Patent Publication Number: US-6713480-B2

Title: Phenylahistin and the phenylahistin analogs, a new class of anti-tumor compounds

Description:
PRIORITY CLAIM 
     This application is a continuation of, and claims priority from, U.S. application Ser. No. 09/440,316, filed Nov. 12, 1999, U.S. Pat. No. 6,358,957, and through which application claims priority from U.S. Provisional Application Serial No. 60/108,211, PHENYLAHISTIN AS AN ANTI-TUMOR COMPOUND, filed Nov. 12, 1998, by Fukumoto et al., and also claimed priority from U.S. Provisional Application Ser. No. 60/108,736, PHENYLAHISTIN AS AN ANTI-TUMOR COMPOUND, filed Nov. 17, 1998, by Fukumoto et al. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to the fields of chemistry and medicine. More specifically, the present invention relates to compounds and procedures useful in the treatment of cancer. 
     2. Description of the Related Art 
     It is thought that a single, universal cellular mechanism controls the regulation of the eukaryotic cell cycle process. See, e.g., Hartwell, L. H. et al., Science (1989), 246: 629-34. It is also known that when an abnormality arises in the control mechanism of the cell cycle, cancer or an immune disorder may occur. Accordingly, as is also known, antitumor agents and immune suppressors may be among the substances that regular the cell cycle. Thus, new eukaryotic cell cycle inhibitors are needed as antitumor and immune-enchancing compounds, and should be useful in the treatment of human cancer as chemotherapeutic, anti-tumor agents. See, e.g., Roberge, M. et al., Cancer Res. (1994), 54, 6115-21. 
     Recently, it has been reported that tryprostatins A and B (which are diketopiperazines consisting of proline and isoprenylated tryptophan residues), and five other structurally-related diketopiperazines, inhibited cell cycle progression in the M phase, see Cui, C. et al., J. Antibiotics (1996), 49, 527-33; Cui, C. et al. J. Antibiotics (1996), 49, 534-40, and that these compounds also affect the microtubule assembly, see Usui, T. et al. Biochem J. (1998) 333, 543-48; Kondon, M. et al. J. Antibiotics (1998) 51, 801-04. Furthermore, natural and synthetic compounds have been reported to inhibit mitosis, thus inhibit the eukaryotic cell cycle, by binding to the colchicine binding-site (CLC-site) on tubulin; which is a macromolecule that consists of two 50 kDa subunits (∀- and ∃-tubulin) and is the major constituent of microtubules. See, e.g., Iwasaki, S., Med. Res. Rev. (1993) 13, 183-198; Hamel, E. Med. Res. Rev. (1996) 16, 207-31; Weisenberg, R. C. et al., Biochemistry (1969) 7, 4466-79. Microtubules are thought to be involved in several essential cell functions, such as axonal transport, cell motility and determination of cell morphology. Therefore, inhibitors of microtubule function may have broad biological activity, and be applicable to medicinal and agrochemical purposes. It is also possible that colchicine (CLC)-site ligands such as CLC, steganacin, see Kupchan, S. M. et al., J. Am. Chem. Soc. (1973) 95, 1335-36, podophyllotoxin, see Sackett, D. L., Pharmacol. Ther. (1993) 59, 163-228, and combretastatins, see Pettit, G. R. et al., J. Med. Chem. (1995) 38, 166-67, may prove to be valuable as eukaryotic cell cycle inhibitors and, thus, may be useful as chemotherapeutic agents. 
     Although diketopiperazine-type metabolites have been isolated from various fungi as mycotoxins, see Horak R. M. et al., J.C.S. Chem. Comm. (1981) 1265-67; Ali M. et al., Toxicology Letters, (1989) 48, 235-41, or as secondary metabolites, see Smedsgaard J. et al., J. Microbiol. Meth. (1996) 25, 5-17, little is known about the specific structure of the diketopiperazine-type metabolites and their antitumor activity, particularly in vivo. Furthermore, even though known antitumor substances isolated from microorganism metabolites (including anthracyclins and mitomycins that exhibit antitumor activity by binding to DNA) have been used as antitumor agents, see  Microorganic Pharmaceutical Chemistry,  revised 2nd edition, edited by Yoshio Ueno &amp; Satoshi Ohmura, Nankohdo Publishing Co., (1986)), and even though anti-tumor substances having non-DNA binding operating mechanism have been isolated from microorganism metabolites, see Minoru Yoshida, M.  Protein Nucleic Acid Enzymes  (1993) 38, 1753; and Iwasaki, N., Chemistry and Living Organisms, (1994) 32, No. 3, 153, there is a particular need for new microorganism metabolite-derived compounds having animal cell-specific proliferation-inhibiting activity and high antitumor activity and selectivity. There is therefore a related need for substantially purified, and structurally and biologically characterized fungal diketopiperazine-type metabolites and fungal diketopiperazine-type metabolite-derivatives. 
     SUMMARY OF THE INVENTION 
     A compound, and any pharmaceutically acceptable salt or pro-drug ester thereof, suitable for use as an anti-tumor agent having the following generic structure:                    
     wherein: 
     R 1 , R 2 , R 5 , R 7 , and R 8  are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C 1 -C 24  alkyl, unsaturated C 1 -C 24  alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl, and substituted phenyl groups, and 
     R 3 , R 4 , and R 6  are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C 1 -C 12  alkyl, unsaturated C 1 -C 12  alkenyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, 
     X 1  and X 2  are separately selected from the group consisting of an oxygen atom, and a sulfur atom, and 
     the dashed bond represents a bond selected from the group consisting of a carbon-carbon single bond and a carbon-carbon double bond. 
     In a preferred embodiment, the invention comprises, in substantially purified form, the compound herein named alternatively “(−)-phenylahistin,” “(−)-NSCL-96F037,” or “(−)-PLH,” which is a diketopiperazine composed of L-phenylalanine and isoprenylated dehydrohistidine. This compound has the following stereo-specific chemical structure:                    
     The invention also comprises a method of treating cancer comprising administering an effective tumor-growth-inhibiting amount of the compound, and any pharmaceutically acceptable salt or pro-drug ester of generic structure (I), and preferably, phenylahistin, and more preferably, (−)-phenylahistin, and pharmaceutically acceptable salt and pro-drug esters thereof. In preferred embodiments of the compound, salt, or pro-drug ester of the present invention, R 3  and R 4  are hydrogen, and each are involved in hydrogen bonds, and/or the dashed bond is a double bond. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The accompanying drawings, which are incorporated in and form part of the specification, merely illustrate embodiments of the present invention. Together with the remainder of the specification, they are meant to serve to explain preferred modes of making certain compounds of the invention to those of skilled in the art. In the drawings: 
     FIG. 1 illustrates Reaction Scheme 1, which shows the preparation of certain reduced derivatives of (−)- and (+)-phenylahistin from the enantiomers of phenylahistin. 
     FIG. 2 illustrates Reaction Scheme 2, which shows a synthetic method for producing a compound of the invention, PLH-Cl, from H 2 N-Phe-Gly-OMe. (The asterisk indicates that racemation occurred; (−):(+)=79:21.). 
     FIG. 3 illustrates Reaction Scheme 3, which shows the preparation of two reduced derivatives of (−)-phenylahistin, compounds identified as (−)-12 and (−)-13, which are respectively, identical to compounds 3 and 5 of FIG.  1 . In FIG. 3, (a) refers to H 2 /10% Pd-C, MeOH, room temperature, reaction time of 2 hours, and (b) refers to H 2 /10% Pd-C, MeOH, room temperature, reaction time of 24 hours. 
     FIG. 4 illustrates Reaction Scheme 4, which shows the preparation of two N-methylated derivatives of (−)-phenylahistin. In FIG. 4, (a) refers to Mel, NaH, DMF, −30° C., reaction time of 2 hours, (b) refers to MeI, NaH, DMF, room temperature, reaction time of 2 hours; and (c) refers to chiral HPLC separation as described herein. 
     FIG. 5 illustrates Reaction Scheme 5, which shows the preparation of two derivatives of (−)-phenylahistin, compounds identified as 19 and 20, which is the (−)-enantiomer of compound 9 of FIG.  2 . In FIG. 5, (a) refers to MeOH at reflux for 14 hours, (b) refers to AcO 2 , AcONa, 80° C., reaction time of 14 hours, and (c-e) refers to reaction in a mixture of compound 18, LDA, HMPA, DMF, at −60° C. for 30 minutes, followed by cooling to room temperature and placing in a solution of Tf 2 O, pyridine for 10 minutes, followed by overnight reaction in NH 4 OH at room temperature. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Numerous references are cited herein. These references are to be considered incorporated by reference into this specification. 
     Objects of this invention include (1) providing a class of new compounds, including pharmaceutically acceptable salts of the class of compounds, that exhibit animal cell-specific proliferation inhibiting, tumor-growth inhibiting activity, and/or cell-cycle inhibiting activity, and (2) providing a class of fungi for producing, and (3) providing a method for producing said class of compounds, as well as a class of pharmaceutically acceptable cell cycle inhibitors and antitumor agents comprising said compounds and/or their derivatives as active ingredients. It is also an object of this invention to provide a method of treating cancer, particularly human cancer, comprising the step of administering an effective tumor-growth inhibiting amount of a member of a class of new anti-tumor compounds. In the preferred embodiment of the present invention, but not necessarily in all embodiments of the present invention, these objectives are simultaneously met. 
     The invention provides the compound represented by Formula (I):                    
     wherein: 
     R 1 , R 2 , R 5 , R 7 , and R 8  are each separately selected from the group hydrogen atom, a halogen atom, saturated C 1 -C 24  alkyl, unsaturated C 1 -C 24  alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, substituted nitro, phenyl, and substituted phenyl groups, all having, where applicable, up to 24 carbon atoms, 
     R 3 , R 4 , and R 6  are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C 1 -C 12  alkyl, unsaturated C 1 -C 12  alkenyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino substituted amino, nitro, and substituted nitro groups, all having, where applicable, up to 12 carbon atoms, 
     X 1  and X 2  are separately selected from the group consisting of an oxygen atom, and a sulfur atom, and the dashed bond represents either a carbon-carbon single bond or a carbon-carbon double bond, of any tertiary conformation, in a particular embodiment of the invention. 
     The invention also provides pharmaceutically acceptable salts and pro-drug esters of the compound of Formula (I). 
     The term “pro-drug ester,” especially when referring to a pro-drug ester of the compound of Formula (I), refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood. The term “pro-drug ester” refers to derivatives of the compound of the present invention formed by the addition of any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of pro-drug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other such groups known in the art, including a (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in “Pro-drugs as Novel Delivery Systems”, Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975); and “Bioreversible Carriers in Drug Design: Theory and Application”, edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for compounds containing carboxyl groups). 
     The term “pharmaceutically acceptable salt,” especially when referring to a pharmaceutically acceptable salt of the compound of Formula (I), refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound. Preferred examples of pharmaceutically acceptable salt are the alkali metal salts (sodium or potassium), the alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from pharmaceutically acceptable organic amines, for example C 1 -C 7  alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane. With respect to compounds of the invention that are basic amines, the preferred examples of pharmaceutically acceptable salts are acid addition salts of pharmaceutically acceptable inorganic or organic acids, for example, hydrohalic, sulfuric, phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluensulfonic or naphthalenesulfonic acid. 
     Preferred pharmaceutical compositions of the present invention include pharmaceutically acceptable salts and pro-drug esters of the compound of Formula (I). Accordingly, if the manufacture of pharmaceutical formulations involves intimate mixing of the pharmaceutical excipients and the active ingredient in its salt form, then it is preferred to use pharmaceutical excipients which are non-basic, that is, either acidic or neutral excipients. 
     In preferred embodiments of the compounds of the present invention, a relatively rigid, planar pseudo three-ring structure is formed. To stable such a relatively rigid, planar pseudo three-ring structure, R 3  and R 4  are hydrogen, each involved in hydrogen bonds. Furthermore, such a relatively rigid, planar pseudo three-ring structure is stabilized wherein the dashed bond is a double bond. 
     The term “halogen atom” means any one of the radio-stable atoms of column 17 of the Periodic Table of the Elements, i.e., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred. 
     The term “alkyl” means any unbranched or branched, substituted or unsubstituted, saturated hydrocarbon, with C 1 -C 6  unbranched, saturated, unsubstituted hydrocarbons being preferred, with methyl, ethyl, iosbutyl, and tert-butyl being most preferred. Among the substituted, saturated hydrocarbons, C 1 -C 6  mono- and di- and per-halogen substituted saturated hydrocarbons and amino-substituted hydrocarbons are preferred, with perfluromethyl, perchloromethyl, perfluoro-tert-butyl, and perchloro-tert-butyl being the most preferred. The term “cycloalkyl” refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring. 
     The term “alkenyl” means any unbranched or branched, substituted or unsubstituted, unsaturated hydrocarbon including polyunsaturated hydrocarbons, with C 1 -C 6  unbranched, mono-unsaturated and di-unsaturated, unsubstituted hydrocarbons being preferred, and mono-unsaturated, di-halogen substituted hydrocarbons being most preferred. In the R 1  and R 8  positions, of the compound of structure (I) a z-isoprenyl moiety is particularly preferred. The term “cycloalkenyl” refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring. 
     The terms “aryl,” “substituted aryl,” “heteroaryl,” and “substituted heteroaryl” refer to aromatic hydrocarbon rings, preferably having five, six, or seven atoms, and most preferably having six atoms comprising the ring. “Heteroaryl” and “substituted heteroaryl,” refer to aromatic hydrocarbon rings in which at least one heteroatom, e.g., oxygen, sulfur, or nitrogen atom, is in the ring along with at least one carbon atom. 
     The term “alkoxy” refers to any unbranched, or branched, substituted or unsubstituted, saturated or unsaturated ether, with C 1 -C 6  unbranched, saturated, unsubstituted ethers being preferred, with dimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl ethers being most preferred. The term “cycloalkoxy” refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring. 
     The terms “purified,” “substantially purified,” and “isolated” as used herein refer to the compound of the invention being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the compound of the invention comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample. 
     The terms “anti-tumor” and “tumor-growth-inhibiting,” when modifying the term “compound,” and the terms “inhibiting” and “reducing”, when modifying the terms “compound” and/or the term “tumor,” mean that the presence of the subject compound is correlated with at least the slowing of the rate of growth of the tumor. More preferably, the terms “anti-tumor,” “tumor-growth-inhibiting,” “inhibiting,” and “reducing” refer to a correlation between the presence of the subject compound and at least the temporary cessation of tumor growth. The terms “anti-tumor,” “tumor-growth-inhibiting,” “inhibiting,” and “reducing” also refer to, particularly in the most preferred embodiment of the invention, a correlation between the presence of the subject compound and at least the temporary reduction in the mass of the tumor. 
     The invention also provides fungi of the genus Aspergillus that are capable of producing the anti-tumor compound of the invention, and specifically, fungi that are capable of producing the anti-tumor compound of the invention wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8  are each a hydrogen atom, and X 1  and X 2  are each separately an oxygen atom. Preferably, the fungi is an  Aspergillus ustus  capable of producing the antitumor compound of the invention in an amount of not less than 2 mg, preferably not less than 8 mg, per liter of the production medium as measured, by high performance liquid chromatography using the compound represented by Formula (I) as a standard with respect to what is obtained by placing, in 18 mm-diameter, 180 mm-length test tubes, 5 ml of production medium (production medium: glucose 5 g/l, glycerin 20 ml/l, cotton seed lees 20 g/l, yeast extract 2 g/l, sodium chloride 2.5 g/l, calcium carbonate 4 g/l (pH6.5)), and then inoculating each test tube with 50 μl of a fungus suspension prepared by suspending conidia of the fungal strain in sterilized water, incubating the culture broth for 5 days at 27° C. under reciprocal shaking (260 rpm), adding 10 ml of acetone to the culture broth in each test tube, effecting extraction for 1 day at room temperature, removing impurities by filtration, vacuum-concentrating the filtrate to distill off the acetone, making three additions of 5 ml of ethyl acetate to the remaining water layer to effect extraction, effecting vacuum-concentration and drying to obtain a solid, and dissolving the solid in methanol. The invention also provides a method of producing the anti-tumor compound of Formula (I) comprising the steps of incubating in a culture medium a microorganism belonging to the genus Aspergillus that is capable of producing the antitumor compound represented of Formula (I) and collecting the compound from the culture. The invention further provides a cell cycle inhibitor and an antitumor agent containing the antitumor substance of Formula (I) as an active ingredient, in combination with a pharmaceutically acceptable carrier or excipient. 
     Most specifically, a preferred embodiment of the invention provides, phenylahistin, which exhibits the following physical and chemical characteristics: 
     (i) molecular weight: 350 (FABMS M/Z 351 (M+H)), 
     (ii) molecular formula: C 20 H 22 N 4 O 2 , 
     (iii) infrared absorption spectrum (IR V max (KBr)(cm −1 )): 3440, 3240, 1670, 1640, 1140; 
     (iv)  1 H-nuclear magnetic resonance spectrum (500 MHz, measured in CDCl 3 , chemical shift value of CHCl 3  set to 7.24 ppm as internal standard) having intensities at (δ (ppm)): 1.47(6H, s), 2.94 (1H, dd, J=14, 10 Hz), 3.45(1H, dd, J=14, 4 Hz), 4.33(1H, brd, J=10 Hz), 5.10(1H, d, J=17 Hz), 5.14(1H, d, J-11 Hz), 5.88(1H, br), 6.00(1H, dd, J=17, 11 Hz), 6.86(1H, s), 7.21-7.26(3H, m), 7.31(2H, t, J=8 Hz), 7.53(1H,s), 9.61(1H, br); 12.08(1H,br); 
     (v)  13 C-nuclear magnetic resonance spectrum (400 MHz, measured in CDCl 3 , chemical shift value of CDCl 3  set to 77.10 ppm as internal standard) having intensities at (δ (ppm)): 28.07(CH 3 ), 28.07(CH 3 ), 37.69(C), 41.33(CH 2 ), 57.24(CH), 105.67(CH), 113.46(CH 2 ), 123.77(CH), 127.56(CH), 129.18(CH), 129.18(CH), 129.62(CH), 129.62(CH), 132.29(C), 132.64(C), 135.55(C), 136.88(C), 144.75(CH), 160.04(C), 164.85(C); 
     (vi)  15 N-nuclear magnetic resonance spectrum (600 MHz, measured in CDCl 3 , chemical shift value of ammonia set to 0 ppm as internal standard) having intensities at (δ (ppm)): 112, 134, 161, 253; 
     (vii) maximum absorption value of ultraviolet spectrum in methanol at 230 nm, and under neutral condition at 323 nm; 
