Synthesis and antiproliferative activity of basic thioanalogues of merbarone

Article abstract of DOI:10.1016/S0968-0896(03)00158-5

Three series of 5-substituted 1,3-diphenyl-6-(ω-dialkyl- and ω-cyclo-aminoalkyl)thio-2-thiobarbiturates (11-13) were synthesized as polysubstituted thioanalogues of merbarone, a topoisomerase II inhibitor acting on the catalytic site. To better understand pharmacophore requirements, a forth series of conformationally constrained analogues 14 was also prepared. Derivatives 11b,e, 14b,e,h,i,j were active in the low micromolar concentration range (IC₅₀: 3.3-4.3 μM), whereas compounds 11a,c,d,f,h,j and 13a,b,d,g,j and 14a,d,f showed IC₅₀ values between 10 and 15.5 μM. In constrast, compounds 12a-c,g-j, 13e,f,h and 14k were inactive. Cytotoxicity data provided from N.C.I. on selected compounds provided evidence that 11b,d, 13d,g and 14b,d,f,h,i,j were endowed with potent antiproliferative activity against leukemia and prostate cell lines (GI₅₀ up to 0.01 μM). In general, bicyclic derivatives 14 were up to 10-fold more potent than monocyclic counterparts against solid tumor-derived cell lines. SAR studies indicated that, in general, a certain tolerability in length of the alkyl side chains and in shape of distal amines is allowed in the four series, but in the monocyclic derivatives (11-13) antiproliferative activity was strongly affected by the nature of the 5-substituents (COOC₂H₅>COCH₃?C₆H ₅). Compounds 11b and 14b were also evaluated against KB cell subclones expressing altered levels of topoisomerases or the multidrug resistance phenotype (MDR). In both cases the above compounds showed a decrease in potency. In enzyme assays, 11b and 14b turned out to be inhibitors of topoisonerase II as merbaron.

Full text of DOI:10.1016/S0968-0896(03)00158-5

Bioorganic & Medicinal Chemistry 11 (2003) 2575–2589
Synthesis and Antiproliferative Activity of Basic Thioanalogues
of Merbarone
Angelo Ranise,a,* Andrea Spallarossa,a Silvia Schenone,a Olga Bruno,a
Francesco Bondavalli,a Alessandra Pani,b Maria Elena Marongiu,b Valeria Mascia,b
Paolo La Collab,* and Roberta Loddob
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Genova, Viale Benedetto XV 3, 16132Genova, Italy
`
Dipartimento di Biologia Sperimentale, Universita di Cagliari, Cittadella Universitaria di Monserrato, 09042Monserrato, Cagliari, Italy
b
Received 3May 2002; revised 21 February 2003; accepted 7 March 2003
Abstract—Three series of 5-substituted 1,3-diphenyl-6-(o-dialkyl- and o-cyclo-aminoalkyl)thio-2-thiobarbiturates (11–13) were
synthesized as polysubstituted thioanalogues of merbarone, a topoisomerase II inhibitor acting on the catalytic site. To better
understand pharmacophore requirements, a forth series of conformationally constrained analogues 14 was also prepared. Deriva-
tives 11b,e, 14b,e,h,i,j were active in the low micromolar concentration range (IC50: 3.3–4.3 mM), whereas compounds 11a,c,d,f,h,j
and 13a,b,d,g,j and 14a,d,f showed IC50 values between 10 and 15.5 mM. In constrast, compounds 12a–c,g–j, 13e,f,h and 14k were
inactive. Cytotoxicity data provided from N.C.I. on selected compounds provided evidence that 11b,d, 13d,g and 14b,d,f,h,i,j were
endowed with potent antiproliferative activity against leukemia and prostate cell lines (GI50 up to 0.01 mM). In general, bicyclic
derivatives 14 were up to 10-fold more potent than monocyclic counterparts against solid tumor-derived cell lines. SAR studies
indicated that, in general, a certain tolerability in length of the alkyl side chains and in shape of distal amines is allowed in the four
series, but in the monocyclic derivatives (11–13) antiproliferative activity was strongly affected by the nature of the 5-substituents
(COOC2H5>COCH3ꢀC6H5). Compounds 11b and 14b were also evaluated against KB cell subclones expressing altered levels of
topoisomerases or the multidrug resistance phenotype (MDR). In both cases the above compounds showed a decrease in potency.
In enzyme assays, 11b and 14b turned out to be inhibitors of topoisonerase II as merbaron.
# 2003Elsevier Science Ltd. All rights reserved.
Introduction
eventually drives cells to death.3,4 In fact, they do not
inhibit the topo II cleavage activity, rather they increase
the physiologic concentration and/or life-time of the
cleavage complexes, thus converting transient DNA
breaks into permanent fractures.3ꢁ7 A consequence of
this peculiar mode of action is that the higher are the
enzyme levels, the more lethal are the effects of topo II
poisons.8,9 This explains why topo II poisons result
particularly efficacious against aggressive cancers which
possess high enzyme levels,9,10 whereas they are ineffec-
tive against slow growing cancers, which possess low
topo II levels.3,4,11
Most of the antitumor agents currently used in the
treatment of human malignancies are targeted at topo-
isomerase II (topo II). The function of this enzyme is
that of relaxing a supercoiled DNA by allowing double-
stranded DNA (dsDNA) chains to pass one through
another following a cleavage generating blunted DNA
ends. Notwithstanding DNA breaks, genome integrity
is maintained because topo II attaches to the newly
generated DNA termini forming transient enzyme–
DNA complexes.1,2
The topo II inhibitors referred to as ‘poisons’ (etopo-
side, doxorubicin, mitoxantrone and amsacrine) convert
this crucial enzyme into a potent cellular toxin that
Compounds interfering with the catalytic activity of the
enzyme [merbarone 1,12 fostriecin 2,13 aclarubicin,14 and
dexrazoxane (ICRF-187, belonging to bis(2,6-dioxopi-
perazine derivatives 315,16 (Fig. 1)] are expected to be
active against both fast and slow growing cancers.11,17
Among them, merbarone is particularly interesting for
the following reasons: (i) it acts primarily by blocking
*Corresponding author. Tel.: +39-010-353-8370(3); fax: +39-010-353-
8358; e-mail: ranise@unige.it (A. Ranise); e-mail: placolla@unica.it
(P. La Colla)
0968-0896/03/$ - see front matter # 2003Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00158-5

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A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
Figure 1. Structures of topisomerase II inhibitors that do not stabilize DNA–topoisomerase II complexes.
topo II-mediated DNA cleavage without intercalating
into DNA or binding to the minor groove;18 (ii) it
inhibits in vitro topo II decatenation and relaxation
activities12 and causes G2/M blockade;19 (iii) it induces
apoptosis20 by a mechanism distinct from that reported
for the topo II poisons etoposide and doxorubicin.21
In a National Cancer Institute (NCI) screening, mer-
barone showed antitumor activity against the murine
Figure 2. Starting compounds 4 and 528 for the synthesis of series 11
and 12.
L1210 leukemia model as well as against B16 melanoma
and M5076 sarcoma22 (optimum dose range 50–100 mg/
kg). The antitumor efficacy was retained when the
tumor implant site was distant from the drug injection
site, but, in spite of its effects in ip- and sc-implanted
L1210 leukemia models, the drug showed only marginal
activity against the ic-implanted tumors. This might
indicate that, because of its acidity (pKa: 4) and high
ionizability at physiological pH, merbarone does not
efficiently penetrate the CNS. However, thanks to its
activity against B16 melanoma and M5076 sarcoma,
merbarone was tested in phase I and II clinical
trials.23ꢁ26 Although no major antitumor effects were
observed, these studies led to the conclusion that the
drug is relatively well tolerated with few constitutional
symptoms.
Figure 3. Calculated LogP and pKa of compounds 1, 4, 5, 6. (a) These
values were obtained through the ACD/I-Lab service; (b) Lit22: 4.00.
Thereby, since merbarone analogues were little investi-
gated,27 we wish to report the synthesis and anti-
proliferative activity of three new series of basic
merbarone thioanalogues 11–13.
5-substituents (ethoxycarbonyl, phenyl and acetyl), this
structural motif providing a wider range of hydrophilic-
lipophilic balance. As a consequence, increase in lipo-
philicity could be expected to facilitate the ability of the
derivatives to cross CNS and, in more general terms,
biological membranes better than 1. Nevertheless, 4, 5,
6 maintain the same excessive acidity of 1 (see their cal-
culated pKa, Fig. 3). In order to both suppress acid
ionizability and hopefully improve pharmacodynamic
and pharmacokinetic properties of title compounds 11,
12, 13, the 6-sulphur atoms of 4, 5, and the 4-sulphur
atom of 6 were alkylated with o-chloroalkyl-dialkyl- or-
cyclo-amines differing in chain length (two or three car-
bon atoms) and/or in terminal (cyclo)aliphatic tertiary
amine. Some synthetic efforts to prepare the corre-
sponding 6-oxygen analogues failed (data not shown).
A few years ago, some of us have described the one-pot
synthesis of 5-substituted 1,3-diphenyl-2,6-dithiobarbi-
turates 4 and 528 (Fig. 2), having a variety of sub-
stituents not readily available by prior-art synthetic
processes at the 1-, 3- and 5-pyrimidine ring positions.
4, 5 and 6 (here described) share the 2-thiobarbiturate
framework with 1. Owing to the presence of the two
phenyl substituents at the 1 and 3positions, these com-
pounds are more lipophilic than merbarone, as evi-
denced by their calculated LogP (Fig. 3). The different
values can be related to the diverse polarity of the

