摘要:

WBiosynthesis of porphyrins and related macrocycles. Part 44.1,2 Synthetic and stereochemical studies on the proposed spiro IIintermediate for biosynthesis of the natural porphyrins am 7i137Mark A. Cassidy, Nigel Crockett, Finian J. Leeper and Alan R. Battersby * University Chemical Laboratory, Lensfield Road, Cambridge CB2 1E W, UK A route is devised for synthesis of both enantiomers of the spiro lactam 4. The enzyme uroporphyrinogen I11 synthase (cosynthetase), which converts hydroxymethylbilane 1 into uroporphyrinogen I11 3, is competitively inhibited more than twenty times more strongly by one enantiomer of 4 than by the other. This finding adds further strong support to the view that cosynthetase acts by generating the spiro pyrrolenine 2 as an intermediate.Haem, chlorophyll and vitamin B,, are all biosynthesised from the same parent macrocycle, uroporphyrinogen I11 3, shortened to uro’gen 111. The formation of this macrocycle from the open- chain hydroxymethylbilane 1 is catalysed by the enzyme cosynthetase (systematically uroporphyrinogen 111 synthase, E.C. 4.2.1.75). Comparison of structures 1 and 3 shows why this process has attracted such strong mechanistic interest; intriguingly, ring D of uro’gen I11 3 has been inverted relative to its position in the bilane 1. Both the building of the hydroxymethylbilane 1 and the ring-closure catalysed by cosynthetase have been extensively studied and this work has been reviewed.3 The key finding relevant to the present paper was that the inversion of ring D occurs by an intramolecular mechanism which only affects ring D.These results, based on a series of 3C-labelling experiments as indicated by the black spots and triangles in Scheme 1,4*5 Cosynthetase1 A NH m HN P A NH m HN p *----------Am. 3 A =CH~COZH,P =CH2CH2C02H Scheme 1 Proposed mechanism for the formation of uro’gen TIT bycosynthetase eliminated most of the 20 or so speculative mechanisms proposed for the ring D inversion and left just two. In one, ring D of the hydroxymethylbilane 1 is detached by cleavage of the bond between C-15 and C-16 but is held in the active site of the enzyme. Ring D is then inverted before rejoining to C-15 and the hydroxymethyl carbon to give 3; there is no experimental support for this mechanism.The other, illustrated in Scheme I, is fundamentally different in that the bond between the hydroxymethyl carbon and C-16 is suggested to be made first, to form the spiro pyrrolenine? 2. This pyrrolenine could then undergo fragmentation-recombination as in Scheme 1 to form uro’gen I11 3 and the chemistry involved has been shown to be both feasible and fa~ile.~ This second mechanism is based on a suggestion by Mathewson and Corwin6 but it is drawn in a simplified form for reasons that have been outlined earlier.7 Strong support for the second mechanism has been provided by the synthesis7 of the spiro lactam 4 which strongly inhibited 0 4 cosynthetase from carrying out its normal conversion of hydroxymethylbilane 1 into uro’gen 111 3; the inhibition was competitive.In addition, other synthetic macrocycles, lacking one or other parts of the five-membered lactam system, were not inhibit~ry.~ This finding was very significant in that the inhibitory spiro lactam 4 is different in structure from both the substrate 1for cosynthetase and the product 3 from the enzyme; the inhibitor only resembles the putative spiro pyrrolenine 2. It should also be emphasised that the two molecules, 2 and 4, only differ around the nitrogen atom of the five-membered spiro ring, otherwise they are identical. The spiro lactam 4 used for the foregoing studies was racemic, but only one enantiomer can accurately match the putative spiro pyrrolenine 2 held in the active site of cosynthetase. Our aim, therefore, was to synthesise both enantiomers of the spiro lactam 4 for testing as inhibitors of cosynthetase.Of the four substituents carried by the chiral centre of the lactam 4, two are extremely similar so a difficult resolution was expected, best carried out early rather than late in the synthesis. IUPAC name: 2H-pyrrole. J. Chem. Soc., Perkin Trans. 1, I996 2079 Results and discussion Trial experiments on the model system 7 Our initial studies were made on the acid 5, which had been synthesised earlier for elaboration to the spiro lactam 4.7 Salts were prepared from 5 using brucine, cinchonine, cinchonidine, quinine and quinidine. Systematic attempts at crystallisation using seven different solvents gave few crystals and no resolution.A totally different approach was needed and we envisaged synthesising the acid 6 so that a variety of chiral auxiliaries could be covalently attached to the carboxy group close to the chiral centre. However, as acid 6 contains a vinylogous malonic acid residue there was concern about its stability. This was checked by synthesising the simpler lactam 7. MeAP~02H v 6 0 7 AM' = CH2C02Me, PMe = CH2CH2C02Me The differentially protected pyrrole 9 was needed as starting material and this was initially prepared by having 2-trimethylsilylethanol present during the thallium-catalysed rearrangement' of the ketone 8 (Scheme 2). The aldehyde 10 was a by-product (a reaction previously observed in a related case 9, and the yield of 9 was too poor for this route to be used for the synthesis of 6; however, it yielded enough material for the preparation of the model 7.Scheme 2 also shows the synthetic route from 9 to 7 via 11, 12 and 14, which was mainly based on similar earlier work,' though the iodination step required new conditions (see Experimental section). Removal of the trimethylsilylethyl group from 14 gave the acid 7 which could be handled without decomposition. The synthesis of the dipyrrolic lactam 6 could, therefore, be confidently undertaken but first the attachment of chiral auxiliaries was tested on the model 7. The esters 15 and 16 were prepared from (R)-l-phenylethanol and (1S)-( -)-endo-borneol, respectively, using N,N-dicyclohexylcarbodiimide(DCC) and 4-dimethylamino- pyridine (DMAP).The former ester 15 was shown by NMR to contain two diastereoisomers in the ratio 1 : 1.25 and the ratio for the latter one 16 was 1 : 1.1. In neither case could the diastereoisomers be separated by TLC or HPLC. Attention therefore turned to the real system 6. Synthesis of the enantiomeric lactams 40a and 40b The first requirement was to develop a practical alternative synthesis of the starting pyrrole 9. This was achieved in good yield (16% over seven steps) on a large scale by the sequence shown in Scheme 3, based mainly on chemistry developed earlier4*'' for similar pyrroles. The key step is reductive alkylation 11*12 of the 4-iodopyrrole 22 using glyoxylic acid as the source of the acetate side-chain. Trial experiments involving some of the later chemistry required for construction of the required lactam 33 then showed that the benzyl ester of 9 caused problems.It was therefore conveniently changed at the stage of 2080 J. Chem. SOC.,Perkin Trans, I, 1996 i PhCH202C PhCH202C H H 8 9 R=Me 10 R=CHOI ii t OCH2CH2SiMe3 C02CH2Ph AcO H&f T YJMe 13 H iv 11 R = C02H iiiG 12 R=I C14 R=CHzCHzSiMe3 7 R=H vi F 15 R=CHMePh 16 R= -& Scheme 2 Reagents: i, T1(NO3),*3H,O, HNO,, Me,SiCH,CH,OH; ii, H,/Pd; iii, KI,, NaHCO,; iv, SnC1, then AgOAc, TsOH, H,O; v, TBAF; vi, DCC, DMAP, ROH the next intermediate 24 to the tert-butyl ester 26 by the steps shown in Scheme 4.This product was then coupled with the a-free pyrrole 29, prepared from the