Polyketides are structurally a very diverse family of natural products with an extremely broad range of biological activities and pharmacological properties. In the first chapter, synthesis of Gerfelin and its analogous compounds; whereas in the second chapter, synthesis of (+)-Ramulosin, (-)-Mellein and some of related naturally occurring compounds will be discussed.

1. Synthesis of gerfelin and related analogous compounds

In the course of screening for an inhibitor of geranylgeranyl diphosphate (GGPP) synthase, Imoto and his co-workers have isolated gerfelin (1) from a fungal strain, Beauveria felina QN22047. For the biological activity they performed an in vitro GGPP synthase assay and reported that gerfelin inhibited GGPP synthase activity with an IC(50) of 3.5 μg/ml. It also inhibits the farnesyl diphosphate (FPP) synthesizing activity of GGPP synthase. I felt interest in this simple structure with an interesting bioactivity and started the synthesis of 1 and its analogs to know the structure-activity relationship (SAR).

It would be possible to form an ether linkage by the Pd-catalyzed Ullmann diaryl ether synthesis between segments A and B (or C and D) which is shown in the retrosynthesis of gerfelin. For the preparation of the left-hand segment, 3,4,5-trihydroxytoluene (8) was converted into acetonide phenol 9 and also into triflate 10. On the other hand, the right-hand segments (12 and 13) were prepared from the known 2,2-dihydoxy-6-methylbenzoic acid (11) by acetalization and triflation.

With both the left- and right-hand segments in hand, firstly I investigated coupling between 9 (≡A) and 13 (≡B). The use of bulkier ligand, 2-(di-tert-butylphosphino)biphenyl instead of triphenylphosphine for Pd catalyzed diaryl ether reaction dramatically improved the yield of coupled product. On the other hand, another combination of the triflate and phenol [10 (≡C) and 12 (≡D)] afforded no desired product even when bulkier ligand was used. Finally, treatment of coupled product with aq. TFA afforded gerfelin (1).

To synthesize some related analogs (2-6) of gerfelin, I treated bromide (14) or triflate (15) with 9 under the basic condition for Michael addition-β-elimination to afford 16. Diels-Alder-decarboxylated product 17 was obtained by reaction of 16 with dimethyl acetylenedicarboxylate. Dimethylester 17 was also converted to half methyl ester 18, diacid 19 and another half ester 20. Acid catalyzed hydrolysis of the acetonide groups of 16-20 afforded 2-6, respectivly. To synthesize biotinylated derivative of gerfelin (7), I adopted diamine 21 as a starting material. Boc-protected amine 22 was coupled with gerfelin to give 23 from which amine-protecting group was removed and reacted with biotinylating agent [Biotin-{NH(CH2)4CH2CO}2-SE] to afford gerfelin-biotin conjugate (7).

Inhibitory activities of the synthetic gerfelin and its analogs (2-5) were estimated by in vitro geranylgeranyl diphosphate synthase assay. Synthetic gerfelin showed similar bioactivity to the natural one; and analogs 3-5 were found to be two to four times less active than gerfelin but 2 exhibited no bioactivity. Although the activity of biotinylated gerfelin analog 7 has not been performed yet, the intermediate gerfelin-amine 23 showed interesting results of activity.

2. Synthesis of (+)-ramulosin, (-)-mellein and related natural compounds

(+)-Ramulosin (25) and (+)-6-hydroxyramulosin (24) were isolated from Pestalotia ramulosa, Botrytis sp. and also from some other sources. Their bioactivity is reported as an inhibition of germination of seeds and spores of microorganisms and antimicrobial activity. (-)-Mellein (26) was isolated from different source including many fungi and several insects after it's first isolation from Aspergillus melleus by Nishikawa in 1933. It is the part of defense secretions in ants and termites. Isolation of (-)-mellein methyl ether (27) was reported from Septoria nodorum.

