AU2003261481A1 - Inhibitors of bacterial NAD synthetase - Google Patents

Description

INHIBITORS OF BACTERIAL NAD SYNTHETASE CROSS-REERENCE TO RELATED

APPLICATIONS

This application is a Continuation-in-Part of International Application No.

PCT/US99/00810, filed January 14, 1999 which claims priority to United States provisional application Serial No. 60/097,880 filed on August 25, 1998 and to United States provisional application Serial No. 60/071,399 filed on January 14, 1998. The contents of each of these referenced patent applications are herein incorporated by this reference in their entireties.

GOVRNMENT NTEREST

STATEMENT

Some research that contributed to the invention herein was supported, in part, by a grant from the United States Department of Defense, Advanced Research Projects Agency.

FIELD OF THE INVENTION SrThe present invention pertains to antibacterial and antimicrobial agents. In particular, the present invention provides methods of synthesizing and screening compounds that are bacterial nicotinamide adenine dinucleotide (NAD) synthetase enzyme inhibitors. The present invention also provides novel compounds that inhibit bacterial NAD synthetase enzyme. The invention also provides libraries of compounds that comprise bacterial NAD synthetase enzyme inhibitors. Further, the present invention provides compounds that exhibit therapeutic activity as antibacterial agents, antimicrobial agents and broad spectrum antibiotics. Still further, the invention provides methods of treating a mammal with bacterial NAD synthetase enzyme inhibitor compounds. The present invention also provides novel disinfecting agents.

BACKGROUND OF THE INVENTION Drug-resistant infectious bacteria, that is, bacteria that are not killed or inhibited by existing antibacterial and antimicrobial compounds, have become an alarmnningly serious worldwide health problem. Ed. Rubenstein, Science, 264,360 (1994)). In fact, a number of bacterial infections may soon be untreatable unless alternative drug treatments are identified.

Antimicrobial or antibacterial resistance has been recognized since the introduction of penicillin nearly 50 years ago. At that time, penicillin-resistant infections caused by Staphylococcus aureus rapidly appeared. Today, hospitals worldwide are facing K" unprecedented crises from the rapid emergence and dissemination of microbes resistant to one or more antimicrobial and antibacterial agents commonly in use today. As stated in the Fact Sheet on Antimicrobial Resistance of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, several strains of antibiotic-resistant bacteria are now emerging and are becoming a threat to human and animal populations, including those summarized below: 1) Strains of Staphylococcus aureus resistant to methicillin and other antibiotics are endemic in hospitals. Infection with methicillin-resistant S. aureus (MRSA) strains may also be increasing in non-hospital settings. Vancomycin is the only effective treatment for MRSA infections. A particularly troubling observation is that S.

aureus strains with reduced susceptibility to vancomycin have emerged recently in Japan Sand the United States. The emergence of vancomycin-resistant strains would present a serious problem for physicians and patients.

2) Increasing reliance on vancomycin has led to the emergence of vancomycin-resistant enterococci (VRE), bacteria that infect wounds, the urinary tract and other sites. Until 1989, such resistance had not been reported in U.S. hospitals. By 1993, however, more than 10 percent of hospital-acquired enterococci infections reported to the SCenters for Disease Control were resistant.

2 3) Streptococcus pneumoniae causes thousands of cases of meningitis and pneumonia, as well as 7 million cases of ear infection in the United States each year.

Currently, about 30 percent ofS. pneumoniae isolates are resistant to penicillin, the primary drug used to treat this infection. Many penicillin-resistant strains are also resistant to other antimicrobial or antibacterial drugs.

4) Strains of multi-drug resistant tuberculosis (MDR-TB) have emerged over the last decade and pose a particular threat to people infected with HIV. Drug-resistant strains are as contagious as those that are susceptible to drugs. MDR-TB is more difficult and vastly more expensive to treat, and patients may remain infectious longer due to inadequate treatment. Multi-drug resistant strains of Mycobacterium tuberculosis have also emerged in several countries, including the U.S. Diarrheal diseases cause almost 3 million deaths a year, mostly in developing countries, where resistant strains of highly pathogenic bacteria such as Shigella dysenteriae, Campylobacter, Vibrio cholerae, Escherichia coli and Salmonella are emerging. Furthermore, recent outbreaks of Salmonella food poisoning have occurred in the United States. A potentially dangerous "superbug" known as Salmonella typhimurium, resistant to ampicillin, sulfa, streptomycin, tetracycline and chloramphenicol, has caused illness in Europe, Canada and the United States.

In addition to its adverse effect on public health, antimicrobial or antibacterial resistance contributes to higher health care costs. Treating resistant infections often requires the use of more expensive or more toxic drugs and can result in longer hospital stays for infected patients. The Institute of Medicine, a part of the National Academy of Sciences, has estimated that the annual cost of treating antibiotic resistant infections in the United States may be as high as $30 billion.

Given the above, it would be highly desirable to develop novel antibacterial and *antimicrobial agents that act by different mechanisms than those agents in use currently.

Further, it would be desirable to be able to synthesize such novel compounds. It would also be desirable to develop libraries of compounds that exhibit inhibitory bacteria] NAD 3 synthetase activity. Such new agents would be useful to counteract antibiotic resistant strains of bacteria and other types of hanmful microbes. It would be even more desirable to develop antibacterial agents that inhibit or block essential bacterial metabolic mechanisms, to result in bacterial death or deactivation, without also affecting the essential metabolic activities of a mammalian host. That is, it would be desirable to develop antibacterial agents that preferentially attack bacteria and other microbes and ill or deactivate the harmful organism without causing any attendant undesirable side effects in a human or animal patient. It would also be desirable to develop methods of rapidly screening potential new antimicrobial and antibacterial agents. It would also be desirable to develop novel disinfecting agents.

SUMMARY OF THE INVENTION In one aspect, the invention provides a NAD synthetase inhibitor compound of the formula: CHr% CH 0 0

X

9 13 174 aa 9 0 0\k~~ CH3 ~190

H

192 H3+ H3'o X X- 0 21321

CHI

0K 228 213

NCIH

0 0 /3 29 225 I' NV -i ltVriC. L~ 4 745 *Aw A ,w I*.

V Vwm..JV 4 VV4 I. I V 0 *76*5; 0 76 0

LX-

769 NO +1 4 g

~X

(48 x- 0 849

X-

850N 853 Sc 0% oto Ch wto fi. A~ 4, ICC

I

I I q -7 0 972 x.

975 976 1 .ti* I *98394 995 96 Still further, the invention provides a bacterial NAD synthetase enzyme inhibitor compound of the structure: Mr, CH\ 0 13 174 213 C)!j 0 CrQr

NH

H

3 C 9 214 x-

HH

315H 349 745 747 V C.2 2rrnf f~%bJ4.4I 9 7IC~ +6 X- 767

N'

NI ~~ydr

C

769 4N 0 848N

N

II 853N 17 .y ~0 C1**

H

869 872 0 K. 875 8876 87 H r- 1.I *a r 84 886

X-

x Ita H N N H 4 lial 888 8919 9894 0 14 C xx *9 Nb oJ 88NK H 906 909 q *jJ H 921 917 924 936 CHjjo~); j o J C l 2 a n r. :trt; t a .fl 1.

9M2 +X r

H

981 982 CH3 ,CH3 X 6 0 7 07 983 '984 CH3 ,CI43 X7CY x 0 0 9 0 985 986 9984 0 gas a 3063 lam a .1

I

0fC1f 2 h 0 {tJ' No 1 1075 a' 0 0 0

N-

O(CHahO

(A

Im GiCH,),O- 0

COOP*

0 rh YNN 0 x+ o 104 (CHJJfD_ m 0 M> moma Pk 1105 .r Q sL 1It In yet a further embodiment, the invention provides a bacterial NAD synthetase enzyme inhibitor of the formula: .1064

+OX-

0ws Tho-O-*r- 1070 NO 'yj r 0 103 303 PhC 1097 t Pb 00 Pb 106 .t

T

O x In a further aspect, the invention provides a bacterial NAD synthetase enzyme inhibitor compound, having Structure 2: R, R 3

R

4 R6 Aryl (linker)- Aryl R4 R7 Structure 2 wherein n is an integer of from 1 to 12, R 1

-R

7 each, independently, is an H, an unsubstituted or a substituted cyclic or aliphatic group, a branched or an unbranched group, wherein the linker is a cyclic or aliphatic, branched or an unbranched alkyl, alkenyl, or an alkynyl group and wherein the linker may also contain heteroatoms.

In yet another aspect, the invention provides a bacterial NAD synthetase enzyme inhibitor compound, having Structure 4: Linker R XRx I R3 B I R7 x x R2X X

R

5 2 A B X Structure 4 wherein X is a C or N within a monocyclic or bicyclic moiety, A and B represent the respective sites of attachment for the linker, n is an integer of from 3 to 12,

R

1 -R7 each, independently, is an H, an unsubstituted or a substituted cyclic group, or an aliphatic group, or a branched or an unbranched group, wherein the linker is a saturated or unsaturated cyclic group or an aliphatic branched or unbranched alkyl, alkenyl or alkynyl 27 group, and wherein the linker may also contain heteroatoms.

Further, the invention provides a method of treating or preventing a microbial infection in a mammal comprising administering to the mammal a treatment effective or treatment preventive amount of a bacterial NAD synthetase enzyme inhibitor compound.

Still further, a method is provided of killing a prokaryote with an amount of prokaryotic NAD synthetase enzyme inhibitor to reduce of eliminate the production of NAD whereby the prokaryote is killed. Moreover, a method is provided of decreasing prokaryotic growth, comprising contacting the prokaryote with an amount of a prokaryotic NAD synthetase enzyme inhibitor effective to reduce or eliminate the production of NAD i whereby prokaryotic growth is decreased. Further provided is a disinfectant compound wherein the compound comprises a bacterial NAD synthetase enzyme inhibitor. Still further, the invention provides a method of disinfecting a material contaminated by a microbe, comprising contacting a contaminated material with a bacterial NAD synthetase enzyme inhibitor compound in an amount sufficient to kill or deactivate the microbe.

In yet another aspect, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. alkylating 5-nitroindole with 6bromohexyl acetate to form a 6-[N-(5-nitroindolyl)] hexyl acetate; b. hydrolyzing the 6hexyl acetate to form N-(5-nitroindolyl)hexan-l-ol; c..esterifying the 6 -[N-(5-nitroindolyl)]hexan-l-ol with nicotinic acid to form nicotinate; and d. N-methylating the 6 -[N-(5-nitroindolyl)]hexyl nicotinate.

Further, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. alkylating 5-nitroindole with bromoalkyl acetate wherein the indole alkyl acetate is converted to indole alkyl alcohol; b. reacting the indole alkyl alcohol with the appropriate reagent to form an indole alkyl ester, and c. Nmethylating the indole alkyl ester.

Moreover, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. reacting indole carboxylic acid with the appropriate reagent to provide an indole carboxylate methyl ester or an indole benzyl carboxylate ester; b. N-alkylating the indole carboxylate methyl ester or the indole carboxylate benzyl ester with bromoalkyl acetate; c. reacting the material from step b above with the appropriate reagent to form an indolealkyl alcohol; d. coupling the indolealkyl alcohol with an aromatic amine; and e. reacting the indolealkyl alcohol with the appropriate reagent to convert the methyl or benzyl indolecarboxylate to the respective indole carboxylic acids.

In another aspect, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. brominating an aniline with Nbromosuccinimide to form a 2-bromo-R'-substituted-aniline or a 2-bromo-R 2 -substitutedaniline; b. reacting the 2-bromo-R'-substituted-aniline or the 2-bromo-R 2 -substitutedaniline using a Heck coupling reaction to form an alkyne-substituted aniline; c. reacting the alkyne-substituted aniline using a cyclization reaction to form an'indole alcohol; d.

quaternizing the indole alcohol with an amine; e. reacting the indole alcohol with methansulfonyl chloride to provide an indole mesylate; and f. reacting the indole mesylate with a carboxylic acid to form an indole ester.

Still further, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. brominating an aniline with Nbromosuccinimide to form a 2-bromo-R'-substituted-aniline or a 2-bromo-R 2 -substitutedaniline; b. reacting the 2-bromo-R'-substituted-aniline or a 2-bromo-R-substituted-aniline using a Heck coupling reaction to form an alkyne-substituted aniline; c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol; d.

quatemizing the indole alcohol with an amine; e.reacting the indole alcohol with triflouromethylsulfonic anhydride to provide a triflate; and f. reacting the indole triflate with an amine to form an indole alkylammonium product.

In a further aspect, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. alkylating a phenol with 7bromo-1-heptanol to provide 7-(phenyloxy)-l-heptanol; b. mesylating 7-(phenyloxy)-Iheptanol to provide 7-(phenyloxy)-l-heptyl methanesulfonate; c. esterifying 7- (phenyloxy)-l-heptyl-methanesulfonate to provide 7-(phenyloxy)-l-heptyl nicotinate; and 'rr4I.. i d. n-methylating 7-(phenyloxy)-l-heptyl nicotinate to provide [7-(phenyloxy)-l-heptyl- (N-methyl) nictotinate] iodide.

In yet another aspect, the invention provides a method of generating a library Scomprising at least one bacterial NAD synthetase enzyme inhibitor compound comprising the steps of: a. obtaining the crystal structure of a bacterial NAD synthetase enzyme; b.

identifying one or more sites of catalytic activity on the NAD synthetase enzyme; c.

identifying the chemical structure of the catalytic sites on the NAD synthetase enzyme; d.

selecting one or more active molecules that will demonstrate affinity for at least one of the catalytic sites on the NAD synthetase enzyme; f synthesizing one or more dimeric compounds comprised of at least one active molecule wherein the active molecule compound are joined by means ofn linker compounds and wherein n is an integer of from 1 to 12, and g. screening the one or more compounds for NAD synthetase inhibitor activity.

In a further aspect of the invention herein, a method is provided for the in vitro screening a compound for bacterial NAD synthetase enzyme inhibitory activity comprising the steps of: a. preparing a bacterial NAD synthetase enzyme solution from pure bacterial NAD synthetase enzyme mixed with a suitable buffer;, b. contacting the bacterial NAD synthetase enzyme solution with a test compound; and c. measuring the rate of the enzyme-catalyzed reaction between the NAD synthetase enzyme and the test K compound, wherein the rate of the enzyme catalyzed reaction comprises a measure of bacterial NAD synthetase enzyme inhibitory activity.

Additional advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included herein.

Before the present methods, compounds, compositions and apparatuses are disclosed and described it is to be understood that this invention is not limited to the specific synthetic methods described herein. It is to be fur-ther understood that the, terminology used herein is forthe purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "an" and "the" include plural referents unless the context clearly dictates otherwise.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, a nother embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about" it will be understood that the particular value forms another enmbodimnent.

Throughout this application, where a chemical diagram has a straight Line emanating from a chemical structure, such a line represents a CH, group. For example, in the following diagram:

COOH

o-methylbenzoic acid is represented.

The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecy, hexadecyl, eicosyl, tetracosyl and the like. The term "cycloalkyl" intends a cyclic alkyl group of from three to eight, preferably five or six carbon atoms.

The term "alkoxy" as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be defined as -OR where R is alkyl as defined above. A "lower alkoxy" group intends an alkoxy group containing from one to six, more preferably from one to four, carbon atoms.

The term "alkylene" as used herein refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for example, methylene ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-), 2-methylpropylene [-CH2-CH(CH3)-CH2-], hexylene and the like. The term "cycloalkylene" as used herein refers to a cyclic alkylene group, typically a 5- or 6-membered ring.

The term "alkene" as used herein intends a mono-unsaturated or di-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Asymmetric structures such as (AB)C=C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present.

The term "alkynyl" as used herein refers to a branched or unbranched unsaturated hydrocarbon group of 1 to 24 carbon atoms wherein the group has at least one triple bond.

The term "cyclic" as used herein intends a structure that is characterized by one or more closed rings. As further used herein, the cyclic compounds discussed herein may be saturated or unsaturated and may be heterocyclic. By heterocyclic, it is meant a closedring structure, preferably of 5 or 6 members, in which one or more atoms in the ring is an element other than carbon, for example, sulfur, nitrogen, etc.

The term "bicyclic" as used herein intends a structure with two closed rings. As further used herein, the two rings in a bicyclic structure can be the same or different.

Either of the rings in a bicyclic structure may be heterocyclic.

By the term "effective amount" of a compound as provided herein is meant a sufficient amount of the compound to provide the desired treatment or preventive effect.

As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount may'be determined by one of ordinary skill in the art using only routine experimentation. It is preferred that the effective amount be essentially nontoxic to the subject, but it is contemplated that some toxicity will be acceptable in some circumstances where higher dosages are required.

By "pharmaceutically acceptable carrier" is meant a material that is not biologically or otherwise undesirable, the material may be administered to an individual along with the compounds of the invention without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

As used herein, "NAD synthetase enzyme" is defined as the enzyme that catalyzes the final reaction in the biosynthesis of NAD, namely, the transformation of NaAD into NAD. As usedherein, the term "catalytic sites" are defined as those portions of the NAD synthetase enzyme that bind to substrates, and cofactors, including nictonic acid dinucleotide (NaAD), NAD, adenosine triphosphate (ATP), adenosine monophosphate (AMP), pyrophosphate, magnesium and ammonia in bacteria or microbes. The term "receptor site" or "receptor subsite" relates to those portions of the bacterial NAD synthetase enzyme in which the bacterial NAD synthetase enzyme inhibitors disclosed herein are believed to bind. For the purposes of this disclosure, the terms "catalytic site," "receptor site" and "receptor subsite" may be used interchangeably.

As used herein, the terms "library" and "library of compounds" denote an intentionally created collection of differing compounds which can be prepared by the synthetic means provided herein or generated otherwise using synthetic methods utilized in the art. The library can be screened for biological activity in any variety of methods, such as those disclosed below herein, as well as other methods useful for assessing the biological activity of chemical compounds. One of skill in the art will recognize that the means utilized to generate the libraries herein comprise generally combinatorial chemical methods such as those described in Gallop, et al, "Applications of Combinatorial Techniques to Drug Discovery," "Part 1 Background and Peptide Combinatorial Libraries," and "Part 2: Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions," J. Med. Chem., Vol. 37(1994) pp. 1233 and 1385. As used herein, the terms "combinatorial chemistry" or "combinatorial methods" are defined as the systematic and repetitive, covalent connection of a set of different "building blocks" of varying structure, such as the active molecules disclosed herein, to provide a large array of diverse molecular entities. As contemplated herein, the large array of diverse molecular entities together form the libraries of compounds of the invention.

As used herein, the term "antibacterial compound" denotes a material that kills or deactivates bacteria or microbes so as to reduce or eliminate the harmful effects of the bacteria on a subject or in a system. Such materials are also known in the art as "bacteriostatic agents" or "bateriocidal agents." The bacteria so affected can be gram positive, gram negative or a combination thereof. The terms "antimicrobial compound" and "broad spectrum antibiotic" denote a material that kills or deactivates a wide variety of microbes, including, but not limited to, one of more of, gram positive or gram negative bacteria, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus viridans, Enterococcus, anaerobic Streptococcus, Pneumococcus, Gonococcus, Meningococcus, Mima, Bacillus anthracis, C. diphtheriae, List. monocytogenes, Streptobacillus monohiliformis, Erysipelothrix insidiosa, E. coli, A. aerogenes, A. faecalis, Proteus mirabilis, Pseudomonas aeruginosa. K. pneumoniae, Salmonella, Shigella, H. influenzae,

I

H. ducreyi, Brucella, Past. pestis, Past. tularensis, Past. multocida, V. comma, Actinobacillus mallei, Pseud. pseudomallei Cl. tetani, Bacteroides, Fusobacterium fusiforme. M. tuberculosis, atypical mycobacteria, Actinomyces israelii, Nocardia, T.

pallidum, T. pernue, Borrelia recurrentis, Peptospira, Rickettsia, and Mycoplasma pneumoniae.

In accordance with the desirability for developing improved antibacterial and antimicrobial agents, with the invention herein novel compounds have been identified that inhibit bacterial NAD synthetase enzymatic activity. Such activity translates into effectiveness as bacteriocidal agents, as well as effectiveness as a broad spectrum antibiotic materials. Novel compounds have been developed that inhibit a previously unrecognized target in prokaryotic organisms, such as bacteria, to block essential biological function and thereby cause bacterial death or deactivation of bacteria or other microbes. Specifically, the invention herein has identified an enzyme found in both gram positive and gram negative.bacteria, NAD synthetase enzyme, which can be utilized as a target for drug design to provide protection from and/or treatment for bacterial and other microbial infections, The NAD synthetase enzyme catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Bacterial NAD synthetase is an ammoniadependent amidotransferase belonging to a family of"N-type" ATP pyrophosphatases; this family also includes asparagine synthetase and argininosuccinate synthetase. NAD synthetase enzyme catalyzes the last step in both the de novo and salvage pathways for NAD* biosynthesis, which involves the transfer of ammonia to the carboxylate of nicotinic acid adenine dinucleotide (NaAD) in the presence of ATP and Mg". The overall reaction is illustrated in Scheme 1.

SCHEME: SCHEME 1: NADSymeacu

ATP

NH'

MAC

2 AMP PPi ATP, Msr Unlike eukaryotic NAD synthetase that found in mammals, which can utilize glutamine as a source of nitrogen, prokaryotic NAD synthetase in bacteria utilizes ammonia as the sole nitrogen source. Through x-ray crystallography and other methods, the invention has identified marked differences in the structures of eukaryotic and prokaryotic forms of the NAD synthetase enzyme. For example, B. subtilis NAD synthetase enzyme, which in the invention has been crystallized and used in the drug design methodologies herein, is a dimeric material with molecular weight around 60,500.

In marked contrast, the eukaryotic form of NAD synthetase found in mammals is multimeric and has a molecular weight of at least 10 times larger.

By utilizing the significant differences between the eukaryotic and prokaryotic forms of NAD synthetase enzyme, the invention herein provides novel compounds that can be utilized as antibacterial and antimicrobial agents that specifically target the prokaryotic NAD synthetase enzyme without also effecting a mammalian host. With the invention herein, it has been found that by specifically inhibiting bacterial NAD synthetase 36 f i*i "M enzymatic activity, bacteria can be deprived of the energy necessary to thrive and replicate. Accordingly, through the invention disclosed and claimed herein, antibacterial and antimicrobial drugs have been developed that preferentially attack the bacteria to kill or deactivate it so as to reduce or eliminate its harmful properties, without appreciably affecting mammalian NAD synthetase enzymatic activity at the same dosage.

Furthermore, novel methods are provided that allow the rapid screening of compounds for "bacterial NAD synthetase enzyme inhibitory activity. Moreover, the invention provides methods of treating microbial infections in a subject. Because of the differences in structure between bacterial and mammalian NAD synthetase enzyme, it would not be "expected that the compounds of the invention would inhibit or otherwise affect mammalian NAD synthetase enzyme in the same manner as the compounds act on bacteria.

Without being bound by theory, through chemical analysis and x-ray crystallography methods, at least two separate catalytic subsites on the bacterial NAD synthetase enzyme in which it is possible to bind at least one or more small molecules ("active molecules") have been characterized. These sites are illustrated below by the cartoon in Figure 2.

37 FIGURE 2: CATALYTIC SITES IN BACTERIAL NAD SYNTHETASE ENZYME Because of the specific structure of these catalytic sites, it has been determined that it is possible to identify small molecules that will demonstrate affinity for at least one of the sites. Small molecules of the proper configuration, the configuration being determined by the structue of the catalytic site(s), will bind with a receptor.site or sites on the bacterial NAD synthetase enzyme, thereby blocking the catalytic activity of the enzyme.

Figure 4 illustrates via cartoon a bacterial NAD synthetase enzyme in which-the catalytic sites are blocked by an example of a compound of the present invention.

FIGURE 4: BACTERIAL NAD SYNTHETASE ENZYME WITH BLOCKED CATALYTICIRECEPTOR SITES Under such circumstances, it is hypothesized that spore-forming bacteria will be unable to undergo germination and outgrowth, and the essential cellular respiratory fbnctions of the vegetative bacteria will be halted, thereby causing cellular death or deactivation, gram positive and gram negative bacteria and other microbes will be killed or prevented from undergoing growth. Accordingly, the invention has found that compounds that exhibit inhibitory activity against the bacterial NAD synthetase enzyme will also exhibit therapeutic activity as antibacterial and antimicrobial compounds, as well as broad spectrum antibiotic materials.

With the invention herein it has been surprisingly found that it is possible to synthesize novel tethered dimeric compounds that will exhibit activity as bacterial NAD synthetase enzyme inhibitors. By linking one or more active molecules through a linker molecule, one or more ends of the tethered dimer can bind in the respective receptor sites or subsites to thereby render the bacterial NAD synthetase enzyme inactive. When more than one active molecule is used, each active molecule can be the same or different. The term "active molecules" as used herein refers to small molecules that may be used alone or tethered together through a linker (tether) fragment to form a tethered dimeric compound.

In the present invention, the active molecules are comprised of substimuent groups as hereinafter disclosed that will bind with at least one of the receptor sites in bacterial NAD synthetase enzyme. In the invention herein one or more active molecules are tethered together to form a dimeric molecule that is capable of inhibiting the bacterial NAD synthetase enzyme.

K' Further, in this invention it has been found that, under some circumstances, different active molecules will be more likely to bind to different locations in the receptor site of a bacterial NAD synthetase enzyme because of the differing chemical make-up of each of these sites. Therefore, in one embodiment, it is beneficial to tether at least two different active molecules to each other wherein each active molecule demonstrates selective affinity for a different subsite in the receptor. Using the tethered dimers herein it is possible to drastically enhance the potency of NAD synthetase enzyme inhibition, as compared to blocking a single site on the bacterial NAD synthetase enzyme. As used herein, the te rnm "selective affinity" means that the active molecule shows enhanced tendency to bind with one subsite with the receptor in the bacterial NAD synthetase enzyme because of a chemical complementarity between the receptor subsite and the active molecule. A tethered dimer compound is illustrated in Scheme 2 below.

SCHEME 2: Active Active Monomer (Linker)-- Monome A

B

In one embodiment, a dimeric inhibitor compound will bind with, for example, the sites of catalytic activity on the bacterial NAD synthetase enzyme, thereby preventing the production of NAD/NADH by the bacteria. As an additional surprising finding in this it a invention, it has been determined that by varying the length of the linker molecule, and, accordingly, the distance between the two active molecules, the affinity of the tethered *inhibitor compound for the NAD synthetase enzyme will also vary.

