Synthesis of [3H,33P]‐phosphoramide and ‐isophosphoramide mustards and metabolites [3H]‐chloroethylaziridine and ‐aziridine for studies of DNA alkylation

Reduction of diethyliminodiacetate with [³H]‐LiAlH₄ and then reaction with SOCl₂ gave bis(2‐chloro‐2‐[³H]‐ethyl)amine hydrochloride. This compound, together with [³³P]‐phosphorus oxychloride, provided for the synthesis of [³H,³³P]‐phosphoramide mustard (as its cyclohexylammonium salt) in three steps over 2 days. Similarly, 2‐[³H]‐ethanolamine was reacted with SOCl₂ to give 2‐chloro‐2‐[³H]‐ethylamine hydrochloride which, along with [³³P]‐POCl₃, was used to synthesize [³H,³³P]‐isophosphoramide mustard in two steps over 1 day. ¹H NMR studies were carried out to determine optimal times for in situ formation and storage of chloroethylaziridine and aziridine. A solution of 10 mM bis(2‐chloroethyl)amine hydrochloride in 0.1 M phosphate/D₂O, pD 7.9 at 37°C for 3 h gave chloroethylaziridine without contamination by starting material or hydrolysis products. For aziridine, the disappearance of 0.2 M 2‐chloroethylamine hydrochloride in 2 M NaOD/D₂O at pD 14, 37°C, gave k = 0.00455 min^?1 (R² = 1.000) and τ_1/2 = 2.55 h; no hydrolysis product was observed over the course of the NMR experiment (4 h). It was concluded that ～13 h (5 half‐lives) of reaction time would yield a solution of aziridine which was relatively free of contaminants. Using these reaction conditions, ³H‐labeled chloroethylamines were used to synthesize 1‐(2‐chloro‐2‐[³H]‐ethyl)‐2‐[³H]‐aziridine and 2‐[³H]‐aziridine in situ. Copyright © 2007 John Wiley & Sons, Ltd.     

Role of DNA minor groove alkylation and DNA cross-linking in the cytotoxicity of polybenzamide mustards

Interstrand DNA cross-links have been considered essential to the activity of current clinical DNA-alkylating antitumour drugs, which generally alkylate in the major groove. However, the relationship between cross-linking adducts located in the minor groove of DNA with cytotoxicity and antitumour activity has not been extensively investigated. Previous studies have shown that cross-linking ability is not correlated with cytotoxicity in a novel series of polybenzamide-linked nitrogen mustard compounds which alkylate DNA at adenines in the minor groove. In the present study the nature of these cross-linking adducts was explored for a related pair of compounds which are both highly effective cross-linkers but which differ in antitumour potential. Both of these drugs effectively interact with adenines in the minor groove, although their sequence specificity differs. However, the cross-linking event was not inhibited by pre-treatment with Hoechst 33258, although this pre-treatment effectively prevented adenine alkylation. The primary cross-links detected may thus represent guanine N7 alkylations in the major groove. Whether minor groove cross-linking adducts can be formed is uncertain, since the effect of background guanine N7 alkylation may complicate analysis. The cytotoxicity of the polybenzamides may therefore be related to other factors such as their interaction with cellular repair systems.     

1,3-Dialkyltriazenes: reactive intermediates and DNA alkylation

The reactions of calf thymus (ct) DNA with 1,3-dimethyltriazene (DMT), N-methyl-N-nitrosourea (MNU), 1,3-diethyltriazene (DET), N-ethyl-N-nitrosourea (ENU), and 1-ethyl-3-methyltriazene (MET) were studied as a function of concentration of the alkylating agents, of various buffers, and of ionic strength. The amount of alkylation at the 7- and O6-positions of guanine increased linearly with dose over a 10-fold concentration range. The slopes of the DMT and MNU curves were identical as were those of DET and ENU. These data suggest that both types of compounds alkylate DNA via a similar intermediate, presumably the corresponding alkanediazonium ion. MET methylates and ethylates DNA, the amount of each product being a function of the competitive formation of the two diazonium ions possible from MET. The MET product ratios could be reproduced by an appropriate mixture of DET and DMT. The alkylation of DNA by DMT and by MET is very sensitive to ionic strength, to the nature of the buffer, and to the identity of the salt used to balance ionic strength. In general, the reaction is favored by low ionic strength, by amine rather than oxy acid buffers, and by doubly charged inert anions. The alkylation of DNA is inversely proportional to the logarithm of the ionic strength over a wide range. The mutagenic activity of triazenes in Salmonella typhimurium is correlated very well with the ability of the triazenes to form adducts, particularly O6-guanine adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