(-)-6-Hydroxymellein (28) was isolated from a wide range of natural source including Daucus carota, Azadirachta indica, Ceratocystis minor etc. and (-)-6-methoxymellein (29) was first isolated by Sondheimer, E. in 1957 from carrots which had developed a bitter taste during storage. Coxon et al. in 1973 and Superchi et al. in 1993, described a differential effect of the dihydroisocoumarins (28 and 29) to the brine shrimp (Artemia salina) and Chinese hamster cells, respectively. ED(50) values of 28 and 29 were 0.66 and 0.43 mM for Artemia salina & 0.66 and 0.46 mM for Chinese hamster cells. To investigate the differences in the toxicity as well as in understanding the physiological and metabolic correlation between 28 and 29 to the producing carrot cells, Marinelli et al. in 1996, reported that ED(50) values for these were 0.07 and 0.032 mM, respectively.

In the present study, I employed one-pot esterification-Michael addition-aldol reaction as shown in the retrosynthesis. To synthesize the one-pot component (R,E)-5-hydroxy-2-hexenal 33 (≡H), I have treated poly-(R)-3-hydroxybutyrate (PHB, 30) for sequential reactions; ethanolysis, TBS protection of the hydroxyl group and then DIBAL reduction to afford aldehyde 31. Reaction of 31 for Wittig olefination (Ph3P=CHCO2Me), and then DIBAL reduction, MnO2 oxidation and finally removal of TBS gave 33. To shorten the steps, I applied aldehyde 31 for Wittig olefination (Ph3P=CHCHO) and then removal of TBS to afford 33.

The next approach was one-pot reaction to the desired bicyclic diol 35 (≡F), which was successful through esterification-Michael addition-aldol reaction of (R,E)-5-hydroxyhex-2-enal (33) and diketene (34). Performing a dilution-controlled one-pot reaction (0.028 M solution of 33 in benzene) afforded the best yield (74%) for 35 and result is reproducible. PTLC separation of the diastereomers (35) afforded (+)-6-hydroxyramulosin (24). To dehydrate 35, I treated it with a various dehydrating agents but best yield of the intermediate diene 36 (≡E) was obtained in high yield (74%) by Martin sulfurane. Selective reduction or aromatization of 36 affored (+)-ramulosin (25) or (-)-mellein (26), respectively. The conversion of phenolic OH group of mellein to methoxy group afforded (-)-mellein methyl ether (27).

Interestingly, when 35 was treated with excess amount of Martin sulfurane (〜3 eq.) it afforded (-)-mellein (72%) just by one-step. Therefore, I felt interest to know what is happening for aromatized. To elucidate the mechanism, I have treated the diene 36 with Martin sulfurane which successfully afforded (-)-mellein. I have isolated and characterize the byproduct diphenyl sulfide which result strongly suggested the mechanism shown in the scheme.

Jones oxidation of 35 gave mixture of ketone (39) and (-)-6-hydroxymellein (28). The conversion of 6-OH group of 28 to a methyl ether afforded (-)-6-methoxymellein (29) along with an overmethylated compound (40).

3. Summary

Gerfelin (1), a novel inhibitor of geranylgeranyl diphosphate synthase, was synthesized for the first time and in a convenient feasible way (3 and 4 steps) where overall yield was 43.6% (total 5 steps). For the SAR study, I have also synthesized six analogous compounds of gerfelin (2-7). In chapter 2, I have described convenient synthetic routes for six naturally occurring 3,4-dihydroisocoumarins (24-29). Overall yields were as following: 49% (5 steps) for 24, 29.3% (7 steps) for 25, 35.2% (7 steps) for 26, 35.2% (8 steps) for 27, 3.9% (6 steps) for 28, 3.5% (7 steps) for 29.

