Rccently we disclosed the isolation, biological properties, and structures of the pyripyropenes which apparently represent the most potent naturally ACAT inhibitors reported to date. Importanly, pyripyropene A 1 proved to be orally active in a hamsters, reducing cholesterol. Herein, we describe the first total synthesis of pyripyropene A 1. The key step is the coupling reaction between α-pyrone-pyridine moiety 4 and the acid chloride of sesquiterpene moiety 3 in the presence of Lewis acid to construct ketone 2. The sesquiterpene moiety has been synthesized via stereoselective reductive formylation, palladium associated carbonylation, and allylic oxidation started from (+)-Wieland-Wiescher ketone. As our point of departure, keto alcohol 7 was treated with tetramethylammonium triacetoxyborohydride to furnish the trans-diol 8 stereoselectively. The benzyl protection of 8 followed by dekatalization gave 9. Treatment of the enol silyl ether derived from 9 (KDA/Me_3SiCl) with methyl iodide and benzyltrimethylammonium fluoride (BTAF) afforded mono-methylated ketone 10. 10 was treated with triflic anhydride in the presence of di-t-butyl-4-methylpyridine to give enol triflate 11. Next, palladium-catalyzed carbonylation (CO atomosphere, Pd(OAc)_2, PPh_3, Et_3N, MeOH, DMF) of 11 as described by Stille obtained the methyl ester 12. Allylic oxidation (SeO_2, in dioxane, reflux) of 12 followed by PCC oxidation gave ketone 13. Stereoselective reduction (NaBH_4, CeCl_3 in MeOH) of 13 afforded 14. Deprotection (H_2, Pd(OH)_2 in MeOH, and 1-propanethiol, NaH in DMF) of 14 followed by per-acetylation (Ac_2O, DMAP in pyridine) gave carboxylic acid 15. 15 was converted to acid chloride 3 in treatment with SOCl_2. Next, the key reaction, the coupling reaction between acid chloride 3 and α-pyrone-pyridine 4 was treated with CF_3COOH to obtain ketone 2. Finally, stereoselective reduction of 2 (NaBH_4, CeCl_3 in MeOH) gave pure (+)-pyripyropene A 1. In summary, the first total synthesis of pyripyropene A 1, which were effective and concise convergent approach, designed to afford easy access to both the natural products and a variety of analogs, has been achieved.
Two conceptually attractive diastereoselective synthetic access to gibberellin A_<12> (GA_<12>, 1), a plant-growth-regurator, are reported. The first approach involves the followings as key steps: (i) palladium-catalyzed cycloalkenylation reaction (11→12), (ii) allylic sulfoxide-sulfenate rearrangement reaction (14→19), (iii) intramolecular Diels-Alder reaction (20→21) and (iv) hydrocyanation reaction with isomerization of double bond (21→22). The second approach involves intramolecular nitrile oxide cycloaddition (INOC) reaction (24→25). We are currently pursuing the accomplishment of total synthesis of GA_<12> (1).
Taxol (1), originally isolated from the Pacific yew (Taxus brevifolia), has shown remarkable potential in recent clinical trials for the treatment of breast and ovarian cancer. Its structural complexity and the limited availability have engendered world-wide intensive studies toward its total synthesis. We describe here 1) biosynthetic approach to taxol BC ring system, 2) convergent approach to taxol skeleton, featuring molecular modeling studies for the rational design of synthetic intermediates. The biosynthetic approach involves the transannular cyclization of 12 membered epoxide 4 for the construction of taxol BC ring system. Rational design of the 12-membered epoxide 4 suitable for providing trans stereochemistry between C-8 methyl and C-3 hydrogen was carried out by applying Monte Carlo conformational search and MM2 force field. The key 12-membered intermediate 4 for the transannular cyclization was constructed by aldol condensation of the 5-membered lactone 7 with aldehyde 8, followed by intramolecular alkylation of the protected cyanohydrin 11. An attempt to construct BC ring system by treatment of 14 with Lewis acid revealed the desired transannular cyclization and additional side reaction leading to bicyclic product 16. Then, a concise method for the formation of C-aromatic taxol system has been developed. In our convergent approach, A, C rings were constructed first and then brought together to form the 8-membered B ring. Construction of A, C rings in the intermediate 19 was carried out by successive Friedel-Crafts reaction and cation-cyclization processes. Formation of B ring was achieved by employing intramolecular alkylation of the protected cyanohydrin 18. X-ray crystallographic analysis of the cyclized product confirmed the stereochemical structure and endo conformation of 26. Next, synthesis of the functionalized C-ring system was carried out based on the intramolecular [3+2] cycloaddition of nitrile oxide derivative 30. MM2 transition state modeling (flexible model) of various nitrile oxide derivatives showed that cycloaddition of oxime 30 having C-2, C-4 six membered acetonide group would give the desired trans stereochemistry between C-8 methyl and C-3 hydrogen. Upon treatment with NaOCl aq., the chiral oxime 30 underwent [3+2] cycloaddition to afford the desired chiral C ring synthon 31.
The enzyme protein kinase C (PKC) is thought to play a major role in cellular signal transduction and has been shown to exist as several isoforms. PKC is activated in cells by diacylglycerol and other diverse tumor promotors such as phorbol 12-myristate-13-acetate (1; PMA). Computer modeling studies on PMA have suggested that carbonyl group at C-3, hydroxyl groups at C-4, C-9, and C-20, and a long-chain hydrophobic portion at C-12 would be important to bind with PKC. However these structural hypotheses have not been confirmed by determining the actual PKC binding affinity or activity relationships of such substituted phorbols. We report here the entire stereospecific syntheses of phorbol analogs with the proposed necessary structural features in PKC activation. In particular, these include the syntheses of the analogs 7 and 10 having long-chain hydrophobic moieties at C-12 of their phorbol C-rings. The key intermediate 18 for the synthesis of these analogs is prepared from (+)-3-carene in a stereocontrolled manner. For the synthesis of 10, treatment of 18 with the phosphonate carbanion generated from 19 resulted in stereospecific formation of the E-stereoisomer of 20. DIBAH reduction of 20 and protection of the resulting primary alcohol, as its tert-butyldiphenylsilyl ether, yielded 21. A three-step series of reactions involving tosylation, iodide substitution, and nitration gave 22. The next crucial step was intramolecular cyclization of 22 to the corresponding isoxazolin 23, which was subsequently hydrogenated to the hydroxy ketone 24 in 85% yield (2 steps). Internal cerium alkoxide assisted propynylation of 24 was then followed by oxidation and conversion of the resulting intermediate into the ethyl-ketone 26. Intramolecular aldol cyclization of 26 and dehydration of the resulting aldol mixture in one-pot, followed by deprotection of the silyl ethers, gave the analog 9. Its allylic alcohol functionality was protected as its trityl ether, and the long-chain myristate group was then introduced at C-12. Selective deprotection of the trityl group of the resulting intermediate afforded the phorbol analog 10. The remaining hydrophobic analog 7 was obtained from 18 using similar methods to those used in the conversion of 18 to 10. The in vitro binding affinity of the phorbol analogs 7 and 10, relative to that of PMA (1), was then evaluated. The analog 7 proved to be very weak with respect to its affinity to bind to PKC, and an approximate 100 fold concentration of 10 was required. These results suggested that either the additional methyl and/or the acetoxyl groups at C-11 and C-13 respectively of 1 are also responsible for its high binding affinity to PKC. They may also be attributed to the presence of too much hydrophobic character due to the long chain at C-12 of the deacetoxy analog 10 resulting in a different hydrophobic interaction with phosphatidyl serine or PKC. In fact, an analog which has octanoyl group at C-12 shows higher binding affinity than that of 10.
Tumor-promoting teleocidins and their core structure (-)-indolactam-V (1) exist as two stable conformers, the twist and the sofa form, in solution at room temperature (Fig. 2). To elucidate the common structural features among several TPA-type tumor promoters with different skeletons and quite similar biological activities (teleocidins, phorbol esters and aplysiatoxins), it is indispensable to know the active conformation of teleocidins. Synthesis of conformationally restricted analogues of 1 is one of the most promising approaches. We have synthesized novel twist-restricted analogues (6a and 7) via aza-Claisen rearrangement of (-)-N^<13>-desmethyl-N^<13>-allylindolactam-V (4) along with a sofa-restricted analogue, (-)-5-methylindolactam-V (2). The tumor-promoting activity of these compounds was examined by two in vitro bioassays closely related to in vivo tumor promotion: binding affinity to the protein kinase C (PKC) regulatory domain and the Epstein-Barr virus early antigen (EBV-EA) inducing ability. The binding affinity to the PKC regulatory domain was measured using peptide C (Fig. 5), a newly introduced and readily available peptide incorporating amino acids 101-151 of the second cysteine-rich repeat of rat brain PKC γ. Both 6a and 7, whose fixed conformation was the twist form, showed significant biological activities comparable to 1. By contrast, the sofa-restricted 2 did not show any biological activities related to tumor promotion. These results clearly showed that the active conformation of (-)-indolactam-V (1) is close to the twist form, not to the sofa form. Although a similar conclusion has been recently reported using newly designed benzolactams, indolactam analogues without the pyrrole moiety, this is the first time to demonstrate the active conformation using the intact indolactam skeleton. Bridge formation between position 5 and 13 of (-)-indolactam-V (1) is one of the most effective methods to fix the molecule to the active conformation and to develop new PKC activators with high isozyme selectivity.
