Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase

Article abstract of DOI:10.1021/jm990358+

N'-Methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048- 619 (1), is a modest inhibitor (IC₅₀ = 180 μM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2- methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (> 1000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S,R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)- 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC₅₀ of 16 ± 2 nM, enhances the oxidation of [¹⁴C]lactate into ¹⁴CO₂ in human fibroblasts with an EC₅₀ of 57 ± 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 μmol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

Full text of DOI:10.1021/jm990358+

236
J . Med. Chem. 2000, 43, 236-249
Secon d a r y Am id es of (R)-3,3,3-Tr iflu or o-2-h yd r oxy-2-m eth ylp r op ion ic Acid a s
In h ibitor s of P yr u va te Deh yd r ogen a se Kin a se
Thomas D. Aicher,*,† Robert C. Anderson,†,‡ J iaping Gao,§ Suraj S. Shetty,§ Gary M. Coppola,†
J ames L. Stanton,† Douglas C. Knorr,† Donald M. Sperbeck,† Leonard J . Brand,† Christine C. Vinluan,
Emma L. Kaplan, Carol J . Dragland, Hollis C. Tomaselli,† Amin Islam,§ Robert J . Lozito,§ Xilin Liu,§
,#
Wieslawa M. Maniara,| William S. Fillers, Dominick DelGrande,§ R. Eric Walter,| and William R. Mann
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, New J ersey 07901
Received J uly 12, 1999
N′-Methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest
inhibitor (IC50 ) 180 µM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of
the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group,
in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic
acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading
to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the
piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased
the potency of the lead compound (>1000-fold). Oral bioavailability of the compounds in this
series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is
substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S,R)-Dimethyl-4-N-(4-cyanoben-
zoyl)piperazine]-(R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the
primary enzymatic assay with an IC50 of 16 ( 2 nM, enhances the oxidation of [14C]lactate
into 14CO2 in human fibroblasts with an EC50 of 57 ( 13 nM, diminishes lactate significantly
2.5 h post-oral-dose at doses as low as 1 µmol/kg, and increases the ex vivo activity of PDH in
muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however,
do not lower glucose in diabetic animal models.
In tr od u ction
states that the oxidations of free fatty acids and glucose
are related in a reciprocal manner.13 Activation of the
PDH complex via inhibition of PDHK would be expected
to result in increased oxidation of pyruvate in muscle
and fat and a concomitant decrease in the conversion
of pyruvate to the gluconeogenic substrates lactate and
alanine (see Figure 1).
The activity of the pyruvate dehydrogenase (PDH)
complex is lower during conditions of reduced oxidative
glucose metabolism such as obesity, starvation, and
diabetes and in patients with congenital lactic acidosis.1-6
The PDH complex catalyzes the decarboxylation of
pyruvate to acetyl-CoA.7 The activity of the PDH
complex is primarily regulated via reversible phospho-
rylation. ATP-dependent phosphorylation of a specific
E1 serine residue by four isozymes of pyruvate dehy-
drogenase kinases (PDHKs)8-10 leads to inactivation of
the complex. Dephosphorylation of the serine residue
by pyruvate dehydrogenase phosphatases reactivates
the complex.11 High intramitochondrial concentrations
of acetyl-CoA, which can be formed from excessive
oxidation of free fatty acids, markedly increase the
activity of the PDHK. It has been proposed that the
reversible acetylation of the lipoamide of the E2 subunit
of the PDH complex, which reversibly binds to PDHK,
is responsible for this end product activation of PDHK.12
This is consistent with the Randle hypothesis, which
Indeed, in early clinical studies, oral administration
of sodium dichloroacetate (DCA), a known inhibitor of
PDHKs, to type 2 diabetic patients lowered fasting
plasma lactate, alanine, and glucose levels.14-16 Al-
though infusion of DCA lowered plasma lactate and
alanine levels in healthy volunteers, no hypoglycemic
effect was observed.17 DCA has proven efficacy as a
therapy for diabetes,14-16 ischemia,18 endotoxic shock,19
hemorrhagic shock,20 lactic acidosis,21 and cardiac insuf-
ficiency.22,23 However, DCA cannot be used in long-term
treatment due to toxicity. The toxic effects presented
by DCA are neuropathic effects, cataract formation, and
testicular degeneration.24-27 The neuropathy caused by
DCA is exhibited primarily by reversible limb motor
weakness and demyelination of cerebral and cerebellar
white matter. The incidence of limb weakness in rats
receiving 1.1 g/kg/day for 7 weeks is ∼6%. The toxic
effects of DCA have been attributed in part to the
accumulation of its main metabolite, oxalic acid. How-
ever, compounds with halides in the R-position to a
carbonyl are known to exhibit toxic effects.28,29 Never-
theless, PDHK inhibition may be a therapeutic ap-
proach to the treatment of type 2 diabetes, ischemia,
* Address correspondence to: Dr. Thomas D. Aicher at Novartis
Institute for Biomedical Research. Phone: (908)-277-5353. Fax: (908)-
277-2405. E-mail: Thomas.Aicher@pharma.novartis.com.
† Chemistry Unit, Metabolic and Cardiovascular Diseases Reasearch.
§ Pharmacology Unit, Metabolic and Cardiovasular Diseases Re-
search.
| DMPK Biotransformation.
Molecular and Cellular Biology Unit, Metabolic and Cardiovas-
cular Diseases Research.
‡ Current address: Sphinx Pharmaceuticals, P.O. Box 13951,
Research Triangle Park, NC 27709.
#
Current address: Biogen Inc., 12 Cambridge Center Bio6/3,
Cambridge, MA 02142.
10.1021/jm990358+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/31/1999

Inhibitors of PDHK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 237
4-substituted piperidines, 2-chloroaniline, cyclohexyl-
amine, and 2-adamantylamine with the appropriate acyl
chloride, sulfonyl chloride, or iso(thio)cyanate in the
presence (method A) or absence (method B) of an
appropriate base afforded the amides, sulfonamides, and
(thio)ureas in excellent yields (see Table 1).
Our efforts soon became focused upon the R-hy-
droxyamides of substituted piperidines/piperazines (see
Tables 1 and 2). The requisite amides of R-hydroxycar-
boxylic acids were synthesized via a number of meth-
ods: for example, by the addition of hydrides or Grig-
nard reagents to pyruvamides (methods C and D). Even
though the chemical scope of these reactions is very
versatile, the choice of the medicinal targets was quickly
limited (discussed below) to hydroxamides prepared
containing small substituents (i.e., Me, CF3, and hydro-
gen). As a result, it was more efficient to couple the few
intact R-hydroxy acids to the appropriate amines. In our
initial work, the coupling agents CDI (method E), DCC/
HOBt (method G), and (benzotriazol-1-yloxy)tripyrroli-
dinophosphonium hexafluorophosphate (PyBOP; method
F) were utilized for this purpose;36 however, the yields
were low and the couplings were generally only effective
with cyclic secondary amines and saturated primary
amines. Utilizing the procedure of Morris et al.,37
anilides of R-hydroxy acids could be produced via
treatment of the carboxylic acid with thionyl chloride
(method H), followed by addition of the aniline.38 The
method is only effective with anilines, resulting in yields
of 30-90% (anilines with electron-donating substituents
afforded higher yields). Primary R-hydroxyamides were
also produced from isocyanides and ketones via the
procedure of Seebach et al. (method I).39
A modification of Kelly’s procedure was the most
general and effective synthetic method for R-hydroxya-
mides (method J ).40 In short, the acyl halide was
prepared by first treating the R-hydroxycarboxylic acid
with bis(trimethylsilyl)urea in CH2Cl2. After filtration
to remove the urea byproduct, the bissilylated acid is
converted to the acyl halide by treatment with oxalyl
chloride in the presence of a catalytic amount of DMF.
In the case of (S)-3,3,3-trifluoro-2-(trimethylsiloxy)-2-
methylpropionyl chloride, produced from (R)-3,3,3-tri-
fluoro-2-hydroxy-2-methylpropionic acid, the acid chlo-
ride can be stored for weeks at room temperature as
the crude reaction mixture and utilized as such in
aliquots with no discernible detrimental effect on the
coupling yields. The crude R-siloxyamides are effectively
desilylated by methanolic HCl. The couplings of the acyl
chloride with the amines in this report was either
effected with Et3N in CH2Cl2 or with a modification of
polymer-supported quench methodology reported by
Hodges and Booth.41 Although the yields of the cou-
plings of bis(trifluoromethyl)-2-(trimethylsiloxy)acetyl
chloride to amines were low (e.g., crude yield of 7f was
35%), method J was the only procedure to afford
product.
F igu r e 1. Rationale for PDHK inhibition. Inhibition of PDHK
should enhance oxidative glycolysis in the muscle and decrease
gluconeogenesis in the liver, thereby aiding in the restoration
of euglycemia.
F igu r e 2. Inhibitors of PDHK.
F igu r e 3. Initial leads.
lactic acidosis, and other indications with reduced
capacity for the oxidative disposal of lactate and pyru-
vate.
Herein is reported a program which resulted in
structurally novel, orally active inhibitors of the PDHK.
Until our recent publications concerning di- and triter-
penes, and a few of the amides elaborated upon in this
paper (see Figure 2),30,31 no compounds other than R,R-
dihalogenated carbonyl compounds were known to
inhibit PDHK.32
While the programming and logistics of screening
compounds through our primary enzymatic assay33,34
in a high-throughput mode on a robotic platform was
being optimized, it was imperative to carefully select
compounds for screening which would maximize diver-
sity in the test set. To achieve this goal, we utilized a
nonhierarchical clustering strategy based upon 2-D
parameters distributed by MDL MACCS software in a
manner similar to that reported by Martin et al.35 Of
the 200 randomly chosen compounds which were dis-
similar to each other by a criteria of 60% SSS, two
compounds, SDZ048-619 (1) and SDZ060-011 (2) (see
Figure 3), presented very modest but interesting inhibi-
tion of PDHK. Subsequent screening identified the
PDHK inhibitor SDZ225-066 (3). On the basis of these
data, a program to investigate the optimization of the
six-membered ring and the acyl moiety of 1 was initi-
ated in the quest for orally active PDHK inhibitors.
Based upon the emerging structure-activity relation-
ships described below, it became desirable to prepare a
variety of 3-substituted and 2,5-disubstituted pipera-
zines. The synthetic routes are outlined in Scheme 2.
Retrosynthetically, the obvious route to the 3-methyl-
substituted piperazines 11a -p was to regioselectively
monoacylate (R)-2-substituted piperazines with (S)-
3,3,3-trifluoro-2-(trimethylsiloxy)-2-methylpropionyl chlo-
Ch em istr y
The desired compounds were prepared by a variety
of methods, as outlined in Scheme 1. Conjugation of

238 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Sch em e 1. Methods of Synthesis of PDHK Inhibitorsa
Aicher et al.
ride. However, monoacetylation of piperazines is diffi-
cult,42 and varied attempts to monoacetylate piperazines
with (S)-3,3,3-trifluoro-2-(trimethylsiloxy)-2-methylpro-
pionyl chloride failed. For systematic monosubstitution
of piperazine analogues at the 3-position with different
substituents, the method of J acobsen et al. was em-
ployed.43 It should be noted that some racemization of
the chiral carbon was observed in this method (see
method M; a final product with ∼90% enantiomeric
excess was typical in our hands). This impurity could
be eliminated via recrystallization and/or chromato-
graphic separation of the desired amide from the
separable diastereomeric impurity produced in the
subsequent coupling with (R)-3,3,3-trifluoro-2-hydroxy-
2-methylpropionic acid.
Employing the protecting moiety developed by Fukaya-
ma et al.,45 (R)-2-methylpiperazine was regioselectively
monosulfonylated with 2-nitrobenzenesulfonyl chloride.
Consequently, large quantities of 11h from (R)-2-me-
thylpiperazine were obtained in excellent overall yield
(51%) and enantiomeric purity (>99%). Reductive cleav-
age of the carbobenzyloxy moiety gave 11a , which was
further functionalized via methods Q, R, and S to afford
11b-p .
2,5-Disubstituted piperazines were synthesized in a
manner similar to method M via the diketopiperazones
(method O). When synthetic work focused on derivatives
of 1-N-[(R,S)-2,5-dimethylpiperazine]-(R)-3,3,3-trifluoro-
2-hydroxy-2-methylpropanamide, the parent compound
14a (see method P) was conveniently prepared from
achiral trans-2,5-dimethylpiperazine by monobenzylat-
ing a 1:1 mixture of the bishydrochloride salt and the
free base in absolute ethanol in a modification of Craig
and Young’s procedure.46 The 1-benzyl-trans-2,5-di-
A second route (see method N) toward 3-methylpip-
erazine analogues, which proved to be more practical
on a large scale, took advantage of the report that
2-substituted piperazines can be monosulfonylated.44

Inhibitors of PDHK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 239
Sch em e 2. Synthesis of Substituted Piperazine PDHK Inhibitorsa
methylpiperazine was efficiently resolved as the bistar-
trate salt in a single recrystallization from methanol to
afford a 90% recovery of material of greater than 90%
enantiomeric excess. A second crystallization afforded
the enantiomerically pure amine. Acylation of the salt
as above produced 14b, followed by reduction over Pd
in ethanolic HCl to afford 14a , which was further
functionalized via methods Q, R, and S to 14c-l.47
acetamides of primary and noncyclic secondary amines
were not inhibitory of PDHK (tested to 1 mM; data not
shown).
The dichloroacetamide analogue 4i (IC50 ) 179 nM)
was the most potent amide of 4-benzylpiperidine tested.
Compound 4i increased lactate oxidation in the cellular
assay (EC50 ) 1.12 µM; assay discussed below). How-
ever, as numerous toxicities have been reported with
substances containing an R,R-dihalogenated carbonyl,24-29
a search to find a suitable replacement for it was begun.
Moieties which were considered for replacement of the
chlorides of 4i were fluoro, hydroxyl, trifluoromethyl,
and methyl. The methyl and hydroxyl moieties are
approximately isosteric, and the hydroxyl moiety is
electron-withdrawing (but has the additional capability
of two hydrogen bonds). The initial synthetic analogues
4f, 4g, and perfluorinated compounds (i.e., 4l) were
>100-fold less potent than 4i.
Resu lts a n d Discu ssion
The thiourea 1 (IC50 ) 180 µM) was found during our
initial screening of 200 dissimilar compounds of the
Novartis compound library, which, due to our previous
interest in HDL elevating agents,48 contains a signifi-
cant number of thioureas of functionalized piperazines.
Upon testing these compounds, it immediately became
apparent that a 4-substituent of the piperidine was
necessary for compounds to exhibit modest inhibition
of PDHK in the primary assay (i.e., compare 1 and 4b
to 4x and 4y) and that the N-methylthiourea of 1 could
be replaced with an acetyl (i.e., 4d ) or N-methylurea
(i.e., 4b) but not larger urea groups (i.e., compare 4b
with 4p and 4q) nor larger acyl groups (i.e., compare
4i and 6a (R) with 4r and 6b). N-Methylureas and
The identification of 3 (IC50 ) 23 µM, EC50 ) 43 µM)
as an inhibitor of PDHK in the primary enzymatic assay
was exciting. Amides of (S)-3,3,3-trifluoro-2-hydroxy-2-
methylpropionic acid had been demonstrated to be
orally bioavailable and are being investigated for the
indication of urinary incontinence.49 The 3,3,3-trifluoro-

