The Novel [4,5-e][1,3]Diazepine-4,8-dione and acyclic carbamoyl imino-ureido derivatives of imidazole: Synthesis, anti-viral and anti-tumor activity evaluations

Article abstract of DOI:10.3390/molecules181113385

In the present paper, we report on the synthesis, and in vitro antiviral and cytostatic activities of a series of novel imidazole[4,5-e][1,3]diazepine-4, 8-dione (compounds 9-11) and acyclic carbamoyl imino-ureido imidazole (compounds 12 and 13) derivatives. These new type of chemical entities showed no significant activity on the broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 μM) with no concomitant cytotoxic effects on human normal fibroblasts (BJ). Importantly, an imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed a selective cytostatic effect toward cervical carcinoma (HeLa) cells (IC50 = 9.5 μM) with no apparent cytotoxicity on human normal fibroblasts (BJ). This compound can be therefore considered as a potential anti-tumor lead compound for further synthetic structure optimization.

Full text of DOI:10.3390/molecules181113385

Molecules 2013, 18, 13385-13397; doi:10.3390/molecules181113385
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Novel [4,5-e][1,3]Diazepine-4,8-dione and Acyclic
Carbamoyl Imino-Ureido Derivatives of Imidazole:
Synthesis, Anti-Viral and Anti-Tumor Activity Evaluations
Karlo Wittine 1, Kristina Poljak 1, Matea Kovač 1, Damjan Makuc 2,3, Janez Plavec 2,3
Jan Balzarini 4, Tamara Martinović 5, Sandra Kraljević Pavelić 5, Krešimir Pavelić 5
and Mladen Mintas 1,*
,
1
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, Zagreb HR-10000, Croatia;
E-Mails: kwittine@fkit.hr (K.W.); polly3112@net.hr (K.P.); matea.kovac@hotmail.com (M.K.)
Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, P. O. B. 660,
Ljubljana SI-1001, Slovenia; E-Mails: damjan.makuc@ki.si (D.M.); janez.plavec@ki.si (J.P.)
EN-FIST Centre of Excellence, Dunajska 156, Ljubljana SI-1001, Slovenia
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10,
Leuven B-3000, Belgium; E-Mail: jan.balzarini@rega.kuleuven.be (J.B.)
Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, Rijeka 51000, Croatia;
E-Mails: tamara.martinovic@uniri.hr (T.M.); sandrakp@biotech.uniri.hr (S.K.);
pavelic@biotech.uniri.hr (K.P.)
2
3
4
5
* Author to whom correspondence should be addressed; E-Mail: mmintas@fkit.hr;
Tel.: +385-1-4597-214; Fax: +385-1-4597-250.
Received: 29 August 2013; in revised form: 5 October 2013 / Accepted: 17 October 2013 /
Published: 30 October 2013
Abstract: In the present paper, we report on the synthesis, and in vitro antiviral
and cytostatic activities of a series of novel imidazole[4,5-e][1,3]diazepine-4,8-dione
(compounds 9–11) and acyclic carbamoyl imino-ureido imidazole (compounds 12 and 13)
derivatives. These new type of chemical entities showed no significant activity on the
broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on
a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed
moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 µM) with no
concomitant cytotoxic effects on human normal fibroblasts (BJ). Importantly, an imidazole
derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed a
selective cytostatic effect toward cervical carcinoma (HeLa) cells (IC50 = 9.5 µM) with

