4-Alkoxy-2-methylthiopyrrolo-[2,3-d]-pyrimidines. A new series of anti-cytokinins

A series of 4-alkoxy-2-methylthiopyrrolo[2,3-d] pyrimidines having alkoxy groups from methoxy to hexyloxy along with the parent 4-hydroxy compound have been tested for cytokinin and anticytokinin activity using tobacco tissue culture. None of the compounds showed any cytokinin activity but several of the compounds were strongly active as anticytokinins, the most active was 4-pentyloxy-2-methyl-thiopyrrolo[2,3-d] pyrimidine. Increasing or decreasing the chain length resulted in a lowering of activity; the hydroxy and methoxy compounds were totally inactive over the concentration range tested. The results are discussed in relation to steric and other considerations.     

Antagonistic Effects of Triazolo[4,5-d]pyrimidine and Pyridylurea Derivatives on Cytokinin-Induced Cytokinin Oxidase/Dehydrogenase Activity in Young Pea Plants

The effect of strong and weak cytokinin antagonists, belonging to the groups of triazolo[4,5-d]pyrimidines (TP), and pyridyl-phenylurea derivatives (PU), on cytokinin oxidase/dehydrogenase activity (CKX) in the tissues of young pea plants was studied. Tested anticytokinins, with the exception of the most efficient one – PU-1, were able to promote increased CKX activity in roots, when applied alone, but they had no significant influence on the enzymatic activity in leaves. N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (CPPU) provoked strong increase in CKX activity in roots, while in leaves considerable inhibition of enzymatic activity was observed. Different types of anticytokinins exhibited diverse preference towards taking off the action of purine and phenylurea cytokinins over CKX activity.     

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.     

Anticytokinin activity of 4-substituted triazolo[4,5-d]pyrimidines and 4-substituted pyrazolo[3,4-d]pyrimidines

The synthesis and physiological activity of some novel 4-substituted triazolo[4,5-d]pyrimidines and 4-substituted pyrazolo[3,4-d]pyrimidines are described. Most of the compounds possessed high anticytokinin activity towards purine (benzyladenine) and phenylurea (4-PU-30) type cytokinins. 1-Benzyl-4-ethoxycarbonylpiperazinyl-1H-1,2,3-triazolo[4,5-d]pyrimidine almost completely removed cytokinin stimulated effects—betacyanin synthesis in Amaranthus caudatus cotyledons; growth of radish cotyledons and retention of chlorophyll in leaf explants. Some chemical structurephysiological activity relationships have been established.     

Use of 5,6-dichloro-l-β-D-ribofuranosylbenzimidazole to distinguish light stimulation from cytokinin stimulation of amaranthin synthesis in Amaranthus tricolor

In Amaranthus tricolor seedlings, amaranthin synthesis can be induced by cytokinins and (or) by light. It was found that 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) was a specific inhibitor of the cytokinin response whereas the light response was unchanged. DRB also inhibited the negative response of pigment production to an anticytokinin, 7-(pentylamino)-3-methylpyrazolo(4,3-d) pyrimidine (PAMPP).DRB can be considered as a specific inhibitor of the physiological responses to cytokinins. The evolution of the inhibition percentage versus cytokinin or anticytokinin concentration shows that DRB is not a `competitor' of cytokinins.The experiments suggest that cytokinins or anticytokinins act as modulators of a fundamental light activated reaction of amaranthin synthesis.Address where proofs should be sent: Laboratoire de Physiologie végétale, Faculté des Sciences et des Techniques, Université de Nice, F-06034 Nice Cedex, France     

Fluorescent anticytokinins as a probe for binding. Isolation of cytokinin-binding proteins from the soluble fraction and identification of a cytokinin-binding site on ribosomes of tobacco callus cells

4-Substituted 2-methylthiopyrido[2,3-d]pyrimidines, a series of recently developed anticytokinins, have been found to fluoresce strongly in water and to be useful as probes for binding studies. The binding activity of the soluble proteins and particulate fraction of tobacco callus cells to the biologically most active member of the family, 4-n-butylamino-2-methylthiopyrido[2,3-d]pyrimidine (BAMPP), was studied fluorimetrically. We found that the binding activity is better monitored in terms of saturable binding rather than in terms of the amount of bound ligand, a conventional method used in isolation studies of hormone receptor proteins. Using this technique we isolated two kinds of high-affinity cytokinin-binding proteins from the soluble fraction and identified a high-affinity binding site on ribosomes.     

