared in several publica-
tions.
Compound
IC50 (mM)
L1210K562
20
21
22
23
24
25
26
27
5
7.5
8
5
3
5
3
5
7
5
5
5
3
5
5
4
8
8
7
Experimental
Chemistry
All reactions were carried out under Argon atmosphere,
unless otherwise described. Standard syringe techniques
were applied for transferring anhydrous solvents. Reac-
tion courses and product mixtures were routinely mon-
itored by thin-layer chromatography on silica gel
(precoated F254 Merk plates), the spots were examined
28
29
8
1020
Distamycin A
IC50, 50% inhibitory concentration represents the mean from dose–
response curves of at least three experiments.
1
with UV light and visualized with aqueous KMnO4. H
NMR spectra were recorded in the given solvent with a
Bruker AC 200 spectrometer. Chemical shifts are
reported as (d) values in parts per million. The splitting
pattern abbreviations are as follows: s (singlet), d
(doublet), dd (double doublet), t (triplet), b (broad), and
m (multiplet). Melting points (mp) were determined
using a Buchi–Tottoli apparatus and are uncorrected.
All products reported showed 1H NMR spectra in
agreement with the assigned structures. Elemental ana-
lyses, conducted by the Microanalytical Laboratory of
the Chemistry Department of the University of Ferrara,
were within 0.4% of the theorical values calculated for
C, H, S, Cl and N. Column chromatography was car-
ried out Merck silica gel (230–240 mesh). All com-
pounds obtained commercially were used without
further purification. Organic solutions were dried over
anhydrous MgSO4. Dioxane was distilled from calcium
hydride, methylene chloride was distilled from calcium
chloride.
The antiproliferative activity of compounds 20–29 has
been tested in vitro on human myeloid leukemia K562
and L1210murine leukemia cell lines and compared to
the antiproliferative effects of the natural product dis-
tamycin A. All the imines 20–29 are active at a con-
centration ranging from 3 to 10 mM and they retain
antiproliferative activities comparable to those exhibited
by distamycin A. The results obtained are summarized
in Table 3.
In conclusion, we have described the antimicrobial and
antitumor activity of a series of 4-chloro-5-hetero-
immine-dithiazoles 20–29 and subsequently we have
also reported a general and attractive route to 2-cyano-
4-methoxy-pyrazole[3,4-d]pyrimidines 30–35 from 1-
substituted-4-cyano-5-amino pyrazoles in two simple
steps. Besides alkosides, this method could be extended
to the addition of other nucleophiles to the cyano group
of the pyrazoleimines 20–25. The conversation of 26
and 28 into 36 and 37, respectively, is worthy of further
investigation. As regards the biological interest of com-
pounds 30–37, biological testing is now in progress and
a complete report will appear elsewhere. Moreover, the
binding affinity of triazolo[3,4-d]- and imidazo[4,5-
d]pyrimidines for the corticotropin-releasing factor
Synthesis of 5-amino-1-phenethylimidazole-4-carbonitrile
(15). Following an already reported procedure;11 dry
ammonia was passed through a suspension of amino-
malononitrile toluene-p-sulphonate (3 g, 15.8 mmol) in
dry acetonitrile (150mL) with stirring and cooling for
20min. Precipitated amonium toluene- p-sulphonate was
removed and the filtrate evaporated to ca. 60mL, then

P. G. Baraldi et al. / Bioorg. Med. Chem. 10 (2002) 449–456
453
triethyl orthoformate (2.65 mL, 15.8 mmol) was added
and the solution refluxed for 10min. The cooled solu-
tion with 2-phenethylamine (2 mL, 15.8 mmol) was set
aside at room temperature overnight. The solution was
then evaporated to a solide residue which was submitted
to purification by flash chromatography (eluent
CH2Cl2/EtOAc 8:2) to give the compound 15 (1.33 g,
39.5%) recrystallized from aqueous ethanol as brown
needles. Mp 197–198 ꢀC (from petroleum ether); IR
(KBr) cmꢁ1: 3352, 3160, 2925, 2210, 1657, 1452, 700;
1H NMR (DMSO-d6 ) d 2.91 (t, 2H, J=7.3 Hz), 4.02
(t, 2H, J=7.3 Hz), 6.25 (bs, 2H), 7.00 (s, 1H), 7.23 (m,
5H).