240 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Aicher et al.
Ta ble 1. Physical, in Vitro, and in Vivo Properties of Amides (Acyl Optimization Study)
empirical
formulaa
% yield
(method)
lactated
(% of control, µmol/kg)
entry
R1
CSNHMe
CONHMe
CONMe2
R2
mp (°C)
IC50 (µM)b
>1000
560 ( 240
22.4 ( 3.1
416 ( 88
74 ( 19
282 ( 38
150 ( 25
310 ( 31
260 ( 60
EC50 (µM)c
130 ( 60
DCAe
4a
4b
4c
4d
4e
4f
4g
4h
70%, 1 mmol/kg
Bn 101-102
Bn oil
Bn oil
Bn oil
Bn 84-85
Bn 177
Bn 73-75
Bn 99-100
Bn 62-63
Bn 65-66
Bn 39-40
Bn oil
Bn oil
Bn 40-42
Bn 188
C
C
14H20N2S
14H20N2O
97 (A)
79 (A)
85 (B)
92 (B)
67 (B)
54 (C)
78 (D)
42 (G)
71 (B)
75 (B)
94 (B)
92 (B)
39 (B)
93 (B)
90 (A)
79 (A)
47 (B)
21 (E)
37 (F)
43 (F)
75 (B)
24 (F)
86 (B)
87 (A)
43 (F)
64 (F)
48 (G)
74 (B)
36 (G)
48 (B)
55 (G)
90 (B)
73 (F)
58 (F)
46 (I)
inactive
C15H22N2O
COMe
COtBu
C
C
14H19NOf
17H25NO
inactive
inactive
COC(OH)(CH3)2
COCH(OH)Me
COC(OH)(cPr)
COCHCl2
COCHClCH3
COCCl3
COCF3
SO2NHMe
COOMe
CONH(p-ClPh)
CONHiPr
COCClCH2iPr
COC(CF3)(OH)Me
R-COC(CF3)(OH)Me
S-COC(CF3)(OH)Me
COC(CF3)(OMe)Me
COC(CF3)(OH)Me
COCH3
C16H23NO2
15H21NO2
16H21NO2
g
C
C
4i
4j
4k
4l
4m
4n
4p
4q
4r
C14H17NOCl2
C15H20NOCl
C14H16NOCl3
C14H16NOF3
C13H20N2O2S
14H19NO2
19H21N2OCl
16H24N2O
C18H26NOCl
C16H20NO2F3
C12H20NO2F3
C12H20NO2F3
C13H22NO2F3
C9H14NO2F3
C7H13NO
0.179 ( 0.039 1.12 ( 0.31 not active, 300 µmol/kg
2.6 ( 0.80
30 ( 3.3
inactive
inactive
∼1000
C
C
C
inactive
inactive
inactive
Bn 93
Bn oil
4sh
4t(R)
4t(S)j
4u
Bn 60-62
Pr 81-83
Pr 82-83
Pr oil
H
H
H
2.16 ( 0.51
3.1 ( 0.5
75 ( 1.1
inactive
23.9 ( 3.2
inactive
29.0 ( 5.5
not active, 300 µmol/kg
not active, 300 µmol/kg
4vh
4x
4y
68-69
oil
oil
CONHMe
R-COC(CF3)(OH)Me
C7H14N2O
inactive
5a (R)
112-114 C10H9NO2ClF3
113-114 C10H9NO2ClF3
0.300 ( 0.028 5.4 ( 0.39
9.8 ( 0.57
5.0 ( 0.80
inactive
20 ( 2.3
inactive
23 ( 2.0
47 ( 7.7
6.2 ( 0.51
200 ( 24
inactive
0.30 ( 0.029
11 ( 0.56
10 ( 0.65
inactive
14 ( 0.94
inactive
17 ( 2.0
inactive
15 ( 1.6
inactive
5a (S)j S-COC(CF3)(OH)Me
5b
5c
5d
5e
5f
5g
6a (R)
COC(OH)(CH3)2
COC(CF3)(OMe)Me
COC(CF3)(OH)Et
SO2NHMe
COC(Et)(Me)OH
COCHCl2
94-96
oil
95-96
oil
C10H12NO2Cl
C11H11NO2ClF3
C11H11NO2ClF3
C7H9N2O2SCl
oil
C
11H14NO2ClF3
107-108 C8H6NOCl3
R-COC(CF3)(OH)Me
79-80
83-84
C10H16NO2F3
C10H16NO2F3
i
i
6a (S)j S-COC(CF3)(OH)Me
6b
7a (R)
COC(CF3)(OPh)Me
R-COC(CF3)(OH)Me
117-118 C15H18NO2F3
152-153 C14H20NO2F3
152-154 C14H20NO2F3
102-104 C14H23NO2
80 (F)
65 (F)
90 (F)
82 (B)
71 (F)
61 (E)
9 (J )
not active, 300 µmol/kg
7a (S)j S-COC(CF3)(OH)Me
7b
7c
7d
7e
7f
7g
7h
8
COC(OH)(CH3)2
COC(CF3)(OMe)Me
COC(CF3)(OH)Et
SO2NHMe
COC(OH)(CF3)2
COCOMe
61-62
C15H22NO2F3
165-166 C15H22NO2F3
134-136 C11H20N2O2S
128-129 C14H17NO2F6
87-90
119-120
67-69
50-52
C
C
C
C
13H19NO2
14H21NO2
11H14NO3F3
11H16NO2F3
5 (G)
COC(OH)(cPr)
60 (G)
83 (K)
17 (L)
k
9
inactive
a
b
Analytical results (C, H, N) were within (0.4% of the theoretical value unless otherwise noted. IC50 (µM ( standard error) in primary
enzymatic assay of PDHK inhibition (ref 34). c EC50 (µM ( standard error) in cellular assay of increased oxidation of lactate (ref 60).d In
vivo study in normal Sprague-Dawley rats (n ) 6/group); animals were orally dosed (µmol/kg) after a 24-h fast. Lactate expressed as
percent of control, 2 h post-dose. e Sodium dichloroacetate. f C: calcd 77.38; found 76.79. C, H: calcd 73.53, 8.87; found 74.30, 9.30.
g
h
i
Prepared with racemic 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Increased lactate conversion to CO2 6-10-fold when dosed at
10× the IC50 (ref 61). Synthesized from (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid of 96% ee. C: calcd 52.58; found 53.12.
j
k
2-hydroxy-2-methylpropionic amide of 4-benzylpiperi-
dine, 4s, was found to be a modest (IC50 ) 2.16 µM) but
promising inhibitor of PDHK. Generally, (R)-3,3,3-
trifluoro-2-hydroxy-2-methylpropanamides are modest
inhibitors of PDHK (i.e., 4s, 5a (R), 6a (R), and 7a (R)
IC50 values from 0.30 to 6.2 µM). In all amides generated
from 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid,
the (R)-enantiomer is substantially (∼30-fold) more
potent than the (S)-enantiomer. Similar to the above-
mentioned consequences of increasing the size of the
dichloroacetyl of 4i or the urea of 4b, increasing the size
of the 3,3,3-trifluoro-2-hydroxy-2-methylpropionic moi-
ety in these PDHK inhibitors afforded significantly less
potent inhibitors. For example, the replacement of the
methyl with an ethyl afforded an approximate 10-fold
decrease in potency (compare 5a (R) and 7a (R) with 5d
and 7d ). Also, substitution of the hydroxyl moiety with
methoxy or modification of the OH via cyclization of the
NH of the amide and the hydroxyl moiety with either
carbonyldiimidazole or formaldehyde afforded com-
pounds which did not inhibit PDHK in the functional
assay (compare 6a with 6b or 8 or 9, compare 5a with

Inhibitors of PDHK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 241
Ta ble 2. Physical, in Vitro, and in Vivo Properties of Piperazine Analogues
empirical
formulaa
% yield
(method)
lactated
(% of control, µmol/kg)
entry
R1
mp (°C)
IC50 (µM)b
EC50 (µM)c
10a
10b
10ce
H
Bn
148-150
oil
156-158
147-149
173-174
oil
137-138
95
97-98
98-100
177-179
oil
C8H13N2O2F3
91 (N) inactive
C
15H19N2O2F3
98 (J ) 19.9 ( 1.5
69 (J ) 31.7 ( 2.0
50 (R) 5.56 ( 0.73
65 (Q) 6.7 ( 0.97
56 (S) 6.0 ( 1.5
69 (J ) 2.84 ( 0.25
69 (Q) 5.02 ( 0.59
29 (G) 4.16 ( 0.18
45 (F) 3.37 ( 0.20
93 (N) inactive
PhCO
C19H19N2O3F3
C14H17N2O4SF3
C16H16N3O3F3
C16H17N4O3F3
C13H15N4O4F3
C19H19N2O3F3
C14H17N2O2F3
C13H16N3O2F3
C9H15N2O2F3
10d e PhSO2
10e
10fe
10g
4-NC(C6H4)CO
PhOC(NCN)
4-NO2-2-pyridyl
10h e 1-naphthoyl
10ie
10je
11a
11b
11c
11d
11e
Ph
2-pyridyl
H
Bn
PhCO
25.2 ( 2.9
13.0 ( 1.2
68%*, 300 µmol/kg
68%*, 300 µmol/kg
C
C
16H21N2O2F3
16H19N2O3F3
65 (M) >1.0
oil
oil
oil
71 (Q) 0.173 ( 0.027
62 (R) 0.037 ( 0.005
45 (Q) 0.079 ( 0.008
PhSO2
4-NC(C6H4)CO
C15H19N2O4F3S
C17H18N3O3F3
f
not active, 30 µmol/kg
67%**, 100 µmol/kg
60%**, 30 µmol/kg
90%, 10 µmol/kgg
0.25 ( 0.067
11f
11g
11h
11i
11j
PhOC(NCN)
4-PhCO(C6H4)CO
CBZ
cHexCO
4-NO2-2-pyridyl
60-70
88-93
oil
186-189
150-152
C17H19N4O3F3
C23H23N2O4F3
51 (S) 0.049 ( 0.005
71 (Q) 0.034 ( 0.006
51 (N) 0.021 ( 0.002
87 (Q) 0.064 ( 0.006
21 (J ) 0.112 ( 0.011
f
65%*, 30 µmol/kg
0.473 ( 0.141 66%*, 30 µmol/kg
C
C
17H21N2O4F3
16H25N2O3F3
f
77%*, 30 µmol/kg
not active, 30 µmol/kg
raises glucose
C14H17N4O4F3
h
h
11k
11l
1-naphthoyl
3,4,5-tri-OMe(C6H2)CO
oil
oil
C
20H21N2O3F3
47 (Q) 0.070 ( 0.005
68 (Q) 0.040 ( 0.014
not active, 100 µmol/kg
C19H25N2O6F3
0.159 ( 0.054 60%*, 300 µmol/kg
71%*, 30 µmol/kg
f
f
f
f
11m
11n
11o
11p
SO2-1-naphth
2,4,6-tri-Me(C6H2)PhSO2 205-207
3,5-di-Cl(C6H3)CO
oil
C19H21N2O4F3S
C18H25N2O4SF3
C16H16N2O3F3Cl2
C19H27N2O4SF3
C25H25N2O4F3
91 (R) 0.025 ( 0.006
66 (R) 0.039 ( 0.011
68 (Q) 0.028 ( 0.004
94 (R) 0.036 ( 0.006
72 (B) inactive
not active, 30 µmol/kg
oil
188
145-146
oil
69-71
161-162
233-235
4-tBu(C6H4)SO2
11qh 4-PhCO(C6H4)CO
11r i
12a
4-PhCO(C6H4)CO
2-MePip
C21H20N2O3Cl2
67 (B) 15 ( 2.5
38 (J ) 2.0 ( 0.12
k
C
10H16NO2F3
13a e 4-Cl-2-pyridyl
13be Bn
C14H17N3O2F3Cl
C16H22N2O2F3Cl
29 (J ) 2.59 ( 0.22
51 (J ) 47 ( 4.8
f
13ce
14a
14b
14c
14d
14e
PhCO
H
Bn
SO2-1-naphth
3,5-di-Cl(C6H3)CO
4-NC(C6H4)CO
160.5-162 C16H19N2O3F3
66 (J ) 5.62 ( 0.52
134-137
126-127
96-100
183-184
172-175
C
C
10H17N2O2F3
17H23N2O2F3
95 (N)l inactive
71 (P, J ) 0.082 ( 0.034
60 (J ) 0.0045 ( 0.0010
68 (Q) 0.0033 ( 0.0003
72 (Q) 0.016 ( 0.002
f
f
not active, 30 µmol/kg
not active, 30 µmol/kg
C20H23N2O4F3S
C17H18N2O3F3Cl2
C18H20N3O3F3
0.057 ( 0.013 74%**, 10 µmol/kg
90%**, 3 µmol/kg
87%**, 1 µmol/kg
14f
14g
14h
14i
14j
14k
14l
15a
16a
16b
PhOC(NCN)
SO2-1-naphth
3,5-di-Cl(C6H3)CO
4-MeO2C(C6H4)CO
4-PhCO(C6H4)CO
CBZ
cHexCO
4-NC(C6H4)CO
Bn
72-80
96-100
C
18H21N4O3F3
80 (S) 0.0091 ( 0.0023
61 (J ) 0.0045 ( 0.0010
68 (Q) 0.0033 ( 0.0003
52 (Q) 0.019 ( 0.0075
58 (Q) 0.0198 ( 0.0033
53 (Q) 0.0143 ( 0.0028
55 (Q) 0.013 ( 0.0013
63 (O) 17 ( 2.9
C20H23N2O4F3S
C17H18N2O3F3Cl2
C19H23N2O5F3
C24H25N2O4F3
f
f
not active, 30 µmol/kg
183-184
149-151
134-138
112-114
172-174
116-117
111-112
166-167
f
C
C
18H23N2O4F3
17H27N2O3F3
m
C20H24N3O3F3
17H23N2O2F3
C20H23N2O4F3S
C
60 (J ) inactive
63 (J ) 49 ( 3.1
SO2-1-naphth
a
b
Analytical results (C, H, N) were within (0.4% of the theoretical value unless otherwise noted. IC50 (µM ( standard error) in primary
enzymatic assay of PDHK inhibition (ref 34). c EC50 (µM ( standard error) in cellular assay of increased oxidation of lactate (ref 60).d In
vivo study in normal Sprague-Dawley rats (n ) 6/group); animals were orally dosed (µmol/kg) after a 24-h fast. Lactate expressed as
percent of control, 2 h post-dose. e Prepared with racemic 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. f Increased lactate conversion
g
to CO2 6-10-fold when dosed at 10× the IC50 (ref 61). No lactate lowering was observed after dosing of 3 and 1 µmol/kg of 11e.
h
i
j
k
l
Hemihydrate. Methyl ether of 11g. Dichloroacetyl analogue of 11g. C: calcd 60.15, found 59.29. Reduction conditions same as the
m
removal of the CBZ moiety in procedure L. Crystals retained 0.25 equiv of hexane; calculated. **p < 0.05
5c, compare 11g with 11q). However, metabolic activa-
tion of these O-functionalized derivatives in vivo can
afford the active moiety: i.e., 11q is converted to 11g
upon oral dosing and consequently is effective in acti-
vating the PDH complex and lowering lactate as a
prodrug.50

