Discovery of a long-chain carbamoyl aminocarnitine derivative, a reversible carnitine palmitoyltransferase inhibitor with antiketotic and antidiabetic activity

Article abstract of DOI:10.1021/jm020979u

The synthesis and pharmacological activity of reversible CPT I inhibitors as potential antiketotic and antidiabetic drugs are reported. Such inhibitors constitute a series of enantiomerically pure aminocarnitine derivatives having the general formula (CH₃)₃N⁺CH₂CH(ZR) CH₂COO^- (with Z = ureido, carbamate, sulfonamide, and sulfamide moieties; R = C₇-C₁₄ linear alkyl chains). A primary pharmacological screening based on the evaluation of CPT I activity in intact rat liver (L-CPT I) mitochondria revealed the best activity for the (R) forms of ureidic derivative 17 (ZR = NHCONHR, R = C₁₄), sulfonamidic derivative 7 (ZR = NHSO₂R, R = C₁₂), and sulfamidic derivative 9 (ZR = NHSO₂NHR, R = C₁₁). The IC₅₀ values are 1.1, 0.7, and 0.8 μM, respectively. For the carbamic derivative 11 (ZR = NHCOOR, R = C₈), an IC₅₀ of 9.5 μM was observed. In addition, an extraordinarily high selectivity toward the liver isoform with respect to the heart isoform (muscle-CPT I M-CPT I) was found for the ureidic compound 17 (IC₅₀(M-CPT I) vs IC₅₀(L-CPTI) = 39.4), as well as for other ureidic or carbamic compounds. Diabetic db/db mice treated orally with 17 and 7 for 45 days at a dose of 50 mg/kg twice a day showed a good reduction of serum glucose levels with respect to the untreated db/db mice (p < 0.01). In addition, 17 showed antiketotic activity in normal fasted rats. 17 has been selected for development as a potential antiketotic and antidiabetic drug.

Full text of DOI:10.1021/jm020979u

J . Med. Chem. 2003, 46, 303-309
303
Discover y of a Lon g-Ch a in Ca r ba m oyl Am in oca r n itin e Der iva tive, a Rever sible
Ca r n itin e P a lm itoyltr a n sfer a se In h ibitor w ith An tik etotic a n d An tid ia betic
Activity
,
†
‡
†
‡
‡
§
Fabio Giannessi,* Pompeo Pessotto, Emanuela Tassoni, Piero Chiodi, Roberto Conti, Francesco De Angelis,
†
†
|
†
‡
Natalina Dell’Uomo, Roberto Catini, Roberto Deias, Maria Ornella Tinti, Paolo Carminati, and
‡
,⊥
Arduino Arduini
Departments of Chemical Research, Endocrinology & Metabolism, and Analytical Research, Sigma Tau Pharmaceutical
Industries S.p.A., Via Pontina Km 30.400, 00040 Pomezia, Italy, and Department of Chemistry, Chemical Engineering and
Materials, University of L’Aquila, L’Aquila, Italy
Received J une 27, 2002
The synthesis and pharmacological activity of reversible CPT I inhibitors as potential antiketotic
and antidiabetic drugs are reported. Such inhibitors constitute a series of enantiomerically
+
-
pure aminocarnitine derivatives having the general formula (CH
3
)
3
N CH
2
CH(ZR)CH
2
COO
(
with Z ) ureido, carbamate, sulfonamide, and sulfamide moieties; R ) C
7
-C14 linear alkyl
chains). A primary pharmacological screening based on the evaluation of CPT I activity in
intact rat liver (L-CPT I) mitochondria revealed the best activity for the (R) forms of ureidic
derivative 17 (ZR ) NHCONHR, R ) C14), sulfonamidic derivative 7 (ZR ) NHSO
2
R, R ) C12),
and sulfamidic derivative 9 (ZR ) NHSO NHR, R ) C11). The IC50 values are 1.1, 0.7, and 0.8
2
µM, respectively. For the carbamic derivative 11 (ZR ) NHCOOR, R ) C ), an IC50 of 9.5 µM
8
was observed. In addition, an extraordinarily high selectivity toward the liver isoform with
respect to the heart isoform (muscle-CPT I ≡ M-CPT I) was found for the ureidic compound 17
(
IC50(M-CPT I) vs IC50(L-CPTI) ) 39.4), as well as for other ureidic or carbamic compounds.
Diabetic db/db mice treated orally with 17 and 7 for 45 days at a dose of 50 mg/kg twice a day
showed a good reduction of serum glucose levels with respect to the untreated db/db mice (p <
0
.01). In addition, 17 showed antiketotic activity in normal fasted rats. 17 has been selected
for development as a potential antiketotic and antidiabetic drug.
In tr od u ction
as liver (L-CPT I) and muscle (M-CPT I) isoforms,
catalyzes the formation of long-chain acylcarnitines.
CPT II, the inner mitochondrial membrane enzyme
present as a single isoform, reconverts long-chain acyl-
carnitines into long-chain acyl coenzyme A thioesters.
A third protein, acylcarnitine translocase, is delegated
to the transport across the membrane. Long-chain acyl-
CoAs are then â-oxidized to acetyl-coenzyme A, which
activates the key gluconeogenetic enzyme pyruvate
Diabetes mellitus is the only noninfectious disease
recognized as epidemic by the World Health Organiza-
tion because of its worldwide diffusion, expecially in
Western life-style countries. It can be divided into two
major categories: type I, or insulin-dependent diabetes
mellitus (IDDM), and the more common type II, or non-
insulin-dependent diabetes mellitus (NIDDM), the latter
accounting for more than 90% of all diabetic patients.
In type II diabetes, characterized by insulin resistance
and inadeguate â-cells activity, increased fatty acid
oxidation contributes greatly to hyperglycemia by for-
mation of high levels of acetyl-coenzyme A, ATP, and
NADH, which increase gluconeogenesis and thus he-
patic glucose production.1 The mitochondrial oxidation
of long-chain fatty acids requires the intervention of two
membrane-bound carnitine-dependent long-chain acyl-
transferases, also known as carnitine palmitoyltrans-
4
carboxylase. CPT inhibitors indirectly reduce liver
gluconeogenesis by lowering the level of acetyl-coenzyme
A and are hence helpful in the treatment of type II
5
,6
diabetes as hypoglycemic agents.
Oxirane carboxylates, such as etomoxir and methyl
,2
5
2
-tetradecylglycidate, are reported to be irreversible
inhibitors of CPT systems, performing good activity as
hypoglycemic compounds. It is worth noting that their
clinical development as hypoglycemic agents was dis-
continued possibly because they are equally active in
inhibiting both L- and M-CPT I isoforms. Side effects
such as cardiac hypertrophy have also been observed.7
Quite recently, scientists from Novartis (Sandoz at the
time) have designed and evaluated carnitine derivatives
as reversible inhibitors of L-CPT I, with a partial
selectivity for L-CPT I versus the M-CPT I isoform
(SDZ-CPI-975 in particular). This results in an im-
proved approach for pharmacological intervention on
fatty acid oxidation, with indirect glycemic control
without induction of myocardial hypertrophy.8
3
ferases (CPT I and CPT II). CPT I, the outer mitochon-
drial membrane enzyme present as two isoforms known
*
To whom correspondence should be addressed. Phone: (+39-
6-91393612). Fax: (+39-06-91393638). E-mail: fabio.giannessi@
sigma-tau.it.
