BRAIN-SPECIFIC DRUG DELIVERY

Field of the Invention;

The present invention relates to a dihydro- pyridine/pyridinium salt type of redox system for the *•* ^■ site-specific or sustained delivery (or both) of a wide variety of drug species to the brain. More especially, this invention relates to the discovery that a biologically active compound coupled to a lipoidal carrier moiety comprising a dihydropyridine nucleus readily and easily penetrates the blood-brain barrier ("BBB") and attains increased levels of concentration in the brain; oxidation of the dihydropyridine carrier moiety j-n vivo to the ionic .pyridiniu salts prevents its elimination from the brain, while elimination from the general circulation is accelerated, resulting in significant and pro- longedly sustained brain-specific drug activity, whether ascribable to the cleavage of the [D-QC] entity and sustained release of the drug in the brain and/or to [D-QC] itself.

Background Art;

The delivery of drug species to the brain is ofttimes seriously limited by transport and metabolism factors and, more specifically, by the functional barrier of the endothelial brain capillary wall deemed the blood-brain barrier, BBB. Site-specific delivery and sustained delivery of drugs to the brain are even more difficult, and to date no useful simple or generic techniques to achieve such phenomena are known to the art.

Indeed, the barriers separating plasma from the brain and cerebrospinal fluid (CSF) are complex systems involving passive and active transport and subserve a number of important functions. The boundary between plasma and the central nervous system (CNS) is much less permeable than that between plasma and other tissue cells to a variety of water soluble substances, such as organic electrolytes, organic acids and bases, as well as to large molecules such as proteins. Such a barrier also provides a path for clearance from the brain of the breakdown products of cellular metabolism. The CNS and its fluids can be considered basically a three-compartment syste i the blood or the plasma, CSF and brain tissue. There is a diffusion-controlled exchange between CSF and the extracellular fluid (CF) of the brain. It has also been suggested that the permeabilities of blood-CSF and blood-brain barriers are practically identical with respect to drugs and other foreign substances. Mayer et al, J. Pharmacol, and Exp. Therap., 125, 185 (1959).

The BBB is, moreover, basically the result of the fact that the endothelial cells in the brain capillaries are joined by _. continuous, tight inter- cellular junctions, such that material has to pass through the cells rather than between them in order to move from blood to brain. It is interesting that • there are areas within the brain, such as the sub- fornical body and the postremia in which the capillary cells are not closely linked so that they lack the characteristics of the BBB. They pro¬ vide the entry of small amounts of compounds which would not ordinarily enter the barriers. Hoffmann and Olszewzki, Neurology (Minneap.) , 11, 1081 (1961) .

Foreign compounds which enter organs other than the central nervous system with ease, may pene¬ trate the CNS slowly or hardly at all. A number of theories concerning the nature of the barrier have been proposed. The widely accepted concept describes the boundary as a fat-like layer interspersed with small pores, although the BBB is not a simple, anatomically well-defined unitary physical entity. Shuttleworth, Prog. Ex . Tumor Res. , 17, 279 (1972) . Penetration of such a barrier may occur by several processes: lipid soluble substances may passively penetrate into the cells, while small molecules such as water and urea may pass through the pores. In addition to these simple physical processes, carrier- mediated and active transport processes govern the movement of many molecules through the BBB. Thus, it is generally accepted that lipid solubility, degree of ionic dissociation or protonation and the ability of temporary combination with membrane constituents affect delivery through the BBB. It has been shown, for example, that in the class of barbiturates, a quantitative correlation could be established between their ease to pass into the brain (as reflected by the different times of onset of anesthetic action) and their lipid/water partition coefficient. Mark et al, J. Pharmacol, and Exp. Therap., 123, 79 (1957). The role of lipid solubility in drug penetration through the BBB is also exemplified by the better absorption of the sparingly water-soluble thiamine propyl disulfide (TPD) as compared to the water- soluble thiamine hydrochloride (THC1) . Thomson et al, Ann. Int. Med. , 74, 529 (1971). Some materials such

as glucose and amino acids are transported by active mechanism, characterized by saturation, bidirectional molecular specificity, bidirectional competitive inhibition and bidirectional countertransport. Fishman, Am J. Physiol., 206, 836 (1964).

Changes in permeability of the BBB can be caused by several pathological and toxicological processes. Pardridge, Connor and Crawford, CRC Crit. Rev. Toxicol., 179. (1975) . A general increase in the barrier permeability, such as a nonspecific breakdown of the barrier has, however, severe consequences, including cerebral edema.

It too is well documented that the BBB is rela¬ tively impermeable to the ionized forms of drugs and other molecules. Drugs which are weak organic electro¬ lytes appear to pass from blood to CSF to reach a steady state ratio characteristic of each molecule according to its pKa and the existence of a normal pH gradient between blood and CSF. It is clear that it is the most difficult for quaternary pyridinium or ammonium salts to penetrate the BBB.

And removal of substances from the brain and CSF is .obviously a significant factor in regulating drug concentrations in the CNS. There are several efflux processes: bulk flow via the arachnoid villi, diffusion of lipid soluble substances into brain and blood, active ' transport and metabolism by adjacent meninges. Once a drug or metabolite enters the CSF from blood or brain by simple diffusion, it may rapidly be removed, either 0 by nonselective bulk flow or by active transport mechanism associated with the choroid plexus or other nondefined structures in the CSF compartment. ^" It is generally accepted that highly lipid-soluble drugs

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leave the CSF more rapidly than poorly lipid-soluble ones, but the barrier to passage of compounds from CSF has only superficial similarity to the blood-CSF barrier. Drug elimination processes from the brain are significantly directly related to drug accumulation in the brain. It is generally assumed that efflux in the opposite direction involves almost the same processes as for entry, except that the role of the bulk flow and the metabolic processes in the brain are not to be overlooked.

The two elimination processes studied in the earlier literature and which can be said to have a certain bearing on the present invention involve elimination from the brain of ionic species. Thus, it is found that non-metabolized inonic species, such as the acetate ion, have a three times slower elimina¬ tion rate from the CSF than from the blood. Freundt, Arz., Forsch., 23, 949 (1973) . An even more dramatic change in the elimination rate was found in the case of a quaternary piperidinium salt. The quaternary salt, formed in_ situ after delivery of a haloalkylamine, which undergoes cyclization to the quaternary salt, in the brain, as well, was found to have an at least ten times slower elimination rate from the brain than from the rest of the body. It was concluded by the authors (Ross and Fro en, Eur. J. Pharmacol., 13, 46 [1970] that the outflow rate of the quaternary salt corresponded to the inflow rate. Similar results were obtained for the erythrocytes: the efflux of the quaternary salt was very slow. Ross, J. Phar . Pharmacol., 27, 322 (1975) .

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And while it too has been suggested to deliver a drug species, specifically N-methylpyridinium-2- carbaldoxime chloride (2-PAM) , into the brain, the active nucleus of which in and of itself constituting a quaternary pyridinium salt, by way of the dihydro¬ pyridine latentiated prodrug form thereof, such approach is conspicuously delimited to relatively small molecule quaternary pyridinium ring-containing drug species and does not provide the overall ideal result of brain- specific, sustained release of the desired drug, with concomitant rapid elimination from the general circula¬ tion, enhanced drug efficacy and decreased toxicity. Hence, no "trapping" in the brain of the 2-PAM formed in situ results, and obviously no brain-speci ic, sustained delivery occurs as any consequence thereof: the 2-PAM is eliminated as fast from the brain as it is from the general circulation and other organs. Compare U.S. Patents Nos. 3, 29^13 and 3.962.447: Bodor et al, J. Pharm. Sci., 67, No. 5, 685 (1978). It has also been speculated to deliver, e.g., an antitumor agent into the brain by utilizing a dihydropyridine/pyridinium redox carrier moiety therefor, but this particular hypothesis necessarily entails derivatizing the dihydropyridine/pyridinium carrier with a substituent R, itself critically designed to control the release rate of the active drug species from the quaternary drivative thereof, as well as being critically functionally coordinated with the particular chemical and therapeutic activity/nature of the anti- tumor drug species itself; Bodor et al, J. Pharm. Sci., supra.

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Accordingly, acutely serious need exists in this art for a truly effective generic but nonethe¬ less flexible method for the site-specific, or sustained delivery, or both, of drug species to the brain, while at the same time avoiding the aforesaid noted and notable disadvantages and drawbacks associated with penetration of the blood-brain barrier, with dihydropyridine latentiated prodrug forms of drug species themselves comprising a pyridinium salt active nucleus, and with the necessity for introducing critically coordinated and designed, release rate- controlling substituents onto any particular drug carrier moiety.

Recently, Bodor et al, Science, Vol. 214, December 18, 1981, pp. 1370-1372, have reported on site-specific sustained release of drugs to the brain. The Science publication outlines a scheme for specific and sustained delivery of drug species to the brain, as depicted in the following Scheme 1:

Scheme 1; BBB, blood-brain barrier.

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According to the scheme in Science, a drug [D] is coupled to a quaternary carrier [QCj ⁺ and the [D-QC] which results is then reduced chemically to the lipoidal dihydro form [D-DHC] . After administra- tion of [D-DHC] in vivo, it is rapidly distributed throughout the body, including the brain. The dihydro form [D-DHC] is then in situ oxidized (rate constant, k* _| _) (by the NAD *^=^= NADH system) to the ideally inactive original [D-QC] ⁺ quaternary salt which, because of its ionic, hydrophilic character, should be rapidly eliminated from the general circulation of the body, while the blood-brain barrier should prevent its elimination from the brain (k^>> _? ; k_>> k_) . Enzymatic cleavage of the [D-QC] ⁺ that is "locked" in the brain effects a sustained delivery of the drug species [D] , followed by its normal elimination (k5) , metabolism. A properly selected carrier [QC] ⁺ will also be rapidly eliminated from the brain (kg >>k2). Because of the facile elimination of - [D-QC] ⁺ from the general circulation, only minor amounts of drug are released in the body (kβ >>k ); [D] will be released primarily in the brain (k4 > k ₂ ) . The overall result ideally will be a brain-specific sustained release of the target drug species.

Bodor et al have reported,in Science, their work with phenylethylamine as the drug model, which was coupled to nicotinic acid, then quaternized to give compounds of the formula

which weresubsequently reduced by sodium dithionite to the corresponding compounds of the formula

Testing of the N-methyl derivative n vivo supported the criteria set forth in Scheme 1. Bodor et al speculated that various types of drugs might possibly be delivered using the depicted or analogous carrier systems and indicated that use of N-methylnicotinic acid esters and amides and their pyridine ring- substituted derivatives was being studied for delivery of amino- or hydroxyl-containing drugs, including small peptides, to the brain. No other possible specific carriers were disclosed.

Other reports of Bodor et al*s work have appeared in The Friday Evening Post, August 14, 1981, Health Center Communications, University of Florida, Gainesville, Florida; Chemical & Engineering News, December 21, 1981, pp. 24-25; and Science News, January 2, 1982, Vol..121, No. 1, page 7. These publications do not suggest any carrier systems other

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than the specific N-methyl and N-benzyl nicotinic acid-type carriers disclosed in the Science publication, Other classes of drugs as well as a few specific drugs are mentioned as possible candidates for 5 derivativization; for example, steroid hormones, cancer drugs and memory enhancers are indicated as targets for possible future work, as are enkephalins, and specifically, dopamine and testosterone. The publications do not suggest how to link such drugs 10 to the carrier, except possibly when the drugs are simple structures containing a single NH ₂ or, perhaps, simple structures containing a single OH, of the primary or secondary type, as is the case, with phenylethylamine or testosterone. There is, 15 for example, no suggestion of how one of ordinary skill in the art would form a drug-carrier combina¬ tion when the drug has a more complicated chemical structure than phenylethylamine, e.g., dopamine or an enkephalin. Thus, except in a very limited 20 area, where only a combination of certain types of drug structures with certain utility classes and certain carrier structures is taught or suggested, the publications simply do not make the broad concept of the invention as represented by Scheme 1 available 25 to the public. Likewise, the many embodiments of the invention as described hereinafter are unsuggested by the art.

It is also known to this art that Parkinsonism, a striatal dopamine deficiency syndrome [H. Ehringer 30 and 0. Hornykiewicz, Klin. sch., 38, 1236 (I960)],

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cannot be treated directly with dopamine, for dopamine and related catechola ines also do not cross the blood-brain barrier [B. E. Roos and G. Steg, Life Sci., 3_, 351 (1964)]. L-Dopa, considered as a prodrug for dopamine, was first discovered to be useful in the treatment of Parkinsonism more than twenty years ago [A. Barbeau, Excepta Medica, Int. Congr. Ser. , 38, 152 (1961) ; W. Birkmayer and 0. Homykiewicz, ien. Klin. ochnenschr. , 73, 787 (1961)]. Indeed, L-Dopa is considered to be the best available treatment for Parkinsonism, but, unfortunately, at the expense of a wide variety of undesirable side effects IA.Barbeau, TIPS, 2 , (11), 29 _. 7 (1981)]. The peripheral side effects of L-Dopa, which range from nausea and vomiting to cardiac arrythmias and hypotension, appear to be due to one or more of the metabolic products thereof, rather than L-Dopa per se. L-Aromatic amino acid decarboxylase enzyme is responsible for the major metabolism of L-Dopa, whether prior, during or after absorption. Concurrent administration of L-Dopa with an inhibitor of aromatic amino acid decarboxylase, which should not be able to penetrate the BBB, reduces ^' the decarboxylation of L-Dopa in peripheral tissues. Such reduction allows higher proportions of L-Dopa to reach the CNS and at the same time diminishes the peripheral side effects considerably, particularly vomiting and cardiac arrythmias, but a number of serious side effects still persist [A.Barbeau, TIPS supra; A.Barbeau and M.Roy, Neurology, 26, 399 [(1976)].

Attempts have also been made to alleviate the well- known dissolution, absorption and metabolism problems of L-Dopa [H. Ninterberger, Biochem. Med., j>, 412 (1971); H. Shindo, T. Komai, K. Tanaka, E. Nakajima and N. Miyakoshi, Chem. Pharm. Bull., 21, 826 (1973); C. 0. Rutledge and M. M. Hoehn, Nature (London) , 244, 447 (1973); R. L. Bronaugh, R. J. McMurty, M. M. Hoehn and C. 0. Rutledge, Biochem. Pharmacol., 24, 1317 (1975)], employing prodrug approaches [N. Bodor, K. B. Sloan, T. Higuchi and K. Sasahara, J. Med. Chem. , 20, 1435 (1977); A. M. Felix, D. P. Winter, S. S. Wang, I. D. Kulesha, W. R. Pool, D. L. Hane and H. Sheppard, J. Med. Chem., 17' 422 (1974)].

Additionally, dopamine agonists, which are used in the treatment of hyperprolactinemia associated with pituitary adenomas or amenorrhea [R. F. Spark and G. Dickenstein, Ann. Int. Med., 90, 949 (1979)], also induce unwanted side effects.

Thus, especially acutely serious need exists in this art to delivery a dopaminergic agent directly and specifically to the brain, in a sustained manner, and there elicit the desired dopaminergic response, e.g., for the treatment of Parkinsonism or hyperprolactinemia. The existence of a blood-testis barrier (BTB) by which some substances are prevented from being carried into the seminiferous tubules long has been suspected P.P.H. Bruyn, R.C. Robertson and R.S. Farr, Anat. Rec, 108, 279 (1950); R.J. Goldacre and B. Sylven, Nature (London) , 184, 63 (1959); R.J. Goldacre and B. Sylven, J. Cancer, 16, 306 (1962); T.S. Ro and H. Busch, Biochem. Biophys Acta (Amst),108, 317 (1965)].

Some investigators have suggested a similarity between the BBB and the BTB [A.T. Cowie, A.K. Lascelle and J.C. Wallace, J. Physiol. (London), 171, 1976 (1964); R.E. Mancini, 0. Vilar, B. Alvarez and A.C. Seiguer, J. Histochem. Cytochem., 13, 376 (1965) ; M. Kormano, Acta Physiol. Scand., 71, 125 (1967) ; M. Kormano, Histochemic, g, 327 (1967)]. Don W. Fawcett, Lee V. Leak and Paul M. Heidger, Jr., J. Reprod. Fert. "Suppl. 10, 105 (1970) have suggested that the permeability barrier is not in the testis capillary walls because these more closely resemble the capillaries of muscle than those involved in the BBB. M. Dym and Don W. Fawcett, Biology of Reproduction, 3_, 308 (1970) concluded that the epithelioid contractile layer around the seminiferous tubules constitutes a significant permeability barrier augmented by an apparently more efficient barrier involving tight cell-to-cell junctions between sertoli cells that inhibits penetration of substances through the germinal epithelium. Despite such histological differences, pharmacokinetic studies [K. Okumura,

I-P. Lee and R.L. Dixon, J. Pharmacol . Ex . Therap. , 194, 89 (1975); I.P. Lee and R.L. Dixon, Environmental Health Perspectives, 24, 117 (1978)] have demonstrated that the functional BTB resembles the BBB in transport characteristics, both depending on lipid solubility and molecular size. Thus, delivery of drug species to the testes is often seriously limited by the blood- testis barrier. Site-specific delivery and sustained delivery of drugs to the testes are even more diffi¬ cult. To date, no useful simple or generic techniques

to achieve such results are known to the art. It is thus apparent that a serious need exists in this art for a method for the site-specific and/or sus¬ tained delivery of drug species to the testes to elicit the desired therapeutic, e.g., hormonal or tumor-inhibiting, response.

Summary and Objects of the Invention:

Accordingly, a major object of the present invention is the provision of a generic method for the specific and/or target enhanced delivery to the brain of a wide variety of drug species and to achieve brain-speci ic drug delivery by effecting the bidirectional transport of the drug species into and out of the brain employing dihydropyridine =^= pyridinium salt carrier type redox systems.

Another object of the invention is to provide for brain specific drug delivery utilizing a dihydro¬ pyridine = ^= pyridinium salt carrier type redox system, which drug/carrier system is characterized by enhanced drug efficacy and decreased toxicity. Indeed, consistent herewith systemic toxicity is significantly reduced by accelerating the elimination of the drug/quaternary carrier system, and even central toxicity is reduced by providing a low level, sustained release of the active drug species in the brain.

Yet another object of this invention is the provision of a chemical delivery system for the site- specific and sustained release of drug species to the brain, and one in which a special pro-prodrug reduced

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form of an active drug species is actually delivered to the body of a patient, not a prodrug as such and not a drug/carrier entity necessarily comprised of critically tailored release rate-controlling substituent(s) .

Still another object of this invention is to provide an effective testicular specific chemical delivery system, for the site-specific and/or sustained eliciting of significant drug response in the testes.

Yet another object of this invention is to provide enhanced delivery to the brain and testes of a wide variety of centrally acting agents which are not themselves able to penetrate the blood-brain and blood-testis barriers to any considerable extent. In accord with the foregoing, the present inven¬ tion provides compounds adapted for the site-specific/ sustained delivery of a centrally acting drug species to the brain, said compounds being: (a) compounds of the formula

[D-DHC] (I) wherein [D] is a centrally acting drug species, and

[DHC] is the reduced, biooxidizable, blood-brain barrier penetrating lipoidal form of a dihydropyridine *^=^ pyridinium salt redox carrier, with the proviso that when [DHC] is

oMH

wherein R is lower alkyl or benzyl and [D] is a drug species containing a single NH ₂ or OH functional group, the single OH group when present being a primary or secondary OH group, said drug species being linked directly through said NH or OH func¬ tional group to the carbonyl function of [DHC] ,. then [D] must be other than a sympathetic stimulant, steroid sex hormone, memory enhancer, long chain alkanol or anticancer or antitumor agent; or (b) non-toxic pharmaceutically acceptable salts of compounds of formula (I) wherein [D] is a centrally acting drug species and [DHC] is the reduced, bio¬ oxidizable, blood-brain barrier penetrating lipoidal form of a dihydropyridine **^=^* pyridinium salt redox carrier. In another aspect, the present invention ^' provides compounds having the formula

[D-QC] ⁺ (II) wherein [D] is a centrally acting drug species and [QC] ⁺ is the hydrophilic, ionic pyridinium salt form of a dihydropyridine = ^= pyridinium salt redox carrier, with the- proviso that when [QC] ^÷ is

wherein R is lower alkyl or benzyl and [D] is a drug species containing a single NH ₂ or OH functional group, the single OH group when present being a primary or

secondary OH group, said drug species being linked directly through said NH ₂ or OH functional group to the carbonyl function of [QC] ⁺ , then [D] must be other than a sympathetic stimulant, steroid sex hormone, memory enhancer, long chain alkanol or anticancer or antitμ or agent. The present invention further provides a generic method for specific and/or target enhanced delivery to the brain of a wide variety of centrally acting drug species, such brain-specific drug delivery being effected via the bidirectional transport of the drug species into and out of the brain by means of dihydropyridine pyridinium salt carrier type redox systems.

In another aspect, the present invention provides, as an effective drug delivery system, compounds having the formula

[D-DHC] (I) and non-toxic pharmaceutically acceptable salts thereof, wherein [D] is a testicularly acting drug species and [DHC] is the reduced, biooxidizable, blood-testis barrier penetrating, lipoidal form of a dihydropyridine = =^= pyridinium salt redox carrier. In still another aspect, the present invention provides compounds having the formula [D-QC] ⁺ (II) wherein [D] is a testicularly acting drug series and [QC] ⁺ is the hydrophilic, ionic pyridinium salt form of a dihydropyridine .. *■> pyridinium salt redox carrier.

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In both of these aspects of the invention, compounds excluded by the provisos set forth in the preceding paragraph are likewise excluded here. Also according to this invention, there is hereby provided a generic method for specific and/or target enhanced delivery to the testes of a wide variety of testicularly acting drug species, such testicular-specific drug delivery being effected via the bidirectional transport of the drug species into and out of the testes by means of dihydropyridine =^= pyridinium salt carrier type redox systems.

In yet another aspect, the present invention provides, as an effective dopaminergic chemical delivery system, compounds having the formula

[D-DHC] (I)

and non-toxic pharmaceutically acceptable salts thereof, wherein [D] is a dopamine having the structural formula

in which each Y is independently hydrogen or a hydro- lytically or metabolically cleavable hydroxyl protec¬ tive group, and [DHC] is the reduced, biooxidizable, blood-brain barrier penetrating, lipoidal form of a dihydropyridine pyridinium salt redox carrier.

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In still another aspect, the present invention provides compounds having the formula

[D-QC] ⁺ (II)

wherein [D] is a dopamine having the structural formula

in which each Y is independently hydrogen or a hydro- lytically or metabolically cleavable hydroxyl protec¬ tive group, and [QC]" ^1* is the hydrophilic, ionic pyridinium salt form of a dihydropyridine -^^

10 pyridinium salt redox carrier.

Briefly, one presently preferred chemical delivery system for dopamine according to this invention has the structure (2_) in the following Scheme 2, and wherein the amino function of dopamine is appropriately linked to

15 the dihydropyridine-type carrier system, while the catechol function is advantageously protected, for example, as a corresponding ester function, e.g., the dipivalyl ester illustrated. The brain-specific delivery of dopamine, or the otherwise eliciting of a

20 dopaminergic response, requires a succession, of processes, including oxidation of the dihydropyridine- ring to the corresponding pyridinium salt (for example, structure 2 ) , which provides the basis for "locking-in" the brain the molecule, hydrolysis of the, e.g., pivalyl esters (see

25 structure 4) likely via the 3- and/or 4-monopivalyl esters and, finally, the release of dopamine (1_) from

4_, which can be either a hydrolysis or a reductive process [a possible reductive release of dopamine was very recently suggested by a model for a pre- synaptic terminal, L. L. Miller, A. N. K. Lau and E. K. Miller, J. Am. Chem. Soc, 104, 5242 (1982)].

