Annelated pyrrolo-pyrimidines from amino-cyanopyrroles and BMMAs as leads for new DNA-interactive ring systems

Article abstract of DOI:10.1016/j.bmc.2004.12.027

The efficient one-pot synthesis of several new tricyclic systems of type 1 and 2, obtained from the reaction of substituted 2-amino-3-cyanopyrroles and 3-amino-4-cyanopyrroles with BMMAs, is reported. The duration and yields of the reaction strongly depen

Full text of DOI:10.1016/j.bmc.2004.12.027

Bioorganic & Medicinal Chemistry 13 (2005) 1545–1553
Annelated pyrrolo-pyrimidines from amino-cyanopyrroles and
BMMAs as leads for new DNA-interactive ring systems
Antonino Lauria, Marcella Bruno, Patrizia Diana, Paola Barraja, Alessandra Montalbano,
Girolamo Cirrincione, Gaetano Dattolo and Anna Maria Almerico*
`
Dipartimento Farmacochimico, Tossicologico e Biologico, Universita di Palermo Via Archirafi 32, 90123 Palermo, Italy
Received 3 August 2004; revised 7 December 2004; accepted 14 December 2004
Abstract—The efficient one-pot synthesis of several new tricyclic systems of type 1 and 2, obtained from the reaction of substituted
2-amino-3-cyanopyrroles and 3-amino-4-cyanopyrroles with BMMAs, is reported. The duration and yields of the reaction strongly
depend on the reactivity of the starting pyrrole and on the size of the ring to be formed. Mechanist features of the reaction were
investigated and proposed by studying also the reactivity of a 3-aminopyrrole-2,4-dicyano substituted. The method reported repre-
sents the first example of the use of BMMA reagents in combination with pyrrole derivatives and allows an easy and versatile entry
to a large number of hitherto unknown pyrrolo-pyrimidines further annelated with nitrogen heterocycles of different sizes. These
new polycondensed heterocycles possess the requisite to interact with DNA.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
linear or angular structure such as acridines, anthracy-
clines, and phenanthridines. For these compounds the
principal driving forces are stacking and charge-transfer
interactions as well as hydrogen bonding and electro-
static forces.7 However in some cases intercalation into
DNA is only the first step in the events that eventually
lead to DNA damage by other mechanisms. Moreover
if the drug candidate bears suitable substituents, such
as a thiomethyl group, interference with cellular detoxi-
fication pathways can be envisaged.
In the search for new anticancer drugs different ap-
proaches are currently undertaken. Several efforts are di-
rected towards the discovery of molecules targeting
angiogenesis, cell-cycle pathways, and cell differentia-
tion. However classical approaches involving the discov-
ery of cytotoxic agents interfering with DNA, either
directly or inhibiting DNA-binding enzymes, have led
to the identification of new promising anticancer agents.1
As our aim is continuously devoted to the study of new
flat polycyclic heteroaromatic systems that can behave
as DNA-interactive drugs, we have successfully pre-
pared and reported the antiproliferative activity of sev-
eral tricyclic and tetracyclic heterocycles that
incorporate the pyrrole or indole moieties. Among them
Therefore in search of suitable lead compounds for the
development of new anticancer drugs, we decided to ex-
plore series of new tricyclic heterocycles as DNA-inter-
active agents and to investigate synthetic methods
leading to annelated pyrrolo-pyrimidines.
indolo[3,2-c]cinnolines,2
indolo[1,2-c]benzo[1,2,3]tri-
Few years ago the use of new reagents for heterocyclic
annelation containing an N-[bis(methylthio)methyl-
ene]amino moiety (= BMMA) was reported. The meth-
od allowed the preparation of condensed pyrimidines in
a one-pot reaction from (hetero)aromatic ortho-amino-
azines,3 pyrrolo[3,4-e][1,2,3]triazolo[1,5-a]pyrimidines,4
pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines,5 and in-
dolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimidine6 can be re-
lated to the well known classes of intercalators with
carbonyl-type
compounds
(ÔcarbonylÕ = COOEt,
COMe, and CN).8 Some authors showed the use
of BMMAs in combination with electron-rich pent-
atomic heterocycles (furan9 and thiophene10) and their
condensed homologues, but nothing has been reported
so far on their employment in reactions with pyrroles
Keywords: Annelated pyrrolo-pyrimidines; Amino-cyanopyrroles;
BMMA reagent; DNA-interactive polycycles.
*
Corresponding author. Tel.: +39 0916161606; fax: +39
0916169999; e-mail: almerico@unipa.it
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.027

1546
A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
or indoles. However our experience on the behavior of
aminopyrroles and aminoindoles11 suggested that the
electron-rich pyrrole and indole should be able to react
with this kind of reagents as well and prompted us to
investigate their use in combination with BMMAs as
an access to hitherto unknown annelated pyrrolo-
pyrimidines.
ethyl ester (4a) in acetic acid under reflux (Scheme 2).
The starting materials were converted into the final
products in 30min. The imidazo[1,2- c]pyrrolo[3,2-e]-
pyrimidinones 6a,b were isolated in very high yields
(85–90%), only in the case of the aminopyrrole 5c, the
tricyclic compound 6c was obtained in 40% yield
because the competing alkylation of the unsubstituted
a-position of the pyrrole ring gave rise to the formation
of a large amount of derivative 7 (35%).
Therefore, in this paper we report the efficient one-pot
synthesis of several new tricyclic systems of type 1 and
2, obtained from the reaction of substituted 2-amino-
3-cyanopyrroles and 3-amino-4-cyanopyrroles with sev-
eral BMMA reagents.
This first approach showed that the BMMAs can be
used to annelate the pyrrole nucleus and that the reac-
tion spontaneously proceeded to the final products
through a sequence that can be envisaged as a domino
reaction in which the primary cyclized bicycle (the pyr-
rolo-pyrimidine 8), because of the presence of a reactive
imine, further cyclizes originating the third ring (Scheme
3). Moreover the 2-amino-3-cyanopyrrole resulted much
more reactive toward BMMAs than the aryl or thio-
phene substrate already studied in literature (shorter
reaction times and higher yields).8 However the method
was not suitable for usage with the pyrrole unsubstituted
in the a-position and therefore the reactivity of deriva-
tive 5c was not investigated any more.
O
SMe
N
O
X
N
N
X
N
N
SMe
N
N
N
1
2
2. Results and discussion
2-Amino-3-cyanopyrroles 5a,b were allowed to react
with all the other BMMAs 4b–e in acetic acid under re-
flux for an appropriate period of time (0.5–1 h). In all
the cases the annelated pyrrolo-pyrimidines 9–12 could
be easily isolated generally in yields from good to high
(Scheme 4).
The starting BMMA reagents 4a–e were prepared in
good yields by reacting adequate amino educts 3 with
CS2/MeI/NEt3 and subsequently accomplishing the
alkylation of the intermediates thus obtained with
MeI/K2CO3 (Scheme 1).