     (viii) excited under a neutral condition in methanol by 320-340 nm ultraviolet light, having a maximum value at 395-400 nm and emitting fluorescence having 350-550 nm wavelength width; 
     (ix) soluble in ethyl acetate, chloroform, methanol, and pyridine, but only slightly soluble in water, benzene, and toluene; 
     (x) negative in ninhydrin reaction, positive in color reaction with nitrous acid (orange); and 
     (xi) substance color: white. 
     According to the examples provided herein, phenylahistin has been isolated from  Aspergillus ustus  NSC-F038 and NSC-F037, two new strains of fungus; this phenylahistin exhibits cytotoxic and cell cycle inhibitory activities. Both of these strains of fungi have been deposited with the National Institute of Bioscience and Human-Technology, Japan, Agency of Industrial Science and Technology, Ministry of International Trade and Industry. These strains exhibit the following mycological characteristics: 
     (1) Growth morphology in various culture media: Growth on Czapek&#39;s yeast extract agar medium (25° C.) is rapid, reaching 45-46 mm in 7 days. Growth on Czapek&#39;s yeast extract agar medium (37° C.) is somewhat slower, reaching 39-41 mm in 7 days. On malt extract agar medium, colony surface is gray and colony rear surface grayish-green. 
     (2) Morphological properties. 
     Morphological properties on Czapek&#39;s agar medium are indicated. 
     Condial heads: Radiate 
     Conidiophore: Smooth surface, brown with length of 100-350:m, 4-7:m diameter 
     Vesicles: Spherical-flask-shaped, 11-15:m diameter, upper 2/3-1/2 forms metulae 
     Metulae: covering upper half to two-thirds of the vesicles, 5-7×4-7 μm 
     Phialides: amplliform, 5-8×3-4:m 
     Conidium: Brown, sperical, rough wall, 3×5:m 
     From these mycological properties,  Aspergillus ustus  NSC-F037 and  Aspergillus ustus  NSC-F038 were found, in accordance with the text Aspergillus, K. B. Raper and D. I. Fennel, Williams and Wilkins (1965), to belong to subphylum Fungi Imperfecti, order monilliales, genus Aspergillus, species  ustus.  The strains were therefore termed  Aspergillus ustus  NSC-F037 and  Aspergillus ustus  NSC-F038. It was concluded that  Aspergillus ustus  NSC-037 and  Aspergillus ustus  NSC-038 were both novel fungal strains, and they were respectively assigned the acquisition numbers FERM P-15829 and FERM P-15930. 
     The compound of Formula (I), the compound of the invention, can be produced by ordinary methods, from a culture medium and via extracting the compound from a culture of either  Aspergillus ustus  NSC-037 or  Aspergillus ustus  NSC-038 in the following manner. Although the culture can be either liquid or solid, industrially advantageous culture can be achieved by inoculating a liquid medium with a fungus suspension of the microorganism and incubating the medium under aeration-mixing. Although the culture medium nutrients are not particularly limited, carbon sources, nitrogen sources and other culture medium ingredients ordinarily used to culture microorganisms may be included and are preferred. Usable carbon sources include starch, glycerin, glucose, sucrose, galactose, and the like. Usable nitrogen sources included peptone, soy bean powder, meat extract, corn-steeped liquor, cotton seed lees, ammonium salt, nitrates, and other organic and inorganic nitrogen compounds. In addition, inorganic salts and trace nutrients may be added as will be deemed appropriate by those of skill in the art. The culture temperature, culture time period, and other culture conditions are preferably chosen as conditions that are appropriate for growth of the selected fungus, and also to maximize production of the compound represented by Formula (1). For instance, the pH of the culture medium is preferably between approximately 4 and approximately 9, and more preferably between 5 and 8, and the culture temperature is preferably between 15 and 35° C., and more preferably between 23 and 28° C. The culture period is preferably between 48 and 192 hours, and more preferably between 72 and 192 hours. However, the culture medium composition, culture medium pH, culture medium temperature, culture period and other culture conditions should naturally be appropriately adjusted according to the type of fungus used, the external conditions, and the like so as to obtain the desired results of producing the compound of the present invention. The compound represented by Formula (1) can be collected from such a culture by appropriate use of means ordinarily used for collecting metabolites. For example, any means utilizing the difference in affinity for and organic solvent between the compound represented by Formula (1) and other substances contained in the culture, means utilizing difference in solubility and means utilizing difference in adsorptive affinity for various resins can be used independently, in appropriate combination, or repeatedly. Specifically, ion-exchange chromatography, chromatography using a nonionic adsorptive resin, gel-filter chromatography, chromatography using an adsorbent such as activated carbon, alumina or silica gel, high-speed liquid chromatography, various other types of liquid chromatography, crystallization, vacuum concentration, freeze-drying and other such means can be used independently, in appropriate combination, or repeatedly. 
     When used as a cell cycle inhibitor or tumor-growth-inhibiting compound, the compound of Formula (I) can be administered by either oral or a non-oral pathways. When administered orally, it can be administered in capsule, tablet, granule, spray, syrup, or other such form. When administered non-orally, it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like, when administered via injection, subcutaneously, intreperitoneally, intravenously, intramuscularly, or the like. Similarly, it may be administered topically, rectally, or vaginally, as deemed appropriate by those of skill in the art for bringing the compound of the invention into optimal contact with a tumor, thus inhibiting the growth of the tumor. Local administration at the site of the tumor is also contemplated, either before or after tumor resection, as are controlled release formulations, depot formulations, and infusion pump delivery. 
     To formulate the compound of Formula (I) as a cell cycle inhibitor or tumor-growth-inhibiting compound, known surface active agents, excipients, smoothing agents, suspension agents and pharmaceutically acceptable film-forming substances and coating assistants, and the like may be used. Preferably alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methyiacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents. In addition to the foregoing preferred ingredients, sweeteners, fragrances, colorants, preservatives and the like may be added to the administered formulation of the compound of the invention, particularly when the compound is to be administered orally. 
     The cell cycle inhibitor and antitumor agent of the invention may be orally or non-orally administered to a human patient in the amount of about 0.001 mg/kg/day to about 10,000 mg/kg/day of the active ingredient, and more preferably about 0.1 mg/kg/day to about 100 mg/kg/day of the active ingredient at, preferably, one time per day or, less preferably, over two to about ten times per day. Alternatively and also preferably, the compound of the invention may preferably be administered in the stated amounts continuously by, for example, an intravenous drip. Thus, for the example of a patient weighing 70 kilograms, the preferred daily dose of the active anti-tumor ingredient would be about 0.07 mg/day to about 700 grams/day, and more preferable, 7 mg/day to about 7 grams/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it may be necessary to administer the anti-tumor compound of the invention in amounts that excess, or even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly advanced or lethal tumors. 
     In the case of using the cell cycle inhibitor of the invention as a biochemical test reagent, the compound of the invention inhibits the progression of the cell cycle when it is dissolved in an organic solvent or hydrous organic solvent and it is directly applied to any of various cultured cell systems. Usable organic solvents include, for example, methanol, methylsulfoxide, and the like. The formulation can, for example, be a powder, granular or other solid inhibitor, or a liquid inhibitor prepared using an organic solvent or a hydrous organic solvent. While a preferred concentration of the compound of the invention for use as a cell cycle inhibitor is generally in the range of about 1 to about 100 μg/ml, the most appropriate use amount varies depending on the type of cultured cell system and the purpose of use, as will be appreciated by persons of ordinary skill in the art. Also, in certain applications it may be necessary or preferred to persons of ordinary skill in the art to use an amount outside the foregoing range. 
     The following examples are meant to describe the preferred modes of making and using the invention, i.e., of isolating, preparing, characterizing, and using certain preferred embodiments of the invention. Variations in the details of the particular methods employed and in the precise chemical compositions obtained will undoubtedly be appreciated by those of skill in the art. 
     EXAMPLE 1 
     Production, Isolation and Purification of Racemic Phenylahistin (PLH) 
     During the course of screening for new cell cycle inhibitors, see Roberge, M.; Tudan, C; Hung, S. M. F.; Harder, K. W.; Jirik, F. R.; Anderson, H.  Cancer Res.  1994, 54, 6115, a novel compound NSCL-96F037 was found in the culture broth of  Aspergillus ustus  NSC-F038. see Fukumoto; K.; Asari, T.; Harada, T. Japanese Patent P409188749, Sep. 4, 1996 (Japanese).  A. ustus  NSC-F038 and NSC-F037 fungal strains were deposited with the National Institute of Bioscience and Human-Technology, Japan, Agency of Industrial Science and Technology, Ministry of International Trade and Industry,  A. ustus  NSC-F037 was designated FERM P-15829 and  A. ustus  NSC-F038 was designated FERM P-15830. The structure and biological activity of the compound initially designated NSCL-96F037 and of several of its derivatives were determined and are herein described. The compound initially designated NSCL-96F037 is herein also termed “phenylahistin” (PLH), and has the following structure:                    
     A. Production, Isolation and Purification of PLH from  Aspergillus ustus  NSC-F037 
     In each of 18 mm-diameter, 180 mm-length test tubes, 5 ml of sterilized production medium (production medium: glucose 5 g/l, glycerin 20 ml/l, cotton seed lees 20 g/l, yeast extract 2 g/l, sodium chloride 2.5 g/l, calcium carbonate 4 g/l(pH6.5)) was placed. The number of so-prepared test tubes was 1,700 (total culture medium: 8.5 liter). Each test tube was inoculated with 50 μl of  Aspergillus ustus  NSC-F037 fungus suspension prepared by suspending conidia of the strain in sterilized water and the culture broth was incubated for 5 days at 27° C. under reciprocal shaking (260 rpm). The culture broth in each test tube was added with 10 ml. of acetone and was extracted for one (1) day at room temperature. Impurities were then removed by filtration and the filtrate (approximately 20 liters) was vacuum-concentrated to distill off the acetate. Nine liters of ethyl acetate was added to the remaining water layer to effect extraction. The ethyl acetate layer was de-watered with sodium sulfate. Vacuum concentration and drying were conducted to obtain a solid which was subjected to silica gel open column chromatography (chloroform-methanol system) to isolate the antitumor activity fraction. This fraction was vacuumed-concentrated and dried to a solid, dissolved in a small amount of 70% methanol, subjected to high performance liquid chromatography using a Senshu Pack ODS-5251-SS 20 mm-diameter×250 mm (Senshu Science Kabushiki Kaisha) to conduct purification with a mobile phase: methanol/water 7/3 condition and to isolate a fraction exhibiting antitumor activity. This fraction was vacuum-concentrated and dried to a solid, subject to high performance liquid chromatography using a Senshu Pack Aquaseal SS5251(60) 20 mm-diameter×250 mm (Senshu Science Kabushiki Kaisha) to conduct purification with a mobile phase: chloroform/methanol 95/5 and to isolate the antitumor activity fraction. This fraction was vacuum-concentrated to obtain a transparent oily substance. This was suspended in a small amount of benzene and the result was vacuum concentrated and dried to a solid, thereby yielding 25 mg of NSCL-96F037, also herein termed phenylahistin (PLH). 
     B. Production, Isolation and Purification of PLH from  Aspereillus ustus  NSC-F038 
     The same steps as above were conducted except  Aspergillus ustus  NSC-F038 was used instead of  Aspergillus ustus  NSC-F037. It was determined that the ability of  Aspergillus ustus  NSC-F038 to produce the antitumor substance NSCL-95F037 was approximately four times that of NSC-F037. Specifically, from NSC-F037, approximately 2.9 mg phenylahistin was purified per liter of culture medium, while from NSC-F038, approximately 11.8 mg of phenylahistin was purified per liter of culture medium. 
     The production of phenylahistin by  A. ustus  NSC-F038 was also determined to be related to the conidia formation of the fungus. Therefore, the fungus was cultured on an agar medium containing glucose (0.5%), glycerol (2%), yeast extract (0.2%), Pharmamedia (Traders Protein) (2%), NaCl (0.25%) and agar (1.5%), adjusted to pH 6.5 before sterilization. This medium was suitable for the conidia formation at 28° C. for 8 days. The cultured agar media (200 plates; 4 L) were then extracted with ethyl acetate. The extract was initially subject to silica gel column chromatography under an ethyl acetate-acetone stepwise gradient, followed by a second silica gel column and eluted with 2% methanol in chloroform. The fractions that exhibited cell cycle inhibitory activity were collected and evaporated, then dissolved in ethyl acetate and left two days at room temperature. The active substance was precipitated to yield 330 mg of a white amorphous powder with the following physical and spectroscopic characteristics: melting point: 233-236° C.; [∀] D   22 +123° (c=0.13, MeOH); UV (MeOH) 8 max (log,): 202 (4.38), 233 sh (4.05), 320 (4.43); IR (KBr pellet)&lt;max: 3440, 3240, 1670, 1640, 1440 cm −1 ; HR-FAB-MS (m/z) 350.1741 (M + , calcd. for C 20 H 22 N 4 O 2 : 350.1743). 
     The fungus  A. ustus  NSC-F038 was, alternatively, cultured is 20 ml of a culture medium comprising 0.5% glucose, 2% glycerin, 0.2% yeast extract, 2% cotton seed lees, 0.25% sodium chloride 2.5 g/l and 1.5% agar (pH6.5) to form a planar medium in a 9 cm-diameter Petri dish. The culture medium was inoculated at 5 points with  Aspergillus ustus  NSC-F038 and incubated in the dark for 7 days at 26° C. to produce spores. The spores were harvested into 5 ml of sterilized water having Tween 20 added to a concentration of 0.05%, thereby obtaining a spore suspension. Each of 400 Petri dishes containing 20 ml of the same culture medium was inoculated with 0.1 ml of the spore suspension. Incubation was conducted in the dark at 26° C. for 8 days. The cultures were comminuted with a mixer, 8 liters of ethyl acetate were added, and the mixture left standing 2 days before extraction. The recovered ethyl acetate was then vacuum-concentrated to obtain 15 g of a brown syrup. The syrup was dissolved in 20 ml of ethyl acetate, and the solution was subject to a silica gel column (bed volume: 8 cm diameter×20 cm length) chromatography under ethyl acetate and eluted with acetone ethyl acetate (1:5). The eluate was fractionated in 500 ml lots in the order eluted. The active compound was eluted in the fifth to tenth lots. These elutes were then vacuum-concentrated to afford a dark-brown powder in a total amount of approximately 4.65 g. The dark-brown powder was then dissolved in 10 ml of chloroform and the solution was subject to silica gel column (bed volume: 5 cm diameter×30 cm length) chromatography under ethyl acetate and eluted first with 500 ml chloroform and then with methanol in chloroform (1:50). 
     The active compound was eluted with methanol in chloroform (1:50) to obtain a brown powder in a total amount of approximately 1.05 g. The brown powder was then thoroughly mixed with ethyl acetate and left to stand 2 days to precipitate out approximately 628 mg of a white powder (A) containing the active compound. A portion of the white powder (A) was hydrolyzed. The so-obtained phenylalanine was analyzed by high performance liquid chromatography using a chiral column. The presence of R-configuration and S-configuration phenylalanine was then confirmed in a standard manner, and it was determined that the white powder (A) was a racemic mixture of the enantiomers of phenylahistin. 
     Another portion of the white powder (A) was dissolved in ethyl acetate containing a small amount of methanol and left to stand for 7 days. As a result a light yellow crystalline solid 187 mg of a purified specimen of (+)-phenylahistin was obtained. These specimens were used in the following four examples, EXAMPLES 2 through 5, as therein described, to verify the cell-proliferation, antimicrobial, cell-cycle inhibiting effects of the compound of the invention. 
     EXAMPLE 2 
     Concentration of Phenylahistin (PLH) Effective in Inhibiting Cell Proliferation 
     Into each well of a 96-well microtiter plate, 100 μl of A-549 cells derived from human lung cancer prepared to 10 5  cells/ml in a culture medium obtained by adding 10% bovine fetus serum to EMEM culture medium (Nissui Seiyaku Co., Ltd.) having antitumor effect against A-549 cells derived from human lung cancer was placed. Methanol solution of racemic NSCL-96F037 obtained as in Example 1 was added to the wells of the uppermost row, specimens were diluted by the half-log dilution method and added, and the plate was incubated in a carbon dioxide gas incubator at 37° C. for 48 hours. The result was added in lots of 10 μl with MTT reagent (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2H-tetra bromide)(1 mg/ml.PBS), followed by incubation in a carbon dioxide gas incubator at 37° C. for 6 hours. The culture medium was discarded and the crystal of produced in the cells was dissolved in 100 μl/well of dimethylsulfoxide. Absorption of 595 nm light was then measured with a microplate reader. By comparing the light absorptions of the untreated cells to that of cells treated with a specimen of a known concentration, the specimen concentration that inhibited cell proliferation 50% (IC 50 ) was calculated, thus obtaining an IC 50 =0.3 μg/ml. 
     EXAMPLE 3 
     Antimicrobial Test of Phenylahistin (PLH) 
     Using colibacillus strain JM109 as a gram-negative bacterium,  Bacillus natto  as a gram-negative bacterium and  Aspergillus niger  IFO6341 as a mold, the following antimicrobial test was conducted by the filter paper agar flat plate method, wherein 100 μg of specimen was placed on 9 mm filter paper, dried in air, and standard agar culture medium was used for bacteria and potato dextrose agar culture medium for mold. No antimicrobial activity was observed. An antimicrobial test was further conducted by the liquid culture medium dilution method using the yeast strain  Saccharomyces cerevisise  HF7C. Again, no antimicrobial activity was observed, indicating that the racemic NSCL-96F037 (phenylahistin) obtained in EXAMPLE 1 had high animal cell-specific proliferation inhibiting activity. 
     EXAMPLE 4 
     Cell Cycle Inhibiting Activity of Phenylahistin (PLH) 
     Cell strain A431 derived from human lung cancer was used. EMEM culture medium containing 10% bovine fetus serum and 1% MEM nonessential amino acid solution (SIGMA M2025) was used to incubate A431 cells at 37° C. in an incubator saturated with 5% carbon dioxide gas and water vapor. The refined specimen of racemic phenylahistin obtained in EXAMPLE 1 was added to the cells in the log-growth phase and progression of the cell cycle was analyzed by flow cytometer and microscopic observation. The results, shown in Table 1, indicate that this phenylahistin was useful as a cell cycle inhibitor. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Cell cycle inhibiting activity 
               
