Patent Application
20080096875
Hepatitis C virus (HCV) infection is estimated to affect 170 million individuals worldwide. This disease is primarily transmitted through contaminated blood products. Although its spread has been slowed as a result of improvement in blood screening in many countries, it remains the leading cause of liver disease-related deaths in the world. For example, it causes about 10,000 deaths annually in the U.S. alone. In the absence of effective therapies, the death rate is expected to triple over the next 2 decades.
Current treatments based on interferon-alpha have low success rates, particularly for genotype-1 infections predominant in Europe, Japan, and the U.S. Also, they are expensive and poorly received by patients. Thus, there is a need to develop better therapeutic agents for treating HCV infection.
This invention is based on the discovery that certain thiourea compounds are effective in treating hepatitis C virus infection.
In one aspect, this invention relates to thiourea compounds of formula (I):
In this formula, each of R1, R2, and R3, independently, is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, aryl, or heteroaryl; or R1 and R2, together with the nitrogen atom to which they are bonded, are C3-C20 heterocycloalkyl; or R2 and R3, together with the two nitrogen atoms to which they are bonded and the carbon atom bonded to both of the two nitrogen atoms, are C3-C20 heterocycloalkyl; each of A1 and A2, independently, is aryl or heteroaryl: each of X, Y, and Z, independently, is O, S, S(O), S(O)2, N(Ra), C(RaRb), C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C1-C20 heterocycloalkyl, aryl, or heteroaryl, in which each of Ra and Rb, independently, is H, C1-C10 alkyl, C3-C20 cycloalkyl, C1-C20 heterocycloalkyl, aryl, or heteroaryl; each of m and n, independently, is 1, 2, 3, 4, or 5; and each of x, y, and z, independently, is 0 or 1.
Referring to formula (I), a subset of the thiourea compounds described above are those in which x is 1, y is 0, and z is 0. In these compounds, X can be O or NH, A1 can be phenylene, A2 can be phenyl, and each of R1, R2, and R3, independently, can be H or C1-C10 alkyl optionally substituted with aryl.
Another subset of the thiourea compounds described above are those in which x is 1, y is 0, and z is 1. In these compounds, X and Z can both be O, each of R1, R2, and R3 can be H, or R1 and R2, together with the nitrogen atom to which they are bonded, can be C3-C20 heterocycloalkyl, A1 can be phenylene, and A2 can be heteroaryl, or aryl optionally substituted with halo, aryl, heteroaryl, CN, OR, COOR, or NRR′, in which each of R and R′ independently, is H, C1-C10 alkyl, or aryl. Referring to formula (I), another subset of the thiourea compounds described above are those in which x is 1, y is 1, and z is 1. In these compounds, X and Z can both be O, Y can be C(RaRb) (in which each of Ra and Rb, independently, can be C1-C10 alkyl), A1 can be phenylene, A2 can be phenyl optionally substituted with aryl, and each of R1, R2, and R3 can be H.
The term “alkyl” refers to a saturated, linear or branched hydrocarbon moiety, such as —CH3, —CH(CH3)2, or —CH2—. The term “alkenyl” refers to a linear or branched hydrocarbon moiety that contains at least one double bond, such as —CH═CH—CH3 or —CH═CH—CH2—. The term “alkynyl” refers to a linear or branched hydrocarbon moiety that contains at least one triple bond, such as —O═C—CH3 or —O═C—CH2—. The term “cycloalkyl” refers to a saturated, cyclic hydrocarbon moiety, such as cyclohexyl or cyclohexylene. The term “cycloalkenyl” refers to a non-aromatic, cyclic hydrocarbon moiety that contains at least one double bond, such as cyclohexenyl. The term “heterocycloalkyl” refers to a saturated, cyclic moiety having at least one ring heteroatom (e.g., N, O, or S), such as 4-tetrahydropyranyl or 4-tetrahydropyranylene. The term “heterocycloalkenyl” refers to a non-aromatic, cyclic moiety having at least one ring heteroatom (e.g., N, O, or S) and at least one double bond, such as pyranyl. The term “aryl” refers to a hydrocarbon, moiety having one or more aromatic rings. Examples of aryl moieties include phenyl (Ph), phenylene, naphthyl, naphthylene, pyrenyl, anthryl, and phenanthryl. The term “heteroaryl” refers to a moiety having one or more aromatic rings that contain at least one heteroatom (e.g., N, O, or S). Examples of heteroaryl moieties include furyl, furylene, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolyl, isoquinolyl and indolyl.
Alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl mentioned herein include both substituted and unsubstituted moieties, unless specified otherwise. Possible substituents on cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl include, but are not limited to, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C10 alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C1-C10 alkylamino, C1-C20 dialkylamino, arylamino, diarylamino, hydroxyl, halo, thio, C1-C10 alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amidino, guanidine, ureido, cyano, nitro, acyl, thioacyl, acyloxy, carboxyl, and carboxylic ester. On the other hand, possible substituents on alkyl, alkenyl, or alkynyl include all of the above-recited substituents except C1-C10 alkyl. Cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl can also be fused with each other.