Gelsemine (1), an alkaloid isolated from Gelsemium sempervirens (Carolina jasmine), has been a popular target for synthetic chemists because of its challenging, close-packed structure. While three total syntheses of (±)-gelsemine via its minor congener, 21-oxogelsemine (2), were reported in 1994, none of these groups has succeeded in controlling the stereochemistry of the spiroindolinone moiety. Herein we report a stereocontrolled total synthesis of (±)-21-oxogelsemine (2). As illustrated in our retrosynthetic analysis of gelsemine (Scheme 1), we planned to construct the critical bicyclo[3.2.1] system 3 by means of a divinylcyclopropane rearrangement of 4 with a complete control of the stereochemistry of the spiroindolinone. The requisite intermediate 7 could be prepared easily by following the protocol reported by Kondo et al. for their prostaglandin synthesis. While condensation of 7 with oxindole furnished a 4:1 mixture of E-and Z-isomers, 8 and 9, use of 4-iodooxindole 10 gave the desired Z-isomer 11 as the sole product. When heated at 90℃ for 30min, the divinylcyclopropane 12 underwent a smooth rearrangement to give exclusively the desired bicyclic product 13 in 98% yield. Since the endo-side of 15 is completely blocked by the indolinone, the Michael addition of MeNH_2 to the α,β-unsaturated ester occurred entirely from the less hindered, exo-side to give the trans-isomer 16. The amine 16 was then converted to the relatively stable carbamoyl chloride 20 in an eight-step sequence. Upon treatment with AgOTf and Ag_2CO_3 in CH_2Cl_2 at 45℃, 20 underwent cyclization to give the lactam 21, which, on acid hydrolysis, provided the aldehyde 22. Olefination with Tebbe reagent followed by oxymercuration^<4b> with Hg(OTf)_2 furnished the 6-membered ether 24. Incidentally, attempted oxymercuration prior to the construction of the lactam ring resulted in the exclusive formation of the undesired 5-membered ether. A two-step deprotection of the N-MOM group yielded (±)-21-oxogelsemine (2), which was identical with an authentic sample (^1H and ^<13>C NMR). Since 21-oxogelsemine can be reduced to gelsemine (1) with AlH_3 or DIBAL, our present work constitutes a formal total synthesis of (±)-gelsemine.
Stereo- and enantiocontrolled syntheses of a variety of natural products have been accomplished using optically pure (+)-norcamphor 1 as a common starting material. Sesquiterpenes (-)β-santalene 2 and (+)-epi-β-santalene 3 are synthesized formally without destroying the bicyclo[2.2.1]heptane framework of the chiral starting material 1. (-)-Semburin 4, a monoterpene from Swertia japonica, (+)-N-benzoylmeroquinene aldehyde 5, (-)-antirhine 6, and (+)-isocorynantheol 7, which are related to the Corynanthe type indole alkaloids, and (-)-kainic acid 8, a marine anthelmintic α-amino acid, are prepared through the oxabicyclo[3.2.1]octanone intermediate 19, obtained from (+)-norcamphor 1 by Baeyer-Villiger oxidation, by employing the formation and the cleavage of α-diketone monothioketal intermediates as key steps. Finally, a new chiral synthon 47 having the bicyclo[3.2.1]octenone framework has been prepared in both enantiomeric forms from (+)-norcamphor 1. Utilizing the new chiral synthon (+)-juvabione 9 and (+)-epijuvabione 10, both possessing juvenile hormone activity, are synthesized in stereo- and enantiocontrolled ways.
FR900482 (1) isolated from the culture broth of Streptomyces sandaensis No. 6897 at Fujisawa Pharmaceutical Co. in 1987, exhibits potent antitumor activity against various types of mammalian solid tumors. 1 shows the antitumor activities equal to or superior to those of mitomycin C (MMC) (2) and is also found to be effective against MMC- and vincristine-resistant P388 leukemia cells. The structure of 1 is quite unique in that the hydroxylamine function forms a hemiacetal array. Its remarkable antitumor activity and unique structure make 1 an exceptionally intriguing and timely target for total synthesis. Although two total syntheses of racemic 1 had been accomplished by Fukuyama and Danishefsky, none of the total syntheses of optically active 1 have been reported to date. We embarked on the project directed at the total synthesis of 1 and its congeners in optically active forms with an aim to elucidate the structure-activity relationships of 1, and succeeded in completing the first enantioselective total synthesis of (+)-1. Our synthetic strategy involves the following three key steps: (i) coupling of the aromatic fragment 7 with the optically active aziridine fragment 8 (7+8→24); (ii) aldol cyclization of dialdehyde 6 to construct the desired eight-membered ring system 27 (6→27); (iii) Epimerization at the C-7 position of hydroxyketone 28 (28→29). We commenced the synthetic studies by pursuing the synthesis of the aromatic fragment 7 and the optically active aziridine fragment 8. Thus, 7 was prepared in fifteen steps starting from commercially available 5-hydroxyisophthalic acid (9). On the other hand, 8 was synthesized from commercially available L-diethyl tartrate (10) by sequential fifteen step operations. Reaction of 7 with 8 proceeded smoothly to give the desired coupling product 24 in a quantitative yield. 24 was further converted to 6 by way of alcohol 25 and the aziridine 26. Crucial aldol cyclization of 6 was effected by employing LiN(TMS)_2 (1.0 equiv.) as a base in dry THE at -78→-5℃, giving rise to 27 in 42% yield after treatment with NaBH_4. Base-catalyzed epimerization at the C-7 position of hydroxyketone 28 derived from 27 followed by reduction with NaBH_4 gave the aziridinobenzazocine 5 possessing the requisite functional groups with correct stereochemistries. With 5 in hand, we further pursued the synthetic studies to complete the total synthesis of (+)-1. Towards this end, 5 was elaborated to the pentacyclic compound 33 in five steps by way of the aniline 30, acetate 31 and hemiacetal 32. Finally, 33 was converted to (+)-1 by sequential six step operations by way of the tetracyclic compound 3 and the phenol 34. The synthesized (+)-1 was identical with a natural sample of 1 in all spectroscopic properties (IR, ^1H-NMR, MS). In summary, we have succeeded in completing the first enantioselective total synthesis of (+)-FR900482 (1) in a convergent manner starting from 5-hydroxyisophthalic acid (9) and L-diethyl tartrate (10). Since the explored synthetic scheme appears to be highly general and flexible to produce various structural types of the congeners of 1, these studies may open an opportunity for developing a novel anticancer agent.
In 1991, penaresidin A (1) and B (2), the actomyosin ATPase activators, were isolated by Kobayashi et al. from the Okinawan marine sponge Penares sp., and characterized as a mixture of the corresponding tetraacetyl derivatives. Because nothing was known about the absolute configuration of penaresidins, we attempted the synthesis of the enantiomerically pure stereoisomers of 1 and 2 so as to determine the absolute configuration of the natural products. Three stereoisomers of penaresidin A, (2S,3R,4S,15R,16S)-, (2S,3R,4S,15S,16S)- and (2S,3R,4S,15R,16R)-1, were synthesized. The corresponding three tetraacetyl derivatives were all dextrorotatory. Because the mixture of tetraacetyl derivatives of the natural products was reported to be dextrorotatory, the stereochemistry of the azetidine ring portion of the natural 1 must be 2S,3R,4S. Then we carefully compared the 500MHz ^1H NMR spectrum of the natural tetraacetyl derivatives with those of the stereoisomers of 15 and concluded that penaresidin A is either (2S,3R,4S,15R,16R)-1 or its (15S,16S)-isomer. As for penaresidin B, we proposed the revised structure as 2' on the basis of a careful study of ^<13>C NMR spectrum of the acetyl derivative of the natural penaresidin B. We then synthesized (2S,3R,4S,15R)- and (2S,3R,4S,15S)-2'. However, we could not determine the absolute configuration of penaresidin B. In the course of the ^1H and ^<13>C NMR analysis of the stereoisomers of 15 and 26, we found each of them to exist as a mixture of two rotational isomers concerning the N-Ac bond. Indeed 15 gave back the starting single isomer of 1 upon hydrolysis. ^1H NMR study of 15 also supports our speculation. The present interpretation of the NMR properties of 15 is different from that proposed by Kamikawa. For the unambiguous determination of the stereochemistry of penaresidin A and B, we must await the reisolation of the pure material so that we shall be able to compare its chiroptical and biological properties with those of our synthetic samples.
