AU780802B2 - Compounds useful for the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor - Google Patents

Description

WO 01/58454 PCTIFI01/00105 1 COMPOUNDS USEFUL FOR THE TREATMENT OR PREVENTION OF A DISEASE MEDIATED BY THE ALPHA-2B-ADRENOCEPTOR The present invention relates to the use of selective alpha-2B-adrenoceptor antagonists for the manufacture of a pharmaceutical preparation useful for the treatment or prevention of diseases mediated by the alpha-2B-adrenoceptor in mammals. The present invention also relates to a method for the treatment or prevention of diseases mediated by the alpha-2B-adrenoceptor in mammals, by administering to said mammal said selective alpha-2B-adrenoceptor antagonist.

BACKGROUND OF THE INVENTION The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

The selective alpha-2B-adrenoceptor antagonists shown in Scheme I below are all previously known. The inventors obtained the compounds A (ordering No AE- 848/34956037), C (ordering No AF-399/36012031) and D (ordering No AH- 034/34347043) from SPECS and BioSPECS Fleminglaan 16, 2289 CP Rijswijk, The Netherlands. The compounds B (ordering No 653716) and E (ordering No 569063) were supplied by ChemBridge Corporation, 16981 Via Tazon, Suite G, San Diego CA 92127.

It is known that alpha-2B-adrenoceptors mediate vascular contractions. Therefore, alpha-2B-antagonists are useful in the treatment or prevention of diseases involving vascular contraction. It has also been found that there is a genetic polymorplusm in the alpha-2B-adrenoceptor gene at certain individuals. It has been observed that the alpha-2B-adrenoceptor protein at some subjects has a deletion of 3 glutamates from the glutamic acid repeat element of 12 glutamates (amino acids 297-309), in an acid -2 stretch of 17 amino acids, located in the third intracellular loop of the receptor polypeptide (Heinonen et al., 1999).

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art 10 publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

.For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

SUM AR OFTEINETO It ha oSenfudta h opud eetdfo h ru ossigo copond

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H:\R~e11\Keep\35510.O1 .doc 04/08/03 2a of which are disclosed in Scheme I, are selective alpha-2B-adrenoceptor antagonists.

Thus, this invention relates to the use of a selective alpha-2B-adrenoceptor antagonist for the manufacture of a pharmaceutical preparation useful for the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal, wherein said antagonist is a compound selected from the group consisting of compounds

A

9* 0...0 e o H:\RBell\Keep\35510.01 .doc 04/08/03 2b disclosed in Scheme I, or a pharmaceutically acceptable salt of said compound.

This invention also relates to the use of a selective alpha-2B-adrenoceptor antagonist in the treatment or prevention for the treatment of a disease mediated by the alpha-2Badrenoceptor in a mammal, wherein said antagonist is a compound selected from the group consisting of compounds

A

see

B

'0

C

S

o o

C

disclosed in Scheme I, or a pharmaceutically acceptable salt of said compound.

for the manufacture of a pharmaceutical preparation for potentiating the clinical Sefficacy of an anesthetic and/or analgetic alpha-2-adrenoceptor agonist, said agonist not being selective for the alpha-2B-adrenoceptor subtype.

This invention also relates to the use of a selective alpha-2B-adrenoceptor antagonist for potentiating the clinical efficacy of an anesthetic and/or analgesic alpha-2adrenoceptor agonist, said agonist not being selective for the alpha-2B-adrenoceptor H:\RBell\Keep\35510.01 .doc 04/08/03 2c subtype.

This invention also relates to a method for the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal, said method comprising administering to said mammal an effective amount of a selective alpha-2B-adrenoceptor antagonist, wherein said antagonist is a compound selected from the group consisting of compounds disclosed in Scheme I, or a pharmaceutically acceptable salt of said compound.