0
†
Department of Chemical Research, Sigma Tau Pharmaceutical
Industries S.p.A.
‡
Department of Endocrinology & Metabolism, Sigma Tau Pharma-
ceutical Industries S.p.A.
§
University of L’Aquila.
Department of Analytical Research, Sigma Tau Pharmaceutical
|
Industries S.p.A.
⊥
Present address: Vascular and Metabolic Disease, Pharmaceutical
Division, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
,9
1
0.1021/jm020979u CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/17/2002

3
04 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Giannessi et al.
Sch em e 2a
a
(
a) 2.5 equiv of CH3(CH2)7OCOCl, 2.5 equiv of NEt3, CH2Cl2,
-
F igu r e 1. Racemic aminocarnitine derivatives.
18 h, 71%; (b) IRA 402 resin (OH ), 94% (66% from 6).
Sch em e 1a
Sch em e 3a
a
(
a) 2 equiv of RNCO, DMSO, 40 °C, 60 h, 47-68% (method
A); or 1 equiv of RNCO, MeOH, 18 h, 52% (method B).
experiments in diabetic mice and in fasted rats, which
allowed the identification of 17, ST 1326, the (R)-4-
trimethylammonio-3-[(tetradecylcarbamoyl)amino]bu-
tyrate, as a candidate for preclinical and clinical devel-
opment as an antiketotic and antidiabetic agent.
a
(a) HCl(g) in isobutyl alcohol, 130 °C, 18 h, 95%; (b) 2.5 equiv
of CH3(CH2)11SO2Cl, 5.5 equiv of NEt3,CH2Cl2, 72 h; (c) NaOH 1
N, 18 h, 44% (from 6); (d) 3 equiv of SO2Cl2, 4 equiv of NEt3,
CH2Cl2, 3 h, then 2 equiv of NEt3, 2 equiv of CH3(CH2)10NH2,18
h; (e) NaOH 1 N, 18 h, 38% (from 6).
Ch em istr y
The (R) and (S) sulfonamide compounds characterized
by a linear alkyl chain of 12 carbons (7 and 8, respec-
tively) were prepared starting from (R)- or (S)-ami-
In our previous work,10 while pursuing the idea that
carnitine analogues, with medium to long alkyl chains,
could occupy the catalytic site of the enzyme, thus
15
nocarnitine 5, as illustrated in Scheme 1, via trans-
formation of the corresponding enantiomeric form into
its isobutyl ester 6, followed by reaction with dodecanoyl
sulfonyl chloride and final hydrolysis. In the same
scheme, we also report the preparation of (R)- and (S)-
sulfamide derivatives with linear alkyl chains, in this
case of 11 carbons (9 and 10, respectively), for a safe
comparison with 7 and 8, having the same overall side
chain length. Thus, 6 was treated with SO Cl in the
slowing down the rate of conversion of long-chain acyl-
CoA into long-chain acylcarnitines,1
1,12
we found that
racemic aminocarnitine derivatives act as good L-CPT
I inhibitors (the relative structures 1-4 are reported
in Figure 1). The most active compounds, sulfamide 3
and sulfonamide 4, were then synthesized in enantio-
merically pure form with a slight modulation of the
chain length. In particular, the optically pure (R)-
sulfonamide 7 and (R)-sulfamide 9 (Scheme 1) confirmed
the expected good L-CPT I inhibition in vitro. In
addition, the only compound selected at that time for
in vivo tests, 7, showed hypoglycemic activity in db/db
mice, a model of type II diabetes, and antiketotic activity
in normal fasted rats.
2
2
presence of triethylamine, followed by reaction with
undecylamine and subsequent hydrolysis to the final
compound.
(R)-Carbamic and (S)-carbamic derivatives with linear
alkyl chains of 8 carbons (11 and 12, respectively) were
prepared as reported in Scheme 2, starting from 6 by
reaction with octylchloroformiate, followed by final
hydrolysis.
Ketoacidosis, in fact, is a serious life-threatening
complication especially in type I diabetes (but also in
type II),13 and it is a major source of morbidity and
(R)-Ureidic and (S)-ureidic derivatives with linear
alkyl chains of 9 carbons (14 and 18, respectively) and
14 carbons (17 and 19, respectively) were prepared as
reported in Scheme 3, by reaction of (R)- or (S)-
aminocarnitine with the appropriate alkyl isocyanate.
For ureidic compounds in the (R) form, a more extensive
modulation of the chain length was performed, also
synthesizing compounds 13 (7-carbon alkyl chain), 14
(9 carbons), 15 (11 carbons), and 16 (12 carbons) always
by reaction of (R)-aminocarnitine with the appropriate
alkyl isocyanate. When the isocyanate was not com-
mercially available, it was prepared from the appropri-
ate carboxylic acid via Curtius reaction, following the
procedure described in the reference reported in the
Experimental Section.
1
4
mortality in diabetic children and adolescents. Since
ketone bodies are the major products of liver fatty acid
oxidation, a liver-specific CPT I inhibitor is actually
expected to significantly reduce their blood levels.
In this paper, we report the results of a more detailed
study into the effects of chirality and chain length, in
aminocarnitine derivatives carrying functional groups
such as those reported in Figure 1, on CPT inhibi-
tion. Actually, the chain length effect was thoroughly
evaluated within the ureido derivative series, while for
1
0
the other compounds it was kept unchanged. For
selected molecules, we also report the results of in vivo

Carnitine Palmitoyltransferase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 305
Ta ble 1. Effect of CPT I Inhibitors on Mitochondrial CPT I
a
Activity
chain length
IC50
IC50
(heart)b
(µM)
b
(no. of carbon (liver)
compd
chirality
atoms)
(µM)
0.7
7
8
9
1
1
1
1
1
1
1
1
1
1
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(R)
(R)
(R)
(R)
(S)
(S)
(R)
12
12
11
11
8
3.4
>10
6
0.8
6
5.8
0
1
2
3
4
5
6
7
8
9
>10
80.1
9.5
8
7
9
11
12
14
9
14
14
>300
>300
>300
46.2
118.4
>120
43.4
>300
167.4
62
43.6
31.3
3.2
1.8
1.1
F igu r e 2. Percent reduction of serum glucose after 45-day
treatment in db/db mice via oral gavage, at doses equivalent
to 50 mg/kg of 17 twice a day, determined after 9 h of fasting
and 9 h from last treatment. Percent reduction is calculated
with respect to untreated db/db mice used as control. The
number of animals per group is 8. Student’s t test indicate (0)
p < 0.05 and (4) p < 0.01 versus control diabetic mice.
