Field of the Invention This invention is directed to η ⁶ metal complexes of 4-aryl-l,4-dihydropyridines. These compounds are calcium channel antagonists and are useful for investigating drug-protein receptor interactions.

Acknowledgement This invention was developed with funding from the National Institutes of Health, Contract No. 1-R15- GM42029-01, and the National Science Foundation, Contract No. Rll-8902065. The United States government may have rights in the invention.

Background of the Invention Muscle contraction and neuronal discharge are regulated by the passage of calcium ions into cells through voltage-dependent channels in the cell membrane. A number of drugs are known that act as agonists or antagonists for the flow of calcium ions through the calcium channel. The 4-aryl-l,4-dihydropyridines are an important class of calcium channel antagonist drugs that inhibit flow of calcium ions through the channels into cells to diminish muscle contraction and neuronal discharge. The 4-aryl-l,4-dihydropyridines have been described previously in the scientific literature, and a number of patents have disclosed these compounds. For instance, Semararo et al.'s U.S. Patent Nos. 4,935,548 and 5,011,848 describe 4-aryl-l,4-dihydropyridines substituted with an aerylate derivative ortho to the dihydropyridine. Stoltefuss et al.'s U.S. Patent No. 5,017,590 and ehinger et al.'s U.S. Patent No. 4,988,717 describe 4-aryl-l,4-dihydropyridines having chiral C-3 ester side chains. Hargreaves et al.'s U.S. Patent No. 4,873,254 describes

4-aryl-l,4-dihydropyridines wherein the arene group is substituted with substituents selected from the group consisting of halogeno, cyano, nitro, alkyl, and

trifluoralkyl. The arene group may also include =N-0-N= attached to the 5- and 6- positions.

Metal complexes have been described for some chemical systems other than the 4-aryl-l,4- dihydropyridines. It is known, for instance, that

Cr(CO) ₆ reacts efficiently with dienamines. Kutney et al. , J. Am. Chem. Soc. , 95:3058-3060 (1973); Kutney et al. , J. Am. Chem. Soc.. 96:7364-7365 (1974). Hegedus et al., "Principles and Applications of Organotransi ion Chemistry," University Science Books, Mill Valley, Ca. (1980) describes a number of aryl, alkenyl, heteroalkenyl, and alkenyl metal complexes.

Furthermore, tricarbonyl metal complexes of biologically important hydrocarbons such as the steroids have been made. Steroids such as progesterone [Jaouen, et al. , Inorσ. Chem.. 27:1850-1852 (1988)] and estrogen [Jaouen et al. , Pure & Appl. Chem.. 57:1865-1874 (1985)], however, do not include highly reactive functional groups, nor do they include more than one six electron region that may react with a metal reagent. Hence, there is no competition between sites at which metallation may occur. Furthermore, this prior work does not disclose how metal reagents will react with highly functionalized molecules that have more than one six electron region capable of reacting with a metal reagent. Moreover, none of the

4-aryl-1,4-dihydropyridines described in previous patents or the scientific literature include η ⁶ metal- ligand complexes of the 4-aryl group. It has also been observed that structural changes in biologically active compounds, especially changes that introduce sterically bulky groups, may perturb drug-receptor interactions or abolish biological activity. For example, a quantitative structure activity relationship directed to the pharmacology of 46 dihydropyridines concluded that steric interactions are important in determining the biological activity of 4- aryl-l,4-dihydropyridine calcium antagonists such as

nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitro-phenyl)- 3,5-pyridinedicarboxylic acid dimethyl ester] analogs. Triggle et al., J. Med. Chem.. 31:2103-2107 (1988). Hence, one would expect that introducing bulky groups into dihydropyridines could diminish calcium channel blocking activity.

Increased biological activity for dihydropyridine analogs may also correlate with the absolute configuration of sterogenic centers, i.e., one enantiomer may be an antagonist while the other is an agonist. Reuter et al., Ann. N.Y. Acad. Sci.. 522:162 (1987) . Although the absolute configuration of the dihydropyridine analogs may be important to the activity of these drugs, it has been difficult to study the relationship of stereochemistry to biological activity in the past because the enantiomers are either difficult to separate or to synthesize.

Although many 4-aryl-l,4-dihydropyridine compounds have been described in the literature, the search continues for novel 4-aryl-l,4-dihydropyridines that are useful calcium channel blockers. Efforts must also be made to find new methods for investigating the structure activity relationship, including the drug- receptor binding interactions, of such compounds. The study of this relationship will help assess which structural molecular changes result in maximal biological activity. Previously described 4-aryl-l,4- dihydropyridines, although they may have biological activity, do not provide information concerning the structure activity relationships involved in binding to calcium channel proteins unless the biological activities of a series of 4-aryl-l,4-dihydropyridines are compared. It would be more efficient to design one molecule or a small class of molecules to provide information concerning the structure activity relationship involved in binding calcium antagonists to calcium channel proteins.

SUMMARY OF THE INVENTION The present invention is directed to η ⁶ metal arene complexes of 4-aryl-l,4-dihydropyridine compounds wherein the metal has an inert gas configuration and a plurality of π acid ligands bound thereto. The π acid ligands are selected from the group consisting of carbonyl (CO) , nitrosyl (NO) , trialkyl phosphines (R ₃ P) or triphenyl phosphine (Ph ₃ P) , phosphites (RO) ₃ P, and carbonyl sulfide, or independently selected from the group consisting of CO and Ph ₃ P. The metal arene complex is more preferably a tricarbonyl metal complex, and most preferably a tricarbonyl chromium complex. The tricarbonyl chromium complexes have been found to have calcium channel antagonist activity, and are also useful in spectroscopic methods for studying binding of the complex to calcium channel receptors. The dihydropyridines of the present invention are particularly useful for spectroscopic studies of the carbon monoxide ligand because the metal shifts the spectral bands of the ligands bound to the metal to a region where there is minimal interference from other spectral absorptions. Hence, the molecular interactions of the ligand and receptor can be observed spectroscopically in a region that is relatively free of interference from other spectral absorptions.

