Prazosin-Conjugated Matrices Based on Biodegradable Polymers and α-Amino Acids--Synthesis, Characterization, and in Vitro Release Study.

Novel and promising macromolecular conjugates of the α1-adrenergic blocker prazosin were directly synthesized by covalent incorporation of the drug to matrices composed of biodegradable polymers and α-amino acids for the development of a polymeric implantable drug delivery carrier. The cyto- and genotoxicity of the synthesized matrices were evaluated using a bacterial luminescence test, protozoan assay, and Salmonella typhimurium TA1535. A new urethane bond was formed between the hydroxyl end-groups of the synthesized polymer matrices and an amine group of prazosin, using 1,1'-carbonyldiimidazole (CDI) as a coupling agent. The structure of the polymeric conjugates was characterized by various spectroscopy techniques. A study of hydrogen nuclear magnetic resonance ((1)H-NMR) and differential scanning calorimetry (DSC) thermodiagrams indicated that the presence of prazosin pendant groups in the macromolecule structures increased the polymer's rigidity alongside increasing glass transition temperature. It has been found that the kinetic release of prazosin from the obtained macromolecular conjugates, tested in vitro under different conditions, is strongly dependent on the physicochemical properties of polymeric matrices. Furthermore, the presence of a urethane bond in the macromolecular conjugates allowed for obtaining a relatively controlled release profile of the drug. The obtained results confirm that the pharmacokinetics of prazosin might be improved through the synthesis of polymeric conjugates containing biomedical polymers and α-amino acids in the macromolecule.

Identification, preparation, and characterization of several polymorphs and solvates of terazosin hydrochloride.

The phenomenon of polymorphism is prevalent in pharmaceuticals, yet it is unusual to identify more than three or four forms for any particular drug. Terazosin hydrochloride has been found to exist at room temperature in four solvent-free forms that can be isolated directly, one solvent-free form that can be prepared by desolvation of a methanolate, a methanol solvate, and a dihydrate. This study presents characterization and methods for preparation of each of these forms. Data are also presented demonstrating the relative stability of these forms.

Synthesis and characterization of a radioiodinated photoaffinity probe for the alpha 1-adrenergic receptor.

A radioiodinated aryl azide analog, 2-[4-(4-azido-3- iodobenzoyl ) piperazin -1-yl]-4-amino-6, 7- dimethoxyquinazoline [(125I] CP65 ,526), of the highly selective alpha 1-adrenergic antagonist prazosin was synthesized and characterized using rat hepatic plasma membranes. Prior to photolysis, this ligand bound with high affinity (Kd 0.3 nM), stereoselectively and in a saturable manner to sites with an alpha 1-adrenergic specificity. When membranes pretreated with [125I] CP65 ,526 were irradiated with ultraviolet light, the ligand incorporated irreversibly into the receptor-binding sites, also with typical alpha 1-adrenergic specificity. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of such labeled membranes followed by radioautography revealed major bands at Mr = 77,000, 68,000, and 59,000 daltons. Labeling of each of these bands was inhibitable by a variety of adrenergic ligands, stereoselectively and with a specificity typical of the alpha 1-adrenergic receptor. Smaller peptides with molecular weights of 42,000 and 31,000 daltons also displayed prazosin-inhibitable [125] CP65 ,526-binding. However, as the labeling of these protein species was not inhibitable by other adrenergic agonists or antagonists, they are unlikely to represent subunits of the receptor. Further evidence that [125I] CP65 ,526 incorporates covalently upon photolysis was the ability to specifically label immunoglobulin heavy and light chains of an antiserum that recognized both this ligand and the parent compound, prazosin. This new, radioiodinated, high-affinity probe should thus be uniquely valuable for the molecular characterization of the alpha 1-adrenergic receptor.

Alkylating prazosin analogue: irreversible label for alpha 1-adrenoceptors.

A series of prazosin analogues comprised of N-acyl derivatives of N'-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine was prepared and the nature of their binding to alpha 1-adrenoceptors was investigated. Derivatives with alpha, beta-unsaturated acyclic acyls had some affinity but no irreversible action at the receptor. Other potent compounds, also without irreversible activity, contained cinnamoyl or (phenylamino)thiocarbonyl residues. High affinity and irreversible binding were obtained with a bicyclo[2.2.2]octa-2,5-dien-2-ylcarbonyl derivative. The conjugated double bond in this compound was in about the same position and distance from the pharmacophore as in some of the above compounds of high affinity but with no irreversible action. Two consecutive recognition steps were thought to be involved in irreversible blockade: reversible binding of the pharmacophore part of the molecule to the binding site of the receptor, followed by reaction of the chemoreactive part with an adjacent nucleophile of the receptor. The present results suggest that for the second step to occur efficiently, some affinity for the receptor must be present even in the chemoreactive part of the molecule; simple spanning of the binding and nucleophile sites of the receptor was insufficient.