A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
2577
1
These structural modifications were made considering
that length, flexibility of the alkyl side chain, basicity
and shape of the distal amine may give an important
contribution to both the anticancer activity and the
solubility of aminoalkyl derivatives under physiological
conditions in respect to parent compounds.29ꢁ35 Efforts
to introduce a cyano group at the pyrimidine ring
5-position, starting from malononitrile unexpectedly
gave bicyclic compound 8. Interestingly, 8 could be
considered, in some extent, as a conformationally con-
strained molecule, broadly related to merbarone, on the
basis of following considerations. 1 (as depicted in Fig.
1), better than 428 and 6 (vide infra), can be con-
formationally stabilised by two intramolecular H-bonds
among the 5-amide moiety and the proximal enolizable
carbonyl groups. The result of these interactions is a
molecule mimicking a phenalene-like ring system with
an outward phenyl moiety. In order to better under-
stand if this conformation is a pharmacophore require-
ment, starting from 8, basic congeners 14 were also
synthesized and tested.
spectral data. The H NMR spectrum exhibited a D2O
exchangeable proton peak as a sharp singlet at d 17.92,
which was assigned to the OH proton of the 6-enol
moiety. Comparison of the 13C NMR of 6 with those of
4 and 528 allowed us to attribute the signals at d 196.94,
187.31, 177.24 and 158.36 to the carbonyl carbon of the
5-acetyl group and to the 2,4-dithione and 6-enol car-
bons, respectively. As a consequence, tautomer 6 is
conformationally stabilized through a strong intramo-
lecular hydrogen bond between the 5-CO group and the
6-OH group. The free rotation of acetyl is hindered by
this bond and, consequently, the resonance correspond-
ing to the 6-enol proton occurred at the above high d
value, being the downfield shift a reflection of the
deshielding effect of the close carbonyl group. In addi-
tion, the lack of OH signal in the IR spectrum could be
due to resonance effects which led to a considerable
strengthening of the hydrogen bond. Its pattern seemed
to be similar to that of enolizable b-dichetones, where
the effects described above led to a remarkable decrease
in the intensity of the OH bond to such an extent that
the identification of the band was rather difficult.36
Chemistry
Synthesis of intermediate 8 (Scheme 2) was started by
reacting malononitrile and phenylisothiocyanate (one
molar equivalent) in anhydrous DMF in the presence of
sodium hydride (1.5 molar equivalents). In this way, an
equilibrated mixture of monoanion A and more reactive
dianion A0 of malononitrile carbothiamide was gener-
ated. The reaction of A and A0 with other two molar
equivalents of phenylisothiocyanate added in a second
time, via the cyclic enaminonitrile B, afforded mono/
disodium salt C, that, following treatment with 1 M
acetic acid, yielded 8. Efforts to isolate intermediate B
were unsucessfull. It should be noted that, unlike the
synthetic procedures for the preparation of 4, 5, and 6,
two molar equivalents of sodium hydride gave a more
complex reaction pattern. Compound 8 can be depicted
in other three tautomeric forms 81-3 (Fig. 5), whose
existence was excluded on the basis of the following
Starting compounds 4 and 5 were prepared according to
previously described synthetic methods,28 which were
partially modified to synthesize 6 (Scheme 1) in order to
improve its purity and yield. Firstly, methyl aceto-
acetate and phenylisothiocyanate (three molar equiv-
alents) were allowed to react under cooling in
anhydrous N,N-dimethylformamide (DMF), in the pre-
sence of sodium hydride (one molar equivalent). Then,
to complete the reaction, another molar equivalent of
sodium hydride was added portionwise at different
times (for mechanistic details see ref 28). The reaction
product was easily separated from by-product
O-methyl-N-phenylthiocarbamate 7, owing to its solu-
bility in 1 M sodium carbonate. Compound 6 was better
represented by a chelated 6-enol form, rather than by
tautomers 61-3 (Fig. 4), on the basis of the following
Scheme 1. Synthesis of intermediate 6. Reagents and reaction conditions: (a) NaH (one equiv), C6H5NCS (three equiv), anhydrous DMF, 5 ꢂC, then
NaH (another equiv), rt, 15 h; (b) H2O, 10 M HCl.
Figure 4. Other three possible tautomers of 6.

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A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
Scheme 2. Synthesis of intermediate 8, of its thiomethyl derivative 10 and of 9 (with the proposed mechanism for formation of 8). Reagents and
reaction conditions: (a) C6H5NCS (1 equiv), NaH (1.5 equiv), anhydrous DMF, 5 ꢂC, then C6H5NCS (2 equiv), rt, 15 h; (b) 1M CH3COOH; (c)
C6H5NCS, CH3I, NaH, anhydrous DMF, heat; (d) C6H5NCS (2 equivs), NaH, anhydrous DMF; (e) CH3I, NaHCO3, DMF, heat.
Figure 5. Other three possible tautomers of 8.
spectral evidences. The 13C NMR spectrum of 8 exhib-
ited the carbon signals at d 182.63, 180.78 and 176.49
which were assigned to the three thione carbons at
positions 4, 7, 2 of the pyrimidopyrimidine scaffold,
respectively; while IR spectrum showed no weak SH
factors for the synthesis of intermediates 4, 5 and 6, it is
interesting to note that the reaction gave 8 in good yields,
even when the dianion formation was uncompleted.
Moreover, if conditions preventing the dianion forma-
tion were adopted by using only one molar equivalent of
sodium hydride, or by blocking one of the sites where the
negative charges are preferentially delocalized, as in the
case of thiomethyl derivative 9, 8 was obtained in poor
yield; or 9 did not cyclize to 10, even though it was
allowed to react in DMF solution with one and two
molar equivalents of sodium hydride and phenyli-
sothiocyanate, respectively. We prepared 937,38 by an
alternative one-pot procedure starting from mal-
ononitrile, phenylisothiocyanate and iodomethane in
anhydrous DMF in the presence of sodium hydride
adsorption within the narrow range of 2600–2550 cmꢁ1
.
1
In the H NMR spectrum two D2O exchangeable pro-
ton peaks were present at d 12.35 and 7.60; the former
corresponded to one proton which was assigned to the
5-imine group (its observed high d value could be due to
the hydrogen bond between the imine moiety and the
4-thione group); the latter was attributed to the 8-NH
proton. The synthesis of 8 deserves some comments.
Since the key intermediate dianions originated from
corresponding initial sodium carbothiamides are decisive

A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
2579
(Scheme 2), while 10 was synthesised by methylation of
8 with iodomethane in DMF in the presence of sodium
bicarbonate (Scheme 2). According to methods A–F
and work up procedure WA and WB (see Experimental),
intermediates 4, 5, 6 and 8 were alkylated at the 6-, 4-
and 7-sulphur atoms, respectively, using o-chloroalkyl-
dialkyl- or -cyclo-amines in the presence of diverse bases
(A: NaHCO3, B: Triton B, C: sodium hydride, D:
K2CO3, E: triethylamine, F: 2 equivalents of chloro-
amine), to afford compounds 11a–f,h,j, 12a–k, 13a,b,d–
h,j and 14a,b,d–f,h–k (Scheme 3). The various synthetic
procedures adopted were due to different reactivity of
the starting intermediates towards alkylation. In some
cases, the reaction gave low to moderate yield or did not
occur. Only 5 reacted with each of the relevant o-chloro-
alkylamines. Regio-alkylation of the 7-sulphur atom of 8
was exemplified by the 13C NMR spectra of 10 and 14b,
where the 7-carbon signals, (at d 180.78 in the 13C NMR
spectrum of 8) were found to resonate at d 169.66 and
169.74, respectively, as a result of the thiourea/iso-
thiourea rearrangement. Similarly, alkylation at position
6 of 4 and at position 4 of 6 was proved by the 13C NMR
spectra of 11a and 13b, where the 6- and 4-thione carbon
signals of the precursors (see 13C NMR spectra of 428 and
6) were lacking, due to formation of the enethioalkyl
substructure. Thus, the 6-carbon signals of 11a and 13b
were found to resonate at d 153.36 and 153.66, respec-
tively. Conversely, alkylation of intermediate 5, as exem-
plified by the 13C NMR spectrum of 12b, brought to a
slight difference in the chemical shift of the C-6 atoms of
12b (d 154.18) and of 5 (d 152.67),28 the latter having a
stable enethiolic tautomeric form.
subpanels of nine different types of cell lines derived
from human cancers.39,40 In these assays merbarone was
used as reference drug (Table 1). Both monocyclic and
bicyclic derivatives showed the same potency against
leukemia cell lines (GI50 range: 0.01–3 mM), and turned
out to be up to 100-fold more potent than merbarone.
On the other hand, bicyclic derivatives 14b,d,f,h,i,j were
more potent than monocyclic counterparts 11b,d, 13d,g
against solid tumor cell lines, showing antiproliferative
activity in the concentration range of 1–10 mM against
NSCLC, colon, CNS, melanoma, renal, prostate and
breast cancer cell lines. By contrast, the monocyclic
derivatives exhibited a comparable potency only against
prostate cancer cells. Interestingly, series 14 emerged as
the most cytotoxic, leading to cell death at concentra-
tions only 2–3-fold higher than those required to inhibit
cell growth by 50%.
Meanwhile, new derivatives of series 11, 13, and 14, in
addition to a new series of compounds 12, were syn-
thesized and tested in our laboratory for anti-
proliferative activity (Table 2). As reference
compounds, one derivative of each of the series tested
at NCI (e.g., 11b, 13d and 14b) was included in the
assays. In general, the IC50 values of 11b, 13d and 14b
against MT-4 cells (Table 2) were about 10-fold higher
than the average GI50 values obtained at NCI against
the leukemia cell line subpanel (Table 1). Among the
new derivatives, none showed antiproliferative potency
higher than that of first generation counterparts.
Compounds 11a,c,e,f,h,j, 13a,b,j and 14e resulted as
potent as 11b, 13d and 14b, respectively, whereas deri-
vatives of series 12 were either marginally active
(12a,b,h–j) or inactive (12c–g,k). Taken together, these
data indicate that the antiproliferative activity decrea-
ses in the following order: 14>11>13>12 within the
series.
Biological results
The first synthesized derivatives 11b,d, 13d,g, and
14b,d,f,h,i,j were evaluated in vitro at N.C.I. against
Scheme 3. Synthesis of the title compounds. Reagents and reaction conditions: (a) o-chloroalkyldialkyl/cyclo-amines, various bases and solvents
(methods A–F), heat.

