JP2007526242A - Tricyclic 1-[(3-indol-3-yl) carbonyl] piperazine derivatives as cannabinoid CB1 receptor agonists - Google Patents

Abstract

The present invention is directed to general formula (I) wherein X is CH ₂ , O or S; R is independent of H, (C _1-4 ) alkyl, (C _1-4 ) alkyloxy and halogen. Represents 1 to 3 substituents selected from: R ₁ is (C _5-8 ) cycloalkyl; R ₂ is H or (C _1-4 ) alkyl; R ₃ , R ₃ ′, R ₄ ′, R ₄ ′, R ₅ , R ₅ ′ and R ₆ ′ are independently hydrogen or (C _1-4 ) alkyl ((C _1-4 ) alkyloxy, OH or halogen; R ₆ is hydrogen or (C _1-4 ) alkyl (optionally substituted with (C _1-4 ) alkyloxy, OH or halogen); or R _6, together with R _7, if a further heteroatom selected from O and S Depending contained are, form a saturated heterocyclic ring of 4-7 membered; R ₇ together with R _6, optionally containing a further heteroatom selected from O and S, a saturated 4 to 7 membered Or form a heterocycle; or R ₇ is H, (C _1-4 ) alkyl or (C _3-5 ) cycloalkyl, wherein the alkyl group is OH, halogen or (C _1-4 ) alkyl. And optionally substituted with oxy.) Tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives or pharmaceutically acceptable salts thereof. The present invention also provides a pharmaceutical composition containing the tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative and pain such as perioperative pain, chronic pain, neuropathic pain, and cancer pain. It also relates to the use of these derivatives in the treatment of pain and spasticity associated with multiple sclerosis.

Description

  The present invention relates to tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives, pharmaceutical compositions containing the derivatives and treatment thereof, in particular in the treatment of pain, these tricyclic 1-[(indole- 3-yl) carbonyl] piperazine derivatives.

  Pain treatment is often limited by the side effects of currently available medications. Opioids are widely used to relieve severe pain. These drugs are cheap and effective, but have serious and life-threatening side effects (most notably respiratory problems and muscle stiffness). In addition, the dose of opioids that can be administered is limited by nausea, vomiting, constipation, pruritus, and urinary retention, resulting in patients receiving suboptimal pain control over suffering from these painful side effects. Often selected. In addition, these side effects often result in patients requiring prolonged hospitalization. Opioids are highly addictive and are designated drugs in many areas. Accordingly, there is a need for new analgesics with improved side effect profiles compared to currently used products at equianalgesic doses.

  There is accumulated evidence that cannabinoid agonists have potential as analgesics and anti-inflammatory agents. Two types of cannabinoid receptors are involved, and the cannabinoid CB1 receptor is predominantly distributed in the central nervous system but is also expressed by peripheral neurons and is rare in other peripheral tissues, and cannabinoid CB2 Receptors are distributed mainly in immune cells (Howlet, A. C. et al .: International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors. Pharmacol. Rev. 54, 161-202, 2002). While CB2 receptors have been implicated in alleviating cannabinoid immune and anti-inflammatory responses, it has recently been suggested that cannabinoid receptor agonists, particularly those acting at the CB1 receptor, are useful in the treatment of pain. (See Iversen, L. and Chapman, V. Current Opinion in Pharmacology, 2, 50-55, 2002 and references therein). WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-[(morpholinyl) methyl] pyrrolo [1,2,3-de] -1,4-benzoxazinyl]- The mesylate salt of (1-naphthalenyl) methanone was disclosed in US Pat. No. 4,939,138 (Sterling Drug Inc.) as an analgesic. This compound is a prototype of an aminoalkylindole (Eissenstat et al., J. Med. Chem. 38, 3094-3105, 1995), which is an animal model of acute pain, persistent inflammatory pain and neuropathic pain. It is a potent cannabinoid CB1 receptor agonist that can produce analgesia with potency comparable to that of morphine.

  Pyrrolo [1,2,3-de] -1,4-benzoxazinecarboxamide derivatives that are structurally closely related have been proposed as cannabinoid receptor modulators for the treatment of respiratory diseases as WO2001 / 58869 (Bristol-Myers Squibb). Comp.). Similar tricyclic 3-carboxamide-indole derivatives are disclosed as 5-HT receptor antagonists in EP 0 393 766 (Duphar Inter. Res. BV).

  Known cannabinoid agonists are generally very lipid soluble and insoluble in water. Accordingly, there is a need for cannabinoid agonists with improved properties for use as therapeutic agents.

  For this purpose, the present invention can be used, for example, in the treatment of pain such as perioperative pain, chronic pain, neuropathic pain, cancer pain and the pain and spasticity associated with multiple sclerosis. General formula (I) as an agonist of the cannabinoid CB1 receptor that can be:

（式中、
Ｘは、ＣＨ_２、Ｏ又はＳであり；
Ｒは、Ｈ、（Ｃ_１−４）アルキル、（Ｃ_１−４）アルキルオキシ及びハロゲンから独立に選択される１個から３個の置換基を表し；
Ｒ_１は、（Ｃ_５−８）シクロアルキルであり；
Ｒ_２は、Ｈ又は（Ｃ_１−４）アルキルであり；
Ｒ_３、Ｒ_３’、Ｒ_４、Ｒ_４’、Ｒ_５、Ｒ_５’及びＲ_６’は、独立に、水素又は（Ｃ_１−４）アルキル（（Ｃ_１−４）アルキルオキシ、ＯＨもしくはハロゲンにより場合によっては置換される。）であり；
Ｒ_６は、水素又は（Ｃ_１−４）アルキル（（Ｃ_１−４）アルキルオキシ、ＯＨもしくはハロゲンにより場合によっては置換される。）であるか；又は
Ｒ_６は、Ｒ_７と一緒に、Ｏ及びＳから選択されるさらなるヘテロ原子を場合によっては含有する、４から７員の飽和複素環を形成し；
Ｒ_７は、Ｒ_６と一緒に、Ｏ及びＳから選択されるさらなるヘテロ原子を場合によっては含有する、４から７員の飽和複素環を形成するか；又は、
Ｒ_７は、Ｈ、（Ｃ_１−４）アルキル又は（Ｃ_３−５）シクロアルキルであり、該アルキル基は、（Ｃ_１−４）アルキルオキシ、ＯＨもしくはハロゲンで場合によっては置換される。）を有する、三環式１−［（インドール−３−イル）カルボニル］ピペラジン誘導体又はそれらの医薬として許容される塩を提供する。

(Where
X is CH ₂ , O or S;
R represents 1 to 3 substituents independently selected from H, (C _1-4 ) alkyl, (C _1-4 ) alkyloxy and halogen;
R ₁ is (C _5-8 ) cycloalkyl;
R ₂ is H or (C _1-4 ) alkyl;
R ₃ , R ₃ ′, R ₄ , R ₄ ′, R ₅ , R ₅ ′ and R ₆ ′ are independently hydrogen or (C _1-4 ) alkyl ((C _1-4 ) alkyloxy, OH or Optionally substituted by halogen));
R ₆ is hydrogen or (C _1-4 ) alkyl (optionally substituted by (C _1-4 ) alkyloxy, OH or halogen); or R ₆ together with R ₇ Forming a 4 to 7 membered saturated heterocycle optionally containing additional heteroatoms selected from O and S;
R ₇ together with R ₆ forms a 4 to 7 membered saturated heterocycle optionally containing additional heteroatoms selected from O and S; or
R ₇ is H, (C _1-4 ) alkyl or (C _3-5 ) cycloalkyl, wherein the alkyl group is optionally substituted with (C _1-4 ) alkyloxy, OH or halogen. A tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative or a pharmaceutically acceptable salt thereof.

The tricyclic core structure of the 1-[(indol-3-yl) carbonyl] piperazine derivative of the present invention is 2,3-dihydro-pyrrolo [3,2,1-ij] quinoline (wherein X is CH ₂ ); 2,3-dihydro-pyrrolo [1,2,3-de] -1,4-benzoxazine (wherein X is oxygen) and 2,3-dihydro-pyrrolo [ 1,2,3-de] -1,4-benzothiazine (X is sulfur).

