Selective inhibitors of human lactate dehydrogenases and lactate dehydrogenase from the malarial parasite Plasmodium falciparum

Article abstract of DOI:10.1021/jm980334n

Derivatives of the sesquiterpene 8-deoxyhemigossylic acid (2,3- dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoic acid) were synthesized that contained altered alkyl groups in the 4-position and contained alkyl or aralkyl groups in the 7-position. These su

Full text of DOI:10.1021/jm980334n

J . Med. Chem. 1998, 41, 3879-3887
3879
Selective In h ibitor s of Hu m a n La cta te Deh yd r ogen a ses a n d La cta te
Deh yd r ogen a se fr om th e Ma la r ia l P a r a site P la sm od iu m fa lcipa r u m
Lorraine M. Deck,† Robert E. Royer,‡ Brian B. Chamblee,†,‡ Valerie M. Hernandez,† Richard R. Malone,†
J ose E. Torres,‡ Lucy A. Hunsaker,‡ Robert C. Piper,§ Michael T. Makler,| and David L. Vander J agt*,‡
Departments of Chemistry and of Biochemistry and Molecular Biology, University of New Mexico School of Medicine,
Albuquerque, New Mexico 87131, Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, and Department
of Pathology, Oregon Health Sciences University and Veterans Administration Medical Center, Portland, Oregon 97201
Received J une 1, 1998
Derivatives of the sesquiterpene 8-deoxyhemigossylic acid (2,3-dihydroxy-6-methyl-4-(1-
methylethyl)-1-naphthoic acid) were synthesized that contained altered alkyl groups in the
4-position and contained alkyl or aralkyl groups in the 7-position. These substituted
dihydroxynaphthoic acids are selective inhibitors of human lactate dehydrogenase-H (LDH-
H) and LDH-M and of lactate dehydrogenase from the malarial parasite Plasmodium falciparum
(pLDH). All inhibitors are competitive with the binding of NADH. Selectivity for LDH-H,
LDH-M, or pLDH is strongly dependent upon the groups that are in the 4- and 7-positions of
the dihydroxynaphthoic acid backbone. Dissociation constants as low as 50 nM were observed,
with selectivity as high as 400-fold.
Gossypol (1), a polyphenolic, binaphthyl disesquiter-
pene found in cottonseed, is an abundant component of
cottonseed oil that exhibits a variety of biological
activities, including antispermatogenic, anticancer, an-
tiparasitic, and antiviral activity.1-9 We demonstrated
previously that gossypol exhibits antimalarial activity
against both chloroquine-sensitive and chloroquine-
resistant strains of Plasmodium falciparum, with IC50
In the present study, we have addressed the question
values about 10 µM.10 Due to the presence of aldehyde
of whether gossypol-related inhibitors can be developed
functional groups, gossypol is toxic. It has been an open
that are selective for lactate dehydrogenase from the
malarial parasite P. falciparum compared to human
question of whether all of the biological activities of
gossypol result from the toxicity associated with the
lactate dehydrogenases LDH-H (LDH-B4) and LDH-M
aldehyde functional groups. However, we demonstrated
(LDH-A4). Although malarial parasites possess mito-
that derivatives of gossypol in which the aldehyde
chondria, they do not have a functional TCA cycle and
groups were converted into other functional groups
depend almost exclusively upon glycolysis for ATP
retained biological activities, including antimalarial
production, which suggests that any of the glycolytic
activity11,12 and antiviral activity.13 Other derivatives
enzymes of P. falciparum represent potential drug
are potent inhibitors of aldose reductase, an enzyme
targets. The amino acid sequence of malarial lactate
implicated in the etiology of diabetic complications.9 The
dehydrogenase (pLDH) revealed many residues at the
synthesis of derivatives of gossypol is limited by the
presence of hydroxyl groups at the 1,1′ (peri) positions.
These phenolic groups complicate the chemistry of
functional groups derived from the aldehydes at the 8,8′-
active site that are unique to pLDH compared to any
other known LDH, including human LDH-M and LDH-
H.16 Therefore, pLDH appears to be an attractive target
for development of antimalarial drugs.
positions. To circumvent this, we recently developed a
Previous studies of the antimalarial activities of
derivatives of gossypol pointed to the cofactor binding
site of lactate dehydrogenase as the target of these
compounds.12 It is not commonly assumed that a
cofactor binding site may be a good target for drug
development, although there have been recent studies
of structure-based drug design targeted at the NAD+
binding site of glyceraldehyde-3-phosphate dehydro-
genase from Trypanosomes.15 In a preliminary study
of the inhibition of pLDH by gossypol-related com-
pounds, derivatives of 8-deoxyhemigossylic acid (3) were
shown to be promising as selective inhibitors of pLDH.17
Here we describe the synthesis and inhibitory properties
de novo synthetic scheme for the synthesis of 1,1′-
dideoxygossypol (2).14 This versatile synthetic scheme
affords the opportunity to deviate from the strict gos-
sypol backbone and to prepare a wide range of gossypol-
related compounds.
* Address correspondence to: Dr. David L. Vander J agt. Phone: 505-
272-5788. E-mail: dlvanderjagt@salud.unm.edu.
† Department of Chemistry, University of New Mexico School of
Medicine.
‡ Department of Biochemistry and Molecular Biology, University of
New Mexico School of Medicine.
§ University of Oregon.
| Oregon Health Sciences University and Veterans Administration
Medical Center.
S0022-2623(98)00334-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/27/1998

3880 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Deck et al.
Sch em e 1
Sch em e 2
of a series of compounds structurally related to 8-deoxy-
hemigossylic acid.