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As per the above Scheme 2, for the brain specific delivery of dopamine (1) , structure 2_ is one chemical delivery system consistent herewith, and 4 _^ is one precursor locked in the brain and eliminated rapidly from the rest of the body. Structures 3 depict inter¬ mediates formed during the stepwise hydrolysis and oxidation processes.

Brief Description of the Drawings:

FIGURE 1 is a graph plotting the time course of l-methyl-3-{N-[£ - (3,4-dipivalyloxyphenyl)-ethyl] }- carbamoy1-1,4-dihydropyridine 5c (O) and its products, monopivalyl-dihydro derivative 11 _^ ( V ) , the dihydro- dopamine derivative _5a ( A ) and the quaternary dopamine precursor 6a (•) in plasma; FIGURE 2 is a graph plotting the time course of l-methyl-3-{N-[ 3 -(3,4-dipivalyloxyphenyl)-ethyl] }- carbamoyl-1,4-dihydropyridine 5c (0) and its products, monopivalyl-dihydro drivative 11 _^ ( V ) and the quaternary dopamine precursor 6a. (•) in whole blood; FIGURE 3 is a graph plotting the time course of l-methyl-3-{N-[β-(3,4-dipivalyloxyphenyl)-ethyl] }- carbamoy1-1,4-dihydropyridine ]5c (O) and its products, monopivalyl-dihydro derivative 11 ( V ) , the dihydro- dopamine derivative 5_a ( A ) and the quaternary dopamine precursor 6a. (t) in 20% brain ho ogenate;

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FIGURE 4 is a graph plotting the time course of l-methyl-3-{N-[β-(3,4-dipivalyloxyphenyl)-ethyl] }- carbamo 1-1,4-dihydroxypyridine 5c (O) and its product, the quaternary dopamine precursor 6a (•) in 20% liver homogenate;

FIGURE 5 is a semilog plot of peak heights of l-methyl-3-{N-[β-(3,4-dipivalyloxyphenyl)-ethyl] }- carbamoyl-l,4-dihydropyridine 5c against time in plasma (•) , brain homogenate (A) , whole blood (O) ^' and liver homogenate (D);

FIGURE 6 is a graph plotting concentrations against time of l-methyl-3-{N-[0~(3,4-dihydroxyphenyl)- ethyl] }carbamoy1 pyridinium cation (6a) in brain (•) and in blood (0) following administration of 1-methyl- 3-{N-[β-(3,4-dipivalyloxyphenyl)-ethy-li } carbamoyl-1, 4-dihydropyridine (5c) , with the error bars indicating SEM;

FIGURE 7 is a graph plotting the effects of compounds j _> c (A) and 6a (•) administered I.V. at 1 mg/kg dose level, on the serum prolactin levels in rats; and

FIGURE 8 is a graph plotting concentrations with standard errors against time for testosterone-17- nicotinate-N-methyl cation, calculated as iodide, in brain (O) and in blood ( □ ) and concentration of released testosterone (ng/g) in brain (T) , all following administration of the corresponding dihydro¬ pyridine compound. Also plotted are concentrations of testosterone in brain (t) and blood ( ■ ) following administration of testosterone, per se.

Detailed Description of the Invention:

More particularly in accord with the present invention, the following definitions are applicable: The term "lipoidal" as used herein is intended to designate a carrier moiety which is lipid-soluble or lipophilic.

The expression "hydroxyl protective group" is intended to designate a group which prevents premature metabolism of an OH group or groups prior to the compound's reaching the desired site in the body. Typical hydroxyl protective groups contemplated by the present invention (e.g., for Y in the case of the dopamine derivatives) are acyl groups and carbonates. When the hydroxyl protective group is acyl (i.e., when it is an organic radical derived from a carboxylic acid by removal of the hydroxyl group) , it preferably represents an acyl radical selected from the group consisting of alkanoyl having 2 to 8 carbon atoms; alkenoyl having one or two double bonds and 3 to 8 carbon atoms;

H cycloalkyl-C H ₂ -C-

wherein the cycloalkyl portion contains 3 to 7 ring atoms and n is zero, one, two or three; phenoxy- acetyl _f pyridinecarbonyl; and

O

II phenyl-CnH~2n-C-

wherein n is zero, one, two or three and phenyl is unsubstituted or is substituted by 1 to 3 alkyl

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each having 1 to 4 carbon atoms, alkoxy having 1 to 4 carbon atoms, halo, trifluoromethyl, dialkyla ino having 2 to 8 carbon atoms or alkanoyl- amino having 2 to 6 carbon atoms. When the acyl group is alkanoyl, there are included both unbranched and branched alkanoyl, for example, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, 2-methylbutanoyl, pivalyl (pivaloy1) , 3-methylpentanoy1 , 3,3-dimethylbutanoy1 ^' , 2,2-dimethylpentanoy1 and the like. Pivalyl and isobutyryl are especially preferred.

When the acyl group is alkenoyl, there are included, for example, σrotonyl, 2,5-hexadienoyl and 3,6-octadienoy1. When the acyl group is Q

II cycloalkyl-C H ₂ -C-

there are included cycloalkanecarbonyl and cyclo- alkanealkanoyl groups wherein the cycloalkane portion can optionally bear 1 or 2 alkyl groups as substituents, e.g. cyclopropanecarbonyl, 1-methylcyclopropanecarbonyl, cyclopropaneacetyl, α-methylcyclopropaneacetyl, 1-methylσyclopropaneacetyl, cyclopropanepropionyl, α-methylcyclopropanepropionyl, 2-isobutylcycloρro- panepropionyl, cyclobutanecarbonyl, 3,3-dimethylcyclo- butanecarbonyl, cyclobutaneacetyl, 2,2-dimethyl-3- ethylcyclobutaneacetyl, cyclopentanecarbonyl, cyclohexaneacetyl, cyσloheptanecarbonyl and cyclo- heptanepropionyl.

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A . ^WIPO

When the acyl group is pyridinecarbonyl, there are included picolinoyl (2-pyridinecarbonyl) , nicotinoyl (3-pyridinecarbonyl) and isonicotinoyl (4-pyridine¬ carbonyl) . When the acyl group is phenyl-C _n H _2n -C-

there are included, for example, benzoyl, phenylacetyl, α-phenylpropionyl, β-phenylpropionyl, p-toluyl, m-toluyl, o-toluyl, o-ethylbenzoyl, p-tert-butylbenzoyl, 3,4-dimethylbenzoy , 2-methyl-4-ethylbenzoyl, 2,4,6- trimethylbenzoyl, m-methylphenylacetyl, p-isobutyl- phenylacety1, β-(p-ethylphenyl)-propionyl, p-anisoyl, m-aπisoyl, o-anisoyl, m-isopropoxybenzoyl, p-metho- xyphenylacetyl, m-isobutoxyphenylacetyl, m-diethyl- aminobenzoyl, 3-methoxy-4-ethoxybenzoyl, 3,4,5- trimethoxybenzoyl, p-dibutylaminobenzoyl, p-n-butoxy- benzoyl, 2,4,6-triethoxybenzoyl, 3,4-diethoxyphenyl- acetyl, β-(3,4,5-trimethoxypheny1)propionyl, o-iodobenzoyl, m-bromobenzoyl, p-chlorobenzoyl, p-fluorobenzoyl, 2-bromo-4-chlorobenzoyl, 2,4,6- trichlorobenzoyl, p-chlorophenylacetyl, α-(m-bromo- pheny1) ropionyl, p-triflύoromethylbenzoyl, 2,4-di(trifluoromethy1)benzoyl, -trifluoromethyl- phenylacetyl, β-(p-trifluoromethylphenyl)propionyl, 2-methyl-4-methoxybenzoyl, 3-chloro-4-ethoxybenzoyl, β-(3-methyl-4-chlorophenyl)propionyl, p-dimethyl- aminobenzoyl, p-(N-methyl-N-ethylamino)benzoyl, o-acetamidobenzoyl, m-propionamidobenzoyl , 3-chloro-4- acetamidophenylacetyl and p-acetamidophenylpropionyl.

When the hydroxyl protective group is a carbonate grouping, it has the structural formula

O II

Y*-O-C-

i.e., it is an organic radical which can be considered to be derived from a carbonic acid by removal of the hydroxyl group from the COOH portion. Y' preferably represents alkyl having 1 to 7 carbon atoms; alkenyl having one or two double bonds and 2 to 7 carbon atoms;

cycloalkyl-CnH2„n-

wherein the cycloalkyl portion contains 3 to 7 ring atoms and n is zero, one, two or three; phenoxy; 2-, 3- or 4-pyridyl; or

phenyl-C _n H _2n -

wherein n is zero, one, two or three and phenyl is unsubstituted or is substituted by 1 to 3 alkyl each having 1 to 4 carbon atoms, alkoxy having 1 to 4 carbon atoms, halo, trifluoromethy1, dialkylamino having 2 to 8 carbon atoms or alkanoylamino having 2 to 6 carbon atoms. Most preferably, Y' is C _j -C- alkyl, particularly ethyl or isopropyl.

Similarly, the expression "carboxyl protective group" is intended to designate a group which prevents premature metabolism of a COOH group or groups prior to the compound's reaching the desired site in the body. Typical carboxyl protecting groups are the

groups encompassed by Y' above, especially C,-C_ alkyl, particularly ethyl or isopropyl.

By "centrally acting" drug species, active agent or compound as utilized herein, there is of course intended any drug species or the like, the principal pharmacological activity of which is CNS and a result of direct action in the brain.

Exemplary .such centrally acting drug species are the CNS-amines and other nervous system agents, whether sympathetic or parasympathetic, e.g., phenylethylamine, dopamine, tyramine, L-DOPA, muscle relaxants, tranquilizers and antidepressants, e.g., benzodiazepine tranquilizers such as diazepam and oxazepam, phenothiazine tranquilizers such as carphenazine, fluphenazine and the like, mild and strong analgesics and narcotics, sedatives and hypnotics, narcotic antagonists, vascular agents, stimulants, anesthetics, small peptides, such as the di-, tri, tetra- and pentapeptides, and other small 6-20 aminoacid unit containing peptides, e.g., the enkephalins (for example, Tyr-Gly-Gly-Phe-Leu) , which, besides being analgesics, initiate epileptic activity in the brain at doses that are about tenfold lower than for effecting analgesic activity, larger peptides, such as pituitary hormones and related agents, anti- epileptic and anticonvulsant drugs generally, including hydantoins such as phenytoin and ethotoin, barbituates such as phenobarbital, hormones, such as the steroid hormones, e.g., estradiol, testosterone, 17 α-ethynyl testosterone, and the like (recent studies on

histological mapping of hormone-sensitive and specific steroid binding cells in the brain have underscored the importance of the steroid action in the brain on sexual behavior) , amphetamine-like drugs, anticancer 5 and anti—Parkinsonism agents, antihypertensives, agents to enhance learning capacity and the memory processes, including treatment of dementias, such as Alzheimer's disease, antibacterials, centrally active hypotensive agents, diagnostic agents, such as radio- 10 pharmaceuticals, monoamine oxidase (MAO) inhibitor drugs, CNS or brain important/essential amino acids, such as tryptophan, and any like centrally acting compounds.

Other illustrative ultimate species of centrally

^•• - ^■■ • ^• active drug entities are: amphetamine, dextroampheta ine, levamphetamine, ethamphetamine, phenmetrazine and phentermine, which are stimulants and appetite suppres¬ sants; codeine, oxycodone, pentazocine, anileridine, hydromorphone, morphine and oxymorphone, which are 0 narcotic analgesics; desipra ine, nortriptyline, opipramol and protriptyline, which are stimulates used, e.g., in endogenous depressions; clonidine and methyldopa, which are sympatholytic agents used, e.g., inhypertension; biperiden, cycrimine and procyclidine, which are centrally 5 acting anticholinergics; tranylcypromine , a sympathomi- metic stimulant and MAO inhibitor; acetophenazine, carphenazine, fluphenazine, perphenazine and piperaceta- zine, which are phenothiazine-type tranquilizers; chlordiazepoxide, clorazepate, nitrazepa and 0 temazepam, which are benzodiazepine-type tran¬ quilizers; haloperidol and clopenthixol, which are transquilizers; norepinephrine, a sympathetic stimulant/ adrenergic agent; nalorphine and naloxone, narcotic

antagonists; hydralazine, a hypotensive; ethotoin, phenobarbital and aminoglutethimide, anticonvulsants; ethami an, a medullary stimulant; bemegride, a barbiturate antagonist; amipheπazole, a stimulant; iopydol, iodopyracet, iodouppurate, iodamide and iopanoic acid, which are radiodiagnosticsr ephedrine, pseudoephedrine, oxymetazoline and phenylephrine, which are sympathomimetic amines and decongestants _? estradiol, estrone and estriol, the natural estrogens γ amoxicillin, oxacillin, carbenicillin and ampicillin, peπcillin-type antibiotics; amobarbital, a sedative; trihexyphenidyl, a centrally acting anticholinergic; hydroxyzine, a tranquilizer; chlortetracycline, tetracycline and methacycline, which are tetracycline- type antibiotics; clindamycin, lincomycin, nalidixic acid, oxolinic acid and phenazopyridine, antibacterials/ antibiotics; flurazepam, bromazepam and lorazepam, tranquilizers; phenytoin, an anticonvulsant; glutethimide, a mild hypnotic/sedative; bethanidine and guanethidine, hypotensives/sympatholytics; methyprylon propranolol, a β-blocker antihypertensive; dicloxacillin, an antibacterial; butalbital, a barbiturate sedative; GABA, γ-vinyl GABA, γ-acetylenic GABA, neurotransmitters for possible use in epilepsy; valproic acid and its metabolites such as 5-hydroxy-2-n-propyl- pentanoic acid, 4-hydroxy—2-n-propylpentanoic acid, 3-hydroxy-2-n-propylpentanoic acid, for use as anticon¬ vulsants; apomorphine, a narcotic depressant; ethotrexate, podophyllotoxiri derivatives (etoposide, teniposide) , doxorubicin, daunomycin and cyclophosphamide, anticancer/antitumor agents; methylphenidate, a stimulant; thiopental, an anesthetic? naloxone, a narcotic antagonist; ethinyl estradiol and mestranel, estrogens;

O PI

norgestrel and norethindrone, progestins; cephalothin, cephalexin and cefoxitin, cephalosporin antibiotics; atenolol and metoprolol, β-blockers/hypotensives; ACTH corticotropin) ; LH-RH, a neurotransmitter; sulfa- diazine and other sulfonamide antibiotics; ribavirin and acyclovir, antiviral agents; chlorambucil and melphalan, nitrogen mustard-type anticancer/antitumor agents; methotrexate and aminopterin, which are folic acid antagonist-type anticancer/antitumor agents; cisplatin-analogue type anticancer/antitumor agents; doxorubicin, daunamycin, dactinomycin and mitomycin C, used in cancer chemotherapy; thioguanine, a purine/ pyrimidine antagonist used in cancer treatment? vincristine and vinblastine, anticancer alkaloids; hydroxyurea and DON, anticancer urea derivatives; FSH and HCS, pituitary gonadotropins; N,N'-bis- (dichloracetyl)-1,8-octamethylene diamine, an agent for male fertility inhibition? levorphanol, a narcotic analgesic; and benzestrol and diethylstilbestrol, synthetic estrogens.

Preferred classes of centrally acting drugs for use herein are the central neurotransmitters, steroids, anticancer and antitumor agents, antiviral agents, tranquilizers, memory enhancers ^• and hypotensives. Among the neurotransmitters, there can be mentioned amino acids, such as GABA, glycine, glutamic acid and aspartic acid; catecholamines, such as dopamine, norepinephrine and epinephrine; serotonin and tryptamine; and peptides such as neurotensin, luteinizing hormone- releasing hormone (LHRH), somatostatin, enkephalins such as met 5-enkephalin and leu5-enkephalin, endorphins such as γ-α- and β-endorphins, oxytocin M and vasopressin.

-y ZEXiT O PI

A ong the steroids, there can be mentioned antiin- flammatory adrenal cortical steroids such as hydrocortisone, betamethasone, cortisone, dexamethasone, flumethasone, fluprednisolone, meprednisone, methyl 5 prednisolone, prednisolone, prednisone, triamcinolone, cortodoxone, fludrocortisone, flurandrenolone acetonide paramethasone and the like; male se hormones, such as testosterone and methyl testosterone; and female sex hormones, both estrogens and progestins, e.g., ethinyl

10 estradiol, norgestrel, mestranol, norethindrone, ethisterone, estradiol, estriol, estrone, dimethisterone, allylestrenol, cingestol, ethynerone, lynestrenol, norgesterone, norvinisterone, ethynodiol, oxogestone, tigestol, quinestrol and the like. Among the anticancer

15 and antitumor agents, there can be mentioned Ara-AC, pentostati (2'-deoxycoformycin) , Ara-C, 3-deazaguanine, dihydro-5-azacytidine, tiazofurin, sangivamycin, Ara-A, 6-MMPR, PCNU, spiromustine, bisbenzimidazole, L-alanosine, DON, L-ICRF, trimethyl TMM, 5-methyl tetrahydrohomofolic

20. acid, glyoxylic acid sulfonylhydrazone, DACH, SR-2555, SR-2508, desmethylmisonidazole, mitoxantrone, menogarol, aclacinomycin A, phyllanthoside, bactobolin, aphidocolin, homoharringtonine, levonantradol, acivicin, streptozotocin, hydroxyurea, chlorambucil, cyclophosphamide, uracil

25 mustard, melphalan, 5-FUDR, vincristine, vinblastine, cytosine arabinoside, 6-mercaptopurine, thioguanine, 5-azacytidine, methotrexate, adriamycin ^' (doxorubicin) , daunomycin, VP-16, largomycine polypeptide, aminopterin, dactinomycin, mitomycin C, and podophyllotoxin

30 derivatives, such as podophyllotoxin etoposide and podophyllotoxin teniposide. Among the antiviral agents.

there can be mentioned ribavirin; acyclovir? amantadine; diarylamidines such as 5-amidino-2-(5- amidino-2-benzofuranyl)indole and 4' ,6-diimidazolino- 2-phenylbenzo(b)thiophene; 2-aminoxazoles such as 2-guanidino-4,5-di-n-propyloxazole and 2-guanidino- 4,5-diphenyloxazole; benzimidazole analogues such as the syn and anti isomers of 6 [ [ (hydroxyimino)phenyl]. methyl]-1-[ (1-methylethyl)sulfonyl]-lH-benzimidazol-2- amine; bridgehead C-nucleosides such as 5,7-dimethyl 2-β-I)-ribofuranosyl-s-triazole(l,5-a)pyrimidine; glycosides such as 2-deoxy-D-glucose, glucosamine, 2-deoxy-2-f.luoro-D-mannose and 6-amino-6-deoxy-E)- glucose; phenyl glucoside derivatives such as phenyl- 6-chloro-6-deoxy-β-D-glucopyranoside; (S)-9-(2,3- dihydroxypropyl)adenine; 6-azauridine; and 5,6- dichloro-1-β-D-ribofuranbsylbenzimidazole. Among the tranquilizers, there can be mentioned benzodiazepine tranquilizers such as diazepa , oxazepam, lorazepam, chlordiazepoxide, flurazepam, bromazepam, clorazepate, nitrazepam and temazepam; hydantoin-type tranquilizers such as phenytoin, ethotoin, mephenytoin ^" ; phenothia- zine-type tranquilizers such as acetophenazine, carphenazine, fluphenazine, perphenazine and piper- acetazine; and others. Among the hypotensives, there can be mentioned clonidine, methyldopa, bethanidine, debrisoquin, hydralazine and guanethidine.

By "testicularly acting drug species" as used herein, there is intended any drug which is capable of exerting a useful pharmacological effect when delivered to the testes. Generally speaking, those

^• EAc OMPI

centrally acting drug species disclosed hereinabove for delivery to the brain can likewise be delivered to and concentrated in the testes utilizing the instant dihydropyridine ^* ^=^ pyridinium salt carrier type redox system? any such centrally acting drugs which exert a therapeutic or other useful biological effect when delivered to the testes are considered to be within the ambit of the expression "testicularly acting drug species". Exemplary of the drugs contemplated for delivery to the testes in accord with the present invention are antibiotics/antibacterials, antiviral agents, anticancer agents, hormonal agents and analgesics. The antibiotics/ antibacterials can be, for example, sulfonamides, penicillins, cephalosporins, tetracyclines, and other antibacterials, such as nalidixic acid and phenazo- pyridine. Among the antiviral agents, there can be mentioned especially ribavirin and acyclovir (ACV) . The drugs used in cancer chemotherapy contem- plated for delivery to the testes include nitrogen mustards, folic acid antagonists, platinum coordination complexes, antibiotic cancer agents, podophyllotoxins, purine and pyrimidine antagonists, alkaloids, urea derivatives, and hormonal anticancer agents. Among the _ hormonal agents contemplated for delivery to the testes are pituitary gonadόtropins, such as follicle stimu¬ lating hormone (FSH) , which maintains spermatogenesis and may be used to treat male infertility and cryptorchism; nonpituitary gonadotropins, such as human chorionic gonadotropin (HCG) , which may be used

for treatment of cryptorchism or hypogonadism in the male; adrenal cortical hormones, particularly glucocorticoids, which may be used in the treatment of inflammation (and possibly also in neoplasm) , such as, for example, hydrocor ^' tisone, betamethasone, dexamethasone, flumethasone, fluprednisolone, mepredi- sone, methyl prednisolone, prednisolone, prednisone and triamcinolone; agents for male fertility inhibition, e.g., agents which impair gametogenesis or otherwise hamper sperm production, such as N,N'-bis (dichloracetyl)- 1,8-octamethylene diamine; androgens, e.g., testosterone and methyltestosterone, agents of this type being useful for treating male hypogonadism, cryptorchism and the male climateric, as well as for maintaining or improving normal sexual function in the male and preventing decline in testicular function; and antiandrogens, which may be of use in male contraception and depres¬ sion of male sexual behavior, especially in veterinary practice. Analgesics contemplated for use herein include narcotic analgesics such as morphine, hydro- morphone., levorphanol and oxy orphone, agents of this kind being of use in the treatment of testicular pain, such as that associated with testicular cancer. Other testicularly acting drug species will be apparent to those skilled in the art. Presently preferred drug species contemplated for administration to the testes utilizing the present dihydropyridine =^= pyridinium salt carrier type redox system are the hormonal agents, principally the androgens, i.e., testosterone and its close analogues such as methyltestosterone; and the anticancer agents, especially those known to be useful

in the treatment of testicular cancer, i.e., chlor- ambucil, melphalan, methotrexate and cisplatin-type compounds.

