Abstract:
Modified actinomycin compounds that incorporate azetidine in the polypeptide component of the actinomycin molecule are disclosed. These compounds are called azetomicins and specifically incorporate an azetidine derivative in the 3&#39; peptide position in at least one of the two polypeptides normally present in actinomycins. The azetomicins are produced by bio-synthetic means from Streptomyces antibioticus that are cultured on a medium including azetidine-2 carboxylic acid and derivatives thereof.

Description:
BACKGROUND OF THE DISCLOSURE 
     The invention disclosed herein was made during the performance of work under a research grant from the U.S. Public Health Service. 
    
    
     This is a division of Ser. No. 605,425, filed Aug. 18, 1975, now U.S. Pat. No. 4,053,460. 
    
    
     Actinomycin drugs have been known since the early 1940&#39;s when Waksman and Woodruff isolated and described the first actinomycin. The actinoycins are a group of antibiotics that occur naturally as metabolic products of the growth of Streptomyces antibioticus (formerly known as Actinomyces antibioticus) and related species, such as S.chrysomallus, S.parvullus. 
     The actinomycins are orange to red antibiotics, that are highly toxic to most animal species. However they have found usage in nontoxic dosages principally because of their antineoplastic effects. Several actinomycins, specifically, Actinomycin D (or C 1 ), and C 3  are highly effective chemotherapeutics in the treatment of Wilms&#39; tumor, trophoblastic tumors and rhabdomyosarcoma. Recently, further successes have been achieved by utilizing the actinomycins in combination with other drugs against various types of neoplastic disease/ disease. 
     In addition, it has been determined that the actinomycin molecule binds to desoxyribonucleic acid (DNA) and thereby interferes with the synthesis of ribonucleic acid (RNA). This phenomenon has also given rise of the use of actinomycins as a tool in the study of molecular and cell biology. 
     Structural studies have revealed that the actinomycin molecule is a chromopeptide, i.e., the molecule contains a chromophore moiety that is linked to a peptide moiety. 
     The chromophore has been shown to a 3-amino-1, 8-dimethyl-2-phenoxazone-4, 5-dicarboxylic acid derivative, or: ##STR1## more commonly referred to as &#34;actinocin&#34;. The chromophore at its two carboxyl sites in turn carries, as amides, two pentapeptides, whose amino acid sequences may be identical, one with the other, in which case the actinomycin is referred to as an &#34;isoactinomycin&#34;; or, when one pentapeptide differs from the other, as an &#34;antisoactinomycin&#34;. 
     While the peptides may have some differences in their amino acid sequences, the actinomycins invariably have but five amino acid moieties in the peptide chains. In addition the amino acids linked to the 4- and 5- carboxyls by amide bonds are always L-threonine, whose hydroxyl is always lactonized with the carboxyl of the fifth amino acid on the peptide chain. The second amino acid may be D-valine or D-allo-isoleucine, while the third may be L-proline, L-γ-hydroxyproline, L-γ-ketoproline, pipecolic acid (not a naturally occurring actinomycin), or sarcosine. The fourth amino acid is always sarcosine, while the fifth may be L-N-methylvaline or L-N-methylisoleucine. As used herein, reference to the free form of an amino acid, e.g., valine, may also be taken to refer to the peptide form, e.g., valyl, etc. Thus, a typical abbreviated structure representation of an actinomycin (D or C 1 ) is: ##STR2## 
     The primed numbers to the right are conventional notations indicating the position of the amino acid on the peptide chain. 
     Although the amino acids within the peptide substituents may be varied only within narrow limits as noted above, the existence of numerous actinomycins is possible because of the unusual structure. Varying the substituents on the chromophore, and/or on the amino acids in the peptide chain can lead to literally hundreds of actinomycin variations and analogues. A great many such variations have been produced both by biosynthesis and by chemical synthesis so that they can be studied as possible valuable materials for chemotherapy applications. Unforturnately, to date, such variations have shown activies of a low order, or no biological activity at all, with a few exceptions of analogues produced by biosynthesis. Generally, however, the early identified naturally produced actinomycins exhibit the highest activities for biological usage. 
