RELATED APPLICATION INFORMATION

This application is a CONTINUATION of application Ser. No. 15/060,179 filed Mar. 3, 2016, which is a CONTINUATION of application Ser. No. 13/479,590 filed May 24, 2012 which is a CIP of International Application PCT/US2011/027974 filed 10 Mar. 2011 entitled “Phenoxy Thiophene Sulfonamides And Their Use As Inhibitors Of Glucuronidase”, which was published in the English language on 15 Sep. 2011, with International Publication Number WO 2011/112858 A1, and which claims priority from U.S. Patent Application No. 61/312,512 filed on 10 Mar. 2010, the content of which is incorporated herein by reference.

This invention was supported in part by funds from the U.S. Government (National Cancer Institute 04-051311 and National Institutes of Health grant 1SC2GM081129). The U.S. Government may have certain rights in the invention.

This invention relates generally to phenoxy thiophene sulfonamides and other drugs that inhibit bacterial glucuronidase. This invention also relates to compositions including one or more of such compounds and methods of using one or more of such compounds as a co-drug in combination with a camptothecin-derived anticancer drug or other drug that, in a patient, is metabolized to form a metabolite that is a substrate for a bacterial β-glucuronidase enzyme. The invention further relates to a method of screening for such compounds. The invention also encompasses a method for selectively inhibiting, in a patient to be treated, bacterial-glucuronidase as compared with mammalian β-glucuronidase, wherein the method comprises administering to the patient an effective amount of a compound selected from the group consisting of nialamide, isocarboxazid, phenelzine, amoxapine, loxapine and mefloquine.

BACKGROUND

Camptothecin, a plant alkaloid derived from the Chinese Camptotheca acuminata tree, was added to the National Cancer Institute's natural products screening set in 1966. It showed strong anti-neoplastic activity but poor bioavailability and toxic side effects. After thirty years of modifying the camptothecin scaffold, two derivatives emerged and are now approved for clinical use. Topotecan (Hycamptin®; GlaxoSmithKline) is currently employed to treat solid ovarian, lung and brain tumors. CPT-11 (also called Irinotecan, and Camptosar®; Pfizer) contains a carbamate-linked dipiperidino moiety that significantly increases bioavailability in mammals. This dipiperidino group is removed from the CPT-11 prodrug in vivo by carboxylesterase enzymes that hydrolyze the carbamate linkage to produce the drug's active metabolite, SN-38. CPT-11 is currently used to treat solid colon, lung, and brain tumors, along with refractory forms of leukemia and lymphoma.

The sole target of the camptothecins is human topoisomerase I. This enzyme relieves superhelical tension throughout the genome and is essential for DNA metabolism, including DNA replication, transcription, and homologous recombination. Topoisomerase I breaks one strand in duplex DNA, forming a covalent 3′-phosphotyrosine linkage, and guides the relaxation of DNA supercoils. It then reseals the single-strand DNA break and releases a relaxed duplex DNA molecule. The camptothecins bind to the covalent topoisomerase I-DNA complex and prevent the religation of the broken single DNA strand, effectively trapping the 91 kDa protein on the DNA. Such immobilized macromolecular adducts act as roadblocks to the progression of DNA replication and transcription complexes, causing double-strand DNA breaks and apoptosis. Because cancer cells are growing rapidly, the camptothecins impact neoplastic cells more significantly than normal human tissues. Structural studies have established that the camptothecins stack into the duplex DNA, replacing the base pair adjacent to the covalent phosphotyrosine linkage. Religation of the nicked DNA strand is prevented by increasing the distance between the 5′-hydroxyl and the 3′-phosphotyrosine linkage to >11 Å.

CPT-11 efficacy is severely limited by delayed diarrhea that accompanies treatment. While an early cholinergic syndrome that generates diarrhea within hours can be successfully treated with atropine, the diarrhea that appears 2-4 days later is significantly more debilitating and difficult to control. CPT-11 undergoes a complex cycle of activation and metabolism that directly contributes to drug-induced diarrhea. CPT-11 administered by intravenous injection can traffic throughout the body, but concentrates in the liver where it is activated to SN-38 by the human liver carboxylesterase hCE1. The SN-38 generated in the liver is conjugated in the liver to yield SN-38 glucuronide (SN-38G). SN-38G is excreted from the liver via the bile duct and into the intestines. Once in the intestines, however. SN-38G serves as a substrate for bacterial glucuronidase enzymes in the intestinal flora that remove the glucuronide moiety and produce the active SN-38. SN-38 in the intestinal lumen produced in this manner contributes to epithelial cell death and the severe diarrhea that limits CPT-11 tolerance and efficacy. This effect has been partially reversed in rats using the relatively weak (IC₅₀=90 μM) β-glucuronidase inhibitor saccharic acid 1,4-lactone.

While broad-spectrum antibiotics have been used to eliminate enteric bacteria from the gastrointestinal tract prior to CPT-11 treatment, this approach has several drawbacks. First, intestinal flora play essential roles in carbohydrate metabolism, vitamin production, and the processing of bile acids, sterols and xenobiotics. Thus, the partial or complete removal of gastrointestinal bacteria is non-ideal for patients already challenged by neoplastic growths and chemotherapy. Second, it is well established that the elimination of the symbiotic gastrointestinal flora from even healthy patients significantly increases the chances of infections by pathogenic bacteria, including enterohemorrhagic E. coli and C. difficile. Third, bacterial antibiotic resistance is a human health crisis, and the unnecessary use of antimicrobials is a significant contributor to this problem. For these reasons, we pursued the targeted inhibition of gastrointestinal bacterial glucuronidases rather than the broad-spectrum elimination of all enteric microflora.

Glucuronidases hydrolyze glucuronic acid sugar moieties in a variety of compounds. The presence of glucuronidases in a range of bacteria is exploited in commonly-used water purity tests, in which the conversion of 4-methylumbelliferyl glucuronide (4-MUG) to 4-methylumbelliferone (4-MU) by glucuronidases is assayed to detect bacterial contamination. Whereas relatively weak inhibitors of glucuronidase have been reported, no potent and/or selective inhibitors of the bacterial enzymes have been presented. Thus, there is a need for selective inhibitors of bacterial glucuronidase with a purpose of reducing the dose-limiting side effect and improving the efficacy of the CPT-11 anticancer drug.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to compounds that are effective as inhibitors of bacterial glucuronidase activity. In this respect, the inventors have found that compounds that have GUS inhibitory activity can be used to prevent dose-limiting diarrhea to the irinotecan therapy.

In another aspect, the present invention relates to a compound for use with camptothecin-derived anticancer drugs. Use of a compound of the invention with an camptothecin-derived anticancer drug like CPT-11 for treating cancer reduces the dose-limiting side effects and improves the efficacy of CPT-11. In an aspect of the invention the compound is of formula (I) as described below, which are phenoxy thiophene sulfonamides. In another aspect of the invention, the compound may be a pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazole sulfonyl, thiophene carbonyl, and/or thiazole carbonyl. In still another aspect of the invention, the compound of formula (I), or a compound that is a pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazole sulfonyl, thiophene carbonyl, and/or thiazole carbonyl, is administered prior to, at the same time as or following administration of CPT-11. The present invention also relates to a method for synthesizing compounds for inhibiting glucuronidases. In an aspect of the invention the compound used of formula (I) as described below, which is a phenoxy thiophene sulfonamide. In another aspect of the invention the compound used may be a pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazole sulfonyl, thiophene carbonyl, and/or thiazole carbonyl.

In a further embodiment, the present invention relates to a compound of the formula (I):

[Image Omitted]

or a pharmaceutically acceptable salt thereof wherein

each of R₁and R₂is the same or different and is selected from H, naphthalene, naphthalene-(C₁-C₄) alkyl, naphthalene-1-ylmethyl, naphthalene-1-ylethyl, naphthalene-1-ylpropyl 3-fluorobenzyl, 3-chlorobenzyl 3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl, 3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl, 3-(triiodomethyl)benzyl, 3-(C₁-C₄alkyl)-benzyl, 3-methylbenzyl, 3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl, 3,5,-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl, 3-fluorophenyl, 3-bromophenyl, 3-iodophenyl, 3-(C₁-C₄alkyoxy) phenyl, 3-methoxyphenyl, 3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl, 4-(C₁-C₄alkyoxy) phenyl, 4-ethoxyphenyl, 4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl, 4-iodobenzyl, 3-(C₁-C₄alkyoxy) benzyl; 3-methoxybenzyl, 4-methoxy-benzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl, 4-(C₁-C₄alkyoxy)phenyl and 4 propoxybenzyl,

each of R₃and R₄is the same or different and is selected from H, F, Cl, Br, and I, and

R₅is selected from 3-(R-1-yl)phenyl, and 4-(R-1-yl)phenyl, wherein R is selected from piperazin, 4-(C₁-C₄alkyl) piperazin, 4-methylpiperazin, 4-ethyl-piperazin, and 4-propylpiperazin.

In yet another embodiment, the invention relates to a method for inhibiting or reducing diarrhea in a patient being treated with a drug that gets metabolized to form a metabolite that is a substrate for a bacterial β-glucuronidase enzyme. The method comprises administering to the patient a compound in an amount effective to inhibit the bacterial β-glucuronidase enzyme, wherein the compound is selected from the group consisting of nialamide, isocarboxazid, phenelzine, amoxapine, and mefloquine. In a preferred embodiment, the drug is irinotecan.

In another aspect, the present invention also relates to a method for inhibiting bacterial β-glucuronidase in a subject in need thereof which comprises administering to the subject one or more compounds that inhibit the glucuronidase. In an aspect of the invention the compound is of formula (I) as described below, which is a phenoxy thiophene sulfonamide. In another aspect of the invention the compound may be a pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazole sulfonyl, thiophene carbonyl, and/or thiazole carbonyl.

In another aspect of the invention, the compound is one which selectively inhibits bacterial glucuronidase. In this connection, nialamide, isocarboxazid, and amoxapine were identified as potent inhibitors of bacterial GUS activity in purified enzyme and whole bacteria cell-based assays, but do not inhibit mammalian GUS. These drugs and their average IC₅₀values for inhibiting GUS include the monoamine oxidase inhibitors nialamide (71 nM) and isocarboxazid (128 nM), the tricyclic antidepressant amoxapine (388 nM) and the antimalarial drug mefloquine (1.2 μM). These drugs had no significant activity (75 μM to >100 μM IC₅₀) against purified mammalian GUS. Nialamide, isocarboxazid and amoxapine also showed potent activity for inhibiting endogenous GUS activity in whole E. coli cells with average IC₅₀values of 17, 336 and 119 nM, respectively. These drugs have potential to be repurposed as therapeutic treatments to reduce diarrhea associated with irinotecan chemotherapy.