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-(1-phenyl-4-cyano-
pyrazol-5-yl)-amine (21). Treatment of 1-phenyl-4-
cyano-5-aminopyrazole13 11 (920mg, 5 mmol) with 1 (1
g, 5 mmol) and pyridine (0.8 mL) in dichloromethane
(20mL), followed by column chromatography (EtOAc/
petroleum ether 1:9, 2:8, and then 1:1) furnished 21 as
green needles (1.06 g, 66% of yield). Mp 195–198 ꢀC
(from petroleum ether); IR (KBr) cmꢁ1: 3447, 2218,
1
1578, 1516, 1497, 1403, 966, 869, 778, 683; H NMR
(CDCl3) d 7.44 (m, 3H), 7.69 d, J=7.6 Hz, 2H), 7.98 (s,
1H). Anal. calcd for C12H6ClN5S2: C, 45.07; H, 1.89;
Cl, 11.09; N, 21.90; S, 20.05; found: C, 44.98; H, 1.81;
Cl, 10.96; N, 21.79; S, 19.96.
Synthesis of 5-amino-1-(2,4-dichlorophenethyl)imidazole-
4-carbonitrile (16). Following the procedure reported
above, starting from aminomalononitrile toluene-p-sul-
phonate (3 g, 15.8 mmol) in dry acetonitrile (150mL),
triethyl orthoformate (2.65 mL, 15.8 mmol) and 2-(2,4-
dichlorophenyl)-ethyl-1-amine (2.38 mL, 15.8 mL), after
work-up, the compound 16 (1.23 g, 27.6%) purified by
flash chromatography (eluent CH2Cl2/EtOAc 8:2) was
recrystallized from aqueous ethanol as green needles.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-(1-benzyl-4-cyano-
pyrazol-5-yl)-amine (22). Treatment of 1-benzyl-4-
cyano-5-aminopyrazole14 12 (990mg, 5 mmol) with 1 (1
g, 5 mmol) and pyridine (0.8 mL) in dichloromethane
(20mL), followed by column chromatography (EtOAc/
petroleum ether 2:8) furnished 22 as brown needles (1.22
g, 73% of yield). Mp 168–171 ꢀC (from petroleum
ether); IR (KBr) cmꢁ1: 3447, 2221, 1583, 1522, 1466,
768, 737, 694; 1H NMR (CDCl3) d 5.31 (s, 2H), 7.28 (m,
5H), 7.83 (s, 1H). Anal. calcd for C14H10ClN5S2: C,
46.77; H, 2.42; Cl, 10.62; N, 20.98; S, 19.21; found: C,
46.68; H, 2.36; Cl, 10.54; N, 20.91; S, 19.12.
Mp 156–158 ꢀC (from petroleum ether); IR (KBr) cmꢁ1
:
3343, 3146, 2910, 2200, 1675, 1435, 710; 1H NMR
(DMSO-d6) d 2.75 (t, 2H, J=7.2 Hz), 4.11 (t, 2H,
J=7.2 Hz), 6.22 (bs, 2H), 7.00 (s, 1H), 7.04 (d, J=7.2
Hz, 1H), 7.22 (d, J=7.2 Hz, 1H), 7.34 (s, 1H).