Saframycin Mx 2 4b was discovered in the culture broth of the myxobacterium, Myxococcus xanthus in 1988. It is interesting that this compound has a quinone moiety on the E-ring and a hydroquinone, because the only successful total synthesis within the saframycin family has been of the bisquinone series. We describe first the transformation of (-)-saframycin A 1a to the saframycin Mx type compound (-)-15 and the synthesis of saframycin E 1e, whose structure has not yet been established because of its instability, from saframycin B 1b. Next, we described the first total synthesis of (-)-N-acetylsaframycin Mx 2 25a from (±)-pentacyclic amine 17, which was a key intermediate of our (±)-saframycin synthesis. The reaction of 17 with Cbz-L-alanine gave a mixture of amides (-)-18a and (-)-18b. Deprotection and acetylation of (-)-18a and (-)-18b produced amides (-)-20a and (-)-20b, respectively. The structure of (-)-20b was determined by X-ray crystallography. The conversion of (-)-20a to the bisquinone (-)-21a and subsequent stereoselective and regioselective introduction of the methoxyl group at position 5 provided (-)-22a. Finally, (-)-22a was subjected to catalytic reduction and regioselective oxidation to give (-)-25a in modest yield. The epi-enantiomer (-)-18b was transformed to (+)-25b in a same four-step sequence. The specific optical rotation and the CD spectra of (-)-25a and (+)-25b were of opposite signs.
In the palladium-mediated vinylations of 4-bromo-1-tosylindole (8) with ethyl acrylate, the C_3-vinylated product (9) was obtained without affecting the carbon-bromine bond when stoichiometric amount of Pd(OAc)_2 was used, while the vinylation occurred at the bromine-substituted position by palladium-catalyzed vinylation (Heck reaction) to give the C_4-vinylated product (10). Those chemoselectivity are synthetically useful, because two different carbon side chains could be introduced regioselectively to aromatic bromides by applying the above reactions. The vinylations of various aromatic bromides with ethyl acrylate in the presence of stoichiometric amount of Pd(OAc)_2 were investigated. 2,4-Dimethoxybenzene (14) and p-bromoacetanilide (16) gave the vinylated product (15 and 18) without affecting the carbon-bromine bond. While the same reaction of bromothiophene (19a and 21) or bromofuran (19b) gave the di-vinyl products (20a, b and 22) as main products. Those results show that the selectivity was lost when the bromine atom was attached at too electron rich position. The synthesis of optically active clavicipitic acid (1) was accomplished in only 6 steps from 4-bromoindole (8). The 4-bromodehydrotryptophan (35) was obtained by applying the above chemoselective vinylation of 8 in the presence of stoichiometric amount of Pd(OAc)_2 in good yield. Asymmetric reduction of 35 gave the optically active 4-bromotryptophan (36). Heck reaction of 36 gave C_4-vinylated product (38) which was synthetic equivalent of an important biosynthetic intermediate, DMAT (3). Deprotection of Boc group of 38 under acidic condition caused the spontaneous cyclization to give the mixture of cis and trans-N-tosylclavicipitic acid methyl esters (40). In this route, the key step is that the two different carbon side chains were selectively introduced at C_3- and C_4-position of 8 by palladium-mediated vinylation. This short step synthesis is the first successful example that employs optically active tryptophan derivative, and this strategy provides an easy access to many optically active indole alkaloids having an tryptophan skeleton which are of synthetic and biosynthetic importance.
α,α-Disubstituted α-amino acids, often found in nature, have attracted substantial synthetic interest because of its importance as enzyme inhibitors, neurotropic factors, and conformational modifier of bioactive peptides. We describe here a new method for the synthesis of optically active α,α-disubstituted α-amino acids by the use of an intramolecular version of asymmetric Strecker reaction. Our synthetic plan is the use of α-amino acid as the amino and chirality transferring group. Strecker reaction of the ketimine intermediate gives CN adducts which would readily be converted to the desired chiral amino acids via an oxidative removal of the chirality transferring group followed by a hydrolysis of the nitrile group (eq 1). After removal of the Boc group of the amino acid ester 6 (R=iPr), the treatment of the obtained ketimine intermediate with NaCN in iPrOH gave, stereoselectively, desired amino nitrile 7a and 7b (72%, 7a:7b=25:1). The reaction involved an imine-enamine equilibrium (between 8 and 9) which was ascertained by the following experiments: (1) the use of 2-PrOD as the solvent gave 7 where significant amounts of D atom were incorporated into its C5 methyl and C6 methylene groups; (2) the treatment of 7a with Na^<13>CN in the presence of lequiv TFA gave a mixture of 7a and ^<13>CN enriched 7a. The amino acid residue was removed by (1) t-BuOCl and (2) conc HCl to give (R)-α-methylserine 1a in 84% yield. The use of D-valine afforded (S)-1b. This method was applyed to the syntheses of α-(hydroxymethyl)phenylalanine (2a, b) and α-(hydroxymethyl)aspartic acid (3a, b). The syntheses of four enantio- and diastereomers of α-methylthreonine (4a, 5a) and its allo-isomer (4b, 5b) were also accomplished based on the above mentioned method starting from dl-acetoin.
α-Substituted serine derivatives have recently attracted so much attention from the view points of the design and the synthesis of enzyme inhibitors. Isolation of natural products having α-substituted serine structures such as conagenin (1), myriocin (2), and lactacystin (3) has also spurred much research on synthetic approaches to this class of α-amino acids due to their potent biological activities. For the purpose of developing an efficient method for the preparation of chiral α-substituted serines, we examined Lewis acid mediated cyclizations of seven imidates 5 prepared from the corresponding epoxy alcohols 4 (Table 1). Upon treatment of imidates 5 either with 0.5 equimolar of BF_3・Et_2O or Et_2AlCl in CH_2Cl_2, the cyclization turned out to occur at the more polarized center of the epoxide with complete inversion of the stereochemistry in the cases of trisubstituted epoxy imidates and the imidate derived from cinnamyl alcohol. In the case of disubstituted epoxy imidates, the reaction took place in 5-exo mode rather than 6-endo mode again with complete inversion of the stereochemistry. Interestingly, BF_3・Et_2O always caused the formation of the trichloroacetamides 8 along with the cyclized products 6 or 7, while Et_2AlCl allowed exclusive formation of the cyclized products 6 or 7. Having developed the new stereo- and regioselective cyclization reaction of epoxy imidates 5 which would be useful for the preparation of α-substituted serines, we then investigated the syntheses of conagenin (1) and myriocin (2). Our synthesis of conagenin (1) took the following convergent approach. The carboxylic acid moiety 12 was prepared starting with propiophenone and acetaldehyde by the sequence involved asymmetric aldol reaction followed by in situ syn-selective reduction, acetylation, and RuO_4 induced oxidative cleavage of the benzene ring. For the synthesis of α-methylserine moiety, the above-mentioned methodology was employed. Thus, Et_2AlCl mediated cyclization of the epoxy imidate 13 prepared from methallyl alcohol gave the dihydrooxazole 15 which, upon sequential functional group interconversions, afforded α-methylserine benzyl ester 19. Condensation of the carboxylic acid 12 with α-methylserine benzyl ester 19 followed by alkaline hydrolysis led to the first total synthesis of (+)-conagenin (1). Furthermore, a stereoselective route to the promising precursor of myriocin 31 has been developed based on the combination of Lewis acid mediated cyclization of an epoxy imidate and the chemistry of allenylmethyltrimethylsilanes which we have recently established. TiCl_4 mediated addition of allenylmethyltrimethylsilane to the aldehyde 21 followed by VO(acac)_2 catalyzed epoxidation gave the epoxy alcohol 24 with excellent stereoselectivity. After conversion of 24 into the epoxy imidate 25, successive treatment of 25 with BF_3・Et_2O and acid hydrolysis afforded the diol 27 having all required asymmetric centers with correct absolute streochemistries. Conversion of the diol 27 into the aldehyde 29 followed by Wittig reaction allowed the formation of 31, a protected form of myriocin (2). The synthesis of myriocin (2) from 31 is now in progress.
Mycestericins D (1), E (3), F (2) and G (4), new immunosuppressants, were isolated from the culture broth of Mycelia sterilia ATCC 20349. The immunosuppressive activities of 1 and 2 exhibited an IC_<50> of 16nM and 120nM against mouse allogeneic mixed lymphocyte reaction (MLR), respectively, while 3 and 4 exhibited an IC_<50> of 13nM and 370nM, respectively. The proposed structures were unambiguously confirmed by spectroscopy, chemical evidence and total synthesis. Their absolute configurations have been determined by comparison of their CD spectra with those of synthetic compounds (R and S)-9 prepared from methyl (2S, 4R)-2-tert-butyl-3-formyl-oxazolidine-4-carboxylate (5) and stearoyl chloride. Thus, mycestericins D (1) and F (2) were assigned to be 2 (S), 3 (S) configurations, while mycestericins E (3) and G (4) were 2 (S), 3 (R) configurations. The first total synthesis of mycestericins have been achieved from 5 and 1,8-octanediole (10). The alkyl chain moiety 21 was prepared in 12 steps from 10 by straightforward reactioin. The key intermediate 22 obtained from 5 and 21 could be converted to the desired final compounds. Stereoselective reduction of the ketone 22 with Zn(BH_4)_2 or NaBH_4 provided the (R)-hydroxy 23, the protecting groups of which were removed with 10% MeOH in CF_3COOH, followed by hydrolysis of 24 to give mycestericin E (3). On the other hand, mycestericin D (1) was synthesized from 22 by deprotection, followed by reduction of 25 with Me_4NBH(OAc)_3 and then hydrolysis of 26. Hydrogenation of mycestericin D (1) and E (3) provided the corresponding F (2) and G (4).
