Patent Grant
11505529
The present invention relates to a series of 8-phenylisoquinoline derivatives used in the treatment of irritable bowel syndrome (IBS).
The 5-HT7 receptor (5-HT7R) is the latest member among the 14 subtypes in 5-HT receptor family. It is widely distributed in both central nervous system (CNS) (most abundant in hypothalamus, thalamus, hippocampus, and cortex) and peripheral organs (e.g. spleen, kidney, intestine, heart and coronary artery), which implicates its role in various physiological functions and pathologic processes. 5-HT7R is positively coupled to adenylate cyclase and has a low sequence homology with other 5-HT receptor subtypes (less than 40%). Based on the studies conducted by using selective 5-HT7R ligands and knock-out mice models, 5-HT7R is involved in circadian rhythm regulation, thermoregulation, sleep disorders, mood disorders, pain, learning and memory. Therefore, the 5-HT7R ligands are potential therapeutic agents for the treatment of a variety of 5-HT7R-related diseases and disorders. The 5-HT7R antagonists may be effective treatment of depression, anxiety, schizophrenia, and dementia, whereas the 5-HT7R agonists could be potential treatment for pain and symptoms of pain (especially neuropathic pain and inflammatory pain).
In addition, 5-HT7R is also a potential drug target for migraine (WO2009029439 A1), hypertention, various mucosal inflammation (WO2012058769 A1), such as irritable bowel syndrome, and urinary incontinence, through its effective smooth muscle relaxation of central and peripheral blood vessels and intestinal, colon, and bladder tissues, respectively. Several therapeutic agents, such as tricyclic antidepressants, typical and atypical antipsychotics and some 5-HT2 receptor antagonists, were found to display moderate to high affinity for 5-HT7R.
In consideration of the versatile therapeutic potential of 5-HT7R ligands, numerous efforts have been focused on the discovery and development of selective 5-HT7R agonists and antagonists. Different structural classes of 5-HT7R ligands 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 of the numerous efforts, there is no 5-HT7R ligand has been used in clinic and still a need to discover and develop novel 5-HT7R ligands with desirable physicochemical and pharmacokinetic properties as potential therapeutic agents for the treatment of 5-HT7R-related diseases and disorders.
Irritable bowel syndrome (IBS) is mainly characterized by recurrent abdominal pain associated with bowel habit changes, in the absence of identifiable organic cause or macroscopic lesions. IBS represents a substantial clinical problem that accounts for 10-40% of gastroenterology outpatients in Asian and Western countries. Severe abdominal pain is the clinical hallmark of IBS, the most likely symptom to result in medical consultation. Subtypes of IBS include diarrhea-predominant IBS-D, constipation predominant IBS-C, or alternating IBS-A. The development of IBS disorder is believed to be related to a disturbed brain-gut axis; however, the pathogenesis is still poorly understood.
Altered intestinal serotonin (5-HT) level in patients is a validated biomarker for IBS. However, clinical drugs targeting 5-HT receptors for IBS treatment are limited nowadays and prescribed only under emergency investigational drug protocol. Alosetron, a 5-HT3R antagonist for treatment of IBS-D, had been withdrawn by FDA for severe side effects (e.g. ischemic colitis, cerebrovascular or cardiovascular ischemia), and reintroduced later for women only with severe symptoms. Other available symptom-relieving agents (e.g. antispasmodics, antidiarrheals, osmotics, sedatives, antidepressants etc.) are not globally effectively for patients. Medical research for IBS pathogenesis relies heavily on analysis of patient biopsy samples. Animal models with visceral hypersensitivity have been established, albeit each with weaknesses and strengths regarding its translational value to IBS. As such, progress in therapeutic development for IBS has been hindered. To date, development of novel targeted drugs for clinical management of IBS is much in need.
Diverse risk factors, including psychological stress, intestinal infection, 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 rate of IBS patients reported past traumatic events in childhood or adulthood. IBS symptoms may begin after a bout of infectious gastroenteritis, termed post-infectious (PI)-IBS. Follow-up studies of a waterborne giardiasis outbreak in Norway reported that more than 40% of patients experience IBS-like symptoms lasting for three years after acute Giardia lamblia infection. The post-infective symptom exacerbation was correlated with the experience of physical or mental stress. Experimental models of post-clearance of pathogen infection and post-resolution of chemical-induced enterocolitis exhibited intestinal hyperalgesia. Moreover, animals subjected to psychological stress also showed visceral hypersensitivity to colorectal distension. Two mouse models with IBS-like visceral hypersensitivity, including dual challenge with Giardia postinfection combined with psychological stress and post-resolution of trinitrobenzene sulfonic acid (TNBS)-induced colitis were used for testing of the analgesic effects of novel 5-HT7R ligands.
Among the receptor subtypes, 5-HT7R is the most recently discovered family member with unknown pathophysiological role. Stimulation of 5-HT7R induces exaggerated relaxation of circular smooth muscle, which has been implicated in ineffective gas propulsion and abdominal bloating. Expression of 5-HT7R has been identified in the enteric neurons (i.e. myenteric afferent neurons and mucosal nerve fibers), smooth muscles, and dendritic cells in the colon, as well as lumbar dorsal root ganglions and brain. The present invention proves a series of 8-phenylisoquinoline derivatives, on alleviation of intestinal pain in two animal models of IBS.
