WO1997014421A1 - Namenamicin, an enediyne antitumor antibiotic from the marine ascidian polysyncraton lithostrotum - Google Patents

Abstract

An antitumor antibiotic, namenamicin, purified from the marine ascidian Polysyncraton lithostrotum is disclosed. An antitumor composition for treating cancerous tumors comprises namenamicin and an inert carrier. A method of treating an individual for cancer comprises administering the composition comprising namenamicin and an inert carrier. A method of purifying namenamicin, a method of inhibiting the growth of a microorganism, and a method of cleaving DNA at a sequence-specific site are also described.

Description

NAMENAMICIN, AN ENEDIYNE ANTITUMOR ANTIBIOTIC FROM THE MARINE ASCIDIAN POLYSYNCRATON LITHOSTROTUM

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S.

Provisional Application No. 60/005,756, filed October 20, 1995.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. CA 36622 and S10 RR06262 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION This invention relates to a composition and method of use thereof for treating cancer. More particularly, the invention relates to an enediyne antibiotic, namenamicin, which has anticancer activity, and a method of use thereof for treating cancer in an individual in need thereof.

Marine organisms, especially invertebrates such as ascidians, sponges, soft corals, and mollusks, produce many secondary metabolites that are not found in the terrestrial world. Recent studies in the field of marine natural products have focused on detection of biomedically important compounds. This research has resulted in the discovery of compounds that have anticancer, antiviral, and antiinflammatory activity. CNS membrane-active toxins, ion channel effectors, and metabolites that interact with DNA and micro-filament processes have also been identified.

Each phylum produces a characteristic distribution of compounds. For example, in the years 1977 to 1985, 85% of the metabolites isolated from coelenterates were terpenoids; 37% and 41% of compounds isolated from sponges were terpenoids and nitrogenous metabolites, respectively; and 89% of compounds isolated from ascidians were nitrogenous compounds, such as amino acid derivatives. CM. Ireland et al . , 13 Proc. Calif. Acad. Sci. 41 (1987) .

The birth of the field of marine natural products was marked with the isolation of several modified arabino nucleosides from the sponge, Cryptoethya crypta . . Bergann & R.J. Feeney, 72 J. Am. Chem. Soc. 2809 (1950) . The first ascidian metabolite, geranyl hydroquinone, was isolated in 1974 from Aplidium sp. W. Fenical, 1974 Food-Drugs Sea 388 (1976) . This compound exhibited activity against some forms of leukemia, Rous sarcoma, and mammary carcinoma in test animals. Since then, ascidians have been targeted for the specific purpose of isolating compounds of biomedical importance. Between 1988 and 1992, about 165 new ascidian metabolites were discovered. CM. Ireland et al . , in D.G. Fautin ed., 13 Biomedical Importance of Marine Organisms 41 (1988) .

Peptides are one of the major structural classes of compounds isolated from ascidians. Ulicylamide and ulithiacyclamide were the first of a series of cyclic peptides isolated from Lissoclinum pa tella . CM.

Ireland & P.J. Scheuer, 102 J. Am. Chem. Soc. 5688 (1980) . The genus Lisεoclinum has proven to be a prolific producer of two classes of cyclic peptides, the heptapeptide lissoclinamides and the octapeptide patella ides/ulithiacyclamides. Each of these classes is characterized by the presence of thiazole and oxazoline amino acids. These peptides exhibit in vi tro toxin activity, with the presence of the oxazoline ring proving important to their potency. T. Shioiri et al . , 36 Biochem. Pharmacol. 4181 (1987) . The first metabolite from an ascidian to enter phase III clinical trials was didemnin B, a cyclic depsipeptide isolated from the Caribbean ascidian, Tridide num solidum. K.L. Rinehart et al . , 212 J. Am. Chem. Soc. 933 (1981); K.L. Rinehart et al. , 212 Science 933 (1981) . Didemnins A, B, and C were first isolated in 1981 and were proposed to contain the unique structural unit, hydroxyisovalerylpropionate (HIP) , and a new allo stereoisomer of statine. K.L. Rinehart et al., 109 J. Am. Chem. Soc. 6846 (1987) . These didemnins were found to inhibit Herpes simplex viruses I and II, Rift Valley Fever virus, Venezuelan equine encephalitis virus, and yellow fever virus. Didemnin H, A. Boulanger et al., 35 Tetrahedron Lett. 4345 (1994) , was found to interact with DNA. J.M. Pezzuto et al . , 54 J. Nat'l Prod. 1522 (1991) .