In another aspect, this invention features thiourea compounds of formula (I), in which R1 is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, aryl, or heteroaryl; each of R2 and R3, independently, is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, aryl, or heteroaryl; or R2 and R3, together with the two nitrogen atoms to which they are bonded and the carbon atom bonded to both of the two nitrogen atoms, are C3-C20 heterocycloalkyl: each of A1 and A2, independently, is aryl or heteroaryl; each of X, Y, and Z, independently, is O, S, S(O), S(O)2, N(Ra), C(RaRb), C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C1-C20 heterocycloalkyl, aryl, or heteroaryl, in which each of Ra and Rb, independently, is H, C1-C10 alkyl, C3-C20 cycloalkyl, C1-C20 heterocycloalkyl, aryl, or heteroaryl; each of m and n, independently, is 0, 1, 2, 3, 4, or 5; and each of x, y, and z, independently, is 0 or 1.
Referring to formula (I), a subset of the thiourea compounds described above are those in which x is 1, y is 0, and z is 0. In these compounds, X can be O, A1 can be phenylene, A2 can be phenyl, R1 can be H or C1-C10 alkyl optionally substituted with aryl, and R2 and R3, together with the two nitrogen atoms to which they are bonded and the carbon atom bonded to both of the two nitrogen atoms, can be C3-C20 heterocycloalkyl;
In another aspect, this invention relates to thiourea compounds of formula (II):
wherein X is O, N(Ra), C(RaRb), or C(O); each of R1, R2, and R3, independently, is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, aryl, or heteroaryl; or R2 and R3, together with the two nitrogen atoms to which they are bonded and the carbon atom bonded to both of the two nitrogen atoms, are C3-C20 heterocycloalkyl; and each of R4, R5, R6, R7, R8, R9, and R10, independently, is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, aryl, heteroaryl halo, N(RcRd), N(Rc)—C(S)—N(RdRe); N(Rc)—C(O)Rd, or N(Rc)—C(O)O—Rd; in which each of Ra, Rb, Rc, Rd, and Re, independently, is H, C1-C10 alkyl, C3-C20 cycloalkyl, C1-C20 heterocycloalkyl, aryl, or heteroaryl; provided that if R10 is at the 3-position, then
is at the 4-position, and; if R10 is at the 4-position, then
is at the 3-position. The 3- and 4-positions of the above formula are delineated below:
An embodiment of the just-described compounds features the following formula:
wherein X, R1, R2, R3, R4, R5, R6, R7, R8, and R9 are as just defined.
Referring the above formula, a subset of the thiourea compounds described above are those in which each of R1, R2, and R3, independently, is H, aryl optionally substituted with C1-C20 heterocycloalkyl, heteroaryl, or C1-C10 alkyl optionally substituted with C1-C10 alkoxy, aryl, N(RR′), in which each of R and R independently, is H or C1-C10 alkyl. In these compounds, each of R4, R5, R6, R7, R8, and R9, independently, can be H, halo, N(RcRd), N(Rc)—C(S)—N(RdRe); N(Rc)—C(O)Rd, or N(Rc)—C(O)O—Rd. For example, each of R4, R5, R7, R8, and R9 can be H and R6 can be H, halo, N(RcRd), N(Rc)—C(S)—N(RdRe), N(Rc)—C(O)Rd, or N(Rc)—C(O)O—Rd.
Another subset of the thiourea compounds described above are those in which each of R1, R2, and R3 is H; or R4 is (CH2)nCH3, in which n is 1, 2, 3, 4, 5, or 6, and each of R2 and R3 is H.
In still another aspect, this invention features a method for treating hepatitis C virus infection. The method includes administering to a subject in need thereof an effective amount of one or more thiourea compounds of formula (I) or (II) shown above. The term “treating” or “treatment” refers to administering one or more thiourea compounds to a subject, who has an above-described infection, a symptom of such an infection, or a predisposition, toward such an infection, with the purpose to confer a therapeutic effect, e.g., to cure, relieve, alter, affect, ameliorate, or prevent the above-described infection, the symptom, of it, or the predisposition toward it.
In addition, this invention encompasses a pharmaceutical composition that contains an effective amount of at least one of the above-mentioned thiourea compounds and a pharmaceutically acceptable carrier.
The thiourea compounds described above include the compounds themselves, as well as their salts, prodrugs, and solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a thiourea compound. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumurate, glutamate, glucuronate, lactate, glutarate, and maleate. Likewise, a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a thiourea compound. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ton, and an ammonium cation such as tetramethylammonium ion. The thiourea compounds also include those salts containing quaternary nitrogen atoms. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active thiourea compounds. A solvate refers to a complex formed between, an active thiourea compound and a pharmaceutically acceptable solvent. Examples of pharmaceutically acceptable solvents include water, ethanol, isopropanol, ethyl acetate, acetic acid, and ethanolamine.
Also within the scope of this invention is a pharmaceutical composition containing one or more of the above-described thiourea compounds for use in treating HCV infection, as well as this therapeutic use and use of the compounds for the manufacture of a medicament for treating HCV infection.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.
