Title: 4-(phenyl-(piperidin-4yl)-amino)-benzamide derivatives and their use for the treatment of pain, anxiety or gastrointestinal disorders
Abstract: Compounds of general formula I[Chemical formula should be inserted here. Please see paper copy] R1 is selected from any one of phenyl, pyridinyl, thienyl, furanyl, imidazolyl, pyrrolyl, triazolyl, thiazolyl and pyridine N-oxide; where each R1 phenyl ring and R1 heteroaromatic ring may optionally and independently be further substituted by 1, 2 or 3 substituents selected from straight and branched C1-C6alkyl, NO2, CF3, C1-C6alkoxy, chloro, fluoro, bromo, and iodo. The substitutions on the phenyl ring and on the heteroaromatic ring may take place in any position on said ring systems; are disclosed and claimed in the present application, as well as salts and pharmaceutical compositions comprising the novel compounds and their use in therapy, in particular in the management of pain, anxiety and functional gastrointestinal disorders.
Patent Number: 7,008,952 Issued on 03/07/2006 to Brown, et al.
| Inventors:
|
Brown; William (Montreal, CA);
Griffin; Andrew (Montreal, CA);
Walpole; Christopher (Montreal, CA)
|
| Assignee:
|
Astrazeneca AB (Sodertalje, SE)
|
| Appl. No.:
|
477636 |
| Filed:
|
May 16, 2002 |
| PCT Filed:
|
May 16, 2002
|
| PCT NO:
|
PCT/SE02/00957
|
| 371 Date:
|
November 13, 2003
|
| 102(e) Date:
|
November 13, 2003
|
| PCT PUB.NO.:
|
WO02/094785 |
| PCT PUB. Date:
|
November 28, 2002 |
Foreign Application Priority Data
| Current U.S. Class: |
514/318; 546/194; 546/209; 546/213 |
| Current Intern'l Class: |
A61K 31/55 (20060101); C07D 211/58 (20060101) |
| Field of Search: |
546/194,209,213
514/318
|
References Cited [Referenced By]
U.S. Patent Documents
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| 4126689 | Nov., 1978 | Sanczuk et al.
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| 4460586 | Jul., 1984 | Berthold.
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| 4680296 | Jul., 1987 | Manoury et al.
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| 5118693 | Jun., 1992 | Toth et al.
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| 5132303 | Jul., 1992 | Toth et al.
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| 5132309 | Jul., 1992 | Toth et al.
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| 5854245 | Dec., 1998 | Duggan et al.
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| 6153626 | Nov., 2000 | Pelcman et al.
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| 6399635 | Jun., 2002 | Pelcman et al.
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| 6455545 | Sep., 2002 | Delorme et al.
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| 6552036 | Apr., 2003 | Boyd et al.
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| 6756387 | Jun., 2004 | Brown et al.
| |
| Foreign Patent Documents |
| 28 22 465 | Nov., 1978 | DE.
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| 206 677 | Aug., 1989 | HU.
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| WO 98/2827/0 | Jul., 1998 | WO.
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| WP08/28275 | Jul., 1998 | WO.
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| WO 99/3380/6 | Jul., 1999 | WO.
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| WO 99/4592/5 | Sep., 1999 | WO.
| |
| WO 0146263 | Jun., 2001 | WO.
| |
Other References
Wei et al N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl0benzamide . . . J.
of Med. Chem. 2000, 43, pp. 3895-3905.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; 1995, Wang, et al: "Opioid.delta.receptor
. . . and 4-(methoxymethyl)fentanyl", Database Accession No. 1995:857886.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Deruiter, et al: "Investigation
of the synthesis . . . 1-substituted 4-(propananilido)perhydroazepines", Database
Accession No. 1992:612296.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Ferrand, et al: "Synthesis
of New 1,2,3-triazin-4-ones as Potential Antidepressants", Database Accession No. 1988:150431.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Adachi, et al: "Aminohaloborane
in organic synthesis. IX. Exclusive . . . N-monoaminoalkylanilines", Database Accession
No. 1987:49715.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Takai, et al: " Synthesis
and Pharmacological Evaluation . . . Heterocyclic Rings at the 4-position", Database
Accession No. 1985:578235.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Sugasawa, et al: " 1-Azacycloalkyl-1,
4-Benzodiazepin-2-ones with antianxiety-antidepressant actions", Database Accession
No. 1985:437458.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Zhu, et al: "Studies on Potent
Analgesics. I. Synthesis . . . Derivatives", Database Accession No. 1981:550311.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Burkartsmaier, et al: "Potential
analgesics. IX: Synthesis . . . acid derivatives", Database Accession No. 1979:54780.
Database ChemABS 'Online', CAS, Columbus, Ohio, US; Obase, et al: "New antihypertensive
agents: III Synthesis and . . . at the 4-position", Database Accession No. 1984:120835.
Bilsky, et al., SNC 80, A Selective Nonpeptidic and Systemically Active Opioid
Delta Agonist, J: Pharmacol. Experi. Ther. 273:359-366(1995).
Takemori, et al., "Selective Natrexone-Drived Opioid Receptor Antagonists," Annu.
Rev. Pharmacol. Toxicol. 32:239-269 (1992).
Enein, et al., abstract #159379n, Synthesis of some 4-substituted amino-1-methyl
piperidines structurally related to antihistaminics, Chem. Abstr. 78:396 (1973).
Laskowska, abstract #105299e, "1-methyl-r-[N-phenyl-N-(2-thienyl)amino]piperidine,"
Chem. Abstr. 81:510 (1974).
Podlogar, et al., "Synthesis and evalation of 4-(N,N-diarylamino)piperidines
with high selectivity to the o-opioid receptor: A combined 3D-QSAR and ligand docking
study," Drug Design and Discovery, 34-50 (2000).
Sarges, et al., "Neuroleptic activity of chiral trans-hexahydro-y-carbvolines,"
J. Med. Chem. 29:8-19 (1986).
|
Primary Examiner: Desai; Rita
Attorney, Agent or Firm: Shen; David, Legaard; Paul K.
Claims
The invention claimed is:
1. A compound of the formula I:
##STR29##
or a salt thereof, wherein:
R
1 is selected from any one of
##STR30##
wherein each said phenyl ring, pyridinyl, thienyl, furanyl, imidazolyl,
triazolyl, pyrrolyl, thiazolyl or pyridyl-N-oxide is optionally substituted by
1, 2 or 3 substituents independently selected from straight and branched C
1-C
6
alkyl, NO
2, CF
3, C
1-C
6 alkoxy, chloro,
fluoro, bromo, and iodo.