     BRIEF DESCRIPTION OF THE INVENTION 
     A new class of actinomycin analogues has been produced and preliminarily tested. The tests indicate that such analogues exhibit excellent biological activity of an order that promises a high probability that this class of actinomycin analogues may be useful in the therapy of human diseases of the neoplastic variety. There is further indication that they may find use an immuno-suppressive drugs. These analogues have also exhibited cytotoxic activity against newly fertilized ova, thus suggesting use in contraceptive therapy. In any event the analogues have already shown significant effect against various types of neoplastic diseases in both mice and virus induced neoplastic disease in chickens. Inhibitory activity against the growth of a cross section of Gm +  bacteria has also been shown by these analogues. 
     Specifically the class of actinomycin analogues of the invention are those analogues wherein azetidine-2- carboxylate and/or its derivatives are substituted for proline normally occupying the 3&#39; position on the peptide moieties attached to actinomycin chromophores. The azetidine substituent may be present in either one or both of the peptide moieties. In an abbreviated representation, such analogues are: ##STR3## 
     The above analogues are referred to as Azetomicin I, with the I indicating the presence of a single azetidine-carboxylate substituent on the molecule. It will be further noted there are two isomeric Azetomicin I&#39;s. 
     The analogues may further comprise the disubstituted or bis-molecules, for example: ##STR4## 
     Such analogues are referred to as Azetomicin II. 
     The Azetomicins are produced biosynthetically by growing Streptomycetes antibioticus in a medium that includes the desired azetidine compound. Such a process yields both Azetomicin I and Azetomicin II compounds. The Azetomicins and associated actinomycins are separated, one from the other, by chromatographic techniques. 
     The Azetomicins show unusually high biological activities when tested against the types of neoplasias used in screening procedures for antinoeplastic drugs. 
     It is therefore an object of the invention to provide Azetomicins. 
     It is another object of the invention to provide mono-azetidine-carboxyl substituted Azetomicins. 
     It is another object of the invention to provide di-azetidine-carboxyl substituted Azetomicins. 
     It is still another object of the invention to provide a process for producing Azetomicins. 
     It is another object of the invention to provide Azetomicins exhibiting high biological activities. 
     Other objects and advantages of the invention will be apparent from the following specification and the claims appended hereto. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The compositions of the invention comprise actinomycin analogues wherein the 3&#39; amino acids on the peptide moieties contain azetidine groups. The azetidine groups may appear in either one or both 3&#39; positions since there are two peptide moieties attached to the actinomycin chromophore. If the azetidine group appears in only one 3&#39; position the compound is referred to as Azetomicin I, or if in both 3&#39; positions, as Azetomicin II. 
     The azetidine substituent group comprise a hetero-cyclic four-membered ring structure with a carbonyl attached to the carbon in the 2 position as follows: ##STR5## wherein the R 1  substituent may be any suitable group that facilitates incorporation of azetidine into the peptide chain. It has been found that the simple carboxylic acid group most easily facilitates incorporation of the azetidine into the peptide chain by biosynthesis with S. antibioticus. Thus L-azetidine-2-carboxylic acid, ##STR6## is the preferred starting material in the biosynthesis of Azetomicins. 
     A number of variations of azetidine-2-carboxylic acid might be utilized to produce azetomicin analogues. Specifically, substituents, such as halogens, ketones, hydroxyl and methyl groups, etc., may be added to the carbon in the third position on the azet ring thus: ##STR7## where x is any of the aforestated substituent groups. However azetomycins already studied and incorporating the unsubstituted azetidine ring exhibit high biological activities. 
     The formula of Azetomicin I is as follows: ##STR8## wherein the double arrows indicate that each peptide chain may occur on either side of the chromophore, i.e., there are 2 Azteomycin I isomers. It should be also noticed that proline occupies the 3&#39; position on the corresponding peptide chain. However there are a great number of proline analogues, including, for instance, ##STR9## wherein y is a halogen, an hydroxyl, or a ketone, etc. 