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no apparent cytotoxicity on human normal fibroblasts (BJ). This compound can be
therefore considered as a potential anti-tumor lead compound for further synthetic
structure optimization.
Keywords: imidazole; [4,5-e][1,3]diazepine; anti-RSV; anti-tumor
1. Introduction
It has been found that infection with respiratory syncytial virus (RSV), which manifests primarily as
bronchiolitis or viral pneumonia, is the leading cause of lower respiratory tract infections (LRTIs) in
infants and young children [1]. Ribavirin still is the only antiviral agent approved for the treatment of
RSV infection, but due to efficacy and toxicity issues, it has only limited utility [2]. There is a clear
need for new anti-RSV therapeutics, with improved efficacy and safety for broader applications [3].
Powell and his colleagues have recently identified a new class of RSV inhibitors, namely
1,4-benzodiazepines [4], which eventually led to identification of RSV604 as a clinical candidate [5].
Furthermore, imidazole is an entity incorporated into many important biological molecules with a
wide range of pharmacological activity. In the field of drug discovery the imidazole scaffold is
widely used in the drug design strategy and imidazoles are generally well known as anticancer agents as
well [6].
Moreover, heterocycles containing an imidazo[4,5-e][1,3]diazepine ring system have already shown
potent in vitro activity at low micromolar concentrations against lung, breast, ovarian and prostate
cancer cell lines [7]. Also, in vitro inhibitory activity of a number of ring-expanded nucleosides
against NTPase/helicases of a family of Flaviviridae have been reported. Compounds containing an
imidazo[4,5-e][1,3]diazepine-4,8-dione ring system have exhibited potent activities against HBV and
HCV [8]. Ring-expanded nucleosides employing a fused imidazo[4,5-e][1,3]diazepine ring system
show promising anti-measles virus activity at submicromolar or micromolar concentration levels with
no apparent toxicity to the host cell line [9].
In the light of these findings we efficiently synthesized a series of new 1H-imidazole-4,5-dicarboxylic
acid/amide derivatives (1–8), imidazo[4,5-e][1,3]diazepine-4,8-dione derivatives (9–11) and
carbamoyl imino-ureido derivatives of imidazole (12 and 13) (Figure 1) and evaluated their antiviral
and cytostatic activity potency.
2. Results and Discussion
2.1. Chemistry
The synthesis of new 1H-imidazole-4,5-dimethyl dicarboxylate (1–4), 1H-imidazole-4,5-diamide
(5–8), imidazole[4,5-e][1,3]diazepine-4,8-dione (9–11) and acyclic carbamoyl imino-ureido imidazole
derivatives (12 and 13) is performed according to the Figure 2.

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Figure 1. New 1H-imidazole-4,5-dicarboxylic acid/amide derivatives (1–8),
imidazo[4,5-e][1,3]diazepine-4,8-dione derivatives (9–11) and carbamoyl imino-ureido
derivatives of imidazole (12 and 13).
Figure 2. Synthesis of imidazo[4,5-e][1,3]diazepine-4,8-dione (9–11) and
ureido-imino-carbamoyl-imidazole derivatives (12–13). Reagents and conditions:
(i) K2CO3, CH3CN, r.t.; (ii) NH3, MeOH, r.t.; (iii) guanidine hydrochloride, NaOMe,
MeOH, r.t.; (iv) 2-fluorophenyl isocyanate, DMF, r.t.
Reaction of 4,5-dimethyl 1H-imidazole-dicarboxylate (dMIdC) with K2CO3 and different
2-chloroalkylamine hydrochlorides, that is 4-(2-chloroethyl)morpholine, 4-(2-chloroethyl)piperidine,
4-(2-chloroethyl)pyrrolidine and 3-dimethylaminopropyl chloride gave 4,5-dimethyl dicarboxylate
derivatives (1–4) with alkylamino chains at the N-1 of the imidazole ring. 1H-imidazo-4,5-diamide
derivatives (5–7) were obtained by ammonolysis of diesters 1–4 in polar protic solvent.

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Time-controlled reaction of compounds 1–3 with guanidine-hydrochloride and sodium-methoxide
gave cyclized imidazo[4,5-e]diazepine-4,8-dione derivatives (9–11) and a mixture of two regioisomeric
guanidino carbamoyl-imidazole derivatives which in subsequent in situ reaction with 2-fluorophenyl
isocyanate in DMF afforded carbamoyl imino-ureido derivatives 12 and 13. It is worth noting that the
reaction of imidazo[4,5-e][1,3]diazepine-4,8-dione derivatives (9–11) with 2-fluorophenyl isocyanate
did not gave the desired ureido-imidazo[4,5-e][1,3]diazepine-4,8-dione derivative. The main reason
obviously lies in the fact that 6-amino-diazepine-4,8-dione molecules (9–11) are predominantly in
solution in their imino tautomeric form as confirmed by NMR spectroscopy, which is inactive in the
final step of the synthesis to create the ureido derivative of imidazo[4,5-e][1,3]diazepine-4,8-dione.
Morpholine derivative 12 for which we believed to have a diazepine-4,8-dione structure and whose
NMR spectrum indicated next to all diagnostic signals for the assumed structure the presence of an
unidentified signal at approximately 3.8 ppm attributable to a OMe group, which is consistent with the
results of the determination of the molecular ion mass by high resolution mass spectrometry.
2.2. Structural Properties
1
The structures of 1–13 have been confirmed by H and 13C-NMR spectra (Experimental). The
analysis of the spectra was achieved on the basis of the chemical shift, signal multiplicity and integral
values. 19F NMR resonances of 12 and 13 were well resolved (Experimental). 1H decoupled 13C-NMR
showed C–F coupling constants that enabled straightforward identification of fluorinated carbon atoms
and their neighbors. Two sets of signals were observed for 9 in the ratio 4:1 as was estimated from the
integral values of 1H-NMR signals (e.g., CH-5 proton at δ 8.05 and 7.69 ppm). Two species were most
probably detected due to presence of dynamic equilibrium between amine and imine tautomers
(C8–NH2 and C8=NH). C8 chemical shifts of the major species showed value of 149.99 ppm, which is
characteristic for the imine form. Therefore, imine form of 9 is the predominant species in solution.
Likewise, broad 13C-NMR signals suggested presence of tautomer forms for 10 and 11. Imine forms
are favored according to C8 chemical shift (δ 149.70 and 150.12 ppm for 10 and 11, respectively).
1
The correlation signals observed in H-13C HSQC and HMBC spectra allowed assignment of C2,
C3 and C5 atoms. Interestingly, major chemical shifts differences between 12 and 13 were observed
for C2 and C3 atoms. The correlation signals between methylene protons CH2-1' and C2 and C5 in
combination with chemical shift values of imidazole carbon atoms suggested that COOCH3 group is
attached to C2 (δ 121.43 ppm) and fluoro-phenyl-ureido-imino-methyl-carbamoyl moiety to C3
(δ 145.53) in 12. On the other hand, COOCH3 in attached to C3 in 13, which is indicated by C2 and C3
chemical shift (δ 127.71 and 134.19 ppm).
These spectroscopic results indicate that compounds 12 and 13 exist as regioisomers with respect to
substitution at positions C-2 and C-3 of the imidazole moiety. This implies that both regioisomers are
formed as indicated in Figure 2 and their subsequent in situ reaction gave compounds 12 and 13.
Structural differences between 12 and 13, which were suggested by distinct NMR chemical shifts of
C2 and C3, were assessed by NOESY experiments. Unfortunately, only trivial NOESY cross-peaks
were observed for 12 and 13. COOCH3 group showed no NOESY signals with methylene protons and
therefore no particular conformational preferences could be established for these compounds.

