Design and synthesis of classical and nonclassical 6-arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as antifolates

The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.     

Novel boron-containing, nonclassical antifolates: synthesis and preliminary biological and structural evaluation

Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.     

Synthesis, antifolate, and antitumor activities of classical and nonclassical 2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines

Classical and nonclassical isosteric C8-N9 bridged analogues of the multitargeted antifolate LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and as antitumor and antiopportunistic infection agents. The syntheses of the analogues were accomplished by reductive amination of the appropriate anilines with 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (28) followed by saponification of the ethyl esters, for the classical analogue 6. The N9-methyl analogues were obtained from the N9-H precursors by reductive methylation. In general, the nonclassical compounds 7-17 were similar in potency to TMP against Toxoplasma gondii DHFR, with selectivity ratios greater than 38 and 21 for 11 and 16, respectively. These compounds were poor inhibitors of Pneumocystis carinii DHFR and rat liver DHFR. The nonclassical analogues were also inactive against TS. The classical analogue 6 was a marginal inhibitor of isolated human TS (IC50 = 46 microM) and of human DHFR (IC50 = 10 microM), however, it was a potent inhibitor of the growth of two human head and neck squamous cell carcinoma cell lines and of CCRF-CEM human lymphoblastic leukemia cells in culture and was similar to LY231514 against ZR-75-1 human breast carcinoma cell line. Evaluation of 6 against MTX-resistant sublines indicated that DHFR is not the major target of 6. Metabolite protection studies of the growth inhibitory activity of 6 suggest that TS is a major target of this drug and that polyglutamyl forms of 6 may serve as the intracellular TS inhibitors. These studies also suggest that 6 has a site of action in addition to sites in the folate pathway.     

Potent dual thymidylate synthase and dihydrofolate reductase inhibitors: classical and nonclassical 2-amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine antifolates

N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3- d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3 H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC 50 = 40 nM) and human DHFR (IC 50 = 20 nM) known to date. The nonclassical analogues 5- 13 were moderately potent against human TS with IC 50 values ranging from 0.11 to 4.6 microM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity.     

Synthesis and biological activities of 5-deaza analogues of aminopterin and folic acid

N-[p-[[(2,4-Diaminopyrido[2,3-d]pyrimidin-6-yl)methyl] amino]benzoyl]-L-glutamic acid (1a, 5-deazaaminopterin) and the 5-methyl analogue (1b) were synthesized in 14 steps from 5-cyanouracil (4a) and 5-cyano-6-methyluracil (4b), respectively, by exploitation of the novel pyrimidine to pyrido[2,3-d]pyrimidine ring transformation reaction. The 5-cyanouracils 4 were treated with chloromethyl methyl ether to the 1,3-bis(methoxymethyl)uracils (5, which were treated with malononitrile in NaOEt/EtOH to give the pyrido[2,3-d]pyrimidines 6. Diazotization of 6 in concentrated HCl afforded the 7-chloro derivatives 8 in high yield. After reduction of 8, the 7-unsubstituted products 9 were reduced in the presence of Ac2O and the products, 6-(acetamidomethyl)pyridopyrimidines 10, were converted into the 6-acetoxymethyl derivatives 12 via nitrosation. After removal of the N-methoxymethyl groups from 12, the 6-(acetoxymethyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones 14 were converted into 2,4-diamino-6-(hydroxymethyl)pyrido[2,3-d]pyrimidine (15a) and its 5-methyl analogue 15b by the silylation-amination procedure. Compounds 15 were brominated to the 6-bromomethyl derivatives 16, which were treated with diethyl (p-aminobenzoyl)-L-glutamate, and the products 17 were saponified to afford 5-deazaaminopterin (1a) and its 5-methyl analogue 1b. Compound 1b was also prepared by an alternative procedure in 10 steps from cyanothioacetamide and ethyl beta-(ethoxymethylene)acetoacetate via 2,4-diamino-6-(hydroxymethyl)-5-methylpyrido[2,3-d]pyrimidine (15b). 5-Deaza-5-methylfolic acid (2) was also prepared in four steps from 15b. The aminopterine analogues 1 showed significant anticancer activity in vitro and in vivo, whereas the folic acid analogue 2 did not exhibit any significant toxicity.     