The present invention relates to a novel compound of the following general formula or a pharmaceutically acceptable salt thereof:
wherein R1 is selected from a group consisting of hydrogen, a C1-10 linear chain alkyl group, a C1-10 branched chain alkyl group, a (CH2)n(Hete)R10R11R12 and a (CH2)nArR10R11R12, wherein the n is an integer from 0 to 6, Hete is a heteroaromatic group, Ar is an aromatic group, and R10, R11 and R12 are independently selected from a group consisting of hydrogen, halo group, a nitro group, an amino group, a cyano group, an acetyl group, a C1-6 linear chain saturated alkyl group, a C1-6 linear chain saturated alkoxy group and a C1-6 linear chain saturated haloalkyl group;
R2 is a hydrogen or a C1-6 linear chain saturated alkyl group; and
X1, X2, X3, X4 and X5 are independently selected from a group consisting of hydrogen, a halo group, a nitro group, an amino group, a cyano group, an acetyl group, a C1-6 linear chain saturated alkyl group, a C1-6 branched chain saturated alkyl group, a C1-6 linear chain saturated alkoxy group, a C1-6 branched chain saturated alkoxy group, a C1-6 linear chain saturated alkylthio group, a C1-6 branched chain saturated alkylthio group, a C1-6 linear chain saturated haloalkyl group and a C1-6 branched chain saturated haloalkyl group.
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. Further, the present invention relates to a method of using the aforementioned pharmaceutical composition in the treatment of irritable bowel syndrome.
The present invention provides a novel compound of the following general formula or a pharmaceutically acceptable salt thereof:
wherein R1 is selected from a group consisting of hydrogen, a C1-10 linear chain alkyl group, a C1-10 branched chain alkyl group, a (CH2)n(Hete)R10R11R12 and a (CH2)nArR10R11R12, wherein n is an integer from 0 to 6, Hete is a heteroaromatic group, Ar is an aromatic group, and R10, R11 and R12 are independently selected from a group consisting of hydrogen, a halo group, a nitro group, an amino group, a cyano group, an acetyl group, a C1-6 linear chain saturated alkyl group, a C1-6 linear chain saturated alkoxy group and a C1-6 linear chain saturated haloalkyl group;
R2 is a hydrogen or a C1-6 linear chain saturated alkyl group; and
X1, X2, X3, X4 and X5 are independently selected from a group consisting of hydrogen, a halo group, a nitro group, an amino group, a cyano group, an acetyl group, a C1-6 linear chain saturated alkyl group, a C1-6 branched chain saturated alkyl group, a C1-6 linear chain saturated alkoxy group, a C1-6 branched chain saturated alkoxy group, a C1-6 linear chain saturated alkylthio group, a C1-6 branched chain saturated alkylthio group, a C1-6 linear chain saturated haloalkyl group and a C1-6 branched chain saturated haloalkyl group.
In one embodiment of the present invention, the halo group of the novel compound is selected from a group consisting of fluorine, chlorine, bromine and iodine. In another embodiment of the present invention, the heteroaromatic group of the novel compound is selected from a group consisting of a pyrrolyl group, a furanyl group, a thiophenyl group, a pyridinyl group, a pyrimidinyl group, a thiazolyl group, an indolyl group, an isoindolyl group, an indazolyl group, a benzofuranyl group, an isobenzofuranyl group, a benzothiophenyl group, a benzimidazolyl group, a benzoxazolyl group and a benzothiazolyl group.
In a preferred embodiment of the present invention, the novel compound is one 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. 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.
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:
wherein R1 is selected from a group consisting of hydrogen, a C1-10 linear chain alkyl group, a C1-10 branched chain alkyl group, (CH2)n(Hete)R10R11R12 and (CH2)nArR10R11R12, wherein n is an integer from 0 to 6, Hete is a heteroaromatic group, Ar is an aromatic group, and R10, R11 and R12 are independently selected from a group consisting of hydrogen, a halo group, a nitro group, an amino group, a cyano group, an acetyl group, a C1-6 linear chain saturated alkyl group, a C1-6 linear chain saturated alkoxy group and a C1-6 linear chain saturated haloalkyl group;
R2 is a hydrogen or a C1-6 linear chain saturated alkyl group; and
X1, X2, X3, X4 and X5 are independently selected from a group consisting of hydrogen, a halo group, a nitro group, an amino group, a cyano group, an acetyl group, a C1-6 linear chain saturated alkyl group, a C1-6 branched chain saturated alkyl group, a C1-6 linear chain saturated alkoxy group, a C1-6 branched chain saturated alkoxy group, a C1-6 linear chain saturated alkylthio group, a C1-6 branched chain saturated alkylthio group, a C1-6 linear chain saturated haloalkyl group and a C1-6 branched chain saturated haloalkyl group.
In one embodiment of the present invention, the halo group of the novel compound of the pharmaceutical composition is selected from a group consisting of fluorine, chlorine, bromine and iodine. In another embodiment of the present invention, the heteroaromatic group of the novel compound of the pharmaceutical composition is selected from a group consisting of a pyrrolyl group, a furanyl group, a thiophenyl group, a pyridinyl group, a pyrimidinyl group, a thiazolyl group, an indolyl group, an isoindolyl group, an indazolyl group, a benzofuranyl group, an isobenzofuranyl group, a benzothiophenyl group, a benzimidazolyl group, a benzoxazolyl group and a benzothiazolyl group.
In a preferred embodiment of the present invention, comprising: 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.
