Characterization of products formed by the reaction of 14C-sulphamethazinediazonium tetrafluoroborate with natural products

1. When bovine serum albumin (BSA) was incubated with 4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphonamido] [U-14C]benzenediazonium tetrafluoroborate (14C-SDTFB) in vitro approx. half of the 14C-activity was bound (14C-BSA). Cysteine, N-ethylmaleimide, p-chloromercuribenzoate and iodoacetamide inhibited the formation of 14C-BSA. 2. When SDTFB was reacted with cysteine four major products were formed. These were identified as 3-(4-[N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamido] diazothio)-2-aminopropionic acid (cys-SDAS), 3-(4-[4,6-dimethyl-2-pyrimidinyl) benzenesulphonamido]thio)-2-aminopropionic acid (cys-Sulmet), 4-hydroxy-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (hydroxy-Sulmet) and N-(4,6-dimethyl-2-pyrimidinyl)benzenesulphonamide (desamino-Sulmet). Diazosulphides were also formed when SDTFB was incubated with thiophenol and glutathione. 3. The diazosulphides reacted with N,N-dimethylaniline (DMA) and 2-naphthol to yield diazo compounds in 22-29% yield; when 14C-BSA was reacted with DMA under the same conditions, a diazo compound was formed-but only in 2% yield. 4. Cys-SDAS when incubated overnight (approx. 16 h) in aqueous solutions (pH 3, 5 and 8) decomposed to yield desamino-Sulmet (30-39%), hydroxy-Sulmet (13-21%), and other unidentified soluble products (24-36%); when 14C-BSA was incubated under the same conditions only 3-4% of the 14C became dissociated from BSA and only a trace amount of desamino-Sulmet was formed. 5. When 14C-SDTFB was incubated with calf thymus DNA at pH3, some of the 14C became associated with the DNA (14C-DNA). However, most of the 14C became dissociated from 14C-DNA when the latter was incubated overnight in aqueous solutions; a minor dissociation product was identified as 14C-desamino-Sulmet.     

Evidence for diazotization of 14C-sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] in swine. The effect of nitrite

Dietary nitrite greatly enhanced the conversion of orally administered 14C-sulfamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide; 14C-sulmet) to 14C-desaminosulfamethazine [N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide; 14C-DA-sulmet] in swine. The disposition of 14C orally administered to swine as 14C-sulfamethazinediazonium tetrafluoroborate (4-[N-(4,6-dimethyl-2-pyrimidinyl)sulfonamido] [U-14C]diazonium tetrafluoroborate) was very similar to the disposition of 14C given to swine as 14C-sulmet in combination with nitrite. These results and other information discussed in the text provide evidence that 14C-sulmet, in the presence of nitrite under the acid conditions in the gastrointestinal tract, was diazotized and that this diazonium intermediate was converted to 14C-DA-sulmet and other unidentified 14C-labeled products.     

In vitro formation of a triazene compound by reaction of sulphadimidine and nitrite

The interaction between the veterinary drug sodium sulphadimidine and nitrite has been studied under acid conditions and the formation of 1,3-di-(4-[N-(4,6-dimethyl-2-pyrimidinyl)sulphamoylphenyl)triazene (DDPSPT) was demonstrated. This compound was not mutagenic when tested on Salmonella typhimurium and Drosophila melanogaster. In addition to the formation of DDPSPT, desaminosulphadimidine was identified as a minor reaction product.     

The isolation and identification of 14C-sulfamethazine (4-amino-n-(4,6-dimethyl-2-pyrimidinyl)[14C]benzenesulfonamide) metabolites in the tissues and excreta of swine

Pigs were given a single oral dose of 14C-sulfamethazine (4-amino-N(I4,6-dimethyl-2-pyrimidinyl)[14C]benzenesulfonamide). Approximately 78% of the 14C was eliminated in the urine and 18% was eliminated in the feces within 192 hr after dosing. The percentage of the 14C remaining in the animals after dosing was as follows: 6 hr, 88%; 24 hr, 49%; 48 hr, 14%; 192 hr, less than 1%. The 14C-labeled compounds in the tissues and excreta were isolated by solvent extraction and by conventional and high-pressure liquid chromatography, and then derivatized and characterized by infrared and mass-spectral analysis. Chemical structures were confirmed by synthesis. The major 14C-labeled compounds in the skeletal muscle, liver and kidney were identified as sulfamethazine, N4-acetylsulfamethazine, the N4-glucose conjugate of sulfamethazine, and N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide (desaminosulfamethazine). The major 14C-labeled compounds in the urine and feces were identified as sulfamethazine and N4-acetylsulfamethazine.     