Other prominent natural products with potent biological properties from marine ascidians include bistramide A, M.P. Foster et al . , 114 J. Amer. Chem. Soc. 1110-14 (1992) , varacin, B.S. Davidson et al . , 113 J. Amer. Chem. Soc. 4709-10 (1991) , and the patellazoles, T.M. Zabriskie et al . , 110 J. Amer. Chem. Soc. 7919-20 (1988) .

Cancer is the leading cause of death in many countries. In the United States and Canada, only diseases of the heart and blood vessels kill more people. About 100 kinds of cancer attack human beings. Drug therapy or chemotherapy is an important method of treating such cancers. More than 50 drugs are used against a variety of cancers, and such drugs have proven especially effective in treating leukemia and lymphoma. Anticancer drugs are designed to destroy cancer cells with as little injury to normal cells as possible. Nevertheless, the drugs tend to injure normal cells to some degree and thus produce various undesirable side effects, ranging from nausea to high blood pressure. There is a need to develop new anticancer drugs that are effective against various kinds of cancers and that are less harmful to normal cells.

In view of the foregoing, it will be appreciated that providing a new antitumor drug and a method of use thereof for treating cancerous tumors would be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION It is an object of the present invention to provide a new anticancer drug.

It is another object of the invention to provide a method of treating cancer in a person in need thereof. It is also an object of the invention to provide a method of purifying namenamicin. It is still another object of the invention to provide a method of cleaving DNA at a sequence-specific site.

These and other objects can be achieved by providing a purified compound represented by the formula

A composition for treating cancer comprises an effective amount of a compound represented by the formula

and an inert carrier. This composition can further comprises a member selected from the group consisting of excipients, wetting agents, emulsifying agents, and buffers .

A method for purifying a compound represented by the formula

comprises the steps of:

(a) collecting specimens of Polysyncraton lithostrotum;

(b) repeatedly extracting the specimens with methanol to yield a methanol extract;

(c) partitioning the methanol extract by successive extractions with solvents of increasing polarity and then selecting solvents containing the compound by biochemical induction assay;

(d) subjecting the solvents containing the compound to silica gel chromatography with step gradient elution with solvents of increasing polarity and selection of first fractions containing the compound by biochemical induction assay; (e) subjecting the first fractions containing the compound to reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity and then selecting second fractions containing the compound by biochemical induction assay; and

(e) rechromatographing the second fractions containing the compound by reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity and then selecting fractions containing the purified compound by biochemical induction assay.

A method for treating cancer in an individual in need of such treatment comprises administering a composition comprising a compound represented by the formula

and an inert carrier. The composition can further comprise a member selected from the group consisting of excipients, wetting agents, emulsifying agents, and buffers. Preferably, the composition is administered by systemic administration. A method of cleaving DNA at a sequence-specific site comprises the step of bringing an aqueous solution containing DNA into contact with a compound represented by the formula

under conditions sufficient for such cleavage to occur. A method of inhibiting growth of a microorganism in an aqueous medium comprises mixing the aqueous medium with an effective amount of a compound represented by the formula

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS FIG. 1 shows the structure of namenamicin. FIG. 2 shows a representation of site selective cleavage of DNA due to namenamicin and calicheamicin y_^ ¹ ; the three major calicheamicin cleavage segments are identified by dashed lines, and the namenamicin cleavage sites are identified by solid arrows.