The compounds of the invention in which X is oxygen are generally described as cannabinoid receptor modulators for treating respiratory diseases in WO2001 / 58869 (supra). These modulators are selectively identified therein as CB2 receptor modulators. The characteristics of 2,3-dihydro-pyrrolo [1,2,3-de] -1,4-benzoxazine derivatives disclosed in WO2001 / 58869 are linked to the 3-position of the benzoxazine ring (4- Morpholinyl) methyl side chain is present. The tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative of the present invention has a (C _5-8 ) cycloalkyl side chain at the corresponding position (the compound has a CB1 agonist activity) Therefore, it is distinguished from that of WO2001 / 58869.

The term (C _1-4 ) alkyl, as used in the definition of formula I, has 1 to 4 carbon atoms, such as butyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl and methyl. Means a branched or unbranched alkyl group.

In the term (C _1-4 ) alkyloxy, (C _1-4 ) alkyl has the meaning as defined above.

The term (C _5-8 ) cycloalkyl means a saturated cyclic alkyl group having 5 to 8 carbon atoms and can thus represent cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

  The term halogen means F, Cl, Br or I.

In the definition of formula I, R ₆ together with R ₇ can form a 4 to 7 membered saturated heterocycle, which is attached to R ₆ together with the carbon atom to which it is attached. It is meant that R ₇ together with the nitrogen atom forms a 4- to 7-membered saturated ring such as azetidine, pyrrolidine, piperidine or 1H-azepine ring. Such a ring may contain additional O or S-heteroatoms to form a ring such as a morpholine, thiomorpholine, tetrahydrothiazole or isothiazole ring.

Preference is given to tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives of the formula I, wherein R is H and R ₁ is cyclopentyl or cyclohexyl.

Particularly preferred are R, R ₂ , R ₃ , R ₃ ′, R ₄ ′, R ₅ , R ₅ ′ and R ₆ ′ are H; R ₄ , R ₆ and R ₇ are independently H Or (C _1-4 ) alkyl; or R ₆ together with R ₇ forms a 5- or 6-membered saturated heterocycle, and R ₄ is H or (C _1-4 ) alkyl. , A tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative of formula I.

  When X is O, an isomer in which the stereochemistry at the 3-position of the benzoxazine ring is (R) is more preferable.

  The tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives of the present invention can generally be prepared by methods known in the art of organic chemistry.

A tricyclic 1-[(indol-3-yl) carbonyl] piperazine of formula I is, for example, a compound of formula II wherein R, R ₁ , R ₂ and X have the meanings already defined, C (O) Y represents a carboxylic acid or an activated derivative thereof (a carboxylic acid ester or a halogenated carboxylic acid, preferably chloride or bromide), and a compound of formula III (wherein R ₃ —R ₇ has the meaning already defined). If R ₇ of the compound of formula III represents hydrogen, the nitrogen atom to which it is attached may have to be temporarily protected during the condensation reaction. Suitable protecting groups for functional groups that are temporarily protected during synthesis are described, for example, in Wuts, P .; G. M.M. And Greene, T .; W. : Protective Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and the like. When C (O) Y represents a carboxylic acid (that is, Y is hydrogen), this condensation reaction is carried out using a coupling agent such as carbonyldiimidazole, dicyclohexylcarbodiimide, and a solvent such as dimethylformamide or dichloromethane. Can be executed in. When C (O) Y represents a halogenated carboxylic acid (ie Y is a halogen), the condensation with an amine derivative of formula III is carried out in a solvent such as dichloromethane in the presence of a base such as triethylamine. obtain. When C (O) Y represents a carboxylic acid ester, direct condensation with the amine derivative of formula III can be carried out at elevated temperatures.

  Compounds of formula III can be obtained from commercial sources and can be prepared by literature procedures or modifications of literature procedures known to those skilled in the art. For example, M.M. E. Jung and J.A. C. By reducing diketopiperazine using a reducing agent such as lithium aluminum hydride or borane-tetrahydrofuran chain, as described by Rohmloff (J. Org. Chem. 50, 4909-4913, 1985), Compounds of formula III can be prepared. Diketopiperazine is a C.I. J. et al. Dinsmore and D.M. C. It can be prepared by various routes described by Bershore (Tetrahedron 58, 3297-3312, 2002).

Compounds of formula II can be prepared by literature means or modifications of literature means known to those skilled in the art. For example, a compound of formula II where C (O) Y represents a carboxylic acid and R ₂ is (C _1-4 ) alkyl can be treated with at least 2 equivalents of a strong base such as n-butyllithium followed by halogen. Can be prepared by alkylation of a compound of formula II where C (O) Y represents a carboxylic acid and R ₂ is hydrogen by treatment with an alkylating agent such as (C _1-4 ) alkylated. .

  Compounds of formula II can be prepared by acylation of compounds of formula IV using an acylating agent. For example, a compound of formula II where Y is hydroxy can be prepared by treatment with trifluoroacetic anhydride in a solvent such as dimethylformamide followed by hydrolysis with aqueous sodium hydroxide at elevated temperature. It can be obtained from a compound. A compound of formula II where Y is chloride is prepared by reaction of a compound of formula IV with oxalyl chloride in a solvent such as 1,1,2,2-tetrachloroethane followed by rearrangement at elevated temperature. be able to. Compounds of formula IV can be prepared from compounds of formula V using Fischer indole synthesis (Chem. Rev. 69, 227-250, 1969).

  Alternatively, the compound of formula II can be prepared by the means described by Wijngaarden et al. (J. Med. Chem. 36, 3693-3699, 1993) or Hwu et al. (J. Org. Chem. 59, 1577-1582, 1994) or It can be prepared from compounds of formula V using variations of these means.

Compounds of formula V can be prepared by literature means or modifications of literature means known to those skilled in the art. For example, a compound of formula V where X is CH ₂ can be prepared from a compound of formula VI by reduction with a reducing agent such as sodium borohydride in the presence of a catalyst such as nickel (II) chloride. . Compounds of formula VI can be prepared by a coupling reaction, such as, for example, the reaction of 2-chloroquinoline with a (C _5-8 ) cycloalkyl Grignard reagent in the presence of a catalyst such as nickel (II) chloride. .

  A compound of formula V in which X is O or S is a reaction of a compound of formula VII in which X is OH or SH with a compound of formula VIII to form an ether or thioether followed by an amine It can be prepared by reduction of the nitro group and reductive cyclization. For example, the etherification reaction can be performed in the presence of a base such as potassium carbonate using a catalyst such as potassium iodide. Reduction and cyclization can be performed using hydrogen gas in the presence of a catalyst such as palladium on charcoal, for example.

  Compounds of formula VII and VIII can be obtained from commercial sources and can be prepared by literature means or modifications of literature means known to those skilled in the art. For example, a compound of formula VIII can be prepared from a compound of formula IX using a brominating agent such as bromine in a solvent such as methanol.

Those skilled in the art will be able to convert various tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives of Formula I to appropriate transformations of functional groups corresponding to certain substituents R and R ₃ -R _7. Will understand that can be obtained. For example, compounds of formula I wherein R ₇ is (C _1-4 ) alkyl or (C _3-5 ) cycloalkyl, the alkyl group of which may be substituted with OH, halogen or (C _1-4 ) -alkyloxy are , in the presence of a base such as potassium carbonate, of a compound of formula I R ₇ is hydrogen, by reaction with a halogenated (C _1-4) alkyl or functionalized halogenated (C _1-4) alkyl, prepared can do.

  Tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives of formula I and their salts contain at least one chiral center and therefore as stereoisomers, including enantiomers and diastereomers. Exists. The invention includes within its scope the aforementioned stereoisomers and the individual R and S enantiomers of compounds of formula I and their salts (substantially free of other enantiomers, ie less than 5% A mixture of such enantiomers in any proportion, including each and a racemic mixture containing substantially equivalent amounts of the two enantiomers, preferably less than 2%, in particular less than 1%. Including.

  Methods for asymmetric synthesis or chiral separation to obtain pure stereoisomers are, for example, chiral induction or synthesis starting from commercially available chiral materials, or using, for example, chromatography on chiral media, or chiral pairs It is well known in the art, such as the separation of stereoisomers by crystallization using ions.

  Pharmaceutically acceptable salts include a free base of a compound of formula I, an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid or ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric It can be obtained by treatment with an organic acid such as acid, glycolic acid, succinic acid, propionic acid, acetic acid or methanesulfonic acid.

