Lead optimization through VLAK protocol: New annelated pyrrolo-pyrimidine derivatives as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2012.07.046

The chemometric protocol VLAK was applied to predict improvement of the biological activity of pyrrolo-pyrimidine derivatives as anticancer agents, by using the NCI ACAM Database as depository of antitumor drugs with a known mechanism of action. Among the selected compounds two of these showed a good increase in the antitumor activity. These new pyrrolo-pyrimidine compounds were demonstrated effective against the full panels of NCI DTP tumour human cell lines. The derivative 8-[3-(piperidino)propyl]-4,10-dimethyl-9-phenyl-6- (methylsulfanyl)-3,4-dihydropyrimido[1,2-c]pyrrolo[3,2-e]pyrimidin-2(8H)-one reveled efficacious against the leukemia subpanel, in particular the RPMI cell line resulted the most sensitive (pGI₅₀ = 6.68). Moreover the derivative 7-(3-Chloropropyl)-9-methyl-5-(methylsulfanyl)-8-phenyl-3H-imidazo[1, 2-c]pyrrolo[3,2-e]pyrimidin-2(7H)-one showed a good antitumor activity against the leukemia subpanel with a low cytotoxic activity, above all against the HCT11 human tumour cell line. The VLAK protocol revealed a good method to design new molecules with good antitumor activity, starting from low active compounds. Moreover this protocol focused on the pyrrolo-pyrimidine derivatives as useful starting point for further development to obtain more potent antitumor agents.

Full text of DOI:10.1016/j.ejmech.2012.07.046

European Journal of Medicinal Chemistry 55 (2012) 375e383
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Lead optimization through VLAK protocol: New annelated pyrrolo-pyrimidine
derivatives as antitumor agents
*
Antonino Lauria , Chiara Patella, Ilenia Abbate, Annamaria Martorana, Anna Maria Almerico
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari “STEMBIO”, Sezione di Chimica Farmaceutica e Biologica, Università di Palermo, Via Cipolla 74D,
I-90123 Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemometric protocol VLAK was applied to predict improvement of the biological activity of
pyrrolo-pyrimidine derivatives as anticancer agents, by using the NCI ACAM Database as depository of
antitumor drugs with a known mechanism of action. Among the selected compounds two of these
showed a good increase in the antitumor activity. These new pyrrolo-pyrimidine compounds were
demonstrated effective against the full panels of NCI DTP tumour human cell lines. The derivative 8-[3-
(piperidino)propyl]-4,10-dimethyl-9-phenyl-6-(methylsulfanyl)-3,4-dihydropyrimido[1,2-c]pyrrolo[3,2-
e]pyrimidin-2(8H)-one reveled efficacious against the leukemia subpanel, in particular the RPMI cell line
resulted the most sensitive (pGI50 ¼ 6.68). Moreover the derivative 7-(3-Chloropropyl)-9-methyl-5-
(methylsulfanyl)-8-phenyl-3H-imidazo[1,2-c]pyrrolo[3,2-e]pyrimidin-2(7H)-one showed a good anti-
tumor activity against the leukemia subpanel with a low cytotoxic activity, above all against the HCT11
human tumour cell line.
Received 20 June 2012
Received in revised form
23 July 2012
Accepted 26 July 2012
Available online 4 August 2012
Keywords:
Anticancer drugs
VLAK protocol
Developmental Therapeutics Program (DTP)
Annelated pyrrolo-pyrimidines
The VLAK protocol revealed a good method to design new molecules with good antitumor activity,
starting from low active compounds. Moreover this protocol focused on the pyrrolo-pyrimidine deriv-
atives as useful starting point for further development to obtain more potent antitumor agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
(Bethesda, USA) in the Developmental Therapeutics Program
(DTP). In the primary anticancer screening assay, tested against
In the last years different chemometric approaches and statis-
tical facilities were applied in the attempt to rationalize and to
correlate biological activities with molecular descriptors. The Prin-
cipal Component Analysis (PCA) applied on a molecular descriptors
matrix resulted to be a robust method to classify HSP90 inhibitors
[1]. Moreover in a previous work the same multivariate approach
was revealed effective in the prediction of mechanism of action for
anticancer drugs [2]. Recently, we have developed a protocol,
named Virtual Lock-And-Key (VLAK), able to predict suitable bio-
logical target for molecular structures with unknown biological
activity, by means molecular descriptors calculation [3]. The VLAK
protocol was applied in the attempt to improve the biological
activity of heterocyclic derivatives with poor antitumor effect.
In particular in a previous work the synthetic use of N-(BisMe-
thylthio)Methylenamino Acids (BMMAs), in domino reactions, had
led to the annelated pyrrolo-pyrimidines depicted in Fig. 1, which
showed no relevant antiproliferative activity [4].
3-cell line panel consisting of the MCF7 (Breast), NCI-H460 (Lung),
and SF-268 (CNS) the annelated pyrrolo-pyrimidines resulted
generally inactive up to 10
mM concentration. The same
compounds tested against the human intestinal adenocarcinoma
(LoVo cell line), although being less active than the reference drug
[doxorubicin (DOXO)], exhibited a moderate antiproliferative
activity with IC50 ¼ 10.5e41.2
mM (Fig. 1) [4].
It is reported in literature as heterocyclic compounds, with poor
biological activity, can improve the anticancer activity against
a large spectrum of human tumour cell lines, if properly decorated
with selected side chains [5]. Therefore with the aim to enforce the
biological activity of the pyrrolo-pyrimidine derivatives (Fig. 1), the
VLAK protocol was applied. Thus an in house database of structures
containing the annelated pyrrolo[3,4-e]pyrimidines (1e6) and
pyrrolo[3,2-e]pyrimidines (7e10), suitably functionalized with
a large number of side chains (A-T), was built (Fig. 2) and submitted
to VLAK protocol in order to identify new potential anticancer
derivatives.
Representative compounds of each of these series were
selected for screening tests by the National Cancer Institute NCI
The selected derivatives were synthesized and biologically
tested to validate the VLAK methodology and to confirm the che-
mometric protocol capability as tool for the identification of new
potential antitumor compounds.
* Corresponding author. Tel.: þ39 0916167210; fax: þ39 0916230923.
E-mail address: antonino.lauria@unipa.it (A. Lauria).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.046

376
A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
O
molecular descriptor value ranges. Each range is defined as
Dj(MA) ꢀ Dj(MA), where Dj(MA) is the molecular descriptor
average value and Dj(MA) is the standard deviation (Fig. 4b,
S
N
Y
O
m
s
m
N
s
N
N
supporting information SI1).