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A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
Table 1. Antiproliferative, cytostatic, cytocide activities (mM)a of selected test compounds against the 9 NCI subpanels of human tumor cells in
culture
Type of tumor
Leukemia
11b
11d
13d
13g
14b
14d
14f
14h
14i
14j
1
b
0.6–2
5
1–31–03.01–nd
5
0.1–0.4
0.3–1.5
1–100
0.4–30
2–60
6–100
1–2
0.04–0.4
0.2–2
5–100
1–10
3–20
6–100
1–2
1–2
4
7–100
1–3
3–10
6–10
1–2
4
6–10
2–8
3–20
5–50
0.3–2
1–4
6–100
1–2
4
7
2
4
7
0.2–1
0.5–3
7
0.6–2
2–5
5–100
1–2
3
5–100
1–5
3–20
5–50
0.2–2
0.5–4
4–100
1–2
3–5
6–100
1–2
10–28
50–100
>100
8–41
20–100
>100
14–47
70–100
>100
25–32
100
5
ndb.
ꢃ100
3–40
10–100
20–100
4–15
15–40
40–100
10–50
30–100
60–100
1–10
1–100
1–20
3–45
10–100
1–10
3–40
6–100
10–30
20–100
50–100
1–15
3–30
10–100
2–50
3–100
6–100
1–15
3ꢄ30
5–50
2
10–100
1–20
4–40
20–100
1–20
4–40
6–100
3–30
10–60
30–100
1–10
3–30
6–70
1–40
4–40
6–100
1–10
2–20
5–50
2
10–100
1–30
3–100
20–100
2–15
4–30
7–80
15–60
30–100
70–100
1–20
3–50
5–100
2–50
5–100
10–100
1–15
3–30
5–50
2–6
NSCLCc
Colon
4
3
4
6–100
2–10
3–30
7–70
1–4
3–15
6–100
1–15
3–30
5–60
1–10
2–20
5–50
2
6–100
2–10
4–30
10–60
6–100
2–5
3–15
5–50
CNS
1–4
3–10
5–50
>100
Melanoma
Ovarian
Renal
1–31–13–2
1–2
1–2
20–50
4–50
3–20
6–100
2–20
3–30
6–60
1–10
3–20
5–50
2
3–5
6–100
1–31–10
3–30
6–60
1–31–31–3
3–10
5–30
2
3–5
6–10
1–10
3–30
5–50
2
3–5
5–10
2
2–5
6–20
2–5
5–30
50–100
>100
10–100
10–50
25–100
100
2–6
3–30
2–20
25–50
3–20
5–50
20–50
2–6
5–20
2
3–30
5–100
70–100
>100
3–7
5–20
2
50–100
>100
14–30
6–50
2–10
Prostate
Breast
4
36–20–20
6
3
25–50
1–20
3–70
6–100
6.16
3
3
3 3 3 830–100
5–15
1–30
3–100
6–100
4.67
5–50
2–40
5–100
60–100
9.33
6
2–6
3–100
6–100
1.82
6
6
2–4
3–100
6–100
2.08
6
6
6
>100
8–30
70–100
>100
1–30
3–20
6–100
4.16
9.77
26.3
2–10
3–100
10–100
1.95
5.12
17.37
0;3–3
3–100
6–100
1.66
3.98
8.91
0.3–3
1–100
6–100
1.54
3.71
10.71
0.3–3
1ꢄ100
6–100
1.66
3.89
12.30
MG-MID GI50
MG-MID TGI
MG-MID LC50
10.9
30.9
15.84
40.73
26.30
56.23
4.78
15.48
5.24
15.13
aIn each column, the GI50, TGI, LC50 range values (mM) (1st, 2nd, 3rd row for each type of tumor, respectively) of the most active compound, are
reported. Likewise, at the bottom of each column are also reported the MG-MID GI50/TGI/LC50 (mM) parameters of the compound, evaluating the
average antitumor activity against the entire panel of cell lines. These data were generously provided by Developmental Therapeutics Program,
N.C.I.
bNot determined.
cNon-small cell lung cancer.
Multidrug resistance (MDR)41,42 and atypical topo-
isomerase-mediated MDR (at-MDR)17,43 are among the
main reasons for chemotherapy failure. Since drug-
resistant cell lines have been reported to express partial17
cross-resistance to merbarone, we deemed it interesting
to investigate the susceptibility of drug-resistant KB
subclones to the basic merbarone thioanalogues. There-
fore, 14b and 11b, representative compounds of the two
most potent series, were tested against cell lines
(KBMDR and KBV20C) over-expressing the drug efflux
pump MDR1/P-glycoprotein responsible for the MDR
phenotype44,45 and against an etoposide-resistant KB cell
line46 (KB7D) that, besides a 2-fold decrease in topo II
levels, over-expresses a protein, referred to as multidrug-
resistance associated protein (MRP), which is known to
reduce the uptake of etoposide and other antineoplastic
agents (MDR phenotype). Compounds 11b and 14b were
also evaluated against a camptothecin-resistant KB cell
line47 (KBCPT300) expressing altered levels of topo I
(approximately 30% of parental KB cells).
Consistently with previous results,46 the KB7D cells
proved highly resistant to the topo II poisons doxo-
rubicin (56-fold) and etoposide (33-fold) and moder-
ately resistant to the antimitotic drug vincristine
(10-fold). By contrast, the KB7D cells showed only
marginal resistance (approximately 2-fold) to merbar-
one, whereas KBMDR cells did not show altered sensi-
tivity to the reference drug.
In order to assess whether the antiproliferative activity
of 11b and 14b correlated with inhibition of topoisome-
rase II catalytic activity, as suggested by the above
results, a cell-free system (see Experimental) was used.48
This inhibition effect was compared with that of the
standard topo-II inhibitor etoposide (VP16) and Mer-
barone. Like VP16, 11b and 14b were able to inhibit the
formation of supercoiled DNA from relaxed DNA (Fig.
6). Interestingly, they turned out to be more potent
inhibitors than Merbarone (Fig. 6).
The title compounds were also randomly assayed
against HIV-1 infected MT-4 cells for their ability to
inhibit the virus-induced cytopathogenicity. However,
none of them was able to prevent the HIV-induced
cytopathogenicity at non cytotoxic concentrations (data
not shown). Furthermore, they were evaluated for their
In general, 11b and 14b were 2–3-fold less active against
the KB resistant subclones (Table 3) than against the
parental KB cell line. The sole exception was the KB7D
subclone, which was 20-fold less susceptible to the test
compounds.

A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
2581
Table 2. Antiproliferative activity of derivatives 11, 12, 13 and 14
against MT-4 cells
Compd
Synthetic route/Work-up procedurea
IC50 (mM)b
11a
11b
11c
11d
11e
11f
11h
11j
12a
12b
12c
12d
12e
12f
12g
12h
12i
A/WA
A/WA
B/WA
A/WA
A/WA
A/WA
A/WA
A/WA
D/WA
A/WA
A/WA
B/WA
A/WA
D/WA
B/WA
A/WA
A/WA
A/WA
B/WA
E/WA
F/WA
B/WA
A/WA
A/WA
F/WA
E/WA
A/WA
B/WB
B/WB
D/WB
C/WB
C/WB
B/WB
B/WB
B/WB
B/WB
12.1ꢅ1.4
4.3ꢅ0.8
11.0ꢅ1.3
NDc
10.5ꢅ1.3
12.7ꢅ1.6
13.0ꢅ0.9
11.5ꢅ1.5
88.3ꢅ5.3
78.5ꢅ6.1
>200
>200
>200
>200
>200
63.6ꢅ4.9
66.6ꢅ5.3
42.4ꢅ3.3
>200
10.3ꢅ1.4
12.1ꢅ1.1
12.0ꢅ2.0
>200
12j
12k
13a
13b
13d
13e
13f
13g
13h
13j
14a
14b
14d
14e
14f
14h
14i
>200
NDc
Figure 6. Inhibitory effects of Etoposide, Merbarone, 11b and 14b on
the catalytic activity of topoisomerase II. The first five lanes represent
controls; lane 1: Marker Linear KDNA, lane 2: Marker Decatenated
KDNA, lane 3: Kinetoplast DNA (KDNA), lane 4: Topo-II activity,
lane 5: Topo-II activity in the presence of DMSO [0.1%]. Lane 6:
Topo-II activity in the presence of VP16 [100 mM]; lane 7: Topo-II
activity in the presence of Merbarone [100 mM]; lane 8: Topo-II
activity in the presence of 11b [100 mM]; lane 9: Topo-II activity in the
presence of 14b [100 mM].
35.4ꢅ2.7
12.2ꢅ1.6
15.5ꢅ1.2
3.3ꢅ0.6
ND
3.6ꢅ0.9
NDc
NDc
NDc
14j
14k
NDc
None of the compounds was active against the tested
bacteria and fungi (data not shown).
109.4ꢅ12
aThe compounds were prepared and isolated according to methods A–
F and work up procedures WA and WB, described in Experimental.
bIC50 (Inhibitory concentration fifty): the values represent the drug
concentration (ꢅS.D.) required to reduce cell growth by 50% with
respect to untreated controls, as measured by the MTT method.
cNot determined.
Discussion
This study is part of an ongoing research program con-
cerning the design of new simple or fused poly-
substituted pyrimidines as lead compounds for the
development of antitumor agents.
capability to inhibit the multiplication of various human
pathogenic fungi (Candida albicans, C. parapsilosis,
C. paratropicalis, Aspergillus fumigatus and Criptococcus
neoformans) and bacteria (Staphylococcus aureus, group
D Streptococcus, Salmonella spp and Shigella spp).
Miconazole and streptomycin were used as reference
compounds in antimicotic and antibacterial assays.
In order to better elucidate pharmacophore require-
ments, some structure–activity relationships can be
made. Many of the active compounds in the tested series
have aminoalkyl side chains with different length and/or
amine shape, although in some instances diethylamino/
pyrrolidino-ethyl, piperidino-ethyl/-propyl appear to
Table 3. Antiproliferative activity of derivatives 11b and 14b in parental and drug-resistant KB cell lines
IC50 (mM)a
Compd
KBwt
KBMDR
KBV20C
KB7D
KBCPT300
11b
14b
2.4ꢅ0.4
0.8ꢅ0.2
6.6ꢅ0.8
3.0ꢅ0.5
20.7ꢅ2.7
1.8ꢅ0.1
0.7ꢅ0.2
>20
7.4ꢅ0.9
2.9ꢅ0.318.0
ND
0.35ꢅ0.1
0.2ꢅ0.09
6.2ꢅ1.8
0.09ꢅ0.01
46.1ꢅ4.9
ꢅ3.1
7.5ꢅ0.8
2.7ꢅ0.4
ND
Merbarone
Doxorubicin
Vincristine
Etoposide
Camptothecin
18.3ꢅ2.5
0.06ꢅ0.01
0.006ꢅ0.001
0.6ꢅ0.1
42.3ꢅ3.7
2.8ꢅ0.30.19
0.05ꢅ0.01
>20
ꢅ0.1
0.018ꢅ0.01
1.5ꢅ0.2
10.0ꢅ2.3
0.03ꢅ0.01
0.03ꢅ0.02
0.19ꢅ0.07
aIC50 (Inhibitory concentration fifty): values represent the drug concentration (ꢅS.D.) required to reduce cell growth by 50% with respect to
untreated controls, as measured by the MTT method.

