ZA200303250B - Treatment of sexual dysfunction. - Google Patents

Treatment of sexual dysfunction. Download PDF

Info

Publication number
ZA200303250B
ZA200303250B ZA200303250A ZA200303250A ZA200303250B ZA 200303250 B ZA200303250 B ZA 200303250B ZA 200303250 A ZA200303250 A ZA 200303250A ZA 200303250 A ZA200303250 A ZA 200303250A ZA 200303250 B ZA200303250 B ZA 200303250B
Authority
ZA
South Africa
Prior art keywords
methyl
indol
cyclohexylmethyl
pyridin
propionamide
Prior art date
Application number
ZA200303250A
Inventor
Gonzalez Maria Isabel
Stock Herman Thijs
Robert Denham Pinnock
Pritchard Martyn Clive
Wayman Christopher Peter
Pieter Hadewijn Van Der Graaf
Naylor Alisdair Mark
Michael Higginbottom
Original Assignee
Warner Lambert Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Warner Lambert Co filed Critical Warner Lambert Co
Publication of ZA200303250B publication Critical patent/ZA200303250B/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Endocrinology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Indole Compounds (AREA)
  • Pyridine Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Description

TREATMENT OF SEXUAL DYSFUNCTION
FIELD OF THE INVENTION
The present invention relates to methods for the treatment of sexual dysfunction and to the preparation of medicaments for the treatment of sexual dysfunction.
BACKGROUND TO THE INVENTION
Both males and females can suffer from sexual dysfunction. Sexual dysfunctions are relatively common in the general population (see O’ Donohue, 1997).
The disorder may relate to seeking sexual behaviour (proceptivity) and/or to acceptance of sexual behaviour, accompanied by sexual arousal (receptivity). The prevalence of sexual problems is higher in populations receiving medicaments, in particular antidepressants and antihypertensives. A need for pharmacotherapy for sexual dysfunction is increasing, but there has been very little research effort directed at finding drugs to treat sexual dysfunction.
Sexual dysfunctions include erectile dysfunctions of organic and psychogenic origin (Benet, 1995) as well as hypoactive sexual desire disorders, sexual arousal disorders, anorgasmy and sexual pain disorders (Berman, 1999, Urology).
In males, impotence can be defined as an inability to achieve penile erection or ejaculation. Its prevalence is claimed to be between 2% and 7% of the human male population, increasing with age up to 50 years and between 18% and 80% between 55 and 80 years of age. In the USA alone, for example, it has been estimated that there g are up to 10 million impotent males, with the majority suffering from problems of organic rather than of psychogenic origin. Although many different drugs have been ) shown to induce penile erection, they were only effective after direct injection into the penis e.g. intraurethrally or intracavernosally (i.c.) and were not approved for erectile dysfunction. US-A-5576290 discloses peptides which are stated to induce erection,
but they have to be given subcutaneously e.g. by injection, and if an excessive dose is ‘ given they produce an exaggerated erectile response and stomach discomfort.
Impotence treatment was revolutionized by the unexpected discovery that cGMP PDE inhibitors, e.g. pyrazolo[4,3-d}pyrimidin-7-ones were useful in the treatment of erectile dysfunction and could be administered orally, therefore obviating the disadvantages associated with ic. administration. One such compound that is currently being manufactured is sildenafil (Viagra).
Thirty to 50% of American women complain of sexual dysfunction. Ageing, menopause, and decline in circulating oestrogen levels significantly increase the incidence of sexual complaints. In a recent publication, Berman J.R. et al. (1999, Int.
J. Impot. Res.) describe methodology for evaluating physiologic and subjective components of the female sexual response in the clinical setting and determine the effects of age and oestrogen status on them. Low or absent sexual drive/desire constitutes the commonest problem in the female population (Laumann et al., 1999), but no therapy is available other than psychotherapy or empirical approaches. In a further publication (Bonney R.C et al., 2000) the causes and management of female sexual dysfunction are discussed, including the use of tibolone (Livial; Organon) : which is a synthetic steroid that mimics the effects of oestrogen and has been reported to have mild androgenic properties, and the use of testosterone.
So far in the UK and the USA no drug has been licensed by the Department of
Health specifically for the treatment of female sexual dysfunction, hence there is an unmet medical need in the treatment of female sexual dysfunction, especially sexual drive problems. } SUMMARY OF THE INVENTION . This invention is based on the realisation that substances that act as bombesin receptor antagonists have utility in the treatment of sexual dysfunction, including the behavioural component thereof, in both male and female subjects. In other words, they can provide a treatment, in males, for erectile dysfunctions of organic and psychogenic origin as well as hypoactive sexual desire disorders, sexual arousal ‘ disorders, anorgasmy and sexual pain disorders in females.
The invention therefore provides a method of treating sexual dysfunction which comprises administering to a subject suffering therefrom and in need of treatment an effective amount of a bombesin (BB) receptor antagonist. ‘The invention further provides the use of a bombesin receptor antagonist in the manufacture of a medicament for preventing or treating male sexual dysfunction or female sexual dysfunction.
Furthermore, many of the compounds that can be used in this invention have both the property of binding to bombesin receptors and the property that an effective dose can be administered orally.
The bombesin antagonists preferably have a Ki against the bombesin receptor of less than 1000nM, preferably less than 500nM, more preferably less than 100 nM, preferably less than 50 nM and most preferably less than 10 nM. Preferably the bombesin antagonists are selective for BB, over the other bombesin receptor subtypes (preferably a selectivity of greater than 10, and more preferably a selectivity greater than 30 and most preferably greater than 100 measurable in vitro by the ratio of their
IC50 or Ki values against the BB1 and BB2 receptors respectively) and has a Ki against the BB, receptor of less than 1000nM, preferably less than 500nM, more preferably less than 100 nM, preferably less than 50 nM and most preferably less than 10 nM. Compounds having the potencies set out above can be identified by the in vitro screen described below.
Thus the invention provides a method of treating drug induced sexual " dysfunction (particularly but not exclusively dysfunction induced by antidepressants) in a male comprising administering to a male suffering therefrom an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist .
The invention further provides a method of treating drug induced sexual ) dysfunction (particularly but not exclusively disfunction induced by antidepressants) in a female comprising administering to a female suffering therefrom an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist.
The invention also provides a method for treating erectile dysfunction in a male comprising administering to a male suffering therefrom an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist .
The invention also provides a method for treating a female patient suffering from hypoactive sexual desire disorder comprising administering to the female patient an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist.
The invention also provides a method for treating a female patient suffering from sexual arousal disorder and/or orgasmic disorder comprising administering to the female patient an effective amount of a bombesin BB1 antagonist or a mixed
BB1/BB2 antagonist.
The present invention additionally comprises the combination of a bombesin receptor antagonist (which may have one of the preferred range of potencies indicated above) for the treatment of male sexual dysfunction as outlined herein (more particularly male erectile dysfunction) and female sexual dysfunction (as outlined herein, more particularly female sexual arousal disorder or female sexual desire disorder) with one or more of the following auxiliary active agents. The combination provides a treatment for erectile dysfunctions of organic, neurogenic and/or psychogenic origin as well as hypoactive sexual desire disorders, sexual arousal
N disorders, anorgasmic and sexual pain disorders ‘ Thus a further aspect of the invention provides a pharmaceutical combination (for simultaneous, separate or sequential administration) of a bombesin receptor antagonist according to the invention and one or more materials selected from (1) to (34) below:
(DH) naturally occurring or synthetic prostaglandins or esters thereof, : 2) o - adrenergic receptor antagonist compounds also known as a - adrenoceptor antaginists or a-receptor antagonists or a-blockers; 3) NO-donor (NO-agonist) compounds; 5 @) potassium channel openers or modulators; (5) dopaminergic agents; (6) vasodilator agents; 7) thromboxane A2 agonists; 8) CNS active agents; (9) ergot alkaloids; (10) compounds which modulate the action of natriuretic factors; . (11) angiotensin receptor antagonists such as losartan. ; (12) substrates for NO-synthase;. (13) calcium channel blockers; (14) cholesterol lowering agents; (15) antiplatelet and antithrombotic agents; (16) insulin sensitising agents and hypoglycaemic agents;. (17) L-DOPA or carbidopa; (18) acetylcholinesterase inhibitors; (19) steroidal or non-steroidal anti-inflammatory agents; (20) estrogen receptor modulators and/or estrogen agonists and/or estrogen antagonists, and pharmaceutically acceptable salts thereof; (21) PDE inhibitors; (22) NPY (neuropeptide Y) inhibitors; (23) NEP inhibitors; (24) vasoactive intestinal proteins (VIP), VIP mimetics, VIP analogues, VIP . receptor agonists or VIP analogues or VIP fragments, or a-adrenoceptor antagonists with VIP combinations; ) (25) melanocortin receptor agonists or modulators or melanocortin ehancers; (26) serotonin receptor agonists, antagonists or modulators; (27) testosterone replacement agents, testosternone, dihydrotestosterone or a testosterone implant;
(28) estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate ’ (MPA) (i.e. as a combination), or estrogen and methyl testosterone hormone replacement therapy agents; (29) modulators of transporters for noradrenaline, dopamine and/or serotonin; (30) purinergic receptor agonists and/or modulators; (31) neurokinin (NK) receptor antagonists; (32) opioid receptor agonists, antagonista or modulators; (33) agonists or modulators for oxytocin/vasopressin receptors; and (34) modulators of cannabinoid receptors.
In particular the invention includes a pharmaceutical composition or a unit dosage form comprising an effective amount of a bombesin receptor antagonist and an effective amount of any of the materials selected from (1) to (34) above.
In the above methods, and in the above combination, composition or dosage form, said antagonists preferably have a Ki against BB1, of of less than 1000nM, preferably less than 500nM, more preferably less than 100 nM, preferably less than 50 nM and most preferably less than 10 nM and/or a selectivity for BB; over the other bombesin receptor subtypes greater than 10, and more preferably greater than 30 and most preferably greater than 100 measurable in vitro by the ratio of their IC50 or Ki values against the BB1 and BB2 receptors respectively.
BRIEF DESCRIPTION OF FIGURES
Figure 1: Effect of (S) 3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexyl- methyl]-2-methyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide (Compound (1)) on ) female rat sexual proceptivity.
Figure 2: Effect of Compound (1) on female rat sexual receptivity.
Figure 3: Effect of repeated administration of Compound (1) on female rat ’ proceptivity.
Figure 4: Effect of intracerebroventricular administration of Compound (1) on female rat sexual proceptivity.
Figure 5: Inhibitory effect of NMB on female rat sexual proceptivity and antagonism of this effect by Compound (1).
Figure 6: Results of an investigation to show whether the effect of Compound (1) on female sexual behaviour is mediated through progesterone.
Figure 7: Results of an investigation to show whether the effect of Compound (1) on female sexual behaviour is mediated through oestradiol.
Figure 8: Results of an investigation to show whether the effect of Compound (1) on female sexual behaviour is mediated through prolactin.
Figure 9: Results of an investigation to show whether the effect of Compound (1) on female sexual behaviour is mediated through LH.
Figure 10: Results of an investigation to show whether the effect of Compound (1) on female sexual behaviour is mediated through FSH.
Figure 11: Effect of Compound (1) on the sexual behaviour of normal male rats (Mount Latency).
Figure 12: Effect of Compound (1) on the sexual behaviour of normal male rats (Intromission Latency).
Figure 13: Effect of Compound (1) on the sexual behaviour of normal male rats (Number of Mounts + Intromission). [
: Figure 14: Effect of Compound (1) on the sexual behaviour of normal male rats (Ejaculation Latency).
Figure 15: Effect of Compound (1) on the sexual behaviour of normal male rats (Refractory Period).
Figure 16: Effect of Compound (1) on the sexual behaviour of sexually dysfunctional male rats (Mount Latency).
Figure 17: Effect of Compound (1) on the sexual behaviour of sexually dysfunctional male rats (Ejaculation Latency).
Figure 18: Effect of Compound (1) on the sexual behaviour of sexually dysfunctional male rats (% animals ejaculating).
Figure 19: Effect of (S)-3-(1H-Indol-3-yl)-N-[ 1-(5-methoxy-pyridin-2-yl)- cyclohexylmethyl]-2-methyl-2-[4-(4-nitro-phenyl)-oxazol-2-ylamino}-propionamide (Compound (2)) in PEG 200 on female rat sexual proceptivity.
Figure 20: Effect of Compound (2) in methylcellulose on female rat sexual proceptivity.
Figure 21: Effect of Compound (2) in PEG 200 on female rat sexual receptivity.
Figure 22: Effect of compound 1 on basal and pelvic nerve-stimulated increases in ] female genital blood flow in the anaesthetised rabbit model of female sexual arousal. . Figure 23: Effect of (25)-N-{[1-(4-aminophenyl)cyclohexyljmethyl}-3-(1H-indol-3- yl)-2-methyl-2-{[(4-nitroanilino)carbonyl]amino} propanamide (Compound 3) on basal and pelvic nerve-stimulated increases female genital blood flow in the anaesthetised rabbit model of female sexual arousal.
: Figure 24: Effect of compound 1 on penile intracavernosal pressure in the conscious male rat.
Figure 25: Effect of compound 3 on penile intracavernosal pressure in the conscious male rat model of penile erection.
Figure 26: Effect of compound 3 alone and in combination with a phosphodiesterase type five inhibitor on basal and pelvic nerve-stimulated increases penile intracavernosal pressure in the anaesthetised rabbit model of penile erection.
DESCRIPTION OF PREFERRED EMBODIMENTS
Suitable subjects
As previously explained the invention provides combinations, compositions and methods for the treatment of male sexual dysfunction or female sexual dysfunction. The inventors believe that there are common mechanisms underlying the pathologies of male and female psychogenic sexual dysfunctions.
Male sexual dysfunction includes male erectile dysfunction (MED). Patients with mild to moderate MED should benefit from treatment with a bombesin antagonist and patients with severe MED should also respond.the ability of bombesin antagonists to return intracavernosal pressure to normal levels in a conscious rat model of penile erection (Example 170, Figs. 24 and 25) and in a pelvic nerve stimulation model (Example 171, Fig 26) has been demonstrated using telemetry. . However, early investigations suggest that the response rate of patients with mild, moderate and severe MED will be greater with a bombesin antagonist/PDES5 inhibitor . combination (see Example 171 and Fig. 26). Mild, moderate and severe MED will be terms known to the man skilled in the art, but guidance can be found in The Journal of
Urology, vol 151, 54-61 (Jan 1994).
~ WO 02/40008 PCT/GB01/05018
Our investigations suggest the below mentioned male sexual dysfunction/
MED patient groups should benefit from treatment with a bombesin antagonist and/or a bombesin antagonist with a phosphoesterase type 5 inhibitor (PDES5i) or other combination set out herein. These patient groups which are described in more detail in
Clinical Andrology vol 23,n0.4, p773-782, and chapter 3 of the book by I. Eardley and K. Sethia "Erectile Dysfunction-Current Investigation and Management”, published by Mosby-Wolfe are as follows: psychogenic, endocrinologic, neurogenic, arteriogenic, drug-induced sexual dysfunction and sexual dysfunction related to cavernosal factors, particularly venogenic causes. The invention finds application in the following sub-populations of patients with sexual dysfunction/MED: the young, the elderly including ageing-related decline in sexual arousability. More particularly, the invention finds application in patients with male sexual dyfunction such as MED arising from:- (1) Arteriogenic/vasculogenic etiologies eg cardiovascular or atherosclerotic diseases, hypercholesterolemia, cigarette smoking, diabetes, hypertension, radiation and perineal trauma, traumatic injury to the iliohypogastric pudendal vacular system. (11) Neurogenic etiologies such as spinal cord injuries or diseases of the central nervous system including multiple sclerosis, diabetes, Parkinsonism, cerebrovascular accidents, peripheral neuropathies, trauma or radical pelvic surgery. (111) Hormonal/lendocrine etiologies such as dysfunction of the hypothalamic/pituitary/gonadal axis, dysfunction of the pancreas, surgical or medical castration, androgen deficiency, high circulating levels of prolactin eg hyperprolactinemia, hyper and hypothyroidism. ] (iv) Psychogenic etiologies such as depression, obsessive-compulsive disorder, anxiety disorder, emotional and relational issues, performance anxiety, marital . discord, dysfunctional attitudes, sexual phobias, religious inhibition or a traumatic past experiences. (v) Drug-induced sexual dysfunction resulting from therapy with selective serotonin reuptake inhibitors (SSRis) and other antidepressant therapies
(tricyclics and major tranquilizers), anti-hypertensive therapies, and sympatholytic drugs.
Drug-induced sexual disfunction in males includes patients whose drug treatment/therapy leads to delayed ejaculation/orgasm, reduced libido and/or erectile : dysfunction. The bombesin antagonists of the invention (more particularly BB1 antagonists) restore ejaculatory/orgasmic, libido and erectile function to normal physiological “levels”. This is supported by the experiments described in Example 8 and Figs 16-18 below.
When the erectile dysfunction is other than drug induced, the bombesin antagonists (more particularly BB1 antagonists) can also be used to treat the erectile dysfunction by potentiating the normal endogenous erectiogenic mechanisms of the male (during normal sexual stimulation) and restoring the erectile function to normal levels during sexual stimulation. Thus Examples 170 and 171 and Figures 24, 25 and 26 hereinafter illustrate that bombesin antagonists (more particularly BBI antagonists) or a bombesin antagonist with a phosphodiesterase type 5 inhibitor potentiate erectiogenic mechanisms in animal models by enhancing intercavernosal pressure and potentiating the effect of the pelvic nerve stimulation-induced increases in intracavernosal pressure.
Early investigations also show that that the invention will help restore the libido/desire in males to normal levels where the desire dysfunction is not drug induced (e.g. psychogenic).
The psychogenic component of male sexual dysfunction has been classified by ] the nomenclature committee of the International Society for Impotence Research (and is illustrated by Sachs B. D., 2000) as generalised type, characterised by a general unresponsiveness or primary lack of sexual arousal, and ageing-related decline in sexual arousability, characterised by generalised inhibition or chronic disorders of sexual intimacy.
The compounds of this invention are useful in the treatment of male sexual ) dysfunction, especially drug-induced male sexual dysfunction and psychogenic male sexual dysfunction associated with generalised unresponsiveness and ageing-related decline in sexual arousability.
Female sexual dysfunction can be grouped into four classes (Scrip’s Complete
Guide to Women’s Healthcare, p.194-205, 2000), which include: e Hypoactive sexual desire disorders, which can be characterised as persistent or recurrent lack of sexual thoughts/fantasies and lack of receptivity to sexual activity, causing personal distress. e Sexual arousal disorders, which can be can be characterised as persistent or recurrent inability to achieve or maintain adequate sexual excitement, causing personal distress. The normal sexual arousal response consists of a number of physiological responses that are observed during sexual excitement. These changes such as vaginal, labial and clitoral engorgement result from increases in genital blood flow. Engorgement leads to increased vaginal lubrication via plasma transudation, increased vaginal compliance (relaxation of vaginal smooth muscle) and increases in vaginal and clitoral sensitivity. Female sexual arousal disorder (FSAD) is a highly prevalent sexual disorder affecting up to 40% of pre-, peri- and postmenopausal (HRT) women. The primary consequence of FSAD is reduced genital engorgement or swelling which manifests itself as a lack of vaginal lubrication and a lack of pleasurable genital sensation. Secondary consequences include reduced sexual desire, pain during intercourse and difficulty in achieving orgasm. The most common cause of FSAD is decreased genital blood flow resulting in reduced vaginal, . labial and clitoral engorgement. (Park, 1997; Goldstein, 1998; Berman, 1999,
Werbin, 1999). ) e Orgasmic disorders can be characterised as persistent or recurrent difficulty or delay in attaining orgasm after adequate sexual stimulation and arousal, causing personal distress.
e Sexual pain disorders can be characterised by dyspareunia, (characterised by recurrent or persistent genital pain associated with sexual intercourse), vaginismus (characterised by recurrent or persistent involuntary spasm of the muscles of the outer third of the vagina which interferes with vaginal penetration, causing personal distress) and other pain disorders (characterised by recurrent or persistent genital pain induced by non coital sexual stimulation).
The compounds of this invention are useful in the treatment of female sexual dysfunction (FSD), and this includes pre-, peri- and post-menopausal female sexual dysfunction associated with hypoactive sexual desire disorders, sexual arousal disorders, orgasmic disorders or anorgasmy, or sexual pain disorders.
Early investigations suggest the below mentioned female sexual dysfunction (FSD) patient groups should benefit from treatement with a bombesin antagonist or a bombesin antagonist and a PDESi (or other combination set out hereinafter). These patient groups are described in more detail in Berman er a/ (Urology, 1999). The invention finds application in the following sub-populations of patients with FSD: the young, the elderly (ageing-related sexual dysfunction), pre-menopausal, peri- menopausal, post-menopausal women with or without hormone replacement therapy.
More particularly the invention finds application in patients with FSD arising from:- (1) Arteriogenic/vasculogenic etiologies eg cardiovascular or atherosclerotic diseases, hypercholesterolemia, cigarette smoking, diabetes, hypertension, radiation and perineal trauma, traumatic injury to the iliohypogastric pudendal vacular system. (11) Neurogenic etiologies such as spinal cord injuries or diseases of the central nervous system including multiple sclerosis, diabetes, Parkinsonism, } cerebrovascular accidents, peripheral neuropathies, trauma or radical pelvic surgery. (iii) Hormonal/endocrine ctiologies such as dysfunction of the hypothalamic/pituitary/gonadal axis, or dysfunction of the ovaries, dysfunction of the pancreas, surgical or medical castration, androgen deficiency, high circulating levels of prolactin eg hyperprolactinemia, natural menopause, premature ovarian failure, hyper and hypothyroidism. (iv) Psychogenic etiologies such as depression, obsessive compulsive disorder, anxiety disorder, postnatal depression/”Baby Blues”, emotional and relational issues, performance anxiety, marital discord, dysfunctional attitudes, sexual phobias, religious inhibition or a traumatic past experiences. wv) Drug-induced sexual dysfunction resulting from therapy with selective serotonin reuptake inhibitors (SSRis) and other antidepressant therapies (tricyclics and major tranquillizers), anti-hypertensive therapies, sympatholytic drugs, chronic oral contraceptive pill therapy.
By drug-induced sexual dysfunction in females we mean to include cases where the drug treatment/therapy leads to delayed orgasm or inability to achieve an orgasm, reduced libido and FSD. The bombesin antagonists (more particularly BB1 antagonists) help restore orgasm, libido and female sexual function to normal physiological “levels”. Furthermore, since bombesin antagonists have been shown to have beneficial effects on sexual function in ovary intact and in ovarectomised animals, it is apparent that bombesin antagonists (more particularly BB1 antagonists) can also be used to treat female sexual arousal disorders (FSAD), hypoactive sexual desire disorders (HSDD) and anorgasmy (FOD) and also sexual pain disorders, especially where these are secondary to arousal disorders. In particular, Example 2 and Figures 2 and 21 and Example 169 and Figures 22 and 23 illustrate that the combinations and methods of treatment of the invention can enhance receptive behaviour and arousal via increased genital blood flow in women with FSAD and FOD respectively. Also, Examples 1, 4 and 5 and Figures 1,3, 4 and 5 illustrate that the combinations and methods of treatment of the invention can increase proceptive behaviour and restore normal desire/libido in women with HSDD.
Bombesin receptor antagonists - General
Bombesin receptors are present in hypothalamic areas. We have found that they can exert a neuromodulatory effect on sexual behaviour.
’ We have tested compounds that are bombesin receptor antagonists using animal models that we have refined and believe are reliable and predictive, in particular with the capacity to make predictions for females. In rodents proceptive behaviour is under hormonal control, progesterone being essential for induction of proceptive behaviour in combination with oestrogen (Johnson M, 1988). The evidence for the hormonal control of proceptive behaviour in primates is conflicting, but on the whole oestrogens and/or androgens appear to enhance proceptive behaviour (Baum
M.J, 1983). The behavioural manifestations of proceptive behaviour in the rat include “hopping and darting” movement, with rapid vibration of the ears. Tests to assess the eagerness to seek sexual contact (sexual motivation) have been reported as the most appropriate way to measure proceptivity (Meyerson, 1973). Receptivity, in the rat, is demonstrated when the female assumes a lordotic position. This occurs when, on mounting, the male exerts pressure with his forepaws on the flanks of the receptive female. The main sites of neuronal control for this behaviour are the ventromedial nucleus (VMN) and the midbrain central grey area (MCG) (for review, see Wilson
C.A,, 1993).
Bombesin is a 14-amino acid peptide originally isolated from the skin of the
European frog Bombina bombina (Anastasi A., 1971). It belongs to a class of peptides which share structural homology in their C-terminal decapeptide region (Dutta A.S.,
Small Peptides; Chemistry, Biology, and Clinical Studies). At present, two mammalian bombesin-like peptides have been identified, the decapeptide neuromedin
B (NMB) and a 23-residue amino acid, gastrin-releasing peptide (GRP).
Bombesin evokes a number of central effects through actions at a heterogeneous population of receptors. The BB, receptor binds neuromedin B (NMB) with higher affinity than gastrin-related peptide (GRP) and neuromedin C (NMC) and
BB; receptors bind GRP and NMC with greater affinity than NMB. More recently evidence has emerged of two more receptor subtypes denoted BB; and BB4 but due to limited pharmacology, little is known of their function at present. BB, and BB, receptors have a heterogeneous distribution within the central nervous system indicating that the endogenous ligands for these receptors may differentially modulate neurotransmission. Among other areas, BB, receptors are present in the ventromedial hypothalamus (Ladenheim E.E, 1990).
Bombesin-like immunoreactivity and mRNA have been detected in mammalian brain (Braun M., et al., 1978, Battey J., et al.1991). NMB and GRP are believed to mediate a variety of biological actions (for a review, see WO 98/07718).
The following patent applications disclose compounds capable of antagonising the effects of NMB and/or GRP at bombesin receptors: CA 2030212, EP 0309297, EP 0315367, EP 0339193, EP 0345990, EP 0402852, EP 0428700, EP 0438519, EP 0468497, EP 0559756, EP 0737691, EP 0835662, JP 07258081, UK 2231051, US 4943561, US 5019647, US 5028692, US 5047502, US 5068222, US 5084555, US 5162497, US 5244883, US 5439884, US 5620955, US 5620959, US 5650395, US 5723578, US 5750646, US 5767236, US 5877277, US 5985834, WO 88/07551, WO 89/02897, WO 89/09232, WO 90/01037, WO 90/03980, WO 91/02746, WO 91/04040, WO 91/06563, WO 92/02545, WO 92/07830, WO 92/09626, WO 92/20363, WO 92/20707, WO 93/16105, WO 94/02018, WO 94/02163, WO 94/21674, WO 95/00542, WO 96/17617, WO 96/28214, WO 97/09347, WO 98/07718, WO 00/09115, WO 00/09116. We believe that compounds disclosed in these applications can be used in the prevention or treatment of male and/or female sexual dysfunction, which is an indication that is not disclosed or suggested by the aforesaid applications, or indeed in any previous scientific publication concerning bombesin receptors.
Bombesin receptor antagonists to which this invention is applicable include both non-peptide compounds and peptide compounds. Compounds that can be formulated into compositions for oral administration, especially human oral administration, without substantial loss of activity are preferred. Many non-peptide compounds having the desired properties fall into this category.
A) Non-peptide bombesin receptor antagonists
One preferred genus of compounds for use in 'the invention comprises bombesin receptor antagonists of the formula (I) 3 vo Tg Nf L al : Src 3 | TTR R?
Ke 0 Ar! ) ® and pharmaceutically acceptable salts thereof, wherein: e jisOorl; eo kisOorl; eo 1is0,1,2, 0r3; e misQorl; e nis0,1o0r2; e Ar is phenyl, pyridyl or pyrimidyl, each unsubstituted or substituted by from 1 to 3 substituents selected from alkyl, halogen, alkoxy, acetyl, nitro, amino, -
CHoNRIORIL cyano, -CF3, -NHCONHp, and -CO7R 12; e Rlis hydrogen or straight, branched, or cyclic alkyl of from 1 to 7 carbon atoms; e R8is hydrogen or forms a ring with R! of from 3 to 7 carbon atoms; e R2ijs hydrogen or straight, branched, or cyclic alkyl of from 1 to 8 carbon atoms which can also contain 1 to 2 oxygen or nitrogen atoms; e RY is hydrogen or forms with R2 a ring of from 3 to 7 carbon atoms which can contain an oxygen or nitrogen atom; or RZ and RY can together be a k carbonyl; o Ar! can be independently selected from Ar and can also include pyridyl-N- : 25 oxide, indolyl, imidazolyl, and pyridyl,
e R4 R5 RO, and R7 are each independently selected from hydrogen and lower alkyl; R4 can also form with RS a covalent link of 2 to 3 atoms which may include an oxygen or a nitrogen atom; e R3 canbe independently selected from Ar or is hydrogen, hydroxy, -NMe2,
N-methyl-pyrrolyl, imidazolyl, N-methyl-imidazolyl, tetrazolyl, N-methyl- tetrazolyl, thiazolyl, -CONRI3R14, alkoxy,
H, ’ L] Ar? ’ Ar 3 o 0
A= oA CFs
Ar © ,or Ar? CF, wherein p is 0, 1 or 2 and Ar2 is phenyl or pyridyl; e RI0 RI RI2 R13 and R14 are each independently selected from hydrogen or straight, branched, or cyclic alkyl of from 1 to 7 carbon atoms.
Preferred compounds are those of Formula (Ia)
RS R7 6
Plier OO
A | N—C—C—N—-oC R3
NY Q H, i 0 Ar! (a) wherein e Ar is phenyl unsubstituted or substituted with 1 or 2 substituents selected from isopropyl, halo, nitro, and cyano; . e R#4 R3, and RY are hydrogen; e R7is methyl or hydrogen; e R3is 2-pyridyl or hydroxy; and eo Ar! is indolyl, pyridyl, pyridyl-N-oxide, or imidazolyl.
Other preferred compounds are those of Formula I wherein ’ e Ar is unsubstituted phenyl; e Rlis cyclopentyl or tert-butyl; e R4and RS are hydrogen; e R7is methyl; e RO is hydrogen; : eo R3is phenyl with two isopropyl substituents, unsubstituted phenyl, or 0] oN ; and
Ar! is indolyl.
Other preferred compounds are those of Formula I wherein e Aris 2,6-diisopropyl-phenyl, 4-nitro-phenyl, and 4-cyano-phenyl; ° R4, RS, and RO are hydrogen; e Ris methyl; eo R2is hydrogen or cyclohexyl; and e R3 is hydroxyl, pyridyl, 0] sel sals nat , , or .
At present, most preferred of the compounds of formula (I) are (S) 3-(1H-
Indol-3-y!)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[3-(4-nitro- phenyl)-ureido}-propionamide (also referred to as Compound 1) and its pharmacologically acceptable salts and (28)-N-{[1-(4- aminophenyl)cyclohexyl]methyl}-3-(1 H-indol-3-yl)-2-methyl-2-{[(4-nitroanilino)-
carbonyl]lamino}propanamide (also referred to as Compound 3) and its : pharmacologically acceptable salts.
Other preferred compounds of Formula (1) are set out below and included also are their pharmaceutically acceptable salts: (S) N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)- 2-methyl-propionamide;
N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-
N-methyl-propionamide;
N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-1-methyl-ureido]-3-(1 H- indol-3-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3-(1-oxy-pyridin-2-yl)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3-pyridin-2-yl-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide; 2-[3-(2-tert-butyl-phenyl)-ureido]}-N-cyclohexylmethyl-3-(1 H-indol-3-yl)-2- methyl-propionamide;
N-cyclohexylmethyl-2-[3-(2,6-dichloro-phenyl)ureido]-3-(1 H-indol-3-yl)-2- methyl-propionamide;
N-cyclohexylmethyl-2-[3-(2,6-dimethoxy-phenyl)ureido]-3-(1 H-indol-3-yl)-2- methyl-propionamide;
N-cyclohexylmethyl-2-[3-(2,6-dimethylamino-phenyl)-ureido]-3-(1 Hindol-3- yl)-2-methyl-propionamide; (S) N-cyclohexylmethyl-3-(1H-indol-3-yl)-2-methyl-2-[3-(4-nitro-phenyl)- ureido]-propionamide;
N-cyclohexylmethyl-2-[3-(2,2-dimethyl- 1 -phenyl)propyl)-ureido]-3-(1H- . indol-3-yl)-2-methyl-propionamide; [S-(R*, R*)] 3-(1 H-indol-3-yl)-2-methyl-2-{3-[1-(4-nitro-phenyl)-ethyl]- . ureido}-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide;
N-(2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-3-(1 H-indol-3-yl)-2-methyl-2-[3- (1-phenyl-cyclopentylmethyl)ureido]-propionamide;
(S)-N-(2,6-diisopropyi-phenyl)-2-[3-(2,2-dimethyl-1-phenyl-propyl)-ureido]- ’ 3-(1H-indol-3-yl)-propionamide; (R)-N-(2,6-diisopropyl-phenyl)-2-[3-(2,2-dimethyl-1-phenyl-propyl)-ureido]- 3-(1H-indol-3-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-N-(2,2-dimethyl-4-phenyl-[ 1,3 Jdioxan- 5-yD)-3-(1 H-indol-3-yl)-2-methyl-propionamide;
N-cyclohexyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2- methyl-propionamide;
N-(2-cyclohexyl-ethyl)-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3- yl)-2-methyl-propionamide; 2-[3~(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl- propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(3- methyl-butyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido}-3-(1 H-indol-3-yl)-2-methyl-N-(3- phenyl-propyl)-propionamide; 2-{3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indoi-3-yl)-2-methyl-N-(1,2,3,4- tetrahydro-naphthalen-1-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(2- phenyl-cyclohexyl)-propionamide; 2-[3~(2,6-diisopropyl-phenyl)-ureido]-N-indan-1-yl-3-(1 H-indol-3-yl)-2- methyl-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-N-(I-hydroxy-cyclohexylmethyl)-3-(1 H- indol-3-yl)-2-methyl-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; . 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(6,7,8,9- tetrahydro-5 H-benzocyclohepten-5-yl)-propionamide; . 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-phenyl- propionamide;
N-(1-hydroxy-cyclohexylmethyl)-3-(1 H-indol-3-yl)-2-methyl-2-[3-(4-nitro- phenyl)-ureido]-propionamide;
2-[3-(4-cyano-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; (S) 3-(1H-indol-3-yl)-2-methyl-2-[3-(4-nitro-phenyl)-ureido]-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide; (S) 3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-[3-(4- trifluoromethyl-phenyl)-ureido}-propionamide; (S) 4-(3-{2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-ureido)-benzoic acid ethyl ester; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-imidazol-4-yl)-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-3-(2-trifluoromethyl-phenyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3-(2-nitro-phenyl)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S) 3-(1 H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2- methyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide; and
N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-ureido}-2-methyl-3- pyridin-2-yl-propionamide.
Another preferred genus of compounds which can be used for the present purpose is of formula (II) and includes pharmaceutically acceptable salts thereof:
R® R® R* R! (Ar), - (CH,), - X), -(CH,), - ! - | - G - | -(CH,), - & (CH,), -R?
NEL wherein: e jis0,1o0r2; eo kisOorl; eo lis0,1,2,0r3; e misQorl; e nis0,1or2;
e qisQorl; ’ e ris0Qorl; whenris 0, Ar is replaced by hydrogen; eo Ar is phenyl, pyridyl, pyrimidyl, thienyl, furyl, imidazolyl, pyrrolyl or thiazolyl each unsubstituted or substituted by from 1 to 3 substituents selected from acetyl, alkoxy, alkyl, amino, cyano, halo, hydroxy, nitro, sulfonamido, sulfonyl, -CF;, -OCF;, -CO;H, -CH,CN, -SO,CF;, -CH,COH and - (CH)NR'R' wherein s is 0, 1, 2 or 3 and R7 and R® are each independently selected from H, straight or branched alkyl of up to 6 carbon atoms, or R’ and
R® together with the nitrogen atom to which they are linked can form a 5- to 7- membered aliphatic ring which may contain 1 or 2 oxygen atoms; e R'is hydrogen, straight or branched alkyl of up to 6 carbon atoms or cycloalkyl of between 5 and 7 carbon atoms which may contain 1 or 2 nitrogen or oxygen atoms; e R® is hydrogen, methyl, or forms with R' an aliphatic ring of from 3 to 7 atoms which can contain an oxygen or nitrogen atom, or together with R' is a carbonyl group;
J Ar is independently selected from Ar or is indoly! or pyridyl-N-oxide; ° rR’, rR, and rR’ are each independently selected from hydrogen and lower alkyl; 20 . R’ is independently selected from Ar or is hydrogen, hydroxy, alkoxy, -NMe», CONRR" wherein rR’ and R" are each independently selected from hydrogen, straight or branched alkyl of up to 6 carbon atoms, or R’ and R!° together with the nitrogen atom to which they are linked can form a 5-to 7- membered aliphatic ring which may contain 1 or 2 oxygen or nitrogen atoms, or RZis
0 0) ~~ 0 NCP
IYER , or Ar? CF, wherein pis 0, 1 or 2 and Ar2 is phenyl or pyridyl; e X is adivalent radical derived from any of the following
N Ng. N N N => ~N > ~~ ENG a © Uo uO
N N _N S N
NN NN NN NT
GQ W@W OO vw 0 9
QO OQ Foy ho
CQ ay o o 0
Oy Oy ° Neso ¢ 0 a a OC
Vi N _N aa 4 a aA oO
N R11 R11 R11
G4 0 0 go 0 oN R12 R12 LIC
R11 R11 R11 R11 R11
DEINE INSEE SIN PE
RIX TN Ry? N RF TO Rai $ Rri2¥ 0 . R11 R11 R11
C00 0) iO
R12 S Rez R12 N R12
R11 Ny R11 NN (20 LC