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-(1-phenethyl-4-cyano-
pyrazol-5-yl)-amine (23). Treatment of 1-phenethyl-4-
cyano-5-aminopyrazole15 13 (1.06 g, 5 mmol) with 1 (1
g, 5 mmol) and pyridine (0.8 mL) in dichloromethane
(20mL), followed by column chromatography (EtOAc/
petroleum ether 2:8) furnished 23 as orange needles
(1.06 g, 61% of yield). Mp 120–122 ꢀC (from petroleum
ether); IR (KBr) cmꢁ1: 3435, 2216, 1583, 1512, 1430,
Synthesis of 1-o-xylyl-4-cyano-5-amino-1H-1,2,3-triazole
(18). Following a procedure previously reported,12 this
compound has been obtained as a white powder. Mp
180–182 ꢀC. IR (KBr) cmꢁ1: 3341, 3183, 2238, 1635,
1553, 752; 1H NMR (DMSO-d6) d 2.35 (s, 3H), 3.36 (bs,
2H), 5.36 (bs, 2H), 6.68 (d, 1H, J=7.15 Hz), 7.08 (s,
1H), 7.18 (m,2H)
1
1188, 1154, 866, 698; H NMR (CDCl3) d 3.06 (t, J=6
Hz, 2H), 4.46 (t, J=6.4 Hz, 2H), 6.93 (m, 2H), 7.06 (m,
3H), 7.04 (s, 1H). Anal. calcd for C14H10ClN5S2: C,
48.34; H, 2.90; Cl, 10.19; N, 20.13; S, 18.44; found: C,
48.26; H, 2.81; Cl, 10.08; N, 20.06; S, 18.35.
General procedure for the reaction of 4,5-dichloro-1,2,3-
dithiazolium chloride (Appel’s salt, 1) with heterocyclic
amines 10–19
To a solution of the heterocyclic amine derivative 10–19
(5 mmol) in dichloromethane (20mL) was added 4,5-
dichloro-1,2,3-dithiazolium chloride 1. The mixture was
stirred at room temperature for 5 h, then pyridine (5
equiv, 10mmol, 0.8 mL) was added and the mixture was
stirred for a further 2 h. After filtration, the product was
purified by column chromatography with a mixture of
ethyl acetate and petroleum ether as eluent.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-[1-(3-phenyl-propyl)-4-
cyano-pyrazol-5-yl)-amine (24). Treatment of 1-(3-phe-
nylpropyl)-4-cyano-5-aminopyrazole15 14 (1.13 g, 5
mmol) with 1 (1 g, 5 mmol) and pyridine (0.8 mL) in
dichloromethane (20mL), followed by column chroma-
tography (EtOAc/petroleum ether 3:7) furnished 24 as
green needles (1.25 g, 69% of yield). Mp 155–160 ꢀC
(from petroleum ether); IR (KBr) cmꢁ1: 3431, 2238,
1739, 1554, 1508, 1484, 1260, 884, 769; 1H NMR
(CDCl3) d 0.78 (m, 2H), 2.12 (t, J=7.4 Hz, 2H), 4.07 (t,
J=7 Hz, 2H), 7.16 (m, 5H), 7.76 (s, 1H). Anal. calcd for
C15H12ClN5S2: C, 49.79; H, 3.34; Cl, 9.80; N, 19.35; S,
17.72; found: C, 49.68; H, 3.28; Cl, 9.71; N, 19.28; S,
17.66.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-(1-methyl-4-cyano-
pyrazol-5-yl)-amine (20). Treatment of 1-methyl-4-
cyano-5-aminopyrazole13 10 (610mg, 5 mmol) with 1 (1
g, 5 mmol) and pyridine (0.8 mL) in dichloromethane
(20mL), followed by column chromatography (EtOAc/
petroleum ether 2:8, 3:7, 1:1 and then only EtOAc)
furnished 20 as brown needles (630mg, 50% of yield).
