8-Phenylisoquinoline and its pharmaceutical composition for treating irritable bowel syndrome

Abstract

A series of 8-phenyl-isoquinoline derivatives (I) exhibit high binding affinity for the 5-HT7 receptor (5HT7R) and show potential analgesic activity in two animal models of irritable bowel syndrome (IBS) by intraperitoneal injection (i.p.) or by oral gavage (p.o.). These 5-HT7 receptor antagonists are a novel class of therapeutic agents for the treatment of IBS. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a series of 8-phenylisoquinoline derivatives for the treatment of irritable bowel syndrome (IBS). Background Art

[0002] The 5-HT7 receptor (5-HT7R) is the most recently identified member of the 14 subtypes of the 5-HT receptor family. It is widely distributed in the central nervous system (CNS) (most abundantly in the hypothalamus, thalamus, hippocampus, and cortex) and in peripheral organs (such as the spleen, kidney, intestine, heart, and coronary arteries), which implies its role in various physiological functions and pathological processes. The 5-HT7R is positively coupled to adenylyl cyclase and has low sequence homology (less than 40%) with other 5-HT receptor subtypes. Based on studies using selective 5-HT7R ligands and knock-out mouse models, the 5-HT7R is involved in circadian rhythm regulation, thermoregulation, sleep disorders, affective disorders, pain, learning, and memory. Thus, 5-HT7R ligands are potential therapeutic agents for treating various 5-HT7R-related diseases and disorders. 5-HT7R antagonists may be effective therapeutic agents for depression, anxiety, schizophrenia, and dementia, while 5-HT7R agonists may be potential therapeutic agents for pain and pain symptoms (especially neuropathic pain and inflammatory pain).

[0003] In addition, the 5-HT7R is also a potential drug target for migraine (WO2009029439A1), hypertension, various mucosal inflammations (WO2012058769A1) (such as irritable bowel syndrome), and urinary incontinence, through its effective smooth muscle relaxation effects on central and peripheral blood vessels and on intestinal, colonic, and bladder tissues, respectively. Several therapeutic agents (such as tricyclic antidepressants, typical and atypical antipsychotic drugs, and some 5-HT2 receptor antagonists) have been found to exhibit moderate to high affinity for the 5-HT7R.

[0004] Given the multiple therapeutic potential of 5-HT7R ligands, many efforts have been focused on the discovery and development of selective 5-HT7R agonists and antagonists. 5-HT7R ligands of different structural types have been reported, including 5-HT7R agonists (such as AS-19, LP-44, LP-12, LP-211, and E-55888) and 5-HT7R antagonists (such as SB-258719, SB-269970, SB-656104, DR-4004, and JNJ-18038683). Despite many efforts, there is still a need to further develop novel 5-HT7R ligands with appropriate physicochemical and pharmacokinetic properties as potential therapeutic agents for treating 5-HT7R-related diseases and disorders.

[0005] Irritable bowel syndrome (IBS) is mainly characterized by recurrent abdominal pain associated with changes in bowel habits in the absence of recognizable organic causes or visible lesions. Among gastroenterology outpatients in Asian and Western countries, approximately 10 to 15% are related to IBS. Severe abdominal pain is a clinical feature of IBS and is the symptom most likely to lead to medical consultation. Subtypes of IBS include diarrhea-predominant IBS-D, constipation-predominant IBS-C, or alternating IBS-A. The development of IBS is thought to be related to disorders of the brain-gut axis; however, the pathogenesis remains poorly understood.

[0006] Altered levels of gut serotonin (serotonin (5-HT)) in patients are a validated biomarker for IBS. However, clinical drugs targeting 5-HT receptors for IBS treatment are currently limited and can only be prescribed under an emergency investigational drug protocol. Alosetron (a 5-HT3R antagonist used to treat IBS-D) has been withdrawn by the FDA due to severe side effects (such as ischemic colitis, cerebrovascular or cardiovascular ischemia) and was later reintroduced only for women with severe symptoms. Other available symptomatic relievers (such as antispasmodics, antidiarrheals, osmotics, sedatives, antidepressants, etc.) are not widely effective for patients. Medical research on the pathogenesis of IBS largely relies on the analysis of patient biopsies. Animal models with visceral hypersensitivity have been established, although each model has its weaknesses and strengths in terms of its translational value for IBS. Therefore, the progress of IBS treatment development has been hindered. To date, the development of novel targeted drugs for clinical treatment of IBS is highly needed.

[0007] A variety of risk factors, including psychological stress, intestinal infections, immune and inflammatory responses, genetic predisposition, and changes in the gut microbiota, have been found to contribute to the development of IBS symptoms. A high proportion of IBS patients report past traumatic events in childhood or adulthood. IBS symptoms may start after an episode of infectious gastroenteritis, known as post-infectious (PI)-IBS. Follow-up studies of a waterborne giardiasis outbreak in Norway reported that over 40% of patients experienced IBS-like symptoms for up to three years after acute Giardia lamblia infection. The exacerbation of these post-infectious symptoms was associated with the experience of physical or mental stress. Experimental models following clearance of pathogen infection and resolution of chemically induced small intestinal colitis have shown intestinal hyperalgesia. In addition, animals subjected to psychological stress also exhibit visceral hypersensitivity to colorectal distension. Two mouse models with IBS-like visceral hypersensitivity, including double stimulation with Giardia post-infection combined with psychological stress and after resolution of colitis induced by trinitrobenzenesulfonic acid (TNBS), were used to test the analgesic effects of novel 5-HT7R ligands.

[0008] Among the 5-HT receptor subtypes, 5-HT7R is the most recently discovered family member with unknown pathophysiological roles. Stimulation of 5-HT7R causes excessive relaxation of circular smooth muscle, which implies ineffective gas propulsion and abdominal distension. Expression of 5-HT7R has been identified in enteric neurons (i.e., myenteric afferent neurons and mucosal nerve fibers), smooth muscle, and dendritic cells in the colon, as well as in the lumbar dorsal root ganglion and the brain. The present invention demonstrates the role of a series of 8-phenylisoquinoline derivatives in alleviating intestinal pain in two IBS animal models. Summary of the Invention

[0009] The present invention relates to a novel compound of the following general formula or a pharmaceutically acceptable salt thereof:

[0010]

[0011] wherein R1 is selected from hydrogen, C 1-10 straight-chain alkyl, C 1-10 branched-chain alkyl, (CH2) n (Hete)R 10 R 11 R 12 and (CH2)n ArR 10 R 11 R 12 , wherein n is an integer from 0 to 6, Hete is an aromatic heterocyclic group, Ar is an aromatic ring group, and R 10 、R 11 and R 12 are independently selected from hydrogen, a halogen group, a nitro group, an amino group, a cyano group, an acetyl group, C 1-6 linear saturated alkyl, C 1- 6 linear saturated alkoxy, and C 1-6 linear saturated haloalkyl; R2 is hydrogen or C 1-6 linear saturated alkyl; and X1, X2, X3, X4 and X5 are independently selected from hydrogen, a halogen group, a nitro group, an amino group, a cyano group, an acetyl group, C 1-6 linear saturated alkyl, C 1-6 branched saturated alkyl, C 1-6 linear saturated alkoxy, C 1-6 branched saturated alkoxy, C 1-6 linear saturated alkylthio, C 1-6 branched saturated alkylthio, C 1-6 linear saturated haloalkyl and C 1-6 branched saturated haloalkyl.

[0012] The present invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of this novel compound or a pharmaceutically acceptable salt thereof. In addition, the present invention relates to a method of using the above pharmaceutical composition to treat irritable bowel syndrome. Description of the Drawings

[0013] Figure 1 、 8 -Synthesis Scheme 1 of new derivatives of phenylisoquinoline.

[0014] Figure 2 、 8 -Synthesis Scheme 2 of new derivatives of phenylisoquinoline.

[0015] Figure 3 、 8 -Synthesis Scheme 3 of new derivatives of phenylisoquinoline.

[0016] Figure 4 、 8 -Synthesis Scheme 4 of new derivatives of phenylisoquinoline.

[0017] Figure 5. Visceral hypersensitivity observed in a murine model of IBS-like following dual stimulation with Giardia combined with stress. (A) The visceromoter response (VMR) to colorectal distension was expressed as the area under the curve (AUC) and measured in each mouse as an index of enteric pain. (B) Representative images of colonic histology in PN and GW mice. (C) Representative images of immunostained 5-HT7R in colonic tissues of PN and GW mice (panel a), and quantification of 5-HT7R immunoreactivity in the muscular / nerve and mucosal layers (panel b and panel c). (D) Western blotting results showed increased 5-HT7R protein levels in GW mice.

[0018] Figure 6. Visceral hypersensitivity noted in a murine model of IBS-like following resolution of TNBS-induced colitis. (A) The visceromoter response (VMR) to colorectal distension was expressed as the area under the curve (AUC) and measured in each mouse as an index of enteric pain. (B) Intestinal myeloperoxidase (MPO) activity was examined as an index of inflammatory leukocyte activation. (C) Histopathological scores of murine colonic tissues. (D) Representative images of colonic histology in sham and TNBS mice. (E) Immunostaining of 5-HT7R in colonic tissues of sham and TNBS-d24 mice. Representative images of 5-HT7R staining (panel a), and quantification of 5-HT7R immunoreactivity in the muscular / nerve and mucosal layers (panel b and panel c). (F) Protein levels of 5-HT7R in murine colon. (G) Transcriptional levels of 5-HT7R in murine colon.

[0019] Figure 7 Analgesic effect of the 5-HT7R antagonist SB269970 (SB7) in a murine model of IBS-like.

[0020] Figure 8 Analgesic effect of oral administration of a novel 8-phenylisoquinoline derivative in GW mice.

