total synthesis of anthramycin nears
TRANSCRIPT
RESEARCH
Total synthesis of anthramycin nears Only conversion of lactam to carbinolamine lacking as chemists pin down absolute configuration of the antitumor agent
CHALLENGE. Hoffmann-La Roche chemists Fausto Schenker, Willy Leimgruber, and Andrew Batcho (left to right) have completely defined the structure and stereochemistry of anthramycin. Total synthesis of the antibiotic, an unmethylated carbinolamine, remains a challenge, however, because its lability does not permit use of conventional procedures for converting a lactam to a carbinolamine
Work on the total synthesis of anthramycin has revealed its absolute stereochemical configuration, according to Dr. Willy Leimgruber of Hoffmann-La Roche, Inc., Nutley, N.J. Speaking to chemists at Stanford University, Palo Alto, Calif., Dr. Leimgruber said that the total synthesis of the new antitumor agent lacks only the conversion of a lactam to a carbinolamine.
Hoffmann-La Roche is interested in anthramycin because it is a new antitumor agent which does not cause bone marrow depression. Preliminary clinical trials show that anthramycin acts against transplantable tumors in mice, and it also arrests a variety of malignant tumors in humans.
Not only Hoffmann-La Roche is looking for antitumor agents, such as anthramycin, from fermentation broths. Charles Pfizer & Co., has confirmed evidence that streptonigrin and mithromycin, both isolated from Strep-tomyces microorganisms, can act as antitumor agents. Merck & Co. has had Dectinomycin (actinomycin D) on the market for almost two years as an agent against Wilm's tumor, a kidney malignancy in children. Other agents that Merck is looking at are hedacedin, tenuozonic acid, and cordy-cepin, all isolated from fermentation broths. Lederle, Lilly, and Upjohn are also exploring the area of antitumor agents from fermentation broths.
Anthramycin comes from an obscure thermophilic actinomycete related to those which produce streptomycin and the tetracyclines. The thermophile was isolated from subtropical soils in the 1950's by Dr. Moses D. Tendler, microbiologist and Talmudic scholar at Yeshiva University, New York City.
Dr. Tendler suspected that thermophilic actinomycetes might produce antitumor substances because of a cancer theory of the German biochemist Otto Warburg. Dr. Warburg believed that the main difference between normal and cancer cells is that normal cells have two energy-producing mechanisms, aerobic and anaerobic, while cancer cells have only one main mechanism, anaerobic.
If this were true, Dr. Tendler reasoned, a substance capable of blocking the anaerobic mechanism might be able to kill cancer cells without damaging normal cells. He speculated fur
ther that a highly aerobic organism, such as a thermophilic actinomycete, might produce materials which inhibit its own anaerobic system.
His speculations bore fruit. Eight out of 10 substances which he obtained from thermophiles showed antitumor activity. The most promising of these was a fermentation broth produced by Streptomyces refluineus, a thermophilic actinomycete discovered by Dr. Tendler. The big hope for anthramycin, the active principle in this broth, is that clinical tests with pure material will show minimal undesirable side effects in human beings.
When Hoffmann-La Roche obtained the active broth from Dr. Tendler, it contained about 0.5% anthramycin. ( Dr. Tendler had named the unknown active principle refuin, from the Hebrew "refuah," meaning a medicine. Dr. Leimgruber renamed it anthramycin, a generic name derived from the anthranilic acid portion of its structure. )
Isolation and characterization of anthramycin were tricky because the material and its derivatives are so labile, Dr. Leimgruber says. The substance is extremely sensitive to heat and is
stable in solution only under essentially neutral conditions. Anthramycin methyl ether is the most stable derivative, and so it was chosen for structure work.
Anthramycin is so reactive that it crystallizes from hot methanol-water as the methyl ether. When it crystallizes from boiling acetone, it dehydrates to form anhydroanthramycin. The first crystals of anthramycin itself were obtained by crystallizing anhydroanthramycin from acetone-water at room temperature. Subsequent investigation showed that these three compounds are interconvertible by different modes of crystallization.