available tert-butyl ester 27, to yield the dipyrromethane 30. The lactam 33 was synthesised R02C PhCH202CMe02>O NOH / H H i2 ivE K 23 RI20 R = C02Bu' vii = R~ = H 21 R=C02H 9 R' = CH2CH2SiMe3, R2 = H V L 22 R=I viii L 24 R' = CHzCHzSiMe3, R2 = OAc Scheme 3 Reagents: i, PhCH,OH, heat; ii, NaNO,, AcOH; iii, MeCOCH2C02Buf,Zn, AcOH; iv, CF,C02H; v, KI,, NaHCO,; vi, OHCCO,H, HI, H3P02,Ac,O; vii, (COCI), then Me,SiCH,CH,OH; viii, SO2CI2 then NaOAc, AcOH C02CH2CH2SiMe3 \ c24 R=CH2Ph 7 25R=H iii C30 R =C02Bu' 31 R=CO2H vi K32 R=I viii K33 R =CH2CH2SiMe3 r 6R=Hvii r 34 R =CHMePh 35 R=41 ix 36 R=42 37 R=43 38 R=44 39 R =44,CH=CBr2 in place of CH2CBr3 0 40 R=Me s Ph 41 42 43 44 Scheme 4 Reagents: i, H,/Pd; ii, Bu'OH, DCC; iii, SnCI,; iv, (COCI), then Br,CCH,OH; v, TsOH, H,O; vi, KI,, NaHCO,; vii, SnCl, then AgOAc, TsOH, H,O; viii, TBAF; ix, Me,C=C(Cl)NMe, then the relevant alcohol; x,NaOMe, MeOH by the steps 30-31 --+32 -33 essentially as earlier for similar system^,^ Scheme 4.Removal of the trimethyl- silylethyl group by fluoride then afforded the acid 6ready for esterification with a range of chiral alcohols.The esters were prepared by reacting the acid 6with l-chloro- 1-dimethylamino-2-methylpropene to give the corresponding acid chloride, which smoothly acylated the various chiral alcohols. Other standard methods for these esterifications failed or gave unacceptable yields.The chosen alcohols were (R)-1- phenylethanol, (1 S)-(-)-endo-borneol (as 41), (R)-(-)-panto-lactone$ (as 42), 2,3-O-isopropylidine-~-(+)-ribonic acid y-lactone l4 (as 43) and the commercially available 3,4-0-benzylidene-D-( +)-ribonic acid Mactone (as 44).In no case could the diastereoisomeric esters 34 to 38 so formed be separated by TLC, but HPLC gave encouraging results for the esters 36 and 38, derived from pantolactone and the ribonic acid &lactone, respectively. However, there were impurities in the former ester which were difficult to separate from the required materials, so the latter ester was selected for further study. HPLC 3 IUPAC name: (R)-3-hydroxy-4,4-dimethyltetrahydrofuran-2-one. QP 3'3 29 i5 il thin Fig.1 HPLC trace for the separation of the diastereoisomers 38a (peak X) and 38b (peak Y) together with 39a (peak P) and 39b (peak Q) analysis of this ribonolactone ester 38 initially showed three pairs of peaks, the four peaks that appear in Fig. 1 together with another pair, smaller than P and Q, having a longer retention time than X and Y.Mass spectrometry and NMR spectroscopy indicated that this slower running pair represented the separated diastereoisomers of material in which the CH,CBr, group had been reduced to CH,CHBr2. Consideration of the earlier chemistry suggested that this reduction was probably due to attack by iodide ion on a bromine atom of the CBr, group during the preparation of the dipyrromethane 32; a CBr, group can act as a source of 'Br+'.Accordingly, the conditions for the iodination step 31 +32 were made as mild as possible and when this product was carried forward to the ester 38,the HPLC trace appeared as in Fig. 1, without the slowest running pair of peaks. Similar analysis of the four remaining peaks in Fig. 1 indicated that peaks X and Y were the desired diastereoisomers 38a and 38b,§ respectively, whereas peaks P and Q were the corresponding diastereoisomers 39a and 39b, respectively, which had lost HBr from the tribromoethyl group at some stage during the synthetic transformations. Again efforts were made to avoid the production of the compounds in peaks P and Q; presumably they are formed by some base-catalysed elimination of hydrogen bromide.Suffice it to say that the various modifications tried either had little effect or they somewhat reduced the yield of the desired compounds in peaks X and Y. Since these by-products were readily separated, the large scale separation was carried out on the mixture as seen in Fig. 1. This effective separation of the diastereoisomers was only possible with very low loading of the column, so automatic HPLC equipment for injection and collection was needed to provide sufficient material for the rest of the synthesis. Analytical HPLC and 400 MHz 'H NMR proved that complete separation of the diastereoisomers had been achieved. The first task was to confirm that the peaks X and Y in Fig. 1 in fact contained the desired diastereoisomers 38a and 38b.Certainly their 'H NMR, I3C NMR and mass spectra Q The two configurations at the starred chiral centre of 40, Scheme4, are not illustrated but are distinguished by a and b attached to the number. The same system with a and b is used for Scheme 5, where one configuration is illustrated but always the other separate enantiomer is being handled as well. J. Chem. SOC.,Perkin Trans. 1,1996 2081 MeAPf+10 40a I I I I 'I I I1 ' ' I II 340 217 Unm Fig. 2 Circular dichroism spectra of the enantiomers 40a derived from peak X (Fig. 1) and 40b derived from peak Y supported that view. Rigorous proof was obtained by treating each diastereoisomer with sodium methoxide in methanol to effect transesterification with loss of the ribonic lactone residue.As had been planned, the benzyl and tribromoethyl esters did not undergo transesterification under the mild conditions used, because they are vinylogous urethanes and thus much less reactive. That the two products, 40a and 40b, were enantiomers was shown by their identical HPLC retention times and 'H NMR and mass spectra; importantly, their circular dichroism (CD) spectra were mirror images with a common crossover point on the zero line (Fig. 2). Synthesis of the enantiomeric spiro lactams 54a and 54b The racemic form 40of the foregoing final products 40a and 40b was the key intermediate for the earlier synthesis of the racemic spiro lactam 54a,b. Now that the pure enantiomers of this intermediate were in hand, each could be carried through the same remaining steps of the synthesis, 40~45-46 -47 ---+ 48 ---,49 --+ 50 --+ 51 --+ 54, shown in Scheme 5.These conversions were carried out as for the racemic series7 and were successfully completed for both enantiomers, but there was one surprise, probably due to small differences in reaction conditions used by different workers. This appeared when the first enantiomer of the acid to be used 48b was decarboxylated by iodination and removal of the iodine by hydrogenation (expected steps 48b 49b --+__+ 50b). The major product was the bis(a-free pyrrole) 53b, not 50b. Evidently both decarboxylation and deformylation had occurred in the iodination step to yield the di-iodo derivative 52b, which on hydrogenation afforded the observed product 53b.Modification of the conditions for the iodination overcame this problem to allow the original steps via 50b (Scheme 5) to be used to build the spiro lactam 54b. In addition the bis(a-free) system 53b was successfully converted in modest yield into the final spiro lactam 54b by acid-catalysed condensation with formaldehyde. The sum of all this synthetic effort, starting from the two enantiomers of the dipyrrolic lactam 40a and 40b, afforded ample quantities of the enantiomeric spiro lactams 54a and 54b for the necessary enzymic experiments. The last ring- closure step, 51a or b ---+ 54a or b (Scheme 5) again yielded small amounts of the macrocyclic dimer 55, readily separable from the monomer, which had been fully characterised in the racemic ~eries;~ these enantiomeric dimers are not further examined here.2082 J. Chem. SOC.,Perkin Trans. I, 1996 0 47 R' = CHO, R2 = C02CH2Ph 48 R' = CHO, R2 = C02H 49 R* = CHO, R* = I 50 R' = CHO, R* = H viiL 51 R' = CHzOH, R2 = H 52 R'=R~=I 53 R'=R~=H viii (from 51) or ix (from53) 0 x c5; ;I; Scheme 5 Reagents: i, Zn, AcOH; ii, CF,CO,H; iii, SnCI,; iv, H,/Pd; v, KI,, NaHCO,; vi, H,/Pt; vii, NaBH,; viii, TsOH; ix, CH,O, CF,CO,H; x, KOH, H,O, MeOH 55 -8000-4-.