The feasibility of the reaction with BMMAs was first
investigated by using 2-amino-3-cyanopyrroles as sub-
strates. For this purpose the pyrrole derivatives 5a–c
were prepared by standard procedures12 and were al-
lowed to react with N-[bis(methylthio)methylene]glycine
Only in the case of the reaction with 4d the yields re-
sulted lower probably because of steric hindrance in
the intermediate 8 (X = CHMe) which can not properly
assume a conformation suitable for the ring closure
(compare 6 vs 11, yields 90–85% vs 60–43%). On the
X
CH2
R
Et
a
b
c
MeS
MeS
i
H2N
X COOR
N
X COOR
CH2CH2
NMe
Et
ii
Et
3a-e
4a-e
CHMe
CHMeCH2
Me
Et
d
e
Scheme 1. Reagents: (i) CS2, MeI, NEt3, CHCl3; (ii) MeI, K2CO3, Me2CO.
O
N
Me
CN
NH2
N
Me
R
CN
NH2
Me
R
MeS
MeS
N
COOEt
SMe
N
H
+
MeS
+
i
ii
SMe
N
HN
N
H
N
H
COOEt
5a-c
4a
6a-c
7
Scheme 2. (a) R = Me; (b) R = Ph; (c) R = H. Reagents: (i) AcOH, D; (ii) NaHCO3 or Na2CO3.

A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
1547
COOR
X
CN
NH2
N
X
COOR
SMe
+
C
N
Y
N
Z
MeS
O
SMe
Y
NH
Z
4
RO
HN
O
N
X
X
N
N
SMe
SMe
Y
Y
N
N
Z
Z
Z = NR', Y = CR'' ; Z = CR'', Y = NR'.
8
Scheme 3. Mechanism of formation of annelated pyrrolo-pyrimidines.
O
O
X
MeS
N
N
X
N
N
H2N
Ph
CN
Ph
X
Me
R'
CN
NH2
Me
R'
N
COOEt
SMe
X
N
+
N
COOR
SMe
+
i
ii
Ph
SMe
MeS
Ph
N
R
N
R
N
MeS
N
H
N
H
13a, R = H
4a-c
14-16
5a R' = Me
4b-e
9-12
13b, R = Me
5b R' = Ph
a, R = H
a, R' = Me
b, R = Me
b, R' = Ph
Scheme 5. Reagents: (i) AcOH, D; (ii) NaHCO3 or Na2CO3.
Scheme 4. Reagents: (i) AcOH, D; (ii) NaHCO3 or Na2CO3.
other hand in the homologous intermediate
(X = CHMeCH2) the formation of the six-membered
ring is easier, as testified by the higher yields, and a
shorter reaction time is required (compare 11a vs 12a,
11b vs 12b).
gives rise to C-2–C-3 bond shorter and with a high p
bond order.11b,14 To verify how this effect could influ-
ence the course of the reaction we decided to investigate
the two possible ring closures in competition by using as
starting material the 3-amino-2,4-dicyanopyrrole 19.
This last was suitably prepared by a modification of
the method reported in literature15 (Scheme 6), from
benzylidenemalonitrile 17.
Thus the reaction of 2-amino-3-cyanopyrroles with
BMMAs seemed to be of general application. Therefore
we decided to investigate the behavior of 3-amino-4-
cyanopyrroles. For this purpose the 3-amino derivatives
13a,b were prepared by known procedure13 and were al-
lowed to react with the BMMAs 4a–c (Scheme 5).
The reaction of the pyrrole 19 with the BMMA 4a led to
a mixture of the annelated pyrrolo-pyrimidines 21 and
23, as expected (Scheme 7). The main product of the
reaction was the imidazo-pyrrolo-pyrimidine 23 (yield
40%), derived from ring closure on the 2- and 3-posi-
tions, confirming thus that the key step of the reaction
sequence is the formation of the intermediate bicycle
and that 22 is formed more efficiently than 20, since it
is reasonable to suppose that there is no significant dif-
ference in the ring closure originating the third ring.
Once again the tricyclic derivatives 14–16 were isolated
after heating in acetic acid under reflux for the appropri-
ate reaction time (4–8 h). It is worthy to note that in this
case the time required to drive the reaction to comple-
tion is longer and the yields are generally lower if com-
pared to those obtained in the case of the reaction with
2,3-disubstituted pyrroles. Therefore it seems that the
annelation on the position 2 and 3 of the pyrroles is fa-
vored with respect to the ring closure between positions
3 and 4 and that the rate limiting step of the domino
reaction is the formation of the pyrimidine leading to
the bicyclic intermediates of type 8 (Scheme 3). This is
not surprising since it is well known that in five-mem-
bered electron-rich heterocycles there is a larger trans-
mission of electronic effects between the 2- and
3-positions (hyper-ortho) than between the 3- and 4-pos-
itions (hypo-ortho) due to the high Ôbond fixationÕ, which
Of course the structure of derivatives 21 and 23 was as-
signed especially on the basis of NMR spectra and in
comparison with the data obtained for the other iso-
meric compounds synthesized herein (6a–c, 14a,b). In
particular very useful resulted the analysis of the 13C
chemical shifts, which were also correlated with theoreti-
cally estimated values.16 In fact, taking into account the
effect of the substituents, the chemical shifts of the car-
bon atoms present analogous values in the two series
in which the pyrrole nucleus is annelated in the same

1548
NC CN
Ph
A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
molecules with p electron systems form charge-transfer
complexes, and also the values of some molecular
descriptors [molecular surface area, ASA (accessible sur-
face area), log P, molar refractivity].
NC
Ph
NH2
CN
NC CN
i
H2N
CN
ii
N
H
H
Ph
Cl
17
18
19
An analysis of the data reported in Table 1 shows that
these values are comparable with those of well known
DNA-intercalators of the acridine or anthracycline clas-
ses such as amsacrine (AMSA) and doxorubicin
(DOXO). Moreover they share the same features of
other tetracyclic systems, which already demonstrated
to be active against a wide range of tumor cell lines,
the indolo[1,2-c]benzo[1,2,3]triazine3 (TRIAZINE) and
the indolo[3,2-c]cinnoline2 (CINNOLINE). Therefore
these pyrrolo-pyrimidine derivatives appear to be good
candidate as DNA-interactive drugs.
Scheme 6. Reagents: (i) Cl2, PyHCl; (ii) NEt3.
positions (compare 6b and 23, 14a and 21) and a good
correlation was found between calculated and found
values in the case of derivatives 21 and 23 (r2 = 0.749
and 0.926, respectively).
Representative compounds of each series were selected
for screening tests by the NCI (Bethesda) in the Devel-
opmental Therapeutics Program. In the primary anti-
cancer screening assay,17 tested against a 3-cell line
panel consisting of the MCF7 (Breast), NCI-H460
(Lung), and SF-268 (CNS) the annelated pyrrolo-pyrim-
idine resulted generally inactive up to 10ꢀ4 M concentra-
tions. However tested against the human intestinal
adenocarcinoma (LoVo),18 although being less active
than the reference drug [doxorubicin (DOXO)], few
compounds exhibited a moderate antiproliferative activ-
ity with IC50 = 10.5–41.2 lM (Fig. 1).
