WO2014052836A2

WO2014052836A2 - Methods and compositions for treating infection

Info

Publication number: WO2014052836A2
Application number: PCT/US2013/062309
Authority: WO
Inventors: Paul M. DUNMAN; Damian J. Krysan; Daniel P. FLAHERTY
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-09-27
Filing date: 2013-09-27
Publication date: 2014-04-03
Anticipated expiration: 2015-03-27
Also published as: US20150238473A1; US10004701B2; US20170065540A1; WO2014052836A3

Description

METHODS AND COMPOSITIONS FOR TREATING INFECTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/706,492, filed on September 27, 2012, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government funding under Grant Nos.

1R01AI1075033-03 and 5P50GM069663 from the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND

Infectious diseases affect the health of people and animals around the world, causing serious illness and death. Thus, an urgent need exists for treatments for infections.

SUMMARY

Provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject

a compound of Formula

wherein R represents hydrogen or hydroxyl and R¹ represents hydrogen, or R and R¹ taken together form a second bond between the carbon atoms bearing R and R¹; R² represents hydrogen or phenyl; n is zero or a positive whole integer of from 1 to 4; X represents CH₂, CHOH, NH, C=0, CHNR³R⁴, where R³ and R⁴ independently are hydrogen or lower alkyl; and Z represents thienyl, pyridinyl, substituted pyridinyl, phenyl or substituted phenyl wherein the substituents on the substituted pyridinyl or substituted phenyl are selected from phenyl, pyridinyl, nitro, a halogen atom, such as chlorine, fluorine, bromine, or iodine, a straight or branched lower alkyl chain of from 1 to 4 carbon atoms, a lower alkoxy group of from 1 to 4 carbon atoms, amino, a mono or di(lower)alkylamino group, a saturated monocyclic heterocyclic group such as pyrrolidino, piperidino, morpholino, or N- (lower)alkylpiperazino, or a group having the structure -COOR⁵, -CR⁶R⁷COOR⁵, -CF₃, CHF₂, CH₂F, or

where R⁵ is hydrogen or lower alkyl, and R⁶ and R⁷ independently are hydrogen or methyl or a pharmaceutically acceptable salt thereof.

Further provided is a method of treating or preventing an infection in a subject with at risk of developing an infection, the method comprising administering to the subject a compound of Formula III

or a pharmaceutically acceptable salt thereof, wherein R¹ and R²are independently selected from ethyl or methyl, n 1 or 2, R³ and R⁴ are both phenyl or substituted phenyl, wherein the substituent can be halo (for example, fluoro-, chloro-, iodo- or bromo-), hydroxyl, a lower alkyl or a substituted lower alkyl wherein the substituent can be halo, hydroxy, or lower alkoxy, and R⁵ is hydrogen, a halogen, a lower alkyl from about 1 to 4 carbon atoms or a substituted alkyl wherein the substituent can be halo, hydroxy, or lower alkoxy.

Also provided is a method of treating or preventing infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: a compound of Formula V

wherein ¾ is a lower alkyl group having from 1 to 4 carbon atoms being substituted with one or several halogen atoms, Z is hydrogen or a lower alkyl group having from 1 to 4 carbon atoms, m is 2 or 3 and X₂ is the ethylene imino roup or the group having the formula:

wherein R is hydrogen or a lower alkyl group having from 1 to 4 carbon atoms which can substituted with a chlorine atom or a hydroxy group, and Z and m have the above-given meaning, a compound of Formula X

wherein R represents a lower alkyl or a lower alkenyl group, a phenyl group which can be substituted by halogen, methyl or lower alkoxy groups, or a benzyl group which can be nuclear substituted by halogen, methyl or lower alkoxy groups,

X represents -0-, -S-, -SO-, or -S0₂-, n represents an integer from 1-4, and

Aryl represents a phenyl group which can be substituted by lower alkoxy or lower alkylmercapto groups; a compound of Formula XII

wherein R and R⁶ are the same or different and are hydroxy, lower alkoxy, lower alkenoxy, dilower alkylamino lower alkoxy (dimethylaminoethoxy), acylamino lower alkoxy

(acetylaminoethoxy), acyloxy lower alkoxy (pivaloyloxymethoxy), aryloxy, such as phenoxy, arloweralkoxy, such as benzyloxy, substituted aryloxy or substituted arloweralkoxy wherein the substitutent is methyl, halo or methoxy, amino, loweralkylamino, diloweralkylamino, hydroxyamino, arloweralkylamino such as benzylamino;

R¹ is hydrogen, alkyl of from 1 to 20 carbon atoms which include branched and cyclic and unsaturated (such as allyl) alkyl groups, substituted loweralkyl wherein the substituent can be halo, hydroxy, lower alkoxy, aryloxy such as phenoxy, amino, diloweralkylamino, acylamino, such as acetamido and benzamido, arylamino, guanidino, imidazolyl, indolyl, mercapto, loweralkylthio, arylthio such as phenylthio, carboxy or carboxamido,

carboloweralkoxy, aryl such as phenyl or naphthyl, substituted aryl such as phenyl wherein the substituent is lower alkyl, lower alkoxy or halo, arloweralkyl, arloweralkenyl,

heteroarlower alkyl or heteroarlower alkenyl such as benzyl, styryl or indolyl ethyl, substituted arloweralkyl, substituted arloweralkenyl, substituted heteroarlower alkyl, or substituted heteroarlower alkenyl, wherein the substituent(s) is halo, dihalo, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino (acetyl amino or benzoylamino) diloweralkylamino, loweralkylamino, carboxyl, haloloweralkyl, cyano or sulfonamido;

arloweralkyl or heteroarloweralkyl substituted on the alkyl portion by amino or acylamino (acetylamino or benzoylamino);

R² and R⁷ are the same or different and are hydrogen or lower alkyl;

R³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, acylamino lower alkyl (such as benzoylamino lower alkyl, acetylamino lower alkyl), amino lower alkyl, dimethylamino lower alkyl, halo lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyllower alkyl, mercapto lower alkyl, lower alkyl thio lower alkyl; R⁴ is hydrogen or lower alkyl;

R⁵ is hydrogen, lower alkyl, phenyl, phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkyl thio lower alkyl; R⁴ and R⁵ can be connected together to form an alkylene bridge of from 2 to 4 carbon atoms, an alkylene bridge of from 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge of from 3 to 4 carbon atoms containing a double bond or an alkylene bridge as above substituted with hydroxy, loweralkoxy, loweralkylor diloweralky; a compound of Formula XIII

wherein Ri is H, alkyl, acyl or silyl(alkyl)₃; R₂ is H and R₃ is OH, O-acyl, O-alkyl or O-silyl (alkyl)₃ or R₃ is H and R₂ is OH. O-acyl, O-alkyl or O-silyl (alkyl)₃; or R₂ and R₃ together represent O; or R₂ and R₃ together represent acetal or cyclic acetal. Ri might also represent a substituted alkyl such as e.g. methoxy ethoxy methyl; a compound of Formula XIV

(XIV) wherein PY is 4- or 3- or 2-pyridinyl or 4- or 3 -or 2-pyridinyl having one or two lower-alkyl substituents, R is hydrogen, lower-alkyl or lower-hydroxyalkyl, and Q is nitro, carbamyl, halo, amino, lower-alkylamino, di(lower-alkyl)amino, or NHAc where Ac is lower-alkanoyl or lower-carbalkoxy;

a compound of Formula XV

a compound of Formula XVI

(XVI)

a compound of Formula XVII

(XVII)

a compound of Formula XVIII

(XVIII) a compound of Formula XIX

(XIX) a compound of Formula (XX)

(XX) or a pharmaceutically acceptable salt thereof.

Further provided is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: Didanosine, Norcyclobenzaprine,

Niridazole, Ifosfamide, Cefalonium, Tamoxifen citrate, Butoconazole, Suloctidil,

Clomiphene, Sulconazole, Miconazole, Mefloquine, Sulfinpyrazone, Terfenadine, Lisinopril, Econzaole, Clofazimine, Equilin, Felodipine, Dacarbazine, Furazolidone, Perhexiline maleate, Oxethazaine, Pimozide, Trifluoperazine, Ellipticine, Fluspirilen, Hexestrol,

Dienestrol, Zidovudine, Metoprolol, Napelline, Methimazole, Amrinone, lopanoic acid, R- Propanolol, Rimexolone and Pyrvinium pamoate, wherein the infection is a bacterial infection. Also provided is a method of removing or preventing biofilm formation on a surface, the method comprising administering to a biofilm containing surface or a surface susceptible to biofilm formation an effective amount of a compound selected from the group consisting of:

a compound of Formula I

{CH-in-X-Z :

(I) wherein R represents hydrogen or hydroxyl and R¹ represents hydrogen, or R and R¹ taken together form a second bond between the carbon atoms bearing R and R¹; R² represents hydrogen or phenyl; n is zero or a positive whole integer of from 1 to 4; X represents CH₂, CHOH, NH, C=0, CHNR³R⁴, where R³ and R⁴ independently are hydrogen or lower alkyl; and Z represents thienyl, pyridinyl, substituted pyridinyl, phenyl or substituted phenyl wherein the substituents on the substituted pyridinyl or substituted phenyl are selected from phenyl, pyridinyl, nitro, a halogen atom, such as chlorine, fluorine, bromine, or iodine, a straight or branched lower alkyl chain of from 1 to 4 carbon atoms, a lower alkoxy group of from 1 to 4 carbon atoms, amino, a mono or di(lower)alkylamino group, a saturated monocyclic heterocyclic group such as pyrrolidino, piperidino, morpholino, or N- (lower)alkylpiperazino, or a group having the structure -COOR⁵, -CR⁶R⁷COOR⁵, -CF₃, CHF₂, CH₂F, or

where R⁵ is hydrogen or lower alkyl, and R⁶ and R⁷ independently are hydrogen or methyl or a pharmaceutically acceptable salt thereof. DESCRIPTION OF DRAWINGS

Figure 1 depicts the adenylate kinase (AK) assay described in the Examples.

Bactericidal agents compromise bacterial cell integrity, releasing cellular adenylate kinase. Extracellular AK is measured by the addition of a commercial ToxiLight AK cocktail containing ADP and luciferase, resulting in luminescence.

Figure 2 shows AK assay development. (A) AK assay measures of E. coli lysed cell supernatants. E. coli DH5a cells (1 x 10⁹) were heat inactivated by boiling and diluted, and the AK assay was used to measure adenylate kinase release. Background signal is also shown (mock-treated cells (right-hand column in Figure 2A). "*" indicates a significant difference between results for boiled and mock treatment (Student's t test; P < 0.05). (B) AK assay results for colistin-treated baumannii strain 98-37-09. Cells were treated with the indicated concentration of colistin, and AK was measured; MIC (4 μg mf¹ is indicated. "*" indicates a significant increase in signal compared to that for mock treatment (0 μg mf¹; Student's t test, < 0.05). Figure 3 shows AK assay measures of S. aureus strain RN4220 treatment with bacteriostatic and bactericidal antibiotics. Standard MIC testing determined the MIC of each antibiotic class (in parentheses). Graphed are the fold changes in AK signal of cells treated with 0.5 x or 1.0^X the MIC value (left hand column and right hand column, respsectively) for each antibiotic, in comparison to untreated control cells; "*" indicates a significant change in signal as determined by Student's t test; P < 0.05 (compared to results for untreated cells).

Figure 4 shows AK assay measures of antibiotic-treated biofilms and small-colony variants. (A) Graphed are AK signals generated by static biofilm-associated cells following mock or antibiotic treatment: colistin (P. aeruginosa) or ciprofloxacin (S. aureus and A. baumannii). (B) Fold change of AK measures of S. aureus SCV UAMS-1112 cells following treatment with l x and 10^x ciprofloxacin, meropenem, and vancomycin, compared to results for mock treated cells. "*" indicates a significant change in signal in comparison to results for mock-treated cells (Student's t test, P < 0.05).

Figure 5 shows AK-based HTS development and screening. (A) Z' factor assay results for Klebsiella pneumoniae. Three-hundred-eighty- four- well microtiter plates were seeded with K. pneumoniae, and alternating rows were mock treated (DMSO) or treated with 50 μΜ colistin. Following 3 h of incubation, AK release was measured and plotted. DMSO-treated well measures are shown at the bottom of Figure 5 A; colistin-treated wells are shown at the top of Figure 5 A. (B) Prestwick library Klebsiella pneumoniae screening results. In total, 26 compounds were determined to result in a 3-fold increase in AK signal, in comparison to results for DMSO-treated cells. Included among this list were polymyxin, cephalosporins, aminoglycosides, fluoroquinolones, and detergents; the complete Prestwick screening results for K pneumoniae and all other organisms screened are provided in Table 4.

Figure 6 shows antimicrobial properties of terfenadine and tamoxifen. (A) Fold changes in AK signal of terfenadine-treated (10^x MIC) S. aureus strain UAMS-1 static bio films and the SCV strain UAMS-1112, compared to those for mock (DMSO)-treated populations, are plotted. "*" indicates a significant increase in signal over that with mock- treated cells (Student's t test, P < 0.05). (B) Plotted are the percent survival of G. mellonella larvae at 48 h post-E.faecium inoculation. Groups of larvae (n = 45) were treated at 2 h and 24 h with either PBS (mock), DMSO, 80 mg kg ¹ tamoxifen, 160 mg kg ¹ tamoxifen, or 20 mg kg^-1 vancomycin.

DETAILED DESCRIPTION

Provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of:

a compound of Formula I

^' M i ) wherein R represents hydrogen or hydroxyl and R¹ represents hydrogen, or R and R¹ taken together form a second bond between the carbon atoms bearing R and R¹; R² represents hydrogen or phenyl; n is zero or a positive whole integer of from 1 to 4; X represents CH₂, CHOH, NH, C=0, CHNR³R⁴, where R³ and R⁴ independently are hydrogen or lower alkyl; and Z represents thienyl, pyridinyl, substituted pyridinyl, phenyl or substituted phenyl wherein the substituents on the substituted pyridinyl or substituted phenyl are selected from phenyl, pyridinyl, nitro, a halogen atom, such as chlorine, fluorine, bromine, or iodine, a straight or branched lower alkyl chain of from 1 to 4 carbon atoms, a lower alkoxy group of from 1 to 4 carbon atoms, amino, a mono or di(lower)alkylamino group, a saturated monocyclic heterocyclic group such as pyrrolidino, piperidino, morpholino, or N- (lower)alkylpiperazino, or a group having the structure -COOR⁵, -CR⁶R⁷COOR⁵, -CF₃, CHF₂, CH₂F, or

where R⁵ is hydrogen or lower alkyl, and R⁶ and R⁷ independently are hydrogen or methyl. The substituents on the substituted phenyl may be attached at the ortho, meta or para positions of the phenyl ring.

Compounds of Formula I include compounds of Formula II,

wherein R represents hydrogen or hydroxyl and R¹ represents hydrogen, or R and R¹ taken together form a second bond between the carbon atoms bearing R and R¹; n is zero or a positive whole integer of from 1 to 4; Z represents thienyl, pyridinyl, substituted pyridinyl, phenyl or substituted phenyl wherein the substituents on the substituted pyridinyl or substituted phenyl are selected from phenyl, pyridinyl, nitro, a halogen atom, such as chlorine, fluorine, bromine, or iodine, a straight or branched lower alkyl chain of from 1 to 4 carbon atoms, a lower alkoxy group of from 1 to 4 carbon atoms, amino, a mono or di(lower)alkylamino group, a saturated monocyclic heterocyclic group such as pyrrolidino, piperidino, morpholino, or N-(lower)alkylpiperazino, or a group having the structure - COOR⁵, -CR⁶R⁷COOR⁵, -CF₃, CHF₂, CH₂F, or

Compounds of Formula I also include terfenadine and derivatives thereof, including, but not limited to the following compounds. The compounds are identified by structure, name and registry number. The registry number for each compound is also set forth in Table 6 as an additional identifier for each compound.

{2-{[i .1'-fei^w^i- -^58!t y!^'^i «*idlf

^' SC-352-0S8

KSC-352- S4

1-(4-c lorop enyl)-4-(4-( ydroxydiphenylmet yl)piperidin-1-yl)butan-1 -ol

KSC-335-015

<1-<2-(fιaf !>ι^ί>i*^^^i^ίdiΛ· -^ si^ph^ylm8th»I^

SC - 38 020

Also provided is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of:

a compound of Formula III

or a pharmaceutically acceptable salt thereof, wherein R¹ and R²are independently selected from ethyl or methyl, n 1 or 2, R³ and R⁴ are both phenyl or substituted phenyl, wherein the substituent can be halo (for example, fluoro-, chloro-, iodo- or bromo-), hydroxyl, a lower alkyl or a substituted lower alkyl wherein the substituent can be halo, hydroxy, or lower alkoxy, and R⁵ is hydrogen, a halogen, a lower alkyl from about 1 to 4 carbon atoms or a substituted alkyl wherein the substituent can be halo, hydroxy, or lower alkoxy. Examples of the compounds of Formula III include, tamoxifen and derivatives

thereof, including but not limited to tamoxifen (Formula IV), 4-hydroxy tamoxifen and clomiphene. In the methods provided herein, wherein a one or more compounds of Formula III are used to treat or prevent infection, the infection can be an infection, wherein the infection is not a fungal infection or a parasitic infection.

Further provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of:

a compound of Formula V

wherein R is hydrogen or a lower alkyl group having from 1 to 4 carbon atoms which may be substituted with a chlorine atom or a hydroxy group, and Z and m have the above-given meaning, or a pharmaceutically acceptable salt thereof.

As used throughout, the term "lower alkyl group containing from 1 to 4 carbon atoms" means methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, and I- methylpropyl. The term "halogen" means chlorine, bromine, fluorine, and iodine. Included herein are compounds of formula IV that correspond to formula VI:

wherein R_ls X₂ and m have the same meaning as in Formula IV. Additional compounds of formula IV, include the compounds of formula VII:

wherein R₂ is a β-chloroethyl or a γ-chloropropyl group, and R₃ is hydrogen, a methyl group or an ethyl group, optionally substituted in the β-position with a chlorine atom or a hydroxy group. Among the compounds of formula VII, are the compounds of formula VIII and IX. The compound of formula VIII is 3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H- 1,3,2-oxazaphosphorine 2-oxide or ifosfamide. Ifosfamide is also known as IFEX.

Further provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: a compound of Formula X

X represents -0-, -S-, -SO-, or -S0₂-, n represents an integer from 1-4, and

Aryl represents a phenyl group which can be substituted by lower alkoxy or lower alkylmercapto groups; or a pharmaceutically acceptable salt thereof.

With respect to Formula X, R represents a lower alkyl or a lower alkenyl group, a phenyl group which can be substituted by halogen, methyl or lower alkoxy groups, or a benzyl group which can be nuclear substituted by halogen, methyl or lower alkoxy groups, X represents -0-, -S-, -SO-, or -S02-, n represents an integer from 1-4, and aryl represents a phenyl group which can be substituted by lower alkoxy or lower alkylmercapto groups.

For example, -R-X-C_nH_2n- can represent the following groups: methoxy-, ethoxy-, propoxy-, isopropoxy-, butoxy-, isobutoxy-, allyloxy-, crotyloxy-. phenoxy-, o,-m- and p- methylphenoxy-, ο,ρ-dimethylphenoxy-, m,p-dimethylphenoxy-, p-chlorophenoxy-, p- bromophenoxy-, -o, m and p-methoxyphenoxy-, p-ethoxyphenoxy-, benzyloxy-, o-, m-, and p-methylbenzyloxy-, p-chlorobenzyloxy-, p-bromobenzyloxy, o, m and p- methoxybenzyloxyand-, p- ethoxybenzyloxy-; methyl, -ethyl, -propyl, -isopropyl, and butyl radicals and -analogous radicals with SO or S0₂ instead of O as divalent group X. Besides the phenyl group, aryl can be, for example, the o- or p-methylmercaptophenyl group, the o- or p-ethyl mercaptophenyl group, the o-; m-, or p-methoxyphenyl group or the 0-, m- or p-ethoxyphenyl group.

An example of the compound of formula X is set forth herein as formula XI. The compound of formula X is sulfmapyrazone. Sulfmapyrazone is also known as Anturane.

Also provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: a compound of Formula XII

R¹ is hydrogen, alkyl of from 1 to 20 carbon atoms which include branched and cyclic and unsaturated (such as allyl) alkyl groups, substituted loweralkyl wherein the substituent can be halo, hydroxy, lower alkoxy, aryloxy such as phenoxy, amino, diloweralkylamino, acylamino, such as acetamido and benzamido, arylamino, guanidino, imidazolyl, indolyl, mercapto, loweralkylthio, arylthio such as phenylthio, carboxy or carboxamido, carboloweralkoxy, aryl such as phenyl or naphthyl, substituted aryl such as phenyl wherein the substituent is lower alkyl, lower alkoxy or halo, arloweralkyl, arloweralkenyl,

R² and R⁷ are the same or different and are hydrogen or lower alkyl;

R³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, acylamino lower alkyl (such as benzoylamino lower alkyl, acetylamino lower alkyl), amino lower alkyl, dimethylamino lower alkyl, halo lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyllower alkyl, mercapto lower alkyl, lower alkyl thio lower alkyl;

R⁴ is hydrogen or lower alkyl;

R⁵ is hydrogen, lower alkyl, phenyl, phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkyl thio lower alkyl;

R⁴ and R⁵ may be connected together to form an alkylene bridge of from 2 to 4 carbon atoms, an alkylene bridge of from 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge of from 3 to 4 carbon atoms containing a double bond or an alkylene bridge as above substituted with hydroxy, loweralkoxy, loweralkylor diloweralky; or a pharmaceutically acceptable salt thereof.

With respect to Formula XII, R and R⁶ are the same or different and are hydroxy, lower alkoxy, lower alkenoxy, dilower alkylamino lower alkoxy (dimethylaminoethoxy), acylamino lower alkoxy (acetylaminoethoxy), acyloxy lower alkoxy (pivaloyloxymethoxy), aryloxy, such as phenoxy, arloweralkoxy, such as benzyloxy, substituted aryloxy or substituted arloweralkoxy wherein the substitutent is methyl, halo or methoxy, amino, loweralkylamino, diloweralkylamino, hydroxy amino, arloweralkylamino such as

benzylamino;

carboloweralkoxy, aryl such as phenyl or naphthyl, substituted aryl such as phenyl wherein the substituent is lower alkyl, lower alkoxy or halo, arloweralkyl, arloweralkenyl, heteroarlower alkyl or heteroarlower alkenyl such as benzyl, styryl or indolyl ethyl, substituted arloweralkyl, substituted arloweralkenyl, substituted heteroarlower alkyl, or substituted heteroarlower alkenyl, wherein the substituent(s) is halo, dihalo, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino (acetyl amino or benzoylamino) diloweralkylamino, loweralkylamino, carboxyl, haloloweralkyl, cyano or sulfonamido;

R² and R⁷ are the same or different and are hydrogen or lower alkyl;

R³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, acylamino lower alkyl (such as

benzoylamino lower alkyl, acetylamino lower alkyl), amino lower alkyl, dimethylamino lower alkyl, halo lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyllower alkyl, mercapto lower alkyl, lower alkyl thio lower alkyl;

R⁴ is hydrogen or lower alkyl; R⁵ is hydrogen, lower alkyl, phenyl, phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkyl thio lower alkyl;

R⁴ and R⁵ may be connected together to form an alkylene bridge of from 2 to 4 carbon atoms, an alkylene bridge of from 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge of from 3 to 4 carbon atoms containing a double bond or an alkylene bridge as above substituted with hydroxy, loweralkoxy, loweralkylor diloweralky.

The loweralkyl or lower alkenyl groups except where noted otherwise represented by any of the variables include straight and branched chain hydrocarbon radicals from one to six carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl or vinyl, allyl, butenyl and the like. The aralkyl groups represented by any of the above variables have from one to four carbon atoms in the alkyl portion thereof and include for example, benzyl, p-methoxy benzyl and the like. Halo means chloro, bromo, iodo or fluoro. Aryl where it appears in any of the radicals except where noted represents phenyl or naphthyl. Heteroaryl groups where they appear include for example pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazoyl and thiazolyl. The R¹, R³ and R⁵ substituted lower alkyl moieties are exemplified by groups such as

R⁴ and R⁵ when joined through the carbon and nitrogen atoms to which they are attached form a 4 to 6 membered ring which may contain one sulfur atom or a double bond. Preferred rings have the formulae:

where Y is CH₂, S, or CHOCH₃.

Compounds of Formula XI include compounds wherein: R and R⁶ can each independently be hydroxy, lower alkoxy, lower alkenoxy, arloweralkyloxy, amino, dilower alkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy wherein the substituent is methyl, halo or methoxy;

R² and R⁷ are hydrogen; R³ is lower alkyl, amino lower alkyl, imidazoyllower alkyl, halo lower alkyl;

R⁴ and R⁵ are joined to form the preferred rings as defined above where Y is C¾, S,

R¹ is as defined previously.

Other compounds include compounds of Formula XI wherein further R¹ is alkyl having from 1 to 8 carbon atoms, substituted lower alkyl wherein the alkyl group has 1-5 carbon atoms and the substituent is amino, arylthio, aryloxy or arylamino, aralkyl or heteroaralkyl wherein the alkyl portion has 1 to 3 carbon atoms such as phenethyl or indolylethyl or substituted arloweralkyl 65 (phenyl lower alkyl or naphthyl lower alkyl) and substituted heteroarloweralkyl wherein the alkyl groups have 1-3 carbons and wherein the substituent(s) is halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl.

Other compounds of Formula XI include compounds wherein Rand R⁶ are hydroxy, lower alkoxy, aralkyloxy;

R² and R⁷ are hydrogen;

R³ is methyl or amino lower alkyl; R⁴ and R⁵ are joined through the carbon and nitrogen atom to form proline, 4- thiaproline or 4-methoxy proline;

R¹ is alkyl having from 1 to 8 carbon atoms, substituted lower alkyl wherein the alkyl group has 1-5 carbon atoms and the substituent is amino, arylthio or aryloxy, aralkyl or heteroaralkyl wherein the alkyl portion has 1 to 3 carbon atoms such as phenethyl or indolylethyl or substituted aralkyl (phenyl lower alkyl or naphthyl lower alkyl) and substituted heteroaralkyl wherein the alkyl groups have 1-3 carbons and wherein the substituent(s) is halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl.

Further examples of compounds of Formula XII include, but are not limited to: N-(l(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline;

N-(l(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline and its maleate salt;

N-(l(S)-ethoxycarbonyl-4-methylpentyl)-L-alanyl-L-proline;

N-(l-carboxy-5-aminopentyl)-L-alanyl-L-proline;

N-. alpha. -(l(S)-carboxy-3-phenylpropyl)-L-lysyl-L-proline (lisinopril);

N-.alpha.(l(S)-ethoxycarbonyl-3-phenylpropyl)-L-lysyl-L-proline;

N-. alpha. [ 1 (S)-carboxy-3-(3-indolyl)propyl]-L-lysyl-L-proline;

N-. alpha. -[l(S)-carboxy-3-(4-chlorophenyl)-propyl]-L-lysyl-L-proline;

N-. alpha. -[l(S)-carboxy-2-phenylthioethyl]-L-lysyl-L-proline;

N- .alpha. - [ 1 (S)-carboxy-3 -(4-chlorophenyl)-propyl] -L-lysyl-L-4.alpha.-metho xyproline; N-. alpha. -[l(S)-carboxy-5-aminopentyl]-L-lysyl-L-proline;

Ethyl N-(l (S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride;

and the like.

Also provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: a compound of Formula XIII

wherein ¾ is H, alkyl, acyl or silyl(alkyl)₃; R₂ is H and R₃ is OH, O-acyl, O-alkyl or O-silyl (alkyl)₃ or R₃ is H and R₂ is OH. O-acyl, O-alkyl or O-silyl (alkyl)₃; or R₂ and R₃ together represent O; or R₂ and R₃ together represent acetal or cyclic acetal. Ri might also represent a substituted alkyl such as e.g. methoxy ethoxy methyl; or a pharmaceutically acceptable salt thereof.

Also provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: a compound of Formula XIV

(XIV) wherein PY is 4- or 3- or 2-pyridinyl or 4- or 3 -or 2-pyridinyl having one or two lower-alkyl substituents, R is hydrogen, lower-alkyl or lower-hydroxyalkyl, and Q is nitro, carbamyl, halo, amino, lower-alkylamino, di(lower-alkyl)amino, or NHAc where Ac is lower-alkanoyl or lower-carbalkoxy; or a pharmaceutically acceptable salt thereof.

With respect to Formula XIII, PY is 4- or 3- or 2-pyridinyl or 4- or 3-or 2-pyridinyl having one or two lower-alkyl substituents, R is hydrogen, lower-alkyl or lower- hydroxyalkyl, and Q is nitro, carbamyl, halo, amino, lower-alkylamino, di(lower- alkyl)amino, or NHAc where Ac is lower-alkanoyl or lower-carbalkoxy, or pharmaceutically- acceptable acid-addition salt thereof. Compounds of formula XIII where Q is amino, lower- alkylamino, di-(lower-alkyl) amino, or NHAc are provided herein. The compounds of formula XIII where Q is nitro or carbamyl are useful as intermediates for preparing the said compounds where Q is amino and those where Q is halo are useful as intermediates in the preparation of the compounds where Q is lower-alkylamino and di-(lower-alkyl)amino.

Other compounds of Formula XIII include compounds where Q is amino, R is hydrogen and PY is 4-pyridinyl or 3-pyridinyl, for example, 3- amino-5-(4-pyridinyl)- 2(lH)-pyridinone (amrinone).

Further provided herein is a method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: a compound of Formula XV

a compound of Formula XVI

a compound of Formula XVII

(XVII) a compound of Formula XVIII

a compound of Formula XIX

(XIX) a compound of Formula (XX)

(XX) or pharmaceutically acceptable salt thereof. Formula XV is fluspirilen, Formula XVI is hexestrol, Formula XVII is dienestrol,

Formula XVIII is napelline, Formula XIX is iopanoic acid and Formula XX is suloctodil.

Dienestrol, Zidovudine, Metoprolol, Napelline, Methimazole, Amrinone, Iopanoic acid, R- Propanolol, Rimexolone and Pyrvinium pamoate, or a pharmaceutically acceptable salt thereof.

Clomiphene, Sulconazole, Miconazole, Mefloquine, Sulfinpyrazone, Terfenadine, Lisinopril, Econzaole, Clofazimine, Equilin, Felodipine, Dacarbazine, Furazolidone, Perhexiline maleate, Oxethazaine, Pimozide, Trifluoperazine, Ellipticine, Fluspirilen, Hexestrol, Dienestrol, Zidovudine, Metoprolol, Napelline, Methimazole, Amrinone, Iopanoic acid, R- Propanolol, Rimexolone and Pyrvinium pamoate, or a pharmaceutically acceptable salt thereof, wherein the infection is a bacterial infection selected from the group consisting of Enter obacterium faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinebacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.

Further provided is a method of treating or preventing a bacterial infection in a subject with or at risk of developing a bacterial infection in a subject comprising administering to the subject a compound that inhibits bacterial DNA gyrase or topoisomerase IV. The compound can be, for example, a compound of Formula I. As set forth above, a compound of Formula I can be terfenadine or a derivative thereof. In the methods set forth herein an inhibitor of bacterial DNA gyrase or topoisomerase IV can be used to treat or prevent Staphylococcus aureus infection.

It is contemplated that one or more, for example, two, three, four, five, etc., of the compounds or derivatives of the compounds set forth herein can be administered to treat or prevent infection. Thus, combinations of the compounds set forth herein are also provided. Pharmaceutically acceptable salts of all of the compounds set forth herein are also provided. The term pharmaceutically acceptable salt as used herein refers to those salts of any of the compounds described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

The infection can be a viral infection, bacterial infection, fungal infection or a parasitic infection, to name a few. All strains and types of pathogenic infection are contemplated herein. The infection can also be a respiratory infection, a gastrointestinal infection or a skin infection, to name a few.

In any of the methods of treating or preventing infection set forth herein, the infection can be any infection, wherein the infection is not a bacterial infection. In any of the methods of treating or preventing infection set forth herein, the infection can be any infection, wherein the infection is not a viral infection. In any of the methods of treating or preventing infection set forth herein, the infection can be any infection, wherein the infection is not a parasitic infection. In any of the methods of treating or preventing infection set forth herein, the infection can be any infection, wherein the infection is not a fungal infection. In any of the methods of treating or preventing infection set forth herein, the infection can be any infection, wherein the infection is not a protozoal infection.

Examples of bacterial infections include, but are not limited to infections caused by the Gram negative or Gram positive bacteria. For example, the infection can be caused by Listeria (sp.), Franscicella tularensis, Enterobacter sp. Enterococcus faecium, other

Enterococcus species, Klebsiella pneumonia, Acinetobacter baumannii, Mycobacterium tuberculosis, Rickettsia (all types), Ehrlichia or Chylamida. Further examples of bacteria include M. tuberculosis, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species, Yersinia pestis, Pasteurella haemolytica,

Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis,

Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Kingella kingae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species. In the methods provided herein, one or more compounds set forth herein can treat or prevent one or more bacterial infections selected from the group consisting of Enter obacterium faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinebacter baumannii, Pseudomonas aeruginosa and Enterobacter sp. In the methods set forth herein, the bacteria can be a small colony variant strain, for example a small colony strain of Staphylococcus aureus. In any of the methods set forth herein, the infection can be a bacterial infection, wherein the bacterial infection is not tuberculosis, for example, Mycobacterium tuberculosis. For example, and not to be limiting any of the compounds set forth herein, including compounds of Formula I and II can be used to treat or prevent infection in a subject with or at risk of developing an infection, wherein the infection is not tuberculosis.

Examples of parasitic infections include, but are not limited to infections caused by the following parasites: Cryptosporidium, Plasmodium (all species), American trypanosomes ( T. cruzi), African trypanosomes, Acanthamoeba, Entaoeba histolytica, Angiostrongylus, Anisakis, Ascaris, Babesia, Balantidium, Baylisascaris, lice, ticks, mites, fleas, Capillaria, Clonorchis, Chilomastix mesnili, Cyclspora, Diphyllobothrium, Dipylidium caninum, Fasciola, Giardia, Gnathostoma, Hetetophyes, Hymenolepsis, Isospora, Loa loa,

Microsporidia, Naegleria, Toxocara, Onchocerca, Opisthorchis, Paragonimus,

Baylisascaris, Strongyloides, Taenia, Trichomonas and Trichuris. In any of the methods set forth herein, the infection can be a parasitic infection, wherein the parasitic infection is not malaria, for example, malaria caused by any species of Plasmodium including Plasmodium falciparum. For example, and not to be limiting any of the compounds set forth herein, including compounds of Formula I and II can be used to treat or prevent infection in a subject with or at risk of developing an infection, wherein the infection is not malaria.

Furthermore, examples of protozoan and fungal species contemplated within the present methods include, but are not limited to, Plasmodium falciparum, other Plasmodium species, Toxoplasma gondii, Pneumocystis carinii, Trypanosoma cruzi, other trypanosomal species, Leishmania donovani, other Leishmania species, Theileria annulata, other Theileria species, Eimeria tenella, other Eimeria species, Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, Penicillium marneffei, and Candida species. In any of the methods set forth herein, the infection can be a protozoan infection, wherein the protozoan infection is not leishmaniasis, for example, leishmaniasis caused by a Leishmania species, for example, Leishmania major. For example, and not to be limiting any of the compounds set forth herein, including compounds of Formula I and III can be used to treat or prevent infection in a subject with or at risk of developing an infection, wherein the infection is not leishmaniasis. Examples of viral infections include but are not limited to, infections caused by RNA viruses (including negative stranded RNA viruses, positive stranded RNA viruses, double stranded RNA viruses and retroviruses) and DNA viruses. All strains, types, subtypes of DNA and RNA viruses are contemplated herein.

Examples of RNA viruses include, but are not limited to picornaviruses, which include aphthoviruses (for example, foot and mouth disease virus O, A, C, Asia 1, SAT1, SAT2 and SAT3), cardioviruses (for example, encephalomycarditis virus and Theiller's murine encephalomyelitis virus), enteroviruses (for example polioviruses 1, 2 and 3, human enteroviruses A-D, bovine enteroviruses 1 and 2, human coxsackieviruses A1-A22 and A24, human coxsackieviruses B1-B5, human echoviruses 1-7, 9, 11-12, 24, 27, 29-33, human enteroviruses 68-71, porcine enteroviruses 8-10 and simian enteroviruses 1-18), erboviruses (for example, equine rhinitis virus), hepatovirus (for example human hepatitis A virus and simian hepatitis A virus), kobuviruses (for example, bovine kobuvirus and Aichi virus), parechoviruses (for example, human parechovirus 1 and human parechovirus 2), rhinovirus (for example, human rhinovirus 1-100 and bovine rhinoviruses 1-3) and teschoviruses (for example, porcine tescho virus).

Additional examples of RNA viruses include calici viruses, which include noroviruses (for example, Norwalk virus), sapoviruses (for example, Sapporo virus), lagoviruses (for example, rabbit hemorrhagic disease virus and European brown hare syndrome) and vesiviruses (for example vesicular exanthema of swine virus and feline calicivirus).

Other RNA viruses include astroviruses, which include mamastorviruses and avastroviruses. Togaviruses are also RNA viruses. Togaviruses include alphaviruses (for example, Chikungunya virus, Sindbis virus, Semliki Forest virus, Western equine

encephalitis, Getah virus, Everglades virus, Venezuelan equine encephalitis virus and Aura virus) and rubella viruses. Additional examples of RNA viruses include the the flaviviruses (for example, tick-borne encephalitis virus, Tyuleniy virus, Aroa virus, Dengue virus (types 1 to 4), Kedougou virus, Japanese encephalitis virus (JEV), West Nile virus (WNV), Kokobera virus, Ntaya virus, Spondweni virus, Yellow fever virus, Entebbe bat virus, Modoc virus, Rio Bravo virus, Cell fusing agent virus, pestivirus, GB virus A, GBV-A like viruses, GB virus C, Hepatitis G virus, hepacivirus (hepatitis C virus (HCV)) all six genotypes), bovine viral diarrhea virus (BVDV) types 1 and 2, and GB virus B). Other examples of RNA viruses are the coronaviruses, which include, human respiratory coronaviruses such as SARS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. Coronaviruses also include bat SARS-like CoV, turkey coronavirus, chicken coronavirus, feline coronavirus and canine coronavirus. Additional RNA viruses include arteriviruses (for example, equine arterivirus, porcine reproductive and respiratory syndrome virus, lactate dehyrogenase elevating virus of mice and simian hemorraghic fever virus). Other RNA viruses include the rhabdoviruses, which include lyssaviruses (for example, rabies, Lagos bat virus, Mokola virus, Duvenhage virus and European bat lyssavirus), vesiculoviruses (for example, VSV-Indiana, VSV-New Jersey, VSV-Alagoas, Piry virus, Cocal virus, Maraba virus, Isfahan virus and Chandipura virus), and ephemeroviruses (for example, bovine ephemeral fever virus, Adelaide River virus and Berrimah virus). Additional examples of RNA viruses include the filoviruses. These include the Marburg and Ebola viruses (for example, EBOV-Z, EBOV-S, EBOV-IC and EBOV-R.