DETAILED DESCRIPTION Before the present composition and method of use thereof for treating cancerous tumors are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a, " "an, " and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "an excipient" includes reference to two or more of such excipients, reference to "an emulsifying agent" includes reference to one or more of such agents, and reference to "a wetting agent" includes reference to two or more of such wetting agents. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, such a "pharmaceutically acceptable" component is one which is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. As used herein, "effective amount" means an amount of an anticancer agent that is nontoxic but sufficient to provide the desired effect and performance against cancer cells at a reasonable benefit/risk ratio attending any medical treatment. An effective amount of namenamicin for inhibiting the growth of a microbial organism is an amount suffient to inhibit such growth to a selected extent under selected time, temperature, and growth condition.

As used herein, "administering" and similar terms mean delivering the compound or composition to the individual being treated such that the compound or composition is capable of being circulated systemically to the parts of the body where the anticancer agent can act on cancer cells. Thus, the composition is preferably administered to the individual by systemic administration, typically by subcutaneous, intramuscular, or intravenous administration, or intraperitoneal administration. Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like can be added.

Example 1 Extraction and Isolation Procedures Specimens of Polysyncraton li thostrotu (Order:

Aplousobranchia, Family: Didemnidae; identified by Dr. Franςoise Monniot, Museum d'Histoire Naturelle, Paris, France) were collected at Namenalala Island, Fiji, and kept frozen until extraction. The frozen tunicates were then freeze dried, pulverized, and repeatedly extracted with methanol. The methanol extract was concentrated and successively partitioned with a series of solvents of increasing polarity. S.M. Kupchan et al . , 38 J. Org. Chem. 178 (1973) , hereby incorporated by reference. The methanol extract was first extracted with hexanes and then with chloroform. Reagent grade solvents were distilled prior to use. An agar based Biochemical Induction Assay (BIA) , R.K. Elespuru & R.J. White, 43 Cancer Res. 2819-30 (1983) , hereby incorporated by reference, was used at each step to determine the fraction containing namenamicin.

The resulting namenamicin-containing extract was then subjected to silica gel chromatography with stepped gradient elution with solvents of increasing polarity, e.g. CHC1₃, 98:2 CHCjL /methanol, 95:5 CHC1 /methanol, 90:10 CHCl₃/methanol . The fractions containing the active molecule were identified by BIA, and these fractions were then subjected to preparative reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity, e.g. 100% H₂0, 50% methanol/H₂0, 70% methanol/H p, 80% methanol/H p, 90% methanol/H₂0, 100% methanol, 100% CHCJ. . The active fractions were again identified by BIA, pooled, and rechromatographed. The resulting active fractions contained pure namenamicin. The yield was about 1 mg per kg of frozen tissue.

Example 2 Mass Spectrometry

Namenamicin prepared according to the procedure of Example 1 was subjected to electrospray mass spectrometry on a VG Fisons Trio quadrupole mass spectrometer according to methods well known in the art. A prominent ion in the electrospray mass spectrum had a mass to charge ratio (m/z) of 991, corresponding to (MH)⁺. This result suggested a molecular formula of C₄₃H₆₂N₂0₁₄S₅.

Another ion consistently observed was at m/z 172, corresponding to a fragment generated by cleavage of the glycosidic bond between the A and C sugars (FIG. 1) . This ion was also reported for the esperamicins, which have an identical carbohydrate unit. J. Golik, in Enediyne Antibiotics as Antitumor Agents 187 (D.B. Borders & T.W. Doyle eds., Marcel Dekker, 1995) . Example 3 NMR Analysis

^XH and ¹³C experiments were conducted on a Varian Unity 500 spectrometer. Detailed analysis of data from the ²H and ¹³ C NMR spectra, a suite of 2D correlation experiments, and comparison with the calicheamicins readily allowed complete assignment of the aglycone shown in FIG. 1. NMR assignments are shown in Table 1. All assignments were completely analogous with the calicheamicins. M.D. Lee et al. , 114 J. Amer. Chem. Soc. 985-97 (1992). Assignments for carbons 3 and 6, though consistent with the calicheamicins, are opposed to those of the esperamicins. J. Golik et al. , 109 J. Am. Chem. Soc. 3462-64 (1987) . A strong HMBC correlation between H8 (6.25 ppm) and a carbon at 83.44 ppm supports the assignment of this acetylene resonance to C6.

