THIARUBRINE ANTIFUNGAL AND ANTIBIOTIC AGENTS 5

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Serial No. 07/447,893, filed December 7, 1989, 0 which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains to a novel group of 5

Thiarubrine substances and closely related derivatives that are useful as antifungal and antibiotic agents. The invention further pertains to a novel method for isolating the Thiarubrine substances. 0

BACKGROUND OF THE INVENTION

A publication entitled "Two Dithiacyclohexadiene

Polyacetylenes from Chaenactis douglasii and Eriphyllum

„,.1anaturn", Norton, R.A. ; Finlayson, A.J. ; Towers, G.H.N.; 25

Phytochemistry, 24(2), 356-7 (1985) discloses two dithiapolyacetylenes isolated from the roots of C_^ douglasii and from the plant roots as well as root cultures of E. lanatum. The two dithiapolyacetylenes have the respective ₃₀ chemical names 3 - (1- -propynyl) 6 (5-hexen-3-yn-l-ynyl)- l,2-dithiacyclohexa-3,5- diene and 3 (pent-3-yn-l-ynyl) 6 (3-buten-l-ynyl)-l,2- dithiacyclohexa-3,5-diene. The trivial names Thiarubrine A and Thiarubrine B, respectively, have been qiven to these compounds.

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Another publication entitled "Production of Antibiotic Thiarubrines by a Crown Gall Tumor Line of Chaenactis douglasii", Cosio, E.G. ; Norton, R.A. ; Towers, E.; Finlayson, A.J. ; Rodriguez, E. ; Towers, G.H.N.; J. Plant Physiol., 124 (1- 2), 155-64 (1986) discloses dithiacyclohexadiene polyacetylenes which display considerable antimicrobial activity. A culture that accumulates these compounds was obtained by selection of red-colored areas from crown gall tumor cultures of Chaenactis douglasii. The tumors were induced by Agrobacterium tumefaciens strain A277. The main acetylenic products found were two Thiarubrines, A and B, and their corresponding thiophenes. Average yields of thiarubrines and thiophenes were 2.5 and o.24 mg/.g dry weight, respectively, which are similar to the values seen in intact plants. The products accumulate in intercellular spaces arranged around a core of vascular tissue, forming red nodules of varying size. The structures crudely resemble the tissue organization of the roots of this plant, the only organ where these compounds accumulate. Formation of rootlets, however, did not take place at any stage during the growth of the tumors. Apparently, accumulation of these polyacetylenes by the tumor line is not a direct result of cellular transformation but a secondary effect of the existing degree of tissue differentiation.

The publication "Thiarubrine Accumulation in Hairy

Root Cultures of Chaenactis douglasii", Constabel, C.P.; Towers, G.H.N.; J. Plant Physiol., 133(1), 67-72 (1988) discloses hairy root cultures of C_^ douglasii established using Agrobacterium rhizogenes strain TR7. One culture line

accumulated twice the revels of the antifungal polyines, c Thiarubrines A and B, compared to non-transformed control root cultures, while maintaining rapid growth. The combination of fast growth and high thiarubrine accumulation could not be duplicated in controls by adding exogenous NAA to the culture medium. Hairy root cultures also produced 0 less Thiarubrine B relative to Thiarubrine A compared to controls.

A publication entitled "Antiviral Properties of Thiarubrine A, a Naturally Occurring Polyine", Hudson J.B.; 5 Graham E.A. ; Fσng R; Finlayson A.J., Towers G.H.N. ; Planta Med 0(1), 1986, 51-54 relates to the naturally occurring polyine, Thiarubrine A. It was evaluated for its antiviral properties in the presence and absence of long wave UV radiation (UV-A) . Four viruses and a mammalian cell line _Q were used as targets. The two mammalian viruses, urine cytomegalovirus and Sindbis virus, both of which possess membranes, were extremely sensitive to the compound, but only in the presence of UV-A radiation. The bacteriophage T4 was slightly affected in UV-A only, whereas the 5 bacteriophage M13 was completely unaffected. Thus

Thiarubrine A is photoactive against membrane containing viruses. In contrast mouse cells were moderately sensitive to the compound in the presence of UV-A, and somewhat less sensitive in the dark. 0

Thiarubrine A is also discussed in "Antibiotic Properties of Thiarubrine A a Naturally Occurring Dithiacyclohexadiene Polyine", Towers G.H.N. ; Abramowski Z; Finlayson A.J. ; Zucconi A; Planta Med 0 (3), 1985, 225-229.