It too will be appreciated that by "dihydro- pyridine carrier" or " [DHC] ", there is intended any nontoxic carrier moiety comprising, containing or including the dihydropyridine nucleus, whether or not a part of any larger basic nucleus, and whether substituted or unsubstituted, the only criterion therefor being capacity for BBB (or BTB) penetration and _in ivo oxidation thereof to the corresponding quaternary pyridinium salt carrier [QC] . As aforesaid, the ionic pyridinium salt drug/carrier prodrug entity [D-QC] which results from such in vivo oxidation is prevented from efflux from the brain (or testes) , while elimination from the general circulation is accel¬ erated. Subsequently _/ the covalent or equivalent bond coupling the drug species [D] to the quaternary carrier [QC] is metabolically cleaved which results in sustained delivery of the drug [D] in the brain (or testes) and facile elimination of the carrier moiety [QC] . Such "covalent or equivalent bond" between the drug and the quaternary carrier can be a simple direct chemical bond, e.g., an amide, an ester, or any other like bond, or same can even be comprised of a linking group or function, e.g., a thiazolidine bridge or a peptide linkage, typically necessitated when the drug species is not susceptible to direct chemical coupling to either the dihydropyridine carrier or the quaternary

-1- carrier. Nonetheless, the bond in the formulae [D-QC] and [D-DHC] is intended to be, and is hereby defined as inclusive of all such alternatives. And the cleavage

of the [D-QC] prodrug to sustainedly delivery the drug species [D] in the brain (or testes) with concomitant facile elimination of the carrier moiety [QC] is characteristically enzymatic cleave, e.g., by estβrase, amidase, cholinesterase, hydrolytic enzyme, or pepti- dase, albeit any type of in brain (or in testis) cleavage which might result, whether enzymatic, metabolic or otherwise, of course remains within the ambit of this invention. Thus, the drug release rate controlling parameter of the subject, pro-prodrugs is imparted simply via the cleavable bonding between drug and carrier, and not by any release rate controlling

_* substituent(s) .

The expression "non-toxic pharmaceutically acceptable salts" as used herein generally includes the nontoxic salts of compounds of formula (I) , wherein [D] is a centrally acting (or testicularly acting) drug species and [DHC] is the reduced, biooxidizable, blood- brain barrier penetrating form of a dihydropyridine = * ⁱ = pyridinium salt redox carrier, formed with nontoxic, pharmaceutically acceptable inorganic or organic acids HX. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulf ric, sulfantic, phosphoric, nitric and the like? and the salts prepared from organic acids such as acetic, propionic, succinic, glycollic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, sali¬ cylic, sulfanilic, fumaric, methanesulfonic, toluene- sulfonic and the like.

In one embodiment according to this invention, simple nontoxic carrier systems [D-QC] = ^■■ =■-■= [D-DHC] are envisaged, utilizing a wide variety of models for D, such as those above outlined. Representative such carrier systems and models are:

R.,0- or R^H-

CONHR,

wherein R ₂ is simply alkyl, e.g., CH,, or benzyl, albeit virtually any other effective substituent is intended. Exemplary of such simple carrier systems are N-alkyl nicotinamide and nicotinate ester derivatives, tethered to such drug species as dopamine, melphalan and testo¬ sterone. The trigonelline (N-methylnicotinic acid) system is quite effective as a carrier; it also is readily eliminated from the circulation and is virtually non-toxic.

Indeed, the present invention provides a flexible arsenal of dihydropyridine =^= pyridinium salt redox carriers for the site-specific/sustained delivery of virtually any centrally/testicularly acting drug species to the brain/testes. Moreover, any dihydropyridine/ pyridinium salt redox carrier entity is contemplated _f

and intended hereby generically, and any such carrier moiety need not be, and is not derivatized with a drug release rate controlling substituent critically tailored to meet, or be coordinated with, the chemical nature and delivery requirements of the particular drug species sought to be preferentially administered to the brain or testes. As utilized herein, the term "carrier" is to be understood as connoting just such a non- derivatized, non-drug/carrier coordinated entity, for consistent herewith it is the "carrier" entity itself and not the nature of any activity or release rate controlling/modifying substituent which is responsible for providing the desired brain-specific or testicular- specific result. Additional examples of such redox carriers include the quaternary pyridinium alcohols (JL) , the analog isoquinoline acid and alcohol systems (2 ) , and multi- charged delivery forms, exemplified by structure 3_ (D represents drug, Z a covalent link) and obviously the corresonding dihydro forms.

3

Yet other redox carriers include those.comprising an acidic chain directly linked to the heterocyclic nitrogen, in quaternary or tertiary amine form. Also the hydroxide type carriers, e;g., ^"~ 'the glucosamine analog indicated below; Representative are:

like

d alkyl

(a> aryl-alkyl, and the like

D « ^■ * drug—NH, or -OH;

Preparation:

OMPI

Method of: H. Lattre et al., Annalen. 579, 123 (1953)

(b)

(c)

Generally preferred dihydropyridine ==- pyridinium salt redox carriers for use in the present invention include the following (where D represents the drug) , and obviously the corresponding dihydro forms:

(a) the pyridinium systems

^• " ^ϊ - ^.EA' tr

O PI

in which the depicted substituent is in the 2-, 3- or 4- position, and R- is C,-C_ alkyl or C--C- _Q aralkyl, preferably methyl or benzyl;

(b) the pyridinium _^ system

v) in which R. is C, to C-. alkylene, i.e., (CH_) where n ^* =l-3;

(c) the isoquinolinium and quinolinium systems

in which R, is defined as above; and

(d) the quinolinium and isoquinolinium systems

(vii) (viii)

in which R_ is defined as above. The corresponding dihydro forms of the foregoing preferred pyridinium salts are depicted below, wherein the position and identity of the structural.variables are as indicated abovet .....

(i) (ii ) (iii )

(iv) (v)

(viii)

In a preferred embodiment of the present inven¬ tion, sustained delivery of drug to the brain in pharmacologically effective concentrations has now been demonstrated, paralleled with much lower concen- trations in the peripheral circulation and other tissues, utilizing dopamine as the target drug species and a trigonelline-type carrier system, with the catechol moiety thereof in certain instances being acylated, e.g., acetylated or pivalylate . According to Scheme 3 which follows, one specific delivery system for dopamine, compound 5_, on administration (e.g. , by injection) is distributed throughout the body and by reason of its lipophilic character facilely penetrates the blood-brain barrier and enters the CNS. Following oxidation both in the brain and in the other tissues, the corresponding hydrophilic quaternary salt (6) is formed. The quaternary salt 6_ is essentially "locked in" the brain and its concentration is considered to increase with time until reaching a maximum, which depends primarily on the relative rates of entrance of the dihydro compound (5_) to the brain (K, ) as compared to K ₂ to the other tissues, the rate of oxidation of the dihydro form to the quaternary

(K, and K_) and- the rates,of its disappearance from the brain (K. +* K_) . At the same time, the very water

4 soluble quaternary form(s) 6^ is/are excreted readily via the kidney and the liver (K _g >>K.). Derivatives j are considered to be essentially inactive forms (K„>>K _g ), and thus systemic activity/toxicity is minimized. Hence, the concentration of 5 and 6 in the blood rapidly increases.

The ratio of the quaternary salt JS in the brain relative to the blood increases to the point where j3, or metabolites thereof-; can only be found in the brain. The quaternary ^■ β ^' ; ^1'1 whether ^•■ in' the- brain, ¹ blood* or other tissues, is 'deemed to release dopamin and" the non-toxic compound, trigonelline, depending upon the rates of site-specific conversion of the precursor 6 _^ to the drug at each of these sites. The concentration of any released dopamine at any time is much higher in the brain than in the blood or other tissues.

Also, as the enzymatic transformation of the quaternary percursor to the drug (dopamine) is relatively slow, same permits a sustained release of dopamine. Too, the simultaneous protection/lipophilic derivatization of the catechol system in dopamine has also now been demonstrated.

It will be appreciated that a compound of formula (I) , such as compound 5_, may be administered as depicted in Scheme 3, or in the form of a non-toxic pharmaceutically acceptable salt thereof, i.e., a salt which can be represented by the formula

efined as in e 3) wherein HX is as defined before; and that, regardless of the actual form in which the compound is administered, it will be converted in vivo to a quaternary salt of the compound 6_ type, the anion X~ being an anion present _j iii vivo. It is not necessary that the compound 6

anion be introduced as part of the compound 5_. And even when the compound 5_ is used in its salt form, the anion of _ is not necessarily the same as that present in compound 5_. jln any:,event/ the exact_identity of the anionic portion.,of. the compound 6_ is immaterial to .-• the depicted enzymatic transformation.

Scheme 3;

R - K (a). COCH ₃ (b) COC(CH ₃ ) ₃ (C)

D str J sut on

Other

Excretion Dopamine•

Trigonelline

Trigonelline 10

Excretion, Metabolism

Excretion, Metabolism

With specific reference to the immediately above, the 1 , 4-dihydropyridine derivatives (_5) were prepared as in the following Scheme 4 :

Scheme 4:

R = H (a), COCH ₃ (b) , COC(CH ₃ ) ₃ (c) DCC = dicyclohexylcarbodiimide

-ςx iiEn

OMPI . WIPO

Similar schemes can be shown for the preparation of the other dopamine derivatives of the invention. The step which introduces the protecting groups is of course only required when it is desired to protect the catechol hydroxyl .groups. Moreover, _, when,carbonate rather than acyl protecting groups are desired, the step of introducing the protecting groups will involve reacting the catechol with a halocarbonate of the type Y'OCOCl or Y'OCOBr (formed by reaction of Y'OH with

10 C0C1 ₂ or COBr ₂ ) , rather than with an acyl halide YCl or YBr, Y and Y' being as generically defined herein- above. Also, the order of steps shown in Scheme 4 may be altered; quaternization, followed by reduction, need not be in the final two steps but may be carried 15 out earlier in the reaction sequence. Yet other reaction schemes and reactants (e.g., using an anhydride rather than an acyl halide to convert 1_ ^° 1- will *° ^e readily apparent to those skilled in the art. Scheme 4 being simply a preferred approach for the specific 20* compounds there depicted. Variations of this approach are likewise applicable to preparing derivatives of other hydroxy-containing amines.

In an attempt to ascertain whether any biotrans- formation of the free catechol is taking place by COMT 25. (catechol-O-methyltransferase) either before or after oxidation, the possible O-methyl metabolites (9 _^ and 10) were synthesized separately following Scheme 4 with 3-methoxytyramine hydrochloride as the starting material.

The stability of the 1, -dihydropyridine deriva¬ tives (5_) was determined in the presence of the oxidizing agents, alcoholic AgN0 ₃ and hydrogen peroxide. The in vitro rates of oxidation of the 1,4-dihydropyri- dine derivative (5_c) in 80% plasma, 20% brain homogenate, 20% liver homogenate and in whole blood were determined. The dihydropyridine derivative (5_c) was then selected for the iji vivo study. A solution in DMSO (dimethylsulfoxide) was injected through the jugular vein to a group of male Sprague-Dawley rats which were then sacrificed at various time intervals; their blood and brains were analyzed for the quaternary percursor of dopamine (6a) . The in vivo dopaminergic activities of the selected compounds 5_c vs. Sa were then determined.

Consistent with the,above, it was found that N-nicotinoyldopamine (7) could be obtained in good yields by coupling dopamine hydrobromide with nicotinic acid in pyridine as a solvent and with dicyclohexyl- carbodiimide as the coupling agent. ^' Attempts to pre¬ pare 7_ by using dopamine free base were largely unsuccessful. As for the catechol protecting groups, the acetyl and pivalyl moieties were selected due to

their rather different steric and partitioning parameters. Acylation could be accomplished with the acyl chlorides by using conventional methods. Reduction of the quaternaries (6 -c _t and 9 ) was . accomplished by using sodium dithio ite in mildly basic aqueous solution, (NaHC0 ₃ ) . It was observed that the dihydro compound obtained in the case of the quaternary j6b gave a faint green color with ferric ions, indicating partial hydrolysis of at least one of the acetyl moieties during reduction, even in the cold, weakly basic solution used as a medium. The dihydropyridine derivatives isolated (_5a-c and lJ were determined to have the expected 1,4-dihydropyridine structure, based on their NMR and uv spectra. Attempts to prepare the β-protonated enamine salts of the isolated dihydro derivatives were also largely unsuccessful, due to acid catalyzed addition reactions. The 1,4-dihydropyridine derivatives (5_a-c) were found to be relatively stable towards oxidation. Compound 5_c was quantitatively oxidized to the corresponding quaternary salt 5c by H ₂ 0 ₂ or alcoholic AgN0 ₃ solution.

The diacetyl derivatives (5b and 6b) appeared to be labile to hydrolysis and therefore were not pursued _in vitro. The dipivalyldihydro derivative (5c) was thoroughly investigated for its in vitro rates of ^" disappearance and metabolic degradation in various biological fluids. It is evident that 5_c represents a rather complex case, as besides oxidation, a two-step hydrolysis will also take place. Scheme 5 illustrates the interconversion of the possible components.

OMPI

Scheme Bt

OMPI _

Figures 1-4 illustrate the results of such an investigation. The apparent half-lives for. the disappearance of 5c in biological fluids at 37 ^C C were calculated. Although the process does not truly follow first order kinetics, the data fit very closely a pseudo first order process (Figure 5) . The obtained values, 51 min (80% plasma) , 17 in (20% brain homogenate) , 18 min (whole citrated blood) and 6 min (20% liver homogenate) , reflect an acceptable stability of the dihydro derivative 5_c. The disappearance of 5_c is accompanied by formation of some monoester (11) and dihydroxy dihydro form (5_a) in all the media except the liver homogenate. The rate of hydrolysis of the first ester moiety is faster than the second and a reasonable amount of monoester 11 builds up with time. The monohydroxy quaternary 12 _^ could not be detected except in the blood as a very small peak which does not change significantly with time. A steady increase in the concentration of the dihydroxy quaternary 6a was observed in all media except liver homogenate. Thus , it is established that this ^• deriva¬ tive, 6a, is forming as the main product of the various interconversion routes and it is the direct precursor thus concluded to be locked in the brain in the ±n vivo experiment. No formation of the methoxy derivatives 9_ and JL could be detected in ay of the biological fluids studied; j>a and 6a. do not appear to be good substrates for COMT.

The first objective of the in vivo studies was to trace the appearance and disappearance of 6a. in blood and brain -following administration of 5_c. Figure 6 summarizes such results, .and is consistent with the mechanism shown in Scheme,3. .After one , single injection of the 1,4-dihydropyridine derivative 5c to the rat, the dihydroxy quaternary 6a. (ion) , which is the only detectable derivative , could be seen to appear and then to disappear quickly from the blood, with a half-life of 27 min. On the contrary, the concentration of ja (ion) is increasing in the brain steadily, reaching a maximum at about 30 min following administration. The descending portion indicates a half-life of disappearance from the brain of about 3.2 h. Nc formation of 0-methyl metabolites (9, 10) could be detected in the brain. This confirms the in vitro results that a (or 5a) is not a good substrate for COMT.

To determine whether dopamine itself was finally released in the brain upon completion of the aforesaid complex delivery process, 5_c was administered intra- jugularly and changes in brain-dopa ine concentrations following that administration were studied. Some of the rats showed up to threefold increase in the dopamine concentrations, others practically none. Since it is possible (and even desired) that the intrinsic brain metabolism of the dopamine does not permit significant build-up of its concentration, specific pharmaco ogic activity was investigated, using changes in the in vivo prolactin secretion. It is known that dopamine and its

agonists decrease prolactin secretion following their binding to stereospecific receptors located on ^" lactophors in the anterior pituitary ^" (AP) gland [G.P. Mueller,

J.W. Simpkins, J. ^' Meites and K.E.. Moore,- Neuroendo- crinology, 20, 121 (1976); . uttke, E. Cassell and J. Meites, Endocrinology, 88, 737 (1971); J.A. Clemens,- E.B. Smalstig and C.J. Shaar, Acta Endocrinol., 79, 230 (1975)3. This effect is dose-dependent and it can also be observed in vitro, incubating anterior pituitaries with dopamine or its agonists [R.M. MacLeod Ed. L. Martini and .F. Ganong, Raven Press] .

It was then determined that exposure of male rats to 17-β-estradiol for two days elevated serum prolactin levels to greater than 150 ng/ l. Intravenous adminis- tration of 5c caused a 79% decrease in serum prolactin concentrations and this dramatic reduction was main¬ tained through 120 min after treatment. In contrast, _6a had no significant effect on the serum prolactin concentrations by 15 min,. and caused a 67% reduction by 3.0 min. Thereafter, serum prolactin levels increased progressively to levels which are not significantly different from vehicle injected controls, by 60 and 120 min. These results are summarized in Figure 7. The rapid onset and prolonged inhibitory effects of 5_c on prolactin secretion is consistent with the time course of the appearance of _6a in the brain following administration of 5c. The "trapping" of 6a. in the brain subsequent to I.V.. injection of 5c provides a constant source of a potent dopaminergic agent, either dopamine or 6a itself. The significantly

lower effect of a when administered I.V. does not unequivocally clarify which alternative is the more responsible. This was. resolved by in vitro- comparison of the relative activities of dopamine versus 6a.

Fresh anterior pituitaries obtained from female rats were incubated with various concentrations of dopamine (DA) and j5a, respectively, and their effects on the rate of release of prolactin were measured.

It was found that at 2x10 —8M concentrations,- neither

_7 DA nor 6a. had any effect,' but at 2x10 M, DA caused a 57% reduction of the prolactin rate secretion, while 6a. had no effect. These results are summarized in the following Table I.

TABLE I: Comparative in vitro activity of jja vs. dopamine'

^a On freshly obtained anterior pituitary (AP) at 37 ^β C. All values are average of 9 separate AP-S.

Prolactin release rate of the incubated AP-S. ^c Weight of the AP-S: Control 4.6+ 0.2mg.

DA treated 4.5+ 0.3mg. heigh of the AP-S: Control 4.6+0.3

6a treated 4.7+0.4

*P<0.05

-^V mlEXfty

OMPI

A- WIPO ^NATIO

These results indicate that if j5a has any activity, it must be significantly less than that of DA. Based on the delayed onset of the activity when ja was administered I.V. and considering the in vitro results, it logically follows that the high and prolonged activity of the 6>a locked in the brain following administration of 5c is due to the fact that 6a is slowly releasing the active DA in the brain. Accordingly, provided hereby is a potent, brain- specific dopaminergic agent comprising a lipophilic dihydropyridine carrier-type chemical delivery system of dopamine ["pro-prodrug" or "pro-pro-prodrug" in the case of the catechol protective group(s) ] , which penetrates the BBB by passive transport. The rapid oxidation in the brain of the carrier moiety to the corresponding quaternary pyridinium salt results in an activated amide of dopamine. The oxidation process is much faster than amide cleavage of the beginning compound 5_ or of j6. Moreover, the ionic nature of the activated quaternary salt results in a significant slowdown of the efflux of this specific form through the BBB, resulting in a selective concentration enhancement of the precursor 6a in the brain. Too, brain-specific dopaminergic activity is assured, logically as dopamine is released from this activated form upon hydrolytic, enzymatic or metabolic cleavage, as is facile excretion of the carrier moiety from the brain.

In yet another embodiment of the invention, like synthesis of the-,analogous rtyramine system-has,been carried out, and.,,the ^* .corresponding determinations made. Such tyramine system is represented as follows:

(CH ₃ ) ₃ CCO-(c)

Naturally, selection of the particular dihydro¬ pyridine =γ= pyridinium salt redox carrier to be used will depend on the chemical structure of the specific drug involved. And not only should the nature of the functional group which is to be linked to the carrier system be considered in selecting the carrier, but the manner in which the ultimate compound is prepared should be tailored to the presence of any other reactive groups in the molecule. The following examples of specific drug/carrier combinations and their manner of synthesis are set forth for the purpose of illus¬ tration only and are not to be considered limitative in any way whatsoever.

Thus, in one specific illustration, the selected drug is testosterone and the selected carrier system is trigonelline =γ^ dihydrotrigonelline; according to this embodiment, testosterone is reacted with nicotinoyl chloride, the resultant ester is then

OMPI

quaternized with methyl iodide, and the quaternary iodide is then reduced with -S^O, to afford the testosterone-CDS (chemical delivery system)-

Other steroids can be similarly derivatized, e.g., 17α-ethynyltestosterone, estradiol and the like.

Another specific illustration involves selecting melphalan and the same type of carrier system as above, but forming an amide rather than an ester linkage. Thus, melphalan is converted to its hydrobromide, which is reacted with nicotinic acid to afford the amide having the formula

which can be esterified, if desired (to increase lipoidal characteristics) , followed by, when the ethyl ester is prepared, quaternizing same'with methyl iodide to form

which can then be reduced to afford the melphalan-CDS

As one of several alternative schemes, melphalan can be derivatized by first esterifying it, e.g., to convert the carboxy function to the ethyl ester, then reacting the resultant melphalan ethyl ester with nicotinoyl chloride to form the amide of the formula

OMPI

which can then be guaternized and ^' the quaternary salt subsequently reduced as indicated above to afford to same melphalan-CDS as depicted.above.- • - • -..-.. -j •

Yet another specific illustration utilizes chlorambucil as the target drug, in which case the desired nicotinic acid carrier system is linked to the drug via a bridging group. Thus, nicotinic acid can be reacted with an appropriate di- or polyhydroxy compound such as ethylene glycol, propylene glycol or inositol and the resultant intermediate is linked via its free hydroxy group(s) to the carboxylic acid function of chlorambucil. That intermediate is then guaternized and the quaternary salt is reduced to afford the chlorambucil-CDS. In the case of nicotinic acid and ethylene glycol starting materials, the chlorambucil-CDS has the formula

On the other hand, when a polyhydroxy compound is reacted with nicotinic, acid in the first step, a variety of products are possible. Thus for example, when inositol is used, the final product may contain anywhere from 1 carrier/5 drug residues to 5 carrier/1 drug residue. In the case of the inositol trinicotin- ate intermediate

conditions for reacting same with chlorambucil can be selected so that one, two or three of the hydroxy functions react with the acid. When all three hydroxys react, the ultimate chlorambucil-CDS has the formula

and contains 3 drug residues and 3 carrier groupings. As another example, methotrexate, which has the structural formula

can be derivatized similarly to chlorambucil via its carboxy function(s), e.g., utilizing the. inositol \ trigonellinates or a glucosamine analogue..

As a further example, podophyllotoxin and its derivatives can be linked to a carrier system of this invention. These drugs can be represented by the structual formula

H or CH.

R. « OH - podophyllotoxin

_R4 (R ₆ « CH ₃ -etoposide

oS.r- teniposide)

and can be derivatized by reacting the hydroxy group in podophyllotoxin (R.=0H) or the hydroxy groups in the glycosidic portions in R. with acidic type redox carriers, e.g., in a manner analogous to the testo- sterone-CDS depicted above. Known cisplatin analogues, in which typically the amino groups have been replaced with organic radicals, can be similarly derivatized according to the invention, the method of •choice depending on the nature of the functional groups in the organic radicals.

OMPI

Similarly, syntheses and like determinations as regards the redox carrier-linked enkephalins can be carried out. ■ -Firs 'synthesized is ^! the known leucine enkephalin XI. The quaternary pyridinium analog XII, the corresponding O-benzyl ether XIII and the amide XIV are next synthesized.

Tyr-Gly-Gly-Phe-Leu XI

OCH ₂ C ₆ H ₅ XIII

OMPI

The O-benzyl pentapeptide ethyl ester derivative of XI is synthesized sequentially.and then coupled with nicotinic acid,-followed bymethylation.* Alternate methods involve introduction of carrier at .earlier stage in the synthesis. The reduction of XII and XIII results in a mixture of products due to the base sensi¬ tivity of the ester. Likewise prepared are the corre¬ sponding leucinol trigonelline ester XV and its dihydro derivative XVI.