Table 1

Atom "C M_CH (HZ) ^{a l}H[mult. , J.Hz) ] HMBC (protons) No.

1 72.13 HI 2

2 100.69 H4, H12

3 88.21 H4, H5

4 124.48 176 5.89(d,10)

5 123.43 176 5.82 (bd,10)

6 83.44 H4, H5, H8

7 100.33 H5, H8

8 67.58 161 6.25 (d,2)

9 131.04^c HI 2

10 136.45^c H12

11 192.34 H12 12 53.42 138 2.78 (d,17)

- 138 3.17 (d,17)

13 139.10^c H12

14 126.36 170 6.39(dd,10,4)

15 39.09 144 3.70(dd,14,4)

- 3.90(dd,14,10)^d

16 22.74 142 2.48 (s)

17 153.14^e

18 52.64 3.66 (bs)

Al 100.33 163 4.53 (d,8) HA2, HA5

A2 74.85^b 3.84 (dd,10,8)^d HA3

A3 74.36 145 4.08(d,10)^d HA2, HA7

A3-OH 4.14 (bs)

A4 61.03 HA6, HA7, HA9

A5 73.96 141 3.97(q,6)^d HA6

A6 15.94 126 1.41(d,6) HA5

A7 84.46 143 3.55(dd,7,7)^d HA3 ,HB1

A8 61.56 150 3.78 (dd,13,7)^d HA7

- 150 3.94 (dd,13,7)^d

A9 14.99 140 2.32 (s)

Bl 98.78 162 4.82 (dd,10,2) HA7

B2ax 37.33 130 1.73 (ddd,14,10,5)^d

B2eq - 130 2.27 (ddd, 14, 5, 2)^d

B3 64.60 151 4.13 (ddd, 5, 5,3)^d

B3-OH 2.75(bs)

B4 55.47 140 2.49(dd,ll,3)^d HB7

B5 69.88 145 3.80(dd,ll,6)^d HB1, HB4

B6 20.04 125 1.37 (d,6) HB4

B7 13.85 139 2.13 (s) HB4

Cl 97.51 177 5.71 (d,4) HA2

C2 34.24 1.48 (ddd, 13,10,4)^d

- 2.30(dd,13,5)^d C3 76.13 139 3.42 (ddd,ll,10,5)^d HCl,HC5eq, HC6

C4 57.76 135 2.67(ddd,12,ll,5)^d

C5eq 62.70 152 3.61 (dd,13,5)^d HCl

C5ax - 3.68 (dd,13,12)^d

C6 56.18 142 3.37(S)

C7 48.04 135 2.77 (multiplet)

C8 22.49 122 1.09(d,6)

C9 23.40 122 1.07 (d,6)

Obtained from a coupled HMQC (heteronuclear multiple quantum coherence) experiment, A. Bax & S.

Subramanian, 69 J. Mag. Reson. 565 (1986) ; L.

Muller, 101 J. Am. Chem. Soc. 4481 (1979) .

These signals were not observed in the carbon spectrum, but were observed as cross peaks in an

HMQC experiment .

These signals were not observed in the carbon spectrum, but were observed as cross peaks in an

HMBC (heteronuclear multiple bond correlation) experiment, M.F. Summers et al . , 108 J. Am. Chem.

Soc. 4285 (1986) .

Coupling constants obtained from a PS DQF COSY

(phase sensitive double quantum filtered correlated spectroscopy) experiment, U. Piantini et al. , 104

J. Am. Chem. Soc. 6800 (1982); M. Ranee et al. , 117

Biochem. Biophys. Res. Commun. 458 (1983) .

