Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
  • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
  • C07C235/66—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings being part of condensed ring systems and singly-bound oxygen atoms, bound to the same carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
  • C07C51/16—Preparation of carboxylic acids or their salts, halides or anhydrides by oxidation
  • C07C51/29—Preparation of carboxylic acids or their salts, halides or anhydrides by oxidation with halogen-containing compounds which may be formed in situ
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
  • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
  • C07C51/377—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by splitting-off hydrogen or functional groups; by hydrogenolysis of functional groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
  • C07C65/105—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic
  • C07C65/11—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic with carboxyl groups on a condensed ring system containing two rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C67/00—Preparation of carboxylic acid esters
  • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
  • C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
  • C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms

Definitions

• the present invention relates to naphthoic acids and derivatives.
• Structure-based drug design is a rapidly expanding field that combines synthetic chemistry, enzymology, modeling and crystallography in the targeted development of new drugs.
• An essential element of structure-based drug design is identification of a lead compound, whether from random screening or from computational procedures, that can be developed into an improved inhibitor through the iterative process of 1) determination of the three-dimensional structure of the complex of the target receptor and lead compound, 2) optimization of inhibitor- receptor interactions through molecular modeling; 3) synthesis of a new inhibitor; and 4) testing of the new inhibitor.
• NAD analogs As potential therapeutics, the anticancer agent tiazofurin (2- ⁇ -D-ribofuranosylthiazole-4 carboxamide) is metabolically converted into the NAD analog thiazole-4-carboxamide adenine dinucleotide which is a potent inhibitor of IMP dehydrogenase type 1 1 , the dominant isoenzyme in neoplastic cells.
• thiazole-4-carboxamide adenine dinucleotide which is a potent inhibitor of IMP dehydrogenase type 1 1 , the dominant isoenzyme in neoplastic cells.
• the nicotinamide part of the dinucleotide binding site of dehydrogenases has not been a target in drug design.
• the present invention provides a compound comprising:
• R includes at least one methylene spacer through which R, is attached to said compound.
• the present invention also provides methods for making hydroxynaphthoic acids.
• Figure 1 illustrates a method for making dihydroxynaphthoic acids of the present invention having a particular radical group at the 7-position
• Figure 2 illustrates methods of making of bromides which can be used as the starting bromide in the method illustrated in Figure 1 ;
• Figure 3 illustrates a method for incorporating specific R 3 radical groups into the naphthoic acids of the present invention
• Figure 4 illustrates a method for incorporating specific R 2 radical groups into the naphthoic acids of the present invention at the 5-position
• Figure 5 illustrates a method for incorporating specific R 5 radical groups into the naphthoic acids of the present invention at the 8-position
• Figure 6 illustrates how monohydroxy bromides useful in the methods of the present invention can be synthesized from a readily available aldehyde starting materials:
• Figure 7 illustrates a method for making compounds of the present invention
• FIG. 8 illustrates methods for making compounds of the present invention
• Figure 9 illustrates methods for making compounds of the present invention
• Figure 10 is a table showing inhibition of human lactate dehydrogenase (LDH- H 4 and LDH-M 4 ) and malarial parasite P. faciparum lactate dehydrogenase (pLDH) by dihydroxynaphthoic acids;
• Figures 11A and 1 1B illustrate the inhibition of pLDH by 7-(p- trifluoromethylbenzyl)-8-deoxyhemigossylicacid (25c);
• Figures 12A and 12B illustrates the inhibition of pLDH-M,, by 2,3-dihydroxy-6- methyl-7-(p-methylbenzyl)-4-( 1 -methylethyl)- 1 -naphthoicacid (25d);
• Figure 13A illustrates the quenching of the intrinsic protein fiuorescence of pLDH by NADH in the presence and absence of ADP
• Figure 13B illustrates the quenching of the intrinsic protein fiuorescence of pLDH by a compound of the present invention in the presence and absence of ADP;
• Figures 14A, 14B, 14C and 14D illustrate the inhibition of various dehydrogenases by compounds of the present invention.
• substituted refers to a radical group which replaces a hydrogen on another radical group or on a compound.
