Dopamine D3 and D4 receptor antagonists: synthesis and structure--activity relationships of (S)-(+)-N-(1-Benzyl-3-pyrrolidinyl)-5-chloro-4- [(cyclopropylcarbonyl) amino]-2-methoxybenzamide (YM-43611) and related compounds

In this study, we synthesized a series of (S)-N-(3-pyrrolidinyl)benzamide derivatives, 1, 2a-d, 5a-1, and 7, and their enantiomers, (R)-1 and (R)-5c-e, and evaluated their binding affinity for cloned dopamine D2, D3, and D4 receptors and their inhibitory activity against apomorphine-induced climbing behavior in mice. The results indicate that D2, D3, and D4 receptors have different bulk tolerance (D4 > D3 > D2) for the substituent of the 4-amino group (R1) on the benzamide nuclei and that cyclopropyl-, cyclobutyl-, and cyclopentylcarbonyl groups likely possess adequate bulkiness with respect to D3 and D4 affinity and selectivity over D2 receptors in this series. The results also suggested that the N-substituent (R2) on the pyrrolidin-3-yl group performs an important role in expressing affinity for D2, D3, and D4 receptors and selectivity among the respective subtypes. One of the compounds, (S)-(+)-N-(1-benzyl-3-pyrrolidinyl)-5-chloro-4-[(cyclopropylcarbonyl+ ++) amino]-2-methoxybenzamide (5c) (YM-43611), showed high affinity for D3 and D4 receptors (Ki values of 21 and 2.1 nM, respectively) with 110-fold D4 selectivity and 10-fold D3 preference over D2 receptors and weak or negligible affinity for representative neurotransmitter receptors. Compound 5c displayed potent antipsychotic activity in inhibiting apomorphine-induced climbing behavior in mice (ED50 value, 0.32 mg/kg sc).     

1-[(4,6-二甲基-2-嘧啶)偶氮]-2-萘酚的合成及其与钴Ⅱ的显色反应

合成了新显色剂 1 [( 4 ,6 二甲基 2 嘧啶 )偶氮 ] 2 萘酚 (DMPAN) ,并研究了该试剂的性质及其与钴显色反应的条件。结果表明 :试剂的离解常数 pka=7 4,λmax=374 0nm ,在 pH6 2 2的磷酸盐缓冲介质中 ,试剂与钴形成红色 1∶1络合物 ,其最大吸收波长λmax=42 6nm ,络合比Co2 ∶R =1∶1 ,摩尔吸光系数ε=2 66× 1 0 4 ,钴量在 0～ 1 2 μg/mL范围内遵守比尔定律 ,在掩蔽剂作用下 ,可不经分离直接用于含钴样品的分析 ,结果满意。     

(3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol: a conformationally restricted analogue of the NR2B subtype-selective NMDA antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)- 1-propanol

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606, 1) is a recently described antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. In the present study, the optimal orientation of compounds of this structural type for their receptor was explored. Tethering of the pendent methyl group of 1 to the phenolic aromatic ring via an oxygen atom prevents rotation about the central portion of the molecule. Several of the new chromanol compounds have high affinity for the racemic [3H]CP-101,606 binding site on the NMDA receptor and protect against glutamate toxicity in cultured hippocampal neurons. The new ring caused a change in the stereochemical preference of the receptor-cis (erythro) compounds had better affinity for the receptor than the trans isomers. Computational studies suggest that steric interactions between the pendent methyl group and the phenol ring in the acyclic series determine which structures can best fit the receptor. The chromanol analogue, (3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1- yl]chroman-4,7-diol (12a, CP-283,097), was found to possess potency and selectivity comparable to CP-101,606. Thus 12a is a new tool to explore the function of the NR2B-containing NMDA receptors.     

Pharmacokinetics of 1-(5-fluoro-2-pyridyl)-6-fluoro-7-(4-methyl-1- piperazinyl)-1,4-dihydro-4-oxoquinolone-3-carboxylic acid hydrochloride (DW-116), a new quinolone antibiotic in rats