            
           
           
               
               
            
               
                 Concentration (μg/ml) 
                 Inhibiting effect 
               
               
                   
               
            
           
           
               
               
            
               
                 0.5 
                 Absent 
               
               
                 1.0 
                 Absent 
               
               
                 2.0 
                 Slight 
               
               
                 4.0 
                 Slight 
               
               
                 8.0 
                 Present 
               
               
                 16.0 
                 Present 
               
               
                 32.0 
                 Present 
               
               
                   
               
            
           
         
       
     
     Inhibiting effect evaluation criteria were as follows: 
     Present: The cell cycle stopped at the G2/M phase in not less that 50% of the cells. 
     Slight: The cell cycle stopped at the G2/M phase in not less than 10% and less than 50% of the cells. 
     Absent: The cell cycle stopped at the G2/M phase in less than 10% of the cells. 
     EXAMPLE 5 
     Cell Proliferation Inhibiting Effect of Phenylahistin (PLH) 
     K-562, human chronic myelogenic leukemia cells were cultured in RPMI164 culture medium (containing 10% bovine fetus serum) and A-431 human pudendal epithelial squamous cancer cells were cultured in DMEM culture medium, containing 10% bovine fetus serum. To these a continuous dilution series of the (+)-phenylahistin obtained in EXAMPLE 1 was added. After 48-hours incubation, a MTT reagent was added to measure growth. The human chronic myelogenic leukemia cells exhibited an IC 50  of 13.3 μg/ml, while the human pudendal epithelial squamous cancer cells exhibited an IC 50  of 2.9 μg/ml, indicate that (+)-phenylahistin was useful as an antitumor agent. 
     EXAMPLE 6 
     Characterization and Resolution of Enantiomers of Phenylahistin (PLH) 
     A. Racemic Phenylahistin 
     Phenylahistin (PLH) had the molecular formula, C 20 H 22 N 4 O 2 , which was determined by HR-FAB-MS (mz found: 350.1741 (M + ), calcd. for C 20 H 22 N 4 O 2 : 350.1743). In the IR spectrum, the absorption at 3440 cm −1  corresponds to the N—H group, and strong absorptions at 1670 and 1640 (cm −1  indicated the existence of amide groups. These findings together with the absence of the amide II band near 1550 cm −1  in the spectrum suggested the presence of the diketopiperazine system in phenylahistin, according to the technique described in Steyn, P. S.  Tetrahedron  1973, 29, 107, which was also supported by the negative ninhydrin reaction. 
     The  13 C-NMR spectrum of phenylahistin, with atomic positions as indicated in Structure (IV), summarized in the first column of Table 2, showed 17 resolved peaks with three overlapping carbon signals. The multiplicity of these peaks was determined by analysis of its DEPT spectra. The  1 H-NMR spectrum displayed 22 proton signals, listed in the second column of Table 2, including three exchangeable protons. All bond connections between proton and carbon were interpreted by  1 H- 13 C COSY. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Table  13 C and  1 H NMR Assignment of Phenylahistin in CDCl 3   
               
            
           
           
               
               
               
            
               
                 Positions 
                 δ C* 
                 δ H** 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1(N H ) 
                 — 
                   
                 9.48 (1H, br s) 
               
               
                 2 
                 132.56 
                 d 
                 7.55 (1H, s) 
               
               
                 4 
                 132.18 
                 s 
                 — 
               
               
                 5 
                 136.83 
                 s 
                 — 
               
               
                 6 
                 105.62 
                 d 
                 6.88 (1H, s) 
               
               
                 7 
                 123.64 
                   
                 — 
               
               
                 8(N H ) 
                 — 
                   
                 12.08 (1H, br s) 
               
               
                 9 
                 164.73 
                 s 
                 — 
               
               
                 10 
                 57.14 
                 d 
                 4.35 (1H, ddd, J = 10, 4, 3# Hz) 
               
               
                 11(N H ) 
                 — 
                   
                 5.82 (1H, br s) 
               
               
                 12 
                 159.94 
                 s 
                 — 
               
               
                 13 
                 41.23 
                 t 
                 2.95 (1H, dd, J = 14, 10 Hz) 
               
               
                   
                   
                   
                 3.49 (1H, dd, J = 14, 4 Hz) 
               
               
                 14 
                 135.45 
                 s 
                 — 
               
               
                 15, 19 
                 129.52 
                 d 
                 7.25 (2H, d, J = 7 Hz) 
               
               
                 16, 18 
                 129.07 
                 d 
                 7.33 (2H, t, J = 7 Hz) 
               
               
                 17 
                 127.45 
                 d 
                 7.27 (1H, t, J = 7 Hz) 
               
               
                 20 
                 37.61 
                 s 
                 — 
               
               
                 21 
                 144.66 
                 d 
                 6.02 (1H, dd, J = 18, 11 Hz) 
               
               
                 22 
                 113.29 
                 t 
                 5.13 (1H, d, J = 18 Hz) 
               
               
                   
                   
                   
                 5.17 (1H, d, J = 11 Hz) 
               
               
                 23,24 
                 27.97 
                 q 
                 1.49 (6H, s) 
               
               
                   
               
               
                 *125 MHz, including multiplicity assignment on the basis of DEPT summary. Chemical shifts in ppm from CDCl 3  as an internal standard (77.00 ppm).  
               
               
                 **500 MHz, Chemical shifts in ppm from TMS as an internal standard (0.00 ppm).  
               
            
           
         
       
     
     #Coupling with 11-H was observed by decoupling experiment.                    
     The  1 H NMR and  1 H - 1 H COSY spectra revealed only the presence of four partial structures: a monosubstituted benzene ring, methylene protons being coupled to a methine proton, which was also coupled to an exchangeable proton, one geminal methyl group and a vinyl group. Since quaternary carbons prevented constructing further partial structures, the PFG-HBMC spectrum were t measured. A partial structure was determined on the basis of the long-range correlations of C-9, C-13, and C-14, which were observed in the spectrum. The carbon chemical shift of C-9 (*164.73) indicated that it was a carbonyl carbon. Therefore, that partial structure was determined to be a phenylalanine residue. In the same manner, a second partial structure was determined from the correlations of C-5, C-20, C-21, C-23 and C-24, which showed an isoprenyl group binding to quaternary sp 2  carbon (*136.83). The diketopiperazine ring was also constructed from the interpretation of correlations between respectively, C-7 and 11-H, C-10 and 8-H, C-12 and 8-H, and C-12 and 10-H in the PFG-HMBC spectrum. The dehydrohistidine moiety was estimated from the remaining three carbons, two nitrogens and three hydrogens, and this structure was supported by correlation signals of C-4, C-5, C-7, and C-12. The correlation signal between C-5 and H-6 indicated that the isoprenyl group was placed in the C-5 of the imidazole ring. This structure was confirmed by analysis of the PFG- 5 N-HMBC spectrum. The nitrogen residues were assigned as follows: N1 (*159); N3 (*253); N8 (*133); N11 (*109) ( 15 N-formamide internal standard at *112.4 ppm). 
     From these data, the planar structure of phenylahistin was established. The stereochemistry of the C-6-C7 double bond was suggested to be Z by the low field shift of 8-H (N H : *12.08), which was further explained by hydrogen-bonding between the 8-H proton and N-3 of the imidazole ring, as in the case of aurantiamine. 
     It was also determined that phenylahistin has a chiral center at the C-10 position. Chiral HPLC analysis of the phenylalanine was conducted according to methods described in Larsen, T. O et al., Phytochemistry (1992) 31, 1613, under the following conditions: HPLC conditions: column; Crownpak CR(+) N4.0×150 mm (Daicel Chemical Industries, Ltd.), mobile phase; H 2 O (pH 2, adjusted with perchloric acid), flow rate; 0.8 ml/mn, detector; UV 8 200 nm. Phenylalanine was obtained from the acidic hydrolysate of the amorphous white powder of phenylahistin, see Yamazaki, M. et al., Tetrahedron Lett. (1975) 27. The phenylalanine obtained from the hydrolysate of phenylahistin was identified using an amino acid analyzer and by measurement of EI-MS [(m+H) + :166.2]); chrial HPLC indicated that the phenylahistin was a mixture of enantiomers in the ratio of approximately R:S=3.1. In order to examine the biological activity of each enantiomer, chiral resolution was carried out by chiral HPLC under the following conditions: column; Chiracel OD N4.6×250 mm (Daicel Chemical Industries, Ltd), mobile phase; n-hexane/ethanol=75/25, flow rate; 1.0 ml/min, temperature; 25° C. Absolute configuration was determined by analysis of phenylalanine obtained from (+)-phenylahistin using chiral HPLC. 
     B. Single-Crystal X-ray Diffraction Analysis of Phenylahistin 
     A pale yellow crystal having approximately dimension 0.4×0.4×0.8 mm was obtained from the ethyl acetate solution of phenuylahistin. All X-ray measurements were made on a Rigaku AFC7R diffractometer with graphite monochromated CuKα radia and a rotating anode generator. Cell constants and an orientation matrixed for data collection were obtained from a least-squares refinement using the setting angles of 25 carefully centered reflections in the range of 77.59&lt;26&lt;79.03°. These crystal data are summarized in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Crystal data of (−)-phenylahistin 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Empirical formula 
                 C 20 H 22 N 4 O 2   
               
               
                   
                 Formula weight 
                 350.42 
               
               
                   
                 Crystal system 
                 tetragonal 
               
               
                   
                 Lattice parameters: 
                 a = 15.3509(7)Å 
               
               
                   
                   
                 o = 8.309(2) Å 
               
               
                   
                   
                 V = 1958.0(2) Å 3   
               
               
                   
                   
                 Z = 4 
               
               
                   
                 Space group 
                 P4 2 (#77) 
               
               
                   
                 D calc. 
                 1.189 g/cm 3   
               
               
                   
                 μ (CuKα) 
                 6.37 cm −1   
               
               
                   
                   
               
            
           
         
       
