COMPOUNDS

Statement as to Federally Sponsored Research

This work was supported by NIA 07723, DE 08649, National Dairy Promotion £_ Research Board Grant administered by the National Dairy Council at Harvard and by the NCI of NIH [2ROCA16903] at Utah. The U.S. government has certain rights to this invention.

Background of the Invention Diphenyleneiodonium cation (DPI) is an organic compound comprising polycoordinated iodine. DPI exhibits a wide range of biological activities including: 1) induction of hypoglycemia and lactic acidosis (Holland et al., J. Biol . Chem . 248:6050, (1973)); 2) inhibition of respiratory burst in neutrophils (Cross et al., Biochem . J. 237:111, (1986)); inhibition of nitric oxide synthase (Stuehr et al., FASEB J. 5:98, (1991)3); and induction of yopathy in rats (Cooper et al., J. Neurol . Sci . 83, 335, (1988); Cooper et al., Biochem . Pharmacol . 37:687, (1988) ) .

PQQ (methoxatin) , is a bis-quinone tricarboxylic acid that is widely distributed in nature; being found in bacteria, animal and plant tissues (Mah et al., FASEB J. Abstract 7:A53, (1993); Paz et al. ASMBBIDBC-ACS Joint Meeting, Addendum, Abstract LB80, (1993) ; Salisbury et al., Nature 280:843, (1979); Paz et al. in Principles and Applications of Quinoproteinε, Davidson, V.L. Ed., Marcell Dekker, Inc.: New York, pp. 381-393 (1992)). PQQ is an essential nutrient in at least one mammal (Kilgore et al., Science 245:850, (1989); Smidt et al., Proc . Soc . Exp. Biol . tied . 197:19, (1991)). PQQ in its reduced form, PQQ(2H), is a nucleophile capable of making charge (i.e., electron) transfer complexes with a variety of

electrophiles, including those associated with biological redox (i.e., oxidation-reduction) cycling (Xu et al. , BBRC 193:434, (1993)).

Administration of a therapeutic composition comprising PQQ affords protection against: 1) hepatotoxicity (Watanabe et al., Curr . Ther. Res . 44:896, (1988)); 2) cataract formation (Nishigori et al., Life Sci . 45: 593, (1989)); 4) inflammation (Hamagishi et al., J. Pharmacol . Exp. Ther. 255:980, (1990)); 5) neurotoxicity (Aizenman et al., J. Neurosci . 12:2362,

(1992)); and 6) stroke-induced brain necrosis (Gardner et al., Society for Neuroscience 18:A321.6, (1992).

U.S. Patent No. 4,513,137 discloses a multi-step reflux method of making iodonium compounds comprising sulfonyloxy ligand transferring groups. Stang et al. (J. Org. Chem. 57:1861-1864, (1992)) disclose the preparation of bis-(phenyliodonium) diyne triflates. Zhadankin et al. (Tetradehedron Letters , 34:6853, (1993)) disclose using aryl(cyano)iodonium triflates. Stang and Zhdankin (J. Am . Chem . Soc . 113:4571, (1991)) disclose iodonium compounds, comprising acetylenyl groups. U.S. patents

3,622,586; 3,944,498; 3,422,152; 3,896,140; 3,734,928;

3,759,989; 3,712,920 each describe iodonium compounds.

Chlorhexidine is a positively charged bis- biguanide with two hydrophobic terminal chlorophenyl rings linked by a hydrophobic hexamethylene chain. Chlorhexidine, a dicationic detergent, is used as an oral rinse, irrigant, or periodontal pack against periodontal disease; the compound exhibits anti-microbial activity against a wide variety of bacteria (reviewed in

Greenstein et al. J. Periodontal. 57, 370-377 (1986)). Chlorhexidine is used for general skin cleaning (e.g. , surgical scrub) , preoperative showering or bathing, and wound cleaning (Physicians' Desk Reference, pp. 1867- 1868; 2374-2375 (1993)).

Summary of the Invention We have found simple methods of making iodonium triflates which do not require reflux conditions or purification (eg. crystallization and filtration) of reactants. The methods are useful for preparing iodonium triflates in high yield from an iodoarene, e.g, a bis- iodoarene. Examples of other iodonium triflates which can be made by these methods include mono-, di-, and higher iodonium triflates, e.g., iodonium compounds including 3-15 (inclusive) iodonium moieties. The claimed methods also provide a simple way of obtaining bis-iodoniu triflates comprising a heterocyclic ring, preferably a heterocyclic ring further comprising sulfur, nitrogen and/or oxygen, most preferably a thienyl, furyl or pyrrolyl ring. By using simple anion substitution steps known in the art, the claimed high yield methods can be used to obtain a variety of iodonium compounds comprising non-triflate anions capable of forming an ionic bound with an iodonium action. Novel bis-iodonium compounds made by these methods include aryl, haloaryl, halobiphenyl, alkylsilylaryl, alkylsilylalkynl, alkylsilylbiphenyl, and alkylsilylthienyl derivatives, as well as aryl and haloaryl bis-iodonium thiophene compounds. We have also found that iodonium compounds are capable of sequestering PQQ and thereby inhibiting PQQ- catalyzed redox reactions in vivo and in vitro . Iodonium compounds, as used herein, include mono-, di-, bis-, and higher iodononium compounds, e.g, iodonium compounds including 3-15 (inclusive) iodonium moieties. Mono- and bis-iodonium compounds are capable of sequestering PQQ and inhibiting critical redox cycling in cells, such as neutrophils, endothelial and muscle cells; cell organelles, such as mitochondria and chloroplasts; enzyme complexes capable of electron transfer reactions such as

nitric oxide synthase, which comprises a nicotinamide adenine dinucleotide (NAD) moiety capable of electron transfer reactions; flavoproteins such as cytosolic diaphorases (e.g., dihydrolipoamide dehydrogenases) which comprise a flavin adenine dinucleotide (FAD) moiety capable of electron transfer reactions, and other enzyme complexes comprising moieties capable of electron transfer reactions, including enzyme complexes capable of redox-cycling reactions. We have also found that certain iodonium compounds can exhibit PQQ sequestring and detergent activities which attact anti-microbial activity.

Iodonium compounds have useful applications. These applications include use as pesticides, e.g., insecticides and herbicides; for example, killing unwanted vegetation, such as the kudzu vine, or damaging insects such as the gypsy moth. Iodonium compounds are also valuable topical fungicides that can be applied to the skin and are rapidly detoxified if absorbed through the skin. Iodonium compounds can be used to kill marine pests such as mussels (i.e., Zebra mussels), starfish, or lamprey eels. Iodonium compounds act as anti- inflammatory agents that reduce the severity of various infectious diseases that trigger excess inflammatory host responses.

Iodonium compounds, e.g., mono-, di-, bis-, and higher iodonium compounds, e.g., iodonium compounds including 3-15 (inclusive) iodonium moieties, are effective anti-microbial agents that can alleviate periodontal disease (e.g., gingivitis, periodontitis, gingivitis-induced inflammation or thrush) . Iodonium compounds can be applied as an oral rinse (i.e., a mouthwash) , oral irrigant, periodontal pack, or topically applied in a suitable vehicle. Iodonium compounds have advantages over the use of oral antiseptics such as

chlorhexidine. For example, iodonium compounds can be inactivated by applying a non-toxic reducing agent (e.g., about 2-4% (w/v) sodium ascorbate or about 2-4% (w/v) sodium thiosulfate in a pharmaceutically acceptable buffer (e.g., phosphate or bicarbonate buffer, each at pH 5-9 (inclusive)). By "non-toxic" is meant a reducing agent which is acceptable for pharmaceutical use. The ability to inactivate an iodonium compound after use reduces the chance of teeth or mouth tissue damage (e.g., teeth staining or undersirable sequestration of PQQ sufficient to kill or damage mouth tissue) .

In another example of the advantages of using an iodonium compound as an anti-microbial agent inside the oral cavity, a cocktail including two or more iodonium compounds can be made where each iodonium compound exhibits a specific or very narrow anti-microbial activity against certain microbes, e.g., the cocktail can be designed to selectively inhibit the growth of specific periodontal pathogen(s) but leave host-compatible bacteria unharmed. Unlike using chlorhexidine as an anti-microbial agent, the cocktail can be tailor-made for a patient suffering from particular combinations of pathogens unique to that patient. If desired, the cocktail can be co-administered with chlorhexidine, where the chlorhexidine is in an amount of equal anti-microbial activity.