More particularly, the present invention is directed to η ⁶ arene-metal complexes of 4-aryl-l,4- dihydropyridines comprising formula (I) below, or biologically active salts thereof. An η6 arene-metal complex is a complex wherein all carbon atoms of the arene ring are bonded to the metal atom. With reference to compound (I) , R _{l r} R ₂ , R ₃ , and R ₄ are lower alkyl chains, either straight chain or branched, wherein lower alkyl is defined as a carbon chain having three carbon atoms or less; R ₅ is a metal-π acid ligand substituent wherein the metal has an inert gas configuration or wherein the outer-shell electrons of the metal, the electrons that are used by the ligand to form bond with

the metal, and the 6 π electrons of the arene group are a total of eighteen electrons. The preferred metals for the present invention are those in Group Via of the periodic chart, namely Cr, Mo, and W. An especially preferred metal is chromium, although any metal-ligand- arene ring combination wherein the metal has an inert gas configuration or that satisfies the eighteen electron count is within the scope of the present

» invention; and R ₆ is selected from the group consisting of hydrogen, electron withdrawing groups and electron donating groups. More specifically, but without limitation, R ₆ may be selected from the group consisting of hydrogen, halogen, lower alkyls, lower alkyl halides, lower alkoxys, and lower alkoxy halides. Formula 1

The metal complexes of the present invention are made from the corresponding 4-aryl-l,4-dihydropyridines. The 4-aryl-l,4-dihydropyridines are synthesized in refluxing ammonia from a mixture of an aromatic aldehyde and 2 equivalents of a 3-ketoester. The resulting 4-aryl-l,4-dihydropyridines are then reacted with a metal-π acid ligand reagent in a regiospecific reaction that attaches the metal-ligand substituent to predominantly the 6 it electrons of the arene system, as opposed to the six electrons of the dienamine.

Regiospecific metallation is achieved by reacting the 4- aryl-l,4-dihydropyridines with metal-carbonyl reagents in refluxing

N-butylether/tetrahydrofuran (9:1) . Steric hindrance is an important factor in determining the biological activity of the 4-aryl-l,4- dihydropyridines. Dihydropyridines having bulky substitutents, especially in the para position, have been found to be less likely to bind to the calcium channel receptor. The etal-ligand complexes of the 4-aryl-l,4-dihydropyridines (MC-DHPs) have an intermediate size, hence it was uncertain whether steric hindrance would prevent the MC-DHPs from binding to channel receptors or other proteins. These steric considerations made it unclear that the MC-DHPs of the present invention would have calcium channel blocking activity or be useful in determining spectroscopic changes that occur when the 4-aryl-l,4-dihydropyridines interact with protein receptors. However, it has been found that the 4-aryl-l,4- dihydropyridine metal complexes are robust calcium antagonists and compare to or exceed the biological activity of previously known calcium channel antagonists. Furthermore, the MC-DHPs of the present invention are the first example of using the spectroscopic characteristics of η ⁶ metal-ligand complexes to study the drug receptor interactions of the 4-aryl-l,4-dihydropyridines. The spectroscopic shifts in the ligands that accompany the binding of the metal- ligand substituent to the 4-aryl-l,4-dihydropyridines and the MC-DHPs to calcium channel receptor, such as the carbonyl shifts in the IR and ¹³ C spectra, and the proton shifts in the proton NMR spectra, are helpful in studying the conformation of the MC-DHPs and the interactions that occur when the MC-DHPs bind to the calcium protein receptor. Furthermore, it has been possible to separate enantiomers of the MC-DHP using either analytical or semi-preparative chiral stationary

phase HPLC techniques. Thus, it is possible to study what effect the different enantiomers have on biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a three-dimensional drawing of the crystal structure of 3,5-dicarboethoxy-l,4-dihydro-2,6- dimethyl-4[T7 ⁶ -tricarbonylchromium-m-methoxy- phenyl]pyridine.

FIG. 2A is a three-dimensional drawing of the boat conformation of the 4-aryl-l,4-dihydropyridines illustrating the nomenclature of the compound.

FIG. 2B is a planar representation of the compound of FIG. 2A showing a plane of symmetry bisecting the molecule. FIG. 3 is a 2-D NOESY NMR spectra of 3,5- dicarboethoxy-1 ,4-dihydro-2,6-dimethyl-4 [η ⁶ - tricarbonylchromium-m-methoxy-pheny1]pyridine showing interactions between the protons of the compound.

FIG. 4 is a graph of the log of K _τ for nifedipine analogs vs. the log of K _τ for several MC-DHPs showing the biological activity of several metallated compounds of the present invention compared to the nonmetallated compound.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compounds of the general formula (I) as well as biologically active salts of such compounds.

Formula 1

A biologically active salt is defined as any salt that does not interfere with the compound's calcium channel antagonist capability. Without limitation, examples of such salts include salts having chlorine or organic compounds, such as acetate or carbonate, as the counter ion.

The compounds represented by formula (I) exist in more than one isomeric form because R ₆ can be attached to either the ortho, eta, or para position of the arene group. When the 4-aryl ring is substituted with both R ₅ and g, and e is in the ortho or meta position, the compounds exist as two enantiomers. If the metal is attached to one face and Re is in the para position, the MC-DHP has a plane of symmetry (FIG. 2B) that bisects the molecule into two identical halves. Hence, these molecules are achiral. However, with R ₆ in the ortho or meta position, the molecules do not have a plane of symmetry and they are chiral. The present invention includes all isomeric and enantiomeric forms of the MC-DHPs, including racemic mixtures and enantiomerically enriched mixtures. ⁿ Enaτvtioτaericall^ enriched" is defined to mean a mixture of stereoisomers having a greater percentage of one enantiomer so that the mixture rotates plane polarized light.

