New insights into DHFR interactions: analysis of Pneumocystis carinii and mouse DHFR complexes with NADPH and two highly potent 5-(omega-carboxy(alkyloxy) trimethoprim derivatives reveals conformational correlations with activity and novel parallel ring stacking interactions

Structural data are reported for two highly potent antifolates, 2,4-diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (PY1011), with 5000-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR), relative to rat liver DHFR, and 2,4-diamino-5-[2-methoxy-5-(4-carboxybutyloxy)benzyl]pyrimidine (PY957), that has 80-fold selectivity for pcDHFR. Crystal structures are reported for NADPH ternary complexes with PY957 and pcDHFR, refined to 2.2 A resolution; with PY1011 and pcDHFR, refined to 2.0 A resolution; and with PY1011 and mouse DHFR (mDHFR), refined to 2.2 A resolution. These results reveal that the carboxylate of the omega-carboxyalkyloxy side chain of these inhibitors form ionic interactions with the conserved Arg in the substrate binding pocket of DHFR. These data suggest that the enhanced inhibitory activity of PY1011 compared with PY957 is, in part, due to the favorable contacts with Phe69 of pcDHFR by the methylene carbons of the inhibitor side chain that are oriented by the triple bond of the 1-pentynyl side chain. These contacts are not present in the PY957 pcDHFR complex, or in the PY1011 mDHFR complex. In the structure of mDHFR the site of Phe69 in pcDHFR is occupied by Asn64. These data also revealed a preference for an unusual parallel ring stacking interaction between Tyr35 of the active site helix and Phe199 of the C-terminal beta sheet in pcDHFR and by Tyr33 and Phe179 in mDHFR that is independent of bound ligand. A unique His174-His187 parallel ring stacking interaction was also observed only in the structure of pcDHFR. These ring stacking interactions are rarely found in any other protein families and may serve to enhance protein stability.     

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.     

Synthesis of new 2,4-Diaminopyrido[2,3-d]pyrimidine and 2,4-Diaminopyrrolo[2,3-d]pyrimidine inhibitors of Pneumocystis carinii,Toxoplasma gondii,and Mycobacterium avium dihydrofolate reductase

A concise new route allowing easy access to five previously unreported 2,4-diamino-6-(substituted benzyl)pyrido[2,3-d]pyrimidines (2a-e) was developed, involving condensation of 2,4-dipivaloylamino-5-bromopyrido[2,3-d]pyrimidine (6) with an organozinc halide in the presence of a catalytic amount of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II).CH(2)Cl(2), followed by removal of the pivaloyl groups with base. Also prepared via a scheme based on the Taylor ring expansion/ring annulation synthesis were three heretofore undescribed 2,4-diamino-5-(substituted benzyl)-7H-pyrrolo[2,3-d]pyrimidines (3b-c). Standard spectrophotometric assays were used to compare the ability of 2a-e and 3b-c to inhibit dihydrofolate reductase (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium, three examples of opportunistic pathogens to which AIDS patients are highly vulnerable because of their immunocompromised state. For comparison, 13 previously untested 2,4-diamino-6-(substituted benzyl)quinazolines (17a-m) were also evaluated as inhibitors of these enzymes, as well as the enzyme from rat liver. None of the quinazolines or pyridopyrimidines tested was more potent against the P. carinii enzyme than the structurally related reference compound piritrexim (1), and none showed selectivity for the P. carinii enzyme over the rat enzyme. One of the pyridopyrimidines (2c) showed 10-fold selectivity for T. gondii versus rat DHFR, and two of them (2b, 2c) showed selectivity for the M. avium enzyme. However, this gain in species selectivity was achieved at the cost of decreased in potency, as has been noted with many other lipophilic DHFR inhibitors.     

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.     

Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo

BACKGROUND AND METHODOLOGY: Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. PRINCIPAL FINDINGS AND CONCLUSIONS: Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. SIGNIFICANCE: JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.     

