J. CHEM. SOC. PERKIN TRANS. I 1989 Studies of Precursor-directed Biosynthesis with Streptomyces. Part 2.‘New and Unusual Manumycin Analogues Produced by Streptomyces parvuhs Ralf Thiericke, Hans-Jorg Langer, and Axel Zeeck * lnstitut fur Organische Chemie, Universitat Gottingen, Tarnmannstrasse 2, D-3400 Gottingen, Federal Republic of Germany The application of a new type of precursor-directed biosynthesis, in which artificial aromatic starter molecules were fed to the manumycin producer Streptomyces parvulus, resulted in new and unusual manumycin analogues for which structures are reported. The new compounds can be divided into three classes; the artificial starter molecule elongated (i) with the triene chain including the C,N moiety, (ii) with the chiral manumycin C,,-side chain only, and (iii) with both substituents.Based on these results it is plausible to differentiate between an amidase linking the chiral C,,-side chain to the artificial precursor, and a CoA-transferase activating the aromatic carboxy group for further elongation via the polyketide pathway. The specificity of the enzymes involved with regard to the structure of the aromatic precursor is discussed. The biogenetic origin of the multifunctional m-C,N unit in the Feeding Experiments and Results manumycin group antibiotics manumycin (1) and asukamycin Following our described method ’ we fed a series of different has recently been el~cidated.~-~ both cases the starter aromatic compounds in unphysiological amounts (55 mM) into In molecule derives from the TCA-cycle intermediate succinate the stationary growing phase of Streptomyces parvulus (strain (C, unit) and the carbohydrate metabolite glycerol (C, unit).Tii 64). The artificial precursors employed included substituted Thus, this biosynthetic pathway differs drastically from those aminobenzoic acids, benzoic acids without an amino function- described for other antibiotics 5--8 containing a m-C,N moiety. ality, aromatic amines, and non-aromatic compounds. The As yet no intermediate has been found on the way from the C, fermentation conditions and the feeding and the work-up pro- and C, units to manumycin/asukamycin. cedures were the same as previously described.’ The resulting As previously reported,’ we observed a strong dependence of crude products were examined by t.1.c.(silica gel, CHC1,-the metabolite pattern on the amount of 3-aminobenzoic acid MeOH, 9: 1 v/v) to obtain the metabolite pattern of each feed- (nzABA) fed to the manumycin producing strain. A 7 mM mABA ing experiment in comparison to an unfed fermentation. The solution suppressed the manumycin biosynthesis, while un- appearance of the red C,, prodigiosins 36-40 h after inocul- physiological concentrations (55 mM) led to a manumycin ation indicates that the secondary metabolism has started and analogue, called 64-mABA (2). It was found that 4-amino- is therefore a conspicuous indicator of normal cell growth. The benzoic acid (PABA) led to formation of 64-pABA (3), in which qualitative estimation of the dye portion in the crude products the chiral C,,-side chain was not linked to the amino group.gave evidence of how strongly the fed substances influence the In this paper we describe the extension of this new type of normal behaviour of the strain used. The results of the feeding precursor-directed biosynthesis and the replacement of the experiments are summarized in Tables 1 and 2. natural n?