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2.3. Biological Results
2.3.1. Antiviral Activity
Compounds 1–3, 5–7 and 9–13 were evaluated for their antiviral activity against a wide variety of
DNA and RNA viruses, including herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia
virus and vesicular stomatitis virus in HEL cells, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4,
and Punta Toro virus in Vero cells, vesicular stomatitis virus, Coxsackie virus B4, and respiratory
syncytial virus in HeLa cells and influenza A (H1N1; H3N2) and influenza B viruses in MDCK
cells. Unfortunately, none of the compounds showed pronounced antiviral activity at subtoxic
concentrations. No cytotoxicity for all evaluated compounds on HEL, Vero, HeLa and MDCK cell
cultures was observed (data not shown).
2.3.2. Cytostatic Activity
Compounds 1–12 were evaluated for their antiproliferative effect against several malignant tumor
cell lines: cervical carcinoma (HeLa), colorectal adenocarcinoma, metastatic (SW 620), breast
epithelial adenocarcinoma, metastatic (MCF-7) and human hepatocarcinoma (HepG2) cells and
compared with their effects on the growth of normal human skin fibroblasts (BJ) (Table 1). The
imidazole[4,5-e][1,3]diazepine-4,8-dione derivative linked with a pyrrolidine ligand (11) showed a
modest cytostatic effect on colon cancer cells (SW620) (IC50 = 20 µM) while imidazole derivative
containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed specific antiproliferative
effect on HeLa cells (IC50 = 9.5 µM). However, both compounds 11 and 3 exerted moderate growth
inhibition only at the highest tested concentrations (10 and 100 µM). Other compounds exerted weak
or no antproliferative effects on tested cell lines.
Table 1. Inhibitory effects of compounds 1–12 on the growth of malignant tumor cell lines
in comparison with their effect on normal human skin fibroblasts (BJ).
Tumor cell growth IC50a (μM)
Cell lines
Compd.
1
Structure
MCF-7 HepG2 SW620 HeLa
BJ
>100
>100
>100
>100
>100
>100
24
>100
2
3
>100 >100
>100
>100
>100
9.5
>100

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Table 1. Cont.
Tumor cell growth IC50 a (μM)
Cell lines
MCF-7 HepG2 SW620 HeLa
Compd.
4
Structure
BJ
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100 >100
5
6
7
32
>100
>100 >100
>100 >100
8
9
>100
>100
>100
>100
>100
>100
41
>100
>100
31
>100 >100
>100 >100
10
11
31
>100
>100
20
>100 >100
>100 >100
12
>100
>100
>100
a
IC50; 50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation
by 50%.