In practice of the invention relating to the design of novel NAD synthetase enzyme inhibitor compounds, a software program can be utilized which facilitates the prediction of the binding affinities of molecules to proteins so as to allow identification of commercially available small molecules with the ability to bind to at least one receptor subsite in the bacterial NAD synthetase enzyme. 'An example of one such computer program is DOCK, available from the Department of Pharmaceutical Chemistry at the University of California, San Francisco. DOCK evaluates the chemical and geometric complementarity between a small molecule and a macromolecular binding site. However, such a program would be useless in the design of a bacterial NAD synthetase enzyme inhibitor in the absence of complete information regarding the enzyme's structure and the chemical makeup of the receptor sites, identified and disclosed fully for the first time herein.

With this invention, the crystal structure of one type of bacterial NAD synthetase enzyme B. subtilis has been for the first time identified fully. The x-ray crystal structure of NAD synthetase enzyme from B. subtilis had been reported in the literature.

Rizzi et aL, The EMBO Journal, 15, 5125, (1996); M. Rizzi et aL, Structure, 1129 (1998)). This was accomplished in free form and in complex with ATP and Mg 2 at 2.6 and 2.0 A, respectively. This structure contained the hydrolyzed form of ATP, namely AM? and Ppi, in the ATP binding site and ATP was present in the NaAD binding site.

However, the prior art was not able to obtain the structure of the enzyme complex containing NaAD due to technical problems that precluded ful identification. Without the structure of the enzyme complex containing NaAD, the structure-based drug design targeted to NAD synthetase enzyme of the present invention could not be developed.

In order to carry out structure-based drug design targeted to bacterial NAD synthetase enzyme, the structure of the enzyme in complex with all substrates, including NaAD has been solved herein. The additional structural information obtained in this invention for the first time clearly defined the interactions between NaAD and the enzyme, which provided information important for guiding combinatorial library design and inhibitor identification. Schematic drawings of crystal structures of the open and blocked receptor/catalytic sites of B. subrilis are set out previously in Figures 2 and 4.

The invention utilizes two approaches reported in the literature (for other biological targets) to help identify lead compounds. (1I Once the structure of a bacterial NA]) synthetase catalytic site was identified, the software DOCK Kunxz et a, J. MoL.

fBIo., 161, 269-288 (1982)) was utilized to search the Available Chemicals Directory database and computationally score the relative binding afffinities for each structure.

]Based on these results and structural information regarding substrate binding, commercially available compounds were selected for purchase and subsequent enzyme kinetics evaluation. Such database se arching strategies in drug discovery are now commonly used by those of sill in the art of drug design. Manallack, Drug Discovery Today, 1, 23 1-238 (1996)). Using the results of biological screening for selected commercially available compounds to identify biologically active molecules, the inventors then designed a combinatorial library consisting of "tethered timers" to rapidly identify more effective inhibitors of NAD synithetase enzyme as antibacterial agents. The use of "tethered dimers" to enhance the binding affinity of two moderately effective small molecule ligauds that interact in the same binding site has been previously described in the literature. Stuker, P.3. Hejduk, RI'. Meadows, and S.W. Fesik, Science, 274, 1531 1534 (1996)). However, this invention involves the first and, therefore, a novel application of database searching coupled with a combinatorial tethered dirter approach that was guided by the structure of and targeted to the bacterial NA]) synthetase enzyme.

Examples from the top scaring small molecules as determined by, for example, DOCK are preferably pre-screened using in vitro enzyme assays as further described herein. As a significant aspect of the invention herein, the preferred screening method utilized should allow the rapid screening of large numbers of compounds for inhibitory activity. In a preferred method of the present invention, the small molecule inhibitor candidate for each site that is most promising as an active molecule, as identified by DOCK (or other programs known to one of skill in the art) and the prescreening method 42 herein, or that were designed based upon the substrate protein complex structure, were synthesized according to the methods disclosed herein below.

In one embodiment, the active molecules are chemically tethered to one another by means of a linker compound. In a further embodiment, the linker comprises one or more CH, or other groups, using a variety of tether lengths, preferably 1 to 12 nonhydrogen atoms, more preferably 3 to 10 nonhydrogen atoms, further more preferably 5 to 9 nonhydrogen atoms and, still more preferably, 6 to 9 nonhydrogen atoms.

S "In another embodiment of the present invention, the novel compounds with preferred structures determined from the methods described above are synthesized by means of rapid, solution phase parallel synthesis of the tethered dimers compounds in a combinatorial fashion. One of skill in the art will recognize such techniques. For each 'class of dimeric compounds designed in accordance with the invention herein, a novel synthetic strategy was developed to allow variation in the length of the linking group through which the active molecules are joined. These synthetic strategies are set forth herein as Schemes 3 through 7 and in Examples I through 5 below. Use of the preferred method of variable linkage greatly increases the number of different tethered dimeric compounds that can be produced from a single pair of the same or different active molecules. The active molecules specifically disclosed herein may be used, as well as any 1 i r pharmaceutically acceptable salts thereof As noted, pharmaceutically acceptable salts of the compounds set out herein below are also contemplated for use in this invention. Such salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylanine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. The reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0 0 C to about 100 C, preferably at room temperature. The molar ratio of compounds of structural formula to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material-a particular preferred embodiment-the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt. When calcium salts are prepared, approximately one-half a molar equivalent of base is used to yield a neutral salt, while for aluminum salts, approximately one-third a molar equivalent of base will be used.

Similarly, salts of aliphatic and/or aromatic amnines are also contemplated for use in *1 this invention. A variety of phannaceutically acceptable salts may be prepared by any of several methods well known to those skilled in the'art. Such methods include treatmnent ofa free aliphatic or aromatic amine with an appropriate carboxylic acid, mineral acid, or alkyl halide, or by con-version of the ammonium salt to another form using ion exchange resins.

Compounds prepared in accordance with the design and synthesis methods of this invention are especially attractive because they may preferably be fur-ther optimized by incorporation of substituents on either the active molecule and/or the linking goup. These latter modifications can also preferably be accomplished using the combinatorial methods disclosed herein.

In a further embodiment of the present invention, selected novel compounds whose structures are designed by the Above methods are synthesized individually using a novel strategy that allows variation in the length of the linking group. An example of a route preferably utilized to synthesize one class of diniers according to the present invention, using a single pair of active molecules, is summarized below in Scheme 3.

SCHEME 3.

02N

H

Nail Br(CHh) nO jCH3 0 (nt 2-7)

(CH

2 )nOCCH, NaOH

(CH

2

)OH"

0 2

N

CH

1

DCC

N

In a preferred embodiment, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. alkylating 5-nitroindole with 6-bromohexyl acetate to form -a nitroindolyl)] hexyl acetate; b. hydrolyzing the 6-[N-(5-nitroindolyl)] hexyl acetate to form nitroindolylflhexan-1-ol; c. *esterifying the 6-[IN-(5-nitroindolyl)]hexan-l-ol with nicotinic acid to form nicotinate; and d. N-methyiating the 6-[N-(5-nitroindolyl)]hexyl nicotinate.

The following compounds were prepared according to Scheme 3 above, wherein n represents the number of linker groups tethering the two active molecules together.

Table 2: SAMPLE COMPOUND PREPARED ACCORDING TO SCHEME 3 Compound N 862 3 863 *4 864 86516 Examples of additional preferred synthetic procedures utilized for preparing the library of the present invention are provided in Schemes 4-7. In Schemes 4-7, it is preferable to utilize combinatorial methods of synthesis using, for exampl e, parallel solution phase synthesis techniques. One of skill in the art will readily recognize the maimer in which the synthetic pathways disclosed below may b.e: varied without departing from the novel and unobvious aspects of the invention.

Schemze4 1) CH 3 50C 'n 2)

N

Aleitlrtvsfy-

R

H, Fi *(CH,)nOCCH3 Remove excess alkyl halide using nucleophilic mein (CH,)n-o (C2)Tamine a>.

'CH,)IOH

CHI

In a preferred embodiment, the invention provides a method of synthesizing a NAT) synthetase inhibitor compound from the route set out in Scheme 4 above, comprising the steps of:.

a. alkylating 5-nitroindole with bromoalkyl. acetate wherein the indole alkyl acetate is convented to indole alkyl alcohol; b. reactn th noeaklacohol with the appropriate reagent to form an indol e alkyl ester; and c. M-methylating the indole alkyl. ester.

In yet another embodiment, the invention provides a method of making a NAD synthetase inhibitor compound from the route set out in Scheme 4 above comprising the steps of: a. alkylating 5-nitroindole with bromoalkyl acetate wherein the indole alkyl acetate is converted to indole alkyl alcohol; b. reacting the indole alkyl alcohol with the appropriate reagent to form an indole alkyl ester; and.

c. reacting the indole alkyl alcohol with mesyl chloride followed by reaction with an amine to generate an ammonium product.

Scheme 5

HO

2

C

R-I

K

2 C0 3 H DMF Br(CH 2 )nOH AcPO pyridie o S II Br(CH 2 nOCCH 3

ROC

NaH 0 \CH2nOCH3 NaOCH I) CHSOCI 2) anine

H

3 Pd-C or NaOH

R

Ih 0

HOC

(CH,)n-N-R 2

R)

In yet a further, still preferred, embodiment, the invention provides a method of making a NAD synthetase inhibitor from the route set out in Scheme 5 above, comprising the steps of: _ZT F1 a. reacting indole carboxylic acid with the appropriate reagent to provide an indole carboxylate methyl ester or an indole benzyl carboxylate ester; b. N-alkylating the indole carboxylate methyl ester or the indole carboxylate benzyl ester with brornoalkyl acetate; c. reacting the material from step b above with the appropriate reagent to form an indolealkyl alcohol; d. coupling the indolealkyl alcohol'with an aromatic amine; and e. reacting the indolealkyl alcohol with the appropriate reagent to convert the methyl or benzyl indolecarboxylate to the respective indole carboxylic acids.

Scheme 6 CH(CI- (CH74y-OH

RI

Br

NBS

DMFNH

R

2

R.

N (CH2).OH

H

NaNH2

(CHA)-OH

Pd(II) Cul, Et 3

N

R, (CHz),0H

NH

2 Ri PdC

CH

1

CN

(CF3SO2)2hO amrnine I) CH 3

SO

2

CI

2) RCOH

R

I R, N (CH2X)-N-R 4 R2 H R S(CH2),-OC-3R In a further preferred embodiment, the invention provides a method of making a NAD synthetase inhibitor from the route set out in Scheme 6 above, comprising the steps of: a. brominating an aniline with N-bromosuccinimide to form a 2-bromo-R'substituted-aniline or a 2-bromo-R-substituted-aniline; b. reacting the 2-bromo-R'-substituted-aniline or the 2-bromo-R2-substitutedaniline using a Heck coupling reaction to form an alkyne-substituted aniline; c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol; d. quaterizing the indole alcohol with an amine; e. reacting the indole alcohol with methansulfonyl chloride to provide an ;indole mesylate; and f. reacting the indole mesylate with a carboxylic acid to form an indole ester.

In yet another preferred embodiment, the invention provides a method of making a NAD synthetase inhibitor compound from the route set out in Scheme 6 above, comprising the steps of: S a. brominating an aniline with N-bromosuccinimide to form a 2-bromo-R'substituted-aniline or a 2-bromo-R 2 -substituted-aniline; b. reacting the 2-bromo-R'-substituted-aniline or a 2-bromo-R 2 -substitutedaniline using a Heck coupling reaction to form an alkyne-substituted aniline; c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol; d. quaternizing the indole alcohol with an amine; e. reacting the indole alcohol with triflouromethylsulfonic anhydride to provide a triflate; and f. reacting the indole triflate with an amine to form an indole alkylammonium product.

SCHEME-7 6r(CH 2 )nOH

IC

3 acetone R Heat

A

CHS,SCI

RI O(CH 2 ),OH tN C l,

CI

-nicotinic acid K2C 3 DMF, 55C 4-(dlmethylamino)phenyacetic acid

KC

2 CO3 OMFI 559C Mel OME D-1 I t E-1 0 D-11 Me} LIME.

0 c c-Il- In a prefered embodiment, the invention p rovides; a method of synthesizing a NM) synthetase inhibitor compound from the route set out in Scheme 7 above, comprising the steps of: a. alicylating a phenol with 7-bromo-1- heptanol to provide 7-(phenyloxy)-1Iheptanol; b. niesylating 7-(phenyloxy)- I -heptanol to provide 7-(phenyloxy)- I -heptyl methanesulfonate; c. esterifying 7-(phenyloxy)-l-heptyl-methanesulfonate to provide 7- (phenyloxy)-l-heptyl nicotinate; and d. n-methylating 7-(phenyloxy)--heptyl nicotinate to provide [7-(phenyloxy)l-heptyl-(N-methyl) nictotinate] iodide.

In a preferred embodiment, the invention provides a compound having the general structure of Structure 2: s STRUCTURE 2: RI R 3

R

4

R

AI eL(likqr)n--Aryf .2 R 5

R

7 wherein: n is an integer of from I to 12, R, R, each, independently, is an H, an unsubstituted or a substituted cyclic or aliphatic group, a branched or an unbranched group, and wherein the linker is a cyclic or aliphatic, branched or an unbranched alkyl, alkenyl, or an alkynyl group and wherein the linker may also contain heteroatoms. By heteroatoms, it is meant that one or more atoms is an element other than carbon.

may also be one of the following groups: an H, alkyl, alkenyl, alknyl, or an aryl. may further be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups.

Note that n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9. The tethered active molecule, in this example denoted "aryl," moieties may.be the same or different.

In a further embodiment, the invention provides a compound of Structure 4: STRUCTURE 4: Linker R I A R6 R3 B R7 X Rs x Structure 4 wherein: X is a C, N, O or S within a monocyclic or bicyclic moiety, A and B represent the respective sites of attachment for the linker, n is an integer of from 1 to 12, Ri- R7 each, independently, is an H, an unsubstituted or a substituted cyclic group, or an aliphatic group, or a branched or an unbranched group, and the linker is a saturated or unsaturated cyclic group or an aliphatic branched or unbranched alkyl, alkenyl or alkynyl group, and wherein the linker may also contain heteroatoms.

R

1

-R

7 may also be one of the following groups: an H, alkyl, alkenyl, alkynyl, or an aryl group. R 1 -R7 may also be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. One of skill in the art would know what moieties are considered to constitute derivatives of these groups. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.

In a further embodiment, the invention provides a compound of Structure 6: i STRUCTURE 6:

A

Linker R1 R 3

R

R

6

B

SA A y R2 R 7 f

B

wherein: X is C, N, O or S, Y is C, N, O, S, carboxy, ester, amide, or ketone, A and B represent the respective sites of attachment for a linker, n is an integer of from 1 to 12, and R 1

-R

7 each, independently, is an H, unsubstituted or substituted cyclic group or an aliphatic group, a branched or an unbranched group, and the linker is a saturated or unsaturated cyclic or aliphatic group, branched or unbranched alkyl, alkenyl, or alkynyl group and wherein the linker may also contain heteroatoms.

Ri-R 7 may also be one of the following groups: an H, alkyl, alkenyl, S alknyl, or an aryl. R 1

-R

7 may further be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. Note that n may also be an integer of from 3 to more preferably 5 to 9 and, still more preferably 6 to 9. The tethered active molecule, in this example denoted "aryl," moieties may be the same or different.

In a further embodiment, the invention provides a compound of Structure 7: R3 R B 2 k4 B t

R

B

Structure 7 wherein: X is C, N, O or S, Y is C, N, O, S, carboxy, ester, amide, or ketone, A and B represent the respective sites of attachment for a linker, n is an integer of from 1 to 12, and R 1

-R

6 each, independently, is an H, unsubstituted or substituted cyclic group or an aliphatic group, a branched or an unbranched group, and the linker is a saturated or unsaturated cyclic or aliphatic group, branched or unbranched alkyl, alkenyl, or alkynyl group and wherein the linker may also contain heteroatoms.

R

1

-R

6 may also be one of the following groups: an H, alkyl, alkenyl, alknyl, or an aryl. Ri-R 6 may further be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. Note that n may also be an integer of from 3 to more preferably 5 to 9 and, still more preferably 6 to 9. The tethered active molecule, in this example denoted "aryl," moieties may be the same or different.

In a further embodiment, the invention provides a.compound of Structure 8: STRUCTURE 8:

R

N

R

2 wherein: n is an integer of from 1 to 12, R, is an H, methoxy, benzylaxy, or itro and R. is 3pyridyI, N-rnethyl-3-pyridyl, 3-quinolinyl, N-methyl-3-quinolinyL, 3- (dirnethylamino~phenyl, 3-(trirnethylafmoflO)phelyl, 4-(dimethylaniino)pheflyl, 4- (trimethylanimonio)pheflyl, 4-(dimethylamino)phenylmethyl, or 4- (trimethylamimonio)phelmelthyl.

In further embodiments, n may also be an integer of from 3 to TO, more preferably to 9 and, still more preferably 6 to 9.

In a further embodimet the invention provides a compound of Structure STRUCTURE

RI

N3 N

R

wherein: nis anmiteger of fromlIto 12,R, isan H, 00,1, -00113, or -OCHyh, R,3 is H, 031, or

CH#CHCO

2 I is H or 'COXH, and Y is N-linked pyridine-3-carboxylic acid, N-linked pyridine, N-linked quinoline, or N-linked isoquinoline. In fuirther embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.

In a further embodiment, the invention provides a compound of Structure 12: STRUCTURE 12: 1(3 I (CH 2 )n-Y

H

56 4 14.4.abl~''-f -1 wherein: n is an integer of from 1 to 12, R, is H, F, or NO 2

R

2 is H, CH 3 CF,, NO., phenyl, n-butyl, isopropyl, F, phenyloxy, triphenylmethyl, methoxycarbonyl, methoxy, carboxy, acetyl, or benzoyl, R, is H or CF, and Y is N-linked pyridine-3-carboxylic acid, N-linked pyridine, N-linked quinoline, or N-linked isoquinoline. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.

In a further embodiment, the invention provides a compound of Structure 14: STRUCTURE 14: 0 II (CH2),-OC-Y R2 Da it 2

H

wherein: n is an integer of from 1 to 12, R is H, phenyloxy, isopropyl, acetyl, or benzoyl, R, is H or CF, and Y is 3-(dimethylamino)phenyl, 3-(trimelthylammonio)phenyl, 4- (dimethylamino)phenyl, 4-(trimethylammonio)phenyl, 2-(phenyl)phenyl, diphenylmethyl, 3-pyridyl, 4-pyridyl, or pyridine-3-methyl-. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9; In a further embodiment, the invention provides a compound of Structure 16: STRUCETRE 16 wherein R is Hor CO 2

CH

3 and n is an integer of from I to4, more preferably 2 to 3, and even more preferably, n is 3.

In a further embodiment the invention provides a compound of Structure 18: I

X

STRUCTURE 18 wherein R is H or CO 2 CH, and n is an integer of from 1 to 4, more preferably 2 to 3, and even more preferably, n is 3.

In further preferred embodiments of the invention herein, compounds of the structures denoted in Tables 102-128 as Compounds 1-274 were synthesized utilizing the methods disclosed herein. For Compounds 1-274, structures denoted in Figure 6 as Fragments I-X each represent an active molecule, as defined previously herein, which can 58 be included in the compounds of the present invention as further described in the respective Tables. In Fragments I-X of figure 6, the point of attachment for the linker compound is at the nitrogen.

In the chemical structures that follow, and as intended for the compounds of this invention, the symbol X" and T designate generally the presence of an anion. As contemplated by the present invention, the type of anion in the compounds of this invention is not critical. The anions present in the compounds of this may be Scomprised of any such moieties known generally to one of skill in the art or that follow from the synthesis methods disclosed herein.

Sy .1 *s Ji ,1 I L 0-J

IV

Vill

V

vi vii Ix x FIGURE 6: FRAGMENTS UTILIZED IN COMPOUNDS 1-274 In preferred embodiments of the invention herein, the compounds of the present invention correspond to Structure 100:

I

n Structure 100 wherein R' is: r4z3 Y n

I,

n V VI dc?' and n is an integer of from I to 12. N may also be from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 100 and as further defined in Table 100. For those compounds that correspond to Structure 100, n may also be an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.

STRUCTURE 100: (~z rS~':r -M~r~A TABLE 100: SUBSTITUENT GROUPS FOR COMPOUNDS 1-24 In the above Table, R' corresponds to a Fragment as previously defned in Figure 6 and ni indicates the number of linker groups separating the two tethered active molecule groups in the compound.

As set out-below in relation to Compounds 25-7274, Fragments A 0 are set out in Figure 8. The group denoted R in Ad] of Figure 8 can be a bcnzy1 group, a methyl group or a hydrogen. The point of attachment of the linker group to Fragments A-G is at the nitrogen group.

in one embodiment, the compounds of the present invention correspond to compounds of Structure 101. For those compounds that correspond to Structure 101, ni is an integer of fr-om 1 to 12, more preferably from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. The paint of attachment of the linker group for both RI and R' is at the respective nitrogen groups of each illustrated fragment.

Structure 101 wherein R' is: 0

IV

VI

Vfl Vill Go A N Ix wherein RI is: 0 4 3 4 3 4 4 33 R"O 4 4 3 S2 OR 7 2 A B C R 2 2O .~o6 0 .0 D E

O

OR

S4 a 3 4a B o R -7a 77al 7 F G wherein the R group in Fragments A-G is a benzyl group, a methyl group or a hydrogen.

In one embodiment of the invention herein, the compounds of the present invention may include the Fragments illustrated below in Figure 8.

64 4 4 4a44. 3 2 0~2 A B C 4 3 4E 0

D

200 6~ 7272 7a 1 F C FIGURE 8: FRAGMENTS A-G IN COMPOUNDS 25-274 In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 102. For those compounds that correspond to Structure 102, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 102, as further set out in Table 102.

STRUCTURE 102: I 1JJ~LN} jtte'Y:Pt t.vpIIvtNri3*rIr~p% ,rAaYr*.'w PPY.* ~rvr.'~~rs 'tI.1fl~.a'n.I 4 r'%R rn I TABLE 102: SUBSTITUENT GROUPS FOR COMPOUNDS 25-48 R' 4 6 8 I 25 26 27 28 29 f II 31 32 33 Jfl* 34 1 35 36 VII 37 38 I 39 VI* 40 41 f 42 VIII .43 44 V[I 46 47 48 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, A corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups. and A in the respective compounds. Groups 11, VII, VIII each have a benzyl group and Groups Jf*, VI*, Vm* each have a hydrogen, respectively, in the position designated'R in Fragment A of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 104. For those compounds that correspond to Structure 104,n is an integer of-from to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 104, as further set out in Table 104.

STRUCTURE 104: V TABLE 104: SUBSTITUENT GROUPS FOR COMPOUNDS 49-66 RI' n= 4 6 8 I 49 1 50 51 1* 52 53 54 VII 1 55 1 56 57 VII* 58 59 Viii 61 62 63 VI* 64 1 7 65 66 6, In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, B corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and B in the respective compounds. Groups I, VII, VHI each have a benzyl group and Groups VII*, VII* each have a hydrogen, respectively, in the position designated R in Fragment B of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 106. For those compounds that correspond to Structure 106, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 106, as further set out in Table 106.

STRUCTURE 106: OiD 67 7 7 M TABLE 106: SUBSTITUENT GROUPS FOR COMPOUNDS 67-90 R' n= 4 6 8 I 67 68 69 I* 70 71 72 II 74 ffI* •76 1 77 78 VII .79 80 81 VII* 82 83 84 VIH 85 86 87 VI* 88 89 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, C corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and C in the respective compounds. Groups I, II, V VIII each have a benzyl group and Groups m VII, VI* each have a hydrogen, respectively, in the position designated R in Fragment C of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 108. For those compounds that correspond to Structure 108, n is an integer of from 1 to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 108, as further set out in Table 108. STRUCTURE 108: Q Fp I A .tn rwcTfl*JgL'LJi-A.. r.

I TABLE 108: R' n=

I

SUBSTITUENT GROUPS FOR COMPOUNDS 91-108 -4 J 6 8 91 92 93 1I* 194 95 96 VII 97 98 99 -vn* 100 o101 102 VIII 103 104 105 VII* 106 J 107 108 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, D corresponds to a fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and D in the compound. Groups I, VII, VIII each have a benzyl group and Groups VI*, VIL* each have a hydrogen, respectively, in the position designated R in Fragment D of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 110. For those compounds that correspond to Structure 110, n is an integer of from 1 to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 110, as further set out in Table 110.

STRUCTURE 110: 69 69 TABLE 110: SUBSTITUENT GROUPS FOR COMPOUNDS 109-126 R' n= .4 6 8 I 109 110 111 1* 112 113 114 VII 115 116 117 VJI* 118 f 119 120 VII 121 122 f 123 VII* 124 125 126 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, E corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and E in the respective compounds. Groups 1, VII, VfII each have a benzyl group and Groups VH*, VffI* each have a hydrogen, respectively, in the position designated R in Fragment E of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structure set out in Structure 112. For those compounds that correspond to Structure 112, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 112, as further set out in Table 112.

STRUCTURE 112:" TABLE 112: SUBSTITUENT GROUPS FOR COMPOUNDS 127-144 R' n= 4 6 8 I 127 1 128 129 1* 130 131 132 VII 133 134 135 VII*.. 136 137 138 Vm 139 140 i 141 142 143 144 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, F corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and F in the respective compounds. Groups I, VII, VIII each have a benzyl group and Groups VI*, VIIIfl* each have a hydrogen, respectively, in the position designated R in Fragment F of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 114. For those compounds that correspond to Structure 114, n is an integer of from 1 to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 114; as further set out in Table 114.

STRUCTURE 114: r TABLE 114: SUBSTITUENT GROUPS FOR COMPOUNDS 145-162 R' n= -4 J 6 8 I 145 146 147 I* 148 149 150 VII 151 152 153 VII[* 154.- 155 156 VIII 157 158 159 VII* j 160 161 162 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, G corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and G in the respective compounds. Groups 1, VH, VIII each have a benzyl group and Groups VII*, VIII* each have a hydrogen, respectively, in the position designated R in Fragment G of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the presdnt invention correspond to the structures set out in Structure 116. For those compounds that correspond to Structure 116, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 116, as further set out in Table 116.

STRUCTURE 116: TABLE 116: SUESTITUENT GROUPS FOR COMPOUNDS 163-1 78 R_ n=3 5- 9 1 163 164 j 16516 1*167 168* 1 169 170 I 171 172 173 J 174 ff* 175 176 .177 t 178 In the above Table, R' corre1-sponds to a Fragment as previously defined in Figure 6, A corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and A inthe respective compounds. Groups 1, 11 each have a methyl group and Groups III* each have a hydrogen, respectively, in the position designated R in Fragment A of Figure 8..

