WO1991017172A1 - Novel mycalamides with antitumor and antiviral activity - Google Patents
Novel mycalamides with antitumor and antiviral activity Download PDFInfo
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- WO1991017172A1 WO1991017172A1 PCT/US1991/002281 US9102281W WO9117172A1 WO 1991017172 A1 WO1991017172 A1 WO 1991017172A1 US 9102281 W US9102281 W US 9102281W WO 9117172 A1 WO9117172 A1 WO 9117172A1
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- 0 C[C@@]([C@@](C)OC(C1)([C@@](C(*)=O)OCc2ccccc2)OC)C1=** Chemical compound C[C@@]([C@@](C)OC(C1)([C@@](C(*)=O)OCc2ccccc2)OC)C1=** 0.000 description 9
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
Definitions
- Such symptoms include weakened condition of the inflicted mammal as evidenced by weight loss, etc.
- the seriousness of cancer is well known since cancer is a major cause of death in man. While certain methods and chemical compositions have been developed which aid in inhibiting, remitting, or controlling the growth of tumors, new methods and antitumor chemical compositions are needed.
- Viral diseases also inflict man, plants, insects and animals.
- the prevention and control of viral diseases has important health and economic implications. Viral diseases contribute to inflictions in humans including the common cold, herpes, acquired immune deficiency syndrome (AIDS), and cancer, so the importance of their control is obvious. Also important is the control of viral diseases in animals for economic and other reasons, e.g., the ability of such animals to become virus reservoirs or carriers which facilitate the spreading of viral diseases to humans.
- Viral plant diseases have been known to have a disruptive effect on the cultivation of fruit trees, tobacco, and various vegetables. Insect viral diseases are also of interest because of the insects' ability to transfer viral diseases to humans. The prevention and control of viral diseases is thus of prime importance to man, and considerable research has been devoted to antiviral measures. Certain methods and chemical compositions have been developed which aid in inhibiting, controlling, or destroying viruses, but additional methods and antiviral compositions are needed.
- the present invention utilizing sponges as a source material and supplemented by novel synthetic production methods, has provided the art with new biologically active compounds and new pharmaceutical compositions useful as antitumor and antiviral agents.
- the invention provides new mycalamide compounds having advantageous biological activities. Also provided are compositions containing such compounds, as well as methods for the preparation and use of the compounds and compositions.
- the invention provides a variety of processes for the production of compounds of the invention.
- a preferred method of producing the new compounds comprises the steps of collecting marine sponges of the genus Mycale. family Mvcalidae. and order Poecilosclerida. contacting such sponges with a selected organic solvent system to obtain an extract, fractionating the extract, and isolating mycalamide compounds from the fractionated extract. These compounds can serve as the starting materials for the preparation of many of the compounds described herein.
- some of the new mycalamide compounds are made by hydrogenation in the presence of a hydrogenation catalyst.
- ion-exchange, hydrolysis, alkylation, acetylation, and other known synthesis type reactions may be used pursuant to known procedures to add, remove, or modify various groups in the original compounds to produce other compounds.
- the compounds of the subject invention are especially important by virtue of the discovery that viruses and tumors can be inhibited by contact with an effective amount of the new compounds.
- the subject invention pertains to novel chemical compounds isolated from marine sponges, and various derivatives and analogs of these compounds.
- suitable organic solvent systems for extraction can be selected from methanol, ethyl acetate, acetone, diethyl ether, t-butyl methyl ether, ethanol, and isopropanol. Mixtures of two or more of such solvents in various ratios and combinations are advantageous.
- Preferred isolation procedures include various chromatography techniques, such as countercurrent chromatography, with suitable columns including multi-layer planetary coil columns.
- suitable columns including multi-layer planetary coil columns.
- solvents are available for use as single or mixed eluents, such as tetrahydrofuran, methanol, ethyl acetate, acetonitrile, n-propanol, n-butanol, water, and equivalent solvents.
- Further purifications using such procedures may also be carried out on the recovered extractions.
- Preferred isolation techniques for further purifications include chromatographic operations such as high-pressure liquid chromatography (HPLC) with suitable columns and suitable solvents.
- novel compounds have been shown to possess antitumor and antiviral activity.
- the subject invention pertains to the compounds themselves, as well as pharmaceutical compositions containing these compounds.
- methods for administering the novel compositions are also disclosed and claimed.
- Various derivatives of these compounds can be produced by known procedures.
- a more complete understanding of the invention can be obtained by reference to preferred embodiments of the invention which are illustrated by the following specific examples of compounds, compositions, and methods of the invention. It will be apparent to those skilled in the art that the examples involve use of materials and reagents that are commercially available from known sources, e.g., chemical supply houses, so no details are given respecting them.
- a new class of compounds having the following structural formula has been identified:
- R 1 and R 2 are the same or different and are hydrogen or lower alkyl, particularly C1-C5 alkyl;
- R 3 " 6 are the same or different and are hydrogen, lower alkyl, acyl, lower alkyl silyl, Bn, or Bz;
- Y is lower alkyl, Bn, or Bz.
- these compounds have been found to have antitumor and antiviral activity.
- the compound known as mycalamide A has the following formula:
- Mycalamide A is described in United States Patent No.4,868,204.
- a New Zealand marine sponge of the genus Mycale (family Mvcalidae. order Poecilosclerida) was collected from the channel at Aquarium Point, Otago Harbour, New Zealand.
- Two hundred grams of frozen sponge were extracted by blending with 3:1 mefhanohtoluene and filtering off the solid. On removing the solvents, the combined extracts from three such steps yielded a brown gum (11.04 g) with antiviral properties.
- Reverse phase flash chromatography (Blunt et al. [1987] J. Natl. Prod.
- the compound known as mycalamide B has the following formula:
- Mycalamide B is described in U.S. Patent No. 4,864,204.
- Mycalamide B an oil having a molecular formula of C ⁇ H ⁇ NO j -*, and a molecular weight of 517, was extracted, using the same general procedure as described in Example 1, from the same sponge, i.e., Mycale sp. (type specimen PML1-9, Chemistry Department, University of Canterbury), family Mycalidae, order Poecilosclerida (Perry et al. [1988] JACS 110:4850-4851; J. Org. Chem. [1990] 55:223).
- Mycalamide A & B co-occur in all samples of the active Mycale sp. of sponge.
- DCIMS (NH 3 ): 535 (5%, M+NH 4 + ), 505 (28%), 504 (38%), 503 (100%, M+NH 4 + - CH 3 OH), 488 (23%), 487 (36%), 486 (89%).
- DCIMS (ND 3 ): 543 (5%), 542 (14%), 541 (10%), 513 (17%), 512 (19%), 511 (34%), 510 (100%), 509 (82%), 508 (29%), 493 (8%), 492 (9%), 491 (15%), 490 (36%), 489 (37%), 488
- Mycalamide A triacetate has a molecular weight of 629 and a molecular formula of (- ⁇ H ⁇ NO jg . Its molecular structure is as follows:
- HREIMS M + -CH 3 OH 590.2800 (-10.6 ppm); M + -CH 3 OH 597.27536 (-5.3 ppm).
- DCIMS (NH- j ): 647 (13%, M+NH 4 ), 617 (29%), 616 (37%), 615 (100%, M+NH 4 + - CH 3 OH), 542 (35%), 318 (42%), 317 (44%), 299 (30%), 286 (58%), 285 (41%), 270 (28%), 269 (60%), 257 (30%).
- DCIMS (CH 4 ): 598 (71%, MH + -CH 3 OH), 538 (100%, MH + -CH 3 OH-CH 3 C0 2 H), 299 (53%), 269 (50%), 240 (41%), 208 (55%).
- Mycalamide A triacetate is prepared as follows: Mycalamide A (2 mg) was dissolved in pyridine (0.5 ml) and acetic anhydride (0.5 ml). After 7 hours at 21°C, water (1 ml) was added and the mixture extracted with CHC1 3 (3 x 1 ml). The solvent was removed and combined organic extracts were subjected to silica gel chromatography (200 mg Davisil, 150 A, 35-70 ⁇ m), developed in steps from hexane to ethyl acetate. A fraction (1.2 mg) eluted with 1:1 hexane:ethyl acetate was pure mycalamide A triacetate, an oil.
- Example 4 Mycalamide B Diacetate
- Mycalamide B diacetate has a molecular weight of 601 and a molecular formula of C ⁇ 9 H 7 N0 12 . Its molecular structure is as follows:
- DCIMS (NH 3 ): 619 (22%, M+NH 4 ), 589 (16%), 588 (32%), 587 (100%, M+NH 4 - CH 3 OH), 570 (25%, MH + -CH 3 OH), 318 (19%), 290 (25%), 258 (62%), 257 (26%), 241 (45%).
- Mycalamide B diacetate is prepared as follows: Mycalamide B (3.5 mg) was dissolved in pyridine (0.1 ml) and acetic anhydride (0.1 ml). After four hours at 21°C, water (0.2 ml) was added and the mixture extracted with CHC1 3 (3 x 2.0 ml). The solvent was removed and the combined organic extracts were subjected to silica gel chromatography (200 mg Davisil, 150 A, 35- 70 ⁇ m), developed in steps from hexane to ethyl acetate. A fraction (3 mg) eluted with 1:1 hexane:ethyl acetate was pure mycalamide B diacetate, an oil.
- Mycalamide B 18-Mono-TBDMS Ether has a molecular weight of 631 and a molecular formula of C 31 H 57 NO 10 Si. Its molecular structure is as follows:
- Mycalamide B 18-Mono-TBDMS Ether is prepared as follows: Mycalamide B (3 mg), t-butyldimethylchlorosilane (12 mg), dimethylaminopyridine (1 mg) and triethylamine (14 mg) were stirred in pyridine (0.2 ml) at room temperature for 20 hours. H 2 0 (0.5 ml) was added, the mixture extracted with CH 2 C1 2 (3 x 0.3 ml), and the solvent removed. The combined organic extract was subjected to silica gel chromatography (200 mg Davisil, 150A, 35-70 ⁇ m), developed in steps from hexane to ethanol/ethyl acetate.
- Mycalamide B 7-monoacetate has a molecular weight of 559 and molecular formula of C 27 H 45 NO.-_.-_. Its molecular structure is as follows:
- DCIMS (NH 3 ) 577 (15%, M+NH 4 + ), 547 (28%), 546 (30%), 545 (100%, M+NH 4 + - CH 3 OH), 530 (10%), 529 (11%), 528 (34%, MH + -CH 3 OH).
- Mycalamide B 7-monoacetate can be prepared as follows: Mycalamide B 18-mono-TMS ether (1 mg ca) was dissolved in pyridine (0.1 ml) and acetic anhydride (0.1 ml). The preparation of mycalamide B 18-mono-TMS ether is described in U.S. Patent No. 4,868,204. After 3 hrs at room temperature, H 2 0 (0.2 ml) was added, the mixture extracted with CH 2 C1 2 (3 x 0.2 ml), and the solvent removed (1 mg). TLC and PMR showed desilylation and about 25% diacetate present in an otherwise clean sample of mycalamide B 7-monoacetate.
- Mycalamide B 18-Monoacetate has a molecular weight of 573 and a molecular formula of C ⁇ H ⁇ NO-,- ⁇ Its molecular structure is as follows:
- DCIMS (NH 3 ): 591 (2%, M+NH 4 + ), 562 (9%), 561 (34%), 560 (32%), 559 (100%, M+NH 4 + -CH 3 OH), 545 (11%), 544 (36%), 543 (30%), 542 (99%, MH + -CH 3 OH).
- mycalamide B 18-monoacetate can be prepared as follows: Mycalamide B (6 mg), Ag 2 0 (40 mg), and Mel (27 mg) were stirred in benzene (0.4 ml) for 2 hours at 90°C. The solution was filtered over celite and the solvent removed. Pyridine (0.1 ml) and acetic anhydride (0.1 ml) were added and the mixture stirred at room temperature for 16 hours. H 2 0 was added (2.5 ml) and the solution extracted with CH 2 C1 2 (2 x 2 ml).
- 7-methoxy mycalamide B has a molecular weight of 531 and a molecular formula of C 26 H 45 NO 10 . Its molecular structure is as follows:
- DCIMS (NH 3 ): 549 (6%, M+NH 4 + ), 520 (4%), 519 (20%), 518 (40%), 517 (100%, M+NH 4 + -CH 3 OH), 502 (13%), 501 (29%), 500 (79%, MH + -CH 3 OH).
- 7-methoxy mycalamide B can be prepared as follows: 7-Methoxy mycalamide B 18-monoacetate (1.8 mg) was stirred in a solution with 1.2 mg K 2 C0 3 in aqueous MeOH (0.4 ml) at room temperature for 5 hours. The solution was concentrated, then H 2 0 (2.5 ml) was added and the solution extracted with CH 2 C1 2 (3 2 ml). The solvent was removed and the sample subjected to prep TLC (developed twice in 1:5 PE:EtOAc) to give pure 7-methoxy mycalamide B (1.2 mg) by NMR, an oil.
- Mycalamide B 7-Monobenzyl Ether has a molecular weight of 607 and a molecular formula of C ⁇ H ⁇ NO-*,,. Its molecular structure is as follows:
- FABMS 631 (9%), 630 (100%, M+Na + ), 598 (7%, M+Na + -CH 3 OH), 577 (8%), 576 (20%, MH + -CH 3 OH).
- Mycalamide B 7-monobenzyl ether can be prepared as follows: Mycalamide B (4.4 mg), powdered KOH (7 mg) and benzyl bromide (12 mg) were stirred in DMSO (0.3 ml) at room temperature for 4 minutes. H 2 0 (2 ml) was added and the mixture extracted with CHC1 3 (3 2 ml). The extract was evaporated to dryness, then subjected to prep TLC (developed in 1:1 PE:EtOAc).
- N-Methyl Mycalamide A has a molecular weight of 559 and a molecular formula of ⁇ _ ⁇ H 49 NO 10 . Its molecular structure is as follows:
- DCIMS (NH- j ): 577 (1%, M+NH 4 + ), 547 (4%), 546 (6%), 545 (19%, M+NH 4 + - CH 3 OH), 531 (5%), 530 (19%), 529 (30%), 528 (100%, MH + -CH 3 OH).
- N-methyl mycalamide A can be prepared as follows: Mycalamide A (5.5 mg), powdered KOH (14 mg) and Mel (23 mg) were stirred in DMSO (0.3 ml) at room temperature for 3.5 hours. H 2 0 was added (0.5 ml) and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to H 2 0), flushed with H 2 0 (6 ml) and then eluted with MeOH (6 ml). The MeOH fraction was evaporated to dryness (5.5 mg), then subjected to prep TLC (developed in EtOAc).
- N-Methyl Mycalamide A has a molecular weight of 545 and a molecular formula of C2 7 H 47 NO 10 . Its molecular structure is as follows:
- DCIMS (NH 3 ): 563 (10%, M+NH 4 + ), 533 (19%), 532 (37%), 531 (100%, M+NH 4 + - CH 3 OH), 516 (17%), 515 (32%), 514 (95%, MH + -CH 3 OH).
- 7,18-dimethoxy N-methyl mycalamide A can be prepared as follows: Mycalamide A (5.5 mg), powdered KOH (14 mg) and Mel (23 mg) were stirred in DMSO (0.3 ml) at room temperature for 3.5 hours. H 2 0 was added (0.5 ml) and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to H 2 0), flushed with H 2 0 (6 ml) and then eluted with MeOH (6 ml). The MeOH fraction was evaporated to dryness (5.5 mg), then subjected to prep TLC (developed in EtOAc).
- N-Methyl Mycalamide A has a molecular weight of 531 and a molecular formula of C ⁇ H ⁇ NO-,,*,. Its molecular structure is as follows:
- DCIMS (NH 3 ): 549 (3%, M+NH 4 + ), 519 (6%), 518 (20%), 517 (66%, M+NH 4 + - CH 3 OH), 502 (10%), 501 (29%), 500 (100%, MH + -CH 3 OH).
- N-methyl mycalamide A can be prepared as follows: Mycalamide A (3 mg), powdered KOH (3 mg) and Mel (7 mg) were stirred in DMSO at room temperature for 4 hours. H 2 0 was added (0.3 ml), the mixture extracted with CH 2 C1 2 (3 x 0.4 ml), and the solvent removed (3.5 mg). Prep RPLC (30% H 2 0 in MeOH) gave two fractions (1.9 mg, 1 mg) which were pure 7-methoxy, N-methyl mycalamide A and a 4:1 mixture of 7,18-dimethoxy, N-methyl mycalamide A and 7,17-dimethoxy, N-methyl mycalamide A by PMR respectively, an oil.
- 6-Ethoxy Mycalamide A has a molecular weight of 517 and a molecular formula of C ⁇ H ⁇ NOJ Q . Its molecular structure is as follows:
- DCIMS (NH- j ): 535 (6%, M+NH 4 + ), 491 (4%), 490 (6%), 489 (100%, M+NH 4 + - CH 3 CH 2 OH), 474 (5%), 473 (5%), 472 (65%, MH + -CH 3 CH 2 OH).
- 6-ethoxy mycalamide A can be prepared as follows: Mycalamide A (20 mg) was dissolved in CDC1 3 containing a trace of ethanol and left for two weeks. PMR showed partial reaction to at least two components and this was confirmed by HPLC. Prep HPLC (35% H 2 0 in MeOH) separated unreacted mycalamide A (4.5 mg) and 6-ethoxy mycalamide A (1.2 mg) in pure form by PMR.
- Mycalamide A 17,18-Di-p-Bromobenzoate has a molecular weight of 869 and a molecular formula of Its molecular structure is as follows:
- Mycalamide A 17,18-Di-p-bromobenzoate can be prepared as follows: Mycalamide A (5.0 mg, 0.01 mmole), p-bromobenzoyl chloride (11 mg, 0.05 mmole), dimethylaminopyridine (1 mg) and triethylamine (7 mg, 0.07 mmole) were stirred in pyridine (0.4 ml) at 75°C overnight. H 2 0 (03 ml) was added, the mixture extracted with CH 2 C1 2 (3 x 0.4 ml), and the solvent removed (17 mg ca.).
- 7-Methoxy Mycalamide A has a molecular weight of 517 and a molecular formula of C 2 5H 43 NO 10 . Its molecular structure is as follows: OCH,
- DCIMS (NH 3 ): 535 (10%, M+NH 4 + ), 505 (28%), 504 (28%), 503 (100%, M+NH 4 + - CH 3 OH), 488 (12%), 487 (9%), 486 (30%, MH + -CH 3 OH).
- 7-methoxy mycalamide A can be prepared as follows: Mycalamide A (7 mg), Ag 2 0 (55 mg) and Mel (21 mg), were stirred in benzene (0.4 ml) at 95°C in a sealed vial for 1.5 hours. The solution was filtered over celite and the solvent removed (7.3 mg). Preparative TLC (developed twice in 1:7 PE:EtOAc) gave four fractions (0.8 mg, 1.4 mg, 2.0 mg, 2.8 mg) which were mycalamide A, 7-methoxy A, 3:2 17-methoxy A:18-methoxy A, and pure 18-methoxy A by nmr respectively. Subsequently 7-methoxy mycalamide A was subjected to further chromatography (prep. TLC, developed 3x in 1:5 PE:EtOAc) to give the pure compound (1.2 mg), an oil.
- Mycalamide A has a molecular weight of 531 and a molecular formula of C- ⁇ H ⁇ NO j --,. Its molecular structure is as follows: OCH,
- DCIMS (NH 3 ): 549 (7%, M+NH 4 + ), 519 (22%), 518 (30%), 517 (100%, M+NH 4 + - CH 3 OH), 502 (11%), 501 (16%), 500 (53%, MH + -CH 3 OH).
- 7,18-dimethoxy mycalamide A can be prepared as follows: Mycalamide A (4.2 mg), Ag 2 0 (25 mg) and Mel (18 mg), were stirred in benzene (03 ml) at 80°C in a sealed vial for 3 days. The solution was filtered over celite and the solvent removed (4.6 mg). Preparative TLC (developed in EtOAc) gave three fractions (0.5 mg, 1.2 mg, 2.0 mg) which were 7,17-dimethoxy mycalamide A, 7,18-dimethoxy A and 7,17,18-trimethoxy A by nmr respectively. These were individually rechromatographed by prep. TLC to give the pure compounds, an oil.
- 18-Methoxy Mycalamide A has a molecular weight of 517 and a molecular formula of C ⁇ H ⁇ NOJ Q . Its molecular structure is as follows:
- DCTMS (NH 3 ): 535 (16%, M+NH 4 + ), 505 (24%), 504 (33%), 503 (100%, M+NH 4 + - CH 3 OH), 487 (19%), 486 (66%, MH + -CH 3 OH).
- 18-methoxy mycalamide A can be prepared as follows: Mycalamide A (7 mg), Ag 2 0 (55 mg) and Mel (21 mg), were stirred in benzene (0.4 ml) at 95°C in a sealed vial for 1.5 hours. The solution was filtered over celite and the solvent removed (7.3 mg). Preparative TLC (developed twice in 1:7 PE:EtOAc) gave four fractions (0.8 mg, 1.4 mg, 2.0 mg, 2.8 mg) which were mycalamide A, 7-methoxy A, 3:2 17-methoxy A18-methoxy A, and pure 18-methoxy A by nmr respectively, an oil.
- N-Methyl Mycalamide A has a molecular weight of 559 and a molecular formula of C- ⁇ oNO-,-*,. Its molecular structure is as follows:
- DCIMS (NH 3 ): 577 (8%, M+NH 4 + ), 547 (15%), 546 (30%), 545 (88%, M+NH 4 + - CH 3 OH), 530 (19%), 529 (37%), 528 (100%, MH + -CH 3 OH).
- N-methyl mycalamide A can be prepared as follows: Mycalamide A (4mg), powdered KOH (7.2mg) and Mel (lOmg) were stirred in DMSO (0.3ml) at room temperature for 20 hours, then at 40°C for 4 hours. H 2 0 was added (0.5ml) and the mixture transferred onto a reverse phase pipette column (200mg C18, equilibrated to H 2 0), flushed with H 2 0 (6ml) and then eluted with MeOH (6ml). The MeOH fraction was evaporated to diyness (43mg), then subjected to prep TLC (developed in EtOAc).
- Mycalamide B Trans-Oxazolidinone has a molecular weight of 487 and a molecular formula of C ⁇ H ⁇ NO- j . Its molecular structure is as follows:
- Mycalamide B trans-oxazolidinone can be prepared as follows: Mycalamide B (2.5 mg) was dissolved in a solution of 1 M NaOMe in methanol (0.3 ml) and stirred at 50°C for 24 hours. The solvent was removed and the residue partitioned in 1:1 CHC1 3 :H 2 0 (5 ml) and then extracted with CHC1 3 (3x 2 ml). Prep TLC of this extract (developed twice in 1:19 EtOH:EtOAc) gave two fractions (1 mg, 0.9 mg), which were cis and trans isomers respectively at C7-C10 of mycalamide B oxazolidinone by nmr, although the second fraction also contained up to 25% unreacted mycalamide B, an oil.
- Mycalamide B cis-Oxazolidinone has a molecular weight of 487 and a molecular formula of C ⁇ H ⁇ NOp. Its molecular structure is as follows:
- Mycalamide B cis-oxazolidinone can be prepared as follows: Mycalamide B (2.5 mg) was dissolved in a solution of 1 M NaOMe in methanol (0.3 ml) and stirred at 50°C for 24 hours. The solvent was removed and the residue partitioned in 1:1 CHC1 3 H 2 0 (5 ml) and then extracted with CHO s (3x2 ml). Prep TLC of this extract (developed twice in 1:19 EtOH:EtOAc) gave two fractions (1 mg, 0.8 mg), which were cis and trans isomers respectively at C7-C10 of mycalamide B oxazolidinone by nmr, although the second fraction also contained up to 25% unreacted mycalamide B, an oil.
- Mycalamide B Bis-Ethylcarbo ⁇ ate Hydrochloride has a molecular weight of 666 and a molecular formula of C 30 H 8 ONO 13 . Its molecular structure is as follows: OCOOC 2 H 5
- Mycalamide B bis-ethylcarbonate hydrochloride can be prepared as follows: Mycalamide B (2.5 mg ca.), K 2 ⁇ 0 3 (8.5 mg) and ethyl chloroformate 4.5 mg) were stirred in benzene (0.3 ml) for 20 hours at room temperature. The solution was then diluted with benzene, filtered over celite and the solvent removed. Prep TLC (developed in 3:2 PE:EtOAc) gave 0.8 mg of the major component, which was pure mycalamide B bis-ethylcarbonate hydrochloride by PMR.
- Mycalamide B 18-Monoacetate has a molecular weight of 559 and a molecular formula of C_ ⁇ AS ⁇ 0 ll . Its molecular structure is as follows:
- DCIMS N%): 577 (1%, M+NH 4 + ), 560 (1%, MH + ), 547 (15%), 546 (35%), 545 (100%, M+NH 4 + -C%OH), 530 (16%), 529 (27%), 528 (88%, MH + -C%OH).
- Mycalamide B 18-monoacetate can be prepared as follows: Mycalamide B diacetate (2.5 mg) was stirred with 0.5 mg K 2 C0 3 in 9:1 MeOH:%0 (0.5 ml) for 1 hour at room temperature. %0 (2.5 ml) was added and the mixture extracted with CH 2 C1 2 (3 x 2 ml). The solvent was removed and the combined organic extracts were subjected to preparative TLC (developed in ethyl acetate).
- Mycalamide B 7-Monoacetate has a molecular weight of 573 and a molecular formula of C ⁇ H ⁇ NO- Q . Its molecular structure is as follows:
- DCIMS (N%): 592 (10%), 591 (32%, M+NH 4 + ), 561 (26%), 560 (33%), 559 (100%, M+NH 4 + -C%OH), 544 (10%), 543 (14%), 542 (43%, MH + -C%OH).
- mycalamide B 7-monoacetate can be prepared as follows: Mycalamide B (6 m g)» Ag 2 0 (40 mg) and Mel (27 mg) were stirred in benzene (0.4 ml) for 2 hours at 90°C. The solution was filtered over celite and the solvent removed. Pyridine (0.1 ml) and acetic anhydride (0.1 ml) were added and the mixture stirred at room temperature for 16 hours. %0 was added (2.5 ml) and the solution extracted with C%C1 2 (2 x 2 ml).
- Mycalamide B 7-Mono-p-Bromobenzoate has a molecular weight of 700 and a molecular formula of CJ-JH ⁇ B ⁇ NOJJ. Its molecular structure is as follows:
- Mycalamide B 18-Mono-p-Bromobenzoate has a molecular weight of 700 and a molecular formula of C 32 H 46 BrN0 11 . Its molecular structure is as follows:
- Mycalamide B 18-mono-p-bromobenzoate can be prepared as follows: Mycalamide B (10- 12 mg), p-bromobenzoyl chloride (26 mg), dimethylaminopyridine (1 mg), and triethylamine (15 mg) were stirred in pyridine (0.8 ml) at 50°C for one week. After concentrating to 0.1 ml, %0 (0.3 ml) was added, the mixture extracted with C%C1 2 (4 x 0.4 ml) and the solvent removed (50 mg ca).
- Mycalamide B Di-p-Bromobenzoate has a molecular weight of 883 and a molecular formula of Its molecular structure is as follows:
- DCIMS N%): 872 (15%), 871 (55%), 870 (32%), 869 (100%, M+NH 4 + -C%OH), 868 (16%), 867 (51%), 854 (17%), 853 (8%), 852 (52%, MH + -C%OH), 850 (18%).
- Mycalamide B di-p-bromobenzoate can be prepared as follows: Mycalamide B (10-12 mg), p-bromobenzoyl chloride (26-mg), dimethylaminopyridine (1 mg), and triethylamine (15 mg) were stirred in pyridine (0.8 ml) at 50°C for one week. After concentrating to 0.1 ml, %0 (0.3 ml) was added, the mixture extracted with C%C1 2 (4 x 0.4 ml) and the solvent removed (50 mg ca).
- 18-Methoxy Mycalamide B has a molecular weight of 531 and a molecular formula of C ⁇ H ⁇ NOJ Q . Its molecular structure is as follows:
- DCIMS N%): 549 (7%, M+NH 4 + ), 520 (6%), 519 (23%), 518 (29%), 517 (99%, M+NH 4 + -C%OH), 503 (5%), 502 (19%), 501 (29%), 500 (100%, MH + -C%OH).