The objectives of this study were to characterize the pharmacokinetics of 1-5-fluoro-2-pyridyl)-6-fluoro-7-(4-methyl-1- piperazinyl)-1,4-dihydro-4-oxoquinolone-3-carboxylic acid hydrochloride (DW-116), a newly developed quinolone antibiotic, and to compare these kinetics with those of ciprofloxacin and rufloxacin, representative quinolone antibiotics, in rats. Rats were subjected to surgery involving catheterization of the femoral vein and artery. DW-116 (4, 20, or 200 mg/kg), ciprofloxacin (20 mg/kg), or rufloxacin (20 mg/kg) was administered either intravenously (iv) or orally. Blood samples were collected at various times and subjected to an HPLC assay for the quinolones. Temporal profiles of plasma concentration after iv administrations of DW-116 at doses of 4, 20, and 200 mg/kg exhibited an apparent multiexponential decline. In the three doses examined, systemic clearance and steady-state volume of distribution of DW-116, calculated by model-independent methods, were in the range 0.17 approximately 0.23 L/h/kg and 2.90 approximately 4.44 L/kg, respectively. When DW-116 was given orally at doses of 4, 20, or 200 mg/kg, the AUC values were nearly identical to those following iv administration, indicating an almost complete absorption (i.e., the percent bioavailability was 90.0 for 4 mg/kg, 99.0 for 20 mg/kg, and 98.3 for 200 mg/kg) in the dose range examined. The absorption of DW-116 appears to be extremely rapid because the mean residence time calculated from the oral administration data was not significantly different from that for the iv administration. At a dose of 20 mg/kg, the mean residence time for iv administered ciprofloxacin and rufloxacin was smaller than that of DW-116, indicating that DW-116 remains in the body longer than the other quinolones. Absolute percent bioavailabilities of ciprofloxacin (69.9%) and rufloxacin (84.9%) were smaller than that obtained for DW-116 (99.0%). Because it has been reported that the in vivo antibacterial activity of DW-116 is comparable or superior to that of rufloxacin and ciprofloxacin, despite the fact that the in vitro activity is significantly lower, the pharmacokinetics of this antibiotic may be responsible, at least in part, for the enhanced in vivo antibacterial activity of DW-116.     

Conformational analysis of methyl 6-O-[(R)- and (S)-1-carboxyethyl]-alpha-D-galactopyranoside by MM and Langevin dynamics simulations

The conformational space of methyl 6-O-[(R)- and (S)-1-carboxyethyl]-alpha-D-galactopyranoside has been investigated. A grid search employing energy minimization at each grid point over the three major degrees of freedom, namely phi, psi and omega, identified low energy regions. The R-isomer shows five low energy conformers within ca. 1 kcal mol(-1) of the global energy minimum. The S-isomer has two conformers within a few tenths of a kcal mol(-1) of the global energy minimum. Langevin dynamics simulations have been have been performed at 300 K for 30 ns of each isomer. The phi dihedral angle has as its major conformer (g-) for the R-isomer whereas it is the (g+) conformer for the S-isomer. For the psi dihedral angle the (t) conformer has the highest population for both isomers. The dihedral angle omega has the (g+) conformer most highly populated, both for the R- and S-isomer. The above five and two conformational states for the R- and S-isomers, respectively, make up 90% in each case of the populated states during the Langevin dynamics (LD) simulations. Rate constants for the omega dihedral angle have been calculated based on a number correlation function. Three bond homo- and heteronuclear, i.e. proton and carbon-13, coupling constants have been calculated from the dynamics trajectories for comparison to experimental values. The heteronuclear coupling constant H2',C6 has been measured for the S-isomer and found to be 3.3 Hz. The J value calculated from the LD simulations, namely 2.6 Hz, is in fair agreement with experiment. A comparison to the X-ray structure of the R-isomer shows that the conformation of the crystalline compound occupies the low energy region most highly populated as a single R-conformer (30%) during the LD simulations.     

Stereochemistry of the in vitro and in vivo methylation of DNA by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and (R)- and (S)-N-nitroso-N-[2H1,3H]methyl-N-methylamine

Reaction of DNA with the carcinogens N-methyl-N-nitrosourea and N-nitroso-N,N-dimethylamine produces several methylated species including the premutagenic O6-methylguanine. The mechanism of methylation is believed to be through a methanediazonium ion. We have studied the mechanism of methylation of DNA by these carcinogens by analyzing the stereochemistry of the methyl transfer. DNA was methylated in vitro by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and in vivo by (R)- and (S)-N-[2H1,3H]methyl-N-methyl-N-nitrosamine and (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea. 7-Methylguanine, 3-methyladenine, O6-methylguanine, and the methylated phosphate backbone were isolated. The methyl groups were converted into acetic acid, and the stereochemistry was analyzed. The identity of the nucleophile did not influence the stereochemistry of the methylation reaction. It was found that the methyl group was transferred with an average of 73% inversion and 27% retention of configuration. The most likely mechanism for the retention of configuration is through multiple methylation events in which nucleophiles which initially react with the methanediazonium ion react as electrophiles with DNA.     