[0021] Figure 9. Dose- and time-response of compound 8 in enteric pain in GW mice. (A) Compound 8 was intraperitoneally administered at various doses 90 minutes before pain analysis. (B) Compound 8 was orally administered at various doses 90 minutes before pain analysis. (C) Compound 8 (5 mg / Kg) was orally administered 1.5, 4, or 12 hours before pain analysis. (D) Compound 8 (3 mg / Kg) was repeatedly orally administered at multiple doses (m.d.) before pain analysis over a 10-day course.

[0022] Figure 10 、 8 The analgesic effect of -phenylisoquinoline derivatives in TNBS mice.

[0023] Figure 11 、Comparison of the analgesic effects and adverse reactions of compound 8 with a reference standard in GW and TNBS mice. (A) Intestinal pain levels in GW mice. (B) Intestinal pain levels in TNBS mice. (C) Representative optical images of colonic histology for each treatment group. Hyperemia (*) and infiltration of granulocytes (arrows) were observed in the ALN group, but not in the other groups. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides a novel compound of the following general formula or a pharmaceutically acceptable salt thereof:

[0026]

[0027] wherein R1 is selected from hydrogen, C 1-10 linear alkyl, C 1-10 branched alkyl, (CH2) n (Hete)R 10 R 11 R 12 and (CH2) n ArR 10 R 11 R 12 , where n is an integer from 0 to 6, Hete is an aromatic heterocyclic group, Ar is an aromatic ring group, and R 10 、R 11 and R 12 are independently selected from hydrogen, halogen, nitro, amino, cyano, acetyl, C 1-6 linear saturated alkyl, C 1- 6 linear saturated alkoxy and C 1-6 linear saturated haloalkyl;

[0028] R2 is hydrogen or C 1-6 linear saturated alkyl; and

[0029] X1, X2, X3, X4 and X5 are independently selected from hydrogen, halogen, nitro, amino, cyano, acetyl, C 1-6 linear saturated alkyl, C 1-6 branched saturated alkyl, C 1-6 linear saturated alkoxy, C 1-6 branched saturated alkoxy, C 1-6 linear saturated alkylthio, C 1-6 branched saturated alkylthio, C 1-6 linear saturated haloalkyl and C 1-6Branched-chain saturated haloalkyl.

[0030] In one embodiment of the present invention, the halo group of the novel compound is selected from fluorine, chlorine, bromine, and iodine. In another embodiment of the present invention, the aromatic heterocyclic group of the novel compound is selected from pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl, thiazolyl, indolyl, isoindolyl, indazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzimidazolyl, benzoxazolyl, and benzothiazolyl.

[0031] In a preferred embodiment of the present invention, the novel compound is selected from 6-methoxy-8-(2-methoxyphenyl)-2-(3-(4-nitrophenyl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol (Compound 7), 6-methoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-4-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol (Compound 8), 6-methoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-3-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol (Compound 9), and 6,7-dimethoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-4-yl)propyl)-1,2,3,4-tetrahydroisoquinoline (Compound 10), or a pharmaceutically acceptable salt thereof. In a more preferred embodiment of the present invention, the novel compound is 6-methoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-4-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol (Compound 8) or a pharmaceutically acceptable salt thereof.

[0032] The present invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a novel compound of the following general formula:

[0033]

[0034] Wherein R1 is selected from hydrogen, C 1-10 linear alkyl, C 1-10 branched alkyl, (CH2) n (Hete)R 10 R 11 R 12 and (CH2) n ArR 10 R 11 R 12 , where n is an integer from 0 to 6, Hete is an aromatic heterocyclic group, Ar is an aromatic ring group, and R 10 , R 11 and R 12 are independently selected from hydrogen, halo group, nitro group, amino group, cyano group, acetyl group, C 1-6 linear saturated alkyl, C 1-6 linear saturated alkoxy and C1-6 Straight-chain saturated haloalkyl;

[0035] R2 is hydrogen or C 1-6 Straight-chain saturated alkyl; and

[0036] X1, X2, X3, X4 and X5 are independently selected from hydrogen, halo, nitro, amino, cyano, acetyl, C 1-6 Straight-chain saturated alkyl, C 1-6 Branched-chain saturated alkyl, C 1-6 Straight-chain saturated alkoxy, C 1-6 Branched-chain saturated alkoxy, C 1-6 Straight-chain saturated alkylthio, C 1-6 Branched-chain saturated alkylthio, C 1-6 Straight-chain saturated haloalkyl and C 1-6 Branched-chain saturated haloalkyl.

[0037] In one embodiment of the present invention, the halo of the novel compound of the pharmaceutical composition is selected from fluorine, chlorine, bromine and iodine. In another embodiment of the present invention, the aromatic heterocyclic group of the novel compound of the pharmaceutical composition is selected from pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl, thiazolyl, indolyl, isoindolyl, indazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl and benzothiazolyl.

[0038] In a preferred embodiment of the present invention, it comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of 6-methoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-4-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol (Compound 8) or a pharmaceutically acceptable salt thereof.

[0039] "Pharmaceutically acceptable carrier" or "excipient" or "pharmaceutically acceptable carrier or excipient" or "bioavailable carrier" or "bioavailable carrier or excipient" includes, but is not limited to, solvents, dispersants, coatings, antimicrobial agents, antifungal agents for preservation, or delayed absorption agents for preparing formulations, and any other known compounds. Generally, these carriers or excipients themselves do not have the activity of treating diseases. The pharmaceutical compositions or formulations prepared by combining the novel compounds or their derivatives disclosed in the present invention with pharmaceutically acceptable carriers or excipients do not cause side effects, allergies or other inappropriate reactions in animals or humans. Therefore, the combination of the novel compounds or their derivatives disclosed in the present invention with pharmaceutically acceptable carriers or excipients can be applied to human clinical practice. The pharmaceutical compositions or formulations containing the novel compounds or their derivatives of the present invention can achieve therapeutic effects through intravenous injection, oral administration, inhalation or topical administration through the nose, rectum, vagina or sublingual. In one embodiment, 0.1 mg to 100 mg of the active ingredient of the compound is administered to patients suffering from different diseases every day.

[0040] The carriers to be used vary according to the pharmaceutical compositions or formulations to be prepared. Sterile injectable compositions can be suspended in sterile intravenous injection diluents or solvents, such as 1,3-butanediol. Acceptable carriers can be mannitol or water. In addition, oil-fixed or synthetic monoglycerides / diglycerides suspension media are commonly used solvents. Fatty acids (such as oleic acid, olive oil, castor oil, glyceride derivatives, especially in polyoxyethylated form) can be prepared for injection and natural pharmaceutically acceptable oils. These oil solutions or suspensions include long-chain alcohol diluents, dispersants, carboxymethyl cellulose or similar dispersants. Other commonly used surfactants include Tweens, Spans, other similar emulsifiers, pharmaceutically acceptable solids, liquids in the pharmaceutical industry, or other bioavailability enhancers for developing formulations.

[0041] The compositions for oral administration are adjusted to orally acceptable compositions or formulations, and the types thereof include capsules, lozenges, troches, emulsions, liquid suspensions, dispersants and solvents. Commonly used carriers for oral administration (such as lozenges) can be, for example, lactose, corn starch, lubricants, magnesium stearate as basic additives. Diluents for capsules include lactose, dry corn starch. The preparation of liquid suspension or emulsion formulations is to suspend or dissolve the active ingredient by combining the oil interface of the emulsifier or suspending agent. Sweeteners, flavoring agents or coloring agents can also be included.

[0042] An aerosol spray or inhalation composition for oral use is prepared by known formulation techniques. For example, the composition is dissolved in physiological saline, and benzyl alcohol, other suitable preservatives or absorbefacients are added to enhance bioavailability. The composition of the compounds provided by the present invention can also be made into suppositories for rectal or vaginal administration.

[0043] Injections include subcutaneous, intraperitoneal, intravenous, intramuscular, intra-articular, intracranial, synovial, intrathecal injection, aortic injection, thoracic injection, lesion injection or other suitable administration techniques.

[0044] In addition, the present invention provides a method for treating irritable bowel syndrome, which comprises the following steps: administering an effective amount of the above-mentioned pharmaceutical composition to a patient in need of such treatment. In one embodiment of the present invention, the halogen group of the pharmaceutical composition is selected from fluorine, chlorine, bromine and iodine. In another embodiment of the present invention, the aromatic heterocyclic group of the pharmaceutical composition is selected from pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl, thiazolyl, indolyl, isoindolyl, indazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzimidazolyl, benzoxazolyl and benzothiazolyl, which provides an antagonistic effect on the 5-HT7 receptor.

[0045] In another embodiment of the present invention, the irritable bowel syndrome is treated by providing an antagonistic effect on the 5-HT7 receptor. In yet another embodiment of the present invention, the irritable bowel syndrome includes pain caused by infection and subsequent stress, and pain caused by chemically induced inflammation.

[0046] In yet another embodiment of the present invention, the irritable bowel syndrome is treated by inhibiting the pain caused by infection and subsequent stress. In another embodiment of the present invention, the irritable bowel syndrome is treated by inhibiting the pain caused by chemically induced inflammation.

[0047] After reviewing the detailed description and the drawings, the above aspects and advantages of the present invention will become apparent to those of ordinary skill in the art. Examples

[0048] The present invention will now be described more specifically with reference to the following examples. It should be noted that the following description of the present invention is presented only for purposes of illustration and description; it is not intended to be exhaustive or limited to the exact form disclosed.

[0049] The present invention provides a library of new derivatives of 8-phenylisoquinoline. The synthesis includes the following 4 schemes.