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3. Experimental
3.1. General Materials and Methods
All commercially available chemicals were purchased from Sigma Aldrich (Hamburg, Germany)
and used without purification. All solvents were analytical grade purity and dried. Methanol (CH3OH)
was stored over 3 Å molecular sieves. Dimethylformamide (DMF) was stored over 4 Å molecular sieves.
Dichloromethane (CH2Cl2) was refluxed over phosphorus pentoxide (P2O5), distilled and stored over
4 Å molecular sieves. Merck silica gel 60 F254 plates were used for thin-layer chromatography.
Column chromatography was performed with Merck silica gel (0.063−0.200 mm), with
1
dichloromethane/methanol as eluent. H and 13C-NMR spectra were recorded on a Varian Gemini
300 spectrometer (Institute Ruđer Bošković, Zagreb, Croatia) and Varian NMR System 600 and
Varian Unity Inova 300 and Agilent Technologies DD2 300 MHz NMR spectrometers (National
Institute of Chemistry, Ljubljana, Slovenia) Samples were measured in CDCl3 and DMSO-d6 solutions
at 25 °C in 5 mm NMR tubes. Chemical shifts (δ) in ppm were referred to TMS. High performance LC
was performed on Agilent 1100 series system with UV detection (photodiode array detector) using
Zorbax C18 reverse-phase analytical column (2.1 × 30 mm; 3.5 µm). All compound used for biological
evaluation showed >95% purity in this HPLC system. The electron impact mass spectra and the purity
of compounds were assessed by using Agilent Technologies 6410 Triple Quad LC/MS instrument
equipped with electrospray interface and triple quadrupole analyzer (LC-MS/MS).
3.2. Procedures for the Preparations of Compounds
3.2.1. General Procedure for Synthesis of 1–4
To a solution of 4,5-dimethyl 1H-imidazole-dicarboxylate (1 g; 5.43 mmol) and K2CO3 (1.5 g;
10.86 mmol) in CH3CN (30 mL) is added 4-(2-chloroethyl)morpholine hydrochloride (for cmpd 1),
4-(2-chloroethyl)piperidine hydrochloride (for cmpd 2), 4-(2-chloroethyl)pyrrolidine hydrochloride
(for cmpd 3) or 3-dimethylaminopropyl chloride hydrochloride (for cmpd 4) (5.43 mmol). Reaction
mixture is stirred at room temperature for 48 h. The solvent is evaporated and the crude product simply
extracted with CH2Cl2 or purified by silica gel column chromatography (CH2Cl2/CH3OH = 20/1).
1-(2-Morpholin-4-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (1): Following general
1
procedure compound 1 is obtained as a yellow oil (1.68 g; 52.23%); MS m/z: 297.1 (M+1). H-NMR
(DMSO-d6): δ 2.37 (4H, m, CH2-4'), 2.58 (2H, t, J = 5.8, CH2-2'), 3.52 (4H, m, CH2-5'), 3.79 (3H, s,
COOCH3-3), 3.84 (3H, s, COOCH3-2), 4.29 (2H, t, J = 5.7, CH2-1'), 7.97 ppm (1H, s, CH-5).
13C-NMR (DMSO-d6): δ 43.63 (C1'), 52.29 (COOCH3-3), 52.86 (COOCH3-2), 53.63 (C4'), 58.72
(C2'), 66.57 (C5'), 124.99 (C2), 136.24 (C3), 141.58 (C5), 160.78 (COOCH3), 163.44 ppm (COOCH3).
1-(2-Piperidin-1-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (2): Following general
procedure compound 2 is obtained as a pale yellow oil (1.32 g; 83.36%); MS m/z: 296.2 (M+1).
1H-NMR (DMSO-d6): δ 1.25–1.45 (6H, m, 2 × CH2-5' and CH2-6'), 2.30 (4H, m, CH2-4'), 2.51 (2H, t,
J = 5.7, CH2-2'), 3.77 (3H, s, COOCH3-3), 3.83 (3H, s, COOCH3-2), 4.25 (2H, t, J = 5.9, CH2-1'),
7.92 ppm (1H, s, CH-5). 13C-NMR (DMSO-d6): δ 24.29 (C6'), 25.94 (C5'), 44.09 (C1'),