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 118S. For those compounds that correspond to Structure I118,'n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to In further embodiments, the compounds herein crrespond to Structure 118, as further set out in Table 118.

STRUaTURE 118: TABLE 118: SUBSTITUENT GROUPS FOR COMPOUNDS 179-194 1179 101112 1*183181816 II187 188 189 190 lgl12t9319 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, B corresponds to a Fragment as previously defined in Figure 8,'and n indicates the number of linker groups separating Groups RZ' and B in the respective compounds. Groups I, I each have a met hyl group and Groups It, M*I"each have a hydrogen, respectively, in the position designated R in Fragment B of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set o -ut in Structure 120. For those compounds that correspond to Structure 120, ni is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In Ibrther embodiments, the compounds herein correspond to Structure 120, as further set out in Table 120.

STRUCTURE 120: TABLE 120: SUBSTITUENT GROUPS FOR COMPOUNDS 195-210 R' n= 3 5 79 I 195 196 j 197 198 I* 199 200 201 202 H 203 204 205 206 fflI 207 '208 209 210 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, C corresponds to a Fragment as previously defined in Figure 8, and n ihdicates the number of linker groups separating Groups R' and C in the respective compounds. Groups I, H1 each have a methyl group and Groups 11* each have a hydrogen, respectively, in the position designated R in Fragment C of Figure 8., In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 122. For those compounds that correspond to Structure 122, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 122, as further set out in Table 122.

STRUCTURE 122: 0Re TABLE 122: SUBSTITIJENT GROUPS FOR COMPOUNDS 211-226 ii' 1 51 1 211 212 213 214 1* 215 1 216 217 218 i 219 220 1 221 222 In*2322 225 1 226 In the above Table, R' corresponds to a Fragment as previously defined in Figure 6, D corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker'groups separating Groups R' and D in the respective compounds. Groups 1 HI each have a inethyi-group and Groups I, III each have a hydrogen, respectively, in the position designated R in Fragment D of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out mn Structure 124. For those compounds that correspond to Structure 124, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 124, as further set out in Table 124.

STRUCTURE 124: TABLE 124: SUBSTITUENT GROUPS FOR COMPOUNDS 227-242 I227 228 229 I 230 P231 232 233 234 II 235 j 236 237 .238 .f*239 240 241 242 In the abo ve Table, R' corresponds to a Fragment as previously defined in Figure 6, E corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups and E in the respective compounds. Groups 1,1H each have a methyl group and Groups II1* each have a hydrogen, respectively, in the position designated R in Fragment E of Figure 8.

In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 126. For those compounds that correspond to Structure 126, n is an integer of from I to 12, from 3 to more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 126, as further set out in Table 126.

STRUCTURE 126:

FR

TABLE 126: SUBSTITUENT GROUPS FOR COMPOUNDS 243-258 RI' n= 3 5 1- 7 9 I243 244 245 j 246 1* 247 1 248 1 249 250 fl.251 I 252 253 254 111* 255 1 256 25 7 258 In the above Table, R' corres ponds to a Fragment as previously defined in Figure 6, F corresponds to a Fragment as previously defined in Figure 8, and n indicates the number of linker groups separating Groups R' and F in the respective compounds. Groups I,1H each have a methyl group and Groups M1* each have a hydrogen, respectively, in the position designated R in Fragment F of Figure 8.

In furthfer preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 128. For those compounds that correspond to Structure 128, n is an integer of from 1 to 12, from 3 to more preferably from 5 to 9, and still more preferably from 61to 9. In further embodimnents, the compounds herein correspond to Structure 128, as further set out in Table 128.

STRUCTURE 128: 7 1t *Z't dsd,.tIL& ei.

TABLE 128: SUBSTITUENT GROUPS FOR COMPOUNDS 259-274 3 K 7 9 1 259 260 261 I 262 1* 263 i 264 t 265 1 266 II267 j 268 1 269 270 111* 271 f 272 273 274 In the above Table, Ri' corresponds to a Fragment as previously defined in Figure 6, G corresponds to a Fragment as previously defined in Figure 6, and n indicates the number of linker groups separating Groups R' and G in the respective compounds. Groups 1, II each have a methyl group and Groups III* each have a hydrogen, respectively, in the position designated R in Fragment G of Figure 8.

As used herein, the following terms are defined as follows: Ph: phenyl; I-propyl= isopropyl; OPh =O-Pheny1; and diNO 2 =dinitric.

In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 130 wherein n is an integer of from i to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. Further preferred embodiments of the compounds corresponding to Structure 130 are set out mn Table 130.

STRUCTURE 130: TABLE 130: COMPOUNDS CORRESPONDING TO STRUCTURE 130 n= 3 4 5 6 7 8 9 275 276 277 278 279 280 281 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 132 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 5-H, 6-CF& 5-CH, 5,7-diF, 5,7-diNO,, 5-Butyl, 5-iPropyl, Phenyl, 5-NO,, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF,, 5-COCH 3 5-OCH3, 5-COOCH, or Further preferred embodiments of the compounds corresponding to Structure 132 are set out in Table 132.

STRUCTURE 132: TABLE 132: COMPOUNDS 282-389 CORRESPONDING TO STRUCTURE 132 R n= 3 4 5 6 7 8 282 283 284 285 286 287 6-CF, 288 289 290 291 292 293 3 294 295 296 297 298 299 5,7-diF 300 301 302 303 304 305 5,7-diNO, 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321. 322 323 J, t V r :3 1 "fl. W 'ir i Dh 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 5-COCH3 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383

COOCH,

384 385 386 387 388 389 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 134 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-H, 6-CF,, 5-CH,, 5,7-diF, 5,7-diNO 2 5-Butyl, 5-iPropyl, Phenyl, 5-NO 2 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF,, 5-COCH, 5-OCH3, 5-COOCH,, or COOH. Further preferred embodiments of the compounds corresponding to Structure 134 are set out in Table 134.

STRUCTURE 134: Jd....:jvj .r 4 TABLE 134: COMPOUNDS 390-497 CORRESPONDING TO STRUCTURE 134 R n= 3 4 5 6 7 8 390 391 392 393 394 395 6-CF 3 396 397 398 399 400 401 402 403 404 405 406 407 5,7-diF 408 409 410 411 412 413 5,7-diNO 2 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 3 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491

COOCH,

492 493 494 495 496 497 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 136 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-H, 6-CF,, 5-CH3, 5,7-diF, 5,7-diNO,, 5-Butyl, 5-iPropyl, Phenyl, 5-NO,, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF,, 5-COCH3, 5-OCH,, 5-COOCH,, or COOH. Further preferred embodiments of the compounds corresponding to Structure 136 are set out in Table 136.

82 STRUCTURE 136: TABLE 136: COMPOUNDS 498-605 CORRESPONDING TO STRUCTURE 136 R a= 3 4 5 6 7 8 498 499 500 501 502 503 6-CF 3 504 505 506 507 508 509 5-CH3 510 511 512 513 514 515 5,7-diF 516 517 518 519 520 521 5,7-diNO 2 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 2 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 3 576 577 578 579 580 581 582 583 584 585 586 587 3 588 589 590 591 592 593 594 595 596 597 598 599; COOCH 3 600 601 602 603 604 605 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 138 wherein n is an integer of from 1 to 12, I I I more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-CF,, 5-OPh, 5-iPropyl, 5-COCH, or 5-COPh and Y is 3-N,Ndimethylaminophenyl (3-NN-diCH,), 4-N,N-dimethylaminophenyl (4-N,N-diCH,), or 2- Ph. Further preferred embodiments of the compounds corresponding to Structure 138 are set out in Table 138.

STRUCTURE 138: TABLE 138: COMPOUNDS 606-650 CORRESPONDING TO STRUCTURE 138 R 4 7 8 Y 3 606 607 608 3-N,N-DiCH, 609 610 611 4-NN-DICH 3 612 613 614 2-Ph 615 616 617 3-N,N-DiCH3 618 619 620 4-N,N-DiCH S-OPh 621 622 623 2-Ph 624 625 626 3-N,N-DiCH, 627 628 629 4-N,N-DiCH, 630 631 632 2-Ph 633 634 635 3-N,N-DICH 636 637 638 4-N,N-DiCH, 639 640 641 2-Ph 642 643 644 3-N,N-DiCH, 645 646 647 4-N,N-DiCH, 1 649 650 2-Ph I In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 140 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-CF,, 5-OPh, 5-iPropyl, 5-COCH, or5-COPh, and Z is CH(Ph) 2 or 3-Pyridyl. Further preferred embodiments of the compounds corresponding to Structure 140 are set out in Table 140.

STRUCTURE 140: TABLE 140: COMPOUNDS 651-680 CORRESPONDING TO STRUCTURE 140 R n= 4 7 8 Z 3 651 652 653 CH(Ph), 3 654 655 656 3-Pyridyl 657 658 659 CH(Ph) 2 660 661 662 3-Pyridyl 663 664 665 CH(Ph), 666 667 668 3-Pyridyl 3 669 670 671 CH(Ph) 3 672 673 674 3-Pyridyl 675 676 677 CH(Ph), 678 679 680 3-Pyridyl In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 142 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF 3 5-OPh, 5-iPropyl, 5-COCH,, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 142 are set out in Table 142.

STRUCTURE 142: TABLE 142: COMPOUNDS 681-695 CORRESPONDING TO STRUCTURE 142 R n= 4 7 8 6-CF, 681 682 683 684 .685 686 687 688 689 690 691 692 693 694 695 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 144 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF3, 5-OPh, 5-iPropyl, 5-COCH,,or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 144 are set out in Table 144.

STRUCTURE 144: t .4wAh qin.r..acrcpa:~rhr.

I

TABLE 144: COMPOUNDS 696-710 CORRESPONDING TO STRUCTURE 144 R =4 7 8 6-CF, 696 697 698 699 700 701 702 703 704

CH

3 705 706 707 708 709 710 In fither embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 146 wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. Further preferred embodiments of the compounds corresponding to Structure 146 are set out in Table 146.

STRUCTURE 146: 0 2

N

TABLE 146: COMPOUNDS 71 1-714 CORRESPONDING TO STRUCTURE 146 n= 1I 711 712 713 714 In fRther embodiments, the compounds of the present invention preferably" correspond to compounds of the Structure 148, as further defined in Table 148.

STRUCTURE 148:

NN

CF

3

H-

TABLE 148: COMPOUND 715 CORRESPONDING TO STRUCTURE 148 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 150 wherein ni is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 t 9.

Further pre ferred embodiments of the compounds corresponding to Structure 150 are set out in Table 150.

STRUCTURE 150: TABLE 150: COMPOUNDS 716-718 CORRESPONDING TO STRUCTURE 150 n= 2 3 4 716 717 718 :.fl.P"wA V. 1 F P7"F1 K X. In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 152 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.

Further preferred embodiments of the compounds corresponding to Structure 152 are set out in Table 152.

STRUCTURE 152:

NI

0N. I I C In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 154 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein Z is CH(DiPh), 4-(N,N-dimnethylamino)phenyl, CH 2

CH

2 -(3-pyridyl), or (2-phenyl)-phenyl. Further preferred embodiments of the compounds corresponding to Structure 154 are set out in Table 154.

89 STRUCTURE 154: 02N.

In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 156 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH 3 or -OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 156 are set out in Table 156.

STRUCTURE 156: R

N-

'i 0

C)

In further embodiments, the compounds of the present invention preferably I

I

1 ,1 correspond to compounds of the Structure 158 wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH 3 or -OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 158 are set out in Table 158.

STRUCTURE 158: 0

NI

In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 160 Wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH 3 or -OCIPh. Further preferred embodiments of the compounds corresponding to Structure 160 are set out in Table 160.

STRUCTURE 160:

N

0 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 162 wherein n is an integer 'of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is OCH 3 or -OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 162 are set out in Table 162.

STRUCTURE 162: In further embodiments, the compounds of the present invention preferably 92 correspond to compounds of the Structure 164 wherein n is an integer of from I to 12, more preferably, from 3 to. 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH3 or -OCHPh. Further preferred embodiments of the compounds corresponding to Structure 164 are set out in Table 164.

STRUCTURE 164: In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 166 wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH or -OCH.Ph. Further preferred-embodiments of the compounds corresponding to Structure 166 are set out in Table 166.

STRUCTURE 166:

I

R N O N+ .0 TABLE 166: COMPOUNDS 780-789 CORRESPONDING TO STRUCTURE 166 R n= 4 5 6 7 8 -OCH, 780 781 782 783 784 -OCHPh 785 786 787 788 789 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 168 wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH, or -OCH,Ph. Further preferred embodiments of the compounds corresponding to Structure 168 are set out in Table 168.

STRUCTURE 168:

N

N f.

I

In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 170 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH 3 or -OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 170 are set out in Table 170.

STRUCTURE 170:

-V

In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 172 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH 3 and -OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 172 are set out in Table 172.

I

STRUCTURE 172: 0 r In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 174 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is -OCH, and -OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 174 are set out in Table 174.

STRUCTURE 174: In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 176 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein Z is 3-quinoline, 3-(N,N-dimethylamino)phenyl, or 4-(N,Ndimethylamino)phenyl. Further preferred embodiments of the compounds corresponding to Structure 176 are set out in Table 176.

STRUCTURE 176: 0 2 N N-pO

Z

0 O TABLE 176: COMPOUNDS 830-847.CORRESPONDING TO STRUCTURE 176 Z n= 4 5 6 7 8 9 3-quinoline 830 831 832 833 834 835 3-(N,N-diCH 3 836 837 838 839 840 841 phenyl 4-(N,N-diCH,) 842 843 844 845 846 847 phenyl In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 178 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.

I

Further preferred embodiments of the compounds corresponding to Structure 178 are set out in Table 178.

STRUCTURE 178:

II

0 2 N TABLE 178: COMPOUNDS 848-853 CORRESPONDING TO STRUCTURE 178 N= 4 5 6 7 8 9 848 849 850 851 852 853 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 180 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.

Further preferred embodiments of the compounds corresponding to Structure 180 are set out in Table 180.

STRUCTURE 180:

I

TABLE 180: COMPOUNDS 854-860 CORRESPONDING TO STRUCTURE 180 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 182 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.

Further preferred embodiments of the compounds corresponding to Structure 182 are set out in Table 182.

STRUCTURE 182: TABLE 182: COMPOUNDS 861-867 CORRESPONDING TO STRUCTURE 182 2 3 4 5 6 7 8 n= In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 184 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 6-CF 3 5-OPh, 5-CH(CH 3 5-COCH, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 184 are set out in Table 184.

STRUCTURE 184: TABLE 184: COMPOUNDS 868-882 CORRESPONDING TO STRUCTURE 184 R n= 4 7 i 8 j 6-CF, 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 186 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF,, 5-OPh, 5-CH(CH) 5-COCH, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 186 are set out in Table 186.

100 I STRUCTURE 186: TABLE 186: COMPOUNDS 883-897 CORRESPONDING TO STRUCTURE 186 R n= 4 7 I 8 6-CF,, 883 884 885 886 887 888 889 890 891 5-COCH3 892 893 894 895 896 897 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 188 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 6-CF,, 5-OPh, 5-CH(CH 3 5-COCH 3 or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 188 are set out in Table 188.

STRUCTURE 188: TABLE 188: COMPOUNDS 898-912 CORRESPONDING TO STRUCTURE 188 R n= 4 7 8 6-CF 3 898 899 900 901 902 903 904 905 906 907 908 909 910 I 911 912 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 190 wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF3, 5-OPh, 5-CH(CH 3 5-COCH3 or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 190 are set out in Table 190.

STRUCTURE 190: CH3 TABLE 190: COMPOUNDS 913-927 CORRESPONDING TO STRUCTURE 190 R n= 4 7 8 6-CF, 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 192 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 6-CF,, 5-OPh, 5-CH(CH) 2 5-COCH, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 192 are set out in Table 192.

STRUCTURE 192: TABLE 192: COMPOUNDS 928-942 CORRESPONDING TO STRUCTURE 192 R n= 4 7 8 6-CF3 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 194 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and R' is an H or -OCH 2 Ph and R 2 is H or COOCH3. Further preferred embodiments of the compounds corresponding to Structure 194 are set out in Table 194.

STRUCTURE 194: n 0 TABLE 194: COMPOUNDS 943-954 CORRESPONDING TO STRUCTURE 194 R1 R2 n= 6, 7 8 9 _I H H 943 944 945 946 H ICOOCH, 947 948 949 950.

-OCH,Ph COOCH, 951 952 953 954 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 196 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R' is an H or a -OCH 2 Ph and R 2 is H or COOCH 3 Further preferred embodiments of the compounds corresponding to Structure 196 are set out in Table 196.

STRUCTURE 196: TABLE 196: COMPOUNDS 955-966 CORRESPONDING TO STRUCTURE 196 R' R 2 n= 6 7 8 9 H H. 955 956 957 958 H JCOOCH, 959 960 961 962 [-OCH2Ph ICOOCH 963 964 965 966 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 198 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R' is an H, -OCHPh or -OCPh 3 and R 2 is H, or COOCH 3 Further preferred embodiments of the compounds corresponding to Structure 198 are set out in Table 198.

STRUCTURE 198: 2 2 R No TABLE 198: COMPOUNDS 967-978 CORRESPONDING TO STRUCTURE 198 R1 R 2 n 6 7 8 9 H H 967 968 969 970 H COOCH, 971 972 973 974 -OCH Ph COOCH 975 976 977 978 -OCPh, COOCH, 1106 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 200 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R' is H or a -OCH 2 Ph and 2 is H or COOCH 3 Further preferred embodiments of the compounds corresponding to Structure 200 are set out in Table 200.

STRUCTURE 200:

T

TABLE 200: COMPOUNDS 979-990 CORRESPONDING TO STRUCTURE 200 R1 R2 n= 6 7 8 9 H H 979 980 981 982 H COOCH, 983 984 985 986 OCH2Ph COOCH, 987 988 989 990 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 202A.

STRUCTURE 202A: 0 -N S

O(CH

2 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 202A wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is H; 4-NO 2 2-CONHPh; 2-NO,; 4-[l'(4'-acetylpiperazine)]; 2- COCH,; 3-OCOCH,; 3-OCH 3 4-COCH 3 3-OCOPh; 2-CONH2; 4-CH=CHCOCH,; 4- OCOPh; 4-CH=CHCOPh; 4-{CO-3'[2'-butylbenzo(b)furan]}; 3-NO 2 phenylhydantoin)]; 2-CH=CHCOPh; 2-OCH 3 4-COPh; 4-CONH 2 3-COCH,; 4-OPh; 4- (N-Phthalimide); 3-(N-Morpholine); 2-(N-pyrrolidine); 2-(N-Morpholine); or 4-OCH 2 Ph.

Further preferred embodiments of the compounds corresponding to Structure 202 are set out in Table 202.

TABLE 202: COMPOUNDS 991-1021 CORRESPONDING TO STRUCTURE 202A R= nw4 n=7 n=8 H 991 993 4-NO 2 992 994 995 2-CONHPb 996 2-NO, 9971 44[l'(4'-acetylpiperazine)] -9 2-COCK 3 999 3-OCOCH 3 ___1000 3 1001 4-COCK 3 H, 10021 3-OCOPh 1003___ 2-CONH 2 1004 4-CH=CHCOCH, 4-OCOPh 10061 4-CH=CHCOPh 10071 4-{CO-3'[2'-butylbenzo(b)firan} 1009 3-NO 1 00 4-[5'(5'phenylhydantoin)I 1010 2-CH=CHCQPh 1011 2-OCH, .__1012J 4-COPh ___1013 4-CONK, 1014 3-COCK 3 ___1015 4-OPh 1016 4-(N-phthalimide) 10171 3-(N-morpholine) 10181 2-(N-pyrrolidine) 1019 2-(N-mozpho line) 1020 4-OCH,Ph 1021 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 204A wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably, from 6 to 9 and wherein R is 4-NO 2 2-CONHPh; 2-NO,; 4-[l'(4'-acetylpiperazine~l; 2-COCK 3 3-OGOCH 3 3-0GW 3 4-GOGH 3 3-OCOPh; 2-CONH,; 4-GH=CHCOGH 3 4-OCOPh; 4- CH=GHCOPh; 4- {GO-3 '[2'-butylbenzo(b)furanfl; 3-NO 2 4-[5'-(Y'-phenylhydantoin)]; 2- CH=CHCOPh; 2-OCH 3 4-GOPh; 4-GONH 2 3-.COGH 3 4-OPh; 4-(N-phthalimide); 3-(Nmorpholine); 2-(N-pyrrolidine); 2-(N-morpholine); or 4-OGH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 204 are set out in Table 204.

STRUCTURE 204A: 0-/

O(GH

2 8 O0 lCH 2 TABLE 204: COMPOUNDS 1022-1048 CORRESPONDING TO STRUCTURE 204A 4-NO 2 1022 2-GONEPh 1023 2-NO 2 1024 1'(4'-acetyjpiperazine)] 1025 2-GOGH 3 1026 3-OGOGH 3 1027 3-OCH 3 1028 4-GOGH 3 10291 3-OCOPh 1030 2-CON! 3 1031 4-GH=CHCOCH 3 1032 4-OGOPh 1033 4-GH=CHGOPh 10341 4- {GO-3'[2'-butylbenzo~b)furan]} 10351 3-NO, 10361 4-[5-(5'pheylhyanton)]1037 2-GH=GHCOPh 1038 2-OGIT, 1039 4-GOPh 1040 4-GONE! 2 1041 3&COCH 3 1042 4-OPh 1043 4-(N-phthalintide) 1044

R=

3-(N-morpholine) 1045 2-(N-pyrroiidine) 1046 2-(N-morpholine) 1047 4-OCH,Ph 1048 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 206 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably, from 6 to 9 and wherein R is H; 4-NO,; 2-CONHPh; 2-NO,; 2-COCH,; 3-OCH,; 4-COCH; 3- OCOPh; 2-CONH,; 4-CH=CHCOCH3; 4-OCOPh; 4-CH=CHCOPh; butylbenzo(b)firan]}; 3-NO 2 2-CH=CHCOPh; 2-OCH; 4-COPh; 3-COCH,; 4-OPh; 4- (N-phthalimide); or 4-OCH,Ph. Further preferred embodiments of the compounds corresponding to Structure 206 are set out in Table 206.

STRUCTURE 206: TABLE 206: COMPOUNDS 1049-1068 CORRESPONDING TO STRUCTURE 206 R= n=4 n=7 n=8 'H 1049 1051 4-NO 2 1050 1052 1053 2-CONHPh 3054 2-NO 2 1055 2-COCH, 1056 3-OCH, 1057 4-COCH, 1058 3-OCOPh 1059 2-CONH, 1060 4-CH=CHCOCH, 1061 4-OCOPh 1062 4-CH=CHCOPh 1063 4- f{CO-3'[2'-butylbenzo(b)furanj} 1064 1065 2-CH=CHCOPh ___1066 '2 10671 1063 3-COCK 3 1069 4-OPh 1070 14-(N-phthalirnide) 1071 4-OCH, Ph 10721 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 208 Wherein n is an integer of from I to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably, from 6 to 9 and wherein R is 4-NO 2 2-CONTIPh; 2-NO,; 2 2 *C0CH 3 3-OCH 3 4-COCK 3 3- OCOPh; 2-CONK 2 4-CH=CHCOCH 3 4-OCOPh; 4-CK=CKCOPh; 4-{CO-311Zbutylbenzo(b)faran]}; 3-NO 2 2-CII=CHCOPh; 2-OCH 3 4-COPh; 3-COCK 3 4-OPh; 4- (N-mlnhaiinuide); 3-(N-xnorpholine); 2-(N-morpholine); or 4-OCH 2 Ph. Further preferred embodiments of the compounds corresponding to Structure 208 are set out in Table 208.

STRUCTURE 208: 0 4 >.7O(CH 2 8 0 1

-CH

2 /7

T

C'l~fflfTTlfQ lfl71IM fl' rPflrPnNflNG Tfl STRUJCTTURE 2fl8 ri AnIT V' lne 4-NO 2 1073 2-CONH~h 1074 2-NO 2 17 2-COCK 3 10761 3-OCH, 1077 4-COCK 3 1078 3-OCOPh 1079.

2-CONH, -1080 4-CH=CHCOCH, 1081 4-OCOPh 1082 4-CH=CHCOPh 1083 4- {CO-3'[2'-butylbenzo(b)furan]} 1084 3-NO, 1085 2-CH=CHCOPh 1086 2-OCH 3 1087 4-COPh 1088 3-COCH 3 1089 4-OPh 1090 4-(N-phthalimide) 1091 3-(N-morpholine) 1092 2-(N-morpholine) 1093 4-OCH,Ph 1094 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 210 wherein R is NH,; NMe; NMe,.I;

NH

2 .HC1; NMe,.HCI. Further preferred embodiments of the compounds corresponding to Structure 210 are set out in Table 210.

STRUCTURE 210: PhCH 2 O COOMe

(CH

2 )OCO /-CH 2

-R

TABLE 210: COMPOUNDS 1095-1099 CORRESPONDING TO STRUCTURE 210

R=

NH,1095 NMe, 1096 NMe3.I- 1097

NH

2 .HCI 1098 NMe.HCI 1099 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 212 wherein R' is PhCONH or Ph 3 C and R" is H or COOCH 3 Further preferred embodiments of the compounds corresponding to Structure 212 are set out in Table 212.

STRUCTURE 212: TABLE 212: COMPOUNDS 1100-1101 CORRESPONDING TO STRUCTURE 212

R"=

PhCONH H 1100 PhC COOCH, 1101 In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 214 wherein P is 4-hydroxyphenyl or 3hydrdxy-4-methylphenyl, Further preferred embodiments of the compounds corresponding to Structure 214 are set out in Table 214.

STRUCTURE 214: TABLE 214: COMPOUNDS 1102-1103 CORRESPONDING TO STRUCTURE 214 4-hydroxyphenyl 1102 3-hydroxy-4-methylphenyl 1103 1 In further embodiments, the compounds of the present invention preferably correspond to compounds of Structure 216 wherein R' is PhCONH and and R" is H or COOCH3 and n= 7 or 8. Further preferred embodiments of the compounds corresponding to Structure 216 are set out in Table 216.

Structure 216:

R"

N

(CH2)nOCOCH2- T TABLE 216: COMPOUNDS 1104-1105 CORRESPONDING TO STRUCTURE 216 n= PhCONH H 8 1104 PhCH,O COOCH, 7 1105 In a particularly preferred embodiment of the invention herein,.the present invention comprises compounds of the structures in Table 301 below.