     It has also been found that pipecolic acid may take the the place of the proline 3&#39; substituent on the peptide chain. The Pipecolic acid substituent is: ##STR10## and in the peptide, the carboxyl is connected to the adjacent 4&#39; sarcosine, while the nitrogen is connected to the adjacent 2&#39; valine. There are many pipecolic based variations, as by way of example: ##STR11## wherein x is a halogen, an hydroxyl, a ketone (═O), a methyl, carboxyl, etc; or: ##STR12## whereby y is a halogen, an hydroxyl, a ketone, or a methyl group, etc. 
     Further variations in the Azetomycins are possible, such as, for example, elsewhere on the peptide chains. There the 2&#39; amino acid, that is most usually D-valine, may be allo-isoleucine on either one or both peptide chains. Additionally, the chromaphore itself can be varied by substituting heteroatoms for carbons in some ring positions, e.g.: ##STR13## while --NO 2  or --NH 2  may be added on to the 7- carbon; or a halogen may be added on to various position carbons. 
     The Azetomicin II molecule is identical with the Azetomicin I molecule except that a second azetidine moiety is substituted in place of the 3&#39; proline on the second peptide chain. Azetomicin II is thus anisomeric with respect to the 3&#39; position since both peptides are identical in structure. However, should different amino acids be present in the other peptide positions, then, of course, isomers will exist. 
     Variations are also possible in the Azetomicin II molecule, in that substituted azetidines may be used; the 2&#39; amino acids may be varied as noted above; and the chromophore itself may have the above indicated substituents added thereto. 
     In all, there appear to be over 1000 possible variants to the azetomycin molecules; and anyone, or all, may exhibit biological activities. 
     The production of azetomycins can be prepared by chemo-synthetic methods or by biosynthesis utilizing Streptomyces antibioticus (or other related species), which is also commonly used in producing the closely related actinomcyins. It has been observed that very often the 3&#39;-prolyl moiety appearing in actinomycins can be replaced by an exogenously added amino acid when S. antibioticus is actively synthesizing actinomycins. This technique is applicable in the production of azetomycins. 
     In brief, S. antibioticus is grown on an enzyme-hydrolyzed protein medium to its maximum growth and the culture is then separated from the growth medium by centrifuging or filtering. The collected culture, S. antibiotieus, is then placed in an actinomycin growth medium to which pure L-azetidine-2 carboxylate (or D,L-azetidine-2-carboxylate) has been added. The S. antibioticus is thus allowed to produce a mixture of actinomycins and azetomycins. The actinomycins and azetomycins are extracted from the growth medium with ethyl acetate and the various compounds are then separated by chromotographic techniques. The individual pure compounds are thus obtained for further use. 
     More specifically, the following is an example of the production of the azetomycins: 
     The initial growth of S. antibioticus was developed on Noble agar-beef extract enzyme-hydrolyzed protein medium (A blood-agar medium can also be used). A typical medium ws 2.5 gm. N-Z-amine plus 1.0 gm. beef extract plus 100 ml. tap water, adjusted to pH 7.1 and autoclaved 7-15 minutes at 121° C. 
     The S. antiobioticus was innoculated into the prepared medium and the culture was incubated for approximately 47 hours, at which time the S. antibioticus was harvested at about 15,000 × g. The cell pellet was washed in isotonic saline and thereafter was placed in the azetomycin production medium which contained 100-250 mc gm/ml L-azetidine-2-carboxylate, L-glutamic acid hydrochloride (2-5 gm/l), D-galactose, D-glucose, Zn ++ , Fe ++ , Ca ++ , and Mg ++ . The medium was buffered to pH 7.2 with potassium phosphate-sodium hydroxide. 
     The culture medium was then incubated for from 3-6 days at which time production was complete and the medium was a clear yellow color. 
     The S. antibioticus was then separated from the production medium by filtration (it can be re-innoculated into a fresh production medium to produce another batch of azetomycins without further growth). The S. antibioticus-free production medium was then extracted with an equal volume of ethyl acetate, and the ethyl acetate was separated and evaporated to a small volume having a deep orange-red color. To store the azetomycin-actinomycin product, the acetate ester solution is evaporated to dryness and the resultant powder is redissolved in acetone and stored in the dark under refrigeration. 