Biphenyl or binaphtyl compounds with an axial chirality are attractive compounds not only as the chiral ligands in asymmeric reactions but also for the synthesis of the biological active natural products. There is a considerable current interest in the development of efficient methodologies for the synthesis of the biphenyl or binapthyl compounds with atropisomers in an optically active form. The cross-coupling reaction of (2,6-disubstituted-1-bromobenzene)chromium complexes 1 with o-substituted phenylboronic acids 2 in the presence of palladium(0) gave mono Cr(CO)_3-complexed biphenyl compounds, whose axial stereochemistry is depending upon the nature of ortho-substituents; phenylboronic acids with ortho alkyl sbustituents 2a〜2c gave kinetically controlled biphenyl mono-Cr(CO)_3 complexes 3, while o-formylphenylboronic acid 2d gave the corresponding axially isomeric coupling products 4. Furthermore, the kinetically controlled coupling products 3 isomerize under thermal condition to the thermodynamically more stable Cr(CO)_3 complexes 4 with central bond rotation. This atropisomerization combined with the diastereoselective cross-coupling reaction provides a promising approach to the formation of the optically pure biphenyl compounds 12 and 14 with axial chirality in both enantiomeric forms from a single chiral arene chromium complex 9. This diastereoselective cross-coupling reaction of the (η^6-arene)Cr(CO)_3 complexes with the arylboronic acids can be applied for the synthesis of steganacinan and korupensamines. We are currently investigating total synthesis of steganacin and korupensamines utilizing enantiomerically pure (arene)chromium complexes 16 and 25.
The furaquinocins constitute a novel class of cytocidal antibiotics isolated from the fermentation broth of Streptomyces sp. KO-3988 by Omura et al. The structure of these compounds comprises two biosynthetically distinct moieties, i.e., the polyketide-derived naphthoquinone and an isoprenoid side chain, which pose synthetic challenges including (a) stereocontrol of three contiguous stereogenic centers at C-2, C-3 (quaternary), and C-10, (b) selective construction of the densely functionalized naphthoquinone, and (c) establishment of the sterically congested aromatic-isoprenoid hybrid structure. We describe herein a convergent total synthesis of racemic furaquinocin D. The key steps include: (1) The BF_3・OEt_2-promoted 1,2-rearrangement of the TMSC≡C-group followed by the in situ reduction of the resulting aldol by Et_3SiH to give stereo-defined 1,3-diol 2 as a single diastereomer. (2) Synthesis of dihydrofuran 8 via Sonogashira coupling of alkyne 3 with aryl iodide 6 followed by Pd(II)-catalyzed cyclization of the C-2 hydroxyl onto the internal triple bond. (3) An efficient protocol for the construction of the furanonaphthalene skeleton effected by the alkaline hydrolysis of methyl ester 8 followed by treatment of the resulting sodium carboxylate with acetyl chloride and 4-DMAP. (4) Highly stereoselective methylene transfer reaction to aldehyde 10 to establish three contiguous stereogenic centers at C-2, C-3, and C-10. The synthetic route to the optically active furaquinocins is also described, which relies on the reductive 1,2-rearrangement of epoxy silyl ether 17 derived from optically active epoxy alcohol 14. The process relies also on the recent finding that the Co-complexation greatly facilitates the 1,2-migration of alkynyl groups.
Gualamycin is a novel water-soluble acaricide isolated from the culture broth of Streptomyces sp. NK11687 in 1995. The structure was deduced to be 1 mainly from the spectral and/or X-ray crystallographic analysis of its disaccharide and aglycone portions (2 and 3). The absolute structure of the disaccharide, however, remained undetermined. We now report the isolation and the first total synthesis of gualamycin to confirm the absolute structure 1. The disaccharide 2 was synthesized from properly protected D-gulosamine and D-galactose derivatives. The aglycone unit 3 was synthesized from t-butyldimethylsilyl 2-azido-2-deoxy-α-L-mannopyranoside 8 through the dihydroxylation of the olefin 11 and S_N2-type cyclization of 15. These syntheses confirmed the absolute structures of both segments. Finally, both segments were combined by reaction of the thioglycoside of the disaccharide 22 with the benzylidenated aglycone 21 to give, after deprotection, gualamycin (1), which is identical with the natural acaricide in all respects.
Kapurimycin A_3 (1) having anthra-γ-pyron ring system with vinyl epoxide side chain is an antitumor antibiotic isolated from culture broth of Streptomyces sp. DO-115. The reaction of 1 with DNA is shown to occur at the C16 epoxide carbon to preferentially form guanine adduct. To get insight into the extraordinary reactivity and selectivity of 1 toward guanine base in DNA synthesis of analogs of 1 and the reaction with DNA were examined. The key step for the synthesis of kapurimycin analogs is 6-endo-digonal cyclization of o-hydroxyarylethynylketone. It was found that under thermodynamic conditions employing KF and 18-crown-6 the cyclization preferentially gave the desired pyrone derivatives. Optically active epoxide segment was prepared from commercially available acetylenic alcohol by asymmetric dihydroxylation using AD-mix-β. Coupling of acetylenic epoxide with aromatic aldehyde followed by oxidation with MnO_2 produced ethynylketone. Treatment of the ethynylketoene under the reaction conditions we developed gave optically active kapurimycin analog. To examine DNA-cleaving activity for those analogs DNA transformation assays were performed using supercoiled pBR322 plasmid DNA. The reaction of kapurimycin analogs with pBR322 was carried out at 37℃ for 5h and unbound agents were removed by ethanol precipitation. The recovered DNA was dissolved and incubated for 24h at the same temperature. Agarose gel electrophoresis clearly showed that the DNA-cleaving activity was highly dependent on the structure of analogs and the presence of epoxide, double bond adjacent to epoxide, and the ring system in 2 was essential for the remarkable DNA cleavage for 1.
Archaebacteria including methanogens, extreme halophiles, extreme thermophiles and thermoacidophiles grow under rather extraordinary conditions and have structurally unique membrane lipids to adapt the extreme environments. The lipids are consisted of hydrophobic isoprenoid chains linked to glycerol with the ether bonds, which are well contrast to the ester linkage of the eubacterial and eukaryotic membrane lipids. The second feature is the stereochemistry of the glycerol portion in the archaebacterial lipids, i.e., the hydrophobic phytanyl groups are attached to the hydroxyl groups at the sn-2- and 3-positions of glycerol. The third and most striking feature of the archaebacterial ether lipid is found in the macrocyclic (36- or 72-membered) ring structures 1 and 2. These unusual lipids have been interested in connection with the physicochemical properties based on the lipid bilayer structure. Our interest in these lipids focuses on the biochemical significance of the macrocyclic molecular structures. Prerequisite is to develop synthetic methods of the macrocyclic lipids, because it is difficult to obtain significant amount of the archaebacterial lipids in pure form from natural sources. Here, we will describe the first synthesis of a 36-membered model compound 3 and the natural archaebacterial cyclic lipid 2 isolated from extreme thermophile Methanococcus jannaschii by the direct closure of the macrocyclic ring using the McMurry coupling. Also described is the synthesis of desmethylated analogs of archaebacterial 72-membered tetraether lipids. The McMurry coupling reaction under high dilution conditions of the dialdehyde 27 and 30 yielded successfully the 72-membered macrocyclic products 28 and 31 in 27 and 36% yileld, respectively. We have developed a highly efficient synthetic method of the macrocyclic lipid based on the McMurry coupling. The properties of the synthesized lipid are currently under study.
Tautomycin (1) is an antibiotic substance found by Isono et al. in Streptomyces spiroverticillatus as a new antibiotic with strong antifungal activity against Sclerotinia sclerotiorum and having inhibitory activity to protein phosphatases type 1 and 2A. Here we describe the syntheses of all Segments, coupling among the segments and the total synthesis of Tautomycin. Synthesis of Segment A. Evans aldol reaction between lithium enolate of chiral N-acetyl-oxazolidinone (15) and the aldehyde (14) provided the aldol adduct (16) with high diastereoselectivity (88:12). Protection of the hydroxy group and removal of the auxiliary gave Segment A (17). Synthesis of Segment B. Sub-segment B1 (6) was synthesized through a diastereoselective cis-hydroxylation as key reaction in 11 steps. The synthesis of Segment B (24) was accomplished by coupling between the epoxide (Sub-segment B1) and the dithiane (Sub-segment B2). Synthesis of Segment C. New heteroconjugate addition strategy allowed stereocontrolled syntheses of 2 Sub-segments, C1 (8) and C2 (9). The sulfone carbanion of the latter was coupled with the former epoxide to furnish Segment C (40). Total synthesis of Tautomycin. Coupling between Segment B and C was achieved between the epoxide and sulfone carbanion in the presence of BF_3・OEt_2 to give 50 which was further coupled by Yamaguchi esterification method with Segment A (17). Final transformation of the furan ring into the maleic anhydride ring and removal of the protecting groups completed the total synthesis of Tautomycin (1).