     
     Of the 2229 reflections which were collected, 2064 were unique (R int =0.019). The intensities of the three representative reflections were measured after every 150 reflections. No decay collection was applied. The linear absorption coefficient: μ for CuKα is 6.37 cm −1 . Azimuthal scans of several reflections indicated no need for an absorption correction. The data were corrected for Lorentz and polarization effects. A correction for secondary extinction was applied. The structure was solved by direct methods, see SAPA91: Fan Hai-Fu (1991). Structure Analysis Programs with Intelligent Control, Rigaku Corporation, Tokyo, Japan, and expanded using Fourier techniques, see DIRDIF94: Beurskens, P. T. et al. (1994) the DIRDIF94 program system, Technical Report of the Crystallography Laboratory, University of Nijmegen, The Netherlands. Nonhydrogen atoms except for C22 were refined anisotropically, while C22 was refined isotropically. Some hydrogen atoms were refined isotropically, the est included in fixed positions. The final cycle of full-matrix least-squares refinement was based on 1724 observed reflections (I&gt;1.50σ(I)) and 242 variable parameters and converged (largest parameter shift was 0.30 times its esd) with unweighted and weighted factors of: R=0.057, R W =0.067. The maximum and minimum peaks on the final difference Fourier map corresponded to 0.20 and −0.12 e − /A 8 , respectively. All calculations were performed using the teXscan crystallographic software package of Molecular Structure Corporation. 
     The stereochemistry of C6-C7 double bond was confirmed to be Z, and the hydrogen bonding between 8(N)—H and N-3 was observed as speculated from low field-shift of 8(N)—H (δ2.08 ppm) in NMR study. Since the absolute configuration of C-10 position could not be determined by this X-ray analysis, we analyzed the configuration of phenylalanine which was obtained from acidic hydrolysate of the crystalline sample using chiral HPLC. The phenylalanine obtained from the crystalline sample was identical with the authentic sample of D-(+)-phenylalanine (R-configuration). Therefore, the absolute configuration of this crystalline sample of phenylahistin was established to be R. 
     Other derivatives of phenylahistin can be synthesized using the foregoing techniques or others well known organic synthesis techniques. 
     EXAMPLE 7 
     Synthesis and Physical Characterization of Phenylahistin Derivatives 
     A. Synthesis of Various Derivatives of Phenylahistin 
     Structural derivatives of phenylahistin were synthesized from the resolved enantiomers according to the following reaction schemes. In Reaction Scheme 1, shown in FIG. 1, the enantioners of phenylahistin were resolved, as described in Example 6, and each was subject to palladium-based catalytic reduction conditions for 2 hours, yielding two mono-reduced phenylahistin derivatives herein designated compounds 3 and 4. These compounds were in turn subject to palladium-based catalytic reduction conditions for 24 hours, yielding di-reduced phenylahistin derivatives, herein designated compounds 5 and 6. 
     In Reaction Scheme 2, as shown in FIG. 2, the synthesis of the phenylahistin derivative designated PLH-Cl, and alternatively designated compound 9, is described. This reaction scheme treats H 2 N-Phe-Gly-OMe, a commercially available di-peptide derivative to yield PLH-Cl, a derivative of PLH in which the isoprenyl moiety is replaced with a methyl group. Since partially racemization was occurred (21%), chiral resolution was carried out to obtain optically pure PLH-Cl. 
     In Reaction Scheme 3, as shown in FIG. 3, the synthesis of (−)-PLH-Cl is described. In the course of synthesizing this compound, another phenylahistin derivative, designated compound 7 is synthesized. 
     B. Physical Characteristics 
     These above-identified compounds were characterized as follows: 
     Compound 3. Colorless solid; mp 224-226° C.; [α] D   25 −295°(c=0.15, MeOH), UV (MeOH) nm 322 (ε 23300), 231 (ε 8830), 203 (ε 16800); IR (KBr) cm −1  3310, 3220, 2970, 1670, 1440;  1 H NMR (270 MHz, CDCl 3 ) δ12.09 (br s, 1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t, J=7 Hz 3H);  13 C NMR (67.5 MHz, CDCl 3 ) δ164.6, 159.9, 138.1, 135.5, 132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3, 36.2, 35.4, 27.9 (2C), 9.2; High-resolution MS m/z 352.1926 (M + ) (Calcd for C 20 H 24 N 4 O 2 : 352.1899). Anal. calculated for C 20 H 24 N 4 O 2 : C,68.16; H,6.86; N,15.90. Found: C,68.34; 6.78; 15.82. 
     Compound 4: Colorless solid; mp 222-223° C.; [α] D   25  +284°(c=0.10, MeOH), UV (MeOH) nm 322 (ε 22800), 231 (ε 8720), 203 (ε 16300); IR (KBr) cm −1 3310, 3220, 2970, 1670, 1440;  1 H NMR (270 MHz, CDCl 3 ) δ12.09 (br s, 1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t, J=7 Hz 3H);  13 C NMR (67.5 MHz, CDCl 3 ) δ164.6, 159.9, 138.1, 135.5, 132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3, 36.2, 35.4, 27.9 (2C), 9.2; High-resolution MS m/z 352.1932 (M + ) (Calcd for C 20 H 24 N 4 O 2 : 352.1899). Anal. calculated for C 20 H 24 N 4 O 2 : C,68.16; H,6.86; N,15.90. Found: C,68.09; 6.87; 15.84. 
     Compound 5: Colorless solid; mp 224-225° C.; [α] D   25  −96°(c=0.16, MeOH), UV (MeOH) nm 257 (ε 194), 205 (ε 18100), IR (KBr) cm −1  3380, 3200, 2970, 1670, 1440;  1 H NMR (270 MHz, DMSO-d 6 ) δ11.55 (br s, 1H), 8.25 (br s 1H), 7.86 (br s 1H), 7.41 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H), 3.94 (brd, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5 Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd, J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz, 3H);  13 C NMR (67.5 MHz, DMSO-d 6 ) δ166.9 165.4, 135.9, 132.5, 131.2, 130.3 (2C), 127.9 (2C), 126.6, 55.7, 54.3, 38.2, 35.0, 34.3, 31.9, 27.6, 27.5, 9.1; High-resolution MS m/z 354.2098 (M + ) (Calcd for C 20 H 26 N 4 O 2 : 354.2055). Anal. calculated for C 20 H 26 N 4 O 2 :1/3H 2 O: C,66.64; H,7.36; N,15.54. Found: C,66.75; 7.41; 15.52. 
     Compound 6: Colorless solid; mp 226-227° C.; [α] D   25  +99°(c=0.10, MeOH), UV (MeOH) nm 257 (ε 174), 205 (ε 18200), IR (KBr) cm −1  3380, 3200, 2970, 1670, 1440;  1 H NMR (270 MHz, DMSO-d 6 ) δ11.55 (br s, 1H), 8.25 (br s. 1H), 7.86 (br s, 1H), 7.41 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H), 3.94 (br d, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5 Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd, J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz, 3H);  13 C NMR (6.75 MHz, DMSO-d 6 ) δ166.9,165.4, 135.9, 132.5, 131.6, 131.2, 130.3 (2C), 127.9 (2C), 126.6, 55.7, 54.3, 38.2, 35.0, 34.3, 31.9, 27.6, 27.5, 9.1; High-resolution MS m/z 354.2110 (M + ) (Calcd for C 20 H 26 N 4 O 2 : 354.2055). Anal. calcd for C 20 H 26 N 4 O 2 :1/2H 2 O: C, 66.09; H,7.49; N,15.41. Found: C,65.80; 7.50; 15.30. 
     Compound 7: Cyclo-Gly-Phe (7). White powder; mp 262-263° C. (decomp.); [α] D   25  +60° (c=0.15,DMSO); UV (MeOH) nm 257 (ε 101), 206 (ε 5770) IR (KBr) cm − 3340, 3200, 3060, 1680, 1470, 1340;  1 H NMR (270 MHz,DMSO-d 6 ) δ8.16 (br s, 1H), 7.90 (br s. 1H), 7.32-7.15 (m, 5H), 4.07 (br dd, J=7, 4 Hz, 1H), 3.35 (dd, J=18, 3 Hz, 1H), 3.10 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5 Hz, 1H), 2.75 (d, J=18 Hz, 1H);  13 C NMR (DMSO-d6) δ167.1,165.7, 136.0, 130.1 (2C), 128.1 (2C), 126.8, 55.5, 43.7, 38.8; Anal. calculated for C 11 H 12 N 2 O 2 :1/5H 2 O: C,63.57; H,6.01; N,13.48. Found: C,63.85; H, 5.86; N,13.40. 
     Compound 8: Colorless solid; mp 84-85° C.; [α] D   25 +7.8° (c=0.52, MeOH); UV (MeOH) nm 209 (ε 20400); IR (KBr) cm −1  1720, 1400, 1380, 1240;  1 H NMR (270 MHz, CDCl 3 ) δ7.33-7.26 (m, 3H), 7.08-7.05 (m, 2H), 5.44 (t, J=5 Hz, 1H), 4.49 (d, J=19 Hz, 1H), 3.35 (dd, J=14, 5 Hz, 1H), 3.20 (dd, J=14, 5 Hz, 1H), 2.58 (s, 3H), 2.55 (s, 3H), 2.48 (d, J=19 Hz, 1H);  13 C NMR (67.5 MHz, CDCl 3 ) δ171.2, 171.0, 167.9, 166.0, 134.3, 129.7 (2C), 129.1 (2C), 128.2, 59.0, 46.0, 38.7, 27.1, 26.8; MS (ESI) m/z 205 (M+H);MS (ESI) m/z 311 (M+Na) + ;Anal. calculated for C 15 H 16 N 2 O 4 : C, 62.49; H, 5.59; N, 9.72. Found: C, 62.50; H, 5.50; N, 9.67. 
     Compound 9: Colorless solid; mp 285-286° C. (decompose); [α] D   25 −267° (c=0.21 DMSO); UV (MeOH) nm 319 (ε 22800); IR (KBr) cm −1  3400, 3180, 1680, 1450;  1 H NMR (270 MHz, DMSO-d 6 ) δ11.50 (br s, 1H), 8.35 (br s, 1H), 7.74 (s, 1H), 7.24-7.14 (m, 5H), 6.20 (s, 1H), 4.48 (m, 1H), 3.33 (br s, 1H), 3.20 (dd, J=14, 4 Hz, 1H), 2.93 (dd, J=14, 5 Hz, 1H), 2.19 (s, 1H);  13 C NMR (67.5 MHz, DMSO-d 6 ) δ164.2, 158.7, 135.6, 134.6, 132.3, 130.0 (2C), 128.0 (2C), 127.5, 126.6, 123.4, 101.7, 55.9, 38.7, 8.9; High-resolution MS m/z 296.1261 (M + ) (Calcd for C 16 H 16 N 4 O 2 : 296.1273). Anal. calculated for C 16 H 16 N 4 O 2 .1/5H 2 O; C,6407; H, 5.51; N, 18.68. Found: C, 64.39; H, 5.65; N, 18.29. 
     EXAMPLE 8 
     Relative Cytotoxic Effects of the Phenylahistin Enantiomers 
     The cytotoxic effects of the enantiomers of phenylahistin on P388 murine leukemia cells were examined. IC 50  values of (−)-phenylahistin (S-configuration) (92.2% e.e.) and (+)-phenylahistin (R-configuration) (97.8% e.e.) were 3.5×10 −7  M and 3.8×10 −5  M, respectively. Judging from the content of (−)-phenylahistin in (+)-phenylahistin, (+)-phenylahistin was considered to have relatively low cytotoxicity to P388 cells. 
     EXAMPLE 9 
     Biological Activity of Phenylahistin and Its Derivatives 
     The biological activity of phenylahistin and its derivatives, synthesized as described EXAMPLE 7 and established according to the methods described in EXAMPLES 5 and 11, are summarized in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Comparative Biological Activity of Phenylahistin and its Derivatives 
               
            
           
           
               
               
               
            
               
                   
                 IC 50  for P388 
                   
               
               
                   
                 Proliferation* 
                 IC 50  for Microtubule Protein 
               
               
                 Compound 
                 (μM) 
                 Polymerization** (μM) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 0.21 
                 25 
               
               
                 2 
                 10 
                 &gt;200 
               
               
                 3 
                 0.23 
                 30 
               
               
                 4 
                 19 
                 &gt;200 
               
               
                 5 
                 &gt;200 
                 &gt;200 
               
               
                 6 
                 &gt;200 
                 &gt;200 
               
               
                 7 
                 &gt;200 
                 &gt;200 
               
               
                 9 
                 7.5 
                 &gt;200 
               
               
                 Cyclo-His-Phe*** 
                 &gt;200 
                 &gt;200 
               
               
                   
               
               
                 *IC 50  was determined by Alamar Blue ™ assay.  
               
               
                 **Microtubule protein was prepared from bovine brain by two cycles of assembly and disassembly method. The concentration of microtubule protein used for the assay was 1.5 mg/ml.  
               
               
                 ***Obtained from SIGMA (code No. C2615)  
               
            
           
         
       