Y
When the molecular descriptor value Dj of a tested structure
N
X (Fig. 4c) falls within the defined range (
m
Dj ꢀ
s
Dj)
a
¼ 1 (i.e. D1,
D3, and Dj), otherwise
a
¼ 0 (i.e. D2, D4) (Fig. 4d). At the end each in
S
house database structure is converted in a binary sequence. As
underlined before, to release a lock all pins must fit; in the proposed
protocol, it is supposed that the higher is the number of fitted pins,
the higher will be the potential anticancer capability of the inves-
tigated compound. Thus, the percentage of affinity A% (Eq. (1)) for
each compound belonging to in house database was defined for
each class (MA) as:
Ph
Ph
N
R2
N
R1
N
H
R1=Ph, Y=CH2
R1=Me, Y=CH2
IC50=26.7 µM
IC50=23.2 µM
R1=Me, Y=CH2CH2 IC50=41.2 µM
R2=H, Y=NMe
R2=Me, Y=CH2
R2=H, Y=CH2CH2 IC50=10.5 µM
IC50=35 µM
IC50=19 µM
Fig. 1. Annelated pyrrolo[3,2-e]pyrimidines and pyrrolo[3,4-e]pyrimidines with low
antitumor activity against the human intestinal adenocarcinoma (LoVo cell line).
X
A% ¼
a
i; jðMAÞ=Dtot*100
(1)
where S
a
i,j(MA) is the sum of all fitted molecular descriptors for
2. Results and discussion
the MA class and Dtot is the total of the molecular descriptors used
in the VLAK protocol.
2.1. Chemometric protocol
In supporting information SI2 is reported the output result for
all in house database against the six lock models representing each
class of drugs (MAs). In Table 2 the best ranked derivatives
expressed in percentage of affinity (A%) are reported: the deriva-
tives E1, H2, I1 showed the higher value of A% for the class of
antimitotic agents (B), while the derivatives N3 and O3 for the
topoisomerase II inhibitors class (D).
In order to release a lock it is necessary that the pins of the lock
fit to the key profile (Fig. 3). In the VLAK protocol the molecular
descriptors are considered the pins of the biological target that, as
Fisher model suggested in his famous Lock-and-Key model [6e9], is
comparable to a lock.
The first step of the VLAK protocol consists in the conversion of
the biological target in a “lock model” in which the keys (the
structures) could be “fitted” [3].
2.2. Chemistry
The NCI AntiCancer Agent Mechanism (ACAM) Database was
chosen as source of biological data for the construction of the VLAK
“lock models”. It consists in a repository of structures with anti-
cancer activity and a reasonably well-known mechanism of action.
Drug screening data are available for each structure as measure-
ment of their growth inhibition ability over a panel of about 60
human tumour cell lines, and all tested molecules are explicitly
designed as a training set for neural network and multivariate
analysis [10e12]. In particular, this database is constituted by 121
antitumor drugs classified for mechanism of action (Table 1) that in
the protocol can be considered like the biological target.
The NCI ACAM Database was chosen because it was demon-
strated as the molecular descriptors, processed by multivariate
analysis, are able to discriminate the mechanisms of action of the
drugs [2]. Thus, for each class of drugs (Alkylating Agents, Anti-
With these premises, the synthesis of the compounds, selected
on the basis of the VLAK protocol [E1, H2, I1, N3, O3], was
successfully achieved through suitable synthetic pathways, as
shown in Scheme 1.
The starting annelated pyrrolo-pyrimidines A1eA3 were
prepared through the classical approach involving the reaction of
2-amino-3-cyanopyrroles 11a,b with N-[bis(methylthio)methy-
lene]amino moiety (BMMA) 12c,d in acetic acid under reflux for an
appropriate period of time (0.5e1 h) (Scheme 1) [4].
Derivatives I1 and H2 were synthesized in two preparative
steps. The reaction of A1 or A2 and 1-Bromo-3-Chloropropane,
using potassium carbonate as base, gave the chloro derivatives B1
and B2 in good yields. These key intermediates, upon heating under
reflux with the appropriate amine in solvent free condition, gave the
targeted compounds I1 and H2 (yields 66% and 76%, respectively).
On the other hand compounds E1, N3, and O3, bearing a suitable
side-chain were prepared through the key intermediates C1 and C3.
Thus, these derivatives were obtained from the sodium salts of A1
or A3, generated in situ by using potassium carbonate, and ethyl
mitotic Agents, Topoisomerase
I Inhibitors, Topoisomerase II
Inhibitors, RNA/DNA Antimetabolites, and DNA Antimetabolites),
a “lock model” was generated starting from own known drugs.
In particular, for each class of drugs with known mechanism of
action (MA) (Fig. 4a), the lock model is defined as a sequence of
O
O
S
N
X#
Y
N
N
Y
N
-H
-(CH2)3Cl
K
L
-(CH2)3NnPr2
A
B
C
D
E
F
G
H
I
N
-(CH2)3CONHCH2COOEt
-(CH2)3CONHCH2COOH
-(CH2)3CONH(CH2)2Im
-(CH2)3CONH(COOMe)CH2Im
-(CH2)3CONH(COOH)CH2Im
-CH2N(CH2CH2)2N(CH2)2OH
-(CH2)2N(CH2CH2)2N(CH2)2OH
-(CH2)2N(CH2CH2)2NMe
-CH2N(CH2CH2)2NMe
S
N
-(CH2)3COOEt
-(CH2)3COOH
-(CH2)5COOEt
-(CH2)5COOH
-(CH2)3NnBu2
-(CH2)3N(CH2)5
-(CH2)3N(CH2CH2)2NMe
-CH2OCH(CH2OH)2
M
N
O
P
Q
R
S
T
R1
Ph
7-10
Ph
N
X
N
1-6
X
R1=Ph, Y=CH2
R1=Ph, Y=CH2CHCH3
R1=Me, Y=CH2
R1=Me, Y=CH2CHCH3
R1=Me, Y=CH2CH2
R1=Ph, Y=CH2CH2
1
2
3
4
5
6
7 Y=CH2
Y=NMe
8
9 Y=CH2CH2
Y=CH CHCH
10
2
3
J
Fig. 2. Annelated pyrrolo[3,2-e]pyrimidines (1e6) and pyrrolo[3,4-e]pyrimidines (7e10) in house database submitted to VLAK protocol. #Im ¼ imidazol-5-yl.

A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
377
Released
Locked
pin
pin
fitted
pin
unfitted
Fig. 3. Lock release mechanism.
4-bromobutyrate in dry DMF. Subsequent hydrolysis with NaOH in
EtOH/H2O mixture quantitatively yielded the corresponding
carboxylic acids D1 and D3. Derivative E1 was synthesized by
reacting D3 with glycine ethyl ester, in dry dichloromethane and
triethylamine (TEA), and using dicyclohexylcarbodiimide (DCC) as
coupling agent.
The amide derivative O3 and N3 were obtained from the same
key intermediate D3 upon reaction with L-histidine methyl ester
dihydrochloride or histamine respectively, in dry dichloromethane
and TEA, and using DCC as coupling agent.
concentration of the compound leading to total inhibition of net
cell growth), and pLC50 value (LC50 is the molar concentration of the
compound that induces 50% net cell death). Moreover, a mean
graph midpoint (MG_MID) is calculated for each of the mentioned
parameters, giving an average activity parameter over all cell lines.
For the calculation of the MG_MID, insensitive cell lines are
included with the highest concentration tested. The discovery of
compounds with new selectivity patterns is one of the targets of
the DTP screening program. Selectivity of a compound with respect
to a certain cell line of the screen is characterized by a high devi-
ation of the particular cell line parameter compared to the MG_MID
value. The pGI50, pTGI, and pLC50 values for these compounds are
reported in Table 4.