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Thiarubrine A, a dithiatyclohexadiene polyine from the roots of Chaenactis douglasii, is disclosed as exhibiting strong antifunqal activity towards Candida albicans and Aspergillus fumigatus at concentrations comparable to those of amphotericin B. It also exhibits cytotoxic activity towards Escherichia coli, Bacillus subtilis and Hycobacteria sp. and the nematode Coenorhabditis elogans. A thiophene derived from thiarubrine A was phototoxic, requiring UV-A liqht for biological activity. The effects of Thiarubrine A, thiophene A and alpha-terthienyl on the above organisms, as well as on CHO cells, were- compared.

Polyacetylene groups and thiophenes are disclosed in "comparison of the antiviral effects of naturally occurring thiophenes and polyacetylenes", Hudson J. B.; Graham E.A. ; Chan G; Finlayson A. . ; Towers G.H.N.; Planta Med 0 (6], 1986 (Reed. 1987), 453-457. Five naturally occurring compounds, containing various thiophene and polyacetylene groups, were compared with respect to their phototoxic activities against two animal viruses, murine cytomegalovirus and Sindbis virus, both of which possess membranes. Alpha-Terthienyl was extremely toxic to both viruses, but only in the presence of long wave ultraviolet radiation. The order of potency was alpha-terthienyl thiarubrine-A > phenylheptatriyne > ACBP-thiophene > thiophene-A (hydrolysis product of thiarubrine A) . The murine-CMV, which had been inactivated by any of these compounds, was still capable of penetrating cultured mouse cells efficiently and reaching the cell nucleus, the normal site of virus replication. The results are discussed in

terms of possible mechanisms of action of phototoxic thiophenes and polyacetylenes.

Canadian Patent No. 1,169,767, issued June 26, 1984, Towers et al., discloses cercaricidal compositions containing naturally occurring conjugated polyacetylenes and method for controlling cercariae using the same.

Canadian Patent No. 1,172,460, granted August 14, 1984, Towers et al., discloses a method for controlling weeds using naturally occurring conjugated polyacetylenes.

Canadian Patent No. 1,173,743, granted September 4, 1984, Towers et al., discloses a method for controlling pests using naturally occurring conjugated polyacetylenes.

SUMMARY OF THE INVENTION

The invention is directed to a novel substance having antifungal and anti-bacteria activity of the formula:

wherein R., is CH_ or CH_R ₃ ;

0 / \ R ₂ is -HC-CH ₂ ; -CH ₃ -CH ₂ R ₃ ; -HCHa-CH _{2 3} ;

-CHR ₃ -CH ₂ Ha ; CH=CH ₂ ;

R 3_ is H; OH; -CHO; COOH or COOR4. wherein R4. is derived from an alcohol;

Ha is F; Cl; Br; or I;

n is 1 or 2; and

m is 1 or 2, and pharmaceutically acceptable salts thereof, provided that when R is CH , n is 1 and m is 2, then R_ is not CH=CH ₂ ; and provided that when R is CH_, n is 2 and m is 1, then R ₂ is not'CH=CH ₂ .

The invention ^* is also directed to the bactericide e or fungicide Thiarubrine D

(CH ₃ -C=C-R-(CsC) ₂ -C SH-CH ₂ ) ;

0 the fungicide Thiarubrine E

(CH ₃ -C=C-R-(C≡C) ₂ -CHOH-CH ₂ OH) ;

the fungicide Thiarubrine F ₅ (CH ₃ -C≡C-R-(C^C) ₂ -CHCl-CH ₂ OH) ;

the fungicide Thiarubrine G

(CH ₃ -C=C-R-(C≡C) ₂ -CHOH-CH ₂ Cl) ; 0

and the fungicide Thiarubrine H

(HOCH ₂ -C=OR-(C=C) ₂ -CH=CH ₂ ) ;

5 where R is

Bacteria may be treated with the Thiarubrine substance in association with light of a wavelength range

35

about 320 to 400 nanometers, or specifically, in association with light of a wavelength about 350 nanometers.