     
     EXAMPLE 10 
     Effect of Phenylahistin on the Cell Cycle Progression of P388 
     The effect of phenylahistin on the cell cycle progression of P388 cells was also investigated using a flow cytometer, as described in Krishan, A., J. Cell Biol. (1975) 66, 188-193. P388 cells in the log growth phase were seeded into flasks and cultured for 14 hours, and then various concentrations of phenylahistin were added to each flask. After an 8-hour incubation, the cells were harvested and fixed with 50% MeOH at −20° C. overnight. The cells were washed with 30% MeOH and then with 10 mM PBS, and treated with 50:g/ml propidium iodide at 4° C. for 2 hours. DNA histograms were obtained using a flow cytometer (Cyto ACE-300:JASCO). 
     (−)-Phenylahistin exhibited cell cycle inhibitory activity at 1×10 −6  M, but (+)-phenylahistin (97.8% e.e.) had no effect at 1×10 −5  M. Therefore, the active configuration of phenylahistin was determined to be S, and it was determined that this configuration most effectively inhibited cell cycle progression in the G2/M phase. 
     EXAMPLE 11 
     Effects of Phenylahistin on Microtubulin Function 
     (−)-Phenylahistin ((−)-PLH) was shown to effect microtubule function, specifically via effecting the proliferation, mitosis, and microtubule structure of A549 cells (human lung carcinoma), and via inhibiting the in vitro polymerization of bovine brain microtubule protein and purified tubulin. In addition, competitive binding experiments using colchicine (CLC) and vinblastine (VLB), two typical antimitotic agents that bind to individual binding sites on tubulin, were used to establish the effect of (−)-PLH on the distinct binding sites on tubulin. 
     A. Materials 
     (−)-PLH was isolated from the agar-culture medium of  A. ustus  NSC-F038 (See, Kanoh, K. et al.: Bioorg. Med. Chem. Lett. (1997), 7, 2847-52, as disclosed in Example 1. CLC was obtained from Sigma (St. Louis, Mo.) and VLB was from Wako Pure Chemicals (Tokyo, Japan). [ 3 H]CLC was purchased from Du Pont/Boston Nuclear (New England, Mass.) and [ 3 H]VLB was from Amersham (Buckinghamshire, UK). A549 cell line (human lung carcinoma) was obtained from American Type Culture Collection (Rockville, Md.). A549 cells were cultured in phenol red free EMEM medium from Nissui Pharmaceuticals (Tokyo, Japan), supplemented with MEM non-essential amino acids (Sigma) and 10% fetal bovine serum from JRH Biosciences, (Lenexa, Kans.). 
     B. Alamar Blue™ Assay 
     Exponentially growing A549 cells were seeded into 96-well tissue culture plates (2×10 3  cells/100 μl/well) and cultured for 16 hours. (−)-PLH or CLC was then added to each well at various concentrations, and the cells were cultured for an additional 48 hours. Live cells were counted using Alamar Blue™ from BioSource International (Camarillo, Calif.) as described in Ahmed, S. A., J. Immunol. Methods, (1994), 170, 211-24. 
     C. Mitotic Index 
     Exponentially growing A549 cells were seeded into 96-well tissue culture plates (2×10 3  cells/100:l/well) and cultured for 16 hours. (−)-PLH or CLC was then added to each well at various concentrations, and the plates were incubated for an additional 24 hours. The number of cells in mitosis (round cells) and total cells in eight randomly selected fields were counted under a phase-contrast microscope. In a series of preliminary studies, it was confirmed that the round cells in this condition were cells in mitosis by using flow cytometry and Hoechst 33258 staining. The mitotic index represented the percentage of round cells among the total number of cells in the eight selected fields. 
     D. Immunocytochemistry 
     Immunocytochemical staining was performed using the method described by Yahara, I. et al., Cell (1978) 15, 251-259. A549 cells were cultured on glass coverslips and incubated with the test drug for 6 hours. The cells were then fixed with 3.7% formaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 5 min, and incubated with a mouse monoclonal antibody against ∀-tubulin (Calbiochem®, Oncogene Research Products, Cambridge, Mass.), followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG from Cosmo Bio Co., (Tokyo, Japan), and the cells were examined under an immunofluorescent microscope. 
     E. Microtubule Protein and Tubulin Preparation 
     Microtubule protein was prepared from bovine brain tissue by two cycles of assembly and disassembly according to the method of Tiwari, S. C. et al. Anal. Biochem, (1993), 215, 96-103. Tubulin was purified from microtubule protein by phosphocellulose chromatography see Algaier, J. et al. Biochim. Biophys. Acta, (1988), 954, 235-43, and tubulin purity was evaluated by polyacrylamide gel electrophoresis, as in Laemmli, U.K., Nature, (1970), 277, 680-85. Essentially, no microtubule-associated proteins were detected in this preparation. Protein concentrations were determined using the Coomassieg Protein Assay Reagent from Pierce (Rockford, Ill.). 
     F. Polymerization Assay 
     Polymerization of microtubule protein was monitored by an increase in turbidity at 37 C. in microtubule assembly buffer containing 100 mM MES, 0.5 mM MgCl 2 , 1 mM EGTA and 1 mM GTP. See Johnson, K. A. et al., J. Mol. Biol., (1977), 117, 1-31. Polymerization of purified tubulin was measured using the same method except the microtubule assembly buffer contained 4 M glycerol. See Lee, J. C. et al., Biochemistry, (1975), 14, 5183-87. Microtubule protein and tubulin polymerization was initiated by a temperature shift from 0° C. to 37° C. and turbidity was measured on a DU-20 thermocontrolled spectrophotometer from Beckman (Fullerton, Calif.) at 360 nm. Drugs were dissolved in dimethyl sulfoxide (DMSO), which was used in all experiments at a final concentration of 2% (v/v). 
     G. Electron Microscopy 
     Microtubule protein (1.5 mg/ml) was polymerized at 37° C. for 20 min in the presence of 100 μM (−)-PLH or vehicle (DMSO). A portion of each sample was diluted 5-fold with 1% glutaraldehyde in the microtubule assembly buffer. Samples were then placed on formvar- and carbon-coated grids, stained with 2% uranyl acetate, and examined using a JEOLJEM-1200 EXII electron microscope JEOL (Tokyo, Japan). 
     H. Competition Assay 
     [ 3 H]CLC binding to tubulin was evaluated by the ultrafiltration method of Takahashi, M., et al., Biochim. Biophys. Acta, (1987) 926, 215-23, with only slight modifications: bovine brain tubulin (0.2 mg/ml) was incubated in the microtubule assembly buffer with various concentrations of [ 3 H]CLC for 20 min at 37° C.; each sample (200 μl) was applied to the reservoir of a UFC3LTK00 ultrafiltration unit from Nihon Millipore Ltd. (Yonezawa, Japan) and centrifuged at 1,500×g for 4 min at room temperature to obtain approximately 60 μl of filtrate. The concentration of unbound [ 3 H]CLC in the filtrates was determined using a EcoLite™(+) liquid scintillator from ICN Pharmaceuticals Inc. (Costa Mesa, Calif.). Specific bound [ 3 H]CLC concentrations were determined by adding an excess (×100) of unlabeled CLC to the reaction mixture. 
     For the competition assay, bovine brain tubulin (0.2 mg/ml) was incubated in the microtubule assembly buffer with 0.5 μM [ 3 H]CLC and various concentrations of competitors for 20 min at 37° C. [ 3 H]CLC binding was measured as described above. DMSO was used as a co-solvent at a concentration that did not affect drug binding to tubulin (final 2% v/v). Measurement of [ 3 H]VLB binding was followed by the DEAE-cellulose filter method of Borisy, G. G., Anal. Biochem. (1972) 373-85, and the competition assay was carried out as described for CLC. 
     I. Inhibitory Effects of (−)-PLH on Proliferation and Mitosis of A549 Cells 
     The effects of (−)-PLH on the proliferation and mitosis of A549 cells was examined. (−)-PLH inhibited the proliferation of A549 cells in a dose-dependent manner with a 50% inhibitory concentration (IC 50 ) value of 0.3 μM, indicating that (−)-PLH was 5-fold less potent than CLC under the assay conditions. However, the mitotic index increased, which correlated with decreased cell proliferation. These results indicated that (−)-PLH arrested the cell cycle during mitosis, which subsequently reduced cell proliferation, similar to the effect observed for other mitotic inhibitors such as CLC and VLB. See Yoshimatsu, K., et al., Cancer Res., (1997), 57, 3208-13. The anti-mitotic effect of (−)-PLH seemed to be reversible, because cells that arrested in M phase by (−)-PLH returned to proliferate again following washing of (−)-PLH and replacement with a fresh medium. 
     J. Immunofluorescence Staining of Microtubules in A549 Cells 
     The effects of (−)-PLH on microtubule structure in A549 cells using an anti α-tubulin antibody and a secondary antibody conjugated with FITC were also investigated. It was determined that (−)-PLH inhibited the microtubule assembly like CLC, but did not hyper-stabilize microtubules in a manner similar to that of paclitaxel (Taxol®). 
     In control cells, a network of cytoskeletal microtubules was clearly visible, and mitotic spindles could be observed in mitotic cells. The addition of (−)-PLH resulted in the disappearance of the microtubule network, and the entire mitotic cell was uniformly stained with α-tubulin antibody, whereas mitotic spindles were never seen. Cells incubated with CLC exhibited a staining profile similar to that of (−)-PLH-treated cells. Paclitaxel, a potent microtubule-stabilizer, produced thick microtubule bundles and multiple bright fluorescent aster formation. See Rowinsky, E. K. et al., J. Natl. Cancer Inst. (1990) 82:1247-59. These results suggested that (−)-PLH inhibited microtubule assembly in A549 cells, but did not stabilize microtubules. 
     K. (−)-PLH Effects Microtubule Protein Polymerization and Purified Tubulin 
     The effect of (−)-PLH on the polymerization of microtubule protein obtained from bovine brain were also examined. Accordingly, the time course of the turbidity change in the presence of various concentrations of (−)-PLH and CLC was examined. (−)-PLH inhibited the in vitro polymerization of microtubule protein in a concentration-dependent manner, 80 μM of (−)-PLH completely inhibited the polymerization of microtubule protein, while (+)-PLH did not affect microtubule assembly at concentrations ranging up to a concentration of 200 μM. 
     It is reported that VLB, see Luduena, R. F.; J. Biol. Chem. (1984) 259:12890-98, or dolastin 10, see Li, Y., et al., Chem. Biol. Interact., (1994), 93, 175-83 increases the turbidity in microtubule protein polymerization at concentrations higher than those required for inhibition. However, (−)-PLH did not increase the turbidity at concentrations up to 200 μM. 
     To determine whether (−)-PLH acts on tubulin directly or on associated proteins, further tests were performed on the assembly using phosphocellulose-purified tubulin, which was free of microtubule associated proteins. Like microtubule protein, (−)-PLH inhibited tubulin polymerization in a concentration-dependent manner. The IC 50  value of CLC was 6.6±1.7 μM under the same experimental conditions. These results indicated that (−)-PLH was as effective as CLC in inhibiting tubulin polymerization, and that (−)-PLH acted on tubulin directly rather than on tubulin-associated proteins. 
     The effect of (−)-PLH on polymerization of the microtubule protein were also studied by electron microscopy. The control sample contained singular microtubules with a normal cylindrical structure, whereas a sample treated with 100 μM (−)-PLH did not contain such structures. These findings confirmed the turbidity measurements and indicated that (−)-PLH inhibited the polymerization of microtubule protein. 
     L. (−)-PLH Inhibited CLC Binding to Tubulin 
     To investigate the binding site of (−)-PLH on tubulin, competitive binding studies using [ 3 H]CLC and [ 3 H]VLB were conducted. [ 3 H]CLC and [ 3 H]VLB are antimitotic agents that bind to CLC and VLB binding sites, respectively. [ 3 H]CLC binding was measured by the ultrafiltration method as described above. Under the experimental conditions, the K d  value of CLC to tubulin was 5.3×10 −7 M, which is in good agreement with the previously reported value, see Sherline, P., et al., J. Biol. Chem., (1975), 250, 5481-86. (−)-PLH inhibited CLC binding to tubulin in a dose-dependent manner, with an estimated K i  value for CLC binding of 7.4×10 −6 M according to a Dixon plot, assuming that (−)-PLH is the competitive inhibitor of CLC binding to tubulin. VLB slightly enhanced CLC binding to tubulin, likely an effect of the stabilization of VLB on the CLC binding activity of tubulin. See Lacey, E. et al., Biochem. Pharmacol. (1987) 36:2133-38. On the other hand, the binding of [ 3 H]VLG was not inhibited by (−)-PLH. CLC enhanced and (−)-PLH slightly enhanced [ 3 H]VLG binding to tubulin. These results suggested that (−)-PLH binds to the CLC binding site (CLC-site) on tubulin or to a site overlapping the CLC-site. 
     (−)-PLH dose-dependently increased the mitotic index in parallel with inhibition of proliferation of A549 cells, indicating that it prevents cell proliferation by arresting the cell cycle in M phase. Most antimitotic agents such as CLC and VLB are known to exert anti-microtubule activity, including disruption of the process of mitotic spindle formation, resulting in cell arrest in mitosis. (−)-PLH also exhibited an anti-microtubule activity, as evident by depolymerization of cytoskeletal microtubule in A549 cells treated with (−)-PLH. To investigate the mechanism underlying the anti-microtubule activity of this compound, an in vitro polymerization assay using a microtubule protein and phosphocellulose-purified tubulin from bovine brain was performed. (−)-PLH inhibited polymerization of both microtubule protein and purified tubulin, suggesting that (−)-PLH directly acted on tubulin, rather than interacting with microtubule associated proteins. Although (−)-PLH was as effective as CLC in inhibiting the polymerization of purified tubulin, it was 5-fold less potent than CLC in inhibiting the proliferation of A549 cells. These differences may be due to a low permeability of the cell membrane to (−)-PLH compared to CLC. Alternatively, the difference may be due to intracellular transformation of (−)-PLH to an inactive form. The competitive binding assay using radiolabeled CLC and VLB showed that (−)-PLH inhibited the binding of CLC to tubulin, suggesting that (−)-PLH is a competitive inhibitor of CLC binding to tubulin and its binding site may be similar or very close to that of CLC. 
     As noted above, several natural and synthetic antimitotic agents have been reported to inhibit mitosis by binding to the CLC-site on tubulin. See Iwasaki, S., et al., Med. Res. Rev., (1993), 13, 183-98; Hamel, E., Med. Res. Rev., (1996), 16, 207-31. It seems likely that these CLC-site ligands such as CLC, steganacin, see Kupchan, S. M., et al., J. Am. Chem. Soc., (1973), 95, 1335-1336, podophyllotoxin, see Sackett, D. L., Pharmacol. Ther., (1993), 59, 163-228 and combretastatins (Pettit, G. R., et al., J. Med. Chem., (1995), 38, 1666-1672, interact at two hydrophobic sites on tubulin with biaryl groups located at appropriate distances and angles, whereas curacin A, see Verdier-Pinard, P., et al., Mol. Pharmacol., (1998), 53, 62-76, probably interacts in a different manner. In the case of (−)-PLH, the spatial arrangement of two aryl groups, the phenyl group and imidazole moiety, is probably important for binding to tubulin, because the enantiomer (+)-PLH showed little or no effect on mitosis and tubulin polymerization (data not shown). 
     Recently, Usui and colleagues, see Usui, T., et al., Biochem, J., (1998), 333, 543-48; Kondoh, M., et al., J. Antibiot, (1998), 51, 801-04, reported that tryprostatin A and its related compounds, which are diketopiperazines consisting of isoprenylated tryptophan and proline, affect the microtubule assembly. They also showed that tryprostatin A inhibited microtubule polymerization by interacting with MAP2/tau-binding site rather than with CLC binding site, at relatively higher concentrations, see Usui, T., et al., Biochem, J., (1998), 333, 543-48; Kondoh, M., et al., J. Antibiot., (1998), 51, 801-04. Although tryprostatin A and (−)-PLH have a similar structural motif, the diketopiperazine ring composed of isoprenylated heterocyclic amino acids, the binding site and effective concentration against microtubule polymerization are different. It is interesting that the two diketopiperazine compounds, which have different constituent amino acids, bind to distinct sites of tubulin molecule and exert a similar biological activity. 
     EXAMPLE 12 
     In Vitro Antitumor Activity of PLH Against Human Cancer Cell Lines 
     For this and the following three examples, EXAMPLES 13, 14 and 15, phenylahistin was prepared as described above in EXAMPLE 1. Agar-cultured medium of  Aspergillus ustus  NSC-F038 was extracted with ethyl acetate, and the extract was purified by silica gel column chromatography twice. PLH was then precipitated in ethyl acetate as a white powder which contained (−) and (+)-PLH. Two separate batches of PLH were used, the (−)-PLH contents of which has been analyzed by chiral HPLC. One contained 24.1% (−)-PLH, which was used for evaluating in vitro antitumor activity and antitumor activity against P388 Leukemia in vivo (Examples 12 and 13), and the other contained 41.7% (−)-PLH, which was used for evaluation of in vivo antitumor activity against Lewis Lung Carcinoma and Colon 26 (Examples 14 and 15). 
     The antitumor activity of PLH was evaluated by the Human Cancer Cell Line Panel (HCC panel) assay, see Yamori T.,  Jap. J. Cancer Chemother.,  (1997), 24, 129-35. This HCC panel consists of 38 tumor cell lines including 7 lung cancer, 6 stomach, 6 colon, 5 ovary, 6 central nerve system, 5 breast, 2 kidney and 1 melanoma cell lines. Each tumor cell was seeded in 96-well tissue culture plates and incubated overnight. Various concentrations (5 doses; final 10 −4 , 10 −5 , 10 −6 , 10 −7  and 10 −8  M) of PLH were added, and the cells were incubated for 48 hours. The number of living cells was then measured using the sulforhodamine B assay, see Keepers Y. P., et al.,  Eur. J. Cancer,  27, 897-900 (1991), and the drug concentration that inhibited cell growth by 50% of control cell growth (GI 50 ), the drug concentration that inhibited cell growth by 100% of control cell growth (TGI) and the drug concentration that reduced the number of living cells by 50% of the initial cell number (LC 50 ) then obtained. All mice were obtained from Japan SLC (Shizuoka, Japan), P388 leukemia cells were obtained from Dainippon Pharmaceuticals Inc. (Osaka, Japan), Lewis Lung Carcinoma from Riken Cell Bank (Tsukuba, Japan), and Colon 26 cells from the National Cancer Center, Japan (Tokyo, Japan). 
     The results of the HCC panel assay (GI 50 , TGI, and LC 50  for 38 cell lines) are shown in Table 5. PLH [(−)-PLH content: 24.1%] exhibited anti-proliferative activity against 38 cell lines with GI 50  values ranging from 2.3×10 −7 M to 4.0×10 −5 M. The mean graph, see Yamori T.,  Jap. J. Cancer Chemother.  (1997) 24, 129-135, of GI 50  was used for analyzing the mode of action of newly found antitumor agents. The GI 50  mean graph of PLH revealed that the inhibition profile, showing a relatively wide antitumor spectrum, was similar to that of the vinca alkaloids or paclitaxel, suggesting that the target of the action of PLH may be the microtubule system. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Cancer 
                 Cell Line 
                 GL 50  (M) 
                 TGI (M) 
                 LC 50  (M) 
               
               
                   
               
             
            
               
                 Breast 
                 HBC-4 
                 4.6 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 BSY-1 
                 2.3 × 10 −7   
                  6.7 × 10 −7   
                 &gt;1.0 × 10 −4   
               
               
                   
                 HBC-5 
                 6.0 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MCF-7 
                 4.7 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MDA-MB-231 
                 6.4 × 10 −7   
                  6.0 × 10 −6   
                 &gt;1.0 × 10 −4   
               
               
                 CNS 
                 U251 
                 4.4 × 10 −7   
                  1.0 × 10 −5   
                 &gt;1.0 × 10 −4   
               
               
                   
                 SF-268 
                 4.1 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 SF-295 
                 3.2 × 10 −7   
                  2.4 × 10 −6   
                 &gt;1.0 × 10 −4   
               
               
                   
                 SF-539 
                 2.3 × 10 −7   
                  6.0 × 10 −7   
                  1.1 × 10 −5   
               
               
                   
                 SNB-75 
                 3.7 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 SNB-78 
                 1.3 × 10 −6   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                 Colon 
                 HCC2998 
                 1.1 × 10 −6   
                  5.3 × 10 −6   
                 &gt;1.0 × 10 −4   
               
               
                   
                 KM-12 
                 3.8 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 HT-29 
                 4.0 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 WiDr 
                 2.7 × 10 −7   
                  7.9 × 10 −7   
                 &gt;1.0 × 10 −4   
               
               
                   
                 HCT-15 
                 4.9 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 HCT-116 
                 4.8 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                 Lung 
                 NCI-H23 
                 9.1 × 10 −7   
                  9.2 × 10 −5   
                 &gt;1.0 × 10 −4   
               
               
                   
                 NCI-H226 
                 4.1 × 10 −7   
                  3.1 × 10 −5   
                 &gt;1.0 × 10 −4   
               
               
                   
                 NCI-H522 
                 2.5 × 10 −7   
                  8.1 × 10 −7   
                 &gt;1.0 × 10 −4   
               
               
                   
                 NCI-H460 
                 4.1 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 A549 
                 9.7 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 DMS273 
                 2.9 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 DMS114 
                 3.3 × 10 −7   
                  2.4 × 10 −6   
                 &gt;1.0 × 10 −4   
               
               
                 Melanoma 
                 LOX-IMVI 
                 1.3 × 10 −6   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                 Ovary 
                 OVCAR-3 
                 30. × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 OVCAR-4 
                 4.0 × 10 −5   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 OVCAR-5 
                 1.2 × 10 −6   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 OVCAR-8 
                 5.2 × 10 −7   
                  5.6 × 10 −5   
                 &gt;1.0 × 10 −4   
               
               
                   