The table below show 183 exemplary compounds of this invention:
The thiourea compounds described above can be prepared by methods well known in the art. Examples 1-183 below provide detailed descriptions of the preparation of compounds 1-183.
Scheme I shown below depicts a typical route for synthesizing certain compounds of the invention. Specifically, 3-nitrophenol can first react with a brominated aromatic compound via a substitution reaction to form an alkoxy-containing compound. The alkoxy-containing compound can then be reduced (e.g., by hydrogen or tin chloride) to convert the nitro group to an amino group. The compound thus formed can then be treated with thiocarbonyl diimidazole (TCDI) and a base (e.g., ammonia) to form a compound of the invention (e.g., compounds 1-14, 21-31, 82-140, and 143-183).
Certain other compounds of the invention can be prepared from benzene-1,3-diamine. For example, as shown in Scheme II below, one of the amino groups on benzene-1,3-diamine can be first protected with a tert-butyloxycarbonyl (BOC) protecting group. The other amino group on benzene-1,3-diamine can then react with a brominated aromatic compound. The compound thus formed can subsequently be deprotected and then treated with thiocarbonyl diimidazole and a base to form compounds of the invention such as compounds 15-20.
Certain other compounds of the invention can be prepared from a monoamino aromatic compound. For example, as shown in Scheme III below, a monoamino aromatic compound can react with thiocarbonyl diimidazole, followed by ammonia or a primary amine, to form a compound of the invention (e.g., compounds 32-38 and 50-71).
Certain other compounds of the invention can be prepared from a diamino aromatic compound. For example, as shown in Scheme IV below, one amino group on 9H-fluorene-2,7-diamine can first be protected with a BOC protecting group. The other amino group 9H-fluorene-2,7-diamine can then react with a halo-containing compound to form either a compound containing a secondary amino group or a compound containing a tertiary amino group. The compound thus formed can be deprotected (e.g., by reacting with trifluoroacetic acid) and then treated with thiocarbonyl diimidazole and a base to form a compound of the invention (e.g., compounds 39-48, 72-75, 141, and 142).
Certain other compounds of the invention containing an imidazolidinyl ring can be prepared by the method shown in Scheme V. Specifically, an amino-containing compound can first react with 1-chloro-2-isothiocyanatoethane to form a chlorine-containing thiourea compound. The thiourea compound can then react with a base (e.g., triethylamine) to form a compound of the invention containing an imidazolidinyl ring (e.g., compounds 76 and 79). The compound thus formed can optionally react with a halo-containing compound to form another compound of the invention (e.g., compounds 77, 78, 80, and 81).
A thiourea compound synthesized above can be purified by a suitable method such as column chromatography, high-pressure liquid chromatography, or recrystallization.
Other thiourea compounds can be prepared using other suitable starting materials through the above synthetic routes and others known in the art. The methods described above may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the thiourea compounds. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable thiourea compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and I., Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
The thiourea compounds mentioned herein may contain a non-aromatic double bond and one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans-isomeric forms. All such isomeric forms are contemplated.
Also within the scope of this invention is a pharmaceutical composition containing an effective amount of at least one thiourea compound described above and a pharmaceutical acceptable carrier. Further, this invention covers a method of administering an effective amount of one or more of the thiourea compounds to a patient having hepatitis C virus infection. “An effective amount” refers to the amount of an active thiourea compound that is required to confer a therapeutic effect on the treated subject. Effective closes will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
To practice the method of the present invention, a composition having one or more thiourea compounds can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term “parenteral” as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.
A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water. Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.
A composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the ease of tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
A nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical, formulation, for example, such a composition can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
A composition having one or more active thiourea compounds can also be administered in the form of suppositories for rectal administration.
The carrier in the pharmaceutical composition must be “acceptable” in the sense that it is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. One or more solubilizing agents can be utilized as pharmaceutical excipients for delivery of an active thiourea compound. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.
The thiourea compounds described above can be preliminarily screened for their efficacy in treating hepatitis C virus infection by an in vitro assay (See Examples 141 and 142 below) and then confirmed by animal experiments and clinic trials. Other methods will also be apparent to those of ordinary skill in the art.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
Potassium carbonate (1.2 g, 8.7 mmol) was added to a stirred suspension of 3-nitrophenol (0.8 g, 5.8 mmol), (5-bromo-pentyl)-benzene (1.32 g, 5.8 mmol), and potassium iodide (0.96 g, 5.8 mmol) in N-methylpyrolidinone (15 mL). The mixture was stirred at 90° C. for 4 hours. After the reaction mixture was cooled to the room temperature, it was quenched with water (30 mL) followed, by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give 1-nitro-3-(5-phenylpentoxy)benzene (1.4 g, 4.93 mmol, yield: 85%) as colorless oil.