R12 R12 where the ring nitrogen atoms may have lower alkyl groups attached thereto, RY ‘ and R'? are independently selected from H, halogen, hydroxy, alkoxy, acetyl, nitro, cyano, amino, CF; and -(CH,)NR"R" where t can be 0 or 1, Rand RY are each independently selected from hydrogen, straight or branched alkyl of up to 6 carbon atoms or cycloalkyl of 5 to 7 carbon atoms, containing up to 2 oxygen or nitrogen atoms.
A preferred species of compounds within the genus defined by formula (II) is represented by the formula (Ila), and includes pharmaceutically acceptable salt thereof:
R3® RS Rr? pox gob eng on
J 0
Ar} RS (11a) wherein: e nisOorl; e Ar is phenyl or pyridyl which may be unsubstituted or substituted with from 1 to 3 substituents selected from halogen, alkoxy, nitro and cyano; e Ar is independently selected from Ar or is pyridyl-N-oxide or indolyl; e R® forms with R' an aliphatic ring of from 3 to 7 atoms which can contain an oxygen or nitrogen atom, or together with R' is a carbonyl group; e R’ is independently selected from Ar or is hydrogen, hydroxy, alkoxy, 9 0 dimethylamino, tetrazolyl or -CONR R" wherein rR and R' are each . independently selected from hydrogen or methyl or R? is any of fo) 0) oN oC ar © , or Ar? CF, wherein p is 0, 1 or 2 and Ar? is phenyl or pyridyl; e R’, R* and R® are each independently selected from hydrogen and methyl; and eo Xs selected from:
Na N PAN Nn Ny S
Oo uw OQ wv 9
N R11 N
Ng Ng 0 o LL N
GO OO Gi OO LL
R11 R11 R11 N R11 N R11
Po 0 Ln 20 00
R13 S$ RZ © R12 5 R12 7 rez #
R'' and R" being independently selected from H, halogen, hydroxy, alkoxy, acetyl, nitro, cyano, amino, CF; and (CH,),NR"’R'* wherein tis 0 or 1 and R"? and R' are independently selected from hydrogen and methyl.
A sub-species of preferred compounds within the general formula (II) has the formula (IIb) or (lc): . \ \ 0800 a
Ar A, R2 Ar py N R2 (11b) (Ikc)
wherein Ar and R? independently represent phenyl or pyridyl which may be : unsubstituted or substituted with from 1 to 3 substituents selected from halogen, alkoxy, nitro and cyano, and pharmaceutically acceptable salts thereof.
A particularly preferred compound falling within formula (II) is (S)-3-(1H- indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[4-(4-nitro- phenyl)-oxazol-2-ylamino]-propionamide (also referred to as Compound 2) and its pharmaceutically acceptable salts.
Other preferred compounds falling within formula (II) are described below in
Examples 10-27 and are included within the invention, as also are their pharmaceutically acceptable salts.
A third genus of bombesin receptor antagonists according to the invention has the formula (111) and include pharmaceutically acceptable salts thereof: 3 5 4 1
EP
Ar 0 R® (111) wherein: e kisO,1o0r2; eo lis0,1,20r3; e misOorl; e nis0,1or2; eo Xis-CO-,-0CO, -SO- and -SO,-; ’ 25 e Ar is benzimidazolyl, benzofuryl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzopyrazinyl, benzotriazolyl, benzoxadiazolyl, furyl, ) imidazolyl, indanyl, indolyl, isoquinolyl, isoxazolyl, naphthyl, oxazolyl, phenyl, pyrazinyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidyl, pyrrolyl, quinolinyl, tetralinyl, tetrazolyl, thiazolyl, thienyl or triazolyl each unsubstituted or substituted with from 1 to 3 substituents selected from amino, ’ acetyl, alkyl (straight chain or branched with from 1 to 6 carbon atoms), alkoxy, cyano, halogen, hydroxy, nitro, phenyl, pyridyl, pyrrolyl, isoxazolyl, phenoxy, tolyloxy, - CF3, -OCF3, -SO,CF3;, -NHCONH,, -CO-H, -CH,CO,H, -CH,CN, SO;Me, SO,NH;, SO;Ph, -(CH),NR'R®, -CONR’R'’, and COR", wherein q is 0, 1 or 2 and R, R® R’, RO R!! are each independently selected from hydrogen or straight or branched alkyl of up to 6 carbon atoms or cyclic alkyl of between 5 to 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms or R” and R® or R® and R'® together with the nitrogen atom to which they are linked can form a 5- to 7-membered aliphatic ring which may contain 1 or 2 oxygen or nitrogen atoms; e Aris independently selected from Ar and can also be pyridyl-N-oxide; e R'is hydrogen or straight or branched alkyl of up to 6 carbon atoms or cyclic alkyl of between 5 and 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms; e R?is independently selected from Ar or is hydrogen, hydroxy, alkoxy, -NMe,, -CONR'R"P, for A
Ac,
AR oN , or ar Op wherein pis 0, 1 or 2, Ar is phenyl or pyridyl; and, R12 and R13 are each . 20 independently selected from hydrogen, straight or branched alkyl of up to 6 carbon atoms or cyclic alkyl of between 5 and 7 carbon atoms; : e R* Rand R® are each independently selected from hydrogen and lower alkyl; and e R® is hydrogen, methyl or forms with R' a ring of from 3 to 7 carbon atoms : which can contain an oxygen or nitrogen atom, or R' and R® can together be carbonyl.
In a preferred group of the compounds of formula (III): eo kisQOorl; s lis]; e misQOorl; eo nisQorl; e Xis-C(O)-, -OC(0O)-, or -SO;,-; eo Ar is benzofuryl, furyl, indolyl, isoquinolyl, naphthyl, phenyl, pyridyl, quinolyl or thienyl each unsubstituted or substituted with 1 or 2 substituents selected from alkoxy, cyano, halogen, nitro, phenyl, phenoxy, -CF3, -(CH2)NR'R®, wherein R’ and R® can form a ring of between 5 to 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms, or R’ and R® can be independently selected from hydrogen, straight or branched alkyl of up to 4 carbon atoms or cyclic alkyl of 5 carbon atoms; e Ar is independently selected from Ar, preferably indolyl, and can also be pyridyl-N-oxide; e Rand R® can form a cyclic alkyl of from 5 to 7 carbon atoms or R' and R® together are carbonyl; e R? is independently selected from unsubstituted or substituted pyridyl or is hydrogen, hydroxy, alkoxy, -NMe,, -CONR'?R"? wherein R'?and R"? are each independently selected from H and CHj; eR’ R*and R’ are each independently selected from hydrogen and methyl. . In another preferred group of the compounds of Formula (III), oe lis; ) e misl; : eo nis0; e RZis 2-pyridyl;
] WQO 02/40008 PCT/GB01/05018 e R® forms a cyclohexyl with R.
A particularly preferred group of compounds is of formula (I111a):
R
0) =
Ar- (CHy), -X- NA oul R < N N oH =
N (11a)
H wherein Ar, k and X have the meanings given above in first, and the pyridine ring is optionally substituted by with 1 or 2 substituents, R and R’, independently selected from alkoxy, cyano, halogen, nitro, phenyl, phenoxy, - CF; -(CH)NR'R?, wherein
R’and R® together with the nitrogen atom to which they are linked can form a 5- to 7-membered aliphatic ring which may contain 1 or 2 oxygen or nitrogen atoms, or R’ and R® can be independently selected from hydrogen or cyclic alkyl of between 5 to 7 carbon atoms, and their pharmaceutically acceptable salts thereof.
In a further set of compounds (Illa), Ar is benzofuryl, furyl, indolyl, isoquinolyl, naphthyl, phenyl, pyridyl, quinolyl or thienyl each unsubstituted or substituted with 1 or 2 substituents selected from alkoxy, cyano, halogen, nitro, phenyl, phenoxy, -CF3, -(CH2)NR'R?, wherein R” and R® can form a ring of between 5 to 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms, or R” or R® can be a independently selected from hydrogen or cyclic alkyl of 5 carbon atoms, and X is —
C(0)-, -OC(0)- or -S0O;.
Preferred N-terminal amide derivatives of the compounds of formula (III)
Amongst N-terminal amide derivatives, i.e. compounds of formula (III) wherein X is -C(O)-, the following compounds are most preferred:
N-{(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- ‘ carbamoyl]-ethyl}-4-nitro-benzamide;
C-dimethylamino-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl}-benzamide; 1 H-indole-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoylj-ethyl}-amide; benzo[b]thiophene-2-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl }-amide;
N-{(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-2-pyrrol-1-yl-benzamide 1 H-indole-5-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; and 1 H-indole-2-carboxylic acid ((S)-2-(1H-indol-3-yl)-1-{[1-(5-methoxy-pyridin- 2-yl)-cyclohexylmethyl]-carbamoyl}-1-methyl-ethyl)-amide.
Other preferred N-terminal amide derivatives of formula (III) include the compounds of Examples 32-35, 37-47, 49-60, 62-80, 82-85 and N-{(S)-2-(1H-indol- 3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl }-2-pyrrol-1- yl-benzamide.
Preferred N-terminal urethane derivatives of the compounds of formula (III)
Amongst N-terminal urethane derivatives, i.e. compounds of formula III wherein X is ~OC(=0)- , the following compounds and theor pharmaceutically acceptable salts are particularly preferred: {(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid naphthalen-1-ylmethyl ester; . {(S)-2-(1H-Indol-3-y1)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-carbamic acid 3,4-dichloro-benzyl ester; i 30 {(8)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4-nitro-benzyl ester; {(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3-nitro-benzyl ester;
{(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- . carbamoyl]-ethyl}-carbamic acid 3-cyano-benzyl ester; {(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-carbamic acid 3-trifluoromethyl-benzyl ester; {(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2,3-dichloro-benzyl ester; and {(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid quinolin-6-ylmethy! ester.
Other preferred N-terminal urethane derivatives of formula (III) include the compounds of Examples 88-90, 92-95, 97-98, 100-102, 104-106 and 108.
Preferred N-terminal sulfonamide derivatives of the compounds of formula (IIT)
Amongst N-terminal sulfonamide derivatives of formula (III) (compounds of formula (III) wherein X is -SO,-) the following compounds are particularly preferred: (8)-3-(1H-indol-3-yl)-2-methyl-2-phenylmethanesulfonylamino-N-(1-pyridin- 2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2-chloro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-2-(naphthalene-1-sulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (quinoline-8-sulfonylamino)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethy!)-2-(2- trifluoromethyl-benzenesulfonylamino)-propionamide; (S)-2-(biphenyl-2-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide; (8)-3-(1H-indol-3-yl)-2-methyl-2-(5-methyl-2-phenoxy-benzenesulfonyl- amino)-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; and (S)-3-(1H-indol-3-y})-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2-p- tolyloxy-benzenesulfonylamino)-propionamide.
Further preferred N-terminal sulfonamide derivatives of formula (III) include the compounds of Examples 112, 114, 116-119, 121-128, 130-151, 155-168 and the following:
(S)-3-(1 H-indol-3-yl)-2-methanesulfonylamino-2-methyl-N-(1-pyridin-2-yl- . cyclohexylmethyl)-propionamide; and (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2,2,2- . trifluoro-ethanesulfonylamino)-propionamide.
The compounds of the general formulae above are optically active. The scope of the invention therefore also includes: e All stereoisomers of the compounds of the above general formulae. e The solvates, hydrates and polymorphs (different crystalline lattice descriptors) of the above compounds. e Pharmaceutical compositions of the above compounds. e Prodrugs of the above compounds such as would occur to a person skilled in the art; see Bundgaard et al (1987).
The alkyl groups contemplated by the invention include straight, branched, or cyclic carbon chains of from 1 to 8 carbon atoms except where specifically stated otherwise. Representative groups are methyl ethyl, propyl, isopropyl, n-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, 2-methylhexyl, n-pentyl, 1-methylbutyl, 2,2-dimethyibutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl, and the like.
The lower alkyl groups include carbon chains of up to 6 carbon atoms. The cycloalkyl groups contemplated by the invention comprise those having 3 to 7 carbon atoms including cyclopentyl and cyclohexyl. They may be substituted with from 1 to 3 groups selected from halogens, nitro, alkyl, and alkoxy.
The alkoxy groups contemplated by the invention comprise both straight and branched carbon chains of from 1to 6 carbon atoms unless otherwise stated. ’ Representative groups are methoxy, ethoxy, propoxy, i-propoxy, t-butoxy, and hexoxy.
The term "halogen" is intended to include fluorine, chlorine, bromine, and iodine.
: The term "amine" is intended to include free amino, alkylated amines, and acylated amines.
The term “subject” includes animals, particularly mammals and more particularly humans.
Optical isomers and salts
The compounds of the above general formulae all have at least one chiral centre and some have multiple chiral centres depending on their structure. In particular, the compounds of the present invention may exist as diastereoisomers, mixtures of diastereoisomers, or as the mixed or the individual optical enantiomers.
The present invention contemplates all such forms of the compounds. The mixtures of diastereoisomers are typically obtained as a result of the reactions described more fully below. Individual diastereoisomers may be separated from mixtures of the diastereoisomers by conventional techniques such as column chromatography or repetitive recrystallization. Individual enantiomers may be separated by conventional methods well known in the art such as conversion to a salt with an optically active compound, followed by separation by chromatography or recrystallization and reconversion to the non-salt form.
Where it is appropriate to form a salt, the pharmaceutically acceptable salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium acetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycoloylarsanilate, . hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, . methylbromide, methylnitrate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, triethiodide, benzathine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.
Preferred salts are made from strong acids. Such salts include hydrochloride, mesylate, and sulfate.
Other non-peptide bombesin antagonists
Other non-peptide bombesin antagonists which are believed to be suitable for use in the present invention are described and claimed in the following documents, the contents of which are incorporated herein by reference: WO 00/09115, WO 00/09116,
WO 92/07830, JP 07258081 and WO 98/07718.
Preparative methods for the compounds of formula (I)
Preparation of the compounds of formula (I) is described in WO 98/07718, the disclosure of which is incorporated herein by reference.
Preparation of the closest reported analogues of Compound 3 - (25)-N-{[1-(4- aminophenyl)cyclohexyl]methyl}-3-(1 H-indol-3-yl)-2-methyl-2-{[(4-nitroanilino)- carbonyl]amino}propanamide are described in Ashwood, V. Brownhill, M.
Higginbottom, D. C. Horwell, J. Hughes, R. A. Lewthwaite, A. T. McKnight, R. D.
Pinnock, M. C. Pritchard, N. Suman-Chauhan, C. Webb and S. C. Williams. Bioorg.
Med. Chem. Lett., 1998, 8, 2589-2594; J. E. Eden, M. D. Hall, D. C. Horwell, W.
Howson, J. Hughes, R. E. Jordan, R. A. Lewthwaite, K. Martin, A. T. McKnight, J.
O’Toole, R. D. Pinnock, M. C. Pritchard, N. Suman-Chauhan and S. C. Williams. } Bioorg. Med. Chem. Lett., 1996, 6, 2617-2622. and in WO98/07718. Compound 3 can be synthesized using methods disclosed in the above publications.
o NH,
Rg $ hid N
OY Ps”
J HN
Preparative methods for compounds of formula (II)
Throughout this application the following abbreviations have the meanings listed below:
NEt; triethylamine
THF tetrahydrofuran
HBTU O-benzotriazol-1-yl-N, N,N’, N -tetramethyluronium hexafluorophosphate
DIPEA N, N-diisopropylethylamine
DMF N,N-dimethylformamide
TEBA benzyltriethylammonium chloride
BOC20 di-tert-butyl dicarbonate
TFA trifluoroacetic acid
DMA N, N-dimethylacetamide
EtOAc ethyl acetate
MeOH methanol
Trp tryptophan
Ph phenyl y HPLC high pressure liquid chromatography
NP normal phase ’ RP reverse phase
DMAP N,N-dimethyl-4-amino pyridine
OAc acetate
OB oestradiol benzoate
The production of compounds of the formula (IT) in which X is oxazolyl is shown in Scheme 1 which illustrates the synthesis of the compounds of Examples 9 to 12 in four steps via Intermediates 4a or 4b. The steps are: e Formation of the p-nitrophenylcarbamate of the methyl ester (Intermediate 1) and subsequent treatment with aqueous ammonia to give a primary urea (Intermediate 2). e Cyclisation of the primary urea with 2-bromo-1-(4-nitro-phenyl)-ethanone to form an oxazole ring (Intermediate 3). e Hydrolysis of the methyl-ester-protecting group gives Intermediates 4a or 4b. ® Reaction of Intermediate 4a or 4b with the amine Z2, using HBTU to form an amide linkage, affords the desired compounds.
Scheme 1: 2 z1 z1 . S N .. 0) N
J i S ii / S on oe AXon Ores o 3 O,N I
Intermediate 1, a-b Intermediate 2, a-b Intermediate 3, a-b a. Z1 = CH,jindole b. Z1 = CH,Ph | i. 11} 2 21 0 > R 0 3
PN 2 v PN po
ON NON = oN NOON 0 lo}
Intermediate 4, a-b
Example 9, 21 = CH,indole, 22 = N N a. Z1 = GH, indole ome b. Z1 = CH,Ph
Example 10, 21 = CH,indole, Z2 = Ld on [o]
Example 11, Z1 = CH,indole, 22 = “O
Example 12, 21 =CH,Ph, 22 = Se i ~~)
OMe
In the above scheme: i) a) 4-Nitrophenylchloroformate, NEt;, THF b) NH; aq. ii) 2-bromo-1-(4-nitro-phenyl)-ethanone in either toluene/dioxan at reflux (3a) or 1,2-dichloroethane at reflux (3b) iii) LiOH, dioxan, H,O iv) HBTU, DIPEA, DMF, 72 . 10 Scheme 2 describes the synthesis of the compounds of Examples 13 to 15 from
Intermediate 2a. e A primary urea 2a is cyclised with an appropriate bromomethyl ketone containing the group Z3 to form an oxazole ring (Intermediate 5). : e Hydrolysis of the methyl ester protecting group of the resulting Intermediate
Sa, 5b or Sc gives the Intermediates 6 a-c. eo Reaction of an Intermediate 6a, 6b or 6c with [1-(5-methoxy-2- pyridyl)cyclohexyljmethanamine in the presence of HBTU to form an amide bond affords the desired compounds.
Scheme 2: o L | Ny 5a, 73 = 4-NCPhen s —_— R 5b, 23 = Phenyl-
A) * Li < o< ALAN \ o< Sc¢,z3= o- le} 0
Intermediate 2a Intermediate 5, a-c
N N
N N o } i oy = oF © ZS oMe 0
Intermediate 6, a-c
In the above scheme: 1) DMF at 30°C i1) LiOH, dioxan, H,O : i11) HBTU, DIPEA, DMF, [1-(5-methoxy-2-pyridyl)cyclohexyl]methanamine (described in WO 98/07718) . Scheme 3 describes a two step synthesis for the compounds of Examples 16-23.
The reactions are preferentially carried out as a “one-pot” process in which: : e An aromatic ring of a compound Z5-Br or Z5-Cl is appended onto the N. terminal of the illustrated amino acid using a copper catalysed reaction. e Formation of an amide linkage between the resulting acid and [1-(5-methoxy- 2-pyridyl)cyclohexyl]methanamine or [1-(2-pyridyl)cyclohexyl]methylamine in the presence of HBTU affords the desired compounds.
Scheme 3:
N N ior ii N mee bo =, LQ © ° #74
Intermediate 7
S ot Da
Example 16 Z4=OMe Z5= on
Oo
Example 17 Z4=H Z5= Qa *
Example 18 Z4=H Z5= N+
N.
Example 19 Z4=H 2Z5= 38 (>)
Example 20 Z4=H Z5= \ 7 * ]
Example 21 Z4=H 25= a
Example 22 Z4=H Z5= <r
Example 23 Z4=H Z5= oe
In the above scheme: i) a) 10% Cul, K,CO;, DMF, 130°C - b) HBTU, DIPEA, DMF, and [1-(5-methoxy-2-pyridyl)cyclohexyljmethan- amine (described in WO 98/07718) or [1-(2-pyridyl)cyclohexylimethylamine ; (described in WO 98/07718) ii) a) 5-10% Cul, K,CO;, TEBA, Pd(P(o-tolyl);)Cl;, DMF, 130°C b) HBTU, DIPEA, DMF, and [1-(5-methoxy-2-pyridyl)cyclohexylmethan- : amine (described in WO 98/07718) or [1-(2-pyridyl)cyclohexylJmethylamine (described in WO 98/07718) 5S * represents the attachment point.
Scheme 4 describes the two step one-pot synthesis of the compound of Example 24: eo The aromatic ring is appended onto the N-terminal of the amino acid (Intermediate 8) using a copper catalysed reaction and then an in situ HBTU amide bond formation reaction affords the desired compound.
Scheme 4:
Co. OC
Xp OX o Oo Z
Intermediate 8 Example 24
In the above scheme: i) 10% Cul, K,CO;, DMA, 90°C it) HBTU, NEt;, DMA, [1-(2-pyridyl)cyclohexyllmethylamine (described in WO 98/07718)
Scheme 5 describes the synthesis of the compounds of Examples 25 - 27 via
Intermediate 10 by the steps of : e N-BOC protection of the amino acid (Intermediate 7) which provides the groups R’ and Ar’.
e Reaction of the protected amino acid with an amine that provides the groups ) R', R?, R* and R® using HBTU to form an amide linkage, and thereby give the
Intermediate 9. e¢ N-BOC deprotection of the Intermediate 9 to give Intermediate 10. e Reductive amination of Intermediate 10 with the appropriate aldehyde
Z6CHO to give the desired compounds.
Scheme 5:
N N N x — xy I~ = (] o . [11] . [o] oO lo] ¥
Intermediate 7 Intermediate 9 Intermediate 10
Exemple 25, Z6= QL iv ng Exemple 26, Z6= at [o] aN $ N. Na » 2 4 o I P Exemple 27, 26= o pe
In the above scheme: 1) BOC,0, K,CO;, dioxane, water 1) HBTU, DIPEA, [1-(2-pyridyl)cyclohexyl methylamine (described in WO 98/07718), DMF iii) TFA, CHCl, iv) NaBH(OAc);, 1,2-dichloroethane. * represents the attachment point.
Scheme 6 describes the synthesis of Intermediate 13.
e The alcohol 11 is methylated using sodium hydride. : e The resulting nitrile is reduced using Raney nickel under an atmosphere of hydrogen.
Scheme 6:
Qo QQ
NC o NC ol N ou .
Intermediate 11 Intermediate 12 Intermediate 13
In the above scheme: i) NaH, CHsl, THF ii) Raney nickel, ethanolic ammonia, H, 345 kPa
Intermediate 13
C-(1-methoxymethyl-cyclohexyl)-methylamine
N. 3 0
Intermediate 13
The above compound was prepared as shown in Scheme 6. 1. Sodium hydride (862mg, 21.5mmol, 60% in oil) was taken up in THF (50ml) under argon at 0°C. To this was added a solution of methyl iodide (1.34ml, 21.6mmol) ) 20 and 1-hydroxy-cyclohexanecarbonitrile (1.0g, 7.18mmol; see J. Frohlich er al., 1994) in THF (30ml) dropwise over 45 minutes. Once addition was complete the reaction mixture was stirred at room temperature overnight, and then quenched with i-propanol followed by water (100ml). The mixture was then extracted with dichloromethane (2x150ml). The combined organic phases were dried (MgSO,) and solvent removed under reduced pressure. Residue was purified by chromatography using heptane/ethyl : acetate (4:1). Removal of solvent under reduced pressure gave 1-methoxymethyl- cyclohexanecarbonitrile (1.1g, 88%) as a pale yellow oil.
IR (film): 2934, 2861, 2832, 2235, 1476, 1452, 1385, 1211, 1187, 1185, 1126, 1102, 978, 932,901,849 cm’; 'H NMR (CDCl): & = 1.13-1.33 (3H, m), 1.57-1.78 (5H, m), 1.94-2.02 (2H, m), 3.36 (1H, s), 3.42 (3H, s); 2. To the 1-methoxymethyl-cyclohexanecarbonitrile (1.1g, 7.2mmol) in ethanolic ammonia (60ml) was added Raney nickel catalyst (0.55g, pre-washed with water and ethanol). Reaction mixture was shaken for 16 hours under hydrogen (345 kPa) at 30°C. The catalyst was filtered off catalyst with extreme caution through a bed of
Kieselguhr and washed with ethanol. Removal of the solvent under reduced pressure gave Intermediate 13 (1.12g, 99%) as a yellow oil.
MS m/e (ES+): 158.2 (M* + H, 100%);
IR (film): 2926, 2857, 1572, 1452, 1378, 1316, 1190, 1140, 966 cm’; 'H NMR (CDCl): 8 = 1.20-1.60 (12H, m), 2.62 (2H, s), 3.23 (2H, s), 3.32 (3H, s)
Preparative methods for compounds of formula (III)
Compounds of the formula (IIT) in which X is —CO- can be prepared by condensing an acid of the formula (ITI-1) a5 Ar - (CH) -COOH (1II-1) or a derivative thereof with an amine of the formula (I11-2) rrroor
HN-C - fi -N- (CH), - (Pn - (CH), - R? » 0 RS 11-2)
in an aprotic polar solvent in the presence of an appropriate catalyst, the values of the : substituents Ar, Ar’ and R! to R® and the parameters k to n being as defined above with reference to formula (III), and optionally converting the resulting product to a pharmaceutically acceptable salt. For example, the condensation may be carried out in dimethylformamide using O-benzotriazol-1-yl-N,N,N’, N -tetramethyluronium hexafluorophosphate (HBTU) and N, N-diisopropyl-ethylamine (DIPEA) as catalyst.
Compounds of the formula (III) in which X is -OC(=0)- can be prepared by forming a carbonate from an alcohol of the formula (111-3)
Ar -(CH,),-OH (111-3) and reacting the carbonate with an amine of the formula (111-2)
I i f i : HN == N= (CH= (Qn = (CH), - RE " 0 R® (111-2) in an aprotic polar solvent in the presence of a base, the values of the substituents Ar,
Ar' and R! to R® and the parameters k to n being as defined above with reference to formula (III), and optionally converting the resulting product to a pharmaceutically acceptable salt. For example, the compound of formula (III-3) may be reacted with 4- nitrophenyl chloroformate in dichloromethane using pyridine as catalyst, and the resulting carbonate may be reacted with the amine of formula (III-2) in dimethyl formamide using N, N-dimethyl-4-amino pyridine as catalyst.
Compounds of the formula (III) in which X is —SO»— can be prepared by . condensing a sulfonyl! chloride of the formula (111-4)
Ar - (CH,),-SO,Cl (111-4)
with an amine of the formula (II1-2) f° I i i
HN -C - f -N-(CH,), - Pn - (CH), -R? " 0) RO (111-2) in an aprotic polar solvent in the presence of a base as catalyst, the values of the substituents Ar, Ar' and R' to R® and the parameters k to n being as defined above with reference to formula (III), and optionally converting the resulting product to a pharmaceutically acceptable salt. For example, the condensation may be carried out in dimethylformamide in the presence of N, N-diisopropylethylamine and N, N-dimethyl- 4-aminopyridine.
In the above methods, the amine of formula (111-2) is preferably a chiral amine of formula (111-5)
R
0 =
R'
Looe ol
HN og
N
H (111-5) wherein the pyridine ring is optionally substituted by with 1 or 2 substituents R and
R’ selected from alkoxy, cyano, halogen, nitro, phenyl, phenoxy, - CF; - (CH2)NR'R?, wherein R” and R® can form a ring of between 5 to 7 atoms, which may contain 1 or 2 oxygen or nitrogen atoms, or R’and R® can be independently selected from hydrogen or cyclic alkyl of from 1 to 5 carbon atoms, methoxy being a particularly preferred substituent, as in the chiral amine (III-6): 0 A eH,
Ha Sy oH (2
N
H (111-6)
‘ B) Peptide bombesin receptor antagonists
Bombesin antagonists which are peptides and which are believed to be suitable for use in the present invention are described in the following documents, the contents of which are incorporated herein by reference:
Publication Publication number number
WO 97/09347 EP 0835662
US 5650395 US 5439884
WO 96/28214 WO 95/00542
EP 0737691 US 5620955
US 5767236 WO 92/02545
WO 91/04040 | EP 0468497
EP 0309297 CA 2030212
EP 0438519 WO 92/20707
EP 0559756 WO 93/16105
WO 89/02897 | US 4943561
WO 90/03980 | US 5019647
WO 91/02746 | US 5028692
WO 92/09626 | US 5047502
WO 92/20363 WO 94/02018
WO 94/02163 WO 88/07551
WO 94/21674 WO 89/09232
WO 96/17617 EP 0315367
US 5084555 EP 0345990
US 5162497 US 5068222
US 5244883 US 5620959
US 5723578 UK 2231051
US 5750646 EP 0339193
US 5877277 WO 90/01037
US 5985834 WO 91/06563
EP 0428700 EP 0402852
Pharmaceutical compositions
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, sachets, and suppositories.
A solid carrier can be one or more substances which may also act as diluents, ’ flavouring 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. The powders and tablets preferably contain 5% to about 70% of the active component. 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.
Liquid form preparations 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 preparations 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, flavouring 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 preparation is in unit dosage form. In such form, the preparation 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 preparation, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be . a capsule, sachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.
For preparing suppository preparations, 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 moulds and allowed to cool and solidify. 3 .
Compositions that are adapted for oral administration to humans are preferred, ¥ especially such compositions in unit dosage form.
Combination therapy
Without wishing to be bound by any particular theory or teaching, the inventors believe that bombesin receptor antagonists could be used as part of a medicament in combination with one or more vasodilator, hormone therapy or neurotransmitter modulator. Such products are used or tested in the treatment of sexual dysfunction.
Vasodilators for the treatment of sexual dysfunctions of organic (rather than psychogenic) origin, act at the penis, clitoris or vagina level on local blood flow or lubricant secretions. Vasodilators useful for the treatment of sexual dysfunction include alprostadil or phentolamine, NO (nitric oxide) enhancers such as L-arginine, and PDES inhibitors such as sildenafil or a pharmaceutically acceptable salt thereof (Scrip's Complete Guide to Women's Healthcare, p.194-205, 2000)(Sachs B.D., 2000,
Benet and Melman, 1995), VIP (Vaso Intestinal Peptide) enhancers (Scrip’s Complete
Guide to Women's Healthcare, p.194-205, 2000) or angiotensin-2 receptor antagonists such as losartan (American Heart Association meeting, New Orleans, 2000).
Hormone therapies useful in the treatment of sexual dysfunction of organic and psychogenic nature include modulators of steroid hormones, steroid hormones or . hormone product (including synthetic hormones) including oestrogen (Scrip’s
Complete Guide to Women's Healthcare, p.194-205, 2000), or androgens such as testosterone (Scrip's Complete Guide to Women's Healthcare, p.194-205, 2000, Sachs
B.D., 2000), which act in areas of the CNS associated with sexual desire and sexual arousal (Wilson CA., 1993).
Neurotransmitter modulators useful in the treatment of both psychogenic and organic sexual dysfunction include neurotransmitter agonists and antagonists such as catecholamine agonists such as the D> agonist quinelorane, SHT> antagonists such as ritanserin, monoamine synthesis modifiers such as treatments that reduce endogenous
SHT activity, including inhibition of SHT synthesis using para-chlorophenylalanine, monoamine metabolism or uptake modifiers that inhibit catecholamine metabolism or reuptake, such as tricyclic antidepressants, e.g. imipramine (Wilson CA., 1993).
The use of this combination therapy includes the preparation of therapies that would allow administration of both components of the medicament, i.e. bombesin receptor antagonists and a vasodilator, hormone therapy medicament or neurotransmitter modulator medicament in a single dose. A preferred formulation would allow oral administration. However, administration by suppository, cream, transdermal patch or injection is also part of this invention. Alternatively the inventors envisage formulations that allow administration of the bombesin receptor antagonist via a separate route to that of the vasodilator, hormone therapy medicament or neurotransmitter modulator medicament. Such routes could include for example oral administration of the bombesin receptor antagonist and transdermal patch application of the vasodilator. Thus there may be provided a kit in which unit doses of bombesin receptor antagonist occur in association with unit doses of the vasodilator, hormone therapy medicament or neurotransmitter modulator medicament.
For example, in the case of a kit where bombesin receptor antagonist is formulated as a tablet capsule or other unit dosage form for oral administration and the vasodilator is provided as a transdermal patch, the two dosage forms could be provided in the form of a two-row tear-off strip in which compartments containing the tablets, etc. occur above compartments containing the transdermal patches. Other forms of packaging in which the two dosage forms are spatially associated so as to make it easy for patients to take them together and to be reminded when they have done so will readily occur to those skilled in the art. The kit will also contain instructions as to when and how the ‘ individual components of the kit should be administered.
More generally, the invention provides a pharmaceutical combination (for simultaneous, separate or sequential administration) of a bombesin receptor antagonist and one or more materials selected from (1) to (34) below: 1) One or more naturally occurring or synthetic prostaglandins or esters thereof.
Suitable prostaglandins for use herein include compounds such as alprostadil, prostaglandin E; prostaglandin Eg, 13, 14 - dihydroprostaglandin E;, prostaglandin E;, eprostinol, natural synthetic and semi-synthetic prostaglandins and derivatives thereof including those described in WO-00033825 and/or US 6,037,346 issued on 14th
March 2000 all incorporated herein by reference, PGE, PGE;, PGA, PGB,, PGF, a, 19-hydroxy PGA,, 19-hydroxy - PGB, PGE,, PGB,, 19-hydroxy-PGA;, 19-hydroxy-
PGB,, PGEs;a, carboprost, tromethamine, dinoprost, dinoprostone, iloprost, gemeprost, metenoprost, sulprostune, tiaprost and moxisylate. 2) One or more « - adrenergic receptor antagonist compounds also known as a - adrenoceptor antagonists or o-receptor antagonists or o-blockers. Suitable compounds for use herein include: the a-adrenergic receptor blockers as described in
PCT application W099/30697 published on 14th June 1998, the disclosure of which relating to a-adrenergic receptors incorporated herein by reference and include, selective aj-adrenoceptor or az-adrenoceptor blockers and non-selective adrenoceptor blockers, Suitable oj-adrenoceptor blockers include: phentolamine, phentolamine mesylate, trazodone, alfuzosin, indoramin, naftopidil, tamsulosin, dapiprazole, phenoxybenzamine, idazoxan, efarxan, yohimbine, rauwolfa alkaloids, Recordati ’ 15/2739, SNAP 1069, SNAP 5089, RS17053, SL 89.0591, doxazosin, terazosin, abanoquil and prazosin; ap-blocker blockers from US 6,037,346 [14th March 2000) dibenarnine, tolazoline, trimazosin and dibenamine; a-adrenergic receptor antagonists as described in US patents: 4,188,390; 4,026,894; 3,511,836; 4,315,007; 3,527,761; 3,997,666; 2,503,059; 4,703,063; 3,381,009; 4,252,721 and 2,599,000 each of which is incorporated herein by reference; or-Adrenoceptor blockers include: clonidine, papaverine, papaverine hydrochloride, optionally in the presence of a cardiotonic agent such as pirxamine. 5.0) One or more NO-donor (NO-agonist) compounds. Suitable NO-donor compounds for use herein include organic nitrates, such as mono- di or tri-nitrates or organic nitrate esters including glyceryl tririnitrate (also known as nitroglycerin), isosorbide 5-mononitrate, isosorbide dinitrate, pentaerythritol tetranitrate, erythrityl tetranitrate, sodium nitroprusside (SNP), 3-morpholinosydnonimine, molsidomine, S- nitroso- N-acetyl penicillamine (SNAP) S-nitroso-N-glutathione (SNO-GLU), N- hydroxy - L-arginine, amylnitrate, linsidomine, linsidomine hydrochloride, (SIN-1) S- nitroso - N-cysteine, diazenium diolates,(NONOQates), 1,5-pentanedinitrate, L- arginine, ginseng, zizphi fructus, molsidomine, Re — 2047, nitrosylated maxisylyte derivatives such as NMI-678-11 and NMI-937 as described in published PCT application WO 0012075 ; and/or (4) One or more potassium channel openers or modulators. Suitable potassium channel openers/modulators for use herein include nicorandil, cromokalim, levecromakalim (lemakalim), pinacidil, diazoxide, minoxidil, charybdotoxin, glyburide, 4-aminopyridine, BaCl,. 5) One or more dopaminergic agents, preferably apomorphine or a selective D2,
D3 or D2/Dsagonist such as, pramipexole and ropirinol (as claimed in WO- 0023056), PNU95666 (as claimed in WO-0040226). (6) One or more vasodilator agents. Suitable vasodilator agents for use herein i include nimodepine, pinacidil, cyclandelate, isoxsuprine, chloropromazine, halo peridol, Rec 15/2739, trazodone. (7) One or more thromboxane A2 agonists. (8) One or more CNS active agents.
: 9 One or more ergot alkaloids. Suitable ergot alkaloids are described in US patent 6,037,346 issued on 14th March 2000 and include acetergamine, brazergoline, bromerguride, cianergoline, delorgotrile, disulergine, ergonovine maleate, ergotamine tartrate, etisulergine, lergotrile, lysergide, mesulergine, metergoline, metergotamine, nicergoline, pergolide, propisergide, proterguride, terguride. (10) One or more compounds which modulate the action of natriuretic factors in particular atrial natriuretic factor (also known as atrial natriuretic peptide), B type and
C type natriuretic factors. (11) One or more angiotensin receptor antagonists such as losartan. (12) One or more substrates for NO-synthase, such as L-arginine. (13) One or more calcium channel blockers such as amlodipine. (14) One or more cholesterol lowering agents such as statins (e.g. atorvastatin/
Lipitor- trade mark) and fibrates. (15) One or more antiplatelet and antithrombotic agents, e.g. tPA, uPA, warfarin, hirudin and other thrombin inhibitors, heparin, thromboplastin activating factor inhibitors. (16) One or more insulin sensitising agents such as triglitazone (rezulin) and hypoglycaemic agents such as glipizide. (17) L-DOPA or carbidopa. (18) One or more acetylcholinesterase inhibitors such as donepezil (Aricept). (19) One or more steroidal or non-steroidal anti-inflammatory agents.
‘ (20) One or more estrogen receptor modulators (SERM) and/or estrogen agonists and/or estrogen antagonists, preferably raloxifene or lasofoxifene, (-)-cis-6-phenyl-5- [4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol and pharmaceutically acceptable salts thereof (compound A below) the preparation of which is detailed in WO 96/21656.
Sa
Compound A (21) One or more PDE inhibitors, more particularly a PDE 2, 5, 7 or 8 inhibitor (for oral or local administration), preferably PDE2 or PDES inhibitor and most preferably a PDES inhibitor (see hereinafter), said inhibitors preferably having an IC50 against the respective enzyme of less than 100nM; and PDE 3 , 4 inhibitor for local administation (e.g. intracavernosal injection). (22) In the case where the combination is for the treatment or prophylaxis of female sexual dysfunction, one or more of an NPY (neuropeptide Y) inhibitor, more particularly NPY1 or NPYS5 inhibitor, preferably NPY1 inhibitor. Preferably said
NPY inhibitors (including NPYY1 and NPYY5) have an IC50 of less than 100 nM, more preferably less than 50 nM. (23) One or more of a neutral endopeptidase (NEP) inhibitor preferably having an
ICS0 for NEP of less than 100nM. Preferably the NEP inhibitor is selective for NEP and has a selectivity over the endothelin converitin enzyme (ECE) and angiotensin converting enzyme (ACE) of greater than 100. However, mixed/dual NEP/ECE and
NEP/ACE inhibitors (such as ompatrilat) are still within the scope of the invention.
(24) One or more of vasoactive intestinal protein (VIP), VIP mimetic, VIP ‘ analogue, more particularly acting through one or more of the VIP receptor subtypes
VPACI1,VPAC or PACAP (pituitory adenylate cyclase activating peptide), one or more of a VIP receptor agonist or a VIP analogue (eg Ro-125-1553) or a VIP fragment, one or more of a a-adrenoceptor antagonist with VIP combination (eg
Invicorp, Aviptadil). (25) One or more of a melanocortin receptor agonist or modulator or melanocortin enhancer, such as melanotan II, PT-14, PT-141 or compounds claimed in WO- 09964002, WO0-00074679, WO0-09955679, WO-00105401, WO-00058361, WO- 00114879, WO-00113112, WO-09954358. (26) One or more of a serotonin receptor agonist, antagonist or modulator, more particularly agonists, antagonists or modulators for SHT1A (including VML 670),
SHT2A, SHT2C, 5HT3 and/or SHT6 receptors, including those described in WO- 09902159, WO-00002550 and/or WO-00028993.. (27) One or more of a testosterone replacement agent (including dehydroandrostendione), testosterone (Tostrelle), dihydrotestosterone or a testosterone implant. (28) One or more of estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate (MPA) (i.e. as a combination), or estrogen and methyl testosterone hormone replacement therapy agent (e.g. HRT especially Premarin, Cenestin, Oestrofeminal, Equin, Estrace, Estrofem, Elleste Solo, Estring, Eastraderm
TTS, Eastraderm Matrix, Dermestril, Premphase, Preempro, Prempak, Premique,