242 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Aicher et al.
ers: substitution with larger moieties (i.e., an isopropyl,
phenyl, or carboxyl moiety) at these positions afforded
inactive or less potent compounds [i.e., compare 14e
(IC50 ) 16 nM) and 15 (IC50 ) 17 µM)]. Indeed, most of
the increase in potency in the in vitro assay of this series
came from this systematic placing of methyl groups on
the piperazine ring. In each substitution there was a
strong stereochemical preference [3-(R) and 2-(S), i.e.,
compare 14b (IC50 ) 82 nM) and 14c (IC50 ) 4.5 nM)
to 16a (IC50 > 300 µM) and 16b (IC50 ) 49 µM)].
F igu r e 4. Effects of methyl substitution on the piperazine.
The methyl moiety favors the syn disposition due to A strain.
This A strain also destabilizes the ground state, possibly
decreasing the energy needed for rotating the amide bond to
the binding conformation.
Of the three possible explanations offered above for
the increased potency of the methyl-substituted com-
pounds over the desmethyl derivatives, the role of A(1,3)
strain in lowering the energy barrier to obtain the
optimal binding conformation of the N-1 and N-4
substituents is the most plausible. The first explanation,
a hydrophobic pocket for the methyl moiety, cannot
alone be responsible, since only 0.8 kcal of energy would
be expected for optimal binding of a methyl moiety.57
Even if optimal fit was realized, this potential binding
energy is not sufficient for the observed increase in
potency. In addition, the 3-methyl substituent increases
the potency of the trifluoromethyl-2-hydroxy-2-methyl-
propionamide derivatives more than that of the dichlo-
roacetamide series. At first glance, one may conclude
that the favoring of the syn disposition of the amide
group is clearly in evidence due to the strong absolute
chirality preference. However, most of the difference is
likely due to an unfavorable steric clash in the undesired
stereoisomer,58 as the four stereoisomeric forms of 1,4-
dibenzoyl-2,5-trans-diethylpiperazine are all signifi-
cantly populated.59
The above data suggested the (R)-3,3,3-trifluoro-2-
hydroxy-2-methylpropionyl moiety was the optimal acyl
moiety for PDHK inhibition. Thus our attention focused
upon the optimization of the amine moiety of the
inhibitors.
Considering that 4s, 5a (R), 6a (R), and 7a (R) were
approximately equal in potency, it is unlikely that the
piperidine ring of 4s would present the optimal 3-D
presentation of the N-1 and the C-4 substituents.
Indeed, the presentation of the 4-substituent of 1 and
4s must be different due to the different degrees of
saturation of the six-membered ring. To obtain an ideal
intermediate angle of presentation and to create a more
versatile target for SAR exploration, 1,4-disubstituted
derivatives of piperazines and homopiperazines were
examined. The initial compounds 10c-j and 13a -c also
displayed modest but significant activity. Compounds
10i and 10j were evaluated for their ability to lower
lactate, the most proximal effect of PDHK inhibition in
24-h fasted normal Sprague-Dawley rats, and both
presented a modest but significant lowering of lactate
2 h post-oral-dose (300 µmol/kg, see Table 2). Encour-
aged by these results, a significant effort focused upon
improving the potency of the piperazine moiety.
It was postulated that the addition of a methyl
substituent on the piperazine ring could increase po-
tency by any of three mechanisms. The first possibility
was a beneficial hydrophobic interaction of the methyl
moiety with the kinase. The second possibility was a
restriction of the preferred ground state of the rotatable
phenyl or acyl moieties. For example, it is known that
in unsymmetrical amides the bulkier substituent prefers
a syn disposition with respect to the carbonyl group
(Figure 4).51,52 The third possibility was, since the
methyl group would be axial in piperazines due to
significant A(1,3) strain,53 that the A value energy of the
methyl moiety could be utilized to lower the energy
required for rotating the acyl moiety of the inhibitor to
that of its binding conformation.54 If for instance the
optimal conformation of the acyl moiety is a rotation of
10° away from the solution ground state, the 2-methyl
moiety will raise the ground state by ∼1.9 kcal/mol. In
an analogous situation, it was reported that the barrier
of rotation of N-acetyl-2-methylpiperidines is lowered
by 1.9 kcal/mol as compared to that of N-acetylpiperi-
dine, which is attributed to the A value of the methyl
group.55,56
The potent inhibitors described above were evaluated
for their ability to increase the conversion of [14C]lactate
into 14CO2 in human fibroblasts as a measure of their
activation of the PDH complex in a modification of
Ofenstein’s assay (Table 1).60 Compounds 4i, 5a , 10j,
11e, 11g, 11l, and 14e had an EC50 in the cellular assay
of less than 15 µM. The typical magnitude of the
maximal increase of lactate conversion to CO2 was 600-
1000% of control. Potency of the compounds in the
cellular assay usually correlated well to their potency
in the primary enzymatic assay. However, the amount
of lactate utilized by cells dropped at higher concentra-
tions of 3-methyl-4-N-aryl-substituted piperazines (ex-
emplified by 11j),61 suggesting an unfavorable secondary
mechanism, not noted with the acyl or sulfonyl deriva-
tives.
The acyl and sulfonyl derivatives were then profiled
for their ability to lower lactate, the most proximal effect
of PDHK inhibition, in 24-h fasted normal animals.62
Lactate levels were measured 2 h post-oral-dosing of
compounds in 24-h fasted normal Sprague-Dawley rats.
A variety of 4-acyl-substituted piperazines were potent
inhibitors of PDHK. Of these, 11k (IC50 ) 70 nM) and
11l (IC50 ) 40 nM) were quite potent and thus were
selected for in vivo evaluation. The naphthoyl derivative
11k did not show any effect. Compound 11l diminished
lactate significantly 2 h post-oral-dose of 300 µmol/kg.
The sulfonamide analogues of the acyl compounds, while
more potent in the primary in vitro assay and the
cellular assay, are not as potent or efficacious at
lowering lactate. The sulfonamide 11n , which possessed
the best in vivo profile, had 2,6-dimethyl substitution
Following this design strategy, monosubstitution of
a methyl group in the 3-position or 2-position markedly
(g30-fold) increased potency [i.e., compare 10e (IC50
)
6.7 µM) to 11e (IC50 )70 nM) and 14e (IC50 ) 16 nM),
compare any of 10b-j to their analogues in 11b-o and
14b-l]. The methyl substitution was superior to oth-

Inhibitors of PDHK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 243
Ta ble 3. Pharmacokinetic Parameters upon Oral Dosing of
PDHK Inhibitors
pharmacokinetic
parameters
11l (300 µmol/kg)
11e (30 µmol/kg)
average ( SD (n ) 6) average ( SD (n ) 4)
AUC (µM‚h)0-24
Tmax (h)
Cmax (µM)
127 ( 21.4
1.8
33.7 ( 5.9
196 ( 51.7
1.25 ( 0.5
23.8 ( 1.42
on the aromatic ring, which may sterically screen the
sulfonamide from hydrogen binding and allow better
transport across cellular and mitochondrial membranes.
To aid in the design of future analogues and to
optimally select further candidates for in vivo studies,
a bioavailability study of 11l was conducted (see Table
3). Although 11l is orally available, its 3,4,5-trimethoxy-
benzoyl ring was demonstrated to be oxidized in vivo
when dosed orally. From a mass spectrophotometric
fragmentation analysis of the metabolites, the only site
of metabolism of 11l observed was on the 3,4,5-tri-
methoxybenzoyl ring (significantly, no metabolism or
derivatization of the 3,3,3-trifluoro-2-hydroxy-2-meth-
ylpropionamide moiety was evident). Two major me-
tabolites were detected. One possessed a parent ion of
M + 17, which is likely due to aromatic hydroxylation.
The other possessed a parent ion of M + 53. It was
hypothesized that aroyl groups with an electron-
withdrawing substituent (i.e., 11e and 11g), although
less potent in the in vitro assay, would be less prone to
oxidative metabolism and therefore may be superior in
overall in vivo properties. In addition, the cyclohexoyl
derivative 11i was selected for in vivo study.
F igu r e 5. PDH complex activation by 11e and 11g in normal
Sprague-Dawley rats. Effect of 11e and 11g on PDH activity
in tibalis anterior muscle, liver, and fat tissues from normal
24-h fasted Sprague-Dawley rats (weight 200 g) 2 h post-oral-
dose, 20 µmol/kg po (n ) 6/CMC group and 11e group, n )
3/11g group). The PDH complex activity was measured in an
arylamine acetyltransferase-coupled spectrophotometric assay
and was normalized for citrate synthase activity (see ref 63).
**p < 0.05 vs the CMC-treated group.
Compounds 11e, 11g, and 11i were equally or more
potent and efficacious in vivo than 11l (see Table 2).
Their effect on lowering lactate was also of longer
duration (data not shown). Compound 11e exhibited a
higher AUC (µM‚h)0-24 (when orally dosed at a 10-fold
lower dose) than 11l (see Table 3, compound 11g and
11i were not profiled).
To confirm that lactate lowering was a consequence
of PDHK inhibition and thereby PDH activation, we
measured PDH activity ex vivo in treated and untreated
control animals. The PDH complex activity was mea-
sured in an arylamine acetyltransferase-coupled spec-
trophotometric assay which was normalized for milli-
units of PDH activity/unit of citrate synthase activity
for compound- and vehicle-treated animals.63 The PDH
activity was elevated in animals treated with 30 µmol/
kg of 11e or 11g versus control animals (see Figure 5).
F igu r e 6. PDH complex activation by 14e in diabetic Zucker
rats. Effect of 14e on PDH activity in tibalis anterior muscle,
liver, and fat tissues from 24-h fasted Zucker diabetic rats (3
µmol/kg/day, 11 days of dosing, 5 h post-oral-dose, n ) 8/group).
The PDH complex activity was measured in an arylamine
acetyltransferase-coupled spectrophotometric assay and was
normalized for citrate synthase activity (see ref 64). **p < 0.05
vs the CMC-treated group.
Two lines of reasoning led us to expect that the
dimethylated analogues 14c-l would exhibit improved
in vivo potency. First, they demonstrated a general 4-8-
fold increased potency in our primary screens versus
the corresponding methylated analogues 11c-o. Sec-
ond, since increased lipophilic steric encumbrance near
an amide bond can decrease the energy of solvation of
an amide, it may be expected that an increased amount
of the orally dosed inhibitor may cross the numerous
membranes necessary to reach the PDHK target in the
physiological setting. The dimethylated analogues were
each 5-10-fold more potent than their monomethylated
analogues (e.g., compare 14e and 14g to 11e and 11g).
However, no increase in efficacy was discernible, sug-
gesting that these compounds may have reached the
limit for PDH activation.
Having demonstrated that the PDHK inhibitors 11g
and 14e are orally active, i.e., lower lactate concentra-
tions in normal animals at low oral doses, we proceeded
to investigate their effects in disease models for type 2
diabetes. In diabetic animal models, both 11g and 14e
elevated PDH activity ex vivo (i.e., see Figure 6). In
studies in Zucker diabetic animals and ob/ ob mice, 14e
with a dose of 30 µmol/kg lowered lactate levels (p <
0.05) by greater than 40% in both animal models 2.5
and 5 h post-oral-dosing on day 1 of the study. After
day 1 of the study, chronic fed and fasting conditions
were studied (data not shown). However, no marked
glucose lowering effect was noted upon oral dosing of
either of these PDHK inhibitors in either animal model
even though the compounds increased PDH complex