The “pharmaceutically acceptable carrier” or “excipient” or “pharmaceutically acceptable carrier or excipient” or “bioavailable carrier” or “bioavailable carrier or excipient” includes but not limited to a solvent, a dispersant, a coating, an antimicrobial agent, an antifungal agent to preserve or a delay-absorbed agent and any other known compound to prepare formulation. In general, these carriers or excipients themselves do not have activity of treating disease. Pharmaceutical compositions or formulations prepared by using the novel compound or its derivatives disclosed in the present invention in combination with a pharmaceutically acceptable carrier or excipient do not cause adverse effect, allergy or other inappropriate reaction of animals or humans. Therefore, the novel compound or its derivatives disclosed in the present invention in combination with a pharmaceutically acceptable carrier or excipient can be applied to human clinically. The pharmaceutical compositions or formulations comprising the novel compound or its derivatives of the present invention can achieve therapeutic effect through intravenous injection, oral administration, inhalation or through local administration of nose, rectum, vagina or hypoglottis. In one embodiment, 0.1 mg to 100 mg of the active ingredient of the compound per day is administered to patients having different diseases.
The carrier to be used is different depending on the pharmaceutical composition or formulation to be prepared. The composition for sterile injection can be suspended in sterile intravenous injection diluents or solvents, such as 1,3-butanediol. The acceptable carrier could be mannitol or water. In addition, the oil fixed or synthesized monoglyceride/diglyceride suspension medium are commonly used solvents. Fatty acids, such as oleic acid, olive oil, castor oil, glyceride derivatives, especially the polyoxyethylenated form could be prepared for injection and natural pharmaceutically acceptable oil. These oil solutions or suspensions include long-chain alcohol diluents, dispersant, carboxymethyl cellulose or similar dispersant. Other surfactants for common use include Tween, Spans, other similar emulsifier, pharmaceutically acceptable solid for pharmaceutical manufacture industry, liquid, or other bioavailable enhancer for formulation development.
The composition for oral administration is adapted to oral acceptable composition or formulation, wherein the types include capsule, lozenge, troche, emulsifier, liquid suspension, dispersant and solvent. The common carrier used for oral administration such as lozenge, for example, can be lactose, corn starch, lubricant, magnesium stearate as basic additives. The diluents used for capsule include lactose, dry corn starch. The preparation for liquid suspension or emulsifier formulation is to suspend or dissolve active ingredients with binding emulsifiers or oil interface of suspending agent. Sweetening agents, flavoring agents or coloring matters can also be included.
The aerosol spray for oral use or inhalation composition is prepared by known formulation technologies. For example, the composition is dissolved in physiological saline, added with benzyl alcohol, other suitable preservatives or absorbefacients to enhance bioavailable properties. The composition of the compound provided by the present invention can also be prepared as a suppository which is administered through rectum or vagina.
The injections include hypodermic, peritoneal cavity, vein, muscle, joint cavity, intracranial, synovial fluid, intrathecal injection, aorta injection, thoracic injection, lesion injection or other suitable administration technologies.
Furthermore, the present invention provides a method for treating irritable bowel syndrome, comprising the step of administering to a subject in need thereof an effective amount of the aforementioned pharmaceutical composition. In one embodiment of the present invention, the halo group of the pharmaceutical composition is selected from a group consisting of fluorine, chlorine, bromine and iodine. In another embodiment of the present invention, the heteroaromatic group of the pharmaceutical composition is selected from a group consisting of a pyrrolyl group, a furanyl group, a thiophenyl group, a pyridinyl group, a pyrimidinyl group, a thiazolyl group, an indolyl group, an isoindolyl group, an indazolyl group, a benzofuranyl group, an isobenzofuranyl group, a benzothiophenyl group, a benzimidazolyl group, a benzoxazolyl group and a benzothiazolyl group, providing an antagonism to a 5-HT7 receptor.
In another embodiment of the present invention, the irritable bowel syndrome is treated by providing an antagonism to 5-HT7 receptors. In yet another embodiment of the present invention, the irritable bowel syndrome comprises a pain induced by infection followed by stress and a pain induced by chemically induced inflammation.
In yet another embodiment of the present invention, the irritable bowel syndrome is treated by inhibiting a pain induced by infection followed by stress. In another embodiment of the present invention, the irritable bowel syndrome is treated by inhibiting a pain induced by chemically induced inflammation.
The above aspects and advantages of the present invention will become apparent to those ordinarily skilled in the art after reviewing the detailed descriptions and accompanying drawing.
The present invention will now be described more specifically with reference to the following examples. It is to be noted that the following descriptions of this invention are presented herein for the purposes of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
The present invention provides a library of novel derivatives of 8-phenylisoquinoline. The synthesis includes the following 4 schemes.
Scheme 1 (as shown in
Scheme 2 (as shown in
Scheme 3 (as shown in
Scheme 4 (as shown in
The specific synthesizing steps of those compounds depicted in the above schemes 1-4 are as follows:
Compound 21: A mixture of compound 20 (100 mg, 0.56 mmol), 2-phenylethyl bromide (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 added slowly 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 afford compound 21 as a white solid (146 mg, 0.52 mmol, 92%).
