PYRROLIDINE SUBSTITUTED FLAVONES FOR THE TREATMENT OF
INFLAMMATORY DISORDERS
FIELD OF THE INVENTION
The present invention relates to use of the compounds of formula 1 (as described
herein), a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt
or a pharmaceutically acceptable solvate thereof, for the treatment of inflammatory
disorders.
BACKGROUND OF THE INVENTION
Inflammation plays a fundamental role in host defenses and the progression of
immune-mediated diseases. The inflammatory response is initiated in response to an
injury (e.g. trauma, ischemia, and foreign particles) and/or an infection (e.g. bacterial
or viral infection) by, including chemical mediators (e.g. cytokines and
prostaglandins) and inflammatory cells (e.g. leukocytes). It is characterized by
increased blood flow to the tissue, causing pyrexia, erythema, induration and pain.
A delicate well-balanced interplay between the humoral and cellular immune
elements in the inflammatory response enables the elimination of harmful agents
and the initiation of the repair of damaged tissue. When this delicately balanced
interplay is disrupted, the inflammatory response may result in considerable damage
to normal tissue and may be more harmful than the original insult that initiated the
reaction. In these cases of uncontrolled inflammatory responses, clinical intervention
is needed to prevent tissue damage and organ dysfunction. Diseases such as
rheumatoid arthritis, osteoarthritis, Crohn's disease, asthma, allergies, septic shock
syndrome, atherosclerosis, inflammatory bowel disease among other clinical
conditions are characterized by chronic inflammation.
Several proinflammatory cytokines, especially TNF-oc (tumor necrosis factor -oc) and
interleukins (IL-1 , IL-6, IL-8) play an important role in the inflammatory process.
Both IL-1 and TNF-oc are derived from mononuclear cells and macrophages and in
turn induce the expression of a variety of genes that contribute to the inflammatory
process.
Rheumatoid arthritis (RA) is an autoimmune disorder. RA is a chronic, systemic,
articular inflammatory disease of unknown etiology. In RA, the normally thin synovial
lining of joints is replaced by an inflammatory, highly vascularized, invasive
fibrocollagenase tissue (pannus), which is destructive to both cartilage and bone.
Areas that may be affected include the joints of the hands, wrists, neck, jaw, elbows,
feet and ankles. Cartilage destruction in RA is linked to aberrant cytokines and
growth factor expression in the affected joints.
Osteoarthritis (OA, also known as degenerative arthritis, degenerative joint disease),
is the most common type of arthritis involving degradation of joints, including articular
cartilage and the subchondral bone next to it. In OA, a variety of potential forces,
hereditary, developmental, metabolic, and mechanical, may initiate processes
leading to loss of cartilage, a strong protein matrix that lubricates and cushions the
joints. When bone surfaces become less well protected by cartilage, subchondral
bone may be exposed and damaged, with regrowth leading to a proliferation of ivory
like, dense, reactive bone in central areas of cartilage loss, a process called
eburnation. Due to decreased movement because of the pain, regional muscles may
atrophy, and ligaments may become more lax. OA is the most common form of
arthritis, and the leading cause of chronic disability.
Inflammatory bowel disease (IBD) is a group of disorders that cause inflammation of
the intestines. The inflammation lasts for a long time and usually relapses. The two
major types of IBD are Crohn's disease and ulcerative colitis. Crohn's disease occur
when the lining and wall of the intestines becomes inflamed resulting in the
development of ulcers. Although Crohn's disease can occur in any part of the
digestive system, it often occurs in the lower part of the small intestine where it joins
the colon. Ulcerative colitis is a chronic auto-immune/inflammatory disease of
unknown etiology afflicting the large intestine and affecting millions of people
worldwide. It is well-established that a dysfunctional immune-response involving
components of normal gastrointestinal gram-negative bacteria and increased
expression of pro-inflammatory cytokines, chemokines, endothelial cell adhesion
molecules (ECAMs) and enhanced leukocyte infiltration into colonic interstitium, play
a key role in the pathogenesis of colitis. The course of the disease may be
continuous or relapsing, mild or severe. The earliest lesion is an inflammatory
infiltration with abscess formation at the base of the crypts of Lieberkuhn.
Coalescence of these distended and ruptured crypts tends to separate the overlying
mucosa from its blood supply, leading to ulceration. Signs and symptoms of the
disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose
discharges consisting mainly of blood, pus, and mucus with scanty fecal particles. A
total colectomy may be required for acute, severe or chronic, unremitting ulcerative
colitis.
Psoriasis is a chronic, non-contagious autoimmune disease which affects the skin
and joints. It commonly causes red scaly patches to appear on the skin. The scaly
patches caused by psoriasis, called psoriatic plaques, are areas of inflammation and
excessive skin production. Psoriasis can also cause inflammation of the joints, which
is known as psoriatic arthritis. Ten to fifteen percent of people with psoriasis have
psoriatic arthritis. There are many treatments available, but because of its chronic
recurrent nature, psoriasis is difficult to treat.
Tumor Necrosis Factor-oc (TNF-oc), a pleiotropic cytokine, is produced mainly by
macrophages, but other types of cells also produce it. TNF-oc demonstrates
beneficial as well as pathological activities. It has both growth stimulating effects and
growth inhibitory properties, besides being self-regulatory. The beneficial functions of
TNF-oc include maintaining homeostasis by regulating the body's circadian rhythm ,
mounting an immune response to bacterial, viral, fungal and parasitic infections,
replacing or remodeling injured tissue by stimulating fibroblast growth and, as the
name suggests, killing certain tumors. TNF-oc is derived from mononuclear cells and
macrophages and in turn induces the expression of a variety of genes that contribute
to various disorders such as inflammatory disorders.
Although TNF-oc plays a critical role in innate and acquired immune responses,
inappropriate production of TNF-oc produce pathological changes resulting in chronic
inflammation and tissue damage. TNF-oc has been shown to play a crucial role in the
pathogenesis of many chronic inflammatory diseases such as inflammatory bowel
disease, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis,
osteoporosis/bone resorption, coronary heart disease, vasculitis, ulcerative colitis,
psoriasis, adult respiratory distress syndrome, diabetes, skin delayed type
hypersensitivity disorders and Alzheimer's disease.
IL-1 (Interleukin 1) is an important part of the innate immune system, which regulates
functions of the adaptive immune system. The balance between IL-1 and IL-1
receptor antagonist (IL-1 ra) in local tissues influences the possible development of
an inflammatory disease and resultant structural damage. In the presence of an
excess amount of IL-1 , inflammatory and autoimmune disorders may be developed
in joints, lungs, gastrointestinal tract, central nervous system (CNS) or blood vessels.
Cellular adhesion molecules intercellular adhesion moleculel (ICAM-1), vascular-cell
adhesion moleculel (VCAM-1 ) , and E-Selectin are responsible for the recruitment of
inflammatory cells, such as neutrophils, eosinophils, and T lymphocytes, from the
circulation to the site of inflammation. The recruitment and retention of leukocyte is a
critical event in the pathogenesis of all chronic inflammatory diseases such as RA.
The first line of treatment for inflammatory disorders involves the use of non-steroidal
anti-inflammatory drugs (NSAIDs) e.g. ibuprofen, naproxen to alleviate symptoms
such as pain. However, despite the widespread use of NSAIDs, many individuals
cannot tolerate the doses necessary to treat the disorder over a prolonged period of
time as NSAIDs are known to cause gastric erosions. Moreover, NSAIDs merely
treat the symptoms of the inflammatory disorder and not the cause. When patients
fail to respond to NSAIDs, other drugs such as methotrexate, gold salts, Dpenicillamine
and corticosteroids are used. These drugs also have significant side
effects.
Monoclonal antibody drugs such as Infliximab, Etanercept and Adalimumab are
useful as anti-inflammatory agents, but have drawbacks such as route of
administration (only parenteral), high cost, allergy induction, activation of latent
tuberculosis, increased risk of cancer and congestive heart disease.
Although phosphodiesterase-4 (PDE4) inhibitors have been developed for the
treatment of asthma, atopic dermatitis, rheumatoid arthritis, multiple sclerosis, and
Crohn's disease, the non-selective inhibition of multiple PDE4s leads to complex
physiological responses. The therapeutic potential of PDE4 inhibitors has been
hampered by their dose-limiting side effects of nausea and emesis as in the case of
Cilomilast (SB 207499, Ariflo™) (Current Opinion in Chemical Biology, 2001 , 5, 432-
438).
Similarly, although p38 mitogen-activated protein kinase (MAPK) inhibitors are
potential anti-inflammatory agents, the side-effects associated with their use include
gastric ulcerations, hepatotoxicity, and nephrotoxicity. For instance, development of
2-[2(S)-Amino-3-phenylpropylamino]-3-methyl-5-(2-naphthyl)-6-(4-pyridyl)pyrimidin-
4(3H)-one (AMG 548, Amgen) was suspended due to random liver enzyme
elevations that were not dose or exposure dependent. The impact on safety profiles
after chronic treatment using p38 MAP kinase inhibitors, needs to be established
(Current Medicinal Chemistry, 2005, 12, 2979-2994).
US-A-4 900 727 discloses benzopyran-4-one derivatives as antiinflammatory agents.
Anti-inflammatory benzopyran-4-one derivative from Dysoxylum binectariferum is
described by R.G.Naik et al in Tetrahedron, 1988, 44 (7), 2081-2086.
Notwithstanding the availability of a number of therapies for the treatment of
inflammatory disorders, there still exists a continuing need for improved and
alternative medicaments for the treatment of inflammatory disorders.
U.S. Patent 7,271 , 1 93, Published US application US20070015802, and published
PCT application, WO20071481 58, all of which are incorporated herewith in all
entirety as references, describe pyrrolidine substituted flavones as CDK inhibitors
with utility in the treatment of different types of cancers. The present inventors have
found that the pyrrolidine substituted flavones designated herein as compounds of
formula 1 find use in the treatment of inflammatory disorders. The scope of the
present invention is to provide a new anti-inflammatory use of the above-mentioned
pyrrolidine substituted flavones.
SUMMARY OF THE INVENTION
According to one aspect of the present invention, there is provided use of a
compound of formula 1 (structure as indicated herein below), a stereoisomer or a
tautomer thereof or a pharmaceutically acceptable salt or a pharmaceutically
acceptable solvate thereof, for the treatment of an inflammatory disorder.
According to a further aspect of the present invention, there is provided a
pharmaceutical composition comprising the compound of formula 1, a stereoisomer
or a tautomer thereof or a pharmaceutically acceptable salt, or a pharmaceutically
acceptable solvate thereof and at least one pharmaceutically acceptable carrier, for
use in the treatment of an inflammatory disorder.
The present invention also provides a method for the treatment of an inflammatory
disorder, comprising administering to a subject in need thereof, a therapeutically
effective amount of a compound of formula 1, a stereoisomer or a tautomer thereof
or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate
thereof.
The present invention in a further aspect concerns use of the compounds of formula
1 or a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a
pharmaceutically acceptable solvate thereof, for the manufacture of a medicament
for the treatment of an inflammatory disorder.
According to another aspect of the present invention, there is provided use of a
compound of formula 1 (as described herein) or a stereoisomer, or a tautomer
thereof or a pharmaceutically acceptable salt, or a pharmaceutically acceptable
solvate thereof, for the treatment of an inflammatory disorder mediated by elevated
levels of one or more inflammatory cytokines such as Tumor Necrosis Factor-alpha
(TNF-oc) and interleukins (IL-1 , IL-6, IL-8) or increased expression of one or more
cell adhesion molecules such as intercellular adhesion moleculel (ICAM-1 ) ,
vascular-cell adhesion moleculel (VCAM-1) and E-Selectin or a combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1 shows western blot analysis for in vitro screening for p38 MAP Kinase
inhibition and degradation
Figure 2A shows the induction of apoptosis in adherent cell population of synovial
fluid mononuclear cells (SFMC) by the representative compound, compound of
example 3
Figure 2B shows the induction of apoptosis in non adherent cell population of
synovial fluid mononuclear cells (SFMC) by the representative compound,
compound of example 3
Figure 3 shows the effect of treatment with representative compound, compound of
example 3, on the articular index of sum of hind paws of arthritic (CIA)DBA/1 J mice
Figure 4 shows the effect of treatment with representative compound, compound of
example 3, on the paw thickness of arthritic (CIA)DBA/1 J mice
Figure 5 shows the histopathological evaluation of animals treated with
representative compound, compound of example 3 in comparison to vehicle (control)
and Enbrel.
DETAILED DESCRIPTION OF THE INVENTION
In an embodiment, the present invention relates to the use of a compound of formula
1:
Formula 1
wherein Ar is a phenyl or a heteroaryl ring, wherein the phenyl or the heteroaryl ring
may be unsubstituted or substituted by 1, 2, or 3 identical or different substituents
selected from: halogen, nitro, cyano, -C4-alkyl, fluoromethyl, difluoromethyl,
trifluoromethyl, hydroxy, C C4-alkoxy, carboxy, C C4-alkoxycarbonyl, C C4-
alkylenehydroxyl, CONH2, CONR1R2, S0 2NR1R2, cycloalkyl, NR-|R2 and SR3;
wherein and R2 are each independently selected from: hydrogen, C C4-alkyl, C
C4-alkylcarbonyl and aryl, or and R2, together with the nitrogen atom to which they
are bonded, form a 5- or 6-membered ring, which may optionally contain at least
one additional heteroatom; and
R3 is selected from hydrogen, Ci-C 4-alkyl, and phenyl; or
a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a
pharmaceutically acceptable solvate thereof; in the treatment of an inflammatory
disorder.
Definitions:
For the purpose of the disclosure, listed below are definitions of various terms used
to describe the present invention. These definitions apply to the terms as they are
used throughout the specification and the appended claims (unless they are
otherwise limited in specific instances) either individually or as part of a larger group.
They should not be interpreted in the literal sense. They are not general definitions
and are relevant only for this application.
The term "alkyi" refers to the radical of saturated aliphatic groups, including straightchain
alkyi groups and branched-chain alkyi groups. Examples of alkyi residues
containing from 1 to 4 carbon atoms are: methyl, ethyl, n-propyl, n-butyl, isopropyl,
isobutyl, sec-butyl, and t-butyl.