This signal was very broad.

The identities of the sugars and the glycosidic linkages between the sugars and the enediyne aglycone were established based on chemical shift, ¹J_CH, and ³tξ_H coupling constants and comparison with calicheamicin β^ and esperamicin A_λ . The chemical shift and ¹J_CH of carbon

Al (δ 100.33, ¹J_CH = 163 Hz) is consistent with a β glycosidic linkage. A. Liptak et al., 36 Tetrahedron

1261-68 (1980) . The upfield shift of the proton at Al

(H-Al, 4.53, d, J = 8 Hz) and the large 8 Hz coupling between H-Al and H-A2 (3.84, dd, J = 10, 8 Hz) further support a β glycosidic linkage bewteen the A ring sugar and the aglycone. H-A3 (4.08, d, J = 10 Hz) is axial based on its couplings to H-A2. The intervening quaternary carbon A4 precluded further analysis of the conformation of the A ring using scalar coupling constants. However, the very strong nuclear Overhauser effect (nOe) correlation between H-Al and H-A5 (3.97, q,

J = 6 Hz) defined a 1,3-diaxial relationship between these protons. The CH(CH₂OH)-0 linkage between A4 and Bl was defined by oxygen bearing carbon resonances at δ

84.46 and 61.56 assigned as A7 and A8, respectively, and the corresponding protons at δ 3.55 (HA7, dd, -7 = 7, 7

Hz), 3.78 (HA8, dd, J = 13, 7 Hz) , and 3.94 (HA8, dd, J = 13, 7 Hz) . The orientation and placement of this unit between the A and B sugars was indicated by heteronuclear multiple bond correlations (HMBC) from A4 to both H-A7 and -8, whereas Bl only showed correlations to H-A7. The conformation of the B ring sugar was established by analysis of proton coupling patterns and nOe interactions. The ¹J_CH (162 Hz) and the coupling of

H-Bl (4.82, dd, J^" = 10, 2 Hz) to H-B2, diastereotopic methylene protons at δ 2.27 and 1.73 indicate that H-Bl is axial and again supports a β glycosidic linkage. The relatively small couplings between the H-B2 protons and

H-B3 (4.13, ddd, J = 5, 5, 3 Hz) suggest H-B3 is equatorial. Since H-B4 (2.49, dd, J = 11, 3 Hz) and H-

B5 (3.80, dd, J^" = 11, 6 Hz) couple very strongly and H-

B5 shows a strong 1,3-diaxial nOe interaction with H-Bl, the conformation of ring B was established as shown and is identical to the corresponding sugar in the calicheamicins aside from the substituent on sulfur at B4. The NMR data for the C ring sugar were essentially identical to the E ring of calicheamicin β_x ^z, which has the same isopropyl amine substituent at the 4 position. Interestingly, the coupling pattern between H-Cl and the H₂-C2 protons along with the observation of nOe coupling between H-Cl and H-C4 suggest that this ring is flattened relative to the corresponding ring in the calicheamicin y series. The flattening of this ring is probably due to steric interactions between C4 isopropyl amine (versus an ethyl amine in the calicheamicin y series) and the A ring and warhead units. The NMR data for the B and C rings of namenamicin were also identical to the corresponding rings of esperamicin Al. J. Golik et al., 109 J. Am. Chem. Soc. 3462-64 (1987) .

These results indicate that namenamicin contains the same "enediyne warhead" as the calicheamicins, M.D-. Lee et al . , 24 Ace. Chem. Res. 235-40 (1991) and references cited therein, however, the attached carbohydrate moiety differs in replacement of the N-O sugar linkage between the A and B sugars with a C-O, an S-methyl substituent at A4 , and the absence of a benzoate ring appended to the B sugar. Enediyne Antibiotics as Antitumor Agents (D.B. Borders & T.W. Doyle eds., Marcel Dekker, 1995) .