     To separte the azetomycins, a two phase mixture of the sodium salt of 3-methyl salicylate with ethyl acetate and butyl ether (4:3:1) was prepared and utilized to resolve the mixed azetomycin-acetinomycin product. The solution was then processed on a descending paper chromatograph. The azetomycin and actinomycin bands separated quite clearly by this method. Column chromatography can also resolve the azetomycins on a larger scale. 
     The individual azetomicin bands were then cut out and the product eluted therefrom with 90% methanol. The azetomicin-methyl salicylate mixture was dried by evaporating the methanol. The resultant dried product was then dissolved in sodium carbonate solution and then shaken with chloroform in a separatory funnel to transfer the azetomicin into the chloroform phase. The chloroform extraction was repeated until the chloroform phase was colorless, the chloroform fractions were washed with water and then combined and evaporated to dryness. The resultant pure azetomicin was redissolved in a minimum amount of acetone for storage. 
     All chromatographic separations and storage of the resultant products were done in the dark. 
     The major products of interest were determined to be Azetomicin I and Azetomycin II. They were stable after storage at 0°-25° C in aqueous solution in the pH range of 5-8 for at least 5 days when maintained in the dark. 
     In the absence of L-azetidine-2-carboxylate in the production medium, the main product contains two 3&#39;-prolyl moieties. Thus the product corresponds to Actinomycin D. In the presence of L-azetidine-2-carboxylate in the production medium additional compounds are produced. In one of these the prolyl-azet ratio is 1:1 and quantitatively there is one less prolyl per molecule. This product corresponds to Azetomicin I. In another product no prolyl moiety is detectable, and there are two azetidinyl-carboxyl moieties per molecule. This product corresponds to Azetomicin II. In both Azetomicin I and II all other analyses are identical to the Actinomycin bis 3&#39;-prolyl compound. 
     BIOLOGICAL TESTING 
     Both Azetomicin I and Azetomicin II, produced by the noted biosynthesis method, were tested for biological activities. Since the related actinomycins have shown utility in treatment of neoplastic disease, both Azetomcyins were subjected to screening tests devised to reveal promising antineoplastic drugs. 
     Some samples of Azetomicin I and Azetomicin II were sent to the National Cancer Institute to be tested in the National Screening Center. 
     One of the most effective preclinical screening devices is a test of the drug agent&#39;s ability to produce an increased life span (% ILS) in mice previously infected with well defined types of cancer. The National Cancer Institute has found that drugs which increase the longevity of mice previously implanted with L-1210 leukemia, or P-388 leukemia, or B-16 melanoma by over 50%, i.e., % ILS = 50+%, tend, with a high probability, to be clinically active and useful in man. It has also been found that drugs which increase mouse life span over 100% are far less frequent and tend to be more frequently adapted for clinical use. Out of literally hundreds of thousands of potential agents screened for anticancer activity, only a small handful have the potency to increase the life span of leukemic mice over 200%, and, to date, almost all of them are accepted among the thirty or so clinically useful anticancer drugs; however many such clinically useful anticancer drugs do not show this much activity. 
     The National Cancer Institute conducted a screening test on samples of both Azetomicin I (3&#39; prolyl, 3&#39; azet) and Azetomicin II (3&#39;, 3&#39; azet) as well as on Dactinomycin (3&#39;, 3&#39; prolyl) that is also known as Actinomycin D or C 1 . The Azetomicin and Dactinomycin molecules are identical, with the exception of the 3&#39; amino acid position on the peptides, wherein the 3&#39; positions, in the case of Dactinomycin are both prolyl; in the case of the Azetomicin I are prolyl and azetidine-2-carboxyl; and in the cae of Azetomicin II are both azetidine-2-carboxyl. 