TABLE 301: A FIRST GROUPING OF BACTERIAL NAD SYNTHETASE INHIBITOR LEAD COMPOUNDS 9 o

CH,

182 CH3Nc

CH

3 9 9

X-

iN.

NJN N

H

H

315 349 745

NN

7460 747 748N

4 11I 0N 848N NO2 0 N 849N

NOX\

6 0

N

850

NO

N f L 9 J c 0 N 853 117

CH

3 .0 go N

CH

3 0-c H 878 879 884 886 887 889 X7

OK

1* 't *11.

'V

975' 976 977 cc- 122

I

In a further preferred embodiment, the present invention comprises one or more compounds from Table 302, below.

TABLE 302: A SECOND GROUPING OF BACTERIAL NAD SYNTHETASE INHIBITOR LEAD COMPOUNDS X- \N I 0 213 I o x- 9 214.

Nk

X-

X.

H

349

'C-

V t 0 t cc 0 ~r L J 7 NX- H

H

921 917 013 ol 0AC 924 936 0 CH H N 939 H X 941 H

+X

942 129

/CH

3 0 A 1

,CH

3 0 i~j NN 975 976 981 982 983 984 131 010-

C

Ph 06 PhO-a0(CHIJ,0- 0 I070 0CH.J 5 0 No 2 1 .132 o O(CH,4hO I 0 1034 0.

o(H.\I x-0 P-.1 0 O(CH,),0 \./1t Ph-/- (CH)8- X1.

1104 \/~r7;o 11 CH,hO -c 7 In a further preferred embodiment, the present invention comprises one or more compounds from Table 303, below.

TABLE 303: A THIRD GROUPING OF BACTERIAL NAD SYNTHETASE INHIBITOR LEAD COMPOUNDS

SX-

N-

o y=a Ph 0(C 1070 Sc N- 0 N0 104$ 1080 0

X-

Pho~ 4/H~ 0 PhJ 0- 107 Ph f 2 a/ 1100 ;Ow Ph IIe~oO 1106 The compounds of the invention may be readily synthesized using techniques generally known to synthetic organic chemists. Suitable experimental methods for making and derivatizing aromatic compounds are described, for example, methods for making specific and preferred compounds of the present invention are described in detail in Examples I to 4 below.

This invention preferably ftirther provides a method of generating a library comprising at least one bacterial NAD synthetase enzyme inhibitor compound comprising the steps of: a. obtaining the crystal structure of a bacterial NAD synthetase enzyme; b. identifying one or more sites of catalytic activity on the NAD synthetase enzyme; c. identifying the chemical structure of the catalytic sites on the NAD synthetase enzyme; d. selecting one or more active molecule compounds that will demonstrate affinity for at least one of the catalytic sites on the NAD synthetase enzyme; e. synthesizing one or more dimeric compounds comprised of at least one active molecule wherein the active molecule compound are joined by means of n linker compounds and wherein n is an integer of from 1 to 12, and f. screening the one or more compounds for NAD synthestase inhibitor activity.

The library further comprises one or more compounds set forth in Table 301 above. In one embodiment, a library of compounds according to the invention herein preferably includes compounds of the structures set out in structures 1 to 1106 above. Further preferably, the library comprises a compound of Structure 2, still preferably, Structure 4, further preferably, Structure 6, and further preferably, Structure 7. In further preferred embodiments, the library comprises at least one compound of StructureS8, Structure Structure 12, Structure 16 or Structure 18.

In another preferred embodiment of the invention herein, the one or more dimeric comipounds comprise at least two active molecules. Still preferably, the active molecules are the same. Alternatively, it is preferable that the active molecules are different.

In the invention herein, a software program that predicts the binding affinities of molecules to proteins is utilized in the active molecule selection step. Further preferably, a software program that evaluates the chemical and geometric cdmplecntarity between a small molecule and macromolecular binding site is utilized in the active molecule selection step.

In yet another preferred embodiment, the compounds are synthesized utilizing a rapid, solution phase parallel synthesis and wherein the compounds are generated in a combinatorial fashion.

In a preferred embodiment, the invention provides a method of treating or preventing a microbial infection in a mnanmnal comprising administering to the mammal a treatment effective or treatment preventive amount of a bacterial NAD synthetase enzyme inhibitor compound. In a particularly preferred embodiment, the compound administered in the method is a compound as set out previously in Table 301. In another embodiment, invention herein preferably includes compounds I to 1106 above. Further preferably, the compound administered comprises at least one compound of Structure 2, still preferably, Structure 4, further preferably, Structure 6. In firther preferred embodiments, the compounds administered in the method comprise compounds of Structure 8, Structure Structure 12, Structure 16 or Structure. 18.

In a preferred embodiment, the invention provides administering a broad spectrum antibiotic to a mammal in need of such treatment or prevention, In a further preferred embodiment, the microbial infection is a bacterial infection, In yet another embodiment of the invention, the bacterial infection is caused by a bacterium that is a gram negative or gram positive bacteria. The bacterial infection may preferably be caused by an antibiotic resistant stain of bacteria.

Further provided by the invention herein is preferably a method of killing a prokaryote with an amount of prokaryotic NAD synthetase enzyme inhibitor compound to reduce Or eliminate the production of NAD whereby the prokaryote is killed. A method of decreasing prokaryotic growth, comprising contacting the prokaryote with an amount of a prokaryotic NAD synthetase enzyme inhibitor effective to reduce or eliminate the production of NAD whereby prokaryotic growth is decreased is also provided. In the method of killing a prokaryote, as well as in the method of decreasing prokaryotic growth, the compound comprises one or more compounds of Table 301, Table 302 or Table 303.

Still preferably, the invention comprises one or more of compounds 1 to 1106 above.

Further preferably, the compound administered is a compound of Structure 2, still preferably, a compound of Structure 4, further preferably, Structure 6. In further preferred embodiments, the compounds administered in the methods compounds of Structure 7, Structure 8, Structure 10, Structure 12, Structure 16 or Structure 18.

In the method of killing a prokaryote, as well as in the method of decreasing prokaryotic growth, the prokaryote is a bacterium. Further preferably, the bacterium is a gram negative or a gram positive bacteria. Still preferably, the prokarjote is an antibiotic resistant strain of bacteria.

Also in the method of killing a prokaryote, as well as in the method of decreasing prokaryotic growth, the NAD synthetase enzyme inhibitor is a compound that selectively binds with catalytic sites or subsites on a bacterial NAD synthetase enzyme to reduce or eliminate the production of NAD by the bacteria.

In the methods discussed above, the compound is preferably administered by oral, rectal, intramuscular, intravenous, intravesicular or topical means of administration. The compounds of this invention can be administered to a cell of a subject either in vivo or ex vivo. For administration to a cell of the subject in vivo, as well as for administration to the subject, the compounds of this invention can be administered orally, parenterally intravenously), by intramuscular injection, by intraperitoneal injection, subcutaneous injection, transdermally, extracorporeally, topically, mucosally or the like.

Depending on the intended mode of administration, the compounds of the present invention can be in pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, 138 suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected composition, possibly in combination with a pharmaceutically acceptable catrer and, in addition, may include other medicinal agents, pharmaceutical agents, catrers, adjuvants, diluents, etc.

Parenteral administration of the compounds of the present invention, if used, is generally characterized by injection. Injeetables can be prepared in conventional forms,.

either as liquid solutions or suspensions, solid farms suitable for solution of suspension in liquid prior to injection, or as emulsions. As used herein, "parenteral administration" includes intradet'mal, subcutaneous, intramuscular, intraperitoneal, intravenous and intratracheal routes. One approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See eg., U.S. Patent No. 3,610,795, which is incorporated by reference herein. These compounds can be present in a pharmaceutically acceptable catrer, which can also include a suitable adjuvant. By "phannaceutically acceptable," it is meant a material that is not biologically or otherwise undesirable, the material may be administered to an individual along with the selected compound without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.

Routes of administration for the compounds herein are preferably in a suitable and pharmacologically acceptable formulation. When administered to a human or an animal subject, the bacterial NAD synthetase enzyme inhibitor compounds of the libraries herein are preferably presented to animals or humans orally, rectally, intramuscularly, intravenously, intravesicularly or topically (including inhalation). The dosage preferably comprises between about 0.1 to about l~g per day and wherein the dosage is administered from about 1 to about 4 times per day. The preferred dosage may also comprise between 0.001 and 1 g. per day, still preferably about 0.01, 0.05, 0.1, and 0.25, 0.5, 0.75 and 1.0 g per day. Further preferably, the dosage may be administered in an amount of about 1, 7.5,10.0, 12.5 and 15.0 g per day. The dosage may be administered at a still preferable rate of about 1, 2, 3, 4 or more times per day. Further, in some circumstances, it may be preferable to administer the compound of the invention continuously, as with, for example, intravenous administration. The exact amount of the compound required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular compound used, its mode of administration and the like.

Thus, it is not possible to specify an exact amount for every compound. However, an appropriate amount can be-detennined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

If ex vivo methods are employed, cells or tissues can be removed and maintained outside the subject's body. according to standard protocols well known in the art. The compounds of this invention can be introduced into the cells via known mechanisms for uptake of small molecules into cells phagocytosis, pulsing onto class I MHCexpressing cells, liposomes, etc.). The cells can then be infused in a pharmaceutically acceptable carrier) or transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

It is fulrther provided a method of disinfecting a material contaminated by a microbe, comprising contacting a contaminated material with a bacterial NAD synthetase enzyme inhibitor compound in an amount sufficient to kill or deactivate the microbe. In yet another embodiment, the compound utilized for contacting comprises one or more compounds of Table 301, Table 302 or Table 303. The compounds utilized for contacting may also comprise one or more of compounds I to 1106. Further preferably, the compound utilized for contacting is a compound of Structure 2, still preferably, a compound of Structure 4, further preferably, Structure 6. In further preferred embodiments, the compounds utilized for contacting in the method comprise compounds of Structure 7, Structure 8, Structure 10, Structure 12, Structure 16 or Structure 18.

In yet a further embodiment of the invention herein, the compounds of the present invention are effective As disinfectant materials for, for example, hard or soft surfaces, fabrics, and other contaminated materials such as those in hospitals, households, schools, nurseri es, and any other location. In yet another embodiment, the invention provides a method for disinfecting comprising contacting a bacterial contaminated material with a bacterial NAD synthetase enzyme inhibitor compound.

In a further aspect of the invention, an in vitro "one-at-a-time" method of screening compounds for bacterial NAD synthetase enzyme inhibitory activity is provided. In a preferred embodiment, this in vitro method of screening compounds for such activity comprises the steps of preparing a solution comprising pure bacterial NAD synthetase enzyme, contacting the solution with the compounds set out herein, and determining the rate of the enzyme-catalyzed reaction. Preferably, measurement of the rate of enzymecatalyzed reaction comprises a measure of NAD synthetase inhibitory activity. In a further embodiment, the rate of enzyme-catalyzed reaction comprises a measure of antibacterial activity. In a still further embodiment, the rate of enzyme-catalyzed reaction corresponds to a measure of antimicrobial activity.

Preferably, the method of preparing the bacterial enzyme solution for use in the in vitro screening method comprises utilizing molecular biological methods to over-express bacterial NAD synthetase enzyme, for example from B. subtilis, in E. coli. One of skill in the art will recognize techniques useful for such a process. A particularly preferable method comprises: a) cloning the Out B gene encoding NAD synthetase enzyme and overexpressing the gene in E. coli; b) purifying the cloned and over-expressed gene by ionexchange; c) purifying further the enzyme material from step b using ion-exchange methods; d) further purifying the material from step c using size exclusion chromatography wherein the bacterial NAD synthetase enzyme is essentially pure; and e) preparing an assay solution in quantities of about 10 to 15 mg pure bacterial NAD synthetase enzyme per liter of fermentation broth. As used herein, "essentially pure" means greater than about 90% purity, more preferably, greater than about 95% purity and, still more preferably, greater than about 99% purity.

In one embodiment of the in vitro screening method, the following procedure is utilized to measure the rate of enzyme catalyzed reaction. A solution ofHEPPS, pH with KCI is prepared containing the following species: ATP, NaAD, MgCI 2

NH

4 Cl, ADH, and ETOH. A stock solution of test inhibitors is then prepared by dissolving solid samples into 100% DMSO. The test compound stock solution is then added to the mixture to give the final test compound concentrations. NAD synthetase enzyme solution is added, the mixture is mixed three times, and the absorbance at 340 nm is then monitored kinetically using an UV-Vis spectrophotometer. The initial kinetics trace after enzyme addition is then fit to a straight line using linear regression, with this rate is then compared to that of a control containing no inhibitor, using the following formula-to calculate Inhibition: 100%, where Vo is the rate of the reaction with no test compound present and V is the rate of the reaction with test the test compound added. Each compound is tested in triplicate, and the resulting values for inhibition were averaged to give the listed value. IC,, (concentration needed to inhibit 50% of the test bacteria) values were obtained for select compounds by assaying six different concentrations of test compound, in triplicate, at concentrations between 0.0 and 2.0 mM, and plotting the resulting inhibition values against the -LOG of the test compound dose to reveal the concentration at which 50% inhibition is observed.

Preferably, the in vitro method can also be adapted to allow screening for compounds with bacterial NAD synthetase enzyme inhibitory activity in other forms.of bacteria, as well as other types of microbes. For example, the above-described procedure can be adapted to screen for inhibitory activity in at least the following bacteria types: BACTERIUM

STRAIN

Escherichia coli K-12 MG1655 (CGSC#6300) Escherichia coli K-12 W3110 (CGSC#4474) Salmonella typhimurium LT2 TT366 Streptococcus pneumonia D39 Streptococcus pneumoniae WU2 Bacillus subtilis A700 In a further embodiment of the in vitro screening method, the method can be used to screen existing compounds commercially available compounds, such as nitroindole and N-methyl nicotinic acid. One of skill in the art will recognize the manner in which the designing and screening methods herein can be utilized to identify commercially available compounds, such as the previous non-exhaustive list, that will exhibit NAD synthetase enzyme inhibitory activity, both in bacteria and other microbes.

In order to test a library of NAD synthetase enzyme inhibitor compounds, such as those of the present invention, it is particularly preferable to utilize a method of rapid (high throughput) screening. To this end, the potential inhibitory activity of the library of synthetic compounds in one embodiment is assessed via a coupled enzymatic assay. The coupled assay involves two steps as summarized below.

Step 1 Mg 2 4 'NH ATP NaAD Mo NAD AMP +Ppi NAD synthetase Step 2 Ethanol NaAD NADH Acetaldehyde H* Alcohol Dehydrogenase In order to rapidly measure the inhibitory activities of the compounds in the library, the invention provides a high through-put screening system (HTS system). The HTS system preferably utilizes an integrated robotic system that coordinates the functions of a liquid handler and a spectrophotometer. The robotic station is preferably responsible for the movement of all hardware and the integration of multiple stations on the worksurface. The liquid handler is preferably programmed to perform all phases of liquid dispensing and mixing. The spectrophotometer is preferably equipped to monitor absorbance in a 96-well plate format.

In one embodiment, the assay is designed for a 96-well plate format reaction buffer containing HEPPS buffer, pH 8.5, MgCl 2 NH4C1 2 KCI, NaAD, n-Octyl--D- Glucopyranoside, ethanol, NAD synthetase, and yeast alcohol dehydrogenase. At the next stage, the liquid handler dispenses DMSO (with or without inhibitor) into the reaction well. The liquid handler mixes these components utilizing a predefined mixing program.

The reaction is initiated by the addition of a solution of ATP dissolved in buffer. The reaction is monitored by measuring the increase in absorbance at 340nm. The linear portion of the reaction is monitored for a period of time. The initial velocity is determined using the software supplied with the spectrophotometer.

The compounds of the library herein are supplied as a stock with a concentration dissolved in 100% DMSO. An initial screen is conducted on all compounds using a 2 or 3 concentration screen. The 2 panel screen used concentrations of 0.2mM and 0. ImM for the compounds. The 3 panel screen used concentrations of 0.2mM, 0.ImM, and 0.05mM.

From the initial screen, "lead compounds" those compounds which demonstrated the greatest inhibitory capacity, are then preferably subjected to a wider screen of concentrations 1mM to 0.001mrnM) to determine the apparent IC-50 values for each compound.

In still a further preferred embodiment of the invention herein, the high throughput method is utilized to screen commercially available compounds for bacterial NAD synthetase enzyme inhibitory activity. In an additional embodiment, the NAD synthetase enzyme inhibitor compounds are tested as inhibitors of bacterial growth against a variety of bacteria types.

In a further embodiment of the invention, compounds within the libraries of NAD synthetase inhibitor compounds are evaluated for antibacterial and antimicrobial activity.

In one embodiment, compounds are preferably evaluated for their potential to inhibit the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Staphoyloccus epidermitis. The inhibitors are preferably initially screened in duplicate at one concentration The test inhibitor compounds are prepared by dissolving the solid samples in DMSO. Aliquots from the inhibitor stocks are placed in sterile 96-well plates by the liquid handler discussed previously. Cultures of B. subtilis, P. aeruginosa and S. epidermitis are prepared in liquid broth (LB) media and incubated in an orbital shaker overnight. Dilutions (with LB media) of the overnight cultures are added to the 96-well plates containing the inhibitors. The plates are incubated and the absorbance measured at 595nrn in a plate reader.

In this embodiment of the invention, a diluted overnight culture without inhibitors serves as one of three controls in the experiments. A positive control, which includes an identical concentration of the drug Tobraniycin as the inhibitors being tested, and a DMSO control are also performed during each inhibitor screen. The DMSO control was included for comparison with the control that contained no inhibitors.

Percent inhibition of each inhibitor was calculated by the following formula: f{(AD AD}*IOO0; where AD =the absorbance at 595nm of the DMSO control and A, =the absorbance of the inhibitor at 595nxn.

In a further embodiment, dose responses are performed on the compounds that inhibited greater than 85% in the initial screen. The dose responses consisted of 5 different concentrations (from 100 mM 0. 1 mM) of each inhibitor and the positive control Tobramycin. The cultures are prepared and grown in the same manner as the inhibitor screens and the same controls were included. The absorbance is measured every hour and a half during the six hours of growth. Percent inhibitions are calculated again for each concentration tested. The lowest concentration that resulted in an 85% inhibition or higher is terned the Minimum Inhibitory Concentration that inhibited bacterial growth (M1C 85 When a NAD synthetase enzyme inhibitor compound of a library herein are to be administered to a humans or an animal a mammal, it is preferable that the compounds show little or no toxicity to the patient. Therefore, in one embodiment of the invention herein, the toxicities of the NAD-synthetase enzyme inhibitors are evaluated using human epithelial cells as set out in Example I I below.

EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions and 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. Efforts have been made to ensure accuracy with respect to numbers amounts, temperature, etc.) but some errors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at room temperature, and pressure is at or near atmospheric.

EXAMPLE 1: EXPERIMENTAL PROCEDURE FOR PREPARING COMPOUNDS SINGLY IN SCHEME 3 The following Example I describes one embodiment of the invention herein for compounds prepared according to the synthetic pathway set out in Scheme 3, described previously. For this particular Example, the linker length n, is equal to 6.

Compounds prepared according this embodiment were prepared, individually, not using parallel solution phase synthesis methods. One of skill in the art will readily recognize the manner in which the following Example may be varied to obtain the linker lengths within the scope of the present invention.

A. Alkylation of 5-nitroindole with 6-bromohexyl acetate. A solution of nitroindole (1.00 g, 6.22 rnmol) in DME (2.0 mL) was added dropwise using an addition funnel to the suspension of NaH '(0.24 g, 0.01 mmol in 2.0 mL DME), previously washed with DME (3 X 3.0 mL). The sides of the addition funnel were rinsed with an additional mL of DME. During the addition, an instantaneous gas evolution occurred. The reaction flask was then immersed into a preheated oil bath at 80°C and allowed to gently reflux for 15 minutes. The flask was then cooled to ambient temperature and a solution of acetate (1.39 g, 6.22 mmol) dissolved in DME (2.0 mL) was added dropwise using the addition funnel. The sides of the funnel were washed with an additional portion ofDME (2.0mL). The reaction flask was then immersed into a preheated oil bath set at 80 0 C and allowed to reflux for 18 hours. Workup consisted of quenching the reaction using saturated NH,C1 (25 mL) and extracting the aqueous layer with ethyl acetate (4 X 25 mL). The organic layers were combined, dried over anhydrous Na 2

SO

4 filtered, and evaporated to dryness under reduced pressure. The product was then purified by flash chromatography on silica gel using hexane-acetone to afford the product (0.39 g) and deacetylated product (1.11 g, for a combined yield The acetylated product was isolated as a yellow colored viscous oil.

The acetylated product from Step A was analyzed, yielding the following confirmatory data: IR (KBr) 1735 'H-NMR (300 MHz) 88.59 1H, H-4, J 2.2 Hz), 8.12 (dd, 1H, H-6, J 9.1, 2.2 Hz), 7.35 1H, H-7, J 9.1 Hz), 7.26 IH, H-2, J 3.2 Hz), 6.68 1H, H-3, J 3.2 Hz); 4.17 2H, N-C.H2, J 7.1 Hz), 4.04 (t, 2H, O-CH2, J 6.6 Hz), 2.03 3H, acetate), 1.90 (quintet, 2H, N-CH 2 -CH, 2H, J 7.2, Hz), 1.61 (quintet, 2H, J 6.8, 7.1 Hz), 1.37 4H, N-CH 2

-CH-CH);

"C-NMR (75 MHz) 8170.8 (acetate), 141.0 138.5, 130.7, 127.4, 117.8, 116.7, 108.9, 103.6, 63.9 (O-IH2), 46.4 29.8, 28.1, 26.2 (CH 3 acetate), 25.3, 20.7; MS (ES, m/z) 327 amu (M Na') (100), 305 (M Anal. Calcd. for CH 20 N,0 4 C,63.14; H, 6.65; N, 9.20. Found: C, 63.09; H, 6.61; N, 9.14.

B. Transesterification of 6-[N-(5-nitroindolyl)]hexyl acetate. The indole acetate from Step A (1.07 g, 3.52 mmol) was dissolved in methanol (25 mL) and anhydrous KCO, (1.46 g, 10.57 mmol) was added. Water (8.0 mL) was then added to this suspension. The contents in the reaction flask were stirred for 20 hours at ambient temperature. The reaction was worked up by evaporation of the solvent under reduced pressure. The residue was then taken up in water (30 mL) and extracted successively with ethyl acetate (2 X 30 mL) and ether (3 X 30 mL). The combined extracts were dried over anhydrous Na2SO 4 filtered, and evaporated under reduced pressure. The crude product was purified using flash chromatography on silica gel using ethyl acetate: hexane to give Compound 862 as a pale yellow solid (0.85 g, 91.6%).

The material from Step B was analyzed yielding the following confirmatory data: m.p. 78.3-78.7 IR (KBr) 3733 (OH) 'H-NMR (300 MHz) 8.60 1H, H-4, J 2.2 Hz), 8.12 (dd, 1H, H-6, J 9.1, 2.2 Hz), 7.36 1H, H-7, J 9,1-Hz), 7.25 1H, H- 2, J 3.3 Hz), 6.68 1H, H-3, J 3.3 Hz), 4.18 2H, N-CH, J 7.1 Hz), 3.63 2H,

O-CH

2 J 6.1, 11.6 Hz), 1.88 (quintet, 2H, N-CH,-CH 2 2H, J 7.2, 7.5 Hz), 1.56 (quintet, 2H, O-CH 2 -CH2, J 6.8, 7.1 Hz), 1.40 2H, N-CH 2 1.25 (t 1H, OH, J 5.4 Hz); "C-NMR (75 MHz) 141.0 138.5, 130.9, 127.4, 118.0, 116.8, 109.0, 103.7, 62.4 (O-CH2), 46.6 32.3, 29.9, 26.5, 25.2,; MS (ES, m/z) 263 amu (M (100), 280 (M 285 (M Anal. Calcd. for C, 4 H,,N,03: C,64.10; H, 6.91; N, 10.68. Found: C, 64.21; H, 6.91; N, 10.69.

C. Esterification of 6-[N-(5-nitroindolyl)]hexan-l-oI using nicotinic acid.

The alcohol from Step B (0.350 g, 1.37 mmol), nicotinic acid (0.210 g, 1.69 mmol), DCC (0.310 g, 1.51 mmol) and DMAP (17.0 mg, 0.140 mmol) were dissolved in dichloromethane (12.0 mL). The suspension was stirred at ambient temperature and monitored by TLC. After 20 hours the reaction was worked up by filtering off the white solid, washing the filter with dichloromethane (15.0 mL), and washing the organic filtrate with brine (3 X 25 mL). The filtrate was then dried over anhydrous Na 2 SO, and evaporated to dryness. Purification of the product was done by flash chromatography on silica gel using ethyl acetate-hexane to give the product as a yellow colored solid (0.45 g, The material from Step C was analyzed yielding the following confirmatory data: m.p. OC; IR (KBr) 1717 'H-NMR (300 MHz) 59.13 1H, J 1.9 Hz), 8.71 (dd, 1H, J 4.8, 1.5, Hz), 8.51 1H, H-4, J 2.2 Hz), 8.20 (dt, 1H, J 6.0 Hz), 8.03 (dd, 1H, H-6, J 9.1, 2.2 Hz), 7.32 (dd, 1H, J 4.8, 1.1 Hz), 7.29 1H, H-7, J 9.1 Hz), 7.17 1H, H-2, J 3.2 Hz), 6.60 1H, H-3, J 3.2 Hz); 4.25 2H, N-CH,, J 6.5 Hz), 4.11 2H, O-CH 2 J 7.0 Hz), 1.83 (quintet, 2H, N-CHI-CH 2H, J 7.2, 7.5 Hz), 1.70 (quintet, 2H, O-CH 2 J 6.8, 7.1 Hz), 1.36 2H, N-CH 2

CH

2 "C-NMR (75 MHz) 8164.9 (nicotinate 153.1 150.5 (C-6'),141.0 138.4(C-7), 136.7 130.8 127.3 125.8 123.1 117.8 and 116.8 108.9 103.6 64.9 (O-CH 2 46.5 (N-CH 2 29.7 (O-CH,

CH

2 28.2 (N-CH 2 CH2), 26.3 (O-CHCH 2 -CH2), 25.4; MS (ES, m/z) 368 amu (M H') (100).

D. N-Methylation of 6-[N-(5-nitroindolyl)]hexyl nicotinate. The ester from Step C (0.104 g, 0.294 mmol) was mixed with iodomethane (0.036 mL, 0.589 mmol). The reaction was heated in an oil bath to 60°C overnight (18 hours). The work-up consisted of evaporating the solvent under reduced pressure then recrystallization of the residue using 2-propanol to give a yellow colofed solid (0.120 g, 82.3%).