Example 4 In Vivo Antitumor Activity

Namenamicin purified according to the procedure of Example 1 was screened for in vivo activity by injecting

CDF1 mice intraperitoneally on day 0 with 1 x IO⁶ P388 tumor cells. Five animals were randomly assigned to a group. On days 1, 5, and 9 after tumor implantation, these mice were treated intraperitoneally with either a placebo or namenamicin. Animals were checked twice daily, and the day of death of each mouse was recorded. A positive drug response is defined as a greater than 25% increase in the mean life span (%ILS) relative to the placebo control. The results of this experiment showed that namenamicin gave a 140% increase in the mean life span at a dose of 3 μg/kg. These results show that namenamicin has in vivo antitumor activity, and is, thus, considered an antitumor agent according to this test viewed by those skilled in the art as reasonably predictive thereof.

Example 5 In Vitro Cytotoxicity

A methanolic extract of Polysyncra ton li thoε tro urn containing namenamicin was subjected to an agar based Biochemical Induction Assay (BIA) , R.K. Elespuru & R.J. White, 43 Cancer Res. 2819-30 (1930) , hereby incorporated by reference. This methanolic extract showed induction of an SOS response in the BIA.

Namenamicin was also tested against a panel of 26 human tumor cell lines. Cells were cultured according to the procedure of M.C. Alley et al . , 48 Cancer Res. 589 (1988) , hereby incorporated by reference.

Screening was performed in a 96-well microtiter plate by the standard 3- [4 , 5-dimethylthiazol-2-yl] -2 , 5- phenyltetrazolium bromide; thiazolyl blue (MTT) cell inhibition assay adopted for anticancer drug screening at NIH. J. Carmichael et al . , 47 Cancer Res. 936 (1987) , hereby incorporated by reference. Positive controls were run simultaneously.

According to this test, namenamicin exhibited potent in vi tro cytotoxicity with a mean IC₅₀ (50% inhibitory concentration) of 3.5 ng/ml. Thus, namenamicin is an antitumor agent according to this test viewed by those skilled in the art as reasonably predictive thereof. Mean Bar Graph analysis of the cytotoxicity against this panel of human tumor cell lines indicated that namenamicin is a DNA cleavage agent .

Example 6 Sequence Specific DNA Cleavage

The sequence specific DNA interactions of namenamicin were mapped on a 142 base pair pBR322 restriction fragment and compared to calicheamicin y^ . G. Krishnamurthy et al., 34 Biochemistry 1001-1010 (1995) , hereby incorporated by reference.