Ch em ica l Syn th eses
The synthetic schemes were designed to provide a
number of compounds that deviate from 8-deoxyhemi-
gossylic acid as follows: (1) the 4-isopropyl group in 3
is replaced by methyl or n-propyl (3a , R1) to test the
role of alkyl groups at the 4-position and (2) hydrogen,
methyl, or benzyl groups are placed at position 7 (3a ,
R2), which is the coupling position in the formation of
disesquiterpenes related to gossypol. The syntheses of
compounds in the 3a series are outlined in Schemes 1
and 2 (compounds 11a -f and 15a -c).
Synthetic Scheme 1 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 1-bromo-3,4-dimethoxy-2-methyl-
benzene (4a ) and 1-bromo-3,4-dimethoxy-2-n-propyl-
benzene (4b) with ethyl 3-methyl-4-oxobut-2-enoate.
These precursors are readily prepared from commer-
cially available starting materials using a procedure
described by Royer et al.14 Because the esters were
difficult to reduce they were saponified, and the acids
were hydrogenolyzed and reduced. The resulting car-
boxylic acids 5a ,b were cyclized with polyphosphoric
ester to give tetralones 6a ,b. The ketone functional
groups of 6a ,b were reduced with sodium borohydride

Selective Inhibitors of Lactate Dehydrogenases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3881
and the intermediate alcohols dehydrated on acidic
workup to form alkenes which were dehydrogenated
with DDQ to form the naphthalenes 7a ,b. 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 8a ,b. Compounds
8a ,b were reacted with n-butyllithium and benzalde-
hyde to form the benzylic alcohols which were hydro-
genolyzed with Pd/C in ethanol to form 9b,d . Formation
of 9a ,c was accomplished by reaction of 8a ,b with
n-butyllithium and methyl iodide. Carbonyl groups
were introduced to compounds 7a ,b, and 9a -d by
formylation with titanium tetrachloride and dichloro-
methyl methyl ether. Oxidation of the aldehyde groups
with sodium hypochlorite formed the carboxylic acids
10a -f. The methyl groups were removed from the
phenolic ethers with boron tribromide to form com-
pounds 11a -f.
The second series of compounds related to 3 addressed
the question of 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. For the 4-methyl derivatives 11a -c, (Table 1), there
is selectivity for pLDH and LDH-M compared to LDH-
H, and affinity increases with the introduction of groups
at the 7-position for all three LDHs, especially for LDH-
M. For the 4-n-propyl derivatives 11d -f (Table 1), the
presence of the n-propyl group at the 4-position has a
marked effect both on affinity and on selectivity. Com-
pound 11e exhibits 190-fold selectivity for LDH-M
compared to LDH-H. The most potent inhibitor of the
compounds tested in this study is 11f which inhibits
LDH-M, KD ) 50 nM. Thus both the 4-position and the
7-position represent sites for modification of the dihy-
droxynaphthoic acid backbone in the development of
LDH inhibitors.
The syntheses of 15a -c are outlined in Scheme 2.
The precursor compound 12 was prepared from 2-iso-
propylphenol using procedures described by Royer et
al.14 The transformations to form compounds 13a -c,
14a -c, and 15a -c were accomplished using the same
procedures used for the corresponding steps in Scheme
1.
Discu ssion
The mechanism of NADH reduction of pyruvate to
lactate catalyzed by LDH is thought to involve direct
hydride transfer of the pro-R (HA) C4-hydrogen from the
reduced nicotinamide ring of NADH to the ketone of
pyruvate to form L-lactate.18 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 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 carboxylate group of pyruvate.19
These catalytic residues are conserved in all LDHs.
The unique structural features of pLDH that separate
it from human LDH as well as from all other known
LDHs involve residues at both the cofactor and sub-
strate sites.16 Residues 98-109 of human LDH-H and
LDH-M (98AGVRQQEGESRL) define the substrate speci-
ficity loop.19 This sequence is quite highly conserved
in all other known LDHs, except pLDH where not only
the sequence differs but there is also a 5-amino acid
insert from residues 104 to 108 (98AGFTKAPGKSD-
KEWNRD) which forms an extended specificity loop.
The recent crystal structure of the pLDH-NADH-
oxamate ternary complex described a closed loop struc-
ture with a cleft at the active site which is not present
in other LDHs.20 Nevertheless, despite these unique
structural features of pLDH, this LDH exhibits high
specificity for pyruvate.17 Additional residues that are
conserved in other LDHs but differ in pLDH include
S163, I250, and T246. In most LDHs, the nitrogen of
the carboxamide group of NADH is H-bonded to the
oxygen of S163, whereas in pLDH residue 163 is leucine.
In addition, I250 normally provides a hydrophobic side
chain 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
In h ibition of p LDH, LDH-H, a n d LDH-M by
Der iva tives of 8-Deoxyh em igossylic Acid
The inhibition of pLDH, LDH-H, and LDH-M by
8-deoxyhemigossylic acid (3), the reference compound
for these studies, is summarized in Table 1. Compound
3 is nonselective in its inhibiton of pLDH and LDH-M,
with dissociation constants in the low-micromolar range.
By comparison, the dissociation constant for inhibition
of LDH-H is 30-fold higher than for LDH-M. The dimer
of 3, namely, 1,1′-dideoxygossylic acid, was shown
previously17 to be nonselective in its inhibition of these
three LDH with dissociation constants about 1 µM. Thus
the dimerization of 3 only affects inhibition of LDH-H,
raising the question of whether only half of the gossypol
backbone is involved in the inhibition of pLDH and
LDH-M by disesquiterpenes related to gossypol. The
inhibition of these three LDHs by 3 is competitive with
the binding of NADH, which is also consistent with
previous observations.17
The first series of derivatives of 3 addressed the
question of 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 3 and its
dimer against pLDH and LDH-M. Compounds 15a ,b
(Table 1) 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. However, introduction of a substi-
tuted benzyl group reverses this pattern; the p-(trifluo-
romethyl)benzyl derivative 15c (Table 1) is more active
against pLDH (Ki ) 0.2 µM) and less active against both
human LDH-M and LDH-H. Compound 15c thus
shows selectivity for pLDH.