>300
58.8
17.4
8
,9
SDZ-CP1975
a
The activity of CPT I was measured radiochemically by using
C]-palmitoyl-CoA and carnitine itself by evaluating the incor-
All the compounds were tested for their aspecific
toxicity on cultures of rat hepatocytes by measuring the
release of LDH (lactate dehydrogenase) after 48 h of
incubation. No toxicity was observed at concentrations
up to 50 µM.
[14
1
4
poration of [ C]-palmitoyl residue on carnitine in intact fresh liver
and heart mithocondria from Sprague-Dawley male rats.
The IC50 values (µM) were calculated from the inhibition curve.
Data are the mean of two determinations in triplicate. The
variation was less than 8%.
1
6,17
1
8
b
The ureido derivatives 13, 16, and 17, together with
the sulfonamide compound 7, were then selected for the
in vivo studies in db/db mice (C57BL/KsJ -db/db), a
To assess the chemical purity of final compounds,
since contamination from long-chain reagents may raise
some concern, HPLC was also extensively used (see
Experimental Section, general procedures). The water
content of these hygroscopic molecules, measured by the
Karl-Fischer method, was considered for screening
purposes. Because the starting material 5 is optically
pure (see ref 15), enantiomeric excess was checked only
in the case of one derivative, namely, compound 17. As
expected, when chiral HPLC (Chirobiotic-Tag column,
1
9
model of type II diabetes, and were tested at oral doses
equimolar to 50 mg/kg, referenced to 17, twice a day
for 45 days (N ) 8 for each group). In particular, 7 and
17 showed a good reduction of serum glucose levels with
respect to untreated db/db mice (Figure 2, -25%; from
673.9 ( 44.09 to 506.6 ( 59.12 mg/dL for 17; in lean
mice, serum glucose level was 202.6 ( 5.91 mg/dL; mean
( SE; p < 0.01). For compounds of the ureido series, a
decreasing activity in vivo, parallel to the decrease in
alkyl chain length, was observed as expected on the
basis of the in vitro L-CPT I inhibition. Treatment with
5
µm, 250 × 4.6 mm) was used, >99% ee was found.
Biology a n d Discu ssion
1
7 showed no significant effects on food intake and body
IC50 (µM) values for L-CPT I and M-CPT I, deter-
weight, whereas water consumption was reduced (-30%),
and a strong improvement in diabetic polyuria was
observed. The reduction of glucose was also accompa-
nied by a significant decrease of serum fructosamine
levels, a measure of total glucose exposure (from 4.80
( 0.17 to 3.36 ( 0.30 mmol/L, -30%; in lean mice,
serum fructosamine level was 3.37 ( 0.16 mmol/L; mean
( SE; p < 0.01). Liver glycogen was also significantly
reduced (-25%), while triglycerides (TG) content was
significantly increased (+38%). Serum insulin levels
were not significantly modified nor were leptin, TG,
urea, alanine aminotransferase, and cholesterol levels;
free fatty acid (FFAs) levels were instead significantly
increased (+20%). It is known that an increase of serum
FFAs is correlated to a deterioration of insulin resis-
tance, but our results indicate that long-term treat-
ment with 17 seems to improve glucose homeostasis in
db/db mice. In any case, further investigation into
different experimental models will be undertaken to
better characterize compound 17.
In treated mice, no effects on heart weight and TG
content were observed, thus confirming the preferential
inhibition of hepatic CPT I activity and therefore its
predictable safety with regard to cardiac hypertrophy.
Furthermore, a kinetic study revealed that compound
17 is a reversible, mixed inhibitor of L-CPT I with
respect to palmitoyl-CoA (apparent Ki ) 0.36 ( 0.04
µM).
mined respectively in rat liver and heart mitochon-
1
6,17
dria,
are reported in Table 1. It should be noted that
the M-CPT I isoform is expressed in both cardiac and
skeletal muscles. For all the functional groups, the
highest activity in vitro was observed for compounds in
the (R) form, thus confirming our early results in the
1
0
case of sulfonamide and sulfamide compounds, as well
as what was already reported for the Sandoz compound
SDZ-CPI-975, which we used as a reference.9
The importance of the chain length was demonstrated
in the ureidic series by testing the (R) compounds
carrying 7 (13), 9 (14), 11 (15), 12 (16), and 14 (17) term
chains, the longer alkyl chain showing the highest
activity. In this series, the IC50 values ranged from 1.1
µM for 17 to 43.6 µM for compound 13. For SDZ-CPI-
75, the IC50 was 17.4 µM. In addition, even if L-CPT I
inhibition was quite similar for ureido 17, sulfonamide
, and sulfamide 9, an unexpected extraordinarily high
2
0
9
7
selectivity for liver with respect to muscle isoform was
observed for 17 (40-fold selectivity against a 4-fold
selectivity for SDZ-CPI-975). It is also worth emphasiz-
ing that such a selectivity is reported as particularly
important in order to avoid undesirable side effects on
7
the cardiac muscle, leading to heart hypertrophy. In
any case, also for 7 and 9, as well as for the other ureido
and carbamate compounds, a good selectivity was
observed.

3
06 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Giannessi et al.
haved differently, with its concentration (determined by
HPLC) beginning to decrease under both conditions just
after 2 h.
In conclusion, in this work we reported the identifica-
tion of compound 17 as an agent able to selectively
inhibit in vitro L-CPT I with respect to M-CPT I and to
depress ketogenesis in vivo (fasted rats). 17 also showed
a good reduction of serum glucose levels in a diabetic
mouse model, without any significant change in heart
weight and TG content. Owing to its encouraging
activity and pharmacological profile, 17 was selected as
a candidate for clinical development as an antiketotic
and antidiabetic drug. Nevertheless, since it has been
reported that development of the aforementioned No-
F igu r e 3. Levels of â-hydroxybutyrate (µmol/L) in 24 h fasted
rats at 2, 4, 6, 8, 10, and 12 h after treatment with compound
1
7 at oral doses of 0 ([) 10 (9), 20 (2), 30 (×), and 40 (/) mg/
kg. Values are the mean ( SE of six animals per group.
vartis compound, SDZ-CPI-975, was stopped owing to
6
“
hepatic mitochondrial aberrations”, this will be con-
sidered during the investigations into the safety of our
compound, before committing to full development.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
determined in open capillary tubes and are uncorrected.
1
H NMR spectra were recorded on a VXR 300S Varian
spectrometer at 300 MHz or on a Gemini 200 Varian at 200
MHz. The chemical shift δ is expressed in parts per million
relative to the standard tetramethylsilane. Ion spray mass
spectra were recorded in positive mode on an ESI LCQ Classic
Thermo-Finnigan ion trap spectrometer. Flash column chro-
matography separations were carried out with Biotage Flash
1
4
F igu r e 4. [ C]-oleoylcarnitine level, as indicator of CPT II
inhibition, after rat hepatocytes incubation with 17 (3 mM)
or (R)-N-palmitoylaminocarnitine (PNH-Cn, 1 mM).