Design, synthesis, and biological evaluation of substituted 3-alkylthio-4,5-diaryl-4H-1,2,4-triazoles as selective COX-2 inhibitors

A new type of 4,5-diaryl-4H-1,2,4-triazole, possessing C-3 thio and alkylthio (SH, SMe or SEt) substituents, was designed and synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors with in vivo anti-inflammatory activity. The compound, 3-ethylthio-5-(4-fluorophenyl)-4-(4-methylsulfonylphenyl)-4H-1,2,4-triazole (10d), exhibited a high in vitro selectivity (COX-1 IC50=20.5 nM; COX-2 IC50=1.8 nM; SI=11.39) relative to the reference drug celecoxib (COX-1 IC50=3.7 nM; COX-2 IC50=2.2 nM; SI=1.68) and also showed good anti-inflammatory activity compared to celecoxib in a carrageenan-induced rat paw edema assay.     

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.     

Synthesis and preliminary biological evaluation of new anti-tubulin agents containing different benzoheterocycles

A new series of compounds, in which the 2-amino-4-methoxyphenyl ring of phenstatin analogue 5 was replaced with 2- or 3-amino-benzoheterocycles, was synthesized and evaluated for antiproliferative activity and inhibition of colchicine binding. The lack of activity of 3',4'-dimethoxy- and 4'-methoxy-benzoyl derivatives (8 and 9, respectively) indicates that the 3',4',5'-trimethoxybenzoyl moiety is critical for the activity. Two compounds, 7 and 11, displayed potent antiproliferative activity, with IC50 values ranging from 25 to 100 nM against a variety of cancer cell lines. Derivative 11 was more active than CA-4 as an inhibitor of tubulin polymerization. The results demonstrated that the antiproliferative activity was correlated with inhibition of tubulin polymerization.     

Structure-based design of new DHFR-based antibacterial agents: 7-aryl-2,4-diaminoquinazolines

Dihydrofolate reductase (DHFR) inhibitors such as trimethoprim (TMP) have long played a significant role in the treatment of bacterial infections. Not surprisingly, after decades of use there is now bacterial resistance to TMP and therefore a need to develop novel antibacterial agents with expanded spectrum including these resistant strains. In this study, we investigated the optimization of 2,4-diamnoquinazolines for antibacterial potency and selectivity. Using structure-based drug design, several 7-aryl-2,4-diaminoquinazolines were discovered that have excellent sub-100 picomolar potency against bacterial DHFR. These compounds have good antibacterial activity especially on gram-positive pathogens including TMP-resistant strains.     

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): a potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC(50)=1.1 nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.     

Synthesis and evaluation of a classical 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine as antifolates

Two classical antifolates, a 2,4-diamino-5-substituted furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine, were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The syntheses were accomplished by condensation of 2,6-diamino-3(H)-4-oxo-pyrimidine with alpha-chloro-ketone 21 to afford two key intermediates 23 and 24, followed by hydrolysis, coupling with l-glutamate diethyl ester and saponification of the diethyl ester to afford the classical antifolates 13 and 14. Compounds 13 and 14 with a single carbon atom bridge are both substrates for folylpoly-gamma-glutamate synthetase (FPGS), the enzyme responsible for forming critical poly-gamma-glutamate antifolate metabolites with increased potency and/or increased cell retention. Compound 14 is a highly efficient FPGS substrate demonstrating that 2,4-diamino-5-substituted furo[2,3-d]pyrimidines are important lead structures for the design of antifolates with FPGS substrate activity. It retains inhibitory potency for DHFR and TS compared to the two atom bridged analog 5. Compound 13 is a poor inhibitor of purified DHFR and TS, and both 13 and 14 are poor inhibitors of the growth of CCRF-CEM human leukemia cells in culture, indicating that single carbon bridged compounds in these series though conducive to FPGS substrate activity were not potent inhibitors.     

Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution

The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.     

Synthesis and biological evaluation of pyrido[3',2':4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110alpha inhibitors

4-Morpholin-4-ylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 1.4 microM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidine derivative 10e was discovered as a p110alpha inhibitor with approximately 400-fold greater potency than 2a. Evaluation of isoform selectivity showed that 10e is a potent inhibitor of p110beta. Furthermore, 10e showed anti-proliferative activity in various cell lines, including multi-drug resistant MCF7/ADR-res cells, and was effective against HeLa human cervical tumor xenografts in nude mice.     