2. A compound according to claim 1, wherein each said phenyl ring, pyridinyl,
thienyl, furanyl, imidazolyl, triazolyl, pyrrolyl, thiazolyl or pyridyl-N-oxide
is optionally substituted by 1, 2 or 3 substituents independently selected from
methyl, CF
3, chloro, fluoro, bromo, and iodo.
3. A compound according to claim 1, wherein each said phenyl ring, pyridinyl,
thienyl, furanyl, imidazolyl, triazolyl, pyrrolyl, thiazolyl or pyridyl-N-oxide
is optionally substituted by a methyl group.
4. A compound according to claim 1, wherein R
1 is pyridinyl, pyrrolyl,
thienyl or furanyl.
5. A compound according to claim 1, selected from any one of 3-[(4-diisopropyl-carbamoyl-phenyl)-(1-furan-3-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3[(1-benzyl-piperidin-4-yl)-(4-diisopropyl-carbamoyl-phenyl)-amino]-4-fluoro-benzamide,
[(4-diisopropyl-carbamoyl-phenyl)-(1-thiazo-2-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3-[(4-diisopropyl-carbamoyl-phenyl)-(1-pyridin-2-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3-[(4-diisopropyl-carbamoyl-phenyl)-(1-thiophen-3-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3-[(4-diisopropyl-carbamoyl-phenyl)-(1-pyridin-4-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3-[(4-diisopropyl-carbamoyl-phenyl)-(1-pyridin-3-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3-[(4-diisopropyl-carbamoyl-phenyl)-(1-thiophen-2-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
3-[(4-diisopropyl-carbamoyl-phenyl)-(1-furan-2-ylmethyl-piperidinyl)amino]-4-fluoro-benzamide, and
3-[(4-Diisopropyl-carbamoyl-phenyl)-(1-pyrrol-2-ylmethyl-piperidin-4-yl)-amino]-4-fluoro-benzamide,
or a salt thereof.
6. A compound according to any one of the preceding claims, wherein said compound
is in form of a hydrochloride, dihydrochloride, sulfate, tartrate, ditrifluoroacetate
or citrate salt.
7. A process for preparing a compound of formula I,
##STR31##
or a salt thereof, wherein:
R
1 is selected from any one of phenyl, pyridinyl, thienyl, furanyl,
imidazolyl, triazolyl, pyrrolyl, thiazolyl or pyridyl-N-oxide, and wherein each
said phenyl, pyridinyl, thienyl, furanyl, imidazolyl, triazolyl, pyrrolyl, thiazolyl
or pyridyl-N-oxide, which is optionally substituted by 1, 2 or 3 substituents independently
selected from strait and branched C
1-C
6 alkyl, NO
2,
CF
3, C
1-C
6 alkoxy, chloro, fluoro, bromo, and iodo;
comprising reacting a compound of the general formula II
##STR32##
wherein PG is selected from Boc, CBZ, benzyl and 2, 4-dimethoxybenzyl,
with N, N-diethyl-4-bromobenzamide, using a palladium catalyst, in the presence
of a base, to give a compound of general formula III,
##STR33##
which is thereafter deprotected and the deprotected product is alkylated
with a compound of the formula of R
1-CHO and hydrolyzed to form a compound
of formula I, or a salt thereof.
8. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically
acceptable carrier.
9. A method of treating pain in a subject comprising administering to said subject
an effective amount of a compound of claim 1.
10. A method of treating anxiety in a subject comprising administering to said
subject an effective amount of a compound of claim 1.
11. A compound of the general formula III
##STR34##
or a salt thereof, wherein PG is selected from Boc, CBZ, benzyl and 2,4-dimethoxybenzyl.
12. A compound of the general formula X
##STR35##
or a salt thereof, wherein R
1 is selected from any one of phenyl,
pyridinyl, thienyl, furanyl, imidazolyl, triazolyl, pyrrolyl, thiazolyl or pyridyl-N-oxide,
and wherein each said phenyl, pyridinyl, thienyl, furanyl, imidazolyl, triazolyl,
pyrrolyl, thiazolyl or pyridyl-N-oxide is optionally substituted by 1, 2 or 3 substituents
independently selected from straight and branched C
1-C
6 alkyl,
NO
2, CF
3, C
1-C
6 alkoxy, chloro, fluoro,
bromo, and iodo.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a US National Stage of International Application No. PCT/SE02/00957
that was filed on May 16, 2002. The International Application claims priority under
35 U.S.C. § 119(a) to Swedish Application No. 0101773-0 filed May 18, 2001.
FIELD OF THE INVENTION
The present invention is directed to novel compounds, to a process for their
preparation, their use and pharmaceutical compositions comprising the novel compounds.
The novel compounds are useful in therapy, and in particular for the treatment
of pain, anxiety, and functional gastrointestinal disorders.
BACKGROUND OF THE INVENTION
The δ receptor has been identified as having a role in many bodily functions
such as circulatory and pain systems. Ligands for the δ receptor may therefore
find potential use as analgesics, and/or as antihypertensive agents. Ligands for
the δ receptor have also been shown to possess immunomodulatory activities.
The identification of at least three different populations of opioid receptors
(μ, δ and κ) is now well established and all three are apparent
in both central and peripheral nervous systems of many species including man. Analgesia
has been observed in various animal models when one or more of these receptors
has been activated.
With few exceptions, currently available selective opioid δ ligands are
peptidic in nature and are unsuitable for administration by systemic routes. One
example of a non-peptidic δ-agonist is SNC80 (
Bilsky E. J. et al., Journal
of Pharmacology and Experimental Therapeutics, 273(1), pp. 359-366 (1995)).
There is however still a need for selective δ-agonists having not only improved
selectivity, but also an improved side-effect profile.
Thus, the problem underlying the present invention was to find new analgesics
having improved analgesic effects, but also with an improved side-effect profile
over current μ agonists, as well as having improved systemic efficacy.
Analgesics that have been identified and are existing in the prior art
have many disadvantages in that they suffer from poor pharmacokinetics and are
not analgesic when administered by systemic routes. Also, it has been documented
that preferred δ agonist compounds, described within the prior art, show
significant convulsive effects when administered systemically.
We have now found certain compounds that exhibit surprisingly improved properties,
i.a. improved δ-agonist potency, in vivo potency, pharmacokinetic, bioavailability,
in vitro stability and/or lower toxicity.
OUTLINE OF THE INVENTION
The novel compounds according to the present invention are defined by the formula
I
##STR1##
wherein
R1 is selected from any one of
##STR2##
where each R1 phenyl ring and R1 heteroaromatic ring
may optionally and independently be further substituted by 1, 2 or 3 substituents
independently selected from straight and branched C1-C6 alkyl,
NO2, CF3, C1-C6 alkoxy, chloro, fluoro,
bromo, and iodo. The substitutions on the phenyl ring and on the heteroaromatic
ring may take place in any position on said ring systems;
When the R
1 phenyl ring and the R
1 heteroaromatic ring(s)
are substituted, the preferred substituents are independently selected from anyone
of CF
3, methyl, iodo, bromo, fluoro and chloro.