-C 6000-2 4000-a vb 2000-I0 Ill I,,,,,i -40 -20 0 20 40 60 [ I]/pnol dm-3 Fig. 3 Dixon plot for the inhibition of cosynthetase by the enantiomeric spiro lactams 4a and 4b.The horizontal line at l/V = 1218 is at I/ V,,, and the straight lines through the experimental points intersect it at [I] = -Ki.Fig. 4 The two enantiomers of the spiro lactam 4 Table 1 Inhibition of cosynthetase by enantiomers of the spiro lactam 4 Inhibition constant Ki (or Kd/Compound pmol dm-3 Enantiomer 4asynthesised from peak X, Fig. 1 Ki 1.8 Racemic 4 Ki 2.5 Enantiomer 4bsynthesised from peak Y,Fig. 1 Ki38 Hydroxymethylbilane 1 KM37 Enzymic studies: inhibition of cosynthetase The two enantiomers of the spiro lactam 54a and 54b were each treated with aqueous methanolic potassium hydroxide under conditions known to hydrolyse only the ester groups to afford the enantiomeric octa-acids 4a and 4b.Cosynthetase isolated from Euglena gracilis,15 was used in standard assays of the enzyme’s activity l5 for catalysing the conversion of hy-droxymethylbilane 1 into uro’gen I11 3. These assays were carried out as for the earlier ones in the racemic series and were run in the presence of varying amounts of each enantiomer of the spiro lactam octa-acids 4a and 4b; kinetic runs using the racemic octa-acid7 4 were also included to act as a standard. The KMvalue for hydroxymethylbilane 1 was determined at the same time by standard assays” at a range of substrate concentrations in the absence of any inhibitor, the KMvalue being determined from a Hanes plot.I6 Dixon plots of the kinetic data from the experiments with the enantiomeric spiro lactams 4a and 4b are shown in Fig.3, from which the inhibition constants Ki shown in Table 1 were derived. They show a striking difference in the effectiveness of the two enantiomers as inhibitors of cosynthetase, with the spiro lactam 4a derived from peak X in Fig. 1 being ca. 20 times more inhibitory than its enantiomer 4b derived from peak Y. Also, the Ki for the strongly inhibiting enantiomer is more than an order of magnitude lower than the KM for the substrate (hydroxymethylbilane 1) of cosynthetase. The fact that one of the two enantiomers of the spiro lactam 4 is a weak inhibitor, rather than having no effect at all, can be understood by setting the lactam ring of these enantiomers in the same orientation, see Fig. 4.This is reasonable since it is the chemistry around ring D of hydroxymethylbilane 1,correspond-ing to the pyrrolenine ring of the putative spiro intermediate 2, that lies at the core of the action of cosynthetase. The three pyrrole rings of the macrocycle will have the same conformation in the two systems and inspection of Fig. 4 shows the only difference between the two systems is that the acetate (A) and propionate (P) side-chains on each pyrrole ring are interchanged. It follows that, whereas the side-chains on the pyrrolic rings of the strongly inhibitory enantiomer presumably fit well into the enzymic active site, the same side-chains of the other enantiomer, though all acidic, are of slightly the wrong size (A for P and P for A).The observed weak inhibition by this enantiomer, rather than a complete lack of inhibition, fits the foregoing analysis. These experiments with the two enantiomers of the spiro lactam, 4a and 4b, add further powerful support to the view that the conversion of hydroxymethylbilane 1 into uro’gen I11 3, catalysed by cosynthetase, goes via the spiro pyrrolenine 2. In addition, determination of the absolute configuration of the strongly inhibiting enantiomer will allow the absolute configuration of the spiro pyrrolenine 2 to be deduced. The following paper l7 describes the way in which that stereochemi- cal problem has been solved. Experimenta1 General directions Most general directions are given in Part 34 of this series.’* In addition, CD spectra were recorded on Jasco J-40CS or 5-600 spectrometers, with a 1 cm pathlength in strain-free cuvettes.HPLC was carried out on Spherisorb S5W silica columns or Spherisorb S5CN nitrile columns with a Waters 6000A pump connected to a Cecil CE272 detector. Solvents for HPLC were filtered through a 0.5 pm sieve. Evaporation was carried out at ca. 15 mmHg on a Biichi rotatory evaporator and residual solvent was removed at high vacuum using an oil pump. Methyl 2-benzyloxycarbonyl-5-methyl-4-(2-trimethylsilyl-ethoxycarbonylmethyl)pyrrole-3-propionate 9 and methyl 2-benzyloxycarbonyl-5-formyl-4-(2-trimethylsilylethoxy-carbonylmethyl)pyrrole-3-propionate10 A solution of acetylpyrrole S19 (1 g, 2.92 mmol) in dimethoxyethane (6 cm3) and 2-trimethylsilylethanol (1.38 g, 11.67 mmol) was treated with a suspension of thallium(rr1) nitrate trihydrate (2.59 g, 5.83 mmol) in dimethoxyethane (1 1 cm3) and concentrated nitric acid (0.2 cm3).The resulting mixture was stirred at room temperature for 28 h and then filtered through Celite. The filtrate was neutralised with 10% aqueous sodium carbonate, mixed with water (10 cm3) and extracted with dichloromethane (3 x 30 cm3). The combined extracts were dried and evaporated under reduced pressure. The residual oil was purified by flash silica chromatography, eluting with dichloromethane-ethyl acetate (24 :l), followed by preparative TLC, eluting with dichloromethane-ethyl acetate (4: l), to give the pyrrole ester 9 (88.6 mg, 779, mp 56-57 “C (from hexane) (see later for spectroscopic data) and the formyl pyrrole 10 (117.5 mg, 11.7%), mp 63-64°C (from dichloromethane-diethyl ether-hexane) (Found: M +,473.1867. C24H3 ,NO,Si requires M, 473.1870); A,,,(CH,Cl,)/nm 302 and 232; v,,,(CH,Cl,)/cm-’ 3395, 2935, 1720, 1650, 1190, 1 165 and 835;dH(CDC1,,400 MHz) -0.01 (9 H, s, SiMe,), 0.96 (2 H, t, J9, CH,Si), 2.54 and 3.01 (each 2 H, t, J8,CH,CH,CO,), 3.60 (3 H, s, OMe), 3.78 (2 H, s, CH,C02), 4.15 (2 H, t, J9,0CH2CH,Si), 5.31 (2 H, s, CH,Ph), 7.30-7.40 (5 H, m, Ph), 9.73 (1 H, s, CHO) and 10.01 (1 H, br s, NH); dc(CDC13, 100 MHz) 1.61, 17.25, 19.66, 29.51, 34.29, 51.53, 63.75, 67.02, 123.89, 125.54, 128.65, 130.60,135.01,159.92,170.61,173.21 and 179.60;m/z(FD)473.3-(2-Methoxycarbonylethyl)-5-methyl-4-(2-~imethy~si~y~-ethoxycarbonylmethyl)pyrrole-2-carboxylicacid 11 To a solution of the pyrrole ester 9 (100 mg, 0.22 mmol) in J.Chem. Soc., Perkin Trans. 