Irreversible enzyme inhibitors. LXXXIII. Candidate active-site-directed irreversible inhibitors of dihydrofolic reductase. VIII. Derivatives of 2,4-diaminopyrimidine. II

2,4-Diamino-6-(p-aminophenethyl)pyrimidines with a 5-phenylbutyl (XIX) and 5-(p-chlorophenyl) (VIII) substituent were synthesized by condensation of the corresponding pyrimidine-6-carboxaldehydes (XVI, X) with the Wittig reagent derived from p-nitro-benzyl bromide, followed by catalytic hydrogenation. Selective bromoacetylation of VIII and XIX afforded the candidate active-site-directed irreversible inhibitors of dihydrofolic reductase, namely, 6-(p-bromoacetamidophenethyl)-2,4-diaminopyrimidine with a 5-(p-chlorophenyl)- (IV) and 5-phenylbutyl- (III) substituents. Although III and IV were excellent reversible inhibitors of dihydrofolic reductase, neither showed any inactivation of the enzyme; in contrast, the corresponding 2-amino-6-(p-bromoacetamidophenethyl)-5-phenylbutyl-4-pyrimidinol (II) - which differs from III only in the 4-substituent (NH₂ vs. OH) - was an excellent active-site-directed irreversible inhibitor of dihydrofolic reductase, but II was a poor reversible inhibitor. Thus the conformations of II and III are most probably different when complexed to dihydrofolic reductase.     

Irreversible enzyme inhibitors. LXXXV. On the mode of pyrimidine binding of 5-alkyl and 5-arylpyrimidines to dihydrofolic reductase

A series of 5-isoamyl- and 5-(p-chlorophenyl)pyrimidines substituted with amino, alkylamino, mercapto, benzyloxy, hydroxy, or hydrogen at the 2- and 4-positions and with amino or methyl at the 6-position have been synthesized for evaluation of the mode of pyrimidine binding to dihydrofolic reductase. The studies were performed in order to determine where a bulky group could be placed on the pyrimidine ring that would still allow good binding; such studies are essential to find a suitable position for placement of a covalent forming group for design of active-site-directed irreversible inhibitors. Two classes of candidate compounds have emerged for further study as irreversible inhibitors, namely, 2-amino-4-mercapto-6-(p-bromoacetamidophenylalkyl)-pyrimidines and 2,4-diamino-6-(p-bromoacetamidophenylalkyl)aminopyrimidines having a group such as phenyl, phenylbutyl or isoamyl at the 5-position that can give strong hydrophobic bonding to the enzyme.     

Irreversible enzyme inhibitors. LXXXIX. candidate active-site-directed irreversible inhibitors of dihydrofolic reductase. X. derivatives of 2-amino-5-benzamidopropyl-4-pyrimidinol

Since 2-amino-5-benzamidopropyl-6-methyl-4-pyrimidinol (VII) was a reasonably good reversible inhibitor of dihydrofolic reductase, the benzamido group was substituted with p-bromomethyl (XVIIa), m-bromomethyl (XVIIIa), and p-bromoacetyl (XIXa) groups; these compounds, and the corresponding 6-phenylpyrimidines, were synthesized from the proper 2-amino-5-aminopropyl-4-pyrimidinol (V) by devising methods that were compatible with the high reactivity of the halogen. Compounds XVII-XIX showed in-activation of dihydrofolic reductase; the fact that p-nitrobenzyl bromide inactivated the enzyme as rapidly as XVII and XVIII and phenacyl bromide inactivated the enzyme as rapidly as XIX indicated that these inactivations proceeded by a random bimolecular mechanism and not the desired active-site-directed mechanism.     