Estratest, Estratest HS, Tibolone). . (29) One or more of a modulator of transporters for noradrenaline, dopamine and/or serotonin, such as bupropion, GW-320659. (30) One or more of a purinergic receptor agonist and/or modulator.
‘ (31) One or more of a neurokinin (NK) receptor antagonist, including those described in WO-09964008. (32) One or more of an opioid receptor agonist, antagonist or modulator, preferably agonists for the ORL-1 receptor. (33) One or more of an agonist or modulator for oxytocin/vasopressin receptors, preferably a selective oxytocin agonist or modulator. (34) One or more modulators of cannabinoid receptors.
Auxiliary Agent PDES inhibitor (I: PDES) :
PDES Inhibitors
Suitable PDESi’s for use in the pharmaceutical combinations according to the present invention are the cGMP PDES5i’s hereinafter detailed. Particularly preferred for use herein are potent and selective cGMP PDESi’s. Suitable cGMP PDES inhibitors for the use according to the present invention include: e pyrazolo [4,3-d]pyrimidin-7-ones disclosed in EP-A-0463756; e pyrazolo [4,3-d]pyrimidin-7-ones disclosed in EP-A-0526004; e pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international patent application WO 93/06104; * isomeric pyrazolo [3,4-d]pyrimidin-4-ones disclosed in published international . patent application WO 93/07149; e quinazolin-4-ones disclosed in published international patent application WO : 93/12095; e pyrido [3,2-d]pyrimidin-4-ones disclosed in published international patent application WO 94/05661;
e purin-6-ones disclosed in published international patent application WO 94/00453; e pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international patent application WO 98/49166,; e pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international patent application WO 99/54333; e pyrazolo [4,3-d]pyrimidin-4-ones disclosed in EP-A-0995751; e pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international patent application WO 00/24745; e pyrazolo [4,3-d]pyrimidin-4-ones disclosed in EP-A-0995750; e the compounds disclosed in published international application W095/19978; e the compounds disclosed in published international application WO 99/24433 and e the compounds disclosed in published international application WO 93/07124.
It is to be understood that the contents of the above published patent applications, and in particular the general formulae and exemplified compounds therein are incorporated herein in their entirety by reference thereto.
Preferred type V phosphodiesterase inhibitors for the use according to the present invention include: 5-[2-ethoxy-5-(4-methyl-1-piperazinylsulphonyl)phenyl]-1-methyl-3-n- propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil) also known as 1- [[3-(6,7-dihydro-1-methyl-7-ox0-3-propyl-1H-pyrazolo[4,3-d}pyrimidin-5-yl)-4- ethoxyphenyl]sulphonyl]-4-methylpiperazine (see EP-A-0463756); 5-(2-ethoxy-5-morpholinoacetylphenyl)- 1 -methyl-3-n-propyl-1,6-dihydro-7H- pyrazolo[4,3-d]pyrimidin-7-one (see EP-A-0526004); 3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-propoxyphenyl}-2-(pyridin- 2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see W(098/49166);
3-ethyl-5-[5-(4-ethylpiperazin- 1-ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3- : yl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see
W099/54333 ); (+)-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxy-1(R)- methylethoxy)pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7- one, also known as 3-ethyl-5-{5-[4-ethylpiperazin-1-ylsulphonyl]-2-([(1R)-2- methoxy-1-methylethyl]oxy)pyridin-3-yl}-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d] pyrimidin-7-one (see W099/54333); 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2- methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, also known as 1-{6- ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3- d]pyrimidin-5-yl]-3-pyridylsulphonyl } -4-ethylpiperazine (see = WO 01/27113,
Example 8); 5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-(1- methylpiperidin-4-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one ~~ (see =~ WO 01/27113, Example 15); 5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2- phenyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO 01/27113, Example 66); 5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6- dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (seeWO 01/27112, Example 124); 5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6- dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO 01/27112, Example 132); (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl) -pyrazino[2',1':6,1]pyrido[3,4-blindole-1,4-dione (IC-351), i.e. the compound of examples 78 and 95 of published international application W095/19978, as well as the compound of examples 1, 3, 7 and 8; 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl}-5-methyl-7- propyl-3H-imidazo[5,1-f](1,2,4]triazin-4-one (vardenafil) also known as 1-[[3-(3,4- dihydro-5-methyl-4-0x0-7-propylimidazo(5,1-f]-as-triazin-2-yl)-4- ethoxyphenyl]sulphonyl]-4-ethylpiperazine, i.e. the compound of examples 20, 19, 337 and 336 of published international application W099/24433; and the compound of example 11 of published international application . WO093/07124 (EISAI); and compounds 3 and 14 from Rotella D P, J. Med Chem., 2000, 43, 1257.
Still other type cGMP PDES inhibitors useful in conjunction with the present invention include: 4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)-propoxy]- 3(2H)pyridazinone; 1-[4-[(1,3-benzodioxol-5-ylmethyl)amiono}-6-chloro-2-quinozolinyl]-4- piperidine-carboxylic acid, monosodium salt; (+)-cis-3,6a,7,9,9,9a-hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-S- methyl-cyclopent-4,5]imidazo[2,1-b]purin-4(3H)one; furazlocillin; cis-2-hexyl-5-methyl-3,4,5,6a,7,8,9,9a- octahydrocyclopent[4,5]-imidazo[2,1- b]purin-4-one; 3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6- carboxylate; 3-acetyl-1- (2-chlorobenzyl)-2-propylindole-6-carboxylate; 4-bromo-5-(3-pyridylmethylamino)-6-(3-(4-chlorophenyl) propoxy)-3- (2H)pyridazinone;
I-methyl-5(5-morpholinoacetyl-2-n-propoxyphenyl)-3-n-propyl-1,6-dihydro- 7H-pyrazolo(4,3-d)pyrimidin-7-one; 1-[4-[(1,3-benzodioxol-5-ylmethyl)arnino]-6-chloro-2- quinazolinyl]-4- piperidinecarboxylic acid, monosodium salt;
Pharmaprojects No. 4516 (Glaxo Wellcome),
Pharmaprojects No. 5051 (Bayer);
Pharmaprojects No. 5064 (Kyowa Hakko; see WO 96/26940);
Pharmaprojects No. 5069 (Schering Plough);
GF-196960 (Glaxo Wellcome);
E-8010 and E-4010 (Eisai); Bay-38-3045 & 38-9456 (Bayer); and . Sch-51866.
The suitability of any particular cGMP PDES inhibitor can be readily determined by evaluation of its potency and selectivity using literature methods followed by evaluation of its toxicity, absorption, metabolism, pharmacokinetics, etc in accordance with standard pharmaceutical practice.
Preferably, the ¢cGMP PDES5 inhibitors have an ICsy at less than 100 nanomolar, more preferably, at less than 50 nanomolar, more preferably still at less than 10 nanomolar. ICso values for the cGMP PDES5 inhibitors may be determined using the PDES assay in the Test Methods Section hereinafter.
Preferably the cGMP PDES inhibitors used in the pharmaceutical combinations according to the present invention are selective for the PDES enzyme.
Preferably they are selective over PDE3, more preferably over PDE3 and PDEA4.
Preferably, the cGMP PDES inhibitors of the invention have a selectivity ratio greater than 100 more preferably greater than 300, over PDE3 and more preferably over
PDE3 and PDE4. Selectivity ratios may readily be determined by the skilled person. ICsp values for the PDE3 and PDE4 enzyme may be determined using established literature methodology, see S A Ballard ef al (1998) and as detailed hereinafter.
Auxiliary Agent : NEP inhibitor (I:NEP)
NEP EC3.424.11 (FEBS Lett., 229(1), 206-210 (1988)), also known as enkephalinase or neprilysin, is a zinc-dependent neutral endopeptidase. This enzyme is involved in the breakdown of several bioactive oligopeptides, cleaving peptide bonds on the amino side of hydrophobic amino acid residues (Reviewed in Turner et al., 1997). The key neuronally released bioactive agents or neuropeptides metabolised by NEP include natriuretic peptides such as atrial natriuretic peptides (ANP) as well as brain natriuretic peptide and C-type natriuretic peptide, bombesin, bradykinin, calcitonin gene-related peptide, endothelins, enkephalins, neurotensin, substance P and vasoactive intestinal peptide. Some of these peptides have potent vasodilatory . and neurohormone functions, diuretic and natriuretic activity or mediate behaviour effects. Background teachings on NEP have been presented by Victor A. McKusick et al on http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following information concerning NEP has been extracted from that source.
A
‘ “Common acute lymphocytic leukemia antigen is an important cell surface marker in the diagnosis of human acute lymphocytic leukemia (ALL). It is present on leukemic cells of pre-B phenotype, which represent 85% of cases of ALL. CALLA is not restricted to leukemic cells, however, and is found on a variety of normal tissues. CALLA is a glycoprotein that is particularly abundant in kidney, where it is present on the brush border of proximal tubules and on glomerular epithelium. Letarte et al. (1988) cloned a cDNA coding for
CALLA and showed that the amino acid sequence deduced from the cDNA sequence is identical to that of human membrane-associated neutral endopeptidase (NEP; EC 3.4.24.11 ), also known as enkephalinase. NEP cleaves peptides at the amino side of hydrophobic residues and inactivates several peptide hormones including glucagon, enkephalins, substance P, neurotensin, oxytocin, and bradykinin. By cDNA transfection analysis, Shipp et al. (1989) confirmed that CALLA is a functional neutral endopeptidase of the type that has previously been called enkephalinase. Barker et al. (1989) demonstrated that the CALLA gene, which encodes a 100-kD type II transmembrane glycoprotein, exists in a single copy of greater than 45 kb which is not rearranged in malignancies expressing cell surface CALLA. The gene was located to human chromosome 3 by study of somatic cell hybrids and in situ hybridization regionalized the location to 3q21-q27. Tran-Paterson et al. (1989) also assigned the gene to chromosome 3 by Southern blot analysis of DNA from human-rodent somatic cell hybrids. D'Adamio et al. (1989) demonstrated that the CALLA gene spans more than 80 kb and is composed of 24 exons.”
Preferred for NEPi’s for use as auxiliary agents in combination with bombesin receptor antagonists according to the present invention are: (2R)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl} cyclopentyl) methyl]pentanoic acid
C
) o o To Me and (28)-2-[(1-{[(5-Ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl}cyclopentyl)- methyl] pentanoic acid
CH, [o o} Ra Me
The title product from stage c) below (824mg) was further purified by HPLC using an AD column and using hexane:iso-propanol:trifluoroacetic acid (85:15:0.2) as elutant to give (2R)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl}- cyclopentyl) methyl]pentanoic acid as a white foam, 400mg, 99.5% ee, 'H NMR (CDCls, 400MHz) 8: 0.90 (t, 3H), 1.36 (m, 6H), 1.50-1.80 (m, 9H), 2.19 (m, 1H), 2.30 (m, 1H), 2.44 (m, 1H), 2.60 (m, 1H), 2.98 (q, 2H), 12.10-12.30 (bs, 1H), LRMS : m/z 338 (MH), [a]p = -9.0° (¢ = 0.1, methanol), and (25)-2-[(1-{[(5- ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl } cyclopentyl)-methyl]pentanoic acid as a white foam, 386mg, 99% ee, 'H NMR (CDCls, 400MH2) 5: 0.90 (t, 3H), 1.38 (m, 1S 6H), 1.50-1.79 (m, 9H), 2.19 (m, 1H), 2.30 (m, 1H), 2.44 (m, 1H), 2.60 (m, 1H), 2.98 (q, 2H), 12.10-12.27 (bs, 1H); LRMS: m/z 338 (MH); and [a]p = +3.8° (c = 0.1, methanol)
Preparation of Starting Materials a) 1-]2-(tert-Butoxycarbonyl)-4-pentyl]-cyclopentane carboxylic acid
CH, . H,C
A mixture of 1-[2-(tert-butoxycarbonyl)-4-pentenyl]-cyclopentane carboxylic ‘ acid (EP 274234) (23g, 81.5mmol) and 10% palladium on charcoal (2g) in dry ethanol (200ml) was hydrogenated at 30psi and room temperature for 18 hours. The reaction mixture was filtered through Arbocel®, and the filtrate evaporated under reduced pressure to give a yellow oil. The crude product was purified by column chromatography on silica gel, using ethyl acetate:pentane (40:60) as the eluant, to provide the desired product as a clear oil, 21g, 91%; 'H NMR (CDCl;, 0.86 (t, 3H), 1.22-1.58 (m, 15H), 1.64 (m, 4H), 1.78 (dd, 1H), 2.00-2.18 (m, 3H), 2.24 (m, 1H);
LRMS : m/z 283 (M-H) b) tert-Butyl 2-[(1-{[(S-ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl}- cyclopentyl)methyl]pentanoate. cH,
Me " 0 o To. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.21mmol), 1-hydroxybenzotriazole hydrate (0.2mmol), N-methylmorpholine (0.3 1mmol) and 2- amino-5-ethyl-1,3,4-thiadiazole (0.22mmol) were added to a solution of the product from stage a) above (150mg, 0.53mmol) in N,N-dimethylformamide (3ml), and the reaction stirred at 90°C for 18 hours. The cooled solution was diluted with ethyl acetate (90ml), washed with water (3x25ml), and brine (25ml), then dried (MgSO,) and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel, using ethyl acetate:pentane (30:70) as the eluant to afford the title compound, 92%; 'H NMR (CDCl, 300MHz) 6: 0.82 (t, 3H), 1.20-1.80 (m, 22H), 1.84 (m, 1H), 2.20 (m, 4H), 3.04 (q, 2H), 9.10 (bs, 1H); LRMS : m/z 396.2 (MH). . c) 2-[(1-{{(5-ethyl-1,3.4-thiadiazol-2-yl)amine]carbonyl}cyclopentyl) methvllpentanoic acid.
CH, - ho) 0 To.
Trifluoroacetic acid (Sml) was added to a solution of the title product from stage b) above (0.31mmol) in dichloromethane (5ml), and the solution stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure and the residue azeotroped with toluene and dichloromethane to afford the title compound as a clear oil, 81%, 'H NMR (CDCl, 400MHz) 6: 0.92 (t, 3H), 1.35 (t, 3H), 1.25-1.80 (m, 11H), 2.20-2.50 (m, 4H), 2.95 (q, 2H), 12.10 (bs, 1H); LRMS : m/z 339.8 (MH); Anal. Found: C, 56.46; H, 7.46; N, 12.36. C6H25N303S requires C, 56.62; H, 7.44; N, 12.37%.
Details on a suitable assay system for identifying and/or studying an I:NEP are presented in the hereinafter in the section entitled NEP Assay. Further examples of
NEP inhibitors are disclosed and discussed in the following review articles:
Pathol. Biol., 46(3), 1998, 191.
Current Pharm. Design, 2(5), 1996, 443.
Biochem. Soc. Trans., 21(3), 1993, 678.
Handbook Exp. Pharmacol., 104/1, 1993, 547.
TiPS, 11, 1990, 245.
Pharmacol. Rev., 45(1), 1993, 87.
Curr. Opin. Inves. Drugs, 2(11), 1993, 1175.
Antihypertens. Drugs, (1997), 113.
Chemtracts, (1997), 10(11), 804.
Zinc Metalloproteases Health Dis. (1996), 105.
Cardiovasc. Drug Rev., (1996), 14(2), 166. . Gen. Pharmacol., (1996), 27(4), 581.
Cardiovasc. Drug Rev., (1994), 12(4), 271.
Clin. Exp. Pharmacol. Physiol., (1995), 22(1), 63.
Cardiovasc. Drug Rev., (1991), 9(3), 285.
Exp. Opin. Ther. Patents (1996), 6(11), 1147. . Yet further examples of NEPi’s are disclosed in the following documents:
EP-509442A
US-192435
US-4929641
EP-599444B
US-884664
EP-544620A
US-798684
J. Med. Chem. 1993, 3821.
Circulation 1993, 88(4), 1.
EP-136883
JP-85136554
US-4722810
Curr. Pharm. Design, 1996, 2, 443.
EP-640594
J. Med. Chem. 1993, 36(1), 87.
EP-738711-A
JP-270957
CAS # 115406-23-0
DE-19510566
DE-19638020
EP-830863
JP-98101565
EP-733642
W09614293 . JP-08245609
JP-96245609
WO09415908
JP05092948 ‘ WO0-9309101
WQ0-9109840
EP-519738
EP-690070
J. Med. Chem. (1993), 36, 2420.
JP-95157459
Bioorg. Med. Chem. Letts., 1996, 6(1), 65.
Further I:NEPs are disclosed in the following documents:
EP-A-0274234
JP-88165353
Biochem.Biophys.Res. Comm.,1989, 164, 58
EP-629627-A
US-77978
Perspect. Med. Chem. (1993), 45.
EP-358398-B
Further examples of I:NEPs are selected from the following structures:
He Compound Structure Mode of Action ‘ References
FXII Me I:NEP
Ord “0 EP-509442A
NS sad US-192435
US-4929641
FXII noc— 2 9 I:NEP 2 cd
Daal (also an ACE inhibitor)
EP-599444B
US-884664
FXIV y I:NEP ee coi EP-544620A lo) 0, 0
US-798684
OH
J. Med. Chem. 1993, 3821.
FXV 0 I:NEP $3 (also an ACE inhibitor)
Me Sal Mixanpril
Ho,c” Me Circulation 1993, 88(4), 1.
FXVI I:NEP us Nod con EP-136883 lo) JP-85136554
US-4722810
FXVII Q I.NEP oO ©O 4 \ ns JL I, Retrothiorphan . H Curr. Pharm. Design, 1996, 2, 443. y
FXVII NEP o 1 _
Hs— JON (also an ACE inhibitor)
MN
: oF J Low EP-640594
FXIX NEP
[8]
HS Neo J. Med. Chem. 1993, 36(1), 87.
FXX ~3 [NEP 9 Ln COH (also an ACE inhibitor)
Se ~n"0 EP-738711-A
Ie on ~( JP-270957
FXXI1 i I:NEP
QL ¢ CAS #
OH x
Hog AN AA on 115406-23-0 0 [0] [0]
FXXII EP
34 } 0 (also an ECE inhibitor) o Hh coer | DE-19510566
DE-19638020
EP-830863
JP-98101565
FXXIII NEP
Sete o (also an ECE inhibitor) 0 SEAN EP-733642 : wo ,c~
FXXIV A NEP
¥ OH 0
RAR EES W096/14293 [o} [¢}
FXXV I:NEP o IS i JP-08245609 h ding hs OH ° ° JP-96245609 + 1 (¢)
FXXVI . I:NEP
H
10, 35d _con W09415908
H o)
FXXVII 0 ©o DO I:NEP rel CoH JP05092948
FXXVII I:NEP
So Jes y WO0-9309101 ~CO,H
FXXIX oF I:NEP 0 hh WO0-9109840 0 CoH
FXXXI N I:NEP 00 5 EP-519738
N Q ort HUN | EP-690070
HO,C
FXXXII Hoc. I:NEP 2° (also an ACE inhibitor)
Acs A fH J. Med. Chem. (1993), or 36, 2420.
FXXXIII eS I:NEP
Cl JP-95157459
HO N_ com Bioorg. Med. Chem. °° Letts., 1996, 6(1), 65.
Preferred additional [:NEPs are selected from the following structures: []
Compound Structure Mode of Action
References
FV \ y o I:INEP o "Oy es EP-A-0274234 o © (Example 300)
FVI \ I:NEP
H Oo pod Oy or EP-A-0274234 o © (Example 379)
FVII OMe I:NEP
N Candoxatrilat
ONG
0 25 EP-274234 o 4 Teo JP-88165353
OH Biochem.Biophys.Res.
Comm. 1989, 164, 58
FVIII I:NEP
SH Omapatrilat
H 0 con
J Ch : (also an inhibitor of ACE)
AJ EP-0629627-A
US-77978
FIX NHSO,Me I:NEP
RENPAC
HANTS yr Sampatrilat
HN
0, 255 (also an inhibitor of ACE) . 0 wd TY Perspect. Med. Chem. oH | (1993), 45. : EP-0358398-B
. FX I:NEP
Me
HO 0 0 CS Phosphoramidon o"oH § com NH } } } ’ HO' (which is commercially available)
FXI 9 on I:NEP
SRR: Thiorphan (which is commercially available)
More preferred additional 1:NEPs are selected from the following structures:
COMPOUN | STRUCTURE
D
F57 Heo
HO N
TL lo} 0 x (0)
F58 pe
HO Ne ~~" " 0 [0] to
H
HO & © © OH
F60 . " .
HO CH, ~ Vas 0 o NN
F61 Heo 3
Abe
HO N
0 o N J ~N
A fe) Oo 0
F62 0
HO
N
H
HO
H,C
F63 o o n—N >
H
H.C
F64 fo) $ 0
N
H
H,C
F65 0 p Q 0 z re he
N
H
H,C
F66 HG
HO (CHg)y o]
Oo (Ic)
BIOAVAILABILITY
Preferably, the compounds of the invention (and combinations) are orally bioavailable. Oral bioavailablity refers to the proportion of an orally administered drug that reaches the systemic circulation. The factors that determine oral bioavailability of a drug are dissolution, membrane permeability and metabolic stability. Typically, a screening cascade of firstly in vitro and then in vivo techniques is used to determine oral bioavailablity.
Dissolution, the solubilisation of the drug by the aqueous contents of the gastro-intestinal tract (GIT), can be predicted from in vitro solubility experiments conducted at appropriate pH to mimic the GIT. Preferably the compounds of the invention have a minimum solubility of 50 mcg/ml. Solubility can be determined by standard procedures known in the art such as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997.
Membrane permeability refers to the passage of the compound through the cells of the GIT. Lipophilicity is a key property in predicting this and is defined by in vitro Log D74 measurements using organic solvents and buffer. Preferably the compounds of the invention have a Log D;4 of -2 to +4, more preferably -1 to +2, a The log D can be determined by standard procedures known in the art such as described in J. Pharm. Pharmacol. 1990, 42:144. ) 25
Cell monolayer assays such as CaCo; add substantially to prediction of favourable membrane permeability in the presence of efflux transporters such as p-
glycoprotein, so-called caco-2 flux. Preferably, compounds of the invention have a caco-2 flux of greater than 2x10cms™, more preferably greater than 5x10°cms™.
The caco flux value can be determined by standard procedures known in the art such as described in J. Pharm. Sci, 1990, 79, 595-600
Metabolic stability addresses the ability of the GIT or the liver to metabolise compounds during the absorption process: the first pass effect. Assay systems such as microsomes, hepatocytes etc are predictive of metabolic liability. Preferably the compounds of the Examples show metabolic stablity in the assay system that is commensurate with an hepatic extraction of less then 0.5. Examples of assay systems and data manipulation are described in Curr. Opin. Drug Disc. Devel., 201, 4, 36-44,
Drug Met. Disp.,2000, 28, 1518-1523
Because of the interplay of the above processes further support that a drug will be orally bioavailable in humans can be gained by in vivo experiments in animals.
Absolute bioavailability is determined in these studies by administering the compound separately or in mixtures by the oral route. For absolute determinations (% absorbed) the intravenous route is also employed. Examples of the assessment of oral bioavailability in animals can be found in Drug Met. Disp.,2001, 29, 82-87; J. Med
Chem, 1997, 40, 827-829, Drug Met. Disp.,1999, 27, 221-226.
By cross reference herein to compounds contained in patents which can be used in accordance with invention, we mean the therapeutically active compounds as defined in the claims (in particular of claim 1) and the specific examples (all of which is incorporated herein by reference).
How the invention may be put into effect will now be described, by way of example only, with reference to the following examples, some of which are . preparative and others of which describe results of biological tests.
Example 1
Effect of (S) 3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexyl-methyl]- 2-methyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide (Compound (1)) on female rat sexual proceptivity
Compound (1) N
O,N “OU 3 8
JIS N N
N N ~~ 0)
Zo”
Ovariectomised adult female Sprague Dawley rats (180-200g, from Charles
River) were housed in groups of 6 in a reversed lighting system of 12h light:dark (lights off 7.00-19.00h). Two weeks after ovariectomy they were used for sexual activity tests. The experiments started at least Sh into the dark period.
Tests were carried out in a circular arena of 90cm diameter, surrounded by a 30cm high wall. Two small cages with wire-mesh front (15x15cm) are fixed into the wall such that the front of the cage is «flush» with the wall and the 2 cages are opposite each other. They contained two stimuli animals: an intact sexually experienced male and a receptive female (ovariectomised, primed with Sug oestradiol benzoate dissolved in corn oil and injected subcutaneously 48 hours before the test and with 0.5 mg of progesterone four hours before the test). Sexually naive test and control animals were used. Forty eight hours before the tests, both the test and control animals were primed with 5ug oestradiol benzoate. For animals used as positive controls, progesterone (0.5mg/0.1ml) was dissolved in corn oil and administered - subcutaneously (s.c.), 4h before the test. Test and control animals were introduced one at atime for 10 minute periods into the arena. During the 10min test, the time that the test or positive control animal spent investigating each stimulus animal was noted.
The arena was thoroughly cleaned between animals. The position of the male/female stimuli boxes was randomised between animals, in order to avoid place preference. : The difference in the percentage of time spent investigating the male minus the female stimuli was calculated, out of the total time spent investigating stimuli animals.
Compound (1) was dissolved in 100% B-cyclodextrin and then diluted with saline to a final solution of 50% 2-hydroxypropyl-B-cyclodextrin. It was administered intraperitoneally (i.p.) at doses of 3 and 10mg/kg, in a dosing volume of 1ml/kg, 1h before tests. Progesterone (0.5mg/0.1ml) was dissolved in corn oil and administered subcutaneously (s.c.), 4h before test, as a positive control.
Compound (1) dose-dependently (3mg/kg-10mg/kg) increased the percentage of time spent investigating the male stimulus, with a MED of 10mg/kg (see Figure 1).
The effect of this dose was similar to the effect of progesterone (prog). (*P<0.05, **P<0.01 Kruskal-Wallis followed by Mann-Whitney test, vs vehicle).
Example 2
Effect of Compound (1) on female rat sexual receptivity
Ovariectomised adult female Sprague Dawley rats (180-200g, from Charles
River) were housed in groups of 6 in a reversed lighting system of 12h light:dark (lights off 7.00-19.00h). Two weeks after ovariectomy they were used for sexual activity tests. The experiments started at least Sh into the dark period.
Compound (1) was dissolved in 100% B-cyclodextrin and then diluted with saline to a final solution of 50% 2-hydroxypropyl-B-cyclodextrin. It was administered intraperitoneally (i.p.) at a dose of 10mg/kg, in a dosing volume of 1ml/kg. ’ Quinelorane (6.25pg/kg) was dissolved in water and administered s.c. as positive control. Forty eight hours before testing, ovariectomised female rats (as described above), were primed with 5ug oestradiol benzoate dissolved in corn oil and injected subcutaneously. This is a low dose of oestrogen that does not re-establish sexual behaviour in an ovariectomised female but provides a minimum hormonal background for pharmacological agents to stimulate sexual behaviour. The females : were placed with a series of vigorous male rats and subjected to 10 mounts.
The lordotic response of the animal was recorded and expressed as a percentage of the mounts (i.e. lordosis quotient, LQ), as previously described.
Animals showing LQ<20 were considered non-receptive and were included in the study. Each rat was tested prior to administration of the compound and then tested similarly post-injection. The pre-treatment times were 1h for Compound (1) and vehicle (50% B-cyclodextrin, i.p.) or 90min for quinelorane.
As shown in Figure 2, a single administration of quinelorane (6.25ug/kg, s.c.) significantly (P<0.01) increased the LQ, 90min after administration, compared to the
LQ shown before administration (paired t test). A single administration of Compound (1) (10mg/kg, i.p.) also had a significant (P<0.05) stimulatory effect on the LQ, 1h after administration, compared to the LQ shown before administration (paired t test).
Example 3
The effect of repeated administration of Compound (1) on female rat proceptivity
In the present study we have investigated whether the repeated administration of a higher dose of Compound (1) (15mg/kg) still results in stimulation of proceptivity.
Ovariectomised adult female Sprague Dawley rats (180-200g) were housed in groups of 5 in a reversed lighting system of 12h light:dark (lights off 5.00-17.00h). - They were used for the experiments at least two weeks after ovariectomy. Forty eight hours before tests, the animals were primed with oestradiol benzoate (5pg/0.1ml in corn oil, s.c). On day 1, progesterone (0.5mg/0.Iml, in corn oil, s.c.) was administered to one of the groups 4h before tests, as a positive control. Compound (1) (15mg/kg, 1.p.) was administered in 50% 2-hydroxypropyl-p-cyclodextrin, 1h before tests. The test lasted 10 minutes and was carried out as described before. The : difference in the percentage of time spent investigating the male minus the female stimuli was calculated, out of the total time spent investigating stimuli animals.
Animals were submitted to a test on day 1 and on day 15. From day 2 to 14 the
Compound (1) group received a daily injection of the compound (15mg/kg, i.p.), while both the vehicle and the progesterone groups received an injection of vehicle.
On day 15 the test took place again, as described for day 1.
On day 1, both progesterone and Compound (1) had a stimulatory effect on proceptivity, compared to the vehicle group (**P<0.01, ANOVA followed by Dunnett's test). On day 15, a similar stimulatory effect was observed (**P<0.01, ANOVA followed by Dunnett’s test) (see Figure 3). No significant difference was observed between the effects on day 1 and day 15 for each treatment group (paired t test). The effects of progesterone and Compound (1) were statistically similar. There were no changes in body weight or general behaviour between groups along the experiment.
From this study we can conclude that Compound (1) (15mg/kg, i.p.) has a stimulatory effect on proceptivity in the female rat, comparable to progesterone, and that such effect is unaffected by the repeated administration of the compound, which seems to be well tolerated.
Example 4
Effect of intracerebroventricular administration of Compound (1) on female rat sexual proceptivity
In order to elucidate the site of action for this effect we have administered the
Compound (1) intracerebroventricularly (i.c.v.).
Ovariectomised female rats (Sprague Dawley, obtained from Charles River,
UK) were stereotaxically implanted (coordinates 0.89mm behind Bregma, 1.3mm lateral and 2.5mm vertical) with stainless steel cannulae (6 mm long, O.D. 0.75mm),
held in place with dental cement. Animals were housed in groups of three and : returned to a reversed lighting system of 12h light:dark (lights off 5.00-17.00h).
Correct placement of the cannulae was assessed post-mortem. Rats were used for tests two weeks after ovariectomy (one week after cannulation). The experiments started at least 5h into the dark period. Forty eight hours before tests, the animals were primed with Sug oestradiol benzoate (s.c, in corn oil) and adapted to the apparatus (in the absence of stimuli animals) for 10min on 2 consecutive days prior testing. The 10min test was carried out as previously described. The difference in the percentage of time spent investigating the male minus the female stimuli was calculated, out of the total time spent investigating stimuli.
Compound (1) was dissolved in 50% 2-hydroxypropyl-B-cyclodextrin in saline. It was administered i.c.v. over a 30sec period, with the aid of a pump set to deliver a flow of 10pul/min. The dosing volume was Sul/rat. The compounds were administered 10min before tests. Progesterone (0.5mg/0.1ml) was dissolved in com oil and administered subcutaneously (s.c.), 4h before test, as a positive control. As shown in Figure 4, Compound (1) dose-dependently (3-30ug/rat) increased the percentage of time spent investigating the male stimulus, with a MED of 10pg. The effect of this dose was similar to the effect of progesterone.
From this study we can conclude that the effect of Compound (1) on female sexual proceptivity is centrally mediated.
In Figure 4 bars represent percentage of time spent investigating male, minus the percentage of time spent investigating the female stimuli+SEM, (n=7-8 per group). *P<0.05, **P<0.01 vs vehicle (Kruskal-Wallis ANOVA test followed by
Mann-Whitney’s test).
Example §
Inhibitory effect of NMB on female rat sexual proceptivity and antagonism of this effect by Compound (1)
We have investigated the potentially inhibitory effect of the BB, agonist neuromedin B (NMB) on female rat sexual proceptivity.
Ovariectomised female rats (Sprague Dawley, obtained from Charles River,
UK) were stereotaxically implanted (coordinates 0.89mm behind Bregma, 1.3mm lateral and 2.5mm vertical) with stainless steel cannulae (6 mm long, O.D. 0.75mm), held in place with dental cement. Animals were housed in groups of three and returned to a reversed lighting system of 12h light:dark (lights off 5.00-17.00h).
Correct placement of the cannulae was assessed post-mortem. Rats were used for tests two weeks after ovariectomy (one week after cannulation). The experiments started at least Sh into the dark period. Forty eight hours before tests, the animals were primed with 5pg oestradiol benzoate (OB) (s.c, in corn oil) and adapted to the apparatus (in the absence of stimuli animals) for 10min on 2 consecutive days prior testing. The
J 10min test was carried out as previously described. The difference in the percentage of time spent investigating male minus female was calculated, out of the total time spent investigating stimuli.
Progesterone (Prog, 0.5mg/0.1ml) was dissolved in corn oil and administered subcutaneously (s.c.), 4h before test, to induce proceptive behaviour. Compound (1) (15mg/kg, i.p.) was dissolved in 50% 2-hydroxypropyl-f-cyclodextrin in saline and administered 1h before the i.c.v. administration. Neuromedin B was obtained from ) Bachem, UK. It was dissolved in isotonic saline and administered i.c.v. over a 30sec period, with the aid of a pump set to deliver a flow of 10ul/min, 10min before tests. - The dosing volume was Sul/rat. Each rat received a total amount of 100ng.
As shown in Figure 5, progesterone (Prog) increased the percentage of time spent investigating the male stimulus, compared to the vehicle group, thus showing stimulation of proceptive behaviour. NMB (100ng, i.c.v.) significantly reduced : proceptivity in progesterone-treated rats. Moreover, pre-treatment with Compound (1) which acts as an antagonist (15mg/kg, i.p.) prevented the inhibitory effect of NMB.
However, the blockade obtained with the dose of Compound (1) used was not total.
From the present study we can conclude that stimulation of BB1 receptors with an agonist results in inhibition of proceptive behaviour. This inhibitory effect may be prevented by the presence of an antagonist. e.g. Compound (1) In figure 5 the bars represent percentage of time spent investigating male, minus the percentage of time spent investigating the female stimuli+SEM, (n=8-12 per group). ***P<0.001 vs progesterone (One-way ANOVA followed by Dunnett’s test).
Example 6
Demonstration that the effect of Compound (1) on female sexual behaviour is not mediated through sexual hormones
Previous examples have shown that Compound (1) (nanomolar affinity “mixed” BB/BB; receptor antagonist) has a dose-dependent stimulatory effect on sexual activity in the female rat, both on proceptivity and receptivity. Although the animals used in that study were ovariectomised, and therefore steroid hormones release can not be expected to occur in response to the compound, there is a possibility that the adrenal glands might secrete steroid hormones in response to
Compound (1). If that was the case, the mediation of the stimulatory effects by progesterone would be relevant for rodents, but it would not be the case for primates.
In the present study, we have investigated the potential effect of the bombesin receptor antagonist Compound (1) on secretion of progesterone. Oestradiol and pituitary hormones (Luteinising hormone (LH), follicle stimulating hormone (FSH) and : prolactin) have also been analysed in the same animals.
Ovariectomised adult female Sprague Dawley rats (180-200g) were housed in groups of 6 in a reversed lighting system of 12h light:dark (lights off 7.00-19.00h).
They were used for the experiments at least two weeks after ovariectomy. Forty eight ‘ hours before tests, the animals were primed with oestradiol benzoate (5ug/0.1ml in corn oil, s.c.). Progesterone (0.5mg/0.1ml, in corn oil, s.c.) was administered 4h before blood collection, as a positive control. Compound (1) (3-10mg/kg, i.p.) was administered in 50% 2-hydroxypropyl-B-cyclodextrin, 1h prior to blood collection.
Blood was collected from the trunk, after decapitation. It was immediately centrifuged (3500r.p.m., 4°C, Smin) and the plasmas frozen until assayed for hormonal content, using commercially available radioimmunoassay kits ('>’I-labelled hormones) for oestradiol, progesterone, LH, FSH and prolactin.
A single administration of progesterone resulted in a significant increase in the progesterone plasma levels (P<0.05), and a significant decrease in LH plasma levels (P<0.01), compared to animals injected with vehicle (Kruskal-Wallis followed by
Mann-Whitney test). However, Compound (1) (3-10mg/kg, i.p.) had no effect on the plasma levels of progesterone (Figure 6, where animals were pre-treated with Sug oestradiol benzoate, s.c., 48h before the test. They were tested 1h or 4h post-injection of Compound (1) (3-10mg/kg, p.o.) or progesterone (0.5mg/0.1ml, s.c.) respectively.
Values represent meantSEM, (n=9 per group). *P<0.05, vs vehicle (Kruskal-Wallis followed by Mann-Whitney test, vs vehicle)), oestradiol (Figure 7, where animals were pre-treated with Spg oestradiol benzoate, s.c., 48h before the test. They were tested 1h or 4h post-injection of Compound (1) (3-10mg/kg, p.o.) or progesterone (0.5mg/0.1ml, s.c.) respectively. Values represent meantSEM, (n=6-7 per group)), prolactin (Figure 8, where animals were pre-treated with Spg oestradiol benzoate, s.c., 48h before the test. They were tested 1h or 4h post-injection of Compound (1) (3- 10mg/kg, p.o.) or progesterone (0.5mg/0.1ml, s.c.) respectively. Values represent meantSEM, (n=10 per group)) LH (Figure 9, where animals were pre-treated with
Sug oestradiol benzoate, s.c., 48h before the test. They were tested lh or 4h post- injection of Compound (1) (3-10mg/kg, p.o.) or progesterone (0.5mg/0.1ml, s.c.) respectively. Values represent mean+SEM, (n=10 per group). **P<0.01, vs vehicle (Kruskal-Wallis followed by Mann-Whitney test, vs vehicle)) or FSH (Figure 10, where animals were pre-treated with Sug oestradiol benzoate, s.c., 48h before the test.
They were tested 1h or 4h post-injection of Compound (1) (3-10mg/kg, p.o.) or ‘ progesterone (0.5mg/0.1ml, s.c.) respectively. Values represent meantSEM, (n=10 per group).
From this experiment we can conclude that Compound (1) did not have an effect on the secretion of sexual hormones, thus suggesting that the effects of the compound on female sexual activity must be mediated by different mechanisms, maybe involving neurotransmitters.
Example 7
Effect of Compound (1) on the sexual behaviour of normal male rats
The potentially stimulatory effect of Compound (1) on male sexual behaviour has been tested on sexually vigorous rats. Sprague Dawley male rats (Charles River,
UK) were kept, 4 rats per cage, in a reversed lighting regime (12:12 hours, lights off at 5.00 h), with free access to food and water. The rats were pre-selected by being presented with a receptive female at 4 days intervals, i.e. every third day (having 2 clear days between presentations) until completing 6-7 days of baseline determination. The animals showing consistently vigorous behaviour (ejaculatory latencies <300s) were chosen for further experiments (n=24). Animals were randomised into three groups. All animals received all three treatments following a latin-square design. Treatments were administered once a week, with a baseline test in between treatments (4 days intervals between baseline and test day). Treatments were
Compound (1) (15mg/kg, dissolved in 50% 2-hydroxypropyl-f3-cyclodextrin in saline), vehicle, or Fluoxetine (20mg/kg dissolved in 100% DMSO). All treatments ‘were administered i.p. in a Iml/kg volume, 1h before tests. . For all the sexual behaviour tests, the males were placed in an observation arena (50-60 cm diameter), starting 5 hours into the dark cycle and observed under red illumination. Three to 4 minutes after placing the male in the arena, a receptive female (ovariectomised, bearing a 7 mm Silastic implant of oestradiol benzoate) was introduced to the arena and the following parameters noted: Mount Latency: time (in ‘ seconds) taken between introduction of female and first mount. A maximum time of 15 minutes (900 seconds) was allowed, and the test terminated if no mounts were recorded within that time (Figure 11), Intromission Latency: time (in seconds) taken between introduction of female and first intromission (Figure 12), Number of
Mounts: to reach ejaculation. When ejaculation was not reached, the number of mounts was not analyzed, Number of Intromissions: to reach ejaculation. When ejaculation was not reached, the number of intromissions was not analyzed (Figure 13 is number of mounts + intromissions), Ejaculation Latency: time (in seconds) taken from first intromission to ejaculation. A maximum time of 30 minutes (1800 seconds) was given, and the test terminated if ejaculation was not achieved in that time (Figure 14), and Refractory Period: time (in seconds) taken from ejaculation to the first mount of the next series of sexual activity. In those animals reaching ejaculation the test was terminated at the end of the refractory period, as indicated by the first mount of the next sexual cycle (Figure 15).