1,1996 2083 tetrahydrofuran (2 cm3) was added 10% palladium-on-charcoal (10 mg). The mixture was hydrogenated at room temperature for 2.5 h, filtered through Celite and evaporated. The residue was recrystallised from diethyl ether-hexane to give thepyrrole acid 11 (79 mg, 98%), mp 105.5-106.5 "C (Found: M', 369.1602. C17H27N06Si requires M, 369.1608);GH(CDC13, 400 MHz) 0.01 (9 H, s, Me3%), 0.92 (2 H, t, J9, CH,Si), 2.23 (3 H, s, CMe), 2.62 and 3.03 (each 2 H, t, J 8, CH,CH,CO,), 3.41 (2 H, s, CH,CO,), 3.65 (3 H, s, OMe), 4.15 (2 H, t, J 9, CH,CH,Si) and 9.22 (1 H, br s, NH); G,(CDCl,, 100 MHz) -1.55 (Me,Si), 11.72 (CMe), 17.33 (CH,Si), 20.56, 30.13 and 34.66 (3 x CH,), 51.49 (OMe), 63.12 (OCH,), 115.12, 116.08, 132.61 and 132.95 (4 x pyrrole-C), 165.65 (C0,H) and 171.89 and 173.91 (2 x C0,Me); m/z (FD) 369 (M', 100%).9-Benzyloxycarbony1-2,7-bis(2-methoxycarbony1ethyl)-8-methoxycarbonylmethyI-4-methyl-3-(2-trimethy~si~y~ethoxy-carbonylmethyl)-4,5ddihydrodipyrrin-l( 10H)-one 14 A solution of pyrrole acid 11 (2.0 g, 5.42 mmol) in dichloromethane (30 cm3) was stirred vigorously with a solution of sodium hydrogen carbonate (1.35 g, 16.07 mmol) in water (25 cm3) under argon. An aqueous solution (60 cm3) of iodine (0.1 mol drn-,) and potassium iodide (0.2 mol dmP3) was added over 5 min and the resulting mixture was stirred for a further 2 min before addition of solid sodium metabisulfite to destroy the excess iodine.The organic layer was separated and the aqueous layer extracted with dichloromethane (3 x 30 cm3). The combined organic layers were dried and evaporated. Flash chromatography on silica, eluting with diethyl ether- hexane (1 :l), gave the or-iodopyrrole 12 as an oil which was used directly in the next step. A stirred solution of the iodopyrrole 12 and acetoxymethyl- pyrrole 13' (2.28 g, 5.41 mmol) in anhydrous dichloromethane (50 cm3) was cooled to 0 "C under argon. Stannic chloride (698 mm3, 5.96 mmol) was added dropwise. After 30 min saturated aqueous sodium hydrogen carbonate (25 cm3) was added and the mixture was stirred for a further 10 min.The organic layer was separated and the aqueous layer extracted with dichloromethane (5 x 25 cm3). The combined organic layers were dried and evaporated. The residual oil was dissolved in tetrahydrofuran (100 cm3) and water (10 cm3) and treated with toluene-p-sulfonic acid (1.5 g, 8.7 mmol) and silver acetate (250 mg, 1.5 mmol). The mixture was stirred under argon for 13 h, then mixed with water (400 cm3) and extracted with dichloromethane (4 x 150 cm3). The combined extracts were dried and evaporated and the residue was purified by flash chromatography on silica, eluting with diethyl ether followed by diethyl ether-ethyl acetate (1 :I), to give the luctam 14 as an oil (1.43 g, 37%) (Found: M', 712.3028.C,,H,,N,O,,Si requires M, 71 2.3027); A,,,(CH,Cl,)/nm 280; V,,,(CH,C~,)/C~-~ 3685, 2940,1720,1690,1600 and 1165;G,(CDC13, 400 MHz) 0.02 (9 H, s, Me,Si), 0.99 (2 H, t, J9, CH2Si), 1.32 (3 H, s, CMe), 2.40-2.70 (8 H, m, 2 x CH,CH,), 2.74 and 3.04 (2 H, ABq, J 15, 5-H,), 3.32 and 3.49 (2 H, ABq, J 17, CH,CO,), 3.51, 3.58 and 3.63 (each 3 H, s, OMe), 3.62 and 3.84 (2 H, ABq, J 17, CH,CO,), 4.18 (2 H, t, J 9, CH2CH2Si), 5.13 and 5.23 (2 H, ABq, J 12, CH2Ph),7.19(1H,s,lactam-NH),7.23-7.34(5H,m,Ph),10.18 (1 H, s, pyrrole-NH); Sc(CDC13, 100 MHz) -1.54 (Me,%), 17.36 (CH,Si), 19.26 and 19.67 (2 x CH,CH,CO,), 23.77 (CMe), 30.86, 31.15, 31.39, 33.36, 34.76 (2 x CH,CH,CO,, 2 x CH2C02, C-5), 51.48 (OMe), 51.77 (2 x OMe), 63.39 (CH,CH,Si), 64.31, 65.67 (C-4 and CH,Ph), 119.16, 121.91, 122.22,127.95,128.19,128.35,128.70,135.30,136.29and 151.53 (W),160.67 (a-C02) and 170.22, 171.84, 171.04, 173.47 and 173.96 (4 x CO, and CONH); m/z (FD) 712 (M', 100%).9-Benzyloxycarbonyl-3-carboxymethyl-2,7-bi~2-methoxy-carbonylethyl)-8-methoxycarbonylmethyl-4ethyl-4,5-dihydro-dipyrrin-l-(lOH)-one 7 Tetrabutylammonium fluoride trihydrate (1 18 mg, 0.37 mmol) 2084 J. Chem. SOC.,Perkin Trans. I, 1996 was added to a solution of the lactam 14 (88.7 mg, 0.12 mmol) in tetrahydrofuran (1 cm3) and the solution was stirred under argon at room temperature for 40 min. Water (6 cm') was added and the pH of the solution adjusted to 3.0-3.5 with dilute sulfuric acid. The solution was extracted with dichloromethane (2 x 5 cm3) and the combined organic extracts were dried and evaporated.The residue was recrystallised from dichlorometh- ane to give the acid 7(46.8 mg, 61%), mp 159-161 "C (Found: MH+, 613.2361. C31H36N2011 requires MH, 613.2397); GH(CDC13, 400 MHz) 1.34 (3 H, s, CMe), 2.34-2.64 (8 H, m, 2 x CH,CH,), 2.83 and 3.13 (2 H, ABq, J 15, 5-H,), 3.32 and 3.53 (2 H, ABq, J 17, CH,CO,), 3.49, 3.56, 3.57 (each 3 H, s, OMe), 3.60 and 3.80 (2 H, ABq, J 17, CH,CO,), 5.19 (2 H, s, CH,Ph), 7.25-7.39 (5 H, m, Ph), 7.51 (1 H, br s, lactam-NH) and 10.20 (1 H, br s, pyrrole-NH); G,(CDCI,, 100 MHz) 19.16 and 19.74 (2 x CH,CH,CO,), 23.66 (CMe), 30.91, 30.97, 31.11,32.75 and 34.86(2 x CH,CH,C02,2 x CH,CO,,C-5), 51.53, 51.69 and 51.82 (3 x OMe), 64.44 and 65.84 (C-4 and CHzPh), 118.95, 121.95, 122.36, 127.94, 128.05, 128.49, 128.89, 134.93, 136.16 and 152.48 (W),161.07 (a-CO,) and 172.36, 172.68, 173.43 and 173.74 (4 x CO, and CONH); m/z (FD) 612 (M', 100%).9-Benzyloxycarbonyl-2,7-bis(2-methoxycarbonylethyl)-& methox y car bon y lme t h yl-4-meth y l-3-(1-phen y le t hoxy- carbonylmethy1)4,5dihydrodipyrrin-l(10H)-one 15 N,N'-Dicyclohexylcarbodiimide (7.6 mg, 37 pmol) and 4- dimethylaminopyridine (0.7 mg, 6 pmol) were added to a solution of the acid 7 (15 mg, 25 Imol) in dichloromethane (1.5 cm3) and (S)-(-)-1-phenylethanol (30 mg, 245 pmol) and the mixture was stirred under argon at room temperature for 20 min. The solvent was evaporated and the residue was purified by preparative TLC, eluting with diethyl ether-methanol (19: l), to give the ester 15 as a foam (10.3 mg, 57%) (Found: M+, 716.2944.C39H44N201 , requires M, 71 6.2945); Amax(CH2- Cl,)/nm 280; G,(CDCl,, 400 MHz, two diastereoisomers in a ratio of cu. 1 :1.25) 1.28 and 1.31 (2 x 3 H, s, 5-Me), 1.54 and 1.55 (2 x 3 H, d, J 7, MeCH), 2.38-2.71 (18 H, m, 4 x CH,CH,, 2 x 5-H,), 2.97 and 3.06 (each 1 H, d, J 15, 5-HB), 3.34 and 3.89(2 H, ABq,J 17, CH,CO,), 3.37and 3.89(2 H,ABq,J17,CHZCO,),3.56(6H,s,2x OMe),3.57,3.59,3.61 and 3.62 (each 3 H, s, OMe), 3.52-3.66 (4 H, m, 2 x CH,CO,), 5.14 and 5.26 (2 H, ABq, J 12, CH2Ph), 5.15 and 5.26 (2 H, ABq, J 12, CH,Ph), 5.85 (1 H, q, J 6, CHMePh), 5.88 (1 H, q, J 7, CHMePh), 6.64 and 6.69 (2 x 1 H, br s, lactam-NH), 7.26-7.37 (20 H, m, 4 x Ph) and 9.88 and 9.89 (2 x 2 H, s, pyrrole-NH); Gc(CDCl3, 100 MHz): 19.27, 19.71, 19.86, 21.84, 21.98, 24.01, 24.13, 30.89, 31.28, 33.04, 33.26, 34.65, 51.62, 51.97, 52.02, 63.62, 65.71, 74.38, 74.48, 119.24, 121.95, 122.03, 122.55, 126.34, 126.46, 128.08, 123.33, 128.42, 128.48, 127.70, 128.74, 135.44, 136.37, 140.47, 140.63, 151.98, 160.54, 169.71, 169.93, 171.67, 172.15, 173.43, 173.55, 174.30 and 174.39; m/z (FD) 716.9-Benzyloxycarbonyl-2,7-bis(2-methoxycar~nylethyl)-~ methoxycarbonylmethyl-4-methyl-3-{(lS,2R,4S)-1,7,7-trimet hylbicyclo [2.2.1 3 heptan-2-yloxycarbonylmethyl}-4,5-dihydrodipyrrin-l(lOH)-one 16 A mixture of N,N'-dicyclohexylcarbodiimide(7.6 mg, 37 pmol), 4-dimethylaminopyridine (0.7 mg, 6 pmol) and a solution of the acid 7 (15 mg, 25 pmol) and (1s)-endo-borneol (75.6 mg, 0.49 mmol) in dichloromethane (1.5 cm3)was stirred under argon at room temperature for 35 min.The solvent was evaporated and the residue was purified by preparative TLC, eluting with diethyl ether-methanol (95:5), to give the ester 16 (13.5 mg, 73%) as an oil (Found: M', 748.3572. C,,H,,N,O,, requires M, 748.3571); A.,,,(CH2C12)/nm 280; d,(CDCl,, 400 MHz, 2 diastereoisomers, ratio 1 :1.1) 0.80 and 0.83 (each 3 H, s, bornyl Me), 0.86 (12 H, s, 4 x bornyl Me), 0.90-0.98, 1.18- 1.21, 1.66-1.90, 2.30-2.33 (14 H, m, 6 x bornyl CH, and 2 x CH), 1.35 (6 H, s, 2 x 4-Me), 2.46-2.75 (18 H, m, 4 x CH2CH2C02 and 2 x 5-HAH,), 3.06 (2 H, d, J 15, 2 x 5-HAHB), 3.38-3.66(4 H, m, 2 x CH2C02), 3.54, 3.62 and 3.65 (each 6 H, s, 2 x OMe), 3.86 and 3.87 (4 H, 2 x ABq, J 17, CH2C02), 4.844.96 (2 H, m, 2 x bornyl OCH) 5.14 and 5.26 (2 H, ABq, J 12, CH2Ph), 5.15 and 5.27 (2 H, ABq, J 12, CH2Ph), 6.84 and 6.87 (each 1 H, br s, lactam-NH), 7.26-7.36 (10 H, m, 2 x Ph) and 10.01 (2 H, br s, 2 x pyrrole-NH); 8,-(CDCI,, 