Compound 30: To a solution of C18H20BrNO2 (50 mg, 0.14 mmol) in 2-propanol (2.0 mL) in a 10-mL thick walled Pyrex reaction vessel, 4-methoxyphenylboronic acid (26 mg, 0.19 mmol) was added. After stirring for 30 min, Pd(OAc)2 (1.3 mg, 0.006 mmol), PPh3 (4.7 mg, 0.02 mmol), 2 M Na2CO3(aq) (0.09 mL, 0.17 mmol), and H2O (0.1 mL) were added. Then the mixture was heated at 140° C. for 10 min in a microwave synthesizer, and H2O (0.35 mL) was added before cooling 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% NaHCO3(aq) and brine. The organic solution was treated with Darco G-60 (100 mg) and stirred at room temperature for 30 min, and then dried over MgSO4, filtered (the sintered glass funnel was charged with Celite to a depth of 1 cm and Florisil was spread evenly on the top of the Celite), and evaporated. The crude residue was chromatographed (silica gel, EtOAc/n-hexane=2/1) to afford an orange oil (40 mg, 0.10 mmol, 73%).
Compounds 29 and 31-40: Table 1 is a parameter table. “Parameter 1” was added into the reaction vessel for microwave-assisted heating and dissolved with “parameter 2” mL 2-propanol. The appearance of the solution was “parameter 3” and the reagent “parameter 4” was added thereinto, and stirred for “parameter 5” minutes. The appearance of the resulting solution was “parameter 6”. The Pd(OAc)2 “parameter 7”, PPh3 “parameter 8”, 2 M Na2CO3(aq) “parameter 9” and “parameter 10” mL H2O were added and heated under the condition of “parameter 11”. Before the temperature of the solution was decreased, “parameter 12” mL H2O was added, stirred in the air until reaching room temperature, diluted with “parameter 13” mL EtOAc, and extracted with “parameter 14” mL H2O. The organic layer was washed with 5% NaHCO3(aq), washed with brine, added in “parameter 15” mg Darco G-60, stirred for “parameter 16” minutes, added in MgSO4 for drying, stirred for “parameter 17” minutes, filtered by the sintered glass funnel covered with about 1 cm of Celite and a thin layer of Florisil, concentrated for drying and purified by flash column chromatography (silica gel, “parameter 18”) to obtain “parameter 19.”
Compound 44: To a solution of C18H20BrNO2 (100 mg, 0.28 mmol) in 2-propanol (1.5 mL) in a 10-mL thick walled Pyrex reaction vessel, 3,5-dimethoxybenzeneboronic acid (62 mg, 0.34 mmol) was added. After stirring for 30 min, Pd(OAc)2 (2.2 mg, 0.01 mmol), PPh3 (8.0 mg, 0.03 mmol), 2 M Na2CO3(aq) (0.17 mL, 0.34 mmol), and H2O (0.7 mL) were added. Then the mixture was heated at 140° C. for 10 min in a microwave synthesizer, and H2O (0.35 mL) was added before cooling 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% NaHCO3(aq) (10 mL) and brine. The organic solution was treated with Darco G-60 (100 mg) and stirred at room temperature for 30 min, and then dried over MgSO4, filtered (the sintered glass funnel was charged with Celite to a depth of 1 cm and Florisil was spread evenly on the top of the Celite), and evaporated. The crude residue was chromatographed (silica gel, EtOAc/n-hexane=1/1) to afford a yellow oil (76 mg, 0.18 mmol, 65%).
Compound 45: To a solution of C18H20BrNO2 (100 mg, 0.28 mmol) in 2-propanol (2.0 mL) in a 10-mL thick walled Pyrex reaction vessel, 2,3-dimethoxyphenylboronic acid (62 mg, 0.34 mmol) was added. After stirring for 30 min, Pd(OAc)2 (2.0 mg, 0.009 mmol), PPh3 (3.7 mg, 0.014 mmol), 2 M Na2CO3(aq) (0.18 mL, 0.36 mmol), and H2O (0.2 mL) were added. Then the mixture was heated at 120° C. for 10 min in a microwave synthesizer, and H2O (0.7 mL) was added before cooling 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% NaHCO3(aq) (5 mL) and brine. The organic solution was treated with Darco G-60 (100 mg) and stirred at room temperature for 30 min, and then dried over MgSO4, filtered (the sintered glass funnel was charged with Celite to a depth of 1 cm and Florisil was spread evenly on the top of the Celite), and evaporated. The crude residue was chromatographed (silica gel, EtOAc/n-hexane=1/1) to afford a yellow oil (82 mg, 0.20 mmol, 71%).
Compounds 15, 60, 85, 95-96, and 98: Table 2 is a parameter table. The starting material “parameter 1” was added into a reaction flask at room temperature under N2, and “parameter 2” mL HOAc was added thereinto. The Pb(OAc)4 “parameter 3” was added, and then the solution was “parameter 4”, poured into a conical flask and added with “parameter 5” mL Na2CO3 (sat) slowly. The pH of the aqueous layer was alkaline (pH=“parameter 6”). The solids produced in neutralization was filtered. The filter cake was washed with CH2Cl2. The filtrate was extracted with “parameter 7” mL CH2Cl2. The organic layer was washed with brine, added with MgSO4 for drying, stirred for 5 minutes, filtered with the sintered glass funnel and concentrated for drying to obtain “parameter 8” product. The crude product was used in the following reaction without further purification.
The solution which was added in HBr “parameter 9” in the room temperature air and the appearance of the solution was “parameter 10.” After stirring for “parameter 11” hours, “parameter 12” mL Na2CO3 (sat) and “parameter 13” mL CH2Cl2 were added slowly to the solution. The pH of the aqueous layer was alkaline (pH=“parameter 14”), and then the “parameter 15” mL CH2Cl2 and “parameter 16” mL H2O were added for extraction. The organic layer was washed with brine, added with MgSO4 for drying, stirred for 5 minutes, filtered with the sintered glass funnel and concentrated for drying to obtain crude product “parameter 17” mg. The “parameter 19” was afforded after flash column chromatography (silica gel, “parameter 18”).
