Descending aortic blood flow during aortic cross-clamp indicates postoperative splanchnic perfusion and gastrointestinal function in patients undergoing aortic reconstruction.

BACKGROUND: The purpose of this observational study was to investigate the relationship between splanchnic and renal blood flow during infrarenal aortic cross-clamp (XC) and postoperative gastrointestinal perfusion and function. METHODS: Descending aortic blood flow (DABF) was continuously monitored with an oesophageal Doppler monitor (Cardio-Q, Deltex Ltd, Chichester, UK) in 31 patients undergoing elective abdominal aortic aneurysm repair. Cardiac output (CO) was determined by indocyanine green dilution before, during, and after XC. Perioperative gastrointestinal perfusion was assessed by gastric intramucosal pH (pHi, Tonocap, GE Healthcare, Helsinki, Finland). Postoperative gastrointestinal recovery was assessed by the number of postoperative days until the patient successfully resumed solid food intake. The relationship between the mean DABF during XC and gastric pHi after XC release and postoperative gastrointestinal recovery was analysed with Spearman's correlation coefficient. RESULTS: accounted for ∼ 55% of CO during XC and significantly decreased during XC, despite arterial pressure remaining within an optimal range. There were two distinct relationships between DABF during XC and gastric pHi after XC release. Gastric pHi steeply and linearly declined when indexed DABF was below 0.82 litre min(-1) m(-2). Above this critical value, there was no linear relationship between them. The duration of postoperative gastrointestinal dysfunction was inversely correlated with the mean DABF during XC. The best cut-off value of the mean indexed DABF during XC to prevent prolonged gastrointestinal dysfunction was 1.2 litre min(-1) m(-2). CONCLUSIONS: Decreased DABF during XC associates splanchnic hypoperfusion after XC release and delayed recovery of gastrointestinal function.

Novel 6,5-fused ring heterocyclic antifolates: biochemical and biological characterization.

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.

Inhibition by phenyl N-tert-butyl nitrone of early phase carcinogenesis in the livers of rats fed a choline-deficient, L-amino acid-defined diet.

Male Wistar rats were fed a choline-deficient, L-amino acid-defined (CDAA) diet alone or in combination with a nitrone-based free radical trapping agent, phenyl N-tert-butyl nitrone (PBN) in the drinking water at the concentrations of 0.013, 0.065, and 0.130% for 12 weeks. PBN inhibited the changes that are normally induced in the livers of rats by the CDAA diet feeding, i.e., development of putative preneoplastic lesions, proliferation of connective tissue, reduction of glutathione S-transferase activity, formation of 8-hydroxyguanine in DNA, and an increase in inducible cyclo-oxygenase (COX2) activity. PBN, however, did not prevent the increases in the COX2 mRNA or protein levels brought on by the CDAA diet These results indicate that the loss of glutathione S-transferase activity and COX2 induction may play significant roles in rat liver carcinogenesis by the CDAA diet and that PBN prevents neoplasia not only by its radical scavenging activity but also by inhibiting COX2 activity at the catalytic level.

Inhibition of NF-kappaB, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl-N-tert-butylnitrone (PBN).

Previously, the spin trapping agent phenyl-N-tert-butylnitrone (PBN) has been shown to decrease the level of nitric oxide synthase mRNA in vivo. This inhibition is suggested to be an underlying mechanism for PBN's wide variety of pharmacological actions in animal models. However, the determination of PBN's cellular pharmacological activities has not been carried out, but is necessary for the understanding of the effects in vivo. Since the known pharmacological effects of PBN are primarily anti-inflammatory in nature, in this study we determined the inhibitory activities of PBN against two inflammatory factors: inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX2). We show here that PBN decreases steady state COX2 mRNA level and COX2 catalytic activity in macrophage cell culture at supra-pharmacological concentrations. While PBN decreases iNOS mRNA, it does not inhibit iNOS catalytic activity, which is consistent with previous in vivo studies. We also studied nuclear factor kappaB (NF-kappaB), a transcription factor that can rapidly activate the expression of genes involved in inflammatory, immune and acute phase responses. The binding of NF-kappaB to iNOS gene has been shown to be critical for iNOS gene expression, and the promoter region of COX2 gene contains NF-kappaB consensus sequence. We show that PBN inhibits lipopolysaccharide-mediated increase of NF-kappaB DNA binding activity with a lower concentration than that for the non-steroidal anti-inflammatory drug (NSAID), salicylate. Furthermore, we show that PBN inhibits COX2 catalytic activity, suggesting that PBN has an NSAID-like function.