2.3. Biological screening
An evaluation of the data reported in Table 4 revealed that B1
and H2, showed antiproliferative activity against all the human
tumour cell lines investigated, generally in the low micromolar
concentration range.
Considering the MG_MID values, the most active compound was
demonstrated to be derivative H2, at both GI50 (5.56) and TGI50
(4.83) level (Table 4).
With respect to the tumour subpanel, H2 resulted particularly
effective against all leukemia cell lines and, in particular, against the
RPMI-8226 cell line with pGI50 of 6.78 (Fig. 5).
It is also of remarkable interest the low number of cell lines
giving pLC50 < 4, highlighting the low toxicity of such compounds
(29/60 for B1 and 27/55 for H2). Moreover the derivative B1
showed a good antitumor effect against the leukemia subpanel
with a low cytotoxic activity above all against the HCT11 tumour
cell line.
All the new pyrrolo-pyrimidines [E1, H2, I1, N3, and O3] and the
intermediates [B1, B2, C1, C3, D1, and D3] were subjected to the
NCI’s disease-oriented human cell lines screening assay to be
evaluated for their in vitro antitumor activity.
Derivative B2 did not pass the selection criteria adopted by DTP
screening data and consequently was excluded from the in vitro test.
A single dose (10 mM) of the accepted compounds was tested
against a panel of approximately 60 tumour human cell lines
grouped in nine disease subpanels, including leukemia, non small-
cell lung, colon, central nervous system, melanoma, ovarian, renal,
prostate, and breast tumour cell lines [13e15] (Table 3, Table G% in
supporting information SI4).
In this biological protocol, each cell line is inoculated and pre-
incubated 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 [16]. Results for each
tested agent are reported as the percent of growth (G%) 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, are passed on for evaluation in the full
panel of 60 cell lines over a 5-log dose range.
The new pyrrolo-pyrimidine derivatives generally showed not
exciting growth inhibition values, with few exceptions: in partic-
ular derivative C3 resulted efficacious against the MDA-MB-468
breast cancer cell line (G% ¼ 55), while I1 was active against SNB-
75 (CNS cancer), SR, and RPMI-8226 (leukemia) showing a G% of
51%, 70%, and 66%, respectively.
Derivatives H2 and B1 recorded several G% values significantly
under the level of 32% and with a G% average of 39% and 60%,
respectively (Table 3). Thus they were passed to five-dose
concentration screening.
3. Conclusions
In this work the VLAK protocol was applied for the optimization
of annelated pyrrolo-pyrimidine derivatives as antitumor agents.
By using the NCI ACAM Database as depository of antitumor drugs
with a known mechanism of action, the VLAK protocol was applied
on an in house database of pyrrolo-pyrimidine derivatives. The
selected compounds were synthesized and screened. Two of these
(B1 and H2) showed a good increase in the antitumor activity with
respect to the starting core structure. In fact the derivative H2
reveled efficacious against the leukemia subpanel and in particular
the RPMI cell line with a pGI50 ¼ 6.68. Both compounds H2 and B1
were demonstrated effective against the full tumour human cell
line panels. The VLAK protocol has proved a good method to opti-
mize the search of compounds with improved antiproliferative
activity. Moreover this protocol had permitted to identify the
pyrrolo-pyrimidine derivatives as useful starting point for further
development to obtain more potent antitumor agents.
The antitumor activity of a tested compound, in the five-dose
screening, is given by three parameters for each cell line: pGI50
value (GI50 is the molar concentration of the compound that
inhibits 50% net cell growth), pTGI value (TGI is the molar

378
A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
Table 1
NCI ACAM database drugs.
Name
NSC ID
MA
Name
NSC ID
MA
Busulfan
Nitrogen mustard
Chlorambucil
Thio-tepa
Melphalan
Triethylenemelamine
Pipobroman
Mitomycin C
Uracil nitrogen mustard
Porfiromycin
Fluorodopan
750
762
3088
6396
8806
9706
25154
26980
34462
56410
73754
79037
95441
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Aa
A
A
A
A
A
A
A
Aa
Aa
Aa
A
A
A
A
A
A
A
Aa
A
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Daunorubicin
606173
606497
606499
606985
610456
610457
610458
610459
618939
629971
643833
82151
122819
123127
141540
164011
249992
267469
268242
269148
301739
308847
337766
349174
355644
366140
740
C
C
C
C
C
C
C
C
C
C
C
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
E
E
E
E
E
E
Ea
E
E
E
E
E
E
E
E
Ea
E
E
F
CCNU
Methyl CCNU
PCNU
Yoshi-864
VM-26
Doxorubicin
VP-16
Rubidazone
95466
102627
119875
132313
135758
142982
167780
172112
178248
182986
241240
256927
271674
296934
329680
338947
344007
348948
353451
357704
363812
409962
757
33410
49842
67574
83265
125973
153858
332598
361792
376128
406042
608832
609395
94600
cis-Platinum
Dianhydrogalactitol
Piperazinedione
Hycanthone
Asaley
Spirohydantoin mustard
Chlorozotocin
AZQ
CBDCA
CHIP
Carboxyphthalatoplatinum
Teroxirone
Hepsulfam
Clomesone
Piperazine ../drugs/mainator
Cyclodisone
Mitozolamide
Cyanomorpholinodoxorubicin
Tetraplatin
BCNU
Colchicine
Colchicine derivative
Vinblastine sulfate
Vincristine sulfate
Trityl cysteine
Taxol
Maytansine
Rhizoxin
Thiocolchicine
Dolastatin 10
m-AMSA
Deoxydoxorubicin
N,N-dibenzyl daunomycin
Menogaril
Mitoxantrone
Amonafide
Bisantrene HCL
Oxanthrazole
Anthrapyrazole derivative
Pyrazoloacridine
Methotrexate
5-Fluorouracil
5-Azacytidine
19893
102816
126771
132483
134033
139105
143095
148958
153353
163501
174121
184692
224131
264880
352122
368390
623017
752
755
1895
27640
32065
51143
63878
71261
71851
Dichlorallyl lawsone
Aminopterin derivative
Aminopterin derivative
Baker’s soluble antifol
Pyrazofurin
Ftorafur (pro-drug)
L-alanosine
Acivicin
Methotrexate derivative
Aminopterin derivative
N-(phosphonoacetyl)-L-aspartate (PALA)
5,6-Dihydro-5-azacytidine
Trimetrexate
Brequinar
An antifol
Thioguanine
Thiopurine
Guanazole
20-Deoxy-5-fluorouridine
Hydroxyurea
Pyrazoloimidazole
Ara-C
Beta-TGDR
Alpha-TGDR
3-HP
5-HP
Inosine glycodialdehyde
5-Aza-20-deoxycytidine
Cyclocytidine
Aphidicolin glycinate
Macbecin II
Allocolchicine
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Taxol derivative
Halichondrin B
Camptothecin
Camptothecin derivative
Camptothecin, Na salt
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Camptothecin derivative
Morpholinodoxorubicin
Camptothecin derivative
Camptothecin derivative
Aminocamptothecin
Camptothecin derivative
95382
100880
107124
176323
249910
295500
295501
354646
364830
374028
603071
606172
C
C
C
C
C
C
C
C
95678
107392
118994
127716
145668
303812
330500
C
MA, mechanism of action; A, Alkylating Agents; B, Antimitotic Agents; C, Topoisomerase I Inhibitors; D, Topoisomerase II Inhibitors; E, RNA/DNA Antimetabolites; F, DNA
Antimetabolites.
a
Excluded in the lock models generation (see Experimental section).