Thus, the site-specific brain delivery of the enkephalins for the treatment of epilepsy is estab¬ lished consistent with the Scheme 1, as is their analgesic activity.

Similarly, as regards the benzodiazepine tranquilizers, e.g.: 1. Oxazepam:

OMPI

2. Diazepam;

1) deliv

2) oxida

sustained brain release in vivo

This reaction scheme utilizes conventional opening of the 7-member ring, accompanied by coupling of the drug to the carrier. The following drugs can be similarly derivatized to the corresponding dihydro derivatives:

OMPI

flurazepam

bromazepam

And in another preferred embodiment of the inven¬ tion, there is provided the effective, selective and nontoxic treatment of epilepsy, based upon the mechanism illustrated in Scheme 1. Indeed, commencing from the "GABA-hypothesis" of epilepsy, the brain-specific, enhanced and sustained release of GABA (γ-aminobutyric acid) itself, and various other compounds either directly or indirectly affecting the concentrations of GABA in the brain, is circumscribed consistent herewith. Model compounds include carboxylic acids, most specifically valproic acid, as well as some of the GABA

OMPI s A- _y , WIPO ,

analogs which inhibit irreversibly the GABA-T, such as γ-vinyl and/or γ-acetylenic GABA. using the aforesaid trigonelline (N-methylnicotinic acid) =^= dihydrotrigonelline system, for example, the selected compounds can be effectively delivered per Scheme 1. Thus, representative target compounds are the dihydro¬ pyridine carrier-drug combinations 1 and the corre¬ sponding pyridinium carrier-drug species, for example, GABA and its esters:

^R l = ^CH 3' ^C 3 ^H 7 ° ^{r CH} 2 ^C 6 ^H 5

Rn = H, C ₂ H ₅ , CH(CH ₃ ) ₂ , etc.

Related derivatives for γ-vinyl and γ-acetylenic GABA are:

R ₃ NH-

n the case of valproic acid, other alternatives are:

X = H, CONH ₂ , CHNOR ₂ , etc.

In another embodiment of like delivery system, applicable for both the GABA and related compounds and for the carboxylic acids, or for any other drug species to be linked to such a carrier, either directly or indirectly, i.e., mediated by a carboxylic acid, e.g., succinic acid, or other linkage, provided is a mono- or poly-substituted nontoxic polyol (such as inositol or sugars) having the trigonelline ---•■•f**-*** dihydrotrigo- nelline system and the compounds to be delivered linked to the same molecule as exemplified by the GABA case (5 =^ 5a) and valproic acid (6 = ^*** =^= 6a) :

-70-

R 4„ == H, GABA or valproic acid, but at least one of _* is:

(in 5a and 6a) (in 5 and 6)

R. can be partially replaced by additional GABA or. valproic acid, changing the carrier/drug ratio as necessary. Some of the valproic acid metabolites can be coupled with carriers of the redox type, via the various hydroxy groups formed during the oxidative degradation:

7 and 7a 8 and 8a

OR,

9 and 9a

OMPI

-71-

Illustrative examples are the corresponding derivatives of the 5-, 4-, and 3-hydroxy-2-n-propyl pentanoic acid derivatives. Additional carrier systems, such as the isoquinoline *= ^= dihydroisoquinoline system can also 5 be developed consistent herewith.

Moreover, based upon the observation that NADH content is significantly reduced in epileptic and like seizures, the use of the subject redox system (in reduced form) will bias the NAD = ^= NADH balance towards 10 NADH during the dihydro carrier →** quaternary transforma¬ tion. Also, the brain-specific delivery of small peptides consistent herewith, e.g., the enkephalins which have been found to initiate epileptic seizures, has led to the design of a variety of long lasting 15 potent antagonists.

And the subject chemical delivery system is also useful for the delivery of other an iconvulsants in a sustained, brain-specific fashion, e.g., the benzo- diazepines and hydantoins, and those compounds, like 20 apomorphine, which are useful in the treatment of photosensitive epilepsy.

It will of course be appreciated in the immediately above regard that the drug treatment of epilepsy has always posed formidable problems. There are many 25 different anticonvulsants available, some more specific for different types of seizures. Indeed, there exist a wide variety of opinions as to which is the most suitable drug for any particular type of seizure, and drug mixtures are typically employed. An inevitable 30 result of the traditional therapy is the development of chronic toxicity, but such result is conspicuously avoided according to the present invention.

-72-

It too will be appreciated that the desired therapeutic effects of all antiepileptic agents investigated, as well as their undesired toxic effects, reflect a statistically significant correlation with the drug levels in plasma. This correlation is based upon a close relationship between the drug concentra¬ tions in plasma and brain tissue. Hence, a primary attribute of this invention is to enable attainment of high and sustained brain levels of the selected active agents, essentially against the plasma-brain concentration gradient and independent of the drug concentration in the blood.

GABA and related compounds are logical candidates. It has been shown that GABA neuron function is impaired in at least certain types of human epilepsy. Animal studies also showed that seizures are induced by reduction of GABA neuron function to a critical degree by (1) inhibition of GABA synthesis, (2) blockade of

GABA receptors or (3) inhibition of GABA-receptor mediated ionic events. In addition, enhancement of GABA synaptic activity (by direct receptor stimulation or by increasing GABA levels in the synapse) has a potent and wide spectrum anticonvulsant effect. These findings foreshadowed that an enhanced and sustained GABA brain delivery or a brain-specific delivery in a sustained manner of a good GABA-agonist would be efficacious in different forms of epilepsy. It is well known that GABA itself, when administered systematically, does not penetrate the normal blood-brain barrier to any significant extent. Among the potential sites at which drugs may act to influence GABA-mediated synaptic function _r the first target is to effect the BBB trans¬ fer of GABA via a redox delivery system. The second

main target is to effect the catabolism of GABA. This invention, accordingly, specifically provides for the efficacious delivery of the GABA-T inhibitors, γ-vinyl and γ-acetylene-GABA, but the delivery of valproic acid, specifically to the brain and without requiring high circulating blood levels, is also envisaged. In order to achieve the required activity, sodium valproate must have a relatively high, 50- 100 μg/ml, level in the blood. The value of valproic acid is well established in most types of epilepsy.

It is evident that valproic acid produces significant increases in both brain and synaptosomal GABA concen¬ trations. Valproic acid itself undergoes extensive metabolism. In capsule summary, the present invention provides for the significantly improved treatment of epilepsy, and concomitant reduction in toxicity of a number of antiepileptic drug species currently in use. And made available to the brain is a variety of important compounds, such as GABA and a wealth of GABA-ergic agents.

It will be apparent from the foregoing discus¬ sion of specific drug-carrier combinations that a wide variety of synthetic- approaches can be utilized, depending on the chemical nature of the selected drug. Various illustrative synthetic schemes as applied to specific centrally acting drugs in accord with this invention are set forth below in the section entitled "Illustrative Synthetic Methods". While the sequence of reaction steps can be varied in many cases, in general, the final step ⁽ except in the case of optional

salt formation) will be reduction of a pyridinium compound of formula (II) to the corresponding dihydropyridine compound of formula (I) . The reduction is usually conducted at a temperature from about -10°C to room temperature, for a period of time ^" from about 10 mins to 2 hrs, conveniently at atmos¬ pheric pressure. Typically, a large excess of reducing agent is employed, e.g., a 1:5 molar ratio of reducing agent to starting [D-QC] compound. The process is conducted in the presence of a suitable reducing agent, preferably an alkali metal dithionite such as sodium di- thionite or an alkali metal borohydride such as sodium borohydride or lithium aluminum borohydride, in a suitable solvent. Sodium dithionite reduction is conveniently carried out in an aqueous solution; the dihydro product [D-DHC] is usually insoluble in water and thus can be readily separated from the reaction medium. In the case of sodium borohydride reduction, an organic reaction medium is employed,- e.g., a lower alkanol such as methanol, an aqueous alkanol or other protic solvent.

The rationale of the present invention as regards testicular delivery can be graphically illustrated with reference to the drug testosterone. The 17β-(1,4- dihydrotrigonelline) ester of testosterone, having formula la depicted in ^" Scheme 2 below, by virtue of its good lipid solubility, crosses not only the BBB but also the BTB in the male. As shown in Scheme 2, biological oxidation to the corresponding quaternary derivative causes a "lock in" of the corresponding ionic, hydrophilic product in the testes of male animals, as well as in the brain. Conversely, oxida¬ tion in locales not involving a permeability barrier would favor rapid clearance from the blood because the

-75-

quaternary derivative (Ila) is excreted more rapidly than the unoxidized form ( a) . Thus, oxidation favors accumulation of (Ila) in the brain and testes while yielding minimal blood levels. A subsequent, 5 slow hydrolysis to free testosterone in the testes (as in the brain) therefore would provide a site- specific, prolonged testosterone action and minimal peripheral effects. Although K _R should be much greater than K _q and K_ should be much greater than K., the

10 slow rates of quaternary hydrolysis should assure a sustained release of testosterone in both testes and brain while yielding minimal, levels in blood and other tissues. It is also expected that trigonelline released in the brain and testes by the hydrolytic

15 process would be excreted easily and at comparable rates.

OMPI

Scheme 2: Testosterone trigonellinate and the BTB, blood-teεtis barrier. -- REA7 OMPI

-77-

Suitable nontoxic pharmaceutically acceptable carriers for use with the topic compounds [D-DHC] , e.g., those less toxic than the target drug species them¬ selves, will be apparent to those skilled in this art. 5 Compare, for example, Remington's Pharmaceutical

Sciences, 4th Edition (1970) . Obviously, the choice of suitable carriers will depend upon the exact nature of the particular dosage form selected, as well as upon the identity of the active drug species [D] . The 10 therapeutic dosage ranges for administration of the compounds according to this invention will generally be the same as, or less than, those characteristically used in this art for administration of the known drug species [D] , per se. Naturally, such therapeutic 15 dosage ranges will vary with the size of the patient, the condition for which the [D-DHC] compound is admin¬ istered, the particular dosage form employed, and the like. The quantity of given dosage form needed to deliver the desired dose of [D] will of course depend 20 upon the concentration of [D-DHC] in any given pharmaceutical composition/dosage form thereof.

The ability of the topic compounds to cross the BBB, as well as the BTB in the male, and to be "locked into" the brain and testes allows administration of the 25 drug in a site-specific manner. A combination of the present dihydropyridine -ST**-***-*-^ pyridinium salt redox . system with a sustained release system will further enhance this site-specificity. Thus, a preferred embodiment of the invention comprises formulating the 30 [D-DHC] compound or the salt of a [D-DHC] compound

"$TJRE OMPI <&,- - WIPO .

utilizing a sustained release carrier system and/or route of administration capable of slowly releasing the chemical, e.g., sustained release tablets and capsules for oral administration; subcutaneous injection, or implantation of drugs in solid pellet form (for example, distributed in a biodegradable polymer) ; intramuscular injection of the compound in solution in oil or suspended in a repository vehicle; a transdermal delivery device or form such as an ointment to be applied locally to the desired site

(when the drug is susceptible of delivery through the skin) and the like. The rate of release of compound from the sustained release system should be comparable to the rate of in vivo oxidation of the dihydro form of the redox system in order to achieve the greatest degree of enhancement of specificity.

-Vg-

Illustrative Synthetic Methods

I. Methods for Derivatizing - H ₂ or -NH- Functions in Drugs

Method A

The drug is reacted with nicotinoyl chloride, with nicotinic anhydride, or with nicotinic acid in the presence of a suitable dehydrating agent such as dicyclohexylcarbodiimide, in an appropriate organic solvent, to afford the corresponding nicotinamide. The nicotinamide is then quaternized, typically by treatment with methyl iodide in a suitable organic solvent, to afford the quaternary derivative [D-QC] , which is then reduced by treatment with sodium dithionite or sodium borohydride as generally described hereinabove to afford the desired compound [D-DHC] . The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds.

OMPI

Starting Material [D-QC] [D-DHC]

I

CD O

I

Starting Material [D-QC] [D-DHC]

clo

bethanidine

trany cyprom ne

Starting Material [D-QC] [D-DHC]

ch

m

phentermine

Starting Material [D-QC] [D-DHC]

phenmetrazine

protriptyline

[D-QC] [ D-DHC 1

Starting Mate rial

oo

**»

dextroa phetamine

Starting Material [D-QC] .[ D-DHC 1

doxorubicin

Starting Material [D-QC] [D-DHC]

amantadine

I

CO

0 ^* 1

I

-87-

Starting Material

mitoxantrone

[D-QC]

[D-DHC]

OMPI

-88-

Method B

This is a variation of Method A used when the drug contains a -COOH function which is to be protected.

The drug is first converted to the corresponding 5 ethyl ester by conventional esterification techniques. That ester is then used as the starting material and Method A is repeated.

Obviously, other esters may be similarly prepared in the first step by use of other 10 esterifying agents.

The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds.

Starting Material [D-DHC]

tryptophan

ampicillin

cephalexin

Starting Material [ D-QC] [D-DHC]

melphalan

avo oτuθT ^Θoe-

-92-

Method C

This is a variation of Method A used when the drug contains one or more OH functions which are to be protected. 5 The drug is first reacted with excess trimethylacetyl chloride to convert the hydroxy group(s) to pivalyloxy grou (s). (This process is generally conducted in the presence of a base? however, strongly acid conditions are used if an amine

10 function is present.) That protected derivative is then used as the starting material and subjected to Method A. Alternatively, the first two steps may be reversed, i.e. the drug may be first converted to the nicotinamide, which may then be reacted with

15 trimethylacetyl chloride to form the protected nicotinamide.

Various other hydroxy protecting groups may be introduced in similar fashion.

The representative drugs listed below may be

20 derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds.

Starting Material [D-QC]

[D-DHC]

Starting Material [D-QC] [ D-DHC]

phenylephrine

Method D

This variation of Method A can be used when the drug contains one or more OH and COOH functions which are to be protected. The protecting groups, typically the ethyl ester and pivalyloxy groups, are introduced as described in Methods B and C, in the sequence considered most convenient. The amine function is derivatized according to Method A.

The representative drugs listed below may be derivatized in this manner to the corresponding _{. [D-QC] and [D-DHC] compounds.

OMPI

Starting Material [D-QC] [D-DHC]

levodopa

Method E .

This method is of particular use when the - H- f nction is part of an amide or imide or a very low pKa primary or secondary amine. The drug is first reacted with an aldehyde

[e.g. formaldehyde, benzaldehyde, acetaldehyde or chloral (Cl ₃ CCHO)] ; for example, in the case"of chloral, one converts the -NH- function to a

Cl-C-CHOH - * ^■ * ^* I

-N-

function and thus forms a suitable bridging group.

The resultant compound is then reacted with nicotinic acid in the presence of a suitable dehydrating agent, or with nicotinoyl chloride or nicotinic anhydride, to form the corresponding nicotinic acid ester of the partial formula

The resultant intermediate is then quaternized and reduced as in Method A.

The representative -starting drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds.

o

OMPI

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

chlortetracycline

glutethiraide

Starting Material [D-QC]

[D-DHC]

uracil mustard

-101-

Method F

Method A is followed, except that in. the- first step, the drug is reacted with 3-quinoline— carboxylic acid or its acid chloride or anhydride 5 instead of nicotinic acid or its acid chloride. The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [Dr-DHC] compounds, as may the remaining drugs listed with Method A. 10 Similarly, Method F may be combined with Methods B, C or D to afford the corresponding N-methyl-3-guinolinecarboxamide derivatives, e.g. of the drugs listed with those methods.

Starting Material [D-QC] [D-DHC]

QMPI

Starting Material [D-QC] [D-DHC]

dextr

< -CH

1-isomer

levamphetamine

ph

-105-

Method G

Method A is followed, except that in the first step, a startingmaterial of the formula

COOCH ₂ COOH

is used in place of nicotinic acid. (That starting material may be prepared by reacting nicotinic anhydride, nicotinoyl chloride or nicotinic acid . with glycolic acid.)

The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds, as may the remaining drugs listed with Method A.

Similarly, Method G may be combined with Methods B, C or D to afford the corresponding derivatives, e.g. of the drugs listed with those methods.

OMPI

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

I

Mdthod H

Method A is followed, except that in the first step, a starting material of the formula

(CH ₂ ) _n C00H

wherein n ^*** - ^* 1-3, preferably 2, is used in place of nicotinic acid. (That starting material may be prepared from nicotinamide, e.g. when n = 2, by reacting 3-iodopropionic acid with nicotinamide.) The drugs listed below may be derivatized in this manner to the corresponding [D-QC] ⁺ and [D-DHC] compounds, as may the remaining drugs listed with Method A.

Similarly, Method H may be combined with Methods B, C or D to afford the corresponding deri- vatives, e.g. of the drugs listed with those methods.

Starting Material [D-QC] [D-DHC]

amphetamine ' *

phentermine

-llO-

II. Methods for Derivatizing -OH and -SH Functions in Drugs

Method I

The drug is reacted with nicotinoyl chloride, with nicotinic anhydride, or with nicotinic acid in the presence of a suitable dehydrating agent such as dicyclohexylcarbodiimide, in an appropriate organic solvent, to afford the corresponding nicotinate. The nicotinate is then guaternized and subsequently reduced as described above in Method A. When the drug contains more than one reactive hydroxyl or thiol functions, reaction conditions may be varied so that more than one hydroxyl or thiol function will be converted to nicotinate groupings. The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds.

Starting Material [D-QC] ⁺ [D-DHC]

pentothal

testosterone

3 ⁾ 2 ^3 ⁾ 2

pentazocine

Starting Material ID-QC] ⁺ [D-DHC]

, norgestrel e M

Starting Mater i a 1 [D-QC]

[D-DHC]

noret ndrone

oxa zepam

Starting Material [D-QC]

[D-DHC]

e sterone

oxycodone

hydromorphone

Starting Material [D-QC] [D-DHC]

OMPI

Starting Material [D-QC] [D-DHC]

tem

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] , +

[D-DHC]

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

OMPI

Starting Material [D-QC] [D-DHC]

b

dexamethasone

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

.- 1 ' ?

Starting Material [D-QC] [D-DHC]

Starting Materials

I .

~

Starting Materials [D-QC] [D-DHC]

streptozotocin

6-mercaptopurine

Starting Material [D-QC] [D-DHC]

OMPI

Starting Material [D-QC] [D-DHC]

I n ω >t~ I

Starting _' Material

Starting Material [D-QC] [D-DHC]

Ara-AC

JTRE-A OMPI

Starting Material [D-QC] [D-DHC]

sangivamycin

Starting Material [D-QC] [D-DHC]

6-MMPR

trijnethyl TMM

Starting Material [D-DHC]

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

5

phenyl-6-chloro- , 6-deoxy-β-D- glucopyranoside ° .-δ-' ^CH *

Starting Material [D-QC]

[ D-DHC 1

ribavirin

(S)-9-(2,3- dihydroxypropy1) adenine

Starting Material [D-QC] [D-DHC]

6-azauridine

**•

CTl

-147-

Method J

This is a variation of ^" Method I used when the drug contains a -COOH function which is to be protected. The drug is first converted to the corresponding ethyl ester by conventional esterification techniques. That ester is then used as the starting material and Method I is repeated ^" . The -COOH group may be similarly converted to other ester groups. The representative drugs listed below may be derivatized in this manner to the corresponding

4-

[D-QC] and [D-DHC] compounds .

Starting Material [D-QC] [D-DHC]

5~hydroxy-2-n- propylpentanoiσ acid

4

3

-149-

Method K

Method I is followed, except that, in the first step, a starting material of the formula*

5 wherein n. = 1-3, preferably 2, is used in place of nicotinic acid.

The representative drugs listed below may be derivatized in. this manner to the corresponding

_|~

[D-QC] and [D-DHC] compounds, as may the remaining 10 drugs listed with Method I.

Similarly, Method K may be combined with Method J to afford the corresponding derivatives, e.g. of the drugs listed with that method.

A starting material of the formula set forth 15 immediately above can also be substituted for nicotinic acid in Method E to afford the corresponding deri¬ vatives, e.g. of the drugs listed with that method.

Starting Material [D-QC] [D-DHC]

norgestrel

Starting Material [D-QC] [D-DHC]

n trazepam

6-

-152-

MethOd L .

Method- I is followed, except that in the first step, the drug is reacted with 3-quinolinecarboxylic acid or its acid chloride or anhydride instead of nicotinic acid or its acid chloride. The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds, as may the remaining drugs listed with Method I. Similarly, Method 3_» may be combined with Method J to afford the corresponding derivatives, e.g. of the drugs listed with that method.

3-Quinolinecarboxylic acid or its acid chloride or anhydride can also be substituted for nicotinic acid or its acid chloride in Method E to afford the corresponding derivatives, e.g. of the drugs listed with that method.

Starting Material [D-QC] ID-DHC]

Starting Material [D-QC] [D-DHC]

oxazepam

Meith d M ^'

Method I is followed, except that in ^" the first step, a starting material of the formula

is used in place of nicotinic acid.

The representative drugs listed below may be derivatized in this manner to the corresponding [D-QC] and [D-DHC] compounds, as may the remaining drugs listed with Method I. Similarly, Method M may be combined with Method J to afford the corresponding derivatives, e.g. of the drugs listed with that method.

A starting material of the formula set forth immediately above can also be substituted for nico- tinic acid in Method E to afford the corresponding derivatives*, e.g. of the drugs listed with that method.

testosterone

naloxone

Starting Material [D-QCl [D-DHC]

thioguanine

∞ I

III, Methods for Derivatizing -COOH Functions in Drugs

Method N

The drug is reacted with excess alcohol of the formula

wherein n = 1-3, preferably 2 , to convert the -COOH function to the corresponding

ester grouping. That ester is then quaternized and subsequently reduced as described above in Method A. When the drug contains more than one reactive carboxyl function, reaction conditions may be varied so that more than one carboxyl function will be converted to ester groupings. (The starting alcohol may be prepared from nicotinamide, e.g. when n = 2, by reacting 2-iodoethanol with nicotinamid .)

The representative drugs listed below may be derivatized in this, manner to the corresponding [D-QC] and- [D-DHC] compounds.

Starting Material [D-QC] [D-DHC]

"M

Starting Material [D-QC] ^' [D-DHC]

iopyracet

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] [D-DHC]

g

Starting Material

5-methyl tetrahydrohomofolic acid

[D-QC]

[D-DHC]

OMPI

Method O

Method N is repeated, except that the starting alcohol employed has the formula

(That starting material may be prepared by reacting nicotinic acid with 1,2-propylene glycol in the presence of dicyclohexylcarbodiimide.)

The representative drugs listed below may be derivatized in this manner to the corresponding

[D-QC] and [D-DHC] compounds, as may the remaining drugs listed with Method N.

TRE TS OMPI

Method P

Method N is- repeated, except that the starting alcohol employed has the formula

(That starting material may be prepared by reacting bromoglucose with nicotinamide.)

The drugs listed below may be derivatized in this manner to the corresponding [D-QC] and

[D-DHC] coijipourids, as may the remaining drugs listed with Method N _*

Starting Material [D-QC] [D-DHC]

Starting Material [D-QC] + [ D-DHC]

chlorambucil

CH

Valproic

IV, Methods for Salt Formation

^" Method Q

An ether solution of [D-DHC] is treated with an equivalent amount of anhydrous p—toluenesulfonic acid dissolved in dry ether. Mixing at room temperature is continued until the imminium salt precipitates out of solution. The salt is then removed by filtration.