-C,N unit in the parent antibiotic manumycin (1) by unusual artificial compounds in order to obtain new manumycin analogues. We were interested in extending our method to unusual artificial precursors as well as obtaining additional information on the specificity of the enzymes involved in the modification of the artificial and subsequently of the natural precursor in the manumycin biosynthesis.I, (81 R3 = OH, R4 = OH (9) R3 = NHNH2 , R4 = H After purification the new compounds were characterized spectroscopically, their molecular formulae were determined by high resolution mass spectra, and their structures were elucid- ated by comparing the ‘H and 13C n.m.r. spectra to those of known manumycin derivative^.'.^,'^ Both, the ‘H and the 13C n.m.r. spectra of (4), (5), (6),and (7) are over-crowded in the aromatic/olefinic region. Therefore, the assignments of the 043 signals in this region were‘ made by increment calculations NH and/or by direct correlation to manumycin (1) and 64-mABA (2), whose signal assignments were established by two dimen- sional n.m.r. method^.'.^ The carbon signals of C-1”,C-3”,C-4”, and C-5” of compounds possessing the C,N moiety are known to be broad at 50.3 MHZ.’,~,’~ We attribute this effect to tautomeric changes occurring on the n.m.r.time-scale, resulting 852 J. CHEM. SOC. PERKIN TRANS. I 1989 R' R' R2 5\b.' NH2 H H OH CH3 NH2 OCH3 NH2 OH OCH3 Scheme 1. Formulae and e.i.-m.s. (70 eV) fragmentation pattern of class 1 and 3 manumycin analogues Table 1. Feeding (55 mol 1-') of different artificial aromatic compounds to Strepptomyces paruulus (strain TU 64) Manumycin (1) Prodigiosins' New product R, Valueb Amount (mg I-') 33 mg 1-' Strong 64-HBA (4) 0.10 7.4 NH Precursor 3-Hydroxybenzoic acid Ferulic acid Vanillic acid 3-Amino-4-methylbenzoic acid 3-Amino-4-methoxybenzoic acid 3-Amino-4-hydroxybenzoic acid 3-Aminobenzohydrazide Very weak 64-VAN (7) 0.39 20.0 Very weak 64-VAN (7) 0.39 23.0 Weak 64-3A4M (5) 0.43 12.1 Weak 64-3A4MO (6) 0.51 8.4 Strong 64-3A4Hy (8) 0.33 13.3 Weak 64-BZH (9) 0.41 23.0 a By observing the colour of the mycelium and the t.1.c. control of the crude products.Silica gel, CHC1,-MeOH (9: 1 v/v). Table 2. Precursor fed to Streptomycesparvulus without the appearance in nearly complete coalescence and hence corresponding line- broadening. The carbon signal at 115 p.p.m. (C-2") definitely Manumycin indicates the existence of the C,N moiety. In addition, the (mg 1-') Prodigiosins a e.i.-mass spectra of compounds (2)-(7) showed a common of new metabolites Precursor (55 mmol 1-') 3-Acetaminobenzoic acid 3-Amino-4-chlorobenzoic acid 3-Aminocyclohexanecarboxylic acid 4-Aminocyclohexanecarboxylic acid 4-Amino-2-hydroxybenzoic acid 5-Amino-2-nitrobenzoic acid 4-Aminophenylacetic acid 5-Aminosalicylic acid Methyl 3-aminobenzoate Benzoic acid 3-Fluorobenzoic acid 4-Fluorobenzoic acid Gallic acid , 2-Hydroxy-3-methoxybenzoic acid Nicotinic acid 3-Nitrobenzoic acid 4-Nitrobenzoic acid Phenylacetic acid Protocatechuic acid Shikimic acid 3-Aminoacetophenone 3-Aminobenzaldehyde 3-Aminobenzoni trile 3-Aminobenzyl alcohol Phenylene- 1,3-diamine Vanillic aldehyde a See Table 1: S = strong, W = weak, VW 8.2 S W S S