In compound (I) , R _x , R ₂ , R ₃ , and R _< , are lower alkyl chains wherein the carbon chain has three or less carbon atoms. The carbon chains can be straight chains or branched chains. Examples include methyl, ethyl, propyl, and isopropyl groups. Furthermore, _x through R can be selected independently from one another such that each substituent is different.

The R ₅ metal ligand of formula (I) is an η ⁶ metal complex of the arene group of the 4-aryl-l,4- dihydropyridines wherein the metal has a plurality of π acid ligands bound thereto. A ligand is a molecule or ion that has at least one electron pair that can be donated to an electron acceptor such as a metal. A π acid ligand forms compounds with transition metal atoms because the metal has d orbitals that can be used in bonding, and the ligand has both donor and acceptor '„ ;bitals. Bonding of CO to a transition metal illustrates the mechanism of π bonding ligands. A filled carbon σ orbital of the CO overlays with a σ orbital on the metal ion. Electron flow from the carbon to the metal in this overlap would lead to an unacceptable concentrar.ion of electron density on a metal ion that does not have at least a +2 charge. The metal reduces the electron density by pushing electrons back to the π orbitals of the ligand, which is possible because the ligand's orbitals can accept electron density.

The 7T acid ligand stabilizes low oxidation states in metals (i.e., low positive, zero or negative formal oxidation states) because these ligands have vacant orbitals of π symmetry that can accept electron density from filled metal orbitals to form a type of π bonding. This π back-bonding is synergistic with the donation of lone-pair electrons from the ligand in forming σ bonds with the metal. This ability of a ligand to accept electron density into low-lying empty π orbitals is referred to as π acidity wherein acidity is used in the Lewis acid sense. The concept of π acid

ligands is well understood in the art, and is described in greater detail in Cotton and Wilkinson, Advanced Inorganic Chemistry, 4th Edition (1980) at pages 62, 82- 95, and 1049-1079, which are incorporated by reference. Examples of such π acid ligands include, without limitation, CO, NO, and Ph ₃ P. A preferred π acid ligand for the present invention is CO, and a preferred R ₅ substituent is a metal-tricarbonyl substituent.

The compounds of the present invention are preferably if metal arene complexes of a 4-aryl-l,4- dihydropyridine wherein the metal has a plurality of π acid ligands bound thereto and has an inert gas configuration. An inert gas configuration is one in which the bonding and nonbonding orbitals resulting from the linear combination of atomic orbitals are filled.

For example, Cr has nine bonding and nonbonding orbitals when it bonds with a π acid ligand. When each bonding and nonbonding orbital is filled with two electrons from the ligands bound to the metal (for a total of 18 electrons) , the Cr assumes an inert gas configuration.

A particular inert gas configuration is achieved in a η ⁶ metal arene complex of a 4-aryl-l,4- dihydropyridine wherein the metal has a plurality of iτ acid ligands bound thereto, and the outer shell electrons of the metal, the electrons used by the π acid ligands to bind to the metal, and the 6 π electrons of the arene group are a total of eighteen electrons. Therefore, the eighteen electrons necessary to provide an inert gas configuration are satisfied by: (1) the 6 π electrons of the arene ring; (2) the number of outer- shell electrons supplied by the particular metal, which depends upon the oxidation state of the metal; and (3) the number of electrons used by the π acid ligands to bind to the metal. Two electrons are used by the iτ acid ligands for each carbonyl and Ph ₃ P, whereas three electrons are required for each NO ligand. Hence, CO and Ph ₃ P can be selected independently from each other so that the metal may have three carbonyl or three

triphenylphosphine ligands, or an appropriate stochiometric combination of CO and Ph ₃ P ligands, such as M(CO) ₂ Ph ₃ P or M(CO) (Ph ₃ P) ₂ .

A specific example of a metal-ligand-arene combination satisfying the eighteen electron count is: (1) Cr (0) , having five 3d electrons and one 4s electron for a total of 6 outer-shell electrons; three carbonyl ligands donating two electrons each for a total of six electrons; and the six π electrons of the arene ring. These eighteen electrons provide an inert gas configuration for chromium in the Cr(CO) ₃ -77 ⁶ arene complex. The outer-shell electron count for the other atoms in Group Via (W and Mo) are the same as Cr, and the W(CO) ₃ -r? ⁶ arene and Mo(CO) ₃ -77 ⁶ arene complexes would satisfy the eighteen electron requirement in the same way.

A second example of a metal-ligand-arene combination satisfying the eighteen electron count is: (1) Cr (0) , having five 3d electrons and one 4s electron for a total of six outer-shell electrons; two NO ligands donating three electrons each for a total of six electrons; and the six π electrons of the arene ring. These eighteen electrons provide an inert gas configuration for chromium in the Cr(NO) ₂ -r7 ⁶ arene complex.

A third example of a metal-ligand-rj ⁶ arene combination satisfying the eighteen electron count is an Fe(0)-ligand-arene combination: Fe(0) has six 3d electrons and two 4s electrons for a total of eight electrons; two carbonyl or triphenylphosphine ligands donating two electrons each for a total of four electrons, or the four π electrons of a diene; and the six n electrons of the arene ring. These eighteen electrons provide an inert gas configuration for Fe(CO) ₂ - arene, Fe[ (Ph ₃ )P] ₂ -arene, or Fe(diene) ₂ -arene complexes. One skilled in the art will realize that any metal-ligand-r? ⁶ arene combination that provides an inert gas configuration for the metal is within the scope of

the present invention. More particularly, for the transition metals, an inert gas configuration may be satisfied by providing a metal-ligand-rj ⁶ arene combination having a total of eighteen electrons. Alternatively, the metals may be those that have six electrons in their outer shell. Therefore, a preferred group of metals are those in Group Via of the periodic chart, namely Cr(3d ⁵ ,4s ¹ ), Mo(4d ⁵ ,5s ^r ), and 6s ² ). More particularly, a preferred embodiment of the present invention employs tricarbonyl chromium as the R ₅ group.