The paramyxoviruses are also RNA viruses. Examples of these viruses are the rubulaviruses (for example, mumps, parainfluenza virus 5, human parainfluenza virus type 2, Mapuera virus and porcine rubulavirus), avulaviruses (for example, Newcastle disease virus), respoviruses (for example, Sendai virus, human parainfluenza virus type 1 and type 3, bovine parainfluenza virus type 3), henipaviruses (for example, Hendra virus and Nipah virus), morbilloviruses (for example, measles, Cetacean morvilliirus, Canine distemper virus, Peste- des-petits-ruminants virus, Phocine distemper virus and Rinderpest virus), pneumoviruses (for example, human respiratory syncytial virus A2, Bl and S2, bovine respiratory syncytial virus and pneumonia virus of mice), metapneumoviruses (for example, human

metapneumovirus and avian metapneumo virus). Additional paramyxoviruses include Fer-de- Lance virus, Tupaia paramyxovirus, Menangle virus, Tioman virus, Beilong virus, J virus, Mossman virus, Salem virus and Nariva virus. Additional RNA viruses include the orthomyxoviruses.

These viruses include influenza viruses and strains (e.g., influenza A (H1N1

(including but not limited to A/WS/33 and A/California/04/2009 strains) H2N2, H3N2, H5N1 , H7N7, H1N2, H9N2, H7N2, H7N3 and H10N7), B and C viruses, as well as avian influenza (for example, strains H5N1 , H5N2, H7N1 , H7N7 and H9N2) thogotoviruses and isaviruses. Orthobunyaviruses (for example, Akabane virus, California encephalitis, Cache Valley virus, Snowshoe hare virus,) nairoviruses (for example, Nairobi sheep virus, Crimean- Congo hemorrhagic fever virus Group and Hughes virus), phleboviruses (for example, Candiru, Punta Toro, Rift Valley Fever, Sandfly Fever, Naples, Toscana, Sicilian and Chagres), and hantaviruses (for example, Hantaan, Dobrava, Seoul, Puumala, Sin Nombre, Bayou, Black Creek Canal, Andes and Thottapalayam) are also RNA viruses. Arenaviruses such as lymphocytic choriomeningitis virus, Lujo virus, Lassa fever virus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, SABV and WWAV are also RNA viruses. Borna disease virus is also an RNA virus. Hepatitis D (Delta) virus and hepatitis E are also RNA viruses. Any of the compounds set forth herein, including, but not limited to the compounds of Formula I and II can be used to treat or prevent a viral infection, wherein the viral infection is not a Lassa fever virus infection.

Additional RNA viruses include reoviruses, rotaviruses, birnaviruses, chryso viruses, cystoviruses, hypoviruses partitiviruses and totoviruses. Orbiviruses such as African horse sickness virus, Blue tongue virus, Changuinola virus, Chenuda virus, Chobar Gorge

Corriparta virus, epizootic hemorraghic disease virus, equine encephalosis virus, Eubenangee virus, Ieri virus, Great Island virus, Lebombo virus, Orungo virus, Palyam virus, Peruvian Horse Sickness virus, St. Croix River virus, Umatilla virus, Wad Medani virus, Wallal virus, Warrego virus and Wongorr virus are also RNA viruses.

Retroviruses include alpharetroviruses (for example, Rous sarcoma virus and avian leukemia virus), betaretroviruses (for example, mouse mammary tumor virus, Mason-Pfizer monkey virus and Jaagsiekte sheep retrovirus), gammaretroviruses (for example, murine leukemia virus and feline leukemia virus, deltraretroviruses (for example, human T cell leukemia viruses (HTLV-1, HTLV-2), bovine leukemia virus, STLV-1 and STLV-2), epsilonretriviruses (for example, Walleye dermal sarcoma virus and Walleye epidermal hyperplasia virus 1), reticuloendotheliosis virus (for example, chicken syncytial virus, lentiviruses (for example, human immunodeficiency virus (HIV) type 1 , human

immunodeficiency virus (HIV) type 2, human immunodeficiency virus (HIV) type 3, simian immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, caprine arthritis encephalitis virus and Visna maedi virus) and spumaviruses (for example, human foamy virus and feline syncytia-forming virus). Examples of DNA viruses include polyomaviruses (for example, simian virus 40, simian agent 12, BK virus, JC virus, Merkel Cell polyoma virus, bovine polyoma virus and lymphotrophic papovavirus), papillomaviruses (for example, human papillomavirus, bovine papillomavirus, adenoviruses (for example, adenoviruses A-F, canine adenovirus type I, canined adeovirus type 2), circoviruses (for example, porcine circovirus and beak and feather disease virus (BFDV)), parvoviruses (for example, canine parvovirus), erythroviruses (for example, adeno-associated virus types 1-8), betaparvoviruses, amdoviruses, densoviruses, iteraviruses, brevidenso viruses, pefudensoviruses, herpes viruses 1,2, 3, 4, 5, 6, 7 and 8 (for example, herpes simplex virus 1, herpes simplex virus 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, Kaposi's sarcoma associated herpes virus, human herpes virus-6 variant A, human herpes virus-6 variant B and cercophithecine herpes virus 1 (B virus)), poxviruses (for example, smallpox (variola), cowpox, monkeypox, vaccinia, Uasin Gishu, camelpox, psuedocowpox, pigeonpox, horsepox, fowlpox, turkeypox and swinepox), and hepadnaviruses (for example, hepatitis B and hepatitis B-like viruses).

One or more of the compounds described herein can be contacted with a cell or populations of cells in vitro, ex vivo or in vivo. For example, the cell or population of cells can be in a subject, or in an in vitro culture. In another example, one or more compounds set forth herein can be used to inhibit bacterial growth, fungal growth, parasitic growth, protozoal growth or viral replication, in vitro, ex vivo or in vivo. Any of the compounds set forth herein can be used alone or in combination with other therapeutic agents such as antiviral compounds, antibacterial agents (for example, antibiotics), antifungal agents, antiparasitic agents, anti-inflammatory agents, anti-cancer agents, etc.

In the methods described herein, the level of infection, for example, in a cell, or a population of cells, or a cell culture, can be assessed by measuring an antigen or other product associated with a particular infection. The level of infection can also be measured in a tissue sample or a culture of cells from a subject, either before or after administration of one or more compounds disclosed herein. For example, the level of viral infection can be measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay (See for example, Payungporn et al. "Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection." J Virol Methods . Sep 22, 2005; Landolt et la. "Use of real-time reverse transcriptase polymerase chain reaction assay and cell culture methods for detection of swine influenza A viruses" Am J Vet Res. 2005 Jan;66(l): 119-24).

Methods of measuring bacterial growth and inhibition of bacterial growth are provided in the Examples. Further, one of skill in the art would know how to determine the concentration of a compound that inhibits bacterial infection (see, for example, Andrews, et al. "Determination of minimum inhibitory concentrations". Journal of Antimicrobial

Chemotherapy 48 (suppl 1): 5-16 (2001)). Other methods for determining antifungal and antibacterial activity are known in the art. See, for example, Hayhoe et al. "Screening for Antibacterial, Antifungal and Anti quorum Sensing Activity," Methods Mol. Biol. 1055: 219- 225 (2013)); Doddanna et al. "Antimicrobial activity of plant extracts on Candida albicans: An in vitro study Indian J. Dent. Res. 24(4): 401-405 (2013), both of which are incorporated by this reference in their entireties.

As used throughout, by subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

As used herein, a biological sample is a sample derived from a subject such as a mammal or human and includes, but is not limited to, any biological fluid, including a bodily fluid. Examples of bodily fluids include, but are not limited to, whole blood, plasma, serum, urine, saliva, ocular fluid, ascites, a stool sample, spinal fluid, tissue infiltrate, pleural effusions, lung lavage fluid, and the like. The biological fluid includes a cell culture medium or supernatant of cultured cells from the subject.

The methods and compounds as described herein are useful for therapeutic treatment. Use of one or more of the compounds set forth herein for the treatment or prevention of infection is also contemplated herein. One or more of the compounds set forth herein for use in a method of treating or preventing infection is also provided herein. Therapeutic treatment involves administering to a subject a therapeutically effective amount of one or more of the agents described herein, optionally, after diagnosis of an infection or risk of infection in the subject. Therefore, all of the methods disclosed herein, can optionally comprise the step of diagnosing a subject with an infection or diagnosing a subject in need of prophylaxis or prevention of infection.

As used herein, the terms treatment, treat, or treating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), or 100%) reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. A control subject can be a subject that has not received a compound set forth herein. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), 100%), or any percent reduction in between 10%> and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

As utilized herein, by preventing infection is meant a method of precluding, delaying, averting, obviating, forestalling, stopping, or hindering the onset, incidence, severity, or recurrence of infection. For example, the disclosed method is considered to be a prevention if there is about a 10%> reduction in onset, incidence, severity, or recurrence of infection, or symptoms of infection (e.g., inflammation, fever, lesions, weight loss, etc.) in a subject exposed to an infection when compared to control subjects exposed to an infection that did not receive a composition for decreasing infection. Thus, the reduction in onset, incidence, severity, or recurrence of infection can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to a control subject. For example, and not to be limiting, if about 10%> of the subjects in a population do not become infected as compared to subjects that did not receive preventive treatment, this is considered prevention.

The compounds set forth herein can also be used to decrease infection in a cell. A decrease or inhibition of infection can occur in a cell, in vitro, ex vivo or in vivo. As utilized throughout, the term "infection" encompasses all phases of pathogenic life cycles including, but not limited to, attachment to cellular receptors, entry, internalization, disassembly, replication, genomic integration of pathogenic sequences, transcription of pathogen R A, translation of pathogen RNA, transcription of host cell mRNA, translation of host cell mRNA, proteolytic cleavage of pathogenic proteins or cellular proteins, assembly of particles, endocytosis, cell lysis, budding, and egress of the pathogen from the cells. The compounds described herein can be provided in a pharmaceutical composition.

Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected agent without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of

pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN^® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

Compositions containing the compound(s) described herein suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol,

polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens,

chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin,

polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

Administration can be carried out using therapeutically effective amounts of the agents described herein for periods of time effective to treat or prevent infection in a subject. The effective amount may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150mg/kg of body weight of active compound per day, about 0.5 to lOOmg/kg of body weight of active compound per day, about 0.5 to about 75mg/kg of body weight of active compound per day, about 0.5 to about 50mg/kg of body weight of active compound per day, about 0.5 to about 25mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about lOmg/kg of body weight of active compound per day, about 20mg/kg of body weight of active compound per day, about lOmg/kg of body weight of active compound per day, or about 5mg/kg of body weight of active compound per day. According to the methods taught herein, the subject is administered an effective amount of the compound. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response. Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

Any appropriate route of administration can be employed, depending on whether local or systemic treatment is desired, and on the area to be treated. The compositions are administered via any of several routes of administration, including topically, orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly,

subcutaneously, intracavity, transdermally, intrahepatically, intracranially,

nebulization/inhalation, or by installation via bronchoscopy. Optionally, the composition is administered by oral inhalation, nasal inhalation, or intranasal mucosal administration.

Administration of the compositions by inhalant can be through the nose or mouth via delivery by spraying or droplet mechanism, for example, in the form of an aerosol. Pharmaceutical compositions can be delivered locally to the area in need of treatment, for example by topical application or local injection. Multiple administrations and/or dosages can also be used. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the composition can also be included.

Also provided is a method of removing biofilm from a surface, comprising

administering an effective amount of one or more of the compounds provided herein to a biofilm-containing surface, wherein the amount is effective to remove biofilm from the surface. Removal of the biofilm from this surface does not have to be complete as this can range from a reduction to complete removal of the biofilm. Thus, in the disclosed methods, removal can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the amount of biofilm on a surface. For example, a method for removing biofilm from a surface is considered to be removal if there is a 10% reduction in the amount of biofilm on the surface as compared to a control. A control surface can be a biofilm containing surface that has not received a compound set forth herein. Thus, the reduction can be a 10%>, 20%>, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to control.

Further provided is a method of preventing biofilm formation on a surface comprising administering an effective amount of one or more of the compounds provided herein to the surface, wherein the amount is effective to prevent biofilm formation. The surface can be susceptible to biofilm formation. The biofilm can be produced by an organism selected from the group consisting of bacteria, algae, fungi and protozoa.

The compound can be, but is not limited to, a compound of Formula I

One or more of the compounds set forth herein can be combined with one or more biodegradable polymers to form a biodegradable antimicrobial composition. These compositions can be applied to a surface or used as a coating. The biodegradable polymers include but are not limited to polylactic acid, polyglycolic acid and copolymers and mixtures thereof such as poly(L-lactide) (PLLA), poly(D,L-lactide) (PLA), polyglycolic acid

[polyglycolide (PGA)], poly(L-lactide-co-D,L-lactide) (PLLA/PLA), poly(L-lactide-co- glycolide) (PLLA/PGA), poly(D,L-lactide-co-glycolide) (PLA/PGA), poly(glycolide-co- trimethylene carbonate) (PGA/PTMC), poly(D,L-lactide-co-caprolactone) (PLA/PCL) and poly(glycolide-co-caprolactone) (PGA/PCL); polyethylene oxide (PEO), polydioxanone (PDS), polypropylene fumarate, poly(ethyl glutamate-co-glutamic acid), poly(tert-butyloxy- carbonylmethyl glutamate), polycaprolactone (PCL), polycaprolactone co-butylacrylate, polyhydroxybutyrate (PHBT) and copolymers of polyhydroxybutyrate, poly(phosphazene), polyphosphate ester), poly(amino acid), polydepsipeptides, maleic anhydride copolymers, polyiminocarbonates, poly[(97.5% dimethyl-trimethylene carbonate)-co-(2.5% trimethylene carbonate)], poly(orthoesters), tyrosine-derived polyarylates, tyrosine-derived

polycarbonates, tyrosine-derived polyiminocarbonates, tyrosine-derived polyphosphonates, polyethylene oxide, polyethylene glycol, polyalkylene oxides, hydroxypropylmethylcellulose, polysaccharides such as hyaluronic acid, chitosan and regenerate cellulose, and proteins such as gelatin and collagen, and mixtures and copolymers thereof, among others as well as PEG derivatives or blends of any of the foregoing.

In the methods of removing or preventing biofilm formation, the surface can be a hard (for example, glass, metal, wood, chrome, plastic, vinyl or formica) or a soft surface (for example, cloth or upholstery). The methods set forth herein can be used to remove or prevent biofilm formation in vitro, ex vivo or in vivo. The methods set forth herein can also be used to remove or prevent biofilm formation on a medical device or a part thereof. For example, the methods set forth herein can be used to remove or prevent biofilm formation on an implantable medical device such as a cardiac rhythm management device (for example, a pacemaker, a defibrillator, an implantable cardioverter defibrillator (ICD) and a cardiac resynchronization therapy defibrillator (CRT device), a neurostimulator, a pulse generator, a drug pump, an infusion device, a physiological monitoring device (for example, a glucose sensor), contact lenses, a stent, a catheter, tubing or a breast implant. Mesh, bandages, and implantable devices, for example, can be coated with a compositions comprising one or more of the compounds set forth herein. Further, organs can be treated with one or more of the compounds set forth herein prior to transplantation in a subject. One or more of the compounds set forth herein can be used to inhibit biofilm formation by one or more of Staphylococcus aureus, Pseudomonas aeuroginosa, Staphylococcus epidermidis, Escherichia coli or Acinetobacter baummanii.

As utilized herein, by preventing biofilm formation is meant a method of precluding, delaying, averting, obviating, forestalling, stopping, or hindering the onset, incidence, severity, or recurrence of biofilm formation. For example, the disclosed method is considered to be prevention if there is about a 10% reduction in onset, incidence, severity, or recurrence of biofilm formation on a surface when compared to a control surface that did not receive a composition for preventing biofilm formation. Thus, the reduction in onset, incidence, severity, or recurrence of biofilm can be about a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to a control surface.

A biofilm can also exist or form in a biological subject, for example, on the teeth or gums of a subject. Therefore, one or more of the compounds set forth herein can be in a toothpaste, mouth rinse, gel, foam, varnish, polish, floss, dental strip, or copolymer membrane in order to remove or prevent biofilm formation on a dental surface.

Further provided herein is a method of identifying an antimicrobial agent comprising contacting a bacterial culture with a test agent and measuring adenylate kinase release in the supernatant of the bacterial culture, wherein an increase in adenylate kinase release as compared to a control indicates that the test compound is an antimicrobial agent. The control can be a bacterial culture that was not contacted with the test compound. The bacterial culture can be a culture of any bacterial strain, for example, a culture of any of the bacteria disclosed herein. The bacterial culture can also be small colony variant bacterial culture or a biofilm associated bacterial culture. Examples of agents identified utilizing this method are provided in the Examples.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention except as and to the extent that th are included in the accompanying claims.

Adenylate kinase (AK) is a ubi uitous intracellular enzyme that is released into the extracel lular space upon ceil lysis. As shown herein, AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth, states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms, The usefulness of tire A assay was validated by screening a Library of off-patent drugs for agents that exhibit antimicrobial properties toward a vari ety of bacterial species, including Escherichia coil and all members of the "ESKAPE" pathogens

(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetohacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs elicited AK release. Examples include, the antihistamine, terfenadine, which was active against S, aureus pi nktonic, SCV population, and bio film -associated cells, to name a few. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Gatteria melhnella infection model. These data demonstrate that the AK assay provides an attracti ve screening approach for identifying new antimicrobial agents. Further, drugs identified using this screening approach, for example, terfenadine and tamoxifen, provide novel antimicrobial drug development scaffolds.

The ESKAPE pathogens frequently cause health care-associated bacterial infections and can escape the effects of most currently available antibiotics. The most successful and widely applied method to identify agents with antibacterial activity has been whole-cell, bacterial growth assays. In this approach, libraries of small molecules or natural products are screened for agents that limit bacterial growth. However, growth-based assays have limitations. For example, the growth or no-growth readout has a limited dynamic range. This is likely to be problematic because growth assays lack the sensitivity required to detect antimicrobial molecules that are present in low concentrations within complex natural product extract libraries or compounds with limited antimicrobial activity. While the latter would obviously not represent a molecule that could be directly translated to clinical use, these low-activity hits could provide structurally novel scaffolds suitable for medicinal chemistry-based optimization. In addition, traditional growth-based assays are not readily amenable to screening for agents that target bacteria within certain clinically relevant bacterial growth states, such as established biofilms and small-colony variants. To address these limitations, provided herein is a high-throughput screen (HT5)- compatible whole-cell assay to detect agents that, directly kill bacteria. The assay is based on the rel ease of intracellular adenylate kinase (A ) into culture medium as a reporter of bacterial cell death . As shown herein., the AK assay exhibits improved sensitivity over that of conventional whole-cell growth assays and displays specificity for bactericidal agents.

Further, the assay can be used to screen for agents that kill small-colony-variant bacteria and bacteria within established biofiims.

To validate the AK assay as an HTS-compatible screening platform, the Prestwick library of off-patent drugs was screened against £^'. coli and each of the ESKAPE pathogens. This library contains representative examples of nearly all classes of antibiotics, and the bactericidal agents within the library that, were expected to be active against, the organism screened were identified. Additionally, agents with no previously reported antibiotic activity were identified. Traditional MIC testing confirmed the antimicrobial, properties of many of these molecules, showing that they could be repurposed as antimicrobials or serve as lead molecules for antibiotic development.. Consistent wi h that prediction, it. was shown that one of these compounds, tamoxifen, is active against E.faecium in a Galleria melloneHa model of infection. Further, it was shown that terfenadine is active against plankionic, small-colony variant, and biofiim-associated S. aureus. Taken together, these data demonstrate that the AK assay provides a general approach to screening for new antimicrobial agents active against a variety of pathogens during plankionic and other disease-associated growth states.

Bacterial growth conditions- The bacterial, species and strains used in these experiments are listed in Table 1. S. aureus strain UAMS- 1112 (generous gift from M. Smeltzer, University of Arkansas Medical Center) is a stable small-colony variant of the common laboratory S. aureus strain 8325-4, which harbors a heniB deletion. Unless otherwise noted, bacteria were grown for 16 h in Mueiler-Hinton (MH) (Becton, Dickinson, Franklin Lakes, NJ) or brain heart infusion (B I) (Becton, Dickinson.) -medium at. 37°C on a rotary shaker at 225 rotations per min (rpm) and then used to inoculate (1 TOO) fresh medium and processed, as described below. Table 1 -Bacterial Strains

a Abbreviations: anipieillin, Amp; colisim, CI; ceftriaxone, Cf; ciprofloxacin, Cp; erythromycin, Erm; kanamycin, Kan; linezolid, Lz; meropenem, Mp; minocycline, Min; Sulfamethoxazole, Sul; vancomycin, Van; not determined, ND; Univ., university.

^''' Clinical isolates were obtained from the University of Rochester School of Medicine and Dentistry.

Chemicals- The Prestwick Chemical Library of molecules with known biological activities was acquired from Prestwick Chemical (Illkireh, Prance). ToxiLight BioAssay kits were obtained from Lonza (Basel, Switzerland). Terfenadine, suloctidil, clomiphene citrate, ceftriaxone, sulfamethoxazole, erythromycin, kanamycin, ciprofloxacin, rifampin, axnpicillhi, minocycline, tamoxifen, and trimethoprim were purchased from Sigma-Aidrich (Si. Louis, MO). Meropenem, linezolid, and vancomycin were purchased from Thermo Fisher

(Waltharn, MA). Colistin was purchased from APP Pharmaceuticals (Schaumb rg, IL). MIC testing- M IC testing was performed to determine the antibiotic susceptibility profile of selected bacterial strains according to Clinical and Laboratory Standards (CLSi) protocols (Hindler et ai. Antimicrobial susceptibility testing, section 5. Clinical microbiology procedures handbook, Vol, 1 America Society of Microbiology, Washington, DC (2010). Briefly, colonies of each, bacterial species were collected from MB agar plates and suspended in individual tubes of MH medium to an optical density (600 nm) of 0.8. The resulting cultures were incubated at 37°C in a rotary shaker at 225 rpm to exponential phase (~1 x 10^s CPU rnb¹) and then diluted in fresh MH medium to a. cell density of ~3 ^x 10^'' CPU mi ^{" 1}. Ten microliters of the diluted cultures was added to 88 μΐ of M B medium in individual wells of a 96-welL round-bottom plate (Corning, Inc.), and 2 μΐ of a stock solution of the indicated reference antibiotic or test compound (0 to 256 μ§ ml^"1) was added to each well. The carrier solvent was either water or dimethyl sulfoxide (DMSO); final DMSO concentrations were less than or equal to 2%. Plates were incubated at 37°C for 24 h, and the IC was defined as the lowest concentration of antibiotic in which there was no visible cell pellet in the wells. Heat-killed bacterial AK release assays- Overnight cultures of E. coil strain 8295 or S. aureus RN4220 were used to inoculate (1 : 100 dilution) 25 ml of fresh MH medium and grown at 37°C on a rotary shaker at 225 rpm to exponential phase (~ 1 x 10 CPU ml^"1). Cells were pelleted by centrifugaiion (2,000 ^>: g) and resuspended in 2.5 ml of sterile water. One milliliter of the resulting suspension was boiled for 3 min and filter sterilized (0.45-μιη filter) to remove cell debris. The filtrate was serially diluted in sterile water, and 100 μί of each dilution was added to individual wells of a white-walled, 96-well plate (Corning, Inc., Corning, NY). To measure the AK activity in the supernatants at each dilution, 100 μΐ of ToxiLight AK reagent was added to each wel l, followed by incubation at room temperature for 30 min, and luminescence was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). Cells were serial ly diluted and plated to enumerate CFU (CFU mf¹) before and after boiling to correlate cell lysis with viability.

AK assay 96-we!I format- Overnight cultures of each bacterial species were used to inoculate (1 : 100 dilution) 25 ml of fresh MH medium and grow at 37°C on a rotary shaker at 225 rpm to exponential phase (~1 x 10^s CPU mi^'"1). Ninety-eight microli ters of MH medium, 2 μΐ of the indicated antibiotic, and 5 x 10⁶ bacteria were added to individual wel ls of a white-walled, 96-well microliter plate. Well components were mixed by pipetting and incubated at 37°C for 3 h. The plate was equilibrated to room temperature for 30 min. Next, 100 μΐ of ToxiLight AKL reagent was added to each well and incubated at room temperature for 30 mm, and luminescence was measured using a SpectraMax M5 plate reader.

AK assay 384~well format and high-throughput screening- Overnight cultures of each bacterial species were used to inoculate (1 :100 dilution) 25 ml of fresh medium and grown at 37°C on a rotary shaker at 225 rpm to exponential phase (-~ 1 ^x 10^s CFXJ mi^'"1 ). in a white- wailed, 384-well plate, 24 μΐ of MH medium, 0.3 ul (50 μΜ) of antibiotic or compound, and 5 10° bacteria were added to individual wells and incubated at 37°C for 3 h. The plates were equilibrated at room temperature for I h. Twenty-five microliters of ToxiLight AK reagent was then added to each well, followed by incubation at room, temperature for 30 roiu, and luminescence was measured using a SpectraMax M5 plate reader.

AK assay of established biofilms- Biofilms were grown as previously described (Beerskers et af. Infect, hnmun. 71 : 4206-4211 (2003); Musken et a . Nat. Protoc. 5: 1460- 1459 (2010); Tomaras et af. Microbiology 149: 3473-3483 (2003)). Briefly, P. aeruginosa and A.

baumannii were cultured overnight in Luria-Bertani medium and then used to seed 96-weIl. flat-bottom plates. Plates were incubated at 37°C in a humidified incubator for 48 h. to allow the formation of static biofilms. Nonadherent ceils were removed by aspiration and washing with sterile phosphate-buffered saline (PBS). Fresh LB medium supplemented with 0, Ix, 10*, or 100 ^x MIC of antibiotic was added to each well and incubated overnight at 37°C. Following treatment, 100 μΐ of each biofilm supernatant was transferred to 96-weli, white- walled plates, 100 μΐ of ToxiLight AK reagent was added to each well, mixtures were incubated for 30 min at room temperature, and luminescence was measured using a

SpectraMax M5 plate reader. Biofi!m-associated bacteria were enumerated by resuspending each biofilm in fresh PBS and plating. For S. aureus UAMS-1 biofilms, 96-weli, flat-bottom plates were first coated with 100 pi of 20% human plasma in carbonate buffer overnight at 4°C. Following coating, the plasma solution was removed and cells were inoculated in each well 1 :200 in 100 pi of tryptic soy broth supplemented with 3% glucose and 0.5% NaCl. Biofilms were cultured for 48 h in a humidified incubator at 37°C. Established S. aureus biofilms were washed once with PBS and then treated with a 100 μΐ of ToxiLight lysis buffer for 3 h, after which the amount of AK. released into supernatants was measured, as described above.

Smali-eoiony variant AK assays- Thirty-six-hour cultures of S. aureus strain UAMS-1112 were used to inoculate (1 :100 dilution) 100 ml of fresh MH medium and grown at 37°C on a rotary shaker at 225 rpra to an optical density (600 nm) of 0.1 to 0.2, corresponding to -- ! ^>; 10° CFU ml ⁱ. Ceils were pelleted by centrifugation and resuspended in 2 ml of fresh MH medium. Ninety-eigh microliters of MH medium containing 5 ^x 10⁶ bacteria and 2 μΐ of the indicated antibiotic were added to individual wells of a white-walled, 96-well microti ter plate. Well, components were mixed by pipetting and incubated a 37°C for 3 h. The plate was equilibrated to room temperature for 30 min. Next, 100 μΐ of ToxiLight AK reagent was added to each well, followed by incubation at room temperature for 30 min, and

luminescence was measured using a SpectraMax M5 plate reader.

Galleria mellonella model of S. aureus infection- A Galleria mellonella model of infection was used to measure the putative antimicrobial properties of tamoxifen against E.faecium and terfenadine against S. aureus. To do so, overnight cultures of E. faecium strain 824-05 or S. aureus strain USA300-0.1 14 were used to inoculate ( 1 : 100 dilution) 25 ml of fresh MH medium and grown at 37°C on a rotary shaker at 225 rpm to exponential phase (~ 1 x 10^s CFU ml^'"1). Cultures were pelleted by centrifugation (2,000 x g), washed with sterile PBS, and resuspended at ~1 κ 1Q CFU ml ' in fresh PBS, Galleria mellonella larvae (Vanderhorst Wholesale, inc.; St. Marys, OH) weighing 200 to 300 mg were inoculated with 5 μΐ of E. faeciim or S. aureus (5 ¾ 10° CFU) into the last left proleg using a 10~f.il Hamilton syringe. Worms were then rnock treated with either DMSO (negative control), vancomycin (20 rng kg ^!; positive control) at 2 h and 24 h postinoculation. For E. faecium studies, groups were also treated with the test compound tamoxifen at 80, .160, or 320 mg kg ^l, whereas groups were treated with the test compound terfenadine (80, 160, or 320 mg kg ^l) for S. aureus studies. Treatments were administered in the same manner as infection, except that each injection was in the next left proleg moving toward the head of the worm. Larvae were housed in petri dishes in the dark at 37°C and monitored for viability at the conclusion of the study (48 h postinoculation); worms were considered dead if they did not respond to physical stimuli. In addition to mock or compound treatment of infected larvae, studies included two additional noninfected negative-control groups: one group that did not receive injections and one group that was injected with PBS to control for the impact of physical trauma. AH experimental groups contained .15 worms, and each experiment was repeated three times. Rationale for adenylate kinase as a reporter of bacterial cell lysis- Adenylate kinase (AK) is a ubiquitous intracellular enzyme that catalyzes the conversion of 2 ADP *→ ATP -?- AMP and is released into the extracellular space upon cell lysis. The premise of the assay is that agents which disrupt cellular integrity, either directly through damage of the membrane/cell wall or indirectly .following the death of tire cell, will induce release of A . into the culture medium. Extracellular AK is subsequently detected by the addition of commercially available ToxiLight AK assay reporter cocktail (Lonza, Basel, Switzerland), which generates a luminescent signal by utilizing AK-generated ATP in the standard luciferase catalyzed reaction (see Fig, 1). As shown herein, an AK assay was developed as a high-throughput screening platform for antibacterial drug discover)'.

T e AK assay provides a sensitive measu e of bacterial lysis- As an initial test of AK release as a reporter of bacterial ceil death for Gram-positive and Gram-negative organisms, the sensitivity with which the assay measures AK. in the culture supernatants of heat-killed E. coll and S. aureus was determined. Each bacterial species was grown to exponential phase, harvested, and resuspended at 1 x 10⁹ CFU per m! in Mueller-Hinton (Ml]) medium.

Bacterial suspensions were heat killed, and a 10-fold dilution series of supernatants was prepared; an aliquot of each heat-killed sample was plated to ensure >99% bacterial death. The AK. acti vity of the dilution series was measured and compared to the AK activity of mock-treated (viable) bacteria by Student's t test. In comparison to untreated cells, a statistically significant increase in AK activity was detected at dilutions containing 1 ,7 x 10* or more heat-killed E. coii supernatants, compared to results for live bacteria (see Fig. 2A). AK activity increased ~433~fold and ~ 1,574-fold in cultures with 1 x 10' and 1 x 10* heat- killed E. coii supernatants, respectively, compared to results for mock-treated bacteria. A comparison of AK released from heat-killed S. aureus to that of mock-treated cells revealed a statistically significant difference in AK activity was detected at dilutions containing 1.8 ^x 10⁴ or more heat-killed S, aureus supernatants, with a maximum 122-fold increase in AK activity observed for 1 ^χ 10 iysed cells. Taken together, these results indicate that the AK assay reproducibly detects AK release following bacterial cel l lysis and that the assay is extremely sensitive, allowing the identification of agents that cause lysis in 0.0001% or 0.001% of the starting inoculum of E, coii or S, aureus cel ls, respectively. Furthermore, the AK assay exhibited a dynamic range of nearly 3 orders of magnitude and an excellent signal- to-noise ratio. The AK assay detects bactericidal molecules that are active against the ESKAPE pathogens- Next, ability of the AK assay to detect the activities of bactericidal antibiotics toward E. coii and each of the ESKAPE pathogens was examined. To do so, the M ICs of six classes of bactericidal antibiotics (penicillin, cephalosporin, quinolone, glycopeptide, carbapenem, and polymyxin) were determined for each organism (see ^'"Fable 2). An A assay was then performed at 0, 0.125 *·, 0.25x, 0.5x, 1 ^x, and 2x MIC of each antibiotic. As discussed below, AK release was detected at antibiotic concentrations below the MIC for most antibiotics tested, showing that the AK assay is more sensitive than growth-based assays for detecting bactericidal agents. As a represe tative example. Fig. 2B presents the AK assay measured polymyxin (eolistm)-mediated killing of A. baumannii strain 98-37-09. The MIC of colistm for this strain is 4 p.g ml^"1, whereas AK release was robustly detected at 0.25 x, 0.5x, and f x MIC value.

TABLE 2

MIC and AK. measures of bacterial species and antibiotic combinations

a Shading indicates significant increase in AK signal (greater than or equal to threefold over vehicle-treated ceils).

The MIC measures for each. ESKAPE pathogen and each organism's corresponding fold increase in AK signal following treatment with 0.5^X and l .G^x the MIC are provided in Table 2 (bactericidal agents). For four of the six bactericidal antibiotics tested, the AK assay proved to be superior to growth-based assays with respect to detecting the killing properties of bactericidal agents at sub- MIC values. More specifically, 100% of the organisms that were determined to be susceptible to the cell wall-active antibiotics ampicillin, meropenem, and colistin exhibited a significant increase in AK signal (>3-fold over that for vehicle-treated cells) at both 0.5 x and 1.0* their MICs. Eighty-three percent and 100% of ceftriaxone- susceptible species exhibited increased AK signal at 0.5 x and I x their MICs, respectively. Five of six (83%) ciproftoxacin-susceptible organisms exhibited AK signal at both 0.5 x and l.Ox MIC. Interestingly, the cell wall-targeted glycopeptide, vancomycin, caused significant AK release at 0.5 and l .Ox their MICs in only 40% and 60% of susceptible species.

To evaluate the specificity of the assay for detecting bactericidal agents, measured AK release by each of the ESKAPE pathogens following exposure to five classes of bacteriostatic agents (sulfonamide, tetracycline, macrolide, aminoglycoside, and oxazolidine) was also measured. To do this, the M IC value of each bacteriostatic antibiotic/organism pair was measured by a conventional growth-based approach. AK release by each bacterial species following treatment with 0.5* or l.Ox MIC of each bacteriostatic agent was subsequently measured. As shown in Fig. 3, S. aureus strain RN4220 was susceptible to all agents tested but bacteriostatic agents generated very low AK release, whereas bactericidal antibiotics generated robust AK. detection, in most instances, a simi lar trend was observed for the other ESKAPE pathogens, indicating that the assay enriches for the identification of bactericidal agents, which are arguably the most valuable antibiotics because they can be used to treat patients with immunological defects or rapidly lethal infections (Table 2, bacteriostatic agents). Two exceptions were noted. Surprisingly, A. ha mannii generated significant AK. signal in response to all the bacteriostatic agents evaluated, showing that AK release may be a fea ture of a more general stress response in this organism. Additionally, the bacteriosta tic agent minocycline generated signal by five of the eight (63%) organisms tested.

Taken together, these results show that the AK assay provides a viable screening approach for identifying bactericidal agents at sub-MICs that could otherwise be missed by growth-based assays. Further, because the AK. assay relies on. bacterial killing as opposed to growth changes to generate its readout, it was hypothesized that it would provide a format to develop screens that are not readily available by conventional, growth-based approaches.

Use of the AK assay to identify agents with antimicrobial activities against established biofilms and small colony variants- Established bacterial biofi lms represent a particularly problematic disease state, in part because biofilm-associated bacteria are recalcitrant to conventional antibiotic therapy. Thus, they ha ve been a focus of antibiotic development. As a result, a number of approaches to screening for molecules with activity toward bacterial biofilms have been developed recently (Benoit et al Environ. Microh. 76: 4136-4142; Perez et al. Lett. Appl. Microb. 51 :331-337 (2010)). Although each has its advantages, they also have a number of limitations, including reproducibility, reliance on specialized equipment, or low throughput. It was hypothesized that the AK. assay would provide a solution to some of these problems because it is rapid, sensitive, and simple to perform and it detects bactericidal molecules, the type of antibiotics required to treat established biofilms.

To determine whether the AK assay could detect agents with activity against established biofilms, S. aureus strain UAMS-.1 , A. baumannii strain 98-37-09, and P.

aeruginosa strain PAOl static biofilms were formed hi 96-weil flat-bottom plates. Forty- eight hours postinoculation, one well corresponding to each organism was stained with crystal violet to verify that biofilm formation had occurred, whereas the remaining wells were treated with 10* the MIC value with either eolistin (A. baumannii biofiims) or ciprofloxacin (P. aeruginosa, and S. aureus biofilms). Following overnight antibiotic treatment, biofilm- associated bacteria were enumerated by plating, and the corresponding superaatants were analyzed by the AK assay. Plating verified that IQx MIC antibiotic treatment resulted in a significant reduction in biofilm-associated P. aeruginosa (a 3.1 -log decrease), S. aureus (a 0.7-iog decrease), and baumannii (a 1.8-log decrease) compared to findings for untreated biofilms. As shown in Fig. 4A, corresponding AK measures indicated that the assay robustly detects the rnikl effects of these antibiotics on each bacterial species tested, showing that it represents a promising approach to identify agents that exhibit bactericidal activity toward established bacterial biofilms.