3. Conclusions
This method represents the first example of the use of
BMMA reagents in combination with pyrrole deriva-
tives and allow an easy and versatile entry to a large
number of hitherto unknown pyrrolo-pyrimidines fur-
ther annelated with nitrogen heterocycles of different
sizes. Preliminary structure activity analysis on these
new classes of tricyclic heterocycles demonstrated that
although not being very active, derivatives of these sys-
tems possess the electronic and steric requisite to inter-
act with DNA and could constitute suitable lead
compounds for new anticancer agents.
From the data shown in Figure 1 it is worthy to note
that generally the triazolo-pyrimidine derivatives re-
sulted inactive or low active regardless to the condensa-
tion position (compare 10b and 16a), whereas the
presence of the imidazole ring enhances the antiprolifer-
ative activity, especially in the case of pyrimidine moiety
condensed on the positions 3 and 4 of the pyrrole nu-
cleus (compare derivatives 6 and 14). Ring enlargement
to give the pyrimido-pyrimidine originates the most ac-
tive compound of these series, derivative 15a. In order to
evaluate the potential ability of the new annelated pyr-
rolo-pyrimidine ring systems to interact with DNA, we
calculated19 the LUMO and HOMO energies, consider-
ing that these variables are of importance when two
4. Experimental
All melting points were taken on a Buchi–Tottoli capil-
lary apparatus and are uncorrected; IR spectra were
determined in bromoform with a Jasco FT/IR 5300
spectrophotometer; 1H and 13C NMR spectra were mea-
sured in DMSO-d6 solution, unless otherwise specified,
(TMS as internal reference) at 200 and 50.3 MHz,
O
N
O
EtO
N
H
COOEt
N
N
Ph
Ph
Ph
N
C
N
SMe
SMe
SMe
N
N
N
N
N
N
H
H
H
H
C
N
C
N
C
N
COOEt
SMe
CN
Ph
N
20
21
+
H
N
MeS
NH2
N
N
N
Ph
C
Ph
C
CN
19
Ph
C
H
4a
N
N
N
N
H
H
H
N
N
H
SMe
C
N
N
SMe
N
N
SMe
N
N
O
EtO
COOEt
O
22
23
Scheme 7. Reaction conditions: AcOH, D.

A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
1549
O
O
N
O
N
N
N
N
Me
Me
Me
Me
N
N
SMe
SMe
SMe
N
N
N
Ph
R
Me
N
H
N
H
N
H
R=Me IC50=26.7 µM
R=Ph IC50=23.2 µM
IC50 >50 µM
IC50=41.2 µM
10b
6a
6b
9a
O
O
O
N
N
N
N
Me
Ph
Ph
Ph
N
N
N
SMe
SMe
N
SMe
N
N
N
N
N
H
Me
H
Ph
Ph
Ph
IC50=10.5 µM
15a
IC50=35 µM
IC50=19 µM
16a
14b
Figure 1. Antiproliferative activity against LoVo cell line18 of annelated pyrrolo-pyrimidines.
Table 1. Molecular descriptors for annelated pyrrolo-pyrimidines
Molecular surface areaa
ASAa
Log Pb
Molar refractivityc
ELUMO
EHOMO
d
d
Compound
6a
6b
234.32
283.78
248.57
297.18
342.48
335.78
341.85
232.31
235.63
458.18
344.32
258.18
318.76
278.48
332.65
404.43
395.29
385.46
262.23
258.17
524.23
398.26
1.9052
3.3393
2.0865
NC
67.506
87.370
72.205
NC
ꢀ0.8254
ꢀ0.8615
ꢀ0.7523
ꢀ1.0205
ꢀ0.6532
ꢀ0.7787
ꢀ1.1165
ꢀ1.5652
ꢀ1.4278
ꢀ1.4032
ꢀ1.2618
ꢀ8.4371
ꢀ8.4130
ꢀ8.3312
ꢀ8.5312
ꢀ8.4339
ꢀ8.2797
ꢀ8.3655
ꢀ8.9688
ꢀ9.2250
ꢀ8.9670
ꢀ8.1802
9a
10b
14b
15a
4.6245
4.5593
NC
112.99
112.79
NC
16a
TRIAZINE
CINNOLINE
DOXO
AMSA
4.5885
2.5766
0.1718
3.1631
80.581
74.209
134.02
107.77
a
2
˚
(A ).
b Predicted values were calculated as a sum of the atomic values determined (see Ref. 20).
c
3
(A ).
˚
d (eV); NC: not calculated.
respectively, using a Bruker AC-E series 200 MHz spec-
trometer. Column chromatography was performed with
Merck silica gel 230–400 mesh ASTM or with a Biotage
FLASH40i chromatography module (prepacked cart-
ridge system).
reflux for 1 h. The solution was cooled, washed with
water (100 mL), and the organic layer was evaporated.
The residue was dissolved in water, extracted with ether
(3 · 100 mL) dried (Na2SO4), and evaporated. The crude
oil was dissolved in acetone (300 mL), K2CO3 (100 g,
0.724 mol), and MeI (85.2 g, 0.6 mol) were added and
the mixture was heated for 3 h under reflux, followed
by stirring at room temperature overnight. The precipi-
tated salt was filtered off, the acetone was removed in
vacuo, water was added, and the product was extracted
with ether, and dried (Na2SO4). Evaporation of the sol-
vent and distillation afforded 4a–e as colorless oils.
Glycine ethyl ester hydrochloride (3a), b-alanine ethyl
ester hydrochloride (3b), ethyl carbazate (3c), L-alanine
methyl ester hydrochloride (3d), and ethyl 3-aminobuty-
rate (3e) were purchased from Aldrich and used without
further purification.
4.1. General procedure for the synthesis of BMMA
reagents 4a–e
4.2. Ethyl N-[bis(methylsulfanyl)methylene]glycinate 4a
The amino ester 3a–e (0.5 mol), CS2 (40.0 g, 0.5 mol),
and triethylamine (106.0 g, 1.050 mol) were reacted at
40 ꢁC in chloroform (500 mL) for 1 h. MeI (177.2 g,
1.250mol) was added and the mixture was heated under
IR identical to an authentic sample.9 The 13C NMR not
reported in literature showed: d 14.0(q, CH ), 14.3 (q,
3
CH3), 14.0(q, CH 3), 53.7 (t, CH2), 60.3 (t, CH2),
162.1 (s, C@N), 169.1 (s, CO).