Continuous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex.

Nitric oxide (NO) generation in murine macrophages was determined in real time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl D-glucamine dithiocarbamate was utilized as the spin trap. This spin trapping compound reacts with NO in solution to form a specific room-temperature stable, mononitrosyl complex which is readily detected and identified by EPR spectroscopy. Mouse peritoneal macrophages were placed in an EPR sample-cell and activated by lipopolysaccharide and gamma-interferon at 37 degrees C, followed by an additional incubation in oxygenated medium without these activation agents. After various incubation periods, spin trap solution was infused to replace the medium in the sample-cell, and the time-evolution of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes of the increase in the EPR intensity with time. In comparison to the NO (or NO2-) generation rate obtained under similar experimental conditions using the Griess reaction assay, the spin trapping method was found to be more sensitive, with a lowest limit of the detection of 3 pmol/min. In addition, by using the spin trapping method, NO generation from the same cells could be measured consecutively during various stages of activation, because infusion of the spin trap solution did not affect the viability of macrophages.

Enhanced expression of Tie2, its ligand angiopoietin-1, vascular endothelial growth factor, and CD31 in human non-small cell lung carcinomas.

Expression of angiogenesis-associated genes was compared in 32 primary non-small cell lung carcinoma samples (14 adenocarcinomas, 17 squamous cell carcinomas, and 1 large cell carcinoma) and paired adjacent noncancerous lung tissues using a multiprobe RNase protection assay. Levels of Tie2, angiopoietin (Ang)-1, vascular endothelial growth factor (VEGF), and CD31 mRNAs were higher in cancers than in adjacent noncancerous tissues, in contrast to the fms-like tyrosine kinase (Flt)-1, Flt-4, Tie1, thrombin receptor, endoglin, and VEGF-C, for which no differences were evident. Overexpression did not seem to differ with histological type and pathological stage. Significant positive correlations were found between mRNA expression of Ang-1 and those of Tie2 and CD31, and that of VEGF and those of Flt-1 and CD31. These findings suggest that Ang-1 and VEGF are important angiogenic factors in human non-small cell lung carcinomas.

Optimal time and dosage of phenyl N-tert-butyl nitrone (PBN) for the inhibition of nitric oxide synthase induction in mice.

We have previously reported that phenyl N-tert-butyl nitrone (PBN) inhibits the induction of inducible nitric oxide synthase (iNOS) and, thus, prevents the overproduction of nitric oxide (NO), resulting in the reduction of endotoxin-mediated death in mice. In this study, to examine the effect of PBN in detail, we investigated the dose- and administration-timing dependence of PBN on endotoxin-induced NO generation in mice. NO generation was monitored in the mouse liver after administration of lipopolysaccharide (LPS) by the in vivo NO-spin trapping method using the iron complex of N-methyl-D-glucamine dithiocarbamate (MGD) as a spin trap, followed by ex vivo EPR measurement of the liver tissue. PBN was effective in reducing liver NO generation monitored 6 h after endotoxin injection when it was administered shortly before or after LPS injection. The maximum inhibition of liver NO was obtained when PBN was administered 30 min before LPS injection. ID50 for the inhibition was estimated to be approximately 200 mg/kg when the LPS dose of 50 mg/kg was used. Expression of mRNA for iNOS in the liver as estimated by reverse transcription polymerase chain reaction was decreased when PBN was given 30 min before LPS injection, indicating that the reduction of expression of iNOS protein by PBN, which has been shown previously, is at least in part caused by a decrease in mRNA expression.