Mp 209–212 ꢀC (from petroleum ether); IR (KBr)
cmꢁ1: 3436, 2227, 1584, 1518, 874, 766; 1H NMR
(CDCl3) d 3.83 (s, 3H), 7.81 (s, 1H). Anal. calcd for
C7H4ClN5S2: C, 32.62; H, 1.56; Cl, 13.76; N, 27.17; S,
24.88; found: C, 32.55; H, 1.47; Cl, 13.68; N, 27.03; S,
24.78.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-[1-t-butyl-4-cyano-
pyrazol-5-yl)-amine (25). Treatment of 1-t-butyl-4-
cyano-5-aminopyrazole15 15 (820mg, 5 mmol) with 1 (1
g, 5 mmol) and pyridine (0.8 mL) in dichloromethane
(20mL), followed by column chromatography (EtOAc/
petroleum ether 1:1) furnished 25 as brown needles (1.02
g, 68% of yield). Mp 142–145 ꢀC (from petroleum

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P. G. Baraldi et al. / Bioorg. Med. Chem. 10 (2002) 449–456
ether); IR (KBr) cmꢁ1: 3448, 2225, 1572, 1449, 1354,
1235, 1210, 992, 880; H NMR (CDCl3) d 1.63 (s, 9H),
7.72 (s, 1H). Anal. calcd for C10H10ClN5S2: C, 40.06; H,
3.36; Cl, 11.83; N, 23.36; S, 21.39; found: C, 40.00; H,
3.29; Cl, 11.77; N, 23.31; S, 21.31.
General procedure for the reaction of N-
heterocyclicimino-1,2,3-dithiazoles (20–26 and 28) with
sodium methoxide
1
A stirred mixture of iminodithiazole (20–26 and 28) (1
mmol) and sodium methoxide (2 mmol) in methanol (6
mL) was heated at reflux for 36–60h. After this time,
the solution was filtered, the solvent was evaporated
from the filtrate and the residue purified by column
chromatography.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-(1-phenethyl-4-cyano-
imidazol-5-yl)-amine (26). Treatment of 1-phenethyl-4-
cyano-5-aminoimidazole 16 (1.06 g, 5 mmol) with 1 (1 g,
5 mmol) and pyridine (0.8 mL) in dichloromethane (20
mL), followed by column chromatography (EtOAc/pet-
roleum ether 7:3) furnished 26 as yellow needles (938
mg, 55% of yield). Mp 85–87 ꢀC (from petroleum
ether); IR (KBr) cmꢁ1: 3437, 2219, 1576, 1508, 1174,
4-Methoxy-1-methyl-1H-pyrazolo[3,4-d]pyrimidine-6-car-
bonitrile (30). Following the general procedure reported
above, treatment of 20 (258 mg, 1 mmol) with MeONa
(108 mg, 2 mmol) in MeOH (6 mL) for 48 h followed by
column chromatography (EtOAc/petroleum ether 1:1)
gave the compound 30 (230mg, 61% of yield) as a white
solid. Mp 192–195 ꢀC, IR (KBr) cmꢁ1: 3436, 1595, 1559,
1369, 1080, 790; 1H NMR (CDCl3) d 4.14 (s, 3H), 4.22 (s,
3H), 8.12 (s, 1H). Anal. calcd for C8H7N5O: C, 50.79; H,
3.73; N, 37.02; found: C, 50.71; H, 3.66; N, 36.93.
1
764, 700; H NMR (CDCl3) d 3.00 (t, J=6.4 Hz, 2H),
4.28 (t, J=6.4 Hz, 2H), 6.97 (m, 3H), 7.15 (m, 2H), 7.44
(s, 1H). Anal. calcd for C14H10ClN5S2: C, 48.34; H,
2.90; Cl, 10.19; N, 20.13; S, 18.44; found: C, 48.28; H,
2.83; Cl, 10.09; N, 20.05; S, 18.38.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-[1-[2-(2,4-dichloro-
phenyl)-ethyl]-4-cyano-imidazol-5-yl)-amine (27). Treat-
ment of 1-[2-(2,4-dichloro-phenyl)-ethyl]-4-cyano-5-
aminoimidazole 17 (1.44 g, 5 mmol) with 1 (1 g, 5
mmol) and pyridine (0.8 mL) in dichloromethane (20
mL), followed by column chromatography (EtOAc/pet-
roleum ether 3:7) furnished 27 as brown needles (1.3 g,
63% of yield). Mp 77–79 ꢀC (from petroleum ether); IR
(KBr) cmꢁ1: 3435, 2219, 1577, 1474, 1171, 823; 1H
NMR (CDCl3) d 3.13 (t, J=6.4 Hz, 2H), 4.32 (t, J=6.2
Hz, 2H), 6.81 (d, J=8.2 Hz, 1H), 7.04 (d, J=9.8 Hz, 1H),
7.28 (m, 1H), 7.47 (s, 1H). Anal. calcd for C14H16Cl3N5S2:
C, 40.35; H, 1.93; Cl, 25.52; N, 16.81; S, 15.39; found: C,
40.28; H, 1.85; Cl, 25.47; N, 16.74; S, 15.31.