3882 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Deck et al.
Ta ble 1. Inhibition of Human Lactate Dehydrogenase (LDH-H and LDH-M) and Malarial Parasite P. falciparum Lactate
Dehydrogenase (pLDH) by Dihydroxynaphthoic Acids
a
From ref 17. All data at pH 7.5, 25 °C.
ternary complex of most LDHs, is replaced by proline
in pLDH. All of these unique features of pLDH 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 re-
lated to 8-deoxyhemigossylic acid (3) can be developed
that are selective inhibitors of pLDH compared to

Selective Inhibitors of Lactate Dehydrogenases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3883
4-(3,4-Dim et h oxy-2-m et h ylp h en yl)-3-m et h ylb u t a n oic
Acid (5a ). Compound 4a (12.0 g, 51.9 mmol) was added to
magnesium turnings (1.26 g, 52.1 mmol) in 100 mL of dry THF
in a 250-mL ground glass Erlenmeyer flask equipped with a
reflux condenser and magnetic stirrer. The mixture was
refluxed with stirring for 1 h, cooled to 0 °C, and added
dropwise to 3-methyl-4-oxobut-2-enoate (7.39 g, 52.1 mmol) in
25 mL of dry THF at 0 °C. The mixture was stirred at ambient
temperature for 1 h and poured onto 100 g of ice containing
25 mL of 6 M HCl. 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 of ice containing 25
mL of 6 M HCl and 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 white crystalline acid:
mp 174-176 °C. 1H NMR: 6.69 (d, 1H), 6.74 (d, 1H), 6.32 (s,
1H), 5.32 (s, 1H), 3.86 (s, 3H), 3.79 (s, 3H), 2.34 (s, 3H), 2.00
(s, 3H), 1.60 (s, 1H). Anal. (C14H18O5) 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 of 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) to give 7.5 g (29.7 mmol,
79% yield) of 5a as an amber oil which crystallized on standing
to form colorless crystals: mp 84-86 °C. 1H NMR: 6.78 (d,
1H), 6.67 (d, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 2.62-2.33 (m, 5H),
2.22 (s, 3H), 0.992 (d, 3H). Anal. (C14H20O4) C,H.
4-(3,4-Dim et h oxy-2-n -p r op ylp h en yl)-3-m et h ylb u t a n -
oic Acid (5b). Compound 4b (10.0 g, 38.4 mmol) and ethyl
bromide (0.6 g, 5.5 mmol) were added to magnesium turnings
(1.2 g, 49.4 mmol) in 100 mL of dry THF in a 250-mL ground
glass Erlenmyer flask and refluxed for 1 h with stirring. After
cooling, the mixture was added dropwise to 3-methyl-4-oxobut-
2-enoate (6.90 g, 48.5 mmol) in 25 mL of dry THF at 0 °C. The
mixture was stirred at ambient temperature for 1 h and poured
onto 100 g of ice containing 25 mL of 6 M HCl. 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 HCl, and
stirred. The semisolid was extracted with ether, washed with
water and brine, and dried over magnesium sulfate. Filtration
and evaporation of the ether gave 9.20 g (31.2 mmol, 95% yield)
of acid as a viscous oil. The acid (9.20 g, 31.2 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 of hydrogen
pressure at 60 °C for 24 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 (180 °C, 1 Torr) to give 7.46 g
(26.6 mmol, 85% yield) of 5b as a pale-yellow oil. 1H NMR:
6.77 (d, 1H), 6.68 (d, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 2.74-2.08
(m, 7H), 1.50 (sex, 2H), 0.96 (t, 3H), 0.94 (d, 3H). Anal.
(C16H24O4) C,H.
human LDH and that these inhibitors appear to be
competitive with cofactor binding. Compound 15c with
a p-(trifluoromethyl)benzyl moiety at the 7-position of
3 shows 75- and 400-fold selectivity for pLDH over
LDH-M and LDH-H, respectively. Surprisingly, how-
ever, some of these substituted dihydroxynaphthoic
acids are highly selective for LDH-M over LDH-H,
despite the high sequence homologies of these two
human LDHs. Generally, these inhibitors show higher
affinities for LDH-M compared to LDH-H, with selec-
tivities ranging from 5- to 190-fold.
The compounds in Table 1 are competitive inhibitors
of cofactor binding. Inhibition with respect to substrate
binding is generally mixed but sometimes appears
competitive. These kinetic observations raise the ques-
tion of whether inhibition of LDH by some of the
substituted dihydroxynaphthoic acids involves complex-
ation at both the cofactor and substrate binding sites
and whether this can be exploited to develop selective
dehydrogenase inhibitors. However, the kinetic pattern
of competitive inhibition against both cofactor and
substrate is problematic. In a recent study of the
inhibition of human aldehyde dehydrogenase (ALDH1
and ALDH2) by the isoflavone prunetin, Sheikh and
Weiner demonstrated that this inhibitor appears to be
competitive against both NAD+ and aldehyde sub-
strate.21 However, this was shown to result from
allosteric inhibition of ALDH. A detailed study of the
binding of substituted dihydroxynaphthoic acids to LDH
will be required to determine whether these inhibitors
bind to both the cofactor and substrate sites of LDH or
to a portion of these two sites.