  The compounds of the present invention may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

  The present invention further relates to tricyclic 1-[(indol-3-yl) carbonyl] piperazines having the general formula I, mixed with pharmaceutically acceptable auxiliaries and optionally mixed with other therapeutic agents. Pharmaceutical compositions containing the derivatives or pharmaceutically acceptable salts thereof are provided. The term “acceptable” means compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The composition is suitable for oral, sublingual, subcutaneous, intravenous, epidural, intrathecal, intramuscular, transdermal, pulmonary, topical or rectal administration, etc., in unit dosage form for administration Includes everything.

  For oral administration, the active agent can be present as discrete units, such as tablets, capsules, powders, granules, solutions, suspensions and the like. For parenteral administration, the pharmaceutical compositions of the invention may be present in unit dose or multi-dose containers, eg, injectable liquids (eg, sealed vials and ampoules) in pre-determined amounts, and water prior to use. Can be stored in a freeze-dried state, requiring only the addition of a sterile liquid carrier such as

  See, for example, the standard reference, Gennaro, A .; R. Et al., Remington: The Science and Practice of Pharmacy (20th Edition, Lippincott Williams & Wilkins, 2000) (especially referred to as Part 5: Pharmaceuticals as pharmacological aids such as those assisted as such). And can be compressed into solid dosage units such as pills, tablets, or processed into capsules, suppositories, or patches. The active substance can be applied as a liquid composition by pharmaceutically acceptable liquids, for example as injectable preparations, in the form of solutions, suspensions, emulsions or as sprays, for example nasal sprays.

  In preparing solid dosage units, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general, any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the active substances of the invention can be administered together as a solid composition include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts. For parenteral administration, aqueous suspensions, isotonic saline solutions and sterile injectable solutions containing pharmaceutically acceptable dispersants and / or wetting agents such as propylene glycol or butylene glycol may be used. it can.

  The present invention further includes a pharmaceutical composition, as already described herein, in combination with a packaging material suitable for the pharmaceutical composition, wherein the packaging material is as previously described herein. Includes instructions for using the composition for use.

  The tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivatives of the present invention were examined in a human CB1 receptor assay using CHO cells and found to be agonists of the CB1 receptor. Methods for examining receptor binding as well as in vitro biological activity of cannabinoid receptor modulators are well known in the art. In general, the expressed receptor is contacted with the compound under test and the binding or stimulation or inhibition of a functional response is measured.

  In order to measure a functional response, isolated DNA encoding the CB1 receptor (preferably human receptor) gene is expressed in a suitable host cell. Such cells can be Chinese Hamster Ovary cells, but other cells are also suitable. Preferably, the cell is of mammalian origin.

  Methods for constructing recombinant CB1-expressing cell lines are well known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition). Expression of the receptor is accomplished by expression of DNA encoding the desired protein. All techniques for further sequence ligation and construction of appropriate expression systems are already well known in the art. Part or all of the DNA encoding the desired protein can be synthetically constructed using standard solid phase techniques, preferably to include restriction sites to facilitate ligation. Appropriate regulatory elements for transcription and translation of the included coding sequence can be provided for the DNA coding sequence. As is well known, expression systems are currently available that are compatible with a wide range of hosts, including prokaryotic hosts such as bacteria and eukaryotics such as yeast, plant cells, insect cells, mammalian cells, and avian cells. A biological host is included.

  A cell expressing the receptor is then contacted with a test compound to observe binding or stimulation or inhibition of a functional response.

  Alternatively, isolated cell membranes containing expressed CB1 (or CB2) receptors can be used to measure compound binding.

Radioactive or fluorescently labeled compounds can be used for binding measurements. The most widely used radiolabeled cannabinoid probe is [ ³ H] CP55940, which has approximately equal affinity for CB1 and CB2 binding sites.

  Another assay involves screening for cannabinoid CB1 agonist compounds by examining a second messenger response, such as measuring receptor-mediated changes in the cAMP or MAP kinase pathways. Thus, such methods involve expression of the CB1 receptor on the cell surface of the host cell and exposing the cell to the test compound. The second messenger response is then measured. Depending on the effect of the test compound on binding of the receptor, the level of the second messenger increases or decreases.

For example, in addition to direct measurement of cAMP levels in exposed cells, in addition to transfection with receptor-encoding DNA, a second DNA may be transfected that encodes a reporter gene whose expression correlates with receptor activation. Cells can be used. In general, reporter gene expression can be regulated by some response element that responds to changes in the level of the second messenger. Suitable reporter genes are for example LacZ, alkaline phosphatase, firefly luciferase and green fluorescent protein. The principles of such transactivation assays are well known in the art and are described, for example, in Stratowa, Ch, Himmel, A. et al. And Czernilosky, A .; P. Curr. Opin. Biotechnol. 6, 574 (1995). In order to select agonist compounds with activity at the CB1 receptor, EC ₅₀ values should be <10 ⁻⁵ M, preferably <10 ⁻⁷ M.

  For example, the compounds may be used in the treatment of pain such as perioperative pain, chronic pain, neuropathic pain, cancer pain, and pain and spasticity associated with multiple sclerosis.

  The cannabinoid agonists of the present invention also potentially have multiple sclerosis, spasticity, inflammation, glaucoma, nausea and vomiting, anorexia, sleep disorders, respiratory diseases, allergies, epilepsy, migraine, cardiovascular diseases, nerves It is useful in the treatment of other diseases including degenerative diseases, anxiety, traumatic brain injury and stroke.

  The compounds can also be used in combination with other drugs, such as opioids and analgesics such as non-steroidal anti-inflammatory drugs (NSAIDs) including COX-2 selective inhibitors.

  The compounds of the present invention may be administered to humans in an amount and for a sufficient period of time to alleviate the symptoms. Illustratively, dose levels for humans can range from 0.001 to 50 mg / kg body weight, preferably 0.01 to 20 mg / kg body weight.

  The invention is illustrated by the following examples.

(R) -3-Cyclohexyl-2,3-dihydro-6- (4-ethylpiperazin-1-ylcarbonyl) pyrrolo [1,2,3-de] -1,4-benzoxazine hydrochloride dimethylformamide To a solution of DN-Boc-cyclohexylglycine (25.0 g, 97.2 mmol) in (200 ml) was added sodium bicarbonate (24.5 g, 291.6 mmol) and methyl iodide (6.66 ml, 106.9 mmol). ) Was added. The mixture was stirred at room temperature for 64 hours in the presence of nitrogen. The resulting mixture was partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The obtained crystals were washed with n-heptane to obtain DN-Boc-cyclohexylglycine methyl ester (25.65 g, 94.5 mmol).

  To a solution of DN-Boc-cyclohexylglycine methyl ester (25.65 g, 94.5 mmol) in methanol (200 ml) and tetrahydrofuran (100 ml) was added calcium chloride (21.0 g, 189 mmol) and sodium borohydride (14 .3 g, 378 mmol) was added. The mixture was stirred at room temperature for 30 minutes in the presence of nitrogen. The resulting mixture was poured into ice water, neutralized with 5N hydrochloric acid, and partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with saturated sodium carbonate solution and brine, dried over sodium sulfate and concentrated. The residue was crystallized from heptane to give crude (R) -N-Boc-2-cyclohexylethanolamine (29.38 g, 94.5 mmol).

  To a mixture of crude (R) -N-Boc-2-cyclohexylethanolamine (29.38 g, 94.5 mmol) and triphenylphosphine (37.2 g, 141.8 mmol) in toluene (150 ml) at 0 ° C. Diisopropyl azodicarboxylate (19.5 ml, 99.2 mmol) was added. After stirring for 1 hour, 2-bromophenol (12.1 ml, 104.0 mmol) was added to the mixture at 0 ° C. The reaction mixture was stirred at 0 ° C. for 2 hours and at room temperature for 20 hours. The resulting mixture was partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with 2N sodium hydroxide solution and brine, dried over sodium sulfate and concentrated.

  The residue was purified by flash chromatography eluting with 0-10% (v / v) ethyl acetate in heptane to give (R) -2- (2-tert-butoxycarbonylamino-2-cyclohexylethoxy) -bromo. Benzene (12.80 g, 32.1 mmol) was obtained.

  (R) -2- (2-tert-Butoxycarbonylamino-2-cyclohexylethoxy) bromobenzene (500 mg, 1.26 mmol), tetrakis (triphenylphosphine) palladium (0) in toluene (4.0 ml) ( 146 mg, 0.126 mmol) and tert-butoxide sodium (181 mg, 1.88 mmol) were exposed to microwave irradiation at 120 ° C. for 10 minutes. The resulting mixture was partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The residue was purified by flash chromatography eluting with 0-17% (v / v) ethyl acetate in heptane to give (R) -4-tert-butoxycarbonyl-3-cyclohexyl-3,4-dihydro -2H-1,4-benzoxazine (270 mg, 0.85 mmol) was obtained. This reaction was repeated 13 times on the same scale to give the same intermediate (3.98 g, 12.5 mmol total).

