Substituted 4-benzyloxy-benzoic acid amide derivatives

The present invention relates to novel substituted 4-benzyloxy-benzoic acid amide derivatives, processes for their preparation, and their use in medicaments, especially for the prophylaxis and treatment of diseases associated with Cold Menthol Receptor 1 (CMR-I) activity, in particular for the treatment of urological diseases or disorders, such as detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor overactivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms; chronic pain, neuropathic pain, postoperative pain, rheumatoid arthritic pain, neuralgia, neuropathies, algesia, nerve injury, ischaemia, neurodegeneration, stroke, and inflammatory disorders such as asthma and chronic obstructive pulmonary (or airways) disease (COPD).

There is abundant direct or indirect evidence that shows the relation between Transient Receptor Potential (TRP) channel activity and diseases such as pain, ischaemia, and inflammatory disorders. Further, it has been demonstrated that TRP channels transduce reflex signals that are involved in the overactive bladder of patients who have damaged or abnormal spinal reflex pathways [De Groat WC: A neurologic basis for the overactive bladder. Urology 50 (6A Suppl): 36-52, 1997]. CMR-I, a nonselective cation channel is such a member of the TRP channel family (TRPM8). Recently, in 2002 the receptor was cloned and it was found to be sensitive to cold temperature and menthol and therefore named as cold menthol receptor - 1 (CMR-I) (McKemy et al, 2002; Peier et al., 2002). This receptor which is activated by 8 — 28°C temperature is expressed on the bladder urothelium and DRG (Dorsal Root Ganglia) and C-fibers. The intravesical ice water or menthol also induce C-fiber mediated spinal micturition reflex in patients with urgency and urinary incontinence (UI). Clinically CMR-I is supposed to mediate the bladder cooling reflex seen after ice water test in overactive patients.

Therefore antagonism of the CMR-I receptor leads to the blockage of neurotransmitter release, resulting in prophylaxis and treatment of the conditions and diseases associated with CMR-I activity.

Antagonists of the CMR-I receptor can be used for prophylaxis and treatment of the conditions and diseases including chronic pain, neuropathic pain, postoperative pain, rheumatoid arthritic pain, neuralgia, neuropathies, algesia, nerve injury, ischaemia, neurodegeneration, stroke, inflammatory disorders, urinary incontinence (UI) such as urge urinary incontinence (UUI), and/or overactive bladder, Lower urinary tract symptoms secondary to or independent of benign prostatic hyperplasia.

UI is the involuntary loss of urine. UUI is one of the most common types of UI together with stress urinary incontinence (SUI) which is usually caused by a defect in the urethral closure mechanism. UUI is often associated with neurological disorders or diseases causing neuronal damages such as dementia, Parkinson's disease, multiple sclerosis, stroke and diabetes, although it also occurs in individuals with no such disorders. One of the usual causes of UUI is overactive bladder (OAB) which is a medical condition referring to the symptoms of frequency and urgency derived from abnormal contractions and instability of the detrusor muscle.

There are several medications for urinary incontinence on the market today mainly to help treating UUI. Therapy for OAB is focused on drugs that affect peripheral neural control mechanisms or those that act directly on bladder detrusor smooth muscle contraction, with a major emphasis on development of anticholinergic agents. These agents can inhibit the parasympathetic nerves which control bladder voiding or can exert a direct spasmolytic effect on the detrusor muscle of the bladder. This results in a decrease in intravesicular pressure, an increase in capacity and a reduction in the frequency of bladder contraction. Orally active anticholinergic drugs which are commonly prescribed have serious drawbacks such as unacceptable side effects such as dry mouth, abnormal visions, constipation, and central nervous system disturbances. These side effects lead to poor compliance. Dry mouth symptoms alone are responsible for a 70% non-compliance rate with oxybutynin. The inadequacies of present therapies highlight the need for novel, efficacious, safe, orally available drugs that have fewer side effects.

In WO 03/037865 and Y. Lu, et al., Bioorg. Med. Chem. Lett. 2004, 14, 3957-3962 related benzyloxy-phenylamide derivatives for the treatment of cancer are described.

The present invention relates to compounds of the general formula (I)

wherein

R ¹ represents hydrogen, halogen or trifluoromethyl,

R ² represents hydrogen or halogen,

R ³ represents hydrogen or halogen,

R ⁴ represents hydrogen, chlorine, fluorine, nitro, trifluoromethyl, trifluoromethoxy, formyl, Ci-C ₄ -alkyl or C _r C ₄ -alkoxy,

R ⁵ represents hydrogen or halogen,

R ⁶ represents hydrogen or Ci-C ₄ -alkyl,

R ⁷ represents C ₃ -C ₈ -alkyl, C ₃ -C ₈ -alkenyl, C ₃ -Cio-cycloalkyl, C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl or diphenylmethyl,

wherein cycloalkyl can be further substituted with one to three identical or different radicals selected from the group consisting of Ci-C ₄ -alkyl,

and

wherein aryl and heteroaryl can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, nitro, amino, hydroxy, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, Ci-Cβ-alkyl, Cj-Cβ-alkoxy, Ci-C ₆ -alkoxy-Ci-C ₆ -alkyl, Ci-Cβ-alkylthio, C _r C ₆ -alkylamino, phenoxy, benzyloxy, phenylthio, benzylthio, phenylcarbonyl, 5- or 6-membered heteroarylmethoxy, Ci-Cβ-alkoxycarbonyl and phenyl,

wherein phenoxy, benzyloxy, phenylthio, benzylthio and phenyl can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, nitro, amino, hydroxy, trifluoro- methyl, trifluoromethoxy, Ci-Cβ-alkyl, C _r C ₆ -alkoxy and Ci-C ₆ -alkyl- amino,

R ⁸ represents hydrogen or Ci-C ₄ -alkyl,

R ⁹ represents Ci-Cβ-alkyl,

wherein alkyl is further substituted with one radical selected from the group consisting of amino, mono-alkylamino, Crd-alkylcarbonylamino or C]-C ₄ -alkoxycarbonylamino,

R ¹⁰ represents hydrogen or Ci-C ₄ -alkyl,

and their salts, hydrates and/or solvates.

Physiologically acceptable salts are preferred in the context of the present invention.

Physiologically acceptable salts according to the invention are non-toxic salts which in general are accessible by reaction of the compounds (I) with an inorganic or organic base or acid conventionally used for this purpose. Non-limiting examples of pharmaceutically acceptable salts of compounds (I) include the alkali metal salts, e.g. lithium, potassium and sodium salts, the alkaline earth metal salts such as magnesium and calcium salts, the quaternary ammonium salts such as, for example, triethyl ammonium salts, acetates, benzene sulphonates, benzoates, dicarbonates, disulphates, ditartrates, borates, bromides, carbonates, chlorides, citrates, dihydrochlorides, fumarates, gluconates, glutamates, hexyl resorcinates, hydrobromides, hydrochlorides, hydroxy- naphthoates, iodides, isothionates, lactates, laurates, malates, maleates, mandelates, mesylates, methylbromides, methylnitrates, methylsulphates, nitrates, oleates, oxalates, palmitates, pantothenates, phosphates, diphosphates, polygalacturonates, salicylates, stearates, sulphates, succinates, tartrates, tosylates, valerates, and other salts used for medicinal purposes.

Hydrates of the compounds of the invention or their salts are stoichiometric compositions of the compounds with water, such as for example hemi-, mono-, or dihydrates.

Solvates of the compounds of the invention or their salts are stoichiometric compositions of the compounds with solvents.

The present invention includes both the individual enantiomers or diastereomers and the corresponding racemates or diastereomeric mixtures of the compounds according to the invention and their respective salts. In addition, all possible tautomeric forms of the compounds described above are included according to the present invention. The diastereomeric mixtures can be separated into the individual isomers by chromatographic processes. The racemates can be resolved into the respective enantiomers either by chromatographic processes on chiral phases or by resolution.

In the context of the present invention, the substituents, if not stated otherwise, in general have the following meaning:

Alkyl in general represents a straight-chain or branched saturated hydrocarbon radical having 1 to 6, preferably 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, iso- propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl. The same applies to radicals such as alkoxy, alkylamino, alkylcarbonylamino, alkoxycarbonylamino, alkoxy-alkyl and the like.

Alkandiyl in general represents a straight-chain or branched saturated alkandiyl radical having 1 to 4 carbon atoms. Non-limiting examples include methylen, ethan-l,2-diyl, ethan-l,l-diyl, propan-

1,3-diyl, propan-l,2-diyl, propan-2,2-diyl, butan-l,4-diyl, butan-l,3-diyl and butan-2,4-diyl.