Imminium salts which may be prepared i this manner are those derived from the following [D-DHC]

(from phenylethylamine)

(from amphetamine)

phent ermine)

In order to further illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that same are intended only as illustrative and in nowise limitative.

In the examples immediately to follow, all melting points were taken on a Mel-Temp apparatus and are not corrected. Elemental analyses were per¬ formed at Atlantic Microlab, Inc., Atlanta, Georgia. Infrared spectra were determined using a Beckman Acculab 1 double-beam recording spectrophotometer. NMR spectra were determined by means of a Varian T60A or FXlOO spectrometer. All chemical shifts reported are in δunits (parts per million) relative to tetra— methylsilane. Ultraviolet absorbance spectra were determined using a Cary Model 219 spectrophotometer. HPLC analysis were performed on Waters Associates Liquid chromatograph with Model 6000A solvent delivery system. Model U6K injector and Model 440 absorbance detector. And in all cases where Anal. C, H, N is indicated, the elementary analysis of the compound was found within +0.4 Of the calculated value.

EXAMPLE 1 Preparation of N-(β-Phenethyl)nicotinamidet To 10.25 g (.083 mol) of nicotinic acid, 27.5 ml of thionyl chloride were gradually added. The mixture was stirred at room temperature for 10 min and then refluxed while stirring for 2 hrs- Excess thionyl chloride was then distilled off under reduced pressure.

Dry benzene (over sodium, 50 ml) was added and then distilled off under reduced pressure (to remove traces of S0C1 ₂ ) . A white crystalline acid chloride hydro- chloride was left, which was used as such for the preparation of amides.

To the solid acid chloride hydrochloride, 150 ml of dry and freshly distilled pyridine were added. To the stirred mixture, 10.45 ml (0.083 mol) of phenethylamine were dropped over 15 min. The mixture was then heated on a water bath while stirring for 2 hrs. Pyridine was distilled off on rotavap. The brown oily residue was poured onto crushed ice. The cream-white solid separated was filtered by suction, washed with cold water and dried in vacuum; yield 13.3 g (70%), m.p. 79-80 ^β C _? ir (KBr) 3320 (NH) and 1630 cm ^"1 (C=0) , NMR (CDCl.) 8.66 (bs, 1H, C. pyridine proton), 8.46 (bd, 1H, C _fi pyridine proton), 8.0-7.6 ( , 1H, C. pyridine proton), 7.33-6.90 (bs, 6H, ^c 6 ^H 5 ^{+ C} 5 PY ^ri dine proton) , 7.0-6.57 (hump, 1H, CONH), 3.73 (q, 2H,- -CH ₂ ), 2.97 (t, 2H, CH ₂ - ) . Anal. ( ₁₄ H ₁₄ N ₂ 0) C, H, N.

EXAMPLE 2

Preparation of l-Benzyl-3-(N-g-phenethyl) carbamoy1- pyridinium bromide; To a solution of 2.26 g (0.01 mol) of N-(β-phen- ethyl) nicotinamide in 5 ml of methanol, 1.4 ml (0.0114 mol) of benzyl bromide were added. ^' The mixture was refluxed for 3 hrs. Methanol was distilled off on rotavap. The yellow, oily residue left was scratched

-175-

when it suddenly solidified into buff, gritty solid. Crystallized from acetone/ether, yield 3.7 g (95%), m.p. 142-144°C, ϋ.V. max (buffer pH 7.4) 210 and 260 nm; ir (KBr) 3180 (NH) and 1670 cm ^"1 (C=0) .

5 NMR (CDCl ₃ /DMSO-d _g ) 6 10.26 (bs, 1H, C ₂ pyridine proton), 9.53-8*.90 (m, 2H, C _g and C . pyridine protons), 8.16-7.13 (m, 12H, 2C _g H ₅ + CONH + C _ς pyridine protons),

. 6.13 (s, 2H, -N-CH ₂ ), 3.96-3.50 ( , 2H, -N-CH ₂ ) ,

3.26-2.83 (m, 2H, CH ₂ ~0) . Anal. (C ₂₁ H ₂₁ BrN ₂ 0) C, H, N.

0 EXAMPLE 3

Preparation of l-Methyl-3-(N-β-phenethyl)- carbamoyl iodide;

To a solution of 2.26 g (0.01 mol) of N-(β- phenethyl)nicotinamide in 5 ml of methanol, 1.3 ml

(0.02 mol) of methyl iodide were added. The mixture 5 was refluxed for 3 hrs. Methanol was distilled off on rotavap and the yellow; oily residue was cooled and scratched when a yellow gritty solid was obtained. Crystallized from acetone,, yield 3.5 g (95%), m.p. 134-136°C, ϋ.V. max (buffer pH 7.4) 210, 225 and 226 nm. 0 Ir (KBr) 3240 (NH) and 1665 cirT ¹ (C=0) . NMR (CDCl-/

DMSO-dg) $ 9.63 (s, 1H, C ₂ pyridine proton), 9.4-8.9

(m, 2H, C4. and C,b. pyridine protons ₊ ), 8.32-8.06 (m, 1H,

^* C ₅ pyridine proton), 4.6 f-s, 3H, -ϊjT-OE ) , 3.9-3.46 (m, 2H, -N-CH ₂ ) , 3.2-2.8 (m, 2H, CH ₂ -0) . Anal. (C ₁₅ H _1? IN ₂ 0) C, H, N.

OMPI

EXAMPLE 4

Preparation of l-Benzyl- ^" 3r--(N-*β-phenethyl)carbamoyl-l,4- dihydropyridine:

To a solution of 3.97 g (0.01 mol) of l-benzyl-3- (N-β-phenethyl)carbamoylpyridinium bromide in 200 ml of deaerated water, 5.0 g ^* (0.06 mol) of. sodium bicarbonate and 200 ml of ether were added. The mixture was stirred in an ice bath and 7.1 g (0.04 ^' mole) of sodium dithionite were added gradually over a period of 5 min. The mixture was ^" stirred for 3 hrs under nitrogen. The ether layer was then separated, washed with water, dried with Na_S0 ₄ and distilled under vacuό. Yield 2.3 g (72%) of bright yellow, viscous oil was obtained which gave positive test for dihydropyridine with alcoholic silver nitrate solution. ϋ.V. max (buffer pH 7.4) 210 and ^' 355 nm. NMR

(CDCl J-)δ two overlapping singlets at 7.2 (10H, 2C b-H 3_) ,

7.1 (bs, 1H, C _j pyridine proton) , 5.68 (doublet of doublets, 1H, J=8 and 2 cps, C _g pyridine proton) , 6.4-5.0(hump, 1H, CONH), 4.84-4.60 (m, 1H, C ₅ pyridine proton), 4.35 (s, 2H, N-CH ₂ ) , 3.5 (q, 2H, J-7.0,

-N-CH ₂ ) , 3.0 (bs, 2H, C. pyridine proton) and 2.8 (t, 2H, -J=7.0, CH ₂ -φ) .

EXAMPLE 5 Preparation of l-Methyl-3-(N-β-phenethyl,)carbamoyl-l _r 4- dihydropyridine:

By the similar method described above, 1-methyl- 3-(N-β-phenethyl)carbamoy1 iodide (3.68 g, 0.01 mol) was reduced using sodium dithionite (7.1 q _t 0.04 mol) and sodium bicarbonate (5.0 g, 0.06 mol). Yield 1.8 g (76%) of bright yellow, viscous oil which reduced alcoholic silver nitrate solution. ϋ.V. max (buffer ph 7.4) 210, 290 and 360 nm; NMR (CDCl ₃ ) δ 7.2 (s, 5H,

C _g H ₅ ) ,.6.9 (bs, 1H,. C ₂ pyridinie proton) , 5.6 (doublet of doublets, 1H,. J=8, 2 cps, C _g pyridine proton), 5.3-5.1 (hump, 1H, CONH), 4.5-4.7 (m, 1H, C ₅ pyridine protons + N-CH ₃ 4 ^* CH ₂ ~φ) . Anal. ^' ( ^c ₁₅ ^H ₁₈ ^N 2 ⁰ - ^{) C} ' ^H ' ^N *

EXAMPLE 6

Preparation of Diethyl 3,5-pyridinedicarbox ate; To suspension of 8.35 g (0.05 mol) of 3,5- pyridinedicarboxylic ac d in 30 ml of absolute ethanol, 10 ml of concentrated sulfuric acid were dropped while stirring. The mixture was then refluxed on a water bath for 5 hrs and poured onto crushed ice. The solution was then made alkaline by the addition of solid KHCO- in small amounts. A white solid separated which was filtered, washed with water and dried. M.p. 42-44°C. The mother liquid was extracted with CH ₂ C1 ₂ when another crop of the diester was obtained. The overall yield of the crude ester was 9.1 g (82%) of sufficient purity for the examples to follow. NMR (CDCl ₃ )δ 9.62 (d, 2H, J-2 Hz, C~ and C _g pyridine protons), 8.76 (t, 1H, J=2 Hz, C. pyridine proton), 4.43 (q, 4H, J=7 Hz, 2 0CH ₂ ) , 1.41 (t, 6H, J=7 Hz, 2 CIT ₃ ).

EXAMPLE 7 Preparation of 5-Carboethoxy-3*-*pyridinecarboxylic acid; To a solution of 10 g (0.045 mol) of diethyl 3,5-pyridinedicarboxylate in 75 ml of ethyl alcohol, 25 ml of 2N alcoholic KOH were added while stirring. Stirring was continued for hour at room temperature. To the mixture, 12.5 ml .of 4N HC1 were added while stirring. The solid which separated was filtered and washed with alcohol. The combined filtrate and washings

- J*E

OMPI

-178-

were distilled on rotovap and the residue was washed with water, filtered and- crystallized from ethanol. Yield 7.5 g (86%) m.p. 180-182°.C, NMR (CDCl ₃ /DMSO-d _g ) δ 10.56 (bs, 1H, COOH), 9.26 (d, 2H, J=2 Hz, C and 5 C pyridine protons), 8.75 (t, 1H," J=2 Hz, C pyridine protons), 4.4 (q, 2H, J=7 Hz, 0-CH ₂ ) , 1.42 (t, 3H, J=7 Hz, CH ₃ ) .

EXAMPLE 8 Preparation of 5-Carbθethoxy-3-(N-β-phenethyl)carbamoyl-

10 pyridine;

To 10 g (0.05 mol) of 5-carboethoxy-3- pyridinecarboxylic acid, 30 ml of thionyl chloride were added and the mixture was warmed .on a water bath while stirring until clear ( =3 hrs) . Excess thionyl chloride

15 was distilled under vacuum. The residue was cooled to room temperature and 50 ml of sodium-dry benzene was added. The solution was cooled in an ice bath and a solution of 6.2 g (0.051 mol) of phenethylamine and 4 ml of pyridine in 50 ml of dry benzene was dropped while

20 stirring over 1 hr and the mixture was left overnight at room temperature. The mixture was then washed with water until free from Cl ^" (tested by AgNO.TS) . The organic layer was dried with Na-SO-. and distilled. The residue was crystallized from ether/pet. ether mixture.

25 Yield 9.0 g (67%), m.p. 159-161°C; ir (KBr) 3300 (NH) , 1725 (ester CO) and 1650 cm ^"1 (amide CO) , NMR (CDC1 ₃ ) δ 9.13-9.96 (two doublets, 2H, J=2 Hz, C ₂ and C _g pyridine protons), 8.53 (t, ^* lH, J=2 Hz, C. pyridine proton), 7.16 (s, 6H, C _g H ₅ + CONH), 4.36 (q, 2H, J=7 Hz, 30 0CH ₂ ) , 3.4 (q, 2H, J=7 Hz, N-CH ₂ ) , 2.9 (5, 2H, J=7 Hz, CH ₂ -φ), 1.33 (t, 3H, J=7 Hz, CH ₃ ) . Anal. (C ₁₇ H ₁₈ N ₂ 0 ₃ > C, H, N.

EXAMPLE 9

Preparation of 5-Carboethoxy-l-methyl-

3-(N-β-phenethyl)carbamoylpyridinium iodide;

To a solution of 2.9 g (0.01 mol) of 5-carbo- ethoxy-3-(N-β-phenethyl)carbamoylpyridine in 5 ml of acetone, 3 ml of methyl iodide were added. The mixture was refluxed while stirring for 8 hrs and then left overnight. The yellow crystalline solid which precipitated was filtered, washed with acetone, dried and crystallized from acetone. Weight 3.5 g (82%), m.p.l68-170°C, ir (KBr) 3250 (NH) , 1725 (ester CO) and 1670 cm ^" (amide CO) , U.V. max (buffer pH 7.4) 268 (weak plateau) and 268 nm (ε=53, 667), NMR (DMS0-d ₆ ) 9.53 (bs, 2H, C ₂ and C _g pyridine protons), 9.33-9.10 (m, 1H, C . pyridine proton), 7.16 (s, 5H, C _g H_) , 4.63-4.26 (complex multiplet, 5H, -Ϊsr-CH ₃ + 0CH ₂ ) , 3.56 (q, 2H, J=6 Hz, -N-CH ₂ ) , 2.90 (t, 2H, J=6, CH ₂ -0) , 1.4 (t, 3H, J=7 Hz, CH ₃ ) . Anal. (C ₁₈ H ₂₁ IN ₂ 0 ₃ ) C, H, N.

EXAMPLE 10

Preparation of 5-Carboethoxy-l-methyl- 3-(N-β-phenethyl)carbamoyJ-1,4-dihydropyridine;

This compound was prepared following the same procedure as in Example 4 using; 1.0 g (0.002 mol) of 5-carboethoxy-l-methyl-3-(N-β-phenethyl)carbamoyl¬ pyridinium iodide, 1.0 g (0.012 mol) sodium bicarbonate and 1.42 g (0.008 mol) sodium dithionite. Yield, .60 g (84%) of orange-yellow viscous oil which reduced alcoholic silver nitrate, but very slowly. ϋ.V. max

(buffer pH 7.4) 205 and 390 nm. NMR (CDC1 ₃ ) 7.33 (s, 5H, C _g H ₅ ), 7.0 (s, 2H, C ₂ and C _g pyridine protons), 5.8-5.3 (hump, 1H, CONH), 4.2 (q, 2H, J=7, 0-CH ₂ ) , 3.66 (q, 2H, J=7 Hz, -N-CH ₂ ) , 3.16 (bs, 2H, C^ pyridine proton), 3.0 (q, 2H, J=7, CH ₂ ~0) , 1.4 (t, 3H, J=7, CH ₃ ) .

EXAMPLE 11

Preparation of 3,4-Di(N-β-phenethyl)carbamylpyridine; To a solution of 2.53 g (0.01 mol) of diethyl 3,5-pyridinedicarboxylate in 10 ml of methanol, 3.0 g (0.025 mol) of phenethylamine were added. The mixture was refluxed overnight and then distilled.. The residue was washed with very dilute HC1 solution and water, dried and crystallized form ethanol. Yield 2.9 g (80%), m.p. 189-190°C. NMR (CDC1 ₃ ) δ 9.00 (d, J=2 Hz, 2H, 2,6-dipyridyl) , 8.33 (5, J=2, 1H, 4-pyridyl) , 7.30 (s, 10H, 2 C _g H ₅ ), 6.93-6.40 (hump, 2H, 2 COHN) , 3.83 (q, J=7, 4H, 2 -N-CH ₂ ) , 3.00 (t, ^" J=7, 4H, 2 -CH ₂ ~0) . Anal. (C ₂₃ H ₂₃ N ₃ 0 ₂ ) C, H, N.

EXAMPLE 12

Preparation of 1-Methyl-3,5-di(N-β-phenethyl)- carbamoy1 -pyridinium iodide; :

To a solution of 2.0 g (5.3 mmol) of 5-carbo- ethoxy-l-methyl-3-N-(β-phenethy1)carbamoyl-1,4- ^" dihydropyridine in 10 ml of acetone, 2 ml of methyl iodide were added and the mixture was refluxed for 24 hrs. The yellow crystalline solid which separated was filtered, washed with acetone and dried. Weight 1.4 g (51%), m.p. 186-188°C. ϋ.V. spectrum of a

solution in phosphate buffer 7.4 showed a plateau at 275 nm, a shoulder at 225 nm and a sharp peak at 203 nm (ε=67,356). Ir (KBr) 3240 (NH) , 1665 and 1650 cm ^"1 (twin band, C=0) . NMR (CDCL ₃ /D ₂ 0) 6 9.35 (d, 2H, J=2, C ₂ and C _g pyridine protons), 8.56- (d, 1H, J=2 Hz, C. pyridine proton), 7.20 (s, 10H, 2 C _g H ₅ ) , 4.56 (s, 3H, -1^-CH ₃ ), 3.66 (t, 4H, J=7 Hz, 2 _T -CH ₂ ) , 2.96 (t, 4H, J=7 Hz, 2 CH ₂ -0). Anal. (C ₂₄ H _2g IN ₃ 0 ₂ ) .

EXAMPLE 13 Preparation of l-Methyl-3,5-di(N-β-phenethyl)«- carbamoy1-1,4-dihydropyridine;

This compound was prepared following the same procedure as in Example 4, using 1 g (0.002 mol) of l-methyl-3,5-di(N-β-phenethyl)carbamoyl pyridinium iodide, 1.0 g (0.012 mol) sodium bicarbonate and 1.4 g (0.008 mol) sodium dithionite. Yield .65 g (86%) of orange-yellow semisolid which could not be crystallized. Its alcoholic solution shows a slow reduction to alcoholic silver nitrate solution. U.V. max (buffer pH 7.4) 388 and 210 nm. NMR (CDC1 ₃ ) 7.13 (s, 5H, ^• C _g H-) , 6.76 (s, 1H, C ₂ pyridine protons), 3.51 (q, 4H, J=7 Hz, 2 -N-CH ₂ ), 3.06-2.60 (m, 9H, 0-CH ₂ + C ₄ pyridine proton + N-CH-) .

EXAMPLE 14

Preparation of N-Nicotinoyldopamine ^" (compound 2) •

To a pyridine solution containing 11.7 g (0.05 mol) dopamine hydrobromide and 6.15 g (0.05 mol) nicotinic acid at 0°C were added 10.3 g (0.05 mol) dicyclohexylcarbodiimide (DCC) . The reaction mixture was stirred at room temperature for 24 hours and the formed dicyclohexylurea was removed by filtration. The pyridine was removed in vacuo and the residue was crystallized from water at 0°C. The product was isolated by filtration and dried over phosphorous pentoxide. Recrystallization from isopropanol gave 9.0 g (0.035 mol), 70% N-nicotinoyldopamine, m.p.

159-162°C; aqueous solution of the compound gave a green color with Fe +3 and reduced AgNO,; ir (KBr)

3300, 2960, 1725, 1630, 1590, 1520, 1430, 1290, 1190,

1115, 720 and 710 cm ^"1 ; NMR (d _g -DMS0) δ 9.25-6.25

(m, 7H) , 3.3 (m, 2H) and 2.65 (m, 2H) ppm. Anal.

^(C 14 ^H 14 ^N 2°3 ^{} C} ' ^H ' ^N*

EXAMPLE 15

Preparation of 3-{N—[β—(3,4-Diacetoxyphenyl)- ethyl] }carbamoylpyridine; ,

To an ice cold suspension of 2.06 g (8 mmol) finely powdered nicotinoyldopamine in 50 ml of chloroform, 1.56 g (10 mmol) of acetylchloride were dropped while stirring. The mixture was refluxed for 3 hrs, then filtered. The filtrate was washed with water until the washing did not give test for chloride ions with AgN0 ₃ T.S. Chloroform was distilled on

rotavap and the residue was crystallized from ether/ pet. ether. Yield 2.2 g (81%) NMR (CDCl ₃ ) 8.90 (bs, IH, C ₂ pyridine proton), 8.56 (bd, IH, C _g pyridine proton), 8.16-7.83 (m, IH, C ₄ pyridine proton), 7.36-7.03 (m, 5H, C _g H ₃ + C ₅ pyridine proton +NH) , 3.60 (q, 2H, J=7 Hz, -N-Cfl ₂ ) , 2.90 (t, 2H, J=7 Hz, -CH ₂ ) .

EXAMPLE 16

Preparation of 3-{N-[β-(3,4-Dipivalyloxyphenyl)*- ethyl] }carbamoylpyridine (compound j3c) ;

To a suspension of 5.16 g (.02 mol) finely powdered nicotinoyldopamine in 100 ml chloroform, 7.23 g (.06 mol) trimethylacetyl chloride were added under stirring. The mixture was refluxed for 6 hrs and then filtered. The filtrate was washed with water free of chloride ions, then washed once with a 5% solution of NaHCO_, then with water. The chloroform was evaporated and the residue was chromatographed by using a silica gel G column and 2% methanol in chloro- form as the eluent. The first fraction was collected and evaporated and the residue was crystallized from ether / petroleum ether. Yield, 6.2 g (73%) of a white crystalline solid, m.p. 112-114°C, NMR (CDC1 ₃ ) δ 9.06 (bs, IH, C ₂ pyridine proton), 8.73 (bd, IH, C _g pyridine proton), 8.30-8.13 (m, IH, C ₄ pyridine proton), 7.46- 7.10 (m, 5H, C _g H ₃ + C ₅ pyridine proton + CONH), 3.66 (q, 2H, J=6.25 Hz, -N-CH ) , 3.0 (t, 2YL , J=6 Hz, -CH ₂ ), 1.41 (s, 18H, 2-C(CH ₃ ) ₃ ). Anal. Calcd for

^C 24 ^H 30 ^N 2°5 ^{: C} ' ⁶⁷ * ^{58; H} ' ⁷ * ⁰⁹ ' ^{* N} ' 6-56. Found; C, 67.61; H, 7.10; N, 6.54.

-* RE tr" .OMPI

EXAMPLE 17

Preparation of l-Methyl-.3-{N-[β-(3,4-dihvdroxy- phenyl)ethyl)] }carbamoyIpyridinium iodide (compound £a); To a solution of 1.26 g (5 mmol) of nicotinoyl- dopamine ( ) in 10 ml of acetone, 1.41 g (10 mmol) of methyl iodide were added and the mixture was refluxed under stirring for 6 hrs. The acetone was removed and the residue was crystallized from methanol/ether.

Yield, 1.7 g (87%), m.p. 155-157 ^e C(dec) . Aqueous solution gave a green color with Fe +3, NMR (D 0)

+ ² δ 9.30-8.28 ( s, 4H, C ₅ H ₄ N) , 7.00 (bs, 3H, C _g H ₃ ) ,

4.60 (s, 3H, -N-CH ₃ ), 3.80 (t, 2H, J=7 Hz, -N-CH ₂ ) , 2.93 (t, 2H, J=7 Hz, CH ₂ ) . Anal. Calcd for C H IN O -H O; C, 43.11; H, 4.55; N, 6.70. Found; C, 43.83; H, 4.23; N, 6.81.