Dr. Leimgruber and his coworkers, Dr. Andrew Batcho and Dr. Fausto Schenker, started their investigation by elucidating the structure of a simpler but similar fermentation product, a yellow pigment. The yellow pigment differs from anthramycin methyl ether in that it contains no methyl, no hy-droxyl, and no methoxyl groups. Anthramycin methyl ether contains one of each.
In anthramycin methyl ether, the positions of the methyl and hydroxyl were determined by hydrolysis, which
42 C&EN OCT. 31, 1966
made the anthranilic acid portion of the molecule. The anthranilic acid contained a hydroxyl at C-3 and a methyl at C-4. The methoxyl, because of its lability toward hydrogénation, was formulated as part of a carbinola-mine function at C- l l . Anthramycin itself is simply the unmethylated car-binolamine.
The antibiotic has one asymmetric center at C- l l and another at the adjacent C-l la . The next task was to determine the relative configuration of these two centers.
Crystallization of anhydroanthramy-cin from dry methanol at room temperature provides a methyl ether (epian-thramycin methyl ether) which differs from the original one by its stereochemistry. Nuclear magnetic resonance analysis showed that the methyl ethers are epimeric at C- l l .
The NMR spectrum of the original methyl ether shows the C- l l proton as a single peak; the NMR spectrum of the epimer shows the C- l l proton as a doublet. Since the C- l l a proton does not split the C- l l proton peak in the spectrum of anthramycin methyl ether, the dihedral angle between the two C—H bonds must be close to 90°. Molecular models show that the only configuration meeting this requirement is the one with the two protons in the cis configuration. Thus, anthramycin methyl ether possesses the cis configuration.
Similar NMR studies with anthramycin revealed that it exists in solution as an equilibrium mixture of epimeric carbinolamines. The absolute configuration of anthramycin—whether the protons are on the front or on the back of the molecule—was established by synthetic studies.
The synthesis begins with a derivative of anthanilic acid, 3-benzyloxy-4-methyl-2-nitrobenzoyl chloride. The acid chloride group is then condensed with the amino group of L-hydroxypro-line methyl ester. This introduces the five-membered ring into the molecule. Reduction of the nitro group (to an amino group), followed by cyclization, gives the tricyclic skeleton of anthramycin.
Oxidation of the hydroxyl to a ketone, which is then condensed with triethylphosphonoacetate, introduces the ring unsaturation and two of the side-chain carbon atoms, one of which is part of a carbethoxy group. Reduction of this ester to an aldehyde and subsequent conversion of the aldehyde to a cyanohydrin provides the third carbon atom of the side chain. Tosyla-tion of the cyanohydrin, followed by elimination of p-toluenesulfonic acid, gives an acrylonitrile side chain. Hydrolysis yields the required unsaturated amide with simultaneous removal of the protective benzyl group.
The result of all these transformations is an optically active, tricyclic dilactam which differs from anthramycin by the lack of only two hydrogen atoms. The dilactam was also obtained by partial synthesis from anthramycin. This proves that anthramycin possesses the same absolute stereochemical configuration (at C- l l a ) as L-hydroxyproline.
Dr. Leimgruber and his colleagues have completely defined the structures and stereochemistry of anthramycin. All that remains to be done is to complete the total synthesis. They regard this as a challenge because the lability of anthramycin does not permit use of conventional procedures for the conversion of the lactam function to a carbinolamine.
Anthramycin's protons at C - l 1 and C- l l a are cis
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Nuclear magnetic resonance analysis reveals that there are two methyl ethers of anthramycin which are epimeric at C - l l . NMR also shows that anthramycin exists in solution as an equilibrium mixture of epimeric carbinolamines
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The synthesis of anthramycin begins with the methyl ester of L-hydroxyproline, which fixes the stereochemistry of the tricyclic dilactam at C- l l a . The final product differs from anthramycin only by the lack of two hydrogen atoms. Conversion of anthramycin to the same tricyclic dilactam shows that anthramycin has the same absolute stereochemistry at C - l l a as L-hydroxyproline
OCT. 31, 1966 C&EN 43
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