4
5
0i apparatus using KP-Sil silica cartridge (32-63 µm, 60 Å,
2
00-550 m /g silica). Thin-layer chromatography (TLC) was
To confirm the inhibition of liver CPT I in an ad-
ditional animal model and to verify its antiketotic effect,
performed on silica gel (5 cm × 10 cm) 60 F254 Merck
prescored plates. Reagents and solvents were purchased from
common suppliers and were used as received. All starting
materials were commercially available with the exception of
(R)- and (S)-aminocarnitine, together with nonylisocyanate,
which were prepared according to literature procedures (see
ref 15 for aminocarnitine; see Org. Synth. 1967, (Collective)
1
7 was orally administered to 24 h fasted normal rats.
Since â-hydroxybutyrate (â-HBA) is a major product of
liver fatty acid oxidation during fasting,21 a CPT I
inhibitor is expected to significantly reduce its level. We
actually observed a strong dose-dependent reduction of
serum â-hydroxybutyrate levels up to12 h from acute
oral dose, with ED50 ) 14.5 mg/kg (Figure 3). The rate
of production of â-hydroxybutyrate seems to increase
after 6-8 h, indicating the recovery of liver â-oxidation.
When 19 ((S)-form) was administered, no effect on
â-HBA levels was observed, again confirming the anal-
ogy between the results of the in vitro and the in vivo
tests and excluding any hypothetical aspecific effect due
to the structure of the molecule.
3
, 846-847 for the synthetic procedure described in the case
of undecylisocyanate and used for nonylisocyanate).
General conditions for HPLC of the final compounds are the
following: column ) Spherisorb S5 SCX, 5 µm, 250 mm × 4.6
4 2 4 3
mm, mobile phase ) 50 mM (NH )H PO /CH CN 60/40 (v/v),
pH 3.7 with H
UV detectors); or Spherisorb-C1, 5 µm, 250 mm × 4.6 mm,
mobile phase ) 50 mM KH PO /CH CN 60/40 (v/v), pH ) as
3 4
PO , T ) 30 °C, flow ) 0.75 mL/min, IR and
2
4
3
it was, T ) 30 °C, flow ) 0.75 mL/min, RI and UV detectors).
To exclude contamination from long-chain impurities, the
following was used: symmetry C18 3 µm, 75 mm × 4.6 mm,
3 2 3 4
mobile phase ) CH CN/H O 80/20 (v/v), pH 3.7 with H PO ,
Because inhibition of CPT II, the CPT isoform com-
mon to all tissues, may at least in part nullify efforts to
obtain a selective liver-specific inhibitor, we also tested
the ability of oleate-treated hepatocytes, preincubated
with compound 17, to transform oleoylcarnitine (origi-
nating from oleate itself) again into oleoyl-CoA. Higher
levels of oleoylcarnitine in treated hepatocytes with
respect to control values are therefore indicators of the
inhibition of CPT II activity. Compound 17 (3 mM) did
not show any variation in oleoylcarnitine levels with
respect to control values (Figure 4), while the known
closely related CPT inhibitor (R)-N-palmitoylaminocar-
nitine22 (1 mM) showed about a 10-fold increment, thus
showing a strong CPT II inhibiting activity.
T ) 25 °C, flow ) 1.50 mL/min, RI and UV detectors).
R)-Am in oca r n itin e Isobu tyl Ester Ch lor id e Hyd r o-
(
ch lor id e ((R)-6, (Isobu tyl (R)-4-Tr im eth yla m m on io-3-
a m in obu tyr a te Ch lor id e Hyd r och lor id e). A suspension of
(R)-aminocarnitine, (R)-5 (3.0 g, 18.72 mmol), in isobutyl
alcohol (120 mL) at 4 °C (ice bath) was saturated with gaseous
HCl, and the resulting solution was refluxed for 18 h (oil bath
temperature ) 130 °C). The solvent was evaporated under
vacuum, and the residue was triturated with ethyl ether to
yield the product as a white solid (5.1 g, 95% yield): mp 115-
1
16 °C (dec); [R]20
+ 6.8° (c 0.5, MeOH); H NMR (200 MHz,
1
D
D
3
2
O) δ 4.35 (m, 1H), 4.00 (d, 2H), 3.88 (d, 2H), 3.28 (s, 9H),
.10 (m, 2H), 1.95 (m, 1H), 0.90 (d, 6H); KF ) 1.0%. Anal.
), C, H, N.
Alternatively, SOCl (molar ratio 6:1) was used instead of
11 2 2 2
(C H26Cl N O
2
gaseous HCl and the amount of isobutyl alcohol was reduced
600 mL for 10 g of aminocarnitine). The residue obtained after
(
In a preliminary stability test, at pH values simulat-
ing those of stomach and gut conditions (pH 2.0 and 8.5,
respectively), 17 showed complete stability. Once again,
the closely related (R)-N-palmitoylaminocarnitine be-
2 2
trituration with ethyl ether was also triturated with CH Cl
to give (R)-6 (96% yield).
(
S)-Am in oca r n itin e Isobu tyl Ester Ch lor id e Hyd r o-
ch lor id e ((S)-6). The compound was prepared as described

Carnitine Palmitoyltransferase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 307
in the case of (R)-6, but starting from (S)-aminocarnitine (S)-
δ 4.56-4.46 (m, 1H), 4.12-4.02 (m, 2H), 3.94-3.88 (m, 2H),
3.66-3.50 (m, 2H), 3.20 (s, 9H), 2.74-2.66 (m, 2H), 2.05-1.86
(m, 1H), 1.68-1.56 (m, 2H), 1.45-1.25 (m, 10H ), 1.05-0.85
2
0
5
0
: mp 116-117 °C (dec); [R]
.8%. Anal. (C11 ), C, H, N.
R)-4-Tr im eth ylam m on io-3-[(dodecan esu lfon yl)am in o]-
bu tyr a te (7). NEt (2.65 mL, 1.92 g, 19 mmol) and dodecane-
sulfonyl chloride (2.33 g, 8.65 mmol, in 3 mL of dry CH Cl
were added to a solution of (R)-aminocarnitine isobutyl ester
chloride hydrochloride (R)-6 (1.0 g, 3.46 mmol) in dry CH Cl
5 mL) at 0 °C. The mixture was left under stirring for 72 h at
D
- 6.9° (c 0.5, MeOH); KF )
2 2 2
H26Cl N O
(m, 9H). This intermediate was transferred into an Amberlite
(
-
IRA-402 column (OH form) and eluted with water. The water
was evaporated under vacuum, and the residue was triturated
with acetone and filtered to give compound 11 as a white solid
3
2
2
)
2
0
in 94% yield (66% yield from 6): mp 155-156 °C (dec); [R]
D
2
2
1
-
14.2° (c 0.5, H
H), 4.05 (t, 2H), 3.52 (d, 2H), 3.20 (s, 9H), 2.42 (d, 2H), 1.60
m, 2H), 1.42-1.22 (m, 10H), 0.90 (brt, 3H); TLC, silica gel,
2 3
O); H NMR (CD OD, 300 MHz) δ 4.40 (m,
(
1
(
room temperature. The solvent was evaporated under vacuum,
the residue was taken up in EtOAc (100 mL), and the white
CHCl3/iPrOH/MeOH/H
.6; MS (ES) m/z 317 (MH ); KF ) 5.7%. Anal. (C16
C, H, N.