Alkenyl in general represents a straight-chain or branched alkenyl radical having 2 to 6, preferably 2 to 4 carbon atoms. Non-limiting examples include vinyl, allyl, n-prop-1-en-l-yl, n-but-2-en-l-yl, 2-methylprop-l-en-l-yl and 2-methylprop-2-en-l-yl.

Alkinyl in general represents a straight-chain or branched alkinyl radical having 2 to 6, preferably 2 to 4 carbon atoms. Non-limiting examples include ethinyl, propargyl (2-propinyl), 1-propinyl, but- 1-inyl, but-2-inyl.

Alkoxy illustratively and preferably represents methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

Alkoxy-alkyl illustratively and preferably represents methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, n-propoxymethyl, n-propoxyethyl, isopropoxymethyl, isopropoxyethyl, terf-butoxymethyl, n-pentoxymethyl and n-hexoxymethyl.

Alkylcarbonylamino in general represents a straight-chain or branched hydrocarbon radical having 1 to 6, preferably 1 to 4 carbon atoms which has a carbonylamino (-CO-NH-) function at the position of attachment and which is bonded to the carbonyl group. Non-limiting examples include formylamino, acetylamino, n-propionylamino, n-butyrylamino, isobutyrylamino, pivaloylamino, n- hexanoylamino.

Alkoxycarbonylamino illustratively and preferably represents methoxycarbonylamino, ethoxy- carbonylamino, n-propoxycarbonylamino, isopropoxycarbonylamino, terf-butoxycarbonylamino, n-pentoxycarbonylamino and n-hexoxycarbonylamino.

Alkylamino represents an alkylamino radical having one or two (independently selected) alkyl substituents, illustratively and preferably representing methylamino, ethylamino, n-propylamino, isopropylamino, ter/-butylamino, n-pentylamino, n-hexylamino, NN-dimethylamino, N,N-diethyl- amino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino, N-tert- butyl-N-methylamino, N-ethyl-N-n-pentylamino and N-n-hexyl-N-methylamino.

Mono-alkylamino represents an alkylamino radical having one alkyl substituents, illustratively and preferably representing methylamino, ethylamino, n-propylamino, isopropylamino, terf-butylamino, n-pentylamino and n-hexylamino.

Cycloalkyl in general represents a cyclic saturated hydrocarbon radical having 3 to 8, preferably 3 to 6 carbon atoms. νon-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclo- hexyl and cycloheptyl.

Aryl in general represents an aromatic mono- or bicyclic radical having 6 to 10 ring atoms, illustratively and preferably representing phenyl and naphthyl.

Heteroaryl per se and in heteroarylmethyl in general represents an aromatic mono- or bicyclic radical having 5 to 10 and preferably 5 or 6 ring atoms, and up to 5 and preferably up to 4 hetero- atoms selected from the group consisting of S, O and N, illustratively- and preferably representing thienyl, furyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidyl, pyridazinyl, indolyl, indazolyl, benzofuranyl, benzo- thienyl, benzothiazolyl, quinolinyl, isoquinolinyl.

Halogen represents fluorine, chlorine, bromine and iodine.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein

R ¹ represents hydrogen or halogen,

R ² represents hydrogen or halogen,

R ³ represents hydrogen,

R ⁴ represents chlorine, nitro, trifluoromethoxy, C _r C ₃ -alkyl or C _r C ₃ -alkoxy,

R ⁵ represents hydrogen,

R ⁶ represents hydrogen,

R ⁷ represents C ₃ -C ₈ -alkyl, C ₃ -C ₇ -cycloalkyI, C ₆ -Ci ₀ -aryl or 5- to 10-membered heteroaryl,

wherein cycloalkyl can be further substituted with one to three identical or different radicals selected from the group consisting of Ci-C ₄ -alkyl,

and

wherein aryl and heteroaryl can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, nitro, amino, hydroxy, trifluoromethyl, trifluoromethoxy, trifiuoromethylthio, C _r C ₆ -alkyl, Ci-C ₆ -alkoxy, Ci-C ₆ -alkylthio, Ci-Cβ-alkylamino, phenoxy, benzyloxy, phenylthio, benzylthio, Ci-Cβ-alkoxycarbonyl and phenyl,

wherein phenoxy, benzyloxy, phenylthio, benzylthio and phenyl can be further substituted with one to three identical or different radicals selected

from the group consisting of halogen, nitro, amino, hydroxy, trifluoromethyl, trifluoromethoxy, Ci-C ₆ -alkyl, CpCβ-alkoxy and C]-C ₆ - alkylamino,

R ⁸ represents hydrogen,

R ⁹ represents d-C ₃ -alkyl,

wherein alkyl is further substituted with one radical selected from the group consisting of amino, mono-alkylamino or Ci-C ₄ -alkoxycarbonylamino,

R ¹⁰ represents hydrogen,

and their salts, hydrates and/or solvates.

In another particularly preferred embodiment, the present invention relates to compounds of general formula (I), wherein

R ¹ represents hydrogen, fluorine or chlorine,

R ² represents hydrogen or fluorine,

R ³ represents hydrogen,

R ⁴ represents methoxy,

R ⁵ represents hydrogen,

R ⁶ represents hydrogen,

R ⁷ represents C ₃ -C ₆ -alkyl, cyclohexyl, cyclopentyl, phenyl, naphthyl, pyridyl, pyrimidyl, thienyl or furyl,

wherein phenyl, naphthyl, pyridyl, pyrimidyl, thienyl and furyl can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, nitro, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, Ci-G»-alkyl, methoxy, ethoxy, methylthio, ethylthio, phenoxy, benzyloxy, phenylthio, ben2ylthio, methoxycarbonyl and phenyl,

wherein phenoxy, benzyloxy, phenylthio, benzylthio and phenyl can be further substituted with one to three identical or different radicals selected

from the group consisting of halogen, nitro, trifluoromethyl, trifluoromethoxy, Ci-C ₄ -alkyl, methoxy and ethoxy,

R ⁸ represents hydrogen,

R ⁹ represents Ci-C ₂ -alkyl,

wherein alkyl is further substituted with one radical selected from the group consisting of amino or terf-butoxycarbonylamino,

R ¹⁰ represents hydrogen,

and their salts, hydrates and/or solvates.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ¹ , R ² and R ³ represent hydrogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ¹ represents halogen, R ² represents hydrogen or halogen and R ³ represents hydrogen or halogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ¹ represents halogen, R ² represents hydrogen or halogen and R ³ represents hydrogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ¹ represents fluorine or chlorine, R ² represents hydrogen or fluorine and R represents hydrogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ⁴ represents trifluoromethoxy or C _r C ₄ -alkoxy.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ⁵ represents hydrogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ⁶ represents hydrogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ⁷ represents phenyl, wherein phenyl can be further substituted with one to three identical or different radicals selected from the group consisting of fluorine, chlorine, nitro, trifluoromethyl, methyl and methoxy.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ⁸ represents hydrogen.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ⁹ represents -CH ₂ NH ₂ or -CH ₂ CH ₂ NH ₂ .

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R ¹⁰ represents hydrogen.

Very particular preference is given to combinations of two or more of the abovementioned preference ranges.

The compounds of general formula (I) can be synthesized by

[A] condensing compounds of general formula (II)

wherein R , R , R and R have the meaning indicated above,

with compounds of general formula (III)

wherein R , R , R , R , R and R have the meaning indicated above, and

X ¹ represents a leaving group, such as halogen, preferably chlorine or bromine, or hydroxy,

in the presence of a base

or

[B] condensing compounds of general formula (IV)

wherein R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ have the meaning indicated above,

with compounds of general formula (V)

wherein R ¹ , R ² , R ³ and R ¹⁰ have the meaning indicated above, and

X ² represents a leaving group, such as halogen, preferably chlorine or bromine,

in the presence of a base.

Amino groups in R ⁹ of compounds of general formula (D) are protected with acid labile groups, preferred is a boc-group. After the synthesis of compounds of general formula (I) this acid labile group can be cleaved via standard procedures known by a person skilled in the art. Compounds of general formula (I) are obtained. Preferred are acidic cleavage conditions.

If a salt of a compound of general formula (I), for example a hydrochloride or trifluoroacetate, is isolated the free base can be obtained by reversed phase chromatography of the salt using a mixture of acetonitile and water as eluent in the presence of a base. Preferably a RP 18 Phenomenex Luna C 18(2) column is used in the presence of diethylamine as base. Or the free base of a compound of general formula (I) can be obtained by neutralizing with a base and extraction.

The process [A] is in general carried out in a temperature range from -20 ⁰ C to boiling point of the solvent, preferably from 0 ⁰ C to +4O ⁰ C.

