Bioactive alkaloids of fungal origin

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 32 © Elsevier B.V. All rights reserved.

549

Bioactive Alkaloids of Fungal Origin Hideo Hayashi Graduate School ofAgriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan ABSTRACT: In order to obtain fungal isolates, which produce bioactive compounds, random screening was carried out using okara (an insoluble residue of the whole soybean homogenate) as a cultural medium. We observed three kinds of activities against silkworms: insecticidal activity, convulsive activity and paralytic activity. A soil isolate, Penicillium simplicissimum ATCC 90288, produced novel insecticidal indole alkaloids that we designated as okaramines. Okaramines were found to be produced by not only this strain but also other strains belonging to P. simplicissimum. The isolate, Aspergillus aculeatus KF-428 also produced two okaramine congeners: okaramines H and I. These data strongly supported the fact that okaramines are widely produced by fungi. Till now, eighteen okaramine congeners have been isolated; their biogenesis and structure-activity relationships are described in this review. We also describe the results of synthetic studies for the okaramines J and N. The isolate, Penicillium expansum MY-57 produced five insecticidal compounds: the new communesin congeners, communesins D, E, and F, and the known communesins A and B. Convulsive compounds, verruculogen and penitrems, were produced by the isolates P. simplicissimum MF-24 and P. simplicissimum ATCC 90288, respectively, indicating that our convenient bioassay system with silkworms could be used to search for convulsive compounds. Novel convulsive compounds, brasiliamides A, B, C, D, and E, were found in the cultural media of Penicillium brasilianum JV-379. Finally, chance observation led to the isolation of new paralytic compounds, asperparalines A, B, and C, from Aspergillus japonicus ATCC 204480. Asperparalines have unique structures consisting of a bicyclo[2,2,2]diazaoctane core and a spirosuccinimide moiety.

INTRODUCTION Combinatorial chemistry has become a pov^erful methodology for the construction of new compounds. Natural products, how^ever, also have enormous potential as a source of new^ compounds. In particular, numerous useful microbial products have been isolated as antibiotics, herbicides, fungicides and enzyme-inhibitors. Moreover, microorganisms have provided various compounds with diverse bioactivities, such as immunomodulatory, antitumor and antihelmintical activities [1].

550

Many efforts have been made to identify strains producing insecticidal components. In the 1960s, piericidins [2,3] and aspochracin [4,5] were isolated from Streptomyces sp. 16-22 and from Aspergillus ochraceus, respectively. In the 1970s, milbemycins were isolated as insecticides and acaricides from Streptomyces hygroscopicus subsp. aureolacrimosus [6-8]. Avermectins were isolated from Streptomyces avermitilis and developed as antiparasitic agents [9]. In the 1980s, our group also isolated a new insecticidal compound, A^-norphysostigmine, from Streptomyces sp. [10]. Spinosyns A, B, C and D (formerly known as A83543A-D), which were isolated from Sacchropolyspora spinosa in 1991, were found to possess potent mosquito larvicidal activity [11]. Spinosyns have also been used for crop defense [12]. However, we should emphasize that, among the various microbial products, only a few, such as the above-described avermectins and spinosyns, have practical applications. In the 1980s, our group began to screen microbes for insecticidal compounds that could be used in practice, or become lead compounds for the generation of new carbon skeletons. As a result, various strains were found to exhibit insecticidal, convulsive and paralytic activities against silkworms. This chapter deals with the procedures used for isolation of bioactive strains and their active principles. An overview of their chemical structures, activities, synthesis, and structurally related compounds is also given. INSECTICIDAL COMPOUNDS Various media were used for culturing fungi, and the fungal secondary metabolites are known to depend on the cultural conditions, such as the medium constituents and temperature. In our study, okara, which is an insoluble residue of whole soybean homogenate and a waste material in tofu (soybean curd) production, was used as a culture medium. This is the first time that this material has been used for culturing fungi. Fungal strains isolated from soil samples in the usual manner were cultured with okara media for about 10 days. The okara and mycelia were soaked in acetone, and aliquots of both acetone extracts were added to an artificial silkworm diet. Third instar silkworm larvae were introduced into a Petri dish containing the artificial diet, and the mortality rate was determined 24 h after initiating the administration. Using this screening method several hundred isolates were checked for their activity, and two strains, Penicillium simplicissimum Thom ATCC 90288 (originally AK-40) and Penicillium expansum Link MY-57, exhibited the insecticidal activity.

551 Okaramines Discovery of Okaramines A (1) andB (2)

The material from an acetone extract of okara fermented with P. simplicissimum ATCC 90288 was partitioned between ethyl acetate and water. The ethyl acetate layer thus obtained exhibited insecticidal activity. The ethyl acetate fraction was repeatedly chromatographed on silica gel with a hexane-acetone mixture, and then on alumina with a hexane-ethyl acetate mixture. Crystallization of the 50% ethyl acetate eluate and of 60-80% ethyl acetate eluates gave two active compounds, which were named okaramines A (1) and B (2), respectively [13-16]. The molecular formula of okaramine A (1) was determined to be C32H32N4O3 by HR-EIMS together with ^^C- and ^H-NMR spectra, implying nineteen degrees of unsaturation. The ^H-NMR spectrum is shown in Fig. (1). The ^^C-NMR spectrum indicated that 1 had two amido carbonyl carbons and twenty olefmic carbons, suggesting that 1 was a heptacyclic compound. The UV absorption maximum at 374 nm indicated the presence of an indole chromophore with an expanded conjugation. Precise analysis of ^H-^H COSY and HMBC spectra led to the planar structure of 1. Acetylokaramine A (3), giving a good crystalline structure for X-ray analysis, and the structure thus established is shown in Fig. (2). Okaramine A (1) was shovra to be composed of two moieties, i.e. 3a-hydroxy-A/^-(reverse-prenyl)-l,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid and 6,6-dimethyl-7//-3,6dihydroazocino[5,4-6]indole-2-carboxylic acid. These two moieties formed a diketopiperazine ring, resulting in the formation of 1.

_A_

10.0

iiiiJk^ 8.0

7.0

6.0

1 5.0

4.0

Fig. (1). 300 MHz 'H-NMR spectrum of okaramine A (1) in acetone-rf^

3.0

uu

ppm

2.0

1.0

552

3a"0R

1, okaramine A R = H 3, acetylokaramine A R = Ac

Fig. (2). ORTEP drawing of acetylokaramine A (3)

Okaramine B (2), C33H34N4O55 seemed to be an analog of 1. The ^H-NMR spectrum shown in Fig. (3) and ^^C-NMR data strongly suggested that the azocinoindole moiety in 1 existed unchanged in 2. The essential difference between the ^H-NMR spectra of 1 and 2 consisted of the absence of a vinyl group and a methine proton, and the appearance of an ethylidene group. Okaramine B (2) also contained an additional

-Ai 8.0

A-A_ 7.0

6.0

.••^•*-^O^i

•

J

ppm

5.0

4.0

Fig. (3). 270 MHz 'H-NMR spectrum of okaramine B (2) in DMS0-<4

3.0

2.0

1.0

553

hydroxyl group and a methoxyl group. To determine the connectivity of each functional group, a long-range 2D ^H-^^C COSY experiment was carried out for 2, and the results indicated that C-11 was bound to C-8a, and that an azetidine ring was newly formed. In addition, it was shown that two hydroxyl groups were located at C-2 and C-3a, and a methoxyl group at C-3. The relative stereochemistry was confirmed by the nuclear Overhauser effect difference spectra of 2, indicating the a-orientation for 2-OH, 3a-0H and H-11 and the P-orientation for 3-OCH3. The absolute configurations at C-2, C-3a and C-8a of 2 were deduced to be the same as those in 1 by comparing the CD spectrum of 2 with that of 1. Okaramine B (2) has two moieties, a hexahydropyrroloindole and a dihydroazocinoindole, and it is of particular interest that the pyrroloindole part is condensed with a newly formed azetidine ring.

'"OH

2, okaramine B

4, okaramine C

Discovery of Okaramine C (4)

The unique structures of okaramines A (1) and B (2) led us to investigate whether okaramines or similar compounds were produced by other strains. First, we examined four strains belonging to P, simplicissimum, i.e. IFO 5762, AHU 8065, AHU 8402 and MF-24, and found that strain AHU 8402 showed the highest activity [17]. From an okara medium fermented with P. simplicissimum AHU 8402, three insecticidal compounds were isolated. Two of them were identified as 1 and 2, and the third one seemed to be a new, related compound, which was thus named okaramine C (4). Okaramine C (4), C32,H36N403, proved to be a tetrahydro-derivative of 1. The ^H-NMR and ^H-^H COSY spectra indicated that 4 had an additional reverse-prenyl group, an exchangeable proton and a -CH2-CH< group instead of -CH=CH- and -CH=C<, suggesting that the C-r=C-2' double bond was saturated and

554

that the N-3'-C-4' bond was reductively cleaved [17]. This finding that P. simplicissimum AHU 8402 produces okaramines A (1), B (2) and C (4) suggests the probability for various strains to produce okaramines. Discovery of Okaramines D (5), E (6), F (7), and G (8)