2
O/CH
3
COOH 42:7:28:10.5:10.5; R
f
)
4
)
precipitate (NEt ‚HCl) thus obtained was filtered off under
3
+
0
H
32
N
2
O
vacuum. The EtOAc solution was evaporated under vacuum
to give 2.8 g of yellow oil; 1 N NaOH (71 mL) was then added
to the oil in order to perform ester hydrolysis, leaving the
suspension under stirring overnight. After evaporation under
vacuum to dryness, the crude product was purified by flash
chromatography using MeOH as eluant to yield 597 mg of 7
(S)-4-Tr im eth yla m m on io-3-[(octyloxyca r bon yl)a m in o]-
bu tyr a te (12). The product was prepared as described in the
case of (R)-11, but starting from (S)-6: mp ) 157-158 °C (dec);
[R]20
H, N.
R)-4-Tr im et h yla m m on io-3-[(t et r a d ecylca r b a m oyl)-
a m in o]bu tyr a te (17). Meth od A. Tetradecyl isocyanate
D
2
32 2 4
+13.6° (c 0.46, H O); KF ) 2.1%. Anal. (C16H N O ) C,
2
0
(
44% yield): mp 157-158 °C (dec); [R]
D
-7° (c 0.265, H
OD, 300 MHz) δ 4.30 (m, 1H), 3.45 (m, 2H), 3.28
s, 9H), 3.15 (m, 2H), 2.45 (d, 2H), 1.78 (m, 2H), 1.45 (m, 2H),
.30 (brs, 16H), 0.90 (brt, 3H); TLC, eluant CHCl /iPrOH/
MeOH/H O/CH COOH 42:7:28:10.5:10.5, R ) 0.6; MS (ES) m/z
93 (MH ); KF 6.9%. Anal. (C19 S) C, H, N.
S)-4-Tr im eth yla m m on io-3-[(d od eca n esu lfon yl)a m in o]-
2
O);
1
H NMR (CD
3
(
(
1
3
(25.10 mL, 21.81 g, 91.12 mmol) was added to a mixture of
2
3
f
(R)-aminocarnitine (R)-5 (7.30 g, 45.56 mmol) in anhydrous
+
3
40 2 4
H N O
DMSO (350 mL), and the reaction mixture was left for 60 h
at 40 °C. The mixture was transferred into an Erlenmeyer
flask (3 L) containing ethyl ether (2.5 L), and the solvent was
(
bu tyr a te (8). The product was prepared as described in the
case of (R)-7, but starting from (S)-6: mp 156-157 °C (dec);
3
separated by decanting. The residue was dissolved in CHCl ,
2
0
[
N.
R]
D
+6° (c 0.245, H
2
O); KF 8.6%. Anal. (C19
H
40
N
2
O
4
S) C, H,
transferred into a flask, and precipitated again with ethyl
ether. The crude product thus obtained was washed several
times with ethyl ether and purified by silica gel chromatog-
(
R)-4-Tr im eth ylam m on io-3-[(u n decylsu lfam oyl)am in o]-
bu tyr a te (9). A solution of SO Cl (1.7 mL, 2.86 g, 21.16 mmol)
in dry CH Cl ) (10 mL) was added dropwise to a solution of
R)-aminocarnitine isobutylester chloride hydrochloride (R)-6
2 g, 6.9 mmol) and NEt (3.84 mL, 2.79 g, 27.6 mmol) in dry
Cl (40 mL) at 0°C. The mixture was left 3 h under stirring
at room temperature. Then, additional NEt (1.92 mL, 1.39 g,
3.8 mmol) and undecylamine (2.97 mL, 2.36 g, 13.8 mmol)
raphy, using a MeOH/CHCl
until elution of impurities with higher R
product with MeOH/CHCl 9:1. Compound 17 was obtained
3
1:9 to MeOH/CHCl
3
8:2 gradient
2
2
f
, then eluting the
2
2
(
(
3
as a white solid (10.38 g, 57% yield): mp 160-162 °C (dec);
3
2
0
1
[
4
2
R]
D
-21.1° (c 0.5, MeOH); H NMR (CD OD, 300 MHz) δ
3
CH
2
2
.52 (m, 1H), 3.60 (dd, 1H), 3.48 (d, 1H), 3.20 (s, 9H), 3.10 (t,
H), 2.40 (m, 2H), 1.45 (m, 2H), 1.28 (brs, 22H), 0.90 (brt, 3H);
3
1
TLC, silica gel, CHCl
0.5:10.5; R ) 0.5; MS (ES) m/z 400 (MH ); KF ) 2.5%. Anal.
) C, H, N.
Meth od B. To a solution of (R)-aminocarnitine (R)-5 (22.0
3 2 3
/iPrOH/MeOH/H O/CH COOH 42:7:28:
were added, and the mixture was left overnight under stirring
at room temperature. The solvent was evaporated under
vacuum, the residue was taken up with EtOAc (100 mL), and
+
1
f
22 45 3 3
(C H N O
the precipitate (NEt ‚HCl) was filtered off under vacuum. The
3
EtOAc solution was evaporated under vacuum to yield 4.8 g
of yellow oil: 1 N NaOH (105 mL) was then added to the oil
in order to perform ester hydrolysis, leaving the suspension
under stirring overnight at room temperature. After evapora-
tion under vacuum to dryness, the crude yellow product was
g, 137.5 mmol) in MeOH (120 mL), tetrabutylammonium
chloride (200 mg) and, slowly, tetradecylisocyanate (44 mL,
38.23 g, 160 mmol) were added under stirring, keeping the
temperature at 5°C (tetradecyl isocyanate was added in about
1.5 h; once the first 10 mL was added, the solution became a
suspension). The suspension was stirred at 5 °C for one night,
and additional MeOH (120 mL) was added. The suspension
was filtered, and the filtrate was evaporated under vacuum.
Acetone (100 mL) was added to the resulting oil to yield a
cream-colored solid. After one night, the solid was separated
by filtration, washed with acetone, and dried under vacuum.