The term "cycloalkyi" refers to a non-aromatic mono or multicyclic ring system of
about 3 to 7 carbon atoms. Examples of cycloalkyi groups include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl and the like. The cycloalkyi groups may be
unsubstituted or substituted by one or more different substituents selected from
halogen, nitro, cyano, CrC 4-alkyl, fluoromethyl, difluoromethyl, trifluoromethyl,
hydroxy, Ci-C4-alkoxy, carboxy, Ci-C4-alkoxycarbonyl, and CONH2.
The term "alkoxy" as used herein refers to an alkyi group, as defined above, having
an oxygen radical attached thereto. Representative alkoxy groups include methoxy,
ethoxy, propoxy, t-butoxy and the like.
The term "halogen" refers to chlorine, bromine, fluorine and iodine.
The term "heteroaryl" as used herein refers to a monocyclic aromatic ring system
containing 1 to 3 identical or different heteroatoms. The term "heteroatom" refers to
nitrogen, oxygen and sulphur. The heteroaryl group can, for example, have 1 or 2
sulphur atoms, 1 or 2 oxygen atoms and/or 1 to 3 nitrogen atoms in the ring. In
monocyclic groups, heteroaryl can be a 3-membered, 4-membered, 5-membered or
6-membered. Heteroaryl groups include pyrrole, thiophene, oxazole, furan, thiazole,
pyrrazole, pyridine and the like.
In the specification where the term "compound of formula 1" is used alone, wherever
appropriate, it is deemed to include a stereoisomer, or a tautomer or a
pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate of the
compound of the formula 1.
It will be understood that "substitution" or "substituted with" includes the implicit
proviso that such substitution is in accordance with permitted valence of the
substituted atom and the substituents, as well as represents a stable compound,
which does not readily undergo undesired transformation such as by rearrangement,
cyclization, or elimination.
The term "pharmaceutically acceptable" means that the carrier, diluent, excipients,
and/or salt must be compatible with the other ingredients of the formulation, and not
deleterious to the recipient thereof.
The term, "therapeutically effective amount" as used herein means an amount of the
compound of formula 1 or a composition comprising said compound, sufficient to
significantly induce a positive modification in the condition to be regulated or treated,
but low enough to avoid side effects if any (at a reasonable benefit/risk ratio), within
the scope of sound medical judgment. The therapeutically effective amount of the
compound of formula 1 or the composition containing said compound will vary with
the particular inflammatory disorder being treated, the age and physical condition of
the subject (patient), the severity of the condition being treated/prevented, the
duration of the treatment, the nature of concurrent therapy, the specific compound or
composition employed, the particular pharmaceutically acceptable carrier utilized,
and like factors.
The term 'treating", "treat" or "treatment" as used herein refers to alleviate, slow the
progression, attenuation or cure of existing disease (for example, rheumatoid
arthritis)
The term "subject" as used herein refers to an animal, preferably a mammal, and
most preferably a human.
The term "inflammatory disorder" as used herein refers to a disease or a condition
characterized by chronic inflammation including but not limited to rheumatoid
arthritis, osteoarthritis, juvenile rheumatoid arthritis, psoriatic arthritis, refractory
rheumatoid arthritis, chronic non-rheumatoid arthritis, osteoporosis/bone resorption,
coronary heart disease, atherosclerosis, vasculitis, ulcerative colitis, psoriasis,
Crohn's disease, adult respiratory distress syndrome, delayed-type hypersensitivity
in skin disorders, septic shock syndrome, and inflammatory bowel disease.
The term "pharmaceutically acceptable salt(s)" is meant to include salt(s) of the
active compounds i.e. the compounds of formula 1, which are prepared with acids or
bases, depending on the particular substituents found on the compounds described
herein. Examples of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, magnesium, ammonium or organic base salt. Examples of
pharmaceutically acceptable organic base addition salts include those derived from
organic bases like lysine, arginine, guanidine, diethanolamine and the like. Examples
of pharmaceutically acceptable acid addition salts include those derived from
inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,
monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogen
phosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the
like, as well as the salts derived from organic acids like acetic, propionic, isobutyric,
oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, glucuronic or
galacturonic acids and the like. The neutral forms of the compounds may be
regenerated by contacting the salt with a base or acid and isolating the parent
compound in the conventional manner.
The term "pharmaceutically acceptable carrier" as used herein means a diluent,
encapsulating material or formulation auxiliary, which is non-toxic, inert, solid, or
semi-solid. Some examples of materials which can serve as pharmaceutically
acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such
as corn starch and potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate, malt, gelatin; talc, as
well as other non-toxic compatible lubricants such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, releasing agents, coating agents,
sweetening, flavoring and perfuming agents; preservatives and antioxidants can also
be present in the composition, according to the judgment of the formulator.
The compounds of formula I may be prepared according to the methods disclosed in
US Patent 7,271 ,193, US Patent Publ. 2007001 5802 and PCT Patent Publication
No. WO20071481 58, which are incorporated herein by reference. The method
described in the following Scheme I can be used to prepare intermediate of formula
(VIA).
SCHEME 1
The preparation steps up to the compound of formula V starting from the compound
of formula (II) are described in US-A-4 900 727, which is incorporated herein by
reference. 1-Methyl-4-piperidone (compound of formula III) was reacted with a
solution of 1,3,5-trimethoxybenzene(compound of formula II) in glacial acetic acid, to
yield 1-methyl-4-(2,4,6-trimethoxyphenyl)-1 ,2,3,6-tetrahydropyridine (compound of
formula IV). Compound of formula IV is reacted with boron trifluoride diethyl etherate,
sodium borohydride and tetrahydrofuran to obtain compound of formula V. In the
conversion of the compound of formula V to that of formula VIA in the above
scheme, the hydroxyl function on the piperidine ring may be converted to a leaving
group such as tosyl, mesyl, triflate or halide by treatment with an appropriate reagent
such as p-toluenesulfonylchloride, methanesulfonylchloride, triflic anhydride or
phosphorous pentachloride in the presence of oxygen nucleophiles such as
triethylamine, pyridine, potassium carbonate or sodium carbonate , followed by ring
contraction in the presence of oxygen nucleophiles such as sodium acetate or
potassium acetate in an alcoholic solvent such as isopropanol, ethanol or propanol.
The ring contraction involved in this step may be effected before flavone formation
as depicted in the above scheme or it may be done after building the flavone with the
desired substitutions.
Enantiomerically pure (-)-trans enantiomer of an intermediate compound of the
formula VIA as defined, is used for the preparation of an enantiomerically pure
compound of the formula I as defined. By using an intermediate having a high
enantiomeric purity as a starting compound in the process, the resultant (+)-trans
enantiomer of pyrrolidines substituted with flavone represented by formula I
produced by the process has a correspondingly high enantiomeric purity.
The process for the preparation of an enantiomerically pure (+)-trans enantiomer of a
compound of formula I, or a pharmaceutically acceptable salt thereof, from the
resolved enantiomerically pure (-)-trans enantiomer of the intermediate compound of
formula VIA comprises following steps:
(a) treating the resolved enantiomerically pure (-)-trans enantiomer of the
intermediate compound of formula VIA,
VIA
with acetic anhydride in the presence of a Lewis acid catalyst to obtain a resolved
acetylated compound of formula VIIA,
(b) reacting the resolved acetylated compound of formula VIIA with an acid of
formula ArCOOH or an acid chloride of formula ArCOCI or an acid anhydride
of formula (ArCO)20 or an ester of formula ArCOOCH 3, wherein Ar is as
defined hereinabove, in the presence of a base and a solvent to obtain a
resolved compound of formula VINA;
VINA
(c) treating the resolved compound of formula VINA with a base in a suitable
solvent to obtain the corresponding resolved -diketone compound of formula
IXA;
IXA
where Ar is as defined.
(d) treating the resolved -diketone compound of formula IXA with an acid such
as hydrochloric acid to obtain the corresponding cyclized compound of
formula XA,
XA
(e) subjecting the compound of formula XA to dealkylation by heating it with a
dealkylating agent at a temperature ranging from 120-1 80 °C to obtain the
(+)-trans enantiomer of the compound of formula I and, optionally, converting
the subject compound into its pharmaceutically acceptable salt.
The Lewis acid catalyst utilized in the step (a) above may be selected from: BF3.
Et20 , zinc chloride, aluminium chloride and titanium chloride.
The base utilized in the process step (b) may be selected from triethylamine, pyridine
and a DCC-DMAP combination (combination of N, N'-dicyclohexyl carbodiimide and
4-dimethylaminopyridine).
It will be apparent to those skilled in the art, the rearrangement of the compound of
formula VINA to the corresponding -diketone compound of formula IXA is known as
a Baker-Venkataraman rearrangement (J. Chem. Soc, 1381 ( 1933) and Curr. Sci.,
4, 214 (1933)).
The base used in the process step (c) may be selected from: lithium hexamethyl
disilazide, sodium hexamethyldisilazide, potassium hexamethyldisilazide, sodium
hydride and potassium hydride. Most preferred base is lithium hexamethyl disilazide.
The dealkylating agent used in process step (e) for the dealkylation of the compound
of formula IXA may be selected from: pyridine hydrochloride, boron tribromide, boron
trifluoride etherate and aluminium trichloride. Most preferred dealkylating agent is
pyridine hydrochloride.
In an aspect of the invention, the compound of formula 1 is selected from the
compounds wherein:
Ar is a phenyl ring, wherein the phenyl ring may be unsubstituted or substituted with
1, 2, or 3 identical or different substituents selected from: halogen, nitro, cyano, C
C4-alkyl, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxy, Ci-C4-alkoxy, carboxy
and NR R2 ; wherein and R2 are each independently selected from: hydrogen, C
C4-alkyl and C C4-alkylcarbonyl or
a stereoisomer or a tautomer thereof or a pharmaceutically acceptable salt or a
pharmaceutically acceptable solvate thereof.
In another aspect of the invention, the compound of formula 1 is selected from the
compounds wherein:
Ar is a phenyl ring, wherein the phenyl ring may be unsubstituted or substituted with
1, 2, or 3 identical or different substituents selected from: halogen, nitro, cyano, C
C4-alkyl, fluoromethyl, difluoromethyl and trifluoromethyl; or a stereoisomer or a
tautomer thereof or a pharmaceutically acceptable salt or a pharmaceutically
acceptable solvate thereof.
In a further aspect of the invention, the compound of formula 1 is selected from:
(+)-irans-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one methane sulfonate;
(+)-irans-2-(2-Chloro-4-cyano-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(4-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-trans- 2-(2-Bromo-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2,4-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-5,7-Dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-2-thiophen-2-ylchromen-
4-one hydrochloride;
(+)-irans-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-2-(3-
nitrophenyl)-chromen-4-one hydrochloride;
(+)-irans-2-(3-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(3-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl
- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-methyl-phenyl)-57-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-pyrrolidin-1-yl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl
- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(3-Bromo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-di
hydroxy-chromen-4-one hydrochloride;
(+)-irans-2-(2,3-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrr
olidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-iodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-isopropylamino-phenyl)-5,7-dihydroxy-8-(2-hydroxy methyl-1 -
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride; and
(+)-irans-2-(3-lodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-
yl)-chromen-4-one hydrochloride.
In yet another further aspect of the invention, the compound of formula 1 is selected
from:
(+)-irans-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-4-cyano-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-trans- 2-(2-Bromo-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2,4-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-57-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-2-(3-
nitrophenyl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-methyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2,3-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrr
olidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-iodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride; and
(+)-irans-2-(2-Chloro-3-isopropylamino-phenyl)-5,7-dihydroxy-8-(2-hydroxy methyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
In an embodiment, the present invention is directed to a method for the treatment of
an inflammatory disorder, involving administering a therapeutically effective amount
of a compound of formula 1 to a subject in need thereof.
In accordance with the present invention, the inflammatory disorders include the
disorders which are mediated by elevated levels of one or more inflammatory
cytokines (TNF-a, IL- , IL-6, IL-8) or increased expression of one or more cell
adhesion molecules (ICAM-1 , VCAM-1 , E-Selectin) or a combination thereof.
In another further embodiment of the invention, the compounds of formula 1 are
TNF-a inhibitors and find use in the treatment of disorders associated with abnormal
TNF-a activity, including: inflammatory bowel disease, rheumatoid arthritis, juvenile
rheumatoid arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis,
chronic non-rheumatoid arthritis, osteoporosis/bone resorption, Crohn's disease,
atherosclerosis, septic shock syndrome, coronary heart disease, vasculitis,
ulcerative colitis, psoriasis, adult respiratory distress syndrome, and delayed type
hypersensitivity in skin disorders.
In another embodiment of the invention, the compounds of formula 1 are IL- , IL-6,
and/or IL-8 inhibitors and find use in the treatment of disorders associated with
abnormal IL- , IL-6, and/or IL-8 activity, including: rheumatoid arthritis,
osteoarthritis and other autoimmune conditions such as multiple sclerosis and
various forms of lupus.
In yet another embodiment of the invention, the compounds of formula 1 are
inhibitors of the expression of one or more cell adhesion molecules such as ICAM-1 ,
VCAM-1 , and E-Selectin and find use in the treatment of inflammatory disorders
associated with increased expression of cell adhesion molecules.
In an embodiment of the invention, there is provided a pharmaceutical composition
comprising a therapeutically effective amount of a compound of formula 1 as an
active ingredient and at least one pharmaceutically acceptable carrier, wherein the
pharmaceutical composition is adapted for the treatment of an inflammatory disorder.
It is currently contemplated that different pharmaceutical compositions adapted for
the treatment of an inflammatory disorder shall contain about 1 to 99 %, for example,
about 5 to 70 %, or about 10 to 30 % by weight of the compound(s) of formula 1 or
the corresponding weight of their pharmaceutically acceptable salts.
According to the present invention, the pharmaceutically acceptable carrier
contained in the pharmaceutical composition may be selected with regard to the
intended route of administration of the compounds of formula 1 and standard
pharmaceutical practice.