Minor congeners of the okaramines were investigated from the culture extracts of R simplicissimum ATCC 90288 in order to elucidate the structure-activity relationship and also to clarify the biosynthesis of okaramines. The congeners were purified by detection using Ehrlich reagent followed by staining with vanillin-sulfuric acid to identify okaramine-related compounds. Four new okaramine congeners were isolated and termed okaramines D (5), E (6), F (7), and G (8) [18, 19]. Okaramine D (5), C33H34N4O6, contained one additional oxygen atom when compared with okaramine B (2). The ^H-NMR spectrum of 5 showed the presence of >CH-CH20H, strongly suggesting that a secondary methyl group at C-12 in 2 had been replaced in 5 by a hydroxymethyl group. AH spectral data and acetylation of 5 led to the conclusion that 5 was 12-hydroxyokaramine B [18]. Okaramine E (6), C32H32N4O4, showed IR and UV spectra quite similar to those of 5, indicating that 6 had the same functionalities and conjugated systems as 5. In the H-NMR spectrum of 6, signals assigned to a hydroxymethyl group at C-12 were observed, suggesting that the substitution on the azetidine ring in 6 was the same as that in 5. The NMR data of 6 also showed the existence of a dihydroazocinoindole moiety and a 1,2-disubstituted benzene ring. Compared with 5, the signals of two hydroxy 1 groups at C-2 and C-3a, a methine group at C-3a, and a methoxyl group at C-3 had disappeared, and signals attributable to a hydroxyl group and a -CH2-CH< linkage were newly observed, suggesting that 6 had a hydroxyl group at C-2 or C-3 a. This assumption helped complete the molecular formula for 6. The location of the hydroxyl group was concluded to be at C-3 a from a comparison of the chemical shifts of C-3b and C-8a between 5 and 6. This conclusion and the configuration of the hydroxyl group were supported by the similarity in the chemical shifts of C-2, C-3 and C-3a between 6 and 1 [18]. The molecular formula of okaramine F (7), C32H30N4O4, was determined by HR-EIMS, and was identical to the molecular formula of okaramine E (6), but with two fewer hydrogen atoms. In the ^H-NMR spectrum of 7, almost all the signals observed in 6 were found, with the exception that the signals assigned to H-2 and H2-3 in 6 were replaced by one singlet signal, indicating that the bond between C-2 and C-3 had become unsaturated. Another possibility that a newly introduced double

555

bond was located between C-3 and C-3a was ruled out by a comparison between the UV spectra of 6 and 7. Okaramines generally show UV absorption at around 380 nm, which is characteristic of the azocinoindole ring, but 7 showed an absorption maximum at 402 nm, strongly suggesting that the double bond was between C-2 and C-3 [18]. The molecular formula of okaramine G (8) was C32H34N4O3, indicating that 8 has two more hydrogen atoms than okaramine A (1) and two less than okaramine C (4). From an inspection of the ^H- and ^^C-NMR spectra of 8, 8 was found to possess a 3a-hydroxy-A^-(reverseprenyl)-!,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid moiety, a 2,3-disubstituted indole and a diketopiperazine ring. The spectra indicated an additional reverse-prenyl and another exchangeable proton in 8, both of them resulting from the reductive cleavage between N-3' and C-4'. This structure was confirmed by HMBC experiments. From the NOESY experiments, the conformation of 8 was deduced to be quite different from that of 1—^namely, 2-(reverse-prenyl)indole moiety in 8 tumed around the C-l'-C-llb' axis and a reverse-prenyl group at C-6a' was closer to a carbonyl at C-12' [19].

«»0H

5, okaramine D

6, okaramine E

lOH

7, okaramine F

lOH

8, okaramine G

556 Discovery of Okaramines H (9) and I (10)

In further screening microbes for insecticides, an isolate Aspergillus aculeatus lizuka KF-428 was obtained from a soil sample. This strain also exhibited the insecticidal activity when grown on okara, and three active principles were isolated. Two of them were identified as okaramines A (1) and B (2). The third one also seemed to be an okaramine-related compound and was named okaramine H (9) [20]. Okaramine H (9) had a molecular formula of C^2H32N403, which was identical to that of okaramine A (1). The ^H- and C-NMR spectra of 9 were very similar to those of 1, suggesting the existence of azocinoindole and pyrroloindole moieties in 9. Signals assignable to a prenyl group in 9 were observed instead of the signals assigned to a reverse-prenyl group in 1. In long-range ^H and ^^C shift-correlated 2D-NMR experiments, the signal of C-7a was correlated with H2-IO, H-6, and H-4, revealing that the prenyl group was located at C-7. The orientations of hydrogen atoms at C-2 and C-8a and of a hydroxyl group at C-3a in 9 were considered to be the same as those of 1, because of the similarity between chemical shifts and coupling constants of H-2 and H-3 with those of 1. Okaramine H (9) might be formed through an aza-Claizen type rearrangement in which a reverse-prenyl group at N-8 of 1 is transferred to C-7 via the formation of a six-membered ring [20]. An inactive okaramine-related compound was isolated from the okara culture of ^. aculeatus KF-428. This compound, okaramine I (10), had a molecular formula of C27H24N4O3. The ^H- and ^^C-NMR spectra of 10 were the same as those of depentenylokaramine A, which was formed by hydrogenolysis of 1 with Pd/C [15, 20].

"lOH

9, okaramine H

10, okaramine I

557 Discovery of Okaramines J (11), K (12), L (13), M (14), N (15), O (16), P (17), Q (18), and R (19)

Okaramines have attracted considerable attention due to their molecular complexity and intriguing biogenesis. We thoroughly searched the fermented material of R simplicissimum ATCC 90288 for new okaramine congeners, with the result that nine members of the okaramine family were isolated. Okaramine J (11) had a molecular formula of C32H36N4O3, which is identical to the molecular formula of okaramine C (4). The ^H-NMR spectrum of 11 is shown in Fig. (4). The critical differences were the absence of one of two reverse-prenyl groups that were observed in 4 and the appearance of a new prenyl group and one exchangeable proton coupled to the methine proton at C-8a. The HMBC spectrum of 11 indicated that the prenyl group was bound to C-7 in the pyrroloindole ring [21]. Okaramine K (12), C32H34N4O3, had a molecular formula identical to that of okaramine G (8) [21]. The essential difference between the ^H-NMR spectra of 8 and 12 was the absence of a reverse-prenyl group

lliUMUL '

10.0

9.0

8.0

7.0

I

a

1 ML] lU

-ppm-

•

6.0

5.0

4.0

Fig. (4). 270 MHz 'H-NMR spectrum of okaramine J (11) in acetone-rf^

3.0

2.0

1.0

558

and the appearance of a prenyl group and one exchangeable proton. The HMBC spectrum of 12 indicated that the prenyl group was bound to C-7 in the pyrroloindole ring. Okaramine K (12) had ^H- and ^"^C-NMR spectra each consisting of signal accompanied by a 1/9-fold weaker signal of the same multiplicity. This suggested that 12 occurred as a 9:1 mixture of isomers. Furthermore, two isomers were obtained in pure form by HPLC, although each pure isomer was found to rapidly convert into a mixture of the original composition. To assign the stereoisomer, we carried out NOESY experiments on 12. The correlation observed between H-N3' and H-IT from the major isomer supports the idea that the major isomer has the (Z) configuration at the C-r=C-2' bond. On the other hand, the minor isomer of 12 was shown to have the (£) configuration at the same bond. It has been reported that aplysinopsin-type indole alkaloids, which are structurally similar to echinulin, underwent photoisomerization in a solution under either UV irradiation or ordinary daylight [22]. Therefore, 12 underwent facile photoisomerization in a solution under UV irradiation to become appreciably enriched by the (E) configurational isomer (Z/E = 6/4). Interestingly, the (Z/E) ratio of the stereoisomers of 12 reverted to a mixture of the original composition in one or two days at room temperature under condition of laboratory daylight. These facts can be interpreted as indicating that (Z)-12 is more thermodynamically stable. However, 8 did not exist as a mixture of (Z/E) configuration because of the steric repulsion between H-ll' of the indole nucleus and I3-CH3, I4-CH3.

•lOH

""OH

11, okaramine J

12, okaramine K

Okaramine L (13) had a molecular formula of C32H36N4O3, which is identical to the formulae of okaramines C (4) and J (11). In the / H - N M R spectrum of 13, the signal at H-N8 that was observed in 4 had disappeared, and a new signal assigned to the benzene ring of the

559

pyrroloindole moiety was observed. Careful comparison of the ^H-NMR spectrum of 13 with that of 11 revealed that methylene protons at C-10 of the prenyl moiety were shifted upfield, suggesting that this moiety was located at N-8. This consideration was confirmed by HMBC experiments, in which correlation was observed between H2-IO and both C-7a and C-8a, and between H-8a and C-10 [21]. Okaramine M (14) had a molecular formula of C29H30N4O3. The presence of a reverse-prenyl group was established by the NMR spectra. The presence of an acetyl group was also indicated in the NMR spectra. The placement of the reverse-prenyl group at C-3a in the indoline moiety was confirmed by HMBC correlations between C-3a and each of I3-CH3, I4-CH3, and H-11. The placement of the acetyl group at N-8 was indicated by the fact that signals of H-8a and H-7 were recognized at a lower-field position than those of the corresponding protons of the other okaramines. Furthermore, ^H and ^^C long-range correlation between H-8a and an acetyl carbon was observed [21]. Okaramine N (15) had a molecular formula of C32H34N4O3, indicating that 15 had two more hydrogen atoms than okaramine A (1). The precise analysis of NMR experiments suggested that the C-r=C-2' double bond

•"OH

14, okaramine M

•••IQH

•••IQH

15, okaramine N

16, okaramine O

560

was saturated, and this assumption was supported by the fact that 15 lacked the UV absorption at 374 nm present in 1. The resuhs of the NOESY experiment indicated that the relative configurations at C-2, C-2', C-3a, and C-8a of 15 were all of cz^-type [23]. Okaramine O (16) had a molecular formula of C32H34N4O4, which was identical to the formula of 15, but with one more oxygen atom. The NMR spectra of 16 indicated the presence of an oxymethine group and a hydroxyl group, and the location of the hydroxyl group was also determined to be at C-l'. The relative configurations at C-2, C-2', C-3a, and C-8a of 16 were determined to be the same as those of 15 on the basis of the NOESY experiment. The hydroxyl group at C-T was determined to have an a-orientation on the basis of the NOESY experiment and the ^H-^H coupling constants [23]. Okaramine P (17), C32H34N4O4, had the same molecular formula as 16. The ^H-NMR spectrum of 17 is characterized by the disappearance of the reverse-prenyl signals in 16 and the appearance of a prenyl group. The HMBC spectrum indicated that the prenyl group was connected to C-7. The relative stereochemistry of 17 was the same as that of 16 at all chiral