244 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Aicher et al.
5H); MS (DCI, isobutane) m/z (rel intensity) 288 (76), 286 (100).
Anal. (C14H17NOCl2) C, H, N.
activity ex vivo (i.e., see Figure 6). In contrast, DCA
treatment effected a significant lowering of blood glu-
cose.
Meth od C. N-(4-Ben zylp ip er id in e)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e (4f). N-(4-Benzylpiperidine)pyruvamide (3.01
g, 12.3 mmol) was added to a 1.0 M solution of methylmag-
nesium bromide in Et2O (24.2 mL, 24.2 mmol) at 0 °C. The
mixture was stirred for 20 min and was poured upon saturated
aqueous NH4Cl. The organic layer was washed with brine,
dried (MgSO4), and concentrated. The white solid was crystal-
lized from a mixture of hexane and CH2Cl2 to afford 4f (1.73,
It was recognized early in the program that DCA also
affected enzymes other than PDHK.23,64,65 DCA can
affect free fatty acid oxidation both directly (by its affect
on short-chain and medium-chain acyltransferases) and
indirectly via the PDH complex’s effect on carnitine
palmitoyltransferase (CPT).66,67,68 DCA’s direct effect on
fatty acid oxidation may be relevant in explaining why
DCA lowered blood glucose, while 14e and 11g did not.
In conclusion, we report the identification of several
potent, orally available PDHK inhibitors. For example,
(+)-1-N-[2,5-(S,R)-dimethyl-4-N-(4-cyanobenzoyl)pipera-
zine]-(R)-3,3,3-trifluoro-2-hydroxy-2-methylpropana-
mide (14e) inhibits PDHK in the primary enzymatic
assay with an IC50 of 16 ( 2 nM, enhances the oxidation
of [14C]lactate into 14CO2 in human fibroblasts with an
EC50 of 57 ( 13 nM, diminishes lactate significantly 2.5
h post-oral-dose at doses as low as 1 µmol/kg, and
increases the ex vivo activity of PDH in muscle, liver,
and fat tissues. However, these efficacious PDHK
inhibitors do not lower blood glucose as was thought to
be possible for the treatment of type 2 diabetes. Nev-
ertheless, they are effective at increasing the utilization
and disposal of lactate and should be of utility to
ameliorate conditions of inappropriate lactate elevation.
1
54%) as white crystals: mp 177 °C; H NMR (CDCl3) δ 1.21
(m, 2H), 1.49 (s, 6H), 1.77 (m, 2H), 1.81 (m, 1H), 2.56 (d, J )
7.0 Hz, 2H), 2.75-2.90 (m, 2H), 4.20-4.44 (m, 2H), 4.79 (bs,
1H), 7.10-7.32 (m, 5H); MS (DCI, NH3) m/z (rel intensity) 263
(11), 262 (100). Anal. (C16H23NO2) C, H, N.
Meth od D. N-(4-Ben zylp ip er id in e)-2-h yd r oxyp r op a n a -
m id e (4g). Sodium borohydride (500 mg, 13.2 mmol) was
added to a solution of N-(4-benzylpiperidine)pyruvamide (2.45
g, 10.0 mmol) in absolute ethanol (25 mL) at 0 °C. The mixture
was warmed to room temperature, stirred for 30 min, and
poured into water. The aqueous layer was extracted twice with
Et2O (50 mL). The combined organic layers were dried (MgSO4)
and concentrated to afford 4g (1.93 g, 78%) as a clear oil: 1H
NMR (CDCl3) δ 1.21 (m, 2H), 1.49 (d, J ) 7.1 Hz, 3H), 1.75-
1.90 (m, 3H), 2.50-2.66 (m, 4H), 2.94 (m, 1H), 3.64 (m, 1H),
4.43 (q, J ) 7.1 Hz, 1H), 4.59 (bs, 1H), 7.10-7.32 (m, 5H); MS
(DCI, NH3) m/z (rel intensity) 249 (11), 248 (100). Anal. (C15H21
-
NO2) C, H, N.
Meth od E. N-(4-Ben zylp ip er id in e)-3,3,3-tr iflu or o-2-h y-
d r oxy-2-m et h ylp r op a n a m id e (4s). 1,1-Carbonyldiimida-
zole (810 mg, 5.0 mmol) was added to a solution of 3,3,3-
trifluoro-2-hydroxy-2-methylpropionic acid (790 mg, 5.0 mmol)
in CH2Cl2 (7 mL). After 15 min, 4-benzylpiperidine (0.88 mL,
5.0 mmol) was added. The mixture was stirred overnight and
was then partitioned between 1 N HCl and CH2Cl2 (15 mL).
The organic layer was dried (MgSO4) and concentrated. The
residue was purified via column chromatography with 1%
MeOH in CH2Cl2 as the eluent. The product was crystallized
from hexane to afford 4s (170 mg, 21%) as white crystals: mp
60-62 °C; 1H NMR (CDCl3) δ 1.18-1.30 (m, 2H), 1.62 (s, 3H),
1.75-1.94 (m, 3H), 2.57 (d, J ) 7.1 Hz, 2H), 2,83-2.95 (m,
2H), 4.35-4.54 (m, 2H), 5.51 (s, 1H), 7.16-7.37 (m, 5H); MS
Exp er im en ta l Section
Ch em istr y. All melting points (mp) were obtained on a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. Proton magnetic resonance spectra were recorded
on a Bruker AC 300-MHz spectrometer. Chemical shifts were
recorded in ppm (δ) and are reported relative to the solvent
peak or TMS. Mass spectra were run on a Finnigan Mat 4600
spectrometer. Elemental analyses, performed by Robertson
Labs, are within 0.4% of theoretical values unless otherwise
indicated. Thin-layer chromatography (TLC) was carried out
on Macherey-Nagel Polygram Sil G/U254 plates. Column chro-
matography separations were carried out using Merck silica
gel 60 (mesh 230-400). Reagents and solvents were purchased
from common suppliers and were utilized as received. All
reactions were conducted under a nitrogen atmosphere. Yields
are of purified product and were not optimized. All starting
materials were commercially available unless otherwise indi-
cated.
(DCI, NH3) m/z (rel intensity) 317 (13), 316 (100). Anal. (C16H20
-
NO2F3) C, H, N.
Meth od F . N-P ip er id in e-3,3,3-tr iflu or o-2-h yd r oxy-2-
m eth ylp r op a n a m id e (4v). A solution of (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate (Py-
BOP) (1.042 g, 2.0 mmol) in CH2Cl2 (2 mL) was added to a
mixture of 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid
(316 mg, 2.0 mmol), piperidine (170 mg, 2.0 mmol), and
N-methylmorpholine (439 µL, 4.0 mmol) in CH2Cl2 (5 mL) at
0 °C. The mixture was warmed to room temperature, stirred
an additional 2 h, and then filtered through SiO2 with 1:1
hexane:ethyl acetate. The eluent was concentrated, and the
residue was crystallized from hexane/CH2Cl2 to afford 4v (109
mg, 24%) as white crystals: mp 68-69 °C; 1H NMR (CDCl3) δ
1.56-1.75 (m, 6H), 3.65-3.73 (m, 4H), 5.54 (bs, 1H); MS (DCI,
NH3) m/z (rel intensity) 227 (8), 226 (100). Anal. (C9H14NO2F3)
C, H, N.
Meth od A. N′-Meth yl-N-(4-ben zylp ip er id in e)th iou r ea
(4a ). Methyl isothiocyanate (2.00 g, 27 mmol) was added to a
solution of 4-benzylpiperidine (4.79 g, 27 mmol) in CH2Cl2 at
0 °C. The mixture was warmed to room temperature, stirred
for 20 min, and concentrated. The white solid was triturated
with hexane to afford 4a as a white powder (6.54 g, 97%): mp
1
101-102 °C; H NMR (CDCl3) δ 1.29-1.45 (dddd, J ) 12.8,
12.5, 11.6, 4.1 Hz, 2H), 1.77 (m, 2H), 1.85 (m, 1H), 2.59 (d, J
) 7.0 Hz, 2H), 3.03 (ddd, J ) 12.8, 12.8, 1.8 Hz, 2H), 3.19 (s,
3H), 3.27 (bs, 1H), 4.54 (m, 2H), 7.13-7.37 (m, 5H); MS (DCI,
NH3) m/z (rel intensity) 251 (3), 250 (12), 249 (100). Anal.
(C14H20N2S) C, H, N.
Meth od G. 1-N-(4-N-P h en ylp ip er a zin e)-3,3,3-tr iflu or o-
2-h yd r oxy-2-m eth ylp r op a n a m id e (10i). To a mixture of
N-phenylpiperazine (324 mg, 2 mmol), racemic 3,3,3-trifluoro-
2-hydroxy-2-methylpropionic acid (316 mg, 2.0 mmol), and
1-hydroxybenzotriazole hydrate (270 mg, 2.0 mmol) in CH2-
Cl2 in a room-temperature bath was added 1,3-dicyclohexyl-
carbodiimide (412 mg, 2.0 mmol). The mixture was kept at
room temperature for 6 h and then filtered. The filtrate was
extracted with 2 N HCl (8 mL). The aqueous layer was
adjusted to pH 10 with 2 N NaOH and extracted with ether
(15 mL). The organic layer was dried (MgSO4) and concen-
trated. The residue was filtered through a plug of SiO2, eluting
with ethyl acetate. The eluent was concentrated an triturated
with hexane to afford 10i (174 mg, 29%) as a white powder:
Met h od B. 4-Ben zylp ip er id in e, Dich lor oa cet a m id e
(4i). Dichloroacetyl chloride (3.0 mL, 31.0 mmol) was added
to a solution of 4-benzylpiperidine (5.26 g, 30.0 mmol) and
triethylamine (4.3 mL, 31 mmol) in CH2Cl2 (200 mL) at 10
°C. The mixture was warmed to room temperature, stirred for
1 h, washed with water, 0.05 M HCl, and water, dried (Na2-
SO4), and concentrated. The residue was crystallized from
ether and hexanes to afford 4i as white crystals (5.7 g, 71%):
1
mp 62-63 °C; H NMR (CDCl3) δ 1.18-1.39 (m, 2H), 1.75-
1.93 (m, 3H), 2.56-2.70 (m, 3H), 3.01-3.12 (m, 1H), 4.15-
4.20 (m, 1H), 4.47-4.55 (m, 1H), 6.21 (s, 1H), 7.10-7.33 (m,
1
mp 97-98 °C; H NMR (CDCl3) δ 1.78 (s, 3H), 3.20-3.28 (m,