Irreversible enzyme inhibitors. XC. candidate active-site-directed irreversible inhibitors of thymidylate synthetase. I. 2-amino-5-(benzenesulfonamidopropyl)-6-methyl-4-pyrimidinol with substituents on the benzene ring

2-Amino-5-[p- (bromoacetamidomethyl)benzenesulfonamidopropyl]-6-methyl-4-pyrimidinol (XV) was synthesized by acylation of 2-amino-5-aminopropyl-6-methyl-4-pyrimidinol (III) with p-cyanobenzenesulfonyl chloride followed by catalytic reduction and reaction of the resultant aminomethyl group with p-nitrophenyl bromoacetate. A second irreversible inhibitor of thymidylate synthetase, namely 2-amino-5-[p-(bromoacetyl)benzene-sulfonamidopropyl]-6-methyl-4-pyrimidinol (XVI), was synthesized by acylation of in with p-acetylbenzenesulfonyl chloride followed by bromination. Both XV and XVI were good reversible inhibitors of thymidylate synthetase and inactivated the enzyme when the candidate compound was incubated with the enzyme. Iodoacetamide, which does not form a complex with enzyme, could inactivate thymidylate synthetase almost as well as XV; therefore it appears that XV inactivated the enzyme by a random bimolecular mechanism rather than by the desired active-site-directed mechanism via an enzyme-inhibitor complex. Similar conclusions were reached with XVI since phenacyl bromide could inactivate the enzyme somewhat more rapidly than XVI.     

Irreversible enzyme inhibitors. XCI. candidate active-site directed irreversible inhibitors of thymidylate synthetase. II. 2-amino-6-methyl-5-(p-tolylsulfonamidopropyl)-4-pyrimidinols with N-substituents on the sulfonamide moiety

Three derivatives of 2-amino-6-methyl-5-(p-tolylsulfonamidopropyl)-4-pyrimidinol (I) with N - substituents on the sulfonamide group, namely bromoacetamidopropyl (XVI), m-bromoacetamidobenzyl (XXIVa), and p-bromoacetamidobenzyl (XXIVb), were synthesized as candidate active-site-directed irreversible inhibitors of thymidylate synthetase. The bromoacetamidopropyl derivative, (XVI), the p-bromoacetamidobenzyl derivative (XXIVb), and iodoacetamide showed irreversible inhibition of thymidylate synthetase, but XXIVa did not. Since iodoacetamide did inactivate the enzyme, but XXIVa did not, it cannot be ascertained whether XXIVb and XVI inactivate the enzyme by a random bimolecular mechanism or by the active-site-directed mechanism without evaluation of additional candidate inhibitors. Two synthetic routes were employed. The key intermediates for the bromoacetamidobenzyl sulfonamides (XXIV) were the corresponding nitrobenzyl sulfonamides (XXI); the latter were best prepared by reductive alkylation of 2-amino-5-aminopropyl-6-methyl-4-pyrimidinol (XXV) with a nitrobenzaldehyde followed by tosylation. The key intermediate for XVI was a toluenesulfonamide with a carbobenzoxyaminopropyl substituent on the nitrogen (XIV); the latter was synthesized via N-carbobenzoxy-N'-tosyl-1,3-diaminopropane (XI).     

A reinvestigation of the stevens rearrangement of 1-benzyl-1,3,4-trimethyl-1,2,5,6-tetrahydropyridinium salts

Authentic samples of 1,3,3-trimethyl-2-(3,4,5-trimethoxyphenyl)-4-methylenepiperidine (Ia) and 2-(p-chlorophenyl)-1,3,3-trimethyl-4-methylenepiperidine (Ib) are prepared by Mannich condensation between 4-methyl-1-methylamino-3-pentanone hydrochloride (VI) and an aromatic aldehyde, followed by a Wittig reaction on the resulting 4-piperidone. Comparing the physical and spectroscopic properties of Ia and Ib with those of the methylene derivatives IIa and IIb obtained as by-products in the Stevens rearrangement of 1-benzyl-1,3,4-trimethyl-1,2,5,6-tetrahydropyridinium salts IIIa and IIIb, respectively, it is shown that the assignment previously made for IIa and IIb is incorrect. Spectroscopic analysis (ir, ¹H nmr, ¹³C nmr, ms) of these compounds and of its hydrogenation products VIII allows the structural and stereochemical assignment of 11a as cis-3-isopropenyl-1,3-dimethyl-2-(3,4,5-trimethoxyphenyl)pyrrolidine and of IIb as cis-2-(p-chlorophenyl)-3-isopropenyl-1,3-dimethylpyrrolidine. The formation of these rearrangement products is mechanistically interpreted as a Stevens [3,2] type process.     