Exp er im en ta l Section
Ch em ica l Syn th esis. Reagent quality solvents were used
without further purification. THF and ether were distilled
from calcium hydride. 1-Bromo-3,4-dimethoxy-2-methylben-
zene (4a ) and 1-bromo-3,4-dimethoxy-2-n-propylbenzene (4b)
were synthesized from o-cresol and 2-n-propylphenol, respec-
tively, according to published procedures.14 Melting points
were determined with a VWR Scientific Electrothermal capil-
lary melting point apparatus and are uncorrected. NMR
spectra were recorded on a Bruker AC250 NMR spectrometer
in CDCl3, unless otherwise stated. Chemical shifts are in ppm
(δ) relative to TMS.
Gen er a l P r oced u r e for Rem ovin g th e Meth yl Gr ou p s
fr om P h en olic Meth yl Eth er s w ith Bor on Tr ibr om id e.
The procedure for removal of methyl groups was identical to
that described by us previously.14
Gen er a l P r oced u r e for F or m yla tion a n d Oxid a tion To
F or m Ca r boxylic Acid s. A reaction mixture with 1 mmol
of the compound to be formylated and 2.6 mmol of dichloro-
methyl 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
containing 10 mL of 6 M HCl, and the organic layer was
washed with water and brine and dried over magnesium
sulfate. Filtration and evaporation of the solvent gave a crude
aldehyde 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 of
water. The reaction mixture was stirred at ambient temper-
ature 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. Filtration and evaporation of the solvent gave a crude
carboxylic acid.
3,4-Dih yd r o-6,7-d im et h oxy-3,5-d im et h yl-1(2H )-n a p h -
th a len on e (6a ). Compound 5a (7.0 g, 27.7 mmol) was added
to 35 g of polyphosphoric ester in 100 mL of methylene chloride
and refluxed for 1 h. The reaction mixture was poured onto
ice and 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 6a as white plates: mp
106-108 °C. 1H NMR: 7.48 (s, 1H), 3.89 (s, 3H), 3.84 (s, 3H),
2.95-2.28 (m, 5H), 2.22 (s, 3H), 1.18 (d, 3H). Anal. (C14H18O3)
C,H.
3,4-Dih yd r o-6,7-d im eth oxy-3-m eth yl-5-n -p r op yl-1(2H)-
n a p h th a len on e (6b). Compound 5b (7.0 g, 24.9 mmol) was

3884 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Deck et al.
added to 35 g of polyphosphoric ester 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 was
evaporated, and the residual oil was chromatographed on silica
gel using ethyl acetate/hexane to give 5.63 g (21.4 mmol, 86%
yield) of 6b as white crystals: mp 69-70 °C. 1H NMR: 7.49
(s, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.00-2.23 (m, 7H), 1.50 (mult,
2H), 1.15 (d, 3H), 1.01 (t, 3H). Anal. (C16H22O3) C,H.
2,3-Dim eth oxy-1,7-d im eth yln a p h th a len e (7a ). Com-
pound 6a (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. 1H NMR: 6.50 (s, 1H),
6.31 (m, H), 5.81 (mult, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 2.84-
2.23 (mult, 3H), 2.19 (s, 3H), 1.10 (d, 3H). Anal. (C14H18O2)
C,H.
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 gel
column chromatography with dichloromethane. Removal of
solvent provided 1.66 g of 7a (7.67 mmol, 86% yield) as
colorless crystals: mp 68-69 °C. 1H NMR: 7.63 (s, 1H), 7.59
(d, 1H), 7.21 (d, 1H), 6.99 (s, 1H), 3.93 (s, 3H), 3.83 (s, 3H),
2.56 (s, 3H), 2.49 (s, 3H). Anal. (C14H16O2) C,H.
(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 residual oil was purified by
silica gel column chromatography using dichloromethane to
give 1.24 g (4.21 mmol, 83% yield) of 8a as buff-colored
crystals: mp 96-97 °C. 1H NMR: 7.89 (s, 1H), 7.67 (s, 1H),
6.89 (s, 1H), 3.94 (s, 3H), 3.84 (s, 3H), 2.54 (s, 6H). Anal.
(C14H15BrO2) C,H.
6-Br om o-2,3-d im eth oxy-7-m eth yl-1-n -p r op yln a p h th a -
len e (8b). Compound 6b (3.5 g, 13.4 mmol) was reduced with
sodium borohydride as described in the synthesis of 7b. The
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 15 min. The solvent was evaporated, and the residue
was taken up in 15 mL of DMF and warmed to 60-70 °C for
1 h. 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, 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (857 mg, 3.78 mmol) was added slowly 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
residual oil was purified by silica gel column chromatography
using dichloromethane to give 1.04 g (3.21 mmol, 85% yield)
of 8b as colorless crystals: mp 64-66 °C. 1H NMR: 7.90 (s,
1H), 7.69 (s, 1H), 6.91 (s, 1H), 3.94 (s, 3H), 3.87 (s, 3H), 2.99
(t, 2H), 2.54 (s, 3H), 1.66 (m, 2H), 1.05 (t, 3H). Anal. (C16H19
BrO2) C,H.
-
2,3-Dim eth oxy-1,6,7-tr im eth yln a p h th a len e (9a ). Com-
pound 8a (646 mg, 2.19 mmol) in 25 mL 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 1 h. The reaction mixture was poured onto 10 g
of ice containing 2 mL of HCl, 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 gel column chro-
matography using dichloromethane to give 426 mg (1.84 mmol,
84% yield) of 9a as buff crystals: mp 59-60 °C. 1H NMR:
7.59 (s, 1H), 7.44 (s, 1H), 6.93 (s, 1H), 3.93 (s, 3H), 3.83 (s,
3H), 2.56 (s, 3H), 2.41 (s, 3H), 2.38 (s, 3H). Anal. (C15H18O2)
C,H.