Compounds 11-12, 63-67, 67-70, 74-75, and 78: Table 3 is a parameter table. The starting material “parameter 1” was added into a flask at room temperature under N2, and then the “parameter 2” mL IPA and “parameter 3” were added thereinto. The starting material was dissolved at “parameter 4” ° C. The appearances of reaction solution were “parameter 5” and “parameter 7” in about “parameter 6” minutes, and then the solution was heated at 110˜120° C. for “parameter 8” hours and concentrated in room temperature. The “parameter 9” mL MeOH was added and the resulting mixture was stirred for “parameter 10” minutes. To the solution, which is “parameter 11” in a ice-bath, NaBH4(s) “parameter 12” was added slowly under N2 and stirred for “parameter 13” minutes. The solution, which is “parameter 14,” was added with “parameter 15” mL H2O and extracted with “parameter 16” mL CHCl3. The organic layer was added with MgSO4 for drying, stirred for “parameter 17” minutes, filtered, and concentrated to obtain “parameter 18”. The “parameter 20” was afforded after flash column chromatography (silica gel, “parameter 19”).
Compound 68: A mixture of C10N11NO2 (300 mg, 1.69 mmol), C8H8Br2 (1.00 g, 3.79 mmol), and 2-propanol (10 mL) was heated to reflux for 23 h. The resulting solution was cooled to room temperature, and evaporated. The crude was dissolved in MeOH (15 mL), cooled to 0° C. in ice-bath, and then NaBH4 (420 mg, 11.1 mmol) was added in portions under N2. The mixture was stirred for another 20 min and then concentrated. The residue was treated with CHCl3 (30 mL) and H2O (30 mL) and then the organic layer was dried over MgSO4, filtered and evaporated. The purification was performed by the precipitation method. The crude product was dissolved with 5 mL of EtOAc, and then the product was precipitates with 10 mL of n-hexane to afford a beige solid (620 mg, 1.71 mmol).
Compound 121: To a solution of C19H23NO2 (250 mg, 0.84 mmol) in HOAc (4.2 mL), Pb(OAc)4 (579 mg, 1.31 mmol) was added and the mixture was stirred at room temperature under N2 for 15 min. The reaction mixture was diluted with CH2Cl2 and Na2CO3 (sat) (20 mL) was added slowly. The solids formed in neutralization were removed by filtration and washed with CH2Cl2. The combined filtrate was extracted with CH2Cl2 (35 mL), and then the organic layer was washed with brine, dried over MgSO4, filtered, and evaporated to afford a brown oil (480 mg, 1.35 mmol), which was used in the following reaction without further purification. To a solution of the crude oil in CH2Cl2 (17 mL), 1,3-dimethoxybenzene (0.17 mL, 1.3 mmol) and trifluoroacetic acid (0.84 mL) were added. The resulting mixture was stirred at room temperature for 30 min, and then Na2CO3 (sat) (20 mL) was added slowly. The resulting solution was extracted with CH2Cl2 (18 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=1/10) to afford a red-brown oil (214 mg, 0.49 mmol, 59%).
Compounds 6, 105-110, 114-115, 120, 122, 123 and 125: Table 4 is a parameter table. The starting material “parameter 1” was added into a flask at room temperature under N2 and dissolved with “parameter 2” mL HOAc. The solution which was “parameter 3” was added with Pb(OAc)4 “parameter 4,” and then the resulting solution which was “parameter 5” was stirred for “parameter 6” minutes, poured into a 125 mL conical flask, stirred and added slowly with “parameter 7” mL Na2CO3(sat). The pH of the aqueous layer was alkaline (pH=8-9). The solid produced by neutralization was filtered and washed with CH2Cl2. The filtrate was extracted with “parameter 8” mL CH2Cl2. The organic layer was washed with brine, added with MgSO4 for drying, stirred for 5 minutes, filtered, and concentrated to afford “parameter 9”. The crude product was used in the following reaction without further purification.
The crude product was dissolved in “parameter 10” mL CH2Cl2 at room temperature under N2. The solution which was “parameter 11” was added with 1,3-dimethoxybenzene “parameter 12” and trifluoroacetic acid “parameter 13”. The color of the solution turned into “parameter 14”. After the solution was stirred for “parameter 15” minutes, “parameter 16” mL Na2CO3(sat) was added slowly. The pH of the aqueous layer was alkaline (pH=8-9), and “parameter 17” mL CH2Cl2 was added for extraction. The organic layer was washed with brine, added with MgSO4 for drying, stirred for 5 minutes, filtered, and concentrated to obtain “parameter 18” mg crude product. The “parameter 20” was afforded after flash column chromatography (silica gel, “parameter 19”).