Plasmid pBR322 DNA was linearized by digestion with restriction endonuclease Hindlll. The terminal 3' phosphates were labeld using α-³ P ATP and the Klenow fragment of DNA polymerase, according to methods well known in the art, e.g. J. Sambrook et al . , Molecular Cloning: A Laboratory Manual (2d ed. , 1989) ; T. Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) ; F. Ausubel et al . , Current Protocols in Molecular Biology (1987) , hereby incorporated by reference. The end-labeled DNA was then digested with restriction endonuclease Neil and subjected to electrophoresis on a preparative 7% polyacrylamide gel, and the 142 basepair fragment of interest (SEQ ID NO:l) was purified by electroelution. The purified DNA was then resuspended in 0.5 ml of 10 mM Tris buffer to yield an estimated DNA stock concentration of 20 nM and a radiochemical concentration of 4 x IO⁷ cpm/ml. Mapping experiments were conducted in the presence of calf thymus DNA as carrier. The labeled DNA was combined with the carrier in a buffer consisting of Tris-HCl and NaCl. Equal volumes of the DNA mixture were transferred to microcentrifuge tubes. To initiate strand scission, dithiothreitol was added to the DNA-namenamicin reaction mixture. The following were the concentrations of the reaction components at the onset of the cleavage reaction: 90:10 of 50 mM Tris buffer (pH 8.1) to ethanol, 7.7 μM of carrier DNA, 50 mM NaCl, 10 mM dithiothreitol. Concentrations of namenamicin were 1.0 or 6.0 μM, and concentrations of calicheamicin were 0.1, 0.2, or 1.0 μM . The reaction was carried out for 1 hour at 37°C DNA cleavage reactions were quenched by increasing the salt concentration to 0.3 M and by addition of three volumes of ice-cold ethanol. The DNA cleavage products were recovered by ethanol precipitation. The recovered DNA was resuspended in formamide/bromophenol blue loading buffer. Prior to electrophoresis on a 7% denaturing polyacrylamide gel, the DNA cleavage fragments were denatured by heating at 90°C for 2 minutes and then were quickly cooled in an ice bath. After electrophoresis, the gel was dried and autoradiographed using preflashed Kodak X-OMAT AR film. FIG. 2 shows a representation of the results of this experiment . Namenamicin produced fewer high specificity cleavage sites than calichaemicin. At comparable concentrations, namenamicin cleaved DNA less efficiently than calicheamicin. There were some similarities in the sequence specific recognition pattens between the two compounds and several distinct differences. For example, TCCT, the primary recognition site for calicheamicin was cleaved by namenamicin, but with diminished cleavage intensity. The primary recognition site for namenamicin in this restriction fragment was a TTT segment, which overlaps the ATCT recognition site of calicheamicin. Both calicheamicin and namenamicin cleave within this region. Interestingly, TTGT, a strong cleavage site for calicheamicin, was not cleaved by namenamicin.

The observations that namenamicin produced fewer high affinity cleavage sites and a slightly altered recognition pattern may be due to its truncated structure compared to calicheamicin y_^z . First, namenamicin lacks the rhamnose sugar (D ring) and the thiobenzoate moiety of calicheamicin y^ . The second difference is the substitution of a C-0 for the N-O glycosidic linkage between the A and B sugars. Thirdly, the A sugar bears an S-methyl group in the 4 position. It is widely recognized that both the thiobenzoate and D sugar contribute to the overall strength of site specific interactions of the calicheamicins.

The cleavage pattern due to namenamicin also bears some similarity to the cleavage pattern reported for esperamicin C M. Uesugi & Y. Sugiura, 32 Biochemistry 4622-27 (1993) . For example, both namenamicin and esperamicin recognize and cleave TCCT residues as well as a run of pyrimidines (TTT) . The lowered cleavage efficiency and the altered selectivity may be attributed to several structural features of the carbohydrate moiety, namely, absence of the rhamnose sugar and thiobenzoate ring, presence of the S-methyl group in the A sugar, and more importantly, the change in the glycosidic linkage between the A and B ring sugars.

Example 7 Antimicrobial Activity

Antimicrobial activity of namenamicin, purified according to the procedures of Example 1, was tested by a broth dilution method against a panel of microorganisms. The broth was inoculated with 1-5 x IO⁵ cfu/ml, except for fungi, which were inoculated at a 10- fold lower concentration. Incubation of bacteria was at 37°C for 36 hours, and incubation of fungi was at 28°C for 36 hours. Namenamicin or control antibiotics, calicheamicin or penicillin G, were added in various concentrations. The results of this experiment are shown in Table 2.

Table 2

Antimicrobial Activity (MIC, μg/ml)

Organism

Namenamicin Calicheamicin Penicillin G

Bacillus subtilis 0.03 0.00005 0.25

Staphylococcus aureus 0.001 0.000001 0.015

Enterococcus faecium 0.03 0.00012 128

Escherichia coli 0.12 0.12 32

Klebsiella pneumonia 0.06 0.25 128

Candida albicans 0.25 0.03 >128

Ustilago aydis 0.004 0.001 >128

Saccharomyces cerevisiae 0.06 0.008 >128

Neurospora crassa 0.25 0.06 >128

These results show that namenamicin also exhibits potent antimicrobial activity.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: CHRIS M. IRELAND AND WILLIAM M. MAISE

(ii) TITLE OF INVENTION: NAMENAMICIN, AN ENEDIYNE

ANTITUMOR ANTIBIOTIC FROM

THE MARINE ASCIDIAN P O L Y S Y N C R A T O N

LITHOSTROTUM

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Thorpe, North & Western, L.L.P.