1550
A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
1
4.3. Ethyl N-[bis(methylsulfanyl)methylene]-b-alaninate 4b
Yield 90%, IR: 1731 (CO) cmꢀ1 1H NMR: d 1.18
(t, 3H, J = 7.4 Hz, CH3), 2.09 (s, 6H, 2 · CH3), 2.40(t,
2H, J = 5.9 Hz, CH2), 3.52 (t, 2H, J = 5.9 Hz, CH2),
4.06 (q, 2H, J = 7.4 Hz, CH2); 13C NMR: d 13.9 (q,
2 · CH3), 14.2 (q, CH3), 31.3 (t, CH2), 51.2 (t, CH2),
59.6 (t, CH2), 157.3 (s, C@N), 172.7 (s, CO). Anal.
Calcd for C8H15NO2S2: C, 43.41; H, 6.83; N, 6.33; S,
28.97. Found: C, 43.19; H, 6.98; N, 6.51; S, 29.31.
(CO) cmꢀ1; H NMR: d 2.21 (s, 6H, 2 · CH3), 2.63 (s,
3H, CH3), 4.25 (s, 2H, H-3), 11.93 (s, 1H, NH); 13C
NMR: d 9.2 (q, CH3), 10.5 (q, CH3), 13.5 (q, CH3),
49.6 (t, C-3), 100.6 (s, C-9a), 107.8 (s, C-9), 128.8 (s,
C-8), 146.6 (s, C-6a), 150.9 (s, C-9b), 164.7 (s, C-5),
183.5 (s, C-2). Anal. Calcd for C11H12N4OS: C, 53.21;
H, 4.87; N, 22.56; S, 12.91. Found: C, 53.39; H, 4.90;
N, 22.71; S, 13.04.
;
4.9. 9-Methyl-5-(methylsulfanyl)-8-phenyl-3H-imi-
dazo[1,2-c]pyrrolo[3,2-e]pyrimidin-2(7H)-one 6b
4.4. Ethyl 2-[bis(methylsulfanyl)methylene]-1-methylhy-
drazinecarboxylate 4c
From 5b and 4a: (reaction time 0.5 h), yields 85%;
mp >300 ꢁC (from methanol); IR: 3360(NH), 1740
Yield 90%, IR: 1698 (CO) cmꢀ1; 1H NMR: d 1.16 (t, 3H,
J = 7.1 Hz, CH3), 2.47 (s, 6H, 2 · CH3), 2.96 (s, 3H,
CH3), 4.04 (q, 2H, J = 7.1 Hz, CH2); 13C NMR: d 13.3
(CO) cmꢀ1 1H NMR: d 2.50(s, 3H, CH 3), 2.68 (s,
;
3H, CH3), 4.31 (s, 2H, H-3), 7.36 (t, 1H, J = 7.4 Hz,
H-40), 7.49 (t, 2H, J = 7.4 Hz, H-30 and H-50), 7.65 (d,
2H, J = 7.4 Hz, H-20 and H-60), 12.45 (s, 1H, NH);
13C NMR (pyridine-d5): d 11.4 (q, CH3), 14.0(q,
CH3), 50.0 (t, C-3), 103.0 (s, C-9a), 111.2 (s, C-9),
127.8 (d, C-40), 127.9 (d, C-20 and C-60), 129.3 (d, C-30
and C-50), 132.2 (s, C-8), 132.8 (s, C-10), 148.4 (s, C-
6a), 152.4 (s, C-9b), 166.5 (s, C-5), 184.2 (s, C-2). Anal.
Calcd for C16H14N4OS: C, 61.92; H, 4.55; N, 18.05 S,
10.33. Found: C, 62.00; H, 4.57; N, 18.01; S, 10.51.
(q, CH3), 14.5 (q, CH3), 14.8 (q, CH3), 36.0(t, CH ),
2
61.1 (q, CH3), 154.1 (s, C@N), 174.2 (s, CO). Anal.
Calcd for C7H14N2O2S2: C, 37.82; H, 6.35; N, 12.60;
S, 28.84. Found: C, 38.19; H, 6.50; N, 12.51; S, 29.11.
4.5. Methyl N-[bis(methylsulfanyl)methylene]alaninate 4d
1
Yield 80%, IR: 1739 (CO) cmꢀ1; H NMR (CDCl3): d
1.43 (d, 3H, J = 6.9 Hz, CH3), 2.42 (s, 3H, CH3), 2.57
(s, 3H, CH3), 3.72 (s, 3H, CH3), 4.51 (q, 1H,
J = 6.9 Hz, CH); 13C NMR (CDCl3,): d 14.7 (q, CH3),
14.8 (q, CH3), 18.4 (q, CH3), 51.9 (q, CH3), 59.9 (d,
CH), 161.4 (s, C@N), 172.9 (s, CO). Anal. Calcd for
C7H13NO2S2: C, 40.56; H, 6.32; N, 6.76; S, 30.93.
Found: C, 41.19; H, 6.40; N, 6.51; S, 31.05.
4.10. 9-Methyl-5-(methylsulfanyl)-3H-imidazo[1,2-c]-
pyrrolo[3,2-e]pyrimidin-2(8H)-one 9a
From 5c and 4a: (reaction time 1 h); the precipitate was
identified as ethyl N-{(5-amino-4-cyano-3-methyl-1H-
pyrrol-1-yl)[bis(methylsulfanyl)]}methyleneglycinate (7),
yields 35%; mp 160 ꢁC (from methanol), IR: 3476–3356
4.6. Ethyl 3-{[bis(methylsulfanyl)methylene]amino}but-
anoate 4e
(NH2), 2925 (NH), 2206 (CN), 1741 (CO) cmꢀ1 1H
;
NMR: d 1.23 (t, 3H, J = 7.4 Hz, CH3), 2.36–2.54 (m,
10H, 3 · CH3 and NH), 4.18 (q, 2H, J = 7.4 Hz, CH2),
4.49 (s, 2H, CH2), 7.49 (s, 2H, NH2), 10.35 (s, 1H,
NH); 13C NMR: d 11.6 (q, CH3), 11.7 (q, CH3), 14.1
1
Yield 85%, IR: 1727 (CO) cmꢀ1; H NMR (CDCl3): d
1.16 (d, 3H, J = 6.0Hz, CH 3), 1.23 (t, 3H, J = 7.0Hz,
CH3), 2.31 (s, 3H, CH3), 2.50(d, 2H, J = 6.0Hz,
CH2), 2.54 (s, 3H, CH3), 4.10(q, 2H, J = 7.0Hz,
CH2), 4.17–4.28 (m, 1H, CH); 13C NMR (CDCl3): d
13.9 (q, CH3), 14.3 (q, CH3), 14.4 (q, CH3), 20.3 (q,
CH3), 42.8 (t, CH2), 54.3 (d, CH), 59.8 (t, CH2), 156.8
(s, C@N), 171.4 (s, CO). Anal. Calcd for C9H17NO2S2:
C, 45.93; H, 7.28; N, 5.95; S, 27.24. Found: C, 46.19;
H, 7.50; N, 6.03; S, 27.51.