fragmentation pattern (Scheme l), consisting of an a-frag-mentation on both sides of the C-13 amide carbonyl group (M -112, M -140) and a selective splitting within the triene chain between C-9 and C-10 in which the aromatic part of the 10.2 W S S S W vw W W vw molecule is stabilized by the uptake of one hydrogen atom (M + 1 -179).The fragment ion at m/z 193 is a direct indicator of the chiral C,,-side chain in the manumycin analogues [e.g. (2), (8), and (9)]. Because of the complexity of the 'H n.m.r. spectra we were unable to analyse the stereochemistry of the triene chains with the exception of the double bond at C-1 1, which is trans in all new manumycin analogues. Since 64-mABA (2) exhibits an all- W trans triene chain, as does the parent antibiotic manumycin (l),' 7.0 25.6 vw S S S vw S vw W we postulate the same configuration for the new manumycin analogues.The stereochemistry of the diene chains in com- pounds (8) and (9)were not examined, but the chemical shifts of the 'H n.m.r. signals are so similar to those of manumycin that we postulate conformity. Compounds 64-3A4Hy (8) and 64-BZH (9) showed similar optical rotation values to 64-mABA (2) and (R)-2,4,6-trimethyldeca-2,4-dienoic acid,' the absolute vw W configurations of which had already been established." It is probable that in the manumycin-analogues (2), (8), and (9) the W vw chiral C,,-side chain has the same chirality as in the parent antibiotic manumycin (1). = very weak. Although the natural m-C,N unit is expected to possess an amino functionality in meta-disposition to a free carboxy group, J.CHEM. SOC. PERKIN TRANS. I 1989 CO2H Q””‘ 1 ONH2 C02H C02H OANH--NH200CH3C02H St r ep to myces par v uIus (Strain TU 64) 0 NH # R’ CLASS 1 CLASS 2 CLASS 3 Scheme 2. Survey on the fed and incorporated aromatic precursors and the three classes of manumycin analogues acid (AHBA) fed in physio- it is noteworthy that precursors with a totally different [7-’3C]-3-amino-5-hydroxybenzoic substitution pattern on the aromatic ring are used by logical amounts was not incorporated into manumycin (l).4 Streptomyces parvulus to produce manumycin analogues. These results are in accordance with the prediction’.4 that Scheme 2 illustrates the biosynthetic variability of our strain. hydroxylated 3-aminobenzoic acids are not intermediates in Based on the structures of the known manumycin analogues we the manumycin biosynthesis.are able to divide them into three classes: Class 1: The precursor is enlarged by the triene chain including the C,N moiety [64-pABA (3),’ 64-HBA (4), 64-3A4M Discussion (5),64-3A4MO(6),and 64-VAN(7)]. Based on the eight known manumycin analogues (2)-(9) Class 2: The precursor is linked to the chiral C13-side chain formed by application of the precursor-directed biosynthetic only [64-3A4Hy (8) and 64-BZH (9)]. method it seems to be possible to differentiate between two Class 3: The precursor is connected to both structural elements, enzymes or enzyme systems which are essential for the incor- thus exhibiting the entire carbon skeleton of the parent poration of the precursor as central starter units.One of them, antibiotic manumycin (1) C64-mABA (2)’]. probably an amidase, is responsible for the connection of the Use of 3-amino-4-hydroxybenzoic acid (3A4Hy) as m-C,N chiral C,,-carboxylic acid to the