[0050] Scheme 1 (as Figure 1 shown): As Figure 1As depicted, the synthesis of N-phenethyl-substituted 8-phenyltetrahydroisoquinolin-7-ol derivatives starts from commercially available 7-hydroxy-6-methoxy-3,4-dihydroisoquinoline (Compound 20). Compound 20 is N-alkylated with phenylethylbromide and then reduced with NaBH4 to give amine 21. Treatment of phenethylamine 21 with Pb(OAc)4 followed by aromatic substitution with HBr yields 8-bromotetrahydroisoquinoline 22. Then, using various substituted arylboranes, the desired target compounds 29 to 45 are synthesized from 22 in moderate yields under Suzuki coupling reaction conditions.

[0051] Scheme 2 (as Figure 2 shown): As shown in Scheme 2, the preparation of N-substituted 8-(2,4-dimethoxyphenyl)-tetrahydroisoquinolin-7-ol also starts from commercially available Compound 20. Compound 20 is treated with various halides and then reduced with NaBH4 to give N-substituted tetrahydroisoquinolin-7-ols 11 and 61 to 75. Compounds 11 and 61 to 75 are oxidized with Pb(OAc)4 in acetic acid and then subjected to TFA-catalyzed aromatic substitution with 1,3-dimethoxybenzene to give the corresponding N-substituted 8-(2,4-dimethoxyphenyl)-6-methoxytetrahydroisoquinolin-7-ols 6 and 101 to 125, respectively.

[0052] Scheme 3 (as Figure 3 shown): As depicted in Scheme 3, Compound 20 is treated with various 3-arylpropyl bromides and then reduced with NaBH4 to give the corresponding N-3-arylpropyl-substituted tetrahydroisoquinolin-7-ol derivatives 12 to 14 and 78. Compounds 12 to 14 and 78 are treated with Pb(OAc)4 and then subjected to aromatic substitution with HBr, respectively, to give bromides 15 to 17 and 98. O-methylation of phenol 16 with methyl iodide in the presence of NaH yields 6,7-dimethoxytetrahydroisoquinoline 18. Aryl coupling reactions of compounds 15 to 18 and 98 with various substituted arylboranes are carried out under Suzuki reaction conditions to give N-3-arylpropyl-substituted 6-methoxy-8-phenyltetrahydroisoquinolin-7-ols 7 to 10 and 142 in moderate yields, respectively.

[0053] Scheme 4 (as Figure 4As shown in the figure: As depicted in Scheme 4, N-phenethyl-substituted 6,7-dimethoxy-8-phenyltetrahydroisoquinoline derivatives 157 to 173 were prepared using N-phenethyl-substituted 8-bromo-6-methoxytetrahydroisoquinolin-7-ol 60 to 96. O-methylation of phenols 60 to 96 with methyl iodide in the presence of NaH gave 6,7-dimethoxytetrahydroisoquinolines 150 to 154, respectively. Under Suzuki reaction conditions, aryl coupling reactions of compounds 150 to 154 with various substituted arylboranes provided 6,7-dimethoxy-8-phenyltetrahydroisoquinolines 157 to 173, respectively.

[0054] The specific synthesis steps of those compounds described in Schemes 1 to 4 above are as follows:

[0055] Compound 21: A mixture of compound 20 (100 mg, 0.56 mmol), 2-bromoethylbenzene (311 mg, 1.68 mmol), and 2-propanol (3.5 mL) was refluxed for 15 hours. The resulting solution was concentrated and MeOH (5 mL) was added to dissolve the residue. The solution was cooled in an ice bath and then NaBH4 (49 mg, 1.29 mmol) was slowly added under N2. The mixture was stirred for another 10 minutes and then concentrated. The residue was treated with H2O (20 mL) and CHCl3 (20 mL), and then the organic layer was washed with brine, dried over MgSO4, filtered, and evaporated. The crude residue was chromatographed (silica gel, MeOH / CH2Cl2 = 1 / 100) to give compound 21 (146 mg, 0.52 mmol, 92%) as a white solid.

[0056] Compound 30: In a 10-mL thick-walled Pyrex reaction vessel, 4-methoxyphenylboronic acid (26 mg, 0.19 mmol) was added to a solution of C 18 H 20 BrNO2 (50 mg, 0.14 mmol) in 2-propanol (2.0 mL). After stirring for 30 minutes, Pd(OAc)2 (1.3 mg, 0.006 mmol), PPh3 (4.7 mg, 0.02 mmol), 2M Na2CO 3(aq) (0.09 mL, 0.17 mmol), and H2O (0.1 mL) were added. Then the mixture was heated in a microwave synthesizer at 140 °C for 10 minutes, H2O (0.35 mL) was added, and then it was cooled to room temperature. The resulting solution was diluted with H2O (5 mL) and extracted with EtOAc (5 mL). The organic layer was washed with 5% NaHCO 3(aq)And brine washing. The organic solution was treated with Darco G-60 (100 mg) and stirred at room temperature for 30 minutes, then dried over MgSO4, filtered (a sintered glass funnel was filled with Celite to a depth of 1 cm and Florisil was evenly coated on top of the Celite), and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 2 / 1) to afford an orange oil (40 mg, 0.10 mmol, 73%).

[0057] Compounds 29 and 31 to 40: Table 1 is a parameter table. "Parameter 1" was added to the reaction vessel for microwave-assisted heating and dissolved in "Parameter 2" mL of 2-propanol. The solution had an appearance of "Parameter 3", to which reagent "Parameter 4" was added and stirred for "Parameter 5" minutes. The resulting solution had an appearance of "Parameter 6". Pd(OAc)2 "Parameter 7", PPh3 "Parameter 8", 2M Na2CO3(aq) "Parameter 9", and "Parameter 10" mL of H2O were added and heated under the condition of "Parameter 11". Before the temperature of the solution decreased, "Parameter 12" mL of H2O was added, stirred in air until reaching room temperature, diluted with "Parameter 13" mL of EtOAc, and extracted with "Parameter 14" mL of H2O. The organic layer was washed with 5% NaHCO3(aq), washed with brine, "Parameter 15" mg of Darco G-60 was added, stirred for "Parameter 16" minutes, MgSO4 was added for drying, stirred for "Parameter 17" minutes, filtered through a sintered glass funnel covered with about 1 cm of Celite and a thin layer of Florisil, concentrated and dried, and purified by flash column chromatography (silica gel, "Parameter 18") to obtain "Parameter 19".

[0058] Table 1: Parameter table for the synthesis of compounds 29 and 31 to 40

[0059]

[0060]

[0061]

[0062] Compound 44: To C in a 10-mL thick-walled Pyrex reaction vessel 18 H 20A solution of BrNO2 (100 mg, 0.28 mmol) in 2-propanol (1.5 mL) was added to 3,5-dimethoxyphenylboronic acid (62 mg, 0.34 mmol). After stirring for 30 minutes, Pd(OAc)2 (2.2 mg, 0.01 mmol), PPh3 (8.0 mg, 0.03 mmol), 2M Na2CO 3(aq) (0.17 mL, 0.34 mmol), and H2O (0.7 mL) were added. Then the mixture was heated in a microwave synthesizer at 140 °C for 10 minutes, H2O (0.35 mL) was added, and then it was cooled to room temperature. The resulting solution was diluted with H2O (10 mL) and extracted with EtOAc (10 mL). The organic layer was washed with 5% NaHCO 3(aq) (10 mL) and brine. The organic solution was treated with Darco G-60 (100 mg) and stirred at room temperature for 30 minutes, then dried over MgSO4, filtered (a sintered glass funnel was filled with Celite to a depth of 1 cm, and Florisil was evenly coated on top of the Celite), and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 1 / 1) to give a yellow oil (76 mg, 0.18 mmol, 65%).

[0063] Compound 45: In a 10-mL thick-walled Pyrex reaction vessel, in C 18 H 20 A solution of BrNO2 (100 mg, 0.28 mmol) in 2-propanol (2.0 mL) was added to 2,3-dimethoxyphenylboronic acid (62 mg, 0.34 mmol). After stirring for 30 minutes, Pd(OAc)2 (2.0 mg, 0.009 mmol), PPh3 (3.7 mg, 0.014 mmol), 2M Na2CO 3(aq) (0.18 mL, 0.36 mmol), and H2O (0.2 mL) were added. Then the mixture was heated in a microwave synthesizer at 120 °C for 10 minutes, H2O (0.7 mL) was added, and then it was cooled to room temperature. The resulting solution was diluted with H2O (5 mL) and extracted with EtOAc (5 mL). The organic layer was washed with 5% NaHCO 3(aq) (5 mL) and brine. The organic solution was treated with Darco G-60 (100 mg) and stirred at room temperature for 30 minutes, then dried over MgSO4, filtered (a sintered glass funnel was filled with Celite to a depth of 1 cm, and Florisil was evenly coated on top of the Celite), and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 1 / 1) to give a yellow oil (82 mg, 0.20 mmol, 71%).

[0064] Compounds 15, 60, 85, 95 to 96, and 98: Table 2 is a parameter table. Under N2 at room temperature, the starting material "Parameter 1" was added to the reaction flask, and "Parameter 2" mL of HOAc was added thereto. "Parameter 3" of Pb(OAc)4 was added, and then the solution was "Parameter 4", poured into a conical flask and slowly "Parameter 5" mL of Na2CO3 (sat) was added. The pH of the aqueous layer was basic (pH = "Parameter 6"). The solid produced by neutralization was filtered. The filter cake was washed with CH2Cl2. The filtrate was extracted with "Parameter 7" mL of CH2Cl2. The organic layer was washed with brine, MgSO4 was added to dry, stirred for 5 minutes, filtered through a sintered glass funnel and concentrated to dryness to obtain the "Parameter 8" product. The crude product was used in the following reaction without further purification.