2582
A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
give the greatest contribution to the antitumor activity.
However, in the pyrimidine series the structural
requirements of 5-substituent seem to be quite stringent.
Thus, the presence of a 5-phenyl ring causes drop or loss
of antiproliferative activity as in derivatives 12, prob-
ably owing to capacity of this moiety to elicit unfavor-
able p–p or lipophilic interactions. Conversely,
replacement of the phenyl ring with polar groups
(ethoxycarbonyl and acetyl in series 11 and 13, respec-
tively) capable of electrostatic interactions, enhances
activity. In series 13, cytotoxicity data also reveal a close
dependence on length and amine size of the basic side
chain. Thus, unlike 11e,f, 13e and 13f, bearing the piper-
idinoethyl and morpholinoethyl moieties, were devoid of
activity. Conversely, homologues 13j,g were active. Fur-
ther comparison of antiproliferative effects induced by
homologous extension of the side chains inside the same
series indicates that, with the exception of 11e,j and
13d,h, compounds carrying the 3-aminopropyl side
chains are either equipotent (11h–11a, 14j–14e) or more
potent than derivatives bearing the 2-aminoethyl moi-
eties [12h>12d, 12jꢀ12e (inactive), 13jꢀ13e (inactive),
14h>14a)].
over anhydrous sodium sulphate and concentrated
using a rotatory evaporator operating at reduced pres-
sure of about 15–20 Torr. TLC systems for routine
monitoring of reaction mixture and confirming the
homogeneity of analytical samples employed alumi-
nium-backed silica gel plates (Merck DC-Alufolien
Kieselgel 60 F254) with chloroform-methanol as devel-
oping solvents. Developed plates were visualized by UV
light and iodine. Melting points were determined on a
Fisher–Johns (mp <300 ꢂC) or an Electrothermal
apparatus (mp >300 ꢂC) and are uncorrected. Micro-
analyses were performed by an EA 1110 Elemental
Analyser, FISON Instruments (Milan).
IR spectra were recorded on a Perkin–Elmer 398 spec-
1
trometer as KBr discs. H and 13C NMR spectra were
recorded in CDCl3 on a Varian Gemini 200 instru-
ments. Chemical shifts were reported in d (ppm) units
relative to the internal reference tetramethylsilane, and
the splitting patterns were described as follows: s (sing-
let), d (doublet), t (triplet), m (multiplet) and brs (broad
singlet). First order values reported for coupling con-
stants were given in hertz.
Side chain elaboration in terms of steric load shows
that: (i) a methyl group at position 2 of the 3-amino-
propyl chains does not cause any difference in anti-
proliferative activity with respect to the unbranched
compounds (compare 14i with 14h and 12i with 12h); (ii)
a 3-chlorophenyl substituent at the piperazine 4-posi-
tion lowers antiproliferative activity, as found in 14k.
This indicates that steric bulk (and/or aromatic char-
acter) of substituents on the distal aliphatic amine ring
could cause adverse effects on activity.
Synthesis of 1-(6-hydroxy-1,3-diphenyl-2,4-dithioxo-
1,2,3,4-tetrahydropyrimidin-5yl)ethanone
(6).
60%
sodium hydride dispersion in mineral oil (8.0 g, 0.2 mol)
was added in a single portion to a stirred, ice-cooled,
anhydrous DMF solution (200 mL) of methyl aceto-
acetate (23.3 g, 0.2 mol). When hydrogen evolution
subsided, neat phenylisothiocyanate (81.23g, 0.6 mol)
was poured into this mixture in a single portion. Then,
60% sodium hydride suspension in mineral oil (8.0 g,
0.2 mol) was added in three portions (5+1+2 g) after
15 min, 1 and 1.5 h, respectively. The resulting mixture
was allowed to react at room temperature for 15 h,
treated with ice-cooled water (600 mL) and extracted
thoroughly with diethyl ether–petroleum ether (bp
40–70 ꢂC) 2:1 (50 mLꢆ2). The extracts were washed
with water (20 mLꢆ5), dried and evaporated under
reduced pressure to give an oily residue which slowly
crystallized. Recrystallization from diethyl ether-petro-
leum ether 7:1 afforded a first crop (5.25 g) of O-methyl-
N-phenylcarbamate 7, mp 96–97 ꢂC.28 The aqueous
solution was cooled with crushed ice and acidified with
10 M HCl (300 mL) to give a red-brown precipitate
which was filtered, washed with water and dissolved in
dichloromethane. The organic phase was extracted with
1 M sodium carbonate (180 mLꢆ3). During the first
extraction the voluminous precipitate formed (sodium
salt of 6) was dissolved upon dilution with water. The
dichloromethane extract was then rinsed with water,
dried and evaporated under reduced pressure. The resi-
due was chromatographed on neutral alumina (eluents:
diethyl ether–dichloromethane 10:1) to yield the bulk of
7 (35.0 g, 74% overall yield). The cooled alkaline aqu-
eous solution furnished by acidification with 10 M HCl
a brown precipitate which was filtered, washed and dis-
solved in dichloromethane. The organic phase was
washed with water (100 mL), dried and evaporated in
vacuo. The solid residue was crystallized from di-
chloromethane–methanol to give 6 (63.0 g, 89% yield)
as an ochre solid. Mp 238–240 ꢂC, IR (KBr) cmꢁ1 1690;
In conclusion, biological data suggest that, in general,
change of the physico-chemical properties of the title
compounds, due to increase of lipophilicity and sup-
pression of acid ionizability, resulting from alkylation of
the reactive sulphur atoms of key-intermediates 4, 5, 6,
and 8 with o-chloro alkyl amines, are beneficial to
activity. Moreover, introduction of conformational
constrain, as in series 14, or replacement of the 5-phe-
nylcarbamoyl moiety of 1 by groups with a higher
degree of polarity (ethoxycarbonyl, acetyl), as in series
11 and 13, are additional structural parameters enhan-
cing potency. These results encourage further investiga-
tion in animal models to establish whether also
pharmacodinamic and pharmacokinetic properties of
the title compounds are improved.
Experimental
Materials and methods
Chemicals (methyl phenylacetate, ethyl malonate,
methyl acetoacetate, malonitrile, phenylisothiocyanate,
60% sodium hydride suspension in mineral oil,
NaHCO3, K2CO3, triethylamine, Triton B, o-chloro-
alkylamines) were purchased from Aldrich Chimica,
Milan (Italy). Solvents were reagent grade. DMF was
dried on molecular sieves. Organic solutions were dried