     The summary results of this test on mice implanted with P388 leukemia are set forth in Table I: 
     
                                           TABLE I__________________________________________________________________________P 388 LEUKEMIA : QD AT DAYS 1-9 TREATMENT            SURVIAL TIME     60 DAYDRUG      Mg/Kg/Inj.            MEDIAN                  (RANGE)                       5 ILS*                            &#34;CURES&#34;__________________________________________________________________________CONTROLS  0      10 DAYS                   (7-15)                       --   0DACTINOMYCIN     0.1    21.5   (8-40)                       115  12%     0.05   28    (13-39)                       180  0AZETOMICIN-II     0.1    23     (6-39)                       130  12%     0.05   22.5  (15-28)                       125  0AZETOMICIN-I     0.2    60+   (24-48)                        500+                            60%     0.1    25.5  (21-38)                       155  0     0.05   25    (19-33)                       150  0ADVANCED P388 LEUKEMIA : Q4D × 3 AT DAYS 5, 9, 13 TREATMENTCONTROLS  0      10 DAYS                   (7-16)                       --   0DACTINOMYCIN     0.15   19.5  (11-29)                        95  0AZETOMICIN-II     0.6    25    (21-33)                       150  0AZETOMICIN-I     0.6    37    (25-55)                       270  0__________________________________________________________________________ *ILS = increased life span. 
    
     From Table I, Azetomcyin II is seen to be roughly comparable in activity to Dactinomycin, but Azetomycin I has clearly greater activity than both. 
     A sample of Azetomicin I was tested on mice infected with advanced Ridgeway osteogenic sarcoma. Of 10 animals treated on a 12th day only injection schedule, complete regression was noted in all 10 animals. These results have been compared with the results obtained in treating Ridgeway osteogenic sarcoma implanted mice with a number of clinically accepted anticancer drugs. Data supplied by Dr. F. Schabel and his coworkers at Southern Research Institute has been collected in Table II below, with the results from Azetomicin I testing also noted: 
     
                                           TABLE II__________________________________________________________________________DRUG-INDUCEDREGRESSION OF ADVANCED RIDGEWAY OSTEOGENIC SARCOMA        (DAYS) REGRESSION (Number/Total)                                &#34;CURES&#34;ANTICANCER DRUG*        SCHEDULE               50+%-PARTIAL                         COMPLETE                                (Number/Total)__________________________________________________________________________ADRIAMYCIN   12     7/8       7/8    0/8CYCLOPHOSPHAMIDE        12     8/8       8/8    3/8L-SARCOLYSIN 12     8/8       7/8    4/8BCNU         12     0/8       0/8    0/8CCNU         12     0/8       0/8    0/8DTIC         12      0/10      0/10   0/10MITOMYCIN-C  12      0/10      0/10   0/10CHLORAMBUCIL 12      0/10      0/10   0/10THIO-TEPA    12      0/10      0/10   0/10L-ASPARAGINASE        12      0/10      0/10   0/10MITHRAMYCIN  12     0/7       0/7    0/7BGG          12      1/10      1/10   0/10VINCRISTINE  12,16,20               10/10      8/10   0/10ARA-C        12,12,20(q3h)                7/10      3/10   0/10PREDNISONE   12,16,20                0/10      0/10   0/10METHOTREXATE 12,16,20,24                0/10      0/10   0/10AZACYTIDINE  12,16,20,24               0/7       0/7    0/7FLUOROURACIL 12,16,20,24                8/10      5/10   0/10MERCAPTOPURINE        12-22   9/10      6/10   0/10DAUNOMYCIN   12-17  9/12       3/12   0/12PROCARBAZINE 12-20   0/10      0/10   0/10AZETOMICIN-I 12     10/10     10/10  10/10__________________________________________________________________________ *All are clinically accepted anticancer drugs except AZETOMYCINS 
    
     In another test, both Azetomicin I and Azetomicin II, were injected once into chicks infected with Rous sarcoma virus. The development of visible tumors was decreased by over 80% as compared with untreated or saline injected controls. 
     In a further test involving a cross section of gram-positive and gram-negative bacteria, Azetomicin I provided to be 70% as active as, but substantially less toxic/mg. than, Actinomicin D, and Azetomicin II proved to be 50% as active, and substantially less toxic/mg., against the growth of Bacillus subtilis, Staphylococcus aureus and Staphylococcus albus.