- 18-methoxy mycalamide B can be prepared as follows: 18-methoxy mycalamide B 7- monoacetate (2 mg) (see Example 23) was stirred in a solution with 0.4 mg K 2 C0 3 in aqueous MeOH (0.4 ml) at room temperature for 1.5 hours. The solution was concentrated, then %0 (2.5 ml) was added and the solution extracted with C%C1 2 (3 x 2 ml). The solvent was removed to give pure 18-methoxy mycalamide B (1.8 mg) by nmr, an oil.
- Mycalamide B 7,N-Dibenzyl Ether has a molecular weight of 697 and a molecular formula of C ⁇ NOJ Q . Its molecular structure is as follows:
- DCIMS N%: 715 (9%, M+NH 4 + ), 686 (13%), 685 (33%), 684 (45%), 683 (100%, M+NH 4 + -C%OH), 668 (22%), 667 (27%), 666 (63%, MH + -C%OH).
- Mycalamide B 7,N-dibenzyl ether can be prepared as follows: Mycalamide B (4.4 mg), powdered KOH (7 mg) and benzyl bromide (12 mg) were stirred in DMSO (0.3 ml) at room temperature for 4 minutes. %0 (2 ml) was added and the mixture extracted with CHC1 3 (3 x 2 ml). The extract was evaporated to diyness, then subjected to prep TLC (developed in 1:1 PE:EtOAc).
- Mycalamide B 7,18-Dibenzyl Ether has a molecular weight of 697 and a molecular formula of CJ- ⁇ O-H ) . Its molecular structure is as follows:
- DCIMS N%: 716 (9%), 715 (19%, M+NH 4 + ), 686 (10%), 685 (28%), 684 (45%), 683 (100%, M+NH 4 + -C%OH), 668 (10%), 667 (16%), 666 (36%, MH + -C%OH).
- Mycalamide B 7,18-dibenzyl ether can be prepared as follows: Mycalamide B (4.4 mg), powdered KOH (7 mg) and benzyl bromide (12 mg) were stirred in DMSO (0.3 ml) at room temperature for 4 minutes. %0 (2 ml) was added and the mixture extracted with CHC1 3 (3 2 ml). The extract was evaporated to diyness, then subjected to prep TLC (developed in 1:1 PE:EtOAc).
- Mycalamide B 7,18,N-Tribenzyl Ether has a molecular weight of 787 and a molecular formula of C ⁇ H ⁇ NOJ Q . Its molecular structure is as follows:
- DCIMS N%: 805 (17%, M+NH 4 + ), 775 (22%), 774 (49%), 773 (100%, M+NH 4 + - C%OH), 758 (8%), 757 (18%), 756 (35%, MH + -C%OH).
- Mycalamide B 7,18,N-tribenzyl ether can be prepared as follows: Mycalamide B (7 mg), powdered KOH (22 mg), and benzyl bromide (36 mg) were stirred in DMSO (0.3 ml) at room temperature for 3 hours. %0 (2 ml) was added and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to %0), flushed with %0 (8 ml), then eluted with MeOH (6 ml). The resulting MeOH fraction was evaporated to diyness, then subjected to prep TLC (5:2 PE:EtOAc).
- Mycalamide B 7,8,18-Tribenzyl Ether has a molecular weight of 787 and a molecular formula of C ⁇ H ⁇ NOJ Q . Its molecular structure is as follows:
- Mycalamide B 7,8,18-tribenzyl ether can be prepared as follows: Mycalamide B (7 mg), powdered KOH (22 mg), and benzyl bromide (36 mg) were stirred in DMSO (03 ml) at room temperature for 3 hours. %0 (2 ml) was added and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to %0), flushed with %0 (8 ml), then eluted with MeOH (6 ml). The resulting MeOH fraction was evaporated to diyness, then subjected to prep TLC (5:2 PE:EtOAc).
- Mycalamide B Bis-TBDMS Ether has a molecular weight of 745 and a molecular formula of C 37 H 71 NO 10 Si 2 . Its molecular structure is as follows:
- Mycalamide B bis-TBDMS ether can be prepared as follows: Mycalamide B (1 mg), t- butyldimethylchlorosilane (12 mg), dimethylaminopyridine (1 mg), and triethylamine (14 mg) were stirred in pyridine (0.2 ml) at 70°C for 20 hours. %0 (0.5 ml) was added, the mixture extracted with C%C1 2 (3 x 0.3 ml), and the solvent removed. The combined organic extract was subjected to silica gel chromatography (200 mg Davisil, 150 A, 35-70 ⁇ ), developed in steps from hexane to ethanol/ethyl acetate. The major fraction (1 mg) eluted with 3:1 PE:EtOAc was pure mycalamide bis-TBDMS ether by PMR, an oil.
- Mycalamide A 7,N-DibenzyI Ether has a molecular weight of 683 and a molecular formula of C gg Hs ⁇ O j . Its molecular structure is as follows:
- DCIMS (N%): 701 (12%, M+NH 4 + ), 672 (17%), 671 (46%), 670 (44%), 669 (100%, M+NH 4 + -CH 3 OH), 654 (19%), 653 (20%), 652 (45%, MH + - ⁇ 3 OH).
- Mycalamide A 7,N-dibenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%C1 2 (2 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg).
- Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component
- Prep TLC developed in 1:1 PE:EtOAc) and appropriate combination of samples gave four pure products (1 mg, 13 mg, 1 mg, 2 mg) which were mycalamide A 7-mono-; 7,18-di-; 7,N-di- and 7,18,N-tri-benzyl ethers by NMR respectively, an oil.
- Mycalamide A 7,18-Dibenzyl Ether has a molecular weight of 683 and a molecular formula of C 38 H53NO 10 . Its molecular structure is as follows:
- DCIMS (N%): 703 (7%), 702 (12%), 701 (28%, M+NH 4 + ), 672 (9%), 671 (27%), 670 (41%), 669 (100%, M+NH 4 + -C%OH), 654 (5%), 653 (8%), 652 (19%, MH + -C%OH).
- Mycalamide A 7,18-dibenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%C1 2 (2 x 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component.
- Mycalamide A 7-Monobenzyl Ether has a molecular weight of 593 and a molecular formula of C ⁇ H ⁇ NO ⁇ Its molecular structure is as follows:
- DCIMS N%: 611 (7%, M+NH 4 + ), 582 (8%), 581 (26%), 580 (34%), 579 (100%, M+NH 4 + -C%OH), 564 (5%), 563 (6%), 562 (17%, MH + -C%OH).
- Mycalamide A 7-monobenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%CI 2 (2 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component.
- Mycalamide A 7,18,N-Tribenzyl Ether has a molecular weight of 773 and a molecular formula of C 45 H 59 NO 10 . Its molecular structure is as follows:
- DCIMS (N%): 791 (8%, M+NH 4 + ), 761 (19%), 760 (49%), 759 (100%, M+NH 4 + - C%OH), 743 (13%), 742 (24%, MH + -0* 3 OH).
- Mycalamide A 7,18,N-tribenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%C1 2 (2 x 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component.
- DCIMS (N%): 550 (3%, M+NH 4 + ), 520 (10%), 518 (61%, M+NH 4 + -C%OH), 503 (23%), 502 (9%), 501 (100%, MH + -O ⁇ 3 OH).
- 7'-deutero, 7'-methoxy, N-methyl mycalamide A can be prepared as follows: 7-methoxy, N-methyl mycalamide A (1.2mg) (see Example 12) was dissolved in 3M NaOCD g CDsOD and stirred at 70°C for 2 days. The crude mixture was then loaded onto a TLC plate (silica gel 60 F ⁇ , 0.2mm) which was developed twice in 1:19 EtOH:EtOAc.
- a trans-oxazolidinone has a molecular weight of 473 and a molecular formula of C 23 H 39 N0 9 . Its molecular structure is as follows:
- 7-epimycalamide A trans-oxazolidinone was prepared as follows: Mycalamide A (7 mg) was stirred in a solution of 2 M NaOH in 50% MeOH-%0 (0.3 ml) at 55°C for 10 hours. The solution was concentrated (0.1 ml) and then loaded onto a C18 pipette column (100 mg). The column was flushed with %0 (8 ml), then a fraction obtained by elution with MeOH (5 ml) was collected and the solvent removed (6 mg). Analytical reverse phase HPLC (45% %0 in MeOH) gave four fractions (1.4 mg, 1.8 mg, 0.7 mg, 0.8 mg).
- Cis-Oxazolidinone has a molecular weight of 473 and a molecular formula of C ⁇ H g oNO,-,. Its molecular structure is as follows:
- 7-epimycalamide A cis-oxazolidinone can be prepared as follows: Mycalamide A (7 mg) was stirred in a solution of 2 M NaOH in 50% MeOH-%0 (03 ml) at 55°C for 10 hours. The solution was concentrated (0.1 ml) and then loaded onto a C18 pipette column (100 mg). The column was flushed with %0 (8 ml), then a fraction obtained by elution with MeOH (5 ml) was collected and the solvent removed (6 mg). Analytical reverse phase HPLC (45% %0 in MeOH) gave four fractions (1.4 mg, 1.8 mg, 0.7 mg, 0.8 mg).
- Neomycalamide A Triacetate has a molecular weight of 597 and a molecular formula of C 2 H 3 N0 12 . Its molecular structure is as follows:
- DCIMS N%): 616 (7%), 615 (34%, M+NH 4 + ), 600 (13%), 599 (31%), 598 (100%, MH + ), 573 (28%), 571 (18%), 557 (24%), 556 (66%, MH + -C%CO), 493 (8%), 419 (35%).
- Neomycalamide A triacetate can be prepared as follows: Mycalamide A triacetate (5 mg) was dissolved in CDC1 3 (0.5 ml) and 10 ⁇ l TFA added After 5 days at room temperature (monitored by PMR), the reaction was quenched and worked up by evaporation of the solvent and partition between 1:1 0 ⁇ 2 ⁇ 2 :%0 (5mg). Prep HPLC (33% %0 in MeOH) gave three fractions (2.5mg, lmg, 0.8mg), where the first was a mixture of more polar compounds, the third was unreacted mycalamide A triacetate, but the second was about 80% pure neomycalamide A triacetate by PMR, an oil.
- Mycalamide A Tri-p-Bromobenzoate has a molecular weight of 1052 and a molecular formula of C 45 H5 0 Br 3 NO 13 . Its molecular structure is as follows:
- Mycalamide A tri-p-bromobenzoate can be prepared as follows: Mycalamide A (5.0mg, O.Olmmole), p-bromobenzoyl chloride (llmg, 0.05mmole), dimethylaminopyridine (lmg) and triethylamine (7mg, 0.07mmole) were stirred in pyridine (0.4ml) at 75°C overnight %0 (0.3ml) was added, the mixture extracted with C% ⁇ 2 (3x0.4ml), and the solvent removed (17mg ca.).
- Cis-Oxazolidinone has a molecular weight of 473 and a molecular formula of Its molecular structure is as follows:
- DCIMS (N%): 491 (5%, M+NH 4 + ), 461 (6%), 460 (30%), 459 (100%, M+NH 4 + - C%OH), 443 (17%), 442 (63%, MH + -C%OH).
- IR mixed isomers 0*C1 3 : 3700-3300, 2910, 1725, 1610, 1380, 1100-1030 cm -1 film 3700- 3100, 2950, 1720, 1380, 1150-1030, 870 cm -1 .
- Mycalamide A cis-oxazolidinone can be prepared as follows: Mycalamide A (3.8mg) was stirred in a solution of IM NaOMe in methanol (0.3ml) at 50°C for 8 hours. The solvent was removed and the residue extracted with CHC1 3 and filtered (33mg). PMR showed a 1:1 mixture of isomers which were separated by analytical reverse phase HPLC (45% %0 in MeOH) to give two fractions (0.9mg, 13mg). PMR and NOE's showed that these were cis and trans isomers respectively at C7-C10 of mycalamide A oxazolidinone, an oil.
- Mycalamide A Trans-Oxazolidinone has a molecular weight of 473 and a molecular formula of C ⁇ H ⁇ O- j . Its molecular structure is as follows:
- DCIMS (N%): 461 (6%), 460 (29%), 459 (100%, M+NH 4 + -C%OH), 443 (7%), 442 (41%, MH + -0* 3 OH).
- IR (mixed isomers) CH0 3 3700-3300, 2910, 1725, 1610, 1380, 1100-1030 cm -1 ; film 3700-3100, 2950, 1720, 1380, 1150-1030, 870 cm -1 .
- Mycalamide A trans-oxazolidinone can be prepared as follows: Mycalamide A (3.8mg) was stirred in a solution of IM NaOMe in methanol (0.3ml) at 50°C for 8 hours. The solvent was removed and the residue extracted with CH0 3 and filtered (33mg). PMR showed a 1:1 mixture of isomers which were separated by analytical reverse phase HPLC (45% %0 in MeOH) to give two fractions (0.9mg, 13mg). PMR and NOE's showed that these were cis and trans isomers respectively at C7-C10 of mycalamide A oxazolidinone, an oil.
- Mycalamide A 17,18-bis-TBDMS ether has a molecular weight of 731 and a molecular formula of C ⁇ H ⁇ - j NO-r ⁇ Si j . Its molecular structure is as follows:
- Mycalamide A 17,18-bis-TBDMS ether can be prepared as follows: Mycalamide A (Z6mg), t-butyldimethylchlorosilane (large excess), dimethylaniinopyridine (2mg) and triethylamine (20mg) were stirred in pyridine (0.4ml) at 60°C for 2 days. %0 (0.5ml) was added and the mixture extracted with CH 2 C1 2 . The organic extract was washed with %0 (3x03ml), the solvent removed, and the combined product subjected to silica gel chromatography (200 mg Davisil, 150A, 35-70 ⁇ m), developed in steps from hexane to ethyl acetate.
- 10-Epimycalamide A 7-Mono-Benzyl Ether has a molecular weight of 593 and a molecular formula of ⁇ H ⁇ NO ⁇ Its molecular structure is as follows:
- DCIMS N%: 611 (2%, M+NH 4 + ), 594 (3%, MH + ), 582 (9%), 581 (26%), 580 (28%), 579 (80%, M+NH 4 + -C%OH), 565 (12%), 564 (37%), 563 (33%), 562 (100%, MH + -C%OH).
- lO-epimycalamide A 7-monobenzyl ether can be prepared as follows: Mycalamide A (6mg), BaO (26mg) and benzyl bromide (15mg) were stirred in DMSO (0.3ml) at 60°C for 2 hours. %0 was added (0.5ml) and the mixture transferred onto a reverse phase pipette column (200mg C18, equilibrated to %0), flushed with %0 (8ml), then eluted with MeOH (6ml). The resulting MeOH fraction was evaporated to diyness, then subjected to prep TLC (1:2 PE:EtOAc).
- 10-Epimycalamide A 7,18-Dibenzyl Ether has a molecular weight of 683 and a molecular formula of C 38 H 53 NO 10 . Its molecular structure is as follows:
- DCIMS N%: 701 (4%, M+NH 4 + ), 672 (5%), 671 (18%), 670 (42%), 669 (100%, M+NH 4 + -C%OH), 654 (11%), 653 (21%), 652 (53%, MH + -C%OH).
- 10-epimycalamide A 7,18-dibenzyl ether can be prepared as follows: Mycalamide A (6mg), BaO (26mg) and benzyl bromide (15mg) were stirred in DMSO (03ml) at 60°C for 2 hours. %0 was added (0.5ml) and the mixture transferred onto a reverse phase pipette column (200mg C18, equilibrated to %0), flushed with %0 (8ml), then eluted with MeOH (6ml). The resulting MeOH fraction was evaporated to dryness, then subjected to prep TLC (1:2 PErEtOAc).
- Mycalamide A Tris-TBDMS Ether has a molecular weight of 845 and a molecular formula of Q ⁇ Hg j NO j oSi- ⁇ . Its molecular structure is as follows:
- Mycalamide A tris-TBDMS ether can be prepared as follows: Mycalamide A (Z6mg), t-butyldimethylchlorosilane (large excess), dimethylaminopyridine (2mg) and triethylamine (20mg) were stirred in pyridine (0.4ml) at 60°C for 2 days. %0 (0.5ml) was added and the mixture extracted with C%C1 2 . The organic extract was washed with %0 (3x03ml), the solvent removed, and the combined product subjected to silica gel chromatography (200 mg Davisil, 150A, 35-70 ⁇ ), developed in steps from hexane to ethyl acetate.
- 7,17,18-Trimethoxy Mycalamide A has a molecular weight of 545 and a molecular formula of C 7 H 47 NO 10 . Its molecular structure is as follows:
- DCIMS N%: 563 (10%, M+NH 4 + ), 533 (18%), 532 (37%), 531 (100%, M+NH 4 + - C%OH), 516 (11%), 515 (22%), 514 (74%, MH + -C%OH).
- 7,17,18-trimethoxy mycalamide A can be prepared as follows: Mycalamide A (4.2 mg), Ag 2 0 (25 mg) and Mel (18 mg), were stirred in benzene (0.3 ml) at 80°C in a sealed vial for 3 days. The solution was filtered over celite and the solvent removed (4.6 mg). Preparative TLC (developed in EtOAc) gave three fractions (03 mg, 1.2 mg, Z0 mg) which were 7,17-dimethoxy mycalamide A, 7,18-dimethoxy A and 7,17,18-trimethoxy A by NMR respectively. These were individually rechromatographed by prep. TLC to give the pure compounds, an oil.
- N-Methyl Mycalamide B has a molecular weight of 545 and a molecular formula of C- ⁇ H ⁇ NO j - ) . Its molecular structure is as follows:
- DCIMS (N%): 563 (2%, M+NH 4 + ), 533 (9%), 531 (34%, M+NH 4 + -C%OH), 516 (25%), 514 (100%, MH + -0* 3 OH).
- N-Methyl Mycalamide B can be prepared as follows: Mycalamide B (2 mg), powdered KOH (2 mg) and Mel (4.6 mg) were stirred in DMSO at room temperature for 24 hours. %0 was added (0.3 ml), the mixture extracted with C% ⁇ 2 (3 x 0.4 ml), and the solvent removed (3 mg). Prep RPLC (30% %0 in MeOH) gave two fractions (1.2 mg, 0.6 mg) which were pure 7-methoxy, N-methyl mycalamide B and 7,18-dimethoxy, N-methyl mycalamide B by PMR respectively, an oil.
- N-Methyl Mycalamide B has a molecular weight of 545 and a molecular formula of C--_7H 47 NO 10 . Its molecular structure is as follows:
- DCIMS N%: 563 (3%, M+NH 4 + ), 533 (5%), 532 (13%), 531 (45%, M+NH 4 + - C%OH), 516 (14%), 515 (28%), 514 (100%, MH + -C%OH).
- N-methyl Mycalamide B can be prepared as follows: 7-Methoxy, N-methyl mycalamide B (1.2mg) was dissolved in a solution of IM NaOMe in MeOH and stirred at 80°C for two days. The solvent was removed, then the residue partitioned in 1:1 %O:0HCl 3 (4ml), extracted in CHC1 3 (3xlml), and dried. The combined organic extract was subjected to silica gel chromatography (200mg Davisil), developed in steps from PE to 5% EtOH:EtOAc. The major fraction eluted with 1:19 EtOH:EtOAc (0.9mg) was a 1:3 mixture of starting material and its epimer at C7 by NMR. (Subsequent mixtures of the two epimers were combined (2mg), treated as above, and purified by prep. TLC (developed twice in EtOAc) to give two fractions containing the pure epimers by PMR), an oil.
- Antitumor effectiveness of the compounds of the subject invention was evaluated using a standardized in vitro P388 mouse leukemia cell assay protocol disclosed in U.S. Patent No. 4,731366 with the antitumor activity being expressed in IC 50 (concentration that results in 50% inhibition of cell replication normalized to untreated cultures) determined with a log-log transform of data and expressed as xx ng/m Results of the P388 antitumor assays are shown in Table 1.
- Antiviral effectiveness of the compounds described in Examples 1-50 were determined against both Herpes simplex type 1 virus (HSV-1) and polio vaccine virus (P VI) replicated in the BSC cell line.
- HSV-1 Herpes simplex type 1 virus
- P VI polio vaccine virus
- Results of the antiviral and cytotoxicity assays are shown in columns labeled 1 through 3 of Table 1.
- the value shown in the column labeled "1" is for HSV, the value in the column labeled "2” is for PVI, and the third reports cytotoxicity (where +* implies diffuse cytotoxicity over the whole well).
- Example 53 Uses. Formulations, and Administrations Therapeutic and prophylactic application of the new compounds, and compositions containing them, can be contemplated to be accomplished by any suitable method and technique presently or prospectively known to those skilled in the ar Further, the compounds of the invention have use as starting materials or intermediates for the preparation of other useful compounds and compositions. The compounds of the invention are useful for various non- therapeutic and therapeutic purposes. It is apparent from the testing that the compounds of the invention are effective for inhibiting viral activity.
- the antiviral properties of the compounds are useful to swab laboratory benches and equipment in a microbiology laboratory to eliminate the presence of viruses, or, in the case of compounds with benzyl or benzoyl groups attached, they can be used as ultraviolet screeners in the plastics industry since they effectively absorb UV rays. As disclosed herein they are also useful prophylactically and therapeutically for treating viral infections in animals and humans.
- the administration of the mycalamide compounds of the invention is useful for treating viral infections and as an antitumor agent.
- pharmaceutical compositions containing compounds of the invention as active ingredients are useful in prophylactic or therapeutic treatment of humans or other mammals infected with or likely to be infected with tumors and virus.
- dosage administered will be dependent upon the identity of the tumor or virus infection; the type of host involved; its age, health, weight, kind of concurrent treatment, if any; frequency of treatment; therapeutic ratio and like considerations.
- dosage levels of the administered active ingredients can be, for examples, dermal, 1 to about 500 mg kg; orally, 0.01 to 200 mg/kg; intranasal 0.01 to about 100 mg/kg; and aerosol 0.01 to about 50 mg kg of animal body weight.
- the active ingredient of the invention can be present in the new compositions for localized use dermaUy, intranasally, bronchially, intramuscularly, intravaginally, intravenously, or orally in a concentration of from about 0.01 to about 50% w/w of the composition, and especially from about 0.1 to about 30% w/w of the composition.
- the compositions of the invention are advantageously used in a variety of forms, e.g., tablets, ointments, capsules, pills, powders, aerosols, granules, and oral solutions or suspensions and the like containing the indicated suitable quantities of the active ingredient.
- compositions are referred to herein and in the accompanying claims generically as "pharmaceutical compositions.”
- they can be in unit dosage form, namely, in physically discrete units suitable as unitary dosages for human or animal subjects, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic or prophylactic effect in association with one or more pharmaceutically acceptable other ingredients, e.g., diluent or carrier.
- the active ingredients can be packaged in pressurized aerosol containers with a propellant, e.g., carbon dioxide, nitrogen, propane, etc. with the usual adjuvants such as cosolvents, wetting agents, etc.
- a propellant e.g., carbon dioxide, nitrogen, propane, etc.
- adjuvants such as cosolvents, wetting agents, etc.
- compositions are ointments
- the active ingredient can be mixed with a diluent vehicle such as cocoa butter, viscous polyethylene glycols, hydrogenated oils, and such mixtures can be emulsified if desired.
- a diluent vehicle such as cocoa butter, viscous polyethylene glycols, hydrogenated oils, and such mixtures can be emulsified if desired.
- pharmaceutical compositions comprise, as an active ingredient, an effective amount of one or more non-toxic, pharmaceutically acceptable ingredient(s).
- examples of such ingredients for use in the compositions include ethanol, dimethyl sulfoxide, glycerol, silica, alumina, starch, calcium carbonate, talc, flour, and equivalent non-toxic carriers and diluents.
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Abstract
Mycalamide compounds are derived from marine sponges of the genus Mycale or prepared by synthetic methods. These compounds, and pharmaceutical compositions containing them as active ingredients, are useful as antitumor and antiviral agents.
Description
DESCRIPTION
NOVEL MYCALAMΓDES WITH ANTΓTUMOR AND ANTΓVΓRAL ACTIVΓΓΎ
Background of the Invention Considerable research and resources have been devoted to oncology and antitumor measures including chemotherapy. Tumors inflict mammals and man with a variety of disorders and conditions including various forms of cancer and resultant cancerous cachexia, which term refers to the symptomatic discomfort that accompanies the infliction of a mammal with a tumor.
Such symptoms include weakened condition of the inflicted mammal as evidenced by weight loss, etc. The seriousness of cancer is well known since cancer is a major cause of death in man. While certain methods and chemical compositions have been developed which aid in inhibiting, remitting, or controlling the growth of tumors, new methods and antitumor chemical compositions are needed.
Viral diseases also inflict man, plants, insects and animals. The prevention and control of viral diseases has important health and economic implications. Viral diseases contribute to inflictions in humans including the common cold, herpes, acquired immune deficiency syndrome (AIDS), and cancer, so the importance of their control is obvious. Also important is the control of viral diseases in animals for economic and other reasons, e.g., the ability of such animals to become virus reservoirs or carriers which facilitate the spreading of viral diseases to humans. Viral plant diseases have been known to have a disruptive effect on the cultivation of fruit trees, tobacco, and various vegetables. Insect viral diseases are also of interest because of the insects' ability to transfer viral diseases to humans. The prevention and control of viral diseases is thus of prime importance to man, and considerable research has been devoted to antiviral measures. Certain methods and chemical compositions have been developed which aid in inhibiting, controlling, or destroying viruses, but additional methods and antiviral compositions are needed.
It has been found that some natural products and organisms are potential sources for chemical molecules having useful biological activity of great diversity. Marine life has been the source for the discovery of compounds having varied biological activities. Some of the United States patents which have issued for such inventions are as follows: U.S. Patent No. 4,548,814 for didemnins, having antiviral activity, were isolated from a marine tunicate; U.S. Patent No. 4,729,996 discloses compounds, having antitumor properties, that were isolated from marine sponges Teichaxinella morchella and Ptilocaulis walpersi; U.S. Patent No. 4,808,590 discloses compounds, having antiviral, antitumor, and antifungal properties, isolated from the marine sponge Theonella sp.; and U.S. Patent No. 4,737,510 discloses compounds, having antiviral and
antibacterial properties, isolated from the Caribbean sponge Aeelas coniferin. Clearly, marine sponges have proved to be a source of biological compounds, and a number of publications have issued disclosing organic compounds derived from marine sponges, including Scheuer, PJ. (ed.) Marine Natural Products. Chemical and Biological Perspectives. Academic Press, New York, 1978- 1983, Vol. I-V; Faulkner, DJ., (1984) Natural Products Reports 1:551-598; Natural Products
Reports (1986) 3:1-33; Natural Products Reports (1987) 4:539-576; Natural Products Report (1988) 5:613-663; J. Am. Chem. Soc. (1985) 107:4796-4798.
It has now been found that certain compounds derived from extracts of marine sponge of the genus Mycale. family Mvcalidae. and order Poecilosclerida. possess useful biological activity. United States Patent No. 4,868,204 describes some of these compounds.
The present invention, utilizing sponges as a source material and supplemented by novel synthetic production methods, has provided the art with new biologically active compounds and new pharmaceutical compositions useful as antitumor and antiviral agents.
Other advantages and further scope of applicability of the present invention will become apparent from the detailed descriptions given herein; it should be understood, however, that the detailed descriptions, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent from such descriptions.
Brief Summary of the Invention
The invention provides new mycalamide compounds having advantageous biological activities. Also provided are compositions containing such compounds, as well as methods for the preparation and use of the compounds and compositions.
The full scope of applicability of the present invention will become apparent from the detailed description given hereinafter; it should be understood, however, that the detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The invention provides a variety of processes for the production of compounds of the invention. A preferred method of producing the new compounds comprises the steps of collecting marine sponges of the genus Mycale. family Mvcalidae. and order Poecilosclerida. contacting such sponges with a selected organic solvent system to obtain an extract, fractionating the extract, and isolating mycalamide compounds from the fractionated extract. These compounds can serve as the starting materials for the preparation of many of the compounds described herein. In further preferred methods of the invention, some of the new mycalamide compounds are made by hydrogenation in the presence of a hydrogenation catalyst. Also, ion-exchange, hydrolysis, alkylation, acetylation, and other known synthesis type reactions may be used pursuant
to known procedures to add, remove, or modify various groups in the original compounds to produce other compounds.
As a result of the discovery of the new compounds, skilled chemists will be able to use known procedures to synthesize these compounds from available stock substances. The compounds of the subject invention are especially important by virtue of the discovery that viruses and tumors can be inhibited by contact with an effective amount of the new compounds.