R ₆ may be hydrogen, an electron withdrawing group (EWG) , or an electron donating group (EDG) . "Electron withdrawing" is defined as any compound or substituent that withdraws electron density to a greater extent than does a hydrogen atom. Examples of electron withdrawing groups that are suitable for R ₆ include halogens and lower alkyl halides. "Electron donating" is defined as any compound or substituent that releases electron density greater than does a hydrogen atom. Examples of electron donating groups that are suitable for R ₆ include methyl, ethyl, and alkoxy. More particularly, R ₆ may be selected from the group consisting of hydrogen, halogen, lower alkyls, lower alkyl halides, lower alkoxys, and ^~~~ lower alkoxy halides. Furthermore, R ₆ may be at any isomeric position on the arene ring, i.e., in the ortho, meta, or para position.

The compounds of the present invention have the metal-carbonyl substituent attached to one face of the arene ring. FIG. 1 shows the crystal structure of 3,5- dicarboethoxy-l,4-dihydro-2,6-dimethyl-4[r? ⁶ - tricarbonylchromium-m-methoxy-phenyl]pyridine having the Cr(CO) ₃ group attached to one face of the arene group and situated over the C-3 or C-5 ester as the compounds are numbered in FIG. 2. The crystal structure indicates that the compounds may adopt a favorable minimum-energy conformation wherein the dihydropyridine is relatively planar and the metal-ligand substituent is in a position

that also allows the compounds to bind to the calcium channel protein receptor.

FIG. 2 shows the boat conformation of the dihydropyridine molecule. FIG. 2 also illustrates the endo-exo and ap-sp terminology that it is used to describe the conformations of the 4-aryl-l,4- dihydropyridines. Endo refers to a conformation wherein the arene group has rotated about the C-4-arene bond so that the R ₆ derivative is opposite the bowsprit hydrogen. Furthermore, the esters can be found in an sp

(synperiplanar, i.e., the esters are on the same (syn) side of the molecule and in the same plane) conformation, or in an ap (antiperiplanar, i.e., on opposite sides (anti) of the molecule but in the same plane) conformation.

The compounds of the present invention are synthesized by first forming the 4-aryl-l,4- dihydropyridines according to the Hantzsch pyridine synthesis. As a typical example of this reaction, an aromatic aldehyde, such as benzaldehyde, is reacted with 2 equivalents of ethyl acetoacetate in refluxing' ammonia. Without limiting the invention to one mechanistic theory, this reaction is believed to proceed — as shown in Scheme 1.

Scheme 1

In this manner, the 4-aryl-l,4-dihydropyridines are formed in a typical yield of about 42-80%.

The Hantzsch pyridine reaction is followed by thin layer chromatography (TLC) , and the disappearance of the aromatic aldehyde is monitored using ultraviolet irradiation. The TLC is typically run in an solvent system comprising a 1:1:1 mixture of hexane/ethyl acetate/ ethylene chloride. The product has a typical Rf of about .15-.35 in this solvent system.

One skilled in the art will realiz< that, by varying the alkyl chains on the esters of the ,9-keto ester starting material, the alkyl chains of the C-3 and C-5 esters may also be varied. Moreover, by lengthening the carbon chain of the _/ 9-ketoester starting material, R _x -R ₂ can also be changed. For instance, by adding one carbon atom to the starting material, the methyl groups at C-2 and C-6 are changed to ethyl groups, as in Scheme 2 below.

Scheme 2

As stated above, R ₆ includes a variety of substituents. 1^ is determined by selecting an aromatic aldehyde as the starting material that has the appropriate substituent attached to the aromatic ring at a desired position relative to the aldehyde functional group. For instance, if the starting aldehyde is ortho, meta, or para anisaldehyde (o-,m-, or p-methoxγ benzaldehyde) the

4-aryl-l,4-dihydropyridines are substituted with a methoxy group at the ortho, meta, or para position of the arene group. This strategy is shown in Scheme 3.

Scheme 3

Similarly, halobenzaldehydes and triflouroalkylbenzaldehydes were used as the aromatic aldehyde starting materials. Tolualdehydes or other alkylbenzaldehydes can also be used as the starting

material. These materials, including the anisaldehydes, can be obtained from chemical companies such as Aldrich Chemical Company, Inc., 1001 West Saint Paul Avenue, Milwaukee, Wisconsin. Once the desired 4-aryl-l,4-dihydropyridine was obtained, the MC-DHPs were formed by dissolving the 4-aryl-l,4-dihydropyridine in n- butlether/tetrahydrofuran (9:1) to form a solution having a concentration of about 0.05-0.20 M. This solution was maintained under an inert atmosphere such as nitrogen or argon. A carbonyl-metal reagent, typically a hexacarbonyl metal, was then added to the solvent and the resulting mixture heated to reflux. Example 1. This reaction is generally allowed to continue for about 72 hours. The product was typically purified by filtering it through celite and then recrystallizing it using hexanes/ethyl acetate. The product can also be purified using silica-gel chromatography. Example 1