[0065] In air at room temperature, "Parameter 9" of HBr was added to the solution and the appearance of the solution was "Parameter 10". After stirring for "Parameter 11" hours, "Parameter 12" mL of Na2CO3 (sat) and "Parameter 13" mL of CH2Cl2 were slowly added to the solution. The pH of the aqueous layer was basic (pH = "Parameter 14"), then "Parameter 15" mL of CH2Cl2 and "Parameter 16" mL of H2O were added for extraction. The organic layer was washed with brine, MgSO4 was added to dry, stirred for 5 minutes, filtered through a sintered glass funnel and concentrated to dryness to obtain the crude product "Parameter 17" mg. After flash column chromatography (silica gel, "Parameter 18"), "Parameter 19" was obtained.

[0066] Table 2: Parameter table for the synthesis of Compounds 15, 60, 85, 95 to 96, and 98

[0067]

[0068]

[0069] Compounds 11 to 12, 63 to 67, 67 to 70, 74 to 75, and 78: Table 3 is a parameter table. Under N2 at room temperature, the starting material "Parameter 1" was added to a flask, and then "Parameter 2" mL of IPA and "Parameter 3" were added thereto. The starting material dissolved at "Parameter 4" °C. The appearance of the reaction solution was "Parameter 5" and "Parameter 7" at about "Parameter 6" minutes, and then the solution was heated at 110 to 120 °C for "Parameter 8" hours and concentrated at room temperature. "Parameter 9" mL of MeOH was added and the resulting mixture was stirred for "Parameter 10" minutes. NaBH4(s) "Parameter 12" was slowly added to the solution at "Parameter 11" in an ice bath under N2 and stirred for "Parameter 13" minutes. The solution at "Parameter 14" was added to "Parameter 15" mL of H2O and extracted with "Parameter 16" mL of CHCl3. The organic layer was added with MgSO4 for drying, stirred for "Parameter 17" minutes, filtered and concentrated to obtain "Parameter 18". After flash column chromatography (silica gel, "Parameter 19"), "Parameter 20" was obtained.

[0070] Table 3: Parameter table for the synthesis of compounds 11 to 12, 63 to 67, 67 to 70, 74 to 75, and 78

[0071]

[0072]

[0073]

[0074] Compound 68: A mixture of C 10 H 11 NO2 (300 mg, 1.69 mmol), C8H8Br2 (1.00 g, 3.79 mmol), and 2-propanol (10 mL) was heated to reflux for 23 hours. The resulting solution was cooled to room temperature and evaporated. The crude product was dissolved in MeOH (15 mL), cooled to 0 °C in an ice bath, and then NaBH4 (420 mg, 11.1 mmol) was added portionwise under N2. The mixture was stirred for another 20 minutes and then concentrated. The residue was treated with CHCl3 (30 mL) and H2O (30 mL), and then the organic layer was dried with MgSO4, filtered, and evaporated. Purification was carried out by precipitation. The crude product was dissolved in 5 mL of EtOAc, and then the product was precipitated with 10 mL of n-hexane to obtain a beige solid (620 mg, 1.71 mmol).

[0075] Compound 121: In C 19 H 23A solution of NO2 (250 mg, 0.84 mmol) in HOAc (4.2 mL) was added to Pb(OAc)4 (579 mg, 1.31 mmol) and the mixture was stirred under N2 at room temperature for 15 minutes. The reaction mixture was diluted with CH2Cl2 and Na2CO3 (sat) (20 mL) was slowly added. The solid formed by neutralization was removed by filtration and washed with CH2Cl2. The combined filtrates were extracted with CH2Cl2 (35 mL), then the organic layer was washed with brine, dried over MgSO4, filtered and evaporated to give a brown oil (480 mg, 1.35 mmol), which was used in the following reaction without further purification. To a solution of this crude oil in CH2Cl2 (17 mL) was added 1,3-dimethoxybenzene (0.17 mL, 1.3 mmol) and trifluoroacetic acid (0.84 mL). The resulting mixture was stirred at room temperature for 30 minutes, then Na2CO3 (sat) (20 mL) was slowly added. The resulting solution was extracted with CH2Cl2 (18 mL), then the organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The crude residue was chromatographed (silica gel, MeOH / CH2Cl2 = 1 / 10) to give a red-brown oil (214 mg, 0.49 mmol, 59%).

[0076] Compounds 6, 105 to 110, 114 to 115, 120, 122, 123 and 125: Table 4 is a parameter table. Under N2 at room temperature, the starting material "Parameter 1" was added to a flask and dissolved in "Parameter 2" mL of HOAc. To this solution of "Parameter 3" was added Pb(OAc)4 "Parameter 4", then the resulting solution of "Parameter 5" was stirred for "Parameter 6" minutes, poured into a 125 mL conical flask, stirred and "Parameter 7" mL of Na2CO3 (sat) was slowly added. The pH of the aqueous layer was basic (pH = 8 to 9). The solid formed by neutralization was filtered off and washed with CH2Cl2. The filtrate was extracted with "Parameter 8" mL of CH2Cl2. The organic layer was washed with brine, MgSO4 was added to dry, stirred for 5 minutes, filtered and concentrated to give "Parameter 9". The crude product was used in the following reaction without further purification.

[0077] The crude product was dissolved in "Parameter 10" mL of CH2Cl2 under N2 at room temperature. To this solution at "Parameter 11", 1,3-dimethoxybenzene "Parameter 12" and trifluoroacetic acid "Parameter 13" were added. The color of the solution turned to "Parameter 14". After stirring the solution for "Parameter 15" minutes, "Parameter 16" mL of Na2CO3 (sat) was slowly added. The pH of the aqueous layer was basic (pH = 8 to 9), and "Parameter 17" mL of CH2Cl2 was added for extraction. The organic layer was washed with brine, MgSO4 was added for drying, stirred for 5 minutes, filtered and concentrated to give "Parameter 18" mg of the crude product. After flash column chromatography (silica gel, "Parameter 19"), "Parameter 20" was obtained.

[0078] Table 4: Parameter Table for the Synthesis of Compounds 6, 105 to 110, 114 to 115, 120, 122, 123 and 125

[0079]

[0080]

[0081]

[0082]

[0083] Compounds 7 and 142: Table 5 is a parameter table. "Parameter 1" and 2-methoxyphenylboronic acid (46 mg, 0.30 mmol) were added to a reaction vessel, and microwave-assisted heating was carried out and dissolved in 2-propanol (2 mL), and stirred for 30 minutes. Pd(OAc)2 "Parameter 2", PPh3 "Parameter 3", 2M Na2CO 3(aq) (0.14 mL, 0.28 mmol), and H2O (0.2 mL) were added, and the mixture was heated at 120 °C for 20 minutes using a microwave synthesizer. Before the solution temperature decreased, H2O (0.7 mL) was added to the solution, stirred in air until reaching room temperature, diluted with 10 mL of EtOAc, and extracted with 10 mL of H2O. The organic layer was washed with 5% NaHCO 3(aq) washed, washed with brine, "Parameter 4" mg of Darco G-60 was added, stirred for 10 minutes, MgSO4 was added for drying, stirred for 10 minutes, filtered through a sintered glass funnel covered with a thin layer of about 1 cm of Celite and Florisil, and concentrated. The crude product was purified by flash column chromatography (silica gel, "Parameter 5") to obtain a yellow oil "Parameter 6". The free base "Parameter 7" was dissolved in CH2Cl2, and then a solution of HCl in CH2Cl2 was added until pH = 1. The resulting mixture was concentrated to obtain the hydrochloride salt "Parameter 8".

[0084] Table 5: Parameter Table for the Synthesis of Compounds 7 and 142

[0085]

[0086] Compound 150: In a solution of C 18 H 20 BrNO2 (100 mg, 0.28 mmol) in DMF (2 mL), trimethylphenylammonium chloride ((CH3)3PhNCl, 102 mg, 0.59 mmol) and t-BuOK (67 mg, 0.60 mmol) were added. The suspension was heated to 60 °C under N2 for 3.5 h, then (CH3)3PhNCl (102 mg, 0.59 mmol) was added and heated to 70 °C for 4.5 h. After cooling to room temperature, the reaction mixture was treated with CHCl3 (10 mL) and 5% NaOH (aq) (20 mL). The organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 1 / 4) to afford a yellow solid (83 mg, 0.22 mmol, 79%).

[0087] Compound 152: In a solution of C 18 H 19 BrN2O4 (406 mg, 1.00 mmol) in DMF (9 mL), NaH (40 mg, 1.67 mmol) in DMF (1 mL) and CH3I (0.06 mL, 0.98 mmol) were added. After stirring for 10 min, NH4Cl (111 mg, 2.08 mmol) was added, then the reaction mixture was treated with diethyl ether (100 mL) and H2O (100 mL). The organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 1 / 2) to afford a yellow solid (123 mg, 0.29 mmol, 30%).

[0088] Compound 153: In C 18 H 19To a solution of BrClNO2 (300 mg, 0.75 mmol) in DMF (6 mL) was added (CH3)3PhNCl (542 mg, 3.16 mmol) and t-BuOK (333 mg, 2.97 mmol). The suspension was heated to 60 °C under N2 for 16 h, then heated to 70 °C for 1 h. After cooling to room temperature, the reaction mixture was treated with Et2O (100 mL) and H2O (100 mL). The organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 1 / 4) to afford a white solid (189 mg, 0.46 mmol, 61%).