EXAMPLE 18

Preparation of l-Methyl-3-{N-[β-(3 ,4-diacetoxy- phenyl)ethyl] }carbamoylpyridinium iodide (compound £b) ; To a solution of 1.71 g (5 mmol) of 3-{N-[β-(3,4- diacetoxyphenyl)ethyl] }carbamoyl pyridine (prepared as compound _8c) , 1.41 g (10 mmol) of methyl iodide were added and the mixture was -refluxed overnight under stirring. The acetone- solution was then decanted from the insoluble oily residue. Ether was added to the acetone solution and the solid separated was crystallized from acetone/ether. Yield, 1.9 g (78%) of yellow crystalline needles, m.p. 171-173°C. ϋ.V. (methanol) 215, 265 nm; NMR (D ₂ 0) δ 8.86-7.63 (ms, 4H, C ₅ H ₄ N) , 6.66 (bs, 3H, C _g H ₃ ) , 4.4 (s, 3H,

-N-CH ₃ ), 3.50 (t, 2H, -N-CH ₂ ) , 3.03 (t, 2H, CH ₂ ) ,

2.21 (bs, 6H, 2 COCH ₃ ) . Anal. Calcd for C^H^IN^

C, 47.12; H, 4.37; N, 5.78. Found; C, 47.23; H, 4.38; N, 5.78.

EXAMPLE 19

Preparation of l-Methyl-3-{N-[β-(3,4-dipivalyloxy- phenyl)ethyl] ^" } carbamoylpyridinium iodide (compound Jjc) ;

To a ^" solution of 5.0 g (11.7 mmol) of compound 8c in 20 ml of acetone, 3.3 g (23.4 mmol) of methyl iodide were added and the mixture was refluxed under stirring for 6 hrs, then cooled. The orange crystalline solid which separated was filtered, washed with ether, and crystallized for acetone/ether. Yield, 5.6 g (85%), m.p. 163-165°C. ϋ.V. (buffer pH 7.4) 270, 215 nm. NMR (DMSO-d O _c ) δ j7..68-7.06 (ms, 7H, CD _C N4.N +

C _g H ₃ -l- NH) , 4.56. (s, 3H, -N-CH ₃ ) , 3.42 (q, 2H, J=7 Hz, -N-CH ₂ ) , 3.19 (t, 2H, J=7 Hz, CH ₂ ) , 1.32 (s, 18H,2 -C(CH ₃ ) ₃ ). Anal. Calcd for C ₂₅ H ₃₃ IN ₂ 0 ₅ C, 52.82; H, 5.85; N, 4.92. Found; C, 52.76; H, 5.87; N, 4.90.

EXAMPLE 20

Preparation of l-Methyl-3-{N-[β-(4-hydroxy-3-methoxy-*- phenyl)ethyl] }carbamoylpyridinium iodide (compound .9) ;

N-nicotinoyl-3-methoxytyramine was prepared by following the procedure used for the preparation of compound 1_- ^τ ** ^e isolated crude amide was quaternized directly with methyl iodide following the method used for the preparation of compound 6a. Crystallization from methanol gave a yellow crystalline compound, m.p. 192-194°C with overall yield of 84%, calculated on the basis of 3-methoxytyramine starting material.

NMR (D-O) closely similar to that of a except for the singlet at δ 3.66 for OCH .

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EXAMPLE 21

^' Preparation of l-Methyl-3-{N-[β-(3,4-dihydroxyphenyl)- ethyL] }carbamoyl-l,4-dihydropyridine (compound 5_a) ; To an ice cold solution of 1.0 g (2.5 mmol) of compound 6a in 200 ml of deaerated water, 1.26 g (15 mmol) sodium bicarbonate were added. Nitrogen was bubbled into the mixture and 1.74 g (10 mmol) of sodium dithionite was added gradually to the mixture under stirring. Stirring was continued for 1 hr and the mixture was then extracted twice with 50 ml of ether. The ether extract was washed with water, dried with anhydrous Na ^' -SO, and evaporated to dryness. Yield, 0.36 g (54%) of a yellow solid, m.p. 90-93°C (dec.) which gave a .green color with ferric chloride test and reduced alcoholic AgNO, instantly. DV (CH ₃ OH) 220, 350 nm. NMR (CDCl ₃ /D ₂ 0) δ 7.2-6.9 (ms, 4H, C _g H ₃ + C- dihydropyridine proton) , 5.6 (m, IH, C _g dihydropyridine proton) 4.6-4.4 (m, IH, C _ς dihydro¬ pyridine proton), 3.4 (m, 2H, -N-CH ₂ ) , 3.1-2.7 (m, 7H, N-CH ₃ + C ₄ dihydropyridine protons + CH ₂ ) . Anal. Calcd for C ₁₅ K ₁₈ N ₂ 0 ₃ ^• %H ₂ 0 C, 63.59; H, 6.76; N, 9.88. Found; C, 63.56; H, 6.85; N, 9.72.

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EXAMPLE 22

Preparation of l-Methyl-3-{N-[β-(3,4-diacetoxyphenyl)- ethyl] }carbamoy1-1,4-dihydropyridine (compound 5_b) ; To an ice cold solution of 1.4 g (3 mmol) of compound J5b in 200 ml of deaerated water, 1.5 g (18 mmol) of sodium bicarbonate was added. A stream of N ₂ was bubbled into the mixture and 2.1 g (12 mmol) of sodium dithionite were gradually added under stirring. Stirring was continued for 30 min and then the mixture was extracted with ethyl acetate. The extract was washed with water, dried with anhydrous Na ₂ S0 ₄ and evaporated to dryness. The yellowish semisolid mass remaining gave a faint green color with ferric chloride test indicating partial hydrolysis of the ester functions. It reduced alcoholic silver nitrate instantly. U.V. (CH ₃ OH) 220, 273 and 355 nm; NMR (CDC1 ₃ /D ₂ 0) δ 7.13-6.80 (ms, 4H, + C x dihydropyridineproton) , 5.53 (doublet of doublets, IH, C, dihydropyridine proton), 4.63-4.46 b (m, IH, 3m, dihydropyridine proton), 3.33 (t, 2H,

J=6.5 Hz, -N-CH ₂ ) , 3.06-2.66 (m, 7H, -N-CH ₃ + C^ dihydropyridine proton + CH ₂ ) , 1.8 (s, s6H, 2C0CH ₃ ) .

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EXAMPLE 23

Preparation of l-Methyl-3-*fN-[β-(3,4-dipivalyloxyphenyl)- ethyl] }carbamoyl-l,4-dihydropyridine (compound jSc) ;

To a cold mixture of 2.0 g ^' (3.5 mmol) of compound 5 6c _f 200 ml of deaerated water and 100 ml of ethyl acetate, 1.14 g (14 mmol) of sodium bicarbonate and 2.43 g (14 mmol) of sodium dithionite were added. The mixture was stirred under N ₂ for 20 mins. The ethyl acetate layer was separated and the aqueous layer was

10 re-extracted with 100 ml of ethyl acetate. The combined ethyl acetate was washed with cold deaerated water, dried over anhydrous Na^SO. and distilled on rotovapor. The viscous yellow oily residue was dissolved in 5 ml of acetone, filtered under N_ atmosphere and then -

15 evaporated under reduced pressure. The solid residue was dried under vacuum over P_0_in N_ atmosphere. It

_£ O __ reduced alcoholic AgN0 ₃ instantaneously and gave no color with FeCl ₃ test. Yield, 1.3 g (83%) m.p. 45-48°C; UV (CH ₃ 0H) 210 and 355 nm; NMR (CDC1 ₃ ) δ 7.04-6.92 (m,

20 4H, C _g H ₃ + C ₂ dihydropyridine proton), 5.71-5.61 (doubletof doublets, IH, C _g dihydropyridine proton), 4.81 (bs, IH, CONH), 4.60-4.51 (m, IH, C _g dihydropyridine proton), 3.53 (q, 2H, J=6.3 Hz, -N-CH ₂ ) , 2.36 (bs, 2H, C ₄ dihydropyridine proton), 2.91 (s, 3H, -CH ₃ ) , 2.79 (t,

25 2H, J=6.3 Hz, CH ₂ ) , 1.33 (s, 18H, CO-C(CH ₃ ) ₃ ) ..

Anal. Calcd for ₂₅ H ₃₄ N ₂ 0 ₅ • 1%H ₂ 0; C, 63.9; H, 7.93; N, 5.96 Found; C, 63.4; H, 7.81; N, 5.94.

EXAMPLE 24

Preparation of l-Methyl-3-{N-[β-(4-hydroxy-3-methoxy- phenyl)ethyl] }carbamoyl-l,4-dihydropyridine

(compound 10) ; This compound was prepared following the same method as for the preparation of compound 5_c. The crude solid obtained showed the same NMR (CDCl ₃ /D ₂ 0) pattern as compound 5_a, except for a peak at δ 3.5 for the 0CH ₃ protons. It was sufficiently pure for the determination of its retention time following the

HPLC method of analysis detailed in Example 37 below. No trials were made for its further crystallization or elemental analysis.

EXAMPLE 25 Preparation of N-Nicotinoyltyramine;

To an ice cold suspension of 3.69 g (0.03 mol) nicotinic acid in a solution of.5.2 g (0.03 mol) tyramine hydrochloride in 100 ml of pyridine, 6.18 g (0.03 mol) of dicyclohexylcarbodiimide (DCC) were gradually added while stirring. Stirring was continued at room temperature for 24 hrs and the formed dicyclohexylurea was removed by filtration. The pyridine was removed by distillation in vacuo and the residue was triturated with cold water, filtered and crystallized from 50% aqueous methanol. Yield, 6.25 g (86%), m.p. 179-181°C. PMR (DMS0-d _g /D ₂ 0) δ 9.0-8.66 (m, 2H, C ₂ and C _g pyridine protons), 8.33-8.10 (m, IH, C ₄ pyridine proton), 7.66-7.46 (m, IH, C _ς pyridine proton), 7.23-6.70 ( , rH, C _g H ₄ ) , 3.56 (t, 2H, -NCH ₂ ) , 2.90 (t, 2H, CH ₂ ) . Anal. (C ₄ H ₁₄ N ₂ O ₂ ) C, H, N-

EXAMPLE 26

Preparation of 3-{N-[β-(4-pivalyloxyphenyl)ethyl]}- carbamoy1pyridine;

To an ice cold suspension of 4.84 g (0.02 mol) N-nicotinyoltyramine in 100 ml chloroform, 3.6 g

(0.03 mol) of trimethylacetyl chloride were dropped while stirring. The mixture was refluxed overnight and the non—reacted nicotinoyltyramine was filtered off. The filtrate was washed with water until free from chloride ions, washed once with 5% solution of

NaHCO-. and then with water. Chloroform was evaporated on rotavap and the residue was crystallized from ether/pet. ether. Yield 3.9 g (60%), m.p. 80-82°C. PMR (CDC1 ₃ ) δ 8.66-6.93 (m, 8H, C^N + C _g H ₄ ) , 3.56 (q, 2H, J=7 Hz, - -CH ₂ ) , 2.86 (5, 2H, =^7 Hz, CH ₂ ) , 1.33 (s, 9H, C-(CH ₃ ) ₃ ).

EXAMPLE 27

Preparation of l-Methyl-3-{N-[β-(4-hydroxyphenyl)•*•- ethyl] }carbamoylpyridinium iodide; To a solution of 1.21 g (5 mmol) of nicotinyoltyramine in 10 ml of acetone, 1.41 g (10 mmol) of methyl iodide were added and the mixture was refluxed while stirring for 6 hrs. The fine, yellow solid separated was filtered and crystallized from methanol ether. Yield 1.78 g (93%) , m.p. 208-210°C. PMR (DMSO-d _c /D O) δ 9.23-8.26 (m, 4H, + 6 2 ₊

C ₅ H ₄ N) , 7.33-6.83 (m, 4H, C _g H ₄ ) , 4.50 (s, 3H, -1S.-CH ₃ ) , 3.70 (t, J=7 Hz, 2H, N-CH ₂ ) , 2.93 (t, J=7 Hz, 2H, CH ₂ ) .

I - IPO .

EXAMPLE 28

Preparation of l-Methyl-3-{N-fβ-(4-pivalyloxyphenyl)- ethyl] }carbamoylpyridinium iodide;

To a solution of 1.63 g (5 mmol) of the product of Example 27 in 10 ml of acetone, 1.41 g (10 mmol) methyl iodide were added and the mixture was re¬ fluxed overnight while stirring. The acetone layer ■was separated by decantation and the yellowish, oily residue was crystallized from methanol/ether. Yield, 1.94 g (83% . 155-157°C PMR (D ₂ 0) δ

9.16-8.00 (m, 4H, , 7.33-6.83 (m, 4H, C _g H ₄ ) , 4.40 (s, 3H, S-CH ₃ 3.5 (t, 2H, J=7 Hz, - -CH ₂ ) , 2.90 (t, 2H, J=7 Hz, CH ₂ ) , 1.30 (s,.9H, C-(CH ₃ ) ₃ ). Anal. (C ₂₀ H ₂₅ N ₂ O ₃ I) C, H, N. EXAMPLE 29

Preparation of l-Methyl-3-{N-[β-(4-hydroxyphenyl)ethyl] } ^• carbamoyl-1,4-dihydropyridine;

To an ice cold solution of 1.15 g (3 mmol) of the product of Example 27 in 200 ml of deaerated water, 1.5 g (18 mmol) sodium bicarbonate were added. While the mixture was bubbled with ₂ gas, 2.09 g (12 mmol) of sodium dithionite were gradually added to the mixture. The mixture was stirred under N ₂ for 1 hr and then extracted twice, each with 100 ml of ethyl acetate. The combined extract was washed with water, dried over anhydrous a ₂ SO. and distilled on rotovap. Yield, 0.38 g (50%) of yellowish semisolid which reduced alcoholic AgN0 ₃ TS instantaneous¬ ly. (PMR as expected.)

r- _r . WIPO

EXAMPLE 30

Preparation of l-Methyl-3-{N-[β-4-pivalyloxyphenyl) }- carbamoy1-1,4-dihydropyridine;

To an ice cold mixture of 2.34 g (5 mmol) of the product of Example 28, 200 ml of deaerated water and 100 ml of ethyl acetate, 1.63 g (20 mmol) sodium bicarbonate and 3.47 g (20 mmol) sodium dithionite were added while stirring the mixture. Stirring was continued under N ₂ gas for 30 min. The ethyl acetate layer was separated and the aqueous layer was extracted with 100 ml of ethyl acetate. The combined ethyl acetate extract was washed with 100 ml cold deaerated water, dried over anhydrous Na-SO. and evaporated on rotavap. The viscous, yellow residue was dissolved in 5 ml of acetone, filtered under N ₂ has through folded filter paper and distilled on rotavap. The solid residue was dried under vacuo over P ₂ O _ς in N- atmosphere. It reduced alcoholic AgNO_, instantaneous¬ ly. Yield, 1.06 g (62%). (PMR as expected.)

EXAMPLE 31

Preparation of 3,5—Pyridinedicarboxylic acid didecyl ester hydrochloride; '_

3,5-Pyridinedicarboxylic acid (9.6 g, 0.06 mole) was converted to the diacid chloride by treatment with excess SOCl ₂ . The mixture was refluxed at 100°C for 6 hrs. Excess S0C1 ₂ was distilled under reduced pressure and 25 ml of decyl alcohol dissolved in benzene were added. The solution was refluxed for 5 hrs after which benzene was distilled and the residue dissolved in ethyl ether. The organic phase was extracted with bicarbonate solution and later dried over Na_SO '4, ^*

"gfR X

OMPI

The ethyl ether solution was acidified with HCl (gas) and 24.2 g of compound (95% yield, m.p. 80-90°C) were obtained. IH (NMR) CDCl ₃ /d _g DMSO δ9.3 (3H, bs) , 8.7 (IH, bs), 4.3 (4H, bT) and 1.4 (38H, bm) ppm.

EXAMPLE 32

Preparation of Didecyl 3,5-dicarboxylate-l-methyl- pyridinium iodide;

The product of Example- 31 (10 g, 0.025 mole) was dissolved in an ethyl ether/bicarbonate solution. The organic phase was rinsed with water and dried over Na ₂ S0 ₄ . The solvent was evaporated and the residue dissolved in acetone and an excess of methyl iodide added. The solution was refluxed for 8 hrs, after which the solvent was evaporated and ethyl ether added to the residue. A yellow solid was obtained which was filtered and rinsed with more ethyl ether.

The solid was recrystallized from a minimum amount of ethyl acetate to yield 12.5 g (85%) m.p. 104-105°C.

Analytical data; Theory; C, 57.04? H, 8.21. Found; C, 57.18; H, 8.09. Spectrophotometrie data

4 in methanol; λ219 ε = 2.7 x 10 1/mol cm; λ277 ε = 3.6 x 10 ³ 1/mol cm.

•/

EXAMPLE 33

(i) Oxidation by Hydrogen Peroxide; To 10 ml of 30% H ₂ 0 ₂ was added 0.2 g of the dihydropyridine derivative (products of Examples

4, 5, 10 or 13) . The mixture was stirred and samples were taken to check the UV spectrum. Complete oxidation to the corresponding quaternary salts was observed. (ϋ) Oxidation by Silver Nitrate;

To 5 ml of saturated methanolic AgN0 ₃ solution was added 1 ml of 5% methanolic solution of the

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dihydropyridine derivative. The mixture was shaken and left for 5 min for complete precipitation of silver, centrifuged and an aliquot was taken to check the UV spectrum. Complete oxidation to the quaternary salts was observed.

(iii) Calibration Curves

UV study of the compounds prepared in Examples 2-5, 9, 10, 12 and 13 revealed that they obey Beer's Law with good coefficients and at a wide range of dilution levels. The study was done at 350 nm for the dihydro derivatives and at 262 and 220 nm for all the quaternary and dihydro.

EXAMPLE 34

Kinetics of Oxidation of the Dihydro Derivatives;

-4 In Plasma; 0.2 ml of (6.25 x 10 M) freshly prepared solution of the dihydro derivative in methyl alcohol was diluted to lb ml with 20% plasma (diluted with phosphate buffer pH 7.4) . The solution was kept at 37°C and UV spectrum was scanned from 400 nm to 300 nm every 10 min for 2 hrs against reference made by dilution of 0.2 ml methyl alcohol with 20% plasma to 10 ml.

In Whole Bloodr In'each of 5 tubes, 0.1 ml of 10 x 10 —4 M methanolic solution of the freshly prepared dihydro derivative, was added 2 ml of fresh heparinized whole human blood and the tubes were kept at 37°C in a water bath. At the end of the time period to be investigated, 8 ml of acetonitrile was added, shaken vigorously and centrifuged. The extension of the supernatant solution at 350 nm was measured. A reference sample was made by addition of 0.1 ml of methyl alcohol instead of the sample solution follow¬ ing the same procedure.

In Brain Homogenate; 2.0 g of rat brain tissue were homogenized in 10 ml of phosphate buffer, pH 7.4. The homogenate was centrifuged for 15 min at 3000 rpm, decanted, heated in a water bath at 50°C for 5 min and then centrifuged again. The supernatant solution was diluted to 100 ml with phosphate buffer, pH 7.4.

Reference Sample; 0.2 ml of methyl alcohol was diluted to 10 ml with the brain homogenate solution, and the solution was used to record the base line on Cary 219 spectrophotometer and as a reference for the dihydro derivative sample solution.

Dihydro Derivative Sample Solutions; 0.2 ml of 6.25 x 10 -4 M methanolic solution of the freshly prepared dihydro derivative was diluted to 10 ml with the brain homogenate solution. The mixture was scanned at 37°C from 400 nm to 300 nm every 10 min for 2 hrs on Cary 210 spectrophotometer. In Liver Homogenate;

Liver Homogenate Solution; 5.0 g of rat liver tissue were homogenized in 50 ml of phosphate buffer, pH 7.4.

The homogenate was centrifuged, decanted, heated in a water bath at 50°C for -.5min and then centrifuged again. The supernatant homogenate was diluted to

250 ml with phosphate buffer, pH 7.4. Reference Sample; 0.2 ml of methyl alcohol was diluted to 10 ml with the liver homogenate solution and the solution was used to record the base line on a

Cary 219 spectrophotometer and as a reference for the dihydro derivative sample solution. Dihydro Derivative Sample Solution; 0.2 ml of

6.25 x 10~ M solution of the freshly prepared dihydro

derivative in methyl alcohol was diluted to 10 ml with liver homogenate solution. The mixture was scanned at 37°C from 400 nm to 300 nm every 5 min for 1 hr.

.OMPI

TABLE II Kinetics of Oxidation

EXAMPLE 35

In Vivo Study on l-Methyl-3-(N-β-phenethyl)carbamoyl-1,4- dihydropyridine;

A group of rats of average weight (about 350 g) was injected through the jugular with a solution of the freshly prepared dihydro derivative in DMSO (0.05 g/ml solution) is a dose level of 125 mg/kg animal body weight.

Γ REX,

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After the appropriate time period, 1 ml of blood was withdrawn from the heart and the animal was perfused with 20 ml of saline solution. The animal was decapi¬ tated. The brains were weighed, kept in the refriger¬ ator overnight and homogenized in 2 ml of water. Acetonitrile, 8 ml, was added and the mixture was homogenized again and then centrifuged. The amount of the quaternary was determined from the HPLC spectrum in relation to a recovery experiment made by

10 adding a specific amount of the quaternary to a blank brain and hybrid in the same manner of homogenization and extraction.

Brain Results;

Normalized value

15 amt in mg/weight lb Normalized in grams t value *

5 .055 40 .1132 5 .0423 47 .125 10 .099 66. .148

20 15 .0553 90 .1626 15 .100 90 .1294 20 .0.935 145 .0949 21 .0743 180 .0838 25 .101 185 .1001

25 30 .1242 210 .0707 32 .095 220 .0753 33 ^' .0778

Blood Concentration; The blood withdrawn was left in the refrigerator overnight and 3 ml of saline 30 was added and the mixture shaken, then 17 ml of aceto¬ nitrilewas added and the mixture was shaken vigorously

for 1 min and then centrifuged. The supernatant solution was injected directly into the HPLC. Results;

EXAMPLE 36

Kinetics of Disappearanceof the Quaternary from

Brain Hornogenate;

A fresh perfused rat brain was homogenized in 20 ml of phosphate buffer, pH 7.4. A solution of 10.0 mg of l-methyl-3-(N-β-phenethyl)carbamoylpyridinium iodide in 2 ml aqueous methanol (lil) was added and the thoroughly mixed mixture was kept at 37°C in a water- bath. At each time period, 1 ml of the mixture was taken and shaken thoroughly with 8 ml of acetonitrile, centrifuged and injected to HPLC. The amount of the quaternary in the sample was determined in comparison with a sample taken at time 0. Linear regression of t against log C shows that K •= 4.8 x 10 -5 sec—1 , h -

3.50 h (in _, vivo exp.) which was found to be =

8.45 x lθ ^""5 sec ^-1 , ^s «■ 2.1 h, r =.957.

FREA

OMPI

Studies of the Dopamine Derivatives EXAMPLE 37

Analytical Methods;

A high pressure liquid chromatography (HPLC) method was developed for the studies of the degrada¬ tion of the dihydropyridine dopamine derivative. The chromatographic analysis was performed on a component system consisting of a Waters Associate Model 6000A solvent delivery system _r Model ϋ6K injector and Model 440 dual channel absorbance detector operated at 254 and 280 nm. A 30 cm x 3.9 mm (internal diameter) reverse phase yBondapak C, _g column (Waters Associates) , operated at ambient temperature, was used for all separations. The mobile phase used for the separation of the dihydropyridine derivative, its degradation products and oxidation products consisted of 0.005 M solution of 1-heptanesulfonic acid sodium salt (PIC B-7 Eastman Kodak) in CH CN; 0.01 M aqueous dibasic ammonium phosphate (2.5:1). At a flow rate of 2.0 ml/min, 6_ had a retention time of 5.1 min; _, 11.8 min; 9 _^r 1.7 min; 11, 3.1 min. A peak was always shown at a retention time of 2.2 min which is believed to be a monodeacylated dihydropyridine derivative, since it eventually did result in 6a..