Stabilities of hydroxyl radical spin adducts of PBN-type spin traps.

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.

Studies on the stability of oxygen radical spin adducts of a new spin trap: 5-methyl-5-phenylpyrroline-1-oxide (MPPO).

A new spin trap, 5-methyl-5-phenylpyrrolin-1-oxide (MPPO), has been evaluated with respect to the intrinsic stabilities of the hydroxyl and superoxide (or hydroperoxyl) radical spin adducts. Hydroxyl or superoxide radicals were generated using various sources in the presence of MPPO, and the hydroxyl or superoxide radical spin adduct of MPPO was detected by EPR spectroscopy. The time course of spontaneous decay of the EPR signal from hydroxyl or superoxide spin adducts followed first-order kinetics and the half-life was dependent on the pH of the medium. At pH 7.4 the half-life times are 76.4 and 5.7 min for the hydroxyl and hydroperoxyl/superoxide spin adducts, respectively. Structural factors which could influence the decay rates are also discussed.

Spin trapping of superoxide radicals following stimulation of neutrophils with fMLP is temperature dependent.

Oxygen radical formation by human neutrophils stimulated with a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), was studied through the use of spin trapping and superoxide dismutase-inhibitable reduction of oxidized cytochrome c. Both methods provided comparable data on temperature-dependent kinetics of superoxide radical formation, but hydroxyl radicals were also detected in spin-trapping experiments. When superoxide generation was monitored at 37 degrees C, the respiratory burst lasted only a few minutes. If the neutrophils were stimulated at 37 degrees C, but superoxide measurements were done at room temperature, the respiratory burst was again transient. However, neutrophils persistently generated superoxide when both stimulation and subsequent measurements were performed at room temperature. In the presence of the actin polymerization inhibitor, cytochalasin B, superoxide generation was persistent, even when measurements were conducted at 37 degrees C. A possible explanation for these observations is that the fMLP receptor complexes quickly aggregate and are internalized at physiological temperature, but not at room temperature. Very little superoxide was formed if cells were kept at a temperature of 4 degrees C for 1 h prior to fMLP addition, which is consistent with decreased expression of the fMLP receptor at cold temperatures.

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.

Determination of the rate of superoxide generation from biological systems by spin trapping: use of rapid oxygen depletion to measure the decay rate of spin adducts.

A method was developed to measure the superoxide generation rate from biological systems using the spin trapping method. Kinetic treatment of the decay rate of the superoxide adduct of 5,5-dimethylpyrroline N-oxide (DMPO) revealed that the EPR signal intensity of the system is proportional to the superoxide generation rate. Rapid depletion of oxygen in the sample was utilized to terminate superoxide generation so that the decay rate of the DMPO superoxide adduct (DMPO-OOH) could be determined. For this decay measurement, a controlled atmosphere EPR cavity was developed and was used with an open-air sample cell. Superoxide generation rates determined with this method for stimulated neutrophils and for the hypoxanthine-xanthine oxidase system were comparable to those obtained with the cytochrome c reduction method. This method is specifically applicable to the system in which dissolved oxygen supplied from the gas phase is utilized as a source of superoxide.

Free radical formation during ketamine anesthesia in rats: a cautionary note.

Ketamine is a useful anesthetic agent with good analgesic properties; however, when ketamine was used to anesthetize rats for spin trapping studies of alcohol-induced free radicals, liver extracts contained a strong electron paramagnetic resonance (EPR) signal of a novel radical. The same EPR signal was observed in liver extracts when rats which had not received alcohol were anesthetized with ketamine. When ketamine was added to liver microsomes and NADPH, a nitroxide radical derived from ketamine could be detected in organic extracts. When the spin trapping agent POBN was also added, microsomes produced both a ketamine nitroxide radical and a spin adduct. Similar results were obtained during ketamine oxidation by hydrogen peroxide in a tungstate-catalyzed reaction, or in a Fenton reaction system. The data suggest that the secondary amine group of ketamine can be oxidized to a stable nitroxide which produces an EPR spectrum in the absence of a spin trapping agent. The POBN spin adduct detected may be from a carbon-centered radical in the cyclohexanone ring of ketamine. Because several types of radicals can be formed from ketamine, this agent may not be appropriate as an anesthetic for many types of in vivo spin trapping experiments.