X^-Yli

HN'"'^ lOH

'•lOH

18, okaramine Q

17, okaramine P

19, okaramine R

561

centers, as judged by the NOESY and the ^^C-NMR chemical shifts [23]. Okaramine Q (18) had a molecular formula of C32H32N4O4. The UV spectrum (Xmax 234, 288, 376 nm) indicated the presence of an indole chromophore with an expanded conjugation. The ^H-NMR spectrum of 18 resembled that of okaramine B (2), except for the absence of the methoxyl proton signal in 2 and the presence of signals due to isolated methylene protons. A precise comparison between the NMR spectra of 18 and 2 led to the conclusion that 18 is a demethoxyl derivative of 2 [23]. Okaramine R (19) appeared to possess the molecular formula of C32H32N4O4 by HREIMS, suggesting the presence of an additional oxygen atom as compared with okaramine A (1). The ^H-NMR spectrum of 19 was identical to that of 1, with the exception that 19 lacked a methine signal at C-8a, and showed a new amide proton signal. In the ^^C-NMR spectrum of 19, the signal at C-8a that was obviously observed in 1 also disappeared, and a new signal assigned to an amide carbonyl

562

carbon was observed. The ^H-NMR signals for 3a-OH and H2-3 were correlated with this carbonyl carbon signal in the HMBC experiments. These data indicated that the carbonyl must be at C-8a, forming an oxyindole moiety [23]. Possible okaramine precursors, i.e. cyclo (Trp-Trp) (20), cyclo (2-(reverse-prenyl)-Trp-Trp) (21), cyclo (A/^^-(reverse-prenyl)-Trp-Trp) (22), cyclo (2-(reverse-prenyl)-Trp-2'-(reverse-prenyl)-Trp) (23), and cyclo (A^^ -(reverse-prenyl)-Trp-2' -(reverse-prenyl)-Trp) (24), were isolated during the course of the investigation of okaramine congeners [21]. Three of these compounds, 21, 22, and 24, are new compounds. cyclo(2-(Reverse-prenyl)-Trp-2' -(reverse-preny 1)-Trp) (23) was synthesized by Schkeryantz and coworkers as a precursor of gypsetin [24], but it was isolated for the first time from natural sources in our study. One year after our findings, Kozlovsky and coworkers reported the isolation of fellutanines A, B, C, and D from Penicilliumfellutanum [25], which were found identical to the compounds 20, 22, and 23, respectively. Absolute Configuration ofOkaramines

In order to clarify the absolute configuration of okaramines, we determined the absolute stereochemistry of the above-mentioned derivatives of cyclo (Trp-Trp) (20). Acid hydrolysis of 20 gave L-tryptophan, which was identified by comparison with standard D,L-tryptophan samples by chiral HPLC analysis. Thus, the absolute configuration at C-2 was proved to be S [21]. The absolute stereochemistries of 21, 22, 23, and 24 were defined by a CD comparison with cyclo (L-Trp-L-Trp) (20). Furthermore, acid hydrolysis of 23 afforded L-tryptophan through the loss of a reverse-prenyl side chain [26, 27]. Hydrolysis of 21, 22, and 24 also gave L-tryptophan. Accordingly, these results elucidated the absolute stereochemistries of 21, 22, 23, and 24 as those depicted [21]. The stereochemistries of okaramine C (4), okaramine J (11), okaramine K (12), okaramine L (13), and okaramine M (14), including the absolute configurations, were then established [21]. The absolute configuration at C-2' of 4, 11, 12, and 13 was determined to be S by chiral HPLC analysis of the acid hydrolysate of each of these compounds. NOESY and NOE difference experiments were carried out to define the stereochemistry of 11. Because NOEs were observed between H-2 and H-2', between H-2 and H-8a, and between H-8a and 3a-0H, the absolute stereochemistry of 11 was determined. Based on NOESY and NOE difference data, the absolute stereochemistry of 11 was found to be identical to those of 4 and 13. The relative configurations at C-2, C-8a,

563

and C-3a in 12 were also determined based on the NOESY and NOE difference experiments. It was assumed that the absolute stereochemistry of 12 was the same as those of 4, 11, and 13, because all these compounds are produced by the same strain. The chiral HPLC analysis of the acid hydrolysate of okaramine M (14) revealed the presence of L-tryptophan. The absolute configuration at C-2' was determined to be S. The J(H,H) coupling observed between H-2 and H-2' of 14 is consistent v^th the cis relationship between these protons. Thus, C-2 of 14 had the S configuration. In the NOE difference spectra of 14, significant NOEs were observed between H-2 and H-2', and between H-2 and H-8a. In addition, NOE enhancement was observed for I3-CH3, I4-CH3, and H-11 upon irradiation of H-8a. Therefore, the absolute stereochemistry of 14 was determined. The absolute stereochemistries of okaramines N (15), O (16), P (17), Q (18), and R (19) were considered to be the same as those of other okaramines because of biogenetic consideration. Insecticidal Activity of Okaramines

Insecticidal activities of okaramines and their derivatives are summarized in Table 1 using the LD50 values. Acetylokaramine A (3) showed the same activity as okaramine A (1). Okaramine C (4), whose azocine ring is cleaved, was also as active as 1, suggesting that the azocine ring moiety does not play an essential role in exhibiting the activity. On the other hand, okaramine G (8), whose azocine ring is also cleaved, showed much less activity than 1. This reduction in activity seems to have been caused by the conformational change in 8. Okaramines H (9) and I (10) exhibited no activity, indicating the importance of a reverse-prenyl group at N-8. Okaramines J (11), K (12), and L (13) exhibited no activity. This Table 1. Insecticidal Activity of Okaramines against Silkworms. Compound

LD50 (^ig/g diet)

okaramine A (1) acetylokaramine A (3) okaramine B (2) okaramine C (4) okaramine D (5) okaramine E (6) okaramine F (7) okaramine G (8) okaramine H (9) okaramine I (10) okaramine J (11) okaramine K (12)

8

8 0.2 8 20 >100 >100

40 >100 >100 >100 >100

Compound

LD5o(^g/gdiet)

okaramine L (13) okaramine M (14) okaramine N (15) okaramine 0 (16) okaramine P (17) okaramine Q (18) okaramine R (19) 4',5'-dihydroxyokaramine B (25) r,2',4',5'-tetrahydroxyokarmaine B (26) derivative of okaramine B (27) derivative of okaramine B (28)

>100 >100 >100 >100 >100

8 >100

6 80 >100 >100

564

fact also strongly suggested that the reverse-prenyl group at N-8 was very important and could not be substituted by a prenyl group. Okaramines N (15) and O (16) showed no activity, indicating that the resulting conformational change of the azocine ring moiety must be one reason for the reduction in activity. The LD50 values of okaramines B (2) and D (5) were 0.2 and 20 |ig/g diet, respectively, indicating that the hydroxylation at C-12 had drastically reduced their activity. Okaramines E (6) and F (7) exhibited no activity at a dose of 100 |ig/g diet, suggesting that the functional groups in the pyrroloindole moiety play an important role in the insecticidal activity. To determine the effects of the azetidine and azocine ring moieties on the activity, chemical modification of 2 was carried out. Hydrogenation of 2 over 10% Pd/C in acetic acid provided 4',5'-dihydrookaramine B (25), r,2',4',5'-tetrahydrookaramine B (26), and two azetidine opened-ring compounds (27 and 28) [28]. Dihydrookaramine B (25) and tetrahydrookaramine B (26) had LD50 values of 6 \xg/g diet and 80 iiig/g diet, respectively, indicating that the reduction of activity was due to conformational change of the azocine ring moiety. Because two azetidine opened-ring derivatives (27 and 28) showed no activity, the azetidine ring was suggested to be the essential component responsible for the activity. The silkworm is a useful insect rather than a pest, and thus it was necessary to determine whether okaramines would also exhibit activity against harmful insects. Okaramines A (1) and B (2) were tested against various harmful insects. As a result, the most active of the 18 okaramines, compound 2 exhibited the same activity against the second instar larvae of the beet armyworm (Spodoptera exigua) as against silkworms, and thus this compound was considered to have potential use in practical applications. Biosynthetic Pathway

Biogenetic consideration of the structures of okaramines and related compounds hitherto isolated leads to the plausible biosynthetic scheme for okaramines outlined in Fig. (5). The basic framework of okaramines is derived from two molecules of L-tryptophan and two isoprene units. The sequence of events in the biosynthesis of okaramines is of crucial importance to the following discussion. According to our proposal, cyclo (L-Trp-L-Trp) (20) derived from L-tryptophan is biosynthetically considered to be an efficient precursor of okaramines. The formation of cyclo (2-(reverse-prenyl)- L-Trp-L-Trp) (21), cyclo(A^^-(reverse-prenyl)L-Trp-L-Trp) (22), and cyclo (A^^-(reverse-prenyl)-L-Trp-2'(reverse-prenyl)-L-Trp) (24) is thought to arise via prenylation of 20.

565

Okaramine C (4) is derived from 24 by intramolecular cyclization and further oxidation at C-3a (Fig. (5) part 1). Intramolecular cyclization of 4 forms a tetrahydroazocine ring, leading to okaramine N (15). Oxidation at C-l' of 15 gives okaramine O (16), which yields okaramine A (1) through dehydration between C-l' and C-2'. On the other hand, aza-Claisen rearrangement of a reverse-prenyl group in 4, 16, and 1 leads to okaramines J (11), P (17), and H (9), respectively (Fig. (5) part 2). Intramolecular cyclization of 1 forms an azetidine ring, resulting in the formation of a biosynthetically significant postulated intermediate (29). Oxidation of this intermediate leads to okaramines Q (18) and E (6). Subsequent modification of 18 leads to okaramine B (2) and okaramine L-Tryptophan I

r

II

•

H

fiV)

H

-^^NV

07'"

20

H

14

reverse- \ prenylatioi\

/

OSJSD 2 1 reverseV prenylation \

H

H

I ^1

H

24

/ cyclizatiion

I cyclization

H

/ N ^-

4

Fig. (5). Proposed biosynthetic pathway for okaramines (part 1)

22

566

D (5) successively. Okaramine D (5) could be formed from the intermediate via 6 (Fig. 5 part 3). Removal of a reverse-prenyl group from 1 leads to okaramine I (10).

=^NJP

rearrangement

'/OH

15 1 hydroxylation

H '^^^ 11 ^ desaturation

HN Hl'hfH'IOH

ur^^'^'^T ""^-L 1

rearrange -ment

Jdehydration

HN

Hl'hrH'JOH O ^ N S ^ rearrange -ment

Fig. (5). Proposed biosynthetic pathway to okaramines (part 2)

rearrangement =^N^ lOH

567

=>^N'

HI'hjqH'lOH '^ '^ elimination H^O >

H\'hH'IOH

Postulated intermediate v hydroxylation ^ .