Maitotoxin (MTX), isolated from the dinoflagellate Gambierdiscus toxicus, is the most toxic and largest non-biopolymer known to date. The toxin possesses a powerful ability to elevate the intracellular Ca^<2+> level of a wide range of cell types. The gross chemical structure and stereochemistry on fused or directly connected ether rings were elucidated on the basis of extensive 2D/3D NMR measurements and negative FAB MS/MS experiments. However, the relative configurations on the acyclic residues (C1-C15, C35-C39, C63-C68, and C134-C142) remained to be determined. Herein we report the relative configurations on the C35-C39 and C63-C68 portions of MTX determined by comparison between stereochemically defined synthetic model compounds (1a-1d and 17) and MTX in their NMR spectra. In order to realize this strategy of stereochemical determination, it was required to assume, and consequently shown, that the preferred conformation of an appropriately designed fragment with the natural configuration in NMR solvents reflects that of the corresponding portion of MTX; the NMR characteristics of the fragment should therefore not be significantly different from those of the corresponding moiety of MTX. Since the stereochemical correlations between rings K/L, O/P, and V/W have already been revealed, the present results led to the complete stereochemical correlation spanning all the ether rings (A-F'; C15-C134) in MTX.
Recently, marine polycyclic ethers as exemplified by brevetoxin B (1), hemibrevetoxin B (2), and maitotoxin (3) have attracted the attention of synthetic organic chemists due to their unusual structural framework, novel functionalities, and potent biological activities. We now report an efficient method for the synthesis of 6- and 7-membered ethers and its application to the synthesis of the C-, CD-, and ABC-ring systems of 2 and the S-, Y-, and ST-ring systems of 3. 1. Synthesis of 6- and 7-membered ethers A novel rearrangement used in the total synthesis of lasalocid A was further investigated using 5- and 6-membered ethers, 6 and 7, having the mesylate group on the side chain. Upon treatment with Zn(OAc)_2 in aq AcOH at reflux, the rearrangement took place with complete stereoselection giving the ring-expanded ethers, 8 and 9, in good yield. 2. Synthetic study of hemibrevetoxin B (2) 2-1. Synthesis of C- and CD-ring systems The present rearrangement was successfully applied to the synthesis of the C- and CD-ring systems of 2. The Sharpless asymmetric epoxidation (AE) of 16 gave α-epoxide 17, which was treated with PhCOOH and Ti(OiPr)_4 followed by Dowex to give 18. The alcohol 18 was subjected to the Sharpless AE and cyclization with PPTS to give 20, which was converted into mesylates 23 and 24. The treatment of 23 and 24 with Zn(OAc)_2 produced the ring-expanded ethers 25 and 26 corresponding to the C-ring system, respectively. The synthesis of the 7, 7-membered ether 30 corresponding to the CD-ring system was also accomplished by the double rearrangement of the 6,6-membered ether 29 prepared from 22 via 27. 2-2. Synthesis of ABC-ring system The synthesis of the ABC-ring system of 2 was investigated using the model compound 11. 6-Endo cyclization of vinyl epoxide 34 prepared from 11 was examined using Nicolaou's procedure but the results were unsatisfactory giving a mixture of 6- and 5-membered ethers, 35 and 36. After several attempts, the 6-endo cyclization was found to be accomplished using styryl epoxide 37. Desilylation of 37 with n-Bu_4NF followed by PPTS treatment gave the desired 6-membered ether 38 with complete stereoselection. Reaction of 39 with allylMgCl-ZnCl_2 produced 40a and 40b in a ratio of 1.7:1. Ozonolysis of 40a followed by treatment with CH_2=C(CH_2OAc)CH_2TMS in the presence of TMSOTf gave 42. α-Alcohol 43 prepared from 40b was also converted into β-alcohol 42 by Jones oxidation followed by L-Selectride reduction. The alkaline hydrolysis of 42 followed by MnO_2 oxidation gave the α, β-unsaturated aldehyde 46 corresponding to the ABC-ring system of 2. The total synthesis of 2 is now in progress in this laboratory. 3. Synthesis of S-. Y- and ST-ring systems of maitotoxin (3) The present rearrangement was also successfully applied to the synthesis of the S-, Y-, and ST-ring systems of maitotoxin (3).
Brevetoxins are powerful neurotoxins, which are produced from the red tide organism, Gymnodinium breve, and regarded as the most important natural cyclic polyether compounds. Hemibrevetoxin-B (1) has molecular size about half that of brevetoxins and has been taken much interest because of its unique structure and the biological activities. The key reaction in the total synthesis of 1 in a chiral form is the conversion of lactone to the alkylated cyclic ether via the enol trifluoromethanesulfonate, which has recently been estabilished in our lavoratory. The starting material is (±)-keto alcohol (2), which is obtained easily from commercially available mixture of decalin-1,5-diols. Comound 2 was allowed to react with a chiral resolution agent and PTS to give a separable 1:1 mixture of the diasteromeric acetals, which was separated and transformed into the chiral keto alcohols [(-)-2 and (+)-2], respectively. These compounds were converted to the common lactone (4), from which the tricyclic lactone (20) was effectively provided by repeating our lactone enol triflate procedure. Compound 21, which was simply obtained form 20, was smoothly converted into 23. When the mesylate of 23 was treated with TBAF, the intramolecular cyclization effected to afford the compound 25 in 83% yield. Compound 25 includes the same stereochemistries in all chiral centers except one carbon as those in 1. We have thus constructed the mother skeleton of 1 over 31 steps in a 1.7% overall yield starting from decalin-1,5-diols.
Hemibrevetoxin B (1), isolated from cultured cells of the red tide organism Gymnodinium breve by Prasad and Shimizu in 1989, has a 6,6,7,7-tetracyclic ether skeleton and contains 10 stereocenters. Much attention has been paid to the synthesis of polycyclic ethers including hemibrevetoxin B owing to their unusual structural framework, novel functionalities, and biological activities. Recently, Nicolaou and coworkers have reported the first total synthesis of hemibrevetoxin B. We have reported stereocontrolled synthesis of the 6,6,7,7-tetracyclic ether skeleton of 1 via the intramolecular allylic tin-aldehyde (and ketone) condensation. Chain elongation to the left-hand side aldehyde from this intermediate was difficult, and therefore we utilized 2 having hydroxypropyl side chain as a starting material. The total synthesis of 1 has been accomplished via the allylic tin methodology. The 6,6-ring system 11 prepared via the modified Nicolaou method was converted to 18. Cyclization of 18 with BF_3・OEt_2, proceeded quite smoothly and stereoselectively to give 19 in 94% yield. No diastereoisomers were detected in the cyclization step. The BF_3・OEt_2 mediated cyclization of 25 prepared from 19 by usual transformation provided the 6,6,7,7 system 26 as a single stereoisomer. Oxidation followed by Grignard reaction gave methyl carbinol derivative as a 1:1 mixture of diasteroisomers. After silyl protection, the desired isomer 27 was isolated by column chromatography. Diene side chain was introduced by Wittig olefination followed by elimination to give 30. Construction of the α-methylene aldehyde moiety was achieved by Mannich reaction with Eschenmoser's salt. ^1H and ^<13>C-NMR spectra of synthetic hemibrevetoxin B(1) was identical with those of natural product.
Phospholipase A_2 (PLA_2), rate-determining enzyme in arachidonic acid cascade, is not only one of the most important hydrolytic enzymes of phospholipids in cell membranes, but also causes cell fusion by the production of high concentrated lisophospholipids. Monoclonal antibody was generally produced by cell culture's method. We investigated the preparation of PLA_2 antibodies, influence against cell membranes, by designed racemic hapten 1 and its structural recognition of several substrates. Hapten 1, synthesized in twelve steps from solketal, was conjugated directly to the carrier protein keyhole limpet hemocyanin (KLH) with water soluble carbodiimide (WSC). Fifty monoclonal antibodies specific for hapten 1 were generated using standard protocols and purified from ascites by protein G affinity chromatography. Homogeneous of each antibody was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Hydrolytic reaction of substrate 7 was assayed in 50mM Tris-HCl buffer (pH 7.6) at 37℃ by enzymatic colorimetric manner for determination of free fatty acid. Twenty-two antibodies were found to accelerate the hydrolytic degradation of 7 to free fatty acid compare with those of uncatalyzed reaction, and the most effective antibody 7B3 was characterized in further detail. The kinetic parameters of 7B3 from Lineweaver-burk plot were afforded values of Km=347μM, Vmax=2.52μM/min and kcat=0.063min^<-1>, respectively. Moreover hydrolytic activity of 7B3 against several substrates was studied for the determination of structural recognition. Substrate 6 (C_1=C_2=C16), 8 (C_1=C10, C_2=C16), and 9 (C_1=C16, C_2=C10) was not hydrolyzed by 7B3. Further hydrolytic activity of 7B3 against substrate 10, replaced choline ammonium group of 7 to bromine, was reduced to about half times. Therefore antibody 7B3 recognized not only the side chains at C_1 and C_2, but also the choline ammonium group in substrate, respectively. From above results, hydrolytic mechanism of 7B3, phospholipase A_2 antibody, was found to occur the efficient three parts recognitions of substrate by catalytic antibody.