  (R) -4-tert-Butoxycarbonyl-3-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazine (3.98 g, 12.5 mmol), 5N hydrochloric acid (10 ml) and ethanol (10 ml). The mixture was stirred at 70 ° C. for 50 minutes. The ethanol was removed in vacuo and the residue was partitioned between dichloromethane and 2N sodium hydroxide solution. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give (R) -3-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazine (2 .72 g, 12.5 mmol).

  (R) -3-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazine (2.72 g, 12.5 mmol) was dissolved in N, N-dimethylformamide (20 ml) and water (3.0 ml) was added. ) In sodium nitrite (949 mg, 13.8 mmol) in 0). Next, 5N hydrochloric acid (6.0 ml) was added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 1 hour and then partitioned between ethyl acetate and water. The aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The resulting residue was dissolved in diethyl ether (50 ml) and lithium aluminum hydride (1.0 M; 9.51 ml, 9.51 mmol) in tetrahydrofuran was added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 1 hour and then quenched with ice water. Ethyl acetate was added to the mixture, the mixture was filtered through a celite plug, and the filter cake was washed with ethyl acetate. The filtrate was partitioned and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The residue was purified by flash chromatography eluting with 0-17% (v / v) ethyl acetate in heptane to give (R) -4-amino-3-cyclohexyl-3,4-dihydro-2H-1, 4-Benzoxazine (1.47 g, 6.33 mmol) was obtained.

  To a solution of (R) -4-amino-3-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazine (1.47 g, 6.33 mmol) in ethanol (40 ml) was added ethyl pyruvate (882 mg). , 7.59 mmol) was added. The reaction mixture was stirred at room temperature for 15 minutes. To the reaction mixture was added sulfuric acid (10% v / v solution in ethanol; 8.0 ml). The reaction mixture was refluxed for 2 hours. The mixture was cooled to room temperature and partitioned between ethyl acetate and sodium carbonate solution. The aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The residue was purified by flash chromatography eluting with 0-10% (v / v) ethyl acetate in heptane and ethyl (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de ] -1,4-benzoxazine-5-carboxylate (1.49 g, 4.76 mmol) was obtained.

  Ethyl (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine-5-carboxylate (1.49 g, 4.76 mmol) in ethanol (50 ml) ) Was added 4N sodium hydroxide (5.94 ml, 23.8 mmol). The mixture was stirred at 70 ° C. for 40 minutes. The ethanol was removed in vacuo and the residue was neutralized with 2N hydrochloric acid and partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The residue was dissolved in quinoline (20 ml) and copper powder (453 mg, 7.13 mmol) was added. The mixture was stirred at 210 ° C. for 1 hour. At room temperature, ethyl acetate and water were added to the mixture, the mixture was filtered through a celite plug, and the filter cake was washed with ethyl acetate. The filtrate was acidified with 5N hydrochloric acid and partitioned. The aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with 1N hydrochloric acid and brine, dried over sodium sulfate and concentrated. The residue was purified by flash chromatography eluting with 0-10% (v / v) ethyl acetate in heptane to give (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3- de] -1,4-benzoxazine (984 mg, 4.08 mmol) was obtained.

(R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine (80 mg, 1,2,2,2-tetrachloroethane (2.0 ml)) To the 0.33 mmol) solution, oxalyl chloride (46 mg, 0.36 mmol) was added with stirring under a nitrogen stream. The mixture was heated at 120 ° C. for 1 hour. The mixture was cooled to room temperature and triethylamine (36 mg, 0.36 mmol) and N-ethylpiperazine (45 mg, 0.40 mmol) were added. The mixture was stirred at room temperature for 18 hours and then partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The resulting brown oil was purified by flash chromatography using 5% (v / v) methanol in dichloromethane as eluent to give the title compound as the free base. Hydrochloride formation was performed by adding hydrogen chloride (2M solution in diethyl ether, 0.5 ml) to a solution of the free base in diethyl ether (2 ml) and ethanol (1 ml). The solvent was removed in vacuo and the precipitate was dried to give the title compound (1: 1 hydrochloride) as a solid (70 mg, 0.17 mmol).

¹ H NMR (400 MHz, CD ₃ OD) δ 1.00-1.40 (6H, m), 1.38 (3H, t, J7.3), 1.58 (1H, d, J12.4), 1 .62-1.70 (1H, m), 1.70-1.80 (2H, m), 1.80-1.90 (1H, m), 3.10-3.70 (6H, m) , 3.25 (2H, q, J7.3), 4.20-4.60 (4H, m), 4.71 (1H, dd, J3.0, 12.6), 6.66 (1H, d, J7.8), 7.08 (1H, t, J7.8), 7.22 (1H, d, J7.8), 7.74 (1H, s); EsIMS: m / z = 382. 2 [M + H] ⁺ , 268.2; [α] _D ²² −18.5 ° (in methanol, c = 1.4 mg / ml).

(R) -3-cyclohexyl-2,3-dihydro-6-[(S) -octahydro-2H-pyrido [1,2, a] pyrazin-2-ylcarbonyl] -pyrrolo [1,2,3-de ] -1,4-benzoxazine hydrochloride To a solution of (S)-(+)-1- (tert-butoxycarbonyl) -2-piperidinecarboxylic acid (2.00 g, 8.72 mmol) in dichloromethane (30 ml) , Glycine methyl ester hydrochloride (1.09 g, 8.72 mmol), 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (2.01 g, 10.46 mmol), 1-hydroxybenzotriazole ( 1.22 g, 9.04 mmol) and triethylamine (2.43 ml, 17.4 mmol) were added. The mixture was stirred for 18 hours under a nitrogen stream. The resulting mixture was washed with 0.5 M hydrochloric acid (20 ml), water (2 × 20 ml), brine (20 ml), dried over sodium sulfate and concentrated to (S) -1- (tert-butoxycarbonyl) piperidine-2- Carboxyglycine methyl ester was obtained as a colorless oil (2.47 g, 8.23 mmol).

  (S) -1- (tert-butoxycarbonyl) piperidine-2-carboxyglycine methyl ester (2.46 g, 8.20 mmol) was dissolved in trifluoroacetic acid (10 ml), and the resulting solution was stirred for 1 hour. The trifluoroacetic acid was then removed to give a colorless oil that was dissolved in methanol (85 ml) and triethylamine (9.0 ml, 64.6 mmol) was added. The resulting mixture was heated under reflux for 4 hours. The solution was then concentrated to give a pale orange oil that was recrystallized from 48% heptane, 48% ether, 4% 2-propanol, and (S) -octahydro-1,4- Dioxo-2H-pyrido [1,2-a] pyrazine was obtained as white crystals (0.66 g, 3.90 mmol).

  (S) -Octahydro-1,4-dioxo-2H-pyrido [1,2-a] pyrazine (0.5 g, 2.98 mmol) was added to lithium aluminum hydride (1M solution in tetrahydrofuran, 11.9 ml, 11 .9 mmol) was added in portions. The resulting mixture was heated at reflux for 0.5 hours. The solution was then cooled to 0 ° C. and treated dropwise with water (1.35 ml), 1M sodium hydroxide solution (0.45 ml) and then water (1.35 ml). Tetrahydrofuran (10 ml) was added and the solution was stirred for 0.5 hours and then filtered. The filter cake was washed with tetrahydrofuran (2 × 5 ml) and the combined filtrate and washings were concentrated to give (S) -octahydro-2H-pyrido [1,2, a] pyrazine as a yellow oil (0.29 g, 2.07 mmol).

(R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine (100 mg, 1,2,2,2-tetrachloroethane (2.0 ml)) To the 0.41 mmol) solution, oxalyl chloride (58 mg, 0.46 mmol) was added with stirring under a nitrogen stream. The mixture was heated at 120 ° C. for 1.5 hours. The mixture was cooled to room temperature and triethylamine (46 mg, 0.46 mmol) and (S) -octahydro-2H-pyrido [1,2-a] pyrazine (70 mg, 0.50 mmol) were added. The mixture was stirred at room temperature for 19 hours and then partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The resulting brown oil was purified by flash chromatography eluting with 50% (v / v) ethyl acetate in n-heptane followed by 0-17% (v / v) methanol in ethyl acetate. The compound was obtained as the free base. Hydrochloric acid salt formation was performed by adding hydrogen chloride (2M solution in diethyl ether, 1 ml) to a solution of the free base dissolved in diethyl ether (2 ml) and ethanol (2 ml). The solvent was removed in vacuo and the precipitate was dried to give the title compound (1: 1 hydrochloride) as a solid (78 mg, 0.18 mmol).