High-performance liquid chromatography study of the pharmacokinetics of various spin traps for application to in vivo spin trapping.

In vivo spin trapping is potentially a very useful tool to investigate the role of free radicals in physiologic processes and disease development. Unfortunately, knowledge on the stability and distribution of spin traps in living systems is limited. Therefore, in our study, we selected 11 acyclic and cyclic nitrone spin traps with diverse properties to determine their pharmacokinetics in mice. At varying times after intraperitoneal administration, we measured the concentration of the spin traps in the liver, heart, and blood. Our results showed that most spin traps were rapidly absorbed and were approximately evenly distributed throughout the mouse body. It was also found that most of the traps were relatively stable in vivo with more than half of the injected amount still available for spin trapping free radicals after an hour. Two of the 11 tested spin traps, however, decomposed after injection. These results indicate that for a successful in vivo spin trapping experiment, the stability of the spin trap is not of major concern, but the time course of distribution may be important.

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.

Spin trapping agent phenyl N-tert-butylnitrone protects against the onset of drug-induced insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the beta-cells. Cytokines and nitric oxide (NO) have been shown to be involved in this destruction. Phenyl N-tert-butylnitrone (PBN) has demonstrated protective effects against several pathological conditions including ischemia-reperfusion injury and endotoxin-induced shock. We report here that PBN co-administration can prevent the onset of the STZ-induced diabetes in mice. PBN co-treatment inhibited the streptozotocin (STZ)-induced hyperglycemia, the elevation in the level of glycated hemoglobin and weight loss in the treated mice. Histological observations indicated destruction of B-cells in the STZ-treated animals and its prevention by PBN co-treatment. EPR spin trapping experiments in the pancreas indicated the in vivo formation of NO in STZ-treated animals and its attenuation by PBN treatment.

Aromatic hydroxylation in PBN spin trapping by hydroxyl radicals and cytochrome P-450.

Phenyl N-tert-butylnitrone (PBN) is widely used as a spin trapping agent, but is not useful detecting hydroxyl radicals because the resulting spin adduct is unstable. However, hydroxyl radicals could attack the phenyl ring to form stable phenolic products with no electron paramagnetic resonance signal, and this possibility was investigated in the present studies. When PBN was added to a Fenton reaction system composed of 25 mM H(2)O(2) and 0.1 mM FeSO(4), 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe(2+) concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxyPBN were also formed. Although PBN is extensively metabolized after administration to animals, its metabolites have not been identified. When PBN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PBN was given to rats, both free and conjugated 4-hydroxyPBN were readily detected in liver extracts, bile, urine, and plasma. Because 4-hydroxyPBN is the major metabolite of PBN and circulates in body fluids, it may contribute to the pharmacological properties of PBN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation.

Pharmacologic properties of phenyl N-tert-butylnitrone.

Phenyl N-tert-butylnitrone (PBN) is the parent of a family of nitrones used as spin-trapping agents to trap free radicals. PBN's pharmacological effects in animal models are extensive, ranging from protection against death after endotoxin shock, protection from ischemia-reperfusion injury, to increasing the life span of mice. Recent additions to the list include protection from bacterial meningitis, thalidomide-induced teratogenicity, drug-induced diabetogenesis, and choline-deficient hepatocarcinogenesis. Because PBN reacts with oxygen radicals to produce less reactive species, it has been suggested that this is the basis of its pharmacological effects. However, there has been no hard evidence for this notation. Nevertheless, many investigators have used the presence of PBN's pharmacologic effect as evidence for free radical involvement in their models. Mechanistic studies on the PBN's antisepsis action revealed that PBN inhibits expression of various pro-inflammatory genes, suggesting that the protective action involves more than a straightforward free radical-scavenging mechanism. Previous and recent developments in the investigations on the pharmacologic properties of PBN are described in this review.