[0089] Compound 154: At C 18 H 19 To a solution of BrFNO2 (400 mg, 1.05 mmol) in DMF (8 mL) was added (CH3)3PhNCl (727 mg, 4.23 mmol) and t-BuOK (468 mg, 4.17 mmol). The suspension was heated to 70 °C under N2 for 16 h. After cooling to room temperature, the reaction mixture was treated with Et2O (100 mL) and H2O (100 mL). The organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The crude residue was chromatographed (silica gel, EtOAc / hexane = 1 / 4) to afford a white solid (247 mg, 0.63 mmol, 60%).

[0090] Compounds 157 to 159, 165 - 168 and 171 - 173: Table 6 is a parameter table. Add "Parameter 1" to the reaction vessel for microwave-assisted heating and dissolve it in "Parameter 2" mL of 2-propanol. Add "Parameter 3" thereto and stir for 30 min. Add Pd(OAc)2 "Parameter 4", PPh3 "Parameter 5", 2M Na2CO 3(aq) "Parameter 6" and "Parameter 7" mL of H2O and heat it to 120 °C for 20 min using a microwave synthesizer. Before the solution temperature drops, add "Parameter 8" mL of H2O, then cool to room temperature, dilute with 10 mL of EtOAc and extract with 10 mL of H2O. Wash the organic layer with 5% NaHCO 3(aq) Wash, then wash with brine, add "Parameter 9" mg of Darco G-60, stir for 10 min, filter through a sintered glass funnel covered with a thin layer of about 1 cm of Celite and Florisil, concentrate and purify by flash column chromatography (silica gel, "Parameter 10") to obtain "Parameter 11".

[0091] Table 6: Parameter Table for the Synthesis of Compounds 157 to 159, 165 to 168, and 171 to 173

[0092]

[0093]

[0094] Table 7: Analytical Data of the Compounds of the Invention

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] 5-HT7 Receptor Affinity of Compounds 6 to 10, 5-HT 2AReceptor affinity and log D data are shown in Table 8.

[0116] Table 8: Receptor affinity and log D of Compounds 6 to 10

[0117]

[0118] Animal

[0119] Studies were conducted using specific pathogen-free C57BL / 6 mice (4 to 6 weeks old) obtained from the Animal Center of National Taiwan University. The animals were housed in a temperature-controlled room (20 ± 2 °C) with a 12 / 12-hour light / dark cycle and fed a standard mouse diet and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan University.

[0120] Reagent

[0121] Novel 8-phenylisoquinoline derivatives were prepared by the following procedure. SB-269970 hydrochloride (SB7), a 5-HT7R antagonist (Sigma #S7389), alosetron hydrochloride (ALN), a 5-HT3R antagonist (Sigma #SML0346), and loperamide hydrochloride (LPM), a μ-opioid receptor agonist (Sigma #L4762), were administered to mice in single or multiple doses intraperitoneally (i.p.) or orally (p.o.) to analyze abdominal pain.

[0122] Two experimental models of visceral hypersensitivity

[0123] (1) Dual stimulation of Giardia infection combined with water avoidance stress

[0124] In this study, two IBS animal models showing visceral hypersensitivity were used, including double stimulation of post-infection combined with psychological stress, and post-inflammation. In the first model, mice were divided into two groups, one group received double stimulation of Giardia infection followed by water avoidance stress (GW), while the other group was pair-fed with saline and untreated (PN) as a normal control group without infection and stress. Axenic Giardia lamblia trophozoites (strain GS / M, ATCC 50581) were cultured in vitro and harvested in the logarithmic phase as described by Singer et al. (T-cell-dependent control of acute Giardia lamblia infections in mice. Infect. Immun. 2000; 68: 170 - 175) and Davids et al. (Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia. J Immunol 2006; 177: 6281 - 6290). Mice were gavaged orally with 10 7Giardia trophozoites, or fed with PBS of the same volume. After 4 to 7 days, the status of Giardia infection was verified by counting motile trophozoites in the small intestine by following the cold-shock protocol (published in Scott KG, Yu LCH, Buret AG. Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury during murine giardiasis. Infect. Immun. 2004;72:3536-3542 and Scott KG, Meddings JB, Kirk DR, et al. Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 2002;123:1179-1190). At the sixth week after infection with trophozoites could not be detected in the small intestine (post-clearance phase), the mice were subjected to chronic psychological stress. The WAS procedure consisted of placing the mice on the central platform (3 x 6 cm) of a container (56 x 50 cm) with 3 cm (vertical height) of room temperature water. The mice were left on this platform for 1 hour to avoid immersion as a psychological stress without physical harm. This 1-hour stress period was carried out for 10 consecutive days to simulate chronic repetitive stress and was carried out between 9:00 and 12:00 to minimize the influence of the circadian rhythm. Uninfected and non-stressed untreated animals were housed in cages as normal control groups. On the last day of this stress period, abdominal pain was measured in the mice.

[0125] To test the analgesic effect in the GW model, a new 5-HT7R ligand was administered to the mice in a single dose 90 or 240 minutes before measuring abdominal pain. In an additional setting, the new ligand was repeatedly administered for 10 consecutive days 30 minutes before the start of each stress period, and abdominal pain was measured immediately after the last stress period.

[0126] (2) Post-inflammatory model

[0127] In the second model, colonic administration of 10% 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich, St. Louis, MO, USA) in 0.2 ml of 50% ethanol was performed via a 22-gauge feeding needle to induce intestinal inflammation. The sham-operated control group was given the same volume of PBS. Intestinal inflammation parameters and pain levels were measured at various time points after the administration of TNBS.

[0128] To test the analgesic effect in this TNBS-induced model, a single dose of the novel 5-HT7R ligand was administered to the mice 90 or 240 minutes prior to or multiple doses of the novel 5-HT7R ligand were repeatedly administered for 10 days prior to measuring visceral pain.

[0129] Assessment of nociception to colorectal distension

[0130] Abdominal pain was measured by the visceral motor response (VMR) to colorectal distension (CRD) in mice with minor modifications according to the previously described method (Lu CL, Hsieh JC, Dun NJ, et al. Estrogen rapidly modulates 5-hydroxytrytophan-induced visceral hypersensitivity via GPR30 in rats. Gastroenterology 2009;137:1040-1050; Hong S, Zheng G, Wu X, et al. Corticosterone mediates reciprocal changes in CB1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat. Gastroenterology 2011;140:627-637e4). Briefly, an electrode made of Teflon-coated stainless steel wire (A-M systems, Carlsborg, WA) was implanted into the external oblique muscle of the abdomen of mice at least 15 days before the VMR experiment. The electrode was exposed to the back of the neck. Before the VMR experiment, mice were habituated to a plastic Plexiglas cylinder for 30 minutes per day for 3 consecutive days. During the CRD experiment, the cylinder was used to partially restrain awake mice. For recording, the electrode was connected to an electromyogram acquisition system (AD instruments, New south wales, Australia). A balloon catheter inserted into the anus and terminating 1.5 cm proximal to the anus was inflated to distend the colon. Mice were subjected to four 10-second distensions (15, 40, and 65 mmHg) with 3-minute rest intervals. The electromyogram (EMG) activity was amplified and digitized using a transducer (AD instruments) and a Powerlab device connected to a P511 AC amplifier (Grass instruments, CA, USA) with Chart 5 software (AD instruments). The EMG activity was rectified, and the response was recorded as the increase in the area under the curve (AUC) of the EMG amplitude during CRD relative to the baseline period.

[0131] Histopathological examination

[0132] Intestinal tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin to properly orient the crypt-villus axis, and then sectioned. 5-μm thick sections were deparaffinized with xylene and graded ethanol, stained with hematoxylin and eosin (H&E), and observed under an optical microscope.

[0133] Reverse transcription polymerase chain reaction

[0134] Total RNA was extracted from tissue samples using Trizol reagent (Invitrogen) according to the manufacturer's instructions. In a 20-μL reaction volume, 2 μg of RNA was reverse transcribed with oligo(dT)15 using the RevertAid TM First Strand cDNA Synthesis Kit (Thermo). Subsequently, the resulting cDNA corresponding to 0.1 μg of the initial RNA was subjected to PCR by adding a mixture containing 1X PCR buffer, 1 U of DreamTaq TMA master mix of DNA polymerase, 0.2 mM dNTP mixture, 0.4 μM forward primer, and 0.4 μM reverse primer. The specific primer pairs for the PCR reaction were as follows: mouse 5-HT7R (forward: 5’-TCTTCGGATGGGCTCAGAATGT-3’ and reverse: 5’-AACTTGTGTTTGGCTGCGCT-3’) and β-actin (forward: 5’-GGGAAATCGTGCGTGAC-3’ and reverse: 5’-CAAGAAGGAAGGCTGGAA-3’) (as disclosed in Forcen R, Latorre E, Pardo J, et al. Toll-like receptors 2 and 4 modulate the contractile response induced by serotonin in mouse ileum: analysis of the serotonin receptors involved. Neurogastroenterol Motil 2015;27:1258-66). The DNA thermal cycler was programmed to perform the following protocol: 1 cycle at 95°C for 3 minutes; 30 cycles of 95°C for 30 seconds (denaturation), 55°C for 30 seconds (annealing), and 72°C for 30 seconds (extension); and a final extension at 72°C for 7 minutes. Negative controls were performed with non-reverse-transcribed samples lacking cDNA. The RT-PCR products were then electrophoresed in a 1.5% agarose gel in the presence of 0.5 μg / mL ethidium bromide, visualized using an ultraviolet transilluminator, and photographed. The intensity of the DNA bands was analyzed using Gel-Pro Analyzer 4.0 software.