A one-way ANOVA followed by Dunnett’s t test was used to compare treated vs vehicle groups each day of testing, for all the sexual behaviour parameters. (*P<0.05, **P<0.01; n=15-16).
Mount latency and intromission latency were significantly increased in the fluoxetine-treated group compared to the vehicle group. Ejaculation latency and refractory period were also increased in this group, showing a decrease in sexual performance as well as the decreased arousal. No changes were seen in the number of mounts and intromissions required to achieve ejaculation. Unlike Fluoxetine,
Compound (1) had no effect on any of the parameters studied, at a dose shown to be stimulatory in sexually dysfunctional males (see example 9). From the present study we can conclude that Compound (1) has no effect on sexual behaviour in sexually . vigorous males.
Example 8
Effect of Compound (1) on the sexual behaviour of sexually dysfunctional male : rats
Fluoxetine induces ejaculation delay, anorgasmy and loss of sexual desire in humans (Crenshaw and Goldberg, 1996). A model of male sexual dysfunction in the rat, induced by daily administration of fluoxetine until a significant detrimental effect on sexual behaviour (arousal and ejaculation) was established. The potentially stimulatory effect of Compound (1) on male sexual behaviour in these sexually dysfunctional male rats was examined. The effects of Compound (1) were compared to those of yohimbine. Preclinical and clinical studies suggest that yohimbine may be an effective treatment for sexual side-effects caused SSRI (Hollander, E., McCarley,
A. (1993) J. Clin. Psychiatry 53:207-209. and Jacobsen).
Sprague Dawley male rats (Charles River, UK) were kept, 4 rats per cage, in a reversed lighting regime (12:12 hours, lights off at 5.00 h), with free access to food and water. The rats were pre-selected by being presented with a receptive female at 4 days intervals, i.e. every third day (having 2 clear days between presentations) until completing 6-7 trials of baseline determination. The animals showing consistently vigorous behaviour (ejaculatory latencies <300s) were chosen for further experiments.
Animals were treated for 3 consecutive days with either vehicle (water) or fluoxetine (20mg/kg, i.p., in a 2ml/kg dosing volume). On the fourth day, the animals treated with water received vehicle (veh+veh) and the animals treated with fluoxetine received one of the three following treatments: Compound (1) (15mg/kg, dissolved in 50% 2-hydroxypropyl-B-cyclodextrin in saline), vehicle (cyclodextrine), or yohimbine (2mg/kg dissolved in water). All treatments were administered i.p. in a ’ Iml/kg volume, 1h before tests.
For all the sexual behaviour tests, the males were placed in an observation arena (50-60 cm diameter), starting 5 hours into the dark cycle and observed under red illumination. Three to 4 minutes after placing the male in the arena, a receptive female (ovariectomised, bearing a 7 mm Silastic implant of oestradiol benzoate) was : introduced to the arena and the following parameters noted: Mount Latency: time (in seconds) taken between introduction of female and first mount. A maximum time of ’ 15 minutes (900 seconds) was allowed, and the test terminated if no mounts were recorded within that time (Figure 16), Ejaculation Latency: time (in seconds) taken from first intromission to ejaculation. A maximum time of 30 minutes (1800 seconds) was given, and the test terminated if ejaculation was not achieved in that time (Figure 17), Percentage of males achieving ejaculation within 30 minutes was calculated (Figure 18).
A one-way ANOVA followed by Dunnett’s t test was used to compare the fluoxetine+vehicle group and other groups for mount and ejaculatory latencies.
Percentage of animals ejaculating was analysed using a Chi-square test followed by
Fisher’s test. (*: P<0.05, **: P<0.01, ***: P<0.001; n=15-19).
Mount latency and ejaculation latency were significantly increased in the fluoxetine-treated groups compared to the vehicle+vehicle group, indicating a decrease in sexual desire as well as sexual performance in these groups. The number of animals ejaculating was significantly lower in the fluoxetine-treated groups, indicating anorgasmy. Compound (1) significantly decreased the mount and ejaculatory latencies at the same time as increasing the percentage of animals ejaculating in the animals rendered sexually dysfunctional by the fluoxetine treatment, to levels comparable to normal animals (veh+veh). Yohimbine followed a similar trend, although this did not reach significance.
From the present study we can conclude a stimulatory effect of Compound (1) on } sexual behaviour in males suffering from sexual dysfunction, at the level of sexual desire, sexual performance and anorgasmy.
Example 9 (S)-3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl- 2-[4-(4-nitro-phenyl)-oxazol-2-ylamino]-propionamide
N x i N° N N
ON’ Oo Z 0 0) 1. To a stirred solution of p-nitrophenylchloroformate (9.27g, 46mmol) in
THF (200ml) at 0°C was added dropwise a solution of H-(S)-aMeTrp-OMe (1a) (10.7g, 46 mmol) and triethylamine (6.4ml, 46mmol) in THF (100ml) over 1 hour.
Stirring was continued for a further 30 minutes at room temperature, after which aqueous ammonia (15ml) was added. IR after 10 minutes indicated bands at 1732 and 1660 cm’. The THF was removed under reduced pressure, and the residue was taken up in EtOAc and washed with IN HCI (x2), Na,CO; solution (until intense yellow colour subsided, ~x8), brine, and dried (MgSO). The solvent was removed under reduced pressure to give 2a as a foam (10.3g, 82%):MS m/e (AP+): 276.16 (M" + H, 100%);
MS m/e (AP-): 274.11 (M"- H, 100%);
IR (film): 3383, 1724, 1657, 1600, 1539, 1456, 1374, 1256, 1108, 743 cm’; 'H NMR (CDC): 8= 1.70 (3H, s), 3.38 (1H, d, J=14.7 Hz), 3.59 (IH, 4, . 20 J=14.7 Hz), 3.71 (3H, 5s), 4.22 (2H, s), 5.16 (1H, s), 6.99 (1H, d, J=2.2 Hz), 7.08-7.20 (2H, m), 7.34 (1H, d, J=8.1 Hz), 7.59 (1H, d, J=7.8 Hz), 8.09 (1H, s). 2. The urea (2a) (6.4g, 23mmol) and 2-bromo-1-(4-nitro-phenyl)-ethanone (6.0g, 23mmol) were stirred in toluene (500ml)/dioxan (100ml) and maintained under reflux for 30 hours, after which solvent was removed under reduced pressure and the residue was purified by chromatography using a 90g Biotage cartridge. 10% EtOAc in . heptane eluted the bromide starting material. 20% EtOAc eluted the desired product.
Removal of solvent under reduced pressure gave 3a as a foam (840mg, 9%):
MS m/e (ES+): 420.56 (M", 100%);
S IR (film): 3394, 1732, 1632, 1605, 1574, 1515, 1456, 1334, 1253, 1210, 1108, 1072, 940, 854, 734 cm™; 'H NMR (CDCl): &= 1.91 (3H, s), 3.46 (1H, d, J=14.6 Hz), 3.69 (3H, s), 3.78 (1H, d, J=14.6 Hz), 5.57 (1H, s), 6.89 (1H, d, J=2.2 Hz), 7.03-7.08 (1H, m), 7.14-7.18 (1H, m), 7.34 (1H, d, J=8.1 Hz), 7.41 (1H, d, J=8.1 Hz), 7.63 (1H, s), 7.85 (2H, d,
J=9.0 Hz), 8.05 (1H, s), 8.24 (2H, d, J=8.6 Hz). 3. The ester (3a) (840mg, 2mmol) was dissolved in dioxan (50ml) and LiOH.H,0 (336mg, 8mmol) in HO (25ml) was added. The mixture was stirred vigorously overnight, and then neutralised with 1M HCI (8ml, 8mmol). The majority of the dioxan was removed under reduced pressure and the product was crystallised, filtered off, washed with water and dried under reduced pressure to give pure 4a (668mg, 82%):
MS m/e (ES+): 407 (M* + H);
IR (film): 1633 cm’; 'H NMR (DMSO-dg) 8= 1.49 (3H, 5), 3.30-3.35 (1H, m, masked by H,0), 3.59 (1H, d, J=14.7 Hz), 6.86-6.90 (1H, m), 6.99-7.03 (2H, m), 7.30-7.36 (2H, m), 7.48 (1H, s), 7.94 (2H, d, J=9.0 Hz), 8.27-8.30 (3H, m), 10.88 (1H, s), (CO;H not seen). 4. The acid (4a) (1.148g, 2.8mmol), O-benzotriazol-1-yl-N,NN’N- tetramethyluronium hexafluorophosphate (HBTU, 1.06g, 2.8mmol), and NN- diisopropylethylamine (DIPEA, 490ul, 2.8mmol) were stirred in DMF (10ml) for 5 minutes before adding DIPEA (490pul, 2.8mmol) and [1-(5-methoxy-2- pyridyl)cyclohexyl}-methanamine (see WO 98/07718, 678mg, 3.1mmol). HPLC . indicated that reaction was complete within 1 hour. Solvent was removed under reduced pressure and the residue was taken up in EtOAc. The organic layer was washed with brine, saturated NaHCO; (x3), brine and dried (MgSO,), after which solvent was removed under reduced pressure. The residue was purified by chromatography using RP silica with 65% MeOH in H,O. Pure fractions were . evaporated to give the desired product as an amorphous solid (1.12g, 66%):
MPt: 100-105°C;
MS m/e (ES+): 609.63 (M* + H, 100%);
IR (film): 3359, 3272, 3054, 2932, 2857, 1628, 1606, 1573, 1515, 1488, 1393, 1336, 1268, 1232, 1181, 1150, 1131, 1097, 1028, 1012, 962, 939, 900, 853, 831, 737 em’; 'H NMR (CDCl3): 8= 1.10-1.60 (8H, m), 1.72 (3H, s), 1.95-2.02 (2H, m), 3.31-3.42 (2H, m), 3.41 (1H, d, J=14.6 Hz), 3.50 (1H, d, J=14.6 Hz), 3.69 (3H, s), 5.34 (1H, s), 6.90-6.97 (2H, m), 7.04-7.09 (2H, m,) 7.14-7.19 (1H, m), 7.33 (1H, d,
J=8.1 Hz), 7.46 (1H, d, J=7.8 Hz), 7.54 (1H, s), 7.77 (2H, d, J=8.8 Hz), 8.00 (1H, d,
J=2.9 Hz), 8.04 (1H, 5), 8.21 (2H, d, J=8.8 Hz); (amide masked by CHCI3)
HPLC A: Rt. 11.86 min, 99.8/100% purity, 20-100% CH3;CN in H,O (+0.1%TFA) over 15 min at ml min", Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
HPLC B: Rt. 14.32 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9, 1mlmin’', Prodigy ODSIII 250x4.6mm SuM, 215 and 254 nm.
Example 10 (S)-3-(1H-Indol-3-yl)-N-(1-methoxymethyl-cyclohexylmethyl)-2-methyl-2-{4-(4- nitro-phenyl)-oxazol-2-ylamino]-propionamide
N x
SING) : ON’ (0)
’ The above compound was synthesized from Intermediate 4a and Intermediate 13 using the same method as used for Example 9. The acid (4a) (203mg, 0.5mmol),
HBTU (190mg, 0.5mmol), and DIPEA (87ul, 0.5mmol) were stirred in DMF (10ml) for 5 minutes before adding DIPEA (87ul x 2, 1.0mmol) and Intermediate 13 (94mg, 0.5mmol, Scheme 6). After 4 hours the solvent was removed under reduced pressure and residue taken up in EtOAc. The organic layer was washed with brine, saturated
NaHCO; (x3), brine, dried (MgSO4) and solvent removed under reduced pressure.
The residue was heated to 60°C in methanol and product filtered off. Drying under reduced pressure gave the desired product as a yellow crystalline solid (214mg, 78%):
MPt: 189-192°C;
MS m/e (ES+): 546.49 (M" + H, 100%);
IR (film): 3285, 2928, 2849, 1637, 1604, 1516, 1453, 1334, 1260, 1108, 1077, 860, 743, 729 cm; 'H NMR (DMSO-dg): = 1.10-1.35 (10H, m), 1.44 (3H, s), 2.91-3.01 (3H, m), 3.06-3.12 (1H, m), 3.07 (3H, s), 3.26-3.31 (1H, m), 3.64 (1H, d, J=14.4 Hz), 6.87-6.93 (2H, m), 7.01 (1H, t, J=7.4 Hz), 7.29-7.37 (3H, m), 7.44 (1H, s), 7.94 (2H, d, J=9.0
Hz), 8.26 (2H, d, J=8.8 Hz), 8.34 (1H, s), 10.84 (1H, s);
HPLC A: Rt. 17.07 min, 100/100% purity, 20-100% CH;CN in H,0 (+0.1%TFA) over 15 min at Imlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
HPLC B: Rt. 14.35 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9,
Imimin’', Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm.
Example 11 , (S)-3-(1H-Indol-3-yl)-2-methyl-2-[4-(4-nitro-phenyl)-oxazol-2-ylamino}-NV-(2-oxe- 2-phenyl-ethyl)-propionamide.
; N x oO . 0
On’ Oo 0)
The above compound was synthesised from Intermediate 4a using the same method as used for Example 9. The acid (4a) (203mg, 0.5mmol), HBTU (190mg, 0.5mmol), and DIPEA (87ul, 0.5mmol) were stirred in DMF (10ml) for 5 minutes before adding DIPEA (87ul, 0.5mmol) and 2-amino-1-phenyl-ethanone (103mg, 0.6mmol). After 4 hours the solvent was removed under reduced pressure and residue taken up in EtOAc, washed with brine, saturated NaHCOj3 (x3), brine, dried (MgSOy4) and solvent removed under reduced pressure. The residue was purified by chromatography using NP 20g Mega Bond Elut cartridge and 40% ethyl acetate in heptane as eluent. Evaporation of pure fractions gave the desired product as a yellow amorphous solid (170mg, 65%):
MPt: 80-90°C;
MS m/e (AP+): 525.83 (16%), 524.44 (M" + H, 100%);
IR (film): 3396, 3059, 2983, 2932, 1694, 1628, 1605, 1575, 1514, 1449, 1336, 1284, 1264, 1225, 1181, 1154, 1096, 1072, 1010, 1001, 940, 853, 737 cm’; 'H NMR (DMSO-dg): = 1.50 (3H, s), 3.39 (1H, d, J=14.7 Hz), 3.64 (1H, d,
J=14.6 Hz), 4.53 (1H, d.d, J=18.1 and 5.4 Hz), 4.66 (1H, d.d, J=18.1 and 5.5 Hz), 6.87 (1H, t, J=7.4 Hz), 6.95 (1H, d, J=2.2 Hz), 7.00 (1H, t, J=7.4 Hz), 7.30 (1H, d, J=8.1
Hz), 7.34 (1H, d, J=8.1 Hz), 7.41 (1H, s), 7.50-7.55 (2H, m), 7.62-7.67 (1H,m), 7.94- 7.99 (4H, m), 8.24 (1H, t, J=5.4 Hz), 8.27 (2H, d, J=9.0 Hz), 8.31 (1H, s), 10.86 (1H, : 5);
HPLC A: Rt. 20.83 min, 98.3/99.6% purity, 20-100% CH;CN in H,0 : (+0.1%TFA) over 25 min at Imlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
HPLC B: Rt. 6.82 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9, Imlmin’ : !, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm.
Example 12 (S)-N-[1-(5-Methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[4-(4-nitro- phenyl)-oxazol-2-ylamino}]-3-phenyl-propionamide } N° N ~
Oy O Z 0
Oo
The above compound was synthesised from 1b and 4b using the same methods as used for Example 9. The acid (4b) (120mg, 0.33mmol), HBTU (124mg, 0.33mmol), and DIPEA (114pl, 0.66mmol), and [1-(5-methoxy-2- pyridyl)cyclohexyl]-methanamine' (86mg, 0.4mmol) were stirred in DMF (4ml) for 18 hours. Solvent removed under reduced pressure and residue taken up in EtOAc.
The organic layer was washed with brine, saturated NaHCO; (x3), brine, dried (MgSO,) and solvent removed under reduced pressure. The residue was purified by chromatography using NP silica with 10-80% ethyl acetate in heptane. Pure fractions were evaporated to give the desired compound as a yellow amorphous solid (90mg, 49%):
MS m/e (AP+): 570.23 (M+ H, 100%);
IR (film): 3363, 2930, 2856, 1658, 1651, 1628, 1574, 1515, 1488, 1334, 1268, : 1232, 1073, 1030, 938, 852 cm™; 'H NMR (DMSO-dg): 6= 0.94-1.46 (11H, m), 1.98-2.10 (2H, m), 3.04-3.14 : (2H, m), 3.25-3.32 (1H, m), 3.57 (1H, d, J=13.6 Hz), 3.73 (3H, s), 6.95-7.00 (3H, m), 7.10-7.24 (5H, m), 7.44 (1H, s), 7.93 (2H, d, J=8.8 Hz), 8.14 (1H, d, J=2.8 Hz), 8.27 (2H, d, J=9.2 Hz), 8.36 (1H, s);
HPLC A: Rt. 5.49 min, 99.76% purity, 20-100% CH;CN in H,0 (+0.1%TFA) - over 7 min at 1.5 mimin’!, Prodigy ODSIII 150x4.6mm 3uM at 40°C, 200-300 nm;
HPLC B: Rt. 5.72 min, 99.46% purity, 20-90% CH;CN/Tris (1mM) over 7 min at 2mlmin”, Prodigy Phenyl-Ethyl, 100x4.6mm 5pM at 30°C, 200-300 nm.
Example 13 (S)-2-[4-(4-Cyano-phenyl)-oxazol-2-ylamino]-3-(1 H-indol-3-yl)-N-[1-(5-methoxy- pyridin-2-yl)-cyclohexylmethyl}-2-methyl-propionamide
N
XN
N° N i D
NZ ?
The above compound was synthesized from 2a via 6a as outlined in Scheme 2 using methods analogous to those used for Example 9. The acid (6a) (309mg, 0.8mmol), HBTU (303mg, 0.8mmol), DIPEA (140ul, 0.8mmol) were stirred in DMF (Sml) for 5 minutes before adding DIPEA (140ul, 0.8mmol) and [1-(5-methoxy-2- pyridyl)cyclohexyl]-methanamine (WO 98/07718) (185mg, 0.84mmol). HPLC indicated reaction complete within 1 hour. Solvent removed under reduced pressure and residue taken up in EtOAc. Washed with brine, saturated NaHCO; (x3), brine, dried (MgSO4) and solvent removed under reduced pressure. Residue purified by chromatography using RP silica with 65% MeOH in H;0O. Pure fractions were evaporated to give Example 13 as a white amorphous solid (320mg, 68%):
MPt: 105-108°C;
MS m/e (ES+): 589.32 (M" + H, 100%), 590.18 (62%);
IR (film): 3355, 2932, 2857, 2225, 1628, 1572, 1521, 1489, 1456, 1328, 1269, 1232, 1096, 1072, 1029, 938, 844, 741 cm’; ; 'H NMR (CDCl): 6= 1.20-1.60 (8H, m), 1.70 (3H, s), 1.93-2.03 (2H, m), 3.30-3.52 (4H, m), 3.68 (3H, s), 5.30 (1H, s), 6.89 (1H, d, J=2.4 Hz), 6.94 (1H, d.d, - J=8.8 and 2.9 Hz), 7.03-7.09 (2H, m,) 7.14-7.19 (1H, m), 7.20-7.25 (1H, m), 7.33 (1H, d, J=8.1 Hz), 7.46 (1H, d, J=7.8 Hz), 7.50 (1H, s), 7.63 (2H, d, J=8.5 Hz), 7.72 (2H, d, J=8.3 Hz); 8.00 (1H, d, J=2.9 Hz), 8.05 (1H, s);
HPLC A: Rt. 11.63 min, 97.7/100% purity, 20-100% CH;CN in HO : (+0.1%TFA) over 15 min at Imlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
HPLC B: Rt. 9.20 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9, 1mimin™, Prodigy ODSIII 250x4.6mm 5pM, 215 and 254 nm.
Example 14 (S)-3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl- 2-(4-phenyl-oxazol-2-ylamino)-propionamide
N
NY
N° N =~ oO Ze)
I
The above compound was synthesised from 2a via 6b as outlined in Scheme 2 using methods analogous to those used for Example 9. The acid (6b) (57mg, 0.148mmol), HBTU (56mg, 0.148mmol), DIPEA (26ul, 0.148mmol) were stirred in
DMF (5ml) for 5 minutes before adding DIPEA (26pl, 0.148mmol) and [1-(5- methoxy-2-pyridyl)cyclohexyl]-methanamine (see WO 98/07718, 34mg, 0.148mmol).
HPLC indicated that the reaction was complete within 2 hours. Solvent was removed under reduced pressure and the residue was taken up in EtOAc, washed with brine, sat. NaHCO; (x3), brine, dried (MgSO) and solvent removed under reduced pressure. . The residue was purified by chromatography using RP silica with 70% MeOH in H,0 as eluent. Repurification using NP 8g Biotage cartridge with 45% ethyl acetate in : heptane as eluent gave the desired product as a glass (20mg, 24%):
MPt: 85-90°C;
MS m/e (ES+): 564.06 (M*, 87%), 564.96 (M+ H, 100%);
IR (film): 3289, 2931, 2857, 1627, 1569, 1520, 1488, 1456, 1337, 1267, 1233, : 1072, 1072, 1030, 939, 739 cm’; 'H NMR (DMSO-dg): 8= 0.95-1.45 (11H, m), 2.00-2.10 (2H, m), 3.10-3.25 (2H, m), 3.21 (1H, d, J=14.6 Hz), 3.59 (1H, d, J=14.6 Hz), 3.71 (3H, s), 6.84-7.14 (7H, m), 7.24-7.40 (5H, m,), 7.70 (2H, d, J=7.6 Hz), 8.05 (1H, s), 8.15 (1H, d, J=2.9
Hz), 10.82 (1H, s);
HPLC A: Rt. 12.01 min, 96.8/95.3% purity, 20-100% CH;CN in HO (+0.1%TFA) over 15 min at 1mlmin”, Prodigy ODSIII 250x4.6mm 5pM, 215 and 254 nm;
HPLC B: Rt. 17.27 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9,
Imlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm.
Example 15 (S)-2-(4-Ethyl-oxazol-2-ylamino)-3-(1 H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2- yl)-cyclohexylmethyl]-2-methyl-propionamide
N
NN
Pp J Soy
NN = 0) Z>0
The above compound was synthesized from 2a via 6c as outlined in Scheme 2 using methods analogous to those used for Example 9. The acid (6c) (188mg, 0.6mmol), HBTU (228mg, 0.6mmol), and DIPEA (105ul, 0.6mmol) were stirred in
DMF (10ml) for 5 minutes before adding DIPEA (105ul, 0.6mmol) and [1-(5- . methoxy-2-pyridyl)cyclohexyl]-methanamine (see WO 98/07718, 150mg, 0.65mmol). HPLC indicated that the reaction was complete within 4 hours. Solvent : was removed under reduced pressure and residue was taken up in EtOAc, washed with brine, sat. NaHCO; (x3), brine, dried (MgS0O,) and solvent removed under reduced pressure. The residue was purified by chromatography using RP silica with 65% MeOH in H,;0. The product was repurified using 20g Mega Bond Elut silica cartridge with 45% ethyl acetate in heptane as eluent. Pure fractions were evaporated : to give the above compound as a glass (30mg, 10%):
MPt: 60-65°C;
MS m/e (ES+): 516.24 (M" + H, 47%), 517.01 (100%), 538.10 (M* + Na, 25%);
IR (film): 3272, 3054, 2931, 2856, 1651, 1622, 1596, 1573, 1520, 1489, 1457, 1358, 1268, 1232, 1206, 1131, 1083, 1028, 949, 830, 740 cm"; 'H NMR (DMSO-dg): 8= 1.10-1.50 (8H, m), 1.11 (3H, t, J=7.4 Hz), 1.29 (3H, s), 2.05-2.15 (2H, m), 2.28-2.34 (2H, m), 3.08-3.18 (3H, m), 3.48 (1H, d, J=14.4 Hz), 3.79 (3H, s), 6.80-6.90 (3H, m), 6.97-7.04 (2H, m,), 7.10-7.20 (3H, m), 7.27-7.30 (2H, m), 8.17 (1H, d, J=2.9 Hz), 10.80 (1H, s);
LCMS: Rt. 1.36 min, 100% purity, 5-100% CH3;CN in H>O (+0.1% Formic acid) over 2 min at 4 mlmin”', Prodigy ODSIII 50x4.6mm 5puM, 215 nm, MS m/e (ES+) 515.95 (100%);
HPLC B: Rt. 12.29 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9, 1 mimin™, Prodigy ODSII 250x4.6mm 5uM, 215 and 254 nm;
Example 16 (S)-3-(1 H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl- 2-[4-(4-nitro-phenyl)-thiazol-2-ylamino]-propionamide
N
SN i NN N
ON Oo = 0
Oo
The above compound was synthesized using a one-pot procedure as outlined in Scheme 3. A suspension of H-S-aMeTrp-OH (Intermediate 7) (437mg, 2mmol), 2-chloro-4-(4-nitro-phenyl)-thiazole (see Peet, Norton P.; Sunder, Shyam.
Reinvestigation of the reported preparation of 3-(4-nitrophenyl)thiazolo[2,3- c][1,2,4]triazepines, J. Heterocycl. Chem. (1986), 23(2), 593-5; 481mg, 2mmol), copper (I) iodide (38mg, 0.2mmol), and K,CO; (415mg, 3mmol) in DMF (12ml) under nitrogen was heated to 130°C for 12 hours. The reaction mixture was cooled to ambient temperature before adding HBTU (759mg, 2mmol) and [1-(5-methoxy-2- pyridyl)cyclohexyl]-methanamine (see WO 98/07718; 441mg, 2mmol). The mixture was stirred overnight, then concentrated in vacuo, after which the residue was partitioned between water (20ml) and dichloromethane (30ml). The organic phase was separated and filtered through silica (3x12cm) using 500ml of dichloromethane and then 500ml of dichloromethane-ether (1:1). Fractions containing product were concentrated under reduced pressure. The residue was absorbed onto 3.5g silica and purified by chromatography (3x1lcm) using heptane-ethyl acetate (1:1.1). The product was repurified using RP chromatography (Biotage KP-C18-HS Flash 12M, i 15ml.min’!, 60-100% methanol in water). Concentration under reduced pressure gave the desired compound as a pale yellow amorphous solid (27mg, 2%):
MPt: 110-114°C;
MS m/e (AP+): 624.88 (M", 100%), 625.70 (M" + H, 52%);
IR (film): 3385, 3279, 2931, 2855, 1654, 1595, 1542, 1509, 1456, 1341, 1268, : 1231, 1108, 1058, 908, 844, 731 cm’'; 'H NMR (CDCls): 8= 1.15-1.55 (8H, m), 1.71 (3H, s), 1.90-2.00 (2H, m), 3.16-3.42 (2H, m), 3.46 (1H, d, J=14.9 Hz), 3.60 (1H, d, J=14.6 Hz), 3.70 (3H, s), 5.51 (IH, s), 6.89-6.93 (3H, m), 6.98 (1H, d, J=8.8 Hz), 7.05-7.10 (1H, m), 7.15-7.25 (2H, m), 7.34 (1H, d, J=8.3 Hz), 7.47 (1H, d, J=7.8 Hz), 7.90 (2H, d, J=9.0 Hz), 7.98 (1H, d, J=2.9 Hz), 9.05 (1H, s), 8.21 (2H, d, J=8.8 Hz);
HPLC A: Rt. 12.30min, 99.4% purity, 20-100% CH3;CN in H,0 (+0.1%TFA) over 15 min at Imlmin™', Prodigy ODSIII 250x4.6mm 5uM, 200-300 nm;
HPLC B: Rt. 15.38min, 99.5% purity, 80:20 methanol/Tris buffer at pH9,
Imlmin’, Prodigy ODSIII 250x4.6mm 5uM, 200-300 nm.
Example 17 (S)-2-(Benzooxazol-2-ylamino)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
N
SN
0) S
CL rs
N 0) YZ 1. The following reagents were combined in the order that they are listed:
Intermediate 7 (545mg, 2.5mmol), 2-chlorobenzoxazole (384mg, 2.5mmol), . potassium carbonate (346mg, 2.5mmol), benzyltriethylammonium chloride (TEBA, 114mg, 0.5mmol), triethylamine (1.04ml, 7.5mmol), DMF (12.5ml), deoxygenated water (1.25ml), copper (I) iodide (24mg, 0.125mmol), trans-dichlorobis(tri-o-tolyl- phosphine)palladium(II) (99mg, 0.125mmol). After heating at 100°C under nitrogen for 24 hours the DMF was removed under reduced pressure. The residue was taken up in ethyl acetate/water and the aqueous phase was acidified to pH 6-6.5 using citric acid. The aqueous phase was extracted with three further portions of ethyl acetate. : The combined organic layers were dried (MgSO,4) and solvent was removed under reduced pressure. The residue was purified by chromatography using 10g NP silica with 0-100% ethyl acetate in heptane. Crystallisation from dichloromethane gave (S)- 2-(benzooxazol-2-ylamino)-3-(1 H-indol-3-yl)-2-methyl-propionic acid (245mg, 29%). MS m/e (ES+) 335.97 (M" + H, 100%), 336.69 (85%). 2. The propionic acid (234mg, 0.7mmol), HBTU (265mg, 0.7mmol), and DIPEA (122pl, 0.7mmol) were stirred in DMF (10ml) for 5 minutes before adding DIPEA (122pl, 0.7mmol) and [1-(2- pyridyl)cyclohexyl]methylamine (WO 98/07718; 140mg, 0.74mmol). After 4 hours at ambient temperature the solvent was removed under reduced pressure. The residue was purified by chromatography using NP silica with 50% ethyl acetate in heptane as eluent. Pure fractions were evaporated to give the desired compound as fine needles (44mg, 3%):
MPt: 198-200°C;
MS m/e (ES*): 508.59 (100%, M* + H), 509.92 (10%);
IR (film): 3381, 3222, 3048, 2929, 2856, 1635, 1581, 1552, 1519, 1458, 1353, 1241, 1096, 742 cm’; 'H NMR (CDCl): § = 1.20-1.60 (8H, m), 1.76 (3H, s), 1.95-2.05 (2H, m), 3.34 (1H, dd, J=13.2 and 4.9 Hz), 3.45 (1H, d.d, J=13.2 and 5.6 Hz), 3.50 (2H, s), 5.67 (1H, s), 6.78-6.82 (1H, m), 6.89 (1H, d, J=2.2 Hz), 6.99-7.35 (10H, m), 7.43 (1H, d, J=8.1 Hz), 8.01 (1H, s), 8.24 (1H, d, J=4.6 Hz),
HPLC A: Rt. 10.54 min, 100/100% purity, 20-100% CH;CN in H,0 (+0.1%TFA) over 15 min at 1mlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
HPLC B: Rt. 10.67 min, 100/100% purity, 80:20 methanol/Tris buffer at pH9,
Imlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
Example 18 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(pyridin-4-ylamino)-N-(1-pyridin-2-yl- ' cyclohexylmethyl)-propionamide
N
=
J N
— 0 z
The above compound was prepared on the same scale and using an analogous method as used for Example 17. 1. The method of Example 17 was repeated except that 4-bromopyridine hydrochloride (486mg, 2.5mmol) was used. 2. The acid from step 1 (30mg, 0.1mmol), HBTU (38mg, 0.1mmol), and DIPEA (18ul, 0.1mmol) were stirred in DMF (10ml) for 5 minutes before adding DIPEA (18ul, 0.1mmol) and [1-(2- pyridyl)cyclohexyl]methylamine (WO 98/07718; 19mg, 0.1lmmol). After 2 hours at ambient temperature the solvent was removed under reduced pressure. The residue was taken up in ethyl acetate and washed with sodium bicarbonate solution (x2), brine, and dried (MgSO,). The solvent was removed under reduced pressure. The crude product was purified by chromatography using 10g ISCO
Redisep cartridge with ethyl acetate as eluent. Repurification using 20g RP-C18 with 70% methanol in water and subsequent evaporation gave the desired product in crystalline form (6mg, 13%):
MPt: 180-195°C; ’ MS m/e (APY): 468.12 (M" + H, 100%), 469.59 (M" + 2H, 20%);
MS m/e (APY): 467.56 (M’, 45%), 466.60 (M" - H, 100%), 465.64 (M" - 2H,
IR (film): 3316, 2930,1651, 1602, 1515, 1430, 1106, 997, 816, 741 cm’; : NMR (CDCL):8 = 1.25-1.70 (8H, m), 1.46 (3H, s), 2.00-2.10 (2H, m), 3.27 (1H, d, J=14.9 Hz), 3.30-3.48 (2H, m), 3.36 (1H, d, J=14.9 Hz), 4.43 (1H, s), 6.22 (2H, d, J=5.6 Hz), 6.85 (1H, d, J=2.0 Hz), 6.89-6.93 (1H, m), 7.11-7.37 (5H, m), 7.46-7.54 (2H, m), 8.08-8.13 (4H, m);
HPLC A: Rt. 7.21 min, 96.1/96.5% purity, 20-100% CH3;CN in HO (+0.1%TFA) over 15 min at 1mimin, Prodigy ODSIII 250x4.6mm 5pM, 215 and 254 nm;
HPLC B: Rt. 6.02 min, 99.1/100% purity, 80:20 methanol/Tris buffer at pH9, 1mlmin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm.
Example 19 (S)-3-(1H-Indol-3-yl)-2-(isoquinolin-4-ylamino)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
N x
N o / A\ N N | Ns
Oo =
Example 19 was prepared on the same scale and using an analogous method as used for Example 17. 1. The method of Example 17 was followed except that 4-bromoisoquinoline (520mg, 2.5mmol) was used. 2. The acid from step 1 (40mg, 0.12mmol), HBTU (46mg, 0.12mmol), and
DIPEA (21pl, 0.12mmol) were stirred in DMF (10ml) for 5 minutes before adding
DIPEA (21pl, 0.12mmol) and [1-(2- pyridyl)cyclohexyl]methylamine (WO 98/07718; 23mg, 0.12mmol). After 2 hours at ambient temperature the solvent was removed under reduced pressure. The residue was taken up in ethyl acetate and ‘ washed with sodium bicarbonate solution (x2) and brine and dried (MgSQO;). The solvent was removed under reduced pressure. The crude product was purified by chromatography using 10g ISCO Redisep cartridge with 80% ethyl acetate in heptane as eluent. Repurification using 20g RP-C18 with 70% methanol in water and subsequent evaporation gave the desired product as a glass (9mg, 14%):
MPt: 98-101°C;
MS m/e (AP): 518.28 (100%, M" + H), 517.40 (M", 50%);
MS m/e (APY): 516.53 (75%, M"), 515.63 (100%, M" - H);
IR (film): 3385, 3278, 3052, 2927, 2849, 1651, 1585, 1520, 1455, 1403, 1343, 781,740 cm’;
NMR (CDCl): = 1.20-1.65 (11H, m), 1.93-2.10 (2H, m), 3.35 (1H, d,
J=14.6Hz), 3.39-3.52 (2H, m), 3.48 (1H, d, J=14.9 Hz), 4.62 (1H, s), 6.55-6.59 (1H, m), 6.90 (1H, d, J=2.0 Hz), 7.00 (1H, d, J=8.1 Hz), 7.17-7.28 (4H, m), 7.37-7.55 (4H, mm), 7.62 (1H, s), 7.70 (1H, d, J=7.6 Hz), 7.74-7.76 (1H, m), 7.87 (1H, d, J=8.1 Hz), 8.15 (1H, s), 8.63 (1H, 5)
HPLC A: Rt. 7.52 min, 100/100% purity, 20-100% CH3;CN in H,0 (+0.1%TFA) over 15 min at Imlmin”, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm;
HPLC B: Rt. 8.33 min, 99.7/100% purity, 80:20 methanol/Tris buffer at pH9, 1mlmin”', Prodigy ODSIII 250x4.6mm 51M, 215 and 254 nm;
Example 20 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (pyrimidin-5-ylamino)-propionamide
N x > Hn N So
N= 0 y
The above compound was prepared on the same scale and using an analogous : method as used for Example 17. 1. The method of Example 17 was followed except that 5-bromopyrimidine (397mg, 2.5mmol) was used. 2. The acid from step 1 (150mg, 0.5mmol), HBTU (190mg, 0.5mmol), and
DIPEA (87ul, 0.5mmol) were stirred in DMF (10ml) for 5 minutes before adding
DIPEA (87ul, 0.5mmol) and [1-(2-pyridyl)cyclohexyl]methylamine (WO 98/07718; 95mg, 0.5mmol). After 2 hours at ambient temperature the solvent was removed under reduced pressure. The residue was taken up in ethyl acetate and washed with sodium bicarbonate solution (x2) and brine and dried (MgSQ,). The solvent was removed under reduced pressure. The crude product was purified by chromatography using 10g ISCO Redisep cartridge with 90% ethyl acctate in heptane as eluent.
Removal of the solvent under reduced pressure gave the desired product as a foam (135mg, 58%):
MPt: 95-98°C;
MS m/e (AP"): 470.60 (25%), 469.58 (M" +H, 100%), 468.77 (M", 92%);
MS m/e (AP): 467.60 (M" - H, 70%), 466.85 (100%);
IR (film): 3291, 3052, 2931, 2857, 1651, 1575, 1519, 1470, 1455, 1427, 1357, 1306, 1265, 1237, 1194, 1156, 1106, 1010, 848, 788, 739 cm’;
NMR (CDCl): 6= 1.20-1.65 (8H, m), 1.48 (3H, s), 2.00-2.10 (2H, m), 3.24- 3.48 (4H, m), 4.14 (1H, s), 6.88-6.92 (2H, m), 7.13-7.24 (3H, m), 7.37 (1H, d, J=8.1
Hz), 7.48-7.55 (3H, m), 7.86 (2H, s), 8.08-8.10 (1H, m), 8.16 (1H, s), 8.57 (1H, s);
HPLC A: Rt. 894 min, 99.3/99.4% purity, 20-100% CH;CN in H,0O (+0.1%TFA) over 15 min at Imlmin”, Prodigy ODSIII 250x4.6mm SuM, 215 and . 254 nm;
HPLC B: Rt. 5.76 min, 95.1/98.7% purity, 80:20 methanol/Tris buffer at pH9, . 1mimin™, Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm.
Example 21 (S)-2-(Biphenyl-2-ylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
N
SN
CS oO =
The above compound was prepared on the same scale and using an analogous method as used for Example 17. 1. The method of Example 18 except for the use of 2-bromo biphenyl (583mg, 2.5mmols). 2. The acid from step 1 (350mg, 0.95mmol), HBTU (400mg, 1mmol), NEt; (0.5ml, 3.5mmol), and 1-(2- pyridyl)cyclohexyl]methylamine (WO 98/07718; 200mg, mmol) were stirred in DMF (15ml). After 1 hour at ambient temperature the reaction mixture was diluted with ethyl acetate (100ml), washed with sodium bicarbonate solution (x2) and dried (MgSO4). The solvent was removed under reduced pressure. The crude product was purified by chromatography using 0-50% ethyl acetate in heptane and then 0-30% dichloromethane in ether as eluent. Removal of the solvent under reduced pressure gave the desired product as a foam (98mg, 19% for step 2): . 20 MS m/e (AP): 565 (M* + Na, 100%), 564 (80%), 542 (M*, 30%)
IR (KBr disc): 3404, 2928, 2855, 1650, 1584, 1508, 1489, 1458, 1432 cm;
NMR (DMSO-dg): 6 = 1.10-1.52 (8H, m), 1.27 (3H, s), 1.95-2.05 (2H, m), 2.95 (1H, d, J=14.4 Hz), 3.02-3.08 (1H, m), 3.08 (1H, d, J=14.6 Hz), 3.28-3.34 (1H,
m), 4.36 (1H, s), 6.37 (1H, d, J=8 Hz), 6.49 (1H, d, J=2.2 Hz), 6.71-6.75 (1H, m), ’ 6.82-6.86 (1H, m), 6.95-7.43 (13H, m), 7.52-7.57 (1H, m), 8.33 (1H, d, J=3.7 Hz), 10.81 (1H, s);
HPLC A: Rt. 12.65min, 99.65% purity, 20-100% CH;CN in H;0O (+0.1%TFA) over 15 min at Imlmin™', Prodigy ODSIII 250x4.6mm 5uM, 200-300 nm;
HPLC B: Rt. 33.05min, 99.89% purity, 80:20 methanol/Tris buffer at pH9,
Imlmin™!, Prodigy ODSIII 250x4.6mm 5pM, 200-300 nm.
Example 22 (S)-3-(1 H-Indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-m- tolylamino-propionamide
N
NS
Or oO | Z
The above compound was prepared using a one-pot procedure analogous to the method used for Example 16. The synthesis was carried out on 1mmol scale using 1-bromo-3-methyl-benzene (171mg, Immol). The crude product was purified by chromatography using 25g NP silica with 25% ethyl acetate in heptane as eluent.
Removal of the solvent under reduced pressure gave the desired compound as a glass (260mg, 54%):
MP1: 70-75°C;
MS m/e (AP): 481.33 (100%, M" + H), 482.37 (40%); ) IR (film): 3385, 3291, 3049, 2929, 2857, 1652, 1607, 1590, 1513, 1456, 1431, 1341, 1302, 1264, 1237, 1177, 1155, 1104, 1010, 774, 741 cm’; . NMR (DMS0-dg): 8= 1.08-1.50 (8H, m), 1.19 (3H, s), 2.00-2.10 (2H, m), 2.16 (3H, s), 3.03 (1H, d.d, J=12.9 and 5.1 Hz), 3.10 (1H, d, J=14.7Hz), 3.22 (1H, d,
J=14.6Hz), 3.24-3.30 (1H, m), 5.43 (1H, s), 6.29 (1H, s), 6.30 and 6.44 (each 1H,
each d, J=7.6 Hz), 6.87-7.07 (6H, m), 7.15-7.19 (1H, m), 7.29 (1H, d, J=8.0 Hz), . 7.33 (1H, d, J=7.8 Hz), 7.48-7.54 (1H, m), 8.31-8.33 (1H, m), 10.81 (1H, s);
HPLC A: Rt. 11.04 min, 98.3% purity, 20-100% CH;CN in H,0 (+0.1%TFA) over 15 min at 1mlmin™', Prodigy ODSIII 250x4.6mm SuM, 200-300 nm;
HPLC B: Rt. 16.87 min, 99.5% purity, 80:20 methanol/Tris buffer at pH9,
Imlmin’!, Prodigy ODSIII 250x4.6mm 5uM, 200-300 nm.
Example 23 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(6-phenyl-pyridin-2-ylamino)-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide
N
SN
2 Wa Ny =N 0 Zz
The above compound was prepared using a one-pot procedure analogous to the method used for Example 16. The synthesis was carried out on 0.4mmol scale using 2-bromo-6-phenyl-pyridine (95mg, 0.4 mmol). The crude product was purified by chromatography using 25g NP silica with 55% ethyl acetate in heptane as eluent.
Removal of the solvent under reduced pressure gave the desired product as a foam (260mg, 54%):
MS m/e (AP") 544.31 (100%, M" + H), 545.35 (35%);
MS m/e (AP) 542.29 (100%, M" - H), 543.31 (M", 40%), . 20 IR (film): 3407, 3276, 3056, 2930, 2857, 1651, 1595, 1576, 1519, 1486, 1467, 1455, 1439, 1339, 1264, 1180, 1157, 1105, 1028, 1009, 991, 804, 763, 739 cm}; : NMR (CDCl) é= 1.03-1.60 (8H, m), 1.53 (3H, s), 1.90-2.03 (2H, m), 3.32- 3.45 (3H, m), 3.65 (1H, d, J=14.6Hz), 4.67 (1H, s), 6.13 (1H, d, J=8.3 Hz), 6.77-7.50 (14H, m), 7.97 (2H, d, J=7.1 Hz), 8.02 (1H, s), 8.23-8.25 (1H, m);
HPLC A: Rt. 4.21 min, 96.8% purity, 20-100% CH;CN in H,0 (+0.1%TFA) : over 7 min at 1.5mlmin’!, Prodigy ODSIII 150x4.6mm 5uM, 200-300 nm.
Example 24 ) (R)-3-Phenyl-2-phenylamino-N-[1-pyridin-2-yl-cyclohexylmethyl)-propionamide
St
NN Ng 0) 2
The above compound was synthesised as a two step process from
Intermediate 8 as shown in Scheme 4. 1. To a solution of Intermediate 8 (0.5g, 3mmol) and bromobenzene (0.35mi, 3.3mmol) in DMA (5ml) under nitrogen was added potassium carbonate (0.6g, 4.3mmol) and copper (I) iodide (SOmg, 0.26mmol) after which the mixture was heated to 90°C for 1.5 hours. Solvent was removed under reduced pressure and the residue was purified by flash chromatography eluting with 5% methanol in dichloromethane. Removal of solvent under reduced pressure gave (R)-3-phenyl-2- phenylamino-propionic acid as an oil (0.41g, 56%):
MS m/e (APY): 242 (M" + H, 100%). 2. The acid from step 1 (0.40g, 1.66mmol), HBTU (0.6g, 1.8mmol), and NE; 3 (0.5ml, 3.5mmol), and 1-(2-pyridyl)cyclohexyl]methylamine (WO 98/07718; 0.35mg, 1.8mmol) were stirred in DMF (15ml). After 1 hour at ambient temperature . the reaction mixture was diluted with ethyl acetate (100ml), washed with sodium bicarbonate solution (x2) and dried (MgSQO,4). The solvent was removed under reduced pressure. The crude product was purified by chromatography using 50% ethyl acetate in heptane and then RP C18 silica with 70% methanol in water as eluent.
Removal of the solvent under reduced pressure gave the desired product as a white . amorphous solid (0.15g, 22%):
MPt: 113-115°C;
MS m/e (AP*): 414.22 (M' + H, 100%);
S IR (KBr disc): 3300, 2931, 2858, 1649, 1605, 1589, 1523, 1498, 1432, 1318, 748 cm’;
NMR (CDCl): 8 = 1.20-1.70 (8H, m), 1. 90-2.15 (2H, m), 2.91 (1H, d.d, J=14.2 and 8.8 Hz), 3.27 (1H, d.d, J=14.2 and 4.4 Hz), 3.38 (1H, d.d, J=13.2 and 5.5 Hz), 3.48 (1H, d.d, J=13.2 and 6.1 Hz), 3.80 (1H, d, J=3.4 Hz), 3.88-3.93 (1H, m), 6.44 (2H, d,
J=7.8 Hz), 6.74 (1H, t, J=11.3 Hz), 6.90-7.45 (11H,m), 8.28 (1H, d, J=3.6 Hz);
HPLC A: Rt. 4.51 min, 100% purity, 20-100% CH;CN in HO (+0.1%TFA) over 10 min at 1.5 mlmin™, Prodigy ODSIII 250x4.6mm 5uM, 200-300 nm;
HPLC B: Rt. 13.15 min, 99.14% purity, 80:20 methanol/Tris buffer at pH9,
Imlmin’, Prodigy ODSIII 250x4.6mm 5uM, 200-300 nm. :
Example 25 (S)-3-(1H-Indol-3-yl)-2-methyl-2-phenylethylamino-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
N
I
A, ~_-N Ny
Oo =
The above compound was prepared as shown in Scheme 5 via Intermediate . 10. 1. To a stirred solution of H-(S)-aMeTrp-OH (7) (10g, 46mmol) and di-t-butyl- dicarbonate (10g, 46mmol) in dioxan (100ml) was added water (20ml) and potassium carbonate (10g, 74mmol). After 4 hours the reaction mixture was acidified with 2N : hydrochloric acid (150ml) and product was extracted with ethyl acetate (2 x 200ml).
The combined organic phases were dried (MgSO,4) and evaporated under reduced pressure. The residue was purified by flash chromatography using ethyl acetate as eluent. Removal of solvent under reduced pressure gave Boc-(S)-aMeTrp-OH as an orange oil (14.5g, 99%). To a stirred solution of Boc-(S)-aMeTrp-OH (7g, 22mmol) in DMF (100ml) was added HBTU (8.0g, 22mmol), triethylamine (5ml, 35mmol), and [1-(2-pyridyl)cyclohexyllmethylamine (WO 98/07718; 4.2g, 22mmol). After 1 hour the reaction mixture was diluted with ethyl acetate (300ml), washed with 2N hydrochloric acid (2 x 200ml), dried (MgSO4) and evaporated under reduced pressure at 60°C. The residue was purified by flash chromatography. Elution with 5% methanol in dichloromethane and subsequent removal of solvent under reduced pressure gave intermediate 9 as yellow oil (8.3g, 77%):
MS m/e (AP+): 491 (M" + H, 100%), 513 (M" + Na, 20%);
IR (film): 3339, 2929, 2858, 1704, 1659, 1651, 1589, 1519, 1487, 1366, 1249, 1164, 1070, 908, 737 cm’;
NMR (CDCl): 6 = 1.20-1.70 (20H, m), 2.00-2.12 (2H, m), 3.25-3.50 (4H, m), 5.05-5.20 (1H, br.s), 6.92 (1H, d, J=2.0 Hz), 7.02-7.32 (6H, m), 7.51 (1H, d, J=8.0
Hz), 7.59-7.64 (1H, m), 8.03 (1H, s), 8.48 (1H, d, J=4 Hz). 2. To a stirred solution of Intermediate 9 (8.2g, 16.5mmol) in dichloromethane (100ml) was added trifluoroacetic acid (3.0ml, 39mmol). After 18 hours the solvent was removed under reduced pressure at 60°C. The residue was treated cautiously with saturated sodium carbonate solution (200ml) before extracting with ethyl acetate (3 x 200ml). The combined organic phases were dried (MgSQO,) and evaporated under reduced pressure at 60°C. The residue was purified by flash chromatography. Elution ] with 0-5% methanol in dichloromethane and subsequent removal of solvent under reduced pressure gave Intermediate 10 as white foam (4.85g, 75%): . MPt: 65-68°C;
MS m/e (AP+): 391 (M' + H, 100%);
IR (KBr disc): 3367, 2926, 2855, 1648, 1589, 1569, 1522, 1455, 1430, 1366, 1341, 1234, 842, 784,742 cm’; oo y
NMR (CDCl3): § = 1.20-1.80 (13H, m), 1.98-2.20 (2H, m), 2.83 (1H, d, : J=14.2 Hz), 3.33 (1H, d, J=14.2 Hz), 3.38 (2H, d, J=5.6 Hz), 6.98-7.20 (6H, m), 7.50- 7.75 (3H, m), 8.05-8.15 (1H, s), 8.49-8.51 (1H, m); 3. To a stirred solution of Intermediate 10 (293mg, 0.75mmol) and phenacetaldehyde (90mg, 0.75mmol) in 1,2-dichloroethane (20m!) was added solid sodium triacetoxyborohydride (316mg, 1.5mmol). After stirring overnight, saturated sodium bicarbonate solution was added — effervescence was observed. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried (MgSO4) and solvent was removed under reduced pressure. The residue was purified by chromatography using 20g RP-C18 with 0-50% methanol in water followed by 20g NP silica with 45% ethyl acetate in heptane. Removal of solvent under reduced pressure gave the desired compound as a glass (60mg, 16%):