The compounds of the invention are useful in eliminating or reducing the diarrhea associated with CPT-11 use for the treatment of cancer.

In yet another embodiment, the method involves screening compounds for their usefulness in reducing diarrhea associated with irinotecan chemotherapy. In one aspect, the method comprises: (a) assaying the compounds for activity in inhibiting purified bacterial β-glucuronidase: (b) assaying the compounds for activity in inhibiting purified mammalian β-glucuronidase; and (c) selecting from the compounds assayed in steps (a) and (b) a compound that inhibits the bacterial β-glucuronidase in step (a) with a potency that is more than 250-fold greater than that with which the compound inhibits the mammalian β-glucuronidase in step (b).

In a preferred embodiment, the assaying in step (a) comprises assaying the compounds for activity in inhibiting purified E. coli bacterial-glucuronidase. In another preferred embodiment, the assaying in step (a) comprises also assaying the compounds for activity in inhibiting endogenous β-glucuronidase activity in a culture comprising intact bacterial cells. In yet another preferred embodiment, the intact bacterial cells are E. coli cells. In still another preferred embodiment, the assaying in step (b) comprises assaying the compounds for inhibiting mammalian β-glucuronidase from B. taurus.

The method for screening can further comprise administering the selected compound to a patient to whom irinotecan chemotherapy is being or will be administered. In a preferred embodiment of the screening method, the selected compound generates an average IC₅₀value of 10 μM or less in an E. coli β-glucuronidase enzyme assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Flow scheme for discovery of drugs with GUS inhibitory activity.

FIG. 2 Structures of studied drugs.

FIG. 3 Potency determinations using the E. coli GUS enzyme assay. Concentration response data for compounds was normalized to controls with and without enzyme and plotted as percent activity. The drugs evaluated were nialamide (●), isocarboxazid (▴), phenelzine (□), amoxapine (▪), loxapine (◯) and mefloquine (♦). The average Hill slope values derived from the IC₅₀curves of all active drugs ranged from 0.9-1.2. Data points represent the average of three determinations per concentration and error bars represent standard deviation. Data are representative of three independent experiments.

FIG. 4 Activities in the E. coli cell-based GUS activity assay. Concentration response data for compounds was normalized to controls with and without whole E. coli cells and plotted as percent activity. The drugs evaluated were nialamide (●), isocarboxazid (▴), phenelzine (□), amoxapine (▪), loxapine (◯) and mefloquine (♦). The average Hill slope values derived from the IC₅₀curves of all active drugs ranged from 0.8-1.0. Data points represent the average of three determinations per concentration and error bars represent standard deviation. Data are representative of three independent experiments.

FIG. 5 Bacterial cytotoxicity assessment of studied drugs. E. coli bacteria were treated with compounds at 10 and 100 μM, as indicated, for two hours. Viability was assessed with MTS viability reagent. Absorbance data was normalized to controls with and without whole E. coli cells and plotted as percent viability, with ‘no cells’ considered as 0% viability and DMSO only treated cells set at 100% viable. Kanamycin, a known cytotoxic antibiotic drug, was used as a control.

DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions

The following abbreviations are used in this specification:

Br=bromine

Cl=chlorine

CPT=camptothecin

DCM=Dichloromethane

DMEM=Dulbecco's Minimal Essential Media

DMF=Dimethylformamide

DMSO=Dimethylsulfoxide

DNA=deoxyribonucleic acid

F=fluorine

FPLC=fast performance liquid chromatography

H=hydrogen

HEPES=(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

I=iodine

kDal=kilodalton

MHz=megahertz

mmol=millimole

μMol=micromolar

NMR=nuclear magnetic resonance

nm=nanometer

OD=optical density

PMB=p-methoxybenzyl

PMSF=phenylmethylsulfonyl fluoride

ppm=parts per million

SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBAI=tetrabutylammonium iodide

TFA=Trifluoroacetic acid

The term “pharmaceutically acceptable salts” refers to the non-toxic, inorganic and organic acid addition salts and base addition salts of compounds of the present invention.

Such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acid; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic, hydroxy-maleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic acid. Pharmaceutically acceptable salts from amino acids may also be used. Such as salts of arginine and lysine.

Pharmaceutically acceptable salts may be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts may be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

As used herein, the terms “treatment” and “therapy” and the like refer to alleviate, slow the progression, prophylaxis, or attenuation of existing disease.

As used herein, the terms “inhibit,” “inhibiting,” and the like means that the activity of glucuronidase is reduced.

As used herein, the term “subject” means an animal or human.

The pharmaceutical compositions of this invention comprise one or more compounds that inhibit glucuronidase and one or more pharmaceutically acceptable carriers, diluents, and excipients.

Pharmaceutical compositions of the present invention may be in a form suitable for use in this invention for examples compositions may be formulated for i) oral use, for example, aqueous or oily suspensions, dispersible powders or granules, elixirs, emulsions, hard or soft capsules, lozenges, syrups, tablets or trouches: ii) parenteral administration, for example, sterile aqueous or oily solution for intravenous, subcutaneous, intraperitoneal, or intramuscular, iii) delivered intracerebrally or iv) topical administration, for example, a suppository or ointment.

As used herein the term “pharmaceutically acceptable” is meant that the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation including the active ingredient(s), and not deleterious to the recipient thereof.

“Pharmaceutically acceptable” also means that the compositions, or dosage forms are within the scope of sound medical judgment, suitable for use for an animal or human without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

A compound can also be used in the manufacture of a medicament. This medicament can be used for the purposes described herein.

The compositions or medicaments normally contain about 1 to 99%, for example, about 5 to 70%, or from about 5 to about 30% by weight of the compound or its pharmaceutically acceptable salt. The amount of the compound or its pharmaceutically acceptable salt in the composition will depend on the type of dosage form and the pharmaceutically acceptable excipients used to prepare it.

The dose of the compounds of this invention, which is to be administered, can cover a wide range. The dose to be administered daily is to be selected to suit the desired effect. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing undue side effects or being toxic to the patient.

The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compounds employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

As used herein, “effective amount” and the like means the amount of the compound or composition necessary to achieve a therapeutic effect.

An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

Compounds of the invention can be formulated into compositions that can be administered to a subject in need of a glucuronidase inhibitor.

The compounds or compositions thereof are used for inhibition of glucuronidase.

The compounds or compositions thereof are used in methods for treating a subject in need of a glucuronidase inhibitor. The compounds or compositions are administered in an amount that is effective to inhibit the glucuronidase. In some embodiments of the invention it is ∃ glucuronidase or bacterial ∃ glucoronidase that is inhibited.

The compounds or compositions described herein can be administered prior to, concurrently with or after administration of a camptothecin-derived anticancer agent such as CPT-11. Administration of the compounds or compositions may result in certain benefits such as decreasing the dose of the anticancer drug, increasing the tolerance of the anticancer drug and alleviating side effects from the use of the anticancer dug. Side effects include gastrointestinal side effects.

For purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements.

Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

In this application, the use of the singular includes the plural and plural encompasses singular, unless specifically stated otherwise. In addition, in this application, the use of “or” means “and/or” unless specifically stated otherwise, even though “and/or” may be explicitly used in certain instances.

The references cited herein are hereby incorporated by reference as fully as if set forth herein.

PREFERRED EMBODIMENTS

In a first aspect of the present invention, seventy-six (76) phenoxythiophene sulfonamides from a 35,000 compound diversity set library were tested for their ability to inhibit the bacterial enzyme β-glucuronidase. The structures and inhibitory activity of the compounds are shown in Table 1.

Eighteen (18) analogs of BRITE-355252 were synthesized and tested to initially explore the structural relationship these compounds display towards inhibition of β-glucuronidase. The structures and inhibitory activity of the 18 analogs of BRITE-355252 are shown in Table 2.

Compounds of formula (I)

[Image Omitted]

wherein R₁, R₂, R₃, R₄and R₅are as defined above can be prepared by a process comprising the steps of

(a) reacting a halo thiophene-sulfonyl halo and R₁—N—H₂, to form a resultant N-monoprotected thiophene sulfonamide having a first N-protecting group comprising R₁.

(b) reacting the resultant N-monoprotected amide with R₂—N-halo and a catalyst in a base, to form a resultant N,N-diprotected thiophene sulfonamide having also a second N-protecting group comprising R₂.

wherein R is selected from piperazin, 4-(C₁-C₄alkyl) piperazin, 4-methylpiperazin, 4-ethyl-piperazin, and 4-propylpiperazin, in a solvent, and then removing the solvent, to obtain a resultant N,N-diprotected phenoxy thiophene sulfonamide, and

(d) reacting the resultant N,N-diprotected phenoxy thiophene sulfonamide with a deprotecting agent that is selective for deprotecting the second N-protecting group, thereby removing the second N-protecting group, and forming a N-monoprotected phenoxy thiophene sulfonamide.

The halogen atom of the halo thiophene-sulfonyl halo compound is selected from bromine, chlorine, fluorine and iodine.

Any base that will in combination with the N-monoprotected amide with R₂—N-halo and a catalyst result in a N,N-diprotected thiophene sulfonamide can be used.

Non-limiting examples of bases that can be used are Et₃N, Na₂CO₃, K₂CO₃and NaH and any base described in the examples.

In an embodiment of the invention the halo thiophene-sulfonyl halo is dichlorothiophene-sulfonyl chloride and R1-N—H₂is naphthylmethylamine. These groups are mixed and cooled to form a N-monoprotected thiophene sulfonamide, having a first N-protecting group that comprises naphthylmethyl.

In an embodiment of the invention the resultant N-monoprotected thiophene sulfonamide, is mixed with methoxybenzyl bromide and a catalyst that can be used in a Finkelstein reaction in sodium hydride, and cooled thereby forming a N,N-diprotected thiophene sulfonamide having also a second N-protecting group that comprises methoxybenzyl, and the resultant N,N-monoprotected thiophene sulfonamide, and Cs₂CO₃and tert-butyl(hydroxyphenyl)piperazine-carboxylate in a solvent, are mixed and heated. The solvent is then removed to obtain a resultant N,N-diprotected phenoxy thiophene sulfonamide.