100 MHz) 13.54, 13.59, 15.26, 18.81, 19.20, 19.65, 23.95, 27.06, 27.98, 30.80, 31.19, 31.28, 33.32, 34.62, 36.63, 36.70, 44.74, 47.88, 48.80, 48.86, 51.51, 51.81, 63.26, 65.59, 65.84, 81.85, 81.97, 119.14, 121.86, 122.34, 127.95, 128.16, 128.35, 135.22, 135.28, 136.28, 151.63, 151.68, 160.53, 170.51, 170.63, 171.66, 172.04, 173.45, 174.06 and 174.10; m/z (FD) 748.Methyl 2-(benzyloxycarbonyl)-4-(tert-butoxycarbonyl)-5-methylpyrrole-3-propionate20 A solution of sodium nitrite (1 7.46 g, 253 mmol) in water (3 1 cm3) was added dropwise to a stirred mixture of 1-benzyl 6-methyl 3-oxohexanedioate 18 (64.25 g, 243 mmol) (made from 17 by the method in ref. 19) in acetic acid (100 cm3) at such a rate that the temperature did not exceed 25 "C.After being stirred overnight at room temperature, the mixture, containing the oxime 19, was added dropwise to a stirred mixture of tert-butyl acetoacetate (45.37 g, 287 mmol) in acetic acid (100 cm3). At the same time a mixture of zinc dust (50 g) and ammonium acetate (50 g) was added at such a rate that it was always in excess and the temperature remained between 50 and 70 "C. The resulting mixture was stirred for 30 min, then diluted with ice-water (750 cm3), stirred for a further 30 rnin and filtered.The residue was washed with water (200 cm3) and dissolved in dichloromethane (250 cm3). The organic solution was washed with water (250 cm3) and then with 5% aqueous sodium carbonate, dried and evaporated. The residue was re- crystallised from diethyl ether to give the pyrrok ester 20 (35.3 g, 36%), mp 95-98°C (Found: M', 401.1829. C,,H,,NO, requires M, 401.1838); G,(CDCl,, 400 MHz) 1.54 (9 H, s, Bu'), 2.46 (3 H, s, 5-Me), 2.53 and 3.35 (each 2 H, t, J 8, CH,CH,CO,), 3.61 (3 H, s, OMe), 5.29 (2 H, s, CH,Ph), 7.30- 7.40 (5 H, m, Ph) and 9.15 (1 H, br s, NH); m/z (FD) 401 (M+, 100%). Methyl 2-benzyloxycarbonyl-4-iodo-5-methylpyrrole-3-propionate 22 A solution of pyrrole ester 20 (19.8 g, 49.3 mmol) in 1,2- dichloroethane (65 cm3) was treated with trifluoroacetic acid (4.94 cm3), heated under reflux with stirring for 75 min and then cooled to room temperature.Water (42 cm3) was added and after 10 rnin the acid 21 was collected by filtration, washed with dichloromethane then water and dried in uocuo for use directly in the next step. The acid 21 was added to a stirred solution of sodium hydrogen carbonate (12.4 g, 148 mmol) in water (82 cm3). When foaming began, 1,2-dichIoroethane (82 cm3) was added and the mixture was heated gently at reflux to dissolve the pyrrole. A solution of iodine (1 3.8 g, 109 mmol) and potassium iodide (16.4 g, 99 mmol) in water (82 cm3) was added over 5 min and the resulting mixture was heated under reflux with stirring for 30 min.Solid sodium metabisulfite was added to destroy excess iodine and the organic layer was separated. The aqueous layer was extracted with dichloromethane (60 cm3) and the combined organic layers were dried and evaporated to give the pale yellow iodopyrrole 22 (16.65 g, 7973, mp 130-131 "C (from diethyl ether) (Found: M', 427.0241. C,,Hl8NO4I requires M, 427.0239); G,(CDCI3, 400 MHz) 2.26 (3 H, s, 5-Me), 2.47 and 3.02 (each 2 H, t, J 8, CH2CH,C02), 3.63 (3 H, s, OMe), 5.28 (2 H, s, CH,Ph), 7.30- 7.39 (5 H, m, Ph) and 9.35 (1 H, br s, NH); m/z (FD) 427 (M', 100%). 5-Benzyloxycarbonyl-4-(2-methoxycarbonylethyl)-2-methyl-pyrrole-3-acetic acid 23 To a stirred solution of 57% aqueous hydriodic acid (125 cm3) at 0 "C was slowly added acetic anhydride (125 cm3) followed by 50% aqueous hypophosphorous acid (24.5 cm3).To this cooled solution was added finely powdered iodopyrrole 22(21.4 g, 50 mmol). The resulting mixture was stirred for 5 min and then glyoxylic acid monohydrate (13.81 g, 150 mmol) was added in 3 portions over 15 min. After a further 20 min at 0 OC, dichloromethane (425 cm3) was added and the solution was washed with water (500 cm3). The organic layer was separated and the aqueous layer was extracted with dichloromethane (3 x 330 cm3). The combined organic layers were washed with 5% aqueous sodium hydrogen sulfite (250 cm3) and then water (250 cm3), dried and evaporated.Crystallisation from dichloromethane-hexane gave the acid 23 (1 7.53 g, 9773 mp 145-146 "C (Found: MH', 360.1454. C,,H,,NO, requires A4 + H, 360.1447); 1,,,(CH,Cl,)/nm 278; v,,,(Nujol)/cm 3700-3000, 3300, 2993, 1735, 1705, 1665 and 1465; G,(CDCI,, 400 MHz) 2.20 (3 H, s, CMe), 2.52 and 2.99 (each 2 H, t, J 8, CH2CH2C02), 3.45 (2 H, s, CH2C02), 3.58 (3 H, s, OMe), 5.27 (2 H, s, CH,Ph), 7.33 (5 H, m, Ph) and 9.26 (1 H, br s, NH); G,-(CDC13, 100 MHz) 11.60 (CMe), 20.64, 29.66, 34.82 (3 x CH,), 51.48 (OMe), 60.06 (CH,Ph), 113.94, 116.72, 128.25, 128.37 (2 C), 128.59 (2 C), 130.94, 132.06 and 135.05 and 161.04, 173.74 and 177.01 (3 x M);(M) m/z (FD) 359 (M', 100%). Methyl 2-benzyloxycarbonyl-5-methyl-4-(2-trimethylsilyl-ethoxycarbonylmethyl)pyrrole-3-propionate 9 To a solution of the acid 23 (3.25 g, 9.05 mmol) in anhydrous dichloromethane (30 cm3) was added oxalyl chloride (3.16 cm3, 4.60 g, 36.24 mmol) dropwise, followed by 3 drops of anhydrous N,N-dimethylformamide.Vigorous effervescence ensued and the red solution was stirred at room temperature under argon for 30 rnin and then evaporated. A solution of the residue in anhydrous dichloromethane (10 cm3) was stirred with 2-(trimethylsily1)ethanol (2.68 g, 22.66 mmol) and 4- dimethylaminopyridine (1.33 g, 10.89 mmol) under argon for 20 min at room temperature and then evaporated. The residue was purified by flash chromatography on silica, eluting with hexane-diethyl ether (9 : I) followed by dichloromethane-ethyl acetate (95 : 5), and crystallisation from hexane to give pyrrole ester 9 (3.85 g, 93%), mp 56-57 "C (Found: M', 459.2076.C,,H,,NO,Si requires M, 459.2077); Ama,(CH2C12)/nm 278; v,,,(CH,Cl,)/cm~' 3425, 2925, 1760-1 640s, 1 165, 1065 and 835; d,(CDCI,, 400 MHz) 0.01 (9 H, s, Me3%), 0.96 (2 H, t, J9, CH2Si), 2.20 (3 H, s, CMe), 2.53 and 3.00 (each 2 H, t, J 8, CH,CH,CO,), 3.39(2H,s,CH,C02),3.60(3H,s,OMe),4.13 (2 H, t, J9, CH,CH,Si), 5.27 (2 H, s, CH,Ph), 7.36 (5 H, m, Ph) and 8.99 (1 H, br s, NH); G,-(CDCl,, 100 MHz) -1.54 (Me,Si), 1 1.66 (CMe), 17.34 (CH,Si), 20.68 ( CH,CH,C02), 30.12 (CH,CO2), 34.86 (CH,CH,CO,), 51.39 (OMe), 63.05 and 65.83 (CH,CH,Si and CH,Ph), 114.59, 116.68, 128.18, 128.30 (2 C), 128.57 (2 C), 130.84, 131.54, 136.23 (CX), 160.76 (2- C02) and 171.87 and 173.62 (2 x C0,Me); m/z(FD) 459 (M+, 100%). Methyl 5-acetoxymethyl-2-benzyloxycarbonyl-4-(2-~imethyl-silylethoxycarbonylmethyl)pyrrole-3-propionate24 A solution of sulfuryl chloride (559 mg, 335 mm3, 4.14 mmol) in anhydrous dichloromethane (4 cm3) was added dropwise over 1 rnin to a stirred solution of the methyl pyrrole 9 (2 g, 4.36 mmol) in anhydrous dichloromethane (16 cm3) under argon at 0°C.After 1 h at OOC, the solution was evaporated and a solution of the residue in acetic acid (20 cm3) was stirred with sodium acetate (1.18 g, 14.38 mmol) under argon at 60 "C for 90 min, then diluted with water (80 cm3) and extracted with dichloromethane (3 x 20 cm3). The combined organic extracts were washed with saturated aqueous sodium hydrogen J.Chem. SOC.,Perkin Trans. 