3,5-dicarboethoxy-l,4-dihydro-2,6-dimethyl-4 η ⁶ - tricarbonylchromiumpheny1] pyridine was obtained from 3,5-dicarboethoxy-l,4-dihydro-2,6-dimethyl-4-phenyl- pyridine by reacting chromium hexacarbonyl in refluxing N-butylether-tetrahydrofuran solution (9:1). The product was obtained in 76% yield as a bright yellow crystalline solid by recrystallization from benzene/hexanes. The spectroscopic properties and combustion analysis of this compound were: IR: 1964, 1884, 1695. 'E NMR: 55.87 (br. s IH) ; 5.6 (d, 2H) ; 5.43 (t, IH) ; 4.98 (t, 2H) ; 4.82 (S, IH) ; 4.22 (q, 4H) ; 2.41 (s, 6H) ; 1.29 (t, 6H) . ¹³ C NMR: 233.3 (CrCO) , 167.1, 145.3, 118.9, 102.9, 97.4, 96.0, 87.7, 60.3, 36.7, 19.55, 14.3. Mass spectrum: M/Z 465 (1.1% rel. intensity, M ⁺ ) , 437 (3), 409 (8.4), 381 (91); 392 (3.2), 336 (2.2), 252 (100). Analysis. (C ₂₂ H ₂₃ N0 ₇ Cr) calculated: c, 56.77; h, 4.98; N, 3.01. Found: c 56.53; h 4.83; n, 3.8.

Once the metal-arene complex is formed, it is possible to substitute a different iτ acid ligand for those bound to the metal. For instance, if the metal has 3 carbonyl ligands bound thereto, one of the carbonyl ligands can be removed and replaced by a Ph ₃ P ligand. The carbonyl ligands can be removed by means known in the art such as photolysis. In this manner, the tricarbonyl complex can be converted into a dicarbonyl-triphenylphosphine metal complex or a carbonyl-bis(triphenylphosphine) metal complex. In a similar fashion, carbonyl or triphenylphosphine ligands can be replaced with an appropriate stoichiometric number of other π acid ligands.

The 4-aryl-l,4-dihydropyridines have two six electron systems: the six electrons of the dihydropyridine's dienamine system; and the 6 π electrons of the phenyl ring. Thus, the metal-7r acid ligand reagent has a choice of six electron systems with which to react. It has been found, however, that regiospecific metallation occurs in the present invention. Regiospecific metallation, defined as attachment of the metal to predominantly the 6 n electrons of the arene group, is unambiguously established as η ⁶ to the 4-aryl substituent by the ^X H and ¹³ C NMR chemical shifts. Assignments also have been made unambiguously by 2D ^-"c correlation, and off resonance decoupling. As an example of how the spectral assignments confirm that metallation is τ?6, the following assignments were made for 3,5-dicarboethoxy- l,4-dihydro-2,6-dimethyl-4-[i76-tricarbonyl chromium phenyl] pyridine. The proton chemical shifts of the 4-phenyl substituent for the starting material (57.0-7.2 ppm) shifted dramatically upfield (54.9 to 5.6) upon substitution with chromium tricarbonyl. Similarly, the carbon signals for the aromatic ring were prominently shifted upfield, from 147.7, 127.8, 127.7, and 126 to 118.9, 97.4, 96.0, and 87.HP LaserJet Series IItional)HPLASEII.PRS and 103.8 (C=C-NH-) for the

starting material to 167.1, 145.3 and 102.9 for the corresponding tricarbonyl chromium complex.

The compounds made according to the synthetic schemes discussed above are listed in Table 1, along with the corresponding IR spectral data, MS molecular ion peak, yield, and melting point. Additional NMR spectroscopic data for the compounds is presented in Tables 2 and 3. The data presented in Tables 1-3 establishes that the compounds as listed were actually made. Moreover, the data provides reference values for comparing the spectral shifts of the compounds upon binding to either model receptors or calcium channel receptors. Table 2 also shows that the ¹³ C absorption for the carbonyl ligands is between about 200 ppm and 235 ppm, well above the absorption for the ester side chain carbonyl from about 165 ppm to about 175 ppm. Hence, the absorption in the carbonyl ligand is not obscured by other spectral absorptions.

TABLE 1

Physical Characterization Data for Tricarbonyl Chromi ^' im Complexes of Hantzsch Esters

"Yield of product after isolation and purification, all compounds gave analytical (C,H,N) and/or accurate mass results within acceptable limits, results of which have been provided to the editor.

^Dichloromethane solvent, unless otherwise noted. ^c Holecular ion unless otherwise noted. ^d Tetrahydrofuran solvent. ^β W-0CH ₃ ]. ^f [M-0CH ₂ CH ₃ I]

TABLE 2

13, C NHR Chemical Shifts, CDCl ₃ unless otherwise noted. (Coupling constants are in Hz)

Entry 1 ,4-Dihydropyridine C-2, 6 CH ₃ 's 4-Aryl CCORCr(CO) ₃

C-2,6 C-3,5 C-4

1 ^a 146.8 101. 31.2 18.0 125.9 .0,-0,-273 166.6, 203.2 146.4 100. 17.7 110.4 (d.Jcp^δ) 166.4

97.4 (d,J _CT =5) 50.8

96.4 (d,J _CT =8)

86.8

78.3 ^,.-^.=23)

144.4 105.6 43.3 19.3 117.2 123.9 167.2 231.1 143.9 103.7 100.1 (^=34) (JCF=276) 166.9 94.7, 93.8 60.5, 60.2 88.3, 84.5 13.9

144.8 105.1 34.8 19.6 119.1. 116.2 166.9 232.1 143.7 104.9 19.4 96.2, 95.0 60.6, 60.1

