Synthesis, biological and modeling studies of 1,3-di-n-propyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purines as adenosine receptor antagonists.

A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)) have been evaluated. In this case the presence of n-propyl groups seems to induce potency at the A(2A) and A(3) adenosine receptor subtypes as opposed to our previously reported series bearing methyl substituents at the 1 and 3 positions. In particular the non-acylated derivative 17 showed affinity at these two receptor subtypes in the micromolar range. Indeed, preliminary molecular modeling investigations according to the experimental binding data indicate a modest steric and electrostatic antagonist-receptor complementarity.

Heterocyclic analogs of DNA minor groove alkylating agents.

It is generally accepted that neoplastic transformation is related to genes alteration or oncogene activation. In particular, DNA minor groove binding drugs have been extensively studied through the years in order to influence the regulation of gene expression by means of specific interactions with DNA bases moieties. Pyrrolo[2,1-c],[1,4].benzodiazepines (PBDs), CC-1065 and distamycins are three classes of minor groove binders which showed interesting cytotoxicity profiles, refined through already reviewed processes of SAR studies. Among the modifications to the three families of antitumor compounds, heterocyclic substitutions have been extensively applied by many groups in order to either modify the reactivity profile or introduce extra interactions within the minor groove, thus changing the binding site or modulating the binding sequence. The updated material related to these modifications has been rationalised and ordered to offer an overview of the argument.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives: potent and selective A(2A) adenosine antagonists.

A series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives (10a-o,q,r), bearing alkyl and aralkyl chains on positions 7 and 8, were synthesized in the attempt to obtain potent and selective antagonists for the A(2A) adenosine receptor subtype. The compounds were tested in binding and functional assays to evaluate their potency for the A(2A) compared with the A1 adenosine receptor subtype. In binding studies in rat brain membranes, most of the compounds showed affinity for A(2A) receptors in the low nanomolar range with a different degree of A(2A) versus A1 selectivity. Comparison of N(7) (10a-d,h-o)- and N(8) (10e-g)-substituted pyrazolo derivatives indicates that N(7) substitution decreases the A1 affinity with the concomitant increase of A(2A) selectivity. Specifically, the introduction of a 3-phenylpropyl group at pyrazolo nitrogen in position 7 (101) increased significantly the A(2A) selectivity, being 210-fold, while the A(2A) receptor affinity remained high (Ki=2.4 nM). With regards to the affinity for A(2A) receptors, also the compound 10n, bearing in the 7-position a beta-morpholin-4-ylethyl group, deserves attention (Ki=5.6 nM) even though the A2A selectivity (84-fold) was not as high as that of 101. Conversely, the compound 10m (N(7)-4-phenylbutyl derivative) showed a remarkable selectivity (A1/a(2A) ratio = 129) associated with lower A(2A) affinity (Ki = 21 nM). In functional studies, most of the compounds examined reversed 5'-(N-ethylcarbamoyl) adenosine-induced inhibition of rabbit platelet aggregation inhibition which is a biological response mediated by the A2A receptor subtype. The compounds are potent and selective A2A antagonists which can be useful to elucidate the pathophysiological role of this adenosine receptor subtype. These compounds deserve to be further developed to assess their potential for treatment of neurodegenerative disorders such as Parkinson's disease.

Synthesis and antitumor activity of a new class of pyrazolo[4,3-e]pyrrolo[1,2-a][1,4]diazepinone analogues of pyrrolo[1,4][2,1-c]benzodiazepines.

A new class of pyrrolo[1,4]benzodiazepine (PBD) analogues featuring a pyrazolo[4,3-e]pyrrolo[1,2-a][1,4]diazepinone ring system has been designed and synthesized. These compounds, 2a-o, are characterized by the substitution of the aromatic A ring, characteristic of the PBDs, with a disubstituted pyrazole ring bearing alkyl and benzyl substituents at N6 or N7 and alkyl or carbomethoxy substituents at C8. Biological evaluation revealed an appreciable in vitro cytotoxic activity for compounds 2a,b,f-i.

Design, synthesis, and biological evaluation of a second generation of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as potent and selective A2A adenosine receptor antagonists.