Tin (II) chloride (5.57 g, 24.7 mmol) was added to a solution of 1-nitro-3-(5-phenylpentoxy)benzene (1.4 g, 4.93 mmol) in 35 mL ethanol. The reaction mixture was stirred at 70° C. for 2 hours. After the reaction mixture was cooled to room temperature, a saturated, sodium bicarbonate aqueous solution (50 mL) was added. The resultant, mixture was extracted with ethyl acetate (2×50 mL). The combined organic phases were washed with brine, dried over anhydrous MgSO4, and concentrated to give a crude product as a white solid. The crude product was purified by silica gel column chromatography eluting with ethyl acetate-n-hexane to give 3-(5-phenyl-pentyloxy)phenylamine (1.03 g, 4.04 mmol, yield: 82%) as a white solid.
A solution of 3-(5-phenyl-pentyloxy)-phenylamine (200 mg, 1.02 mmol) and thiocarbonyl diimidazole (TCDI, 1.90 mg, 1.06 mmol) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. After a 25% aqueous ammonia solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was removed and then the residue thus obtained was purified by silica gel column chromatography eluting with methanol-dichloromethane to give [3-(5-phenyl-pentyloxy)phenyl]-thiourea (compound 1) (273 mg, 0.87 mmol, yield: 85%) as a white solid.
EI-MS (M+1): 315.
Compound 2 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 301.
Compound 3 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 287.
Compound 4 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 329.
Compound 5 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 343.
Compound 6 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 357.
Potassium carbonate (10.35 g, 75.0 mmol) was added to a stirred suspension of phenol (4.7 g, 50.0 mmol), 1,5-dibromopentane (12.65 g, 55.0 mmol), and potassium iodide (0.83 g, 5.0 mmol) in N-methylpyrolidinone (100 mL). The reaction mixture was stirred at 90° C. for 4 hours. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give (5-bromopentyloxy)benzene (12.0 g, 49.38 mmol, yield: 98%) as yellow oil.
Potassium carbonate (10.35 g, 75.0 mmol) was added to a stirred suspension of (5-bromopentyloxy)benzene (12.0 g, 49.38 mmol), 3-nitrophenol (6.95 g, 50.0 mmol), and potassium iodide (0.83 g, 5.0 mmol) in N-methylpyrolidinone (100 mL). The reaction mixture was stirred at 90° C. for 4 hours. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give 1-nitro-3-(5-phenoxypentoxy) benzene (11.89 g, 39.5 mmol, yield: 80%) as colorless oil.
Tin (II) chloride (19.78 g, 87.89 mmol) was added to a solution of 1-nitro-3-(5-phenoxypentoxy) benzene (5.29 g, 17.58 mmol) in 100 mL ethanol. The reaction mixture was stirred at 70° C. for 2 hours. After the reaction mixture was cooled to room temperature, a saturated sodium bicarbonate aqueous solution (50 mL) was added. The solution was extracted with ethyl acetate (3×50 mL), and the combined organic phases were washed with brine, dried over anhydrous MgSO4, and concentrated to give a crude product as a white solid. The crude product was purified by silica gel column chromatography eluting with ethyl acetate-n-hexane to give 3-(5-phenoxy-pentyloxy)phenylamine (4.67 g, 17.22 mmol, yield: 98%) as a light yellow solid.
A solution of 3-(5-phenoxy-pentyloxy)phenylamine (200 mg, 0.74 mmol) and thiocarbonyl diimidazole (TCDI, 158 mg, 0.89 mmol) in dichloromethane (3 mL) was stirred at room temperature for 2 hours. After a 25% ammonia aqueous solution (2 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was then removed and the residue thus obtained was purified by silica gel column chromatography eluting with methanol-dichloromethane to give [3-(5-phenoxy-pentyloxy)-phenyl]-thiourea (compound 7) (126 mg, 0.38 mmol yield: 52%) as a white solid.
EI-MS (M+1): 331.
Compound 8 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 403.
Compound 9 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 409, 411.
Compound 10 was prepared in a manner similar to that, described in Example 7.
EI-MS (M+1): 345.
Compound 11 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 359.
Compound 12 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 407.
Compound 13 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 14 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 435.
(BOC)2O (10.1 g, 46.3 mmol) was added to a solution of benzene-1,3-diamine (5.0 g, 46.3 mmol) in dichloromethane (80 mL). The reaction mixture was stirred at room temperature for 60 hours. The reaction mixture was quenched with, water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give (3-aminophenyl)-carbamic acid tert-butyl ester (4.34 g, 20.8 mmol, yield: 45%) as a white solid.
Potassium carbonate (0.6 g, 4.35 mmol) was added to a stirred suspension of (3-amino-phenyl)-carbamic acid tert-butyl ester (0.6 g, 2.9 mmol), (5-bromo-pentyl)benzene (0.66 g, 2.9 mmol), and potassium iodide (0.48 g, 2.9 mmol) in N-methylpyrolidinone (14 mL). The reaction mixture was stirred at 90° C. for 4 hours. It was quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give [3-(5-phenyl-pentylamino)-phenyl]-carbamic acid tert-butyl ester (802 mg, 2.26 mmol, yield: 78%) as yellow oil.
