U.S. patent application number 14/692846 was filed with the patent office on 2016-03-17 for pyrazole-amide compounds and pharmaceutical use thereof.
The applicant listed for this patent is JAPAN TOBACCO INC.. Invention is credited to Takahisa Motomura, Gakujun Shomi.
Application Number | 20160074364 14/692846 |
Document ID | / |
Family ID | 51536938 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160074364 |
Kind Code |
A1 |
Motomura; Takahisa ; et
al. |
March 17, 2016 |
PYRAZOLE-AMIDE COMPOUNDS AND PHARMACEUTICAL USE THEREOF
Abstract
A compound represented by the following formula: ##STR00001##
wherein n is 1 or 2, ##STR00002## or a pharmaceutically acceptable
salt thereof, and a pharmaceutical use thereof.
Inventors: |
Motomura; Takahisa;
(Takatsuki, JP) ; Shomi; Gakujun; (Takatsuki,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JAPAN TOBACCO INC. |
Tokyo |
|
JP |
|
|
Family ID: |
51536938 |
Appl. No.: |
14/692846 |
Filed: |
April 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14210746 |
Mar 14, 2014 |
9040717 |
|
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14692846 |
|
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61791164 |
Mar 15, 2013 |
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Current U.S.
Class: |
514/406 |
Current CPC
Class: |
A61P 13/00 20180101;
A61K 31/415 20130101; A61P 27/02 20180101; A61P 3/00 20180101; A61P
3/04 20180101; A61P 3/06 20180101; A61P 9/00 20180101; A61P 27/00
20180101; A61P 3/10 20180101; A61P 25/00 20180101; A61P 9/06
20180101; A61P 27/12 20180101; A61P 13/12 20180101; A61P 9/12
20180101; A61P 11/00 20180101; A61P 35/00 20180101; C07D 231/12
20130101; A61P 9/04 20180101; A61P 9/10 20180101; A61P 43/00
20180101 |
International
Class: |
A61K 31/415 20060101
A61K031/415 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2013 |
JP |
2013-053195 |
Jun 18, 2013 |
JP |
2013-127318 |
Claims
1.-11. (canceled)
12. A method for the prophylaxis of diabetes, insulin resistance
syndrome, metabolic syndrome, hyperglycemia, hyperlactacidemia,
diabetic complications, cardiac failure, cardiomyopathy, myocardial
ischemia, myocardial infarction, angina pectoris, dyslipidemia,
atherosclerosis, peripheral arterial disease, intermittent
claudication, chronic obstructive pulmonary disease, brain
ischemia, cerebral apoplexy, mitochondrial disease, mitochondrial
encephalomyopathy, cancer or pulmonary hypertension in a mammal,
comprising administering a pharmaceutically effective amount of a
compound selected from the group consisting of: i) a compound
represented by formula [I]: ##STR00043## wherein n is 1 or 2, ii) a
compound represented by formula [II]: ##STR00044## iii) a compound
represented by formula [IIh]: ##STR00045## and iv) a compound
represented by formula [III]: ##STR00046## and pharmaceutically
acceptable salts thereof, to the mammal.
13. The method according to claim 12, wherein the diabetes is type
1 diabetes or type 2 diabetes.
14. The method according to claim 12, wherein the diabetic
complications are selected from the group consisting of diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy and
cataract.
15. The method according to claim 12, wherein the cardiac failure
is acute cardiac failure or chronic cardiac failure.
16. The method according to claim 13, wherein the diabetes is type
2 diabetes.
17. The method according to claim 12, wherein the compound is
represented by formula [I].
18. The method according to claim 12, wherein the compound is
represented by formula [II].
19. The method according to claim 12, wherein the compound is
represented by formula [IIh].
20. The method according to claim 12, wherein the compound is
represented by formula [III].
Description
TECHNICAL FIELD
[0001] The present invention provides a pyrazole-amide compound and
a pharmaceutical use thereof. More particularly, the present
invention relates to a pyrazole-amide compound or a
pharmaceutically acceptable salt thereof having a pyruvate
dehydrogenase kinase (hereinafter to be abbreviated as PDHK)
inhibitory activity, a pharmaceutical composition containing the
same, a prophylactic or therapeutic agent containing the same for
diabetes (type 1 diabetes, type 2 diabetes etc.), insulin
resistance syndrome, metabolic syndrome, hyperglycemia,
hyperlactacidemia, diabetic complications (diabetic neuropathy,
diabetic retinopathy, diabetic nephropathy, cataract etc.), cardiac
failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer, pulmonary hypertension, or
Alzheimer disease, and the like.
BACKGROUND ART
[0002] In tissues, for reactions using energy such as biosynthesis,
active transport, muscle contraction and the like, the energy is
supplied by hydrolysis of adenosine triphosphate (ATP). ATP is
produced by oxidation of metabolic fuel which yields much energy,
such as glucose and free fatty acids. In oxidative tissues such as
muscle, ATP is mostly produced from acetyl-CoA that enters citric
acid cycle. Acetyl-CoA is produced by oxidation of glucose via
glycolytic pathway or .beta. oxidation of free fatty acid. An
enzyme that plays a pivotal role in controlling acetyl-CoA
production from glucose is pyruvate dehydrogenase (hereinafter to
be abbreviated as PDH). PDH catalyzes reduction of nicotinamide
adenine dinucleotide (NAD) to NADH, simultaneously with oxidation
of pyruvic acid to acetyl-CoA and carbon dioxide (e.g., non-patent
documents 1, 2).
[0003] PDH is a multienzyme complex consisting of three enzyme
components (E1, E2 and E3) and some subunits localized in
mitochondrial matrix. E1, E2 and E3 are responsible for
decarboxylation from pyruvic acid, production of acetyl-CoA and
reduction of NAD to NADH, respectively.
[0004] Two classes of enzyme having regulatory function bind to
PDH. One is PDHK, which is a protein kinase having specificity to
PDH. The role thereof is to inactivate E1.alpha. subunit of the
complex by phosphorylation. The other is PDH phosphatase, which is
a specific protein phosphatase that activates PDH via
dephosphorylation of E1.alpha. subunit. The proportion of PDH in
its active (dephosphorylated) state is determined by the balance of
kinase activity and phosphatase activity. The kinase activity is
regulated by the relative concentration of metabolic substrates.
For example, the kinase activity is activated by an increase in
NADH/NAD, acetyl-CoA/CoA and ATP/adenosine diphosphate (ADP)
ratios, and inhibited by pyruvic acid (e.g., non-patent document
3).
[0005] In the tissues of mammals, 4 kinds of PDHK isozymes are
identified. Particularly, PDHK2 is expressed in a wide range of
tissues including the liver, skeletal muscles and adipose tissues
involved in glucose metabolism. Furthermore, since PDHK2 shows
comparatively high sensitivity to activation by increased NADH/NAD
or acetyl-CoA/CoA and inhibition by pyruvic acid, involvement in a
short-term regulation of glucose metabolism is suggested (e.g.,
non-patent document 4).
[0006] In addition, PDHK1 is expressed in large amounts in cardiac
muscle, skeletal muscle, pancreatic .beta. cell and the like.
Furthermore, since expression of PDHK1 is induced via activation of
hypoxia inducible factor (HIF) 1 in ischemic state, its involvement
in ischemic diseases and cancerous diseases is suggested (e.g.,
non-patent document 5).
[0007] In diseases such as insulin-dependent (type 1) diabetes,
non-insulin-dependent (type 2) diabetes and the like, oxidation of
lipids is promoted with simultaneous reduction in glucose
utilization. This reduction in glucose utilization is one of the
factors causing hyperglycemia. When the oxidative glucose
metabolism decreases in type 1 and type 2 diabetes and obesity, PDH
activity also decreases. It suggests involvement of reduced PDH
activity in the reduced glucose utilization in type 1 and type 2
diabetes (e.g., non-patent documents 6, 7).
[0008] On the contrary, hepatic gluconeogenesis is enhanced in type
1 and type 2 diabetes, which also forms one factor causing
hyperglycemia. The reduced PDH activity increases pyruvic acid
concentration, which in turn increases availability of lactic acid
as a substrate for hepatic gluconeogenesis. It suggests possible
involvement of reduced PDH activity in the enhanced gluconeogenesis
in type 1 and type 2 diabetes (e.g., non-patent documents 8, 9).
When PDH is activated by inhibition of PDHK, the rate of glucose
oxidation is considered to rise. As a result, glucose utilization
in the body is promoted and hepatic gluconeogenesis is suppressed,
whereby hyperglycemia in type 1 and type 2 diabetes is expected to
be improved (e.g., non-patent documents 10, 11, 12). Another factor
contributing to diabetes is impaired insulin secretion, which is
known to be associated with reduced PDH activity in pancreatic
.beta. cells, and introduction of PDHK1, 2 and 4 (e.g., non-patent
documents 13, 14). In addition, sustained hyperglycemia due to
diabetes is known to cause complications such as diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy and the
like. Thiamine and .alpha.-lipoic acid contribute to activation of
PDH as coenzymes. Thiamine and .alpha.-lipoic acid, or thiamine
derivative and .alpha.-lipoic acid derivative are shown to have a
promising effect on the treatment of diabetic complications. Thus,
activation of PDH is expected to improve diabetic complications
(e.g., non-patent documents 15, 16).
[0009] Under ischemic conditions, limited oxygen supply reduces
oxidation of both glucose and fatty acid and reduces the amount of
ATP produced by oxidative phosphorylation in the tissues. In the
absence of sufficient oxygen, ATP level is maintained by promoted
anaerobic glycolysis. As a result, lactic acid increases and
intracellular pH decreases. Even though the body tries to maintain
homeostasis of ion by energy consumption, abnormally low ATP level
and disrupted cellular osmolarity lead to cell death. In addition,
adenosine monophosphate-activating kinase, activated during
ischemia, phosphorylates and thus inactivates acetyl-CoA
carboxylase. The levels of total malonyl-CoA in the tissue drop,
carnitine palmitoyltransferase-I activity is therefore increased
and fatty acid oxidation is favored over glucose oxidation by
allowing the transport of acyl-CoA into mitochondria. Oxidation of
glucose is capable of yielding more ATP per molecule of oxygen than
is oxidation of fatty acids. Under ischemic conditions, therefore,
when energy metabolism becomes glucose oxidation dominant by
activation of PDH, the ability to maintain ATP level is considered
to be enhanced (e.g., non-patent document 17).
[0010] In addition, since activation of PDH causes oxidation of
pyruvic acid produced by glycolysis, and reducing production of
lactic acid, the net proton burden is considered to be reduced in
ischemic tissues. Accordingly, PDH activation by inhibition of PDHK
is expected to protectively act in ischemic diseases such as
cardiac muscle ischemia (e.g., non-patent documents 18, 19).
[0011] A drug that activates PDH by inhibition of PDHK is
considered to decrease lactate production since it promotes
pyruvate metabolism. Hence, such drug is expected to be useful for
the treatment of hyperlactacidemia such as mitochondrial disease,
mitochondrial encephalomyopathy and sepsis (e.g., non-patent
document 20).
[0012] In cancer cells, the expression of PDHK1 or 2 increases. In
cancer cells, moreover, ATP production by oxidative phosphorylation
in mitochondria decreases, and ATP production via the anaerobic
glycolysis in cytoplasm increases. PDH activation by inhibition of
PDHK is expected to promote oxidative phosphorylation in
mitochondria, and increase production of active oxygen, which will
induce apoptosis of cancer cells. Therefore, the PDH activation by
PDHK inhibition is useful for the treatment of cancerous diseases
(e.g., non-patent document 21).
[0013] Pulmonary hypertension is characterized by high blood
pressure caused by partial narrowing of the pulmonary artery due to
promoted cell proliferation therein. In pulmonary hypertension,
therefore, activation of PDH in the pulmonary artery cell is
expected to promote oxidative phosphorylation in mitochondria,
increase production of active oxygen, and induce apoptosis of the
pulmonary artery cells. Therefore, the PDH activation by PDHK
inhibition is considered to be useful for the treatment of
pulmonary hypertension (e.g., non-patent document 22).
[0014] Energy production and glucose metabolism in the cerebrum
decrease in Alzheimer disease, and also, PDH activity declines.
When the PDH activity declines, production of acetyl CoA decreases.
Acetyl CoA is utilized for ATP production in the electron transport
system via the citric acid cycle. Acetyl CoA is also a starting
material for synthesizing acetylcholine, which is one of the
neurotransmitters. Therefore, reduced brain PDH activity in
Alzheimer disease is considered to cause neuronal cell death due to
the decreased ATP production. Moreover, it is considered that
synthesis of acetylcholine, which is the transmitter for
cholinergic nerve, is inhibited to induce deterioration of memory
and the like. Activation of PDH in the brain is expected to enhance
energy production and acetylcholine synthesis in Alzheimer disease.
Therefore, activation of PDH by the inhibition of PDHK is
considered to be useful for the treatment of Alzheimer disease
(e.g., non-patent documents 23, 24).
[0015] It has been shown that dichloroacetic acid, which is a drug
having a PDH activating action, provides promising effects for the
treatment of diabetes, myocardial ischemia, myocardial infarction,
angina pectoris, cardiac failure, hyperlactacidemia, brain
ischemia, cerebral apoplexy, peripheral arterial disease, chronic
obstructive pulmonary disease, cancerous disease, and pulmonary
hypertension (e.g., non-patent documents 10, 18, 20, 22, 25, 26,
27).
[0016] From the foregoing findings, a PDHK inhibitor is considered
to be useful for the prophylaxis or treatment of diseases relating
to glucose utilization disorder, for example, diabetes (type 1
diabetes, type 2 diabetes etc.), insulin resistance syndrome,
metabolic syndrome, hyperglycemia, hyperlactacidemia, diabetic
complications (diabetic neuropathy, diabetic retinopathy, diabetic
nephropathy, cataract etc.). Furthermore, a PDHK inhibitor is
considered to be useful for the prophylaxis or treatment of
diseases caused by limited energy substrate supply to the tissues,
for example, cardiac failure (acute cardiac failure, chronic
cardiac failure), cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia and cerebral
apoplexy.
[0017] Therefore, a PDHK inhibitor is considered to be useful for
the treatment or prophylaxis of diabetes (type 1 diabetes, type 2
diabetes etc.), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract
etc.), cardiac failure (acute cardiac failure, chronic cardiac
failure), cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer,
pulmonary hypertension or Alzheimer disease.
DOCUMENT LIST
Non-Patent Documents
[0018] non-patent document 1: Reed L J, Hackert M L.