MS m/e (ES): 496.56 (28%), 495.5 (52%, M* + H), 364.43 (22%), 269.34 (51%), 268.90 (88%), 248.37 (100%);
IR (film): 3274, 3058, 2928, 2856, 1651, 1588, 1568, 1519, 1469, 1454, 1431, 1355, 1263, 1236, 1155, 1117, 1053, 1030, 1009, 992, 930, 782, 742 cm; 'H NMR (CDCls): 8 = 1.20-1.65 (11H, m), 2.00-2.20 (2H, m), 2.40-2.75 (4H, m), 2.94 and 3.05 (each 1H, each d, J=14.4 Hz), 3.41 (2H, d, J=6.1 Hz), 6.74 (1H, d,
J=2.2 Hz), 7.04-7.25 (9H, m), 7.32 (1H, d, J=7.8 Hz), 7.55-7.60 (3H, m), 7.90 (1H, s), 8.55-8.58 (1H, m);
HPLC A: Rt. 8.52 min, 99.0/98.6% purity, 20-100% CH3;CN in H,0 (+0.1%TFA) over 15 min at Imlmin”, Prodigy ODSIII 250x4.6mm 5puM, 215 and 254 nm;
HPLC B: Rt. 23.84 min, 99.6/100% purity, 80:20 methanol/Tris buffer at pH9, 1mlmin™', Prodigy ODSIII 250x4.6mm 5uM, 215 and 254 nm.
Example 26 (S)-2-[(Benzofuran-2-ylmethyl)-amino]-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin- } 2-yl-cyclohexylmethyl)-propionamide
N x = N 0) ] N N N
Oo Z
The above compound was prepared as shown in Scheme 5 via Intermediate 10.
To a stirred solution of Intermediate 10 (150mg, 0.38mmol) and benzofuran-2- carbaldehyde (56mg, 0.38mmol) in 1,2-dichloroethane (5ml) was added solid sodium triacetoxyborohydride (162mg, 0.77mmol). After stirring at room temperature for 48 hours saturated sodium bicarbonate solution was added — effervescence was observed.
The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried (MgSO,) and solvent removed under reduced pressure. The residue was purified by chromatography using 60% ethyl acetate in heptane. Removal of solvent under reduced pressure gave the desired product as an amorphous white solid (29mg, 15%):
MS m/e (ES): 521.08 (M+ H, 100%), 391.06 (50%);
IR (film): 3268, 3056, 2930, 2856, 1656, 1588, 1569, 1519, 1469, 1454, 1431, 1355, 1342, 1255, 1171, 1105, 1052, 1009, 909, 788, 740 cm’; ’ 'H NMR (CDCl3): § = 1.20-2.20 (14H, m), 3.08 (1H, d, J=14.4 Hz), 3.14 (1H, d, J=14.8 Hz), 3.45-3.49 (2H, m), 3.66 (1H, d, J=14.4 Hz), 3.76 (1H, d, J=14.8 Hz), ’ 6.33 (1H, s), 6.84-6.88 (1H, m), 7.00-7.65 (12H, m), 8.32 (1H, s), 8.39 (1H, d, J=4.0
Hz),
HPLC A: Rt. 8.86 min, 99.7/99.1% purity, 20-100% CH;CN in HO ' (+0.1%TFA) over 15 min at Imimin™, Prodigy ODSIII 250x4.6mm 5pM, 215 and 254 nm.
Example 27 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(4-nitro-benzylamino)-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
N x
N J So on I ®) | Z 0)
The above compound was prepared as shown in Scheme 5 via Intermediate 10. To a stirred solution of Intermediate 10 (150mg, 0.38mmol) and 4- nitrobenzaldehyde (58mg, 0.38mmol) in 1,2-dichloroethane (5ml) was added solid sodium triacetoxyborohydride (114mg, 0.54mmol). After stirring at room temperature for 24 hours saturated sodium bicarbonate solution was added — effervescence was observed. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried (MgSO,) and solvent removed under reduced pressure. The residue was purified by chromatography using 60% ethyl acetate in heptane. Repurifcation using RP silica with 45% methanol in water (+ 1% acetic acid) gave pure product.
The pure fractions were combined, basified (sodium carbonate), and extracted with ethyl acetate. Removal of solvent under reduced pressure gave the desired compound ] 20 asa glass (10.5mg, 5%):
MPt: 58-60°C; . MS m/e (ES"): 526.15 (M" + H, 100%), 527.14 (33%);
IR (film): 3365, 2924, 2856, 1652, 1513, 1429, 1346, 1257, 1048 cm’; 'H NMR (DMSO-dg): 8 = 1.10-1.55 (8H, m), 1.19 (3H, s), 1.88-2.08 (2H, m), 2.25- 2.30 (1H, m), 2.95-3.02 (2H, m), 3.10-3.20 (1H, m), 3.17-3.27 (1H, m), 3.50-3.80
(2H, m), 6.93-7.63 (11H, m), 8.12 (2H, d, J=8.8 Hz), 8.42 (1H, d, J=3.6 Hz), 10.86 : (1H, s).
Example 28
BB1 and BB) Binding Assays
In the following experiments, measurement of BB; and BB binding was as follows. CHO-K1 cells stably expressing cloned human NMB (for (BB assay) and
GRP receptors (for BB) assay) were routinely grown in Ham's F12 culture medium supplemented with 10% foetal calf serum and 2 mM glutamine. For binding experiments, cells were harvested by trypsinization, and stored frozen at -70°C in
Ham's F12 culture medium containing 5% DMSO until required. On the day of use, cells were thawed rapidly, diluted with an excess of culture medium, and centrifuged for 5 minutes at 2000 g. Cells were resuspended in 50 mM Tris-HCI assay buffer (pH 7.4 at 21°C, containing 0.02% BSA, 40pg/mL bacitracin, 2pg/mL chymostatin, 4ug/mL leupeptin, and 2uM phosphoramidon), counted, and polytronned (setting 5, 10 sec) before centrifuging for 10 minutes at 28,000 g. The final pellet was resuspended in assay buffer to a final cell concentration of 1.5 x 105/mL. For binding assays, 200pL aliquots of membranes were incubated with [1251}[ Tyr4]bombesin (<0.1 nM) in the presence and absence of test compounds (final assay volume 250uL) for 60 minutes and 90 minutes for NMB and GRP receptors, respectively. Nonspecific binding was defined by 1uM bombesin. Assays were terminated by rapid filtration under vacuum onto Whatman GF/C filters presoaked in 0.2% PEI for >2 hours, and washed 50 mM Tris-HCl (pH 6.9 at 21°C; 6 x 1 mL). Radioactivity bound was determined using a gamma counter.
All competition data was analysed using nonlinear regression utilizing ) iterative curve-plotting procedures in Prism® (GraphPad Software Inc., San Diego,
USA). ICs, values were corrected to K; values using the Cheng-Prusoff equation (Cheng Y., Prusoff W. H., Biochem. Pharmacol. 22: 3099-3108, 1973).
The results obtained are listed in Table 1.
Table 1 : Human NMB and GRP receptor binding affinities
I EE
I EA
ILL EE ER
IE ES
IE SS E—
IEA FC I wwe ww =e
IE ES I
IE A
I A mw
IEA RA EE
Example 29
Effect of (S)-3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]- . 2-methyl-2-[4-(4-nitro-phenyl)-oxazol-2-ylamino]-propionamide (Compound (2)) in PEG200 on female rat sexual proceptivity
Ovariectomised adult female Sprague Dawley rats (180-200g, from Charles : River) were housed in groups of 6 in a reversed lighting system of 12h light:dark (lights off 7.00-19.00h). Two weeks after ovariectomy they were used for sexual activity tests. Animals were adapted to the apparatus (in the absence of stimuli animals) for 10min on 2 consecutive days prior to testing. The experiments started at least Sh into the dark period.
Tests were carried out in a circular arena of 90cm diameter, surrounded by a 30cm high wall. Two small cages with wire-mesh front (15x15cm) are fixed into the wall such that the front of the cage is “flush” with the wall and the 2 cages are opposite each other. They contain two stimuli animals: an intact sexually experienced male and a receptive female (ovariectomised, primed with 5ug oestradiol benzoate dissolved in corn oil and injected subcutaneously 48 hours before the test and with 0.5 mg of progesterone four hours before the test). Sexually naive test and control animals were used. Forty eight hours before the tests, both the test and control animals were primed with Sug oestradiol benzoate. Test animals were treated with the above compound (30-100mg/kg) which was dissolved in PEG 200 vehicle and administered orally in a ml/kg volume 1h before each test. For animals used as positive controls, progesterone (0.5mg/0.1ml) was dissolved in com oil and administered subcutaneously (s.c.), 4h before the test. Test and control animals were introduced one at a time for 10 minute periods into the arena. During the 10min test, the time that the test or positive control animal spent investigating each stimulus animal was noted.
The arena was thoroughly cleaned between animals. The position of the male/female stimuli boxes was randomised between animals, in order to avoid place preference.
The difference in the percentage of time spent investigating male minus female was calculated, out of the total time spent investigating stimuli animals.
It was found (see Figure 19) that the above compound dose-dependently (30- . 100) increased the percentage of time spent investigating the male stimulus, with a
MED of 100mg/kg (see below). The effect of this dose was similar to the effect of progesterone (maximal). (*P<0.05, **P<0.01 Kruskal-Wallis followed by Mann-
Whitney test, vs vehicle).
. Example 30 . Effect of Compound (2) in methyl cellulose on female rat sexual proceptivity.
Example 29 was repeated except that the above compound (3-30 mg/kg) was dissolved in 0.5% methyl cellulose and was administered p.o. in a dosing volume of 3ml/kg 1h before tests. Progesterone, (0.5mg/0.1ml) was dissolved in corn oil and administered s.c., 4h before test, as a positive control.
The above compound dose-dependently (3-30mg/kg) increased the percentage of time spent investigating the male stimulus, with a MED of 10mg/kg. This represents a 10-fold increase in potency compared to the oral results obtained in the
PEG200 vehicle (MED=100mg/kg). The results are shown in Figure 20 in which bars represent percentage of time spent investigating male, minus the percentage of time spent investigating the female stimuli+SEM, (n=6-9 per group). *P<0.05, **P<0.01 vs vehicle (One-way ANOV A followed by Dunnett’s test vs vehicle group).
Example 31
Effect of Compound (2) in PEG 200 on female rat sexual receptivity.
Ovariectomised adult female Sprague Dawley rats (180-200g, from Charles
River) were housed in groups of 6 in a reversed lighting system of 12h light:dark (lights off 7.00-19.00h). Two weeks after ovariectomy they were used for sexual activity tests. The experiments started at least Sh into the dark period. ’ The above compound was dissolved in PEG200 vehicle and administered orally. Quinelorane dihydrochloride (LY 163,502, 6.25pg/kg) was dissolved in water and administered subcutaneously (s.c.), as a positive control. Both compounds were administered in a 1ml/kg volume.
Forty eight hours before tests, the animals were primed with Sug oestradiol benzoate (Sigma Chemical. Co. Ltd., UK) dissolved in com oil and injected subcutaneously. The females were placed with a series of vigorous male rats and subjected to 10 mounts. The lordotic response of the animal was recorded and expressed as a percentage of the mounts (i.e. lordosis quotient, LQ). Treatment induced L.Q=0-10% in most of the animals, which were considered non-receptive (NR). Animals showing higher LQ were not included in the study. Each rat was tested prior to administration of the compound and then tested similarly at 1h and 90min post-injection of the above compound or quinelorane respectively.
A single administration of quinelorane (6.25ug/kg) significantly (P<0.01) increased the LQ, 90min after administration, compared to the LQ shown before administration (paired t test). A single oral administration of the above compound dose-dependently (10-100mg/kg) increased the LQ 1h after administration, with a
MED of 100mg/kg (P<0.01) compared to the LQ shown before administration (paired t test). The effect of the above compound (100mg/kg) was similar to the effect of quinelorane (6.25ug/kg) as is shown in Figure 21.
Synthesis Example (compounds of formula (III)) (S)-2-Amino-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)- propionamide (Intermediate 111-7) and (S)-2-Amino-3-(1 H-indol-3-yl)-2-methyl-N-(1-(5-methoxy-pyridin-2-yl)- cyclohexylmethyl)-propionamide (Intermediate 111-6)
In reaction scheme 7 below, Intermediates III-6 and III-7 are made by (i) ) protecting the amino group of the starting amino acid a with di-r-butyl carbonate and potassium carbonate in dioxane/water, (ii) forming an amide by reaction of the N- : protected amino acid with an amine bl or b2 in dimethylformamide in the presence of
O-benzotriazol-1-yl-N,N,N’,N -tetramethyluronium hexafluorophosphate (HBTU) and
N, N-diisopropyl-ethylamine (DIPEA), and (iii) deprotecting the amino group of the product ¢1 or ¢2 by reaction with trifluoroacetic acid in dichloromethane.
Scheme 7 . = R i. R (0) H,N N 0) =
H,N H 2 Son bl R=H boc” Sy N b2 R=OMe H = i. i, =
N N
H H a c1 R=H c2 R= OMe 0 = | R
Has Ay SN —_— “ H ii A
N
H
Intermediate lil-7 R=H
Intermediate Hfl-6 R = OMe
L BOC,0, K,CO;, dioxane, water il. HBTU, DIPEA, DMF iii. TFA, CH,Cl, {(S)-2-(1-H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid zert-butyl ester (cl) ¢)) To a stirred solution of H-(S)-aMeTrp-OH (a) (10g, 46mmol) and di-t-butyl- . dicarbonate (10g, 46mmol) in dioxane (100ml) was added water (20ml) and potassium carbonate (10g, 74mmol). After 4 hours the reaction mixture was acidified with 2N hydrochloric acid (150ml) and product extracted with ethyl acetate (2 x 200ml). The combined organic phases were dried (MgSO4) and evaporated under reduced pressure. The residue was purified by flash chromatography, eluting with : ethyl acetate. Removal of solvent under reduced pressure gave Boc-(S)-aMeTrp-OH as orange oil (14.5g, 99%). (2) To a stirred solution of Boc-(S)-aMeTrp-OH (7g, 22mmol) in DMF (100ml) was added HBTU (8.0g, 22mmol), triethylamine (5ml, 35mmol), and [1-(2- pyridyl)cyclohexyl]methylamine (bl, 4.2g, 22mmol, described in WO 98/07718).
After 1 hour the reaction mixture was diluted with ethyl acetate (300m!) and washed with 2N hydrochloric acid (2 x 200ml), dried (MgSO) and evaporated under reduced pressure at 60°C. The residue was purified by flash chromatography. Elution with 5% methanol in dichloromethane and subsequent removal of solvent under reduced pressure gave cl as yellow oil (8.3g, 77%):
IR (film): 3339, 2929, 2858, 1704, 1659, 1651, 1589, 1519, 1487, 1366, 1249, 1164, 1070, 908, 737 cm™;
NMR (CDCl): 6 = 1.20-1.70 (20H, m), 2.00-2.12 (2H, m), 3.25-3.50 (4H, m), 5.05-5.20 (1H, br.s), 6.92 (1H, d, J=2.0 Hz), 7.02-7.32 (6H, m), 7.51 (1H, d, J=8.0
Hz), 7.59-7.64 (1H, m), 8.03 (1H, s), 8.48 (1H, d, J=4 Hz);
MS m/e (AP+): 491 (M" + H, 100%), 513 (M" + Na, 20%). (3) (S)-2-Amino-3-(1 H-indol-3-yl)-2-methyl-/V-(1-pyridin-2-yl-cyclohexyl methyl)-propionamide (Intermediate 111-7)
To a stirred solution of ¢l (8.2g, 16.5mmol) in dichloromethane (100ml) was added trifluoroacetic acid (3.0ml, 39mmol). After 18 hours the solvent was removed under reduced pressure at 60°C. The residue was treated cautiously with saturated sodium carbonate solution (200ml) before extracting with ethyl acetate (3 x 200ml).
The combined organic phases were dried (MgSO,) and evaporated under reduced pressure at 60°C. The residue was purified by flash chromatography. Elution with 0- . 5% methanol in dichloromethane and subsequent removal of solvent under reduced pressure gave Intermediate II1-7 as white foam (4.85g, 75%).
MPt: 65-68°C;
IR (KBr disc): 3367, 2926, 2855, 1648, 1589, 1569, 1522, 1455, 1430, 1366, : 1341, 1234, 842, 784, 742 cm’;
NMR (CDCl): 8 = 1.20-1.80 (13H, m), 1.98-2.20 (2H, m), 2.83 (1H, d,
J=14.2 Hz), 3.33 (1H, d, J=14.2 Hz), 3.38 (2H, d, J=5.6 Hz), 6.98-7.20 (6H, m), 7.50-
S 7.75(3H, m), 8.05-8.15 (1H, s), 8.49-8.51 (1H, m);
MS m/e (AP+): 391 (M* + H, 100%). {(S)-2-(1-H-Indol-3-yl)-1-methyl-1-[(1-(5-methoxy-pyridin-2-yl)- cyclohexylmethyl)-carbamoyl]-ethyl}-carbamic acid tert-butyl ester (c2)
To a stirred solution of Boc-(S)-aMeTrp-OH (1.44g, 4.5Smmol) in DMF (50ml) was added HBTU (1.72g, 4.5mmol), DIPEA (2.38ml, 13.6mmol), and [1-(5- methoxy-2-pyridyl)cyclohexyljmethanamine (1g, 4.5mmol). After over night the reaction mixture was diluted with ethyl acetate (300ml) and water, dried (MgSO,) and evaporated under reduced pressure. The residue was purified by flash chromatography. Elution with ethylacetate/heptane (1:1) and subsequent removal of solvent under reduced pressure gave ¢2 as an oil (2.207g, 94%).
NMR (CDCl): 8 = 1.24-1.60 (8H, m), 1.39 (9H, s), 1.52 (3H, s), 2.00-2.18 (2H, m), 3.20-3.43 (4H, m), 3.82 (3H, s), 6.92 (1H, d, J=2.4 Hz), 7.02-7.20 (6H, m), 7.30(1H, d, J=6.0 Hz), 7.51 (1H, d, J=8Hz), 8.00 (1H, s), 8.17 (1H, d, J=2.8Hz).
MS m/e (ES+): 521.36 (M” + H, 100%), 543.25 (M' + Na).
Intermediate 111-6
To astirred solution of ¢2 (2.2g, 4.2mmol) in dichloromethane (10ml) was added trifluoroacetic acid (Sml, excess). After stirring over night the reaction mixture was ) taken up in IN HCI and extracted with diethylether. Organic phase discarded. The aqueous phase was basified cautiously with saturated sodium carbonate solution . before extracting with ethyl acetate (3 x 50ml). The combined organic phases were dried (MgSO,) and evaporated under reduced pressure at 60°C to give Intermediate
IT1-6 as a glass (1.253g, 71%).
IR (film): 3272, 2930, 2857, 1651, 1595, 1573, 1520, 1489, 1478, 1455, 1393, 1358, ‘ 1291, 1268, 1232, 1181, 1150, 1131, 1030, 1012, 831, 741 cm™;
NMR (DMSO): 6 =1.10-1.65 (13H, m), 1.80-1.90 (1H, m), 2.00-2.10 (1H, m), 2.70 (1H, d, J=13.9 Hz), 3.10 (1H, d, J=13.9 Hz), 3.10-3.22 (2H, m), 3.77 (3H,s), 6.93- 7.07 (4H, m), 7.16-7.19 (1H, m), 7.32 (1H, d, J=8.1 Hz), 7.48-7.55 (2H, m), 8.21 (1H, d, J=3.2 Hz), 10.88 (1H, s);
MS nv/e (ES+): 421.27 (M" + H, 100%), 443.26 (M* + Na).
Examples 32-86
N-acyl derivatives of Intermediate 111-6 and III-7
Scheme 8 describes the synthesis of N-acyl derivatives of Intermediates III-7 and
HI-6.
Scheme 8 =
SSE 1 A
R1” “oH @ ON N
Intermediate 1-7 ——— > , 1 H 2
N
H
Examples 32-85 0) = < [ES UY
R1” oH (@ ZN N
Intermediate 111-6 EE —— LC H i. /
N
H
. Example 86 1. HBTU, DIPEA, DMF
In scheme 8, R1 represents the rest of the carboxylic acid d molecule. These intermediates d are listed in table 2.
: N-acyl derivatives of Intermediate 111-7
To acid d (0.18 mmol) was added 0.50 M HBTU in DMF (300 pL, 0.15 mmol), 1.0 M diisopropylethylamine in DMF (300 pL, 0.30 mmol) and 0.40 M
Intermediate III-7 in DMF (375 pL, 0.15 mmol). The solution was shaken on an orbital shaker at room temperature for 18 h. Water (1.0 mL) was added and the mixture was loaded onto a LC-18 SPE cartridge (0.5 g sorbent) and the cartridge was eluted with water (3 mL), 25% methanol/water (3 mL), 50% methanol/water (4 mL) and methanol (4.5 mL)). The methanol fraction was concentrated and analysed by
LCMS. When the purity was <90% the product was further purified by prep. HPLC (column: Phenomenex primesphere 10 p C18-HC 110A, 100x21.20 mm; mobile phase: methanol / water 10 to 100% gradient). The products were characterised and analysed by LCMS (column: 50x4.6 mm Prodigy ODSIII (5p) column; mobile phase: acetonitrile / water (0.1% formic acid) 5 to 100% gradient over 2 min, held at 100% acetonitrile for 1 min; flow rate 4 mL/min; UV detection at 215 nm; mass spec: 150- 900 Da full scan APCI+ centroid data)
The following products were made by the above method, with the starting material listed in Table 2 and gave the test results indicated in Table 3:
TABLE 2 al co 33 [#Methyl-bensoic acid 34 [+Chloro-benzoic acid . | 35 [#-Methoxy-benzoic acid 36 [#Niwo-benaoic acid 4-Methanesulfonyl-benzoic acid
I
I i
I
I
I i
I a
TABLE 3
Example Product MH" Purity | LCMS | BB1 | BB2 : No % | Ret IC50 | IC50 time (aM) | (nM) min 32 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 494,64 100 1.71 | 2499 | IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-benzamide 33 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 508,67 95 1.76 | 2499 | IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-4-methyl-benzamide 34 4-Chloro-N-{(S)-2-(1H-indol-3-yl)- 529,09 94 1.84 | 1349 | IA 1-methyl-1-[(1-pyridin-2-yl- cyclohexyl-methyl)-carbamoyl]- ethyl}-benzamide
EE
1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-4-methoxy-benzamide 36 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 539,64 1.79 343 IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl}- ethyl }-4-nitro-benzamide 37 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 572,73 95 2272 | 1A 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-4-methanesulfonyl-benzamide 38 3-Cyano-N-{(S)-2-(1H-indol-3-yl)-1- | 519,65 91 1.71 | 2042 | 1A methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-benzamide 39 3-Chloro-N-{(S)-2-(1H-indol-3-yl)- 529,09 97 1.84 | 1269 | 1A 1-methyl-1-[(1-pyridin-2-yl- . cyclohexyl-methyl)-carbamoyl]- ethyl}-benzamide : 40 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 524,67 1.73 | 2859 | 1A 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-3-methoxy-benzamide
41 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 572,73 95 3051 | IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-3-methanesulfonyl-benzamide ‘ 42 Dimethylamino-N-{(S)-2-(1H-indol- | 537,71 1.74 | 2518 | IA 3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexyl-methyl)-carbamoyl]- ethyl}-benzamide 43 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 508,67 1.79 | 2351 IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-3-methyl-benzamide 44 2-Chloro-N-{(S)-2-(1H-indol-3-yl)- 529,09 1.79 | 3229 | 1A
I-methyl-1-[(1-pyridin-2-yl- cyclohexyl-methyl)-carbamoyl]- ethyl}-benzamide 45 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 539,64 91 1.71 | 4581 IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-2-nitro-benzamide
N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 524,67 100 1.73 | 2559 | IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-2-methoxy-benzamide 47 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 508,67 1.79 | 3283 | IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-2-methyl-benzamide 48 C-Dimethylamino-N-{(S)-2-(1H- 537,71 93 1.79 716 IA indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexyl-methyl)-carbamoyl]- ethyl}-benzamide 49 2-Fluoro-N-{(S)-2-(1H-indol-3-yl)- 512,63 1.76 | 3949 | 1A
I-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-benzamide 50 (S)-3-(1H-Indol-3-yl)-2-methyl-N- 522,70 94 1.76 944 IA (1-pyridin-2-yl-cyclohexylmethyl)-2- ; (2-p-tolyl-ethanoylamino)- propionamide 51 (S)-3-(1H-Indol-3-yl)-2-methyl-N- 522,70 1.76 944 1A (1-pyridin-2-yl-cyclohexylmethyl)-2- (2-o-tolyl-ethanoylamino)- propionamide
52 (S)-2-[2-(4-Hydroxy-phenyl)- 524,67 1.50 | 3135 | IA ethanoylamino]-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide : 53 (8)-2-[2-(3-Hydroxy-phenyl)- 524,67 1.52 1437 | IA ethanoylamino]-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 54 (S)-3-(1H-Indol-3-yl)-2-methyl-N- 522,70 95 1.76 817 IA (1-pyridin-2-yl-cyclohexylmethyl)-2- (2-m-tolyl-ethanoylamino)- propionamide 55 (S)-2-[2-(2-Fluoro-phenyl)- 526,66 94 1.71 878 | 1546 ethanoylamino]-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 56 (S)-3-(1H-Indol-3-yl)-2-methyl-N- 514,70 93 1.65 | 1437 | 1A (1-pyridin-2-yl-cyclohexylmethyl)-2- (2-thiophen-3-yl-ethanoylamino)- propionamide 57 Pyridine-2-carboxylic acid {(S)-2- | 495,63 3709 | IA (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide 58 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 495,63 1.47 | 1365 | 1A 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl }-isonicotinamide 59 Furan-3-carboxylic acid {(S)-2-(1H- | 484,60 97 1204 | 1A indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]- ethyl}-amide
Furan-2-carboxylic acid {(S)-2-(1H- | 484,60 1204 | 1A indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]- ethyl}-amide 1H-Indole-2-carboxylic acid {(S)-2-| 533,68 1.79 289 | 527 (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- . carbamoyl]-ethyl}-amide 62 5-Methyl-isoxazole-3-carboxylic 499,62 94 146 | 4127 | 1A } acid {(S)-2-(1H-indol-3-yl)-1- methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-amide
63 1-Methyl-1H-pyrrole-2-carboxylic 497,65 1.46 | 4819 ] acid {(S)-2-(1H-indol-3-yl)-1- methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- : ethyl}-amide 64 Thiophene-2-carboxylic acid {(S)-2-| 500,67 100 1.42 1437 | 1A (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide 65 Thiophene-3-carboxylic acid {(S)-2- | 500,67 100 1.39 | 2201 | 1A (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-amide 1H-Indole-6-carboxylic acid {(S)-2- | 533,68 1.42 1604 | IA (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide : 67 1H-Indole-5-carboxylic acid {(S)-2-| 533,68 100 1.35 1881 | IA (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-amide 1H-Indole-4-carboxylic acid {(S)-2-| 533,68 1.35 | 4503 | 1A pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide 1H-Indole-7-carboxylic acid {(S)-2-| 533,68 1369 | 1A (1H-indol-3-yl)-1-methy!-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide 70 1-Methyl-1H-indole-2-carboxylic 547,71 100 1.70 | 1233 | 1A acid {(S)-2-(1H-indol-3-y1)-1- methyl-1-{(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl }-amide 71 Benzo[b]thiophene-2-carboxylic acid | 550,73 100 1.63 IA {(S)-2-(1H-indol-3-yl)-1-methyl-1- [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide 72 Benzothiazole-6-carboxylic acid | 551,72 95 1.35 897 | 1495 } {(S)-2-(1H-indol-3-yl)-1-methyl-1- [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide : 73 1H-Benzotriazole-5-carboxylic acid | 535,65 95 1.25 | 3167 {(S)-2-(1H-indol-3-yl)-1-methyl-1- [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl} -amide
74 3-Methyl-thiophene-2-carboxylic 514,70 100 1.53 744 1A acid {(S)-2-(1H-indol-3-yl)-1- methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- : ethyl}-amide 75 5-Methyl-thiophene-2-carboxylic 514,70 1663 | IA acid {(S)-2-(1H-indol-3-yl)-1- methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]- ethyl}-amide 76 6-Methyl-pyridine-2-carboxylic acid | 509,66 1.6 2816 | IA {(S)-2-(1H-indol-3-yl)-1-methyl-1- [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl } -amide 77 Isoquinoline-3-carboxylic acid {(S)- | 545,69 1.71 1363 2-(1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide
Quinoxaline-2-carboxylic acid {(S)- 2-(1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-amide 79 Quinoline-8-carboxylic acid {(S)-2-| 545,69 1.57 | 4479 | 1A (1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-amide 5-Phenyl-oxazole-4-carboxylic acid | 561,69 95 2660 IA {(S)-2-(1H-indol-3-yl)-1-methyl-1- [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-amide 81 N-{(S)-2-(1H-Indol-3-yl)-1-methyl- 559,72 1.71 361 IA 1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl}- ethyl}-2-pyrrol-1-yl-benzamide 82 (S)-3-(1H-Indol-3-yl)-2-[2-(4- 538,70 1.71 1694 | IA methoxy-phenyl)-ethanoylamino}-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 83 (S)-2-[2-(4-Dimethylamino-phenyl)- | 551,74 100 1.36 | 2708 | IA ‘ ethanoylamino]-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- . cyclohexylmethyl)-propionamide 84 (S)-3-(1H-Indol-3-yl)-2-methyl-2-[2- | 553,67 95 1.5 1979 | 1A (2-nitro-phenyl)-ethanoylamino]-N- (1-pyridin-2-yl-cyclohexylmethyl)- propionamide
ME wd ) methoxy-phenyl)-ethanoylamino]-2- methyl-N-(1-pyridin-2-yl-
X cyclohexylmethyl)-propionamide
IA: IC50 > 10000 nM
N-acyl derivative of Intermediate 111-6
Example 86 1H-Indole-2-carboxylic acid ((S)-2-(1H-indol-3-yl)-1-{[1-(5-methoxy-pyridin-2- yl)-cyclohexylmethyl|-carbamoyl}-1-methyl-ethyl)-amide
To a solution of 1-A-Indole-2-carboxylic acid (38 mg, 0.24 mmol), Intermediate I1I- 6 (100 mg, 0.19 mmol) and diisopropylethylamine (61 mg, 0.47 mmol) in DMF (5 mL) was added HBTU (90 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the residue was diluted with ethyl acetate, washed with brine, dried (MgSO;) and concentrated under reduced pressure. The residue was purified by column chromatography (60% ethyl acetate/heptane) to give Example 86 as an amorphous white solid (65 mg, 61%).
IR (film): 3285, 2931, 2855, 1651, 1537, 1489, 1456, 1420, 1342, 1310, 1267, 1028, 908, 744 cm’;
NMR (CDCl3): 6 =1.10-1.61 (11H, m), 1.95-2.04 (2H, m), 3.29-3.52 (4H, m), 3.43 (3H, s), 6.47 (1H,s), 6.86-6.90 (1H, m), 6.98-6.99 (2H, m), 7.09-7.42 (8H, m), 7.52-7.58 (2H, m), 7.73-7.74 (1H, m) 8.05 (1H, s), 9.11 (1H, s);
MS m/e (ES+): 564 (M" + H, 100%).
Binding studies of Example 86 to the bombesin receptors gave the following results (ICs0): BB1: 11 nM, BB2: 119 nM. ) Examples 87-110
N-terminal urethane derivatives of Intermediate 111-7
Scheme 9 describes the synthesis of urethane derivatives of Intermediate III-7:
— Conversion of alcohol into 4-nitrophenyl carbonates — N-terminal urethane formation
Scheme 9 2 0 ry NO, ro © N Aol
R2-OH — R2., A Intermediate li1-7 T “ H € ii. yZ
Examples 87-110 ’ i. 4-nitrophenyl chloroformate, pyridine, THF ii. DMAP, DMF
In scheme 9, R2 represents the rest of the intermediate e. These intermediates e are listed in table 4.
To a stirred solution of alcohol e (10 mmol) and 4-nitrophenyl chloroformate (2.01 g, 10 mmol) in dichloromethane (50 mL) at 0°C was added dropwise a solution of pyridine (0.81 mL, 10 mmol) in dichloromethane (10 mL). The reaction mixture was allowed to slowly warm to room temperature and was stirred at room temperature for 16 h. The solvent was removed under reduced pressure and the residue was taken up in ethyl acetate (50 mL) and was washed successively with 10% citric acid (2x30 mL), water (30 mL), sat.
NaHCOj; solution (2x50 mL) and brine (50 mL). The organic phase was dried (MgSQy) and was concentrated under reduced pressure. The crude product was recrystallised from typically ethyl acetate, diethyl ether or heptane to give pure carbonate f. The product was characterised by IR (see Table 4 for carbonate signals).
To carbonate f (0.21 mmol) was added DMF (0.4 mL) followed by 0.50 M DMAP in
DMF (400 pL, 0.20 mmol) and 0.50 M Intermediate III-7 in DMF (200 pL, 0.10 mmol). : The solution was shaken on an orbital shaker at room temperature for 42 h. Water (1.0 mL) was added and the mixture was loaded onto a LC-18 SPE cartridge (0.5 g sorbent) and the cartridge was eluted with 25% methanol/water (3.4 mL) and methanol (4 mL). The methanol fraction was concentrated and purified by prep. HPLC (column: Phenomenex primesphere 10 p C18-HC 110A, 100x21.20 mm; mobile phase: methanol/water 10 to
100% gradient). The products were characterised and analysed by LCMS (column: 50x4.6 : mm Prodigy ODSIII (Su) column; mobile phase: acetonitrile/water (0.1% formic acid) 5 to 100% gradient over 2 min, held at 100% acetonitrile for 1 min; flow rate 4 mL/min; UV ’ detection at 215 nm; mass spec: 150-900 Da full scan APCI+ centroid data).
The following products were made by the above method, with the starting material listed in Table 4 and gave the test results indicated in Table 5:
TABLE 4
IR cm’)
I I
CE fe ms
I NS
[ee | we
I CL NL
I IJ
I GL NL
(3-Trifluoromethyl-phenyl)-methanol 1770 . p-Tolyl-methanol 1759 109] (2,3-Dichloro-phenyl)-methanol 1758
Quinolin-6-yl-methanol 1761
TABLES
Examp MH Purity | LCMS | BB1 | BB2 le % | Ret IC50 | ICSO
No time (nm) | (nm) min 87 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 574,73 100 1.67 239 1A [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl ]-ethyl}-carbamic acid naphthalen-1-ylmethyl ester 88 {(S)-2-(1H-Indol-3-y1)-1-methyl-1- 584,72 95 1.41 | 1758 | 1A [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3,4- dimethoxy-benzyl ester {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 574,73 100 1.67 | 1001 | IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-carbamic acid naphthalen-2-ylmethyl ester {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 550,71 91 1.59 955 1A [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid indan-2-yl ester {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 593,56 93 1.73 202 IA [(1-pyridin-2-yl-cyclohex ylmethyl)- carbamoyl]-ethyl}-carbamic acid 3,4- dichloro-benzyl ester 92 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 554,70 93 1.49 | 1610 | IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4- ) methoxy-benzyl ester 93 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 559,11 1.62 IA : [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4- chloro-benzyl ester
. 94 {(S)-2-(1H-Indol-3-y!)-1-methyl-1- 542,66 1.52 923 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2- ‘ fluoro-benzyl ester 95 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 559,11 1.62 624 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2- chloro-benzyl ester {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 569,67 97 1.51 41 463 [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-carbamic acid 4- nitro-benzyl ester 97 {(8)-2-(1H-Indol-3-yl)-1-methyl-1- 538,70 94 751 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-carbamic acid 2- methyl-benzyl ester {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 580,78 100 1.86 | 1986 | IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4- tert-butyl-benzyl ester {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 569,67 97 1.51 17 612 [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3- nitro-benzyl ester 100 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 554,70 1.52 818 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-carbamic acid 2- methoxy-benzyl ester 101 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 592,67 97 1.7 1102 | IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-carbamic acid 4- trifluoromethyl-benzyl ester 102 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 568,72 1065 | IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3- ethoxy-benzyl ester 103 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 549,68 1.43 85 1A . [(1-pyridin-2-yl-cyclohexylmethyl)- . carbamoyl]-ethyl}-carbamic acid 3- cyano-benzyl ester
104 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 593,56 95 1.78 450 IA . [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl } -carbamic acid 2,4-dichloro-benzyl ester 105 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 538,70 1.59 841 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3- methyl-benzyl ester 106 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 616,77 1.78 | 1350 | IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3- phenoxy-benzyl ester 107 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 592,67 1.67 182 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3- trifluoromethyl-benzyl ester 108 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 538,70 97 1.60 | 1084 | 1A [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4- methyl-benzyl ester 109 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 593,56 1.73 152 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2,3-dichloro-benzyl ester 110 {(S)-2-(1H-Indol-3-yl)-1-methyl-1- 575,72 97 1.22 171 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid quinolin-6-ylmethyl ester
Examples 111-168
N-terminal sulfonamide derivatives of Intermediate 111-7 2 0 Oo =
R3=S-Cl (9) Ra_L H
Intermediate ilI-7 —_— 5 pn N i. x =z . N
H
Scheme 10 Examples 111-168
In scheme 10, R3 represents the rest of the intermediate g. These intermediates ‘ g are listed in table 6.
To sulfonyl chloride g (0.14 mmol) was added 0.143 M Intermediate I1I-7 in
DMF (700 pL, 0.10 mmol) followed by 300 pL of a solution containing a mixture of diisopropylethylamine (0.667 M in DMF, 0.20 mmol) and 4-dimethylaminopyridine (0.033 M in DMF, 0.01 mmol). The reaction mixture was shaken in an orbital shaker at 70°C for 16 h. The crude reaction mixture was loaded onto a 5 g silica cartridge and the cartridge was eluted with ethyl acetate in heptane (30 to 100% gradient). Removal of the solvent under reduced pressure gave the sulfonamides (Examples 111-168).
The purity of the sulfonamide was checked by LCMS. Those samples that were less than 95% pure were further purified by prep HPLC (column: YMC-Pack ODS-AM,
Sum, 150x20 mm; mobile phase: acetonitrile / water 40 to 100% gradient). The products were characterised and analysed by LCMS (column: 150x4.6 mm Prodigy
ODS3 (3u) column; mobile phase: acetonitrile (0.085% TFA) / water (0.1% TFA) 20 to 100% gradient over 7 min, held at 100% acetonitrile (0.085% TFA) for 1 min; flow rate 1.5 mL/min; detection: diode array 200-300 nm; mass spec: 150-900 Da full scan
APCI+ centroid data) (see Table 7).
The following examples were made by the above method, with the starting material listed in Table 6 and gave the test results indicated in Table 7:
N
TABLE 6
I eT re ester
ON GE
IC
TGC rome herenesiony coils
I
- TABLE 7
J Example MH"' Purity | LCMS | BB1 |BB2
No % | Ret IC50 | IC50 time (nm) | (om) min 111 (S)-3-(1H-Indol-3-yl)-2-methyl-2- 544,72 100 4.64 186 IA phenylmethanesulfonylamino-N-(1- pyridin-2-yl-cyclohexylmethyl)- propionamide 112 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 544,72 4.74 557 IA pyridin-2-yl-cyclohexylmethyl)-2- (toluene-4-sulfonylamino)- propionamide 113 (S)-2-(2-Chloro- 565,14 4.71 257 IA benzenesulfonylamino)-3-(1H-indol- 3-y)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 114 (S)-2-(2-Fluoro- 548,68 100 454 267 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 115 (S)-3-(1H-Indol-3-yl)-2-methyl-2- 580,76 4.98 185 1576 (naphthalene- 1-sulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)- propionamide 116 (S)-2-(4-Chloro- 565,14 97 4.89 373 | 4386 benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 117 (S)-2-(5-Dimethylamino- 623,82 4.39 1302 IA naphthalene-1-sulfonylamino)-3-(1H- indol-3-yl)-2-methyl-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide 118 (S)-3-(1H-Indol-3-yl)-2-methyl-2- 580,76 100 5.01 322 1A , (naphthalene-2-sulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethy!)- propionamide ’ 119 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 536,72 4.39 232 pyridin-2-yl-cyclohexylmethyl)-2- (thiophene-2-sulfonylamino)- propionamide
120 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 581,74 4.53 108 IA . pyridin-2-yl-cyclohexylmethyl)-2- (quinoline-8-sulfonylamino)- propionamide ‘ 121 (8)-3-(1H-Indol-3-yl)-2-methyl-2-(3- | 575,69 4.58 208 1960 nitro-benzenesulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)- propionamide 122 (S)-2-(4-Fluoro- 548,68 4.60 560 | 4165 benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 123 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(4- | 575,69 4.65 515 IA nitro-benzenesulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)- propionamide 124 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 599,58 5.03 440 | 2246 pyridin-2-yl-cyclohexylmethyl)-2-(3- trifluoromethyl- benzenesulfonylamino)- propionamide 125 (S)-2-(3,4-Dichloro- 599,58 547 216 IA oo benzenesulfonylamino)-3-(1H-indol- HEAR 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 126 (S)-2-(3-Fluoro- 548,68 4.65 407 | 2761 benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 127 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 598,69 95 5.31 553 IA pyridin-2-yl-cyclohexylmethyl)-2-(4- trifluoromethyl- benzenesulfonylamino)- propionamide 128 (S)-2-(5-Chloro-thiophene-2- 571,17 4.94 404 IA sulfonylamino)-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 129 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 598,69 5.11 134 ) pyridin-2-yl-cyclohexylmethyl)-2-(2- trifluoromethyl- benzenesulfonylamino)- ’ propionamide 130 (S)-2-(3-Chloro- 565,14 5.05 331 2687 benzenesulfonylamino)-3-(1H-indol- 3-yD)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
131 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 544,72 4.93 393 1019 . pyridin-2-yl-cyclohexylmethyl)-2- (toluene-3-sulfonylamino)- propionamide : 132 (S)-2-(3,4-Dimethoxy- 590,75 4.50 608 IA benzenesulfonylamino)-3-(1H-indol- 3-yI)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 133 (S)-2-(4-Cyano- 555,70 4.61 766 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 134 (S)-2-(2-Cyano- 555,70 97 4.62 408 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 135 (S)-2-(5-Chloro-1,3-dimethyl-1H- 583,16 4.38 1252 IA pyrazole-4-sulfonylamino)-3-(1H- indol-3-yl)-2-methyl-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide 136 (8)-2-(3,5-Dimethyl-isoxazole-4- 549,70 4.54 515 IA sulfonylamino)-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 137 (S)-2-(Benzo[ 1,2,5]thiadiazole-4- 588,76 97 4.67 256 IA sulfonylamino)-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 138 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(1- | 534,69 3.60 3667 IA methyl-1H-imidazole-4- sulfonylamino)-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 139 (S)-2-(Benzo[1,2,5]Joxadiazole-4- 572,69 100 4.70 507 IA sulfonylamino)-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 140 3-{(S)-2-(1H-Indol-3-yl)-1-methyl-1- | 594,76 100 4.79 167 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethylsulfamoyl }- . thiophene-2-carboxylic acid methyl ester 141 (S)-3-(1H-Indol-3-yl)-2-(5-isoxazol- | 603,77 4.60 534 IA : 3-yl-thiophene-2-sulfonylamino)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
