Design, synthesis and evaluation of 2,4-diaminoquinazolines as inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper describes the design, synthesis and evaluation of a series of 2,4-diaminoquinazolines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were designed by a generating virtual library of compounds and docking them into the enzyme active site. Following their synthesis, they were found to be potent and selective inhibitors of leishmanial dihydrofolate reductase. The compounds were also found to have potent activity against Trypanosoma brucei rhodesiense, a causative organism of African trypanosomiasis and also against Trypanosoma cruzi, the causative organism of Chagas disease. There was significantly lower activity against Leishmania donovani, one of the causative organisms of leishmaniasis.     

2,4-Diaminopyrimidines as inhibitors of Leishmanial and Trypanosomal dihydrofolate reductase

This paper describes the synthesis of 4'-substituted and 3',4'-disubstituted 5-benzyl-2,4-diaminopyrimidines as selective inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were then assayed against the recombinant parasite and human enzymes. Some of the compounds showed good activity. They were also tested against the intact parasites using in vitro assays. Good activity was found against Trypanosoma cruzi, moderate activity against Trypanosoma brucei and Leishmania donovani. Molecular modeling was undertaken to explain the results. The leishmanial enzyme was found to have a more extensive lipophilic binding region in the active site than the human enzyme. Compounds which bound within the pocket showed the highest selectivity.     

Novel non-classical C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines as potential inhibitors of dihydrofolate reductase and as anti-opportunistic agents

Six novel C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines 18-23 were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and as anti-opportunistic agents. These compounds represent the only examples of 9-methyl substitution in the carbon-carbon bridge of 2,4-diaminopyrrolo[2,3-d]pyrimidines. The analogs 18-23 were synthesized in a concise eight-step procedure starting from the appropriate commercially available aromatic methyl ketones. The key step involved a Michael addition reaction of 2,4,6-triaminopyrimidine to the appropriate 1-nitroalkene, followed by ring closure of the nitro adducts via a Nef reaction. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma) and rat liver (rl). The biological result indicated that some of these analogs are potent inhibitors of DHFR and some have selectivity for pathogen DHFR. Compound 23 was a two digit nanomolar inhibitor of tgDHFR with 9.6-fold selectivity for tgDHFR.     

Synthesis and biological evaluation of biguanide and dihydrotriazine derivatives as potential inhibitors of dihydrofolate reductase of opportunistic microorganisms

Twenty-one biguanide and dihydrotriazine derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms: Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma), and rat liver (rl). The most potent compound in the series was B2-07 with 12?nM activity against tgDHFR. The most striking observation was that B2-07 showed similar potency to trimetrexate, ～233-fold improved potency over trimethoprim and ～7-fold increased selectivity as compared to trimetrexate against tgDHFR. Molecular docking studies in the developed homology model of tgDHFR rationalized the observed potency of B2-07. This molecule can act as a good lead for further design of molecules with better selectivity and improved potency.     

Synthesis of 2,4-diamino-6-(thioarylmethyl)pyrido[2,3-d]pyrimidines as dihydrofolate reductase inhibitors

Six 2,4-diaminopyrido[2,3-d]pyrimidines with a 6-methylthio bridge to an aryl group were synthesized and biologically evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). The syntheses of analogues 3-8 were achieved by nucleophilic displacement of 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine 14 with various arylthiols. The alpha-naphthyl analogue 4 showed the highest selectivity ratios of 3.6 and 8.7 against pcDHFR and tgDHFR, respectively, versus rat liver (rl) DHFR. The beta-naphthyl analogue 5 exhibited the highest potency within the series with an IC(50) value against pcDHFR and tgDHFR of 0.17 and 0.09 microM, respectively. Analogue 4 was evaluated for in vitro antimycobacterium activity and was shown to inhibit the growth of Mycobacterium tuberculosis H(37)Rv cells by 58% at a concentration of 6.25 microg/mL.     

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6–16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6–16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6–16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.     