Inhibitors of PDHK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 245
4H), 3.92-4.00 (m, 4H), 5.13 (bs, 1H), 6.91-6.96 (m, 3H),
7.23-7.30 (m, 2H); MS (DCI, NH3) m/z (rel intensity) 304 (15),
303 (100). Anal. (C14H17N2O2F3) C, H, N.
and concentrated, and the residue was eluted through SiO2
with 0.5% MeOH in CH2Cl2. The major fraction was concen-
trated, and the residue was crystallized from a mixture of
hexane and CH2Cl2 to afford 9 (174 mg, 17%) as colorless
crystals: mp 50-52 °C; 1H NMR (CDCl3) δ 1.14 (m, 1H), 1.22-
1.44 (m, 4H), 1.59 (s, 3H), 1.76 (m, 1H), 1.82-1.93 (m, 4H),
5.04 (d, J ) 3 Hz, 1H), 5.12 (d, J ) 3 Hz, 1H); MS (DCI, NH3)
m/z (rel intensity) 253 (21), 252 (100). Anal. (C11H16NO2F3)
Calcd: C 52.58, H 6.42, N 5.58. Found: C 53.12, H 6.14, N
5.63.
Meth od M. (-)-1-N-[3-(R)-Meth yl-4-(3,4,5-tr im eth oxy-
ben zoyl)p ip er a zin e]-(R)-3,3,3-tr iflu or o-2-h yd r oxy-2-m e-
th ylp r op a n a m id e (11l). 3,4,5-Trimethoxybenzoyl chloride
(180 mg, 0.79 mmol) was added to a solution of 3-(R)-methyl-
1-benzylpiperazine (150 mg, 0.79 mmol) and triethylamine
(180 mg, 0.95 mmol) in CH2Cl2 (5 mL). The mixture was stirred
overnight, concentrated, and filtered through silica gel with
2% MeOH in CH2Cl2 to afford 1-N-[2-(R)-methyl-4-benzylpip-
erazine]-3,4,5-trimethoxybenzamide as a clear oil (280 mg,
92%): 1H NMR (CDCl3) δ 1.39 (d, J ) 6.1 Hz, 3H), 2.07 (m,
1H), 2.19 (m, 1H), 2.66 (m, 1H), 2.83 (m, 1H), 3.39 (m, 1H),
3.43 (d, J ) 12.7 Hz, 1H), 3.54 (d, J ) 12.7 Hz, 1H), 3.83 (s,
3H), 3.84 (s, 6H), 6.09 (s, 2H), 7.22-7.35 (m, 5H).
A mixture of the benzamide (270 mg, 0.70 mmol), a catalytic
amount of 10% palladium hydroxide on carbon (10 mg), and
ethanol (5 mL) was shaken overnight in a Parr shaker under
hydrogen (50 psi). The mixture was filtered and the filtrate
was concentrated to afford 1-N-[2-(R)-methylpiperazine]-3,4,5-
trimethoxybenzamide as a clear oil (210 mg, 100%): 1H NMR
(CDCl3) δ 1.36 (d, J ) 6.1 Hz, 3H), 2.43 (bs, 1H), 2.45 (m, 1H),
2.75 (ddd, J ) 12.3, 11.9, 3.3 Hz, 1H), 2.86 (m, 1H), 2.95 (dd,
11.9, 3.3 Hz, 1H), 3.03 (m, 1H), 3.19 (m, 1H), 3.85 (s, 3H), 3.86
(s, 6H), 4.40 (b, 2H), 6.58 (s, 2H).
Meth od H. N-(2-Ch lor oa n ilin e)-2-h yd r oxy-2-m eth yl-
bu tyr a m id e (5f). Thionyl chloride (3.8 mL, 52 mmol) was
added dropwise to a solution of 2-hydroxy-2-methylbutyric acid
(5.90 g, 50 mmol) in DMA (50 mL) at -10 °C. After stirring
for 30 min, 2-chloroaniline (3.95 mL, 37 mmol) was added and
the mixture was stirred for 3 h at room temperature. The
mixture was diluted in ether, washed with water, 2 N HCl,
water, and saturated NaHCO3, dried, and concentrated. The
oil was filtered through SiO2 with 1:1 hexane/ethyl acetate.
The eluent was concentrated to afford 5f (4.74 g, 55%) as a
colorless oil: 1H NMR (CDCl3) δ 0.98 (t, J ) 7.0 Hz, 3H), 1.56
(s, 3H), 1.88 (m, 1H), 2.03 (m, 1H), 2.32 (bs, 1H), 7.03 (m, 1H),
7.26 (m, 1H), 7.35 (m, 1H), 8.43 (m, 1H), 9.22 (bs, 1H); MS
(DCI, NH3) m/z (rel intensity) 230 (31), 229 (12), 228 (100).
Anal. (C11H14NO2ClF3) C, H, N.
Meth od I. N-(Cycloh exyla m in e)-3,3,3-tr iflu or o-2-h y-
d r oxy-2-p h en ylp r op a n a m id e (6b). A 1.0 M solution of
titanium tetrachloride in CH2Cl2 (7.0 mL, 7.0 mmol) was added
to a solution of cyclohexyl isocyanide (0.73 g, 5.84 mmol) in
CH2Cl2 at 0 °C. After stirring for 1 h, 2,2,2-trifluoroacetophe-
none (1.12 g, 6.42 mmol) was added to the mixture. The
mixture was stirred an additional 2 h, 1 N HCl (50 mL) was
added, and the stirring was continued for 1 h. The organic
layer was washed with saturated NaHCO3 and brine, dried,
and concentrated. The solid was recrystallized from a mixture
of hexane and CH2Cl2 to afford 6b (818 mg, 46%) as white
1
crystals: mp 117-118 °C; H NMR (CDCl3) δ 1.02-2.00 (m,
10H), 3.82 (m, 1H), 4.94 (s, 1H), 5.90 (bs, 1H), 7.36-7.43 (m,
3H), 7.60-7.64 (m, 2H); MS (DCI, NH3) m/z (rel intensity) 303
(14), 302 (100). Anal. (C15H18NO2F3) C, H, N.
Meth od J . (S)-3,3,3-Tr iflu or o-2-(tr im eth ylsiloxy)-2-m e-
th ylp r op ion yl Ch lor id e. 1,3-Bis(trimethylsilyl)urea (4.10 g,
20 mmol) was added to a solution of (R)-(+)-3,3,3-trifluoro-2-
hydroxy-2-methylpropionic acid (3.16 g, 20 mmol) in CH2Cl2
(40 mL) at room temperature. The mixture was stirred
overnight and filtered. The solids were washed with CH2Cl2
(2 × 10 mL). A few drops of DMF were added to the combined
filtrates; the solution was cooled to 0 °C, followed by the
dropwise addition of oxalyl chloride (1.75 mL, 20 mmol) over
10 min. After stirring an additional 1 h at 0 °C, the mixture
was warmed to room temperature and stirred for 2 h. The
solution of the acid chloride can be stored indefinitely and was
assumed to have been prepared in quantitative yield.
Gen er a l a cyla tion con d ition s: A 0.83 M solution of (S)-
3,3,3-trifluoro-2-(trimethylsiloxy)-2-methylpropionyl chloride
(1.1 equiv) in CH2Cl2 was added to a 1 M solution of the amine
(1 equiv) in CH2Cl2 and Et3N (2.5 equiv). The mixture was
stirred for 4 h, washed with water, and concentrated. To a 1
M solution of the crude silyl ether in MeOH was added 1 N
HCl (10% of the volume of the MeOH). The mixture was stirred
for 5 h and then diluted with an equal volume of water. The
mixture was concentrated in vacuo to remove MeOH and then
extracted three times with CH2Cl2 (200 mL, then 2 × 100 mL).
The crude products were purified via recrystallization from
CH2Cl2/hexane or via chromatography through silica gel.
(S)-3,3,3-Trifluoro-2-(trimethylsiloxy)-2-methylpropionyl chlo-
ride (54 mg, 0.22 mmol) was added to a stirred mixture of 1-N-
[2-(R)-methylpiperazine]-3,4,5-trimethoxybenzamide (43 mg,
0.15 mmol) and polystyrene-supported morpholine42 (50 mg,
4.3 mmol/g, 0.22 mmol) in CH2Cl2 (3 mL). The mixture was
shaken for 3 h, and tris(2-aminoethyl)ethylamine polystyrene42
(65 mg, 1.7 mmol/g, 0.11 mmol) was added. The mixture was
shaken overnight and filtered. The resin bed was washed with
CH2Cl2. The combined filtrates were concentrated, and the
residue was dissolved in MeOH (5 mL) and 1 M hydrochloric
acid (1 mL). After stirring overnight, the solvents were
evaporated to afford 11l as a crude oil (61 mg, purity 83%)69
which was purified via column chromatography eluting with
1:1 hexane/ethyl acetate (43 mg, 68%): 1H NMR (CDCl3) δ (all
peaks are broad due to interconversion of rotamers) 1.36 (d, J
) 6.1 Hz, 3H), 1.73 (s, 3H), 3.07-3.30 (m, 3H), 3.85 (s, 3H),
3.86 (s, 6H), 4.07 (m, 1H), 4.30-4.60 (m, 3H), 6.60 (s, 2H); MS
(DCI, NH3) m/z (rel intensity) 436 (18), 435 (100). Anal.
(C19H25N2O6F3‚0.5H2O) C, H, N. Compound 11l was also
prepared in larger quantities from 11a via method Q.
Meth od N. (-)-1-N-[3-(R)-Meth yl-4-(ca r boben zyloxy)-
p ip er a zin e]-(R)-3,3,3-t r iflu or o-2-h yd r oxy-2-m et h ylp r o-
p a n a m id e (11h ). NaHCO3 (72 g, 860 mmol) and acetone (160
mL) were added to a mixture of (R)-(-)-2-methylpiperazine
(20.0 g, 200 mmol) in water (250 mL). At 0-5 °C, a solution of
2-nitrobenzenesulfonyl chloride (53.2 g, 240 mmol) in acetone
(80 mL) was added dropwise. The mixture was stirred over-
night at ambient temperature. Water (200 mL) was added, and
the resulting mixture was concentrated in vacuo to 450 mL
and then acidified to pH 3 by the addition of 2 N HCl. The
mixture was extracted with CH2Cl2 (2 × 150 mL). The organic
layers were discarded, and the aqueous layer was made basic
to pH 9 with 2 N NaOH. The mixture was extracted with
EtOAc (3 × 150 mL). The combined organic layers were dried
(MgSO4) and concentrated to afford 6 as a pale yellow oil: 1H
NMR (CDCl3) δ 1.03 (d, J ) 6.1 Hz, 3H), 2.35 (dd, J ) 10.1,
11.8 Hz, 1H), 2.64-3.05 (m, 4H), 3.62 (m, 1H), 7.55-7.73 (m,
3H), 7.90-7.96 (m, 1H); MS (DCI, NH3) m/z (rel intensity) 287
(8), 286 (100).
Meth od K. (R)-3-Cycloh exyl-5-m eth yl-5-tr iflu or om eth yl-
2,4-oxa zolid in ed ion e (8). 1,1-Carbonyldiimidazole (324 mg,
2.0 mmol) was added to a solution of 6a (478 mg, 2.0 mmol)
in CH2Cl2 (10 mL). The mixture was stirred for 72 h and
filtered through SiO2 with 1:1 hexane/ethyl acetate. The eluent
was concentrated, and the residue was triturated with hexane
1
to afford 8 (442 mg, 83%) as a white solid: mp 67-69 °C; H
NMR (CDCl3) δ 1.17-1.43 (m, 3H), 1.60-1.70 (m, 3H), 1.71
(s, 3H), 1.81-1.90 (m, 2H), 2.03-2.14 (m, 2H), 3.92 (m, 1H);
MS (DCI, NH3) m/z (rel intensity) 267 (13), 266 (100). Anal.
(C11H14NO3F3) C, H, N.
Meth od L. (R)-3-Cycloh exyl-5-m eth yl-5-tr iflu or om eth yl-
4-oxa zolid in on e (9). Paraformaldehyde (1.50 g, 50 mmol) was
added to a solution of 6a (1.00 g, 4.18 mmol) in toluene (100
mL) with a catalytic amount of p-toluenesulfonic acid mono-
hydrate (25 mg). The mixture was refluxed for 16 h, cooled,
A solution of benzyl chloroformate (33 mL) in acetone (40
mL) was added to a mixture of 6, acetone (200 mL), water (200