• substituted refers to a radical group including an alkyl. alkenyl, or alkynyl substituent or a functional group substituent such as halide, e.g. fluoride, chloride, bromide or iodide; carboxylate; nitro, etc.
• halide e.g. fluoride, chloride, bromide or iodide
• carboxylate nitro, etc.
• halogen refers to any of F, Cl, Br, or I.
• methylene spacer refers to a -CH 2 -, -CHX,-, or -CX,X 2 - group where each of X, and X 2 is a substituent or a radical group.
• C ⁇ 8 alkyl refers to a straight or branched chain alkyl radical group having one to eight carbon atoms including for example, methyl, ethyl, propyl, isopropyl. butyl, sec-butyl, isobutyl, pentyl, dimethyl-propyl, hexyl, t-octyl and octyl, and cognate terms (such as "C,. 8 alkoxy") are to be construed accordingly.
• C,. 5 alkyl refers to a straight or branched chain alkyl radical group having one to five carbon atoms (such as methyl or ethyl).
• C 2 . 8 alkenyl refers to a straight or branched chain alkyl radical group having one to eight carbon atoms and having in addition at least one double bond of either E or Z stereochemistry where applicable. This term would include, for example, vinyl, 1-propenyl, 1- and 2-butenyl and 2-methyl-2-propenyl.
• C 2 . 8 alkynyl refers to a straight or branched chain alkyl radical group having one to eight carbon atoms and having in addition at least one triple bond. This term would include, for example, propargyl, 1 - and 2-butynyl, etc.
• C 3.8 cycloalkyl refers to a saturated cyclic radical group having from 3 to 8 carbon atoms arranged in a ring and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, etc.
• C 3 . 8 cycloalkenyl refers to an unsaturated cyclic radical group having from 3 to 8 carbon atoms arranged in a ring and includes, for example, cyclohexenyl, cyclohexadienyl, etc..
• aryl refers to a radical group having the ring structure characteristic of benzene, naphthalene, phenanthrene, anthracene, pyrene, benzopyrene, etc.
• aralkyl refers to a substituted aryl radical group having one or more C, .g alkyl substituents regardless of whether the link to a compound or radical group is through the alkyl or the aryl of the aralkyl radical group.
• heterocyclic refers to a radical group having one or more ring structures in which one or more atoms in the ring structure is an element other than carbon such as sulfur, nitrogen, oxygen, etc.
• Scheme 1 in Figure 1 shows how to synthesize dihydroxynaphthoic acids of the invention having a particular radical group R 4 at the 7- position (I L).
• a known l -bromo-3,4-dimethoxy-benzene (1A) such as bromo-3,4- dimethoxy-2-isopropylbenzene
• a known ethyl 4-oxobut-2-enoate (IB) such as ethyl 3-methyl-4-oxobut-2-enoate
• the resulting carboxylic acid is cyclized with polyphosphoric ester to give tetralone (I E).
• I E tetralone
• the ketone function of I E is reduced with sodium borohydride and the intermediate alcohol dehydrated on acidic workup to form an alkene (IF).
• IF alkene
• Addition of bromine to the 1 F forms a dibromide which is immediately dehydrohalogenated with dimethylformamide to form vinyl bromide (1G) which is dehydrogenated with 2,3- dichloro-5 ,6-dicyano- 1 ,4-benzoquinone to form a bromonaphthalene ( 1 H) .
• Scheme 2 in Figure 2 illustrates efficient syntheses of bromides (2D) and (2H) which can be used as the starting bromide (1A) of scheme 1 having a specific radical group R,
• radical groups R a , R b , and R c can be almost any radical group, just as groups R,, R 2 , R 3 , R 4 , and R 5 can be almost any radical group.
• Compound 2A is synthesized from 3-methoxysalicylic acid by methylation using dimethylsulfate and potassium carbonate in acetone.
• Grignard reaction of 2A with a Grignard reagent, such as methylmagnesium bromide affords alcohol (2B).
• Scheme 3 in Figure 3 illustrates a method for incorporating specific R 3 radical groups into the naphthoic acids of the present invention.
• a substituted glyoxal (3 A) is converted to an acetals (3B) by reaction with methanol in the presence of acid.