Detailed Description of the Invention The subject invention pertains to novel chemical compounds isolated from marine sponges, and various derivatives and analogs of these compounds. In order to isolate the new compounds by extraction from marine sponges, suitable organic solvent systems for extraction can be selected from methanol, ethyl acetate, acetone, diethyl ether, t-butyl methyl ether, ethanol, and isopropanol. Mixtures of two or more of such solvents in various ratios and combinations are advantageous.
Compounds of the invention can be synthesized and/or isolated by various fractionation and chromatographic techniques from the extracts obtained as disclosed. Preferred isolation procedures include various chromatography techniques, such as countercurrent chromatography, with suitable columns including multi-layer planetary coil columns. A variety of solvents are available for use as single or mixed eluents, such as tetrahydrofuran, methanol, ethyl acetate, acetonitrile, n-propanol, n-butanol, water, and equivalent solvents. Further purifications using such procedures may also be carried out on the recovered extractions. Preferred isolation techniques for further purifications include chromatographic operations such as high-pressure liquid chromatography (HPLC) with suitable columns and suitable solvents. The novel compounds have been shown to possess antitumor and antiviral activity. Thus, the subject invention pertains to the compounds themselves, as well as pharmaceutical compositions containing these compounds. Also disclosed and claimed are methods for administering the novel compositions. Various derivatives of these compounds can be produced by known procedures. A more complete understanding of the invention can be obtained by reference to preferred embodiments of the invention which are illustrated by the following specific examples of compounds, compositions, and methods of the invention. It will be apparent to those skilled in the art that the examples involve use of materials and reagents that are commercially available from known sources, e.g., chemical supply houses, so no details are given respecting them. As will be seen in some of the examples below, a new class of compounds having the following structural formula has been identified:
wherein R1 and R2 are the same or different and are hydrogen or lower alkyl, particularly C1-C5 alkyl; R3"6 are the same or different and are hydrogen, lower alkyl, acyl, lower alkyl silyl, Bn, or Bz; X is =CH2, -CH3, or -0-CH2; and Y is lower alkyl, Bn, or Bz.
Advantageously, these compounds have been found to have antitumor and antiviral activity.
Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1
The compound known as mycalamide A has the following formula:
Mycalamide A is described in United States Patent No.4,868,204. A New Zealand marine sponge of the genus Mycale (family Mvcalidae. order Poecilosclerida) was collected from the channel at Aquarium Point, Otago Harbour, New Zealand. Two hundred grams of frozen sponge were extracted by blending with 3:1 mefhanohtoluene and filtering off the solid. On removing the solvents, the combined extracts from three such steps yielded a brown gum (11.04 g) with antiviral properties. Reverse phase flash chromatography (Blunt et al. [1987] J. Natl. Prod. 50:290) gave bioactive fractions on eluting with 1:1, 3:1, and 9:1 mixtures of H20:methanol. These were combined to give a brown oil (307 mg). A subsample of this material (140 mg) was applied to a
column of Fractogel PGM 2000 (120 g Fractogel, column 43 cm x 2 cm). Eluting with 1:4 H20:methanol (0.5 ml/min) gave bioactive fraαions at around 1.5 void volumes, which were combined (brown oil, 50 mg). Silica gel column chromatography (DAVISIL, 35-60 /.m, 5g) starting with CH2C12, then increasing amount of methanol, gave 1.7 mg of mycalamide A in the most bioactive fraction, eluted with 1:9 methanol:CH2Cl2.
Spectral data:
[ ]365 nm + 110° (c 0.2 gm/100 ml, CHC13)
MS (El): M+, measured 503.27220 daltons, calculated for C-^H^NOj.-, 503.27305 (-1.7 ppm). M+-methanol, measured 471.24824 daltons, calculated for C^H^NO-*, 471.24683 (+3.0 ppm).
IR, film (cm-1): 3700-3100, 2690, 1740, 1700, 1540, 1470, 1390, 1100, 1080, 1040.
1H NMR (CDC13): δ 7.49 (NH9, d, 9.8), 5.87 (H10, t, 9.8), 5.13 (10-OCH2, d, 6.9), 4.84 (4=CH2, m), 4.73 (4=CH2, m), 4.30 (H7, s), 4.22 (H12, dd, 6.7, 10.3), 3.98 (H2, dq, 2.7, 6.6), 3.86 (Hll, dd, 6.7, 9.8), 3.74 (H17, m), 3.60 (H15, dd, 4.0, 5.5), 3.55 (13-OCH3, s), 3.55 (H18, m, hidden), 3.46 (H13, d, 10.3), 3.38 (H18, dd, 6.2, 11.2), 3.29 (6-OCH3, s), 2.36 (H^, m), 2.24 (H3, dq, 2.7, 7.0), 1.54 (H216, m), 1.19 (2-CH3, d, 6.6), 0.99 (3-CH3, d, 7.0), 0.98 (14-CH3(eq), s), 0.87 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 171.52 (C8), 145.40 (C4), 110.41 (4=CH2), 99.66 (C6), 86.71 (10- OCH-j), 79.01 (C13), 78.91 (C15), 74.30 (C12), 73.62 (CIO), 72.77 (C7), 71.51 (C17), 71.16 (Cll), 69.70 (C2), 66.41 (C18), 61.75 (B-OCΗj), 48.88 (6-OCH3), 41.61 (C14), 41.31 (C3), 33.70 (C5), 31.95 (C16), 23.10 (14-CH3(eq)), 17.89 (2-CH3), 13.51 (14-CH3(ax)), 12.03 (3-CH3).
Example 2
The compound known as mycalamide B has the following formula:
OCH,
Mycalamide B is described in U.S. Patent No. 4,864,204. Mycalamide B, an oil having a molecular formula of C^H^NOj-*, and a molecular weight of 517, was extracted, using the same general procedure as described in Example 1, from the same sponge, i.e., Mycale sp. (type
specimen PML1-9, Chemistry Department, University of Canterbury), family Mycalidae, order Poecilosclerida (Perry et al. [1988] JACS 110:4850-4851; J. Org. Chem. [1990] 55:223). Experience indicates that mycalamide A & B co-occur in all samples of the active Mycale sp. of sponge.
Spectral data: [ ]D + 39° (c 0.2, CHC13).
HREΓMS: M+-CH30 486.26993 (-0.8 ppm), M+-CH3OH 485.26422 (+3.5 ppm).
DCIMS (NH3): 535 (5%, M+NH4 +), 505 (28%), 504 (38%), 503 (100%, M+NH4 +- CH3OH), 488 (23%), 487 (36%), 486 (89%).
DCIMS (ND3): 543 (5%), 542 (14%), 541 (10%), 513 (17%), 512 (19%), 511 (34%), 510 (100%), 509 (82%), 508 (29%), 493 (8%), 492 (9%), 491 (15%), 490 (36%), 489 (37%), 488
(16%).
DCIMS (CH4): 488 (16%), 487 (32%), 486 (100%, MH+CH3OH), 456 (16%).
IR (film): 3700-3100, 2950, 1700, 1540, 1470, 1390, 1100, 1080, 1040, 750 cm-"1.
IR (CHC13): 3600-3300, 2900, 1690, 1390, 1100, 1040 cm-1. XH NMR CDCy: δ 7.54 (NH9, d, 10.0), 5.79 (H10, t, 9.7), 5.12 (10-O-CH2, d, 7.0), 4.85
(10-O-CH2, d, 6.9), 4.85 (4=CH2, t, 2.0), 4.72 (4=CH2, t, 1.9), 4.29 (H7, s), 4.21 (H12, dd, 6.7, 10.4), 4.02 (H2, dq, 2.8, 6.6), 3.79 (Hll, dd, 6.7, 9.7), 3.65 (H18, dd, 3.3, 11.9), 3.55 (13-0-CH3, s), 3.47 (H18, dd, 5.7, 11.9), 3.44 (H13, d, 10.5), 3.41 (H15, dd, 3.2, 9.1), 3.29 (6-O-CHa, s), 3.24 (17-0-CH3, s), 3.2 (H17, m), 2.36 (H5(eq), d, 13.9), 2.22 (H5(ax), td, 2.0, 13.9), 2.24 (H3, dq, Z4, 6.9), 1.5 (H2, m), 1.20 (2-CH3, d, 6.6), 1.01 (3-CH*j, d, 7.1), 0.97 (14-CH3(eq), s), 0.85 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 171.88 (C8), 145.10 (C4), 111.02 (4=CH2), 99.95 (C6), 86.49 (10-
O-CH-j), 79.27 (C13), 78.84 (C17), 75.46 (C15), 74.44 (C12), 73.90 (CIO), 71.73 (C7), 70.94 (Cll),
69.64 (C2), 63.48 (C18), 61.78 (13-0-CH3), 56.64 (17-0-CH3), 48.57 (6-0-CH3), 41.47 (C14), 41.27 (C3), 33.64 (C5), 29.63 (C16), 23.13 (14-CH3(eq)), 17.93 (2-CH3), 13.32 (14-CH3(ax)), 12.13 (3-
CH3).
Example 3 — Mycalamide A Triacetate
Mycalamide A triacetate has a molecular weight of 629 and a molecular formula of (-^H^NOjg. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
HREIMS: M+-CH3OH 590.2800 (-10.6 ppm); M+-CH3OH 597.27536 (-5.3 ppm).
DCIMS (NH-j): 647 (13%, M+NH4), 617 (29%), 616 (37%), 615 (100%, M+NH4 +- CH3OH), 542 (35%), 318 (42%), 317 (44%), 299 (30%), 286 (58%), 285 (41%), 270 (28%), 269 (60%), 257 (30%).
DCIMS (CH4): 598 (71%, MH+-CH3OH), 538 (100%, MH+-CH3OH-CH3C02H), 299 (53%), 269 (50%), 240 (41%), 208 (55%).
1H NMR (CDC13): <5 732 (NH9, d, 9.4), 5.76 (H10, t, 9.0), 5.47 (H7, s), 5.06 (10-OCH2, d, 9.4), 5.76 (H10, t, 9.0), 5.47 (H7, s), 5.06 (10-OCH2, d, 6.9), 4.98 (H17, m), 4.87 (4=CH2, m), 4.86 (10-OCH2, d, 7.0), 4.76 (4=CH2, m), 4.27 (H18, dd, 2.7, 12.4), 4.14 (H18, dd, 5.2, 12.4), 4.10 (H12, dd, 6.0, 9.4), 3.99 (H2, dq, 2.8, 6.6), 3.79 (Hll, dd, 6.0, 8.7), 3.52 (13-OCH3, s), 3.36 (H13, d, 9.4), 3.45 (H15, dd, 2.4, 9.8), 3.18 (6-OCH3, s), 2.4 (H^, m), 2.25 (H3, dq, 2.7, 7.0), 2.20 (7- OCOCH3, s), 2.05 and 2.00 (17-OCOCH3, s and 18-OCHCH3, 2xs), 1.7-1.8 (H216, m), 1.20 (2- CH3, d, 6.6), 1.03 (3-CH3, d, 7.1), 1.01 (14-CH3(eq), s), 0.86 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDQ3): δ 170.60 (C8), 169.85, 167.64 and 167.01 (7-OCO, 17-OCO, and 18- OCO), 145.08 (C4), 110.85 (4=CH->), 99.26 (C6), 86.53 (lO-OCB-j), 79.73 (C13), 75.53 (C15), 74.06 (C12), 73.72 (CIO), 71.66 (C7), 70.01 (C17), 69.91 (Cll), 69.73 (C2), 63.57 (C18), 61.55 (13- OCH3), 48.60 (6-OCH3), 41.24 (C14, C3), 34.18 (C5), 30.08 (C16), 23.69 (14-CH3(eq)), 21.06, 20.80 and 20.65 (7-OCOCH3, 17-OCOCH3, and 18-OCOCH3), 17.87 (2-CH3), 14.4 (14-CH3(ax)), 12.04 (3-CH3) ppm.
Mycalamide A triacetate is prepared as follows: Mycalamide A (2 mg) was dissolved in pyridine (0.5 ml) and acetic anhydride (0.5 ml). After 7 hours at 21°C, water (1 ml) was added and the mixture extracted with CHC13 (3 x 1 ml). The solvent was removed and combined organic extracts were subjected to silica gel chromatography (200 mg Davisil, 150 A, 35-70 ^m), developed in steps from hexane to ethyl acetate. A fraction (1.2 mg) eluted with 1:1 hexane:ethyl acetate was pure mycalamide A triacetate, an oil.
Example 4 — Mycalamide B Diacetate
Mycalamide B diacetate has a molecular weight of 601 and a molecular formula of C^9H 7N012. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (NH3): 619 (22%, M+NH4), 589 (16%), 588 (32%), 587 (100%, M+NH4- CH3OH), 570 (25%, MH+-CH3OH), 318 (19%), 290 (25%), 258 (62%), 257 (26%), 241 (45%).
IR (CHC13): 3400, 2950, 2900, 1750, 1710, 1380, 1100, 1030, 910 cm-1.
1H NMR (CDC13): <5 7.27 (NH9, d, 9.4), 5.75 (H10, t, 9.4), 5.45 (H7, s), 5.07 (10-OCH2, d, 7.0), 4.88 (4=CH2, m), 4.85 (10-OCH2, d, 6.9), 4.75 (4=CH2, m), 4.28 (H18, dd, 2.5, 12.3), 4.17 (H12, dd, 6.6, 10.2), 4.07 (H18, dd, 4.7, 12.5), 4.02 (H2, dq, 2.8, 6.7), 3.77 (Hll, dd, 6.5, 9.4), 3.53 (13-OCH3, s), 3.39 (H13, d, 9.8), 3.3 (H17, m), 33 (H15, hidden), 3.25 (17-OCH3, s), 3.17 (6- OCH3, s), 2.4 (H-25, m), 2.28 (H3, dq, 2.7, 73), 2.20 (7-OCOCH3, s), 2.08 (18-OCOCH3, s), 1.6-1.7 (H216, m), 1.22 (20CH3, d, 6.5), 1.04 (3-CH3, d, 7.2), 0.97 (14-CH3(eq), s), 0.86 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.85 (C8), 169.68 and 166.67 (7- and 18-OCO), 144.75 (C4), 111.26 4=CH2), 99.15 (C6), 86.53 (lO-OCH--*), 79.43 (C13), 77.93 (C17), 75.68 (C15), 74.18 (C12), 74.02 (CIO), 71.48 (C7), 70.64 (Cll), 69.95 (C2), 63.47 (C18), 61.70 (13-OCH3), 56.88 (17-OCH3), 48.49 (6-OCH3), 41.24 (C14), 41.16 (C3), 34.39 (C5), 30.33 (C16), 23.38 (14-CH3(eq)), 20.97 and 20.65 (7-OCOCH3 and 18-OCOCH3), 17.92 (2-CH , 13.8 (14-CH3(ax), broad), 12.19 (3-CH3) ppm.
Mycalamide B diacetate is prepared as follows: Mycalamide B (3.5 mg) was dissolved in pyridine (0.1 ml) and acetic anhydride (0.1 ml). After four hours at 21°C, water (0.2 ml) was added and the mixture extracted with CHC13 (3 x 2.0 ml). The solvent was removed and the combined organic extracts were subjected to silica gel chromatography (200 mg Davisil, 150 A, 35- 70 μm), developed in steps from hexane to ethyl acetate. A fraction (3 mg) eluted with 1:1 hexane:ethyl acetate was pure mycalamide B diacetate, an oil.
Example 5 - Mycalamide B 18-Mono-TBDMS Ether
Mycalamide B 18-Mono-TBDMS Ether has a molecular weight of 631 and a molecular formula of C31H57NO10Si. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
1H NMR (CD2Cl2): δ 7.51 (NH9, d, 9.4), 5.78 (H10, t, 9.4), 5.12 (lO-OCH-j, d, 7.0), 4.86 (4=CH2, t, 2.0), 4.84 (10-OCH2, d, 7.0), 4.70 (4=CH2, t, 2.0), 4.24 (H7, d, 2.7), 4.14 (H12, dd, 6.3, 9.5), 4.03 (H2, dq, 2.9, 6.6), 3.86 (7-OH, d, 2.7), 3.79 (Hll, dd, 63, 9.0), 3.67 (H18, dd, 2.8, 11.5), 3.57 (H18, dd, 4.1, 11.5), 3.53 (13-OCH3, s), 3.41 (H13, d, 9.3), 3.36 (H15, dd, 1.5, 9.8), 3.28 (6- OCH3, s), 3.21 (17-OCH3, s), 3.12 (H17, m), 2.30 (H5(eq), d, 13.8), 2.27 (H3, dq, 2.6, 7.0), 2.15 (H5(ax), td, 2.1, 14.2), 1.76 (H16, ddd, 2.0, 9.5, 14.2), 1.5 (H16, m), 1.21 (2-CH3, d, 6.6), 1.02 (3- CH3, d, 7.2), 1.02 (14-CH3(eq), s), 0.91 (18-OSiC(CH3)3, s), 0.87 (14-CH3(ax), s), 0.08 and 0.09 (18-OSi(CH3)2, 2xs) ppm (couplings in Hz).
13C NMR (CD2α2): δ 145.88 (C4), 110.74 (4=CH-2), 100.14 (C6), 86.53 (10-OCH_), 79.96 (C13), 79.09 (C17), 76.49 (C15), 74.23 (C12, CIO), 71.59 (C7), 69.70 (C2), 62.48 (C18), 61.53 (13-OCH3), 56.74 (17-OCH3), 48.46 (6-OCH3), 41.57 (C3), 33.70 (C5), 29.91 (C16), 25.89 (18- OSiC(CH3)3), 23.70 (14-CH3(eq)), 17.90 (2-CH3), 12.34 (3-CH3), -5.43 (18-OSi(CH3)2). NB: C8, Cll, C14, 14-CH3(ax) not observed.
Mycalamide B 18-Mono-TBDMS Ether is prepared as follows: Mycalamide B (3 mg), t-butyldimethylchlorosilane (12 mg), dimethylaminopyridine (1 mg) and triethylamine (14 mg) were stirred in pyridine (0.2 ml) at room temperature for 20 hours. H20 (0.5 ml) was added, the mixture extracted with CH2C12 (3 x 0.3 ml), and the solvent removed. The combined organic extract was subjected to silica gel chromatography (200 mg Davisil, 150A, 35-70 μm), developed in steps from hexane to ethanol/ethyl acetate. The major fraction (1.7 mg) which eluted with 1:19 EtOH:EtOAc was unreacted mycalamide B, but a fraction (1.5 mg) which eluted with 1:1 PE:EtOAc was pure mycalamide B 18-mono-TBDMS ether by PMR, an oil.
Example 6 — Mycalamide B 7-Monoacetate
Mycalamide B 7-monoacetate has a molecular weight of 559 and molecular formula of C27H45NO.-_.-_. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (NH3) 577 (15%, M+NH4 +), 547 (28%), 546 (30%), 545 (100%, M+NH4 +- CH3OH), 530 (10%), 529 (11%), 528 (34%, MH+-CH3OH).
1H NMR (CD2CI2): δ 7.44 (NH9, d, 9.8), 5.77 (H10, t, 9.5), 5.47 (H7, s), 5.12 (10-OCH2, d, 7.0), 4.89 (4=CH2, m), 4.85 (10-OCH2, d, 7.1), 4.74 (4=CH2, m), 4.20 (H12, dd, 6.7, 10.7), 4.02 (H2, dq, 2.9, 6.5), 3.78 (Hll, dd, 6.7, 9.6), 3.66 (H18, dd, 2.6, 12.2), 3.54 (13-OCH3, s), 3.45 (H13, d, 10.3), 3.44 (H15, dd, 1.7, 10.0), 3.35 (H18, dd, 6.2, 12.2), 3.23 (17-OCH3, s), 3.16 (6-OCH3, s),
3.13 (H17, m), 2.39 (H-J5, m), 230 (H3, dq, 2.9, 7.1), 2.20 (7-OCOCH3, s), 1.58 (H16, m), 1.41 (H16, ddd, 1.6, 10.2, 14.3), 1.24 (2-CH3, d, 6.6), 1.05 (3-CH3, d, 7.1), 0.98 (14-CH3(eq), s), 0.85 (14- CH3(ax), s) ppm (couplings in Hz).
*H NMR (CDC13): ό 7.46 (NH9, d, 10.0), 5.77 (H10, t, 9.3), 5.52 (H7, s), 5.09 (10-OCH2, d, 7.1), 4.88 (4=CH2, m), 4.87 (10-OCH2, d, 7.1), 4.77 ( =0^ ), 4.22 (H12, dd, 7.1, 10.2), 4.04 (H2, dq, 2.8, 6.6), 3.75 (Hll, dd, 6.6, 9.6), 3.71 (HIS, dd, 2.1, 123), 3.55 (13-OCH3, s), 3.44 (H15, broad d, 10.0), 3.42 (H13, d, 10.4), 3.41 (H18, dd, 6.7, 123), 3.26 (17-OCH3, s), 3.17 (6-OCH3, s),
3.14 (H17, m), 2.43 (H25, m), 2.29 (H3, dq, 2.9, 7.1), 2.21 (7-OCOCH3, s), 1.54 (H16, m), 138 (H16, m), 1.24 (2-CH3, d, 6.7), 1.05 (3-CH3, d, 7.1), 0.96 (14-CH3(eq), s), 0.85 (14-CH3(ax), s) ppm (couplings in Hz).
13CNMR (CD-ClrT): δ 145.17 (C4), 111.11 (4=CH2), 99.46 (C6), 86.78 (lO-OCH^, 79.91 (C17), 79.40 (C13), 75.86 (C15), 74.75 (C12), 74.29 (CIO), 71.45 (C7), 70.30 (C2), 63.81 (C18), 61.75 (13-OCH3), 56.67 (17-OCH3), 48.50 (6-OCH3), 41.50 (C14), 41.37 (C3), 34.73 (C5), 30.18 (C16), 23.13 (14-CH3(eq)), 20.54 (7-OCOCH3), 17.84 (2-CH3) 1332 (14-CH3(ax), broad), 12.20 (3-CH3). NB: C8, 7-OCO and Cll not observed.
Mycalamide B 7-monoacetate can be prepared as follows: Mycalamide B 18-mono-TMS ether (1 mg ca) was dissolved in pyridine (0.1 ml) and acetic anhydride (0.1 ml). The preparation of mycalamide B 18-mono-TMS ether is described in U.S. Patent No. 4,868,204. After 3 hrs at
room temperature, H20 (0.2 ml) was added, the mixture extracted with CH2C12 (3 x 0.2 ml), and the solvent removed (1 mg). TLC and PMR showed desilylation and about 25% diacetate present in an otherwise clean sample of mycalamide B 7-monoacetate. Subsequently this was purified by subjecting the combined sample to silica gel chromatography (200 mg Davisil, 150A, 35-70 μm), developed in steps from hexane to ethyl acetate. The major fraction (0.7mg) which eluted with 100% ethyl acetate was pure mycalamide B 7-monoacetate, an oil.
Example 7 - 7-Methoxy. Mycalamide B 18-Monoacetate
7-Methoxy, Mycalamide B 18-Monoacetate has a molecular weight of 573 and a molecular formula of C^H^NO-,-^ Its molecular structure is as follows:
The compound is characterized as follows:
DCIMS (NH3): 591 (2%, M+NH4 +), 562 (9%), 561 (34%), 560 (32%), 559 (100%, M+NH4 +-CH3OH), 545 (11%), 544 (36%), 543 (30%), 542 (99%, MH+-CH3OH).
1H NMR (CDC13): δ 7.11 (NH9, d, 9.9), 5.79 (H10, t, 9.7), 5.11 (10-OCH2, d, 6.9), 4.83 (10-OCH2, d, 6.7), 4.82 (4=CH2, t, 1.7), 4.71 (4=CH2, t, 1.8), 4.27 (H18, dd, 2.5, 12.1), 4.20 (H12, dd, 6.7, 10.2), 4.03 (H18, dd, 5.3, 12.1), 3.94 (H2, dq, 2.8, 6.6), 3.85 (H7, s), 3.81 (Hll, dd, 6.8, 9.6), 3.54 (13-OCH3, s), 3.54 (7-OCH3, s), 3.43 (H13, d, 10.2), 3.37 (H17, m), 3.36 (H15, dd, 4.5, 7.6), 3.28 (17-OCH3, s), 3.27 (6-OCH3, s), 2.41 (H5(ax), td, 2.0, 14.1), 2.30 (H5(eq), d, 14.3), 2.21 (H3, dq, 2.9, 7.1), 2.08 (18-OCOCH3, s), 1.63 (H216, m), 1.17 (2-CH3, d, 6.6), 0.98 (3-CH3, d, 7.1), 0.97 (14-CH3(eq), s), 0.87 (14-CH3(ax), s) ppm (couplings in Hz).
13CNMR (CDC13): δ 170.87 (C8), 169.69 (18-OCO), 145.95 (C4), 110.25 (4=CH--_), 99.91 (C6), 86.45 (lO-OCH-j), 82.72 (C7), 79.42 (C13), 75.53 (C15), 74.31 (C12), 73.31 (CIO), 70.75 (Cll, broad), 69.45 (C2), 64.12 (C18), 61.77 (13-OCH3), 60.06 (7-OCH3), 56.91 (17-OCH3), 48.94 (6- OCH3), 41.43 (C14), 41.38 (C3), 34.15 (C5), 30.20 (C16), 2331 (14-CH3(eq)), 20.95 (18- OCOCH3), 17.87 (2-CH3), 13.55 (14-CH3(ax), broad), 11.87 (3-CH3). NB: C17 not observed.
7-methoxy, mycalamide B 18-monoacetate can be prepared as follows: Mycalamide B (6 mg), Ag20 (40 mg), and Mel (27 mg) were stirred in benzene (0.4 ml) for 2 hours at 90°C. The solution was filtered over celite and the solvent removed. Pyridine (0.1 ml) and acetic anhydride
(0.1 ml) were added and the mixture stirred at room temperature for 16 hours. H20 was added (2.5 ml) and the solution extracted with CH2C12 (2 x 2 ml). Prep TLC of this extract (3:1 EtOAc:PE) gave three fractions (1.7 mg, 2.0 mg, 2.4 mg) which were pure 7-methoxy mycalamide B 18-acetate, 18-methoxy mycalamide B 7-acetate, and mycalamide B diacetate respectively by nmr, an oil.
Example 8 — 7-Methoxy Mycalamide B
7-methoxy mycalamide B has a molecular weight of 531 and a molecular formula of C26H45NO10. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (NH3): 549 (6%, M+NH4 +), 520 (4%), 519 (20%), 518 (40%), 517 (100%, M+NH4 +-CH3OH), 502 (13%), 501 (29%), 500 (79%, MH+-CH3OH).
1H NMR (CDC13): δ 7.25 (NH9, d, 9.7), 5.82 (H10, t, 9.7), 5.13 (10-OCH2, d, 6.9), 4.85 (10-OCH2, d, 7.0), 4.83 (4=CH2, t, 1.9), 4.71 (4=CH2t, 2.0), 4.23 (H12, dd, 6.8, 10.5), 3.94 (H2, dq, 2.7, 6.6), 3.89 (H7, s), 3.80 (Hll, dd, 6.8, 10.0), 3.69 (H18, dd, 3.0, 12.0), 3.57 (7-OCH3, s), 3.56 (13-OCH3, s), 3.45 (H13, d, 10.5), 3.45 (H18, ), 3.43 (H15, dd, 1.7, 10.0), 330 (17-OCH3, s), 3.27 (6-OCH3, s), 3.22 (H17, m), 2.41 (H5(ax), td, 1.9, 143), 232 (H5(eq), d, 14.4), 2.21 (H3, dq, 2.5, 7.0), 1.57 (H16, m), 1.46 (H16, m), 1.17 (2-CH3, d, 6.6), 0.98 (3-CH3, d, 7.0), 0.97 (14-CH3(eq), s), 0.86 (14-CH3(ax), s) ppm (couplings in Hz).
13CNMR (CDC13): δ 170.33 (C8), 145.77 (C4), 110.37 (4=CH , 100.00 (C6), 86.61 (10- OCH2), 82.39 (C7), 79.72 (C17), 79.27 (C13), 75.49 (C15), 74.64 (C12), 73.51 (CIO), 71.41 (Cll), 69.52 (C2), 64.69 (C18), 61.86 (13-OCH-j), 6039 (7-OCH3), 56.87 (17-OCH3), 48.98 (6-OCH3), 41.59 (C14), 4136 (C3), 34.19 (C5), 29.94 (C16), 23.12 (14-CH3(eq)), 17.85 (2-CH3), 13.18 (14- CH3(ax), broad), 11.88 (3-CH3).