6-Ben zyl-2,3-d im eth oxy-1,7-d im eth yln a p h th a len e (9b).
Compound 8a (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
15 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 HCl and stirred for 1 h. The mixture was
filtered to give a buff solid: mp 174-175 °C. 1H NMR: 7.85
(s, 1H), 7.60 (s, 1H), 7.34-7.25 (m, 5H), 7.03 (s, 1H), 6.09 (s,
1H), 3.94 (s, 3H), 3.84 (s, 3H), 2.55 (s, 3H), 2.34 (s, 3H), 2.28
(br s, 1H, disappears upon shaking with D2O).
2,3-Dim eth oxy-7-m eth yl-1-n -p r op yln a p h th a len e (7b).
Compound 6b (5.00 g, 19.0 mmol) was dissolved in 20 mL of
2-propanol, sodium borohydride (1.08 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 HCl 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 evapo-
ration, and the residue was distilled bulb-to-bulb (190 °C, 1
Torr) to give 4.46 g (18.0 mmol, 95% yield) of an alkene. 1H
NMR: 6.51 (s, 1H), 6.31 (dd,1H), 5.80 (dd, 1H), 3.81 (s, 3H),
3.80 (s, 3H), 2.86-2.35 (m, 5H), 1.50 (m, 2H), 1.10 (d, 3H),
0.99 (t, 3H).
The alkene (4.46 g, 18.0 mmol) was dissolved in 25 mL of
benzene, was treated while stirring with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (4.10 g, 18.0 mmol), and was stirred
for an additional 2 h. The reaction mixture was filtered
through a short column of alumina and washed with benzene.
The solvent was evaporated, and the residual oil was purified
by silica gel column chromatography using dichloromethane
to give 4.24 g (17.4 mmol, 96% yield) of 7b as buff-colored
crystals: mp 48-49 °C. 1H NMR: 7.64 (s, 1H), 7.60 (d, 1H),
7.21 (d, 1H), 7.01 (s, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 3.01 (t,
2H), 2.50 (s, 3H), 1.67 (m, 2H), 1.06 (t, 3H). Anal. (C16H20O2)
C,H.
6-Br om o-2,3-d im eth oxy-1,7-d im eth yln a p h th a len e (8a ).
Compound 6a (4.5 g, 19.2 mmol) was converted into the alkene
as described in the synthesis of 7a . The alkene (1.48 g, 6.78
mmol) was dissolved in 100 mL of dry dichloromethane, and
bromine (1.08 g, 6.78 mmol) in 5 mL of 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 1 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 75-76
°C. 1H NMR: 6.65 (s, 1H), 6.43 (s, 1H), 3.82 (s, 3H), 3.77 (s,
3H), 3.03-2.62 (m, 3H), 2.18 (s, 3H), 1.11 (d, 3H). Anal.
(C14H17BrO2) C,H.
The alcohol was dissolved in ethanol and hydrogenated on
a Parr hydrogenator with 10% palladium on carbon and 60
psi of 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 gel column chromatog-
raphy using dichloromethane to give 522 mg (1.70 mmol, 88%
yield) of 9b as white crystals: mp 85-86 °C. 1H NMR: 7.63
(s, 1H), 7.41 (s, 1H), 7.30-7.14 (m, 5H), 6.95 (s, 1H), 4.11 (s,
2H), 3.93 (s, 3H), 3.84 (s, 3H), 2.56 (s, 3H), 2.36 (s, 3H). Anal.
(C21H22O2) C,H.
2,3-Dim et h oxy-6,7-d im et h yl-1-n -p r op yln a p h t h a len e
(9c). Compound 8b (700 mg, 2.16 mmol) in 25 mL of dry ether
was cooled to 0 °C under nitrogen. n-Butyllithium (3.0 mmol)
The vinylic bromide (1.51 g, 5.08 mmol) was dissolved in
25 mL of benzene, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone

Selective Inhibitors of Lactate Dehydrogenases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3885
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 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 was evaporated to give an oil which was purified by silica
gel column chromatography to give 446 mg (1.73 mmol, 80%
yield) of 9c as a colorless oil. 1H NMR: 7.89 (s, 1H), 7.44 (s,
1H), 6.93 (s, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.01 (t, 2H), 2.40
(s, 3H), 2.37 (s, 3H), 1.67 (m, 2H), 1.05 (t, 3H). Anal.
(C17H22O2) C,H.
6-Ben zyl-2,3-d im eth oxy-7-m eth yl-1-n -p r op yln a p h th a -
len e (9d ). Compound 8b (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 15 min at 0 °C, and then benzaldehyde (424
mg, 4 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 was evaporated to give a semisolid which
was dissolved in ethanol and hydrogenated on a Parr hydro-
genator with 10% palladium on carbon and 60 psi of 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 gel column chromatography
using dichloromethane to give 727 mg (2.17 mmol, 88% yield)
of 9d as a white crystalline solid: mp 83-85 °C. 1H NMR:
7.64 (s, 1H), 7.39 (s, 1H), 7.14-7.29 (m, 5H), 6.95 (s, 1H), 4.09
(s, 2H), 3.91 (s, 3H), 3.86 (s, 3H), 3.01 (t, 2H), 2.36 (s, 3H),
1.68 (m, 2H), 1.05 (t, 3H). Anal. (C23H26O2) C,H.
procedure for oxidation gave a solid that was purified by silica
gel column chromatography using dichloromethane to give 1.18
g (4.08 mmol, 72% yield) of 10d as off-white crystals: mp 123-
125 °C. 1H NMR: 8.10 (d, 1H), 7.71 (s, 1H), 7.33 (d, 1H), 4.06
(s, 3H), 3.94 (s, 3H), 3.06 (t, 2H), 2.52 (s, 3H), 1.70 (m, 2H),
1.09 (t, 3H). Anal. (C17H20O4) C,H.