Structure elucidation and conformational properties of synthetic cannabinoids (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibe nzo [b,d]pyranyl)-2-hexyl-1,3-dithiolane and its methylated analog

The synthetic cannabinoid (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]+ ++pyranyl)-2-hexyl-1,3-dithiolane (AMG-3) is a cannabimimetic molecular probe with one of the highest binding affinities reported to date. Therefore, due to its potential pharmacological importance, its structure was sought to be elucidated and its conformational properties were studied using a combination of 1D, 2D NMR spectroscopy and molecular modelling. The structure of its methylated analog (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H dibenzo [b,d]pyranyl-1-methoxy)-2-hexyl-1,3 dithiolane (AMG-18), was also studied and its conformational properties were compared with AMG-3. AMG-18 lacks of the phenolic hydroxyl group a strict requirement for cannabimimetic activity and is almost devoid of any biological activity. The conformational analysis studies showed that 1',1' dithiolane ring restricted the orientation preferences of alkyl chain. This may account for the high binding affinity of AMG-3 to cananbinoid receptors. Grid scan search studies showed different preferences of possible adopting dihedral values of phenolic hydroxyl group and its methyl ether. These observations may account for their differences in biological activity.     

Synthesis and evaluation of 2-[(5-methylbenz-1-ox-4-azin-6-yl)imino]imidazoline, a potent, peripherally acting alpha 2 adrenoceptor agonist

We have synthesized 2-[(5-methylbenz-1-ox-4-azin-6-yl)imidazoline, 3, a potent, peripherally acting alpha 2 adrenoceptor agonist. The agent is conveniently prepared in five steps from 2-amino-m-cresol. The agent has demonstrated good selectivity for alpha 2 adrenoceptors in binding and functional studies. When applied topically to eyes, the agent is efficacious for the reduction of intraocular pressure. The agent does not penetrate the blood-brain barrier and, as a consequence, does not lower blood pressure or induce sedation when administered topically or intravenously. We have determined the pKa and log P in water versus both octanol and dodecane of 3 and a set of related agents. The best physical parameter to explain its lack of central nervous system penetration appears to be log P measured in octanol versus water.     

1-(2-羟基3,5-二硝基苯基)-3-[4-(苯基偶氮)苯基]-三氮烯与镍的显色反应及其分析应用

研究了新合成的 1-( 2-羟基-3,5- 二硝基苯基 )- 3-[4 -(苯基偶氮 )苯基 ] - 三氮烯 (HDNPAPT)试剂与镍的显色反应。在Na2 B4 O7 NaOH介质中 (pH10. 0～ 10. 6)及乳化剂OP存在下 ,镍与HDNPAPT形成稳定的 1:1红色络合物 ,λmax=5 45nm ,表观摩尔吸光系数ε545=1.45× 10 5,镍浓度在 0～ 8μg/2 5mL范围内符合比尔定律。拟定的新方法已应用于铝合金中微量镍的测定 ,结果满意     

Pharmacological studies on the new quinoline derivative 1-(2-methylphenyl)-4-[(3-hydroxypropyl) amino]-6-trifluoromethoxy-2,3-dihydropyrrolo[3,2-c] quinoline with potent anti-ulcer effect

The effects of the novel acylquinoline derivative, 1-(2-methylphenyl)-4-[(3-hydroxypropyl)amino]-6-trifluoromethoxy-2,3- dihydropyrrolo[3,2-c]quinoline (AU-006) on experimental ulcer models and on gastric secretion were examined. AU-006 prevented dose dependently gastric lesions induced by 95% ethanol when given orally (30-300 mg/kg). Similarly, gastric lesions caused by 0.3 N NaOH were inhibited by oral pretreatment with AU-006. To investigate the anti-ulcer mechanism of AU-006, the effect of AU-006 on gastric acid secretion was tested. Intraduodenal administration of AU-006 reduced in vivo gastric acid secretion. The protective effect of AU-006 against gastric lesions induced by ethanol was not affected by a nitric oxide synthase inhibitor, NG-nitro-L-arginine-methyl ester (l-NAME). In addition, the ethanol-induced mucus reduction was not recovered upon AU-006 administration. These results suggest that AU-006 is effective in the treatment of gastric ulcers by inhibiting gastric acid secretion, and that its activity is not related to either nitric oxide production or mucus secretion.     

