NZ710770B2 - Novel fxr (nr1h4) binding and activity modulating compounds - Google Patents
Novel fxr (nr1h4) binding and activity modulating compounds Download PDFInfo
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- NZ710770B2 NZ710770B2 NZ710770A NZ71077012A NZ710770B2 NZ 710770 B2 NZ710770 B2 NZ 710770B2 NZ 710770 A NZ710770 A NZ 710770A NZ 71077012 A NZ71077012 A NZ 71077012A NZ 710770 B2 NZ710770 B2 NZ 710770B2
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- alkyl
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- fxr
- compounds
- compound
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Classifications
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- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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Abstract
Disclosed herein are compounds of formula 1 where the substituents are as defined herein. Further disclosed is the use of the compounds in the manufacture of a medicament for the prophylaxis and/or treatment of chronic conditions selected from the group consisting of Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC). The compounds also have activity as FXR binding and activity modulating compounds and are therefore intended for use in the treatment of conditions such as liver diseases, inflammatory bowel diseases, diabetes, obesity, myocardial infarction, stroke and hyperproliferative disorders. and Primary Sclerosing Cholangitis (PSC). The compounds also have activity as FXR binding and activity modulating compounds and are therefore intended for use in the treatment of conditions such as liver diseases, inflammatory bowel diseases, diabetes, obesity, myocardial infarction, stroke and hyperproliferative disorders.
Description
Novel FXR (NR1H4) binding and activity modulating compounds
This application is a divisional application out of New Zealand patent application 620177, filed
12 July 2012, the contents of which are incorporated herein by reference in their entirety.
The present invention generally relates to compounds which bind to the NR1H4 receptor (FXR)
and act as agonists or modulators of FXR. The invention further relates to the use of the
compounds in the preparation of medicaments for the treatment and/or prophylaxis of diseases
and/or conditions through binding of said nuclear receptor by said compounds.
Multicellular organisms are dependent on advanced mechanisms of information transfer
between cells and body compartments. The information that is transmitted can be highly
complex and can result in the alteration of genetic programs involved in cellular differentiation,
proliferation, or reproduction. The signals, or hormones, are often low molecular weight
molecules, such as peptides, fatty acid, or cholesterol derivatives.
Many of these signals produce their effects by ultimately changing the transcription of specific
genes. One well-studied group of proteins that mediate a cell's response to a variety of signals
is the family of transcription factors known as nuclear receptors, hereinafter referred to often as
"NR". Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis
and trans retinoic acid, thyroid hormone, bile acids, cholesterol-derivatives, fatty acids (and
other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are
structurally similar to other members of this group, but for which no ligands are known. Orphan
receptors may be indicative of unknown signalling pathways in the cell or may be nuclear
receptors that function without ligand activation. The activation of transcription by some of these
orphan receptors may occur in the absence of an exogenous ligand and/or through signal
transduction pathways originating from the cell surface (D. J. Mangelsdorf et al., Cell 1995, 83,
835; R. M. Evans, Mol. Endocrinol. 2005, 19, 1429).
In general, three functional domains have been defined in NRs. An amino terminal domain is
believed to have some regulatory function. It is followed by a DNA-binding domain hereinafter
referred to as "DBD" which usually comprises two zinc finger elements and recognizes a
specific Hormone Responsive Element hereinafter referred to as "HRE" within the promoters of
responsive genes. Specific amino acid residues in the "DBD" have been shown to confer DNA
sequence binding specificity (M. Schena and K. R. Yamamoto, Science 1988, 241, 965). A
ligand-binding-domain hereinafter referred to as "LBD" is at the carboxy-terminal region of
known NRs.
In the absence of hormone, the LBD appears to interfere with the interaction of the DBD with its
HRE. Hormone binding seems to result in a conformational change in the NR and thus opens
this interference (A. M. Brzozowski et al., Nature 1997, 389, 753). A NR without the LBD
constitutively activates transcription but at a low level.
Coactivators or transcriptional activators are proposed to bridge between sequence specific
transcription factors, the basal transcription machinery and in addition to influence the chromatin
structure of a target cell. Several proteins like SRC-1, ACTR, and Grip1 interact with NRs in a
ligand enhanced manner (D. M. Heery et al., Nature 1997, 387, 733; T. Heinzel et al., Nature
1997, 387, 43; K. W. Nettles and G. L. Greene, Annu. Rev. Physiol. 2005, 67, 309).
Nuclear receptor modulators like steroid hormones affect the growth and function of specific
cells by binding to intracellular receptors and forming nuclear receptor-ligand complexes.
Nuclear receptor-hormone complexes then interact with a HRE in the control region of specific
genes and alter specific gene expression (A. Aranda and A. Pascual, Physiol. Rev. 2001, 81,
1269).
The Farnesoid X Receptor alpha (hereinafter also often referred to as NR1H4 when referring to
the human receptor) is a prototypical type 2 nuclear receptor which activates genes upon
binding to promoter region of target genes in a heterodimeric fashion with Retinoid X Receptor
(B. M. Forman et al., Cell 1995, 81, 687). The relevant physiological ligands of NR1H4 are bile
acids (D. J. Parks et al., Science 1999, 284, 1365; M. Makishima et al., Science 1999, 284,
1362). The most potent one is chenodeoxycholic acid (CDCA), which regulates the expression
of several genes that participate in bile acid homeostasis. Farnesol and derivatives, together
called farnesoids, are originally described to activate the rat orthologue at high concentration
but they do not activate the human or mouse receptor. FXR is expressed in the liver, throughout
the entire gastrointestinal tract including the esophagus, stomach, duodenum, small intestine,
colon, ovary, adrenal gland and kidney. Beyond controlling intracellular gene expression, FXR
seems to be also involved in paracrine and endocrine signalling by upregulating the expression
of the cytokine Fibroblast Growth Factor 15 (rodents) or 19 (monkeys, humans, J. A. Holt et al.,
Genes Dev. 2003, 17, 1581; T. Inagaki et al., Cell Metab. 2005, 2, 217).
Small molecule compounds which act as FXR modulators have been disclosed in the following
publications: , , , , WO
2007/092751, , , , , WO
2009/005998, , and . Further small
molecule FXR modulators have been recently reviewed (M. L. Crawley, Expert Opin Ther. Pat.
2010, 20,1047; D. Merk et al., Future Med. Chem. 2012, 4, 1015).
In we disclosed chiral cyclopropylidene compounds of the following general
formula
wherein the variables are defined similar as in this application.
The problem underlying the present invention is to generate FXR-agonists with improved
physicochemical properties in general, and reduced hydrophobicity, improved aqueous solubility
and better membrane permeability, in particular, compared to compounds claimed in
In one aspect, the invention relates to the use of a compound according to the following
Formula (1), an enantiomer, diastereomer, tautomer, solvate or pharmaceutical acceptable salt
thereof
wherein
R is selected from the group consisting of COOR , CONR R , tetrazolyl, SO NR R , C alkyl,
6 7 8 2 7 8 1-6
SO -C alkyl and H, with R independently selected from the group consisting of H or C alkyl,
2 1-6 6 1-6
and R and R independently from each other selected from the group consisting of H, C alkyl,
7 8 1-6
halo-C alkyl, C alkylene-R , SO -C alkyl, wherein R is selected from the group consisting
1-6 1-6 9 2 1-6 9
of COOH, OH and SO H;
A is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazolyl, indolyl, thienyl,
benzothienyl, indazolyl, benzisoxazolyl, benzofuranyl, benzotriazolyl, furanyl, benzothiazolyl,
thiazolyl, oxadiazolyl, each optionally substituted with one or two groups independently selected
from the group consisting of OH, O-C alkyl, O-halo-C alkyl, C alkyl, halo-C alkyl, C
1-6 1-6 1-6 1-6 3-6
cycloalkyl and halogen;
Q is phenyl, optionally substituted with one or two groups independently selected from the
group consisting of C alkyl, halo-C alkyl, halogen and CF ;
1-6 1-6 3
Y is selected from N or CH;
Z is selected from
, , or
wherein
X = CH, N, NO;
R is selected from the group consisting of hydrogen, C alkyl, C cylcoalkyl, C
1 1-3 3-6 4-5
alkylcycloalkyl, wherein C1-3 alkyl is optionally substituted with 1 to 3 substituents independently
selected from halogen, hydroxy or C alkoxy;
R and R are independently selected from the group consisting of hydrogen, C alkyl, C
2 3 1-3 1-3
haloalkyl, C alkoxy, C haloalkoxy and halogen,
1-3 1-3
in the preparation of a medicament for the prophylaxis and/or treatment of chronic intrahepatic
conditions selected from the group consisting of Primary Biliary Cirrhosis (PBC) and Primary
Sclerosing Cholangitis (PSC).
Certain statements that appear below are broader than what appears in the statements of the
invention above. These statements are provided in the interests of providing the reader with a
better understanding of the invention and its practice. The reader is directed to the
accompanying claim set which defines the scope of the invention.
Described herein is a compound according to the following Formula (1), an enantiomer,
diastereomer, tautomer, solvate, prodrug or pharmaceutical acceptable salt thereof
wherein
R is selected from the group consisting of COOR , CONR R , tetrazolyl, SO NR R , C alkyl,
6 7 8 2 7 8 1-6
SO -C alkyl and H, with R independently selected from the group consisting of H or C alkyl,
2 1-6 6 1-6
and R and R independently from each other selected from the group consisting of H, C alkyl,
7 8 1-6
halo-C1-6 alkyl, C1-6 alkylene-R9, SO2-C1-6 alkyl, wherein R9 is selected from the group consisting
of COOH, OH and SO3H;
A is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazolyl, indolyl, thienyl,
benzothienyl, indazolyl, benzisoxazolyl, benzofuranyl, benzotriazolyl, furanyl, benzothiazolyl,
thiazolyl, oxadiazolyl, each optionally substituted with one or two groups independently selected
from the group consisting of OH, O-C alkyl, O-halo-C alkyl, C alkyl, halo-C alkyl, C
1-6 1-6 1-6 1-6 3-6
cycloalkyl and halogen;
Q is selected from the group consisting of phenyl, pyridyl, thiazolyl, thiophenyl, pyrimidyl, each
optionally substituted with one or two groups independently selected from the group consisting
of C alkyl, halo-C alkyl, halogen and CF ;
1-6 1-6 3
Y is selected from N or CH;
Z is selected from
, , or
wherein
X = CH, N, NO;
R is selected from the group consisting of hydrogen, C alkyl, C cylcoalkyl, C
1 1-3 3-6 4-5
alkylcycloalkyl, wherein C alkyl is optionally substituted with 1 to 3 substituents independently
selected from halogen, hydroxy or C alkoxy;
R and R are independently selected from the group consisting of hydrogen, C alkyl, C
2 3 1-3 1-3
haloalkyl, C alkoxy, C haloalkoxy and halogen.
1-3 1-3
In another embodiment in combination with any of the above or below embodiments, R-A in the
compound according to Formula (1) is selected from
, , , , ,
, , , , ,
, or .
In another embodiment in combination with any of the above or below embodiments, Q in the
compound according to Formula (1) is
.
In another embodiment in combination with any of the above or below embodiments, Z in the
compound according to Formula (1) is
In another embodiment in combination with any of the above or below embodiments, the
compound according to Formula (1) is selected from
, ,
, ,
, ,
, ,
, ,
, ,
, ,
, ,
, ,
, ,
, ,
or .
In another embodiment in combination with any of the above or below embodiments, the
compound according to Formula (1) is
wherein R is selected from the group consisting of CO H, CONHSO Me, and tetrazolyl.
In another embodiment, described is a compound according to Formula (1) for use as a
medicament.
In another embodiment, described is a compound according to Formula (1) for use in the
prophylaxis and/or treatment of diseases mediated by FXR.
In another embodiment, described is the use of a compound according to Formula (1) for the
preparation of a medicament for the prophylaxis and/or treatment of diseases mediated by FXR.
In another embodiment in combination with any of the above or below embodiments, the
disease is selected from chronic intrahepatic or some forms of extrahepatic cholestatic
conditions; liver fibrosis; obstructive or chronic inflammatory disorders of the liver; liver cirrhosis;
liver steatosis and associated syndromes, cholestatic or fibrotic effects that are associated with
alcohol-induced cirrhosis or with viral-borne forms of hepatitis; liver failure or liver ischemia after
major liver resection; chemotherapy associated steatohepatitis (CASH); acute liver failure;
and/or Inflammatory Bowel Diseases.
In another embodiment in combination with any of the above or below embodiments, the
disease is selected from lipid and lipoprotein disorders; Type Il Diabetes and clinical
complications of Type I and Type Il Diabetes, including diabetic nephropathy, diabetic
neuropathy, diabetic retinopathy and other observed effects of clinically manifest long term
Diabetes; conditions and diseases which result from chronic fatty and fibrotic degeneration of
organs due to enforced lipid and specifically triglyceride accumulation and subsequent
activation of profibrotic pathways, such as Non-Alcoholic Fatty Liver Disease (NAFLD), or Non-
Alcoholic Steatohepatitis (NASH); obesity or metabolic syndrome (combined conditions of
dyslipidemia, diabetes or abnormally high body-mass index); and/or cute myocardial infarction,
acute stroke or thrombosis which occurs as an endpoint of chronic obstructive atherosclerosis.
In another embodiment in combination with any of the above or below embodiments, the
disease is selected from non-malignant hyperproliferative disorders and malignant
hyperproliferative disorders, specifically of hepatocellular carcinoma, colon adenoma and
polyposis, colon adenocarcinoma, breast cancer, pancreas adenocarcinoma, Barrett's
esophagus or other forms of neoplastic diseases of the gastrointestinal tract and the liver.
The improved physico-chemical properties have been achieved by the introduction of a polar
hydroxyl group on a 1,3-cyclobutylidene or 1,3-azetidinylidene group replacing the former 1,2-
cyclopropylidene ring.
Surprisingly, the resulting compounds maintained their activity on the FXR receptor but
demonstrated improved physico-chemical properties, such as higher aqueous solubility and/or
membrane permeability.
The compounds described herein share a common chemical structure according to Formula (1)
in claim 1.
In a preferred embodiment in combination with any of the above or below embodiments,
described is an enantiomer, diastereomer or pharmaceutically acceptable salt of a compound
according to Formula (1).