The material from Step D was analyzed yielding the following confirmatory data: m.p. 109.1-109.9°C; IR (KBr) 1717 'H-NMR (300 MHz) 89.42 (sm 1H,H- 9.11 1H, J 6.1 Hz), 8.63 1H, J 8 Hz), 8.50 1H, H-4, J Hz), 8.19 (dt, IH, H-6, J 6.3, 7.9 Hz), 8.01 (dd, 1H, J 2.3, 9.1 Hz), 7.56 1H, H-7, J 9.1 Hz), 7.50 1H, H-2, J 3.1 Hz); 6.69 1H, H-3, J 330 Hz); 4.50 3H, 4.42 2H, N-CHI, J 6.4 Hz), 4.31 2H, O-CH 2 J 6.9 Hz), 1.95 (quintet, 2H, N-CH 2

-CH

2 2H, J 7.2, 7.5 Hz), 1.84 (quintet, 2H, O-CH 2 J 6.8, 7.1 Hz), 1.46 2H, N-CH 2

-CH

2 "C-NMR (75 MHz) 8162.7 (nicotinate 149.8 148.2 (C-),142.7 140.5 133.2 132.2 129.4 129.3 118.8 and 117.8 111.1 104.8 67.7 49.6 (N t 47.5 (N- 30.9 (O-CH, CH,, 29.2 (N-CH, CH2), 24.2 (O-CH 2

CH

2 MS (ES, m/z) 368 amu (100), 127 (100); Anal. Calcd. for C 20 H,,NO,I: C, 48.50; H, 4.48; N, 8.48.

Found: C, 48.36; H, 4.46; N, 8.34.

The synthetic procedures described below with respect to Schemes 4-6 were developed for use as combinatorial chemical methods using, for example, parallel solution phase synthesis techniques. One of skill in the art would recognize the meaning of these terms.

EXAMPLE 2: GENERAL EXPERIMENTAL PROCEDURES USED FOR PREPARING SOLUTION PHASE COMBINATORIAL LIBRARIES DESCRIBED IN SCHEME 4.

The following Example 2 describes a preferred embodiment of the invention herein for compounds prepared according to the synthetic pathway set out in Scheme 4, described previously. One of skill in the art will recognize that many possible variations of this embodiment exist that will not result in deviation from the novel and unobvious aspects of the invention.

A. Alkylation of 5-nitroindole with the bromoalkyl acetate and conversion of the indole alkyl acetate to the alcohol. A solution of 5-nitroindole (Ig, 6.17mmol) in DMF (10.0mL) was prepared in 4 dram vials (size 28 X 57mm) This solution was then transferred to a second 4 dram vial containing a suspension of NaH (0.22g, 9.25mmol) in DMF (8.OmL). During the addition, an instantaneous gas evolution was observed and a nitrogen inlet was used to prevent gas pressure build up. The robotic synthesizer was then used to dispense 3.0mL of the indole sodium salt solution into 5 culture tubes (16 X 125mm) in a synthesizer block. The bromoalkyl acetate (1.36mmol) was dissolved in DMF (8mL total volume, 1.36M solution) in a 4 dram vial, ImL of this solution was transferred into designated test tubes using the robotic synthesizer. After allowing the reaction to block shake for 15 hours at ambient temperature, tris(2-aminoethyl)amine resin (0.15g, 0.329mmol) was added and the reaction was shaken for 12 hours with heating at OC. The resin was filtered using 3cc syringes each with a cotton plug and connected to a 24-port manifold and a water aspirator provided vacuum suction. The filtrate was collected in culture tubes (16 X 125mm) and the resin was washed using MeOH Prior to placing the tubes in the reaction block a catalytic amount of NaH (10-12mg) was added to each tube and allowed to shake at ambient temperature for 12 hours. Work-up consisted of adding ethyl acetate (4.0mL) and water (3.0mL) to each sample, shaking and removing the organic layer then subsequently washing the organic layer using brine (2 X The organic layer was dried over anhydrous Na 2

SO

4 filtered vide supra, and the solvent was transferred to 4 dram vials evaporated using a speed vac to give the alcohol as a solid residue whose weight range was from 100mg to 226mg.

B. Formation of the indole alkyl ester. The alcohol (0.100g, 0.38 mmol) was purged with argon and dissolved in dichloromethane (5.0mL), ImL aliquots were transferred to 5 culture tubes (13 X 100mm), triethylamine (1061L) was added to each tube, and the tubes were then capped and placed in an ice bath for 15 minutes.

Methanesulfonyl chloride (38 1 L) was then added to each vial, then shaken by hand for seconds and placed in the refrigerator at 1.8 0 C for 12 hours. Each sample was worked up by the addition of ethyl acetate (5mL), and washed with water (2 X 3mL) and brine (3 mL). The organic layer was dried by passing it through a B-D 3cc syringe containing anhydrous NaSO using the manifold described above and collecting the filtrate in culture test tubes (16 X 125mm). The solvent was then transferred to 4 dram vials and evaporated vide supra to give residue weights of 0.128g to 0.159g. The residue in the vials (0.128g, 0.498mmol) was then purged with argon and dissolved in anhydrous DMF (3mL). This solution was then transferred to culture tubes (13 X 100mm) containing 2 equiv. of nicotinic acid (457mg, 1.72mmol) and 1 equiv. KCO, (120mg, 0.858mmol) in DMF The tubes were shaken and heated in a digitally controlled heating block at for 15 hours. The reactions were worked up by pouring the contents of the tubes into 4 dram vials containing ethyl acetate (5mL), and this was washed with water (2 X 5mL) and brine (2 X 5mL). The organic layer was dried by passing it through a 3cc syringe containing anhydrous Na2SO 4 vide supra. The filtrate was collected into culture tubes (16 X 125mm) and transferred to 4 dram vials and evaporated under reduced pressure to give the ester as a residue whose weight range was 27mg to 59mg.

C. N-Methylation. The ester from Step B above, (32mg, 0.108mmol) was transferred into culture tubes (13 X 100mm) and dissolved in DME (1.5mL) then followed by the addition of 5 equiv. ofiodomethane (36iL, 0.077mmol). The tubes were shaken and heated in a digital heating block at 50 0 C for 12 hours. Work-up consisted of transferring the contents of the tubes into 1 dram vials and evaporating the solvent under reduced pressure to give the N-methyl derivative as a solid product (weight range 17mg to 39mg) which was isolated by filtration.

EXAMPLE 3: GENERAL EXPERIMENTAL PROCEDURES USED FOR PREPARING SOLUTION PHASE COMBINATORIAL LIBRARIES DESCRIBED IN SCHEME The following Example 3 describes a preferred embodiment of the invention herein for compounds prepared according to the synthetic pathway set out in Scheme 5, described previously. One of skill in the art will recognize that many possible variations on this embodiment exist that will not result in deviation from the novel and unobvious aspects of the invention A. Methyl and benzyl esters of indole carboxylates. Potassium carbonate (0.55 eq) was added to indole carboxylic acid (6.1 nmmol) stirred in dry DMF (10 mL) at r.t. After 10 min., the alkyl iodide (benzyl or methyl) (1.1 eq) was added. This was worked up after 24 hours in 30 mL centrifuge tubes by taking the RM, diluting in EtOAc mL), and washing with NaHCO 3 (2x10 mL), HO0 (2x10 mL), and brine (10 mL). The resulting solution was dried (Na 2

SO

4 evaporated to dryness, and recrystallized from EtOAc-hexanes.

B. N-Alkylation of indole esters with bromoalkyl acetates. NaH (2.93 nmmol) was washed with dry DMF(4 mL), re-suspended in dry DMF (7 mL) and cooled at 0 °C under a nitrogen atmosphere. A solution of the dry indolecarboxylate ester (1.95 mmol) in dry DMF (7 mL) was slowly added, dropwise, to the NaH suspensions contained in 20 mL vials. This was under mixed on an orbital shaker and warmed to r.t. After 1 hour, 2 mL of each 14 mL solution was dispensed into 7 100x13 cultures tubes (7x7=49 tubes containing 0.285 nmmol each).

For each linker size n=5 to the bromoalcohol acetate (7.7 eq) was diluted to to 3.5 mL with dry DMF. A portion of this solution (0.5 mL, 1.1 eq., 0.313 mmol) was slowly added to the reaction mixture containing the indole anions. The mixtures were shaken at r.t. for 15 hours. TLC for product revealed Rf=-0.3 to 0.7 (3:7 EtOAc-hexanes).

Each culture tube was treated with a polymer supported trapping resin, tris(2aminoethyl)amine (0.16 eq, 0.046 mmol), and the tubes were shaken at 50°C for 6.5 hours.

The mixture was filtered through cotton in 1 mL syringes using a 24 port manifold, the filter was washed with dry MeOH (2 mL), and the filtrate was collected and concentrated in 100x13 mm culture tubes to provide the product.

C. Formation of the alcohol from the indolealkyl acetate. For the methyl esters, a MeOH-MeONa solution was prepared as follows: NaH (2.85 mmol) was washed with dry DMF (2x2mL), suspended in dry DMF (2mL), cooled at 0°C, and dry MeOH (8 mL) was slowly added. The resulting mixture was then shaken for 30 min at r.t. A portion (0.2 mL, 0.2 eq) of this solution was dispensed to each tube containing the indolealkyl acetate, and the resulting mixture was shaken at r.t. for 16 hours. The mixtures were diluted with EtOAC (6 mL) and extracted with H20 (5 mL) in 30 mL centrifuge tubes. The aqueous washes were re-extracted with EtOAc (3x2 mL). The combined EtOAc layers were washed with H0O (2x4 mL) and brine (2 mL), dried (Na 2 and filtered into 20 mL vials. The solvent was removed in a speed-vac under reduced pressure to provide the product: Rf=0.05 to 0.35 (3:7 EtOAc-hexanes).

For the benzyl esters, a 1 N NaOH solution (5 eq) was added to the indolealkyl acetate and the mixture was shaken for 2 days at r.t. The mixtures were diluted with EtOAC (6 mL) and extracted with H20 (5 mL) in 30 mL centrifuge tubes. The aqueous I washes were re-extracted with EtOAc (3x2 mL). The combined EtOAc layers were washed with HIO (2x4 mL) and brine (2 mL), dried (Na 2 and filtered into 20 mL vials. The solvent was removed in a speed-vac under reduced pressure to provide the product: Rf=0.05 to 0.35 (3:7 EtOAc-hexanes).

D. Coupling of the indole alcohol with aromatic amines. To the alcohol (0.1 mmol) in dry CHCl, (1 mL) was added the aromatic amine (10 eq. pyridine, quinoline, isoquinoline, or methyl nicotinate; 4 eq. benzyl 3-quinolinecarboxylate). The resulting mixture was cooled at 0 °C and trifluoromethanesulfonic anhydride (1.3 eq.) was slowly added. The mixture was shaken for 2 hours at 0 OC, and then at r.t. for 14 hours.

The reaction mixture was diluted with EtOAc (3mL) and washed with 1 N HCI (3x1 mL), 153 water (2x1 mL) and brine (1 mL). The solution was dried (Na 2

SO

4 and concentrated on the speed-vac under reduced pressure to provide the product.

E. Conversion of the methyl and benzyl indolecarboxylates to the carboxylic acids. For methyl esters, the methyl indolecarboxylate (0.1 mmol) was solubilized in MeOH-H 2 O 0.8 mL) and 1 NNaOH (7 eq for diesters, 5 eq for monoesters) was added. The reaction mixture was then heated at 45 0 C on an orbital platform shaker for 14 hours. The solution was evaporated to dryness on a speed-vac and the residue dissolved in DMSO for biological evaluation.

Benzyl esters (0.04-0.09 nmmol) were solubilized in a mixture of MeOH-CHCl,-

HO

2 0 (1.5 mL) were hydrogenated using Pd/C (50 mg) in 100x13 mm culture tubes containing 10 glass beads (diameter=3 mm) under 40 psi H 2 at r.t. for 8 hours.

Under these conditons, 14 tubes could be placed in a 500 mL PAR apparatus bottle.

Filtration through a celite pad and concentration on a speed-vac under reduced pressure afforded the carboxylic acids. Products containing the reduced pyridinium ring were also produced.

EXAMPLE 4: GENERAL EXPERIMENTAL PROCEDURES USED FOR PREPARING SOLUTION PHASE COMBINATORIAL LIBRARIES DESCRIBED IN SCHEME 6 The following Example 4 describes a preferred embodiment of the invention herein for compounds prepared according to the synthetic pathway set out in Scheme 6, described previously. One of skill in the art will recognize that many possible variations on this embodiment exist that will not result in deviation from the novel and unobvious aspects of the invention.

A. Bromination of anilines. An anhydrous dimethyl formamide (DMF) solution (40 mL) of a commercially available aniline (0.02 mol) was treated with Nbromosuccinimide (NBS, 1.1 eq.) at room temperature overnight. The resulting mixture was quenched by pouring it onto ice and extracted with ethyl acetate (EtOAc, 2 x 30 mL).

The combined organic layers were washed with water (30 mL), brine (30 mL), dried over MgSO,, filtered and concentrated to give the product.

B. Heck coupling. To an anhydrous triethylamine solution (TEA, 3 mL) of 2bromo-R'-substituted-aniline (0.006 mol) (1 eq), in 10 x 1.3 cm test tubes, was added bistriphenylphosphine palladium chloride (2 mol%) at room.temperature followed by the addition of copper iodide (2 mol%). To this heterogeneous mixture, the corresponding terminal alkynol (1.5 eq.) and glass beads were added. The resulting mixture was allowed to react for 6h. at 80 0 C under vigorous vortex shaking. Upon cooling, the reaction mixture was filtered through a celite bed (in 5 mL disposable syringes). Concentration under high vacuum (speed-vac) afforded the product.

C. Cyclization to form indoles. To an anhydrous acetonitrile (3 mL) solution of alkyne-substituted aniline in 10 x 1.3 cm test tubes at room temperature was added palladium chloride (2mol%) followed by the addition of glass beads. The resulting mixture was heated to 60 °C for lh under vigorous vortex shaking. Upon cooling, the reaction mixture was filtered through a bed ofcelite (in 5 mL disposable syringes). The solvent was evaporated under high vacuum (speed-vac) to afford the products.

D. Quaternization with amines. To a cooled (0 solution of the indole alcohol in aromatic amine (pyridine, quinoline, or isoquinoline) (2 mL), under a nitrogen Satmosphere in 10 x 1.3 cm test tubes, was added trifluoromethanesulfonyl anhydride (TfO) (1.3 The resulting solution was allowed to react for 6 h. The reaction mixture was quenched by the addition of an ice-cold 1.5N HCI solution (3 mL) followed by the addition of EtOAc (4 mL). The organic layer was washed with water (3 mL), brine (3 mL), dried over MgSO 4 and filtered through a silica gel column (I x 2 cm, in 5 mL syringes) in order to remove unreacted organic materials. The column was then flushed with a dichloromethane:methanol (19:1) solution (4 mL). This extract was concentrated to afford the products.

E. Formation of isolated mesylate. To an anhydrous DCM solution (2 mL) of the indole alcohol was added TEA (1.5 eq.) at room temperature in 10 x 1.3 cm test tubes. The resulting solution was cooled to 0 OC and treated with methanesulfonyl.

chloride (1.1 eq.) for I h. The reaction mixture was quenched by the addition of water (3 mL), followed by DCM (3 mL). The organic layer was washed with brine (3 mL), dried over MgSO,, filtered through a celite bed (in 5 mL disposable syringes) and concentrated under high vacuum (speed-vac) to give the indole mesylates.

F. Formation of ester. To an anhydrous DMF solution (2 mL) of the indole mesylate (1 eq.) in 10 x 1.3 cm test tubes was added the corresponding carboxylic acid (R,-COOH, 2 eq.) followed by K 2 CO (2 eq.) and glass beads at room temperature. The resulting suspension was heated to 55 *C for 16 h under vigorous vortex shaking. Upon cooling the reaction mixture was quenched by adding water (3 mL) followed by ethyl acetate (3 mL). The organic layer was washed with brine (4 mL), dried under MgSO,, filtered through a cotton bed (in 5 mL disposable syringes) and concentrated under high vacuum (speed-vac) to give the final ester.

EXAMPLE 5: GENERAL EXPERIMENTAL PROCEDURES.USED FOR PREPARING LIBRARIES DESCRIBED IN SCHEME 7 A. EXPERIMENTAL PROCEDURE FOR PREPARING COMPOUNDS SINGLY IN SCHEME 7 (n 7) A. Alkylation of Phenol with 7-bromo-l-heptanol Phenol (0.098g, 1.04mmol) and 7-bromo-1-heptanol (0.243g, 1.246mmol) were dissolved in acetone (25mL) in a 50mL round bottomed flask. Potassium carbonate (0.86g, 6.23mmol) was added to this solution and the flask was fitted with a condenser and refluxed using an oil bath at 70C for a period of 26 hours. The reaction flask was then cooled to room temperature. The contents of the flask was then filtered through a fluted filter paper and washed with acetone (10mL). The combined filtrate was then evaporated to dryness under reduced pressure. The residue obtained was dissolved in ethyl acetate The ethyl acetate solution was then washed with 1N. NaOH (3 X 5mL), water (2 X 5mL) and brine (2 X 5mL). The organic layer was dried over NaSO 4 Removal of solvent from the dried extract under reduced pressure afforded the product. The product

I

was then purified by flash chromatography (Si gel, 12 X 2.5cm) using ethyl acetate hexane to afford the pure 7-(phenyloxy)- 1-heptanol (0.22g, quantitative yield). The product alcohol from step A was analyzed yielding the following confirmatory data: 'H- NMR (CDC1I) 6 1.31-1.52 6H), 1.52-1.63 2H), 1.73-1.89 2H), 1.97 (bs, 1H), 3.61 2H, J=6.57Hz), 3.93 2H, J=6.50Hz), 6.80-6.97 3H) and 7.20-7.28 2H); "C-NMR (CDCI 3 6 25.5, 25.8, 29.0, 29.1, 32.4, 62.5, 67.7, 114.3, 120.3, 129.2 and 158.8; IR (neat): 3348 cm'; MS 209 B. Mesylation of 7-(phenyloxy)-l-heptanol A solution of 7-(phenyloxy)-l-heptanol (0.21g, 1.Olmmol) in anhydrous methylene chloride (20mL), taken in a 50 mL round bottomed flask was cooled to 0 C using an ice bath. Methanesulfonyl chloride (0.177g, 1.55mmol) was added to this followed by a dropwise addition of triethylamine (0.28mL, 2.1 1mmol). The reaction mixture was then stirred at 0 0 C for Ihour and allowed to attain room temperature in 2hours. It was then quenched with IN. HCI (10mL) and diluted with methylene chloride The organic layer was separated and the aqueous layer was extracted with methylene chloride (2X10mL). The combined organic layer was washed with IN. HCI (3X10mL), water (2X10mL) and brine (2XmL). The organic layer was dried over Na2SO 4 Removal of solvent from the dried extract under reduced pressure afforded the product 7- (phenyloxy)-l-heptyl methanesulfonate (0.25g, The product from the step B was analyzed yielding the following confirmatory data: 'H-NMR (CDC1,) 1.31-1.52 6H), 1.71-1.84 4H), 2.98 3H), 3.94 2H, J=6.43Hz), 4.21 2H, J=6.51Hz), 6.80-6.94 3H) and 7.24-7.30 2H); "C-NMR (CDCl 3 25.2, 25.7, 28.6, 28.9, 29.0, 37.1, 67.5, 69.9, 114.3, 120.3, 129.2 and 158.9; IR (neat): 1349 MS 287 C. Esterification of 7-(phenyloxy)-l-heptyl methanesulfonate A solution of 7-(phenyloxy)-l-heptyl methanesulfonate (0.2g, 0.699mmol) and nicotinic acid (0.173g, 1.41nmuol) in anhydrous dimethyl formamide (15mL) was placed in a 25mL round bottomed flask. Potassium carbonate (0.097g, 0.702mmol) was added to this solution and the reaction mixture was heated at 50-55'C using an oil bath for a period of 16 hours. It was then allowed to attain room temperature diluted with ethyl acetate and quenched with saturated NH 4 CI containing ice (20mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3X10mL). The combined organic layer was washed with saturated NaHCO, (3X10mL), water (2X10mL) and brine (2X 10mL). The organic layer was dried over NaSO,. Removal of solvent from the dried extract under reduced pressure afforded the product 7-(phenyloxy)-l-heptyl nicotinate (0.176g, The product ester from step C was analyzed yielding the following confirmatory data: 'H-NMR (CDCI,) 5 1.38-1.62 6H), 1.76-1.92 4H), 3.95 2H, J=6.45Hz), 4.35 2H, J=6.64Hz), 6.85-6.98 3H), 7.22-7.38 2H), 7.38 (dd, 1H, J,=7.93Hz, J,=4.93Hz), 8.29 (dt, 1H, J,=7.91Hz, J2=1 .92Hz), 8.77 (dd, 1H, J,=4.83Hz, J=1 .70Hz) and 9.23 1H, J=2.00Hz), 3 C-NMR (CDCI 3 5 25.8, 25.9, 28.5, 28.9, 29.1, 65.4, 67.6, 114.3, 120.4, 123.2, 126.2, 129.3, 136.9, 150.8, 153.2, 158.9 and 165.2; JR (neat): 1712 cm'; MS 314 Anal. Calcd. for C,, 9

H

2 NO: C, 72.80; H, 7.40 and N, 4.47. Found: C, 72.94; H, 7.54 and N, 4.51.

D. N-Methylation of 7-(phenyloxy)-l-heptyl nicotinate 7-(phenyloxy)-l-heptyl nicotinate (0.05g, 0.159mmol) was dissolved in anhydrous dimethoxyethane (5mL) taken in a I OmL round bottomed flask fitted with a condenser.

Methyl iodide (0.2ml, 3.21mmol) was added to this and heated at reflux for 16 hours. It was then allowed to attain room temperature. The solid product formed was filtered, washed with hexanes and dried to obtain the product [7-(phenyloxy)-l-heptyl (N-methyl) nicotinate] iodide (.04g, The product from the step D was analyzed yielding the following confirmatory data. mp. 82C; 'H-NMR (MeOH-d,) 8 1.41-1.58 6H), 1.76- 1.88 4H), 3.96 2H, J=6.54Hz), 4.47 2H, J=6.74), 4.51 3H), 6.82-6.91(m, 3H), 7.21-7.27 2H), 8.22 (dd, 1H, J,=7.89Hz, Jz=6.31Hz), 9.03 1H, J=8.10Hz), 9.12 (d, 1H, J=6.09Hz) and 9.48 1H) "C-NMR (MeOH-d 4 6 27.1, 27.2, 29.6, 30.2, 30.4, 68.3, 68.9, 115.6, 121.6, 129.4, 130.5, 132.3, 146.4, 148.2, 149.8, 160.67 and 162.9; IR (neat): 1719cm"; MS 328 Anal. Calcd. for C 2 0

H

2 ,NO,I: C, 52.74; H, 5.76; N, 3.08. Found: C, 52.51; H, 5.77; N, 2.96.

B. GENERAL EXPERIMENTAL PROCEDURES USED FOR PREPARING SOLUTION PHASE COMBINATORIAL LIBRARIES DESCRIBED IN SCHEME 7 (n 8) A. General considerations The solvents used were puchased as anhydrous in Sure-Seal T M bottles from Aldrich chemical company. The starting phenols and reagents were purchased from Aldrich, Lancaster or Acros chemical companies and used as such. Reactions were carried out in an orbital shaker purchased from Digi-Block laboratory devices company. Evaporation of solvents were carried out in 25ml wide mouthed vials using a Savant SC210 speedvac plus instrument. Parallel filtrations were carried out using a Burdick Jackson 24-port manifold. Preparative parallel chromatography were performed on Baker flash silica gel packed in 5ml plastic disposable syringes.

B. Synthesis of alcohols (B) To the solution of commercially available phenols (1-1.5mmol) and 8-bromo- 1-octanol eq) in acetone (5ml) taken in screwcap vials (10ml capacity), KICO 3 (6eq) was added. The reaction mixtures were then capped and heated with orbital shaking (225 rpm) at 70C in a digiblock orbital shaker for 36h. They were allowed to attain room temperature and filtered in parallel through syringe tubes fitted with cotton using the filtration manifold and washed with 5ml of acetone each. The filtrates were concentrated using the speedvac. The residues obtained were dissolved in EtOAc (8ml) each, washed K with IN. NaOH (2 x 2ml), water (2 x 2ml) and dried (NaSO). These were filtered in parallel through syringe tubes fitted with cotton using the filtration manifold. The filtrates were then evaporated using the speedvac to obtain the product alcohols (70-97%yield).

C. Synthesis of mesylates (C) To the solutions of the alcohols and Et,N (2eq) in CH 2 Cl 2 (6ml) at 0 C, MsCI was added and kept at 0°C using an ice bath for 3h with occasional stirring. They were diluted with CH 2

CI

2 (5ml) and washed with IN. HCI (3 x 2ml), water (1 x Iml) and brine (I x Iml) and dried (NaSO4). These were filtered in parallel through syringe tubes fitted with cotton using the filtration manifold. Evaporation of solvent from the filtrates using the speedvac afforded the mesylates (90-100% yield).

D. Synthesis of esters D-II) To the solutions of the mesylates and the acid (2eq) in DMF (6ml) taken in screwcap vials (10ml capacity), KCO, (leq) was added. The reaction mixtures were then capped and heated with orbital shaking (225 rpm) at 55 0 C in a digiblock shaker for 24h.

They were diluted with EtOAc (20ml) and quenched with sat. NH 4 Cl (5ml). The organic layers were separated and the aqueous layers were extracted with 10 ml more of EtOAc.

The combined organic extracts for each reaction were then washed with sat. NaHCO, (2 x and brine (2 x 5ml) and dried (Na 2 SO). These were filtered in parallel through syringe tubes fitted with cotton using the filtration manifold. Removal of solvent from the filtrates using a speedvac furnished the esters D-II) (59-91%yield).

E. Synthesis of the quarternary salts E-II) To the solution of the esters D-II) in DME (6ml) in screwcap vials capacity), Mel (30 eq) was added. The reaction mixtures were capped and heated with orbital shaking (225 rpm) at 85*C in a digiblock shaker for 36h. Then they were allowed to attain room temperature and solvent was completely evaporated using a speedvac. The crude products obtained were purified in parallel chromatography over Si gel columns (5 x 1cm). The columns were first eluted with CHCl, (20ml), EtOAc (20ml) and then with in CH 2

CI

2 (30ml) to obtain the quarternary salts E-II) (40-92% yield).