In an embodiment of the invention the resultant N,N-diprotected phenoxy thiophene sulfonamide is mixed with a deprotecting agent that is selective for deprotecting the second N-protecting group, thereby removing the methoxy benzyl that is the second N-protecting group, and thereby forming a N-alkyl or N-aryl phenoxy thiophene sulfonamide.

Examples of non-limiting embodiments of the invention are where: the dichlorothiophene-sulfonyl chloride is 4,5-dichlorothiophene-2-sulfonyl chloride; the naththylmethylamine is 1-naphthylmethylamine: the methoxybenzyl bromide is 4-methoxybenzyl bromide; the catalyst is tetrabutyl-ammonium iodide; the butyl (hydroxyphenyl)piperazine-carboxylate is tert-butyl-4-(3-hydroxyphenyl)piperazine-1-carboxylate; the solvent is dimethyl formamide and/or the selective deprotecting agent comprises dichloromethane and triflouroacetic acid: or a combination thereof.

In addition to dimethyl formamide, non-limiting examples of solvents that can be used are DMSO and dioxane and the solvents described in the examples.

The following reaction Scheme 1 illustrates the preparation of compounds within the scope of the present invention:

[Image Omitted]

Scheme 1 refers to the preparation of compounds of formula I. Referring to Scheme 1, compounds of the formula I are prepared by reacting commercially available 4,5-dichlorothiophene-2-sulfonyl chloride 1 with an amine to generate dichlorothiophene sulfonamide 2. PMB (p-methoxybenzyl) protected 4,5-dichlorothiophene sulfonamide 3 is generated by reacting compound 2 with NaH in DMF, pmethoxybenzyl bromide and a catalytic amount of TBAI. Nucleophilic displacement of the C-5 chlorine with a phenol in the presence of Cs₂CO₃produce N,N-diprotected 5-(3-phenoxy)-thiophene-2-sulfonamide 3. In the final step, the protecting group is removed using TFA in DCM (1:1) to give the desired compound.

In another aspect of the present invention, known compounds were assayed for their ability to inhibit bacterial glucuronidase. The Prestwick collection of FDA-approved drugs were screened with the GUS enzyme assay to validate the GUS enzyme assay for HTS. This screen of 1,120 compounds resulted in 40 actives having ≥50% inhibition for a hit rate of 3.6% and all plates had Z′-factors of ≥0.8 (average Z-factor was 0.90). Since the collection was screened at 10 μM compound, a high concentration relative to in vivo drug levels, a cut-off of 91% inhibition was applied as criteria for selecting initial compounds for follow-up studies. This requirement allowed us to focus on the more potent actives, resulting in a short list of 7 compounds. Furthermore, antibiotics and antiseptics were eliminated since the desire is to identify drugs that do not disrupt the gut microbial flora, but instead only inhibit bacterial GUS activity. This further triaging of actives resulted in 4 compounds. We observed that two of these actives belong to the monoamine oxidase inhibitor (MAOI) class of drugs, though another MAOI while active (62% inhibition), did not quite meet the 91% inhibition criteria. So to test more examples of this class of inhibitors, we also included this compound (phenelzine) in our studies. Thus, a total of five compounds were selected for follow-up studies which included IC₅₀confirmation and E. coli cell-based assays. A flow chart providing an overview of this screening process is depicted in FIG. 1. The materials and method we used to assay these compounds are described in Example 3. infra.

The five compounds that remained after triage were nialamide, isocarboxazid, phenelzine, amoxapine and mefloquine (FIG. 1). Nialamide, isocarboxazid and phenelzine are all irreversible hydrazine-class MAOI drugs, though nialamide is no longer on the market. Amoxapine is a tricyclic antidepressant and mefloquine is an antimalarial drug. Concentration response data for each of the five compounds was generated using the purified E. coli GUS enzyme assay from which IC₅₀values and Hill slopes were calculated (FIG. 3). The average IC₅₀values and standard deviations (SDs) for the MAOIs nialamide, isocarboxazid and phenelzine were 71±31, 128±56, and 2,282±1041 nM (see Table 3 for all compound IC₅₀data).

Amoxapine generated an average IC₅₀value and SD of 388±98 nM in the E. coli GUS enzyme assay. Loxapine is another tricyclic antidepressant drug that has the identical structure as amoxapine, except that loxapine has a methyl group, instead of hydrogen, on the secondary amine of the piperazine ring (FIG. 1). We tested loxapine as a specificity control and this compound resulted in an average IC₅₀value of >100 μM in the GUS enzyme assay. Thus, the methyl group on the piperazine ring of loxapine resulted in >250-fold loss in potency. The antimalarial drug mefloquine hydrochloride generated an average IC₅₀value of 1,212±234 nM in this GUS enzyme assay.

Compound aggregation has been reported as a common non-specific inhibitor mechanism for purified enzyme assays. The Hill slopes calculated from concentration response data can be used to eliminate many non-specific inhibitors in enzyme assays. For single site binding, the Hill slope of an IC₅₀curve should be 1.0. IC₅₀curves with steep slopes, i.e. significantly greater than 1.0, can be an indicator of non-specific mechanisms, including compound aggregation. The IC₅₀curves for all the tested compounds (with measurable IC₅₀values) had average Hill slope values that ranged from 0.93 to 1.26 in the E. coli GUS enzyme assay, which is close to the ideal value expected when measuring inhibition of a single enzyme. To assess whether the compounds were inhibiting signal by merely quenching fluorescence of the product formed. GUS enzyme assays were done in which compound (100 μM) was added after the enzyme reaction was stopped and then fluorescence was measured as usual. Adding the compounds at the end of the assay resulted in no inhibition of signal for any of the studied compounds (data not shown), indicating that the observed activity is not due to fluorescence quenching, color quenching or other assay artifact. Thus, these compounds produced data consistent with specific binding to a single site on GUS and not inhibition by non-specific mechanisms or assay artifact.

Tumor-derived mammalian GUS activity may be important for optimal anti-tumor efficacy of irinotecan. Recent evidence suggests that mammalian GUS may convert SN-38G back to SN-38 within the tumor and thus increase the concentration of active drug (SN-38) in the tumor. Therefore, any inhibitor of bacterial GUS used therapeutically should not inhibit the mammalian GUS since this may decrease the efficacy of irinotecan at the site of the tumor. Therefore, we tested the three most potent drugs from the screen—nialamide, isocarboxazid and amoxapine—in enzyme assays identical to the E. coli GUS enzyme assay, except for the use of mammalian GUS purified from Bovine taurus liver. Nialamide generated an average IC₅₀of 74.8 μM, while isocarboxazid and amoxapine had IC₅₀values >100 μM. Thus, nialamide was over 1,000-fold more potent against E. coli GUS than mammalian GUS, while the other two drugs where >250-fold more selective for the E. coli GUS.

An E. coli cell based assay was developed in order to assess the activity of these drugs against whole cells, instead of purified enzyme. We took advantage of the well-known specificity and sensitivity of the 4MUG substrate to detect GUS activity in E. coli cells. This assay mimicked the enzyme assay in format, with the GUS enzyme replaced by live log-phase E. coli cells and the assay incubated for a longer time (2 hr) to detect GUS activity in these un-modified cells. Four experimental results confirmed that this cell-based assay was measuring GUS activity and no other E. coli cell enzymes. First, The K_mvalue for the substrate was determined with this cell-based assay to be 151 μM (data not shown), which is similar to the 125 μM K_mvalue we previously reported for the purified enzyme assay. Secondly, the Hill slopes derived from concentration-response data for all 5 active dugs tested in this cell-based assay were in the 0.8-1.1 range, close to the expected value of 1.0 for inhibition of a single enzyme. Thirdly, maximal inhibition was achieved by all active compounds (FIG. 4), which would be expected if 100% of the observed activity was coming from a single enzyme rather than a mixture of enzymes. Finally, the overall rank order of potencies in the cell based assay is similar to the E. coli GUS assay and absolute IC₅₀values derived from the cell-based assay for the test compounds are within 5-fold of the purified bacterial GUS enzyme assay (see Table 3).

The potencies of the five hits from the Prestwick collection and the one control compound were determined using the E. coli cell-based assay (FIG. 4 and Table 3). In the irreversible MOM class of drugs, nialamide potently inhibited the cell-based assay with an IC₅₀of 17 t 2 nM, while isocarboxazid generated an IC₅₀of 336±120 nM. Thus, the potency of nialamide shifted 4-fold more potent in the cell-based assay compared to the E. coli GUS enzyme assay, while isocarboxazid was about 3-fold less potent in the cell-based assay. The other irreversible MOM drug, phenelzine, was also tested in the cell-based assay and resulted in an IC₅₀value of 7.123 nM, which is 2.6-fold less potent compared to the enzyme assay. Amoxapine was also tested in this assay resulting in an IC₅₀of 119±61 nM, which is 3.3-fold more potent than the 388 nM IC₅₀value generated using the enzyme assay. As a specificity control, loxapine was also tested, but was completely inactive (>100 μM) in this assay, consistent with its inactivity in the enzyme assay. Mefloquine generated an average IC₅₀value of 5.961±1.526 nM and thus shifted over 4.9-fold less potent in the cell-based assay compared to the enzyme assay.

One possible explanation for the inhibitory activity of the drugs in the cell-based assay is E. coli cell toxicity and/or bacteriostatic activity resulting in reduced GUS activity. Therefore, the viability of drug-treated E. coli cells was assessed with a metabolic viability assay (MTS kit, Promega). Each compound was tested at 100 and 10 μM for 2 hrs and data normalized to solvent (DMSO) controls with and without cells (FIG. 4). Kanamycin was used as a control to demonstrate assay sensitivity to a known bactericidal antibiotic. Kanamycin treatment (50 μg/ml) reduced activity in this assay by over 90%. Amoxapine, isocarboxazid, loxapine, and nialamide showed no signs of toxicity in this assay at up to 100 μM drug. In contrast, the 100 μM concentration of mefloquine completely inhibited viability in this assay while the 10 μM concentration was at maximum control levels (100% viability). Thus, the IC₅₀value of mefloquine in the cell-based GUS assay may be a blend of GUS inhibitory activity and bactericidal activity at higher concentrations. Phenelzine showed some inhibitory activity (˜10-20%) at both the 100 and 10 μM concentrations, but not enough to explain all of its cell-based activity.