The process is generally carried out at normal pressure. However, it is also possible to carry it out at elevated pressure or at reduced pressure (for example in a range from 0.5 to 5 bar).

Suitable solvents for the process are ethers such as dioxan or tetrahydrofuran, or halogeno-hydro- carbons such as dichloromethane, dichloroethane or trichloromethane, or other solvents such as dimethylformamide, ethyl acetate or acetonitrile. It is also possible to use mixtures of the above- mentioned solvents. Preferred for the process is tetrahydrofuran or dichloromethane.

Suitable bases for the process are generally inorganic or organic bases. These preferably include alkali carbonates such as sodium or potassium carbonate or hydrogen carbonate, cyclic amines such as, for example, N-methylmorpholine, N-methylpiperidine, pyridine or 4-N,N-dimethylamino- pyridine, or (Ci-C ₄ )-trialkylamines such as, for example, triethylamine or diisopropylethylamine. Preference is given to triethylamine.

If X ¹ is hydroxy, a coupling agent is added to the reaction mixture such as a carbodiimide, for example ν,ν'-diethyl-, N,N,'-dipropyl-, N,N'-diisopropyl-, NjN'-dicyclohexylcarbodiimide, N-(3-di- methylaminoisopropyl)-N'-ethylcarbodiimide-hydrochloride (EDC), N-cyclohexylcarbodiimide- N'-propyloxymethyl-polystyrene (PS-carbodiimide) or O-(benzotriazol-l-yl)-N,N,N',N'-tetra- methyluronium-hexafluorophosphate (HBTU), 2-(2-oxo-l -(2H)-pyridyl)- 1 , 1 ,3,3-tetramethyl- uroniumtetrafluoroborate (TPTU) or O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyl-uronium- hexafluorophosphate (HATU), or 1-hydroxybenztriazole (HOBt), or benzotriazol-1- yloxytris(dimethylamino)-phosphoniumhexafluorophosphate (BOP), or mixtures of these reagents.

The process [B] is in general carried out in a temperature range from 0 ⁰ C to boiling point of the solvent, preferably from 2O ⁰ C to boiling point of the solvent.

The process is generally carried out at normal pressure. However, it is also possible to carry it out at elevated pressure or at reduced pressure (for example in a range from 0.5 to 5 bar).

Optionally an alkali iodide such as sodium or potassium iodide can be added to the reaction mixture.

Suitable solvents for the process are ethers such as dioxan or tetrahydrofuran, or halogeno-hydro- carbons such as dichloromethane, dichloroethane or trichloromethane, or other solvents such as dimethylformamide, dimethylsulfoxide, ethyl acetate or acetonitrile. It is also possible to use mixtures of the above-mentioned solvents. Preferred for the process is acetonitrile.

Suitable bases for the process are generally inorganic or organic bases. These preferably include alkali carbonates such as sodium or potassium carbonate or hydrogen carbonate, cyclic amines such as, for example, N-methylmorpholine, N-methylpiperidine, pyridine or 4-N,N-dimethylamino- pyridine, or (Ci-C ₄ )-trialkylamines such as, for example, triethylamine or diisopropylethylamine. Preference is given to potassium carbonate.

The compounds of the general formulas (ID) and (V) are known per se, or they can be prepared by customary methods.

The compounds of general formula (II) can be synthesized by condensing compounds of general formula (VI)

wherein R ⁶ and R ⁷ have the meaning indicated above,

with compounds of general formula (VII)

wherein R ⁸ and R ⁹ have the meaning indicated above,

under conditions of a reductive amination.

The process is in general carried out in a temperature range from -20 ⁰ C to boiling point of the solvent, preferably from 0°C to +40 ⁰ C.

The process is generally carried out at normal pressure. However, it is also possible to carry it out at elevated pressure or at reduced pressure (for example in a range from 0.5 to 5 bar).

Suitable solvents for the process are halogeno-hydrocarbons such as dichloromethane, dichloro- ethane or trichloromethane, or alcohols such as methanol, ethanol, n-propanol, iso-propanol, n- butanol or ferf-butanol, or a mixture of alcohol and water. Preferred for the process is methanol or a mixture of methanol and water.

Suitable reducing agents for the process are sodium borohydride or triacetoxyborohydride.

The compounds of the general formulas (VI) and (VII) are known per se, or they can be prepared by customary methods.

The compounds of general formula (TV) can be synthesized by treating compounds of general formula (Ia)

wherein R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ have the meaning indicated above, under reducing conditions.

The process is in general carried out in a temperature range from -20 ⁰ C to boiling point of the solvent, preferably from 0 ⁰ C to +40 ⁰ C.

The process is generally carried out at normal pressure. However, it is also possible to carry it out at elevated pressure or at reduced pressure (for example in a range from 0.5 to 5 bar).

Suitable solvents for the process are alcohols such as methanol, ethanol, n-propanol, iso-propanol, n-butanol or tert-butano\, or tetrahydrofuran, or a mixture of alcohol and water. Preferred for the process is methanol, ethanol, tetrahydrofuran or a mixture of ethanol and water.

Suitable reducing agents for the process are metals such as palladium, platin, nickel or ruthenium or oxides thereof in the presence of hydrogen such as palladium on charcoal and hydrogen. Preferred for the process is palladium on charcoal and hydrogen.

The compounds of general formula (Ia) can be synthesized via process [A] or [B].

The above-mentioned process can be illustrated by the following scheme:

Scheme 1

sodium borohydride

triethylamine

isolation as free base or salt (TFA or HCI)

isolation as free base or salt (TFA or HCI)

lternatively above mentioned process can be conducted on solid support using polymer bound diamines. Initially the diamines are attached to the resin via an acid labile linkage. In the final step of the synthesis the products are released from the solid support. The following scheme illustrates the process on solid phase:

Scheme 2

The compounds according to the invention exhibit an unforeseeable, useful pharmacological activity spectrum. They are therefore suitable for use as medicaments for the treatment and/or prophylaxis of disorders in humans and animals.

Surprisingly, the compounds of the present invention show excellent CMR-I antagonistic activity. They are, therefore suitable especially for the prophylaxis and treatment of diseases associated with CMR-I activity, in particular for the treatment of urological diseases or disorders, such as detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor oeractivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms.

The compounds of the present invention are also effective for treating or preventing a disease selected from the group consisting of chronic pain, neuropathic pain, postoperative pain, rheumatoid arthritic pain, neuralgia, neuropathies, algesia, nerve injury, ischaemia, neuro- degeneration and/or stroke, as well as respiratory diseases and inflammatory diseases such as asthma, COPD and allergic rhinitis since the diseases also relate to CMR-I activity.

The compounds of the present invention are also useful for the treatment and prophylaxis of neuropathic pain, which is a form of pain often associated with herpes zoster and post-herpetic neuralgia, painful diabetic neuropathy, neuropathic low back pain, posttraumatic and postoperative neuralgia, neuralgia due to nerve compression and other neuralgias, phantom pain, complex regional pain syndromes,χinfectious or parainfectious neuropathies like those associated with HTV

infection, pain associated with central nervous system disorders like multiple sclerosis or Parkinson disease or spinal cord injury or traumatic brain injury, and post-stroke pain.

Furthermore, the compounds of the present invention are useful for the treatment of musculoskeletal pain, forms of pain often associated with osteoarthritis or rheumatoid arthritis or other forms of arthritis, and back pain.

In addition, the compounds of the present invention are useful for the treatment of pain associated with cancer, including visceral or neuropathic pain associated with cancer or cancer treatment.

The compounds of the present invention are furthermore useful for the treatment of visceral pain, e.g. pain associated with obstruction of hollow viscus like gallstone colik, pain associated with irritable bowel syndrome, pelvic pain, vulvodynia, orchialgia or prostatodynia, pain associated with inflammatory lesions of joints, skin, muscles or nerves, and orofascial pain and headache, e.g. migraine or tension-type headache.

The present invention further provides medicaments containing at least one compound according to the invention, preferably together with one or more pharmacologically safe excipient or carrier substances, and also their use for the above-mentioned purposes.

The active component can act systemically and/or locally. For this purpose, it can be applied in a suitable manner, for example orally, parenterally, pulmonally, nasally, sublingually, lingually, buccally, rectally, transdermally, conjunctivally, otically or as an implant.

For these application routes, the active component can be administered in suitable application forms.

Useful oral application forms include application forms which release the active component rapidly and/or in modified form, such as for example tablets (non-coated and coated tablets, for example with an enteric coating), capsules, sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, solutions and aerosols.

Parenteral application can be carried out with avoidance of an absorption step (intravenously, intraarterially, intracardially, intraspinally or intralumbarly) or with inclusion of an absorption (intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). Useful parenteral application forms include injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates and sterile powders.