H:\RBell\Keep\35510.01 .doc 04/08/03 2d This invention also relates to a method for the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal, said method comprising administering to said mammal an effective amount of a selective alpha-2B-adrenoceptor antagonist, wherein the disease is a vascular disease, which is selected from the group consisting of: vasoconstriction of hypoxic brain damage subsequent to subarachnoid haemorrhage, S- migraine, Raynaud's disease or cold intolerance, 10 pre-eclampsia, male erectile dysfunction, or obesity.

This invention also relates to a method for the treatment or prevention of a disease 15 mediated by the alpha-2B-adrenoceptor in a mammal, said method comprising administering to said mammal an effective amount of a selective alpha-2B-adrenoceptor oo 1antagonist, wherein the alpha-2B-adrenoceptor antagonist is administered to a mammal to potentiate the clinical efficacy of an anesthetic and/or analgetic alpha-2Badrenoceptor agonist, said agonist not being selective for the alpha-2B-adrenoceptor subtype.

DETAILED DESCRIPTION OF THE INVENTION Alpha-2B-adrenoceptor antagonists are useful in the treatment and/or prevention of 25 many diseases.

Individuals having a deletion in the alpha-2B-adrenoceptor protein (Heinonen et al., S: 1999), particularly the deletion/deletion genotype (D/D genotype) is an important S H.\RBell\Keep\35510.01 .doc 04/08/03 WO 01/58454 PCT/FI01/00105 3 target group which benefits from administration of selective alpha-2B-adrenoceptor antagonists.

It has been found that in a population-based cohort of Finnish middle-aged men that subjects with a D/D genotype of the alpha-2B-adrenoceptor gene have a significantly elevated risk for acute myocardial infarction (AMI) in a five-year follow-up study. The risk for AMI was increased in subjects who had no previously diagnosed coronary heart disease (CHD) at the study outset. Therefore, it has been postulated that the D/D genotype is related to an impaired capacity to down-regulate alpha-2B-adrenoceptor function during sustained receptor activation. Therefore, alpha-2B-adrenoceptors are believed to be involved in the pathogenesis of a significant fraction of all cases of AMI, especially in subjects with the D/D genotype, but also in I/D and I/I subjects (I means "insertion" and stands for the "normal" allele).

The alpha-2B-adrenoceptor antagonists as disclosed in this invention would be particularly useful in the treatment or prevention of coronary heart diseases. As examples can be mentioned a) Acute AMI If alpha-2B-adrenoceptor dependent vasoconstriction is a causative factor in some cases of AMI, then antagonism of these receptors should restore coronary circulation and reduce the ischemic myocardial damage.

b) Unstable angina pectoris An alpha-2B-adrenoceptor antagonist will relieve the vasoconstrictive component in the sustained ischemic episode, thus alleviating the symptoms and preventing AMI.

WO 01/58454 PCTIFI01/00105 4 c) Prinzmetal's variant form of angina pectoris Vasoconstriction is a key factor in the pathogenesis of Prinzmetal's angina, and an alpha-2B- adrenoceptor antagonist may resolve and prevent attacks.

d) Other forms of chronic angina pectoris and CHD An alpha-2B-adrenoceptor antagonist will help to alleviate the vasoconstrictive component in all types of CHD, providing both symptomatic relief and protection from AMI. A general reduction in vascular tone will contribute to this by reducing venous return, cardiac workload and oxygen consumption (a nitrate-type effect; see below).

e) Prevention of restenosis after coroary angioplasty in cases where vasoconstriction plays a role in restenosis.

Furhermore, the alpha-2B-adrenoceptor antagonists as disclosed in this invention would be useful in the treatment or prevention of essential hypertension, especially in subjects with increased sympathetic activity and a hyperdynamic circulatory system.

In the study mentioned above, the D/D variant of the alpha-2B-adrenoceptor gene was not clearly associated with blood pressure. The inventors believe that this was due to two main factors, 1) antihypertensive treatment, and 2) complex regulation of systemic blood pressure. In another study (Hcinonen et it was observed that the D/D genotype was associated with reduced basal metabolic rate and reduced heart rate. These associations probably reflect increased vascular resistance in tmese subjects.