New A2A adenosine receptor antagonists in the series of pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidines, bearing oxygenated substituents on the phenylalkyl chains on the 7-position, have been synthesized. The compounds were tested in binding and functional assays to evaluate affinity, potency, and selectivity for rat A2A compared to rat A1 and human A3 receptor subtypes. The most interesting compounds (5d,e,h) were tested also in binding to human A1 and A2A adenosine receptors. They showed very good affinity (Ki = 0.94 nM for compound 5h) and interesting selectivity with respect to both rA1 and hA3 (compound 5h: rA1/rA2A = 787, hA3/rA2A > 10 000). These important findings make this new series of compounds the first really selective for A2A adenosine receptors. Thermodynamic parameters were evaluated; all the tested compounds displayed an enthalpy-driven binding as expected for antagonists. Moreover, compound 5h showed a negative entropy value. The highly negative enthalpic and entropic contributions could mean that 5h fits very well in the binding site where, probably, an electrostatic interaction is present associated to a scarce solvent reorganization around the receptor binding site. These compounds deserve to be further developed to assess their potential for treatment of neurodegenerative disorders such as Parkinson's disease.

Novel N6-(substituted-phenylcarbamoyl)adenosine-5'-uronamides as potent agonists for A3 adenosine receptors.

A series of adenosine-5'-uronamide derivatives bearing N6-phenylurea groups have been synthesized and tested for their affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells. Some N6-arylcarbamoyl derivatives, N6-((2-chlorophenyl)carbamoyl)-, N6-((3-chlorophenyl)carbamoyl)-, and N6-((4-methoxyphenyl)carbamoyl)adenosine-5'-ethyluronamide (4l-n), were found to have affinity at A3 receptors in the low nanomolar range (Ki values < 10 nM). In CHO cells stably transfected with the rat A3 receptor, compound 4n was found to be a full agonist in inhibiting adenylate cyclase activity. The present study represents the first example of N6-acyl-substituted adenosine analogs having high affinity at adenosine receptors and, in particular, at the A3 receptor subtype.

Fluorosulfonyl- and bis-(beta-chloroethyl)amino-phenylamino functionalized pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives: irreversible antagonists at the human A3 adenosine receptor and molecular modeling studies.

A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A(3) adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl group at the N(8) pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N(5) position, displayed a K(i) value at the hA(3) receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(beta-chloroethyl)amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA(3) receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N(8) pyrazole nitrogen. The bis-(beta-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A(3) receptor was built and analyzed to help interpret these results. The model of the A(3) transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.

Effect of A-ring modifications on the DNA-binding behavior and cytotoxicity of pyrrolo[2,1-c][1,4]benzodiazepines.

Several A-ring-modified analogues of the DNA-binding antitumor agent DC-81 (5) have been synthesized in order to study structure-reactivity/cytotoxicity relationships. For two molecules (23 and 30) the modifications required the addition of a fourth ring to give the novel dioxolo[4,5-h]- and dioxano[5,6-h]pyrrolo[2,1-c][1, 4]benzodiazepin-11-one (PBD) ring systems, respectively. Another three analogues (34, 38, and 48) have the native benzenoid A-ring replaced with pyridine, diazine, or pyrimidine rings to give the novel pyrrolo[2,1-c][1,4]pyridodiazepine, pyrrolo[2,1-c][1, 4]diazinodiazepine, and pyrrolo[2,1-c][1,4]pyrimidinodiazepine systems, respectively. The other new analogues (16a,b) have extended chains at the C8-position of the DC-81 structure. During the synthesis of these compounds, a novel tin-mediated regiospecific cleavage reaction of the dioxole intermediate 18 was discovered, leading to the previously unknown iso-DC-81 (20). In addition, an unusual simultaneous nitration-oxidation reaction of 4-(3-hydroxypropoxy)-3-methoxybenzoic acid (8) was found to produce 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid (9), a key intermediate, in high yield. In general, the results of cytotoxicity and DNA-binding studies indicated that none of the changes made to the A-ring of the PBD system significantly improved either binding affinity or cytotoxicity in comparison to DC-81. This result suggests that the superior potency of natural products such as anthramycin (1), tomaymycin (2), and sibiromycin (3) is due entirely to differences in C-ring structure, and in particular exo or endo unsaturation at the C2-position and C2-substituents containing unsaturation. This study also provided information regarding the influence of A-ring substitution pattern on the relative stability of the interconvertible N10-C11 carbinolamine, carbinolamine methyl ether, and imine forms of PBDs.

Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo[2,1-c][1, 4]benzodiazepine and minor-groove-binding oligopyrrole carriers.

The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.

Synthesis and biological activity of a new series of N6-arylcarbamoyl, 2-(Ar)alkynyl-N6-arylcarbamoyl, and N6-carboxamido derivatives of adenosine-5'-N-ethyluronamide as A1 and A3 adenosine receptor agonists.

A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ +-b earing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors expressed in CHO cells. The derivatives contained the 5' substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ + (NECA). While the carboxamido derivatives (9-13) showed affinity for A1 receptors, the urea derivatives (30-45) showed different degrees of affinity and selectivity for the A3 adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (Ki = 9 nM) and selectivity for the A3 receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-be ta-D-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A3 adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A2A and/or A3 receptors but rather showed a dramatic reduction of A2A affinity, resulting in compounds with good A2A/A3 selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A1 and A3 receptor subtypes, excluding the A2A receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A3 receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [35S]GTP-gamma-S.

Binding affinity of adenosine receptor agonists and antagonists at human cloned A3 adenosine receptors.

The A3 adenosine receptor is one of the four adenosine receptors which have thus far been identified. Cloning of the A3 receptor from animal species such as rat, sheep and human has shown that there are interspecies differences in its peripheral distribution, and binding affinity for various adenosine receptor ligands. The adenosine derivative, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA), is a potent A3 receptor agonist which is used as a reference drug. In this report we have characterized the binding of selected adenosine receptor agonists and antagonists to HEK 293 cells transfected with the human A3 adenosine receptor using [125I]AB-MECA as radioligand. HE-NECA and NECA were the most potent compounds showing Ki values in the low nanomolar range, while the recently discovered non-xanthine A2A receptor antagonists ZM 241385, SCH 58261 and SCH 63390 showed affinity values in the micromolar range. These data further indicate the need to examine the affinity of new adenosine receptor ligands directly in human A3 receptors.

Chemical synthesis of [(13)c]daidzein.

Pharmacokinetic and metabolic studies of phytoestrogens of the isoflavone class have been hampered by the lack of suitable stable-isotope-labeled analogs. A method for preparation of a [(13)C]-labeled analog of daidzein is described. [2-(13)C]Daidzein was synthesized by reaction of [(13)C]diethoxydimethylaminomethane with 2,4-dihydroxybenzoin. The final product was purified to more than 99% by reverse-phase high-performance liquid chromatography and structural analysis confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Because [2-(13)C]daidzein is analytically and metabolically stable, it is a suitable analog for use as an internal standard for quantifying daidzein in biological fluids using isotope dilution mass spectrometry. This nonradioactive tracer is also ideal for investigating the pharmacokinetics of daidzein in humans because it is biologically indistinguishable from the unlabeled form.

[2,1-c][1,4]benzodiazepine (PBD)-distamycin hybrid inhibits DNA binding to transcription factor Sp1.

We designed and synthesized the hybrid 6, prepared combining the minor groove binders distamycin A and pyrrolo [2,1-c][1,4] benzodiazepine (PBD) 4, related to the natural occurring anthramycin (2) and DC-81 (3). In this paper, the effects of the compound 6 on molecular interactions between DNA and transcription factor Sp1 were studied. The results obtained demonstrate that PBD-distamycin hybrid is a powerful inhibitor of Sp1/DNA interactions.

Selective adenosine A2A receptor antagonists.