The invention is also directed to an antifungal composition comprising a Thiarubrine substance of the formula:

wherein R. is CH ₃ or CH

R„ is -HC

">-CH.,; -CHR -CH _^ R ; -HCHa-CH R„ ^_C "2' 3 "2"3' "~" "2"3'

-CHR ₃ -CH ₂ Ha; CH=CH,

R3_ is H; OH; -CHO; COOH or COOR4. wherein R4. is derived from an alcohol;

Ha is F; Cl; Br; or I;

n is 1 ^* or 2; and

is 1 or 2

and pharmaceutically acceptable derivatives thereof, provided that when R. is CH ₃ , n is 1 and m is 2, then R ₂ is not CH=CH ₂ ; and provided that when R is CH ₃ , n is 2 and m is 1, then R is not CH=CH ₂ in association with a pharmaceutically acceptable carrier.

In the composition, Ha may be chlorine, R_ may be OH, R ₃ may be OH, n may be 1 and may be 2. The carrier may be distilled water, saline or any other pharmaceutically acceptable carrier.

The composition can be used to control E^ coli. Staphylococcus aureus, Streptococcus faecalis. Mycobacterium sp. or Enterobacter aerogenes in dark and more effectively in the presence of light having a wavelength of about 320 to 400 nanometers, or specifically, having a wavelength of about 350 nanometers. The composition can be used to control Candida albicans, Aspergillus fu igatus or Cryptococcus neoformans as well.

The foregoing Thiarubrine substance is present in the composition at a concentration of about 1 to 100 nanograms/ml of carrier, or specifically, at a concentration of about 10 nanograms/ml of carrier.

The invention* is also directed to a method of controlling Candida albicans or Aspergillus which comprises exposing a host infected with the Candida albicans or Aspergillus to an antifungal effective amount of a compound of the formula:

wherein R- is CH-_$ or -CH«__£OH; and R^ is

CHOH-CH ₂ OH; -HCC1-CH ₂ 0H; CH0H-CH ₂ C1 or CH=CH ₂

Candida albicans may be treated at a concentration of Thiarubrine to Candida albicans in the range of about 1.0 to about loo nanograms/ml of distilled water. 5

The invention pertains to an antifungal composition comprising Thiarubrine D and a pharmaceutically acceptable carrier. The Thiarubrine D can be present in the composition at a concentration of 7 nanograms/ml of carrier. _Q The carrier in the composition can be water or any other pharmaceutically acceptable carrier.

5

4. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

4.1 NOVEL THIARUBINES

The present invention is based on the discovery of a group of novel dithiacyclohexadiene compounds isolated from extracts of a common ragweed Ambrosia chamissonis Asteraceae (tribe Heliantheae). found in coastal areas ranging from southern California to British Columbia, Canada. The novel compounds, isolated from extracts of root tissues, have been found to possess remarkable antifungal and antibactericidal activity.

The compounds which are somewhat similar in structure to dithacyclohexadienes, i.e., Thiarubrine A and B, previously isolated from members of the Asteraceae family including Chaenactis douglasii, A. chamissonis and other species have been generically designated by the coined name Thiarubrine.

The novel Thiarubrine substances, and their closely related ho ologues which have been discovered, can be represented by the following generic formula:

-12-

wherein R- is- CH or CH ₂ R ₃ ;

R„ is:

-HC Λ

^• JO -CH~-; -CHR-J-CH--.RJ-; -HCHa-CH-RJ_; -CHR ό-CH.Ha; -CH=CH Δ_;

R 3- is H; OH; -CHO; COOH or COOR4. wherein R4. is derived from an alcohol; 15

Ha is F; Cl; Br; I;

n is 1 or 2; and

20

m is 1 or 2, provided that when R. is CH ₃ , n is 1 and m is 2, then R_ is not CH=CH _? ; and provided that when R- is CH_, n is 2 and m is 1, then is not CH=CH ₂ .