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K_i 49 nM) compared to hDHFR (K_i 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K_i values of 593 and 617 nM, respectively; these K_i values are well above both the K_i for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.     

Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC50 = 2.26 microM; COX-2 IC50 = 0.012 microM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 microM; COX-2 IC50 = 0.15 microM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 microM; COX-2 IC50 = 0.978 microM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 microM; COX-2 IC50 = 1.00 microM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 microM; COX-2 IC50 = 1.14 microM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.     

Spasmolytic flavonoids from Syzygium samarangense (Blume) Merr. & L.M. Perry

The hexane extract of Syzygium samarangense (Ss.Hex) dose-dependently (10-1000 microg/ ml) relaxed the spontaneously contracting isolated rabbit jejunum. Four rare C-methylated flavonoids with a chalcone and a flavanone skeleton were isolated from Ss.Hex and were subsequently tested for spasmolytic activity. All flavonoids, identified as 2'-hydroxy-4',6'-dimethoxy-3'-methylchalcone (1), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (2), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (3), and 7-hydroxy-5-methoxy-6,8-dimethyl-flavanone (4), showed dose-dependent spasmolytic activity in the rabbit jejunum with IC50 values of 148.3 +/- 69.4, 77.2 +/- 43.5, 142.4 +/- 58.6 and 178.5 +/- 37.5 microg/ml (mean +/- SEM), respectively. The dihydrochalcone derivative of compound 1, 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (5), when tested for spasmolytic activity, did not significantly relax the smooth muscle relative to the other compounds. Verapamil, a standard spasmolytic, has an IC50 value of 0.16 +/- 0.04 microg/ml. This is the first report of the relaxant activity of chalcones, specifically of compounds 1-3.     

1H/15N HSQC NMR studies of ligand carboxylate group interactions with arginine residues in complexes of brodimoprim analogues and Lactobacillus casei dihydrofolate reductase

1H and 15N NMR studies have been undertaken on complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with analogues of the antibacterial drug brodimoprim (2,4-diamino-5-(3', 5'-dimethoxy-4'-bromobenzyl)pyrimidine) in order to monitor interactions between carboxylate groups on the ligands and basic residues in the protein. These analogues had been designed by computer modeling with carboxylated alkyl chains introduced at the 3'-O position in order to improve their binding properties by making additional interactions with basic groups in the protein. Specific interactions between ligand carboxylate groups and the conserved Arg57 residue have been detected in studies of 1H/15N HSQC spectra of complexes of DHFR with both the 4-carboxylate and the 4, 6-dicarboxylate brodimoprim analogues. The spectra from both complexes showed four resolved signals for the four NHeta protons of the guanidino group of Arg57, and this is consistent with hindered rotation in the guanidino group resulting from interactions with the 4-carboxylate group in each analogue. In the spectra of each complex, one of the protons from each of the two NH2 groups and both nitrogens are considerably deshielded compared to the shielding values normally observed for such nuclei. This pattern of deshielding is that expected for a symmetrical end-on interaction of the carboxylate oxygens with the NHeta12 and NHeta22 guanidino protons. The differences in the degree of deshielding between the complexes of the two structurally similar brodimoprim analogues and the methotrexate indicates that the shielding is very sensitive to geometry, most probably to hydrogen bond lengths. The 1H/15N HSQC spectrum of the DHFR complex with the brodimoprim-6-carboxylate analogue does not feature any deshielded Arg NHeta protons and this argues against a similar interaction with the Arg57 in this case. It has not proved possible to determine whether the 6-carboxylate in this analogue is interacting directly with any residue in the protein. 1H/15N HSQC spectra have been fully assigned for the complexes with the three brodimoprim analogues and chemical shift mapping used to explore interactions in the binding site. The 1H signals of the bound ligands for all three brodimoprim analogues have been assigned. Their 1H chemical shifts were found to be fairly similar in the different complexes indicating that the 2, 4-diaminopyrimidine and the benzyl ring are binding in essentially the same binding sites and with the same overall conformation in the different complexes. The rotation rate about the NepsilonCzeta bond in the brodimoprim-4,6-dicarboxylate complex with DHFR has been determined from a zz-HSQC exchange experiment, and its value is quite similar to that observed in the DHFR.methotrexate complex (24 +/- 10 s-1 at 8 degrees C and 50 +/- 10 s-1 at 15 degrees C, respectively). The 1H and 15N chemical shift differences of selected amide and guanidino NH groups, measured between the DHFR complexes, provided further evidence about the interactions involving Arg57 with the 4-carboxylate and 4,6-dicarboxylate brodimoprim analogues.     

