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New alkylating derivatives of oligonucleotides carrying a steroid (cholesterol, testosterone or ergosterol) residue have been synthesized, the residue being introduced via its hydroxyl group into the triester oligonucleotide block in the presence of triisopropylbenzenesulphonyl chloride and N-methylimidazole. Covalent attachment of steroids to oligonucleotides increases their hydrophobicity and does not influence the melting temperature of their complementary complexes. The data obtained showed that the oligonucleotide derivatives, bearing both an alkylating group of nitrogen mustard and a steroid residue, can be used as reagents for specific modification of nucleic acids.

A series of DNA-targeted aniline mustards have been prepared, and their chemical reactivity and in vitro and in vivo cytotoxicity have been evaluated and compared with that of the corresponding simple aniline mustards. The alkylating groups were anchored to the DNA-intercalating 9-aminoacridine chromophore by an alkyl chain of fixed length attached at the mustard 4-position through a link group X, while the corresponding simple mustards possessed an electronically identical small group at this position. The link group was varied to provide a series of compounds of similar geometry but widely differing mustard reactivity. Variation in biological activity should then largely be a consequence of this varying reactivity. Rates of mustard hydrolysis in the two series related only to the electronic properties of the link group, with attachment of the intercalating chromophore having no effect. The cytotoxicities of the simple mustards correlated well with group electronic properties (with a 200-300-fold range in IC50S). The corresponding DNA-targeted mustards were much more potent (up to 100-fold), but their IC50 values varied much less with linker group electronic properties. Most of the DNA-targeted mustards showed in vivo antitumor activity, being both more active and more dose-potent than either the corresponding untargeted mustards and chlorambucil. These results show that targeting alkylating agents to DNA by attachment to DNA-affinic units may be a useful strategy.

Efficiency of the intracomplex alkylation of octadecadeoxyribonucleotide d(pC5A8C5) (target) by Rp- and Sp-individual diastereomers of the methylphosphonate octathymidylate 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide (-pNHCH2RCl) derivatives bearing an additional N-(2-hydroxyethyl)phenazinium residue (phn), viz. ClRCH2NHpTp.(TpTp)3TpNH(CH2)2NHPhn (I) and PhnNH(CH2)2NHpTp(TpTp)3TpNHCH2RCl (II) (p = -OP(O) (CH3)O-), has been investigated. Stabilisation of the complementary complex formed by the target oligonucleotide and methylphosphonate oligonucleotide derivatives by the Phn group considerably rose the efficiency of the intracomplex alkylation of the target as compared with alkylation by reagents without Phn. RP-isomeric derivatives of (I) and (II) proved to be the most effective alkylating reagents. Specificity of alkylation of nucleic acid target by reagents (I) and (II) is studied.

Methylating agents, such as streptozotocin, procarbazine, N-methyl-N-nitrosourea, dimethyl sulfate, 1,2-dimethylhydrazine, and a series of 1,2-bis(sulfonyl)-1-methylhydrazines synthesized in this laboratory, were evaluated and shown to be therapeutically active against murine models of African trypanosomiasis. At high dose levels, methylating agents halted trypanosome proliferation and transformed cells into bizarre forms containing multiple nuclei and kinetoplasts. These cells disappeared from the bloodstream of mice bearing these organisms in 48-72 h. When administered at repetitive low doses, methylating agents induced the entire population of trypanosomes to differentiate into biochemically distinct short-stumpy forms in a synchronous manner. These results suggest that methylating agents may be used as biochemical tools in the study of trypanosome differentiation.

The synthesis of three novel prodrugs, 4-[bis[2-(mesyloxy)ethyl]amino]benzoyl-L-glutamic acid (7), 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]amino]benzoyl-L-glutamic acid (8), and 4-[bis(2-chloroethyl)amino]benzoyl-L-glutamic acid (9), for use as anticancer agents, is described here. Each is a bifunctional alkylating agent in which the activating effect of the ionized carboxyl function is masked through an amide bond to the glutamic acid residue. These relatively inactive prodrugs are designed to be activated to their corresponding nitrogen alkylating agents (10, 11, and 12, respectively) at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2). The viability of two different tumor cell lines was monitored with each prodrug in the presence of CPG2. All three compounds showed substantial prodrug activity--with conversion to the corresponding active drug leading to greatly increased cytotoxicity.