87.7, 85.3 14.4, 14.3

145.0 104.9 34.8 19.6 118.6, 116.2 167.2 232.1 144.3 104.4 19.2 96.1, 95.0 167.1

87.8, 85.3 51.5, 51.2

145.3 102.9 36.7 19.6 118.9 167.1 233.3 97.4 60.3 96.0 14.3 87.7

155.1 101.9 35.9 16.3 125.6 176.5 214.9 104.9 51.7 102.2 94.0

144.5 104.0 32.3 19.3 142.4, 110.3 55.4 167.2 233.5 143.9 103.8 19.2 98.8, 95.1 60.1 , 59.9 82.7, 71.6

146. 101. 31.7 17.8 143.0. 110.1 55.8 167.2 202.2 145. 100. 17.7 99.0, 97.0 166.7 84.6, 74.4 40.7, 50.6

145, 102.6 36.9 19.6 139.2, 121.4 55.9 167.1 233.7 145, 102.4 90.0, 89.9 167.0 84.0, 80.2 60.3 14.3

10 145.3 102.8 35.6 19.6 143.7, 113.6 55.4 167.2 233.6 19.5 96.7, 76.6 60.3

14.3

DHSO-d ₆

TABLE 3

H NMR Chemical Shifts, CDCl ₃ solvent unless otherwise noted.

^a co ₂ cι ₂

^HSO-d ^c Decoupled 1 "9 NHR, compared to -118.3 for the corresponding "free ligand." decoupled ¹⁹ F NHR, compared to -55.2 for the corresponding "free ligand."

The preferred compounds of the present invention are: 3,5-dicarbomethoxy-l, -dihydro-2,6-dimethyl- [η ⁶ - tricarbonylchromium-o-fluoropheny1] pyridine; 3,5-dicarboethoxy-l,4-dihydro-2 ,6-dimethyl-4[τ? ⁶ - tricarbonylchromium-o-trifluoromethylphenyl]

pyridine; 3,5-dicarboethoxy-l,4-dihydro-2 ,6-dimethyl- 4[77 ⁶ -tricarbonylchromium-o-chlorophenyl] pyridine; 3,5-dicarbomethoxy-l,4-dihydro-2,6-dimethyl- 4[T7 ⁶ -tricarbonylchromium-o-chlorophenyl] pyridine; 3,5-dicarboethoxy-l,4-dihydro-2,6-dimethyl- 4[77 ⁶ -tricarbonylchromiumphenyl] pyridine; 3,5-dicarbomethoxy-l,4-dihydro-2,6-dimethyl-4[ η ^e - tricarbonylchromiumphenyl] pyridine; 3,5-dicarboethoxy-l,4-dihydro-2,6-dimethyl-4[r ⁶ - tricarbonylchromium-o-methoxyphenyl] pyridine;

3,5-dicarbomethoxy-l,4-dihydro-2,6-dimethyl-4[ η ⁶ - tricarbonylchromium-o-methoxypheny1] pyridine; 3,5-dicarboethoxy-l,4-dihydro-2,6-dimethyl-4[ η ⁶ - tricarbonylchromium-m-methoxyphenyl] pyridine; and 3,5-dicarbomethoxy-l,4-dihydro-2,6-dimethy1-4 [η ⁶ - tricarbonylchromium-p-methoxyphenyl] pyridine.

A particularly preferred embodiment of the compounds of the present invention are 3,5- dicarbomethoxy-1,4-dihydro-2,6-dimethyl-4 [η ⁶ - tricarbonylchromium-o-fluorophenyl] pyridine, 3,5- dicarboethoxy-1,4-dihydro-2,6-dimethyl-4 [η ⁶ - tricarbonylchromium-o-trifluoromethylphenyl] pyridine, 3,5-dicarboethoxy-l,4-dihydro-2,6-dimethyl-4[ η ⁶ - tricarbonylchromium-o-chlorophenyl] pyridine, and 3,5-dicarbomethoxy-l,4-dihydro-2,6-dimethyl-4[ η ⁶ - tricarbonylchromium-o-chloropheny1] pyridine.

Chiral MC-DHP compounds result from the synthesis described above when R ₆ is other than hydrogen and is in the ortho or meta position. The stereoisomers can be separated by HPLC chiral stationary phase techniques. The particular chiral stationary phase column used for separating the MC-DHP enantiomers was either a Chiralcel-OJ column or a Chiracel-OD column, both manufactured by Daicel Chemical Industries, Inc., Exton, Pennsylvania. The stationary phase is silica impregnated with a modified cellulose derivative, the

Chiralcel-OD column being a carbonate derivative, and the Chiralcel-OJ column being an ester derivative.

To separate the enantiomers, the compound was dissolved in 10% isopropanol-heptane and injected into the HPLC Chiralcel-OJ or Chiralcel-OD column. The compounds separated according to this method are presented below in Table 4. The t _R entry is the retention time of the compounds in minutes on the chiral HPLC column, and is the chromatographic separation factor, where an value of 1.05 represents a baseline separation. The enantiomers can be separated in either analytical quantities (equal to or less than a mg) or, if a semi-preparative 20 x 250 mm OJ column is used, then preparative amounts (5-10 mg) can be separated on each HPLC injection.

TABLE 4

HPLC-CSP separation of Tricarbonyl Chromium complexes of Hantzsch esters, Entry numbers correspond to compounds in Table 1.

Compound R G t _R ' ^a o ^b

(min.)

CH o-F t _R1 .=92.2 1.08 tø.=100.0

CH ₂ CH ₃ o-Cl t _R1 .=76.0 1.26 tR2'=95.5

CH ₃ o-Cl t _R1 .=97.0 1.15 tR2.=111.4

t _R1 .=32.2 3.03°

^.=97.6 8 CH ₃ o-OCH ₃ t _R1 .=172.0 1.09

^.=183.2

CH ₂ CH ₃ m-OCH ₃ t _R1 .=70.6 1.10

^.=77.8 t _Rl .=151.9 1.25° tR2.=190.3

"Adjusted retention times.