WO 01/58454 PCTIFI01/00105 In transgenic mice with targeted inactivation of the alpha-2B-adrenoceptor gene, intravenously administered alpha-2-adrenoceptor agonists fail to induce the characteristic blood pressure elevation which is seen in normal animals and also in humans after large doses of such drugs (Link et al., 1996). The hypotensive effect of these drugs was markedly accentuated. This demonstrates that alpha-2Badrenoceptors mediate vascular contraction. Thus, an antagonist should reduce blood pressure. This effect has not been seen with alpha-2B-nonselective alpha-2adrenoceptor antagonists, because antagonism of alpha-2A-adrenoceptors increases sympathetic outflow, cardiac output and blood pressure. In mice with dysfunctional alpha-2A-adrenoceptors, alpha-2-adrcoccptor agonists caused an accentuated hypertensive response and no hypotension (MacMillan ct al., 1996).

An alpha-2B-adrenoceptor antagonist is postulated to have favourable effects in hypertensive subjects through their effects on renal function, muscle blood flow, and also on vascular resistance in other vascular beds. The anti-AMI effect of such a drug will be an additional benefit, as hypertension is a significant risk factor for AMI. This protection is due to three factors: 1) a reduction in systemic blood pressure, 2) decreased risk of coronary vasoconstriction, and 3) a nitrate-like effect on venous return, myocardial workload and oxygen consumption.

Moreover, the alpha-2B-adrenoceptor antagonists as disclosed in this invention would be useful in the treatment or prevention of other vascular diseases.

Specifically, benefits can be expected in the treatment or prevention of vasoconstriction and hypoxic brain damage subsequent to subarachnoid haemorrhage, migraine, Raynaud's disease and cold intolerance, pre-eclampsia, male erectile dysfynction, and obesity and the metabolic syndrome.

WO 01/58454 PCT/FI01/00105 6 The last mentioned effect is due to the fact that reduced muscle blood flow and reduced basal metabolic rate contribute to the development of obesity and hypertension. An alpha-2B-adrenoceptor antagonist will, by increasing the muscle blood flow, increase energy expenditure and shift the caloric balance to a favourable direction.

The alpha-2B-adrenoceptor antagonists disclosed in this invention are also useful in anesthesia and analgesia to potentiate the clinical efficacy of alpha-2-adrenoceptor agonists which are not selective for the alpha-2B-adrenoceptor subtype. By blocking the vasoconstriction induced by these agonists, a simultaneously administered alpha-2B-adrenoceptor antagonist will allow the use of larger doses of said agonists, up to anesthetic dose levels which have not previously been possible in man, only in veterinary anesthetic practice.

EXPERIMENTAL SECTION Binding affinity human alpha-2-adrenoceptor The affinity of test compounds for the three human c 2 -adrenoceptor subtypes (CtA, X2B and t 2 c) was determined in competition binding assays with 3H-rauwolscine.

The biological material for these experiments consisted of membranes from Shionogi S115 cells stably transfected with either of the three human a 2 subtypes (Marjamaki et al. 1992). Membrane (5-10 gg of total protein per sample) and I nM 2 nM 3H-rauwolscine (specific activity 78 Ci/mmol) were incubated in 50 mM

KH

2

PO

4 pH 7.5 with 6 concentrations of the compounds. Each concentration was run in duplicate. Nonspecific binding was defined by 100 pIvi oxyimIeiazlin anud corresponded to 5 15% of total binding. After 30 min at room temperature, incubations were terminated by rapid vacuum filtration through GF/B glass fiber filter and three 5 ml washes with icecold incubation buffer. The filters were then WO 01/58454 PCTIFI01/00105 7 dried, impregnated with scintillate and their radioactivity was measured by scintillation counting. The analysis of the experiments was carried out by nonlinear least square curve fitting. Experimentally determined IC50 values were converted to Ki's by making use of the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

Experiments were repeated a minimum of three times.