In another example of the advantages of using an iodonium compound as anti-microbial agent (either inside or outside the oral cavity) , the anti-microbial activity of an iodonium compound can be enhanced by co- administering a boric acid solution (e.g. , about 2% (w/v) boric acid in a pharmaceutically acceptable buffer (e.g., phosphate or bicarbonate buffer, each at pH 5-9 (inclusive)). Alternatively, the boric acid buffer solution can be administered prior to application of an

iodonium compound in order to enhance the anti-microbial activity of the compound. Preferably, the application of the boric acid solution is immediately prior to the application of the iodonium compound. Without wishing to be bound to any specific theory, it appears that boric acid is capable of forming a chelate complex with endogenous ascorbate (i.e., ascorbate naturally present in tissue) , thereby preventing endogenous ascorbate from inactivating an applied iodonium compound. By "enhanced" is meant that the boric acid solution increases the anti¬ microbial activity of an iodonium compound by at least 1.5 fold as determined by tests described herein, e.g., the agar dilution method described in part IX, (1) , below. Another advantage of using an iodonium compound as anti-microbial agent (either inside or outside the oral cavity) is the ability to stabilize the anti-microbial activity by complexing the iodonium compound with an anion of low nucleophilicity (e.g., triflate, perchlorate, tetrafluoroborate, hexafluorophosphate, halide, or nitrate) , rather than an anion of higher nucleophilicity (e.g. , acetate or benzoate) . The advantages of using a triflate anion to stablize the anti-microbial activity of an iodonium compound are at least two-fold, e.g., 1) an iodonium triflate has superior solubility, and 2) the triflate anion is biologically innocuous.

Iodonium compounds, e.g., mono-, di-, bis-, and higher iodonium compounds, e.g. , iodonium compounds including 3-15 (inclusive) iodonium moieties, are also useful for general skin cleaning (e.g., surgical scrub), preoperative showering or bathing, and wound cleaning. The anti-microbial activity of the iodonium compound can be enhanced or inactivated as described above.

In general, the invention features a method of making a bis-(aryl) iodonium triflate, the method including adding a diiodoarene of the following general formula:

I-Ar-I

I

R ¹

wherein: a) Ar is any one of phenyl, biphenyl, or an aryl optionally substituted by one or more R ¹ groups independently selected from any one of a halogen, loweralkyl, loweralkoxy, haloalkyl-, cycloalkyl, aryl, heteroatom substituted aryl, aryloxy, or heterocyclic group;

combining said diiodoarene with an oxidizing agent capable of forming an oxidized iodoarene, preferably peracetic acid, more preferably pertrifluoracetic acid;

reacting said oxidized diiodoarene with a loweralkylsilyl compound of the general formula:

(R ² ) ₃ Si-R ³ wherein: a) R ² is any one of methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, iso-butyl and each R ² is the same or different;

b) R ³ is any one of a CN-, aryl, preferably 4-phenyl, haloaryl-, preferably 4-C ₆ H ₄ F, 4-C ₆ H ₄ I, loweralkylphenyl-, haloloweralkylphenyl-, preferably

4-(CF ₃ )C ₆ H ₄ , 3,5-(CF ₃ ) ₂ C ₆ H ₃ , halobiphenyl-, preferably 4-C ₆ H ₄ -C ₆ H ₄ -I, alkenyl, loweralkylsilylalkenyl- , alkynyl, loweralkylsilylalkynyl-, loweralkylsilylaryl-, preferably 4-C ₆ H ₄ SiMe ₃ ,loweralkylsilylbiphenyl-, thienyl, preferably loweralkylsilylthienyl-, most preferably 2-(tri ethylsilyl)thienyl- or loweralkylalkynyl-, preferably Me ₃ -C≡C-; and a loweralkyIsilyltriflate, preferably trimethylsilyltriflate (Me ₃ SiOS0 ₂ CF ₃ ) , of the general formula

(R ⁴ ) ₃ -SiOS0 ₂ CF ₃

wherein: a) R ⁴ is any one of methyl, ethyl, propyl, iso-propyl, sec-butyl, iso-butyl, preferably butyl, and each R ⁴ is the same or different;

under conditions capable of forming a bis-(aryl) iodonium triflate of the following general formula:

R ³ -I ⁺ -Ar-I ⁺ -R ³ I R ¹

wherein: a) each R ¹ and R ³ is the same or different; and b) each R ¹ and R ³ is as defined above.

A loweralkyl group, as used herein, is 1-12 carbon atoms, preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl group. A lower

alkoxy group, as used herein, is 1-12 carbon atoms, preferably methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, or tert-butoxy group. A haloalkyl group, as used herein, contains 1-5 carbon atoms, and at least one halogen, preferably fluoromethyl, chloromethyl, bromomethyl, fluorochloromethyl, fluorobromomethyl, chlorobromomethyl, fluorochlorobromomethyl trifluoromethyl, trichloromethyl, or tribromomethyl group. A halogen is a fluoro, chloro, bromo, or iodo group. A cycloalkyl group, as used herein, can be 3-20 carbon atoms, preferably 3 to 10 carbon atoms, most preferably 3 to 5 carbon atoms, including a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or a cyclooctyl group. An aryl group, as used herein, can be 6 to 40 carbons, preferably 6 to 20 carbons, and can be any mono- or poly-cyclic (i.e., polycondensed) aromatic ring system, including phenyl, benzyl, tolyl, cumyl, alpha- and beta- naphthyl, anthracenyl, phenanthrenyl, azulenyl, pyrenyl, or napthyl substituted with one or more loweralkyl, haloalkyl or cycloalkyl groups. A haloaryl as used herein, can be any aryl group substituted with one or more halogen groups. Preferred haloaryl groups include 4-C ₆ H ₄ F and 4-C ₆ H ₄ I. A heteroatom substituted aryl group, as used herein, can be an aryl group substituted with one or more nitro, nitroso, cyano, carboxyl, aldehydo, hydoxy, or loweralkoxy groups. An aryloxy group, as used herein, can be phenoxy or benzoxy. A heterocyclic group, as used herein, includes a pyridiyl, furyl, thienyl, pyridazinyl, or pyrrolyl group. A complex heterocyclic group, as used herein, can be a mono- or polycyclic (i.e., polycondensed) hydrocarbon ring or ring system comprising N, 0, or S, for example, a pyrazinyl, acridinyl, or phenanthridinyl group. A loweralkylphenyl group, as used herein, is a phenyl group substituted with one or more

loweralkyl groups. A haloloweralkylphenyl group, as used herein, is a phenyl group substituted with one or more loweralkyl groups, the loweralkyl groups further substituted by one or more halogen groups. A preferred haloloweralkylphenyl group is a 4-(CF ₃ )C ₆ H ₄ , or 3,5-

(CF ₃ ) ₂ C ₆ H ₃ group. A halobiphenyl group, as used herein, is a biphenyl group substituted by one or more halogen groups. A preferred halobiphenyl group is 4-C ₆ H ₄ -C ₆ H ₄ -I. An alkenyl group, as used herein, is a linear or branched hydrocarbon of 1-5 carbon atoms with one or more carbon- carbon double bonds. A loweralkylsilylalkenyl group, as used herein, can be an alkenyl group subsituted with a Si atom, the Si atom further substituted with one or more loweralkyl groups. An alkynyl group, as used herein, is a linear or branched hydrocarbon of 1-5 carbon atoms with one or more carbon-carbon triple bonds, preferably an acetylenyl group. A loweralkylsilylalkynyl group, as used herein, can be any linear or branched hydrocarbon with at least one carbon-carbon triple bond substituted with a Si atom, the Si atom further substituted with one or more loweralkyl groups. A preferred loweralkylsilylalkynyl group is a -C≡CSiMe ₃ group. A loweralkylsilylaryl group, as used herein, is an aryl group substituted with a Si atom, the Si atom further substituted with one or more loweralkyl groups. A preferred loweralkylsilylaryl group is a 4-C ₆ H ₄ SiMe ₃ group. A loweralkylεilylbiphenyl, as used herein, is a biphenyl group substituted with a Si atom, the Si atom further substituted with one or more lower alkyl groups. A thieynl group, as used herein, can be a thienyl group substituted with one or more loweralkyl, aryl, alkenyl or alkynyl groups. A loweralkylsilylthienyl, as used herein, is a thienyl group substituted with a Si atom, preferably the Si atom is linked to the number two position of a thienyl ring, the Si atom further substituted with one or more

loweralkyl groups. A preferred loweralkylsilylthienyl group is a 2-(trimethylsilyl)thienyl group. A loweralkylalkynyl group, as used herein, is an alkyne substituted with one or more loweralkyl groups. A preferred loweralkylalkynyl group is Me-O≡C-.

In a related aspect, the invention features a method of making a bis-(aryl) iodonium triflate, wherein a diiodoarene is of the following general formula:

wherein: a) n=l; and R ⁴ is attached to any one of an ortho, or meta ring position; or

b) n=2, 3, or 4 and each R ⁴ is individually attached to two or more ortho or meta ring positions and each R ⁴ is the same of different; and c) R ⁴ is any one of a hydrogen, loweralkyl, loweralkoxy, haloalkyl-, cycloalkyl, aryl,

heteroatom substituted aryl, aryloxy, or heterocyclic group.