Another context in which the AK assay can be particularly valuable is in the identification of molecules that exhibit bactericidal activity toward bacterial sma! [-colony variants (SCV). SCV are slow-growing populations of bacterial species that have been hypothesized to cause latent or recurrent infections and are tolerant of standard antibiotic treatment regimens. Based on the aberrant SCV growth characteristics, typical growth-based FITS assays would be difficult to employ. Accordingly, the ability of the A . assay to identify agents that kill S. aureus SCV strain UAMS- 1112 was tested. To do so, 1 ^χ 10° UAMS- 1 112 cells were treated with 1 >^: or 10^χ MIC ciprofloxacin, meropenem, or vancomycin for 3 h, Following treatment, suspensions were plated to measure the antimicrobial properties of each antibiotic, and the AK assay was performed to measure adenylate kinase release. Plating revealed that ciprofloxacin and vancomycin had no effect on SCV viability at any concentration tested and exhibited no change in AK release in comparison to results for mock- treated cells (Fig. 4B), supporting the observation that S. aureus small-colony variants are recalcitrant to antibiotic treatment. Meropenem treatment resulted in a 0.5-log decrease in SCV viability, which corresponded to a 2, 5-fold increase in AK signal compared to that for mock-treated cells (Fig. 4B). Taken together, these results show that the AK assay provides the sensitivity needed to detect the slight antimicrobial effects of antibiotics, such as meropenem, toward S. aureus SCV. Validation of AK as an HTS-cempatible assa of antibacterial activity- To test the viability of the AK assay as a general tool in HTS-based antibacterial small-molecule discovery, optimized assay parameters, including inoculum, drug incubation time, and AK. reaction time, for screening pianktonic ES APE species as well as E. coli in a 384-weil format were determined. Based on these experiments, a standardized assay, as described above, was developed. As part of this optimization process, control assays in a 384- weli format were performed to measure the signal to noise and reproducibility of the assay in an HTS manner, For this, plates were seeded with E. coli or an ESKAPE pathogen. Alternating columns of the plate were then mixed with either 2% DM SO (negative control) or a bactericidal antibiotic (positive control), and AK signal was detected; a representative result for DMSO- and colistin-treated K. pneumoniae is shown in Fig. 5 A. Comparisons of the variance in signal between positive- and negative-control measures indicated that the AK assay provides Z '-factor scores between 0.59 and 0.82 (depending on the specific organism), indicating that the assay is sufficiently robust for HTS.

AK~based screening of library of off-patent drags and biologically active molecules- To further validate the AK assay protocol, E. coli and each of the ESKAPE pathogens was screened against the Prestwick library of FDA-approved drugs and biologically active molecules (1,120 compounds, total; 50 μΜ final drug concentration). The Prestwick library contains representatives of nearly ail classes of antibiotics currently in clinical use. making it art ideal library for testing the ability of the AK assay to detect bactericidal agents in a high- throughput screening format. Accordingly, the library was screened for antimicrobial agents that were active against planktonic E. coli and each of the ESKAPE pathogens using the AK assay; the cutoff for positive-scoring molecules was set at a 3-fold increase in extracellular AK activity, corresponding to the detection limit of statistically significant increases in AK activity for planktonic bacteria. The hit rates for the different organisms ranged from 1 A% to 4.8%; a representative example of raw screening data for Klebsiella pneumoniae is shown in Fig. SB. Screening results for all ESKAPE pathogens are summarized in Table 3, whereas results for all compounds within the Prestwick library are provided in Table 4.

Table 3- Preswick library screening results

" Tetracyclines e.ve identified.

Table 4 AK-based Prestwick Library Screening Results.

Hydrochlorothiazide Na+ CI- transport inhibitor Diuretic

Ursolic acid Diuretic

Inhibitor of folic acid

Sulfaguanidine Antibacterial synthesis

Pindolol Beta adrenergic antagonist Antiarrhythmic Isoniazid Antibacterial Mexiletine hydrochloride Na+ channel blocker Antiarrhythmic

Pentylenetetrazole GABA antagonist CNS stimulant Flavoxate hydrochloride Phosphodiesterase inhibitor Antispasmodic Chlorzoxazone Muscle relaxant Bufexamac Antiinflammator Ornidazole Bacterial DNA damage Antibacterial Glutethimide, para-amino Aromatase inhibitor Antineoplasic

Ca++ channel inhibitor

Ethosuximide Anticonvulsant voltage dependant

Dropropizine (R,S) Antitussive

Inhibitor of folic acid

Mafenide hydrochloride Antibacterial biosynthesis

K+ channel Ca++ dependant

Pinacidil Vasodilatator activator

Riluzole hydrochloride Glutamate antagonist Neuroprotective Albendazole

Nitrofurantoin Bacterial DNA damage Urinary antisepti

Clonidine hydrochloride Alpha2 agonist Antihypertensor

Hydralazine hydrochloride Adrenergic antagonist Antihypertensor

Bupropion hydrochloride Antidepressant

Phenelzine sulfate MAO inhibitor Antidepressant

Alprenolol hydrochloride Betal antagonist Antihypertensor

Meticrane Na+ channel blocker Antihypertensor

Khellin Phototherapeutic a

Benzonatate Antitussive

Zimelidine dihydrochloride

5-HT uptake inhibitor Antidepressant monohydrate

Hydroflumethiazide Na+ CI- transport inhibitor Antihypertensor

Azacyclonol HI antagonist Anxiolytic

Sulfacetamide sodic hydrate Antipsoriasic

Azathioprine Antimetabolite Immunosuppressa

Heptaminol hydrochloride Antihypotensive

Lynestrenol Antioestrogen Progestogen

Inhibitor of folic acid

Sulfathiazole Antibacterial synthesis

Guanabenz acetate Alpha agonist Antihypertensor

Tyrosine aminotransferase

Levodopa Antiparkinsonian inhibitor

Dopamine beta-hydroxylase

Disulfiram Alcohol deterren inhibitor

Nucleic acid synthesis

Idoxuridine Antiviral inhibitors

Acetylsalicylsalicylic acid Cyclooxygenase inhibitor Antiinflammator

Angiotensive converting

Captopril Antihypertensor enzyme inhibitor

Mianserine hydrochloride 5-HT antagonist Antidepressant Minoxidil K+ channel activator Antihypertensor Nocodazole Microtubule poison Antineoplastic Tranexamic acid Plasminogen inhibitor Hemostatic Chlorothiazide Na+ CI- transport inhibitor Antihypertensor Etofylline Phosphodiesterase inhibitor Cardiac analepti Diphenidol hydrochloride Antivertigo Tranylcypromine hydrochloride MAO inhibitor Antidepressant Norethindrone Progestogen Alverine citrate salt Anticholinergic Spasmolytic

Norepinephrine uptake

Nortriptyline hydrochloride Antidepressant inhibitor

Aceclofenac Cyclooxygenase inhibitor Antiinflammator

Prostaglandine synthesis

Niflumic acid Analgesic inhibitor

Monoamine oxydase

Iproniazide phosphate Antidepressant inhibitor (non selective)

Isotretinoin Cystic acne

0.7 1.0 0.7 0.6 Sulfamethoxazole

0.7 1.0 0.8 0.6 Retinoic acid eratolytic 0.7 1.0 0.8 0.7 Mephenesin Muscle relaxant 0.7 1.0 0.8 0.6 Antazoline hydrochloride HI antagonist Antihistaminic 0.8 1.0 0.7 0.6 Phenformin hydrochloride Neoglucogenese inhibitor Antidiabetic 0.7 1.3 1.4 0.6 Ethacrynic acid Na+ CI- uptake inhibitor Diuretic

Androgenic receptor

0.7 1.0 0.4 0.6 Flutamide Anticancer antagonist

Modulates cell membrane

0.9 1.0 0.8 0.6 Praziquantel Anthelmintic permeability

Inhibitor of folic acid

0.8 0.8 0.8 0.7 Sulfaphenazole Antibacterial synthesis

R(-) Apomorphine hydrochloride

0.8 0.8 0.9 0.8 Dl agonist Emetic hemihydrate

0.9 0.8 0.6 0.7 Panthenol (D) Vitamin

Dopamine-reuptake

1.0 0.9 0.7 0.8 Amoxapine Antidepressant inhibitor

Inhibitor of folic acid

1.0 0.8 0.7 0.7 Sulfadiazine Antibacterial synthesis

0.9 0.9 0.6 0.6 Cyproheptadine hydrochloride 5-HT antagonist Antipruritic 0.9 0.9 0.7 0.7 Norethynodrel Progestogen 0.9 0.8 0.5 0.8 Famotidine H2 histaminic antagonist Antiulcerative

Ribosomal protein synthesis

0.9 0.9 0.4 Thiamphenicol Antibacterial inhibitor

0.6 0.9 0.8 Danazol Estrogen antagonist Antigonadotropi

0.7 0.8 0.8 Cimetidine H2 antagonist Antiulcer 0.8 0.8 0.7 Nicorandil Antihypertensor 0.6 0.8 0.8 Doxylamine succinate HI antagonist Antihistaminic 0.7 1.0 0.8 Tomatine Antifungal 0.7 0.8 0.7 Ethambutol dihydrochloride Chelating agent Antibacterial 0.8 0.8 0.8 Nomifensine maleate Dopamine uptake inhibitor Antidepressant 0.7 1.0 0.8 Antipyrine Analgesic 0.7 0.8 0.9 Dizocilpine maleate Probe for NMDA receptors

0.7 0.8 0.8 Antipyrine, 4-hydroxy Antipyrine metabol 0.7 0.8 0.9 Acenocoumarol Vitamin K antagonist Anticoagulant 0.7 0.9 1.0 Am py rone Analgesic 0.8 0.9 1.0 Ethisterone Progestogen

Alkaline phosphatase

0.7 1.1 1.0 Levamisole hydrochloride Immunomodulat inhibitor

0.7 0.9 1.0 Triprolidine hydrochloride HI antagonist Antihistaminic

Monoamine oxidase

0.8 0.9 1.0 Pargyline hydrochloride Antihypertensor inhibitor

0.8 0.9 1.1 Doxepin hydrochloride Adrenaline uptake inhibitor Anticonvulsant 0.6 0.9 0.9 Methocarbamol Muscle relaxant 0.7 0.9 1.0 Dyclonine hydrochloride Na+ channel blocker Local anesthesic 2.6 1 Aztreonam Antibacterial

Bacterial transpeptidase

inhibitor

1.0 0.8 0.7 0.8 Dimenhydrinate HI antogonist Antihistaminic

Bacterial transpeptidase

1.0 0.7 1.3 Cloxacillin sodium salt Antibacterial inhibitor

0.9 2.7 0.6 0.9 Disopyramide Na+ channel blocker Antiarrhythmic 1.0 0.8 0.8 0.8 Catharanthine

Specific inhibitor of Ca2+

1.3 2.0 1.0 Clotrimazole Antibacterial activated K+ channels

0.9 0.7 0.7 1.0 Pentolinium bitartrate Ganglionic blocking agent Antihypertensor 1.0 0.8 0.8 0.9 Vinpocetine Phosphodiesterase inhibitor Nootropic drug

Inhibitor of respiratory

0.9 0.7 0.8 0.8 Aminopurine, 6-benzyl

kinase in plants

Noradrenaline reuptake

1.1 0.8 0.7 1.0 Clomipramine hydrochloride Antidepressant inhibitor

K+ channel ATP dependant

1.0 0.7 0.8 0.9 Tolbutamide Hypoglycemic inhibitor

1.1 0.8 0.8 1.5 Fendiline hydrochloride Ca++ channel activator Antianginal

Ribosomal

0.8 0.2 1.0 Chloramphenicol Antibacterial peptidyltransferase inhibitor

0.8 0.7 0.6 0.8 Naloxone hydrochloride Opiate antagonist Opioate antidote 0.8 2.2 0.7 0.9 Epirizole Antiinflammator 0.8 0.7 0.6 0.8 Metolazone Diuretic 0.9 0.7 0.6 0.8 Diprophylline Phosphodiesterase inhibitor Cardiac analepti 1.2 1.5 0.8 2.2 Ciprofloxacin hydrochloride Anti-bacterial

Non competitive

Triamterene Diuretic aldosterone antagonist

Ampicillin trihydrate Antibacterial Dapsone Folic acid antagonist Antiinflammator Haloperidol Dopamine antagonist Antipsychotic

Ribosomal protein synthesis

Troleandomycin Antibacterial inhibitor

Naltrexone hydrochloride

Opioid antagonist Analgesic dihydrate

Pyrimethamine Antimalarial

Chlorpheniramine maleate HI antagonist Antihistaminic

Hexamethonium dibromide

Ganglion blocking agent Antihypertensor dihydrate

Nalbuphine hydrochloride Opioid ligand Analgesic

Diflunisal Cyclooxygenase inhibitor Antiinflammator

Eicosenoid receptor

Picotamide monohydrate Antithrombotic antagonist

Niclosamide Helmintic DNA damage Anthelmintic Triamcinolone Antiinflamatory Midodrine hydrochloride Alpha adrenergic Hypotensor Vincamine Cerebral antianox Thalidomide Immunosuppressa Indomethacin Cyclooxygenase inhibitor Antiinflammator

Oxolinic acid Topoisomerase II inhibitor Antibacterial

Cortisone Antiinflammator Nimesulide Cyclooxygenase 2 inhibitor Antiinflammator Prednisolone Glucocorticoid Hydrastinine hydrochloride Dopamine receptor blocker Cardiotonic Fenofibrate Lipoprotein lipase activator Hypolipidemiant Pentoxifylline Phosphodiesterase inhibitor Vasodilatator

Vascular cyclooxygenase

Bumetanide Diuretic activator

Metaraminol bitartrate Adrenergic agonist Vasoconstrictor

Labetalol hydrochloride

Salbutamol Beta adrenergic agonist Bronchodilatato

Cinnarizine HI antagonist Antihistaminic

Prilocaine hydrochloride Na+ channel blocker Local anesthesic

Methylprednisolone, 6-alpha Glucocorticoid

Camptothecine (S,+) Topoisomerase I inhibitor Antitumor agent

Quinidine hydrochloride

Heme polymerase inhibitor Antiarrhythmic monohydrate

Procaine hydrochloride Na+ channel blocker Local anesthesic

Bromocryptine mesylate Prolactin inhibitor Antiparkinsonian

Moxisylyte hydrochoride Alpha antagonist Vasodilatator

Metanephrine hydrochloride DL

Isoprenaline uptake inhibitor

extraneuronal

Betazole hydrochloride Histamine analog Gastric secretion stim Dehydrocholic acid Choleretic Isoxicam Cyclooxygenase inhibitor Antiinflammator Hesperetin P450 inhibitor

Naproxen Cyclooxygenase inhibitor Antiinflammator Perphenazine Dopamine antagonist Antipsychotic Naphazoline hydrochloride Adrenergic ligand Vasoconstrictor Mefloquine hydrochloride Heme polymerase inhibitor Antimalarial Ticlopidine hydrochloride ADP antagonist Platelet antiaggreg

Sterol 14-demethylase

Isoconazole Antibacterial inhibitor

Dicyclomine hydrochloride Anticholinergic Antispasmodic Spironolactone Aldosterone antagonist Diuretic Amyleine hydrochloride Na+ channel blocker Local Anesthesic

Pirenzepine dihydrochloride Ml antagonist Antiulcerative

Lidocai^'ne hydrochloride Na+ channel blocker Local anesthesic

Dexamethasone acetate Antiinflammator

Ranitidine hydrochloride H2 antagonist Antiulcerative

Fludrocortisone acetate Mineralocorticoi

Tiratricol, 3,3',5-

Hypocholesterolemic triiodothyroacetic acid

Fenoterol hydrobromide Beta adrenergic agonist Bronchodilatato

Flufenamic acid Cyclooxygenase inhibitor Analgesic

Homochlorcyclizine

HI antagonist Antihistaminic dihydrochloride

Flumequine Topoisomerase II inhibitor Antibacterial

Diethyicarbamazine citrate Lipoxygenase inhibitor Antihelmintic

Tolfenamic acid Cyclooxygenase inhibitor Antiinflammator

Chenodiol Detergent Anticholelithogen

Meclofenamic acid sodium salt

Cyclooxygenase inhibitor Antiinflammator monohydrate

Perhexiline maleate Ca2+ blocking agent Vasodilatator

Kawain Ca++ channel blocker Antiaggregant

Oxybutynin chloride Anticholinergic Spasmolytic

Trimethoprim Folic acid antagonist Antibacterial

Spiperone D2 antagonist Antipsychotic

Metoclopramide

5-HT3 antagonist Antiemetic monohydrochloride

Pyrilamine maleate HI antagonist Antihistaminic

Microtubule formation

Fenbendazole Antihelmintic inhibitor

Sulfinpyrazone Uricosuric Trichlorfon Cholinesterase inhibitor Antihelminthic Glipizide

Anticonvulsivant

Anxiolytic Antipsychotic Antihistaminic Antiinflammator

Antianginal

Antidiabetic (type noninsulin-depend

Antipsychotic Antihistaminic Local anesthesic

Antibacterial

Local anesthesic Antihistaminic Antihistaminic

Antibacterial

Vasodilatator

Antibacterial

1.2 1.1 Piroxicam Cyclooxygenase inhibitor Antiinflammator 1.0 1.0 Dantrolene sodium salt Blocker of Ca2+ release Skeletal muscle rela

Neuromuscular depolarizing

1.3 1.1 Pyrantel tartrate Anthelmintic agent

0.9 1.0 Trazodone hydrochloride 5-HT uptake inhibitor Antidepressant 1.3 1.0 Fenspiride hydrochloride Bradykinin antagonist Antiinflammator 1.2 1.1 Glafenine hydrochloride Analgesic 1.3 1.0 Gemfibrozil Antihyperlipoprotein 1.4 1.1 Pimethixene maleate Anticholinergic Antihistaminic 1.2 1.0 Mefexamide hydrochloride Psychoanaleptic 1.3 1.0 Pergolide mesylate Dopaminergic agonist Antiparkinsonian 1.4 1.1 Tiapride hydrochloride Dopamine antagonist Antidyskinetic 1.2 1.0 Acemetacin Cyclooxygenase inhibitor Antiinflammator 0.8 0.9 Mebendazole

1.3 1.1 Benzydamine hydrochloride 5-HT receptor antagonist Analgesic 0.8 0.8 Fenbufen Cyclooxygenase inhibitor Antiinflammator 1.3 1.1 Fipexide hydrochloride Glutamatergic Nootropic 1.1 0.9 Ketoprofen Cyclooxygenase inhibitor Antiinflammator

Progesterone receptor

1.3 1.1 Mifepristone Abortifacient antagonist

1.1 1.1 Indapamide Diuretic 1.4 1.1 Diperodon hydrochloride Local anesthesic

Fumarate reductase

Morantel tartrate Anthelmintic inhibitor

Verapamyl hydrochloride Alphal antagonist Antyhypertensiv Homatropine hydrobromide (R,S) Muscarinic antagonist Antispasmodic Dipyridamole

Nifedipine L-type Ca2+ channels blocker Antihypertensor Chlorhexidine Detergent Bacteriostatic Chlorpromazine hydrochloride Dopamine antagonist Antiemetic

Loperamide hydrochloride Ca2+ channel antagonist Antidiarrhoeic Diphenhydramine hydrochloride HI receptor antagonist Antihistaminic

Ribosomal protein synthesis

Chlortetracycline hydrochloride Antibacterial inhibitor

Minaprine dihydrochloride Dopamine agonist Antidepressant

Oestrogen receptor

Tamoxifen citrate

antagonist

Sterol 14-demethylase

Miconazole Antifungal inhibitor

Nicergoline Alpha agonist Vasodilatator Isoxsuprine hydrochloride beta adrenergic agonist Vasodilatator Canrenoic acid potassium salt Detergent Antihypercholesterol

Acebutolol hydrochloride Betal antagonist Antianginal Thioproperazine dimesylate Dopamine antagonist Antipsychotic Tolnaftate Antifungal

1.0 1.0 0.9 1.5 1.2 Dihydroergotamine tartrate Serotonine antagonist Antimigraine 1.5 1.8 0.8 I Norfloxacin Topoisomerase II inhibitor Antibacterial 1.0 I 1.0 1.3 1.2 Lisinopril Converting enzyme inhibitor Antihypertensor

Inhibitor of mitochondrial

1.1 1.0 1.0 1.4 1.1 Antimycin A Antifungal electron transport

Ribosomal protein synthesis

1.3 0.9 1.2 1.2 Lincomycin hydrochloride Antibacterial inhibitor

1.0 1.2 0.9 1.3 1.2 Xylometazoline hydrochloride Vasoconstrictor 1.1 1.3 1.0 1.6 1.2 Telenzepine dihydrochloride Ml muscarinic antagonist Antiulcerative 1.0 1.1 1.1 1.4 1.2 Oxymetazoline hydrochloride Partial alpha2A agonist Vasoconstrictor

Ergosterol synthesis

1.5 mi 1.3 1.3 Econazole nitrate Antifungal inhibition

1.0 1.3 1.0 1.6 1.2 Nifenazone Cyclooxygenase inhibitor Analgesic 1.0 1.1 1.0 1.4 1.2 Bupivacaine hydrochloride Na+ channel blocker Local anesthesic 1.1 1.4 0.9 1.4 1.3 Griseofulvin Enzymatic inductor Antifungal 1.0 1.2 1.1 1.5 1.2 Clemastine fumarate HI antagonist Antihistaminic 1.2 1.6 1.0 1.6 1.2 Clemizole hydrochloride HI antagonist Antihistaminic

Ribosomal protein synthesis

0.9 0.2 0.5 Oxytetracycline dihydrate Antibacterial inhibitor

1.0 1.1 1.1 1.2 Tropicamide Muscarinic antagonist Mydriatic 1.3 2.3 1.3 1.3 Pimozide Dopamine antagonist ? Antipsychotic 1.0 1.1 0.9 1.2 Nefopam hydrochloride Analgesic 1.1 1.3 0.9 1.2 Heme polymerase inhibitor Antimalarial

Amodiaquin dihydrochloride

dihydrate

Phentolamine hydrochloride Alpha adrenergic antagonist Antihypertensor Mebeverine hydrochloride Antispasmodic

Todralazine hydrochloride Antihypertensor

Ribosomal protein synthesis

Erythromycin Antibacterial inhibitor

Imipramine hydrochloride 5-HT transport inhibitor Antidepressant

Ribosomal protein synthesis

Oleandomycin phosphate Antibacterial inhibitor

Sulindac Cyclooxygenase inhibitor Antiinflammator

Transrcriptase inverse

Didanosine Antiviral inhibitor

Amitryptiline hydrochloride Alpha 1 antogonist Antidiabetic

Ribosomal protein synthesis

Josamycin Antibacterial inhibitor

Adiphenine hydrochloride Anticholinergic Local anesthesic Paclitaxel Tubuline inhibitor Antineoplastic Dibucaine Na+ channel blocker Local anesthesic Ivermectin GABA ligand Anthelmintic Prednisone Glucocorticoid Gallamine triethiodide M2 antagonist allosteric Muscle relaxant Thioridazine hydrochloride Ca2+ channel antagonist Neuroleptic Neomycin sulfate Antibacterial

Ribosomal protein synthesis

inhibitor

Diphemanil methylsulfate Anticholinergic Bronchodilatato

Ribosomal protein synthesis

Dihydrostreptomycin sulfate Antibacterial inhibitor

Trimethobenzamide

D2 antagonist Antiemetic hydrochloride

Ribosomal protein synthesis

Gentamicine sulfate Antibacterial inhibitor

Etodolac Cyclooxygenase inhibitor Antiinflammator

Ifenprodil tartrate Adrenergic antagonist Vasodilatator

Scopolamin-N-oxide

Anticholinergic Antiparkinsonia hydrobromide

Flunarizine dihydrochloride Na+ channel blocker Vasodilatator

Hyoscyamine (L) Cholinergic Antispasmodic

Trifluoperazine dihydrochloride Dopamine antagonist Psycholeptic

Chlorphensin carbamate Muscle relaxant

Enalapril maleate Converting enzyme inhibitor Antihypertensor

Bacterial transpeptidase

Metampicillin sodium salt Antibacterial inhibitor

Ribosomal protein synthesis

Minocycline hydrochloride Antibacterial inhibitor

Dilazep dihydrochloride Adenosine uptake inhibitor Vasodilatator

ATP-dependent K+ channel

Glibenclamide Antidiabetic inhibitor

1.4 2.8 1.0 Ofloxacin Topoisomerase II inhibitor Antibacterial 1.1 1.1 1.1 1.3 1.4 Guanethidine sulfate Catecholamine depletor Antihypertensor 1.5 I 1.0 Lomefloxacin hydrochloride Topoisomerase II inhibitor Antibacterial

Quinacrine dihydrochloride Monoamine oxydase

1.0 1.2 1.0 1.3 0.9 Antimalarial dihydrate inhibitor

1.0 1.0 1.2 1.3 1.1 Orphenadrine hydrochloride HI antogonist Antihistaminic 1.4 1.2 1.5 1.6 1.3 Clofilium tosylate K+ channel blocker Antiarrhythmic 1.0 1.1 1.2 1.3 1.1 Proglumide Gastrin inhibitor Antiulcerative 1.1 1.0 1.5 1.6 1.1 Fluphenazine dihydrochloride Dopamine antagonist Antipsychotic

Ribosomal protein synthesis

0.9 0.4 0.3 0.4 0.4 Streptomycin sulfate Antibacterial inhibitor

0.9 0.3 0.4 0.5 0.4 Testosterone propionate Androgen

Alpha 1- adrenergic

0.9 0.3 0.4 0.4 0.4 Alfuzosin hydrochloride Antihypertensor antagonist

0.9 0.4 0.4 0.4 0.4 Arecoline hydrobromide Cholinergic Anthelmintic

Glucagon secretogen,

0.9 0.4 0.4 0.3 0.3 Chlorpropamide Antidiabetic somatostatin secretogen

1.0 0.2 0.4 0.4 0.4 Thyroxine (L) Thyroid hormone Hypocholesterolemic

Phenylpropanolamine

0.8 0.3 0.5 0.4 0.4 Alpha adrenergic agonist Decongestant hydrochloride

0.9 0.4 0.4 0.4 0.4 Tocopherol (R,S) Anti-oxidant 0.9 0.4 0.5 0.4 0.3 Ascorbic acid Vitamin

Aspartic proteases

0.8 0.4 0.5 0.4 0.4 Pepstatin A Antiviral irreversible inhibitor

L-aromatic aminoacid

0.6 0.9 0.4 0.6 0.5 0.4 Methyldopa (L,-) Antihypertensor decarboxylase inhibitor

0.5 0.8 0.4 0.5 0.5 0.4 SR-95639A Ml agonist receptor

Bacterial transpeptidase

0.9 0.7 2.1 0.7 Cefoperazone dihydrate Antibacterial inhibitor

Inhibition of viral uncoating

and viral assembly,

0.5 0.9 0.4 0.5 0.5 0.4 Adamantamine fumarate Antiviral alteration of dopamine

release and reuptake

0.7 0.8 0.4 0.6 0.4 0.4 Zoxazolamine Uric acid uptake inhibitor Muscle relaxant 0.7 ill 0.6 0.7 2.0 0.5 Butoconazole nitrate Ergosterol inhibitor Antifungal 0.5 0.9 0.5 0.4 0.5 0.5 Tacrine hydrochloride hydrate Cholinesterase inhibitor Cognition enhanc

Na+ channel blocker, K+

channel blocker, non¬

0.7 1.7 0.7 0.6 2.2 0.5 Amiodarone hydrochloride Antiarrhythmic competitive beta-adrenergic

blocker

0.5 0.9 0.6 0.5 0.5 0.5 Bisoprolol fumarate Betal antagonist Antihypertensor 0.5 0.9 0.5 0.7 0.5 0.4 Amphotericin B Ergosterol ligand Antibacterial 0.8 0.9 0.6 0.6 0.6 0.6 Serotonin hydrochloride 5-HT agonist Neurotransmitte

Tubocurarine chloride

0.7 0.9 0.5 0.6 0.7 0.6 Curarising Muscle relaxant pentahydrate (+)

0.9 2.3 0.9 0.6 Cefotiam hydrochloride Antibacterial

5-HT antagonist, partial

0.7 0.9 0.5 0.6 0.7 0.6 Dihydroergocristine mesylate adrenergic agonist, partial Vasodilatator dopaminergic agonist

0.6 0.9 0.4 0.5 0.5 0.6 Azathymine, 6 Antimetabolite Anticancer

Inhibitor of carbachol-

0.5 0.5 0.6 0.5 Noscapine stimulated phosphoinositide Antitussive turnover

Dopamine antagonist, 5-HT

0.5 0.5 0.5 0.6 Benperidol Antipsychotic antagonist

0.3 0.5 0.6 0.5 Syrosingopine Catecholamine depletor

Bacterial transpeptidase

0.5 2.5 1.5 0.5 Cefaclor Antibacterial inhibitor

0.5 0.5 0.5 0.5 Atropine sulfate monohydrate Muscarinic antagonist Anticholinergic

Performs membrane

0.6 11 m. Colistin sulfate Antibacterial ionophores

Eserine sulfate, physostigmine

0.4 0.5 0.5 0.6 Cholinesterase inhibitor Ophtalmic agent sulfate

2.1 0.5 0.6 0.5 Daunorubicin hydrochloride DNA intercaling Antineoplastic 0.5 0.5 0.6 0.5 Aconitine Open TTX-Na+ channel Analgesic 0.5 0.5 0.5 0.5 Dosulepin hydrochloride Antidepressant 0.3 0.6 1.1 0.4 Rescinnamin Catecholamine depletor Antihypertensor 0.4 3.2 0.5 I Ceftazidime pentahydrate Antibacterial

High affinity GABA A

0.4 0.5 0.5 0.4 Dihydroergotoxine mesylate receptor CI- channel, Anticonvulsant prolactin inhibitor

0.5 0.5 0.5 0.4 lobenguane sulfate Antineoplastic if radiol

Protein synthesis inhibitor,

0.5 0.6 0.5 0.5 Emetine dihydrochloride Antiamebic

5-HT Iigand

0.5 0.6 0.7 0.6 Tremorine dihydrochloride Cholinergic Convulsant

Androsterone Antihypertensor

Practolol Beta antagonist Antihypertensor

Acetylcholine esterase

Anisomycin Antiprotozoal inhibitor

Transcriptase reverse

Zidovudine, AZT Antiviral inhibitor

Carbarsone Antiamebic

Inhibitor of folic acid

Sulfisoxazole biosynthesis, Eta endothelin Antibacterial receptor antagonist

Apigenin MAP kinase inhibitor Antiproliferative cGMP phosphodiesterase

Zaprinast inhibitor, phosphodiesterase Erectogen

5 inhibitor

Aspartic acid, N-acetyl (R,S)

Chlormezanone Skeletal muscle rela

Bacterial transpeptidase

Bacampicillin hydrochloride Antibacterial inhibitor

Alpha antagonist,

Procainamide hydrochloride antinuclear antibodies, Antiarrhythmic anticholinergic

Apoptosis inducer, PLA2

Betulinic acid Antimalarial inhibitor

N6-methyladenosine Antimetabolite Anticancer Biotin Vitamin H

Guanfacine hydrochloride Alpha 2A agonist Antihypertensor

Bisacodyl Na+ uptake inhibitor Cathartic Domperidone Dopamine Antagonists Antiemetic

Stimulator of calcium and

Calciferol Vitamin D phosphate absorption

Metixene hydrochloride Anticholinergic Antiparkinsonian Tetracaine hydrochloride Local anesthesic Nitrofural Bacterial DNA damage Antibacterial Mometasone furoate Antiinflammator

Non competitive ATPase H+

Omeprazole Antiulcerative pump inhibitor

Tomatidine Cholinesterase activity Antifungal Propylthiouracil Antimetabolite Antihyperthyroid Dacarbazine Alkylating agent Antineoplastic

Sterol 14-demethylase

Terconazole Antifungal inhibitor

Ipratropium bromide Antimuscarinic agent Bronchodilatato Tiaprofenic acid Cyclooxygenase inhibitor Antiinflammator Acetopromazine maleate salt Dopaminergic antagonist ? Tranquilizer

Bacterial mucopeptide

Vancomycin hydrochloride Antibacterial biosynthesis inhibitor

Rauwolscine hydrochloride Alpha2 antagonist Antidepressant Artemisinin Oxidant Antimalarial

Alpha 1-adrenoreceptor

Corynanthine hydrochloride Anti-leishmania dr antagonist

Propafenone hydrochloride Beta adrenergic antagonist Antiarrhythmic Palmatine chloride Anticholinestrase activity Uterine contracta Ethamivan Respiratory analep Trimethylcolchicinic acid Tubuline inhibitor ? Anticancer agent

Na+ CI- uptake inhibitor,

Furosemide Diuretic carbonic anhydrase inhibitor

Suloctidil Vasodilatator Methapyrilene hydrochloride Histamine HI antagonist Antihistaminic Carcinine Anti-oxidant

Adrenergic transport

Desipramine hydrochloride inhibitor, 5-HT transport Antidepressant inhibitor

Carisoprodol Muscle relaxant

Monoamine oxidase A

Clorgyline hydrochloride Antidepressant inhibitor

Bacterial transpeptidase

Cephalosporanic acid, 7-amino Antibacterial inhibitor

Clenbuterol hydrochloride Beta adrenergic agonist Bronchodilatato

Competitive glutamate

Chicago sky blue 6B

uptake inhibitor

Noradrenaline uptake

Maprotiline hydrochloride inhibitor, 5-HT uptake Antidepressant inhibitor

0.8 0.8 Buflomedil hydrochloride Vasodilatator 0.8 0.9 Thioguanosine Antimetabolite Anticancer

Glucose-6-phosphate

0.8 0.9 Chlorogenic acid Antiinflammator translocase inhibitor

D2 dopamine receptor

1.0 1.0 Chlorprothixene hydrochloride antagonist, GABAA receptors Neuroleptic antagonist

0.8 1.0 Roxatidine Acetate HCI Antiulcerative 0.8 0.9 Ritodrine hydrochloride Beta2 agonist Tocolytic 1.0 0.9 Cholecalciferol Vitamin

5-HT antagonist, dopamine

0.9 0.9 Clozapine Antipsychotic antagonist, GABA ligand

0.9 0.9 Cisapride 5-HT antagonist Peristaltic stimula 1.0 0.9 Vigabatrin GABA transaminase inhibitor Anticonvulsant 0.9 1.0 Hydrastine hydrochloride GABAa antagonist Hypotensor 1.0 0.9 Biperiden hydrochloride Anticholinergic Antiparkinsonian 1.0 0.9 Lobelanidine hydrochloride Nicotinic ligand

0.9 0.9 Cetirizine dihydrochloride HI antagonist Antihistaminic 0.9 1.0 Papaverine hydrochloride Phosphodiesterase inhibitor Vasodilator 1.1 0.9 Etifenin Chelating agent Diagnostic agent 0.8 0.9 Yohimbine hydrochloride Alpha antagonist Mydriatic

Metaproterenol sulfate,

1.0 0.9 Beta-adrenergic agonist Bronchodilatato orciprenaline sulfate

Lobeline alpha (-) hydrochoride Nicotinic receptor ligand Respiratory stimul

Ribosomal protein synthesis

Sisomicin sulfate Antibacterial inhibitor

Anti-HIV reverse

Berberine chloride transcriptase, cholinesterase Antibacterial inhibitor

Quercetine dihydrate Lipoxygenase inhibitor Antimalarial Cilostazol Antithrombotic Resveratrol Antiinflammator

Cholinesterase inhibitor,

Galanthamine hydrobromide Alzheimer treatme nicotinic receptor agonist

Bromperidol Dopamine antagonist Antipsychotic Bicuculline (+) GABAa receptor antagonist Convulsant Cyclizine hydrochloride HI antagonist Antiemetic Yohimbinic acid monohydrate

Na+ uptake inhibitor,

Chlorthalidone Diuretic carbonic anhydrase inhibitor

Coralyne chloride hydrate Topoisomerase I inhibitor Anti-leukemic Dobutamine hydrochloride Betal, Beta2 agonist Bronchodilatato Corticosterone Glucocorticoid

Mono amine oxidase

Moclobemide Antidepressant inhibitor (Type A)

Cyanocobalamin Vitamin Clopamide Gonad stimuling agent Antihypertensor

Bacterial transpeptidase

Cefadroxil Antibacterial inhibitor

Hycanthone DNA intercaling agent Anthelmintic

IL 2 synthesis inhibitor,

Cyclosporin A calcineurine phosphatase Immunosuppressa inhibitor

Ca++ channel block,

Adenosine 5'-monophosphate adenosine receptor

Nutrient monohydrate activation, activation of

outward K+ current

Digitoxigenin Na+ K+ ATPase inhibitor Cardiotonic

Bacterial transpeptidase

Amoxicillin Antibacterial inhibitor

Digoxin Na+ K+ ATPase inhibitor Cardiotonic

Bacterial transpeptidase

Cephalexin monohydrate Antibacterial inhibitor

Doxorubicin hydrochloride DNA intercalant Antibacterial

Dextromethorphan hydrobromide

Opioid ligand Antitussive monohydrate

Carbimazole Iodine oxidazing inhibitor Antityroidic hormo

Alpha adrenergic antagonist,

Droperidol 5-HT antagonist, dopamine Antipsychotic antagonist

Epiandrosterone Anabolic steroid Fluoxetine hydrochloride 5-HT uptake inhibitor Antidepressant Laudanosine (R,S) Convulsant

lohexol Diagnostic aid

Ajmalicine hydrochloric! Alpha antagonist Antihypertensor Norcyclobenzaprine Antiulcerative Trigonelline Antihyperglycaemic agent

Pyrazinamide Antibacterial Diclofenac sodium Cyclooxygenase inhibitor Antiinflammator

Ca++ channel blocker

Trimethadione Anticonvulsant voltage dependant ?