4. Experimental
Database entries containing cations or consisting of a mix of two
structures were excluded. Thus the starting database was constituted
from 114 compounds (supporting information SI3) classified in six
mechanism of action: 30, Alkylating Agents; 13, Antimitotic Agents;
24, Topoisomerase I Inhibitors; 15, Topoisomerase II Inhibitors; 16,
RNA/DNA Antimetabolites; 16, DNA Antimetabolites (Table 1).
CODESSA PRO software was used for the molecular descriptors
4.1. Computational details
The compounds contained in the NCI AntiCancer Agents Mecha-
nism Database (NCI ACAM Database) [10e12], were drawn and
optimized in vacuo by ligprep of MAESTRO SUITE [17]. The NCI ACAM

A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
379
Fig. 4. VLAK protocol applied on ACAM database.
calculation [18]. For all structures 286 molecular descriptor belonging
Yield 50.3%. Mp 156.5e157.1 ꢁC. IR: 1656 (CO) cmꢂ1
.
1H NMR:
to different classes were calculated (supporting information SI1).
d
1.81e1.99 (m, 2H, CH2CH2CH2), 2.24 (s, 3H, CH3), 2.71 (s, 2H, CH2),
3.46 (t, 2H, J ¼ 6.0 Hz, CH2Cl), 4.28 (t, 2H, J ¼ 6.0 Hz, NCH2), 4.33 (s,
3H, CH3), 7.47e7.62 (m, 5H, Ph). 13C NMR:
10.2 (q, CH3), 13.6 (q,
4.2. Chemistry
d
CH3), 32.2 (t, CH2CH2CH2), 42.3 (t, C-3), 49.6 (t, CH2Cl), 59.4 (t,
NCH2), 100.2 (s, C-9a), 111.0 (s, C-9), 128.6 (d, C-40), 128.8 (d, C-20, C-
60), 129.7 (s, C-8), 130.2 (d, C-30, C-50), 133.8 (s, C-10), 145.5 (s, C- 6a),
146.7 (s, C-9b), 152.4 (s, C-5), 165.3 (s, C-2). Anal. Cald. for
C19H19ClN4OS: C, 58.98; H, 4.95; N, 14.48; S, 8.29. Found: C, 59.09;
H, 4.97; N, 14.45; S, 8.39.
All melting points (ꢁC) were determined on a BüchieTottoli
capillary apparatus and are uncorrected; IR spectra were deter-
mined in bromoform with a Jasco FT/IR 5300 spectrophotometer;
1H NMR, 13C NMR spectra were recorded in DMSO-d6 solution (TMS
as internal reference) at 200 and 50.3 MHz, respectively, using
a Bruker AC-E series 200 MHz spectrometer. Microanalyses were in
agreement with theoretical values ꢀ0.4%. Thin layer chromatog-
raphy was performed on precoated (0.25 mm) silica gel GF254
plates; compounds were detected with 254 nm UV lamp. Column
chromatography was performed with Merck silica gel 230e400
mesh ASTM or with a Biotage FLASH40i chromatography module
(prepacked cartridge system).
4.2.2. 8-(3-Chloropropyl)-4,10-dimethyl-6-(methylsulfanyl)-9-
phenyl-3,4-dihydropyrimido[1,2-c] pyrrolo[3,2-e]pyrimidin-2(8H)-
one (B2)
To a stirred suspension of A2 (0.5 g, 1.5 mmol) in dry N,N-dime-
thylformamide(5mL),potassiumcarbonate(0.62g,4.5mmol)and1-
Bromo-3-Chloro-propane (0.44 mL, 4.5 mmol), were added. The
mixturewasheatedat80ꢁCinanoilbathfor1h,cooledtoroomtemper-
atureandpouredintoiceandcoldwater.Thecruderesiduewascollected
byfiltrationand,afterdrying,purifiedbycolumnchromatographyusing
adichloromethane/ethylacetatemixtureingradientaseluent.Recrys-
tallizedfromdiethylether.
4.2.1. 7-(3-Chloropropyl)-9-methyl-5-(methylsulfanyl)-8-phenyl-
3H-imidazo[1,2-c]pyrrolo[3,2-e]pyrimidin-2(7H)-one (B1)
To a stirred suspension of A1 (1 g, 3.2 mmol) in dry N,N-dime-
thylformamide (10 mL), potassium carbonate(1.40 g,10.1 mmol) and
1-Bromo-3-Chloro-propane (1 mL, 10.0 mmol), were added. The
mixture was heated at 80 ꢁC in an oil bath for 1 h, cooled to room
temperature and poured into ice and cold water. The crude residue
was collected by filtration and, after drying, purified by column
chromatography using a dichloromethane/ethyl acetate mixture in
gradient as eluent.
Yield 66.6%. Mp 191.7e192.6 ꢁC. IR: 1660 (CO) cmꢂ1 1H NMR:
.
d
1.50 (d, 3H, J ¼ 7.4 Hz, CH3),1.82e1.98 (m, 2H, CH2CH2CH2), 2.41 (s,
3H, CH3), 2.60 (dd, 1H, H-3), 2.70 (s, 3H, CH3), 2.89 (dd, 1H, H-3),
3.34 (t, 2H, J ¼ 6.6Hz, CH2Cl), 4.07 (t, 2H, J ¼ 6.6 Hz, NCH2),
4.87e4.93 (m, 1H, H-4), 7.08e7.50 (m, 5H, Ph). 13C NMR:
d 11.5 (q,
CH3), 14.7 (q, CH3), 17.0 (q, CH3), 32.8 (t, CH2CH2CH2), 36.3 (t, C-3),
39.9 (t, CH2Cl), 41.8 (t, NCH2), 51.1 (d, C-4), 104.3 (s, C-10a), 113.9 (s,
C-10), 126.6 (d, C-40), 128.8 (d, C-20, C-60), 130.4 (d, C-30, C-50), 131.0
(s, C-9), 131.5 (s, C-10), 145.9 (s, C-7a), 154.07 (s, C-10b), 154.13 (s, C-
6), 174.3 (s, C-2). Anal. Cald. For C21H23ClN4OS: C, 60.79; H, 5.59; N,
13.50; S, 7.73. Found: C, 60.79; H, 5.59; N, 13.50; O, 3.86; S, 7.73.
Table 2
In house database virtual screening selected hits (A%).
