Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy

A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79/V79-NTR(puro)) and murine (EMT6/EMT6-NTR(puro)) cell line pairs, and were compared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN 29427. The low solubility of the prodrugs (from 3 to 39 microM) precluded the determination of IC(50) values against the parent cell lines in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24h period, although release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold). Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel (1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold) but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs.     

Nitrobenzyl carbamate prodrugs of enediynes for nitroreductase gene-directed enzyme prodrug therapy (GDEPT)

The synthesis and evaluation of the 4-nitrobenzylcarbamate enediyne 6 and related compounds as prodrugs activated by a nitroreductase enzyme (NTR) from E. coli B is described. Expression of NTR in three different cell lines gives increases in cytotoxicity of 21- to 135-fold for 6 (IC₅₀ values 13–24 nM in the NTR-expressing lines), indicating its potential as a prodrug for NTR-mediated Gene-Directed Enzyme Prodrug Therapy. The cytotoxicity of 6 and related enediynes is shown to be oxygen-dependent, especially in nucleotide excision repair-proficient cells, which might limit activity in hypoxic regions of tumours.     

Nitrobenzylcarbamate prodrugs of cytotoxic acridines for potential use with nitroreductase gene-directed enzyme prodrug therapy

The synthesis, solvolytic behaviour and cytotoxicity of novel 4-nitrobenzyl carbamates and carbonates derived from 3-amino-4-hydroxymethylacridine 1 are described. Compounds 2 and 6 are both substrates for Escherichia coli nitroreductase and the highly active lead structure 1 is liberated upon incubation of the two compounds in the presence of NTR and its cofactor NADH. Additionally, the cytostatic activity of 2 and 6 against human HT29 colon carcinoma cell lines is decreased 80-fold and 360-fold, respectively, indicating their suitability and potency as prodrugs for either gene-directed enzyme prodrug therapy or antibody-directed enzyme prodrug therapy.     

Synthesis of L-(+)-3-(3-hydroxy-4-pivaloyloxybenzyl)-2,5-diketomorpholine as potential prodrug of L-dopa

The synthesis and in vitro chemical and enzymatic stability of L-(+)-3-(3-hydroxy-4-pivaloyloxybenzyl)-2,5-diketomorpholine (9) as L-Dopa prodrug are described. Prodrug 9 possesses a good lipophilicity (log P = 2.153 +/- 0.017), is stable in aqueous buffer solutions (pH 1.3 and 7.4), and in 80% rat and human plasma it is turned into L-Dopa.     

2-(3-Amino-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide: a new tiazofurin analogue with potent antitumour activity

A new tiazofurin analogue, 2-(3-amino-3-deoxy-beta-d-xylofuranosyl)thiazole-4-carboxamide (3), was synthesized starting from d-glucose and evaluated for its in vitro antiproliferative activity against a panel of human tumour cell lines. Compound 3 exhibited the most powerful cytotoxicity against K562 cells, being approximately 100-fold more potent than tiazofurin. This analogue was also active against Jurkat, HT-29 and HeLa malignant cells, with respective IC(50) values being ca. 2-, 27- and 17-fold lower than those observed for tiazofurin. Remarkably, compound 3 did not exhibit any significant cytotoxicity towards normal foetal lung MRC-5 cell line.     

2-nitroimidazol-5-ylmethyl as a potential bioreductively activated prodrug system: reductively triggered release of the PARP inhibitor 5-bromoisoquinolinone.

5-Chloromethyl-1-methyl-2-nitroimidazole reacted efficiently with the anion derived from 5-bromoisoquinolin-1-one to give 5-bromo-2-((1-methyl-2-nitroimidazol-5-yl)methyl)isoquinolin -1-one. Biomimetic reduction effected release of the 5-bromoisoquinolin-1-one. The 2-nitroimidazol-5-ylmethyl unit thus has potential for development as a general prodrug system for selective drug delivery to hypoxic tissues.     

9-[2-(R)-(Phosphonomethoxy)propyl]-2,6-diaminopurine (R)-PMPDAP and its prodrugs: Optimized preparation,including identification of by-products formed,and antiviral evaluation in vitro

New large-scale synthetic approach to antiretroviral agent 9-[2-(R)-(phosphonomethoxy)propyl]-2,6-diaminopurine, (R)-PMPDAP, was developed. Reaction of (R)-propanediol carbonate with 2,6-diaminopurine afforded exclusively (R)-9-(2-hydroxypropyl)-2,6-diaminopurine which was subsequently used for introduction of a phosphonomethyl residue using TsOCH₂P(O)(OiPr)₂ or BrCH₂P(O)(OiPr)₂ followed by deprotection of ester groups. All minor ingredients and by-products formed during the process were identified and further studied. The final product was obtained in high yield and its high enantiomeric purity (>99%) was confirmed by chiral capillary electrophoretic analysis using β-cyclodextrin as a chiral selector. Antiretroviral activity data of (R)-PMPDAP and its diverse prodrugs against HIV and FIV were investigated. Akin to (R)-PMPDAP, both prodrugs inhibit FIV replication in a selective manner. Compared to the parent molecule, the amidate prodrug was 10-fold less active against FIV in cell culture, whereas the alkoxyalkyl ester prodrug was 200-fold more potent in inhibiting FIV replication in vitro.     