　多様な構造を有するポリケチド類は天然に広く存在し、様々な生物活性を示すものが多い。本研究では生物活性を有する天然のフェノール性ポリケチドに着目し、これらの効率的な合成と類縁体合成を行っている。本論文では、第一章でgerfelinおよびその類縁体の合成研究と構造活性相関研究について、第二章ではmelleinとramulosinおよびこれらの類縁体の合成研究について論じている。

　第一章では、ヒトのゲラニルゲラニル二リン酸(GGPP)の生合成阻害剤であるgerfelin (1)の合成研究と類縁体合成による構造活性相関研究を行っている。Gerfelinは、2003年にGGPPシンターゼを阻害する物質としてカビの二次代謝産物として単離、構造決定されたフェノール性ポリケチドである。本化合物の合成はパラジウムを触媒としたUllmann型のジアリールエーテル形成を鍵反応として行っている。まずgerfelinの左側部分に相当するフェノール2を、安価な3,4,5-トリメトキシトルエンを原料として効率的に合成した。一方右側部分に相当するトリフラート3は、オルシノールを原料としてVilsmeier反応などを用いて高収率で合成した。両者のカップリングによるジアリールエーテルの形成であるが、種々のパラジウム触媒を用いて検討を行った結果、2-(di-tert-butylphosphino)biphenylをリガンドとして用いた場合に高収率でカップリング体が得られることを見いだした。これにより6工程44％でgerfelinの全合成に成功している。

　また同様の手法を用いて5種類の類縁体4-8を効率的に合成し、このうち4種類を用いて構造活性相関を検討した。その結果、類縁体4は全く活性を示さなかったが、類縁体5-7は天然物よりやや弱いながらもGGPPシンターゼ阻害活性を示した。以上のことからヒドロキシ安息香酸部分が活性に重要な役割を持つことが示唆された。

　さらにgerferin結合タンパクの同定を目的として、gerferinのカルボン酸部分にビオチンを導入した誘導体9も合成している。

　第二章では、天然に広く存在する光学活性なイソクマリン型化合物の効率的合成研究を行っている。Mellein、ramulosinおよびその類縁体は、種々のカビの培養液などから単離され、抗菌活性フェロモン様活性、細胞毒性、ファイトアレキシン活性など様々な活性をを有する。合成標的としてmellein(10)、ramulosin(11)、mellein methyl ether(12)、6-hydroxymellein(13)、6-methoxymellein(14)、6-hydroxyramulosin(15)を選択し、これらの化合物をone-pot反応を鍵反応として合成した。

　短工程で調製できる光学活性な不飽和ヒドロキシアルデヒド17とジケテン16のone-pot反応により、エステル化-マイケル付加-アルドール反応が連続して進行し、イソクマリン型骨格を有する二環性化合物18を簡便に高収率で合成した。中間体18は分離可能なジアステレオマー混合物として得られるが、主生成物は6-hydroxyramulosin(15)であった。この中間体18を用い、他の5種類の化合物も合成している。中間体18を脱水後に還元することによりramulosin(11)を、脱水後芳香化することによりmellein(10)やmellein methyl ether(12)を短工程で得た。この脱水と芳香化の過程においてMartinのスルフランを過剰に用いると、脱水に引き続き芳香化が進行するという興味深い反応も発見した。また中間体18を酸化的に芳香化させることにより6-hydroxymellein(13)、6-methoxymellein(14)への誘導にも成功している。総収率はそれぞれmellein(6工程35％)、ramulosin(7工程29%)、mellein methyl ether(7工程35%)、6-hydroxymellein(6工程4%)、6-methoxymellein(7工程4%)、6-hydroxyramulosin(5工程38%)であった。

　さらにこのone-pot反応を応用し、カビから単離されたフタリド骨格を有する抗腫瘍物質19の合成にも成功した。

　以上本論文は、フェノール性ポリケチドの合成研究の成果をまとめたものであるが、類似の様々な化合物の効率的合成やより高活性な化合物の創製を可能にしており、学術上ならびに応用上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。

2

3

gerfelin(1)

4

mellein(10)

ramulosin(11)

mellein methyl ether(12)

6-hydroxymellein(13)

6-methoxymellein(14)、

6-hydroxyramulosin(15)

16

17

18

19