(B) STREET: 9035 South 700 East, Suite 200

(C) CITY: Sandy

(D) STATE: Utah (E) COUNTRY: USA

(F) ZIP: 84070

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage

(B) COMPUTER: AST Ascentia 9OON

(C) OPERATING SYSTEM: DOS 6.22

(D) SOFTWARE: Word Perfect 6.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: U.S. Serial No. 60/005,756

(B) FILING DATE: 20-OCT-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Alan J. Howarth

(B) REGISTRATION NUMBER: 36,553

(C) REFERENCE/DOCKET NUMBER: T3265.PCT (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (801)566-6633

(B) TELEFAX: (801)566-0750

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 142 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : AGCTTTAATG CGGTAGTTTA TCACAGTTAA ATTGCTAACG CAGTCAGGCA CCGTGTATGA 60 AATCTAACAA TGCGCTCATC GTCATCCTCG GCACCGTCAC CCTGGATGCT GTAGGCATAG 120 GCTTGGTTAT GCCGGTACTG CC 142

Claims

CLAIMS We claim:

1. A purified compound represented by the formula

2. A composition for treating cancer comprising an effective amount of a compound represented by the formula

and an inert carrier

3. The composition of claim 2 further comprising a member selected from the group consisting of excipients, wetting agents, emulsifying agents, and buffers.

4. A method for purifying a compound represented by the formula

comprising the steps of: (a) collecting specimens of Polysyncraton lithostroturn;

(b) repeatedly extracting said specimens with methanol to yield a methanol extract;

(c) partitioning the methanol extract by successive extractions with solvents of increasing polarity and then selecting the solvents containing the compound by biochemical induction assay;

(d) subjecting the solvents containing the compound to silica gel chromatography with step gradient elution with solvents of increasing polarity and selection of first fractions containing the compound by biochemical induction assay;

(e) subjecting the first fractions containing the compound to reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity and then selecting second fractions containing the compound by biochemical induction assay; and

(f) rechromatographing the second fractions containing the compound by reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity and then selecting fractions containing the purified compound by biochemical induction assay.

5. A compound purified from Polysyncraton lithostrotum by a process comprising the steps of:

(a) collecting specimens of Polysyncraton lithostrotum;

(b) repeatedly extracting said specimens with methanol to yield a crude homogenate;

(c) partitioning the methanol extract by successive extractions with solvents of increasing polarity and then selecting solvents containing the compound by biochemical induction assay; (d) subjecting the solvents containing the compound to silica gel chromatography with step gradient elution with solvents of increasing polarity and then selecting first fractions containing the compound by biochemical induction assay; (e) subjecting the first fractions containing the compound to reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity and then selecting second fractions containing the compound by biochemical induction assay; and

(e) rechromatographing the second fractions containing the compound by reverse phase chromatography with stepped gradient elution with solvents of decreasing polarity and then selecting fractions containing the purified compound by biochemical induction assay.

6. A method for treating cancer in an individual in need of such treatment comprising administering a composition comprising a compound represented by the formula

and an inert carrier.

7. The method of claim 6 wherein said compound further comprises a member selected from the group consisting of excipients, wetting agents, emulsifying agents, and buffers.

8. The method of claim 6 wherein said composition is administered by systemic administration.

9. A method of cleaving DNA at a sequence- specific site comprising the step of bringing an aqueous solution containing DNA into contact with a compound represented by the formula

10. A method of inhibiting growth of a microorganism in an aqueous medium comprising mixing the aqueous medium with an effective amount of a compound represented by the formula