To summarize, we screened a collection of FDA-approved drugs, the Prestwick collection, using our high throughput GUS enzyme assay. The hit rate was high, with 40 actives displaying ≥50% inhibition at the screening concentration of 10 μM. Raising the cut-off to 91% and elimination of antiseptics/antibiotics resulted in a short list of 4 actives for follow-up. We also included a compound (phenelzine) that did not meet the activity cut-off. It was also chosen for follow-up since it was in the same class as two on the short list and it had >50% inhibition. Thus, the actives were nialamide, isocarboxazid, phenelzine, amoxapine and mefloquine and all of these compounds confirmed by IC₅₀determinations in the E. coli GUS enzyme assay. The five hits can be categorized into three dug classes: irreversible MAOI, tricyclic antidepressant and antimalarial.

In the irreversible MAOI class, nialamide was a very potent inhibitor of GUS activity with an IC₅₀of 71 nM in the GUS enzyme assay. Surprisingly, this is more potent than its reported in vitro IC₅₀values of 2.6-13 μM for inhibiting monoamine oxidase (in rat brain homogenates), its original intended target. When tested for inhibitory potency against purified mammalian GUS, nialamide had an IC₅₀of approximately 75 μM. Thus, nialamide displayed a dramatic 1,000-fold selectivity for inhibiting E. coli GUS over mammalian GUS. Furthermore, nialamide had more potent activity for inhibiting endogenous GUS in the E. coli cell-based assay, generating an IC₅₀of 17 nM. This activity was not due to acute toxicity of nialamide. This unusual increased potency in a cell-based assay (also observed with amoxapine) may be due to a unique mechanism of action or due to compound concentration inside the bacterial cell. This same phenomenon was observed previously for some, but not all, GUS inhibitor compounds. The other compound in this same class, isocarboxazid, was also relatively potent with an IC₅₀of 128 nM, which is more potent than its reported potency of 4.8 μM IC₅₀for MAO in rat brain homogenates. Isocarboxazid was >780-fold more selective for inhibiting bacterial GUS compared to its activity against mammalian GUS, which was not measurable (>100 μM IC₅₀). This dug also inhibited in the cell-based assay with an IC₅₀of 336 nM, 2.6-fold less potent compared to the purified enzyme assay. Phenelzine is also an MAOI that is structurally similar to nialamide and isocarboxazid in that it contains a hydrazine group and is irreversible against its original target. Phenelzine was a much weaker inhibitor of GUS at 2.2 μM IC₅₀, in contrast to its IC₅₀for MAO that was reported to be 70-900 nM (depending on subclass of MAO-A or MAO-B, or total activity). Thus, the phenelzine results indicated that inhibition by the MAOIs was not solely due to the presence of a hydrazine group or the irreversible nature of these drugs. Phenelzine showed some toxicity to E. coli at 10 and 100 μM, but not enough to account for all of its GUS inhibitory activity.

The tricyclic antidepressant amoxapine potently inhibited purified GUS with an IC₅₀of 388 nM. In comparison, amoxapine had no measurable IC₅₀against mammalian GUS (>100 μM) thus resulting in a >250-fold selectivity for inhibiting bacterial GUS over mammalian GUS. Furthermore, amoxapine had more potent activity in the cell-based assay with an IC₅₀of 119 nM. Loxapine was used as a control compound for this class since it has an identical structure to amoxapine except that loxapine has a methyl group on the nitrogen of the piperazine group. Despite this very small structural difference, loxapine had no measurable IC₅₀value (>100 μM) for both the GUS enzyme assay and the cell-based assay. Thus, the amoxapine/loxapine pair served to illustrate the exquisite structural selectivity for inhibiting signal in these assays and demonstrated that a free amine in the piperazine group is critical for inhibiting GUS.

Finally, mefloquine is an antimalarial drug that was also identified in our screen. This drug had only weak activity for inhibiting purified GUS (IC₅₀=1.2 μM) and its potency worsened by 5-fold when tested in the cell-based assay (IC₅₀=6 μM). Since this compound resulted in complete inhibition in the toxicity assay at 100 μM, though none evident at 10 μM, it is possible that some of the cell-based activity is due to toxicity. Nialamide, the most potent of these drugs for inhibiting bacterial GUS, has a number of issues with respect to its use as a therapeutic. First, nialamide is no longer on the market. Nialamide was withdrawn from the market in 1963 due to interactions with food products containing high levels of tyramine. Ingestion of certain foods high in tyramine (e.g. aged cheese) resulted in sometimes severe tyramine toxicity in patients taking nialamide, a general problem with all the non-selective irreversible MAOIs. Therefore, toxicity of nialamide is a major concern, even if it were available on the market again. However, given the high potency of this drug for inhibiting GUS, may be possible to use lower, and thus safer, doses of nialamide that would have acceptable side effects. Special diets, especially avoiding intake of tyramine-enriched foods, help reduce food toxicity side effects of MAOIs. It is also conceivable that nialamide could be re-formulated for low-dose time release in the intestine. The food-induced toxicity reported for nialamide is assumed to be due to inhibition of MAO in the intestine. Thus, we believe that dosing with nialamide may result in sufficient concentrations of nialamide in the GI tract to effectively inhibit GUS. In contrast to nialamide, isocarboxazid is still on the market for treatment of major depression. Like all drugs in the MAOI class, isocarboxazid has toxicity/side effect concerns and can be problematic in combination with many other medicines due to drug-drug interactions. Phenelzine had relatively weak activity in our assays and so it is not clear if effective in vivo concentrations could be achieved. It should be recognized that any GUS inhibitor would only be needed short term (weeks) and perhaps even intermittently. Thus, we believe that the long term toxicity of nialamide or isocarboxazid is avoidable with strategic, short term dosing regimens to minimize long term drug exposure with the accompanying toxicity/side effects.

Amoxapine is a marketed member of an older class of antidepressant drugs with significantly fewer toxicity concerns compared to the MAOI drug class. It also has a safer side effect profile and far fewer drug-drug interactions than the MAOI class of drugs in general. Antidepressant use in general is common in cancer patients and thus amoxapine could also treat cancer-induced depression. According to a recent report, amoxapine and loxapine have been discovered to be potent non-competitive inhibitors of β-glycoprotein, a transporter responsible for multidrug resistance displayed by some cancer cells. Thus, the use of amoxapine as a GUS inhibitor could also have the added benefit of enhancing the sensitivity of multidrug resistant cancer cells to irinotecan and/or other chemotherapeutic drugs given in combination with irinotecan. Tricyclic antidepressants typically take about three weeks to reach peak efficacy for treatment of depression. Unlike the long term, slow acting mechanism of amoxapine for depression, amoxapine as a GUS inhibitor will only be needed short term and should act immediately to prevent re-activation of SN-38G. Thus, some of the side effects encountered with chronic use of amoxapine may be minimized with strategic intermittent dosing. The combination of its potency for inhibiting GUS and its safer profile suggests that amoxapine is the preferred drug as a therapeutic treatment of irinotecan induced diarrhea. Moreover, based on the potency of amoxapine for inhibiting GUS, compounds that get metabolized in vivo to form amoxapine as a metabolite would similarly be potent GUS inhibitors. In particular, loxapine undergoes metabolism that includes some of the drug being de-methylated at the piperazine ring—essentially generating amoxapine and amoxapine-like molecules in vivo. Thus, loxapine, though it lacked activity in our in vitro assays, would similarly be expected to have GUS inhibitory activity in vivo due to its metabolites.

In short, we have identified five known drugs that inhibit E. coli GUS activity in enzyme assays: nialamide isocarboxazid, phenelzine, amoxapine and mefloquine. These compounds displayed IC₅₀values ranging from 71 nM to 2.3 μM against purified E. coli GUS. Furthermore, nialamide, isocarboxazid and amoxapine had no significant activity against purified mammalian GUS. All five compounds also had activity in an E. coli cell-based assay with IC₅₀values for inhibiting endogenous GUS ranging from 17 nM to 7.1 μM.

Each of these drugs, or drugs that get metabolized in vivo to form these drugs as metabolites, can be administered to a patient selectively to inhibit bacterial β-glucoronidase (as compared with mammalian β-glucoronidase) in the patient whereby to reduce diarrhea associated with, for example, irinotecan chemotherapy. Each of these drugs can be administered to the patient in an amount effective to inhibit the bacterial β-glucoronidase with the dosages being controlled within the following limits:

Amoxapine: less than or equal to 400 mg/day (or 600 mg/day for a hospitalized patient):

Isocarboxazid: less than or equal to 60 mg/day;

Nialamide: less than or equal to 3.3 mg/kg (from FDA web site):

Loxapine: less than or equal to 250 mg/day;

Mefloquine: less than or equal to 1250 mg/day; and

Phenelzine: less than or equal to 90 mg/day.

EXAMPLES

The invention is further understood by reference to the following Examples, which are intended to be purely exemplary of the invention. The present invention is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only. Any methods that are functionally equivalent to those described in the Examples are within the scope of the invention. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications fall within the scope of the appended claims.

Example 1
β-Glucuronidase Activity Assay

Expression and Purification of E. coli β-Glucuronidase. The full-length E. coli β-glucuronidase gene was obtained from bacterial genomic DNA and was cloned into the pET-28a expression plasmid (Novagen) with an N-terminal 6×-Histidine tag. BL21-DE3 competent cells were transformed with the expression plasmid and grown in the presence of kanamycin (25 ug/ml) in LB medium with vigorous shaking at 37° C. until an OD₆₀₀of 0.6 was attained. The expression was induced with the addition of 0.3 mM isopropyl-1-thio-D-galactopyranoside (IPTG) and further incubated at 37° C. for 4 hours. Cells were collected by centrifugation at 4500×g for 20 min at 4° C. Cell pellets were resuspended in Buffer A (20 mM Potassium Phosphate, pH 7.4, 25 mM Imidazole, 500 mM NaCl), along with PMSF (2 μL/mL from 100 mM stock) and 0.05 μL/mL of protease inhibitors containing 1 mg/mL of aprotinin and leupeptin. Resuspended cells were sonicated and centrifuged at 14,500×g for 30 min to clarify the lysate. The cell lysate was flowed over a pre-formed Ni-NTA His-Trap gravity column and washed with Buffer A. The Ni-bound protein was eluted with Buffer B (20 mM Potassium Phosphate, pH 7.4, 500 mM Imidazole, 500 mM NaCl). Collected fractions were then tested for initial purity by SDS-PAGE. Relatively pure (˜85%) fractions were combined and loaded into the Åktaxpress FPLC system and passed over a HiLoad™ 16/60 Superdex™ 200 gel filtration column. The protein was eluted into 20 mM HEPES, pH 7.4, and 50 mM NaCl for crystallization and activity assays. Two milliliter fractions were collected based on highest ultraviolet absorbance at 280 nm. Fractions were analyzed by SDS-PAGE (which indicated >95% purity), combined, and concentrated to 10 mg/mL for long-term storage at −80° C. In addition, some experiments were performed with purified E. coli β-glucuronidase enzyme purchased from Sigma-Aldrich.