4-Methoxy-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-6-car-
bonitrile (31). Following the general procedure reported
above, treatment of 21 (320mg, 1 mmol) with MeONa
(108 mg, 2 mmol) in MeOH (6 mL) for 36 h followed by
column chromatography (EtOAc/petroleum ether 2:8)
gave the compound 31 (168 mg, 67% of yield) as a white
solid. Mp 120–123 ꢀC, IR (KBr) cmꢁ1: 2924, 1592, 1550,
1483, 1372, 1193, 762. 1H NMR (CDCl3) d 4.25 (s, 3H),
7.44 (t, J=7.2 Hz, 1H), 7.57 (t, J=7.56 Hz, 2H), 8.16
(d, J=8 Hz, 2H), 8.30(s, 1H). Anal. calcd for
C13H9N5O: C, 62.15; H, 3.61; N, 27.87; found: C, 62.08;
H, 3.53; N, 27.79.
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-[1--(2-methyl-benzyl)-
4-cyano-1,2,3-triazol-5-yl)-amine (28). Treatment of 1-o-
xylyl-4-cyano-5-amino-1,2,3-triazole 18 (1.06 g, 5 mmol)
with 1 (1 g, 5 mmol) and pyridine (0.8 mL) in dichloro-
methane (20mL), followed by column chromatography
(EtOAc/petroleum ether 2:8) furnished 28 as yellow
needles (1 g, 58% of yield). Mp 146–149 ꢀC (from pet-
roleum ether); IR (KBr) cmꢁ1: 3448, 2240, 1739, 1554,
4-Methoxy-1-benzyl-1H-pyrazolo[3,4-d]pyrimidine-6-car-
bonitrile (32). Following the general procedure reported
above, treatment of 22 (334 mg, 1 mmol) with MeONa
(108 mg, 2 mmol) in MeOH (6 mL) for 48 h followed by
column chromatography (EtOAc/petroleum ether 3:7)
gave the compound 32 (188 mg, 71% of yield) as a white
solid. Mp 116–118 ꢀC, IR (KBr) cmꢁ1: 1595, 1561, 1473,
1395, 1375, 1248, 1059, 971, 791, 719.1H NMR (CDCl3)
d 4.19 (s, 3H), 5.63 (s, 2H), 7.31 (m, 5H), 8.11 (s, 1H).
Anal. calcd for C14H11N5O: C, 63.39; H, 4.18; N, 26.40;
found: C, 63.31; H, 4.11; N, 27.69.
1
1485, 1260, 884, 769. H NMR (CDCl3) d 2.42 (s, 3H),
5.53 (s, 2H), 7.17 (m, 4H). Anal. calcd for
C13H9ClN6S2: C, 44.76; H, 2.60; Cl, 10.16; N, 24.09; S,
18.39; found: C, 44.71; H, 2.52; Cl, 10.08; N, 24.00; S,
18.31.
4-Methoxy-1-phenethyl-1H-pyrazolo[3,4-d]pyrimidine-6-
carbonitrile (33). Following the general procedure
reported above, treatment of 23 (348 mg, 1 mmol) with
MeONa (108 mg, 2 mmol) in MeOH (6 mL) for 48 h
followed by column chromatography (EtOAc/petro-
leum ether 1:1) gave the compound 33 (188 mg, 71% of
(4-Chloro-[1,2,3]dithiazol-5-ylidene)-(6-methyl-3-cyano-
4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-2-yl)-amine (29).