Calycanthine Calcium channel blocker

HMG CoA reductase

Lovastatin Antihypercholesterol inhibitor

Convolamine hydrochlori Cholinergic ligand Vasodilatator

Performs membrane

Nystatine Antifungal ionophores

Isocorydine (+) Ganglioblocker Hypotensor Budesonide Antiinflammator Xylazine Alpha-2 adrenergic agonist Antinociceptive

Bacterial transpeptidase

Imipenem Antibacterial inhibitor

Seneciphylline Antitumor activit

15- hydroxydehydrogenase

Sulfasalazine Treatment of ulcerativ inhibitor

Boldine Smooth muscle relaxant Choleretic

Beta2 adrenergic receptor

Bambuterol hydrochloride Bronchodilatato agonist

Estradiol-17 beta Estrogen Betamethasone Glucocorticoid

Dopamine beta hydroxylase

Fusaric acid Antibacterial inhibitor

Tubulin polymerisation

Colchicine Antiinflammator inhibitor

Gabazine Antagonist GABA

5-HT1 antagonist, D2

Metergoline Antiprolactin agonist, 5-HT2 antagonist

Ginkgolide A Cholinergic antagonist Alzheimer treatme Brinzolamide Carbonic anhydrase inhibitor Antiglaucoma dru Cyclobenzaprine hydrochloride Muscle relaxant Ambroxol hydrochloride Expectorant Carteolol hydrochloride Antihypertensor

Increase of glucose

penetration and use by cells,

Benfluorex hydrochloride Hypolipedimic decrease of triglyceride

intestinal absorption

Hydrocortisone base Glucocorticoid Bepridil hydrochloride Ca++ channel blocker Antianginal

Acetylcholine esterase

Hydroxytacrine maleate (R,S)

inhibitor

Meloxicam Cyclooxygenase inhibitor Anti-inflammator

Pilocarpine nitrate Cholinergic Antiglaucoma dru

Benzbromarone Uric acid transport inhibitor Coronarodilatato

Bacterial transpeptidase

Dicloxacillin sodium salt Antibacterial inhibitor

Glycocholic acid Detergent Anticholelithogen

Dopamine antagonist,

Scoulerine Antiemetic alphal antagonist

Thiostrepton Antibacterial

Inhibitor of glucose uptake

Ajmaline Antihypertensor by heart tissue

Glutamine synthetase

Methionine sulfoximine (L)

inhibitor

For inducing pulmo

Monocrotaline

diseases in rats

Tiabendazole Microtubule inhibitor Anthelmintic Piperlongumine Antifungal Rifampicin RNA polymerase inhibitor Antibacterial Hydrocotarnine hydrobromide Hemostatic Ethionamide Antibacterial (-)-Cinchonidine Antimalarial

Tenoxicam Cyclooxygenase inhibitor Antiinflammator Eburnamonine (-) Cerebral vasodilata Triflusal Cyclooxygenase inhibitor Antithrombotic

Cinchonine Heme polymerase inhibitor Antimalarial

Mesoridazine besylate D antagonist Antipsychotic

Canavanine sulfate monohydrate Nitric oxide inductible

Anticancer agent (L,+) synthetase inhibitor

Trolox Vitamin E analog

Harmaline hydrochloride Monoamine oxydase

Antihelminthic dihydrate inhibitor

Ketotifen fumarate HI antagonist Antihistaminic

Alizapride HCI Antiemetic

Debrisoquin sulfate Catecholamine depletor Antihypertensor

Lactobionic acid Antithrombonic

Dehydrofolate reductase

Amethopterin (R,S) Antineoplastic inhibitor

Microtubule

Lumicolchicine gamma

depolymerizating agent

5-HT antagonist, alpha

Methylergometrine maleate Oxytocic adrenergic agonist

Lysergol 5-HT antagonist Antipsychotic

5-HT autoreceptor

antagonist, 5-HTlc

Methiothepin maleate antagonist, 5-HT release

inhibitor electrical or K+

induced

Mebhydroline 1,5-

HI antagonist Antihistaminic naphtalenedisulfonate

1.2 1.0 1.1 1.0 Clofazimine Antileprosy

Ribosomal protein synthesis

0.8 1.0 0.3 mi 0.5 Meclocycline subsalicylate Antibacterial inhibitor

Phosphodiesterase inhibitor

1.0 1.0 0.9 1.0 1.0 1.1 Nafronyl oxalate ?, 5-HT antagonist, Vasodilatator bradykinine antagonist

1.2 1.2 2.1 0.9 1.9 1.2 Meclozine dihydrochloride Histamine antagonist Antiemetic

0.9 0.8 1.1 0.7 0.8 1.0 Bezafibrate Lipoprotein lipase activator Antihyperlipoprotein

0.9 1.0 1.3 0.8 1.2 1.1 Melatonin Melatonin receptor ligand Immunostimulan

Cell cycle blocker, apoptosis

1.0 1.0 1.4 0.9 0.9 1.1 Mimosine Anticancer agent inducer

1.0 1.1 1.4 1.0 1.0 1.2 Menadione Vitamin K3

1.0 1.1 1.2 1.0 1.0 0.9 Clebopride maleate Spasmolytic

1.0 1.0 1.4 1.0 1.2 1.0 Dinoprost trometamol Protaglandin agonist Smooth muscle activ

1.1 1.1 1.1 1.0 1.2 1.2 Pirenperone 5-HT2 antagonist

1.2 1.0 0.8 0.8 1.1 1.2 Harmalol hydrochloride dihydrate Vasorelaxant

Isoquinoline, 6,7-dimethoxy-l-

1.3 0.9 1.0 1.0 1.0 1.2 methyl-l,2,3,4-tetrahydro,

hydrochloride

Harmol hydrochloride

1.4 1.0 0.9 0.9 1.1 1.1 Liver conjugation probe Anxiolytic monohydrate

1.2 0.9 0.8 0.9 1.1 0.9 Phenacetin Cyclooxygenase inhibitor Analgesic

1.1 1.1 0.9 0.7 1.0 1.2 Harmine hydrochloride Topoisomerase II inhibitor Antibacterial

1.1 1.0 1.2 0.8 1.2 1.0 Atovaquone Antipneumocysti

0.8 1.0 m 1.2 Ellipticine Intercaling agent Anticancer 0.9 1.0 1.0 1.2 Methoxamine hydrochloride Alpha adrenergic agonist Antihypotensive 0.1 0.8 0.8 1.0 Chrysene-l,4-quinone

Blokage of the action of

1.2 0.9 1.1 1.1 (S(-(-)-Atenolol adrenergic mediators on Antihypertensor beta receptors

0.8 1.0 1.1 1.3 Demecarium bromide Cholinergic (ophtal 1.0 1.0 0.9 1.1 Piracetam Nootropic drug 1.0 0.8 1.0 1.2 Quipazine dimaleate salt 5-HT agonist, 5-HT3 ligand 1.1 1.1 1.1 0.9 Phenindione Antivitamin K Anticoagulant 1.1 1.0 1.0 1.0 Sparteine (-) Ganglioplegic Antiarrhythmic 1.2 0.8 0.9 1.0 Thiocolchicoside GABA agonist ? Muscle relaxant 1.1 1.0 0.9 1.0 Diflorasone Diacetate Anti-inflammatory (to 1.5 1.0 1.0 1.1 Clorsulon Anthelmintic 1.3 0.6 1.0 0.9 Harmane hydrochloride Imidazoline receptors ligand Vasorelaxant 1.4 1.1 2.9 1.3 Lidoflazine Ca++ channel activator Coronary vasodilata 0.9 0.8 1.3 1.1 Tropisetron HCI 5 HT3 antagonist Antiemetic 0.9 1.0 1.4 1.1 Betaxolol hydrochloride Beta adrenergic antagonist Antihypertensor 1.2 1 1.8 II Cefixime Antibacterial 0.6 1.0 1.3 1.1 Nicardipine hydrochloride Ca2+ channel antagonist Antihypertensor 1.2 1.0 1.3 1.2 Metrizamide Contrasting produ 0.6 0.9 1.4 1.1 Probucol Antihyperlipoprotein

Constituent of bacterial

Muramic acid, N-acetyl

peptidoglycan

DNA topoisomerase II

Mitoxantrone dihydrochloride Antineoplastic inhibitor

Neutral endopeptidase

Myricetin

inhibitor

GBR 12909 dihydrochloride Dopamine reuptake inhibitor Antidepressant Naringenine Antiestrogenic Carbetapentane citrate Sigmal receptor ligand Antitussive Naringin hydrate Anti-oxidant

Blocker of the apamin- sensitive small conductance

Dequalinium dichloride Antibacterial

Ca2+ activated K+ channel,

detergent

Neostigmine bromide Cholinesterase inhibitor Spinal analgesic

Cytochrome P450cl7

Ketoconazole inhibitor, sterol 14- Antifungal demethylase inhibitor

Niridazole Helminthic DNA damage Anthelmintic

Protein synthesis inhibitor

Fusidic acid sodium salt Antibacterial

GTPase coupled

Ceforanide Antibacterial

Cell membrane proteins

Ciclopirox ethanolamine Antifungal synthesis inhibitor

Benzodiazepine receptor

Methoxy-6-harmalan Psoriasis treatme ligand

Probenecid Uric acid uptake inhibitor Uricosuric

Potent inhibitor of

Stachydrine hydrochloride

tyrosinase

Betahistine mesylate HI agonist, H3 antagonist Vasodilatator Pyridoxine hydrochloride Vitamin

Ribosomal protein synthesis

Tobramycin Antibacterial inhibitor

Partial nicotinic receptor

Cytisine (-) Antiinflammator antagonist

Alkaline phosphatase

Tetramisole hydrochloride Antihelminthic inhibitor

Pseudopelletierine hydrochloride

Pregnenolone Estrogen Racecadotril Enkephalinase inhibitor Antidiarrhoeic Molsidomine NO° production ? Vasodilatator Folic acid Vitamin Be or M Chloroquine diphosphate Heme polymerase inhibitor Antipaludic Salsolinol hydrobromide Dopamine analog

Trimetazidine dihydrochloride Antianoxic Gramine Cholinesterase inhibitor Antibacterial Parthenolide MAP kinase inhibitor Antiinflammator Dimethisoquin hydrochloride Local anesthesic Terbutaline hemisulfate Beta-2 adrenergic agonist Bronchodilatato

Na+ K+ ion dependant

1.2 1.0 1.1 0.9 1.2 1.1 Strophantine octahydrate Cardiotonic

ATPase inhibitor

5-HT1, 5-HT2, 5-HT2A

1.3 1.0 1.0 1.0 1.2 1.0 Ketanserin tartrate hydrate Antihypertensor antagonist

Pantothenic acid calcium salt

1.1 1.1 1.1 1.0 1.1 1.0 Nutritional facto monohydrate

Acetylcholine stores

1.1 0.9 1.0 1.0 1.2 0.9 Hemicholinium bromide depleter, acetylcholine

uptake inhibitor

mm 1.2 2.5 2.0 1.2 Cefotetan Antibacterial

Ribosomal protein synthesis

1.2 0.9 2.5 1.0 1.1 1.0 Kanamycin A sulfate Antibacterial inhibitor

0.4 0.3 0.2 0.3 0.4 0.4 Piperine Enzyme inhibitor Antidiarrhoeic mm mm Ribosomal protein synthesis

1.0 1.3 Amikacin hydrate Antibacterial inhibitor

HI Antagonist,

1.4 1.0 1.1 1.1 1.2 1.1 Brompheniramine maleate Antihistaminic anticholinergic

Topoisomerase II inhib

1.2 1.1 1.4 1.0 1.2 1.0 Etoposide Antineoplastic kinase inhibitor

1.2 0.9 2.1 1.0 1.1 1.0 Primaquine diphosphate Heme polymerase inhibitor Antimalarial

Antioestrogen, gonad-

2.0 1.6 1.4 Clomiphene citrate (Z,E) Ovulation inducto stimulating agent

1.0 1.1 1.1 1.1 1.1 1.0 Progesterone Progestogen

Fumarate reductase

1.1 1.0 1.3 1.0 1.1 1.0 Oxantel pamoate inhibitor, neuromuscular Anthelmintic depolarizing agent

Felodipine L-type Ca2+ channels blocker Antihypertensor

Prochlorperazine dimaleate Dopamine antagonist Antiemetic

M ethoxy-8-psora I en Apoptosis inducer with UV Psoriasis treatme Hesperidin Anticancer Puromycin dihydrochloride Antimetabolite Antiprotozoal Hexetidine Detergent Antifungal Thiamine hydrochloride Vitamin

Monoamine B oxydase

Selegiline hydrochloride Antiparkinsonian inhibitor

Dipivefrin hydrochloride Antiglaucoma dru Pentamidine isethionate Antiparasitic

Neutral endopeptidase

Thiorphan Antinociceptive inhibitor

ATP-sensitive K+ ion

Tolazamide Antidiabetic channels blocker

Riboflavine Vitamin

Nifuroxazide Bacterial DNA damage Antibacterial

Hydroquinine hydrobromide

Preventor of muscular hydrate

Inosine monophosphate

Mycophenolic acid Antineoplastic dehydrogenase inhibitor

Epivincamine Antiaggregant

Ribosomal protein synthesis

Dirithromycin Antibacterial inhibitor

Retrorsine Antineoplastic

K+ channel voltage

Gliclazide Antidiabetic dependant inhibitor

Conessine Antiamebic DO 897/99 D3 antagonist

Acetylcholinesterase release

Protoveratrine A Antihypertenso stimulant

Prenylamine lactate Ca++ channel activator Vasodilatator

Butyrylcholinesterase

Solanine alpha Antifungal inhibitor

Phosphodiesterase III

Sulmazole Cardiotonic inhibitor

Althiazide Na+ CI- transport inhibitor Diuretic Epicatechin-(-) Antidiarrhoeic Isopyrin hydrochloride Cyclooxygenase inhibitor Antipyretic Flunisolide Glucocorticoid

Bacterial transpeptidase

Phenethicillin potassium salt Antibacterial inhibitor

N-Acetyl-DL-homocysteine

Disulfure bridge breaker Expectorant Thiolactone

Inhibitor of folic acid

Sulfamethoxypyridazine Antibacterial synthesis

Flurandrenolide Glucocorticoid Deferoxamine mesylate Iron chelating agent

Helveticoside Inhibitor of membrane ATP Cardiotonic

Alpha-adrenergic receptor

Mephentermine hemisulfate Antihypotensive agonist

Myosmine Cholinergic

Ergocryptine-alpha Vasoconstrictor Betonicine

Inhibitor of folic acid

Sulfadimethoxine Antibacterial biosynthesis

Antineoplastic adju Etanidazole DNA damage

(radiosensitizer)

Inhibitor of folic acid

Sulfanilamide Antibacterial biosynthesis

Ribosomal protein synth

Butirosin disulfate salt Antibacterial inhibitor

Balsalazide Sodium Anti-inflammator Carbinoxamine maleate salt Histamine antagonist Antihistaminic Niacin Antihyperlipoprotein Methazolamide Anhydrase carbonic inhibitor Diuretic Bemegride Respiratory stimul Pyrithyldione Sedative

Mainly used as a non-

Digoxigenin Diagnosis isotopic label for DNA

Ribosomal protein synthesis

Spectinomycin dihydrochloride Antibacterial inhibitor

0.5 0.8 0.5 0.7 0.5 Meglumine Expectorant 0.6 0.9 0.7 0.6 0.5 Piromidic acid Topoisomerase II inhibitor Antibacterial 0.6 0.8 0.5 0.6 0.5 Cantharidin Aphrodisiac noradrednaline and 5-HT

uptake inhibitor, alpha

0.6 0.9 0.5 0.6 0.6 Trimipramine maleate salt antagonist, anticholinergic,

Dopamine and Histamine

antagonist

0.5 0.9 0.2 0.6 1.1 Clioquinol Anti-infective 0.7 0.9 0.5 0.6 0.6 Chloropyramine hydrochloride HI antagonist Antihistaminic 0.5 0.7 0.4 0.4 0.4 Oxybenzone Ultraviolet scree

Monoamine oxidase

0.8 0.9 0.8 0.4 Furazolidone inhibitor and protozoal DNA Antiinfective damage

0.6 0.8 0.5 0.5 0.6 Promethazine hydrochloride Antihistaminic 0.6 1.1 0.5 0.5 0.5 Dichlorphenamide Carbonic anhydrase inhibitor

0.2 0.3 0.2 0.2 0.2 Chrysin 5-lipoxygenase inhibitor Antifungal

Ergosterol synthesis

0.8 0.7 0.9 2.6 Sulconazole nitrate Antifungal inhibition

0.5 0.9 0.5 0.5 0.6 Proxyphylline Vasodilator 0.5 1.4 0.5 0.7 0.7 Glimepiride Antidiabetic

Inhibitor of folic acid

0.6 0.9 0.7 0.6 1.0 Sulfaquinoxaline sodium salt Antibacterial synthesis

0.6 1.0 0.6 0.6 0.7 Picrotoxinin GABA channel blocker Analeptic 0.7 2.6 1.2 1.4 0.7 Streptozotocin Antineoplastic

Anticholinergic and

0.7 Mepenzolate bromide

muscarinic antagonist

7.0 Metoprolol-(+,-) (+)-tartrate salt Beta adrenergic antagonist

0.8 Benfotiamine Vitamin 0.8 Flumethasone Antiinflammator 0.8 Halcinonide Antiinflammator 0.7 Flecainide acetate Antiarrhythmic 1.0 Lanatoside C Na+ K+ ATPase inhibitor Cardiotonic

Bacterial transpeptidase

0.9 Cefazolin sodium salt Antibacterial inhibitor

Na+ channel blocker and

0.7 Benzamil hydrochloride blocker of Na+/Ca++

exchanger

Nucleotide transport

0.8 Atractyloside potassium salt Anticancer inhibitor

0.8 Suxibuzone Antiinflammator 0.7 Folinic acid calcium salt Antianemic 0.9 6-Furfurylaminopurine Plant growth acceler 0.7 Levonordefrin Adrenergic receptor agonist Vasoconstrictor 0.9 Avermectin Bl Ligand GABA receptors Antihelmetic 0.7 Ebselen Cyclooxygenase inhibitor Antiinflammator 1.1 Bergenin monohydrate Antiarrhythmic 1.1 3-Acetylcoumarin

Cromolyn disodium salt Antiasthmatic

Esculin Hydrate Skin protectant Bucladesine sodium salt Adenylate cyclase modulator Cardiotonic Felbinac Analgesic

Bacterial transpeptidase

Cefsulodin sodium salt Antibacterial inhibitor

Butylparaben Antifungal Fosfosal Cyclooxygenase inhibitor Analgesic Aminohippuric acid Diagnostic aid (renal fu

Suprofen Cyclooxygenase inhibitor Analgesic N-Acetyl-L-leucine Vertigo Catechin-(+,-) hydrate Antidiarrhoeic Pipemidic acid Antibacterial

Nadolol Adrenergic beta antagonist Antihypertensor Dioxybenzone Ultraviolet scree

Bacterial transpeptidase

Moxalactam disodium salt Antibacterial inhibitor

Adrenosterone Androgenic activi

Mastocytes degranulation

Aminophylline inhibitor and Vasodilatator benzodiazepines antagonist

Methylatropine nitrate Mydriatic Vitexin Antiviral

Coenzyme nicotinamide

Nadide

adenine dinucleotide

Gelsemine Tumour inhibito

Inhibitor of folic acid

Sulfamethizole Antibacterial synthesis

Solasodine Cytotoxic Medrysone Glucocorticoid Delcorine Nicotinic ligand Antiarrhythmic Flunixin meglumine Analgesic Evoxine Glycine receptor antagonist Sedative

Ribosomal protein synthesis

Spiramycin Antibacterial inhibitor

Nisoldipine Antihypertensiv Glycopyrrolate Antispasmodic Foliosidine Anticonvulsant

Bacterial transpeptidase

Cefamandole sodium salt Antibacterial inhibitor

Skimmianine 5- HT ligand Sedative

Performs membrane

onensin sodium salt Antibacterial ionophores

Anabasine Insecticide Isoetharine mesylate salt Beta adrenergic agonist Bronchodilator Tetrandrine Analgesic

Mevalonic-D, L acid lactone HMG CoA substrate

Bacterial transpeptidase

Azlocillin sodium salt Antibacterial inhibitor

Hymecromone Antispasmodic Clidinium bromide Anticholinergic Spasmolytic Caffeic acid Antineoplastic

Inhibitor of folic acid

Sulfamonomethoxine Antiseptic synthesis

Diloxanide furoate Antiamebic Benzthiazide Na+ CI- transport inhibitor Diuretic

Diagnostic aid (pitui

Metyrapone

function)

Trichlormethiazide Na+ CI- transport inhibitor Diuretic Urapidil hydrochloride Antihypertensor Oxalamine citrate salt Antiinflammator

Fluspirilen Antipsychotic Propantheline bromide Muscarinic antagonist Antiulcerative S-(+)-ibuprofen Analgesic

Membrane ionophores

Lasalocid sodium salt Antibacterial producer

Ethynodiol diacetate Progestogen Dimethadione Anticonvulsant Nabumetone Analgesic

Ethaverine hydrochloride Antispasmodic

Nisoxetine hydrochloride Inhibitor of noradrenaline

Dydrogesterone Progestogen

Treatment of benign pr

Terazosin hydrochloride Alpha adrenergic antagonist

hyperplasia

(dJ)-Tetrahydroberberine Sedative Phenazopyridine hydrochloride Analgesic Deltaline Nicotinic receptor ligand Antiarrhythmic

Ribosomal protein synthesis

Demeclocycline hydrochloride Antibacterial inhibitor

topoisomerase II inhibitor?

Graveoline Antihypotensive and CNS stimulant

Fenoprofen calcium salt dihydrate Cyclooxygenase inhibitor Antiinflammator Hippeastrine hydrobromide Hypotensor

Bacterial transpeptidase

Piperacillin sodium salt Antibacterial inhibitor

Beta-Escin Peripheral vascular dis Diethylstilbestrol Estrogen

Gossypol Ca2+ uptake inhibitor Local contracepti Chlorotrianisene Nuclear receptor ligand Non-steroidal estro Ricinine

Ribosomal protein synthesis

Ribostamycin sulfate salt Antibacterial inhibitor

Nicotinic receptor antagonist

and Ganglioblocker

Cholinergic agonist

Muscle relaxant

Nicotinic receptor antagonist Spasmolytic Na+ channel blocker Local anesthesic

Sympathomimetic (

Bacterial transpeptidase

Antibacterial inhibitor

Depigmentor Antineoplastic

Ligand and potent inhibitor

of carbonic anhydrase B

DNA topoisomerase IV

Antibacterial inhibitor

Estrogen Keratolytic

CCKa ligand

Cyclooxygenase inhibitor Analgesic

Antihypertensor

Mucus stimulating synthesis Antiulcerative

Anti-inflammator

Diagnostic aid (radiop locetamic acid

medium)

Nimodipine Vasodilator Ganciclovir Antimetabolite Antiviral Bacitracin Antibacterial Ethopropazine hydrochloride Anticholinergic Antiparkinsonian

Potent inhibitor of vesicular

L(-)-vesamicol hydrochloride

acetylcholine storage

Austricine hydrate Antiatheroscleroti

Butamben Na+ channel blocker Anesthetic beta- Belladonnine Muscarinic receptor and

dichloroethylate nicotinic receptor lignad

Sulfa pyridine Antibacterial

Nicotinic acetylcholine

Pempidine tartrate Antihypertensor receptor antagonist

Meclofenoxate hydrochloride Acetylcholine precursor Cerebral stimulan

Model for hepatitis and

Heliotrine

cirrhosis in liver

Furaltadone hydrochloride Bacterial DNA damage Antibacterial Nitrarine dihydrochloride Hypotensor Ethoxyquin Antifungal

Inhibitor of protein

Lycorine hydrochloride Antitumoral translation

Tinidazole Antiprotozoal

Protozoal and bacterial DNA

damage

1.0 1.2 0.9 1.0 1.1 Karakoline Inhibitor of ACE Hypotensor 1.0 1.2 0.7 1.0 1.1 Guanadrel sulfate Antihypertensor 1.0 1.1 1.0 1.0 1.1 Estropipate Menopause 1.1 1.5 0.7 1.0 1.0 Vidarabine Adenosine antimetabolite Antiviral 1.0 1.2 1.0 1.1 1.1 Ungerine nitrate Enhancer of analgesics Sedative

Inhibitor of folic acid

1.0 1.2 1.0 2.3 1.1 Sulfameter Antibacterial synthesis

1.0 1.4 1.0 1.0 1.1 Napelline Anticholinergic agent Antiarrhythmic 1.1 1.2 1.1 1.0 1.2 Isopropamide iodide Anticholinergic

1.0 1.1 0.8 1.1 1.2 Securinine CNS stimulant 1.0 1.2 0.9 1.0 1.2 Nizatidine H2 antagonist Antiulcerative 1.0 1.1 0.9 1.1 1.2 Trimeprazine tartrate Histamine antagonist Antipruritic 1.0 1.1 1.0 1.0 1.3 Thioperamide maleate H3 antagonist Antiemetic

Nafcillin sodium salt Bacterial transpeptidase

1.0 mi 1.0 1.5 1.3 Antibacterial monohydrate inhibitor

beta 1-Adrenoceptor

1.0 1.1 0.8 1.0 1.2 Xamoterol hemifumarate Cardiotonic selective partial agonist

1.1 1.2 0.9 1.1 1.2 Procyclidine hydrochloride Muscarinic antagonist Antiparkinsonian 1.0 1.3 1.0 1.2 1.2 Rolipram Nootropic drug 1.1 1.3 0.9 1.2 1.1 Amiprilose hydrochloride Immunomodulat

MM 1.5 Thonzonium bromide Detergent

Ethynylestradiol 3-methyl ether Estrogen

Idazoxan hydrochloride Alpha2 agonist Antiparkinsonian (-) -Levobunolol hydrochloride renergic beta antagonist Antiglaucoma dru Quinapril HCI Angiotensin

Diagnostic aid (radiop lodixanol

medium)

Nilutamide Antineoplastic

Ribosomal protein synthesis

Rolitetracycline Antibacterial inhibitor

Ketorolac tromethamine Analgesic Equilin Estrogen

Protriptyline hydrochloride Antidepressant

Fillalbin

Alclometasone dipropionate Corticoi^'de Anti-inflammator Citalopram Hydrobromide 5HT uptake inhibitor

Inhibitor of T and B cell

Leflunomide Immunosuppressi proliferation

Dopamine receptor

Promazine hydrochloride Antipsychotic antagonist

Norgestrel-(-)-D Progestogen Oral contraceptiv

Inhibitor of folic acid

Sulfamerazine Antibacterial synthesis

Fluocinonide Antiinflammator

Inhibitor of glutathione

Acacetin reductase and of Antitumor agent topoisomerase I

Inhibitor of folic acid

Sulfamethazine sodium salt Antibacterial synthesis

Ethotoin Anticonvulsant

Guaifenesin Expectorant

3-alpha-Hydroxy-5-beta- androstan-17-one

Alexidine dihydrochloride Detergent Antibacterial

Tetrahydrozoline hydrochloride Adrenergic Vasoconstrictor

Cytochrome P450 mono-

Proadifen hydrochloride oxygenases inhibitor, Na+ Local anesthesic channel blocker

Hexestrol Nuclear receptor ligand Estrogen antineopla Zomepirac sodium salt Cyclooxygenase inhibitor

Bacterial transpeptidase

Cefmetazole sodium salt Antibacterial inhibitor

Cinoxacin Topoisomerase II inhibitor Antibacterial Paroxetine Hydrochloride 5-HT uptake inhibitor

Propofol Anesthetic (intraven

Ribosomal protein synthesis

Doxycycline hyclate Antibacterial inhibitor

S(-)Eticlopride hydrochloride

Liothyronine Thyroid hormone Thyroidic drug

Primidone Anticonvulsant

Ribosomal protein synthesis

Roxithromycin Antibacterial inhibitor

Flucytosine Antifungal Beclomethasone dipropionate Antiinflammator (-)-MK 801 hydrogen maleate NMDA antagonist Anticonvulsant Tolmetin sodium salt dihydrate Cyclooxygenase inhibitor Antiinflammator Bephenium hydroxynaphthoate Antihelmintic (+) -Levobunolol hydrochloride Beta adrenergic antagonist Antiglaucoma dru Dehydroisoandosterone 3-acetate Menopausal syndro

Alpha 1 adrenergic

Doxazosin mesylate Antihypertensor antagonist

Inhibitor of L-aromatic In combination with le Benserazide hydrochloride

amino acid decarboxylase as antiparkinsonia

HMG CoA reductase

Fluvastatin sodium salt Anti- hyperlipoprotei inhibitor

Diagnostic aid (radiop lodipamide

medium-cholecystogr

Methylhydantoin-5-(L)

Esculetin Antifungal

Trihexyphenidyl-D,L

Anticholinergic Antiparkinsonian Hydrochloride

Clobetasol propionate Glucocorticoid

Succinylsulfathiazole Antibacterial

Inhibitor of folic acid

Antiviral

Antipyretic r

Antihyperlipoprotein

Antiemetic

Diuretic

Antibacterial

Anticoagulant

Antihistaminic

Antihyperthyroid

Antihistaminic

Antibacterial

Local anesthesic

Pigmentation age (photosensitizer)

Spasmolytic

Cardiotonic

Antibacterial

Anticonvulsant

Raloxifene hydrochloride

Bretylium tosylate Antiadrenergic Etidronic acid, disodium salt Calcium regulato Pralidoxime chloride

Methylhydantoin-5-(D)

Nonselective alpha-

Phenoxybenzamine hydrochloride Antihypertensor adrenergic blockade

HMG-CoA reductase

Simvastatin Antihyperlipidemi inhibitor

beta 2 adrenergic

Salmeterol Bronchodilator agonist

Azacytidine-5 Antimetabolite Antineoplastic Altretamine

Ribosomal protein synthesis

Paromomycin sulfate Antiamebic inhibitor

Prazosin hydrochloride Antihypertensor

Acetaminophen Cyclooxygenase inhibitor Antipyretic Timolol maleate salt Antiglaucoma dru

Inhibitor of folic acid

Phthalylsulfathiazole Antibacterial synthesis

beta-3 Adrenergic receptor

(+,-)-Octopamine hydrochloride Adrenergic agonist

Luteolin Expectorant (±)-Nipecotic acid

Activates GABA-like ion

channels

Inhibitor of folic acid

1.1 1.2 1.1 1.6 Sulfabenzamide Antibacterial synthesis

1.0 1.0 1.0 1.1 1.7 ( ) -Naproxen sodium salt Cyclooxygenase inhibitor Antiinflammator 1.0 1.2 0.9 1.2 1.6 Benzocaine Na+ channel blocker Anesthetic 0.8 1.4 0.8 1.1 1.6 Propidium iodide Detergent Antibacterial 1.0 1.4 1.1 1.1 1.4 Dipyrone Cyclooxygenase inhibitor Antipyretic 1.1 1.3 1.2 1.4 2.0 Cloperastine hydrochloride Histamine antagonist Antitussive 1.0 1.2 1.0 1.1 1.8 Isosorbide dinitrate Nitric oxide (NO) donor Antianginal 1.1 1.3 0.9 1.1 1.6 Eucatropine hydrochloride Anticholinergic

Inhibitor of folic acid

1.0 1.1 1.1 2.1 1.5 Sulfachloropyridazine Antibacterial synthesis

1.0 1.5 1.1 1.1 1.5 Isocarboxazid Antidepressant 1.0 1.3 1.1 1.1 1.5 Pramoxine hydrochloride Local anesthesic

Anticholelithogenic,

1.2 1.1 0.8 1.0 1.7 Lithocholic acid Cholagogue gastrointestinal agent

1.1 1.0 0.9 1.2 1.8 Finasteride Antialopecia age 1.0 1.2 1.0 1.1 1.8 Methotrimeprazine maleat salt Analgesic 1.0 1.2 1.0 1.3 1.7 Fluorometholone Glucocorticoid 1.3 0.4 0.9 II Dienestrol Nuclear receptor ligand Non-steroidal estro

Bacterial transpeptidase

1.0 1.1 2.0 Cephalothin sodium salt Antibacterial inhibitor

1.1 1.2 0.9 1.1 1.6 Pridinol methanesulfonate salt Anticholinergic Antiparkinsonia

Bacterial transpeptidase

Cefuroxime sodium salt Antibacterial inhibitor

Amrinone TNF production inhibitor

Diagnostic aid (radio lopamidol

medium)

Crotamiton Scabicide lopromide Contrast molecule

Propranolol hydrochloride Beta blocking agent Antiarrhythmic

Mastocyte degranulation

Theophylline monohydrate Bronchodilatato inhibitor in vitro

(R)-(+)-Atenolol

Adenosine receptor

Theobromine Cardiotonic antagonist

Tyloxapol Mucolytic Reserpine

Florfenicol Antibacterial Arcaine sulfate Lowers blood sugar

Megestrol acetate Progestogen selective muscarinic

Scopolamine hydrochloride Antiemetic antagonist

Deoxycorticosterone Corticoide Antiinflammator

Diagnostic aid (radio loversol

medium)

Urosiol

Capsaicin Vanilloid receptor ligand Topical analgesic

Proparacaine hydrochloride Local anesthesic (V

Cholinergic agonist,

Carbachol Myotic

Cholinesterase inhibitor ?

Aminocaproic acid Antiallergic

Bittering agent to prevent

Denatonium benzoate

poisoning

Etomidate Hypnotic Scopoletin Eicosanoid release inhibitor Antispasmodic Tridihexethyl chloride Antispasmodic Enilconazole Antifungal Penbutolol sulfate Antiarrhythmic Methacycline hydrochloride Antibacterial Prednicarbate Glucocorticoid (topi Gibberellic acid Plant growth regula Sertaconazole nitrate Antibacterial Sotalol hydrochloride Beta-blocker

Repaglinide Antidiabetic 6-Hydroxytropinone

Piretanide Antihypertensor Decamethonium bromide Muscle relaxant (skel Piperacetazine Antipsychotic

0.4 3-Acetamidocoumarin Antiinflammator

0.5 Oxyphenbutazone Inhibition of cyclo-oxygenase Anti-inflammator

Control of enteric infe

0.5 Roxarsone To improve growth an efficiency (VET)

0.4 Quinethazone Diuretic 0.8 Remoxipride Hydrochloride Dopaminergic antagonist Antipsychotic 0.7 Moricizine hydrochloride Antiarrhythmic 0.8 THIP Hydrochloride GABAergic Agonist

■i t. lopanoic acid Contrast molecule

Highly selective reversible

Anti depressant

0.7 Pirlindole mesylate inhibitor of monoamine

Seizures oxidase type A

0.8 Pivmecillinam hydrochloride Antibacterial 0.9 Pronethalol hydrochloride Antihypertensor 0.7 Levopropoxyphene napsylate Antitussive 0.9 Naftopidil dihydrochloride Alphal agonist Antihypertensor 0.7 Piperidolate hydrochloride Antispasmodic 0.7 Tracazolate hydrochloride GABA receptor ligand

0.7 Trifluridine Antiviral (ophthalm

Phosphodiesterase III & IV

0.9 Zardaverine

inhibitor

Blocking beta-adrenergic

0.9 Oxprenolol hydrochloride Antiarrhythmic receptors

Memantine Hydrochloride NMDA receptor antagonist Altzheimer diseas

Antagonist of 5- hydroxytryptamine

Ondansetron Hydrochloride Antiemetic

(serotonin) subtype 3 (5-HT

3 ) receptors

Thromboxane synthase

Ozagrel hydrochloride Antianginal inhibitor

Propoxycaine hydrochloride Local anesthesic Piribedil hydrochloride Dopaminergic agonist Vasodilator (periph Oxaprozin Analgesic Nitrocaramiphen hydrochloride Muscarinic antago Phensuximide Anticonvulsant Nandrolone Anabolic

Low-osmolar, ionic contrast Diagnostic aid (radiop loxaglic acid

medium medium)

Histamine H2 receptor

Dimaprit dihydrochloride

agonist

Naftifine hydrochloride Antifungal Reserpinic acid hydrochloride

Meprylcaine hydrochloride Local anesthesic Beta-sistosterol Anticholesteremi

Milrinone Cardiotonic Proscillaridin A Glucoside cardioto Methantheline bromide Antispasmodic

Sanguinarine Potent cytotoxic Antimicrobial dru

Ticarcillin sodium Antibacterial Harpagoside Antiinflammator Thiethylperazine malate adrenergic antagonist Antiemetic Asiaticoside Antibiotic

Anti-inflammator

Mesalamine

(gastrointestinal)

Betulin Antineoplastic alpha-Santonin Anthelmintic Gliquidone Antidiabetic Imidurea

Pizotifen malate Antihistaminic Lansoprazole Antiulcerative Ribavirin Antiviral Bethanechol chloride Cholinergic

Cyclopenthiazide Diuretic Cyproterone acetate Antiandrogen Fluvoxamine maleate Antidepressant

Beta-adrenergic receptor

(R)-Propranolol hydrochloride Antihypertensor blocking agent

Prophylactic use in m Cefalonium

and dry udder ther

Ciprofibrate Antihyperlipoprotein

Fluticasone propionate Anti-inflammator Tropine

Blocking of Dl and D2

Zuclopenthixol hydrochloride Schizophrenia dopaminergic receptors

Benzylpenicillin sodium Antibacterial

Inhibition of dihydrofolate

Proguanil hydrochloride

reductase

Chlorambucil Antineoplastic Lymecycline Antibiotic Methiazole

In combination with alf

Alfadolone acetate

as anesthesic (intrave

Beta-adrenergic blocking

(S)-propranolol hydrochloride Antihypertensor agent

In combination with alf

Alfaxalone acetate as anesthe

(intravenous)

Anti-acetylcholinesterase

(-)-Eseroline fumarate salt activity & opiate agonist Potent analgesic activity

Azapropazone Cyclooxygenase inhibitor Analgesic Condelphine

Meptazinol hydrochloride Analgesic (narcoti Leucomisine

Apramycin Antibacterial

Dubinidine

Epitiostanol Antineoplastic

Competitive inhibition of

D-cycloserine alanin racenase and D-alanin

synthase (bacterial cell wall)

Fursultiamine Hydrochloride Altzheimer's disea 2-Chloropyrazine Cardiotonic Gabexate mesilate Anticoagulant (+,-)-Synephrine Hypertensive

Pivampicillin Antibacterial (S)-(-)-Cycloserine Tuberculosis Talampicillin hydrochloride Antibacterial Homosalate Tool for uv screen

Flucloxacillin sodium Antibacterial Spaglumic acid NMDA receptor ligands

Trapidil Vasodilator (corona Ranolazine Antianginal Deptropine citrate Chronic bronchiti

D2-dopamine

(-)-Quinpirole hydrochloride receptor agonist, some

selectivity for D3 sites

Sertraline 5-HT uptake inhibitor

Inhibition of

1.0 1.3 Sulfadoxine Antibacterial dihydropteroate synthase

1.0 0.8 Ethamsylate Retinopathy 1.1 1.3 Cyclopentolate hydrochloride Mydriatic 1.1 1.8 Moxonidine Imidazoline receptor ligand Antihypertensor 1.0 1.4 Estriol Estrogen 1.1 1.5 Etilefrine hydrochloride Adrenergic agonist Antihypotensive

Cardiovascular drug (Sic

1.1 2.3 (-(-Isoproterenol hydrochloride Beta Adrenergic Agonist

Syndrome)

1.1 1.4 Alprostadil Vasodilator

Topoisomerase I inhibitor,

0.6 1.6 Kaempferol tyrosin kinase, xanthin Anti-inflammator oxidase

1.1 1.5 Tribenoside Antihaemorrhoidic 1.2 1.4 Nialamide Antidepressant 1.0 4 ¾ Rimexolone Corticoid Anti-inflammatory (l

Dietary factor for cont

1.1 1.6 Vitamin K2

blood pressure

1.1 1.4 Isradipine Antianginal 1.2 1.3 Perindopril Antihypertensiv 1.1 1.4 Tiletamine hydrochloride Anesthetic 1.0 1.3 Fexofenadine HCI Antihistaminic 1.1 1.2 Isometheptene mucate Adrenergic Sympathomimetic (

Quinic acid

Nifurtimox Antiprotozoal (Trypano Clonixin Lysinate Analgesic Letrozole Antineoplastic

Treatment of age-rel

Verteporfin

macular degenerati

Tyrosinase inhibitor and

Arbutin Melanin biosynthesis Antibacterial inhibitor

Meropenem Antibacterial Tocainide hydrochloride Antiarrhythmic Ramipril Converting enzyme inhibitor Antihypertensor Benzathine benzylpenicillin Antibacterial Mephenytoin Anticonvulsant Risperidone 5-HT2 antagonist Antipsychotic

Inhibition of RNA-

Rifabutin Antibacterial polymerase DNA-dependent

Torsemide Diuretic Parbendazole Anthelmintic (VE Halofantrine hydrochloride Antimalarial Mecamylamine hydrochloride Antihypertensor Articaine hydrochloride Local Anesthesic Procarbazine hydrochloride DNA depolymerization

1.1 1.2 1.0 1.1 1.1 1.1 Nomegestrol acetate Progestin 1.0 1.0 1.3 0.9 1.1 0.9 Viomycin sulfate Antibacterial

Competitive antagonist of

1.1 1.0 1.1 1.0 1.1 0.9 Pancuronium bromide autonomic cholinergic Neuromuscular blockin receptors

1.3 1.2 0.9 1.1 1.5 1.0 Saquinavir mesylate Protease inhibitor

1.1 1.0 1.1 1.0 1.1 0.9 Molindone hydrochloride Antipsychotic 1.1 1.1 1.3 1.7 1.6 1.0 Ronidazole Antimicrobial 1.4 1.0 1.2 1.0 1.4 1.0 Alcuronium chloride Neuromuscular blockin 1.1 2.3 1.0 1.1 1.2 0.9 Dorzolamide hydrochloride Antiglaucoma dru

Reverse transcriptase

1.3 1.1 1.1 1.1 1.2 1.0 Zalcitabine Antiviral (HIV) inhibitor

1.1 1.0 1.2 1.0 1.2 1.0 Azaperone Tranquilizer 1.2 1.1 1.2 1.2 1.4 1.1 Methyldopate hydrochloride Antihypertensor

I 1.0 II 1 I Cefepime hydrochloride Antibacterial

1.1 1.0 1.1 1.1 1.2 1.0 Levocabastine hydrochloride Antihistaminic 1.0 1.1 1.1 1.0 1.2 1.0 Clocortolone pivalate Glucocorticoid

Anthelmintic (Nemat

0.8 1.5 1.4 0.8 0.9 0.7 Pyrvinium pamoate

enterobiasis)

II: 2.9 Nadifloxacin Antibacterial

Consistent with expectations, a detailed assessment of the hits revealed that AK screening enriches for the identification of bactericidal compounds (Table 3). More specifically, 100% of the antibiotics that were identified to be active against P. aeruginosa or K. pneumoniae represented bactericidal agents. Similarly, 96%>, 94%>, and 80%> of the antibiotics that were active against E. coli, E. cloacae, and E.faecium, respectively, were bactericidal antibiotics. The assay identified 71% and 64% bactericidal antibiotics for A. baumannii and S. aureus.