K EXAMPLE 6: GENERAL PROCEDURE FOR CRYSTALLIZATION, DATA COLLECTION AND DETERMINAITON OF STRUCTURAL RELATIONSHIP BETWEEN NAD SYNTHETASE INHIBITOR COMPOUNDS AND THE NAD SYNTHETASE ENZYME.

A. Crystallization. Protein was expressed and purified as described in the literature. (Nessi, Albertini, Speranza, Galizzi, A. The out B gene of Bacillus subtilis codesfor NAD' synthetase. J. Biological Chemistry 270, 6181-6185).

Crystals were grown by vapor diffusion at 280 C from 21 23% polyethylene glycol (PEG) 400, 100 mM acetate buffer, pH 5.2, 50 mM MgCl,, 2.5 mM P -mercapto ethanol.

Inhibitors were dissolved in minimal volume of PEG 400 and then mixed with crystallization medium to final concentration of 5 10 mM in 23% v/v PEG 400. 10 pl of protein solution (16 mg/mi in crystallization buffer) were mixed with 10 pL of inhibitor in crystallization medium incubated at 28 C. The crystals of NAD synthetase complexed with inhibitors obtained belonged to space group P21 as described previously in the literature. (Rizzi, Nessi, Matteve, Coda, A. Galizzi, A. Crystal structure of NH,-dependent NAD* synthetase from Bacillus subtilis. EMBO Journal 15, 5125-5134 (1996)).

B. Data collection. Diffraction data for the different complexes of NAD synthetase with inhibitors were collected at ambient temperature or at 120" K with use of R-axisII and R-axisIV image plates and a rotating anode X-ray source, using Xstream Cryosystem device. Data were processed with DENZO and SCALEPACK as described.

(Otwinowski, Minor, W. Processing of X-ray data collected in oscillation mode. in Carter C.W Jr. and Sweet M.M eds.), Methods of Enzymology, v. 76, 307-326, Academic Press, New York (1996)). All subsequent calculations were performed with CCP4 program suite. (CCP4. The SERC (UK) Collaborative Computing Project No. 4, A suite of Programs for Protein Crystallography, SERC Daresbury Laboratory, Warrington, UK, 1979.) C. Refinement. All complexes ofNAD synthetase with inhibitors were isomorphous with the recently solved structure NAD synthetase complexed with AMP, SPPi, ATP and Mg 2 (Rizzi, Nessi, Bolognesi, Coda, A. Galizzi, A.

Crystallization ofNAD' synthetase from Bacillus subtilis. Proteins 26, 236-238 (1996).

The coordinates from this structure excluding ligands and water molecules were used as a starting model for the free enzyme at 2.0 A resolution. Rigid-body refinement followed by simulated annealing were carried out with X-PLOR (Brunger, X-PLOR Version 3.1. A system for X-ray Crystallography and NMR Yale Univ Press, New Haven, CT, 1992)) until convergence was reached using all reflections to 2.0 A resolution. The model of the free enzyme was subsequently used for phasing and refinement of the complexes of NAD synthetase with inhibitors. The procedure for refinement with X-PLOR of a particular model included first simulated annealing cycle and positional refinement of the protein. Inhibitors were manually built into difference Fourier maps using QUANTA (Molecular Simulations (Jones, Zou, Cowan, S. Kjeldgaard, M.

Improved method for building protein models in electron density maps and the location of the errors in these models. Acta Crystallogr. A 47, 110-119 (1991)) and O and refinement continued. A bulk solvent correction were then applied and ordered water molecules added following standard criteria.

EXAMPLE 7: "ONE-AT-A-TIME" IN-VITRO SCREENING METHOD The "one-at-a-time" in vitro bacterial NAD synthetase enzyme activity assay described below was used to test for relative activities of selected active molecules and synthetic dimers. The method was used to test selected NAD synthetase inhibitor compounds of the library herein, as well as commercially available compounds predicted to have bacterial NAD synthetase enzyme activity inhibitor capabilities.

A solution (1014 L) of 60 mM HEPPS pH 8.5 with 20 mM KC1 was prepared containing the following species: 0.210 mM ATP, 0.152 mM NaAD, 4 mM MgCI 2 mM NH 4 C1, 0.21 mg/mL ADH, and 1% ETOH. A stock solution of test inhibitors was then prepared by dissolving solid samples into 100% DMSO. 20 L of the test compound stock solution was then added to the mixture to give the final test compound concentrations listed. To start the enzyme assay, 16 L of a 65 g/mL NAD Synthetase solution were added, the mixture was mixed three times, and the absorbance at 340 nm was then monitored kinetically for 400 s using an Aviv 14DS UV-Vis spectrophotmeter.

The initial kinetics trace from 30 to approximately 250 seconds after enzyme addition was then fitted to a straight line using linear regression, and this rate was then compared to that of a control containing no inhibitor, using the following formula to calculate Inhibition: *100%, where Vo is the rate of the reaction with no test compound present and V is the rate of the reaction with test the test compound added. Each compound was tested in triplicate, and the resulting values for inhibition were averaged to give the listed value. ICo, values were obtained for select compounds by assaying six different concentrations of test compound, in triplicate, at concentrations between 0.0 and 2.0 mM, and plotting the resulting inhibition values against the -LOG of the test compound dose to reveal the concentration at which 50% inhibition was observed.

EXAMPLE 8: COMPARISON OF BACTERIAL NAD SYNTHETASE ACTIVITY IN DIFFERENT BACTERIA TYPES To determine initially if a compound found active in the assays, Compound 864, was also an effective inhibitor of a variety of different bacteria, a standard antibiotic assay was performed. The results are summarized in Table 306. In this assay 250 pg of Compound 864 (25 Rg/ml in DMSO) was spotted on 6 or 7 mm paper disks. Each disk was placed on separate 30 ml solid-medium plates layered with bacteria. Blood agar plates were used for Streptococcus, and minimal-glucose plates were used for the other microorganisms in Table 306. DMSO controls provided negative results.

TABLE 306: INHIBITION OF GRAM BACTERIA BY COMPOUND 864 GRAM ZONE OF BACTERIUM STRAIN OR INHIBITION (mun) Escherichia coli K-12 MG1655 (CGSC#6300) Escherichia coli K-12 W3110 (CGSC#4474) Salmonella typhimurium LT2 TT366 Streptococcus pneumonia D39 12 Streptococcuspneumoniae WU2 Bacillus subtilis A700 19.5 Compound 864 demonstrates inhibitory activity from which bacterial NAD synthetase inhibitory activity in a variety of bacteria may be extrapolated. Further, it is evident from this data that inhibition of bacterial NAD synthetase enzyme corresponds to inhibition of both gram positive and gram negative bacteria. Such data also demonstrates the effectiveness of the compounds herein as bacteriacidal agents, antimicrobial agents and disinfectants.

163 EXAMPLE 9: ADAPTATION OF ENZYME ASSAY TO HIGH THROUGH-PUT SCREENING OF INHIBITORS The enzyme kinetics assay for bacterial NAD synthetase enzyme inhibitory activity utilized as the primary biological screen, discussed previously as the "one-at-a-time" in vitro assay, was adapted to a microtiter plate format so that many compounds could be screened in a short time in a high-throughput system.

The final reaction mixture included 0.2ml of 60mM HEPPS buffer, pH 8.5, MgCl1, 19mM NH 4 CI,, 20mM KCI, 0.ImM NaAD, 0.3% n-Octyl--D-Glucopyranoside, 1% ethanol, Ig/ml NAD synthetase, 62.5g/mI yeast alcohol dehydrogenase, 0.2mM ATP and 2.5% DMSO.

The measurement of inhibitory activities of the test compounds was conducted using a high through-put screening system (HTS system). The HTS system utilizes an integrated Sagian 2M ORCA robotic system coordinating the functions of a Beckman Biomek 2000 liquid handler and a Molecular Devices SpectraMax Plus spectrophotometer. The 2M ORCA robotic station was responsible for the movement of all hardware and the integration of multiple stations on the worksurface. The Biomek 2000 is programmed to perform all phases of liquid dispensing and mixing. The SpectraMax Plus spectrophotometer was equipped to monitor absorbance in a 96- well plate format.

The present assay was designed for a 96-well plate format and begun with the dispensing of 0.170ml of reaction buffer containing 60mM HEPPS buffer, pH 8.5, MgCI, I9mM NH 4 Cl 2 20mM KCI, 0.118mM NaAD, 0.3% n-Octyl--D-Glucopyranoside, 1.18% ethanol, 1.18g/ml NAD synthetase, and 73.75g/ml yeast alcohol dehydrogenase.

Once the Biomek 2000 has completed this stage of the liquid handling, a 0.005ml volume of test compound in 100% DMSO or a 0.005ml of DMSO was dispensed in the reaction well. The Biomek 2000 mixed these components utilizing a predefined mixing program.

The reaction was initiated by the addition of 0.025ml of a solution of 1.6mM ATP dissolved in 60mM HEPPS buffer, pH 8.5, 10mM MgCl,, 19mM NH 4

CI

2 20mM KCI, DMSO, and 0.3% n-Octyl--D-Glucopyranoside. The reactions were monitored by 164 measuring the increase in absorbance at 304nm. The linear portion of the reaction was monitored for 180sec. The initial velocity was determined using Softmax Pro, the software supplied with the Molecular Devices SpectraMax Plus spectrophotometer.

The compounds were supplied as a stock with a concentration of 50mM dissolved in 100% DMSO. An initial screen was conducted on all compounds using a 2 or 3 concentration screen. The 2 panel screen used concentrations of 0.2mM and O.1mM for the compounds. The 3 panel screen used concentrations of 0.2mM, O.1mM, and 0.05mM.

From the initial screen, lead compounds which indicated the greatest inhibitory capacity were then subjected to a wider screen of concentrations (0.1mM to 0.005mM) to determine the apparent IC-50 values for each compound.

Double reciprocal plots of initial velocities have yielded the kinetic parameters given in the following table for the 2 mL cuvette assay. Also included in the table are the Km values obtained in the 0.2 mL microtiter plate assay. In this latter assay, a Beckmann/Sagian automated robotic system was applied in for high through-put screening in one preferred embodiment of the method.

TABLE 308: KINETIC DATA FOR HIGH THROUGH-PUT SCREENING METHOD 2 mL Assay 0.2 mL Assay Substrate Km (mM) Vmax (nM/sec) Km (mM) K- Mg+ 2 2.6 120 2.9 NH3 2.88 137 ATP 0.12 436 0.152 NaAD 0.075 286 0.076 With the preferred high through-put system and the adapted enzymatic screening assay for bacterial NAD synthetase inhibitory enzyme activity described previously, large numbers of compounds can be screened in a short period.

EXAMPLE 10: NAD SYNTHETASE INHIBITORY ACTIVITY OF COMPOUNDS Compounds of the libraries herein were screened using the high through-put enzyme kinetics assays described above in Example 9. Tables 310, 312A, 312B, 314 and 316 below present NAD synthetase enzyme inhibition data for a number of compounds of the libraries herein tested at 0.25 mM, 0.2 mM, 0.1 mM and 0.05 mM doses, respectively.

c

I

TABLE 310 COMPOUND ACTIVITIES AT 0.25 mM Table 310 0.25Mm COMPOUND NUMBER INHIBITION 86 13.5 63.1 871 81.9 873 98.0 874 97.0 877 98.3 880 [96.7 885 198.0 888 99.0 891 99.9 892 97.7 893 13.8 895 95.7 897 50.9 898 51.8 900 84.9 901 32.8 903 95.5 907 20.6 910 88.5 912 27.6 913 7.6 915 917 88.9 918 98.6 919 90.2 922 87.4 927 93.1 928 930 96.7 931 933 934 98.5 937 88.8 938 98.8 939 97.8 940 88.7 TABLE 312A COMPOUND ACTIVITIES AT 0.2Mm Table 31 2A 0.2 mM Compound %Compound Inhibition Number Inhibition Number 6 5.67 334 41.67 9 28.02 335 3.28 13 80.80 339 42.87 14 78.85 341 3.54 23 27.12 342 11.92 164 5.47 343 10.82 165 90.97 344 4.58 166 87.68 348 44.42 173 73.86 351 65.08 213 90.67 354 2.96 222 52.98 355 2.08 227 91.19 356 1.95 236 9.59 357 67.58 238 38.21 358 23.19 246 92.19 359 35.55 254 73.91 360 3.46 262 88.76 363 75.01 267 26.97 364 29.20 268 11.23 365 16.45 284 13.92 367 9.72 285 32.45 369 35.33 287 16.01 370 41.34 289 9.28 371 43.85 291 71.94 373 14.13 292 44.36 377 30.12 293 87.66 379 6.27 296 16.79 380 10.09 299 49.13 382 42.85 300 11.79 .383 2.76 301 6.12 384 4.10 302 21.48 385 61.62 303 50.56 386 28.75 305 54.83 388 25.86 306 33.93 389 12.44 307 4.40 392 10.89 Table 312A 0.2 mM Compound %Compound Inhibition- Number Iniiin Number 308 33.71 394 4.62 310 38.29. 399 15.22 311 29.67 401 14.26 318 14.94 403 5.07 322 14.40 405 6.07 323 28.08 406 10.96 324 34.99 407 24.14 329 30.77 408 7.04 330 23.96 409 19.02 410 8.77 474 2.45 411 8.84 476 17.49 413 4.76 477 10.15 414 6.91 478 9.76 415 7.72 482 17.07 417 14.59 483 7.31 418 5.95 484 39.95 419 24.28 486 4.97 420 9.16 488 17.65 421 1.86 489 5.87 422 16.23 .490 2.96 423 12.09 491 8.24 425. 19.12 492 2.59 428 26.53 493 9.12 429 13.01 494 17.44 430 1.20 495 6.80 431 10.77 1496 36.97 432 13.21 497 29.10 434 5.36 498 47.31 435 17.24 .499 25.59 436 11.57 501 4.98 437 .6.91 502 44.08 438 9.45 503 37.04 440 12.69 505 25.51 441 11.80 506 21.74 443 5.51 507 26.18 445 5.43 508 51.84 13.78 509 78.00 2.30 510 20.99 2.92 511 1 11.02 8.67 512 I 17.50 Table 312A 0.2 mM Compound %Compound Inhibition Number Inhibition Number 450 1 7.90 1513 23.66 452 20.04 514 22.32 454 7.95 515 30.39 455 2.69 516 29.95 457 3.31 517 34.72 458 15.72 519 16.27 460 4.17 520 55.83 461 17.92 521 29.59 462 3.84 522 35.74 464 1 13.50 523 18.12 457.92 1524 30.81 466 1 5.79 525 8.39 467 F1 15.08 1 526 42.77____ 473 j 15.06 528 I 65.81 683___ 529 15.50 58j87 530 20.5258174 531 36.55 5620 532 53.8058564 533 24.68 588 2.49_ 534 26.99 590 28-82 535 12.61 591___ 18.59__ 536 32.49 18.70____ 537 10.69 60.19___ 53840.95 539 16.80 595___ 17.94___ 540 20.20 56.49___ 542 15.89 597 19.76_ 543 28.06 598___ 544 19.66 19.31____ 545 32.18 6031 549 14.08 550 28.1860591 551 50.05 603__51.72_ 555 30.89 604 34.10 557 10.46 605 68.65 558 1.27 608 6.75 559 33.24 611 15.13 560 46.91 614 8.32 561 24.70 617 2.02 Table 312A 0.2 mM Compound %Compound Inhibition Number Inhibition Number 562 46.44 619 19.53 563 22.68 620 19.03 564 26.95 627 11.19 565 15.63 630 10.72 566 29.72 636 17.36 567 22.51 640 1.45 568 18.95 645 4.31 569 34.84 648 5.82 571 17.47 653 20.59 572 31.02 654 2.11 573 26.24 659 25.47 574 11.95 662 8.39 575 42.01 663 15.43 576 2.05 672 2.63 577 20.58 673 1.81 578 30.96 682 6.65 579 12.57 685 7.81 581 21.66 697 .4.28 582 6.13 700 2.54 712 12,59 864 19.33 740 7.23 865 46.43 741 30.47 867 70.33 742 28.20 967 19.51 744 95.85 968 88.52 745 85.38 969 83.16 760 2.28 970 96.65 761 6.88 979 38.72 762 40.05 980 74.86 763 66.50 981 95.16 764 79.25 982 93.74 862 9.05 9092.16 I- a. iAWr 1 .ji* IY"h I' 1 j *4 I TABLE 312B Activities of compounds at 0.2mM

TABLE

312B3 0.2mM Compound No %Inhibition Cornpound No %Inhibition 995 27 1064 92 1012 2 1065 81 1013 29 1014 14 1067 28 1015 15 1068 92 1016 29 1069 36 1017 .23 1070 82 1018 17 1071 1019 3 1073 78 1020 8 1074 91 1024 45 79 1025 1076 74 1026 23 1077 73 1027 2 68 1030 17 97 1031 23 1080 83 1034 30 1081 .84 1036 6 1082 81 1038 22 1084 106 1039 30 1085 87 1040 3 1086 81 1045 14 1087 61 1046 2 1088 100 1047 45' 1089 56 1053 '72 1090 96 3054 86 1091 1055 46 1092 1056 18 1093 66 1057 59 1095 161 1058 18 1096 140 1059 85 1097 98 1060 19 1098 1061 61 1099 67 1062 18 1105 167 1063 132 TABLE 314 COMPOUND ACTIVITIES AT 0.1 mM Table 314 0.1 mM Compound Inhibition Compound Inhibition Number Number 989 54.83 670 4.65 988 84.32- 638 2.31 978 80.31 637 4.42 977 87.61' 589 24.98 976 70.96 556 18.19 975 55.17 554 68.22 974 88.21 553 49.49 973 97.77 552 15.24 972 96.76 548 24.59 971 100.00 547 8.32 965 8.48 546 4.72 943 21.38 541 10.26 942 97.79 518 30.39 941 100.00 500 18.25 936 97.75 487 9.88 924 97.67 472 12.76 921 96.65 451 2.94 909 97.17 444 8.55 904 47.68 439 2.57 894 91.13 433 1.79 889 93.60 426 5.49 886 94.50 402 4.71 .882 94.50 397 4.51 881 90.89 396 3.64 879 99.58 395 20.85 878 96.43 387 29.97 876 95.41 381 25.05 875 93.56 376 37.32 872 98.31 375 60.14 853 73.46 374 31.84 850 87.46 373 7.72 849 90.92 368 21.10 848 70.02 362 8.31 832 78.64 361 16.08 831 26.21 355 3.31 769 98.31 352 32.69 768 98.64 349 86.56 767 95.96 346 42.57 766 91.22 345 37.00 765 89.99 344 54.05 749 98.19 344 10.78 748 98.38 338 27.28 747 97.81 337 35.94 746 91.27 336 18.02 743 94.10 333 26.29 715 20.73 332 12.27 676 1.46 328 47.85 327 55.31 297 50.83 326 16.11 295 25.05 325 53.22 290 12.89 321 37.25 288 42.38 320 44.72 269 51.12 319 16.99 245 7.01 317 25.04 230 93.06 316 41.58 229 99.35 315 77.23 228 95.08 314 9.19 214 82.84 313 27.37 182 95.41 312 10.25 154 9.24 309 41.47 82 9.68 304 29.48 12 62.22 TABLE 316 COMPOUND ACTIVITIES AT 0.05 mM Table 316 0.05mM Compound Number Inhibition 944 2.06 948 2.52 950 7.32 960 6.37 964 1.18 966 8.25 983 92.49 984 87.50 985 92.14 986 30.80 Tables 318A and 318B set out IC, values for compounds of the invention herein.

Table 318A sets out values for the various potent compounds ("lead compounds") of the NAD synthetase enzyme inhibitor compound libraries disclosed herein. Table 318B sets out values for a number of compounds of the invention herein, some of which are also considered to be potent compounds. The potency of the compounds is expressed according to ICso values. The IC 0 s value is that amount of NAD synthetase enzyme inhibitor compound required to inhibit the enzyme by 175 TABLE 318A IC 50 DATA LEAD COMPOUNDS Table Compound IC 5O(pMil Compound IC Number _______Number 13 50 879 174 40 882 182 60, 884 190 50 8680 213 65 887 214 30 889 228 60 891 229 25 894 s0 230 12.5 906 270 60 909 315 100 917 349 75 921 745 85 924 746 50 936 747 70 939 748 30 941 749 25 942 765 90 970 766 65 972 767 60 973 45 768 30 974 769 20 975 38 832 90 976 848 90 977 849 70 981 850 80 982 853 45 983 869 40 984 872 50 985 875 45 986 876 75 988 878 80 990 I~~&Pd(i.h 1 8 *4 1

JI

1 .I..Ib S i~I TABLE 318B ICo DATA FOR SELECTED COMPOUNDS

TABLE

318B COMPOUND ICSO of NUMBER Compound 1031 36.3 1064 37.5 1065 37.5 1068 62.5 1070 36.2 1074 1075 1076 135 1078 122.5 1079 36.2 1080 100 1081 67.5 1082 1084 12.5 1085 137.5 1086 27.5 1087 1090 21.2 1095 100 1096 68.9 1097 63.7 1099 100 1104 61 Table 320 sets out screening results for a selection of compounds from the libraries of bacterial NAD synthetase enzyme inhibitor compounds of the invention herein. As apparent from the table, all compounds tested exhibit some inhibitory activity against Staphylococcus epidennitis and, accordingly, the compounds exhibit effectiveness as antimicrobial agents, antibacterial agents and disinfecting agents.

TABLE 320: SCREENING RESULTS OF NM) SYNTHETASE INHIBITOR COMPOUNDS AGAINST S. EPIDERMITIS (Sorted by Percent Inhibition) Table 320 EPIDERMITIS Compound [Concentration Inhibition Number Screened 237 l0uM 100.00 593 1 O0uM 100.00 587 iQ0uM 100.00 518 1 0uM 100.00 375 ___IQuM 100.00 37 lO00uM 100.00 369 OuM 100.00 363 IQ0uM 100.00 362 IQ0uM 100.00 357 I0uM 100.00 339 1IQ0uM 100.00 333 IQ0uM 100.00 327 1lODUM 100.00 321 lO0uM 100.00 315 lO0uM 100.00 303 IQ0uM 100.00 297 lO0uM 100.00 291 IQ0uM 100.00 345 lO0uM 99.86 809 1buM 99.81 512 lO0uM 99.71 288 b0uM 99.65 524 1IQ0uM 99.57 351 IQ0uM 99.57 254 b OuM 99.39 238 1lOuM 99.39 839 1lOuM 99.35 12 l0uM 99.22 500 1iD0uM 99.14 309 1 00IQuM 99.07 835 1lOuM 99.03 851 98.90 841 1buM 98.90 840 98.77 749 1Ib0uM 98.73 174 1buM 98.71 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 506 1 OOuM 98.64 829 10uM 98.64 853 lOuM 98.58 852 10uM 98.58 14 10uM 98.58 285 100uM 98.57 13 O1uM 98.54 222 10uM 98.50 560 100uM 98.43 381 100UM 98.43 748 100uM 98.38 173 lOuM 98.30 566 100uM 98.21 214 100uM 98.13 834 1OuM 98.13 808 lOuM 98.06 833 10uM 98.00 229 100uM 97.95 747 lOuM 97.93 602 100uM 97.93 13 10uM 97.80 578 100uM 97.79 744 10uM 97.74 190 100uM 97.71 182 100uM 97.69 824 1OuM 97.67 743 1OuM 97.67 387 100uM 97.64 380 100uM 97.64 270 10uM 97.48 764 1OuM 97.48 554 100uM 97.43 213 lOuM 97.41 828 1OuM 97.29 804 10uM 97.29 807 10uM 97.22 823 1OuM 97.03 542 100uM 96.93 746 10uM 96.83 228 100uM 96.78 13 10uM 96.77 536 100uM 96.64 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 572 100uM 96.57 227 10uM 96.46 548 00OuM 96.43 596 100uM 96.36 386 l00uM 96.36 14 10uM 96.19 768 100uM 96.11 584 100uM 96.07 769 l00uM 95.94 827 10uM 95.86 12 10uM 95.73 262 10uM 95.50 230 100uM 95.48 745 10uM 95.09 821 O1uM 95.02 553 10uM 94.92 832 10uM 94.70 295 1OuM 94.48 590 100uM 94.43 865 100uM 94.40 826 10uM 92.57 261 10uM 92.51 767 100uM 91.76 368 100uM 91.36 766 10uM 90.50 246 10uM 90.26 296 10uM 87.47 864 100uM 87.03 831 1OuM 84.68 281 1OuM 84.23 825 1OuM 83.13 165 10uM 76.36 372 10uM 67.34 820 1OuM 65.74 556 10uM 60.18 267 10uM 56.54 850 10uM 56.50 805 10uM 50.87 865 1OuM 50.42 552 10uM 50.00 861 1OuM 49.58 855 10uM 49.58 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 865 10uM 48.55 862 10uM 48.29 822 lOuM 46.41 191 1OuM 46.32 269 10uM 46.12 605 100uM 44.29 663 1OOuM 44.28 599 100uM 44.14 405 10uM 44.01 538 10uM 43.05 830 10uM 42.99 727 10uM 42.23 180 10uM 40.33 661 10uM 39.31 657 100uM 37.12 464 10uM 35.45 623 100uM 34.17 640 10uM 33.86 610 10uM 33.51 682 10uM 32.56 9 1OuM 31.80 453 1OuM 31.13 439 10uM 30.57 589 10uM 29.35 530 100uM 27.36 654 10uM 25.20 243 1OuM 23.43 458 10uM 22.77 680 10uM 22.48 632 100uM 22.42 486 10uM 22.28 431 1OuM 22.28 686 10uM 22.14 166 1OuM 21.66 235 10uM 20.50 659 100uM 20.41 614 100uM 20.35 627 100uM 20.10 847 10uM 19.84 617 100uM 19.54 356 100uM 19.29 624 100uM 19.16 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 704 10uM 19.07 513 100uM 17.86 838 10uM 17.65 423 10uM 16.85 726 10uM 16.76 426 1OuM 16.57 573 10uM 16.09 459 10uM 16.09 215 1OuM 15.87 507 100uM 15.86 411 10uM 15.74 643 10uM 15.46 545 10uM 15.39 171 10uM 15.33 342 1OuM 14.97 648 100uM 14.57 687 1OuM 14.44 693 1OuM 14.37 626 100uM 14.26 471 10uM 14.07 630 100uM 13.69 203 10uM 13.62 651 100uM 13.51 647 100uM 13.38 728 1OuM 13.28 709 10uM 12.94 665 100uM 12.75 620 100uM 12.69 631 10uM 12.19 677 100uM 12.12 437 10uM 11.84 615 100uM 11.81 621 100uM 11.75 655 10uM 11.51 675 100uM 11.37 519 10uM 11.35 669 1OOuM 11.24 445 10uM 11.21 491 10uM 11.14 476 10uM 11.14 618 100uM 11.06 492 10uM 11.00 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 684 10uM 10.83 638 1OOuM 10.55 612 100uM 10.24 529 lOuM 10.15 634 10uM 10.08 690 10uM 10.01 608 100uM 9.86 422 10uM 9.82 611 100uM 9.67 392 10uM 9.61 562 10uM 9.44 765 lOuM 9.31 683 lOuM 9.26 199 10uM 9.26 645 100uM 9.23 200 10uM 9.20 606 100uM 9.17 432 10uM 8.91 642 l00uM 8.86 598 10uM 8.86 531 10uM 8.77 440 10uM 8.77 10uM 8.66 653 100uM 8.42 729 10uM 8.24 452 10uM 8.22 641 100uM 8.10 465 10uM 8.08 344 10uM 8.08 622 10uM 7.97 501 100uM 7.93 503 10uM 7.82 417 lOuM 7.80 625 10uM 7.77 24 10uM 7.76 449 10uM 7.73 412 10uM 7.73 650 100uM 7.73 674 10uM 7.70 443 10uM 7.66 350 10uM 7.59 635 100uM 7.47 Table 320 1 EPIDERMITIS Compound Concentration Inhibition Number Screened 450 l0uM 7.45 639 1iQ0uM 7.41 609 lO0uM 7.35 236 I0uM 7.29 394 I0uM 7.03 710 1buM 6.95 636 1IQ0uM 6.72 706 1 uM 6.68 629 lO0uM 6.66 455 l0uM 6.55 406 b0uM 6.41 225 I0uM 6.40 326 l0uM 6.27 300 6.20 188 IbuM 6.06 543 I0uM 5.99 390 10uM 5.92 444 1buM 5.85 428 1 uM 5.85 397 1lOuM 5.85 355 1 uM 5.78 671 1lO0uM 5.72 434 b0uM 5.64 367 1OuM 5.64 670 I0uM 5.59 616 1buM 5.59 579 1 uM 5.57 451 1lOuM 5.57 361 I0uM 5.57 633 1 __bOuM 5.53 676 5.52 400 5.50 537 1 uM 5.43 438 1buM 5.29 391 5.29 367 1buM 5.22 740 l0uM 5.17 403 1lOuM 4.87 780 1 OuM 4.78 863 lOuM 4.72 539 I0uM 4.67 457 1 uM 4.67 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 312 10uM 4.60 550 10uM 4.40 482 10uM 4.39 306 10uM 4.32 703 10uM 4.29 681 10uM 4.22 644 100uM 4.02 701 10uM 3.88 664 10uM 3.88 477 10uM 3.69 456 10uM 3.69 446 10uM 3.69 707 10uM 3.61 700 10uM 3.61 220 10uM 3.61 181 10uM 3.47 662 10uM 3.27 549 10uM 3.13 462 1OuM 3.13 568 10uM 3.10 668 lOuM 3.07 429 10uM 3.06 318 1OuM 2.99 488 lOuM 2.72 694 10uM 2.59 656 10uM 2.59 652 10uM 2.52 284 10uM 2.51 167 1OuM 2.45 409 10uM 2.44 208 10uM 2.32 843 10uM 2.20 364 10uM 2.16 742 lOuM 2.13 585 10uM 2.09 416 lOuM 2.09 415 10uM 1.95 223 10uM 1.91 408 10uM 1.88 338 10uM 1.67 603 10uM 1.60 540 10uM 1.60 Table 320 S. EPIDERMITIS Compound Concentration Inhibition Number Screened 672 10uM 1.57 219 10uM 1.57 396 10uM 1.53 373 10uM 1.53 673 10uM 1.50 658 10uM 1.43 613 10uM 1.36 483 10uM 1.25 424. 10uM 1.11 646 10uM 1.09 698 10uM 1.02 359 00OuM 1.01 Table 322 sets out the MIC=, (minimum inhibitory concentration to achieve inhibition) values against B. subtilis (gram positive bacteria) for a number of lead compounds within a library of bacterial NAD synthetase enzyme inhibitor compounds from Table 301 above and of the invention herein. This table demonstrates that the compounds of the invention herein are useful as antibacterial agents, antimicrobial agents and disinfecting agents.