A method of making an organo-substituted iodonium triflate, the method including: obtaining an organotin compound of the general formula:

R ⁶ -Sn(R ⁵ ) ₃

wherein: a) R ⁵ is any one of methyl, ethyl, propyl, iso-propyl, or butyl, and each R ⁵ is the same or different;

b) R ⁶ is any one of an aryl, preferably phenyl, haloaryl-, preferably 4-C ₆ H ₅ F, 4-C ₆ H ₅ I loweralkylphenyl-, haloloweralkylphenyl-, preferably

4-(CF ₃ )C ₆ H ₅ , 3,5-(CF ₃ ) ₂ C ₆ H ₃ halobiphenyl-, preferably 4-C ₆ H ₄ -C ₆ H ₅ I, alkenyl, loweralkylsilylalkenyl-, alkynyl, loweralkylsilylalkynyl-, loweralkylsilylaryl-, preferably -C ₆ H ₄ SiMe ₃ loweralkylsilylbiphenyl-, thienyl, preferably loweralkylsilylthienyl-, most preferably 2-(trimethylsilyl)thienyl- or loweralkylalkynyl-, preferably Me-C≡C-;

combining the organotin compound with an iodonium trifate of the following general formula:

R ⁷ -I ⁺ -CN[OS0 ₂ CF ₃ ;T

wherein: a) R ⁷ is any one of an aryl, preferably phenyl, haloaryl-, preferably 4-C ₆ H ₅ F, 4-C ₆ H ₅ I loweralkylphenyl-, haloloweralkylphenyl-, preferably

4-(CF ₃ )C ₆ H ₅ , 3,5-(CF ₃ ) ₂ C ₆ H ₃ halobiphenyl-, preferably 4-C ₆ H ₄ -C ₆ H ₅ I alkenyl, loweralkylsilylalkenyl-, alkynyl, loweralkylsilylalkynyl-, loweralkylsilylaryl-, preferably -C ₅ H ₄ SiMe ₃ loweralkylsilylbiphenyl-, thienyl, preferably loweralkylsilylthienyl-, most preferably 2-(trimethylεilyl)thienyl- or loweralkylalkynyl-, preferably Me-C≡C-;

under conditions capable of forming an organo-substituted iodonium triflate of the following general formula:

R ⁶ -I ⁺ -R ⁷ [OS0 ₂ CF ₃ ]~

wherein: a) each R ⁶ and R ⁷ is the same or different; and

b) each R ⁶ and R ⁷ is as defined above.

In a related aspect, the invention features a method of making an organo-substituted bis-iodonium triflate, the method including: obtaining an organostannane of the general formula:

wherein: a) X is either S or O; or X is NH;

b) R ^8a is any one of methyl, ethyl, propyl, iso-propyl, or butyl, and each R ^8a is the same or different;

c) n=l; and R ^9a is attached to any one of the number 3 or 4 ring positions; or n=2 and each R ^9a is individually attached to both the number 3 and 4 ring positions and each R ^9a is the same or different;

d) R ^9a is any one of hydrogen, halogen, loweralkyl, loweralkoxy, haloalkyl-, cycloalkyl, aryl, heteroatom substituted aryl, aryloxy, or heterocyclic group; and combining said organostannane with an iodonium triflate of the general formula:

R ⁸ -I ⁺ -CN[OS0 ₂ CF ₃ ] ^'

wherein: a) R ⁸ is any one of an aryl, preferably phenyl, haloaryl-, preferably 4-C ₆ H ₅ F, 4- C ₆ H ₅ I loweralkylphenyl-, haloloweralkylphenyl-, preferably

4-C ₆ H ₅ I loweralkylphenyl-, haloloweralkylphenyl-, preferably 4-(CF ₃ )C ₆ H ₅ , 3,5-(CF ₃ ) ₂ C ₆ H ₃ halobiphenyl-, preferably 4-C ₆ H ₄ -C ₆ H ₅ I alkenyl, loweralkylsilylalkenyl-, alkynyl, loweralkylsilylalkynyl-, loweralkylsilylaryl-, preferably -C ₆ H ₄ SiMe ₃ loweralkylsilylbiphenyl-, thienyl, preferably loweralkylsilylthienyl-, most preferably 2-(trimethylsilyl)thienyl- or loweralkylalkynyl-, preferably Me-C≡C-;

under conditions capable of forming an organo- substituted bis-iodonium triflate of the following general formula:

wherein: a) X is either S or O; or X is NH;

b) each R ⁸ and R ^9a is the same or different; and

c) each R ⁸ and R ^9a i? as defined above.

In another aspect, the invention features a bis- (aryl) iodonium salt represented by the following formula:

R ¹⁰ -I ⁺ -Ar-I ⁺ -R ¹⁰ 2 [ Z ^_ ] R ⁹

wherein: a) Ar is any one of a phenyl, biphenyl, or an aryl group optionally substituted by one or more R ⁹ groups independently selected from any one of loweralkyl, loweralkoxy, haloalkyl-, cycloalkyl, aryl, heteroatom substituted aryl, aryloxy, or heterocyclic group;

b) R ⁹ is any one of a loweralkyl,loweralkoxy, haloalkyl-, cycloalkyl, aryl, heteroatom substituted aryl, aryloxy, or heterocyclic group;

c) R ¹⁰ is any one of a CN-, aryl, preferably 4-phenyl, haloaryl-, preferably 4-C ₆ H ₄ F, 4-C ₆ H ₄ I, loweralkylphenyl-, haloloweralkylphenyl-, preferably 4-(CF ₃ )C ₆ H ₄ , 3,5-(CF ₃ ) ₂ C ₆ H ₃ , halobiphenyl-, preferably 4-C ₆ H ₄ -C ₆ H ₄ -I, alkenyl, loweralkylsilylalkenyl-, alkynyl, loweralkylsilylalkynyl-, loweralkylsilylaryl-, preferably 4-C ₆ H ₄ SiMe ₃ , loweralkylsilylbiphenyl-thienyl, preferably loweralkylsilylthienyl-, most preferably 2-(trimethylsilyl)thienyl- or loweralkylalkynyl-, preferably Me ₃ -C≡C-;

d) Z ^~ is an anion capable of forming an ionic bond sufficient to form said bis-(aryl) iodonium salt; and

e) each R ⁹ and R ¹⁰ is the same or different.

Z ^~ , as used herein, is an anion capable of forming an ionic bond with an iodonium cation sufficient to form a salt. Examples of preferred anions include triflate (OS0 ₂ CF ₃ ) , chloride, bromide, iodide, acid sulfate, nitrate, tetrafluoroborate, lactate (CH ₃ CHOHC0 ₂ ) , sulfonate, organnosulfonate, disulfide, sulfite, disulfite, thiosulfite, sulfide, hydroxide, hexafluorophosphate, alkoxide, chlorite, hypochlorite, nitrite, nitrate, azide, triiode, cyanide, hydrazide, perchlorate, haloacetate, preferably fluoroacetate, chloroacetate, bromoacetate, iodoacetate, fluorochloroacetate, fluorobromoacetate, chlorobromoacetate, fluorochlorobromoacetate, loweralkanoate, haloloweralkanoate, preferably fluoroloweralkanoate, chloroloweralkanoate, bromoloweralkanoate, iodoloweralkanoate, fluorochloroaloweralkanoate, fluorobromoloweralkanoate, chlorobromoloweralkanoate, or fluorochlorobromoloweralkanoate anion.

In another aspect, the invention features a bis- iodonium salt represented by the following formula:

wherein: a) X is either S or O; or X is NH;

b) n=l; R ^lla is attached to any one of the number 3 or 4 ring position; or n=2 and each _R ^li a ^ _s individually attached to both the number 3 and 4 ring positions and each R ^lla is the same or different; and c) R ^lla is any one of hydrogen, halogen, loweralkyl, loweralkoxy, haloalkyl-, cycloalkyl, aryl, heteroatom substituted aryl, aryloxy, or heterocyclic group;

d) R ¹¹ is any one of a CN-, aryl, preferably 4-phenyl, haloaryl-, preferably 4-C ₆ H ₄ F,

4-C ₆ H ₄ I, loweralkylpheny1-, haloloweralkylphenyl-, preferably 4-(CF ₃ )C ₆ H ₄ , 3,5-(CF ₃ ) ₂ C ₆ H ₃ , halobiphenyl-, preferably 4-C ₆ H ₄ -C ₆ H ₄ -I, alkenyl, loweralkylsilylalkenyl-, alkynyl, loweralkylsilylalkynyl-, loweralkylsilylaryl-, preferably 4-C ₆ H SiMe ₃ , loweralkylsilylbiphenyl-, thienyl, preferably loweralkylsilylthienyl-, most preferably 2-(trimethylsilyl)thienyl- or loweralkylalkynyl-, preferably Me ₃ -C≡C-;

e) Z ^~ is an anion capable of forming an ionic bond sufficient to form said bis-iodonium salt.

Those skilled in the art will appreciate that the above-mentioned synthetic methods can be adapted to make mono-, di-, other bis-, and higher iodonium compounds, e.g., iodonium compounds which include 3-15 (inclusive) iodonium moieties.