A series of five novel substances have been chemically identified, assigned the generic trivial name 2 _Q Thiarubrine and have been given letter designations as follows:

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1. Thiarubrine D: _-C ΛH-CH _? )

2. Thiarubrine E: (CH ₃ -C*C-R-(c=C) -CHOH-CH OH) ;

3. Thiarubrine F: (CH ₃ -C-C-R-(C-C) ₂ -CHC1-CH_0H) ;

4. Thiarubrine G: (CH ₃ -CsC- -(0*c) -CHOH-CH Cl) ; and

5. Thiarubrine H: (HOCH ₂ -C*C-R-(C _* C) ₂ ~CH=CH ₂ ) ;

wherein R is

4.2 METHOD FOR PREPARING THE NOVEL THIARUBRINES

The therapeutically effective thiarubrine antifungal and antibacterial agents are prepared by a method comprising steps as follows:

(a) tissues of plant(s) of the Asteraceae family that contain thiarubrines, e.g., Ambrosia chamissonis (var.), including the whole plant, plant parts, or tissue cultures thereof, are rinsed with water and are either air-

dried, frozen, or stored in various organic solvents (i.e. hexane, methanol, etc.) ;

(b) the roots and or various plant parts (i.e. roots, leaves, stems, etc.) obtained as in (a) are either lyophilized directly or the root bark is peeled and/or lyophilized, or blended or used directly; (c) the plant parts as in (a) or (b) are extracted by either percolation with various organic solvents (i.e. hexane, ethyl acetate, methanol, etc.), or by cold organic solvent extraction using various solvents (hexane, methanol, etc.) followed by filtration or centrifugation, or by supercritical fluid extraction (SFE) (i.e. carbon dioxide or ammonia as extractants) ;

(d) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, and subjected to gel filtration (e.g. on Sephadex) with water + water + miscible organic solvents as the mobile phase; or alternatively

(e) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to reversed-phase TLC, column or HPLC chromatography, using water, and/or water + water-miscible organic solvents, all with or without buffers, as the mobile phase; or alternatively,

(f) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to silica gel TLC, column or HPLC chromatography with various organic solvents (i.e. freon, low-boiling hydrocarbons, alone or in mixture with polar modifiers, e.g. diethyl ether, ethyl acetate, etc.) as the mobile phase; or alternatively

(g) the orgaxiic solvent-containing extracts _ obtained from step (c) are concentrated in vacuo, subjected to SFE (supercritical fluid extraction) or SFC (supercritical fluid chromatography) with various gases alone or in mixture with polar modifiers (i.e. water, methanol, etc.); or alternatively

(h) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, subjected to counter-current partition chromatography using anhydrous biphasic systems (i.e. hexane/acetonitrile, etc) ; or alternatively -c (i) the organic solvent-containing extracts obtained from step (c) are concentrated in vacuo, and subjected to gas chromatography;

(j) the thiarubrine-containing fractions display a red or red/brown chromophoric band and are detected by uv ₂₀ or visible λ = 200-550 nm) detectors;

(k) the thiarubrines are obtained by collecting, and concentrating the uv/vis-active fractions as in step (d) ; or alternatively

(1) the thiarubrines are obtained by collecting, 25 and concentrating the uv/vis-active fractions as in step (e) ; or alternatively

(m) the thiarubrines are obtained by collecting, and concentrating the uv/vis-active fractions as in step (f) ; or alternatively 30 (n) the thiarubrines are obtained by collecting, and concentrating the uv/vis-active fractions as in step (g) ; or alternatively

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(o) the thiarubrines are obtained by collecting, and concentrating the uv/vis-active fractions as in step (h) ; or alternatively

(p) the thiarubrines are obtained by collecting, and concentrating the uv/vis-active fractions as in step (i). 0

4.3 APPLICATIONS OF THIARUBRINES

The novel Thiarubrine compounds of the invention J5 display strong antifungal activity in the dark and strong antifungal and bactericidal activity in association with ultra-violet A light in the wave length range 320 to 400 nm, and notably 350 nm.

20 These substances are effective in killing strains of Candida albicans. Aspergillus fumigatus and Cryptococcus neoformans in the dark. They are effective in killing Escherichia coli, Staphylococcus aureus, Streptococcus faecalis, Mycobacterium sp, and Enterobacter aerogenes.

25

Thus, the Thiarubrine compounds are advantageously used for treatment of infections induced by the above mentioned fungal and bacterial agents. For example, the epoxide compound, designated Thiarubrine D, is 30 a novel complex sulphur-containing substance. Thiarubrine D can be described by the specific formula:

35

Its very high antifungal activity in extremely low concentrations indicates a usefulness in vivo, i.e. for human patients.

The chemical, Thiarubrine D, is light sensitive and breaks down under light to the corresponding thiophene. It is therefore necessary to store the chemical in the dark and for effectiveness to conduct antifungal therapy in the absence of blue and ultraviolet light.