Id
MA
A
B
C
D
E
F
E1
H2
I1
N3
O3
49.7
46.9
46.9
40.2
39.9
91.6
91.3
91.3
71.7
78.3
59.1
49.0
55.6
57.0
42.3
81.5
68.2
75.9
90.6
92.0
55.2
53.5
59.4
68.9
53.1
32.9
30.4
31.8
34.6
33.6
4.2.3. 7-[3-(N-methylpiperazine)propyl]-9-methyl-5-(methylsul-
fanyl)-8-phenyl-3H-imidazo[1,2-c]pyrrolo[3,2-e]pyrimidin-2(7H)-
one (I1)
To a stirred solution of B1 (0.12 g, 0.3 mmol) in dry dioxane
(5 mL), N-methyl-piperazine (0.07 mL, 0.6 mmol) was added. The
mixture was heated at reflux for 6 h and, after cooling, the
solvent was evaporated under reduced pressure. The crude
MA, mechanism of action; A, Alkylating Agents; B, Antimitotic Agents; C, Topo-
isomerase I Inhibitors; D, Topoisomerase II Inhibitors; E, RNA/DNA Antimetabolites;
F, DNA Antimetabolites. In bold are reported the highest A% values for the selected
compounds.

380
A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
A1
B1
B2
I1
A2
A3
H2
C1
C3
E1
N3
D3
D1
O3
Scheme 1. Reagents and conditions: (i) AcOH,
D; (ii) NaHCO3 or NaCO3; (iii) K2CO3, DMF, Cl(CH2)3Br; (iv) dioxane, N-methyl-piperazine or piperidine; (v) K2CO3, DMF, ethyl 4-
bromobutyrate; (vi) NaOH, EtOH/H2O; (vii) DCC, DCM, 0 ꢁC, glycine ethyl ester hydrochloride/TEA; (viii) DCC, DCM, 0 ꢁC, histamine/TEA; (ix) DCC, DCM, 0 ꢁC, L-histidine methyl
ester hydrochloride/TEA.
residue was purified by column chromatography using
dichloromethane/ethyl acetate 98:2 as eluent. Recrystallized
from diethyl ether.
9a), 110.4 (s, C-9), 128.5 (d, C-40), 128.8 (d, C-20, C-60), 130.0 (s, C-8),
130.2 (d, C-30, C-50), 133.9 (s, C-10), 146.6 (s, C-6a), 152.1 (s, C-9b),
165.3 (s, C-5), 183.7 (s, C-2). Anal. Cald. for C24H30N6OS: C, 63.97; H,
6.71; N, 18.65; S, 7.11. Found: C, 64.06; H, 6.75; N, 18.61; S, 7.18.
Yield 66.5%. Mp 181.4e182.3 ꢁC. IR: 1665 (CO) cmꢂ1 1H NMR:
.
d
1.24 (s, 3H, CH3), 1.58 (t, 2H, J ¼ 6.0 Hz, CH2N), 2.10e2.22 (m, 10H,
CH2-200, CH2-300, CH2-500, CH2-600, CH2CH2CH2), 2.24 (s, 3H, CH3), 2.70
4.2.4. 8-[3-(piperidino)propyl]-4,10-dimethyl-9-phenyl-6-
(methylsulfanyl)-3,4-dihydropyrimido[1,2-c]pyrrolo[3,2-e]
pyrimidin-2(8H)-one (H2)
(s, 3H, CH3), 4.16 (t, 2H, J ¼ 6.0 Hz, NCH2), 4.32 (s, 2H, CH2);
7.45e7.60 (m, 5H, Ph). 13C NMR:
d 10.2 (q, CH3), 13.6 (q, CH3), 26.2 (t,
CH2CH2CH2), 40.8 (t, C-3), 45.5 (t, CH2N), 49.6 (t, NCH2), 52.1 (t,
To a stirred solution of B2 (0.15 g, 0.4 mmol) in dry dioxane
(5 mL), piperidine (0.08 mL, 0.8 mmol) was added. The mixture was
CH2-200, CH2-600), 54.4 (t, CH2-300, CH2-500), 54.5 (s, CH3), 100.2 (s, C-

A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
381
Table 3
Table 4
Overview of one dose screening test results (G%average for each human tumor cell
Five-dose screening for B1 and H2 derivatives.
lines pannel).
Panel
Cell line
B1
H2
Pannel
C3
D3 C1
84
D1 O3 N3 B1 I1
92 102 102 36
H2
pGI50 pTGI pLC50 pGI50 pTGI pLC50
Leukemia
100 105
81
3
91 53
98 26
89 44
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
5.29 <4.00 <4.00 5.57 4.92 <4.00
5.47 <4.00 <4.00 5.74 5.41 5.08
Non-small cell lung cancer 106 105 101 101 100 100 69
Colon cancer
CNS cancer
122 116 113 106 104 118 48
107 108 96 95 96 102 56
110 110 103 101 104 109 48 100 44
120 110 105 100 107 116 76
112 108 109 99 106 110 77
126 108 101 105 101 125 82 101 48
5.24 <4.00 <4.00 NT
5.41 <4.00 <4.00 5.70 5.29 4.69
5.43 4.18 <4.00 6.78 5.92 5.37
5.46 <4.00 <4.00 NT NT NT
NT
NT
Melanoma
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
95 47
98 63
Non-small cell A549/ATCC
lung cancer EKVX
HOP-62
5.11
5.16
4.96
4.94
4.96
5.08
5.01
5.43
5.41
4.18 <4.00 5.53 4.97 4.10
4.51 <4.00 5.52 4.70 <4.00
100 102 101 100 103 105 61
110 108 102 100 103 108 60
80 15
92 39
a
G%average
4.20 <4.00 5.5
4.43 <4.00 NT
4.58 <4.00
NT <4.00
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
a
Calculated on all G% values (table G% in SI4).
4.53 4.09 5.45 <4.00 <4.00
4.33 <4.00 5.53 <4.00 <4.00
4.61 4.23 5.21 4.05 <4.00
4.82 4.25 5.49 4.92 4.17
heated at reflux for 4 h and, after cooling, the solvent was evapo-
rated under reduced pressure. The crude residue was purified by
column chromatography using dichloromethane/methanol 98:2 as
eluent. Recrystallized from diethyl ether.
NCI-H522
4.72 4.12 NT
NT
NT
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
5.38
5.34
5.50
5.39
5.41
5.38
5.32
4.82 4.32 5.90 5.60 5.30
4.71 4.19 5.49 4.79 4.21
4.32 <4.00 5.62 <4.00 <4.00
4.67 <4.00 5.35 4.55 <4.00
4.71 <4.00 5.79 5.14 <4.00
4.73 4.09 5.50 4.96 4.47
4.27 <4.00 5.62 4.98 4.49
Yield 76.3%. Mp 203.6e204.0 ꢁC. IR: 1659 (CO) cmꢂ1 1H NMR
.