amino group, and the other starter unit resulted in 64-3A4Hy (8), in contrast to 5-amino- activates the carboxy group (CoA-transferase) for the subse- salycilic acid which only blocked the manumycin biosyntheses quent chain extension process via the polyketide pathway.12 without the production of a new metabolite. In addition, The latter may be part of a multienzyme complex. Because of 854 the appearance of three different classes of metabolites one may conclude that the amidase and the CoA-transferase act in non- coupled processes (Scheme 2).It seems likely that the amidase is more sensitive to the structure of the artificial precursor than the transferase activating the aromatic carboxy group. It is not, as yet, clear why elongation of 3-amino-4-hydroxybenzoic acid at the carboxy group fails. In the case of 3-hydroxybenzoic acid, the parent antibiotic manumycin (1) (33 mg/l) was isolated together with (4) (7.5 mg/l). Thus, 3-hydroxybenzoic acid does not compete very successfully with the natural m-C,N precursor. This contrasts with the other feeding experiments (Table 1) in which the manumycin biosynthesis was suppressed totally. In none of our fermentations to produce manumycin or manumycin analogues did we observe any compounds exhibit- ing variations in the length of the triene chain.It therefore seems likely that the polyketide synthetase involved in the chain formation is highly specific in its action. This was confirmed by feeding experiments with vanillic acid (4-hydroxy-3-methoxy- benzoic acid, VAN) and ferulic acid (4-hydroxy-3-methoxy- cinnamic acid, FER) (Table 1 and Scheme 2) in which it was found that both starter molecules initiate the formation of the same product, 64-VAN (7).With the exception of C-ll/C-12 the configuration of the double bonds has not been determined because of signal overlapping in the ‘H n.m.r. spectrum. The termination of the triene chain elongation seems to be under the strict control either of the polyketide synthetase itself or of a second amidase which is responsible for the connection of the activated triene carboxylic acid to the C,N unit.This seems to be valid for all other manumycin-group antibiotics with the exception of colabomycin,” in which a tetraene chain was formed by the producing micro-organism. Besides the aromatic compounds, which led to the manu- mycin analogues described above (Scheme 2), we carried out further feeding experiments using different acids, amines, and amino acids (Table 2). Most of these precursors suppressed the manumycin biosynthesis totally, but did not result in any new metabolite. At this state of our investigation we cannot decide whether the negative results are due to the specificity of the enzymes involved (amidase, CoA-transferase) or to other effects, e.g.transport into the cells, inhibition of the secondary metabol- ism, toxic side effects, and wrong timing and/or concentrations during the feeding experiments. All in all the production of the manumycin analogues described here does highlight the variability of the precursor- directed biosynthetic method developed for the manumycin producer. The replacement of a central starter unit in other antibiotics (not only natural products containing a m-C,N unit) by increasing concentrations of suitable precursors may lead to interesting derivatives produced by micro-organisms and may reinforce our method as an alternative to chemical derivatization, especially in the case of high-cost drugs. Experimental General procedure was as described in ref.1. Fermentation and Feeding Experiments.