2-β-(N-arylpiperazino) ethyl indandiones-1,3 and indandiols-1,3

Conclusion By the action of N-arylpiperazines on the tosylates of 2--hydroxyethyl-2-phenyl and 2-methylsubstituted indandiones-1,3 one obtains 2--(N-arylpiperazino) ethyl derivatives of 2-substituted indandiones-1,3 which are reduced by sodium borane to the corresponding indandiol-1,3 derivatives. The arylpiperazino derivatives of 2-substituted indandiones-1,3 and indandiols-1,3 have neurotropic properties. A certain association between structure, toxicity, and pharmacological action of these compounds was noted.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 10, pp. 25–29, October, 1968.     

Cognitive sequelae of 1,4- and 1,5-benzodiazepines

Two studies were carried out comparing the effects of clobazam (a 1,5-benzodiazepine), and clonazepam (a 1,4-benzodiazepine), with placebo, in a double-blind crossover design. Ten and nine healthy male volunteers, respectively, participated in each study. Performance on a series of automated psychological tests was assessed on four occassions: prior to any treatment; at the end of the first 2-week treatment period; following a 2-week washout phase, and again at the end of a second 2-week treatment period. Blood samples were collected at the last three occasions for analysis of drug levels. Minimal impairment in cognitive functioning was found with clobazam, only two of the more complex tasks being affected. In contrast, the pattern of findings with clonazepam suggests that it may have adverse affects on a broader range of functions. A relationship was found between performance on three measures and serum level of clobazam, such that response latency decreased with increasing serum level. In contrast, for clonazepam, a significant correlation was found on one task, in the direction of increased response latency with increasing drug level. These findings are discussed in the light of clinical literature about the use and efficacy of these two benzodiazepines, as antiepileptic agents.     

Synthesis, reactions and biological activity of some new thieno[2,3-f]-1,3-benzodioxoles.

The reaction of 7-chlorothieno[2,3-f]-1,3-benzodioxole-6-carbonyl chloride (2) with some aromatic or heterocyclic amines gave the corresponding 6-(aryl or heterocyclyl) carbamoyl-7-chlorothieno [2,3-f]-1,3-benzodioxoles (3a-c, 4a, b and 5). Compound 2 was also reacted with potassium thiocyanate, ethanol or sodium azide to afford the isothiocyanto compound 6, the ester 7 and the acid azide 9, respectively. Hydrazinolysis of 7 gave the carbohydrazide 8. The compounds 6, 8 and 9 were used as precursors in the synthesis of the target heterocycles, 7-chlorothieno[2,3-f]-1,3-benzodioxoles substituted with a variety of moieties at position-6 (10-15, 17, 19-26, 28-31). Also, 2-methyl-1,3-dixolo[5,6][1]benzothieno[2,3-c]quinolin- 6(5 H)-one (33) was prepared. The antibacterial and antifungal activities of some selected compounds were also reported.     