29

HOH2C-''-I;HJ'»OH

4-CT OCH3

0CH3

N^O hydroxylation •

2 Fig. (5). Proposed biosynthetic pathway to okaramines (part 3)

Synthetic Study of Okaramines N (15) and J (11)

In 2003, Corey and coworkers described a remarkably simple synthesis of okaramine N (15) that took advantage of the new and

568

powerful Pd-promoted construction of the tetrahydroazocinoindole subsystem (Fig. (6)) [29]. (5)-iV-Boc-tryptophan methyl ester (30) was converted to the known indoline (31). Introduction of a reverse-prenyl group and oxidation furnished 32. The removal of the Boc-protecting group from 32 and saponification gave an amino acid. Schotten-Baumann acylation of the amino acid with FmocCl afforded H COOMe

a) NaBH4CN, AcOH b) i) CuCl, z-PrjNEt, 2-acetoxy-2-methyl-3-butyne ii) DDQ iii) H2, Pd/C, quinoline c) i) SOClj ii) LiOH iii) FmocCl d) 3-methyl-2-butenal,NaBH4 e) /-Pr2NEt, bis(2-oxo 3-oxazolidyl)phosphinic chloride f) Pd(0Ac)2 g) EtjNH h) A^-methyltriazolidinedione Fig. (6). Synthesis of okaramine N (15) by Corey [29]

569

reverse-prenylated indole (33). A^-Alkyl tryptophan methyl ester (34) was acylated with 33 to afford a tetracyclic intermediate (35). Treatment of 35 with Pd(0Ac)2 provided tetrahydroazocinoindole (36). Exposure of 36 to excess diethylamine in THF resulted in Fmoc cleavage and cyclization to furnish diketopiperazine (37). The bisindole (37) underwent highly

Anth = 9-anthracenyl a) i) AnthSOjCl, Et3N ii) tert-buty\ isourea b) i) NBS, EtjN ii) 3,3-dimethyldioxirane iii) NaBH c)l,l-dimethylpropargyl bromide, CuCl,/-Pr2NEt d)H2,Pd/Al203 e)TFA f) TMSOTf, 2,6-lutidine g) PyBop, Et3N h) Al/Hg i) i) KOHMeOH ii) HBTU, /-Pr^NEt Fig. (7). Synthesis of okaramine J (11) by Ganesan [30]

570

selective reaction with the commercially available "ene" reaction reagent A^-methyltriazolinedione to form the ene product at C-3 of the A^-unsubstituted indole subunit. Subsequent photooxidation followed by reduction of the resulting product afforded the hydroxylated octacycle (38). Finally, thermolysis of 38 furnished okaramine N (15). Total synthesis of okaramine J (11) was achieved by Ganesan and coworkers in 2003 [30]. A key reaction in their synthesis was the acid-catalyzed, room-temperature, aza-Claisen rearrangement of an A^-reverse-prenylated hex^ydro[2,3-6]pyrroloindole to a C-prenylated derivative (Fig. (7)). Hexahydro[2,3-6]pyrroloindole (40) was obtained by oxidative Witkop cyclizatipn of L-tryptophan tert-hutyl ester. The alkylation of 40 afforded alkyne (41). The resulting alkyne (41) was hydrogenated to afford alkene (42). Treatment of 42 with TFA produced rearranged 43, indicating that this transformation was a charge-accelerated, aza-Claisen rearrangement. Removal of the /^r/-butyl ester provided acid (44). The indole C-2 reverse-prenylated derivative (45) was made in four steps from L-tryptophan according to the procedure described for the total synthesis of gypsetin [24]. Coupling 44 and 45 afforded pentacycle (46). Reductive removal of the anthracenylsulfonamide protecting group afforded 47. The methyl ester was hydrolyzed to the free amino acid, which underwent cyclization under peptide-coupling conditions to give okaramine J (11). Okaramine-Related Compounds

Okaramine A (1) is a novel heptacyclic compound containing a hexahydropyrroloindole moiety and a dihydroazocinoindole moiety. The azocinoindole moiety has been reported to constitute only two compounds: a metabolite (48) of Aspergillus ustus [31] and cycloechinulin (49) produced by ^. ochraceus [32] (Fig. (8)). One of the structural characteristics of the okaramine family is the presence of a reverse-prenylated hexahydro[2,3-6]pyrroloindole moiety. Some related

H3C0

48 Fig. (8). Compounds with azocinoindole moiety

49, cycloechinuHn

571

compounds are shown in Fig. (9). Brevianamide E (50) was isolated from the culture medium of Penicillium brevicompactum by Birch and Wright in 1970 [33]. Amauromine (51) was isolated as a vasodilator from the culture broth of Amauroascus sp. No. 6237 by Takase and coworkers in 1984 [34, 35]. A family of new compounds, including ardeemin (52), iV^-acetylardeemin (53), and 15b-hydroxy-A^^-acetylardeemin (54), were isolated from the fermentation broth and mycelia of a strain of Aspergillus Jischeri var. brasiliensis by Karwowski and coworkers in 1993 [36. 37]. A^^-Acetylardeemin (53) potentiates the cytotoxicity of the anticancer agent vinblastine in multi-drug resistant human tumor cells [36]. In 1994, Shinohara and coworkers isolated gypsetin (55) as an inhibitor of acyl-CoA:cholesterol acyltransferase from the cultured broth of Nannizzia gypsea var. incurvata IFO 9229 [38, 39]. In 2000, Kozlovsky and coworkers reported the isolation of fellutanine D (56) from the cultures of Penicillium fellutanum; and since then it has been reported cytotoxic [25].

OH

H

50, brevianamide E

52, ardeemin R = H 53, A^'^-acetylardeemin R = Ac OH H I

51, amauromine

54, 15b-hydroxy-A^acetylardeemin

^^H

H i s ^ 5 H OH

55, gypsetin

56, fellutanine D

Fig. (9). Compounds with hexahydro[2,3-^]pyrroloindole moiety

572

Each of the tryptophan metaboUtes shown in Fig. (10) has prenyl groups, reverse-prenyl groups, and a diketopiperazine ring. EchinuHn (57), which contains a tryptophan moiety, was isolated by Birch and Farrar in 1963 [40]. Neoechinulin (58), which contains a dehydrotryptophan moiety, was also isolated as a pigment from the same molds that produced 57 by Barbeta and coworkers in 1969 [41]. Neoechinulins A (59), B (60), and C (61) were isolated as ivory crystals, yellow crystals, and yellow crystals, respectively, from sugar beet molasses cultures of Aspergillus amstelodami by Dossena and coworkers in 1974 [42]. Neoechinulins D (62) and E (63) were also isolated from the neoechinulins A-, B-, and C-producing strains by Marchelli and coworkers in 1977 [43]. Cryptoechinulin A, which is identical to compound 61, was isolated in small amounts from cultures of A. amstelodami along with a large quantity of 57 by Cardillo and coworkers in 1974 [44], and cryptoechinulin G (64) was isolated from the same strain by Gatti in 1978 [45]. In 1976, Nagase and coworkers isolated isoechinulins A (65), B (66), and C (67) from the course of their search

59, neoechinulin A

60, neoechinulin B H

61, cryptoechinuUn A neoechinulin C Fig. (10). Structures of echinulin family (part 1)

62, neoechinuUn D

573

for indole metabolites in the mycelia oi Aspergillus rubber [46]. In 1999, Fujimoto and coworkers reported the isolation of tardioxopiperazines A (68) and B (69) as immunomodulatory constituents from an Ascomycete Microascus tardifaciens [47]. It is noteworthy that all compounds shown in Fig. 10 have a reverse-prenyl group at C-2 in the indole ring.

63, neoechinulin E

65, isoechinulin A

67, isoechinulin C

64, cryptoechinuline G

66, isoechinulin B

68, tardioxopiperazine A

69, tardioxopiperazine B Fig. (10). structures of echinulin family (part 2)

574

Communesins Identification of Communesins A (70) andB (71) and Discovery of Communesins D (72), E (75), and F (74)

The acetone extract of okara fermented with Penicillium expansum Link MK-57 was found to exhibit the insecticidal activity against silkworms. The acetone extract of okara fermented with this strain was purified by solvent extractions, column chromatography, HPLC, and crystallization to yield five active compounds—i.e., two known compounds, communesins A (70) and B (71), and three new ones, communesins D (72), E (73), and F (74) [48]. The structures of the known compounds, 70 and 71, were assigned by comparing their physicochemical properties and spectral data with those reported in the literature [49].

70, communesin A

71, communesin B

Communesin D (72) was obtained as colorless needles and gave a protonated molecular ion [M+H]^ at m/z 523.2687 by HRFABMS, consistent with the molecular formula of C32H34N4O3. The UV spectrum showed an absorption maximum at 266 nm, suggesting that 72 had the same chromophore as 71. The ^H- and ^^C-NMR data are similar to those for 71, indicative of the presence of a 1,2-disubstituted benzene and a 1,2,3-trisubstituted benzene ring moieties. The NMR data also strongly suggested that 72 had the same carbon skeleton—including the seven-ring system—as 71. Communesin D (72) was also found to have a (2£',4£)-2,4-hexadienoyl moiety by the ^H-NMR signals. The methyl signal at N-15 in 71 was not observed in 72, and a new signal assignable to an aldehyde proton was observed at 5H 8.91 (IH, d, J = 0.5). This fact, together with the difference in molecular formula between 71 and 72, suggested that the methyl group in 71 was substituted by a formyl group in 72. Key HMBC correlations between H-l' and C-6, and between H-5 and each of C-6, C-4, and C-8a, clearly established the location of this formyl group as N-15 and allowed the planar structure of 72 to be fully assigned [48]. Communesin E (73) had a molecular formula of C27H30N4O2, as

575

determined by HRFABMS and NMR data, suggesting that 73 was a demethyl compound of 70. The ^H-NMR spectra of 73 and 70 were nearly superimposable, but 73 lacked the signal for an A^-methyl observed in 70, indicating that 73 was an A^-15 demethyl derivative of 70. In addition, the presence of an acetyl group at N-16 and a 2-methyl-l,2-epoxypropyl moiety at C-11 was also suggested by the ^H-NMR data. Consequently, 73 was elucidated to be the A/^^^-demethylconmiunesin A [48]. Communesin F (74) was found to have the molecular formula of C28H31N4O from the HRFABMS and NMR data. The ^H-NMR (Fig. (11)) and ^^C-NMR spectra of 74 differed from those of 70 only by the absence of the epoxyl group and the appearance of a double bond, consistent with the difference in molecular formula between 74 and 70. The whole structure of 74, including the heptacyclic skeleton, an acetyl group at N-16 and a methyl group at N-15, was determined by a precise analysis of the ^H-^H COSY, HSQC, HMBC, and NOESY spectra of 74 [48]. The relative stereochemistry of 72, 73, and 74 was deduced to be the same as that of 70 and 71 at all chiral centers on the basis of the close similarity of the spectral parameters, especially the ^^C-NMR chemical shifts, with the corresponding values for 70 and 71.