In a preferred embodiment in combination with any of the above or below embodiments, R in
Formula (1) is selected from the group consisting of COOR6, CONR7R8, SO2NR7R8, and SO2-C1-
alkyl.
In a preferred embodiment in combination with any of the above or below embodiments, R in
Formula (1) is H.
and
In a preferred embodiment in combination with any of the above or below embodiments, R7
R in Formula (1) are independently from each other selected from the group consisting of H
and SO -C alkyl.
2 1-6
In a preferred embodiment in combination with any of the above or below embodiments, R in
Formula (1) is H.
In a preferred embodiment in combination with any of the above or below embodiments, R in
Formula (1) is SO -C alkyl.
2 1-6
In a preferred embodiment in combination with any of the above or below embodiments, A is
selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazolyl, indazolyl, and
oxadiazolyl.
In a preferred embodiment in combination with any of the above or below embodiments, A is
substituted with one or two groups independently selected from C alkyl, more preferably C
1-6 1-3
alkyl. In another preferred embodiment in combination with any of the above or below
embodiments, A is unsubstituted.
In a preferred embodiment in combination with any of the above or below embodiments, Q is
phenyl.
In a preferred embodiment in combination with any of the above or below embodiments, Q is
substituted with one or two groups independently selected from halogen, more preferably one
group selected from halogen, in particular Cl.
In a preferred embodiment in combination with any of the above or below embodiments, Z is
In a preferred embodiment in combination with any of the above or below embodiments, X =
CH.
In a preferred embodiment in combination with any of the above or below embodiments, R is
C cylcoalkyl, in particular cyclopropyl.
In a preferred embodiment in combination with any of the above or below embodiments, R and
R are independently selected from halogen, in particular Cl.
The compounds described is can be in the form of a prodrug compound. "Prodrug compound"
means a derivative that is converted into a compound as described herein by a reaction with an
enzyme, gastric acid or the like under a physiological condition in the living body, e.g. by
oxidation, reduction, hydrolysis or the like, each of which is carried out enzymatically. Examples
of the prodrug are compounds, wherein the amino group in a compound as described is
acylated, alkylated or phosphorylated to form, e.g., eicosanoylamino, alanylamino,
pivaloyloxymethylamino or wherein the hydroxyl group is acylated, alkylated, phosphorylated or
converted into the borate, e.g. acetyloxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy,
alanyloxy or wherein the carboxyl group is esterified or amidated. These compounds can be
produced from compounds as described according to well-known methods. Other examples of
the prodrug are compounds, wherein the carboxylate in a compound as described is, for
example, converted into an alkyl-, aryl-, choline-, amino, acyloxymethylester, linolenoylester.
Metabolites of compounds of the present invention are also contemplated herein.
Where tautomerism, like e.g. keto-enol tautomerism, of compounds of the present invention or
their prodrugs may occur, the individual forms, like e.g. the keto and enol form, are each
contemplated herein as well as their mixtures in any ratio. Same applies for stereoisomers, like
e.g. enantiomers, cis/trans isomers, conformers and the like.
If desired, isomers can be separated by methods well known in the art, e.g. by liquid
chromatography. Same applies for enantiomers by using e.g. chiral stationary phases.
Additionally, enantiomers may be isolated by converting them into diastereomers, i.e. coupling
with an enantiomerically pure auxiliary compound, subsequent separation of the resulting
diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of a
compound useful in the present invention may be obtained from stereoselective synthesis using
optically pure starting materials. Another way to obtain pure enantiomers from racemic mixtures
would use enantioselective crystallization with chiral counterions.
The compounds useful in of the present invention can be in the form of a pharmaceutically
acceptable salt or a solvate. The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases
or acids and organic bases or acids. In case the compounds useful in the present invention
contain one or more acidic or basic groups, the invention also comprises the use of their
corresponding pharmaceutically or toxicologically acceptable salts, in particular their
pharmaceutically utilizable salts. Thus, the compounds which contain acidic groups can be
present on these groups and can be useful according to the invention, for example, as alkali
metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts
include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or
organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino
acids. The compounds useful in the present invention which contain one or more basic groups,
i.e. groups which can be protonated, can be present and can be useful according to the
invention in the form of their addition salts with inorganic or organic acids. Examples of suitable
acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid,
methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic
acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic
acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic
acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric
acid, adipic acid, and other acids known to the person skilled in the art. If the compounds useful
in the present invention simultaneously contain acidic and basic groups in the molecule, also
contemplated are, in addition to the salt forms mentioned, inner salts or betaines (zwitterions).
The respective salts can be obtained by customary methods which are known to the person
skilled in the art like, for example, by contacting these with an organic or inorganic acid or base
in a solvent or dispersant, or by anion exchange or cation exchange with other salts. Also
contemplated herein are all salts of the compounds described herein which, owing to low
physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be
used, for example, as intermediates for chemical reactions or for the preparation of
pharmaceutically acceptable salts.
Further the compounds useful in the present invention may be present in the form of solvates,
such as those which include as solvate water, or pharmaceutically acceptable solvates, such as
alcohols, in particular ethanol.
Also described are pharmaceutical compositions comprising at least one compound as
described herein, or a prodrug compound thereof, or a pharmaceutically acceptable salt or
solvate thereof as active ingredient together with a pharmaceutically acceptable carrier.
"Pharmaceutical composition" means one or more active ingredients, and one or more inert
ingredients that make up the carrier, as well as any product which results, directly or indirectly,
from combination, complexation or aggregation of any two or more of the ingredients, or from
dissociation of one or more of the ingredients, or from other types of reactions or interactions of
one or more of the ingredients. Accordingly, the pharmaceutical compositions described herein
encompass any composition made by admixing at least one compound as described and a
pharmaceutically acceptable carrier.
The pharmaceutical composition may additionally comprise one or more other compounds as
active ingredients like a prodrug compound or other nuclear receptor modulators.
The compositions are suitable for oral, rectal, topical, parenteral (including subcutaneous,
intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation) or
nasal administration, although the most suitable route in any given case will depend on the
nature and severity of the conditions being treated and on the nature of the active ingredient.
They may be conveniently presented in unit dosage form and prepared by any of the methods
well-known in the art of pharmacy.
The compounds useful in the present invention can be prepared by a combination of methods
described in Schemes I to III. As depicted in Scheme I a 4-membered cyclic ketone, substituted
with substituent A in the 3-position can react with a metalated aromatic or heteroaromatic ring
M-Q-O-CH Z (M = metal, e.g. Li) in aprotic solvents and preferably at low temperatures to afford
a hydroxyl substituted 4-membered ring bearing the substituents A and Q. In the case where Y
is CH two isomers can form (A and Q transannular cis or trans to each other). Under optimized
conditions the formation of mainly one of the two isomers can be achieved. The two isomers
can be separated by appropriate methods known in the art like e.g. silica gel chromatography or
preparative RP-HPLC.
Scheme I
In Scheme II the methods are summarized which are used to prepare the 4-membered cyclic
ketones needed for the synthesis of the compounds described herein. In option a) a vinyl
bearing intermediate, e.g. prepared by vinylation of a corresponding halogen-containing starting
material R-A-X (X = halogen) can react with in situ formed α, α-dichloro ketene to form a 2,2-
dichlorocyclobutanone. After dehalogenation, e.g. with Zn in acetic acid under reflux, the
desired 3-substituted cyclobutanones are obtained. Alternatively, the vinyl-intermediates can
react with in situ generated unsubtituted ketene to afford in one step the desired cyclobutanone
intermediates. In option b) 3-methylenecyclobutanecarbonitrile is used as starting material.
Substituted heterocycles can be built up from the cyano group in several steps by methods
known to those skilled in the art. The desired cyclobutanones can be obtained by oxidative
cleavage of the exocyclic double bond using conditions and reagents known to those skilled in
the art, e.g. by the use of OsO , ozone or RhCl /NaIO as oxidants. Option c) shows the
4 3 4
methods used to prepare the substituted azetidinones. Cu- or Pd-catalysed C-N cross coupling
between 3-hydroxy-azetidine and halo-aromatic or halo-heteroaromatic rings afford the
corresponding N-substituted 3-hydroxy-azetidines which can be transformed into the desired
azetidinones by oxidation.
Scheme II
Scheme III illustrates some possibilities to perform modifications of the substituents at the A
group after the formation of the 4-membered hydroxy-bearing rings. For example, a leaving
group X (e.g. bromide) can be substituted by a cyano group, a carboxylic ester, methylsulfonyl
or thioether by transition metal catalysed cross coupling reactions. The obtained derivatives can
be further transformed into other derivatives by methods known to those skilled in the art. For
example, the cyano and the ester group can be hydrolysed under basic conditions to the afford
a carboxylic acid which in turn can be transformed into acyl-sulfonamides. A benzyl thioether
can be chlorinated to afford the chlorosulfonyl intermediate which reacts with ammonia to the
corresponding sulfonamides.
Scheme III
for R = carboxylic ester:
substitution of
leaving group X hyrolysis
Q OCH Z
2 Q OCH Z
Q OCH Z 2
for R = S-benzyl thioether:
chlorination
acyl sulfonamide
formation
NHR R
H A Y
A Y Q OCH Z
Q OCH Z
Q OCH Z
ClO S
R R NO S 2
8 7 2
As a result, described herein are compounds according to the general Formula (1) which bind to
FXR and act as agonists or modulators of FXR.
Also described is the use of said compounds for the treatment and/or prophylaxis of diseases
and/or conditions through binding of said nuclear receptor by said compounds. Also described
is the use of said compounds for the preparation of a medicament for the treatment and/or
prophylaxis of diseases and/or conditions through binding of said nuclear receptor by said
compounds. Also described is the use of compounds according to Formula (1) in the
preparation of a medicament for the prophylaxis and/or treatment of chronic intrahepatic or
some forms of extrahepatic cholestatic conditions, of liver fibrosis, of acute intraheptic
cholestatic conditions, of obstructive or chronic inflammatory disorders that arise out of improper
bile composition, of gastrointestinal conditions with a reduced uptake of dietary fat and fat-
soluble dietary vitamins, of inflammatory bowel diseases, of lipid and lipoprotein disorders, of
Type II Diabetes and clinical complications of Type I and Type II Diabetes, of conditions and
diseases which result from chronic fatty and fibrotic degeneration of organs due to enforced lipid
and specifically triglyceride accumulation and subsequent activation of profibrotic pathways, of
obesity and metabolic syndrome (combined conditions of dyslipidemia, diabetes and abnormally
high body-mass index), of acute myocardial infarction, of acute stroke, of thrombosis which
occurs as an endpoint of chronic obstructive atherosclerosis, of persistant infections by
intracellular bacteria or parasitic protozoae, of non-malignant hyperproliferative disorders, of
malignant hyperproliferative disorders, of colon adenocarcinoma and hepatocellular carcinoma
in particular, of liver steatosis and associated syndromes, of liver failure or liver malfunction as
an outcome of chronic liver diseases or of surgical liver resection, of Hepatitis B infection, of
Hepatitis C infection and/or of cholestatic and fibrotic effects that are associated with alcohol-
induced cirrhosis or with viral-borne forms of hepatitis.
Medicaments as referred to herein may be prepared by conventional processes, including the
combination of a compound as described herein and a pharmaceutically acceptable carrier.
FXR is proposed to be a nuclear bile acid sensor. As a result, it modulates both, the synthetic
output of bile acids in the liver and their recycling in the intestine (by regulating bile acid binding
proteins). But beyond bile acid physiology, FXR seems to be involved in the regulation of many
diverse physiological processes which are relevant in the etiology and for the treatment of
diseases as diverse as cholesterol gallstones, metabolic disorders such as Type II Diabetes,
dyslipidemias or obesity, chronic inflammatory diseases such as Inflammatory Bowel Diseases
or chronic intrahepatic forms of cholestasis and many others diseases (T. Claudel et al.,
Arterioscler. Thromb. Vasc. Biol. 2005, 25, 2020; Y. D. Wang et al., Cell Res. 2008, 18, 1087.
FXR regulates a complex pattern of response genes in the liver and in the gastrointestinal tract.
The gene products have impact on diverse physiological processes. In the course of functional
analysis of FXR, the first regulatory network that was analyzed was the regulation of bile acid
synthesis. While the LXRs induce the key enzyme of the conversion of cholesterol into bile
acids, Cyp7A1, via the induction of the regulatory nuclear receptor LRH-1, FXR represses the
induction of Cyp7A1 via the upregulation of mRNA encoding SHP, a further nuclear receptor
that is dominant repressive over LRH-1. Since FXR binds the end products of this pathway,
primary bile acids such as cholic acid (CA) or CDCA, this can be regarded as an example of
feedback inhibition on the gene expression level (B. Goodwin et al., Mol. Cell 2000, 6, 517; T. T.
Lu et al., Mol. Cell 2000, 6, 507). Parallel to the repression of bile acid synthesis via SHP, FXR
induces a range of so-called ABC (for ATP-binding cassette) transporters that are responsible
for the export of toxic bile acids from the hepatocyte cytosol into the canaliculi, the small bile
duct ramifications where the bile originates. This hepatoprotective function of FXR became first
apparent with the analysis of FXR knockout mice (C. J. Sinal et al., Cell 2000, 102, 731). where
under- or overexpression of several ABC-transporters in the liver was shown. Further detailed
analysis revealed that the major bile salt excretory pump BSEP or ABCB11 (M.
Ananthanarayanan et al., J. Biol. Chem. 2001, 276, 28857; J. R. Plass et al., Hepatology 2002,
35, 589) as well as the key enzyme which mediates lipid transfer from lipoproteins to
phospholipids, PLTP (N. L. Urizar et al., J. Biol. Chem. 2000, 275, 39313), and the two key
canalicular membrane transporters for phospholipids, MRP-2 (ABCC4) (H. R. Kast et al., J. Biol.
Chem. 2002, 277, 2908) and MDR-3 (ABCB4); L. Huang et al., J. Biol. Chem. 2003, 278,
51085) are direct targets for ligand-directed transcriptional activation by FXR (summarized in:
M. Miyata, J. Pharmacol. Exp. Ther. 2005, 312, 759; G. Rizzo et al., Curr. Drug Targets Immune
Endocr. Metabol. Disord. 2005, 5, 289).