High Throughput Screening β-Glucuronidase Assay The β-glucuronidase assay was performed by the addition of 0.5 μl of compound (or DMSO) to the well of a black 384-well plate followed by the addition of 30 μl of diluted β-glucuronidase enzyme. The enzyme was diluted in assay buffer (50 mM HEPES, pH 7.4) plus 0.0166% Triton X-100 for a final enzyme concentration of 50 μM and final detergent concentration of 0.01%. After a 15 minute incubation at room temperature (23° C.), 20 ul of substrate, 4-Methylumbelliferyl β-D-glucuronide hydrate (4MUG) diluted into assay buffer, was added to the reaction for a final concentration of 125 uM. β-glucuronidase hydrolyzes the non-fluorescent 4MUG resulting in a fluorescent product, 4-methylumbelliferyl. After a 30 minute incubation at room temperature, the reaction was stopped by the addition of 20 ul 1 M Na₂CO₃. The fluorescence (in relative fluorescence units, RFU) was measured using a 355 nm excitation filter and 460 nm emission filter using a Victor V (Perkin Elmer) plate reader. Minimum (min) controls were performed using reactions with no enzyme. Maximum (max) controls were performed using no compound. 1% DMSO was maintained in all reactions. Percent inhibition was calculated using RFU data by the following formula: [1−(assay readout-average of min)/(Average of Max-Average of Min)]×100. The known-glucuronidase inhibitor, D-Glucaric acid-1,4-lactone monohydrate, was used to validate the assay and serve as a positive control. IC₅₀value was defined as the concentration of inhibitor calculated to inhibit 50% of the assay signal based on a serial dilution of compound. Values were calculated using either a four or three-parameter dose response (variable slope) equation in GraphPad Prism™ or ActivityBase™. For the IC₅₀determinations, serial dilutions of compounds were performed in 100% DMSO with a two-fold dilution scheme resulting in 10 concentrations of compound. These results are shown in Tables 1 and 2.

Example 2
Preparation of Analogs of BRITE-355252
General Procedures for the Preparation of Analogs of BRITE-355252

All solvents and reagents were obtained from commercial sources and used without further purification unless otherwise stated. All reactions were performed in oven-dried glassware (either in RB flasks or 20 ml vials equipped with septa) under an atmosphere of nitrogen and the progress of reactions was monitored by thin-layer chromatography and LC-MS. Analytical thin-layer chromatography was performed on precoated 250 prn layer thickness silica gel 60 F₂₅₄plates (EMD Chemicals Inc.). Visualization was performed by ultraviolet light and/or by staining with phosphomolybdic acid (PMA) or p-anisaldehyde. All the silica gel chromatography purifications were carried out by using Combiflash® Rf (Teledyne Isco) and CombiFlash® Companion® (Teledyne Isco) either with EtOAc/hexane or MeOH/CH₂Cl₂mixtures as the eluants. Melting points were measured on a MEL-TEMP® capillary melting point apparatus and are uncorrected. Proton nuclear magnetic resonance (¹H NMR) spectra and carbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on a Varian VNMRS-500 (500 MHz) spectrometer. Chemical shifts (δ) for proton are reported in parts per million (ppm) downfield from tetramethylsilane and are referenced to it (TMS 0.0 ppm). Coupling constants (J) are reported in Hertz. Multiplicities are reported using the following abbreviations: br=broad; s=singlet d=doublet; t=triplet; q=quartet: m=multiplet. Chemical shifts (δ) for carbon are reported in parts per million (ppm) downfield from tetramethylsilane and are referenced to residual solvent peaks: carbon (CDCl₃77.0 ppm). Mass spectra were recorded on an Agilent 1200 Series LC/MS instrument equipped with a XTerra® MS (C-18, 3.5 μm) 3.0×100 mm column.

Representative Procedure for the Preparation of 4,5-Dichloro-N(Aryl/Alky) Thiophene-2-Sulfonamides

To a solution of 4,5-dichlorothiophene-2-sulfonyl chloride (1.000 g, 4.002 mmol) in anhydrous CH₂Cl₂(20 mL) was added 1-naphthylmethylamine (0.630 g, 4.007 mmol) followed by Et₃N (0.84 mL, 6.027 mmol) and stirred at room temperature for 2 h. The reaction mixture was diluted with water (20 mL) and extracted with CH₂Cl₂(100 mL), washed with brine, dried (Na₂SO₄) and concentrated under vacuo. The residue was purified by recrystallization from CH₂Cl₂-hexane to afford the pure 4,5-dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide (1.350 g, 91%) as a white crystalline product.

Representative Procedure for the PMB Protection of 4,5-dichloro-N (aryl/alkyl)thiophene-2-sulfonamides

Sodium hydride (0.081 g, 3.375 mmol) was slowly added in portions to a solution of 4,5-dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide (1.250 g, 3.358 mmol) in anhydrous DMF (10 mL) at 0° C., and stirred for 15 min. Then, 4-methoxybenzyl bromide (PMBBr) (0.675 g, 3.357 mmol), and a catalytic amount of TBAI (0.030 g, 0.081 mmol) were added at 0° C., and allowed to stir at room temperature for 2 h. After completion of the reaction, it was quenched by slow addition of water (5 mL) and extracted with EtOAc (100 mL), washed with water and brine, dried (Na₂SO₄), concentrated under vacuo and the residue purified by flash silica gel column chromatography (Combiflash® Rf) using EtOAc-hexane (1:9) as eluant to afford 4,5-dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl) thiophene-2-sulfonamide (1.500 g, 91%) as a white solid.

Representative Procedure for the Coupling of Phenols with PMB Protected 4,5-dichloro-N-(aryl/alkyl)thiophene-2-sulfonamides

A mixture of 4,5-dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide (0.100 g, 0.203 mmol), tert-butyl 4-(3-hydroxyphenyl)piperazine-1-carboxylate (0.068 g, 0.244 mmol) and Cs₂CO₃(0.099 g, 0.304 nmol) in anhydrous DMF (2 mL) was heated at 80° C. for 2.5 h. The solvent was removed under vacuo and the residue was purified by Combiflash® Rf (Isco) using EtOAc-hexanes (1:9) to obtain a white solid (0.140 g, 94%).

Representative Procedure for the Deprotection PMB Group

To a solution of tert-butyl 4-(3-(3-chloro-5-(N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)sulfamoyl)thio-phen-2-yloxy)phenyl)piperazine-1-carboxylate (0.085 g, 0.116 mmol) in anhydrous CH₂Cl₃(2 mL) was added TFA (2 mL) and stirred at room temperature for 3 h. The solvent mixture was removed under vacuo and the residue was re-dissolved in CH₂Cl₂(20 mL), washed with aqueous sat. NaHCO₃followed by brine, dried (Na₂SO₄), and concentrated under vacuo.

BRITE-355252
4-Chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The crude product was purified by flash silica gel column chromatography using MeOH—CH₂Cl₂(1:9) to afford a light orange solid (0.055 g, 92%). ¹H NMR (500 MHz, DMSO-d₆): δ (ppm): 2.81 (t, 4H, J=5.0 Hz), 3.09 (t, 4H, J=5.0 Hz), 4.56 (s, 2H), 6.43 (dd 1H, J=2.0, 8.0 Hz), 6.75 (t, 1H, J=2.5 Hz), 6.83 (dd, 1H, J=2.5, 8.0 Hz), 7.26 (t, 1H, J=8.0 Hz), 7.43-7.48 (m, 3H), 7.54-7.58 (m, 2H), 7.87 (dd, 1H, J=1.5, 7.5 Hz), 7.93-7.96 (m, 1H), 8.06-8.09 (m, 1H). APCI/ESI MS: m/z 513.9 [M+H]⁺

BRITE-492796
4-Chloro-N-methyl-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 89%% yield: White solid, mp: 144-146° C.;

¹H NMR (500 MHz, DMSO-d): δ (ppm): 2.55 (s, 3H), 2.86 (t, 4H, J=4.5 Hz), 3.14 (t, 4H, J=4.5 Hz), 4.63 (s, 2H), 6.62-6.66 (m, 1H), 6.84-6.88 (m, 2H), 7.30 (t, 1H, J=8.0 Hz), 7.48-7.62 (m, 4), 7.91 (s, 1H), 7.94 (d, 1H, J=8.0 Hz), 7.98 (d, 1H, J=9.0 Hz), 8.29 (d, 1H, J=8.0 Hz). APCI/ESI MS m/z 527.9 [M+H]⁺

BRITE-492794
4-Chloro-5-(3-(4-methylpiperazin-1-yl)phenoxy)-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide

The product was prepared in 89%% yield: White solid, mp: 156-158C; ¹H NMR (500 MHz, DMSO-d): δ (ppm): 2.21 (s, 3H), 2.43 (t, 4H, J=5.0 Hz), 3.17 (t, 4H, J=5.0 Hz), 4.55 (d, 2H, J=4.5 Hz), 6.44 (dd, 1H, J=2.0, 8.0 Hz), 6.78 (t, 1H, J=2.0 Hz), 6.84 (dd, 1H, J=2.0, 8.0 Hz), 7.27 (t, 1H, J=8.0 Hz), 7.43-7.48 (m, 3H), 7.53-7.58 (m, 211), 7.88 (dd, 1H, J=1.5, 7.5 Hz), 7.93-7.97 (m, 1H), 8.05-8.09 (m, 1H), 8.52 (t, 1H, J=4.5 Hz, NH). APCI/ESI MS Im/z 527.9 [M+H]⁺

BRITE-492809
4-Chloro-N-(3-fluorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 75% yield, White solid, mp: 86-88° C. (decomposed): 1H NMR (500 MHz, DMSO-d): δ (ppm): 3.06 (t, 4H J=5.0 Hz), 3.20 (t, 4H, J=5.0 Hz), 4.23 (s, 211), 6.54 (dd, 1H, J=2.5, 8.0 Hz), 6.67 (t, 1H, J=2.5 Hz), 6.77 (dd, 1H, J=2.5, 8.0 Hz), 6.95-7.02 (m, 2H), 7.04 (d, 1H, J=7.0 Hz), 7.23-7.32 (m, 211), 7.33 (s, 1H).