Among the bactericidal antibiotics detected, β-lactam, cephalosporin, polymyxin, and fluoroquinolone antibiotics were active against Gram-negative pathogens (E. coli and the ESKAPE pathogens A. baumannii, P. aeruginosa, and K. pneumoniae), and penicillins, cephalosporins, quinolines, glycopeptides, and carbapenems were active toward S. aureus (see Table 4). It was also determined that clofazimine, which was initially developed as an antimycobacterial agent and was recently shown to exhibit bactericidal activity toward S. aureus, was indeed active against S. aureus. Bacteriostatic compounds, such as clindamycin, and macrolides, such as erythromycin, were not identified as being active toward any of the species tested. Interestingly, many tetracyclines were identified as killing S. aureus strain USA300-0114 and A. baumannii strain 98-37-09. In addition, the library contains membrane- active antiseptics, such as chlorhexidine, and with the exception of E. cloacae, these were also strong hits against all organisms tested. Screening results also revealed that the E.

faecium strain tested proved to be resistant to most classes of antibiotics within the Prestwick library by MIC testing (Table 2), and this was also observed in the AK assay, indicating that the assay exhibits a low false-positive rate. Interestingly, the assay also detected 38 drugs with no known antimicrobial properties (denoted as "other" in Table 3) that were active against each organism; see Table 4 for a complete list of these compounds.

Antimicrobial activities of nonantibiotic drugs that induce AK release- Recently, the exploration of the so-called "off-target" activities of previously developed drugs has emerged as an approach to identify chemical scaffolds that could be exploited for new therapeutic indications. In that regard, Prestwick library screening results revealed that 4% to 56% (depending on the organism) of the members that generated significant AK signal were compounds with no previously reported antimicrobial activity (see Table 4). To determine whether the 38 nonantibiotics identified in the screen have potential for repurposing as anti-infectives, 4 nonantibiotic drugs that were commercially available were further evaluated by two secondary assays. First, dose-response assays were performed to validate that they induced AK activity, and all were reconfirmed. Second, the in vitro antimicrobial activity for each drug was measured by standard MIC testing. With the exception of one drug/organism pair, all drugs exhibited in vitro activity toward each organism. More specifically, tamoxifen, suloctidil, and clomiphene exhibited MICs of 8 μg mf¹ against E.faecium. S. aureus and A. baumannii were susceptible to terfenadine (16^g mf¹ MIC and 64^g mF¹ MIC, respectively). Suloctidil was also detected to be active against P. aeruginosa by both primary and confirmatory AK screens, but the drug did not elicit an antimicrobial response by MIC measures. Some strains of P. aeruginosa secrete AK at high cell density, and it is possible that suloctidil may trigger a similar response.

Terfenadine and tamoxifen, which exhibited antimicrobial properties toward planktonic S. aureus and E.faecium cells, respectively, were characterized further.

Terfenadine was evaluated for activity against S. aureus biofilms and small-colony variants using the AK assays described above. Treatment of 48-h S. aureus strain UAMS-1 biofilms with lOx-MIC terfenadine elicited a modest 2.7-fold increase in AK release (see Fig. 6A); plating-based viability assays determined that this correlated with a 1.1-log reduction in biofilm cell viability, which was comparable to the activity of ciprofloxacin under the same assay conditions (see Fig. 4A). Similarly, treatment of S. aureus small-colony-variant UAMS-1112 cells with 10^x MIC terfenadine elicited a 3.3-fold increase in AK signal in comparison to that for mock-treated cells (see Fig. 6A). Conventional MIC testing subsequently verified that terfenadine is active against UAMS-1112 at 2 μg mf¹.

Next, the in vivo antimicrobial properties of terfenadine and tamoxifen were evaluated using a Galleria mellonella model of S. aureus and E. faecium infection, respectively. For terfenadine, groups of larvae (n = 45) were infected with 1.0 x 10⁶S. aureus USA300-1114 cells. Worms were then treated at 2 h and 24 h with a range of terfenadine concentrations (20 to 160 mg kg ¹), vehicle (DMSO; negative control), or 20 mg kg ¹ vancomycin (positive control), and larval survival was assessed 48 h postinoculation. For tamoxifen studies, experiments were performed exactly as described above except that larvae were inoculated with 1.4 x 10ⁱ E.faecium strain 824-05 cells and larvae were treated with either 80 or 160 mg kg ¹ tamoxifen. Terfenadine-treated larvae did not reproducibly exhibit increased survival relative to vehicle-treated larvae. However, tamoxifen treatment of E. faecium- fQctcd larvae resulted in a dose-dependent increase in survival. As shown in Fig. 6B, treatment with 80 or 160 mg kg ¹ resulted in a 4.2-fold- or 7-fold-higher survival, respectively, than was found with vehicle-treated controls. Taken together, these results indicate that terfenadine exhibits in vitro activity toward S. aureus planktonic, SCV, and biofilm populations and that tamoxifen is active toward planktonic E. faecium and against the organism in a simple animal model of infection. As discussed below, these data show that tamoxifen and terfenadine represent attractive new chemical scaffolds for antibacterial optimization.

As set forth herein, Prestwick library screening revealed that each bacterial species studied was susceptible to members of the library with no previously reported antimicrobial activity. The antimicrobial properties of two of these drugs was studied in more detail:

terfenadine and tamoxifen. Terfenadine is a nonsedating antihistamine based on a 4- substituted piperidine scaffold. Based on further studies, it was determined that terfenadine acts as a topoisomerase inhibitor and is structurally similar to certain topoisomerase inhibitors, including for example novel bacterial topoisomerase II inhibitor (NBTI). Based on the structural similarity between terfenadine and these molecules, it is likely that terfenadine is acting as a DNA gyrase and topoisomerase inhibitor. More specifically, terfenadine and derivatives thereof act as topoisomerase 4 inhibitors. Furthermore, it was found that terfenadine has activity against S. aureus small-colony variants and biofilms, properties not previously reported for this scaffold.

A screen of E. faecium identified two structurally related nonsteroid estrogen receptor antagonists, tamoxifen and clomiphene. Tamoxifen is used to treat some forms of estrogen- receptor-positive breast cancer, while clomiphene is used in fertility treatment regimens. These two compounds are members of the triarylethylene class of estrogen receptors.

Since the number of agents with activity toward enterococcus is quite limited, tamoxifen's in vivo activity was investigated using a Galleria model of enterococcus infection. Although it was not as active as vancomycin, tamoxifen did impart a survival benefit, indicating that it has in vivo antimicrobial activity. High-dose tamoxifen therapy has been used in experimental treatment of refractory human cancers, and dosing results in micromolar serum concentrations of tamoxifen corresponding to the levels of drug associated with antienterococcal activity observed in the studies provided herein. In addition to providing a powerful new HTS approach to identify antimicrobial agents active against planktonic bacteria, an AK assay that is easily amenable to screening bacteria in disease states that cannot be readily screened via conventional approaches is provided herein. In that regard, the AK assay is capable of measuring the killing properties of bactericidal agents administered to biofilms formed by both Gram-negative and Gram- positive representatives of the ESKAPE pathogens. Similarly, the AK assay can detect the bactericidal properties of antibiotics toward a phenotypically stable S. aureus small-colony variant. These features can be exploited to develop corresponding high-throughput screening assays for the identification of agents that kill established biofilms and small-colony variants or provide powerful secondary assays aimed at characterizing the potential antimicrobial properties of molecules of interest.

EXAMPLE II

Antimicrobial properties of terfenadine and terfenadine derivaties.

Terfenadine derivatives were synthesized as described below.

General Method A

KSC-335-007

l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one. To a vial was added the diphenyl(piperidin-4-yl)methanol (1.190 g, 4.45 mmol), l-(4-(tert- butyl)phenyl)-4-chlorobutan-l-one (1.012 g, 4.24 mmol), sodium bicarbonate (0.427 g, 5.09 mmol) with water:2-butanone (18 mL, 1 :5). The reaction stirred at 85 °C for 16 h and was then cooled to rt and water (50 mL) was added. The reaction was extracted with EtOAc (3 x 50 mL). The EtOAc layer was dried with MgS0₄, filtered, concentrated and purified by MPLC (20 min, 0 - 10% MeOH:DCM) to produce pure l-(4-(tert-butyl)phenyl)-4-(4- (hydroxydiphenylmethyl) piperidin-l-yl)butan-l-one (1.37 g, 2.92 mmol, 69%> yield) as a colorless oil. 1H NMR (500 MHz, CDC1₃): δ 7.90 (d, J= 8.4 Hz, 2H), 7.49 - 7.45 (m, 6H), 7.31 - 7.25 (m, 4H), 7.20 - 7.14 (m, 2H), 2.97 - 2.92 (m, 4H), 2.45 - 2.36 (m, 3H), 2.09 (br s, 1H), 2.00 - 1.87 (m, 4H), 1.49 - 1.32 (m, 4H), 1.34 (s, 9H). ¹³C NMR (125 MHz, CDC1₃) δ 199.7, 156.5, 146.0, 134.5, 128.1, 128.0, 126.4, 125.7, 125.4, 79.4, 57.9, 43.9, 43.4, 44.1, 36.2, 35.0, 31.0, 26.2, 21.9. LCMS Retention time: 4.207 min. LCMS purity 99.5%. HRMS (ESI): m/z calcd for C₃₂H₃₉N0₂ [M+H]⁺ 470.2981, found 470.3054.

KSC-335-005

4-(4-(Hydroxydiphenylmethyl)piperidin-l-yl)-l-(4-isopropylphenyl)butan-l-one.

Method A: diphenyl(piperidin-4-yl)methanol (0.620 g, 2.317 mmol), 4-chloro-l-(4- isopropylphenyl)butan-l-one (0.496 g, 2.207 mmol), sodium bicarbonate (0.222 g, 2.65

mmol) with water (3 mL) and 2-butanone (15 mL, Ratio: 5). to produce pure

(hydroxydiphenylmethyl)piperidin-l-yl)-l-(4-isopropylphenyl)butan-l-one (0.481 g, 1.056 mmol, 48% yield) as a colorless oil. 1H NMR (500 MHz, CDC1₃): δ 7.89 (d, J = 8.4 Hz, 2H), 7.49 - 7.45 (m, 4H), 7.31 - 7.26 (m, 6H), 7.20 - 7.14 (m, 2H), 2.98 - 2.91 (m, 4H), 2.45 - 2.35 (m, lH), 2.38 (t, J = 6.8 Hz, 2H), 2.08 (br s, 1H), 1.99 - 1.89 (m, 4H), 1.61 (br s, 1H), 1.48 - 1.33 (m, 4H), 1.27 (d, J= 6.8 Hz, 6H).

KSC-335-006

l-(4-(tert-butyl)phenyl)-5-(4-(hydroxydiphenylmethyl)piperidin-l-yl)pentan-l-one.

Method A: diphenyl(piperidin-4-yl)methanol (0.544 g, 2.035 mmol), l-(4-(tert-butyl)phenyl)- 5-chloropentan-l-one (0.490 g, 1.938 mmol), sodium bicarbonate (0.195 g, 2.326 mmol) with water:2-butanone (18 mL, 1 :5) to produce pure l-(4-(tert-butyl)phenyl)-5-(4- (hydroxydiphenylmethyl)piperidin-l-yl)pentan-l-one (0.600 g, 1.240 mmol, 64.0 % yield) as a colorless oil. 1H NMR (500 MHz, CDC1₃): δ 7.89 (d, J = 8.4 Hz, 2H), 7.49 - 7.45 (m, 6H), 7.32 - 7.26 (m, 4H), 7.20 - 7.14 (m, 2H), 2.98 - 2.92 (m, 4H), 2.48 - 2.32 (m, 3H), 2.14 (br s, 1H), 1.99 - 1.89 (m, 2H), 1.77 - 1.62 (m, 2H), 1.60 - 1.42 (m, 6H), 1.34 (s, 9H).

KSC-335-008

l-(4-(tert-butyl)phenyl)-3-(4-(hydroxydiphenylmethyl)piperidin-l-yl)propan-l-one.

Method A: diphenyl(piperidin-4-yl)methanol (0.543 g, 2.032 mmol), l-(4-(tert- butyl)phenyl)-3-chloropropan-l-one (0.435 g, 1.936 mmol), sodium bicarbonate (0.195 g, 2.323 mmol) with water:2-butanone (18 mL, 1 :5) to produce pure l-(4-(tert-butyl)phenyl)-3- (4-(hydroxydiphenyl methyl)piperidin-l-yl)propan-l-one (0.838 g, 1.84 mmol, 95% yield) as a colorless oil. 1H NMR (500 MHz, CDC1₃): δ 7.89 (d, J = 8.4 Hz, 2H), 7.50 - 7.45 (m, 6H), 7.32 - 7.25 (m, 4H), 7.21 - 7.14 (m, 2H), 3.15 (t, J = 7.1 Hz, 2H), 3.01 - 2.96 (m, 2H), 2.81 (t, J = 7.1 Hz, 2H), 2.49 - 2.42 (m, 1H), 2.15 - 2.04 (m, 3H), 1.56 - 1.46 (m, 4H), 1.33 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 198.9, 171.1 , 156.8, 145.8, 134.3, 128.2, 128.0, 126.5, 125.8, 125.5, 79.4, 60.4, 54.2, 53.4, 44.0, 36.3, 35.1 , 31.1 , 26.4, 21.1 , 14.2. LCMS Retention time: 3.968 min. LCMS purity 98.1%. HRMS (ESI): m/z calcd for C₃iH₃₇N0₂ [M+H]⁺ 456.2824, found 456.2897.

KSC-335-009

l-(4-chlorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one. Method A: diphenyl(piperidin-4-yl)methanol (0.626 g, 2.341 mmol), 4-chloro-l-(4-chlorophenyl) butan- 1-one (0.484 g, 2.229 mmol), sodium bicarbonate (0.225 g, 2.68 mmol) with water:2- butanone (18 mL, 1 :5) to produce pure l-(4-chlorophenyl)-4-(4-

(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one (0.394 g, 0.879 mmol, 39.4 % yield) as a colorless oil. 1H NMR (400 MHz, CDC1₃): δ 7.90 (d, J = 8.4 Hz, 2H), 7.48 - 7.44 (m, 4H), 7.42 (d, J = 8.6 Hz, 2H), 7.31 - 7.26 (m, 4H), 7.20 - 7.15 (m, 2H), 3.96 - 2.89 (m, 4H), 2.45 - 2.34 (m, 3H), 2.08 (br s, 1H), 1.99 - 1.87 (m, 4H), 1.50 - 1.30 (m, 4H).

General Method B KSC-335-014, Terfenadine l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol. To a vial was added the l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l- yl)butan-l-one (KSC-335-007 (0.198 g, 0.422 mmol) and MeOH (2 mL). The sodium borohydride (0.032 g, 0.844 mmol) was then added and the reaction stirred at rt for 3 h. The reaction was concentrated to dryness, water (5 mL) was added and a white precipitate formed. The precipitate was filtered out and then dissolved in DCM (10 mL), dried with MgS0₄, filtered and concentrated to produce pure l-(4-(tert-butyl)phenyl)-4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol (0.151 g, 0.320 mmol, 76% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.52 - 7.46 (m, 4H), 7.33 - 7.25 (m, 8H), 7.21 - 7.15 (m, 2H), 4.61 - 4.56 (m, 1H), 3.16 - 3.1 1 (br m, 1H), 3.00 - 2.94 (m, 1H), 2.51 - 2.34 (m, 4H), 2.10 - 1.88 (m, 4H), 1.83 - 1.75 (m, 1H), 1.70 - 1.45 (m, 6H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.4, 146.1 , 146.0, 142.7, 128.2, 128.1 , 126.4, 126.3, 125.7, 125.6, 125.3, 125.0, 79.2, 73.4, 58.9, 54.7, 53.3, 44.2, 39.7, 34.4, 31.4, 26.0, 25.9, 24.1. LCMS Retention time: 4.137 min. LCMS purity 97.5%. HRMS (ESI): m/z calcd for C₃₂H₄iN0₂ [M+H]⁺ 472.3144, found 472.3219.

KSC-335-012

4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(4-isopropylphenyl)butan-l-ol. Method B : 4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)- 1 -(4-isopropylphenyl)butan- 1 -one (KSC- 335-005) (0.154 g, 0.338 mmol) and MeOH (2 mL) sodium borohydride (0.026 g, 0.676 mmol) to produce pure 4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(4- isopropylphenyl)butan-l-ol (0.146 g, 0.319 mmol, 94% yield) as on oil. 1H NMR (400 MHz, CDCI₃): δ 7.52 - 7.46 (m, 4H), 7.32 - 7.23 (m, 6H), 7.20 - 7.14 (m, 4H), 4.61 - 4.58 (m, 1H), 3.18 - 3.11 (br m, 1H), 3.00 - 2.94 (br m, 1H), 2.87 (septet, J = 6.9 Hz, 1H), 2.50 - 2.33 (m, 3H), 2.29 (br s, 1H), 2.10 - 1.89 (m, 3H), 1.82 - 1.44 (m, 8H), 1.22 (d, J= 6.9 Hz, 6H). ¹³C NMR (125 MHz, CDC1₃): δ 147.2, 146.1, 146.0, 128.2, 128.1, 126.5, 126.4, 126.1, 125.7, 125.7, 79.2, 73.5, 58.9, 54.7, 53.3, 44.2, 39.9, 33.7, 26.0, 25.9, 24.2, 24.1, 24.0. LCMS Retention time: 4.056 min. LCMS purity 98.6%. HRMS (ESI): m/z calcd for C₃₁H₃₉NO₂ [M+H]⁺ 458.2986, found 458.3062.

KSC-335-013

l-(4-(tert-butyl)phenyl)-5-(4-(hydroxydiphenylmethyl)piperidin-l-yl)pentan-l-ol.

Method B : 1 -(4-(tert-butyl)phenyl)-5 -(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)pentan- 1 - one (KSC-335-006) (0.204 g, 0.422 mmol) and MeOH (2 mL) and sodium borohydride (0.032 g, 0.844 mmol) to produce pure l-(4-(tert-butyl)phenyl)-5-(4-

(hydroxydiphenylmethyl)piperidin-l-yl)pentan-l-ol (0.156 g, 0.321 mmol, 76 %> yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.50 - 7.45 (m, 4H), 7.36 (d, J= 8.4 Hz, 2H), 7.31 - 7.24 (m, 6H), 7.19 - 7.14 (m, 2H), 4.65 - 4.60 (m, 1H), 2.98 - 2.90 (br m, 1H), 2.48 - 2.38 (m, lH), 2.30 (t, J= 7.2 Hz, 2H), 2.23 (br s, 1H), 1.97 - 1.87 (m, 2H), 1.84 - 1.60 (m, 4H), 1.55 - 1.35 (m, 8H), 1.31 (s, 9H). ¹³C NMR (125 MHz, CDCI₃): δ 150.3, 146.0, 142.0, 128.1, 126.4, 125.8, 125.5, 125.3, 79.4, 74.1, 58.4, 54.1, 54.0, 44.2, 38.5, 34.5, 31.4, 31.3, 26.5, 26.3, 26.2, 23.7. LCMS Retention time: 4.254 min. LCMS purity 96.4%. HRMS (ESI): m/z calcd for C₃₃H₄₃NO₂ [M+H]⁺ 486.3294, found 486.3370.

KSC-335-015

l-(4-chlorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol. Method B: 1 -(4-chlorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan-l -one (KSC-335-009) (0.156 g, 0.348 mmol) and MeOH (2 mL) sodium borohydride (0.026 g, 0.696 mmol) to produce pure 1 -(4-chlorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -ol (0.1 18 g, 0.262 mmol, 75% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.32 - 7.24 (m, 8H), 7.20 - 7.15 (m, 2H), 4.61 - 4.56 (m, 1H), 3.16 - 3.10 (br m, 1H), 2.97 - 2.91 (m, 1H), 2.51 - 2.33 (m, 4H), 2.12 - 1.88 (m, 3H), 1.83 - 1.75 (m, 1H), 1.78 - 1.46 (m, 8H). ¹³C NMR (125 MHz, CDC1₃): δ 146.0, 145.9, 144.5, 139.1 , 128.19, 128.18, 128.1 , 127.1 , 126.49, 126.45, 125.62, 125.57, 79.2, 72.9, 58.8, 54.7, 53.2, 44.2, 40.2, 26.0, 25.9, 24.1. LCMS Retention time: 3.845 min. LCMS purity 98.4%. HRMS (ESI): m/z calcd for C₂₈H₃₂C1N0₂ [M+H]⁺ 450.2122, found 450.2194.

KSC-335-016 l-(4-(tert-butyl)phenyl)-3-(4-(hydroxydiphenylmethyl)piperidin-l-yl)propan-l-ol.

Method B : 1 -(4-(tert-butyl)phenyl)-3 -(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)propan- 1 - one (KSC-335-008) (0.1 16 g, 0.255 mmol) and MeOH (2 mL) and sodium borohydride (0.019 g, 0.509 mmol) to produce pure l-(4-(tert-butyl)phenyl)-3-(4-

(hydroxydiphenylmethyl)piperidin-l-yl)propan-l-ol (0.106 g, 0.232 mmol, 91%> yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.49 - 7.44 (m, 4H), 7.36 - 7.25 (m, 8H), 7.22 - 7.16 (m, 2H), 6.72 (br s, 1H), 4.90 - 4.85 (m, 1H), 3.21 - 3.15 (br m, 1H), 3.1 1 - 3.05 (br m, 1H), 2.70 - 2.62 (m, 1H), 2.57 - 2.40 (m, 2H), 2.14 - 2.06 (m, 2H), 1.91 - 1.79 (m, 3H), 1.57 - 1.45 (m, 4H), 1.31 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.6, 145.8, 145.7, 141.9, 128.2, 128.1 , 126.6, 126.5, 125.8, 125.7, 125.2, 125.0, 79.4, 75.3, 57.3, 55.2, 53.2, 44.1 , 34.4, 33.7, 31.4, 26.7, 26.4. LCMS Retention time: 4.006 min. LCMS purity 97.7%. HRMS (ESI): m/z calcd for C₃iH₃₉N0₂ [M+H]⁺ 458.2991 , found 458.3066.

KSC-335-018

4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-phenylbutan-l-one. Method A:

diphenyl(piperidin-4-yl)methanol (0.524 g, 1.961 mmol), 4-chloro-l-phenylbutan-l-one (0.300 ml, 1.868 mmol), sodium bicarbonate (0.188 g, 2.241 mmol) with water:2-butanone (18 mL, 1 :5) to produce pure 4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-phenylbutan-l- one (0.177 g, 0.428 mmol, 23% yield) as a colorless oil. 1H NMR (500 MHz, CDC1₃): δ 7.98

- 7.94 (m, 2H), 7.57 - 7.52 (m, 1H), 7.49 - 7.42 (m, 6H), 7.32 - 7.26 (m, 4H), 7.20 - 7.15 (m, 2H), 3.00 - 2.90 (m, 4H), 2.46 - 2.35 (m, 3H), 2.09 (br s, 1H), 2.00 - 1.87 (m, 4H), 1.50

- 1.33 (m, 4H).

KSC-335-020

l-(tert-butyl)-4-(4-chlorobutyl)benzene. To a vial was added the l-(4-(tert-butyl)phenyl)- 4-chlorobutan-l-one (0.266 g, 1.114 mmol) and triethylsilane (0.518 g, 0.712 ml, 4.46 mmol) with TFA (4 mL). The reaction stirred at 75 °C for 18 h, was cooled to rt and concentrated in vacuo. The residue was then dissolved in DCM (5 mL) and washed with water (4 mL). The DCM layer was collected and washed with water (1 x 5 mL), dried with MgS04, filtered and adsorbed to silica and purified by Teledyne ISCO Combiflash chromatography (20 min, 0 - 40% EtOAc:Hex) and fractions 4 and 5 were collected to produce pure l-(tert-butyl)-4-(4- chlorobutyl)benzene (0.155 g, 0.690 mmol, 61.9 % yield) as an oil. 1H NMR (400 MHz, CDC1₃): δ 7.32 (d, J= 7.6 Hz, 2H), 7.12 (d, J= 7.6 Hz, 2H), 3.56 (t, J= 6.5 Hz, 2H), 2.62 (t, J= 7.5 Hz, 2H), 1.87 - 1.74 (m, 4H), 1.32 (s, 9H).

KSC-335-021

4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-phenylbutan-l-ol. Method B: 4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)-l-phenylbutan-l-one (KSC-335-018) (0.065 g, 0.157 mmol) and MeOH (2 mL) and sodium borohydride (0.012 g, 0.314 mmol) to produce pure 4-(4-(hydroxydiphenylmethyl) piperidin-l-yl)-l-phenylbutan-l-ol (0.048 g, 0.116 mmol, 73% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.37 - 7.33 (m, 2H), 7.32 - 7.26 (m, 5H), 7.23 - 7.14 (m, 3H), 4.65 - 4.61 (m, 1H), 3.15 (d, J= 11.7 Hz, 1H), 2.96 (d, J= 11.7 Hz, 1H), 2.52 - 2.31 (m, 4H), 2.11 - 1.90 (m, 3H), 1.84 - 1.74 (m, 2H), 1.70 - 1.44 (m, 7H). ¹³C NMR (125 MHz, CDC1₃): δ 146.0, 145.9, 145.8, 128.2, 128.1, 128.0, 126.6, 126.5, 126.4, 125.6, 125.6, 79.2, 73.6, 58.9, 54.7, 53.3, 44.2, 40.1, 26.0, 25.9, 24.1. LCMS Retention time: 3.711 min. LCMS purity 99.8%. HRMS (ESI): m/z calcd for C₂₈H₃₃N0₂ [M+H]⁺ 416.2517, found 416.2592.

KSC-335-022

Methyl 2-methyl-2-(4-(4-((methylsulfonyl)oxy)but-l-yn-l-yl)phenyl)propanoate. To a vial was added the methyl 2-(4-(4-hydroxybut-l-yn-l-yl)phenyl)-2-methylpropanoate (0.051 g, 0.207 mmol) and dry DCM (2 mL). The methanesulfonyl chloride (0.047 g, 0.032 mL, 0.414 mmol) and pyridine (0.147 g, 0.151 mL, 1.864 mmol) were each added and the reaction and stirred at rt for 16 h. The reaction was then diluted with DCM (5 mL) and washed with 1%> w/v sulfuric acid in water (3 x 7 mL), saturated NaHC03 (7 mL) and brine (7 mL). The organic layer was dried with MgS04, filtered and concentrated to produce methyl 2-methyl-2-(4-(4-((methylsulfonyl) oxy)but-l-yn-l-yl)phenyl)propanoate (0.063 g,

0.194 mmol, 94 % yield). 1H NMR (400 MHz, CDC1₃): δ 7.35 (d, J= 8.6 Hz, 2H), 7.26 (d, J = 8.6 Hz, 2H), 4.38 (t, J= 6.8 Hz, 2H), 3.64 (s, 3H), 3.06 (s, 3H), 2.87 (t, J= 6.8 Hz, 2H), 1.56 (s, 6H).

KSC-335-023

4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(4-methoxyphenyl)butan-l-one. Method A: diphenyl(piperidin-4-yl)methanol (0.490 g, 1.832 mmol), 4-chloro-l-(4- methoxyphenyl)butan-l-one (0.371 g, 1.744 mmol), sodium bicarbonate (0.176 g, 2.093 mmol) with water (3 mL) and 2-butanone (15 mL) to produce pure 4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)-l-(4-methoxyphenyl)butan-l-one (0.282 g, 0.636 mmol, 36% yield) as a colorless oil. 1H NMR (400 MHz, CDC1₃): δ 7.94 (d, J= 9.0 Hz, 2H), 7.49 - 7.45 (m, 4H), 7.31 - 7.26 (m, 4H), 7.17 (tt, Ji = 7.3 Hz, J₂ = 1.3 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 3.87 (s, 3H), 2.97 (br s, 1H), 2.93 (t, J= 7.3 Hz, 2H), 2.48 - 2.35 (m, 3H), 2.02 - 1.88 (m, 4H), 1.63 - 1.40 (m, 4H).

KSC-335-024

l-(4-Fluorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one. Method A: diphenyl(piperidin-4-yl)methanol (0.385 g, 1.439 mmol), 4-chloro-l-(4- fluorophenyl)butan-l-one (0.275 g, 1.371 mmol), sodium bicarbonate (0.138 g, 1.645 mmol) with water (3 mL) and 2-butanone (15 mL) to produce pure 1 -(4-fluorophenyl)-4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one (0.174 g, 0.403 mmol, 29% yield) as a colorless oil. 1H NMR (400 MHz, CDC1₃): δ 8.00 - 7.96 (m, 2H), 7.48 - 7.44 (m, 4H), 7.31 - 7.26 (m, 4H), 7.20 - 7.15 (m, 2H), 7.14 - 7.08 (m, 2H), 2.97 - 2.94 (m, 4H), 2.46 - 2.36 (m, 3H), 2.15 (br s, 1H), 2.01 - 1.88 (m, 4H), 1.52 - 1.35 (m, 4H).

KSC-335-025

l-(4-Bromophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one. Method A: diphenyl(piperidin-4-yl)methanol (0.439 g, 1.642 mmol), l-(4-bromophenyl)-4- chlorobutan-l-one (0.409 g, 1.564 mmol), sodium bicarbonate (0.158 g, 1.877 mmol) with water (3 mL) and 2-butanone (15 mL) to produce pure l-(4-bromophenyl)-4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)butan-l-one (0.245 g, 0.498 mmol, 32% yield) as a colorless oil. 1H NMR (400 MHz, CDC1₃): δ 7.83 - 7.79 (m, 2H), 7.60 - 7.56 (m, 2H), 7.48 - 7.45 (m, 4H), 7.31 - 7.26 (m, 4H), 7.20 - 7.15 (m, 2H), 2.95 - 2.90 (m, 4H), 2.45 - 2.35 (m, 3H), 2.17 (br s, 1H), 2.00 - 1.88 (m, 4H), 1.51 - 1.33 (m, 4H).

KSC-335-030

4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(4-methoxyphenyl)butan-l-ol. Method B: 4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)- 1 -(4-methoxyphenyl)butan- 1 -one (KSC-335- 023) (0.113 g, 0.255 mmol) and MeOH (2 mL) and sodium borohydride (0.019 g, 0.509 mmol) to produce pure 4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(4- methoxyphenyl)butan-l-ol (0.062 g, 0.139 mmol, 55% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.32 - 7.24 (m, 6H), 7.20 - 7.15 (m, 2H), 6.84 (d, J= 7.0 Hz, 2H), 4.60 - 4.56 (m, 1H), 3.78 (s, 3H), 3.14 (d, J= 11.7 Hz, 1H), 2.95 (d, J= 11.7 Hz, 1H), 2.50 - 2.35 (m, 4H), 2.10 - 2.02 (m, 1H), 1.99 - 1.86 (m, 2H), 1.80 - 1.73 (m, 1H), 1.69 - 1.45 (m, 7H). ¹³C NMR (125 MHz, CDC1₃): δ 158.3, 146.1, 146.0, 138.1, 128.1, 128.1, 126.7, 126.4, 126.4, 125.7, 125.6, 113.5, 79.2, 73.2, 58.9, 55.2, 54.7, 53.2, 44.2, 40.0, 26.0, 25.9, 24.1. LCMS Retention time: 3.620 min. LCMS purity 98.3%. HRMS (ESI): m/z calcd for C₂₉H₃₅N0₃ [M+H]⁺ 446.2652, found 446.2728.

KSC-335-031

l-(4-fluorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol. Method B: 1- (4-fluorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -one (KSC-335-024) (0.064 g, 0.148 mmol) and MeOH (2 mL) and sodium borohydride (0.011 g, 0.297 mmol) to produce pure 1 -(4-fluorophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -ol (0.059 g, 0.136 mmol, 92% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.33 - 7.25 (m, 6H), 7.20 - 7.14 (m, 2H), 6.96 (t, J= 8.8 Hz, 2H), 4.61 - 4.57 (m, 1H), 3.14 (d, J= 11.7 Hz, 1H), 2.94 (d, J= 11.7 Hz, 1H), 2.51 - 2.35 (m, 4H), 2.13 - 2.02 (m, 1H), 2.02 - 1.86 (m, 2H), 1.78 - 1.45 (m, 7H). ¹³C NMR (125 MHz, CDC1₃): δ 162.6, 160.7, 146.0, 145.9, 141.7, 141.6, 128.2, 128.1, 127.2, 127.1, 126.5, 126.4, 125.6, 125.5, 114.9, 114.7, 79.2, 73.0, 58.8, 54.8, 53.1, 44.2, 40.3, 30.9, 26.0, 25.9, 24.1. LCMS Retention time: 3.691 min. LCMS purity 98.5%. HRMS (ESI): m/z calcd for C₂8H₃₂FN0₂ [M+H]⁺ 434.2406, found 434.2482.

KSC-335-032

l-(4-bromophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol. Method B: 1 -(4-bromophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -one (KSC-335-025) (0.083 g, 0.169 mmol) and MeOH (2 mL) and sodium borohydride (0.013 g, 0.337 mmol) to produce pure 1 -(4-bromophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -ol (0.053 g, 0.107 mmol, 63.6 % yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.41 (d, J= 6.8 Hz, 2H), 7.32 - 7.26 (m, 4H), 7.22 (d, J= 6.6 Hz, 2H), 7.20 - 7.15 (m, 2H), 4.60 - 4.56 (m, 1H), 3.14 (d, J= 11.7 Hz, 1H), 2.93 (d, J= 11.7 Hz, 1H), 2.50 - 2.42 (m, 1H), 2.41 - 2.34 (m, 2H), 2.11 - 2.05 (m, 1H), 2.01 - 1.95 (m, 1H), 1.94 - 1.86 (m, 1H), 1.78 - 1.45 (m, 8H). ¹³C NMR (125 MHz, CDC1₃): δ 146.0, 145.9, 145.1, 131.1, 128.2, 128.1, 127.5, 126.5, 126.4, 125.6, 125.6, 120.2, 79.2, 72.9, 58.8, 54.7, 53.2, 44.1, 40.1, 26.0, 25.9, 24.1. LCMS Retention time: 3.902 min. LCMS purity 99.1%. HRMS (ESI): m/z calcd for C₂₈H₃₂BrN0₂ [M+H]⁺ 494.1614, found 494.1673.

KSC-335-041

(l-(4-(4-(tert-butyl)phenyl)butyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.153 g, 0.571 mmol), l-(tert-butyl)-4-(4- chlorobutyl)benzene (KSC-335-020) (0.154 g, 0.685 mmol) and potassium carbonate (0.473 g, 3.43 mmol) in acetonitrile. The reaction stirred overnight at 85 °C and for 18 h and was then cooled to rt and filtered. The filtrate was then diluted with brine and extracted with diethyl ether (3 x 15 mL). The ether layers were combined, dried with MgS04, filtered and purified by reverse-phase MPLC (20 min, 10 - 100% MeCN:H20) to produce pure (l-(4-(4- (tert-butyl)phenyl)butyl)piperidin-4-yl)diphenylmethanol (0.180 g, 0.395 mmol, 69% yield) as an oil. 1H NMR (400 MHz, CDC1₃): δ 7.38 - 7.34 (m, 4H), 7.19 - 7.14 (m, 6H), 7.07 - 7.02 (m, 2H), 6.99 - 6.95 (m, 2H), 2.86 - 2.80 (m, 2H), 2.45 (t, J= 7.3 Hz, 2H), 2.35 - 2.26 (m, 1H), 2.22 - 2.17 (m, 2H), 1.84 - 1.76 (m, 3H), 1.52 - 1.30 (m, 8H), 1.18 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 171.1, 148.4, 146.0, 139.4, 128.1, 128.0, 126.4, 125.8, 125.1, 79.5, 60.4, 58.8, 54.1, 44.2, 35.2, 34.3, 31.4, 29.5, 26.8, 26.4, 21.0, 14.2. LCMS Retention time: 3.928 min. LCMS purity 97.8%. HRMS (ESI): m/z calcd for C₃₂H₄iNO [M+H]⁺ 456.3188, found 456.3261.