¹ H NMR (400 MHz, CD ₃ OD) δ1.00-1.35 (6H, m), 1.50-2.05 (11H, m), 3.00-3.10 (1H, m), 3 .10-3.30 (3H, m), 3.40-3.55 (3H, m), 4.20-4.30 (2H, m), 4.50-4.70 (2H, m) , 4.71 (1H, dd, J3.0, 12.6), 6.67 (1H, d, J7.2), 7.08 (1H, t, J7.8), 7.21 (1H, d, J 7.2), 7.74 (1H, S); EsIMS: m / z = 408.2 [M + H] ⁺ , 268.2; [α] _D ²² -27.5 ° (in methanol, c = 5.8 mg / ml).

(S) -3-cyclohexyl-2,3-dihydro-6-[(S) -octahydro-2H-pyrido [1,2-a] pyrazin-2-ylcarbonyl] pyrrolo [1,2,3-de] -1,4-Benzoxazine hydrochloride The title compound was prepared from (S) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,4] prepared from LN-Boc-cyclohexylglycine according to the procedure of Example 1. Prepared using the procedure described in Example 2 using 3-de] -1,4-benzoxazine.

¹ H NMR (400 MHz, CD ₃ OD) δ1.00-1.35 (6H, m), 1.50-2.05 (11H, m), 3.05 (1H, t, J10.4), 3 .10-3.30 (3H, m), 3.40-3.55 (3H, m), 4.20-4.30 (2H, m), 4.30-4.60 (2H, m) , 4.71 (1H, dd, J3.0, 12.6), 6.66 (1H, d, J8.0), 7.08 (1H, t, J8.0), 7.22 (1H, d, J 8.0), 7.73 (1H, S); EsIMS: m / z = 408.2 [M + H] ⁺ , 268.2; [α] _D ²² + 14.4 ° (in methanol, c = 1) .3 mg / ml).

(R) -3-cyclohexyl-2,3-dihydro-6-[(S) -3,4-dimethylpiperazin-1-ylcarbonyl] pyrrolo [1,2,3-de] -1,4-benzoxazine Hydrochloride (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine in N, N-dimethylformamide (5.0 ml) at 0 ° C. To a solution of 600 mg, 2.49 mmol) was added trifluoroacetic anhydride (0.311 ml, 2.73 mmol). The mixture was stirred at room temperature for 5 hours and then partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash chromatography eluting with 0-25% (v / v) ethyl acetate in heptane to give (R) -3-cyclohexyl-6-trifluoromethylcarbonyl-2,3-dihydropyrrolo [1. , 2,3-de] -1,4-benzoxazine (628 mg, 1.86 mmol) was obtained.

(R) -3-cyclohexyl-6-trifluoromethylcarbonyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine (628 mg, in 1,4-dioxane (20 ml). To a 1.86 mol) solution was added 4N NaOH (5.0 ml). The mixture was refluxed for 42 hours, then acidified to pH 1 using 5N hydrochloric acid and partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ , concentrated and purified (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3. -De] -1,4-benzoxazine-6-carboxylic acid (572 mg) was obtained.

(R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine-6-carboxylic acid in N, N-dimethylformamide (3.0 ml) ( 120 mg, 0.421 mmol) and (S) -1,2-dimethylpiperazine (62 mg, 0.547 mmol) in a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (97 mg, 0.505 mmol) and 1-Hydroxybenzotriazole (68 mg, 0.505 mmol) was added. The mixture was stirred at room temperature for 18 hours and then partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by flash chromatography eluting with 0-20% (v / v) methanol in ethyl acetate to give the title compound as the free base. Hydrochloride formation was performed by adding hydrogen chloride (2M solution in diethyl ether; 0.5 ml) to a solution of the free base in diethyl ether (2 ml) and ethanol (1 ml). The solvent was removed in vacuo and the precipitate was dried to give the title compound (1: 1 hydrochloride) as a solid (84 mg, 0.20 mmol).

¹ H NMR (400 MHz, CD ₃ OD) δ1.00-1.35 (5H, m), 1.39 (3H, d, J4.8), 1.58 (1H, d, J12.0), 1 .60-1.70 (1H, m), 1.70-1.82 (3H, m), 1.82-1.90 (1H, m), 2.96 (3H, s), 3.20 -3.70 (5H, m), 4.20-4.30 (2H, m), 4.40-4.70 (2H, m), 4.71 (1H, d, J10.0), 6 .67 (1H, d, J8.2), 7.08 (1H, t, J8.2), 7.21 (1H, d, J8.2), 7.74 (1H, s); EsIMS: m /Z=382.1 [M ⁺ H] ⁺ , 268.1.

  The following compounds were prepared using the method of Example 4 further.

  5A: Instead of (S) -1,2-dimethylpiperazine, (S) -2-methyl-piperazine was used, and (R) -3-cyclohexyl-2,3-dihydro-6-[(S)- 3-Methylpiperazin-1-ylcarbonyl] pyrrolo- [1,2,3-de] -1,4-benzoxazine hydrochloride was prepared.

EsIMS: m / z = 368 [M + H] ⁺ , 267.8; [α] _D ²² -44.4 ° (c = 2.3 mg / ml in methanol).

5B: In place of (S) -1,2-dimethylpiperazine, cis-2,6-dimethylpiperazine was used, and (R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -3 , 5-Dimethylpiperazin-1-ylcarbonyl] pyrrolo [1,2,3-de] -1,4-benzoxazine hydrochloride was prepared. EsIMS: m / z = 382.1 [M + H] ⁺ , 267.6; [α] _D ²² −19.6 ° (c = 2.8 mg / ml in methanol).

  5C: (R) -3-cyclohexyl-2,3-dihydro-6- [4-methylpiperazin-1-ylcarbonyl was substituted with N-methylpiperazine instead of (S) -1,2-dimethylpiperazine. Pyrrolo [1,2,3-de] -1,4benzoxazine hydrochloride was prepared.

EsIMS: m / z = 368 [M + H] ⁺ , 268; [α] _D ²² -20.3 ° (in methanol, c = 3.0 mg / ml).

(R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -2,6-dimethylpiperazin-1-ylcarbonyl] pyrrolo- [1,2,3-de] -1,4-benzo Oxazine hydrochloride To a solution of (cis) -2,6-dimethylpiperazine (900 mg, 8.49 mmol) and sodium bicarbonate (saturated solution 0.2 ml) in THF (5 ml) was added benzyl bromide (1.02 ml, 8 ml). .49 mmol) was added. This mixture was exposed to microwave irradiation at 80 ° C. for 15 minutes. The solvent was removed in vacuo and the residue was washed with sodium bicarbonate solution and extracted with dichloromethane. The residue is purified by flash chromatography eluting with 5-10% (v / v) methanol in dichloromethane to give 1-N-benzyl- (cis) -3,5-dimethylpiperazine as a clear oil. (900 mg, 4.40 mmol).

  A solution of (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine-6-carboxylic acid (362 mg, 1.23 mmol) in dichloromethane (20 ml) To this was added oxalyl chloride (0.215 ml, 2.46 mmol). The dark blue mixture was stirred at room temperature for 2 hours, then the solvent was removed in vacuo to give (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4- Benzoxazine-6-carboxylic acid chloride (380 mg, 1.23 mmol) was obtained as a blue solid.

  (R) -3-cyclohexyl-2,3-dihydropyrrolo [1,2,3-de] -1,4-benzoxazine-6-carboxylic acid chloride (380 mg, 1.23 mmol) in dichloromethane (20 ml) And to a solution of N, N diisopropylethylamine (0.2 ml, 1.3 mmol) was added 1-N-benzyl- (cis) -3,5-dimethylpiperazine (250 mg, 1.2 mmol) in dichloromethane (5 ml). The mixture was stirred at room temperature for 16 hours. The mixture was partitioned between sodium bicarbonate solution and dichloromethane. The organic layer was separated and the solvent was removed in vacuo. The residue was purified by flash chromatography eluting with 0-5% (v / v) methanol in dichloromethane. Hydrochloride formation was carried out by adding hydrogen chloride (2M solution in diethyl ether, 2 ml) to the free base in dichloromethane (1 ml). The precipitate was filtered and dried, and (R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -4-benzyl-2,6-dimethylpiperazin-1-ylcarbonyl] pyrrolo [1, 2,3-de] -1,4-benzoxazine hydrochloride was obtained as a light blue powder (240 mg, 0.51 mmol).