Synthesis and identification of the major metabolites of prazosin formed in dog and rat.

The 6-O-demethyl and 7-O-demethyl analogues of the new antihypertensive drug prazosin [2-[4-(2-furoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline hydrochloride] have been unequivocally synthesized via separate ten-step reaction sequences starting from isovanillin and vanillin, respectively. The 6-O-demethyl derivative was found to be identical with the major prazosin metabolite formed in dog and rat, while the 7-O-demethyl derivative was identical with another, less prevalent but significant metabolite. Two minor metabolites of prazosin, 2-(1-piperazinyl)-4-amino-6,7-dimethoxyquinazoline and 2,4-diamino-6,7-dimethoxyquinazoline, are also described. All four metabolites are less potent blood pressure lowering agents in dogs than prazosin but may contribute to its antihypertensive effect, since they account for a major portion of the administered dose.

Structure-activity relationships in prazosin-related compounds. Effect of replacing a piperazine ring with an alkanediamine moiety on alpha 1-adrenoreceptor blocking activity.

Several prazosin-related compounds were synthesized in which the piperazine ring of prazosin (1) was replaced by an alkanediamine chain and were evaluated for their blocking activity on alpha 1- and alpha 2-adrenoreceptors in isolated rat vas deferens. All the compounds investigated proved highly selective toward the alpha 1-adrenoreceptor owing to a very low affinity for alpha 2-adrenoreceptors. Furthermore, compounds 2, 9, and 13 were also investigated in vivo to determine their hypotensive effect on anesthetized rats, which were compared with that of prazosin (1). It was confirmed that the piperazine moiety of 1 is not essential for potency. However, optimum activity depends on two parameters: carbon-chain length of the alkanediamine moiety and N-methylation of both the amide and the 2-amino functions. In the desmethyl series, optimum activity was associated with the lower homologues (2-4) bearing a chain of two to four methylenes whereas in the N,N'-dimethyl series peak potency was observed with a six-carbon chain as in 13. Compound 13 proved the most active of the series and was more potent than prazosin (1) in both in vivo and in vitro assays. It is hypothesized that the alpha 1-adrenoreceptor incorporates a lipophilic area that is located between the binding sites for the quinazoline and the furoyl moieties and is able to accommodate a polymethylene chain.

The furoxan system as a useful tool for balancing "hybrids" with mixed alpha 1-antagonist and NO-like vasodilator activities.

The design of new vasodilator derivatives in which two different pharmacophoric groups are present in a single molecule has been pursued by substitution of NO-prodrug furoxan moieties for the furanylcarbonyl function in Prazosin, a well-known alpha 1-receptor antagonist. The aim was to obtain new antihypertensive agents in which two vasodilation mechanisms, alpha 1-antagonist and NO-mediated, can operate in an appropriate balance. The alpha 1-antagonist activity was assessed on rat aortic strips in the presence and in the absence of oxyhemoglobin (HbO2), a well-known scavenger of nitric oxide. The resulting hybrids displayed different pharmacological behaviors. When the 4-furoxanylcarbonyl system, bearing an ester or an amide function at the 3-position, was present (derivatives 7a,b), hybrids with predominant alpha 1-antagonist activity were obtained. By contrast, in the derivative 7c, in which the nitrile function is linked to the 3-position of the furoxan ring, the NO-mediated vasodilating properties are predominant. Finally, the (furoxanylsulfonyl)piperidine derivatives 13a,b showed NO vasodilation and alpha 1-antagonist activities in an appropriate balance. For the furoxan derivatives, the NO-dependent vasodilating ability, assessed on the K(+)-depolarized aortic strip, and the NO release features under the action of thiol cofactors are also discussed.

Analogues of prazosin that bear a benextramine-related polyamine backbone exhibit different antagonism toward alpha1-adrenoreceptor subtypes.

Hybrid tetraaamine disulfides 4-9 were synthesized by combining the structural features of prazosin (1), a competitive alpha1-adrenoreceptor antagonist, and benextramine (2), an irreversible alpha1/alpha2-adrenoreceptor antagonist, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D). To verify the role of the disulfide moiety on the interaction with alpha1-adrenoreceptor subtypes, carbon analogues 10-15 were included in this study. All quinazolines lacking the disulfide bridge behaved, like 1, as competitive antagonists, whereas all polyamine disulfides displayed a nonhomogeneous mechanism of inhibition at the three subtypes since they were, like 2, noncompetitive antagonists at the alpha1A and alpha1B subtypes while being, unlike 2, competitive antagonists at the alpha1D. In particular, the blocking effects were characterized by a decrease of the maximal response to noradrenaline that was affected only slightly by washings. Probably the alpha1A and alpha1B subtypes bear in the binding pocket a suitable thiol function that would suffer an interchange reaction with the disulfide moiety of the antagonist and which is missing, or not accessible, in the alpha1D subtype. Polyamines 8, 9, and 14, among others, emerged as promising tools for the characterization of alpha1-adrenoreceptors, owing to their receptor subtype selectivity. Finally, the effect of nonbasic substituents on the phenyl ring of prazosin analogues 16-28 on potency and selectivity for the different subtypes can hardly be rationalized.