Considering the inherent high binding specificity of antibodies, catalytic antibodies in which the substrate specificity and the reaction pathway can be programmed by the hapten could provide powerful tools for regio- and stereoselective organic synthesis. In particular, regioselective protection and/or deprotection of carbohydrates in oligosaccharide synthesis are interesting targets for testing the potential of catalytic antibodies. Recently, we have reported the generation of catalytic antibody 17E11 which is capable of discriminating between chemically identical functional groups in the same molecule, catalyzing regioselective deprotection of acylated carbohydrates. Examination of the substrate specificity of antibody 17E11 showed the desired specificity as well as the applicability to a wide range of substrates. Thus, antibody 17E11 is capable of recognizing the regio- and stereochemistry at C-3 and C-4, and only the ester group at C-4 is hydrolyzed exclusively in acylated gluco-type carbohydrates possessing a variety of substituents at C-1, C-2, and C-6. To extend catalytic antibody technology for practical use, we have attempted to utilize antibody 17E11 at several steps associated in oligosacchride synthesis. As a result, it has been demonstrated that these catalytic antibodies efficiently hydrolyze a variety of carbohydrate esters and resulting 4-OH products can be used as glycosyl acceptors in syntheses of oligosaccharide and glycopeptide. The potentiality of the antibody-catalyst for synthesis of some oligosaccharides will be discussed.
The jelly coat of egg starfish, Asterias amurensis, contains a glycoprotein, the acrosome reaction-inducing substance (ARIS), with carbohydrate moieties that are essential for biological activity. Previously a trisaccharide with the sequence of Xyl(1-3)Gal(1-3 or 4)[4 or 3(SO_3-)]Fuc, had been suggested to be one of the major structural units of ARIS. To elucidate the structure of the interglycosidic linkages and position of sulfation on the fucose, trisaccharides 1-3 were synthesized and compared to the natural product which was obtained by acid hydrolysis of pronase digests of ARIS (P-ARIS). Trisaccharides 1 and 2 were synthesized by coupling of the disaccharide glycosyl donors 6 and 11, respectively, with L-fucoside acceptor 14 in the presence of AgClO_4-SnCl_2. The 3-O-sulfated regioisomer 3 was synthesized using glycosyl donor 11 and methyl α-fucoside acceptor 15. Examination of the ^1H-NMR spectra of the sulfated trisaccharides 1-3 showed that the spectrum of 2 was identical to that of the natural material. This confirms that the interglycosidic anomeric linkages are β-xylose and α-galactose, respectively. The position of sulfation was confirmed to be at OH-4 of fucose. Wimalasiri et al., recently showed that another oligosaccharide structure present on P-ARIS contained 4-linked xylopyranosyl, 4-linked fucopyranosyl, 3,4-linked fucopyranosyl and 3-linked galactopyranosyl residues in molar ratios of 1:1:2:1 by methylation analysis. Further analysis of the ^1H-NMR spectrum of this oligosaccharide on P-ARIS suggests it is the pentasaccharide repeating unit shown in Fig 2. We have therefore targeted the synthesis of tetrasaccharides 17 and 18 to aid in the elucidation of the natural pentasaccharide structure. Model studies by FAB mass spectrometry suggest that the sulfation position can be elucidiated by a comparison of the fragmentation patterns of sulfated monosaccharide standards and sulfated oligosaccharides.
L-Threonine aldolases which catalyze the aldol cleavage of threonine to glycine and acetaldehyde in metabolic pathways also catalyze the aldol condensation using glycine as enolate source; however, the enzymes have not been widely used in organic synthesis since Yamada succeeded in the isolation and crystallization of the enzyme from Candida humicola (AKU 4586). The enzyme accepts a broad range of aldehydes and produced multifunctional β-hydrox-α-amino acids, which are constituents of many naturally occurring compounds with interesting biological activities. The substrate specificities of aldehydes acceptors are summarized in Table I. It was found that the aldolase from Candida humicola catalyzes the condensation of glycine and acetaldehyde to form a 93:7 mixture of L-allo-threonine and L-threonine, and moreover, high erythrolthreo ratio was also observed when an oxygen is at the β-position of the aldehydes. A peptide-like mimetic of sialyl Lewis x, which mediates the early stage of adhesion between leukocyte and activated endotherial cells, and Mycesterin D, which is isolated from Isaria sinclairii and shows novel type of immune supress activity, were synthesized by using L-threonine aldolase catalyzed reaction as a key step. The details will be discussed.
Stemona alkaloids are a general term for polycyclic alkaloids, which have been isolated from the roots of Stemonaceous plants. The extracts from the plants have long been used as anthelmintics for domestic animals and as cough remedies for humans. Tuberostemonine (1), isolated from Stemona tuberosa of these plants, has an unusual skeleton containing ten chiral centers and two α-methyl-γ-lactone rings and possesses inhibitory activity on the excitatory transmission at the crayfish neuromuscular junction. Furthermore, tuberostemonine A (2) and stenine (3) consisting of the same A, B, C, D-ring system as 1 have been also isolated from these plants. In this simposium we report synthetic studies on these alkaloids 1, 2, and 3 employing an intramolecular asymmetric Diels-Alder reaction as a key step. The key intramolecular asymmetric Diels-Alder reaction of (E,E,E)-triene 6 proceeded smoothly in the presence of 1.3 equivalent of Me_2AlCl as a Lewis acid to afford the desired bicyclic compound 14a in high yield with good stereoselectivity (Scheme 2). Manipulation of 14a under the conditions described in scheme 3 gave stereoselectively the common intermediate 20 for these alkaloids, from which first total synthesis of (-)-stenine (3) has been accomplished as shown in sheme 4. An introduction of E-ring has been carried out by TMSOTf catalyzed condensation reaction of acetal 20 with 3-methyl-2-trimethylsilyloxyfuran in diethyl ether at -78℃ to provide 23a and 23b with the ratio of ca. 1:1 (Scheme 5). An approach from these compounds to 1 will be also presented.
The diastereofacial discrimination in the addition of organometallics to carbonyl and imino compounds constitutes one of the most reliable tools for the stereoselective construction of the multiple asymmetric carbons. Herein we would like to report a highly diastereoselective addition of acetylide to a chiral aldehyde 1d derived from L-cysteine leading to a new route to (+)-deoxybiotin 8, a precursor of (+)-biotin 9. Diastereoface discrimination addition reaction of acetylide to a chiral imine 1a, b, or aldehyde 1c, d was examined, and the addition to the chiral imine 1a or 1b afforded syn-2a or 2b, an undesired addition product for deoxybiotin 8, respectively, regardless of the acetylide species. We next turned our attention to the inversion of the chiral center by the S_N2 type displacement of the hydroxyl functionality with a nitrogen nucleophile. For such a purpose stereodivergent synthesis of syn-amino alcohol 2d was needed. The addition of the the lithium acetylide in THF gave anti-2d as a major isomer, whereas the desired amino alcohol syn-2d was obtained as a sole product in 86% yield by using the chlorozinc acetylide. The addition product was successfully used for a short step synthesis of (+)-deoxybiotin 8, a precursor of (+)-biotin 9, in enantiomerically pure form in 12 steps starting from L-cysteine: The transformation involves deprotection, imidazolidone formation, cyclization of the thiol functionality to the triple bond, and hydrogenation to give (+)-deoxybiotin 8 in enantiomerically pure form, which has been reported to be converted readily into (+)-biotin 9 by a microbiological oxidation. The present method has several advantages; the starting aldehyde 1d was readily available on a large quantity without any difficulty; the creation of the all cis-stereochemistry of the substituents was conducted by the chelation-controlled addition to aldehyde and the hydrogenation of double bonds; other functional group manipulations all proceeded well to give enantiomerically pure (+)-deoxybiotin 8 in 15% overall yield in 12 steps starting from L-cysteine hydrochloride.
Docking studies of staurosporine and three inhibitor molecules with the isoquinolinesulphonamide structure, H7, H8 and H89, were performed to cAMP-dependent protein kinase, in order to interpret their competitivity between them and ATP. The crystal structure of the enzyme complexed with a peptidyl inhibitor, ATP and Mn^<2+> was taken from PDB. The program ADAM, which was developed for searching stable docking models automatically covering all possible binding modes and ligand conformations, was used for this purpose. Inter- and intramolecular energies were used in all pruning steps of the program and several stable models were yielded from the program. For each inhibitor molecule, the most stable docking model, which was obtained after energy minimization of the models considering protein flexibilities and water molecules, was used for later discussion. Based on these docking models, the complicated inhibitory behaviors of the molecules were well explained by the spaces in the active site of the enzyme occupied by them.
Cholic acid is biosynthesized from cholesterol in liver via β-oxidation of 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid (THCA). Stereochemical aspects of this β-oxidation have now been investigated by preparation of the potential biosynthetic intermediate carboxylic acids including their ^2H- or ^<13>C-labelled forms, followed by their incubation with rat liver homogenate in the presence of CoA and ATP. Analysis of the incubation products by HPLC, NMR and MS revealed the followings: (1) (25R)-THCA, (25S)-THCA and Δ^<24E>-THCA were converted to cholic acid and four C-24/C-25 diastereoisomers of 24-hydroxy-THCA. Incubation of the latters produced cholic acid and Δ^<24E>-THCA. Thus all these compounds in the form of CoA esters, including the recently found 24-oxo-THCA-CoA seem to be intermediates of cholic acid biosynthesis. (2) Stereochemically defined four deuteriated (at C-24) THCA were chemically prepared. Their incubation and the FAB-MS spectra of the resulting Δ^<24E>-THCA indicated that deuterium was retained in the Δ^<24E>-THCAs derived from [24-pro-S-^2H]-substrates, while deuterium was lost during conversion from [24-pro-R-^2H]-substrates. We proposed that the dehydrogenation occurs in syn-mode, wherin (25R)-THCA-CoA, not (25S)-epimer, is a substrate for the oxidase; the (25S)-isomer would isomerize into (25R)-epimer prior to the dehydrogenation. (3) Hydration of Δ^<24E>-THCA-CoA appears to be non-stereospecific because the four 24-hydroxy-THCAs were produced from Δ^<24E>-THCA. Although we have no definite explanation of this phenomenon, the possibility of interconversion of the four alcohols via 24-oxo compound was excluded by the observation that deuterium of [24-^2H]-Δ^<24E>-THCA were retained in all the four 24-hydroxy-THCAs.