EXAMPLE 38

Determination of the Enzymatic Hydrolitic Cleavage and Rate of Oxidation of Compound 5_c; In Human Plasma;

The freshly collected plasma used was obtained at he Civitan Regional Blood Center, Inc. (Gainesville,

Florida) and contained about 80% plasma diluted with anticoagulant citrate phosphate dextrose solution U.S.P. The plasma was stored in a refrigerator and used the next day. One hundred μl of a freshly prepared 0.61 M solution of compound 5_c in methanol was added to 20 ml of plasma, previously equilibrated to 37°C in a water bath and mixed thoroughly to result in an initial concentration of 3.05 x 10 moles/liter. One ml samples of plasma were withdrawn from the test medium, added immediately to 5 ml of ice cold aceto- nitrile, shaken vigorously and placed in a freezer. When all samples had been collected, they were centri¬ fuged and the supernatants were filtered through Whatman 1 filter papers and analyzed to HPLC.

In Human Blood;

The freshly collected heparinized blood was obtained at the Civitan Regional Blood Center, Inc. (Gainesville, Florida) . The blood was stored in a refrigerator and used the next day. One hundred μl of a freshly prepared 0.19 solution of compound 5c in methanol was added to 20 ml of blood, previously equilibrated to 37°C in a water bath and mixed thoroughly to result in an initial concentration of

-4 9 x 10 moles/liter. One ml samples of blood? were withdrawn from the test medium every 5 minutes, added immediately to 5 ml of ice cold acetonitrile, shaken vigorously and placed in a freezer. When all samples had been collected, they were centrifuged and the supernatants were filtered using Whatman 4 filter paper and analyzed by HPLC.

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In Rat Brain Homogenate;

The brain homogenate was prepared by the following method. Five Sprague-Dawley rats were killed by decapita¬ tion and the brains were removed, weighed (total weight 9.85 g) and homogenized in 49.3 ml of aqueous 0.11 M phosphate buffer, pH 7.4. The homogenate was centri¬ fuged and the supernatant was used for the test. 100 μl of 0.18 M solution of compound 5_c was mixed with 10 ml of homogenate, previously equilibrated to 37°C in a

10 water bath, to result in an initial concentration of

-3 1.8 x 10 moles/liter. Samples of 1.0 ml were withdrawn every 10 minutes from the test medium, added immediately to 5 ml of ice cold acetonitrile and placed in a freezer. When all samples had been collected, they were centri¬

15 fuged. Each supernatant was filtered through two Whatman 1 filter papers and analyzed by HPLC.

In Rat Liver Homogenate:

The liver homogenate was prepared by the following method. Three Sprague-Dawley rats were killed by 20 decapitation and the livers were removed, weighed and homogenized by tissue homogenizer in 0.11 M aqueous phosphate buffer, pH 7.4, to make 20% liver homogenate. The homogenate was centrifuged and the supernatant was used for the test. 100 μl of 0.1 M solution of 25 compound 5c in methanol were mixed with 20 ml of the homogenate, previously equilibrated to 37°C in a water bath, to result in an initial concentration of 9 x 10 -4 moles/liter. Samples of 1.0 ml were withdrawn every 5 minutes from the test medium, added immediately to 5 ml

30 of ice cold acetonitrile, shaken vigorously and placed in a freezer. When all samples had been collected, they were centrifuged and each supernatant was filtered through Whatman 1 filter paper and analyzed by HPLC.

Rates of disappearance (overall oxidation and degradation) of compound 5_c:

(i) in Plasma: R = 2.25 x lθ ^"4 sec ^"1 t% = 51.3 min r = 0.998 n = (3 x 6)

(ii) In 20% Brain Homogenate: R » 6.7 x lθ ^"4 sec ~ ¹ = 17.2 min r = 0.996 n = (3 6)

(iii) In Blood: R 6.3 x 10 -4 ϊs 18.2 min r 0.997 n (3 x 7)

(iv) In Liver: R 1.93 x 10 -3 t% 5.9 min r 0.950 n (3 x 5)

EXAMPLE 39

Determination of Concentration of Compound 6a in Brain and Blood after Parenteral Administration of 5_c:

Male Sprague-Dawley rats of average weight of 150 ± lOg were used. The rats were anesthetized with IM injection of Inovar and the jugular was exposed. Com- pound 5_c was injected intrajugularly in the form of 10% solution in DMSO at a dose of 64.2 mg/kg (equivalent to 50 mg/kg compound 6a) . The injection was given at a rate of 24 μl/min using a calibrated infusion pump.

After appropriate time periods, 1 ml of blood was withdrawn from the heart and dropped immediately into a tared tube containing 3 ml acetonitrile, which was afterwards weighed to determine the weight of the blood taken. The animal was then perfused with 20 ml of saline solution, decapitated and the brain was removed. The weighed brain was homogenized with 0.5 ml of distilled water, 3 ml of acetonitrile was added and the mixture was rehomogenized thoroughly, centri¬

10 fuged, filtered and then analyzed for compound 6a using the HPLC method. The tubes containing the blood were shaken vigorously, centrifuged, decanted and also analyzed for compound 6a. using the HPLC method. Quantitation was done by using a recovery standard 15 curve obtained by introducing a known amount of 6a in either brain homogenate or blood and then treated in the same manner. See FIGURE 6 and the discussion thereof hereinabove.

EXAMPLE 40

20 Pharmacological studies:

In vivo effect on pituitary prolactin secretion:

Adult male rats (Cha'rles Rivers, CD-I) weighing 200 to 225 g were provided food and water ad libitum for at least one week period to experimentation. To

25 elevate serum prolactin levels, each rat received a single s.c. implant of a Silastic tube (1.57 mm interior diameter, 5 mm x 3.15 mm overall size) packed with crystalline 17-β-estradiol. Two days later the rats were lightly anesthetized with ether and a small

30 incision was made over the right jugular vein for intravenous (I.V.) administration of the test drugs.

Compound j6a was injected at a_ dose of 1 mg/kg body weight/ml saline andgroups of six rats were decapitated at 15, 30, 60 and 120 min later to collect blood samples. Control rats (time 0) received an I.V. 5 injection of the saline vehicle and were decapitated 30 min later. Compound 5lc was dissolved in 10% ethanol in saline and was injected IV. Rats were decapitated at 15, 30 and 120 min later. Control .(time 0) animals received the 10% ethanol vehicle 10 and were sampled 30 min later.

Trunk blood was collected, allowed to clot for 2h and the serum was separated and stored at -20°C for subsequent assay for prolactin concentrations. Each serum sample was assayed in duplicate by the double- 15 antibody radioimmunoassay procedure described by the National Pituitary Agency Hormone Distribution Program. Serum prolactin concentrations are expressed in terms of the PRL-RP-2 reference preparation provided. The intraassay coefficient of variation for 10 replicate 20 samples ofpooled serum obtained from male rats was 13.8%.

The effects of compounds 5_c and 6a. on serum prolactin concentrations were evaluated by one-way analysis of variance and Student-Newman Keuls tests. 5 A probability level of less than 0.05 was selected for significance. See FIGURE 7 and the discussion thereof hereinabove.

In vitro evaluation of the prolactin inhibitory effect of j5a: 0 Adult female rats (Charles Rivers Lab.) weighing 225-250 g were maintained on food and water ad libitum. Animals were sacrificed by decapitation; their pituitary

k

glands were quickly removed from the cranium. The anterior pituitary (AP) of each animal was dissected into two equal halves and placed into incubation media. (Gibco's Minimal Essential Media supplied by Grand Island Biological Co. was used.) The incubation was conducted at 37°C, under continuous aeration (95% 0 ₂ , 5% C0 ₂ ) ; the pH was 7.2. After one hour of preincubation, the media were discarded and replaced with fresh ones containing either DA ( 2 x 10 —8M) ,

—8 —4 6a ( 2 x 10 ) or ascorbic acid (10 M) . In all cases, one-half of AP received the test drug _? the other, the ascorbate control. After one hour, samples were taken from the media and the remaining media were discarded.

Fresh media containing DA ( 2 x 10 —7) , 6a (2 x 10—7) and ascorbate, respectively, were then added. One hour later, the second samples were taken. After the 3 h incubation period, each half AP's were weighed. The samples were diluted 1:50 with phosphate buffered saline and then assayed in triplicate by the radioimmunoassay method described. The data are given as ng prolactin released/mg wet weight/h. Paired Student's T-test was used to evaluate the significance of the inhibitory effects of the test drugs on pro¬ lactin secretion. The control AP half and the drug treated half were employed in each paired comparison. See TABLE I and the discussion thereof hereinabove.

In the examples immediately to follow, all melting points were taken on a Mel-Temp apparatus and are not corrected. Elemental analyses were per¬ formed at Atlantic Microlab, Inc., Atlanta, Georgia. Infrared spectra were determined by using a Beckman Acculab 1 double-beam recording spectrophotometer. NMR spectra were determined by means of Varian 60A or FX100 spectrometers. All chemical shifts reported are in 6 units (parts per million) relative to tetra- methylsilane. Ultraviolet absorbance spectra were determined using a Cary Model 210 spectrophotometer. HPLC analyses were performed on a Beckman 345 ternary liquid chromatograph with Model 112 solvent delivery system. Model 210 injector. Model 160 absorbance detector and Model 421 controller.

EXAMPLE 41 Preparation of Testosterone nicotinate (compound 41) : Thionyl chloride (2 ml) was added to 0.7 g (5.7 mmol) of nicotinic acid and the mixture was refluxed for 3 hrs. Excess thionyl chloride was removed under reduced pressure. To the cold residue, 10 ml of dry pyridine was added, followed with 1.44 g (5.0 mmol) of testosterone. The mixture was heated with continuous stirring at 100°C over a water bath for 4 hrs. Py- ridine was removed in vacuo and 5 ml of methanol was added to the oily residue. The mixture was cooled and the solid that crystallized was filtered and re¬ crystallized from methanol/acetone mixture to give 1.4 g of 41 as white crystals (yield 71%) , m.p. 187-188°C. This intermediate was used directly for the synthesis of the chemical delivery system.

EXAMPLE 42

Preparation of 17β-[(l-Methyl-3-pyridiniumcarbonyl)***» oxy]androst-4-en-3-one iodide (compound *J2) (Testo- sterone-17-nicotinate N-methyl iodide) ; To a solution of 1.0 g (2.5 mmol) of testo¬ sterone nicotinate 41 in 15 ml of acetone, 1 ml of methyl iodide was added and the mixture was refluxed overnight. The yellow solid that separated was re¬ moved by filtration, washed with acetone and crys- tallized from methanol/ether to yield 1.25 g ^" (92% yield) of pure 4_2 as yellow crystals, m.p. 215-220°C (dec). U.V. (CH ₃ OH) λ 270 nm (shoulder) ε = 4579; 240 (shoulder), ε = 19375. NMR (CDCI3) 610.0-8.3 (ms, 4H, pyridinium protons), 5.73 (s, IH, c ₄ testo- sterone proton), 4.86 (s, 3H, +N-CH ₃ ) ,.2.40-1.06 (ms, 26H, testosterone skelton protons) . Analysis calcu¬ lated for C26H34INO3: C, 58.32; H, 6.40; N. 2.62. Found: C _r 58.17; H, 6.48; N, 2.60.

EXAMPLE 43 Preparation of 17 β-[ (l,4-Dihydro-l-methyl-3-pyri- dinylcarbonyl)oxy]androst-4-en-3-one (compound £_3) :

To an ice cold solution of 1.1 g (2 mmol) of testosterone nicotinate N-methyl iodide 42_ in 150 ml of deaerated 10% aqueous methanol, 0.67 g (8 mmol) of sodium bicarbonate and 1.37 g (8 mmol) of sodium dithionite were added. The mixture was stirred for 20 minutes and the pale yellow solid which separated was filtered, washed with water and dried over 2O5 under vacuum. Wt. 0.82 g (98% yield), m.p. 172- 175°C. UV (CH3OH) λ 356 nm, ε ___ 9511; ir (KBr)

1700, 1660 cm ^"3 - (two C=0 stretching). NMR (dg-DMSO)

δ 6.90 (bs, IH, C ₂ dihydropyridine proton), 5.83- 5.70 (m, IH, C _g dihydropyridine proton) ,.5.56 (s, IH, C ₄ testosterone proton), 4.7-4.33 (m, IH, C ₅ dihydropyridine proton) , 3.26 (bs, 2H, C ₄ dihydro- pyridine protons), 2.93 (s, 3H, N-CH3) , 2.5-.83

(m, 26H, testosterone skelton protons with the angular methyl protons at 1.16 and .83). Analysis calculated for C ₂₆ H ₃₅ N0 ₃ : C, 76.25; H, 8.61; N, 3.42. Found: C, 76.07; H, 8.65; N,3.38.

EXAMPLE 44

Analytical Methods:

A high pressure liquid chromatograph (HPLC) method was developed for the studies of the degrada¬ tion of the quaternary 4_2 and dihydropyridine de- rivative 4_3. The chromatographic analyses were per¬ formed on the Beckman described hereinabove. The absorbance detector was operated at 254 nm. A 15 cm x 4.6 mm (internal diameter) , 5 μ particle size ultrasphere reverse phase C-^g column (Altex) , oper- ated at ambient temperature, was used for all separa¬ tions. The mobile phase used for the separation of the dihydropyridine derivative, its degradation products and oxidation products consisted of 0.002 M solution of 1-heptanesulfonic acid sodium salt (PIC B-7 Eastman Kodak) in CH ₃ CN, 0.01 M aqueous dibasic ammonium phosphate (7:3). At a flow rate of 2.0 ml/min, compound 4_2 _^ had a retention time of 12 min and compound 43_, 5 min. For the analysis of testosterone in the ±n vivo brain delivery studies, a solvent system consisted of 0.002 M solution of PIC B-7 in CH3CN, 0.1 M aqueous dibasic ammonium

yx yf^A lT

phosphate (1:1) . At a flow rate of 2.0 ml/min, -tes¬ tosterone had a retention of 3.3 min and compound 42 had a retention time of 36.5 min (very broad peak) .

EXAMPLE 45 Chemical Oxidation Studies:

(i) By Silver Nitrate: 1 ml of 5% methanolic solu¬ tion of the dihydropyridine compound 4_3_ was added to 5 ml of saturated methanolic AgNθ3 solution. The mixture was shaken, left 10 minutes for complete oxi- dation, centrifuged and the UV spectrum checked.

(ii) By Hydrogen Peroxide: To a standardized solution of ^H 2°2 t .18 M) contained in a UV cuvette equilibrated at 37°C, a solution of dihydropyridine compound 4_3 was added to the sample cuvette to make a concentra-

—6 tion of approximately 5 x 10 M. The mixture was thoroughly mixed and monitored for the disappearance of the dihydropyridine maximum at 356 nm using a Cary

210 interfaced with an Apple II microprocessor and using an enzyme kinetic software package. (iii) By Diphenylpicrylhydrazyl Free Radical: To 2 ml of 9.3 x 10 M solution of 2,2-diphenyl-l-picryl- hydrazyl free radical in aceonitrile, equilibrated

-2 at 26°C, 20 ml of 1.5 x 10 M solution of the di- hydropydine compound 43 in acetonitrile was added to

-4 make a final concentration of 1.48 x 10 M. The mixture was monitored at 515 nm against a reference cuvette containing the same.mixture in exactly the same concentrations, but previously prepared and left for at least 10 minutes and used as reference for A∞. The instrument used was a Cary 210 interfaced with an

Apple II microprocessor and using an enzyme kinetic software package.

OMI

-211- .

EXAMPLE 46

Determination of. Zn. Vitro Rates.of Oxidation • of Compound 43 in Biological Media: -

In Human Plasma? 5 The freshly collected plasma used was obtained at the Civitan Regional Blood Center, Inc. (Gaines¬ ville, Florida) and contained about 80% plasma diluted with anticoagulant citrate phosphate dextrose solution U.S.P. The plasma was stored in a refrigerator and 10 used the next day. 100 μl of a freshly prepared 0-024 M solution of compound 4_3 in DMSO were added to 10 ml plasma, previously equilibrated to 37°C in a water bath and mixed thoroughly to result in an initial concentration of 2.4 x 10 -4 moles/liter. One ml

15 samples of plasma were withdrawn every 20 minutes from the test medium, added immediately to 5 ml of ice cold acetonitrile, shaken vigorously and placed in a freezer. When all samples had been collected, they were centrifuged and the supernatants were filtered

20 through nitrocellulose membrane filters (por 0.45) and analyzed by HPLC, following appearance of 4_2_ (Method A) . In Human Blood:

The freshly collected heparinized blood was ob—

25 tained at the Civitan Regional Blood Center, Inc. (Gainesville, Florida) . The blood was stored in a refrigerator and used the next day. 100 μl of a freshly prepared 0.048 M solution of compound 4_3 in DMSO were added to 20 ml blood,- previously equili-

30 brated to 37°C in a water bath and mixed thoroughly,

-4 to result in an initial concentration of 2.4 x 10 moles/liter. One ml samples of blood were withdrawn from the test medium every 10 minutes, added immedi¬ ately to 5 ml of ice cold acetonitrile, shaken vig- 35 orously and placed in a freezer. When all samples had

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been collected, they were centrifuged and the super¬ natants were filtered using nitrocellulose membrane filters (por 0.45) and analyzed by HPLC, following appearance of 2_ and disappearance of 43. 5 In Rat Brain Homogenate:

The brain homogenate was prepared by the following method. Five female Sprague-Dawley rats were killed by decapitation and the brains were re¬ moved, pooled, weighed (total weight 9.2 g) and 0 homogenized in 36.8 ml of aqueous 0.11 M phosphate buffer, pH 7.4. 100 μl of 0.024 M solution of compound 4_3_ in DMSO were mixed with 20 ml of the homogenate, previously equilibrated to 37°C in a water bath, to result in an initial concentration

-4 5 of 2.4 x 10 moles/liter. Samples of 1.0 ml were withdrawn every 10 minutes from the test medium, added immediately to 5 ml of ice cold acetonitrile, shaken vigorously and placed in a freezer. When all samples had been collected, they were centri- 0 fuged and the supernatants were filtered through nitrocellulose membrane filter (por 0.45) and analyzed by HPLC (Method A) .

EXAJVgLE 47 5 In Vitro Determination of the Site-Specific

Conversion of the Prodruσ j2 to Testosterone:

A fresh brain homogenate was prepared as above described. 100 μl of Q.0.17 M solution of the quaternary compound 42 in methanol were mixed with 10 0 ml of the brain homogenate, previously equilibrated to 37 ^β C to result in an initial concentration of 1.7 x

—4 10 M. Samples of 1.0 ml were withdrawn every 20

minutes from the test-medium, added immediately to 5 ml of ice cold acetonitrile and placed in a freezer. When all the samples had been collected they were centrifuged and the supernatant was filtered through 5 nitrocellulose membrane filter (por 0.45) and analyzed for the quaternary compound 42.

EXAMPLE 48

In Vivo Brain.Delivery of Testosterone Following Administration of the Dihydro Compound 43:

10 Female Sprague-Dawley rats of average weight of 225110 g were used. The rats were anaesthetized with IM injection of Innovar ^R (0.13 ml/kg) and the external jugular was exposed. Compound 4_3 was injected intra¬ jugularly in the form of 2.5% solution in DMSO at a 15 dose of 40 mg/kg (equivalent to 52.3 mg quaternary 42 or 28.2 mg testosterone). The injection was given at a rate of 44.4 μl/minute using a calibrated infusion pump. After appropriate time periods, 1 ml of blood was withdrawn from the heart and dropped immediately 20 into a tared tube containing 5 ml acetonitrile . which was later weighed to determine the weight of the blood taken. The animal was then perfused with 20 ml of saline solution, deqapitated and the brain was removed. The weighed brain was homogenized with 1 ml 25 of distilled water, 5 ml of acetonitrile was added and the mixture was rehomogenized thoroughly, centrifuged, filtered and then analyzed using the HPLC method. The tubes containing the blood were shaken vigorously, centrifuged, filtered and also analyzed using the 30 HPLC method described at 0.05 sensitivity limit for determination' of the quaternary 4_2_ and at 0.001 sensi¬ tivity limit for determination of liberated testo-

• ^**

-214-

sterone. Quantitation was done using a recovery standard curve obtained by introducing a known amount of either compound _^ 42 or testosterone in either brain homogenate or blood and then treated in the same manner 5 of extraction and analysis.

EXAMPLE 49

In Vivo Brain Delivery of Testosterone Following its Administration

Female Sprague-Dawley rates with an average weight 10 of 225+10 g were injected with testosterone at a dose level of 28.2 mg/kg following the same procedure pre¬ viously described. Samples of brain and blood collected were analyzed for testosterone using HPLC.

EXAMPLE 50

15 In Vivo Brain Delivery of Quaternary 42 Following its Administration:

Following the same procedure, female Sprague- Dawley rats were injected I.V. with the quaternary solution (0.18%) in DMSO at a dose level of 13.0 20 mg/kg (it was found to be toxic at higher doses) . The brain samples collected were analyzed for presence of the quaternary 42_ using HPLC.

EXAMPLE 51 Results of Experiments of Examples 45-50: 25 The rates of oxidation of the dihydro deriva¬ tive 43 _^ with silver nitrate, hydrogen peroxide and diphenylpicrylhydrazyl free radical (DPP*) were determined. The reactions were carried out under pseudo first order conditions, either with-higher con-

-215-

centrations of the oxidant in the case of hydrogen peroxide or higher concentrations of 43_ in the case of the picryl reagent. With DPP ^# , a reference sample was made using the same amounts as the test sample, 5 but prepared 10 minutes before mixing and monitoring the test sample. This reference is used as a measure of Ac and these were the data used to calculate the kinetic parameters. The in vitro rates of oxidation of the dihydro derivative were also determined in

10 biological fluids, e.g. 80% plasma, whole blood, 20% brain homogenate and 20% liver homogenate. The rate of disappearance of the ester 42 _^ and appearance of testosterone in the medium was also determined. Finally, the in vivo brain delivery and blood concen-

15 tration profile of the quaternary derivative and testosterone released, against time, was determined following a single injection of the dihydropyridine derivative 43 to female rats. These results were compared to blood and brain kinetics of testosterone

20 following administration of such.

Chemical Oxidation of the Dihydropyridine Derivative 43;

(i) By Silver Nitrate; It was observed that this di¬ hydro derivative 43_ is more stable towards oxidation

25 than the monophenethylamine type derivatives re¬ ported hereinabove; it takes a few minutes standing for the silver to form. The product is exclusively the quaternary salt 42_, as verified by the change in the UV and NMR spectra.

30 (ϋ) By Hydrogen Peroxide: At low concentrations of the dihydro compound 43 (5 x 10 M) , compared to the high concentration of the peroxide (0.18 M) , the oxi¬ dation proceeds according to a first order kinetics.