Separation of enantiomers was performed on a Chiralcel-CO colum. ^c Chiralcel-OJ, at 50°C.

The arene metal group is an electron withdrawing moiety. Electron withdrawing groups on dihydropyridine rings often correlate with greater calcium channel antagonist biological activity. However, the steric bulk of the tricarbonyl metal substituent must be presented in a favorable orientation in the receptor "cavity" in order for the compounds to bind with the protein receptor. Prior to making and isolating the compounds of the present invention, it was not known what effect the relatively bulky metal-ligand substituent would have on the compound's binding to the protein receptor. It was possible that the metal would be excessively bulky and therefore diminish or eliminate

calcium channel blocking activity. The crystal structure of the 2-methoxy derivative (FIG. 1) shows that the tricarbonyl chromium substituent is positioned over one of the ester groups at either the C-3 or C-5 positions. This structure apparently and unexpectedly allows the compound to assume a favorable topographic configuration that allows it to bond to the calcium channel. While the solid state crystal structure may not correlate well with the solution dynamics of the compounds, it at least indicates that a favorable, minimum-energy conformation may exist that allows the 4- aryl-l,4-dihydropyridine-metal complexes to bind to the protein receptor.

Protein Receptor Binding Studies were performed to assess the specificity of binding and expected biological activity of the compounds of the present invention. Specific binding occurs when the calcium channel antagonist binds to the calcium channel region of the protein receptor. Nonspecific binding is binding of the antagonist to a location on a protein other than the calcium channel receptor site. Specificity of binding refers to the predominance of specific over nonspecific binding. To investigate the specificity of the MC-DHP compounds and their use as probes for drug receptor interaction, fresh membrane preparations were treated with a [(hydroxymethyl)amino] methane (tris) buffer solution of the compounds. Microsomal membranes from guinea pig heart ventricles were prepared as described in J. Pharmacological Experimental Therapy . 225:291 (1983) , and J. Pharmacological Experimental Therapy. 231:291 (1984), incorporated herein by reference. Briefly, membrane protein (40-120 micrograms) was incubated in 5 ml of 50 mM tris buffer at pH 7.2 for 90 minutes at 25° with 5 x 10 ^"11 (+) [ ³ H]PN 200 110

(isopropyl-4-(2,l,3-benzoxydizol-4-yl)l,4-dihydro-5- methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylate) and varying concentrations of the competing MC-DHPs of the

present invention. [ ³ H]PN 200 110 is a standard radiolabeled calcium antagonist used to test the binding efficacy of potential calcium channel blockers. (+) [ ³ H]PN 200 110 with a specific activity of 70 Ci/Mol [Ci = curies = 3.7 x 10 ¹⁰ Bq; becquerel (Bq) = 1 nuclear transformation/second] was purchased from DuPont-New England Nuclear of Boston, Massachusetts. Duplicate tubes containing IO ^"7 M (+) [ ³ H]PN 200 110 were used to define nonspecific binding. All tubes, including the duplicate tubes, were filtered and washed rapidly with 2-5 ml portions of ice-cold tris buffer in a Brandell cell harvester [Model M-24R, Brandell Instruments Ltd. , Gaithesburg, Maryland] . The trapped radioactivity was then counted by liquid scintillation spectrometry at an efficiency of 40-45% to determine whether or to what extent binding occurred.

Competitive binding of the MC-DHPs was performed by binding the [ ³ H]PN 200 110 to a calcium channel receptor and measuring the degree to which it was displaced from the receptor by varying concentrations of the MC-DHPs. The competing MC-DHPs were prepared in 100% ethanol as 10 ^"3 M stock solutions. Subsequent dilutions were made using 50% ethanol (10 ^" * M) or distilled water (10 ^"5 and greater) . All dilutions were prepared on the day of the experiment. Concentrations of ethanol up to 0.2% (v:v) did not affect specific binding. Binding data were analyzed by iterative curve fitting programs (BDATA, CDATA, EMF software, Knoxville, Tennessee) . Iterative curve fitting is a mathematical procedure for matching an observed series of data to a function, and estimating the level of confidence in the correlation.

After allowing for specific binding to the membrane preparation containing the calcium channel protein receptor, FT-IR, ¹³ C-NMR, 2D-NMR and NOE can be performed on the receptor and bound antagonist. The shift of the carbonyl absorption would be monitored between 2200-1800 cm ^"1 . The spectral shifts of the carbonyls on the metal atom will change sufficiently on binding to the protein

receptor to provide information about how the protein binds to the MC-DHPs. For example, if the carbonyl substituents hydrogen bond to the amino acids of the protein receptor, the bond order of the carbonyl is effectively reduced. As a result, the FT-IR bands corresponding to the carbonyl substituents shift to lower frequencies.

Experiments using characteristic ¹³ C NMR carbonyl absorptions of the metal carbonyl groups were conducted with the isolated compound not bound to the calcium channel protein. See Table 2. Due to both the low natural abundance of ¹³ C and limitations due to the concentrations of MC-DHP, the MC-DHPs can be enriched in a stable isotope at the metal-carbonyl functional group. The metal-carbonyl substituent can be enriched in ¹³ C by performing a ¹³ C photochemical exchange with CO. See, for instance, Bitterwolf et al., Polyhedron. 7:1377-82 (1988) , incorporated herein by reference. ¹³ CO can be purchased from chemical companies such as Aldrich Chemical Company of Milwaukee, Wisconsin. As with the IR spectra, the shifts of the carbon atom of the carbonyl group correlate with the chemical environment of the MC-DHPs. If the carbonyl is hydrogen bonded, the conjugation of the C-0 is reduced. Therefore, the corresponding peak in the ¹³ C NMR shifts from about 250 ppm to about 200 ppm. Initial binding and spectral studies involved model - (ARG-X-X) _n - ARG peptides, which were also prepared enriched in ¹³ C.