Trifluoroacetic acid (TFA, 2.0 mL, 26.3 mmol) was added to a solution of [3-(5-phenyl-pentylamino)-phenyl]-carbamic acid tert-butyl ester (802 mg, 2.26 mmol) in 10 mL dichloromethane. The reaction mixture was stirred at room temperature for 1 hour. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give N-(5-phenyl-pentyl)-benzene-1,3-diamine (529 mg, 2.08 mmol, yield: 92%) as light yellow solid.
A solution of N-(5-phenyl-pentyl)-benzene-1,3-diamine (89 mg, 0.4 mmol) and thiocarbonyl diimidazole (TCDI, 74 mg, 0.42 mmol) in dichloromethane (4 mL) was stirred at room temperature for 2 hours. After a 25% aqueous ammonia solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was then removed and the residue thus obtained was purified by silica gel column chromatography eluting with methanol-dichloromethane to give [3-(5-phenyl-pentylamino)-phenyl]-thiourea (compound 17) (113 mg, 0.36 mmol, yield: 90%) as a white solid.
EI-MS (M+1): 314.
Compound 16 was prepared in a manner similar to that described in Example 15.
EI-MS (M+1): 300.
Compound 17 was prepared in a manner similar to that described in Example 15.
EI-MS (M+1): 286.
Compound 18 was prepared in a manner similar to that described in Example 15.
EI-MS (M+1): 328.
Compound 19 was prepared in a manner similar to that described in Example 15.
EI-MS (M+1): 342.
Compound 20 was prepared in a manner similar to that described in Example 15.
EI-MS (M+1): 356.
Compound 21 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 329.
Compound 22 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 343.
Compound 23 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 357.
Compound 24 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 371.
Compound 25 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 385.
Compound 26 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 399.
Compound 27 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 413.
Compound 28 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 427.
Compound 29 was prepared in a manner similar to that described in Example 1,
EI-MS (M+1): 419.
Compound 30 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 433.
Compound 31 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 447.
A solution of 7-bromo-9H-fluoren-2-ylamine (0.3 g, 1.0 mmol) and thiocarbonyl diimidazole (TCDI, 0.2 g, 1.2 mmol) in dichloromethane (10 mL) was stirred at room temperature for 2 hours. After a 25% aqueous ammonia solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was then removed and the residue thus obtained was purified by silica gel column chromatography dining with methanol-dichloromethane to give (7-bromo-9H-fluoren-2-yl)-thiourea (compound 32) (297 rag, 0.93 mmol, yield 93%) as a white solid.
EI-MS (M+1): 320.
Compound 33 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 270.
Compound 34 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 255.
Compound 35 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 332, 334.
Compound 36 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 255.
Compound 37 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 241.
Compound 38 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 273.
Sodium carbonate (1.06 g, 10.0 mmol) was added to a solution of 9H-fluorene-2,7-diamine (1.0 g, 5.0 mmol) and (BOC)2O (1.4 mL, 7.5 mmol) in 1,4-dioxane (20 mL) and H2O (10 mL). The reaction mixture was stirred at room temperature overnight. It was then quenched with saturated ammonium chloride aqueous solution (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give (7-amino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (640 mg, 2.16 mmol, yield: 43%) as a yellow solid.
Potassium carbonate (120 mg, 0.87 mmol) was added to a stirred suspension of (7-amino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (200 mg, 0.67 mmol), n-propyl iodide (114 mg, 0.67 mmol) in acetonitrile (20 mL). The reaction mixture was stirred at refluxing temperature for 4 hours. It was then quenched with a saturated ammonium chloride aqueous solution (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give ((7-propylamino-9H-fluoren-2-yl)-carbamic acid, tert-butyl ester (91 mg, 0.27 mmol, yield: 40%) as a light brown solid and (7-dipropylamino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (114 mg, 0.30 mmol, yield: 45%) as a light brown solid.
Trifluoroacetic acid (TFA, 2.0 mL, 26.3 mmol) was added to a solution of (7-dipropylamino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (270 mg, 0.71 mmol) in 20 mL dichloromethane. The reaction mixture was stirred at room temperature for 1 hour. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give N,N-dipropyl-9H-fluorene-2,7-diamine (220 mg, 0.78 mmol, yield: 91%) as a light brown solid.
A solution of N,N-dipropyl-9H-fluorene-2,7-diamine (220 mg, 0.78 mmol) and thiocarbonyl diimidazole (TCDI, 163 mg, 0.92 mmol) in dichloromethane (5 mL) was stirred at room temperature for 2 hours. After a 25% ammonia aqueous solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was then removed and the residue thus obtained was purified by silica gel column chromatography eluting with methanol-dichloromethane to give (7-dipropylamino-9H-fluoren-2-yl)-thiourea (compound 39) (231 mg, 0.69 mmol, yield: 88%) as a white solid.
EI-MS (M+1): 340.
Compound 40 was prepared in a manner similar to that described in Example 39.
EI-MS (M+1): 312.
Compound 41 was prepared in a manner similar to that described in Example 39.
EI-MS (M+1): 284.