7-methoxy mycalamide B can be prepared as follows: 7-Methoxy mycalamide B 18-monoacetate (1.8 mg) was stirred in a solution with 1.2 mg K2C03 in aqueous MeOH (0.4 ml) at room temperature for 5 hours. The solution was concentrated, then H20 (2.5 ml) was added and the solution extracted with CH2C12 (3 2 ml). The solvent was removed and the sample
subjected to prep TLC (developed twice in 1:5 PE:EtOAc) to give pure 7-methoxy mycalamide B (1.2 mg) by NMR, an oil.
Example 9 - Mycalamide B 7-Monobenzyl Ether
Mycalamide B 7-Monobenzyl Ether has a molecular weight of 607 and a molecular formula of C^H^NO-*,,. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
HRFABMS: 6303202 (M+Na+, -8.3 ppm).
FABMS: 631 (9%), 630 (100%, M+Na+), 598 (7%, M+Na+-CH3OH), 577 (8%), 576 (20%, MH+-CH3OH).
1H NMR (CDC13): -3 7.4-7.3 (φ, m), 7.35 (NH9, d, 9.8), 5.85 (H10, t, 9.8), 5.13 (10- OCH2, d, 6.9), 4.84 (10-OCH2, d, 7.1), 4.82 (4=CH2, m), 4.81 (7-OCH2ø, d, 11.5), 4.72 (4=CH2, m), 4.64 (7-OCH2ø, d, 11.3), 4.23 (H12, dd, 6.9, 10.6), 4.11 (H7, s), 3.91 (H2, dq, 2.8, 6.6), 3.77 (Hll, dd, 6.7, 9.9), 3.71 (H18, dd, 2.9, 12.1), 3.56 (13-OCH3, s), 3.48 (H18, dd, 6.6, 11.9), 3.46 (H13, d, 10.4), 3.45 (H15, broad d, 10.0), 3.27 (17-OCH3, s), 3.27 (H17, m), 3.10 (6-OCH3, s), 2.43 (H25, m), 2.20 (H3, dq, 2.7, 7.0), 1.55 (H16, m), 1.45 (H16, ddd, 1.6, 9.8, 143), 1.17 (2-CH3, d, 6.6), 0.98 (14-CH3(eq), s), 0.98 (3-CH3, d, 7.0), 0.88 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.52 (C8), 146.00 (C4), 136.98, 128.57, 128.24 and 128.20 (7- OCH2 ), 110.30 (4=CH2), 100.10 (C6), 86.58 (lO-OCH^, 80.15 (C7), 79.71 (C17), 79.29 (C13), 75.45 (C15), 74.64 (C12), 74.22 (CIO), 73.48 ( -OCH^, 71.38 (Cll, broad), 69.50 (C2), 64.69 (C18), 61.85 (13-OCH3), 56.86 (17-OCH3), 48.95 (6-OCH3), 41.59 (C14), 41.35 (C3), 34.53 (C5), 29.85 (C16), 23.14 (14-CH3(eq)), 17.84 (2-CH3), 13.22 (14-CH3(ax), broad), 11.86 (3-CH3).
Mycalamide B 7-monobenzyl ether can be prepared as follows: Mycalamide B (4.4 mg), powdered KOH (7 mg) and benzyl bromide (12 mg) were stirred in DMSO (0.3 ml) at room temperature for 4 minutes. H20 (2 ml) was added and the mixture extracted with CHC13 (3 2 ml). The extract was evaporated to dryness, then subjected to prep TLC (developed in 1:1 PE:EtOAc). Three bands of silica were recovered and eluted with ethyl acetate to give three pure
products (1.8 mg, 0.8 mg, 0.9 mg), which were mycalamide B 7-mono-; 7,N-di- and 7,18-di-benzyl ethers by NMR respectively, an oil.
Example 10 - 7.17.18-Trimethoxy. N-Methyl Mycalamide A
7,17,18-Trimethoxy, N-Methyl Mycalamide A has a molecular weight of 559 and a molecular formula of <_^H49NO10. Its molecular structure is as follows:
The compound is characterized as follows:
DCIMS (NH-j): 577 (1%, M+NH4 +), 547 (4%), 546 (6%), 545 (19%, M+NH4 +- CH3OH), 531 (5%), 530 (19%), 529 (30%), 528 (100%, MH+-CH3OH).
1H NMR (CDC13): δ 6.26 (H10, d, 10.1), 5.15 (10-OCH2, d, 7.0), 4.85 (10-OCH2, d, 7.0), 4.81 (4=CH2, t, 2.0), 4.70 (4=CH2, t, 2.0), 4.26 (H12, dd, 6.8, 10.5), 4.25 (H7, s), 4.18 (Hll, dd, 6.8, 10.1), 3.91 (H2, dq, 2.8, 6.6), 3.55 (13-OCH3, s), 3.53 (H18, m), 3.49 (H13, d, 10.5), 3.44 (7- OCH3, s), 338 (H15, broad d, 8.7), 337 (lβ-OCH^ s), 3.36 (H18, m), 333 (H17, m), 3.29 (6- OCH3, s), 3.27 (17-OCH3, s), 3.20 (N-CH^ s), 2.65 (H5(ax), td, 2.0, 14.2), 2.26 (H5(eq), d, 14.2), 2.18 (H3, dq, 2.8, 7.0), 1.70 (H16, ddd, 1.8, 9.6, 14.1), 1.51 (H16, ddd, 2.2, 8.9, 14.2), 1.12 (2-CH3, d, 6.6), 0.97 (14-CH3(eq), s), 0.97 (3-CH3, d, 7.0), 0.87 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.75 (C8), 146.76 (C4), 109.75 (4=0*2), 100.74 (C6), 86.37 (10- OCH-j), 84.10 (C7), 79.35 (C13), 77.33 (CIO, C17), 75.82 (C15), 74.31 (C12), 72.49 (C18), 69.40 (C2), 66.78 (Cll), 61.76 (13-OCH3), 59.07 (18-OCH3), 58.83 (7-OCH3), 56.36 (17-OCH3), 48.93 (6-OCH3), 41.64 (C14), 4135 (C3), 34.06 (C5), 30.43 (C16), 28.79 (N-CH3), 23.15 (14-CH3(eq)), 17.68 (2-CH3), 13.21 (14-CH3(ax)), 11.97 (3-CH3).
7,17,18-Trimethoxy, N-methyl mycalamide A can be prepared as follows: Mycalamide A (5.5 mg), powdered KOH (14 mg) and Mel (23 mg) were stirred in DMSO (0.3 ml) at room temperature for 3.5 hours. H20 was added (0.5 ml) and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to H20), flushed with H20 (6 ml) and then eluted with MeOH (6 ml). The MeOH fraction was evaporated to dryness (5.5 mg), then subjected to prep TLC (developed in EtOAc). Two fractions of silica were recovered and each eluted with 1:9 EtOH:EtOAc (4 ml) to give two fractions on evaporation (1 mg, 3.5 mg) which were
7,18-dimethoxy, N-methyl mycalamide A and 7,17,18-trimethoxy, N-methyl mycalamide A by nmr respectively, an oil.
Example 11 - 7.18-Dimethoxy, N-Methyl Mycalamide A
7,18-Dimethoxy, N-Methyl Mycalamide A has a molecular weight of 545 and a molecular formula of C27H47NO10. Its molecular structure is as follows:
The compound is characterized as follows:
DCIMS (NH3): 563 (10%, M+NH4 +), 533 (19%), 532 (37%), 531 (100%, M+NH4 +- CH3OH), 516 (17%), 515 (32%), 514 (95%, MH+-CH3OH).
1H NMR (CDC13): δ 6.35 (H10, d, 9.5), 5.16 (10-OCH2, d, 6.7), 4.88 (10-OCH2, d, 6.6), 4.82 (4=CH2, m), 4.72 (4=CH2, t, 2.0), 4.29 (H12, dd, 7.2, 10.0), 4.25 (H7, s), 4.19 (Hll, dd, 7.1, 9.5), 3.92 (H2, dq, 2.8, 6.5), 3.81 (H17, m), 3.69 (H15, dd, 2.5, 10.0), 3.57 (13-OCH3, s), 3.54 (H13, d, 10.1), 3.46 (7-OCH3, s), 3.35 (18-OCH3, s), 3.31 (6-OCH3, s), 3.28 (H218, m), 3.19 (N-CH3, s), 2.70 (H5(ax), broad d, 14.4), 2.29 (H5(eq), d, 14.5), 2.19 (H3, dq, 3.0, 6.9), 1.71 (H16, broad d, 14.4), 1.47 (H16, m), 1.13 (2-CH3, d, 6.4), 1.00 (3-CH3, d, 6.8), 0.99 (14-CH3(eq), s), 0.90 (14- CH3(ax), s) ppm (couplings in Hz).
13C NMR: δ 171.24 (C8), 147.01 (C4), 109.52 (4=0^, 100.87 (C6), 86.68 (10-OCH-j), 83.39 (C7), 79.15 (C13), 78.64 (C15), 76.44 (C18), 74.40 (C12), 69.58 (C17), 69.36 (C2), 67.01 (Cll), 61.83 (13-OCH3), 59.12 (18-OCH3), 58.90 (7-OCH3), 48.90 (6-OCH3), 41.90 (C14), 41.43 (C3), 33.83 (C5), 33.34 (C16), 28.70 (N-CH3), 22.95 (14-CH3(eq)), 17.65 (2-CH3), 13.20 (14- CH3(ax)), 12.12 (3-CH3). NB: CIO not observed.
7,18-dimethoxy N-methyl mycalamide A can be prepared as follows: Mycalamide A (5.5 mg), powdered KOH (14 mg) and Mel (23 mg) were stirred in DMSO (0.3 ml) at room temperature for 3.5 hours. H20 was added (0.5 ml) and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to H20), flushed with H20 (6 ml) and then eluted with MeOH (6 ml). The MeOH fraction was evaporated to dryness (5.5 mg), then subjected to prep TLC (developed in EtOAc). Two fractions of silica were recovered and each eluted with 1:9 ElOH:EtOAc (4 ml) to give two fractions on evaporation (1 mg, 3.5 mg) which were
7,18-dimethoxy, N-methyl mycalamide A and 7,17,18-trimethoxy, N-methyl mycalamide A by nmr respectively, an oil.
Example 12 - 7-Methoxy. N-Methyl Mycalamide A
7-Methoxy, N-Methyl Mycalamide A has a molecular weight of 531 and a molecular formula of C^H^NO-,,*,. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (NH3): 549 (3%, M+NH4 +), 519 (6%), 518 (20%), 517 (66%, M+NH4 +- CH3OH), 502 (10%), 501 (29%), 500 (100%, MH+-CH3OH).
1H NMR (CDC13): δ 6.34 (H10, d, 9.9), 5.17 (10-OCH2, d, 6.6), 4.89 (10-OCH2, d, 6.7), 4.83 (4=CH2, m), 4.73 (4=CH2, m), 4.28 (H12, dd, 7.1, 10.6), 4.27 (H7, s), 4.15 (Hll, dd, 7.2, 10.0), 3.93 (H2, dq, 2.6, 63), 3.74 (H17, m), 3.70 (H15, dd, 3.2, 9.8), 3.60 (H18, m), 3.58 (13-OCH3, s), 3.53 (H13, d, 10.7), 3.45 (7-OCH3, s), 3.34 (H18, m), 3.31 (6-OCH3, s), 3.19 (N-CH3, s), 2.68 (H5(ax), broad d, 14.5), 2.29 (H5(eq), d, 143), 2.20 (H3, dq, 2.4, 6.8), 1.58 (H216, broad td, 3.0, 10.1), 1.14 (2-CH3, d, 6.5), 1.01 (3-CH3, d, 7.0), 0.99 (14-CH3(eq), s), 0.88 (14-CH3(ax), s) ppm (couplings in Hz).
13CNMR (CDC13): δ 146.76 (C4), 109.76 (4=CH--_), 100.82 (C6), 86.49 (lO-OCH^, 82.56 (C7), 79.16 (C13), 78.14 (C15), 77.26 (CIO), 74.40 (C12), 71.07 (C17), 69.39 (C2), 67.09 (Cll), 66.86 (C18), 61.86 (13-OCH3), 58.89 (7-OCH-j), 48.90 (6-OCH3), 41.84 (C14), 41.39 (C3), 33.88 (C5), 32.97 (C16), 28.75 (N-CH3), 22.98 (14-CH3(eq)), 17.66 (2-CH3), 13.11 (14-CH3(ax)), 12.16 (3-CH3). NB: C8 not observed.
7-methoxy, N-methyl mycalamide A can be prepared as follows: Mycalamide A (3 mg), powdered KOH (3 mg) and Mel (7 mg) were stirred in DMSO at room temperature for 4 hours. H20 was added (0.3 ml), the mixture extracted with CH2C12 (3 x 0.4 ml), and the solvent removed (3.5 mg). Prep RPLC (30% H20 in MeOH) gave two fractions (1.9 mg, 1 mg) which were pure 7-methoxy, N-methyl mycalamide A and a 4:1 mixture of 7,18-dimethoxy, N-methyl mycalamide A and 7,17-dimethoxy, N-methyl mycalamide A by PMR respectively, an oil.
Example 13 - 6-Ethoxy Mycalamide A
6-Ethoxy Mycalamide A has a molecular weight of 517 and a molecular formula of C^H^NOJQ. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (NH-j): 535 (6%, M+NH4 +), 491 (4%), 490 (6%), 489 (100%, M+NH4 +- CH3CH2OH), 474 (5%), 473 (5%), 472 (65%, MH+-CH3CH2OH).
1H NMR (CDC13): δ 7.50 (NH9, d, 10.0), 5.87 (H10, t, 9.8), 5.14 (10-OCH2, d, 6.9), 4.87 (10-OCH2, d, 6.9), 4.83 (4=0^ m), 4.73 (4=CH2, m), 4.26 (H7, s), 4.22 (H12, dd, 6.6, 10.2), 3.99 (H2, dq, 2.8, 6.5), 3.85 (Hll, dd, 6.8, 9.7), 3.74 (H17, m), 3.63 (H15, m), 3.6 (H18, m), 3.56 (13- OCH3, s), 3.55 (6-OCH2, q, 7.0), 3.46 (H13, d, 10.2), 3.38 (H18, dd, 6.1, 11.2), 2.38 (H25, m), 2.23 (H3, dq, 3.0, 7.1), 1.57 (H216, m), 1.18 (6-OCH2CH3, t, 7.0), 1.17 (2-CH3, d, 6.6), 1.00 (3-CH3, d, 7.1), 0.98 (14-CH3(eq), s), 0.88 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 110.49 (4=CH2), 99.74 (C6), 86.83 (lO-OCH^, 79.16 (C13), 78.96 (C15), 74.42 (C12), 73.77 and 73.70 (CIO and C7), 71.49 (C17), 71.28 (Cll), 69.84 (C2), 66.53 (C18), 61.82 (13-OCH3), 56.74 (6-OCH2), 41.41 (C3), 33.90 (C5), 32.05 (C16), 23.23 (14-CH3(eq)), 17.92 (2-CH3), 15.18 (6-OCH2CH3), 13.53 (14-CH3(ax)), 12.08 (3-CH3). NB: C8, C4, C14 not observed.
6-ethoxy mycalamide A can be prepared as follows: Mycalamide A (20 mg) was dissolved in CDC13 containing a trace of ethanol and left for two weeks. PMR showed partial reaction to at least two components and this was confirmed by HPLC. Prep HPLC (35% H20 in MeOH) separated unreacted mycalamide A (4.5 mg) and 6-ethoxy mycalamide A (1.2 mg) in pure form by PMR.
Example 14 — Mycalamide A 17.18-Di-p-Bromobenzoate
Mycalamide A 17,18-Di-p-Bromobenzoate has a molecular weight of 869 and a molecular formula of
Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
1H NMR (CDOj): δ 7.83 and 7.82 (17- and 18-OCOC6H4Br, 2xd, 8.6), 7.55 (NH9, d, hidden), 7.54 (17- and 18-OCOC6H4Br, d, 8.7), 5.85 (H10, t, 9.2), 5.42 (H17, qd, 4.5, 9.0), 5.1210- OCH2, d, 6.9), 4.86 (10-OCH2, d, 6.8), 4.82 (4=0*2, t, 1.9), 4.70 (4=CH2, t, 1.7), 4.56 (H218, d, 4.5), 4.27 (H7, d, 2.9), 4.15 (H12, dd, 6.0, 9.5), 4.00 (H2, dq, 2.9, 6.6), 3.93 (7-OH, d, 2.9), 3.87 (Hll, dd, 6.2, 8.7), 3.59 (H15, dd, 2.2, 9.9), 3.53 (13-OCH3, s), 3.41 (H13, d, 9.5), 3.28 (6-00 s), 2.39 (H5(eq), d, 143), 2.26 (H5(ax), broad d, 14.2), 2.22 (H3, dq, 2.4, 7.1), 1.97 (H16, ddd, 2.1, 9.0, 14.6), 1.85 (H16, ddd, 3.5, 10.5, 14.5), 1.19 (2-CH3, d, 6.7), 1.01 (14-CH3(eq), s), 0.98 (3-0 d, 7.1), 0.88 (14-CH3(ax), s) ppm (couplings in Hz).
Mycalamide A 17,18-Di-p-bromobenzoate can be prepared as follows: Mycalamide A (5.0 mg, 0.01 mmole), p-bromobenzoyl chloride (11 mg, 0.05 mmole), dimethylaminopyridine (1 mg) and triethylamine (7 mg, 0.07 mmole) were stirred in pyridine (0.4 ml) at 75°C overnight. H20 (03 ml) was added, the mixture extracted with CH2C12 (3 x 0.4 ml), and the solvent removed (17 mg ca.). Prep RPLC (10% H20 in MeOH) gave four fractions (2 mg, 1 mg, 1.2 mg, 1.1 mg) which were mycalamide A 18-mono-p-bromobenzoate and mycalamide A 7,18-di-p-bromobenzoate, obtained previously, and two new compounds, identified by PMR as pure mycalamide A 17,18-di-p-bromobenzoate and mycalamide A tri-p-bromobenzoate respectively, a white solid.
Example 15 — 7-Methoxy Mycalamide A
7-Methoxy Mycalamide A has a molecular weight of 517 and a molecular formula of C25H43NO10. Its molecular structure is as follows:
OCH,
OCH,
The compound is characterized as follows:
DCIMS (NH3): 535 (10%, M+NH4 +), 505 (28%), 504 (28%), 503 (100%, M+NH4 +- CH3OH), 488 (12%), 487 (9%), 486 (30%, MH+-CH3OH).
1H NMR (CDCI3): δ 7.18 (NH9, d, 10.0), 5.91 (H10, t, 10.0), 5.14 (10-OCH2, d, 6.9), 4.85 (10-OCH2, d, 6.9), 4.82 (4=CH2, t, 1.9), 4.71 (4=CH2, t, 1.8), 4.24 (H12, dd, 6.9, 10.6), 3.90 (H2, dq, 2.6, 6.7), 3.88 (H7, s), 3.87 (Hll, dd, 6.9, 10.0), 3.75 (H17, m), 3.64 (H15, dd, 5.1, 6.5), 3.60 (H18, dd, 3.4, 11.0), 3.56 (7- and 13-OCH3, s), 3.48 (H13, d, 10.6), 3.38 (H18, dd, 6.0, 11.0), 3.28 (6-OCH3, s), 2.43 (H5(ax), td, 2.0, 143), 232 (H5(eq), d, 14.1), 2.19 (H3, dq, 2.6, 7.0), 1.56 (H216, m), 1.17 (2-CH3, d, 6.6), 0.97 (14-CH3(eq), s), 0.96 (3-CH3, d, 7.0), 0.88 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.56 (C8), 146.27 (C4), 109.95 (4=0^, 100.02 (C6), 86.83 (10- OCH^, 82.99 (C7), 79.22 (C13), 78.99 (C15), 74.55 (C12), 73.22 (CIO), 71.65 (C17), 71.60 (Cll), 69.40 (C2), 66.68 (C18), 61.89 (13-OCH3), 60.50 (7-OCH3), 49.18 (6-OCH3), 41.81 (C14), 41.40 (C3), 34.14 (C5), 32.03 (C16), 22.98 (14-CH3(eq)), 17.81 (2-CH3), 13.24 (14-CH3(ax)), 11.77 (3- CH3).
7-methoxy mycalamide A can be prepared as follows: Mycalamide A (7 mg), Ag20 (55 mg) and Mel (21 mg), were stirred in benzene (0.4 ml) at 95°C in a sealed vial for 1.5 hours. The solution was filtered over celite and the solvent removed (7.3 mg). Preparative TLC (developed twice in 1:7 PE:EtOAc) gave four fractions (0.8 mg, 1.4 mg, 2.0 mg, 2.8 mg) which were mycalamide A, 7-methoxy A, 3:2 17-methoxy A:18-methoxy A, and pure 18-methoxy A by nmr respectively. Subsequently 7-methoxy mycalamide A was subjected to further chromatography (prep. TLC, developed 3x in 1:5 PE:EtOAc) to give the pure compound (1.2 mg), an oil.
Example 16 - 7.18-Dimethoxy Mycalamide A
7,18-Dimethoxy Mycalamide A has a molecular weight of 531 and a molecular formula of C-^H^NOj--,. Its molecular structure is as follows:
OCH,
OCH,
The compound is characterized as follows:
DCIMS (NH3): 549 (7%, M+NH4 +), 519 (22%), 518 (30%), 517 (100%, M+NH4 +- CH3OH), 502 (11%), 501 (16%), 500 (53%, MH+-CH3OH).
XH NMR (CDC13): δ 7.13 (NH9, d, 9.9), 5.90 (H10, t, 10.0), 5.14 (10-OCH2, d, 7.0), 4.83 (10-OCH2, d, 7.0), 4.81 (4=CH2, t, 1.8), 4.70 (4=CH2, t, 1.9), 4.23 (H12, dd, 6.8, 10.5), 3.89 (H2, dq, 2.7, 6.6), 3.88 (H7, s), 3.88 (Hll, dd, 6.7, 9.8), 3.82 (H17, m), 3.58 (H15, m), 3.55 (13-OCH3, s), 3.54 (7-OCH3, s), 3.47 (H13, d, 10.8), 335 (18-OCH3, s), 330 (H18, dd, 4.6, 9.6), 3.28 (6-OCH3, s), 3.26 (H18, dd, 5.7, 9.7), 2.44 (H5(ax), td, 1.9, 143), 231 (H5(eq), d, 14.5), 2.18 (H3, dq, 2.6, 7.2), 1.6 and 1.52 (H216, m), 1.16 (2-CH3, d, 6.6), 0.97 (14-CH3(eq), s), 0.96 (3-CH3, d, 7.2), 0.88 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDClg): δ 170.43 (C8), 146.45 (C4), 109.79 (4=CH^, 99.97 (C6), 86.74 (10- OCH^, 83.16 (C7), 79.38 (C13), 79.08 (C15), 76.19 (C18), 74.47 (C12), 73.07 (CIO), 71.26 (Cll), 69.96 (C17), 69.32 (C2), 61.86 (13-OCH3), 60.45 (7-OCH3), 59.10 (18-OCH3), 49.14 (6-OCH3), 41.81 (C14), 41.45 (C3), 34.20 (C5), 32.48 (C16), 23.03 (14-CH3(eq)), 17.80 (2-CH3), 1335 (14- CH3(ax)), 11.73 (3-CH3).
7,18-dimethoxy mycalamide A can be prepared as follows: Mycalamide A (4.2 mg), Ag20 (25 mg) and Mel (18 mg), were stirred in benzene (03 ml) at 80°C in a sealed vial for 3 days. The solution was filtered over celite and the solvent removed (4.6 mg). Preparative TLC (developed in EtOAc) gave three fractions (0.5 mg, 1.2 mg, 2.0 mg) which were 7,17-dimethoxy mycalamide A, 7,18-dimethoxy A and 7,17,18-trimethoxy A by nmr respectively. These were individually rechromatographed by prep. TLC to give the pure compounds, an oil.
Example 17 — 18-Methoxy Mycalamide A
18-Methoxy Mycalamide A has a molecular weight of 517 and a molecular formula of C^H^NOJQ. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCTMS (NH3): 535 (16%, M+NH4 +), 505 (24%), 504 (33%), 503 (100%, M+NH4 +- CH3OH), 487 (19%), 486 (66%, MH+-CH3OH).
1H NMR (CDC13): δ 7.49 (NH9, d, 9.8), 5.85 (H10, t, 9.6), 5.13 (10-OCH2, d, 6.9), 4.87 (10-OCH2, d, 6.9), 4.84 (4=CH2, t, 1.6), 4.74 (4=CH2, t, 1.5), 4.29 (H7, s), 4.21 (H12, dd, 6.6, 10.2), 4.00 (H2, dq, 2.7, 6.5), 3.84 (Hll, dd, 6.6, 9.6), 3.79 (H17, m), 3.55 (13-OCH3, s), 3.54 (H15, m), 3.44 (H13, d, 10.1), 3.35 (18-OCH3, s), 3.32 (H18, dd, 4.2, 9.5), 3.30 (6-00 s), 3.23 (H18, dd, 6.2, 9.6), 2.38 (H5(eq), d, 13.9), 2.31 (H5(ax), td, 1.7, 14.0), 2.24 (H3, dq, 2.6, 7.1), 1.60 and 1.54 (H216, m), 1.19 (2-CH3, d, 6.6), 1.01 (3-CH3, d, 7.1), 0.99 (14-CH3(eq), s), 0.87 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 171.73 (C8), 145.50 (C4), 110.71 (4=0*2), 99.85 (C6), 86.72 (10- OCH_), 79.34 (C13), 78.56 (C15), 76.05 (C18), 74.31 (C12), 73.83 (CIO), 72.52 (C7), 70.87 (Cll, broad), 69.71 (C17), 69.48 (C2), 61.77 (13-OCH3), 59.05 (18-OCH3), 48.86 (6-OCH3), 41.54 (C14), 41.35 (C3), 33.72 (C5), 32.49 (C16), 23.25 (14-CH3(eq)), 17.93 (2-CH3), 13.72 (14-CH3(ax), broad), 12.07 (3-CH3).
18-methoxy mycalamide A can be prepared as follows: Mycalamide A (7 mg), Ag20 (55 mg) and Mel (21 mg), were stirred in benzene (0.4 ml) at 95°C in a sealed vial for 1.5 hours. The solution was filtered over celite and the solvent removed (7.3 mg). Preparative TLC (developed twice in 1:7 PE:EtOAc) gave four fractions (0.8 mg, 1.4 mg, 2.0 mg, 2.8 mg) which were mycalamide A, 7-methoxy A, 3:2 17-methoxy A18-methoxy A, and pure 18-methoxy A by nmr respectively, an oil.
Example 18 - 7M7.18-Trimethoxy. N-Methyl Mycalamide A
7',17,18-Trimethoxy, N-Methyl Mycalamide A has a molecular weight of 559 and a molecular formula of C-^^oNO-,-*,. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (NH3): 577 (8%, M+NH4 +), 547 (15%), 546 (30%), 545 (88%, M+NH4 +- CH3OH), 530 (19%), 529 (37%), 528 (100%, MH+-CH3OH). αH NMR (CDC13): δ 6.33 (H10, d, 10.2), 5.13 (10-OCH2, d, 6.7), 4.85 (10-OCH2, d, 6.7), 4.82 (4=CH2, t, 2,0), 4.73 (4=CH2, t, 2.0), 4.27 (H12, dd, 6.9, 10.6), 4.16 (H7, s), 4.12 (Hll, dd, 6.9, 10.3), 3.94 (H2, dq, 2.6, 6.6), 3.56 (7-OCH3, s), 3.55 (13-OCH3, s), 3.52 (H13, d, 10.7), 3.49 (H18, dd, 3.0, 10.1), 3.37 (H18, dd, 3.6, 10.0), 335 (18-OCH3, s), 3.33 (H15, broad d, 9.2), 330 (17- OCH3, s), 3.26 (6-OCH3, s), 3.23 (H17, m), 3.11 (N-CH3, s), 2.80 (H5(ax), td, 2.0, 14.6), 2.63 (H5(eq), d, 14.7), 2.21 (H3, dq, 2.4, 7.2), 1.83 (H16, broad dd, 9.4, 143), 1.42 (H16, m), 1.14 (2- CH3, d, 6.6), 1.04 (3-CH3, d, 7.0), 0.97 (14-CH3(eq), s). 0.86 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 109.72 (4=0*2), 101-78 i06), 87.00 (IO-OCH2), 81.15 (C7), 79.11 (C13), 773 (CIO), 76.09 (C15), 74.69 (C12), 72.01 (C18), 69.18 (C2), 67.27 (Cll), 61.85 (13- OCH3), 59.22 (18-OCH3), 56.42 (17-00*3), 50.48 (6-OCH3), 41.41 (C3), 33.24 (C5), 30.06 (C16), 28.90 (N-CH3), 23.01 (14- J3(eq)), 17.99 (2-OΪ3), 12.97 (14-CH3(ax)), 12.08 (3-CH3). NB: C4, C6, C8, 7-OCH3, C14, C17 not observed.