Forms suitable for other application routes include for example inhalatory pharmaceutical forms (including powder inhalers, nebulizers), nasal drops/solutions, sprays; tablets or capsules to be

administered lingually, sublingually or buccally, suppositories, ear and eye preparations, vaginal capsules, aqueous suspensions (lotions, shake mixtures), lipophilic suspensions, ointments, creams, milk, pastes, dusting powders or implants.

The active components can be converted into the recited application forms in a manner known per se. This is carried out using inert non-toxic, pharmaceutically suitable excipients. These include inter alia carriers (for example microcrystalline cellulose), solvents (for example liquid polyethylene glycols), emulsifiers (for example sodium dodecyl sulphate), dispersing agents (for example polyvinylpyrrolidone), synthetic and natural biopolymers (for example albumin), stabilizers (for example antioxidants such as ascorbic acid), colorants (for example inorganic pigments such as iron oxides) or taste and/or odor corrigents.

For human use, in the case of oral administration, it is recommendable to administer doses of from 0.001 to 50 mg/kg, preferably of 0.01 mg/kg to 20 mg/kg. In the case of parenteral administration, such as, for example, intravenously or via mucous membranes nasally, buccally or inhalationally, it is recommendable to use doses of 0.001 mg/kg to 0.5 mg/kg.

In spite of this, it can be necessary in certain circumstances to depart from the amounts mentioned, namely as a function of body weight, application route, individual behaviour towards the active component, manner of preparation and time or interval at which application takes place. It can for instance be sufficient in some cases to use less than the aforementioned minimum amount, while in other cases the upper limit mentioned will have to be exceeded. In the case of the application of larger amounts, it can be advisable to divide them into a plurality of individual doses spread through the day.

The percentages in the tests and examples which follows are, unless otherwise stated, by weight; parts are by weight. Solvent ratios, dilution ratios and concentrations reported for liquid/liquid solutions are each based on the volume.

A. Examples

Abbreviations;

aq. aqueous boc tert-butoxycarbonyl

CDCl ₃ deutero chloroform cone. concentrated

DCI direct chemical ionisation (for MS)

DIC diisopropylcarbodiimid

DIEA diisopropylethylamine (Hϋnig's base)

DMAP 4-N,N-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

EI electron impact ionisation (for MS)

ESI electro-spray ionisation (for MS) h hour(s)

HBTU 2-( 1 H-benzotriazole-1 -yl)-l , 1 ,3,3-tetramethyluronium hexafluorophosphate

HOBT hydroxybenzotriazole

HPLC high pressure liquid chromatography

LC-MS liquid chromatography coupled with mass spectroscopy min minute(s)

Mp. melting point

MS mass spectroscopy

NMR nuclear magnetic resonance spectroscopy ofth. of theoretical (yield)

RP reverse phase (for HPLC) rt room temperature

R. retention time (for HPLC) sat. saturated

TBABH tetrabutylammonium borohydride

TMOF trimehtylorthoformiate

TFA trifluoroacetic acid

THF tetrahydrofuran

LC-MS / HPLC methods;

method 1 (HPLQ: Instrument: HP 1100 with DAD-detection, column: Kromasil 100 RP-18, 60 mm x 2.1 mm, 3.5 μm; eluent A: 5 ml HClO ₄ (70 %) /1 water, eluent B: acetonitrile; gradient: 0 min 2% B -» 0.5 min 2% B -> 4.5 min 90% B -> 6.5 min 90% B -> 6.7 min. 2% B -> 7.5 min 2 % B; flow: 0.75 ml/min; column temperature: 3O ⁰ C; UV detection: 210 nm.

method 2 (LC-MS1: Instrument MS: Micromass Quattro LCZ; Instrument HPLC: HP 1100 Series; UV DAD; column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; eluent A: 1 1 water + 0.5 ml 50% formic acid, eluent B: 1 1 acetonitrile + 0.5 ml 50% formic acid; gradient: 0.0 min 90% A → 2.5 min 30% A → 3.0 min 5% A → 4.5 min 5% A; flow: 0.0 min 1 ml/min → 2.5 min/3.0 min/4.5 min 2 ml/min; oven: 50 ⁰ C; UV detection: 210 nm.

method 3 CLC-MSt: Instrument MS: Micromass ZQ; Instrument HPLC: Waters Alliance 2795; column: Merck Chromolith SpeedROD RP-18e 50 mm x 4.6 mm; eluent A: 1 1 water + 0.5 ml 50% formic acid; eluent B: 1 1 acetonitrile + 0.5 ml 50% formic acid; gradient: 0.0 min 10% B-> 3.0 min 95% B-> 4.0 min 95% B; flow: 0.0 min 1.0 ml/min-» 3.0 min 3.0 ml/min-> 4.0 min 3.0 ml/min; oven: 35°C; UV detection: 210 nm.

method 4 (LC-MS): Instrument MS: Micromass ZQ; Instrument HPLC: Waters Alliance 2795; column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; eluent A: 1 1 water + 0.5 ml 50% formic acid, eluent B: 1 1 acetonitrile + 0.5 ml 50% formic acid; gradient: 0.0 min 90% A -» 2.5 min 30% A → 3.0 min 5% A → 4.5 min 5% A; flow: 0.0 min 1 ml/min → 2.5 min/3.0 min/4.5 min 2 ml/min; oven: 50 ⁰ C; UV detection: 210 nm.

method 5 (LC-MSV Instrument: Micromass Quattro LCZ; Instrument HPLC: Agilent Serie 1100; column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm x 4 mm; eluent A: 1 1 water + 0.5 ml 50% formic acid, eluent B: 1 1 acetonitrile + 0.5 ml 50% formic acid; gradient: 0.0 min 90% A -> 2.5 min 30% A -> 3.0 min 5% A -> 4.5 min 5% A; flow: 0.0 min 1 ml/min, 2.5 min/3.0 min/4.5 min 2 ml/min; oven: 50 ⁰ C; UV detection: 208- 400 nm.

method 6 (HPLQ: Instrument: HP 1100 with DAD-detection; column: Kromasil 100 RP-18, 60 mm x 2.1 mm, 3.5 μm; eluent A: 5 ml HClO ₄ (70 %) /1 water, eluent B: acetonitrile; gradient: 0 min 2% B -» 0.5 min 2% B ^ 4.5 min 90% B -* 9 min 90% B -> 9.2 min. 2% B -> 10 min 2 % B; flow: 0.75 ml/min; column temperature: 30 ⁰ C; UV detection: 210 nm.

method 7 (LC-MS): instrument MS: Micromass LCZ; instrument HPLC Agilent Serie 1100; column: Thermo Hypersil GOLD 3μ 20 mm x 4 mm; eluent A: 1 1 water + 0.5 ml 50% formic acid, eluent B: 1 1 acetonitrile + 0.5 ml 50% formic acid; gradient: 0.0 min 100% A -> 0.2 min 100% A

-> 2.9 min 30% A -> 3.1 min 10% A -> 5.5 min 10% A; oven: 50 ⁰ C; flow: 0.8 ml/min; UV- Detection: 210 nm.

method 8 (LC-MS): Instrument MS: Micromass TOF (LCT); Instrument HPLC: 2-column-method, Waters2690; column: YMC-ODS-AQ, 50 mm x 4.6 mm, 3.0 μm; eluent A: water + 0.1% formic acid, eluent B: acetonitrile + 0.1% formic acid; gradient: 0.0 min 100% A -> 0.2 min 95% A -> 1.8 min 25% A -> 1.9 min 10% A -> 2.0 min 5% A -> 3.2 min 5% A; flow: 3.0 ml/min; oven: 40 ⁰ C; UV detection: 210 nm.

Starting Materials and Intermediates:

Example IA

tert-butyl {2-[(2-thienylmethyl)amino]ethyl} carbamate

5 g (43.7 mmol) 2-thiophencarboxaldehyde and 7.7 g (48.1 mmol) tert-butyl (2-aminoethyl)- carbamate are dissolved in 100 ml methanol and stirred for 2 h at room temperature. The solution is cooled to 0 ⁰ C and 8.27 g (218.5 mmol) sodium borohydride are carefully added. Water is added until a clear solution is formed and the mixture is stirred at room temperature overnight. The mixture is concentrated under vacuum, the residue is diluted with dichloromethane and the organic phase is washed with brine, dried over magnesium sulfate and concentrated under vacuum. The crude is purified by column chromatography over silicagel (cyclohexane/ethyl acetate 7/3) to yield 6.7 g (62% of th.) of the title compound.

