total synthesis of anthramycin nears

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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 stereo- chemistry of anthramycin. Total synthesis of the antibiotic, an unmethylated car- binolamine, 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 anthra- mycin has revealed its absolute stereo- chemical 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 an- titumor agent lacks only the conversion of a lactam to a carbinolamine. Hoffmann-La Roche is interested in anthramycin because it is a new anti- tumor agent which does not cause bone marrow depression. Prelimi- nary clinical trials show that anthra- mycin acts against transplantable tu- mors in mice, and it also arrests a vari- ety 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 con- firmed 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 antitu- mor 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 thermo- philic actinomycetes might produce an- titumor 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 mecha- nisms, aerobic and anaerobic, while cancer cells have only one main mecha- nism, anaerobic. If this were true, Dr. Tendler rea- soned, a substance capable of blocking the anaerobic mechanism might be able to kill cancer cells without damag- ing 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 thermo- philic actinomycete discovered by Dr. Tendler. The big hope for anthra- mycin, 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 He- brew "refuah," meaning a medicine. Dr. Leimgruber renamed it anthra- mycin, a generic name derived from the anthranilic acid portion of its struc- ture. ) Isolation and characterization of an- thramycin were tricky because the ma- terial and its derivatives are so labile, Dr. Leimgruber says. The substance is extremely sensitive to heat and is stable in solution only under essen- tially neutral conditions. Anthramycin methyl ether is the most stable deriva- tive, 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 ob- tained by crystallizing anhydroanthra- mycin from acetone-water at room temperature. Subsequent investiga- tion showed that these three com- pounds are interconvertible by dif- ferent 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. An- thramycin 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

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Page 1: Total synthesis of anthramycin nears

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

Page 2: Total synthesis of anthramycin nears

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

°H H H ori

OH o c H * Η Ι .Η

Λ hv^dAewXhui^CMi^

CoMHj.

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

Xr <aH \ 7M7

Μ k ocoMe

QuoUL* -Ù^L jtfttA.

M CH Z CI 2 ^

OH ^pw^G^jatA-citu^t* w "V^oH

ΟΝα^Ο^ΤΗΡ/μ^ί

H

CONH,

Co*w.pOA^<M. fc>* m . p . ( 3 z 3 iz> 3 2 5 t f c )

"\3 C '^ N

OH H H 0ÇL

0 COMHz O A c Ο CONHi

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

Page 3: Total synthesis of anthramycin nears

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RESEARCH BRIEFS

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A human heart may be replaced to­tally by a mechanical device made at Indiana University's school of medi­cine, Indianapolis. Development is far enough along to warrant early clinical trials in the event that a cardiac patient at the medical center has absolutely no other chance of survival, according to Dr. Harris B. Shumacker, head of the medical engineering group which de­veloped the device during the past three years. Unlike other artificial heart pumps, which temporarily ease the work load of a heart, the Indiana device completely replaces a damaged or worn-out heart, which is removed from the body. In early animal tests, the device performed as expected. It neither produced clots nor damaged the blood. The device consists of two flexible silicone rubber chambers en­closed in a rigid container. Hydraulic fluid rhythmically pumped in the space between the chambers and the con­tainer squeezes the chambers, thus pumping the blood. Power for the in­ternal, electrically driven hydraulic pump is supplied from an external battery via a wire through the body wall.

Mars has more hydrogen compounds in its atmosphere than was thought previously. New spectral data show that these compounds, probably in­cluding substituted methanes, are pres­ent in a concentration of about one part per thousand in the planet's atmos­phere, according to Dr. Lewis D. Kap­lan of California Institute of Technol­ogy's Jet Propulsion Laboratory in Pas­adena. By comparison, the concentra­tion of methane in earth's atmosphere is about 1 p.p.m. Dr. Kaplan revealed the Mars data at the Second ACS Western Regional Meeting in San Francisco. The data are unexpected because Mars' low gravitational field was considered by some incapable of retaining hydrogen and its derivatives. The results pose the question of how these hydrogen compounds are replen­ished. Biological activity is one possi­bility. The study was a joint effort be­tween Dr. Kaplan and Dr. Pierre and Dr. Jannine Connes of the French National Center of Scientific Research. Dr. Kaplan and the French scientists plan a new series of spectral studies early next year when Mars will again be close to earth.

Laser memory may control satellites or fly aircraft as a result of the devel­opment of an "optical maze runner" by the Air Force Avionics Laboratory (Wright-Patterson AFB, Ohio). The device is modeled after biological nerve patterns that store information for learning and decision.

44 C&EN OCT. 31, 1966

NEW!