Synthesis and the Biological Activity of Phosphonylated 1,2,3-Triazolenaphthalimide Conjugates

A novel series of diethyl {4-[(5-substituted-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates designed as analogues of amonafide was synthesized. All phosphonates were assessed for antiviral activity against a broad range of DNA and RNA viruses and several of them showed potency against varicella-zoster virus (VZV) [EC50 (50% effective concentration) = 27.6–91.5 μM]. Compound 16b exhibited the highest activity against a thymidine kinase-deficient (TK−) VZV strain (EC50 = 27.59 μM), while 16d was the most potent towards TK+ VZV (EC50 = 29.91 μM). Cytostatic properties of the compounds 14a–i–17a–i were studied on L1210, CEM, HeLa and HMEC-1 cell lines and most of them were slightly cytostatic for HeLa [IC50 (50% inhibitory concentration) = 29–130 µM] and L1210 cells [IC50 (50% inhibitory concentration) = 14–142 µM].

The concept of combining two pharmacophoric fragments of biologically active compounds into a single molecule is commonly applied aiming at improvement of activities and eventually to avoid serious side effects of the known candidates. Having this idea in mind several structural analogues of amonafide have been synthesized over the years (Figure 1).    Qian and Li reported the synthesis of 6-(1,2,3-triazole)-1,8-naphthalimides 1 and proved their cytotoxic activity [33]. Furthermore, 5-substituted analogues 2 were also obtained and their cytotoxicity against MCF-7, HeLa and 7721 cells was evaluated [34]. Among all tested compounds 2, derivatives having a 2-(N,N-dimethylamino)ethyl group at the imide nitrogen and phenyl at C4 in the 1,2,3-triazole ring or alternatively lacking this substituent were found the most active (IC50 in the 0.258-0.725 μM range) with inhibition abilities higher than that of amonafide, used as a control. On the other hand, antifungal and antimicrobial properties of 1,2,4-triazole derivatives 3 were examined and several compounds exhibited even better activity against some tested strains than orbifloxacin, chloromycin and fluconazole used as reference drugs [35]. Among bis(1,2,3-triazole)-conjugates of naphthalimides 4, a derivative substituted with 3,4-dichlorophenyl groups exhibited better inhibitory activity toward Escherichia coli than norfloxacin and chloromycin with a minimum inhibitory concentration (MIC) value of 1 μg/mL [36].
These achievements prompted us to propose a new modification at the imide nitrogen of amonafide 14-17 by installation of N1-substituted 1,2,3-triazoles decorated at the end of the alkyl chain with phosphonoalkyl groups. We aimed to understand the influence of the phosphonate group on the biological activity of the designed amonafide analogues. On the other hand, compounds 14-17 resemble analogues of acyclic nucleotides in which the phosphate group is replaced with a phosphonate moiety and a naphthalimide fragment serves as a modified nucleobase. Thus, in principle compounds 14-17 may primarily act as intercalators (through a naphthalimide ring) but also by a phosphonate activation and termination of DNA/RNA synthesis. Qian and Li reported the synthesis of 6-(1,2,3-triazole)-1,8-naphthalimides 1 and proved their cytotoxic activity [33]. Furthermore, 5-substituted analogues 2 were also obtained and their cytotoxicity against MCF-7, HeLa and 7721 cells was evaluated [34]. Among all tested compounds 2, derivatives having a 2-(N,N-dimethylamino)ethyl group at the imide nitrogen and phenyl at C4 in the 1,2,3-triazole ring or alternatively lacking this substituent were found the most active (IC 50 in the 0.258-0.725 µM range) with inhibition abilities higher than that of amonafide, used as a control. On the other hand, antifungal and antimicrobial properties of 1,2,4-triazole derivatives 3 were examined and several compounds exhibited even better activity against some tested strains than orbifloxacin, chloromycin and fluconazole used as reference drugs [35]. Among bis(1,2,3-triazole)-conjugates of naphthalimides 4, a derivative substituted with 3,4-dichlorophenyl groups exhibited better inhibitory activity toward Escherichia coli than norfloxacin and chloromycin with a minimum inhibitory concentration (MIC) value of 1 µg/mL [36].
These achievements prompted us to propose a new modification at the imide nitrogen of amonafide 14-17 by installation of N1-substituted 1,2,3-triazoles decorated at the end of the alkyl chain with phosphonoalkyl groups. We aimed to understand the influence of the phosphonate group on the biological activity of the designed amonafide analogues. On the other hand, compounds 14-17 resemble analogues of acyclic nucleotides in which the phosphate group is replaced with a phosphonate moiety and a naphthalimide fragment serves as a modified nucleobase. Thus, in principle compounds 14-17 may primarily act as intercalators (through a naphthalimide ring) but also by a phosphonate activation and termination of DNA/RNA synthesis.