Structural studies on bioactive compounds. Part 36: design,synthesis and biological evaluation of pyrimethamine-based antifolates against Pneumocystis carinii

As part of a research effort to improve the quality of current chemotherapy of Pneumocystis carinii pneumonia, we report a structure-based design project to optimise activity, species selectivity and pharmaceutical properties of the triazenyl-pyrimethamine TAB (4) (IC(50)=0.17 microM; rat liver DHFR IC(50)/P. carinii DHFR IC(50)=114). This has led us to design, synthesise and evaluate four new series of pyrimethamine derivatives bearing triazole, triazolium, triazinium and amino moieties at the 3'-position of the p-chlorophenyl ring. Such stabilised 'triazene' derivatives address the potentially compromised pharmaceutical profile of TAB and the 3'-amine substituted agents afford conformationally flexible substitutes. The benzylamino-pyrimethamine derivative (24a) (IC(50)=0.12 microM, rat liver DHFR IC(50)/P. carinii DHFR IC(50): 5.26) was the most potent and the only P. carinii-selective antifolate of the new series.     

Design and synthesis of new water-soluble tetrazolide derivatives of celecoxib and rofecoxib as selective cyclooxygenase-2 (COX-2) inhibitors

In an attempt to prepare a new water-soluble, parenteral COX-2 inhibitor, rofecoxib (9) and celecoxib (13) analogues were designed and synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors with in vivo anti-inflammatory activity. In this experiment, respective SO(2)Me and SO(2)NH(2) hydrogen-bonding pharmacophores were replaced by a tetrazole ring. Molecular modeling (docking) studies showed that the tetrazole ring of these two analogues (9 and 13) was inserted deep into the secondary pocket of the human COX-2 binding site where it undergoes electrostatic interaction with Arg(513). The rofecoxib (9) and celecoxib (13) analogues exhibited a high in vitro selectivity (9, COX-1 IC(50) = 3.8 nM; COX-2 IC(50) = 1.8 nM; SI = 2.11; 13, COX-1 IC(50) = 4.1 nM; COX-2 IC(50) = 1.9 nM; SI = 2.16) relative to the reference drug celecoxib (COX-1 IC(50) = 3.7 nM; COX-2 IC(50) = .2 nM; SI=1.68) and also showed high aqueous solubility at pH higher than 7 and good anti-inflammatory activity in a carrageenan-induced rat paw edema assay. However, 9 and 13 had no significant damage on gastric mucosa.     

Cytotoxic lignans from Larrea tridentata

Six lignans, including the cyclolignan 3,4'-dihydroxy-3',4'-dimethoxy-6,7'-cyclolignan, were isolated from the flowering tops of Larrea tridentata. Additionally the flavanone, (S)-4',5-dihydroxy-7-methoxyflavanone, was isolated for the first time from L. tridentata or any member of the family Zygophyllaceae. All of the compounds were assessed for their growth inhibitory activity against human breast cancer, human colon cancer and human melanoma cell lines. The lignans had IC50 values of 5-60 microM with the linear butane-type lignans being the most potent, and it was found that colon cancer cells were the least sensitive cell type tested. The relative potency of linear butane type lignans against human breast cancer appears to correlate positively with the number of O-methyl groups present on the molecule.