KSC-335-053

Methyl 4-(l,3-dithian-2-yl)benzoate. A flame dried vial was evaporated 3 times with argon and methyl 4-formylbenzoate (0.50 g, 3.05 mmol) was added with anhydrous DCM (8.70 mL) followed by 1,3-propanedithiol (0.339 mL, 3.35 mmol). The reaction began to stir at rt for 1.5 h. The reaction was then cooled to 0 °C and the BF3 Et₂ (0.425 ml, 3.35 mmol) was added dropwise. The reaction was then warmed slowly to rt and stirred overnight. The reaction was then diluted with DCM (15 mL) and quenched with saturated NaHC0₃ (15 mL) and the DCM layer was dried with MgS0₄, filtered and adsorbed to silica and purified by MPLC (20 min, 0 - 35% EtOAc:Hex) to produce methyl 4-(l,3-dithian-2-yl)benzoate (0.669 g, 2.63 mmol, 86% yield). 1H NMR (400 MHz, CDC1₃): δ 8.01 (d, J= 8.4 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 3.91 (s, 3H), 3.12 - 3.03 (m, 2H), 2.96 - 2.90 (m, 2H), 2.23 - 2.16 (m, 1H), 2.01 - 1.89 (m, 1H), 1.55 (s, 2H).

KSC-335-054

Methyl 2-(4-(4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)but-l-yn-l-yl)phenyl)-2- methylpropanoate. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.528 g, 1.974 mmol), methyl 2-methyl-2-(4-(4-((methylsulfonyl)oxy)but-l-yn-l- yl)phenyl)propanoate (KSC-335-022) (0.582 g, 1.794 mmol) and potassium carbonate (0.744 g, 5.38 mmol) with acetonitrile (10 mL). The reaction stirred at 70 °C for 18 h and cooled to rt and filtered to remove the potassium carbonate. The filtrate was adsorbed to silica gel and purified by reverse-phase MPLC (20 min, 10 - 100% MeCN:H20) to produce pure methyl 2- (4-(4-(4-(hydroxydiphenylmethyl) piperidin- 1 -yl)but- 1 -yn- 1 -yl)phenyl)-2-methylpropanoate (0.434 g, 0.876 mmol, 49% yield). 1H NMR (400 MHz, CDC1₃): δ 7.49 - 7.46 (m, 4H), 7.34 - 7.27 (m, 6H), 7.25 - 7.15 (m, 4H), 3.64 (s, 3H), 3.03 - 2.97 (m, 2H), 2.68 - 2.63 (m, 2H), 2.59 - 2.54 (m, 2H), 2.48 - 2.40 (m, 1H), 2.14 - 2.06 (m, 2H), 1.63 (br s, 1H), 1.55 (s, 6H), 1.55 - 1.45 (m, 4H).

KSC-335-056

Methyl 4-(2-(3-chloropropyl)-l,3-dithian-2-yl)benzoate. To a dry vial was added the methyl 4-(l,3-dithian-2-yl)benzoate (KSC-335-053) (0.256 g, 1.01 mmol) and this was evacuated with argon 3 times. The dry THF (7 mL) was added and the reaction was cooled to -78 °C at and the NaHMDS (1.258 mL, 1.258 mmol) was added. After 30 minutes the 1- chloro-3-iodopropane (0.531 mL, 5.03 mmol) was added. The reaction was then allowed to warm to rt overnight. The mixture was quenched with the addition of saturated NH₄C1 (10 mL) at and diluted with EtOAc (15 mL) and shaken. The EtOAc layer was collected, dried with MgS0₄, filtered and adsorbed to silica and purified by MPLC (20 min, - 25% EtOAc:Hex) to produce pure methyl 4-(2-(3-chloropropyl)-l ,3-dithian-2-yl)benzoate (0.102 g, 0.308 mmol, 31% yield). ). 1H NMR (400 MHz, CDC1₃): δ 8.07 - 8.03 (m, 2H), 8.01 -

7.98 (m, 2H), 3.93 (s, 3H), 3.41 (t, J = 6.4 Hz, 2H), 2.74 - 2.62 (m, 4H), 2.19 - 2.13 (m, 2H),

1.99 - 1.92 (m, 2H), 1.78 - 1.70 (m, 2H).

KSC-335-059

(S)-l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-ol. To a flame-dried vial was added dry THF (2 mL) and then cooled to 0 °C. The 1.0 M i?-5,5-Diphenyl-2-methyl-3,4-propano-l ,3,2- oxazaborlidine (0.105 mL, 0.105 mmol) in THF was added followed by the 2.0 M borane- methyl sulfide complex (0.654 mL, 1.309 mmol) in THF. The reaction began to stir at 0 °C for 30 minutes. To another flame-dried vial was added the l-(4-(tert-butyl)phenyl)-4- chlorobutan-l-one (0.250 g, 1.047 mmol) and this was evacuated with argon 3 times then dissolved in dry THF (5 mL) and the oxazaborlidine solution was added dropwise at 0 °C and the reaction was allowed to warm to rt stirred for 2 h. The reaction was quenched with MeOH (10 mL) extracted with EtOAc (20 mL) then was washed with 1.0 M HC1 (3 x 25 mL). The EtOAc layer was dried with MgS04, filtered and concentrated to produce pure

(S)-l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-ol (0.248 g, 1.030 mmol, 98% yield).

[alpha]25₅₈₉ = -24.0° (c = 10 in CHCI3). 1H NMR (400 MHz, CDC1₃): δ 7.38 (d, J= 8.4 Hz, 2H), 7.28 (d, J= 8.4, 2H), 4.71 - 4.67 (m, 1H), 3.61 - 3.53 (m, 2H), 1.98 - 1.79 (m, 4H), 1.32 (s, 9H).

KSC-335-060

(R)-l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-ol. Prepared the same as KSC-335-059 with 1.0 M (5)-5,5-diphenyl-2-methyl-3,4-propano-l ,3,2-oxazaborlidine (0.105 ml, 0.105 mmol), 2.0 M Borane -methyl sulfide complex (0.654 ml, 1.309 mmol) and l-(4-(tert-butyl)phenyl)- 4-chlorobutan-l-one (0.250 g, 1.047 mmol) to produce pure (R)-l-(4-(tert-butyl)phenyl)-4- chlorobutan-l-ol (0.216 g, 0.897 mmol, 86% yield). [alpha]25₅₈₉ = +23. Γ (c = 10 in CHCI₃). 1H NMR (400 MHz, CDC1₃): δ 7.38 (d, J= 8.4 Hz, 2H), 7.28 (d, J

- 4.67 (m, 1H), 3.61 - 3.53 (m, 2H), 1.98 - 1.79 (m, 4H), 1.32 (s, 9H).

KSC-335-061

Methyl 4-(4-chlorobutanoyl)benzoate. To a vial was added the methyl 4-(2-(3- chloropropyl)-l,3-dithian-2-yl)benzoate (KSC-335-056) (0.102 g, 0.308 mmol) and acetonitrile (1.5 mL) with water (0.2 mL). The (bis(trifluoroacetoxy)iodo)benzene (0.199 g, 0.462 mmol) was then added and the reaction stirred at rt for 1 h. The reaction was quenched with saturated NaHC0₃ (7 mL) then diluted with EtOAc (10 mL) and extracted. The EtOAc was collected and washed with water (2 x 8 mL) and then dried with MgS0₄, filtered and adsorbed to silica and purified by MPLC (20 min, 0 - 25% EtOAc:hex to produce pure methyl 4-(4-chlorobutanoyl)benzoate (0.0512 g, 0.213 mmol, 69% yield). 1H NMR (400 MHz, CDCI3): δ 8.11 (d, J= 8.00 Hz, 2H), 8.00 (d, J= 8.6 Hz, 2H), 3.93 (s, 3H), 3.67 (t, J = 6.1 Hz, 2H), 3.19 (t, J = 6.1 Hz, 2H), 2.22 (quintet, J= 6.3 Hz, 2H).

(S)-l-(4-(tert-butyl)phenyl)-4-chlorobutyl acetate. To a vial was added the (S)-l-(4-(tert- butyl)phenyl)-4-chlorobutan-l-ol (KSC-335-059) (0.248 g, 1.030 mmol) and diethyl ether (5.15 ml). The TEA (0.215 mL, 1.545 mmol) was added followed by the acetyl chloride (0.073 mL, 1.030 mmol) and the reaction began to stir at rt for 2 h. A white precipiate formed immediately. Water (5 mL) was added the reaction after 2 h and the ether layer was extracted. The aqueous was extracted again with more ether and the combined organics were dried with MgS0₄, filtered and concentrated to produce pure (S)-l-(4-(tert-butyl)phenyl)-4- chlorobutyl acetate (0.250 g, 0.884 mmol, 86 % yield). [alpha]25₅₈₉ = -42.11, (c = 10, CH₂C1₂). 1H NMR (400 MHz, CDC1₃): δ 7.36 (d, J= 8.4 Hz, 2H), 7.25 (d, J= 8.4 Hz, 2H), 5.78 - 5.73 (m, 1H), 3.53 (t, J= 6.4 Hz, 2H), 2.06 (s, 3H), 2.02 - 1.68 (m, 4H), 1.31 (s, 9H).

KSC-336-063

(R)-l-(4-(tert-butyl)phenyl)-4-chlorobutyl acetate. Prepared the same as KSC-335-062 with (R)-l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-ol (KSC-335-060) (0.216 g, 0.897 mmol) and diethyl ether (4.5 mL), TEA (0.188 ml, 1.346 mmol) and acetyl chloride (0.064 ml, 0.897 mmol) to produce pure (R)-l-(4-(tert-butyl)phenyl)-4-chlorobutyl acetate (0.249 g, 0.880 mmol, 98% yield). [alpha]25₅₈₉ = +54.57, (c = 10, CH₂C1₂). 1H NMR (400 MHz, CDC1₃): δ 7.36 (d, J= 8.4 Hz, 2H), 7.25 (d, J= 8.4 Hz, 2H), 5.78 - 5.73 (m, 1H), 3.53 (t, J= 6.4 Hz, 2H), 2.06 (s, 3H), 2.02 - 1.68 (m, 4H), 1.31 (s, 9H).

KSC-335-064

(S)-l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl acetate.

To a vial was added the diphenyl(piperidin-4-yl)methanol (0.154 g, 0.578 mmol), (S)-l-(4- (tert-butyl)phenyl)-4-chlorobutyl acetate (0.196 g, 0.693 mmol) (KSC-335-062) and potassium carbonate (0.319 g, 2.310 mmol) in acetonitrile ( 10 mL). The reaction stirred overnight at 70 °C for 18 h and was then cooled to rt and filtered. The filtrate was then diluted with DCM and washed with water (10 mL) and brine (10 mL). The DCM layers were combined, dried with MgS0₄, filtered and purified by reverse-phase MPLC (20 min, 10 - 100% MeCN: water) to produce pure (S)-l-(4-(tert-butyl)phenyl)-4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)butyl acetate (0.135 g, 0.263 mmol, 46%> yield) as a brown oil. 1H NMR (400 MHz, CDC1₃): δ 7.48 - 7.45 (m, 4H), 7.35 - 7.14 (m, 10H), 5.73 - 5.68 (m, 1H), 2.93 - 2.87 (m, 2H), 2.46 - 2.37 (m, 1H), 2.87 (t, J= 7.7 Hz, 2H), 2.11 (br s, 1H), 2.04 (s, 3H), 1.95 - 1.84 (m, 3H), 1.80 - 1.72 (m, 1H), 1.53 - 1.38 (m, 6H), 1.29 (s, 9H).

KSC-335-065

(R)-l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl acetate.

Prepared the same way as KSC-335-064 with diphenyl(piperidin-4-yl)methanol (0.196 g, 0.734 mmol), (R)-l-(4-(tert-butyl)phenyl)-4-chlorobutyl acetate (KSC-335-063) (0.249 g, 0.880 mmol) and potassium carbonate (0.406 g, 2.93 mmol) in acetonitrile to produce pure (R)- 1 -(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butyl acetate (0.237 g, 0.461 mmol, 63% yield) as a brown oil. 1H NMR (400 MHz, CDC1₃): δ 7.48 - 7.45 (m, 4H), 7.35 - 7.14 (m, 10H), 5.73 - 5.68 (m, 1H), 2.93 - 2.87 (m, 2H), 2.46 - 2.37 (m, 1H), 2.87 (t, J= 7.7 Hz, 2H), 2.11 (br s, 1H), 2.04 (s, 3H), 1.95 - 1.84 (m, 3H), 1.80 - 1.72 (m, 1H), 1.53 - 1.38 (m, 6H), 1.29 (s, 9H).

KSC-335-066

Methyl 2-(4-(4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butanoyl)phenyl)-2- methylpropanoate. To a vial was added the methyl 2-(4-(4-(4-

(hydroxydiphenylmethyl)piperidin- 1 -yl)but- 1 -yn- 1 -yl)phenyl)-2-methylpropanoate (KSC- 335-054) (0.074 g, 0.149 mmol). The mercuric oxide (1.493 ml, 0.045 mmol) was made into a 0.03 M solution in 4% w/v sulfuric acid and added to the starting material then heated to 55 °C and stirred for 3.5 h. The reaction turned a milky white color upon addition of the mercuric oxide solution. The reaction was removed from heat and diluted with saturated NaHC0₃ (10 mL) and extracted with DCM (3 x 10 mL). The DCM layers were combined and dried with MgS04, filtered and concentrated then purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce methyl 2-(4-(4-(4-(hydroxydiphenylmethyl) piperidin-1- yl)butanoyl)phenyl)-2-methylpropanoate (0.0217 g, 0.042 mmol, 28.3 % yield). 1H NMR (400 MHz, CDC1₃): δ 7.93 - 7.91 (m, 2H), 7.48 - 7.45 (m, 4H), 7.42 - 7.39 (m, 2H), 7.30 - 7.26 (m, 4H), 7.19 - 7.14 (m, 2H), 3.63 (s, 3H), 2.96 - 2.88 (m, 4H), 2.44 - 2.34 (m, 3H), 2.08 (br s, 1H), 1.60 (s, 6H), 1.62 - 1.56 (m, 4H), 1.46 - 1.30 (m, 4H).

KSC-335-069

(S)-l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol. To a vial was added the (S)-l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l- yl)butyl acetate (0.135 g, 0.263 mmol) and this vial was evacuated with nitrogen 3 times. The dry THF (9 mL) was then added. The 1.0 M lithium aluminum hydride (0.263 mL, 0.263 mmol) in THF was added dropwise at rt and the reaction stirred for 5 h. The reaction was quenched slowly with water (10 mL) and then extracted with diethyl ether (2 x 10 mL). The ether layer was dried with MgS0₄, filtered, concentrated and purified by reverse-phase MPLC (20 min, 10 - 100%, MeCN:H20) to produce pure (S)-l-(4-(tert-butyl)phenyl)-4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol (0.100 g, 0.212 mmol, 81 % yield).

[alpha]25₅₈₉ = . 38.8°. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.33 - 7.24 (m, 8H), 7.20 - 7.14 (m, 2H), 4.59 (dd, J= 8.2 Hz, 2.8 Hz, 1H), 3.13 (br d, J = 1 1.2 Hz, 1H), 2.97 (br d, J = 1 1.2 Hz, 1H), 2.50 - 2.34 (m, 3H), 2.10 - 1.88 (m, 4H), 1.84 - 1.74 (m, 1H), 1.68 - 1.44 (m, 6H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.4, 146.1 , 146.0, 142.7, 128.2, 128.1 , 126.45, 126.43, 125.7, 125.6, 125.4, 125.0, 79.3, 73.4, 58.9, 54.7, 53.3, 44.2, 39.7, 34.4, 31.4, 26.1 , 26.0, 24.1. LCMS Retention time: 4.159 min. LCMS purity 99.7%. HRMS (ESI): m/z calcd for C₃₂H₄iN0₂ [M+H]⁺ 472.3137, found 472.3210.

KSC-335-070

(R)-l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l-ol. To a vial was added the (i?)-l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l- yl)butyl acetate (0.237 g, 0.461 mmol) and this vial was evacuated with nitrogen 3 times. The dry THF (Volume: 4.61 ml) was added and then the 1.0 M lithium aluminum hydride (0.461 mL, 0.461 mmol) in THF was added portionwise at rt for 5 h. The reaction was quenched slowly with water (10 mL) and then extracted with diethyl ether (2 x 10 mL). The ether layer was dried with MgSC"4, filtered, concentrated and purified by reverse-phase MPLC (20 min, 10 - 100%, MeCN:J¾0) to produce pure (R)-l-(4-(tert-butyl)phenyl)-4-(4- (hydroxydiphenylmethyl) piperidin-l-yl)butan-l-ol (0.116 g, 0.246 mmol, 53% yield).

[alpha]25₅₈₉ = + 38.6°. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.33 - 7.24 (m, 8H), 7.20 - 7.14 (m, 2H), 4.59 (dd, J= 8.2 Hz, 2.8 Hz, 1H), 3.13 (br d, J= 11.2 Hz, 1H), 2.97 (br d, J= 11.2 Hz, 1H), 2.50 - 2.34 (m, 3H), 2.10 - 1.88 (m, 4H), 1.84 - 1.74 (m, 1H), 1.68 - 1.44 (m, 6H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.4, 146.1, 146.0, 142.7, 128.2, 128.1, 126.45, 126.43, 125.7, 125.6, 125.4, 125.0, 79.3, 73.4, 58.9, 54.7, 53.3, 44.2, 39.7, 34.4, 31.4, 26.1, 26.0, 24.1. LCMS Retention time: 4.156 min. LCMS purity 97.8%. HRMS (ESI): m/z calcd for C₃₂H₄iN0₂ [M+H]⁺ 472.3137, found 472.3210.

KSC-335-077

4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(p-tolyl)butan-l-ol. Method B: 4-(4- (hydroxydiphenylmethyl)piperidin-l-yl)-l-(p-tolyl)butan-l-one (0.073 g, 0.171 mmol) and MeOH (Volume: 2 mL) and SODIUM BOROHYDRIDE (0.013 g, 0.341 mmol) to produce pure 4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)-l-(p-tolyl)butan-l-ol (0.070 g, 0.163 mmol, 95% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.46 (m, 4H), 7.32 - 7.09 (m, 10H), 4.59 (dd, J= 8.0, 2.8 Hz, 1H), 3.15 - 3.09 (m, 1H), 2.99 - 2.92 (m, 1H), 2.52 - 2.32 (m, 6H), 2.32 (s, 3H), 2.09 - 1.87 (m, 3H), 1.82 - 1.73 (m, 1H), 1.68 - 1.44 (m, 6H). ¹³C NMR (125 MHz, CDC1₃): δ 146.1, 146.0, 142.9, 136.0, 128.7, 128.11, 128.10, 128.06, 126.39, 126.37, 125.7, 125.6, 125.5, 79.2, 73.3, 58.9, 54.6, 53.4, 53.3, 44.2, 39.9, 30.9, 26.0, 25.9, 26.0, 21.0. LCMS Retention time: 3.809 min. LCMS purity 94.1%. HRMS (ESI): m/z calcd for C₂₉H₃₅N0₂ [M+H]⁺ 430.2668, found 430.2741.

KSC-335-080

Methyl 2-(4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl)phenyl)-2- methylpropanoate. Method B: methyl 2-(4-(4-(4-(hydroxydiphenylmethyl) piperidin-1- yl)butanoyl)phenyl)-2-methylpropanoate (KSC-335-066)(0.148 g, 0.288 mmol) and MeOH (1 mL) and sodium borohydride (0.016 g, 0.432 mmol) to produce pure methyl 2-(4-(l- hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl)phenyl)-2-methylpropanoate (0.108 g, 0.209 mmol, 73% yield). 1H NMR (400 MHz, CDC1₃): δ 7.54 - 7.49 (m, 4H), 7.35 - 7.27 (m, 8H), 7.23 - 7.17 (m, 2H), 4.62 (dd, J = 8.0, 2.8 Hz, 1H), 3.65 (s, 3H), 3.16 (d, J = 1 1.7 Hz, 1H), 2.98 (d, J = 1 1.7 Hz, 1H), 2.53 - 2.40 (m, 3H), 2.29 (br s, 1H), 2.14 - 2.06 (m, 1H), 2.01 - 1.93 (m, 2H), 1.84 - 1.50 (m, 14H). LCMS Retention time: 3.786 min. LCMS purity 98.2%. HRMS (ESI): m/z calcd for C₃₃H₄iN0₄ [M+H]⁺ 516.3036, found 516.3108.

KSC-335-081

2-(4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl)phenyl)-2- methylpropanoic acid. To a vial was added the methyl 2-(4-(l-hydroxy-4-(4- (hydroxydiphenylmethyl)piperidin- 1 -yl)butyl)phenyl)-2-methylpropanoate (KSC-335-080) (0.094 g, 0.182 mmol) and THF (Volume: 3 mL,). The LiOH (0.022 g, 0.91 1 mmol) was dissolved in water (3.00 mL) and then added to the reaction stirred at 80°C for 18 h. The reaction was removed from heat and cooled to rt and 1.0 M HCl in water was added to adjust to pH to 4 and a gummy off-white solid formed. DCM (5 mL) was added to the mixture and it was sonicated to break up the solid. The DCM layer was removed and the aqueous was extracted with DCM (2 x 5 mL). The DCM layer was concentrated and the residue was purified by reverse-phase MPLC (20 min, 10 - 100%) MeCN:water) to produce the product with impurities. This was submitted to the purification core. The pure sample was recovered to produce 2-(4-( 1 -hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butyl)phenyl)-2- methylpropanoic acid (0.0378 g, 0.075 mmol, 41% yield). 1H NMR (400 MHz, DMSO-d₆): δ 8.22 (s, 1H), 7.52 - 7.49 (m, 4H), 7.29 - 7.26 (m, 8H), 7.15 7.10 (m, 2H), 4.47 (t, J= 5.9 Hz, 1H), 2.94 - 2.86 (m, 3H), 2.35 - 2.30 (m, 2H), 2.05 - 1.95 (m, 2H), 1.60 - 1.35 (m, 12H), 1.28 - 1.22 (m, 2H). ¹³C NMR (125 MHz, CDC1₃): δ 177.7, 163.7, 147.2, 144.4, 143.4, 127.8, 125.8, 125.7, 125.6, 125.1, 78.4, 71.8, 57.7, 53.4, 53.2, 45.5, 43.1, 37.3, 26.5, 25.6, 22.6. LCMS Retention time: 2.612 min. LCMS purity 100%. HRMS (ESI): m/z calcd for C₃₂H₃₉N0₄ [M+H]⁺ 502.2879, found 502.2952.

KSC-342-006

l-(4-(tert-butyl)phenyl)-2-(4-(hydroxydiphenylmethyl)piperidin-l-yl)ethanone. Method A: diphenyl(piperidin-4-yl)methanol (0.4 g, 1.496 mmol), l-(4-(tert-butyl)phenyl)-2- chloroethanone (0.300 g, 1.425 mmol), sodium bicarbonate (0.144 g, 1.710 mmol) with water (3 mL) and 2-butanone (Volume: 15 mL) to produce pure l-(4-(tert-butyl)phenyl)-2-(4- (hydroxydiphenylmethyl)piperidin-l-yl)ethanone (0.452 g, 1.024 mmol, 71.8 % yield) as a colorless oil. . 1H NMR (500 MHz, CDC1₃): δ 7.93 (d, J= 8.6 Hz, 2H), 7.50 - 7.43 (m, 6H), 7.32 - 7.26 (m, 4H), 7.20 - 7.15 (m, 2H), 3.78 (s, 2H), 3.07 - 3.01 (m, 2H), 2.47 (tt, J= 11.8 Hz, 3.5 Hz, 1H), 2.26 - 2.18 (m, 2H), 1.66 - 1.45 (m, 5H), 1.33 (s, 9H). ). ¹³C NMR (125 MHz, CDC1₃): δ 196.1, 157.0, 145.8, 133.5, 129.8, 128.2, 128.0, 126.5, 125.8, 125.5, 125.1, 79.5, 64.2, 54.2, 43.8, 35.1, 31.2, 31.0, 26.2. LCMS Retention time: 3.987 min. LCMS purity 98.8%. HRMS (ESI): m/z calcd for C₃₀H₃₅NO₂ [M+H]⁺ 442.2668, found 442.2741.

KSC-342-010

l-(4-(tert-butyl)phenyl)-2-(4-(hydroxydiphenylmethyl)piperidin-l-yl)ethanol. Methon B: l-(4-(tert-butyl)phenyl)-2-(4-(hydroxydiphenylmethyl)piperidin-l-yl)ethanone (0.113 g, 0.256 mmol) and MeOH (1 mL) and SODIUM BOROHYDRIDE (0.019 g, 0.512 mmol) to produce pure 1 -(4-(tert-butyl)phenyl)-2-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)ethanol (0.106 g, 0.239 mmol, 93% yield) as a solid. 1H NMR (400 MHz, CDC1₃): δ 7.50 - 7.46 (m, 4H), 7.37 - 7.26 (m, 8H), 7.22 - 7.17 (M, 2H), 4.67 (m, 1H), 4.01 (br s, 1H), 3.20 (m, 1H), 2.86 (m, 1H), 2.50 - 2.45 (m, 3H), 2.37 - 2.30 (m, 1H), 2.10 - 2.02 (m, 1H), 1.60 - 1.45 (m, 5H), 1.31 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 150.3, 145.82, 145.78, 139.1, 128.20, 128.19, 126.59, 126.57, 125.7, 125.6, 125.2, 79.5, 68.6, 66.3, 55.8, 53.4, 52.2, 44.1, 34.5, 31.3, 26.8, 26.5. LCMS Retention time: 4.083 min. LCMS purity 94.2%. HRMS (ESI): m/z calcd for C₃₀H₃₇NO₂ [M+H]⁺ 444.2824, found 444.2897.

KSC-342-014

Methyl 4-(4-chloro-l-hydroxybutyl)benzoate. Method B: methyl 4-(4- chlorobutanoyl)benzoate (KSC-335-061) (0.042 g, 0.175 mmol) and MeOH with sodium borohydride (0.013 g, 0.349 mmol). to produce methyl 4-(4-chloro-l-hydroxybutyl)benzoate (0.037 g, 0.152 mmol, 87 % yield). 1H NMR (400 MHz, CDC1₃): δ 8.00 (d, J= 8.4 Hz, 2H), 7.40 (d, J= 8.2 Hz, 2H), 4.8 - 4.6 (m, 1H), 3.90 (s, 3H), 3.58 - 3.52 (m, 2H), 2.19 (br s, 1H), 1.96 - 1.78 (m, 4H)

KSC-342-017

Methyl 4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl)benzoate. To a vial was added the methyl 4-(4-chloro-l-hydroxybutyl)benzoate (KSC-342-014) (0.037 g, 0.152 mmol), diphenyl(piperidin-4-yl)methanol (0.122 g, 0.457 mmol), SODIUM

BICARBONATE (0.026 g, 0.305 mmol), SODIUM IODIDE (1.143 mg, 7.62 μιηοΐ) and the vial was evacuated with argon 3 times. Dry acetonitrile (2 ml) was added and the reaction stirred overnight at reflux and was then cooled to rt after 18 h and the solvent was concentrated. The residue was dissolved in DCM (5 mL) and washed with 0.1 N HC1 (5 mL), water (5 mL) and brine (5 mL). The product was purified by MPLC (0 - 10 %>

MeOH:DCM) to produce pure methyl 4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin- l-yl)butyl)benzoate (0.0268 g, 0.057 mmol, 37% yield). 1H NMR (400 MHz, CDC1₃): δ 7.97 (d, J= 8.4 Hz, 2H), 7.52 - 7.47 (m, 4H), 7.42 (d, J= 7.9 Hz, 2H), 7.32 - 7.29 (m, 4H), 7.20 - 7.14 (m, 2H), 4.66 (m, 1H), 3.90 (s, 3H), 3.14 (m, 1H), 2.94 (m, 1H), 2.78 (br s, 1H), 2.52 - 2.43 (m, lH), 2.39 (t, J= 4.8 Hz, 2H), 2.13 - 2.06 (m, 1H), 2.04 - 1.92 (m, 2H), 1.77 - 1.47 (m, 8H). ¹³C NMR (125 MHz, CDC1₃): δ 167.2, 151.4, 146.0, 145.9, 129.5, 128.4, 128.2, 128.2, 126.51, 126.48, 125.64, 125.59, 79.2, 73.2, 58.8, 54.7, 51.9, 44.2, 40.0, 26.0, 25.9, 24.0. LCMS Retention time: 3.652 min. LCMS purity 100%. HRMS (ESI): m/z calcd for C₃₀H₃₅NO₄ [M+H]⁺ 474.2566, found 474.2639.

KSC-342-021

4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butyl)benzoic acid. To a vial was added the methyl 4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l- yl)butyl)benzoate (0.0195 g, 0.041 mmol) and THF (1 mL). The LiOH (6.90 mg, 0.288 mmol) was dissolved in water (1 mL) and added to the reaction. The reaction stirred at overnight and was then acidified with 1.0 M HCl to pH 2 - 3 then extracted with DCM (3 x 5 mL). The DCM layer was concentrated and purified by reverse-phase MPLC (10 - 100 % MeCN: water) to produce pure 4-(l-hydroxy-4-(4-(hydroxydiphenylmethyl)piperidin-l- yl)butyl)benzoic acid (0.009 g, 0.020 mmol, 47% yield). 1H NMR (400 MHz, CD₃OD): δ 7.85 (d, J= 8.2 Hz, 2H), 7.53 - 7.49 (m, 4H), 7.34 - 7.26 (m, 6H), 7.19 - 7.14 (m, 2H), 4.70 (m, 1H), 3.46 (m, 1H), 3.35 (s, 2H), 3.01 - 2.96 (m, 2H), 2.92 - 2.77 (m, 3H), 1.84 - 1.64 (m, 8H). ¹³C NMR (125 MHz, CD₃OD): δ 174.7, 148.2, 147.2, 137.6, 130.4, 129.1, 127.6, 127.0, 126.4, 79.9, 74.0, 53.9, 49.8, 36.9, 25.5, 21.8. LCMS Retention time: 2.490 min. LCMS purity 100%. HRMS (ESI): m/z calcd for C₂₉H₃₃N0₄ [M+H]⁺ 460.2410, found 460.2482.

KSC-342-074

1- (Tert-butyl)-4-(2-chloroethyl)benzene. To a vial was added the l-(4-(tert-butyl)phenyl)-

2- chloroethanone (0.171 g, 0.812 mmol) and triethylsilane (0.519 mL, 3.25 mmol) with TFA (4 mL). The reaction stirred at 75 °C for 17 h and was then concentrated in vacuo. The residue was dissolved in DCM (5 mL) and washed with water (4 mL). The DCM layer was collected and washed with water (1 x 5 mL), dried with MgS0₄, filtered and adsorbed to silica and purified by MPLC (20 min, 0 - 40% EtOAc:hex) to produce pure l-(tert-butyl)-4- (2-chloroethyl)benzene (0.1 14 g, 0.580 mmol, 71% yield). 1H NMR (400 MHz, CDC1₃): δ 7.34 (d, J= 8.3 Hz, 2H), 7.16 (d, J= 8.3 Hz, 2H), 3.71 (t, J= 7.5 Hz, 2H), 3.05 (t, J= 7.6 Hz, 2H), 1.32 (s, 9H).

KSC-342-080

(l-(3-(4-(Tert-butyl)phenyl)propyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.142 g, 0.530 mmol), l-(tert-butyl)-4-(3- chloropropyl)benzene (0.134 g, 0.636 mmol) and POTASSIUM CARBONATE (0.439 g, 3.18 mmol) in acetonitrile. The reaction stirred overnight at 85 °C for 18 h and then filtered. The filtrate was then diluted with brine and extracted with diethyl ether (3 x 15 mL). The ether layers were combined, dried with MgS0₄, filtered and purified by reverse-phase MPLC (20 min, 10 - 100% MeCN: water) to produce pure (l-(3-(4-(tert- butyl)phenyl)propyl)piperidin-4-yl)diphenylmethanol (0.152 g, 0.344 mmol, 65%> yield) as an oil. 1H NMR (400 MHz, CDC1₃): δ 7.49 - 7.45 (m, 4H), 7.31 - 7.24 (m, 6H), 7.19 - 7.14 (m, 2H), 7.1 1 - 7.08 (m, 2H), 2.96 (m, 2H), 2.57 (t, J= 7.7 Hz, 2H), 2.48 - 2.32 (m, 3H), 2.22 (br s, 1H), 1.97 - 1.90 (m, 2H), 1.83 - 1.75 (m, 2H), 1.53 - 1.45 (m, 4H), 1.29 (s, 9H). LCMS Retention time: 2.965 min. LCMS purity 97.2%. HRMS (ESI): m/z calcd for C₃iH₃₉NO

[M+H]⁺ 442.3032, found 442.3104.

KSC-342-081

(l-(4-(Tert-butyl)phenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.150 g, 0.561 mmol), l-(tert-butyl)-4-(2- chloroethyl)benzene (KSC-342-074) (0.1 10 g, 0.561 mmol) and potassium carbonate (0.465 g, 3.37 mmol) in acetonitrile. The reaction stirred overnight at 85 °C for 18 h and then filtered. The filtrate was then diluted with brine and extracted with diethyl ether (3 x 15 mL). The ether layers were combined, dried with MgS04, filtered and purified by reverse-phase MPLC (20 min, 10 - 100% MeCN:water) to produce pure (l-(4-(tert- butyl)phenethyl)piperidin-4-yl)diphenylmethanol (0.180 g, 0.421 mmol, 75% yield) as an oil. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.47 (m, 4H), 7.33 - 7.27 (m, 6H), 7.21 - 7.16 (m, 2H), 7.14 - 7.11 (m, 2H), 3.09 - 3.03 (m, 2H), 2.80 - 2.74 (m, 2H), 2.60 - 2.55 (m, 2H), 2.51 - 2.42 (m, 1H), 2.28 (br s, 1H), 2.08 - 2.00 (m, 2H), 1.57 - 1.50 (m, 4H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 148.8, 145.9, 137.3, 128.3, 128.1, 126.5, 125.8, 125.2, 79.5, 60.8, 54.0, 44.2, 40.9, 34.3, 33.1, 31.4, 26.4. LCMS Retention time: 4.384 min. LCMS purity 99.7%. HRMS (ESI): m/z calcd for C₃₀H₃₇NO [M+H]⁺ 428.2875, found 428.2948.

KSC-342-088

(l-(4-amino-4-(4-(tert-butyl)phenyl)butyl)piperidin-4-yl)diphenylmethanol. To a vial was added the l-(4-(tert-butyl)phenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l- one (0.200 g, 0.426 mmol), Ammonium acetate (0.328 g, 4.26 mmol) and Sodium

cyanoborohydride (0.040 g, 0.639 mmol) with MeOH (Volume: 4 ml). The reaction stirred at rt at 2:56:22 PM. The reaction was stirred overnight and then concentrated and diluted with dilute aqueous ammonium hydroxide and extracted with DCM. The DCM layer was concentrated and the crude NMR showed product and starting material. The reaction then purified by reverse-phase Teledyne ISCO Combiflash chromatography (10 - 100%)

MeCN:basic water) and fractions 5 and 6 were collected. These were then subjected to normal phase purification (0 - 10% MeOH (5% NH30H):DCM) to produce pure (l-(4- amino-4-(4-(tert-butyl)phenyl)butyl)piperidin-4-yl)diphenylmethanol (0.010 g, 0.021 mmol, 5% yield). 1H NMR (400 MHz, CDC1₃): δ 7.48 - 7.44 (m, 4H), 7.34 - 7.26 (m, 6H), 7.22 - 7.14 (m, 4H), 3.84 (t, J= 6.7 Hz, 1H), 2.95 - 2.87 (m, 2H), 2.46 - 2.37 (m, 1H), 2.28 (t, J = 7.6 Hz, 2H), 1.95 - 1.85 (m, 2H), 1.70 - 1.60 (m, 5H), 1.55 - 1.40 (m, 6H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.7, 146.0, 143.3, 128.1, 126.5, 125.9, 125.8, 125.3, 79.5, 58.7, 55.8, 54.2, 54.0, 44.2, 37.5, 34.4, 31.4, 26.4, 24.2. LCMS Retention time: 2.397 min. LCMS purity 100%. HRMS (ESI): m/z calcd for C₃₂H₄2N₂0 [M+H]⁺ 471.3297, found 471.3370.

KSC-348-001

l-([l,l'-Biphenyl]-4-yl)-4-(4-(hy(iroxy(iiphenylmethyl)piperi(iin-l-yl)butan-l-one. To a vial was added the l-(4-bromophenyl)-4-(4-(hydroxydiphenylmethyl)piperidin-l-yl)butan-l- one (0.098 g, 0.199 mmol), Ι,Γ-bis (di-t-butylphosphino)ferrocene palladium dichloride, (2.71 mg, 3.98 μηιοΐ) and phenylboronic acid (0.029 g, 0.239 mmol) followed by acetonitrile (1.5 mL). The potassium carbonate (0.041 g, 0.299 mmol) was dissolved in water (1.5 mL) and added the reaction. The reaction stirred at 60 °C for 18 h. The reaction was stopped and the organic layer was diluted with EtOAc and extracted then washed with brine. The EtOAc layer was collected, dried with MgS0₄, filtered and purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce the desired l-([l,l'-biphenyl]-4-yl)-4-(4- (hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -one (0.035 g, 0.071 mmol, 36% yield). 1H NMR (400 MHz, CDC1₃): δ 8.03 (d, J= 8.6 Hz, 2H), 7.67 (d, J= 8.4 Hz, 2H), 7.64 - 7.61 (m, 2H), 7.50 - 7.45 (m, 6H), 7.42 - 7.38 (m, 1H), 7.31 - 7.26 (m, 4H), 7.19 - 7.14 (m, 2H), 3.02 - 2.91 (m, 4H), 2.46 - 2.37 (m, 3H), 2.10 (br s, 1H), 1.97 - 1.92 (m, 4H), 1.50 - 1.35 (m, 4H).