.—_*«-.

-3 -1 k = 2.7+0.3 x 10 ^" sec ^; tx. ^' = 3.98±0.7 min r - .995

At higher concentrations, the dihydro compound is in¬ soluble in H ₂ 0 ₂ .

(iii) By Diphenylpicrylhydrazl (DPP*) Free Radical: The reaction was carried out under pseudo first order conditions using excess of the dihydropyridine deriv¬ ative. With the concentrations used, all runs gave good first order plots ^' over 3 half lives, with corre¬ lation coefficient better than 0.9998. k « ^■ 4.87±0.31 x 10 -2 sec-1 t^ - 14.1±0.6 seconds

Trials to determine the second order rate constant using different concentrations of DPP* were unsuccess¬ ful.

(iv) In Vitro Oxidation and Hydrolysis in Biological Media:

Table III shows the rates, half-lives and corre¬ lation coefficient for the process of oxidation of the 1,4-dihydropyridine derivative 43 in different bio¬ logical media.

The rate of hydrolysis of the quaternary 42.in 20% brain homogenate was also determined and it was found to be 3.6 x 10 sec , corresponding to a half- life, tL, of 5:16 h.

Table III. Kinetics of in vitro oxidation of the dihydropyridine ester 43 to the quaternary derivative 42 in biological fluids.'

Medium k sec ^"1 ) t .(min.) r Method? JJ

80% Plasma 8.12x10 -5 142 .959 A -4

20% Brain Homogenate 1.72x10 67 .997 A -4

Whole Blood 1.74x10 66 .997 A,B

- -4

^ t 37°C, initial concentration of [43] = 2.4 x 10 M

^• ^Method A: Following appearance of [42]

Method B: Following disappearance of [43]

(v) In Vivo Administration of Compound 43 and Testo¬ sterone:

FIGURE 8 illustrates the concentration of the quaternary derivative 42 in brain and blood and con¬ centration of testosterone released in the brain, following intravenous administration of the 1,4- dihydropyridine derivative 43. Also, FIGURE 8 shows the concentration of testosterone in brain and blood following administration of testosterone. Statistical analysis of the descending portions of the curves shown in FIGURE 8 provides the following results: (1) Rates of disappearance of the quaternary compound

.833 .833

(2) Rate of disappearance of released testosterone following administration of dihydro compound 43 =

2.65xlθ ^"3 min ^"1 t, = 4.4 h r = .768 h (Results analyzed for up to 5 hrs, the data shown in Fig. 8 are for 3 hrs)

(3) Rate of disappearance of testosterone following administration of testosterone: from brain = 5.5x10 - ^■ * ^* ? ^■ - min-1 t±, = 12.6 min r = .994 from blood = 4.74x10 -'->'min- t^ = 14.5 min r = .959

Thus, 17β-X(1,4-dihydro-l-methyl-3-pyridinyl- carbonyl)oxy]androst-4-en-3-one 43 could be obtained in

-g R Λ ^" ^ _t OMPI ⁰

a high yield (more thaii 90%) from testosterone 17 - '■ nicotinate by simple chemical procedures. The dihydro product obtained directly from the reduction reaction medium was found by HPLC to be quite pure and a single crystallization from hot methanol afforded an ana¬ lytically pure product. No signs of oxidation were observed during crystallization, even from hot meth¬ anol, filtration or drying. The crystalline solid dihydro compound did not show signs of oxidation, de- composition or polymerization when tested by HPLC, during the 2-month shelf storage at ambient temperature under nitrogen. This compound 3_ was found to be quantitatively oxidizable to the corresponding quaternary derivative 4r2_, as identified by UV spec- troscopy, whether by silver nitrate or hydrogen peroxide. The process of oxidation with silver nitrate is slower than that with the dihydropyridine deriva¬ tive of phenethylamine reported hereinabove. Oxi¬ dation with hydrogen peroxide or DPP* , at pseudo first order conditions,was found to take place at measurable rates (t- = 3.98 + 0.7 min and 14.1 ± 0.6 seconds, respectively) compared to the rates of oxi¬ dation of the corresponding phenethylamine and dopamine derivatives which were found to be too fast to be monitored using the same procedure. The iii vitro investigation in biological fluids indicated a facile oxidative conversion of the dihydro, form ^3 to the corresponding quaternary 421, but at a slower rate than that of the analogous amides of phenethylamine or dopamine.

Insofar as concerns the in vivo studies of compound 43, the results shown in Fig. 8 indicate that the dihydro

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derivative penetrates the BBB and is readily oxidized in the brain to the quaternary precursor 42. The in • vivo rate of oxidation of the dihydro seems faster than that obtained from the in vitro experiment. No di- hydro derivative could be detected in the brain without the sensitivity limits of the procedure. After 42 reaches its maximum concentration, within about 15 minutes, its concentration starts a decline phase corresponding to overall excretion and/or metabolism- hydrolysis. The overall rate of this phase was cal- culated to be 2 x 10 -3 min~1 (tr = 5.7 h). In the same time, the concentration of 42 in blood was de¬ creasing progressively from the beginning at a

-2 -1 rate 1.27 x 10 min or with a half life of 54 min. Equimolar administration of testosterone using the same solvent (DMSO) and the same route of adminis¬ tration showed a rapid absorption of testosterone into the brain, reaching a maximum concentration within 5 minutes, followed by fast clearance from both brain and blood (t^ = 12.6 min and 14.5 min respec¬ tively) . The ratio of brain/blood concentration for . testosterone was found to be 1.6 at 5 minutes and 1.8 at 15 minutes from administration. The ratio of brain/blood concentration of the quaternary 42 _^ was found to increase progressively with time (3.23 at 15 min, 6.33 at 45 min and 12 at 3 hrs from administration) This indicates the predicted "lock in" property for the quaternary 42_. Testosterone was found to be re¬ leased from the quaternary ester 42 and could be de- tected in the brain following administration of the dihydro derivative 43_. Analysis of the time concen¬ tration curve for release of testosterone indicated two

phase kinetics for disappearance from the brain. The first phase is a fast descending one at a rate of

-2 -1 1.2 x 10 min followed by a slow clearance phase with a rate of 5.8 x 10 -4 min-1 and a half life of about 20 hrs which corresponds to about 130 hrs for complete clearance from the brain. This result, if compared to that obtained by H. Frey, A. Aadvaag, D. Saahum and J. Falch, Eur. J. Clin. Pharmacol., 16, 345 (1979) , for the clearance of testosterone from plasma after oral administration (about 6 hrs) , is very promising. Although the concentrations of testosterone in the brain following administration of compound 43 are low compared to that following administration of testo¬ sterone, this is by no means a disadvantage because such high concentration of testosterone may not be needed for receptor saturation. By dose manipulation of the dihydro derivative, a concentration of testo¬ sterone just sufficient for receptor saturation for a delayed period could be attained. Similarly designed in vivo studies of compound 43_ in male rats should show analogous "locking in" of the quaternary 42 in the testes following administra¬ tion of the dihydro compound, as well as release of testosterone from the quaternary and detection of testosterone in the testes following administration of the dihydro derivative.

In the examples immediately to follow, all melting points were taken on a Mel-Temp apparatus and are not corrected. Elemental analyses were performed at Atlantic Microlabs, Inc., Atlanta, Georgia. Infrared spectra were determined by means of a Beckman Acculab 1 double- beam recording spectrophotometer. NMR spectra were determined by means of a Varian T60A spectrometer. All chemical shifts reported are in δ (parts per million) relative to tetramethylsilane. Ultraviolet absorbance spectra were determined using a Carey Model 219 spectro¬ photometer. HPLC analyses were performed on a Waters Associates Liquid chromatograph with Model 6000A solvent delivery system, Model U6K injector and Model 440 absorbance detector.

EXAMPLE 52

Preparation of the l-Methyl-3-carbamoylpyridinium derivative of Tyr-Gly-Gly-Phe-Leu-OC ₂ H ₅ ,l-Methyl-3-carbamoylpyridinium derivative of leu -enkephalin ethyl e≤ter) ; N-α-t-Butoxycarbonyl-O-bem∑- l-L-tyrosine

(7 g, 0.019 mol) was dissolved in tetrahydrofuran in a three-neck round bottom flask which was cooled to approximately -10°C in an ice/acetone bath under a nitrogen atmosphere. N-methylmorpholine (6.3 ml, 0.06 mol) was added to the stirred solution, followed by 2.5 ml (0.019 mol) of isobutyl chloroformate. .Immediately after the addition of isobutyl chloro— formate, N-methyl morpholine hydrochloride precipi¬ tated. After 5 min, 3.7 g (0.019 mol) of L-leucine ethyl ester hydrochloride, dissolved in dimethylforma- mide, were added. The reaction mixture was stirred at

OMPI

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this temperature for an hour, after which the solvent was evaporated. The residue obtained was dissolved inethyl acetate/water and the organic layer was extracted with sodium bicarbonate solution, water, .01 N HCl and water. The organic layer was dried over Na ₂ S0 ₄ and after evaporation of the solvent an oil was obtained. Crystallization from CHC1,/petroleum ether yielded 7.4 g (0.014 mol, 76%) m.p.104-107°C, of N-α-t-butoxycarbonyl-O-benzyl-L-tyrosylglycylglycine ethyl ester. ^Η NMR (CDC1 ₃ ) δ 7.2 (5H, s) , 6.9 (4H, doublet of doublets), 5.0 (2H, s) , 1.1 (12H, m) . The ethyl ester was cleaved by treating 6.2 g (0.012 mol) of it with an equivalent amount of 2N NaOH in methanol. The solution was stirred at room temperature for half an hour after which the solvent was evaporated. An equivalent amount of 2N HCl was added to the cooled residue and the solid obtained was filtered and dried to yield 3.5 g (96%), m.p. 118-122°C, of the free (t-butoxycarbonyl-O-benzyl)tyrosylglycylglycine. t-Butoxycarbonylphenylalanylleucine ethyl ester was prepared starting with 6g (0.019 mol) of t-butoxy- carbonyl-L-phenylalanine, and 3.7 g (0.019 mol) of leucine ethyl ester hydrochloride. Work up and crystallization from CHC1 ₃ /petroleum ether yielded 6.5 g (84%) , m.p. 109-112°C, of the desired compound. H NMR (CDC1 ₃ ) δ 7.2 (5H, s) , 6.4 (IH, bm) , 5.1 (IH, bm), 4.3 (4H, b ) , 3.1 (2H, bm) , 1.3 (20H, m) .

The t-butoxycarbonyl protecting group was cleaved by treatment of 4.9 g (0.012 mol) of t-butoxycarbonyl- phenylalanylleucine ethyl ester with 60 ml of 33% trifluoroacetic acid/CH 2.Cl2.,. The solution was stirred

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at room temperature for half an hour, after which the solvent was evaporated and the residue was treated with a bicarbonate solution which resulted in the formation of a solid. ^' The solid, phenylalanylleucine 5 ethyl ester, was filtered, rinsed with water and dried to yield 5.6 g (97%), m.p. 150-154 ^β C. t-Butoxycarbonyl-O-benzyltyrosylglycylglycyl- phenylalanylleucine ethyl ester was prepared by the same method using 0.01 mol of starting materials, 10 (t-butoxycarbonyl-O-benzyl)tyrosylglycylglycine and phenylalanylleucine ethyl ester. A white solid was obtained which was recrystallized from methyl alcohol/ water to yield 4.9 g (63%), m.p. 149-152°C.

The t-butoxycarbonyl group of t-butoxycarbonyl— 15 O-benzyltyrosylglycylglycylphenylalanylleucine ethyl ester was cleaved as previously described to give 0-benzyl-tyr-gly-gly-phe-leu-OEt • TFA (trifluoroacetic acid) salt. Anal. calc. of ^C _3Q H ₄₈ O _Q N ₅ F ₃ • H ₂ 0: C, 58.13; H, 6.25; N, 8.69. Found: C, 58.06; 20 H, 6.26; N, 8.69) .

Nicotinic acid (160 mg, 1.3 mmole) and O-benzyl- tyr-gly-gly-phe-leu-OEt • TFA salt (Ig, 1.3 mmole) were dissolved in pyridine and 268 mg (1.3 mmole) of dicyclohexylcarbodiimide were added. The mixture was 25 stirred at room temperature for 24 hrs, after which the dicyclohexylurea was filtered and the pyridine distilled in vacuo. Water was added to the residue and the solid obtained was filtered and washed with more water. The solid N-nicotinoyl-O-benzylpentapeptide 30 ethyl ester was recrystallized by methanol/water. H NMR gave the expected pattern.

The N-nicotinoyl pentapeptide derivative (500 mg, 0.64 mmol) obtained above was dissolved in 10% formic acid/methanol, followed by addition of 500 mg of palladium black. The mixture was stirred overnight at room temperature, after which the solvent was evaporated. The residue was neutralized with a saturated NaHC0 ₃ solution and extracted with ethyl acetate. The solvent was evaporated and the residue recrystallized from ethyl acetate/ethyl ether to yield 370 mg (0.54 mmol), 84% of product. H NMR gave the expected pattern, corresponding to

Anal. calc. for C-gH.-OgNg- H ₂ 0: C, 56.83? H, 6.89;

N, 11.04. Found: C, 56.88; H, 6.56; N, 10.48. That product (30 mg, 0.44 mmol) was dissolved in acetone and an excess of methyl iodide was added. The solution was refluxed for 8 hrs, after which the solvent was evaporated and the residue was filtered from ethyl ether.

A yellowish (260 rag, 0.31 mm), 71%, product was obtained, corresponding to the l-methyl-3-carbamoylpyridinium

5 derivative of leu -enkephalin ethyl ester. Anal. calc. for C ₃₇ H ₄ _0gN ₆ I: C, 53.50; H, 5.70; N, 10.12.

Found: C, 53.44; H, 4.77; N, 10.07.

EXAMPLE 53

Preparation of N-[2-(3-Indolyl)ethyl]nicotinamide:

To a solution of 1.97 g (10 mmol) of tryptamine hydrochloride and 1.23 g (10 mmol) of nicotinic acid in 10 ml of dry pyridine at 0°C were added 2.20 g (10.7 mmol) of dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for 24 hrs, and the formed dicyclohexylurea was removed by filtration (2.34 g) . The pyridine was removed in vacuo, and 10 ml of methanol were added to the residue. Insoluble dicyclohexylurea in methanol was removed by filtration (0.05 g) . The methanol was removed in vacuo and 10 ml of methylene chloride was added to the residue. Insoluble compound in methylene chloride was removed by filtration (0.04 g) . The methylene chloride was removed in vacuo and the residue was crystallized from isopropanol. Recrystallization from methanol/isopropanol gave 1.92 g (72.5%) of N-[2-(3-indolyl)ethyl]nicotin¬ amide as pale brown plates, m.p. 150-152°C. IR (KBr) 3280, 3050, 2940', 1646, 1526, 1412, 1302, 1102, 740, 697 cm ^" . Anal. calc. for ^c i6 ^H i5 ^N 3 ^{0: C f} 72.42; H, 5.91; N, 15.84. Found: C, 72.51; H, 5.74; N, 15.77.

EXAMPLE 54 preparation of l-Methyl-3-{ [N-2-(3-indόlyl)ethyl] }- carbamoylpyridinium iodide:

To a solution of 1.06 g (4 mmol) of N-[2-(3- indolyl)ethyl]nicotinamide in 5 ml of methanol, 1 ml (16 mmol) of methyl iodide was added. The mixture

was refluxed for 5 hrs. The methanol and excess methyl iodide were removed in vacuo. The residue was recrystallized from methanol/isopropanol to yield 1.42 g (87.4%) of l-methyl-3{[N-2-(3-indolyl)ethyl] }- carbamoyl pyridinium iodide as yellow needles, m.p. 215-217°C. IR (KBr): 3280, 3000, 2940, 1660, 1540, 1500, 1316, 1220, 735 cm ^"1 . Anal. calc. for ^C Ϊ7 ^H 18 ^N 3 ^{OIϊ C} ' ⁵⁰ ' ¹³ ' ^H ' -46; _N/ χo.32; I, 31.16. Found: C, 50.22; H, 4.49; N, 10.27; I, 31.06.

EXAMPLE 55

Preparation of l-Methyl-3-{N[2-(3-indolyl)ethyl] }- carbamoyl-1,4-dihydropyridine:

To a solution of 0.61 g (1.5 mmol) of 1-methyl- 3{ [N-2-(3-indolyl)ethyl] }carbamoylpyridinium iodide in 50 ml of deaerated water and 50 ml of ethyl acetate, 1.00 g (12 mmol) of sodium bicarbonate was added. The mixture was stirred in an ice bath and 1.65 g (8 mmol) of sodium dithionite was added gradually under nitrogen. The mixture was stirred for 6 hrs, the ethyl acetate layer was decanted and the water layer was extracted with ethyl acetate. The combined solution was washed with water, dried with anhydrous sodium sulfate and the solvent removed in vacuo. A yield of l-methyl-3{ [2-(3-indolyl)ethyl] }carbamoyl-l,4- dihydropyridine of 0.29 g (69%) was obtained as a yellow semisolid, m.p. 40-70°C. IR (KBr) 3250, 2900, 1670 cm ^"1 . Anal. calc. for C ₁₇ H ₁₉ N ₃ 0 * -H ₂ 0: C, 70.32; H, 6.94; N, 14.47. Found: C, 70.47; H, 6.76; N, 14.52.

MPI

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EXAMPLE 56

Preparation of 5-Benzyloxygramine:

A solution of 8.90 g (0.04 mol) of 5-benzyloxy- indole in 40 ml of dioxane was added dropwise, over 5 the course of 30 mins, to an ice-cooled, stirred mixture of 40 ml of dioxane, 40 ml of acetic acid, 3.2 ml of

37% aqueous formaldehyde (0.04 mol) and 8.8 ml of

25% aqueous dimethylamine (0.05 mol) . The solution was stirred and cooled for two hrs and then allowed to warm

10 to room temperatue over night. The next day, 500 ml of water were added, and the turbid mixture which resulted was filtered after the addition of charcoal. The filtrate was made alkaline (to pH 8-9) with 400 ml of 10% sodium hydroxide solution. The gramine

15 quickly solidified and was filtered off after cooling in the refrigerator overnight. Washing with water, and drying gave 9.20 g (82.0%) of coarse powder, m.p. 125-128°C. Recrystallization from ethyl acetate gave slightly green glittering cubes, m.p. 136-137°C,

20 of the desired 5-benzyloxygramine. IR (KBr) 3110,

3020, 2920, 2840, 2800, 2755, 1610, 1575, 1470, 1455, 1480, 1210, 1190, 1000 and 780 cm ^"1 .

EXAMPLE 57

Preparation of 5-Benzyloxyindole~3-acetamide; 25 A solution of 8.41 g (0.03 mol) of 5-benzyloxy- gramine, 7.5 g (0.15 mol) of sodium cyanide, 120 ml of ethanol and 30 ml of water was refluxed for 90 hours.

The solution, which contained some precipitate, was diluted with 200 ml of water and cooled in the refrigerator. The crystalline material which separated was washed thoroughly with water and dried, giving 4.40 g (52.3%) of a slightly brown sticky tan powder,m.p. 137-140°C. Recrystallization from methanol/benzene gave small needles, m.p. 156-158°C, of 5-benzyloxyindole-3-acetamide. IR (KB) 3400, 3290, 3180, 1645, 1610, 1580, 1485, 1450, 1275, 1210, 1200 and 795 cm ^"1 .

EXAMPLE 58

Preparation of 5-Benzyloxytryptamine hydrochloride:

4.21 g (0.015 mol) of 5-benzyloxyindole-3— acetamide which were dissolved in 200 ml of tetrahydro- furan were added gradually to a solution of 3.80 g

(0.1 mol) of lithium aluminum hydride in 200 ml of ether over a 30 minute period and under a nitrogen atmosphere. The solution was refluxed for 24 hrs.

The excess hydride was decomposed with ethanol and then water was added to ensure complete decomposition of the precipitated complex. The ether layer was decanted and the residue was washed with fresh ether. The combined solution was washed with water and dried over solid potassium hydroxide. The solvent was evaporated in vacuo and the oily residue was taken up in ether and precipitated with hydrogen chloride gas. The pale purple 5—benzyloxytryptamine hydrochloride was recrystallized from ethanol/ether, yield 3.00 g (66.0%), m.p. 263-265°C. IR (KBr) 3290, 3010, 2910, i600, 1580, 1480, 1200, 1100 and 1000 cm ^"1 .

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EXAMPLE 59

Preparation of N-{2-[3-(5-benzyloxy)- indolyl]ethyi}nicotinamide:

To a solution of 303 mg (1 mmol) of 5-benzyloxy— tryptamine hydrochloride and 123 mg (1 mmol) of nicotinic acid in 5 ml of pyridine at 0°C was added 220 mg (1.07 mmol) of dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for . 24 hrs and the formed dicyclohexylurea was removed by filtration. The pyridine was removed in vacuo, and the residue was recrystallized from methanol/isopropanol. Yield 218 mg (58.9%), m.p. 192-194 ^β C of N-{2-[3-(5- benzyloxy)indolyl]ethyl}ήicotinamide. IR (KBr) 3280, 3050, 2900, 1655, 1590, 1535, 1480, 1310, 1220, 1200, 1185, 1020, and 710 cm ^"1 .

EXAMPLE 60

Preparation of l-Methyl-3-N-{2-[3-(5-benzyloxy)- ^' indolyl]ethyl}carbamo lpyridinium iodide:

To a solution of 185 mg (0.5 mmol) of N-{2-[3-(5-benzyloxy)indolyl]ethyl}nicotinamide in 2 ml ' of methanol there was added 0.2 ml (3.2 mmol) of methyl iodide. The mixture was refluxed for 3 hrs. The methanol and excess methyl iodide were removed in vacuo. The residue of yellow solid gradually turned purplish. Yield 128 mg (50.0%), m.p. 228-230°C, IR (KBr) 3210, 3020, 1670, 1495, 1480, 1190, 1025, 1000, 770 cm ^"1 .

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Catatylic hydrogenolysis, using palladium- on-charcoal catalyst, of l-methyl-3-N-{2-[3-(5- benzyloxy)indol]ethyl}carbamoylpyridinium iodide affords l-methyl-3-N-{2-[3-(5-hydroxy)indolyl]ethyi}- carbamoylpyridinium iodide. ^" Subsequent esterifica¬ tion witfr trimethylacetyl chloride _. - affords the corresponding pivalyl ester of the formula

which can then be reduced as described hereinabove to the corresponding dihydro derivative of the formula

Accordingly, provided hereby are not only a generic method and novel class of pro-prodrugs for the specific and/or target enhanced delivery to the brain (andtestes) of a wide variety of drug species via the bidirectional transport of' the drug species into and out of the brain (and testes) employing dihydropyridine =^= pyridinium salt carrier redox systems, but also a system providing insight into the basic transport processes (both active and passive) of, and enzymatic activities in, the blood-brain (and blood-testis barrier) , as well as into the various processes

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specific to the function of the brain (and testes) . Again, another very significant aspect of the biore- versible redox delivery system according to this invention is the toxicity implication, for significantly reduced is systemic toxicity by accelerating the elimination of the drug/quaternary carrier system. And even central toxicity is reduced by providing for- low level, sustained release of the active drug species in the brain (and testes) . Low toxicity is provided both as regards the quaternary carrier and in combina¬ tion with the drug.

While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitu- tions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims.