CA2288199A1 - Hydroxynaphthoic acids and derivatives - Google Patents

Abstract

In one embodiment, the present invention provides a compound comprising formula (I), wherein A = H or OH, X = OH, a halogen, OR, NHR, NR', R'' where R, R', and R'' = H, C1-8alkyl, C2-8alkenyl, or C2-8alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or heterocyclic, substituted or unsubstituted; and R1,R2,R3,R4,R5 = H, C1-8alkyl, C2-8alkenyl, or C2-8alkynyl, cycloalkyl, cycloalkenyl, aryl or heterocyclic, substituted or unsubstituted, wherein R1 includes at least one methylene spacer through which R1 is attached to said compound. The present invention also provides methods for making hydroxynaphthoic acids.

Description

RELATED APPLICATION
S The present application is based on U.S. Provisional Patent Application No.
60/045,083 filed April 29, 1997, the entire disclosure and contents of which is hereby incorporated by reference. The present application is also a continuation-in-pan of U.S.
Patent Application No. 08/988,472 filed December 10, 1997 which itself is a continuation application of U.S. Patent Application No. 08/431,294 filed April 28.
1995. The entire disclosures and contents of both these applications are hereby incorporated by reference.
BACKGROUND OF T'HE INVENTION
Field of the Invention The present invention relates to naphthoic acids and derivatives.
Description of the Prior Art 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.
Most drugs are targeted at the active sites of enzymes and other proteins.
There are numerous dehydrogenases with critical metabolic roles that represent potential drug targets. The NAD(H) or NADP(H) cofactor binding sites of dehydrogenases have not been widely developed as drug targets, mainly because the structures of cofactor binding sites of dchydrogcnases, as determined by x-ray crystallography, are often quite similar, which implies that development of selective dehydrogenase inhibitors will be difficult. There has been some success in developing selective inhibitors that compete for the adenosine part of the cofactor binding site. For example, structure based design of adenosine-related inhibitors of glyceraldehyde-3-P
dehydrogenase from trypanosomes has been reported. There has also been some success in developing NAD analogs as potential therapeutics. For example, the anticancer agent tiazofurin (2-I S ~3-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. Generally, however, the nicotinamide part of the dinucleotide binding site of dehydrogenases has not been a target in drug design.

2 WO 98/49130 PC'r/US98/08544 SUMMARY OF THF; INVENTION
It is therefore an object of the present invention to provide Pan-Active Site inhibitors of dehydrogenases.
S
In one embodiment, the present invention provides a compound comprising:
HO Ra A R;
I S wherein:
A=HorOH
X = OH, a halogen, OR, NHR, NR'R" where R, R', and R" = H, C,_8 alkyl, C,_~
alkenyl, or C,_8 alkynyl, cycloalkyl, cycloa.lkenyl, aryl, aralkyl or heterocyclic, substituted or unsubstituted; and R,, R,, R1, R4, R; = H, C,_ft alkyl, C,_H alkenyl, or C~_H alkynyl, cycloalkyl, cyloalkenyl, aryl or heterocyclic, substituted or unsubstituted, wherein R, includes at least one methylene spacer through which R, is attached to said compound.
In another embodiment, the present invention also provides methods for making hydroxynaphthoic acids.
Other objects and features of the present invention will be apparent from the following detailed description of the preferred embodiments.

3 R~ R., BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described in conjunction with the accompanying drawings, in which:
Figure I 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 l;
Figure 3 illustrates a method for incorporating specific R~ radical groups into the naphthoic acids of the present invention;
I S Figure 4 illustrates a method for incorporating specific R, radical groups into the naphthoic acids of the present invention at the 5-position;
Figure 5 illustrates a method for incorporating specific RS 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;
Figure 8 illustrates methods for making compounds of the present invention;
Figure 9 illustrates methods for making compounds of the present invention;

4 Figure 10 is a table showing inhibition of human lactate dehydro<~enase (LDf-1-1-1, and LDH-M4) and malarial parasite P..Jecipururm lactate dehydrogenase (pLDI--1) by dihydroxynaphthoic acids;
Figures 1 I A and 1 I B illustrate the inhibition of pLDH by 7-(p-trifluoromethylbenzyl)-8-deoxyhemigossyIicacid (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 fluorescence of pLDI I
by NADH in the presence and absence of ADP;
Figure I 3B illustrates the quenching of Uhe intrinsic protein fluorescence of pLDI-1 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions For the purposes of the present specification and claims, the following terms, unless specifically indicated otherwise, have the following meanings:
The term "substituent" refers to a radical group which replaces a hydrogen on another radical group or on a compound.

5 The term ''substituted" refers to a radical group including an alkyl. alkenyl, or alkynyl substitucnt or a functional group substituent such as halide, e.g.
fluoride.
chloride, bromide or iodide; carboxylate; nitro, etc. For the purposes of the present specification and claims, where the terms "substituted" or "unsubstituted"
follow a list of radical groups, these terms refer to all of the preceding radical groups in the list.
The term "halogen" refers to any of F, Ci, Br, or I.
The term ''methylene spacer" refers to a -CH,-, -CHX,-, or -CX,X,- group where each of X, and X, is a substituent or a radical group.
The term "C,_g 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,_H alkoxy") are to be construed accordingly.
Similarly.
the term "C,_; alkyl" refers to a straight or branched chain alkyl radical group having one to five carbon atoms (such as methyl or ethyl).
The term "C,_g 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, I-propenyl, I- and 2-butenyl and 2-methyl-2-propenyl.
T'he term "C,_R 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.
The term ''C~_g 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.

6 The term "C;_g cycloalkenyl" refers to an unsaturated cyclic radical group having from 3 to 8 carbon atoms arranged in a ring and includes, for example, cyclohexenyl, cyclohexadicnyl, etc..
The term "aryl" refers to a radical group having the ring structure characteristic of benzene, naphthalene, phenanthrene, anthracene, pyrene, benzopyrene, etc.
The term "aralkyl" refers to a substituted aryl radical group having one or more C,_g alkyl substituents regardless of whether thc: link to a compound or radical group is through the alkyl or the aryl of the aralkyl radical group.
The term "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 I S carbon such as sulfur, nitrogen, oxygen, etc.
For the purposes of the present invention, including the accompanying drawing figures. the possible values for the radicals R,, R,, R;, R4, and R5 are set forth above in the summary of the invention section.
Descrintiion Various methods can be used to synthesize the compounds of the present rnventron:
For example, Scheme I in Figure i shows how to synthesize dihydroxynaphthoic acids of the invention having a particular radical group R~
at the 7-

7 position ( I L). A known I -bromo-3,4-dimethoxy-benzene ( 1 A), such as bromo-3,4-dimethoxy-2-isopropylbenzene, is reacted with a known ethyl 4-oxobut-2-enoate (IB}, such as ethyl 3-methyl-4-oxobut-2-enoate, to form an ester ( I C). Because the ester, 1 C.
is difficult to reduce it is saponified to the acid (1 D), which is hydrogenolyzed and reduced. T'he resulting carboxylic acid is cyclized with polyphosphoric ester to give tetralone ( 1 E). The ketone function of 1 E is reduced with sodium borohydride and the intermediate alcohol dehydrated on acidic workup to form an alkene ( 1 F).
Addition of bromine to the 1 F .forms a dibromide which is immediately dehydrohalogenated with dimethylformamide to form vinyl bromide ( 1 G) which is dehydrogenated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form a bromonaphthalene (IH).
Compound IH is reacted with n-butyl lithium and benzaldehyde to form a benzylic alcohol which is hydrogenolyzed with palladium on charcoal in ethanol to form lI where R,, is benzyl.
Formation of the compound I I where R~ is methyl is accomplished by reaction of 1 H
with n-butyl lithium and methyl iodide. Carbonyl groups are introduced to the I 5 compound 1 I by formylation with titanium tetrachloride and dichloromethyl methyl ether to form 1 J. Oxidation of the aldehydes with sodium hypochlorite form a carboxylic acids 1 K. The methyl groups are removed from the phenolic ether 1 K with boron tribromide to form compound 1 L.
Scheme 2 in Figure 2 illustrates efficient syntheses of bromides (2D) and (2H) which can be used as the starting bromide ( 1 A) of scheme 1 having a specific radical group R, (ln Scheme 2, radical groups R~, Rh, and R~ can be almost any radical group, just as groups R,, R2, R~, R4, and RS 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). Hydrogenolysis of 2B with IO%
palladium on charcoal in ethyl acetate afforded compound (2C). Bromination using bromine in carbon tetrachloride at -10 C afforded compound (2D). Compound (2H) can be synthesized by the same methodology except that 2,3-dimethoxybenzaldehyde is the

8 starting material. In scheme 2, the synthesis of (2D) represents a pathway for introducing two radical groups (R;, and R~), while the synthesis of (2H) represents the introduction one radical group (R~) attached to bromide by a methylene spacer.
Furthermore, by varying the radical group R~ of the starting bromide ( 1 A), the method of scheme 1 can be used to synthesize families of dihydroxynaphthoic acids having different radical groups R,.
Scheme 3 in Figure 3 illustrates a method for incorporating specific R~
radical groups into the naphthoic acids of the present invention. A substituted glyoxal (3A) is converted to an acetals (3B) by reaction with m~°thanol in the presence of acid. Reaction of 3B with triethylphosphonoacetate (3C) usin~; a modification of the Wadsworth Emmons reaction affords compound 3D. Hydrolysis in dilute acid affords the aldehyde ( 1 B). Compounds 1 B can be used to form napht:hoic acids as described in scheme 1.
IS
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 lA, is reacted with a y-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 Vii, y unsaturated ester (4C). 4B is a substituted y-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. The resulting carboxylic acid is cyclized with polyphosphoric ester to give tetralone (4E). The ketone function of 4E is reduced with sodium borohydride and the intermediate alcohol dehydrated on acidic workup to form an alkene (4F). Addition of bromine to the 4F forms a dibromide which is immediately dehydrohalogenated with dimethylformamide to form vinyl bromide (4G) which is dehydrogenated with 2,3-dichloro-~,6-dicyano-1,4-benzoquinone to form a bromonaphthalene (4H, which is equivalent to 1 H in scheme 1 ). Compound of the

9 present invention {4I) can then be formed by following the steps from 1 H to I
L in scheme 1.
Scheme 5 in Figure 5 illustrates a method for incorporating specific R;
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 I 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 (lI) in scheme I.
Although 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 ( 1 A) in schemes I through 5 to produce monohydroxynaphthoic acids of the present invention having any of the radical groups R,, R,, R;, Ra, and RS at the appropriate positions on the naphthoicalene ring.
Although the methods illustrated in schemes I through 6 only specifically show how to synthesize naphthoic acids of the present invention, it should be clear that 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. For example, ethyl and methyl ester derivatives can be formed using the Fischer esterification process on the naphthoic acids of the present invention. Similarly, 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. As is well known. any suitable amine can be used in this procedure.
In addition to providing hydroxynaphthoic acids and derivatives in a purified form, the present invention also provides hydroxynaphthoic acids and derivatives as present in a combinatorial library. In its broadest form, 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 I S libraries, combine the power of genetics to screen libraries while oligonucleotide libraries utilize PCR (polymerase chain reaction) to amplify candidate ligands. 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. Therefore, it is preferable to develop encoded libraries where the library is synthesized on beads. The chemical history of each bead is recorded by cosynthesis of an easily analyzable ''tag"
on each bead . Genetic algorithms can also be use in the design of the combinatorial library.
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 connpound. 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 modif ed to develop potent selective inhibitors. By incorporating a substrate analog into a hydroxynaphthoic acid at position 4, 5, 6, 7. or 8 the combinatorial library method of the presern; 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, I S 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 trues 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.
The invention will now be described by way of example. The following examples are illustrative and are not meant to limit the scope of the invention which is set forth by the appended claims.
EXAMPLES
The syntheses of compounds 21 a-21 f and 25a-25g of the present are outlined in scheme 8 of Figure 8 and scheme 9 of Figure 9, re:;pectively. Synthetic scheme 8 features the incorporation of the carbon atoms for the second rin<J of the naphthalene system in one step by the reaction of the Grignard reagent formed from 1-bromo-3.4-dimethoxy-methylbcnzene ( 14a) and 1-bromo-3,4-dimethoxy-2-n-propylbenzene( 14b) with ethyl 3-methyl-4-oxobut-2-enoate. These precursors are readily prepared from commercially available starting materials using a procedure described in Royer eml., ",Symhe.si,s and Anti-HIV Activity of 1. I '-Dideoxygossypol and Related Compounds ", J. Med.
Chem.
1995, 38, 2427-2432. Because the esters were difficult to reduce they were saponified, and the acids 15a and 15b were hydrogenolyzed and reduced. The resulting carboxylic acids were cyclized with polyphosphoric ester to give tetralones 16a and 16b.
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 deh~~drogenated with DDQ to afford the I5 bromonaphthalencs 18a and 18b. Compound; 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. Formation of 19a and 19c was accomplished by reaction of 18a and 18b with n-butyl lithium and methyl iodide. Carbonyl groups were introduced to compounds 17a, 17b, and 19a-19d by formylation with titanium tetrachloride and dichloromethyl methyl ether. Oxidation of the aldehyde groups with sodium hypochlorite formed the carboxylic acids 20a-20f. The methyl groups were removed from the phenolic ethers with boron tribromide to form compounds 21a-21f Scheme 9 outlines the syntheses of 25a-25g. Tlne precursor compound 22a was prepared from 2-isopropyl phenol using procedures described in Royer et al., ( "Synthesis and Anty-HIVActivityo~~l,I '-Dideox~~gossypoland RelatcedCompounds", J. Med. Chem.
1993, 38, 2427-2432). The transformationsto form compounds 23a-23g, 24a-24g, and 25a-25g were accomplished using the same procedures used for the corresponding steps in scheme 8.
Inhibition of Parasite Lactate DehydrogE:nase (pLDH) and Human Lactate Dehydrogenases(LDH-H and LDH-M) by Derivatives of 8-DeoxyhemigossylicAcid.

The inhibitionofpLDH, LDH-H and LDH-M by 8-deoxyhemigossylicacid (13) OOH
HO~ Y

HO ~ /
the reference compound for these studies. is summarized in Table I of Figure

10.
Compound 13 is nonselective in its inhibiton of pLDH and LDH-M, with dissociation constants in the low p molar range. By comparison, the dissociation constant for inhibition of LDH-H is 30 fold higher than for LDH-M. The inhibition of these three LDH
by 13 is competitive with the binding of NADH.
IS
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 I 3 and its diner against pLDH and LDH-M. Compounds 2~a and 25b (Table I ) show the effects of methyl or benzyl groups in the 7-position on inhibition of LDI-I.
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. Surprisingly, however, introduction of a substituted benzyi group has a major effect on all three LDH; the p-trifluoromethylbenzyl derivative (25c, Table 1 ) is highly selective in favor of pLDH (K; = 0.2 ~tM) compared to either human LDH.
Clearly, introduction of the appropriate group at the 7-position can provide selective inhibitors 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 methy I 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. For the 4-methyl derivatives (21 a, 21 b, and 21 c, Table 1 ), there is selectivity for pLDI-I and LDl-I-M compared to LDH-H, and affinity increases with the introduction of groups at the '7-position for all three LDH, especially for LDH-M. For the 4-n-propyl derivatives (21 d, :? 1 e, and 21 f, Table 1 ), the presence of the n-propyl group at the 4-position has a marked effect both on affinity and on selectivity.
Compound 2 I a exhibits 190 fold selectivity for LDH-M compared to LDH-H. The most potent inhibitor of the compounds tested in this study is 25d which inhibits LDH-M, Kp =
30 nM. Thus both the 4-position and the 7-position represent sites for modification of the dihydroxynaphthoicacid backbone in the development of LDH inhibitors.
Discussion The mechanism of NADH reduction oil pyruvate to lactate catalyzed by LDH is thought to involve direct hydride transfer of the pro-R (H~) Ca-hydrogen from the reduced nicotinamide ring of NADH to the ketone of pyruvate to form L-lactate. In this mechanism, 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 D 168/H :l 95 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 o1° the ketone group by 8109. 8171 acts to anchor the substrate through interaction with tl7e carboxylate group of pyruvate. These catalytic residues are conserved in all LDH.
The unique structural features of pLDH that separate it from human LDH as well as from all other known LDH involve residues both at the cofactor and substrate sites.
Residues 98-109 of human LDH-H and LDH-M (~BAGVRQQEGESRL) define the substrate specificity loop. This sequence is quite highly conserved in all other known LDH. except pL.DH where not only the sequence differs but there is also a 5 amino acid insert from residues 104 to 108 (''~AGFTKAPGKSDKEWNRD)which forms an extended specificity loop. 'rhe 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 5163, I250 and T246. In most LDH, the nitrogen of the carboxamide group of NADH is H-bonded to the oxygen of S 163, whereas in pLDH residue 163 is leucine. In addition, I250 normally provides a hydrophobic sidechain that stacks against the nicotinamide ring; in pLDH residue 250 is proline. T246, which is adjacent to both the nicotinamide group and the substrate in the ternary complex of most LDH, is replaced by proline in pL.DH. All of these unique features of pLDI-I
suggest that the active site of pLDH may be a selective drug target.
The results of the present study demonstrate that substituted dihydroxynaphthoic acids structurally related to 8-deoxyhemigossylic acid (13) can be developed that are selective inhibitors of pLDH compared to human LDH and that these inhibitors appear to be competitive with cofactor binding. Compound 2~c with a p-trifluoromethylbenzyl moiety at the 7-position of 13 shows 75 and 400 fold selectivity for pLDH over LDH-M
and LDH-I-I, respectively. Surprisingly, however, some of these substituted dihydroxynaphthoic acids are highly selective for LDH-M over LDH-H, in spite of the high sequence homologies of these two human LDH. Generally, these inhibitors show higher affinities for LDH-M compared to LDH-H, with selectivities ranging from 5 to 190 fold.
The compounds in Table I are competitive inhibitors of cofactor binding, as shown in Figures 11 a 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.
Experimental Section Chemical Synthesis. Reagent quality solvents were used without further purification. THF and ether were distilled from calcium hydride. 1-Bromo-3,4-dimethoxy-2-methylbenzene ( 14a) and 1-bromo- 3.4-dimethoxy-2-n-propylbenzene( 14b) were synthesized from o-cresol and 2-n-propylphenol respectively according to published procedures ( 14}. Melting points were determined with a VWR Scientific Electrothermal capillary melting point apparatus and are uncorrected. NMR spectra were recorded on a Bruker AC250 NMR spectrometer in CDCL,, unless otherwise stated. Chemical shifts are in ppm relative to TMS.
General Procedure for Removing the Methyl Groups from Phenolic Methyl Ethers with Boron Tribromide. The procedure for removal of methyl groups was identical to that described previously.
General Procedure for Formylation and (Jxidation to Form Carboxylic Acids. A
reaction mixture with I mmol of the compound to be formylated and 2.6 mmol of dichloromethyl methyl ether in 20 mL of dichloromethane under nitrogen was cooled in an ice bath. Titanium tetrachloride (1.5 mmol) was added slowly with stirring.
The mixture was allowed to come to ambient temperature and was stirred for 2 h.
The mixture was added with stirring to 100 g of ice with I 0 m:L of 6 M HCI, and the organic layer was washed with water and brine and dried over magnesium sulfate. Filtration and evaporation of the solvent gave a crude oil which was dissolved in 15 mL of acetonitrile and cooled in an ice bath. Sodium dihydrogen phosphate (0.2 mmol) and 30%
hydrogen peroxide ( 1.1 mmol) were added followed by 1.4 mmol of sodium chlorite dissolved in 5 mL water. The reaction mixture was stirred at ambient temperature for 2 h and then poured onto 100 g of ice containing 10 mL of 6 M HCl and extracted with ether.
The ether layer was washed with water and brine and dried over magnesium sulfate.
4-[3,4-Dimethoxy-2-methylphenyl)-3-methylbutanoicAcid (lSa). Compound 14a S ( 12.0 g, S 1.9 mmol ) was added to magnesium turnings ( 1.26 g, S2. I mmol) in 100 mL dry THF in a 250 mL ground glass Erlenmeyer I7ask equipped with a reflux condenser and magnetic stirrer. The mixture was rel7uxed with stirring for 1 h, cooled to 0°C, and added dropwise to 3-methyl-4-oxobut-2-enoate(7.39 g, S2.1 mmol) in 2S mL of dry THF
at 0°C.
The mixture was stirred at ambient temperature for 1 h and poured onto 100 g ice and 2S
mL of 6 M HCI. The organic layer was extracted into ether, washed with water and brine, dried over magnesium sulfate and filtered. The ether was removed by rotary evaporation to give 12.3 g (41.8 mmol, 80% yield) of ester as a crude oil which was dissolved in 100 mL of ethanol with 4.70 g of KOH. Water, 20 mL, was added, and the mixture was refluxed for 3 h. The mixture was poured onto 100 g ice and and 2S mL 6 M HCI
and 1 S stirred. The white solid was filtered, washed with water, dried and recrystallized from ethyl acetate to give 10.0 g (37.5 mmol, 90% yield) of acid: mp 174-176°C. 'H
NMR:(ppm) 6.69 (d, I H), 6.74 (d, 1 H), 6.32 (s, 1 H), 5.32 (s, 1 H), 3.86 (s, 3H), 3.79 (s, 3H), 2.34 (s, 31 I), 2.00 (s, 3H), 1.60 (s, 1 H). Anal. (C,,,H,~O;) C,H. 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.S g (29.7 mmol, 79% yield) of 1 Sa as an amber oil which crystallized on standing to form colorless crystals: mp 84-86°C. 'H
NMR:(ppm) 6.78 (d, 2S 1 H), 6.67 (d, 1 H), 3.83 (s, 3H), 3.77 (s, 3H), 2.62-2.33 (mutt, SH), 2.22 (s, 3H), 0.992 (d, 3H). Anal. (C,4H2oO4) C,H.
4-[3,4-Dimethoxy-2-n-propylphenyl]-3-methylbutanoicAcid ( 1 Sb). Compound 14b (10.0 g, 38.6 mmol) and ethyl bromide (0.6 g, S.5 mmol) were added to magnesium turnings (1.2 g. 49.4.mmo1) in 100 mL of dry THF in a 2S0 mL ground glass Erlenmeyer filask and was reiluxed 1 h with stirring. After cooling. the mixture was added dropwise to 3-methyl-4-oxobut-2-enoate (6.90 g, 48.5 mmol j in 25 mL of dry TI-IF at 0"C.
The mixture was stirred at ambient temperature for 1 h and poured onto 100 g ice and 25 mL
of 6 M HC'l. The organic layer was extracted into ether, washed with water and brine, and dried over magnesium sulfate. The ether was evaporated to give 10.6 g (32.9 mmol, 85%
yield) of ester as a crude oil which was dissolved in 100 mL of ethanol with 4.50 g of KOH. Water, 20 mL, was added, and the mixture was refluxed for 3 h, then poured onto 100 g of ice and 25 mL of 6 M HCI and stirred. The semi-solid was extracted with ether, washed with water and brine, and dried over magnesium sulfate. Filtration and evaporation of the ether gave 9.20 g (31.3 mmoll, 95% yield) of acid as a viscous oil. The acid (9.20 g, 31.3 mmol) in 100 mL of a<:etic 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 24 h. The reaction mixture was vacuum filtered through eelite, and the celite was washed with ether. The solvent was evaporated in a fume hood, and the residual oil was distilled bulb to bulb (180°C, 1 Torr) to give 7.46 g (26.6 mmol, 85%
yield) of 15b as a pale yellow oil. 'H NMR:(ppm) 6.77 (d, 1 H), 6.68 (d, 1 H), 3.79 (s, 3H), 3.77 (s, 3H), 2.74-2.08 (m, 7H), 1.50 (sex, 2H), 0.96 (t, 3H), 01.94 (d, 3H). Anal.
(C,GH,aOa) C,H.
3,4-Dihydro-6,7-dimethoxy-3,5-dimethyl-1 (2H)-naphthalenone ( 16a). Compound 1 Sa (7.0 g, 27.7 mmol) was added to 35 g of pol;yphosphoricester in 100 mL of methylene chloride and refluxed for 1 h. T'he reaction mixture was poured onto ice, stirred to hydrolyze the polyphosphoric ester, and the resulting solid was filtered and recrystallized from methanol to give 5.4 g (23.0 mmol, 83°/~ yield) of 16a as white plates: mp 106-108°C. 'H NMR:(ppm) 7.48 (s, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 2.95-2.28 (mull, SH), 2.22 (s, 3H), 1.18 (d, 3H). Anal. (C,4H,~0,) C,H.
3,4-Dihydro-6,7-dimethoxy-3-methyl-5-n-propyl-1 (2H)-naphthalenone ( 16b).
Compound 15b (7.0 g, 25 mmol) was added to 35 g of polyphosphoricester in 100 mL of methylene chloride and refluxed for 1 h. The reaction mixture was poured onto ice, stirred, and extracted with ether. The ether eras evaporated, and the residual oil was PCTlUS98/08544 chromatographed on silica gel using ethyl acetate/hexane to give 5.63 g (21.5 mmol, 86%
yield) of 16b as white crystals: 'H NMR:(ppm) 7.49 (s, 11-I), 3.89 (s, 3H), 3.87 (s, 3H), 3.00-2.23 (molt. 71-I). 1.50 (molt, 2H), 1.15 (d, 3H), 1.01 (t, 3H). Anal.
(C,~fl"O;) C,H.
2,3-Dimethoxy-1,7-dimethylnaphthalene ( 17a). Compound 16a (4.5 g, 19.2 mmol) was dissolved in 20 mL of 2-propanol, sodium borohydride (1.08 g, 28.8 mmol) was added, and the mixture was stirred at reflux for 1 h. The cooled solution was acidified by dropwise addition of 6 M HCl with stirring, then refluxed for 1 h, poured onto ice and extracted with ether. The ether layer was washed with water and brine and dried over magnesium sulfate. The ether was removed by :rotary evaporation, and the residue was distilled bulb to bulb ( 170°C, 1 Torr) to give 3.31 g ( 15.2 mmol, 79%
yield) of alkene which crystallized on standing. 'H NMR:(ppm) E~.50 (s, 1 H), 6.31 (molt, 1-1), 5.81 (molt, 1H), 3.83 (s, 3H), 3.77 (s, 3H}, 2.84-2.23 (mull, 31-I), 2.19 (s, 3H), 1.10 (d, 3H). Anal.
(C,,,l-l,y0,) C,I-I. A mixture of alkene ( 1.95 g, 8.93 mmol) in 25 mL of benzene and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(2.03 g, 8.93 mmol) was stirred for 2 h, and the resulting mixture was filtered through a short column of alumina. The solvent was evaporated, and the residual oil was purified by silica column chromatography with dichloromethane. Removal of solvent provided 1.66 g of 17a (7.67 mmol. 86%
yield) as colorless crystals: mp 68-69°C. 'H NMR:(ppm) 7.63 (s, 1 H), 7.59 (d, I
H), 7.21 (d, 1 H), 6.99 (s, 1 H), 3.93 (s, 3H), 3.83 (s, 3H), 2.56 (s, 3H), 2.49 (s, 3H). Anal.
(C,aH,~,O,) C,H.
2,3-Dimethoxy-7-methyl-I-n-propylnaphthalene(I7b). Compound 16b (5.00 g, 19.1 mmol) was dissolved in 20 mL of 2-propanol, sodium borohydride { I .08 g, 28.8 mmol) was added, and the reaction mixture was stirred with refluxing for 1 h.
After cooling, the mixture was acidified by dropwise addition of 6 M HC1 with stirring. The acidified mixture was refluxed for 1 h, poured onto ice and extracted with ether. The ether extract was washed with water and brine and was dried over magnesium sulfate.
The ether was removed by rotary evaporation, and the residue was distilled bulb to bulb (190°C, 1 Torr) to give 4.46 g (18.1 mmol, 95% yield) of an alkene. 'H
NMR:(ppm) 6.51 (s, I H), 6.3 I (dd, l H), 5.80 (dd, 1 H), 3.81 (s, 3H), :3.80 (s, 3H), 2.86-2.35 (molt, SH), I .50 WO 98!49130 PCT/US98/08544 (m. 2H), 1.10 (d, 3H). 0.99 (t. 3H}. The alkenc: (4.46 g. 18.1 mmol) was dissolved in 25 mL. of benzene and was treated while stirring with 2,3-dichloro-5.6-dicyano-1.4-benzoquinone (4.10 g, 18.0 mmol) and was stirred an additional 2 h. The reaction mixture was filtered through a short column of alumin,~ and washed with benzene. The solvent S was evaporated, and the residual oil was purified by silica column chromatography using dichloromethane to give 4.24 g ( 17.4 mmol, 96~% yield) of 17b as an oil. 'H
NMR:(ppm) 7.64 (s, 1 H). 7.60 (d, I H), 7.21 (d, 1 H), 7.01 (:~, 1 H), 3.95 (s, 3H), 3.87 (s, 3 H), 3.01 (t.
2H), 2.50 (s. 31-1), 1.67 (m, 2H), 1.06 (t. 3I-1). Anal. (C,~,H,°O~) C,H.
6-Bromo-2,3-dimethoxy-l,7-dimethylnaphthalene(18a). Compound 16a (4.5 g, 19.2 mmol) was converted into the alkene as described in the synthesis of 17a.
The alkene ( 1.48 g. 6.78 mmol } was dissolved in I 00 mI, of dry dichloromethane, and bromine ( 1.08 g, 6.78 mmol) in 5 mL. dichloromethane was added dropwise with stirring over a 15 min period. The solvent was evaporated, and the residue was taken up in 25 mL of DMF and warmed to 60-70"C for i h. The reaction mixture was poured onto ice and stirred. The solid was filtered, dried and recrystallized from petroleum ether to give 1.51 g (5.08 mmol, 75% yield) of colorless crystals: mp 75-75°C. 'H NMR:(ppm) 6.65 (s, 1 H), 6.43 (s, 1H), 3.82 (s, 3~I), 3.77 (s, 3H), 3.03-2.62 (mult, 3H), 2.18 (s, 3H), 1.11 (d, 3H). Anal.
(C,4H"BrO,) C,1-I. The vinylic bromide (I.51 g, 5.08 mmol) was dissolved in 25 mL of benzene, and 2.3-dichloro-5.6-dicyano-1,4-benzo-quinone ( 1.14 g, 5.02 mmol) was added with stirring, and stirring was continued for 2 h. The reaction mixture was filtered through a short column of alumina and eluted with benzene. The solvent was evaporated, and the residua! oil was purified by silica column chromatography using dichloromethane to give 1.24 g (4.21 1'1111101, 83% yield) of 18a as colorless crystals: mp 96-97°C. 'H NMR:(ppm) 7.89 (s, I H). 7.67 (s, l I1), 6.89 (s, 1 H). 3.94 (s, 3I-I), 3.84 (s, 3H).
2.54 (s. 6H). Anal.
(C~aH,;BrO,) C.H.
6-Bromo-2,3-dimethoxy-7-methyl-1-n-propylnaphthalene(18b). Compound 16b (3.5 g, 13.4 mmol) was reduced with sodium borohydride as described in the synthesis of 17b. 'rhe alkene (2.5 g, 10.1 mmol) was dissolved in 100 mL of dry dichloromethane,and bromine ( 1.62 g, 10,1 mmol) in 5 mL of dichloromethane was added dropwise with stirring over a period of I 5 min. The solvent was evaporated, and the residue was taken up in 15 mI, of DMF and warmed to 60-70°C for 1 hr. The reaction mixture was poured onto ice and stirred, after which the solid was filtered, dried and recrystallized from petroleum ether to give 2.46 g (7.56 mmol, 75% yield) of white crystals. The vinylic bromide (1.23 g, 3.78 mmol) was dissolved in 25 mL. of benzene, and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(857 mg, 3.78 mmol) was added slowly with stirring, followed by continued stirring for 2 h. The reaction mixture was filtered through a short column of alumina and eluted with benzene. The solvent was evaporated, and the residual oil was purified by silica column chromatography using dichloromethane to give 1.04 g (3.21 mmol. $s% yield) of I 8b as colorless crystals, mp 64-66°C. 'H
NMR:(ppm) 7.90 (s, 1 H), 7.69 (s, 1 I-1), 6.91 (s, 1 H), 3.94 (s, 3H), 3.87 (s, 3II), 2.99 (t, 2H), 2.54 (s, 3H), 1.66 (m, 2H), 1.05 {t, 3IV). Anal. (C,~H,~BrO,) C,H.
2,3-Dimethoxy-1,6,7-trimethylnaphthalene(19a). Compound 18a (646 mg, 2.19 mmol) in 25 mI, of dry ether was cooled to 0°C under nitrogen. n-Butyllithium (2.50 mmol) was added as a solution in hexane. The mixture was stirred for 15 min at 0°C, and then methyl iodide (0.38 g, 3.0 mmol) was added. The mixture was stirred at ambient temperature under nitrogen for I h. 1'he reaction mixture was poured onto 10 g of ice containing 2 mL of HCI, and the organic layer was separated. washed with water and brine and dried over magnesium sulfate. After filtration, the ether was evaporated to give an oil which was purified by silica column chromatography using dichloromethane to give 426 mg ( 1.84 mmol. 84% yield) of 19a as buff crystals, mp 59-60°C. 'H
NMR:(ppm) 7.59 (s, 1 H), 7.44 (s, 1 H). 6.93 (s, I H), 3.93 (s. 3H), 3.8 3 (s, 3H), 2.56 (s, 3H), 2.41 (s, 3H), 2.38 (s, 3H). Anal. (C,;H,80,) C,H.
6-Benzyl-2,3-dimethoxy-1,7-dimethylnaphthalene (19b). Compound 18a (570 mg, 1.93 mmol) in 25 mL of dry ether was cooled to 0°C under nitrogen.
n-Butyllithium (2.2 mmol) was added as a solution in hexane. The mixture was stirred for I 5 min at 0°C, and then benzaldehyde (265 mg, 2.5 mmol) was added. The mixture was stirred at ambient temperature under nitrogen for 1 h. The reaction mixture was poured onto ice containing HC1 and stirred for 1 h. The mixti.~re was filtered to give a buff solid. 'H
NMR:(ppm) 7.85 (s, 1H), 7.60 (s, 1H), 7.34-7.25 (m, SH), 7.03 (s, IH), 6.09 (s, IH), 3.94 (s, 3H), 3.84 (s, 3H}, 2.55 (s, 3H), 2.34 (s, 3H;~, 2.28 (brs, 1H, disappears upon shaking with D,O). The alcohol 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 dichloromethane to give 522 mg (1.70 mmol, 88% yield) of 19b as a crystalline solid, 'H NM(R:(ppm) 7.63 (s, 1 H), 7.41 (s, 1 H), 7.30-7.14 (m, SH), 6.95 (s, 1H), 4.11 (s, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 2.56 (s, 3H), 2.36 (s, 3H).
2,3-Dimethoxy-6,7-dimethyl-1-n-propylnaphthalene(19c). Compound 18b (700 mg, 2.16 mmol) in 25 mL of dry ether was cooled to 0°C under nitrogen.
n-Butyllithium (3.0 mmol) was added as a solution in hexane. The mixture was stirred for I S
min at 0°C, and then methyl iodide (0.38 g, 3 mmol) was added. The mixture was stirred at ambient temperature under nitrogen for I h. The reacticm mixture was acidified, and the organic layer was separated, washed with water and brine, and dried over magnesium sulfate.
After filtration, the ether was evaporated to giive an oil which was purified by silica column chromatography to give 446 mg (1.73 mmol, 80% yield) of 19c as an amber oil.
'H NMR:(ppm) 7.89 (s, 1 H), 7.44 (s, I H), 6.93 (s, 1 H), 3.92 (s, 3H), 3.86 (s, 3H), 3.01 (t, 2H), 2.40 (s, 3H), 2.37 (s, 3H), I .67 (m, 2H), 1.0'.> (t, 3H). Anal.
(C"H,zO,) C,H.
6-Benzyl-2,3-dimethoxy-7-methyl-1-n-propylnaphthalene(19d). Compound 18b (800 mg, 2.47 mmol) in 25 mL of dry ether was cooled to 0°C under nitrogen. n-Butyllithium (3.0 mmol) was added as a solution in hexane. The mixture was stirred for I S min at 0°C, and then benzaldehyde (424 rng, 4 mmol) was added. The mixture was stirred at ambient temperature under nitrogen for I h. The reaction mixture was acidified.
and the organic layer was separated, washed with water and brine, and was dried over magnesium sulfate. After filtration, the ether 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 evaporated, and the residual oil was purified by silica column chromatography using dichloromethane to give 727 mg (2. I 7 mmol, 88% yield) of 19d as a crystalline solid, mp 83-85°C. 'H NMR:(ppm) 7.64 (s, 1 H), 7.39 (s, 1 I-I), 7.14-7.29 (m, SH), 6.95 (s, 1 H), 4.09 (s. 2H). 3.91 (s, 3H). 3.86 (s, 3H), 3.01 (t, 2H). 2.36 (s, 3H), 1.68 (m, 2H), I.OS (t, 3H).
Anal. (C,~H~~O,) C,1-I.
2,3-Dimethoxy-4,6-dimethyl-I-naphthoicacid (20a). Compound 17a (1.38 g, 6.38 mmol) was formylated by the general procedure for formylation to give 1.51 g (6.18 mmol, 97% yield) of solid aldehyde. 'H NMR:(ppm) 10.74 (s, 1H), 9.15 (d, 1 H), 7.72 (s, 1H), 7.40 (d, 1H), 4.04 (s, 3H), 3.87 (s, 3H), 2.62 (s, 3H), 2.50 (s, 3H).
Oxidation by the I S general procedure for oxidation gave a solid that was purified by silica column chromatography using dichloromethane to give 1.18 g (4.53 mmol, 72% yield) of 20a as white crystals. 'H NMR:(ppm) 8.07 (d, 1 H), 7.70 (s, 1 H), 7.35 (d, 1 H), 4.06 (s, 3H), 3.89 (s, 3H), 2.60 (s, 3H), 2.51 (s, 3H). Anal. (C,SH,~,O,,) C,H.
2, 3-Dimethoxy-4,6,7-trimethyl- I -naphthoic acid (20b). Compound I 9a (317 mg, 1.37 mmol) was formylated by the general procedure for formylation to give 318 mg ( 1.23 mmol, 89% yield) of solid aldehyde. 'H NMR:(ppm) 10.75 (s, I H), 9.05 (s, 1 H), 7.67 (s, IH), 4.0~ (s, 3H), 3.88 (s, 3H), 2.63 (s, 3H), 2.48 (s, 3H), 2.44 (s, 3H).
Oxidation by the general procedure for oxidation gave a solid that was purified by silica column chromatography using dichloromethaneto give 306 mg (1.12 mmol, 81% yield) of 20b as white crystals, mp 138-140°C. 'H NMR:(ppm) 8.08 (s, 1H), 7.68 (s, 1H), 4.07 (s, 3H), 3.89 (s, 3H), 2.61 (s, 3H), 2.44 (s, 6H). Anal. (C,6H,804) C,H.
7-Benzyl-2,3-dimethoxy-4,6-dimethyl-I-naphthoic acid (20c). Compound 19c (250 mg, 0.816 mmol) was formylated by the general procedure for formylation to give 250 mg (0.748 mmol, 92% yield) oFsolid aldeinyde. 'H NMR:{ppm) 10.74 (s, 1H), 9.16 (s. 1 H), 7.66 (s, 1 H), 7.18-7.10 (m. 5H), 4.13 (s, 2H). 4.02 (s, 3H), 3.84 (s, 3H), 2.59 (s, 3H). 2.31 (s. 3H). Oxidation by the general procedure for oxidation gave a solid that was purified by silica column chromatography using dichloromethane to give 207 mg (0.591 mmol, 79% yield) of 20c.
2,3-Dimethoxy-6-methyl-4-n-propyl-I-naphthoic acid (20d). Compound 17b (1.38 g, 6.38 mmol) was formylated by the gent°ral procedure for formylation to give 1.51 g (6.18 mmol, 97% yield) of solid aldehyde. Oxidation by the general procedure for oxidation gave a solid that was purified by silica chromatography using dichloromethane to give 1.18 g (4.53 mmol, 72% yield) of 20d as white crystals, mp 123-125°C. 'H
NMR:(ppm) 8.10 (d. 11-1), 7.71 (s. IH), 7.33 (d. IH), 4.06 (s, 3H}. 3.94 (s, 3H), 3.06 (t, 2H), 2.52 (s. 3H), 1.70 (m, 2H), I .09 (t, 3H).
Ana(. (C"I l,"O,) C.H.
2,3-Dimethoxy-6,7-dimethyl-4-n-propyl-1-naphthoicacid (20e). Compound 19c (317 mg, 1.38 mmol) was formylated by the general procedure for formylation to give 318 mg ( 1.23 mmol, 89% yield) of solid aldehyde. Oxidation by the general procedure for oxidation gave a solid that was purified by silica column chromatography to give 306 mg ( 1.12 mmol, 81 % yield) of 20e as white crystals.
7-Benzyl-2,3-dimethoxy-6-methyl-4-n-propyl-I-naphthoicacid (20f). Compound 19d (250 mg, 0.816 mmol) was formylated by the general procedure for formylation to give 250 mg (0.748 mmol, 92% yield) of solid aldehyde. Oxidation by the general procedure for oxidation gave 207 mg (0.591 mmol, 79% yield) of 20f as white crystals, mp 143-145°C. '> i NMR:(ppm) 8.03 (s, 1 H), 7.70 (s, 1 H), 7.27-7.10 (m, 5H). 4.15 (s, 2H), 4.05 (s, 3H), 3.93 (s, 3H), 3.05 (t, 2H), 2.34 (s, 3H), 1.69 (m, 2H), 1.08 (t, 3H). Anal.
(C~aH~c~O:~) C,H.

2,3-Dihydroxy-4,6-dimethyl-I-naphthoic acid (21a). Compound 20a (500 mg, 1.92 mmol} was demethylated by the general procedure for demethylation. The solvent was evaporated, and the product was recrystallized from ether/petroleum ether to give 350 mg ( 1.51 mmol, 79% yield) of 21 a as buff colored crystals. Anal. (C"H,,O~) C,H.
2,3-Dihydroxy-4,6,7-trimethyl-1-naphthoicacid (21b). Compound 20b (400 mg, 1.46 mmol) was demcthylated. and the product was purified as above to give 270 mg ( 1.10 mmol, 7S% yield) of 21 b as buff colored crystals.
7-Benzyl-2,3-dihydroxy-4,6-dimethyl-1-naphthoic acid (21 c). Compound 20c (634 mg, I .81 mmol} was demethylated, and the product was purified as above to give 420 mg (1.30 mmol, 72% yield) of 21c as buff colored crystals, mp 179-180°C. Anal.
(C~°H,gO~) C,H.
2,3-Dihydroxy-6-methyl-4-n-propyl-1-naphthoicacid (21d). Compound 20d (600 mg. 2.08 mmol) was demethylated, and the product was purified as above to give 390 mg ( 1.50 mmol. 72% yield) of 21 d as buff colored crystals, mp I 59-160°C. Anal. (C,SH~~,Oa) C.l-I .
2,3-Dihydroxy-6,7-dimethyl-4-n-propyl-I-naphthoicacid (21e). Compound 20e (550 mg, 1.82 mmol) was demethylated, and the product was purified as above to give 380 mg ( 1.27 mmol, 70% yield) of 21 a as buff colored crystals, mp 170-172°C. Anal.
(CIGH1804} CeH.
7-Benzyl-2,3-dihydroxy-6-methyl-4-n-propyl-1-naphthoicacid (21f). Compound 20f (634 mg, I .67 mmol) was demethylated, and the product was purified as above to give 417 mg ( 1. I 9 mmol, 71 % yield) of 21 f as buff colored crystals, mp 183-I
84°C.
'NMR:(ppm, DMSO-d~) 8.63 (s, 1H), 7.61 (s, IH), 7.27-7.12 (m, 5H), 4.01 (s, 2H), 2.97 (t. 2H), 2.28 (s, 3H), 1.57 (m, 2H), 0.99 (t, 3H). Anal. (C~,H"OQ) C,H.

2,3-Dimethoxy-6.7-dimethyl-1-(1-metlnylethyl)-naphthalen~(23a). Compound 22a ( 14) ( 1.40 g, 4.32 mmol) in 25 mL dry ether was cooled to 0°C under nitrogen. n-Butyllithium (6.0 mmol) was added as a solution in hexane. The mixture was stirred for 15 min at 0°C, and then methyl iodide (1.14 g, 8.0 mmol) was added. The mixture was stirred at ambient temperature under nitrogen for 1 h, then acidified, and the organic layer was separated, washed with water and brine ;end dried over magnesium sulfate.
After filtration, the ether was evaporated to give an oil which was purified by silica column chromatography using dichloromethane to give 892 mg (3.46 mmol, 80% yield) of 23a as huff colored crystals, mp 75-77"C. ' H NMR:(ppm) 7.85 (s, 1 H), 7.44 (s, I I-I), 6.93 (s, 11-I), 3.89 (m, I11). 3.88 (s. 3I-1), 3.85 (s, 3H), 2.39 (s, 3H), 2.35 (s, 3H), 1.51 (d, 6H). Anal.
(C "H"O=) C,H.
6-Benzyl-2,3-dimethoxy-7-methyl-1-(1-mcthylethyl)-naphthalene (23b}.
Compound 22a ( I .14 g, 3.86 mmol) in 25 rr~L of dry ether was cooled to 0°C under nitrogen. n-Butyllithium (4.5 mmol) was added as a solution in hexane. The mixture was stirred for 15 min at 0°C, and then benzaldehyde (1.06 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 faker layer was evaporated to give a white solid, mp 163-165°C. 'H NMR:(ppm) 7.84 (s, 1 H), 7.25-7.34 (m, 6H), 7.03 (s, 1 H), 6.08 (s. 1 t-I), 3.93 (s, 3I1), 3.91 (Ill, 11-I). 3.88 (s, 31-I), 2.34 (s. 3H), 2.25 (broad s, 1 H, which disappeared upon shaking with D,O), 1.50 (d, 6H). The alcohol 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 caher. The solvent was removed to afford a pale yellow solid that was recrystallized from ethyl acetate to give 1.04 g (3.1 1 mmol, 88% yield) of 23b as a white crystalline solid, mp 159-161°C. 'H
NMR:(ppm) 7.88 (s.
I H), 7.40 (s, 1 H), 7.32-7.15 (m, 5H), 6.96 (s, 1 H), 4.09 (s, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 2.37 (s, 3H), 1.50 (d, 6H). Anal. (Cz3Hz60,) C,H.

WO 98!49130 PCT/US98/08544 6-p-'f ri fl uoromethyl benzyl-2, 3-di methoxy-7-methyl- I -( I -methylethyl )-naphthalene (23c). Compound 22a ( 1.00 g, 3.09 mmol) in 25 mL of dry ether was cooled to 0°C.' cinder nitrogen. n-Butyllithium (4 mmol) was added as a solution in hexane. The mixture was stirred for 1 ~ min at 0"C. and then p-trilluoromethylbenzaldehyde(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. 'H NMR: (ppm) 7.90 (s, IH), 7.55 (d, 2H), 7.26 (d, 2H), 6.98 (s, 1 H). 4.1 S (s, 2H), 3.93 (s. 3H), 3.88 (s, 3H), 3.48 (rn, 1 H), 2.04 (s, 3H), I .50 (d, 6H).
2.3-Dimethoxy-6,7-dimethyl-4-(1-methylethyl)-I-naphthoic acid (24a).
Compound 2 3a (550 mg. 2.13 mmol) was formylated by the general procedure for formylation to give 506 mg ( I .77 mmoi, 83% yield) of solid aldehyde.
Oxidation by the general procedure for oxidation gave a solid that was purified by silica column chromatography using dichloromethane to give 450 mg ( 1.49 mmol, 84% yield) of 24a as white crystals, mp 191-193°C. 'H NMR:(ppm) 7.96 (s, IH), 7.93 (s, IH), 4.05 (s, 3H), 3.9~ (m, I H), 3.93 (s, 3H), 2.44 (brs, 6H), 1.52 (d, 6H). Anal. (C,8H,z04) C,H.
7-Benzyl-2.3-dimethoxy-6-methyl-4-(1-methylethyl)-naphthoic acid (24b).
Compound 23b (500 mg, 1.49 mmol) was formylated by the general procedure for formylation to give 446 mg (1.23 mmol, 82% yield) of solid aldehyde. 'H
NMR:(ppm) 10.7 (s, 1 H), 9.16 (s, I H), 7.93 (s, 1 H), 7.32-7.13 (m, SH), 4.16 (s, 2H), 4.02 (s, 3H), 3.95 (m, I H), 3.92 (s, 31-I), 2.35 (s, 3H), 1.52 (d, 6H). Oxidation by the general procedure described for oxidation gave a solid which was purified by silica column chromatography using dichloromethane to give 391 mg ( I .03 nrmol, 84% yield) of 24b as white crystals.
'H NMR:(ppm) 8.00 (s, 1 H), 7.94 (s, 1 H}, 7.24-7.1 I (m, SH), 4.1 S (s, 2H), 4.04 (s, 3H}, 3.95 (m, 1 H), 3.93 (s, 3H), 2.34 (s, 3H), 1.52 (d, 6H).
Anal. (C~~H,o04) C,HI.
S
2,3-Dimethoxy-6-methyl-4-( 1-methyletluyl)-7-p-trifluoromethylbenzyl- I -naphthoic acid (24c). Compound 23c (600 mg, 1.49 mmol) was formylated by the general procedure for formylation to give 526 mg (1.2='. mmol, 82% yield) of solid aldehyde. 'H
NMR:(ppm) 10.74 (s, I H), 9.18 (s, 1 H), 7.97 (s, 1 H), 7.49 (d, 2H), 7.22 (d, 2H), 4.19 (s.
2H). 4.02 (s. 311), 3.90 (m, 1 H), 3.91 (s, 3H), 2.30 (s, 3H}, 1.50 (d, 6H).
Oxidation by the general procedure for oxidation gave a solicj which was purified by silica column chromatography to give 458 mg ( I .02 mmol. 84% yield) of 24c as white crystals.
2,3-Dihydroxy-6,7-dimethyl-4-(1-methylethyl)-1-naphthoic acid (25a).
Compound 24a (450 mg, 1.49 mmol) was demethylated, and the product was recrystallized from ether/petroleum ether to gives 302 mg ( 1.10 mmol, 68%
yield) of 25a as buff colored crystals.
7-Benzyl-2,3-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoic acid (25b).
Compound 24b (652 mg, 1.72 mmol) wa.s demethylated, and the product was recrystallized from ether/petroleum ether to give 416 mg ( 1. I 9 mmol, 69%
yield) of 2~b as buff colored crystals. 'H NMR:(ppm) 8.61 (s, 1 H), 7.89 (s, 1 H}, 7.25-7-15 (m, SH).
6.25 (s, 1 H), 4.15 (s, 2H), 3.90 (m, I H), 2.36 (s, 3H), I .51 (d, 6H). Anal.
(C,ZH"04) C,H.
2~ 7-p-Trifluoromethylbenzyl-2,3-dihydrox.y-6-methyl-4-( I-methylethyl)-1-naphthoic acid (25c). Compound 24c (300 mg, 0.672 mmol) was demethylated, and the product was recrystallized from ether/petroleurn ether to give 196 mg (0.47 mmol, 70%
yield) of 25c as buff colored needles. 'H NMR:(ppm) 8.97 (s, 11-1), 7.87 (s, 1 H), 7.86 (d, 2H), 7.14 (d, 2H), 6.25 (s, 1 H), 4.20 (s, 2H), 3.86 (m, 1 H), 2.32 (s, 3H), I
.52 (d, 6H).
Anal. (C,~H,,F;04) C,H.

Compounds 25d, 25e, 25f, and 25g were made by procedures similar to that used to make compound 25c.
Purl fication of Recombinant Parasite Lactate Dehydrogenase.
Recombinant pLDH was produced in E. toll, similar to the procedure described by Bzik et al. in "Expression of Plasmodium falciparum Lactate Dehydrogenase in Escherida toll." in Mol. l3iochenz I'urusitol., 1993, 59, 155-166 and was purified by Cibacron blue affinity chromatography and chromatofocusing as described previously. Human LDH-H,, and LDH-M., were from Sigma.
Enzyme Assays and Kinetics.
LDH activity in the direction of NADH reduction of pyruvate to L-lactate was determined spectrophotometrically in pH 7.5 Tris buffer, 100 mM, containing 10 mM
pyruvate and 1 mM NADI-1, 25"C. 340nm, s = 6.:? mM-'em-'. Michaelis constants were determined by nonlinear regression analysis of initial rate data using the Enzfitter program (Elsevier-Biosoft). K; values were determined from double reciprocal plots by linear regression analysis.
Those hydroxynaphthoic acids of the present invention which have been structurally designed to occupy the substrate site such as 25c and 25d (see Figure 9) lead to a Pan-Active Site, potent and selective inhibition as shown in Figures 1 1 A, 11 B, 12A
and 12B. 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 11 A
and I 1 B show that inhibition of pLDH by 7-(p-trifluoromethylbenzyl)-8-deoxyhemigossyliracid (25c) is competitive against both cofactor and substrate. Fil;ures 12A and 12B show that inhibition of pLDH-M,, by 2,3-dihydroxy-6-methyl-7-(p-methylbenzyl)-4-(1-methylethyl)-1-naphthoic acid (25d) is competitive against both cofactor and substrate.
To test the Pan-Active Site inhibitor model for the compounds of the present invention, the binding of dihydroxynaphthoic; acids to pLDH and human LDH was examined using fluorescence techniques. The effects of ADP on the binding of inhibitors was compared with the effects of ADP on the binding of NADH to determine which part of the cofactor binding site was the target of ohe inhibitors. Representative results are shown in Figures 13A and 13B for the binding of compound 25c to pLDH. In Figure 13A
is shown the quenching of intrinsic protein fluorescence of pLDH by NADH in the presence or absence of ADP, demonstrating that the presence of ADP competes with the binding of NADH (as shown by the shift to the right of the quenching curve).
In figure 13B is shown the quenching of pLDH fluorescence by compound 25c in the presence and absence of ADI'. The presence of ADP actually enhances the binding of inhibitor (as shown by the shift to the left of the quenching c:urve). From these results, it is clear that compound 25c is not competing with the ADP part of the NADH binding site, suggesting that the compound 25c binds to the nicotinamidc: part of the NADH site. The same results were obtained with LDI-I-M4.
The results described in Table 1 of Figure 10 and in Figures 11A through 13B
support the conclusion that substituted dihydroxynaphthoicacids inhibit LDH
and pLDH
as competitive inhibitors of cofactor binding at the nicotinamide site and are consistent with the idea that inhibition may also involve the substrate site. These results support the concept of Pan Active Site inhibition.
Other studies with other dehydrogenases that contain the classic Rossman fold, in addition to LDH, were carried out to test that ini~ibition of dehydrogenases by substituted didhydroxynaphthoic acids is not limited to inhibition of LDH. A number of dehydrogenases were examined as shown in Fil;ures 14A (2,3-dihydroxy-6-methyl-7-(0-methylbenzyl)-4-(1-methylethyl)-1-naphthoic acid (25e) inhibition D-lactate dehydrogenase)- 1413 (2,3-dihydroxy-6-methyl-7--(m-methylbenzyl}-4-(1-methylethyl)-1-naphthoic acid (?sf1 inhibition of malate dehydrohenase), 14C (compound 25b inhibition of glucose 6-phosphate dehydrogenase) and 14D~ (compound 21 a inhibition of alcohol dehydrogenase).
Although the present invention has been fully described in conjunction with the preferred embodiment thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom.

Claims (27)

WHAT IS CLAIMED IS:

1. A compound comprising:

wherein:

A = H or OH
X = OH, a halogen, OR, NHR, NR'R" where R, R', and R" = H, C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, cycloalkyl, cycloalkenyl aryl or heterocyclic, substituted or unsubstituted; and R1, R2, R3, R4, R5 = H, C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or heterocyclic, substituted or unsubstituted, wherein R1 includes at least one methylene spacer through which R1 is attached to said compound.

2. The compound of claim 1, wherein A = H.

3. The compound of claim 1, wherein A = OH.

4. The compound of claim 1, wherein X = OH.

5. The compound of claim 1, wherein: R1, R2, R3, R4, R5 = H or C1-8 alkyl, C1-8 cycloalkyl, C2-8 alkenyl, or C2-8 alkynyl, unsubstituted, wherein R1 includes at least one methylene spacer through which R1 is attached to said compound.

6. The compound of claim 1, wherein A = OH, X = OH, R1 = CH3, R2 = H, R3 = CH3, R4 = H, and R5 = H.

7. The compound of claim 1, wherein A = OH, X = OH, R1 = CH3, R2 = H, R3 = CH3, R4 = CH3, and R5 = H.

8. The compound of claim 1, wherein A = OH, X = OH, R1 = CH2CH2CH3, R2 = H, R3 = CH3, R4 = H, and R5 = H.

9. The compound of claim 1, wherein A = OH, X = OH, R1 = CH2CH2CH3, R2 = H, R3 = CH3, R4 = CH3, and R5 = H.

10. The compound of claim 1, wherein A = OH, X = OH, R1 = CH2CH2CH3, R2 = H, R3 = CH3, R4 = CH2C6H5, and R5 = H.

11. The compound of claim 1, wherein A = OH, X = OH, R1 = CH(CH3)2, R2 = H, R3 = CH3, R4 = CH3, and R5 = H.

12. The compound of claim 1, wherein A = OH, X = OH, R1 = CH(CH3)2, R2 = H, R3 = CH3, R4 = CH2C6H5, and R5 = H.

13. The compound of claim 1, comprising 7-p-Trifluoromethylbenzyl-2,3-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoicacid.

14. The compound of claim 1, comprising 2,3-dihydroxy-6-methyl-7-(o-methylbenzyl)-4-(1-methylethyl)-1-naphthoicacid.

15. The compound of claim 1, comprising 2.3-dihydroxy-6-methyl-7-(m-methylbenzyl)-4-(1-methylethyl)-1-naphthoic-acid.

16. The compound of claim 1, comprising 2,3-dihydroxy-6-methyl-7-(p-methylbenzyl)-4-(1-methylethyl)-1-naphthoicacid.

17. The compound of claim 1, comprising 7-p-chlorobenzyl-2,3-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoicacid.

18. A method for making a compound comprising:

wherein:
A = H or OH; and R1, R2, R3, R4, R5, H, C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or heterocyclic, substituted or unsubstituted, wherein R1 includes at least one methylene spacer through which R1 is attached to said compound said method comprising the steps of:
reacting the following compound:

with boron tribromide to form the following compound:

wherein A, is CH3O or H.

19. The method of claim 18 further comprising the steps of:
reacting the following compound:
with titanium tetrachloride and dichloromethyl methyl ether to form the following compound:
37~

and reacting 1j with sodium hypochlorite to form compound 1k.

20. The method of claim 19 further comprising the steps of:
reacting the following compound with a Grignard reagent comprising R5MgBr to form compound 1i.

21. The method of claim 19, further comprising the steps of:
reacting the following compound:

with the following compound:

to form the following compound:

saponifying compound 1c to form the following compound::

hydrogenolyzing and reducing 1d to form a hydrogenolyzed and reduced carboxylic acid;

cyclizing the hydrogenolyzed and reduced carboxylic acid to form the following compound:

reducing compound 1e to form an intermediate alcohol:
dehydrating the intermediate alcohol to form the following compound:

reacting compound 4f with bromine to form a dibromide;
dehydrohalogenating the dibromide to form the following compound:
dehydrogenating compound 1g to form the following compound:
~~
reacting compound 1h with n-butyl lithium and benzaldehyde to form a benzylic alcohol; and hydrogenolyzing the benzyl alcohol to form compound 1i.

22. The method of claim 21, further comprising the steps of:
reacting the following compound:
with Grignard reagent to form the following compound:
hydrogenolysizing compound 2b to form the following compound:

and brominating compound 2c to form compound 1a, wherein R a and R b are each one of H, C1-8 alkyl, C2-8 alkenyl, or C2-8 alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or heterocyclic, substituted or unsubstituted.

42 The method of claim 21, further comprising the steps of:
reacting the following compound:

with Grignard reagent to form the following compound::

hydrogenolyzing compound 2b to form the following compound:

and brominating compound 2g to form compound 1a wherein R c is H, C1-8 alkyl, C1-8 alkenyl, or C2-8 alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or heterocyclic, substituted or unsubstituted.

24. The method of claim 21, further comprising the steps of:

reacting R3COCHO with methanol to turn R3COCH(OCH3)2;
reacting R3COCH(OCH3)2 with CH3CH2OCOCH2PO(OEt)2 to form (CH3O)2CHCR3CHCOOEt; and hydrolyzing (CH3O)2CHCR3CHCOOEt to form compound 1b.

25. The method of claim 19, further comprising the steps of:
reacting the following compound:

with the following compound:

to form the following compound:

saponifying compound 4c to form the following compound:

hydrogenolyzing and reducing 4d to form a hydrogenolyzed and reduced carboxylic acid;
cyclizing the hydrogenolyzed and reduced carboxylic acid to form the following compound:

reducing compound 4e to form an intermediate alcohol;

dehydrating the intermediate alcohol to form the following compound:

reacting compound 4f with bromine to form a dibromide;
dehydrohalogenating the dibromide to form the following compound:

dehydrogenating compound 4g to form the following compound:

reacting compound 4h with n-butyl lithium and benzaldehyde to form a benzylic alcohol; and hydrogenolyzing the benzyl alcohol to form compound 1i.

26. The method of claim 18, wherein A1 is CH3O and A is OH.

27. The method of claim 18, wherein A1 and A are each H.