Preferential alkylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of guanines with guanines as neighboring bases in DNA

The base sequence of DNA has been shown to influence the kinds and amounts of alkylation of purine bases by N-methyl-N-nitrosourea [W. T. Briscoe and L-E. Cotter, Chem. Biol. Interact. 56, 321 (1985)]. In the present study, the alkylation of DNA polymers of defined sequence by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has been investigated. The assay involved treating poly (dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), and calf thymus DNA with BCNU, followed by hydrolysis to release the modified purine bases and separation and quantitation of these by HPLC. Analysis of the results revealed that there was a 24-fold increase of 7-(beta-hydroxyethyl)guanine (HOEtG) in poly(dG).poly(dC) relative to poly(dA-dG).poly(dC-dT). There was also a 3-fold increase in HOEtG in poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and calf thymus DNA relative to poly(dA-dG).poly(dC-dT). A 2- to 4-fold increase of 7(beta-aminoethyl)guanine (AmEtG) was observed in poly(dG-dC).poly(dG-dC) relative to the other polymers tested. This study has determined that guanines in certain base sequences in polydeoxyribonucleotides are more susceptible to BCNU alkylation at the N-7 position than guanines in other sequences.     

Synthesis,properties, and reactions of derivatives of 1,3-dimethyl-6-(5′-aminopyrimidylthio-6′)uracils

Derivatives of 1,3-dimethyl-6-(5′-aminopyrimidylthio-6′)uracils were synthesized and some of their properties and reactions were studied. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 1, pp. 13–16, January, 2008. Communication No. 58 in a series entitled “Studies of nitrogen-and sulfur-containing heterocyclic compounds”; for No. 57 see [1].     

Quantitation of biomarkers of exposure to nitrogen mustards in urine from rats dosed with nitrogen mustards and from an unexposed human population

The nitrogen mustards bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2), and tris(2-chloroethyl)amine (HN3) have the potential to be used as chemical terrorism agents because of their extreme vesicant properties. We modified a previously reported method to incorporate automated solid-phase extraction, improve chromatography, and include the urinary metabolite for HN3. The improved method was used to measure levels of the urinary metabolites N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA), and triethanolamine (TEA) in rats dosed with HN1, HN2, and HN3, respectively, and to establish background levels of EDEA, MDEA, and TEA in human urine samples from a population with no known exposure to nitrogen mustards. Rat dosing experiments confirmed that EDEA, MDEA, and TEA could be detected in urine for at least 48 h after exposure to HN1, HN2, and HN3, respectively. Substantial amounts of EDEA (89 ng/mL), MDEA (170 ng/mL), and TEA (1105 ng/mL) were measured in the urine of rats exposed to 10 mg HN1, HN2, and HN3, respectively, 48 h after exposure. The background concentrations for TEA in the human population ranged from below the limit of detection (LOD 3 ng/mL) to approximately 6500 ng/mL. Neither EDEA (LOD 0.4 ng/mL) nor MDEA (LOD 0.8 ng/mL) was detected above the LOD in the human samples.     

10-Piperazinyl-4H-theino[3,2-b][1,5]- and -[3,4-b][1,5]benzodiazepines as potential neuroleptics

The synthesis of 10-piperazinyl-4H-thieno[3,2-b][1,5]benzodiazepines is described. The activity of these compounds has been assessed on the basis of their ability to produce hypothermia in mice and block a conditioned avoidance response (CAR) and produce catalepsy in rats, and the results are compared with various classical and nonclassical neuroleptic drugs. A number of compounds (6, 17, 21, and 22) demonstrate potency greater than clozapine and also show low degree of catalepsy. It is believed that this profile of activity, unlike standard neuroleptics, is associated with the relative lack of extrapyramidal side effects in the clinic. The corresponding 9-piperazinyl-4H-thieno[1,4]benzodiazepines (12 and 35, limited analogues prepared in the respective series, were inactive.     

2-(Arylpiperazinyl)-1,3-indanediones and -1, 3-indanediols

By the action of N-arylpiperazines on 2-bromo-2-methyl- and 2-bromo-2-phenyl-1,3-indanediones derivatives of 2-(N-phenylpiperazinyl)-2-substituted-1,3-indanediones have been obtained, and these have been reduced with sodium borohydrite to the corresponding 1,3-indanediol derivatives. The 2-(arylpiperazinyl)-2-substituted-1,3-indanediones and the corresponding 1,3-indanediols possess marked neurotropic properties. Some connection has been established between the structure, toxicity, and pharmacological effect of these substances.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 6, pp. 9–13, June, 1967.     