H

N £ N H H H

H..I ^'CHO

72, communesin D

73, communesin E

74, communesin F

576

[L^_JLJLJL^ 7.0

6.0

5.0

k

i k JJALM 4.0

3.0

mil

pprrr

2.0

Fig. (11). 400 MHz iH-NMR spectrum of communesin F (74) in CDCI3

The insecticidal activity of communesins A (70), B (71), D (72), E (73), and F (74) against third instar larvae of silkworms was examined by an oral administration. The LD50 values for 71 and 74 were 5 and 80 |Lig/g of diet, respectively. Communesins A (70), D (72), and E (73) exhibited lower insecticidal activity than did 71 and 74, with the LD50 values for 70, 72, and 73 being 130, 130, and 200 |ig/g of diet, respectively.

Biosynthetic Pathway of Communesins

May and coworkers proposed the plausible biosynthetic root to communesin A (70) shown in Fig. (12) (part 1) [50]. A quinone methide (75) derived from tryptamine and the related natural product, aurantioclavine (76), undergo a Diels-Alder reaction to form a polycyclic intermediate (77). This highly twisted lactam (77) should be easily cleaved by the residual primary amine to produce spiro lactam (78). Reduction of 78 and aminal closure afford a common intermediate (79) of communesins. Epoxidation and acylation of 79 afford 70. Expansion

577

HOOC

Tryptamine

^

+

R 76 R = H (aurantioclavine) Quinone methide R = Me imine H ^ ^^

NH epoxidation H and I acylation

79 Fig. (12). Plausible biosynthetic pathway to communesins (part 1) [50]

of this pathway suggests the pathway to communesins B (71), D (72), E (73), and F (74) (Fig. (12) part 2.). Acylation of 79 affords 74. On the other hand, epoxidation and acylation afford 71. Elimination of a methyl group at N-15 of 70 generates 73, while oxidation of a methyl group at N-15 in 71 generates 72. Communesin-Related Compounds

Communesins A (70) and B (71) were originally isolated from the mycelia of a strain of Penicillium sp. adhering to the marine alga, Enteromorpha intestinalis, and reported to exhibit cytotoxic activity in the P-388 lymphocytic leukemia test system in cell cultures [49]. The ED50 values for 70 and 71 are reported to be 3.5 and 0.45 |ig/ml, respectively, in the test system.

578

NH

NH

I ^

1

epoxidation

^

acylation

79

acylation

70 jdemethylation

Fig. (12). Plausible biosynthetic pathway to communesins (part 2)

Communesin-related compounds are shown in Fig. (13). Jadulco and coworkers quite recently isolated communesins B (71), C (80), and D (72) from the fungus Penicillium sp. derived from the Mediterranean sponge Axinella verrucosa [51]. These three communesins have been shown to exhibit moderate antiproHferative activity in several bioassays performed on different leukemia cell lines. Nomofungin (81), which had a pyran oxygen instead of an NH in 71, was isolated from the fermentation broth of an unidentified endophytic fungus obtained from the bark of Ficus microcarpa [52]. Later synthetic studies of nomofungin revealed that this compound was identical to 71 [53, 54]. Perophoramidine (82) was isolated from the Philippine ascidian Perophora namei [55]. Perophoramidine is a hexacyclic substructure of

579

N = N H H H

80, communesin C

81, nomoflingin

82, perophoramidine Fig. (13). Communesin-related compounds

the core heptacyclic ring system of commimesins; it exhibits cytotoxicity toward the HCT116 colon carcinoma cell line and induces apoptosis via PARP cleavage. CONVULSIVE COMPOUNDS Verruculogen Identification of Verruculogen (83)

During the course of searching for okaramine-related compounds OH O z CHI

83, verruculogen

580

produced by strains belonging to P. simplicissimum, we observed interesting convulsive activity against silkworms in a strain of P. simplicissimum MF-24. The purification guided by the convulsive effect on silkworms led to the isolation of an active principle. The active principle, C27H33N3O7, was identified as verruculogen (83) [56]. Verruculogen (83) was originally isolated from the culture of P. verruculosum as an agent responsible for the tremor producer activity in mice or 1-day old cockerels [57]. Verruculogen (83) caused convulsive activity in the silkworms at a dose of 0.1 |Lig/g diet. Verruculogen-Related Compounds OH

O OH O r OH I

°^oV 84, acetoxyverruculogen 85, fumitremorgin B o

o

o

r OH

^ . H3C0

HJ

H*I

II H

86, fumitremorgin A OH O r OHl

88, 12,13-dihydroxyfumitremorgin C Fig. (14). Verruculogen-related compounds

87, fumitremorgin C o

91, demethoxyfumitremorgin C

581

Verruculogen-related compounds are summarized in Fig. (14). In 1982, Uramoto and coworkers reported the isolation and structural elucidation of acetoxyverruculogen (84) from P. verruculosum as a tremorgenic metabolite [58]. In 1974, Yamazaki and coworkers reported the planar structure of fumitremorgin B (85) [59], which had been isolated as one of two toxins (fumitremorgins A and B) from Aspergillus fumigatus, growing on rice and miso (soybean paste) [60]. The structures of fuitremorgins A (86) and B (85) were determined in 1980 [61, 62]. These two compounds cause severe tremors and convulsion in experimental animals. Fumitremorgin C (87), the simplest member of the fumitremorgin family, was isolated from A. fumigatus by Cole and coworkers in 1977 [63]. Hermkens' group reported the total synthesis of 87 in 1988 [64], and Hino's group also reported the synthesis of 87 in 1989 [65]. Abraham and Argmann described the isolation of 12,13-dihydroxyfumitremorgin C (88) from A. fumigatus DSM 790 [66]. In 1995, Cui and coworkers reported the isolation of trypanostatins A (89) and B (90) from a marine fungal strain of .4. fumigatus BM939 [67]. Trypanostatins completely inhibit the cell-cycle progression of tsFT210 cells in the G2/M phase [68]. Cui and coworkers also isolated demethoxyfumitremorgin C (91), 83, 85, 87, and 88, showing the co-occurrence of these compounds, and 89 and 90 in the secondary metabolite of the strain BM939 [69, 70]. These findings suggested the

H3CO

89, tryprostatin A R = OCH3 90, tryprostatin B R = H

94, cyclotryprostatin C Fig. (15). Tryprostatin-related compounds

92, cyclotryprostatin A R = H 93, cyclotryprostatin B R = CH3 o o

95, cyclotryprostatin D

582

possible intermediacy of 91 in the biogenesis of the verruculogen and fumitremorgins. In 1997, Cui and coworkers also isolated cyclotryprostatins A (92), B (93), C (94), and D (95) as new inhibitors of the mammalian cell cycle from the same strain, A. fumigatus BM939 [71]. The structures of the tryprostatin family are shown in Fig. (15). Penitrem A and 6-Bromopenitrem E Identification of Penitrem A (96) and Discovery of 6 Bromopenitrem E (98)

As mentioned earlier, R simplicissimum ATCC 90288 produced insecticidal okaramines. Moreover, this strain induced the same effect on the silkworms as verruculogen (83). The acetone extract obtained from the mycelia and media of this strain was concentrated and the aqueous residue was extracted with dichloromethane. The dichloromethane extract was partitioned between hexane and methanol, containing 10% water. The activity was found only in the lower layer. The active ethyl acetate extract obtained from the lower layer was chromatographed on silica gel with a hexane-ethyl acetate mixture. The 40--60% ethyl acetate eluates were rechromatographed on silica gel with a hexane-chloroform mixture. HPLC of the active 90^-100% chloroform eluates on a Capcell pack Ci8 column, using 65.7% aqueous methanol with a flow rate of 1.0 ml/min, yielded two active compounds, AC 1 and AC 2. The convulsive principle AC 1, C37H44CINO6, was determined to be penitrem A (96) by means of the spectral data (MS, UV, IR, ^H-, and ^C-NMR) [72], which were indistinguishable from those reported previously for 96 [73]. The convulsive principle AC 2, C37H44BrN06, showed spectroscopic characteristics quite similar to those of 96. The only difference between the two principles was that AC 2 had a bromine atom in the place of the chlorine atom of 96. The ^H-NMR spectrum of AC 2 is shown in Fig. (16). In the ^"^C-NMR spectrum of 96, signals assignable to C-6 and C-7

\/^^^X H

96, penitrem A

H

98, 6-bromopenitrem E

583

1 T—I—f—t

10.0

nM, 1

t "I—!—r—<—I—"n—r—r—»—r—\—r—i—t—i—j—*-

9.0

8.0

7.0

6.0

IAIIUUUI

All A.

ai«-'-f'-«''-»"«*|Wi

5.0

4.0

3.0

.