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52.28 (COOCH3-3), 52.84 (COOCH3-2), 54.47 (C4'), 59.10 (C2'), 125.17 (C2), 136.05 (C3),
141.47 (C5), 160.79 (COOCH3), 163.42 ppm (COOCH3).
1-(2-Pyrrolidin-1-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (3): Following general
procedure compound 3 is obtained as a pale yellow oil (1.11 g; 72.98%); MS m/z: 282.1 (M+1).
1H-NMR (DMSO-d6): δ 1.69 (4H, m, CH2-5'), 2.43 (4H, m, CH2-4'), 2.70 (2H, t, J = 6.1, CH2-2'),
3.78 (3H, s, COOCH3-3), 3.83 (3H, s, COOCH3-2), 4.27 (2H, t, J = 6.1, CH2-1'), 7.96 ppm (1H, s,
CH-5). 13C-NMR (DMSO-d6): δ 23.64 (C5'), 45.64 (C1'), 52.30 (COOCH3-3), 52.87 (COOCH3-2),
53.96 (C4'), 56.20 (C2'), 125.18 (C2), 136.06 (C3), 141.41 (C5), 160.78 (COOCH3), 163.39 ppm
(COOCH3).
1-(3-Dimethylamino-propyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (4): Following general
procedure compound 4 is obtained as a pale yellow oil (0.75 g; 75.0%); MS m/z: 270.1 (M+1).
1H-NMR (DMSO-d6): δ 1.84 (2H, m, CH2-2'), 2.16 (6H, s, 2 × CH3), 2.29 (2H, t, J = 6.5, CH2-3'), 3.76
(3H, s, COOCH3-3), 3.81 (3H, s, COOCH3-2), 4.18 (2H, t, J = 7.1, CH2-1'), 7.96 ppm (1H, s, CH-5).
13C-NMR (DMSO-d6): δ 28.21 (C2'), 44.74 (C1'), 45.04 (CH3) 52.34 (COOCH3-3), 52.99 (COOCH3-2),
55.73 (C3'), 124.89 (C2), 136.37 (C3), 141.29 (C5), 160.67 (COOCH3), 163.39 ppm (COOCH3).
3.2.2. General Procedure for Synthesis of 5–8
To a stirred solution of compounds 1–4 (500 mg; 1.68 mmol) in anhydrous CH3OH (15 mL)
ammonia is introduced at 0 °C. After saturation a reaction mixture is stirred overnight at room
temperature. The solvent and excess ammonia are removed under reduced pressure and the crude
product purified by silica gel column chromatography (CH2Cl2/CH3OH = 2/1).
1-(2-Morpholin-4-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid diamide (5): According to the general
procedure compound 5 is obtained as a white crystals (262 mg; 58.17%; m.p. = 196–197 °C); MS m/z:
1
267.1 (M+1). H-NMR (DMSO-d6): δ 2.39 (4H, m, CH2-4'), 2.59 (2H, t, J = 6.1, CH2-2'),
3.51 (4H, m, CH2-5'), 4.53 (2H, t, J = 6.1, CH2-1'), 7.53 (1H, s, CONH2-2), 7.75 (1H, s, CONH2-3),
7.88 (1H, s, CH-5), 7.95 (1H, s, CONH2-3), 10.65 ppm (1H, s, CONH2-2). 13C-NMR (DMSO-d6):
δ 43.93 (C1'), 53.25 (C4'), 58.51 (C2'), 66.18 (C5'), 126.16 (C2), 136.90 (C3), 140.53 (C5), 160.66
(CO-2), 165.80 ppm (CO-3).
1-(2-Piperidin-1-il-etil)-1H-imidazol-4,5-diamide (6): According to the general procedure compound 6
is obtained as a white crystals (194 mg; 53.93%; m.p. = 200–201 °C); MS m/z: 265.2 (M+1).
1H-NMR (DMSO-d6): δ 1.35 (2H, m, CH2-6'), 1.43 (4H, m, CH2-5'), 2.34 (4H, m, CH2-4'), 2.54 (2H, t,
J = 6.2, CH2-2'), 4.51 (2H, t, J = 6.2, CH2-1'), 7.51 (1H, s, CONH2-2), 7.74 (1H, s, CONH2-3),
7.85 (1H, s, CH-5), 7.94 (1H, s, CONH2-3), 10.64 ppm (1H, s, CONH2-2). 13C-NMR (DMSO-d6):
δ 24.41 (C6'), 26.08 (C5'), 44.90 (C1'), 54.55 (C4'), 59.30 (C2'), 126.64 (C2), 135.36 (C3), 140.98
(C5), 161.14 (CO-2), 166.31 ppm (CO-3).
1-(2-Piperidin-1-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid diamide (7): According to the general
procedure compound 7 is obtained as a white crystals (182 mg; 45.50%; m.p. = 205–210 °C); MS m/z:
252.2 (M+1). 1H-NMR (DMSO-d6): δ 1.65 (4H, m, CH2-5'), 2.45 (4H, m, CH2-4'), 2.72 (2H, t, J = 5.6,