In another aspect, the invention also features a method of inhibiting a PQQ-catalyzed redox reaction, the method including obtaining an iodonium compound capable of sequestering PQQ; contacting PQQ with the iodonium compound under conditions capable of forming a PQQ-iodonium complex; and sequestering PQQ in a PQQ-iodonium complex sufficient to inhibit said PQQ-catalyzed redox reaction. In another aspect the invention also features a method of killing unwanted vegetation, preferably unwanted kudzu vine, the method including; obtaining an iodonium compound capable of sequestering PQQ; and dispersing the iodonium compound on the surface of the unwanted vegetation sufficient to sequester PQQ and kill the vegetation.

In another aspect the invention features a method of killing marine pests, preferably zebra mussels, lamprey eels and starfish, the method including: obtaining an iodonium compound capable of sequestering PQQ; dissolving said iodonium compound in an aqueous solvent; and dispersing said solution over an area subject to marine pest infestation in an amount sufficient to kill the marine pests.

The invention also features a topical fungicide comprising an iodonium compound capable of being inactivated after skin penetration.

In a related aspect, the invention features a method of inhibiting inflammation in a mammal, preferably a human, the method including: obtaining an iodonium compound capable of sequestering PQQ;

administering said iodonium compound; contacting PQQ with said iodonium compound under conditions capable of forming a PQQ-iodonium complex; and sequestering PQQ in a PQQ-iodonium complex sufficient to inhibit said inflammation.

In another aspect, the invention features a method of inhibiting microbial growth on a tissue surface or teeth from a human or veterinary patient, the method including: providing an iodonium compound exhibiting anti¬ microbial activity; and topically applying the iodonium compound to a tissue surface or teeth in an amount sufficient to inhibit growth of susceptible microbes on the tissue surface or teeth. By "microbial growth" is meant the undesired proliferation of organisms such as bacteria, yeast or fungus. By "topically applying" is meant the administration of an iodonium compound to teeth or a tissue surface (either inside or outside the oral cavity) in a pharmaceutically acceptable vehicle. "Susceptible microbes", as used herein, are those organisms, e.g., pathogenic oral bacteria; whose growth is completely inhibited in a test described herein, e.g., the agar dilution method described in part IX (1) , below. In one embodiment, the tissue surface is in the oral cavity (e.g., gums or oral ucosa) , preferably in an oral cavity characterized by at least one of dental caries, plaque, gingivitis, periodontitis (e.g. refracting periodontitis) , or gingivitis-induced inflammation. A particular application of the invention is the inhibitation of microbial growth in immunodeficient individuals, e.g. HIV-infected patients suffering from the undesired proliferation of oral microbes, e.g., oral fungus accompanying thrush. In another embodiment, the tissue surface is skin outside

the oral cavity, e.g., a skin wound outside the oral cavity. In another embodiment, the tissue surface or teeth have been subjected to dental surgery and the application of the iodonium compound is in the form of an oral rinse, oral irrigant, periodontal pack, or it is topically applied in a pharmaceutically acceptable vehicle. In another embodiment, after topically applying an iodonium compound to a tissue surface or teeth, the iodonium compound is inactivated by applying a non-toxic reducing agent, preferably about 2-4% (w/v) (inclusive) ascorbate or about 2-4% (w/v) (inclusive) sodium thiosulfate in a pharmaceutically acceptable buffer (e.g. , phosphate or bicarbonate buffer, each at pH 5-9 (inclusive)). In another embodiment, either prior to or during topically applying the iodonium compound to a tissue surface or teeth, a boric acid solution, preferably about 2% (w/v) boric acid in a pharmaceutically acceptable buffer (e.g., phosphate or bicarbonate buffer at pH 5-9 (inclusive)); is administered to the tissue surface or teeth in an amount sufficient to enhance the anti-microbial activity of the iodonium compound. Preferably, the boric acid solution is administered immediately prior to the application of the iodonium compound. The enhancement of the anti- microbial activity of an iodonium compound is determined by tests described below, e.g., the agar dilution method described in part IX (1) , below.

In a related aspect, the method further includes providing and applying chlorhexidine and an iodonium compound in an amount sufficient to inhibit the growth of susceptible microbes on a tissue surface or teeth. Preferably, the chlorhexidine and iodonium compound are each applied in amounts of equal anti-microbial activity as determined by methods described herein, e.g., the agar dilution method described in part IX (1) , below.

In another related aspect, the method further includes providing and applying two or more iodonium compounds, either by themselves, or in combination with chlorhexidine, where each compound is in an amount of equal anti-microbial activity as defined by methods described herein, e.g., the agar dilution method described in part IX (1) , below.

Brief Description of the Drawings Fig. 1 depicts chemical formulae of PQQ(l), DPI(2), and selected mono-(3,4) and bis(5-8)-iodonium compounds synthesized by triflate-mediated synthetic methods of the invention.

Fig. 2a and Fig. 2b show general schemes for preparing iodonium compounds: Fig. 2a) Scheme 1: bis- iodonium triflate prepared from diiodobenze, and Fig. 2b) Scheme 2: bis-(aryliodonium) thiophene triflate prepared from reaction of an aryl(cyano) iodonium triflate and 2,5- bis(tributyltin)thiophene.

Table 1 shows the inhibition of PQQ-catalyzed redox cycling by selected mono- and bis- iodonium compounds.

Tables 2 and 3 show the activity of iodonium compounds against oral bacteria.

Tables 4 and 5 show the activity of iodonium compounds against streptococci isolated from patients with refractory peritonitis.

Description of Preferred Embodiments I. Preparation of Bis-iodonium Triflates - Commercial diiodobenzene (9, Fig. 2) was oxidized to a bis(trifluoroacetate) (10, Fig. 2) by CF ₃ C0 ₃ H in 85% yield. CF ₃ C0 ₃ H (perfluoroucetic acid) is prepared by mixing 1.5 ml of 80% H ₂ 0 ₂ and 20ml trifluoroacetic anhydride. Reaction of (10) with two equivalents of a trimethylsilyl compound RSiMe ₃ (11, Fig. 2) and Me ₃ SiOS0 ₂ CF ₃ , in CH ₂ Cl ₂ , gave a bis-diiodoniu triflate (5, Fig. 2) in 66-97% yields. Examples of bis-iodonium triflate derivatives synthesized from the corresponding trimethylsilyl compound are shown in Fig. 1 (5a-h) . The synthesis of diyne bis-iodonium triflates (6a-b, 7) has been described (Stang et al., J. Org. Chem. 57, 1861- 1864, (1992)). The bis-iodonium triflate derivative (5h, Fig. 2) was prepared as outlined, except 2,5- bis(trimethylsilyl) thiophene was used as the appropriate trimethylsilyl derivative. Bis-iodonium triflates (8a-b, Fig. 1) were prepared by reacting 2,5- bis(tributyryl)thiophene (13, Fig. 2) with an aryl(cryano) iodonium triflate (14, Fig. 2). i) Analysis of Iodonium compounds- Iodonium compounds can be analyzed by HRMS (High Resolution Mass spectroscopy) . The expected spectral values of the proposed iodonium compounds can be found below. The spectral values of related iodonium compounds have been published (Watanabe et al., Curr. Ther. Res . 44:896, (1988); Hobara et al., Pharmacology 37:264, (1988); Nishigori et al., Life Sci . 45:593, (1989); Hamagishi et al., J. Pharmacol . Exp. Ther. 255:980, (1990); Aizenman et al., J. Neurosci 12:2362, (1992); Salisbury et al., Nature 280: 843, (1979); Mah et al., FASEB J. , Abstract, 7:A53, (1993); Paz et al., ASBMB/DBC-ACS Joint Meeting, Addendum, Abstract, LB89, (1993); Gardner et al. , Society for Neuroscience Abstract, 18:A321.6, (1992); Fliickiger

et al., In Principles and Applications of Quinoproteins , Davidson, Ed. ; Marcel1 Dekker, Inc. : New York, pp. 331- 341, 1992; Paz et al., J. Biol . Chem . 266:689, (1991)).

II. POO Inhibition Studies with Mono- and Bis-iodonium compounds

PQQ inhibition studies were carried out via a PQQ- catalyzed redox-cycling assay (Fliickiger et al., In Principles and Applications of Quinoproteins, Davidson, Ed.; Marcell Dekker, Inc.: New York, pp. 331-341, 1992; Paz et al., J ^" . Biol . Chem . 266:689, (1991); each reference herein incorporated by reference) using 13.3 nM PQQ. Several concentrations of each iodonium compound were used in order to determine the IC ₅₀ _ A stock solution of each iodonium compound was prepared with DMSO and then diluted in water to obtain the desired concentration. The results of the assay without, and with, various concentrations of an added iodonium compound are shown in Table I. Each assay was run in parallel in order to control for any non-PQQ catalyzed reduction of 4-nitro blue tetrazolium.