142 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(2- | 589,72 4.65 430 IA . nitro-phenylmethanesulfonylamino)-
N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide : 143 (8)-2-(3-Cyano- 555,70 4.55 460 IA benzenesulfonylamino)-3-(1H-indol- 3-yD)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 144 (S5)-2-(1,2-Dimethyl-1H-imidazole-4- | 548,71 3.55 2482 IA sulfonylamino)-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 145 (S)-3-(1H-Indol-3-yl)-2-(3-methoxy- | 560,72 4.75 295 3686 benzenesulfonylamino)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)- propionamide 146 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(8- | 625,75 4.89 177 IA nitro-naphthalene- 1 -sulfonylamino)-
N-(1-pynidin-2-yl- cyclohexylmethyl)-propionamide 147 (S)-2-(2-Chloro-5-nitro- 610,14 5.00 374 Ia benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 148 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 634,03 100 5.45 215 Ia pyridin-2-yl-cyclohexylmethyl)-2- (2,4,6-trichloro- benzenesulfonylamino)- propionamide 149 (S)-2-(4-Chloro-2-nitro- 610,14 100 5.13 513 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 150 (5)-2-(5-Benzenesulfonyl-thiophene- | 676,88 5.03 297 IA 2-sulfonylamino)-3-(1H-indol-3-yl)- 2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 151 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 614,69 5.35 635 IA pyridin-2-yl-cyclohexylmethyl)-2-(4- , triflnvoromethoxy- benzenesulfonylamino)-propion amide ' 152 (S)-3-(1H-Indol-3-yl)-2-methyl-2-(5- | 636,82 97 5.79 76 IA methyl-2-phenoxy- benzenesulfonylamino)-N-(1-pyridin- 2-yl-cyclohexylmethyl)- propionamide
153 (8)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 636,82 97 5.79 IA ; pyridin-2-yl-cyclohexylmethyl)-2-(2- p-tolyloxy-benzenesulfonylamino)- propionamide - 154 (S)-2-(Biphenyl-2-sulfonylamino)-3- | 606,79 97 5.52 IA (1H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)- propionamide 155 2-{(S)-2-(1H-Indol-3-yl)-1-methyl-1- | 588,73 4.84 242 IA [(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethylsulfamoyl}-benzoic acid methyl ester 156 (S)-2-(3-Chloro-4-fluoro- 583,13 95 5.12 284 1216 benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 157 (S)-2-(2,5-Dichloro-thiophene-3- 605,61 5.23 214 1A sulfonylamino)-3-(1H-indol-3-y})-2- cyclohexylmethyl)-propionamide 158 (S)-2-(3-Chloro-4-methyl- 579,17 97 5.28 299 | 3939 benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 159 (S)-3-(1H-Indol-3-yl)-2-(2-methoxy- | 574,75 4.92 445 IA 4-methyl-benzenesulfonylamino)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 160 (S)-3-(1H-Indol-3-yl)-2-methyl-N-(1- | 613,81 4.79 344 IA pyridin-2-yl-cyclohexylmethyl)-2-(5- pyridin-2-yl-thiophene-2- sulfonylamino)-propionamide 161 (S)-2-(5-Bromo-6-chloro-pyridine-3- | 645,02 95 5.09 187 IA sulfonylamino)-3-(1H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 162 (S)-2-(2,4-Dinitro- 620,69 4.97 475 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 163 (S)-3-(1H-Indol-3-yl)-2-(4- 608,78 4.20 1043 IA methanesulfonyl- - benzenesulfonylamino)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)- propionamide
164 (S)-2-(4-tert-Butyl- 586,80 5.65 IA } benzenesulfonylamino)-3-(1H-indol- 3-yD)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide : 165 (S)-2-(2,4-Dichloro-5-methyl- 613,61 97 5.64 172 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 166 (S)-2-(Chloro-trifluoromethyl- 633,14 100 5.33 627 IA benzenesulfonylamino)-3-(1H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide 167 (S)-3-(1H-Indol-3-yl)-2-methyl-2- 643,69 100 5.34 758 IA (nitro-trifluoromethyl- benzenesulfonylamino)-N-(1-pyridin- 2-yl-cyclohexylmethyl)- propionamide 168 (S)-2-(4-Butyl- 586,80 5.84 492 IA benzenesulfonylamino)-3-(1 H-indol- 3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide
Example 169
Bombesin antagonists potentiate pelvic nerve-stimulated increases in female genital blood flow in the anaesthetised rabbit model of sexual arousal.
Bombesin anatgonists used = Compound 1 and (25)-N-{[1-(4-aminophenyl) cyclohexyl] methyl}-3-(1H-indol-3-yl)-2-methyl-2-{[(4-nitroanilino) carbonyl] amino} propanamide (Compound 3). 0] or NH,
H H
N N ry hig ib - H
OL + 0 . N
Ney 5 hBB, Ki 0.25 nM : hBB; Ki 46 nM
Female New Zealand rabbits (~2.5kg) were pre-medicated with a combination of Medetomidine (Domitor®) 0.5ml/kg i.m., and Ketamine (Vetalar®) 0.25ml/kg i.m. whilst maintaining oxygen intake via a face mask. The rabbits were tracheotomised using a Portex™ uncuffed endotracheal tube 3 ID., connected to ventilator and maintained at a ventilation rate of 30-40 breaths per minute, with an approximate tidal volume of 18-20 ml, and a maximum airway pressure of 10 cm H,O. Anaesthesia was then switched to Isoflurane and ventilation continued with O, at 2l/min. The right marginal ear vein was cannulated using a 23G or 24G catheter, and Lactated Ringer solution perfused at 0.5ml/min. The rabbit was maintained at 3% Isoflurane during invasive surgery, dropping to 2% for maintenance anaesthesia.
The left groin area of the rabbit was shaved and a vertical incision was made approximately Scm in length along the thigh. The femoral vein and artery were exposed, isolated and then cannulated with a PVC catheter (17G) for the infusion of drugs and compounds. Cannulation was repeated for the femoral artery, inserting the catheter to a depth of 10cm to ensure that the catheter reached the abdominal aorta.
This arterial catheter was linked to a Gould system to record blood pressure. Samples for blood gas analysis were also taken via the arterial catheter. Systolic and diastolic pressures were measured, and the mean arterial pressure calculated using the formula (diastolic x2 + systolic) +3. Heart rate was measured via the pulse oxymeter and Po- ne-mah data acquisition software system (Ponemah Physiology Platform, Gould
Instrument Systems Inc). A ventral midline incision was made into the abdominal cavity. The incision was about Scm in length just above the pubis. The fat and muscle : was bluntly dissected away to reveal the hypogastric nerve which runs down the body cavity. It was essential to keep close to the side curve of the pubis wall in order to ; avoid damaging the femoral vein and artery which lie above the pubis. The sciatic and pelvic nerves lie deeper and were located after further dissection on the dorsal side of the rabbit. Once the sciatic nerve is identified, the pelvic nerve was easily located.
The term pelvic nerve is loosely applied; anatomy books on the subject fail to identify : the nerves in sufficient detail. However, stimulation of the nerve causes an increase in vaginal and clitoral blood flow, and innervation of the pelvic region. The pelvic nerve was freed away from surrounding tissue and a Harvard bipolar stimulating electrode was placed around the nerve. The nerve was slightly lifted to give some tension, then the electrode was secured in position. Approximately 1ml of light paraffin oil was placed around the nerve and electrode. This acts as a protective lubricant to the nerve and prevents blood contamination of the electrode. The electrode was connected to a Grass S88 Stimulator. The pelvic nerve was stimulated using the following parameters:- 5V, pulse width 0.5ms, duration of stimulus 10 seconds and a frequency range of 2 to 16Hz. Reproducible responses were obtained when the nerve was stimulated every 15-20 minutes. A frequency response curve was determined at the start of each experiment in order to determine the optimum frequency to use as a sub-maximal response, normally 4Hz. A ventral midline incision was made, at the caudal end of the pubis, to expose the pubic area.
Connective tissue was removed to expose the tunica of the clitoris, ensuring that the wall was free from small blood vessels. The external vaginal wall was also exposed : by removing any connective tissue. One laser Doppler flow probe was inserted 3cm into the vagina, so that half the probe shaft was still visible. A second probe was positioned so that it lay just above the external clitoral wall. The position of these probes was then adjusted until a signal was obtained. A second probe was placed just above the surface of a blood vessel on the external vaginal wall. Both probes were clamped in position.
Compound 1 and compound 3 were dissolved in 50% p-cyclodextrin in saline. They were administered at a dose of 15mg/kg subcutaneously (sc). Vaginal } and clitoral blood flow was recorded either as numbers directly from the Flowmeter using Po-ne-mah data acquisition software (Ponemah Physiology Platform, Gould
Instrument Systems Inc), or indirectly from Gould chart recorder trace. Calibration was set at the beginning of the experiment (0-125ml/min/100g tissue). All data are reported as mean + s.e.m.. Significant changes were identified using Student’s t-tests. /
The non-selective BB1/BB2 bombesin receptor antagonist (Compound 1; ; 15mg/kg sc) acts as a potent enhancer of pelvic-nerve stimulated (PNS) increases in genital blood flow in the anaesthetised rabbit (Figure 22). Compound 1 had no effect ) on basal genital blood flow in the absence of PNS (Figure 22). This reinforces our view that antagonising/blocking BB1/BB2 receptors will enhance the arousal response by potentiating the central mechanism(s) that control sexual arousal/genital blood flow, and will not induce arousal in the absence of sexual stimulation.
The selective BB1 receptor antagonist (Compound 3; 15mg/kg sc) acts as a potent enhancer of pelvic-nerve stimulated (PNS) increases in vaginal and clitoral blood flow in the anaesthetised rabbit (Figure 23). The potentiation was significant 45mins after sc dosing and remained elevated for circa 1hr. Compound 3 had no effect on basal genital blood flow in the absence of PNS (Figure 23). This reinforces our view that a selective BBI receptor antagonist will enhance the arousal response by potentiating the central mechanism(s) that control sexual arousal/genital blood flow, thereby treating FSAD, and will not induce arousal in the absence of sexual stimulation. Since these agents also enhance clitoral blood flow it is likely that they will be effective in the treatment of orgasmic disorders.
At the level of the genitalia, the enhancement observed is similar to the beneficial effects observed with apomorphine, and consequently we believe that a centrally mediated potentiation of the descending neuronal pathways that control genital blood flow is responsible.
Example 170
Bombesin antagonists (compounds 1 and 3) induce increases in penile intracavernosal pressure in the conscious male rat.
Bombesin anatgonists used = Compound 1 and (2S)-N-{[1-(4-aminophenyl) cyclohexyl] methyl}-3-(1H-indol-3-yl)-2-methyl-2-{[(4-nitroanilino) carbonyl] amino} propanamide (Compound 3).
In addition to treating women with FSD, bombesin antagonists will be useful in treating male erectile dysfunction (MED. Both the non-selective BB1/2 antagonists (Compound 1; 10mg/kg sc) and the selective BB1 receptor antagonist (Compound 3; 15mg/kg sc) is pro-erectile in a conscious rat model of penile erection (Figures 24 and 25). Erectile responses were recorded by measuring intracavernosal pressure using surgically implanted telemetric device. The specific details the surgical procedures, data acquisition and analysis can be found in detail in Bernabe 1999.
Compound 1 and compound 3 were dissolved in 50% pB-cyclodextrin in saline. They were administered at a dose of 15mg/kg subcutaneously (sc). One hour after 10mg/kg subcutaneous administration of Compound 1 and 45 minutes after a subcutaneous administration of 15mg/kg compound 3 one observes significant increases in intracavernosal pressure. These increases equate to penile erection. Both
Compounds 1 and 3 induced a number of erections in a similar manner to those observed with apomorphine or melanotan-1I - both of which are clinically proven agents that are effective in the treatment of MED. Moreover the amplitude of the increases observed were similar to those observed with apomorphine or melanotan-II.
The mechanism of action is thought to be similar to the effects on female genital flow ie CNS potentiation of the descending neuronal pathways that control penile erection.
Example 171
Concomitant administration of a Bombesin antagonists with a PDES inhibitor enhance pelvic nerve stimulated increases in penile intracavernosal pressure in an anaesthetised rabbit model of erection
In addition to treating women with FSD, bombesin antagonists will be useful in treating male erectile dysfunction (MED) either alone or in combination with a selective PDES inhibitor.
Male New Zealand rabbits (~2.5kg) were pre-medicated with a combination of Medetomidine (Domitor®) 0.5ml/kg i.m., and Ketamine (Vetalar®) 0.25ml/kg i.m. whilst maintaining oxygen intake via a face mask. The rabbits were tracheotomised using a Portex™ uncuffed endotracheal tube 3 ID., connected to ventilator and maintained at a ventilation rate of 30-40 breaths per minute, with an approximate tidal volume of 18-20 ml, and a maximum airway pressure of 10 cm HO. Anaesthesia was then switched to Isoflurane and ventilation continued with OQ; at 21/min. The right marginal ear vein was cannulated using a 23G or 24G catheter, and Lactated Ringer solution perfused at 0.5ml/min. The rabbit was maintained at 3% Isoflurane during invasive surgery, dropping to 2% for maintenance anaesthesia. The left jugular vein was exposed, isolated and then cannulated with a PVC catheter (17G) for the infusion of drugs and compounds.
The left groin area of the rabbit was shaved and a vertical incision was made approximately 5cm in length along the thigh. The femoral vein and artery were exposed, isolated and then cannulated with a PVC catheter (17G) for the infusion of ] drugs and compounds. Cannulation was repeated for the femoral artery, inserting the catheter to a depth of 10cm to ensure that the catheter reached the abdominal aorta. - This arterial catheter was linked to a Gould system to record blood pressure. Samples for blood gas analysis were also taken via the arterial catheter. Systolic and diastolic pressures were measured, and the mean arterial pressure calculated using the formula
(diastolic x2 + systolic) +3. Heart rate was measured via the pulse oxymeter and Po- ’ ne-mah data acquisition software system (Ponemah Physiology Platform, Gould
Instrument Systems Inc).
A ventral midline incision was made into the abdominal cavity. The incision was about 5cm in length just above the pubis. The fat and muscle was bluntly dissected away to reveal the hypogastric nerve which runs down the body cavity. It was essential to keep close to the side curve of the pubis wall in order to avoid damaging the femoral vein and artery which lie above the pubis. The sciatic and pelvic nerves lie deeper and were located after further dissection on the dorsal side of the rabbit. Once the sciatic nerve is identified, the pelvic nerve was easily located.
The term pelvic nerve is loosely applied; anatomy books on the subject fail to identify the nerves in sufficient detail. However, stimulation of the nerve causes an increase in intracavernosal pressure and cavernosal blood flow, and innervation of the pelvic region. The pelvic nerve was freed away from surrounding tissue and a Harvard bipolar stimulating electrode was placed around the nerve. The nerve was slightly lifted to give some tension, then the electrode was secured in position. Approximately 1ml of light paraffin oil was placed around the nerve and electrode. This acts as a protective lubricant to the nerve and prevents blood contamination of the electrode.
The electrode was connected to a Grass S88 Stimulator. The pelvic nerve was stimulated using the following parameters:- 5V, pulse width 0.5ms, duration of stimulus 20 seconds with a frequency of 16Hz. Reproducible responses were obtained when the nerve was stimulated every 15-20 minutes. Several stimulations using the above parameters were performed to establish a mean control response. The compound(s) to be tested were infused, via the jugular vein, using a Harvard 22 infusion pump allowing a continuous 15 minute stimulation cycle. The skin and connective tissue around the penis was removed to expose the penis. A catheter set (Insyte-W, Becton-Dickinson 20 Gauge 1.1 x 48mm) was inserted through the tunica . albica into the left corpus cavernosal space and the needle removed, leaving a flexible catheter. This catheter was linked via a pressure transducer (Ohmeda 5299-04) to a
Gould system to record intracavernosal pressure. Once an intracavernosal pressure was established, the catheter was sealed in place using Vetbond (tissue adhesive, 3M).
Heart rate was measured via the pulse oxymeter and Po-ne-mah data acquisition software system (Ponemah Physiology Platform, Gould Instrument Systems Inc).
Intracavernosal blood flow was recorded either as numbers directly from the
Flowmeter using Po-ne-mah data acquisition software (Ponemah Physiology
Platform, Gould Instrument Systems Inc), or indirectly from Gould chart recorder trace. Calibration was set at the beginning of the experiment (0-125ml/min/100g tissue). All data are reported as mean + s.e.m.. Significant changes were identified using Student’s t-tests.
Compound 1 and compound 3 were dissolved in 50% p-cyclodextrin in saline. They were administered at a dose of 15mg/kg subcutaneously (sc).
Concomitant inhibition of BB1 receptors with compound 3 and PDES enzyme with a PDES inhibitor produced a marked enhancement of intracavernosal pressure, or the erectile process.
Figure 26 demonstrate that concomitant inhibition of Compound 3 (10mg/kg sc) and a selective inhibitor of PDES (3-ethyl-5-{5-[4-ethylpiperzino)sulphonyl-2- propoxyphenyl}-2-(2-pyridylmethyl)-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-7-one also known as 3-ethyl-5-{5-[4-ethylpiperzin-1-ylsulphonyl)-2-n-propoxyphenyl}-2- (2-pyridyl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one; See
WO098/491066; mg/kg iv) produced a marked enhancement of the ICP, or the erectile process, than was achievable with the same dose of the same Compound 3 inhibitor alone. BBI antagonists and PDES inhibitors or combinations of the two, have no significant effect on un-stimulated intracavernosal pressure ie they do not } induce an increase in the absence of sexual drive/arousal. This data illustrates that there are a number of clinical benefits of concomitant administration of a PDE5 . inhibitor and a bombesin antagonist over PDES inhibitor therapy alone. These include increased efficacy and opportunities to treat MED subgroups that do not respond to
PDES inhibitor therapy.
: Test Assays: Auxiliary Compounds
NEP ENZYME ASSAY
THE PREPARATION AND ASSAY OF SOLUBLE (NEP) NEUTRAL
ENDOPEPTIDASE FROM CANINE, RAT, RABBIT AND HUMAN KIDNEY
CORTEX.
Soluble NEP is obtained from the kidney cortex and activity is assayed by measuring the rate of cleavage of the NEP substrate Abz-D-Arg-Arg-Leu-
EDDnp to generate its fluorescent product, Abz-D-Arg-Arg.
EXPERIMENTAL PROCEDURE:- 1. MATERIALS
All water is double de ionised. 1.1 Tissues
Human Kidney IIAM (Pennsylvania. U.S.A.)
Rat Kidney
Rabbit Kidney
Canine Kidney 1.2 Homogenisation medium 100mM Mannitol and 20mM Tris @ pH 7.1 2.42g Tris (Fisher T/P630/60) is diluted in 1 litre of water and the pH adjusted to 7.1 using 6M HCI at room temperature. To this 18.22g Mannitol (Sigma M-9546) is . added. 1.3 Tris buffer (NEP buffer).
50m] of 50mM Tris pH 7.4 (Sigma T2663) is diluted in 950ml of water. 1.4 Substrate (Abz-D-Arg-Arg-Leu-EDDnp)
Made to order from SNPE, and is stored as a powder at —20°C. A 2mM stock is made by gently re-suspending the substrate in Tris buffer, this should not be vortexed or sonicated. 600ul aliquots of the 2mM stock are stored at —20 for up to one month. (Medeiros, M.A.S., Franca, M.S.F. et al., (1997), Brazilian Journal of Medical and
Biological Research, 30, 1157-1162). 1.5 Total product
Samples corresponding to 100% substrate to product conversion are included on the plate to enable the % substrate turnover to be determined. The total product is generated by incubating ml of 2mM substrate with 20ul of enzyme stock for 24 hours at 37°C. 1.6 Stop solution.
A 300puM stock of Phosphoramidon (Sigma R7385) is made up in NEP buffer and stored in 50pul aliquots at -20. 1.7 Dimethyl sulphoxide (DMSO). 1.8 ~~ Magnesium Chloride -MgCl,.6H,0 (Fisher M0600/53). 1.9 Black 96 well flat bottom assay plates (Costar 3915). 1.10 Topseal A (Packard 6005185). 1.11 Centrifuge tubes 2. SPECIFIC EQUIPTMENT 2.1 Sorvall RC-5B centrifuge (SS34 GSA rotor, pre-cooled to 4°C). 2.2 Braun miniprimer mixer. : 2.3 Beckman CS-6R centrifuge. 24 Fluostar galaxy. 2.5 Wesbart 1589 shaking incubator.
3. METHODS 3.1 TISSUE PREPARATION ) 32 Dog, rat, rabbit, and human NEP is obtained from the kidney cortex using a method adapted from Booth, A.G. & Kenny, A.J. (1974) Biochem. J. 142, 575-581. 3.3 Frozen kidneys are allowed to thaw at room temperature and the cortex is dissected away from the medulla. 3.4 The cortex is finely chopped and homogenised in approximately 10 volumes of homogenisation buffer (1.2) using a Braun miniprimer (2.2). 3.5 Magnesium chloride (1.8) (20.3mg/gm tissue) is added to the homogenate and stirred in an ice-water bath for 15 minutes. 3.6 The homogenate is centrifuged at 1,500g (3,820rpm) for 12 minutes in a
Beckman centrifuge (2.3) before removing the supernatant to a fresh centrifuge tube and discarding the pellet. 3.7 The supernatant is centrifuged at 15,000g (12,100rpm) for 12 minutes in a
Sovall centrifuge (2.1) and the supernatant is discarded. 3.8 The pale pink layer -on the top of the remaining pellet is removed and re- suspended in homogenisation buffer containing magnesium chloride (9mg MgCl in
Sml buffer per 1g tissue). 3.9 The suspension is centrifuged at 2,200g (4,630rpm) for 12 minutes in a
Beckman centrifuge (2.3) before discarding the pellet. 3.10 The supernatant is centrifuged at 15,000g (12,100rpm) for 12 minutes using the Sorvall centrifuge (2.1) and the supernatant is discarded. 3.11 The final pellet is resuspended in homogenisation buffer containing ~ 25 magnesium chloride (0.9mg MgCl in 0.5ml buffer per 1g tissue). A homogenous suspension is obtained using a Braun miniprimer (2.2). This is then frozen down in 100ul aliquots to be assayed for NEP activity. ; 4.0 DETERMINATION OF NEP ACTIVITY
The activity of the previously aliquoted NEP is measured by its ability to cleave the
NEP specific peptide substrate. 41 A 4% DMSO/NEP buffer solution is made (4mls DMSO in 96mls NEP buffer). 4.2 Substrate, total product, enzyme, and Phosphoramidon stocks are left on ice to thaw. 43 50ul of 4% DMSO/NEP buffer solution is added to each well. 44 The 2mM substrate stock is diluted 1:40 to make a 50uM solution. 100ul of 50uM substrate is added to each well (final concentration 25uM). 45 50ul of a range of enzyme dilutions is added to initiate the reaction (usually 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200 are used). 50ul of NEP buffer is added to blank wells. 4.6 The 2mM total product is diluted 1:80 to make a 25uM solution. 200ul of 25uM product is added to the first four wells of a new plate. 4.7 Plates are incubated at 370C in a shaking incubator for 60 minutes. 4.8 The 300uM Phosphoramidon stock is diluted 1:100 to 300nM. The reaction is stopped by the addition of 100ul 300nM Phosphoramidon and incubated at 37°C in a shaking incubator for 20 minutes before being read on the Fluostar (ex320/em420). 5.NEP INHIBITION ASSAYS 5.1 Substrate, total product, enzyme and Phoshoramidon stocks are left on ice to thaw. 5.2 Compound stocks are made up in 100% DMSO and diluted 1:25 in NEP buffer to give a 4% DMSO solution. All further dilutions are carried out in a 4%
DMSO solution (4mls DMSO in 96mls NEP buffer). 53 50ul of compound in duplicate is added to the 96 well plate and 50ul of 4%
DMSO/NEP buffer is added to control and blank wells. . 54 The 2mM substrate stock is diluted 1:40 in NEP buffer to make a 50uM solution (275p1 2mM substrate to 10.73ml buffer is enough for 1 plate). 5.5 The enzyme stock diluted in NEP buffer (determined from activity checks).
5.6 The 2mM total product stock is diluted 1:80 in NEP buffer to make a 25uM : solution. 200pl is added to the first four wells of a separate plate. 5.7 The 300uM Phosphoramidon stock is diluted 1:1000 to make a 300nM stock (11p1 Phosphoramidon to 10.99ml NEP buffer. 5.8 To each well in the 96 well plate the following is added:
Table Reagents to be added to 96 well plate.
Compound/ Tris Substrate | NEP Total
DMSO Buffer enzyme | product 2pl compound | 50pl 100u! 50ul 5.9 The reaction is initiated by the addition of the NEP enzyme before incubating at 37°C for 1 hour in a shaking incubator. 5.10 The reaction is stopped with 100pl 300nM Phosphoramidon and incubated at 37°C for 20 minutes in a shaking incubator before being read on the Fluostar (ex320/em420). 6. CALCULATIONS
The activity of the NEP enzyme is determined in the presence and absence of compound and expressed as a percentage. % Control activity (turnover of enzyme): : Mean FU of controls — Mean FU of blanks X 100
Mean FU of totals — Mean FU of blanks
% Activity with inhibitor:
Mean FU of compound — Mean FU of blanks X 100
Mean FU of totals ~ Mean FU of blanks
Activity expressed as % of control: % Activity with inhibitor X 100 % Control activity
A sigmoidal dose-response curve is fitted to the % activities (% of control) vs compound concentration and IC50 values calculated using LabStats fit-curve in
Excel.
ACE ASSAY
THE PREPARATION AND ASSAY OF SOLUBLE ANGIOTENSIN
CONVERTING ENZYME (ACE), FROM PORCINE AND HUMAN KIDNEY
CORTEX.
Soluble ACE activity is obtained from the kidney cortex and assayed by : measuring the rate of cleavage of the ACE substrate Abz-Gly-p-nitro-Phe-Pro-
OH to generate its fluorescent product, Abz-Gly. 1. MATERIALS
All water is double de ionised. 1.1 Human Kidney [IAM (Pennsylvania. U.S.A.) or UK Human } Tissue Bank (UK HTB) 1.2 Porcine kidney ACE Sigma (A2580) 1.3 Homogenisation buffer-1
100mM Mannitol and 20mM Tris @ pH 7.1 2.42¢ Tris (Fisher T/P630/60) is diluted in 1 litre of water and the pH adjusted to 7.1 using 6M HCI at room temperature. To this 18.22g Mannitol (Sigma M-9546) is added. 1.4 Homogenisation buffer-2 100mM Mannitol, 20mM Tris @ pH7.1 and 10mM MgCl, 6H,0O (Fisher M0600/53)
To 500ml of the homogenisation buffer 1 (1.4) 1.017g of MgCl, is added. 1.5 Tris buffer (ACE buffer). 50mM Tris and 300mM NaCl @ pH 7.4 50ml of SOmM Tris pH 7.4 (Sigma T2663) and 17.52g NaCl (Fisher S/3160/60) are made up to 1000ml in water. 1.6 Substrate (Abz-D-Gly-p-nitro-Phe-Pro-OH) (Bachem M-1100)
ACE substrate is stored as a powder at —20°C. A 2mM stock is made by gently re- suspending the substrate in ACE buffer, this must not be vortexed or sonicated. 400pul aliquots of the 2mM stock are stored at —20°C for up to one month. 1.7 Total product
Samples corresponding to 100% substrate to product conversion are included on the plate to enable the % substrate turnover to be determined (see calculations). The total product is generated by incubating ml of 2mM substrate with 20ul of enzyme stock for 24 hours at 37°C. 1.8 Stop solution. 0.5M EDTA (Promega CAS[6081/92/6]) is diluted 1:250 in ACE buffer to make a 2mM solution. 1.9 Dimethyl sulphoxide (DMSO). 1.10 Magnesium Chloride -MgCl,.6H,0 (Fisher M0600/53). 1.11 Black 96 well flat bottom assay plates (Costar 3915 or Packard). 1.12 Topseal A (Packard 6005185). 1.13 Centrifuge tubes
2. SPECIFIC EQUIPTMENT 2.1 Sorvall RC-5B centrifuge (SS34 GSA rotor, pre-cooled to 4°C). 2.2 Braun miniprimer mixer. 23 Beckman CS-6R centrifuge. 24 BMG Fluostar Galaxy. 2.5 Wesbart 1589 shaking incubator. 3. METHODS 3.1 TISSUE PREPARATION 3.3 Human ACE is obtained from the kidney cortex using a method adapted from
Booth, A.G. & Kenny, A.J. (1974) Biochem. J. 142, 575-581. 33 Frozen kidneys are allowed to thaw at room temperature and the cortex is dissected away from the medulla. 3.4 The cortex is finely chopped and homogenised in approximately 10 volumes of homogenisation buffer-1 (1.4) using a Braun miniprimer (2.2). 3.5 Magnesium chloride (1.11) (20.3mg/gm tissue) is added to the homogenate and stirred in an ice-water bath for 15 minutes. 3.6 The homogenate is centrifuged at 1,500g (3,820rpm) for 12 minutes in a
Beckman centrifuge (2.3) before removing the supernatant to a fresh centrifuge tube and discarding the pellet. 3.7 The supernatant is centrifuged at 15,000g (12,100rpm) for 12 minutes in a
Sovall centrifuge (2.1) and the supernatant is discarded. 3.8 The pale pink layer on the top of the remaining pellet is removed and re- suspended in homogenisation buffer-2 (1.5) (Sml buffer per 1g tissue). 3.9 The suspension is centrifuged at 2,200g (4,630rpm) for 12 minutes in a
Beckman centrifuge before discarding the pellet. . 3.10 The supernatant is centrifuged at 15,000g (12,100rpm) for 12 minutes using the Sorvall centrifuge and the supernatant is discarded.
3.11 The final pellet is resuspended in homogenisation buffer-2 (0.5ml buffer per : 1g tissue). A homogenous suspension is obtained using a Braun miniprimer. This is then frozen down in 100ul aliquots to be assayed for NEP activity. 4.0 DETERMINATION OF ACE ACTIVITY
The activity of the previously aliquoted ACE is measured by its ability to cleave the
ACE specific peptide substrate.
Porcine ACE (1.2) is defrosted and resuspended in ACE buffer (1.6) at 0.004U/ul, this is frozen down in 50pul aliquots. 4.1 A 4% DMSO/ACE buffer solution is made (4mls DMSO in 96mls ACE buffer). 4.2 Substrate (1.7), total product (1.8) and enzyme (1.1, 1.2, 1.3), are left on ice to thaw. 4.3 50ul of 4% DMSO/ACE buffer solution is added to each well. 4.4 The 2mM substrate stock is diluted 1:100 to make a 20uM solution. 100ul of 20uM substrate is added to each well (final concentration in the assay 10uM). 4.5 50pul of a range of enzyme dilutions is added to initiate the reaction (usually 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200 are used). 50ul of ACE buffer is added to blank wells. 4.6 The 2mM total product is diluted 1:200 to make 10uM solution. 200p1 10uM product is added to the first four wells of a new plate. 47 Plates are incubated at 37°C in a shaking incubator for 60 minutes. 438 The enzyme reaction is stopped by the addition of 100ul 2mM EDTA in ACE buffer and incubated at 37°C in a shaking incubator for 20 minutes before being read on the BMG Fluostar Galaxy (ex320/em420). . 5. ACE INHIBITION ASSAYS 5.1 Substrate, total product, and enzyme stocks are left on ice to thaw.
52 Compound stocks are made up in 100% DMSO and diluted 1:25 in ACE : buffer to give a 4% DMSO solution. All further dilutions are carried out in a 4%
DMSO/ACE buffer solution (4mls DMSO in 96mls ACE buffer). 53 50ul of compound, in duplicate, is added to the 96 well plate and 50ul of 4%
DMSO/ACE buffer is added to control and blank wells. 54 Steps 5.2 and 5.3 can be carried out either by hand or using the Packard multiprobe robots 5.5 The 2mM substrate stock is diluted 1:100 in ACE buffer to make a 20uM solution (10uM final concentration in the assay) (110ul of 2mM substrate added to 10.89ml buffer is enough for 1 plate). 5.6 The enzyme stock is diluted in ACE buffer, as determined from activity checks (4.0). 5.7 The 2mM total product stock is diluted 1:200 in ACE buffer to make a 10puM solution. 200p1 is added to the first four wells of a separate plate. 58 The 0.5mM EDTA stock is diluted 1:250 to make a 2mM stock (44ul EDTA to 10.96ml ACE buffer). 59 To each well of the 96 well plate the following reagents are added:
Table 1: Reagents added to 96 well plate.
Compound/ Tris Substrate | ACE Total
Sl MY fl eg 5.10 50ul of the highest concentration of each compound used in the assay is added . in duplicate to the same 96 well plate as the totals (5.7). 150ul of ACE buffer is added to determine any compound fluorescence.
5.11 The reaction is initiated by the addition of the ACE enzyme before incubating : at 37°C for 1 hour in a shaking incubator. 5.12 The reaction is stopped by the addition of 100ul 2mM EDTA and incubated at 37°C for 20 minutes in a shaking incubator, before being read on the BMG Fluostar 5S Galaxy (ex320/em420). 6. CALCULATIONS
The activity of the ACE enzyme is determined in the presence and absence of compound and expressed as a percentage.
FU = Fluorescence units (1) % Control activity (turnover of enzyme):
Mean FU of controls — Mean FU of blanks X 100
Mean FU of totals — Mean FU of blanks (ii) % Activity with inhibitor:
Mean FU of compound — Mean FU of blanks X 100
Mean FU of totals — Mean FU of blanks (iil) Activity expressed as % of control: % Activity with inhibitor X 100 % Control activity
OR Mean FU of compound — Mean FU of blanks X 100
Mean FU of controls — Mean FU of blanks
: (iv) % Inhibition = 100 - % control } Vv) For fluorescent compounds the mean FU of blanks containing compound (5.10) is deducted from the mean FU of compound values used to calculate the % Activity.
A sigmoidal dose-response curve is fitted to the % activities (% of control) vs compound concentration and ICs values calculated using LabStats fit-curve in Excel.
PDES inhibitor — TEST METHODS
Phosphediesterase (PDE) inhibitory activity
In vitro PDE inhibitory activities against cyclic guanosine 3°,5’- monophosphate (cGMP) and cyclic adenosine 3’,5’-monophosphate (cAMP) phosphodiesterases were determined by measurement of their ICsy values (the concentration of compound required for 50% inhibition of enzyme activity).
The required PDE enzymes were isolated from a variety of sources, including human corpus cavernosum, human and rabbit platelets, human cardiac ventricle, human skeletal muscle and human and canine retina, essentially by the method of
W.J. Thompson and M.M. Appleman (Biochem., 1971, 10, 311). In particular, the c¢GMP-specific PDE (PDES) and the c¢cGMP-inhibited cAMP PDE (PDE3) were obtained from human corpus cavernosum or human platelets; the cGMP-stimulated
PDE (PDE2) was obtained from human corpus cavernosum and human platelets; the calcium/calmodulin (Ca/CAM)-dependent PDE (PDE1) from human cardiac ventricle; the cAMP-specific PDE (PDE4) from human skeletal muscle and human . recombinant, expressed in SF9 cells; and the photoreceptor PDE (PDE6) from human or canine retina. Phosphodiesterases 7-11 were generated from full length human recombinant clones transfected into SF9 cells.
: Assays can be performed either using a modification of the “batch” method of
W.J. Thompson et al. (Biochem., 1979, 18, 5228) or using a scintillation proximity assay for the direct detection of AMP/GMP using a modification of the protocol described by Amersham plc under product code TRKQ7090/7100. In summary, the effect of PDE inhibitors was investigated by assaying a fixed amount of enzyme in the presence of varying inhibitor concentrations and low substrate, (cGMP or cAMP in a 3:1 ratio unlabelled to H]-labeled at a conc ~1/3 K,,) such that ICs¢ = K;. The final assay volume was made up to 100ul with assay buffer [20 mM Tris-HCI pH 7.4, 5 mM MgCl,, 1 mg/ml bovine serum albumin]. Reactions were initiated with enzyme, incubated for 30-60 min at 30°C to give <30% substrate turnover and terminated with 50 pl yttrium silicate SPA beads (containing 3 mM of the respective unlabelled cyclic nucleotide for PDEs 9 and 11). Plates were re-sealed and shaken for 20 min, after which the beads were allowed to settle for 30 min in the dark and then counted on a TopCount plate reader (Packard, Meriden, CT) Radioactivity units were converted to % activity of an uninhibited control (100%), plotted against inhibitor concentration and inhibitor ICsq values obtained using the ‘Fit Curve’ Microsoft Excel extension (or in-house equivalent). Results from these tests show that the compounds of the present invention are inhibitors of cGMP-specific PDES.
Functional activity
This can be assessed in vitro by determining the capacity of a compound of the invention to enhance sodium nitroprusside or electrical field stimulation-induced relaxation of pre-contracted rabbit corpus cavernosum tissue strips, using methods based on that described by S.A. Ballard et al. (Brit. J. Pharmacol., 1996, 118 (suppl.), abstract 153P) or S.A. Ballard et al. (J. Urology, 1998, 159, 2164-2171). - Compounds can be screened in anaesthetised dogs to determine their capacity, after i.v. administration, to enhance the pressure rises in the corpora cavernosa of the penis induced by intracavernosal injection of sodium nitroprusside, using a method based : on that described by Trigo-Rocha et al. (Neurourol. and Urodyn., 1994, 13, 71).
NPY assay:
An assay for identifying NPY inhibitors is presented in WO-A-98/52890 (see page 96, lines 2 to 28).
Other References
Anastasi A. et al., Experientia, 1971; 27: 166
S A Ballard ef al, Journal of Urology, 1998, vol. 159, pages 2164-2171
Barker, P. E.; Shipp, M. A.; D'Adamio, L.; Masteller, E. L.; Reinherz, E. L. The common acute lymphoblastic leukemia antigen gene maps to chromosomal region 3(q21-q27). J. Immun. 142: 283-287, 1989.
Battey J., et al., TINS, 1991;14:524
Baum M.J., J. Biosci., 1983; 33:578-582
Benet AE and Melman A, 1995, Urol. Clin. N. Amer. 22: 699-709
Berman et al., 1999, hypoactive sexual desire disorders, sexual arousal disorders, anorgasmy and sexual pain disorders, Urology 54: 385-391.
Berman, J., Goldstein, I., Werbin, T. ef al. (1999a). Double blind placebo controlled . study with crossover to assess effect of sildenafil on physiological parameters of the female sexual response. J. Urol., 161, 805.
Berman J.R. et al. Int. J. Impot. Res., 1999, 11: S31-38
Bernabe J, Rampin O, Sachs BD, Giuliano F. Intracavernous pressure during erection in rats: an integrative approach based on telemetric recording. Am J Physiol. 1999 Feb;276(2 Pt 2):R441-9.
Bonney R.C et al., Scrip’s Complete Guide to Women's Healthcare, PJB Publications
Ltd, London, 2000
Braun M,, et al., Life. Sci., 1978; 23: 2721
Bundgaard et al., Acta Pharm. Suec., 1987; 24: 233-246
D'Adamio, L.; Shipp, M. A.; Masteller, E. L.; Reinherz, E. L. : Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5-prime untranslated regions. Proc. Nat.
Acad. Sci. 86: 7103-7107, 1989.
Dutta A.S., Small Peptides; Chemistry, Biology, and Clinical Studies, Chapter 2, pp 66-82
J. Frohlich et al., Heterocycles 1994, 37, 1879-91
Goldstein, I. & Berman, J.R. (1998). Vasculogenic female sexual dysfunction: vaginal engorgement and clitoral erectile insufficiency syndromes. Int. J. Impot. Res., 10,
S84-S90.
Johnson M and Everitt B., Essential Reproduction (3 edn), Blackwell, Oxford, 1988.
Ladenheim E.E et al, Brain Res., 1990; 537: 233-240
Laumann et al., 1999 JAMA 281:537-544)
Letarte, M.; Vera, S.; Tran, R.; Addis, J. B. L.; Onizuka, R. J.; Quackenbush, E. J;
Jongeneel, C. V.; McInnes, R. R. : Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J. Exp. Med. 168: 1247-1253, 1988.
Meyerson B.J, Lindstrom L.H., Acta Physiol. Scand., 1973; 389 (Suppl.): 1-80.
O’Donohue W, et al, Clin. Psychol. Rev. 1997; 17: 537-566.
Park, K., Goldstein, I, Andry, C., er al. (1997). Vasculogenic female sexual dysfunction: The hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. Int. J. Impotence Res., 9, 27-37.
Sachs B. D., Neuroscience and Biobehavioral Review , 2000, 24 541-560.
Shipp, M. A.; Vijayaraghavan, J.; Schmidt, E. V.; Masteller, E. L.; D'Adamio, L.;
Hersh, L. B.; Reinherz, E. L. : Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 (‘enkephalinase'): direct evidence by cDNA transfection analysis. Proc. Nat. Acad. Sci. 86: 297-301, 1989.
Tran-Paterson, R.; Willard, H. F.; Letarte, M. : The common acute lymphoblastic leukemia antigen (neutral endopeptidase--3.4.24.11) gene is located on human chromosome 3. Cancer Genet. Cytogenet. 42: 129-134, 1989.
Werbin, T., Salimpour, P., Berman, L., et al. (1999). Effect of sexual stimulation and age on genital blood flow in women with sexual stimulation. J. Urol., 161, 688. ) Wilson C.A., Pharmacological targets for the control of male and female sexual behaviour, In: Sexual Pharmacology, Riley A.J. et al, (Eds), Clarendon Press, Oxford, 1993: 1-58