Molecules 2013, 18
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CH2-2'), 4.52 (2H, t, J = 6.1, CH2-1'), 7.53 (1H, s, CONH2-2), 7.75 (1H, s, CONH2-3), 7.88 (1H, s,
CH-5), 7.94 (1H, s, CONH2-3), 10.65 ppm (1H, s, CONH2-2). 13C-NMR (DMSO-d6): δ 23.16 (C5'),
46.03 (C1'), 53.52 (C4'), 55.97 (C2'), 126.11 (C2), 134.98 (C3), 140.42 (C5), 160.61 (CO-2), 165.80
ppm (CO-3).
1-(3-Dimethylamino-propyl)-1H-imidazole-4,5-dicarboxylic acid diamide (8): According to the
general procedure compound 8 is obtained as a white crystals (144 mg; 48.0%; m.p. = 205–210 °C);
MS m/z: 240.1 (M+1). 1H-NMR (DMSO-d6): δ 1.83 (2H, m, CH2-2'), 2.11 (6H, s, 2 × CH3), 2.14 (2H,
t, J = 6.9, CH2-3'), 4.42 (2H, t, J = 7.0, CH2-1'), 7.57 (1H, s, CONH2-2), 7.83 (1H, s, CONH2-3), 7.89
13
(1H, s, CH-5), 7.98 (1H, s, CONH2-3), 10.66 ppm (1H, s, CONH2-2). C-NMR (DMSO-d6): δ 29.09
(C2'), 45.50 (CH3), 45.93 (C3'), 56.31 (C1'), 126.71 (C2), 135.60 (C3), 140.61 (C5), 161.02 (CO-2),
166.25 ppm (CO-3).
3.2.3. General Procedure for Synthesis of 9–11
To a solution of guanidine hydrochloride (5.38 mmol) in anhydrous methanol (10 mL) cooled to
0 °C is added a solution of sodium methoxide (25 wt%; 13 mmol). The reaction mixture is stirred for
30 min at 0 °C. The sodium salt formed is removed by filtration, and the filtrate thus obtained is added
to a solution of compounds 1–3 (1.35 mmol) in anhydrous methanol (5 mL). Reaction mixture is
stirred for 72 h at room temperature. The solvent is evaporated and the crude product purified by silica
gel column chromatography (CH2Cl2/CH3OH = 2/1).
6-Imino-1-(2-morpholin-4-yl-ethyl)-6,7-dihydro-1H,5H-1,3,5,7-tetraaza-azulene-4,8-dione (9): (55 mg;
1
13.99%; m.p. > 300 °C); MS m/z: 292.1 (M+1). H-NMR (DMSO-d6): δ 2.38 (4H, m, CH2-4'), 2.58
(2H, m, CH2-2'), 3.51 (4H, m, CH2-5'), 4.48 (2H, m, CH2-1'), 6.64 (1H, b, NH),
7.37 (1H, b, NH), 8.04 (1H, s, CH-5), 10.66 ppm (1H, b, NH). 13C-NMR (DMSO-d6): δ 43.18 (C1'),
53.19 (C4'), 58.27 (C2'), 66.15 (C5'), 129.7 (C2), 135.2 (C3), 143.41 (C5), 149.99 (C8), 160.27 and
161.63 ppm (C6 and C10).
6-Imino-1-(2-piperidin-1-yl-ethyl)-6,7-dihydro-1H,5H-1,3,5,7-tetraaza-azulene-4,8-dione (10): (22 mg;
1
2.23%; m.p. > 300 °C); MS m/z: 290.2 (M+1). H-NMR (DMSO-d6): δ 1.36 (2H, m, CH2-6'), 1.43
(4H, m, CH2-5'), 2.35 (4H, m, CH2-4'), 2.55 (2H, t, J = 6.2, CH2-2'), 4.46 (2H, t, J = 6.2, CH2-1'), 6.48
13
(1H, b, NH), 7.55 (1H, b, NH), 8.02 (1H, s, CH-5), 10.60 ppm (1H, b, NH). C-NMR (DMSO-d6): δ
23.86 (C6'), 25.51 (C5'), 43.62 (C1'), 53.98 (C4'), 58.50 (C2'), 130.8 (C2), 133.8 (C3), 143.35 (C5),
149.70 (C8), 161.92 and 162.16 ppm (C6 and C10).
6-Imino-1-(2-pyrrolidin-1-yl-ethyl)-6,7-dihydro-1H,5H-1,3,5,7-tetraaza-azulene-4,8-dione (11): (55 mg;
1
13.99%; m.p. > 300 °C); MS m/z: 292.1 (M+1). H-NMR (DMSO-d6): δ 1.90 (4H, m, CH2-5'), 3.24
(4H, m, CH2-4'), 3.54 (2H, m, CH2-2'), 4.68 (2H, t, J = 6.1, CH2-1'), 6.57 (1H, b, NH), 7.68 (1H, b, NH),
8.19 (1H, s, CH-5), 10.74 ppm (1H, b, NH). 13C-NMR (DMSO-d6): δ 22.65 (C5'), 43.06 (C1'), 53.79 (C4'),
54.14 (C2'), 130.7 (C2), 134.4 (C3), 143.5 (C5), 150.12 (C8), 159.15 and 161.97 ppm (C6 and C10).