PPm

2.0

1.0

Fig. (16). 500 MHz ^H-NMR spectrum of 6-bromopenitrem E (98) in acetone-^ig

were observed at 124.5 and 111.8 ppm, respectively. Penitrem E (97) [73], v^hich had no chlorine atom at C-6, showed C-6 and C-7 signals at 120.3 and 111.6 ppm, respectively. AC 2 showed signals corresponding to those at 114.7 and 115.0 ppm. If a bromine atom was at C-7, signals of a quaternary carbon and a methine carbon should have been observed in a higher field than 111.6 ppm and in a lower field than 120.3 ppm, respectively. Therefore, it was deduced that a bromine atom was located at C-6 of AC 2, indicating that AC 2 is 6-bromopenitrem E (98), a new congener of the penitrems [74]. Penitrem A (96) and 6-bromopenitrem E (98) showed convulsive activity against silkworms at a dose of 0.3 |Lig/g diet. Penitrems and a Related Compound

In 1983, de Jesus and coworkers isolated tremorogenic mycotoxins named penitrems A (96), B (99), C (100), D (101), E (97), and F (102) from Penicillium cructosum [73, 75]. Penitrems and related compounds are shown in Fig. (17). Penitrems are indole alkaloids combined with

584

H../Nf.-V

H

H

99,penitremB R = H 102, penitrem F R = C1

97, penitrem E

%/^-M^

Hx.ASf-V

H

H

100, penitrem C R = CI 101, penitrem D R = H

H

H

103, pennigritrem

Fig. (17). Structures of penitrems and pennigritrem

terpenoid moieties. Penitrems A (96), C (100), and F (102) are chlorinated at a benzene ring, de Jesus and coworkers also investigated the biosynthesis of 96 both with ^^C- and '^H-labeled precursors and showed that 96 is derived from tryptophan, geranylgeranylpyrophosphate, and two isopentenylpyrophosphate units [76]. In 1992, Penn and coworkers isolated an analog of 96, pennigritrem (103), from Penicillium nigricans [77]. Pennigritrem (103) is a fungal indole-diterpenoid, but 103 has an oxetane ring, like taxol [78] and cephalomannine [79], unique among the members of penitrem family. Brasiliamides Discovery of Brasiliamides A (104), B (105), C (106), D (107), andE (108)

The convulsive effect induced by penitrem A (96), 6-bromopenitrem E (98), or verruculogen (83) in silkworms suggested that this bioassay using silkworms may be valuable as an initial screening in the search for compounds that act on the nervous system. Further random screening using this bioassay resulted in the finding that the isolate Penicilium

585

brasilianum Batista JV-379 exhibited remarkable convulsive activity [80]. The strain P. brasilianum JV-379 was cultured with okara. After cultivation for two weeks, the cultured okara was soaked in methanol. The methanol extract was concentrated and the aqueous residue was extracted with hexane and ethyl acetate successively. The active ethyl acetate extract was chromatographed on silica gel with a solvent system (hexane-ethyl acetate-methanol). The 100% ethyl acetate and 5% methanol eluates were combined and chromatographed fiirther to afford brasiliamides A (104) and C (106). The 70% ethyl acetate eluate was purified by column chromatography to afford brasiliamide B (105). The 10% methanol eluate was rechromatographed on silica gel and fiirther flash-chromatographed on Chromatorex ODS to yield brasiliamides D (107) and E (108).

r—I—I

I

10.0

I

I

I—I—I

9.0

1 I

I—I

1 '

8.0

ii I

'

I

'—'

7.0

'

'

I

'

6.0

'

'

• '

5.0

I

UIL •

'

'

4.0

Fig. (18). 270 MHz iH-NMR spectrum of brasiliamide A (104) in CDCI3

'

I

3.0

2.0

ppm

1.0

586

The molecular formula of brasiliamide A (104) was detemiined to be C24H26N2O6 from the HR-EIMS and NMR data, indicative of thirteen degrees of unsaturation. The IR spectrum revealed the presence of aromatic rings, a ketone carbonyl and amide groups. The ^H-NMR (Fig. (18)), ^"^C-NMR, and 2D NMR spectra indicated the presence of a phenyl group, a 3-methoxy-4,5-methylenedioxyphenyl group, two acetoamides, and a trisubstituted double bond. The presence of three isolated methylenes was also suggested from the NMR data. An olefmic proton and an amide proton were mutually coupled, indicative of the linkage between a trisubstituted double bond and NH in the acetamide group. The ketone carbonyl should form -CH2-CO-CH2- comprising two isolated methylenes. The remaining methylene protons were correlated to three non-oxygenated carbons in the 3-methoxy-4,5-methylenedioxyphenyl group, suggesting that 104 had a 3-methoxy4,5-methylenedioxybenzyl moiety. The methylene protons were also correlated to olefinic carbons, indicative of the linkage between the 3-methoxy-4,5-methylenedioxybenzyl group and a quatemary carbon in the >C=CH-NH-C0CH3 moiety. The signals for an isolated methylene showed cross peaks with carbons in the phenyl group together with a ketone carbonyl, indicating the presence of a ^-CH2-CO-CH2-. The geometry of the double bond (C-8=C-r) was confirmed as Z-configuration by the NOE correlation between an olefinic proton and aromatic protons (H-2 and H-6). Consequently, the structure of 104 was elucidated to be N^ ,A^^-diacetyl-A^'^-(2-oxo-3 -phenylpropyl)-3 -(3 methoxy-4,5-methylenedioxyphenyl)-1,2-(Z)-propenediamine [80]. Ov^7^8 7

r

1,1

?^

OCH3

OCH3

,,Ao 104, brasiliamide A

105, brasiliamide B

Brasiliamide B (105) had a molecular formula of C24H26N2O5, indicative of thirteen degrees of unsaturation as in the case of brasiliamide A (104). In the ^H-NMR spectrum of 105, the signals were complicated, with almost all being doubled or broadened in CDCI3 at 20^C. This phenomenon was observed in various deuterated solvents, e.g., acetone-J6, C6D6, CD3OD, and DMSO-c/6. These observations suggested that conformational isomers of 105 were present in the solutions.

587

lUl 9.0

8.0

7.0

IL

jLxjiA^ljuAnjJ\^

ppm

6.0

5.0

4.0

3.0

2.0

1.0

Fig. (19). 270 MHz ^H-NMR spectrum of brasiliamide B (105) in acetone-J^ at -60 °C

Lowering the temperature to -60^C in CDCI3 sharpened all signals, and some signals derived from minor conformers were observed as shown in Fig. (19). The alteration of an acetyl methyl region was particularly remarkable, and four pairs of acetyl methyl signals were observed in the spectrum. The ratio of these signals was approximately 76:15:7:2, suggesting that 105 existed in equilibrium with four conformational isomers in solution. We therefore tried to elucidate the structure of 105 with a major conformer in CDCI3. The ^H-NMR, ^^C-NMR, and ^H-^H COSY spectra revealed the presence of the same partial structures as in 104, a phenyl group, a 3-methoxy-4,5-methylenedioxyphenyl group, a trisubstituted double bond, and two acetoamides. The spectra also indicated the presence of a -CH2-CH-CH2- moiety. These partial structures were connected from the COLOC spectrum, and the whole structure of 105 was determined to be l,4-diacetyl-2-benzyl-5-(3-methoxy-4,5-methylenedioxybenzyl)-

588

Fig. (20). ORTEP drawing of dihydrobrasiliamide B (109)

be l,4-diacetyl-2-benzyl-5-(3-methoxy-4,5-methylenedioxybenzyl)1,2,3,4-tetrahydropyra-zine. This structure was completely supported by the following X-ray crystallographic data. Hydrogenation of 105 with 5% Pd/C afforded reductive products. The major product was purified by reversed-phase ODS column chromatography to give a dihydroderivative, C24H28N2O5. Crystallization of dihydrobrasiliamide B (109) from methanol yielded rhombic prisms that were suitable for an X-ray crystallographic analysis. The ORTEP drawing of 109 shown in Fig. (20) established the complete structure of 109 as rrara-l,4-diacetyl2-benzyl-5-(3-methoxy-4,5-methylenedioxybenzyl)piperazine [80]. Brasiliamide C (106), C94H26N2O5. had the same molecular formula as brasiliamide B (105). The %-NMR, ^^C-NMR, and ^H-^H COSY spectra indicated the presence of a 3-methoxy-4,5-methylenedioxyphenyl group, a phenyl group, two acetamide groups, a trisubstituted double bond, an isolated methylene and a -CH2-CH-CH2- linkage. The HMBC spectrum indicated that the phenyl group was bound to the double bond and that the 3-methoxy-4,5-methylenedioxyphenyl group was located at C-l". The geometry of the double bond in the benzylidene moiety was determined to be an E-configuration, based on the NOE correlation between the acetyl methyl (8-CH3) and the olefinic proton (H-T) [81]. The molecular formula of brasiliamide D (107) was determined to be C24H28N2O5 from the HR-EIMS and NMR data, indicative of twelve degrees of unsaturation. Precise analysis of the NMR data revealed that 107 had the partial structures of a benzyl group, a 3-methoxy-4,5methylenedioxybenzyl group, two acetoamides, and a disubstituted piperazine ring. These partial structures were connected from the HMBC spectrum. The planar structure was determined to be the same as that of dihydrobrasiliamide B (109), but the spectral data for 107 were completely different from those of 109. The piperazine ring conformation of 107 was not a chair-form but rather a twist boat-form conformation based on the coupling constants between the methine and methylene protons (J2,3a = 6.4 Hz, J2,3b = 11.3 Hz and J5,6a = 6.4 Hz, J5,6b =11.3 Hz) in the piperazine ring, while that of 109 was a typical chair-form conformation. These observations suggested that both methine protons

589 (H-2 and H-5) were pseudo-axial in configuration and that the relative configuration of these protons was 2,5'Cis [81]. The molecular formula of brasiliamide E (108) was determined to be C22H26N2O4. The ^^C-NMR spectrum closely resembled to that of 107, except for the absence of one acetyl group, strongly suggesting that 108 was a deacetyl derivative of 107. The position of an acetyl group was inferred from the HMBC spectrum to be at N-1 [81].