                 SK-OV-3 
                 3.7 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                 Renal 
                 RXF-6311 
                 1.9 × 10 −6   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 ACHN 
                 2.8 × 10 −5   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                 Stomach 
                 St-4 
                 4.0 × 10 −5   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MKN1 
                 4.0 × 10 −7   
                  2.1 × 10 −5   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MKN7 
                 3.9 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MKN28 
                 4.2 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MKN45 
                 1.5 × 10 −6   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
                 MKN74 
                 4.8 × 10 −7   
                 &gt;1.0 × 10 −4   
                 &gt;1.0 × 10 −4   
               
               
                   
               
            
           
         
       
     
     The TGI values were relatively high, i.e., even at 100 μM PLH, and proliferation of 25 of the 38 cell lines was not completely suppressed. PLH did not exhibit intrinsic cytotoxicity under these experimental conditions, and significant LC 50  values were not obtained except for SF-539 cells. 
     EXAMPLE 13 
     In Vivo Antitumor Activity Against P388 Leukemia of Phenylahistin (PLH) 
     Male CDFl mice (4 weeks old) were inoculated intraperitoneally with P388 leukemia cells (1×10 6  cells/body) on day 0. PLH [(−)-PLH content; 24%] was suspended in 10% ethanol-saline, and administered daily at a dose of 4.2 mg/kg, 12.5 mg/kg, 42 mg/kg or 125 mg/kg intraperitoneally, as a single injection (0.2 ml/body), from days 1 to 12. Each group consisted of six mice. A T/C (%) was calculated according to the following formula: 
     
       
           T/C  (%)=[(mean number of survival days of treated group)/(means number of survival days of control group)]×100 
       
     
     The antitumor activity of PLH [(−)-PLH content: 24.1%] in mice inoculated intraperitoneally with murine P388 leukemia is shown in Table 6. PLH showed significant antitumor activity with a T/C of 129% and 151% at a daily dosage of 41.5 (the calculated dose of (−)-PLH) and 124 (30) mg/kg respectively, for 12 days. The increases in body weight seen in the control group were also suppressed by daily administrations of PLH 41.5 and 124 mg/kg. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                   
                 4.2 
                 12.5 
                 42 
                   
               
               
                   
                   
                 mg/kg 
                 mg/kg 
                 mg/kg 
                   
               
               
                   
                   
                 (1 mg/ 
                 (3 mg/ 
                 (10 mg/ 
                 125 mg/kg 
               
               
                   
                 Control 
                 kg)# 
                 kg)# 
                 kg)# 
                 (30 mg/kg)# 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Mean Body 
                 25.2 ± 0.6 
                 25.3 ± 
                 24.1 ± 
                 23.0 ± 
                 22.3 ± 0.3 
               
               
                 weight on 
                   
                 0.4 
                 0.6 
                 0.5 
               
               
                 day 12 (g) 
               
               
                 Mean Number 
                 12.5 ± 0.4 
                 14.6 ± 
                 14.6 ± 
                 16.1 ± 
                 18.9 ± 0.9** 
               
               
                 of Survival 
                   
                 0.6* 
                 0.5** 
                 0.8** 
               
               
                 days T/C (%) 
                 100 
                 117 
                 117 
                 129 
                 151 
               
               
                   
               
               
                 # Calculated dose of (−)-PLH. *p &lt; 0.05, **p &lt; 0.01, Mann-Whitney U-Test  
               
            
           
         
       
     
     Many fungal diketopiperadine metabolites isolated as micotoxins, for example, fumitremorgin B, a diketopiperazine metabolite consisting of proline and isoprenylated tryptophane and identified as cell cycle inhibitors, exert tremorgenic activity when administered to mice. In spite of the structural and biological similarities between PLH and fumitremorgin B, PLH did not induce such side effects, even at the highest dose (124 mg/kg/day). This indicates that the difference of constituents in diketopiperazines may be responsible for the distinct pharmacological effect of PLH. Also, PLH seems not to be long-acting in vivo, as a single administration of PLH (415 mg/kg PLH [(−)-PLH 100 mg/kg]) was not particularly effective (T/C=115%). 
     EXAMPLE 14 
     In Vivo Antitumor Activity Against Lewis Lung Carcinoma 
     Male BDFl mice (6 weeks old) were inoculated subcutaneously in the right flank region with Lewis Lung Carcinoma cells (1×10 5  cells/body) on day 0. PLH [(−)-PLH content; 41.7%] was suspended in 10% ethanol-saline, and was administered daily at a dose of 24 mg/kg, 72 mg/kg, or 240 mg/kg intraperitoneally, as a single injection (0.2 ml/body), from days 1 to 14. Each group consisted of six mice. Mice were weighed, and the length (L) and the width (W) of the tumor were measured three times a week. On day 15, mice were sacrificed, and the tumor was excised and weighed. The estimated tumor weight (ETW) was calculated according to the following formula: 
     
       
           ETW ( mg )= L ( mm )× W   2  ( mm   2 )/2, 
       
     
     where L represents the length of the tumor and W represents the width of the tumor. The suppression rate was calculated according to the following formula: 
     
       
         Suppression rate (%)=[1−( TWt/TWc )]×100, 
       
     
     where TWt represents the mean tumor weight of the treated group and TWc indicates that of the control group. Average and standard errors were calculated for each group. Variance analysis was performed by the Bartlett method (significance level: 5%), and if the variance was uniform, a Dunnett&#39;s multiple comparison test was performed. However, if the variance was not uniform, rank conversion was performed followed by variance analysis again by the Bartlett method (significance level: 5%). Significance was established at the p&lt;0.05 level. Based on the weight of the excised tumors in the control group, the suppression rate was calculated for each group, and the ED 50  and 95% confidence limit were then determined by linear regression analysis. 
     None of the control and 24 mg/kg/day group mice died. In the 72 mg/kg/day group, 1 mouse died on day 11, and in the 240 mg/kg/day group, 1 mouse died on day 13. 
     In the control group, body weight changed very little during drug administration. Similarly, little change in body weight was observed in the 24 and 72 mg/kg/day groups. However, in the 240 mg/kg/day group, the body weight was markedly lower than control values after day 3, and significantly lower on day 6. 
     In the control group, the estimated tumor weight (ETW) was 105 mg on day 7, and gradually increased to 2,306 mg on day 15. The ETW was slightly lower in the 24 mg/kg/day group, in which it increased from 86 mg on day 7 to 2,244 mg on day 15. Similarly, the ETW was slightly lower in the 72 mg/kg/day group, in which it increased from 34 mg on day 7 to 2,142 mg on day 15. In the 240 mg/kg/day group, the ETW was the lowest, increasing from 34 mg on day 7 to 617 mg on day 15. Significant suppression of the ETW was observed on days 10, 13 and 15 (81.8, 82.3 and 73.2%, respectively). 
     The weight of the excised tumor on the fifteenth day was 3,264 mg for the control group, and 2,941 and 2,380 mg for 24 and 72 mg/kg/day groups, respectively, indicating a rate of suppression of 9.9% and 27.1%, respectively. In the 240 mg/kg/day group, the excised tumor weight was 619 mg, indicating a rate of suppression of 81.0%. The ED 50  and 95% confidence limit was 105.3 mg/kg/day [calculated dose of (−)-PLH: 43.9 mg/kg/day] and 22.0˜504.1 mg/kg/day [(−)-PLHL: 9.2˜210.2 mg/kg/day], respectively. 
     EXAMPLE 15 
     In Vivo Antitumor Activity Against Colon 26 
     Male CDFl mice (6 weeks old) were inoculated subcutaneously in the right flank region with Colon 26 cells (1×10 5  cells/body) on day 0. Each group consisted of six mice. Preparation and administration of PLH, and the evaluation of antitumor activity was carried out as described for Lewis Lung Carcinoma, in EXAMPLE 14. 
     The antitumor activity of PLH ((−)-PLH content: 41.7%) in mice subcutaneously inoculated Colon 26 cells was determined. No mice died during the experimental period in the control, 24 mg/kg/day and 72 mg/kg/day groups. However, in the 240 mg/kg/day group, one mouse died on day 12, two died on day 13 and one died on day 14. No marked changes in the weight of the control was observed, 24 mg/kg/day and 72 mg/kg/day groups, whereas, in the 240 mg/kg/day group, the body weight was lower than that of the control group from day 3, and was lower from day 3 to day 10. 
     In the control group, the ETW gradually increased from 51 mg on day 7 to 1067 mg on day 15. In the 24 mg/kg/day and 72 mg/kg/day groups, the ETWs increased but the increments were slightly lower than those of the control group. In the 240 mg/kg/day group, the increments of ETW were significantly lower at 14 mg on day 7 and 254 mg on day 15. The suppression rates were 64.4% on day 10, 78.8% on day 13 and 85.4% on day 15. 
     The weight of the excised tumor on the fifteenth day was 1,159 mg for the control group, and 1,113 and 843 mg for 24 and 72 mg/kg./day groups, respectively, indicating a rate of suppression of 4.0% and 27.3%, respectively. In the 240 mg/kg/day group, the excised tumor weight was 211 mg, indicating a rate of suppression of 81.8%. The ED 50  and 95% confidence limit was 107.3 mg/kg/day [calculated dose of (−)-PLH: 44.7 mg/kg/day] and 29.0˜397.7 mg/kg/day [(−)-PLH: 12.1˜165.8 mg/kg/day], respectively. 
     The in vitro antitumor activity of a scalemic mixture of (−)-PLH and (+)-PLH using HCC panel assay consisting of 38 human cell lines and in vivo antitumor activity using P388 Leukemia, Lewis Lung Carcinoma, and Colon 26 cells were also analyzed in light of the data illustrating that (−)-PLH exhibits anti-proliferative activity against P388 cells with an IC 50  value of 3.5×10 −7  M and completely inhibits the cell cycle progression of P388 cells in G2/M phase at 1×10 −6  M, while (+)-PLH exhibited little or no effect on proliferation or cell cycle progression even at higher concentrations. See EXAMPLES 3, 4, 5, and 12, 13, 14. It was concluded that the antitumor activity of PLH was due to the active S-configuration enantiomer, (−)-PLH. 
     EXAMPLE 16 
     Activity of (+)- and (−)-PHL Against Human Cancer Cell Lines 
     In order to examine detailed biological activity, especially antitumor activity of both (+)- and (−)-phenylahistin, resolution of (+)- and (−)-phenylahistin using chiral HPLC was conducted. By repeating chiral separation twice or three times, each enantiomer with an optical purity over 99.8% was obtained, and each enantiomer was recrystallizes from ethanol-hexane mixture. Antitumor activity of each enanriomer is summarized in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Antiproliferative activities of (−)-PLH and (+)-PLH 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 IC 50  (M) 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Cell Line (position) 
                 (−)-phenylahistin 
                 (+)-phenylahistin 
               
               
                   
                   
               
               
                   
                 A-431 (damal) 
                 2.2 × 10 −7   
                 2.0 × 10 −5   
               
               
                   
                 A549 (lung) 
                 3.0 × 10 −7   
                 3.0 × 10 −5   
               
               
                   
                 Hela (ovary) 
                 2.0 × 10 −7   
                 1.0 × 10 −5   
               
               
                   
                 MCF7 (breast) 
                 3.3 × 10 −7   
                 1.1 × 10 −5   
               
               
                   
                 TE-671 (CNS) 
                 3.7 × 10 −6   
                 1.3 × 10 −4   
               
               
                   
                 WiDr (colon) 
                 1.8 × 10 −7   
                 8.5 × 10 −6   
               
               
                   
                 K562 (leukemia) 
                 1.9 × 10 −7   
                 1.0 × 10 −5   
               
               
                   
                   
               
               
                   
                 *IC 50  values were determined by MTT assay.  
               
            
           
         
       
     
     Antitumor activity of (+)- and (−)-phenylahistin was examined against seven human cancer cell lines including A431, WiDr, TE-671, Hela, MCF7, A-549 and K562 cells. The IC 50  values of each enantiomer were shown in Table 7. (−)-Phenylahistin exhibited antitumor activity with the IC 50  values ranging from 1.8×10 −7  (against WiDr cells) to 3.7×10 −5  (against TE-671). While (+)-PLH exhibited approximately 33 to 100-fold less activity than (−)-PLH. 
     EXAMPLE 17 
     Syntheses and Biological Activity of Phenylahistin Derivatives: Criterion to Distinguish Biologically Active Derivatives from Relatively Less-Active Derivatives 
     To characterize the anti-tumor phenylahistin derivatives of the present invention, the structural factors that are required to exhibit the anti-microtubule activity of these compounds have been determined. These factors serve to distinguish active derivatives of the invention from relatively less-active compounds. 
     In this and the following example, X-ray crystallographic analyses of phenylahistin, the synthesis and physical characterization of various phenylahistin derivatives, and analyses of the structural factors necessary for anti-microtubule activity were conducted. These analyses, which were focused on the structure of unique isoprenylated dehydrohistidine, indicate that this isoprenylated dehydrohistidine forms a rigid planar pseudo three-ring structure, composed of the diketopiperazine and imidazole rings through a hydrogen bond between N8-H and N3 and α, β-unsaturated bond (C6-C7). It is concluded that this structural feature is important for the activity of the anti-tumor activity of the compounds of the present invention. 
     The derivatives analyzed in this and the following example were prepared in the following manner. As shown in FIG. 3 (Reaction Scheme 3), simple alkylation and reduction of phenylahistin (labeled “11” in FIG. 3) and the modification of cyclo(Gly-Phe) with imidazole derivatives were useful to synthesized the derivatives of phenylahistin for analyzing the structural factors. Derivatives 12-15 were synthesized from phenylahistin by alkylation or reduction. As shown in FIG. 3, Reaction Scheme 2, compounds 12 and 13 were synthesized by hydrogenation of each enantiomer of phenylahistin over 10% palladium on carbon in MeOH under atmospheric hydrogen at room temperature. Two (2) hours of hydrogenation yielded derivative 12, in which the 1,1-dimethyl-2-propenyl group of phenylahistin was reduced, and further hydrogenation (24 h) of compound 12 yielded compound 13, in which the dehydrohistidine part was reduced. In this second reduction, only one diastereomer, which has the same polarity in optical rotation as that of compound 12, was obtained. The diastereoselective hydrogenation of compound 13 may be due to the steric hindrance of the phenyl ring of the Phe residue. 
     As shown in FIG. 4, Reaction Scheme 4, methylation of phenylahistin (labeled “1” in FIG. 4) with Mel and NaH in DMF gave mono- and tri-methylated compounds. The extent of the methylation could be controlled by reaction temperature rather than amounts of the reagents. Mono-methylated derivative 14 at the τ nitrogen of the imidazole ring, whose methylation site was detected by NMR analysis (note that the low filed shift of 8(N)—H (δ12.20 ppm) in the  1 H NMR spectrum was maintained in compound 14) and was predominantly obtained with equivalent of the reagents at −30° C., and a tri-methylated derivative 15 was obtained in the reaction with 30 equivalents of the reagents at room temperature. Compound 14 and 15 with the L-phenylalanine residue were separated by HPLC with a chiral column. 
     As shown in FIG. 5, Reaction Scheme 5, compounds 19 and 20 were synthesized from cyclo(Gly-Phe), compound 16, that was prepared by cyclization of H-Phe-Gly-OMe. After acetylation of the two amide nitrogens of the diketopiperazine ring in 16, 4(5)-imidazolecarboxaldehyde or 4-methyl-5-imidazolecarboxaldehyde (18) was introduced in the presence of lithium diisopropylamide (LDA) and hexamethylphosphoramide (HMPA), according to the method of Bond, R., et al.  Synthetic Commun.,  19, 2551 (1989), and subsequent dehydration with triflic anhydride-pyridine and deacetylation with aqueous NH 4 OH gave compounds 19 and 20, although less than 20% of racemization was observed in these steps. 
     According to X-ray crystallographic analyses of (+)-phenylahistin, as provided in Table 8, the stereochemistry of C6-C7 double bond was confirmed to be Z, and the existence of a hydrogen bond between N8-H and N3 was observed. This result was in good agreement with the observation in the NMR studies (low field-shift of N8-H (δ12.08 ppm). These findings suggested that two heterocycles, i.e., the diketopiperazine and imidazole rings, were fixed in the same plane by forming a pseudo three-ring structure. The benzyl group of the Phe residue was stacked over the diketopiperazine ring by sticking out from this plane, whose conformation is reported as the energetically most favored one in diketopiperazine with an aromatic amino acid residue, according to the method of Liwo, A. et al.  Tetrahedron Lett.,  26, 1873 (1985). The 1,1-dimethyl-2-propenyl group at the imidazole ring was also restricted its movement by the steric hindrance of a hydrogen atom at the β-position (C6) of the α,β-unsaturated His derivative. Therefore, it is indicated that the conformation of (+)-phenylahistin is highly restricted. Since the (−)-form of 11, i.e., the biologically active enantiomer, also takes the same conformational feature as that of its (+)-form only with the opposite configuration at the α-position of the Phe residue, this rigid conformation of phenylahistin may be important for the binding to the microtubule protein. 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Data of the x-ray crystallographic analysis of (+)-phenylahistin 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Crystal parameters 
                 — 
               