7',17,18-trimethoxy, N-methyl mycalamide A can be prepared as follows: Mycalamide A (4mg), powdered KOH (7.2mg) and Mel (lOmg) were stirred in DMSO (0.3ml) at room temperature for 20 hours, then at 40°C for 4 hours. H20 was added (0.5ml) and the mixture transferred onto a reverse phase pipette column (200mg C18, equilibrated to H20), flushed with H20 (6ml) and then eluted with MeOH (6ml). The MeOH fraction was evaporated to diyness (43mg), then subjected to prep TLC (developed in EtOAc). Three fractions of silica were recovered and each eluted with 1:9 EtOH:EtOAc (4ml) to give three fractions on evaporation (0.5mg, 1.5mg, lmg) which were 7,18-dimethoxy, N-methyl mycalamide A, 7,17,18-trimethoxy, N-methyl mycalamide A and 7',17,18-trimethoxy, N-methyl mycalamide A by nmr respectively, an oil.
Example 19 - Mycalamide B Trans-Oxazolidinone
Mycalamide B Trans-Oxazolidinone has a molecular weight of 487 and a molecular formula of C^H^NO-j. Its molecular structure is as follows:
The compound is characterized as follows:
HRFABMS: 510.26340 (M+Na+, -8.8 ppm).
FABMS: 511 (26%), 510 (100%, M+Na+), 457 (22%), 456 (67%, MH+-Cf*3OH). lH NMR (CDC13): δ 5.57 (H10, dd, 2.2, 6.1), 4.84 (4=CH2, t, 2.0), 4.72 (4=CH2, t, 1.8), 4.38 (H7, d, 2.2), 3.97 (H12, dd, 5.1, 7.4), 3.94 (H2, dq, 2.6, 6.6), 3.80 (Hll, dd, 5.1, 6.1), 3.78 (H18, dd, 4.6, 9.7), 3.62 (H18, dd, 5.9, 9.9), 3.54 (13-OCH3, s), 3.5 (H17, m), 3.43 (17-OCH3, s), 3.40 (H15, dd, 2.6, 10.9), 3.30 (6-OCH3, s), 2.93 (H13, d, 7.3), 2.47 (H5(ax), td, 2.0, 13.7), 2.32 (H5(eq), d, 13.8), 2.21 (H3, dq, 2.6, 6.7), 1.85 (H16, m), 1.7 (H16, m), 1.18 (2-CH3, d, 6.6), 1.04 (3-CH3, d, 6.8), 1.03 (14-CH3(eq), s), 0.88 (14-CH3(ax), s) ppm (couplings in Hz). NB: NH9 not observed.
Mycalamide B trans-oxazolidinone can be prepared as follows: Mycalamide B (2.5 mg) was dissolved in a solution of 1 M NaOMe in methanol (0.3 ml) and stirred at 50°C for 24 hours. The solvent was removed and the residue partitioned in 1:1 CHC13:H20 (5 ml) and then extracted with CHC13 (3x 2 ml). Prep TLC of this extract (developed twice in 1:19 EtOH:EtOAc) gave two fractions (1 mg, 0.9 mg), which were cis and trans isomers respectively at C7-C10 of mycalamide B oxazolidinone by nmr, although the second fraction also contained up to 25% unreacted mycalamide B, an oil.
Example 20 — Mycalamide B Cis-Oxazolidinone
Mycalamide B cis-Oxazolidinone has a molecular weight of 487 and a molecular formula of C^H^NOp. Its molecular structure is as follows:
The compound is characterized as follows:
FABMS: 511 (17%), 510 (100%, M+Na+), 456 (82%, MH+-CH3OH).
XH NMR (CD03): δ 6.65 (NH9, broad s), 5.52 (H10, dd, 1.6, 8.8), 4.82 (4=CH2, t, 1.8), 4.70 (4=0*2, t, 1.9), 4.34 (H7, d, 1.6), 3.94 (H12, dd, 6.7, 9.6), 3.93 (H2, dq, 2.3, 6.6), 3.84 (Hll, dd, 6.6, 8.7), 3.79 (H18, dd, 4.6, 11.7), 3.69 (H18, dd, 5.1, 11.7), 3.64 (H17, m), 3.59 (13-OCH3, s), 3.46 (H15, dd, 4.0, 83), 338 (17-OCH3, s), 330 6-OCH3, s), 2.95 (H13, d, 9.8), 2.39 (H5(ax), td, 1.8, 13.7), 2.33 (H5(eq), d, 13.8), 2.19 (H3, dq, 2.4, 7.0), 1.65 (H216, m), 1.19 (2-CH3, d, 6.6), 1.04 (3-CH3, d, 7.0), 0.97 (14-CH3(eq), s), 0.89 (14-CH3(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): <5109.64 (4=CH^, 98.32 (C6), 88.02 (C13), 80.84 (CIO), 78.92 (C17), 75.63 (C15), 74.96 (C7), 69.18 (C2), 69.04 (C12), 63.00 (13-00%, 62.94 (C18), 56.94 (17-OC%), 48.62 (6-OC%), 41.77 (C3), 33.81 (C5), 29.45 (C16), 23.41 (14-C%(eq)), 17.89 (2-C%), 13.67 (14-CH3(ax)), 11.77 (3-C%). NB: C4, C8, Cll, C14 not observed.
Mycalamide B cis-oxazolidinone can be prepared as follows: Mycalamide B (2.5 mg) was dissolved in a solution of 1 M NaOMe in methanol (0.3 ml) and stirred at 50°C for 24 hours. The solvent was removed and the residue partitioned in 1:1 CHC13H20 (5 ml) and then extracted with CHOs (3x2 ml). Prep TLC of this extract (developed twice in 1:19 EtOH:EtOAc) gave two fractions (1 mg, 0.8 mg), which were cis and trans isomers respectively at C7-C10 of mycalamide B oxazolidinone by nmr, although the second fraction also contained up to 25% unreacted mycalamide B, an oil.
Example 21 — Mycalamide B Bis-Ethylcarbonate Hydrochloride
Mycalamide B Bis-Ethylcarboπate Hydrochloride has a molecular weight of 666 and a molecular formula of C30H 8ONO13. Its molecular structure is as follows:
OCOOC2H5
OCH,
The compound is characterized as follows:
*H NMR (CDC13): δ 5.67 (H10, t, 9.4), 5.35 (H7, d, 1.0), 5.15 (H5, d, 1.1), .5.14 (10- OCH2, d, 7.1), 4.88 (10-OC% d, 6.9), 4.22 (H12, dd, 6.7, 10.6), 4.19 (H18, dd, 3.2, 11.7), 4.18 (H2, dq, 2.8, 6.5), 4.15 (7- and 18-OCOOCH2, q, 7.0), 4.04 (H18, dd, 5.1, 11.7), 3.94 (Hll, dd, 6.6, 9.7), 3.56 (13-OCH3, s), 3.44 (H13, d, 10.2), 3.36 (H17, m), 3.35 (H15, dd, 2.4, 8.1), 3.26 (17-OCH3, s), 2.53 (H3, broad dq, 2.8, 6.9), 1.7 and 1.62 (H216, m), 1.58 (4-C%, s), 1.30 (2-C%, d, 6.5), 1.27 (2x-OCH2C%, t, 7.1), 1.03 (3-C%, d, 7.0), 0.99 (14-C%(eq), s), 0.87 (14-C%(ax), s) ppm (couplings in Hz). NB: NH9 not observed.
Mycalamide B bis-ethylcarbonate hydrochloride can be prepared as follows: Mycalamide B (2.5 mg ca.), K2θ03 (8.5 mg) and ethyl chloroformate 4.5 mg) were stirred in benzene (0.3 ml) for 20 hours at room temperature. The solution was then diluted with benzene, filtered over celite and the solvent removed. Prep TLC (developed in 3:2 PE:EtOAc) gave 0.8 mg of the major component, which was pure mycalamide B bis-ethylcarbonate hydrochloride by PMR.
Example 22 — Mycalamide B 18-Monoacetate
Mycalamide B 18-Monoacetate has a molecular weight of 559 and a molecular formula of C_η ASϋ0ll. Its molecular structure is as follows:
DCIMS (N%): 577 (1%, M+NH4 +), 560 (1%, MH+), 547 (15%), 546 (35%), 545 (100%, M+NH4 +-C%OH), 530 (16%), 529 (27%), 528 (88%, MH+-C%OH).
1H NMR (CDCI3): δ 7.50 (NH9, d, 9.6), 5.78 (H10, t, 9.5), 5.11 (10-OC% d, 7.0), 4.87 (4=CH2, t, 2.0), 4.85 (10-OCH2, d, 7.0), 4.73 (4=0*2, t, 1.9), 4.26 (H7, s), 4.20 (H18, dd, 2.6, 123), 4.20 (H12, dd, 6.5, 10.0), 4.09 (H18, dd, 53, 12.2), 4.05 (H2, dq, 2.9, 6.5), 3.90 (7-OH, broad s), 3.78 (Hll, dd, 6.7, 9.5), 3.55 (13-OC%, s), 3.42 (H13, d, 10.1), 338 (H15, m), 331 (6-OC% s), 3.30 (H17, m), 3.24 (17-OC% s), 2.36 (H5(eq), d, 14.0), 2.27 (H3, dq, 2.7, 7.1), 2.17 (H5(ax), td, 2.1, 14.1), 2.08 (18-OCOC%, s), 1.60 (H216, m), 1.21 (2-C%, d, 6.6), 1.02 (3-0*3, d, 7.1), 0.97 (14-C%(eq), s), 0.86 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 171.69 (C8), 171.04 (18-OCO), 144.95 (C4), 111.25 (4=0%, 100.00 (C6), 86.43 (lO-OCHj), 79.27 (C13), 76.26 (C17), 75.61 (C15), 74.26 (C12), 74.05 (CIO), 71.11 (C7), 70.80 (Cll, broad), 69.54 (C2), 64.14 (C18), 61.76 (13-OC%, 56.98 (17-OC%), 48.51 (6-OC%), 41.40 (C14), 4131 (C3), 33.59 (C5), 30.29 (C16), 23.28 (14-C%(eq)), 20.97 (18- OCOC%), 18.01 (2-CH3), 13.60 (14-C%(ax), broad), 12.22 (3-C%.
Mycalamide B 18-monoacetate can be prepared as follows: Mycalamide B diacetate (2.5 mg) was stirred with 0.5 mg K2C03 in 9:1 MeOH:%0 (0.5 ml) for 1 hour at room temperature. %0 (2.5 ml) was added and the mixture extracted with CH2C12 (3 x 2 ml). The solvent was removed and the combined organic extracts were subjected to preparative TLC (developed in ethyl acetate). Two bands of silica were recovered (Rf=0.7 and 0.2) and eluted with 13 EtOH:EtOAc, giving two fractions on evaporation (1.7 mg, 0.5 mg) which were pure mycalamide B 18- monoacetate and mycalamide B respectively by nmr, an oil.
Example 23 — 18-Methoxy. Mycalamide B 7-Monoacetate
18-Methoxy, Mycalamide B 7-Monoacetate has a molecular weight of 573 and a molecular formula of C^H^NO-Q. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 592 (10%), 591 (32%, M+NH4 +), 561 (26%), 560 (33%), 559 (100%, M+NH4 +-C%OH), 544 (10%), 543 (14%), 542 (43%, MH+-C%OH).
XH NMR (CDC13): δ 7.38 (NH9, d, 9.4), 5.78 (H10, t, 9.5), 5.48 (H7, s), 5.08 (10-OC%, d, 6.9), 4.88 (4=C%, t, 1.7), 4.85 (10-OC%, d, 6.9), 4.76 (4=CH2, t, 1.7), 4.18 (H12, dd, 6.5, 10.0), 4.03 (H2, dq, 2.8, 6.7), 3.75 (Hll, dd, 6.4, 9.3), 3.54 (13-OC%, s), 3.46 (H18, dd, 2.4, 10.5), 3.39 (H13, d, 10.1), 337 (18-OC%, s), 3.34 (H15, m), 3.32 (H18, dd, 5.0, 10.5), 3.26 (17-OC%, s), 3.24 (H17, m), 3.17 (6-OC%, s), 2.43 (H5(ax), td, 1.8, 14.2), 2.36 (H5(eq), d, 14.2), 2.28 (H3, dq, 2.9, 7.2), 2.20 (7-OCOC%, s), 1.65 (H16, m), 1.60 (H16, m), 1.22 (2-C%, d, 6.6), 1.05 (3-C%, d, 7.1), 0.98 (14-C%(eq), s), 0.86 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 169.60 (C8), 166.79 (7-OCO), 144.73 (C4), 111.31 (4=0%, 99.26 (C6), 86.48 (10-00%, 79.55 (C13), 77.34 (C17), 76.08 (C15), 74.22 (C12), 73.79 (CIO), 72.21 (C18), 71.45 (C7), 70.58 (Cll, broad), 70.00 (C2), 61.71 (13-OC%), 59.11 (18-OC%), 56.84 (17- OC%), 48.50 (6-OCH3), 41.32 (C14), 41.17 (C3), 34.36 (C5), 30.00 (C16), 23.46 (14-CH3(eq)), 20.63 (7-OCOC%), 17.94 (2-C%), 13.81 (14-C%(ax), broad), 12.26 (3-C%).
18-methoxy, mycalamide B 7-monoacetate can be prepared as follows: Mycalamide B (6 mg)» Ag20 (40 mg) and Mel (27 mg) were stirred in benzene (0.4 ml) for 2 hours at 90°C. The solution was filtered over celite and the solvent removed. Pyridine (0.1 ml) and acetic anhydride (0.1 ml) were added and the mixture stirred at room temperature for 16 hours. %0 was added (2.5 ml) and the solution extracted with C%C12 (2 x 2 ml). Prep TLC of this extract (3:1 EtOAc:PE) gave three fractions (1.7 mg, 2.0 mg, 2.4 mg) which were pure 7-methoxy mycalamide B 18-acetate, 18-methoxy mycalamide B 7-monoacetate, and mycalamide B diacetate respectively by nmr, an oil.
Example 24 — Mycalamide B 7-Mono-p-Bromobenzoate
Mycalamide B 7-Mono-p-Bromobenzoate has a molecular weight of 700 and a molecular formula of CJ-JH^BΓNOJJ. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
1H NMR (CDC13): δ 7.98 (7-OCOC6H4Br, d, 8.7), 7.62 (7-OCOC6H4Br, d, 8.6), 7.48 (NH9, d, 9.8), 5.79 (H10, t, 9.5), 5.74 (H7, s), 5.11 (10-OC%, d, 7.1), 4.94 (4=C% m), 4.89 (10- OC%, d, 7.0), 4.83 (4=C% m), 4.24 (H12, dd, 7.0, 10.5), 4.09 (H2, dq, 2.9, 6.3), 3.78 (Hll, dd, 7.0, 9.5), 3.65 (H18, dd, 1.9, 12.2), 3.56 (13-OC%, s), 3.42 (H13, d, 10.7), 3.4 (H15, m), 336 (H18, dd, 6.8, 12.4), 3.28 (17-OC%, s), 3.20 (6-OC%, s), 3.2 (H17, m), 2.59 (H5(ax), broad d, 14.0), 2.53 (H5(eq), d, 13.8), 233 (H3, dq, 2.5, 6.9), 1.5-1.6 (%16, m), 1.28 (2-C%, d, 6.5), 1.10 (3-C%, d, 7.1), 0.95 (14-C%(eq), s), 0.86 (14-C%(ax), s) ppm (couplings in Hz).
A minor product (0.8mg), isolated from the p-bromobenzoylation of mycalamide B (see Example 25), was found to be mycalamide B 7-mono-p-bromobenzoate by PMR, a white solid.
Example 25 — Mycalamide B 18-Mono-p-Bromobenzoate
Mycalamide B 18-Mono-p-Bromobenzoate has a molecular weight of 700 and a molecular formula of C32H46BrN011. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
1H NMR (CDC13): δ 7.91 (18-OCOC6H4Br, d, 8.5), 7.59 (18-OCOC6H4Br, d, 8.6), 7.51 (NH9, d, 9.7), 5.82 (H10, t, 9.5), 5.13 (10-OC% d, 7.0), 4.88 (4=C%, m), 4.87 (10-OC%, d, 6.9), 4.73 (4=C%, t, 1.9), 4.48 (H18, dd, 23, 12.5), 436 (H18, dd, 5.0, 12.2), 4.25 (H7, d, 2.1), 4.21 (H12, dd, 6.6, 10.2), 4.06 (H2, dq, 2.8, 6.5), 3.83 (7-OH, d, 2.4), 3.82 (Hll, dd, 6.7, 9.5), 3.56 (13- OC%, s), 3.47 (H15, broad d, 9.5), 3.45 (H17, m), 3.44 (H13, d, 10.0), 3.31 and 3.30 (6-OC% and 17-OC%, 2xs), 236 (H5(eq), d, 14.1), 2.29 (H3, dq, 2.9, 7.2), 2.17 (H5(ax), td, 1.9, 14.1), 1.69 (H16, ddd, 2.0, 9.6, 14.8), 1.6 (H16, m), 1.21 (2-C%, d, 6.6), 1.03 (3-C%, d, 7.1), 0.98 (14- C%(eq), s), 0.88 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 171.69 (C8), 145.01 (C4), 131.78, 131.14, 129.03 and 128.18 (18- OCOC6H4Br), 111.19 (4=C%, 99.93 (C6), 86.40 (10-OC%, 7933 (C13), 76.47 (C17), 75.67 (C15), 74.19 (C12), 74.00 (CIO), 7131 (C7), 70.70 (Cll, broad), 69.55 (C2), 64.48 (C18), 61.73 (13- OC%), 57.09 (17-OC%), 48.57 (6-OC%), 41.50 (C14), 4131 (C3), 33.58 (C5), 30.49 (C16), 23.39
(14-C%(eq)), 18.00 (2-C%), 13.78 (14-C%(ax), broad), 12.20 (3-C%). NB: 18-OCO not observed.
Mycalamide B 18-mono-p-bromobenzoate can be prepared as follows: Mycalamide B (10- 12 mg), p-bromobenzoyl chloride (26 mg), dimethylaminopyridine (1 mg), and triethylamine (15 mg) were stirred in pyridine (0.8 ml) at 50°C for one week. After concentrating to 0.1 ml, %0 (0.3 ml) was added, the mixture extracted with C%C12 (4 x 0.4 ml) and the solvent removed (50 mg ca). Prep RPLC (10% %0 in MeOH, then 18% %0 in MeOH) gave four fractions overall (1.2 mg, 0.8 mg, 5.2 mg, 2.4 mg) which were mycalamide B, mycalamide B 7- and 18-mono-, and mycalamide B di-p-bromobenzoates by PMR respectively, a white solid.
Example 26 — Mycalamide B Di-p-Bromobenzoate
Mycalamide B Di-p-Bromobenzoate has a molecular weight of 883 and a molecular formula of
Its molecular structure is as follows:
The compound is characterized as follows:
DCIMS (N%): 872 (15%), 871 (55%), 870 (32%), 869 (100%, M+NH4 +-C%OH), 868 (16%), 867 (51%), 854 (17%), 853 (8%), 852 (52%, MH+-C%OH), 850 (18%).
1H NMR (CDC13): δ 7.90 and 7.85 (18- and 7-OCOC6H4Br, 2xd, 8.5), 7.57 and 7.54 (18- and 7-OCOC6H4Br, d, 8.5), 7.28 (NH9, d, 9.6), 5.79 (H10, t, 9.4), 5.63 (H7, s), 5.08 (10-OC%, d, 6.7), 4.91 (4=0*2, m), 4.87 (10-OC% d, 6.9), 4.79 (4=0*2, m), 4.54 (H18, dd, 3.0, 12.3), 4.28 (H18, dd, 4.2, 12.4), 4.17 (H12, dd, 6.5, 9.8), 4.05 (H2, dq, 2.6, 6.6), 3.80 (Hll, dd, 6.3, 8.9), 3.53 (13-OC%, s), 3.46 (HIS, broad d, 9.1), 3.45 (H17, m), 3.38 (H13, d, 9.7), 3.31 (17-OC%, s), 3.19
(6-OC%, s), 2.52 (H5(ax), broad d, 13.5), 2.45 (H5(eq), d, 13.9), 2.30 (H3, dq, 2.7, 6.9), 1.7 (H16, m), 1.58 (H16, m), 1.23 (2-C%, d, 6.6), 1.07 (3-C%, d, 7.1), 0.96 (14-C%(eq), s), 0.87 (14- C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 166.63 (C8), 144.74 (C4), 131.91, 131.69, 131.41, 131.14, 129.16, 128.71, 128.15 and 128.01 (7- and 18-OCOC6H4Br), 111.37 (4=0%, 99.27 (C6), 86.52 (10-
OC%, 79.52 (C13), 76.48 (C17), 75.78 (C15), 74.10 (C12), 74.08 (CIO), 72.30 (C7), 70.50 (Cll, broad), 70.04 (C2), 63.68 (C18), 61.67 (13-OC%), 57.01 (17-OC%), 48.62 (6-OC%), 41.22 (C3),
41.16 (C14), 34.54 (C5), 30.51 (C16), 23.56 (14-C%(eq)), 17.93 (2-C%), 14.03 (14-C%(ax), broad), 12.19 (3-C%). NB: 7-OCO and 18-OCO not observed.
Mycalamide B di-p-bromobenzoate can be prepared as follows: Mycalamide B (10-12 mg), p-bromobenzoyl chloride (26-mg), dimethylaminopyridine (1 mg), and triethylamine (15 mg) were stirred in pyridine (0.8 ml) at 50°C for one week. After concentrating to 0.1 ml, %0 (0.3 ml) was added, the mixture extracted with C%C12 (4 x 0.4 ml) and the solvent removed (50 mg ca). Prep RPLC (10% %0 in MeOH, then 18% %0 in MeOH) gave four fractions overall (1.2 mg, 0.8 mg, 5.2 mg, 2.4 mg) which were mycalamide B, mycalamide B 7- and 18-mono-, and mycalamide B di-p-bromobenzoates by PMR respectively, a white solid.
Example 27 — 18-Methoxy Mycalamide B
18-Methoxy Mycalamide B has a molecular weight of 531 and a molecular formula of C^H^NOJQ. Its molecular structure is as follows:
The compound is characterized as follows:
DCIMS (N%): 549 (7%, M+NH4 +), 520 (6%), 519 (23%), 518 (29%), 517 (99%, M+NH4 +-C%OH), 503 (5%), 502 (19%), 501 (29%), 500 (100%, MH+-C%OH).
1H NMR (CDC13): δ 7.55 (NH9, d, 9.5), 5.81 (H10, t, 9.6), 5.13 (10-OC%, d, 6.9), 4.86 (10-OC%, d, 6.8), 4.86 (4=C%, t, 1.8), 4.74 (4=C%, t, 1.8), 4.28 (H7, d, 2.5), 4.20 (H12, dd, 6.6, 10.2), 4.05 (H2, dq, 2.9, 6.6), 3.92 (7-OH, d, 2.6), 3.79 (Hll, dd, 6.6, 9.6), 3.56 (13-OC%, s), 3.46 (H18, dd, 2.2, 103), 3.44 (H13, d, 10.3), 337 (18-OC%, s), 337 (H15, m), 332 (6-OC%, s), 3.31 (H18, dd, 5.2, 10.3), 3.25 (17-OC%, s), 3.24 (H17, m), 2.37 (H5(eq), d 143), 2.27 (H3, dq, 2.5,
7.2), 2.20 (H5(ax), td, 1.9, 14.1), 1.62 and 1.57 (%16, m), 1.21 (2-C%, d, 6.6), 1.03 (3-C%, d, 7.1), 0.99 (14-C%(eq), s), 0.87 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDCy: δ 171.78 (OS), 145.00 (C4), 111.23 (4=C%, 100.01 (C6), 86.47 (10-
00*2), 79.43 (C13), 75.99 (C15), 7433 (C12), 73.94 (CIO), 72.63 (C18), 71.45 (C7), 70.62 (Cll, broad), 69.58 (C2), 61.76 (13-OC%), 59.20 (18-OC%), 56.87 (17-OC%), 48.56 (6-OC%), 41.47
(C14), 4130 (C3), 33.61 (C5), 29.95 (C16), 2334 (14-C%(eq)), 18.01 (2-C%), 13.66 (14-C%(ax), broad), 12.28 (3-C%). NB: C17 not observed.
18-methoxy mycalamide B can be prepared as follows: 18-methoxy mycalamide B 7- monoacetate (2 mg) (see Example 23) was stirred in a solution with 0.4 mg K2C03 in aqueous MeOH (0.4 ml) at room temperature for 1.5 hours. The solution was concentrated, then %0 (2.5 ml) was added and the solution extracted with C%C12 (3 x 2 ml). The solvent was removed to give pure 18-methoxy mycalamide B (1.8 mg) by nmr, an oil.
Example 28 - Mycalamide B 7.N-Dibenzyl Ether
Mycalamide B 7,N-Dibenzyl Ether has a molecular weight of 697 and a molecular formula of C^^NOJQ. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 715 (9%, M+NH4 +), 686 (13%), 685 (33%), 684 (45%), 683 (100%, M+NH4 +-C%OH), 668 (22%), 667 (27%), 666 (63%, MH+-C%OH).
1H NMR (CDC13): δ 7.4-7.2 (2x , m), 6.26 (H10, d, 8.9), 5.20 (10-OC% d, 6.8), 5.11 (N-C%ø, d, 17.2), 4.87 (10-OC%, d, 6.6), 4.78 (4=C%, t, 1.9), 4.68 (4=C%, m), 4.61 (N-C%ø, d, 17.6), 4.39 (7-OC%ø, d, 11.5), 4.25 (H12, dd, 7.4, 9.9), 4.21 (H7, s), 4.03 (Hll, broad t, 8.0), 3.97 (7-OC%ø, d, 12.1), 3.89 (H2, dq, 2.8, 6.5), 3.86 (H18, dd, 3.0, 11.8), 3.69 (H15, dd, 2.5, 8.3), 3.60 (H18, dd, 5.5, 11.8), 3.54 (13-OC%, s), 3.46 (H17, m), 3.42 (H13, d, 9.7), 3.34 (17-OC%, s), 2.96 (6-OC%, s), 2.59 (H5(ax), broad d, 14.1), 2.23 (H5(eq), d, 14.2) 2.17 (H3, dq, 2.3, 6.8), 1.62 (H16, m), 1.56 (H16, m), 1.18 (2-C%, d, 6.4), 1.02 (14-C%(eq), s), 0.98 (3-C%, d, 6.7), 0.87 (14- C%(ax), s) ppm (couplings in Hz).
Mycalamide B 7,N-dibenzyl ether can be prepared as follows: Mycalamide B (4.4 mg), powdered KOH (7 mg) and benzyl bromide (12 mg) were stirred in DMSO (0.3 ml) at room temperature for 4 minutes. %0 (2 ml) was added and the mixture extracted with CHC13 (3 x 2 ml). The extract was evaporated to diyness, then subjected to prep TLC (developed in 1:1 PE:EtOAc). Three bands of silica were recovered and eluted with ethyl acetate to give three pure products (1.8 mg, 0.8 mg, 0.9 mg), which were mycalamide B 7-mono-; 7,N-di- and 7,18-di-benzyl ethers by NMR respectively, an oil.
Example 29 - Mycalamide B 7.18-Dibenzyl Ether
Mycalamide B 7,18-Dibenzyl Ether has a molecular weight of 697 and a molecular formula of CJ-^^O-H). Its molecular structure is as follows:
The compound is characterized as follows:
DCIMS (N%: 716 (9%), 715 (19%, M+NH4 +), 686 (10%), 685 (28%), 684 (45%), 683 (100%, M+NH4 +-C%OH), 668 (10%), 667 (16%), 666 (36%, MH+-C%OH).