(q, CH3), 18.0(q, CH ), 47.2 (t, CH2), 61.1 (t, CH2),
3
77.1 (s, C(SCH3)2), 112.6 (s, CN), 115.4 (s, C-3), 117.2
(s, C-4), 140.0 (s, C-2), 147.4 (s, C-5), 169.2 (s, CO). Anal.
Calcd for C13H20N4O2S2: C, 47.54; H, 6.14; N, 17.06; S,
19.52. Found: C, 47.49; H, 6.20; N, 17.16; S, 19.70.
The aqueous layer was extracted with EtOAc. The com-
bined extracts were dried with anhydrous Na2SO4 and
the solvent was removed under vacuum to give 9-
methyl-5-(methylsulfanyl)-3H-imidazo[1,2-c]pyrrolo[3,2-e]-
pyrimidin-2(7H)-one (6c): yields 40%; mp >300 ꢁC; IR:
4.7. General procedure for the reaction of amino-cyano-
pyrroles and BMMAs
1
3370(NH), 1705 (CO) cm ꢀ1; H NMR: d 2.28 (s, 3H,
The mixture of amino-cyanopyrrole 5 or 13 (3 mmol)
and BMMA 4 (3 mmol) in AcOH (10mL) was heated
under reflux for the appropriate time, with stirring.
After cooling, the reaction mixture was added to ice/
water and neutralized with solid NaHCO3 or Na2CO3.
The solid precipitate was filtered off and crystallized or
purified by chromatography.
CH3), 2.64 (s, 3H, CH3), 4.26 (s, 2H, H-3), 6.96 (s,
1H, H-8), 11.97 (s, 1H, NH); 13C NMR: d 10.8 (q,
CH3), 13.5 (q, CH3), 49.6 (t, C-3), 100.1 (s, C-9a),
110.2 (s, C-9), 112.8 (d, C-8), 120.0 (s, C-6a), 147.6 (s,
C-9b), 151.9 (s, C-5), 183.7 (s, C-2). Anal. Calcd for
C10H10N4OS: C, 51.27; H, 4.30; N, 23.91, S, 13.69.
Found: C, 51.25; H, 4.34; N, 23.99, S, 13.75.
4.8. 8,9-Dimethyl-5-(methylsulfanyl)-3H-imidazo[1,2-
c]pyrrolo[3,2-e]pyrimidin-2(7H)-one 6a
4.11. 9,10-Dimethyl-6-(methylsulfanyl)-3,4-dihydropyr-
imido[1,2-c]pyrrolo[3,2-e]pyrimidin-2(8H)-one 9a
From 5a and 4a: (reaction time 0.5 h), yields 90%;
mp >300 ꢁC (from methanol); IR: 3429 (NH), 1702
From 5a and 4b: (reaction time 0.5 h), yields 70%;
mp >300 ꢁC (from methanol); IR: 3285 (NH), 1705

A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
1551
1
(CO) cmꢀ1; H NMR: d 2.17 (s, 3H, CH3), 2.23 (s, 3H,
CH3), 4.31 (q, 1H, J = 6.9 Hz, H-3), 11.95 (s, 1H,
NH); 13C NMR: d 9.2 (q, CH3), 10.4 (q, CH3), 13.6
(q, CH3), 15.7 (q, CH3), 56.4 (d, C-3), 100.7 (s, C-9a),
108.0 (s, C-9), 128.9 (s, C-8), 146.4 (s, C-6a), 151.1 (s,
C-9b), 164.1 (s, C-5), 186.6 (s, C-2). Anal. Calcd for
C12H14N4OS: C, 54.94; H, 5.38; N, 21.36; S, 12.22.
Found: C, 55.05; H, 5.44; N, 21.39; S, 12.18.
CH3), 2.52 (t, 2H, J = 6.0Hz, H-3), 2.58 (s, 3H, CH 3),
4.18 (t, 2H, J = 6.0Hz, H-4), 11.71 (s, 1H, NH); 13C
NMR: d 10.0 (q, CH3), 10.5 (q, CH3), 14.7 (q, CH3),
29.4 (t, C-3), 43.2 (t, C-4), 103.4 (s, C-10a), 108.8 (s,
C-10), 128.1 (s, C-9), 145.3 (s, C-7a), 153.4 (s, C-10b),
153.9 (s, C-6), 173.7 (s, C-2). Anal. Calcd for
C12H14N4OS: C, 54.94; H, 5.38; N, 21.36; S, 6.10.
Found: C, 54.88; H, 5.48; N, 21.50, S, 6.05.
4.16. 3,9-Dimethyl-5-(methylsulfanyl)-8-phenyl-3H-imi-
dazo[1,2-c]pyrrolo[3,2-e]pyrimidin-2(7H)-one 11b
4.12. 10-Methyl-6-(methylsulfanyl)-9-phenyl-3,4-dihydro-
pyrimido[1,2-c]pyrrolo[3,2-e]pyrimidin-2(8H)-one 9b
From 5b and 4d: (reaction time 1 h), yields 43%;
mp >300 ꢁC (from toluene); IR: 3446 (NH), 1704 (CO)
1
From 5b and 4b: (reaction time 0.5 h), yields 60%;
mp >300 ꢁC (from methanol); IR: 3280(NH), 1710
cmꢀ1; H NMR: d 1.58 (d, 3H, J = 7.2 Hz, CH3), 2.49
(s, 3H, CH3), 2.68 (s, 3H, CH3), 4.36 (q, 1H,
J = 7.2 Hz, H-3), 7.37 (dt, 1H, J = 7.2 and 0.8 Hz, H-
40), 7.48 (dt, 2H, J = 7.2 and 0.8 Hz, H-30 and H-50),
7.63 (dd, 2H, J = 7.2 and 0.8 Hz, H-20 and H-60), 12.44
(CO) cmꢀ1 1H NMR: d 2.53 (s, 3H, CH3), 2.56 (t,
;
2H, J = 6.1 Hz, H-3), 2.65 (s, 3H, CH3), 4.23 (t, 2H,
J = 6.1 Hz, H-4), 7.33–7.62 (m, 5H, Ph), 12.16 (s, 1H,
NH); 13C NMR: d 11.5 (q, CH3), 14.8 (q, CH3), 29.3,
(t, C-3), 43.3 (t, C-4), 104.1 (s, C-10a), 111.0 (s, C-10),
127.1 (d, C-40), 127.5 (d, C-20 and C-60), 128.6 (d, C-30
and C-50), 130.8 (s, C-9), 131.5 (s, C-10), 146.2 (s, C-
7a), 154.4 (s, C-10b), 155.1 (s, C-6), 173.7 (s, C-2). Anal.