TABLE 322: MIC85 RESULTS OF NAD SYNTHETASE ENZYME INHIBITOR LEAD COMPOUNDS AGAINST B. SUBTILLIS (Sorted by TABLE 322 B. SUB TILIS COMPOUND MIC85 (yM)

NUMBER

769 3 749 3 977 986 TABLE 322 B. SUBTILIS COMPOUND MIC85 (pM)

NUMBER

988 990 230 976 985 984 Table 324 sets out the MIC, 5 (minimum inhibitory concentration to achieve inhibition) against Staphylococcus epidermitis for a number of compounds within a library of bacterial NAD synthetase enzyme inhibitor compounds of the invention herein. This table demonstrates that the compounds of the invention herein are useful as antibacterial agents, antimicrobial agents and disinfecting agents TABLE 324: M1C 85 RESULTS OF NAD SYNTHETASE ENZYME INHIBITOR COMPOUNDS AGAINST S. EPII)ERMITIS (Sorted by MICE, Values) Table 324

S.

EPIDEBMITS

Compound Number Micas (PM) 190 3 229 3 230 3 238 3.3 824 3.7 826 3.7 827 3.7 828 3.7 834 3.7 835 3.7 14 173 174 182 213 214 228 237 254 262 270 295 553 554 743 746 747 10__ 748 767 768 Table 324

EPIDERMITS

Compound Number M1C 5 (1,M) 769 807 809 823___ 833 840 841 12 13 222 227 246 261 288 291 296 297 .315 362 363 _374 512 518 552 7 744 745 764 766 804 808 821 831 832 839 851 852 853 EXAMPLE 11: IN VITRO TOXICITY IN HUMAN CELLS OF SELECTED COMPOUNDS WITHIN THE LIBRARY OF COMPOUNDS Using the K(562 human myeloid ccli line, stock solutions of inhibitors in were adddd to the cell culture in RPMI 1640 medium which contained 10% fetal calf serum and was kept under a 10% CO 2 atmosphere. Th 'e final concentration of DM50 was less than and a DMSO control was included. The mixtures were incubated at doubling dilutions (approximately 1000 piM 10 jiM range) of inhibitor for 15 hours at 37C. At this time propidium iodide was added (I pg/mL) and the mixture incubated at min. at 4*C. The cells were washed once with medium, centrifuged, and resuspended in 2% bovine serum albuniinlphosphate-buffered saline. The cell suspension was then run through a FAC Saliber flow cytometer and approximately 5000 cells were counted. Theproportion of dead (stained) cells was determined, and the percent of live cells was expressed as controls. The minimum toxic concentration was the lowest tested concentration of inhibitor which caused a significantly lower percentage of live cells as compared to controls.

TABLE 326: HUMAN CELL TOXICITY OF SELECTED LEAD COMPOUNDS Table 326 man Cell Toxicity Compound Number Minimum Toxic

(M)

1000 200 500 409 200 948 200 270 200 939 500 947 200 953 100' 274 It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention.

Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

In the specification the term "comprising" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply theinclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term "comprising" such as "comprise" and "comprises".

Claims (2)