Claims (47)

1. A pharmaceutical combination (for simultaneous, separate or sequential ‘ administration) of a bombesin receptor antagonist and one or more materials selected from (1) to (34) below: (1) naturally occurring or synthetic prostaglandins or esters thereof; 2) o - adrenergic receptor antagonist compounds also known as ao - adrenoceptor antaginists or a-receptor antagonists or a-blockers; (3) NO-donor (NO-agonist) compounds; 4) potassium channel openers or modulators; 5) dopaminergic agents; 6) vasodilator agents; 0) thromboxane A2 agonists; 8) CNS active agents; ©) ergot alkaloids; (10) compounds which modulate the action of natriuretic factors; . (11) angiotensin receptor antagonists such as losartan.; (12) substrates for NO-synthase;. (13) calcium channel blockers; (14) cholesterol lowering agents, (15) antiplatelet and antithrombotic agents; (16) insulin sensitising agents and hypoglycaemic agents;. (17) L-DOPA or carbidopa; (18) acetylcholinesterase inhibitors; (19) steroidal or non-steroidal anti-inflammatory agents; (20) estrogen receptor modulators and/or estrogen agonists and/or estrogen antagonists, and pharmaceutically acceptable salts thereof; (21) PDE inhibitors; . (22) NPY (neuropeptide Y) inhibitors; (23) NEP inhibitors;
(24) vasoactive intestinal proteins (VIP), VIP mimetics, VIP analogues, ’ VIP receptor agonists or VIP analogues or VIP fragments, or a-adrenoceptor antagonists with VIP combinations; (25) melanocortin receptor agonists or modulators or melanocortin ehancers; (26) serotonin receptor agonists, antagonists or modulators; (27) testosterone replacement agents, testosternone, dihydrotestosterone or a testosterone implant; (28) estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate (MPA) (i.e. as a combination), or estrogen and methyl testosterone hormone replacement therapy agents; (29) modulators of transporters for noradrenaline, dopamine and/or serotonin; (30) purinergic receptor agonists and/or modulators; (31) neurokinin (NK) receptor antagonists; (32) opioid receptor agonists, antagonista or modulators; (33) agonists or modulators for oxytocin/vasopressin receptors; and (34) modulators of cannabinoid receptors.
2. The combination of claim 1, wherein the bombesin receptor antagonist is a selective bombesin BB1 antagonist.
3. The combination of claim 2, wherein the bombesin BB1 antagonist has a selectivity for BB, over the other bombesin receptor subtypes greater than 10.
4. The combination of claim 2, wherein the bombesin BB1 antagonist has a selectivity for BB, over the other bombesin receptor subtypes greater than 30. .
5. The combination of claim 2, wherein the bombesin BB1 antagonist has a selectivity for BB) over the other bombesin receptor subtypes greater than 100.
6. The combination of claim 1, wherein the bombesin receptor antagonist is a : mixed BB1/BB2 antagonist.
7. The combination of any preceding claim, wherein the bombesin receptor antagonist has a Ki against BB1 of less than 1000nM.
8. The combination of any of claims 1-6, wherein the bombesin receptor antagonist has a Ki against BB1 of less than 500nM.
9. The combination of any of claims 1-6, wherein the bombesin receptor antagonist has a Ki against BB1 of less than 100nM.
10. The combination of any of claims 1-6, wherein the bombesin receptor antagonist has a Ki against BB1 of less than 50nM.
11. The combination of any of claims 1-6, wherein the bombesin receptor antagonist has a Ki against BB1 of less than 10nM.
12. The combination of any preceding claim, comprising an effective amount of a non-peptide bombesin receptor antagonist.
13. The combination of any of claims 1-11, wherein there is administered to the subject an effective amount of a bombesin receptor antagonist which is a peptide
14. The combination of any preceding claim, wherein the non-peptide bombesin receptor antagonist is a compound that is absorbable when administered orally.
15 The combination of any preceding claim, wherein the bombesin receptor . antagonist is a compound of the formula (I)
3 reblog fF ) : N . rc 3 | ~ Cee RO oo Re 0 Ar! ) ® or a pharmaceutically acceptable salt thereof, wherein: e jisOorl; o kisOorl; S e 1is0,1,2,0r3; e misOorl; e nis, lor2; e Ar is phenyl, pyridyl or pyrimidyl, each unsubstituted or substituted by from 1 to 3 substituents selected from alkyl, halogen, alkoxy, acetyl, nitro, amino, - CHoNRI0R 11, cyano, -CF3, -NHCONH3, and -COpR 12; o Rlis hydrogen or straight, branched, or cyclic alkyl of from 1 to 7 carbon atoms; eo R8is hydrogen or forms a ring with R1 of from 3 to 7 carbon atoms; oe RZis hydrogen or straight, branched, or cyclic alkyl of from 1 to 8 carbon atoms which can also contain 1 to 2 oxygen or nitrogen atoms, e RY is hydrogen or forms with R2a ring of from 3 to 7 carbon atoms which can contain an oxygen or nitrogen atom; or R2 and R? can together be a carbonyl; e Ar! can be independently selected from Ar and can also include pyridyl-N- oxide, indolyl, imidazolyl, and pyridyl; ° R4, RS, RO, and R7 are each independently selected from hydrogen and lower alkyl; R4 can also form with R3 a covalent link of 2 to 3 atoms which - may include an oxygen or a nitrogen atom; e R3 can be independently selected from Ar or is hydrogen, hydroxy, -NMe,, N-methyl-pyrrolyl, imidazolyl, N-methyl-imidazolyl, tetrazolyl, N-methyl- tetrazolyl, thiazolyl, -CONRI3R14 alkoxy,
fo A Ace, Ar oN , or aw © CF, wherein p is 0, 1 or 2 and Ar? is phenyl or pyridyl; o RIO RII RI2 RI3 and R14 are each independently selected from hydrogen or straight, branched, or cyclic alkyl of from 1 to 7 carbon atoms.
16. The combination of any of claims 1-14, wherein the bombesin receptor antagonist is a compoundof Formula (Ia) «PV (OO Ath N—C—C—N—C R® TL lo} Ar (1a) wherein e Ar is phenyl unsubstituted or substituted with 1 or 2 substituents selected from isopropyl, halo, nitro, and cyano; ) R4, RS, and RO are hydrogen; e R7 is methyl or hydrogen; e R3 is 2-pyridyl or hydroxy; and e Arl is indolyl, pyridyl, pyridyl-N-oxide, or imidazolyl.
17. The combination of claim 15, wherein the bombesin receptor antagonist is a compound of Formula | wherein e Ar is unsubstituted phenyl; e Rl iscyclopentyl or tert-butyl; e R#%and RS are hydrogen;
eo R7is methyl; e ROis hydrogen; e R3is phenyl with two isopropyl substituents, unsubstituted phenyl, or 0 oO \ ; and Arlis indolyl.
18. The combination of claim 15, wherein the bombesin receptor antagonist is a compound of Formula I wherein e Aris 2,6-diisopropyl-phenyl, 4-nitro-phenyl, and 4-cyano-phenyl;
10 . R4, RS, and RS are hydrogen; e R7 is methyl; e RZis hydrogen or cyclohexyl; and e R3 is hydroxyl, pyridyl, 0 oO oh, Oo s y Or .
19. The combination of claim 1, wherein the bombesin receptor antagonist is (S)3-(1H-Indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2- methyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide (also referred to as Compound 1) or one of its pharmaceutically acceptable salts or is . (2S)-N-{[1-(4-aminophenyl) cyclohexyl] methyl}-3-(1H-indol-3-yl)-2-methyl- 2-{[(4-nitroanilino) carbonyl] amino} propanamide (also knowm as Compound 3) or one of its pharmaceutically acceptable salts.
20. The combination of claim 1, wherein the bombesin receptor antagonist is a d compound set out below or a pharmaceutically acceptable salt thereof: (S) N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)- 2-methyl-propionamide; N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)- N-methyl-propionamide; N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-1-methyl-ureido]}-3-(1 H- indol-3-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3-(1-oxy-pyridin-2-yl)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3-pyridin-2-yl-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide; 2-[3-(2-tert-butyl-phenyl)-ureido}-N-cyclohexylmethyl-3-(1 H-indol-3-yl)-2- methyl-propionamide; N-cyclohexylmethyl-2-[3-(2,6-dichloro-phenyl)ureido]-3-(1 H-indol-3-yl)-2- methyl-propionamide; N-cyclohexylmethyl-2-[3-(2,6-dimethoxy-phenyl)ureido]-3-(1 H-indol-3-yl)-2- methyl-propionamide; N-cyclohexylmethyl-2-[3-(2,6-dimethylamino-phenyl)-ureido]-3-(1 Hindol-3- yl)-2-methyl-propionamide; (S) N-cyclohexylmethyl-3-(1 H-indol-3-yl)-2-methyl-2-[3-(4-nitro-phenyl)- ureido}-propionamide; N-cyclohexylmethyl-2-[3-(2,2-dimethyl- 1 -phenyl)propyl)-ureido]-3-(1H- indol-3-yl)-2-methyl-propionamide; [S-(R*, R*)] 3-(1 H-indol-3-yl)-2-methyl-2-{3-[1-(4-nitro-phenyl)-ethyl]- ureido}-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; N-(2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-3-(1 H-indol-3-yl)-2-methyl-2-[3- (1-phenyl-cyclopentylmethyl)ureido]-propionamide; . (S)-N-(2,6-diisopropyl-phenyl)-2-[3-(2,2-dimethyl-1-phenyl-propyl)-ureido]- 3-(1H-indol-3-yl)-propionamide;
(R)-N-(2,6-diisopropyl-phenyl)-2-[3-(2,2-dimethyl-1-phenyl-propyl)-ureido}- ‘ 3-(1H-indol-3-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-N-(2,2-dimethyl-4-phenyl-[ 1,3]dioxan- 5-y1)-3-(1 H-indol-3-y1)-2-methyl-propionamide; N-cyclohexyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2- methyl-propionamide; N-(2-cyclohexyl-ethyl)-2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3- yD-2-methyl-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido}-3-(1 H-indol-3-yl)-2-methyl- propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(3- methyl-butyl)-propionamide; 2-[3-(2,6-d1isopropyl-phenyl)-ureido]}-3-(1 H-indol-3-y})-2-methyl-N-(3- phenyl-propyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(1,2,3 4- tetrahydro-naphthalen- 1-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-(2- phenyl-cyclohexyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-N-indan-1-yl-3-(1 H-indol-3-yl)-2- methyl-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-N-(I-hydroxy-cyclohexylmethyl)-3-(1 H- indol-3-yl)-2-methyl-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]}-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido}-3-(1 H-indol-3-yl)-2-methyl-N-(6,7,8,9- tetrahydro-5H-benzocyclohepten-5-yl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-indol-3-yl)-2-methyl-N-phenyl- propionamide; . N-(1-hydroxy-cyclohexylmethyl)-3-(1 H-indol-3-yl)-2-methyl-2-[3-(4-nitro- phenyl)-ureido]-propionamide;
2-[3-(4-cyano-phenyl)-ureido}-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl- : cyclohexylmethyl)-propionamide; (S) 3-(1 H-indol-3-yl)-2-methyl-2-[3-(4-nitro-phenyl)-ureido]-N-(1-pyridin-2- ’ yl-cyclohexylmethyl)-propionamide; (S) 3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-[3-(4- trifluoromethyl-phenyl)-ureido]-propionamide; (S) 4-(3-{2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-ureido)-benzoic acid ethyl ester; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-3-(1 H-imidazol-4-yl)-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-3-(2-trifluoromethyl-phenyl)-propionamide; 2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3-(2-nitro-phenyl)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S) 3-(1 H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2- methyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide; and N-cyclohexylmethyl-2-[3-(2,6-diisopropyl-phenyl)-ureido]-2-methyl-3- pyridin-2-yl-propionamide.
21. The combination of any of claims 1-14, wherein the bombesin receptor antagonist is a compound of formula (II) or a pharmaceutically acceptable salt thereof: R} RS ~~ R* R! (Ar), - (CH), - (X), -(CH,), - ! - | - " - | -(CH,),- & (CH,), -R? oo © Le an ‘ 25 wherein: oe jisO,1o0r2; ; e kisOorl; eo lis0,1,2,0r3; e¢ misOorl;
e nis0,1or2; . e qisOorl; e risOor 1; whenrisO, Ar is replaced by hydrogen; ® Ar is phenyl, pyridyl, pyrimidyl, thienyl, furyl, imidazolyl, pyrrolyl or thiazolyl each unsubstituted or substituted by from 1 to 3 substituents selected from acetyl, alkoxy, alkyl, amino, cyano, halo, hydroxy, nitro, sulfonamido, sulfonyl, -CF3, -OCF;, -COH, -CH,CN, -SO,CF;, -CH,CO,H and - (CH»)NR'R® wherein s is 0, 1, 2 or 3 and R” and R® are each independently selected from H, straight or branched alkyl of up to 6 carbon atoms, or R’ and RS together with the nitrogen atom to which they are linked can form a 5- to 7- membered aliphatic ring which may contain 1 or 2 oxygen atoms; ° R is hydrogen, straight or branched alkyl of up to 6 carbon atoms or cycloalkyl of between 5 and 7 carbon atoms which may contain 1 or 2 nitrogen or oxygen atoms; e RC is hydrogen, methyl, or forms with R' an aliphatic ring of from 3 to 7 atoms which can contain an oxygen or nitrogen atom, or together with R' is a carbonyl group; J Ar is independently selected from Ar or is indoly! or pyridyl-N-oxide; ° Rr’, rR, and rR’ are each independently selected from hydrogen and lower alkyl; ° R’ 1s independently selected from Ar or is hydrogen, hydroxy, alkoxy, -NMe», -CONRR" wherein R and R'* are each independently selected from hydrogen, straight or branched alkyl of up to 6 carbon atoms, or R? and R'® together with the nitrogen atom to which they are linked can form a 5-to 7- membered aliphatic ring which may contain 1 or 2 oxygen or nitrogen . atoms, or R?is i H, i 2 Ar 2 Ar y 0 10]
a. oC “Fs Ar? 0 > Or Ar? CF, wherein p is 0, 1 or 2 and Ar? is phenyl or pyridyl; e X is a divalent radical derived from any of the following gi! J @ @ (J J N N N S N 2 NTS x NTS NSD OW QO ww 0 WU N N N. D) Sy N=0 X N N =O Qo Cre 0 o 0 GQ OO ¢ : Sow OH 1a OO Yi N N A {1 <A A a J N R11 R11 R11 4 OD CO ° R12 R1Z R12 R11 R114 R11 R11 R11 CL LO 0 on 20 R12 N R12 N R12 0 RZ S Ri ° ~ R11 R11 R11 N N N R11 0 00 2) LO : R12 S riz R12 N R12 R11 N R11 RS SN (20 20 R12 R12
: where the ring nitrogen atoms may have lower alkyl groups attached thereto, R'! and R" are independently selected from H, halogen, hydroxy, alkoxy, acetyl, ' nitro, cyano, amino, CF3 and -(CH,){NR'’R'* where t can be 0 or 1, R'> and R™ are each independently selected from hydrogen, straight or branched alkyl of up to 6 carbon atoms or cycloalkyl of 5 to 7 carbon atoms, containing up to 2 oxygen or nitrogen atoms.
22 The combination of any of claims 1-14, wherein the bombesin receptor antagonist is a compound of the formula (IIa), or a pharmaceutically acceptable salt thereof: R* RS R4 RI oxobtog bon. cn ow © R¢ (11a)
wherein: e nisOorl; e Aris phenyl or pyridyl which may be unsubstituted or substituted with from 1 to 3 substituents selected from halogen, alkoxy, nitro and cyano; e Ar is independently selected from Ar or is pyridyl-N-oxide or indolyl; e R® forms with R' an aliphatic ring of from 3 to 7 atoms which can contain an oxygen or nitrogen atom, or together with R' is a carbonyl group; e Ris independently selected from Ar or is hydrogen, hydroxy, alkoxy, . dimethylamino, tetrazolyl or -CONRR" wherein R’ and R' are each independently selected from hydrogen or methyl or R? is any of
CI) AO) £0 HW | AP ar 0 Oo ~~ oN Fs ar © , OF Ar CF, wherein p is 0, 1 or 2 and Ar? is phenyl! or pyridyl; e R’ R* and R’ are each independently selected from hydrogen and methyl; and eo X is selected from: Na N N N s Aq D A A Oo Ga OO 9 0 N R11 N Na Na o 0 A GG oO oOo KD R11 R11 R11 N R11 N R11 CD OD DD D0 oo R12 S$ R12 0 R12 s R12 7 Rez 7 R! and R" being independently selected from H, halogen, hydroxy, alkoxy, i0 acetyl, nitro, cyano, amino, CF; and (CH,){NR"R" wherein tis 0 or 1 and R" and R'* are independently selected from hydrogen and methyl.
23. The combination of any of claims 1-14, wherein the bombesin receptor antagonist is a compound has the formula (IIb) or (IIc) or is a pharmaceutically acceptable salt thereof: N N ‘ 2400 0 0 PP N R2 Ar BY N R2 (11b) (Iie)
’ wherein Ar and R® independently represent phenyl or pyridyl which may be unsubstituted or substituted with from 1 to 3 substituents selected from halogen, : alkoxy, nitro and cyano, and pharmaceutically acceptable salts thereof.
24. The combination of claim 1, wherein the bombesin receptor antagonist is (S)- 3-(1H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[4- (4-nitro-phenyl)-oxazol-2-ylamino]-propionamide (also referred to as Compound 2) or a pharmaceutically acceptable salt. 25 The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a pharmaceutically acceptable salt thereof: (S)-3-(1H-indol-3-yl)-N-(1-methoxymethyl-cyclohexylmethyl)-2-methyl-2-[4- (4-nitro-phenyl)-oxazol-2-ylamino]-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-2-[4-(4-nitro-phenyl)-oxazol-2-ylamino]-N-(2- oxo-2-phenyl-ethyl)-propionamide; (S)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[4-(4-nitro- phenyl)-oxazol-2-ylamino]-3-phenyl-propionamide; (S)-2-[4-(4-cyano-phenyl)-oxazol-2-ylamino]-3-(1 H-indol-3-yl)-N-[1-(5- methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-propionamide; (S)-3-(1H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2- methyl- 2-(4-phenyl-oxazol-2-ylamino)-propionamide; (S)-2-(4-ethyl-oxazol-2-ylamino)-3-(1 H-indol-3-yl)-N-[1-(5-methoxy-pyridin- 2-yl)-cyclohexylmethyl]-2-methyl-propionamide; (S)-3-(1H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2- methyl-2-[4-(4-nitro-phenyl)-thiazol-2-ylamino]-propionamide; . (S)-2-(benzooxazol-2-ylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; . (S)-3-(1H-indol-3-yl)-2-methyl-2-(pyridin-4-ylamino)-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide;
(8)-3-(1H-indol-3-yl)-2-(isoquinol-4-ylamino)-2-methyl-N-(1-pyridin-2-yl- ! cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (pyrimidin-5-ylamino)-propionamide; (S)-2-(biphenyl-2-ylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; (8)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-m- tolylamino-propionamide; (5)-3-(1H-indol-3-yl)-2-methyl-2-(6-phenyl-pyridin-2-ylamino)-N-(1-pyridin- 2-yl-cyclohexylmethyl)-propionamide; (R)-3-phenyl-2-phenylamino-N-(1-pyridin-2-yl-cyclohexylmethyl)- propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-2-phenylethylamino-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; (S)-2-[(benzofuran-2-ylmethyl)-amino]-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide, and (S)-3-(1H-indol-3-yl)-2-methyl-2-(4-nitro-benzylamino)-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide.
26. The combination of any of claims 1-14, wherein the bombesin receptor antagonist is a compound of formula (III) or a pharmaceutically acceptable salt thereof: 3 5 4 1 ed : clon R2 Ar 8) R¢ (111) wherein: . 25 eo kis0,1o0r2; eo lisO,1,2o0r3; ’ e misOQorl; e nis0,1o0r2; eo Xis-CO-,-0CO,-SO- and -SO,-;
e Ar is benzimidazolyl, benzofuryl, benzothiadiazolyl, benzothiazolyl, ) benzothienyl, benzopyrazinyl, benzotriazolyl, benzoxadiazolyl, furyl, imidazolyl, indanyl, indolyl, isoquinolyl, isoxazolyl, naphthyl, oxazolyl, phenyl, pyrazinyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidyl, pyrrolyl, quinolinyl, tetralinyl, tetrazolyl, thiazolyl, thienyl or triazolyl each unsubstituted or substituted with from 1 to 3 substituents selected from amino, acetyl, alkyl (straight chain or branched with from 1 to 6 carbon atoms), alkoxy, cyano, halogen, hydroxy, nitro, phenyl, pyridyl, pyrrolyl, isoxazolyl, phenoxy, tolyloxy, - CF, -OCF;, -SO,CF;, -NHCONH,, -CO,H, -CH,CO,H, -CH,CN, SO;Me, SO,NH,, SO,Ph, -(CH;)qNR'R’, -CONR’R", and COR", wherein q is 0, 1 or 2 and R”, R®, R%, R'°, R!' are each independently selected from hydrogen or straight or branched alkyl of up to 6 carbon atoms or cyclic alkyl of between 5 to 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms or R’ and R® or R® and R'® together with the nitrogen atom to which they are linked can form a 5- to 7-membered aliphatic ring which may contain 1 or 2 oxygen or nitrogen atoms; e Ar is independently selected from Ar and can also be pyridyl-N-oxide; e R'is hydrogen or straight or branched alkyl of up to 6 carbon atoms or cyclic alkyl of between 5 and 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms; e R?is independently selected from Ar or is hydrogen, hydroxy, alkoxy, -NMe,, -CONR'?R", foe 0 Loe, AP oN , or LC wherein p is 0, 1 or 2, Ar is phenyl or pyridyl; and, R12 and R13 are each ‘ independently selected from hydrogen, straight or branched alkyl of up to 6 carbon atoms or cyclic alkyl of between 5 and 7 carbon atoms; eR? R*and R’ are each independently selected from hydrogen and lower alkyl; and e RS is hydrogen, methyl or forms with R' a ring of from 3 to 7 carbon atoms which can contain an oxygen or nitrogen atom, or R' and R® can together be carbonyl.
27. The combination of claim 26, wherein the bombesin receptor antagonist is a compound formula (IIT) in which: eo kisOorl; oe lisl, oe misQOorl; e nisOorl; eo Xis-C(O)-, -OC(O)-, or -SO»-; e Ar is benzofuryl, furyl, indolyl, isoquinolyl, naphthyl, phenyl, pyridyl, quinolyl or thienyl each unsubstituted or substituted with 1 or 2 substituents selected from alkoxy, cyano, halogen, nitro, phenyl, phenoxy, -CFs;, -(CHy)gNR'R?, wherein R” and R® can form a ring of between 5 to 7 atoms which may contain 1 or 2 oxygen or nitrogen atoms, or R” and R® can be independently selected from hydrogen, straight or branched alkyl of up to 4 carbon atoms or cyclic alkyl of 5 carbon atoms; e Ar is independently selected from Ar, preferably indolyl, and can also be pyridyl-N-oxide; e R! and R® can form a cyclic alkyl of from 5 to 7 carbon atoms or R' and R® together are carbonyl; e R? is independently selected from unsubstituted or substituted pyridyl or is hydrogen, hydroxy, alkoxy, -NMe;, -CONR'*R" wherein R'? and R"? are each independently selected from H and CH3; e RR? R*and R’ are each independently selected from hydrogen and methyl.
‘ 28. The combination of claim 27, wherein the bombesin receptor antagonist is a compound of Formula (IIT) in which, oe lisl; e mis; e nis; } o R’is 2-pyridyl; e R°formsa cyclohexyl with R'.
29. The combination of any of claims 1-14, wherein the bombesin receptor antagonist 1s a compound of formula (IIIa) or a salt thereof: 0] =~ i Ar - (CH,), -X- eho 28 N (Ila) H wherein Ar, k and X have the meanings given above in first, and the pyridine ring is optionally substituted by with 1 or 2 substituents, R and R’, independently selected from alkoxy, cyano, halogen, nitro, phenyl, phenoxy, - CFs, «(CH,){NR'R?, wherein R’ and R® together with the nitrogen atom to which they are linked can form a 5- to 7-membered aliphatic ring which may contain 1 or 2 oxygen or nitrogen atoms, or R’ and R® can be independently selected from hydrogen or cyclic alkyl of between 5 to 7 carbon atoms, and their pharmaceutically acceptable salts thereof.
30. The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a salt thereof : N-{(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-4-nitro-benzamide; C-dimethylamino-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl}-benzamide;
1H-indole-2-carboxylic acid {(S)-2-(1 H-indol-3-y1)-1-methyl-1-[(1-pyridin-2- ’ yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; benzo[b]thiophene-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl }-amide; N-{(S)-2-(1H-Indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-2-pyrrol-1-yl-benzamide 1 H-indole-5-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; and 1 H-indole-2-carboxylic acid ((S)-2-(1H-indol-3-yl)-1-{[1-(5-methoxy-pyridin- 2-yl)-cyclohexylmethyl]-carbamoyl}-1-methyl-ethyl)-amide.
31. The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a salt thereof N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-4-methyl-benzamide; 4-chloro-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl}-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-4-methoxy-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-4-methanesulfonyl-benzamide; 3-cyano-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl}-benzamide; 3-chloro-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- A cyclohexylmethyl)-carbamoyl}-ethyl}-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- . carbamoyl]-ethyl}-3-methoxy-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-3-methanesulfonyl-benzamide;
dimethylamino-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- : cyclohexylmethyl)-carbamoyl]-ethyl} -benzamide; N-{(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-3-methyl-benzamide; 2-chloro-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl } -benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-2-nitro-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-2-methoxy-benzamide; N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-2-methyl-benzamide; 2-fluoro-N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl } -benzamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2-p- tolyl-ethanoylamino)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2-o- tolyl-ethanoylamino)-propionamide; (S)-2-[2-(4-hydroxy-phenyl)-ethanoylamino]-3-(1 H-indol-3-yl)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (8)-2-[2-(3-hydroxy-phenyl)-ethanoylamino]-3-(1 H-indol-3-yl)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2-m- tolyl-ethanoylamino)-propionamide; (8)-2-[2-(2-fluoro-phenyl)-ethanoylamino]-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2- thiophen-3-yl-ethanoylamino)-propionamide; . pyridine-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide N-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)-
carbamoyl]-ethyl }-isonicotinamide; ’ furan-3-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl } -amide; furan-2-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl- cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 5-methyl-isoxazole-3-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-{(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl } -amide; 1-methyl-1H-pyrrole-2-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1- [(1-pyridin-2-yl-cyclohexylmethyl)-carbamoyl}-ethyl } -amide; thiophene-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl }-amide; thiophene-3-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 1 H-indole-6-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 1H-indole-5-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 1H-indole-4-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl }-amide; 1 H-indole-7-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 1-methyl-1H-indole-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl}-ethyl} -amide; benzothiazole-6-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 1H-benzotriazole-5-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1- . pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 3-methyl-thiophene-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1- ‘ pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; 5-methyl-thiophene-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide;
6-methyl-pyridine-2-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1- } pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; isoquinoline-3-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin- 2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; quinoxaline-2-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin- 2-yl-cyclohexylmethyl)-carbamoyl]-ethyl } -amide; quinoline-8-carboxylic acid {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2- yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; S-phenyl-oxazole-4-carboxylic acid {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1- pyridin-2-yl-cyclohexylmethyl)-carbamoyl]-ethyl}-amide; (S)-3-(1 H-indol-3-yl)-2-[2-(4-methoxy-phenyl)-ethanoylamino]-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-[2-(4-dimethylamino-phenyl)-ethanoylamino]-3-(1 H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-2-[2-(2-nitro-phenyl)-ethanoylamino]-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-[2-(2-methoxy-phenyl)-ethanoylamino]-2-methyl-N- . (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; and N-{(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl}-ethyl}-2-pyrrol-1-yl-benzamide.
32. The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a salt thereof {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid naphthalen-1-ylmethyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- } carbamoyl]-ethyl}-carbamic acid 3,4-dichloro-benzyl ester; {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- . carbamoyl]-ethyl}-carbamic acid 3-nitro-benzyl ester; {(S)-2-(1H-indo!l-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3-trifluoromethyl-benzyl ester;
{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid quinolin-6-ylmethyl ester; {(S)-2-(1 H-indol-3-y1)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4-nitro-benzyl ester; and {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3-cyano-benzyl ester.
33. The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a salt thereof: {(S)-2-(1 H-indol-3-yl)-1-methyi-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3,4-dimethoxy-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid naphthalen-2-ylmethyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid indan-2-yl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-carbamic acid 4-methoxy-benzyl ester; {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4-chloro-benzyl ester; {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2-fluoro-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl }-carbamic acid 2-chloro-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2-methyl-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- ] carbamoyl]-ethyl}-carbamic acid 4-tert-butyl-benzyl ester; {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- . carbamoyl]-ethyl}-carbamic acid 2-methoxy-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4-trifluoromethyl-benzyl ester;
{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- ‘ carbamoyl]-ethyl}-carbamic acid 3-ethoxy-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 2,4-dichloro-benzyl ester; {(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3-methyl-benzyl ester; {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 3-phenoxy-benzyl ester; and {(S)-2-(1H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethyl}-carbamic acid 4-methyl-benzyl ester. 34 The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a salt thereof: (S)-3-(1 H-indol-3-yl)-2-methyl-2-phenylmethanesulfonylamino-N-(1-pyridin- 2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2-chloro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-2-(naphthalene-1-sulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (quinoline-8-sulfonylamino)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2- trifluoromethyl-benzenesulfonylamino)-propionamide; (S)-2-(biphenyl-2-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2- yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-2-(5-methyl-2-phenoxy-benzenesulfonyl- . amino)-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; and (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2-p- : tolyloxy-benzenesulfonylamino)-propionamide.
35. The combination of claim 1, wherein the bombesin receptor antagonist is one of the following compounds or a salt thereof: (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (toluene-4-sulfonylamino)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methanesulfonylamino-2-methyl-N-(1-pyridin-2-yl- cyclohexylmethyl)-propionamide; (S)-2-(2-fluoro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(4-chloro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2,2,2- trifluoro-ethanesulfonylamino)-propionamide; (S)-2-(5-dimethylamino-naphthalene-1-sulfonylamino)-3-(1 H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (5)-3-(1 H-indol-3-yl)-2-methyl-2-(naphthalenc-2-sulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (thiophene-2-sulfonylamino)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-2-(3-nitro-benzenesulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(4-fluoro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (8)-3-(1 H-indol-3-yl)-2-methyl-2-(4-nitro-benzenesulfonylamino)-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(3- trifluoromethyl-benzenesulfonylamino)-propionamide; } (S)-2-(3,4-dichloro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; . (S)-2-(3-fluoro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; !
(8)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(4- trifluoromethyl-benzenesulfonylamino)-propionamide; (S)-2-(5-chloro-thiophene-2-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(3-chloro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2- (toluene-3-sulfonylamino)-propionamide; (8)-2-(3,4-dimethoxy-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(4-cyano-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2-cyano-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (8)-2-(5-chloro-1,3-dimethyl- 1 H-pyrazole-4-sulfonylamino)-3-(1 H-indol-3- y1)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (8)-2-(3,5-dimethyl-isoxazole-4-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(benzo[1,2,5]thiadiazole-4-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (8)-3-(1H-indol-3-yl)-2-methyl-2-(1-methyl-1 H-imidazole-4-sulfonylamino)- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(benzo[1,2,5]oxadiazole-4-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; 3-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1 -pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethylsulfamoyl}-thiophene-2-carboxylic acid methyl ester; (S)-3-(1 H-indol-3-yl)-2-(5-isoxazol-3-yl-thiophene-2-sulfonylamino)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; . (S)-3-(1H-indol-3-yl)-2-methyl-2-(2-nitro-phenylmethanesulfonylamino)- N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide;
(S)-2-(3-cyano-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- : pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(1,2-dimethyl-1 H-imidazole-4-sulfonylamino)-3-(1 H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-(3-methoxy-benzenesulfonylamino)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-methyl-2-(8-nitro-naphthalene-1-sulfonylamino)-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2-chloro-5-nitro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (8)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(2,4,6- trichloro-benzenesulfonylamino)-propionamide; (S)-2-(4-chloro-2-nitro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N- (1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(5-benzenesulfonyl-thiophene-2-sulfonylamino)-3-(1 H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1 -pyridin-2-yl-cyclohexylmethyl)-2-(4- trifluoromethoxy-benzenesulfonylamino)-propionamide; 2-{(S)-2-(1 H-indol-3-yl)-1-methyl-1-[(1-pyridin-2-yl-cyclohexylmethyl)- carbamoyl]-ethylsulfamoyl}-benzoic acid methyl ester; (S)-2-(3-chloro-4-fluoro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (5)-2-(2,5-dichloro-thiophene-3-sulfonylamino)-3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(3-chloro-4-methyl-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1H-indol-3-yl)-2-(2-methoxy-4-methyl-benzenesulfonylamino)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-2-(5- pyridin-2-yl-thiophene-2-sulfonylamino)-propionamide;
(S)-2-(5-bromo-6-chloro-pyridine-3-sulfonylamino)-3-(1 H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2,4-dinitro-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-(4-methanesulfonyl-benzenesulfonylamino)-2-methyl- N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(4-tert-butyl-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2,4-dichloro-5-methyl-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2- methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-2-(2-chloro-5-trifluoromethyl-benzenesulfonylamino)-3-(1 H-indol-3-yl)- 2-methyl-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; (S)-3-(1 H-indol-3-yl)-2-methyl-2-(2-nitro-4-trifluoromethyl-benzenesulfonyl- amino)-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide; and (S)-2-(4-butyl-benzenesulfonylamino)-3-(1 H-indol-3-yl)-2-methyl-N-(1- pyridin-2-yl-cyclohexylmethyl)-propionamide.
36. The combination of any preceding claim, which is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.
37. The combination of any preceding claim, adapted for oral administration.
38. Use of a combination as claimed in any preceding claim in the preparation of a medicament for the treatment or prophylaxis of male sexual dysfunction (more particularly male erectile dysfunction) and/or female sexual dysfunction (more particularly hypoactive sexual desire disorders, sexual arousal disorders, anorgasmic disorders or sexual pain disorders.
39. A pharmaceutical combination (for simultaneous, separate or sequential administration) of a bombesin receptor antagonist and a PDE 5 inhibitor.
: 40. A pharmaceutical combination (for simultaneous, separate or sequential administration) of a bombesin receptor antagonist and a NEP inhibitor.
41. A pharmaceutical combination (for simultaneous, separate or sequential administration) of a bombesin receptor antagonist and one or more estrogen receptor modulators (SERM) and/or estrogen agonists and/or estrogen antagonists.
42. A pharmaceutical combination (for simultaneous, separate or sequential administration) of a bombesin receptor antagonist and lasofoxifene.
43. A method of treating drug induced sexual dysfunction (particularly but not exclusively dysfunction induced by antidepressants) in a male comprising administering to a male suffering therefrom an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist.
44. A method of treating drug induced sexual dysfunction (particularly but not exclusively disfunction induced by antidepressants) in a female comprising administering to a female suffering therefrom an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist.
45. A method for treating erectile dysfunction in a male comprising administering to a male suffering therefrom an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist .
46. A method for treating a female patient suffering from hypoactive sexual desire } disorder comprising administering to the female patient an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist.
47. A method for treating a female patient suffering from sexual arousal disorder ’ and/or orgasmic disorder comprising administering to the female patient an effective amount of a bombesin BB1 antagonist or a mixed BB1/BB2 antagonist.
ZA200303250A 2000-11-17 2003-04-25 Treatment of sexual dysfunction. ZA200303250B (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/GB2000/004380 WO2002040022A1 (en) 2000-11-17 2000-11-17 Treatment of sexual dysfunction using bombesin antagonist