Structure-function relationships in dihydrolipoamide
acyltransferases. J Biol Chem. 1990 Jun. 5; 265(16):8971-4. [0019]
non-patent document 2: Patel M S, Roche T E. Molecular biology and
biochemistry of pyruvate dehydrogenase complexes. FASEB J. 1990
November; 4(14):3224-33. [0020] non-patent document 3: Sugden M C,
Holness M J. Recent advances in mechanisms regulating glucose
oxidation at the level of the pyruvate dehydrogenase complex by
PDK5. Am J Physiol Endocrinol Metab. 2003 May; 284(5):E855-62.
[0021] non-patent document 4: Bowker-Kinley M M, Davis W I, Wu P,
Harris R A, Popov K M. Evidence for existence of tissue-specific
regulation of the mammalian pyruvate dehydrogenase complex. Biochem
J. 1998 Jan. 1; 329 (Pt 1):191-6. [0022] non-patent document 5: Kim
J W, Tchernyshyov I, Semenza G L, Dang C V. HIF-1-mediated
expression of pyruvate dehydrogenase kinase: a metabolic switch
required for cellular adaptation to hypoxia. Cell Metab. 2006
March; 3(3):177-85. [0023] non-patent document 6: Morino K,
Petersen K F, Dufour S, Befroy D, Frattini J, Shatzkes N, et al.
Reduced mitochondrial density and increased IRS-1 serine
phosphorylation in muscle of insulin-resistant offspring of type 2
diabetic parents. J Clin Invest. 2005 December; 115(12):3587-93.
[0024] non-patent document 7: Caterson I D, Fuller S J, Randle P J.
Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic
acid on pyruvate dehydrogenase complex activity in starved and
alloxan-diabetic rats. Biochem J. 1982 Oct. 15; 208(1):53-60.
[0025] non-patent document 8: Boden G, Chen X, Stein T P.
Gluconeogenesis in moderately and severely hyperglycemic patients
with type 2 diabetes mellitus. Am J Physiol Endocrinol Metab. 2001
January; 280(1):E23-30. [0026] non-patent document 9: Shangraw R E,
Fisher D M. Pharmacokinetics and pharmacodynamics of
dichloroacetate in patients with cirrhosis. Clin Pharmacol Ther.
1999 October; 66(4):380-90. [0027] non-patent document 10:
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 Mar. 9; 298(10):526-30.
[0028] non-patent document 11: Mayers R M, Leighton B, Kilgour E.
PDH kinase inhibitors: a novel therapy for Type II diabetes?
Biochem Soc Trans. 2005 April; 33(Pt 2):367-70. [0029] non-patent
document 12: Jeoung N H, Rahimi Y, Wu P, Lee W N, Harris R A.
Fasting induces ketoacidosis and hypothermia in
PDHK2/PDHK4-double-knockout mice. Biochem J. 2012 May 1;
443(3):829-39. [0030] non-patent document 13: Zhou Y P, Berggren P
O, Grill V. A fatty acid-induced decrease in pyruvate dehydrogenase
activity is an important determinant of beta-cell dysfunction in
the obese diabetic db/db mouse. Diabetes. 1996 May; 45(5):580-6.
[0031] non-patent document 14: Xu J, Han J, Epstein P N, Liu Y Q.
Regulation of PDK mRNA by high fatty acid and glucose in pancreatic
islets. Biochem Biophys Res Commun. 2006 Jun. 9; 344(3):827-33.
[0032] non-patent document 15: Benfotiamine. Monograph. Altern Med
Rev. 2006 September; 11(3):238-42. [0033] non-patent document 16:
Vallianou N, Evangelopoulos A, Koutalas P. Alpha-lipoic Acid and
diabetic neuropathy. Rev Diabet Stud. 2009 Winter; 6(4):230-6.
[0034] non-patent document 17: Ussher J R, Lopaschuk G D. The
malonyl CoA axis as a potential target for treating ischaemic heart
disease. Cardiovasc Res. 2008 Jul. 15; 79(2):259-68. [0035]
non-patent document 18: Wargovich T J, MacDonald R G, Hill J A,
Feldman R L, Stacpoole P W, Pepine C J. Myocardial metabolic and
hemodynamic effects of dichloroacetate in coronary artery disease.
Am J Cardiol. 1988 Jan. 1; 61(1):65-70. [0036] non-patent document
19: Taniguchi M, Wilson C, Hunter C A, Pehowich D J, Clanachan A S,
Lopaschuk G D. Dichloroacetate improves cardiac efficiency after
ischemia independent of changes in mitochondrial proton leak. Am J
Physiol Heart Circ Physiol. 2001 April; 280(4):H1762-9. [0037]
non-patent document 20: Stacpoole P W, Nagaraja N V, Hutson A D.
Efficacy of dichloroacetate as a lactate-lowering drug. J Clin
Pharmacol. 2003 July; 43(7):683-91. [0038] non-patent document 21:
Bonnet S, Archer S L, Allalunis-Turner J, Haromy A, Beaulieu C,
Thompson R, et al. A mitochondria-K+ channel axis is suppressed in
cancer and its normalization promotes apoptosis and inhibits cancer
growth. Cancer Cell. 2007 January; 11(1):37-51. [0039] non-patent
document 22: McMurtry M S, Bonnet S, Wu X, Dyck J R, Haromy A,
Hashimoto K, et al. Dichloroacetate prevents and reverses pulmonary
hypertension by inducing pulmonary artery smooth muscle cell
apoptosis. Circ Res. 2004 Oct. 15; 95(8):830-40. [0040] non-patent
document 23: Saxena U. Bioenergetics breakdown in Alzheimer's
disease: targets for new therapies. Int J Physiol Pathophysiol
Pharmacol. 2011; 3(2):133-9. [0041] non-patent document 24:
Stacpoole P W. The pyruvate dehydrogenase complex as a therapeutic
target for age-related diseases. Aging Cell. 2012 June;
11(3):371-7. [0042] non-patent document 25: Marangos P J, Turkel C
C, Dziewanowska Z E, Fox A W. Dichloroacetate and cerebral
ischaemia therapeutics. Expert Opin Investig Drugs. 1999 April;
8(4):373-82. [0043] non-patent document 26: Calvert L D, Shelley R,
Singh S J, Greenhaff P L, Bankart J, Morgan M D, et al.
Dichloroacetate enhances performance and reduces blood lactate
during maximal cycle exercise in chronic obstructive pulmonary
disease. Am J Respir Crit Care Med. 2008 May 15; 177(10):1090-4.
[0044] non-patent document 27: Flavin D F. Non-Hodgkin's Lymphoma
Reversal with Dichloroacetate. J Oncol. Hindawi Publishing
Corporation Journal of Oncology, Volume 2010, Article ID 414726, 4
pages doi:10.1155/2010/414726.
SUMMARY OF THE INVENTION
[0045] The present invention is as follow.
[1] A compound represented by the formula [I]:
##STR00003##
wherein n is 1 or 2, or a pharmaceutically acceptable salt thereof,
[2] a compound represented by the formula:
##STR00004##
[3] the compound of the above-mentioned [2], which is represented
by the formula [II]:
##STR00005##
[4] the compound of the above-mentioned [2], which is represented
by the formula [IIh]:
##STR00006##
[5] a compound represented by the formula [III]:
##STR00007##
[6] a pharmaceutical composition comprising the compound of any of
the above-mentioned [1] to [5], or a pharmaceutically acceptable
salt thereof, and a pharmaceutically acceptable carrier, [7] a PDHK
inhibitor comprising the compound of any of the above-mentioned [1]
to [5], or a pharmaceutically acceptable salt thereof, [8] a PDHK1
inhibitor comprising the compound of any of the above-mentioned [1]
to [5], or a pharmaceutically acceptable salt thereof, [9] a PDHK2
inhibitor comprising the compound of any of the above-mentioned [1]
to [5], or a pharmaceutically acceptable salt thereof, [10] a
hypoglycemic agent comprising the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof, [11] a lactic acid-lowering agent comprising the compound
of any of the above-mentioned [1] to [5], or a pharmaceutically
acceptable salt thereof, [12] an agent for the prophylaxis or
treatment of diabetes, insulin resistance syndrome, metabolic
syndrome, hyperglycemia, hyperlactacidemia, diabetic complications,
cardiac failure, cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer or
pulmonary hypertension, which comprises the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof, [12'] an agent for the prophylaxis or treatment of
diabetes, insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications, cardiac
failure, cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer,
pulmonary hypertension or Alzheimer disease, which comprises the
compound of any of the above-mentioned [1] to [5], or a
pharmaceutically acceptable salt thereof, [13] the prophylactic or
therapeutic agent of the above-mentioned [12], wherein the diabetes
is type 1 diabetes or type 2 diabetes, [14] the prophylactic or
therapeutic agent of the above-mentioned [12], wherein the diabetic
complications are selected from the group consisting of diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy and
cataract, [15] the prophylactic or therapeutic agent of the
above-mentioned [12], wherein the cardiac failure is acute cardiac
failure or chronic cardiac failure, [16] a pharmaceutical
composition comprising (a) the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof, and (b) at least one other medicament effective for the
prophylaxis or treatment of a disease selected from the group
consisting of diabetes (type 1 diabetes, type 2 diabetes), insulin
resistance syndrome, metabolic syndrome, hyperglycemia,
hyperlactacidemia, diabetic complications (diabetic neuropathy,
diabetic retinopathy, diabetic nephropathy, cataract), cardiac
failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
diseases, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer and pulmonary hypertension,
[16'] a pharmaceutical composition comprising (a) the compound of
any of the above-mentioned [1] to [5], or a pharmaceutically
acceptable salt thereof, and (b) at least one other medicament
effective for the prophylaxis or treatment of a disease selected
from the group consisting of diabetes (type 1 diabetes, type 2
diabetes), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract),
cardiac failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
diseases, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer, pulmonary hypertension and
Alzheimer disease, [17] a combination drug comprising (a) the
compound of any of the above-mentioned [1] to [5], or a
pharmaceutically acceptable salt thereof, and (b) at least one
other medicament effective for the prophylaxis or treatment of a
disease selected from the group consisting of diabetes (type 1
diabetes, type 2 diabetes), insulin resistance syndrome, metabolic
syndrome, hyperglycemia, hyperlactacidemia, diabetic complications
(diabetic neuropathy, diabetic retinopathy, diabetic nephropathy,
cataract), cardiac failure (acute cardiac failure, chronic cardiac
failure), cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer and
pulmonary hypertension, which are administered simultaneously,
separately or continuously. [17'] a combination drug comprising (a)
the compound of any of the above-mentioned [1] to [5], or a
pharmaceutically acceptable salt thereof, and (b) at least one
other medicament effective for the prophylaxis or treatment of a
disease selected from the group consisting of diabetes (type 1
diabetes, type 2 diabetes), insulin resistance syndrome, metabolic
syndrome, hyperglycemia, hyperlactacidemia, diabetic complications
(diabetic neuropathy, diabetic retinopathy, diabetic nephropathy,
cataract), cardiac failure (acute cardiac failure, chronic cardiac
failure), cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer,
pulmonary hypertension and Alzheimer disease, which are
administered simultaneously, separately or continuously. [18] a
method of inhibiting PDHK in a mammal, comprising administering a
pharmaceutically effective amount of the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof to said mammal, [19] a method of inhibiting PDHK1 in a
mammal, comprising administering a pharmaceutically effective
amount of the compound of any of the above-mentioned [1] to [5], or
a pharmaceutically acceptable salt thereof to said mammal, [20] a
method of inhibiting PDHK2 in a mammal, comprising administering a
pharmaceutically effective amount of the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof to said mammal, [21] a method for the prophylaxis or
treatment of diabetes (type 1 diabetes, type 2 diabetes), insulin
resistance syndrome, metabolic syndrome, hyperglycemia,
hyperlactacidemia, diabetic complications (diabetic neuropathy,
diabetic retinopathy, diabetic nephropathy, cataract), cardiac
failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer or pulmonary hypertension
in a mammal, comprising administering a pharmaceutically effective
amount of the compound of any of the above-mentioned [1] to [5], or
a pharmaceutically acceptable salt thereof to said mammal, [21'] a
method for the prophylaxis or treatment of diabetes (type 1
diabetes, type 2 diabetes), insulin resistance syndrome, metabolic
syndrome, hyperglycemia, hyperlactacidemia, diabetic complications
(diabetic neuropathy, diabetic retinopathy, diabetic nephropathy,
cataract), cardiac failure (acute cardiac failure, chronic cardiac
failure), cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer,
pulmonary hypertension or Alzheimer disease in a mammal, comprising
administering a pharmaceutically effective amount of the compound
of any of the above-mentioned [1] to [5], or a pharmaceutically
acceptable salt thereof to said mammal, [22] a method of decreasing
the blood glucose level in a mammal, comprising administering a
pharmaceutically effective amount of the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof to said mammal, [23] a method of decreasing the lactate
level in a mammal, comprising administering a pharmaceutically
effective amount of the compound of any of the above-mentioned [1]
to [5], or a pharmaceutically acceptable salt thereof to said
mammal, [24] use of the compound of any of the above-mentioned [1]
to [5], or a pharmaceutically acceptable salt thereof for the
production of a PDHK inhibitor, [25] use of the compound of any of
the above-mentioned [1] to [5], or a pharmaceutically acceptable
salt thereof for the production of a PDHK1 inhibitor, [26] use of
the compound of any of the above-mentioned [1] to [5], or a
pharmaceutically acceptable salt thereof for the production of a
PDHK2 inhibitor, [27] use of the compound of any of the
above-mentioned [1] to [5], or a pharmaceutically acceptable salt
thereof for the production of a blood glucose level-lowering agent,
[28] use of the compound of any of the above-mentioned [1] to [5],
or a pharmaceutically acceptable salt thereof for the production of
a lactate level-lowering agent, [29] use of the compound of any of
the above-mentioned [1] to [5], or a pharmaceutically acceptable
salt thereof for the production of a prophylactic or therapeutic
agent for diabetes (type 1 diabetes, type 2 diabetes), insulin
resistance syndrome, metabolic syndrome, hyperglycemia,
hyperlactacidemia, diabetic complications (diabetic neuropathy,
diabetic retinopathy, diabetic nephropathy, cataract), cardiac
failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer or pulmonary hypertension,
[29'] use of the compound of any of the above-mentioned [1] to [5],
or a pharmaceutically acceptable salt thereof for the production of
a prophylactic or therapeutic agent for diabetes (type 1 diabetes,
type 2 diabetes), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract),
cardiac failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer, pulmonary hypertension or
Alzheimer disease, [30] the use of any of the above-mentioned [24]
to [29], in combination with at least one other medicament
effective for the prophylaxis or treatment of a disease selected
from the group consisting of diabetes (type 1 diabetes, type 2
diabetes), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract),
cardiac failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer and pulmonary hypertension,
and [30'] the use of any of the above-mentioned [24] to [29], in
combination with at least one other medicament effective for the
prophylaxis or treatment of a disease selected from the group
consisting of diabetes (type 1 diabetes, type 2 diabetes), insulin
resistance syndrome, metabolic syndrome, hyperglycemia,
hyperlactacidemia, diabetic complications (diabetic neuropathy,
diabetic retinopathy, diabetic nephropathy, cataract), cardiac
failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer, pulmonary hypertension and
Alzheimer disease, and the like.