Cytokinin-agonistic and antagonistic activities of 4-substituted-2-methylpyrrolo[2,3-d] pyrimidines, 7-deaza analogs of cytokinin-active adenine derivatives

Sixteen 4-substituted-2-methylpyrrolo[2,3-d]pyrimidines were tested for their activities as cytokinin agonists and antagonists in three bioassay systems. With the systematic variation of the 4-substituent, the activity changed consecutively from agonistic to antagonistic in the tobacco callus test, and this agonist-antagonist relationship was analysed by a steric substituent parameter as a common quantitative measure. Two compounds were found to possess bud-forming activity in the absence of added cytokinins. Eight compounds promoted betacyanin synthesis'in Amaranthus and 4 promoted germination in lettuce seeds. The 4-hydroxyethyl derivative, which was inactive in the tobacco test, suppressed betacyanin synthesis at 100 μM against 100 μM 6-(3-methyl-2-butenylamino)- purine. The 4-phenyl derivative showed antagonist activity in the tobacco test, little activity in the Amaranthus bio- assay and significant agonist activity in the lettuce seed germination. The 4-cyclobutyl derivative exerted the strongest anticytokinin activity (I₅₀ at 0.06 μM against 0.05 μM kinetin) in the tobacco callus assay, while little activity was observed in the other two assay systems.     

Biosynthesis and cytokinin activity of 8-hydroxy and 2,8-dihydroxy derivatives of zeatin and N-6(increment-2-isopentenyl)adenine.

8-Hydroxy and 2,8-dihydroxy derivatives of the cytokinins, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine and N-6-(increment -2-isopentenyl)adenine, have been biosynthesized by xanthine oxidase oxidation. 8-Hydroxy derivatives have been shown to be the major intermdeiates. These compounds were tested for cytokinin activity in the tobacco bioassay. The results suggest that substitution of the 8 position with a hydroxyl group causes less decrease of cytokinin activity than substitution of both the 2 and 8 positions with hydroxyl groups.     

Design, synthesis, and structure-activity relationships of pyrazolo[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors

A series of pyrazolo[3,4-d]pyrimidines were synthesized and their antiviral activity was evaluated in a plaque reduction assay. It is very interesting that this class of compounds provide remarkable evidence that they are very specific for human enteroviruses, in particular, coxsackieviruses. Some derivatives proved to be highly effective in inhibiting enterovirus replication at nanomolar concentrations. SAR studies revealed that the phenyl group at the N-1 position and the hydrophobic diarylmethyl group at the piperazine largely influenced the in vitro antienteroviral activity of this new class of potent antiviral agents. It was found that the pyrazolo[3,4-d]pyrimidines with a thiophene substituent, such as compounds 20-24, in general exhibited high activity against coxsackievirus B3 (IC(50) = 0.063-0.089 microM) and moderate activity against enterovirus 71 (IC(50) = 0.32-0.65 microM) with no apparent cytotoxic effect toward RD (rhabdomyosarcoma) cell lines (CC(50) > 25 microM).     

Pyrazolo[4,3-d]pyrimidines as new generation of cyclin-dependent kinase inhibitors

A search among analogues of anti-CDK purines led to the identification of substituted pyrazolo[4,3-d]pyrimidines as novel inhibitors of CDK1/cyclin B. Some of these derivatives also show antiproliferative activity on cancer cell line K-562, thus may find an application as anticancer agents.     

Antimicrobial and antitumor activity of N-heteroimmine-1,2,3-dithiazoles and their transformation in triazolo-, imidazo-, and pyrazolopirimidines.