In vitro aldose reductase inhibitory activity of 5-benzyl-2,4-thiazolidinediones

Several 5-benzyl-2,4-thiazolidinediones (5-7) were synthesised and tested as in vitro aldose reductase (ALR2) inhibitors. Most of them, particularly N-unsubstituted 5-benzyl-2,4-thiazolidinediones 5 and (5-benzyl-2,4-dioxothiazolidin-3-yl)acetic acids 7, displayed moderate to high inhibitory activity levels. In detail, the insertion of an acetic chain on N-3 significantly enhanced ALR2 inhibitory potency, leading to acids 7 which proved to be the most effective among the tested compounds. In addition, in N-unsubstituted derivatives 5 the presence of an additional aromatic ring on the 5-benzyl moiety was generally beneficial. In fact, the ALR2 inhibition results of compounds 5-7, compared to those of the previously assayed corresponding 5-arylidene-2,4-thiazolidinediones, indicated that N-unsubstituted derivatives 5b, c and d, which bore an additional aromatic group in the para position of the 5-benzyl residue, were significantly more effective than their 5-arylidene counterparts; in all other cases, the saturation of the exocyclic double bond CC in 5 brought about a moderate decrease in activity.     

Synthesis and Testing of 5-Benzyl-2,4-diaminopyrimidines as Potential Inhibitors of Leishmanial and Trypanosomal Dihydrofolate Reductase

Dihydrofolate reductase is a drug target that has not been thoroughly investigated in leishmania and trypanosomes. Work has previously shown that 5-benzyl-2,4-diaminopyrimidines are selective inhibitors of the leishmanial and trypanosome enzymes. Modelling predicted that alkyl/aryl substitution on the 6-position of the pyrimidine ring should increase enzyme activity of 5-benzyl-2,4-diaminopyrimidines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Various compounds were prepared and evaluated against both the recombinant enzymes and the intact organisms. The presence of a substituent had a small or negative effect on activity against the enzyme or intact parasites compared to unsubstituted compounds.     

Synthesis and antiplasmodial activity of novel 2,4-diaminopyrimidines

Two sets of diaminopyrimidines, totalling 45 compounds, were synthesized and assayed against Plasmodium falciparum. The SAR was relatively shallow, with only the presence of a 2-(pyrrolidin-1-yl)ethyl group at R² significantly affecting activity. A subsequent series addressed high Log D values by introducing more polar side groups, with the most active compounds possessing diazepine and N-benzyl-4-aminopiperidyl groups at R¹/R². A final series attempted to address high in vitro microsomal clearance by replacing the C6-Me group with CF₃, however antiplasmodial activity decreased without any improvement in clearance. The C6-CF₃ group decreased hERG inhibition, probably as a result of decreased amine basicity at C2/C4.     

Design and synthesis of guanylthiourea derivatives as potential inhibitors of Plasmodium falciparum dihydrofolate reductase enzyme

A new class of compounds based on S-benzylated guanylthiourea has been designed as potential PfDHFR inhibitors using computer aided methods (molecular electrostatic potential, molecular docking). Several compounds in this class have been synthesized starting from guanylthiourea and alkyl bromides. In vitro studies showed that two compounds from this class are active with the IC₅₀ value of 100 μM and 400 nM.     

Selective inhibitors of dihydrofolate reductase

The Nobel Foundation 1989.     

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas’ disease. We have undertaken a detailed structure–activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme–ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60–70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.     

Quantitative structure-activity analysis of 5-arylidene-2,4-thiazolidinediones as aldose reductase inhibitors

Design of aldose reductase (ALR2) inhibitors has received considerable attention. Aldose reductase inhibitors, when administered from the onset of hyperglycemia, prevent the progression of polyol accumulation-linked complications. The feasibility that inhibition of aldose reductase provides a pharmacologically direct treatment for diabetic complications that is independent of the control of blood sugar levels has spurred the development of structurally diverse aldose reductase inhibitors. In this work, we report quantitative structure-activity relationship (QSAR) analysis performed by 3D-QSAR analysis, Hansch analysis, and Fujita-Ban analysis on a series of 5-arylidene-2,4-thiazolidinediones as aldose reductase inhibitors. The 2D & 3D-QSAR models were generated using 18 compounds and Fujita-Ban analysis models were obtained using 23 compounds. The predictive ability of the resulting 2D and 3D models was evaluated against a test set of 5 compounds. Analyses of results from the present QSAR study inferred that 3rd position of the phenyl ring and acetic acid substitution at N-position of thiazolidinediones play a key role in the aldose reductase inhibitory activity.     