1,1996 2085 carbonate (2 x 50 cm3) and then with water (30 cm3), dried and evaporated to give the acetoxyrnethylpyrrole 24 as fine needles (2.12 g, 9473, mp 64.5-65.5 "C (from diethyl ether- hexane) (Found: M', 517.2121. C,6H,,N08Si requires M, 5 17.21 32); &,ax(CH2C12)/nm 269; v,,,(CH,Cl,)/~rn-~ 3420, 2940, 1760-1650~, 1200, 1170, 1070 and 835; &(CDCl,, 400 MHz) 0.00 (9 H, s, Me,%), 0.95 (2 H, t, J9, CH,Si), 2.04 (3 H, s, Ac), 2.52 and 2.99 (each 2 H, t, J8, CH,CH,CO,), 3.49 (2 H, s, CH,CO,), 3.60 (3 H, s, OMe), 4.12 (2 H, t, J9, CH,CH,Si), 5.03 (2 H, s, CH,OAc), 5.28 (2 H, s, CH,Ph), 7.35 (5 H, m, Ph) and 9.18 (1 H, br s, NH); G,(CDCl,, 100 MHz) -1.56 (Me,Si), 17.33 (CH,Si), 20.40 (CH,CH,CO,), 20.85 (COMe), 29.81 (CH,C02), 34.70 (CH,CH,CO,), 51.44 (OMe), 56.91 (CH,OAc), 63.28 and 66.14 (CH2CH2Si and CH,Ph), 117.17, stannic chloride (140 mm3, 1.20 mmol) dropwise.The mixture was stirred for 2 h, then treated with 10% aqueous sodium acetate (20 cm3) and stirred for a further 10 min. The layers were separated and the aqueous layer extracted with dichloromethane (2 x 50 cm3). The combined organic layers were extracted with 10% aqueous sodium carbonate (3 x 50 cm3) and these aqueous extracts were acidified with concentrate hydrochloric acid to pH 1. The precipitated solid was collected by filtration and dissolved in dichloromethane (4 x 50 cm3). The solution was dried and evaporated and the residue was crystallised from dichloromethane-diethyl ether- hexane to give the acid 28 (233 mg, 78%), mp 140-142OC (Found: MH', 270.0963.C12HlSN06 requires M + H, 270.0978); 1,,,(CH,Cl,)/nm 268; v,,,(CH,Cl,)/~rn-~ 3685, 119.08, 128.30, 128.39 (2 C), 128.61 (2 C), 128.79, 129.97 and 1735,1660,1605,1200 and 1175; dH(CDCl,, 400 MHz) 2.62 and 135.93(C=C)and 160.43, 171.45, 171.51 and 173.49(4 x M);3.04 (each 2 H, t, J 8, CH,CH,CO,), 3.52 (2 H, s, CH,C02), m/z (FD) 517 (M+, 100%). 5-Acetoxymethyl-3-(2-methoxycarbonylethyl)~-(2-trimethyl-silylethoxycarbonylmethyl)pyrrole-2-carboxylicacid 25 A solution of the pyrrole ester 24 (1 g, 1.93 mmol) in tetrahydrofuran (7 cm3) was stirred with 10% palladium-on-charcoal (0.1 g) under an atmosphere of hydrogen at room temperature for 90 min and then filtered through Celite.The residue was washed with tetrahydrofuran (3 cm3) and the combined filtrate and washings were evaporated and the residue was recrystallised from methyl acetate-hexane to give the acid 25 (0.81 g, 98%), mp 108-1 10 "C (decomp.) (Found: M', 427.1660. C19H,,N0,Si requires M,427.1662);;Imax(CH2-Cl,)/nm 276; v,,,(CH,Cl,)/cm-' 3420, 3300-2500, 2940, 1725 br, 1660, 1170 and 835; dH(CDCl,, 400 MHz) 0.02 (9 H, s, Me,Si), 0.98 (2 H, t, J8, CH,Si), 2.07 (3 H, s, Ac), 2.61 and 3.03 (each 2 H, t, J 8, CH,CH,CO,), 3.52 (2 H, s, CH,CO,), 3.65 (3 H, s, OMe), 4.15 (2 H, t, J 8, CH,CH,Si), 5.07 (2 H, s, CH,OAc) and 9.30 (1 H, br s, NH); G,(CDCl,, 100 MHz) -1.56 (Me,%), 17.33 (CH,Si), 20.27 (CH,CH,CO,), 20.85 56.97 (CH,OAc), 63.36 (CH,CH,Si), 117.41, 118.42, 129.98 and 131.59 (CX) and 165.43, 171.55 (2 C) and 173.71 (4 x M);m/z (FD) 427 (M', 100%).Methyl 5-acetoxymethyl-2-tevt-butoxycarbonyl-4-(2-trimethyl-silylethoxycarbonylmethyl)pyrrole-3-propionate26 A solution of the acid 25(1 g, 2.34 mmol) in tert-butyl alcohol (8 cm3) and dichloromethane (8 cm3) was stirred with a solution of N,N'-dicyclohexylcarbodiimide (0.58 g, 2.8 1 mmol) in tert- butyl alcohol (2 cm3) under argon for 90 min and then evaporated. The residue was purified by flash chromatography on silica, eluting with diethyl ether-hexane (1 :l), and crystallisation from diethyl ether-hexane to give the pyrrole ester 26 (1.09 g, 96%) as needles, mp 69-71 "C (Found: M ,+ 483.2297.C23H37N08Si requires M, 483.2288); Amax(CH2- Cl,)/nm 269; ~,,,(CH,C~,)/C~-~ 3430, 1723s br, 1690, 1170 and 835; dH(CDC1,, 400 MHz) 0.01 (9 H, s, Me,Si), 0.97 (2 H, t, J9, CH,Si), 1.54 (9 H, s, Bu'), 2.05 (3 H, s, Ac), 2.55 and 2.97 (each 2 H, t, J8, CH,CH,CO,), 3.49 (2 H, s, CH,CO,), 3.64 (3 H, s, OMe), 4.13 (2 H, t, J 9, CH,CH,Si), 5.04 (2 H, s, CH,OAc) and 9.13 (1 H, br s, NH); Gc(CDC13, 100 MHz) -1.55 (Me,Si), 17.34 (CH,Si), 20.50 (CH2CH,C02), 20.87 (COMe), 28.38 (CMe,), 29.85 (CH,CO,), 34.95 (CH,-CH,CO,), 51.44 (OMe), 56.95 (CH,OAc), 63.24 (CH,CH,- Si), 81.28 (CMe,), 116.84, 120.81, 127.81 and 128.30 (C=C) and 160.36, 171.46, 171.63, 173.60 (4 x C=O); m/z (FD) 483 (M', 100%). 3-(2-Methoxycarbonylethyl)-4-(methoxycarbonylmethyl)-pyrrole-Zcarboxylicacid 28 A solution of the pyrrole ester 27,' (360 mg, 1.11 mmol) in dry dichloromethane (20 cm3) was cooled to 0 "C and treated with 2086 J.Chem. Soc., Perkin Trans. 1,1996 (Found: MH', (COMe),29.81(CH,CO2),34.54(CH,CH,CO,),51.53(OMe),53 1.8608); R,,,(CH,Cl,)/nm 3.65and3.69(each3H,s,OMe),6.91(lH,d,J3,5-H)and9.27 (1 H, br s, NH); Gc(CDC13, 100 MHz) 20.21 (CH,CH,CO,), 30.48 (CH,CO,), 34.56 (CH,CH,CO,), 51.56 and 52.06 (OMe), 117.69, 118.54 and 131.31 (W),122.95 (C-5) and 165.72, 172.38 and 173.84 (3 x C=O). Methyl4-methoxycarbonylmethyl-2-(2,2,2-tribromoethoxy-carbonyl)pyrrole-3-propionate29 A solution of acid 28 (150 mg, 0.558 mmol) in dry dichloromethane (4 cm3) was cooled to 0 "C, treated with oxalyl chloride (146 mm3, 1.67 mmol) and N,N-dimethylforma- mide (3 drops) (vigorous effervescence), stirred under argon at 0 "C for 30 min and evaporated.The residue was dissolved in dichloromethane (2 cm3), stirred with 2,2,2-tribromoethanol (1.18 g, 4.1 8 mmol) and 4-dimethylaminopyridine (8 1.7 mg, 0.669 mmol) under argon for 30 min and then evaporated under reduced pressure. The residue was purified by flash chromatography on silica, eluting with diethyl ether-hexane (1 :I), to give the tribromoethyl ester 29 as an oil (198 mg, 67%) 531.8643. C,4H,,79Br,N0, requires M + H, 274; v,,x(CH,Cl,)/cm-' 3450, 1740, 1570, 1510, 1410, 1120 and 940; GH(CDCl,, 400 MHz) 2.64 and 3.09 (each 2 H, t, J 8, CH,CH,CO,), 3.53 (2 H, s, CH,CO,), 3.63 and 3.69 (each 3 H, s, OMe), 5.10 (2 H, s, CH,CBr,), 6.96 (1 H, d, J 3, 5-H) and 9.03 (1 H, br s, NH); Gc(CDCl,, 100 MHz) 20.24 (CH,CH,CO,), 30.44 (CH,CO,), 34.98 (CH,CH2CO2), 35.83 (CH2CBr3), 51.52 and 52.07 (OMe), 76.87 (CH,CBr,), 117.78, 117.85 and 130.97 (C=C), 123.29 (C-5) and 159.00, 172.28 and 173.55 (3 x C=O).Dimethyl 1-(?er?-butoxycarbonyl)-7-methoxycarbonylmethyl-9-(2,2,2-tribromoethoxycarbonyl)3-(2-trimethylsi~ylethoxy-carbonylmethyl)-5,1O-dihydrodipyrrin-2,8-dipropionate30 A solution of the a-free pyrrole 29(599 mg, 1.12 mmol) and the acetoxymethylpyrrole 26 (542 mg, 1.I2 mmol) in dichlorometh- ane (7 cm3) was stirred with toluene-p-sulfonic acid (21 mg, 0.11 mmol) under argon at room temperature for 2 h, then washed with 5% aqueous sodium carbonate (2 x 15 cm3) followed by water (1 x 15 cm3), dried and evaporated.The residue was purified by flash chromatography on silica, eluting with diethyl ether-hexane (1 :l), to give the dihydrodipyrrin 30 as a foam (893 mg, 84%) (Found: M', 954.0590. C35H49Br3- N,O,,Si requires M, 954.0605); d,(CDCl,, 400 MHz) 0.05 (9 H, s, Me,Si), 1.02 (2 H, t, J 8, CH,Si), 1.51 (9 H, s, But), 2.52, 2.58, 2.98 and 3.10 (each 2 H, t, J 8, 2 x CH,CH,CO,), 3.52 and 3.59(each 2 H,s,CH,CO,), 3.62, 3.63 and 3.77(each 3 H, s, OMe), 3.82 (2 H, s, 5-H,), 4.26 (2 H, t, J 8, CH,CH,Si), 5.06 (2 H, s, CH,CBr,) and 10.1 1 and 10.47 (each 1 H, br s, NH); dc(CDC13, 100 MHz) -1.44 (Me,Si), 17.27 (CH,Si), 20.40 and 22.58 (2 x CH,CH,CO,), 28.39 (CMe,), 29.24 and 29.76 (2 x CH,CO,), 35.00 (2 C)and 35.19 (2 x CH,CH,C02 and CH,CBr,), 51.41 (2 C) and 52.65 (3 x OMe), 64.02 (CH,CH,Si), 77.00 (CH,CBr,), 80.49 (CMe,), 113.67, 114.22, 117.04, 119.90, 128.43, 130.63, 130.78 and 134.09 (C=C) and 158.84, 160.20, 173.56, 173.66 (2 C) and 173.74 (6 x GO);m/z (FD)954,956,958 and 960 (1 :3 :3 :1, M+, 100%). 