,0

OCH3

109, dihydroxybrasiliamide B (2,5-trans)

106, brasiliamide C

V OCH3

OCH3

108, brasiUamide E i2,5-cis)

107, brasiUamide D (2,5-cis)

Conformational Analysis

ofBrasiliamides

^V

^^ ^^

Y^

x"D Xy Xy Xi - ^ O

^

El form

Zl form ^

Ratio

42%

O

O

^

O

72 form . 35%

^

E2 form ' 23%

Fig. (21). Rotaitional properties of/raA25-l,4-diacetyl-2,5-dimethylpiperazine (112)

590 brasiliamide B (105) (at -60 °C in CHCI3) Oy-

\^0

O ^

^ °

'"Xl ''^\ ^a\ ''XK Ao

Ao

o^

°i

76% 15% 7% dihydroxybrasiliamide B (109) (at room temperature in CHCI3)

ph-^'Y"^

p'^^"r%

p^^"r^

''^"T'I ^-J^^

^N^R

^ N ^

^N^R

A

Ao

o^

°^

35%

18%

''4%

33%

V

Y°

brasiliamide C (106) (at -15 °C in CHCI3)

V

v°

Ph

"tx -rx -XX -xx 1%

330/^

2%

64%

brasiliamide D (107) (at -15 °C in CHCI3)

V 51%

V

°v

^°

10% '''"

° 7%

22%

Fig. (22). Rotaitiona] properties of brasiliamides (R = 3-methoxy-4,5-methylenedioxybenzyl)

Brasiliamides B (105), C (106), D (107), and E (108) showed conformational change due to the restricted rotation of amide bonds in a solution, and four or two sets of conformers appeared in the NMR spectra. To clarify their conformational properties, the conformational change

591

was first examined using a model compound, transA,4' diacetyl-2,5-dimethylpiperazine (110). Three sets of signals due to each rotamer were completely assigned, and the direction of the acetamide group on each rotamer was determined from differential NOE measurements. In these experiments, the chemical shifts of the ring protons were markedly changed due to the direction of the carbonyl group in acetamides. When the carbonyl group in the acetamide (N-1) is directed to the C-2 side, the neighboring methine proton (H-2) is deshielded. On the other hand, when the carbonyl group is directed to the C-6 side, the equatorial proton of methylene (H2-6) is deshielded. The ratio of three rotamers, El, Zl (Z2) and E2, was evaluated to be approximately 42:35:23, respectively, on the basis of integral values of the ring methyls (Fig. 21). In the case of 105, 106, 107, 108, and 109, the same method was used to analyze conformational changes. The results are shown in Fig. (22). Bioactivity ofBrasiliamides

The convulsive activity of brasiliamides against the third instar larvae of silkworm was also examined. The activity of brasiliamides A (104) and B (105) was evaluated according to the ED50 values of 300 and 50 |ig/g diet, respectively, upon oral administration. The functional moiety for the activity was expected to be a 3-methoxy4,5-methylenedioxybenzyl moiety, since natural compounds having a 3-methoxy-4,5methylenedioxybenzyl group, such as myristicin [82], have been reported as insecticides. Dihydrobrasiliamide B (109), brasiliamide C (106) and brasiliamide D (107) showed weaker activity than brasiliamide B (105), indicating that a double bond in the piperazine ring was more significant than a 3-methoxy-4,5-methylenedioxybenzyl group. Brasiliamide E (108), which is equivalent to deacetoxybrasiliamide D, showed no activity, suggesting that the acetyl group is essential to exhibit the activity. Biosynthesis of Brasiliamides

Brasiliamides are comprised of two phenylpropane moieties and acetates. The plausible biosynthetic pathway of brasiliamides starting with the formation of diketopiperazine ring is outlined in Fig. (23). Phenylalanine and 3-methoxy-4,5-methylenedioxyphenylalanine are combined into a diketopiperazine (HI). Reduction of ketones, dehydration reaction, and rearrangement of the double bond lead to intermediates 112 and 113. Acetylation of the two compounds 112 and 113 leads to brasiliamide C (106) and brasiliamide B (105), respectively. The two compounds 112

592

and 113 give an intermediate (114) after reduction of each double bond. Acetylation of 114 leads to brasiliamide E (108) and brasiliamide D (107) successively. On the other hand, oxidative cleavage of the tetrahydropiperazine ring in 105 affords 104.

\

^

^

Ao

^ N ^ ' '

J113 ^ 113

112 \ \ 106

^N' '

^ N ^ ' '

^

H

^

'N' H

"'/^

I

114

A°

R

105

^

Ph

y"..M^

Ph

t..x.

HN Ph'

104

H

I 108

Ph

V •^°

107

Fig. (23). Proposed biosynthetic pathway to brasiliamides (R = 3-methoxy-4,5-methyIenedioxyphenyl) The configuration shown is relative one.

593 Brasiliamide-Related Compounds

Piperazine-containing compounds are shown in Fig. (24). In 1969, Caesar and coworkers reported the isolation of nigragillin (115) from Aspergillus phoenicis [83]. Piperazinomycin (116) was isolated as an antifungal antibiotic from the cultured broth of Streptoverticillium olivoreticuli subsp. neoenacticus by Tamai and coworkers in 1982 [84, 85]. Piperazinomycin (116) has a unique cyclic structure in which two benzene rings and one piperazine ring are linked together by three

115, nigragillin

116, piperazinomycin o

118, nigerazine B

117, nigerazine A

i^^yy^ 119, dragmacidon

120, dragmacidon A H

121, dragmacidon B Fig. (24) Compounds with piperazine ring

122,2,5-bis(6'-bromo-3'-indolyl)piperazine

594

separate atoms (one oxygen atom and two carbon atoms). Iwamoto and coworkers isolated nigerazines A (117) and B (118) as new metabolites positive to Dragendorff's reagent from Aspergillus niger 1-639 [86, 87]. Nigerazines reduce the root elongation of lettuce seedlings. A number of cytotoxic bis-indole alkaloids have been discovered in marine organisms. Dragmacidon (119) was isolated from deep water Caribbean sponge, Dragmacidon sp., by Kohmoto and coworkers in 1988 [88]. Dragmacidons A (120) and B (121) were isolated from the Pacific Ocean sponge Hexadella sp., collected off the coast of British Columbia by Morris and coworkers in 1990 [89]. Dragmacidon A (120) shows cytotoxic activity, while 121 shows no activity. In 1991 Fahy and coworkers isolated 2,5-bis(6'-bromo-3'-indolyl)piperazine (122) from the encrusting grey tunicate Didemnum candidum collected in the southem Gulf of Califomia [90]. PARALYTIC COMPOUNDS Asperparalines Discovery ofAsperparalines A (123), B (124), and C (125)

The hint, which led us to this finding, was the accidental observation of paralytic syndrome in silkworms that had digested an extract of a certain isolate. We had been screening numerous soil isolates for their insecticidal activity against silkworms upon oral administration in the usual manner. It was of great interest that the strain Aspergillus japonicus Saito JV-23 resulted in paralysis in silkworms. The isolate A. japonicus JV-23 was also cultured on okara as in other experiments mentioned earlier. The fermented okara together with mycelia was soaked in methanol. The methanol extract was extracted with dichloromethane after removal of methanol. The dichloromethane extract was purified by solvent partition, column chromatography, and crystallization, to finally yield three active compounds, asperparalines A (123), B (124), and C (125) [91, 92]. Asperparaline A (123) was shown to have the molecular formula of C20H29N3O3 by HR-EIMS together with ^H-NMR and ^^C-NMR spectra, indicative of eight degrees of unsaturation. Resonances at 171.9, 175.3, and 181.6 ppm in the ^C-NMR spectrum of 123 indicated the presence of three carbonyls, one of which had to be an amide carbonyl, while two other carbons in the five-membered ring were imido carbonyls based on the absorption bands at 1773 and 1698 cm"^ in the IR spectrum, revealing 123 to be pentacyclic. The ^H-NMR spectrum shown in Fig. (25) confirmed the presence of five methyl groups (two A^-methyls, two

595

m Mi^lU

_iljL,...JULJ

J

IL ppm

3.0

2.5

2.0

1.5

1.0

Fig. (25). 270 MHz ^H-NMR spectrum of asperparaline A (123) in acctonc-d^

tertiary methyls, and a secondary methyl), three isolated methylenes, a -CH2-CH< linkage, and a -CH2-CH2-CHCH3- linkage. For the connectivity of partial structures, HMBC experiments were carried out. Consequently, the planar structure of 123 was determined. Furthermore, the conformation of the structure of 123 was obtained by the application of X-ray crystallographic analysis. The ORTEP drawing of 123 is shown in Fig. (26) [91, 92]. The spectral features of asperparaline B (124), C19H27N3O3, were quite similar to those of asperparaline A (123). In the H-NMR spectrum of 124, one of two A^-methyl groups in 123 disappeared, and a signal assignable to NH was newly observed, suggesting that 124 lacked either 22-CH3 or 23-CH3. In the HMBC spectrum of 124, an A^-methyl signal at

5 O 16

17

123, asperparaline A Fig. (26). ORTEP drawing of asperparaline A (123)

596 5H 3.15 was correlated with signals of C-7 and C-14, confirming that 124 was A^^^-demethylasperparaline A [92]. Another analog, asperparaline C (125), had the molecular formula of C19H27N3O3, which was the same as that of asperparaline B (124). In the ^H-NMR spectrum of 125, a doublet methyl signal assigned to 3-CH3 in asperparaline A (123) disappeared, and a -CH2-CH2-CH2- linkage was observed, indicating that 125 was C^-demethylasperparaline A. This assumption was supported by the HMBC and NOESY experiments [92]. The structures of asperparalines A (123), B (124), and C (125) were determined to be spire compounds made up of an A^methyl succinimide and a cyclopent[flindolizine having an iV-methyl amide bridge. Another structural characteristic of asperparalines is a bicyclo[2,2,2]diazaoctane core, and various compounds, such as those of the paraherquamide family mentioned in the following section, contain the same core in their structures. However, all these compounds have an indole moiety in their structures- so, it is of great interest that asperparalines have no indole part in their structures.