(SI5):
d
1.23e1.45 (m, 6H, CH2-300, CH2-400, CH2-500), 1.32 (d, 3H,
KM12
SW-620
J ¼ 7.4 Hz, CH3), 1.69 (m, 2H, CH2CH2CH2), 2.00e2.40 (m, 7H, H-3,
CH2N, CH2-200, CH2-600), 2.26 (s, 3H, CH3), 2.69 (s, 3H, CH3), 2.87 (dd,
1H, H-3), 4.10 (t, 2H, NCH2), 4.87e4.90 (m, 1H, H-4), 7.45e7.51 (m,
CNS cancer
Melanoma
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
5.07
5.39
5.47
5.40
5.55
5.41
4.32 <4.00 5.47 4.85 4.34
4.57 <4.00 5.59 4.99 4.16
4.85 4.40 5.19 4.06 <4.00
4.75 4.34 5.53 <4.00 <4.00
4.93 4.17 5.57 4.66 <4.00
4.79 4.27 5.61 4.95 4.47
5H, Ph). 13C NMR:
d 10.9 (q, CH3), 14.8 (q, CH3), 17 (q, CH3), 24.2 (t,
CH2), 25.3 (t, CH2), 30.5 (d, CH), 35.2 (t, CH2), 35.9 (t, CH2), 40.5 (t,
CH2), 50.6 (t, CH2), 52.9 (t, CH2), 102.9 (s, C-10a), 111.7 (s, C-10),
128.6 (d), 129.9 (d), 130.1 (d), 133.3 (s, C-10), 145.0 (s, C-7a), 153.0 (s,
C-10b), 154.0 (s), 159.0 (s), 173.2 (s). Anal. Cald. for C26H33N5OS: C,
67.36; H, 7.17; N, 15.11; S, 6.91. Found: C, 67.36; H, 7.17; N, 15.11; S,
6.91.
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
5.38
5.08
5.48
5.69
4.98
4.99
5.46
4.90
5.68
4.76 4.30 5.52 4.89 4.45
4.63 4.23 5.63 5.14 4.27
4.82 <4.00 5.44 4.76 4.19
5.33 4.76 5.54 4.98 4.23
4.56 4.13 5.67 5.28 4.68
4.60 4.20 5.54 4.88 4.44
4.85 4.42 5.70 5.31 4.83
4.3. General procedure for the preparation of 8-substituted ethyl 4-
[9-methyl-5-(methylsulfanyl)-2-oxo-3H-imidazo-[1,2-c]pyrrolo
[3,2-e]pyrimidin-7-yl] butyrate (C1, C3)
4.55 4.19 NT
NT
NT
4.94 4.42 5.55 4.85 4.42
To a stirred suspension of A1 or A3 (2.0 mmol) in dry N,N-
dimethylformamide (5 mL), potassium carbonate (0.85 g,
6.0 mmol) and ethyl-4-bromo-butyrate (0.86 mL, 6.0 mmol) were
added. The mixture was heated at 80 ꢁC in an oil bath for 3 h (in the
case of A1) and for 11 h (in the case of A3) and after cooling to room
temperature was poured into ice and cold water. The crude residue
was collected by filtration and, after drying, purified by column
chromatography using dichloromethane/methanol 98:2 as eluent.
Recrystallized from diethyl ether.
Ovarian cancer IGROV1
OVCAR-3
5.45
5.08
5.17
4.94
4.79
4.67 4.02 NT NT NT
4.44 <4.00 5.62 5.07 4.27
4.35 <4.00 5.59 4.75 <4.00
4.42 <4.00 4.72 <4.00 <4.00
4.14 <4.00 5.43 4.51 <4.00
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
5.37 <4.00 <4.00 4.77 4.13 <4.00
Renal cancer
786-0
5.07
5.41
5.02
5.34
4.95
5.20
5.07
4.98
4.21 <4.00 5.45 4.32 <4.00
4.82 4.27 5.50 5.04 <4.00
4.42 <4.00 5.52 4.65 <4.00
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
4.55 <4.00 NT
4.73 <4.00
C1: Yield 84%. Mp 225.4e226.0 ꢁC. IR: 1733 (CO), 1701 (CO)
4.53 4.10 5.82 5.33 4.6
4.41 <4.00 5.64 4.97 <4.00
4.11 <4.00 5.54 5.03 <4.00
4.54 4.11 5.30 4.62 4.12
cmꢂ1. 1H NMR:
d
1.18 (t, 3H, J ¼ 7.4 Hz, COOCH2CH3), 1.82e1.94 (m,
2H, CH2CH2CH2), 2.07 (t, 2H, CH2CO), 2.35 (s, 3H, CH3), 2.72 (s, 3H,
CH3), 4.04 (q, 2H, J ¼ 7.4 Hz, COOCH2CH3), 4.20 (t, 2H, J ¼ 7.0 Hz,
Prostate cancer PC-3
DU-145
5.06 <4.00 <4.00 5.86 5.45 5.04
NCH2), 4.23 (s, 2H, H-3), 7.34e7.55 (m, 5H, Ph). 13C NMR:
d 10.4 (q,
4.94
4.57 4.21 5.47 4.56 <4.00
CH3),14.10 (q, CH3),14.15 (q, COOCH2CH3), 24.9 (t, CH2CH2CH2), 31.0
(t, CH2CO), 42.1 (t, NCH2), 49.5 (t, C-3), 60.4 (t, COOCH2CH3), 101.2
(s, C-9a), 112.6 (s, C-9), 128.7 (d, C-40), 128.8 (d, C-20, C-60), 130.1 (d,
C-30, C-50), 130.4 (s, C-8), 134.6 (s, C-10), 147.3 (s, C-6a), 152.0 (s, C-
9b), 166.0 (s, C-5), 172.3 (s, CO), 183.6 (s, C-2). Anal. Cald. for
C22H24N4O3S: C, 62.25; H, 5.70; N, 13.20; S, 7.55. Found: C, 62.35; H,
5.73; N, 13.15; S, 7.60.
Breast cancer
MCF7
5.47
4.78 4.11 5.54 4.95 4.21
4.62 4.11 5.74 5.02 <4.00
MDA-MB-231/ATCC 5.25
HS 578T
BT-549
T-47D
MDA-MB-468
5.20
5.31
5.58
5.50
4.46 <4.00 5.64 5.1
4.01
4.45 <4.00 5.58 5.02 4.07
4.96 4.02 5.70 5.12 <4.00
4.88 4.22 5.57 5.00 <4.00
MG_MID
5.26
4.51 4.11 5.56 4.83 4.23
C3: Yield 71%. Mp 163.9e165.0 ꢁC, IR: 1725 (CO),1697 (CO) cmꢂ1
.
1H NMR:
d
1.24 (t, 3H, J ¼ 7.0 Hz, COOCH2CH3), 2.07 (t, 2H, CH2CO),
2.27e2.32 (m, 2H, CH2CH2CH2), 2.30 (s, 3H, CH3), 2.35 (s, 3H, CH3),
2.69 (s, 3H, CH3), 4.04 (q, 2H, J ¼ 7.4Hz, COOCH2CH3), 4.16 (s, 2H,
C-9a), 110.2 (s, C-9), 130.0 (s, C-8), 146.9 (s, C-6a), 151.0 (s, C-9b),
165.0 (s, C-5), 172.5 (s, CO), 183.6 (s, C-2). Anal. Cald. for
17H22N4O3S: C, 56.34; H, 6.12; N, 15.46; S, 8.85. Found: C, 56.41; H,
H-3), 4.19 (t, 2H, J ¼ 7.0 Hz, NCH2). 13C NMR:
d 9.7 (q, CH3), 9.8 (q,
CH3), 14.0 (q, CH3), 14.1 (t, COOCH2CH3), 25.1 (t, CH2CH2CH2), 31.1 (t,
CH2CO), 41.4 (t, NCH2), 49.5 (t, C-3), 60.6 (t, COOCH2CH3), 100.9 (s,
C
6.15; N, 15.47; S, 8.91.