• Reaction of 3B with triethylphosphonoacetate (3C) using a modification of the Wadsworth Emmons reaction affords compound 3D.
• Hydrolysis in dilute acid affords the aldehyde (IB).
• Compounds IB can be used to form naphthoic acids as described in scheme 1.
• Scheme 4 in Figure 4 illustrates a method for incorporating specific R, radical groups into the naphthoic acids of the present invention at the 5-position.
• Grignard reagent (4A) which can be derived from 1A, is reacted with a ⁇ -keto ester (4B) under kinetic conditions and reaction takes place at the more reactive ketone site to form an alcohol which is then dehydrated to form resulting ⁇ , ⁇ unsaturated ester (4C).
• 4B is a substituted ⁇ -keto-ethyl ester which is readily available or which can be prepared easily using well known methods. Because the ester (4C) is difficult to reduce it is saponified to the acid (4D), which is hydrogenolyzed and reduced.
• Scheme 5 in Figure 5 illustrates a method for incorporating specific R 5 radical groups into the naphthoic acids of the present invention at the 8-position.
• a tetralone like compound 5A whose preparation is described in scheme 1 is reacted with a Grignard reagent to form an alcohol which will dehydrate upon workup to afford an alkene having the structure of intermediate compound 5B.
• Intermediate compound (5B) can then be used to synthesize a naphthoic acid of the present invention (5C) by substituting intermediate compound (5B) for intermediate compound (II) in scheme 1.
• schemes 1 through 5 only specifically illustrate how to synthesize dihydroxynaphthoic acids of the present invention, the present invention also provides monohydroxynaphthoic acids as well.
• Scheme 6 in Figure 6 illustrates how monohydroxy bromide (6B) can be synthesized from a readily available aldehyde starting material (6A). Benzaldehyde (6A) is reacted with a Grignard reagent, dehydrated, hydrogenolyzed, and then brominated to form bromide (6B).
• Bromide (6B) can then be substituted for (1A) in schemes 1 through 5 to produce monohydroxynaphthoic acids of the present invention having any of the radical groups R R 2 , R 3 , R 4 , and R 5 at the appropriate positions on the naphthoicalene ring.
• corresponding naphthoic acid derivatives of the present invention can be readily formed by derivatizing the carboxylic acid group of the naphthoic acids by well known methods.
• ethyl and methyl ester derivatives can be formed using the Fischer esterification process on the naphthoic acids of the present invention.
• amide derivatives of the present invention can be formed by reacting the naphthoic acids of the present invention with dicyclohexylcarbodiimide (DCC) in the solvent methylene chloride.
• DCC dicyclohexylcarbodiimide
• any suitable amine can be used in this procedure.
• the present invention also provides hydroxynaphthoic acids and derivatives as present in a combinatorial library.
• a combinatorial library can be defined as any ensemble of molecules.
• Most progress has been made in developing and, especially, in screening very large peptides or oligonucleotide libraries for ligands with high selectivity for a designated target. Numerous methodologies have been developed to prepare and to screen these libraries. In principle, many of these technologies are applicable to the development of libraries of small organic chemicals. However, screening of very complex mixtures of organic molecules is difficult compared to screening peptides or oligonucleotide libraries.
• Peptide libraries such as phage display libraries, combine the power of genetics to screen libraries while oligonucleotide libraries utilize PCR (polymerase chain reaction) to amplify candidate ligands.
• PCR polymerase chain reaction
• One widely used strategy to develop chemical libraries is the split-synthesis method, where a starting material is divided into aliquots that are treated separately with different reagents, pooled, then split into the desired number of samples for the next reaction cycle. This split-pool-react cycle can be repeated at multiple steps in the chemical synthesis scheme, generating complex final mixtures. Deconvolution of these complex pools to identify lead compounds can be done using iterative screening and resynthesis of smaller libraries. However, this can be time consuming.
• the rationale for synthesis and screening of very complex mixtures is to increase the probability that one or more high affinity ligand is present in the mixture, thereby improving the chances of identifiying a lead compound.
• the present invention's method for making dehydrogenase inhibitors represents a different situation since these already are lead compounds. Therefore, the combinatorial library method of the present invention uses screening to identify specific dihydroxynaphthoic acids that exhibit selectivity for the nicotinamide site of a target dehydrogenase. These lead compounds can then be selectively modified to develop potent selective inhibitors.
• the combinatorial library method of the present invention is able to produce a Pan- Active Site Inhibitor.
• a number of the reactions shown in schemes 1 through 6 can be used in the combinatorial library method of the present invention as shown in scheme 7 in Figure 7.
• Bromide (7A) represents a family of common intermediates with different R, groups at the 4-position (schemes 1 and 2).
• Each of these bromides can be modified with mixtures of alkylhalides (R'X') or with mixtures of substituted benzaldehydes (R"CHO) to form libraries of dihydroxynaphthoic acids with alkyl (7C) or aralkyl (7E) groups at the 7-position.
• These libraries can be small libraries or complex libraries, depending on the complexity of bromide (7A), R'X', and R"CHO.
• the essential reactions of bromides (7A) with R'X' or R"CHO, involving initial reaction of 7A with n-butyllithium, are quantitative. The same is true for a number of reactions in schemes 1 through 6 that involve Grignard reactions. Thus, there are multiple points where synthesis of mixtures is feasible. Therefore, scheme 7 is representative of this approach to production of limited sized libraries.
• Synthetic scheme 8 features the incorporation of the carbon atoms for the second ring of the naphthalene system in one step by the reaction of the Grignard reagent formed from l -bromo-3,4-dimethoxy-2- mefhylbenzene (14a) and l -bromo-3,4-dimethoxy-2-n-propylbenzene(14b) with ethyl 3- methyl-4-oxobut-2-enoate.
• the ketone functional groups of 16a and 16b were reduced with sodium borohydride, and the intermediate alcohols dehydrated on acidic workup to form alkenes which were dehydrogenated with DDQ to form the naphthalenes 17a and 17b.
• Addition of bromine to the alkenes formed dibromides which were immediately dehydrohalogenated with DMF to form vinyl bromides which were dehydrogenated with DDQ to afford the bromonaphthalenes 18a and 18b.
• Compounds 18a and 18b were reacted with n-butyl lithium and benzaldehyde to form the benzylic alcohols which were hydrogenolyzed with Pd/C in ethanol to form 19b and 19d.
• the precursor compound 22a was prepared from 2-isopropyl phenol using procedures described in Royer et al, ( "Synthesis andAnty- HIV Activity of 1, 1 '-Dideoxygossypol and Related Compounds ", J. Med. Chem. 1995, 38, 2427-2432).
• the transformations to form compounds 23a-23g, 24a-24g, and 25a-25g were accomplished using the same procedures used for the corresponding steps in scheme 8.
• the first series of derivatives of 13 addressed the question whether addition of groups at the 7-position has any effect on binding, in view of the fact that this is the coupling position in the gossypol series and in view of the similar inhibitory properties of 13 and its dimer against pLDH and LDH-M.
• Compounds 25a and 25b show the effects of methyl or benzyl groups in the 7-position on inhibition of LDH. There is little change in the dissociation constants by introduction of a methyl group. Introduction of a benzyl group results in markedly stronger inhibition of LDH-M and LDH-H but not of pLDH.
• the second series of compounds related to 13 addressed the question whether modification of the alkyl group in the 4-position affects inhibition of LDH.
• Two groups of compounds were compared, one with methyl at the 4-position and one with n-propyl at the 4-position. both groups of compounds containing hydrogen, methyl, or benzyl at the 7- position.
• the results are shown in Table 1.
• 4-methyl derivatives 21a, 21b, and 21 c, Table 1
• the mechanism of NADH reduction of pyruvate to lactate catalyzed by LDH is thought to involve direct hydride transfer of the pro-R (H A ) C 4 -hydrogen from the reduced nicotinamide ring of NADH to the ketone of pyruvate to form L-lactate.
• the ordered formation of the LDH-NADH binary complex and LDH-NADH- pyruvate ternary complex is followed by rate determining closure of a substrate specificity loop to encase the reactants in a desolvated environment before hydride transfer occurs.
• Hydride transfer is facilitated by the D168/H195 proton donor dyad which transfers a proton to the ketone functional group of pyruvate in concert with hydride transfer, a process that is also facilitated by polarization of the ketone group by R109.