According to the present invention, the pharmaceutical composition can be
administered orally, for example in the form of pills, tablets, coated tablets, capsules,
granules or elixirs. Administration, however, can also be carried out rectally, for
example in the form of suppositories, or parenterally, for example intravenously,
intramuscularly or subcutaneously, in the form of injectable sterile solutions or
suspensions, or topically, for example in the form of solutions or ointments, or
transdermally in the form of patches, or in other ways, for example in the form of
aerosols or nasal sprays.
The pharmaceutical compositions according to the present invention are prepared in
a manner known per se and familiar to one skilled in the art e.g., by means of
conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or tabletting processes.
For the production of pills, tablets, coated tablets and hard gelatin capsules it is
possible to use, for example, lactose, corn starch or derivatives thereof, gum arabic,
magnesia or glucose, etc. Carriers for soft gelatin capsules and suppositories are, for
example, fats, waxes, natural or hardened oils, etc. Suitable carriers for the
production of solutions, for example injectable sterile solutions, or of emulsions or
syrups are, for example, water, physiological sodium chloride solution or alcohols, for
example, ethanol, propanol or glycerol, sugar solutions, such as glucose solutions or
manifold solutions, or a mixture of the various solvents which have been mentioned.
In addition to the compound of formula 1 and the pharmaceutically acceptable
carriers, the pharmaceutical compositions can also contain additives such as, for
example, fillers, antioxidants, dispersants, emulsifiers, defoamers, flavors,
preservatives, solubilizers or colorants.
The dose of the compounds of this invention, which is to be administered, can cover
a wide range. The dose to be administered daily is to be selected to suit the desired
effect. A dosage of about 1 to 500 mg/m2 of the compound of the present invention
may be administered per day. If required, higher or lower daily doses can also be
administered. The selected dosage level will depend upon a variety of factors
including the activity of the particular compound of formula 1 employed, the route of
administration, the time of administration, the rate of excretion of the particular
compound being employed, the duration of the treatment, other drugs, compounds
and /or materials used in combination with the particular compounds employed, the
age, sex, weight, condition, general health and prior medical history of the subject
(patient) being treated, and like factors well known in the medical arts.
The present invention also envisages the use of a compound of formula 1 in
combination with other pharmaceutically active compounds. For instance, a
pharmaceutical composition, including a compound of the formula 1 can be
administered to a subject, in particular a human, with another pharmaceutically
active compound known to be useful in treating an inflammatory disorder such as
NSAIDs, gold sodium thiomalate (GST), methotrexate (MTX), and dexamethasone
(DEX) in the form of pharmaceutical preparations.
In accordance with the present invention, certain compounds of formula 1 can exist
in unsolvated forms as well as solvated forms, including hydrated forms. Certain
compounds of formula 1 may exist in multiple crystalline or amorphous forms. In
general, all physical forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present invention.
Those skilled in the art will recognize that stereocentres exist in compounds of
formula 1. Accordingly, the present invention includes all possible stereoisomers and
geometric isomers of formula 1 and includes not only racemic compounds but also
the optically active isomers as well. When a compound of formula 1 is desired as a
single enantiomer, it may be obtained either by resolution of the final product or by
stereospecific synthesis from either isomerically pure starting material or
intermediate. Resolution of the final product, an intermediate or a starting material
may be effected by any suitable method known in the art for example Chiral reagents
for Asymmetric Synthesis by Leo A. Paquette; John Wiley & Sons Ltd (2003).
Additionally, in situations wherein tautomers of the compounds of formula 1 are
possible, the present invention is intended to include all tautomeric forms of the
compounds.
It is understood that modifications that do not substantially affect the various
embodiments of this invention are included within the invention disclosed herein.
The compounds of this invention can be prepared by standard organic chemistry as
illustrated by the accompanying working examples. The following examples are set
forth to illustrate the synthesis of some particular compounds of the present invention
and to exemplify general processes. The invention is explained in detail in the
examples given below and should not be construed to limit the scope of the
invention.
The following abbreviations or terms are used herein:
CD3OD Deuterated methanol
CDCI3 Deuterated chloroform
DCM Dichloromethane
DMF N,N-Dimethylformamide
DMSO Dimethylsulfoxide
DMSO-de Deuterated dimethylsulfoxide
EDTA Ethylene diamine tetra acetic acid
FCS Fetal calf serum
HCI Hydrochloric acid
NaHC0 3 Sodium bicarbonate
Na2C0 3 Sodium carbonate
NaH Sodium hydride
n-BuLi : n-butyl Lithium
NMR Nuclear Magnetic Resonance
PBS Phosphate buffered saline
THF Tetrahydrofuran
EXAMPLES
The compounds corresponding to examples 1-22 have been prepared by the method
as disclosed in published US application US20070015802, and/or published PCT
application, WO2007148158, both of which are incorporated herewith in all entirety
as reference.
Example 1:
(-)-trans-(3-(3-acetyl-2-hydroxy-4,6-dimethoxyphenyl)-1-methylpyrrolidin-2-
yl)methyl acetate
The compound was prepared by the method disclosed in example 11 of published
US application US2007001 5802, and example 6 of published PCT application,
WO20071481 58.
Example 2 :
(+)- frans-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-
1 -methylpyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 43 of
published PCT application, WO20071481 58.
Example 3 :
(+)- frans-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 44 of
published PCT application, WO20071481 58.
Example 4 :
(+)- frans-2-(2-Chloro-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 49 of
published PCT application, WO20071481 58.
Example 5 :
(+)- frans-2-(2-Chloro-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hyclroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 50 of
published PCT application, WO20071481 58.
Example 6 :
(+)- frans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 14 of published
US application US2007001 5802, and example 9 of published PCT application,
WO20071481 58.
Example 7 :
(+)- frans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 15 of published
US application US2007001 5802, and example 10 of published PCT application,
WO20071481 58.
Example 8 :
(+)- frans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 140 of
published US application US2007001 5802, and example 15 of published PCT
application, WO20071481 58.
Example 9 :
(+)-frans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 16 of published
PCT application, WO20071481 58.
Example 10:
(+)-frans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one methane sulfonate
The compound was prepared by the method as disclosed in example 17 of published
PCT application, WO20071481 58.
Example 1 1 :
(+)-frans-2-(2-Chloro-4-cyano-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 39 of published
PCT application, WO20071481 58.
Example 12:
(+)-frans-2-(2-Chloro-4-cyano-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 40 of published
PCT application, WO20071 481 58.
Example 13:
(+)-frans-2-(4-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 100 of
published US application US2007001 5802, and example 34 of published PCT
application, WO20071481 58.
Example 14:
(+)-frans-2-(4-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 35 of published
PCT application, WO20071481 58.
Example 15:
(+)-trans- 2-(2-Bromo-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 42 of published
US application US20070015802
Example 16:
(+)-trans- 2-(2-Bromo-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 43 of published
US application US20070015802.
Example 17:
(+)- frans-2-(2,4-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 30 of published
PCT application, WO20071 481 58.
Example 18:
(+)- frans-2-(2,4-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 3 1 of published
PCT application, WO20071481 58.
Example 19:
(+)- frans-2-(2-Chloro-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
The compound was prepared by the method as disclosed in example 27 of published
US application US20070015802.
Example 20:
(+)- frans-2-(2-Chloro-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 28 of published
US application US20070015802.
Example 2 1 :
(+)- frans-5,7-Dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-2-
thiophen-2-yl-chromen-4-one
The compound was prepared by the method as disclosed in example 68 of published
US application US20070015802.
Example 22:
(+)- frans-5,7-Dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-2-
thiophen-2-yl-chromen-4-one hydrochloride
The compound was prepared by the method as disclosed in example 69 of published
US application US20070015802.
Example 23:
(+)-trans- 3-Nitro-benzoic acid 2-(2-acetoxymethyl-1-methyl-pyrrolidin-3-yl)-6-
acetyl -3,5-dimethoxy-phenyl ester
3-nitrobenzoic acid ( 1 .42 g, 8.49 mmol) was converted to its acid chloride using
oxalyl chloride (0.998 ml_, 11.38 mmol). Triethylamine ( 1 .586 ml_, 11.38 mmol) was
added to a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM (20
ml_). To this, a solution of the acid chloride dissolved in dry DCM (10 ml.) was added
dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture was
poured over crushed ice, basified with saturated sodium carbonate solution (pH 10),
extracted with chloroform (3 x 200 ml_), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Yield: 2.6 g (61 .25 %).
Example 24:
(+)-frans-8-(2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-2-(3-nitrophenyl)-
chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 5.32 mL, 12.48 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (2.63 mL,
12.48 mmol) was added dropwise and stirred for 15 minutes. To this, a solution of
compound of example 23 (2.5 g, 4.97 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
yield frans-acetic acid 3-{2-hydroxy-4,6-dimethoxy-3-[3-(3-nitro-phenyl)-3-oxopropionyl]-
phenyl}-1 -methyl-pyrrolidin-2-ylmethyl ester as an oil (2.2 g), which was
dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation. At the end
of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The
aqueous layer was extracted with chloroform (50 x 3 mL) and washed with water (25
mL) and brine (25 mL). The organic layer was dried over anhydrous Na2S0 4,
concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 1. 1 5 g (52.51 %) ; 1H NMR (CDCI3, 300MHz): 8.81 (t, 1H), 8.45 (d, 1H),
8.35 (d, 1H), 7.58 (t, 1H), 6.76 (s, 1H), 6.47 (s, 1H), 4.50 (m, 1H), 4.01 (s, 3H), 4.02
(s, 3H), 3.81 (dd, 1H), 3.41 (m, 2H), 2.90 (m, 2H), 2.55 (s, 3H), 2.1 9 (m, 2H); MS
(ES+): m/z 441 (M+1).
Example 25:
(+)-frans-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-2-(3-
nitrophenyl)-chromen-4-one
A mixture of compound of example 24 (0.5 g, 1.13 mmol) and pyridine hydrochloride
( 1 .2 g, 10.38 mmol) was heated at 180 °C for a period of 2.5 hours. The reaction
mixture was diluted with methanol (60 mL) and basified with solid Na2C0 3 to pH 10.
The reaction mixture was filtered, and washed with methanol. The organic layer was
concentrated and the residue purified by column chromatography using 0.01 %
ammonia and 4.5 % methanol in chloroform as eluent to afford the title compound.
Yield: 0.250 g (53.53 %) ; 1H NMR (CDCI3, 300MHz): 12.44 (s, 1H), 8.86 (s, 1H),
8.38 (d, 1H), 8.1 5 (d, 1H), 7.72 (t, 1H), 6.67 (s, 1H), 6.31 (s, 1H), 4.39 (dd, 1H),
4.07 (dd, 1H), 3.86 (m, 1H), 3.33 (m, 2H), 2.85 (m, 1H), 2.66 (s, 3H), 2.56 (m, 1H),
2.01 (m, 1H); MS(ES+): m/z 413 (M+1).
Example 26:
(+)-frans-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-2-(3-
nitrophenyl)-chromen-4-one hydrochloride
The compound of example 25 (0.225 g, 0.545 mmol) was suspended in methanol (2
ml.) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title salt.
Yield: 0.225 g (92.21 %) ; 1H NMR (DMSO-d 6, 300MHz): 12.87 (s, 1H), 11.63 (s,
1H), 8.81 (s, 1H), 8.55 (d, 1H), 8.42 (d, 1H), 7.85 (t, 1H), 7.22 (s, 1H), 6.47 (s, 1H),
4.1 5 (m, 1H), 3.69 (m, 5H), 2.92 (s, 3H), 2.42 (m, 1H), 2.22 (m, 1H).
MS (ES+): m/z 4 13 (M+1) (corresponding to freebase).
Example 27:
(+)-trans 3-Bromo-2-chloro-benzoic acid 2-acetyl-6-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-3,5-di methoxy-phenyl ester
3-Bromo-2-chloro-benzoic acid (2.0 g, 8.53 mmol) was converted to its acid chloride
using oxalyl chloride (0.99 ml_, 11.3 mmol). Triethylamine ( 1.586 ml_, 11.3 mmol)
was added to a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM
( 15 ml_). To this, a solution of the acid chloride dissolved in dry DCM ( 10 ml.) was
added drop wise and the reaction stirred at 25 °C for 2 hours. The reaction mixture
was poured over crushed ice, basified with saturated sodium carbonate solution (pH
10), extracted with chloroform (3 x 200 ml_), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Yield: 3.0 g (66.99 %).
Example 28:
(+)-trans- 2-(3-Bromo-2-chloro-phenyl)-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-
3-yl)-5,7-dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 5.54 mL, 13.10 mmol) in THF (30
mL), maintained at 0 °C under nitrogen atmosphere, hexamethyldisilazane (2.764
mL, 13.10 mmol) was added drop wise and stirred for 15 minutes. To this, a solution
of compound of example 27 (3.0 g, 5.27 mmol) in THF (30 mL) was added drop
wise, maintaining the temperature at 0 °C. After the addition, the reaction was
allowed to warm to room temperature and stirred for 2.5 hours. The reaction mixture
was acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8-9.
The aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
yield acetic acid 3-{3-[3-(3-bromo-2-chloro-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
dimethoxy-phenyl}-1 -methyl- pyrrolidin-2-ylmethyl ester as an oil (2.4 g, 4.21 mmol),
which was dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation.
At the end of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 -
9. The aqueous layer was extracted with chloroform (50 x 3 mL) and washed with
water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous
Na2S0 4, concentrated under reduced pressure and dried over vacuum. The residue
was purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 0.423 g ( 15.74 %); 1H NMR (CDCI3, 300MHz): 7.81 (dd, 1H), 7.62 (dd, 1H),
7.34 (dd, 1H), 6.46 (s, 1H), 6.43 (s, 1H), 4.27 (m, 1H), 4.02 (s, 6H), 3.85 (m, 2H),
3.74 (m, 2H), 3.01 (m, 1H), 2.87 (s, 3H), 2.48 (m, 1H), 2.26 (m, 1H); MS(ES+): m/z
509 (M+1 ) .