Table 1: Binding affinities on human cL 2 -adrenoceptor subtypes Data is presented as Ki's in nM (Mean SEM), n 3 unless indicated otherwise .0 Compound alpha-2A alpha-2B alpha-2C A 4100 200 30 4 >4700 C >30000 1860 >30000 (n=2) D >30000 530 90 >30000 B >30000 215 ±60 >30000 E >100000 2900 300 >100000 Binding affinity rat cortical a-adrenoceptor The affinity for rat neocortical acx-adrenoceptors was determined in competition binding assays with 3 H-prazosin. The biological material for these assays consisted of membranes from rat neocortex. Membrane suspensions (100-200 -ig of total protein per sample) and 0.2 nM-0.25nM of 3 H-prazosin (specific activity 74 Ct/mmol) were incubated with b concentrations of compounds in a totai volume of 0.25 ml (50 mM Tris plI 7.7 at 25 0 Each concentration was run in duplicate.

Nonspecific binding was defined by 10 pM phentolamine methanesulfonate and corresponded to 25-30 of total binding. After 30 min at room temperature, WO 01/58454 PCT/FI01/00105 8 incubations was terminated by rapid filtration through GF/B glass-fiber filter mats and three washes with ice-cold 10 mM Tris (pH 7.7 at 4 0 After drying, a solid scintillate was melted onto the filter mats, and their radioactivity was measured by scintillation counting.

Result At concentrations of up to 30 pM, compound A caused insufficient displacement of 3 H-prazosin to allow the estimate of an 1Co 5 value. It is therefore concluded that the

IC

5 0 and the Ki of compound A must be >30 000 nM.

Antagonist activity on human az-adrenoceptor subtypes Antagonist potencies were determined as the ability of test compounds to competitively inhibit epinephrine-stimulated 3sS-GTPyS binding to G proteins (Tian et al., 1993; Wieland and Jakobs, 1994; Jasper et al., 1998) in membranes of CHO cells stably transfected with one of the three human a 2 subtypes (Pohjanoksa et al., 1997; Marjamdiki et al., 1998). Membranes (2-6 pg of protein per sample) and 12 concentrations of test compound were preincubated for 30 min with a fixed concentration fo epinephrine (5 pM for (X2A., 15 pM for t 2 B, 5 pM for a2c) in mM Tris, 5 mM MgCI 2 150 mM NaCI, 1 mM DTT, 1 mM EDTA, 10 pM GDP, pM ascorbic acid, pH 7.4 at room temperature. Binding of radiolabel was started by the addition of trace amounts of 35 S-GTPyS (0.08 nM- 0.15 nM, specific activity 1250 Ci/mmol) to the incubation mixture. After an additional 60 min at room temperature, the incubation was terminated by rapid vacuum filtration through glass fiber filter. Filters were washed three times with 5 ml icecold wash buffer (20 mM Tris, 5 mM MgC1 2 1 mM EDTA pH 7.4 at room temperature), dried and counted for radioactivity in a scintiallation counter. Analysis of experiments was carried out by nonlinear least square fitting. Experiments were repeated at least three times.

WO 01/58454 PCT/F101/00105 9 Table 2: Antagonist effect of compound A and compound B on the human a 2 adrenoceptor subtypes Data is presented as KB's in nM (Mean SEM), n is a minimum of three experiments.