[0135] Immunofluorescence staining of 5-HT7R

[0136] The deparaffinized tissue sections were incubated in 10 mM sodium citrate buffer (pH 6.0) containing 0.05% Tween-20 and boiled in a microwave. The sections were cooled at room temperature. After quenching the reaction with 1 mg / ml NaBH4 (pH 8.0) in PBS for 15 minutes at room temperature, the tissue was blocked with 1% bovine serum albumin for 2 hours at room temperature. The tissue sections were incubated overnight at 4 °C with primary antibodies, rabbit polyclonal anti-5-HT7R (1:300, Abcam), rabbit PGP9.5 antibody (1:250, GeneTex), or isotype control. The sections were washed with PBS and incubated with secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:250, Molecular Probes) for 1 hour at room temperature. The tissue was then incubated with Hoechst dye (1 μg / ml in PBS) (Sigma) for an additional 30 minutes. The slides were observed under a fluorescence microscope and images were taken.

[0137] Western Blotting

[0138] Intestinal mucosal proteins were extracted with complete radioimmunoprecipitation (RIPA) buffer and subjected to SDS / polyacrylamide gel electrophoresis (PAGE) (4 to 13% polyacrylamide) (as described in Kuo WT, Lee TC, Yang HY, et al. LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis. Cell Death Differ 2015;22:1590 - 1604; Wu LL, Peng WH, Kuo WT, et al. Commensal Bacterial Endocytosis in Epithelial Cells Is Dependent on Myosin Light Chain Kinase-Activated Brush Border Fanning by Interferon-gamma. Am J Pathol 2014;184:2260 - 2274; and Yu LC, Shih YA, Wu LL, et al. Enteric dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistant and commensal bacteria through epithelial transcytosis. Am J Physiol Gastrointest Liver Physiol 2014;307:G824 - 35). The resolved proteins were then electrotransferred to PVDF or nitrocellulose membranes in a semi-dry blotter. The blots were blocked with 5% (w / v) non-fat dry milk in Tris-buffered saline (TBS) or 5% (w / v) bovine serum albumin in TBS containing Tween 20 (TBS-T; 0.1% (v / v) Tween-20 in TBS) for 1 hour, washed with TBS-T, and incubated overnight at 4°C with primary antibodies. The membranes were washed and incubated with secondary antibodies for 1 hour. After washing, the membranes were incubated with chemiluminescent solution and signals were detected. The primary antibodies used included rabbit polyclonal anti-5-HT7R (1:500, Abcam) and anti-β-actin (1:10,000, Sigma).The secondary antibody used was goat anti-rabbit IgG conjugated to horseradish peroxidase (1:1000, Cell Signaling).

[0139] Statistical analysis

[0140] All values are expressed as mean ± SEM and were compared by paired Student's t-test. Significance was established at P < 0.05. In two murine models of IBS, intestinal hypernociception was associated with upregulation of colonic 5-HT7R expression

[0141] Two animal models of IBS visceral hypersensitivity were utilized to examine the analgesic effects of a series of 8-phenylisoquinoline derivatives as novel 5-HT7R ligands. Mice were divided into two groups, one group received Giardia infection followed by water avoidance stress (GW), and the other group was pair-fed and untreated (PN) to serve as an uninfected non-stressed normal control. The visceromotor response (VMR) to colorectal distension was expressed as the area under the curve (AUC) and measured in each mouse as an index of intestinal pain

[0142] In the first model, increased abdominal pain was observed in response to dual stimuli of Giardia infection combined with psychological stress (GW) compared to the normal control group (Figure 5(A)). Figure 5B Representative images showing colonic histology in PN and GW mice are presented. The colonic morphology was similar between GW mice and the normal control group (Figure 5(B)). 5-HT7R immunostaining was performed on colonic tissues from PN and GW mice. Figure 5(C) shows representative images of 5-HT7R staining (panel a) and quantification of 5-HT7R immunoreactivity in the muscular / nerve and mucosal layers (panels b and c). Figure 5(D) shows the results of protein immunoblotting, which shows increased 5-HT7R protein levels in GW mice. Upregulation of 5-HT7R expression was observed in colonic tissues of GW mice (Figure 5(C) and Figure 5(D)), with higher levels in the smooth muscle, enteric nerves, and mucosal regions (Figure 5(C)).

[0143] In the second model, a bolus of the colitogenic chemical TNBS or PBS was administered into the colon of mice on day 0, and intestinal inflammation and pain were examined and measured on various different days. These animals showed increased abdominal pain on days 7, 14, and 24 after TNBS administration (Figure 6(A)). However, the colonic inflammation indices such as myeloperoxidase activity and histopathological scores peaked on day 2 and showed elimination on day 7 ( Figure 6B to Figure 6D ). Therefore, day 24 after TNBS administration was used as the time point to examine class IBS visceral hypersensitivity. Upregulation of 5-HT7R expression was observed in the colonic tissues of TNBS mice, with higher levels in the smooth muscle, enteric nerves, and mucosal regions ( Figure 6E and Figure 6F ).

[0144] 5-HT7R activation is involved in visceral hypersensitivity in the IBS model

[0145] To verify the role of 5-HT7R in visceral hypersensitivity for proof-of-concept, a putative 5-HT7R antagonist for research (SB-269970) was intraperitoneally (i.p., 0.5 mg / Kg) injected into this animal model and intestinal pain was measured by VMR. Intestinal pain levels in mice were significantly inhibited by intraperitoneal administration of SB7 ( Figure 7 ).

[0146] Analgesic effects of novel 5-HT7R ligands

[0147] Novel 8-phenylisoquinoline derivatives (Compound I) targeting 5-HT7R with high binding affinity and water solubility were synthesized (Compounds 6 to 10, shown in Table 8). In the initial experiment, Compounds 6 to 10 (5-HT7R ligands) were orally (p.o.) administered to GW mice at 5 mg / kg to evaluate the inhibitory effect on abdominal pain. A single dose of 5 mg / Kg was administered 90 minutes before VMR analysis. All tested compounds showed analgesic effects, and Compound 8 showed the strongest inhibition of intestinal pain relative to the baseline level ( Figure 8 ).

[0148] To examine the dose response of the analgesic effect, Compound 8 was intraperitoneally (i.p.) injected at 0.05 and 0.5 mg / kg, or orally (p.o.) injected at 1.5 and 5 mg / kg into GW mice. A dose-dependent analgesic effect of Compound 8 was observed in GW mice ( Figure 9A and Figure 9B)。To verify whether this analgesic effect is persistent, 5 mg / kg of CYY1005 was orally administered to GW mice 1.5, 4, or 12 hours before pain measurement. A decrease in pain level was observed at the three time points ( Figure 9C )。In addition, repeated administration of Compound 8 (multiple doses) also reduced visceral pain in GW mice in a dose-dependent manner ( Figure 9D )。

[0149] A vehicle or a novel 5-HT7R ligand was orally (p.o.) injected into TNBS mice to evaluate the inhibitory effect on abdominal pain. A single dose of 5 mg / kg was administered 90 minutes before VMR analysis. In these TNBS mice, oral administration of these novel 5-HT7R ligands at a single dose alleviated visceral pain ( Figure 10 (A)). Similarly, repeated administration of Compound 8 at multiple doses also reduced visceral pain in TNBS mice ( Figure 10 (B)).

[0150] Compare the analgesic effects and adverse reactions between the novel 8-phenylisoquinoline derivatives and the reference standard

[0151] Compound I (Compounds 6 to 10) and the reference standard were orally administered in two animal models to compare their analgesic potencies. These compounds and the reference standard included SB7 (a 5-HT7R antagonist), alosetron (ALN, a 5-HT3R antagonist), and loperamide (LPM, a μ-opioid receptor agonist), which were administered at 5 mg / Kg 90 minutes before pain analysis. In GW mice, oral administration of ALN reduced visceral pain but was less efficient compared to Compound 8 in GW mice ( Figure 8 (A)). On the other hand, oral administration of SB7 and LPM had no effect on intestinal pain sensation in GW mice ( Figure 11 (A)). In the second animal model, administration of ALN, SP7, or LPM had no effect on visceral pain in TNBS mice ( Figure 11 (B)).

[0152] All mice administered the vehicle or the compound showed normal colon histology, except for those administered ALN. Among the 14 mice administered ALN, 2 (14%) showed hyperemia and granulocytic infiltration in the colon tissue ( Figure 11 (C)).

[0153] The newly FDA-approved drugs eluxadoline, a mixed μ-opioid agonist, and rifamixin, an oral non-absorbable gut-selective antibiotic, have recently been added to the treatment options for IBS-D. These agents represent molecular mechanisms or environmental factors different from the 5-HT7R target. Notably, any opioid agonist carries a risk of drug addiction after long-term treatment. This series of 8-phenylisoquinoline derivatives, i.e., 5-HT7R antagonists, are more advantageous compared to traditional painkillers (such as non-steroidal anti-inflammatory drugs and anticholinergics) or antidiarrheal opioid agonists (such as loperamide), as they may act peripherally and selectively on the hyperalgesic gut.

[0154] In the present invention, the 8-phenylisoquinoline derivatives (I) (Compounds 6 to 10) in an IBS animal model showed stronger analgesic effects compared to alosetron without side effects, and thus they are suitable for male and female patients as a new option for IBS treatment.

[0155] References

[0156] The entire background of the following articles is incorporated herein by reference in their entirety.

[0157] Part I

[0158] 1. To, Z.P.; Bonhaus, D.W.; Eglen, R.M.; Jakeman, L.B. Characterization and distribution of putative 5-HT7 receptors in the guinea pig brain. Br J Pharmacol. 1995, 115, 107 - 116.

[0159] 2. Leopoldo, M. Serotonin(7) receptors (5-HT(7)Rs) and their ligands. Curr Med Chem. 2004, 11, 629 - 661.

[0160] 3. Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R. 5-HT(7) receptor modulators: a medicinal chemistry survey of recent patent literature (2004 - 2009). Expert Opin Ther Pat. 2010, 20, 739 - 754.