A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
2583
1H NMR (CDCl3) d 2.83(s, 3H, CH 3/acetyl-H), 6.92–
7.23(m, 10H, arom H), 17.92 (sharp s, 1H, exchange-
able, enol-H); 13C NMR (CDCl3) d 28.07, 107.82, 128.1,
128.64, 128.88, 129.51, 139.42, 142.16, 158.36 (6-C),
177.24 (2-C), 187.31 (4-C), 196.94 (CO). Elemental
analysis: calcd for C18H14N2O2S2; C, 61.00; H, 3.98; N,
7.90; S, 18.09%; found; C, 61.02; H, 3.99; N, 7.98; S,
18.28%.
1485, 1430; 1H NMR d 1.70 (s, 3H, thiomethyl-H),
6.95–7.30 (m, 15H, arom H), 11.75 (s, 1H, exchange-
able, imine-H); 13C NMR (CDCl3) d 15.41, 128.91,
129.08, 129.16, 129.86, 130.28, 130.60, 137.34, 141.26,
144.72, 150.12, 155.70 (5-C), 169.66 (7-C), 179.69 (2-C),
184.84 (4-C). Elemental analysis: calcd for C25H19N5S3;
C, 61.83; H, 3.94; N, 14.42; S, 19.80%; found C, 61.85;
H, 3.93; N, 14.22; S, 19.63%.
Synthesis of 5-imino-1,3,6-triphenyl-5,8-dihydropyri-
mido[4,5-d] pyrimidine-2,4,7(1H,3H,6H)-trithione (8).
To a stirred, ice-cooled anhydrous DMF solution (150
mL) of malonitrile (6.9 g, 0.1 mol) and phenylisothio-
cyanate (13.7 g, 0.1 mol), 60% sodium hydride disper-
sion in mineral oil (6 g, 0.15 mol) and, after 10 min, neat
phenylisothiocyanate (27.04 g, 0.2 mol) were added in a
single portion. The resulting mixture was stirred at
0–5 ꢂC for 15 min and then at room temperature for 15
h. Reaction was quenched by adding ice-cooled 1 M
acetic acid (250 mL). The orange precipitate formed was
filtered and dissolved in dichloromethane. The organic
phase was washed with water (40 mL), 4 M sodium
hydroxyde (20ꢆmL) and dried. Concentration in vacuo
yielded 8 (33.1 g, 70.2% yield), which was crystallized
from dichlomethane–methanol 3:2 as a yellow-orange
solid; mp 314–316 ꢂC; IR (KBr) cmꢁ1 3410, 3280, 1610,
General procedure A for the preparation of compounds
11a,b,d,e,f,h,j, 12b–e,h,j, 13e,f,j. To a stirred mixture of
4 (3.85 g, 10 mmol) or 5 (3.89 g, 10 mmol) or 6 (3.54 g,
10 mmol) and sodium bicarbonate (1.0 g, 12 mmol) in
DMF (50 mL), the proper o-chloroalkylamine (12
mmol) was added at room temperature after 15 min.
Then, the reaction mixture was heated at 55–60 ꢂC for 4
h under stirring.
Work-upA. The solid, that separated after treatment
with water (300 mL), was filtered, dissolved in di-
chloromethane, dried and evaporated in vacuo to dry-
ness. The oily or solid residues were purified by
crystallization from proper solvent mixtures.
Physical and chemical data of compounds 11a,b,d,e,f,h,j
1
1590, 1540, 1305; H NMR (DMSO-d6) d 6.90–7.75 (m,
15H, arom H), 7.90 (s, 1H, exchangeable, NH), 12.35 (s,
1H, exchangeable, imine-H); 13C NMR (CDCl3) d
97.91; 128.06, 128.14, 128.73, 129.01, 129.35, 129.71,
130.68, 137.77, 140.75, 144.08, 149.05, 156.95 (5-C),
176.49 (2-C), 180.78 (7-C), 182.63(4-C). Elemental
analysis: calcd for C24H17N5S3; C, 61.14; H, 3.64; N,
14.86; S, 20.36%; found; C, 61.00; H, 3.76; N, 14,71; S,
20.16%.
Ethyl 6-[(2-dimethylaminoethyl)thio]-4-oxo-1,3-diphenyl-
2 - thioxo - 1,2,3,4 - tetrahydropyrimidine - 5 - carboxylate
(11a). 2.87 g (63% yield), mp 208–210 ꢂC (from di-
1
chloromethane/ethanol). IR (KBr) cmꢁ1 1730, 1685; H
NMR (CDCl3) d 1.35 (t, 3H, J=8 Hz, CH3/ethoxy-H),
2.09 (s, 6H, 2 CH3/dimethylamino-H), 2.40 (t, 2H, J=7
Hz, S-CH2/ethyl-H), 2.90 (t, 2H, J=7 Hz, N-CH2/ethyl-
H), 4.40 (q, 2H, J=8 Hz, CH2/ethoxy-H), 7.02–7.74 (m,
10H, arom H). 13C NMR (CDCl3) d 14.53, 35.80, 45.34,
57.84, 63.02, 119.62, 128.46, 129.32, 129.91, 130.17,
139.64, 141.81,153.36 (6-C), 157.26 (4-C), 163.95
(COOC2H5), 179.83(2-C). Elemental analysis: calcd for
C23H25N3O3S2; C, 64.64; H, 5.53; N, 9.22; S, 14.07%;
found C, 64.40; H, 5.50; N, 9.04; S, 13.94%.
Synthesis of 3-Anilino-2-cyano-3-methylthioacrylonitrile
(9). 60% sodium hydride dispersion in mineral oil (0.88
g, 20 mmol) was added in a single portion to a stirred, ice-
cooled anhydrous DMF solution (25 mL) of mal-
ononitrile (1.32 g, 20 mmol), phenylisothiocyanate (2.70 g,
20 mmol) and iodomethane (2.84 g, 20 mmol). The
resulting mixture was stirred at room temperature for 1 h,
then at 50–55 ꢂC for 15 min. The solid separated after
adding water (150 mL) was filtered, dissolved in dichloro-
methane and dried. Evaporating in vacuo to dryness gave
a residue that was crystallized from methanol–dichloro-
methaneꢂ4:1 to give colorless needles (3.87 g, 90%); mp
178–179 C (literature values 176 ꢂC,37 177 ꢂC38).
Ethyl 6-[(2-diethylaminoethyl)thio]-4-oxo-1,3-diphenyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (11b).
3.96 g (82% yield), mp 159–161 ꢂC (from dichloro-
methane/2-propanol). IR (KBr) cmꢁ1 1725, 1675; 1H
NMR (CDCl3) d 0.9 (t, 6H, J=7.0 Hz, 2CH3/diethyila-
mino-H), 1.37 (t, 3H, J=7.0 Hz, CH3/ethoxy-H), 2.05–
3.18 (m, 8H, S-CH2, N-CH2/ethyl-H and 2CH2/diethyl-
amino-H), 4.40 (q, 2H, J=7.0 Hz, CH2/ethoxy-H),
7.03–7.40 (m, 10H, arom H). Elemental analysis: calcd
for C25H29N3O3S2; C, 62.09; H, 6.04; N, 8.69; S,
13.26%; found C, 62.08; H, 5.91; N, 8.53; S, 13.35%.
Synthesis of 5-imino-7-(methylthio)-1,3,6-triphenyl-5,6-
dihydropyrimido[4,5-d] pyrimidine-2,4(1H,3H)-dithione
(10). A mixture of 8 (4.72 g, 10 mmol), sodium bicar-
bonate (0.84 g, 10 mmol) and iodomethane (1.42 g, 10
mmol) in DMF (20 mL) was stirred at room tempera-
ture for 1 h and then heated at 55 ꢂC for 2 h. After
treatment with water (100 mL) the precipitated formed
was collected by filtration and dissolved in dichloro-
methane. The organic phase was dried and evaporated
under reduced pressure to give a residue which, after
crystallization from dichloromethane–methanol–diethyl
ether 1:2:2, afforded 10 (3.7 g, 76%) as a yellow-orange
solid; mp 265–267 ꢂC; IR (KBr) cmꢁ1 3180, 1615, 1590,
Ethyl 4-oxo-1,3-diphenyl-6-[(2-pyrrolidin-1-ylethyl)thio]-
2 - thioxo - 1,2,3,4 - tetrahydropyrimidine - 5 - carboxylate
(11d). 4.24 g (88% yield), mp 190–191 ꢂC (from di-
1
chloromethane/ethanol). IR (KBr) cmꢁ1 1720, 1680, H
NMR (CDCl3) d 1.37 (t, 3H, J=7.0 Hz, CH3/ethoxy-
H), 1.58–2.08 (m, 4H, 2CH2/pyrrolidine-H), 2.19–3.12
(m, 8H, S-CH2, N-CH2/ethyl-H and 2CH2/pyrrolidine-
H), 4.41 (q, 2H, CH2/ethoxy-H), 7.05–7.80 (m, 10H,
arom H). Elemental analysis: calcd for C25H27N3O3S2;

2584
A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
C, 62.35; H, 5.65; N, 8.72; S, 13.31%; found C, 62.51;
H, 5.54; N, 8.50; S, 13.08%.
2.27 (m, 4H, S-CH2, N-CH2/ethyl-H), 7.23–7.7 (m, 15H,
arom H). 13C NMR (CDCl3) d 12.27, 34.82, 47.08,
51.31, 121.90, 128.62, 128.71, 128.98,129.68, 129.80,
129.98, 131.16, 133.43, 140.51,142.96, 154.18 (6-C),
159.56 (4-C), 163.95, 179.33 (2-C). Elemental analysis:
calcd for C28H29N3OS2; C, 68.96; H, 5.99; N, 8.62; S,
13.15%; found C, 69.11; H, 6.01; N, 8.85; S, 12.94%.
Ethyl 4-oxo-1,3-diphenyl-6-[(2-piperidin-1-ylethyl)thio]-2
- thioxo - 1,2,3,4 - tetrahydropyrimidine - 5 - carboxylate
(11e). 4.31 g (87% yield), mp 187–188 ꢂC (from di-
1
chloromethane/ethanol). IR (KBr) cmꢁ1 1720, 1685; H
NMR(CDCl3) d 1.14–1.89 (m, 9H, CH3/ethoxy-H and 3
CH2/piperidine-H), 2.04–2.59 (m, 4H, 2 CH2/piper-
idine-H), 2.43(t, 2H, J=6.0 Hz, S-CH2/ethyl), 2.93(t,
2H, J=6.0, N-CH2/eyhyl-H), 4.42 (q, 2H, J=6.0 Hz,
CH2/ethoxy-H), 7.10–7.84 (m, 10H, arom H). Elemental
analysis: calcd for C26H29N3O3S2; C, 63.00; H, 5.90; N,
8.48; S, 12.94%; found C, 62.86; H, 5.87; N, 8.36; S,
12.72%.
6-[(2-Diisopropylaminoethyl)thio]-1,3,5-triphenyl-2-thi-
oxo-2,3-dihydropyrimidin-4-(1H)-one (12c). 3.61 g (70%
yield), mp 203–204 ꢂC (from dichloromethane/metha-
nol). IR (KBr) cmꢁ1 1670, 1390, 1320; 1H NMR
(CDCl3) d 0.77 (d, 12H, J=6.0 Hz, 4CH3/diisopropyl-
amino-H), 2.15–2.4 (m, 4H, S-CH2, N-CH2/ethyl-H),
2.35–297 (m, 2H, 2CH/diisopropylamino-H), 7.05–7.72
(m, 15H, arom H). Elemental analysis: calcd for
C30H33N3OS2; C, 69.87; H, 6.45; N, 8.15; S, 12.43%;
found C, 70.00; H, 6.45; N, 8.30; S, 12.31%.
Ethyl 6-[(2-morpholin-4-ylethyl)thio]-4-oxo-1,3-diphenyl-
2 - thioxo - 1,2,3,4 - tetrahydropyrimidine - 5 - carboxylate
(11f). 4.18 g (84% yield), mp 201–202 ꢂC (from di-
1
chloromethane/ethanol). IR (KBr) cmꢁ1 1720, 1685; H
1,3,5-Triphenyl-6-[(2-piperidin-1-ylethyl)thio]-2-thioxo-
2,3-dihydropyrimidin-4-(1H)-one (12e). 4.15 g (83%
yield), mp 231–233 ꢂC (from dichloromethane/metha-
NMR (CDCl3) d 1.34 (t, 3H, J=7.0 Hz, CH3/ethoxy-
H), 2.04–2.69 (m, 6H, S-CH2/ethyl-H and 2CH2/mor-
pholine-H) 2.92 (t, 2H, J=6.0 Hz, N-CH2/ethyl-H),
3.19–3.86 (m, 4H, 2CH2/morpholine-H), 4.40 (q, 2H,
J=7.0 Hz, CH2/ethoxy-H), 7.06–7.82 (m, 10H, arom
H). Elemental analysis: calcd for C25H27N3O4S2; C,
60.34; H, 5.47; N, 8.44; S, 12.89%; found C, 60.40; H,
5.48; N, 8.37; S, 12.97%.
1
nol). IR (KBr) cmꢁ1 1675, 1555, 1390, 1330; H NMR
(CDCl3) d 1.30–1.60 (m, 6H, 3 CH2/piperidine-H),
1.98–2.35 (m, 8H, S-CH2, N-CH2/ethyl-H and 2 CH2/
piperidine-H), 7.25–7.67 (m, 15H, arom H). Elemental
analysis: calcd for C29H29N3OS2; C, 69.71; H, 5.85; N,
8.41; S, 12.83%; found C, 69.74; H, 5.99; N, 8.65; S,
12.97%.
Ethyl 6-[(3-dimethylaminopropyl)thio]-4-oxo-1,3-diphenyl
- 2 - thioxo - 1,2,3,4 - tetrahydropyrimidine - 5 - carboxylate
(11h). 3.00 g (64% yield), mp 137–139 ꢂC (from di-
6-[(3-Dimethylaminopropyl)thio]-1,3,5-triphenyl-2-thi-
oxo-2,3-dihydropyrimidin-4-(1H)-one (12h). 3.69 g (78%
yield), mp 194–195 ꢂC (from dichloromethane/metha-
1
chloromethane/ethanol). IR (KBr) cmꢁ1 1725, 1680; H
1
NMR (CDCl3) d 1.36 (t, 3H, J=7.0 Hz, CH3/ethoxy-
H), 1.48–1.91 (m, 2H, CH2/propyl-H), 1.97–2.38 (m,
2H, S-CH2/propyl-H), 2.13(s, 6H, 2CH 3/dimethyl-
amino-H), 2.78 (t, 2H, J=7.0, N-CH2/propyl-H), 4.40
(q, 2H, J=7.0 Hz, CH2/ethoxy-H), 7.02–7.84 (m, 10H,
arom H). Elemental analysis: calcd for C24H27N3O3S2;
C, 61.38; H, 5.80; N, 8.95; S, 13.65%; found C, 61.19;
H, 5.69; N, 8.70; S, 13.44%.
nol). IR (KBr) cmꢁ1 1672, 1560, 1390, 1327; H NMR
(CDCl3) d 1.2–1.75 (m, 2H, CH2/propyl-H) 1.77–2.28
(m, 4H, S-CH2, N-CH2/propyl-H), 2.09 (s, 6H, 2CH3/
dimethylamino-H), 7.3–7.7 (m, 15, arom H). Elemental
analysis: calcd for C27H27N3OS2; C, 68.47; H, 5.75; N,
8.87; S, 13.54%; found C, 68.24; H, 5.75; N, 8.81; S,
13.74%.
6-{[3-(Dimethylamino)-2-methylpropyl]thio}-1,3,5-triphe-
nyl-2-thioxo-2,3-dihydropyrimidin-4-(1H)-one (12i). 4.34
g (89% yield), mp 168–170 ꢂC (from dichloromethane/
Ethyl 1,3-diphenyl-6-[(3-piperidin-1-ylpropyl)thio]-4-oxo-
2 - thioxo - 1,2,3,4 - tetrahydropyrimidine - 5 - carboxylate
(11j). 4.08 g (80% yield), mp 143–145 ꢂC (from di-
1
methanol). IR(KBr) cmꢁ1 1670, 1555, 1388, 1325; H
1
chloromethane/ethanol). IR (KBr) cmꢁ1 1725, 1690; H
NMR (CDCl3) d 0.63(d, 3H, J=6.0 Hz, methyl-H),
1.37–1.88 (m, 3H, CH- and S-CH2/propyl-H),
1.90–2.10 (m, 2H, N-CH2/propyl-H), 2.06 (s, 6H,
2CH3/dimethylamino-H), 7.2–7.73(m, 15H, arom H).
Elemental analysis: calcd for C28H29N3OS2; C, 68.98;
H, 5.99; N, 8.62; S, 13.15%; found C, 68.72; H, 6.11;
N, 8.62; S, 12.95%.
NMR (CDCl3) d 1.07–1.9 (m, 11H, CH3/ethoxy-, CH2/
propyl-H and 3CH2/piperidine-H), 1.99–2.50 (m, 6H,
S-CH2/propyl-H and 2CH2/piperidine-H), 2.75 (t, 2H,
J=7.0 Hz, N-CH2/propyl-H), 4.40 (q, 2H, J=7.0 Hz,
CH2/ethoxy-H), 7.05–7.77 (m, 10H, arom H). Elemental
analysis: calcd for C27H31N3O3S2; C, 63.63; H, 6.13; N,
8.24; S, 12.58%; found C, 63.41; H, 6.07; N, 8.13; S,
12.39%.
1,3,5-triphenyl-6-[(3-piperidin-1-ylpropyl)thio]-2-thioxo-
2,3-dihydropyrimidin-4-(1H)-one (12j). 4.42
g (86%
yield), mp 155–156 ꢂC (from dichloromethane/metha-
Physical and chemical data of compounds 12b,c, e, h, i, j
1
nol). IR (KBr) cmꢁ1 1680, 1555, 1385, 1330; H NMR
6-[(2-diethylaminoethyl)thio] -1,3,5-triphenyl-2-thioxo-
2,3-dihydropyrimidin-4-(1H)-one (12b). 3.66 g (75%
yield), mp 190–191 ꢂC (from dichloromethane/metha-
nol). IR (KBr) cmꢁ1 1670, 1390, 1325; 1H NMR
(CDCl3) d 0.82 (t, 6H, J=7.6 Hz, 2CH3/diethylamino-
H), 2, 25 (q, 4H, J=7.6 Hz, 2CH2/diethylamino-H),
(CDCl3) d 1.3–1.73 (m, 8H, CH2/propyl-H and 3CH2/
piperidine-H), 1.82–2.38 (m, 8H, S-CH2, N-CH2/pro-
pyl-H and 2CH2/piperidine-H), 7.20–7.65 (m, 15H,
arom H). Elemental analysis: calcd for C30H31N3OS2;
C, 70.14; H, 6.08; N, 8.18; S, 12.48%; found C, 70.01;
H, 6.06; N, 8.14; S, 12.20%.