382
A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
added and the mixture was stirred at room temperature for 24 h.
Evaporation of the solvent under reduced pressure gave a solid that
was purified by column chromatography using dichloromethane/
methanol 8:2 as eluent. Recrystallized from ethanol.
Yield 63.6%. Mp 189.6e191.0 ꢁC. IR: 1753 (CO), 1695 (CO) cmꢂ1
.
1H NMR:
d
1.86e1.94 (m, 2H, CH2CH2CH2), 2.03 (t, 2H, J ¼ 7.4 Hz,
CH2CO), 2.21 (s, 6H, 2 CH3), 2.63 (s, 3H, CH3), 3.22 (t, 2H, J ¼ 6.6 Hz,
NHCH2CH2), 3.37-3.51 (m, 2H, NHCH2), 4.13 (t, 2H, J ¼ 7.4 Hz, NCH2),
4.21 (s, 2H, H-3), 6.76 (s, 1H, CH), 7.50 (s, 1H, CH), 7.85 (t, 1H,
J ¼ 6.04 Hz, NH). 13C NMR:
d 9.3 (q, CH3), 9.5 (q, CH3), 13.5 (q, CH3),
25.4 (t, CH2CH2CH2), 26.8 (t, NHCH2CH2), 32.2 (t, CH2CO), 38,6 (t,
NHCH2), 41.1 (t, NCH2), 49.5 (t, C-3), 99.3 (s, C-9a), 108.3 (s, C-9),
130.1 (s, C-8), 131.2 (C-50), 134.5 (d, CH) 134.8 (d, CH) 146.1 (s, C-6a),
150.9 (s, C-9b), 164.6 (s, C-5), 171.0 (s, CONH), 173.9 (s, C-2). Anal.
Cald. for C20H25N7O2S: C, 56.19; H, 5.89; N, 22.93; S, 7.50. Found: C,
56.29; H, 5.85; N, 22.98; S, 7.45.
Fig. 5. Doseeresponse leukemia panel graph for H2 derivative.
4.4.4. Methyl 3-(1H-imidazol-4-yl)-2-[8,9-dimethyl-5-
(methylsulfanyl)-2-oxo-3H-imidazo-[1,2-c]pyrrolo[3,2-e]pyrimidin-
7-yl-butyramide]propanoate (O3)
4.4. General procedure for the preparation of 8-substituted 4-[9-
methyl-5-(methylsulfanyl)-2-oxo-3H-imidazo-[1,2-c]pyrrolo[3,2-e]
pyrimidin-7-yl] butanoic acid (D1, D3)
To a stirred suspension of carboxylic acid D3 (0.6 mmol) in dry
dichloromethane (5 mL), cooled to 0 ꢁC, DCC (0.14 g, 0.66 mmol)
was added. After 1 h, L-histidine methyl ester dihydrochloride
To a stirred suspension of C1 or C3 (1.4 mmol), in dry ethanol
(5 mL), a solution of potassium hydroxide (0.23 g, 4.0 mmol) in
water (10 mL) was added. The mixture was stirred at room
temperature for 12 h (in the case of C1) and 24 h (in the case of C3).
After cooling, the ethanol was evaporated under reduced pressure
and the aqueous layer was then carefully adjusted to pH 5. The
resulting solid was filtered off, air-dried, and purified by column
chromatography using dichloromethane/methanol 98:2 as eluent.
Recrystallized from diethyl ether.
(0.22 g, 0.9 mmol) and triethylamine (0.13 mL, 0.9 mmol) were
added and the mixture was stirred at room temperature for 24 h.
Evaporation of the solvent under reduced pressure gave a solid that
was purified by column chromatography using dichloromethane/
methanol 98:2 as eluent. Recrystallized from ethanol.
Yields 54.7%. Mp 148.1e148.9 ꢁC. IR: 1751 (CO), 1724 (CO), 1696
(CO) cmꢂ1 1H NMR:
. d 1.81e1.88 (m, 2H, CH2CH2CH2), 2.11 (t, 2H,
J ¼ 7.4 Hz, CH2CO), 2.24 (s, 6H, 2 CH3), 2.65 (s, 3H, CH3), 2.90 (t, 1H,
J ¼ 7.0 Hz, CHCO), 3.03 (d, 2H, CH2), 3.59 (s, 3H, OCH3), 4.25 (s, 2H,
H-3), 4.49 (t, 2H, J ¼ 7.4 Hz, NCH2), 6.90 (s, 1H, CH), 7.80 (s, 1H, CH),
4.4.1. 4-[9-methyl-5-(methylsulfanyl)-2-oxo-8-phenyl-3H-
imidazo-[1,2-c]pyrrolo[3,2-e]pyrimidin-7-yl] butanoic acid (D1)
Yield 64%. Mp 212.2e213.5 ꢁC. IR: 3412 (OH), 1753 (CO), and
8.35 (d, 1H, NH). 13C NMR:
d 9.3 (q, CH3), 9.5 (q, CH3), 13.5 (q, CH3),
25.3 (t, CH2CH2CH2), 28.3 (t, CH2), 31.8 (t, CH2CO), 41.1 (t, NCH2),
49.5 (t, C-3), 51.8 (d, CH), 52.2 (q, OCH3), 99.6 (s, C-9a),107.9 (s, C-9),
116.6 (d, CH), 129.0 (s, C-8), 130.1(s, C-50), 134.6 (d, CH), 146.0 (s, C-
6a), 151.0 (s, C-9b), 164.5 (s, C-5), 171.4 (s, CO), 171.9 (s, CONH), 183.5
(s, C-2). Anal. Cald. for C22H27N7O4S: C, 54.42; H, 5.60; N, 20.19; S,
6.60. Found: C, 54.55; H, 5.57; N, 20.15; S, 6.62.
1697 (CO) cmꢂ1. 1H NMR:
d 1.60e1.68 (m, 2H, CH2CH2CH2), 1.99 (t,
2H, CH2CO), 2.23 (s, 3H, CH3), 2.70 (s, 3H, CH3), 4.16 (t, 2H, J ¼ 7.0 Hz,
NCH2), 4.22 (s, 2H, H-3), 7.45e7.55 (m, 5H, Ph), 12.05e12.09 (bs, 1H,
OH). 13C NMR:
d 10.6 (q, CH3), 14.0 (q, CH3), 25.0 (t, CH2CH2CH2),
30.8 (t, CH2CO), 41.5 (t, NCH2), 49.5 (t, C-3), 101.2 (s, C-9a), 112.6 (s,
C-9),128.5 (d, C-40),128.8 (d, C-20, C-60),130.1 (d, C-30, C-50),130.6 (s,
C-8), 134.3 (s, C-10), 147.2 (s, C-6a), 152.8 (s, C-9b), 165.8 (s, C-5),
173.9 (s, CO), 184.1 (s, C-2). Anal. Cald. for C20H20N4O3S: C, 60.59; H,
5.08; N, 14.13; S, 8.09. Found: C, 60.74; H, 5.14; N, 14.16; S, 8.16.