Design, synthesis, and biological activity of prazosin-related antagonists. Role of the piperazine and furan units of prazosin on the selectivity for alpha1-adrenoreceptor subtypes.

Prazosin-related quinazolines 4-20 were synthesized, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D) and by binding assays in CHO cells expressing human cloned alpha1-adrenoreceptor subtypes. The replacement of piperazine and furan units of prazosin (1) by 1, 6-hexanediamine and phenyl moieties, respectively, affording 3-20, markedly affected both affinity and selectivity for alpha1-adrenoreceptor subtypes in functional experiments. Cystazosin (3), bearing a cystamine moiety, was a selective alpha1D-adrenoreceptor antagonist being 1 order of magnitude more potent at alpha1D-adrenoreceptors (pA2, 8.54 +/- 0.02) than at the alpha1A- (pA2, 7.53 +/- 0.01) and alpha1B-subtypes (pA2, 7.49 +/- 0. 01). The insertion of substituents on the furan ring of 3, as in compounds 4 and 5, did not improve the selectivity profile. The simultaneous replacement of both piperazine and furan rings of 1 gave 8 which resulted in a potent, selective alpha1B-adrenoreceptor antagonist (85- and 15-fold more potent than at alpha1A- and alpha1D-subtypes, respectively). The insertion of substituents on the benzene ring of 8 affected, according to the type and the position of the substituent, affinity and selectivity for alpha1-adrenoreceptors. Consequently, the insertion of appropriate substituents in the phenyl ring of 8 may represent the basis of designing new selective ligands for alpha1-adrenoreceptor subtypes. Interestingly, the finding that polyamines 11, 16, and 20, bearing a 1,6-hexanediamine moiety, retained high affinity for alpha1-adrenoreceptor subtypes suggests that the substituent did not give rise to negative interactions with the receptor. Finally, binding assays performed with selected quinazolines (2, 3, and 14) produced affinity results, which were not in agreement with the selectivity profiles obtained from functional experiments. This rather surprising and unexpected finding may be explained by considering neutral and negative antagonism.

Traceless solid-phase synthesis of 2,4-diaminoquinazolines.

[reaction: see text] The solid-phase synthesis of 2,4-diaminoquinazolines is presented. The chemistry involves the sequential condensation of 2-aminobenzonitriles and amines starting from an acyl isothiocyanate resin via a traceless cleavage and cyclization. The alpha-1 antagonist prazosin was synthesized, as well as several other examples, in good yields and purity.

A new photoaffinity probe, 4-amino-2-[4-(4-azidocinnamoyl)piperazino]-6,7-dimethoxyqu inazoline, for alpha 1-adrenoceptors.

A photoaffinity probe for alpha 1-adrenoceptors was synthesized and its properties examined on rat brain membrane preparations. The binding of 4-amino-2-[4-(4-azidocinnamoyl)piperazino]-6,7-dimethoxyquin azoline (ACP) to these receptors was of high affinity (KD = 1.05 nM) and reversible in the dark. A dose-dependent decrease in the concentration of [3H]prazosin binding sites without a change in KD was observed when membranes were preincubated with ACP, photolyzed, and then extensively washed prior to assay. This reduction in receptor concentration was prevented by alpha 1-adrenergic ligands. The specificity of ACP for alpha 1-receptors was further demonstrated by its inability to compete with [3H]dihydroalprenolol and [3H]yohimbine binding in these same membranes. Also, the concentrations and affinity constants of beta-adrenoceptors and alpha 2-adrenoceptors were unaffected in membranes which had been photolyzed after preincubation with ACP. No reduction in concentration of alpha 1-adrenoceptors was detected if ACP was photolyzed prior to incubation with receptors or if ACP was maintained in darkness throughout the experiment. The results suggest that ACP is a specific and sensitive photoprobe that may be useful for further studies on alpha 1-adrenoceptor coupled systems and that may be particularly suited for use in cell culture work.

Pharmacochemistry of the furoxan ring: recent developments.