A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
2585
Physical and chemical data of compounds 13e,f,j
dichloromethane/2-propanol). IR (KBr) cmꢁ1 3190,
1620; H NMR (CDCl3) d 1.86–2.21 (m, 2H, S-CH2/
1
5-Acetyl-1,3-diphenyl-6-[(2-piperidin-1-ylethyl)thio]-2-thi-
oxo-2,3-dihydropyrimidin-4(1H)-one (13e). 2.42 g (52%
yield), mp 182–184 ꢂC (from dichloromethane/ethanol).
ethyl-H), 2.01(s, 6H, 2CH3/dimethylamino-H), 2.31–2.58
(m, 2H, N-CH2/ethyl-H), 7.1–7.78 (m, 15H, arom H),
11.45 (s,1H, exchangeable, imine-H). Elemental analysis:
calcd for C28H26N6S3; C, 61.97; H, 4.83; N, 15.48; S,
17.72%; found C, 61.75; H, 4.72; N, 15.29; S, 17.57%.
1
IR (KBr) cmꢁ1 1680, 1385, 1330; H NMR (CDCl3) d
1.25–1.55 (m, 6H, 3CH2/piperidine-H), 2.05–3.0 (m, 8H,
S-CH2, N-CH2/ethyl-H and 2CH2/piperidine-H), 2.60
(s, 3H, acetyl-H), 7.15–7.65 (m, 10H, arom H). Ele-
mental analysis: calcd for C25H27N3O2S2; C, 64.49; H,
5.84; N, 9.02; S, 13.77%; found C, 64.26; H, 5.98; N,
8.94; S, 13.45%.
7-[(2-Diethylaminoethyl)thio]-5-imino-1,3,6-triphenyl-5,6
-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dithione
(14b). 1.48 g (26% yield), mp 193–195 ꢂC (from di-
chloromethane/2-propanol/petroleum ether). IR (KBr)
cmꢁ1 3190, 1625; 1H NMR (CDCl3) d 0.86 (t, 6H,
J=7.0 Hz, 2 CH3/diethylamino-H), 2.29 (q, 4H, J=7.0
5-Acetyl-6-[(2-morpholin-4-ylethyl)thio]-1,3-diphenyl-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one (13f). 3.60
g
Hz, 2 CH2/diethylamino-H), 1.98–2.63(m, 4H, S-CH ,
2
(77% yield), mp 240–242 ꢂC (from dichloromethane/eth-
N-CH2/ethyl-), 7.05–7.78 (m, 15H, arom H), 11.50 (s,
1H, exchangeable, imine-H). 13C NMR (CDCl3) d
31.93, 45.67, 57.67, 104.31, 128.91, 129.01, 129.09,
129.19, 129.99, 130.28, 130.53, 130.64, 137.34, 141.35,
144.75, 150.16, 155.78 (4-C), 169.74 (2-C), 176.73(7-C),
184.73(5-C). Elemental analysis: calcd for C 30H30N6S3;
C, 63.13; H, 5.30; N, 14.72; S, 16.85%; found C, 63.18;
H, 5.26; N, 14.74; S, 16.50%.
1
anol). IR (KBr) cmꢁ1 1704, 1670, 1385, 1320; H NMR
(CDCl3) d 2.08–2.70 (m, 6H, S-CH2/ethyl-H and 2CH2/
morpholine-H), 2.65 (s, 3H, acetyl), 2.85 (t, 2H, J=7.0
Hz, N-CH2/ethyl-H), 3.40–3.75 (m, 4H, 2CH2/morpho-
line-H), 7.15–7.70 (m, 10H, arom H). Elemental analysis:
calcd for C24H25N3O3S2; C, 61.45; H, 5.39; N, 8.99; S,
13.71%; found C, 61.35; H, 5.44; N, 8.74; S, 13.53%.
5-Acetyl-1,3-diphenyl-6-[(3-piperidin-1-ylpropyl)thio]-2-
7-[(3-Dimethylaminopropyl)thio]-5-imino-1,3,6-triphenyl-
thioxo-2,3-dihydropyrimidin-4(1H)-one (13j). 3.60
g
5,6-dihydropyrimido[4,5-d]
thione (14h). 2.81 g (54% yield), mp 218–220 ꢂC (from
dichloromethane/2-propanol/petroleum ether). IR
(KBr) cmꢁ1 3190, 1620; H NMR (CDCl3) d 1.05–1.45
(m, 2H, methylene-H), 1.67–260 (m, 4H, S-CH2, CH2/
N-propyl-H), 2.10 (s, 6H, dimethylamino-H), 7.0–
7.8(m, 15H, arom H), 11.65 (s, 1H, exchangeable,
imine-H). Elemental analysis: calcd for C29H28N6S3; C,
62.56; H, 5.07; N, 15.09; S, 17.27%; found C, 62.53; H,
5.11; N, 14.97; S, 17.10%.
pyrimidine-2,4(1H,3H)-di-
(75% yield), mp 127–129 ꢂC (from dichloromethane/
ethanol). IR (KBr) cmꢁ1 1705, 1675, 1390, 1325; 1H
NMR(CDCl3) d 1.32–1.90 (m, 8H, CH2/propyl-H and
3CH2/piperidine-H), 2.04–2.45 (m, 6H, S-CH2/propyl-
H and 2CH2/piperidine-H), 2.50–2.54 (m, 10H, arom
H). Elemental analysis: calcd for C26H29N3O2S2; C,
65.11; H, 6.09; N, 8.76; S, 13.77%; found C, 64.95; H,
5.98; N, 8.76; S, 13.57%.
1
General procedure B for the preparation of compounds
14a,b,h,i,j,k, 11c, 12d,g,k, 13d. A 40% Triton methanol
solution (4.4 mL, 10 mmol) was evaporated in vacuo to
dryness and the viscous residue was dissolved in DMF
(40 mL). To the solution, stirred for 15 min, 8 (4.72, 10
mmol) or 4 (3.85 g, 10 mmol) or 5 (3.89 g, 10 mmol) or
6 (3.54 g, 10 mmol) and the proper o-chloroalkylamine
(12 mmol) were added at room temperature. The mix-
ture was then heated under stirring at 65–70 ꢂC for 4 h.
Compounds 11c, 12d,g,k, 13d were isolated according to
work-upA, while those of series 14 as follows:
7 - {[3 - (dimethylamino) - 2 - methylpropyl]thio} - 5 - imino -
1,3,6 - triphenyl - 5,6 - dihydropyrimido[4,5 - d]pyrimidine -
2,4(1H,3H)-dithione (14i). 1.48 g (26% yield), mp
234–236 ꢂC (from dichloromethane/petroleum ether).
1
IR (KBr) cmꢁ1 3190, 1620; HNMR (CDCl3) d 0.63(d,
3H, J=6.0 Hz, methyl-H), 1.50–1.93(m, 3H, methine-H
and S-CH2/propyl-H), 2.06 (s, 6H, 2 CH3/dimethyl-
amino-H), 2.35–2.65 (m, 2H, N-CH2/propyl-H),
6.95–7.73(m, 15H, arom H), 11.55 (brs, 1H, exchange-
able, imine-H). Elemental analysis: calcd for
C30H30N6S3; C, 63.13; H, 5.30; N, 14.72; S, 16.85%;
found C, 62.93; H, 5.23; N, 14.56; S, 16.51%.
Work-upB. After treatment with water (200 mL), the
oily or solid phase separated was dissolved and extrac-
ted with dichlorometane. The combined organic layers
were extracted with 1 M HCl (25 mLꢆ3) and discarded.
The acid solution was made alkaline with 4 M NaOH
(40 mL) and extracted thoroughly with dichloro-
methane. The combined extract was dried and evapo-
rated in vacuo to dryness to give a residue that was
purified by crystallization.
5-Imino-1,3,6-triphenyl-7-[(3-piperidin-1-ylpropyl)thio]-
pyrimidine-2,4(1H,3H)-di-
5,6-dihydropyrimido[4,5-d]
thione (14j). 4.00 g (67% yield), mp 201–203 ꢂC (from
dichloromethane/2-propanol/petroleum
1
ether).
IR
(KBr) cmꢁ1 3185, 1620; H NMR (CDCl3) d 1.06–1.66
(m, 8H, CH2/propyl-H and 3CH /piperidine-H), 1.78–
2
2.61 (m, 8H, S-CH2, N-CH2/propyl-H and 2 CH2/
piperidine-H), 11.45 (s, 1H, exchangeable, imine-H).
Elemental analysis: calcd for C32H32N6S3; C, 64.40; H,
5.40; N, 14.08; S, 16.12%; found C, 64.21; H, 5.44; N,
13.98; S, 16.28%.
Physical and chemical data of compounds 14a,b,h,i,j,k
7-[(2-Dimethylaminoethyl)thio]-5-imino-1,3,6-triphenyl-
5,6 - dihydropyrimido[4,5 - d]pyrimidine - 2,4(1H,3H) - di-
thione (14a). 2.98 g (55% yield), mp 228–230 ꢂC (from
7-({3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}thio)-5-
imino-1,3,6-triphenyl-5,6-dihydropyrimido[4,5-d] pyrimi-