About 40 g of product 17 thus obtained were dissolved in
MeOH (73 mL) and filtered on Celite. The filtrate was
transferred into a flask containing acetone (690 mL) under
stirring, giving at first a suspension that at the end of the
operation became a clear solution. After 3 days at 5 °C, 28.8 g
of a light cream-colored solid was obtained (yield ) 52%;
additional crops, usually slightly less pure, can be obtained
from the liquor by standing at 5°C).
purified by flash chromatography using CHCl
/1 to 1/9) as eluant. The product thus obtained was further
3
/MeOH (from
9
purified by flash chromatography using MeOH as eluant, to
yield, after solvent evaporation under vacuum, compound 9
2
0
in 38% yield: mp 157-159 °C (dec); [R]
D
-4° (c 0.25, H
OD, 300 MHz) δ 4.10 (m, 1H),
.45 (d, 2H), 3.25 (s, 9H), 2.95 (m, 2H), 2.50 (m, 2H), 1.55
brm, 2H), 1.35 (brs, 16H), 0.90 (brt, 3H); TLC, silica gel,
2
O,
1
pH 2 with HCl); H NMR (CD
3
3
(
CHCl
.7; MS (ES) m/z 394 (MH ); KF 4.7%. Anal. (C18
C, H, N.
S)-4-Tr im eth yla m m on io-3-[(u n d ecylsu lfa m oyl)a m in o]-
3
/iPrOH/MeOH/H
2
O/CH
3
COOH 42:7:28:10.5:10.5, R
f
)
S)
+
0
H
39
N
3
O
4
(
bu tyr a te (10). The product was prepared as described in the
case of (R)-9, but starting from (S)-6: mp 152-154 °C (dec);
2
0
[
R]
D
+4° (c 0.25%, H
39 3 4
N O S) C, H, N.
2
O, pH 2 with HCl); KF 2.9%. Anal.
Alternatively, the crude product obtained by evaporation of
the reaction solvent (MeOH) was purified by chromatography
as described in method A.
(C
18
H
(
R)-4-Tr im eth yla m m on io-3-[(octyloxyca r bon yl)a m in o]-
bu tyr a te (11). Octyl chloroformate (2.52 g, 13 mmol) and NEt
1.8 mL, 1.31 g, 13 mmmol) were added to a solution of (R)-
aminocarnitine isobutyl ester chloride hydrochloride (R)-6 (1.5
g, 5.2 mmol) in anhydrous CH Cl (30 mL). The reaction was
3
(R)-4-Tr im eth yla m m on io-3-[(h eptylca r ba m oyl)a m in o]-
bu tyr a te (13). The compound was prepared as described in
the case of 17 (method A), starting from heptyl isocyanate,
and was further purified after chromatography by crystalliza-
(
2
2
2
0
left 18 h under stirring at room temperature, the solvent was
evaporated under vacuum, and the residue was dissolved in
EtOAc and filtered. The filtrate was evaporated under vacuum
to give a residue that was purified by silica gel flash chroma-
tion (CH
3
CN), giving a 47% yield: mp 149-150 °C (dec); [R]
D
1
-34.0° (c 0.97, MeOH); H NMR (CD
3
OD, 300 MHz) δ 4.52
(m, 1H), 3.60 (dd, 1H), 3.48 (d, 1H), 3.20 (s, 9H), 3.10 (t, 2H),
2.40 (m, 2H), 1.45 (m, 2H), 1.30 (brs, 8H), 0.90 (brt, 3H); TLC,
tography, using a CHCl
3
to CHCl
3
/CH
3
OH 9:1 gradient to give
silica gel, CHCl
3
/iPrOH/MeOH/H
) 0.5; MS (ES) m/z 302 (MH ); KF ) 6.2%. Anal.
) C, H, N.
2 3
O/CH COOH 42:7:28:10.5:
+
1
8
.5 g (71% yield) of the intermediate ester compound: mp 80-
10.5, R
f
2
0
1
2 °C, [R]
D
-15.2° (c 0.45, H
2
O); H NMR (CD
3
OD, 300 MHz)
(C15
H
31
N
3 3
O

3
08 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
R)-4-Tr im eth yla m m on io-3-[(n on ylca r ba m oyl)a m in o]-
Giannessi et al.
(
Deter m in a tion of â-Hyd r oxybu tyr a te Levels in Ser u m
of F a sted Ra ts Tr ea ted w ith CP T I In h ibitor s in Vivo.
Normally fed Sprague-Dawley male rats (provided by Charles
River Italia) (200-225 g) maintained at 22 ( 2 °C on a 12 h
light-dark cycle fasted for 24 h and subsequently treated
orally with 17 (10, 20, 30, and 40 mg/kg). At 0, 2, 4, 6, 8, 10,
12 h after treatment, blood was collected from the tail vein
and serum was obtained after centrifugation. â-Hydroxybu-
tyrate concentrations were determined by commercial kits
(Sigma) in a Cobas Mira (Roche) autoanalyzer. Results are
expressed as the mean ( SE of six animals per group.
Eva lu a tion of CP T II Activity In h ibition in Vitr o.
Hepatocytes were isolated from 24 h fasted Sprague-Dawley
male rats (225-250 g), provided by Harlan Italia. Liver cells
were incubated for 30 min at 37 °C in 0.7 mL of gassed (95:5
bu tyr a te (14). The compound was prepared as described in
the case of 17 (method A), starting from nonyl isocyanate,
2
0
giving a 68% yield: mp 145-147 °C (dec); [R]
D
-15.4° (c 0.5,
O, 300 MHz) δ 4.52 (m, 1H), 3.60 (dd, 1H),
.45 (d, 1H), 3.18 (s, 9H), 3.10 (t, 2H), 2.40 (d, 2H), 1.45 (m,
H), 1.28 (brs, 12H), 0.90 (brt, 3H); TLC, silica gel, CHCl
O/CH COOH 42:7:28:10.5:10.5, R ) 0.5; MS
ES) m/z 330 (MH ); KF ) 2.8%. Anal. (C17 ) C, H, N.
R)-4-Tr im eth yla m m on io-3-[(u n d ecylca r ba m oyl)a m i-
1
2 2
H O); H NMR (D
3
2
iPrOH/MeOH/H
(
3
/
2
3
f
+
35 3 3
H N O
(
n o]bu tyr a te (15). The compound was prepared as described
in the case of 17 (method A), starting from undecyl isocyanate,
and further purified after chromatography by crystallization
2
0
(CH
3
CN), giving a 50% yield: mp 150-151 °C (dec); [R]
D
1
-
21.2° (c 1, MeOH); H NMR (CD OD, 300 MHz) δ 4.52 (m,
3
1
H), 3.60 (dd, 1H), 3.48 (d, 1H), 3.20 (s, 9H), 3.10 (t, 2H), 2.40
m, 2H), 1.45 (m, 2H), 1.28 (brs, 16H), 0.90 (brt, 3H); TLC,
silica gel, CHCl /iPrOH/MeOH/H O/CH COOH 42:7:28:10.5:
) 0.5; MS (ES) m/z 358 (MH ); KF ) 2.3%. Anal.
) C, H, N.
O
2
/CO
2
) Krebs medium containing 2% defatted bovine serum
albumin (BSA), 1 mM glucose, 1 mM pyruvate, 9 mM lactate,
(
1
4
3
2
3
+
100 mM [ C]-L-carnitine (125700 dpm/nmol), and 17 (3 mM)
or (R)-N-palmitoylaminocarnitine (1 mM). At these concentra-
tions, 17 and (R)-N-palmitoylaminocarnitine markedly de-
creased ketone body production. After 30 min of preincubation
with test compounds, 1 mM sodium oleate was added to the
incubation mixture, and 2 h later the incubation was stopped
1
0.5, R
f
(C
19
H
39
N
3 3
O
(
R)-4-Tr im eth yla m m on io-3-[(d od ecylca r ba m oyl)a m i-
n o]bu tyr a te (16). The compound was prepared as described
in the case of 17 (method A), starting from dodecyl isocyanate,
2
0
giving a 55% yield: mp 147-149 °C (dec); [R]
D
-24.6° (c 0.48,
OD, 300 MHz) δ 4.52 (m, 1H), 3.60 (dd,
H), 3.48 (d, 1H), 3.20 (s, 9H), 3.10 (t, 2H), 2.40 (m, 2H), 1.45
m, 2H), 1.28 (brs, 18H), 0.90 (brt, 3H); TLC, silica gel, CHCl
iPrOH/MeOH/H O/CH COOH 42:7:28:10.5:10.5, R ) 0.6; MS
ES) m/z 372 (MH ); KF ) 5.4%. Anal. (C20 ) C, H, N.
S)-4-Tr im eth yla m m on io-3-[(n on ylca r ba m oyl)a m in o]-
with 5% HClO
4
. The pelleted sample, containing long-chain
1
14
MeOH); H NMR (CD
3
acylcarnitine (oleoyl-[ C]-L-carnitine), was washed twice with
1
4
1
(
4
5% HClO and was dissolved in 1 N KOH, and then the [ C]-
3
/
L-carnitine radioactivity was measured. Values are the mean
( SE for three determinations done in duplicate.
2
3
f
+
(
H
41
N
3
O
3
Deter m in a tion of LDH Levels in Vitr o. Levels of LDH
were evaluated using the method described in ref 18 by
commercial kits with a Cobas Mira S autoanalyzer after
incubation for 48 h of test compounds at different concentra-
tions (10, 50, 100 µM) on hepatocytes obtained from Wistar
male rats liver. These last were isolated and prepared for
experiment as described in ref 23.
(
bu tyr a te (18). The compound was prepared as described in
the case of 17 (method A), starting from nonyl isocyanate and
2
0
(
H
S)-aminocarnitine: mp 146-147 °C (dec); [R]
O); KF ) 1.8%. Anal. (C17 ) C, H, N.
S)-4-Tr im eth ylam m on io-3-[(tetr adecylcar bam oyl)am i-
D
+16.7° (c 0.43,
2
35 3 3
H N O
(
n o]bu tyr a te (19). The compound was prepared as described
in the case of 17 (Method A), starting from tetradecyl isocy-
K
i
Assa y Deter m in a tion on CP T I Exp r essed in P ich ia
pa stor is. K and K values were determined by measuring
L-CPT 1 activity radiochemically at different concentrations
i
m
2
0
anate; mp 166-167 °C (dec); [R]
1.7%. Anal. (C22 ) C, H, N.
P h a r m a cology. Deter m in a tion of CP T I Activity In -
D
+20.7° (c 0.5, MeOH); KF
)
H
45
N
3
O
3
of palmitoyl-CoA (0-200 µmol/L) and 17 (0.5 and 1.0 µmol/L)
1
4
24
and at fixed [ C]-L-carnitine (500 µmol/L) concentrations on
h ibition in Vitr o. CPT I inhibition was evaluated with minor
modifications as described in ref 17 on fresh intact liver or
heart mitochondrial preparations obtained from normally fed
Sprague-Dawley male rats (provided by Charles River Ita-
lia). Mitochondria were isolated from liver or heart and
suspended in a buffer containing 75 mM (for heart) or 225 mM
yeast extract expressing L-CPT 1, obtained as described in ref
1
4
25. The [ C]-palmitoylcarnitine formed was measured. Specific
-1
-1
activity of CPT 1 was expressed as nmol min mg of protein.
Data were fitted according to the Michaelis-Menten equation
to obtain K or according to the equation for mixed inhibition
m
of 17 at two concentrations (0.5 and 1.0 µmol/L) to obtain K .
i
1
6
(
for liver) sucrose, 1 mM EGTA (pH 7.5), mannitol 225 mM,
Curve fitting was performed using GraphPad Prisma software
with nonlinear regression analysis and Excel software using
linear regression analysis. K values were obtained by plotting
i
and 5 mM Tris-HCl. The assay medium (100 µL) containing
1
2
20 mM KCl, 1 mM EGTA (pH 7.5), 10 mM HEPES (pH 7.5),
1
4
0 mM sucrose, 1 mM DTT, 8.3 µM BSA, 50 mM [ C]-
inhibitor concentrations vs Kmapp/Vmaxapp ratio.
palmitoyl-CoA (specific activity 10000 DPM/mole), and 10 mM
L-carnitine was incubated at 37 °C in the presence of scalar
concentrations of the test compound (0-3 mM). Reaction time
Data are expressed as mean ( standard deviation, and the
2
correlation coefficient (r ) was used to evaluate the quality of
the fitting.
1
min. Data are the mean of two determinations in triplicate.
Ack n ow led gm en t. The authors thank Giuseppina
Calvisi, Enrico De Fusco, Emanuele Pani, Alessandro
Peschechera, Federico Serafini, and Ornella Petrella for
technical assistence. They also thank Drs. Enrico Am-
mannati (Argus Chemicals) and Angelo De Luca for
their contribution to the chemical development of (R)-5
and 17, and Ms. Marlene Deutsch for her copyreading
skills.
The variation between experiments was less than 8%.
An tid ia betic Activity in Vivo. Male C57BL/KsJ -db/db
mice were obtained at 6 weeks of age from J ackson Labora-
tories and maintained at 25 ( 2 °C on a 12 h light-dark cycle.
Animals were given standard laboratory food and water, ad
libitum, and were used for experiments at 8 weeks of age.
Eight animals were used in each treatment group, and test
compounds were administered orally at doses equimolar to 17
(50 mg/kg), twice a day for 45 days. The control animals were
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
final compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
given vehicle carboxymethylcellulose (0.5% in deionized H O,
2
dose of 10 mL/kg). At sacrifice, blood samples were collected
after 9 h from the last treatment (animals in postabsorptive
state fasted for 9 h). The glucose serum levels were determined
in a Cobas Mira S (Roche) autoanalyzer using commercial kits
Refer en ces
(
Roche), and the percent reduction values with respect to
(1) DeFronzo, R. A.; Bonadonna, R. C.; Ferranini, E. Pathogenesis
of NIDDM: a balanced overview. Diabetes Care 1992, 15, 318-
untreated control db/db mice were calculated as follows:
3
68.