NH ₂ -CH ₂ -COCr -j PQQH ₂ -| - 2 <V "I P" ™ NH-CH-COO- -J L pQQ J L 0a -* •- T*

In the redox-cycling assay (picture above) , glycine present in large excess is oxidized at pH 10 in air. This chemical reaction is catalyzed by small amounts of PQQ. As reduced PQQ (PQQH ₂ ) is generated, it is oxidized by dioxygen to oxidized PQQ (PQQ) and two

superoxide radicals are generated. Superoxide is capable of reducing 4-nitroblue tetrazolium (T ⁺ ) to visible formazan (TH) . In twenty minutes at 37°C, about 2000 redox cycles occur such that nanomolar concentrations of PQQ generate micromolar amounts of visible formazan. Iodonium compounds that sequester PQQ inhibit the PQQ redox-cycling assay by preventing the reduction of PQQ to PQQH ₂ . Alkynl and aryl monoiodonium triflates effectively inhibit PQQ-catalyzed redox reactions on a micromolar scale, while bis-iodonium including bis- iodonium triflates, inhibit PQQ-catalyzed reactions on a nanomolar scale.

The results are summarized in Table 1.

Table 1. Iodonium Compounds Sequester PQQ

Experiment Compound ^Ic so*

1.5LtM

6.0μM

3.0μM

19μM

437nM

667nM

7nM

13nM

33nM

77nM

333nM

667nM

63nM

36nM

1.3xM

667/xM

2. OμM

29nM

13nM

The data in Table 1 indicate that bis-iodonium compounds are capable of sequestering PQQ, thereby inhibiting electron transfer in the redox cycling assay. Bis-iodonium compounds are more effective PQQ sequestering agents than mono-iodonium compounds. For example, Table 1 shows that redox-cycling assay inhibition by alkynl mono-iodonium compounds (3a-d) is comparable to inhibition of PQQ by Ph ₂ I ⁺ (BPI) and DPI. However, the bis-iodonium compounds (5a-h, 6a-b, 7, 8a-b, in Fig. 1) are the most effective in inhibiting redox- cycling. The data indicate that bis-iodonium salts 5a-c, 8a-b are the most effective at sequestering PQQ and inhibiting electron transfer in the redox cycling assay. Compounds 5a-c, 8a-b are 100-lOOOX better than mono- iodonium compounds at sequestering PQQ and inhibiting electron transfer in the redox-cyciing assay.

PQQ-catalyzed redox-cycling reactions are essential to maintain life and require the transfer of electrons down an electrochemical gradient from one component of the redox cycle to the next (see above) . NAD- or FAD-catalyzed electron transport in cells and cell organelles such as mitochondria, require electron transfer from one component of an electron transport chain to the next component down an electrochemical gradient. Electron transfer reactions also occur in plant chloroplasts and bacteria (see Stryer, Biochemistry 3rd Ed. pp. 397-407; Alberts, et al. The Cell pp. 341- 387; both references herein incorporated by reference). Two bis-iodonium compounds (5a, 5b) strongly inhibit NAD- catalyzed electron transport in the mitochondria (see Paz et al., ASBMB/DBC-ACS Joint Meeting, Addendum, Abstract LB80, (1993)). The inhibition of mitochondrial electron transport by bis-iodonium compounds is reversed by the addition of PQQ. Those skilled in the art of biochemistry would know from these results that bis-

iodonium compounds would also inhibit electron transport in a wide range of bacteria, plants, fungi, and animals.

Given that DPI is selective for muscle cells, the chemical moieties present in iodonium compounds described above afford selective cell permeability to these compounds. Given that iodonium compounds are capable of selective cell permeability and are capable of sequestering PQQ, thereby inhibiting redox-cycling reactions such as NAD- or FAD-dependent electron transport, iodonium compounds preferably bis-iodonium compounds, are useful as effective and selective inhibitors of electron transfer reactions involving PQQ. Such iodonium compounds inhibit in vitro and in vivo enzymatic reactions involving electron transfer in a wide variety of bacteria, fungi, plants, and animals, thereby serving as effective biocidal agents. The in vitro redox-cycling assay described above can be used to screen iodonium compounds of the invention for the ability to sequester PQQ and inhibit redox-cycling. Iodonium compounds that sequester PQQ and inhibit redox-cycling in vitro will serve as effective biocidal agents.

III. Experimental Methods. Melting points of iodonium compounds were obtained with a Mel-Temp capillary melting point apparatus. Infrared spectra were recorded on a Mattson FT-IR εpectrophotometer. NMR spectra were recorded on a Varian XL 300 spectrometer at 300 MHz ( ^X H NMR) , 75 MHz ( ¹³ C NMR) , 282 MHz ( ¹⁹ F NMR) . Chemical shifts for ¹ H and ¹³ C NMR are reported in parts per million (PPM) relative to internal tetramethylsilane or the proton resonance due to the residual in protons in the deuterated NMR solvent; the chemical shifts for ¹⁹ F NMR are relative to external CFC1 ₃ Mass spectra were obtained with a VG Micro ass 7050E double focusing high resolution mass spectrometer with the VG data system 2000

under positive ion fast atom bombardment (FAB) conditions at 8 keV.3-Nitrobenzyl alcohol was used as a matrix in CH ₂ C1 ₂ or CHC1 ₃ as solvent, polypropylene glycol was used as a reference for peak matching. Microanalysis were performed by Atlantic Microlab Inc. , Norcross, Georgia.

IV. Materials. All commercial reagents were ACS reagent grade and used without further purification. Aryl(cyano) iodonium triflates (14, Fig. 2) were prepared from bis(trifluoroacetoxy) iodoarenes, trimethylsilyltriflate and cyanotrimethylsilane (Zhdankin et al., Tetrahedron Letters 34:6853, (1993)). Iodonium compounds 3, 4, 6 and 7 in Fig. 1 were prepared by published methods (Bachi et al., J. Org. Chem . 56:3912, (1991); Stang et al., J. Heterocyclic . Chem . 29:815, (1992); Stang et al. , J. Org. Chem . , 57:1861, (1992); Stang et al. , J. Am . Chem . Soc . 114:4411, (1992)). All solvents used were dried by distillation over CaH ₂ . The reaction flasks were flame-dried and flushed with nitrogen. p-TTetra(trifluroracetoxy ^' )diiodolbenzene: (10 in Fig. 2a) p-Diiodobenzene 9 (3.3 g, 10 mmol) was added by small portions during 30 min to a stirred mixture of CF ₃ C0 ₃ H [prepared from trifluoroacetic anhydride (10 ml, 71 mmol) and 80% hydrogen peroxide (2 ml, 47 mmol) by known procedure. An additional oxidizing agent can be prepared by mixing similar amounts of peracetic acid and hydrogen peroxide.] The reaction mixture was stirred for 0.5 h at -78° C, then 2 h at -20° C and left overnight at room temperature. Concentration of the resulting clear solution and crystallization of the product by addition of ether afforded analytically pure 10 as a white microcrystalline solid, yield 6.64 g (85%); mp 195-197° C (deco p.). IR (CCl ₄ , cm ^-1 ): 3084, 3061 (C ₆ H ₄ ) , 1664, 1146, 982 (all CF ₃ C0 ₂ ) . ¹ H NMR ( δ , CF ₃ C0 ₂ H/CDC1 ₃ 1/10): 8.43 (s, C ₆ H ₄ ) . ¹⁹ F NMR ( δ , CF ₃ C0 ₂ H/CDC1 ₃ 1/10): -78.9

(s, CF ₃ C0 ₂ ) . ¹³ C{ ¹ H} NMR (<S, CF ₃ C0 ₂ H/CDC1 ₃ 1/10) : 118.7 (q, CF ₃ ); 126.0 (ε, C _ipsoAr ) ; 137.9 (s, CH _Ar ) , 162.4 (q,

C=0) . Anal. Calcd for C ₁₄ H ₄ I ₂ 0 ₈ F ₁₂ ^{: C} ' 21.50; H, 0.52.

Found C, 21.16; H, 0.78. V. Detailed Procedure for the Preparation of

(p-Phenylene ¹ ,bisiodonium Triflates (5; Fig. 2) : To a stirred solution of 10 (0.78 g, 1 mmol) in

CH ₂ C1 ₂ (20 ml) the corresponding silylated arene 11 (2.5-3 mmol) and Me ₃ SiOTf 12 (0.5 ml, 2.5 mmol) were added at - 78°C under N ₂ . The reaction mixture was allowed to warm to room temperature and additionally stirred for 3-5 h.