Another embodiment of the present invention is a compound according to figure
I wherein R
1 is as defined above and each R
1 phenyl ring
and R
1 heteroaromatic ring may independently be further substituted
by a methyl group
A further embodiment of the present invention is a compound according to figure
I wherein R
1 is phenyl, pyrrolyl, pyridinyl, thienyl or furanyl, optionally
with 1 or 2 of the preferred substituents on the R
1 phenyl or R
1
heteroaromatic ring.
A further embodiment of the present invention is a compound according to figure
I wherein R
1 is phenyl, pyrrolyl or pyridinyl, optionally with 1 or
2 of the preferred substituents on the R
1 phenyl or R
1 heteroaromatic ring.
Another embodiment of the present invention is a compound according to figure
I wherein R
1 is thienyl or furanyl, optionally with 1 or 2 of the preferred
substituents on the R
1 heteroaromatic ring.
Within the scope of the invention are also salts and enantiomners of the compounds
of the formula I, including salts of enantiomers.
Reaction step b in Scheme 1, vide infra, is performed by reacting an intermediate
compound of the general formula II
##STR3##
wherein PG is a urethane protecting group, such as Boc and CBZ, or benzyl
or substituted benzyl protecting group, such as 2,4-dimethoxybenzyl; with N,N-diisopropyl-4-bromobenzamide,
using a palladium catalyst, e.g. tris(dibenzylideneacetone) dipalladium(0) (Pd
2(dba)
3),
in the presence of a base, e.g. tert-BuONa and a phosphine ligand such as bis-diphenylphosphanyl-dimethyl-9H-xanthene
(xantphos), to give the compounds of general formula III,
##STR4##
which is thereafter deprotected, under standard conditions hydrolysed under
basic conditions and alkylated using either:
i) a compound of the general formula R1—CH2—X,
wherein R1 is as defined above and X is a halogen, preferably bromine
or chlorine and a suitable base, or
ii) a compound of the general formula R1—CHO, wherein R1
is as defined above, and a suitable reducing agent,
to give compounds of the general formula I.
Suitable bases to be used in the standard alkylation step i) above include,
but are not limited to, triethylamine and potassium carbonate.
Suitable reducing agents to be used in the standard reduction step ii) include,
but are not limited to, sodium cyanoborohydride and sodium triacetoxyborohydride.
The novel compounds of the present invention are useful in therapy, especially
for the treatment of various pain conditions such as chronic pain, neuropathic
pain, acute pain, cancer pain, pain caused by rheumatoid arthritis, migraine, visceral
pain etc. This list should however not be interpreted as exhaustive.
Compounds of the invention are useful as immunomodulators, especially for
autoimmune diseases, such as arthritis, for skin grafts, organ transplants and
similar surgical needs, for collagen diseases, various allergies, for use as anti-tumour
agents and anti-viral agents.
Compounds of the invention are useful in disease states where degeneration
or dysfunction of opioid receptors is present or implicated in that paradigm. This
may involve the use of isotopically labeled versions of the compounds of the invention
in diagnostic techniques and imaging applications such as positron emission tomography (PET).
Compounds of the invention are useful for the treatment of diarrhoea, depression,
anxiety and stress-related disorders such as post-traumatic stress disorders, panic
disorder, generalized anxiety disorder, social phobia, and obesessive compulsive
disorder; urinary incontinence, various mental illnesses, cough, lung oedema, various
gastro-intestinal disorders, e.g. constipation, functional gastrointestinal disorders
such as Irritable Bowel Syndrome and Functional Dyspepsia, Parkinson's disease
and other motor disorders, traumatic brain injury, stroke, cardioprotection following
miocardial infarction, spinal injury and drug addiction, including the treatment
of alcohol, nicotine, opioid and other drug abuse and for disorders of the sympathetic
nervous system for example hypertension.
Compounds of the invention are useful for the treatment of diarrhoea, depression,
anxiety, urinary incontinence, various mental illnesses, cough, lung oedema, various
gastro-intestinal disorders, spinal injury and drug addiction, including the treatment
of alcohol, nicotine, opioid and other drug abuse and for disorders of the sympathetic
nervous system for example hypertension.
Compounds of the invention are useful as an analgesic agent for use during
general anaesthesia and monitored anaesthesia care. Combinations of agents with
different properties are often used to achieve a balance of effects needed to maintain
the anaesthetic state (eg. amnesia, analgesia, muscle relaxation and sedation).
Included in this combination are inhaled anaesthetics, hypnotics, anxiolytics,
neuromuscular blockers and opioids. Also within the scope of the invention is the
use of any of the compounds according to the formula I above, for the manufacture
of a medicament for the treatment of any of the conditions discussed above.
A further aspect of the invention is a method for the treatment of a subject
suffering
from any of the conditions discussed above, whereby an effective amount of a compound
according to the formula I above, is administered to a patient in need of such treatment.
A further aspect of the present invention is intermediates of the general formula
II and III,
##STR5##
wherein PG is a urethane protecting group, such as Boc and CBZ or benzyl
or substituted benzyl protecting group, such as 2,4-dimethoxybenzyl.
A further aspect of the present invention is intermediates of Formula X;
##STR6##
wherein R
1 is as described above in relation to Formula I.
In another alternative synthetic route, reaction step b in Scheme 2, vide infra,
is performed by reacting an intermediate compound of the general formula IV
##STR7##
wherein PG is a urethane protecting group, such as Boc and CBZ or benzyl
or substituted benzyl protecting group, such as 2,4-dimethoxybenzyl, with N,N-diisopropyl-4-bromobenzamide,
using a palladium catalyst, e.g. tris(dibenzylideneacetone) dipalladium(0) [Pd
2(dba)
3],
in the presence of a base, e.g. tert-BuONa and a phosphine ligand such as bis-diphenylphosphanyl-dimethyl-9H-xanthene
(xantphos), to give the compounds of general formula V,
##STR8##
which is thereafter deprotected and alkylated by means described above either
reductively with a compound of the general formula R
1—CHO, of
directly, using a compound of general formula R
1—CH
2—X,
followed by conversion of the ketal functionality to a primary amide under standard
conditions via i) hydrolysis of the ketal to the aldehyde (VI), followed by ii)
oxidation of the aldehyde to the corresponding carboxylic acid (VII), followed
by iii) amidation with ammonium chloride to the primary amide) to give compounds
of the general formula I.
Suitable hydrolysis conditions to be used in the standard hydrolysis step
(i) include, but are not limited to aqueous hydrochloric acid in tetrahydrofuran.
##STR9##
Suitable conditions for the oxidation step (ii) include, but are not limited
to stirring at 0° C. in aqueous sodium dihydrogen phosphate and sodium chlorite
in the presence of excess 2-methyl-2-butene.
##STR10##
Suitable conditions for the amidation step (iii) include but are not limited
to treatment with excess ammonium chloride in the presence of a coupling agent
such as benzotriazole-1-yloxy-trisphosphonium hexafluorophospiate (hereinafter
Py-BOP) and 1-hydroxybenzotriazole (HOBT) in the presence of an acid scavenger
such as diisopropylethylamine (DIPEA).
##STR11##
METHODS OF PREPARATION
EXAMPLES
The invention will now be described in more detail by the following Schemes and
Examples, which are not to be construed as limiting the invention.
##STR12##
Intermediate 1: 3-(1-Benzyl-piperidin-4-ylamino)-4-fluoro-benzonitrile.
To a dry flask containing 3-bromo-4-fluoro benzonitrile (6.56 g, 1 eq) was added
dry toluene (100 mL), 4-amino-1-benzyl piperidine (1.2 eq), racemic BINAP (612
mg, 0.03 eq), palladium acetate (220 mg, 0.02 eq) and sodium tert-butoxide (4.4
g, 1.4 eq). The reaction was heated to 80° C. under an inert atmosphere for
20 hours, then cooled, diluted with ethyl acetate (200 mL) and washed with water
(100 mL). The combined organics were dried (MgSO
4), filtered and concentrated.
Purification by flash chromatography, eluting 3% methanol in dichloromethane yielded
amine (1) as a yellow solid (6.655 g, 66%).
Intermediate 2: [(1-Benzyl-piperidin-4-yl)-(5-cyano-2-fluoro-phenyl)-amino]-N,N-diisopropyl-benzamide.
To a solution of amine (1) (3.625 g, 1 eq.) in dry toluene (50 mL) was added
aryl
bromide (5.03 g, 1.5 eq), xantphos (546 mg, 0.08 eq), Pd
2(dba)
3
(433 mg, 0.04 eq) and sodium tert-butoxide (1.70 g, 1.4 eq). The reaction was heated
to reflux for 14 hours then was cooled and diluted with ethyl acetate (100 mL).
The solution was washed with water (100 mL) then the organics were dried (MgSO
4),
filtered and concentrated. Purification by flash chromatography, eluting 50% ethyl
acetate 50% hexanes yielded amine (2) as an orange foam (5.16 g, 85%).
Intermediate 3: [(5-Cyano-2-fluoro-phenyl)-piperidin-4-yl-amino]-N,N-diisopropyl-benzamide.
To a solution of amine (2) (5.16 g, 1 eq) in 1,2-dichloroethane (80 mL) at 0°
C. was added 1-chloroethyl chloroformate (1.25 mL, 1.15 eq). After 15 minutes at
0° C. the reaction was warmed to room temperature then was heated to 70°
C. for 90 minutes. The reaction was then cooled and concentrated. The residue was
dissolved in methanol (80 mL) then heated to 70° C. for 1 hour. The solution
was then cooled and concentrated. Purification by flash chromatography, eluting
20% methanol in dichloromethane, rising to 40% methanol in dichloromethane gave
amine (3) as a yellow foam (3.301 g, 76%).
##STR13##
##STR14##
Intermediate 4: (1-Benzyl-piperidin-4-yl)-(5-[1,3]dioxolan-2-yl-2-fluoro-phenyl)-amine.
To a dry flask containing 2-(3-bromo-4-fluorophenyl)-1,3-dioxaolane (1.0 eq)
and
4-amino-N-benzylpiperidine, (1.2 eq) in dry toluene is added BINAP (0.03 eq), palladium
acetate (0.02 eq) and sodium tert-butoxide (1.4 eq). The reaction is heated to
80° C. under nitrogen. After 2 hours the solution is cooled, diluted with
ethyl acetate and washed with one portion water. The organics are dried over anhydrous
magnesium sulfate, filtered and concentrated. The residue is purified by flash
chromatography, eluting with a methanol in dichloromethane gradient.
Intermediate 5: [(1-Benzyl-piperidin-4-yl)-(5-[1,3]dioxolan-2-yl-2-fluoro-phenyl)-amino]-N,N-diisopropyl-benzamide.
To a dry flask containing amine 4 in dry toluene (about 6 mL per millimole of
4) is added aryl bromide (1.4 eq), xantphos (0.06 eq), Pd
2(dba)
3
(0.03 eq) and sodium tert-butoxide (1.4 eq). The reaction is heated to 110°
C. under nitrogen. After about 24 hours the solution is cooled, diluted with ethyl
acetate and washed with one portion water. The organics are dried over anhydrous
magnesium sulfate, filtered and concentrated. The residue is purified by flash
chromatography, eluting with a methanol in dichloromethane gradient.
Intermediate 6: [(1-Benzyl-piperidin-4-yl)-(2-fluoro-5-formyl-phenyl)-amino]-N,N-diisopropyl-benzamide.
To a solution of acetal 5 in tetrahydrofuran is added 2N HCl solution (2.0 eq).
After 16 hours at room temperature, dichloromethane is added and the aqueous layer
is neutralized with aqueous saturated sodium bicarbonate solution. The organic
layer is removed and the aqueous layer extracted with two portions of dichloromethane.
The combined organic extracts are dried (MgSO
4), filtered and concentrated
and the residue is purified by flash chromatography, eluting with ethyl acetate.
Intermediate 7: [(1-Benzyl-piperidin-4-yl)-(4-diisopropylcarbamoyl-phenyl)-amino]-fluoro-benzoic acid.
To a solution of aldehyde 6 (1.0 eq) in tert-butanol is added 2-methyl-2-butene
(10.0 eq) and the solution is cooled to 0° C. A solution of sodium dihydrogen
phosphate (9 eq) and sodium chlorite (9 eq) in water is added and the reaction
is stirred for 30 minutes at 0° C. The tert-butanol is removed and the reaction
mixture is extracted with dichloromethane (5×). The combined organic extracts
are dried (MgSO
4), filtered and concentrated and the residue is purified
by flash chromatography, eluting with a methanol/dichloromethane gradient.
Example 2
[(1-Benzyl-piperidin-4-yl)-(4-diisopropylcarbamoyl-phenyl)-amino]-fluoro-benzoic acid.
To a solution of acid 7 (1.0 eq) in DMF is added pyBOP (1.5 eq); HOBt (1.5 eq),
diisopropylethylamine (4.0 eq) and ammonium chloride (2 eq). After 16-24 hours
at room temperature the reaction is concentrated. The residue is dissolved in ethyl
acetate and is washed with two portions of water and one portion of saturated sodium
bicarbonate solution. The organic layer is dried (MgSO
4), filtered and
concentrated and the residue is purified by flash chromatography, eluting with
a methanol in dichloromethane gradient. Additional examples were synthesized via
the general procedures described below.
A. Reductive Amination of Intermediate 3:
##STR15##
To a solution of the amine, 3, in dry tetrahydrofuran (THF) or dichloroethane
is added the aldehyde (1-1.5 eq.), followed by sodium triacetoxy borohydride (1-1.6
eq.). The reaction is stirred at room temperature under a nitrogen atmosphere for
an extended period of time (6-48 hours) to ensure complete reaction. The reaction
mixture is then subjected to a standard work-up procedure and a standard purification.
The amount of THF (or dichloroethane) is not crucial. An amount corresponding to
about 1 mL/30 mg is preferred.
Procedure 1A in the synthesis of Example 1 below is typical.
B. Hydrolysis of the Intermediate Cyano Compound:
##STR16##
To a solution of the cyano intermediate in tert-butanol, is added ground potassium
hydroxide (KOH) (2.5 eq.) and the resulting mixture is heated to reflux for about
two hours. The mixture was then cooled to room temperature and subjected to a standard
work-up procedure and a standard purification. The amount of tert-butanol is not
crucial. An amount corresponding to about 1 mL/30 mg is preferred.
Procedure 1B in the synthesis of Example 1 below is typical.
Example 1
[(4-Diisopropylcarbamoyl-phenyl)-(1-furan-3-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide.
##STR17##
1A: [(5-Cyano-2-fluoro-phenyl)-(1-furan-3-ylmethyl-piperidin-4-yl)-amino]-N,N-diisopropyl-benzamide
To a solution of amine (406 mg) in dry 1,2-dichloroethane (12 mL) was added 3-furaldehyde
(110 μL; 1.3 eq) and sodium triacetoxyborohydride (286 mg; 1.4 eq). The reaction
was stirred at room temperature under nitrogen. After 24 hours the reaction was
diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate.
The aqueous was extracted with two portions dichloromethane and the combined organics
were dried over anhydrous magnesium sulfate, filtered and concentrated. The residue
was purified by flash chromatography eluting 50% ethyl acetate 50% hexanes, rising
to 90% ethyl acetate 10% hexanes. A colorless foam was obtained (347.2 mg; 72%).
##STR18##
1B: [(4-Diisopropylcarbamoyl-phenyl)-(1-furan-3-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide
To a solution of nitrile (347.2 mg) in tert-butanol (8 mL) was added ground potassium
hydroxide (97 mg; 2.5 eq) and the reaction was heated to reflux. After 90 minutes
the solution was cooled, diluted with dichloromethane and washed with one portion
water. The organics was removed and the aqueous neutralized and extracted with
two portions dichloromethane. The combined organics were dried over anhydrous magnesium
sulfate, filtered and concentrated. The residue was purified by flash chromatography,
eluting with 7% methanol in dichloromethane. A colorless foam was obtained (264.1
mg; 73%). The foam was dissolved in ether (8 mL) containing some dichloromethane
and 1N HCl/ether (760 μL; ≅1.5 eq) was added After 1 hour the solution
was concentrated and the solid dried under high vacuum.
Additional Examples are prepared analogously. Analytical data for synthetic
Examples is shown in Table 1 on the following pages.
| TABLE 1 |
|
| Analytical data for synthetic Examples. |
| Ex. |
R1 |
Name |
NMR data (400 MHz, CD3OD) |
|
| 1 |
##STR19##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-furan-3-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
1.40 (br s, 12H, CH3); 1.75-1.82 (m, 2H, CH2);2.39 (d,
J = 14Hz, 2H, CH2); 3.27-3.31 (m, 2H,NCH2); 3.63 (d, J =
13Hz, 2H, NCH2); 3.88 (brs, 2H, NCH); 4.26 (s, 2H, NCH2Ar); 4.46-4.52
(m, 1H, NCH); 6.65 (d, J = 1.5Hz, 1H, Ar-H); 6.76 (d, J = 9Hz, 2H, Ar-H); 7.23-7.28
(m,2H, Ar-H); 7.46 (t, J = 9Hz, 1H, Ar-H); 7.68 (t,J = 1.5Hz, 1H, Ar-H); 7.83 (s,
1H, Ar-H); 7.89(dd, J = 2.5, 7Hz, 1H, Ar-H); 8.05-8.08 (m, 1H,Ar-H). |
| |
| 2 |
##STR20##
|
[(1-Benzyl-piperidin-4-yl)-(4-diisopropyl-carbamoyl-phenyl)-amino]-fluoro-benzamide |
1.30 (br s, 12H, CH3); 1.64-1.73 (m, 2H, CH2);2.30 (d,
J = 12Hz, 2H, CH2); 3.24 (t, J = 12Hz,2H, NCH2); 3.51 (d,
J = 13Hz, 2H, NCH2); 3.80(br s, 2H, NCH); 4.28 (s, 2H, NCH2Ar);
4.30-4.44 (m, 1H, NCH); 6.67 (d, J = 8.5Hz, 2H, Ar-H); 7.16-7.18 (m, 2H, Ar-H);
7.38 (t, J = 9Hz,1H, Ar-H); 7.46 (s, 5H, Ar-H); 7.80 (dd, J = 2, 7.5Hz, 1H, Ar-H);
7.96-8.00 (m, 1H, Ar-H). |
| |
| 3 |
##STR21##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-thiazol-2-ylmethyl-piperidin-4-yl) amino]-fluoro-benzamide |
1.36 (br s, 12H, CH3); 1.82-1.84 (m, 2H, CH2);2.41 (d,
J = 14Hz, 2H, CH2); 3.44 (t, J = 12Hz,2H, NCH2); 3.78 (d,
J = 12Hz, 2H, NCH2); 3.90(br s, 2H, NCH); 4.47-4.53 (m, 1H, NCH);4.78
(s, 2H, NCH2Ar); 6.75 (d, J = 8.5Hz, 2H,Ar-H); 7.21-7.24 (m, 2H, Ar-H);
7.45-7.48 (m,1H, Ar-H); 7.84 (d, J = 3.5Hz, 1H, Ar-H); 7.90(dd, J = 2.5, 7.5Hz,
1H, Ar-H); 8.00 (d,J = 3.5Hz, 1H, Ar-H); 8.05-8.08 (m, 1H, Ar-H). |
| |
| 4 |
##STR22##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-pyridin-2-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
1.41 (br s, 12H, CH3); 1.89-1.96 (m, 2H, CH2);2.39 (d,
J = 14Hz, 2H, CH2); 3.47-3.52 (m, 2H,NCH2); 3.71 (d, J =
12.5Hz, 2H, NCH2); 3.92(br s, 2H, NCH); 4.54-4.57 (m, 1H, NCH)4.60 (s,
2H, NCH2Ar); 6.79 (d, J = 8.5Hz, 2H,Ar-H); 7.27-7.29 (m, 2H, Ar-H);
7.48 (t,J = 9Hz, .1H, Ar-H); 7.62-7.64 (m, 1H, Ar-H);7.71 (d,. J = 8Hz, 1H, Ar-H);
7.92 (dd, J = 2,7.5Hz, 1H, Ar-H); 8.08-8.11 (m, 2H, Ar-H);8.76-8.78 (m, 1H, Ar-H). |
| |
| 5 |
##STR23##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-thiophen-3-yl)-amino]-fluoro-benzamide |
1.36 (br s, 12H, CH3); 1.74-1.81 (m, 2H, CH2);2.38 (d,
J = 14Hz, 2H, CH2); 3.30-3.36 (m, 2H,NCH2); 3.59 (d, J =
12Hz, 2H, HCH2); 3.86 (brs, 2H, NCH); 4.39 (s, 2H, NCH3Ar);
4.46-4.51(m, 1H, NCH); 6.75 (d, J = 9Hz, 2H, Ar-H);7.22-7.28 (m, 3H, Ar-H); 7.46
(t, J = 9Hz, 1H,Ar-H); 7.63 (dd, J = 2, 5Hz, 1H, Ar-H); 7.73-7.74 (m, 1H, Ar-H);
7.88 (dd, J = 2.5, 7.5Hz,1H, Ar-H); 8.04-8.07 (m, 1H, Ar-H). |
| |
| 6 |
##STR24##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-pyridin-4-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
1.41 (br s, 12H, CH3); 1.99 (q, J = 12Hz, 2H,CH2); 2.41
(d, J = 14Hz, 2H, CH2); 3.51 (t,J = 12Hz, 2H, NCH2); 3.69
(d, J = 11.5Hz, 2H,NCH2); 3.90 (br s, 2H, NCH); 4.54-4.58 (m,1H, NCH);
4.76 (s, 2H, NCH2Ar); 6.78 (d,J = 8Hz, 2H, Ar-H); 7.27 (d, J = 8.5Hz,
2H, ArH); 7.48 (t, J = 9Hz, 1H, Ar-H); 7.92 (d,J = 5.5Hz, 1H, Ar-H); 8.07-8.09
(m, 1H, Ar-H); 8.38 (d, J = 6.5Hz, 2H, Ar-H); 9.06 (d,J = 6.5Hz, 2H, Ar-H). |
| |
| 7 |
##STR25##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-pyridin-3-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
1.38 (br s, 12H, CH3); 1.92-1.98 (m, 2H,CH2); 2.41 (d,
J = 13.5Hz, 2H, CH2); 3.49 (t,J = 12Hz, 2H, NCH2); 3.71 (d,
J = 12Hz, 2H,NCH2); 3.88 (br s, 2H, NCH); 4.54-4.58 (m,1H, NCH); 4.70
(s, 2H, NCH2Ar); 6.79 (d,J = 8.5Hz, 2H, Ar-H); 7.27 (d, J = 8.5Hz, 2H,Ar-H);
7.47 (t, J = 9Hz, 1H, Ar-H); 7.91 (d,J = 5.5Hz, 1H, Ar-H); 8.06-8.09 (m, 1H, Ar-H);
8.24 (dd, J = 6.8Hz, 1H, Ar-H); 8.88 (d,J = 8Hz, 1H, Ar-H); 9.06 (d, J = 6Hz, 1H,
Ar-H); 9.23 (s, 1H, Ar-H). |
| |
| 8 |
##STR26##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-thiophen-2-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
1.41 (br s, 12H, CH3); 1.77-1.81 (m, 2H,CH2); 2.42 (d,
J = 14Hz, 2H, CH2); 3.33-3.38(m, 2H, NCH2); 3.66 (d, J =
12.5Hz, 2H,NCH2); 3.90 (br s, 2H, NCH); 4.49-4.54 (m,1H, NCH); 4.64
(s, 2H, NCH2Ar); 6.78 (d,J = 9Hz, 2H, Ar-H); 7.23 (dd, J = 3.5, 5Hz,
1H,Ar-H); 7.27 (d, J = 9Hz, 2H, Ar-H); 7.41-7.42(m, 1H, Ar-H); 7.48 (t, J = 9Hz,
Ar-H);7.71 (dd, J = 1, 5Hz, 1H, Ar-H); 7.92 (dd, J = 2,7.5Hz, 1H, Ar-H); 8.07-8.10
(m, 1H, Ar-H). |
| |
| 9 |
##STR27##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-furan-2-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
1.42 (br s, 12H, CH3); 1.75-1.83 (m, 2H,CH2); 2.42 (d,
J = 14Hz, 2H, CH2); 3.33-3.38(m, 2H, NCH2); 3.63 (d, J =
12.5Hz, 2H,NCH2); 3.85 (br s, 2H, NCH); 4.47 (s, 2H,NCH2Ar);
4.48-4.52 (m, 1H, NCH); 6.61(d, J = 2, 3.5Hz, 1H, Ar-H); 6.79 (d, J = 9Hz,Ar-H);
6.80 (d, 33Hz, LR, Ar-H); 7.27(d, J = 9Hz, 2H, Ar-H); 7.47-7.50 (m, 1H, Ar-H);
7.74 (dd, J = 1, 2Hz, 1H, Ar-H); 7.91 (dd,J = 2, 7.5Hz, 1H, Ar-H); 8.07-8.10 (m,
1H, Ar-H). |
| |
| 10 |
##STR28##
|
[(4-Diisopropyl-carbamoyl-phenyl)-(1-pyrrol-2-ylmethyl-piperidin-4-yl)-amino]-fluoro-benzamide |
|
Pharmaceutical Compositions
The novel compounds according to the present invention may be administered orally,
sublingually, intramuscularly, subcutaneously, topically, intranasally, intraperitoneally,
intrathoracially, intravenously, epidurally, intrathecally, intracerebroventricularly
and by injection into the joints.
A Preferred Route of Administration is Orally, Intravenously or Intramuscularly.
The dosage will depend on the route of administration, the severity of the disease,
age and weight of the patient and other factors normally considered by the attending
physician, when is determining the individual regimen and dosage level as the most
appropriate for a particular patient.
For preparing pharmaceutical compositions from the compounds of this invention,
inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid
form preparations include powders, tablets, dispersible granules, capsules, cachets,
and suppositories.
A solid carrier can be one or more substances which may also act as diluents,
flavoring
agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating
agents; it can also be an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with
the
finely divided active component. In tablets, the active component is mixed with
the carrier having the necessary binding properties in suitable proportions and
compacted in the shape and size desired.
For preparing suppository compositions, a low-melting wax such as a mixture of
fatty acid glycerides and cocoa butter is first melted and the active ingredient
is dispersed therein by, for example, stirring. The molten homogeneous mixture
is then poured into convenient sized molds and allowed to cool and solidify.
Suitable carriers are magnesium carbonate, magnesium stearate, talc, lactose,
sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl
cellulose, a low-melting wax, cocoa butter, and the like.
Salts include, but are not limited to, pharmaceutically acceptable salts. Examples
of pharmaceutically acceptable salts within the scope of the present invention
include: acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,
calcium acetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate,
edisylate, estolate, esylate, fumarate, glucaptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,
isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate,
phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate,
sulfate, tannate, tartrate, teoclate, triethiodide, and benzathine.
Examples of pharmaceutically unacceptable salts within the scope of the
present invention include: hydroiodide, perchlorate, tetrafluoroborate. Pharmaceutically
unacceptable salts could be of use because of their advantageous physical and/or
chemical properties, such as crystallinity.
Preferred pharmaceutically acceptable salts are hydrochlorides, sulfates
and bitartrates. The hydrochloride and sulfate salts are particularly preferred
The term composition is intended to include the formulation of the active component
with encapsulating material as a carrier providing a capsule in which the active
component (with or without other carriers) is surrounded by a carrier which is
thus in association with it. Similarly, cachets are included.
Tablets, powders, cachets, and capsules can be used as solid dosage forms
suitable for oral administration.
Liquid from compositions include solutions, suspensions, and emulsions. Sterile
water or water-propylene glycol solutions of the active compounds may be mentioned
as an example of liquid preparations suitable for parenteral administration. Liquid
compositions can also be formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions for oral administration can be prepared by dissolving the
active component in water and adding suitable colorants, flavoring agents, stabilizers,
and thickening agents as desired. Aqueous suspensions for oral use can be made
by dispersing the finely divided active component in water together with a viscous
material such as natural synthetic gums, resins; methyl cellulose, sodium carboxymethyl
cellulose, and other suspending agents known to the pharmaceutical formulation art.
Preferably the pharmaceutical compositions is in unit dosage form. In
such form, the composition is divided into unit doses containing appropriate quantities
of the active component. The unit dosage form can be a packaged preparation, the
package containing discrete quantities of the preparations, for example, packeted
tablets, capsules, and powders in vials or ampoules. The unit dosage form can also
be a capsule, cachet, or tablet itself, or it can be the appropriate number of
any of these packaged forms.
Biological Evaluation
In Vitro Model
Cell Culture
A. Human 293S cells expressing cloned human μ, δ, and κ
receptors and neomycin resistance were grown in suspension at 37° C. and 5%
CO2 in shaker flasks containing calcium-free DMEM10% FBS, 5% BCS, 0.1%
Pluronic F-68, and 600 μg/ml geneticin.
B. Mouse and rat brains were weighed and rinsed in ice-cold PBS (containing
2.5 mM EDTA, pH 7.4). The brains were homogenized with a polytron for 15 sec (mouse)
or 30 sec (rat) in ice-cold lysis buffer (50 mM Tris, pH 7.0, 2.5 mM EDTA, with
phenylmethylsulfonyl fluoride added just prior use to 0.5 MmM from a 0.5 M stock
in DMSO:ethanol).
Membrane Preparation
Cells were pelleted and resuspended in lysis buffer (50 mM Tris, pH 7.0, 2.5
mM EDTA, with PMSF added just prior to use to 0.1 mM from a 0.1 M stock in ethanol),
incubated on ice for 15 min, then homogenized with a polytron for 30 sec. The suspension
was spun at 1000 g (max) for 10 min at 4° C. The supernatant was saved on
ice and the pellets resuspended and spun as before. The supernatants from both
spins were combined and spun at 46,000 g (max) for 30 min. The pellets were resuspended
in cold Tris buffer (50 nM Tris/Cl, pH 7.0) and spun again. The final pellets were
resuspended in membrane buffer (50 mM Tris, 0.32 M sucrose, pH 7.0). Aliquots (1
ml) in polypropylene tubes were frozen in dry ice/ethanol and stored at -70°
C. until use. The protein concentrations were determined by a modified Lowry assay
with SDS.
Binding Assays
Membranes were thawed at 37° C., cooled on ice, passed 3 times through
a 25-gauge needle, and diluted into binding buffer (50 mM Tris, 3 mM MgCl
2,
1 mg/ml BSA (Sigma A-7888), pH 7.4, which was stored at 4° C. after filtration
through a 0.22 m filter, and to which had been freshly added 5 μg/ml aprotinin,
10 μM bestatin, 10 μM diprotin A, no DTT). Aliquots of 100 μl
were added to iced 12×75 mm polypropylene tubes containing 100 μl of
the appropriate radioligand and 100 μl of test compound at various concentrations.
Total (TB) and nonspecific (NS) binding were determined in the absence and presence
of 10 μM naloxone respectively. The tubes were vortexed and incubated at
25° C. for 60-75 min, after which time the contents are rapidly vacuum-filtered
and washed with about 12 ml/tube iced wash buffer (50 mM Tris, pH 7.0, 3 mM MgCl
2)
through GF/B filters (Whatman) presoaked for at least 2 h in 0.1% polyethyleneimine.
The radioactivity (dpm) retained on the filters was measured with a beta counter
after soaking the filters for at least 12 h in minivials containing 6-7 ml scintillation
fluid. If the assay is set up in 96-place deep well plates, the filtration is over
96-place PEI-soaked unifilters, which were washed with 3×1 ml wash buffer,
and dried in an oven at 55° C. for 2 h. The filter plates were counted in
a TopCount (Packard) after adding 50 μl MS-20 scintillation fluid/well.
Functional Assays
The agonist activity of the compounds is measured by determining the degree to
which the compounds receptor complex activates the binding of GTP to G-proteins
to which the receptors are coupled. In the GTP binding assay, GTP[γ]
35S
is combined with test compounds and membranes from HEK-293S cells expressing the
cloned human opioid receptors or from homogenised rat and mouse brain. Agonists
stimulate GTP[y]
35S binding in these membranes. The EC
50
and E
max values of compounds are determined from dose-response curves.
Right shifts of the dose response curve by the delta antagonist naltrindole are
performed to verify that agonist activity is mediated through delta receptors.
Procedure for Rat Brain GTP
Rat brain membranes are thawed at 37° C., passed 3 times through a 25-gauge
blunt-end needle and diluted in the GTPγS binding (50 mM Hepes, 20 mM NaOH,
100 mM NaCl, 1 mM EDTA, 5 mM MgCl
2, pH 7.4, Add fresh: 1 mM DTT, 0.1%
BSA). 120 μM GDP final is added membranes dilutions. The EC50 and Emax of
compounds are evaluated from 10-point dose-response curves done in 300 μl
with the appropriate amount of membrane protein (20 μg/well) and 100000-130000
dpm of GTPγ
35S per well (0.11-0.14 nM). The basal and maximal
stimulated binding are determined in absence and presence of 3 μM SNC-80
Data Analysis
The specific binding (SB) was calculated as TB-NS, and the SB in the presence
of various test compounds was expressed as percentage of control SB. Values of
IC
50 and Hill coefficient (n
H) for ligands in displacing
specifically bound radioligand were calculated from logit plots or curve fitting
programs such as Ligand, GraphPad Prism, SigmaPlot, or ReceptorFit. Values of K
i
were calculated from the Cheng-Prussoff equation. Mean±S.E.M. values
of IC
50, K
i and n
H were reported for ligands tested
in at least three displacement curves. Biological activity of the compounds of
the present invention is indicated in Table 2.
| |
HDELTA (nM) |
RAT BRAIN (nM) |
MOUSE BRAIN (nM) |
| Ex.# |
IC50 |
EC50 |
% EMax |
EC50 |
% EMax |
EC50 |
% EMax |
|
| 1-9 |
0.4-1.155 |
0.38-4.558 |
86.77-106.11 |
3.203-59.76 |
115-176.433 |
3.509-51.477 |
99.535-155.587 |
|
Receptor Saturation Experiments
Radioligand K
δ values were determined by performing
the binding assays on cell membranes with the appropriate radioligands at concentrations
ranging from 0.2 to 5 times the estimated K
δ (up to 10 times if
amounts of radioligand required are feasible). The specific radioligand binding
was expressed as pmole/mg membrane protein. Values of K
δ and B
max
from individual experiments were obtained from nonlinear fits of specifically bound
(B) vs. nM free (F) radioligand from individual according to a one-site model.
Determination of Mechano-Allodynia Using Von Frey Testing
Testing was performed between 08:00 and 16:00 h using the method described
by Chaplan et al. (1994). Rats were placed in Plexiglas cages on top of a wire
mesh bottom which allowed access to the paw, and were left to habituate for 10-15
min. The area tested was the mid-plantar left hind paw, avoiding the less sensitive
foot pads. The paw was touched with a series of 8 Von Frey hairs with logarithmically
incremental stiffness (0.41, 0.69, 1.20, 2.04, 3.63, 5.50, 8.51, and 15.14 grams;
Stoelting, Ill., USA). The von Frey hair was applied from underneath the mesh floor
perpendicular to the plantar surface with sufficient force to cause a slight buckling
against the paw, and held for approximately 6-8 seconds. A positive response was
noted if the paw was sharply withdrawn. Flinching immediately upon removal of the
hair was also considered a positive response. Ambulation was considered an ambiguous
response, and in such cases the stimulus was repeated.
Testing Protocol
The animals were tested on postoperative day 1 for the FCA-treated group. The
50% withdrawal threshold was determined using the up-down method of Dixon (1980).
Testing was started with the 2.04 g hair, in the middle of the series. Stimuli
were always presented in a consecutive way, whether ascending or descending. In
the absence of a paw withdrawal response to the initially selected hair, a stronger
stimulus was presented; in the event of paw withdrawal, the next weaker stimulus
was chosen. Optimal threshold calculation by this method requires 6 responses in
the immediate vicinity of the 50% threshold, and counting of is these 6 responses
began when the first change in response occurred, e.g. the threshold was first
crossed. In cases where thresholds fell outside the range of stimuli, values of
15.14 (normal sensitivity) or 0.41 (maximally allodynic) were respectively assigned.
The resulting pattern of positive and negative responses was tabulated using the
convention, X=no withdrawal; O=withdrawal, and the 50% withdrawal threshold was
interpolated using the formula:
50%
g threshold=10
(Xf+kδ)/10,000
where Xf=value of the last von Frey hair used (log units); k=tabular value
(from Chaplan et al. (1994)) for the pattern of positive/negative responses; and
δ=mean difference between stimuli (log units). Here δ=0.224.
Von Frey thresholds were converted to percent of maximum possible effect (% MPE),
according to Chaplan et al. 1994. The following equation was used to compute % MPE:
%
MPE=Drug treated threshold (
g)-allodynia threshold (
g)×100
Control threshold (g)-allodynia threshold (g)
Administration of Test Substance
Rats were injected (subcutaneously, intraperitoneally, intravenously or orally)
with a test substance prior to von Frey testing, the time between administration
of test compound and the von Frey test varied depending upon the nature of the
test compound.
Writhing Test
Acetic acid will bring abdominal contractions when administered intraperitoneally
in mice. These will then extend their body in a typical pattern. When analgesic
drugs are administered, this described movement is less frequently observed and
the drug selected as a potential good candidate.
A complete and typical Writhing reflex is considered only when the following
elements
are present: the animal is not in movement; the lower back is slightly depressed;
the plantar aspect of both paws is observable. In this assay, compounds of the
present invention demonstrate significant inhibition of writhing responses after
oral dosing of 1-100 μmol/kg.
(i) Solutions Preparation
Acetic acid (AcOH): 120 μL of Acetic Acid is added to 19.88 ml of distilled
water in order to obtain a final volume of 20 ml with a final concentration of
0.6% AcOH. The solution is then mixed (vortex) and ready for injection.
Compound (drug): Each compound is prepared and dissolved in the most suitable
vehicle according to standard procedures.
(ii) Solutions Administration
The compound (drug) is administered orally, intraperitoneally (i.p.), subcutaneously
(s.c.) or intravenously (i.v.)) at 10 ml/kg (considering the average mice body
weight) 20, 30 or 40 minutes (according to the class of compound and its characteristics)
prior to testing. When the compound is delivered centrally Intraventricularly (i.c.v.)
or intrathecally (i.t.) a volume of 5 μL is administered.
The AcOH is administered intraperitoneally (i.p.) in two sites at 10 ml/kg (considering
the average mice body weight) immediately prior to testing.
(iii) Testing
The animal (mouse) is observed for a period of 20 minutes and the number of occasions
(Writhing reflex) noted and compiled at the end of the experiment. Mice are kept
in individual "shoe box" cages with contact bedding. A total of 4 mice are usually
observed at the same time: one control and three doses of drug.
For the anxiety and anxiety-like indications, efficacy has been established in
the geller-seifter conflict test in the rat.
For the functional gastrointestina disorder indication, efficacy can be established
in the assay described by Coutinho S V et al, in American Journal of Physiology—Gastrointestinal
& Liver Physiology. 282(2):G307-16, 2002 February, in the rat.
*