HPLC (method 1): R, = 3.59 min; MS (ESIpos): m/z = 257 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ = 7.30 (m, IH), 6.90 (m, 2H), 6.70 (m, IH), 3.80 (s, 2H), 3.00 (m, 2H), 2.55 (m, 2H), 2.20 (bs, IH), 1.40 (s, 9H)

Example 2A

tert-butyl {2-[(4-chlorobenzyl)amino]ethyl}carbamate

5 g (35.6 mmol) 4-chlorobenzaldehyde and 6.3 g (39.2 mmol) tert-butyl (2-aminoethyl)carbamate are dissolved in 120 ml methanol and stirred for 2 h at room temperature. The solution is cooled to 0 ⁰ C and 6.73 g (177.9 mmol) sodium borohydride are carefully added. Water is added until a clear solution is formed and the mixture is stirred at room temperature overnight. The mixture is concentrated under vacuum, the residue is diluted with dichloromethane and the organic phase is washed with brine, dried over magnesium sulfate and concentrated under vacuum. The crude

product is re-crystallized from petroleum ether to yield 5.8 g (57% of th.) of the title compound as a solid.

HPLC (method 1): R, = 3.97 min; MS (ESIpos): m/z = 285 (M+H) ⁺

¹ H-NMR (300 MHz, DMSO-d ₆ ): δ = 7.35 (m, 4H), 6.70 (m, IH), 3.60 (s, 2H), 3.00 (m, 2H), 2.55 (m, 2H), 2.20 (bs, 1 H), 1.40 (s, 9H).

Example 3A

tert-butyl [2-(benzylamino)ethyl]carbamate

2.0 g (18.4 mmol) benzaldehyde and 3.32 g (20.7 mmol) N-Boc-ethylendiamine are dissolved in 25 ml methanol. The mixture is stirred 2 h at rt before cooled to 0 ⁰ C and 3.57 g (94.2 mmol) sodium borohydride and water are added to generate a solution, which is stirred over night at rt. Solvents are removed in vacuo and the residue is dissolved in dichloromethane and washed with brine, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by reverse phase HPLC (water / acetonitrile) to afford 2.4 g (51% of th.) of the title compound.

HPLC (method 1): R, = 3.70 min; MS (ESIpos): m/z = 251 (M+H) ⁺

¹ H-NMR (400 MHz, DMSOd ₆ ): δ = 7.30 (m, 4H), 7.21 (m, IH), 6.72 (m, IH), 3.68 (s, 2H), 3.02 (m, 2H), 2.53 (m, 2H), 2.08 (bs, IH), 1.37 (s, 9H).

Example 4A

tert-butyl {3-[(4-chlorobenzyl)amino]propyl}carbamate

10 g (71.1 mmol) 4-chlorobenzaldehyde and 13.6 g (78.3 mmol) tert-butyl (2-aminopropyl)- carbamate are dissolved in 200 ml methanol and stirred for 2 h at room temperature. The solution is cooled to 0 ⁰ C and 13.5 g (355.7 mmol) sodium borohydride are carefully added. Water is added

until a clear solution is formed and the mixture is stirred at room temperature overnight. The mixture is concentrated under vacuum, the residue is diluted with dichloromethane and the organic phase is washed with brine, dried over magnesium sulfate and concentrated under vacuum. The crude product is re-crystallized from petroleum ether to yield 8.9 g (42% of th.) of the title compound as a solid.

HPLC (method 1): R, = 4.03 min; MS (ESIpos): m/z = 299 (M+H) ⁺

¹ H-NMR (300 MHz, DMSO-d ₆ ): δ = 7.35 (m, 4H), 6.70 (m, IH), 3.60 (s, 2H), 2.95 (m, 2H), 2.40 (m, 2H), 2.10 (bs, IH), 1.50 (m, 2H), 1.40 (s, 9H).

Example 5A

tert-butyl {3-[(4-fluorobenzyl)amino]propyl}carbamate

7.9 g (45.3 mmol) tert-butyl (2-aminopropyl)carbamate are dissolved in 40 ml methanol. 6.19 g (49.9 mmol) fluorobenzaldehyde are added and the resulting suspension is stirred for 2 h at rt. After cooling to 0 ⁰ C 8.57 g (227 mmol) sodium borohydride are added in portions. After addition of 1 ml water the reaction mixture is stirred over night at rt, before water is added. The aqueous layer is saturated with sodium chloride and extracted three times with dichloromethane. The combined organic layers are dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by chromatography on silica gel (gradient dichloromethane / methanol 100: 1 to 20:1) to yield 9.50 g (74% of th.) of the title compound.

LC-MS (method 2): R, = 1.43 min; m/z = 283 (M+H) ⁺

¹ H-NMR (300 MHz, DMSO-Cl ₆ ): δ = 7.33 (2d, 2H), 7.10 (t, 2H), 6.80 (t, IH), 3.62 (s, 2H), 2.95 (q, 2H), 2.45 (t, 2H), 2.15 (bs, IH), 1.51 (m, 2H), 1.35 (s, 9H).

Example 6A

tert-butyl (3 - { [ 1 -(4-fluoropheny l)ethy 1] amino } propy l)carbamate

To a mixture of 3.15 g (24 mmol) 4-fluoroacetophenone, 3.49 g (24 mmol) tert-butyl (2- aminopropyl)carbamate in 15 ml THF are added at rt 11.37 g (40 mmol) Ti(CMPr) ₄ . The mixture is stirred 6 to 8 h at rt, before 2.27 g (60 mmol) sodium borohydride are added followed by 20 ml ethanol (cooling bath required, foaming!). The mixture is stirred over night at rt, before poured onto water. The pH is adjusted to pH 9 with aq. ammonia-solution and the mixture is filtrated. The filter cake is washed three times with 200 ml methyl-tert-butylether. The filtrate is extracted two times with methyl-tert-butylether. The organic layers are combined, washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude product is purified by chromatography (gradient dichloromethane / methanol 100:1 to 20:1) to yield 4.35 g (73% of th.) of the title compound.

LC-MS (method 3): R ₁ = 1.46 min; m/z = 297 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ = 7.34 (2d, 2H), 7.10 (t, 2H), 6.75 (t, IH), 3.65 (q, IH), 2.92 (m, 2H), 2.30 (m, IH), 2.22 (m, IH), 1.97 (bs, IH), 1.45 (m, 2H), 1.35 (s, 9H), 1.19 (d, 3H).

Example 7A

tert-butyl {2-[( 1 -phenylethyl)amino]ethyl } carbamate

To a mixture of 2.88 g (24 mmol) acetophenone, 3.20 g (24 mmol) tert-butyl (2-amino- ethyl)carbamate in 15 ml THF are added at rt 11.37 g (40 mmol) Ti(CMPr) ₄ . The mixture is stirred 6 to 8 h at rt, before 2.27 g (60 mmol) sodium borohydride are added followed by 20 ml ethanol (cooling bath required, foaming!). The mixture is stirred over night at rt, before poured onto water. The pH is adjusted to pH 9 with aq. ammonia-solution and the mixture is filtrated. The filter cake is washed with three times with 200 ml methyl-tert.butylether. The filtrate is extracted two times with methyl-tert.butylether. The organic layers are combined, washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude product is purified by chromatography

(gradient dichloromethane / methanol 100:1 to 20:1) to yield 1.36 g (26% of th.) of the title compound.

LC-MS (method 4): R, = 1.16 min; m/z = 265 (M+H) ⁺

¹ H-NMR (400 MHz, DMSOd ₆ ): δ = 7.30 (m, 4H), 7.20 (m, IH), 6.71 (t, IH), 3.65 (q, IH), 2.95 (m, 2H), 2.35 (m, 2H), 2.05 (bs, IH), 1.35 (m, 9H), 1.21 (d, 3H).

Example 8A

4-(benzyloxy)-3-methoxybenzoic acid

8.83 g (36.4 mmol) 4-benzyloxy-3-methoxybenzaldehyde are dissolved in 150 ml acetone and cooled to 0 ⁰ C. 5.76 g (36.4 mmol) potassium permanganate are added at 0 ⁰ C in portions. The rapidly stirred mixture is allowed to warm slowly to rt. After 1 to 2 h the reaction is terminated by addition of ethanol (50 ml). After excess of potassium permanganate is destroyed the mixture is treated with IN hydrochloric acid and filtered over celite. The residual solid is washed with ethanol. All filtrates are combined and concentrated, the residual solution is poured onto water. The precipitate is collected by filtration and washed three times with water. Additional crops of product can be obtained: the filter cake is washed with dichloromethane / methanol mixtures or ethanol, concentration to remove the bulk of the solvent and precipitation by addition of water. In total 7.48 g (79% of th.) of the title compound can be obtained.