Colorless microcrystalline products 5a-f (Fig.l) precipitated in analytically pure form, i) Physical and Spectral Data: 5a: Yield 0.05 g (6%); mp 252-260° C (decomp.) IR

(CC1 ₄ , cm ^-1 ): 3086 (Ar) , 1245, 1167, 1028 (all CF ₃ S0 ₃ ) . H NMR (5, d ₆ -DMS0) : = 7.25 (dd, 4H, J=7.0 Hz), 8.12 (m,

8H) ; ¹⁹ F NMR (δ , d ₆ -DMS0) : -78.5 (s, CF ₃ S0 ₃ ) , -106.1 (s,

ArF) . ¹³ C{ ¹ H} NMR (δ , d ₆ -DMSO) ; 121.3 (q, J = 318 Hz, CF ₃ S0 ₃ ~) ; 120.1, 120.4, 137.3, 138.4, 138.7, 139.2 (all

Ar) .

5b: Yield 0.7 g (90%); mp 280-290° C (decomp.). 5c: Yield 0.95 g (92%); mp 270-275° C (decomp.).

IR (CC1 ₄ , cm ^-1 ): 3082 (Ar) , 1265, 1170, 1024 (all CF ₃ C0 ₃ ) . ^X H NMR (δ, d ₆ -DMS0) : 7.82 (d, 4H, J = 7.3 Hz), 7.87 (d,

4H, J = 7.4 Hz), 8.12 (s, 4H) . ¹⁹ F NMR (δ , d _g -DMSO) : -

77.8 (s, CF ₃ S0 ₃ ) . ¹³ C{ ¹ H} NMR (<5, d ₆ -DMSO) : δ = 121.3 (q,

J = 318 Hz, CF ₃ S0 ₃ ~) ; 96.5, 120.1, 120.5, 138.4, 138.9,

141.2 (all s, Ar) . HRMS (FAB) for C ₁₉ H ₁₂ SI ₄ 0 ₃ F ₃ [M]TfO ^" ] ⁺ calcd 88.4663334, found 884.661143. Anal Calcd for

C ₂₀ H ₁₂ I ₄ 0 ₆ F ₆ S ₆ : C, 23.23; H, 1.17; I, 49.09. Found: C,

32.24; H, 1.69; I, 42.61.

5d: Yield 0.79 g (66%); mp 275-278° C (decomp.).

IR (CC1 ₄ , cm ^-1 ) : 3082 (Ar) , 1247, 1168, 1026 (all CF ₃ S0 ₃ ) . ¹ H NMR (<S, d ₆ -DMS0) : 7.36 (d, 4H, J = 7.5 Hz) , 7.70 (d,

4H, J = 7.6 Hz) , 7.80 (d, 4H, J = 7.5 Hz), 8.12 (s, 4H) ,

8.14 (d, 4H, J = 7.5 Hz) . ¹⁹ F NMR (δ , d ₆ -DMSO) : -78.4 (s, CF ₃ S0 ₃ ) . ¹³ C{ ¹ H} NMR ( δ , d ₆ -DMSO) : 121.0 (q, J = 318 Hz, CF ₃ S0 ₃ ~) ; 95.2, 120.3, 120.6, 131.4, 136.5, 136.8, 136.9, 137.5, 138.4, 138.6 (all Ar) . HRMS (FAB) for C ₃₁ H ₂₀ SI ₄ 0 ₃ F ₃ [M-TfO ^" ] ⁺ calcd 1036.725934, found

1036.723128. Anal. Calcd for C ₃₂ H ₂₀ I ₄ O ₆ F ₆ S ₂ : C, 32.40; H, 1.70; I, 42.79. Found: C, 32.24; H, 1.69; I, 42.61.

5e: Yield 0.9 g (97%) ; p 255-257° C (decomp.) . 5f: Yield 0.7 g (85%) ; mp 183-185° C (decomp.) . 5g: Yield 0.93 g (86%) ; mp 256-258° C (decomp.) .

IR (CC1 ₄ , cm ^-1 ) : 3082 (Ar) , 2957 (Me ₃ Si) , 1245, 1167, 1027 (all CF ₃ S0 ₃ ) . ^λ NMR (5, d ₆ -DMSO) : 0.21 (s, 18H) , 7.62 (M, 8H) , 7.74 (d, 4H, J = 7.6 Hz) , 8.12 (ε, 4H) , 8.15 (d, 4H, J = 7.5 Hz) . ¹⁹ F NMR (δ , D ₆ -DMS0) : -78.2 (ε, CF ₃ S0 ₃ ) . ¹³ C{ ¹ H} NMR (δ, d ₆ -DMSO) : -1.5 (ε) , 121.0 (q, J = 318 Hz, CF ₃ S0 ₃ ~) ; 120.1, 120.7, 131.4, 136.5, 136.8, 136.9, 137.5, 138.4, 138.6, 141.5 (all Ar) . HRMS (FAB) for C ₃₇ H ₃₈ SSi ₂ I ₂ 0 ₃ F ₃ [M-TfO ^" ] ⁺ calcd 929.011939, found 929.012254. 5h: Yield 0.8 g (85%) ; mp 225-230° C (decomp.) .

IR (CC1 ₄ , cm ^-1 ) : 3075, 2965, 1474, 1384, 1282, 1235, 1165, 1026, 911. ^λ K NMR (δ , CD ₃ CN) : 8.03 (s, 4H) , 7.90 (d, J = 8.0 Hz, 2H) , 7.24 (d, J = 8.0 Hz, 4H) , 0.21 (ε, 18H, 2Me ₃ Si) . ¹⁹ F NMR (δ , CD ₃ CN) : -78.3 (s, CF ₃ S0 ₃ ) . HRMS (FAB) for C ₂₁ H ₂₆ S ₃ Si ₂ I ₂ 0 ₃ F ₃ [M-TfO-] ⁺ calcd 788.862184, found 788.861207.

VI. Reaction of 2.5-bisftributyltin thiophene (13. Fig. 2b) with aryl(cyano) iodonium triflate (14. Fig. 2b).

To a stirred solution of reagent 14 (1 mmol) a solution of the appropriate tributyltin derivative 13 (1-

1.5 equivalents) in CH ₂ C1 ₂ (15 ml) was added at -40° C.

The mixture was warmed to room temperature and stirred until the formation of a clear solution. The products

(8, Figs. 1, 2b) were precipitated from the reaction mixture by the addition of dry hexane (20-30 ml) . The microcrystalline iodonium triflate εalt waε filtered under nitrogen, washed with dry hexane (30 ml) and dried in vacuo. Analytically pure materials were obtained by recryεtallization from a concentrated solution of the iodonium εalt in CH ₂ C1 ₂ by addition of hexane and ether. Other organotin derivatives, for example methylethynyltributytin and phenyltributyltin, can be εubεtituted for 13 (Fig. 2b) in order to obtain the corresponding bis(aryliodonium) theophene. i) Physical and Spectral Data

8a: Yield 0.39 g (49%), mp 193-194° C (decomp.). IR (CC1 ₄ , cm ^-1 ): 3097, 3066, 1241, 1159, 1023. H NMR (5, d ₆ -DMSO/CD ₃ CN) : 7.55 (t, J = 8.0 Hz, 4H) , 7.71 (t, J = 8.0 Hz, 2H) , 7.83 (s, 2H) , 8.19 (d, J = 8.0 Hz, 4H) . ¹⁹ F NMR ( δ , d ₆ -DMSO/CD ₃ CN) : -78.74 (ε, CF ₃ S0 ₃ ~) . ¹³ C{ ¹ H} NMR (6, d ₆ -DMSO/CD ₃ CN) : 111.0, 119.4, 121.2 (q, J = 320.7 Hz, CF ₃ S0 ₃ ~) , 132.3, 132.9, 135.1, 140.9. Anal. Calcd for C ₁₈ H ₁₂ I ₂ 0 ₆ F ₆ S ₃ : C, 27.43; H, 1.53; S, 12.20. Found: C, 27.35; H. 1.56; S, 12.12. 8b: Yield 0.21 g (25%), mp 170° C (dec). IR

(CC1 ₄ , cm ^-1 ): 3098, 1575, 1246, 1170, 1027 cm ^-1 ; H NMR ( δ , CD ₃ CN) : 8.15 (dd, 4H) , 7.85 (s, 2H) , 7.3 (dd, 4H) ; ¹⁹ F NMR (<S, CD ₃ CN) : -78.1 (CF ₃ S0 ₃ ~) , -105.0 (FC ₆ H ₄ ) . ¹³ C NMR (<S, CD ₃ CN) : 162.5 (d) , 138.7, 136.3, 136.2, 121,0 (q, CF ₃ S0 ₃ ) , 117.7, 110.5. FAB HRMS m/z 674.804664 [M-CF ₃ S0 ₃ - ] ⁺ , calcd for C ₁₇ H ₁₀ I ₂ S ₂ F ₅ 0 ₃ : 675.807858.