2,&Bis(2-methoxycar~nylethyl)-7-methoxycarbony~ethyl-9-(2,2,2-tribromoethoxycarbonyl)-3-(2-trimethylsilylethoxy-carbonylmethyl)-5,lO-dihydrodipyrrin-l-carboxylicacid 31 A solution of the dipyrromethane 30 (4.71 g, 4.92 mmol) in dry dichloromethane (33 cm3) was stirred under argon at between -10 and -15 "C and stannic chloride (432 mm3, 3.69 mmol) was added dropwise over 1 min.The solution was allowed to warm to 5 "C over 40 rnin and then treated with 5% aqueous sodium acetate (47 cm3). After a further 5 min, the organic layer was separated and the aqueous layer was extracted with dichloromethane (5 x 50 cm3). The combined organic layers were washed with brine (2 x 50 cm3), dried and evaporated. The residue was purified by flash chromatography on silica, eluting with dichloromethane-methanol (19 :l), to give the acid 31 (2.78 g, 63%), mp 156155 "C (from methyl acetate- hexane); L,,,(CH,Cl,)/nm 287; v,,,(CH,Cl,)/cm-' 3400-2800, 3310, 2950, 1750-163Os, 1230, 1165 and 890; GH(CDCl3, 400 MHz) 0.05 (9 H, s, Me,Si), 1.01 (2 H, t, J 8, CH,Si), 2.56, 2.60, 3.02 and 3.10 (each 2 H, t, J 8, CH,CH,CO,), 3.54 and 3.61 (each 2 H, s, CH,C02), 3.62, 3.63 and 3.77 (each 3 H, s, OMe), 3.88 (2 H, s, 5-H,), 4.24 (2 H, t, J 8, CH,CH,Si), 5.05 (2 H, s, CH,CBr,) and 10.34 and 10.66 (each 1 H, br s, NH); m/z (FD) 898, 900, 902 and 904 (1:3:3:1,M+, 100%). 4-[5-Benzyloxycarbonyl-3-(2-methoxycarbonylethyl)-4-met hox ycar bon y lmethylp yrr ol-2-y lmeth yl ]-2,8-bis( 2-met hoxy- carbonylethyl)-7-methoxycarbonylmethyl-9~2,2,2-t~bromo-ethoxycarbony1)-3-(2-trimethylsil ylethox ycarbonylmet hy1)- 4,5dihydrodipyrrin-l(lOH)-one 33 A solution of the acid 31 (250 mg, 0.278 mmol) in dichloromethane (1.7 cm3) was stirred vigorously with a solution of sodium hydrogen carbonate (69.9 mg, 0.832 mmol) in water (1.3 cm3) and an aqueous solution (2.78 cm3) of iodine (0.1 mol dmP3) and potassium iodide (0.2 mol dm-3) was added over 5 min.After a further 35 min, solid sodium metabisulfite was added to destroy the excess iodine, the organic layer was separated and the aqueous layer was extracted with dichloromethane (3 x 1 cm'). The combined organic layers were dried and evaporated under reduced pressure. The residual oil was purified by flash silica chromatography on silica, eluting with diethyl ether-hexane (3:2), to give the iododihydrodipyrrin 32 as a foam (241 mg) which was used directly in the next step. A stirred solution of the iododihydrodipyrrin 32 (239 mg, 0.243 mmol) and the acetoxymethylpyrrole8 13 (105 mg, 0.243 mmol) in dry dichloromethane (2.5 cm3) under argon at 0 "C and was treated with a solution of stannic chloride (28.4 mm3, 0.243 mmol) in dichloromethane (1 ~rn-~)and then, after 30 min, with saturated aqueous sodium hydrogen carbonate (2.6 cm3).After a further 10 min, the organic layer was separated and the aqueous layer extracted with dichloromethane (3 x 5 cm3). The combined organic layers were dried and evaporated under reduced pressure. The residual oil was dissolved in tetrahydrofuran (3.4 an3)and water (0.34 cm3) and toluene- p-sulfonic acid monohydrate (66.2 mg, 0.35 mmol) and silver acetate (21.9 mg, 0.13 mmol) were added. The mixture was stirred under argon for 17 h, then diluted with water (1 8 cm3) and extracted with dichloromethane (4 x 18 cm3). The combined organic extracts were dried and evaporated and the residual oil was purified by flash chromatography on silica, eluting with diethyl ether-hexane (97 :3) followed by diethyl ether, to give the dipyrrolic lactam 33 as a foam (104 mg, 30%); &,,,(CH,Cl,)/nm 284; v,,,(CH,Cl,)/cm-' 34 10, 3290, 2940, 1710s, br, 1190,1170 and 1075;GH(CDC13, 400 MHz) 0.05 (9 H, s, Me3Si), 1.05 (2 H, t, J 8, CH,Si), 2.43-2.55 and 2.69-2.71 (each 6 H, m, 3 x CH,CH,CO,), 2.74 and 3.04 (2 H, ABq, J 15) and 2.85 and 3.13 (2 H, ABq, J 15, CH,CCH,), 3.14 and 3.49 (2 H, ABq, J 16), 3.38 and 3.66 (2 H, ABq, J 18) and 3.73 and 3.83 (2 H, ABq, J 17, 3 x CH,CO,), 3.54, 3.59, 3.59, 3.60 and 3.63 (each 3 H, s, OMe), 4.29 (2 H, t, J 8, CH,CH,Si), 5.01 and 5.11 (2 H, ABq, J 12) and 5.18 and 5.27 (2 H, ABq, J 12, CH,CBr, and CH,Ph), 7.29-7.39 (5 H, m, Ph), 7.54 (1 H, br s, lactam-NH) and 9.22 and 10.36 (each 1 H, br s, pyrrole-NH); SC(CDCl3, 100 MHz) -1.47 (Me3Si), 17.34 (CH,Si), 19.28, 19.92 and 20.39 (3 x CH2CH2C02), 29.02, 30.46 (2 C), 30.58, 30.93, 32.95, 34.73, 34.98 and 36.07 (3 x CH,CO,, 3 x CH,-CH,C02, CH,CCH, and CH,CBr,), 51.47, 51.58, 51.80, 51.88 and 52.34 (5 x OMe), 65.17 and 65.73 (2 C) (CH,CH,Si, CH,Ph and C-4), 76.88 (CH,CBr,), 115.99, 116.83, 119.44, 122.18, 122.51, 127.60, 128.18, 128.42, 128.49, 130.09, 136.00, 138.51 and 149.10 (W),158.54 and 160.1 1 (pyrrole-CO,) and 171.60, 171.75, 172.02, 173.35, 173.42 and 173.59 (6 x CO, and CONH); m/z (FD) 1241, 1243, 1245 and 1247 (1: 3:3: 1, M+, 100%).4-[5-Benz ylox ycarbonyl3-(2-methoxycar bon ylet hyl)-4- methoxycarbonylmethyIpyrrol-2-ylmethyl]-7-carboxymethyl-2,&.bis(2-methoxycarbonylethyl)-9-(2,2,2-~ibrom~~oxy-carbonyl)-3-(2-trimethylsilylethoxycarbonylmethyl)~,~ dihydrodipyrrin-l(lOH)-one 6 A solution of ester 33 (419 mg, 0.33 mmol) in tetrahydrofuran (12 cm3) was stirred with tetrabutylammonium fluoride trihydrate (316 mg, 1.00 mmol) under argon at room temperature for 1 h, then mixed with dichloromethane (15 cm3) and washed with dilute sulfuric acid (pH 3.0-3.5; 2 x 10 cm3) followed by water (10 cm3), dried and evaporated.The residue was purified by flash chromatography on silica, eluting with dichloromethane-methanol (9 :l), to give the acid 6 as a glass (271 mg, 71%) which was used without further purification to make the following series of esters; G,(CDCl,, 400 MHz) 2.21-3.93 (37 H, series of br m, 3 x CH,CO,, 3 x CH,CH,CO,, CH,CCH, and 5 x OMe), 4.95-5.33 (4 H, br m, CH,Ph and CH,CBr,), 7.20-7.46 (5 H, m, Ph), 8.00 (1 H, br s, lactam-NH) and 9.45 (2 H, br s, 2 x pyrrole-NH); m/z (FD) 1141, 1143, 1145 and 1147 (1:3:3:1, M+, 50%) and 1097, 1099, 1101 and 1103 (1:3:3:1,M -C02 100%).4-[5-Benzyloxycarbonyl-3-(2-methoxycarbonylethyl)-4-methoxycarbonylmethyIpyrrol-2-ylmethyl]-2,8-bis(2-methoxy-carbonylethyl)-7-methoxycarbonylmethyl-3-[(1 S)-1-phenyl- ethoxycarbonylmethyl]-9-(2,2,2-tribromoethoxycarbonyl)-4,5-dihydrodipyrrin-l( 10H)-one 34 A solution of acid 6 (1 4.6 mg, 13 pmol) in dry dichloromethane (0.5 cm3) was stirred with 1-chloro-1-dimethylamino-2-methylprop-1-ene l3 (3.42 mg, 20 pmol) under argon at room temperature for 20 rnin and then treated with (S)-(-)-1- phenylethanol (18.0 mg, 147 pmol).After a further 2.5 h, the solvent was evaporated under reduced pressure and the residue was purified by preparative TLC, eluting with dichloromethane-methanol(l9: l), to give recovered acid 6 (3.2 mg, 22%) and ester 34 (6.0 mg, 36%) as an oil, shown by 'H NMR to be a mixture of two diastereoisomers (1 : l), contaminated with byproducts giving NMR signals indicating that they had a 2,2-dibromoethenyl group and a 2,2-dibromoethyl group, respectively, replacing the 2,2,2-tribromo- ethyl residue of the main products, This interpretation is supported by the isolation and characterisation of analogous byproducts in a later experiment; A,,,(CH,Cl,)/nm 279 and 219; GH(CDC13, 400 MHz) 1.59-1.63 (6 H, m, 2 x PhCHMe), 2.33-3.14 and 3.32-3.85 (44 H, series of m, 6 x CH,CH2CO2, 2 x CH,CCH, and 6 x CH,C02), 3.47-3.64 (30 H, m, 10 x OMe), 5.00-5.12 (4 H, m, 2 x CH2CBr3) and 5.17 and 5.27 (each 2 H, ABq, J 13, 2 x CH,Ph), 5.98-6.05 (2 H, m, 2 x PhCHMe), 7.29-7.37 (20 H, m, 4 x Ph), 7.48 and 7.53 J.Chem. SOC.,Perkin Trans. 