1H NMR (CDC13): δ 7.4-73 (2xø, m), 7.22 (NH9, d, 9.8), 5.81 (H10, t, 9.8), 5.11 (10- OC%, d, 6.9), 4.81 (10-OC%, d, 6.7), 4.80 (4=C%, m), 4.72 (7-OC%ø, d, 113), 4.69 (4=C%, t, 1.8), 4.62 (18-OC%ø, d, 12.1), 4.54 (18-OC%ø, d, 12.0), 4.52 (7-OC%ø, d, 11.3), 4.19 (H12, dd, 6.5, 10.2), 4.01 (H7, s), 3.87 (H2, dq, 2.7, 6.6), 3.80 (Hll, dd, 6.7, 9.7), 3.61 (H18, m), 3.54 (13- OC%, s), 3.45 (H18, m), 3.44 (H15, m), 3.38 (H13, d, 10.2), 3.28 (H17, m), 3.27 (17-OC%, s), 3.06 (6-OC%, s), 2.43 (H5(ax), td, 1.8, 14.3), 2.35 (H5(eq), d, 14.3), 2.18 (H3, dq, 2.9, 7.1), 1.65 (H216, ), 1.16 (2-C%, d, 6.6), 0.95 (3-C%, d, 7.1) 0.93 (14-C%(eq), s). 0.87 (14-C%(ax), s) ppm (couplings in Hz).
^C NMR (CDC13): δ 170.17 (C8), 146.40 (C4), 138.58, 137.06, 128.51, 128.28, 128.10, 127.79 and 12739 (7- and 18-OC%ø), 109.94 (4=C%, 99.95 (C6), 86.42 (10-OC%, 80.73 (C7), 79.55 (C13), 77.79 (C17), 75.84 (C15), 7433 (C12), 73.98 (CIO), 73.25 and 73.13 (7- and 18- 00%, 70.54 (C18), 69.34 (C2), 61.75 (13-OC%), 57.10 (17-OC%), 48.94 (6-OC%), 41.46 (C14), 41.41 (C3), 34.42 (C5), 30.11 (C16), 23.22 (14-C%(eq)), 17.83 (2-OΪ3), 13.60 (14-C%(ax), broad), 11.82 (3-C%). NB: Cll not observed.
Mycalamide B 7,18-dibenzyl ether can be prepared as follows: Mycalamide B (4.4 mg), powdered KOH (7 mg) and benzyl bromide (12 mg) were stirred in DMSO (0.3 ml) at room temperature for 4 minutes. %0 (2 ml) was added and the mixture extracted with CHC13 (3 2 ml). The extract was evaporated to diyness, then subjected to prep TLC (developed in 1:1 PE:EtOAc). Three bands of silica were recovered and eluted with ethyl acetate to give three pure products (1.8 mg, 0.8 mg, 0.9 mg), which were mycalamide B 7-mono-; 7,N-di- and 7,18-di-benzyl ethers by NMR respectively, an oil.
Example 30 - Mycalamide B 7,18,N-Tribenzyl Ether
Mycalamide B 7,18,N-Tribenzyl Ether has a molecular weight of 787 and a molecular formula of C^H^NOJQ. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%: 805 (17%, M+NH4 +), 775 (22%), 774 (49%), 773 (100%, M+NH4 +- C%OH), 758 (8%), 757 (18%), 756 (35%, MH+-C%OH). αH NMR (CDC13): δ 7.4-7.25 (3xø, m), 6.10 (H10, d, 7.8), 5.15 (10-OC%, d, 6.2), 5.07 (N-C% , d, 173), 4.89 (10-OC%, d, 6.2), 4.77 (4=C%, m), 4.66 (4=C%, m), 4.65 (N-C% , d, 16.8), 4.59 (18-00*20, d, 12.0), 4.53 (18-OC%ø, d, 12.0), 4.37 (7-OC%ø, d, 11.6), 4.25 (H12, m), 4.23 (H7, s), 4.07 (Hll, broad t, 6.8), 3.93 (7-OC%ø, d, 11.5), 3.85 (H2, m), 3.71 (H18, dd, 1.7, 10.0), 3.61 (HIS, dd, 3.2, 10.9), 3.51 (13-OC%, s), 3.50 (H15, m), 3.47 (H17, m), 3.31 (H13, d, 10.4), 3.30 (17-OC%, s), 2.89 (6-OC%, s), 2.86 (H5(ax), broad d, 14.7), 2.28 (H5(eq), d, 14.5), 2.15 (H3, m), 1.85 (H16, m), 1.65 (H16, m), 1.14 (2-C%, d, 7.0), 0.98 (3-C%, d, 7.0), 0.98 (14- C%(eq), s), 0.85 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 171.63 (C8), 146.97 (C4), 139.01, 138.30, 138.17, 128.63, 128.28, 128.14, 127.88, 127.51, 127.26 and 126.67 (7-0-, 18-0- and N-C%ø), 109.65 (4=C%, 101.44 (C6), 86.04 (10-00%, 80.69 (C13), 79.60 (CIO), 77.19 (C17), 7633 (C15), 75.98 (C7), 73.42 (C12), 73.30 (18-00*2), 71.91 (7-OC%, 69.45 (C2), 69.05 (C18), 67.67 (Cll), 61.39 (13-OC%), 56.44 (17-OC%), 47.81 (6-OC%), 46.32 (N-C%, 41.53 (C3), 40.46 (C14), 33.46 (C5), 30.66 (C16), 24.11 (14-C%(eq)), 17.76 (2-C%), 14.84 (14-C%(ax)), 12.18 (3-C%).
Mycalamide B 7,18,N-tribenzyl ether can be prepared as follows: Mycalamide B (7 mg), powdered KOH (22 mg), and benzyl bromide (36 mg) were stirred in DMSO (0.3 ml) at room temperature for 3 hours. %0 (2 ml) was added and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to %0), flushed with %0 (8 ml), then eluted with MeOH (6 ml). The resulting MeOH fraction was evaporated to diyness, then subjected to prep TLC (5:2 PE:EtOAc). Two bands of silica (Rf 0.6, 0.7) were recovered and each eluted with ethyl acetate to give two pure products (2.2 mg, 5.2 mg), which were mycalamide B 7,8,18-tri- and 7,18,N-tri- benzyl ethers by NMR respectively, an oil.
Example 31 - Mycalamide B 7.8.18-Tribenzyl Ether
Mycalamide B 7,8,18-Tribenzyl Ether has a molecular weight of 787 and a molecular formula of C^H^NOJQ. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%: 789 (60%), 788 (100%, MH+).
1H NMR (CDO^: δ 7.4-7.2 ( , m), 5.28 (8-OC%ø, d, 12.5), 5.13 (10-OC%, d, 6.1), 5.09 (8-OC%ø, d, 12.6), 5.00 (H10, d, 2.0), 4.76 (4=0*2, t, 2.0), 4.69 (4=C%, t, 2.0), 4.68 (10- OC%, d, 6.1), 4.67 (H7, s), 4.60 (18-OC%ø, d, 12.2), 4.51 (7-OC%ø, d, 12.1), 4.47 (18-OC%ø, d, 12.2), 439 (7-00*20, <*> 17Λ)> 3sπ ( l2, dd, 1.8, 3.2), 3.76 (H2, dq, 2.6, 6.4), 3.63 (H18, dd, 2.7, 10.4), 3.52 (H18, dd, 43, 10.6), 3.51 (Hll, t, 1.9), 3.51 (H15, dd, 2.5, 12^), 339 (H17, m), 3.36 (13-OC%, s), 334 (17-OC%, s), 3.13 (6-OC%, s), 3.09 (H5(ax), td, 2.0, 14.7), 2.86 (H13, d, 3.2), 2.34 (H5(eq), d, 14.8), 2.31 (H16, m), 2.12 (H3, dq, 2.4, 7.1), 1.71 (H16, m), 1.20 (14-C%(ax), s), 1.02 (2-0*3, d, 6.5), 0.91 (14-C%(eq), s), 0.84 (3-C%, d, 7.0) ppm (couplings in Hz).
13C NMR (CD03): δ 147.53 (C4), 128.41, 128.34, 128.25, 128.01, 127.87, 127.69, 127.56 and 127.31 (7-, 8- and 18-OC%ø), 109.11 (4=0*2), W132 i*06)' 86^0 (10-OC%, 84.03 (C13), 82.56 (CIO), 78.65 (C15), 78.30 (C17), 7332 and 73.29 (18-OC% and C7), 71.71 (7-00%, 69.77 (C18), 69.09 (C2), 68.70 (C12), 67.43 (8-00%, 64.79 (Cll), 5932 (13-OC%), 56.98 (17-OC%, 48.24 (6-00*3), 41.59 (C3), 36.66 (C14), 33.83 (C5), 28.46 (C16), 27.68 (14-C%(ax)), 22.25 (14- C%(eq)), 17.67 (2-C%), 11.73 (3-C%). NB: 3xø, C8 not observed.
Mycalamide B 7,8,18-tribenzyl ether can be prepared as follows: Mycalamide B (7 mg), powdered KOH (22 mg), and benzyl bromide (36 mg) were stirred in DMSO (03 ml) at room temperature for 3 hours. %0 (2 ml) was added and the mixture transferred onto a reverse phase pipette column (200 mg C18, equilibrated to %0), flushed with %0 (8 ml), then eluted with MeOH (6 ml). The resulting MeOH fraction was evaporated to diyness, then subjected to prep TLC (5:2 PE:EtOAc). Two bands of silica (Rf 0.6, 0.7) were recovered and each eluted with ethyl acetate to give two pure products (2.2 mg, 5.2 mg), which were mycalamide B 7,8,18-tri- and 7,18,N-tri- benzyl ethers by NMR respectively, an oil.
Example 32 - Mycalamide B Bis-TBDMS Ether
Mycalamide B Bis-TBDMS Ether has a molecular weight of 745 and a molecular formula of C37H71NO10Si2. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
XH NMR (CD03): δ 7.27 (NH9, d, 9.9), 5.76 (H10, dd, 8.8, 9.8), 5.10 (10-OC% d, 7.0), 4.82 (10-OC%, d, 7.0), 4.81 (4=C% t, 2.0), 4.71 (4=CH2, t, 2.0), 4.22 (H7, s), 4.14 (H12, dd, 6.2, 9.2), 3.87 (H2, dq, 2.8, 6.5), 3.74 (Hll, dd, 6.1, 8.7), 3.70 (H18, dd, 2.8, 11.5), 3.57 (H18, dd, 3.7, 11.6), 3.52 (13-OC%, s), 338 (H13, d, 93), 3.35 (H15, dd, 2.0, 9.6), 3.29 (6-OC%, s), 3.26 (17- OC%, s), 3.16 (H17, m), 2.51 (H5(eq), d, 14.4), 2.36 (H5(ax), td, 2.0, 14.6), 2.19 (H3, dq, 2.9, 7.1), 1.79 (H16, ddd, 2.0, 9.8, 14.2), 1.55 (H16, m), 1.17 (2-C%, d, 6.6), 1.00 (14-C%(eq), s), 0.99 (3- C%, d, 7.0), 0.94 (7-OSiC(C%)3, s), 0.88 (18-OSiC(C%3, s), 0.87 (14-0*3(ax), s), 0.16 and 0.14 (7-OSi(C%)2, 2xs), 0.06 (18-OSi(C%2- s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.69 (OS), 146.62 (C4), 109.97 (4=C%, 99.59 (OS), 86.34 (10- OC%, 80.07 (C13), 78.87 (C17), 77.2 (C7), 76.31 (C15), 73.92 (C12), 73.38 (CIO), 70.1 (Cll, broad), 69.56 (C2), 6Z03 (C18), 61.51 (13-OC%), 56.69 (17-OC%), 50.00 (6-OC%), 4132 (C3), 41.04 (C14), 35.59 (C5), 29.70 (C16), 25.97 (7-OSiC(C%)3), 25.87 (18-OSiC(C%3), 23.89 (14- C%(eq)), 17.85 (2-C%), 14.3 (14-OI3(ax), broad), 11.85 (3-C%), -4.74 (7-OSi(C%)2), -5.26 (18-OSi(C%2.
Mycalamide B bis-TBDMS ether can be prepared as follows: Mycalamide B (1 mg), t- butyldimethylchlorosilane (12 mg), dimethylaminopyridine (1 mg), and triethylamine (14 mg) were stirred in pyridine (0.2 ml) at 70°C for 20 hours. %0 (0.5 ml) was added, the mixture extracted with C%C12 (3 x 0.3 ml), and the solvent removed. The combined organic extract was subjected to silica gel chromatography (200 mg Davisil, 150 A, 35-70 μ ), developed in steps from hexane to ethanol/ethyl acetate. The major fraction (1 mg) eluted with 3:1 PE:EtOAc was pure mycalamide bis-TBDMS ether by PMR, an oil.
Example 33 - Mycalamide A 7,N-Dibenzyl Ether
Mycalamide A 7,N-DibenzyI Ether has a molecular weight of 683 and a molecular formula of CggHs^Oj . Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 701 (12%, M+NH4 +), 672 (17%), 671 (46%), 670 (44%), 669 (100%, M+NH4 +-CH3OH), 654 (19%), 653 (20%), 652 (45%, MH+- Ϊ3OH).
1H NMR (CDα^: δ 7.7-7.25 (2xø, m), 6.40 (H10, d, 9.2), 5.21 (10-OC% d, 7.0), 5.14 (N-O*20, d, 17.0), 4.90 (10-OC%, d, 6.9), 4.78 (4=C% t, 1.9), 4.69 (4=C% t, 1.8), 4.63 (N- C%ø, d, 17.1), 433 (7-OC%ø, d, 12.0), 4.24 (H7, s), 4.24 (H12, dd, 7.0, 9.2), 4.00 (Hll, dd, 7.0, 9.2), 3.91 (H2, dq, Z9, 6.5), 3.86 (7-OC%ø, d, 12.5), 3.8 (H17, m), 3.78 (H15, m), 3.69 (H18, dd, 2.8, 11.1), 3.56 (13-OC%, s), 3.48 (H13, d, 10.0), 3.29 (H18, dd, 6.5, 11.0), 2.96 (6-OC%, s), 2.64 (H5(ax), broad d, 14.4), 2.28 (H5(eq), d, 14.1), 2.19 (H3, dq, 2.5, 7.0), 1.55 (H216, m), 1.19 (2-C%, d, 6.5), 1.01 (3-C%, d, 7.0), 1.00 (14-C%(eq), s), 0.87 (14-C%(ax), s) ppm (couplings in Hz).
Mycalamide A 7,N-dibenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%C12 (2 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component Prep TLC (developed in 1:1 PE:EtOAc) and appropriate combination of samples gave four pure products (1 mg, 13 mg, 1 mg, 2 mg) which were mycalamide A 7-mono-; 7,18-di-; 7,N-di- and 7,18,N-tri-benzyl ethers by NMR respectively, an oil.
Example 34 — Mycalamide A 7.18-Dibenzyl Ether
Mycalamide A 7,18-Dibenzyl Ether has a molecular weight of 683 and a molecular formula of C38H53NO10. Its molecular structure is as follows:
OCH 3
The compound is characterized as follows:
DCIMS (N%): 703 (7%), 702 (12%), 701 (28%, M+NH4 +), 672 (9%), 671 (27%), 670 (41%), 669 (100%, M+NH4 +-C%OH), 654 (5%), 653 (8%), 652 (19%, MH+-C%OH).
1H NMR (CDC13): δ 7.4-7.25 (2xø, m), 7.22 (NH9, d, 10.0), 5.92 (H10, t, 10.0), 5.13 (10- OC%, d, 7.1), 4.82 (10-OC% d, 7.0), 4.80 (4=C%, m), 4.75 (7-OCH2ø, d, 11.2), 4.70 (4=0*2, m), 4.63 (7-OC%ø, d, 10.8), 4.59 (18-OC%ø, d, 11.7), 4.52 (18-OC%ø, d, 12.1), 4.22 (H12, dd, 6.8, 10.5), 4.08 (H7, s), 3.92 (H17, m), 3.86 (H2, dq, 2.9, 6.6), 3.81 (Hll, dd, 6.7, 10.1), 3.60 (H15, dd, 1.8, 10.4), 3.55 (13-OC%, s), 3.46 (H13, d, 10.3), 3.44 (H18, dd, 4.7, 9.6), 3.39 (H18, dd, 5.6, 9.6), 3.10 (6-OC%, s), 2.47 (H5(ax), broad d, 14.5), 2.40 (H5(eq), d, 14.4), 2.18 (H3, dq, 2.9, 7.1), 1.7 (H16, m), 1.55 (H16, m), 1.16 (2-C% d, 6.6), 0.97 (14-C%(eq), s), 0.95 (3-C%, d, 7.0), 0.88 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.43 (CS), 146.62 (C4), 138.86, 137.15, 128.46, 128.41, 128.34, 128.07, 127.64 and 127.53 (7- and 18-OC%ø), 109.77 (4=C%, 100.03 (C6), 86.76 (lO-OCHj), 80.91 (C7), 79.23 (C13), 79.13 (C15), 74.48 (C12), 74.07 (CIO), 73.78 (C18), 73.20 and 73.11 (7- and 18-OC%, 71.29 (Cll), 70.09 (C17), 69.29 (C2), 61.84 (13-OC%), 49.15 (6-OC%), 41.78 (C14), 41.41 (C3), 34.65 (C5), 32.58 (C16), 23.05 (14-C%(eq)), 17.80 (2-C%), 13.41 (14-C%(ax)), 11.74 (3-C%).
Mycalamide A 7,18-dibenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%C12 (2 x 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component. Prep TLC (developed in 1:1 PE:EtOAc) and appropriate combination of samples gave four pure products (1 mg, 1.3 mg, 1 mg, 2 mg) which were mycalamide A 7-mono-; 7,18-di-; 7,N-di- and 7,18,N-tri-benzyl ethers by NMR respectively, an oil.
Example 35 — Mycalamide A 7-Monobenzyl Ether
Mycalamide A 7-Monobenzyl Ether has a molecular weight of 593 and a molecular formula of C^H^NO^ Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%: 611 (7%, M+NH4 +), 582 (8%), 581 (26%), 580 (34%), 579 (100%, M+NH4 +-C%OH), 564 (5%), 563 (6%), 562 (17%, MH+-C%OH).
*H NMR (CDC13): δ 7.4-7.25 (ø, m), 7.24 (NH9, d, 10.0), 5.93 (H10, t, 10.1), 5.13 (10- OC%, d, 7.0), 4.84 (10-00*2, d, 7.0), 4.82 (4=C% t, 1.8), 4.77 (7-OC%ø, d, 11.2), 4.72 (4=0%, t, 1.9), 4.68 (7-OC%ø, d, 11.2), 4.23 (H12, dd, 6.8, 10.5), 4.10 (H7, s), 3.88 (H2, dq, 2.7, 6.6), 3.82 (H17, m), 3.80 (Hll, dd, 6.8, 10.0), 3.68 (H15, dd, 5.8, 6.9), 3.62 (H18, dd, 3.5, 113), 3.56 (13-OC%, s), 3.49 (H13, d, 10.5), 339 (H18, dd, 6.0, 11.2), 3.14 (6-OC%, s), 2.48 (H5(ax), td, 1.9, 14.4), 2.41 (H5(eq), d, 143), 2.19 (H3, dq, 2.5, 7.0), 1.55 (H216, m), 1.17 (2-C%, d, 6.6), 0.98 (14- C%(eq), s), 0.97 (3-CH3, d, 7.0), 0.88 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (0DO3): δ 170.59 (C8), 146.48 (C4), 128.55, 128.38 and 128.19 (7-OC%ø), 109.91 (4=C%, 100.09 (OS), 86.86 (10-00*2), 80.81 (C7), 79.24 (C13), 78.98 (C15), 74.56 (C12), 74.19 (CIO), 73.24 (7-00%, 71.70 (C17), 71.66 (Cll), 6938 (C2), 66.74 (C18), 61.88 (13-OC%, 49.22 (6-00*3), 41.82 (C14), 41.38 (C3), 34.61 (C5), 32.02 (C16), 22.98 (14-C%(eq)), 17.80 (2- C%), 13.26 (14-C%(ax)), 11.76 (3-0*3). NB: lxø not observed.
Mycalamide A 7-monobenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%CI2 (2 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component. Prep TLC (developed in 1:1 PE:EtOAc) and appropriate combination of samples gave four pure products (1 mg, 13 mg, 1 mg, 2 mg) which were mycalamide A 7-mono-; 7,18-di-; 7,N-di- and 7,18,N-tri-benzyl ethers by NMR respectively, an oil.
Example 36 - Mycalamide A 7,18.N-Tribenzyl Ether
Mycalamide A 7,18,N-Tribenzyl Ether has a molecular weight of 773 and a molecular formula of C45H59NO10. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 791 (8%, M+NH4 +), 761 (19%), 760 (49%), 759 (100%, M+NH4 +- C%OH), 743 (13%), 742 (24%, MH+-0*3OH).
1H NMR (CD03): δ 7.4-7.25 (3xø, m), 6.33 (H10, d, 9.1), 5.19 (10-OC%, d, 6.0), 5.11 (N-C%ø, d, 17.1), 4.91 (10-OC% d, 6.0), 4.76 (4=C% t, 1.9), 4.66 (4=0*2, m)» 4-65 (N-CH^, d, 17.2), 4.56 (18-00*20, d, 12.4), 4.52 (18-OC%ø, d, 12.0), 4.36 (7-OC%ø, d, 11.5), 4.27 (H7, s), 4.24 (H12, dd, 6.9, 10.0), 4.03 (Hll, dd, 7.1, 8.7), 3.92 (H17, m), 3.90 (7-OC%ø, d, 11.5), 3.88 (H2, dq, 2.7, 6.5), 3.77 (H15, m), 3.55 (13-OC%, s), 3.47 (H13, d, 10.0), 3.44 (H18, m), 336 (H18, dd, 5.1, 9.1), 2.90 (6-OC% s), 2.86 (H5(ax), broad d, 14.0), 233 (H5(eq), d, 14.0), 2.16 (H3, dq, 2.7, 7.0), 1.83 (H16, broad d, 14.0), 1.5 (H16, m), 1.16 (2-Oi3, d, 6.5), 1.01 (3-C%, d, 7.0), 1.00 (14-C%(eq), s), 0.87 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CD03): -5 138.45, 128.68, 128.32, 128.15, 127.70, 127.60, 127.48, 127.35 and 126.95 (3xø), 109.52 (4=C%, 86.23 (10-OC%, 79.84 (C13), 78.68 (C15), 77.80 (CIO), 76.22 (C7), 74.35 (C12), 74.16 (18-OC%, 73.32 (7-OC%, 71.79 (C18), 69.28 (C17), 69.11 (C2), 68.44 (Cll), 61.67 (13-OC%), 47.92 (6-OC%), 45.96 (N-C%, 41.48 (C3), 34.29 (C5), 33.60 (C16), 23.32 (14-C%(eq)), 17.70 (2-C%), 13.70 (14-C%(ax)), 12.46 (3-OΪ3). NB: C4, C6, CS and C14 not observed.
Mycalamide A 7,18,N-tribenzyl ether can be prepared as follows: Mycalamide A (5 mg), powdered KOH (8 mg) and benzyl bromide (15 mg) were stirred in DMSO at room temperature for 3 minutes. %0 (2 ml) was added and the mixture extracted with C%C12 (2 x 2 ml). This extract was washed with %0 (2 x 2 ml) and the solvent removed (7.5 mg). Prep RPLC (15% %0 in MeOH) gave four fractions (1 mg, 0.7 mg, 2.5 mg, 2 mg), but the third fraction was a mixture of the second and another component. Prep TLC (developed in 1:1 PE:EtOAc) and appropriate combination of samples gave four pure products (1 mg, 1.3 mg, 1 mg, 2 mg) which
were mycalamide A 7-mono-; 7,18-di-; 7,N-di- and 7,18,N-tri-benzyl ethers by NMR respectively, an oil.
Example 37 — 7'-Deutero. 7 '-Methoxy. N-Methyl Mycalamide A
7'-Deutero, 7'-Methoxy, N-Methyl Mycalamide A has a molecular weight of 532 and a molecular formula of C26H44DNO10. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 550 (3%, M+NH4 +), 520 (10%), 518 (61%, M+NH4 +-C%OH), 503 (23%), 502 (9%), 501 (100%, MH+-OΪ3OH).
1H NMR (CDCls): δ 6.40 (H10, d, 10.3 ), 5.15 (10-OC%, d, 6.7), 4.86 (10-OC% d, 6.8), 4.84 (4=C% t, 2.0), 4.74 (4=C%, t, 2.0), 4.27 (H12, dd, 7.0, 10.7), 4.14 (Hll, dd, 7.0, 10.4), 3.94 (H2, dq, 2.7, 6.5), 3.67 (H17, m), 3.66 (H18, m), 3.64 (H15, dd, 1.5, 9.8), 3.58 and 3.56 (7- and 13- OC%, 2xs), 3.53 (H13, d, 10.7), 331 (6-OC% s), 3.26 (H18, dd, 6.9, 11.0), 3.14 (N-C%, s), 2.84 (H5(ax), td, 2.0, 14.6), 2 1 (H5(eq), d, 14-5), 2.21 (H3, dq, 2.6, 6.8), 133 (%16, m), 1.13 (2-0*3, d, 6.6), 1.03 (3-0%, d, 7.0), 0.96 (14-C%(eq), s), 0.87 (14-0*3(ax), s) ppm (couplings in Hz).
7'-deutero, 7'-methoxy, N-methyl mycalamide A can be prepared as follows: 7-methoxy, N-methyl mycalamide A (1.2mg) (see Example 12) was dissolved in 3M NaOCDg CDsOD and stirred at 70°C for 2 days. The crude mixture was then loaded onto a TLC plate (silica gel 60 F^, 0.2mm) which was developed twice in 1:19 EtOH:EtOAc. The two bands of silica were recovered and each eluted with 13 EtOH:EtOAc giving two fractions (0.4mg, 0.7mg) where the first was a mixture but the second was pure 7'-methoxy, N-methyl mycalamide A with quantitative inclusion of deuterium at the epimerised C7 centre by PMR and MS, an oil.
Example 38 - 7-Epimvcalamide A Trans-Oxazolidinone
7-epimycalamide A trans-oxazolidinone has a molecular weight of 473 and a molecular formula of C23H39N09. Its molecular structure is as follows:
The compound is characterized as follows:
HRFABMS: 442.24300 (MH+-C%OH, -2.4 ppm).
FABMS: 443 (28%), 442 (100%, MH+-C%OH).
1H NMR (CDO-j): δ 6.67 (NH9, broad s), 5.67 (H10, dd, 2.2, 8.6), 4.83 (4=C% t, 2.)), 4.71 (4=C% t, 2.1), 4.28 (H7, d, 2.1), 3.97 (H12, dd, 6.9, 9.7), 3.94 (H2, dq, 2.4, 6.6), 3.86 (H17, m), 3.84 (Hll, dd, 6.9, 8.7), 3.62 (H15, m), 3.60 (13-OCH3, s), 3.58 (H18, m), 3.52 (H18, m), 3.43 (6-OC%, s), 195 (H13, d, 9.7), 2.92 (H5(ax), td, 2.0, 14.4), 2.20 (H3, dq, 2.4, 6.9), 2.13 (H5(eq), d, 14.3), 1.53 (%16, m), 1.17 (2-C%, d, 6.5), 1.02 (3-C%, d, 7.0), 0.97 (14-C%(eq), s), 0.88 (14- C%(ax), s) ppm (couplings in Hz).
7-epimycalamide A trans-oxazolidinone was prepared as follows: Mycalamide A (7 mg) was stirred in a solution of 2 M NaOH in 50% MeOH-%0 (0.3 ml) at 55°C for 10 hours. The solution was concentrated (0.1 ml) and then loaded onto a C18 pipette column (100 mg). The column was flushed with %0 (8 ml), then a fraction obtained by elution with MeOH (5 ml) was collected and the solvent removed (6 mg). Analytical reverse phase HPLC (45% %0 in MeOH) gave four fractions (1.4 mg, 1.8 mg, 0.7 mg, 0.8 mg). PMR and NOE's showed that the first two were cis and trans isomers respectively at C7-C10 of mycalamide A oxazolidinone, but the second two were at least 70% pure trans and cis isomers, respectively, of 7-epimycalamide A oxazolidinone, with the major impurity being unreacted mycalamide A by PMR and HRFABMS, an oil.