Formation of a diazonium cation intermediate in the metabolism of sulphamethazine to desaminosulphamethazine in the rat

14C-Sulphamethazinediazonium tetrafluoroborate (14C-SDTFB) when orally administered to rats was converted primarily to 14C-labelled desaminosulphamethazine (desaminosulmet) and methanol-insoluble residues in the gastrointestinal tract (gut). 14C-labelled sulphamethazine (sulmet), N4-acetylsulmet, the N4-glucose conjugate of sulmet and other unidentified products were also observed in the tissues and urine of rats given 14C-SDTFB. 2. When 14C-sulmet, nitrite and dimethylaniline were simultaneously administered to a rat by the oral route, one of the 14C-labelled products formed in the stomach was isolated and identified as 4-dimethylaminophenyl [4-(N-4,6-dimethyl-2-pyrimidinyl)sulphamidophenyl] diazene, providing evidence that 14C-sulmet was diazotized in the stomach of the animal. 3. SDTFB was weakly mutagenic when evaluated by the Ames test. 4. The methanol-insoluble 14C-labelled residues in the gut of rats dosed orally with 14C-SDTFB and 14C-sulmet + nitrite were partially converted to 14C-labelled desaminosulmet, sulmet, N4-acetylsulmet and other unidentified products when fed to recipient rats.     

The disposition of N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide in the rat

Rats given a single oral dose of N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide (14C-DAS) excreted 64.2% of the 14C in the urine and 22.4% in the feces within 96 hr. Compounds accounting for 86% of the 14C in the 0-24-hr urine were isolated by a variety of chromatographic techniques and identified by IR, NMR, and MS analysis. Approximately 4% of the 14C in the urine was the parent compound. The structures of 14C-metabolites in the urine indicated that 14C-DAS was metabolized by at least three pathways which included: 1) hydroxylation and glucuronic acid conjugation at the 4-position of the benzene ring; 2) hydroxylation, and sulfate ester and glucuronic acid conjugation at the 5-position on the heterocyclic ring; and 3) hydroxylation and glucuronic acid conjugation of one methyl group on the heterocyclic ring.     

The effect of dietary nitrite and nitrate on the metabolism of sulphamethazine in the rat

Rats given 100 p.p.m. of 14C-sulphamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulphonamide] in the diet excreted less 14C-activity in the urine as the amount of nitrite (0 to 1000 p.p.m.), but not nitrate (3730 p.p.m.), in the diet was increased. As the level of nitrite, but not nitrate, was increased, there was a concomitant increase in the amount of 14C-desaminosulphamethazine (4-[(N-4,6-dimethyl-2-pyrimidinyl)benzene-[U-14C]-sulphonamide in the blood, liver, skeletal muscle and gastrointestinal tract. As the level of nitrite, but not nitrate, supplementation was increased, the amount of methanol-insoluble 14C-activity in the gastrointestinal tract increased but the amount of insoluble 14C-activity in the blood, skeletal muscle and liver was not changed.     

The effect of nitrite on 14C-sulphathiazole (4-amino-N-2-thiazolyl[U-14C]benzenesulphonamide) metabolism in the rat

1. Rats given a meal containing 613 p.p.m. of 14C-sulphathiazole (4-amino-N-2-thiazolyl[14C]benzenesulphonamide) excreted less 14C-activity in urine and more 14C-activity in faeces as nitrite in the meal was increased (0, 10, 100 or 1000 p.p.m.). As nitrite in the meal was increased from 0 to 1000 p.p.m. the total 14C-residues in the gastrointestinal tract six hours after dosing increased, but decreased in other tissues. 2. High nitrite in the meal resulted in increased methanol insoluble 14C-activity in the gastrointestinal tract but had little or no effect on the methanol-insoluble activity in liver and blood. 3. Conversion of 14C-sulphathiazole to 14C-desaminosulphathiazole (N-2-thiazolyl[U-14C]benzenesulphonamide) in the rat was greatly increased by nitrite in the meal.     