LC-MS (method 5): R, = 2.09 min; m/z = 259 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-(I ₆ ): δ = 12.68 (bs, IH) 7.54 (dd, IH), 7.51-7.31 (m, 6H), 7.14 (d, IH), 5.18 (s, 2H), 3.81 (s, 3H).

Example 9A

4-[(4-fluorobenzyl)oxy]-3-methoxybenzoic acid

To a suspension of 5.0 g (29.3 mmol) 4-hydroxy-3-methoxybenzoic acid in 15 ml THF are added

39 ml 2N sodium hydroxide solution. The resulting colored solution is cooled to 0 ⁰ C and 6.68 g

(97.0 mmol) 4-fluorobenzyl bromide, dissolved in 10 ml THF, are added. The mixture is stirred 15 min at 0°C, then allowed to warm up to rt and stirred over night at reflux. The cooled reaction mixture is extracted two times with n-hexane (discarded), the remaining THF-layer is concentrated in vacuo to dryness. The residue is treated with water and acidified to ca. pH 1 with cone, hydrochloric acid to afford a precipitate which is collected by filtration and washed with water. The crude solid is dried and crystallized with diethyl ether and filtered. After drying at high vacuum 3.47 g (38% of th.) of the title compound are obtained.

HPLC (method 6): R ₄ = 4.26 min; MS (ESIpos): m/z = 277 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ = = 12.68 (bs, IH) 7.60-7.42 (m, 4H), 7.23 (t, 2H), 7.14 (d, IH), 5.15 (s, 2H), 3.80 (s, 3H).

Using an analogous procedure the following compounds are prepared:

Example 14A

methyl 4-(benzyloxy)-3-chIorobenzoate

To a mixture of 5 g (26.8 mmol) methyl-3-chloro-4-hydroxybenzoate are added 50 ml acetonitrile,

8.73 g (26.8 mmol) powdered cesium carbonate and 0.89 g (5.36 mmol) potassium iodide. The mixture is treated at rt with 4 ml (33.5 mmol) benzyl bromide. Gradually a white solid precipitated. After reaction is complete (1 to 2 h) the mixture is poured onto water, the aqueous layer is extracted three times with ethyl acetate. The combined organic layers are washed with buffer solution (pH 7) and brine, dried over magnesium sulfate and concentrated in vacuo. The residual oil is crystallized with ethanol, the precipitate is collected by filtration and the filtrate is concentrated in vacuo and a another crystallization with ethanol yielded a second crop of product. Both product batches are combined to afford 6.4 g (86% of th.) of the title compound.

LC-MS (method 2): R ₄ = 2.99 min; m/z = 277 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-dβ): δ = 7.95 (d, IH), 7.91 (dd, IH), 7.53-7.32 (m, 6H), 5.31 (s, 2H), 3.83 (s, 3H).

Example 15A

ethyl 4-(benzyloxy)-3-nitrobenzoate

Following analogous procedure as for example 14A ethyl 4-(benzyloxy)-3-nitrobenzoate is prepared from corresponding, commercially available phenol.

LC-MS (method 2): R ₄ = 2.78 min; m/z = 302 (M+H) ⁺

¹ H-NMR (400 MHz, DMSOd ₆ ): δ = 8.40 (d, IH), 8.20 (dd, IH), 7.60(d, IH), 7.50-7.35 (m, 5H), 5.42 (s, 2H), 4.33 (q, 2H), 1.34 (t, 3H).

Example 16A

4-(benzyloxy)-3-chlorobenzoic acid

3.0 g (10.84 mmol) methyl 4-(benzyloxy)-3-chlorobenzoate are dissolved in a mixture of 10 ml THF and 10 ml methanol. At rt a solution of 864 mg (21.7 mmol) sodium hydroxide in 10 ml water is added. Stirring is continued for 1 h before the mixture is acidified with IN hydrochloric acid. The white precipitated solid is collected by filtration, washed with water and dried under vacuum (ca. 50 ⁰ C) until weight constancy is observed. The filtrate can be extracted with diethyl ether to yield after evaporation a second batch of pure product. Both batches are combined to afford 2.83 g (99% of th.) of the title compound.

LC-MS (method 4): R, = 2.19 min; m/z = 263 (M+H) ⁺

¹ H-NMR (300 MHz, DMSO-d ₆ ): δ = 8.42 (d, IH), 8.38 (dd, IH), 7.55-7.30 (m, 6H), 5.31 (s, 2H).

Example 17A

4-(benzyloxy)-3-nitrobenzoic acid

Following analogous procedure as for example 16A 4-(benzyloxy)-3-nitrobenzoic acid is prepared from ethyl 4-(benzyloxy)-3-nitrobenzoate.

LC-MS (method 2): R, = 2.30 min; m/z = 291 (M+H+H ₂ O) ⁺

¹ H-NMR (300 MHz, DMSOd ₆ ): δ = 13.38 (bs, IH), 8.38 (d, IH), 8.18 (dd, IH), 7.58 (d, IH), 7.50-7.33 (m, 5H), 5.40 (s, 2H).

Example 18A

4-(benzyloxy)-3-methoxybenzoyl chloride

1.0 g (3.87 mmol) 4-(benzyloxy)-3-methoxybenzoic acid are suspended in 20 ml dichloromethane. After a drop of DMF is added 1.84 g (15.5 mmol) thionylchloride is added carefully in portions. The suspension dissolves and the mixture is stirred at it for 1 h, then at 4O ⁰ C till evolution of gas has ceased. The solvents and excess of reagents are removed in vacuo and the resulting acid chloride can be used without further purification.

Using an analogous procedure the following acid chlorides are prepared:

Example 22A

tert-butyl {3-[(4-fluorobenzyl)(3-formyl-4-hydroxybenzoyl)amino]propyl} carbamate

1.5 g (5.59 mmol) tert-butyl {3-[(4-fluorobenzyl)amino]propyl}carbamate are dissolved in 15 ml THF. 1.02 g (6.15 mmol) 3-formyl-4-hydroxybenzoic acid, 906 mg (6.71 mmol) HOBT and

2.24 ml (12.86 mmol) diisopropylethylamine are added. The stirred mixture is cooled to 0-5 ⁰ C and

1 ml (6.43 mmol) N,N-diisopropylcarbodiimid are added. The mixture is allowed to warm up to rt and is stirred over night, before poured onto water. The aqueous layer is acidified with IN hydrochloric acid and then extracted three times with ethyl acetate. The combined organic layers are washed with brine, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by column chromatography on silica gel followed by preparative reverse phase HPLC

(water - acetonitrile) to afford 610 mg (25% of th.) of the title compound.

LC-MS (method 5): R, = 2.23 min; m/z = 331 (M+H-Boc) ⁺

¹ H-NMR (300 MHz, DMSO-(I ₆ ): δ = 11.12 (bs, IH), 10.26 (s, IH), 7.68 (d, IH), 7.57 (dd, IH), 7.49-7.13 (m, 4H), 7.04 (d, IH), 6.70 (m, IH), 4.58 (m, 2H), 3.19 (m, 2H), 2.80 (m, 2H), 1.65 (m, 2H), 1.30 (s, 9H).

Example 23A

tert-butyl {2-[(4-hydroxy-3-methoxybenzoyl)(2-thienylmethyl)amino]ethyl }carbamate

2.2 g (8.6 mmol) tert-butyl {2-[(2-thienylmethyl)amino]ethyl} carbamate are dissolved in 50 ml THF. 1.59 g (9.44 mmol) 4-hydroxy-3-methoxybenzoic acid, 1.58 g (10.3 mmol) HOBT, 3.3 ml (19.7 mmol) diisopropylethylamine and 1.53 ml (9.9 mmol) N,N-diisopropylcarbodiimid are

added. The mixture is stirred over night, before diluted with ethyl acetate and water. The aqueous layer is acidified with IN hydrochloric acid and then extracted with ethyl acetate. The combined organic layers are washed with brine, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by column chromatography on silica gel (gradient cyclohexane / ethyl acetate 3 : 1 to 1 : 1 ) to afford 2.64 g (76% of th.) of the title compound.

LC-MS (method 4): R, = 1.96 min; m/z = 407 (M+H) ⁺

¹ H-NMR (300 MHz, DMSOd ₆ ): δ = 9.41 (s, IH), 7.46 (d, IH), 7.10-6.74 (m, 6H), 4.74 (m, 2H), 3.77 (s, 3H), 3.29 (m, 2H), 3.14 (m, 2H), 1.38 (s, 9H).

