Pyrimidines, purified

P5N has two isozymes, P5N-I (pyrimidine nucleotidase) and P5N-II (deoxyri-bonucleotidase) (H6, P2). P5N-I is active principally with pyrimidine substrates at an optimal neutral pH P5N-II activity occurs with both purine and pyrimidine substrates and was maximal with deoxy analogues at an acidic pH optimum. This enzyme was partially purified from human red blood cells and had a molecular weight of 28,000 (T19). The primary structures of both isozymes have not been... 

At the end of the heating period the contents of the flask will have solidified. To the cold mixture 40 ml. of water is added to hydrolyze the potassium methoxide and precipitate the pyrimidine the fine crystals are filtered and dried. The crude product is placed in a 500-ml. distilling flask with 250 ml. of purified kerosene (Note 3). On distilling the kerosene, the pyrimidine codistils and solidifies in the receiving flask to a snow-white mass of crystals. These are filtered, washed well with petroleum ether, and dried in an oven at 100°. The yield of pure material, melting at 182-183°, is 27.5-28.7 g. (67-70%) (Note 4). 

Dimethyl-l,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (41) is also an old product [7,11, 31] that has recently been found to react with GSH to give S-nitrosogluta-thione, NO and HNO [32]. It stimulates partially purified rat lung soluble guanylate cyclase, but not the heme-deficient enzyme. The activation is inhibited by ODQ. The product also displays significant vasodilator activity on rat thoracic aorta rings at nanomolar concentrations. Finally, [l,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxide derivatives (42, R,Ri=CH3,H) release NO, detected as nitrite, in the presence of thiols. A mechanism for this release has been proposed [33]. 

Conversion of 4-aminopyrazolo [3,4-d] pyrimidine (VIII) to its ribonucleotide by mouse tumours and host tissues has been observed [118,119]. Although no evidence of the anabolism of A -methyladenine (111) [120] to the ribonucleotide was obtained in mice with Ehrlich ascites carcinoma [121, 122], it is anabolized by bacteria [123. 124] and the enzyme responsible was partially purified from Salmonella typhimurium [125]. Human epidermoid carcinoma No. 2 cells resistant to 2-fluoroadenine (H.Ep.-2/FA) have lost adenine phosphoribosyl-... 

Recently the hydroxy metabolites of various sulfonamides could be Isolated and purified, so that specific HPLC techniques could be developed (22,23). As shown in Figure 1, sulfadimidine can be metabolized by hydroxylation at the 5 and 6 position of the pyrimidine ring and by the acetylation- deacetylation pathway (21). After hydroxylation, the metabolites may become glucuronidated and also acetylated (Figure 2). The hydroxy metabolites are microbiologically active and they can be potentiated by trimethoprim (13). 

If smaller reaction products are present, purify the full-length RNA from the gel to avoid the selection and amplification of shorter sequences. Alternatively, for generation of nuclease-resistant transcripts substitute CTP and GTP by 2 -fiuoro- or 2 -amino-pyrimidines (TriLink BioTechnologies, San Diego, CA) at final concentrations of 1.2 mM. 

Pyrimido[4,5-d]pyrimidine derivatives undergo a wide variety of nucleophilic displacement reactions. Pyrimido[4,5-d]pyrimidin-4-ones are readily chlorinated in either phosphorus oxychloride (62JOC4211) or a mixture of phosphorus pentachloride and thionyl chloride (74JMC451) to give the corresponding 4-chloropyrimido[4,5-d]pyrimidines. These chloro compounds are generally not purified but are isolated crude and used for subsequent nucleophilic displacements. 

Resin 12 (110 mg, 0.054 mmol) is treated with TFA as previously described for resin 9. The crude product (purity 88%) is subjected to chromatography on silica using EtOAc/ petroleum ether as the mobile phase. The purified product 4-amino-2-benzylamino-6-piperidin-l-yl pyrimidine is obtained in >70% yield. 

When dimethyl hex-2-en-4-yne-l,6-dioate 62, a dimerized product of methyl propiolate, was reacted with 2-aminopyridine in refluxing methanol for 44 hours (E)-3-(2-oxo-2//-pyrido[ 1,2-a]pyrimidin-4-yl)prop-2-enoate 63 was obtained, but the product could not be purified either by chromatography or by recrystallization [82JCS(P1)1905]. 