Antiviral Activity and Cytostatic/Cytotoxic Evaluation
Phosphonates 14a-i-17a-i were evaluated for their antiviral activities against a wide variety of DNA and RNA viruses using the following cell-based assays: (a) human embryonic lung (HEL) cell cultures: herpes simplex virus-1 (KOS strain), herpes simplex virus-2 (G strain), vaccinia virus, vesicular stomatitis virus, thymidine kinase-deficient herpes simplex virus-1 (TK − KOS ACV r ) and adenovirus Hippeastrum hybrid agglutinin (HHA), Urticadioica agglutinin (UDA), dextran sulfate (molecular weight 5000, DS-5000), ribavirin, oseltamivir carboxylate, amantadine and rimantadine were used as the reference compounds. The antiviral activity was expressed as the EC 50 : the compound concentration required to reduce virus-induced cytopathogenicity by 50% (other viruses).
Among the synthesized compounds, several phosphonates 14, 15 and 16 slightly inhibited the replication of both TK + and TK − VZV strains with EC 50 in the 27.6-91.5 µM range, however with lower potency than that of acyclovir and brivudine, used as reference drugs (Table 1). The cytotoxicity of the tested compounds toward the uninfected host cells was defined as the minimum cytotoxic concentration (MCC) that causes a microscopically detectable alteration of normal cell morphology. The 50% cytotoxic concentration (CC 50 ), causing a 50% decrease in cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay system. None of the tested compounds affected cell morphology of HEL, HeLa, Vero, MDCK and CRFK cells at concentrations up to 100 µM.
The cytostatic activity of the tested compounds was defined as the 50% cytostatic inhibitory concentration (IC 50 ) causing a 50% decrease in cell proliferation and was determined against murine leukaemia L1210, human lymphocyte CEM, human cervix carcinoma HeLa and human dermal microvascular endothelial HMEC-1 cells (Table 2). Table 2. The inhibitory effect of the tested compounds against the proliferation of murine leukemia (L1210), human T-lymphocyte (CEM) and human cervix carcinoma cells (HeLa).

Compound
IC 50 a (µM) Among all tested compounds 1,2,3-triazole-amonafide conjugates 15a-i having a bromine atom at C6 of the naphthalimide unit were the most cystostatic toward the tested tumor cell lines at concentrations as low as 14 µM being especially effective for L1210 (IC 50 = 14-42 µM). Conjugates 16a-i containing the nitro group at C5 were slightly less active and showed moderate cytostatic effects toward HeLa cells (IC 50 = 29-132 µM). The replacement of the nitro by an amino group at C5 of the naphthalimide skeleton resulted in the decrease or even loss of the inhibitory capacity of the respective analogues (15a-i vs. 17a-i). Similarly, negligible inhibitory properties against the proliferation of the tested cell lines were noticed for the series of naphthalimide phosphonates 14a-i (R = H).

General Information
All microwave irradiation experiments were carried out in a RM 800 microwave reactor (Plazmatronika, Wrocław, Poland). (7) A suspension of a compound 5 (1.00 mmol), propargyl amine (1.05 mmol) in ethanol (15 mL) was stirred at 78 • C for 3 h. The reaction mixture was cooled to room temperature and filtered to give pure

Antiviral Activity Assays
The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK − ) HSV-1 KOS strain resistant to ACV (ACV r ), herpes simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK − VZV strain 07−1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, Parainfluenza 3, Influenza virus A (subtypes H1N1, H3N2), influenza virus B, Reovirus-1, Sindbis, Reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain III B and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID 50 of virus (1 CCID 50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (VZV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC 50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%.

Cytostatic Activity Assays
All assays were performed in 96-well microtiter plates. To each well were added (5-7.5) × 10 4 tumor cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37 • C in a humidified CO 2 -controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC 50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.
The synthesized phosphonates 14a-i-17a-i were evaluated against a variety of DNA and RNA viruses and several of them appeared slightly active against VZV (EC 50 = 27.6-91.5 µM). Among them, the compound 16b, which showed no potency toward the TK + VZV strain, was found the most active against the TK − VZV strain (EC 50 = 27.59 µM), with EC 50 values comparable to reference drugs. On the other hand, compound 16d exhibited the highest activity against TK + VZV (EC 50 = 29.91 µM), athough lower than that of reference compounds.
Cytostatic properties of compounds 14a-i-17a-i were studied on L1210, CEM, HeLa and HMEC-1 cell lines and most of them were only slightly cytostatic for HeLa (IC 50 = 29-130 µM) and L1210 cells (IC 50 = 14-142 µM). Among all tested compounds 14a-i-17a-i derivatives substituted with a bromine atom at C6 (15b and 15d) were the most active. Based on a preliminary SAR analysis it was established that the presence of the 1,2,3-triazole unit is essential for the cytostatic activity. Furthermore, compounds with longer linkers [(CH 2 ) 3 , (CH 2 ) 4 and CH 2 CH 2 OCH 2 CH 2 )] showed the higher cytostatic potency than those having shorter fragments [CH(OH)CH 2 and CH 2 NHC(O)CH 2 ].