Example 24A

tert-butyl {3-[(4-chlorobenzyl)(4-hydroxy-3-methoxybenzoyl)amino]propyl }carbamate

6.81 g (40.5 mmol) 3-methoxy-4-hydroxybenzoic acid, 11.0 g (36.8 mmol) tert-butyl {3-[(4- chlorobenzyl)amino]propyl}carbamate, 5.97 g (44.2 mmol) HOBT and 14 ml (84.7 mmol) diiso- propylethylamine are dissolved in 200 ml THF. 6.6 ml (42.3 mmol) N,N-diisopropylcarbodiimid are added at rt. The mixture is stirred over night, before diluted with ethyl acetate and water. The aqueous layer is acidified with IN hydrochloric acid and then extracted with ethyl acetate. The combined organic layers are washed with brine, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by column chromatography on silica gel (gradient cyclohexane / ethyl acetate from 3:1 to 1 :1) to afford 14.4 g (87% of th.) of the title compound.

LC-MS (method 5): R, = 2.24 min; m/z = 449 (M+H) ⁺

¹ H-NMR (300 MHz, DMSO-dβ): δ = 9.41 (s, IH), 7.45-7.25 (m, 4H), 6.96 (s, IH), 6.87-6.72 (m, 3H), 5.50 (d, IH), 4.58 (bs, 2H), 3.75 (bs, 3H), 3.24 (bs, 2H), 2.85 (bs, 2H), 1.68 (m, 2H), 1.35 (s, 9H).

Example 25A

p-nitrophenyl-carbonate Wang resin

30 g (30 mmol) of Wang-OH resin (Novabiochem, 1 mmol/g) and 35.60 g (450 mmol, 15 eq) pyridine in 300 ml dichloromethane are cooled to 0 ⁰ C in an ice bath. A solution of 30.24 g p-nitro- phenyl-chloroformiat (150 mmol, 4 eq) in 120 ml dichloromethane are added dropwise during 20 min. The mixture is stirred 3 h at rt afterwards. The resin is washed once with isopropanol, twice with DMF, once with isopropanol and three times alternately with methanol and dichloromethane. The resin is dried in vacuo.

Preparation Examples;

Example 1

tert-butyl {2-[[4-(benzyloxy)-3-methoxybenzoyl](2-thienylmethyl)amino]e thyl}carbamate

To a mixture of 1010 mg (3.94 mmol) tert-butyl {2-[(2-thienylrnethyl)amino]ethyl}carbamate in 15 ml dichloromethane is added a solution of 1200 mg (4.2 mmol) 4-(benzyloxy)-3-methoxy- benzoyl chloride in 15 ml dichloromethane. 0.82 ml (5.9 mmol) triethylamine are added and the resulting mixture is stirred at rt for 2 h, before water is added. The organic layer is washed with IN sodium hydroxide solution and brine, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by preparative RP HPLC (water acetonitrile) to afford 992 mg (51% of th.) of the title compound.

HPLC (method 6): R ₁ = 5.02 min; MS (ESIpos): m/z = 497 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ = 7.50-7.30 (m, 6H), 7.14-6.80 (m, 6H), 5.10 (s, 2H), 4.74 (m, 2H), 3.78 (s, 3H), 3.28 (m, 2H), 3.15 (m, 2H), 1.37 (s, 9H).

Using an analogous procedures the following compounds are prepared:

Example 7

tert-butyl {2-[{4-[(2-chlorobenzyl)oxy]-3-methoxybenzoyl}(2-thienylmeth yl)amino]ethyl}- carbamate

150 mg (0.51 mmol) 4-[(2-chlorobenzyl)oxy]-3-methoxybenzoic acid, 72.7 mg (0.54 mmol) HOBT and 103 mg (0.54 mmol) N-(3-dimethylaminopropyl)-N-ethylcarbodiimid hydrochloride are dissolved in 10 ml dichloromethane and stirred at rt for 2 h, before 131 mg (0.51 mmol) tert-butyl {2-[(2-thienylmethyl)amino]ethyl}carbamate are added and stirring is continued at rt over night. The reaction mixture is diluted with dichloromethane, washed with brine, dried over magnesium sulfate and concentrated in vacuo. The crude product is purified by preparative RP HPLC (water acetonitrile) to afford 80 mg (18% of th.) of the title compound.

LC-MS (method 5): R, = 2.84 min; m/z = 531 (M+H) ⁺

¹ H-NMR (300 MHz, DMSO-(I ₆ ): δ = 7.64-7.35 (m, 6H), 7.15-6.85 (m, 5H), 5.17 (s, 2H), 4.75 (m, 2H), 3.78 (s, 3H), 3.42-3.06 (m, 4H), 1.36 (s, 9H).

Using similar procedures the following compounds are prepared:

General procedure A; Amide bond formation

The carboxylic acid (1.0 eq.) is dissolved in anhydrous DMF (0.1 to 0.2 mol/1), amine derivative (1.0 to 1.3 eq.), HOBT (0 to 1.2 eq.), 4-DMAP (0.1 to 1.2 eq.), diisoppropylethylamine (1.5 to 2.0 eq.) are added. The mixture is stirred at rt for 10 min, cooled to O ⁰ C, before l-(3-dimethyl- aminopropyl)-3-ethylcarbodiimid hydrochloride (1.1 eq.) is added in one portion. The cooling bath is removed and stirring is continued at rt (typically over night). The reaction mixture is poured onto water and extracted three times with ethyl acetate. The combined organic layers are washed (IN hydrochloric acid, saturated sodium bicarbonate solution, pH 7 buffer solution and / or brine), dried with magnesium sulfate or sodium sulfate and concentrated in vacuo. The residue is purified by chromatography on silica gel (mixtures of cyclohexane and ethyl acetate or dichloromethane and methanol) or reverse phase HPLC (gradients of water and acetonitrile) to afford the title compound.

The following compounds are prepared according to general procedure A:

(d,

General procedure B; Alleviation of phenols

To a mixture of phenol (1.0 eq.) in acetonitrile (ca. 150 mg/ml) is added at rt cesium carbonate (1.0 to 2.0 eq.) and a catalytic amount of potassium iodide. To the heterogeneous mixture a benzyl bromide derivative (ca. 1.1 eq.) is added and stirring is continued at rt (1 h to over night). The reaction mixture is poured onto water and extracted two to three times with ethyl acetate. The combined organic layers are washed (pH 7 buffer solution and/or brine) dried over magnesium sulfate and concentrated in vacuo. The product can be further purified by column chromatography on silica gel (mixtures of dichloromethane and methanol).

The following compounds are prepared according to general procedure B:

Example 28

N-(2-aminoethyl)-4-(benzyloxy)-3-methoxy-N-(2-thienylmeth yl)benzamide trifluoroacetate

500 mg (1.01 mmol) tert-butyl {2-[[4-(benzyloxy)-3-methoxybenzoyl](2-thienylmethyl)amino]- ethyl} carbamate are dissolved in dichloromethane, cooled to 0 ⁰ C and treated with TFA. The mixture is stirred 1 h at 0 ⁰ C and 1 h at rt, before volatile components are removed at reduced pressure. The residual oil is treated with petroleum ether, the supernatant layer is decanted and the oily product dried at high vacuum. Crystalline material can be obtained from treatment with diethyl ether; yield 468 mg (91% of th.) of the title compound.

LC-MS (method 5): R, = 1.77 min; m/z = 397 (M+H) ⁺

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ = 7.78 (bs, 3H), 7.51 (d, IH), 7.48-7.31 (m, 5H), 7.18-6.90 (m, 5H), 5.11 (s, 2H), 4.72 (s, 2H), 3.75 (s, 3H), 3.55 (m, 2H), 3.02 (m, 2H).

Using analogous procedures the following compounds are prepared, optionally oily trifluoro- acetate salts are further purified by preparative reverse phase HPLC:

(s,

General procedure C: cleavage of the Boc group and isolation of hydrochloride salts after precipitation

The Boc derivative is treated with a large excess of hydrochloric acid in 1,4-dioxane (4 molar solution) at rt. After completion of the reaction the mixture is concentrated in vacuo. The residual oil is treated first with petroleum ether (supernatant layer discarded), then with diethyl ether and the title compounds can be obtained after crystallization (at low temperatures) and filtration of the suspension.

The following examples are prepared according to general procedure C from the corresponding Boc protected starting materials (examples cf. above):

General procedure D: cleavage of the Boc group and isolation of hydrochloride salts bγ preparative RP HPLC

A stirred solution of Boc derivative in 1,4-dioxane (ca. lg/ml) is treated with ca. 10 equivalents of hydrochloric acid in 1,4-dioxane (4 molar solution) at rt for ca. 1 h. Methanol is added to the mixture until a homogeneous solution is obtained. The product is isolated from the crude solution after purification by reverse phase HPLC (water-acetonitrile gradient) and concentration in vacuo as a hydrochloric acid salt.