  (R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -4-benzyl-2,6-dimethylpiperazin-1-ylcarbonyl] pyrrolo [1,2,3 in ethanol (5 ml) To a solution of -de] -1,4-benzoxazine hydrochloride (200 mg, 0.42 mmol), 10% palladium on charcoal (10 mg) was added. The mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. The mixture was filtered and the solvent removed in vacuo to give the title compound as a white solid (100 mg, 0.26 mmol).

¹ H NMR (400 Hz, CD ₃ OD) δ 1.03-1.39 (5H, m), 1.49 (6H, d, J7.2), 1.55-1.87 (6H, m), 3 .47 (4H, m), 4.29 (2H, m), 4.72 (1H, d, J9.8), 4.80 (2H, m), 6.66 (1H, d, J7.5) ), 7.09 (1H, m, J7.7), 7.19 (1H, d, J8.9), 7.66 (1H, s); EsIMS: m / z = 382.3 [M + H] ⁺ , 268; [α] _D ²² -17.0 ° (in methanol, c = 2.4 mg / ml).

(R) -3-Cyclohexyl-2,3-dihydro-6-[(cis) -3,4,5-trimethylpiperazin-1-ylcarbonyl] pyrrolo [1,2,3-de] -1,4- Benzoxazine hydrochloride (R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -3,5-dimethylpiperazin-1-ylcarbonyl] pyrrolo [1,2,3 in ethanol (10 ml) -De] -1,4-benzoxazine hydrochloride (100 mg, 0.26 mmol) in a solution of formaldehyde (37% aqueous solution; 1 ml, 12.5 mmol) and sodium triacetoxyborohydride (200 mg, 0.93 mmol). Added. The mixture was stirred at room temperature for 30 minutes. The solvent was removed in vacuo. The residue was purified by flash chromatography eluting with 1-10% (v / v) methanol in dichloromethane to give the title compound as the free base. Hydrochloride formation was carried out by adding hydrogen chloride (2M solution in diethyl ether; 2 ml) to the free base in dichloromethane (1 ml). The precipitate was filtered and dried to give the title compound (1: 1 hydrochloride). EsIMS: m / z = 396.1 [M + H] ⁺ , 268; [α] _D ²² −19.6 ° (in methanol, c = 2.1 mg / ml).

(R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -3,5-dimethyl-4-ethylpiperazin-1-ylcarbonyl] pyrrolo [1,2,3-de] -1, 4-Benzoxazine hydrochloride Following the procedure described in Example 7, the title compound was prepared using acetaldehyde instead of formaldehyde. EsIMS: m / z = 410.3 [M + H] ⁺ , 268; [α] _D ²² −17.8 ° (in methanol, c = 2.0 mg / ml).

(R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -3,5-dimethyl-4- (2-hydroxyethyl) piperazin-1-ylcarbonyl] pyrrolo [1,2,3- de] -1,4-benzoxazine hydrochloride (R) -3-cyclohexyl-2,3-dihydro-6-[(cis) -3,5-dimethylpiperazin-1-ylcarbonyl in acetonitrile (3 ml) To a solution of pyrrolo [1,2,3-de] -1,4-benzoxazine hydrochloride (120 mg, 0.31 mmol) was added 2-bromoethanol (0.024 ml, 0.34 mmol). This mixture was exposed to microwave irradiation at 150 ° C. for 30 minutes. The mixture was filtered and the solvent was removed in vacuo. Using HPLC (Waters Xterra [RP18, 5 μm] 30 mm × 10 mm column, 10-100% [v / v] acetonitrile solution in water, gradient over 25 min, 0.1% trifluoroacetic acid buffer, detection at 254 nm UV) The residue was purified to give the title compound as a trifluoroacetic acid (TFA) salt. Hydrochloride formation was carried out by adding hydrogen chloride (2M solution in diethyl ether; 2 ml) to the TFA salt in dichloromethane (1 ml). The precipitate was filtered and dried to give the title compound (20 mg, 0.04 mmol).

¹ H NMR (400 Hz, CD ₃ OD) δ 1.01-1.39 (5H, m), 1.44 (6H, d, J5.3), 1.56-1.85 (6H, m), 3 .32 (2H, d, J14.2), 3.54 (2H, s), 3.72 (2H, m), 3.82 (0.5H, m), 3.93 (1.5H, d) , J4.5), 4.24-4.28 (3H, m), 4.36-4.40 (0.5H, d, J11.6), 4.52 (1.5H, d, J13. 1), 4.69 (1H, d, J10.1), 6.66 (1H, d, J7.5), 7.06-7.10 (1H, m), 7.20 (1H, d, J8.0), 7.75 (1H, s); EsIMS: m / z = 426.1 [M + H] ⁺ , 268; [α] _D ²² −18.3 ° (in methanol, c = 2.2 mg / ml).

(R) -3-Cyclohexyl-2,3-dihydro-6-[(cis) -3,5-dimethyl-4- (2-methoxyethyl) piperazin-1-ylcarbonyl] pyrrolo [1,2,3- de] -1,4-benzoxazine hydrochloride The title compound was prepared using the procedure described in Example 9 using 2-bromoethyl methyl ether instead of 2-bromoethanol.

EsIMS: m / z = 440 [M + H] ⁺ , 268; [α] _D ²² -20.8 ° (in methanol, c = 2.5 mg / ml).

(Rac) -4-cyclopentyl-5,6-dihydro-1- (4-ethylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] -quinoline hydrochloride Cooled in an ice / methanol bath To a solution of 2-chloroquinoline (8.2 g, 50 mmol) and [1,2-bis (diphenylphosphino) -ethane] dichloronickel (II) (200 mg, 0.38 mmol) in diethyl ether (20 ml) , Cyclopentylmagnesium bromide (2M solution in diethyl ether; 25.5 ml, 51 mmol) was added over 15 minutes. The resulting brown solution was then stirred for 30 minutes, the ice bath was removed and the mixture was stirred for an additional 10 minutes. The reaction mixture was then cooled again to 0 ° C. and saturated ammonium chloride solution (40 ml) was added slowly. The resulting reaction mixture was poured into a separatory funnel and further diluted with diethyl ether (60 ml) and saturated ammonium chloride solution (60 ml). The organic layer was separated and the aqueous layer was washed with diethyl ether (2 × 100 ml). The combined organic layers were dried (MgSO ₄ ), filtered and the solvent removed in vacuo to give 2-cyclopentylquinoline (9.98 g, 50.4 mmol) as an oil.

To a solution of 2-cyclopentylquinoline (7.95 g, 40.3 mmol) and nickel (II) chloride hexahydrate (1.63 g, 6.85 mmol) in methanol (120 ml) cooled in an ice / methanol bath, Sodium borohydride (6.1 g, 161.2 mmol) was added in portions over 1.5 hours. The cooling layer was removed and the reaction was stirred for an additional 30 minutes. The methanol was then removed in vacuo. To the resulting black precipitate was added 2M hydrochloric acid (100ml) and then basified with 10M potassium hydroxide. The mixture was poured into a separatory funnel and extracted with ether (4 × 200 ml). The combined organic layers were dried (MgSO ₄ ), filtered and the solvent removed in vacuo to give 2-cyclopentyl-1,2,3,4-tetrahydroquinoline as a light brown oil (7.45 g, 37 .1 mmol).

To a solution of 2-cyclopentyl-1,2,3,4-tetrahydroquinoline (4.0 g, 20 mmol) in tetrahydrofuran (15 ml), ethyl bromide pyruvate (˜90% purity; 1.38 ml, 9.9 mmol) Was added and the reaction was stirred for 15 hours. The resulting precipitate was filtered and washed with tetrahydrofuran (20 ml). The filtrate was evaporated in vacuo. The resulting residue was dissolved in tetrahydrofuran (10 ml) and 2-methoxyethanol (10 ml) and this solution was added to a refluxing solution of magnesium (II) chloride (1.05 g, 11 mmol) in 2-methoxyethanol (10 ml). Added dropwise. The reaction mixture was refluxed for 2 hours, then more magnesium (II) chloride (1.05 g, 11 mmol) was added and reflux was continued overnight. The reaction was cooled and the solvent removed in vacuo. The resulting brown oil was dissolved in dichloromethane (150 ml) and washed with 2M HCl (50 ml), saturated potassium carbonate solution (50 ml) and brine (50 ml). The organic layer was dried (MgSO ₄ ), filtered and the solvent removed in vacuo. The resulting oil was purified by flash chromatography using 33-67% (v / v) dichloromethane in heptane to give 4-cyclopentyl-5,6-dihydro-4H-pyrrolo [3,2,1-ij. Quinolin-1-carboxylic acid ethyl ester (1.45 g, 4.9 mmol) was obtained.