The fact that FXR seems to be the major metabolite sensor and regulator for the synthesis,
export and re-circulation of bile acids suggested the use of FXR ligands to induce bile flow and
change bile acid composition towards more hydrophilic composition. With the development of
the first synthetic FXR ligand GW4064 (P. R. Maloney et al., J. Med. Chem. 2000, 43, 2971; T.
M. Willson et al., Med. Res. Rev. 2001, 21, 513) as a tool compound and of the semi-synthetic
artificial bile acid ligand 6-alpha-ethyl-CDCA, the effects of superstimulation of FXR by potent
agonists could be analyzed. It was shown that both ligands induce bile flow in bile duct ligated
animals. Moreover, in addition to choleretic effects, also hepatoprotective effects could be
demonstrated (R. Pellicciari et al., J. Med. Chem. 2002, 45, 3569; Y. Liu et al., J. Clin. Invest.
2003, 112, 1678). This hepatoprotective effect was further narrowed down to an anti-fibrotic
effect that results from the repression of Tissue Inhibitors of Matrix-Metalloproteinases, TIMP-1
and 2, the induction of collagen-deposit resolving Matrix-Metalloproteinase 2 in hepatic stellate
cells and the subsequent reduction of alpha-collagen mRNA and Transforming growth factor
beta (TGF-beta) mRNA which are both pro-fibrotic factors by FXR agonists (S. Fiorucci et al.,
Gastroenterology 2004, 127, 1497; S. Fiorucci et al., J. Pharmacol. Exp. Ther. 2005, 314, 584).
Furthermore, anti-cholestatic activity was demonstrated in bile-duct ligated animal models as
well as in animal models of estrogen-induced cholestasis (S. Fiorucci et al., J. Pharmacol. Exp.
Ther. 2005, 313, 604).
Genetic studies demonstrate that in hereditary forms of cholestasis (Progressive Familiar
Intrahepatic Cholestasis = PFIC, Type I – IV) either nuclear localization of FXR itself is reduced
as a consequence of a mutation in the FIC1 gene (in PFIC Type I, also called Byler´s Disease)
(F. Chen et al., Gastroenterology 2004, 126, 756; L. Alvarez et al., Hum. Mol. Genet. 2004, 13,
2451) or levels of the FXR target gene encoding MDR-3 phospholipid export pump are reduced
(in PFIC Type III). Taken together there is a growing body of evidence that FXR binding
compounds will demonstrate substantial clinical utility in the therapeutic regimen of chronic
cholestatic conditions such as Primary Biliary Cirrhosis (PBC) or Primary Sclerosing Cholangitis
(PSC) (reviewed in: G. Rizzo et al., Curr. Drug Targets Immune Endocr. Metabol. Disord. 2005,
, 289; G. Zollner et al., Mol. Pharm. 2006, 3, 231; S. Y. Cai et al., Expert Opin. Ther. Targets
2006, 10, 409).
The deep impact that FXR activation has on bile acid metabolism and excretion is not only
relevant for cholestatic syndromes but even more directly for a therapy against gallstone
formation. Cholesterol gallstones form due to low solubility of cholesterol that is actively pumped
out of the liver cell into the lumen of the canaliculi. It is the relative percentage of content of the
three major components, bile acids, phospholipids and free cholesterol that determines the
formation of mixed micelles and hence apparent solubility of free cholesterol in the bile. FXR
polymorphisms map as quantitative trait loci as one factor contributing to gallstone disease (H.
Wittenburg, Gastroenterology 2003, 125, 868). Using the synthetic FXR tool compound
GW4064 it could be demonstrated that activation of FXR leads to an improvement of the
Cholesterol Saturation Index (CSI) and directly to an abolishment of gallstone formation in C57L
gallstone susceptible mice whereas drug treatment in FXR knockout mice shows no effect on
gallstone formation (A. Moschetta et al., Nature Medicine 2004, 10, 1352).
These results qualify FXR as a good target for the development of small molecule agonists that
can be used to prevent cholesterol gallstone formation or to prevent re-formation of gallstones
after surgical removal or shockwave lithotripsy (discussed in: S. A. Doggrell, Curr. Opin.
Investig. Drugs 2006, 7, 344).
Thus, in one embodiment, the compound according to Formula (1) and pharmaceutical
compositions comprising said compound is used for the prophylaxis and/or treatment of
obstructive or chronic inflammatory disorders that arise out of improper bile composition such as
cholelithiasis also known as cholesterol gallstones.
Beyond its strong hepatoprotective and choleretic as well as anti-fibrotic effects that FXR shows
upon small molecule stimulated activation in the liver, FXR seems to have a role in protecting
the intestine from neoplastic transformation and from the development of polyps and their
transition into adenocarcinoma in the gut (S. Modica et al., Cancer Res. 2008, 68, 9589 and R.
R. Maran et al., J. Pharmacol. Exp. Ther. 2009, 328, 469). Similar to the situation in the intestine
absence of FXR leads to a high increase in the formation of Hepatocellular Cacrcinoma (HCC),
the most prominent form of liver cancer (I. Kim et al., Carcinogenesis 2007, 28, 940 and F. Yang
et al., Cancer Res. 2007, 67, 863). Whereas a functional FXR prevents the formation of colon
adenocarcinoma and hepatocellular carcinoma, FXR activation induces liver regeneration after
hepatectomy (W. Huang et al., Science 2006, 312, 233).
The combined hepatoprotective, anti-neoplastic and liver regenerative effects associated with
FXR activation can be therapeutically exploited for the use of FXR agonists in the treatment of
sever liver diseases. In one embodiment, the compounds as described herein and
pharmaceutical compositions comprising said compounds are used in the treatment of liver
diseases such as HCC, stimulation of liver regrowth and amelioration of side effects associated
with major liver resection, liver cirrhosis independent of the etiology and prevention or treatment
of liver ischemia in the course of liver transplantation or major liver surgery.
Since the discovery of the first synthetic FXR agonist and its administration to rodents it became
evident that FXR is a key regulator of serum triglycerides (P. Maloney et al., J. Med. Chem.
2000, 43, 2971; T. Willson et al., Med. Res. Rev. 2001, 21, 513). Over the past six years
accumulating evidence has been published that activation of FXR by synthetic agonists leads to
significant reduction of serum triglycerides, mainly in the form of reduced VLDL, but also to
reduced total serum cholesterol (H. R. Kast et al., Mol. Endocrinol. 2001, 15, 1720; N. L. Urizar
et al., Science 2002, 296, 1703; G. Lambert et al., J. Biol. Chem. 2003, 278, 2563; M.
Watanabe et al., J. Clin. Invest. 2004, 113, 1408; A. Figge et al., J. Biol. Chem. 2004, 279,
2790; S. Bilz et al., Am. J. Physiol. Endocrinol. Metab. 2006, 290, E716).
But the lowering of serum triglycerides is not a stand alone effect. Treatment of db/db or ob/ob
mice with synthetic FXR agonist GW4064 resulted in marked and combined reduction of serum
triglycerides, total cholesterol, free fatty acids, ketone bodies such as 3-OH Butyrate. Moreover,
FXR activation engages with the intracellular insulin signaling pathway in hepatocytes, resulting
in reduced output of glucose from liver gluconeogenesis but concomitant increase in liver
glycogen. Insulin sensitivity as well as glucose tolerance were positively impacted by FXR
treatment (K. R. Stayrook et al., Endocrinology 2005, 146, 984; Y. Zhang et al., PNAS 2006,
103, 1006; B. Cariou et al., J. Biol. Chem. 2006, 281, 11039; K. Ma et al., J. Clin. Invest. 2006,
116, 1102; D. Duran-Sandoval et al., Biochimie 2005, 87, 93). An effect on reduction of body
weight was also recently observed in mice overfed with a high lipid diet (C. Lihong et al.,
American Diabetes Association (ADA) 66 annual scientific sessions, June 2006, Abstract
Number 856-P). This weight loss effect might results from FXR´s induction of FGF-19, a
fibroblast growth factor that is known to lead to weight loss and athletic phenotype (J. Holt et al.,
Genes Dev. 2003, 17, 1581; E. Tomlinson et al., Endocrinology 2002, 143, 1741). In recent
patent applications, the effect of FXR agonist on reduction of body weight was demonstrated
(; ).
Taken together, these pharmacological effects of FXR agonists can be exploited in different
therapeutic ways: FXR binding compounds are thought to be good candidates for the treatment
of Type II Diabetes because of their insulin sensitization, glycogenogenic, and lipid lowering
effects.
In one embodiment, the compounds described herein and pharmaceutical compositions
comprising said compounds are used in the prophylaxis and/or treatment of Type II Diabetes
which can be overcome by FXR-mediated upregulation of systemic insulin sensitivity and
intracellular insulin signalling in liver, increased peripheral glucose uptake and metabolisation,
increased glycogen storage in liver, decreased output of glucose into serum from liver-borne
gluconeogenesis.
In a further embodiment, said compounds and pharmaceutical compositions are used for the
prophylaxis and/or treatment of chronic intrahepatic, such as PBC, PSC, progressive familiar
cholestasis (PFIC), alcohol-induced cirrhosis and associated cholestasis, and some forms of
extrahepatic cholestatic conditions, or liver fibrosis.
Also described is a compound of Formula (1) or to a pharmaceutical composition comprising
said compound for the prophylaxis and/or treatment of gastrointestinal conditions with a
reduced uptake of dietary fat and fat-soluble dietary vitamins which can be overcome by
increased intestinal levels of bile acids and phospholipids.
In a further embodiment, said compound or pharmaceutical composition is used for preventing
and/or treating a disease selected from the group consisting of lipid and lipoprotein disorders
such as hypercholesterolemia, hypertriglyceridemia, and atherosclerosis as a clinically manifest
condition which can be ameliorated by FXR´s beneficial effect on lowering total plasma
cholesterol, lowering serum triglycerides, increasing conversion of liver cholesterol into bile
acids and increased clearance and metabolic conversion of VLDL and other lipoproteins in the
liver.
In one further embodiment, said compound and pharmaceutical composition are used for the
prophylaxis and/or treatment of diseases where the combined lipid lowering, anti-cholestatic
and anti-fibrotic effects of FXR-targeted medicaments can be exploited for the treatment of liver
steatosis and associated syndromes such as NASH, or for the treatment of cholestatic and
fibrotic effects that are associated with alcohol-induced cirrhosis, or with viral-borne forms of
hepatitis.
In conjunction with the hypolipidemic effects it was also shown that loss of functional FXR leads
to increased atherosclerosis in ApoE knockout mice (E. A. Hanniman et al., J. Lipid Res. 2005,
46, 2595). Therefore, FXR agonists might have clinical utility as anti-atherosclerotic and
cardioprotective drugs. The downregulation of Endothelin-1 in Vascular Smooth Muscle Cells
might also contribute to such beneficial therapeutic effects (F. He et al., Circ. Res. 2006, 98,
192).
Also described is a compound according to Formula (1) or a pharmaceutical composition
comprising said compound for preventive and posttraumatic treatment of cardiovascular
disorders such as acute myocardial infarction, acute stroke, or thrombosis which occur as an
endpoint of chronic obstructive atherosclerosis.
Beyond controlling intestinal and colonic polyp formation, FXR seems to be expressed in breast
cancer tissue and cell lines but not in healthy breast tissue and seems to interact with the
Estrogen Receptor in ER positive breast cancer cells (K. E. Swales et al., Cancer Res. 2006,
66, 10120 and F. Journe et al., Breast Cancer Res. Treat. 2009, 115, 523).
This would allow to regard FXR also as a potential target for the treatment of proliferative
diseases, especially metastasizing cancer forms that express a small molecule responsive form
of FXR.
In a further embodiment, said compounds and pharmaceutical compositions are used for the
prophylaxis and/or treatment of malignant hyperproliferative disorders such as different forms of
cancer, specifically certain forms of breast, liver or colon cancer where interference with an FXR
ligand will have a beneficial impact.
Finally, FXR seems also to be involved in the control of antibacterial defense in the intestine (T.
Inagaki et al., PNAS. 2006, 103, 3920) although an exact mechanism is not provided. From
these published data, however, one can conclude that treatment with FXR agonists might have
a beneficial impact in the therapy of Inflammatory Bowel Disorders (IBD), in particular those
forms where the upper (ileal) part of the intestine is affected (e.g. ileal Crohn´s disease)
because this seems to be the site of action of FXR´s control on bacterial growth. In IBD the
desensitization of the adaptive immune response is somehow impaired in the intestinal immune
system. Bacterial overgrowth might then be the causative trigger towards establishment of a
chronic inflammatory response. Hence, dampening of bacterial growth by FXR-borne
mechanisms might be a key mechanism to prevent acute inflammatory episodes.
Also described is a compound according to Formula (1) or a pharmaceutical composition
comprising said compound for preventing and/or treating a disease related to Inflammatory
Bowel Diseases such as Crohn´s disease or Colitis ulcerosa. FXR-mediated restoration of
intestinal barrier function and reduction in non-commensal bacterial load is believed to be
helpful in reducing the exposure of bacterial antigens to the intestinal immune system and can
therefore reduce inflammatory responses.
Also described is a compound or pharmaceutical composition for the prophylaxis and/or
treatment of obesity and associated disorders such as metabolic syndrome (combined
conditions of dyslipidemias, diabetes and abnormally high body-mass index) which can be
overcome by FXR-mediated lowering of serum triglycerides, blood glucose and increased
insulin sensitivity and FXR-mediated weight loss.
In a further embodiment, the compounds or pharmaceutical composition as described are useful
in preventing and/or treating clinical complications of Type I and Type II Diabetes. Examples of
such complications include Diabetic Nephropathy, Diabetic Retinopathy, Diabetic Neuropathies,
or Peripheral Arterial Occlusive Disease (PAOD). Other clinical complications of Diabetes are
also contemplated herein.