(
2) Golay, A.; Swislocki, A. L. M.; Chen, Y. D.; Reaven, G. M.
Relationships between plasma-free fatty acid concentration,
endogenous glucose production, and fasting hyperglycemia in
normal and non-insulin-dependent diabetic individuals. Metabo-
lism 1987, 36, 692-696.
(serum glucose in treated mice) -
(
serum glucose in control mice)
×
100.
serum glucose in control mice

Carnitine Palmitoyltransferase Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 309
(
3) McGarry, J . D.; Brown, N. F. The mitochondrial carnitine
palmitoyltransferase system. From concept to molecular analy-
sis. Eur. J . Biochem. 1997, 244, 1-14.
(16) (a) Kerner, J .; Bieber, L. Isolation of a malonyl-CoA sensitive
CPT/â-oxidation enzyme complex from heart mitochondria.
Biochemistry 1990, 29, 4326-4334. (b) Hovius, R.; Lambrescts,
H.; Nicolay, K.; De Kruijff, B. Improved methods to isolate and
subfractionate rat liver mitochondria. Lipid composition of the
inner and outer membrane. Biochim. Biophys. Acta 1990, 1021,
217-226.
(
4) Attwood, P. A.; Keech, D. B. Pyruvate carboxylase. Curr. Top.
Cell. Regul. 1984, 23, 1-55.
(
5) Foley, J . E. Rationale and Application of Fatty Acid Oxidation
Inhibitors in Treatment of Diabetes Mellitus. Diabetes Care
1
992, 15, 773-784.
(
17) Fiol, C. J .; Kerner, J .; Bieber, L. L. Effect of malonyl-CoA on
the kinetics and substrate cooperativity of membrane-bound
carnitine palmitoyltransferase of rat heart mitochondria. Bio-
chim Biophys. Acta 1987, 916 (3), 482-492.
(
6) Anderson, R. Carnitine Palmitoyltransferase: A Viable Target
for the Treatment of NIDDM? Curr. Pharm. Des. 1998, 4, 1-15.
7) Bressler, R.; Gay, R.; Copeland, J . G.; Bahl, J . J .; Bedotto, J .;
Goldman, S. Chronic inhibition of fatty acid oxidation: New
model of diastolic dysfunction. Life Sci. 1989, 44, 1897-1906.
8) Deems, R. O.; Anderson, R. C.; Foley, J . E. Hypoglycemic effects
of a novel fatty acid oxidation inhibitor in rats and monkeys.
Am. J . Physiol. 1998, 274, R524-R528.
(
(
(
(
18) Vassault, A. UV-method with pyruvate and NADH. In Methods
of Enzymatic Analysis, 3rd ed.; Bergmeyer, H. U., Ed.; Verlag
Chemie: Weinheim, Germany, 1983; Vol. 3, pp 118-126.
19) Kodama, H.; Fujita, M.; Yamaguchi, I. Development of hyper-
glycaemia and insulin resistance in conscious genetically diabetic
(
(
9) Anderson, R. C.; Balestra, M.; Bell, P. A.; Deems, R. O.; Fillers,
W. S.; Foley, J . E.; Fraser, J . D.; Mann, W. R.; Rudin, M.;
Villhauer, E. B. Antidiabetic Agents: A New Class of Reversible
Carnitine Palmitoyltranferase I Inhibitors. J . Med. Chem. 1995,
(C57BL/KsJ db/db) mice. Diabetologia 1994, 37, 739-744.
20) Boden, G.; Shulman, G. I. Free fatty acids in obesity and type 2
diabetes: defining their role in the development of insulin
resistance and â-cell dysfunction. Eur. J . Clin. Invest. 2002, 32
3
8, 3448-3450.
(Suppl. 3), 14-23.
(
10) Giannessi, F.; Chiodi, P.; Marzi, M.; Minetti, P.; Pessotto, P.;
De Angelis, F.; Tassoni, E.; Conti, R.; Giorgi, F.; Mabilia, M.;
Dell’Uomo, N.; Muck, S.; Tinti, M. O.; Carminati, P.; Arduini,
A. Reversible Carnitine Palmitoyltransferase Inhibitors with
Broad Chemical Diversity as Potential Antidiabetic Agents. J .
Med. Chem. 2001, 44, 2383-2386.
11) J enkins, D. L.; Griffith, O. W. Antiketogenic and Hypoglycemic
Effects of Aminocarnitine and Acylaminocarnitines. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 290-294.
(21) Zammit, V. A. Regulation of ketone body metabolism. Diabetes
Rev. 1994, 2, 132-154.
(
22) Shinagawa, S.; Kanamaru, T.; Harada, S.; Asai, M.; Okazaki,
H. Chemistry and inhibitory activity of long chain fatty acid
oxidation of emeriamine and its analogues. J . Med. Chem. 1987,
3
0, 1458-1463.
(
(
23) Venerando, R.; Miotto, G.; Kadowaki, M.; Siliprandi, N.; Mor-
timore, G. E. Multiphasic control of proteolysis by leucine and
alanine in the isolated rat hepatocyte. Am. J . Physiol. 1994, 266,
C455-C461.
(
12) Savle, P. S.; Pande, S. V.; Lee, T. S.; Gandour, R. D. Stereoiso-
meric Acylamidomorpholinium Carnitine Analogues: Selective
Inhibitors of Carnitine Palmitoyltransferase I and II. Bioorg.
Med. Chem. Lett. 1999, 9, 3099-3102.
(24) Zammit, V. A.; Fraser, F.; Padovese, R. Distinct Kinetics of
Carnitine Palmitoyltransferase I in Contact Sites and Outer
Membranes of Rat Liver Mitochondria. J . Biol. Chem. 2001, 276
(23), 20182-20185.
(
(
(
13) Carrol, M. F.; Schade, D. S. Ten Pivotal Questions about Diabetic
Ketoacidosis. Postgrad. Med. 2001, 110 (5), 89-95.
14) Felner, E. I.; White, P. C. Improving Management of Diabetic
Ketoacidosis in Children. Pediatrics 2001, 108 (3), 735-740.
15) Castagnani, R.; De Angelis, F.; De Fusco, E.; Giannessi, F.;
Misiti, D.; Meloni, D.; Tinti, M. O. Stereospecific Synthesis of
(25) J ackson, V.; Cameron, J . M.; Fraser, F.; Zammit, V. A.; Price,
N. T. Use of Six Chimeric Proteins To Investigate the Role of
Intramolecular Interactions in Determining the Kinetics of
Carnitine Palmitoyltransferase I Isoforms. J . Biol. Chem. 2000,
275 (26), 19560-19566.
(
R)-Aminocarnitine (Emeriamine) Starting from (R)-Carnitine
via Double Inversion of Configuration. J . Org. Chem. 1995, 60,
318-8319.
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