Effect of the Invention
[0046] The compound of the present invention or a pharmaceutically
acceptable salt thereof inhibits a PDHK activity, and is useful as
a therapeutic or prophylactic agent for diabetes (type 1 diabetes,
type 2 diabetes), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract),
cardiac failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer, pulmonary hypertension or
Alzheimer disease, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 shows an effect of test compounds on the liver PDH
activity (percentage of active liver PDH activity to the total
liver PDH activity) in non-fasting SD(IGS) rats (mean.+-.standard
deviation (n=3)).
[0048] FIG. 2 shows an effect of test compounds on the adipose
tissue PDH activity (percentage of active adipose tissue PDH
activity to the total adipose tissue PDH activity) in non-fasting
SD(IGS) rats (mean.+-.standard deviation (n=3)).
DESCRIPTION OF EMBODIMENTS
[0049] The present invention is explained in detail in the
following.
[0050] The compound of the present invention is a compound
represented by the formula [I]:
##STR00008##
wherein n is 1 or 2, (hereinafter to be also referred to as
compound (1)), or a pharmaceutically acceptable salt thereof.
[0051] The compound of the present invention is a compound
represented by the formula [II]:
##STR00009##
(2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)--
9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide) (hereinafter
to be also referred to as compound (2)).
[0052] The compound of the present invention is a compound
represented by the formula [IIh]:
##STR00010##
(2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)--
9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide monohydrate)
(hereinafter to be also referred to as compound (2h)).
[0053] The compound of the present invention is a compound
represented by the formula [III]:
##STR00011##
(2-{4-[(9R)-9-hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)-
-9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide)
(hereinafter to be also referred to as compound (3)).
[0054] A pharmaceutically acceptable salt of the compound of the
present invention may be any salt as long as it forms a nontoxic
salt with the compound of the present invention. Examples thereof
include salts with inorganic acids, salts with organic acids, salts
with amino acids and the like.
[0055] Examples of the salt with inorganic acid include a salt with
hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid,
hydrobromic acid and the like.
[0056] Examples of the salt with organic acid include salts with
oxalic acid, maleic acid, citric acid, fumaric acid, lactic acid,
malic acid, succinic acid, tartaric acid, acetic acid,
trifluoroacetic acid, gluconic acid, ascorbic acid, methanesulfonic
acid, benzenesulfonic acid, p-toluenesulfonic acid and the
like.
[0057] Examples of the salt with amino acid include salts with
lysine, arginine, aspartic acid, glutamic acid and the like.
[0058] A pharmaceutically acceptable salt of the compound of the
present invention is preferably a salt with an inorganic acid.
[0059] In addition, the compound of the present invention or a
pharmaceutically acceptable salt thereof may be labeled with an
isotope (e.g., .sup.3H, .sup.14C, .sup.35S etc.).
[0060] As the compound of the present invention or a
pharmaceutically acceptable salt thereof, compound (1) or a
pharmaceutically acceptable salt thereof, each of which is
substantially purified, is preferable. More preferred is the
compound of the present invention or a pharmaceutically acceptable
salt thereof, each of which is purified to a purity of not less
than 80%.
[0061] The compound of the formula [I] or a pharmaceutically
acceptable salt thereof may exist as a solvate. The term "solvate"
refers to the compound of the formula [I] or a pharmaceutically
acceptable salt thereof with which a solvent molecule is
associated, and also includes hydrates. Such solvates are
preferably pharmaceutically acceptable solvates. Such solvates
include, for example, hydrate, ethanol solvate,
dimethylsulfoxide-solvate and the like of the compound of the
formula [I] or a pharmaceutically acceptable salt thereof. Specific
examples include hemihydrate, monohydrate, dihydrate or
mono(ethanol)solvate of the compound of the formula [I] or a
monohydrate of the compound of the formula [I], 2/3(ethanol)solvate
of dihydrochloride of the same and the like. Such solvates can be
produced according to conventional methods.
[0062] Examples of the "pharmaceutical composition" include oral
preparations such as tablet, capsule, granule, powder, troche,
syrup, emulsion, suspension and the like, and parenteral agents
such as external preparation, suppository, injection, eye drop,
nasal preparation, pulmonary preparation and the like.
[0063] The pharmaceutical composition of the present invention is
produced according to a method known per se in the art of
pharmaceutical preparations, by mixing the compound of the present
invention or a pharmaceutically acceptable salt thereof with a
suitable amount of at least one kind of pharmaceutically acceptable
carrier and the like as appropriate. While the content of the
compound of the present invention or a pharmaceutically acceptable
salt thereof in the pharmaceutical composition varies depending on
the dosage form, dose and the like, it is, for example, 0.1 to 100
wt % of the whole composition.
[0064] Examples of the "pharmaceutically acceptable carrier"
include various organic or inorganic carrier substances
conventionally used as preparation materials, for example,
excipient, disintegrant, binder, fluidizer, lubricant and the like
for solid preparations, and solvent, solubilizing agent, suspending
agent, isotonicity agent, buffering agent, soothing agent and the
like for liquid preparations. Where necessary, moreover, additives
such as preservative, antioxidant, colorant, sweetening agent and
the like are used.
[0065] Examples of the "excipient" include lactose, sucrose,
D-mannitol, D-sorbitol, cornstarch, dextrin, microcrystalline
cellulose, crystalline cellulose, carmellose, carmellose calcium,
sodium carboxymethyl starch, low-substituted
hydroxypropylcellulose, gum arabic and the like.
[0066] Examples of the "disintegrant" include carmellose,
carmellose calcium, carmellose sodium, sodium carboxymethyl starch,
croscarmellose sodium, crospovidone, low-substituted
hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline
cellulose and the like.
[0067] Examples of the "binder" include hydroxypropylcellulose,
hydroxypropylmethylcellulose, povidone, crystalline cellulose,
sucrose, dextrin, starch, gelatin, carmellose sodium, gum arabic
and the like.
[0068] Examples of the "fluidizer" include light anhydrous silicic
acid, magnesium stearate and the like.
[0069] Examples of the "lubricant" include magnesium stearate,
calcium stearate, talc and the like.
[0070] Examples of the "solvent" include purified water, ethanol,
propylene glycol, macrogol, sesame oil, corn oil, olive oil and the
like.
[0071] Examples of the "solubilizing agents" include propylene
glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine,
sodium carbonate, sodium citrate and the like.
[0072] Examples of the "suspending agent" include benzalkonium
chloride, carmellose, hydroxypropylcellulose, propylene glycol,
povidone, methylcellulose, glycerol monostearate and the like.
[0073] Examples of the "isotonic agent" include glucose,
D-sorbitol, sodium chloride, D-mannitol and the like.
[0074] Examples of the "buffering agent" include sodium
hydrogenphosphate, sodium acetate, sodium carbonate, sodium citrate
and the like.
[0075] Examples of the "soothing agent" include benzyl alcohol and
the like.
[0076] Examples of the "preservative" include ethyl
parahydroxybenzoate, chlorobutanol, benzyl alcohol, sodium
dehydroacetate, sorbic acid and the like.
[0077] Examples of the "antioxidant" include sodium sulfite,
ascorbic acid and the like.
[0078] Examples of the "colorant" include food colors (e.g., Food
Color Red No. 2 or 3, Food Color yellow No. 4 or 5 etc.),
.beta.-carotene and the like.
[0079] Examples of the "sweetening agent" include saccharin sodium,
dipotassium glycyrrhizinate, aspartame and the like.
[0080] The pharmaceutical composition of the present invention can
be administered orally or parenterally (e.g., topical,
intramuscular, subcutaneous, rectal, intravenous administration
etc.) to human as well as mammals other than human (e.g., mouse,
rat, hamster, guinea pig, rabbit, cat, dog, swine, bovine, horse,
sheep, monkey etc.). The dose varies depending on the subject of
administration, disease, symptom, dosage form, administration route
and the like. For example, the daily dose for oral administration
to an adult patient (body weight: about 60 kg) is generally within
the range of about 1 mg to 1 g, based on compound (1) as the active
ingredient. This amount can be administered in one to several
portions.
[0081] Since the compound of the present invention or a
pharmaceutically acceptable salt thereof has a PDHK (PDHK1 and/or
PDHK2) inhibitory activity, it is considered to be advantageous for
the treatment or prophylaxis of the diseases relating to an
impairment of glucose utilization, for example, diabetes (type 1
diabetes, type 2 diabetes etc.), insulin resistance syndrome,
metabolic syndrome, hyperglycemia, hyperlactacidemia, diabetic
complications (diabetic neuropathy, diabetic retinopathy, diabetic
nephropathy, cataract etc.). In addition, the PDHK inhibitor is
considered to be advantageous for the treatment or prophylaxis of
diseases wherein supply of an energy substrate to a tissue is
limited, for example, cardiac failure (acute cardiac failure,
chronic cardiac failure), cardiomyopathy, myocardial ischemia,
myocardial infarction, angina pectoris, dyslipidemia,
atherosclerosis, peripheral arterial disease, intermittent
claudication, chronic obstructive pulmonary disease, brain ischemia
and cerebral apoplexy. Furthermore, the PDHK inhibitor is
considered to be advantageous for the treatment or prophylaxis of a
mitochondrial disease, mitochondrial encephalomyopathy, cancer
pulmonary hypertension or Alzheimer disease, and the like.
[0082] Diabetes is, for example, type 1 diabetes or type 2
diabetes.
[0083] Examples of the diabetic complications include diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy and
cataract.
[0084] Cardiac failure is, for example, acute cardiac failure or
chronic cardiac failure.
[0085] To "inhibit PDHK" means to inhibit the function of PDHK and
eliminate or attenuate the activity. To "inhibit PDHK", human PDHK
is preferably inhibited. As a "PDHK inhibitor", preferred is a
"human PDHK inhibitor".
[0086] To "inhibit PDHK1" means to inhibit the function of PDHK1
and eliminate or attenuate the activity. For example, it means to
inhibit the function as PDHK1 based on the conditions in the
below-mentioned Experimental Example 1. To "inhibit PDHK1", human
PDHK1 is preferably inhibited. As a "PDHK1 inhibitor", preferred is
a "human PDHK1 inhibitor". More preferred is a "PDHK1 inhibitor for
human target organ".
[0087] To "inhibit PDHK2" means to inhibit the function of PDHK2
and eliminate or attenuate the activity. For example, it means to
inhibit the function as PDHK2 based on the conditions in the
below-mentioned Experimental Example 1. To "inhibit PDHK2", human
PDHK2 is preferably inhibited. As a "PDHK2 inhibitor", preferred is
a "human PDHK2 inhibitor". More preferred is a "PDHK2 inhibitor for
human target organ".
[0088] To "activate PDH" means to activate PDH in a target organ
(e.g., liver, skeletal muscle, adipose tissue, heart, brain) and
the like, cancer or the like.
[0089] To "decrease blood glucose level" means to decrease the
glucose concentration in blood (including in serum and plasma),
preferably to decrease high blood glucose level, more preferably,
to decrease the blood glucose level to a therapeutically effective
normal level for human.
[0090] To "decrease lactic acid level" means to decrease the lactic
acid concentration in blood (including in serum and plasma),
preferably to decrease high lactic acid level, more preferably, to
decrease the lactic acid level to a therapeutically effective
normal level for human.
[0091] The compound of the present invention or a pharmaceutically
acceptable salt thereof can be used in combination with one or a
plurality of other medicaments (hereinafter to be also referred to
as a concomitant drug) according to a method generally employed in
the medical field (hereinafter to be referred to as combined
use).
[0092] The administration period of the compound of the present
invention or a pharmaceutically acceptable salt thereof, and a
concomitant drug is not limited, and they may be administered to an
administration subject as combination preparation, or the both
preparations may be administered simultaneously or at given
intervals. In addition, the pharmaceutical composition of the
present invention and a concomitant drug may be used as a
medicament in the form of a kit. The dose of the concomitant drug
is similar to the clinically-employed dose and can be appropriately
selected according to the subject of administration, disease,
symptom, dosage form, administration route, administration time,
combination and the like. The administration form of the
concomitant drug is not particularly limited, and it only needs to
be combined with the compound of the present invention or a
pharmaceutically acceptable salt thereof.
[0093] Examples of the combination drug include therapeutic agents
and/or prophylaxis agents for diabetes (type 1 diabetes, type 2
diabetes etc.), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract),
cardiac failure (acute cardiac failure, chronic cardiac failure),
cardiomyopathy, myocardial ischemia, myocardial infarction, angina
pectoris, dyslipidemia, atherosclerosis, peripheral arterial
disease, intermittent claudication, chronic obstructive pulmonary
disease, brain ischemia, cerebral apoplexy, mitochondrial disease,
mitochondrial encephalomyopathy, cancer, pulmonary hypertension or
Alzheimer disease, and the like, and one or more agents therefrom
and the compound of the present invention or a pharmaceutically
acceptable salt thereof can be used in combination.
[0094] Examples of the "agent for the treatment and/or prophylaxis
of diabetes" include insulin preparation, sulfonylurea hypoglycemic
agent, metformin, DPP-4 inhibitor, insulin resistance improving
agent (for example, thiazolidine derivative), GLP-1 receptor
agonist and the like.
EXAMPLES
[0095] The production method of the compound of the present
invention or a pharmaceutically acceptable salt thereof is
specifically explained by Examples. However, the present invention
is not limited by these Examples.
[0096] Even if no description is found in the present production
method, steps may be modified for efficient production, such as
introduction of a protecting group into a functional group where
necessary with deprotection in a subsequent step, using a
functional group as a precursor in each step, followed by
conversion to a desired functional group at a suitable stage,
changing the order of production methods and steps, and the
like.
[0097] The treatment after reaction in each step may be performed
by a conventional method, where isolation and purification can be
performed as necessary according to a method appropriately selected
from conventional methods such as crystallization,
recrystallization, distillation, partitioning, silica gel
chromatography, preparative HPLC and the like, or a combination
thereof. All reagents and solvents have quality of commercially
available products, and were used without purification.