Phosphorylation, "aging" and possible alkylation reactions of saligenin cyclic phosphorus esters with alpha-chymotrypsin.

Saligenin cyclic phosphonates, phosphates and N-alkylphosphoramidates readily phosphorylate α-chymotrypsin at the hydroxyl group of serine-195. The phosphoenzyme undergoes rapid further reaction (aging) both to release saligenin and form bound phenolic residues without regeneration of esteratic activity. The bound phenolics include about equal parts of trapped saligenin, possibly in the region of the active site, and material which may result from enzyme alkylation. The ratio between these types of bound phenolics is altered by pretreatment of the enzyme with ethoxyformic anhydride but not with diisopropyl-fluorophosphate. N- ε2 of the imidazole portion of histidine-57 may participate in the aging reaction by stabilizing a potential benzyl carbonium ion intermediate. This intermediate is subsequently trapped either by a hydroxyl nucleophile to produce saligenin or possibly by the stabilizing imidazole to yield N-alkylated enzyme. Trypsin with an esteratic site similar in configuration to chymotrypsin undergoes analogous phosphorylation and aging reactions.     

Enzymatic conjugation of hexachloro-1,3-butadiene with glutathione. Formation of 1-(glutathion-S-yl)-1,2,3,4,4-pentachlorobuta-1,3-diene and 1,4-bis(glutathion-S-yl)-1,2,3,4-tetrachlorobuta-1,3-diene

The glutathione-dependent metabolism of the nephrotoxin and nephrocarcinogen hexachloro-1,3-butadiene (HCBD) was investigated in subcellular fractions from rat liver and kidney. HCBD was metabolized by hepatic glutathione S-transferases to (E)- and (Z)-1-(glutathion-S-yl)-pentachlorobuta-1,3-diene (GPCB) in a ratio of 20:1, which were identified by secondary ion MS and by GC-MS after acid hydrolysis. The formation of GPCB was dependent on time and on protein and glutathione concentrations. Microsomal glutathione S-transferases from rat liver catalyzed GPCB formation more efficiently than did cytosolic glutathione S-transferases; very low rates of GPCB formation were observed in kidney subcellular fractions. GPCB is also a substrate for glutathione S-transferases and is metabolized to a diglutathione conjugate, which was identified by secondary ion MS and 13C NMR spectrometry as 1,4-bis(glutathion-S-yl)-1,2,3,4-tetrachlorobuta-1,3-diene (BTCB). BTCB formation from GPCB was dependent on time and on protein, glutathione, and GPCB concentrations. Hepatic cytosol catalyzed BTCB formation more efficiently than did hepatic microsomes; significant amounts of BTCB were also formed in kidney cytosol. Hepatic formation of glutathione S-conjugates, translocation of the S-conjugates to the kidney, and renal processing to form reactive intermediates may be the cause of HCBD-induced nephrotoxicity and, perhaps, nephrocarcinogenicity.     

4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2, 4-dichlorophenyl)pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bioavailable CRF(1) receptor antagonist

Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that compound 11i (DMP696) is a potent hCRF(1) receptor antagonist (K(i) = 1.7 nM vs 7.5 nM for alpha-hel-CRF(9-41), hCRF(1) adenylate cyclase IC(50) = 82 nM vs 286 nM for alpha-hel-CRF(9-41)). Compound 11i has excellent oral pharmacokinetic profiles in rats and dogs (37% and 50% oral bioavailabilities, respectively). This compound displays good activity in the rat situational anxiety model (MED = 3 mg/kg (po)), whereas a literature standard 1 (CP154526-1) was inactive (MED > 30 mg/kg (po)). Analogue 11i reduced stereotypical mouth movements in rhesus monkeys by 50% at 21 mg/kg (po) using the human intruder paradigm. Overall, the profile of pyrazolotriazine 11i indicates that hCRF(1) receptor antagonists may be anxiolytic agents, which have reduced motor side effect profiles.