A variety of organoselenium alkylating agents were synthesized, using 2-hydroxyethyl and 3-hydroxypropyl selenocyanate intermediates, and studied to determine their chemical reactivities with 4-(4-nitrobenzyl)pyridine (NBP) and cytotoxicities against CCRF-CEM, L1210/0, and L1210/L-PAM cells. The comparison between the 2-chloroethyl sulfides and selenides 1-4 revealed the markedly enhanced nucleophilicity of selenium (Se) over sulfur (S) by two or more orders of magnitude. This finding indicates that a major consideration in the design of antitumor alkylating organoselenides is the reactivity of selenium. A Taft plot of the experimental first-order rate constant, knbp, and sigma* in a series of 2-chloroethylseleno compounds gave a slope of -1.73 (rho*), with the exception of 2-chloroethyl 2-nitrophenyl selenide (10). The anomalous behavior of 10 is explained in terms of the ortho-nitro stabilization effect directly interacting with the selenium atom of ethyleneselenonium ion to form a 5-membered cyclic intermediate. In the same series, a 5000-fold difference in alkylating reactivity offered only a sixfold variation in cytotoxicity against CCRF-CEM cells. Increasing the alkylating chain length from ethlene to propylene units markedly reduced alkylating reactivities. In the CH3Se(CH2)n Cl series, 16 (n = 3) was 1.5 X 10(5) times slower than 2 (n = 2) in NBP alkylation, revealing that 3-chloro-n-propyl selenides are not chemically reactive enough to be biological alkylating agents despite the presence of the highly nucleophilic selenium atom. Replacement of chloride with mesylate in 3-substituted propyl selenides, such as 17 and 20, restored desirable reactivities and cytotoxicities.

Secondary disulfonamides and secondary tetrasulfondiamides were prepared for antineoplastic activity testing. For the disulfonamides, one alkyl series (n-butyl), three aralkyl series (benzyl, phenethyl, and phenpropyl), and one diaralkyl series (diphenpropyl) were prepared. Two series of tetrasulfondiamides were made from diamines, one in which the central methylene chain was varied from four to six and another using the tetrasubstituted sulfonyl derivative of xylylenediamine. Thirteen final compounds were synthesized. Ten of these compounds and four intermediates have not been reported previously. Preliminary screening results for the final compounds are given.

Solid tumors contain hypoxic cells which are relatively resistant to radiation treatment and to most forms of chemotherapy. These cells can be preferentially targeted using chemotherapeutic agents that are specifically activated by cellular reductase enzymes in the absence of oxygen. A new class of bioreductive alkylating agents based on the 2-nitroimidazole nucleus has been developed which contains a haloalkyl substituent on the N-1 position of the imidazole ring. Compounds of this series were readily reduced by mammalian NADPH-cytochrome c reductase, and reduction led to the production of an electrophilic center. This reactive component was hypothesized to be responsible for the preferential cytotoxicity of the agents of this class to hypoxic tumor cells through alkylation of cellular components.

A number of 2-chloromethyl and 2-bromomethyl derivatives of naphthoquinones, quinolinediones, and naphthazarins were designed and synthesized as potential bioreductive alkylating agents, and the antitumor activity of these compounds was assessed in mice bearing Sarcoma 180 ascites cells. The results indicated that, with the exception of 3-benzamido-2-chloromethyl-1,4-naphthoquinone, which was inactive, all newly synthesized naphthoquinones possessed strong antitumor activity against this neoplasm. 6,7-Bis(bromomethyl)quinoline-5,8-dione had moderate inhibitory activity against Sarcoma 180 at its optimal daily dosage level of 15 mg/kg. 3-Bromo-2-bromomethyl- and 3-bromo-2-chloromethylnaphthazarin produced a moderate extension of the life span of tumor-bearing mice; whereas, in contrast, 6,7-dimethyl analogs of these agents were inactive when employed in daily doses up to 40 mg/kg body weight.