Since the discovery of anticancer activity of taxol, much attention has been paid for the isolation of new taxane diterpenoids from various species of yews. In our continuing search for bioactive natural products, we examined extracts of the Japanese yew Taxus cuspidata Sieb. et Zucc. and obtained five new taxane diterpenoids, named taxuspines A〜H (1〜8) and J (9), together with known taxoids. Some of the taxoids increased cellular accumulation of vincristine in multidrug-resistant tumor cells. Here we describe isolation and structure elucidation of taxuspines A〜H (1〜8) and J (9) and the structure-activity relationships of the taxoids (1〜36). The structures of taxuspines A〜H (1〜8) and J (9) were determined by extensive analyses of ^1H-^1H COSY, HMQC, and HMBC spectra and chemical means and the relative stereochemistries of compounds 1〜5 were elucidated by NOESY spectra. Taxuspines A〜C (1〜3), H (8), and J (9) possess different skeletons from usual taxoids consisting a 6/8/6-membered ring system. Taxuspines A (1) and J (9) is a rare example of taxoids involving of a 5/7/6-membered ring system, of which the skeleton has been recently reported for taxchinins. Taxuspine B (2) is the second example of taxane diterpenoid involving a 6/10/6-membered ring system. On the other hand, taxuspines C (3) and H (8) possesses an unusual 6/5/5/6-membered ring system. Taxuspine D (4) is the first taxane diterpenoid containing an enoleacetate moiety. Some of the taxoids (2,3,9,14,15,17,18, and 36) increased the vincristine accumulation in multidrug-resistant tumor 2780AD cells as potent as verapamil, while taxol (26) decreased the vincristine accumulation in the cells. These results imply that the taxoids could be substrates of P-glycoprotein and useful for overcoming of multidrug-resistance. Taxuspine D (4) exhibited taxol-like activity to inhibit markedly Ca^<2+>-induced depolymerization of microtubules, although it lacks the oxetane ring and N-acylphenylisoserine moiety which are often associated with unique activity of taxol (26). It is noted that taxuspine E (5) lacking the side chain at C-13 showed potent cytotoxicity against KB cells, since the baccatin III-type compounds having an oxetane ring but no C-13 side chain were reported to be ca. 1700-fold less cytotoxic against KB cells than taxol (26).
On the basis of the existence of many naturally occurring cyclic peptides with unique structures and biological activities, we have focused our attention on various cyclic peptides, such as RA and Astin series, with antitumor activities, from higher plants. Despite their importance, surprisingly few studies of higher plants occurring cyclic peptides exist in the literature. As a part of our continuing studies in search of new bioactive cyclic peptides from higher plants, we have isolated 16 novel cyclic peptides, named pseudostellarins A-H (1-8), from the roots of Pseudostellaria heterophylla, named dichotomin A-E (9-13), from the roots of Stellaria dichotoma L. var. lanceolata Bge., and named yunnanins A-C (14-16), from the roots of Stellaria yunnanensis, all of them belonging to Caryophyllaceae family. Some of them showed tyrosinase inhibitory and cell growth inhibitory activities. These structures were elucidated by extensive 2D NMR such as COSY, HOHAHA, HMBC, HMQC, phase sensitive NOESY and ROESY spectra, chemical degradation and ESI MS/MS methods. Synthetic and naturally occurring cyclic peptides have been the subject of many conformational studies with the cyclic pentapeptides, hexapeptides and octapeptides, receiving the most attention. However there have been no systematic examinations of cyclic heptapeptides. In order to determine the importance of the specific functional groups for the overall conformation of cyclic heptapeptides, and in an attempt to understand the relationships between chemical composition and three-dimensional structure, conformational analyses of cyclic heptapeptides, pseudostellarin D (4), yunnanins A and C (14 and 16) using X-ray crystallography, high field NMR and MD calculations were undertaken. The dominant solution conformations of 4, 14 and 16 analyzed by high field NMR and MD calculations employing distance constraints were, on the whole, homologous to thoes observed in the solid state.
Most people are familiar with the sight of a young seedling bending towards a window or the brightest source of light to which it is exposed. This directional growth response is known as phototropism. Went (1928) proposed that phototropism is caused by a lateral gradient of growth-promoting auxin in the bending organ. This led to the Cholodny-Went theory of photo- and geotropic curvatures (1937). In contrast to the C.-W. theory, Brinsma and Hasegawa recently found that the shaded half did not contain more auxin than the illuminated ones. Instead it was found that the even distribution of auxin was accompained by a lateral gradient of growth-inhibitory substances during phototropic curvature. We have isolated some photoinduced growth inhibitory substances related to phototropism, raphanusanins from radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls, 6-methoxy-2-benzoxazolinone from light-grown maize (Zea mays L.) shoots, 3-hydroxy-β-ionone from light-grown dwarf bean (Phaseolus vulgaris) shoots, and p-hydroxy cinnamic acid from light-grown bean (Vigno mungo L.) shoots, and carried out some chemical studies on these precursors, 4-methylthio-3-butenyl-isothiocyanate (4-MTBI), 2,4-dihydroxy-7-methoxy-2H-1,4-benzozazin-3(4H)-one (DIMBOA) and related compounds. From a view point of phototropism, 4-methylthio-3-butenyl glucosinolate could be initially hydrolyzed with endogeneous myrosinase (thioglucosidase) to afford 4-MTBI at the light exposure side, which is spontaneously converted into some five-membered thiolactams in radish hypocotyls. 2β-Glucoside of DIMBOA is initially hydrolyzed with endogeneous glucosidase to afford DIMBOA at the light exposure side, further emzymatic acylation of which immediately affords MBOA, wherein the resulting MBOA interestingly inhibits the coleoptile growth of maize.
Recently, marine polyether compounds have attracted much attention because of their extremely potent bioactivities, For the structure elucidation of these polyether groups, two-dimensional nuclear magnetic resonance (2D-NMR) experiments have been regarded as the sole tool. Yet the NMR methodology alone is sometimes insufficient to elucidate the structure with a molecular weight over 2000Da, or those with repetitive structure units which give rise to heavy overlapping of NMR signals. Fast atom bombardment tandem mass spectrometry (FAB MS/MS) played an essential role in the structural determination of polyhydroxy-polyether compound which have repetitive or similar structure moieties. Negative-ion FAB spectra of these compounds are typically dominated by molecular-related ion species due to the presence of a negative charge moiety such as a sulfate ester group. Collision induced dissociation (CID) of these molecular-related ion species gave charge remote fragmentation patterns which were very useful for structural determination. Maitotoxin, with a molecular weight of 3422Da, is one of the most potent and complex marine toxins. Conventional negative-ion FAB CID MS/MS spectra could not show simplified and prominent product ions of maitotoxin. Because of the intensity of the precursor ion of molecular related ion species corresponding to the ^<12>C monoisotopic mass is less than that expected from the natural abundance of ^<13>C. However, the negative ion FAB CID MS/MS spectrum measured with reduced selectivity for precursor ions gave a series of prominent product ions for the determination of structure, despite the location of one of the sulfate esters being in the middle of the molecule. The interpretation of the CID spectrum agreed with the structure of maitotoxin.
To obtain the useful informations on the structure of minute amounts of natural glycosphingolipids, we have been developing the mass spectrometrical technique. In this time, we wish to report the usefulness of the CAD-MS/MS of [M+Na]^+ ions obtained in the (+)FABMS to the structure elucidation of the cerebrosides. First, the mechanism of the fragmentation of the [M+Na]^+ ions were discussed. The CAD-MS/MS spectra of [M+Na]^+ ions obtained in the (+)FABMS of the synthetic ceramides 1, 2, 3, and 4 were investigated (Fig. 1). No significant fragment ion was observed in the spectra of 3 and 4, on the other hand, the high intense fragment ions due to fission of the amide bond were detected in the spectra of 1 and 2 possessing 2'-OH group. It means that the conjugation of amide is spread by the neighboring 2'-OH group in 1 and 2 followed by the amide N atom becomes sp3-like, which is able to coordinate to Na^+, and therefore the amide linkage is easily cleaved by means of the formation of the five membered ring proposed as scheme 1. Next, the application of the method to the structure elucidation of the ceramide moiety of the glycosphingolipids were attempted. The prominent fragment ions originated by cleavage of amide bond were observed in CAD-MS/MS spectra of [M+Na]^+ ions appeared in the (+)FABMS of the cerebrosides 5 and 6 (Fig. 2), and which indicate that the method provide the useful information for the structures of the ceramide moieties of the cerebrosides possessing 2'-OH group without chemical degradation. Furthermore, the FAB-CAD-MS/MS method was applied to the cerebroside molecular species obtained from starfish, namely, the molecular mass and the fatty acid composition of the main constituents of the species are estimated without acid hydrolysis (Fig. 3). In addition, the method was proved to be applicable to the structure elucidation of lactosylceramide (Fig. 4).