2586
A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
dine-2,4(1H,3H)-dithione (14k). 3.19 g (45% yield), mp
160–162 ꢂC (from dichloromethane/ethanol). IR (KBr)
cmꢁ1 3190, 1620; 1H NMR (CDCl3) d 1.06–1.42 (m, 2H,
CH2/propyl-H), 1.86–2.66 (m, S-CH2, N-CH2/propyl-H
and piperazine-H), 2.96–3.26 (m, 4H, piperazine-H),
6.56–7.76 (m, 19H, arom H), 11.62 (brs, 1H, exchange-
able, imine-H). Elemental analysis: calcd for
C37H34ClN7S3; C, 62.74; H, 4.84; N, 13.84; S, 13.58%;
found C, 62.94; H, 4.64; N, 13.99; S, 13.82%.
(66% yield), mp 170–172 ꢂC (from dichloromethane/eth-
anol). IR (KBr) cmꢁ1 1680, 1380, 1330;1H NMR (CDCl3)
d 1.57–1.9 (m, 4H, 2 CH2/pyrrolidine-H), 2.22–2.53(m,
6H, S-CH2/ethyl-H and 2 CH2/pyrrolidine-H), 2.59 (s,
3H, CH3/acetyl-H), 2.82 (t, 2H, J=7.0 Hz, N-CH2/ethyl-
H), 7.2–7.65 (m, 10H, arom H). Elemental analysis: calcd
for C24H25N3O2S2; C, 63.83; H, 5.58; N, 9.30; S, 14.20%;
found C, 63.67; H, 5.52; N, 9.20; S, 14.40%.
Procedure C for the preparation of compounds 14e,f. To
a stirred mixture of 8 (4.72 g, 10 mmol) and the proper
o-chloroalkylcycloalkylamine (12 mmol) in anhydrous
DMF (30 mL), 60% sodium hydride dispersion in
mineral oil (0.4 g, 10 mmol) was added. The reaction
mixture was heated at 60–65 ꢂC under stirring, and,
after 4 h, treated according to Work-upB.
Physical and chemical data of compound 11c
Ethyl 6-[(2-diisopropylaminoethyl)thio]-4-oxo-1,3-diphe-
nyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(11c). 3.07 g (60% yield), mp 182–184 ꢂC (from di-
chloromethane/2-propanol/petroleum ether). IR (KBr)
1
cmꢁ1 1725, 1690; H NMR (CDCl3) d 0.85 (d, 12H,
J=8.0 Hz, 4 CH3/diisoproylamine-H),1.35 (t, 3H,
J=7.0 Hz, CH3/ethoxy-H), 2.30–3.05 (m, 6H, S-CH2,
N-CH2/ethyl-H and 2 CH/diisopropylamine-H), 4.35
(q, 2H, J=7.0 Hz, CH2/ethoxy-H), 7.05–7.65 (m, 10H,
arom H). Elemental analysis: calcd for C27H33N3O3S2;
C, 63.38; H, 6.50; N, 8.21; S, 12.53%; found C, 63.54;
H, 6.50; N, 8.36; S, 12.61%.
Physical and chemical data of compounds 14e,f
5-Imino-1,3,6-triphenyl-7-[(2-piperidin-1-ylethyl)thio]-
5,6-dihydropyrimido[4,5-d]
pyrimidine-2,4(1H,3H)-di-
thione (14e). 3.32 g (57% yield), mp 213–215 ꢂC (from
dichloromethane/diethylether). IR (KBr) cmꢁ1 3190,
1
1620; H NMR (CDCl3) d 1.30–1.78 (m, 6H, 3 CH2/
piperidine-H), 1.92–2.7 (m, 8H, S-CH2, N-CH2/ethyl-H
and 2 CH2/piperidine-H), 7.05–7.85 (m, 15H, arom H),
11.57 (brs, 1H, exchangeable, imine-H). Elemental ana-
lysis: calcd for C31H33N6S3; C, 63.89; H, 5.19; N, 14.42;
S, 16.50%; found C, 63.89; H, 5.14; N, 14.20; S,
16.40%.
Physical and chemical data of compounds 12d,g,k
1,3,5-Triphenyl-6-[(2-pyrrolidin-1-ylethyl)thio]-2-thioxo-
2,3-dihydropyrimidin-4-(1H)-one (12d). 4.47 g (92%
yield), mp 234–235 ꢂC (from dichloromethane/metha-
1
nol). IR (KBr) cmꢁ1 1670, 1555, 1390, 1320; H NMR
(CDCl3) d 1.50–2.02 (m, 4H, 2 CH2/pyrrolidine-H), 2.04–
2.45 (m, 8H, S-CH2, N-CH2/ethyl-H and 2 CH2/pyrroli-
dine-H), 7.20–7.74 (m, 15H, arom H). Elemental analysis:
calcd for C28H27N3OS2; C, 69.25; H, 5.90; N, 8.95; S,
13.20%; found C, 69.07; H, 5.58; N, 8.63; S, 12.50%.
5-Imino-7-[(2-morpholin-4-ylethyl)thio]-1,3,6-triphenyl-
5,6-dihydropyrimido[4,5-d]
pyrimidine-2,4(1H,3H)-di-
thione (14f). 3.68 g (63% yield), mp 238–240 ꢂC (from
dichloromethane/diethylether). IR (KBr) cmꢁ1 3.200,
1
1620; H NMR (CDCl3) d 1.61–2.71 (m, 8H, S-CH2,
N-CH2/ethyl-H and 2 CH2/morpholine-H), 3.40–3.85
(m, 4H, morpholine-H), 6.95–8.0 (m, 15H, arom H),
11.58 (s, 1H, exchangeable, imine-H). Elemental analy-
sis: calcd for C30H28N6OS3; C, 61.62; H, 4.83; N, 14.37;
S, 16.45%; found C, 61.60; H, 4.83; N, 14.13; S,
16.28%.
6-[(2-Azepan-1-ylethyl)thio]-1,3,5-triphenyl-2-thioxo-2,3-
dihydropyrimidin-4-(1H)-one (12g). 4.37 g (85% yield),
mp 196-197 ꢂC (from dichloromethane/methanol). IR
(KBr) cmꢁ1 1669, 1555, 1390, 1325; 1H NMR (CDCl3) d
1.17–1.75 (m, 8H, 4 CH2/homopiperidine-H), 2.05–2.54
(m, 8H, S-CH2, N-CH2/ethyl-H and 2 CH2/homo-
piperidine-H), 7.25–7.67 (m, 15H, arom H). Elemental
analysis: calcd for C30H31N3OS2; C, 70.14; H, 6.08; N,
8.18; S, 12.48%; found C, 69,95; H, 6.02; N, 8.14; S,
12.19%.
Procedure D for the preparation of compounds 14d,
12a,f. To a stirred mixture of 8 (4.72, 10 mmol) or 5
(3.89 g, 10 mmol) and anhydrous potassium carbonate
(1.52 g, 11 mmol) in DMF (50 mL), the proper
o-chloroalkylamine (12 mmol) was added in a single
portion after 15 min at room temperature. The reaction
mixture was then heated at 60–65 ꢂC and, after 4 h, was
treated according to Work-upB for 14d or Work-upA for
12a,f.
6-({3 - [4 - (3 - Chlorophenyl)piperazin -1 -yl]propyl}thio) -
1,3,5-triphenyl-2-thioxo-2,3-dihydropyrimidin-4-(1H)-one
(12k). 3.44 g (55% yield), mp 116–118 ꢂC (from aceto-
1
nitrile/methanol). IR (KBr) cmꢁ1 1665, 1590; H NMR
(CDCl3) d 1.1–1.5 (m, 2H, CH2/propyl-H), 1.83–2.65
(m, 8H, S-CH2, N-CH2/-propyl-H and 2 CH2/piper-
azine-H), 2.83-3.17 (m, 4H, piperazine-H), 6.73–7.6 (m,
19H, arom H). Elemental analysis: calcd for
C35H33ClN4OS2; C, 67.24; H, 5.32; N, 8.96; S, 10.26%;
found C, 66.99; H, 5.42; N, 9.16; S, 10.40%.
5-Imino-1,3,6-triphenyl-7-[(2-pyrrolidin-1-ylethyl)thio]-
5,6-dihydropyrimido[4,5-d]
pyrimidine-2,4(1H,3H)-di-
thione (14d). 3.97 g (70% yield), mp 231–233 ꢂC (from
dichloromethane/ethanol). IR (KBr) cmꢁ1 3190, 1620;
1H NMR (CDCl3) d 1.5–1.9 (m, 4H, 2 CH2/pyrrolidine-
H), 2.05–2.69 (m, 8H, S-CH2, CH2-N/ethyl-H and 2
CH2/pyrrolidine-H), 7.10–7.72 (m, 15H, arom H), 11.54
(brs, 1H, exchangeable, imine-H). Elemental analysis:
calcd for C30H28N6S3; C, 65.35; H, 4.96; N, 14.78; S,
16.91%; found C, 63.15; H, 4.96; N, 14.62; S, 16.70%.
Physical and chemical data of compound 13d
5-acetyl-1,3-diphenyl-6-[(2-pyrrolidin-1-ylethyl)thio]-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one (13d). 2.98
g