  4-Cyclopentyl-5,6-dihydro-4H-pyrrolo [3,2,1-ij] quinoline-1-carboxylic acid ethyl ester (1.45 g, 4.9 mmol) in water (10 ml) and ethanol (15 ml) To the solution was added sodium hydroxide (1.96 g, 19 mmol) and the reaction mixture was heated at reflux overnight (˜14 hours). The reaction was cooled and acidified with 5M HCl. The resulting white precipitate was filtered and dried under vacuum to give 4-cyclopentyl-5,6-dihydro-4H-pyrrolo [3,2,1-ij] quinoline-1-carboxylic acid (1.13 g, 4.2 mmol). ) Was obtained as a white solid.

To a solution of 4-cyclopentyl-5,6-dihydro-4H-pyrrolo [3,2,1-ij] quinoline-1-carboxylic acid (342 mg, 1.27 mmol) in dichloromethane (20 ml) was added oxalyl chloride (218 μl, 3.18 mmol) was added and the reaction mixture was stirred for 1 hour. Then more oxalyl chloride (436 μl, 6.36 mmol) was added and the reaction was stirred overnight. Solvent and excess reagent were removed in vacuo leaving a green solid. This green solid was dissolved in dichloromethane (20 ml) and N-ethylpiperazine (323 μl, 2.54 mmol) was added dropwise. The resulting reaction mixture was stirred for 1 hour and then poured into a separatory funnel. The organic layer was washed with saturated potassium bicarbonate solution (20 ml) and brine (20 ml), dried (MgSO ₄ ) and the solvent removed in vacuo leaving a brown oil. The oil was purified by flash chromatography eluting with 0-5% (v / v) methanol in dichloromethane to give the title compound (400 mg, 1.09 mmol). ¹ H NMR (400 MHz, CD ₃ OD) δ _H 1.31-1.43 (4H, m), 1.45-1.79 (6H, m), 1.87-1.96 (1H, m) , 2.05-2.24 (2H, m), 2.30-2.37 (1H, m), 2.90 (1H, dt, J16.6, 3.9), 3.03-3. 20 (3H, m), 3.26 (2H, q, J7.5), 3.45-3.65 (4H, m), 4.16-4.23 (1H, m), 4.57 ( 2H, d, J14, 7), 6.99 (1H, d, J7.1), 7.12 (1H, t, J8.0), 7.48 (1H, d, J8.0), 7. 76 (1H, s); EsIMS: m / z = 366.3 [M + H] ⁺ , 252.1.

The product obtained in Example 11 was subjected to chiral HPLC separation on Chiracel ^(R) OD column (2cm x25cm), using isohexane / isopropanol 92/8 to (v / v), at a flow rate of 15ml / min Eluted. When the product was detected using a UV detector at a wavelength of 240 nm, enantiomer 1 (retention time 24.18 minutes, enantiomeric excess> 89%) and enantiomer 2 (retention time 33.6 minutes). Enantiomeric excess> 92%).

12A: (+)-4-cyclopentyl-5,6-dihydro-1- (4-ethylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] quinoline hydrochloride in dichloromethane (5 ml) To a solution of enantiomer 1 of (147 mg, 0.38 mmol) was added hydrochloric acid (2M solution in diethyl ether; 0.5 ml). Excess reagent and solvent were removed in vacuo leaving the title compound as a white solid.

EsIMS: m / z = 366.1 [M + H] +, 252.1; [α] _D ²² + 25.8 ° (in methanol, c = 2.6 mg / ml).

12B: (−)-4-cyclopentyl-5,6-dihydro-1- (4-ethylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] quinoline hydrochloride in dichloromethane (5 ml) To a solution of enantiomer 2 of (143 mg, 0.38 mmol) was added hydrochloric acid (2M solution in diethyl ether; 0.5 ml). Excess reagent and solvent were removed in vacuo leaving the title compound as a white solid.

EsIMS: m / z = 366.0 [M + H] ⁺ , 252.1; [α] _D ²² -21.3 ° (in methanol, c = 2.4 mg / ml)

(+)-4-Cyclocyclohexyl-5,6-dihydro-1- (4-ethylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] -quinoline hydrochloride Chlorobenzene (300 ml) To a solution of quinoline (11.84 ml, 100 mmol) in water (300 ml), cyclohexanecarboxylic acid (35.88 g, 280 mmol), silver nitrate (1.36 g, 8.0 mmol), ammonium persulfate (22.82 g, 100 mmol) And trifluoroacetic acid (7.67 g, 100 mmol) were added in succession. The mixture was heated to 140 ° C. with stirring for 3 hours. The mixture was then cooled to room temperature, basified with solid sodium hydroxide and the solvent removed in vacuo. The residue was then subjected to 18 hours of continuous extraction with isohexane, the solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography eluting with 20% (v / v) ethyl acetate in isohexane. 2-cyclohexylquinoline was obtained as an oily substance (2.66 g, 12.61 mmol).

  A solution of 2-cyclohexylquinoline (2.66 g, 12.61 mmol) in glacial acetic acid (25 ml) was treated with sodium cyanoborohydride (2.38 g, 37.82 mmol), 4 hours at room temperature, then 40 ° C. For 18 hours. The mixture was then treated with 2M sodium hydroxide (200ml), stirred for 30 minutes and extracted into ethyl acetate (3x100ml). The combined organic layers were then washed with water (3 × 100 ml), dried over sodium sulfate, evaporated under reduced pressure and the residue removed by flash chromatography eluting with 0-3% (v / v) ethyl acetate in isohexane. Purification gave 2-cyclohexyl-1,2,3,4-tetrahydroquinoline as a yellow oil (1.61 g, 7.49 mmol).

2-Cyclohexyl-1,2,3,4-tetrahydroquinoline (2.61 g, 12.14 mmol) in ethanol (75 ml) was added to (R)-(−)-2-hydroxy-5,5-dimethyl-4. -Treated with phenyl-1,3,2-dioxaphosphorinane-2-oxide (2.94 g, 12.14 mmol) and stirred the mixture at 50 ° C. until completely dissolved. The mixture was then evaporated under reduced pressure until the total volume was 30 ml and left to crystallize for 3 hours. The suspension was filtered and the colorless precipitate was recrystallized from ethanol to give a colorless crystalline solid (2.1 g). This was treated with saturated sodium carbonate solution (50 ml) and extracted into dichloromethane (2 × 50 ml). The combined organic layers were then dried over sodium sulfate and evaporated under reduced pressure to give non-racemic 2-cyclohexyl-1,2,3,4-tetrahydroquinoline as a colorless oil (0.98 g 4.56 mmol). Isohexane / ethanol 97: by chiral HPLC on a Chiralcel ^(R) OJ column eluting with a flow rate of 1 ml / min using a 3 (v / v), the enantiomeric excess was determined to 94%. Enantiomers were detected at a wavelength of 230 nm using a UV detector. The enantiomer eluted with retention times of 8.4 minutes (97%) and 9.4 minutes (3%).

According to the procedure of Example 1, instead of (R) -3-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazine, the above non-racemic 2-cyclohexyl-1,2,3, 4-Tetrahydroquinoline was used to give the title compound as a colorless solid (0.05 g, 0.12 mmol). ¹ H NMR (400 MHz, CDCl ₃ ) δ _H 1.05-1.26 (5H, m), 1.48-1.87 (9H, m), 2.01-2.11 (1H, m), 2.30-2.39 (1H, m), 2.70-3.12 (6H, m), 3.44-3.52 (2H, m), 3.96-4.13 (3H, m ), 4.53 (2H, d, br, J14.1), 7.00 (1H, d, J7.5), 7.16 (1H, t, J7.4), 7.44 (1H, d , J8.1), 7.59 (1H, s). EsIMS: m / z 380 [M + H] ⁺ , 266; [α] _D ²² + 42.6 ° (in methanol, c = 2.7 mg / ml).