A mixture of 3-(2-bromoethyl)-2-methyl-pyrido[l,2-a]pyrimidin-4-one monohydrobromide, 3-furan-2-yl-methyl-(3H-imidazo[4,5-b]pyridine-2yl)-4-piperidinyl)-amine dihydrobromide, of sodium carbonate and of N,N-dimethylformamide was stirred and heated overnight at about 70°C. The reaction mixture was poured onto water. The product was extracted with trichloromethane. The extract was dried, filtered and evaporated. The residue was purified by column chromatography over silica gel using a mixture of trichloromethane and methanol (94 6 by volume), saturated with ammonia, as eluent. The pure fractions were collected and the eluent was evaporated. The residue was crystallized from acetonitrile, yielding 3-(2-(4-((3-(2-furanylmethyl)-3H-imidazo[4,5-b]pyridin-2-yl)amino)-l-piperidinyl)ethyl)-2-methyl-4H-pyrido[l,2-a]pyrimidin-4-one melting point 202°C. 

Several thieno[3,2-d]pyrimidine-2,4-diones 267 were evaluated in vitro for their ability to inhibit enzymatic activity on partially purified bovine lens aldose reductase. A high level of in vitro activity was demonstrated (ICjo 10"6 to 4 x 10 8 M) (91JMC1492 93MI1). 

In the first step, the pyrimidine is hydrogenated at the double bond by dihydrouracil dehydrogenase (EC 1.3.1.2) to a dihydropyrimidine (Scheme 1.6.2). The enzymes obtained from rat [4] and human [5] liver have been purified and characterized. They were later subjected to molecular cloning [6], The hydrogens are added to the double bond at the Si face of C5 and C6 in an anti-addition reaction. This was deduced from NMR spectra recorded from the isolated degradation products after administration of [5-2H]- and [6-2H]uracil and 2H20 to a mammalian enzyme system [7]. 

To monitor tumor response to capecitabine therapy noninvasively, Zheng and co-workers, from the Indiana University School of Medicine, developed the synthesis of the fluorine- 18-labeled capecitabine as a potential radiotracer for positron emission tomography (PET) imaging of tumors.28 Cytosine (20) was nitrated at the C-5 position with nitric acid in concentrated sulfuric acid at 85°C, followed by neutralization to provide 5-nitrocytosine (27) in moderate yield. This nitro pyrimidine was then carried through the glycosylation and carbamate formation steps, as shown in the Scheme below, to provide the 6/s-protected 5-nitro cytidine 28 in 47% for the three-step process. Precursor 28 was then labeled by nucleophilic substitution with a complex of 18F-labeled potassium fluoride with cryptand Kryptofix 222 in DMSO at 150 °C to provide the fluorine-18-labe led adduct. This intermediate was not isolated, but semi-purified and deprotected with aqueous NaOH in methanol to provide [l8F]-capecitabine in 20-30% radiochemical yield for the 3-mg-scale process. The synthesis time for fluorine-18 labeled capecitabine (including HPLC purification) from end of bombardment to produce KI8F to the final formulation of [18F]-1 for in vivo studies was 60-70 min. 

The purified E. coli protein has a molecular weight of 49 kD. It does not require any divalent cation for activity. It contains two different noncovalently bound chromophores that absorb light. One chromophore is flavin adenine dinucleotide (FADH- or FADH2). The other is 5,10-methenyltetrahydrofolyl polyglutamate (MTHF). The absorption of light by the chromophores is essential for the enzymatic reversal of the pyrimidine dimer back to the original pyrimidine monomers. However,... 

After cooling, remove the solvent at reduced pressure using a rotary evaporator. Purify the crude reaction mixture by column chromatography on silica gel, eluting with diethyl ether to give pyrimidine (117 mg, 77%) as a white solid. 

A phosphorylase from Escherichia coli has been purified it is specific for 2-deoxy-D-ribosyl phosphate, but can use uracil, 2-thiouracil, 5-amino-uracil, 5-bromouracil, and 2-thiothymine as a pyrimidine. Deaminases of adenosine, 2-deoxyadenosine, cytidine, and 2-deoxycytidine have been detected in Escherichia coli. 

To a suspension of NaH (76 mg) in 15 ml THF was added the product from Step 2, the mixture stirred 2 hours, 2-chloro-pyrimidine (71 mg) added, and the mixture stirred 42 hours. The solvent was removed and the residue partitioned between 50 ml 10% HO Ac and 50 ml EtOAc. The residue was purified by chromatography on silica gel eluting with a gradient of 5-10% methyl alcohol and CH2CI2. MS data supplied. 

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