               
                   
                 Empirical formula 
                 C 20 H 22 N 4 O 2   
               
               
                   
                 Formula weight 
                 350.42 
               
               
                   
                 Crystal system 
                 tetragonal 
               
               
                   
                 Lattice parameters: 
                 a = 15.3509(7) Å 
               
               
                   
                   
                 c = 8.309(2) Å 
               
               
                   
                   
                 V = 1958.0(2) Å 3   
               
               
                   
                   
                 Z = 4 
               
               
                   
                 Space group 
                 P4 2  (#77) 
               
               
                   
                 Dcalc. 
                 1..189 g/cm 3   
               
               
                   
                 μ (CuKα) 
                 6.37 cm −1   
               
               
                   
                 Refinement parameters 
                 — 
               
               
                   
                 Reflections measured 
                 2229 
               
               
                   
                 Nonzero reflections 
                 1724 
               
               
                   
                 R-indexResiduals: R a   
                 0.057 
               
               
                   
                 Residuals: RW b   
                 0.067 
               
               
                   
                 Goodness of fit indicator c   
                 4.31 
               
               
                   
                   
               
               
                   
                   a Σ||Fo| − |Fc||Σ|Fo| 
               
               
                   
                   b [Σ w ((|Fo| − |Fc|) 2 /ΣwFo 2 )] 1/2   
               
               
                   
                   c [(Σ w (|Fo| − |Fc|) 2 /(No-Nv)] 1/2   
               
            
           
         
       
     
     To study the relationship between the ridgid plane structure of (−)-phenylahistin an d that compound&#39;s biological activity, the derivatives of (−)-phenylahistin were synthesized and the anti-microtubule effect on the polymerization of microtubule protein prepared from bovine brain and the anti-proliferative effect on P388 cells were studied, as described in prior examples. As described above, (−)-phenylahistin exhibits the colchicine-like inhibition of microtubule polymerization (IC 50 =25 μM). This inhibitory activity of (−)-phenylahistin was almost the same as that of colchicine (IC 50 =16 μM), although the anti-proliferative activity of (−)-phenylahistin (IC 50 =0.21 μM) was about 10 times less active than that of colchicine (IC 50 =0.03 μM). The difference of these activities is due either to the unaccounted-for biological activities of colchicine or to lower cellular permeability of (−)-phenylahistin in anti-proliferative assay, since (−)-phenylahistin has more hydrophilic structure than colchicine. Furthermore, as described above, (−)-phenylahistin with the L-Phe residue was 50 times more active than (+)-phenylahistin in anti-proliferative assays. (+)-Phenylahistin also exhibited relatively weak inhibitory activity against microtubule polymerization (15% inhibition at 200 μM) compared with (−)-phenylahistin. Thus, the orientation of the benzyl group of the phenylalanine residue is important to the biological activity of the compounds of the present invention. 
     As shown in Table 9, compound 12, in which the 1,1-dimethyl-2-propenyl group of phenylahistin was reduced, showed the same activity as phenylahistin, indicating that this double bond is not important for the anti-microtubule activity. However, compound (−)-13, in which the dehydrohistidine of (−)-12 was reduced, completely lost the inhibitory activity. Since this modification disrupts the substantially planar characteristic of the pseudo three-ring structure in the same plane as the phenylahistin backbone, this result suggests that, for the inhibitory activity, it is important that the two rings, i.e., diketopiperazine and imidazole rings, are fixed in the same plane. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Biological activity of phenylahistin, its derivatives, and selected 
               
               
                 structurally-related compounds. 
               
            
           
           
               
               
               
            
               
                   
                   
                 IC 50  (μM) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Microtubule Protein a   
                 P388 
                   
               
               
                   
                 compound 
                 polymerization 
                 proliferation 
                   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 (−)-11 
                 25 
                 0.21 
                 (±0.02) b   
               
               
                   
                 (+)-11 
                 &gt;200 [15%] c   
                 10 
                 (±1.5) 
               
               
                   
                 (−)-12 
                 30 
                 0.23 
                 (±0.05) 
               
               
                   
                 (+)-12 
                 &gt;200 [12%] 
                 19 
                 (±4.2) 
               
               
                   
                 (−)-13 
                 &gt;200 
                 &gt;200 
                   
               
               
                   
                 (+)-13 
                 &gt;200 
                 &gt;200 
                   
               
               
                   
                 14 
                 100 
                 0.95 
                 (±0.03) 
               
               
                   
                 15 
                 &gt;200 
                 160 
                 (±5.5) 
               
               
                   
                 19 
                 &gt;200 [11%] 
                 &gt;200 
                   
               
               
                   
                 20 
                 &gt;200 [9%] 
                 15 
                 (±4.5) 
               
               
                   
                 Cyclo(His-Phe) d   
                 &gt;200 
                 &gt;200 
                   
               
               
                   
                 colchicine d   
                 16 
                 0.031 
                 (±0.01) 
               
               
                   
                   
               
               
                   
                   a Microtubule protein was prepared from bovine brain by two cycle of assembly and disassembly method. The concentration of microtubule protein used for the assay was 1.5 mg/mL.  
               
               
                   
                   b Values are means (±SEM) of three experiments.  
               
               
                   
                   c The inhibition percentage at 200 μM was indicated in the parenthesis when the inhibition was observed, since the diketopiperazine derivatives are insoluble in the concentration more than 200 μM.  
               
               
                   
                 19 and 20 were chiral-purified.  
               
               
                   
                   d Cyclo(His-Phe) and colchicine are each commercially available; each was purchased from Sigma.  
               
            
           
         
       