-The fermentation conditions for StreptomjJces parvulus (strain Tii 64) have been described in detaiL9 Feeding experiments with various pre- cursors (see Tables 1 and 2) were carried out using 100 ml of culture medium (2% mannitol; 2% degreased soybean meal) in 1000 ml Erlenmeyer flasks which were shaken for 72 h; 36-40 h after inoculation the precursor, dissolved in a small amount of sterile water and adjusted to pH 7.0 with 2~ NaOH, was added under sterile conditions. The cultures were harvested as described for manumycin9 and the crude products were screened by t.1.c. in various solvent systems (see Tables 1 and 2).J. CHEM. SOC. PERKIN TRANS. I 1989 N-(2-Hydroxy-5-0x0 cyclopent-1 -en yI )-7-(3-hydr oxyp hen y1)-hepta-2,4,6-trienamide (64-HBA), (4).-The dark red crude product was chromatographed on a silica gel column [35 x 2.5 cm; CHC1,-MeOH(9: 1 v/v)], and the resulting brown product was stirred with ethanol (20 ml) at room temperature and filtered. The filter cake was rechromatographed on a silica gel column [20 x 1.5 cm; CHC1,-MeOH (4: 1 v/v)] to give the pure amide (4) (7.5 mg I-’), m.p. 263 “C; R, 0.76 [CHCl,-MeOH (4: 1 v/v)]; v,,,,(KBr) 3 420, 3 260, 1 610, 1 580sh, 1 545sh, and 1 005 cm-’; h,,,,(MeOH) 357 (E 39 400), 262 (24 600), and 202 nm (34 000); h,,,.(MeOH-HCI) 363 (E 42 300), 263 (19 700), and 202 nm (32 300); h,,,,(MeOH-NaOH) 343 (E 57 300), 361 (53 SOO), and 210 nm (144 600); Gc[(CD,),SO] 166.0 (s, C-13), 157.5(s, C-2), 142.3 (d, C-ll), 141.0 (d, C-9), 137.7 (s, C-4), 136.4 (d, C-7), 121.5 (d, C-12), 117.8 (d, C-5), 115.6 (d, C-3), 114.8 (s, C-2”), 113.1 (d, C-l), and 28.8 (br t, C-4” and C-5”) [the signals at 130.4 (d), 129.6 (d) and 128.2 (d) could not be assigned definitely to C-6, C-8, and C-10; the signals fr C-1” and C-3” were not observed at 50.3 MHz]; G,[(CD,),SO] 2.06 (br s, 4”-H, and 5”-H2),6.48-7.23 (9 H,m),7.30(dd,J15 and 12 Hz, 11-H), 9.41 (br s, NH or OH), 9.91 (br s, NH or OH), and 13.80 (br s, OH); m/z 311.1157 (26%, M+;C18H1,N04) 199 (42), 198 (lo), 171 (43), 153 (41), and 132 (100).7-( 3- Amino-4-methylphenyl)-N-(2-hydro.uy-5-oxocq,clopent-l-enyl)hepta-2,4,6-trienamide(64-3A4M), @).--The dark red crude product was chromatographed twice on a silica gel column [35 x 2.5 cm; CHC1,-MeOH (9:l v/v)] and was further purified on a Sephadex LH-20 column (50 x 2.5 cm; CHCI,) to yield the yellow amorphous product (5)(12.1 mg I-’), m.p.289 “C (decomp.); v,,,.(KBr) 3 480,3 260,l 594, and 1 006 cm-’; h,,,.(MeOH) 355 (E 21 400) and 260 (11 000); h,,,,(MeOH-HCl) 353 (E 43900) and 256 (10000); h,,,.(MeOH-NaOH) 348 (E 34700) and 260 (20900); GJ(CD,),SO] 166.1 (s, C-13), 146.6 (s, C-2), 142.6 (d, C-ll), 141.5 (d, C-9), 137.5 (d, C-7), 134.7 (s, C-4), 130.2 (d, C-lo), 129.4 (d, C-6), 126.6 (d, C-8), 122.2 (s, C-l), 120.9 (d, C-12), 115.2 (d, C- 5), 114.9 (s, C-2”), 112.0 (d, C-3), 28.9 (br t, C-4” and C-5”), and 17.2 (9,CH,) (the signals for C-1” and C-3” were not observed at 50.3 MHz); G,[(CD,),SO] 1.26 (s, Me), 2.07 (br s, 4”-H,), 2.49 (br s, 5”-H,), 3.30 (br s, NH overlapped by HOD), 6.46-7.02 (8 H, m), 7.33 (dd, J 15 and 11.5 Hz, 11-H), and 9.90 (br s, OH); m/z 324.1474 (61%, M+;C,,H,ON,O,), 212 (20), 197 (5), 184 (34), 169 (20), and 146 (51).7-(3-Amino-4-methoxyphenyl)-N-(2-hydrox~-5-oxu~~cfopent-l-enyl)hepta-2,4,6-trienamide(64-3A4MO), (6).