In the early 1990s it became clear that the A2A adenosine receptor had characteristics that made it distinct from the other A1, A2B and A3 adenosine receptors. Great progress has been made with the discovery of selective A2A receptor antagonists. A variety of synthetic substitutions on the xanthine moiety led the chemists of Kyowa-Hakko to discover that introduction of the styryl group in the 8 position of xanthines was critical in achieving compounds endowed with selective A2A receptor antagonistic properties. One compound, KW 6002, (E)1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine, is currently being developed for treatment of Parkinson's disease. A number of non-xanthine heterocycles have also been synthesized starting from the non-selective adenosine antagonist CGS 15943, a triazoloquinazoline. Thus, replacement of the phenyl ring of CGS 15943 with a heterocyclic ring such as pyrazole or imidazole, led to a series of interesting compounds whose prototype, SCH 58261, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, has become a reference A2A receptor antagonist. Modification of N7 substituents has progressed to optimize A2A receptor selectivity and pharmacokinetic characteristics. A related class of compounds having a bicyclic instead of the tricyclic ring structure is also of interest. The prototype of these triazintriazolo derivatives, ZM 241385, is a potent A2A receptor antagonist; however, it also shows interactions with A2B receptors. The relevance of the A2A receptors in specific disease states, especially in the central nervous system, makes this class of adenosine receptor blockers of interest for treatment of neurodegenerative disorders such as Parkinson's disease.

DNA minor-groove binders: results and design of new antitumor agents.

DNA minor-groove binding drugs have been extensively studied in the last years in order to influence the regulation of gene expression in neoplastic disorders by means of specific interactions with DNA bases. Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), CC-1065 and distamycins are three classes of minor-groove alkylating agents which showed interesting cytotoxicity profiles, but they cannot be used in humans for various toxicity problems. For this reason many groups applied heterocyclic substitutions extensively, in order to either modify the reactivity profile or introduce extra interactions within the minor groove, thus changing the binding site or modulating the binding sequence.

Synthesis, solvolytic stability and cytotoxicity of a modified derivative of CPzI, a pyrazole analog of the alkylation subunit of the antitumor agent CC-1065: effect of the nitrogen substitution on the functional reactivity.

The synthesis and the comparative preliminary biological evaluation of a new pyrazole analog (16) of the CC-1065 alkylating unit (CPI) are described. This new derivative showed low cytotoxicity against L1210 murine leukemia (IC50 3064 nM) with respect to reference compound, but contrarily to literature data, was found to be more stable to solvolysis than the natural derivative (+/-)-N-Boc-CPI (pH 3, t1/2 = 212 h vs. 37 h). The results of such investigation showed that alkylation of the pyrazole nitrogen caused a loss of cytotoxic activity in vitro against tumor cells. This experimental observation allowed us to confirm the importance of free N-H for the anticellular activity.

Synthesis, cytotoxicity and antitumor activity of some new simplified pyrazole analogs of the antitumor agent CC-1065. Effect of an hydrophobic group on antitumor activity.

Three simplified pyrazole analogs (7-9) of the antitumor agents CC-1065, were synthesized. In in vitro assays, against L1210 cell lines all derivatives showed a cytotoxicity in a pM range, values close to the natural target compound (+)-CC-1065. In in vivo tests, against disseminate L1210 leukemia cells, synthesized compounds showed a good potency (O.D. 300 micrograms/Kg) but no activity. These observations further validate the effect of the hydrophilic and/or hydrophobic characteristics of the substituents present on the molecules, confirming the relevance of this phenomena on in vivo activity. In fact in this case the increase of hydrophobic characteristics of the molecules produce the loss of activity, probably due to a worse bioavailability of the drugs in animals.

Synthesis, antibacterial activity and structure-activity relationships of N-substituted 4-diazo-pyrazole-5-carboxamides. 2.

A series of 4-diazopyrazole-5-carboxamides 1 d-n has been synthesized and their antibacterial activity against a number of Gram-negative and Gram-positive strains has been tested. Some of the compounds resulted quite active and the whole set allowed to further study the SAR of the class. Substituents in position 5 affect Gram-negative and Gram-positive activities via bulk and electronic properties respectively; position 3 mostly affects the Gram-negative activity, while the presence of the charged diazo group in position 4 is crucial for both antibacterial activities.