Publications (1)

Publication Number Publication Date
ZA200303250B true ZA200303250B (en) 2004-04-26

Family

ID=9886325

Family Applications (1)

Application Number Title Priority Date Filing Date
ZA200303250A ZA200303250B (en) 2000-11-17 2003-04-25 Treatment of sexual dysfunction.

Country Status (13)

Country Link
EP (1) EP1333829A1 (en)
JP (1) JP2004525864A (en)
KR (1) KR20040062416A (en)
CN (1) CN1479618A (en)
AU (1) AU2001214046A1 (en)
BR (1) BR0017374A (en)
CA (1) CA2426521A1 (en)
HU (1) HUP0303500A2 (en)
IL (1) IL155814A0 (en)
MX (1) MXPA03003481A (en)
PL (1) PL366002A1 (en)
WO (1) WO2002040022A1 (en)
ZA (1) ZA200303250B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10123163A1 (en) 2001-05-09 2003-01-16 Gruenenthal Gmbh Substituted cyclohexane-1,4-diamine derivatives
US7244743B2 (en) 2002-06-05 2007-07-17 Solvay Pharmaceuticals Gmbh Non-peptidic BRS-3 agonists
CN117665174B (en) * 2024-02-01 2024-04-23 巴中市产品质量检验检测中心 Method for detecting PDE5 and SSRI and application thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5369094A (en) * 1990-11-29 1994-11-29 The Administrators Of The Tulane Educational Fund Polypeptide bombesin antagonists
US5650395A (en) * 1995-03-13 1997-07-22 Hurel; Steven Treatment of pulmonary hypertension
TR199900364T2 (en) * 1996-08-22 1999-05-21 Warner-Lambert Company Non-peptide bombesin receptor antagonists.
AU2180600A (en) * 1998-12-18 2000-07-12 Warner-Lambert Company Non-peptide nk1 receptors antagonists
WO2000067708A2 (en) * 1999-05-07 2000-11-16 Board Of Regents, The University Of Texas System Oral steroidal hormone compositions and methods of use
ES2183500T3 (en) * 1999-06-04 2003-03-16 Jordanian Pharmaceutical Mfg A NEW COMPOUNDS AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM.

Also Published As

Publication number Publication date
PL366002A1 (en) 2005-01-24
CA2426521A1 (en) 2002-05-23
JP2004525864A (en) 2004-08-26
KR20040062416A (en) 2004-07-07
EP1333829A1 (en) 2003-08-13
WO2002040022A1 (en) 2002-05-23
IL155814A0 (en) 2003-12-23
MXPA03003481A (en) 2004-09-10
CN1479618A (en) 2004-03-03
BR0017374A (en) 2003-09-30
AU2001214046A1 (en) 2002-05-27
HUP0303500A2 (en) 2004-01-28

Similar Documents

Publication Publication Date Title
EP1333824B1 (en) Treatment of sexual dysfunction with bombesin receptor antagonists
US20020169101A1 (en) Treatment of sexual dysfunction
US20060041014A1 (en) Treatment of male sexual dysfunction
TWI242005B (en) Ortho, ortho-substituted nitrogen-containing bisaryl compounds, processes for their preparation, their use as medicaments, and pharmaceutical preparations comprising them
WO2006027680A1 (en) 3-(1-carbamoylcyclohexyl) propionic acid derivatives as inhibitors of neutral endopeptidase enzyme
US20160297748A1 (en) Compounds, compositions, and methods for modulating ferroptosis and treating excitotoxic disorders
KR20090094374A (en) 1-substituted imidazole derivatives and their use as aldosterone synthase inhibitors
JP2010501573A (en) Condensed imidazole derivatives for the treatment of diseases mediated by aldosterone synthase and / or 11-β-hydroxylase and / or aromatase
CZ20024167A3 (en) Glutaramide derivatives substituted with a cyclopentyl group and functioning as neural endopeptidase inhibitors
JP2008266338A (en) Selective dopamine d3 receptor agonist for treating sexual dysfunction
US7547716B2 (en) Sulfonamide derivatives
US20040087561A1 (en) Treatment of sexual dysfunction
EP1501800A1 (en) Bombesin antagonists
ZA200303250B (en) Treatment of sexual dysfunction.
CA2426089A1 (en) Bombesin receptor antagonists
US20020058606A1 (en) Treatment of sexual dysfunction
CA2431747A1 (en) Treatment of male sexual dysfunction
WO2007071840A2 (en) Compounds based on four aromatic rings, preparation and uses thereof
ZA200303249B (en) Treatment of sexual dysfunction using bombesin antagonist.
EP1258474A2 (en) Alkylamide compounds
US20040185094A1 (en) Pharmaceutical
ZA200300120B (en) Treatment of Male sexual dysfunction.
ZA200304460B (en) Treatment of male sexual dysfunction.