VII. Iodonium Salts as Biocidal Agents

Iodonium compounds of the invention are useful as herbicides which kill unwanted vegetation, e.g., the kudzu vine; aε insecticides which kill insectε, e.g., the gypsy moth; and as agents which kill marine pests, e.g. , zebra musεels, starfiεh, and lamprey eels. For such uses, an iodonium triflate or other iodonium salt of the invention can be dispersed on an inert finely divided solid (e.g., silica) and employed aε a dust. Such a mixture can be used directly on plants or insectε. Alternatively, an iodonium triflate or other iodonium salt of the invention can be employed as a spray or emulsion by diεεolving in water, preferably a εolution of 90% by weight water and 10% by weight dimethylsulfoxide (DMSO) or 10% by weight acetonitrile or 10% by weight dimethylformanide. Preferred compositions contain from 0.0001% to 50% by weight of an iodonium triflate or other iodonium salt of the invention. i) Use as a topical fungicide - An iodonium triflate or other iodonium salt of the invention, can be delivered in a pharmaceutically acceptable carrier to a skin surface subject to fungal infection. Suitable carriers include water, oils such as mineral oils, soybean oil and the like, creams or gels comprising glycerol and/or potasεium, ammonium or sodium stearate, and water. Preferably an iodonium triflate or other iodonium salt of the invention can be used in a carrier at 0.05% by weight. Examples of other pharmaceutically acceptable carriers for topical administration can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co.: Easton, PA, 1980.

VIII. Iodonium Compounds as Anti-Inflammatory Agents

Macrophages and neutrophilε mediate inflammatory reactions asεociated with trauma, pulmonary emphyεema, cystic fibrosis, bronchitis, psoriasiε, arthritiε, and

rheumatoid arthritis, among other inflammatory diseases. Tissue destruction by macrophages and neutrophilε has been extensively reviewed (Weiss, S.J. N . Engl . J. Med . 320, 365 (1989); Lehrer, R.I. et al. Ann. Intern . Med . 109, 127, (1988); Malech, H.L. et al. Ann. Intern . Med . 317, 687, (1987)). Both cell types expose phagocytosed cells and/or tiεεue to free radical εpecieε εuch as superoxide (0 ₂ -) , hydroxyl radical and hypochlorite. Macrophages and neutrophils produce free radical species by using known redox-cycling reactions. Without wishing to bind ourselves to any particular theory, it is proposed that free radical generation involves PQQ. Given that iodonium compounds of the invention inhibit redox- cycling by sequeεtering PQQ, iodonium compounds modulate the toxicity of free radical species produced by macrophages and neutrophils which is associated with inflammation.

I. Testing Iodonium Compounds - Methods of isolating neutrophilε and in vitro and in vivo model syεtemε for testing the activity of agents that inhibit free radical formation by neutrophils are well known (reviewed in Weiss, S.J. N . Engl . J. Med . 320, 365 (1989); Lehrer, R.I. et al. Ann . Intern . Med . 109, 127, (1988); Malech, H.L. et al. Ann . Intern . Med . 317, 687, (1987)). In general, an iodonium compound of the invention can be dissolved in water, preferably water with 10% DMSO or other suitable solvent and added to a an in vitro model syεte comprising neutrophilε in order to εee if the production of damaging free radicalε haε been reduced. Preferably, an iodonium compound is at a concentration of at least one microgram per ml in these systems. In vivo model systems for testing the activity of agents that inhibit neutrophils or macrophage free radical generation are also known in the art, for example

rodent models are known and can be uεed to teεt iodonium compoundε of the invention for anti-inflammatory activity in vivo.

IX. Iodonium Compounds Are Anti-Microbial Agents Suitable For Topical Application

It is an object of the invention to use one or more iodonium compounds, e.g., mono-, di-, bis-, and higher iodonium compounds, e.g., compounds which include 3-15 (inclusive) iodonium moieties; whenever chlorhexidine administration is indicated, but with superior results. Like chlorhexidine, iodonium compounds are anti-microbial agents which can alleviate juvenile and adult periodontal diseases such as gingivitis, periodontitis (e.g., refractory peritontitis) , gingivitiε-induced inflammation, oral thrush, and acute necrotizing gingivitis. Like chlorhexidine, iodonium compounds are also effective anti-microbial agents for use in general or preoperative skin cleaning, preoperative showering or bathing, wound cleaning, or as a εterilizing agent for the treatment of dental caries.

However unlike chlorhexidine, iodonium compounds can be inactivated after use by briefly applying a non-toxic reducing agent. The ability to inactivate an iodonium compound after use reduces the potential for teeth or mouth tissue damage (e.g., teeth staining). Exampleε of non-toxic reducing agentε suitable for use include 2-4% (w/v) sodium ascorbate or 2-4% sodium thiosulfate in a pharmaceutically acceptable buffer (e.g., phosphate or bicarbonate buffer at pH 5-9 (inclusive)). Other pharmaceutically acceptable salts of ascorbate and thiosulfate can also be used, e.g., potassium ascorbate and potassium thiosulfate. Other useful non-toxic reducing agents can be identified by combining a molar excess (e.g., a 1.5 fold molar excess) of the reducing

agent with a particular iodonium compound and evaluating the anti-microbial activity of the treated iodonium compound in the agar dilution method described in part IX (1) , below. Generally, an acceptable non-toxic reducing agent will eliminate the anti-microbial activity of the iodonium compound. The anti-microbial activity of an iodonium compound can be enhanced by administering, either prior to or during the topical application of the iodonium compound, a 2-4% (w/v) (inclusive) boric acid solution in a pharmaceutically acceptable buffer (e.g. , phosphate or bicarbonate at pH 5-9 (inclusive)). Preferably, the boric acid solution is administered immediately prior to the application of the iodonium compound. The enhancement of the anti-microbial activity of an iodonium compound iε at leaεt 1.5 fold and can be determined by tests described herein, e.g., the agar dilution method described in part IX (1) , below.

An iodonium compound, e.g., a mono-, di-, bis-, or higher iodonium compound, e.g., a compound including 3-15 (inclusive) iodonium moieties, can be uεed with one or more other iodonium compoundε or optionally, combined with chlorhexidine, each compound in amounts of equal anti-microbial activity (determined by methods described below, e.g. , the agar dilution method described in part IX (1) , below) . The combination of one or more iodonium compoundε or optionally, chlorhexidine, will reduce the danger of selecting resistant oral flora. An iodonium compound, either alone, with one or more other iodonium compounds, or optionally combined with chlorhexidine, can also be used in: 1) general or preoperative skin cleaning

(e.g., surgical scrub); 2) preoperative showering or bathing; 3) wound cleaning; or 4) as a εterilizing agent for the treatment of dental caries, e.g., to sterilize a cavity prior to performing dental restoration, e.g. , insertion of amalgam to fill a cavity.

₁ . _I o _d onium compounds Inhibit Oral Bacteria

Growth

_A ) _A gar Dilution Method: Iodonium compounds were teste _{d f} or anti-microbial activity against reference strains of oral bacteria. These reference strains inclu _d ed ₄ 8 subgingival species (Tables 2 and 3) and 26 strains of streptococci isolated from patients with refractory periodontitis (Tables 3 and 4) . By "refractory" is meant that the periodontitis was resistant to conventional intervention, e.g. , antibiotic treatment; oral antiseptic treatment, including chlorhexidine use. Several of these bacteral strains are known oral pathogens, e.g. Porphyromonas gingivitis . The Minimum inhibitory Concentration (i.e. MIC) was independently determined for bis-iodonium compounds 6a, 8a, and 8b (see Fig. 1) . The MIC was defined as the minimum concentration of an iodonium compound necessary to completely inhibit the growth of a reference strain of bacteria. 1) Preparation of Agar Medium

T _h e composition of the agar medium is disclosed below:

_A gar medium was sterilized in individual test tubes ( ₁ test tube per Petri plate) by autoclaving at 121°C for 15 min. The sterilized agar medium was

subsequently cooled in a 50°C water bath. The volume in each tube was adjusted with water so that the final volume approximate 40 ml when the filter-sterilized bis- iodonium compounds were added to the agar medium. 2) Preparation of test media

Bis-iodonium compounds 6a, 8a, and 8b (see Fig. 1) were each dissolved in 5 ml of dimethyl sulfoxide, then distilled water was added to adjust the final concentration of bis-iodonium compound (in 40 mis of agar medium) to 2μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32μg/ml or 64 μg/ml. As a control, ^* a solution was prepared without any added bis-iodonium compound. Each solution was subεequently filter εterilized by paεsage through a 0.22 μm Nalgene filter. After addition of control or bis- iodonium compound containing εolutionε into about 40 mlε agar medium, the agar medium waε aεeptically dispensed into plastic microtiter dishes. The agar medium was allowed to set overnight.