In the present work recent results obtained in the pharmacochemistry of the furoxan system are reported. In particular, after a brief description of the salient points of the furoxan chemistry, the synthesis and the properties of a series of Nifedipine and Prazosin analogues, containing this heterocyclic system, are described. Since we observed that a few furoxan derivatives are able to elicit both a dose-dependent rise in platelet cGMP levels and to promote a dose-dependent inhibition of AA-induced [Ca++] rise, and that many substituted furoxans show potent vasodilating and antiaggregatory activity, the possibility of using the furoxan system as a lead in the design of new vasodilators is also discussed.

Some new prazosin analogues.

The synthesis and the pharmacological properties of some new prazosin analogues are described.

A modified synthesis of iodoazidoaryl prazosin.

The antihypertension agent iodoazidoaryl prazosin (IAAP) has been made using a convergent route involving addition of an acylated piperazine 7 to 2-chloroquinazoline 5. IAAP has been shown to function as a multidrug resistance (MDR) reversal agent and bind to P-glycoprotein, a transmembrane transport protein. A study is also reported involving palladium-catalyzed substitution with amine heterocycles. With N,N-bis(2,6-diisopropyl)dihydroimidazolium chloride (10) as the ligand (2 mol %) for palladium(II) acetate (2 mol %) in THF at room temperature, morpholine added to 5 in 81% yield.

Synthesis of poly(hydroxypropylglutamine-prazosin carbamate) and release studies.

Prazosin, an antihypertensive drug with postsynaptic alpha 1-adrenergic blocking activity, has been coupled to poly-N5-(3-hydroxypropyl-L-glutamine) (PHPG) via a carbamate linkage. PHPG was activated by p-nitrophenyl chloroformate and then reacted with prazosin to form p(HPG-prazosin carbamate) conjugate. Drug loading was 23.9% (w/w). Activated polymer and conjugates were characterized by infrared spectroscopy and differential scanning calorimetry. In vitro studies proceeded in pH 7.4 isotonic phosphate-buffered saline solution. Prazosin was released at a rate of 0.92 mg/day/100 mg conjugate from p(HPG-prazosin carbamate) particles. In vivo studies were performed with New Zealand White rabbits. P(HPG-prazosin carbamate) conjugate particles (100 mg) were suspended in 2 ml saline and injected subcutaneously into both flanks of rabbits. P(HPG-prazosin carbamate) conjugates, following an initial burst, demonstrated a nearly constant plasma prazosin concentration profile above 2 ng/ml, which was maintained for 10 days.

Photoaffinity cross-linking of a radioiodinated probe, 125I-A55453, into alpha 1-adrenergic receptors.

We have synthesized and characterized a high-affinity alpha 1-adrenergic receptor probe, 4-amino-6,7-dimethoxy-2[4'- [5"(3"'-125I-iodo-4"'-aminophenyl)pentanoyl]-1'-piperazinyl] quinazoline (125I-A55453). This ligand binds reversibly to rat hepatic plasma membranes with high affinity (KD = 77 +/- 6 pM), and it labels the same number of "specific" prazosin-competable sites as the alpha 1-adrenergic receptor-selective radioligand [125I] iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone. Specific binding is stereoselective and competed for by alpha-adrenergic agents with an alpha 1-adrenergic receptor specificity. 125I-A55453 can be covalently photoincorporated into peptides of rat hepatic and splenic membranes using the bifunctional photoactive cross-linker, N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. Following photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled hepatic membranes reveals a major "specifically" labeled peptide of Mr = 82,000 (+/- 1,000) with minor peptides at Mr = 50,000 (+/- 500), and 40,000 (+/- 300). Covalent incorporation of 125I-A55453 into the Mr = 82,000 peptide is inhibited by adrenergic drugs with an alpha 1-adrenergic receptor specificity. Labeled splenic membranes demonstrate a broad band of photoincorporated radioactivity centered at Mr = 82,000, and covalent incorporation into this peptide is also attenuated with an alpha 1-adrenergic receptor specificity. This new high-affinity radioiodinated probe has features which should make it useful for the molecular characterization of alpha 1-adrenergic receptors in tissues.

The cyano-NNO-azoxy function in the design of an irreversible label for alpha 1 adrenoreceptors.

A potential alpha 1-adrenergic irreversible antagonist 6, containing the cyano-NNO-azoxy function was synthesized and tested. The effects of norepinephrine on rat thoracic aorta were irreversibly blocked by this compound at the concentration of 1 x 10(-5) M after 60 minutes. Binding studies showed that 6, at 1 x 10(-6) M, did not modify the KD of Prazosin and caused a 30% decrease of the Bmax. Substitution in 6 of the bis (2-chloroethyl)amino moiety for the cyano-NNO-azoxy function afforded 7 which behaves as an irreversible antagonist able to change KD of Prazosin without influencing Bmax.