Treatment of 2-amino-3-cyano-6-methyl-4,5,6,7-tetra-
hydro-thieno[2,3-c]pyridine16 19 (965 mg, 5 mmol) with
1 (1 g, 5 mmol) and pyridine (0.8 mL) in dichloro-
methane (20mL), followed by column chromatography
(EtOAc/petroleum ether 8:2) furnished 29 as green nee-
dles (625 mg, 38% of yield). Mp 151–153 ꢀC (from pet-
roleum ether); IR (KBr) cmꢁ1: 3334, 2199, 1734, 1694,
1559, 1526, 1457, 1374, 1251, 1150, 1047, 792; 1H NMR
(CDCl3) d 2.46 (m, 2H), 2.58 (m, 2H), 2.80(s, 3H), 3.67
(s, 2H). Anal. calcd for C11H9ClN4S3: C, 40.17; H, 2.76;
Cl, 10.78; N, 17.04; S, 29.25; found: C, 40.08; H, 2.71;
Cl, 10.72; N, 16.96; S, 29.18.
yield) as a white solid. Mp 98–100 ꢀC, IR (KBr) cmꢁ1
:
1
1600, 1560, 1476, 1397, 1378, 1305, 968, 788, 696; H
NMR (CDCl3) d 3.25 (t, J=7.4 Hz, 2H), 4.19 (s, 3H),
4.72 (t, J=7.8 Hz, 2H), 7.22 (m, 5H), 8.11 (s, 1H). Anal.
calcd for C15H13N5O: C, 64.51; H, 4.69; N, 25.08;
found: C, 64.42; H, 4.62; N, 25.00.
4-Methoxy-1-(3-phenyl-propyl)-1H-pyrazolo[3,4-d]pyri-
midine-6-carbonitrile (34). Following the general proce-
dure reported above, treatment of 24 (362 mg, 1 mmol)

P. G. Baraldi et al. / Bioorg. Med. Chem. 10 (2002) 449–456
455
with MeONa (108 mg, 2 mmol) in MeOH (6 mL) for 42
h followed by column chromatography (EtOAc/petro-
leum ether 3:7) gave the compound 34 (164 mg, 54% of
dilution method. Mueller–Hinton Agar (BBL) and
Sabouraud Dextrose Agar (Difco) were employed for
bacterial and fungal growth, respectively. Stock solu-
tions of tested compounds were prepared in N,N-dime-
thylformamide (DMF). Inocula containing 105–106 cfu/
mL of bacteria and 104–105 cfu/mL of fungi were pre-
pared from broth cultures in log phase growth. Bacterial
and fungal plates were made in triplicate and incubated
at 37 and 28 ꢀC for 48 and 72 h, respectively.
yield) as a white solid. Mp 93–95 ꢀC, IR (KBr) cmꢁ1
:
2925, 1593, 1557, 133, 1244, 962, 790. 1H NMR
(CDCl3) d 2.94 (t, J=7.2 Hz, 2H), 2.64 (t, J=7.8 Hz,
2H), 4.20(s, 3H), 4.51 (t, J=7 Hz, 2H), 7.23 (m, 5H),
8.10(s, 1H). Anal. calcd for C 16H15N5O: C, 65.52; H,
5.15; N, 23.88; found: C, 65.46; H, 4.53; N, 23.81.