Example 39 - 7-Epimvcalamide A Cis-Oxazolidinone
7-Epimycalamide A Cis-Oxazolidinone has a molecular weight of 473 and a molecular formula of C^HgoNO,-,. Its molecular structure is as follows:
The compound is characterized as follows:
HRFABMS: 442.24100 (MH+-C%OH, -6.9 ppm).
FABMS: 443 (26%), 442 (100%, MH+-OΪ3OH).
1H NMR (CDO-j): δ 8.04 (NH9, broad s), 5.66 (H10, dd, 1.4, 6.9), 4.82 (4=C% t, 2.1), 4.70 (4=C% t, 2.0), 433 (H7, d, 13), 4.06 (Hll, t, 63), 4.01 (H12, dd, 6.1, 83), 3.95 (H2, dq, 2.6, 6.6), 3.88 (H17, m), 3.59 (13-OC%, s), 3.57 (H218, m), 3.47 (H15, broad d, 9.5), 336 (6-OC% s), 3.05 (H5(ax), td, 2.1, 14.3), 3.04 (H13, d, 8.1), 2.19 (H3, dq, 23, 6.7), 2.11 (H5(eq), d, 14.2), 1.60 and 1.54 (%16, m), 1.15 (2-C% d, 6.5), 0.99 (3-C% d, 6.7), 0.98 (14-C%(eq), s), 0.86 (14- Oi3(ax), s) ppm (couplings in Hz).
7-epimycalamide A cis-oxazolidinone can be prepared as follows: Mycalamide A (7 mg) was stirred in a solution of 2 M NaOH in 50% MeOH-%0 (03 ml) at 55°C for 10 hours. The solution was concentrated (0.1 ml) and then loaded onto a C18 pipette column (100 mg). The column was flushed with %0 (8 ml), then a fraction obtained by elution with MeOH (5 ml) was collected and the solvent removed (6 mg). Analytical reverse phase HPLC (45% %0 in MeOH) gave four fractions (1.4 mg, 1.8 mg, 0.7 mg, 0.8 mg). PMR and NOE's showed that the first two were cis and trans isomers respectively at C7-C10 of mycalamide A oxazolidinone, but the second two were at least 70% pure trans and cis isomers, respectively, of 7-epimycalamide A oxazolidinone, with the major impurity being unreacted mycalamide A by PMR and HRFABMS, an oil.
Example 40 — Neomvcalamide A Triacetate
Neomycalamide A Triacetate has a molecular weight of 597 and a molecular formula of C2 H 3N012. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 616 (7%), 615 (34%, M+NH4 +), 600 (13%), 599 (31%), 598 (100%, MH+), 573 (28%), 571 (18%), 557 (24%), 556 (66%, MH+-C%CO), 493 (8%), 419 (35%).
1H NMR (CDC13): δ 8.11 (NH9, d, 9.0), 6.02 (H5, q, 1.4), 5.86 (H10, t, 9.2), 5.15 (10- OC%, d, 7.0), 5.11 (H17, m), 4.86 (10-OC% d, 7.0), 432 (H2, dq, 2.9, 6.5), 4.28 (H18, dd, 2.7, 12.6), 4.21 (H12, dd, 63, 10.4), 4.10 (H18, dd, 5.5, 12.8), 3.88 (Hll, dd, 6.2, 9.7), 3.56 (13-OC% s), 3.47 (H15, m), 3.40 (H13, d, 10.1), 2.25 (H3, m), 2.25 (7-OCOC%, s), 2.10 and 2.01 (17-and 18-OCOC%, 2xs), 1.94 (4-C%, d, 13), 1.6-1.8 (H216, m), 1.44 (2-C% d, 6.5), 1.04 (3-0*3, d, 7.0), 0.97 (14-C%(eq), s), 0.88 (14-C%(ax), s) ppm (couplings in Hz).
Neomycalamide A triacetate can be prepared as follows: Mycalamide A triacetate (5 mg) was dissolved in CDC13 (0.5 ml) and 10 μl TFA added After 5 days at room temperature (monitored by PMR), the reaction was quenched and worked up by evaporation of the solvent and partition between 1:1 0ϊ2α2:%0 (5mg). Prep HPLC (33% %0 in MeOH) gave three fractions (2.5mg, lmg, 0.8mg), where the first was a mixture of more polar compounds, the third was unreacted mycalamide A triacetate, but the second was about 80% pure neomycalamide A triacetate by PMR, an oil.
Example 41 — Mycalamide A Tri-p-Bromobenzoate
Mycalamide A Tri-p-Bromobenzoate has a molecular weight of 1052 and a molecular formula of C45H50Br3NO13. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
XH NMR (CDOa): δ 7.97 and 7.84 (7-, 17-, and 18-OCOC6H4Br, 2xd, 8.7), 7.59, 7.58 and 7.56 (7-, 17- and 18-OCOC6H4Br, 3xd, 8.8), 7.45 (NH9, d, 9.2), 5.80 (H10, t, 8.6), 5.64 (H7, s), 5.33 (H17, m), 5.10 (10-OC% d, 7.0), 4.89 (10-OC% d, 7.0), 4.87 (4=C% t, 1.8), 4.75 (4=CH2, t, 1.8), 4.61 (H18, dd, 2.6, 12.5), 4.54 (H18, dd, 4.9, 12.6), 4.11 (H12, dd, 5.9, 9.0), 4.03 (H2, dq, 2.8, 6.6), 3.86 (Hll, dd, 5.7, 8.0), 3.62 (H15, dd, 1.4, 10.2), 3.51 (13-OC% s), 3.28 (H13, d, 92), 3.20 (6-OC%, s), 2.58 (H5(ax), td, 1.7, 14.1), 2.51 (H5(eq), d, 143), 2.28 (H3, dq, 2.7, 6.9), 2.02 (H16, m), 1.88 (H16, m), 1.23 (2-C% d, 6.6), 1.05 (3-0*3, d, 7.1), 1.03 (14-0*3(eq , s), 0.88 (14- C%(ax), s) ppm (couplings in Hz).
Mycalamide A tri-p-bromobenzoate can be prepared as follows: Mycalamide A (5.0mg, O.Olmmole), p-bromobenzoyl chloride (llmg, 0.05mmole), dimethylaminopyridine (lmg) and triethylamine (7mg, 0.07mmole) were stirred in pyridine (0.4ml) at 75°C overnight %0 (0.3ml) was added, the mixture extracted with C%α2 (3x0.4ml), and the solvent removed (17mg ca.). Prep RPLC (10% %0 in MeOH) gave four fractions (2mg, lmg, 1.2mg, Llmg) which were mycalamide A 18-mono-p-bromobenzoate and mycalamide A 7,18-di-p-bromobenzoate, obtained previously, and two new compounds, identified by PMR as pure mycalamide A 17,18-di-p-bromobenzoate and mycalamide A tri-p-bromobenzoate respectively, a white solid.
Example 42 — Mycalamide A Cis-Oxazolidinone
Mycalamide A Cis-Oxazolidinone has a molecular weight of 473 and a molecular formula of
Its molecular structure is as follows:
The compound is characterized as follows:
HRFABMS: 496.25270 (M+Na+, +0.9 ppm).
DCIMS (N%): 491 (5%, M+NH4 +), 461 (6%), 460 (30%), 459 (100%, M+NH4 +- C%OH), 443 (17%), 442 (63%, MH+-C%OH).
IR (mixed isomers) 0*C13: 3700-3300, 2910, 1725, 1610, 1380, 1100-1030 cm-1 film 3700- 3100, 2950, 1720, 1380, 1150-1030, 870 cm-1.
1H NMR (CD03): 6.58 (NH9, broad s), 5.59 (H10, dd, 1.6, 8.9), 4.83 (4=C%, t, 1.9), 4.73 (4=C%, t, 1.9), 439 (H7, d, 1.5), 3.97 (H12, dd, 6.7, 9.8), 3.95 (H2, dq, 2.5, 6.6), 3.94 (H17, m), 3.88 (Hll, dd, 6.8, 8.9), 3.61 (13-OC%, s), 3.6 (H18, m), 3.6 (H15, m, hidden), 3.56 (H18, dd, 4.1, 11.2), 3.30 (6-OC%, s), 2.96 (H13, d, 9.6), 2.40 (H5(ax), td, Z0, 13.8), 2.28 (H5(eq), d, 13.9), 2.21 (H3, dq, Z 7.0), 1.57 (H16, ddd, Z5, 10.6, 14.7), 1.51 (H16, m), 1.20 (2-C%, d 6.6), 1.05 (3-C%, d, 6.9), 0.98 (14-C%(eq), s) 0.89 (14-C%(ax), s) ppm (couplings in Hz).
1H NMR (CD3OD): δ 5.65 (H10, dd, 1.8, 8.5), 4.80 (4=0% t, 2.0), 4.66 (4=C%, t, 2.1), 4.39 (H7, d, 1.7), 3.91 (H2, dq, 3.0, 6.5), 3.9 (H17, m), 3.89 (H12, dd, 6.6, 9.6), 3.73 (Hll, dd, 6.6, 8.6), 3.6 (H15, m), 3.59 (HIS, dd, 3.8, 11.2), 3.58 (13-00*3, s), 3.51 (H18, dd, 4.8, 11.3), 3.27 (6- OC%, s), 3.04 (H13, d, 9.5), Z37 (H5(ax), td, 2.0, 13.8), 2.32 (H5(eq), d, 14.3), 2.19 (H3, dq, Z7, 7.1), 1.74 (H16, ddd, 1.7, 5.5, 14.4), 1.48 (H16, m), 1.17 (2-C%, d, 6.6), 1.02 (3-C% d, 7.0), 0.97 (14-C%(eq), s), 0.86 (14-C%(ax), s) ppm (couplings in Hz).
13C MR (CDC13): δ 170.02 (08), 146.07 (04), 110.03 (4=C%, 98.82 (OS), 88.02 (C13), 80.83 (CIO), 78.58 (C15), 77.58 (Cll), 75.68 (C7), 72.04 (C17), 69.31 (C2), 68.89 (C12), 66.74 (C18), 63.09 (13-OC%), 48.54 (6-OC%), 41.72 (C3), 41.50 (C14), 33.76 (C5), 31.44 (C16), 23.42 (14-C%(eq)), 17.87 (2-C%), 13.78 (14-C%(ax)), 11.70 (3-0%).
Mycalamide A cis-oxazolidinone can be prepared as follows: Mycalamide A (3.8mg) was stirred in a solution of IM NaOMe in methanol (0.3ml) at 50°C for 8 hours. The solvent was removed and the residue extracted with CHC13 and filtered (33mg). PMR showed a 1:1 mixture of isomers which were separated by analytical reverse phase HPLC (45% %0 in MeOH) to give
two fractions (0.9mg, 13mg). PMR and NOE's showed that these were cis and trans isomers respectively at C7-C10 of mycalamide A oxazolidinone, an oil.
Example 43 — Mycalamide A Trans-Oxazolidinone
Mycalamide A Trans-Oxazolidinone has a molecular weight of 473 and a molecular formula of C^H^O-j. Its molecular structure is as follows:
The compound is characterized as follows:
HRFABMS: 496.25390 (M+Na+, +3.3 ppm).
DCIMS (N%): 461 (6%), 460 (29%), 459 (100%, M+NH4 +-C%OH), 443 (7%), 442 (41%, MH+-0*3OH).
IR (mixed isomers) CH03: 3700-3300, 2910, 1725, 1610, 1380, 1100-1030 cm-1; film 3700-3100, 2950, 1720, 1380, 1150-1030, 870 cm-1.
1H NMR (CDO^: δ 7.87 (NH9, broad s), 5.62 (H10, dd, 23, 53), 4.85 (4=C% t, Z0), 4.74 (4=0*2, *. 1S)> 4-45 (H7' d» 2-4)» 4-°° ( l2, dd, 5.6, 7.4), 3.95 (H2, dq, Z6, 6.5), 3.94 (Hll, t, 5.4), 3.92 (H17, m), 338 (%18, m), 3.56 (13-00*3, s), 3.54 (H15, m, hidden), 331 (6-OC%, s), 2.98 (H13, d, 7.5), Z48 (H5(ax), td, 1.9, 13.7), 232 (H5(eq), d, 13.7), Z22 (H3, dq, Z8, 7.0), 1.68 and 137 (%16, m), 1.19 (2-0*3, d, 6.6), 1.04 (3-C%, d, 6.9), 1.03 (14-C%(eq), s), 0.88 (14- C%(ax), s) ppm (couplings in Hz).
1H NMR (CD3OD): δ 531 (H10, dd, Z6, 3.4), 4.82 (4=0*2, t, 1.9), 4.67 (4=C%, t, ZO), 4.45 (H7, d, Z6), 3.92 (H12, dd, 5.1, 6.5), 3.91 (HZ dq, Z6, 6.5), 3.78 (H17, m), 3.85 (Hll, dd, 3.6, 5.0), 3.62 (H15, dd, Z9, 10.2), 3.54 (H18, dd, 43, 11.2), 331 (13-OC%, s), 3.49 (H18, dd, 4.8, 113), 3.28 (6-OC%, s), 3.10 (H13, d, 6.6), 2.41 (H5(ax), td, ZO, 14.1), Z28 (H5(eq), d, 13.8), Z21 (H3, dq, Z6, 7.0), 1.82 and 1.68 (%16> m), -17 (2-0%, d, 6.6), 1.06 (14-C%(eq), s), 1.01 (3-C% d, 7.0), 0.87 (14-Oϊ3(ax), s) ppm (couplings in Hz).
13CNMR (CD03): δ 171.63 (C8), 14637 {CA), 109.75 (4=C%, 99.28 (OS), 86.88 (C13), 86.41 (C18), 7936 (C15), 77.12 (C7), 74.12 (Cll), 7Z48 (C17), 69.29 (C2), 68.89 (C12), 66.76
(C18), 61.73 (13-OC%, 48.81 (6-OC%), 41.61 (C3), 39.81 (014), 33.65 (C5), 31.01 (C16), 24.43 (14-C%(eq)), 17.86 (2-C%, 16.28 (14-C%(ax)), 11.80 (3-C%).
Mycalamide A trans-oxazolidinone can be prepared as follows: Mycalamide A (3.8mg) was stirred in a solution of IM NaOMe in methanol (0.3ml) at 50°C for 8 hours. The solvent was removed and the residue extracted with CH03 and filtered (33mg). PMR showed a 1:1 mixture of isomers which were separated by analytical reverse phase HPLC (45% %0 in MeOH) to give two fractions (0.9mg, 13mg). PMR and NOE's showed that these were cis and trans isomers respectively at C7-C10 of mycalamide A oxazolidinone, an oil.
Example 44 - Mycalamide A 17.18-Bis-TBDMS Ether
Mycalamide A 17,18-bis-TBDMS ether has a molecular weight of 731 and a molecular formula of C^H^-jNO-røSij. Its molecular structure is as follows:
The compound is characterized as follows:
1H NMR (CDC13): <5 738 (NH9, d, 9.9), 5.74 (H10, t, 9.1), 10-OC%, d, 6.9), 4.88 (4=C%, m), 4.84 (10-OC% d, 6.8), 4.75 (4=0*2, t, 1.8), 4.23 (H7, s), 4.15 (H1Z dd, 6.3, 9.7), 4.03 (H2, dq, Z9, 6.6), 3.77 (Hll, dd, 6.3, 8.8), 3.66 (H17, m), 3.59 (H18, dd, 4.2, 10.8), 3.54 (13- OC%, s), 3.51 (H18, dd, Z5, 10.7), 3.41 (H15, broad d, 9.9), 338 (H13, d, 9.5), 3.31 (6-OC%, s), 2.38 (H5(eq), d, 14.1), 2.26 (H3, dq, 2.9, 7.0), 2.17 (H5(ax), broad d, 14.4), 1.80 (H16, ddd, 1.7, 9.9, 14.1), 135 (H16, m), 1.21 (2-C%, d, 6.5), 1.02 (3-C%, d, 7.0), 1.01 (14-C%(eq , s), 0.89 (18- OSiC(C%)3, s), 0.86 (14-C%(ax), s), 0.83 (17-OSiC(C%)3, s), 0.06 (18-OSi(C%)2, s), -0.02 (17- OSi(C%)2, 2xs) ppm (couplings in Hz).
13C NMR (CD03): δ 171.34 (08), 144.91 (C4), 11135 (4=C%, 99.93 (Co), 86.28 (10- OC%, 79.86 (C13), 76.15 (C15), 74.17 (CIO), 74.05 (C12), 71.80 (C7), 70.38 (C17), 70.10 (Cll, broad), 69.71 (C2), 65.81 (C18), 6138 (13-OC%), 48.71 (6-OC%), 41.42 (C3), 41.10 (C14), 33.49 (C5), 3331 (C16), 26.03 and 25.89 (17- and 18-OSiC(C%)3), 23.86 (14-C%(eq)), 17.98 (2-C%), 14.25 (14-C%(ax), broad), 11.97 (3-C%), -4.32 and -4.67 (17-OSi(C%)2), -5.22 (18- OSi(CH3)2).
Mycalamide A 17,18-bis-TBDMS ether can be prepared as follows: Mycalamide A (Z6mg), t-butyldimethylchlorosilane (large excess), dimethylaniinopyridine (2mg) and triethylamine (20mg) were stirred in pyridine (0.4ml) at 60°C for 2 days. %0 (0.5ml) was added and the mixture extracted with CH2C12. The organic extract was washed with %0 (3x03ml), the solvent removed, and the combined product subjected to silica gel chromatography (200 mg Davisil, 150A, 35-70 μm), developed in steps from hexane to ethyl acetate. A minor fraction (13mg) which eluted with 9:1 PE:EtOAc was mycalamide A tris-TBDMS ether, while the major fraction (23 mg) which eluted with 3:1 PE:EtOAc was pure mycalamide A 17,18-bis-TBDMS ether by PMR, an oil.
Example 45 — 10-Epimvcalamide A 7-Mono-Benzyl Ether
10-Epimycalamide A 7-Mono-Benzyl Ether has a molecular weight of 593 and a molecular formula of ^H^NO^ Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%: 611 (2%, M+NH4 +), 594 (3%, MH+), 582 (9%), 581 (26%), 580 (28%), 579 (80%, M+NH4 +-C%OH), 565 (12%), 564 (37%), 563 (33%), 562 (100%, MH+-C%OH).
XH NMR (CDO^: <5 7.77 (NH9, d, 93), 7.4-7.2 (ø, m), 5.46 (H10, dd, Zl, 9.3), 5.07 (10- OC%, d, 6.6), 4.83 (10-OC% d, 6.9), 4.81 (4=C% m), 4.80 (7-OC%ø, d, 1Z0), 4.71 (4=C% t, 1.9), 4.53 (7-OC%ø, d, 113), 4.02 (H7, s), 3.89 (HZ dq, Z7, 63), 3.81 (Hll, t, 1.9), 3.75 (HIZ dd, 1.7, Z4), 3.65 (H17, m), 3.64 (H15, dd, 3.0, 1Z9), 3.51 (H18, dd, 3.4, 11.0), 3.38 (13-OC%, s), 333 (H18, dd, 73, 11.0), 3.19 (6-OC%, s), Z92 (H13, d, Z4), Z45 (H5(ax), td, 1.9, 14.4), 232 (H5(eq), d, 14.6), Z25 (H16, ddd, 7.1, 1Z1, 15.1), Z19 (H3, dq, Z9, 7.0), 1.44 (H16, ddd, 3.1, 5.0, 15.4), 1.21 (14-C%(ax), s), 1.17 (2-0*3, d, 6.6), 0.99 (3-0*3, s, 7.0), 0.91 (14-Oi3(eq), s) ppm (couplings in Hz).
13C NMR (CDOa): δ 169.81 (C8), 146.46 (C4), 137.26, 128.49, 12838 and 127.98 (7- OC%ø), 110.08 (4=0*2), 99.65 (OS), 9139 (10-OC%, 83.74 (C13), 81.28 (C7), 80.92 (C15), 77.2 (CIO), 73.43 (7-0 *2), 72-69 (C12), 71.50 (C17), 69.66 (C2), 66.31 (C18), 61.81 (Cll), 59.25
(13-OC%), 49.59 (6-OC%), 41.37 (C3), 36.65 (C14), 34.39 (C5), 30.41 (C16), 2735 (14-C%(ax)), 22.47 (14-C%(eq)), 17.74 (2-C%), 11.82 (3-C%). lO-epimycalamide A 7-monobenzyl ether can be prepared as follows: Mycalamide A (6mg), BaO (26mg) and benzyl bromide (15mg) were stirred in DMSO (0.3ml) at 60°C for 2 hours. %0 was added (0.5ml) and the mixture transferred onto a reverse phase pipette column (200mg C18, equilibrated to %0), flushed with %0 (8ml), then eluted with MeOH (6ml). The resulting MeOH fraction was evaporated to diyness, then subjected to prep TLC (1:2 PE:EtOAc). Four bands of silica were recovered and each eluted with ethyl acetate to give four fractions (lmg, lmg, 13mg, 13mg) which were mycalamide A and 10-epimycalamide A 7-monobenzyl ethers, and 10-epimycalamide A and mycalamide A 7,18-dibenzyl ethers by NMR respectively, an oil.
Example 46 - 10-Epimvcalamide A 7.18-Dibenzyl Ether
10-Epimycalamide A 7,18-Dibenzyl Ether has a molecular weight of 683 and a molecular formula of C38H53NO10. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%: 701 (4%, M+NH4 +), 672 (5%), 671 (18%), 670 (42%), 669 (100%, M+NH4 +-C%OH), 654 (11%), 653 (21%), 652 (53%, MH+-C%OH).
*H NMR (CDC13): δ 7.91 (NH9, d, 9.5), 7.4-7.2 (2xø, m), 5.43 (H10, dd, 2.1, 9.1), 5.05 (10-OC%, d, 6.6), 4.82 (10-OC%, d, 6.6), 4.81 (7-OC%ø, d, 11.6), 4.78 (4=C%, t, 1.9), 4.67 (4=C%, t, 1.9), 4.50 (18-OC%ø, d, 1Z0), 4.48 (7-OC%ø, d, 11.4), 4.43 (18-OC%ø, d, 12.0), 3.99 (H7, s), 3.87 (H2, dq, 2.8, 6.6), 3.82 (H17, m), 3.78 (Hll, t, 2.0), 3.74 (HIZ dd, 1.9, 2.4), 3.58 (H15, dd, 3.2, 11.8), 3.40 (H18, dd, 3.6, 9.5), 3.37 (13-OC%, s) 330 (H18, dd, 7.1, 9.4), 3.07 (6- OC%, s), 2.91 (H13, d, Z3), 2.44 (H5(ax), td, 1.9, 14.6), 2.35 (H5(eq), d, 14.1), Z22 fΗl6, ddd, 6.7, 11.9, 15.0), Z16 (H3, dq, 3.0, 7.3), 1.54 (H16, ddd, 3.3, 5.9, 15.2), 1.20 (14-C%(ax), s), 1.13 (2- C%, d, 6.6), 0.99 (3-C%, d, 7.1), 0.91 (14-C%(eq), s) ppm (couplings in Hz).
13C NMR (CDCI3): δ 170.03 (OS), 146.57 (C4), 137.71, 128.53, 128.40, 128.33, 127.89, 127.72 and 127.66 (7- and 18-OC%ø), 109.87 (4=C%, 99.72 (OS), 91.42 (10-OC%, 83.84 (C13), 80.48 (C15), 79.95 (07), 77.26 (CIO), 73.85 (C18), 73.31 and 73.25 (7- and 18-OC%, 7Z71
(C12), 69.69 (C17), 69.46 (C2), 61.54 (Cll), 59.26 (13-OC%, 48.83 (6-OC%), 4130 (C3), 36.64 (C14), 34.18 (C5), 30.71 (C16), 27.41 (14-C%(ax)), 2230 (14-C%(eq)), 17.77 (2-C%), 11.81 (3- C%). NB: lxø not observed.
10-epimycalamide A 7,18-dibenzyl ether can be prepared as follows: Mycalamide A (6mg), BaO (26mg) and benzyl bromide (15mg) were stirred in DMSO (03ml) at 60°C for 2 hours. %0 was added (0.5ml) and the mixture transferred onto a reverse phase pipette column (200mg C18, equilibrated to %0), flushed with %0 (8ml), then eluted with MeOH (6ml). The resulting MeOH fraction was evaporated to dryness, then subjected to prep TLC (1:2 PErEtOAc). Four bands of silica were recovered and each eluted with ethyl acetate to give four fractions (lmg, lmg, 13mg, 13mg) which were mycalamide A and 10-epimycalamide A 7-monobenzyl ethers, and 10-epimycalamide A and mycalamide A 7,18-dibenzyl ethers by NMR respectively, an oil.
Example 47 - Mycalamide A Tris-TBDMS Ether
Mycalamide A Tris-TBDMS Ether has a molecular weight of 845 and a molecular formula of QβHgjNOjoSi-}. Its molecular structure is as follows:
The compound is characterized as follows:
1H NMR (CDCI3): δ 730 (NH9, d, 9.7), 5.74 (H10, dd, 8.5, 9.7), 5.09 (10-OC% d, 6.9), 4.81 (4=C%, t, 1.9), 4.81 (10-OC% d, 6.8), 4.71 (4=C% t, 1.9), 4.20 (H7, s), 4.11 (HIZ dd, 6.0, 9.3), 3.86 (HZ dq, Z6, 6.5), 3.71 (Hll, dd, 6.1, 83), 3.71 (H17, m), 3.64 (H18, dd, 3.8, 113), 333 (H18, dd, Z 113), 3.52 (13-OC% s), 3.41 (H15, dd, 1.6, 10.0), 336 (H13, d, 92), 3.29 (6-OC%, s), Z55 (H5(eq), d, 14.4), Z36 (H5(ax), td, Z0, 14.4), Z19 (H3, dq, Z5, 7.0), 1.83 (H16, ddd, 1.7, 10.2, 13.8), 13 (H16, m), 1.17 (2-C% d, 6.6), 1.01 (14-0*3(eq), s), 0.99 (3-0*3, d, 7.1), 0.94 (7- 0SiC(O*3)3, s), 0.88 (18-0SiC(O*3)3, s), 0.87 (14-C%(ax), s), 0.84 (π-OS^CH^ s), 0.17 and 0.13 (7-0Si(O*3)2, 2xs)' °-05 and -°-01 ( 7- and 18-OSi(C%2, 2xs) ppm (couplings in Hz).
13C NMR (CD03): <5 17038 (CS), 146.65 (C4), 109.96 (4=C%, 9938 (C6), 86.13 (10- OC%, 80.12 (C13), 77.13 (C7), 76.20 (C15), 73.76 (C12), 73.54 (CIO), 7038 (C17), 70.03 (Cll, broad), 6934 (C2), 65.60 (C18), 61.44 (13-OC%, 50.10 (6-OC%, 4136 (C3), 40.89 (C14, broad), 35.79 (C5), 33.05 (C16), 26.02 and 25.90 (7-, 17- and 18-0810(0*3)3), 24.06 (14-C%(eq)), 17.86
(2-C%), 14.6 (14-C%(ax), broad), 11.77 (3-C%), --4.27, -4.43, --4.74, -4.84, -5.13 and -5.26 (7-, 17- and 18-OSi(C%2).
Mycalamide A tris-TBDMS ether can be prepared as follows: Mycalamide A (Z6mg), t-butyldimethylchlorosilane (large excess), dimethylaminopyridine (2mg) and triethylamine (20mg) were stirred in pyridine (0.4ml) at 60°C for 2 days. %0 (0.5ml) was added and the mixture extracted with C%C12. The organic extract was washed with %0 (3x03ml), the solvent removed, and the combined product subjected to silica gel chromatography (200 mg Davisil, 150A, 35-70 μ ), developed in steps from hexane to ethyl acetate. A minor fraction (13mg) which eluted with 9:1 PE:EtOAc was mycalamide A tris-TBDMS ether, while the major fraction (Z5 mg) which eluted with 3:1 PE:EtOAc was pure mycalamide A 17,18-bis-TBDMS ether by PMR, an oil.
Example 48 — 7.17.18-Trimethoxy Mycalamide A
7,17,18-Trimethoxy Mycalamide A has a molecular weight of 545 and a molecular formula of C 7H47NO10. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%: 563 (10%, M+NH4 +), 533 (18%), 532 (37%), 531 (100%, M+NH4 +- C%OH), 516 (11%), 515 (22%), 514 (74%, MH+-C%OH).