Compounds 7 and 142: Table 5 is a parameter table. “Parameter 1” and 2-methoxyphenylboronic acid (46 mg, 0.30 mmol) were added into the reaction vessel for microwave-assisted heating and dissolved with 2-propanol (2 mL), and stirred for 30 minutes. Pd(OAc)2 “parameter 2”, PPh3 “parameter 3”, 2 M Na2CO3(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 temperature of the solution was decreased, the solution was added with H2O (0.7 mL), stirred in the 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% NaHCO3(aq), washed with brine, added in “parameter 4” mg Darco G-60, stirred for 10 minutes, added in MgSO4 for drying, stirred for 10 minutes, filtered by the sintered glass funnel covered with about 1 cm of Celite and a thin layer of Florisil, and concentrated. The crude product was purified by flash column chromatography (silica gel, “parameter 5”) to obtain a yellow oil “parameter 6.” 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 hydrochloride salt “parameter 8”.
Compound 150: To a solution of C18H20BrNO2 (100 mg, 0.28 mmol) in DMF (2 mL), trimethylphenyl-ammonium 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, and 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/n-hexane=1/4) to afford a yellow solid (83 mg, 0.22 mmol, 79%).
Compound 152: To a solution of C18H19BrN2O4 (406 mg, 1.00 mmol) in DMF (9 mL), which was cooled to 0° C. and degassed, NaH (40 mg, 1.67 mmol) and CH3I (0.06 mL, 0.98 mmol) in DMF (1 mL) were added. After stirring for 10 min, NH4Cl (111 mg, 2.08 mmol) was added, and 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/n-hexane=1/2) to afford a yellow solid (123 mg, 0.29 mmol, 30%).
Compound 153: To a solution of C18H19BrClNO2 (300 mg, 0.75 mmol) in DMF (6 mL), (CH3)3PhNCl (542 mg, 3.16 mmol) and t-BuOK (333 mg, 2.97 mmol) were added. The suspension was heated to 60° C. under N2 for 16 h, and 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/n-hexane=1/4) to afford a white solid (189 mg, 0.46 mmol, 61%).
Compound 154: To a solution of C18H19BrFNO2 (400 mg, 1.05 mmol) in DMF (8 mL), (CH3)3PhNCl (727 mg, 4.23 mmol) and t-BuOK (468 mg, 4.17 mmol) were added. 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/n-hexane=1/4) to afford a white solid (247 mg, 0.63 mmol, 60%).
Compounds 157-159, 165-168 and 171-173: Table 6 is a parameter table. “Parameter 1” was added into a reaction vessel for microwave-assisted heating and dissolved with “parameter 2” mL 2-propanol. “Parameter 3” was added thereinto, and stirred for 30 minutes. Pd(OAc)2 “parameter 4”, PPh3 “parameter 5”, 2 M Na2CO3(aq)“parameter 6” and “parameter 7” mL H2O were added and heated to 120° C. for 20 min using a microwave synthesizer. Before the temperature of the solution was decreased, “parameter 8” mL H2O was added, and then cooled to room temperature, diluted with 10 mL EtOAc, and extracted with 10 mL H2O. The organic layer was washed with 5% NaHCO3(aq) followed by brine, added in “parameter 9” mg Darco G-60, stirred for 10 min, filtered by the sintered glass funnel covered with about 1 cm of Celite and a thin layer of Florisil, concentrated, and purified by flash column chromatography (silica gel, “parameter 10”) to obtain “parameter 11.”
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The 5-HT7 receptor binding affinity, 5-HT2A receptor binding affinity, and log D data of compounds 6-10 are shown in Table 8.
Animals
Specific pathogen free C57BL/6 mice (4-6 weeks of age) obtained from the Animal Center of the National Taiwan University were used for the study. Animals were raised in a temperature-controlled room (20±2° C.) with 12/12-h light/dark cycles, and fed with regular mice chow and water ad libitum. All experimental procedures were approved by the Animal Care and Use Committee of the National Taiwan University.
Reagents
Novel 8-phenyl-isoquinoline derivatives were prepared by the procedures described below. 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 intraperitoneally (i.p.) or perorally (p.o.) administered by a single dose or multiple doses to mice for the analysis of intestinal pain.
Two Experimental Models of Visceral Hypersensitivity
(1) Dual Challenge of Giardia Postinfection Combined with Water Avoidance Stress
Two animal models of IBS that had shown visceral hypersensitivity were used in the study, including dual challenge of postinfection combined with psychological stress, and post-inflammation. In the first model, mice were divided into two groups, including one group subjected to dual triggers of Giardia postinfection and water avoidance stress (GW) and the one group pair-fed with saline and non-handled (PN) as uninfected unstressed normal controls. Axenic Giardia lamblia trophozoites (strain GS/M, ATCC 50581) were cultured in vitro and harvested at log-phase as described in 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 orally gavaged with 107 Giardia trophozoites suspended in 0.2 ml of sterile phosphate-buffered saline (PBS) or pair-fed with the same volume of PBS. The status of Giardia infection was verified after 4-7 days by enumeration of motile trophozoites in the small intestine following a cold-shock protocol (disclosed in Scott K G, Yu L C H, Buret A G. Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury during murine giardiasis. Infect. Immun. 2004; 72:3536-3542 and Scott K G, Meddings J B, Kirk D R, et al. Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 2002; 123:1179-1190). On the sixth week postinfection in which the trophozoites could not be detected in the small intestine (post-clearance phase), mice were subjected to chronic psychological stress. The procedure of WAS involved placing the mouse on a platform (3×6 cm) in the center of a container (56×50 cm) with 3 cm (vertical height) of room temperature water. Mice remained on the platform for 1 hr to avoid water immersion as a psychological stress without physical harm. The 1-hr stress sessions were carried out for 10 consecutive days to mimic chronic repeated stress, and were performed between 9:00 and 12:00 to minimize the effect of the circadian rhythm. Uninfected and unstressed non-handled animals were kept in their cages as normal controls. On the last day of the stress session, intestinal pain was measured in mice.