The following examples are prepared according to general procedure D from the corresponding Boc protected starting materials (examples cf. above):

(m,

Starting from hydrochlorides or trifluoroacetates (after deprotection of boc groups) corresponding free amines can be prepared simply by washing mixtures of the salts in dichloromethane with saturated sodium hydrogencarbonate solution and evaporation of dichloromethane. Free amines can be purified by chromatography on silica gel (dichloromethane/methanol) or preparative RP HPLC (water / acetonitrile):

General procedure E: AIkylation of phenols with benzyl halides and deprotection of Boc with TFA

A mixture of tert-butyl {3-[(4-chlorobenzyl)(4-hydroxy-3-methoxybenzoyl)amino]propyl }carbamate (0.1 mmol), potassium carbonate (0.2 mmol) and the corresponding benzylhalide (0.1 mmo) is heated for 8 h at 80°-C in DMF. The mixture is filtered and purified by preparative HPLC. The remaining solids are treated with TFA (200 μl) for 0.5 h and the solvent is evaporated in vacuum, to yield the target molecules as TFA salts.

The following compounds are prepared according to general method E starting from tert-butyl {3- [(4-chlorobenzyl)(4-hydroxy-3-rnethoxybenzoyl)amino]propyl} carbamate and respective benzyl halides:

=

=

Example 65

N-(2-aminoethyl)-4-(benzyloxy)-N-(4-chlorobenzyl)-3-metho xybenzamide

General procedure F: Solid phase synthesis

Step 1 : diamine

1.00 g of p-nitrophenyl-carbonate Wang resin (ca. 1 mmol/g, 1 eq) is treated with 601 mg (10 mmol, 10 eq.) ethane- 1,2-diamine and 1.29 g (10 mmol, 10 eq.) DIEA in 25 ml DMF for 3 h at rt. The resin is washed three times with DMF and three times alternately with methanol and dichloromethane and dried in vacuo.

Step 2: aldehyde

100 mg of this resin is treated with 2.5 ml of a 2:l-mixture of TMOF/dichloromethane and 281 mg (5.00 mmol, 5 eq.) p-chlorobenzaldehyde over night at rt. The resin is washed three times with DMF and suspended again in 1 ml DMF. 74.1 mg TBABH (3.00 mmol, 3 eq.) are added. After one hour at rt the mixture is cooled to -40 ⁰ C and 519 mg acetic acid (60 mmol, 60 eq.) are added dropwise while shaking, followed by shaking over night at rt. The resin is washed three times with DMF, methanol and dichloromethane and dried in vacuo.

Step 3: benzoic acid

The resin is suspended in 2.5 ml DMF and treated with 61.9 mg 4-benzyloxy-3-methoxybenzoic acid (5.00 mmol, 5 eq) and 121 mg (10.00 mmol, 10 eq.) DIC over night at rt. The resin is washed three times with DMF, and alternately three times with methanol and dichloromethane.

Step 4: cleavage

The product is cleaved from the resin by four times treatment with 0.5 ml TFA/dichloromethane = 1:1. The cleaving solutions are collected and evaporated. The crude product is purified by preparative LC-MS.

Analytical data for N-(2-aminoethyl)-4-(benzyloxy)-N-(4-chlorobenzyl)-3-methoxyb enzamide: LC-MS (method 8): R, = 1.62 min, m/z = 425 (M+H) ⁺

Using an analogous procedure the following compounds are prepared:

Hydrochloric salts in examples 69-72 are prepared by treatment of the purified sample with 0.5 ml dichloromethane and a 0.5 M solution of hydrogen chloride in dioxane followed by evaporation of the solvent.

B. Evaluation of physiological activity

The potential Cold Menthol Receptor - 1 (CMR-I) antagonistic activity of the compounds of the invention may be demonstrated, for example, using the following assays:

Measurement of the menthol-induced Ca ²⁺ influx in HEK293 Cell expressing CMR-I receptor (Assay 1)

A cell-based calcium influx assay using HEK293 cells stably expressing human CMR-I is used to identify CMR-I receptor-antagonists. Menthol, a CMR-I specific agonist, is used for stimulation of these cells, inducing an increase in intracellular calcium. This menthol-induced Ca ²⁺ increase is traced by fluorescence measurement. Therefore the cells are loaded with fluo4-AM prior to stimulation. For testing inhibitors the cells are preincubated with various concentrations of the compound before menthol stimulation. The potency of potential CMR-I inhibitors is quantified by measuring decrease of fluorescence .

table A

Measurement of the menthol-induced Ca ²⁺ influx in primary cultured rat dorsal root ganglia neurons (Assay 2)

Since CMR-I is expressed on DRG (C-fibers), in which this receptor mediates the altered afferent information in overactive bladder; primary cultures of rat DRG are used as functional in vitro test. Stimulation of the cells is done with menthol and cold and the induced calcium influx is quantified by fluorescence in the presence or absence of CMR-I inhibitors.

Preparation of primary cultured rat DRG neurons: DRG are prepared from Zucker rats (30 days in age) and neuronal cells are dispersed in 0.1% collagenase. After removal of Schwann cells by adhering to a culture plate, non-adherent neuronal cells are recovered and cultured on laminin- and

poly-D-lysine coated 384 well plates for 2 days in the presence of 50 ng/ml rat NGF and 50 μM 5- fluorodeoxyuridine.

Measurement of Ca ²⁺ : Rat DRG neurons are suspended in a culture medium and seeded into 384- well plates (black walled clear-base / Nalge Nunc International). Following the culture for 48 hrs the medium is changed to 2 μM Fluo-4 AM (Molecular Probes) and 0.02% Puronic F- 127 in assay buffer (Hank's balanced salt solution (HBSS), 17 mM HEPES (pH7.4), 1 raM Probenecid, 0.1% bovine serum albumin (BSA)) and the cells are incubated for 60 min at 25 ⁰ C. After washing twice with assay buffer the cells are incubated with a test compound or vehicle (dimethylsulfoxide) for 20 min at 25°C. The fluorescence change indicating mobilization of cytoplasmic Ca ²⁺ is measured for 60 sec after the stimulation with 50 μM menthol. The fluorescence change is calculated in the samples treated with a test compound and vehicle respectively. Inhibitory effect of the compound is calculated by a comparison of the values.

Measurement of the micturition frequency in guinea pigs in vivo (Assay 3)

Experiments are performed according to the principles of the national law for the protection of laboratory. Female Guinea Pigs (300-350g) are anaesthetized with urethane (1 mg/kg i.p.). A midline abdominal incision is performed, both ureters are exposed and ligated, a catheter is implanted in the bladder pole and the abdomen is closed. For administration of the compounds the vena jugularis is exposed and canulated with a catheter. After this surgery the bladder catheter is connected via a t-shaped tube to an infusion pump (Braun Perfusor® compact) and to a pressure transducer (BioResearch Center, MLT0698, Nagoya). Saline is infused and intrabladder pressure is registered. After 1 h of equilibration period and the establishment of constant voiding cycles, menthol (0.6 mM) is added to the infused saline. At this point also vehicle (control group) or

CMR-I inhibitors are administered i.v. as bolus injection. The effect of treatment on the micturition interval (corresponding to bladder capacity) and micturition pressure is calculated and compared between vehicle-treated and compound-treated groups.

C _; Operative examples relating to pharmaceutical compositions

The compounds according to the invention can be converted into pharmaceutical preparations as follows:

Tablet

Composition:

100 mg of the compound of Example 1, 50 mg of lactose (monohydrate), 50 mg of maize starch (native), 10 mg of polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.

Tablet weight 212 mg, diameter 8 mm, curvature radius 12 mm.

Preparation:

The mixture of active component, lactose and starch is granulated with a 5% solution (m/m) of the PVP in water. After drying, the granules are mixed with magnesium stearate for 5 min. This mixture is moulded using a customary tablet press (tablet format, see above). The moulding force applied is typically 15 kN.

Orally administrable suspension

Composition:

1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of Rhodigel (xanthan gum from FMC, Pennsylvania, USA) and 99 g of water.

A single dose of 100 mg of the compound according to the invention is provided by 10 ml of oral suspension.

Preparation:

The Rhodigel is suspended in ethanol and the active component is added to the suspension. The water is added with stirring. Stirring is continued for about 6h until the swelling of the Rhodigel is complete.

Solution for intravenous administration:

Composition:

1 mg of the compound of Example 1, 15 g of polyethylene glycol 400 and 250 g of water for injection.

Production:

The compound of Example 1 is dissolved with polyethylene glycol 400 in the water with stirring., The solution is sterilized by filtration (pore diameter 0.22 μm) and dispensed under aseptic conditions into heat-sterilized infusion bottles. These are closed with infusion stoppers and crimped caps.