3) Preparation of bacterial inocula Individual bacterial species were maintained on blood agar plates under an atmosphere of 80% N ₂ , 10% H ₂ and 10% C0 ₂ at 35°C for 3 days. Bacterial inocula was aseptically harvested from each plate and suspended in 1 ml of sterile mycoplasma broth (Baltimore Biological Labratorieε) . Each bacterial εuεpenεion was adjusted to an optical density approximating a MacFarland 0.5 standard (see generally, Manual of Microbiological Methods , American Society for Microbiology, McGraw-Hill, N.Y. (1957) ) . 4) Inoculation of test media

100 μl of each bacterial strain suspension was placed in the microtiter plate containing either control agar medium or agar medium with 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32μg/ml or 64 μg/ml of a bis-iodonium compound. Because of the large number of individual

bacterial species tested, an MIC 2000 inoculator was used to transfer 1.5 μl of each inoculum to the surface of a microtiter plates. Control plateε were inoculated at the beginning and end of the εerieε of inoculations to verify aseptic experimental conditions. These plates were used to indicate that the inoculum was present and viable throughout the series. This was confirmed for all species.

5) Results Agar plates were incubated under an atmosphere of 80% N ₂ , 10% H ₂ and 10% C0 ₂ at 35°C. The MIC for each iodonium compound and reference strain was determined after an incubation period of 5 days. The data are presented in Tables 2, 3, 4, 5, below.

TABLE 2: IODONIUM COMPOUNDS INHIBIT ORAL BACTERIA GROWTH

Note: A "*" in the table indicates that a bacterial strain was able to grow in the presence of at least 64 μg/ml of an iodonium compound. An MIC of 2 indicates that a bacterial strain was able to grow in the presence of 2 μg/ml or less of a iodonium compound.

Iodonium test compounds 6a, 8a, and 8b are shown in Fig. 1.

TABLE 3: IODONIUH COMPOUNDS INHIBIT ORAL BACTERIA GROWTH

Note: A "*" in the table indicates that a bacterial strain was able to grow in the presence of at least 64 μg/ml of an iocsniuα coαpound. An MIC of 0.25 indicates that a bacterial strain was able to grow in the presence of 0.25 μg/al or less of an iodonium compound.

Iodonium test ₊ compounds 3c, 4, and 5b are shown in Fig. Compound 4a is the mono-iodonium triflate P _j l ^" OSO-CF _j . "Chlor" is chlorhexidine

TABLE 4

IODONIUM COMPOUNDS INHIBIT THE GROWTH OF STREPTOCOCCI ISOLATED FROM PATIENTS WITH REFRACTORY PERIODONTITIS

Note: A "*" in the table indicates that a bacterial strain was able to grow in the presence of at least 64 μg/ml of a iodonium compound.

Iodonium compounds 6a, 8a, and 8b are shown in Fig. 1

TABLE 5

Note: A "*" in the table indicates that a bacterial strain was able to grow in the presence of at least 64 μg/ml of an iodonium compound.

Iodonium compounds 3c, 4, and 5b are shown in Fig. 1. Compound 4a is the mono-iodonium triflate Ph _j l OSO _j C _j . "Chlor" is chlorhexidine.

Tables 2, 3, 4 and 5 show that iodonium compounds inhibit the growth of a wide variety of bacteria; several iodonium compoundε exhibit anti-microbial activity which iε superior to that of chlorhexidine. For example, table 2 shows that iodonium compoundε inhibit the growth of εeveral εubgingival bacterial εpecieε below 64 μg/ml. Oral bacteria εuch as B . forsythus, C . rectus, F . nucleatum subspecies, P . gingivalis, P . intermedia, P. nigrescens and P. micros, were each sensitive to compound 8b ( a bis-iodonium compound) at low levels (< _. 16 μg/ml) . P. intermedia and P. nigrescens were especially εensitive to iodonium compounds 6a and 8b. Generally, each of these bacteria are extremely difficult to eliminate from the oral cavity and each are suspected oral pathogens. Table 4 shows that iodonium compounds inhibit the growth of streptococci isolated from patients with refractory periodontitis. For example, εtrain HA45 waε inhibited by iodonium compounds 6a, 8a, and 8b. Tables 3 and 5 point out that several iodonium compounds exhibit a lower MIC (i.e. better anti-microbial activity) than chlorhexidine. For example, compoundε 4a, 5b and 4 (a mono-iodonium compound) inhibit the growth of C. concisus at levelε 2-4 fold lower than chlorhexidine. The data indicate that mono-, di-, bi-, and higher iodonium compounds, e.g., compounds including 3-15 (inclusive) iodonium moieties, can be used to inhibit the growth of a variety of oral bacteria on a tissue surface or teeth from a human or veterinary patient.

B) Additional tests: Iodonium compounds can be tested for anti-microbial activity by other well-known methods. Such tests are useful for evaluating the anti¬ microbial activity of an iodonium compound. For example, a paper disc can be impregnated with an iodonium compound and placed on the surface of an agar plate which includeε a lawn of any gram-negative or gram-positive bacteria,

for example, any bacteria of the genus streptococci (gram negative) or εtaphylococci (gram poεitive) . Such bacteria can include pathogenic bacteria found inεide the oral cavity or outside the oral cavity, e.g. , on a skin surface. The concentration of a particular Iodonium compound in the paper diεc can be varied by εerial dilution in order to determine the MIC. Standard criteria for interpreting paper diεc teεtε have been published (Bauer et al., Am. J. Clin. Path. 45, 493-496 (1966)). The method of Hennessey can also be used to determine the anti-microbial activity of an iodonium compound against streptococci or staphylococci strains (J. Periodont. Res. 8, 61, (1973)).

The anti-fungal activity of an iodonium compound against opportunistic infectionε of the oral cavity, for example, thrush fungus (i.e. Candida albicans) can be examined by the method of Budtz-Jorgensen E. and Loe, H. (Scand. Dent. J. Dent. Res. 80, 457 (1972) . In another teεt, anti-plaque activity of an iodonium compound can be evaluated by topically applying (e.g. rinsing) about 10 ml of a 0.2% (w/v) solution of the iodonium compound, followed by an examination of plague depoεits or salivary bacteria (Loe, H. and Schiott C.R., J. Periodont. Res 5, 79 (1970); Schiott, C.R. et al. J. Periodont Res. 5, 84 (1970)).

In any above test method, chlorhexidine can be co- administered (at a concentration giving equivalent anti¬ microbial activity) with an iodonium compound in order to detect synergistic anti-microbial effects between chlorhexidine and the iodonium compound.

C) Administration: An iodonium compound which inhibits the growth of susceptible microbes by any above- described test (e.g., compounds 6a, 8a, 3c, 4a, 5b, 4 and 8b) can be used as an oral rinsing agent, oral irrigating agent, subgingival irrigating agent, or periodontal pack.

For oral use, an iodonium salt (e.g., the gluconate salt of compounds 6a, 8a or 8b) can be combined with water, about 10% alcohol, a sorbitan εtearate, and a flavoring agent. Generally, the effective final concentration of an iodonium compound is at least 1 μg/ml (between 0.001% and 2% w/v (inclusive)). If desired, administration of an iodonium compound is followed by applying a non-toxic reducing agent, e.g. about 2-4% (w/v) sodium ascorbate or about 2-4% (w/v) sodium thiosulfate in a pharmaceutically acceptable buffer (e.g., phosphate or bicarbonate buffer, each at pH 5-9 (inclusive)) in order to inactivate (i.e. chemically reduce) the compound. The non-toxic reducing agent is desirably in molar excess with respect to the applied iodonium compound (at least 1.5-fold molar excess) . If desired, the activity of an iodonium compound can be enhanced by topically applying (either before or during application of the iodonium compound) about 2% boric acid in pharmaceutically acceptable buffer (e.g., phosphate or bicarbonate buffer, each at pH 5-9 (inclusive)). Generally, in order to inactivate or enhance iodonium compound activity, a non-toxic reducing agent or boric acid solution (respectively) is applied for at leaεt 1 minute, followed by rinεing with a pharmaceutically acceptable buffer or water. For example, an oral rinεe which includes an iodonium compound can be inactivated by rinsing 3 times (about 30 seconds each) with a 2% (w/v) ascorbate solution, followed by one (about 10 second) water rinse.

For use as an agent which inhibits the growth of suεceptible microbes outεide the oral cavity, e.g., use as a skin cleanser (e.g., surgical scrub), preoperative showering or bathing, or wound cleaner; an iodonium salt (e.g. , the gluconate salt of compounds 6a, 8a or 8b) can be dissolved in 4% isopropyl alcohol at a final concentration of at least 1 μg/ml or 0.1%-5% (w/v)

(inclusive) in a pharmaceutically acceptable carrier (vehicle) which includes a detergent and/or emollient. As discusεed previouεly, the anti-microbial activity of the applied iodonium compound can be inhibited or enhanced, if deεired. Chlorhexidine or another antiεeptic may be optionally added in an amount of equal anti-microbial activity (determined by methodε deεcribed herein, e.g., the agar dilution method deεcribed in part IX, (1) , above) . Examples of well-known pharmaceutically acceptable vehicles for oral or external use can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980) .

All publications and patent applications mentioned in the specification are indicative of the level of skill of those in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent aε if each individual publication or patent application were εpecifically and individually εtated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one skilled in the art will easily ascertain that certain changes and modifications can be practiced without departing from the spirit and εcope of the appended claims.