• R171 acts to anchor the substrate through interaction with the carboxy late group of pyruvate.
• Residues 98-109 of human LDH-H and LDH-M define the substrate specificity loop. This sequence is quite highly conserved in all other known LDH. except pLDH where not only the sequence differs but there is also a 5 amino acid insert from residues 104 to 108 ( 98 AGFTKAPGKSDKEWNRD) which forms an extended specificity loop.
• the recent crystal structure of the pLDH-NADH-oxamate ternary complex described a closed loop structure with a cleft at the active site which is not present in other LDH. Nevertheless, in spite of these unique structural features of pLDH, this LDH exhibits high specificity for pyruvate.
• Additional residues that are conserved in other LDH but differ in pLDH include SI 63, 1250 and T246.
• SI 63, 1250 and T246 In most LDH, the nitrogen of the carboxamide group of NADH is H-bonded to the oxygen of SI 63, whereas in pLDH residue 163 is leucine.
• 1250 normally provides a hydrophobic sidechain that stacks against the nicotinamide ring; in pLDH residue 250 is proline.
• the compounds in Table 1 are competitive inhibitors of cofactor binding, as shown in Figures 1 la and 12a. Inhibition with respect to substrate binding is generally mixed, but sometimes appears competitive, as shown in Figures 1 1 b and 12b for inhibition of pLDH by 25c. These kinetic studies raise the question whether inhibition of LDH by substituted dihydroxynaphthoic acids involves complexation at both the cofactor and substrate binding sites and whether this can be exploited to develop selective dehydrogenase inhibitors.
• the acid (10.0 g, 37.6 mmol) in 100 mL of acetic acid was hydrogenated on a Parr hydrogenator with 0.4 g of 10%) palladium on carbon and 60 psi hydrogen pressure at 60°C for 20 h.
• the reaction mixture was vacuum filtered through celite, and the celite was washed with ether.
• the solvent was evaporated in a fume hood, and the residual oil was distilled bulb to bulb (170°C, 1 Torr Hg) to give 7.5 g (29.7 mmol. 79% yield) of 15a as an amber oil which crystallized on standing to form colorless crystals: mp 84-86°C.
• Compound 22a (1.00 g, 3.09 mmol) in 25 mL of dry ether was cooled to 0"C under nitrogen.
• n-Butyllithium (4 mmol) was added as a solution in hexane. The mixture was stirred for 15 min at 0°C, and then p-trifluoromethylbenzaldehyde(0.87 g, 5.0 mmol) was added. The mixture was stirred at ambient temperature under nitrogen for 1 h.
• the reaction mixture was acidified, and the organic layer was separated, washed with water and brine, and dried over magnesium sulfate. After filtration, the ether layer was evaporated to give a semi-solid which was dissolved in ethanol and hydrogenated on a Parr hydrogenator with 10%> palladium on carbon and 60 psi hydrogen pressure at room temperature for 2 h.
• the reaction mixture was vacuum filtered through celite, and the celite was washed with ether. The solvent was removed, and the residual oil was purified by silica column chromatography using dichloromethaneto give 1.09 g (2.72 mmol, 88% yield) of 23c as a crystalline solid.
• Recombinant pLDH was produced in E. coli, similar to the procedure described by
• Pan-Active Site inhibitors are defined as inhibitors that occupy all or parts of both the substrate binding site and the cofactor binding site.
• Figures 11A and 1 IB show that inhibition of pLDH by 7-(p-trifluoromethylbenzyl)-8-deoxyhemigossylicacid (25c) is competitive against both cofactor and substrate.
• Figures 12A and 12B show that inhibition of pLDH-M 4 by 2,3-dihydroxy-6-methyl-7-(p-methylbenzyl)-4-(l-methylethyl)-l- naphthoic acid (25d) is competitive against both cofactor and substrate.
• 14B (2,3-dihydroxy-6-methyl-7-(m-methylbenzyl)-4-( 1 -methylethyl)- 1 - naphthoic acid (25f) inhibition of malate dehydrogenase
• 14C compound 25b inhibition of glucose 6-phosphate dehydrogenase
• 14D compound 21 e inhibition of alcohol dehydrogenase

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