MPP+ analogs acting on mitochondria and inducing neuro-degeneration.

This review focuses on the mechanisms of action and the injurious effect of complex I inhibitors, of which 1-methyl-4-phenylpyridinium ion (MPP(+)) is a well studied example. These compounds can be divided into two groups, i.e. competitive inhibitors with respect to ubiquinone, such as piericidine A, and non-competitive inhibitors such as rotenone. Complex I inhibitors such as MPP(+) have been reported to induce anatomical, behavioral, and biochemical changes similar to those seen in Parkinson's disease, which is characterized by nigrostriatal dopaminergic neuro-degeneration. Spectroscopic analyses and structure-activity relationship studies have indicated that the V-shaped structure of the rotenone molecule is critical for binding to the rotenone binding site on complex I. Many isoquinoline derivatives, some of them endogenous, are also complex I inhibitors. Many lines of evidence show that complex I inhibitors elicit neuronal cell death. Recently, it was reported that chronic and systemic exposure to low-dose rotenone reproduces the features of Parkinson's disease. This work further focused attention on compounds acting on mitochondria, such as MPP(+). In Guadeloupe, the French West Indies, patients with atypical parkinsonism or progressive supranuclear palsy are frequently encountered. These diseases seem to be associated with ingestion of tropical herbal teas or tropical fruits of the Annonaceae family, which contain complex I inhibitors such as benzylisoquinoline derivatives and acetogenins. Complex I inhibitors may not simply result in reactive oxygen species generation or ATP exhaustion, but may influence complex downstream signal transduction processes. An understanding of these changes would throw light on the ways in which complex I inhibitors induce a wide range of abnormalities.

Synthesis and antitumor activities of novel 6-5 fused ring heterocycle antifolates: N-[4-[omega-(2-amino-4-substituted-6,7-dihydrocyclopenta [d]pyrimidin-5-yl)alkyl]benzoyl]-L-glutamic acids.

Novel antifolates with a 6-5 fused ring system, 6,7-dihydrocyclopenta [d]pyrimidine, (3a,b and 4a,b) were synthesized on the basis of combined modification of the heterocycle and bridge regions of the folate molecule. The synthetic method involves (1) synthesis of key intermediates of tert-butyl 4-[omega-(2-substituted-3-oxocyclopentanyl) alkyl]benzoates (8a,b and 9a,b) by a carbon-carbon radical coupling of tert-butyl 4-(omega-iodoalkyl)benzoates (7a,b) with 2-substituted-2-cyclopenten-1-ones (5 and 6) utilizing tributyltin hydride, (2) cyclization of either the methyl enol-ethers derived from the 2-cyanocyclopentanones (8a,b) or the 2-(methoxycarbonyl)cyclopentanones (9a,b) themselves by treatment with guanidine which leads to 6,7-dihydrocyclopenta [d]pyrimidines with a 4-(tert-butoxycarbonyl)phenylalkyl group (11a,b and 14a,b), (3) deprotection to the corresponding carboxylic acids (12a,b and 15a,b), and (4) amidation with diethyl glutamate and deesterification. Potent dihydrofolate reductase inhibition and highly potent cell growth inhibition were found with 2,4-diaminopyrimidine-fused cyclopentene compounds containing the trimethylene (3a) or ethylene bridge (3b) but not with the corresponding 2-amino-4-hydroxy analogs (4a,b). Compounds 3a and 3b were more growth inhibitory to several tumor cell lines (P388, colon 26, colon 38, and KB) than was methotrexate, with 3a being the most potent. Both 3a and 3b gave increases in the lifespan of P388 leukemic mice comparable to that observed with MTX. Both compounds were therapeutic against colon 26 colorectal carcinoma in mice. Compound 3a was highly effective against LC-6 non-small cell lung carcinoma in nude mice.