4.4.5. Ethyl N-[4-(9-methyl-5-(methylsulfanyl)-2-oxo-8-phenyl-
3H-imidazo[1,2-c]pyrrolo[3,2-e]pyrimidin-7-yl)-butanoyl]glycinate
(E1)
To a stirred suspension of carboxylic acid D1 (0.4 mmol) in dry
dichloromethane (5 mL), cooled to 0 ꢁC, DCC (0.093 g, 0.45 mmol)
was added. After 1 h, glycine ethyl ester hydrochloride (0.085 g,
0.61 mmol) and triethylamine (0.085 mL, 0.61 mmol) were added
and the mixture was stirred at room temperature for 24 h. Evap-
oration of the solvent under reduced pressure gave a solid that was
purified by column chromatography using dichloromethane/
methanol 98:2 as eluent. Recrystallized from ethanol.
4.4.2. 4-[8,9-dimethyl-5-(methylsulfanyl)-2-oxo-3H-imidazo-[1,2-
c]pyrrolo[3,2-e]pyrimidin-7-yl] butanoic acid (D3)
Yield 97.3%. Mp 220.6e221.5 ꢁC. IR: 3446 (OH), 1750 (CO), and
1695 (CO) cmꢂ1. 1H NMR:
d 1.79e1.85 (m, 2H, CH2CH2CH2), 2.00 (t,
2H, CH2CO), 2.24 (s, 3H, CH3), 2.30 (s, 3H, CH3), 4.04 (t, 2H,
J ¼ 7.0 Hz, NCH2), 4.20 (s, 3H, CH3), 4.44 (s, 2H, H-3), 12.05e12.09
(bs, 1H, OH). 13C NMR:
d
9.8 (q, CH3),
d
10.1 (q, CH3), 14.1 (q, CH3),
Yield 78%. 173.1e174.7 ꢁC. IR: 1756 (CO), 1733 (CO), 1695 (CO)
25.0 (t, CH2CH2CH2), 30.9 (t, CH2CO), 41.3 (t, NCH2), 49.5 (t, C-3),
101.1 (s, C-9a), 111.4 (s, C-9), 130.3 (s, C-8), 147.0 (s, C-6a), 151.7 (s,
C-9b), 165.5 (s, C-5), 173.6 (s, CO), 182.6 (s, C-2). Anal. Cald. for
C15H18N4O3S: C, 53.88; H, 5.43; N, 16.75; S, 9.59. Found: C, 53.96; H,
5.45; N, 16.77; S, 9.65.
cmꢂ1. 1H NMR (SI5):
d
1.15 (t, 3H, J ¼ 8.0 Hz, COOCH2CH3), 1.65e1.72
(m, 2H, CH2CH2CH2), 1.91 (t, 2H, CH2CO), 2.24 (s, 3H, CH3), 2.69 (s,
3H, CH3), 3.70 (d, 2H, NHCH2), 4.04 (q, 2H, J ¼ 8.0 Hz, COOCH2CH3),
4.12 (t, 2H, J ¼ 7.0 Hz, NCH2), 4.32 (s, 2H, H-3), 7.39-7.56 (m, 5H, Ph),
8.14 (t, 1H, J ¼ 6.0 Hz, CONH). 13C NMR (SI5):
d 10.2 (q, CH3), 13.6 (q,
SCH3), 14.0 (q, COOCH2CH3), 25.2 (t, CH2CH2CH2), 31.8 (t, CH2CO),
40.7 (t, NHCH2), 41.9 (t, NCH2), 49.6 (t, C-3), 60.3 (t, COOCH2CH3),
100.1 (s, C-9a), 110.6 (s, C-9), 128.5 (d), 128.8 (d), 130.1 (d), 133.9 (s,
C-8), 134.6 (s, C-10), 146.7 (s, C-6a), 152.2 (s, C-9b), 165.3 (s, C-5),
169.8 (s, CONH), 171.4 (s, CO), 183.6 (s, C-2). Anal. Cald. for
C24H27N5O4S: C, 59.86; H, 5.65; N, 14.54; S, 6.66. Found: C, 59.98; H,
5.61; N, 14.57; S, 6.54.
4.4.3. N-[2-(1H-imidazol-4-yl)ethyl]-4-[8,9-dimethyl-5-
(methylsulfanyl)-2-oxo-3H-imidazo[1,2-c]pyrrolo[3,2-e]pyrimidin-
7-yl]butyramide (N3)
To a stirred suspension of carboxylic acid D3 (0.3 mmol) in dry
dichloromethane (5 mL), cooled to 0 ꢁC, DCC (0.07 g, 0.3 mmol) was
added. After 1 h, histamine-free-base (0.05 g, 0.45 mmol) was

A. Lauria et al. / European Journal of Medicinal Chemistry 55 (2012) 375e383
383
4.5. Biology
parameters if the level of activity is reached; however, if the effect is
not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum
concentration tested.
The human tumour cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and
2 mM -glutamine. For a typical screening experiment, cells are
inoculated into 96 well microtiter plates in 100 L at plating
L
m
Acknowledgements
densities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 ꢁC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line
at the time of drug addition (Tz). Experimental drugs are solubi-
lized in dimethyl sulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate is thawed and
diluted to twice the desired final maximum test concentration with
Thanks are due to the NCI, Bethesda, MD (USA) for performing
the antitumor testing of the synthesized compounds.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.07.
046. These data include MOL files and InChiKeys of the most
important compounds described in this article.
complete medium containing 50
10-fold or ½ log serial dilutions are made to provide a total of five
drug concentrations plus control. Aliquots of 100 l of these
different drug dilutions are added to the appropriate microtiter
wells already containing 100 l of medium, resulting in the
required final drug concentrations.
Following drug addition, the plates are incubated for an addi-
tional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative humidity. For
adherent cells, the assay is terminated by the addition of cold TCA.
mg/ml gentamicin. Additional four,
References
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Cells are fixed in situ by the gentle addition of 50 ml of cold 50%
(w/v) TCA (final concentration, 10% TCA) and incubated for 60 min
at 4 ꢁC. The supernatant is discarded, and the plates are washed five
times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 ml) at 0.4% (w/v) in 1% acetic acid is added to each well, and
plates are incubated for 10 min at room temperature. After staining,
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½ðTi ꢂ TzÞ=Tzꢃ ꢄ 100 for concentrations for which Ti < Tz:
Three dose response parameters are calculated for each exper-
imental agent. Growth inhibition of 50% (GI50) is calculated from
[(Ti ꢂ Tz)/(C ꢂ Tz)] ꢄ 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug incu-
bation. The drug concentration resulting in total growth inhibition
(TGI) is calculated from Ti ¼ Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning) indicating
a net loss of cells following treatment is calculated from [(Ti ꢂ Tz)/
Tz] ꢄ 100 ¼ ꢂ50. Values are calculated for each of these three