In-vitro cytotoxicity and cell cycle analysis of two novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC(50) of 3-5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G(1) phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

Synthesis and evaluation of in vitro antiviral activity of 2-[3-(substituted phenyl)-4,5-dihydro-1H-5-pyrazolyl]benzofuran-3-yl chloride derivatives

3-Chlorobenzofuran-2-carbaldehyde was condensed with substituted acetophenone by using the Claisen-Schmidt condensation to obtain 3-(3-chlorobenzofuran-2-yl)-1-(substituted phenyl)-2-propen-1-one (2a-m) which upon further treatment with hydrazine hydrate gave 2-[3-(substituted phenyl)-4,5-dihydro-1H-5- pyrazolyl)benzofuran-3-yl chloride derivatives (3a-m). All the newly synthesized derivatives were evaluated in vitro for cytotoxicity and antiviral activity in Crandell-Rees Feline Kidney cell, human embryonic lung (HEL) cell, HeLa cell and Vero cell cultures against different viruses. Several compounds, i.e. 2f, 2g, 2i, 2m, 3b, 3d, 3g, 3h and 3m proved quite cytotoxic to the host cells (minimum cytotoxic concentration: 1-10 μg/mL). No specific antiviral activity [50% antivirally effective concentration (EC₅₀) ≥ 5-fold lower than the minimum cytototoxic concentration] was observed for any of the compounds.     

Cancer chemotherapy: a SN-38 (7-ethyl-10-hydroxycamptothecin) glucuronide prodrug for treatment by a PMT (Prodrug MonoTherapy) strategy

A glucuronide-based prodrug of SN-38 (7-ethyl-10-hydroxycamptothecin) has been synthesized for use in a Prodrug MonoTherapy Strategy (PMT). Since this prodrug is significantly less cytotoxic than SN-38 itself and efficiently releases the drug in vitro in the presence of beta-D-glucuronidase, it can be considered as an appropriate candidate for cancer treatment by a PMT strategy.     

Synthesis of 2-deoxy-2-(4-nitroimidazol-1-yl)-D-alditols

Small molecules possessing defined configuration at centres of chirality provide a valuable chiral pool. Among different strategies applied for modification of chiral compounds, the most common is to begin with a single stereoisomer and use a synthesis that does not affect the chiral centres. The ANRORC type reaction has been applied for conversion of unprotected 2-amino-2-deoxy-D-hexopyranoses into 2-deoxy-2-(4-nitroimidazol-1-yl)-D-hexopyranoses in a reaction of some 2-aminosugars with 1,4-dinitroimidazoles. The reaction occurs with retention of configuration at C-2 of sugar ring. The products of the reaction were obtained as anomeric mixtures and separated into anomers after acetylation followed by column chromatography. 2-Deoxy-2-(4-nitroimidazol-1-yl)-D-hexopyranoses treated with sodium borohydride in methanolic solution gave the corresponding 2-deoxy-2-(4-nitroimidazol-1-yl)-D-hexitols, characterised as per-O-acetylated derivatives.     

In-Vitro Cytotoxicity and Cell Cycle Analysis of Two Novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC₅₀ of 3–5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G₁ phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

N-alkylated cyclen cobalt(III) complexes of 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol DNA alkylating agent as hypoxia-activated prodrugs

A series of cobalt complexes of the potent DNA minor groove alkylator 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (seco-6-azaCBI-TMI) were prepared from a series of N-substituted cyclen ligands. The final N-substituted complexes carried formal overall charges ranging from +2 to -2 and showed limited improvements in solubility. They showed similar stabilities to that of the complex with the unsubstituted cyclen ligand, and large but variable attenuation of the cytotoxicity of the free alkylator (2-30-fold), compared to 150-fold for the unsubstituted ligand. However, they had oxic/hypoxic ratios (2-22-fold) comparable to that of the unsubstituted cyclen complex (5).     

Synthesis and antipicornavirus activity of (R)- and (S)-1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one

The new pyridyl imidazolidinone derivative, 1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one (+/-)-1a, was synthesized and found to have an excellent antiviral activity against EV71 (IC50 = 0.009 microM). Therefore, both the enantiomers, (S)-(+)-1a and (R)-(-)-1a, have been prepared starting from readily available monomethyl (R)-3-methylglutarate (7) as a useful chiral building block and their antiviral activity was evaluated in a plaque reduction assay. Interestingly, we observed that the enantiomer (S)-(+)-1a was 10-fold more active against enterovirus71 (EV71) (IC50 = 0.003 microM) than the corresponding enantiomer (R)-(-)-1a (IC50 = 0.033 microM). Similar results were found against all five strains (1743, 2086, 2231, 4643, and BrCr) of EV71 tested. This demonstrated that the absolute configuration of the chiral carbon atom at the 3-position of the alkyl linker considerably influenced the anti-EV71 activity of these pyridyl imidazolidinones.     