Compound 42 was prepared in a manner similar to that described in Example 39.
EI-MS (M+1): 368.
Trifluoroacetic acid (TFA, 2.0 mL, 26.3 mmol) was added, to a solution of (7-propylamine-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (91 mg, 0.27 mmol) prepared in Example 39 in 10 mL dichloromethane. The reaction mixture was stirred at room temperature for 1 hour. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give N2-propyl-9H-fluorene-2,7-diamine (60 mg, 0.25 mmol, yield: 92%) as a light brown solid.
A solution of N2-propyl-9H-fluorene-2,7-diamine (60 mg, 0.25 mmol) and thiocarbonyl diimidazole (53 mg, 0.30 mmol) in dichloromethane (5 mL) was stirred at room temperature for 2 hours. After a 25% ammonia aqueous solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was then removed and the residue was purified by silica gel column chromatography eluting with methanol-dichloromethane to give (7-propylamino-9H-fluoren-2-yl)thiourea (compound 43) (68 mg, 0.23 mmol, yield: 90%) as a white solid.
EI-MS (M+1): 298.
Compound 44 was prepared in a manner similar to that described in Example 43.
EI-MS (M+1): 284.
Compound 45 was prepared in a manner similar to that, described in Example 43,
EI-MS (M+1): 270.
Compound 46 was prepared in a manner similar to that described in Example 43.
EI-MS (M+1): 312.
Compound 47 was prepared in a manner similar to that described in Example 43.
EI-MS (M+1): 374.
Compound 48 was prepared in a manner similar to that described in Example 43.
EI-MS (M+1): 492.
Sodium carbonate (1.06 g, 10.0 mmol) was added to a solution of 9H-fluorene-2,7-diamine (1.0 g, 5.0 mmol) and (BOC)2O (1.4 mL, 7.5 mmol) in dioxane (20 mL) and H2O (10 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. It was then quenched with water (30 mL) followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give (7-amino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (640 mg, 2.16 mmol, yield: 43%) as a yellow solid.
A solution of (7-amino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (1.16 mg, 0.39 mmol) and thiocarbonyl diimidazole (81 mg, 0.45 mmol) in dichloromethane (5 mL) was stirred at room temperature for 2 hours. Alter a 25% ammonia aqueous solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was then removed and the residue thus obtained was purified by silica gel column chromatography eluting with methanol-dichloromethane to give (7-thioureido-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (118 mg, 0.33 mmol, yield: 85%) as a white solid.
Trifluoroacetic acid (TFA, 2.0 mL, 26.3 mmol) was added to a solution of (7-thioureido-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (75 mg, 0.21 mmol) in 2 ml, dichloromethane. The reaction mixture was stirred at room temperature for 1 hour. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give (7-amino-9H-fluoren-2-yl)-thiourea (compound 49) (51 mg, 0.20 mmol, yield: 95%) as a white solid.
EI-MS (M+1): 256.
Compound 50 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 315.
Compound 51 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 333, 335.
Compound 52 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 347, 349.
Compound 53 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 361, 363.
Compound 54 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 375, 377.
Compound 55 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 389, 391.
Compound 56 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 403, 405.
Compound 57 was prepared in a manner similar to that described in Example 32,
EI-MS (M+1): 417, 419.
Compound 58 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 431, 433.
Compound 59 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 391, 393.
Compound 60 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 375, 377.
Compound 61 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 390, 392.
Compound 62 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 418, 420.
Compound 63 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 404, 406.
Compound 64 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 423, 425.
Compound 65 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 437, 439.
Compound 66 was prepared in a manner similar to that described in Example 32,
EI-MS (M+1): 451, 453.
Compound 67 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 430, 432.
Compound 68 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 394, 396.
Compound 69 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 395, 397.
Compound 70 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 480, 482.
Compound 71 was prepared in a manner similar to that described in Example 32.
EI-MS (M+1): 445, 447.
Triethylamine (37 mg, 0.37 mmol) was added to a solution of (7-amino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (100 mg, 0.34 mmol) and n-butyryl chloride (36 mg, 0.34 mmol) in dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 4 hours. It was then quenched with excess saturated ammonium chloride aqueous solution (30 mL), followed by extraction with dichloromethane (30 mL×3). The organic layers were combined, washed, with brine, and concentrated under vacuum. The residue was subjected to column chromatography on silica gel to give (7-butylamino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (99 mg, 0.27 mmol, yield: 80%) as a white solid.
Trifluoroacetic acid (TFA, 2.0 mL, 26.3 mmol) was added to a solution of (7-butyrylamino-9H-fluoren-2-yl)-carbamic acid tert-butyl ester (99 mg, 0.27 mmol) in 2 mL dichloromethane. The reaction mixture was stirred at room temperature for 1 hour. It was then quenched with water (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give N-(7-amino-9H-fluoren-2-yl)-butyramide (69 mg, 0.26 mmol, yield: 95%) as a yellow solid.