KSC-348-002

l-([l,l'-biphenyl]-4-yl)-4-(4-(hydroxydiphenylmethyl)piperi(iin-l-yl)butan-l-ol. Method B : 1 -([ 1 , 1 '-biphenyl]-4-yl)-4-(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)butan- 1 -one (KSC- 348-001) (0.035 g, 0.071 mmol) and MeOH (2 mL) and sodium borohydride (5.41 mg, 0.143 mmol) to produce pure l-([l, -biphenyl]-4-yl)-4-(4-(hydroxydiphenylmethyl)piperidin-l- yl)butan-l-ol (0.033 g, 0.067 mmol, 94% yield) as on oil. 1H NMR (400 MHz, CDC1₃): δ 7.59 - 7.55 (m, 2H), 7.53 - 7.46 (m, 6H), 7.43 - 7.38 (m, 4H), 7.34 - 7.25 (m, 5H), 7.19 - 7.13 (m, 2H), 4.65 (dd, J= 8.2 Hz, 2.7 Hz, 1H), 3.16 - 3.10 (m, 1H), 2.99 - 2.93 (m, 1H), 2.56 (br s, 1H), 2.50 - 2.34 (m, 3H), 2.10 - 1.92 (m, 3H), 1.86 - 1.76 (m, 1H), 1.70 - 1.45 (m, 6H). ). ¹³C NMR (125 MHz, CDC1₃): δ 146.1, 146.0, 145.0, 141.1, 139.4, 128.6, 128.2, 128.1, 127.01, 126.96, 126.8, 126.44, 126.41, 126.1, 125.7, 125.6, 79.2, 73.3, 58.9, 54.7, 53.3, 44.2, 40.0, 26.0, 25.9, 24.1. LCMS Retention time: 4.049 min. LCMS purity 97.6%. HRMS (ESI): m/z calcd for C₃₄H₃₇NO₂ [M+H]⁺ 492.2824, found 492.2897.

KSC-348-049

(4-(tert-butyl)phenyl)(4-(hydroxydiphenylmethyl)piperidin-l-yl)methanone. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.232 g, 0.868 mmol), acetonitrile (3 mL) and TEA (0.181 ml, 1.30 mmol). The 4-(tert-butyl)benzoyl chloride (0.173 mL, 0.954 mmol) was added and the reaction stirred at 70 °C for 6 h and was diluted with EtOAc (15 mL) and washed with saturated NaHC0₃ (15 mL). The EtOAc was collected, dried with MgS04, filtered and adsorbed to silica and purified by MPLC (20 min, 0 - 30 % EtOAc:Hex) to produce pure (4-(tert-butyl)phenyl)(4-(hydroxydiphenylmethyl)piperidin- 1 -yl)methanone (0.304 g, 0.711 mmol, 82% yield) as a white solid. 1H NMR (400 MHz, CDC1₃): δ 7.50 - 7.44 (m, 4H), 7.38 - 7.14 (m, 10H), 4.77 (br s, 1H), 3.84 (br s, 1H), 3.05 - 2.63 (m, 3H), 2.25 - 2.17 (m, 1H), 1.75 - 1.35 (m, 4H), 1.29 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 170.4, 152.7, 145.4, 133.2, 128.3, 126.7, 125.7, 125.2, 79.5, 44.5, 34.7, 31.2. LCMS Retention time: 3.774 min. LCMS purity 100%. HRMS (ESI): m/z calcd for C₂₉H₃₃NO₂ [M+H]⁺ 428.2511, found 428.2584.

KSC-348-050

(l-(4-(tert-butyl)benzyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.085 ml, 0.393 mmol), acetonitrile (2 mL) and TEA (0.082 ml, 0.589 mmol). The p-tert-butylbenzyl bromide (0.098 g, 0.432 mmol) was then added and the reaction stirred at 70 °C and stirred for 5 h then diluted with EtOAc (15 mL) and washed with saturated NaHC0₃ (15 mL). The EtOAc was collected, dried with MgS04, filtered and adsorbed to silica and purified by reverse phase MPLC (20 min, 10 - 100 % MeCN: water) to produce pure (l-(4-(tert-butyl)benzyl)piperidin-4-yl)diphenylmethanol (0.131 g, 0.317 mmol, 81% yield) as a brown oil. 1H NMR (400 MHz, CDCI₃): δ 7.48 - 7.45 (m, 4H), 7.32 - 7.25 (m, 6H), 7.22 - 7.14 (m, 4H), 3.47 (s, 2H), 2.96 - 2.90 (m, 2H), 2.46 - 2.38 (m, 1H), 2.02 - 1.95 (m, 1H), 1.51 - 1.44 (m, 4H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDCls): δ 149.8, 146.0, 135.1, 128.9, 128.1, 126.4, 125.8, 125.0, 79.5, 62.8, 53.9, 44.2, 34.4, 31.4, 26.5, 21.0. LCMS Retention time: 4.186 min. LCMS purity 99.7%. HRMS (ESI): m/z calcd for C₂9H₃₅NO [M+H]⁺ 414.2719, found 414.2791.

KSC-348-058

(l-(4-methoxyphenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.381 g, 1.425 mmol), acetonitrile (5 mL) and TEA (0.298 ml, 2.138 mmol). The l-(2-bromoethyl)-4-methoxybenzene (0.245 ml, 1.568 mmol) was then added and the reaction stirred at 75 °C and the stirred for 16 h then was quenched with saturated NaHC0₃, extracted with EtOAc, dried with MgS0₄, filtered and adsorbed to silica. The product was purified by MPLC (20 min, 0 - 10% MeOH:DCM) to produce pure (1- (4methoxyphenethyl) piperidin-4-yl)diphenylmethanol (0.289 g, 0.720 mmol, 50% yield) as a sticky solid. 1H NMR (400 MHz, CDC1₃): δ 7.49 - 7.46 (m, 4H), 7.32 - 7.28 (m, 4H), 7.19 (tt, J= 7.3 Hz, 1.9 Hz, 2H), 7.12 (d, J= 8.6 Hz, 2H), 6.82 (d, J= 8.5 Hz, 2H), 3.77 (s, 3H),

3.29 - 3.24 (m, 2H), 2.95 - 2.90 (m, 2H), 2.80 - 2.75 (m, 2H), 2.58 - 2.50 (m, 1H), 2.42 -

2.30 (m, 3H), 1.90 - 1.77 (m, 2H), 1.65 - 1.57 (m, 2H). ¹³C NMR (125 MHz, CDC1₃): δ 158.3, 145.5, 129.6, 128.3, 126.7, 125.6, 114.0, 79.3, 55.3, 53.6, 53.4, 43.4, 31.5, 25.1.

LCMS Retention time: 3.695 min. LCMS purity 100%. HRMS (ESI): m/z calcd for

C₂₇H₃iN0₂ [M+H]⁺ 402.2355, found 402.2353.

KSC-352-055

4-(4-Benzoylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-one. To a vial was added the l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-one (0.060 g, 0.252 mmol) and potassium iodide (0.063 g, 0.377 mmol) with acetonitrile (2 mL). The reaction stirred at 85 °C for 1 h then the phenyl(piperidin-4-yl)methanone (0.050 g, 0.264 mmol) along with potassium carbonate (0.052 g, 0.377 mmol) was added. The reaction was then heated back to 85 °C for 48 h. The reaction was cooled to rt and diluted with water then extracted with EtOAc (3 x 15 mL). The EtOAc layer was dried with MgS0₄, filtered and adsorbed to silica. The product was purified by reverse-phase MPLC (20 min, 10 - 100% MeCN:water) to produce 4-(4-benzoylpiperidin- l-yl)-l-(4-(tert-butyl)phenyl)butan-l-one (0.026 g, 0.066 mmol, 26% yield). 1H NMR (400 MHz, CDCls): δ 7.95 - 7.90 (m, 4H), 7.57 - 7.53 (m, 1H), 7.49 - 7.44 (m, 4H), 3.27 - 3.18 (m, 1H), 3.03 - 2.96 (m, 4H), 2.44 (t, J= 7.04 Hz, 2H), 2.13 - 2.06 (m, 2H), 1.98 - 1.91 (m,

2H), 1.85 - 1.76 (m, 4H), 1.34 (s, 9H).

KSC-352-060

(l-(4-nitrophenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin -4-yl)methanol (0.513 g, 1.919 mmol), l-(2-bromoethyl)-4-nitrobenzene (0.401 g, 1.744 mmol) and acetonitrile (10 mL). The TEA (0.365 ml, 2.62 mmol) was then added and the reaction stirred at 85 °C for 18 h then cooled to rt. The reaction was diluted with water and extracted with EtOAc (3 x 15 mL). The EtOAc layer was collected and dried with MgS04, filtered and adsorbed to silica then purified by MPLC (15 min, 0 - 10 %> MeOH:DCM). to produce pure (l-(4-nitrophenethyl)piperidin-4-yl)diphenylmethanol (0.145 g, 0.348 mmol, 20% yield). 1H NMR (400 MHz, CDC1₃): δ 8.13 (d, J= 8.7 Hz, 2H), 7.49 - 7.46 (m, 4H), 7.35 (d, J= 8.7 Hz, 2H), 7.32 - 7.27 (m, 4H), 7.21 - 7.16 (m, 2H), 3.11 - 3.05 (m, 2H), 2.97 - 2.91 (m, 2H), 2.69 - 2.63 (m, 2H), 2.52 - 2.43 (m, 1H), 2.25 (br s, 1H), 2.20 - 2.10 (m, 2H), 1.63 - 1.53 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 147.9, 146.5, 145.7, 129.5, 128.2, 126.6, 125.7, 123.7, 79.4, 59.4, 53.9, 43.9, 33.2, 26.1, 21.1. LCMS Retention time: 3.654 min. LCMS purity 98.8%. HRMS (ESI): m/z calcd for C26H28N2O3 [M+H]⁺ 417.2162, found 417.2173.

KSC-352-061

(l-(4-bromophenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.496 g, 1.855 mmol), l-bromo-4-(2-bromoethyl)benzene (0.258 ml, 1.69 mmol) and acetonitrile (10 mL). The TEA (0.353 mL, 2.53 mmol) was then added and the reaction stirred at 85 °C for 18 h then cooled to rt. The reaction was diluted with water and extracted with EtOAc (3 x 15 mL). The EtOAc layer was collected and dried with MgS04, filtered and adsorbed to silica then purified by MPLC (15 min, 0 - 10 %

MeOH:DCM). to produce pure (l-(4-bromophenethyl)piperidin-4-yl)diphenylmethanol (0.488 g, 1.083 mmol, 64% yield). 1H NMR (400 MHz, CDC1₃): δ 7.50 - 7.46 (m, 4H), 7.38 (d, J= 8.3 Hz, 2H), 7.34 - 7.27 (m, 4H), 7.18 (tt, J= 7.3 Hz, 1.8 Hz, 2H), 7.06 (d, J= 8.4 Hz, 2H), 3.06 - 3.00 (m, 2H), 2.77 - 2.72 (m, 2H), 2.58 - 2.52 (m, 2H), 2.50 - 2.41 (m, 1H), 2.15 - 2.00 (m, 3H), 1.56 - 1.49 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 145.8, 139.3, 131.4, 130.4, 128.2, 126.6, 125.8, 119.8, 79.5, 60.4, 54.0, 44.1, 33.1, 26.4, 21.1 LCMS Retention time: 3.969 min. LCMS purity 99.8%. HRMS (ESI): m/z calcd for C₂6H₂₈BrNO [M+H]⁺ 450.1354, found 450.1427.

KSC-352-063

(l-(2-([l,l'-biphenyl]-4-yl)ethyl)piperidin-4-yl)(iiphenylmethanol. To a vial was added the phenylboronic acid (0.019 g, 0.152 mmol), l, -bis(di-tert-butylphosphino)ferrocene palladium dichloride (4.12 mg, 6.33 μιηοΐ), (l-(4-bromophenethyl)piperidin-4- yl)diphenylmethanol (KSC-352-061) (0.057 g, 0.127 mmol) and potassium carbonate (0.035 g, 0.253 mmol). The vial was then evacuated with argon 3 times and acetonitrile (1 mL) was added followed by water (1 mL). The reaction then stirred at 60 °C for 18 h then was diluted with EtOAc (5 mL) and saturated NaHC0₃ (5 mL). The EtOAc layer was collected and the aqueous layer was extracted with more EtOAc (2 x 5 mL). The EtOAc layers were combined and dried with MgS04, filtered and concentrated. The reaction was purified by MPLC (10 - 100%) MeCN:water) to produce pure (l-(2-([l,l'-biphenyl]-4-yl)ethyl)piperidin-4- yl)diphenylmethanol (0.045 g, 0.101 mmol, 79% yield) as a clear oil. 1H NMR (400 MHz, CDC1₃): δ 7.59 - 7.45 (m, 2H), 7.53 - 7.48 (m, 6H), 7.45 - 7.40 (m, 2H), 7.35 - 7.25 (m, 7H), 7.22 - 7.16 (m, 2H), 3.11 - 3.05 (m, 2H), 2.87 - 2.81 (m, 2H), 2.65 - 2.59 (m, 2H), 2.53 - 2.44 (m, 1H), 2.22 (br s, 1H), 2.12 - 2.04 (m, 2H), 1.59 - 1.52 (m, 4H). ¹³C NMR (125 MHz, CDCls): δ 145.9, 141.0, 139.5, 139.0, 129.1, 128.7, 128.2, 127.1, 127.03, 126.96, 126.5, 125.8, 79.5, 60.7, 54.0, 50.8, 44.2, 33.3, 26.4. LCMS Retention time: 4.086 min.

LCMS purity 99%. HRMS (ESI): m/z calcd for C₃₂H₃₃NO [M+H]⁺ 448.2562, found

448.2635.

KSC-352-064

Diphenyl(l-(4-(pyridin-4-yl)phenethyl)piperidin-4-yl)methanol. To a vial was added the (l-(4-bromophenethyl)piperidin-4-yl)diphenylmethanol (KSC-352-061) (0.050 g, 0.111 mmol), pyridin-4-ylboronic acid (0.018 g, 0.133 mmol), 1 , l'-bis(di-tert- butylphosphino)ferrocene palladium dichloride (3.62 mg, 5.55 μιηοΐ) and potassium carbonate (0.031 g, 0.222 mmol). The vial was evacuated 3 times with argon and then acetonitrile (1 mL) followed by water (1 mL) was added. The reaction stirred at 60 °C for 18 h and was then cooled to rt and diluted with EtOAc (5 mL) and saturated NaHC0₃ (5 mL). The EtOAc layer was collected and the aqueous layer was extracted with more EtOAc (2 x 5 mL). The EtOAc layers were combined and dried with MgS0₄, filtered and concentrated. The reaction was purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce pure diphenyl(l-(4-(pyridin-4-yl)phenethyl)piperidin-4-yl)methanol (0.015 g, 0.033 mmol, 30%> yield) as an oil. 1H NMR (400 MHz, CDCI3): δ 8.64 - 8.62 (m, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.51 - 7.46 (m, 6H), 7.32 - 7.28 (m, 6H), 7.18 (tt, J= 7.3 Hz, 1.8 Hz, 2H), 3.10 - 3.04 (m, 2H), 2.88 - 2.82 (m, 2H), 2.65 - 2.58 (m, 2H), 2.51 - 2.43 (m, 1H), 2.19 (br s, 1H), 2.12 - 2.03 (m, 2H), 1.58 - 1.51 (m, 4H). ¹³C NMR (125 MHz, CDCI3): δ 150.2, 148.1, 145.9, 141.7, 135.8, 129.5, 128.2, 127.0, 126.5, 125.8, 121.4, 79.5, 60.5, 54.1, 44.1, 41.0, 33.4, 26.4. LCMS Retention time: 3.585 min. LCMS purity 93.3%. HRMS (ESI): m/z calcd for

C3₁H3₂N₂O [M+H]⁺ 449.2515, found 449.2587.

KSC-352-065

Diphenyl(l-(4-(pyridin-3-yl)phenethyl)piperidin-4-yl)methanol. To a vial was added the (l-(4-bromophenethyl)piperidin-4-yl)diphenylmethanol (0.057 g, 0.127 mmol), pyridin-3- ylboronic acid (0.019 g, 0.152 mmol), l ,l'-Bis(di-tert-butylphosphino)ferrocene palladium dichloride (4.12 mg, 6.33 μιηοΐ) and potassium carbonate (0.035 g, 0.253 mmol). The vial was evacuated with argon 3 times and then acetonitrile (1 mL) followed by water (1 mL) was added. The reaction stirred at 60 °C for 18 h and then cooled to rt and diluted with EtOAc (5 mL) and saturated NaHC0₃ (5 mL). The EtOAc layer was collected and the aqueous layer was extracted with more EtOAc (2 x 5 mL). The EtOAc layers were combined and dried with MgS0₄, filtered and concentrated. The reaction was purified by RP MPLC (10 - 100% MeCN: water) to produce pure diphenyl(l-(4-(pyridin-3-yl)phenethyl)piperidin-4-yl)methanol (0.05 g, 0.1 1 1 mmol, 88% yield) as an oil. 1H NMR (400 MHz, CDC1₃): δ 8.79 (dd, J = 2.4 Hz, 0.9 Hz, 1H), 8.53 (dd, J = 4.8 Hz, 1.6 Hz, 1H), 7.86 - 7.83 (m, 1H), 7.51 - 7.47 (m, 6H), 7.36 - 7.27 (m, 7H), 7.20 - 7.15 (m, 2H), 3.10 - 3.03 (m, 2H), 2.87 - 2.81 (m, 2H), 2.71 (br s, 1H), 2.63 - 2.58 (m, 2H), 2.51 - 2.43 (m, 1H), 2.1 1 - 2.03 (m, 2H), 1.58 - 1.52 (m, 4H). ). ¹³C NMR (125 MHz, CDC1₃): δ 148.04, 147.97, 146.0, 140.5, 136.5, 135.5, 134.3, 129.4, 128.1 , 127.1 , 126.4, 125.8, 123.5, 79.4, 60.5, 54.1 , 44.1 , 33.2, 26.2. LCMS Retention time: 3.582 min. LCMS purity 98.8%. HRMS (ESI): m/z calcd for C₃iH₃₂N₂0 [M+H]⁺ 449.2515, found 449.2587.

KSC-352-066

l-(4-(tert-butyl)phenyl)-4-(4-(hydroxy(phenyl)methyl)piperidin-l-yl)butan-l-ol. Method B: 4-(4-benzoylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-one (KSC-352-055) (0.026 g, 0.066 mmol) and MeOH (2 mL) and sodium borohydride (10.05 mg, 0.266 mmol) to produce pure 1 -(4-(tert-butyl)phenyl)-4-(4-(hydroxy(phenyl)methyl)piperidin- 1 -yl)butan- 1 -ol (0.019 g, 0.048 mmol, 72% yield) as a mixture of diastereomers. 1H NMR (400 MHz, CDC1₃): δ 7.36 - 7.24 (m, 9H), 4.63 - 4.57 (m, 1H), 4.33 (d, J= 7.6 Hz, 1H), 3.23 - 2.83 (m, 2H), 2.42 - 2.35 (m, 2H), 2.13 - 1.89 (m, 3H), 1.86 - 1.52 (m, 6H), 1.46 - 1.16 (m, 4H), 1.31 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.4, 143.4, 142.8, 128.3, 127.63, 127.61, 126.6, 125.4, 125.0, 78.74, 78.67, 73.4, 58.9, 54.30, 54.26, 52.8, 52.7, 43.21, 43.2, 39.9, 34.4, 31.4, 28.4, 28.3, 28.2, 24.2, 24.1. LCMS Retention time: 3.707 min. LCMS purity 97.1%. HRMS (ESI): m/z calcd for C₂₆H₃₇N0₂ [M+H]⁺ 396.2824, found 396.2897.

KSC-352-069

(l-(4-aminophenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the (l-(4- nitrophenethyl) piperidin-4-yl)diphenylmethanol (KSC-352-060) (0.135 g, 0.324 mmol) with MeOH (1 mL) and DCM (1 mL). The reaction was cooled to 0 °C and the Raney Nickel (1.902 mg, 0.032 mmol) was added. The sodium borohydride (0.031 g, 0.810 mmol) was then added portionwise and the reaction stirred at rt for 28 h and the Raney Nickel filtered through celite. The reaction was diluted with DCM and washed with water and the DCM layer was dried with MgS0₄, filtered and adsorbed to silica then purified by MPLC (0 - 15%> MeOH:DCM) to produce pure (l-(4-aminophenethyl)piperidin-4-yl)diphenylmethanol (0.077 g, 0.199 mmol, 61% yield). 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.47 (m, 4H), 7.32 - 7.27 (m, 4H), 7.18 (tt, J= 7.3 Hz, 1.8 Hz, 2H), 6.97 (d, J= 8.3 Hz, 2H), 6.61 (d, J= 8.3 Hz, 2H), 3.52 (br s, 2H), 3.07 - 3.01 (m, 2H), 2.70 - 2.65 (m, 2H), 2.54 - 2.41 (m, 3H), 2.22 (br s, 1H), 2.07 - 1.98 (m, 2H), 1.56 - 1.49 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 145.9, 144.4, 130.4, 129.4, 128.1, 126.5, 125.8, 115.2, 79.5, 61.2, 54.1, 44.2, 32.8, 26.4, 21.0.

LCMS Retention time: 3.328 min. LCMS purity 93.1%. HRMS (ESI): m/z calcd for

C₂6H₃oN₂0 [M+H]⁺ 387.2358, found 387.2431.

KSC-352-075

(l-(4-(Dimethylamino)phenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the (l-(4-aminophenethyl)piperidin-4-yl)diphenylmethanol (KSC-352-069) (0.030 g, 0.078 mmol) and acetic acid (1 mL). The paraformaldehyde (0.058 mL, 0.776 mmol) solution in water followed by sodium cyanoborohydride (0.015 g, 0.233 mmol) was then added and the reaction stirred at rt for 20 h. The reaction was concentrated and diluted with saturated NaHC0₃ and extracted with EtOAc (3 x 5 mL). The EtOAc layer was dried with MgS04, filtered and concentrated. The product was purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce (l-(4-(dimethylamino)phenethyl)piperidin-4-yl)diphenylmethanol (0.027 g, 0.065 mmol, 84% yield). 1H NMR (400 MHz, CDC1₃): δ 7.50 - 7.46 (m, 4H), 7.31 - 7.26 (m, 4H), 7.17 (tt, J= 7.3 Hz, 1.8 Hz, 2H), 7.06 (d, J= 8.3 Hz, 2H), 6.68 (d, J= 8.3 Hz, 2H), 3.08 - 3.02 (m, 2H), 2.90 (s, 6H), 2.72 - 2.67 (m, 2H), 2.56 - 2.50 (m, 2H), 2.48 - 2.42 (m, 1H), 2.30 (br s, 1H), 2.07 - 2.00 (m, 2H), 1.56 - 1.51 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 149.1, 146.0, 129.2, 128.4, 128.1, 126.4, 125.8, 112.9, 79.4, 61.1, 54.0, 44.2, 41.0, 40.8, 32.6, 26.4. LCMS Retention time: 2.50 min. LCMS purity 96.1%. HRMS (ESI): m/z calcd for C₂8H₃4N₂0 [M+H]⁺ 415.2671, found 415.2744.

KSC-352-082

Diphenyl(l-(4-(trifluoromethyl)phenethyl)piperidin-4-yl)methanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.100 g, 0.374 mmol), l-(2-bromoethyl)-4- (trifluoromethyl)benzene (0.057 ml, 0.340 mmol) and acetonitrile (10 mL). The TEA (0.071 mL, 0.510 mmol) was then added and the reaction stirred at 85 °C for 18 h. The reaction was diluted with water and extracted with EtOAc (3 x 15 mL). The EtOAc layer was collected and dried with MgS0₄, filtered and adsorbed to silica then purified by MPLC (15 min, 0 - 10 % MeOH:DCM) to produce pure diphenyl(l-(4-(trifluoromethyl)phenethyl)piperidin-4- yl)methanol (0.074 g, 0.168 mmol, 49% yield) as an oil. 1H NMR (400 MHz, CDC1₃): δ 7.54 - 7.47 (m, 4H), 7.33 - 7.27 (m, 6H), 7.19 (tt, J= 7.3 Hz, 1.8 Hz, 2H), 3.06 - 3.00 (m, 2H), 2.86 - 2.80 (m, 2H), 2.60 - 2.55 (m, 2H), 2.51 - 2.42 (m, 1H), 2.14 (br s, 1H), 2.10 - 2.03 (m, 2H), 1.57 - 1.47 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 145.9, 144.6, 129.0, 128.3 (q, J= 32 Hz), 128.2, 126.5, 125.8, 125.2 (q, 3.8 Hz), 124.2 (q, J= 271.8 Hz), 79.5, 60.2, 54.1, 44.1, 33.6, 26.4, 21.0. LCMS Retention time: 3.877 min. LCMS purity 99.1%. HRMS (ESI): m/z calcd for C₂₇H₂₈F₃NO [M+H]⁺ 440.2123, found 440.2196.

KSC-352-088

(l-(4-Fluorophenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.099 g, 0.370 mmol), l-(2-bromoethyl)-4-fluorobenzene (0.047 rriL, 0.337 mmol) and acetonitrile (10 mL). The TEA (0.070 mL, 0.505 mmol) was then added and the reaction stirred at 85 °C at for 19 h and was cooled to rt. The reaction was diluted with water and extracted with EtOAc (3 x 15 mL). The EtOAc layer was collected and dried with MgS0₄, filtered and adsorbed to silica then purified by MPLC (15 min, 0 - 10 % MeOH:DCM) to produce pure (l-(4-fluorophenethyl)piperidin-4-yl)diphenylmethanol (0.123 g, 0.316 mmol, 94% yield). 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.47 (m, 4H), 7.32

- 7.27 (m, 4H), 7.21 - 7.11 (m, 4H), 6.98 - 6.92 (m, 2H), 3.06 - 3.00 (m, 2H), 2.78 - 2.72 (m, 2H), 2.56 - 2.51 (m, 2H), 2.50 - 2.42 (m, 1H), 2.21 (br s, 1H), 2.08 - 1.99 (m, 2H), 1.57

- 1.48 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 161.2 (d, J= 243.6 Hz), 145.9, 136.5 (d, J = 3.2 Hz), 129.9 (d, J= 7.9 Hz), 128.1, 126.5, 125.8, 115.0 (d, J= 21.2 Hz), 79.5, 60.8, 54.1, 44.2, 33.0, 26.4, 21.0. LCMS Retention time: 3.733 min. LCMS purity 96.8%. HRMS (ESI): m/z calcd for C₂6H₂₈FNO [M+H]⁺ 390.2155, found 390.2228.

KSC-352-090

2-(3-(Tert-butyl)phenyl)ethanol. To a vial was added the l-bromo-3-(tert-butyl)benzene (0.107 g, 0.502 mmol) and dry THF. The reaction was then cooled to -78 °C and the BuLi (0.221 mL, 0.552 mmol) (2.5 M in hexanes) was added dropwise and the reaction stirred for 30 min at -78 °C and then ethylene oxide (0.502 mL, 1.255 mmol) (2.5 - 3.3M solution in THF) was added dropwise and the reaction stirred for 10 minutes at -78 °C then warmed to rt and stirred for 1 h. The reaction was then quenched with 1.0 M HC1 (2 mL) and extracted with EtOAc (3 x 5 mL). The EtOAc layer was combined, concentrated and purified by reverse-phase MPLC (10 - 100 % MeCN: water) to produce pure 2-(3-(tert- butyl)phenyl)ethanol (0.035 g, 0.196 mmol, 39% yield). 1H NMR (400 MHz, CDC1₃): δ 7.31 - 7.27 (m, 3H), 7.10 -7.07 (m, 1H), 3.90 (t, J= 6.6 Hz, 2H), 2.91 (t, J= 6.5 Hz, 2H), 1.52 (br s, 1H), 1.36 (s, 9H).

KSC-352-093

3-(Tert-butyl)phenethyl 4-methylbenzenesulfonate. To a vial was added the 2-(3-(tert- butyl)phenyl)ethanol (KSC-352-090) (0.035 g, 0.196 mmol), TEA (0.082 mL, 0.589 mmol) and DCM (2 mL) followed by p-toluenesulfonyl chloride (0.056 g, 0.294 mmol). The reaction began to stir at rt for 20 h and was then diluted with saturated NaHC0₃ and extracted with EtOAc. The EtOAc was then dried with MgS0₄, filtered and concentrated then purified by reverse-phase MPLC (10 - 100% MeCN: water) to provide pure 3-(tert-butyl)phenethyl 4- methylbenzenesulfonate (0.060 g, 0.180 mmol, 92% yield). 1H NMR (400 MHz, CDC1₃): δ 7.70 (d, J= 8.4 Hz, 2H), 7.30 - 7.24 (m, 3H), 7.19 (t, J= 7.52 Hz, 1H), 7.14 - 7.12 (m, 1H), 4.22 (t, J= 7.2 Hz, 2H), 2.96 (t, J= 7.2 Hz, 2H), 2.43 (s, 3H), 1.29 (s, 9H).

KSC-352-097

(l-(3-(Tert-butyl)phenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the 3- (tert-butyl)phenethyl 4-methylbenzenesulfonate (KSC-352-093) (0.060 g, 0.180 mmol), diphenyl(piperidin-4-yl)methanol (0.048 g, 0.180 mmol) and acetonitrile (1 mL). The TEA (0.038 mL, 0.271 mmol) was added and the reaction stirred at 85 °C for 18 h and was cooled to rt then diluted with water and extracted with EtOAc. The EtOAc layer was concentrated and the crude product was purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce the pure (l-(3-(tert-butyl)phenethyl)piperidin-4-yl)diphenylmethanol (0.065 g, 0.152 mmol, 84% yield). 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.47 (m, 4H), 7.32 - 7.27 (m, 4H), 7.23 - 7.16 (m, 5H), 7.01 - 6.98 (m, 1H), 3.09 - 3.04 (m, 2H), 2.81 - 2.76 (m, 2H), 2.61 - 2.55 (m, 2H), 2.51 - 2.43 (m, 1H), 2.28 (br s, 1H), 2.10 - 2.02 (m, 2H), 1.30 (s, 9H), 1.32 - 1.26 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 151.2, 145.9, 140.0, 128.1, 128.0, 126.5, 125.8, 125.71, 125.68, 123.0, 79.5, 54.1, 44.2, 34.6, 34.0, 31.6, 31.4, 26.4, 22.6. LCMS Retention time: 4.214 min. LCMS purity 96.4%. HRMS (ESI): m/z calcd for C₃₀H₃₇NO

[M+H]⁺ 428.2875, found 428.2948.

KSC-352-099

3-((Phenylsulfonyl)methylene)oxetane. To an oven-dried vial was added

(methylsulfonyl)benzene (0.570 g, 3.65 mmol) and the vial was evacuated with argon 3 times. The dry THF (17 mL) was added and the reaction was cooled to 0 °C. The 2.5 M BuLi in hexanes (3.21 mL, 8.03 mmol) was added dropwise and the reaction began to stir at 0 °C and stirred for 45 minutes. The diethyl chlorophosphate (0.528 mL, 3.65 mmol) was then added at 0 °C and the reaction stirred for 30 minutes. The reaction was then cooled to -78 °C and the oxetan-3-one (0.330 mL, 5.15 mmol) was then added dropwise and the reaction stirred for 2 h. The reaction was then warmed to rt and filtered through a silica plug. The reaction was then concentrated onto silica and purified by MPLC (20 min, 0 - 40%

EtOAc:hex) to provide pure 3-((phenylsulfonyl)methylene)oxetane (0.579 g, 2.75 mmol,

75% yield). 1H NMR (400 MHz, CDC1₃): δ 7.91 - 7.87 (m, 2H), 7.69 - 7.64 (m, 1H), 7.60 - 7.55 (m, 2H), 6.12 (quintet, J= 2.3 Hz, 1H), 5.66 - 5.63 (m, 2H), 5.30 - 5.27 (m, 2H).

KSC-367-003

2-(4-(3-((Phenylsulfonyl)methyl)oxetan-3-yl)phenyl)ethanol. To a vial was added the chloro(l,5-cyclooctadiene)rhodium(I), dimer (0.012 g, 0.025 mmol) and 1,4-dioxane (10 mL). The 1.5 M aqueous KOH (0.496 mL, 0.745 mmol) was then added and the reaction stirred for 1 minute at rt. Then the (4-(2-hydroxyethyl)phenyl)boronic acid (0.103 g, 0.621 mmol) and 3-((phenylsulfonyl)methylene)oxetane (KSC-352-099) (0.052 g, 0.248 mmol) in 1 mL dioxane was added and the reaction stirred for 30 minutes at 100 °C in μwaves. The reaction was then cooled to rt and diluted with EtOAc and washed with 1.0 M HC1. The water layer was extracted with EtOAc (3 x 15 mL). The EtOAc layer was collected and the water was extracted with EtOAc again. The EtOAc layers were combined and dried with MgS0₄, filtered and concentrated then purified by reverse-phase MPLC (30 min, 10 - 100% MeCN: water) to produce pure 2-(4-(3-((phenylsulfonyl)methyl) oxetan-3-yl)phenyl)ethanol (0.063 g, 0.190 mmol, 76% yield). 1H NMR (400 MHz, CDC1₃): δ 7.55 - 7.52 (m, 2H), 7.50 - 7.46 (m, 1H), 7.36 - 7.31 (m, 2H), 7.09 (d, J= 8.4 Hz, 2H), 7.01 (d, J= 8.3 Hz, 2H), 5.03 (d, J= 6.4 Hz, 2H), 4.93 (d, J= 6.4 Hz, 2H), 4.03 (s, 2H), 3.82 (t, J= 7.3 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H).

KSC-367-027

4-(diphenylmethylene)piperidine. To a vial was added the diphenyl(piperidin-4- yl)methanol (0.514 g, 1.922 mmol) and TFA (4 mL). The reaction stirred at 75 °C at for 24 h and was concentrated in vacuo. The residue was then dissolved in DCM (5 mL) and washed with water (4 mL). The DCM layer was collected and washed with water (1 x 5 mL), dried with MgS0₄, filtered concentrated then purified by reverse-phase MPLC (10 - 100%

MeCN:Hex) to produce pure 4-(diphenylmethylene)piperidine (0.296 g, 1.187 mmol, 62%> yield). 1H NMR (400 MHz, CDC1₃): δ 7.34 - 7.28 (m, 4H), 7.25 - 7.20 (m, 2H), 7.18 - 7.14 (m, 4H), 2.96 - 2.92 (m, 4H), 2.37 - 2.33 (m, 4H), 1.75 (br s, 1H).

KSC-367-032

4-(4-Benzylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-one. To a vial was added the 4-benzylpiperidine (0.25 ml, 1.422 mmol), l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-one (0.407 g, 1.707 mmol), acetonitrile and TEA (0.297 mL, 2.133 mmol). The reaction stirred at 85 °C for 17 h. The reaction was cooled to rt and diluted with saturated NaHC0₃ then extracted with EtOAc. The EtOAc was dried with MgS0₄, filtered and concentrated. The crude material was purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce 4- (4-benzylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-one (0.291 g, 0.771 mmol, 54% yield). 1H NMR (400 MHz, CDC1₃): δ 7.91 (d, J= 8.6 Hz, 2H), 7.47 (d, J= 8.5 Hz, 2H), 7.29 - 7.24 (m, 2H), 7.20 - 7.16 (m, 1H), 7.14 - 7.11 (m, 2H), 2.96 (t, J= 7.2 Hz, 2H), 2.90 - 2.84 (m, 2H), 2.50 (d, J= 7.0 Hz, 2H), 2.36 (t, J= 7.3 Hz, 2H), 1.96 - 1.82 (m, 4H), 1.63 - 1.56 (m, 2H), 1.53 - 1.46 (m, 1H), 1.34 (s, 9H), 1.28 - 1.18 (m, 2H).

KSC-367-033

3-(4-(Hydroxydiphenylmethyl)piperidin-l-yl)propan-l-ol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.502 g, 1.878 mmol), 3-bromopropan-l-ol (0.204 mL, 2.253 mmol), acetonitrile and TEA (0.393 mL, 2.82 mmol). The reaction stirred at 85 °C for 3 h. The reaction was cooled to rt and diluted with saturated NaHC0₃ and extracted with EtOAc. The EtOAc layer was concentrated and purified by reverse-phase MPLC (10 - 100 % MeCN: water) to provide 3-(4-(hydroxydiphenylmethyl)piperidin-l-yl)propan-l-ol (0.395 g, 1.214 mmol, 65% yield) as a white solid. 1H NMR (400 MHz, CDC1₃): δ 7.48 - 7.44 (m, 4H), 7.31 - 7.27 (m, 4H), 7.20 - 7.15 (m, 2H), 5.45 (br s, 1H), 3.75 (t, J= 5.2 Hz, 2H), 3.12 - 3.06 (m, 2H), 2.57 (t, J= 5.7 Hz, 2H), 2.47 - 2.36 (m, 1H), 1.98 - 1.91 (m, 2H), 1.70 - 1.65 (m, 3H), 1.54 - 1.42 (m, 4H).

KSC-367-036

3-(4-(Hydroxydiphenylmethyl)piperidin-l-yl)propyl 4-methylbenzenesulfonate. Prepared with same method at KSC-352-093 using 3-(4-(hydroxydiphenylmethyl)piperidin-l- yl)propan-l-ol (KSC-367-033) (0.395 g, 1.214 mmol), 4-methylbenzene-l-sulfonyl chloride (0.347 g, 1.821 mmol), DCM (5 mL) and TEA (0.508 mL, 3.64 mmol) to produce pure 3-(4- (hydroxydiphenylmethyl)piperidin-l-yl)propyl 4-methylbenzenesulfonate (0.193 g, 0.402 mmol, 33% yield). 1H NMR (400 MHz, CDC1₃): δ 7.69 - 7.66 (m, 2H), 7.46 - 7.41 (m, 4H), 7.24 - 7.19 (m, 4H), 7.15 - 7.05 (m, 4H), 4.26 - 4.12 (m, 4H), 3.73 - 3.65 (m, 2H), 3.27 - 3.18 (m, 2H), 2.68 - 2.60 (m, 1H), 2.47 - 2.37 (m, 2H), 2.30 (s, 3H), 1.88 - 1.76 (m, 2H), 1.52 - 1.43 (m, 2H).