The reaction of Appel's salt with o-amino nitrile heterocycles 10-19 gave the corresponding 4-chloro-5-heteroimmine-1,2,3-dithiazoles 20-29 which were evaluated for their antibacterial, antifungal and antitumor activity. Although all these N-heteroimines were devoid of significant antibacterial activity, they showed significant antifungal activity. Moreover, the same derivatives represent highly versatile intermediates in heterocyclic synthesis, in fact the pyrazoleimino dithiazoles 20-26 can be converted in one step into 2-cyano derivatives of the corresponding 4-methoxy-pyrazolo[3,4-d]pyrimidines 30-35 by sodium methoxide in refluxing methanol. This provides a general and attractive route to 4-methoxy-6-cyano pyrazolo[3,4-d]pyrimidines from 1-substituted 5-amino pyrazoles 10-19 in two simple steps. Finally, the isosteric replacement of the pyrazole ring atoms to give the imidazole[3,4-d]pyrimidine and triazole [4,5-d] pyrimidine ring systems was examined.     

Abiotic synthesis of purines and other heterocyclic compounds by the action of electrical discharges

Summary The synthesis of purines and pyrimidines using Oparin-Urey-type primitive Earth atmospheres has been demonstrated by reacting methane, ethane, and ammonia in electrical discharges. Adenine, guanine, 4-aminoimidazole-5-carboxamide (AICA), and isocytosine have been identified by UV spectrometry and paper chromatography as the products of the reaction. The total yields of the identified heterocyclic compounds are 0.0023%. It is concluded that adenine synthesis occurs at a much lower concentration of hydrogen cyanide than has been shown by earlier studies. Pathways for the synthesis of purines from hydrogen cyanide are discussed, and a comparison of the heterocyclic compounds that have been identified in meteorites and in prebiotic reactions is presented.     

8-Azapurines as new inhibitors of cyclin-dependent kinases

Purine inhibitors of cyclin-dependent kinases (CDK) seem to be a potential anticancer drug candidate as one of the first representatives, roscovitine, is passing Phase II clinical trials for cancer and glomerulonephritis. In this article, we describe a novel modification of the purine scaffold influencing CDK2 inhibitory activities as well as anticancer properties in cell lines of different histopathological origin. The introduced N at position 8 of the purine ring generally lowered CDK2 inhibitory activity of new 8-azapurines (1,2,3-triazolo[4,5-d]pyrimidines) in comparison to the model trisubstituted purines, whereas the antiproliferative potential of some derivatives remained very high, reflecting their ability to activate p53 tumor suppressor.     

6-Dimethylamino 1H-pyrazolo[3,4-d]pyrimidine derivatives as new inhibitors of inflammatory mediators in intact cells

The synthesis of 6-dimethylamino 1H-pyrazolo[3,4-d]pyrimidines substituted at positions 1 and 4, and their effects on murine macrophage and human neutrophil functions are described. Several compounds and especially 4b-6b are potent inhibitors of PGE(2) generation in murine macrophages. This action is related to a direct effect on COX-2 activity without affecting the enzyme expression. Some of these compounds also inhibited COX-1 and COX-2 in human monocytes and 4b showed selectivity for COX-2 inhibition.     

New pyrazolo[3,4-d]pyrimidines endowed with A431 antiproliferative activity and inhibitory properties of Src phosphorylation

New 4-aminopyrazolo[3,4-d]pyrimidines bearing various substituents at the position 1 and 6, were synthesized. The new compounds showed antiproliferative activity toward A431 cells, were found to be inhibitors of Src phosphorylation, and induced apoptotic cell death. In particular, 2h was a better inhibitor of Src phosphorylation than the reference compound PP2.     

Novel non-classical C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines as potential inhibitors of dihydrofolate reductase and as anti-opportunistic agents

Six novel C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines 18-23 were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and as anti-opportunistic agents. These compounds represent the only examples of 9-methyl substitution in the carbon-carbon bridge of 2,4-diaminopyrrolo[2,3-d]pyrimidines. The analogs 18-23 were synthesized in a concise eight-step procedure starting from the appropriate commercially available aromatic methyl ketones. The key step involved a Michael addition reaction of 2,4,6-triaminopyrimidine to the appropriate 1-nitroalkene, followed by ring closure of the nitro adducts via a Nef reaction. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma) and rat liver (rl). The biological result indicated that some of these analogs are potent inhibitors of DHFR and some have selectivity for pathogen DHFR. Compound 23 was a two digit nanomolar inhibitor of tgDHFR with 9.6-fold selectivity for tgDHFR.     