[0161] 4. Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R.; Hedlund, P. Serotonin 5-HT7 Receptor Agents: Structure-Activity Relationships and Potential Therapeutic Applications in Central Nervous System Disorders. Pharmacol Ther. 2011, 129, 120-148.

[0162] 5. Tokarski, K.; Bobula, B.; Grzegorzewska-Hiczwa, M.; Kusek, M.; Hess, G. Modification of 5-HT(7) Receptor Function in the Rat Brain Induced by Stress and Antidepressant Treatment. Pharmacol Rep. 2012, 64, 1305-1315.

[0163] 6. Tokarski, K.; Zelek-Molik, A.; Duszynska, B.; Satala, G.; Bobula, B.; Kusek, M.; Chmielarz, P.; Nalepa, I.; Hess, G. Acute and Repeated Treatment with the 5-HT7 Receptor Antagonist SB 269970 Induces Functional Desensitization of 5-HT7 Receptors in the Rat Hippocampus. Pharmacol Rep. 2012, 64, 256-265.

[0164] 7. Medina, R.A.; Vazquez-Villa, H.; Gomez-Tamayo, J.C.; Benhamu, B.; Martin-Fontecha, M.; de la Fuente, T.; Caltabiano, G.; Hedlund, P.B.; Pardo, L.; Lopez-Rodriguez, M.L. The Extracellular Entrance Provides Selectivity for In Vivo Antidepressant-Like Behavior of Serotonin 5-HT7 Receptor Antagonists. J Med Chem. 2014, 57, 6879-6884.

[0165] 8. Mnie-Filali, O.; Faure, C.; Lambas-Senas, L.; El Mansari, M.; Belblidia, H.; Gondard, E.; Etievant, A.; Scarna, H.; Didier, A.; Berod, A.; Blier, P.; Haddjeri, N. Pharmacological Blockade of 5-HT7 Receptors as a Putative Fast-Acting Antidepressant Strategy. Neuropsychopharmacology. 2011, 36, 1275-1288.

[0166] 9. Nikiforuk, A. Selective blockade of 5-HT7 receptors facilitates attention shifting in stressed and control rats. Behav Brain Res. 2012, 226, 118 - 123.

[0167] 10. Canale, V.; Kurczab, R.; Partyka, A.; Satala, G.; Lenda, T.; Jastrzebska-Wiesek, M.; Wesolowska, A.; Bojarski, A. J.; Zajdel, P. New 5-HT7 antagonists among arylsulfonamide derivatives of (aryloxy)ethyl-alkylamines: Multi-target-based design, synthesis, antidepressant and anxiolytic properties. Eur J Med Chem. 2016, 108, 334 - 346.

[0168] 11. Zagorska, A.; Kolaczkowski, M.; Bucki, A.; Siwek, A.; Kazek, G.; Satala, G.; Bojarski, A. J.; Partyka, A.; Wesolowska, A.; Pawlowski, M. Structure - activity relationships and molecular studies of novel arylpiperazinylalkyl purine-2,4-diones and purine-2,4,8-triones with antidepressant and anxiolytic activities. Eur J Med Chem. 2015, 97, 142 - 154.

[0169] 12. Roth, B. L.; Craigo, S. C.; Choudhary, M. S.; Uluer, A.; Monsma, F. J., Jr.; Shen, Y.; Meltzer, H. Y.; Sibley, D. R. Binding of typical and atypical antipsychotic drugs to serotonin-6 and serotonin-7 receptors. J Pharmacol Exp Ther. 1994, 268, 1403 - 1410.

[0170] 13. Andressen, K. W.; Manfra, O.; Brevik, C. H.; Ulsund, A. H.; Vanhoenacker, P.; Levy, F. O.; Krobert, K. A. Atypical antipsychotics clozapine and olanzapine facilitate downregulation and show functional selectivity for the human 5-HT7 receptor. Br J Pharmacol. 2015, 172, 3846 - 3860.

[0171] 14. Waters, K.A.; Stean, T.O.; Hammond, B.; Virley, D.J.; Upton, N.; Kew, J.N.; Hussain, I. Role of the selective 5-HT(7) receptor antagonist SB-269970 in psychiatric and cognitive animal models. Behav Brain Res. 2012, 228, 211-218.

[0172] 15. Ivachtchenko, A.V.; Lavrovsky, Y.; Okun, I. AVN-101: A multi-target candidate drug for the treatment of central nervous system diseases. J Alzheimers Dis. 2016, 53, 583-620.

[0173] 16. Brenchat, A.; Rocasalbas, M.; Zamanillo, D.; Hamon, M.; Vela, J.M.; Romero, L. Assessment of 5-HT(7) receptor agonist selectivity using nociception and thermoregulation tests in knockout and wild-type mice. Adv Pharmacol Sci. 2012, 2012, 312041.

[0174] 17. Yesilyurt, O.; Seyrek, M.; Tasdemir, S.; Kahraman, S.; Deveci, M.S.; Karakus, E.; Halici, Z.; Dogrul, A. Critical role of spinal 5-HT7 receptors in opioid- and non-opioid drug stress-induced analgesia. Eur J Pharmacol. 2015, 762, 402-410.

[0175] 18. Brenchat, A.; Nadal, X.; Romero, L.; Ovalle, S.; Muro, A.; Sanchez-Arroyos, R.; Portillo-Salido, E.; Pujol, M.; Montero, A.; Codony, X.; Burgueno, J.; Zamanillo, D.; Hamon, M.; Maldonado, R.; Vela, J.M. Pharmacological activation of 5-HT7 receptors reduces mechanical and thermal hypersensitivity induced by nerve injury. Pain. 2010, 149, 483-494.

[0176] 19. Terron, J.A. Role of 5-HT7 receptors in the serotonin-induced long-lasting hypotensive response in the rat. Br J Pharmacol. 1997, 121, 563-571.

[0177] 20. Perrone-Capano, C.; Adriani, W. Editors: Further insights into the neuro-psycho-pharmacology of serotonin 7 receptors. Front Behav Neurosci. 2015, 9, 307.

[0178] 21. Kim, J.J.; Bridle, B.W.; Ghia, J.E.; Wang, H.; Syed, S.N.; Manocha, M.M.; Rengasamy, P.; Shajib, M.S.; Wan, Y.; Hedlund, P.B.; Khan, W.I. Targeted inhibition of serotonin 7 (5-HT7) receptor function modulates immune responses and reduces the severity of intestinal inflammation. J Immunol. 2013, 190, 4795 - 4804.

[0179] 22. De Ponti, F.; Tonini, M. Irritable bowel syndrome: new drugs targeting serotonin receptor subtypes. Drugs. 2001, 61, 317 - 332.

[0180] 23. Yu, F.Y.; Huang, S.G.; Zhang, H.Y.; Ye, H.; Chi, H.G.; Zou, Y.; Lv, R.X.; Zheng, X.B. Comparison of 5-hydroxytryptophan signaling pathway characteristics in diarrhea-predominant irritable bowel syndrome and ulcerative colitis. World J Gastroenterol. 2016, 22, 3451 - 3459.

[0181] 24. Read, K.E.; Sanger, G.J.; Ramage, A.G. Evidence that central 5-HT7 receptors are involved in the micturition reflex of anesthetized female rats. Br J Pharmacol. 2003, 140, 53 - 60.

[0182] 25. Schoeffter, P.; Ullmer, C.; Bobirnac, I.; Gabbiani, G.; Lubbert, H. Functional, endogenous expression of serotonin 5-HT(7) receptors in human vascular smooth muscle cells. British Journal of Pharmacology. 1996, 117, 993 - 994.

[0183] 26. Terron, J.A. Is the 5-HT7 receptor involved in the pathogenesis and prophylactic treatment of migraine? European Journal of Pharmacology. 2002, 439, 1 - 11.

[0184] 27. Di Pilato, P.; Niso, M.; Adriani, W.; Romano, E.; Travaglini, D.; Berardi, F.; Colabufo, N. A.; Perrone, R.; Laviola, G.; Lacivita, E.; Leopoldo, M. Selective agonists of serotonin 7 (5-HT7) receptor and their applications in preclinical models: an overview. Rev Neurosci. 2014, 25, 401-415.

[0185] 28. Nikiforuk, A. Serotonin 5-HT7 receptor in therapeutic targets for central nervous system diseases: rationale and progress to date. CNS Drugs. 2015, 29, 265-275.

[0186] Part Two

[0187] 1. Jung IS, Kim HS, Park H, et al. Clinical course of infectious irritable bowel syndrome: a five-year follow-up study. J Clin Gastroenterol. 2009;43:534-540.

[0188] 2. Chang FY, Lu CL, Chen TS. Current prevalence of irritable bowel syndrome in Asia. J Neurogastroenterol Motil. 2010;16:389-400.

[0189] 3. Elsenbruch S. Abdominal pain in irritable bowel syndrome: a review of putative psychological, neural and neuroimmune mechanisms. Brain Behav Immun 2011;25:386-94.

[0190] 4. Stasi C, Bellini M, Bassotti G, et al. Serotonin receptors and their role in the pathophysiology and treatment of irritable bowel syndrome. Tech Coloproctol 2014;18:613-21.

[0191] 5. Ford AC, Brandt LJ, Young C, et al. Efficacy of 5-HT3 antagonists and 5-HT4 agonists in irritable bowel syndrome: systematic review and meta-analysis. Am J Gastroenterol 2009;104:1831-43; quiz 1844.

[0192] 6. Ross CA. Child sexual abuse and psychosomatic symptoms in irritable bowel syndrome. J Child Sex Abus 2005; 14: 27-38.

[0193] 7. Qin HY, Cheng CW, Tang XD, et al. Influence of psychological stress on irritable bowel syndrome. World J Gastroenterol 2014; 20: 14126-31.