KSC-367-039

4-(4-Benzylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-ol. Method B: 4-(4- benzylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-one (KSC-367-032) (0.291 g, 0.771 mmol) and MeOH (5 mL) and sodium borohydride (0.117 g, 3.08 mmol) to produce pure 4- (4-benzylpiperidin-l-yl)-l-(4-(tert-butyl)phenyl)butan-l-ol (0.232 g, 0.611 mmol, 79% yield). 1H NMR (400 MHz, CDC1₃): δ 7.35 - 7.25 (m, 6H), 7.21 - 7.12 (m, 3H), 4.64 - 4.60 (m, 1H), 3.13 - 3.07 (m, 1H), 2.94 - 2.88 (m, 1H), 2.54 (d, J= 7.0 Hz, 2H), 2.44 - 2.34 (m, 2H), 2.03 - 1.93 (m, 2H), 1.90 - 1.38 (m, 10H), 1.31 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.4, 142.9, 140.7, 129.1, 128.2, 125.8, 125.4, 125.0, 73.5, 59.0, 54.6, 52.9, 42.9, 40.1, 38.0, 34.4, 31.8, 31.6, 31.4, 24.3. LCMS Retention time: 4.330 min. LCMS purity 99.1%. HRMS (ESI): m/z calcd for C₂₆H₃₇NO [M+H]⁺ 380.2875, found 380.2948.

KSC-367-043

(l-(3-((4-(Tert-butyl)phenyl)amino)propyl)piperidin-4-yl)diphenylmethanol. To a vial was added the 3-(4-(hydroxydiphenylmethyl)piperidin-l-yl)propyl 4-methylbenzenesulfonate (KSC-367-036) (0.088 g, 0.183 mmol), 4-(tert-butyl)aniline (0.035 mL, 0.220 mmol) and TEA (0.038 mL, 0.275 mmol) with acetonitrile (3 mL). The reaction began to stir at 85 °C for 18 h and was then cooled to rt and diluted with saturated NaHC0₃ then extracted with EtOAc. The EtOAc was concentrated and purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce pure (l-(3-((4-(tert-butyl)phenyl)amino) propyl)piperidin-4- yl)diphenylmethanol (0.038 g, 0.083 mmol, 45% yield). 1H NMR (400 MHz, CDC1₃): δ 7.50 - 7.46 (m, 4H), 7.33 - 7.28 (m, 4H), 7.21 - 7.16 (m, 4H), 6.51 (d, J= 8.7 Hz, 2H), 3.13 (t, J = 6.4 Hz, 2H), 3.02 - 2.96 (m, 2H), 2.49 - 2.41 (m, 3H), 2.15 (br s, 1H), 2.00 - 1.93 (m, 2H), 1.80 - 1.73 (m, 2H), 1.57 - 1.43 (m, 5H), 1.27 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 146.4, 145.9, 139.7, 128.2, 126.5, 125.9, 125.8, 112.4, 79.6, 57.4, 54.2, 44.2, 43.7, 33.8, 31.5, 26.6, 26.2. LCMS Retention time: 4.208 min. LCMS purity 100%. HRMS (ESI): m/z calcd for C₃₀H₃₈N₂O [M+H]⁺ 457.6620, found 457.3194.

KSC-367-044

(l-(3-(4-(Tert-butyl)phenoxy)propyl)piperidin-4-yl)diphenylmethanol. To a vial was added the 3-(4-(hydroxydiphenylmethyl)piperidin-l-yl)propyl 4-methylbenzenesulfonate (KSC-367-036) (0.105 g, 0.219 mmol), 4-(tert-butyl)phenol (0.039 g, 0.263 mmol) and TEA (0.046 mL, 0.328 mmol) with acetonitrile (3 mL). The reaction stirred at 85 °C for 18 h then diluted with saturated NaHC0₃ and extracted with EtOAc. The EtOAc layer was

concentrated and purified by RP MPLC (10 - 100% MeCN: water) to produce pure (l-(3-(4- (tert-butyl)phenoxy)propyl)piperidin-4-yl)diphenylmethanol (0.015 g, 0.033 mmol, 15 %> yield). 1H NMR (400 MHz, CDC1₃): δ 7.49 - 7.46 (m, 4H), 7.31 - 7.26 (m, 6H), 7.19 - 7.14 (m, 2H), 6.81 (d, J= 8.8 Hz, 2H), 3.96 (t, J= 6.4 Hz, 2H), 3.01 - 2.94 (m, 2H), 2.51 - 2.40 (m, 3H), 1.99 - 1.89 (m, 5H), 1.52 - 1.45 (m, 4H), 1.28 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 156.7, 145.9, 143.2, 128.1, 126.5, 126.1, 125.7, 116.3, 113.9, 79.5, 66.3, 60.4, 55.4, 54.1, 44.1, 34.0, 31.5, 29.7, 27.0, 26.4. LCMS Retention time: 4.293 min. LCMS purity 95.2%. HRMS (ESI): m/z calcd for C₃₀H₃₇NO₂ [M+H]⁺ 458.6470, found 458.3068.

KSC-367-047

2-(6-(tert-butyl)pyridin-3-yl)ethanol. Prepared by same method as KSC-352-090 with 5- bromo-2-(tert-butyl)pyridine (0.303 g, 1.415 mmol), dry THF (10 mL), 2.5 M BuLi in hexanes (0.623 mL, 1.557 mmol) and ethylene oxide (1.415 ml, 3.54 mmol) (2.5 - 3.3M solution in THF) to produce pure 2-(6-(tert-butyl)pyridin-3-yl)ethanol (0.211 g, 1.177 mmol, 83% yield). ). 1H NMR (400 MHz, CDC1₃): δ 8.40 (br d, J= 2.4 Hz, 1H), 7.48 (dd, J= 8.2 Hz, 2.4 Hz, 1H), 7.26 (dd, J= 8.2 Hz, 0.8 Hz, 1H), 3.85 (br t, J= 6.0 Hz, 2H), 2.82 (t, J= 6.5 Hz, 2H), 2.29 (br s, 1H), 1.33 (s, 9H).

KSC-367-049

2-(6-(tert-butyl)pyridin-3-yl)ethyl 4-methylbenzenesulfonate. Prepared by same method at KSC-352-093 with 2-(6-(tert-butyl)pyridin-3-yl)ethanol (KSC-367-047) (0.211 g, 1.177 mmol), TEA (0.492 mL, 3.53 mmol) and DCM (2 mL) followed by p-toluenesulfonyl chloride (0.337 g, 1.766 mmol) to provide pure 2-(6-(tert-butyl)pyridin-3-yl)ethyl 4- methylbenzenesulfonate (0.260 g, 0.780 mmol, 66% yield). 1H NMR (400 MHz, CDC1₃): δ 8.29 (br d, J= 2.4 Hz, 1H), 7.69 (d, J= 8.3 Hz, 2H), 7.40 (dd, J= 8.2 Hz, 2.4 Hz, 1H), 7.30 - 7.27 (m, 2H), 7.23 (dd, J= 8.2 Hz, 0.8 Hz, 1H), 4.19 (t, J= 6.9 Hz, 2H), 2.92 (t, J= 6.8 Hz, 2H), 2.43 (s, 3H), 1.34 (s, 9H).

KSC-367-052

(l-(2-(6-(Tert-butyl)pyridin-3-yl)ethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.250 g, 0.936 mmol), 2-(6-(tert-butyl)pyridin- 3-yl)ethyl 4-methylbenzenesulfonate (KSC-367-049) (0.260 g, 0.780 mmol) and acetonitrile (5 mL). The TEA (0.163 mL, 1.170 mmol) was then added and the reaction stirred at 50 °C for 20 h and was then cooled to rt and diluted with saturated NaHC0₃ then extracted with DCM. The DCM layer was concentrated and purified by reverse-phase MPLC (10 - 100% MeCN: water) to produce pure (l-(2-(6-(tert-butyl)pyridin-3-yl)ethyl)piperidin-4- yl)diphenylmethanol (0.308 g, 0.719 mmol, 92% yield). 1H NMR (400 MHz, CDC1₃): δ 8.39 - 8.38 (m, 1H), 7.51 - 7.48 (m, 4H), 7.42 (dd, J= 8.1 Hz, 2.4 Hz, 1H), 7.32 - 7.27 (m, 4H), 7.25 - 7.22 (m, 1H), 7.20 - 7.15 (m, 2H), 3.07 - 3.01 (m, 2H), 2.76 - 2.71 (m, 2H), 2.57 - 2.52 (m, 2H), 2.50 - 2.42 (m, 1H), 2.31 (br s, 1H), 2.09 - 2.01 (m, 2H), 1.57 - 1.48 (m, 4H), 1.35 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 166.9, 148.7, 145.9, 136.3, 132.3, 128.1, 126.5, 125.8, 118.6, 79.4, 60.2, 54.0, 44.1, 37.0, 30.3, 30.2, 26.4. LCMS Retention time: 3.802 min. LCMS purity 99.5%. HRMS (ESI): m/z calcd for C₂9H₃₆N₂0 [M+H]⁺ 429.2828, found 429.2900.

KSC-367-053

l-(4-(Tert-butyl)phenyl)-4-(4-(diphenylmethylene)piperidin-l-yl)butan-l-one. To a vial was added the 4-(diphenylmethylene)piperidin-l-ium 2,2,2-trifluoroacetate (KSC-367-027) (0.0475 g, 0.131 mmol), l-(4-(tert-butyl)phenyl)-4-chlorobutan-l-one (0.037 g, 0.157 mmol) and TEA (0.046 mL, 0.327 mmol) in acetonitrile. The reaction stirred at 75 °C for 20 h. The reaction was removed from heat and diluted with saturated NaHC0₃, extracted with EtOAc. The EtOAc layer was concentrated and purified by reverse-phase MPLC (10 - 100%

MeCN: water) to produce pure l-(4-(tert-butyl)phenyl)-4-(4-(diphenylmethylene)piperidin-l- yl)butan-l-one (0.019 g, 0.042 mmol, 32% yield). 1H NMR (400 MHz, CDC1₃): δ 7.91 (d, J = 8.2 Hz, 2H), 7.46 (d, J= 8.3 Hz, 2H), 7.30 - 7.25 (m, 4H), 7.21 - 7.17 (m, 2H), 7.13 - 7.10 (m, 4H), 2.99 (t, J= 7.2 Hz, 2H), 2.51 - 2.32 (m, 10H), 1.98 - 1.91 (m, 2H), 1.34 (s, 9H).

KSC-367-055

(l-(4-Chlorophenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(piperidin-4-yl)methanol (0.221 g, 0.827 mmol), l-(2-bromoethyl)-4-chlorobenzene (0.165 g, 0.752 mmol), acetonitrile (4 mL) and TEA (0.157 mL, 1.128 mmol). The reaction stirred at 75 °C for 18 h then was cooled to rt and diluted with saturated NaHC0₃ and extracted with EtOAc. The EtOAc layer was concentrated and purified by reverse-phase MPLC (10 - 100%) MeCN: water) to produce pure (l-(4-chlorophenethyl)piperidin-4- yl)diphenylmethanol (0.224 g, 0.552 mmol, 73% yield). 1H NMR (400 MHz, CDC1₃): δ 7.39 - 7.36 (m, 4H), 7.20 - 7.15 (m, 4H), 7.12 - 7.09 (m, 2H), 7.08 - 7.03 (m, 2H), 6.99 - 6.96 (m, 2H), 2.92 - 2.87 (m, 2H), 2.64 - 2.58 (m, 2H), 2.43 - 2.38 (m, 2H), 2.37 - 2.30 (m, 1H), 2.22 (br s, 1H), 1.95 - 1.88 (m, 2H), 1.44 - 1.35 (m, 4H). ¹³C NMR (125 MHz, CDC1₃): δ 145.9, 145.8, 138.9, 131.6, 129.9, 128.3, 128.0, 126.4, 125.7, 79.4, 79.3, 60.4, 57.0, 44.1, 33.0, 26.4. LCMS Retention time: 3.903 min. LCMS purity 99.6%. HRMS (ESI): m/z calcd for C₂₆H₂₈C1N0 [M+H]⁺ 406.1859, found 406.1932.

KSC-367-058

l-(4-(Tert-butyl)phenyl)-4-(4-(diphenylmethylene)piperidin-l-yl)butan-l-ol. Method B: l-(4-(tert-butyl)phenyl)-4-(4-(diphenylmethylene)piperidin-l-yl)butan-l-one (KSC-367-053) (0.019 g, 0.042 mmol) and sodium borohydride (6.37 mg, 0.168 mmol) to produce pure l-(4- (tert-butyl)phenyl)-4-(4-(diphenylmethylene)piperidin-l-yl)butan-l-ol (0.010 g, 0.022 mmol, 52% yield). 1H NMR (400 MHz, CDC1₃): δ 7.35 - 7.26 (m, 8H), 7.23 - 7.18 (m, 2H), 7.13 - 7.10 (m, 4H), 4.68 - 4.65 (m, 1H), 2.66 - 2.60 (m, 2H), 2.55 - 2.42 (m, 8H), 2.00 - 1.94 (m, 1H), 1.90 - 1.81 (m, 1H), 1.73 - 1.63 (m, 3H), 1.30 (s, 9H). ¹³C NMR (125 MHz, CDC1₃): δ 149.5, 142.7, 142.3, 136.4, 134.6, 129.7, 128.0, 126.4, 125.3, 125.0, 73.3, 58.7, 55.0, 39.6, 34.4, 31.6, 31.4, 31.1, 24.0, 22.6. LCMS Retention time: 4.454 min. LCMS purity 97.1%. HRMS (ESI): m/z calcd for C₃₂H₃₉NO [M+H]⁺ 454.3032, found 454.3104.

KSC-367-066

4-(3-((phenylsulfonyl)methyl)oxetan-3-yl)phenethyl 4-methylbenzenesulfonate. Prepared by the same method as KSC-352-093 with 2-(4-(3-((phenylsulfonyl)methyl)oxetan-3- yl)phenyl)ethanol (KSC-367-003) (0.104 g, 0.313 mmol), TEA (0.131 mL, 0.939 mmol) and DCM (2 mL) followed by p-toluenesulfonyl chloride (0.089 g, 0.469 mmol) to produce 4-(3- ((phenylsulfonyl)methyl)oxetan-3-yl)phenethyl 4-methylbenzenesulfonate (0.041 g, 0.084 mmol, 27% yield). 1H NMR (400 MHz, CDC1₃): δ 7.75 (d, J= 8.3 Hz, 2H), 7.57 - 7.46 (m, 4H), 7.36 - 7.32 (m, 4H), 7.02 - 6.96 (m, 3H), 5.00 (d, J= 6.4 Hz, 2H), 4.93 (d, J= 6.4 Hz, 2H), 4.17 (t, J = 7.0 Hz, 2H), 4.01 (s, 2H), 2.90 (t, J= 7.0 Hz, 2H), 2.45 (s, 3H).

KSC-367-069

Diphenyl(l-(4-(3-((phenylsulfonyl)methyl)oxetan-3-yl)phenethyl)piperidin-4- yl)methanol. To a vial was added the 4-(3-((phenylsulfonyl)methyl)oxetan-3-yl)phenethyl 4- methylbenzenesulfonate (KSC-367-066) (0.041 g, 0.084 mmol) and diphenyl(piperidin-4- yl)methanol (0.025 g, 0.093 mmol) in acetonitrile. The TEA (0.018 ml, 0.126 mmol) was then added and the reaction stirred at 50 °C for 18 h. The reaction was removed from heat and cooled to rt then diluted with saturated NaHC0₃. The reaction was then extracted with EtOAc (3 x 5 mL) and the EtOAc layer was dried with MgS0₄, filtered and concentrated. The crude residue was purified by reverse-phase MPLC (15 min, 10 - 100% MeCN: water) to produce diphenyl(l-(4-(3 ((phenylsulfonyl) methyl)oxetan-3-yl)phenethyl)piperidin-4- yl)methanol (0.024 g, 0.041 mmol, 49% yield). 1H NMR (400 MHz, CDC1₃): δ 7.53 - 7.43

(m, 6H), 7.33 - 7.27 (m, 7H), 7.21 - 7.16 (m, 2H), 7.04 (d, J= 8.2 Hz, 2H), 6.96 (d, J= 8.2 Hz, 2H), 5.02 (d, J= 6.4 Hz, 2H), 4.92 (d, J= 6.4 Hz, 2H), 4.01 (s, 2H), 3.08 - 3.02 (m, 2H), 2.74 - 2.69 (m, 2H), 2.56 - 2.44 (m, 3H), 2.10 - 2.02 (m, 2H), 1.60 - 1.50 (m, 5H).

KSC-367-072

(l-(4-(3-Methyloxetan-3-yl)phenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the diphenyl(l -(4-(3-((phenylsulfonyl)methyl)oxetan-3-yl)phenethyl)piperidin-4- yl)methanol (KSC-367-069) (0.024 g, 0.041 mmol) and MeOH (10 mL) and the reaction as heated to 50 °C. The magnesium was added in 3 additions (0.016 g, 0.066 mmol, 16 equiv) 1.5 h apart. The reaction was removed from heat 1.5 h after the final magnesium addition, cooled to rt and poured into 1.0 M HCl with ice. The aqueous layer was then extracted with DCM (3 x 10 mL) and the organic layers were combined and dried with MgS0₄, filtered and concentrated then purifed by reverse-phase MPLC (15 min, 10 - 100% MeCN: water). To produce (l-(4-(3-methyloxetan-3-yl)phenethyl)piperidin-4-yl)diphenylmethanol (0.0045 g, 10.19 μιηοΐ, 24.70 % yield) as a light brown solid. 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.47

(m, 4H), 7.32 - 7.27 (m, 4H), 7.21 - 7.16 (m, 4H), 7.13 - 7.10 (m, 2H), 4.95 (d, J= 5.5 Hz, 2H), 4.61 (d, J= 5.5 Hz, 2H), 3.09 - 3.02 (m, 2H), 2.81 - 2.75 (m, 2H), 2.59 - 2.54 (m, 2H), 2.50 - 2.42 (m, 1H), 2.16 (br s, 1H), 2.09 - 2.00 (m, 2H), 1.71 (s, 3H), 1.56 - 1.48 (m, 4H). C NMR (125 MHz, CDC1₃): δ 145.9, 144.1, 138.4, 128.8, 128.2, 126.5, 125.8, 125.1, 83.8, 79.5, 60.7, 54.1, 44.1, 43.1, 33.3, 27.8, 26.4. LCMS Retention time: 3.652 min. LCMS purity 97.9%. HRMS (ESI): m/z calcd for C₃oH₃₅N0₂ [M+H]⁺ 442.2668, found 442.2741.

KSC-367-088

2-(Tert-butyl)phenyl trifluoromethanesulfonate. To a vial was added the 2-(tert- butyl)phenol (1.0 ml, 6.51 mmol) and DCM (4 mL) followed by pyridine (1.053 mL, 13.02 mmol). The reaction was then cooled to 0 °C and the triflic anhydride (1.320 mL, 7.81 mmol) was added dropwise and the reaction stirred for 2 h. The reaction was then allowed to warm to rt and was diluted with DCM and quenched with 1.0 M HC1. The DCM layer was collected and washed with saturated NaHC0₃ and brine. The organic layer was then dried (MgS0₄), filtered and adsorbed to silica and purified by MPLC (0 - 25% EtOAc:hex) to produce pure 2-(tert-butyl)phenyl trifluoromethanesulfonate (1.66 g, 5.88 mmol, 90%> yield).

1H NMR (400 MHz, CDC1₃): δ 7.49 - 7.46 (m, 1H), 7.37 - 7.34 (m, 1H), 7.31 - 7.27 (m, 2H), 1.43 (s, 9H).

KSC-381-009

2-(2-(Tert-butyl)phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane. To a flame-dried vial was added molecular sieves and 2-(tert-butyl)phenyl trifluoromethanesulfonate (KSC-367- 088) (0.475 g, 1.683 mmol) and l, -Bis(diphenylphosphino)ferrocenedichloropalladium(II) (0.042 g, 0.050 mmol). The vial was evacuated with argon 3 times and then dioxane (1 mL), TEA (0.704 mL, 5.05 mmol) and 4,4,5,5-tetramethyl-l,3,2-dioxaborolane (0.732 mL, 5.05 mmol) were added via syringe. The reaction stirred at reflux (100 °C) for 2 h. The reaction was then removed from heat and diluted with water then extracted with DCM (3 x 5 mL). The DCM layer was then washed again with water (3 x 10 mL). The DCM layer was dried with MgS0₄, filtered and concentrated. The residue was diluted with hexanes and filtered through MgS0₄ to remove the residual Pd complex. The hexanes layer was then

concentrated to produce pure 2-(2-(tert-butyl)phenyl)-4,4,5,5-tetramethyl-l,3,2- dioxaborolane (0.093 g, 0.357 mmol, 86% yield). 1H NMR (400 MHz, CDC1₃): δ 7.45 (dd, J = 7.2 Hz, 1.2 Hz, 1H), 7.41 - 7.39 (m, 1H), 7.29 (td, J= 7.6 Hz, 2.0 Hz, 1H), 7.14 (td, J= 7.6 Hz, 2.0 Hz, 1H), 1.41 (s, 9H), 1.38 (s, 12H).

KSC-381-011

l-Bromo-2-(tert-butyl)benzene. To a vial was added the 2-(2-(tert-butyl)phenyl)-4,4,5,5- tetramethyl-l,3,2-dioxaborolane (KSC-381-009) (0.180 g, 0.692 mmol) and MeOH (1.5 mL). The copper (II) bromide (0.464 g, 2.075 mmol) was then dissolved in water and added to the reaction then stirred at 80 °C for 24 h. The reaction was then removed from heat and diluted with water and extracted with EtOAc. The EtOAc was dried with MgS0₄, filtered and concentrated to produce l-bromo-2-(tert-butyl)benzene (0.103 g, 0.483 mmol, 70% yield) as a brown liquid. 1H NMR (400 MHz, CDC1₃): δ 7.59 (dd, J= 8.0 Hz, 1.6 Hz, 1H), 7.44 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.24 (td, J= 7.6 Hz, 2.0 Hz, 1H), 7.02 (td, J= 7.6 Hz, 2.0 Hz, 1H), 1.51 (s, 9H).

KSC-381-015

2-(2-(Tert-butyl)phenyl)ethanol. Prepared by same method as KSC-352-090 with 1-bromo- 2-(tert-butyl)benzene (KSC-381-011) (0.147 g, 0.690 mmol), dry THF, 2.5 M BuLi in hexanes (0.303 ml, 0.759 mmol) and then ethylene oxide (0.690 ml, 1.724 mmol) (2.5 - 3.3M solution in THF) to produce pure 2-(2-(tert-butyl)phenyl)ethanol (0.018 g, 0.101 mmol, 14.64 % yield) as a clear oil. 1H NMR (400 MHz, CDC1₃): δ 7.43 - 7.37 (m, 1H), 7.23 - 7.19 (m, 1H), 7.18 - 7.14 (m, 2H), 3.90 (t, J= 7.6 Hz, 2H), 3.19 (t, J = 7.6 Hz, 2H), 1.44 (s, 9H).

KSC-381-018

2-(Tert-butyl)phenethyl 4-methylbenzenesulfonate. Prepared according to same procedure as KSC-352-093 with 2-(2-(tert-butyl)phenyl)ethanol (KSC-381-015) (0.018 g, 0.101 mmol), TEA (0.042 mL, 0.303 mmol) and DCM (2 mL) followed by p-toluenesulfonyl chloride (0.029 g, 0.151 mmol) to provide pure 2-(tert-butyl)phenethyl 4- methylbenzenesulfonate (0.022 g, 0.066 mmol, 66% yield) and a clear oil. 1H NMR (400 MHz, CDCls): δ 7.76 (d, J= 8.0 Hz, 2H), 7.37 - 7.31 (m, 3H), 7.17 - 7.04 (m, 3H), 4.19 (t, J = 7.6 Hz, 2H), 3.25 (t, J= 7.6 Hz, 2H), 2.44 (s, 3H), 1.33 (s, 9H).

KSC-381-020

(l-(2-(Tert-butyl)phenethyl)piperidin-4-yl)diphenylmethanol. To a vial was added the 2- (tert-butyl)phenethyl 4-methylbenzenesulfonate (KSC-381-018) (0.022 g, 0.066 mmol), diphenyl(piperidin-4-yl)methanol (0.018 g, 0.066 mmol) and acetonitrile. The TEA (0.014 mL, 0.099 mmol) was added and the reaction stirred at 85 °C for 20 h. The reaction was cooled to rt and diluted with water then extracted with EtOAc. The EtOAc layer was concentrated and the crude product was purified by reverse-phase MPLC (10 - 100%

MeCN: water) to produce the pure (l-(2-(tert-butyl)phenethyl) piperidin-4- yl)diphenylmethanol (0.013 g, 0.030 mmol, 46% yield). 1H NMR (400 MHz, CDC1₃): δ 7.51 - 7.48 (m, 4H), 7.37 - 7.28 (m, 5H), 7.21 - 7.09 (m, 5H), 3.15 - 3.04 (m, 4H), 2.65 - 2.60 (m, 2H), 2.52 - 2.42 (m, 1H), 2.17 - 2.08 (m, 3H), 1.60 - 1.53 (m, 4H), 1.41 (s. 9H). ¹³C NMR (125 MHz, CDC1₃): δ 147.7, 145.9, 138.5, 132.1, 128.2, 126.5, 126.1, 125.9, 125.84, 125.79, 79.5, 61.7, 54.2, 44.2, 35.7, 31.7, 29.7, 26.4. LCMS Retention time: 4.200 min. LCMS purity 99.3%. HRMS (ESI): m/z calcd for C₃₀H₃₇NO [M+H]⁺ 428.2875, found 428.2948.

Bacterial Strains and Conditions

All strains used to evaluate the antimicrobial activity of terfenadine and

corresponding structural derivatives are shown below in Table 5. S. aureus strain UAMS-1 is an osteomyelitis clinical isolate (Gillaspy et al. "Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect. Immun. 63:3373-3380 (1995)), whereas cipro floxacin-resistant strains CRC118 and CRC61 are spontaneous ciprofloxacin-resistant derivatives of UAMS-1 that were selected by growth on Mueller-Hinton agar (MHA) (Becton, Dickinson & Company, Franklin Lakes, NJ) at 1.5X MIC ciprofloxacin (0.75 μg/ml).

Species Strain Source

S. aureus UAMS-1 1

CRC61 Dunman Lab, URMC

CRC118 Dunman Lab, URMC

Minimum Inhibitory Concentration (MIC) Testing

Minimum Inhibitory Concentration (MIC) testing was performed to determine the minimum concentration of test compound that is necessary to inhibit visible growth of bacteria according to Clinical and Laboratory Standards (CLSI) guidelines (Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard— Ninth Edition. CLSI document M07-A9. 2012. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA.) To do so, 10⁵ colony forming units of an overnight bacterial culture were seeded into individual wells of 96-well round- bottom microtiter plates containing 88 μΐ of MHB medium. To the first column, 2 μΐ of the test compound's corresponding solvent was also added to each well (negative control). To the next ten columms, 2 μΐ of the test compound (dissolved in DMSO for terfenadine and its derivatives or sterile water for ciprofloxacin) was added in increasing 2-fold increments of 0.5 μg/ml to 256 μg/ml to each successive well. Each test compound was evaluated in duplicate. Plates were incubated at 37°C incubator for 16 h at which point the minimum inhibitory concentration was determined to be the lowest concentration of test compound that inhibited bacterial growth, as judged by the unaided eye.

S. aureus DNA Gyrase Supercoiling Assay (TopoGEN)

The gyrase supercoiling assays were performed to determine if test compounds interfered with S. aureus DNA gyrase activity, following the manufacturer's recommendations (TopoGEN). Reactions (20 μΐ) contained kit provided assay buffer, ATP, potassium glutamate, relaxed plasmid DNA (0.4 μg/ml), 2 Units of S. aureus DNA gyrase, and various amounts of test compound. Reactions were incubated at 37°C for 30 min and then stopped by the addition of 10% SDS, filtered through 0.025 μιη Millipore membrane filters in a 10 mM Tris-HCl buffer (pH 8), and electrophoresed in a 1% agarose TAE gel. Gels were stained with 0.5 μg/ml ethidium bromide and images were analyzed using densitometry (Image J, NIH). The IC₅₀ values for each test compound was determined to be the compound concentration that inhibited S. aureus DNA gyrase activity by 50%. S. aureus Topoisomerase IV Decatenation Assay (Inspiralis)

A Topoisomerase IV assay was performed on test compounds to determine if they interfered with the ability of S. aureus topoisomerase IV to decatenate kDNA, according to the recommendations of the manufacturer (Inspiralis). To do so, 0.25 U S. aureus topoisomerase IV enzyme was mixed with 200 ng kDNA in kit provided reaction buffer, in the absence or presence of various concentrations of test compounds at 37°C for 30 min. The reaction was stopped by the addition of STEB stop buffer and 30 μΐ of 24: 1

chlorform:isoamyl alcohol (total volume 90 μΐ). Reaction products then electrophoresed in a 1%) agarose TAE gel, stained with 0.5 μg/ml ethidium bromide and images were analyzed using densitometry (Image J, NIH). The IC₅₀ value for each test compound was determined as the compound concentration that inhibited S. aureus topoisomerase IV activity by 50%.

The results are set forth in Table 6.

Claims

What is claimed is:

1. A method of treating or preventing an infection in a subject with or at risk of

developing an infection, the method comprising administering to the subject a a compound of Formula I

R

wherein R represents hydrogen or hydroxyl and R¹ represents hydrogen, or R and R¹ taken together form a second bond between the carbon atoms bearing R and R¹; R² represents hydrogen or phenyl; n is zero or a positive whole integer of from 1 to 4; X represents CH₂, CHOH, NH, C=0, CHNR³R⁴, where R³ and R⁴ independently are hydrogen or lower alkyl; and Z represents thienyl, pyridinyl, substituted pyridinyl, phenyl or substituted phenyl wherein the substituents on the substituted pyridinyl or substituted phenyl are selected from phenyl, pyridinyl, nitro, a halogen atom, such as chlorine, fluorine, bromine, or iodine, a straight or branched lower alkyl chain of from 1 to 4 carbon atoms, a lower alkoxy group of from 1 to 4 carbon atoms, amino, a mono or di(lower)alkylamino group, a saturated monocyclic heterocyclic group such as pyrrolidino, piperidino, morpholino, or N-(lower)alkylpiperazino, or a group having the structure -COOR⁵, -CR⁶R⁷COOR⁵, -CF₃, CHF₂, CH₂F, or

where R⁵ is hydrogen or lower alkyl, and R⁶ and R⁷ independently are hydrogen methyl or a pharmaceutically acceptable salt thereof.

A method of treating or preventing an infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound of Formula III

3. A method of treating or preventing infection in a subject with or at risk of developing an infection, the method comprising administering to the subject a compound selected from the group consisting of:

a compound of F

(V) wherein Ri is a lower alkyl group having from 1 to 4 carbon atoms being substituted with one or several halogen atoms, Z is hydrogen or a lower alkyl group having from 1 to 4 carbon atoms, m is 2 or 3 and X₂ is the ethylene imino group or the group having the formula:

wherein R is hydrogen or a lower alkyl group having from 1 to 4 carbon atoms which may be substituted with a chlorine atom or a hydroxy group, and Z and m have the above-given meaning, a compound of Formula X

X represents -0-, -S-, -SO-, or -S0₂-, n represents an integer from 1-4, and

wherein R and R⁶ are the same or different and are hydroxy, lower alkoxy, lower alkenoxy, dilower alkylamino lower alkoxy (dimethylaminoethoxy), acylamino lower alkoxy (acetylaminoethoxy), acyloxy lower alkoxy (pivaloyloxymethoxy), aryloxy, such as phenoxy, arloweralkoxy, such as benzyloxy, substituted aryloxy or substituted arloweralkoxy wherein the substitutent is methyl, halo or methoxy, amino, loweralkylamino, diloweralkylamino, hydroxy amino, arloweralkylamino such as benzylamino;

R¹ is hydrogen, alkyl of from 1 to 20 carbon atoms which include branched and cyclic and unsaturated (such as allyl) alkyl groups, substituted loweralkyl wherein the substituent can be halo, hydroxy, lower alkoxy, aryloxy such as phenoxy, amino, diloweralkylamino, acylamino, such as acetamido and benzamido, arylamino, guanidino, imidazolyl, indolyl, mercapto, loweralkylthio, arylthio such as phenylthio, carboxy or carboxamido, carboloweralkoxy, aryl such as phenyl or naphthyl, substituted aryl such as phenyl wherein the substituent is lower alkyl, lower alkoxy or halo, arloweralkyl, arloweralkenyl, heteroarlower alkyl or heteroarlower alkenyl such as benzyl, styryl or indolyl ethyl, substituted arloweralkyl, substituted arloweralkenyl, substituted heteroarlower alkyl, or substituted heteroarlower alkenyl, wherein the substituent(s) is halo, dihalo, lower alkyl, hydroxy, lower alkoxy, amino,

aminomethyl, acylamino (acetyl amino or benzoylamino) diloweralkylamino, loweralkylamino, carboxyl, haloloweralkyl, cyano or sulfonamido; arloweralkyl or heteroarloweralkyl substituted on the alkyl portion by amino or acylamino

(acetylamino or benzoylamino);

R² and R⁷ are the same or different and are hydrogen or lower alkyl;

R³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxy phenyl lower alkyl, hydroxy lower alkyl, acylamino lower alkyl (such as benzoylamino lower alkyl, acetylamino lower alkyl), amino lower alkyl,

dimethylamino lower alkyl, halo lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyllower alkyl, mercapto lower alkyl, lower alkyl thio lower alkyl;

R⁴ is hydrogen or lower alkyl;

R⁴ and R⁵ may be connected together to form an alkylene bridge of from 2 to 4 carbon atoms, an alkylene bridge of from 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge of from 3 to 4 carbon atoms containing a double bond or an alkylene bridge as above substituted with hydroxy, loweralkoxy, loweralkylor diloweralky; a compound of Formula XIII

wherein ¾ is H, alkyl, acyl or silyl(alkyl)₃; R₂ is H and R₃ is OH, O-acyl, O-alkyl or O-silyl (alkyl)₃ or R₃ is H and R₂ is OH. O-acyl, O-alkyl or O-silyl (alkyl)₃; or R₂ and R₃ together represent O; or R₂ and R₃ together represent acetal or cyclic acetal. Ri might also represent a substituted alkyl such as e.g. methoxy ethoxy methyl; a compound of Formula XIV

(XIV) wherein PY is 4- or 3- or 2-pyridinyl or 4- or 3 -or 2-pyridinyl having one or two lower-alkyl substituents, R is hydrogen, lower-alkyl or lower-hydroxyalkyl, and Q is nitro, carbamyl, halo, amino, lower-alkylamino, di(lower-alkyl)amino, or NHAc where Ac is lower-alkanoyl or lower-carbalkoxy; a compound of Formula XV

a compound of Formula XVI

a compound of Formula XVII

(XVII) a compound of Formula XVIII

(XVIII) a compound of Formula XIX

(XIX) a compound of Formula (XX)

(XX) or a pharmaceutically acceptable salt thereof.

4. The method of claim 3, wherein the compound is selected from the group consisting of ifosfamide, sulfinpyrazone, lisinopril, equilin, fluspirilen, hexestrol, dienestrol, napelline, amrinone and iopanoic acid.

5. The method of any of claims 1-4, wherein the infection is a bacterial infection, a viral infection, a parasitic infection or a fungal infection.

6. The method of any one of claims 1-5, wherein the infection is a respiratory infection.

7. The method of any one of claims 1-5, wherein the infection is a gastrointestinal

infection.

8. The method of any one of claims 1-5, wherein the infection is a skin infection.

9. The method of claim 5 wherein the bacterial infection is selected from the group consisting of Enter obacterium faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinebacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.

10. The method of claim 5, wherein the bacterial infection is a small colony variant bacterial infection.

11. A method of treating or preventing an infection in a subject with or at risk of

developing an infection, the method comprising administering to the subject a compound selected from the group consisting of: Didanosine, Norcyclobenzaprine, Niridazole, Ifosfamide, Cefalonium, Tamoxifen citrate, Butoconazole, Suloctidil, Clomiphene, Sulconazole, Miconazole, Mefloquine, Sulfinpyrazone, Terfenadine, Lisinopril, Econzaole, Clofazimine, Equilin, Felodipine, Dacarbazine, Furazolidone, Perhexiline maleate, Oxethazaine, Pimozide, Trifluoperazine, Ellipticine, Fluspirilen, Hexestrol, Dienestrol, Zidovudine, Metoprolol, Napelline, Methimazole, Amrinone, Iopanoic acid, R-Propanolol, Rimexolone and Pyrvinium pamoate, wherein the infection is a bacterial infection.

12. The method of claim 11, wherein the bacterial infection is selected from the group consisting of Enter obacterium faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinebacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.

13. A method of removing or preventing bio film formation on a surface, the method

comprising administering to a biofilm containing surface or a surface susceptible to bio film formation an effective amount of a compound of Formula I

(CHiln-X-Zl

(I) wherein R represents hydrogen or hydroxyl and R¹ represents hydrogen, or R and R¹ taken together form a second bond between the carbon atoms bearing R and R¹; R² represents hydrogen or phenyl; n is zero or a positive whole integer of from 1 to 4; X represents CH₂, CHOH, NH, C=0, CHNR³R⁴, where R³ and R⁴ independently are hydrogen or lower alkyl; and Z represents thienyl, pyridinyl, substituted pyridinyl, phenyl or substituted phenyl wherein the substituents on the substituted pyridinyl or substituted phenyl are selected from phenyl, pyridinyl, nitro, a halogen atom, such as chlorine, fluorine, bromine, or iodine, a straight or branched lower alkyl chain of from 1 to 4 carbon atoms, a lower alkoxy group of from 1 to 4 carbon atoms, amino, a mono or di(lower)alkylamino group, a saturated monocyclic heterocyclic group such as pyrrolidino, piperidino, morpholino, or N-(lower)alkylpiperazino, or a group having the structure -COOR⁵, -CR⁶R⁷COOR⁵, -CF₃, CHF₂, CH₂F, or

14. The method of claim 13, wherein the bio film comprises one or more of

Staphylococcus aureus, Pseudomonas aeuroginosa, Staphylococcus epidermidis, Escherichia coli or Acinetobacter baummanii.

15. A method of identifying an antimicrobial agent comprising:

a) contacting a bacterial culture with a test agent;

b) and measuring adenylate kinase release in the supernatant of the bacterial culture, wherein an increase in adenylate kinase release as compared to a control indicates that the test compound is an antimicrobial agent.

16. The method of claim 15, wherein the bacterial culture is selected from the group consisting of Staphylococcus aureus, Pseudomonas aeuroginosa, Staphylococcus epidermidis, Escherichia coli or Acinetobacter baummanii.