Amino acid catalyzed condensation of purines and pyrimidines with 2-deoxyribose.

Mild heating of aqueous mixtures containing 2-deoxyribose, amino compounds, and purines or pyrimidines produces derivatives of the purines and pyrimidines in high yield. Among the major products formed are 2,3-dideoxy-3-(1'-pyrimidino)pentose and 2,3-dideoxy-3-(9'-purino)pentose. The mechanism of the reaction includes amine-catalzyed dehydration of the alpha, beta positions of the sugar followed by addition of the purine or pyrimidine to the double bond. Rapid addition of purines and pyrimidines to alpha, beta-unsaturated carbonyl compounds (such as acrolin) is a general phenomenon which does not require an amine catalyst. While multiple derivatization of the purines will take place, the N-9 derivative is formed first.     

Conformational analysis of cytokinins and analogs

The conformational energy surfaces of 12 active cytokinins and analogs are studied with the aid of PCILO quantum mechanical calculations. The resulting conformational energy maps indicate that cytokinin activity is associated with the ability of the above molecules to attain a specific conformation, presumably related to their conformation at the active site of cytokinin receptor(s). The calculations locate the conformational energy minima and describe the flexibility of the studied molecules in terms of conformational barriers and transition paths. An approximate relation is found between cytokinin activity and the values of energy barriers to transitions between certain local minima. According to this relation, active compounds should have rotational barriers within 4–12 kcal/mol, besides the known hitherto constitutional requirements for high physiological activity.     

A rapid and sensitive auxin binding system for detecting N6-substituted adenines, and some urea and thiourea derivatives, that show cytokinin activity in cell division tests.

A selective, sensitive and rapid (2 min or less) method for detecting compounds with potential for cytokinin activity is described. The method does not measure cytokinesis; instead, it determines the ability of cytokinin-active agents to (i) activate the intake of either L-tryptophan or indoleacetic acid by germinated spores of the water-mould Achlya, while inhibiting the energy-dependent transport of all L-amino acids usually found in proteins; (ii) inhibit the energy-dependent transport of nucleosides and sugars by the same organism. The compounds with cytokinin activity generally activate auxin (tryptophan) intake at 10(-8) M or greater and inhibit at 10(-6) M or greater. The most effective activating compounds were N6-(delta2-isopentenyl)adenine, N6-benzyladenine. N6-furfuryladenine, and N6-(trans-hydroxy-3-methyl-but-2-enyl)adenine. These compounds are classed generally as cytokinins in plant growth studies. A cell membrane - localized glycopeptide of molecular weight 6000 was isolated from this organism and shown to be the site at which cytokinins, auxin, and tryptophan bind. An earlier study had also established that calcium ions bind to this entity as well. Tryptophan binding to the glycopeptide was enhanced by cytokinins, suggesting that this may be the way in which whole cells display enhanced tryptophan binding in the bioassay. On the other hand, calcium binding was antagonized by cytokinin. The results suggest that this may be an important experimental system for use in studying one possible way in which cytokinin may regulate plant growth.     

Synthesis of aromatic compounds containing a 1,1-dialkyl-2-trifluoromethyl group, a bioisostere of the tert-alkyl moiety

1,1-Dialkyl-2-perfluoroalkyl compounds, which are potential metabolically stable bioisosteres of the tert-alkyl moiety, have been synthesized from the corresponding tertiary alcohols using titanium (IV) chloride-dimethylzinc or trimethylaluminium as the source of the methyl group. The synthetic methods proved to be versatile for synthesizing 1,1-dimethyl-2,2,2-trifluoroethyl compounds and analogs, including compounds containing aromatic and heterocyclic rings.