APCI/ESI MS m/z 481.9 [M+H]⁺

BRITE-354873
4-Chloro-5-(3-(piperazin-1-yl)phenoxy)-N-(3-(trifluoromethyl)benzyl)thiophene-2-sulfonamide

The product was prepared in 71% yield: White solid, mp: 58-60° C. (decomposed): ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.03 (t, 4H, J=5.0 Hz), 3.17 (t, 4H, J=5.0 Hz), 4.29 (s, 2H), 6.53 (dd, 1H, J=2.5, 8.0 Hz), 6.66 (t, 1H, J=2.5 Hz), 6.76 (dd, 1H, J=2.5, 8.0 Hz), 7.22-7.25 (m, 1H), 7.31 (s, 1H), 7.43-7.50 (m, 3H), 7.56 (d, 1H, J=7.0 Hz).

APCI/ESI MS m/z 531.9 [M+H]⁺

BRITE-492808
4-Chloro-N-(3-methylbenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 71% yield; White solid, nip: 122-124C: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 2.33 (s, 3H), 3.03 (t, 4H, J=5.0 Hz), 3.17 (t, 4H, J=5.0 Hz), 4.20 (s, 2H), 6.52 (dd, 1H, J=2.5, 8.0 Hz), 6.66 (t, 1H, J=2.5 Hz), 6.76 (dd, 1H, J=2.5, 8.0 Hz), 7.02 (d, 1H, J=8.0 Hz), 7.04 (s, 1H), 7.11 (d, 1H, J=7.5 Hz), 7.20 (d, 1H, J=8.0 Hz), 7.23 (d, 1H, J=8.0 Hz), 7.32 (s, 1H). APCI ESI-MS m/z 477.9 [M+H]⁺

BRITE-492807
4-Chloro-N-(3,5-dichlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 89% yield; Yellowish syrup: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.06 (t, 4H, J=5.0 Hz), 3.20 (t, 4H, J=5.0 Hz), 4.19 (s, 2H), 6.56 (dd, 1H, J=1.5, 8.0 Hz), 6.68 (s, 1H), 6.77 (dd, 1H, J=1.5, 8.0 Hz), 7.14 (d, 2H, J=0.5 Hz), 7.24-7.29 (m, 2H), 7.31 (s, 1H). APCI/ESI MS m/z 531.8 [M+H]⁺

BRITE-354909
4-Chloro-N(4-methoxyphenyl)-S-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 89% yield; White solid, mp: 118-120° C.: ¹H NMR (500 MHz, CDCl₃+CD₃OD): δ (ppm): 3.02 (t, 4H, J=5.0 Hz), 3.16 (t, 4H, J=5.0 Hz), 3.79 (s, 3H), 6.48 (dd, 1H, J=1.5, 8.0 Hz), 6.60 (t, 1H, J=1.5 Hz), 6.74 (dd, 1H, J=2.0.8.5 Hz), 6.81-6.85 (m, 2H), 7.05-7.09 (m, 2H), 7.18 (s, 1H), 7.22 (t, 1H, J=8.0 Hz). APCI/ESI MS m/z 479.9 [M+H]⁺

BRITE-492806
4-Chloro-N-(naphthalen-1-ylmethyl)-5-(4-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 97% yield: Light orange solid, mp: 156-158° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.04-3.07 (m, 4H), 3.13-3.16 (m, 4H), 4.65 (s, 2H), 6.89-6.92 (m, 2H), 7.02-7.05 (m, 2H), 7.32 (s, 1H), 7.37-7.42 (m, 2H), 7.52-7.55 (m, 2H), 7.83 (dd, 1H, J=2.0, 7.0 Hz), 7.86-7.89 (m, 1H), 7.92-7.94 (m, 1H).

APCI/ESI MS m/z 514.0 [M+H]⁺

BRITE-492805
4-Chloro-N-(2-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 83% yield; Light orange solid, mp: 99-101° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.02-3.04 (m, 4H), 3.15-3.18 (m, 4H), 4.35 (s, 2H), 6.49 (dd, 1H, J=2.0.8.0 Hz), 6.64 (t, 1H, J=2.0 Hz), 6.75 (dd, 1H, J=2.5, 8.5 Hz), 7.20 (s, 1H), 7.22 (d, 1H, J=0.5 Hz), 7.24 (s, 1H), 7.30 (s, 1H), 7.32-7.35 (m, 2H).

APCI/ESI MS m/z 497.9 [M+H]⁺

BRITE-355123
4-Chloro-N-(3-chlorobenzyl)-1-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 85% yield: White solid, nip: 123-125° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.01-3.04 (m, 4H), 3.15-3.18 (m, 4H), 4.21 (s, 2H), 6.54 (dd, 1H, J=2.5, 8.0 Hz), 6.67 (t, 1H, J=2.5 Hz), 6.77 (dd, 1H, J=2.5, 8.0 Hz), 7.13-7.16 (m, 1H), 7.21-7.30 (m, 4H), 7.32 (s, 1H), APCI/ESI MS: 497.9 [M+H]⁺

BRIT,-492802
4-Chloro-N-(4-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 78% yield, Light orange solid, mp: 118-120° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.01-3.04 (m, 4H), 3.15-3.18 (m, 4H), 4.20 (s, 2H), 6.53 (ddd, 1H, J=0.5, 2.0, 8.0 Hz), 6.67 (t, 1H, J=2.0 Hz), 6.77 (dd, 1H, J=2.0, 8.0 Hz), 7.18-7.21 (m, 21), 7.22 (s, 1H), 7.29-7.32 (m, 2), 7.34 (s, 1H).

APCI/ESI MS m/z 497.9 [M+H]⁺

BRITE-492803
4-Chloro-N-(4-methoxybenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 48% yield; White solid, mp: 106-108C: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 2.97 (t, 4H, J=5.0 Hz), 3.12 (t, 4H, J=5.0 Hz), 3.77 (s, 3H), 4.14 (s, 2H), 6.51 (dd, 1H, J=2.0.8.0 Hz), 6.64 (t, 1H, J=2.0 Hz), 6.73 (dd, 1H, J=2.0, 8.0 Hz), 6.82 (d, 2H, J=8.5 Hz), 7.14 (d, 2H, J=8.5 Hz), 7.23 (t, 1H, J=8.5 Hz), 7.28 (s, 1H). APCI/ESI MS m/z 494.0 [M+H]⁺

BRITE-355227
4-Chloro-N-(3-methoxybenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 31% yield; White solid, nip: 60-62° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 2.97 (t, 4H, J=5.0 Hz), 3.12 (t, 4H, J=5.0 Hz), 3.76 (s, 311), 4.18 (s, 2H), 6.52 (dd, 1H, J=2.0, 8.0 Hz), 6.64 (t, 1H, J=2.0 Hz), 6.73 (dd, 1H, J=2.0, 8.5 Hz), 6.76 (s, 1H), 6.78-6.83 (m, 2H), 7.22 (ABq. 2H, J=8.5 Hz), 7.28 (s, 1H). APCI/ESI MS m/z 494.1 [M+H]⁺

BRITE-492800
4-Chloro-N-(3-chlorophenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 78% yield; White solid, mp: 154-156° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.17 (t, 4H, J=5.0 Hz), 3.28 (t, 4H, J=5.0 Hz), 6.49 (dd, 1H, J=2.0, 8.0 Hz), 6.74 (s, 1H), 6.78 (d, 1H, J=7.5 Hz), 6.83 (dd, 1H, J=2.0, 8.0 Hz), 6.86 (dd, 1H, J=1.0, 7.5 Hz), 6.96 (s, 1H), 7.10 (t, 1H, J=8.0 Hz), 7.23 (s, 1H), 7.26 (t, 1H, J=8.0 Hz), 8.34 (br s, 1H, NH). APCI/ESIMS m/z 484.0 [M+H]⁺

BRITE-492799
4-Chloro-N-(3-methoxyphenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 78% yield; White solid, mp: 180-182° C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.03 (t, 4H, J=5.0 Hz), 3.14 (t, 4H, J=5.0 Hz), 3.78 (s, 3H), 6.47 (dd, 1H, J=2.0.8.0 Hz), 6.59 (t, H, J=2.0 Hz), 6.65-6.68 (m, 1H), 6.71-6.76 (m, 31), 7.18-7.23 (m, 2H), 7.27 (s, 1H). APCI/ESI MS m/z 480.0 [M+H]⁺

BRITE-492798
4-Chloro-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

To a solution of tert-butyl 4-(3-(5-(N,N-bis(4-methoxybenzyl) sulfamoyl)-3-chlorothiophen-2-yloxy)phenyl)piperazine-1-carboxylate (0.430 g, 0.602 mmol) in anhydrous CH₂Cl₂(0.5 mL) was added TFA (4.5 mL) and stirred at room temperature for 4 h. The solvent mixture was removed under vacuo and the residue was re-dissolved in CH₂Cl₂(30 mL), washed with aqueous sat. NaHCO₃followed by brine, dried (NaSO₄), and concentrated under vacuo. The residue was purified by Combiflash® Rf (Isco) using MeOH—CH₂Cl₂(1:5) to give a white solid (0.180 g, 80%). ¹H NMR (500 MHz, CD₃OD): δ (ppm): 3.02-3.05 (m, 4H), 3.19-3.22 (m, 4H), 6.56 (dd, 1H, J=2.0, 8.0 Hz), 6.73 (t, 1H, J=2.0 Hz), 6.84 (dd, 1H, J=2.0, 8.5 Hz), 7.27 (t, 1H, J=8.5 Hz), 7.40 (s, 1H).