-The crude product was chromatographed on a silica gel column [35 x 2.5 cm; CHC1,-MeOH (9:l v/v)] and was further purified on a Sephadex LH-20 column (90 x 2.5 cm; MeOH) and on prepar- ative t.1.c. [silica gel on glass; CHC1,-MeOH (9 :1 v/v)] to yield the yellow amorphous product (6)(28.4 mg 1-’), m.p.258 “C; v,,,. 3 450, 3 360, 3 230, 1 688sh, 1 608, 1 570, and 1 002 cm-’; h,,,,(MeOH) 366 (E 38 300) and 260 (26 200); h,,,.(MeOH-HCl365 (E 49 900) and 261 (13 000); h,,,.(MeOH-NaOH) 365 (E 38 600), 260 (26 000), and 206 (69 200); G,[(CD,),SO] 166.2 (s, C-13), 147.2 (s, C-l), 142.7 (d, C-11), 141.8 (d, C-9), 137.7 (s, C-2), 137.4 (d, C-7), 129.3 (s, C-4), 128.7 (d, C-lo), 125.4 (d, C-8), 120.4 (d, C-12), 116.4 (s, C-2”)) 114.9 (d, C-5), 111.2 (d, C-3), 110.5 (d, C-6), 55.3 (9, OCH,), and 28.7 (br t, C-4” and C-5”) (the signals for C-1” and C-3” are not observable at 50.3 MHz); GH[(CD,),SO] 3.30 (6 H, br m,4”-H2, 5”-H,,NH), 3.81 (s, OCH,), 6.42-7.04 (9 H, m), 7.33 (dd, J 15 and 11.5 Hz, 1 1-H), 9.92 (br s, OH), and 13.82 (br s, OH); m/z 340.1424 (18%, Mf; C19H,oN,O,), 292 (7), 243 (lo), 228 (12), 200 (29), 162 (loo), and 97 (22).7-(4-Hydroxy-3-methox~phenyl)-N-(2-hydroxy-5-oxoc~clo-pent- 1 -enyZ)hepta-2,4,6-trienamide(64-VAN), (7).-The dark J. CHEM. SOC. PERKIN TRANS. I 1989 red crude product was chromatographed twice on a silica gel column [35 x 2.5 cm; CHC1,-MeOH (9:l v/v)] and was further purified on a Sephadex LH-20 column (90 x 2.5 cm, CHCI,) to yield 23 mg 1-’ (20 mg 1-’ ferulic acid feeding experi- ment) of yellow amorphous amide (7), m.p. 214 OC; v,,,. 3 440, 2 930,l 618sh, 1 600sh, 1 588, and 1 003 cm-l; h,,,.(MeOH) 364 (E 26 700) and 260 (16 900); h,,,.(MeOH-HCl) 384 (E 28 000) and 268 (10 100); h,,,.(MeOH-NaOH) 417 (E 23 loo), 260 (17 loo), and 210 (13 300); G,[(CD,),SO] 188.9 (br s, C-1”)) 165.1 (s, C-13), 165.0 (br s, C-3”)) 147.8 (s, C-1 or C-2), 147.5 (s, C-1 or C-2), 141.0 (d, C-11), 140.3 (d, C-9), 136.2 (d, C-7), 129.1 (d, C-8 or C-lo), 128.2 (s, C-4), 127.7 (d, C-8 or C-lo), 125.7 (d, C-5), 122.4 (d, C-12), 120.7 (d, C-6), 115.6 (s, C-2’7, 110.0 (d, C-3), 55.6 (t, OCH,), 29.7 (t, C-4“ or C-5’9, and 28.8 (t, C-4” or C-5”); G,[(CD,),SO] 2.08 (br s, 4”-H,), 2.32 (br s, 5”-H,), 3.50 (br s, OH), 2.81 (s, OMe), 6.38-7.03 (7 H, m), 7.12 (s, 3-H), 7.23 (dd, J 15 and 12 Hz, 11-H), 8.82 (br s, NH or OH), 9.61 (br s, NH or OH), and 13.78 (br s, OH); m/z 341.1263 (3.6%, Mf; C,,H,,NO,), 229 (24), 201 (lo), 197 (13), 169 (66), 163 (loo), and 131 (67).313 (E 8 7001, 254 (16 700), and 221 (18 600); G,(CDCl,) 166.0 (S, C-1’)) 163.5 (S, C-7), 146.9 (s, C-2), 143.0 (d, C-5’), 140.7 (d, C-3’), 132.5 (s, C-4), 129.9 (s, C-2’ or C-4’), 129.7 (s, C-2’ or C-4’1, 125.1 (4 C-6), 118.7 (d, C-l), 116.7 (d, C-5), 113.6 (d, C-3), 37.0 (t, C-7’), 32.9 (d, C-6’), 29.8 (t, C-S’), 22.8 (t, C-9’), 20.8 (4, c-13’), 16.5 (9, C-12’), 14.1 (q, C-lo), and 13.7 (1, C-11‘); GH[(CD,),CO] 0.88 (t, J 6 Hz, 10’-H,), 0.97 (d, J 6.5 Hz, 13’-H3), 1.25 (br m, 7’-H,, 8’-H,, and 9’-H,), 1.82 (d, J 1.5 Hz, 12’-H3), 2.07 (d, J 1.5 Hz, 11’-H,), 