(+)-(4-Cyclohexyl-5,6-dihydro-1- (4-methylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] -quinoline hydrochloride as described in Example 13 According to the procedure described, the title compound was prepared using N-methylpiperazine instead of N-ethylpiperazine: ¹ H NMR (400 MHz, CDCl ₃ ) δ _H 1.05-1.28 (6H, m) , 1.51-1.87 (5H, m), 2.02-2.11 (1H, m), 2.31-2.39 (1H, m), 2.76-3.10 (7H, m), 3.41-3.50 (2H, m), 3.92-4.10 (3H, m), 4.55 (2H, d, br, J13.8), 7.00 (1H, d, J7.1), 7.16 (1H, t, J7.0), 7.44 (1H, d, J8.1) ), 7.59 (1H, s) EsIMS: m / z 366 [M + H] ⁺ , 266; [α] _D ²² + 19.3 ° (in methanol, c = 1.5 mg / ml).

(−)-(4-Cyclohexyl-5,6-dihydro-1- (4-ethylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] quinoline hydrochloride as described in Example 13 In place of (R)-(−)-2-hydroxy-5,5-dimethyl-4-phenyl-1,3,2-dioxaphosphorinane-2-oxide, (S)-(+ ) -2-Hydroxy-5,5-dimethyl-4-phenyl-1,3,2-dioxaphosphorinane-2-oxide using non-racemic 2-cyclohexyl-1,2,3,4-tetrahydro Quinoline was prepared.

The enantiomeric excess of the 2-cyclohexyl-1,2,3,4-tetrahydroquinoline intermediates, isohexane / ethanol 97: 3 (v / v) and eluted at a flow rate of 1 ml / min using a Chiracel ^(R) OJ column Was 86% by chiral HPLC. This enantiomer was detected using a UV detector at a wavelength of 230 nm with retention times of 8.4 minutes (7%) and 9.4 minutes (93%). According to the procedure of Example 1, in place of (R) -3-cyclohexyl-3,4-dihydro-2H-1,4-benzoxazine, this non-racemic 2-cyclohexyl-1,2,3,4-tetrahydro The title compound was prepared using quinoline. ¹ H NMR (400 MHz, CDCl ₃ ) δ _H 1.05-1.26 (5H, m), 1.48-1.87 (9H, m), 2.01-2.11 (1H, m), 2.30-2.39 (1H, m), 2.70-3.12 (6H, m), 3.44-3.52 (2H, m), 3.96-4.13 (3H, m ), 4.53 (2H, d, br, J14.1), 7.00 (1H, d, J7.5), 7.16 (1H, t, J7.4), 7.44 (1H, d , J8.1), 7.59 (1H, s). EsIMS: m / z 380 [M + H] ⁺ , 266; [α] _D ²² -28.4 ° (in methanol, c = 1.6 mg / ml).

(−)-(4-Cyclohexyl-5,6-dihydro-1- (4-methylpiperazin-1-ylcarbonyl) -4H-pyrrolo [3,2,1-ij] -quinoline hydrochloride as described in Example 15 According to the procedure described, the title compound was prepared using N-methylpiperazine instead of N-ethylpiperazine.

¹ H NMR (400 MHz, CDCl ₃ ) δ _H 1.05-1.28 (6H, m), 1.51-1.87 (5H, m), 2.02-2.11 (1H, m), 2.31-2.39 (1H, m), 2.76-3.10 (7H, m), 3.41-3.50 (2H, m), 3.92-4.10 (3H, m ), 4.55 (2H, d, br, J13.8), 7.00 (1H, d, J7.1), 7.16 (1H, t, J7.0), 7.44 (1H, d , J8.1), 7.59 (1H, s). EsIMS: m / z 366 [M + H] ⁺ , 266; [α] _D ²² -46.2 ° (in methanol, c = 2.1 mg / ml).

  In vitro measurement of efficacy and potency at the human CB1 receptor expressed in CHO cells.

Chinese hamster ovary (CHO) cells expressing human CB1 receptor and luciferase reporter gene were transformed into phenol red / serum-free DMEM / F-12Nut containing penicillin / streptomycin (50 U / 50 μg / ml) and fungizone (1 μg / ml). Suspended in Mix and seeded into 96-well plates at a density of 3 × 10 ⁴ cells per well (100 μl final volume). The cells were incubated overnight (about 18 hours at 37 ° C., 5% CO ₂ /95% air) before the assay.

  Test compounds (10 mM solution in dimethyl sulfoxide) were diluted with F12 Nut Mix to make stock solutions in the range of 0.11 mM to 0.11 nM. The stock solution (10 μl) was added directly to the appropriate wells. The plate was incubated at 37 ° C. for 5 hours to allow agonist-induced expression of the luciferase enzyme. Under reduced illumination, LucLite substrate (Packard; reconstituted according to manufacturer's instructions; 100 μl) was added to each well. Plates were covered with a top seal and then incubated at room temperature for 5 minutes before counting on a Packard TopCount (single photon counting, counting time 0.01 minutes, counting delay 5 minutes).

To obtain EC ₅₀ values, a “best fit” curve was fitted by the least sum of squares method on a plot of counts per second (CPS) versus compound concentration (M). Table 1 shows the pEC ₅₀ values obtained for some representative compounds of the invention.

Tail flick latency in mice While measuring tail flick latency, mice were trained to be in the tail flick device (Ugo, Basile, Italy). The tail was exposed to a focused beam of radiant heat at about 2.5 cm from the tip. Tail flick latency was defined as the interval between application of thermal stimulus and tail withdrawal. To prevent tissue damage, a 12 second interruption was performed. Four groups of 8 mice were administered intravenously with one of three doses of vehicle or test compound (vehicle: saline 9 g / l; injection volume 10 ml / kg). Tail flick latency was measured before test compound administration and at regular intervals (usually 20 minutes, 40 minutes and 60 minutes) after compound administration. ED ₅₀ was calculated at _{T max.}

The compounds of Examples 2, 4, 14, 15, and 16 significantly increased tail flick latency, with an ED ₅₀ of <5 μmol / kg.

Claims (7)

A tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative having the general formula (I),

(Where
X is CH ₂ , O or S;
R represents 1 to 3 substituents independently selected from H, (C _1-4 ) alkyl, (C _1-4 ) -alkyloxy and halogen;
R ₁ is (C _5-8 ) cycloalkyl;
R ₂ is H or (C _1-4 ) alkyl;
R ₃ , R ₃ ′, R ₄ , R ₄ ′, R ₅ , R ₅ ′ and R ₆ ′ are independently hydrogen or (C _1-4 ) alkyl ((C _1-4 ) alkyloxy, OH or Optionally substituted by halogen));
R ₆ is hydrogen or (C _1-4 ) alkyl (optionally substituted by (C _1-4 ) alkyloxy, OH or halogen); or R ₆ together with R ₇ Forming a 4 to 7 membered saturated heterocycle optionally containing additional heteroatoms selected from O and S;
R ₇ together with R ₆ forms a 4 to 7 membered saturated heterocycle optionally containing additional heteroatoms selected from O and S; or
R ₇ is H, (C _1-4 ) alkyl or (C _3-5 ) cycloalkyl, wherein the alkyl group is optionally substituted with OH, halogen or (C _1-4 ) alkyloxy. Or a pharmaceutically acceptable salt thereof. The tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative according to claim ₁ , wherein R is H and R ₁ is cyclopentyl or cyclohexyl. The tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative according to claim 1 or 2, wherein X is CH ₂ or O. R, R ₂ , R ₃ , R ₃ ′, R ₄ ′, R ₅ , R ₅ ′ and R ₆ ′ are H; R ₄ , R ₆ and R ₇ are independently H or (C _{1- 4} ) is alkyl; or R ₆ together with R ₇ forms a 5- or 6-membered saturated heterocycle, and R ₄ is H or (C _1-4 ) alkyl. The tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative according to any one of the above.   5. A tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative according to any one of claims 1 to 4 for use in therapy.   A pharmaceutical composition comprising a tricyclic 1-[(indol-3-yl) carbonyl] -piperazine derivative according to any one of claims 1 to 4 together with a pharmaceutically acceptable carrier.   Use of a tricyclic 1-[(indol-3-yl) carbonyl] piperazine derivative of formula I as defined in claim 1 in the preparation of a medicament for the treatment of pain.