[0194] 8. Spiller R, Garsed K. Post-infectious irritable bowel syndrome. Gastroenterology 2009; 136: 1979-1988.

[0195] 9. Beatty JK, Bhargava A, Buret AG. Post-infectious irritable bowel syndrome: mechanistic insights into chronic disorders following intestinal infection. World J Gastroenterol 2014; 20: 3976-3985.

[0196] 10. Dizdar V, Gilja OH, Hausken T. Increased visceral sensitivity in post-infectious irritable bowel syndrome and functional dyspepsia caused by Giardia. The role of the 5HT3-antagonist ondansetron. Neurogastroenterol Motil 2007; 19: 977-982.

[0197] 11. Morch K, Hanevik K, Rortveit G, et al. Fatigue and abdominal symptoms are high 2 years after a Giardia outbreak. Trans R Soc Trop Med Hyg 2009; 103: 530-532.

[0198] 12. Wensaas KA, Langeland N, Hanevik K, et al. Irritable bowel syndrome and chronic fatigue 3 years after acute giardiasis: a historical cohort study. Gut 2012; 61: 214-9.

[0199] 13. Halliez MC, Motta JP, Feener TD, et al. Giardia duodenalis induces paracellular bacterial translocation and causes post-infectious visceral hypersensitivity. Am J Physiol Gastrointest Liver Physiol 2016: ajpgi00144 2015.

[0200] 14. Lapointe TK, Basso L, Iftinca MC, et al. TRPV1 sensitization mediates postinflammatory visceral pain after acute colitis. Am J Physiol Gastrointest Liver Physiol 2015;309:G87-99.

[0201] 15. Feng B, La JH, Tanaka T, et al. Altered colorectal afferent function is associated with TNBS-induced visceral hypersensitivity in mice. Am J Physiol Gastrointest Liver Physiol 2012;303:G817-24.

[0202] 16. Annahazi A, Dabek M, Gecse K, et al. Protease-activated receptor-4 induces colorectal analgesia in mice: a feedback loop of endogenous activation in visceral inflammatory pain. Neurogastroenterol Motil 2012;24:76-85,e13.

[0203] 17. Larauche M, Gourcerol G, Million M, et al. Altered visceral sensitivity and colonic motor function induced by repeated psychological stress in mice: effects of surgery and postoperative single housing on visceral motor responses. Stress 2010;13:343-54.

[0204] 18. Hsu LT, Hung KY, Wu HW, et al. Enterogenous cholecystokinin contributes to visceral hypersensitivity via nerve growth factor-dependent neurite outgrowth. J Gastroenterol Hepatol 2016.

[0205] 19. Salvioli B, Serra J, Azpiroz F, et al. Origin of gas retention and symptoms in patients with bloating. Gastroenterology 2005;128:574-9.

[0206] 20. Tonini M, Vicini R, Cervio E, et al. 5-HT7 receptors regulate peristalsis and modulation in guinea pig ileum. Gastroenterology 2005;129:1557-66.

[0207] 21. Dickson EJ, Heredia DJ, Smith TK. Critical roles of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex. Am J Physiol Gastrointest Liver Physiol 2010; 299: G144-57.

[0208] 22. Hemedah M, Coupar IM, Mitchelson FJ. [3H]-Mesulergine labels 5-HT7 sites in rat brain and guinea-pig ileum but not in rat jejunum. Br J Pharmacol 1999; 126: 179-88.

[0209] 23. Kim JJ, Bridle BW, Ghia JE, et al. Targeted inhibition of serotonin type 7 (5-HT7) receptor function modulates immune responses and reduces the severity of intestinal inflammation. J Immunol 2013; 190: 4795-804.

[0210] 24. Yaakob NS, Chinkwo KA, Chetty N, et al. Distribution of 5-HT3, 5-HT4, and 5-HT7 receptors along the human colon. J Neurogastroenterol Motil 2015; 21: 361-9.

[0211] 25. Meuser T, Pietruck C, Gabriel A, et al. Involvement of the 5-HT7 receptor in mediating 5-HT-induced activation of rat primary afferent neurons. Life Sci 2002; 71: 2279-89.

[0212] 26. Hansen HD, Herth MM, Ettrup A, et al. Radiosynthesis and in vivo evaluation of a novel radioligand for PET imaging of the brain 5-HT7 receptor. J Nucl Med 2014; 55: 640-6.

[0213] 27. Zou BC, Dong L, Wang Y, et al. Expression and role of 5-HT7 receptor in the brain and intestine of rats with irritable bowel syndrome. Chin Med J (Engl) 2007; 120: 2069-74.

[0214] 28. Singer SM, Nash TE. T cell-dependent control of acute Giardia infection in mice. Infect. Immun. 2000; 68: 170-175.

[0215] 29. Davids BJ, Palm JE, Housley MP, et al. The polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia lamblia. J Immunol 2006;177:6281-6290.

[0216] 30. Scott KG, Yu LCH, Buret AG. Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury in murine giardiasis. Infect. Immun. 2004;72:3536-3542.

[0217] 31. Scott KG, Meddings JB, Kirk DR, et al. Giardia intestinalis infection reduces epithelial barrier function in a myosin light chain kinase-dependent manner. Gastroenterology 2002;123:1179-1190.

[0218] 32. Lu CL, Hsieh JC, Dun NJ, et al. Estrogen rapidly modulates serotonin-induced visceral hypersensitivity in rats via GPR30. Gastroenterology 2009;137:1040-1050.

[0219] 33. Hong S, Zheng G, Wu X, et al. Corticosterone mediates reciprocal changes between CB1 and TRPV1 receptors in primary sensory neurons of chronically stressed rats. Gastroenterology 2011;140:627-637e4.

[0220] 34. Forcen R, Latorre E, Pardo J, et al. Toll-like receptors 2 and 4 regulate serotonin-induced contractile responses in the murine ileum: analysis of the serotonin receptors involved. Neurogastroenterol Motil 2015;27:1258-66.

[0221] 35. Kuo WT, Lee TC, Yang HY, et al. LPS receptor subunits have opposing roles in epithelial apoptosis and colorectal carcinogenesis. Cell Death Differ 2015;22:1590-1604.

[0222] 36. Wu LL, Peng WH, Kuo WT, et al. Symbiotic bacterial endocytosis in epithelial cells depends on interferon-γ effects on myosin light chain kinase-activated brush borders. Am J Pathol 2014;184:2260-2274.

[0223] 37. Yu LC, Shih YA, Wu LL, et al. Gut dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistance and commensal bacteria via epithelial cell transcytosis. Am J Physiol Gastrointest Liver Physiol 2014; 307: G824-35.

[0224] 38. Nee J, Zakari M, Lembo AJ. Current and emerging drug options for the treatment of diarrhea-predominant irritable bowel syndrome. Expert Opin Pharmacother 2015; 16: 2781-92.

[0225] 39. Dunlop SP, Hebden J, Campbell E, et al. Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndrome. Am J Gastroenterol. 2006; 101: 1288-1294.

[0226] 40. Dunlop SP, Jenkins D, Neal KR, et al. The relative importance of enterochromaffin cell hyperplasia, anxiety, and depression in postinfectious IBS. Gastroenterology 2003; 125: 1651-1659.

[0227] 41. Atkinson W, Lockhart S, Whorwell PJ, et al. Altering serotonin signaling in patients with constipation- and diarrhea-predominant irritable bowel syndrome. Gastroenterology 2006; 130: 34-43.

[0228] 42. Qin HY, Wu JC, Tong XD, et al. Systematic review of animal models of postinfectious / postinflammatory irritable bowel syndrome. J Gastroenterol 2011; 46: 164-74.

[0229] 43. Li CP, Ling C, Biancani P, et al. Effects of progesterone on colonic motility and fecal output in diarrheic mice. Neurogastroenterol Motil 2012; 24: 392-e174.

[0230] 44. Julio-Pieper M, O'Mahony CM, Clarke G, et al. Altered colonic 5-HT and defecation responses induced by chronic stress in mice are strain-dependent. Stress 2012; 15: 218-26.

[0231] 45. Camilleri M. Pharmacology and clinical experience with alosetron. Expert Opin Investig Drugs 2000; 9: 147 - 59.

[0232] 46. Berman SM, Chang L, Suyenobu B, et al. Conditional specific inactivation of brain regions by the 5-HT3 receptor antagonist alosetron. Gastroenterology 2002; 123: 969 - 77.

[0233] 47. Mayer EA, Berman S, Derbyshire SW, et al. Effect of the 5-HT3 receptor antagonist alosetron on responses to visceral stimulation in patients with irritable bowel syndrome. Aliment Pharmacol Ther 2002; 16: 1357 - 66.

[0234] 48. Bradesi S, Lao L, McLean PG, et al. Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia. Pain 2007; 130: 56 - 65.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is 6-methoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-4-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol, 6-methoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-3-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-7-ol, or 6,7-dimethoxy-8-(2-methoxyphenyl)-2-(3-(pyridin-4-yl)propyl)-1,2,3,4-tetrahydroisoquinoline.

2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

3. Use of a pharmaceutical composition for the preparation of a medicament for the treatment of irritable bowel syndrome, the pharmaceutical composition comprising an effective amount of the compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

4. The use as claimed in claim 3, wherein the irritable bowel syndrome is treated by providing an antagonistic effect on the 5-HT7 receptor.

5. The use as claimed in claim 3, wherein the irritable bowel syndrome is treated by inhibiting pain caused by infection and subsequent stress.

6. The use as claimed in claim 3, wherein the irritable bowel syndrome is treated by inhibiting pain caused by chemically induced inflammation.

Images