4-Methoxy-1-t-butyl-1H-pyrazolo[3,4-d]pyrimidine-6-car-
bonitrile (35). Following the general procedure reported
above, treatment of 25 (300 mg, 1 mmol) with MeONa
(108 mg, 2 mmol) in MeOH (6 mL) for 48 h followed by
column chromatography (EtOAc/petroleum ether 6:4)
gave the compound 35 (120mg, 52% of yield) as a white
solid. Mp 100–103 ꢀC, IR (KBr) cmꢁ1: 2995, 1598, 1546,
Inhibition of in vitro tumour cell growth
The human chronic myeloid leukaemia K562 and the
murine lymphocytic L1210leukemia cell lines were
grown in vitro as a stationary suspension culture in
RPMI 1640medium (GIBCO BRL) supplemented with
10% FCS (GIBCO BRL), 2 mM l-glutamine (GIBCO
BRL), 100 U/mL penicillin and 100 mg/mL streptomy-
cin. To determine survival after drug exposure, expo-
nentially growing K562 and Jurkat cells were cultured
in the presence of various concentrations of the drugs
and the antiproliferative activity of the drugs was eval-
uated after 4 days by counting surviving cells in a model
ZBI Coulter Counter (Coulter Electronics, Hialeah, FL,
USA). Results were expressed as IC50 (dose causing
50% inhibition of cell growth in treated cultures relative
to untreated controls). All experiments were repeated at
least twice. For each drug concentration, duplicate cell
cultures were used. Vehicle or solvent controls were run
with each experiment. All the compounds tested were
dissolved in DMSO at 1 mg/mL immediately before the
use and diluted just before addition to the cell culture.
1
1373, 1240, 1043, 963, 788; H NMR (CDCl3) d 1.81 (s,
9H), 4.19 (s, 3H), 8.05 (s, 1H). Anal. calcd for
C11H13N5O: C, 57.13; H, 5.67; N, 30.28; found: C,
57.04; H, 5.59; N, 30.19.
6-Methoxy-9-phenethyl-9H-purine-2-carbonitrile
(36).
Following the general procedure reported above, treat-
ment of 26 (348 mg, 1 mmol) with MeONa (108 mg, 2
mmol) in MeOH (6 mL) for 48 h followed by column
chromatography (EtOAc/petroleum ether 6:4) gave the
compoundꢀ36 (140mg, 52% of yield) as a white solid.
Mp 85–87 C, IR (KBr) cmꢁ1: 1612, 1556, 1388, 1356,
1
1290, 945, 777, 692. H NMR (CDCl3) d 3.18 (t, J=7.2
Hz, 2H), 4.21 (s, 3H), 4.52 (t, J=7.2 Hz, 2H), 7.04 (m,
2H), 7.28 (m, 3H), 7.68 (s, 1H). Anal. calcd for
C15H13N5O: C, 64.51; H, 4.69; N, 25.08; found: C,
64.43; H, 4.59; N, 24.98.
Acknowledgements
7-Methoxy-3-(2-methyl-benzyl)-3H-[1,2,3]triazolo[4,5-d]pyr-
imidine-5-carbonitrile (37). Following the general proce-
dure reported above, treatment of 28 (348 mg, 1 mmol)
with MeONa (108 mg, 2 mmol) in MeOH (6 mL) for 60
h followed by column chromatography (EtOAc/petro-
leum ether 6:4) gave the compound ꢀ36 (194 mg, 69% of
2924, 1590, 1507, 1376, 1250, 1093, 1053, 739; 1H NMR
(CDCl3) d 2.53 (s, 3H), 4.21 (s, 3H), 5.71 (s, 2H), 7.20
(m, 4H). Anal. calcd for C14H12N6O: C, 59.99; H, 4.32;
N, 29.98; found: C, 59.88; H, 4.25; N, 29.92.
We wish to thank Pharmacia & Upjohn for providing
distamycin A and Ministero Universita e Ricerca Sci-
entifica (MURST) (60%) for generous financial support
of this work. Maria del Carmen Nunez is the recipient of
a Fundacion Ramon Areces fellowship. R.G. received
grants by CNR PF Biotechnology, CNR Agenzia 2000
and Finalized Research funds (2001) from the Italian
Ministry of Health.
yield) as a white solid. Mp 105–110 C, IR (KBr) cmꢁ1
:
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