Synthesis and evaluation of curcumin analogues as potential thioredoxin reductase inhibitors

Series of curcumin derivatives were synthesized; the inhibitory activities on thioredoxin reductase (TrxR) of all analogues were evaluated by DTNB assay in vitro. It is found that most of the analogues can inhibit TrxR in the low micromolar range; Structure-activity relationship analysis reveals that analogues with furan moiety have excellent inhibitory effect on TrxR in an irreversible manner, indicating that the furan moiety may serve as a possible pharmacophore during the interaction of curcumin analogues with TrxR. The effect of selected curcuminoids on growth of different TrxR overexpressed cancer cell lines was also investigated and discussed.     

Novel inhibitors of Leishmanial dihydrofolate reductase

The program DOCK3.5 was used to search the Cambridge Structural Database for novel inhibitors of Leishmanial dihydrofolate reductase. A number of compounds were obtained and screened against the enzyme and against the intact parasite Leishmania donovani and the related organisms Trypanosoma brucei and Trypanosoma cruzi. The compounds screened showed weak activity in both the enzyme assays and the in vitro assays.     

Novel 5-substituted, 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors with antiangiogenic and antitumor activity

Recent evidence suggests that combination therapy of cancer with receptor tyrosine kinase (RTK) inhibitors, which are usually cytostatic, with conventional chemotherapeutic agents, which are usually cytotoxic, provide an improved treatment option. We have designed, synthesized, and evaluated a series of novel 2,4-diamino-5-substituted furo[2,3-d]pyrimidines with RTK and dihydrofolate reductase (DHFR) inhibitory activity in single molecules, as potential cytostatic and cytotoxic agents with antitumor activity. These compounds were synthesized from 2,4-diamino-5-chloromethyl furo[2,3-d]pyrimidine and aryl methyl ketones using the Wittig reaction to afford the C-8-C-9 unsaturated analogs followed by catalytic reduction to the corresponding saturated compounds. The saturated and unsaturated C-8-C-9 bridged compounds were evaluated as inhibitors of vascular endothelial growth factor receptor (VEGFR-2, Flk, KDR), epidermal growth factor receptor, and platelet-derived growth factor receptor-beta (PDGFR-beta). Selected analogs were also evaluated as antiangiogenic agents in the chicken embryo chorioallantoic membrane (CAM) assay. The compounds were also evaluated as inhibitors of human (h) DHFR and Toxoplasma gondii (tg) DHFR. In each evaluation, a known standard compound was used as a comparison. Of the compounds evaluated, compound 32 was as potent as the standard compounds against VEGFR-2 and PDGFR-beta, showing dual inhibitory activity against RTK. This analog was also highly effective in the CAM assay. A second analog 18 also demonstrated dual VEGFR-2 and PDGFR-beta inhibitory activity as well as potent antiangiogenic activity in the CAM assay. Four additional analogs were also effective against PDGFR-beta and in the CAM assay. An unsaturated C-8-C-9 moiety was necessary for RTK inhibitory activity. Compound 32 also showed inhibitory activity against hDHFR and tgDHFR, illustrating the multitarget inhibitory potential of these analogs. The biological activity of these analogs also suggests the necessity of an unsaturated C-8-C-9 bridge for dual RTK and DHFR inhibitory activity. Compounds 18 and 32 were also evaluated in a B16 melanoma mouse model and were found to be more active as antitumor agents than methotrexate. In addition, both 18 and 32 were also active in decreasing lung metastases in a mouse model of B16 melanomas.     

Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.     

Structure-activity relationships and molecular modelling of 5-arylidene-2,4-thiazolidinediones active as aldose reductase inhibitors

The structure-activity relationships (SARs) of 5-arylidene-2,4-thiazolidinediones active as aldose reductase inhibitors (ARIs) were extended by varying the substitution pattern on the 5-arylidene moiety and on N-3. In particular, the introduction of an additional aromatic ring or an H-bond donor group on the 5-benzylidene ring enhanced ALR2 inhibitory potency. Moreover, the presence of a carboxylic anionic chain on N-3 was shown to be an important, although not essential, structural requisite to produce high levels of ALR2 inhibition. The length of this carboxylic chain was critical and acetic acids 4 were the most effective inhibitors among the tested derivatives. Molecular docking simulations into the ALR2 active site accorded with the in vitro inhibition data. They allowed the rationalization of the observed SARs and provided a pharmacophoric model for this class of ARIs.     

Interactions between inhibitors of dihydrofolate reductase.

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.