Compound alpha-2A* alpha-2B alpha-2C* A 2400 ±700 73 ±23 2400+ 900 B >10 000 250 80 >10 000 only incomplete dose-response curves could be obtained, KB numbers are minimum estimates For the purpose of the invention, the alpha-2B-adrenoceptor antagonist as disclosed in Scheme I or its pharmaceutically acceptable salt can be administered by various routes. The suitable administration forms include, for example, oral formulations; parenteral injections including intravenous, intramuscular, intradermal and subcutanous injections; transdcrmal or rectal administration forms. The required dosage of the compounds of the alpha-2B-adrcnoceptor antagonist will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the administration route and the specific compound being employed.

The suitable dose varies in the range 5 Lg to 100 mg per kg body weight and day for an adult person.

It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

WO 01/59454 PCT/FIOI/00105 Scheme I Compound

A

B

00

CC

0 H H. WO 01/58454 PCTIFIOI/0O105

REFERENCES

Chen-, and Prusoff, 1973. Biochem. Pharinacol. 22: 3099 Jasper, Lesnick, Chang, Yamanashi, Chang, Hsu, Daunt, Bonhaus, and E-en, 1998. Biochern. Pharmnacol.

1035 Marjanaki, Ala-Uotila, Luomala, Perjl1a. Jansson, Jalkanen, M., Regan, and Scheinin, 1992. Biochem. IBiophys. Acta 1134: 169 Marjamdki, Pihiavisto, Cockcroft, Heinonen, Savola, and Scheinin, 1998. Mol. Phunnacol. 53: 370 Pohjanoksa, Jansson, Luomala, Marjam~ki, Savola, and Scheinin, 1997. Eur. J. Pharmacol. 35: 53 Tian, Duzic, Lanier, and Deth, 1993. Mol. Phannacol. 524 Wieland, and Jakobs, 1994. Meth. Enzymol. 237: 3 He]inonen et al. 1999, The Journal of Clinical Endocrinology Metabolism, 84: 24 29 Link R E et al., 1996. Science 273:803 MacMillan L B et al., 1996, Science 273:80 1

Claims (25)

1. Use of a selective alpha-2B-adrenoceptor antagonist for the manufacture of a pharmaceutical preparation for the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal, wherein said antagonist is a compound selected from the group consisting of compounds: A B C E e~L- _c o or a pharmaceutically acceptable salt thereof.

2. Use of a selective alpha-2B-adrenoceptor antagonist in the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor antagonist in a mammal, wherein said antagonist is a compound selected from the group consisting of cnmnniindsr H:\marieag\Keep\Speci\35510.01 .doc 16/09/04 13 or a pharmaceutically acceptable salt thereof.

3. A use according to claim 1 or claim 2, wherein the disease is a coronary heart disease (CHD).

4. A use according to claim 3, wherein the disease is selected from the group consisting of: S- acute myocardial infarction (AMI); unstable angina pectoris; 10 Prinzmetal's variant form of angina pectoris; other forms of chronic angina pectoris and CHD; and restenosis after coronary angioplasty.

5. A use according to claim 1 or claim 2, wherein the disease is essential 15 hypertension.

6. A use according to claim 1 or claim 2, wherein the disease is a vascular disease, which is selected from the group consisting of: -vasoconstriction or hypoxic brain damage subsequent to subarachnoid hemorrhage; \\melb~fileshomeS\marieag\Keep\Speci\355lO.l .doc 16/09/04 14 -migraine; Raynaud's disease or cold intolerance; -pre-eclampsia; male erectile dysfunction; and obesity.

7. Use of a selective alpha-2B-adrenoceptor antagonist for the manufacture of a pharmaceutical preparation for potentiating the clinical efficacy of an anesthetic and/or analgetic alpha-2B-adrenoceptor agonist, said agonist not being selective for the alpha-2B-adrenoceptor subtype, wherein said antagonist is a compound selected from the group A, B, C and E as defined in claim 1.