Irreversible enzyme inhibitors. LXXXI. Further observations on the mode of binding of the anilino moiety of 2-amino-5-(anilinopropyl)-6-methyl-4-pyrimidinol to dihydrofolic reductase and thymidylate synthetase

Replacement of phenyl group of 2-amino-5-(anilinopropyl)-6-methyl-4-pyrimidinol (III) by benzyl (XI) led to a large loss in binding to both dihydrofolic reductase and thymidylate synthetase; the binding by XI returned when the protonated benzylamino group was N-acetylated to XII, which removes the charge at pH 7.4 and changes the ground-state conformation of the benzene ring. Replacement of the benzyl group of the acetamide, XII, with the polar 2-, 3-, or 4-, picolyl groups also led to a loss in binding. Substitution of p-fluoro or m-trifluoromethyl on the anilino group of III, or replacement of the anilino of III by 3-pyridylamino, gave little - if any - enhancement in binding to the enzymes.     

Irreversible enzyme inhibitors. XCIV. inhibition of dihydrofolic reductase with derivatives of 2,6-diaminopurines

In order to gain additional information on the hydrophobic bonding region of dihydrofolic reductase, some derivatives of 2, 6-diaminopurine with aryl or aralkyl groups at the N⁶, C⁸ and N⁹-positions were investigated as inhibitors. Since none of the six compounds gave an increment in binding over the parent 2, 6-diaminopurine, hydrophobic bonding to dihydrofolic reductase could not be detected with this ring system. Furthermore, 2, 6-diaminopurine bridged from its 8-position with methylene groups to the amino group of p-aminobenzoic acid also failed to show an increment in binding. The 8-substituted 2, 6-diaminopurines were synthesized by base-catalyzed cyclodehydration of the appropriate 5-acylamido-2, 4, 6-triaminopyrimidines; the latter compounds were readily prepared by selective acylation of tetraaminopyrimidine.     

Diazepines I. A new synthesis of 6-phenyl-4H-imidazo[1,2-a] [1,4] benzodiazepines

6-Phenyl-4H-imidazo[1,2-a][1,4]benzodiazepines are obtained on reaction of 2-amino-7-chloro-5-phenyl-3H-1,4-benzodiazepine with α-bromoketones. In the cases of 3-bromo-2-butan-one and of 3-bromo-2-pentanone, 2-alkylimidazobenzodiazepine but not 1,2-dialkyl compound is the major product. A mechanism for the imidazole ring formation is presented.     

11. Anil-Synthese. 16. Mitteilung. Über die Herstellung von styryl-derivaten des 2-phenyl-imidazo [1,2-a]pyridins

Preparation of styryl derivatives of 2-phenyl-imidazo [1, 2-a]pyridine 2-(p-Tolyl)-imidazo [1, 2-a]pyridines and 7-methyl-2-phenyl-imidazo [1,2-a]-pyridines can be converted, in dimethylformamide, on reaction with anils of aromatic aldehydes in the presence of potassium hydroxide or potassium t-butoxide, into the corresponding 2-(stilben-4-yl)- and 2-phenyl-7-styry1-imidazo [1, 2-a]-pyridines (‘Anil-Synthesis’). The 2-(p-tolyl)-imidazo [1,2-a]pyridines react far less readily than the 7-methyl-2-phenyl-imidazo[1,2-a]pyridines.     