Search for chemical substances regulating the biofilm formation has been carried out. The assay system has been developed by using the marine bacterium possessing the attaching properties (Rhodospirillum salexigens SCRC 113). This bioassay system in laboratory is suitable for screening the crude extracts from marine organisms and the synthetic compounds. First, the adhesion inhibitory of samples was determined by monitoring amounts of polysaccharide produced by the bacteria as evaluation of the biofilm formation. Substances regulating the biofilm formation have been isolated from marine organisms. For example, bisdeacetylsolenolide D, a biofilm formation inhibitor and butenolide derivative (Hateramine), a biofilm formation accelator have been obtained from marine sponges, respectively. Ethyl N-(2-phenethyl) carbamate (1) isolated from the marine bacterium SCRC3P79 (Cytophaga sp.), remarkably inhibited the biofilm formation. The synthetic compound p-nitro derivative (2), exhibited significant inhibition of the biofilm formation. Furthermore, (2) exhibited antibacterial activity. However this assay system was replaced to new method, because procedure described here did not afford enough reproducibility of the data. Secondly, growth inhibition with tested samples against the attaching bacteria was indicated by measurement of absorbance of the culture medium and the biofilm formation was estimated. Aeroplysinin-I isolated from marine sponge Pssamaplysilla purpurea, exhibited inhibition of the biofilm formation. New nitroalkanes, (3) and (4), have been obtained from an unidentified marine sponge. The synthetic compounds (5), (6), (7) and (8) exhibited significant inhibition of the biofilm formation. As expected, the compounds (5), (6), (7) and (8) exhibited remarkable inhibition of the biofilm formation in the field experiment.
Constituents of the Japanese sea hare Dolabella auricularia collected in Mie Prefecture, Japan were examined by using bioassay, and new cytotoxic depsipeptides, dolastatins G (1) and H (14) and isodolastatin H (15) were isolated. Dolastatin G (1) showed cytotoxicity against HeLa-S_3 cells with an IC_<50> of 1.0μg/mL. On the basis of 2D NMR technique, dolastatin G (1) has proved to be a 35-membered cyclic depsipeptide which consists of a hexapeptide and two new hydroxy acids. The absolute stereochemistry of the hexapeptide moiety was determined by the chiral HPLC analysis of amino acids obtained by acidic hydrolysis of dolastatin G (1). The absolute stereochemistry of two hydroxy acid parts was determined by the enantioselective synthesis of two corresponding fragments obtained by degradation of dolastatin G (1). For the purpose of confirming the stereostructure of dolastatin G (1), synthetic studies on dolastatin G (1) have been carried out. Three subunits, 7, 10, and 11, were synthesized, coupling of which gave seco acid 13. The synthesis of dolastatin G (1) from seco acid 13 is in progress. A 1:1 mixture of dolastatin H (14) and isodolastatin H (15) showed potent cytotoxicity against HeLa-S_3 cells with an IC_<50> of 0.00381μg/mL. On the basis of spectroscopic data, dolastatin H (14) has proved to be a linear tetrapeptide which contains two unusual amino acids and is esterified at the C-terminus by the primary hydroxyl group of 3-phenyl-1,2-propanediol. Isodolastatin H (15) is the structural isomer of dolasatin H (15), in which the tetrapeptide is esterified at the C-terminus by the secondary hydroxyl group of 3-phenyl-1,2-propanediol. The absolute stereochemistry of dolastatin H (14) and isodolastatin H (15) was unambiguously determined by the enantioselective total synthesis.
The manzamines, first described by Higa and coworkers in 1986, are an intriguing group of marine alkaloids, characterized by a fused and bridged tetra- or pentacyclic ring system that is joined to a tryptophan-derived β-carboline. The biogenesis of the manzamines was initially baffling. Baldwin's ingenious suggestion of an intramolecular Diels-Alder reaction in a macrocyclic bisdihydropyridine is eminently reasonable. We now present the structure of kauluamine (1), which is an unsymmetrical manzamine dimer, thus adding yet another level of complexity to this fascinating group of alkaloids. The dichloromethane-ethanol (5:1)-soluble part of the ethanol extracts of an Indonesian sponge, Prianos sp., was subjected to an Al_2O_3 column followed by HSCCC to give kauluamine (1) as an unstable solid of composition C_<72>H_<94>N_8O_3. Structural elucidation proved difficult because of instability and severe overlapping of signals in ^<13>C and ^1H NMR spectra. Many of these difficulties could be overcome by formation of a monoacetate (2, C_<74>H_<96>N_8O_4). 2D NMR techniques, especially HOHAHA and HMQC-HOHAHA experiments, made structural elucidation feasible.
In our search for biomedically important metabolites from Japanese marine invertebrates, the hydrophilic extract of the marine sponge Penares aff. incrustans strongly inhibited the binding of ^<125>I-labeled ω-conotoxin to rat brain synaptic plasma membranes. Bioassay-guided isolation afforded an inseparable mixture of closely-related diacylated unusual polyamines. The EtOH extract of the frozen sponge (1kg) was partitioned between H_2O and Et_2O, and the aqueous layer was further extracted with n-BuOH. The MeOH-soluble material of the n-BuOH fraction was subjected to various chromatographies to yield a mixture of penaresamides (1, 20.3mg, 2.0×10^<-3>% yield). The mixture of penaresamides was positive to both ninhydrin and Dragendorff reagents, suggesting their basic nature. Interpretation of the HOHAHA spectrum indicated that penaresamides were acylated polyamine. Penaresamides were hydrolyzed with 2N HCl at 110℃ followed by solvent partitioning to afford penaresamine (2) and a mixture of fatty acids. The structure of 2 was determined by spectral data. GC-MS analysis of the methylated fatty acid fraction disclosed the presence of five C_<11> fatty acids. Penaresamides exhibited a FABMS peak at m/z 773, corresponding to a molecular formula of C_<44>H_<94>^<35>Cl_2N_6O_2. Spectral data led to the structures of the bisamides of penaresamine (2) containing two C_<11>-fatty acids. Therefore, the structures of penaresamides (1) were elucidated to be diacylated polyamine. The proposed structures were confirmed by a total synthesis of panaresamide A, which posseses two unbranched acyl groups. The natural penaresamides (1) and the synthetic penaresamide A inhibited the binding of ^<125>I-ω-conotoxin to N-type Ca^<2+> channels with IC_<50> values of 1.3μM and 5.8μM, respectively.
First enzyme which catalyzes Diels-Alder reaction has been partially purified. This crude enzyme is able to catalyze [4+2]-cycloaddition of prosolanapyrone III to the exo adduct solanapyrone A and the endo adduct solanapyrone D in a 7:1 ratio differing substantially from the ratio found in a background reaction (exo/endo, 1:23). The optical purity of the enzymatic reaction product solanapyrone A was estimated as 92%ee by HPLC analysis monitored with CD spectrometer. The enzyme also exhibited an oxidase activity of prosolanapyrone II to III. Two-step conversion of prosolanapyrone II to solanapyrones A and B by the crude enzyme gave better diastereoselectivity and enantioselectivity (A: 99%ee). A high reaction rate of the background reaction for prosolanapyrone III caused some losses in both diastereo- and enantioselectivity. Thus, we suggest that a specific enzyme catalyzes both oxidation and Diels-Alder reaction producing enantiomerically pure solanapyrones.
Portulal 1 is a diterpene isolated from Portulaca grandiflora Hook. as a plant-growth regulator and have a unique [7.5]-ring system. From doubt to the proposed biosynthetic scheme and interest in the chemosystematics we have investigated extensively the constituents of related Portulaca species as well as the minor congeners of 1. From Portulaca grandiflora we characterized six compounds 2-7 with the [7.5]-ring system, in which four constituents were new, three compounds 8-10 with novel [7.6]-ring system, and one clerodane diterpene 11. For a gardening variant, P. cv. Jewel it is said that the consideration as an independent species would be pertinent from the standpoint of genetics. Examination of this plant disclosed the occurrence of trans-clerodane diterpenoids 12-16 ([6.6]-ring system) as major constituents and that of a diterpenoid 17 with [7.6]-ring system as minor one. This results strongly support the proposition that P. cv. Jewel should be an independent species. Next we examined the constituents of P. pilosa L. endemic to Iriomote island, Okinawa Pref.. We clarified the occurrence of three diterpenoids 19-21 with [7.6]-ring system in the aerial parts and that of three compounds 22-24 with [6.6]-ring system in the roots, a fact which would be biologically significant. P. cv. Jewel, P. pilosa and P. grandiflora have been disclosed to have the constituents with the biosynthetically more advanced structures in this order, which would imply the linear evolutional transition of the enzyme systems in Portulaca plants. At the same time we have proposed a chemically rational biosynthetic scheme based on the functionalization pattern of the diterpenoid components.