2,3-Dim et h oxy-6,7-d im et h yl-4-n -p r op yl-1-n a p h t h oic
Acid (10e). Compound 9c (317 mg, 1.22 mmol) was formyl-
ated by the general procedure for formylation to give 318 mg
(1.11 mmol, 91% yield) of solid aldehyde. Oxidation by the
general procedure for oxidation gave a solid that was purified
by silica gel column chromatography to give 306 mg (1.01
mmol, 80% yield) of 10e as buff-colored crystals: mp 144-
145 °C. 1H NMR (DMSO-d6): 7.92 (s, 1H), 7.68 (s, 1H), 4.05
(s, 3H), 3.90 (s, 3H), 3.05 (t, 2H), 2.43 (s, 6H), 1.68 (m, 2H),
1.08 (t, 3H). Anal. (C18H22O4) C,H.
7-Ben zyl-2,3-d im et h oxy-6-m et h yl-4-n -p r op yl-1-n a p h -
th oic Acid (10f). Compound 9d (250 mg, 0.75 mmol) was
formylated by the general procedure for formylation to give
250 mg (0.69 mmol, 92% yield) of solid aldehyde. Oxidation
by the general procedure for oxidation gave 207 mg (0.55 mmol,
73% yield) of 10f as off-white crystals: mp 143-145 °C. 1H
NMR: 8.03 (s, 1H), 7.70 (s, 1H), 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. (C24H26O4) C,H.
2,3-Dih yd r oxy-4,6-d im et h yl-1-n a p h t h oic Acid (11a ).
Compound 10a (500 mg, 1.92 mmol) was demethylated by the
general procedure for demethylation.14 The solvent was
evaporated, and the product was recrystallized from ether/
petroleum ether to give 350 mg (1.51 mmol, 79% yield) of 11a
as an off-white solid: mp 196-197 °C. 1H NMR (DMSO-d6):
8.58 (d, 1H), 7.60 (s, 1H), 7.20 (d, 1H), 2.44 (s, 3H), 2.40 (s,
3H). Anal. (C13H12O4) C,H.
2,3-Dim et h oxy-4,6-d im et h yl-1-n a p h t h oic Acid (10a ).
Compound 7a (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. 1H NMR: 10.74 (s, 1H), 9.15 (d,
1H), 7.72 (s, 1H), 7.40 (d, 1H), 4.04 (s, 3H), 3.87 (s, 3H), 2.62
(s, 3H), 2.50 (s, 3H).
2,3-Dih yd r oxy-4,6,7-tr im eth yl-1-n a p h th oic Acid (11b).
Compound 10b (400 mg, 1.46 mmol) was demethylated,14 and
the product was purified as above to give 270 mg (1.10 mmol,
75% yield) of 11b as a tan solid: mp 162-163 °C. 1H NMR
(DMSO-d6): 8.43 (s, 1H), 7.60 (s, 1H), 2.44 (s, 3H), 2.34 (s, 6H).
Anal. (C14H14O4) C,H.
Oxidation by the general procedure for oxidation gave a solid
that was purified by silica gel column chromatography using
dichloromethane to give 1.18 g (4.53 mmol, 72% yield) of 10a
as buff-colored crystals. 1H NMR: 8.07 (d, 1H), 7.70 (s, 1H),
7.35 (d, 1H), 4.06 (s, 3H), 3.89 (s, 3H), 2.60 (s, 3H), 2.51 (s,
3H). Anal. (C15H16O4) C,H.
2,3-Dim eth oxy-4,6,7-tr im eth yl-1-n a p h th oic Acid (10b).
Compound 9a (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. 1H NMR: 10.75 (s, 1H), 9.05 (s,
1H), 7.67 (s, 1H), 4.05 (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 gel column chromatography using
dichloromethane to give 306 mg (1.12 mmol, 81% yield) of 10b
as white crystals: mp 138-140 °C. 1H NMR: 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. (C16H18O4) C,H.
7-Ben zyl-2,3-d im eth oxy-4,6-d im eth yl-1-n a p h th oic Acid
(10c). Compound 9c (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. 1H NMR: 10.74 (s, 1H),
9.16 (s, 1H), 7.66 (s, 1H), 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 gel column chromatography using
dichloromethane to give 207 mg (0.591 mmol, 79% yield) of
10c as white crystals: mp 179-180 °C. 1H NMR (DMSO-d6):
8.14 (s, 1H), 7.70 (s, 1H), 7.13-7.25 (m, 5H), 4.16 (s, 2H), 4.07
(s, 3H), 3.89 (s, 3H), 2.61 (s, 3H), 2.35 (s, 3H). Anal. (C22H22O4)
C,H.
2,3-Dim eth oxy-6-m eth yl-4-n -p r op yl-1-n a p h th oic Acid
(10d ). Compound 7b (1.38 g, 5.63 mmol) was formylated by
the general procedure for formylation to give 1.51 g (5.52
mmol, 98% yield) of solid aldehyde. Oxidation by the general
7-Ben zyl-2,3-d ih yd r oxy-4,6-d im eth yl-1-n a p h th oic Acid
(11c). Compound 10c (634 mg, 1.81 mmol) was demethyl-
ated,14 and the product was purified as above to give 420 mg
(1.30 mmol, 72% yield) of 11c as a white solid: mp 179-180
°C. 1H NMR (DMSO-d6): 8.49 (s, 1H), 7.62 (s, 1H), 7.10-7.28
(m, 5H), 4.06 (s, 2H), 2.52 (s, 3H), 2.45 (s, 3H). Anal.