APCI/ESI MS m/z 374.0 [M+H]⁺

BRITE-492797
N-(Naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 87% yield; Light orange solid, mp: 65-67C: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 2.92 (t, 4H, J=5.0 Hz), 3.08 (1, 4H, J=5.0 Hz), 4.62 (s, 2H), 6.43 (d, 1H, J=4.5 Hz), 6.56-6.60 (m, 1H), 6.65 (t, 1H, J=2.5 Hz), 6.71 (dd, 1H, J=2.0, 8.5 Hz), 7.23 (t, 1H, J=8.5 Hz), 7.37 (d, 2H, J=4.5 Hz), 7.39 (d, 1H, J=4.0 Hz), 7.48-7.54 (m, 2H), 7.79 (t, 1H, J=4.5 Hz), 7.82-7.86 (m, 1H), 7.95 (dd, 1H, J=1.0, 7.5 Hz), APCI/ESI MS m/z 480.1 [M+H]⁺

Example 3
Materials And Methods

Material & Methods

All common reagents such as HEPES, Triton X-100, carbenicillin, and dimethyl sulfoxide (DMSO) were reagent-grade quality and obtained from Thermo Fisher Scientific (Waltham Mass.) or Sigma-Aldrich (St. Louis, Mo.). 4-methylumbelliferyl glucuronide (4MUG) was obtained from Sigma-Aldrich (St. Louis, Mo.). The solid black 96-well plates (cat#3915) for the assay and 96 well clear plates (cat#9017) for cytotoxicity assay were from Corning Incorporated (Corning, N.Y.). Falcon polypropylene plates (cat#1190) used for serial dilution of compounds were obtained from Becton Dickinson (Franklin Lake, N.J.). Amoxapine, nialamide, isocarboxazid and other compounds for follow-up studies were obtained from Sigma-Aldrich. The Prestwick Chemical Collection was obtained from Prestwick Chemical Company (Washington D.C.). E. coli DH5α (Zymo Research, Irvine, Calif.) was used for the cell-based assay. The expression and purification of GUS enzyme from E. coli carrying an expression plasmid containing the full-length E. coli GUS gene has been previously described (26). Bovine taurus GUS enzyme was purchased from Sigma-Aldrich. In addition, some experiments were performed with purified E. coli β-glucuronidase enzyme purchased from Sigma-Aldrich.

GUS Enzyme Assay—Manual Version

The semi-automated GUS high throughput enzyme assay was performed as previously described [25] and was used to screen the Prestwick Chemical Collection. The follow-up studies were performed manually in a similar manner with the exception of plate type and volumes, as briefly outlined here. Compound stock solutions were made in 100% DMSO. Serial dilutions of compounds for IC₅₀determinations were initially performed in 100% DMSO in 96 well polypropylene plates, then each compound concentration diluted into assay buffer (50 mM HEPES, pH 7.4 and 0.017% Triton X-100), producing a constant 5% DMSO in all wells. Subsequently, 20 μl of this aqueous diluted compound (or just 5% DMSO for controls) was added to the wells of a solid black 96-well plate followed by 40 μl of GUS enzyme (83 μM GUS) diluted in assay buffer. After addition of enzyme, the reaction was initiated by addition of 40 μl of 4MUG substrate (312.5 μM 4MUG) diluted in 50 mM HEPES. pH 7.4, 4MUG stock solutions were prepared in the same buffer. Final concentrations in the assembled assay were 50 mM HEPES. pH 7.4, 0.01% Triton X-100, 1% DMSO. 125 μM 4MUG and 33 μM GUS. The enzyme reaction was allowed to proceed for 20 minutes at 23° C., and was terminated by the addition of 40 μl of a 1M sodium carbonate solution. Fluorescence at 460 nm was determined using 355 nm excitation wavelength with a 0.1 s/well read time in a BMG PheraStar (BMG LABTECH. Cary, N.C.). Fluorescence data, expressed in relative fluorescence units (RFU), were normalized to DMSO (100% activity) and “no enzyme” (0% activity) controls as maximum and minimum responses, respectively. The Bovine taurus GUS enzyme assay was performed in an identical manner except Bovine taurus GUS enzyme (1 nM) was used instead of bacterial GUS. The IC₅₀values and Hill slopes were calculated from concentration response data using GraphPad Prism software (GraphPad Software Inc., La Jolla, Calif.) employing either four-parameter or a three parameter (fixed bottom) curve fit.

GUS Cell Based Assay

Cultures of E col (DH5α) carrying the empty expression vector pCMV5 were grown over night in LB containing carbenicilin (50 μM) and then used to initiate fresh LB/carbenicillin cultures adjusted to an initial OD of 0.1. These cultures were allowed to reach an OD of 0.6 and then washed twice with 50 mM HEPES, pH 7.4 containing carbenicillin 50 μM, and concentrated by centrifugation to an OD of 1 for use in the assay. The GUS cell based assay was performed in an identical manner as the enzyme assay except the Triton X-100 was left out of the assay buffer, the E. coli cells replaced the enzyme and the reaction was allowed to proceed for 2 hrs at 37° C. The resulting data was analyzed as outlined for the enzyme assay.

Toxicity Assay

Compounds were tested for cytotoxicity in E. coli cells. The cells were grown and prepared for assay as described above, and plated in clear 96-well plates. Cells were treated with 100 μM and 10 μM concentrations (1% DMSO) of test compounds and incubated for 2 hours at 37° C. Subsequently, 25 uL of MTS viability reagent (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit, Promega Corp., Madison. WS) was added to the wells and incubation continued for 5 minutes. The plates were then analyzed for absorbance at 490 nm on a SpectraMax Plus 384 (Molecular Devices, Sunnyvale, Calif.). Controls included DMSO only (considered 100% viability), “no cells” (representing 0% viable cells), and the cytotoxic positive control compound kanamycin at 50 μg/ml.

REFERENCES

Pommier, Y. (2006) Topoisomerase I inhibitors: camptothecins and beyond, Nat Rev Cancer 6.789-802

Pizzolato, J. F., and Saltz, L. B. (2003) The camptothecins, Lancet 361, 2235-2242.

Smith, N. F., Figg, W. D., and Sparreboom, A. (2006) Pharmacogenetics of irinotecan metabolism and transport: an update, Toxicol In Vitro 20, 163-175.

Ma. M. K., and McLeod. H. L. (2003) Lessons learned from the irinotecan metabolic pathway, Curr Med Chem 10, 41-49.

Mathijssen, R. H, J., van Alphen. R. J., Verweij, J., Loos, W. J., Nooter, K., Stoter, G., and Sparreboom. A. (2001) Clinical Pharmacokinetics and Metabolism of Irinotecan (CPT-11). Clin Cancer Res 7, 2182-2194.

Miley, M. J., Zielinska, A. K., Keenan, J. E., Bratton, S. M., Radoinska-Pandya. A., and Redinbo. M. R. (2007) Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7, J Mol Biol 369, 498-511.

Nagar, S., and Blanchard, R. L. (2006) Pharmacogenetics of uridine diphosphoglucuronosyltransferase (UGT) 1A family members and its role in patient response to irinotecan, Drug Metab Rev 38, 393-409.

Stein, A., Voigt, W., and Jordan. K. Review: Chemotherapy-induced diarrhea: pathophysiology, frequency and guideline-based management. Therapeutic Advances in Medical Oncology, pp. 51-63 vol. 2.

Tobin, P. J., Dodds, H. M., Clarke, S., Schnitzler, M., and Rivory. L. P. (2003) The relative contributions of carboxylesterase and beta-glucuronidase in the formation of SN-38 in human colorectal tumours, Oncology reports 10, 1977-1979.

Hi. Z. P., Yang, X. X., Chan, S. Y., Xu, A. L., Duan, W., Zhu. Y. Z., Sheu, F. S., Boelsterli, U. A., Chan E., Zhang, Q. Wang, J. C., Ee, P. L., Koh, H. L., Huang, M., and Zhou, S. F. (2006) St. John's wort attenuates irinotecan-induced diarrhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis. Toxicology and applied pharmacology 216, 225-237.

Kurita, A., Kado, S., Matsumoto, T., Asakawa, N., Kaneda, N., Kato, I., Uchida, K., Onoue, M., and Yokokura, T. Streptomycin alleviates irinotecan-induced delayed-onset diarrhea in rats by a mechanism other than inhibition of beta-glucuronidase activity in intestinal lumen, Cancer chemotherapy and pharmacology.

Basinska, A., and Florianczyk, B. (2003) Beta-glucuronidase in physiology and disease, Annales Uiversiatis Mariae Curie-Skldowvska 58, 386-389.

Famleitner, A. H., Hocke, L., Beiwl, C., Kavka, G. C., and Mach, R. L. (2002) Hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide in differing sample fractions of river waters and its implication for the detection of fecal pollution., Water Res. 36, 975-981.

Wallace, B. D., Wang, H., Lane, K. T., Scott, J. E., Orans, J., Koo. J. S., Venkatesh, M., Jobin, C., Yeh, L. A., Mani, S., and Redinbo, M. R. (2010) Alleviating cancer drug toxicity by inhibiting a bacterial enzyme, Science 330, 831-835.

Ahmad S., Hughes, M. A., Lane, K. T., Redinbo, M. R., Yeh. L. A., and Scott, J. E. (2011) A High Throughput Assay for Discovery of Bacterial beta-Glucuronidase Inhibitors. Curr Chem Genomics 5, 13-20.

McGovern, S. L., Caselli, E., Grigorieff, N., and Shoichet, B. K. (2002) A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening, J Med Chem 451712-1722.

Feng. B. Y. Simeonov, A., Jadhav, A., Babaoglu, K., Inglese, J., Shoichet, B. K., and Austin, C. P. (2007) A high-throughput screen for aggregation-based inhibition in a large compound library. J Med Chem 50, 2385-2390.

Tobin, P., Clarke, S., Seale. J. P., Lee. S. Solomon, M., Aulds, S., Crawford, M., Gallagher, J., Eyers, T., and Rivory, L. (2006) The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and beta-glucuronidase in human colorectal tumours, Br J Clin Pharmacol 62, 122-129.

Prijovich, Z. M., Chen, K. C., and Roffler, S. R. (2009) Local enzymatic hydrolysis of an endogenously generated metabolite can enhance CPT-11 anticancer efficacy, Mol Cancer Ther 8, 940-946.

Huang, P. T., Chen, K. C., Prijovich, Z. M., Cheng, T. L., Leu. Y. L., and Roffler, S. R, (2011) Enhancement of CPT-11 antitumor activity by adenovirus-mediated expression of beta-glucuronidase in tumors, Cancer Gene Ther 18, 381-389.

Ulus, L H., Maher, T. J., and Wurtman, R. J. (2000) Characterization of phentermine and related compounds as monoamine oxidase (MAO) inhibitors. Biochem Pharmacol 59, 1611-1621.

Maxwell. D. R., Gray, W. R., and Taylor, E. M. (1961) Relative activity of some inhibitors of mono-amine oxidase in potentiating the action of tryptamine in vitro and in vivo, Br J Pharmacol Chemotherapy 17, 310-320.

Lexchin, J. (2005) Drug withdrawals from the Canadian market for safety reasons, 1963-2004, CMAJ 172, 765-767.