A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
2587
Physical and chemical data of compounds 12a,f
oxo-2,3-dihydropyrimidin-4(1H)-one (13b). 1.81 g (40%
yield), mp 127–129 ꢂC (from dichloromethane/ethanol).
IR (KBr) cmꢁ1 1715, 1680; 1H NMR (CDCl3) d 0.92 (t,
6H, J=7.0 Hz, 2 CH3/dietyhlamino-H), 2.58 (s, 3H,
acetyl-H), 7.15–765 (m, 10H, arom H). 13C NMR
(CDCl3) d 32.34, 37.34, 45.34, 57.64, 125.01, 128.45,
129.36, 129.84, 129.95, 130.24, 139.61, 141.81, 153.66
6-[(2-Dimethylaminoethyl)thio]-1,3,5-triphenyl-2-thioxo-
2,3-dihydropyrimidin-4-(1H)-one (12a). 2.39 g (52%
yield), mp 200–201 ꢂC (from dichloromethane/metha-
1
nol). IR (KBr) cmꢁ1 1673; H NMR (CDCl3) d 1.95 (s,
6H, 2 CH3/dimethylamino-H), 2.07–2.35 (m, 4H,
S-CH2, N-CH2/ethyl-H), 7.2–7.68 (m, 15H, arom H).
Elemental analysis: calcd for C26H25N3OS2; C, 67.94;
H, 5.48; N, 9.14; S, 13.95%; found C, 67.75; H, 5.35; N,
9.11; S, 13.76%.
(6-C), 157.99 (4-C), 179.64 (2-C), 198.23(COCH ). Ele-
3
mental analysis: calcd for C24H27N3O2S2; C, 63.55; H,
6.00; N, 9.26; S, 14.14%; found C, 63.55; H, 5.84; N,
9.01; S, 13.98%.
6-[(2-Morpholin-4-ylethyl)thio]-1,3,5-triphenyl-2-thioxo-
2,3-dihydropyrimidin-4-(1H)-one (12f). 4.66 g (93%
yield), mp 228–229 ꢂC (from dichloromethane/metha-
5-acetyl-6-[(2-azepan-1-ylethyl)thio]-1,3-diphenyl-2-thi-
oxo-2,3-dihydropyrimidin-4(1H)-one (13g). 1.97 g (41%
yield), mp 150–152 ꢂC (from dichloromethane/2-propa-
1
1
nol). IR (KBr) cmꢁ1 1672, 1590, 1390, 1327; H NMR
nol). IR (KBr) cmꢁ1 1702, 1675, 1386, 1325; H NMR
(CDCl3) d 1.98–2.38 (m, 8H, S-CH2, N-CH2/ethyl-H and
2 CH2/morpholine-H), 3.40–3.75 (m, 4H, 2 CH2/mor-
pholino-H), 7.25–7.75(m, 15H, arom H). Elemental ana-
lysis: calcd for C28H27N3O2S2; C, 67.04; H, 5.42; N, 8.38;
S, 12.78%; found C, 67.07; H, 5.37; N, 8.51; S, 12.50%.
(CDCl3) d 1.47 (m, 8H, homopiperidine-H), 1.92–2.94
(m, 8H, S-CH2/,N-CH2/ethyl- and homopiperidine-H),
2.57 (s, 3H, acetyl-H), 7.07–7.78 (m, 10H, arom H).
Elemental analysis: calcd for C26H29N3O2S2; C, 65.11;
H, 6.09; N, 8.76; S, 13.37%; found C, 65.34; H, 6.05; N,
8.83; S, 13.32%.
Procedure E for the preparation of compounds 13a,h. To
a stirred mixture of 6 (3.54, 10 mmol) and triethylamine
(1.01 g, 10 mmol) in 1,4-dioxane (80 mL), the proper
o-chloroalkylamine (12 mmol) was added in a single
portion, after 15 min at room temperature. The reaction
mixture was heated at 85–90 ꢂC for 4 h and then treated
according to Work-upA.
Antiproliferative assays performed at NCI. The NCI
high-flux anticancer drug screen39,40 utilized a panel of
60 human tumor cell lines in culture derived from nine
clinically isolated neoplastic diseases, namely non-small
cell lung, colon, CNS, ovarian, renal, prostate, breast
cancer, melanoma and leukemia. Cell lines were
exposed to test agents in 96-well plates for the last 48 of
a 72 h incubation and were stained for total protein with
sulforhodamine B according to the standard NCI pro-
tocol. The cytotoxic effects of each test agent were
evaluated through the growth inhibition parameters
GI50, TGI and LC50, which represent the molar drug
concentration required to produce half (GI50), or total
(TGI) growth inhibition, and 50% of cytocidal effect
(LC50), respectively. A standard dose–response curve
for individual agent, tested at concentrations of 10ꢁ8 to
10ꢁ4 M, was provided for each of the 60 cell lines. The
relative responsiveness of each cell line compared with
the average responsiveness of all of the lines was pro-
vided in graphic form as mean-graph mid-point
(MG-MID). Compounds which did not inhibit cell
growth by 50% at 10ꢁ4 M were still employed in the
calculation of cytotoxicity.
Physical and chemical data of compounds 13a,h
5-Acetyl-6-[(2-dimethylaminoethyl)thio]-1,3-diphenyl-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one (13a). 2.68
g
(63% yield), mp 186–187 ꢂC (from dichloromethane/
ethanol). IR (KBr) cmꢁ1 1673; 1H NMR (CDCl3) d 2.06
(s, 6H, 2 CH3/dimethylamino-H), 2.37 (t, 2H, J=6.0
Hz, S-CH2/ethyl-H), 2.58 (s, 3H, CH3/acetyl-H), 2.85 (t,
2H, J=6.0 Hz, N-CH2/ethyl-H), 7.15–7.65 (m, 10H,
arom H). Elemental analysis: calcd for C22H23N3O2S2;
C, 62.06; H, 5.45; N, 9.87; S, 15.07%; found C, 61.90;
H, 5.38; N, 9.71; S, 14.80%.
5-Acetyl-6-[(3-dimethylaminopropyl)thio]-1,3-diphenyl-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one (13h). 2.81
g
(64% yield), mp 163–164 ꢂC (from dichloromethane/
ethanol). IR (KBr) cmꢁ1 1705, 1678, 1390, 1330; 1H
NMR (CDCl3) d 1.3–1.85 (m, 2H, CH2/propyl-H) 2.15
(s, 6H, 2 CH3/dimethylamino-H), 2.0–2.25 (m, 2H,
S-CH2/propyl-H), 2.6 (s, 3H, acetyl-H), 7.3–7.7 (m, 15,
arom H). Elemental analysis: calcd for C23H25N3O2S2;
C, 62.84; H, 5.73; N, 9.56; S, 14.59%; found C, 62.66;
H, 5.55; N, 9.50; S, 14.29%.
Assays performed in our laboratory
Test compounds were dissolved in DMSO at an initial
concentration of 200 mM and were then serially diluted
in culture medium. Tumor cell growth at each drug
concentration was expressed as percentage of untreated
controls and the concentrations resulting in 50% (EC50
and IC50, respectively) growth inhibition was deter-
mined by linear regression analysis.
Procedure F for the preparation of compounds 13b,g. A
stirred solution of 6 (3.54 g, 10 mmol) and the proper o-
chloroalkylamine (24 mmol) in DMF (40 mL) was
heated at 70–75 ꢂC for 4 h and then treated according to
Work-upA.
Cells. MT4 lymphoblastoid T cells were from American
Type Culture Collection (ATCC, USA). The nasophar-
ingeal carcinoma KB cell line and the resistant mutant
subclones KBMDR KBV20C, KB7D and KBCPT300 were
generous gift of Prof. Y. C. Cheng, Yale University,
USA. All resistant KB cell lines were maintained in
Physical and chemical data of compounds 13b,g
5-acetyl-6-[(2-diethylaminoethyl)thio]-1,3-diphenyl-2-thi-

2588
A. Ranise et al. / Bioorg. Med. Chem. 11 (2003) 2575–2589
growth medium supplemented with 0.02 mM vincristine
for KBV20C, 7 mM etoposide for KB7D, 0.3 mM camp-
tothecin for KBCPT300, 0.09 mM doxorubicin for
KBMDR. MT-4 [grown in RPMI 1640 containing 10%
foetal calf serum (FCS), 100 UI/mL penicillin G and
100 mg/mL streptomicin] were also used for anti-HIV-1
assays. All cell cultures were checked periodically for
the absence of mycoplasma contamination with a
MycoTect Kit (Gibco).
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Acknowledgements
This work was supported by MURST and CNR
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