     
     In further syntheses, the nitrogen atoms of phenylahistin were modified by methylation. Compound 14, in which the τ nitrogen of the imidazole ring of (−)-phenylahistin was methylated, was about 5 times less active than (−)-phenylahistin, suggesting this imidazole nitrogen probably participates in the binding with the microtubule protein or this methylation affords the structural hindrance to the conformation of the 1,1-dimethyl-2-propenyl group on the imidazole ring. Tri-methylated compound 15 almost lost the inhibitory activity. This result also suggests that the rigid plane conformation of (−)-phenylahistin is important to the anti-microtubule activity, since the methylation of a diketopiperazine nitrogen (N8) disrupts the formation of hydrogen bond on N8-H necessary for the rigid pseudo three-ring conformation. 
     The importance of an alkyl group at the 5-position on the imidazole ring to the biological activity of the compounds of the present invention was analyzed via the following analyses of compounds 19 and 20, which were also synthesized from cyclo(Phe-Gly), compound 16. In compounds 19 and 20, the 1,1-dimethyl-2-propenyl group of (−)-phenylahistin was replaced with a hydrogen atom or a methyl group, respectively. Compound 19, in which the 1,1-dimethyl-2-propenyl group was replaced with the hydrogen atom at this position, showed no inhibitory activity. Compound 20, in which the 1,1-dimethyl-2-propenyl group was replaced with a methyl group at this position, also showed no activity in the anti-microtubule assay, although weak inhibition on P388 cell proliferation was observed. This drastic decreased in biological activity in 19 indicates that an alkyl group with a proper length or a quaternary carbon at the 5-position of the imidazole ring is important for the activity. 
     Accordingly, the results of the x-ray crystallographic analysis and the biological evaluation of the phenylahistin derivatives elucidate a structural factor of phenylahistin necessary for its anti-microtubule activity. This factor is that the rigid and planar pseudo three-ring structure formed by a hydrogen bond are important for the biological activity of (−)-phenylahistin. 
     For this example, the microtubule protein polymerization assay was performed as follows. Microtubule protein was prepared from bovine brain tissue by two cycles of assembly and disassembly. Polymerization of microtubule protein was monitored by an increase in turbidity at 37° C. in microtubule assembly buffer containing 100 mM MES, 0.5 mM MgCl 2 , 1 mM EGTA and 1 mM GTP. The polymerization of microtubule protein was initiated by a temperature shift from 0° C. to 37° C. Turbidity was measured on a thermo-controlled spectrophotometer (Beckman DU-20, Fullerton, Calif.) at 360 nm. Compounds were dissolved in DMSO, which was used in all experiments at a final concentration of 2% (v/v). 
     For this example, the Alamar Blue™ assay was performed as follows. Exponentially growing P388 cells were seeded into 96-well tissue culture plates (5×10 3  cells/100 μl/well) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum for 16 h. Compounds (DMSO solution) were then added to each well at various concentrations, and the cells were cultured for an additional 48 h. Living cells were counted using Alamar Blue™ (BioSource International, Camarillo, Calif.), according to the method of Ahmed, S. A. et al.,  J. Immunol. Methods,  170, 211 (1994). 
     For this example, the Single-Crystal X-ray Diffraction Analysis was performed as follows. A pale yellow crystal of (+)-phenylahistin having approximately dimension 0.4×0.4×0.3 mm, which was crystallized from EtOAc, was used for single-crystal x-ray diffraction analysis. All x-ray measurements were made on a Rigaku AFC7R diffractometer with graphite monochromated Cukα radiation and a rotating anode generator. The crystal data and refinement parameters were summarized in Table 8. Of the 2229 reflections which were collected, 2064 were unique (Rint=0.019). The data were corrected for Lorentz and polarization effects. A correction for secondary extinction was applied. The structure was solved by direct methods and expanded using standard Fourier techniques. All calculations were performed using the teXscan crystallographic software package of Molecular Structure Corporation. 
     EXAMPLE 18 
     Syntheses and Physical Characterization of Phenylahistin Derivatives 
     The melting points of the phenylahistin derivatives described in the previous example were determined on a Mettler FP62 apparatus and are uncorrected.  1 H-NMR and  13 C-NMR spectra were recorded on JEOL GSX27OJ spectrometer. The spectra were recorded with tetramethylsilane (δ=0.0 for  1 H); DMSO-d 6  (δ=39.5 for  13 C); CDCl 3  (δ=77.0 for  13 C) as internal reference. Mass spectra (electrospray ionization, methanol as the mobile phase) were analyzed with Finnigan SSQ 7000 spectrometer. High-resolution fast atom bombardment mass spectra were analyzed with JEOL JMS-DX303 spectrometer. Infrared spectra were recorded on a JEOL JIR-5500 infrared spectrophotometer in KBr pellets. A silica-gel column chromatography was performed using Merck 70-230 mesh silica gel 60. Optical rotations were measured on a Horiba SEPA-200 polarimeter, and are given in units of 10 −1  deg cm 2  g −1 . Elemental analyses were performed by Fisons EA 1108 elemental analyzer. 
     The precise synthetic routes and physical characteristics of the phenylahistin derivatives described in the previous example are as follows. 
     (−)-Cyclo-[5-(1,1-dimethylpropyl)dehydrohistidinyl-L-phenylalanine] (−)-12: To a solution of (−)-phenylahistin (30 mg, 0.086 mmol) in MeOH (10 mL) was added 10 mg of 10% palladium on carbon and the mixture was stirred at room temperature for 2 h under hydrogen at atmospheric pressure. After the catalyst was filtered off and the filtrate was concentrated under reduced pressure, the residue was purified by column chromatography on silica (5 g) using CHCl 3 -MeOH (50:1) as an eluent. Desired fractions were collected and the solvent was evaporated. The residual white powder was recrystallized from EtOH-hexane to give 12 mg (39% of (−)-12 as a colorless solid: mp 224-226° C.; [α] D   25 −295(c=0.15, MeOH), UV (MeOH) nm 322 (ε 23300), 231 (ε 8830), 203 (ε 16800); IR (KBr) cm −1  3310, 3220, 2970, 1670, 1440;  1 H NMR (270 MHz, CDCl 3 ) δ12.09 (br s, 1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t, J=7 Hz 3H);  13 C NMR (67.5 MHz, CDCl 3 ) δ164.6, 159.9, 138.1, 135.5, 132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3, 36.2, 35.4, 27.9 (2C), 9.2; HRMS m/z 352.1926 (M + ) (calcd for C 20 H 24 N 4 O 2 : 352.1899). Anal. calcd for C 20 H 24 N 4 O 2 : C,68.16; H,6.86; N,15.90. Found: C,68.34; 6.78; 15.82. 
     (+)-Cyclo-[5-(1,1-dimethylpropyl)dehydrohistidinyl-L-phenylalanine] (+)-12: This compound was prepared according to the same procedure for the preparation of (−)-12. 44% yield from (+)-1; colorless solid; mp 222-223° C.; [α] D   25  +284(c=0.10, MeOH), UV (MeOH) nm 322 (ε 22800), 231 (ε 8720), 203 (ε 16300); IR (KBr) cm −1  3310, 3220, 2970, 1670, 1440;  1 H NMR (270 MHz, CDCl 3 ) δ12.09 (br s, 1H), 8.98 (br s. 1H), 7.56 (s, 1H), 7.29-7.38 (m, 5H), 6.87 (s, 1H), 5.67 (s, 1H), 4.34 (ddd, J=2, 3, 10 Hz, 1H), 3.51 (dd, J=3, 14 Hz, 1H), 2.29 (dd, J=10, 14 Hz, 1H), 1.74 (q, J=7 Hz, 2H), 1.40 (s, 6H), 0.74 (t, J=7 Hz 3H);  13 C NMR (67.5 MHz, CDCl 3 ) δ164.6, 159.9, 138.1, 135.5, 132.2, 132.1, 129.5 (2C), 129.1 (2C), 127.4, 123.6, 105.4, 57.2, 41.3, 36.2, 35.4, 27.9 (2C), 9.2; HRMS m/z 352.1932 (M + ) (calcd for C 20 H 24 N 4 O 2 : 352.1899). Anal. calcd for C 20 H 24 N 4 O 2 : C,68.16; H,6.68; N,15.90. Found: C,68.09; 6.87; 15.84. 
     (−)-Cyclo-[5-(1,1-dimethylpropyl)histidinyl-L-phenylalanine] (−)-13: This compound was prepared from (−)-12, according to the same procedure for the preparation of (−)-12, with 24 h of reaction time. 60% yield from (−)-12; white powder; mp 224-225° C.; [α] D   25 −96(c=0.16, MeOH), UV (MeOH) nm 257 (ε 194), 205 (ε 18100), IR (KBr) cm −1  3380, 3200, 2970, 1670, 1440;  1 H NMR (270 MHz, DMSO-d6) δ11.55 (br s, 1H), 8.25 (br s. 1H), 7.86 (br s, 1H), 741 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H), 3.94 (br d, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14,5 Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd, J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz 3H);  13 C NMR (67.5 MHz, CDCl 3 ) δ166.87,165.42, 135.93, 132.54, 131.58, 131.23, 130.32 (2C), 127.94 (2C), 126.61, 55.70, 54.31, 38.16, 35.04, 34.31, 31.85, 27.63, 27.57, 9.05; HRMS m/z 354.2098 (M + ) (calcd for C 20 H 24 N 4 O 2 : 354.2055). Anal. calcd for C 20 H 24 N 4 O 2 :1/3 H 2 O: C,66.64; H,7.46; N,15.54. Found: C,66.75; 7.41; 15.52. 
     (+)-Cyclo-[5-(1,1-dimethylpropyl)histidinyl-L-phenylalanine] (+)-13: This compound was prepared from (+)-12 according to the same procedure for the preparation of (−)-13. 46% yield from (+)-12; white powder; mp 226-227° C.; [α] D   25  +99(c=0.10, MeOH), UV (MeOH) nm 257 (ε 174), 205 (ε 18200), IR (KBr) cm −1  3380, 3200, 2970, 1670, 1440;  1 H NMR (270 MHz, DMSO-d 6 ) δ11.55 (br s, 1H), 8.25 (br s. 1H), 7.86 (br s, 1H), 7.41 (s, 1H), 7.29-7.14 (m, 5H), 4.24 (br s, 1H), 3.94 (br d, J=11 Hz, 1H), 3.11 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14,5 Hz, 1H), 2.82 (dd, J=15, 2 Hz, 1H), 1.45 (q, J=7 Hz, 2H), 1.32 (dd, J=15, 11 Hz, 1H), 1.13 (s, 3H), 1.12 (s, 3H), 0.60 (t, J=7 Hz 3H);  — C NMR (67.5 MHz, CDCl 3 ) δ166.87,165.42, 135.93, 132.54, 131.58, 131.23, 130.32 (2C), 127.94 (2C), 126.61, 55.70, 54.31, 38.16, 35.04, 34.31, 31.85, 27.63, 27.57, 9.05; HRMS m/z 354.2110 (M + ) (calcd for C 20 H 24 N 4 O 2 : 354.2055). Anal. calcd for C 20 H 24 N 4 O 2 :1/2 H 2 O: C,66.09; H,7.49; N,15.41. Found: C,65.80; 7.50; 15.30. 
     (−)-Cyclo-[(N im -methyl-5-(1,1-dimethyl-2-propenyl))dehydrohistidinyl-L-phenylalanine] (−)-14: To a solution of phenylahistin (200 mg, 0.57 mmol) in DMF (15 mL) was added 45 mg (1.88 mmol) of sodium hydride (NaH) (60% in mineral oil) in portions and the mixture was stirred at −30° C. for 10 min. To this mixture, 1.0 mL (17.1 mmol) of MeI was added dropwise and stirred at −30° C. for 2 h. 20 mL of saturated aqueous NH 4 Cl was added to the reaction mixture and the mixture was extracted three times with 50 mL of EtOAc. The combined organic layers were washed with saturated NaCl, dried over NA 2 SO 4  and concentrated in vacuo. The residual white powder (150 mg) was chromatographed over 20 g of silica gel and eluted with CHCl 3 -MeOH (50:1) as an eluent to give 130 mg (63%) of 14 as white powder: The (−)-form of 14 was isolated by HPLC using chiral column (CHIRALCEL OD, 10×250 mm) eluted with a mixture of hexane and ethanol (3:1) at a flow rate of 6.0 mL/min on a Waters system (600E series). 19% yield from 1; white powder; mp 214-215° C.; [α] D   25 −285(c=0.30, MeOH), UV (MeOH) nm 317 (ε 25400), 232 (sh, ε 8640), 204 (ε 16800); IR (KBr) cm −1  3200, 2980, 1680, 1440;  1 H NMR (270 MHz, CDCl 3 ) δ12.20 (br s, 1H), 7.39 (s. 1H), 7.38-7.24 (m, 5H), 7.09 (s, 1H), 6.01 (dd, J=18, 9 Hz, 1H), 5.68 (br s, 1H), 5.14 (d, J=9 Hz, 1H), 5.00 (d, J=18 Hz, 1H), 4.33 (m, 1H), 3.66 (s. 3H), 3.50 (dd, J=14, 3 Hz, 1H), 2.92 (dd, J=14, 10 Hz, 1H), 1.59 (s, 6H);  13 C NMR (67.5 MHz, CDCl 3 ) δ164.6, 160.1, 145.6, 138.5, 136.5, 135.6, 134.6, 129.5 (2C), 129.1 (2C), 127.4, 124.0, 112.7, 106.6, 57.1, 41.3, 39.2, 34.9, 28.9, 28.8; HRMS m/z 364.1909 (M + ) (calcd for C 21 H 24 N 4 O 2 : 364.1899). Anal. calcd for C 21 H 24 N 4 O 2 .1/4 H 2 O: C,68.36; H,6.69; N,15.19. Found: C,6844; H, 6.67; N, 15.01. 
     (−)-Cyclo-N,N′-dimethyl-[(N im -methyl-5-(1,1-dimethyl-2-propenyl))dehydro-histidinyl-L-phenylalanine] (−)-15: This compound was prepared from phenylahistin with 10 equivalents of NaH and 30 equivalent of Mel at room temperature according to the same procedure for the preparation of (−)-14. For the purification of (−)-15, a mixture of hexane and ethanol (4:1) was used as an eluent in the chiral column HPLC mentioned in the preparation of (−)-14. 8% yield from phenylahistin: white powder; mp 95-98° C.; [α] D   25 −632(c=0.50, MeOH), UV (MeOH) nm 287 (ε 11100), 203 (ε 15900); IR (KBr) cm −1  2900, 1690, 1640, 1380;  1 H NMR (270 MHz, CDCl 3 ) δ8.76 (br s, 1H), 7.33-7.21 (m, 5H), 7.08 (s, 1H), 6.00 (dd, J=18, 11 Hz, 1H), 5.28 (d, J=11 Hz, 1H), 5.08 (d, J=18 Hz, 1H), 4.19 (dd, J=10,4 Hz, 1H), 3.86 (s, 3H), 3.38 (dd, J=14, 4 Hz, 1H), 3.13 (dd, J=14, 10 Hz, 1H), 2.92 (s, 3H), 2.55 (s, 3H), 1.60 (s, 3H), 1.57 (s, 3H);  13 C NMR (67.5 MHz, CDCl 3 ) δ166.8, 160.2, 143.5, 137.8, 136.5, 134.8, 129.4 (2C), 128.8 (2C), 127.2, 124.6, 114.4, 105.6, 65.9, 40.6, 38.8, 36.6, 34.9, 28.4, 28.1; HRMS m/z 392.2223 (M + ) (calcd for C 23 H 28 N 4 O 2 : 392.2212). Anal. calcd for C 23 H 28 N 4 O 2 : C,70.38; H,7.19; N,14.27. Found: C,70.26; H, 7.30; N, 14.27. 
     Cyclo-[glycinyl-L-phenylalanine] 16: To a solution of H-Phe-Gly-OMe, which was prepared from Boc-Phe-Gly-OMe with 4N HCl-dioxane (20 g, 59 mmol), in MeOH (100 mL) was refluxed for 16 h. The white precipitate occurred during reflux was washed with 10 mL of MeOH for three times and collected to give 7.5 g (62%) of 16 as a white powder, mp 262-263° C., (decomp.); [α] D   25 +60 (c=0.15, DMSO), UV (MeOH) nm 257 (ε 101), 206 (ε 5770); IR (KBr) cm −1  3340, 3200, 3060, 1680, 1470, 1340;  1 H NMR (270 MHz, DMSO-d 6 ) δ8.16 (br s, 1H), 7.90 (br s. 1H), 7.32-7.15 (m, 5H), 4.07 (br dd, J=7,4 Hz 1H), 3.35 (dd, J=18, 3 Hz, 1H), 3.10 (dd, J=14, 4 Hz, 1H), 2.88 (dd, J=14, 5 Hz, 1H), 2.75 (d, J=18 Hz, 1H),  13 C NMR (67.5 MHz, DMSO-d 6 ) δ167.1,165.5, 136.0, 130.1 (2C), 128.1 (2C), 126.8, 55.5, 43.7, 38.8; MS (ESI) m/z 205 (M+H) + ; Anal. calcd for C 11 H 12 N 2 O 2 .1/5H 2 O: C,63.57; H,6.01; N,13.48. Found: C,63.85; H, 5.86; N, 13.40. 
     Cyclo-N,N′-diacetyl-[glycinyl-L-phenylalanine] 17: The mixture of compound 16 (0.5 g, 2.45 mmol) and fused sodium acetate (201 mg, 2.45 mmol) in acetic anhydride (10 mL) was heated for 16 h at 100° C. under nitrogen. After acetic anhydride was removed in vacuo at 45° C., the residue was solved in EtOAc and the resulted organic layer was washed with saturated NaCl, dried over Na 2 SO 4  and 17 was crystallized from EtOAc. 88% yield (0.62 g); colorless solid; mp 84-85° C.; [α] D   25  +7.8 (c=0.52, MeOH); UV (MeOH) nm 209 (ε 20400); IR (KBr) cm −1  1720, 1400, 1380, 1240;  1 H NMR (270 MHz, CDCl 3 ) δ7.33-7.26 (m, 3H), 7.08-7.05 (m, 2H), 5.44 (t, J=5 Hz, 1H), 4.49 (d, J=19 Hz, 1H), 3.35 (dd, J=14, 5 Hz, 1H), 3.20 (dd, J=14, 5 Hz, 1H), 2.58 (s, 3H), 2.55 (s, 3H), 2.48 (d, J=19 Hz, 1H);  13 C NMR (67.5 MHz, CDCl 3 ) δ171.2, 171.0, 167.9, 166.0, 134.3, 129.7 (2C), 129.1 (2C), 128.2, 59.0, 46.0, 38.7, 27.1, 26.8; MS (ESI) m/z 311 (M+Na) + ; Anal. calcd for C 15 H 15 N 2 O 4 : C, 62.49; H, 5.59; N, 9.72. Found: C, 62.50; H, 5.50; N, 9.67. 
     Cyclo-[dehydrohistidinyl-L-phenylalanine] 19: To a solution of 17 (311 mg, 0.93 mmol) in dimethoxyethanol (DME) (10 mL) was added 0.6 mL of 2M lithium diisopropylamide (LDA, 1.2 mmol) and the mixture was stirred at −70° C. for 10 min. To this solution was added a solution of 4(5)-formylimidazole (120 mg, 1.25 mmol, Maybridge Chemical Co. Ltd., Cornwall, U.K.) in HMPA (4 mL)-DME (6 mL) at −70° C. This mixed solution was allowed to warn to −30° C. After stirring for 30 min at this temperature, a triflic anhydride (370 μL, 2.2 mmol) and pyridine (180 μL, 2.2 mmol) were added and the solution was allowed to warm to room temperature. After additional stirring for 1 h, aqueous ammonia (3 mL) was added and stirred for 14 h at room temperature. The reaction mixture was extracted with CHCl 3  for three times, dried over Na 2 SO 4  and concentrated in vacuo. The resultant residue was purified by HPLC with a reverse phase column (Waters, μBondasphere 19×150 mm, 10 μm, C-18) employing a gradient from 60 to 80% CH 3 CN in 0.1% TFA at a flow rate of 17 mL/min on a Waters system (600E series). However, since purified compound 19 contained 28% of a racemized compound with D-phenylalanine (44% ee), further purification by HPLC with a chiral column was performed using the same procedure described for the purification of (−)14. 8% yield from 7; white powder; mp 208-209° C. [α] D   25  −257 (c=0.21, DMSO); UV (MeOH) nm 307 (ε 16700); IR (KBr) cm −1  3400, 3120, 1680, 1440;  1 H NMR (270 MHz, DMSO-d 6 ) δ12.88 (br s, 1H), 11.09 (br s, 1H), 8.44 (s, 1H), 8.13 (br s, 1H), 7.48 (s, 1H), 7.24-7.11 (m, 5H), 6.25 (s, 1H), 4.48 (m, 1H), 3.19 (dd, J=14, 4 Hz, 1H), 2.95 (dd, J=14, 5 Hz, 1H);  13 C NMR (67.5 MHz, DMSO-d 6 ) δ164.8, 158.6, 135.8, 135.5, 132.3, 130.0 (2C), 128.0 (2C), 126.7, 125.6, 118.3, 101.8, 55.9, 38.9; (overlapping DMSO-d 6 ); HRMS m/z 282.1084 (M + ) (calcd for C 15 H 14 N 4 O 2 : 282.1117). Anal. calcd for C 15 H 14 N 4 O 2 —CF 3 COOH: C, 51.52; H, 3.81; N, 14.14, Found: C, 51.15; H, 3.62; N, 1384. 
     Cyclo-[(5-methyl)dehydrohistidinyl-L-phenylalanine] 20: This compound was prepared from compound 17 with 4-methyl-5-imidazolecarboxaldehyde according to the same procedure for the preparation of 19. 3% yield from 17; colorless solid; mp 285-286° C. (decomp); [α] D   25  −267° (c=0.21, DMSO); UV (MeOH) nm 319 (ε 22800); IR (KBr) cm −1  3400, 3180, 1680, 1450;  1 H NMR (270 MHz, DMSO-d 6 ) δ11.50 (br s, 1H), 8.35 (br s, 1H), 7.74 (s, 1H), 7.24-7.14 (m, 5H), 6.20 (s, 1H), 4.48 (m, 1H), 3.33 (br s, 1H), 3.20 (dd, J=14, 4 Hz, 1H), 2.93 (dd, J=14, 5 Hz, 1H), 2.19 (s, 1H);  13 C NMR (67.5 MHz, DMSO-d 6 ) δ164.2, 158.7, 135.6, 134.6, 132.3, 130.0 (2C), 128.0 (2C), 127.5, 126.6, 123.4, 101.7, 55.9, 38.7, 8.9; High-resolution MS m/z 296.1261 (M + ) (calcd for C 16 H 16 N 4 O 2 : 296.1273). Anal. calcd for C 16 H 16 N 4 O 2  1/5H 2 O; C, 64.07; H, 5.51; N, 18.68. Found: C, 64.39; H, 5.65; N, 18.28. 
     EXAMPLE 19 
     Exemplary Formulation Administered Intravenously, by Drip, Injection, or the Like 
     Vials containing 5 g of powdered glucose are each added aseptically with 10 mg of the invention compound and sealed. After being charged with nitrogen, helium or other inert gas, the vials are stored in a cool, dark place. Before use, the contents are dissolved in ethanol and added to 100 ml of a 0.85% physiological salt water solution. The resultant solution is administered as a method of inhibiting the growth of a cancerous tumor in a human diagnosed as having such a tumor at between approximately 10 ml/day to approximately 1000 ml/day, intravenously, by drip, or via a subcutaneous or intraperitoneal injection, as deemed appropriate by those of ordinary skill in the art. 
     EXAMPLE 20 
     Exemplary Formulation to be Administered Orally 
     A mixture obtained by thoroughly blending 1 g of the compound of the invention, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method. The granules are thoroughly dried and sifted to obtain a granule preparation suitable for packaging in bottles or by heat sealing. The resultant granule preparations are orally administered at between approximately 100 m/day to approximately 1000 ml/day, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans. 
     EXAMPLE 21 
     Use of Phenylahistin to Treat a Human Suffering from Cancer 
     PLH is administered according to the formulation of EXAMPLE 17 to a human patient afflicted with lung cancer. PLH is administered at between approximately 10 mg per day to approximately 1000 mg per day, and in an preferred amount of 50 mg per day, until the growth of the tumor is inhibited. 
     EXAMPLE 22 
     Use of (−)-Phenylahistin to Treat Human Suffering from Cancer 
     (−)-PLH is isolated according to the method of EXAMPLE 6, substantially purified, and administered according to the formulation of EXAMPLE 17 to a human patient, afflicted with ovarian cancer, at between approximately 10 mg per day to approximately 100 mg per day, and in a preferred amount of 50 mg per day, until the growth of the tumor is inhibited. 
     EXAMPLE 23 
     Anti-tumor Effect of 2-Isopropyl-(−)-Phenylahistin 
     2-isopropyl-(−)-phenylahistin, i.e., compound 3 of EXAMPLE 7, is synthesized according to the method of EXAMPLE 7, substantially purified, and administered according to the formulation of EXAMPLE 17 to a human patient afflicted with breast cancer at between approximately 10 mg per day to approximately 100 mg per day, and in a preferred amount of 50 mg per day, until the growth of the tumor is inhibited. 
     EXAMPLE 24 
     Synthesis of 2-Dichloroisopropyl-(−)-Phenylahistin 
     2-dichloroisopropyl-(−)-phenylahistin is synthesized in the following manner: (−)-phenylahistin, compound 1 of EXAMPLE 7, is placed in the presence of Cl 2  and in the presence of water and heat to yield 2 dichloroisopropyl-(−)-phenylahistin. 
     EXAMPLE 25 
     Anti-tumor Effect of 2-Dichloroisopropyl-(−)-Phenylahistin 
     2-dichloroisopropyl-(−)-phenylahistin, as synthesized in EXAMPLE 22, is substantially purified and administered according to the formulation of EXAMPLE 17 to a human patient afflicted with Lewis Lung Carcinoma at between approximately 10 mg per day to approximately 100 mg per day, and in a preferred amount of 50 mg per day, until the growth of the tumor is inhibited. 
     EXAMPLE 26 
     Use of a Combinatorial Chemistry Screening Library 
     Synthetic studies of PLH are conducted to obtain more potent and less toxic agent. According to the method described by Gordon and Steele, J. Bioorg. Med. Chem. Letters (1995)., 5, 47-50, phenylahistin are its derivatives are screened using an efficient solid phase combinatorial synthesis for a typical diketopiperazine library. By using such technique, phenylahistin is utilized as a prototype for the development of promising new antitumor agents.