2.47 (br s, 6’-H), 3.87 (br s, NH), 5.38 (d, J 9 Hz, 5’-H), 6.86 (m, 6-H), 6.96 (s, 3-H), and 7.16-7.27 (m, 1-H, 5-H, and 3’-H); m/z 343.2260 (28%, M+; C20H29N302), and 258 (7), 218 (5), 193 (68), 151 (4), and 120 (100%).Acknowledgements We are grateful to Professor H. Zahner, Institut fur Biologie der Universitat Tiibingen for providing us with Streptomyes parvulus (strain TU 64) and to M. Lackner and H. Miiller for 4-Hydroxy-3 -(2,4,6-trimethyldeca-2,4-dienyZcarbonylamino)-benzoic Acid (64-3A4Hy), @).-The crude product was chro- matographed on a silica gel column [35 x 2.5 cm; CHC1,- MeOH (9: 1 v/v)] and was further purified on a Sephadex LH-20 column (90 x 2.5 cm, MeOH) to yield (8) as a yellow oil (13.3 mg l-’), [sc]k2 -15.2”(c 0.60 in CHCI,); v,,,,(KBr) 3 440, 2 920,2 860,l 650sh, 1 629, and 1 596sh cm-’; h,,,,(MeOH) 407 (E 18 500), 270 (26 200), and 212 (120 100); h,,,.(MeOH-HCl) 407 (E 18 200), 270 (25 700), and 212 (119 500); h,,,,(MeOH-NaOH) 408 (E 17000), 269 (25200), and 214 (127200); G,(CDCI,) 0.88 (t, J 6.5 Hz, 10’-H3), 0.98 (d, J 6.5 Hz, 13’-H3), 1.16-1.40 (br m, 7’-H,, 8’-H2, and 9’-H,), 1.86 (d, J 1.6 Hz, 12’- H3), 2.14 (d, J 1.6 Hz, ll’-H3), 2.36-2.60 (br m, 6’-H), 5.44 (d, J 10 Hz, 5’-H), 6.53 (br s, NH or OH), 6.98 (s, 3’-H), 7.51 (d, J8.8 Hz, 6-H), 8.14 (dd, J 8.5 and 2 Hz, 5-H), 8.27 (d, J 2 Hz, 3-H), 8.52 (s, NH or OH), and 9.07 (br s, COOH); m/z 346 (4.3%, MH’), 193 (9.5), and 153 (8.4).3 -(2,4,6- Trimethyldeca-2,4-dienylcarbonylamino)benzo-hydrazidr (64-BZH), (9).-The dark red crude product was chromatographed on a silica gel column [20 x 2.5 cm; CHC1,- MeOH (9: 1 v/v)] and was further purified on a Sephadex LH-20 column (90 x 2.5 cm; MeOH) to yield pure (9) as an oil (23.0 mg 1-’), [m]h2 -42” (c 0.95 in acetone); v,,,,(KBr) 3 420, 3 260, 2 960, 2 930, 1660, 1 630, 1610, and 1 590 cm-’; h,,,.(MeOH) 254 (E 20 800) and 223 (20 100); h,,,.(MeOH- HCl) 262 (E 16 400) and 222 (14 300); h,,,,(MeOH-NaOH) excellent technical assistance.This work was supported by the Fonds der Chemischen Industrie. References I Part 1, R. Thiericke and A. Zeeck, J. Chem. Soc., Perkin I, 1988,2123. 2 H. G. Floss, in ‘Recent Advances in Phytochemistry,’ ed. E. E. Conn, Plenum Press, New York, 1986, 20, p. 13. 3 H. G. Floss, P. J. Keller, and J. M. Beale, J. Nut. Prod., 1986,49,957. 4 J. M. Beale, R. Harrold, H. G. Floss, R. Thiericke, A. Zeeck, A. Nakagawa, and S. Omura, J. Am. Chern. Soc., 1988, 110,4435. 5 0.Ghisalba and J. Niiesch, J. Antibiotics, 1981, 34, 64. 6 K. L. Rinehart, Jr., M. Potgieter, D. L. Delaware, and H. Seto, J. Am. Chem. SOC.,1981, 103, 2099. 7 U. Degwert, R. van Hiilst, H. Pape, R. Herrold, J. M. Beale, P. J. Keller, J. P. Lee, and H. G. Floss, J. Antibiotics, 1987, 40, 855. 8Y. Sat0 and S. J. Goule, J. Am. Clzem. Soc., 1986, 108, 4625. 9 A. Zeeck, K. Schroder, K. Frobel, R. Grote, and R. Thiericke, J. Antibiotics, 1987, 40, 1530. 10 A. Zeeck, K. Frobel, C. Heusel, K. Schroder, and R. Thiericke, J. Antibiotics, 1987, 40, 1541. 11 R. Thiericke, M. Stellwaag, A. Zeeck, and G. Snatzke, J. Antibiotics, 1987, 40, 1549. 12 R. Thiericke and A. Zeeck, J. Antibiotics, 1988, 41, 694. 13 R. Grote, A. Zeeck, H. Drautz, and H. Zahner, J.Antibiotics, 1988, 41, 1178. Received 6th April 1988; Paper 8/01352G
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