246 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Aicher et al.
mL), and NaHCO3 (50 g) at 0-5 °C. The mixture was stirred
at room temperature overnight. Water (200 mL) was added,
and the mixture was concentrated to approximately 400 mL.
The mixture was then extracted with EtOAc (2 × 150 mL).
The combined organic layers were dried (MgSO4) and concen-
trated to afford a yellow oil (66.6 g): 1H NMR (CDCl3) δ 1.25
(d, J ) 7.0 Hz, 3H), 2.77 (ddd, J ) 3.5, 12.3, 12.4 Hz, 1H),
2.97 (dd, J ) 3.7, 12.5 Hz, 1H), 3.27 (ddd, J ) 3.5, 12.3, 12.4
Hz, 1H), 3.61 (d, J ) 12.5 Hz, 1H), 3.80 (dd, J ) 1.7, 12.3 Hz,
1H), 4.05 (m, 1H), 4.46 (m, 1H), 5.12 (s, 2H), 7.30-7.40 (m,
5H), 7.60-7.75 (m, 3H), 7.93-7.96 (m, 1H); MS (DCI, NH3)
m/z (rel intensity) 421 (9), 420 (100).
To a solution of the yellow oil in DMF (200 mL) was added
thiophenol (18.7 mL, 182 mmol) followed by K2CO3 (62.5 g,
452 mmol). The mixture was stirred at room temperature for
24 h and then filtered with the aid of Celite. The solids were
washed with CH2Cl2 (4 × 200 mL), and the combined filtrates
were acidified to pH 2 with 1 N HCl (300 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 100 mL), and then the
aqueous layer was made basic to pH 10 with 2 N NaOH. The
aqueous layer was extracted with EtOAc (3 × 100 mL). The
combined organic layers were dried (MgSO4) and concentrated
to a yellow oil (27.6 g): 1H NMR (CDCl3) δ 1.21 (d, J ) 7.0
Hz, 3H), 2.64 (ddd, J ) 3.7, 12.1, 12.2 Hz, 1H), 2.73 (dd, J )
1.0, 12.5 Hz, 1H), 2.80-2.95 (m, 2H), 3.02 (ddd, J ) 3.3, 12.2,
12.5 Hz, 1H), 3.83 (dd, J ) 1.0, 12.5 Hz, 1H), 4.22 (m, 1H),
4.46 (m, 1H), 5.09 (s, 2H), 7.30-7.40 (m, 5H); MS (DCI, NH3)
m/z (rel intensity) 236 (11), 235 (100).
A 0.83 M solution of (S)-3,3,3-trifluoro-2-(trimethylsiloxy)-
2-methylpropionyl chloride (150 mL, 124.5 mmol) in CH2Cl2
was added to a solution of the above oil (27.6 g) in CH2Cl2 (200
mL) and Et3N (43 mL, 310 mmol). The mixture was stirred
for 4 h, and then the mixture was washed with water and
concentrated to a pale yellow oil. To a solution of the silyl ether
in MeOH (200 mL) was added 1 N HCl (10 mL). The mixture
was stirred for 5 h, diluted with water (150 mL), concentrated
in vacuo to 160 mL, and then extracted with CH2Cl2 (1 × 200
mL, then 2 × 100 mL). The combined organic layers were dried
(MgSO4) and concentrated to an orange/brown oil. The major
product was isolated via a filtration of the residue through
SiO2 with 3:2 hexane/ethyl acetate. Concentration of the major
fraction afforded 11h (38.7 g, 51%) as a clear oil: [R]D ) -20.0
(c ) 1.05, MeOH); 1H NMR (CDCl3) δ (all peaks are broad due
to interconversion of rotamers) 1.12 (bd, J ) 7.0 Hz, 3H), 1.71
(s, 3H), 3.07-3.28 (m, 3H), 4.00 (m, 1H), 4.27-4.48 (m, 3H),
4.79 (s, 1H), 5.16 (s, 2H), 7.33-7.43 (m, 5H); MS (DCI, NH3)
m/z (rel intensity) 376 (14), 375 (100). Anal. (C17H21N2O4F3)
C, H, N.
eluting with 1% MeOH in CH2Cl2 to afford the amide (10.3 g,
40%) as a white foam: 1H NMR (CDCl3) δ 0.91 (d, J ) 6.7 Hz,
3H), 0.92 (d, J ) 6.7 Hz, 3H), 1.36 (d, J ) 7.0, 3H), 2.06 (m,
1H), 3.68 (s, 3H), 3.99 (q, J ) 7.0 Hz, 1H), 4.21-4.28 (m, 1H),
4.32-4.47 (m, 2H), 4.56 (dd, J ) 9.3, 6.0 Hz, 1H), 4.66-4.72
(m, 2H), 5.52 (d, J ) 9.1 Hz, 1H), 7.20-7.46 (m, 9H), 7.62 (d,
J ) 7.3 Hz, 2H), 7.78 (d, J ) 7.3 Hz, 2H).
A mixture of N-R-(9-fluorenylmethoxycarbonyl)-L-valinyl-N-
â-benzyl-L-alanine methyl ester (10.3 g, 20.0 mmol) in MeOH
(400 mL) and concentrated aqueous ammonia (75 mL) was
refluxed overnight and then concentrated. The residue was
purified via flash chromatography eluting with 2% MeOH in
CH2Cl2 to afford the diketopiperazine (3.8 g, 73%) as a white
foam. An analytical sample was crystallized from ether and
hexanes: mp 88-89 °C; [R]D ) -20.2 (c ) 1.0, MeOH); 1H
NMR (CDCl3) δ 0.90 (d, J ) 6.7 Hz, 3H), 1.06 (d, J ) 6.7 Hz,
3H), 1.47 (d, J ) 7.0 Hz, 3H), 2.69 (m, 1H), 3.85 (quartet, J )
7.0 Hz, 1H), 3.96 (d, J ) 7.0 Hz), 3.97 (d, J ) 14.9 Hz, 1H),
5.36 (d, J ) 14.9 Hz, 1H), 6.19 (bs, 1H), 7.18-7.40 (m, 5H).
A solution of the diketopiperazine (3.00 g, 11.5 mmol) in
THF (25 mL) was added dropwise to a refluxing mixture of
LiAlH4 (2.0 g, 52 mmol) in THF (50 mL). The mixture was
refluxed for 3 h, then stirred overnight at room temperature.
The mixture was quenched with saturated MgSO4 solution,
and the aluminate salts were filtered. The salts were extracted
several times with ether. The combined filtrates were com-
bined and concentrated to the crude piperazine as an oil (2.5
g, 94%): 1H NMR (CDCl3) δ 0.87 (d, J ) 6.7 Hz, 3H), 0.92 (d,
J ) 6.7 Hz, 3H), 1.16 (d, J ) 6.1 Hz, 3H), 1.89 (t, J ) 11.0 Hz,
1H), 2.25-2.45 (m, 2H), 2.66 (m, 1H), 2.81 (dd, J ) 11.9, 2.6
Hz, 1H), 3.08 (dd, J ) 11.9, 2.8 Hz, 1H), 3.17 (d, J ) 13.5 Hz,
1H), 4.10 (d, J ) 13.5 Hz), 7.24-7.40 (m, 5H).
The crude oil was acylated via method J to afford the benzyl
amine: mp 116-117 °C; [R]D ) 46.8 (c ) 1.00, MeOH); 1H
NMR δ 0.75 (d, J ) 6.4 Hz, 3H), 0.76 (d, J ) 6.7 Hz, 3H), 0.99
(d, J ) 6.7 Hz, 3H), 1.66 (s, 3H), 2.47-2.68 (m, 3H), 3.06 (m,
1H), 3.44 (d, J ) 13.2 Hz, 1H), 3.52 (m, 1H), 3.64 (d, J ) 13.2
Hz), 3.87 (m, 1H), 4.25 (m, 1H), 5.28 (bs, 1H), 7.22-7.38 (m,
5H). Anal. (C19H27N2O2F3) Calcd: C 61.28, H 7.31, N 7.52.
Found: C 61.46, H 7.52, N 7.45.
This benzyl amine was reduced via method N to afford the
amine: mp 136-137 °C; [R]D ) 16.0 (c ) 1.00, MeOH); 1H
NMR (CDCl3) δ 0.79 (d, J ) 6.6 Hz, 3H), 0.99 (d, J ) 6.6 Hz,
3H), 1.21 (d, J ) 7.0 Hz, 3H), 1.65 (s, 3H), 2.40 (m, 1H), 2.92
(d, J ) 12.9 Hz, 1H), 3.15 (dd, J ) 13.6, 4.0 Hz, 1H), 3.24 (m,
1H), 3.44 (m, 1H), 3.94 (d, J ) 13.3 Hz, 1H), 4.27 (m, 1H);
yield 2.70 g, 63%.
Acylation of the amine with 4-cyanobenzoyl chloride via
method Q afforded 15a as white crystals: mp 116-117 °C;
[R]D ) 46.8 (c ) 1.0, MeOH); 1H NMR (CDCl3) δ 0.60-1.70
(m, 9H), 2.02 (m, 1H), 2.82-5.04 (m, 7H), 7.43 (m, 2H), 7.71
(d, J ) 8.4 Hz, 2H); MS (DCI, NH3) m/z (rel intensity) 413
(13), 412 (100). Anal. (C20H24N3O3F3) C, H, N, F.
N-1-[3-(R)-Meth ylp ip er a zin e]-(R)-3,3,3-tr iflu or o-2-h y-
d r oxy-2-m eth ylp r op a n a m id e (11a ). A catalytic amount of
palladium on carbon support (10% palladium by weight, 200
mg) was added to a solution of 11h (12.5 g, 33.3 mmol) in
absolute EtOH (100 mL) in a Parr shaker bottle (500 mL
bottle). The vessel was evacuated and filled with hydrogen
three times and finally placed under a hydrogen atmosphere
of 42 psi. The mixture was shaken for 3 h, then filtered with
the aid of Celite. The reaction was repeated twice under
identical conditions, and the combined filtrates were concen-
trated to afford 11a (22.3 g, 93%) as a white powder: mp 177-
Meth od P . Resolu tion of tr a n s-2,5-Dim eth yl-4-ben -
zylp ip er a zin e. To a solution of racemic trans-2,5-dimethyl-
4-benzylpiperazine (59 g, 0.29 mol) in MeOH (150 mL) was
added a solution of (-)-tartaric acid (87 g, 0.58 mol) in MeOH
(250 mL) dropwise for 5 min. Crystallization began upon
addition, and the solution was stored at 0 °C overnight. The
mixture was filtered, washed with cold MeOH (100 mL), and
dried to afford the ditartaric acid salt of (R,S)-2,5-dimethyl-
4-benzylpiperazine (73.9 g) as white crystals: mp 103-104 °C;
[R]D ) -41.0 (c ) 1.00, MeOH).
A single recrystallization from MeOH (cooling to room
temperature) afforded the salt with >98% ee as white crystals
(58.0 g, 79%): mp 118.5-120 °C; [R]D ) -45.7 (c ) 1.00, H2O);
1H NMR (D2O) δ 1.18 (d, J ) 6.5 Hz, 3H), 1.51 (d, J ) 6.3 Hz,
3H), 1.92 (bs, 1H), 2.89 (t, J ) 12.9 Hz, 1H), 3.18 (t, J ) 13.8
Hz, 1H), 3.29 (dd, J ) 13.8, 3.0 Hz, 1H), 3.47 (m, 1H), 3.53
(m, 1H), 3.62 (dd, J ) 12.9, 3.0 Hz, 1H), 3.98 (d, J ) 13.1 Hz,
1H), 4.41 (s, 4H), 4.71 (d, J ) 13.1 Hz, 1H), 7.39-7.48 (m,
5H); MS (DCI, NH3) m/z (rel intensity) 206 (14), 205 (100).
Anal. (C21H32N2O12) C, H, N.
1
179 °C; H NMR (CD3OD) δ 1.08 (d, J ) 6.5 Hz, 3H), 2.30-
3.10 (m, 6H), 4.38 (m, 1H); MS (DCI, NH3) m/z (rel intensity)
242 (9), 241 (100). Anal. (C9H15N2O2F3) C, H, N.
Meth od O. (+)-1-N-[2-(S)-Isop r op yl-5-(R)-m eth yl-4-N-
(4-cyan oben zoyl)piper azin e]-(R)-3,3,3-tr iflu or o-2-h ydr oxy-
2-m eth ylp r op a n a m id e (15a ). Oxalyl chloride (4.5 mL, 50
mmol) was added to a solution of N-(9-fluorenylmethoxylcar-
bonyl)-L-valine (17.0 g, 50 mmol) in CH2Cl2 (100 mL) and DMF
(0.1 mL) at 0 °C. The mixture was warmed slowly to room
temperature for 2 h. The crude acid chloride was added to a
solution of N-benzyl-L-alanine methyl ester (9.65 g, 50 mmol)
in CH2Cl2 (100 mL) and Et3N (11.0 mL, 80 mmol) at 10 °C.
The mixture was allowed to warm to room temperature over
1 h. The mixture was washed with water, dried, and concen-
trated. The residue was subjected to column chromatography

Inhibitors of PDHK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 247
Meth od Q. 1-N-[4-N-(4-Cya n oben zoyl)p ip er a zin e]-(R)-
3,3,3-t r iflu or o-2-h yd r oxy-2-m et h ylp r op a n a m id e (10e).
4-Cyanobenzoyl chloride (448 mg, 2.7 mmol) was added to a
suspension of 10a (560 mg, 2.48 mmol) in CH2Cl2 (10 mL) and
Et3N (320 mg, 3.16 mmol) at 15 °C. The mixture was stirred
for 1 h, washed with water and 0.5 N HCl, dried, and
concentrated. The residue was crystallized from ether to afford
vehicle. Blood samples were taken 2 h after the dosing and
were collected in microcentrifuge tubes containing heparin and
fluoride to prevent blood clotting and inhibit glycolysis. Blood
lactate and glucose concentrations were determined using a
YSI 2700 dual channel biochemistry analyzer (Yellow Springs
Instrument Co., Yellow Springs, OH). A two-tailed and un-
paired t-test was used to compare the differences between the
two groups.
(4) Eva lu a tion of P h a r m a cok in etic P a r a m eter s. Nor-
mal Sprague-Dawley rats were dosed as above. Two ad-
ditional rats provided plasma for a calibration curve. Blood
samples were taken prior to dosing (0 h) and 0.25, 0.5, 1, 2, 4,
8, and 24 h post-oral-dosing. Samples collected in heparinized
tubes were centrifuged, and the concentration of compound
in the supernatant was determined by LC/MS.
(5) Ar yla m in o Acetyltr a n sfer a se-Cou p led Sp ectr op h o-
tom etr ic ex Vivo Assa y. Pyruvate dehydrogenase activity
in tissue extracts was determined spectophotometrically ac-
cording to a modification of described methods63 allowing for
microtiter plate measurement as follows: For each assay, a
10-µL portion of tissue extract [diluted in 0.1 M tris(hy-
droxymethyl)aminomethane-HCl, pH 7.8] was added to a well
of a 96-well microtiter plate. To this was added a 170-µL
portion of assay buffer [100 mM tris(hydroxymethyl)ami-
nomethane-HCl, pH 7.8, 0.5 mM EDTA, 1 mM magnesium
chloride, 50 mM sodium fluoride, 500 µM nicotinamide adenine
dinucleotide, 1 mM thiamine pyrophosphate, 100 µM coenzyme
A, 20 µg/mL p-(p-aminophenylazo)benzenesulfonic acid] con-
taining arylamine acetyltransferase. The mixture was incu-
bated for 10 min in a Molecular Devices Thermomax or
Spectromax 250 spectrophotometer set at 30 °C in order to
deplete any acetyl-CoA present in the tissue extract. The PDH
assay was initiated by the addition of 1 mM pyruvate (20 µL/
well), and conversion of p-(p-aminophenylazo)benzenesulfonic
acid to acetyl-p-(p-aminophenylazo)benzenesulfonic acid was
monitored at 450 nm (kinetic mode) for 30 min. The PDH
complex activity was normalized for citrate activity. Citrate
synthase activity was determined essentially according to
Srere et al.72 For each assay, a 10-µL portion of tissue extract
[diluted in 0.1 M tris(hydroxymethyl)aminomethane-HCl, pH
7.8] was added to 190 µL of a buffer containing 100 mM tris-
(hydroxymethyl)aminomethane-HCl (pH 7.4), 50 µM acetyl-
CoA, 100 µM oxaloacetate, and 100 µM dithiobis(2-nitrobenzoic
acid). The production of mercaptide ion was monitored at 405
nm (kinetic mode) for 10 min using a Molecular Devices
Thermomax or Spectromax 250 spectrophotometer set at 30
°C.
10e (570 mg, 65%) as white crystals: mp 173-174 °C; [R]D
)
-2.0 (c ) 0.83, MeOH); 1H NMR (CDCl3) δ 1.69 (s, 3H), 3.35-
3.90 (broad peaks, 8H), 4.63 (s, 1H), 7.50 (d, J ) 7.9 Hz, 2H),
7.73 (d, J ) 7.9 Hz, 2H); MS (DCI, NH3) m/z (rel intensity)
375 (25), 356 (19), 166 (89), 143 (100). Anal. (C16H16N3O3F3)
C, H, N, F.
Meth od R. (-)-1-N-[3-(R)-Meth yl-4-N-(4-ter t-bu tylben -
zen esu lfon yl)p ip er a zin e]-(R)-3,3,3-t r iflu or o-2-h yd r oxy-
2-m eth ylp r op a n a m id e (11p ). To a solution of 4-tert-butyl-
benzenesulfonyl chloride (116 mg, 0.50 mmol) in CH2Cl2 (3 mL)
was added N-1-[2-(R)-methylpiperazine]-(R)-3,3,3-trifluoro-2-
hydroxy-2-methylpropanamide (120 mg, 0.50 mmol) followed
by Et3N (100 µL). The mixture was set aside overnight and
filtered through SiO2 with 1:1 hexane:ethyl acetate. The eluent
was concentrated to a paste. The residue was triturated with
a mixture of hexanes and ether and filtered to afford 11p (198
mg, 94%) as white crystals: mp 188 °C; [R]D ) -10.6 (c ) 1.00,
1
MeOH); H NMR (CDCl3) δ 1.02 (d, J ) 7.0 Hz, 3H), 1.33 (s,
9H), 1.66 (s, 3H), 3.02-3.22 (m, 3H), 3.63-3.72 (m, 1H), 4.15-
4.37 (m, 3H), 4.45 (s, 1H), 7.50 (d, J ) 8.4 Hz, 2H), 7.71 (d, J
) 8.4 Hz, 2H). Anal. (C19H27N2O4SF3) C, H, N.
Meth od S. (-)-1-N-[4-N-((R)-3,3,3-Tr iflu or o-2-h yd r oxy-
2-m et h ylp r op ion yl)-(R,S)-2,5-d im et h ylp ip er a zin e]ca r -
boxim id ic Acid , N-Cya n op h en yl Ester (14f). A solution of
N-1-[2,5-(S,R)-dimethylpiperazine]-(R)-3,3,3-trifluoro-2-hydroxy-
2-methylpropanamide (80 mg, 0.32 mmol) and diphenyl cy-
anocarbonimidate (78 mg, 0.32 mmol) in THF (2 mL) was
heated for 16 h at 60 °C. The mixture was concentrated and
the residue was eluted through SiO2 with 2% MeOH in CH2-
Cl2. The major fraction was concentrated to afford 14f (100
mg, 80%) as a white solid: mp 72-80 °C; [R]D ) -31.4 (c )
1
0.70, MeOH); H NMR (CDCl3) δ 1.20-1.35 (b, 6H), 1.67 (bs,
3H), 3.15-5.00 (m, 7H), 7.07 (d, J ) 7.8 Hz, 2H), 7.29 (m, 1H),
7.41 (m, 2H); MS (DCI, NH3) m/z (rel intensity) 400 (20), 399
(100). Anal. (C18H21N4O3F3) C, H, N.
Biologica l P r oced u r es. (1) P DHK P r im a r y En zym a tic
Assa y. The primary enzymatic assay was performed using
commercially available PDH complex as described previously
by spectrophotometrically measuring the production of NADH
at 340 nM in the presence and absence of ATP.34,35,70
(6) E va lu a t ion of Com p ou n d s in Dia b et ic An im a l
Mod els.73 Fed adult male C57BL ob/ ob mice (J ackson Labs,
Bar Harbor, ME) were distributed in groups of 10 and matched
for blood glucose on day 0. Food was withheld for 4.5 h prior
to dosing with either vehicle (0.5% carboxymethylcellulose with
0.2% Tween-80, 1 mL/100 g of body weight) or compound in
vehicle. Blood samples were taken 1, 2.5, and 5 h after the
dosing and were collected in microcentrifuge tubes containing
heparin and fluoride to prevent blood clotting and inhibit
glycolysis. Blood lactate and glucose concentrations were
determined using a YSI 2700 dual channel biochemistry
analyzer (Yellow Springs Instrument Co., Yellow Springs, OH).
A two-tailed and unpaired t-test was used to compare the
differences between the two groups.
(2) Cellu la r P DH Assa y. The cellular PDH assay was
performed as described previously60 with the exception that
PDH activity was assayed in cells in suspension rather than
in confluent cells. Cells were harvested using trypsin 2-4 days
after reaching confluence. Aliquots of cells (200 µL, (3.5-5) ×
105 cells) were transferred to plastic tubes containing 25 µL
of test compound solution or solvent control. The tubes were
equilibrated to 37 °C and the PDH assay was initiated by the
addition of 25 µL of DMEM-S medium containing 25 µM
lactate and 0.2 µCi/mL d,l-[1-14C]lactate. Each tube was briefly
gassed (95% O2, 5% CO2) and a plastic collection cup was
introduced into each tube, suspended from a rubber septum
used to seal the neck of the tube. The tubes were slowly shaken
for 25 min at 37 °C. The assay was terminated by addition of
250 µL of 12% w/v trichloroacetic acid. The tubes were chilled
on a block of ice and 100 µL of Solvable (NEN Research
Products, Boston, MA) was added to the plastic collection cups
in order to collect released 14CO2. After a 30-min incubation
collection cups containing Solvable were transferred to vials
containing scintillation fluid, and radioactivity was determined
by liquid scintillation counting.
Ack n ow led gm en t. The authors thank Dr. Michael
J . Shapiro and Bertha Owens for NMR and MS analy-
ses. We also thank Fariborz Firooznia for helpful
comments while reviewing this manuscript.
Refer en ces
(3) Mea su r em en t of La ct a t e Low er in g in Nor m a l
An im a ls. Normal male Sprague-Dawley rats weighing ap-
proximately 200 g were randomly assigned to vehicle- or
compound-treated groups. After a 24-h fast the animals were
dosed with either vehicle (0.5% carboxymethylcellulose with
0.2% Tween-80, 1 mL/100 g of body weight) or compound in
(1) Caterson, I. D.; Kerby, A. L.; Cooney, G. J .; Frankland, R.;
Denyer, G. S.; Nicks, J .; Williams, P. F. Inactivation of Pyruvate
Dehydrogenase Complex in Heart Muscle Mitochondria of Gold-
Thioglucose-Induced Obese Mice is not Due to a Stable Increase
in Activity of Pyruvate Dehydrogenase Kinase. Biochem. J . 1988,
253, 291-294.