Wimbiscus, M., Kostenko, O., and Malone, D. (2010) MAO inhibitors: risks, benefits, and lore. Cleve Clin J Med 77, 859-882.

Fisch, M. (2004) Treatment of depression in cancer, J Nat Cancer Inst Monogr, 105-111.

Palmeira, A., Rodrigues, F., Sousa, E., Pinto, M., Vasconcelos, M. H., and Fernandes, M. X. (2011) New uses for old drugs: pharmacophore-based screening for the discovery of P-glycopotein inhibitors, Chem Biol Drug Des 78, 57-72.

Leonard, B. E. (1984) Pharmacology of new antidepressants. Prog Neuropsychopharmacol Biol Psychiatry 8, 97-108.

Badway, M. A., and Dugas, J. E. (1984) Loxapine yields amoxapine. J Clin Psychopharmacol 4, 363-364.

Cheung, S. W., Tang, S. W., and Remington, G. (1991) Simultaneous quantitation of loxapine, amoxapine and their 7- and 8-hydroxy metabolites in plasma by high-performance liquid chromatography, J Chromatogr 564, 213-221.

Hsiang, Y. H., Hertzberg, R., Hecht, S. & Liu, L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260, 14873-8 (1985).

Redinbo, M. R., Champoux, J. J. & Hol, W. G. Structural insights into the function of type IB topoisomerases. Curr Opin Struct Biol 9, 29-36 (1999).

Redinbo, M. R., Stewart, L., Kuhn. P., Champoux, J. J. & Hol, W. G. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279, 1504-13 (1998).

Stewart, L., Redinbo, M. R., Qiu, X., Hol, W. G. & Champoux J. J. A model for the mechanism of human topoisomerase I, Science 279, 1534-41 (1998).

Chrencik, J. E. et al. Mechanisms of camptothecin resistance by human topoisomerase I mutations. J Mol Biol 339, 773-84 (2004).

Staker, B. L. et al. The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc Natl Acad Sci USA 99, 15387-92 (2002).

Fittkau, M., Voigt, W., Holzhausen, H. J. & Schmoll, H. J. Saccharic acid 1,4-lactone protects against CPT-11-induced mucosa damage in rats. J Cancer Res Clin Oncol 130, 388-94 (2004).

Flieger, D. et al. Phase H clinical trial for prevention of delayed diarrhea with cholestyramine/levofloxacin in the second-line treatment with irinotecan biweekly in patients with metastatic colorectal carcinoma. Oncology 72, 10-6 (2007).

Cummings, J. H. & Macfarlane, G. T. Role of intestinal bacteria in nutrient metabolism. JPEN J Parenter Enerl Nutr 21, 357-65 (1997).

Guarner, F. & Malagelada, J. R. Gut flora in health and disease. Lancet 361, 512-519 (2003).

Job, M. L. & Jacobs, N. F., Jr. Drug-induced Clostridium difficile-associated disease. Drug Saf 17.37-46 (1997).

Levy, S. B. & Marshall, B. Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 10. S122-9 (2004).

Nord, C. E., Kager. L. & Heimdahl, A. Impact of antimicrobial agents on the gastrointestinal microflora and the risk of infections. Am J Med 76, 99-106 (1984).

Settle, C. D. & Wilcox, M. H. Review article: antibiotic-induced Clostridium difficile infection. Aliment Pharmacol Ther 10, 835-41 (1996).

Sears, S., McNally, P., Bachinski, M. S. & Avery, R. Irinotecan (CPT-11) induced colitis: report of a case and review of Food and Drug Administration MEDWATCH reporting. Gastrointest Endosc 50, 841-4 (1999).

Stamp, D. Antibiotic therapy may induce cancers in the colon and breasts through a mechanism involving bile acids and colonic bacteria. Med Hypotheses 63, 555-6 (2004).

Yang, L. & Pei, Z. Bacteria, inflammation, and colon cancer. World J Gastroenterol 12, 6741-6 (2006).

Russell, W. M. & Klaenhammer, T. R. Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH. Appl Environ Microbiol 67, 1253-61 (2001).

TABLE 1

β-Glucuronidase Inhibitory activity of phenoxythiophene sulfonamides

Compound ID
Structure
IC₅₀(μm)

BRITE-354972

[Image Omitted]

0.000

BRITE-355123

[Image Omitted]

0.090

BRITE-355252

[Image Omitted]

0.090

BRITE-354975

[Image Omitted]

0.120

BRITE-354989

[Image Omitted]

0.150

BRITE-354909

[Image Omitted]

0.170

BRITE-354725

[Image Omitted]

0.190

BRITE-354969

[Image Omitted]

0.200

BRITE-355417

[Image Omitted]

0.210

BRITE-355006

[Image Omitted]

0.210

BRITE-355017

[Image Omitted]

0.220

BRITE-354979

[Image Omitted]

0.230

BRITE-355004

[Image Omitted]

0.240

BRITE-354667

[Image Omitted]

0.270

BRITE-354966

[Image Omitted]

0.270

BRITE-355262

[Image Omitted]

0.280

BRITE-354965

[Image Omitted]

0.290

BRITE-354873

[Image Omitted]

0.290

BRITE-354615

[Image Omitted]

0.310

BRITE-355016

[Image Omitted]

0.320

BRITE-354517

[Image Omitted]

0.320

BRITE-354958

[Image Omitted]

0.330

BRITE-355360

[Image Omitted]

0.340

BRITE-355227

[Image Omitted]

0.350

BRITE-354948

[Image Omitted]

0.390

BRITE-355336

[Image Omitted]

0.410

BRITE-355423

[Image Omitted]

0.430

BRITE-355468

[Image Omitted]

0.500

BRITE-355202

[Image Omitted]

0.510

BRITE-355003

[Image Omitted]

0.510

BRITE-354946

[Image Omitted]

0.560

BRITE-354983

[Image Omitted]

0.570

BRITE-355014

[Image Omitted]

0.600

BRITE-355015

[Image Omitted]

0.630

BRITE-354627

[Image Omitted]

0.710

BRITE-354764

[Image Omitted]

0.730

BRITE-354993

[Image Omitted]

0.730

BRITE-354956

[Image Omitted]

0.750

BRITE-354984

[Image Omitted]

0.750

BRITE-354974

[Image Omitted]

0.760

BRITE-354947

[Image Omitted]

0.770

BRITE-354392

[Image Omitted]

0.790

BRITE-355045

[Image Omitted]

0.880

BRITE-354994

[Image Omitted]

0.900

BRITE-354957

[Image Omitted]

0.920

BRITE-355074

[Image Omitted]

0.970

BRITE-354565

[Image Omitted]

1.000

BRITE-354998

[Image Omitted]

1.000

BRITE-354955

[Image Omitted]

1.310

BRITE-355296

[Image Omitted]

1.370

BRITE-355008

[Image Omitted]

1.390

BRITE-355005

[Image Omitted]

6.700

BRITE-355192

[Image Omitted]

16.140

BRITE-354839

[Image Omitted]

20.030

BRITE-354428

[Image Omitted]

20.560

BRITE-355224

[Image Omitted]

22.910

BRITE-355018

[Image Omitted]

23.330

BRITE-355240

[Image Omitted]

28.570

BRITE-355329

[Image Omitted]

31.510

BRITE-355250

[Image Omitted]

50.060

BRITE-355339

[Image Omitted]

76.560

BRITE-355319

[Image Omitted]

84.560

BRITE-355243

[Image Omitted]

92.780

BRITE-355180

[Image Omitted]

101.980

BRITE-355244

[Image Omitted]

123.470

BRITE-355214

[Image Omitted]

134.850

BRITE-355030

[Image Omitted]

149.400

BRITE-355234

[Image Omitted]

151.550

BRITE-355169

[Image Omitted]

152.600

BRITE-354458

[Image Omitted]

175.430

BRITE-355211

[Image Omitted]

192.490

BRITE-355201

[Image Omitted]

201.630

BRITE-355233

[Image Omitted]

375.580

BRITE-355221

[Image Omitted]

401.950

BRITE-355253

[Image Omitted]

443.900

BRITE-354502

[Image Omitted]

965.440

TABLE 2

Structure and β-Glucuronidase InhibitoryActivity of BRITE-355252 analogs

Compound ID
Structure
IC50 (μM)

BRITE-354873

[Image Omitted]

0.030

BRITE-354909

[Image Omitted]

0.060

BRITE-355123

[Image Omitted]

0.020

BRITE-355227

[Image Omitted]

0.050

BRITE-355252

[Image Omitted]

0.020

BRITE-492794

[Image Omitted]

10.170

BRITE-492796

[Image Omitted]

0.120

BRITE-492797

[Image Omitted]

0.090

BRITE-492798

[Image Omitted]

0.330

BRITE-492799

[Image Omitted]

0.070

BRITE-492800

[Image Omitted]

0.070

BRITE-492802

[Image Omitted]

0.030

BRITE-492803

[Image Omitted]

0.100

BRITE-492805

[Image Omitted]

0.040

BRITE-492806

[Image Omitted]

0.300

BRITE-492807

[Image Omitted]

0.130

BRITE-492808

[Image Omitted]

0.030

BRITE-492809

[Image Omitted]

0.040

TABLE 3

Summary of GUS Inhibitory Activity of Studied Drugs

E. coli GUS
B. taurus GUS
E. coli

Currently
Enzyme Assay^a
Enzyme Assay^a
Cell-Based Assay^a

Drug
Drug Class
Marketed
IC₅₀± SD (nM)
IC₅₀± SD (nM)
IC₅₀± SD (nM)

Nialamide
Irreversible MAOI
No
71 ± 32
74,813 ± 1,841
17 ± 2

Isocarboxazid
Irreversible MAOI
Yes
128 ± 56
>100,000
336 ± 120

Phenelzine
Irreversible MAOI
Yes
2,282 ± 1041
ND^b
7,123 ± 1650

Amoxapine
Tricyclic Antidepressant
Yes
388 ± 98
>100,000
119 ± 61

Loxaspine^c
Tricyclic Antidepressant
Yes
>100,000
ND
>100,000

Mefloquine
Antimalarial
Yes
1,212 ± 234
ND
5,961 ± 1526

^aIC₅₀value determinations were performed at least three times, with average IC₅₀values and standard deviations (SD) shown. The range of average Hill slopes for all measurable IC₅₀curves (where at least 50% inhibition was obtained) was 0.84 to 1.26.

^bND = not determined;

^cThis drug was included as a study control