Chemistry of sulfonyl isocyanates and sulfonyl isothiocyanates. XI. Cyclizations with epoxides

4-Toluenesulfonyl isocyanate cyclized with 1,2-epoxy-3-phenoxypropane and 2,3-epoxypropyl 4-methoxyphenyl ether, respectively, to give 3-(4-toluenesulfonyl)-5-phenoxymethylene-2-oxazolidone ( I ) and 3-(4-toluenesulfonyl)-5-(4-methoxyphenoxymethylene)-2-oxazolidone ( II ). Compounds I and II were hydrolyzed in 2 M sodium hydroxide solution to the corresponding uncyclized hydroxy amides, VII and VIII. Compound I was remarkably stable toward 6 M hydrochloric acid and amines. Styrene oxide, 1,2-epoxybutane, 3-chloro-1,2-epoxypropane, and 1-methoxy-2-methylpropylene oxide reacted with the isocyanate to afford 3-(4-toluene-sulfonyl)-4-phenyl-2-oxazolidone (III), 3-(4-toluenesulfonyl)-4-ethyl-2-oxazolidone ( IV ), 3-(4-toluenesulfonyl)-5-chloromethyl-2-oxazolidone ( V ), and 3-(4-toluenesulfonyl)-4,4-dimethyl-5-methoxy-2-oxazolidone ( VI ), respectively. The yield of VI was constant over a temperature range of 25–90°.     

Synthesis of 3-(α-chlorophenylhydrazono)heteroarylmethyl-2-oxo-1,2-dihydroquinoxalines with antimicrobial activity

The reactions of 3-(α-chlorophenylhydrazono)hydrazinocarbonylmethyl-2-oxo-1,2-dihydroquinoxalines 4a,c with triethyl orthoesters resulted in the intramolecular cyclization to give the 3-(α-chlorophenylhydrazono-1,3,4-oxadiazol-2-ylmethyl)-2-oxo-1,2-dihydroquinoxalines 5a-d , but not the 1,2,4,5-tetrazepinylquinoxalines 6a-d . The cyclization mode into the 1,3,4-oxadiazole ring was confirmed by the alternate syntheses of 5a,c from the reactions of the 3-(1,3,4-oxadiazol-2-ylmethylene)-2-oxo-1,2,3,4-tetrahydroquinoxalines 7a,b with o-chlorobenzenediazonium chloride. Moreover, the reactions of 3-(benzimidazol-2-ylmethylene)-2-oxo-1,2,3,4-tetrahydroquinoxaline hydrochloride 8 with o-, m- and p-chlorobenzenediazonium chlorides afforded the 3-(α-chlorophenylhydrazonobenzimidazol-2-ylmethy])-2-oxo-1,2-dihydroquinoxa]ine hydrochlorides 9a-c , respectively. Compounds 5a-d and 9a-c were found to exhibit antimicrobial activities.     

Synthesis of 6-substituted pyrimidines by the wittig reaction. III . Via 2-amino-4-hydroxy-5-phenylbutyl-6-pyrimidylmethyl triphenyl phosphonium bromide

2-Amino-6-bromomethyl-5-phenylbutyl-4-pyrimidinol (IV) smoothly alkylated triphenyl phosphine, resulting in a 95% yield of the phosphonium salt (V). This Wittig reagent (V) readily condensed with p-nitrobenzaldehyde, p-nitrocinnamaldehyde, or cinnamalde-hyde in N,N-dimethylformamide to give the 6-(p-nitrostyryl) (X), 6-(p-nitrophenyl-1,3-butadien-1-yl) (VIH), and 6-(phenyl-1,3-butadien-1-yl) (IX) pyrimidinols in 72, 67 and 44% yields, respectively. Catalytic reduction of VIII and IX afforded the corresponding 6-(p-aminophenylethyl) (XII) and 6-(p-aminophenylbutyl) (XI) 4-pyrimidinols.     

Reaction of carbonyl compounds with dimethyl 3-ketoglutarate. Synthesis of bicyclic furan derivatives

Acid catalyzed reaction of cyelododecane-1,2-dione VI with dimethyl,3-ketoglutarate II led to the formation of methyl (E)-6,7,8,9,10,11,12,13-octahydro-3-(methoxycarbonyl)cyclododeca-[b]furan-2-aeetate IX, presumably formed through the dihydrodihydroxy furan VIII. This dihydrofuran intermediate VIII was shown to be characteristic of the reaction of 2-substituted carbonyl-compounds with 11 for both α-chlorocyclohexanone and α-chlorocyclopentanone furnished furans XVIa and XX, respectively, when treated with 11. The effect of dihydrodihydroxy-furan intermediates on the course of such reactions is discussed.     