[0098] Percentage % shows wt %. Other abbreviations used in the
example section mean the following.
[0099] s: singlet
[0100] d: doublet
[0101] t: triplet
[0102] q: quartet
[0103] m: multiplet
[0104] br: broad
[0105] dd: double doublet
[0106] td: triple doublet
[0107] ddd: double double doublet
[0108] J: coupling constant
[0109] CDCl.sub.3: deuterated chloroform
[0110] DMSO-D.sub.6: deuterated dimethyl sulfoxide
[0111] .sup.1H NMR: proton nuclear magnetic resonance
[0112] HPLC: high performance liquid chromatography
[0113] DPPA: diphenylphosphoryl azide
[0114] .sup.1H-NMR spectrum was measured in CDCl.sub.3 or
DMSO-D.sub.6 using tetramethylsilane as an internal standard, and
all .delta. values are shown in ppm.
(10 mM Phosphate Buffer (pH 2.0))
[0115] Sodium dihydrogen phosphate (3.60 g) was dissolved in water
(3000 ml), and adjusted to pH 2.0 with phosphoric acid to give the
title buffer.
HPLC Analysis Conditions
Analysis Condition 1
[0116] Measurement device: HPLC system SHIMADZU CORPORATION
high-performance liquid chromatograph Prominence Column: DAICEL
CHIRALCEL OD-3R 4.6 mm.PHI..times.150 mm Column temperature:
40.degree. C. Mobile phase: (SOLUTION A) 10 mM phosphate buffer (pH
2.0), (SOLUTION B) acetonitrile The composition of the mobile phase
(SOLUTION A:SOLUTION B) was linearly changed from 50:50 to 20:80
over 20 min and then maintained at 20:80 for 5 min. Flow rate: 0.5
ml/min
Detection: UV (220 nm)
Analysis Condition 2
[0117] Measurement device: HPLC system SHIMADZU CORPORATION
high-performance liquid chromatograph Prominence Column: DAICEL
CHIRALCEL OJ-RH 4.6 mm.PHI..times.150 mm Column temperature:
40.degree. C. Mobile phase: (SOLUTION A) 10 mM phosphate buffer (pH
2.0), (SOLUTION B) acetonitrile The composition of the mobile phase
(SOLUTION A:SOLUTION B) was linearly changed from 70:30 to 40:60
over 20 min and then maintained at 40:60 for 10 min. Flow rate: 0.5
ml/min
Detection: UV (220 nm)
[0118] Analysis condition 3 Measurement device: HPLC system
SHIMADZU CORPORATION high-performance liquid chromatograph
Prominence Column: DAICEL CHIRALPAK AD-3R 4.6 mm.PHI..times.150 mm
Column temperature: 40.degree. C. Mobile phase: (SOLUTION A) 10 mM
phosphate buffer (pH 2.0), (SOLUTION B) acetonitrile The
composition of the mobile phase (SOLUTION A:SOLUTION B) was
linearly changed from 50:50 to 20:80 over 20 min and then
maintained at 20:80 for 5 min. Flow rate: 0.5 ml/min
Detection: UV (220 nm)
Example 1
Synthesis of
2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9-
H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (compound
(2))
Step 1
Ethyl 2'-chloro-4'-methoxybiphenyl-2-carboxylate
##STR00012##
[0120] Under an argon atmosphere, 1-bromo-2-chloro-4-methoxybenzene
(44.3 g) was dissolved in toluene (220 ml), ethyl
2-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)benzoate (60.8 g),
water (132 ml), sodium hydrogen carbonate (33.6 g) and
dichlorobis(triphenylphosphine)palladium(II) (2.8 g) were added,
and the mixture was stirred at an oil bath temperature of
120.degree. C. for 7 hr. To the reaction mixture was added ethyl
2-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)benzoate (5.2 g),
and the mixture was further stirred for 2 hr. The reaction mixture
was cooled to room temperature, toluene (100 ml) and water (200 ml)
were added, and the mixture was stirred overnight. To the reaction
mixture was added activated carbon (3 g), and the mixture was
further stirred for 1 hr. The insoluble material was filtered off
through celite, and the insoluble material was washed with toluene
(100 ml) and water (200 ml). The obtained filtrates were combined
to allow for layer separation. The obtained organic layer was
washed with water (100 ml), and the solvent was evaporated to give
the title compound (67.7 g).
[0121] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 7.88-7.86 (1H,
m), 7.63 (1H, td, J=7.6, 1.4 Hz), 7.51 (1H, td, J=7.6, 1.4 Hz),
7.27 (1H, dd, J=7.6, 0.9 Hz), 7.18 (1H, d, J=8.6 Hz), 7.06 (1H, d,
J=2.6 Hz), 6.95 (1H, dd, J=8.6, 2.6 Hz), 4.01 (2H, m), 3.80 (3H,
s), 0.96 (3H, t, J=7.1 Hz).
Step 2
2'-Chloro-4'-methoxybiphenyl-2-carboxylic acid
##STR00013##
[0123] Ethyl 2'-chloro-4'-methoxybiphenyl-2-carboxylate (67.7 g)
was dissolved in ethanol (100 ml), 4N aqueous sodium hydroxide (100
ml) was added, and the mixture was stirred at an oil bath
temperature of 110.degree. C. for 4.5 hr. The reaction mixture was
cooled to room temperature, water (200 ml) and toluene (100 ml)
were added, and the mixture was stirred overnight. To the reaction
mixture was added activated carbon (3.6 g), and the mixture was
further stirred for 1 hr. The insoluble material was filtered off
through celite, and the insoluble material was washed with toluene
(30 ml) and water (300 ml). The obtained filtrates were combined to
allow for layer separation. The obtained aqueous layer was washed
with toluene (100 ml), the aqueous layer was acidified with
concentrated hydrochloric acid (40 ml), and stirred at room
temperature for 1 hr. The precipitated solid was collected by
filtration. The obtained solid was air-dried for 3 hr, and dried
under reduced pressure at 60.degree. C. overnight to give the title
compound (50.2 g).
[0124] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 12.57 (1H, s),
7.90-7.88 (1H, m), 7.60 (1H, td, J=7.6, 1.3 Hz), 7.49 (1H, td,
J=7.6, 1.3 Hz), 7.24 (1H, dd, J=7.6, 1.0 Hz), 7.19 (1H, d, J=8.4
Hz), 7.06 (1H, d, J=2.4 Hz), 6.95 (1H, dd, J=8.5, 2.4 Hz), 3.81
(3H, s).
Step 3
4-Chloro-2-methoxy-9H-fluoren-9-one
##STR00014##
[0126] Under an argon atmosphere, to
2'-chloro-4'-methoxybiphenyl-2-carboxylic acid (65.4 g) was added
an Eaton reagent (phosphorus pentoxide-methanesulfonic acid (weight
ratio 1:10) solution, 330 ml), and the mixture was stirred at an
oil bath temperature of 100.degree. C. for 1 hr. The reaction
mixture was ice-cooled, water (650 ml) was slowly added dropwise,
and the mixture was stirred at room temperature for 1 hr. The
precipitated solid was collected by filtration, and washed with
water (500 ml). The obtained solid was air-dried overnight to give
the title compound (92.0 g).
[0127] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.01 (1H, d,
J=7.4 Hz), 7.64-7.60 (2H, m), 7.36 (1H, td, J=7.4, 0.9 Hz), 7.17
(2H, dd, J=8.4, 2.3 Hz), 3.85 (3H, s).
Step 4
4-Chloro-2-hydroxy-9H-fluoren-9-one
##STR00015##
[0129] Under an argon atmosphere, to
4-chloro-2-methoxy-9H-fluoren-9-one (92.0 g) were added
N-methylpyrrolidone (120 ml) and pyridine hydrochloride (144 g).
The reaction mixture was stirred at an oil bath temperature of
200.degree. C. for 3 hr with removing water by a Dean-Stark
apparatus. The reaction mixture was cooled to 90.degree. C., water
(600 ml) was added dropwise, and the mixture was stirred at room
temperature for 2 hr. The precipitated solid was collected by
filtration, and washed with water (400 ml). The obtained solid was
air-dried for 3 days, a mixed solvent of hexane and ethyl acetate
(hexane:ethyl acetate 1:1, 300 ml) was added, and the mixture was
stirred at room temperature for 1 hr. The solid was collected by
filtration, and washed with a mixed solvent of hexane and ethyl
acetate (hexane:ethyl acetate=1:1, 500 ml). The obtained solid was
dried under reduced pressure at 50.degree. C. for 3 hr to give the
title compound (48.6 g).
[0130] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 10.56 (1H, s),
7.96 (1H, d, J=8.4 Hz), 7.61-7.57 (2H, m), 7.32 (1H, td, J=7.4, 0.9
Hz), 6.97 (1H, d, J=2.2 Hz), 6.94 (1H, d, J=2.2 Hz).
Step 5
Ethyl 4-(4-chloro-9-oxo-9H-fluoren-2-yloxy)butyrate
##STR00016##
[0132] 4-Chloro-2-hydroxy-9H-fluoren-9-one (48.6 g) was dissolved
in N,N-dimethylformamide (150 ml), potassium carbonate (58.3 g) and
ethyl 4-bromobutyrate (33.5 ml) were added, and the mixture was
stirred at 60.degree. C. for 2 hr. The reaction mixture was cooled
to 40.degree. C., and toluene (300 ml) and water (300 ml) were
added to allow for layer separation. The obtained aqueous layer was
extracted again with toluene (100 ml). The obtained organic layers
were combined, washed twice with water (100 ml), anhydrous sodium
sulfate and activated carbon (2.5 g) were added, and the mixture
was stirred at room temperature for 5 min. The insoluble material
was filtered off through celite, and the solvent in the filtrate
was evaporated. To the obtained residue was added hexane (220 ml),
and the mixture was stirred at 50.degree. C. for 10 min and at room
temperature for 1 hr. The precipitated solid was collected by
filtration, and washed with hexane. The obtained solid was dried
under reduced pressure to give the title compound (66.9 g). In
addition, the solvent in the obtained filtrate was evaporated, to
the residue were added ethyl acetate (5 ml) and hexane (20 ml), and
the mixture was stirred at room temperature for 1 hr. The
precipitated solid was collected by filtration, and washed with
hexane. The obtained solid was dried under reduced pressure to
further give the title compound (2.5 g).
[0133] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.01 (1H, d,
J=7.6 Hz), 7.65-7.61 (2H, m), 7.37 (1H, t, J=7.6 Hz), 7.17-7.14
(2H, m), 4.13-4.05 (4H, m), 2.47 (2H, t, J=7.3 Hz), 2.02-1.95 (2H,
m), 1.19 (3H, td, J=7.2, 0.7 Hz).
Step 6
Ethyl
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]bu-
tyrate
##STR00017##
[0135] Under an argon atmosphere, ethyl
4-(4-chloro-9-oxo-9H-fluorene-2-yloxy)butyrate (69.4 g) was
dissolved in THF (700 ml), and N-(4-tert-butylbenzyl)cinchonidium
4-methoxyphenoxide (6.4 g) was added. To the reaction mixture was
added dropwise a solution of trimethyl(trifluoromethyl)silane (52.0
ml) in THF (140 ml) at -16.degree. C., and the mixture was stirred
at the same temperature for 15 min. To the reaction mixture were
successively added acetic acid (23.0 ml) and 1M tetrabutylammonium
fluoride/THF solution (222 ml), and the mixture was stirred at room
temperature for 1 hr. The solvent in the reaction mixture was
evaporated, and to the obtained residue were added toluene (500 ml)
and saturated aqueous sodium hydrogen carbonate (200 ml) to allow
for layer separation. The obtained organic layer was washed
successively with saturated aqueous sodium hydrogen carbonate (150
ml, twice), 1N aqueous sodium hydroxide (100 ml), water (100 ml),
1N hydrochloric acid (100 ml), water (100 ml) and saturated brine
(100 ml). To the obtained organic layer were added anhydrous
magnesium sulfate and silica gel (150 g), and the mixture was
stirred for 10 min. The insoluble material was filtered off, and
the insoluble material was washed successively with toluene (300
ml) and ethyl acetate (800 ml). The obtained filtrate and the
toluene washing were combined and the solvent was evaporated to
give the title compound (72.1 g). Also, the solvent in the ethyl
acetate washing was evaporated, to the obtained residue were added
silica gel (40 g) and a mixed solvent of hexane and ethyl acetate
(ethyl acetate:hexane 2:1, 300 ml), and the mixture was stirred at
room temperature. The insoluble material was filtered off, and the
insoluble material was washed with a mixed solvent of hexane and
ethyl acetate (ethyl acetate:hexane=2:1, 300 ml). The solvent in
the obtained filtrate was evaporated to further give the title
compound (20.3 g).
[0136] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.14 (1H, d,
J=7.7 Hz), 7.66 (1H, d, J=7.5 Hz), 7.53 (1H, t, J=7.6 Hz),
7.42-7.38 (2H, m), 7.14 (2H, s), 4.11-4.05 (4H, m), 2.47 (2H, t,
J=7.5 Hz), 2.03-1.96 (2H, m), 1.19 (3H, td, J=7.1, 0.8 Hz).
(Absolute Configuration)
[0137] Identification of the absolute configuration of
4-chloro-2-methyl-9-(trifluoromethyl)-9H-fluoren-9-ol in the
after-mentioned step 10 confirmed that the title compound obtained
in this step is an (R) form. The optical purity was 52.9% e.e.
[0138] The optical purity was determined under the HPLC analysis
condition 1. Retention time of (S) form 19.6 min, retention time of
(R) form 23.0 min.
Step 7
4-[(9R)-4-Chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid
##STR00018##
[0140] Ethyl
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyrat-
e (92.2 g) was dissolved in ethanol (100 ml), 4N aqueous sodium
hydroxide (100 ml) was added, and the mixture was stirred at
80.degree. C. overnight. The reaction mixture was cooled to room
temperature, water (200 ml) was added, and the mixture was washed
twice with toluene (100 ml). The obtained aqueous layer was
neutralized with concentrated hydrochloric acid (40 ml), and
extracted twice with ethyl acetate (300 ml). The obtained ethyl
acetate extract was washed successively with water (100 ml, twice),
and saturated brine (100 ml), anhydrous magnesium sulfate and
activated carbon (4.2 g) were added, and the mixture was stirred at
room temperature for 10 min. The insoluble material was filtered
off, and the solvent in the filtrate was evaporated. To the
obtained residue was added chloroform (80 ml), and the mixture was
heated to 50.degree. C. Hexane (400 ml) was added dropwise, and the
mixture was stirred at the same temperature for 30 min, and at room
temperature for 2 hr. The precipitated solid was collected by
filtration, washed with a mixed solvent of hexane and chloroform
(hexane:chloroform=9:1, 50 ml), and dried under reduced pressure at
80.degree. C. for 2 hr to give the title compound (72.5 g).