A solution of N-(7-amino-9H-fluoren-2-yl)-butyramide (69 mg, 0.26 mmol) and thiocarbonyl diimidazole (55 mg, 0.30 mmol) in dichloromethane (2 mL) was stirred at room temperature for 2 hours. After a 25% ammonia aqueous solution (2.0 mL, excess) was added, the reaction mixture was stirred at room temperature overnight. The solvent was removed and then the residue thus obtained was purified by silica gel column chromatography eluting with methanol-dichloromethane to give N-(7-thioureido-9H-fluoren-2-yl)-butyramide (compound 72) (75 mg, 0.23 mmol, yield: 90%) as a white solid.
EI-MS (M+1): 326.
Compound 73 was prepared in a manner similar to that described in Example 72.
EI-MS (M+1): 366.
Compound 74 was prepared in a manner similar to that described in Example 72.
EI-MS (M+1): 351.
Compound 75 was prepared in a manner similar to that described in Example 72.
EI-MS (M+1): 356.
2-Chloroethyl isothiocyanate (293 mg, 2.4 mmol) was added to a solution of 3-benzyloxy-phenylamine (398 mg, 2.0 mmol) in dichloromethane (4 mL). The reaction mixture was stirred at room temperature overnight, it was quenched with water (30 mL), followed by extraction with dichloromethane (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give 1-(3-benzyloxyphenyl)-3-(2-chloro-ethyl)-thiourea (627 mg, 1.96 mmol, yield: 98%) as colorless oil.
Triethylamine (2.0 mL, excess) was added to a solution of 1-(3-benzyloxyphenyl)-3-(2-chloro-ethyl)-thiourea (187 mg, 0.58 mmol) in dry THF (3 mL). The reaction mixture was stirred at refluxing temperature for 6 hours, it was then, quenched with a saturated ammonium chloride aqueous solution (30 mL), followed by extraction with ethyl acetate (30 mL×3). The organic layers were combined, washed with brine, and concentrated under vacuum. The residue thus obtained was subjected to column chromatography on silica gel to give 1-(3-benzyloxy-phenyl)-imidazolidine-2-thione (compound 76) as a white solid (150 mg, 0.52 mmol, yield: 90%).
EI-MS (M+1): 285.
A suspension of Compound 76, i.e., 1-(3-benzyloxy-phenyl)-imidazolidine-2-thione (71 mg, 0.25 mmol) and potassium tert-butoxide (56 mg, 0.50 mmol) in acetonitrile (1 mL) was cooled in an ice bath and stirred at 0° C. for 30 minutes, followed by addition of a solution of n-butyl bromide (41 mg, 0.30 mmol) in acetonitrile (1 mL). After 5 minutes, the ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction was then quenched with water, followed by extraction with ethyl acetate (20 mL×3). The organic layers were combined and washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude mixture thus obtained was purified with silica gel column chromatography to yield 1-(3-benzyloxy-phenyl)-3-butyl-imidazolidine-2-thione (compound 77) as yellow oil (59 mg, 0.18 mmol, yield: 72%).
EI-MS (M+1): 341.
Compound 78 was prepared in a manner similar to that described in Example 77.
EI-MS (M+1): 403.
Compound 79 was prepared in a manner similar to that, described in Example 76.
EI-MS (M+1): 341.
Compound 80 was prepared in a manner similar to that described in Example 77.
EI-MS (M+1): 397.
Compound 81 was prepared in a manner similar to that, described in Example 77.
EI-MS (M+1): 459.
Compound 82 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 400.
Compound 83 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 349.
Compound 84 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 395.
Compound 85 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 365.
Compound 86 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 367.
Compound 87 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 418.
Compound 88 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 332.
Compound 89 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 332.
Compound 90 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 333.
Compound 91 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 375.
Compound 92 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 374.
Compound 93 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 402.
Compound 94 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 416.
Compound 95 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 414.
Compound 96 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 429.
Compound 97 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 361.
Compound 98 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 361.
Compound 99 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 100 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 400.
Compound 101 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 437.
Compound 102 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 103 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 441.
Compound 104 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 485, 487.
Compound 105 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 381.
Compound 106 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 381.
Compound 107 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 413.
Compound 108 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 356.
Compound 109 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 356.
Compound 110 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 356.
Compound 111 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 414.
Compound 112 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 399.
Compound 113 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 303.
Compound 114 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 317.
Compound 115 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 345.
Compound 116 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 359.
Compound 117 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 379.
Compound 118 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 393.
Compound 119 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 120 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 435.
Compound 121 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 359.
Compound 122 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 387.
Compound 123 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 399.
Compound 124 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 401.
Compound 125 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 414.
Compound 126 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 382.
Compound 127 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 382.
Compound 128 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 382.
Compound 129 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 382.
Compound 130 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 382.
Compound 131 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 382.
Compound 132 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 370.
Compound 133 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 397.
Compound 134 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 397.
Compound 135 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 413.
Compound 136 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 447.
Compound 137 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 423.
Compound 138 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 423.
Compound 139 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 407.
Compound 140 was prepared in a manner similar to that described in Example 1.
EI-MS (M+1): 407.
Compound 141 was prepared in a manner similar to that described in Example 39.