Synthesis of pyrido[1,2-a]pyrimido[4,5-d]pyrimidin-5-ones. Cyclization reaction of 4-[(3-hydroxy-2-pyridyl)amino]-2-phenyl-5-pyrimidinecarboxylic acid with acetic anhydride

Treatment of 4-[(3-hydroxy-2-pyridyl)amino]-2-phenyl-5-pyrimidinecarboxylic acid (X) with acetic anhydride under refluxing conditions afforded 10-hydroxy-2-phenyl-5H-pyrido[1,2-a]-pyrimido[4,5-d]pyrimidin-5-one acetate (IX). The intermediate X was prepared from 4-chloro-2-phenyl-5-pyrimidinecarboxylic acid ethyl ester (V). The reaction of V with the sodium salt of 2-amino-3-hydroxypyridine at room temperature gave 4-(2-amino-3-pyridyloxy)-2-phenyl-5-pyrimidinecarboxylic acid ethyl ester (VI). Treatment of VI with a hot aqueous sodium hydroxide solution and subsequent acidification gave X. Involvement of 4-[(3-hydroxy-2-pyridyl)amino]-2-phenyl-5-pyrimidinecaroboxylic acid ethyl ester (VIII) (Smiles rearrangement product) as an intermediate in the above alkaline hydrolysis reaction of VI to X was demonstrated by the isolation of VIII and its subsequent conversion into X under alkaline hydrolysis conditions. Acetylation of VIII with acetic anhydride in pyridine solution gave 4-[(3-hydroxy-2-pyridyl)amino]-2-phenyl-5-pyrimidinecarboxylic acid ethyl ester acetate (XI), which afforded IX on fusion at 220°. This alternative synthesis of IX from XI supported the structural assignment of IX. Fusion of VI gave 10-hydroxy-2-phenyl-5H-pyrido[1,2-a]pyrimido]4,5-d]pyrimidin-5-one (VII). The latter was also obtained when VIII was fused at 210°. Acetylation of VII with acetic anhydride afforded IX.     

Anil-Synthese 18. Mitteilung. Über die Herstellung von Styryl-Derivaten des 3-Phenyl-benzisoxazols

Preparation of styryl derivatives of 3-phenyl-benzisoxazole 3-(p-Tolyl)-1,2-or 2, 1-benzisoxazoles and 6-methyl-3-phenyl-1,2-benzisoxazoles react with anils of aromatic aldehydes in the presence of dimethylformamide and potassium hydroxide or potassium t-butoxide to yield the corresponding 3-(stilben-4-yl)-1,2- or 2,1-benzisoxazoles and the 3-phenyl-6-styryl-1,2-benzisoxazoles respectively (‘Anil Synthesis’). Further, the Schiff's bases derived form chloroanilines and 3-(p-formylphenyl)-1,2-benzisoxazoles yield, with methyl-and p-tolyl substituted heterocyles the corresponding heterocyclic substitued styryl and stilbenyl derivatives.     

The photochemical synthesis of novel heterocyclic compounds from s-triazolo[4,3-b]pyridazine (II)

s-Triazolo[4,3-b Jpyridazine (I) photochemically reacted with dihydropyran; 2,3-dihydro-p-dioxin; 2,5-dihydrofuran; 2,5-dimethoxy-2,5-dihydrofuran; and 1,3-dioxep-5-ene to give a new series of substituted pyrrolo[1,2-b]-.s-triazoles (II-IX). In most reactions, two or more products were formed. The following compounds have been prepared from I: 9-methylene-4a,5,6,7,8a,9-hexahydropyrano[2,3 :4,5]pyrrolo[1,2-b]-s-triazole (Ha), the corresponding 9-cyanomethyl product (III), and 9-methylene-4a,7,8,8a-tetrahydro-6H,9H-pyrano[3′,2′:4,5]pyrrolo[1,2-b]-s-triazole (IIb) from dihydropyran; 9-methylene-4a,6,7,8a-tetrahydro-9H-p-dioxino[2′,3′:4,5]-pyrrolo[1,2-6]-s-triazole (IV) from 2,3-dihydro-p-dioxin; 8-methylene-4a,5,7a,8-tetrahydro-7H-furo[3′,4′:4,5]pyrrolo[1,2-b]-s-triazole (V) and the corresponding 8-cyanomethyl product (VI) from 2,5-dihydrofuran; 8-cyanomethyl-5,7-dimethoxy-4a,5,7a,8-tetrahydro-7H-furo[3′,4′:4,5]-pyrrolo[1,2-6]-s-lriazole (VII) from 2,5-dimethoxy-2,5-dihydrofuran; and 10-methylene-4a,5,9a,10-tetrahydro-9H-[1,3]dioxepino[5′,6′:4,5]pyrrolo[1,2-b]-s-triazole (VIII) and the corresponding 10-cyanomethyl product (IX) from 1,3-dioxep-5-ene. The addition of several other compounds (1,2,3,6-tetrahydropyridine, 1-acetylimidazole, 3-sulfolene, 2,3-dihydro-p-dithiin, and vinylene carbonate) was attempted, but no reactions were observed.     

Kinetics of oxidation of butane 2,3-diol by osmium(VIII)

The oxidation of butane 2,3-, propane 1,2-, ethane diol and 2-methoxy ethanol in aqueous alkaline medium by Os(VIII) has been studied. The reaction is base catalyzed and shows first-order kinetics in Os(VIII), whereas the order is less than 1 in butane 2,3-diol [BD]. The rate of oxidation is BD > propane 1,2 > ethane diol ≈ 2-methoxy ethanol. The change in ionic strength has no effect on the rate of reaction. Activation parameters ΔE, PZ, and ΔS* have been evaluated.     

The synthesis of benzofuroquinolines. I. Some benzofuro[2,3-b]quinoline and benzofuro[3,2-c]quinoline derivatives

Benzofuro[2,3-b]quinoline ( Ia ) and its 11-methyl derivative ( Ib ) were synthesized by demethylcyclization of 3-(o-methoxyphenyl)-1,2-dihydroquinolin-2-ones (VIa,b). Benzofuro[2,3-b]quinoline-11-carboxylic acid (Id) was synthesized by chlorination followed by the action of potassium hydroxide of a lactone (IX) prepared by demethyl-cyclization of 3-(o-methoxyphenyl)-2-oxo-1,2-dihydroquinoline-4-carboxylic acid (VIII). Isomeric benzofuro[3,2-c]quinoline (Ha) and its 6-methyl derivative (IIb) were synthesized by demethyl-cyclization of 3-(o-methoxyphenyl)-1,4-dihydroquinolin-4-ones (XIa,b). Both the methyl derivatives (Ib and IIb) were converted to the carboxylic acids (Id and IId) through condensation with benzaldehyde followed by oxidation. The benzofuroquinolines (Ia,b,d and IIa,b) thus obtained were oxidized to the corresponding N-oxides (IIIa,b,d and IVa,b).     

Potential cerebral perfusion agents. Synthesis and evaluation of berberine analogues

The synthesis and tissue distribution studies in rats of tetra[³H]-hydroberberine ([³H] 2 ) and 8-(p-[¹²⁵I]iodobenzyl)tetrahydroberberine ([¹²⁵I] 6 ) are described. Compound 2 was synthesized by sodium borohydride reduction of berberine hydrochloride ( 1 ). Treatment of berberine hydrochloride with p-bromo-benzylmagnesium bromide gave 8-(p-bromobenzyl)dihydroberberine ( 4 ) which after sodium borohydride reduction and iodine-125 bromine exchange gave [¹²⁵I] 6 . The unsubstituted tetrahydro compound [³H] 2 showed significantly higher brain uptake (2.2% dose/gm after 5 minutes) as compared to the corresponding 8-substituted derivative [¹²⁵I] 6 . Both radiolabeled compounds washed out from the brain relatively quickly.