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3.2.4. General Procedure for Synthesis of 12–13
To a solution of guanidine hydrochloride (514 mg; 5.38 mmol) in anhydrous methanol (8 mL)
cooled to 0 °C is added a solution of sodium methoxide (25 wt%; 0.75 mL; 13.12 mmol). The reaction
mixture is stirred for 30 min at 0 °C. The sodium salt formed is removed by filtration, and the filtrate
thus obtained is added to a solution of compound 1 or 2 (400 mg; 1.35 mmol) in anhydrous methanol
(5 mL). Reaction mixture is stirred for 8 h at room temperature, solvent evaporated and in reaction
mixture in situ dissolved in DMF (3 mL) is added 2-fluorophenyl isocyanate (0,02 mL; 0,18 mmol).
Reaction mixture is stirred for 24 h at room temperature. The solvent is evaporated and the crude
product purified by silica gel column chromatography (CH2Cl2/CH3OH = 60/1) to give:
5-({[3-(2-Fluoro-phenyl)-ureido]-imino-methyl}-carbamoyl)-1-(2-morpholin-4-yl-ethyl)-1H-imidazole-
4-carboxylic acid methyl ester (12) as a white crystals (6.5 mg; 1.63%; m.p. > 164–165 °C); MS m/z
1
462.1915 (M+1). H-NMR (DMSO-d6): δ 2.48 (4H, t, J = 4.7, CH2-4'), 2.73 (2H, t, J = 6.0, CH2-2'),
3.66 (4H, t, J = 4.7, CH2-5'), 4.10 (3H, s, COOCH3-3), 4.55 (2H, t, J = 6.0, CH2-1'), 6.97 (1H, m, F-
C6H4), 7.06 (1H, m, F-C6H4), 7.11 (1H, m, F-C6H4), 7.37 (1H, b, NH), 7.71 (1H, s, CH-5), 8.29 (1H,
m, F-C6H4), 8.83 (1H, b, NH), 9.57 (1H, b, NH), 12.48 ppm (1H, b, NH). 13C-NMR (DMSO-d6): δ
24.11 (C6'), 25.71 (C5'), 43.49 (C1'), 54.29 (C4'), 59.05 (C2'), 115.10 (F-C6H4, d, JCF = 18.0), 116.5
(F-C6H4), 121.43 (C2), 124.80 (F-C6H4, d, JCF = 3.2), 136.3 (F-C6H4), 139.97 (C5), 145.53 (C3),
150.95 (F-C6H4, d, JCF = 236.4), 158.20, 161.73 and 163.60 (2×C=O and C=NH), 167.55 (COOCH3).
19F NMR (DMSO-d6): δ −125.27 (F-C6H4, b).
5-({[3-(2-Fluoro-phenyl)-ureido]-imino-methyl}-carbamoyl)-3-(2-piperidin-1-yl-ethyl)-3H-imidazole-
1
4-carboxylic acid methyl ester (13) as a yellow oil (7.2 mg; 0.72%); MS m/z 460.2 (M+1). H-NMR
(CDCl3): δ 1.34 (2H, m, CH2-6'), 1.42 (4H, m, CH2-5'), 2.30 (4H, m, CH2-4'), 2.5 (2H, m, CH2-2'),
3.73 (3H, s, COOCH3-2), 4.17 (2H, t, J = 6.2, CH2-1'), 6.5–7.0 (4 × 1H, m, F-C6H4), 7.69 (1H, s,
CH-5), 7.7–8.6, 12.3 ppm (4 × 1H, b, NH). 13C-NMR (CDCl3): δ 46.15 (C1'), 53.79 (C4'), 53.86
(COOCH3), 58.60 (C2'), 66.90 (C5'), 114.70 (F-C6H4, d, JCF = 19.2), 120.47 (F-C6H4), 122.66 (F-C6H4,
d, JCF = 7.1), 124.36 (F-C6H4, d, JCF = 3.3), 127.69 (F-C6H4, d, JCF = 16.5), 127.71 (C2), 134.19 (C3),
142.33 (C5), 152.25 (F-C6H4, d, JCF = 242.8), 158.04, 160.43 and 162.98 (2×C=O and C=NH), 165.70
(COOCH3). 19F NMR (CDCl3): δ −124.37 (F-C6H4, b).
3.3. Biological Methods
3.3.1. Cell Culturing
The cell lines HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic), MCF-7
(breast epithelial adenocarcinoma, metastatic), HepG2 (hepatocellular carcinoma) and BJ (normal
diploid human fibroblasts), were cultured as monolayers and maintained in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL
penicillin and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C.