Discovery of novel antitubercular 1,5-dimethyl-2-phenyl-4-([5-(arylamino)-1,3,4-oxadiazol-2-yl]methylamino)-1,2-dihydro-3H-pyrazol-3-one analogues

In search of potential therapeutics for tuberculosis, we describe herewith the synthesis, characterization and antimycobacterial activity of 1,5-dimethyl-2-phenyl-4-([5-(arylamino)-1,3,4-oxadiazol-2-yl]methylamino)-1,2-dihydro-3H-pyrazol-3-one analogues. Among the synthesized compounds, 4-[(5-[(4-fluorophenylamino]-1,3,4-oxadiazol-2-yl)methylamino]-1,2-dihydro-1,5-dimethyl-2-phenylpyrazol-3-one (4a) was found to be the most promising compound active against Mycobacterium tuberculosis H(37)Rv and isoniazid resistant M. tuberculosis with minimum inhibitory concentrations, 0.78 and 3.12μg/mL, respectively, free from any cytotoxicity (>62.5μg/mL).     

Evaluation of 5-[1-(2-halo(or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridines as inhibitors of herpes simplex virus

A series of 5-[1-(2-haloethyl(or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridines (3-7) and related uracil analogs (9-10) were prepared using 5-vinyl-2-deoxyuridine (2) and 5-vinyl uracil (8) as starting materials. The regiospecific reaction of 2 and 8 with iodine monochloride and an alcohol provided the target compounds 3-10. These analogs were evaluated in vitro for inhibitory activity against thymidine-kinase (TK) positive and negative strains of herpes simplex virus type-1. The compounds 3-10 were either weak or non-inhibitory to HSV-1 replication. All compounds investigated exhibited low host cell cytotoxicity.     

Synthesis of [18F]-labeled EF3 [2-(2-nitroimidazol-1-yl)-N-(3,3,3-trifluoropropyl)-acetamide], a marker for PET detection of hypoxia

[18F]-2-(2-Nitroimidazol-1-yl)-N-(3,3,3-trifluoropropyl)-acetamide ([18F]-EF3) has been prepared, in 65% chemical yield and 5% radiochemical yield, by coupling 2,3,5,6-tetrafluorophenyl 2-(2-nitroimidazol-1-yl) acetate 1 with [18F]-3,3,3-trifluoropropylamine 7. This original radiolabelled key-synthon was obtained in 40% overall chemical yield by oxidative [18F]-fluorodesulfurization of ethyl N-phthalimido-3-aminopropane dithioate 4, followed by deprotection with hydrazine of the resulting [18F]-N-phthalimido-3,3,3-trifluoropropylamine 5. All the process was performed within 90 min, from the [18F]-HF production in the cyclotron to the purification of the final target.     

Muscarinic M(3) receptor antagonists with (2R)-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxyphenylacetamide Structures. Part 2

Optimization of the amine part of our original muscarinic M(3) receptor antagonist 1 was performed to identify M(3) receptor antagonists that are superior to 1. Compounds carrying a variety of diamine moieties without hydrophobic substituent on the nitrogen atom were screened against the binding affinity for the M(3) receptor and the selectivity for M(3) over the M(1) and M(2) receptors. This process led to a 4-aminopiperidinamide (2l) with a K(i) value of 5.1 nM and with a selectivity of the M(3) receptor that was 46-fold greater than that of the M(2) receptor. Further derivatization of 2l by inserting a spacer group or by incorporating alkyl group(s) into the amine part resulted in the identification of an 4-(aminoethyl)piperidinamide 2l-b with a K(i) value of 3.7 nM for the M(3) receptor and a selectivity for the M(3) receptor that was 170-fold greater than that of the M(2) receptor.     

Synthesis and in vitro antimycobacterial activity of N1-nicotinoyl-3-(4'-hydroxy-3'-methyl phenyl)-5-[(sub)phenyl]-2-pyrazolines

A series of N1-nicotinoyl-3- (4'-hydroxy-3'-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv (MTB) and INH-resistant M. tuberculosis (INHR-MTB) using the agar dilution method. Among the synthesized compounds, compound (i) N1-nicotinyl-3-(4'-hydroxy-3'-methyl phenyl)-5-(1'-chlorophenyl)-2-pyrazoline was found to be the most active agent against MTB and INHR-MTB, with minimum inhibitory concentration of 0.26 microm. When compared to INH-compound i was found to be 2.8- and 43.7-fold more active against MTB and INHR-MTB, respectively.     

Synthesis and antimicrobial activity of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a–g (7b–f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure–activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity.