U.S. patent application number 10/714448 was filed with the patent office on 2004-07-15 for novel compounds.
This patent application is currently assigned to AstraZeneca AB. Invention is credited to Brown, William, Plobeck, Niklas, Walpole, Christopher.
Application Number | 20040138225 10/714448 |
Document ID | / |
Family ID | 20418210 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040138225 |
Kind Code |
A1 |
Brown, William ; et
al. |
July 15, 2004 |
Novel compounds
Abstract
Compounds of general formula (I), wherein R.sup.1 is selected
from phenyl, pyridinyl, thiophenyl, furanyl, imidazolyl and
triazolyl; where each R.sup.1 phenyl ring and R.sup.1
heteroaromatic ring optionally and independently be further
substituted by 1, 2 or 3 substituents selected from straight and
branched C.sub.1-C.sub.6 alkyl, NO.sub.2, CF.sub.3, C.sub.1-C.sub.6
alkoxy, chloro, fluoro, bromo, and iodo.
Inventors: |
Brown, William; (St.
Laurent, CA) ; Walpole, Christopher; (St. Laurent,
CA) ; Plobeck, Niklas; (St. Laurent, CA) |
Correspondence
Address: |
Michael A. Sanzo
Fitch, Even, Tabin & Flannery
Suite 401L
1801 K Street, N.W.
Washington
DC
20006-1201
US
|
Assignee: |
AstraZeneca AB
|
Family ID: |
20418210 |
Appl. No.: |
10/714448 |
Filed: |
November 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10714448 |
Nov 17, 2003 |
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10149981 |
Oct 21, 2002 |
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6680318 |
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10149981 |
Oct 21, 2002 |
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PCT/SE00/02562 |
Dec 15, 2000 |
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Current U.S.
Class: |
514/253.1 ;
514/254.07; 514/254.11; 544/360; 544/370; 544/376 |
Current CPC
Class: |
C07D 409/12 20130101;
A61P 31/12 20180101; A61P 25/04 20180101; A61P 25/18 20180101; C07D
405/12 20130101; A61P 25/02 20180101; A61P 25/36 20180101; A61P
25/32 20180101; A61P 11/00 20180101; C07D 307/79 20130101; A61P
1/04 20180101; A61P 29/00 20180101; A61P 35/00 20180101; A61P 25/24
20180101; A61P 25/34 20180101; A61P 1/12 20180101; A61P 43/00
20180101; A61P 25/16 20180101; A61P 13/02 20180101; A61P 19/08
20180101; A61P 25/22 20180101; A61P 37/02 20180101; A61P 37/08
20180101; A61P 23/00 20180101 |
Class at
Publication: |
514/253.1 ;
514/254.07; 514/254.11; 544/360; 544/370; 544/376 |
International
Class: |
A61K 031/496; C07D
49/14; C07D 45/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 1999 |
SE |
9904674-0 |
Claims
1. A compound according to formula I 10wherein R.sup.1 is selected
from (i) phenyl; (ii) pyrdinyl 11(iii) thiophenyl 12(iv) furanyl
13(v) imidazolyl 14(vi) triazolyl 15where each R.sup.1 phenyl ring
and R.sup.1 heteroaromatic ring may optionally and independently be
further substituted by 1, 2 or 3 substituents selected from
straight and branched C.sub.1-C.sub.6 alkyl, NO.sub.2, CF.sub.3,
C.sub.1-C.sub.6 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; as well as
pharmaceutically acceptable salts and isomers thereof.
2. A compound according to claim 1, wherein the optional
substituent(s) on the aromatic or the heteroaromatic ring(s) is
selected from anyone of NO.sub.2, iso-butyl, CF.sub.3, methoxy,
methyl, or chloro.
3. A compound according to claim 1 or 2, selected from any one of
4-[(4-benzyl-1-piperazinyl)(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)me-
thyl]-N,N-diethylbenzamide dihydrochloride (compound 6);
4-{(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)[4-(4-iodobenzyl)-1-pipera-
zinyl]methyl}-N,N-diethylbenzamide dihydrochloride (compound 7);
4-{(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)[4-(3-pyridinylmethyl)-1-p-
iperazinyl]methyl}-N,N-diethylbenzamide dihydrochloride (compound
8); and
4-{(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)[4-(2-pyridinylmethyl)-1-p-
iperazinyl]methyl}-N,N-diethylbenzamide ditrifuroacetae (compound
9).
4. A compound according to any one of claims 1-3, which compound is
present as the (+)-enantiomer.
5. A compound according to anyone of claims 1-3, which compound is
present as the (-)-enantiomer.
6. A compound according to any of the preceding claims, in form of
its hydrochloride, sulfate, tartrate or citrate salts.
7. A compound according to any of claims 1-6 for use in
therapy.
8. A compound according to claim 7, wherein the therapy is pain
management.
9. A compound according to claim 7, wherein the therapy is directed
towards gastrointestinal disorders.
10. A compound according to claim 7, wherein the therapy is
directed towards spinal injuries.
11. A compound according to claim 7, wherein the therapy is
directed to disorders of the sympathetic nervous system.
12. Use of a compound according to formula I of claim 1 for the
manufacture of a medicament for use in the treatment of pain.
13. Use of a compound according to formula I of claim 1 for the
manufacture of a medicament for use in the treatment of
gastrointestinal disorders.
14. Use of a compound according to formula I of claim 1 for the
manufacture of a medicament for use in the treatment of spinal
injuries.
15. A pharmaceutical composition comprising a compound of the
formula I according to claim 1 as an active ingredient, together
with a pharmacologically and pharmaceutically acceptable
carrier.
16. A method for the treatment of pain, whereby an effective amount
of a compound of the formula I according to claim 1 is administered
to a subject in need of pain management.
17. A method for the treatment of gastrointestinal disorders,
whereby an effective amount of a compound of the formula I
according to claim 1, is administered to a subject suffering from
said gastrointestinal disorder.
18. A method for the treatment of spinal injuries, whereby an
effective amount of a compound of the formula I according to claim
1, is administered to a subject suffering from said spinal injury.
Description
FIELD OF THE INVENTION
[0001] 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.
BACKGROUND AND PRIOR ART
[0002] The .delta. receptor has been identified as having a role in
many bodily functions such as circulatory and pain systems. Ligands
for the .delta. receptor may therefore find potential use as
analgesics, and/or as antihypertensive agents. Ligands for the
.delta. receptor have also been shown to possess immunomodulatory
activities.
[0003] The identification of at least three different populations
of opioid receptors (.mu., .delta. and .kappa.) 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.
[0004] With few exceptions, currently available selective opioid
.delta. ligands are peptidic in nature and are unsuitable for
administration by systemic routes. One example of a non-peptidic
.delta.-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
.delta.-agonists having not only improved selectivity, but also an
improved side-effect profile.
[0005] 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 .beta. agonists,
as well as having improved systemic efficacy.
[0006] 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
.delta. agonist compounds, described within the prior art, show
significant convulsive effects when administered systemically.
[0007] We have now found that certain compounds not specifically
disclosed by, but included within the scope of WO 98/28270, exhibit
surprisingly improved .delta.-agonist properties and in vivo
potency relative to compounds disclosed in WO98/28270, when
administered systemically. The compounds of the present invention
exhibit significant and unexpected increased levels of delta
receptor agonism and metabolic stability.
[0008] Outline of the Invention
[0009] The novel compounds according to the present invention are
defined by the formula I 1
[0010] wherein
[0011] R.sup.1 is selected from
[0012] (i) phenyl;
[0013] (ii) pyridinyl 2
[0014] (iii) thiophenyl 3
[0015] (iv) furanyl 4
[0016] (v) imidazolyl 5
[0017] (vi) triazolyl 6
[0018] where each R.sup.1 phenyl ring and R.sup.1 heteroaromatic
ring may optionally and independently be further substituted by 1,
2 or 3 substituents selected from straight and branched
C.sub.1-C.sub.6 alkyl, NO.sub.2, CF.sub.3, C.sub.1-C.sub.6 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.
[0019] Within the scope of the invention are also pharmaceutically
acceptable salts of the compounds of the formula I, as well as
isomers thereof.
[0020] When the phenyl ring and the heteroaromatic ring(s) are
substituted the preferred substituents are selected from anyone of
CF.sub.3, methyl, iodo, bromo, fluoro and chloro.
[0021] In a preferred embodiment of the invention, the compounds of
formula I are present as the (+)enantiomer, or as the
(-)-enantiomer.
[0022] By "isomers" we mean compounds of the formula I, which
differ by the position of their functional group and/or
orientation. By "orientation" we mean stereoisomers,
diastereoisomers, regioisomers and enantiomers.
[0023] 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.
[0024] 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.
[0025] 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 labelled versions of the compounds of the invention in
diagnostic techniques and imaging applications such as positron
emission tomography (PET).
[0026] 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.
[0027] 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 hypnotica,
anxiolytics, neuromuscular blockers and opioids.
[0028] 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.
[0029] 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.
[0030] Also included within the scope of the present invention, is
any novel intermediate as described in Scheme I hereinafter useful
in the synthesis of compounds of formula I above.
[0031] Methods of Preparation
[0032] The compounds according to the present invention may be
prepared by following the synthetic procedure described in Scheme I
below. This known procedure is described in Katritsky, A. R., Lan,
X. Chem. Soc. Rev., pp. 363-373 (1994), which is hereby
incorporated by reference. 7
EXAMPLES
[0033] The invention will now be described in more detail by the
following Examples, which are not to be construed as limiting the
invention.
[0034] The compounds according to Examples 1-3 were prepared by
following the synthetic procedure described in Scheme I below.
8
Example 1
Preparation of
4-[(4-benzyl-1-piperazinyl)(2,2-dimethyl-2,3dihydro-1-benzo-
furan-7-yl)methyl]-N,N-diethylbenzamide dihydrochloride (compound
6)
(i) Preparation of 2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-yl
trifluoromethanesulfonate (compound 1)
[0035] 2,2-dimethyl-2,3-dihydro-1-benzofuran-7-ol (19 g, 0.11 mol)
and pyridine (18 mL, 0.23 mol) was dissolved in CH.sub.2Cl.sub.2 at
0.degree. C. Triflic anhydride (23 mL, 0.14 mol) was added
dropwise. After stirring 1 h at 25.degree. C. the mixture was
diluted with CH.sub.2Cl.sub.2 and washed with HCl (aq.), dried (Mg
SO.sub.4) and evaporated in vacuo.
[0036] Yield 32 g (96%) of compound 1, which did not need
purification but was used directly in the following step.
[0037] .sup.1H NMR (CDCl.sub.3) .delta. 1.50 (s, 6H), 3.09 (s, 2H),
6.81 (mn, 1H), 7.03 (m, 1H), 7.11 (m, 1H), MS (EI) m/e 296, 163,
135, 107.
(ii) Preparation of Methyl
2,2-dimethyl-2,3-dihydro-1-benzofuran-7-carboxy- late (compound
2)
[0038] Compound I prepared in the previous step above (32 g, 0.11
mol) was dissolved in DMSO (200 mL), MeOH (100 mL) and Et.sub.3N
(34 mL, 0.25 mol). Carbon monoxide was passed through the solution
2-3 min, then palladium acetate (0.24 g) and dppf (1.1 g) was added
and the mixture heated at 70.degree. C. under CO atmosphere. After
4 h, more palladium acetate (0.10 g) and dppf (0.50 g) was added.
After 12 h, EtOAc and water was added and the organic phase was
washed with HCl (aq.), brine, dried (MgSO.sub.4) and evaporated.
chromatography on silica (0-20% EtOAc in heptane) gave 12 g (52%)
of compound 2.
[0039] .sup.1H NMR (CDCl.sub.3) .delta. 1.52 (s, 6H), 3.00 (s, 2H),
3.88 (s, 3H), 6.82 (m, 1H), 7.27 (m, 1H), 7.70 (m, 1H). MS (EI) m/e
206, 174, 159, 146, 131.
(iii) Preparation of
2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-carbaldehyde (compound
3)
[0040] Compound 2 (5.0 g,24 mmol) was dissolved in toluene (100 mL)
and DIBAL in toluene (33 mL, 1.5 M, 50 mmol) was added at
-78.degree. C. under nitrogen atmosphere. After 30 min, the
reaction was worked up by addition of HCl (aq.), the organic phase
was dried (MgSO.sub.4) and evaporated in vacuo. The residue was
dissolved in CH.sub.2Cl.sub.2 (50 mL) and finely ground pyridinium
dichromate (PDC) (11 g, 29 mmol) was added in portions. The mixture
was heated at 40.degree. C. and portions of PDC (1 g) was added
until reaction was complete. Dilution with heptane, filtering
through silca and evaporation gave a crude product which was
purified by chromatography on silica (0-20% EtOAc in heptane) to
give compound 3 (3.3 g, 19 mmol, 67% from compound 2. .sup.1H NMR
(CDCl.sub.3) .delta. 1.54 (s, 6H), 3.03 (s, 2H), 6.88 (m, 1H), 7.34
(m, 1H), 7.58 (m, 1H), 10.22 (s, 1H). MS (EI) m/e 176, 161, 147,
130.
(iv) & (v) Preparation of
4-[(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)(-
hydroxy)methyl]-N,N-diethylbenzamide (compound 4) and
4-[(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)(1-piperazinyl)methyl]-N,N-
-diethylbenzamide (compound 5)
[0041] N,N-Diethyl-4-iodobenzamide (compound I) (14 g, 47 mmol) was
dissolved in THF (150 mL) and cooled to -78.degree. C. under
nitrogen atmosphere. n-BuLi (21 mL, 2.2 M solution in hexane, 47
mmol) was added dropwise. Stirring was continued for 30 min at
-78.degree. C. The aldehyde (compound 3) (4.1 g, 24 mmol) was added
dropwise dissolved in THF (2 mL). NH.sub.4Cl (aq.) was added after
30 min. After concentration in vacuo, extraction with EtOAc/water,
drying (MgSO.sub.4) and evaporation of the organic phase, the
residue was purified by chromatography on silica to (compound 4)
(6.1 g, 17 mmol). After treatment with SOCl.sub.2 (1.5 mL, 20 mmol)
in dry CH.sub.2Cl.sub.2 (200 mL) at 0 to 25.degree. C. for 1 h, the
solvent was evaporated in vacuo. The residue was dissolved in MeCN
(100 mL) and reacted with piperazine (5.8 g, 68 mmol) at 80.degree.
C. for 12 h. After concentration in vacuo and chromatography on
silica (0 to 15% MeOH in CH.sub.2Cl.sub.2, with 1% NH.sub.4OH) gave
(compound 5) (4.9 g, 11 mmol). Dihydrochloride made with HCl (aq)
and lyophilization. mp 130-40.degree. C. (di HCl salt).
[0042] IR (KBr, v.sub.max) 2982, 2722, 2481, 1628, 1450, 1371,
1292, 1140. .sup.1H NMR (CD.sub.3OD) .delta. 1.1, 1.2 (2 m, 6H),
1.36, 1.43 (2 s, 6H), 2.72 (m, 4H), 2.9 (m, 2H), 3.25 (m, 6H), 3.5
(m, 2H), 4.8 (s, 1H), 6.74 -7.60 (m, 7H). Anal.
(C.sub.26H.sub.35N.sub.3O.sub.2) C, H, N.
(vi) Preparation of the title compound
4-[(4-benzyl-1-piperazinyl)(2,2-dim-
ethyl-2,3-dihydro-1-benzofuran-7-yl)methyl]-N,N-diethylbenzamide
dihydrochloride (compound 6)
[0043] Compound 5 (0.62 g, 1.5 mmol)and triethylamine (0.41 mL, 2.9
mmol) was dissolved in MeCN (5 mL) and reacted with benzyl bromide
(0.17 mL, 1.5 mmol) at 25.degree. C. After 2 h a second portion
benzyl bromide was added, and after 4 h the reaction was worked up
by concentration in vacuo and chromatography on silica (0 to 10%
MeOH in CH.sub.2Cl.sub.2) to give the title compound 6 (0.49 g,
0.95 mmol). Dihydrochloride made with HCl (aq) and lyophilization.
MS (ES) 512.08 (MH+).
[0044] IR (NaCl, free amine, v.sub.max) 2969, 2806, 2360, 1630,
1451, 1368, 1289, 1135 cm.sup.1. .sup.1H NMR (CDCl.sub.3, free
amine) .delta. 1.1, 1.2 (2 m, 6H, amide-Me), 1.36, 1.46 (2 s, 6H,
Me.sup.2C), 2.5 (m, 8H, piperazine-H), 2.92 (m, 2H, ArCH2), 3.2,
3.5 (2 m, amide-CH2), 3.51 (s, 2H, ArCH2N), 4.62 (s, 1H, Ar2CH),
6.72-7.52 (m, 7H, Ar--H). Anal.
(C.sub.33H.sub.41N.sub.3O.sub.2.times.3.4 HCl) C, H; N: calcd,
6.61; found, 7.19.
Example 2
[0045] Preparation of
4-{(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)[4-(4-
-iodobenzyl)-1-piperazinyl]methyl}-N,N-diethylbenzamide
dihydrochloride (compound 7)
[0046] Procedure as for compound 6. Compound 5 (0.12 g, 0.29 mmol)
was reacted with 4-iodobenzyl bromide, (96 mg, 0.32 mmol) for 48 h
to give the title compound 7 (56 mg, 88 .mu.mol).
[0047] MS (ES) 638.24 (MH+). IR (NaCl, free amine, v.sub.max) 2969,
2810, 1630, 1451, 1288, 1135, 1007 cm.sup.-1. .sup.1H NMR
(CDCl.sub.3, free amine) .delta. 1.1, 1.2 (2 m, 6H, amide-Me),
1.36, 1.45 (2 s, 6H, Me2C), 2.4 (m, 8H, piperazine-H), 2.94 (m, 2H,
ArCH2), 3.2, 3.5 (2 m, amide-CH2), 3.43 (s, 2H, ArCH2N), 4.62 (s,
1H, Ar2CH), 6.73 (m, 1H, Ar--H), 6.94 (d, J=7.3 Hz, 1H, ArH), 7.05
(d, J=8.0 Hz, 2H, ArH), 7.19 (d, J=6.6 Hz, 1H, ArH), 7.25 (d, J=8.0
Hz, 2H, ArH), 7.48 (d, J=8.0 Hz, 2H, ArH), 7.61 (d, J=8.0 Hz, 2H,
ArH). Anal. (C.sub.26H.sub.37Cl.sub.2N.s- ub.3O.sub.2) C, H, N.
Example 3
Preparation of
4-{(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)[4-(3-pyridi-
nylmethyl)-1-piperazinyl]methyl}-N,N-diethylbenzamide
dihydrochloride (compound 8)
[0048] Compound 5 (0.20 g, 0.47 mmol) was dissolved in MeOH (2 mL
with 3-pyridine carboxaldehyde(90 .mu.L, 0.95 mmol) and HOAc (3
.mu.L, 50 .mu.mol). Sodium cyanoborohydride (60 mg, 0.95 mmol) was
added at 0.degree. C. and reaction stirred 48 h at 25.degree. C.
Reaction was worked up by concentration in vacuo, extraction
(CH.sub.2Cl.sub.2/K.sub.2- CO.sub.3(aq)) and chromatography on
silica (0 to 10% MeOH in CH.sub.2Cl.sub.2) to give the title
compound 8 (82 mg, 0.16 mmol). Dihydrochloride made with HCl (aq)
and lyophilization.
[0049] MS 513.25 (MH+). IR (NaCl, free amine, v.sub.max) 2970,
2808, 2360, 1631, 1452, 1425, 1290, 1135, 1096, 1009 cm.sup.1.
.sup.1H NMR (CDCl.sub.3, free amine) .delta. 1.1, 1.2 (2 m, 6H,
amide-Me), 1.36, 1.46 (2 s, 6H, Me2C), 2.5 (m, 8H, piperazine-H),
2.94 (m, 2H, ArCH2), 3.2, 3.5 (2 m, amide-CH2), 3.51 (s, 2H,
ArCH2N), 4.64 (s, 1H, Ar2CH), 6.72-7.66 (m, 9H, Ar--H), 8.44 -8.54
(m, 2H, Ar--H). Anal. (C.sub.32H.sub.42Cl.sub.- 2N.sub.4O.sub.2) C,
H, N.
Example 4
Preparation of
4-{(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)[4-(2-pyridi-
nylmethyl)-1-piperazinyl]methyl}-N,N-diethylbenzamide
ditrifuroacetae (compound 9).
[0050] 9
[0051] The title compound 9 was prepared by dissolving compound 5
(0.45 g, 0.98 mmol) in MeOH (10 mL) with 2-pyridine carboxaldehyde
(110 .mu.L, 1.18 mmol) and HOAc (3 .mu.L, 50 .mu.mol). Sodium
cyanoborohydride (70 mg, 1.18 mmol) was added at 0.degree. C. and
reaction stirred 48 h at 25.degree. C. Reaction was worked up by
concentration in vacuo, extraction (CH.sub.2Cl.sub.2
/K.sub.2CO.sub.3(aq)) and chromatography by reverse phase HPLC to
give the title compound 9, 461 mg(63%).
[0052] MS 513.04 (MH+).
[0053] Pharmaceutical Compositions
[0054] The novel compounds according to the present invention may
be administered orally, intramuscularly, subcutaneously, topically,
intranasally, intraperitoneally, intrathoracially, intravenously,
epidurally, intrathecally, intracerebroventricularly and by
injection into the joints.
[0055] A preferred route of administration is orally, intravenously
or intramuscularly.
[0056] 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
determining the individual regimen and dosage level as the most
appropriate for a particular patient.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] Pharmaceutically acceptable salts are 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, iodide, 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, benzathine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine, procaine, aluminium,
calcium, lithium, magnesium, potassium, sodium, and zinc. Preferred
pharmaceutically acceptable salts are the hydrochlorides, and
bitartrates. The hydrochloride salts are particularly
preferred.
[0063] 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.
[0064] Tablets, powders, cachets, and capsules can be used as solid
dosage forms suitable for oral administration.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] Biological Evaluation
[0069] In Vitro Model
[0070] Cell Culture
[0071] Human 293S cells expressing cloned human .mu., .delta., and
.kappa. receptors and neomycin resistance were grown in suspension
at 37.degree. C. and 5% CO.sub.2 in shaker flasks containing
calcium-free DMEM10% PBS, 5% BCS, 0.1% Pluronic F-68, and 600
.mu.g/ml geneticin.
[0072] Membrane Preparation
[0073] 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.degree. 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 mM 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.degree. C. until use. The protein
concentrations were determined by a modified Lowry assay with
SDS.
[0074] Binding Assays
[0075] Membranes were thawed at 37.degree. C., cooled on ice,
passed 3 times through a 25-gauge needle, and diluted into binding
buffer (50 mM Tris, 3 mM MgCl.sub.2, 1 mg/ml BSA (Sigma A-7888), pH
7.4, which was stored at 4.degree. C. after filtration through a
0.22 m filter, and to which had been freshly added 5 .mu.g/ml
aprotinin, 10 .mu.M bestatin, 10 .mu.M diprotin A, no DTT).
Aliquots of 100 .mu.l, (for .mu.g protein, see Table 1) were added
to iced 12.+-.75 mm polypropylene tubes containing 100 .mu.l of the
appropriate radioligand (see Table 1) and 100 .mu.L of test
peptides at various concentrations. Total (TB) and nonspecific (NS)
binding were determined in the absence and presence of 10 .mu.M
naloxone respectively. The tubes were vortexed and incubated at
25.degree. 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.sub.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.times.1 ml
wash buffer, and dried in an oven at 55.degree. C. for 2 h. The
filter plates were counted in a TopCount (Packard) after adding 50
.mu.l MS-20 scintillation fluid/well.
[0076] Data Analysis
[0077] The specific binding (SB) was calculated as TB-NS, and the
SB in the presence of various test peptides was expressed as
percentage of control SB. Values of IC.sub.50 and Hill coefficient
(n.sub.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.sub.i were calculated from the Cheng-Prussoff equation. Mean.+-.
S.E.M. values of IC.sub.50, K.sub.i and n.sub.H were reported for
ligands tested in at least three displacement curves. Biological
data are reported below in Table 1.
1TABLE 1 Summary of biological data. HDelta Rat Brain Mouse Brain
MLM RLM % % % 10000 100000 10000 100000 Example # HDelta EC50 EMAX
EC50 EMAX EC50 EMAX % rem. % rem. % rem. % rem. 3 3.519 19.47 103.1
133.72 92.97 4 3.264 7.38 103.9 72.59 118.04 144.13 118.5 44.5 93
42.5 90.5
[0078] Receptor Saturation Experiments
[0079] Radioligand K.delta. 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.delta. (up to 10 times if amounts of radioligand
required are feasable). The specific radioligand binding was
expressed as pmole/mg membrane protein. Values of K.delta. and
B.sub.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.
[0080] Determination of Mechano-Allodynia Using Von Frey
Testing
[0081] 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.
[0082] Testing Protocol
[0083] 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 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.sup.(Xf+k.sup..sub..delta..sup.)/10,000
[0084] 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 .delta.=mean difference between
stimuli (log units). Here .delta.=0.224.
[0085] 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: 1 % MPE = Drug
treated threshold ( g ) - allodynia threshold ( g ) Control
threshold ( g ) - allodynia threshold ( g ) .times. 100
[0086] Administration of Test Substance
[0087] Rats were injected (subcutaneously, intraperitoneally, 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.
[0088] Writhing Test
[0089] 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.
[0090] A complete and typical Writhing reflexe 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.
[0091] (i) Solutions Preparation
[0092] Acetic acid (AcOH): 120 .mu.L 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.
[0093] Compound (drug): Each compound is prepared and dissolved in
the most suitable vehicle according to standard procedures.
[0094] (ii) Solutions Administration
[0095] 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 .mu.L is administered.
[0096] The AcOH is administered intraperitoneally (i.p.) in two
sites at 10 ml/kg (considering the average mice body weight)
immediately prior to testing.
[0097] (iii) Testing
[0098] 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.
* * * * *