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3.3.2. Proliferation Assays
The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at
3,000 to 5,000 cells per well according to the doubling times of specific cell line. Test agents were then
added in 10-fold dilutions (0,01 to 100 µM) and incubated for further 72 h. Working dilutions were
freshly prepared on the day of testing in the growth medium. The solvent (DMSO) was also tested
for eventual inhibitory activity by adjusting its concentration to be the same as in the working
concentrations (DMSO concentration never exceeded 0.1%). After 72 h of incubation, the cell growth
rate was evaluated by performing the MTT assay: experimentally determined absorbance values were
transformed into a cell percentage growth (PG) using the formulas proposed by NIH and described
previously [10]. This method directly relies on control of untreated cells at the day of substances
addition because it compares the growth of treated cells with the growth of untreated cells in control
wells on the same plate—the results are therefore a percentile difference from the calculated expected
value. The IC50 values for each compound were calculated from dose-response curves using linear
regression analysis by fitting the mean test concentrations that give PG values above and below the
reference value. If, however, all of the tested concentrations produce PGs exceeding the respective
reference level of effect (e.g., PG value of 50) for a given cell line, the highest tested concentration is
assigned as the default value (in the screening data report that default value is preceded by a “>” sign).
Each test point was performed in quadruplicate in three individual experiments. The results were
statistically analyzed (ANOVA, Tukey post-hoc test at p < 0.05). Finally, the effects of the tested
substances were evaluated by plotting the mean percentage growth for each cell type in comparison to
control on dose response graphs.
3.3.3. Antiviral Activity Assays
The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced
cytopathicity in HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and
vesicular stomatitis virus], Vero (parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro
virus), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus) or MDCK
[influenza A (H1N1; H3N2) and influenza B] cell cultures. Confluent cell cultures (or nearly confluent
for MDCK cells) in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50
being the virus dose to infect 50% of the cell cultures). The cell cultures were incubated at the time of
infection in the presence of varying concentrations (200, 40, 8, … µM) of the test compounds.
Viral cytopathicity was recorded microscopically as soon as it reached completion in the control
virus-infected cell cultures that were not treated with the test compounds. The methodology of the
anti-HIV assays was as follows: human CEM cells (~3 × 105 cells/mL) were infected with 100 CCID50
of HIV(IIIB) or HIV-2(ROD)/mL and seeded in 200-µL wells of a microtiter plate containing
appropriate dilutions of the test compounds. After 4 days of incubation at 37 °C, HIV-induced giant
cell formation was examined microscopically.

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4. Conclusions
The main objective of this study was synthesis of a molecule that would be closely related to
RSV604. None of the compounds showed pronounced antiviral activity at subtoxic concentrations. No
cytotoxicity for all evaluated compounds on HEL, Vero, HeLa and MDCK cell cultures was observed.
From the structure-activity point of view, we believe that the main reason for lack of antiviral
activity is probably the absence of either sugar moiety at N-1 of imidazole or a closer resemblance to
the active compound RSV604, that was chemically caused and constrained by the formation of the
inactive imino form of the cyclized product instead of the targeted amino form. The 1H-imidazole-4,5-
dicarboxylic acid dimethyl ester derivative having an ethyl morpholino ligand (compound 1) and the
1H-imidazole-4,5-diamide derivative with morpholino moiety bound to an imidazole ring (compound 5)
have also exerted selective but modest cytostatic effect towards human cervix carcinoma (HeLa) cells
(IC50 = 24 and 32 µM), while moderate non-selective antiproliferative activity was observed for
compound 10. The imidazole[4,5-e][1,3]diazepine-4,8-dione derivative linked with a pyrrolidine
ligand (11) showed only a modest cytostatic effect on colon cancer cells (SW620) (IC50 = 20 µM).
These compounds showed no cytotoxic effects on human normal fibroblasts.
Synthetic chemistry is often full of surprises and presented compounds containing a carbamoyl
imino-ureido moiety are structurally very interesting, e.g., compounds 12 and 13. Moreover,
1H-imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer to N-1 of the
imidazole ring (compound 3) exerted a more pronounced selective cytostatic effect towards human
cervix cancer (HeLa) cells (IC50 = 9.5 µM) in comparison with other tested compounds with no
apparent cytotoxicity on normal human skin fibroblasts (BJ) as well. This compound might therefore
be suited for further exploration as a potential lead compound and chemical modifications.
Acknowledgments
Support for this study was provided by the Ministry of Science of the Republic of Croatia
(Projects #125-098-2464-2922, #335-0982464-2393, #335-0000000-3532). The research was
supported by the KU Leuven (GOA no. 10/014).
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–11 are available from the authors.
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distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).