1H NMR (0>α3): δ 7.13 (NH9, d, 9.6), 5.81 (H10, t, 9.8), 5.13 (10-OC%, d, 7.0), 4.82 (10-OC%, d, 7.0), 4.82 (4=C%, t, 1.9), 4.71 (4=C%, t, 1.9), 4.21 (H12, dd, 6.6, 10.4), 3.91 (H2, dq, 2.7, 6.5), 3.87 (H7, s), 3.84 (Hll, dd, 6.6, 9.9), 3.55 (7- and 13-OC%, s), 3.45 (H18, m), 3.44 (H12, d, 10.2), 337 (18-OC%, s), 3.32 (H15, m), 3.31 (H18, m), 33 (H17, m), 3.29 (17-OC%, s), 3.28 (6-OCH3, s), 2.43 (H5(ax), td, Z0, 13.2), Z30 (H5(eq), d, 14.2), 2.19 (H3, dq, 2.6, 7.2), 1.62 (%16, m), 1.16 (2-C%, d, 6.6), 0.97 (14-C%(eq), s), 0.97 (3-C%, d, 7.0), 0.87 (14-C%(ax), s) ppm (couplings in Hz).
13C NMR (CDC13): δ 170.06 (C8), 146.20 (C4), 110.02 (4=C%, 99.91 (OS), 86.49 (10- OC%, 8Z98 (07), 79.48 (C13), 77.66 (C17), 75.81 (C15), 74.41 (C12), 73.11 (CIO), 72.87 (C18), 70.59 (Cll, broad), 6939 (C2), 61.80 (13-OC%), 60.20 (7-OC%), 59.19 (18-OC%), 56.81 (17-
OC%), 49.01 (6-OC%, 41.57 (C14), 41.42 (C3), 34.16 (C5), 29.83 (C16), 23.18 (14-C%(eq)), 17.82 (2-0*3), 13.45 (14-CH3(ax), broad), 11.80 (3-C%.
7,17,18-trimethoxy mycalamide A can be prepared as follows: Mycalamide A (4.2 mg), Ag20 (25 mg) and Mel (18 mg), were stirred in benzene (0.3 ml) at 80°C in a sealed vial for 3 days. The solution was filtered over celite and the solvent removed (4.6 mg). Preparative TLC (developed in EtOAc) gave three fractions (03 mg, 1.2 mg, Z0 mg) which were 7,17-dimethoxy mycalamide A, 7,18-dimethoxy A and 7,17,18-trimethoxy A by NMR respectively. These were individually rechromatographed by prep. TLC to give the pure compounds, an oil.
Example 49 — 7-Methoχy. N-Methyl Mycalamide B
7-Methoxy, N-Methyl Mycalamide B has a molecular weight of 545 and a molecular formula of C-^H^NOj-). Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 563 (2%, M+NH4 +), 533 (9%), 531 (34%, M+NH4 +-C%OH), 516 (25%), 514 (100%, MH+-0*3OH).
1HNMR (CDC13): <5 6.26 (H10, d, 10.2), 5.16 (10-OC% d, 6.7), 4.87 (10-OC%, d, 6.7), 4.83 (4=C% m), 4.74 (4=OΪ2, m), 4.29 (H12, dd, 7.0, 10.0), 4.25 (H7, s), 4.16 (Hll, dd, 6.8, 9.8), 3.94 (HZ dq, Z7, 63), 3.72 (H18, dd, Z7, 11.8), 3.57 (13-OC%, s), 3.52 (H18, dd, 5.9, 113), 3.50 (H15, m), 3.48 (H13, d, 10.0), 3.46 (7-OC% s), 331 and 3.30 (6-OC% and 17-OC%, 2xs), 33 (H17, m), 3.22 (N-C%, s), 2.71 (H5(ax), broad d, 14.2), Z28 (H5(eq), d, 14.1), Z20 (H3, dq, Z9, 7.0), 1.54 (%16, m), 1.14 (20O*3, d, 6.5), 1.00 (3- I3, d, 7.0), 0.99 (14-C%(eq), s), 0.87 (14- α*3(ax), s) ppm (couplings in Hz).
7-Methoxy, N-Methyl Mycalamide B can be prepared as follows: Mycalamide B (2 mg), powdered KOH (2 mg) and Mel (4.6 mg) were stirred in DMSO at room temperature for 24 hours. %0 was added (0.3 ml), the mixture extracted with C%α2 (3 x 0.4 ml), and the solvent removed (3 mg). Prep RPLC (30% %0 in MeOH) gave two fractions (1.2 mg, 0.6 mg) which were
pure 7-methoxy, N-methyl mycalamide B and 7,18-dimethoxy, N-methyl mycalamide B by PMR respectively, an oil.
Example 50 - 7 '-Methoxy. N-Methyl Mycalamide B
7'-Methoxy, N-Methyl Mycalamide B has a molecular weight of 545 and a molecular formula of C--_7H47NO10. Its molecular structure is as follows:
OCH,
The compound is characterized as follows:
DCIMS (N%): 563 (3%, M+NH4 +), 533 (5%), 532 (13%), 531 (45%, M+NH4 +- C%OH), 516 (14%), 515 (28%), 514 (100%, MH+-C%OH).
1H NMR (0DO3): <5 6.32 (H10, d, 10.3), 5.13 (10-OC% d, 6.8), 4.85 (10-OC%, d, 6.8), 4.83 (4=0% t, 2.0), 4.73 (4=C%, t, 2.1), 4.28 (H12, dd, 7.0, 10.7), 4.22 (H7, s), 4.09 (Hll, dd, 7.1, 10.2), 3.94 (H2, dq, 2.6, 6.6), 3.72 (H18, dd, 2.5, 11.8), 3.61 (7-OC%, s), 3.55 (13-OC%, s), 3.54 (H18, m, hidden), 330 (H13, d, 10.8), 3.47 (H15, dd, 1.6, 8.6), 336 (17-OC%, s), 3.29 (6- OC%, s), 3.14 (N-C%, s), 3.12 (H17, m), 2.84 (H5(ax), td, 2.0, 14.9), 2.54 (H5(eq), d, 15.0), 2.20 (H3, dq, Z3, 7.0), 138 and 1.45 (%16, m), 1.14 (2-C%, d, 6.5), 1.02 (3-C%, d, 7.0), 0.96 (14- C%(eq), s), 0.85 (14-C%(ax), s) ppm (couplings in Hz).
13CNMR (CDC13): δ 146.98 (C4), 110.10 (4=C%, 102.04 (C6), 86.85 (10-OC%, 81.09 (C7), 79.73 (C17), 79.05 (C13), 75.84 (C15), 74.74 (C12), 69.63 (C2), 67.24 (Cll), 65.70 (C18), 61.88 (13-OC%), 60.39 (7-OC%), 56.94 (17-OC%), 50.55 (6-OC%), 41.66 (C14), 41.35 (C3), 32.34 (C5), 30.83 (C16), 29.09 (N-C%), 23.00 (14-C%(eq)), 18.00 (2-C%), 12.94 (14-C%(ax)), 11.96 (3-0%). NB: 08, CIO not observed.
7'-methoxy, N-methyl Mycalamide B can be prepared as follows: 7-Methoxy, N-methyl mycalamide B (1.2mg) was dissolved in a solution of IM NaOMe in MeOH and stirred at 80°C for two days. The solvent was removed, then the residue partitioned in 1:1 %O:0HCl3 (4ml), extracted in CHC13 (3xlml), and dried. The combined organic extract was subjected to silica gel chromatography (200mg Davisil), developed in steps from PE to 5% EtOH:EtOAc. The major fraction eluted with 1:19 EtOH:EtOAc (0.9mg) was a 1:3 mixture of starting material and its
epimer at C7 by NMR. (Subsequent mixtures of the two epimers were combined (2mg), treated as above, and purified by prep. TLC (developed twice in EtOAc) to give two fractions containing the pure epimers by PMR), an oil.
Example 51 — Antitumor Assay
Antitumor effectiveness of the compounds of the subject invention was evaluated using a standardized in vitro P388 mouse leukemia cell assay protocol disclosed in U.S. Patent No. 4,731366 with the antitumor activity being expressed in IC50 (concentration that results in 50% inhibition of cell replication normalized to untreated cultures) determined with a log-log transform of data and expressed as xx ng/m Results of the P388 antitumor assays are shown in Table 1.
Example 52 — Antiviral Activity
Antiviral effectiveness of the compounds described in Examples 1-50 were determined against both Herpes simplex type 1 virus (HSV-1) and polio vaccine virus (P VI) replicated in the BSC cell line. In this protocol, bioassay discs (6.0 mm Scleicher and Schuell) are soaked with the desired amount of test solution, then air dried at ambient temperature for 20 minutes. The discs are then pushed through the overlay to sit directly on the monolayer contained in a 16 mm diameter well and incubated in 5% C02 enriched atmosphere at 37°C for 24 hours. Wells are examined, using an inverted microscope, for the size of antiviral and/or cytotoxic zones to evaluate the wells for the antiviral and cytotoxicity results according to the scale: — = no discernable antiviral or cytotoxic effects, + = antiviral or cytotoxic zone 1-2 mm excess radius from disc, ++ = zone of excess radius 2-4 mm, +++ = zone of excess radius above 4 mm, WW — effect over the whole well
Results of the antiviral and cytotoxicity assays are shown in columns labeled 1 through 3 of Table 1. The value shown in the column labeled "1" is for HSV, the value in the column labeled "2" is for PVI, and the third reports cytotoxicity (where +* implies diffuse cytotoxicity over the whole well).
Example 53 — Uses. Formulations, and Administrations Therapeutic and prophylactic application of the new compounds, and compositions containing them, can be contemplated to be accomplished by any suitable method and technique presently or prospectively known to those skilled in the ar Further, the compounds of the invention have use as starting materials or intermediates for the preparation of other useful compounds and compositions. The compounds of the invention are useful for various non- therapeutic and therapeutic purposes. It is apparent from the testing that the compounds of the invention are effective for inhibiting viral activity. Because of the antiviral properties of the compounds, they are useful to swab laboratory benches and equipment in a microbiology
laboratory to eliminate the presence of viruses, or, in the case of compounds with benzyl or benzoyl groups attached, they can be used as ultraviolet screeners in the plastics industry since they effectively absorb UV rays. As disclosed herein they are also useful prophylactically and therapeutically for treating viral infections in animals and humans. The administration of the mycalamide compounds of the invention is useful for treating viral infections and as an antitumor agent. Thus, pharmaceutical compositions containing compounds of the invention as active ingredients are useful in prophylactic or therapeutic treatment of humans or other mammals infected with or likely to be infected with tumors and virus. The dosage administered will be dependent upon the identity of the tumor or virus infection; the type of host involved; its age, health, weight, kind of concurrent treatment, if any; frequency of treatment; therapeutic ratio and like considerations. Advantageously, dosage levels of the administered active ingredients can be, for examples, dermal, 1 to about 500 mg kg; orally, 0.01 to 200 mg/kg; intranasal 0.01 to about 100 mg/kg; and aerosol 0.01 to about 50 mg kg of animal body weight.
Expressed in terms of concentration, the active ingredient of the invention can be present in the new compositions for localized use dermaUy, intranasally, bronchially, intramuscularly, intravaginally, intravenously, or orally in a concentration of from about 0.01 to about 50% w/w of the composition, and especially from about 0.1 to about 30% w/w of the composition. The compositions of the invention are advantageously used in a variety of forms, e.g., tablets, ointments, capsules, pills, powders, aerosols, granules, and oral solutions or suspensions and the like containing the indicated suitable quantities of the active ingredient. Such compositions are referred to herein and in the accompanying claims generically as "pharmaceutical compositions." Typically, they can be in unit dosage form, namely, in physically discrete units suitable as unitary dosages for human or animal subjects, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic or prophylactic effect in association with one or more pharmaceutically acceptable other ingredients, e.g., diluent or carrier.
Where the pharmaceutical compositions are aerosols, the active ingredients can be packaged in pressurized aerosol containers with a propellant, e.g., carbon dioxide, nitrogen, propane, etc. with the usual adjuvants such as cosolvents, wetting agents, etc.
Where the pharmaceutical compositions are ointments, the active ingredient can be mixed with a diluent vehicle such as cocoa butter, viscous polyethylene glycols, hydrogenated oils, and such mixtures can be emulsified if desired. In accordance with the invention, pharmaceutical compositions comprise, as an active ingredient, an effective amount of one or more non-toxic, pharmaceutically acceptable ingredient(s). Examples of such ingredients for use in the compositions include ethanol, dimethyl
sulfoxide, glycerol, silica, alumina, starch, calcium carbonate, talc, flour, and equivalent non-toxic carriers and diluents.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
Table 1.
Mycalamide B 7-Monoacetate
7-Methoxy Mycalamide B
7,17,18-Trimethoxy Mycalamide A
7-Methoxy, N-Methyl Mycalamide B Mycalamide B 7-Monobenzyl Ether
7,17,18-Trimethoxy, N-Methyl Mycalamide A
7,18-Dimethoxy, N-Methyl Mycalamide A
7-Methoxy, N-Methyl Mycalamide A
6-Ethoxy Mycalamide A
Mycalamide A 17,18-Di-p-Bromobenzoate
7-Methoxy Mycalamide A
7,18-Dimethoxy Mycalamide A
18-Methoxy Mycalamide A
Mycalamide B 18-Mono-TBDMS Ether
* - HSV activity — = no discemable antiviral or cytotoxic effects b = PV1 activity + = antiviral or cytotoxic zone 1-2 mm excess radius from disc c = cytotoxicity ++ = zone of excess radius 2-4 mm NT = not tested +++ = zone of excess radius above 4 mm
WW or +* = effect over the whole well t ngdisk $ ng/ml
Table 1 Continued AT Assay AV Assay P388 ICgpj: Conct 1_ Z t
7',17,18-Trimethoxy, N-Methyl Mycalamide A 5000 20000 ++ +++ +* 40000 WW ww +*
Mycalamide B Trans-Oxazolidinone NT NT
Mycalamide B Cis-Oxazolidinone NT NT
Mycalamide B Bis-Ethylcarbonate Hydrochloride NT NT
Mycalamide B 18-Monoacetate 0.9 10 20
18-Methoxy Mycalamide B 7-Monoacetate 1.2 20 50
7-Methoxy Mycalamide B 18-Monoacetate 500 5000 Mycalamide B 7-Mono-p-Bromobenzoate 24 20 50
Mycalamide B 18-Mono-p-Bromobenzoate 20 5 20
Mycalamide B Di-p-Bromobenzoate 360 2000 4000
18-Methoxy Mycalamide B 0.07 0.5 1.0
Mycalamide B 7,N-Dibenzyl Ether 130 1000 2000
Mycalamide B 7,18-Dibenzyl Ether 75 1000 2000
Mycalamide B 7,18 ,N-Tribenzyl Ether 900 5000 10000
Mycalamide B 7,8,18-Tribenzyl Ether 375 2000 5000 ww +++ +*
Mycalamide B Bis-TBDMS Ether 160 500 2000 +++ ++ +*
Mycalamide A 7,N-Dibenzyl Ether 800 2000 + + +* 5000 +++ ww +*
* = HSV activity — as no discemable antiviral or cytotoxic effects b = PV1 activity + = antiviral or cytotoxic zone 1-2 mm excess radius from disc c = cytotoxicity + + = zone of excess radius 2-4 mm NT = not tested +++ = zone of excess radius above 4 mm
WW or +* = effect over the whole well t ng disk $ ng ml
Table 1 Continued
Mycalamide A 7,18-Dibenzyl Ether
Mycalamide A 7-Monobenzyl Ether
Mycalamide A 7,18,N-Tribenzyl Ether
7'-Deutero, 7'-Methoxy, N-Methyl Mycalamide
7-Epimycalamide A Trans-Oxazolidinone 7-Epimycalamide A Cis-Oxazolidinone Neomycalamide A Triacetate
Mycalamide A Tri-p-Bromobenzoate
Mycalamide A Cis-Oxazolidinone
Mycalamide A Trans-Oxazolidinone
Mycalamide A 17,18-Bis-TBDMS Ether
10-Epimycalamide A 7-Monobenzyl Ether
10-Epimycalamide A 7,18-Dibenzyl Ether
Mycalamide A Tris-TBDMS Ether
7'-Methoxy, N-Methyl Mycalamide B NT NT
' - HSV activity — = no discemable antiviral or cytotoxic effects b = PV1 activity + = antiviral or cytotoxic zone 1-2 mm excess radius from disc c = cytotoxicity + + = zone of excess radius 2- nun NT = not tested +++ = zone of excess radius above 4 nun
WW or +* = effect over the whole well t ng/disk; t ng ml
Claims
1. A compound selected from the group consisting of (a) mycalamide B 7-monobenzyl ether; (b) 7,7,18-trimethoxy, N-methyl mycalamide A; (c) 7,18-dimethoxy, N-methyl mycalamide A (d) 7-methoxy, N-methyl mycalamide A; (e) mycalamide A 17,18-Di-p-bromobenzoate; (f) 7',17,18-trimethoxy, N-methyl mycalamide A; (g) mycalamide B trans-oxazolidinone; (h) mycalamide B cis-oxazolidinone; (i) mycalamide B bis-ethylcarbonate hydrochloride; (j) mycalamide B 7-mono-p-bromobenzoate; (k) mycalamide B 18-mono-p-bromobenzoate; (1) mycalamide B Di-p-bromobenzoate; (m) mycalamide B 7,N-dibenzyl ether, (n) mycalamide B 7,18-dibenzyl ether; (o) mycalamide B 7,18,N-tribenzyl ether, (p) mycalamide B 7,8,18-tribenzyl ether, (q) mycalamide A 7,N-dibenzyl ether, (r) mycalamide A 7,18-dibenzyl ether (s) mycalamide A 7-monobenzyl ether, (t) mycalamide A 7,18,N-tribenzyl ether, (u) 7'-deutero, 7'-methoxy, N-methyl mycalamide A; (v) 7-epimycalamide A trans-oxazolidinone; (w) 7-epimycalamide A cis-oxazolidinone; (x) neomycalamide A triacetate; (y) mycalamide A tri-p-bromobenzoate; (z) mycalamide cis-oxazolidinone; (aa) mycalamide A trans-oxazolidinone; (bb) 10-epimycalamide A 7-monobenzyl ether, (cc) 10-epimycalamide A 7,18-dibenzyl ether, (dd) 7-methoxy, N-methyl mycalamide B; and (ee) 7'-methoxy, N-methyl mycalamide B.
Z The compound, according to claim 1, wherein said compound is mycalamide B 7- monobenzyl ether.
3. The compound, according to claim 1, wherein said compound is 7,7,18-trimethoxy, N- methyl mycalamide A
4. The compound, according to claim 1, wherein said compound is 7,18-dimethoxy, N- methyl mycalamide A
5. The compound, according to claim 1, wherein said compound is 7-methoxy, N-methyl mycalamide A
6. The compound, according to claim 1, wherein said compound is mycalamide A 17,18- Di-p-bromobenzoate.
7. The compound, according to claim 1, wherein said compound is 7',17,18-trimethoxy, N-methyl mycalamide A
8. The compound, according to claim 1, wherein said compound is mycalamide B trans- oxazolidinone.
9. The compound, according to claim 1, wherein said compound is mycalamide B cis- oxazolidinone.
10. The compound, according to claim 1, wherein said compound is mycalamide B bis- ethylcarbonate hydrochloride.
11. The compound, according to claim 1, wherein said compound is mycalamide B 7- mono-p-bromobenzoate.
12. The compound, according to claim 1, wherein said compound is mycalamide B 18- mono-p-bromobenzoate.
13. The compound, according to claim 1, wherein said compound is mycalamide B Di-p- bromobenzoate.
14. The compound, according to claim 1, wherein said compound is mycalamide B 7,N- dibenzyl ether.
15. The compound, according to claim 1, wherein said compound is mycalamide B 7,18- dibenzyl ether.
16. The compound, according to claim 1, wherein said compound is mycalamide B 7,18^-1- tribenzyl ether.
17. The compound, according to claim 1, wherein said compound is mycalamide B 7,8,18- tribenzyl ether.
18. The compound, according to claim 1, wherein said compound is mycalamide A 7,N- dibenzyl ether.
19. The compound, according to claim 1, wherein said compound is mycalamide A 7,18- dibenzyl ether.
20. The compound, according to claim 1, wherein said compound is mycalamide A 7- monobenzyl ether.
21. The compound, according to claim 1, wherein said compound is mycalamide A 7,18,N- tribenzyl ether.
2Z The compound, according to claim 1, wherein said compound is 7'-deutero, 7'- methoxy, N-methyl mycalamide A
23. The compound, according to claim 1, wherein said compound is 7-epimycalamide A trans-oxazolidinone.
24. The compound, according to claim 1, wherein said compound is 7-epimycalamide A cis-oxazolidinone.
25. The compound, according to claim 1, wherein said compound is neomycalamide A triacetate.
26. The compound, according to claim 1, wherein said compound is mycalamide A tri-p- bromobenzoate.
27. The compound, according to claim 1, wherein said compound is mycalamide cis- oxazolidinone.
28. The compound, according to claim 1, wherein said compound is mycalamide A trans- oxazolidinone.
29. The compound, according to claim 1, wherein said compound is 10-epimycalamide A 7-monobenzyl ether.
30. The compound, according to claim 1, wherein said compound is 10-epimycalamide A 7,18-dibenzyl ether.
31. The compound, according to claim 1, wherein said compound is 7-methoxy, N-methyl mycalamide B.
32. The compound, according to claim 1, wherein said compound is 7'-methoxy, N- methyl mycalamide B.
33. A compound having the following structural formula:
wherein R1 and R2 are the same or different and are hydrogen or lower alkyl, particularly C1-C5 alkyl; R3"6 are the same or different and are hydrogen, lower alkyl, acyl, lower alkyl silyl, Bn, or Bz; X is =C% -C%, or -0-C% and Y is lower alkyl, Bn, or Bz.
34. A process for inhibiting or killing viruses, said process comprising treating the virus with a compound selected from the group consisting of (a) mycalamide B 7-monoacetate; (b) 7-methoxy mycalamide B; (c) 7,17,18-trimethoxy mycalamide A; (d) mycalamide B 7-monobenzyl ether, (e) 7,17,18-trimethoxy, N-methyl mycalamide A; (f) 7,18-dimethoxy, N-methyl mycalamide A; (g) 7-methoxy, N-methyl mycalamide A; (h) 6-ethoxy mycalamide A (i) mycalamide A 17,18-Di-p-bromobenzoate; (j) 7-methoxy mycalamide A (k) 7,18-dimethoxy mycalamide A; (1) 18-methoxy mycalamide A; (m mycalamide B 18-mono-TBDMS ether, (n) 7\17,18-trimethoxy, N-methyl mycalamide A (o) mycalamide B 18-monoacetate; (p) 18-methoxy mycalamide B 7-monoacetate; (q) 7-methoxy mycalamide B 18-monoacetate; (r) mycalamide B 7-mono-p-bromobenzoate; (s) mycalamide B 18-mono-p-bromobenzoate; (t) mycalamide B Di-p-bromobenzoate; (u) 18-methoxy mycalamide B; (v) mycalamide B 7,N-dibenzyl ether; (w) mycalamide B 7,18-dibenzyl ether, (x) mycalamide B 7,18,N-tribenzyl ether, (y) mycalamide B 7,8,18-tτibenzyl ether, (z) mycalamide B bis-TBDMS ether, (aa) mycalamide A 7,N-dibenzyl ether, (bb) mycalamide A 7,18-dibenzyl ether, (cc) Mycalamide A 7-Monobenzyl Ether; (dd) mycalamide A 7,18,N-tribenzyl ether; (ee) 7'-deutero, 7'-methoxy, N-methyl mycalamide A (ff) neomycalamide A triacetate; (gg) mycalamide A tri-p-bromobenzoate; (hh) mycalamide A cis-oxazolidinone; (ii) mycalamide A trans-oxazolidinone; Qj) mycalamide A 17,18-bis-TBDMS ether, (kk) 10-epimycalamide A 7-monobenzyl ether, (11) 10-epimycalamide A 7,18-dibenzyl ether, and (mm) mycalamide A tris-TBDMS ether.
35. The process, according to claim 34, wherein said compound is selected from the group consisting of (a) mycalamide B 7-monoacetate; (b) 6-ethoxy mycalamide A; (c) 18-methoxy mycalamide A; (d) mycalamide B 18-mono-TBDMS ether, (e) mycalamide B 18-monoacetate; (f) 18-mefhoxy mycalamide B 7-monoacetate; (g) mycalamide B 7-mono-p-bromobenzoate; (h) mycalamide B 18-mono-p-bromobenzoate; and (i) 18-methoxy mycalamide B.
36. A method of treating tumor cells comprising contacting the cells with a compound selected from the group consisting of (a) mycalamide B 7-monoacetate; (b) 7-methoxy mycalamide B; (c) 7,17,18-trimethoxy mycalamide A (d) mycalamide B 7-monobenzyI ether, (e) 7,17,18-tτimethoxy, N-methyl mycalamide A (f) 7,18-dimethoxy, N-methyl mycalamide A; (g) 7-methoxy, N-methyl mycalamide A; (h) 6-ethoxy mycalamide A; (i) mycalamide A 17,18-Di-p-bromobenzoate; (j) 7-methoxy mycalamide A; (k) 7,18-dimethoxy mycalamide A; (1) 18-methoxy mycalamide A; (m) mycalamide B 18-mono-TBDMS ether; (n) 7',17,18-trimethoxy, N-methyl mycalamide A; (o) mycalamide B 18-monoacetate; (p) 18-methoxy mycalamide B 7-monoacetate; (q) 7-methoxy mycalamide B 18-monoacetate; (r) mycalamide B 7-mono-p-bromobenzoate; (s) mycalamide B 18-mono-p-bromobenzoate; (t) mycalamide B Di-p-bromobenzoate; (u) 18-methoxy mycalamide B; (v) mycalamide B 7,N-dibenzyl ether; (w) mycalamide B 7,18-dibenzyl ether; (x) mycalamide B 7,18,N-tribenzyl ether; (y) mycalamide B 7,8,18-tribenzyl ether; (z) mycalamide B bis-TBDMS ether, (aa) mycalamide A 7,N-dibenzyl ether, (bb) mycalamide A 7,18-dibenzyl ether, (cc) Mycalamide A 7-Monobenzyl Ether, (dd) mycalamide A 7,18,N-tτibenzyl ether, (ee) 7'-deutero, 7'-methoxy, N-methyl mycalamide A; (ff) neomycalamide A triacetate (gg) mycalamide A tri-p-bromobenzoate; (hh) mycalamide A cis-oxazolidinone; (ii) mycalamide A trans-oxazolidinone; (jj) mycalamide A 17,18-bis-TBDMS ether; (kk) 10-epimycalamide A 7-monobenzyl ether, (11) 10-epimycalamide A 7,18-dibenzyl ether, and (mm) mycalamide A tris-TBDMS ether.
37. The process, according to claim 36, wherein said compound is selected from the group consisting of (a) mycalamide B 7-monoacetate; (b) 6-ethoxy mycalamide A; (c) 18-methoxy mycalamide A; (d) mycalamide B 18-monoacetate; (e) 18-methoxy mycalamide B 7-monoacetate; and (f) 18-methσxy mycalamide B.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51869590A | 1990-05-03 | 1990-05-03 | |
| US518,695 | 1995-08-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1991017172A1 true WO1991017172A1 (en) | 1991-11-14 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1991/002281 WO1991017172A1 (en) | 1990-05-03 | 1991-04-04 | Novel mycalamides with antitumor and antiviral activity |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO1991017172A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5298523A (en) * | 1992-12-14 | 1994-03-29 | Harbor Branch Oceanographic Institution, Inc. | Method for treating transplant patients using mycalamide compounds |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4868204A (en) * | 1987-04-29 | 1989-09-19 | Harbor Branch Oceanographic Institution, Inc. | Mycalamide compounds, compositions thereof and methods of preparation and use |
-
1991
- 1991-04-04 WO PCT/US1991/002281 patent/WO1991017172A1/en unknown
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4868204A (en) * | 1987-04-29 | 1989-09-19 | Harbor Branch Oceanographic Institution, Inc. | Mycalamide compounds, compositions thereof and methods of preparation and use |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5298523A (en) * | 1992-12-14 | 1994-03-29 | Harbor Branch Oceanographic Institution, Inc. | Method for treating transplant patients using mycalamide compounds |
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