We have demonstrated in a series of tests which are tabulated below that Thiarubrines D, E, F, G and H will inhibit the qrowth of Candida albicans, at very low levels, i.e. in the nanogram/ml range.

The following Examples are presented to illustrate the present invention and in no way intended to be a limitation thereon.

5. EXAMPLES

5.1. PREPARATION OF NOVEL THIARUBRINES

A^ chamissonis were collected from Marin County,

CA and from Tiaswwassen, British Columbia.

Freeze-dried roots (350 - 500 g) were extracted with cold MeOH, concentrated in vaccuo at 30 ° C and

redissolved in MeCN. The combined extracts were taken up in CHC1 ₃ , dried and evaporated. The residue was chromatographed on a silica gel 60 column (Merck 70 -230 mesh), eluted with n-heptane-n-hexane (1:4) gradually changed to n-hexane-EtOAc (2:3) and finally EtOAc. Extracts and fractions for HPLC analysis were taken to dryness and resuspended in MeCN. Samples (20 and 100 μl) were injected on a Varian 5000 liquid chromatograph with a MCH-10 reverse phase column (4 X 300 mm) using MeCN-H_0 (18:7) at a flow rate of 1.0 ml mm -1. A much better resolution of

Thiarubrines F and G and H was achieved with MeCN-H_0 (2:3 and 1:1) and each compound isolated by extraction of the recovered HPLC fractions with CHC1 ₃ . All procedures were performed in dim light to prevent photodegradation of the compounds.

MeCN

Thiarubrine D: UV λ_max, nm: 484, 344, 272; MS m/'z

(rel. int.): 244 (M) + (100), 214, (75), 182 (21), 170 (98) .

MeCN Thiarubrine E: UV _maχ nm: 482, 342,326(sh): MS m/z (rel. int.): 262 (M) + (100), 231, (M-CH OH) + (80), 199 (M-CH ₂ OH-S) +(15).

MeCN

Thiarubrine F: UV-vis λ nm: 484, 344, 230; MS m/z mes _ _L

(rel. int.) 279.9783 (M) + (80), 249 [M-CH OH] (30),

248 (M-S) ⁺ (50) , -217 [M-S-CH_OH] ⁺ , (48) .

MeCN

Thiarubrine G: UV-vis-λme„s nm: 484,' 342, 228,' MS m/'z

(rel. int.): 279.9780 (M) , (100), 248 (M-S) , (18) 0 244 (M-HC1) ⁺ (50), 231, [M-CH ₂ C1] ⁺ (98), 199 (M-CH_C1-S) ⁺ (30).

MeCN _C Thiarubrine H: UV-vis λ „ nm: 490, 354, 232, MS ID mes . m/z (rel. int.): 244.0009 (M) , (100), 227 (M-OH) (5), 212 (M-S) ⁺ (65), 195 (M-S-OH) ⁺ (6).

0 5.2. BIOLOGICAL ACTIVITY OF THIARUBRINES

The. following experiments demonstrate the biological activity of the Thiarubrines of the present invention.

25

30

35

TABLE 1

Comparison of Concentrations of Thiarubrines and

Fungizone Causing 50% Growth Inhibition (μg/ml)

During 24 Hours Exposure

Thiarubrines

C. albicans 0.028 p815 0.035 L1210 0. 035 WEH1-3 0.027 3T3 0.45

P815 = Mouse mastocytoma

L1210 = Mouse lymphocytic leukemia

WEH1-3 = Mouse myelomonocyte

3T3 = Mouse fibroblasts

TABLE 2

Toxicity Index of Thiarubrines and Fungizone for Different Animal Cell Lines

Thiarubrines D E Fungizone

LC JP815

LC '5,_ _Λ 0 —C. albicans 200 17 > 135

LC WEHI-3 LC 5_0_ C. albicans 83 28 51

LC ₅₀ ^T

LC,_U_ C. albicans 12 800 86 > 135

Table 2 shows how many times the concentrations needed to inhibit growth in 50% of animal cells are higher than doses required to inhibit growth in 50% of C^ albicans.