For testing of anti-nociceptive effects in the GW model, mice were administered novel 5-HT7R ligands by a single dose 90 or 240 minutes prior to intestinal pain measurement. In additional settings, the novel ligands were repeatedly administered for 10 consecutive days 30 minutes before the start of each stress session and intestinal pain was measured immediately after the last stress session.
(2) Postinflammation Model
In the second model, intestinal inflammation was induced by intracolonic administration of 10% 2,4,6-trinotrobenzene sulfonic acid (TNBS) in 0.2 ml of 50% ethanol (Sigma-Aldrich, St. Louis, Mo., USA) via a 22-gauge feeding needle. Sham controls were given PBS in the same volume. Intestinal inflammatory parameters and pain levels were measured on various time points after TNBS administration.
For testing of anti-nociceptive effects in the post-TNBS model, mice were administered with novel 5-HT7R ligands by a single dose at 90 or 240 minutes before or by repeated administration of multiple doses for 10 consecutive days prior to intestinal pain measurement.
Assessment of Pain Sensation to Colorectal Distension
Abdominal pain was measured by visceromoter response (VMR) to colorectal distension (CRD) in mice following previously described methods with slight modification (Lu C L, Hsieh J C, Dun N J, 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 CB 1 and TRPV1 receptors in primary sensory neurons in the chronically stressed rat. Gastroenterology 2011; 140:627-637 e4). Briefly, electrodes made from Teflon-coated stainless steel wire (A-M systems, Carlsborg, Wash.) were implanted in the abdominal external oblique muscles of mice at least 15 days prior to VMR experiments. The electrodes were exteriorized onto the back of the neck. Mice were habituated in the plexiglass cylinder for 30 minutes per day for 3 consecutive days before VMR experiments. The cylinder was used for partial restraint of conscious mice during the CRD experiments. For recording, electrodes were connected to an electromyogram acquisition system (AD instruments, New south wales, Australia). The colon was distended by inflating a balloon catheter inserted intra-anally such that it ended 1.5 cm proximal to the anus. Mice were subjected to four 10-second distensions (15, 40, and 65 mmHg) with 3-min rest intervals. The electromyographic (EMG) activity was amplified and digitized using a transducer (AD instruments) connected to a P511 AC amplifier (Grass instruments, Calif., USA) and Powerlab device 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 versus the baseline period.
Histopathological Examination
Intestinal tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin wax with proper orientation of the crypt to villus axis before sectioning. Sections of 5-μm thickness were deparaffinized with xylene and graded ethanol, stained with hematoxylin and eosin (H&E), and observed under a light microscope.
Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from tissue samples using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The RNA (2 μg) was reversely transcribed with oligo(dT)15 using RevertAid™ First Strant cDNA Synthesis kit (Thermo) in 20 μL reaction volume. The resulting cDNA corresponding to 0.1 μg of initial RNA was then subjected to PCR by the addition of master mix containing 1X PCR buffer, 1 U DreamTaq™ DNA Polymerase, 0.2 mM dNTPs mixture, 0.4 μM upstream primer, and 0.4 μM downstream primer. The specific primer pairs for 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 a protocol as follows: 95° C. for 3 min for 1 cycle; 95° C. for 30 sec (denaturation), 55° C. for 30 sec (annealing), and 72° C. for 30 sec (extension) for 30 cycles; and 72° C. for 7 min for final extension. Negative controls were performed with samples lacking cDNA that was not reversely transcribed. RT-PCR products were then electrophoresed in a 1.5% agarose gel in the presence of 0.5 μg/mL ethidium bromide, visualized with an ultraviolet transilluminator, and photographs were taken. The intensity of the DNA bands was analyzed using the Gel-Pro Analyzer 4.0 software.
Immunofluorescent Staining of 5-HT7R
Deparaffinized histological slides were incubated with 10 mM Tri-sodium citrate buffer (pH 6.0) containing 0.05% Tween-20 and boiled in microwave. Sections were left at room temperature to cool down. After quenching with 1 mg/ml NaBH4 in PBS (pH 8.0) for 15 minutes at room temperature, tissues were blocked with 1% bovine serum albumin for 2 hours at room temperature. Tissue sections were incubated with primary antibodies, rabbit polyclonal anti-5-HT7R (1:300, Abcam), rabbit PGP9.5 antibody (1:250, GeneTex) or isotype controls overnight at 4° C. The sections were washed with PBS and incubated with a secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:250, Molecular Probes) for one hour at room temperature. The tissues were then incubated with a Hoechst dye (1 μg/ml in PBS) (Sigma) for another 30 minutes. The slides were observed under a fluorescent microscope and the images were captured.
Western Blotting
Intestinal mucosal proteins were extracted with complete radio-immunoprecipitation (RIPA) buffer and subjected to SDS/polyacrylamide gel electrophoresis (PAGE) (4-13% polyacrylamide) (as described in Kuo W T, Lee T C, Yang H Y, et al. LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis. Cell Death Differ 2015; 22:1590-1604; Wu L L, Peng W H, Kuo W T, 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 L C, Shih Y A, Wu L L, 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 onto PVDF or nitrocellulose membranes in a semi-dry blotter. Blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline (TB S) or 5% (w/v) bovine serum albumin in TBS with Tween 20 (TBS-T; 0.1% (v/v) Tween-20 in TBS) for 1 h, washed with TBS-T, and incubated with a primary antibody at 4° C. overnight. The membrane was washed and incubated with a secondary antibody for 1 h. After washing, the membranes were incubated with chemiluminescent solution and signals detected. The primary antibodies used included rabbit polyclonal anti-5-HT7R (1:500, Abcam) and anti-β-actin (1:10000, Sigma). The secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000, Cell Signaling).