(C20H18O4) C,H.
2,3-Dih yd r oxy-6-m eth yl-4-n -p r op yl-1-n a p h th oic Acid
(11d ). Compound 10d (600 mg, 2.08 mmol) was demethy-
lated,14 and the product was purified as above to give 390 mg
(1.50 mmol, 72% yield) of 11d as a white solid: mp 160-161
°C. 1H NMR (DMSO-d6): 8.57 (d, 1H), 7.64 (s, 1H), 7.20 (d,
1H), 2.97 (t, 2H), 2.42 (s, 3H), 1.55 (m, 2H), 0.98 (t, 3H). Anal.
(C15H16O4) C,H.
2,3-Dih ydr oxy-6,7-dim eth yl-4-n -pr opyl-1-n aph th oic Acid
(11e). Compound 10e (550 mg, 1.82 mmol) was demethyl-
ated,14 and the product was purified as above to give 380 mg
(1.27 mmol, 70% yield) of 11e as a white solid: mp 171-172
°C. 1H NMR: 8.43 (s, 1H), 7.60 (s, 1H), 2.96 (t, 2H), 2.33 (s,
3H), 2.31 (s, 3H), 1.53 (m, 2H), 0.96 (t, 3H). Anal. (C16H18O4)
C,H.
7-Ben zyl-2,3-d ih yd r oxy-6-m et h yl-4-n -p r op yl-1-n a p h -
th oic Acid (11f). Compound 10f (634 mg, 1.67 mmol) was
demethylated,14 and the product was purified as above to give
417 mg (1.19 mmol, 71% yield) of 11f as a white solid: mp
183-184 °C. 1H NMR (DMSO-d6): 8.63 (s, 1H), 7.61 (s, 1H),
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. (C22H22O4) C,H.
2,3-Dim et h oxy-6,7-d im et h yl-1-(1-m et h ylet h yl)n a p h -
th a len e (13a ). Compound 12a 14 (1.40 g, 4.33 mmol) in 25
mL of 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 and then

3886 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Deck et al.
acidified, 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 gel column chromatography using dichloro-
methane to give 892 mg (3.46 mmol, 80% yield) of 13a as white
crystals: mp 75-77 °C. 1H NMR: 7.85 (s, 1H), 7.44 (s, 1H),
6.93 (s, 1H), 3.89 (m, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 2.39 (s,
3H), 2.35 (s, 3H), 1.51 (d, 6H). Anal. (C17H22O2) C,H.
6-Ben zyl-2,3-d im et h oxy-7-m et h yl-1-(1-m et h ylet h yl)-
n a p h th a len e (13b). Compound 12 (1.14 g, 3.53 mmol) in 25
mL 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 fo 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 white solid: mp 163-165 °C. 1H NMR:
7.84 (s, 1H), 7.25-7.34 (m, 6H), 7.03 (s, 1H), 6.08 (s, 1H), 3.93
(s, 3H), 3.91 (m, 1H), 3.88 (s, 3H), 2.34 (s, 3H), 2.25 (br s, 1H,
disappeared upon shaking with D2O), 1.50 (d, 6H).
The alcohol was dissolved in ethanol and hydrogenated on
a Parr hydrogenator with 10% palladium on carbon and 60
psi of 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 to
afford a pale-yellow solid that was recrystallized from ethyl
acetate to give 1.04 g (3.11 mmol, 88% yield) of 13b as a white
crystalline solid: mp 159-161 °C. 1H NMR: 7.88 (s, 1H), 7.40
(s, 1H), 7.32-7.15 (m, 5H), 6.96 (s, 1H), 4.09 (s, 2H), 3.93 (s,
3H), 3.87 (s, 3H), 2.37 (s, 3H), 1.50 (d, 6H). Anal. (C23H26O2)
C,H.
6-[p-(Tr iflu or om eth yl)ben zyl]-2,3-d im eth oxy-7-m eth -
yl-1-(1-m eth yleth yl)n aph th alen e (13c). Compound 12 (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-(trifluoromethyl)benzaldehyde (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 semisolid which was dissolved
in ethanol and hydrogenated on a Parr hydrogenator with 10%
palladium on carbon and 60 psi of 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 gel column chromatography using dichloromethane
to give 1.09 g (2.71 mmol, 88% yield) of 13c as a pale-yellow
oil. 1H NMR: 7.90 (s, 1H), 7.55 (d, 2H), 7.26 (d, 2H), 6.98 (s,
1H). 4.15 (s, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3.48 (m, 1H), 2.04
(s, 3H), 1.50 (d, 6H). Anal. (C24H25O2F3) C,H.
84% yield) of 14b as a buff-colored crystalline solid: mp 187-
188 °C. 1H NMR: 8.00 (s, 1H), 7.94 (s, 1H), 7.24-7.11 (m, 5H),
4.15 (s, 2H), 4.04 (s, 3H), 3.95 (m, 1H), 3.93 (s, 3H), 2.34 (s,
3H), 1.52 (d, 6H). Anal. (C24H26O4) C,H.