8 8 K) n NJ

01 01

TABLE 3

Comparison of Minimal Concentration Causing 100% Growth Inhibition (μg/ml) During 24 Hours Exposure

Thiarubrines Thiophenes

A D E D E Fungizone Gentamycin

0.625 0.078 0.019 5.0 >5.0 1.25

1

2.5 0.625 0.625 >5.0 >5.0 2.5

0.16 0.0024 0.0048 >5.0 >5.0 0.16 0.625 1.25 0.3125 >5.0 >5.0

This table shows that minimal concentration causing 100% growth inhibition of animal tumourcells - P815 line is about 500 times greater than the concentration needed for 100% growth inhibition of C. albicans in the case of Thiarubrine D, 65 time greater for Thiarubrine E, and about 4 times for Thiarubrine A.

According to these data, concentration needed to inhibit growth of animal cells - P815 in 50% is 200 times higher than for C. albicans in the case of Thiarubrine D, 17 times higher for Thiarubrine E and the same for Thiarubrine A.

Doses inhibiting growth in 50% were estimated by plotting concentration on a logarithmic scale and percent of growth inhibition for 24 hours exposure on a probability scale.

TABLE 5

Thiarubrine D Minimal Inhibitory

Organism

1. Staphylococcus aureus

2, Streptococcus faecalis

3. Escherichia coli

4. Pseudomonas aeruginosa

10 5. Staphylococcus aureus

6. Enterobacter aerogenes

7. Klebsiella pneumoniae

8. Staphylococcus epidermis

9. Cryptococcus neoformans 10. Candida pseudotropicalis 11. Staphylococcus aureus -

(metacylliu res.)

15 12. Pseudomonas aeruginosa 13. Pseudomonas aeruginosa 14. Pseudomonas aeruginosa 15. Zanthomonas maltophilia 16. Staphylococcus aureus

(metacylliu res.)

Acinetobacter

Acinetobacter

Enterobacter cloacoe

Enterobacter cloacoe

Aspergillus

Candida

Candida

Candida

Candida

Enterobacter sp.

Serratia marscenes

Listeria monocytogenes

Listeria monocytogenes

Enterococcus faecalis

Klebsiella sp.

Candida

Enterococcus (gentamycin res.

Aspergillus Enterococcus faecalis

Results were obtained by agar dilution method. Mueller _ ₅ Hinton agar plates (pH - 7.4) with different doses of ThD

were inoculated with 104 cfu/spot and incubated 21 hours at

34°. MIC is the lowest concentration with no growth.

Results were obtained by agar diffusion method, Disc diameter - 6 mm. -* = no zone over disc diameter.

TABLE 7

LC _5Q (24 hrs.) Data Obtained by Different Method

Doses Required to Inhibit Growth of C. albicans in 50% During 24 hrs. (ng/ml)

Organism

C. albicans P815 mastocytoma

Data on degradation of Thiarubrine D (ThD) expressed as changes of concentration required to inhibit growth of C_. albicans in 50% during 24 hrs. exposure (Lc ₅ 24 hrs.).

TABLE 8A

Degradation of ThD under Exposure to "Bench" Cool White Light

Time of Exposure LC -5,- _n 0 (24 hrs.) ng/ml

24 hrs. ¹¹ 5,000

6 hrs. " 5,000 0.5 hrs. 140 15 ins. 26

5 mins. 12

0 mins. 7

TABLE 8B

Temperature and Time Dependent Degradation of ThD in Water Solution (Saboraud Dextrose Medium

LC ₅₀ 24 Hrs. ng/ml Temperature Time of Ex osure 37°C Room

210 110 85 42

^' 33 22

20 16 7 7

TABLE 9

Influence of Exposure of ThD for Two Hrs. to Different pH (5, 6, 7, 8) of Sodium Phosphate Buffer

_EH C _5Q (ng/ml)

5 40

6 34

7 20

8 52

TABLE 10

Changes in LC Addition of Fetal

Bovine Serum (FBS) to

Saboraud ^* Dextrose Medium (SAB)

LC _5Q (ng/ml)

7 14 16

TABLE 11 Decomposition of ThD in 95% Ethanol in -20"C

Days

0 21

Comparison of Doses of ThD Required to Inhibit Different Numbers of C. albicans Cells

Cell Density LC _5Q (ng/ml)

10 ⁴ /ml 7

10 ⁵ /ml 16

10 ⁶ ml 42

TABLE 13

Legend

H=high; L=low; T=thiophene; Y=yes; y=less active; ?=not teste

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention

without departing from the spirit or scope thereof. Accordingly, the scope, of the invention is to be construed in accordance with the substance defined by the following claims.