Calcd for C17H16N4OS: C, 62.94; H, 4.97; N, 17.27; S,
9.88. Found: C, 62.91; H, 4.91; N, 17.37; S, 9.90.
(s, 1H, NH); 13C NMR: d 11.0(q, CH ), 13.8 (q, CH3),
3
15.7 (q, CH3), 56.5 (d, C-3), 101.8 (s, C-9a), 110.0 (s,
C-9), 127.3 (d, C-40, C-20 and C-60), 128.7 (d, C-30 and
C-50), 131.2 (s, C-8), 131.4 (s, C-10), 147.3 (s, C-6a),
152.6 (s, C-9b), 164.9 (s, C-5), 186.8 (s, C-2). Anal. Calcd
for C17H16N4OS: C, 62.94; H, 4.97; N, 17.27; S, 9.88.
Found: C, 63.05; H, 4.94; N, 17.25; S, 9.75.
4.13. 5-(Methylsulfanyl)-3,8,9-trimethyl-3H-pyrrolo-
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2(7H)-one 10a
4.17. 6-(Methylsulfanyl)-4,9,10-trimethyl-3,4-dihydropy-
rimido[1,2-c]pyrrolo[3,2-e]pyrimidin-2(8H)-one 12a
From 5a and 4c: (reaction time 0.5 h), yields 65%;
mp >300 ꢁC (from methanol); IR: 3305 (NH), 1705
From 5a and 4e: (reaction time 0.5 h), the residue was
purified by column chromatography using DCM–
MeOH 95:5 as eluant: yields 72%; mp 238 ꢁC; IR:
1
(CO) cmꢀ1; H NMR: d 2.23 (s, 6H, 2 · CH3), 2.69 (s,
3H, CH3), 3.52 (s, 3H, CH3), 11.94 (s, 1H, NH); 13C
3150(broad NH), 1655 (CO) cm ꢀ1; H NMR: d 1.29
1
NMR: d 9.1 (q, CH3), 10.5 (q, CH3), 14.0(q, CH ),
(d, 3H, J = 7.4 Hz, CH3), 2.17 (s, 3H, CH3), 2.18 (s,
3H, CH3), 2.24–2.29 (m, 1H, H-3), 2.75 (s, 3H, CH3),
2.83 (dd, 1H, J = 5.9 and 1.4 Hz, H-3), 4.82–4.86 (m,
1H, H-4), 11.74 (s, 1H, NH); 13C NMR: d 9.9 (q,
CH3), 10.4 (q, CH3), 14.6 (q, CH3), 17.0(q, CH 3),
36.0 (t, C-3), 50.5 (d, C-4), 103.5 (s, C-10a), 109.0 (s,
C-10), 128.2 (s, C-9), 145.1 (s, C-7a), 152.6 (s, C-10b),
152.7 (s, C-6), 173.1 (s, C-2). Anal. Calcd for
C13H16N4OS: C, 56.50; H, 5.84; N, 20.27; S, 11.60.
Found: C, 56.47; H, 5.93; N, 20.18; S, 11.75.
3
37.8 (q, CH3), 100.1 (s, C-9a), 107.0 (s, C-9), 129.4 (s,
C-8), 143.5 (s, C-6a), 144.6 (s, C-9b), 153.9 (s, C-5),
167.4 (s, C-2). Anal. Calcd for C11H13N5OS: C, 50.18;
H, 4.98; N, 26.60; S, 12.18. Found: C, 50.28; H, 4.87;
N, 26.49, S, 12.23.
4.14. 3,9-Dimethyl-5-(methylsulfanyl)-8-phenyl-3H-pyr-
rolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2(7H)-one 10b
From 5b and 4c: (reaction time 0.5 h), yields 60%;
mp >300 ꢁC (from methanol); IR: 3330(NH), 1731
4.18. 4,10-Dimethyl-6-(methylsulfanyl)-9-phenyl-3,4-di-
hydropyrimido[1,2-c]pyrrolo[3,2-e]pyrimidin-2(8H)-one 12b
(CO) cmꢀ1
;
1H NMR: d 2.52 (s, 3H, CH3), 2.75 (s,
3H, CH3), 3.56 (s, 3H, CH3) 7.33–7.67 (m, 5H, Ph),
12.46 (s, 1H, NH); 13C NMR: d 10.9 (q, CH3), 14.2
(q, CH3), 37.9 (q, CH3), 101.0 (s, C-9a), 108.9 (s, C-9),
127.3 (s, C-8), 127.4 (d, C-40), 127.5 (d, C-20 and C-60),
128.7 (d, C-30 and C-50), 131.3 (s, C-10), 131.8 (s, C-
6a), 144.4 (s, C-9b), 154.5 (s, C-5), 167.4 (s, C-2). Anal.
Calcd for C16H15N5OS: C, 59.06; H, 4.65; N, 21.52; S,
9.85. Found: C, 59.23; H, 4.66; N, 21.60, S, 9.94.
From 5b and 4e: (reaction time 0.5 h), the residue was
purified by column chromatography using DCM–MeOH
95:5 as eluant: yields 56%; mp 244 ꢁC; IR: 3411 (NH),
1
1652 (CO) cmꢀ1; H NMR: d 1.32 (d, 3H, J = 7.4 Hz,
CH3), 2.34–2.40(m, 1H, H-3), 2.54 (s, 3H, CH ), 2.66
3
(s, 3H, CH3), 2.86–2.93 (m, 1H, H-3), 4.80–4.93 (m, 1H,
H-4), 7.35 (dt, 1H, J = 8.8 and 1.5 Hz, H-40), 7.47 (dt,
2H, J = 8.8 and 1.5 Hz, H-30 and H-50), 7.60(dd, 2H,
J = 8.8 and 1.5 Hz, H-20 and H-60), 12.24 (s, 1H, NH);
13C NMR: d 11.5 (q, CH3), 14.7 (q, CH3), 17.0(q,
CH3), 35.9 (t, C-3), 50.6 (d, C-4), 104.2 (s, C-10a), 111.1
(s, C-10), 127.1 (d, C-40), 127.5 (d, C-20 and C-600), 128.5
(d, C-30 and C-50), 131.0(s, C-9), 131.5 (s, C-1 ), 146.0
(s, C-7a), 153.2 (s, C-10b), 154.1 (s, C-6), 173.2 (s, C-2).
Anal. Calcd for C18H18N4OS: C, 63.88; H, 5.36; N,
16.56; S, 9.47. Found: C, 63.92; H, 5.44; N, 26.49; S, 9.55.