2,3-Dim eth oxy-6-m eth yl-4-(1-m eth yleth yl)-7-[p-(tr iflu -
or om eth yl)ben zyl]-1-n a p h th oic Acid (14c). Compound
13c (600 mg, 1.49 mmol) was formylated by the general
procedure for formylation to give 526 mg (1.22 mmol, 82%
yield) of solid aldehyde. 1H NMR: 10.74 (s, 1H), 9.18 (s, 1H),
7.97 (s, 1H), 7.49 (d, 2H), 7.22 (d, 2H), 4.19 (s, 2H), 4.02 (s,
3H), 3.90 (m, 1H), 3.91 (s, 3H), 2.30 (s, 3H), 1.50 (d, 6H).
Oxidation by the general procedure for oxidation gave a solid
which was purified by silica gel column chromatography to
give 458 mg (1.02 mmol, 84% yield) of 14c as white crystals:
mp 177-178 °C. 1H NMR (DMSO-d6): 8.05 (s, 1H), 7.96 (s,
1H), 7.48 (d, 2H), 7.23 (d, 2H), 4.19 (s, 2H), 4.04 (s, 3H), 3.93
(s, 3H), 2.30 (s, 3H), 1.52 (d, 6H). Anal. (C25H25O4F3) C,H.
2,3-Dih yd r oxy-6,7-d im eth yl-4-(1-m eth yleth yl)-1-n a p h -
th oic Acid (15a ). Compound 14a (450 mg, 1.49 mmol) was
demethylated,14 and the product was recrystallized from ether/
petroleum ether to give 302 mg (1.01 mmol, 68% yield) of 15a
as a white solid: mp 192-193 °C. 1H NMR (DMSO-d6): 8.47
(s, 1H), 7.82 (s, 1H), 3.86 (m, 1H), 2.33 (s, 3H), 2.32 (s, 3H),
1.41 (d, 6H). Anal. (C16H18O4) C,H.
7-Ben zyl-2,3-d ih yd r oxy-6-m eth yl-4-(1-m eth yleth yl)-1-
n a p h th oic Acid (15b). Compound 14b (652 mg, 1.72 mmol)
was demethylated,14 and the product was recrystallized from
ether/petroleum ether to give 416 mg (1.19 mmol, 69% yield)
of 15b as a buff-colored solid: mp 151-152 °C. 1H NMR: 8.61
(s, 1H), 7.89 (s, 1H), 7.25-7-15 (m, 5H), 6.25 (s, 1H), 4.15 (s,
2H), 3.90 (m, 1H), 2.36 (s, 3H), 1.51 (d, 6H). Anal. (C22H22O4)
C,H.
7-[p-(Tr iflu or om eth yl)ben zyl]-2,3-d ih yd r oxy-6-m eth yl-
4-(1-m eth yleth yl)-1-n a p h th oic Acid (15c). Compound 14c
(300 mg, 0.672 mmol) was demethylated,14 and the product
was recrystallized from ether/petroleum ether to give 196 mg
(0.47 mmol, 70% yield) of 15c as a white solid: mp 216-218
°C. 1H NMR (DMSO-d6): 8.60 (s, 1H), 7.86 (s, 1H), 7.82 (d,
2H), 7.20 (d, 2H), 4.12 (s, 2H), 3.87 (m, 1H), 2.27 (s, 3H), 1.41
(d, 6H). Anal. (C23H21F3O4) C,H.
P u r ifica tion of Recom bin a n t P a r a site La cta te Deh y-
d r ogen a se. Recombinant pLDH was produced in Escherichia
coli, similar to the procedure of Bzik et al.,16 and was purified
by Cibacron blue affinity chromatography and chromatofocus-
ing as descibed previously.17 Human LDH-H4 and LDH-M4
were from Sigma.
En zym e Assa ys a n d Kin etics. 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 NADH, 25 °C,
340 nm, ꢀ ) 6.2 mM-1 cm-1
. Michaelis constants were
determined by nonlinear regression analysis of initial rate data
using the Enzfitter program (Elsevier-Biosoft). Ki values were
determined from double-reciprocal plots by linear regression
analysis, as described previously.17
2,3-Dim eth oxy-6,7-d im eth yl-4-(1-m eth yleth yl)-1-n a p h -
th oic Acid (14a ). Compound 13a (550 mg, 2.12 mmol) was
formylated by the general procedure for formylation to give
506 mg (1.77 mmol, 83% yield) of solid aldehyde. Oxidation
by the general procedure for oxidation gave a solid that was
purified by silica gel column chromatography using dichlo-
romethane to give 450 mg (1.49 mmol, 84% yield) of 14a as
white crystals: mp 191-193 °C. 1H NMR: 7.96 (s, 1H), 7.93
(s, 1H), 4.05 (s, 3H), 3.95 (m, 1H), 3.93 (s, 3H), 2.44 (br s, 6H),
1.52 (d, 6H). Anal. (C18H22O4) C,H.
Ack n ow led gm en t. This work was supported by
Grant GM52576 from the Minority Biomedical Sciences
Support (MBRS) program, NIH, and by SBIR Contract
DAMD17-94C4037 from the Department of Defense.
Refer en ces
7-Ben zyl-2,3-d im et h oxy-6-m et h yl-4-(1-m et h ylet h yl)-
n a p h th oic Acid (14b). Compound 13b (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. 1H
NMR: 10.75 (s, 1H), 9.16 (s, 1H), 7.93 (s, 1H), 7.32-7.13 (m,
5H), 4.16 (s, 2H), 4.02 (s, 3H), 3.95 (m, 1H), 3.92 (s, 3H), 2.35
(s, 3H), 1.52 (d, 6H).
Oxidation by the general procedure described for oxidation
gave a solid which was purified by silica gel column chroma-
tography using dichloromethane to give 391 mg (1.03 mmol,
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