In addition to the FT-IR and ¹³ C NMR studies described above, both NOE and 2-D NOESY NMR experiments can be used to study the conformation of the MC-DHPs and their interaction with both the model peptides and putative receptor protein. FIG. 3 shows a 2-D NOESY spectra of 3,5-dicarbomethoxy-l,4-dihydro-2,6-dimethyl- 4[n ⁶ -tricarbonylchromium-m-methoxyphenyl] pyridine. The 2-D NOESY spectra provides information concerning the conformation of the MC-DHPs. In FIG. 3, the

correlations of the protons labelled a-e and b-e correspond to the m-OCH ₃ . The e-f correlation arises from interactions between the m-OCH ₃ group and the C-2/6 methyl groups of the DHP, and therefore provides evidence that the MC- DHPs exist in the endo conformation (see FIG. 2A) . The f-g correlation is between ethyl esters and arises from an ap ester conformation. The g-e correlation probably arises from the m-OCH ₃ group in the endo conformation and an ap ester.

NOE experiments were also used to determine the conformation of the MC-DHPs. NOE experiments determine the proximity of nuclei, and are more generally used to determine the proximity of protons on the same molecule. When protons absorb energy in an NMR experiment, they are excited to a higher energy state. The excited nuclei must thereafter relax and release the excited state energy to the surrounding chemical environment. If an excited proton is within about 4 A of another proton, then the excited proton may release its energy to the proton proximate to it. Thus, the NOE experiments provided further information concerning the conformation of the MC-DHPs. The conformation information obtained from the 2-D NOESY NMR spectra and the NOE experiments can be compared with the spectral absorptions of the MC-DHPs upon binding to the model peptides or the calcium channel receptor. This information can be used to investigate the conformational changes that occur when the MC-DHPs bind to model peptides or the calcium channel receptor.

Ligands other than carbonyl may be used to form the MC-DHPs. These ligands can have nuclei suitable for NMR experiments such as ( ¹⁵ N0) . Therefore, a heteronuclear NMR (an NMR that excites heteroatoms as opposed to exciting hydrogen) can focus on a particular heteroatom such as N. The heteronuclear NMR spectra of the ligand will provide information concerning the chemical

environment of each heteroatom examined spectroscopically. Furthermore, proton coupling to nuclei such as ¹⁵ N and ³¹ P can be determined spectroscopically. Therefore, if the carbonyl ligand is replaced with a nitrosyl ligand ( ¹⁵ NO) or a Ph ₃ ³¹ P ligand, then additional NMR experiments can be conducted which would provide information regarding the chemical environment of the ligands. For example, the coupling of protons from the calcium channel receptor to the ligands would result in spectral shifts in the NMR, both for the heteroatom in heteroatom NMR spectra and for protons coupled to the heteroatom in the proton spectra.

The effect of MC-DHPs on the binding of [ ³ H]PN 200 110 is shown below in Table 5. The K _r values of the MC-DHPs compare favorably with the K _r value for the 4-phenyl-l,4-dihydropyridines that are not substituted with the R ₅ metal-ligand complex (see Fig. 4) . K _τ is the concentration at which 50% of the radiolabeled calcium antagonist is displaced from a membrane preparation that contains the protein receptor. Therefore, a lower K _τ value correlates with greater MC- DHP-receptor binding activity. nH is the Hill coefficient and is a measure of whether the drug- — receptor interaction is unimolecular, biomolecular etc. The Hill coefficients shown below indicate that the interaction between the MC-DHPs and the protein receptor is unimolecular.

TABLE 5

Effect of MC-DHPs on I 3H]PN 200110 binding to guinea pig heart membranes.28 Entry numbers correspond to compounds in Table 1.

N = number of individual experiments each performed in duplicate.

Within the limitations of this series, it appears that binding follows the order EWG greater than H greater than EDG. For instance, the O-Cl derivative has a lower K~ value than does the m-methoxy derivative.

Hence, Table 5 indicates that a lower concentration of the electron withdrawing derivatives is required to effectively compete with [ ³ H]PN 200 110 than the electron donating derivatives. FIG. 4 is a substantially straight line graph of the log of the K _τ for nifedipine versus the log K _x of the MC-DHPs shown on the graph. FIG. 4 shows that the electron withdrawing groups are much more active than the electron releasing groups. For example, the CF ₃ - chromium derivative [CF ₃ is a strong electron withdrawing substituent] has a - log [K _x ] value of about 10, whereas the 2-methoxy derivative (an electron donating substituent) has a -log [Kj] value of about 8. The smaller the value of K _τ , the more active a compound is in competitive binding studies. The -log of a small K _x value is a larger positive number than the -log of a large K _x value. Therefore, FIG. 4 shows that the 2-CF ₃ . chromium derivative has a greater activity than the 2- methoxy-chromium derivative. Moreover, the least reactive is the 4-methoxy derivative, presumably because

the 4-substituted compounds interfere with binding at the protein receptor.

A robust calcium channel antagonist is one having a K__ < IO ^"6 , and the results shown in FIG. 4 and Table 5 indicate that the MC-DHPs of the present invention are robust calcium channel antagonists. Moreover, these results suggest that the MC-DHPs readily bind to the protein receptor. Thus, the MC-DHPs of the present invention are efficient calcium channel antagonists, and moreover, because of their unique spectroscopic properties, provide detailed spectroscopic information concerning the solution and solid state conformation of the MC-DHPs and how they may bind to the protein receptor. Having illustrated and described the principles of the invention in several preferred embodiments, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications coming within the spirit and scope of the following claims.