Oligo-heptathymidylates covalently linked to porphyrins bind to complementary sequences and can induce local damages on the target molecule. In dark reactions, iron porphyrin derivatives exhibited various chemical reactivities resulting in base oxidation, crosslinking and chain scission reactions. Reactions induced by reductants, such as ascorbic acid, dithiothreitol or mercapto-propionic acid, led to very localised reactions. A single base was the target for more than 50% of the damages. Oxidising agents such as H2O2 and its alkyl derivatives induced reactions that extended to a wider range of altered bases. The specificity of the chemical modifications observed in these systems is discussed from a mechanistic point of view.

Chlornaltrexamine (CNA) produces ultralong-lasting (3--6 days) narcotic antagonism in mice and persistent stereospecific binding to rat-brain homogenate. Protection studies in mice suggest that CNA mediates its narcotic antagonist effects by interacting with the same receptors that are occupied by naloxone. A single icv dose of CNA also has been found to inhibit the development of physical dependence in mice for at least 3 days. These studies suggest that CNA exerts its sustained effects by selective covalent association with opioid receptors.

Examples of a new class of alkylating agents, selenium mustards, were prepared for study of their chemical kinetic properties and cytotoxicities against human lymphoblastoid CCRF-CEM cells. In a series of para-substituted aryl 2-chloroethyl selenides, a linear free energy relationship between the first-order rate constant, k'nbp and sigma p gave a rho value of -1.3, indicating that formation of a cyclic ethylene selenonium ion is the rate-controlling step for alkylation of 4-(4-nitrobenzyl)pyridine (NBP). Consistent with the ethyleneselenonium ion pathway, rates of solvolyses were extremely sensitive to increasing water content, and a positive correlation was found between reactivity with NBP and nucleophilic selectivity (Swain-Scott s constant). The s constant, which predicts for variation in intracellular product spread, varied from 0.53 up to 0.95, equal to aliphatic nitrogen mustards. Alkylating activities based on extent of NBP alkylation, however, showed relatively low values, 8-23% of that of mechlorethamine, possibly due to hydrolysis occurring by a separate pathway from nucleophilic substitution. Reactivities and nucleophilic selectivities both showed positive correlations with cytotoxicities, suggesting that the rate and extent of alkylation of relatively strong nucleophilic centers mediate the biologic effects of these compounds. Two bifunctional selenium mustards were substantially more cytotoxic than monofunctional aromatic selenides. No additional cytotoxicity due to the selenium atom was observed, with the exception of diselenide (-SeSe-) compounds. Thus, selenium alkylating agents kinetically and biologically resemble classical, mustard-type alkylating agents.

Affinity-labeling reagents are useful for the inactivation of proteases in vivo but are apparently limited in application by the possibility of side reactions. In addition, increased specificity would be desirable. Substrate-derived chloromethyl ketones, one class of protease inhibitors, have been examined with the hope that replacement of the departing group halogen by other substituents might lead to improved inhibitor characteristics. Analogues of Z-Phe-Ch2X were synthesized in which X is a sulfonate or carboxylate substituent and examined as inactivators of chymotrypsin. The sulfonate esters were found to be more reactive than the previously studied halogen derivatives.

A series of 3-amino-4-(p-aminophenyl)isoquinolines bearing the bis(2-chloroethyl)amino group was synthesized as potential CNS antitumor agents. Diol precursors 1e and 1f were prepared by the treatment of 1b and 1c with ethylene oxide. Diol precursors 5a-c and 9 were prepared by the treatment of 4a-c and 8 with diethanolamine. The reaction of these diols with SOCl2 yielded target mustards 10-15 which were evaluated in the intraperitoneal murine L1210 tumor. No intermediates or target mustards were active in this tumor system.

3,3'-Diacetoxy-alpha, beta-dialkylstilbenes (alkyl = CH3 to C4H9, 1-4) 3,3'-dihydroxy-alpha, beta-diethylstilbene (5), and their corresponding stilbene oxides (6-10) were synthesized. Compounds 1-10 competitively antagonized in vitro the interaction of [3H]estradiol with its receptor. 3,3'-Diacetoxy-alpha, beta-diethylstilbene (2), 3,3'-diacetoxy-alpha, beta-diethylstilbene oxide (7), and their phenolic analogues (5 and 10) were most effective. Shortening or lengthening the alkyl side chains led to a decrease in receptor affinity. Among the stilbenes and epoxides, those with C2H5 and C3H7 groups (2, 3, 5 and 7, 8, 10) caused the strongest inhibition of the growth of a hormone-dependent postmenopausal human mammary carcinoma serially implanted in nude mice. The strong antitumor activity of 5 and 10 was confirmed by experiments on the 9,10-dimethyl-1,2-benzanthracene-induced, hormone-dependent mammary carcinoma of the Sprague-Dawley rat.

N-Nitroso compounds may well rank high among the genotoxic carcinogens present in our environment. Small amounts of such compounds may be formed in the human stomach after consumption of high-nitrate vegetables. Volatile nitrosamines can be conveniently determined but reliable methods of analysis for non-volatile N-nitroso compounds are still lacking. In this study we have used the 4-(4-nitrobenzyl)pyridine test to look for the formation of alkylating compounds such as N-nitroso-N-methylurea in a wide range of food products after incubation with nitrite under simulated gastric conditions. Our results indicate that many food products do not form alkylating compounds in appreciable amounts, even though the nitrite concentration used was five to ten times that found in saliva after a high-nitrate meal. Comparatively strong alkylating activity, however, was detected after incubation of samples of sauerkraut, certain dairy products (yoghurt, biogarde, quark, buttermilk and milk), wine and smoked mackerel. Samples of sauerkraut incubated with simulated gastric fluid, but without (added) nitrite, also displayed appreciable alkylating activity. The formation of alkylating substances in non-fat yoghurt was markedly inhibited by addition of ascorbic acid. The identity of the alkylating agents remains unknown. The isolation procedure was optimized for N-nitroso-N-methylurea, but will certainly result in the isolation of other compounds, such as C-nitroso-, C-nitro- or perhaps even C-nitroso-C'-nitro-compounds as well. Biogenic amines, glucosinolates, indole derivatives or other compounds may be involved as precursors. If alkylating agents are formed in vivo after ingestion of high-nitrate vegetables or drinking water, this is likely to occur only when the food products mentioned above are ingested simultaneously with or shortly after the nitrate load and not appreciably (except perhaps in the case of sauerkraut) when they are ingested alone, without a nitrate source. The health implications of these findings cannot yet be established. Many alkylating agents, however, have strong carcinogenic properties and continued investigation of food products (and their interaction products with nitrite) is indicated.

A model to produce a new type of alkylating agents having anti-cancer activity is described. They consist of a halomethyl-azol group of the benzyl type as alkylating center, and a sugar moiety as a carrier fragment. Alkylating nucleosides were produced as halomethyl-pyrazole, -imidazole, 1.2.3-triazole, and 1.2.4-triazole derivatives following the model, and tested upon Ehrlich ascites cancer and P-388 lymphocytic leukemia.

Two alkylating glucocorticoids have been synthesized in order to test the possibility of alkylating glucocorticoid receptors. The title compounds are 9alpha-fluoro-11beta,16alpha,17alpha,21-tetrahydroxypregna-1,4-diene-3,20-dione 21-[bis(2-chloroethyl)carbamate] 16,17-acetonide (I) and 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione 21-[bis(2-chloroethyl)carbamate] (II), prepared from triamcinolone acetonide and cortisol, respectively, through the reaction of the C-21 hydroxyl group with phosgene and di-2-chloroethylamine in the presence of triethylamine. Both compounds are biologically active as inhibitors of the growth of cultured mouse fibroblasts and are able to compete for the specific binding of radiolabeled triamcinolone acetonide to the L929 cell receptor. The bis(2-chloroethyl)carbamate moiety is capable of reacting with nucleophilic groups as evidenced by the colorimetric reaction with 4-(p-nitrobenzyl)pyridine. Both the interaction with the receptor and inhibition of cell growth by these two glucocorticoids are reversible.

6-Ethynyluracil (3) was prepared by two different synthetic procedures. In one approach, 6-formyluracil was reacted with (dibromomethylene)triphenylphosphorane to give 6-(2,2-dibromovinyl)uracil (2), which was silylated and treated with phenyllithium to yield 3. Alternatively, silylated 6-iodouracil was reacted with trimethylsilylacetylene in dry triethylamine in the presence of a palladium/copper catalyst to give 6-[(trimethylsilyl)ethynyl]uracil (5). Compound 5 was converted to 3 in refluxing methanol. At neutral pH, 3 reacted with thiols, such as glutathione, 2-mercaptoethanol, and L-cysteine, but did not react with glycine or L-lysine. This reaction was accompanied by a shift in the UV maximum of 3 from 286 nm to 321-325 nm. The reaction of 3 with 2-mercaptoethanol gave cis-6-[2[(2-hydroxyethyl)-thio]vinyl]uracil as the predominant product. Compounds 2 and 3 inhibited the growth of leukemia L1210, B-16 melanoma, and lewis lung carcinoma cells at concentrations ranging from 1 x 10(-6) to 2 x 10(-5) M. As determined with L1210 cells, the inhibition of growth caused by 2 and 3 was not prevented by the natural pyrimidines, indicating that the agents do not act as antimetabolites.

Three general methods for the synthesis of derivatives of the bis-alkylating agent, myleran [1,4-bis(methane-sulfonoxy)butane (1)], are presented. These derivatives contain a lipophilic group attached to the 2 position of 1,4-bis(methanesulfonoxy)butane by means of a sulfonyl function. As examples of the scope of the methods, the synthesis of seven such derivatives (12a-g) is presented. All seven were inactive against L1210 and P388 leukemia in the mouse.

Several 1,2-bis(arylsulfonyl)-1-methylhydrazines were synthesized and evaluated for antineoplastic activity against the L1210 leukemia. The most active compound to emerge from this study, 2-[(4-chlorophenyl)sulfonyl]-1-methyl-1-(4-tolylsulfonyl)hydrazine , increased the survival time of tumor-bearing mice by 88%. The alkylating activity of the synthesized analogues and several compounds reported earlier was determined by measuring the absorbance at 540 nm of the alkylated product of 4-(4-nitrobenzyl)pyridine. The results obtained support the concept that the ability to alkylate is a necessary but not a sufficient condition for the expression of antitumor activity by agents of this class.

In an effort to develop opiate receptor-site-directed alkylating agents, a series of 3-hydroxy-17-aralkylmorphinans containing reactive groups was synthesized and tested for analgesic and opiate antagonist activity. Many of the target compounds exhibited the characteristics of agonists and, among this group, some were found to be active blockers of morphine analgesia. One of the more potent antagonists (41) was investigated further and it was found that while its action is specifically associated with opiate receptors, 41 could not be classified either as a competitive or noncompetitive antagonist in the classical sense. The duration of antagonist action in vivo of 41 and its in vitro receptor binding characteristics suggest that covalent association with opiate receptors is not not an important factor.

The synthesis, structure and biological activities of a series of derivatives of normorphine, noracetylmethadol (IV a), 6-amino-4,4-diphenyl-3-heptanol acetate (V a), and norpropoxyphene (VI a), in which the corresponding nitrogen supports an alkylating group (chloroethyl or fumaroyl) are reported. Structural identification was achieved spectroscopically. 13C Nuclear Magnetic Resonance proved the most useful tool in this task. N-Chloroethylnoracetylmethadol (IV c), acted as a potent long-lasting analgesic. Although some compounds (IV c), (V c) and (V d) showed substantial cytostatic activity, no antineoplastic activity in mice with P388 leukaemia was detected in the series.

Functional groups such as nitro, chloro, bromo, and methoxy were introduced in the meta and para positions of the phenyl ring of the antineoplastic agent 2-chloromethyl-3-phenyl-1,4-naphthoquinone. Tests for tumor-inhibitory potency of these derivatives against Sarcoma 180 ascites cells in mice indicated that the para-substituted methoxyphenyl, chlorophenyl, and bromophenyl derivatives possessed antitumor activity comparable to that of the parent compound 2-chloromethyl-3-phenyl-1,4-naphthoquinone, whereas meta-substituted nitro and bromo derivatives were either inactive or only weakly active anticancer agents in this system.

Maleimidohippurates and maleimidobenzoates were synthesized that possess a carboxy group for active uptake into renal proximal tubular cells and a reactive maleimide moiety to covalently bond with proximal tubular components. The reactivity of the maleimide moiety in each series was progressively reduced by substitution of methyl groups in place of the vinyl hydrogens. In contrast to N-ethylmaleimide (NEM), the resulting maleimidohippurates and maleimidobenzoates did not possess significant diuretic activity in the dog following renal arterial administration. However, as predicted, the nephrotoxicity of the maleimidohippurates paralleled their in vitro alkylating ability and was quite specifically located in the proximal portion of the canine renal tubule.

Synthetic studies on new antitumor drugs in Japan are mainly oriented toward analogs of known active structures. In the nitrosourea area ACNU, a pyrimidine analog of CCNU, is currently under clinical investigation and has myelosuppression as a side effect Nitrosoureas with a sugar moiety are of great interest and two compounds called GANU and MCNU are now ready for phase I study in Japan. Among new alkylating agents are included a series of bis-methanesulfonate of aminoglycols, an analog of cyclophosphamide called 4-hydroperoxy-cyclophosphamide, and aziridine derivatives which include the now commercially available carbazilquinone. In the antimetabolite field cyclocytidine is an analog of arabinosyl cytosine which has been extensively studied. A newer analog called N4-behenoyl-cytosine arabinoside is now being studied experimentally. In the fluorinated pyrimidine area Ftorafur has been extensively studied and a new compound FD-1 is of interest.

A number of simple silyl enol ethers and vinyl trifluoromethanesulfonates, a relatively new class of organic compounds capable of undergoing alkylation by a nucleophilic addition-elimination process, were evaluated in the P388 lymphocytic leukemia system. No activity (ILS = 8-22%) was observed in the simple vinyl derivatives. Some activity (ILS = 20-42%) was observed for a series of siloxy and sulfonate (CH3SO2 and CF3SO3) functionalized alpha-methylene lactone systems. The enhanced activity of the functionalized systems over the parent methylene lactone is ascribed to a possible irreversible alkylation by cellular nucleophiles via a nucleophilic addition-elimination process.

New alkylating ligands derived from indole with high affinity for beta-adrenoceptors were synthesized and their properties examined. N8-(Bromoacetyl)-N1-[3-(4-indolyloxy)-2-hydroxypropyl]-(Z)-1,8-dia mino-p- menthane (8) and its N1,N8 isomer (9) were prepared by the reaction of bromoacetyl bromide with a product of the condensation of 4-indolyl glycidyl ether with (Z)-1,8-diamino-p-menthane. A similar reaction employing 2-cyano-4-indolyl glycidyl ether yielded the respective cyano derivatives 10 and 11. Apparent affinities (Ki, M) for beta-adrenoceptors on membrane preparations from rat heart and lung were 4.6 X 10(-10) and 1.34 X 10(-9) for 8, 2.3 X 10(-8) and 4.5 X 10(-9) for 9, 6.1 X 10(-10) and 1.49 X 10(-9) for 10, and 1.83 X 10(-9) and 2.78 X 10(-9) for 11, respectively. When membranes were preincubated with the above ligands (1 X 10(-8) M, 30 min, 30 degrees C) and then washed extensively, reduction in the concentration of specific binding sites of [3H]dihydroalprenolol ranged from 7% to 76% and there was no change in KD of the remaining binding sites. (+/-)-Alprenolol and (-)-isoproterenol, but not (+)-isoproterenol, when included with the alkylating ligands in the preincubation mixtures, prevented the reduction in concentration of [3H]dihydroalprenolol binding sites. Compounds 8-11 alone did not stimulate adenylate cyclase activity in rat heart homogenates. However, these compounds inhibited (-)-isoproterenol-stimulated adenylate cyclase activity with Ki values ranging between 5 X 10(-9) and 60 X 10(-9) M. These results suggest that high-affinity irreversible beta-adrenergic antagonists were obtained that may be useful for in vivo studies of beta-adrenoceptors.

5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.

The synthesis of succinimide derivatives in which alkylating groups have been attached to the imide nitrogen or to the 3 position of the ring is described. The synthesis of one bis-alkylating derivative 19 is also described. The alkylating groups used were (a) alpha-haloacetyl, (b) alpha-haloacetamido, (c) maleamyl, and (d) maleimido. These compounds were prepared as potential long-acting anticonvulsants. None of the compounds showed activity against maximal electroshock or metrazole-induced seizures.

A series of chemically reactive derivatives of the nonsteroidal estrogen hexestrol have been synthesized as potential affinity labels for the estrogen receptor or as cytotoxic agents with selective activity against receptor-containing cells. These compounds are hexestrol ethers with halo ketone, halohydrin, or epoxide functions or 4-substituted deoxyhexestrols with halo ketone, benzyl halide, nitro, azide, sulfonyl fluoride, or sulfonyl azide groups. The alkylating activity of the electrophilic derivatives was measured using the colorimetric reagent nitrobenzylpyridine, the bromo derivatives being considerably more reactive than the chloro ones. Their reversible binding to the lamb uterine estrogen receptor was measured by competitive binding assays, and their irreversible reaction with receptor was measured by exchange assays that determine the rate and extent of receptor inactivation. In general, monoetherification of hexestrol or substitution of deoxyhexestrol produces compounds with relatively low affinity for the estrogen receptor (0.3-10% that of estradiol). Most of the electrophilic derivatives are rapid and effective inactivators of receptor (24-70% inactivation within 0.5-5 h at 25 degrees C). Of the photosensitive derivatives, 4-azidodeoxyhexestrol appears to be the most efficient receptor inactivator (49%). The high reactivity of these compounds toward the estrogen receptor and the lack of interference by their reaction with other cellular nucleophiles suggest that these compounds may be useful as affinity-labeling agents or as selective cytotoxic agents in intact systems.

Several potentially bis(alkylating) bis(quinones) (3-5) and 1,4- and 1,3-bis(alkylating) monoquinones (6-13) belonging to general structure 2,2'-ethylenebis[5-[(leaving group)methyl]-1,4-benzoquinone] (3-5) and 2,5- and 2,6-bis[(leaving group)methyl]-1,4-benzoquinone water-soluble and -insoluble classes were prepared by oxidative demethylation of the corresponding tetramethoxydiphenylethanes (17-19) and dimethoxybenzenes (24, 27, 36-39), respectively. Methods employed for the preparation of tetramethoxydiphenylethane intermediates involved (1) arylmethyl bromide coupling and (2) catalytic hydrogenation of stilbene intermediates derived via Wittig reaction of (arylmethyl)phosphonium salts with aryl aldehydes. However, in biological investigations using a subcutaneous B16 (hypoxic) melanoma tumor in BDF1 hybrid mice with cyclophosphamide as positive control the most interesting series of structurally related analogues were the potentially monoalkylating monoquinones of the 2-[(leaving group)methyl]-1,4-benzoquinone type (i.e., 14 and 15) having water-insoluble (acetoxy) and water-solubilizing (succinate) groups. Serial measurements of tumor size, and evaluation of increased life span, in response to drug treatment also revealed potentially 1,4-bis(alkylating) (bromomethyl)-1,4-quinone 7 and 1,3-bis(alkylating) (hydroxymethyl)-1,4-quinone 10 to have variable activity, but none of the potentially bis(alkylating) bis(quinones) showed antitumor properties in this model.

A new group of steroidal alkylating agents has been synthesized for potential anticancer activity. The compounds contain a sulfonic ester, a mono- or a bifunctional nitrogen mustard function attached to an ethyl chain and forming ether linkages at the 3- or 17 beta-position of the steroids selected. The products were tested for their in vitro anabolic and catabolic properties by measuring their effects on the bovine pancreatic ribonuclease.

N-[4-[(2-Chloroethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidone (3) and N-[4-[(2-bromoethyl)methylamino]-2-butynyl]-5-methyl-2- pyrrolidone (4) were synthesized. Compounds 3 and 4 cyclized in neutral aqueous solution to an aziridinium ion (4A). The rate constants for the cyclization of 3 and 4 at 37 degrees C were 0.025 and 0.89 min-1, respectively. The aziridinium ion was equipotent with carbachol as a muscarinic agonist on the isolated guinea pig ileum. It was more potent than the corresponding 2-pyrrolidone derivative (2A) in alkylating muscarinic receptors in homogenates of the rat cerebral cortex. This higher potency was due to greater receptor affinity of 4A as compared to 2A rather than to greater rate constant for alkylation of muscarinic receptors. These properties of 3 and 4 and their low toxicity should make them valuable tools for receptor inactivation studies in vivo and in vitro.

Several 5-diethylaminomethyl derivatives and nitrogen mustards of uracil and 2-thiouracil have been synthesized and tested for their potential anticancer activity in vitro on KB cells and in vivo on Ehrlich carcinoma. Among the alkylating derivatives tested several showed cytotoxic activity in vitro and compound V [5-[bis(2-chloroethyl) amino] methyl-6-propyluracil hydrochloride] showed both in vitro and in vivo anticancer activity.

A number of derivatives of 2-amino-2-deoxysaccharides and 1-methylamino-1-deoxypolyols acylated by cytotoxic phenylalkanic and amino acids were synthesized and experimentally tested for antitumor activity. Some compounds showed a high antitumor activity against plasmacytoma MOPC-406: a 2-fold increase in experimental animals survival and a 80--100% cure rate were observed. Physicochemical properties, particularly, stability in aqueous solution and antitumor activity of one of the most potent compounds--1-methylamino-1-deoxy-1-N[p-di(2-chloroethyl) aminophenylacetyl]-D-glucitol (Agluphen)--are described in detail.

Hypoxic cells of solid tumors are an obstacle to effective cancer therapy. Since hypoxic cells remote from the tumor blood supply may have a greater capacity for reductive reactions than well-oxygenated cells, we have prepared a series of anthraquinone prodrugs which may be capable of generating a reactive quinonemethide species following enzymatic reduction to the hydroquinone and loss of the substituent on the methylene group in the 2 position. The synthesized 2-methyl-substituted anthraquinone derivatives have first half-wave reduction potentials of -0.52 to -0.56 V at pH 7.0, which are the lowest oxidation-reduction potentials of quinone bioreductive alkylating agents synthesized by this laboratory to date. Tests of the cytotoxicity of these agents to oxygenated and chronically hypoxic EMT6 tumor cells in culture demonstrated that 2-(hydroxymethyl)anthraquinone, 2-[(N-methylcarbamyl)-methyl]anthraquinone, 2-[[(p-toluenesulfonyl)oxy]methyl]anthraquinone, and 2-(methoxymethyl)anthraquinone were significantly more toxic to hypoxic cells than to their normally aerated counterparts. The findings demonstrate differences between various leaving groups in the 2 position for the expression of differential cytotoxicity.

The writers report the results of synthesis and experimental studies of 2-amino-2-desoxy-1, 3, 4, 6-tetraacetyl-D-glucopyranose derivatives acylated by n-di-/2-chloroethyl/aminophenyl acetic acid (chlorophenacyl), n-di-/2-chloroethyl/aminophenyl-N-acetyl-DL-alanine (acetylsarcolysin) and n-di-/2-chloroethyl/aminophenyl-N-acetyl-DL-alanyl-DL-valine (acetylsarcolysin valine). Studies on the antitumor activity were carried out on mice with 8 different transplantable tumors. The addition of glucosamine to alkylating substances containing biologically active components was found to change their properties. The new type derivatives are watersoluble, show less molar toxicity and mainly high activity on plasmocytoma MOPC-406 (from 97% of the survival rate to 50% of cure).