248 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Aicher et al.
(2) (a) Wieland, O.; Siess, E.; Schulze-Wethmar, F. H.; von Funcke,
H. G.; Winton, B. Active and Inactive Forms of Pyruvate
Dehydrogenase in Rat Heart and Kidney: Effect of Diabetes,
Fasting, and Refeeding on Pyruvate Dehydrogenase Intercon-
version. Arch. Biochem. Biophys. 1971, 143, 593-601. (b) De-
nyer, G. S.; Lam, D.; Cooney, G. J .; Caterson, I. D. Effect of
Starvation and Insulin In Vivo on the Activity of the Pyruvate
Dehydrogenase Complex in Rat Skeletal Muscles. FEBS Lett.
1989, 250, 464-468. (c) Imura, M.; Takatani, O. ShortOLINIT-
Term Regulation of Pyruvate Dehydrogenase Complex by Insu-
lin. Horm. Metab. Res. 1981, 13, 267-272.
(20) Granot, H.; Steiner, I. Successful Treatment of Irreversible
Hemorrhagic Shock in Dogs with Fructose-1,6-Diphosphate and
Dichloroacetate. Circ. Shock 1985, 15, 163-173.
(21) Henderson, G. N.; Curry, S. H.; Derendorf, H.; Wright, E. C.;
Stacpoole, P. W. Pharmacokinetics of Dichloroacetate in Adult
Patients with Lactic Acidosis. J . Clin. Pharmacol. 1997, 37, 416-
425.
(22) Bersin, R.; Kwasman, M.; DeMarco, T.; Lau, D.; Klinski, C.;
Golonka, J .; Chatterjee, K. Improved Hemodynamic Function
in Congestive Heart Failure with the Metabolic Agent Sodium
Dichloroacetate (DCA). J . Am. Coll. Cardiol. 1990, 15, 157A.
(23) Dichloroacetic acid showed efficacy in these indications. For a
review of the pharmacology of DCA, see: Stacpoole, P. W. The
Pharmacology of Dichloroacetate. Metabolism 1989, 38, 1124-
1144.
(3) Mondon, C. E.; J ones, I. R.; Azhar, S.; Hollenbeck, C. B.; Reaven,
G. M. Lactate Production and Pyruvate Dehydrogenase Activity
in Fat and Skeletal Muscle From Diabetic Rats. Diabetes 1992,
41, 1547-1554.
(24) Stacpoole, P. W.; Moore, G. W.; Kornhauser, D. M. Toxicity of
Chronic Dichloroacetate. N. Engl. J . Med. 1979, 300, 372.
(25) Yount, E. A.; Felten, S. Y.; O’Connor, B. L.; Peterson, R. G.;
Powell, R. S.; Yum, M. N.; Harris, R. A. Comparison of the
Metabolic and Toxic Effects of 2-Chloropropionate and Dichlo-
roacetate. J . Pharmacol. Exp. Ther. 1982, 222, 501-508.
(26) Katz, R.; Tai, C. N.; Diener, R. M.; McConnell, R. F.; Semonick,
D. E. Dichloroacetate, Sodium: 3-Month Oral Toxicity Studies
in Rats and Dogs. Toxicol. Appl. Pharmacol. 1987, 57, 273-287.
(27) Toth, G. P.; Kelty, K. C.; George, E. L.; Read, E. J .; Smith, M.
K. Adverse Male Reproductive Effects Following Subchronic
Exposure of Rats to Sodium Dichloroacetate. Fundam. Appl.
Toxicol. 1992, 19, 57-63.
(4) Mandarino, L. J .; Consoli, A.; Kelly, D. E.; Reilly, J . J .; Nurjhan,
N. Fasting Hyperglycemia Normalizes Oxidative and Nonoxi-
dative Pathways of Insulin-Stimulated Glucose Metabolism in
Noninsulin-Dependent Diabetes Mellitus. J . Clin. Endocrinol.
Metab. 1990, 71, 1544-1551.
(5) Del Prato, S.; Bonadonna, R. C.; Bonora, E.; Gulli, G.; Solini,
A.; Shank, M.; DeFronzo, R. A. Characterization of Cellular
Defects of Insulin Action in Type 2 (Non-Insulin-Dependent)
Diabetes Mellitus. J . Clin. Invest. 1993, 91, 484-494.
(6) Henry, R. R.; Thorburn, A. W.; Beerdson, P.; Gumbiner, B.
Dose-Response Characteristics of Impaired Glucose Oxidation
in Non-Insulin-Dependent Diabetes Mellitus. Am. J . Physiol.
1991, 261, E132-E140.
(7) For a review of the structural components of the PDH multien-
zyme complex, see: Mattevi, A.; De Kok, A.; Perham, R. N. The
Pyruvate Dehydrogenase Multienzyme Complex. Curr. Opin.
Struct. Biol. 1992, 2, 877-887.
(8) Sugden, P. H.; Kerbey, A. L.; Randle, P. J .; Waller, C. A.; Reid,
K. B. Amino Acid Sequences Around the Sites of Phosphorylation
in the Pig Heart Pyruvate Dehydrogenase Complex. Biochem.
J . 1979, 181, 419-426.
(28) Halpert, J . R.; Balfour, C.; Miller, N. E.; Kaminsky, L. S.
Dichloromethyl Compounds as Mechanism-Based Inactivators
of Rat Liver Cytochromes P 450 In Vitro. Mol. Pharmacol. 1986,
30, 19-24.
(29) Bookstaff, R. C.; Moore, R. W.; Ingall, G. B.; Peterson, R. E.
Androgenic Deficiency in Male Rats Treated with Perfluorode-
canoic acid. Toxicol. Appl. Pharmacol. 1990, 104, 322-333.
(30) Aicher, T. D.; Anderson, R. C.; Bebernitz, G. R.; Coppola, G. M.;
Gao, J .; Shetty, S. S.; J ewell, C. F.; Maniara, W. M.; Knorr, D.
C.; Liu, C.; Sperbeck, D. M.; Brand, L. J .; Vinluan, C. C.;
DelGrande, D.; Walter, R. E.; Mann, W. R. (R)-3,3,3-Trifluoro-
2-hydroxy-2-methylpropionamides are Orally Active Inhibitors
of Pyruvate Dehydrogenase Kinase. J . Med. Chem. 2741-2746.
(31) Aicher, T. D.; Damon, R. E.; Koletar, J .; Vinluan, C. C.; Brand,
L. J .; Gao, J .; Shetty, S. S.; Mann; W. R. Triterpene and
Diterpene Inhibitors of Pyruvate Dehydrogenase Kinase (PDHK).
Bioorg. Med. Chem. Lett. 2223-2228.
(9) Yeaman, S. J .; Hutcheson, E. T.; Roche, T. E.; Pettit, F. H.;
Brown, J . R.; Reed, L. J .; Watson, D. C.; Dixon, G. H. Sites of
Phosphorylation on Pyruvate Dehydrogenase from Bovine Kid-
ney and Heart. Biochemistry 1978, 17, 2364-2370.
(10) (a) Popov, K. M.; Kedishvili, N. Y.; Zhao, Y.; Shimomura, Y.;
Crabb, D. W.; Harris, R. A. Primary Structure of Pyruvate
Dehydrogenase Kinase Establishes a New Family of Eukaryotic
Protein Kinases. J . Biol. Chem. 1993, 268, 26602-26606. (b)
Popov, K. M.; Kedishvili, N. Y.; Zhao, Y.; Gudi, R.; Harris, R. A.
Molecular Cloning of the p45 Subunit of Pyruvate Dehydroge-
nase Kinase. J . Biol. Chem. 1994, 269, 29720-29724. (c) Gudi,
R.; Bowker-Kinley, M. M.; Kedishvili, N. A.; Zhao, Y.; Popov, K.
M. Diversity of the Pyruvate Dehydrogenase Kinase Gene
Family in Humans. J . Biol. Chem. 1995, 270, 28989-28994.
(11) Linn, T. C.; Pettit, F. H.; Reed, L. J . R-Keto Acid Dehydrogenase
Complexes. X. Regulation of the Activity of the Pyruvate
Dehydrogenase Complex from Beef Kidney Mitochondria by
Phosphorylation and Dephosphorylation. Proc. Natl. Acad. Sci.
U.S.A. 1969, 62, 234-241.
(12) Cate, R. L.; Roche, T. E. A Unifying Mechanism for Stimulation
of Mammalian Pyruvate Dehydrogenasea Kinase by Reduced
Nicotinamide Adenine Dinucleotide, Dihydrolipoamide, Acetyl
Coenzyme A, or Pyruvate. J . Biol. Chem. 1978, 253, 496-503.
(13) Randle, P. J .; Kerbey, A. L.; Espinal, J . Mechanisms of Decreas-
ing Glucose Oxidation on Diabetes and Starvation: Role of Lipid
Fuel and Hormones. Diabetes Metab. Rev. 1988, 4, 623-638.
(14) Pratt, M. L.; Roche, T. E. Mechanism of Pyruvate Inhibition of
Kidney Pyruvate Dehydrogenasea Kinase and Synergistic Inhi-
bition by Pyruvate and ADP. J . Biol. Chem. 1979, 254, 7191-
7196.
(32) Espinal, J .; Leesnitzer, T.; Hassman, A.; Beggs, M.; Cobb, J .
Inhibition of Pyruvate Dehydrogenase Kinase by Halogenated
Acetophenones. Drug Dev. Res. 1995, 35, 130-136.
(33) Available from Sigma Aldrich Corp., Milwaukee, WI (cat.#
P-7032).
(34) The primary enzymatic assay is identical to the “functional PDK
assay” previously described by our group; see: J ackson, J . C.;
Vinluan, C. C.; Dragland, C. J .; Sundararajan, V.; Yan, B.;
Gounarides, J . S.; Nirmala, N. R.; Topiol, S.; Ramage, P.; Blume,
J . E.; Aicher, T. D.; Bell, P. A.; Mann, W. R. Heterologously
Expressed Inner Lipoyl Domain of Dihydrolipoyl Acetyltrans-
ferase Inhibits ATP-Dependent Inactivation of Pyruvate Dehy-
drogenase Complex: Identification of Important Amino Acid
Residues. Biochem. J . 1998, 334, 703-711.
(35) (a) Martin, Y. C.; Brown, R. D.; Bures, M. G.; Pavlik, P. A.
Strategies and Concerns Applying Diversity or Similarity Mea-
sures to Large Databases. 210th ACS National Meeting, Chi-
cago, IL, 1995; Abstr. 210, MEDI 126. (b) Brown, R. D.; Bures,
M. G.; Martin, Y. C. A Comparison of Some Commercially
Available Structural Descriptors and Clustering Algorithms.
Proc. First Electron Comput. Chem. Conf. 1995, Paper 12. (c)
Brown, R. D.; Martin, Y. C. The Information Content of 2D and
3D Structural Descriptors Relevant to Ligand-Receptor Bind-
ing. J . Chem. Inf. Comput. Sci. 1997, 37, 1-9.
(36) Adapted from: Patino, N.; Frerot, E.; Galeotti, N.; Poncet, J .;
Coste, J .; Dufour, M.-N.; J ouin, P. Total Synthesis of the
Proposed Structure of Dolastatin 15 Tetrahedron 1992, 48,
4115-4122.
(37) Morris, J . J .; Hughes, L. R.; Glen, A. T.; Taylor, P. J . Non-
Steroidal Antiandrogens. Design of Novel Compounds Based on
an Infrared Study of the Dominant Conformation and Hydrogen-
Bonding Properties of a Series of Anilide Antiandrogens. J . Med.
Chem. 1991, 34, 447-455.
(15) Whitehouse, S.; Cooper, R. H.; Randle, P. J . Mechanism of
Activation of Pyruvate Dehydrogenase by Dichloroacetate and
other Halogenated Carboxylic Acids. Biochem J . 1974, 141, 761-
774.
(16) Stacpoole, P. W.; Moore, G. W.; Kornhauser, D. M. Metabolic
Effects of Dichloroacetate in Patients with Diabetes Mellitus and
Hyperlipoproteinemia. N. Engl. J . Med. 1978, 298, 526-530.
(17) (a) Curry, S. H.; Chu, P. I.; Baumgartner, T. G.; Stacpoole, P.
W. Plasma Concentrations and Metabolic Effects of Intravenous
Sodium Dichloroacetate. Clin. Pharmacol. Ther. 1985, 37, 89-
93. (b) Wells, P. G.; Moore, G. W.; Rabin, D.; Wilkinson, G. R.;
Oates, J . A.; Stacpoole, P. W. Metabolic Effects and Pharmaco-
kinetics of Intravenously Administered Dichloroacetate in Hu-
mans. Diabetologia 1980, 19, 109-113.
(38) The acid chloride, if produced, does not acylate saturated primary
or cyclic secondary amines. The reaction may proceed through
a N-sulfinylamine intermediate; see: Shin, J . M.; Kim, Y. H.
New Facile Synthesis of R-Hydroxyamides: Intermolecular and
Intramolecular Catalysis in the Reaction of R-Hydroxycarboxylic
Acids with N-Sulfinylamines. Tetrahedron Lett. 1986, 27, 1971-
1974.
(18) Bersin, R. M.; Stacpoole, P. W. Dichloroacetate as Metabolic
Therapy for Myocardial Ischemia and Failure. Am. Heart J .
1997, 134, 841-855.
(19) Preiser, J . C.; Moulart, D.; Vincent, J . L. Dichloroacetate
Administration in the Treatment of Endotoxic Shock. Circ. Shock
1990, 36, 221-228.
(39) (a) Schiess, M.; Seebach, D. N-Methyl-C-(trichlortitanio)fromin-
imidoylchlorid. Ein Effizientes Reagenz zur Homologisierung
Von Aldehyden und Ketonen zu R-Hydroxy-Carbonsaureamiden.