4.15. 5-(Methylsulfanyl)-3,8,9-trimethyl-3H-imidazo[1,2-
c]pyrrolo[3,2-e]pyrimidin-2(7H)-one 11a
From 5a and 4d: (reaction time 1 h), the residue was
purified by column chromatography using DCM–
MeOH 95:5 as eluant: yields 60%; mp > 300 ꢁC; IR:
1
3430(NH), 1701 (CO) cm ꢀ1; H NMR: d 1.55 (d, 3H,
J = 6.9 Hz, CH3), 2.20(s, 6H, 2 · CH3), 2.63 (s, 3H,

1552
A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
4.19. 7,9-Diphenyl-5-(methylsulfanyl)-3H-imidazo[1,2-
c]pyrrolo[3,4-e]pyrimidin-2(8H)-one 14a
8.35 (d, 2H, J = 7.3 Hz, H-200 and H-600), 12.65 (s, 1H,
NH); 13C NMR: d 14.4 (q, CH3), 38.2 (q, CH3), 99.6
(s, C-9a), 122.2 (s, C-9), 125.7 (d, C-40), 126.7 (s, C-
6a), 126.8 (d, C-20 and C-60), 128.3 (d, C-400), 128.4 (d,
C-30 and C-50), 128.7 (d, C-200 and C-600), 129.0(s, C-
10), 129.3 (d, C-300 and C-500), 129.6 (s, C-100), 136.6 (s,
C-7), 145.1 (s, C-9b), 155.8 (s, C-5), 166.3 (s, C-2). Anal.
Calcd for C21H17N5OS: C, 65.10; H, 4.42; N, 18.08; S,
8.27. Found: C, 65.22; H, 4.51; N, 18.38; S, 8.38.
From 13a and 4a: (reaction time 4 h), yields 35%;
mp >30 ꢁC (methanol); IR: 3232 (NH), 1749 (CO)
1
cmꢀ1; H NMR: d 2.68 (s, 3H, CH3), 4.33 (s, 2H, H-
3), 7.21–7.53 (m, 6H, H-40, H-400, H-20, H-60, H-200 and
H-600, 8.14 (dd, 2H, J = 8.8 and 1.5 Hz, H-30 and H-
50), 8.30(dd, 2H, J = 7.4 and 1.5 Hz, H-300 and H-500,
12.68 (s, 1H, NH); 13C NMR: d 13.6 (q, CH3), 49.3 (t,
C-3), 100.2 (s, C-9a), 121.7 (s, C-9), 125.5 (d, C-20 and
C-60), 126.2 (d, C-40), 127.9 (d, C-30 and C-50), 128.0
(d, C-400), 128.4 (d, C-200 and C-600), 129.2 (d, C-300 and
C-500), 130.0 (s, C-7), 130.6 (s, C-10), 130.8 (s, C-100),
133.8 (s, C-6a), 148.6 (s, C-9b), 167.3 (s, C-5), 184.3 (s,
C-2). Anal. Calcd for C21H16N4OS: C, 67.72; H, 4.33;
N, 15.04; S, 8.61. Found: C, 67.79; H, 4.40; N, 15.09;
S, 8.68.
4.23. 3-Amino-2,4-dicyano-5-phenylpyrrole 19
Benzylidenemalonitrile 17 (154 g, 1 mol) and pyridinium
chloride (0.7 g, 6 mmol) were heated at 160 ꢁC for 3 h in
a three-neck round bottom flask under a gaseous chlo-
ride stream. The mixture was cooled to room tempera-
ture and the crude product 18 was quickly
recrystallized from propanol avoiding overboiling. To
a mixture of 18 (19 g, 0.1 mol) and aminoacetonitrile
bisulfate (15.5 g, 0.1 mol) in absolute ethanol (80 mL)
NEt3 (0.15 mol) was added dropwise and the mixture
was stirred at room temperature for 30min. Then
NEt3 (0.5 mol) was added and the reaction was heated
under reflux for 1.5 h. After cooling it was added to
water/ice and neutralized with HCl 4 N to pH 6–7.
The precipitate was filtered off and purified by column
chromatography using DCM–MeOH 9:1 as eluant.
Recrystallization from CH3CN gave 19, yield 41%, mp
300 ꢁC [lit.15 295 ꢁC]; IR: 3454 and 3359 (NH2), 3197
4.20. 7,9-Diphenyl-8-methyl-5-(methylsulfanyl)-3H-imi-
dazo[1,2-c]pyrrolo[3,4-e]pyrimidin-2(8H)-one 14b
From 13b and 4a: (reaction time 4 h), yields 40%;
mp >300 ꢁC (methanol); IR: 1724 (CO) cmꢀ1
;
1H
NMR: d 1.91 (s, 3H, CH3), 3.72 (s, 3H, CH3), 4.29 (s,
H-3), 7.40–7.77 (m, 10H, 2 · Ph); 13C NMR: d 13.2 (q,
CH3), 35.4 (q, CH3), 49.3 (t, C-3), 99.9, (s, C-9a),
124.8 (s, C-9), 127.4 (d, C-40), 127.9 (d, C-20 and C-60),
128.4 (d, C-30 and C-50), 128.6 (d, C-400, 129.1 (s, C-
10), 129.5 (s, C-100, 129.7 (d, C-200and C-600, 131.3 (d, C-
300 and C-500, 131.6 (s, C-7), 132.7 (s, C-6a), 148.5 (s,
C-9b), 166.7 (s, C-5), 184.6 (s, C-2). Anal. Calcd for
C22H18N4OS: C, 68.37; H, 4.69; N, 14.50; S, 8.30.
Found: C, 68.29; H, 4.72; N, 14.46; S, 8.28.
1
(NH), 2212 (CN), 2210(CN) cm ꢀ1; H NMR: d 5.93
(s, 2H, NH2), 7.43–7.58 (m, 3H, H-40, H-30 and H-50),
7.76 (dd, 2H, J = 7.3 and 1.5 Hz, H-20 and H-60),
12.34 (s, 1H, NH); 13C NMR: d 79.8 (s, C-4), 84.8 (s,
C-2), 114.2 (s, CN), 115.4 (s, CN), 126.1 (d, C-20 and
C-60), 128.6 (s, C-10), 129.1 (d, C-30 and C-50), 129.6
(d, C-40), 139.6 (s, C-3), 148.3 (s, C-5).
4.21. 8,10-Diphenyl-6-(methylsulfanyl)-3,4-dihydropy-
rimido[1,2-c]pyrrolo[3,4-e]pyrimidin-2(9H)-one 15a
4.24. Reaction of 3-amino-2,4-dicyano-5-phenylpyrrole
and BMMA
From 13a and 4b: (reaction time 5 h), yields 40%;
mp >300 ꢁC (methanol); IR: 3411 (NH), 1665 (CO)
cmꢀ1
;
1H NMR: d 2.50(s, 3H, CH 3), 2.59 (d, 2H,
The mixture of 19 (3.27 mmol) and BMMA 4a
(6.54 mmol) in AcOH (10mL) was stirred under reflux
for 18 h. After cooling, the reaction mixture was added
to ice/water. The solid was filtered off and purified by
J = 7.3 Hz, H-3), 4.21 (d, 2H, J = 7.3 Hz, H-4), 7.24–
7.49 (m, 6H, H-40, H-400, H-30, H-50, H-300 and H-500),
7.86 (dd, 2H, J = 7.3 and 1.4 Hz, H-20 and H-60), 8.27
(d, 2H, J = 7.3 Hz, H-200 and H-600), 12.53 (s, 1H, NH);
column
chromatography
using
DCM–MeOH
13C NMR: d 15.0(q, CH ), 29.5 (t, C-3), 42.7 (t, C-4),
98:2. The first compound to be eluted was 5-(meth-
ylsulfanyl)-8-oxo-2-phenyl-7,8-dihydro-1H-imidazo[1,2-c]-
pyrrolo[2,3-e]pyrimidine-3-carbonitrile (23), yield 40%,
mp >300 ꢁC; IR: 3375 (NH), 2229 (CN), 1685 (CO)
3
120.2 (s, C-10a), 125.1 (d, C-20 and C-60), 126.0(d, C-
40), 126.2 (s, C-10), 127.3 (d, C-30 and C-50), 127.7 (s,
C-10), 127.8 (d, C-400), 128.1 (s, C-100), 128.5 (d, C-200
and C-600), 129.8 (s, C-8), 130.5 (d, C-300 and C-500),
130.6 (s, C-7a), 130.9 (s, C-10b), 152.2 (s, C-6), 174.7
(s, C-2). Anal. Calcd for C22H18N4OS: C, 68.37; H,
4.69; N, 14.50; S, 8.30. Found: C, 68.42; H, 4.71; N,
14.48; S, 8.38.