(+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl-N- hydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (CMI-392), a potent dual 5-lipoxygenase inhibitor and platelet-activating factor receptor antagonist

By incorporating an N-hydroxyurea functionality onto diaryltetrahydrofurans, a novel series of compounds was investigated as dual 5-lipoxygenese (5-LO) inhibitor and platelet-activating factor (PAF) receptor antagonist. These dual functional compounds were evaluated in vitro for 5-LO inhibition in RBL cell extracts and human whole blood, and PAF receptor antagonism in a receptor binding assay. PAF-induced hemoconcentration and arachidonic acid- and TPA-induced ear edema in mice were used to determine in vivo activities. The structure-activity relationship analysis to define a preclinical lead is presented. (+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl- N-h ydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (40, CMI-392) was selected for further study. In the arachidonic acid-induced mouse ear edema model, 40 was more potent than either zileuton (a 5-LO inhibitor) or BN 50739 (a PAF receptor antagonist), and it demonstrated the same inhibitory effect as a physical combination of the latter two agents. These results suggest that a single compound which both inhibits leukotriene synthesis and blocks PAF receptor binding may provide therapeutic advantages over single-acting agents. The clinical development of compound 40 is in progress.     

The antiherpesvirus activity of H2G [(R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine] is markedly enhanced by the novel immunosuppressive agent mycophenolate mofetil

Mycophenolate mofetil (MMF) has been approved as an immunosuppressive agent in kidney transplant recipients and may thus be used concomitantly with antiherpetic agents, which are used for the treatment of intercurrent herpesvirus infections. We have recently demonstrated that MMF and its parent compound mycophenolic acid (MPA), which is a potent inhibitor of IMP dehydrogenase, potentiate the antiherpesvirus activity of acyclovir, ganciclovir, and penciclovir. We have now evaluated the antiviral efficacy of the combination of MPA and the novel antiherpesvirus agent H2G [(R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine]. When combined with H2G, MPA (at concentrations ranging from 0.25 to 10 microgram/ml, which are readily attainable in human plasma) markedly potentiated the antiviral efficacy of H2G against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), as reflected by a 10- to 150-fold decrease in the 50% effective concentration. Moreover, the activity of H2G against a thymidine kinase-deficient strain of HSV-1 (TK- HSV-1) was increased more than 2,500-fold when combined with MPA. MPA by itself had little or no effect on the replication of these viruses. Similar observations were made for varicella-zoster virus. Also, ribavirin (another inhibitor of IMP dehydrogenase) caused a marked enhancement of the activity of H2G against HSV-1 (10-fold), HSV-2 (10-fold), and TK- HSV-1 (>185-fold). Exogenously added guanosine reversed the potentiating effects of MPA on the antiviral activity of H2G, indicating that this potentiating effect resulted from a depletion of the endogenous dGTP pools, thus favoring the inhibitory action of the H2G triphosphate on the viral DNA polymerase.     

S 18126 ([2-[4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)piperazin-1-yl methyl]indan-2-yl]), a potent, selective and competitive antagonist at dopamine D4 receptors: an in vitro and in vivo comparison with L 745,870 (3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2, 3b]pyridine) and raclopride