Inhibitors of PDHK
Helv. Chim. Acta 1983, 66, 1618-1623. (b) Seebach, D.; Adam,
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 249
(57) Andrews, P. R.; Craik, D. J .; Martin, J . L. Functional Group
Contributions to Drug-Receptor Interactions. J . Med. Chem.
1984, 27, 1648-1657.
(58) In the anilide series reported in the following paper, a strong
unfavored steric interaction is seen at the 2-, 5-, and 6-positions
of the anilide.
(59) Similarily, all four stereoisomeric forms of 1,4-dibenzoyl-2,5-
trans-diethylpiperazine are populated; see: Tsuboyama, S.;
Tsuboyama, K.; Uzawa, J .; Koda, R.; Nakamaru, M.; Kobayashi,
K.; Sakurai, T. Conformation of 1,4-Dibenzoyl-2,5-dialkylpip-
erazine. Twist-Boat Ring Conformation. Tetrahedron Lett. 1977,
18, 2895-2898.
(60) Ofenstein, J . P.; Kiechle, F. L.; Dandurand, D. M.; Belknap, W.
M.; Moore, K. H.; Holmes, R. D. Lactate Oxidation for the
Detection of Mitochondrial Dysfunction in Human Skin Fibro-
blasts. Anal. Biochem. 1993, 210, 332-336.
(61) In addition to the compounds whose EC50’s are listed in the table,
several sulfonylated, acetylated, and arylated derivatives were
tested for their ability to increase the conversion of [14C]lactate
into 14CO2 in human fibroblasts at 1×, 4×, and 10× their EC50’s.
While the acetylated and sulfonylated compounds each increased
the conversion of lactate to 600-1000% of control, the arylated
compound 11j and other arylated compounds not shown had a
lower increase at 10× their IC50 value than at 4× their IC50
value.
(62) Glucose lowering in normal animals was not expected to be
evident in normal animals on the basis of Stacpoole’s report (ref
17) and was not observed with compounds of this series in
normal animals.
(63) (a) Coore, H. G.; Denton, R. M.; Martin, B. R.; Randle, P. J .
Regulation of Adipose Tissue Pyruvate Dehydrogenase by
Insulin and Other Hormones. Biochem. J . 1971, 125, 115-127.
(b) Kaempfer, S.; Blackman, M.; Christiansen, M.; Wu, K.; Cesar,
D.; Vary, T.; Hellerstein, M. K. Fraction of Hepatic Cytosolic
Acetyl-CoA Derived From Glucose In Vivo: Relation to PDH
Phosphorylation State. Am. J . Physiol. 1991, 260, E865-E875.
(64) Man, K. C.; Brosnan, J . T. Inhibition of Medium and Short-chain
Fatty Acid Oxidation in Rat Heart Mitochondria by Dichloro-
acetate. Metab. Clin. Exp. 1982, 31, 744-748.
(65) Latipaa, P. M.; Hiltunen, J . K.; Peuhkurinen, K. J .; Hassinen,
I. E. Regulation of Fatty Acid Oxidation in Heart Muscle. Effects
of Pyruvate and Dichloroacetate. Biochim. Biophys. Acta 1983,
752, 162-171.
G.; Gees, T.; Schiess, M.; Weigand, W. Scope and Limitations of
the TiCl4-Mediated Additions of Isocyanides to Aldehydes and
Ketones with Formation of R-Hydroxycarboxylic Acid Amides.
Chem. Ber. 1988, 121, 507-517.
(40) Kelly, S. E.; LaCour, T. G. A One Pot Procedure For the
Synthesis of R-Hydroxyamides from the Corresponding R-Hy-
droxyacids. Synth. Commun. 1992, 22, 859-869.
(41) Booth, R. J .; Hodges, J . C. Polymer-Supported Quenching
Reagents for Parallel Purification. J . Am. Chem. Soc. 1997, 119,
4882-4886.
(42) Desai, M.; Watthey, J . W. H.; Zuckerman, M. A Convenient
Preparation of 1-Aroylpiperazines. Org. Prep. Proced. Int. 1976,
8, 85-86.
(43) Mickelson, J . W.; Belonga, K. L.; J acobsen, E. J . Asymmetric
Synthesis of 2,6-Methylated Piperazines. J . Org. Chem. 1995,
60, 4177-4183.
(44) (a) Bolos, J .; Gubert, S.; Anglada, L.; Planas, J . M.; Burgarolas,
C.; Castello, J . M.; Sacristan, A.; Ortiz, J . A.7-[3-(1-Piperidinyl)-
propoxy]chromenones as Potential Atypical Antipsychotics. J .
Med. Chem. 1996, 39, 2962-2970. (b) Morikawa, A.; Sone, T.;
Asano, T. 5-Isoquinolinesulfonamide Derivatives. III. Synthesis
and Vasodilatory Activity of 1-(5-Isoquinolinesulfonyl)piperazine
Derivatives. Chem. Pharm. Bull. 1992, 40, 770-773.
(45) For the use of 2-nitrobenzenesulfonyl moiety as a protecting
group for amines, see: (a) Fukayama, T.; J ow, C.-K.; Cheung,
M. 2- and 4-Nitrobenzenesulfonamides: Exceptionally Versatile
Means for Preparation of Secondary Amines and Protection of
Amines. Tetrahedron Lett. 1995, 36, 6373-6374. (b) Fukuyama,
T.; Cheung, M.; J ow, C.-K.; Hidai, Y.; Kan, T. 2,4-Dinitroben-
zenesulfonamides: A Simple and Practical Method for the
Preparation of a Variety of Secondary Amines and Diamines.
Tetrahedron Lett. 1997, 38, 5831-5834.
(46) Craig, J . C.; Young, R. J . 1-Benzylpiperazine. Organic Syntheses;
Wiley: New York, 1962; Coll. Vol. V, pp 88-91.
(47) The exception: Methanesulfonyl chloride sulfonylated the alcohol
of 11a to a significant extent ∼10-20% (data not shown).
(48) (a) Coppola, G. M.; Damon, R. E., II. Preparation of N-
Phenylthioureas as Antiatherosclerotics. Can. Pat. Appl. CA 92-
2072704 920629. (b) Coppola, G. M.; Anjaria, H.; Damon, R. E.
Correlation of Oxidation Potential and Toxicity in Thiobenza-
mides. Bioorg. Med. Chem. Lett. 1996, 6, 9-142.
(49) Ohnmacht, C. J .; Russell, K.; Empfield, J . R.; Frank, C. A.;
Gibson, K. H.; Mayhugh, D. R.; McLaren, F. M.; Shapiro, H. S.;
Brown, F. J .; Trainor, D. A.; Ceccarelli, C.; Lin, M. M.; Masek,
B. B.; Forst, J . M.; Harris, R. J .; Hulsizer, J . M.; Lewis, J . J .;
Silverman, S. M.; Smith, R. W.; Warwick, P. J .; Kau, S. T.; Chun,
A. L.; Grant, T. L.; Howe, B. B.; Li, J . H.; Trivedi, S.; Halterman,
T. J .; Yochim, C.; Dyroff, M. C.; Kirkland, M.; Neilson, K. L.
N-Aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamides: KATP
Potassium Channel Openers. Modifications on the Western
Region. J . Med. Chem. 1996, 39, 4592-4601.
(50) Compound 11q is rapidly metabolized to 11g upon oral dosing.
Compound 11g is the only metabolite identified in plasma taken
from the dosed animals.
(51) LaPlanche, L. A.; Rogers, M. T. Configuration in Unsymmetri-
cally N, N-Disubstituted Amides. J . Am. Chem. Soc. 1963, 85,
3728-3730.
(52) For reviews of the conformations of N-acyl groups in heterocycles,
see: (a) Benassi, R.; Folli, U.; Schenetti, L.; Taddei, F. The
Conformations of Acyl Groups in Heterocyclic Compounds. Adv.
Heterocycl. Chem. 1987, 41, 75-186. (b) Rubiralta, M.; Giralt,
E.; Diez, A. Piperidine Structure, Preparation, Reactivity, and
Synthetic Application of Piperidine and its Derivatives; Elsevi-
er: New York, 1991; pp 194-224.
(53) (a) J ohnson, F.; Malhotra, S. K. Steric Interference in Allylic
and Pseudo-Allylic Systems. I. Two Stereochemical Theorems.
J . Am. Chem. Soc. 1965, 87, 5492-5493. (b) J ohnson, F. Allylic
Strain in Six-Member Rings. Chem. Rev. 1968, 68, 375-412.
(54) For a speculated increased ability of benzamides versus aceta-
mides to bind to biological systems due to A(1,3) strain, see:
J ohnson, R. A. Conformations of Alkylpiperidine Amides. J . Org.
Chem. 1968, 33, 3627-3632.
(55) Fraser, R. R.; Grindley, T. B. The “A” Value for the Methyl Group
in 2-Methylpiperidines. Tetrahedron Lett. 1974, 15, 4169-4172.
(56) The replacement of a CH3/H synaxial interaction with a CH3/
lone pair interaction should not have a large effect on the ∆G of
the system: i.e., 3-methylpiperidine and 1-methylcyclohexane
have nearly identical ∆G; see: Crowley, P. J .; Robinson, M. J .
T.; Ward, M. G. Conformational Effects in Compounds with six-
membered rings. XII. The Conformational Equilibrium in N-
Methylpiperidine. Tetrahedron 1977, 33, 915-925.
(66) The indirect effect of DCA on fatty acid oxidation via CPT is via
changes of levels of malonyl-CoA. The concentration of malonyl-
CoA can be influenced both by inhibition of PDHK, i.e., by 14e
or DCA, and/or by inhibition of the short- or medium-chain
acyltransferases by either DCA or other inhibitors (see ref 67).
(67) Compounds which sequester CoA and inhibit the short- to
medium-chain acyltransferases inhibit CPT indirectly; see:
Aicher, T. D.; Bebernitz, G. R.; Bell, P. A.; Brand, L. J .; Dain, J .
G.; Deems, R.; Fillers, W. S.; Foley, J . E.; Knorr, D. C.; Nadelson,
J .; Otero, D. A.; Simpson, R.; Strohshein, R. J .; Young, D. A.
Hypoglycemic Prodrugs of 4-(2,2-Dimethyl-1-oxopropyl)benzoic
Acid. J . Med. Chem. 1999, 42, 153-163.
(68) The compounds of this report do not inhibit any enzymes other
than PDHK to the best of our knowledge. Other enzymes profiled
include Ser/Thr/Tyr kinases (i.e., cAMPk or p38Map kinase) and
KATP channels (see ref 31).
(69) This crude oil was of sufficient purity to be tested in the primary
enzymatic assay. The resulting IC50’s of crude substances made
this way were in acceptable agreement (within 25%; i.e., 40 nM
vs 50 nM) with the values of the purified substances. All IC50’s
reported within this paper are of purified substances.
(70) A reviewer suggested that the compounds may directly activate
the PDH complex. The compounds do not directly activate
PDH: i.e, they do not increase the production of NADH from
PDHactive (see Figure 1). In addition, in a direct assessment of
PDHK activity in the PDC preparation via measuring the initial
rate of phosphorylation of the E1 subunit of the complex, the
IC50 for 14e remains as it is in the primary enzymatic assay
(see ref 35 for the details of this assay).
(71) Srere, P. A.; Brazil, H.; Gonen, L. The Citrate Condensing
Enzyme of Pigeon Breast and Moth Flight Muscle. Acta Chem.
Scand. 1963, 17 (Suppl. 1), S129-S134.
(72) Male ZDF rats at 8 weeks of age in groups of 6 were profiled via
this same procedure.
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