Synthesis and anti-HIV activity of 4-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene) amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulfonamide and its derivatives.

4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene) amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its derivatives were synthesized by reaction of isatin and its derivatives with sulphadimidine. Their chemical structures have been confirmed by IR, (1)H NMR data and elemental analysis. Investigation of anti-HIV activity of compounds were tested against replication of HIV-1 (IIIB) and HIV-2 (ROD) strains in acutely infected MT-4 cells and the activity compared with standard azidothymidine. Among the compounds tested, 4-[(1,2-dihydro-2 oxo-3H-indol-3-ylidene)amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its N-acetyl derivative were the most active compounds.     

Synthesis and anti-HIV activity of 4-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene) amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulfonamide and its derivatives

4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene) amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its derivatives were synthesized by reaction of isatin and its derivatives with sulphadimidine. Their chemical structures have been confirmed by IR, ¹H NMR data and elemental analysis. Investigation of anti-HIV activity of compounds were tested against replication of HIV-1 (IIIB) and HIV-2 (ROD) strains in acutely infected MT-4 cells and the activity compared with standard azidothymidine. Among the compounds tested, 4-[(1,2-dihydro-2 oxo-3H-indol-3-ylidene)amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its N-acetyl derivative were the most active compounds.     

The effect of nitrite on 14C-sulphathiazole (4-amino-N-2-thiazolyl[U-14C]benzenesulphonamide) metabolism in the rat

1. Rats given a meal containing 613 p.p.m. of ¹⁴C-sulphathiazole (4-amino-N-2-thiazolyl[¹⁴C]benzenesulphonamide) excreted less ¹⁴C-activity in urine and more ¹⁴C-activity in faeces as nitrite in the meal was increased (0, 10, 100 or 1000 p.p.m.). As nitrite in the meal was increased from 0 to 1000 p.p.m. the total ¹⁴C-residues in the gastrointestinal tract six hours after dosing increased, but decreased in other tissues.

2. High nitrite in the meal resulted in increased methanol insoluble ¹⁴C-activity in the gastrointestinal tract but had little or no effect on the methanol-insoluble activity in liver and blood.

3. Conversion of ¹⁴C-sulphathiazole to ¹⁴C-desaminosulphathiazole (N-2-thiazolyl[U-¹⁴C]benzenesulphonamide) in the rat was greatly increased by nitrite in the meal.     

Lactose conjugation of sulphonamide drugs in the lactating dairy cow.

1. 14C-Sulphamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene-[U-14C]-sulphonamide; 220 mg/kg of body weight) was given orally or i.v. to lactating dairy cows. Milk collected from 0-48 h after dosing accounted for 2.0% (oral dose) and 1.1% (i.v. dose) of the total 14C-activity administered. 2. Sulphamethazine accounted for 70-79% (oral dose) and 54-75% (i.v. dose) of the total 14C in milk samples collected from 0-48 h after dosing. N4-acetylsulphamethazine accounted for 1-2% (oral dose) and 1-4% (i.v. dose) of the 14C in milk. 3. The major 14C-labelled metabolite in the milk was isolated and identified as the N4-lactose conjugate of sulphamethazine, a unique type of metabolite not previously reported. This metabolite accounted for 10-14% (oral dose) and 9-20% (i.v. dose) of the 14C-activity in the milk collected from 0-48 h after dosing with 14C-sulphamethazine. 4. N4-lactose conjugates of sulphapyridine, sulphamerazine, sulphathiazole, sulphadimethoxine and sulphaquinoxaline were present in the milk from cows orally dosed with these five sulphonamide drugs.     