Furthermore, conditions and diseases which result from chronic fatty and fibrotic degeneration
of organs due to enforced lipid and specifically triglyceride accumulation and subsequent
activation of profibrotic pathways may also be prevented and/or treated by applying the
compounds or pharmaceutical composition described herein. Such conditions and diseases
encompass NASH and chronic cholestatic conditions in the liver, Glomerulosclerosis and
Diabetic Nephropathy in the kidney, Macula Degeneration and Diabetic Retinopathy in the eye
and Neurodegenerative diseases such as Alzheimer´s Disease in the brain, or Diabetic
Neuropathies in the peripheral nervous system.
In practical use, the compounds as described can be combined as the active ingredient in
intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier may take a wide variety of forms depending on the form of
preparation desired for administration, e.g., oral or parenteral (including intravenous). In
preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be
employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like in the case of oral liquid preparations, such as, for example,
suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in
the case of oral solid preparations such as, for example, powders, hard and soft capsules and
tablets, with the solid oral preparations being preferred over the liquid preparations.
Because of their ease of administration, tablets and capsules represent the most advantageous
oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If
desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such
compositions and preparations should contain at least 0.1 percent of active compound. The
percentage of active compound in these compositions may, of course, be varied and may
conveniently be between about 2 percent to about 60 percent of the weight of the unit. The
amount of active compound in such therapeutically useful compositions is such that an effective
dosage will be obtained. The active compounds can also be administered intranasally as, for
example, liquid drops or spray.
The tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth,
acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent
such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule,
it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.
Various other materials may be present as coatings or to modify the physical form of the dosage
unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may
contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and
propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
Since the compounds described herein mostly represent carboxylic acids or similar anionic
isosters thereof, and since it is well known that salt forms of ionic drug compounds can
substantially affect the bioavailability of drug compounds, the compounds may also be used as
salts with various countercations to yield an orally available formulation. Such pharmaceutically
acceptable cations may be amongst others mono- or bivalent ions such as ammonium, the
alkaline metals sodium or potassium or the alkaline earth metals magnesium or calcium, certain
pharmaceutically acceptable amines such as tris(hydroxymethyl)aminomethane,
ethylendiamine, diethylamine, piperazine or others, or certain cationic amino acids such as
lysine or arginine.
The compounds described herein may also be administered parenterally. Solutions or
suspensions of these active compounds can be prepared in water suitably mixed with a
surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid
polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent
that easy syringability exists. It must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of microorganisms such as bacteria
and fungi. The carrier can be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof, and vegetable oils.
Any suitable route of administration may be employed for providing a mammal, especially a
human, with an effective dose of a compound as described herein. For example, oral, rectal,
topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms
include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments,
aerosols, and the like. Preferably compounds as described are administered orally.
The effective dosage of active ingredient employed may vary depending on the particular
compound employed, the mode of administration, the condition being treated and the severity of
the condition being treated. Such dosage may be ascertained readily by a person skilled in the
art.
When treating or preventing FXR mediated conditions for which compounds as described
herein are indicated, generally satisfactory results are obtained when the compounds are
administered at a daily dosage of from about 0.1 milligram to about 100 milligram per kilogram
of animal body weight, preferably given as a single daily dose or in divided doses two to six
times a day, or in sustained release form. For most large mammals, the total daily dosage is
from about 1.0 milligrams to about 1000 milligrams, preferably from about 1 milligram to about
50 milligrams. In the case of a 70 kg adult human, the total daily dose will generally be from
about 7 milligrams to about 350 milligrams. This dosage regimen may be adjusted to provide
the optimal therapeutic response.
The compounds described herein can be prepared according to the procedures of the following
Schemes and Examples, using appropriate materials and are further exemplified by the
following specific examples. Moreover, by utilizing the procedures described herein, in
conjunction with ordinary skills in the art, additional compounds can be readily prepared. The
compounds illustrated in the examples are not, however, to be construed as forming the only
genus that is considered as useful in the invention. The examples further illustrate details for the
preparation of the compounds useful in the present invention. Those skilled in the art will readily
understand that known variations of the conditions and processes of the following preparative
procedures can be used to prepare these compounds. The instant compounds are generally
isolated in the form of their pharmaceutically acceptable salts, such as those described above.
The amine-free bases corresponding to the isolated salts can be generated by neutralization
with a suitable base, such as aqueous sodium hydrogen carbonate, sodium carbonate, sodium
hydroxide and potassium hydroxide, and extraction of the liberated amine-free base into an
organic solvent, followed by evaporation. The amine-free base, isolated in this manner, can be
further converted into another pharmaceutically acceptable salt by dissolution in an organic
solvent, followed by addition of the appropriate acid and subsequent evaporation, precipitation
or crystallization. The carboxylic free acids corresponding to the isolated salts can be generated
by neutralization with a suitable acid, such as aqueous hydrochloric acid, sodium hydrogen
sulfate, sodium dihydrogen phosphate, and extraction of the liberated carboxylic-free acid into
an organic solvent, followed by evaporation. The carboxylic acid, isolated in this manner, can be
further converted into another pharmaceutically acceptable salt by dissolution in an organic
solvent, followed by addition of the appropriate base and subsequent evaporation, precipitation
or crystallization.
An illustration of the preparation of compounds as described herein is shown below. Unless
otherwise indicated in the schemes, the variables have the same meaning as described above.
The examples presented below are intended to illustrate particular embodiments. Suitable
starting materials, building blocks and reagents employed in the synthesis as described below
are commercially available from Sigma-Aldrich or Acros Organics, for example, or can be
routinely prepared by procedures described in the literature, for example in "March's Advanced
Organic Chemistry: Reactions, Mechanisms, and Structure", 5 Edition; John Wiley & Sons or
T. Eicher, S. Hauptmann "The Chemistry of Heterocycles; Structures, Reactions, Synthesis and
Application", 2 edition, Wiley-VCH 2003; Fieser et al. “Fiesers´ Reagents for organic
Synthesis” John Wiley & Sons 2000.
Examples
Example 1: Methyl 3-((1s,3s)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)benzoate (1)
Step 1: 4-((4-Bromochlorophenoxy)methyl)cyclopropyl(2,6-dichlorophenyl)-isoxazole
(1a)
To a solution of (5-cyclopropyl(2,6-dichlorophenyl)isoxazolyl)methanol (13 g, 45.8 mmol)
in CH Cl (DCM) (200 mL) was added dropwise SOCl (40 mL, 336 mmol). The resulting
2 2 2
mixture was stirred at rt for 2 h and the solvents were removed under reduced pressure. The
residue was dissolved in N,N-dimethylformamide (DMF) (200 ml) and 4-bromochlorophenol
(9.7 g, 47 mmol), K CO (40 g, 290 mmol) and NaI (12 g, 80 mmol) were added to this solution.
The mixture was stirred at 60°C overnight, then cooled to rt, diluted with water (1000 mL) and
extracted with ethyl acetate (EA) (500 mL × 3). The combined organic phases were washed
with brine (500 mL × 3), dried over Na SO and concentrated in vacuo. The residue was purified
by flash chromatography on silica gel (CC) to give the title compound 1a (19 g, 88%) as a white
solid.
Step 1: Methyl 3-(2,2-dichlorooxocyclobutyl)benzoate (1b)
To a 3-necked round bottomed flask, under a nitrogen atmosphere, fitted with a condenser, an
overhead stirrer and pressure equalised dropping funnel was dissolved methyl 3-vinylbenzoate
(5 g, 31 mmol) in dry Et O (150 mL). To this flask was added zinc dust (6 g, 3 eq) and the
reaction was sonicated for 30 min. After this time a solution of trichloroacetylchloride (8.7 mL,
2.5 eq) in dry Et O (50mL) was added dropwise whilst continuing the sonication over the next
min. During the process the reaction mixture was heated to 35°C. The sonication was
continued for 2.5 h at reflux and the reaction appeared to be complete by H NMR analysis. The
reaction was allowed to cool to rt and quenched with water (~50 mL). This was done in a
dropwise manner interspersed sveral times by a few minutes since a delayed exothermic
reaction occurred. After 20 min stirring in water the reaction mixture was filtered through a pad
of celite and rinsed through with Et O. The organic layer was washed with portions of water (2 x
250 mL), saturated sodium bicarbonate (2 x 250 mL) and brine (1 x 250 mL), dried over sodium
sulfate, filtered and concentrated under reduced pressure to afford the crude product 1b as a
dark yellow thick oil (crude 8.7 g).
Step 2: Methyl 3-(3-oxocyclobutyl)benzoate (1c)
Crude compound 1b (8.7 g) was dissolved in glacial acetic acid (55 mL) in a round bottomed
flask under a nitrogen atmosphere. To this flask was added zinc dust (4.6 g, 2.2 eq) and the
reaction was stirred and heated to 120°C for 3 h. After cooling to rt the mixture was filtered
though a pad of celite, this was washed with portions of EA. The combined solution was
concentrated under reduced pressure before being dissolved in EA (500 mL), washed with brine
(150 mL x 2) and then dried over sodium sulfate, filtered and concentrated again. The crude
mixture was stirred for 5 min in chloroform (250 mL) and filtered through a sintered funnel. The
filtrate was concentrated to give the crude product as a pale yellow oil. The crude product was
purified by CC in (PE/EA = 9:1, PE = petroleum ether) to give the desired product 1c (2.5 g,
38% for 2 steps) as a pale yellow oil.
Step 3: Methyl 3-(3-(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)benzoate (1)
To a stirring solution of compound 1a (1.67 g, 3.5 mmol) in dry THF (30 mL) was added n-BuLi
(2.5 M in hexane, 1.2 eq, 1.69 mL) dropwise over 10 min at −78°C under a nitrogen
atmosphere. This was stirred for 1 h at this temperature before adding a solution of compound
1c (0.72 g, 1 eq) in dry THF (10 mL) dropwise and stirred for 1 h at this temperature. The
reaction mixture was allowed to warm to rt slowly and left stirring overnight. The reaction was
quenched with a solution of saturated ammonium chloride solution (50 mL) and EA (250 mL).
The organic layer was separated and the aq. layer was washed with EA (2 x 100 mL). The
combined organic extracts were dried over sodium sulfate, filtered and concentrated to give the
crude product as a brown oil. The product was isolated following CC with PE/EA (19:1 to 3:1).
The reaction and purification was repeated twice on the same scale and the combined product
(3.13 g) was repurified under the same conditions to afford the final product 1 (1.7 g, 19%). H
NMR (CDCl ): 7.93 (m, 1H), 7.90-7.85 (m, 1H), 7.50-7.30 (m, 5H), 6.88 (s, 1H), 6.75-6.72 (m,
1H), 4.80 (s, 2H), 3.88 (s, 3H), 3.20-3.10 (m, 1H), 3.00-2.91 (m, 2H), 2.60-2.49 (m, 2H), 2.15-
2.08 (m, 1H), 1.30-1.25 (m, 2H), 1.15-1.10 (m, 2H).
Example 2: 3-((1s,3s)(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)benzoic acid (2)
Compound 1 (1.7 g, 2.84 mmol) was dissolved in THF (100 mL) at rt. A solution of LiOH (285
mg, 4.2 eq) in water (20 mL) was added and the solution was stirred and warmed to 35°C for
three days. After this time the THF was removed under reduced pressure. The remaining aq.
solution was diluted with water (25 mL) and washed with Et O (2 x 50 mL). The aq. layer was
then transferred to a round bottomed flask and acidified to pH 6 using 1N HCl. The formed white
precipitated was filtered off and dried under reduced pressure at 50°C to give title compound 2
(1.3 g, 78%, single isomer by H-NMR and LC-MS) as white solid. H NMR (400 MHz, CD OD)
δ: 7.98 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.58-7.46 (m, 5H), 7.41 (t, J = 7.6 Hz, 1H), 6.91 (d, J =
2.4 Hz, 1H), 6.80 (dd, J = 8.8, 2.4 Hz, 1H), 4.95 (s, 2H), 3.29-3.25 (m, 2H), 2.96 (m, 1H), 2.55-
2.49 (m, 2H), 2.37 (m, 1H), 1.24-1.22 (m, 4H). MS (ESI ) m/z: 584 (582) [M −1] .
Relevant intensive NOEs (obtained from the ROESY spectra; arrows below) indicate that the
two aromatic moieties are 1,3-trans oriented in Example 2.
Alternative route to Example 2
Step 1: 3-(3-Bromophenyl)cyclobutanone (2a)
N,N-Dimethylacetamide (9.0 g, 103 mmol) was dissolved in 1,2-dichloroethane (200 mL). The
solution was cooled to 0°C before trifluoromethanesulfonic anhydride (63 g, 223 mmol) was
added. The reaction was stirred for an additional 60 min at 0°C. Then 1-bromovinylbenzene
(15 g, 81.9 mmol) and 2,4,6-collidine (10.5 g, 86.6 mmol) were added. The reaction was heated
to reflux overnight, quenched by addition of water (300 mL) and stirred for 2 hr at rt. The mixture
was extracted with DCM (300 mL × 3). The combined organic layers were dried over Na SO
and concentrated in vacuo. Purification by CC (EA/PE = 1:20) gave the title compound 2a (5.0
g, 27%) as a pale yellow solid.
Step 2: 3-(3-Bromophenyl)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)cyclobutanol (2b)
To a solution of compound 1a (14 g, 29.6 mmol) in dry THF (500 ml) at −78°C was added
dropwise n-BuLi (18.5 mL, 1.6 M in hexane, 29.6 mmol). The mixture was stirred for an
additional 1 h at −78°C and a solution of compound 2a (6.5 g, 28.9 mmol) in dry THF (50 mL)
was added dropwise. The resulting mixture was stirred at −78°C for 1 h and then warmed to rt
and quenched with saturated aq. NH Cl (500 mL). The mixture was extracted with EA (500 mL
× 2), the combined organic layers were washed with brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by CC (EA/PE = 1:5) to give the title compound 2b (6.5 g,
37%) as a white solid.
Step 3: 3-(3-Cyanophenyl)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)cyclobutanol (2c)
To a solution of compound 2b (3.1 g, 5 mmol) in DMF (50 mL) were added under argon
atmosphere Zn(CN)2 (500 mg, 4.3 mmol), Pd2(dba)3 (300 mg, 0. 33 mmol) and Xantphos (150
mg, 0.31 mmol). The mixture was stirred for 10 h at 115°C under microwave irradiation. After
cooling to rt the reaction mixture was diluted with water (250 mL) and extracted with EA (250 mL
× 2). The combined organic layers were washed with brine (100 mL × 3) and dried over Na SO .
The residue was purified by CC (EA/PE) to give the title compound 2c (1.2 g, 42%) as a pale
yellow solid.
Step 4: 3-((1s,3s)(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)benzoic acid (2)
To a solution of compound 2c (15 g, 24.2 mmol) in EtOH (750 mL) was added aq. NaOH (40 g
in 100 mL of water). The resulting mixture was heated to reflux overnight and then cooled to rt.
The reaction was concentrated in vacuo to remove the volatile solvent, diluted with water (1000
mL) and the pH was adjusted to 2 with diluted aq. HCl (1N). The formed precipitate was
collected by filtration to give the crude product as a yellow solid (13.8 g). Purification by
preparative preversed phase HPLC (RP-HPLC) afforded the title compound 2 (8.0 g, 56%,
single isomer by H-NMR) as a white solid.
Preparative Example 3
Step 1: Methyl 3-(3-hydroxyazetidinyl)benzoate (3a)
To a solution of methyl 3-iodobenzoate (4.5 g, 17.2 mmol) in DMSO (30 mL) was added 3-
azetidinol hydrogen chloride salt (1.3 g, 11.8 mmol), Cs CO (9.5 g, 29.2 mmol), CuI (446
mg, 2.3 mmol) and L-proline (540 mg, 4.7 mmol) and then the mixture was heated at 90°C for
18 h under argon atmosphere. The solution was diluted with EA and water and the organic layer
was washed with brine three times, concentrated under reduced pressure and purified by CC
(PE/EA = 2:1) to give compound 3a (1.6 g, 66%) as a yellow solid.
Step 2: Methyl 3-(3-oxoazetidinyl)benzoate (3)
To a solution of compound 3a (1.60 g, 7.7 mmol) in dry DCM (30 mL) was added Dess-Martin
periodinane (6.5 g, 15.4 mmol) at 0°C and the mixture was stirred at rt for 2 h under N
atmosphere. The mixture was quenched with saturated sodium bicarbonate solution and diluted
with EA. The organic portion was washed with brine, dried over Na2SO4, filtered, concentrated
under reduced pressure and purified by CC (PE/EA = 4:1) to give compound 3 (1.2 g, 75%) as a
white solid.
Example 4: 3-((1s,3s)(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)-N-(methylsulfonyl)benzamide (4)
To the solution of compound 2 (100 mg, 0.17 mmol) in DCM (5 mL) were added EDCI·HCl (100
mg, 0.52 mmol), DMAP (100 mg, 0.81 mmol) and MeSO NH (40 mg, 0.42 mmol). The mixture
was stirred at 30°C overnight and then diluted with EA and washed by H O, brine and dried over
Na SO . Concentration in vacuo and purification by prep-TLC gave crude target compound as a
light yellow solid. RP-HPLC purification afforded the title compound 4 (38 mg, 33%) as a white
solid. H NMR (400 MHz, CD OD) δ: 7.87 (s, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.61-7.53 (m, 4H),
7.50-7.46 (m, 2H), 6.91 (d, J = 2.4 Hz, 1H), 6.80 (dd, J = 8.8, 2.4 Hz, 1H), 4.95 (s, 2H), 3.38(s,
3H), 3.30-3.26 (m, 2H), 3.01 (m, 1H), 2.57-2.51 (m, 2H), 2.37 (m, 1H), 1.25-1.23 (m, 4H). MS
(ESI ) m/z: 659 [M −1] .
Example 5: 3-(3-(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)benzenesulfonamide (5)
OH Cl
H NO S
Step 1: 3-(3-(Benzylthio)phenyl)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)cyclobutanol (5a)
To a solution of compound 2b (619 mg, 1 mmol) in toluene (20 mL) under argon atmosphere
were added K CO (276 mg, 2 mmol), phenylmethanethiol (125 mg, 1 mmol), Pd (dba) (200
2 3 2 3
mg, 0. 22 mmol) and Xantphos (75 mg, 0.16 mmol). Then the mixture was stirred at 115°C for 4
h. After being cooled to rt, the reaction was diluted with water (100 mL) and extracted with EA
(100 mL × 2). The combined organic layers were washed with brine (100 mL × 2), dried over
Na2SO4 and concentrated to dryness. Purification by CC gave compound the compound 5a
(200 mg; 30%) as a pale yellow solid. H NMR (400 MHz, CDCl3) δ: 7.36-7.32 (m, 3H), 7.28-
7.07 (m, 9H), 7.01 (d, J = 7.2 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.66 (dd, J = 8.8, 2.0 Hz, 1H),
4.75 (s, 2H), 4.04 (s, 2H), 3.06-3.00 (m, 2H), 2.84-2.78 (m, 2H), 2.44-2.38 (m, 3H), 2.09 (m,
1H), 1.24-1.18 (m, 2H), 1.11-1.08 (m, 2H). MS (ESI ) m/z: 662 [M+1] .
Step 2: 3-(3-(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazolyl)methoxy)phenyl)
hydroxycyclobutyl)benzenesulfonyl chloride (5b)
To a solution of compound 5a (34 mg, 0.05 mmol) in CH CN/HOAc/H O (1 mL/37 µL/25 µL)
was added 2,4-dichloro-5,5-dimethylhydantion (20 mg, 0.1 mmol). The mixture was stirred at 0-
°C for 2 h. The reaction was diluted with water and extracted with CH Cl . The combined
organic layers were washed with a 5% NaHCO solution, brine and dried over Na SO .
3 2 4
Concentration to dryness afforded the crude product 5b (30 mg) as a colorless oil, which was
used directly in the next step.
Step 3: 3-(3-(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazolyl)methoxy)phenyl)
hydroxycyclobutyl)benzenesulfonamide (5)
To the solution of compound 5b (30 mg) in CH CN (2 mL) was added NH OH (0.3 mL). The
mixture was stirred at rt for 1 h. Concentration to dryness and purification by prep. RP-HPLC
gave the title compound 5 (3.5 mg, 10% for two steps) as a white solid. H NMR (400 MHz,
CDCl3) δ: 7.85 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.54-7.41 (m, 5H), 7.35 (d, J = 8.4 Hz, 1H), 6.90
(s, 1H), 6.75 (d, J = 8.4 Hz, 1H), 4.83 (s, 2H), 4.77 (s, broad, 2H), 3.20 (t, J = 10.4 Hz, 2H), 3.04
(m, 1H), 2.58 (t, J = 10.6 Hz, 2H), 2.17 (m, 1H), 1.31-1.30 (m, 2H), 1.20-1.16 (m, 2H). MS (ESI
m/z: 617 [M −1] .
Example 6: 1-(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazolyl)methoxy)phenyl)-
3-(3-(methylsulfonyl)phenyl)cyclobutanol (6)
HO Cl
O Cl
To the solution of compound 2b (200 mg, 0.32 mmol) in DMSO, sodium methanesulfinate (50
mg, 0.46 mmol), CuI (20 mg, 0.1 mmol), L-proline (37 mg, 0.32 mmol) and
diisopropylethylamine (DIEA) (41 mg, 0.32 mmol) was added. The mixture was stirred at 95°C
overnight and then diluted with water and extracted with EA. The combined organic layers were
washed with water and dried over Na SO Concentration to dryness under reduced pressure
2 4.
and purification by prep. RP-HPLC gave the title compound 6 as a white solid (35 mg, 21%,
single isomer by H NMR and LC-MS). H NMR (400 MHz, CDCl ) δ: 7.84 (s, 1H), 7.79 (d, J =
7.6 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.44-7.41 (m, 3H), 7.34 (t, J = 7.2
Hz, 1H), 6.90 (d, J = 2.8 Hz, 1H), 6.75 (dd, J = 8.4, 2.0 Hz, 1H), 4.83 (s, 2H), 3.24-3.19 (m, 2H),
3.08-3.04 (m, 4H), 2.62-2.56 (m, 2H), 2.17 (m, 1H), 1.31-1.29 (m, 2H), 1.20-1.16 (m, 2H). MS
(ESI+) m/z: 618 (620) [M+1] , 600 (602) [M −H O+1] .
Example 7: Methyl 5-((1s,3s)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)isopropyl-1H-pyrazolecarboxylate (7)
Step 1: Methyl 1-isopropylvinyl-1H-pyrazolecarboxylate (7a)
A suspension of methyltriphenylphosphonium bromide (2.69 g, 7.52 mmol) in dry THF (40 mL)
was cooled to –78°C and n-butyllithium (1.6 M solution in hexane, 3.7 mL, 5.91 mmol) was
added dropwise. The yellow-orange suspension was stirred at –78°C for 50 min and then a
solution of methyl 5-formylisopropyl-1H-pyrazolecarboxylate (prepared as described in
, 1.05 g, 5.37 mmol) in dry THF (10 mL) was added dropwise. The mixture
was stirred at –78°C for 1.75 h, the cooling bath was removed and the mixture (off-white
suspension) was stirred at rt for 1 h. The mixture was then partitioned between diluted aq.
NaHCO solution (150 mL) and EA (150 mL). The aq. layer was extracted twice with EA (50 mL
each) and the combined organic layer was washed twice with water (50 mL each) and
concentrated without drying to give 2.74 g of a yellow oil which slowly crystallized. The crude
product was purified by CC (preadsorption with CH Cl , hexane/EA 4:1) to give alkene 7a
(590 mg, 57%) as a colorless oil. H NMR (DMSO-d ) δ: 7.02 (s, 1H), 6.87 (dd, J = 17.3, 11.2
Hz, 1H), 5.94 (dd, J = 17.3, 1.3 Hz, 1H), 5.45 (dd, J = 11.2, 1.3 Hz, 1H), 4.80 (sept, J = 6.6 Hz,
1H), 3.79 (s, 3H), 1.38 (d, J = 6.6 Hz, 6H). C H N O (194.23). LC-MS (ESI): 195 [M+H] .
14 2 2
Step 2: Methyl 1-isopropyl(3-oxocyclobutyl)-1H-pyrazolecarboxylate (7b)
The reaction was performed in two dry sealed tubes (two batches of equal quantity). The
batches were combined for workup and purification. Single batch procedure: To a solution of
N,N-dimethylacetamide (0.22 mL, 2.34 mmol) in 1,2-dichloroethane (12 mL) under nitrogen at –
to –20°C was added dropwise trifluoromethanesulfonic anhydride (0.43 mL, 2.57 mmol),
forming an opaque suspension. The mixture was stirred at –15°C for 10 min, and a solution of
alkene 7a (151 mg, 0.78 mmol) and sym.-collidine (0.42 mL, 3.12 mmol) in 1,2-dichloroethane
(3 mL) was added dropwise (yellow solution formed). Upon completion of the addition the
cooling was bath removed, the mixture was allowed to warm to rt (orange turbid solution) and
the tube was sealed. The mixture was then stirred at 90°C for 15 h (brown mixtures). Water
(5 mL) was added at rt and the mixtures were stirred at 100°C for 2 h (turbid two-phase
solutions). After cooling to rt, the mixtures were combined and partitioned between diluted aq.
NaHCO solution and CH Cl and the aq. layer was extracted three times with CH Cl (30 mL
3 2 2 2 2
each). The combined organic layer was dried (Na SO ), filtered and concentrated to give a
brown oil (2.2 g). Purification by CC (6×13 cm, preadsorption with CH Cl , toluene/EA 3:1) gave
cyclobutanone 7b (115.5 mg, 31%) as a yellow oil. H NMR (DMSO-d ) δ: 6.81 (s, 1H), 4.58
(sept, J = 6.5 Hz, 1H), 3.78 (s, 3H), 3.85-3.73 (m, 1H), 3.59-3.45 (m, 2H), 3.37-3.24 (m, 2H,
partially overlapped by water signal), 1.39 (d, J = 6.6 Hz, 6H). C H N O (236.27). LC-MS
12 16 2 3
(ESI): 237 [M+H] .
Step 3: Methyl 5-((1s,3s)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)isopropyl-1H-pyrazolecarboxylate (7)
A solution of bromide 1a (368 mg, 0.78 mmol) in dry THF (6 mL) was cooled to –78°C and a
1.6M n-butyllithium solution in hexanes (0.48 mL, 0.76 mmol) was added dropwise. The mixture
was stirred at –78°C for 20 min and a solution of cyclobutanone 7b (164 mg, 0.69 mmol) in dry
THF (4 mL) was added dropwise. The mixture was stirred at –78°C for 2.5 h and saturated aq.
NH Cl solution (1 mL) was added dropwise at this temperature. The cooling bath was removed
and the mixture was allowed to warm to rt and stirred at rt for 0.5 h. The mixture was then
added to diluted aq. NH4Cl solution and extracted three times with EA. The combined organic
layer was dried (Na2SO4), filtered and concentrated to give 516 mg of an almost colorless oil.
Purification by CC (4.5×23 cm, preadsorption with CH Cl , eluent hexane/acetone = 2:1)
afforded recovered cyclobutanone 7b (31.3 mg, 19%, slightly yellow oil) and impure product
(333 mg). Repurification by CC (4×22 cm, hexane/EA = 1:1) or prep-TLC gave pure product 7
(210 mg, 48%) as white foam. H NMR (DMSO-d ) δ: 7.65 (d, J = 2.1 Hz, 1H), 7.62 (s, 1H),
7.59-7.48 (m, 2H), 6.92 (d, J = 2.4 Hz, 1H), 6.76 (dd, J = 8.6, 2.6 Hz, 1H), 6.66 (s, 1H), 5.49 (s,
1H), 4.92 (s, 2H), 4.42 (quint-like m, J = 6.5 Hz, 1H), 3.78 (s, 3H), 3.24-3.11 (m, 2H, partially
overlapped by water signal), 3.04-2.90 (m, 1H), 2.54-2.33 (m, 3H, partially overlapped by DMSO
signal), 1.32 (d, J = 6.5 Hz, 6H), 1.26-1.08 (m, 4H). C H Cl N O (630.95). LC-MS (ESI): 630,
31 30 3 3 5
632 [M+H] .
Example 8: 5-((1s,3s)(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)isopropyl-1H-pyrazolecarboxylic acid (8)
Ester 7 (98.3 mg, 0.156 mmol) was dissolved in a mixture of THF (7.5 mL), MeOH (2.5 mL) and
water (2.5 mL) and LiOH·H O (65 mg, 1.56 mmol) was added at rt. The mixture was stirred at rt
for 18 h. The mixture was partitioned between diluted aq. NH Cl solution and EA and the
organic layer was washed once with water. The combined aq. layer was extracted twice with
EA. The combined organic layer was dried (Na2SO4), filtered and concentrated to give 103 mg
of an almost white solid. The product was purified by CC (3×3.5 cm, EA/EtOH = 10:1 to 1:4) to
afford 8 (94.8 mg, 99%) as a white solid. H NMR (DMSO-d ) δ: 7.66-7.60 (m, 1H), 7.62 (s, 1H),
7.59-7.49 (m, 2H), 6.91 (d, J = 2.5 Hz, 1H), 6.76 (dd, J = 8.6, 2.4 Hz, 1H), 6.38 (s, 1H), 5.51 (s,
1H, exchangeable with D O), 4.92 (s, 2H), 4.31 (quint-like m, J = 6.5 Hz, 1H), 3.25-3.08 (m, 2H,
partially overlapped by water signal), 2.93-2.77 (m, 1H), 2.57-2.43 (m, 1H, hidden by DMSO
signal), 2.43-2.29 (m, 2H, partially overlapped by DMSO signal), 1.29 (d, J = 6.5 Hz, 6H), 1.26-
1.08 (m, 4H). The CO H signal does not appear in the spectrum. C H Cl N O (616.92). LC-
2 30 28 3 3 5
MS (ESI): 616, 618 [M+H] .
Alternative route to Example 8
Step 1: 1-(3-Methylenecyclobutyl)ethanone (8a)
Methylene cyclobutane carbonitrile (5.0 g, 53.7 mmol) was dissolved in dry diethylether (25 mL),
cooled in an ice bath and MeMgBr (26.8 mL, 80.5 mmol, 3 M in ether) was added dropwise. The
mixture was left stirring overnight at rt, cooled to 0°C, quenched carefully with 15% NaHSO aq.
sol. (100 mL). The mixture was stirred at rt for 30 min. and the layers were separated. The aq.
phase was extracted with pentane (50 mL) and diethylether (50 mL). The combined organic
layers were washed with brine and dried over Na SO . The solvents were removed under
vacuum at rt and the crude product was obtained as a yellowish liquid.
Step 2: Ethyl 4-(3-methylenecyclobutyl)-2,4-dioxobutanoate (8b)
Sodium (1.15 g, 49.9 mmol) was dissolved in dry EtOH (30 mL, denaturated with 5%
diethylether). Compound 8a (5.5 g, 49.9 mmol, crude) was dissolved in dry EtOH (45 mL) and
the above prepared sodium ethoxide solution was added. This mixture was stirred at rt for 15
min and then diethyl oxalate (6.8 mL, 49.9 mmol) was added dropwise. The reaction mixture
was placed in a pre-heated (to 67 °C) oil bath and stirred at this temperature for 4.5 h. The
mixture was left at rt overnight. The solvent was removed, EA (100 mL) and 1 M HCl (70 mL)
were added and organic phase was separated. The aq. phase was re-extracted with EA (50
mL). The combined organic phases were washed with water, brine and dried over anh. Na SO .
The solvent was removed under reduced pressure and the residue was purified on silica using
hexanes/MTBE 9:1 as eluent giving pure product 8b. Yield: 6.29 g, 56% over two steps. H-
NMR (CDCl3), δ (ppm): 6.36 (s, 1H), 4.85-4.80 (m, 2H), 4.34 (q, J = 8.0 Hz, 2H), 3.35-3.25 (m,
1H), 3.05-2.85 (m, 4H), 1.36 (t, J = 8.0 Hz, 3H).
Step 3: Ethyl 1-isopropyl(3-methylenecyclobutyl)-1H-pyrazolecarboxylate (8c)
Compound 8b (6.29 g, 29.9 mmol) was dissolved in dry EtOH (65 mL, denaturated with 5% of
MeOH) and isopropyl hydrazine hydrochloride (3.97 g, 35.9 mmol) was added. The reaction
mixture was stirred for 3 h at rt. The solvent was removed and to the oily residue were added
EA (100 mL), water (50 mL) and sat. NaHCO3 (50 mL) sequentially. The layers were separated
and the aq. phase was re-extracted with EA (50 mL). The combined organic phases were
washed with brine (70 mL) and dried over anh. Na SO . The solvent was removed under
vacuum and the residue was dried under reduced pressure. Yield: 7.23 g (contains 3.4% of
EtOAc by NMR, recalculated pure yield: 6.98 g, 94%). Crude product 8c is 98% pure by HPLC
and NMR. H-NMR (CDCl ), δ (ppm): 6.62 (s, 1H), 4.88-4.82 (m, 2H), 4.42-4.32 (m, 3H), 3.56-
3.45 (m, 1H), 3.17-3.07 (m, 2H), 2.88-2.79 (m, 2H), 1.49 (d, J = 8.0 Hz, 6H), 1.37 (t, J = 8.0 Hz,
3H).
Step 4: Ethyl 1-isopropyl(3-oxocyclobutyl)-1H-pyrazolecarboxylate (8d)
Compound 8c (6.45 g, 26.0 mmol) was dissolved in a mixture of MeCN (77 mL) and water (13
mL) and cooled in an ice-bath. To this solution RuCl ×H2O (0.19 g, 0.86 mmol) was added,
followed by portion-wise addition of NaIO (19.35 g, 90.9 mmol). An exotherm was observed
during this addition. The obtained thick slurry was stirred at rt for 45 min. The reaction mixture
was diluted with Na S O aq. sol. (10%, 260 mL), water (50 mL) and DCM (100 mL). The
2 2 3
phases were separated and the aq. phase was extracted with DCM (2×70 mL). The combined
organic phases were washed with Na S O aq. sol. (10%, 50 mL), water (100 mL), brine (100
2 2 3
mL) and dried over anh. Na SO .The crude product (6.5 g) was purified on silica, eluting with
hexanes/MTBE to give pure product as an oil that solidified upon storage at –20°C. Yield: 5.8 g
(78% over two steps). H-NMR (DMSO-d ), δ (ppm): 6.78 (s, 1H), 4.57 (h, J = 8.0 Hz, 1H), 4.26
(q, J = 8.0 Hz, 2H), 3.85-3.75 (m, 1H), 3.58-3.45 (m, 2H), 3.35-3.25 (m, 2H), 1.39 (d, J = 8.0 Hz,
6H), 1.28 (t, J = 8.0 Hz, 3H).
Step 5: 4-((4-Bromochlorophenoxy)methyl)cyclopropyl(2,6-dichlorophenyl)isoxazole
(8e)
3-Chlorobromophenol (3.8 g, 18.3 mmol) was mixed with (5-cyclopropyl(2,6-
dichlorophenyl)isoxazolyl)methanol (3.47 g, 12.2 mmol) and triphenylphosphine (6.41 g, 24.4
mmol) in toluene (150 mL). The mixture was cooled in an ice-bath and DIAD (4.8 mL, 24.4
mmol) as a solution in toluene (10 mL) was added drop-wise. The reaction was stirred at rt for
21 h and the solvents were removed on a rotavap leaving a yellow oily residue. This was
dissolved in DCM (200 mL), silica (~20 g) was added and the mixture was evaporated to
dryness. This material was loaded on the top of a silica column and purified eluting with
hexanes/MTBE 9:1. The product containing fractions were pooled and the solvent removed
under reduced pressure, leaving pure product 8e as a colourless oil that crystallized upon
drying under vacuum overnight. Yield: 5.07 g (88%). H-NMR (CDCl ), δ (ppm): 7.45-7.30 (m,
4H), 6.90 (s, 1H), 6.60-6.55 (m, 1H), 2.15-2.07 (m, 1H), 1.32-1.25 (m, 2H), 1.20-1.11 (m, 2H).
Step 6: Ethyl 5-((1s,3s)(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)isopropyl-1H-pyrazolecarboxylate (8f)
LiCl (0.684 g, 16.15 mmol) was dissolved in THF (20 mL) at rt and iPrMgCl (2.0 M in THF, 8.1
mL, 16.15 mmol) was added. The mixture was stirred for 10 min at rt, cooled in an ice-bath and
a solution of compound 8e (2.55 g, 5.38 mmol) in THF (20 mL) was added over 5 min. The
cooling bath was removed and the mixture was stirred at rt for 4 h. The mixture was cooled to –
°C and a solution of compound 8d (1.48 g, 5.92 mmol) in THF (16 mL) was added rapidly.
The mixture was stirred at rt for 90 min. and then 0.5 M NaHSO aq. (35 mL) and EA (50 mL)
were added. The resulting mixture was stirred for 10 min., the layers were separated and the
aq. layer was extracted with EA (30 mL). The combined organic phases were washed with
NaHCO aq. (50 mL), brine (50 mL) and dried over anh. Na SO . The crude product (3.79 g)
3 2 4
was obtained after removal of the solvent as a white foam. 3.6 g of this crude was purified on
silica column, eluting with hexanes/EA 3:2 to give pure product 8f as a solid foam. Yield: 1.62 g
(49%). H-NMR (DMSO-d ), δ (ppm): 7.65-7.47 (m, 4H), 6.93-6.91 (m, 1H), 6.79-6.72 (m, 1H),
6.65 (s, 1H), 5.48 (s, 1H), 4.92 (s, 2H), 4.42 (h, J = 8.0 Hz, 1H), 4.26 (q, J = 8.0 Hz, 2H), 3.32 (s,
2H), 3.22-3.14 (m, 2H), 3.05-2.90 (m, 1H), 2.45-2.35 (m, 2H), 1.35-1.10 (m, 14H).
Step 7: 5-((1s,3s)(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)isopropyl-1H-pyrazolecarboxylic acid (8)
Compound 8f (1.60 g, 2.48 mmol) was dissolved in THF (100 mL), then MeOH (50 mL), water
(50 mL) and LiOH×H2O (1.04 g, 24.8 mmol) were added sequentially. The mixture was stirred
for 4.5 h at rt and then concentrated under reduced pressure to remove MeOH and THF. The
remaining aq. solution was acidified by addition of 1 M HCl aq. (24 mL) to reach pH of 4.05 (pH
electrode control). Already at approx. pH 7 a precipitate started to form. The formed solid was
filtered off, washed on the filter with water and dried under vacuum at rt to give product 8 as a
white powder. Yield: 1.40 g (92%). H-NMR (CDCl ), δ (ppm): 7.44-7.32 (m, 4H), 6.91 (d, J = 4.0
Hz, 1H), 6.78 (s, 1H), 6.75 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 4.83 (s, 2H), 4.35-4.20 (m, 1H),
3.25-3.14 (m, 2H), 3.04-2.90 (m, 1H), 2.62-2.54 (m, 2H), 2.21-2.11 (m, 1H), 1.46 (d, J = 8.0 Hz,
6H), 1.34-1.28 (m, 2H), 1.20-1.14 (m, 2H). C-NMR (CDCl ), δ (ppm): 172.7, 164.8, 159.2,
158.4, 147.2, 141.3, 135.8, 134.1, 132.8, 131.3, 128.1, 127.6, 127.3, 117.7, 113.3, 110.0,
106.3, 73.1, 59.8, 51.1, 41.7, 22.6, 22.0, 8.5, 7.8. MS (ESI ) m/z: 616.4 [M+1] .
Example 8A: 5-((1r,3r)(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)isopropyl-1H-pyrazolecarboxylate (8A)
Example 8A can be prepared by subjecting the crude product 8f to the ester hydrolysis as
described for 8 and isolation from the crude product 8 as a minor isomer by preparative RP-
HPLC. H-NMR (CDCl ), δ (ppm): 7.42-7.30 (m, 2H), 7.11 (d, J = 8.0 Hz, 1H), 6.75-6.65 (m, 1H),
6.57 (s, 1H), 4.79 (s, 2H), 4.50-4.41 (m, 1H), 3.96-3.85 (m, 1H), 2.98-2.90 (m, 2H), 2.67-2.57
(m, 2H), 2.20-2.09 (m, 1H), 1.51 (d, J = 8.0 Hz, 6H), 1.32-1.14 (m, 4H). C-NMR (CDCl ), δ
(ppm): 172.6, 166.2, 159.2, 158.4, 147.4, 141.2, 135.7, 134.6, 132.8, 131.3, 128.1, 127.7,
127.5, 116.8, 113.5, 110.0, 105.8, 75.1, 59.8, 51.2, 41.8, 25.4, 22.6, 8.5, 7.8. MS (ESI ) m/z:
616.3 [M+1] .
The transannular configuration of the major isomer (compound 8) and the minor isomer
(compound 8A) was confirmed by NOE experiments. The detected indicative NOEs between
protons are indicated in the following pictures by double arrows:
NOEs detected for example 8 with 1,3-trans transannular configuration of the aromatic moieties
NOEs detected for example 8A with 1,3-cis transannular configuration of the aromatic moieties
Example 9: Methyl 6-(3-(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)methyl-1H-indazolecarboxylate (9)
HO Cl
Step 1: Methyl 1-methylvinyl-1H-indazolecarboxylate (9a)
To the solution of methyl 6-bromomethyl-1H-indazolecarboxylate (60 mg, 0.22 mmol) in
DMF (10 mL), tributyl(vinyl)tin (99 µL, 0.34 mmol), Pd(Ph ) (11 mg, 9 µmol) was added. After
the addition was completed, the mixture was stirred at 90°C for 4 h under Ar. Then the solvent
was removed under reduced pressure. Purification by CC afforded compound 9a (52 mg, 88%).
Step 2: Methyl 1-methyl(3-oxocyclobutyl)-1H-indazolecarboxylate (9b)
Following the procedure as described in Example 7/Step 2, compound 9b was obtained from 9a
in 57% yield. H NMR (400 MHz, CDCl ) δ: 8.14 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.23 (d, J =
8.8 Hz, 1H), 4.13 (s, 3H), 3.99 (s, 3H), 3.87-3.79 (m, 1H), 3.58-3.51 (m, 2H), 3.33-3.26 (m, 2H).
m/z: 259 [M+1] .
Step 3: Methyl 6-(3-(2-chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)methyl-1H-indazolecarboxylate (9)
Following the procedure as described in Example 7/Step 3, compound 9 was obtained from 9b
in 40% yield.
Example 10: 6-(3-(2-Chloro((5-cyclopropyl(2,6-dichlorophenyl)isoxazol
yl)methoxy)phenyl)hydroxycyclobutyl)methyl-1H-indazolecarboxylic acid (10)
HO Cl
Following the procedure as described in Example 8, compound 10 was obtained from
compound 9 in 45% yield as a white solid. H NMR (400 MHz, CDCl ) δ: 8.14 (d, J = 8.0 Hz, 1H),
7.48 (d, J = 8.8 Hz, 1H), 7.43-7.32 (m, 4H), 7.29 (m, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.76 (dd, J =
7.2 Hz, 2.4 Hz, 1H), 4.84 (s, 2H), 4.18 (s, 3H), 3.45-3.40 (m, 1H), 3.28-3.23 (m, 2H), 3.19-3.10
(m, 1H), 2.68-2.63 (m, 2H), 2.21-2.14 (m, 1H), 1.33-1.29 (m, 2H), 1.20-1.15 (m, 2H). m/z: 638
[M+1] .
Preparative Example 11
Step1: Methyl 5-(3-hydroxyazetidinyl)nicotinate (11a)
A mixture of methyl 5-bromonicotinate (2.00 g, 9.26 mmol), azetidinol (1.01 g, 9.26 mmol),
Cs CO (9.06 g, 27.8 mmol), BINAP (1.15 g, 1.85 mmol) and Pd(OAc) (0.44 g, 1.85 mmol) in
2 3 2
dry dioxane (115 mL) was heated overnight at 85°C under N atmosphere. The resulting mixture
was filtrated, concentrated under reduced pressure and purified by prep-HPLC to give
compound 11a (250 mg, 13%) of as a yellow solid.
Step 2: Methyl 5-(3-oxoazetidinyl)nicotinate (11)
To a solution of compound 11a (250 mg, 1.20 mmol) in dry DCM (15 mL) was added Dess-
Martin periodinane (1.014 g, 2.40 mmol) at 0°C under N atmosphere and the solution was
stirred at rt for 2 h. The resulting solution was quenched with saturated sodium bicarbonate
solution and diluted with EA. The organic portion was washed with brine, dried over Na SO ,
filtered, concentrated under reduced pressure and purified by CC (DCM/MeOH = 150:1) to give
compound 11 (140 mg, 57%) of as a yellow solid.
Preparative Example 12
Using a similar procedure as that described in Preparative Example 11 the following compound
has been prepared:
Example 13/1 to 13/9
The following table lists further examples prepared according the above mentioned preparative
examples and examples. All listed compounds were prepared as single isomers.
# Structure Analytical data
# Structure Analytical data
H NMR (400 MHz, DMSO-d ) δ: 1.15-1.25 (m, 4H),
2.35-2.50 (m, 5H, partially under solvent signal),
2.80-2.91 (m, 1H), 3.11-3.20 (m, 2H), 4.93 (s, 2H),
13/1
6.72-6.81 (m, 1H), 6, 93 (s, 1H), 7.40-7.51 (m, 2H),
7.52-7.60 (m, 2H), 7.62-7.66 (m, 2H), 7.85-7.90 (m,
2H). MS Calcd.: 583; MS Found: 584 [M+H] .
H NMR (400 MHz, CD OD) δ: 1.05-1.12 (m, 4H),
2.18-2.34 (m, 9H), 2.62-2.71 (m, 1H), 2.99-3.09 (m,
13/2
2H), 4.78 (s, 2H), 6.60-6.64 (m, 1H), 6.72-6.78 (m,
1H), 6.85 (s, 2H), 7.30-7.42 (m, 4H). MS Calcd.:
611; MS Found: 612 [M+H] .
H NMR (400 MHz, CD OD) δ: 1.10-1.23 (m, 4H),
2.36-2.49 (m, 3H), 3.00-3.12 (m, 1H), 3.15-3.25 (m,
2H), 3.87 (s, 3H), 4.95 (s, 2H), 6.72-6.80 (m, 1H),
13/3
6.89 (s, 1H), 6.90-7.00 (m, 1H), 7.42-7.60 (m, 4H),
7.89-7.93 (m, 1H), 7.98 (s, 1H). MS Calcd.: 613; MS
Found: 612 [M −H] .
H NMR (400 MHz, CD OD) δ: 1.20-1.30 (m, 4H),
2.27 (s, 3H), 2.30-2.55 (m, 3H), 2.98-3.10 (m, 1H),
3.25-3.40 (m, 2H, partially under solvent signal),
13/4
4.95 (s, 2H), 6.76-6.84 (m, 1H), 6.91 (s, 1H), 7.20-
7.25 (m, 1H), 7.43-7.63 (m, 4H), 7.75-7.82 (m, 1H),
8.03 (s, 1H). MS Calcd.: 597; MS Found: 596 [M −H]
H NMR (400 MHz, CD3OD) δ: 1.20-1.25 (m, 4H),
2.33-2.43 (m, 4H), 2.46-2.56 (m, 2H), 2.88-2.97 (m,
1H) 3.22-3.30 (m, 2H), 4.94 (s, 2H), 6.78-6.82 (m,
13/5
1H), 6.90 (s, 1H), 7.37 (s, 1H), 7.43-7.60 (m, 4H),
7.69 (s, 1H), 7.78 (s, 1H). MS Calcd.: 597; MS
Found: 596 [M −H] .
H NMR (400 MHz, CD OD) δ: 1.17-1.23 (m, 4H),
2.31-2.40 (m, 4H), 2.42-2.50 (m, 2H), 2.83-2.92 (m,
1H) 3.19-3.26 (m, 2H), 4.92 (s, 2H), 6.74-6.80 (m,
13/6
1H), 6.88 (s, 1H), 7.19-7.22 (m, 1H), 7.43-7.57 (m,
4H), 7.83 (s, 1H). MS Calcd.: 597; MS Found: 598
[M+H] .
H NMR (400 MHz, CD OD) δ: 1.09-1.11 (m, 4H),
2.19-2.26 (m, 1H), 4.08-4.10 (m, 2H), 4.19-4.21 (m,
13/7
2H), 4.80 (s, 2H), 6.64-6.67 (m, 2H), 6.75 (s, 1H),
7.16-7.21 (m, 2H), 7.28-7.39 (m, 6H); MS Calcd.:
584; MS Found: 585 (M+1).
# Structure Analytical data
H NMR (300 MHz, CDCl3) δ: 1.11 (m, 2H), 1.24 (m,
2H), 2.11 (m, 1H), 4.33 (m, 2H), 4.46 (m, 2H), 4.78
(s, 2H), 6.67 (dd, J = 1.2Hz, 8.4 Hz, 1H), 6.77 (d, J =
13/8
2.4 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 7.28-7.35 (m,
3H), 7.55 (d, J = 1.2 Hz, 1H), 7.94 (s, 1H), 8.56 (d, J
= 3.6 Hz, 1H); MS Calcd.: 585; MS Found: 586
(M+1).
H NMR (300 MHz, DMSO-d6) δ: 1.13-1.23 (m, 4H),
2.50 (m, 1H), 4.23 (d, J = 8.4 Hz, 2H), 4.51 (d, J =
9.3 Hz, 2H), 4.96 (s, 2H), 6.24 (s, 1H), 6.80 (d, J =
13/9
7.5 Hz, 1H), 6.88 (s, 1H), 6.97 (s, 1H), 7.07 (s, 1H),
7.44 (d, J = 8.4 Hz, 1H), 7.58-7.66 (m, 3H), 8.25 (s,
1H); MS Calcd.: 585; MS Found: 586 (M+1).
Example 14/1 and 14/2
Using a similar procedure as described in the Examples 1 to 13 and Schemes above, the
following compounds were obtained by using the appropriate building blocks.
# Structure Analytical data
H NMR (400 MHz, DMSO-d ): δ 1.13~1.23 (m, 4H),
1.33 (d, J = 6.4 Hz, 6H), 2.37~2.47 (m, 3H), 2.90-
2.95 (m, 1H), 3.14-3.19 (t, J = 8.8 Hz, 2H), 3.57 (d,
J = 4.0 Hz, 2H), 4.38 (m, 1H), 4.92 (s, 2H), 5.51 (s,
14/1
1H ), 6.51 (s, 1H ), 6.76 (dd, J = 2.4 Hz, J = 8.4 Hz,
1H), 6.91 (d, J = 2.4 Hz, 1H), 7.51-7.58 (m, 2H),
7.62-7.65 (m, 2H), 7.69 (s, 1H); MS Calcd.: 672; MS
Found: 673 [M+H] .
H NMR (400 MHz, DMSO-d ): δ 1.13~1.20 (m, 4H),
1.31 (d, J = 6.0 Hz, 6H), 2.36~2.47 (m, 3H), 2.59-
2.63 (m, 2H), 2.89-2.93 (m, 1H), 3.17-3.19 (m, 2H),
3.47-3.52 (m, 2H), 4.33-4.39 (m, 1H), 4.92 (s, 2H ),
14/2
.51 (s, 1H ), 6.50 (s, 1H ), 6.76 (dd, J = 2.4 Hz, J =
8.4 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 7.51-7.58 (m,
2H), 7.62-7.65 (m, 2H), 8.18-8.20 (m, 1H); MS
Calcd.: 744; MS Found: 721 [M–Na] .
H NMR (400 MHz, DMSO-d ): δ 1.10~1.25 (m, 4H),
1.34 (d, J = 6.4 Hz, 6H), 3.03-2.95 (m, 1H), 2.50-
2.30 (m, 3H), 3.20-3.10 (m, 2H), 4.50-4.35 (m, 1H),
14/3
4.92 (s, 2H), 5.5 (s, 1H), 6.92 (s, 1H), 6.78-6.70 (m,
2H), 7.70-7.49 (m, 4H), 11.44 (s, 1H); MS Calcd.:
694; MS Found: 695 [M+H] .
The following compound can be prepared in the same manner by using similar procedures as
described above:
Assays
FRET activity assay
Determination of a ligand mediated cofactor peptide interaction to quantify ligand binding to the
nuclear receptor FXR was performed as follows: Preparation of human FXR alpha ligand
binding domain: The human FXRalpha LBD was expressed in E. coli strain BL21(DE3) as an N-
terminally GST tagged fusion protein. The DNA encoding the FXR ligand binding domain was
cloned into vector pDEST15 (Invitrogen). Expression was under control of an IPTG inducible T7
promoter. The amino acid boundaries of the ligand binding domain were amino acids 187–472
of Database entry NM_005123 (RefSeq). Expression and purification of the FXR-LBD: An
overnight preculture of a transformed E.coli strain was diluted 1:20 in LB-Ampicillin medium and
grown at 30°C to an optical density of OD =0.4–0.6. Gene expression was then induced by
addition of 0.5 mM IPTG. Cells were incubated an additional 6 h at 30°C, 180 rpm. Cells were
collected by centrifugation (7000 x g, 7 min, rt). Per liter of original cell culture, cells were
resuspended in 10 mL lysis buffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4
mg/mL lysozyme) and left on ice for 30 min. Cells were then subjected to sonication and cell
debris removed via centrifugation (22000 x g, 30 min, 4°C). Per 10 mL of supernatant 0.5 mL
prewashed Glutathione 4B sepharose slurry (Qiagen) was added and the suspension kept
slowly rotating for 1 h at 4°C. Glutathione 4B sepharose beads were pelleted by centrifugation
(2000 x g, 15 sec, 4°C) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl
and 1M NaCl). The pellet was resuspended in 3 mL elution buffer per liter of original culture
(elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl and 80 mM glutathione added immediately
prior to use as powder). The suspension was left rotating for 15 min at 4°C, the beads pelleted
and eluted again with half the volume of elution buffer than the first time. The eluates were
pooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM
MgCl as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analysed by SDS-
Page.
The method measures the ability of putative ligands to modulate the interaction between the
purified bacterial expressed FXR ligand binding domain (LBD) and a synthetic biotinylated
peptide based on residues 676–700 of SRC-1 (LCD2, 676–700). The sequence of the peptide
used was B-CPSSHSSLTERHKILHRLLQEGSPS-COOH where the N-terminus was biotinylated
(B). The ligand binding domain (LBD) of FXR was expressed as fusion protein with GST in BL-
21 cells using the vector pDEST15. Cells were lysed by sonication, and the fusion proteins
purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions.
For screening of compounds for their influence on the FXR-peptide interaction, the Perkin Elmer
LANCE technology was applied. This method relies on the binding dependent energy transfer
from a donor to an acceptor fluorophor attached to the binding partner of interest. For ease of
handling and reduction of background from compound fluorescence LANCE technology makes
use of generic fluorophore labels and time resolved detection Assays were done in a final
volume of 25 µL in a 384 well plate, in a Tris-based buffer (20 mM Tris-HCl pH 7.5; 60 mM KCl,
mM MgCl ; 35 ng/µL BSA), containing 20–60 ng/well recombinantly expressed FXR-LBD
fused to GST, 200–600 nM N-terminally biotinylated peptide, representing SRC1 aminoacids
676–700, 200 ng/well Streptavidin-xlAPC conjugate(Prozyme) and 6–10 ng/well Eu W1024 –
antiGST (Perkin Elmer). DMSO content of the samples was kept at 1%. After generation of the
assay mix and diluting the potentially FXR modulating ligands, the assay was equilibrated for 1
h in the dark at rt in FIA-plates black 384 well (Greiner). The LANCE signal was detected by a
Perkin Elmer VICTOR2VTM Multilabel Counter. The results were visualized by plotting the ratio
between the emitted light at 665 and 615 nm. A basal level of FXR-peptide formation is
observed in the absence of added ligand. Ligands that promote the complex formation induce a
concentration-dependent increase in time-resolved fluorescent signal. Compounds which bind
equally well to both monomeric FXR and to the FXR-peptide complex would be expected to give
no change in signal, whereas ligands which bind preferentially to the monomeric receptor would
be expected to induce a concentration-dependent decrease in the observed signal.
To assess the inhibitory potential of the compounds, EC -values were determined for example
compounds as listed below in Table 1 (A = EC < 25 nM; B = 25 ≤ EC < 100 nM; C = EC ≥
50 50 50
100 nM).
Table 1
Group Example #
A 4, 8, 10, 13/8, 13/9, 14/1, 14/2
B 1, 2, 5, 6, 8A, 13/1, 13/3, 13/4, 13/5, 13/7
C 13/2, 13/6
Mammalian one hybrid (M1H) assay
Determination of a ligand mediated Gal4 promoter driven transactivation to quantify ligand
binding mediated activation of FXR was performed as follows: The cDNA part encoding the FXR
ligand binding domain was cloned into vector pCMV-BD (Stratagene) as a fusion to the yeast
GAL4 DNA binding domain under the control of the CMV promoter. The amino acid boundaries
of the ligand binding domain were amino acids 187–472 of Database entry NM_005123
(RefSeq). The plasmid pFR-Luc (Stratagene) was used as the reporter plasmid, containing a
synthetic promoter with five tandem repeats of the yeast GAL4 binding sites, driving the
expression of the Photinus pyralis (American firefly) luciferase gene as the reporter gene. In
order to improve experimental accuracy the plasmid pRL-CMV (Promega) was cotransfected.
pRL-CMV contains the constitutive CMV promoter, controlling the expression of the Renilla
reniformis luciferase. All Gal4 reporter gene assays were done in HEK293 cells (obtained from
DSMZ, Braunschweig, Germany) grown in MEM with L-Glutamine and Earle's BSS
supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium
pyruvate, and 100 units Penicilin/Streptavidin per mL at 37°C in 5% CO . Medium and
supplements were obtained from Invitrogen. For the assay, 5 x 10 cells were plated per well in
96well plates in 100 µL per well MEM without Phenol Red and L-Glutamine and with Earle's
BSS supplemented with 10% charcoal/dextran treated FBS (HyClone, South Logan, Utah), 0.1
mM nonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, and 100 units Penicilin/
. The following day the cells were >90%
Streptavidin per mL, incubated at 37 °C in 5% CO2
confluence. Medium was removed and cells were transiently transfected using 20 µL per well of
a OptiMEM - polyethylene-imine-based transfection-reagent (OptiMEM, Invitrogen;
Polyethyleneimine, Aldrich Cat No. 40,827-7) including the three plasmids described above.
MEM with the same composition as used for plating cells was added 2–4 h after addition of
transfection mixture. Then compound stocks, prediluted in MEM were added (final vehicle
concentration not exceeding 0.1%). Cells were incubated for additional 16 h before firefly and
renilla luciferase activities were measured sequentially in the same cell extract using a Dual-
Light-Luciferase-Assay system (Dyer et al., Anal. Biochem. 2000, 282, 158–161). All
experiments were done in triplicates.
To assess the FXR agonistic potency of the example compounds, potency ranges were
determined in the M1H assay as listed below in Table 2 (A = EC < 25 nM; B = 25 ≤ EC < 100
50 50
nM; C = EC ≥ 100 nM).
Table 2
Group Example #
A 13/4, 13/5, 13/6
B 2, 8, 8A, 10, 13/1, 13/3, 13/7
C 1, 4, 5, 6, 13/2, 13/8, 13/9, 14/1
Aqueous solubility assay
The aq. solubility in PBS, pH 7.4 was determined as follows. A 10 mM compound stock solution
in DMSO was added to PBS (pH 7.4) to reach a theoretical final concentration of 200 µM. The
resulting solution/suspension was shaken at 1250 rpm for 1 h and then stored in the dark at rt
for 23 h. At this time any precipitate is separated from the solution by centrifugation at 3900 rpm
for 30 min. The aq. solubility was determined by comparing the peak area of the principle peak
in a calibration standard (200 µM) in an organic solvent (methanol/water 60:40, v/v) with the
peak area of the corresponding peak in the buffer sample. As detection method was used
HPLC-UV/VIS at 230 nm.
Parallel Artificial Membrane Permeation Assay (PAMPA)
For the PAMPA, 5 mM stock solutions of test items were prepared in DMSO. 5 mM stock
solutions of reference items were prepared in EtOH (carbamazepine, guanabenz) or in
EtOH:H O 1:1 (v/v) (ceftriaxone), respectively. Compounds were diluted in PBS (pH 7.4) to
obtain the starting solutions containing 5% of the respective organic solvent and 250 µM
reference compounds or 10 µM test items, respectively. For the assay, a modified procedure of
the PAMPA as described by Kansy et al. Kansy et al. (J. Med. Chem. 1998, 41, 1007) was
used. The reference compounds for low (ceftriaxone), medium (guanabenz) and high
permeation (carbamazepine) were included as internal controls.
Permeation experiments were carried out in a Multiscreen 96 well tray (donor) covered by a
96-well Multiscreen Immobilon (acceptor). The hydrophobic filter material of the Immobilon plate
was pre-wetted with 70% ethanol and treated with a solution of lipids (lecithin dissolved in
dodecane). The donor plate was filled with test compounds and reference compounds and both
plates were inserted into each other and placed onto an orbital shaker for 15 min at 100 rpm.
The transport study was started by applying 150 µL PBS-buffer containing the test and
reference compounds to the donor plate. After 15 – 16 h of diffusion at rt, the contents of the
acceptor and donor plate were collected and quantified using LC/MS-detection (test items) or by
UV spectroscopy using a Spectramax Plus (Molecular Devices) (reference items). The
absorption maxima for the reference items ceftriaxone, guanabenz and carbamazepine were
240 nm, 270 nm and 286 nm, respectively. Recovery samples were prepared as described for
the permeation assay samples and were incubated in representative vials during the
permeation period under the same conditions.
For LC/MS analysis of the test items, 100 µL incubate were removed from acceptor and donor
compartment and processed for acetonitrile (ACN) precipitation as described below.
Additionally, test item samples from the lipid layer were extracted by flushing each well two
times with 150 µL EA. The solutions were collected in 1.5 mL reaction tubes and the solvent
was evaporated. The dried residues were resuspended in a PBS/DMSO/ACN mixture reflecting
the composition of the acceptor and donor samples (i.e. 100 µL buffer supplemented with 5%
DMSO, 200 µL ACN+ISTD). The final solvent content of each sample was 66% ACN.
Samples from donor and acceptor compartments and calibration standards were precipitated by
addition of 200 µL ACN/ISTD or 400 µL ACN/ISTD, respectively. After vigorous shaking
(10 seconds) and centrifugation (5 min at 4800 x g, rt), the particle free supernatants were
subjected to LC-MS/MS. Membrane compartments were extracted as described above. After
reconstitution, samples were vigorously shaken (10 seconds) and spun down (5 min at 4800 x
g, rt). The particle free supernatants were subjected to LC-MS/MS.
For analysis of compounds as described, the HPLC system consisted of an Accela U-HPLC
pump and an Accela auto sampler (Thermo Fisher Scientific, USA). Mass spectrometry was
performed on an Exactive mass spectrometer (orbitrap technology with accurate mass)
equipped with an heated electrospray (H-ESI2) interface (Thermo Fisher Scientific, USA)
connected to a PC running the standard software Xcalibur 2.1.
The LC was performed in the gradient mode (Table 3) using ACN/0.1% formic acid as organic
phase (A) and 10 mM ammonium formate/0.1% formic acid as aq. phase (B); and the pump flow
rate was set to 500 µL/min. Separation was performed on a Gemini C6-Phenyl, 3 µm, 50x2.0
mm (Phenomenex, Germany) analytical column with a pre-column (Gemini C6-Phenyl, 3 µm,
4x2.0 mm).
Table 3: HPLC gradients
Mobile phase 0 min 0.1 min 1.2 min 2.6 min 2.7 min 3.5 min
A (%) 5 5 97 97 5 5
B (%) 95 95 3 3 95 95
As MS tune file a generic tune file was used for all analytes applying the positive or negative ion
mode. As lock mass for internal mass calibration the [M+H] ion of diisooctyl
phthalate(m/z 391.28429), which is ubiquitously present in the solvent system, was used.
Analyte was acquired by scanning ±1 Thomson around the expected mass of the monoisotopic
[M+H] or [M −H] ion. The mass resolution of the Orbitrap was set to 50,000. The accurate mass
of each analyte was used for peak integration. Further instruments settings were as follows:
HCD-Gas off, AGC high dynamic range, max. trap injection time 100 ms, sheath gas 30, aux
gas 8, sweep gas 2, spray voltage 4 kV, capillary temperature 250°C, ESI 2 heater temperature
250°C.
The objective of the present invention was to generate FXR-agonists with improved physico-
chemical properties compared to compounds claimed in ; and/or to provide
the public with a useful choice. This was achieved by the introduction of a polar hydroxyl group
on a 1,3-cyclobutylidene or 1,3-azetidinylidene group replacing the former 1,2-cyclopropylidene
ring.
Surprisingly, the resulting compounds maintained their activity on the FXR receptor but
demonstrated improved physico-chemical properties, such as higher aq. solubility and/or
membrane permeability. A direct comparison of the corresponding compounds of the two series
is given in Table 4.
Table 4
Aqueous PAMPA, clogD
Structure solubility Membrane permeability (ChemAxon
(PBS, pH 7.4) in % Flux * software)
Cl 20 µM 13.6 5.1
192 µM 24.0 4.4
195 µM n.d.** 4.4
Aqueous PAMPA, clogD
Structure solubility Membrane permeability (ChemAxon
(PBS, pH 7.4) in % Flux * software)
72 µM 20.7 5.2
192 µM 21.1 4.5
158 µM 28.9 4.2
HO Cl
171 µM 46.1 3.5
HO N
* Flux (%) = (c acceptor well) / sum (c donor well + c acceptor well) × 100 × 2
** n.d. = not determined
In each case either the aqueous solubility or the PAMPA membrane permeability or both are
significantly improved by the introduction of the hydroxy-cyclobutyl or hydroxy-azetidyl moiety.
As most nuclear receptor active molecules, FXR agonists are generally very lipophilic (M. L.
Crawley, Expert Opin. Ther. Patents 2010, 20, 1047). Therefore, better aqeous solubility and
membrane permeability are supposed to result in a higher oral bioavailability and in general in a
better suitability for clinical development of those compounds as drugs (L. Huang, J. Dong, S.
Karki in Evaluation of drug candidates for preclinical development (Eds. C. Han, C. B. Davis, B.
Wang), Wiley & Sons, Hoboken 2010, 187-217).
The term ‘comprising’ as used in this specification and claims means ‘consisting at least in part
of’. When interpreting statements in this specification and claims which includes the
‘comprising’, other features besides the features prefaced by this term in each statement can
also be present. Related terms such as ‘comprise’ and ‘comprised’ are to be interpreted in
similar manner.
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless specifically stated otherwise,
reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of the
common general knowledge in the art.
We
Claims (8)
1. Use of a compound according to the following Formula (1), an enantiomer, diastereomer, tautomer, solvate or pharmaceutical acceptable salt thereof 5 wherein R is selected from the group consisting of COOR , CONR R , tetrazolyl, SO NR R , C alkyl, 6 7 8 2 7 8 1-6 SO -C alkyl and H, with R independently selected from the group consisting of H or C alkyl, 2 1-6 6 1-6 and R independently from each other selected from the group consisting of H, C alkyl, and R7 8 1-6 halo-C alkyl, C alkylene-R , SO -C alkyl, wherein R is selected from the group consisting 1-6 1-6 9 2 1-6 9 10 of COOH, OH and SO H; A is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazolyl, indolyl, thienyl, benzothienyl, indazolyl, benzisoxazolyl, benzofuranyl, benzotriazolyl, furanyl, benzothiazolyl, thiazolyl, oxadiazolyl, each optionally substituted with one or two groups independently selected from the group consisting of OH, O-C alkyl, O-halo-C alkyl, C alkyl, halo-C alkyl, C 1-6 1-6 1-6 1-6 3-6 15 cycloalkyl and halogen; Q is phenyl, optionally substituted with one or two groups independently selected from the group consisting of C alkyl, halo-C alkyl, halogen and CF ; 1-6 1-6 3 Y is selected from N or CH; Z is selected from 20 , , or wherein X = CH, N, NO; R is selected from the group consisting of hydrogen, C alkyl, C cylcoalkyl, C 1 1-3 3-6 4-5 alkylcycloalkyl, wherein C alkyl is optionally substituted with 1 to 3 substituents independently 25 selected from halogen, hydroxy or C alkoxy; R and R are independently selected from the group consisting of hydrogen, C alkyl, C
2 3 1-3 1-3 haloalkyl, C alkoxy, C haloalkoxy and halogen, 1-3 1-3 in the preparation of a medicament for the prophylaxis and/or treatment of chronic intrahepatic conditions selected from the group consisting of Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC). 5 2. The use according to claim 1 wherein R-A is selected from the group consisting of , , , , , , , , , , , or . 10
3. The use according to claim 1 or claim 2 wherein Q is
4. The use according to any one of claims 1 to 3 wherein Z is
5. The use according to any one of claims 1 to 4 wherein the compound is selected from the group consisting of , , , , , , , , , , 5 , , , , , , , , , , , , 5 , or .
6. The use according to any one of claims 1 to 5, the compound having the following structure or a pharmaceutically acceptable salt thereof.
7. The use according to any one of claims 1 to 5, the compound having the following 5 structure HO Cl O Cl or a pharmaceutically acceptable salt thereof.
8. The use according to any one of claims 1 to 7, substantially as herein described with 10 reference to any example thereof.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161507153P | 2011-07-13 | 2011-07-13 | |
EP11005722.1 | 2011-07-13 | ||
EP11005722A EP2545964A1 (en) | 2011-07-13 | 2011-07-13 | Novel FXR (NR1H4) binding and activity modulating compounds |
US61/507,153 | 2011-07-13 | ||
NZ620177A NZ620177B2 (en) | 2011-07-13 | 2012-07-12 | Novel fxr (nr1h4) binding and activity modulating compounds |
Publications (2)
Publication Number | Publication Date |
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NZ710770A NZ710770A (en) | 2017-02-24 |
NZ710770B2 true NZ710770B2 (en) | 2017-05-25 |
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