124, asperparaline B

125, asperparaline C

Bioactivity of Asperparalines

The biological activities of asperparalines against several insects were also examined. Asperparaline A (123) induced paralysis in silkworms at a dose of 10 |j,g/g diet within 1 h of oral administration, and the paralysis lasted for 7 to 10 h. When injected with a microsyringe, 123 induced paralysis at a dose of 3 ^ig/g body weight within 20 min, and the paralysis lasted for 4 to 5 h. Asperparalines B (124) and C (125) exhibited almost the same effect on silkworms. Asperparaline A (123) showed remarkable insecticidal activity against third instar larvae of Nilaparvata lugens, third instar larvae of Nephotettix cincticeps, and the adults of Musca domestica. Biosynthetic Study

The paraherquamide family and other related compounds cited in the

597

following section have a bicyclo[2,2,2]diazaoctane core. Biosynthetic studies on these compounds support the notion that this structural motif is formed by a biosynthetic intramolecular [4+2] cycloaddition of the isoprene-derived olefin across a preformed azadiene moiety derived from an oxidized piperazine-dione as shown in Fig. (27). In 2003, Williams and coworkers performed feeding experiments on A. japonicus JV-23 to determine the primary amino acid building blocks that comprise asperparaline A (123) [93]. Incorporation of acetate, L-methionine,

OH

; \ N N

X

V

Fig. (27). Formation of bicyclo[2,2,2]diazaoctane core

H

O

'•AjA OH

GPP +

NH2 L-Isoleucine

^^ L-Tryptophan

dimethylallyl pyrophosphate

oxidation

127

126

^.. OH

oxidation and methylation

128 Fig. (28). Possible biosynthetic pathway to asperparaline A (123) [93]

598 L-isoleucine, and L-tryptophan was observed, suggesting that 123 likely shares a common biosynthetic pathway with the paraherquamides as shown in Fig. (28). Prenylation of the cyc/o-L-tryptophanL-p-methylproline and intramolecular [4+2] cyclization (via 126) would provide the putative bicyclo[2,2,2] core (127). Williams and coworkers have already demonstrated that 127 serves as a biosynthetic precursor to paraherquamide A (146) shown in Fig. (33). Oxidation of 127 leads to the catechol derivative (128). Oxidative cleavage of four carbon atoms from the oxygenated aromatic ring in 128 could fumish the spirosuccinimide ring of 123. Synthetic

Study

The first synthesis of the model compound of asperparaline A (123) was reported in 1999 by Williams and coworkers [94], as shown in Fig. (29). They developed a novel synthetic approach to a 3-spirosuccinimide system from a 2,3-disubstituted pyrrole. The synthesis was begun with the commercially available 3,3,5,5-tetramethylcyclohexanone (129), which was transformed into oxime (130). The pyrrole (131) was obtained from 130 through a Trofimov reaction. The pyrrole (131) was then A^-methylated with iodomethane to afford A^-methylpyrrole (132). The A^-methylpyrrole (132) was oxidized through a photooxygenation reaction using Rose Bengal as a photosensitizer under UV light irradiation to afford hydroxypyrrolidone (133). Treatment of 133 with

H

a)

129

130

¥>

c)

131

/

dl

132

133

134

a) HjNOH-HCl b) C2H2 c) Mel d) Oj, hv, Rose Bengal e) NaH, A Fig. (29). Williams' model study on the spirosuccinimide ring system of asperparaline A (123) [94]

599

So-

a) 135 ^^^

c)

137 COOH

d)

/ N

e).

V^'

-COOH

138

139

140

a) CHj(CN)2, piperidine, PhCOOH b) NaCN, AcOH c) HBr d) AcCl e) MeNH^ Fig. (30). Tanimori's approach to the spirosuccinimide ring system of asperparaline A (123) [95]

sodium hydride in DMSO at 180°C furnished the desired spirosuccinimide (134). In 2000, Tanimori and coworkers also reported a method for synthesizing the spirosuccinimide moiety of asperparaline A (123) [95], as shown in Fig. (30). 2,2-Dimethylcyclopentanone (135) was treated with malononitrile in the presence of piperidine and benzoic acid to /^NH

a)

'''COOH

GOGH

CGGMe

CGGMe

141 C02(CO)6 e) CGGMe

142 NHMe

N'

~N NV

H>=0

COOMe

143 a) ref. [97] b) SOCl^, MeOH c) propargyl bromide, Lil d) C0j(C0)8, Ar e) 48% aq. MeNHj, MeNH-HCl Fig. (31). Tanimori's approach to asperparaline C (125) [96]

600

afford unsaturated dinitrile (136). Michael addition of the cyanide anion to 136 proceeded smoothly to provide trinitrile (137). Acid-catalyzed hydrolysis of 137 was accompanied by decarboxylation to give acid (138). The acid (138) was converted into anhydride, a crude product reacted with methylamine to afford the desired spirocyclic A'-methylsuccinimide (140). In 2001, Tanimori and coworkers described a Pauson-Khand cyclization reaction to construct the tetracyclic indolidine core of asperparaline C (125) [96], as shown in Fig. (31). The starting enyne (141) was synthesized from L-proline by the standard procedure [97]. The [2+2+1] cycloaddition of 141 with Co(CO)8 gave tricyclic indolidinone (142) as a single diastereomer. Condensation of 142 with methylamine resulted in conjugate addition of the amine to the enone moiety followed by ring closure to provide bridged tetracyclic lactam (143). Although some model compounds have been synthesized, total synthesis of asperparalines has not been reported. Asperparaline'Related

Compounds

The unique bicyclo[2,2,2]diazaoctane ring system constitutes one of the structural characteristics of brevianamides (Fig. (32)). Brevianamide A (144) was originally isolated from cultures of Penicillium brevicompactum by Birch and Wright in 1969 [98]. Brevianamide A (144) was also isolated from cultures of Penicillium viridicatum by Wilson and coworkers in 1973 [99]. Bird and coworkers observed that 144 is formed only after conidiation has begun in solid cultures of P. brevicompactum [100]. Birch and Russell isolated brevianamide B (145)

144, (+)-brevianamide A

145, (+)-brevianamide B

ent'l45, (-)-brevianamide B Fig. (32). Structures of brevianamides

601

from the culture of P, brevicompactum and reported that 145 is a stereoisomer of 144 [101]. In 1987 and 1990, Paterson and coworkers reported that 144 is a potent antifeedant against pests [102, 103]. The structure and absolute configuration of 144 was determined by Coetzer in 1974 through X-ray crystallography on a semisynthetic derivative, 5-bromobrevianamide A [104]. Williams and coworkers achieved the first total synthesis of 145 in 1988 [105] and also reported that P. brevicompactum constructs 144 and 145 in optically pure form and that the natural 145 and semi-synthetic one {ent-XAS) derived from 144 are of the opposite absolute configuration [106]. The paraherquamide family is another group of compounds containing a bicyclo[2,2,2]diazaoctane ring system; the structures of the members are shovm in Fig. (33). Paraherquamide A (146) was isolated as a toxic metabolite fi*om Penicillium paraherquei by Yamazaki and coworkers [107, 108]. In 1990, Ondeyka and coworkers reported the structural determination and antihelmintic activity of paraherquamides B (147), C (148), D (149), E (150), F (151), and G (152) isolated from the fermentation of Penicillium charlesii [109]. Paraherquamides A (146), E (150), F (151), and G (152) were also isolated fi'om Penicillium sp. by Blanchflower and coworkers in 1991 [110]. The new paraherquamide congeners VM 55595 (153), VM 55596 (154), VM 55597 (155), and VM 55599 (156) were isolated from Penicillium sp. IMI 331995 by

146, paraherquamide A R = OH 150, paraherquamide E R = H (VM54159)

147, paraherquamide B

148, paraherquamide C

149, paraherquamide D

Fig. (33). Structures of paraherquamide family (part 1)

602

151, paraherquamide F R = H (VM55594) 152, paraherquamide G R = OH (VM54158) o

153, VM55595

154, VM55596

156, VM55599 157, sclerotiamide Fig. (33). Structures of paraherquamide family(part 2)

Blachflower and coworkers in 1993 [111]. Sclerotiamide (157), shown in Fig. (33), was isolated from the sclerotia of Aspergillus sclerotiorum NRRL 5167 by Whyte and Gloer in 1996 [112]. Sclerotiamide (157) causes significant mortality and unusual physiological effects against the com earworm Helicoverpa zea. In 1997, Banks and coworkers isolated antihelmintic metabolites from Aspergillus sp. IMI 337664, and described structures of aspergillimide (VM5598) identical to asperparaline A (123), 16-keto aspergillimide (SB202327) (158) and the paraherquamides VM54159 (159), SB203105 (160), and SB200437 (161). The structures of these compounds are shown in Fig. (34) [113]. SB203105 (160) is the first example of a 4-substituted paraherquamide.

603

158,16-keto aspergillimide (SB202327) o.

160, SB203105

159,VM54159

161, SB200437

Fig. (34). Structuers of 16-keto asperlillimide and paraherquamides

CJ-17,665 (162) was isolated from the fermentation broth of Aspergillus ochraceus CL41582 by Sugie and coworkers in 2001 [114]. CJ-17,665 (162) inhibits the growth of multi-drug resistant Staphylococcus aureus, Streptomyces pyogenes, and Enterococcus faecalis. In 2002, Qian-Cutrone and coworkers isolated stephacidins A (163) and B (164) from A, ochraceus WC76466 [115]. Stephacidins A

o A

H

163, stephacidin A Fig. (35). Structures of CJ-17,665 and stephacidins

164, stephacidin B

604

(163) and B (164) show in vitro cytotoxic activity against various antitumor cell lines, but 164 exhibits more potent and selective antitumor activities, especially against testosterone-dependent prostate cancer cell line, LNCaP. The structures of 162,163, and 164 are shown in Fig. (35). Marcfortines A (165), B (166), and C (167) were isolated from the mycelium of Penicillium roqueforti by Polonsky and coworkers in 1980 (Fig. (36)) [116, 117]. Marcfortines contain a piperidine ring instead of a pyrrolidine ring in paraherquamides and sclerotiamide. Biogenetically, the basic skeleton of 165 is clearly derived from a dioxopiperazine formed from tryptophan and pipecolic acid. In 1999, Kuo and coworkers elucidated the biosynthetic pathway of the pipecolic acid moiety of 165 [118]. In 2002, Williams reviewed studies on total synthesis and biosynthesis of the paraherquamide family, with a focus on the biological Diels-Alder construction of the bicyclo [2,2,2] diazaoctane ring system [119].

"III

165, marcfortine B R = CH3 166, marcfortine B R = H

167, marcfortine C

Fig. (36). Structuers of marcfortines

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