The novel benzoindane S 18126 possessed > 100-fold higher affinity at cloned, human (h) D4 (Ki = 2.4 nM) vs. hD2 (738 nM), hD3 (2840 nM), hD1 (> 3000 nM) and hD5 (> 3000 nM) receptors and about 50 other sites, except sigma1 receptors (1.6 nM). L 745,870 similarly showed selectivity for hD4 (2.5 nM) vs. hD2 (905 nM) and hD3 (> 3000 nM) receptors. In contrast, raclopride displayed low affinity at hD4 (> 3000 nM) vs. hD2 (1.1 nM) and hD3 receptors (1.4 nM). Stimulation of [35S]-GTPgammaS binding at hD4 receptors by dopamine (DA) was blocked by S 18126 and L 745,870 with Kb values of 2.2 and 1.0 nM, respectively, whereas raclopride (> 1000 nM) was inactive. In contrast, raclopride inhibited stimulation of [35S]-GTPgammaS binding at hD2 sites by DA with a Kb of 1.4 nM, whereas S 18126 (> 1000 nM) and L 745,870 (> 1000 nM) were inactive. As concerns presynaptic dopaminergic receptors, raclopride (0.01-0.05 mg/kg s.c. ) markedly enhanced DA synthesis in mesocortical, mesolimbic and nigrostriatal dopaminergic pathways. In contrast, even high doses (2. 5-40.0 mg/kg s.c.) of S 18126 and L 745,870 were only weakly active. Similarly, raclopride (0.016 mg/kg i.v.) abolished inhibition of the firing rate of ventrotegmental dopaminergic neurons by apomorphine, whereas even high doses (0.5 mg/kg i.v.) of S 18126 and L 745,870 were only weakly active. As regards postsynaptic dopaminergic receptors, raclopride potently (0.01-0.3 mg/kg s.c.) reduced rotation elicited by quinpirole in rats with unilateral lesions of the substantia nigra, antagonized induction of hypothermia by PD 128, 907, blocked amphetamine-induced hyperlocomotion and was effective in six further models of potential antipsychotic activity. In contrast, S 18126 and L 745,870 were only weakly active in these models (5.0-> 40.0 mg/kg s.c.). In six models of extrapyramidal and motor symptoms, such as induction of catalepsy, raclopride was likewise potently active (0.01-2.0 mg/kg s.c.) whereas S 18126 and L 745,870 were only weakly active (10.0-80.0 mg/kg s.c.). In freely moving rats, raclopride (0.16 mg/kg s.c.) increased levels of DA by + 55% in dialysates of the frontal cortex. However, it also increased levels of DA in the accumbens and striatum by 70% and 75%, respectively. In contrast to raclopride, at a dose of 0.16 mg/kg s.c. , neither S 18126 nor L 745,870 modified frontal cortex levels of DA. However, at a high dose (40.0 mg/kg s.c.), S 18126 increased dialysate levels of DA (+ 85%) and noradrenaline (+ 100%), but not serotonin (+ 10%), in frontal cortex without affecting DA levels in accumbens (+ 10%) and striatum (+ 10%). In conclusion, S 18126 and L 745,870 behave as potent and selective antagonists of cloned, hD4 vs. other dopaminergic receptor types in vitro. However, their in vivo effects at high doses probably reflect residual antagonist actions at D2 (or D3) receptors. Selective blockade of D4 receptors was thus associated neither with a modification of dopaminergic transmission nor with antipsychotic (antiproductive) or extrapyramidal properties. The functional effects of selective D4 receptor blockade remain to be established.     

Orally active cephalosporins. II. Synthesis and structure-activity relationships of new 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-cephalospori ns with 1,2,3-triazole in C-3 side chain

The synthesis, antibacterial activity and oral absorbability of 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3 -(1H-1,2, 3-triazol-4-yl)thiomethylthio-3-cephem-4-carboxylic acid (1g) and related compounds are described. Their oral absorbability was influenced by the spacer length between C-3 of a cephem nucleus and C-4' of 1,2,3-triazole. The SCH2S structure was also found to contribute to their good oral absorption. The quantitative relationship between the bioavailability and the spacer length of cephalosporins (1a-1n) is discussed.     

Thiopyrano[2,3,4-cd]indoles as 5-lipoxygenase inhibitors: synthesis, biological profile, and resolution of 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5 -dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]butanoic acid

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.     

Modulation of the antitumor activity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a by O(6)-methyl-2'-deoxyguanosine--a new inhibitor of O(6)-alkylguanine-DNA-alkyltransferase

O6-Methyl-2'-deoxyguanosine (O6-MedG), a novel inhibitor of O6-alkylguanine-DNA alkyltransferase (O6-AGT), has been synthesized. The ability of O6-MedG to deplete the O6-AGT activity in leukemia L1210 and melanoma B16 cells in vivo has been studied. After intraperitoneal administration of O6-MedG to mice bearing leukemia L1210 or melanoma B16, the activity of O6-AGT in tumour cells decreased by 50%. Pretreatment of leukemia L1210 bearing mice with O6-MedG (200 mg/kg) 24 hours prior to ACNU (15 mg/kg) administration resulted in six out of seven 60-day survivors. Treatment of mice with ACNU (15 mg/kg) alone increased the life span by 200%. Treatment of melanoma B16 bearing mice with O6-MedG and 3 hours thereafter with ACNU resulted in a 50% inhibition of tumour growth, whereas the inhibiting effect of ACNU alone was 16%. There was no difference in leukemia growth when L1210/BCNU bearing mice were treated with O6-MedG followed by ACNU treatment. In vivo ACNU (15 mg/kg) produced a deep and prolonged inhibition of DNA, RNA and protein synthesis in leukemia L1210 cells. The DNA synthesis in leukemia L1210/BCNU cells was shown to recover more rapidly than in L1210 cells. The activities of DNA-polymerases alpha and beta and, especially, of O6-AGT were elevated in ACNU-resistant leukemia cells as compared with ACNU-sensitive cells. The activation of some repairing enzymes, such as O6-AGT, DNA-polymerases alpha and beta as well as increased levels of GSH may play a role in the development of drug resistance to ACNU.