Statistical Analysis
All values were expressed as mean±SEM, and compared by paired Student's t test. Significance was established at P<0.05.
Intestinal Hypernociception Correlated with Upregulation of Colonic 5-HT7R Expression in two IBS-Like Mouse Models
Two animal models of IBS-like visceral hypersensitivity were utilized to examine anti-nociceptive effects of a series of 8-phenylisoquinoline derivatives which were novel 5-HT7R ligands. Mice were divided into two groups, one group was subjected to Giardia postinfection and water avoidance stress (GW) and another group was pair-fed and non-handled (PN) as uninfected unstressed normal controls. The visceromoter response (VMR) to colorectal distension was expressed as the area under a curve (AUC), and was determined in each mouse as an indicator of intestinal pain.
In the first model, by dual challenge of Giardia postinfection combined with psychological stress (GW) an increased abdominal pain was observed compared to normal controls (
In the second model, mice were given one bolus of colitogenic chemical TNBS or PBS intracolonically on day 0 and intestinal inflammation and pain were examined and measured on various days. These animals displayed an increased abdominal pain 7, 14 and 24 days post-TNBS (
5-HT7R Activation is Involved in Visceral Hypersensitivity in the IBS Models
To verify the role of 5-HT7R on visceral hypersensitivity for proof-of-concept, a putative 5-HT7R antagonist for research use (SB-269970) was intraperitoneally (i.p., 0.5 mg/Kg) injected into the animal models and intestinal pain was measured by VMR. Administration of SB7 through i.p. significantly inhibited intestinal pain levels in mice (
Anti-Nociceptive Effects of Novel 5-HT7R Ligands
Novel 8-phenyl-isoquinoline derivatives (compounds I) targeting 5-HT7R with high binding affinity and water solubility were synthesized (Compounds 6-10 shown in Table 8). In the initial experiments, compounds 6-10 (5-HT7R ligands) were perorally (p.o.) administered at 5 mg/kg in GW mice to assess the inhibitory effect on abdominal pain. A single dose at 5 mg/Kg was administered 90 minutes before the analysis of VMR. All of the compounds tested showed anti-nociceptive effects, among which compound 8 exhibited the strongest inhibition of intestinal pain to baseline levels (
To examine the dose response on anti-nociceptive effects, compound 8 was injected intraperitoneally (i.p.) at 0.05, and 0.5 mg/kg, or perorally (p.o.) at 1.5, and 5 mg/kg to GW mice. Dose-dependent analgesic effects were observed in GW mice by compound 8 (
TNBS mice were perorally (p.o.) injected with vehicle or novel 5-HT7R ligands to assess the inhibitory effect on abdominal pain. A single dose at 5 mg/kg was administered 90 minutes before the analysis of VMR. In these TNBS mice, these novel 5-HT7R ligands attenuated intestinal pain at a single dose by p.o. administration (
Comparison of analgesic effects and adverse response between 8-phenylisoquinoline derivatives and reference standards
The anti-nociceptive potency of compounds I (compounds 6-10) was compared with reference standards by p.o. administration in the two animal models. These compounds and reference standards 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 the GW mice, p.o. administration of ALN reduced intestinal pain but was less efficient compared to compound 8 in GW mice (
All mice administered vehicle or compounds displayed normal colonic histology, except those given ALN. In 2 out of 14 mice (14%) administered ALN, hyperemia and granulocyte infiltration were observed in the colonic tissues (
Newly FDA-approved agents, eluxadoline (a mixed μ-opioid agonist) and rifamixin (a nonabsorbable gut-specific antibiotic) had been recent additions to the treatment options for IBS-D. These pharmaceutic agents represented molecular mechanisms or environmental factors different from the 5-HT7R targets. It was noteworthy that any opioid agonist would pose a risk for drug addiction following long-term treatment. Compared to traditional pain-killers (e.g. non-steroidal anti-inflammatory drugs and anticholinergic agents) or anti-diarrheal opioid agonists (e.g. loperamide), this series of 8-phenyl-isoquinoline derivatives, i.e., 5-HT7R antagonists, were more beneficial because they might peripheral-selectively act at the hypernociceptive intestine.
In this invention, 8-phenyl-isoquinoline derivatives (I) (Compounds 6-10) exhibited stronger analgesic actions without adverse effects compared to alosetron in IBS animal models, and therefore they were suitable to be used in both male and female patients as new therapeutic options for IBS treatment.
The entire context of the following articles is incorporated into the present context by references in their entirieties.
Part I
This patent application is a U.S. National Stage Application of PCT/CA2018/000043 filed Mar. 2, 2018 and claims the benefit of priority from U.S. Provisional Application Ser. No. 62/466,370, filed Mar. 3, 2017, the contents of each of which is incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2018/000043 | 3/2/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/157233 | 9/7/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 8552028 | Su | Oct 2013 | B2 |
| Number | Date | Country | |
|---|---|---|---|
| 20200071274 A1 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62466370 | Mar 2017 | US |