Synthesis of classical, three-carbon-bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates

Bridge homologation of the previously reported classical two-carbon-bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) [which is a 6-regioisomer of LY231514 (Alimta)] and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine, afforded the three-carbon-bridged antifolates analogues 4 and 5, with enhanced inhibitory activity against tumor cells in culture (EC(50) values in the 10(-8)-10(-7) M range or less). These two analogues were synthesized via a 10-step synthetic sequence starting from methyl 4-bromobenzoate (14), which was elaborated to the alpha-chloromethyl ketone (8) followed by condensation with 2,6-diamino-pyrimidin-4-one (7) to afford the substituted furo[2,3-d]pyrimidine 9 and the pyrrolo[2,3-d]pyrimidine 10. Subsequent coupling of each regioisomer with diethyl-l-glutamate followed by saponification afforded 4 and 5. The biological results indicate that elongation of the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine are highly conducive to antitumor activity in vitro, despite a lack of increase in inhibitory activity against the target enzymes. This supports our original hypothesis that truncation of the B-ring of a highly potent 6-6 ring system to a 6-5 ring system can be compensated by bridge homologation to restore the overall length of the molecule.     

Synthesis of classical, four-carbon bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates

We report, for the first time, the biological activities of four-carbon-atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpolyglutamate synthetase (FPGS) as well as antitumor activity. Extension of the bridge homologation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2), afforded two four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity and inhibitory activity against tumor cells in culture (EC(50) < or = 10(-7) M) compared with the two-carbon bridged analogues. These results support our original hypothesis that the distance and orientation of the side chain p-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine ring are a crucial determinant of biological activity. In addition, this study demonstrates that, for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated target enzymes is not necessarily a predictor of a lack of antitumor activity.     

Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: design, synthesis, and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates(1)

We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC(50) = 90 nM) and human DHFR (IC(50) = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were only inhibitors of human TS with IC(50) values of 0.23-26 microM.     

Folate analogues. 35. Synthesis and biological evaluation of 1-deaza, 3-deaza, and bridge-elongated analogues of N10-propargyl-5,8-dideazafolic acid

Structural modifications at the pyrimidine ring and at the C9,N10-bridge region of the thymidylate synthase (TS) inhibitors N10-propargyl-5,8-dideazafolate (1; PDDF; CB 3717), 2-desamino-N10-propargyl-5,8-dideazafolate (2, DPDDF), and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (3, DMPDDF) have been carried out. Methods for the synthesis of 2-desamino-N10-propargyl-1,5,8-trideazafolate (4), 2-desamino-2-methyl-N10-propargyl-3,5,8-trideazafolate (5a), and 2-desamino-2-methyl-N10-propargyl-5,8-dideaza-1,2-dihydrofolate (6) have been developed. The bridge-extended analogues isohomo-PDDF (7) and isohomo-DMPDDF (8) contain an additional methylene group interposed between N10 and the phenyl ring of 1 and 3, respectively. All new compounds were evaluated as inhibitors of TS and the growth of tumor cells in culture. Selected analogues were tested as substrates of folylpolyglutamate synthetase (FPGS) and striking differences in substrate activity were observed among these compounds, indicating that structural modifications at the pyrimidine ring of classical antifolates profoundly influence their polyglutamylation. Enzyme inhibition data established that both N1 and N3-H of the pyrimidine ring are essential for efficient binding of quinazoline-type antifolates to human TS.     

The effect of 5-alkyl modification on the biological activity of pyrrolo[2,3-d]pyrimidine containing classical and nonclassical antifolates as inhibitors of dihydrofolate reductase and as antitumor and/or antiopportunistic infection agents

Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I2. The condensation of alpha-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC50 = 60 nM) and 4 (IC50 = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50 500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.     

Nonsteroidal cardiotonics. 2. The inotropic activity of linear, tricyclic 5-6-5 fused heterocycles

We previously reported the structure-activity relationships (SAR) of adibendan (1), a potent and long-acting cardiotonic. This paper describes the synthesis of a novel series of linear, tricyclic fused heterocycles of the 5-6-5 type. The compounds were evaluated for positive inotropic activity in anesthetized rats, cats, and dogs. Changes in left ventricular dP/dt were measured as an index of cardiac contractility. The increase in contractility was not mediated via stimulation of beta-adrenergic receptors. The data revealed the intrinsic positive inotropic activity of the parent compound of this series, 5,7-dihydro-7,7-dimethylpyrrolo[2,3-f]benzimidazol-6(1H)-one (2). The structural features that impart optimal inotropic activity are presented and compared with those of the 4,5-dihydro-3(2H)-pyridazinone series. The most potent compounds were evaluated orally in conscious dogs with implanted Konigsberg pressure transducers to measure ventricular pressures, and their effect on left ventricular dP/dt was compared with that of 1, pimobendan, and indolidan. After administration of 1 mg/kg, 1, 3, 7, 19, 22, 24, 31, 54, pimobendan, and indolidan were equipotent, but only with 1, 31, pimobendan, and indolidan, durations of action exceeded 6 h.     

Synthesis of classical and a nonclassical 2-amino-4-oxo-6-methyl-5-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of thymidylate synthase.

Compounds 2-5 were designed as potential antifolate nonpolyglutamatable inhibitors of thymidylate synthase (TS). These analogues are structurally related to 2-amino-4-oxo-5-substituted quinazolines and 2-amino-4-oxo-5-substituted pyrrolo[2, 3-d]pyrimidines which have shown excellent inhibition of TS and, for the quinazoline, significant promise as clinically useful antitumor agents. Compounds 2-4 were synthesized by appropriate amine exchange reactions on pivaloyl-protected 5-dimethylaminomethyl-substituted 6-methyl pyrrolo[2,3-d]pyrimidine 7 which in turn was obtained from the Mannich reaction of pivaloylated-6-methyl pyrrolo[2, 3-d]pyrimidine 6. In instances where the amine exchange reaction was sluggish, the Mannich base was quaternized with methyl iodide which afforded much faster exchange reaction with improved yields. For compound 5, 4-mercaptopyridine was used as the nucleophile and reacted with 7. The analogues 2-4 inhibited Lactobacillus casei (lc) TS and recombinant human (h) TS with IC50 in the 10(-4) to 10(-5) M range. Compound 5 inhibited lcTS and hTS 20% at 26 and 25 microM, respectively. In addition, compound 5 inhibited the growth of Pneumocystis carinii and Toxoplasma gondii cells in culture by 76% at 32 x 10(-6) M and 50% at 831 x 10(-6) M, respectively.     

Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid

Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.     

Inhibition of dihydrofolate reductase from bacterial and vertebrate sources by folate, aminopterin, methotrexate and their 5-deaza analogues

The inhibition of dihydrofolate reductases from Escherichia coli and chicken liver by folate, methotrexate, aminopterin and their 5-deaza analogues was investigated to examine the importance of the N-5 nitrogen in slow-binding inhibition. Methotrexate, aminopterin and their 5-deaza analogues acted as slow, tight-binding inhibitors of both enzymes. Inhibition by methotrexate and 5-deazamethotrexate conformed to a mechanism in which there is an initial rapid formation of an enzyme-NADPH-inhibitor complex followed by a slow isomerization of this complex (Mechanism B). Aminopterin exhibited the same type of inhibition with the enzyme from E. coli. With the chicken-liver enzyme, however, the inhibition by aminopterin conformed to another type of slow-binding mechanism which involves only the slow interaction of the inhibitor with the enzyme to form an enzyme-NADPH-inhibitor complex (Mechanism A). The inhibition of both enzymes by 5-deazaaminopterin was also described by Mechanism A. Folate behaved as a classical, steady-state inhibitor of both enzymes, whereas 5-deazafolate exhibited slow-binding inhibition (Mechanism B) with the enzyme from E. coli and classical, steady-state inhibition with the enzyme from chicken liver. The substitution of a carbon for a nitrogen at the 5-position of methotrexate and aminopterin did not affect the tightness of binding of these compounds. By contrast, 5-deazafolate was bound about 4000 times more tightly than folate to the enzyme from E. coli and about 30 times more tightly than folate to the chicken-liver enzyme. Reasons for the differences in the binding of folate and 5-deazafolate are discussed.     

新型三环酮内酯TE-802的合成

目的研究新型三环酮内酯TE-802的合成方法。方法以克拉霉素为起始原料，经水解、乙酰化、环碳酸酯化、氧化、脱碳酸酯化、酰化、环氨基甲酸酯化和环合等反应合成目标化合物。结果成功地合成了TE-802，总收率为22．1％。目标化合物的结构经红外光谱、核磁共振氢谱、质谱及元素分析确证。结论该合成路线中使用的原料和试剂价廉易得、总成本低，收率高，各步反应条件温和、操作简便。     

Potentially CNS-active tricyclic compounds with a bridged central ring. 3. Synthesis of partially hydrogenated 9,10-epithioanthracenes

The synthesis of 9,10-epithio-9,10-dihydroanthracenes is presented. Since the direct synthesis from benzo[c]thiophene and dehydrobenzene was not possible, benzo[c]thiophene and 1,4-benzoquinone were reacted by means of Diels-Alder cyclization to 9,10-epithio-1,4,9,10-tetrahydro-1,4-dioxoanthracene, which we intended to convert into 9,10-epithio-9,10-dihydroanthracene after intermediate transfer to suitable derivatives. The configurations at the connection points 4a and 9a of the partly or completely hydrogenated derivatives in one ring were ascertained, as were those in positions 1 and 4, Sp2-hybridisation of both the C-atoms 4a and 9a resulted in the elimination of the sulphur atom.     

Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents: part I

Antifolates that inhibit the key enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR) have found clinical utility as antitumor and antiopportunistic agents. Methotrexate {MTX, (1)} and 5-fluorouracil (5-FU) were among the first clinically useful DHFR and TS inhibitors, respectively. The development of resistance to 5-FU, its occasional unpredictable activity and toxicity resulted in the search of novel antifolates. Pemetrexed (4) and raltitrexed (5) specifically inhibit TS, and are clinically useful as antitumor agents. A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Novel antifolates have been developed that do not need to be polyglutamylated and include plevitrexed (6) and GW1843 (7). Nonclassical antifolates for antitumor and parasitic chemotherapy, such as nolatrexed (8), trimethoprim {TMP, (11)} and piritrexim {PTX, (12)}, can passively diffuse into cells and hence do not have to depend on FPGS or the reduced folate carrier (RFC). Variations in the structures of antifolates have helped delineate the structural influence on the interaction with TS, DHFR, FPGS, and RFC utilization. The differences in the active site of human and pathogen DHFR have also been exploited. The literature contains excellent reviews on the design and synthesis of antifolates prior to 1996. This two-part review discusses the design, synthesis and structural requirements for TS and DHFR inhibition and their relevance to antitumor and parasitic chemotherapy, since 1996. Monocyclic and 6-5 fused bicyclic antifolates will be discussed in Part I, while 6-6 bicyclic and tricyclic antifolates will be discussed in Part II.     

Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents: Part II

Antifolates that inhibit the key enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR) have found clinical utility as antitumor and antiopportunistic agents. Methotrexate {MTX, (1)} and 5-fluorouracil (5-FU) were among the first clinically useful DHFR and TS inhibitors, respectively. The development of resistance to 5-FU, its occasional unpredictable activity and toxicity resulted in the search of novel antifolates. Pemetrexed (4) and raltitrexed (5) are newer antifolates that specifically inhibit TS, and are clinically useful as antitumor agents. A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Recently, classical antifolates that do not need to be polyglutamylated have also been developed and include plevitrexed (6) and GW1843 (7). Nolatrexed (8), trimethoprim {TMP, (11)} and piritrexim {PTX, (12)} are nonclassical antifolates for antitumor and parasitic chemotherapy that passively diffuse into cells and hence do not have to depend on FPGS or the reduced folate carrier (RFC). Structural requirements for inhibition with antifolates have been studied extensively and novel agents that exploit key interactions in the active site of TS, DHFR, FPGS, and RFC have been proposed. This two-part review discusses the design, synthesis and structural requirements for TS and DHFR inhibition and their relevance to antitumor and parasitic chemotherapy, since 1996. Monocyclic and 6-5 fused bicyclic antifolates were discussed in Part I. The 6-6 bicyclic and tricyclic antifolates will be discussed here in Part II.     

Synthesis of tricyclic analogs of incasane

Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 22, No. 9, pp. 1060–1063, September, 1988.     

Synthesis and antitumor activity of thieno-separated tricyclic purines

The purine ring system is undoubtedly one of the most ubiquitous heterocyclic ring systems in nature as it has the distinction of being the parent ring in countless derivatives of biological relevance. It is not surprising then that modified purines possess the potential to impact several areas, including a better understanding of the biological effects of DNA damaging agents, enzyme/substrate interactions, and in the development of more potent medicinal agents. One focus for our research at Georgia Tech has centered around the design and synthesis of a series of extended purine analogues containing a heterocyclic spacer ring, with sites set on investigations into their use as (i) potential anticancer and antiviral agents, (ii) dimensional probes for enzyme and coenzyme binding sites, and (iii) structural probes of the minor groove of DNA. The synthesis and preliminary antitumor activity of two thieno-separated purine analogues are described herein. Tricyclic 1 was synthesized in 12 steps from tribromoimidazole and with an overall yield of 7%. Tricyclic 2 was synthesized in 9 steps with an overall yield of 13%. Both 1 and 2 exhibited growth inhibitory effects on HCT116 colorectal cancer cells in vitro.