Example 29:
(+)-frans-2-(3-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -
methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of compound of example 28 (0.280 g, 0.550 mmol) and pyridine
hydrochloride (0.780 g, 6.74 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.125 g (47.34 %); 1H NMR (CD30D, 300MHz): 7.92 (dd, 1H), 7.67 (dd, 1H),
7.40 (t, 1H), 6.32 (s, 1H), 6.13 (s, 1H), 3.93 (m, 1H), 3.74 (dd, 1H), 3.61 (dd, 1H),
3.44 (m, 1H), 3.27 (m, 1H), 3.07 (m, 1H), 2.71 (s, 3H), 2.20 (m, 2H);
MS(ES+): m/z 480.
Example 30:
(+)- frans-2-(3-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 29 (0.120 g, 0.24 mmol) was suspended in methanol (2
ml.) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title salt.
Yield: 0.1 20 (93.02 %)
Example 3 1 :
(+)- frans-3-Chloro-benzoic acid 2-(2-acetoxymethyl-1-methyl-pyrrolidin-3-yl)-6-
acetyl-3,5-dimethoxy-phenyl ester
3-chlorobenzoic acid ( 1 . 15 g, 7.39 mmol) was converted to its acid chloride using
thionylchloride (0.81 ml_, 10.9 mmol). Triethylamine (3.85 ml_, 27.7 mmol) was
added to a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM ( 15
ml_). To this, a solution of the acid chloride dissolved in dry DCM (5.0 ml.) was
added dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture
was poured over crushed ice, basified with saturated sodium carbonate solution (pH
10), extracted with chloroform (3 x 200 ml_), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Yield: 3.8 g ( 100 %).
Example 32:
(+)- frans-2-(3-Chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-
dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 8.2 mL, 19.25 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (4.06 mL,
19.9 mmol) was added drop wise and stirred for 15 minutes. To this, a solution of
compound of example 3 1 (3.8 g, 7.75 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(3-chloro-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (2.7 g), which T- i
was dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation. At the
end of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The
aqueous layer was extracted with chloroform (50 x 3 mL) and washed with water (25
mL) and brine (25 mL). The organic layer was dried over anhydrous Na2S0 4,
concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 0.565 g ( 16.95 %) ; 1H NMR (CDCI3, 300MHz): 8.02 (d, 1H), 7.82 (d, 1H),
7.42 (t, 2H), 6.6 (s, 1H), 6.42 (s, 1H), 4.32 (m, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.78
(d, 1H), 3.25 (m, 2H), 2.75 (m, 2H), 2.55 (m, 2H), 2.40 (s, 3H); MS(ES+): m/z 431 . 1
(M+1 ) .
Example 33:
(+)-frans-2-(3-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one
A mixture of compound of example 32 (0.565 g, 1.31 mmol) and pyridine
hydrochloride ( 1 .2 g, 10.38 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.150 g (19.96 %) ; 1H NMR (CD3OD, 300MHz): 7.83 (d, 1H), 7.71 (d, 1H),
7.49 (d, 1H), 7.43 (t, 1H), 6.53 (s, 1H), 6.28 (s, 1H), 4.31 (m, 1H), 4.05 (d, 1H), 3.82
(m, 1H), 3.31 (m, 2H), 2.85 (m, 3H), 2.66 (s, 3H); 2.53 (m, 1H),
2.01 (m, 1H); MS(ES+): m/z 402.1 (M+1).
Example 34:
(+)- frans-2-(3-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride
The compound of example 33 (0.140 g, 0.349 mmol) was suspended in methanol (2
mL) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title compound, the hydrochloride salt.
Yield: 0.1 00 g (65.40 %)
Example 35:
(+)- frans-2-Chloro-3-nitro-benzoic acid 2-(2-acetoxymethyl-1-methyl-pyrrolidin-
3-yl)-6-acetyl-3,5-dimethoxy-phenyl ester
2-chloro-3-nitro-benzoic acid ( 1 .5 g, 7.44 mmol) was converted to its acid chloride
using oxalyl chloride (0.83 mL, 9.9.45 mmol). Triethylamine (2.80 mL, 20.1 9 mmol)
was added to a solution of compound of example 1 in dry DCM (15 mL). To this, a
solution of the acid chloride dissolved in dry DCM (10 mL) was added dropwise and
the reaction stirred at 25 °C for 2 hours. The reaction mixture was poured over
crushed ice, basified with saturated sodium carbonate solution (pH 10), extracted
with chloroform (3 x 200 mL), and the solvent removed under reduced pressure to
afford the title compound as a viscous oil.
Crude yield: 3.8 g.
Example 36:
(+)- frans-2-(2-Chloro-3-nitro-phenyl)-8-(2-(hydroxymethyl)-1-methyl-pyrrolidin-
3-yl)-5,7-dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 8.2 mL, 19.25 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (4.02 mL,
19.25 mmol) was added drop wise and stirred for 15 minutes. To this, a solution of
compound of example 35 (3.8 g, 7.1 1 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
yield acetic acid 3-{3-[3-(2-chloro-3-nitro-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (2.5 g), which was
dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation. At the end
of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The
aqueous layer was extracted with chloroform (50 x 3 mL) and washed with water (25
mL) and brine (25 mL). The organic layer was dried over anhydrous Na2S0 4,
concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 0.90 g (26.59 %) ; 1H NMR (CDCI3, 300MHz): 7.93 (dd, 1H), 7.87 (dd, 1H),
7.54 (t, 1H), 6.48 (s, 1H), 6.43(s, 1H), 4.1 1 (m, 1H), 3.978 (s, 3H), 3.94 (s, 3H), 3.63
(m, 1H), 3.29 (m, 2H), 2.65 (m, 1H), 2.47 (m, 2H), 2.29 (s, 3H), 2.01 (m, 1H); MS
(ES+): m/z 475.1 (M+1).
Example 37:
(+)-frans-2-(2-Chloro-3-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of compound of example 36 (0.900 g, 1.88 mmol) and pyridine
hydrochloride (0.600 g, 5.1 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.300 g (35.6 %) ; 1H NMR (DMSO-d6, 300MHz): 8.26 (dd, 1H), 8.07 (dd
1H), 7.77 (t, 1H), 6.56 (s, 1H), 6.1 1 (s, 1H), 3.72 (m, 1H), 3.47 (d, 2H), 2.86 (m, 2H),
2.71 (m, 1H), 2.41 (s, 3H), 2.1 1 (m, 1H); 2.79 (m, 1H);
MS(ES+): m/z 447.1 (M+1 ) ; MS(ES-): m/z 445.1 (M-1).
Example 38:
(+)-frans-2-(2-Chloro-3-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 37 (0.250 g, 0.560 mmol) was suspended in methanol (2
mL) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title compound, the hydrochloride salt.
Yield: 0.200 g (74.01 %)
Example 39:
(+)-trans- 2-Chloro-3-trifluoromethyl-benzoic acid 2-(2-acetoxymethyl-1 -methylpyrrolidin-
3-yl)-6-acetyl-3,5-dimethoxy-phenyl ester
2-Chloro-3-trifluoromethyl-benzoic acid (10.0 g, 44.5 mmol) was converted to its acid
chloride using oxalyl chloride (4.6 mL, 50.7 mmol). Triethylamine (7.2 mL, 52.0
mmol) was added to a solution of compound of example 1 ( 13.6 g, 38.70 mmol) in
dry DCM (60 mL). To this, a solution of the acid chloride dissolved in dry DCM ( 10.0
mL) was added drop wise and the reaction stirred at 25 °C for 2 hours. The reaction
mixture was poured over crushed ice, basified with saturated sodium carbonate
solution (pH 10), extracted with chloroform (3 x 200 mL), and the solvent removed
under reduced pressure to afford the title compound as a viscous oil.
Crude yield: 25.0 g.
Example 40:
(+)-frans-2-(2-Chloro-3-trifluoromethyl-phenyl)-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-5,7-di methoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 4.2 mL, 9.8 mmol) in THF (30 mL),
maintained at 0 °C under nitrogen atmosphere, hexamethyldisilazane (2.06 mL, 9.8
mmol) was added drop wise and stirred for 15 minutes. To this, a solution of
compound of example 39 (2.2 g, 3.9 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
yield acetic acid 3-{3-[3-(2-chloro-3-trifluoromethyl-phenyl)-3-oxo-propionyl]-2-
hydroxy-4,6-dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (2.0 g),
which was dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation.
At the end of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 -
9. The aqueous layer was extracted with chloroform (50 x 3 mL) and washed with
water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous
Na2S0 4, concentrated under reduced pressure and dried over vacuum. The residue
was purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 0.560 g (28.89 %); 1H NMR (MeOD, 300MHz): 8.05 (d, 1H), 7.99 (d, 1H),
7.72 (dd, 1H), 6.74 (s, 1H), 6.41 (s, 1H) , 4.04 (s, 3H), 4.01 (s, 3H), 3.86 (m, 1H),
3.53 (m, 2H), 3.33 (m, 1H), 2.99 (t, 1H), 2.75 (t, 1H), 2.36 (s, 3H), 2.08 (m, 2H).
Example 4 1 :
(+)-frans-2-(2-Chloro-3-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of compound of example 40 (0.560 g, 1.12 mmol) and pyridine
hydrochloride (0.700 g, 6.05 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.1 00 g (18.96 %) ; 1H NMR (CD3OD, 300MHz): 8.04 (d, 1H), 7.98 (dd, 1H),
7.70 (t, 1H), 6.37 (s 1H), 6.14 (s, 1H), 3.93 (m, 1H), 3.72 (d, 1H), 3.62 (d, 1H), 3.33
(m, 1H), 2.68 (s, 3H), 2.62 (m, 2H); 2.19 (m, 2H); MS(ES-): m/z 468 (M-1 ) .
Example 42:
(+)-frans-2-(2-Chloro-3-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 4 1 (0.090 g, 0.1 92 mmol) was suspended in methanol
( 1 .5 mL) and treated with ethereal HCI and the organic solvent was evaporated to
afford the title compound, the hydrochloride salt.
Yield: 0.085 g (87.58 %)
Example 43:
(+)-trans- 2-Chloro-3-methyl-benzoic acid 2-(2-acetoxymethyl-1 -methylpyrrolidin-
3-yl)-6-acetyl-3,5-dimethoxy-phenyl ester
2-Chloro-3-methyl-benzoic acid ( 1 .42 g, 8.3 mmol) was converted to its acid chloride
using oxalyl chloride (0.893 mL, 10.0 mmol). Triethylamine ( 1.37 mL, 9.6 mmol) was
added to a solution of compound of example 1 (2.26 g, 6.4 mmol) in dry DCM ( 15
mL). To this, a solution of the acid chloride dissolved in dry DCM (10 mL) was added
dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture was
poured over crushed ice, basified with saturated sodium carbonate solution (pH 10),
extracted with chloroform (3 x 200 mL), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Crude yield: 3.0 g.
Example 44:
(+)-trans- 2-(2-Chloro-3-methyl-phenyl)-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-
3-yl)-5,7-dimethoxy-chromen-4-one
NaH (0.840 g, 29.7 mmol) was suspended in dry DMF ( 15 mL) and a solution of
compound of example 43 (3.0 g, 5.95 mmol) in 15 mL of dry DMF was added
dropwise. Reaction was stirred at room temperature in nitrogen atmosphere for 2
hours. The reaction mixture was quenched with methanol ( 1 .0 mL). Ammonium
chloride solution was added to it followed by extraction in DCM (3 x 20 mL) and the
solvent removed under reduced pressure to obtain acetic acid 3-{3-[3-(2-chloro-3-
methyl-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-dimethoxy-phenyl}-1-methylpyrrolidin-
2-ylmethyl ester as an oil (2.5 g, 5.6 mmol), which was dissolved in cone.
HCI ( 15 mL) and stirred for 3 hours to effect cyclisation. At the end of 3 hours, the
reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The aqueous layer was
extracted with chloroform (20 x 3 ml.) and washed with water (20 ml.) and brine (20
ml_). The organic layer was dried over anhydrous Na2S0 4, concentrated under
reduced pressure and dried over vacuum. The residue was purified by column
chromatography with 3 % methanol in chloroform and 0.01 % ammonia as eluent to
afford the title compound.
Yield: 1.5 g ( 56.9 %) ; 1H NMR (CDCI3, 300MHz): 7.37 (dd, 1H), 7.24 (t, 1H), 6.39
(s, 1H), 6.10 (s, 2H), 3.92 (s, 3H), 3.84 (m, 1H), 3.76 (s, 6H), 3.55 (d, 1H), 3.33 (m,
1H), 3.19 (m, 1H), 2.69 (m, 1H), 2.58 (m, 2H) , 2.37 (s, 3H), 2.20 (m, 1H); MS(ES+):
m/z 444.2 (M+1 ) .
Example 45:
(+)-frans-2-(2-Chloro-3-methyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of compound of example 44 ( 1 .0 g, 2.25 mmol) and pyridine hydrochloride
(3.0 g, 25.95 mmol) was heated at 180 °C for a period of 2.5 hours. The reaction
mixture was diluted with methanol (60 ml.) and basified with solid Na2C0 3 to pH 10.
The reaction mixture was filtered, and washed with methanol. The organic layer was
concentrated and the residue purified by column chromatography using 0.01 %
ammonia and 4.5 % methanol in chloroform as eluent to afford the title compound.
Yield: 0.300 g (32.1 2 %) ; 1H NMR (DMSO-d 6 300MHz): 7.58 (d, 1H), 7.56 (d 1H),
7.41 (t, 1H), 6.41 (s, 1H), 6.1 3 (s, 1H) , 3.71 (m, 1H), 3.40 (m, 2H), 2.79 (m, 2H),
2.40 (m, 1H), 2.41 (s, 3H), 2.39 (m, 3H), 2.10 (m, 1H), 1.78 (m, 1H); MS(ES+): m/z
4 16 (M+1 ) .
Example 46:
(+)-frans-2-(2-Chloro-3-methyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 45 (0.300 g, 0.72 mmol) was suspended in methanol ( 1 .5
ml.) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title compound, the hydrochloride salt.
Yield: 0.250 g (76.9 %)
Example 47:
(+)-trans- 2-Chloro-3-pyrrolidin-1-yl-benzoic acid 2-(2-acetoxymethyl-1-methylpyrrolidin-
3-yl)-6-acetyl-3,5-dimethoxy-phenyl ester
2-Chloro-3-pyrrolidin-1 -yl-benzoic acid ( 1 .6 g, 7.1 5 mmol) was converted to its acid
chloride using thionyl chloride (0.79 mL, 10.7 mmol). Triethylamine ( 1 .13 mL, 8.5
mmol) was added to a solution of compound of example 1 (2.0 g, 5.6 mmol) in dry
DCM ( 15 mL). To this, a solution of the acid chloride dissolved in dry DCM ( 10 mL)
was added dropwise and the reaction stirred at 25 °C for 2 hours. The reaction
mixture was poured over crushed ice, basified with saturated sodium carbonate
solution (pH 10), extracted with chloroform (3 x 200 mL), and the solvent removed
under reduced pressure to afford the title compound as a viscous oil.
Yield: 3.5 g (84.87 %)
Example 48:
(+)-fraA7s-(2-Chloro-3-pyrrolidin-1-yl-phenyl)-8-(2-hydroxymethyl-1-methylpyrrolidin
-3-yl)-5,7-dimethoxy-chromen-4-one
To a solution of n-BuLi ( 15 % solution in hexane, 6.85 mL, 15.6 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (3.36 mL,
15.6 mmol) was added drop wise and stirred for 15 minutes. To this, a solution of
compound of example 47 (3.5 g, 6.4 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(2-chloro-3-pyrrolidin-1-yl-phenyl)-3-oxo-propionyl]-2-
hydroxy-4,6-dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (4.0 g),
which was dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect
cyclisation. At the end of 3 hours, the reaction mixture was basified with solid
NaHC0 3 to pH 8 - 9. The aqueous layer was extracted with chloroform (50 x 3 mL)
and washed with water (25 mL) and brine (25 mL). The organic layer was dried over
anhydrous Na2S0 4, concentrated under reduced pressure and dried over vacuum.
The residue was purified by column chromatography with 3 % methanol in
chloroform and 0.01 % ammonia as eluent to afford the title compound.
Yield: 0.650 g (22.96 %) ; 1H NMR (CDCI3, 300MHz): 7.23 (d, 1H), 7.03 (dd, 2H),
6.46 (s, 1H), 6.36 (s, 1H), 4.02 (s, 3H), 3.97 (s, 3H), 3.91 (dd, 2H), 3.8 (dd, 2H), 3.63
(m, 1H), 3.40 (m, 4H), 3.05 (dd, 2H), 2.82 (m, 1H), 2.62 (dd, 1H), 2.46 (m, 2H), 2.24
(s, 3H), 1.96 (m, 1H); MS(ES+): m/z 499.2 (M+1 ) .
Example 49:
(+)-fraA7s-2-(2-Chloro-3-pyrrolidin-1 -yl-phenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of compound of example 48 (0.650 g, 1.30 mmol) and pyridine
hydrochloride ( 1 .5 g, 12.90 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to yield the title
compound as a yellow solid.
Yield: 0.075 g (12.0 %) ; 1H NMR (CD3OD, 300MHz): 7.32 (d, 1H), 7.1 0 (d, 1H),
7.09 (d, 1H), 6.98 (s, 1H), 6.62 (s, 1H), 4.97 (m, 1H), 4.25 (m, 1H), 3.90 (t, 2H), 3.34
(s, 2H), 2.90 (m, 2H), 2.50 (s, 3H), 2.10 (s, 4H), 1.33 (s, 4H);
MS(ES+): m/z 471 .2 (M+1).
Example 50:
(+)-frans-2-(2-Chloro-3-pyrrol idin-1 -yl-phenyl)-5,7-dihydroxy-8-(2-
hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 49 (0.075 g, 0.1 59 mmol) was suspended in methanol ( 1
mL) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title compound, the hydrochloride salt.
Yield: 0.070 g (86.92 %)
Example 5 1 :
(+)-trans- 3-Bromo-benzoic acid 2-(2-acetoxymethyl-1 -methyl-pyrrolidin-3-yl)-6-
acetyl-3,5-dimethoxy-phenyl ester
3-bromobenzoic acid ( 1 .37 g, 6.8 mmol) was converted to its acid chloride using
oxalyl chloride (0.76 mL, 8.8 mmol). Triethylamine (4.04 mL, 28 mmol) was added to
a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM (20 mL). To this,
a solution of the acid chloride dissolved in dry DCM (10 mL) was added dropwise
and the reaction stirred at 25 °C for 2 hours. The reaction mixture was poured over
crushed ice, basified with saturated sodium carbonate solution (pH 10), extracted
with chloroform (3 x 200 mL), and the solvent removed under reduced pressure to
afford the title compound as a viscous oil.
Crude yield: 4.6 g
Example 52:
(+)-frans-2-(3-Bromo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-
dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 9.18 mL, 2 1.5 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (4.51 mL,
2 1 .4 mmol) was added dropwise and stirred for 15 minutes. To this, a solution of
compound of example 5 1 (4.6 g, 8.6 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S04. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(3-bromo-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (5.0 g), which was
dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation. At the end
of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The
aqueous layer was extracted with chloroform (50 x 3 mL) and washed with water (25
mL) and brine (25 mL). The organic layer was dried over anhydrous Na2S0 4,
concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 1.4 g (35.3 %) ; 1H NMR (CDCI3, 300MHz): 9.21 (d, 1H), 8.90 (d, 1H), 8.70
(d, 1H), 8.48 (t, 1H), 7.72 (s, 1H), 7.53 (s, 1H ) 5.65 (m, 1H), 4.14 (s, 3H), 4.09 (s,
3H), 4.69 (m, 2H), 4.33 (m, 2H), 4.18 (m, 2H), 3.88 (s, 3H), 3.36 (m, 2H);
MS(ES+): m/z 474.0 (M+1 ) .
Example 53:
(+)-frans-2-(3-Bromo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-
dihydroxy-chromen-4-one
A mixture of compound of example 52 ( 1 .40 g, 3.04 mmol) and pyridine
hydrochloride (2.0 g, 17.30 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.540 g (39.4 %) ; 1H NMR (CDCI3, 300MHz): 8.04 (t, 1H), 7.78 (d, 1H), 7.65
(d, 1H), 7.39 (,m, 1H), 6.56 (s, 1H), 6.30(s, 1H), 4.32 (dd, 1H), 4.03 (dd, 1H), 3.84
(dd, 1H), 3.32 (m, 2H), 2.91 (m, 1H), 2.68 (s, 3H), 2.55 (m, 2H), MS(ES+): m/z
447.68 (M+1 ) .
Example 54:
(+)-frans-2-(3-Bromo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-
dihydroxy-chromen-4-one hydrochloride
The compound of example 53 (0.540 g, 1.2099 mmol) was suspended in methanol
(2 mL) and treated with ethereal HCI and the organic solvent was evaporated to
afford the title compound, the hydrochloride salt.
Yield: 0.500 g (85.64 %)
Example 55:
(+)-trans- 2,3-Dichloro-benzoic acid 2-(2-acetoxymethyl-1 -methyl-pyrrolidin-3-
yl) -6-acetyl-3,5-di methoxy-phenyl ester
2,3-dichloro benzoic acid ( 1 .3 g, 6.82 mmol) was converted to its acid chloride using
oxalyl chloride (0.76 ml_, 8.87 mmol). Triethylamine (4.04 ml_, 28 mmol) was added
to a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM (20 ml_). To
this, a solution of the acid chloride dissolved in dry DCM (10 ml.) was added
dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture was
poured over crushed ice, basified with saturated sodium carbonate solution (pH 10),
extracted with chloroform (3 x 200 ml_), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Crude yield: 4.35 g
Example 56:
(+)-frans-2-(2,3-Dichloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5
,7-dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 8.15 ml_, 19.1 mmol) in THF (30
ml_), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (4.0 ml_,
19.1 mmol) was added dropwise and stirred for 15 minutes. To this, a solution of
compound of example 55 (4.35 g, 8.29 mmol) in THF (30 ml.) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 ml_). The organic layer was
washed with water (50 ml_), brine (50 ml.) and dried over anhydrous Na2S04. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(2,3-dichloro-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (4.5 g), which was
dissolved in cone. HCI (60 ml.) and stirred for 3 hours to effect cyclisation. At the end
of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The
aqueous layer was extracted with chloroform (50 x 3 ml.) and washed with water (25
ml.) and brine (25 ml_). The organic layer was dried over anhydrous Na2S0 4,
concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 0.670 (25.47 %) ; 1H NMR (CD3OD, 300MHz): 8.77 (d, 1H), 8.66 (d, 1H),
8.55 (t, 1H), 7.51 (s, 1H), 7.56 (s, 1H), 5.37 (m, 2H), 5.1 3 (s, 3H), 5.1 1 (s, 3H), 4.95
(m, 1H), 4.83 (dd, 3H), 4.70 (m, 2H), 3.37 (d, 1H), 3.84 (s, 3H), 3.47 (m, 2H);
MS(ES+): m/z 464.1 (M+1 ) .
Example 57:
(+)-frans-2-(2,3-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one
A mixture of compound of example 56 (0.670 g, 1.44 mmol) and pyridine
hydrochloride (2.0 g, 17.30 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 mL) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.31 0 g (49.34 %) ; 1H NMR (DMSO-d6, 300MHz): 77.78 (d, 1H), 7.71 (d,
1H), 7.49 (m, 1H), 6.52 (s, 1H), 6.38 (s, 1H), 4.21 (m, 1H), 3.87 (m, 2H), 3.68 (m,
1H), 3.60 (dd, 1H), 3.37 (m, 1H), 2.97 (s, 3H), 2.51 (m, 1H), 2.27 (m, 1H);
MS(ES-): m/z 434.1 (M -1).
Example 58:
(+)-frans-2-(2,3-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride
The compound of example 57 (0.31 0 g, 0.714 mmol) was suspended in methanol
( 1 .5 mL) and treated with ethereal HCI and the organic solvent was evaporated to
afford the title compound, the hydrochloride salt.
Yield: 0.300 g (89.1 1 %)
Example 59:
(+)-trans- 2-Chloro-3-iodo-benzoic acid 2-(2-acetoxymethyl-1 -methyl-pyrrolidin-
3-yl)-6-acetyl-3,5-dimethoxy-phenyl ester
2-Chloro-3-iodo-benzoic acid ( 1 .92 g, 6.8 mmol) was converted to its acid chloride
using oxalyl chloride (0.76 mL, 8.8 mmol). Triethylamine (4.04, 28 mmol) was added
to a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM (20 ml_). To
this, a solution of the acid chloride dissolved in dry DCM (10 ml.) was added
dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture was
poured over crushed ice, basified with saturated sodium carbonate solution (pH 10),
extracted with chloroform (3 x 200 ml_), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Crude yield: 5.2 g.
Example 60:
(+)-frans-2-(2-Chloro-3-iodo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-
yl)-5,7-dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 9.01 ml_, 2 1.0 mmol) in THF (30
ml_), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (4.44 ml_,
2 1 .0 mmol) was added dropwise and stirred for 15 minutes. To this, a solution of
compound of example 59 (5.2 g, 8.4 mmol) in THF (30 ml.) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 ml_). The organic layer was
washed with water (50 ml_), brine (50 ml.) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(2-chloro-3-iodo-phenyl)-3-oxo-propionyl]-2-hydroxy-4,6-
dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (4.8 g), which was
dissolved in cone. HCI (60 ml.) and stirred for 3 hours to effect cyclisation. At the end
of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The
aqueous layer was extracted with chloroform (50 x 3 ml.) and washed with water (25
ml.) and brine (25 ml_). The organic layer was dried over anhydrous Na2S0 4,
concentrated under reduced pressure and dried over vacuum. The residue was
purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 0.430 g (9.1 5 %) ; 1H NMR (CDCI3, 300MHz): 8.04 (d, 1H), 7.63 (d, 1H), 7.14
(t, 1H), 6.40 (s, 1H), 6.47 (s, 1H), 4.19 (m, 1H), 4.02 (s, 3H), 4.01 (s, 3H), 3.89 (d,
1H), 3.50 (m, 2H), 2.95 (m, 2H), 2.56 (s, 3H), 2.25 (m, 2H).
Example 6 1 :
(+)-frans-2-(2-Chloro-3-iodo-phenyl)-5,7-dihydroxy-8-(2-hyclroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of compound of example 60 (0.430 g, 0.77 mmol) and pyridine
hydrochloride (2.0 g, 17.30 mmol) was heated at 180 °C for a period of 2.5 hours.
The reaction mixture was diluted with methanol (60 ml.) and basified with solid
Na2C0 3 to pH 10. The reaction mixture was filtered, and washed with methanol. The
organic layer was concentrated and the residue purified by column chromatography
using 0.01 % ammonia and 4.5 % methanol in chloroform as eluent to afford the title
compound.
Yield: 0.1 20 g (29.4 %) ; 1H NMR (CD3OD, 300MHz): 8.1 5 (d, 1H), 7.68 (d, 1H),
7.23 (t, 1H), 6.30 (s, 1H), 6.15 (s, 1H), 3.94 (m, 1H), 3.78 (d, 2H), 3.41 (m, 2H), 3.05
(m, 1H), 2.71 (s, 3H), 2.20 (m, 2H); MS(ES+): m/z 528.0 (M+1); MS(ES-): m/z
526.02 (M-1 ) .
Example 62:
(+)-frans-2-(2-Chloro-3-iodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 6 1 (0.1 20 g, 0.227 mmol) was suspended in methanol ( 1
ml.) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title compound, the hydrochloride salt.
Yield: 0.1 15 g (89.90 %)
Example 63:
(+)-trans- 2-Chloro-3-isopropylamino-benzoic acid 2-(2-acetoxymethyl-1 -
methyl-pyrrolidin-3-yl)-6-acetyl-3,5-dimethoxy-phenyl ester
2-Chloro-3-isopropylamino-benzoic acid ( 1 .8 g, 8.19 mmol) was converted to its acid
chloride using oxalyl chloride (0.80 g, 9.0 mmol). Triethylamine ( 1 .87 ml, 18.5 mmol)
was added to a solution of compound of example 1 (2.4 g, 6.8 mmol) in dry DCM (20
ml_). To this, a solution of the acid chloride dissolved in dry DCM (10 ml.) was added
dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture was
poured over crushed ice, basified with saturated sodium carbonate solution (pH 10),
extracted with chloroform (3 x 200 mL), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Crude yield: 3.0 g.
Example 64:
(+)-frans-2-(2-Chloro-3-isopropylamino-phenyl)-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-5,7-dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 5.85 mL, 13.7 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (2.87 mL,
13.7 mmol) was added dropwise and stirred for 15 minutes. To this, a solution of
compound of example 63 (3.0 g, 5.4 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(2-chloro-3-isopropylamino-phenyl)-3-oxo-propionyl]-2-
hydroxy-4,6-dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl ester as an oil (3.0 g),
which was dissolved in cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation.
At the end of 3 hours, the reaction mixture was basified with solid NaHC0 3 to pH 8 -
9. The aqueous layer was extracted with chloroform (50 x 3 mL) and washed with
water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous
Na2S0 4, concentrated under reduced pressure and dried over vacuum. The residue
was purified by column chromatography with 3 % methanol in chloroform and 0.01 %
ammonia as eluent to afford the title compound.
Yield: 1.0 g (37.5 %) ; 1H NMR (CDCI3, 300MHz): 7.50 (d, 1H), 7.67 (d, 1H), 6.40
(s, 1H), 6.10 (s, 1H), 5.95 (s, 1H), 3.94 (m, 1H), 3.87 (s, 3H), 3.89 (s, 3H), 3.56 (m,
2H), 3.43 (m, 1H), 3.23 (m, 2H), 2.75 (m, 2H), 2.57 (s, 3H); 2.46 (s, 3H), 2.44 (s, 3H);
MS(ES+): m/z 487.2 (M+1 ) .
Example 65:
(+)-fraA7s-2-(2-Chloro-3-isopropylamino-phenyl)-5,7-dihydroxy-8-(2-hydroxy
methyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one
A mixture of example 64 (0.900 g, 1.98 mmol) and pyridine hydrochloride (2.0 g,
17.30 mmol) was heated at 180 °C for a period of 2.5 hours. The reaction mixture
was diluted with methanol (60 mL) and basified with solid Na2C0 3 to pH 10. The
reaction mixture was filtered, and washed with methanol. The organic layer was
concentrated and the residue purified by column chromatography using 0.01 %
ammonia and 4.5 % methanol in chloroform as eluent to afford the title compound.
Yield: 0.070 g (8.08 %) ; 1H NMR (CD3OD, 300MHz): 7.73 (d, 1H), 7.05 (d, 1H),
6.91 (d, 1H), 6.58 (s, 1H), 6.37 (s, 1H), 7.72 (d, 1H), 7.05 (d, 1H), 6.93 (d, 1H), 6.58
(s, 1H), 6.37 (s, 1H), 4.39 (m, 1H), 4.1 5 (m,1 H), 3.98 (m,1 H), 3.58 (m, 1H), 2.65 (m,
2H), 3.50 (m, 2H), 2.37 (m, 1H), 3.03 (s, 3H), 1.30-1 .28 (d, 6H);
MS(ES+): m/z 459.2 (M+1 ) ; MS(ES-): m/z 457.2 ( M-1 ) .
Example 66:
(+)-frans-2-(2-Chloro-3-isopropylamino-phenyl)-5,7-dihydroxy-8-(2-hydroxy
methyl-1 -methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
The compound of example 65 (0.070 g, 0.1 53 mmol) was suspended in methanol ( 1
mL) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title compound, the hydrochloride salt.
Yield: 0.065 g (85.91 %)
Example 67:
(+)-trans- 3-lodo-benzoic acid 2-(2-acetoxymethyl-1 -methyl-pyrrolidin-3-yl)-6-
acetyl-3,5-dimethoxy-phenyl ester
3- lodo-benzoic acid ( 1 .83 g, 7.37 mmol) was converted to its acid chloride using
oxalyl chloride (0.98 mL, 11.06 mmol), triethylamine ( 1 .52 mL, 11.06 mmol) was
added to a solution of compound of example 1 (2.0 g, 5.69 mmol) in dry DCM (20
mL). To this, a solution of the acid chloride dissolved in dry DCM (10 mL) was added
dropwise and the reaction stirred at 25 °C for 2 hours. The reaction mixture was
poured over crushed ice, basified with saturated sodium carbonate solution (pH 10),
extracted with chloroform (3 x 200 mL), and the solvent removed under reduced
pressure to afford the title compound as a viscous oil.
Crude yield: 4.6 g.
Example 68:
(+)-frans-2-(3-lodo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-
dimethoxy-chromen-4-one
To a solution of n-BuLi (15 % solution in hexane, 8.4 mL, 19.77 mmol) in THF (30
mL), maintained at 0 ° under nitrogen atmosphere, hexamethyldisilazane (4.14 mL,
19.77 mmol) was added dropwise and stirred for 15 minutes. To this, a solution of
compound of example 67 (4.6 g, 7.9 mmol) in THF (30 mL) was added drop wise,
maintaining the temperature at 0 °C. After the addition, the reaction was allowed to
warm to room temperature and stirred for 2.5 hours. The reaction mixture was
acidified with dilute HCI, and basified with 10 % sodium bicarbonate to pH 8 - 9. The
aqueous layer was extracted with chloroform (3 x 75 mL). The organic layer was
washed with water (50 mL), brine (50 mL) and dried over anhydrous Na2S0 4. The
organic layer was concentrated under reduced pressure and dried under vacuum to
obtain acetic acid 3-{3-[3-(3-iodophenyl)-3-oxo-propionyl]-2-hydroxy-4,6-dimethoxyphenyl}-
1 -methyl-pyrrolidin-2-ylmethyl ester as an oil (5.0 g), which was dissolved in
cone. HCI (60 mL) and stirred for 3 hours to effect cyclisation. At the end of 3 hours,
the reaction mixture was basified with solid NaHC0 3 to pH 8 - 9. The aqueous layer
was extracted with chloroform (50 x 3 mL) and washed with water (25 mL) and brine
(25 mL). The organic layer was dried over anhydrous Na2S0 4, concentrated under
reduced pressure and dried over vacuum. The residue was purified by column
chromatography with 3 % methanol in chloroform and 0.01 % ammonia as eluent to
afford the title compound.
Yield: 0.6 g (14.5 %) ; 1H NMR (CDCI3, 300MHz): 8.39 (d, 1H), 7.97 (d, 1H), 7.85
(d, 1H), 7.25 (t, 1H), 6.65 (d, 1H), 6.48 (s, 1H), 4.46 (m, 1H), 4.10 (s, 3H), 4.07 (s,
3H), 3.94 (d, 1H), 3.47 (m, 2H), 2.95 (m, 2H), 2.60 (s, 3H), 2.23 (m, 2H).
Example 69:
(+)-frans-2-(3-lodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one
A mixture of compound of example 68 (0.4 g , 0.70 mmol) and pyridine hydrochloride
(0.327 g, 2.83 mmol) was heated at 180 °C for a period of 2.5 hours. The reaction
mixture was diluted with methanol (60 ml.) and basified with solid Na2C0 3 to pH 10.
The reaction mixture was filtered, and washed with methanol. The organic layer was
concentrated and the residue purified by column chromatography using 0.0 1 %
ammonia and 4.5 % methanol in chloroform as eluent to afford the title compound.
Yield: 0.15 g (42.97 %) ; 1H NMR (CD 3OD, 300MHz) : 8.38 (d, 1H), 8.02 (d, 1H),
7.94 (d, 1H), 733 (t, 1H), 6.65 (s, 1H), 6.15 (s, 1H), 4.25 (m, 1H), 3.75 (d, 2H), 3.42
(m, 2H), 3.33 (m, 1H), 2.80 (s, 3H), 2.35 (m, 2H) ; MS(ES+) : m/z 494.21 (M+ 1) .
Example 70:
(+)-frans-2-(3-lodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride
The compound of example 69 (0.050 g, 0.10 1 mmol) was suspended in methanol ( 1
ml ) and treated with ethereal HCI and the organic solvent was evaporated to afford
the title salt.
Yield: 0.045 g (84. 14 %).
Biological Screening of Compounds:
The efficacy of the compounds of formula 1 in inhibiting inflammatory cytokines
comprising Tumor Necrosis Factor-alpha (TN F-oc) and interleukins ( IL-1 , IL-6, IL-8),
and/or inhibiting the expression of cell adhesion molecules comprising intercellular
adhesion moleculel (ICAM-1 ) , vascular-cell adhesion molecule l (VCAM- 1) and ESelectin
can be determined by a number of pharmacological assays well known in
the art. The exemplified pharmacological assays, which follow, have been carried out
with the compounds of formula 1 and/or their salts.
Assays:
Example 7 1 :
In vitro screening to identify inhibitors of TNF- a, IL-1 , IL-6 and IL-8
Cytokine production by lipopolysaccharides (LPS) in hPBMCs (human peripheral
blood mononuclear cells) was measured as in reference, Physiol. Res. 2003, 52,
593-598, the disclosure of which is incorporated by reference for the teaching of the
assay.
Blood was collected from healthy donors into Potassium EDTA vacutainer tubes (BD
vacutainer). The PBMC were isolated using gradient centrifugation in Histopaque-
1077 solution (Sigma). Isolated PBMC were suspended in RPMI 1640 (Rosewell
Park Memorial Institute) culture medium (Sigma-Aldrich Fine Chemicals, USA)
containing 10 % fetal bovine serum (FBS) (J RH, USA), 100 U/m L penicillin (Sigma
Chemical Co. St Louis, MO, USA) and 100 g/mL streptomycin (Sigma Chemical
Co. St Louis, MO). The cell concentration was adjusted to 1x 106 cells/mL. The
viability as determined by trypan blue dye exclusion was uniform Ly > 98 %. The cell
suspension ( 100 ) was added to the wells of a 96-well culture plate. Following cell
plating , 79 of the culture medium and 1 of various concentrations of test
compounds (compounds of formula 1) (final concentration 0.001 , 0.003, 0.005, 0.01 ,
0.025, 0.03, 0.05, 0.1, 0.25, 0.3 and 1 ) dissolved in DMSO (dimethyl sulfoxide,
Sigma, MO, USA) were added to the cells. The final concentration of DMSO was
adjusted to 0.5 %. The vehicle (0.5 % DMSO) was used as control . Rolipram (300
) , a PDE4-selective inhibitor (synthesized in Medicinal Chemistry Department,
Piramal Life Sciences Limited (PLSL)), was used as a standard TNF-oc inhibitor,
whereas SB 203580 (an inhibitor of p38 mitogen-activated protein (MAP) kinase) ( 10
) (Sigma) was used as a standard for IL- (interleukin- 1beta), IL-6 (interleukin-
6), and IL-8 (interleukin-8) inhibition. The plates were incubated for 30 minutes at 37
°C in an atmosphere of 5 % C0 2. Finally, 20 ( 10 g/mL) per well of LPS,
(Escherchia coli 0 127:B8, Sigma Chemical Co. , St. Louis, MO) was added to the
wells of the incubated plates, for a final concentration of 1 g/m L. The plates were
incubated at 37 °C for 5 hours in an atmosphere of 5 % C0 2. To assess the cytotoxic
effect of the compounds of formula 1, the cellular viability test was performed using
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2Htetrazolium)
reagent after 5 hours of incubation. Supernatants were harvested and
assayed for TNF-oc, IL-1 , IL-6 and IL-8 by ELISA as described by the manufacturer.
(OptiE IA ELISA sets, BD Biosciences, Pharmingen). The 50 % inhibitory
concentration (IC 5o) values were calculated by a nonlinear regression method using
GraphPad software (Prism 3.03).
The following Table(s) 1 and 1A show(s) results of the above experiments.
TABLE 1: IC50 values for inhibition of TNF-a
Test Compounds Activity (TNF-a)
ICso 
Compound of example 3 1.0
Compound of example 5 0.4
Compound of example 7 0.2
Compound of example 9 0.9
Compound of example 12 1.5
Compound of example 16 0.3
Compound of example 18 0.3
Compound of example 20 0.7
Compound of example 22 2.3
Compound of example 26 0.4
Compound of example 30 4.1
Compound of example 34 2.5
Compound of example 38 1.3
Compound of example 42 2.4
Compound of example 46 0.5
Compound of example 58 1.5
Compound of example 62 1.3
Compound of example 66 0.9
TAB LE 1A: IC5ovalues of representative compound, compound of example 3,
for inhibition of LPS-induced TNF-oc, IL-1 , IL- 6 and IL- 8 release
from hPBMCs
Based on the above data it can be seen that the compound of example 3, a
representative compound of formula 1, effectively blocked the expression of
proinflammatory cytokines (TNF-oc, IL-1 , IL-6 and IL-8) by LPS-stimulated freshly
isolated human peripheral blood mononuclear cells.
Example 72:
In vitro screening method to identify inhibitors of TNF-a release
Synovial tissue setup
Cytokine production in synovial cells obtained from rheumatoid arthritis patients
undergoing knee replacement surgery, was measured as per the method described
in reference, Lancet, 1989, 29, 244-247, the disclosure of which is incorporated
herein by reference for the teaching of the assay.
The synovial membrane tissue was digested by incubating at 37 °C for 3 hours in an
atmosphere of 5 % C0 2, in Roswell Park Memorial Institute (RPM I) medium
containing penicillin-G ( 100 U/m L), streptomycin (100 g/mL), amphotericin B (50
ng/m L) (GIBCO), 1.33 mg/m L collagenase Type I (Worthington Biochemical
Corporation, New Jersey, USA), 0.5 g/m L DNAse Type I (SIG MA) and 8.33 U/mL
heparin (Biological E. Limited, India) . The digested tissue was then filtered through a
membrane (mesh size 70 micron) (SIGMA), the cells were washed 3 times and
suspended in complete medium (RPM I supplemented with 5% FCS and 5% human
serum) . The cell concentration was adjusted to 1x 106 cells/m L. The viability as
determined by trypan blue dye exclusion was uniform Ly > 98%. The cell suspension
( 100 ) was added to the wells of a 96-well culture plate. Following cell plating, 100
of the culture medium and 1 of various concentrations of the compound of
example 3 (final concentration 0.001 , 0.003, 0.0 1, 0.03, 0.1, 0.3, 1 and 3 )
dissolved in DMSO (dimethylsulfoxide, Sigma, MO, USA) were added to the cells.
The final concentration of DMSO was adjusted to 0.5 %. The vehicle (0.5 % DMSO)
was used as control. SB 203580 (20 ) was used as a standard. The plates were
incubated for 16 hours at 37°C in 5 % C0 2 atmosphere. The culture plates were
centrifuged (2,500 rpm for 10 minutes) and the supernatants collected and stored at
-70°C. The amount of T N F-oc, lnterleukin-1 (IL- ) , lnterleukin-6 ( IL-6) and
lnterleukin-8 ( IL-8) in the supernatants were assayed using the ELISA protocol
recommended by the manufacturers (OptiE IA ELISA sets, BD Biosciences
Pharmingen). The 50 % inhibitory concentration (IC5o) values were calculated by a
nonlinear regression method using the Graph Pad software (Prism 3.03).
The following Table 2 shows results of the above experiment.
TABLE 2 : IC5o values of representative compound, compound of example 3,
for inhibition of spontaneous release of TNF-oc, IL- 6 and IL- 8 from
freshly isolated cells obtained from RA patients
Based on the above data it can be seen that the compound of example 3, a
representative compound of formula 1, blocked the expression of proinflammatory
cytokines (TNF-oc, IL-6 and IL-8) in freshly isolated synovial cells.
Example 73:
In vitro screening to identify inhibitors of adhesion molecule expression
The assay was designed as in reference, Transplantation, 1997, 63(5) , 759-764, the
disclosure of which is incorporated herein by reference for the teaching of the assay.
Human umbilical vein endothelial cells (HUVECs) were obtained from Cascade
Biologies (Oregon-USA). Cells were cultured in M-200 medium (Cascade Biologies)
supplemented with low serum growth supplements (LSGS) (Cascade Biologies),
penicillin-G ( 100 U/mL), streptomycin ( 100 ) and amphotericin B (50 ng/mL)
(GIBCO). Confluent HUVECs in 96-well fibronectin-coated plates were pretreated
with various concentrations of the compound of example 3 (final concentration 0.003,
0.005, 0.01 , 0.025, 0.05, 0.1 , 0.3 and 1 ) dissolved in DMSO (dimethylsulfoxide,
Sigma, MO, USA). The final concentration of DMSO was adjusted to 0.5 %. The
vehicle (0.5 % DMSO) was used as control. BAY 11-7082 (inhibitor of cytokineinduced
-phosphorylation)^. 5 and 1 ) (Calbiochem) was used as a standard
inhibitor of endothelial cell adhesion molecule (ECAMs) expression. The plates were
incubated at 37 ° in an atmosphere of 5 % C0 2. After TNF-oc stimulation ( 1 ng/mL)
(R&D Systems (Minneapolis, MN)) for 4 hours at 37 °C in an atmosphere of 5 %
C0 2, supernatants were harvested and assayed for the expression of ICAM-1
(intercellular cell adhesion molecules), whereas the expressions of VCAM-1
(vascular cell adhesion molecules) and E-selectin were evaluated after 6 hours of
stimulation. To assess the cytotoxic effect of the compound, the cellular viability test
was performed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfonyl)-2H-tetrazolium) reagent. To quantitatively measure the amount of cell
surface expressions of ICAM-1 , VCAM-1 and E-selectin, ELISA assay was
performed using anti-ICAM-1 (clone BBIG-11), anti-VCAM-1 (clone BBIG-V1), anti-Eselectin
(clone BBIG-E4) antibodies, isotype control mouse Ig (clone 1171 1. 1 1)
and the secondary antibody (anti-mouse IgG-HRP antibody) which were procured
from R&D Systems (Minneapolis, MN, USA). The 50 % inhibitory concentration (IC50)
values were calculated by a nonlinear regression method using GraphPad software
(Prism 3.03)
The results of this study are presented in the following Table 3.
TABLE 3 : IC5o values of representative compound, compound of example 3, for
inhibition of TNF- a induced cell surface expression of ICAM-1, VCAM-
1 and E-selectin on HUVECs
Test compound(s) ICso ()
ICAM-1 VCAM-1 E-selectin
Compound of example 3 0.67 0.28 0.65
Based on the results presented in the above Table 3, it can be seen that the
compound of example 3, a representative compound of formula 1, effectively
blocked the expression of endothelial cell adhesion molecules ICAM-1 , VCAM-1 and
E-selectin, in TNF-a stimulated HUVEC (n=2).
Example 74:
In vitro screening for p38 MAP Kinase inhibition and degradation
The method for identifying inhibitors of p38 mitogen-activated protein (MAP) Kinase
was designed as in reference, Journal of Lipid Research, 1999, 40, 19 1 1-1 9 19, the
disclosure of which is incorporated herein by reference for the teaching of the assay.
The method for degradation was designed as in reference, The Journal of
Immunology, 1999, 103, 6800-6809, the disclosure of which is also incorporated by
reference for the teaching of the assay.
Materials
Human Jurkat T-cells from American Type Culture Collection (ATCC) were cultivated
in Rosewell Park Memorial Institute (RPMI) medium supplemented with 10 % fetal
bovine serum (FBS), penicillin ( 100 U/mL) and streptomycin ( 100 g/mL) at 37 ° in
humidified atmosphere of 95 % air-5 % C0 2. Culture medium was changed every 2-3
days and always 24 hours before harvest. RPMI medium, FBS, CellLytic, anti--
actin, sodium orthovanadate and anisomycin were purchased from Sigma-Aldrich
(USA). TNF-a was purchased from R & D Systems (USA). Complete Protease
inhibitor cocktail was procured from Roche.
Methods
Preparation of cytoplasmic extracts for western blotting
Jurkat cells were preincubated with vehicle or the compound of example 3 (0.1 )
for 1 hour in a 37 °C, C0 2 incubator. For MAPK experiments, the cells were
stimulated with anisomycin (10 g/mL) for 30 minutes. SB 203580 was used as a
standard ( 1 ) . For studies pertaining to degradation of and (phospho)
the cells were stimulated with 0.1 nM TNF-a for 0, 5, 15, 30, 60 and 90 minutes. The
cells were harvested, rapidly washed with ice-cold PBS and lysed with cold CellLytic
buffer supplemented with complete protease inhibitor cocktail and sodium
orthovanadate. The protein extracts were obtained after centrifugation at 15,000 rpm
at 4 ° (20 minutes). Aliquots of the resulting extracts were analyzed for their protein
content using the Coomassie Plus Protein Assay Reagent (Pierce) following
manufacturer's instructions.
Western Blotting
Primary antibodies were as follows:
p38 MAPK, phospho- p38 MAPK, and phospho- were purchased from
Calbiochem (USA). Anti - -actin was bought from Sigma (USA).
In all the experiments, equivalent amounts of protein (10 g) was loaded on SDS/
12.5 %-polyacrylamide electrophoresis gels and resolved at 150 V for 2 hours in a
buffered solution (24.9 mM Tris base, 250 mM glycine, 0.1% SDS (sodium dodecyl
sulfate)). After electrophoresis, the proteins were transferred from the gel to a
nitrocellulose membrane (Sigma-Aldrich) at 25 V for 45 minutes in transfer buffer
(47.9 mM Tris base, 38.6 mM glycine, 0.037 % SDS, 20 % methanol; pH 9.2-9.4).
Blots were blocked in Tris-buffered saline (TBS) (20 mM Tris base, 0.9 % NaCI; pH
7.4) containing 5 % nonfat dry milk (Santa Cruz Biotechnology) for 1 hour 15 minutes
at room temperature, and incubated with the primary antibody which was prepared in
SuperBlock Blocking Buffer in TBS (Pierce) at 4°C overnight with gentle rocking.
Membranes were washed and then probed with horse-radish peroxidase (HRP)-
conjugated secondary antibody. Bands were visualized using chemiluminescent
peroxidase substrate (Sigma-Aldrich) and a Kodak Imaging station. Blots were
stripped with stripping buffer (50 mM Tris-HCI pH 6.8, 1 % SDS and 100 mM -
mercaptoethanol) for 20 minutes at 50 °C, washed and reprobed with a primary
antibody to the housekeeping protein -actin as a loading control.
The results of this study are depicted in Figure 1.
Based on the results of the above experiment, it can be said that the compound of
example 3, a representative compound of formula 1, blocks TNFoc - induced IkBoc
phosphorylation and degradation but is not an inhibitor of p38 MAP kinase
Example 75:
In vitro screening: PDE4 inhibition
Human histiocytic lymphoma (U937) cells from the American Type Culture Collection
were grown in plastic flasks in Roswell Park Memorial Institute 1640 culture medium
(RPMI, Gibco BRL, UK) supplemented with 10 % heat inactivated fetal calf serum
(FBS), penicillin (100 U/mL) and streptomycin ( 100 g/mL) at 37 ° in humidified
atmosphere of 95 % air-5 % C0 2. Culture medium was changed every 2-3 days and
always 24 hours before harvest. RPMI medium, FBS and Salbutamol were
purchased from Sigma-Aldrich (USA). Rolipram was synthesized in the Medicinal
Chemistry Department, Piramal Life Sciences Limited.
Method
In all the experiments, culture medium was removed by centrifugation (21 6 x g for 5
minutes) and the cells were suspended in Krebs-Ringer-Henseleit buffer of the
following composition (millimolar): sodium chloride (NaCI), 118; potassium chloride
(KCI), 4.6; sodium bicarbonate (NaHC0 3), 24.9; (potassium dihydrogen phosphate
(KH2P0 4) , 1; D-glucose, 11. 1 ; calcium chloride (CaCI2) , 1; magnesium chloride
(MgCI2) , 1. 1 ; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5; (pH 7.4). To
induce PDE4 activity, 490 aliquots of cells ( 1 .02 X 106 cells /mL) were
preincubated with vehicle or various concentrations of the compound of example 3
( 1 , 3 and 10 ) for 15 minutes in a 37 °C in humidified atmosphere of 95 % air-5 %
C0 2, before being stimulated with salbutamol ( 1 ) for 7 minutes. Rolipram was
used as a standard (0.3, 1 and 3 ) . Salbutamol was dissolved in deionized water,
the compound of example 3 and Rolipram, were dissolved in dimethyl sulfoxide
(DMSO) and the final concentration of DMSO in the assay never exceeded 0.5 %
cAMP assay was performed using Bridge-It cAMP Designer Fluorescence Assay
(Mediomics LLC, USA) in 96-well, black, flat-bottom plates, according to
manufacturer's instructions. Fluorescence measurements were taken with Polarstar
Optima (BMG Labtech-Germany).
Data Analysis
Relative fluorescence was calculated using the following formula where:
RF= Relative Fluorescence,
F0= fluorescent intensity of the blank or buffer control,
F= fluorescence of cAMP or sample:
Data was converted to RF values as presented in the following table 4. A standard
curve was plotted using the RF values of the given standards and the values of
cAMP corresponding to the test compounds were determined using the standard
plot.
TABLE 4 : Study of the effect of representative compound, compound of
example 3, on PDE4 inhibition
From the results presented in the above table 4, it can be seen that the compound of
example 3, a representative compound of formula 1, is not a PDE4 inhibitor.
Example 76:
Induction of Apoptosis in Synovial Fluid mononuclear cells (SFMC):
The assay was designed as in reference, Arthritis and Rheumatism, Vol. 52, No. 1,
January 2005, the disclosure of which is incorporated herein by reference for the
teaching of the assay.
Synovial fluid was collected from rheumatoid arthritis patients under going knee
aspiration. The fluid was collected in vacutainers and processed within 2 hours of
collection. SFMC were isolated using gradient centrifugation using Ficoll-Hypaque
solution (Sigma). Isolated SFMC were suspended in RPMI 1640 (Rosewell Park
Memorial Institute) culture medium (Sigma-Aldrich Fine Chemicals, USA) containing
20 % fetal bovine serum (FBS) (JRH, USA), 100 U/ml penicillin (Sigma Chemical Co.
St Louis, MO) and 100 g/mL streptomycin (Sigma Chemical Co. St Louis, MO).
The cell concentration was adjusted to 1x106 cells/mL. The viability as determined by
trypan blue dye exclusion was uniformly > 98%. 4 mL of this cell suspension was
added to each well of a 6-well plate. The SFMC were incubated for 3-4 hours. Non
adherent cells were removed and cultured separately in 6-well plates. Fresh medium
was added to the adherent cells. After 24 hours incubation in a humidified
atmosphere at 37 in 5 % C0 2, non-adherent cells were removed and pooled with
previous days samples. These cells were plated at a density of 2x1 06 cells/well. The
adherent cell population was re-fed fresh complete medium. Following cell plating,
both the adherent as well as non-adherent cell population were treated with 5 
and 10 of compound of example 3, dissolved in DMSO (dimethylsulfoxide,
Sigma, MO, USA). The final concentration of DMSO was adjusted to 0.5 %. The
plates were incubated for 24 hours at 37 in a humidified atmosphere of 5 % C0 2.
The cells thus treated were harvested and stained with Annexin-V/Propidium iodide
(AV/PI) to check for induction of apoptosis.
Harvesting and staining of cells
Cells were collected in 15 mL tubes. Adherent cells were dislodged by gentle
scraping. After washing in PBS, the cell pellet was resuspended in 1 X binding buffer
(BD Pharmingen Cat#51-661 2 1E). Cell count was adjusted to 1x1 06 cells/mL. 100
of this cell suspension was transferred to a FACS tube. 5 of Annexin-V was
added followed by 5 of 50 g/mL propidium iodide. This suspension was
incubated in the dark for 15-20 minutes. The volume was made up with 400 
binding buffer. Acquisition was done using a flow cytometer.
The results of the study are depicted in Figure 2A and Figure 2B.
Conclusion:
The compound of example 3 induces apoptosis in both adherent as well as non
adherent cell population of mononuclear cells isolated from synovial fluid acquired
from rheumatoid arthritis patients.
Example 77:
Anti-arthritic activity:
Anti-arthritic potential of the compound of example 3, a representative compound of
formula 1, was evaluated in vivo by using two different models.
1. LPS-induced TNF-a release in Balb/c mice.
2. Collagen-induced arthritis in mice.
A) LPS-induced TNF-a release in Balb/c mice
The assay was designed as in reference, J . Med. Bio. Res., 1997, 30, 1199-1207,
the disclosure of which is incorporated herein by reference for the teaching of the
assay.
Protocol:
Balb/c mice (originally procured from National Institute of Nutrition, Hyderabad,
India), of either sex weighing between 18-22 g were orally administered the
compound of example 3 at doses of 12.5, 50, 75, 100 mg/kg. All suspensions were
freshly prepared in 0.5 % CMC. One hour later, LPS ( 1mg/kg) (Escherchia coli,
serotype 0127:B8, Sigma Chemical Co., St. Louis, MO) dissolved in sterile pyrogenfree
saline was administered intra-peritoneally to the control group, standard
treatment group (Rolipram, 30 mg/kg, p.o.) and test groups (compound of example
3), except the negative control group, which received normal saline.
Blood samples were collected from anesthetized mice, with heparin as an ant i
coagulant (25 IU per sample) 1.5 hours post LPS challenge. These were then
centrifuged at 10000 rpm for 10 minutes and plasma samples were analysed for
levels of TNF-a by ELISA, as described by the manufacturer (OptiEIA ELISA sets,
BD Biosciences Pharmingen).
Percent inhibition of TNF-a release was calculated by comparing the TNF-a levels of
the treatment groups with those of the control group.
The results of this study are presented in the following Table 5.
TABLE 5 : % Inhibition of LPS-induced TNF- a release in Balb/c mice for
representative compound, compound of example 3
It can be seen from the data presented in the above Table 5 that the compound of
example 3, a representative compound of formula 1, inhibits LPS-induced TNF-o
release in Balb/c mice in a dose-dependent manner.
B) Collagen induced arthritis in mouse:
The assay was designed as in reference, J . Exp. Med. , 1985, 162, 637-646, the
disclosure of which is incorporated herein by reference for the teaching of the assay.
Protocol:
Male DBA/1 J mice (originally procured from Jackson Laboratories, USA), aged 8-1 0
weeks were immunized with an emulsion equivalent to 200 g of type II collagen in
Freund's Complete Adjuvant, injected intradermally at the base of the tail. A booster
shot with the same emulsion was given 2 1 days later. A group of naive mice was
maintained alongside.
From day 23 to day 56, mice were examined daily once for the signs of rheumatoid
arthritis, using the Articular Index and paw thickness as parameters. Articular Index
scoring was performed employing the following criteria:
Forelimbs: Scale 0-3
0: No redness or swelling
1: Redness, but no swelling
2: Redness and swelling of the paw
3: Redness and severe swelling of the paw
Hind limbs: Scale 0-5
0: No redness or swelling
1: Redness and mild swelling of paw
2: Redness and moderate swelling of paw and/or swelling of at least one of the
digits.
3: Redness and moderate/severe swelling of paw, swelling of ankle joint and/or
swelling of one or more digits.
4: Redness and severe swelling of paw, digits and ankle joint, with joint stiffness.
5: Redness and severe swelling of paw, digits and ankle joint, with joint stiffness
and altered angle of digits.
Mice with a minimum hind paw score of 2 were inducted into the study.
Mice were randomized into the various study groups and orally administered the
vehicle (0.5 % CMC, 1 mL/kg), test compound (compound of example 3, 50 mg/kg
twice daily) and standard compound (Enbrel, 3 mg/kg, s.c, once daily). Each group
had a minimum of 8 mice. The dosing of the compounds was done for 23 days.
The following parameters were observed and recorded daily,
1. Body weight
2. Articular index
3. Paw thickness in mm using a tension free caliper
4. Any significant observation regarding the condition of the animal.
On the last day (24th day of dosing), one hour after the compound treatment, the
animals were sacrificed, blood withdrawn, and plasma collected for drug level
analyses. Also, the limbs of all the animals were preserved for histopathological
evaluations.
The results are graphically depicted in Figure 3 (Articular index versus days of
treatment) and Figure 4 (Paw thickness versus days of treatment). Data obtained
during the course of this study (articular index and paw thickness) was statistically
evaluated by applying Mann-Whitney U test.
Enbrel treated mice showed a significant reduction in the severity of the disease as
assessed by articular index and paw thickness.
The compound of example 3, a representative compound of formula 1, also showed
a trend towards ameriolation of the disease.
Histopathological analysis:
Mice were humanely euthanized and the hind paws were harvested from each
animal, fixed in 10 % neutral buffered formalin, decalcified in 5 % formic acid and
embedded in paraffin. Section () were stained with either hematoxylin and eosin
or safranin O and evaluated microscopically. Histopathological changes were scored
as follows: mild (score = 1), moderate (score = 2) or severe (score = 3) for the
parameters of cellular infiltration, bone erosions and cartilage damage, graded
separately. Cartilage depletion was indicated visually by diminished safranin O
staining of proteoglycan matrix. The mean total score was compared to that of
vehicle treated group. In case of histological scoring, Kruskal-Wallis analysis was
followed by Dunn's multiple comparison tests to evaluate the statistical difference
between two groups. Values of p < 0.05 were considered significant. Histological
evaluation showed reduced joint inflammation, cartilage damage and bone
destruction.
The results of this experiment as shown in Figure 5, indicated that the compound of
example 3, a representative compound of formula 1, has a potential anti-arthritic
effect.
It should be noted that, as used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless the content clearly
dictates otherwise. Thus, for example, reference to a composition containing "a
compound" includes a mixture of two or more compounds. It should also be noted
that the term "or" is generally employed in its sense including "and/or" unless the
content clearly dictates otherwise.
All publications and patent applications in this specification are indicative of the level
of ordinary skill in the art to which this invention pertains.
The invention has been described with reference to various specific and preferred
embodiments and techniques. However, it should be understood that many
variations and modifications may be made while remaining within the spirit and
scope of the invention.
We claim:
1. A compound of formula 1 for use in the treatment of an inflammatory disorder,
Formula 1
wherein Ar is a phenyl or a heteroaryl ring, wherein the phenyl or the heteroaryl ring
may be unsubstituted or substituted by 1, 2, or 3 identical or different substituents
selected from: halogen, nitro, cyano, CrC 4-alkyl, fluoromethyl, difluoromethyl,
trifluoromethyl, hydroxyl, Ci-C 4-alkoxy, carboxy, Ci-C 4-alkoxycarbonyl, C C4-
alkylenehydroxyl, CONH2, CONRiR 2, S0 2NRiR 2, cycloalkyl, NR-|R2 and SR3;
wherein and R2 are each independently selected from: hydrogen, Ci-C 4-alkyl, C
C4-alkylcarbonyl and aryl, or and R2, together with the nitrogen atom to which they
are bonded, form a 5- or 6-membered ring, which may optionally contain at least
one additional heteroatom; and
R3 is selected from hydrogen, Ci-C 4-alkyl, and phenyl; or
a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a
pharmaceutically acceptable solvate thereof.
2. The compound for use in the treatment of an inflammatory disorder according to
claim 1, wherein Ar is a phenyl ring, wherein the phenyl ring may be unsubstituted or
substituted with 1, 2, or 3 identical or different substituents selected from: halogen,
nitro, cyano, Ci-C 4-alkyl, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxyl, -
C4-alkoxy, carboxy and NR^ ; wherein and R2 are each independently selected
from: hydrogen, Ci-C 4-alkyl and Ci-C 4-alkylcarbonyl or
a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt or a
pharmaceutically acceptable solvate thereof.
3. The compound for use in the treatment of an inflammatory disorder according to
claim 1 or claim 2, wherein the compound of formula 1 is selected from:
(+)-irans-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one methane sulfonate;
(+)-irans-2-(2-Chloro-4-cyano-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(4-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-trans- 2-(2-Bromo-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2,4-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-5,7-Dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-2-thiophen-2-ylchromen-
4-one hydrochloride;
(+)-irans-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-2-(3-
nitrophenyl)-chromen-4-one hydrochloride;
(+)-irans-2-(3-Bromo-2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(3-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl
- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-methyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-pyrrolidin-1-yl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl
- 1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(3-Bromo-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-di
hydroxy-chromen-4-one hydrochloride;
(+)-irans-2-(2,3-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrr
olidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-iodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-isopropylamino-phenyl)-5,7-dihydroxy-8-(2-hydroxy methyl-1 -
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride; and
(+)-irans-2-(3-lodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-
yl)-chromen-4-one hydrochloride.
4. The compound for use in the treatment of an inflammatory disorder according to
claim 1 or claim 2, wherein the compound of formula 1 is selected from:
(+)-irans-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Bromo-5-fluoro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-4-cyano-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-trans- 2-(2-Bromo-4-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2,4-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-5-fluoro-phenyl)-57-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methyl-pyrrolidin-3-yl)-2-(3-
nitrophenyl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-nitro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-methyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2,3-Dichloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl- 1-methyl-pyrr
olidin-3-yl)-chromen-4-one hydrochloride;
(+)-irans-2-(2-Chloro-3-iodo-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1 -methylpyrrolidin-
3-yl)-chromen-4-one hydrochloride; and
(+)-irans-2-(2-Chloro-3-isopropylamino-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl- 1-
methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
5. The compound for use in the treatment of an inflammatory disorder, according to
any one of the preceding claims 1 to 4 , wherein the inflammatory disorder is either
mediated by elevated levels of one or more inflammatory cytokines selected from
Tumor Necrosis Factor-alpha (TN F-oc) and interleukins (IL- , IL-6, IL-8) or by
increased expression of one or more cell adhesion molecules selected from
intercellular adhesion molecule l (ICAM- 1) , vascular-cell adhesion molecule l
(VCAM-1 ) and E-Selectin or a combination thereof.
6. The compound for use in the treatment of an inflammatory disorder according to
any one of the preceding claims 1 to 5, wherein the inflammatory disorder is selected
from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile rheumatoid
arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis, chronic nonrheumatoid
arthritis, osteoporosis/bone resorption, coronary heart disease,
atherosclerosis, vasculitis, ulcerative colitis, psoriasis, Crohn's disease, adult
respiratory distress syndrome, delayed-type hypersensitivity in skin disorders, septic
shock syndrome, and inflammatory bowel disease.
7 . Use of a compound of formula 1 as defined in any one of the preceding claims 1
to 4, for the manufacture of a medicament for the treatment of an inflammatory
disorder.
8. The use according to claim 7, wherein the inflammatory disorder is either
mediated by elevated levels of one or more inflammatory cytokines selected from
Tumor Necrosis Factor-alpha (TNF -oc) and interleukins (IL- , IL-6, IL-8) or by
increased expression of one or more cell adhesion molecules selected from
intercellular adhesion moleculel (ICAM-1 ) , vascular-cell adhesion moleculel
(VCAM-1 ) and E-Selectin or a combination thereof.
9. The use according to 7 or claim 8, wherein the inflammatory disorder is selected
from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile rheumatoid
arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis, chronic nonrheumatoid
arthritis, osteoporosis/bone resorption, coronary heart disease,
atherosclerosis, vasculitis, ulcerative colitis, psoriasis, Crohn's disease, adult
respiratory distress syndrome, delayed-type hypersensitivity in skin disorders, septic
shock syndrome, and inflammatory bowel disease.
10. A method for the treatment of an inflammatory disorder, comprising administering
to a subject in need of such treatment a therapeutically effective amount of the
compound of formula 1 as defined in any one of the preceding claims 1 to 4.
11. The method according to claim 10, wherein the inflammatory disorder is either
mediated by elevated levels of one or more inflammatory cytokines selected from the
group consisting of Tumor Necrosis Factor-alpha (TNF -oc) and interleukins (IL-1 , IL-
6, IL-8) or by increased expression of one or more cell adhesion molecules selected
from a group consisting of intercellular adhesion moleculel (ICAM-1), vascular-cell
adhesion moleculel (VCAM-1 ) and E-Selectin, or a combination thereof.
12. The method according to claim 10 or claim 11, wherein the inflammatory disorder
is selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile
rheumatoid arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis,
chronic non-rheumatoid arthritis, osteoporosis/bone resorption, coronary heart
disease, atherosclerosis, vasculitis, ulcerative colitis, psoriasis, Crohn's disease,
adult respiratory distress syndrome, delayed-type hypersensitivity in skin disorders,
septic shock syndrome, and inflammatory bowel disease.
13. A pharmaceutical composition comprising a therapeutically effective amount of a
compound of formula 1 as defined in any one of the preceding claims 1 to 4 in
combination with at least one pharmaceutically acceptable carrier, wherein said
pharmaceutical composition is adapted for use in the treatment of an inflammatory
disorder.
14. A pharmaceutical composition, comprising a therapeutically effective amount of
the compound of formula 1 as defined in any one of the preceding claims 1 to 4, and
at least one further pharmaceutically active compound, together with at least one
pharmaceutically acceptable carrier, wherein said pharmaceutical composition is
adapted for use in the treatment of an inflammatory disorder.
15. The pharmaceutical composition according to claim 13 or claim 14, wherein the
inflammatory disorder is either mediated by elevated levels of one or more
inflammatory cytokines selected from the group consisting of Tumor Necrosis Factoralpha
(TNF-a) and interleukins (IL-1 , IL-6, IL-8) or increased expression of one or
more cell adhesion molecules selected from a group consisting of intercellular
adhesion moleculel (ICAM-1), vascular-cell adhesion moleculel (VCAM-1) and ESelectin
or a combination thereof.
16. The pharmaceutical composition according to any one of the preceding claims 13
to 15, wherein the inflammatory disorder is selected from the group consisting of
rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis, psoriatic arthritis,
osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis,
osteoporosis/bone resorption, coronary heart disease, atherosclerosis, vasculitis,
ulcerative colitis, psoriasis, Crohn's disease, adult respiratory distress syndrome,
delayed-type hypersensitivity in skin disorders, septic shock syndrome, and
inflammatory bowel disease.