EI-MS (M+1): 436.
Compound 142 was prepared in a manner similar to that described in Example 39.
EI-MS (M+1): 346.
Compound 143 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 361.
Compound 144 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 391.
Compound 145 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 382.
Compound 146 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 382.
Compound 147 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 397.
Compound 148 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 432.
Compound 149 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 409.
Compound 150 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 423.
Compound 151 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 451.
Compound 152 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 315.
Compound 153 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 451.
Compound 154 was prepared in a manner similar to mat described in Example 7.
EI-MS (M+1): 315.
Compound 155 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 422.
Compound 156 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 435.
Compound 157 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 437.
Compound 158 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 159 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 353.
Compound 160 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 367.
Compound 161 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 381.
Compound 162 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 411.
Compound 163 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 395.
Compound 164 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 365.
Compound 165 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 415.
Compound 166 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 395.
Compound 167 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 168 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 441.
Compound 169 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 379.
Compound 170 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 393.
Compound 171 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 379.
Compound 172 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 340.
Compound 173 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 367.
Compound 174 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 425.
Compound 175 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 475.
Compound 176 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 437.
Compound 177 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 178 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 421.
Compound 179 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 443.
Compound 180 was prepared in a manner similar to that described in Example 7,
EI-MS (M+1): 380.
Compound 181 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 413.
Compound 182 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 413.
Compound 183 was prepared in a manner similar to that described in Example 7.
EI-MS (M+1): 397.
Dulbecco's modified Eagle's medium (DMEM) high glucose, fetal bovine serum (FBS), G4.18 (geneticin), and blasticidin were purchased from Invitrogen (Carlsbad, Calif.). A reporter cell line, Ava5-EG(Δ4AB)SEAP, for HCV drug screening was derived from HCV replicon cells (Ava5). See, e.g., Lee et al., Anal. Biochem. 316:162-70 and Lee et at, J. Virol. Methods 116:27-33. EG(Δ4AB)SEAP is a reporter gene consisting of enhanced green fluorescent protein (EG), an NS3-NS4A protease decapeptide recognition sequence (Δ4AB), and secreted alkaline phosphatase (SEAP). See, e.g., Lee et al., Anal. Biochem. 316: 162-70. A reporter gene, EG(Δ4AB)SEAP, was stably integrated in the Ava5 cells to generate Ava5-EG(Δ4AB)SEAP cells. The cells were cultured in a medium containing 500 μg/ml G418 (geneticin) and 10 μg/ml blasticidin in a 5% CO2 incubator.
Ava5-EG(Δ4AB)SEAP cells were seeded in 96-well plates (5×103 cells/100 μl/well). After incubation for 1 day, the cells were treated with various concentrations of a test compound for 48 hours. Each culture medium was replenished with a fresh medium containing the test compound at the same concentration to remove the accumulated SEAP. The cells were then incubated for another 24 hours. The culture medium was collected and subjected to SEAP activity assays. The SEAP activities were measured using the Phospha-Light assay kit (Tropix, Foster, Calif., USA) according to manufacturer's instructions. Of note, SEAP activity in the culture medium, can be used to reflect anti-HCV activity. See, e.g., Lee et al., J. Virol. Methods 116:27-33.
Compounds 1-42, 45-62, 64-91, 93-135, and 137-183 were tested for their efficacy in inhibiting HCV replication. Unexpectedly, 119 test compounds showed low EC50 values (i.e., the concentration of a test compound at which 50% HCV replication is inhibited) between 0.001 μM and 1 μM. Among them, 63 test compounds showed EC50 values as low as between 0.001 μM and 0.1 μM.
Cell viability was determined by the MTS assay similar to that described in Cory et al., Cancer Commun. 3:207-12. In short, Ava5-EG(Δ4AB)SEAP cells were seeded in 96-well plates (5×103 cells/100 μl/well). 100 μL/well solution containing phenol red-free DMEM, MTS (tetrazolium compound [3-(4,5-dimethylthiozol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]; Promega, Madison, Wis.) and phenazine methosulfate (PMS; Sigma, St. Louis, Mo.) at a ratio of 80:20:1 to each well. The cells were incubated with test compounds for 1-4 hours at 37° C. in a humidified, 5% CO2 incubator and the absorbance was then measured at 490 nm.
Compounds 1-42, 45-62, 64-91, 93-135, and 137-183 were tested in the above cytotoxicity assay. Unexpectedly, all test compounds showed CC50 values (i.e., the concentration of a test compound at which 50% of the cells are killed) above 1 μM. Specifically, 67 of the tested compounds showed CC50 values above 50 μM, 88 of the tested compounds showed CC50 values between 10 μM and 50 μM, and 23 of the test compounds showed CC50 values between 1 μM and 10 μM. Most of the effective compounds exerted little cytotoxicity.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.
Pursuant to 35 U.S.C. § 119(e), this application claims priority to U.S. Provisional Application 60/837,782, filed on Aug. 15, 2006. The contents of the provisional application are incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60837782 | Aug 2006 | US |