1
cmꢀ1; H NMR: d 2.71 (s, 3H, CH3), 4.43 (s, 2H, H-
7), 7.59–7.62 (m, 3H, H-30, H-40 and H-50), 7.98–8.02
(m, 2H, H-20 and H-60); 13C NMR: d 13.63 (q, CH3),
50.48 (t, C-7), 84.49 (s, C-3), 111.70 (s, C-3a), 114.72
(s, CN), 127.53 (d, C-20), 127.53 (d, C-60), 128.22 (s,
C-10), 129.25 (d, C-30), 129.25 (d, C-50), 130.86 (d, C-
40), 147.30(s, C-2), 148.27 (s, C-9b), 153.49 (s, C-9a),
159.10(s, C-5), 182.98 (s, C-8). Anal. Calcd for
C16H11N5OS: C, 59.80; H, 3.45; N, 21.79; S, 9.98.
Found: C, 59.77; H, 3.50; N, 21.83; S, 9.95.
4.22. 7,9-Diphenyl-3-methyl-5-(methylsulfanyl)-3H-pyr-
rolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidin-2(8H)-one 16a
From 13a and 4c: (reaction time 8 h), yields 30%;
mp >300 ꢁC (methanol); IR: 3410(NH), 1712 (CO)
cmꢀ1
;
1H NMR: d 2.83 (s, 3H, CH3), 3.64 (s, 3H,
Further elution gave 5-(methylsulfanyl)-2-oxo-9-phenyl-
2,8-dihydro-3H-imidazo[1,2-c]pyrrolo[3,4-e]pyrimidine-7-
carbonitrile (21), yield 10%, mp 235 ꢁC; IR: 3467 (NH),
CH3), 7.51–7.65 (m, 6H, H-40, H-400, H-30, H-50, H-300
and H-500), 8.25 (d, 2H, J = 7.3 Hz, H-20 and H-60),

A. Lauria et al. / Bioorg. Med. Chem. 13 (2005) 1545–1553
1553
1
2227 (CN), 1655 (CO) cmꢀ1; H NMR: d 2.09 (s, 3H,
12. (a) Shvedov, V. I.; Mezentseva, M. V.; Grinev, A. N.
Khim. Geterotsikl. Soedin. 1975, 9, 1217; (b) Johnson, R.
W.; Mattson, R. J.; Sowell, J. W. J. Heterocycl. Chem.
1977, 14, 383; (c) Wamhoff, H.; Wehling, B. Synthesis
1976, 51.
13. (a) Dattolo, G.; Cirrincione, G.; Almerico, A. M.; Presti,
G.; Aiello, E. Heterocycles 1983, 20, 829; (b) Almerico, A.
M.; Cirrincione, G.; Aiello, E.; Dattolo, G. J. Heterocycl.
Chem. 1989, 26, 1631.
CH3), 3.39 (s, 2H, H-3), 7.22–7.80(m, 5H, Ph),
12.40(s, 1H, NH); 13C NMR: d 22.88 (q, CH3), 54.85
(t, C-3), 89.65 (s, C-7), 115.40(s, CN), 119.98 (s,
C-6a), 126.78 (d, C-20), 126.78 (d, C-60), 126.89 (s, C-
9a), 128.85 (s, C-10), 128.93 (d, C-30), 128.93 (d, C-50),
129.26 (d, C-40), 138.02 (s, C-9), 146.48 (s, C-9b),
160.64 (s, C-5), 169.04 (s, C-2). Anal. Calcd for
C16H11N5OS: C, 59.80; H, 3.45; N, 21.79; S, 9.98.
Found: C, 59.85; H, 3.42; N, 21.73; S, 10.04.
14. Consiglio, G.; Spinelli, D.; Gronowitz, S.; Hornfeldt, A.
B.; Noto, R. Chem. Scripta 1982, 19, 46.
15. Gewald, K.; Schafer, H.; Bellman, P.; Hain, U. J. Prakt.
Chem. 1992, 334, 491.
References and notes
16. Semi-empirical molecular orbital calculations were run on
an Indigo-2 Silicon Graphics work station by using the
Vamp (V 6.5) software, supplied by Oxford Molecular—
Accelrys. The structures of the molecules were fully
optimized in vacuo and in DMSO by SCF calculation
with PM3 method according to Stewart, J. J. P. J. Comput.
Chem. 1989, 10, 209–221. A prediction of the 13C NMR
chemical shifts by the neural-net technique was obtained
according to Clark, T.; Ruahut, G.; Breindl, A. J. Mol.
Mod. 1955, 1, 22. Tables with full data are available as
supplementary material.
17. In the protocol, each cell line is inoculated and preincu-
bated on a microtiter plate. Test agents are then added at a
single concentration and the culture incubated for 48 h.
End-point determinations are made with alamar blue
(Gray, G. D.; Wickstrom, E. Biotechniques 1996, 21, 780)
Results for each test agent are reported as the percent of
growth of the treated cells when compared to the
untreated control cells. Compounds which reduce the
growth of any one of the cell lines to approximately 32%
or less (negative numbers indicate cell kill) are passed on
for evaluation in the full panel of 60cell lines over a 5-log
dose range.
18. Test compounds, dissolved in (CH3)2SO at an initial
concentration of 200 mM and serially diluted in culture
medium, were incubated in the presence of LoVo cells for
72 h at 37 ꢁC. The number of viable cells was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) method (Denizot, F.; Lang, R. J.
Immunol. Methods 1986, 89, 271). Tumor cell growth at
each drug concentration was expressed as percentage of
untreated controls and the concentration resulting in 50%
(IC50) growth inhibition was determined by linear regres-
sion analysis. For doxorubicin, used as reference drug,
IC50 = 2.5 lM.
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