Depletion kinetics of 14C-sulfamethazine [4-amino-N-(4, 6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] metabolism in swine

Swine weighing 60-70 kg were orally administered 14C-sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] at 12-hr intervals for 7 days (165 mg/dose; 0.126-5.04 mCi/mmol). The animals were sacrificed at 8 hr or 2, 5, or 10 days after the last dose was given and tissues were assayed for total 14C activity. The presence of 14C-labeled sulfamethazine, N4-acetylsulfamethazine, desaminosulfamethazine, and the N4-glucose conjugate of sulfamethazine in blood, liver, kidney, skeletal muscle, and adipose tissue was verified by HPLC and GC-MS analysis. Total 14C residue levels in all tissues examined had dropped to less than 0.1 ppm sulfamethazine equivalents by day 10 of the depletion period. The mean half-life (t1/2) for sulfamethazine, the N4-glucose conjugate of sulfamethazine, and N4-acetylsulfamethazine was estimated to be 0.8 day. In some tissues, the depletion of the N4-glucose conjugate of sulfamethazine and N4-acetylsulfamethazine had decreased significantly between days 5 and 10, resulting in an approximate doubling of the t1/2 for that period. In contrast, the half-life of desaminosulfamethazine varied from a mean of 0.96 day during the 8-hr-5-day depletion period to 3.7-9.1 days during the 5- 10-day depletion period. In most tissues, the t1/2 for the 14C-activity in the methanol-insoluble fraction increased by 3-5-fold between days 5 and 10 of the depletion period. No predictable relationship was observed between blood sulfamethazine or metabolite levels and total residue levels in the tissues.     

2-氨基-4,6-二氯-5-甲酰胺基嘧啶的合成工艺研究

目的合成硫酸阿巴卡韦中间体2-氨基-4,6-二氯-5-甲酰胺基嘧啶。方法以氨基丙二酸二乙酯盐酸盐和碳酸胍为原料,经过环合、Vilsmeier两步反应,制备2-氨基-4,6-二氯-5-甲酰胺基嘧啶。结果合成了2-氨基-4,6-二氯-5-甲酰胺基嘧啶,收率82.62%,纯度99.61%。结论获得了一条高效、简洁的2-氨基-4,6-二氯-5-甲酰胺基嘧啶制备路线,期望能够应用于工业化生产。     

Steady state kinetics of 14C-sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] metabolism in swine

Swine were dosed orally with 14C-sulfamethazine [4-amino-N-(4, 6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulfonamide] for 3, 5, or 7 days (two 165-mg doses/day; 0.46 muCi/mg) and killed 8 hr after the last dose. The concentration of carbon-14 in the tissues increased by an average of 21% from day 3 to day 5 of dosing. However, there was no further increase from day 5 to day 7, indicating that a steady state level of carbon-14 in the tissues was attained by dosing on 5 consecutive days. Liver, kidney, skeletal muscle, blood, and adipose tissue from all animals were analyzed for 14C-labeled sulfamethazine, N4-acetylsulfamethazine, desaminosulfamethazine [N-(4, 6-dimethyl-2-pyrimidinyl)benzenesulfonamide], and the N4-glucose conjugate of sulfamethazine. The identity of these compounds (the hydrolysis product of N4-glucose conjugate) was confirmed by HLPC and gas-liquid chromatography/mass spectral analysis after methylation. The relative distribution of 14C-sulfamethazine and these metabolites varied somewhat among the tissues analyzed but did not vary within a tissue after different periods of dosing.     

Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid

The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.     

Formation of a diazonium cation intermediate in the metabolism of sulphamethazine to desaminosulphamethazine in the rat

¹⁴C-Sulphamethazinediazonium tetrafluoroborate (¹⁴C-SDTFB) when orally administered to rats was converted primarily to ¹⁴C-labelled desaminosulphamethazine (desaminosulmet) and methanol-insoluble residues in the gastrointestinal tract (gut). ¹⁴C-labelled sulphamethazine (sulmet), N⁴-acetylsulmet, the N⁴-glucose conjugate of sulmet and other unidentified products were also observed in the tissues and urine of rats given ¹⁴C-SDTFB.

2. When ¹⁴C-sulmet, nitrite and dimethylaniline were simultaneously administered to a rat by the oral route, one of the ¹⁴C-labelled products formed in the stomach was isolated and identified as 4-dimethylaminophenyl[4-(N-4,6-dimethyl-2-pyrimidinyl)sulphamidophenyl]diazene, providing evidence that ¹⁴C-sulmet was diazotized in the stomach of the animal.

3. SDTFB was weakly mutagenic when evaluated by the Ames test.

4. The methanol-insoluble ¹⁴C-labelled residues in the gut of rats dosed orally with ¹⁴C-SDTFB and ¹⁴C-sulmet + nitrite were partially converted to ¹⁴C-labelled desaminosulmet, sulmet, N⁴-acetylsulmet and other unidentified products when fed to recipient rats.     