1. A bacterial NAD synthetase enzyme inhibitor compound of the structure: CH3, 19 o 182 7 X" 190 K? N 6q 6 4~ HH

315. 349 745 X7 746 N,- 747 7 i 748 N 8y Cit I+ 3+ N' x- A 3 Nx NO N 832 4 848 NO 849 X 6 0 850 9 O N N' 853 195 CF{- L j 7 Nl V L N Y+X H Y NX 869 872 14 N 875 Z 876 H 7 x- N 878 H 879 H 882 H N 884 886 H jl H 887 889 0 CH 3 1-0 A HH 891 894 H H 906 N H 921 H HNi 924 936 H+ 939 H 941 0A X712 'N HN 1 )8 X- X- +V I X 975 976 983 984 985 986 or 988 2. A method of treating or inhibiting a bacterial infection in a mammal comprising administering to the' mammal, a treatment effective or treatment preventative amount of a bacterial NAD synthetase enzyme inhibitor compound, having Structure 2: R\ R3 R4 /R6 Aryl (linker) Aryl I I R4 R5 R7 Structure 2 wherein the Aryl groups may be the same or different and are selected from the group consisting of phenyl, fused pyridinyl, indolyl, pyridinyl, and pyridinium, R 1 R7 each, independently, is H, aryl, hydroxy, ketone, nitro, amino, amidino, guanidine, carboxylate, amide, sulfonate, halogen, or substituted, or unsubstituted straight, branched, or cyclic alkyl, alkenyl or alkynyl, and wherein the linker is an alkyl chain which may optionally include an ether, ester or an amide group and has .a length of n non-hydrogen atoms, n being an integer from 3 to 12. 3. The method of Claim 2 wherein n is an integer of from 3 to 4. The method of Claim 2 wherein n is an integer of from 5 to 9. The method of Claim 2 wherein n is an integer of from 6 to 9. 6. The method of Claim 2 wherein R 1 -R 7 each, independently, is an H, alkyl, alkenyl, alkynyl, or an aryl group. 7. The method of Claim 2 wherein R 1 -Ry, each, independently, is a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. 8. A method of treating or inhibiting a bacterial infection in a mammal comprising administering to the mammal, a treatment effective or treatment preventative amount of a bacterial NAD synthetase enzyme inhibitor compound, having Structure 4: Linker x "x I 4 X X R3 B R7 7( x R2 X A 5 X B Structure 4 wherein X is a C or N, within a monocyclic or bicyclic moiety, A and B represent the respective sites of attachment for the linker, n is an integer of from 3 to 12, R 1 -R7 each, independently, is an H, aryl, hydroxy, ketone, nitro, amino, amidino, guanidine, carboxylate, amide, sulfonate, halogen, or substituted, or unsubstituted straight, branched, or cyclic alkyl, alkenyl or alkynyl the linker has a length of 3 12 non-hydrogen atoms and is a saturated or unsaturated cyclic or aliphatic group, branched or unbranched alkyl, alkenyl or alkynyl group that may optionally be substituted with one or more groups R 4 or R 5 as hereinbefore defined, wherein the linker may also contain heteroatoms 9. The method of Claim 8 wherein n is an integer of from 3 to The method of Claim 8 wherein n is an integer of from 5 to 9. 11. The method of Claim 8 wherein n is an integer of from 6 to 9. 12. The method of Claim 8 wherein R 1 -R 7 each, independently, is an H, alkyl, alkenyl, alkynyl, or an aryl group. 13. The method of Claim 8 wherein R 1 -R 7 each, independently, is a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. 202 14. A method of treating or inhibiting a bacterial infection in a mammal comprising administering to the mammal, a treatment effective or treatment preventative amount of a bacterial NAD synthetase enzyme inhibitor compound, having Structure 6: A I Linker R' R R6 B F? 4 A B Y N R R7 A R 7 B Structure 6 wherein X is C or N, Y is C, N, O, S, carboxy, ester, amide, or ketone, A and B represent the respective sites of attachment for a linker, n is an integer of from 3 to 12, and R, R 7 each independently, is an H, aryl, hydroxy, ketone, nitro, amino, amidino, guanidine, carboxylate, amide, sulfonate, halogen, or substituted, or unsubstituted straight, branched, or cyclic alkyl, alkenyl or alkynyl H, an unsubstituted or a substituted cyclic group, or an aliphatic group, or a branched or an unbranched group and the linker has a length of 3-12 non- hydrogen atoms and is a saturated or unsaturated cyclic group or an aliphatic branched group, branched or unbranched alkyl, alkenyl or alkynyl group that may optionally be substituted with one or more groups R 4 or R5 as hereinbefore defined, and wherein the linker may also contain heteroatoms. The method of Claim 14 wherein n is an integer of from 3 to 16. The method of Claim 14 wherein n is an integer of from 5 to 9. 17. The method of Claim 14 wherein n is an integer of from 6 to 9. 18. The method of Claim 14 wherein R, R7 each independently, is an alkyl, alkenyl, or alkynyl, or an aryl group. 203 19. The method of Claim 14 wherein R 1 R 7 each independently, is an H, hydroxyl, ketone, nitro, amino, amidno, guanidono, carboxylate, amide, sulfonate, or halogen and the common derivatives of these groups. A bacterial NAD synthetase enzyme inhibitor compound having Structure 8: 203a Structure 8 wherein n is an integer of from3 to 12, R, is an H, methoxy, benzyloxy, or nitro and R, is 3-pyridyl, N-methyl-3;pyridyl, 3-quinolinyl, N-methyl-3-quinolinyl, 3- (dimethylamino)phenyl, 3-(trimethylammonio)phenyl, 4-(dimethylamino)phenyl, 4-(trimethylammonio)phenyl, 4-(dimethylamino)phenylmethyl, or 4- (trimethylammonio)phenylmethyl. 21. The compound of Claim 20 wherein n is an integer of from 3 to 22. The compound of Claim 20 wherein n is an integer of from 5 to 9. 23. The compound of Claim 20 wherein n is an integer of from 6 to 9. 24. A bacterial NAD synthetase enzyme inhibitor compound 2, having Structure R2 RN (CH 2 Y Structure wherein n is an integer of from3 to 12, R, is an H, CO,H, -OCH 3 or -OCH 2 Ph, R 2 is H, COH, or CH=CHCOH, R 3 is H or COH, and Y is N-linked pyridine-3- carboxylic acid, N-linked pyridine, N-linked quinoline, or N-linked isoquinoline. The compound of Claim 24 wherein n is an integer of from 3 to 26. The compound of Claim 24 wherein n is an integer of from 5 to 9. 27. The compound of Claim 24 wherein n is an integer of from 6 to 9. 28. A bacterial NAD synthetase enzyme inhibitor compound having Structure 12: R2 (CH 2 )n-Y N N H Rl Structure 12 wherein n is an integer offrom3to 12, R, is H, F, or NO 2 R, is H, CH3, CF,, NO 2 phenyl, n-butyl, isopropyl, F, phenyloxy, triphenylmethyl, methoxycarbonyl, methoxy, carboxy, acetyl, or benzoyl, R 3 is H or CF, and Y is N-linked pyridine-3- carboxylic acid, N-linked pyridine, N-linked quinoline, or N-linked isoquinoline. 29. The compound of Claim 28 wherein n is an integer of from 3 to The compound of Claim 28 wherein n is an integer of from 5 to 9. 31. The compound of Claim 28 wherein n is an integer of from 6 to 9. 32. A bacterial NAD synthetase enzyme inhibitor compound having Structure 14: 0 R (CH 2 )n-OC-Y R 2 H Structure 14 wherein n is an integer of from3 to 12, R 1 is H, phenyloxy, isopropyl, acetyl, or benzoyl, R, is H or CF,, and Y is 3-(dimethylamino)phenyl, 3- (trimelthylammonio)phenyl, 4-(dimethylamino)phenyl, 4- (trimethylammonio)phenyl, 2-(phenyl)phenyl, diphenylmethyl, 3-pyridyl, 4- pyridyl, or pyridine-3-methyl. The compound of Claim 32 wherein n is an integer of from 3 to The compound of Claim 32 wherein n is an integer of from 5 to 9. The compound of Claim 32 wherein n is an integer of from 6 to 9. A bacterial NAD synthetase enzyme inhibitor compound Structure 16: having STRUCTURE 16 wherein R is H or CO 2 CH 3 and n is an integer of from I to 4. The compound of Claim 36. wherein n is an integer of from 2 to 3. The compound of Claim 36 wherein n is 3. SA bacterial NAD synthetase enzyme inhibitor compound Structure 18: >u0 8 S C H 2 0 ^s L trIn. Structure 18 having 206 wherein R is H or CO,CH, and n is an integer of from 1 to 4. The compound of Claim 39 wherein n is an integer of from 2 to 3. The compound of Claim 39 wherein n is 3. A bacterial NAD synthetase enzyme inhibitor compound' Structure 100: N +O n Structure 100 having wherein R' is: 0 IV Vill V VI H3CO Ix Ix SVII x x and n is an integer of from 1 to 12. The compound of Claim 42 wherein n is an integer of from 3 to The compound of Claim 42 wherein n is an integer of from 5 to 9. The compound of Claim 42 wherein n is an integer of from 6 to 9. bacterial NAD synthetase enzyme inhibitor compound Structure 101: R St e 11 R Structure 101 having wherein R' is: U, v vr V VI H 3 CO dA- IX wherein RI is: SVII Vill 0 4 3 4 3 4 4 R 4a 4a 0 3 6 a 6a 0 -R 6 C7a 1 7 7ai 77 A B C ,O 2 6O R R0Y 6 7 [7a -R 0 D E O SOR 0 4 4a 3 4 3 0 5 2 F G. wherein the R group in Fragments A-G is a benzyl group, a methyl group or a hydrogen and wherein n is an integer of from3 to 12. 47. The compound of Claim 46 wherein n is an integer of from 3 to 48. The compound of Claim 46 wherein n is an integer of from 5 to 9. 49. The compound of Claim 46 wherein n is an integer of from 6 to 9. bacterial NAD synthetase enzyme inhibitor compound having Structure 130: O O02N- 1" Structure 130 wherein n is an integer of from3 to 12. 51. The compound of Claim 50 wherein n is an integer of from 3 to 52. The compound of Claim 50 wherein n is an integer of from 5 to 9. 53. The compound of Claim 50 wherein n is an integer of from 6 to 9. 54. A compound of Structure 132: R NT NH T- Structure 132 wherein n is an integer of from3to 12 and R is 5-H, 6-CF,, 5-CH,, 5,7-diF, 5,7- diNO 2 5-Butyl, 5-iPropyl, 5-Phenyl, 5-NO,, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-OCH 3 5-COOCH, or The compound of Claim 54 wherein n is an integer of from 3 to 56. The compound of Claim 54 wherein n is an integer of from 5 to 9. 57. The compound of Claim 54 wherein n is an integer of from 6 to 9. 58. bacterial NAD synthetase enzyme inhibitor compound having Structure 134: Structure 134 wherein n is an integer of from3 to 12 and R is 5-H, 6-CF,, 5-CH,, 5,7-diF, 5,7- diNO,, 5-Butyl, 5-iPropyl, 5-Phenyl, 5-NO,, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF 3 5-OCH 3 5-COOCH,,or 59. The compound of Claim 58 wherein n is an integer of from 3 to The compound of Claim 58 wherein n is an integer of from 5 to 9. 61. The compound of Claim 58 wherein n is an integer of from 6 to 9, 62. #4 bacterial NAD synthetase enzyme inhibitor compound having Structure 136: N R Structure 136 wherein n is an integer of from3to 12 and R is 5-H, 6-CF,, 5-CH,,5,7-diF, 5,7- diNO,, 5-Butyl, 5-iPropyl, 5-Phenyl, 5-NO 2 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-OCH,, 5-COOCH3, or 63. The compound of Claim 62 wherein n is an integer of from 3 to 64. The compound of Claim 62 wherein n is an integer of from 5 to 9. The compound of Claim 62 wherein n is an integer of from 6 to 9. 66. 4 bacterial NAD synthetase enzyme inhibitor compound having Structure 138: 01 R CH2 >O Y Structure 138 wherein n is an integer of from3to 12 and R is 5-CF,, 5-OPh, 5-iPropyl, COCH 3 or 5-COPh and Y is 3-N,N-dimethylamino(phenyl), 4-N,N- dimethylamino(phenyl), or 2-Ph. 67. The compound of Claim 66 wherein n is an integer of from 3 to 68. The compound of Claim 66 wherein n is an integer of from 5 to 9. 69. The compound of Claim 66 wherein n is an integer of from 6 to 9. A bacterial NAD synthetase enzyme inhibitor compound having Structure 140: 0 R CH 2 ONH Z Structure 140 wherein n is an integer of from 3to 12, R is 5-CF 3 5-OPh, 5-iPropyl, 5-COCH 3 or and Z is CH(Ph), or 3-Pyridyl. 71. The compound of Claim 70 wherein n is an integer of from 3 to 72. The compound of Claim 70 wherein n is an integer of from 5 to.9. 73. The compound of Claim 70 wherein n is an integer of from 6 to 9. 74. /q bacterial NAD synthetase enzyme inhibitor compound .having Structure 142: 0 R I CH 2 >0 NH n I N Structure 142 wherein n is an integer of from3to 12 and R is 6-CF 3 5-OPh, 5-iPropyl, COCH,, or The compound of Claim 74 wherein n is an integer of from 3 to 76. The compound of Claim 74 wherein n is an integer of from 5 to 9. 77. The compound of Claim 74 wherein n is an integer of from 6 to 9. 78. q bacterial NAD synthetase enzyme inhibitor compound having Structure 144: 0 N NH n O Structure 144 wherein n is an integer of from3to 12 and R is 6-CF3, 5-OPh, 5-iPropyl, COCH, or 79. The compound of Claim 78 wherein n is an integer of from 3 to The compound of Claim 78 wherein n is an integer of from 5 to 9. 81. The compound of Claim 78 wherein n is an integer of from 6 to 9. 82. A bacterial NAD synthetase enzyme inhibitor compound having Structure 146: 0 2 N T H Structure 146 wherein n is an integer of from 3to 12. 83. The compound of Claim 82 wherein n is an integer of from 3 to 84. The compound of Claim 82 wherein n is an integer of from 5 to 9. The compound of Claim 82 wherein n is an integer of from 6 to 9. 86. f bacterial NAD synthetase enzyme inhibitor compound Structure 148: xx^ T CF 3 N Structure 148. 87. bacterial NAD synthetase enzyme inhibitor compound Structure 150: having having NHAc Structure 150 wherein R is an integer of from 1 to 12. The compound of Claim 87 wherein n is an integer of from 3 to The compound of Claim 87 wherein n is an integer of from 5 to 9. The compound of Claim 87 wherein n is an integer of from 6 to 9. A bacterial NAD synthetase enzyme inhibitor compound Structure 152: having Structure 152 wherein n is an integer of from3 to 12. 92. The compound of Claim 91 wherein n is an integer of from 3 to 93. The compound of Claim 91 wherein n is an integer of from 5 to 9. 94. The compound of Claim 91 wherein n is an integer of from 6 to 9. f bacterial NAD synthetase enzyme inhibitor compound of having Structure 154: OzN z 0 2 N H Structure 154 wherein Z is CH(diPh), 4-(N,N-dimethylamino)phenyl, CHCH,-(3-pyridyl), or (2- phenyl)-phenyl. 96. P bacterial NAD synthetase enzyme inhibitor compound having Structure 156: RO N Structure 156 wherein n is an integer offrom3to 12 and R is -OCH, or -OCHPh. 97. The compound of Claim 96 wherein n is an integer of from 3 to 98. The compound of Claim 96 wherein n is an integer of from 5 to 9. 99. The compound of Claim 96 wherein n is an integer of from 6 to 9. 100. I bacterial NAD synthetase enzyme inhibitor compound having Structure 158: I Structure 158 wherein n is an integer of from3to 12 and R is -OCH or -OCHPh. 101. The compound of Claim 100 wherein n is an integer of from 3 to 102. The compound of Claim 100 wherein n is an integer of from 5 to 9. 103. The compound of Claim 100 wherein n is an integer of from 6 to 9. 104. ,f bacterial NAD synthetase enzyme inhibitor compound Structure 160: N N R Nmo O Structure 160 wherein n is an integer of from3 to 12 and R is -OCH, or -OCHPh. 105. The compound of Claim 104 wherein n is an integer of from 3 to 106. The compound of Claim 104 wherein n is an integer of from 5 to 9. 107. The compound of Claim 104 wherein n is an integer of from 6 to 9. 108. bacterial NAD synthetase enzyme inhibitor compound Structure 162: having having Structure 162 wherein n is an integer of from3 to 12 and R is -OCH, or -OCH,Ph. The compound of Claim 108 wherein n is an integer of from 3 to The compound of Claim 108 wherein n is an integer of from 5 to 9. The compound of Claim 108 wherein n is an integer of from 6 to 9. I bacterial NAD synthetase enzyme inhibitor compound Structure 164: having Structure 164 wherein n is an integer of from3. to 12 and R is -OCH 3 or -OCH,Ph. 113. The compound of Claim 112 wherein n is an integer of from 3 to 114. The compound of Claim 112 wherein n is an integer of from 5 to 9. 115. The compound of Claim 112 wherein n is an integer of from 6 to 9. 116. P bacterial NAD synthestase inhibitor compound having Structure 166: IN R N in+ 0 Structure 166 wherein n is an integer of from I to 12 and R is -OCH, or -OCH 2 Ph. 117. The compound of Claim 114 wherein n is an integer of from 3 to 118. The compound of Claim 114 wherein n is an integer of from 5 to 9. 119. The compound of Claim 114 wherein n is an integer of from 6 to 9. 120. t bacterial NAD synthetase enzyme inhibitor compound having Structure 168: 218 Structure 168 wherein n is an integer of from 3 to 12 and R is -OCH, or -OCH 2 Ph. 121. The compound of Claim 120 wherein n is an integer of from 3 to 122. The compound of Claim 120 wherein n is an integer of from 5 to 9. 123. The compound of Claim 120 wherein n is an integer of from 6 to 9. 124. A bacterial NAD synthetase enzyme inhibitor compound having Structure 170: "N+ R N 0h O Structure 170 wherein n is an integer of from3 to 12 and R is -OCH, or -OCHzPh. 125. The compound of Claim 124 wherein n is an integer of from 3 to 126. The compound of Claim 124 wherein n is an integer of from 5 to 9. 127. The compound of Claim 124 wherein n is-an integer of from 6 to 9. 128. A bacterial NAD synthetase enzyme inhibitor compound having Structure 172: Structure 172 wherein n is an integer of from 3 to 12 and R is -OCH, or -OCHPh. 129. The compound of Claim 128 wherein n is an integer of from 3 to 130. The compound of Claim 128 wherein n is an integer of from 5 to 9. 131. The compound of Claim 128 wherein n is an integer of from 6 to 9. 132. A bacterial NAD synthetase enzyme inhibitor compound having Structure 174: I-- R N O Structure 174 wherein n is an integer of from3 to 12 and R is -OCH, or -OCHPh. 133. The compound of Claim 132 wherein n is an integer of from 3 to 134. The compound of Claim 132 wherein n is an integer of from 5 to 9. 135. The compound of Claim 132 wherein n is an integer of from 6 to 9. 136. A bacterial NAD synthetase enzyme inhibitor compound having Structure 176: 0 2 N -C NmOr Z 0 Structure 176 wherein n is an integer of from3 to 12 and Z is 3-quinoline, 3-(N,N- dimethylamino)phenyl, or 4-(N,N-dimethylamino)phenyl. 137. The compound of Claim 136 wherein n is an integer of from 3 to 138. The compound of Claim 136 wherein n is an integer of from 5 to 9. 139. The compound of Claim 136 wherein n is an integer of from 6 to 9. 140. A bacterial NAD synthetase enzyme inhibitor compound having Structure 178: 0 2 N NmO I N 0 N Structure 178 wherein n is an integer of from 3 to 12. 141. The compound of Claim 140 wherein n is an integer of from 3 to 142. The compound of Claim 140 wherein n is an integer of from 5 to 9. 143. The compound of Claim 140 wherein n is an integer of from 6 to 9. 144.. A bacterial NAD synthetase enzyme inhibitor compound having Structure 180; Structure 180 wherein n is an integer of from 3 to 12. 145. The compound of Claim 144 wherein n is an integer of from 3 to 146. The compound of Claim 144 wherein n is an integer of from 5 to 9. 147. The compound of Claim 144 wherein n is an integer of from 6 to 9. 148. A' bacterial NAD synthetase enzyme inhibitor compound having Structure 182: Structure 182 wherein n is an integer of from3 to 12. 149. The compound of Claim 148 wherein n is an integer of from 3 to 150. The compound of Claim 148 wherein n is an integer of from 5 to 9. 151. The compound of Claim 148 wherein n is an integer of from 6 to 9. 152. A bacterial NAD synthetase enzyme inhibitor compound having Structure 184: Structure 184 O CHO- 1 T R CH 2 O N n H H3C CH 3 CH 3 wherein n is an integer of from 3to 12 and R is 6-CF 3 5-OPh, 5-CH(CH,) 2 COCH, or 153. The compound of Claim 152 wherein n is an integer of from 3 to 154. The compound of Claim 152 wherein n is an integer of from 5 to 9. 155. The compound of Claim 152 wherein n is an integer of from 6 to 9. 156. bacterial NAD synthetase enzyme inhibitor compound. Structure 186: having NN CH3 H lH CH3 H 3 C Structure 186 wherein n is an integer of fron3 to 12 and R is 6-CF,, 5-OPh, 5-CH(CH 3 COCH, or The compound of Claim 156 wherein n is an integer of from 3 to The compound of Claim 156 wherein n is an integer of from 5 to 9. The compound of Claim 156 wherein n is an integer of from 6 to 9. A bacterial NAD synthetase enzyme inhibitor compound having Structure 188: Structure 188 wherein n is an integer of from3 to 12 and R.is 6-CF,, 5-OPh, 5-CH(CH 3 COCH 3 or The compound of Claim 160 wherein n is an integer of from 3 to The compound of Claim 160 wherein n is an integer of from 5 to 9. The compound of Claim 160 wherein n is an integer of from 6 to 9. A bacterial NAD synthetase enzyme inhibitor compound Structure 190: having Structure 190 165. 166. 167. 168. wherein n is an integer of from3 to 12 and R is 6-CF 3 5-OPh, 5-CH(CH 3 2 COCH, or The compound of Claim 164 wherein n is an integer of from 3 to The compound of Claim 164 wherein n is an integer of from 5 to 9. The compound of Claim 164 wherein n is an integer of from 6 to 9. A bacterial NAD synthetase enzyme inhibitor compound having Structure 192: Structure 192 wherein n is an integer of from 3 to 12 and R is 6-CF3, 5-OPh, 5-CH(CH,) 2 COCH, or 169. The compound of Claim 168 wherein n is an integer of from 3 to 170. The compound of Claim 168 wherein n is an integer of from 5 to 9. 171. The compound of Claim 168 wherein n is an integer of from 6 to 9. 172. A bacterial NAD synthetase enzyme inhibitor compound having Structure 194: R 2 R N ON Structure 194 wherein n is an integer of from3 to 12 and R' is an H or -OCH2Ph and R 2 is H or COOCH3. 173. The compound of Claim 172 wherein n is an integer of from 3 to 174. The compound of Claim 172 wherein n is an integer of from 5 to 9. 175. The compound of Claim 172 wherein n is an integer of from 6 to 9. 176. A bacterial NAD synthetase enzyme inhibitor compound having Structure 196: 2 R R O n 0 CH Structure 196 wherein n is an integer of from3 to 12 and R' is H or -OCH 2 Ph and RZ is H or COOCH,. 177. The compound of Claim 176 wherein n is an integer of from 2 to 12. 178. The compound of Claim 176 wherein n is an integer of from 5 to 9. 179. The compound of Claim 176 wherein n is an integer of from 6 to 9. 180. A bacterial NAD synthetase enzyme inhibitor compound having Structure 198: -R R 'NGo-) T- S, CH3 Structure 198 wherein n is an integer of from3 to 12, and R 1 is H, -OCH 2 Ph, or -OCPh 3 and R 2 is H or COOCH,. 181. The compound of Claim 180 wherein n is an integer of from 3 to 182. The compound of Claim 180 wherein n is an integer of from 5 to 9. 183. The compound of Claim 180 wherein n is an integer of from 6 to 9. 184. oacterial NAD synthetase enzyme inhibitor compound having Structure 200: R' S/ NOCH2 T Structure 200 wherein n is an integer of from3 to 12 and R' is'H or a -OCH 2 Ph and R 2 is H or COOCH,. 185. The compound of Claim 184 wherein n is an integer of from 3 to 186. The compound of Claim 184 wherein n is an integer of from 5 to 9. 187. The compound of Claim 184 wherein n is an integer of from 6 to 9. 188. A bacterial NAD synthetase inhibitor compound having Structure 202A: O -N R O(CHN 2 )nO Structure 202A wherein n is an integer of from3 to 12 and wherein R is H; 4-NO,; 2-CONHPh; 2-NO 2 4- [1'(4'-acetylpiperazine)]; 2-COCH,; 3-OCOCH,; 3-OCH 3 4-COCH 3 3-OCOPh; 2- CONH,; 4-CH=CHCOCH 3 4-OCOPh; 4-CH=CHCOPh; 4- {CO-3'2'- butylbenzo(b)furan] 3-NO,; 4-[5'-(5'-phenylhydantoin)]; 2-CH=CHCOPh; 2-OCH,; 4- COPh; 4-CONH,; 3-COCH 3 4-OPh; 4-(N-phthalimide); 3-(N-morpholine); 2-(N- pyrrolidine); 2-(N-morpholine); or 4-OCH,Ph. 189. The compound of Claim 188 wherein n is an integer of from 3 to 190. The compound of Claim 188 wherein n is an integer of from 5 to 9. 191. The compound of Claim 188 wherein n is an integer of from 6 to 9. 192. A bacterial NAD synthetase inhibitor compound having Structure 204A: 0 R O(CH 2 )80 CH 2 Structure 204A wherein n is an integer of from I to 12 and wherein R is H; 4-NO,; 2-CONHPh; 2-NO,; 4- [1'(4'-acetylpiperazine)]; 2-COCH,; 3-OCOCH 3 3-OCH 3 4-COCH; 3-OCOPh; 2- CONH,; 4-CH=CHCOCH,; 4-OCOPh; 4-CH=CHCOPh; 4-{CO-3'12'- butylbenzo(b)furan]}; 3-NO,; 4-[5'-(5'-phenylhydantoin)]; 2-CH=CHCOPh; 2-OCHz; 4- COPh; 4-CONH 2 3-COCH 3 4-OPh; 4-(N-phthalimide); 3-(N-morpholine); 2-(N- pyrrolidine); 2-(N-morpholine); or 4-OCH,Ph. 193. The compound of Claim 192 wherein n is an integer of from 3 to 194. The compound of Claim 192 wherein n is an integer of from 5 to 9. 195. The compound of Claim 192 wherein n is an integer of from 6 to 9. 196. A bacterial NAD synthetase inhibitor compound having Structure 206: O \r+ RA O(CH 2 )nO Structure 206 229 wherein n is an in teger of from3 to 12 and R. is H; 4-NO,; 2-CONHPh; 2-NO,22-COCH,; 3-OCR 3 4-COCH 3 3-OCOPh; 2-CONK,; 4-CH=CHCOCHt; 4-OCOPh; 4-C~HC-COPh; 4-{CO-3'[2'-butyibenzo(b)fisanh}; 3-NO 2 2-CH=CHCOPh; 2-OCH 3 4-GOPh; 3-COCH- 3 4-OPh; 4-(N-phtbalimide); or 4-OCH 2 Ph. 197. The compound of Claim 196 wherein n is an integer of from 3 to J9.The compound of Claim 196 wherein nis an integer of from.5 to 9. 199. The compound of Claim 196 wherein n is an integer of from 6 to 9. 200, compound; having Structure 208: O(CH2M 8 O CHZ T Structure 208 wherein R is 4-NO 2 2-CONB~h; 2-NO 2 2-COCH,; 3-OCH 3 4-COCH 3 3-OCOPh; 2- CON}1 2 4-CH--CHCOCE 3 4-OCOPh; 4-CH=CHCOPh; 4- f{CO-3'(2'- butylbenzo(b)ftiranj}; 3-NO 2 2-CH=CHCOPh; 2-OCR 3 4-COPh; 3-COCH,; 4-0Th; 4- (N-plithalimide); 3-(N-rnorpholine); 2-(N-morpholine); or 4-OCH 2 Ph. 201. 4, bacterial NAD synthetase inhibitor compound having Structure 210: PhCH 2 O (CH 2 h7oCoO /CHf-R Structure 210 wherein R is NH 2 NMe 2 NI'e 3 NH 2 .HCl; NI'A; or HG!. 202- A bacterial NAD synthetase inhibitor compound having Structure 212: R R N N (CH 2 8 OCO-- Structure 212 wherein R' is PhCONH or Ph 3 C and R" is H or COOCH 3 203. A bacterial NAD synthetase inhibitor compound having Structure 214: 0 2 N (CH2)80CO- Structure 214 wherein R is 4-hydroxyphenyl or 3-hydroxy-4-methylphenyl. 204. A method of treating or preventing a microbial infection in a mammal comprising 7 administering to the mammal a treatment effective or treatment preventive amount of a bacterial NAD synthetase enzyme inhibitor compound. 205. The method of Claim 204 wherein the compound comprises a compound of Claim 1. 206. The method of Claim 204 wherein the compound comprises a compound of any one of claims 20 to 203. 207. The method of any one of Claims 204 to 206 wherein the microbial infection is a bacterial infection. 208. The method of any one of Claims 204 to 206 wherein the microbe is a gram negative or gram positive bacteria. 209. The method of any one of Claims 204 to 206 wherein the microbial infection comprises an infection caused by an antibiotic resistant strain of bacteria. 210. The method of any one of Claims 204 to 210 comprising oral, rectal, intramuscularly, intravenous, intravesicular or topical administration. 211. The method of any one of Claims 204 to 211 wherein the compound is administered in a dosage of between about 0.1 to about 15g per day and wherein the dosage is administered from about 1 to about 4 times per day. 212. The method of any one of Claims 204 to 212 further comprising administering a broad spectrum antibiotic. 213. A method of killing a prokaryote with an amount of prokaryotic NAD synthetase enzyme inhibitor to reduce or eliminate the production of NAD whereby the prokaryote is killed and the inhibitor comprises at least one compound of any one of claims 1 or 20 to 203, a compound having Structure 2 as described in any one of claims 2 to 7, a compound of Structure 4 as described in any one of claims 7 to 13 or a compound of Structure 6 as described in any one of claims 14 to 19. 214. A method of decreasing prokaryotic growth, comprising contacting the prokaryote with an amount of a prokaryotic NAD synthetase enzyme inhibitor effective to reduce or eliminate the production of NAD whereby prokaryotic growth is decreased, wherein the inhibitor comprises at least one compound of any one of claims 1 or 20 to 203, a compound having Structure 2 as described in any one of claims 2 to 7, a compound of Structure 4 as described in any one of claims 7 to 13 or a compound of Structure 6 as described in any one of claims 14 to 19. 232 215. The method of Claim 213 or claim 214 wherein the inhibitor comprises a compound of Claim 1. 216. The method of Claim 213 or claim 214 wherein the prokaryote is a bacterium. 217. The method of Claim 216 wherein the bacterium is a gram negative or a gram positive bacteria. 218. The method of Claim 213 or claim 214 wherein the prokaryote is an antibiotic resistant strain of bacteria. 219. The method of Claim 213 wherein the NAD synthetase enzyme inhibitor is a compound that selectively binds with catalytic sites on a bacterial NAD synthetase enzyme to reduce or eliminate the production of NAD by the bacteria. 220. The method of Claim 214, wherein the NAD synthetase enzyme inhibitor is a compound that selectively binds with catalytic sites on a bacterial NAD synthetase enzyme to reduce or eliminate the production of NAD by the bacteria. 221. The method of any one of Claims 214 to 220, wherein inhibitor is administered to a. mammal and the administering step comprises oral, rectal, intramuscularly, intravenous, intravesicular or topical administration. 222. The method of Claim 221, wherein the compound is administered in a dosage of between about 0.1 to about 15 g per day and wherein the dosage is administered from about 1 to about 4 times per day. 223. The method of Claim 221 or Claim 222 further comprising administering a broad spectrum antibiotic. 224. A disinfectant compound comprising a bacterial NAD synthetase enzyme inhibitor wherein the inhibitor comprises at least one compound of any one of claims 1 or 20 to 203, a compound having Structure 2 as described in any one of claims 2 to 7, a compound of Structure 4 as described in any one of claims 7 to 13 or a compound of Structure 6 as described in any one of claims 14 to 19. 225. A method of disinfecting a material contaminated by a microbe, comprising contacting a contaminated material with a bacterial NAD synthetase enzyme inhibitor compound in an amount sufficient to kill or deactivate the microbe, wherein the inhibitor comprises at least one compound of any one of claims 1 or 20 to 203, a compound having Structure 2 as described in any one of claims 2 to 7, a compound of Structure 4 as described in any one of claims 7 to 13 or a compound of Structure.6 as described in any one of claims 14 to 19. 226. The method of claim 225 wherein the compound comprises a compound of claim 1. 227. The method of claim 225 or claim 226 wherein the microbe is a bacterium. 228. A method of making a bacterial NAD synthetase inhibitor compound of the formula: 0 2 N N S(CH 2 )n--O I- CH 3 where n is an integer from 3 to 12, the method comprising the steps of: a. alkylating 5-nitroindole with 6-bromohexyl acetate to form a nitroindolel)] hexyl acetate; b. hydrolyzing the 6-[N-(5-nitroindolyl)] hexyl acetate to form nitroindolyl)]hexan-1 -ol; 234 c. esterifying the 6-[N-(5-nitroindolyl)]hexan-1-ol with nicotinic acid to form 6-[N-(5-nitrindolyl)]hexyl nicotinate; and d. N-methylating the 6-[N-(5-nitrindolyl)]hexyl nicotinate. 229. A method of making a bacterial NAD synthetase inhibitor compound of the formula: R 0 (CH2)n-O N CH 3 wherein R may be one of the following groups: an H, alkyl, alkenyl, aryl, hydroxyl, ketone, nitro, amino, amidino, guanido, carboxylate, amide, sulfonate, halogen or common derivatives of these groups and n is an integer from 3 to 12, the method comprising the steps of: a. alkylating a 5-nitroindole with bromoalkyl acetate wherein the indole alkyl acetate is converted to indole alkyl alcohol; b. reacting the indole alkyl alcohol with the appropriate agent to form an indole alkyl ester; and c. N-methylating the indole alkyl ester. 230. A method of making a bacterial NAD synthetase inhibitor compound of the formula: O HOC N, R, (CHn2)n N--+R2 R3 wherein R 1 -R 3 each independently, is an H,'alkyl, alkenyl, aryl, hydroxyl, ketone, nitro, amino, amidino, guanido, carboxylate, amide, sulfonate, halogen or common derivatives of these groups and n is an integer from 3 to 12, the method comprising the steps of: a. reacting indole carboxylic acid with the appropriate reagent to provide an indole carboxylate methyl ester or an indole benzyl carboxylate ester, b. N-alkylating the indole carboxylate methyl ester or the indole carboxylate benzyl ester with bromoalkyl acetate; c. reacting the material from step b above with the appropriate reagent to form an indolealkyl alcohol; d. coupling the indolealkyl alcohol with an aromatic amine; and e. reacting the indolealkyl alcohol with the appropriate reagent to convert the methyl or benzyl indolecarboxylate to the respective indole carboxylic acids. 236 231. A method of making a bacterial NAD synthetase inhibitor compound of the formula: R1 N (CH 2 OC- R 3 R 2 H wherein R 1 -R 3 each independently, is an H, alkyl, alkenyl, aryl, hydroxyl, ketone, nitro, amino, amidino, guanido, carboxylate, amide, sulfonate, halogen or common derivatives of these groups and n is an integer from 3 to 12, the method comprising the steps of: a. brominating an analine with N-bromosuccinimide to form a 2-bromo- Ri-substituted-aniline or a 2-bromo-R 2 -substituted-aniline; b. reacting the 2-bromo-R 1 -substituted-aniline or the 2-bromo-R 2 substituted-aniline using a Heck coupling reaction to form an alkyne- substituted aniline; c. reacting the alkyne substituted aniline using a cyclization reaction to form an indole alcohol; d. quaternizing the indole alcohol with an amine; e. reacting the indole alcohol with methansulfonyl chloride to provide an indole mesylate; and f. reacting the indole mesylate with a carboxylic acid to form an indole ester. 232. A method of making a bacterial NAD synthetase inhibitor compound of the formula: R1 "N N/ ^(CH 2 R 3 R 2 H wherein Ri-R 3 each independently, is an H, alkyl, alkenyl, aryl, hydroxyl, ketone, nitro, amino, amidino, guanido, carboxylate, amide, sulfonate, halogen or 237 common derivatives of these groups and n is an integer from 1 to 12, the method comprising the steps of: a. brominating an analine with N-bromosuccinimide to form a 2-bromo- Ri-substituted-aniline or a 2-bromo-R 2 -substituted-aniline; b. reacting the 2-bromo-R 1 -substituted-aniline or the 2-bromo-R 2 substituted-aniline using a Heck coupling reaction to form an alkyne- substituted aniline; c. reacting the alkyne substituted aniline using a cyclization reaction to form an indole alcohol; d. quaternizing the indole alcohol with an amine; e. reacting the indole alcohol with triflouromethylsulfonic anhydride to provide a triflate; and f. reacting the indole triflate with an amine to form an indole alkylammonium product. 233. A method of making a bacterial NAD synthetase inhibitor compound comprising the steps of a. alkylating a phenol with 7-bromo-l-heptanol to provide 7-(phenyloxy)- 1-heptanol; b. mesylating 7-(phenyloxy)-1-heptanol to provide 7-(phenyloxy)-1-heptyl methanesulfonate; c. esterifying 7-(phenyloxy)-1-heptyl-methanesulfonate to provide 7- (phenyloxy)-l-heptyl nicotinate; and d. n-methylating 7-(phenyloxy)-1-heptyl nicotinate to provide [7- (phenyloxy)-l-heptyl-(N-methyl) nictotinate] iodide. 234. A method of generating a library comprising at least one bacterial NAD synthetase enzyme inhibitor compound comprising the steps of: a. obtaining the crystal structure of a bacterial NAD synthetase enzyme; b. identifying one or more sites of catalytic activity on the NAD synthetase enzyme; c. identifying the chemical structure of the catalytic sites on the NAD synthetase enzyme; d. selecting one or more active molecules that will demonstrate affinity for at least one of the catalytic sites on the NAD synthetase enzyme; 238 e. synthesizing one or more dimeric compounds comprised of at least one active molecule compound wherein the active molecule compound are joined by means of n linker compounds and wherein n is an integer of from 3 to 12, and f. screening the one or more compounds for bacterial NAD.synthetase inhibitor activity. 235. The method of Claim 234 wherein the library comprises one or more compounds of Claim 1. 236. The method of Claim 234 wherein the library comprises one or more compounds of structure 2 as defined in Claim 2. 237. The method of any one of Claims 234 to 236 comprising at least two active molecule compounds. 238. The method of any one of Claims 234 to 236 wherein the active molecules are the same. 239. The method of any one of Claims 234 to 236 wherein the active molecules are different. 240. The method of any one of Claims 234 to 239 wherein a software program that predicts the binding affinities of molecules to proteins is utilized in the active molecule selection step. 241. The method of any one of Claims 234 to 239 wherein a software program that evaluates the chemical and geometric complementarity between a small molecule and macromolecular binding site is utilized in the active molecule selection step. 242. The method of any one of Claims 234 to 241 wherein the compounds are synthesized utilizing a rapid, solution phase parallel synthesis and wherein the compounds are generated in a combinatorial fashion. 243. A method for the in vitro screening a compound for bacterial NAD synthetase enzyme inhibitory activity comprising the steps of: 239 a. preparing a bacterial NAD synthetase enzyme solution from pure bacterial NAD synthetase enzyme mixed with a suitable buffer; b. contacting the bacterial NAD synthetase enzyme solution with a test compound; measuring the rate of the enzyme-catalyzed reaction between the NAD synthetase enzyme and the test compound, and d. evaluating any toxicity of the test compound to a mammalian cell, wherein the rate of the enzyme catalyzed reaction comprises a measure of bacterial NAD synthetase enzyme inhibitory activity 244. The method of Claim 243 wherein the rate of the enzyme catalyzed reaction comprises a measure of the antibacterial properties of the test compound. 245. The method of Claim 243 wherein the rate of the enzyme catalyzed reaction comprises a measure of the antimicrobial properties of the test compound. 246. The method of Claim 243 wherein the bacterial NAD synthetase enzyme comprises a gram positive bacteria, a gram negative bacteria or a combination thereof. 247. The method of Claim 243 wherein the assay volume is about 2.0 mL. 248. The method of Claim 243 wherein the assay volume is about 0.2 ml. 249. The method of Claim 243 wherein the test compound is applied in an amount of greater than about 500 pL. 250. The method of Claim 243 wherein the test compound is applied in an amount of greater than or equal to about 200 pL. 251. The method of Claim 243 wherein the test compound is applied in an amount of equal to or less than about 200 puL. 252. A method of treating or inhibiting a bacterial infection in a mammal comprising administering to the mammal a treatment effective or a treatment 240 preventative amount of a bacterial NAD synthetase enzyme inhibitor compound of Structure 7 Linker R, v3 A- 0 H-7B ,Y B Structure 7 wherein X is C or N, Y is C, N, O, S, carboxy, ester, amide or ketone, A and B represent the respective sites of attachment for a linker, n is an integer of from 3 to 12, and R 1 -R 6 each, independently, is H, aryl, hydroxy, ketone, nitro, amino, amidino, guanidine, carboxylate, amide, sulfonate, halogen, or substituted, or unsubstituted straight, branched, or cyclic alkyl, alkenyl or alkynyl, the linker has a length of 3-12 non-hydrogen atoms and is a saturated or unsaturated cyclic or aliphatic group, branched or unbranched alkyl, alkenyl or alkynyl group that may optionally be substituted with one or more groups R 3 or R 4 as hereinbefore defined, and wherein the linker may also contain heteroatoms. 253. "The method of Claim 252 wherein n is an integer of from 3 to 254. The method of Claim 252 wherein n is an integer of from 5 to 9. 255. The method of Claim 252 wherein n is an integer of from 6 to 9. 256. The method of Claim 252 wherein R1.R 6 each, independently, is an H, alkyl, alkenyl, or alkynyl, or an aryl group. 257. The method of Claim 252 wherein R 1 -R 6 each, independently, is an H, hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen and the common derivatives of these groups. 258. A bacterial NAD synthetase enzyme inhibitor compound of the structure: CF tl 7 N7 H AH 869 872 H +X H 8+) 875 876 CH 3 o A CH 1 3 H N A>H N7 N H X 882AN 242 N H 7 NI H) 917 921 0H 0 CHI Na 8 N 924 936 CH3 O<CH H 939 H 941 H 942 244 /CH 3 0 97D 972 /CHI /CH 0 0- 975 976 245. 981 982 983 984 985 986 1064 Ph 0 WAS pho--O-O(CH2)mO-F 0 x N ~-O'(~1d3J 9011 9' V svaoo~..K~ICIj&. Id ate OTM) \Jo 0 I.N cIOJYN L601 0 4 otcao- 0601 0 -COO-F 04d% 0 4 a'Cn~ zo[ 0 0 0 q -0 259. A bacterial NAD synthetase enzyme inhibitor of the formula: 4- O(CHJ&O~ 0 10M6P 0 I1G69 1010 'C- 0 NO, 107S 4/ 0h I M0 A 0- 0- 0N I084 -4 1080 loss PI.U O N X-\ o 1090 Ph YNH,-. 14 o A -or Ph 0 o N- X-/ 260. The method of Claim 234 wherein the library comprises one or more compounds of Claim 258. 261. The method of Claim 234 wherein the library comprises one or more compounds of Claim 259. 262. A bacterial NAD synthetase enzyme inhibitor having Structure 216: T- (CH 2 )NOCOCH 2 N Structure 216 wherein R' is PhCONH and R" is H or COOCH 3 and n is an integer of from 7 to 8. 263. A NAD bacterial synthetase enzyme inhibitor substantially as hereinbefore described with reference to any one of the compounds listed in any one of Tables 310, 312A, 312B, 314, 316, 318A, 318B, 320, 322, 324 or 326. 264. A bacterial NAD synthetase enzyme inhibitor having the following structure: Linker A R4 B Rs R6 B R7 B wherein Y is an ester or an amide, A and B represent the respective sites of attachment for a linker, n is an integer of from 3 to 12, and Ri R7 each independently is an H, aryl, hydroxy, ketone, nitro, amino, amidino, guanidine, carboxylate, amide, sulfonate, halogen, or substituted, or unsubstituted straight, branched, or cyclic alkyl, alkenyl or alkynyl H, an unsubstituted or a substituted cyclic group, or an aliphatic group, or a branched or an unbranched group and the linker has a length of 3-12 non-hydrogen atoms and is a saturated or unsaturated cyclic group or an aliphatic branched group, branched or unbranched alkyl, alkenyl or alkynyl group that may optionally be substituted with one or more groups R 4 or R 5 as hereinbefore defined, and wherein the linker may also contain heteroatoms. 265. A method of treating or inhibiting a bacterial infection in a mammal comprising administering to the mammal, a treatment effective or treatment preventative amount of a bacterial NAD synthetase enzyme inhibitor compound, as defined in anyone of claims 258 to 264. DATED THIS SIXTH DAY OF NOVEMBER 2003 THE UAB RESEARCH FOUNDATION BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS 252