8. Use of a selective alpha-2B-adrenoceptor antagonist for potentiating the clinical efficacy of an anesthetic and/or analgetic alpha-2-adrenoceptor agonist, said agonist not being selective for the alpha-2B-adrenoceptor subtype, wherein said antagonist is a compound selected from the group A, B, C and E as defined in claim 1. o

9. A method for the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal, said method comprising administering to said mammal an effective amount of a selective alpha-2B-adrenoceptor antagonist, wherein said antagonist is a compound selected from the group consisting of compounds: A c E Or-o o H:\yvteuc\keep\Specifications\.3 5 1001 Amendedftgesdoc 14.02.05 15 or a pharmaceutically acceptable salt thereof.

A method according to claim 9, wherein the disease is a coronary heart disease (CHD).

11. A method according to claim 9 or claim 10, wherein the disease is: acute myocardial infarction (AMI), unstable angina pectoris, Prinzmetal's variant form of angina pectoris, other forms of chronic angina pectoris and CHD, and restenosis after coronary angioplasty.

12. A method according to claim 9 or claim 10, wherein the disease is essential hypertension.

13. A method according to claim 9, wherein the disease is a vascular disease, which is selected from the group consisting of: vasoconstriction or hypoxic brain damage subsequent to subarachnoid hemorrhage, 20 migraine, Raynaud's disease or cold intolerance, pre-eclampsia, male erectile dysfunction, and obesity.

14. A method according to any one of claims 9 to 13, wherein said alpha-2B- .adrenoceptor antagonist is administered to a mammal to potentiate the clinical efficacy of an anesthetic and/or analgetic alpha-2-adrenoceptor agonist, said agonist not being selective for the alpha-2B-adrenoceptor subtype.

A method according to any one of claims 9 to 14, wherein said alpha-2B- adrenoceptor antagonist is administered to an individual having a deletion of 3 glutamates from the glutamic acid repeat element of 12 glutamates (amino acids 297 to 309), in an acid stretch of 17 amino acids, located in the third intracellular loop of the receptor polypeptide.

16. A method according to claim 15, where said individual is a H:\yvettec\keep\Specilcations\355 10 01 AmendedPagesdoc 14 0205 16 deletion/deletion genotype.

A compound selected from the group consisting of compounds: e 5 or a pharmaceutically acceptable salt thereof, when used in the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal.

18. A compound selected from the group consisting of compounds: H:\marieag\Keep\Speci\35510.01 .doc 16/09/04 17 A B C E o o 0 0 or a pharmaceutically acceptable salt thereof, when used as a pharmaceutical preparation in the treatment or prevention of a disease mediated by the alpha-2B- adrenoceptor in a mammal.

19. A compound according to claim 17 or claim 18, wherein the disease is a coronary heart disease (CHD).

20. A compound according to claim 19, wherein the disease is selected from the group consisting of: acute myocardial infarction (AMI), unstable angina pectoris, Prinzmetal's variant form of aneina oectoris. other forms of chronic angina pectoris and CHD, and 15 restenosis after coronary angioplasty.

21. A compound according to claim 17 or claim 18, wherein the disease is essential hypertension.

22. A compound according to claim 17 or claim 18, wherein the disease is a \\melb_files\home$\marieag\Keep\Speci\35510.01 .doc 16/09/04 18 vascular disease, which is selected from the group consisting of: vasoconstriction or hypoxic brain damage subsequent to subarachnoid hemorrhage, migraine, Raynaud's disease or cold intolerance, pre-eclampsia, male erectile dysfunction, and obesity.

23. A use according to any one of claims 1, 2, 7 or 8, substantially as herein described with reference to the written description.

24. A method according to claim 9, substantially as herein described with reference to the written description. A compound according to claim 17 or claim 18 when used in the treatment or prevention of a disease mediated by the alpha-2B-adrenoceptor in a mammal, substantially as herein described with reference to the written description. Dated this 14 th day of February 2005 OY JUVANTIA PHARMA LTD By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and

25 Trade Mark Attorneys of Australia S. H:\yvettec\keep\Specificaions\35001 AmendedPagesdoc 14.02.05