Synthesis of carbon-14 labelled spiro-piperidyl-rifamycins (LM 118) and rifabutin (LM 427)

Two syntheses of radiolabelled spiro-piperidyl-rifamycins are described.

• 1 . A five steps synthesis (Scheme 1) was performed to give 4-deoxo-3,4-[2-spiro-(N-[¹⁴C]methyl-4-piperidyl)]-(1H)-imidazo-(2,5-dihydro)-rifamycin S ([¹⁴C]LM 118), in an overall radiochemical yield of ∼ 20%, 98% radiochemically pure and with a specific activity of 440 MBq/mmol (11.9 mCi/mmol) starting from [¹⁴C]methyl iodide 1 .
• 2 . A three steps synthesis (Scheme 2) was performed to give 4-deoxo-3,4-[2-spiro-(N-2-methyl[1-¹⁴C]propane-4-piperidyl)]-(1H)-imidazo-(2,5-dihydro)-rifamycin S ([¹⁴C] rifabutin) in an overall radiochemical yield of ∼38%, 97% radiochemically pure with a specific activity of 1.27 GBq/mmol (34.32 mCi/mmol). 1-(2-Methyl[1-¹⁴C]propanoyl)-piperazin-4-one ethylene ketal was employed as starting material.

     

Polyamine metabolism I: Synthesis of dansyl derivatives of N-(monoaminoalkyl)- and N-(polyaminoalkyl)acetamides and elucidation in urine of a cancer patient.

The dansyl derivatives of N-(monoaminoalkyl)- and N-(polyaminoalkyl)acetamides were synthesized and unequivocally characterized. TLC of the dansyl derivatives obtained from human urine indicated the presence of N-[3-[(4-aminobutyl)amino]propyl]acetamide (N1-acetylspermidine, N-[4-[(3-aminopropyl)amino]butyl]acetamide (N8-acetylspermidine), and N-(4-aminobutyl)acetamide (N-acetylputrescine) in appreciable amounts. The dansyl derivatives of N1-acetylspermidine, N8-acetylspermidine, and N-acetylputrescine were isolated and purified using various chromatographic methods. The mass spectra of these compounds were similar to those of authentic samples, which confirmed the identity of these compounds and established the presence of N8-acetylspermidine as well as N1-acetylspermidine and N-acetylputrescine in human urine.     

Inhibition of the hepatic O6-alkylguanine-DNA alkyltransferase in vivo by pretreatment with antineoplastic agents

The mammalian DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT) is inactivated during the repair process and its activity can only be restored by de novo synthesis. We have made use of this property to determine whether and to what extent various chemotherapeutic agents alkylate DNA in the O6-position of guanine, ie. produce lesions susceptible to AT repair. Adult female Fischer rats received a single i.p. injection of a high dose (LD50) of the respective agent and, 5 hr later, a chasing dose of N-nitroso-[14C]dimethylamine (0.2 mg/kg; 4 hr survival). The amount of 7-[14C]methylguanine formed was approximately 95 mumol/mol guanine and not significantly altered by pretreatment with any of the drugs. The ratio of O6-[14C]methylguanine/7-[14C]methylguanine was 0.019 for control animals, indicating that during the observation period of 4 hr, 83% of the O6-[14C]methylguanine produced had been removed by the hepatic AT. Little or no effect was found in rats that received spirohydantoin mustard, hexamethylmelamine, cis-platinum or mitomycin C. A significant increase in the O6-/7-[14C]methylguanine ratio was found after pretreatment with AZQ (0.026) and cyclophosphamide (0.028), agents for which lesions involving the O6-position of guanine have not yet been identified. N-(2-Hydroxyethyl)-N-nitrosourea and the cytostatic haloethylinitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), and N-chloroethyl-N-hydroxyethylnitrosourea (HECNU) inhibited the hepatic AT, producing a ratio of 0.025-0.035. Considerably higher ratios of 0.059 and 0.101 were observed after administration of the methylating agents procarbazine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), respectively. Complete saturation of the repair system (O6-/7-[14C]methylguanine ratio, 0.11) was only achieved with N-methyl-N-nitrosourea.