[0141] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 12.17 (1H, br
s), 8.14 (1H, d, J=7.7 Hz), 7.66 (1H, d, J=7.5 Hz), 7.54 (1H, td,
J=7.7, 1.2 Hz), 7.42-7.30 (2H, m), 7.18-7.15 (2H, m), 4.09 (2H, t,
J=6.4 Hz), 2.41 (2H, t, J=7.3 Hz), 2.00-1.93 (2H, m).
Step 8
(1S)-1-(4-Methylphenyl)ethylamine salt of
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid
##STR00019##
[0143] Under a nitrogen atmosphere,
(1S)-1-(4-methylphenyl)ethylamine (19.5 g) was dissolved in ethyl
acetate (720 ml), and
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid (72.5 g) was added. The reaction mixture was stirred at
60.degree. C. for 2 hr, and at room temperature overnight. The
precipitated solid was collected by filtration, and washed with
ethyl acetate (100 ml). The obtained solid was dried under reduced
pressure at 60.degree. C. for 5 hr to give the title compound (68.6
g). In addition,
4-[(9S)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid could be obtained from the filtrate.
(Optical Purity)
[0144] The optical purity of
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid was determined under the HPLC analysis condition 1 (optical
purity 90.2% e.e.). Retention time of (R) form 12.9 min, retention
time of (S) form 10.4 min.
[0145] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.14 (1H, d,
J=7.7 Hz), 7.66 (1H, d, J=7.7 Hz), 7.53 (1H, td, J=7.6, 1.1 Hz),
7.40 (1H, td, J=7.6, 1.0 Hz), 7.26 (2H, d, J=7.9 Hz), 7.16-7.10
(4H, m), 4.08 (2H, t, J=6.5 Hz), 4.01 (1H, q, J=6.7 Hz), 2.32 (2H,
t, J=7.3 Hz), 2.26 (3H, s), 1.98-1.91 (2H, m), 1.26 (3H, d, J=6.7
Hz).
Step 9
4-[(9R)-4-Chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid
##STR00020##
[0147] To (1S)-1-(4-methylphenyl)ethylamine salt of
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyric
acid (68.6 g) were added ethyl acetate (500 ml) and 2N hydrochloric
acid (300 ml), and the mixture was stirred at room temperature for
10 min. The mixture was allowed for layer separation. The obtained
organic layer was washed successively with water (250 ml) and
saturated brine (200 ml). The obtained organic layer was dried over
anhydrous magnesium sulfate, the insoluble material was filtered
off, and the solvent in the filtrate was evaporated to give the
title compound (60.0 g).
[0148] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 12.17 (1H, br
s), 8.14 (1H, d, J=7.7 Hz), 7.66 (1H, d, J=7.5 Hz), 7.54 (1H, td,
J=7.7, 1.2 Hz), 7.42-7.30 (2H, m), 7.18-7.15 (2H, m), 4.09 (2H, t,
J=6.4 Hz), 2.41 (2H, t, J=7.3 Hz), 2.00-1.93 (2H, m).
Step 10
(9R)-4-Chloro-9-(trifluoromethyl)-9H-fluorene-2,9-diol
##STR00021##
[0150] To
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-ylox-
y]butyric acid (50 g) were added N-methylpyrrolidone (200 ml) and
pyridine hydrochloride (298 g), and the mixture was stirred at an
oil bath temperature of 200.degree. C. for 2 days. The reaction
mixture was cooled to room temperature, diluted with ethyl acetate
(500 ml), and washed twice with water. The obtained aqueous layer
was extracted again with ethyl acetate (300 ml). The combined
organic layer was washed successively with water, 1N hydrochloric
acid and saturated brine. To the obtained organic layer were added
anhydrous magnesium sulfate and activated carbon (10 g), and the
mixture was stirred at room temperature. The insoluble material was
filtered off through celite. The solvent in the obtained organic
layer was evaporated, hexane was added to the residue, and the
mixture was stirred at room temperature. The precipitated solid was
collected by filtration, and dried under reduced pressure at room
temperature. The obtained crude product was dissolved in ethyl
acetate (500 ml), washed 3 times with water, dried over anhydrous
magnesium sulfate. The insoluble material was filtered off, and the
solvent in the filtrate was evaporated. To the residue was added
hexane, and the mixture was stirred at room temperature. The
precipitated solid was collected by filtration, dried under reduced
pressure at room temperature to give the title compound (22.4
g).
[0151] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 10.37 (1H, br
s), 8.09 (1H, d, J=7.5 Hz), 7.63 (1H, d, J=7.5 Hz), 7.50 (1H, td,
J=7.6, 1.0 Hz), 7.36 (1H, td, J=7.6, 1.0 Hz), 7.32 (1H, br s), 7.06
(1H, s), 6.91 (1H, br d, J=2.0 Hz).
(Absolute Configuration)
[0152] The absolute configuration of the title compound was
determined by HPLC analysis using the optically active column of
compound (100A) and compound (100B) prepared in the following steps
(Step A-1 to Step A-2 and Step B-1).
Step A-1
##STR00022##
[0154] 4-Chloro-2-methyl-9H-fluoren-9-one was subjected to
trifluoromethylation, reaction with ethyl bromoacetate, and
hydrolysis to give
[4-chloro-2-methyl-9-(trifluoromethyl)-9H-fluorene-9-yloxy]acetic
acid. This compound was optically resolved using
(1R)-1-phenylethylamine, and the absolute configuration was
determined to be (R) by single crystal X-ray structural analysis of
the obtained (1R)-1-phenylethylamine salt (100AA).
Step A-2
##STR00023##
[0156] (9R)-4-Chloro-2-methyl-9-(trifluoromethyl)-9H-fluoren-9-ol
(compound (100A)) was synthesized from compound 100AA by an acid
treatment and the like.
Step B-1
##STR00024##
[0158] The hydroxyl group at the 2-position of
4-chloro-9-(trifluoromethyl)-9H-fluorene-2,9-diol obtained in step
10 was converted to a methyl group by the above-mentioned method to
give 4-chloro-2-methyl-9-(trifluoromethyl)-9H-fluoren-9-ol
(compound (100B)).
(HPLC Analysis Using Optically Active Column)
[0159] Both enantiomers of compound (100) were separated by HPLC
using an optically active column (HPLC analysis condition 3). HPLC
analysis of compound (100A) confirmed that the retention time of
(R) form was 18.4 min, and the retention time of (S) form was 17.0
min. Compound (100A) and compound (100B) were analyzed under the
HPLC condition to find that the retention time matches.
[0160] It is considered that the absolute configuration of the
asymmetric carbon does not convert during the production of the
above-mentioned compound (100A) and compound (100B). The results
have confirmed that
4-chloro-9-(trifluoromethyl)-9H-fluorene-2,9-diol obtained in step
10 has an absolute configuration of (R).
Step 11
(9R)-4-Chloro-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9H-fluore-
n-9-ol
##STR00025##
[0162] Under a nitrogen atmosphere,
(9R)-4-chloro-9-(trifluoromethyl)-9H-fluorene-2,9-diol (55.5 g) was
dissolved in N,N-dimethylformamide (550 ml),
3-hydroxy-3-methylbutyl toluene-4-sulfonate (49.6 g) and potassium
carbonate (39.5 g) were added, and the mixture was stirred at an
oil bath temperature of 70.degree. C. overnight. To the reaction
mixture was added a solution of 3-hydroxy-3-methylbutyl
toluene-4-sulfonate (4.0 g) in N,N-dimethylformamide (5 ml), and
the mixture was further stirred at the same temperature for 9.5 hr.
The reaction mixture was ice-cooled, water (800 ml) was added, and
the mixture was extracted with ethyl acetate (900 ml). The obtained
organic layer was washed with water (500 ml, 3 times) and saturated
brine (500 ml). The obtained organic layer was dried over anhydrous
sodium sulfate, the insoluble material was filtered off, and the
solvent in the filtrate was evaporated. The obtained residue was
purified by silica gel column chromatography (a mixture of hexane
and ethyl acetate was used as an elution solvent, first eluted with
a mixture of (hexane:ethyl acetate) at a mixing ratio 3:1, and then
with the mixture at a mixing ratio 2:1, and further with the
mixture at a mixing ratio 3:2) to give the title compound (49.5
g).
[0163] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.12 (1H, d,
J=7.6 Hz), 7.64 (1H, d, J=7.4 Hz), 7.52 (1H, td, J=7.6, 0.9 Hz),
7.40-7.36 (2H, m), 7.15-7.13 (2H, m), 4.41 (1H,$), 4.16 (2H, t,
J=7.1 Hz), 1.85 (2H, t, J=7.1 Hz), 1.17 (6H, s).
Step 12
Ethyl
2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluorometh-
yl)-9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionate
##STR00026##
[0165] Under an argon atmosphere,
(9R)-4-chloro-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9H-fluor-
en-9-ol (49.5 g) was dissolved in toluene (445 ml), ethyl
2-methyl-2-[4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-pyrazol-1--
yl]propionate (59.2 g), water (149 ml), tripotassium phosphate
(54.3 g), palladium acetate (2.9 g) and
2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos) (10.5 g)
were added, and the mixture was stirred at an oil bath temperature
of 100.degree. C. for 3.5 hr. The reaction mixture was cooled to
room temperature, and water (300 ml) was added. The insoluble
material was filtered off through celite, and the insoluble
material was washed with toluene (150 ml) and water (50 ml). The
obtained filtrates were combined to allow for layer separation. The
obtained organic layer was washed successively with water (500 ml)
and saturated brine (500 ml). The obtained organic layer was dried
over anhydrous sodium sulfate, the insoluble material was filtered
off, and the solvent in the filtrate was evaporated. The obtained
residue was purified by silica gel column chromatography (a mixture
of hexane and ethyl acetate was used as an elution solvent, first
eluted with a mixture of (hexane:ethyl acetate) at a mixing ratio
2:1, and then with the mixture at a mixing ratio 1:1, and further
with the mixture at a mixing ratio 1:2), and further purified by
silica gel column chromatography (a mixture of hexane and acetone
was used as an elution solvent, first eluted with a mixture of
(hexane:acetone) at a mixing ratio 2:1, and then with the mixture
at mixing ratios 4:1, 3:1, 2:1, 1:1, 2:3, and further with the
mixture at a mixing ratio 1:2) to give the title compound (68.4
g).
[0166] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.18 (1H, s),
7.65 (1H, s), 7.59-7.57 (1H, m), 7.25-7.21 (4H, m), 7.13 (1H, br d,
J=1.6 Hz), 6.84 (1H, d, J=2.3 Hz), 4.38 (1H, s), 4.16-4.11 (4H, m),
1.86 (2H, t, J=7.1 Hz), 1.84 (6H, s), 1.16 (6H, s), 1.13 (3H, t,
J=7.0 Hz).
Step 13
2-{4-[(9R)-9-Hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9H-
-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionic acid
##STR00027##
[0168] Ethyl
2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9-
H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionate (68.4 g) was
dissolved in ethanol (256 ml), 4N aqueous sodium hydroxide (128 ml)
was added, and the mixture was stirred at room temperature for 2.5
hr. The reaction mixture was ice-cooled, 2N hydrochloric acid (333
ml) was added dropwise, and the mixture was extracted with ethyl
acetate (500 ml). The obtained organic layer was washed
successively with water (400 ml, twice) and saturated brine (400
ml). The obtained organic layer was dried over anhydrous sodium
sulfate, the insoluble material was filtered off, and the solvent
in the filtrate was evaporated to give the title compound (70.0
g).
[0169] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.:13.06 (1H, br
s), 8.14 (1H, s), 7.62 (1H, s), 7.57 (1H, dd, J=6.4, 0.6 Hz),
7.27-7.19 (4H, m), 7.12 (1H, s), 6.84 (1H, d, J=2.3 Hz), 4.38 (1H,
s), 4.14 (2H, t, J=7.2 Hz), 1.85 (2H, t, J=7.2 Hz), 1.82 (3H, s),
1.81 (3H, s), 1.16 (6H, s).
Step 14
2-{4-[(9R)-9-Hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9H-
-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (compound
(2))
##STR00028##
[0171] Under a nitrogen atmosphere,
2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9-
H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionic acid (66.7 g)
was dissolved in N,N-dimethylformamide (480 ml),
1-hydroxybenzotriazole (HOBt) 1 hydrate (27.6 g),
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (WSC) hydrochloride
(34.6 g) and 28% aqueous ammonia (24.5 ml) were added, and the
mixture was stirred at room temperature overnight. The reaction
mixture was ice-cooled, water (630 ml) and 2N hydrochloric acid
(330 ml) were added dropwise, and the mixture was extracted with
ethyl acetate (800 ml). The obtained aqueous layer was extracted
again with ethyl acetate (500 ml). The obtained organic layers were
combined, and washed successively with water (500 ml, twice),
saturated aqueous sodium hydrogen carbonate (500 ml), and saturated
brine (500 ml). The obtained organic layer was dried over anhydrous
sodium sulfate, the insoluble material was filtered off, and the
solvent in the filtrate was evaporated to give the title compound
(60.0 g).
[0172] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.08 (1H, s),
7.66 (1H, s), 7.58-7.56 (1H, m), 7.32-7.30 (1H, m), 7.25-7.22 (4H,
m), 7.12 (1H, br s), 6.96 (1H, br s), 6.87 (1H, d, J=2.3 Hz), 4.38
(1H, s), 4.14 (2H, t, J=7.2 Hz), 1.85 (2H, t, J=7.2 Hz), 1.78 (3H,
s), 1.78 (3H, s), 1.17 (6H, s).
Step 15
2-{4-[(9R)-9-Hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9H-
-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide monohydrate
(compound (2h))
##STR00029##
[0174]
2-{4-[(9R)-9-Hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromet-
hyl)-9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide
(compound (2)) (60.0 g) obtained in the previous step was dissolved
in ethyl acetate (109 ml), water (2 ml) was added, and the mixture
was heated to 50.degree. C. To this mixture were successively added
dropwise hexane (226 ml), and a mixed solvent of hexane and ethyl
acetate (hexane:ethyl acetate 2:1, 150 ml), and the mixture was
allowed to cool to room temperature and stirred overnight. The
precipitated solid was collected by filtration, and washed with a
mixed solvent of hexane and ethyl acetate (hexane:ethyl
acetate=2:1, 180 ml). The obtained solid was dried under reduced
pressure at room temperature overnight to give the title compound
(52.2 g, optical purity 98.6% e.e.). The optical purity was
determined under the HPLC analysis condition 2. Retention time of
(R) form 11.3 min, retention time of (S) form 13.9 min.
[0175] Specific optical rotation [.alpha.].sub.D+37.9.degree.
(c=1.01 MeOH 25.degree. C.)
[0176] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.08 (1H, s),
7.66 (1H, s), 7.58-7.56 (1H, m), 7.32-7.30 (1H, m), 7.25-7.22 (4H,
m), 7.12 (1H, br s), 6.96 (1H, br s), 6.87 (1H, d, J=2.3 Hz), 4.38
(1H, s), 4.14 (2H, t, J=7.2 Hz), 1.85 (2H, t, J=7.2 Hz), 1.78 (3H,
s), 1.78 (3H, s), 1.17 (6H, s).
(Elemental Analysis Measurement)
[0177] The results of the elemental analysis matched well with the
theoretical value of compound (2h) calculated.
[0178] Calculated: C, 59.88; H, 5.80; N, 8.06 (Calculated as
monohydrate).
[0179] Found: C, 59.86; H, 5.74; N, 8.00.
Step 16
2-{4-[(9R)-9-Hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoromethyl)-9H-
-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (compound
(2))
##STR00030##
[0181] To
2-{4-[(9R)-9-hydroxy-2-(3-hydroxy-3-methylbutyloxy)-9-(trifluoro-
methyl)-9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide
monohydrate (compound (2h)) (22.63 g) obtained in the previous step
was added toluene (340 ml). The reaction mixture was stirred at an
oil bath temperature of 130.degree. C. for 2 hr under a nitrogen
atmosphere with removing water by a Dean-Stark apparatus. The
reaction mixture was further stirred at an oil bath temperature of
70.degree. C. for 1.5 hr, allowed to cool to room temperature, and
stirred overnight. The precipitated solid was collected by
filtration, and washed with toluene (100 ml). The obtained solid
was dried under reduced pressure at room temperature for 3 days,
and further dried under reduced pressure at 60.degree. C. for 1 day
to give the title compound (21.5 g).
[0182] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.08 (1H, s),
7.66 (1H, s), 7.58-7.56 (1H, m), 7.32-7.30 (1H, m), 7.25-7.22 (4H,
m), 7.12 (1H, br s), 6.96 (1H, br s), 6.87 (1H, d, J=2.3 Hz), 4.38
(1H, s), 4.14 (2H, t, J=7.2 Hz), 1.85 (2H, t, J=7.2 Hz), 1.78 (3H,
s), 1.78 (3H, s), 1.17 (6H, s).
(Elemental Analysis Measurement)
[0183] The results of the elemental analysis matched well with the
theoretical value of compound (2) calculated.
[0184] Calculated: C, 62.02; H, 5.61; N, 8.35 (Calculated as
anhydrous).
[0185] Found: C, 62.17; H, 5.60; N, 8.47.
Step C-1
Preparation of N-(4-tert-butylbenzyl)cinchonidium bromide
##STR00031##
[0187] Cinchonidine (10.6 g) was dissolved in tetrahydrofuran (200
ml), 4-tert-butylbenzylbromide (10.1 g) and tetrabutylammonium
iodide (0.66 g) were added, and the mixture was stirred at
70.degree. C. overnight. The reaction mixture was cooled to room
temperature, the solid was collected by filtration, and washed with
ethyl acetate (50 ml). The obtained solid was dried under reduced
pressure overnight to give the title compound (18.5 g).
[0188] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.99 (1H, d,
J=4.4 Hz), 8.27 (1H, d, J=8.2 Hz), 8.11 (1H, dd, J=8.5, 1.0 Hz),
7.89-7.79 (2H, m), 7.78-7.71 (1H, m), 7.63 (2H, d, J=8.4 Hz), 7.59
(2H, t, J=8.4 Hz), 6.72 (1H, d, J=4.2 Hz), 6.57-6.51 (1H, br s),
5.67 (1H, ddd, J=17.0, 10.4, 6.4 Hz), 5.14 (1H, d, J=17.2 Hz), 5.08
(1H, d, J=12.6 Hz), 5.00-4.90 (2H, m), 4.30-4.18 (1H, m), 3.91 (1H,
t, J=8.7 Hz), 3.74-3.64 (1H, m), 3.35-3.18 (2H, m), 2.76-2.65 (1H,
m), 2.18-1.94 (3H, m), 1.90-1.78 (1H, m), 1.40-1.22 (1H, m), 1.34
(9H, s).
Step C-2
Preparation of N-(4-tert-butylbenzyl)cinchonidium
4-methoxyphenoxide
##STR00032##
[0190] N-(4-tert-Butylbenzyl)cinchonidium bromide (18.5 g),
AMBERLYST(registered trademark) A26 (strong basic ion exchange
resin of styrene, divinylbenzene matrix) (18.5 g) and methanol (280
ml) were added, and the mixture was stirred at room temperature
overnight. The insoluble material was filtered off through celite,
and washed with methanol (100 ml). To the filtrate was added
4-methoxyphenol (4.8 g), and the solvent was evaporated. The
residue was azeotropically evaporated 3 times with toluene (100
ml), and toluene (20 ml) was added. Then, diisopropyl ether (200
ml) was added dropwise, and the mixture was stirred at room
temperature for 3 hr. The precipitated solid was collected by
filtration, washed with diisopropyl ether (50 ml) and the mixture
was dried under reduced pressure at room temperature overnight to
give the title compound (21.8 g).
[0191] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.91 (1H, d,
J=4.4 Hz), 8.17 (1H, d, J=8.2 Hz), 8.07 (1H, d, J=8.4 Hz), 7.89
(1H, d, J=4.4 Hz), 7.79 (1H, t, J=7.6 Hz), 7.64 (1H, t, J=7.5 Hz),
7.57-7.52 (5H, m), 6.56-6.55 (2H, m), 6.43-6.42 (3H, m), 5.67-5.59
(1H, m), 5.28 (1H, d, J=12.1 Hz), 5.12 (1H, d, J=17.2 Hz), 4.92
(1H, d, J=10.6 Hz), 4.84 (1H, d, J=12.1 Hz), 4.65-4.53 (1H, m),
3.80 (1H, t, J=8.8 Hz), 3.65-3.63 (1H, m), 3.57 (3H, s), 3.25 (1H,
t, J=11.6 Hz), 3.10-3.07 (1H, m), 2.67 (1H, br s), 2.07-2.02 (2H,
m), 1.95 (1H, br s), 1.79-1.76 (1H, br m), 1.33 (9H, s), 1.16-1.11
(1H, m).
Step D
Preparation of 3-hydroxy-3-methylbutyl toluene-4-sulfonate
##STR00033##
[0193] Under a nitrogen atmosphere, 3-methylbutane-1,3-diol (300 g)
was dissolved in pyridine (900 ml), and a solution of
4-methylbenzenesulfonyl chloride (500 g) in toluene (900 ml) and
acetonitrile (125 ml) was added dropwise over 2 hr. The reaction
mixture was stirred at room temperature for 4 hr, and toluene (500
ml) and water (1800 ml) were added to allow for layer separation.
The obtained organic layer was washed successively with aqueous
sulfuric acid and water (twice). The solvent in the obtained
organic layer was evaporated, and the residue was azeotropically
evaporated with toluene (500 ml) to give the title compound (535
g).
[0194] .sup.1H-NMR (CDCl.sub.3) .delta.: 7.81-7.76 (2H, m),
7.36-7.31 (2H, m), 4.20 (2H, td, J=6.8, 1.6 Hz), 2.44 (3H, s), 1.85
(2H, td, J=6.8, 1.6 Hz), 1.33 (1H, s), 1.21 (6H, s).
Example 2
Synthesis of
2-{4-[(9R)-9-hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)--
9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (compound
(3))
Step 1
Ethyl
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]bu-
tyrate
##STR00034##
[0196] (9R)-4-Chloro-9-(trifluoromethyl)-9H-fluorene-2,9-diol (200
mg) obtained in step 10 of example 1 was dissolved in
N,N-dimethylformamide (2 ml), potassium carbonate (185 mg) and
ethyl 4-bromobutyrate (105 .mu.l) were added, and the mixture was
stirred at room temperature for 7 hr. To the reaction mixture was
added water, and the mixture was extracted twice with ethyl
acetate. The obtained organic layer was washed successively with
water (twice) and saturated brine. The obtained organic layer was
dried over anhydrous magnesium sulfate, the insoluble material was
filtered off, and the solvent in the filtrate was evaporated. The
obtained residue was purified by silica gel column chromatography
(a mixture of hexane and ethyl acetate was used as an elution
solvent, first eluted with a mixture at a mixing ratio 5:1
(hexane:ethyl acetate), then successively with a mixture at a
mixing ratio 3:1, and further with a mixture at a mixing ratio 2:1)
to give the title compound (197 mg).
[0197] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.19 (1H, d,
J=7.7 Hz), 7.66 (1H, d, J=7.7 Hz), 7.46 (1H, td, J=7.6, 1.0 Hz),
7.32 (1H, td, J=7.6, 1.0 Hz), 7.16 (1H, br s), 6.93 (1H, d, J=2.1
Hz), 4.14 (2H, q, J=7.1 Hz), 4.05 (2H, t, J=7.1 Hz), 2.82 (1H, s),
2.50 (2H, t, J=7.1 Hz), 2.15-2.06 (2H, m), 1.25 (3H, t, J=7.1
Hz).
Step 2
(9R)-4-Chloro-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)-9H-fluor-
en-9-ol
##STR00035##
[0199] Under a nitrogen atmosphere, ethyl
4-[(9R)-4-chloro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-2-yloxy]butyrat-
e (197 mg) was dissolved in THF (2 ml), and methyllithium/diethyl
ether solution (1.07 M, 2.2 ml) was added dropwise at 0.degree. C.
The reaction mixture was stirred at the same temperature for 2 hr,
water was added, and the mixture was extracted with ethyl acetate
(twice). The obtained organic layer was washed successively with
water (twice) and saturated brine. The obtained organic layer was
dried over anhydrous magnesium sulfate, the insoluble material was
filtered off, and the solvent in the filtrate was evaporated. The
obtained residue was purified by silica gel column chromatography
(a mixture of hexane and ethyl acetate was used as an elution
solvent, first eluted with a mixture at a mixing ratio 3:1
(hexane:ethyl acetate), then with a mixture at a mixing ratio 2:1)
to give the title compound (169 mg).
[0200] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.19 (1H, d,
J=7.7 Hz), 7.66 (1H, d, J=7.7 Hz), 7.47 (1H, td, J=7.7, 1.0 Hz),
7.32 (1H, td, J=7.7, 1.0 Hz), 7.17 (1H, br s), 6.93 (1H, d, J=2.3
Hz), 4.02 (2H, t, J=6.4 Hz), 2.82 (1H, s), 1.92-1.85 (2H, m),
1.65-1.62 (2H, m), 1.26 (3H, s), 1.25 (3H, s).
Step 3
Ethyl
2-{4-[(9R)-9-hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromet-
hyl)-9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionate
##STR00036##
[0202] Under an argon atmosphere,
(9R)-4-chloro-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)-9H-fluo-
ren-9-ol (169 mg) was dissolved in 1,4-dioxane (1.5 ml), ethyl
2-methyl-2-[4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-pyrazol-1--
yl]propionate (194 mg), water (0.5 ml), tripotassium phosphate (178
mg), palladium acetate (9 mg), and SPhos (33 mg) were added, and
the mixture was stirred at 100.degree. C. for 4.5 hr. The reaction
mixture was cooled to room temperature, water was added, and the
mixture was extracted with ethyl acetate (twice). The obtained
organic layer was washed successively with water (twice) and
saturated brine. The obtained organic layer was dried over
anhydrous magnesium sulfate, the insoluble material was filtered
off, and the solvent in the filtrate was evaporated. The obtained
residue was purified by silica gel column chromatography (a mixture
of hexane and ethyl acetate at a mixing ratio 1:1 (hexane:ethyl
acetate) was used as an elution solvent) to give the title compound
(218 mg).
[0203] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 7.69 (1H, s),
7.63-7.62 (2H, m), 7.21-7.19 (4H, m), 6.81 (1H, d, J=2.3 Hz), 4.21
(2H, q, J=7.1 Hz), 4.04 (2H, t, J=6.3 Hz), 2.82 (1H, s), 1.92 (3H,
s), 1.91 (3H, s), 1.89-1.88 (2H, m), 1.66-1.64 (2H, m), 1.26 (6H,
s), 1.26-1.23 (3H, m).
Step 4
2-{4-[(9R)-9-Hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)-9-
H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionic acid
##STR00037##
[0205] Ethyl
2-{4-[(9R)-9-hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)--
9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionate (218 mg) was
dissolved in ethanol (2.2 ml), 4N aqueous sodium hydroxide (320
.mu.l) was added, and the mixture was stirred at room temperature
overnight. The reaction mixture was neutralized with 1N
hydrochloric acid, and extracted with ethyl acetate (twice). The
obtained organic layer was washed successively with water (twice)
and saturated brine. The obtained organic layer was dried over
anhydrous magnesium sulfate, the insoluble material was filtered
off, and the solvent in the filtrate was evaporated to give the
title compound (179 mg).
[0206] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 7.73 (1H, s),
7.68 (1H, s), 7.63-7.62 (1H, m), 7.23-7.09 (4H, m), 6.78 (1H, d,
J=2.6 Hz), 4.02 (2H, t, J=6.3 Hz), 1.93 (6H, s), 1.89-1.86 (2H, m),
1.65-1.61 (2H, m), 1.25 (6H, s).
Step 5
2-{4-[(9R)-9-Hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)-9-
H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (compound
(3))
##STR00038##
[0208] Under a nitrogen atmosphere,
2-{4-[(9R)-9-hydroxy-2-(4-hydroxy-4-methylpentyloxy)-9-(trifluoromethyl)--
9H-fluoren-4-yl]-1H-pyrazol-1-yl}-2-methylpropionic acid (89 mg)
was dissolved in N,N-dimethylformamide (1 ml), ammonium chloride
(28 mg), N,N-diisopropylethylamine (148 .mu.l) and
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridin-1-ium
3-oxide hexafluorophosphate (HATU) (99 mg) were added, and the
mixture was stirred at room temperature overnight. To the reaction
mixture was added water, and the mixture was extracted with ethyl
acetate (twice). The obtained organic layer was washed successively
with diluted brine (twice) and saturated brine. The obtained
organic layer was dried over anhydrous magnesium sulfate, the
insoluble material was filtered off, and the solvent in the
filtrate was evaporated. The obtained residue was purified by
silica gel thin layer chromatography (a mixture of chloroform and
methanol at a mixing ratio 9:1 (chloroform:methanol) was used as an
elution solvent) to give the title compound (48 mg, optical purity
96.9% e.e.). The optical purity was determined under the HPLC
analysis condition 2. Retention time of (R) form 13.0 min,
retention time of (S) form 14.4 min.
[0209] Specific optical rotation [.alpha.].sub.D+37.5.degree.
(c=1.04 MeOH 25.degree. C.)
[0210] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) .delta.: 8.07 (1H, s),
7.66 (1H, s), 7.57-7.55 (1H, m), 7.34-7.31 (1H, m), 7.24-7.23 (3H,
m), 7.18 (1H, s), 7.11 (1H, br s), 6.94 (1H, br s), 6.86 (1H, d,
J=2.3 Hz), 4.16 (1H, s), 4.03 (2H, t, J=6.5 Hz), 1.80 (3H, s), 1.79
(3H, s), 1.80-1.75 (2H, m), 1.51-1.47 (2H, m), 1.11 (6H, s).
Preparation Example of Crystal of Compound (3)
[0211] To compound (3) (40 mg) synthesized by the above-mentioned
example steps was added a mixture of MeOH and water (volume ratio
1:3 (0.5 mL)). Then, to this solution was added a crystal (0.5 mg)
of compound (2h) and the mixture was stirred at room temperature
for 3 days. The precipitated solid was collected by filtration to
give a crystal (41 mg) of compound (3).
(Preparation of Compounds (A), (B), (C) and (D))
[0212] Compound (A), compound (B), compound (C) and compound (D),
which are represented by the following formulas, were each obtained
as an optically active form according to the production method
described in WO 2010/041748.
##STR00039##
Compound (A)
2-(4-{(9R)-9-Hydroxy-2-[2-(3-hydroxyadamantan-1-yl)ethoxy]-9-(trifluoromet-
hyl)-9H-fluoren-4-yl}-1H-pyrazol-1-yl)acetamide
##STR00040##
[0213] Compound (B)
(9R)-2-(2-Hydroxy-2-methylpropoxy)-4-(1-methyl-1H-pyrazol-4-yl)-9-(trifluo-
romethyl)-9H-fluoren-9-ol
##STR00041##
[0214] Compound (C)
(9R)-4-[1-(2-Hydroxyethyl)-1H-pyrazol-4-yl]-2-(2-hydroxy-2-methylpropoxy)--
9-(trifluoromethyl)-9H-fluoren-9-ol
##STR00042##
[0215] Compound (D)
2-{4-[(9R)-2-Fluoro-9-hydroxy-9-(trifluoromethyl)-9H-fluoren-4-yl]-1H-pyra-
zol-1-yl}-2-methylpropanamide
[0216] As a Formulation Example of the present invention, the
following preparation can be mentioned. However, the present
invention is not limited by these Formulation Examples.
Formulation Example 1
Production of Capsule
TABLE-US-00001 [0217] 1) compound of Example 1 (compound (2)) 30 mg
2) microcrystalline cellulose 10 mg 3) lactose 19 mg 4) magnesium
stearate 1 mg
[0218] 1), 2), 3) and 4) are mixed and filled in a gelatin
capsule.
Formulation Example 2
Production of Tablet
TABLE-US-00002 [0219] 1) compound of Example 1 (compound (2)) 10 g
2) lactose 50 g 3) cornstarch 15 g 4) carmellose calcium 44 g 5)
magnesium stearate 1 g
[0220] The total amount of 1), 2), 3) and 30 g of 4) are kneaded
with water, vacuum dried, and sieved. The sieved powder is mixed
with 14 g of 4) and 1 g of 5), and the mixture is punched by a
tableting machine. In this way, 1000 tablets each containing 10 mg
of the compound of Example 1 (compound (2)) per tablet are
obtained.
Experimental Example 1
Inhibitory Action of PDHK Activity In Vitro
[0221] The inhibitory action of PDHK activity was assessed
indirectly by measuring the residual PDH activity after kinase
reaction in the presence of a test compound.
(Inhibitory Action of PDHK1 Activity)
[0222] In the case of human PDHK1 (hPDHK1, Genbank Accession No.
L42450.1), a 1.3 kbp fragment encoding this protein was isolated
from human liver cDNA by polymerase chain reaction (PCR). Modified
hPDHK1 cDNA wherein FLAG-Tag sequence was added to the N terminus
was prepared by PCR and cloned into a vector (pET17b-Novagen). The
recombinant construct was transformed into Escherichia coli
(DH5.alpha.-TOYOBO). The recombinant clones were identified, and
plasmid DNA was isolated and subjected to the DNA sequence
analysis. One clone which had the expected nucleic acid sequence
was selected for expression work.
[0223] For expression of hPDHK1 activity, Escherichia coli strain
BL21(DE3) cells (Novagen) were transformed with the pET17b vector
containing modified hPDHK1 cDNA. The Escherichia coli were grown to
an optical density 0.6 (600 nmol/L) at 30.degree. C. Protein
expression was induced by the addition of 500 .mu.mol/L
isopropyl-.beta.-thiogalactopyranoside. The Escherichia coli were
cultured at 30.degree. C. for 5 hr and harvested by centrifugation.
Resuspension of the Escherichia coli paste was disrupted by a
microfluidizer. FLAG-Tagged protein was purified using FLAG
affinity gel (Sigma).
[0224] The gel was washed with 20 mmol/L
N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-sodium
hydroxide (HEPES-NaOH), 500 mmol/L sodium chloride, 1% ethylene
glycol, and 0.1% polyoxyethylene-polyoxypropylene block copolymer
(Pluronic F-68, pH 8.0), and the binding protein was eluted with 20
mmol/L HEPES-NaOH, 100 .mu.g/mL FLAG peptide, 500 mmol/L sodium
chloride, 1% ethylene glycol, and 0.1% Pluronic F-68 (pH 8.0).
[0225] The eluted fractions containing FLAG-Tagged protein were
pooled, dialyzed against 20 mmol/L HEPES-NaOH, 150 mmol/L sodium
chloride, 0.5 mmol/L ethylenediamine tetraacetic acid (EDTA), 1%
ethylene glycol, and 0.1% Pluronic F-68 (pH 8.0), and preserved at
-80.degree. C. Upon the assay, the hPDHK1 enzyme concentration was
set at a minimum concentration giving over 90% inhibition of PDH
activity.
[0226] 0.05 U/mL PDH (porcine heart PDH complex, Sigma P7032) and
1.0 .mu.g/mL hPDHK1 were mixed in a buffer (50 mmol/L
3-morpholinopropane sulfonic acid (pH 7.0), 20 mmol/L dipotassium
hydrogen phosphate, 60 mmol/L potassium chloride, 2 mmol/L
magnesium chloride, 0.4 mmol/L EDTA, 0.2% Pluronic F-68, 2 mmol/L
dithiothreitol), and the mixture was incubated at 4.degree. C.
overnight to obtain a PDH/hPDHK1 complex.
[0227] The test compounds were diluted with dimethyl sulfoxide
(DMSO). The PDH/hPDHK1 complex (20 .mu.L), test compound (1.5
.mu.L) and 3.53 .mu.mol/L ATP (diluted with buffer, 8.5 .mu.L) were
added to a half area 96 well UV-transparent microplate (Corning
3679), and PDHK reaction was performed at room temperature for 45
min. DMSO (1.5 .mu.L) was added to control wells instead of test
compound. In order to determine maximum rate of the PDH reaction,
DMSO (1.5 .mu.L) was added to blank wells instead of test compound
in absence of hPDHK1.
[0228] Then, 10 .mu.L of substrates (5 mmol/L sodium pyruvate, 5
mmol/L Coenzyme A, 12 mmol/L NAD, 5 mmol/L thiamin pyrophosphate,
diluted with buffer) were added. The mixture was incubated at room
temperature for 90 min, and the residual PDH activity was
measured.
[0229] The absorbance at 340 nm before and after PDH reaction was
measured using a microplate reader to detect NADH produced by the
PDH reaction. The hPDHK1 inhibition rate (%) of the test compound
was calculated from the formula [{(PDH activity of the test
compound-PDH activity of control)/PDH activity of blank-PDH
activity of control)}.times.100]. The IC.sub.50 value was
calculated from the concentrations of the test compound at two
points enclosing 50% inhibition of the hPDHK1 activity.
[0230] The results obtained using compound (2), compound (2h) and
compound (3) as test compounds are shown in the following Table
1.
(Inhibitory Action of PDHK2 Activity)
[0231] In the case of human PDHK2 (hPDHK2, Genbank Accession No.
BC040478.1), modified hPDHK2 cDNA wherein FLAG-Tag sequence was
added to the N terminus of hPDHK2 cDNA clone
(pReceiver-M01/PDK2-GeneCopoeia) was prepared by PCR and cloned
into a vector (pET17b-Novagen). The recombinant construct was
transformed into Escherichia coli (DH5.alpha.-TOYOBO). The
recombinant clones were identified, and plasmid DNA was isolated
and subjected to the DNA sequence analysis. One clone which had the
expected nucleic acid sequence was selected for expression
work.
[0232] For expression of hPDHK2 activity, Escherichia coli strain
BL21(DE3) cells (Novagen) were transformed with the pET17b vector
containing modified hPDHK2 cDNA. The Escherichia coli were grown to
an optical density 0.6 (600 nmol/L) at 30.degree. C. Protein
expression was induced by the addition of 500 .mu.mol/L
isopropyl-.beta.-thiogalactopyranoside. The Escherichia coli were
cultured at 30.degree. C. for 5 hr and harvested by centrifugation.
Resuspension of the Escherichia coli paste was disrupted by a
microfluidizer. FLAG-Tagged protein was purified using FLAG
affinity gel. The gel was washed with 20 mmol/L HEPES-NaOH, 500
mmol/L sodium chloride, 1% ethylene glycol, and 0.1% Pluronic F-68
(pH 8.0), and the binding protein was eluted with 20 mmol/L
HEPES-NaOH, 100 .mu.g/mL FLAG peptide, 500 mmol/L sodium chloride,
1% ethylene glycol, and 0.1% Pluronic F-68 (pH 8.0). The eluted
fractions containing FLAG-Tagged protein were pooled, dialyzed
against 20 mmol/L HEPES-NaOH, 150 mmol/L sodium chloride, 0.5
mmol/L EDTA, 1% ethylene glycol, and 0.1% Pluronic F-68 (pH 8.0),
and preserved at -80.degree. C. Upon the assay, the hPDHK2 enzyme
concentration was set to a minimum concentration giving over 90%
inhibition of PDH activity.
[0233] 0.05 U/mL PDH and 0.8 .mu.g/mL hPDHK2 were mixed in a buffer
(50 mmol/L 3-morpholinopropanesulfonic acid (pH 7.0), 20 mmol/L
dipotassium hydrogen phosphate, 60 mmol/L potassium chloride, 2
mmol/L magnesium chloride, 0.4 mmol/L EDTA, and 0.2% Pluronic F-68,
2 mmol/L dithiothreitol), and the mixture was incubated at
4.degree. C. overnight to obtain a PDH/hPDHK2 complex. The test
compounds were diluted with DMSO. The PDH/hPDHK2 complex (20
.mu.L), test compound (1.5 .mu.L) and 3.53 .mu.mol/L ATP (diluted
with buffer, 8.5 .mu.L) were added to a half area 96 well
UV-transparent microplate, and PDHK reaction was performed at room
temperature for 45 min. DMSO (1.5 .mu.L) was added to control wells
instead of the test compound. In order to determine maximum rate of
the PDH reaction, DMSO (1.5 .mu.L) was added to blank wells instead
of compound in absence of hPDHK2. Then, 10 .mu.L of substrate (5
mmol/L sodium pyruvate, 5 mmol/L Coenzyme A, 12 mmol/L NAD, and 5
mmol/L thiamine pyrophosphate, diluted with buffer) were added. The
mixture was incubated at room temperature for 90 min, and the
residual PDH activity was measured. The absorbance at 340 nm before
and after PDH reaction was measured using a microplate reader to
detect NADH produced by the PDH reaction. The hPDHK2 inhibition
rate (%) of the test compound was calculated from the formula
[{(PDH activity of test compound-PDH activity of control)/PDH
activity of blank-PDH activity of control)}.times.100]. The
IC.sub.50 value was calculated from the concentrations of the test
compound at two points enclosing 50% inhibition of the hPDHK2
activity.
[0234] The results obtained using compound (2), compound (2h),
compound (3), compound (A), compound (B), compound (C) and compound
(D) as test compounds are shown in the following Table 1.
TABLE-US-00003 TABLE 1 hPDHK1 IC.sub.50 hPDHK2 IC.sub.50 Compound
(.mu.mol/L) (.mu.mol/L) Compound (2) 0.0047 0.0046 Compound (2h)
0.0066 0.0049 Compound (3) 0.0035 0.0042 Compound (A) -- (not
tested) 0.0051 Compound (B) -- (not tested) 0.0074 Compound (C) --
(not tested) 0.0067 Compound (D) -- (not tested) 0.0051
Experimental Example 2
Ex Vivo PDH Activation Assay
(Experimental Method)
[0235] The action of test compound on tissue PDH activity was
evaluated. NADH production was detected via p-iodonitrotetrazolium
violet (INT)-coupled system to measure PDH activity.
[0236] Normal male Sprague-Dawley rats were randomly allocated to
the vehicle group and the test compound groups. The vehicle (0.5%
aqueous methylcellulose solution, 5 mL/kg) or the test compound was
orally administered to the rats. At 5 or 20 hr after
administration, the rats were anesthetized with an intraperitoneal
injection of sodium pentobarbital (60 mg/kg), and liver slices and
epididymal adipose tissues were collected.
[0237] To the liver slices were rapidly added 9 volumes of ice-cold
homogenization buffer (0.25 mol/L sucrose, 5 mmol/L
tris(hydroxymethyl)aminomethane hydrochloride (pH 7.5), 2 mmol/L
EDTA), and the mixtures were homogenized using a Polytron
homogenizer. The homogenates were centrifuged at 600.times.g,
4.degree. C. for 10 min to obtain the supernatant. The supernatants
(1 mL) were centrifuged at 16,000.times.g, 4.degree. C. for 10 min
to collect the precipitates. The precipitates were washed by
resuspension in the homogenization buffer (1 mL) and centrifuged in
the same manner. The precipitates were frozen with liquid nitrogen
and stored at -80.degree. C. as the liver mitochondrial
fraction.
[0238] To the adipose tissues were rapidly added 3 volumes of an
ice-cold homogenization buffer, and the mixtures were homogenized
using a Polytron homogenizer. The homogenates were centrifuged at
600.times.g, 4.degree. C. for 10 min to obtain the supernatant. The
supernatants were centrifuged at 16,000.times.g, 4.degree. C. for
10 min to collect the precipitates. The precipitates were washed by
resuspension in the homogenization buffer (1 mL) and centrifuged in
the same manner. The precipitates were frozen with liquid nitrogen
and stored at -80.degree. C. as the adipose tissue mitochondrial
fraction.
[0239] The mitochondrial fractions were thawed and suspended with
the sample buffer (0.25 mol/L sucrose, 20 mmol/L
tris(hydroxymethyl)aminomethane hydrochloride (pH 7.5), 50 mmol/L
potassium chloride, and 1 mL/L
4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton
X-100)). Active PDH activity (PDHa activity) and total PDH activity
(PDHt activity) were measured to evaluate the PDH activity. For the
measurement of the PDHt activity, equal amounts of the
mitochondrial suspension and the activation buffer (0.25 mol/L
sucrose, 20 mmol/L tris(hydroxymethyl)aminomethane hydrochloride
(pH 7.5), 50 mmol/L potassium chloride, 1 mL/L Triton X-100, 4
mmol/L calcium chloride, 40 mmol/L magnesium chloride, 10 mmol/L
sodium dichloroacetate) were mixed, and the mixtures were incubated
at 37.degree. C. for 10 min. Forty microliters of the mitochondrial
suspensions diluted with a sample buffer were added to a 96-well
microplate for activity measurement and blank measurement. Then 180
.mu.L of the reaction mixture (0.056 mmol/L potassium phosphate
buffer (pH 7.5), 5.6 mmol/L DL-carnitine, 2.8 mmol/L NAD, 0.22
mmol/L thiamin pyrophosphate, 0.11 mmol/L Coenzyme A, 1.1 mL/L
Triton X-100, 1.1 mmol/L magnesium chloride, 1.1 g/L bovine serum
albumin, 0.67 mmol/L INT, 7.2 .mu.mol/L phenazine methosulfate, 28
mmol/L sodium oxamate) was added to each well, and then 20 .mu.L of
50 mmol/L sodium pyruvate for activity measurement or water for
blank measurement were added. The mixtures were incubated at room
temperature under shading. The absorbances at 500-750 nm, which
were attributable to reduction of INT, the final electron acceptor,
were measured using a microplate reader over time and the changes
in the absorbance were calculated. The PDH activity was calculated
by subtraction of the change in absorbance of the blank well from
that of the activity measurement well. The percentage of the PDHa
activity to the PDHt activity was calculated and taken as an index
of the PDH activation.
[0240] The results obtained using compound (2h), compound (3),
compound (A), compound (B), compound (C) and compound (D) as test
compounds are shown in the following Table 2, FIG. 1 (Liver) and
FIG. 2 (Adipose tissue). In addition, the results obtained using
compound (2) are shown in the following Table 3.
TABLE-US-00004 TABLE 2 PDHa activity (% of PDHt activity) Liver
Adipose tissue 5 hr 20 hr 5 hr 20 hr 3 3 3 3 Compound Vehicle mg/kg
Vehicle mg/kg Vehicle mg/kg Vehicle mg/kg Compound 13 .+-. 3 59
.+-. 6 13 .+-. 3 31 .+-. 5 31 .+-. 10 59 .+-. 2 31 .+-. 10 44 .+-.
12 (2h) Compound 13 .+-. 3 56 .+-. 8 13 .+-. 3 32 .+-. 9 31 .+-. 10
70 .+-. 9 31 .+-. 10 42 .+-. 3 (3) Compound 13 .+-. 3 35 .+-. 6 13
.+-. 3 16 .+-. 10 31 .+-. 10 32 .+-. 14 31 .+-. 10 24 .+-. 7 (A)
Compound 13 .+-. 3 43 .+-. 7 13 .+-. 3 10 .+-. 3 31 .+-. 10 59 .+-.
5 31 .+-. 10 30 .+-. 4 (B) Compound 13 .+-. 3 44 .+-. 5 13 .+-. 3
13 .+-. 3 31 .+-. 10 53 .+-. 4 31 .+-. 10 28 .+-. 7 (C) Compound 13
.+-. 3 41 .+-. 15 13 .+-. 3 20 .+-. 1 31 .+-. 10 43 .+-. 7 31 .+-.
10 24 .+-. 3 (D) mean .+-. S.D. (n = 3)
TABLE-US-00005 TABLE 3 PDHa activity (% of PDHt activity) Liver
Adipose tissue 5 hr 20 hr 5 hr 20 hr 3 3 3 3 Compound Vehicle mg/kg
Vehicle mg/kg Vehicle mg/kg Vehicle mg/kg Compound 28 .+-. 6 74
.+-. 12 28 .+-. 6 50 .+-. 16 42 .+-. 4 88 .+-. 11 42 .+-. 4 61 .+-.
15 (2) mean .+-. S.D. (n = 3)
Experimental Example 3
Effect of Repeated Administration of Test Compound on HbA1c in ZDF
Rats
(Experimental Method)
[0241] Zucker Diabetic Fatty rats (male, 7-week-old, CHARLES RIVER
LABORATORIES JAPAN INC.), an animal model for type 2 diabetes,
given a purified diet (5.9% fat diet, Oriental Yeast Co., Ltd.)
were allocated to the vehicle group and the test compound groups so
that no bias occurred in the plasma glucose and insulin levels,
HbA1c levels and body weights. Repeated oral doses of the test
compound (1 mg/kg/5 mL) were administered to the rats once daily at
3 hr before the dark period. A 0.5% aqueous methylcellulose
solution was orally administered in the same manner to the rats of
the vehicle group. On day 14 of administration, blood samples were
collected from the tail vein and HbA1c level (%) was measured.
Statistical analysis was performed by Dunnett's test. Values of
p<0.05 were considered statistically significant.
[0242] The results obtained using compound (2) and compound (3) as
test compounds are shown in the following Table 4.
TABLE-US-00006 TABLE 4 HbA1c (%) Compound Vehicle 1 mg/kg Compound
(2) 3.6 .+-. 0.3 3.2 .+-. 0.1* Compound (3) 3.7 .+-. 0.2 3.4 .+-.
0.1* day 14 of administration mean .+-. S.D. (n = 10) *p < 0.05
vs. vehicle group (Dunnett's test)
Experimental Example 4
hERG (Human Ether-a-go-go Related Gene) Whole Cell Patch Clamp
Test
(Experimental Method)
[0243] Using human ether-a-go-go related gene (hERG)-transfected
HEK293 cells (Cytomyx Limited), an influence on hERG current was
examined according to the whole cell patch clamp technique. The
hERG-transfected HEK293 cells were passaged using a CO.sub.2
incubator (BNA-111, TABAI ESPEC CORP.) under the set conditions of
37.degree. C., 5% CO.sub.2, saturated humidity. Culture containers
used were Collagen Type I Coated 75 cm.sup.2 flask (4123-010, AGC
TECHNO GLASS CO., Ltd.) and Collagen Type I Coated 35 mm culture
dish (4000-010, AGC TECHNO GLASS CO., Ltd.). The culture medium
used was E-MEM (Eagle Minimum Essential Medium (Earle's Salts,
Nikken biomedical laboratory) added with 10% FCS (Fetal calf serum,
BioWest, L.L.C.) and 1% MEM Non-Essential Amino Acids Solution
(NEAA, Invitrogen Corporation). Geneticin for the selection of hERG
gene expressing cells was added thereto to a concentration of 400
.mu.g/mL. As the cells for the measurement, 3.times.10.sup.4
hERG-transfected HEK293 cells were plated on a 35 mm culture dish 4
to 7 days before measurement of the hERG current. The culture dish
produced for the measurement contained the above-mentioned culture
medium without geneticin (Invitrogen Corporation).
[0244] The highest evaluation concentration of each compound was
determined from the highest concentration at which precipitation
was not found in the standard extracellular fluid (NaCl: 140
mmol/L, KCl: 2.5 mmol/L, MgCl.sub.2: 2 mmol/L, CaCl.sub.2: 2
mmol/L, HEPES: 10 mmol/L, glucose: 10 mmol/L (adjusted to pH 7.4
with Tris-base)). As the application method, each solution to be
applied was ejected from a Y-tube having a tip diameter of about
0.25 mm, which was adjacent (about 2 mm) to the cells, and applied
to the cells. The ejection rate was about 0.4 mL/min.
[0245] The experiment was performed at room temperature under a
phase contrast microscope. The 35 mm culture dish plated with the
cells was set on a measurement apparatus, and the standard
extracellular fluid was continuously applied to the cells from the
Y-tube. A glass electrode for the measurement was filled with an
intracellular fluid (Potassium Gluconate: 130 mmol/L, KCl: 20
mmol/L, MgCl.sub.2: 1 mmol/L, ATP-Mg: 5 mmol/L, EGTA: 3.5 mmol/L,
HEPES: 10 mmol/L (adjusted to pH 7.2 with Tris-base)). A
conventional whole cell patch clamp method was applied to the
cells, and the maintenance electric potential was set to -80 mV.
Under a fixed electric potential, the whole cell current was
amplified by an amplifier for patch clamp (AXOPATCH-200B, Axon
Instruments, Inc.), and the data was loaded into a computer
(IMC-P642400, Intermedical Co., Ltd.) using a data acquisition
analysis software (pCLAMP 9.2, Axon Instruments, Inc.).
[0246] The measurement of the hERG current was performed in the
following two steps. In both cases, the hERG current was initiated
by giving a command potential (maintenance electric potential -80
mV, prepulse+20 mV, 1.5 sec, test-pulse -50 mV, 1.5 sec).
[0247] Step (1): The above-mentioned command potential was given at
0.1 Hz for 2 min.
[0248] Step (2): The above-mentioned command potential was
subjected to P/3 subtraction of pCLAMP 9.2 to remove leak current.
This was repeated three times and an average thereof was taken as
hERG current.
[0249] Subsequent to step (1), Step (2) was performed (about 3
min), and the maximum tail current obtained by applying a
test-pulse to the hERG current obtained by the method of step (2)
was taken as hERG current value. Hereafter, the operations of (1)
and (2) were alternately repeated until completion of the
experiment and the hERG current value was measured.
[0250] Stable hERG current value was recorded three times (about 10
min), and the standard extracellular fluid was instantaneously
exchanged with each application fluid. The hERG current value was
measured three times (about 10 min) in the same manner during
perfusion of the application fluid, and the current value obtained
by the 3rd measurement was taken as hERG current value after
perfusion of the application fluid.
[0251] The data for each cell was converted to a relative value
with an average of the three hERG current values recorded in about
10 min before perfusion of the application fluid (Before value) as
100%. This was measured for two cells, and an average thereof was
calculated as Relative current (%).
Relative current (%)=100.times.A/B [0252] A: hERG current value
after perfusion of application fluid [0253] B: average of three
hERG current values recorded in about 10 min before perfusion of
application fluid (Before value)
[0254] In addition, a suppression rate on the DMSO group was
calculated according to the following formula.
Suppression rate (%)=100-(C/D).times.100 [0255] C: average of
Relative current (%) of respective test compound groups [0256] D:
average of Relative current (%) of DMSO group
[0257] The results obtained using compound (2), compound (3),
compound (A), compound (B), compound (C) and compound (D) as test
compounds are shown in the following Table 5.
TABLE-US-00007 TABLE 5 Concentration.sup.a) Inhibition IC.sub.50
value Test compound (.mu.mol/L) rate (%) (.mu.mol/L) Compound (2)
30 24.4 >30 Compound (3) 30 27.3 >30 Compound (A) 10 11.8
>10 Compound (B) 1 17.4 3.6 10 72.9 Compound (C) 3 11.5 13.2 30
69.0 Compound (D) 3 9.4 14.2 30 67.5 .sup.a)The highest evaluation
concentration of each compound was set from the highest
concentration at which precipitation in the standard extracellular
fluid was not found.
Experimental Example 5
Metabolic Stability Test in Liver Microsome
(Experimental Method)
[0258] Human liver microsome (manufactured by Xenotech, H0620,
final concentration (after dilution), 0.2 mg protein/mL) was
suspended in 100 mM potassium phosphate buffer (pH 7.4, containing
.rho.-nicotinamide adenine dinucleotide phosphate: 1.3 mM,
D-glucose-6-phosphate: 3.3 mM, magnesium chloride: 3.3 mM,
glucose-6-phosphate dehydrogenase: 0.45 U/mL), and further mixed
with a test compound dissolved in MeCN/DMSO (95/5) (final
concentration 5 .mu.M). The mixture was incubated at 37.degree. C.
for 10 min and 60 min, acetonitrile containing formic acid (final
concentration 0.1%) was added, and the mixture was centrifuged. The
test compound (unmodified) in the supernatant was measured by high
performance liquid chromatography/mass spectrometry (LC/MS)
(manufactured by Waters, LC: Acquity UPLC, MS:SQ Detector or TQ
Detector). The residual ratio (%) was calculated from the obtained
measurement value.
[0259] The results obtained using compound (2), compound (3),
compound (A), compound (B), compound (C) and compound (D) as test
compounds are shown in the following Table 6.
TABLE-US-00008 TABLE 6 Stability in liver microsome (residual ratio
%) human rat Test compound 10 min 60 min 10 min 60 min Compound (2)
98.8 96.5 98.8 100.0 Compound (3) 98.4 85.9 102.7 95.8 Compound (A)
34.8 0.0 24.8 0.0 Compound (B) 98.0 88.2 101.1 92.1 Compound (C)
94.0 75.1 94.2 85.3 Compound (D) 105.4 101.5 105.2 105.1
INDUSTRIAL APPLICABILITY
[0260] Since the compound of the present invention or a
pharmaceutically acceptable salt thereof has a PDHK inhibitory
activity, it is useful as an active ingredient of a medicament for
the prophylaxis or treatment of diabetes (type 1 diabetes, type 2
diabetes etc.), insulin resistance syndrome, metabolic syndrome,
hyperglycemia, hyperlactacidemia, diabetic complications (diabetic
neuropathy, diabetic retinopathy, diabetic nephropathy, cataract
etc.), cardiac failure (acute cardiac failure, chronic cardiac
failure), cardiomyopathy, myocardial ischemia, myocardial
infarction, angina pectoris, dyslipidemia, atherosclerosis,
peripheral arterial disease, intermittent claudication, chronic
obstructive pulmonary disease, brain ischemia, cerebral apoplexy,
mitochondrial disease, mitochondrial encephalomyopathy, cancer,
pulmonary hypertension or Alzheimer disease.
* * * * *