1,1996 2087 (each 1 H, br s, lactam-NH), 9.17 and 9.27 (each 1 H, br s, pyrrole-NH) and 10.16 (2 H, br s, 2 x pyrrole-NH) [byproducts: 4.63-4.74 (m, 2 x CH,CHBr,) and 5.83 (m, 2 x CHBr,) (ca. 22%); 8.01 and 8.06 (each s, 2 x CHSBr,) (ca.22%)];rn/z(FD)1245,1247,1249and1251 (1:3:3:1,M+, 100%).4-[5-Benzyloxycarbonyl-3-( 2-me thoxycarbonylethyl)-4- methoxycarbonylmethyIpyrrol-2-ylmethyl]-2,8-bis(2-methoxy-carbonylethyl)-7-methoxycarbonylmethyl-9-(2,2,2-tribromo-ethoxycarbony1)-3-{( lS,2R,4S)-l,7,7-trimethylbicyclo[2.2.1 ]-heptan-2-yloxycarbonylmethyl}-4,5-dihydrodipyrrin-1(1OH)-one 35 A solution of acid 6(1 1 mg, 10 pmol) in dry dichloromethane (0.5 cm3) was stirred with 1-chloro-1 -dimethylamino-2- methylprop-1-ene (2.6 mg, 19 pmol) under argon at room temperature for 30 min and then treated with (1 S)-endo-borneol (27 mg, 220 pmol). After a further 2 h, the solvent was evaporated under reduced pressure and the residue was purified by preparative TLC, eluting with dichloromethane-methanol (24: l), to give recovered acid 6 (3.2 mg, 29%) and ester 35 (4.1 mg, 32%) as an oil, shown by 'H NMR to be a mixture of two diastereoisomers (1 : 1) contaminated with byproducts anal- ogous to those reported in the foregoing experiment; J.max(CH,Cl,)/nm 285 and 218; GH(CDC13, 400 MHz) 0.84 and 0.86 (each 3 H, s, 2 x bornyl-Me), 0.87 (12 H, s, 4 x bornyl-Me), 0.98-1.07, 1.24-1.43, 1.58-1.83, 1.93-2.03 and 2.31-2.42 (14 H, m, 6 x bornyl-CH, and 2 x bornyl-CH), 2.42-2.57, 2.70-2.74 and 2.82-3.05 (28 H, m, 6 x CH,CH,C02 and 2 x 5-CH2), 2.85 and 3.17 (each 1 H, ABq, J 15, 5-CH2), 2.85 and 3.17 (each 1 H, ABq, J l5,5-CH2), 3.16 and 3.43 (each 1 H, ABq, J 15, CH,CO,), 3.16 and 3.44 (each 1 H, ABq, J 15, CH,CO,), 3.48-3.75 (6 H, m, 3 x CH,CO,), 3.54-3.64(30 H, m, 10 x OMe), 3.65 and 3.66 (each 1 H, ABq, J 15, CH,CO,), 4.97-5.16 (6 H, m, 2 x CH,CBr3 and 2 x bornyl-CHO), 5.18-5.27 (4 H, 2 x ABq, J 12, 2 x CHZPh), 7.26-7.39 (10 H, m, 2 x Ph), 7.54 (2 H, s, 2 x lactam-NH), 9.28 and 10.28 (each 2 H, br s, 4 x pyrrole-NH) [impurities: 4.604.76 (ABq of d, J 11 and 7, 2 x CH2CHBr2) and 5.79 and 5.80 (each t, J 7, 2 x CHBr,) (ca.21%); 8.01 (s, 2 x CH=CBr,) (ca.22701; m/z (FD) 1277,1279, 1281 and 1283 (1:3:3: 1, M+, 100%). 4-[5-Benzyloxycarbonyl-3-( 2-methoxycar bony le thyl)-4- methoxycarbonylmethylpyrrol-2-ylmethyl]-3-[(3R)-4,4-dimethyl-2-oxotetrahydrofuran-3-yloxycarbony~methyl]-2,8-bis(2-methoxycarbonylet hyl)-9-methoxycarbonylmethyl-9-(2,2,2-tribromoethoxycarbonyl)-4,5-dihydrodipyrrin-l(1OH)-one 36 A solution of acid 6(21.1 mg, 22 pmol) in dry dichloromethane (1 cm3) was stirred with 1-chloro-1 -dimethylamino-2- methylprop-1 -ene (12.1 mg, 90 pmol) under argon at room temperature for 20 min, then treated with (R)-(-)-pantolac-tonel (80 mg, 0.61 mmol).After a further 30 min, the solvent was evaporated and the residue was purified by preparative TLC, eluting with dichloromethane-methanol (9 : I), to give recovered acid 6(7 mg, 33%) and the ester 36(6 mg, 25%) as an oil, shown by 'H NMR to be a mixture of two diastereoisomers (1 : 1) contaminated with small amounts of dibromo byproducts analogous to those seen earlier; Amax(CH2C12)/nm 284 and 218. This mixture was purified by HPLC on a Spherisorb S5CN semi-preparative column, eluting with diethyl ether+thyl acetate (3 : I), to give the pure separate diastereoisomers of esler 36as oils.First eluted diastereoisomer: G,(CDCl,, 400 MHz) 1.19 and 1.22(each 3 H, s, CMe,), 2.49-2.57 and 2.68-3.23 (1 7 H, series of m, 3 X CH2CH2CO2, CH,CCH2 and CHAHBC02), 3.50 (1 H, d, J 17, CHAHBCO,), 3.57, 3.58, 3.59, 3.61 and 3.64 (each 3 H, s, OMe), 3.64-3.88 (4 H, m, 2 x CH,CO,), 4.04-4.09 (2 H, ABq, J 9, lactone-CH,), 5.06 (2 H, s, CH,CBr,) and 5.18-5.28 (2 H, ABq, J 12, CH,Ph), 5.46 (1 H, s, lactone-CH), 7.28-7.39 2088 J. Chem. SOC.,Perkin Trans. I, 1996 (5 H, m, Ph), 7.43 (1 H, br s, lactam-NH) and 9.35 and 9.92 (each 1 H, br s, pyrrole-NH); m/z (FD) 1253, 1255, 1257 and 1259(1:3:3:1,Mf, 100%). Second eluted diastereoisomer: G,(CDCl,, 400 MHz) 1.22 and 1.27 (each 3 H, s, CMe,), 2.45-2.56, 2.71-2.78 and 2.95- 3.21 (17 H, series of m, 3 x CH,CH,CO,, CH,CCH2 and CHAHBCO,),3.50(1 H, d, J 16, CHAHBCO~), 3.56, 3.58, 3.60, 3.61 and 3.63 (each 3 H, s, OMe), 3.69-3.96 (4 H, m, 2 x CH2C02), 4.05-4.10 (2 H, ABq, J 9, lactone-CH,), 5.05 (2 H, s, CH2CBr3) and 5.19-5.28 (2 H, ABq, J 12, CH,Ph), 5.50(1 H, s, lactone-CH), 7.30-7.42 (5 H, m, Ph), 7.67 (1 H, br s, lactam-NH) and 9.09 and 10.14 (each 1 H, br s, pyrrole-NH); m/z(FD) 1253, 1255, 1257and 1259(1:3:3:1,M+, 100%).4-[5-Benzyloxycarbonyl-3-(2-methoxycarbonylethyI)-4-methoxycarbonylmethyIpyrrol-2-ylmethyl]-3-[(2R,3R,4R)-3,4-isopropylidenedioxy-50x0tetrahydrofuran-2-ylmethoxy-carbonylmethyl]-2,8-bis(2-methoxycarbonylethyl)-7-methoxy-carbonylmethyl-9-(2,2,2-tribromoethoxycar~ny~)-4,5dihydro-dipyrrin-l(lOH)-one 37 A solution of acid 6 (8.39 mg, 8 pmol) in dry dichlorometh- ane (0.5 cm3) was stirred with 1 -chloro-1 -dimethylamino-2- methylprop-1-ene l3 (2.95 mg, 22 pmol) under argon at room temperature for 10 min, then treated with 2,3-0-isopropylidene-D-(+)-ribono- 1 ,$-lactone (24.4 mg, 0.10 mmol).After a further 1 h, the solvent was evaporated and the residue purified by preparative TLC, eluting with dichloromethane-methanol(l9:I), to give the ester 37(4.3 mg, 43%) as an oil, shown by 'H NMR to be a mixture of two diastereoisomers (1 :1) contaminated with small amounts of dibromo byproducts analogous to those seen earlier; A,,,(CH,Cl,)/nm 284 and 218 nm; GH(CDC13, 400 MHz) 1.40 and 1.49 (12 H, 2 x s, 2 x CMe,), 2.33-2.56, 2.68-2.72, 2.83- 2.86, 2.98-3.06, 3.14-3.19 and 3.41-3.79 (78 H, series of m, 6 x CH,CH,CO,, 2 x CH,CCH,, 6 x CH,CO, and 10 x OMe), 4.22-4.40 (4 H, m, 2 x lactone-CH,), 4.76-4.83 (4 H, m, 2 x lactone-3- and -4-H), 4.99 and 5.01 (2 H, ABq, J9, CH,CBr,), 5.04-5.06 (4 H, m, CH,CBr, + 2 x lactone-2-H), 5.17 and 5.26 (2 H, ABq, J 12, CH,Ph), 5.18 and 5.26 (2 H, ABq, J 12, CH,Ph), 7.27-7.38 (10 H, m, 2 x Ph), 7.51 and 7.67 (each 1 H, br s, lactam-NH) and 9.18,9.41,9.96 and 10.17 (each 1 H, br s, pyrrole-NH) [impurities: 4.65-4.71 (m, CH,CHBr,) and 5.04-5.06 (m, CHBr,) (ca.15%); 8.01 and 8.02 (each s, CHXBr,) (ca. 25%)]; m/z (FD) 1311, 1313, 1315 and 1317 (1:3:3:l,Mf, 100%).3-{(3R,4R,5R)-4,5-[(R)-Benzylidenedioxy]-2-oxotetrahydro-pyran-3-yloxycarbonylmethyl}-4-[5-benz y lox ycarbonyl-3-(2-methoxycarbonylethyl)-4-methoxycarbonylmethy~pyrrol-2-ylmet hyl] -2,8-b~s(2-methoxycarbonylethyl)-7-methoxycarbony~-methyl-9-(2,2,2-tribromoethoxycarbonyl)~,5-dihydrodipyrrin-1(1OH)-one 38 A solution of acid 6 (500 mg, 0.438 mmol) in dry dichloromethane (20 cm3) was stirred with l-chloro-l-dimethylamino-2-methylprop-1 -ene (221 mg, 1.58 1 mmol) under argon at room temperature for 9 min, then treated with 3,4-0-benzylidene-~-ribonic6-lactone (550 mg, 2.328 mmol). After a further 3 h, the solvent was evaporated and the residue purified by flash chromatography on silica, eluting with dichloromethane-methanol (1 9 :1), to give a mixture of the diastereoisomeric esters 38as an oil (537 mg, 90%).An aliquot (106 mg) was purified by HPLC on a Spherisorb S5CN semi- preparative column, eluting with diethyl ether-ethyl acetate (3 :l), to give four fractions, as follows. (i) The ester 38b (peak Y in Fig. 1) as an oil (27.6 mg, 23%) (Found: MH+, 1360.1447. C,,H,,Br,N,O,, requires M + H, 1360.1349); A,,,(MeCN)/nm 283; &(CDCl,, 400 MHz) 2.35- 2.67 (10 H, m, CH,CH,CO,), 2.82 and 3.07 (2 H, ABq, J 15) and 2.86 and 3.10 (2 H, ABq, J 15, CH,CCH,), 3.00 (2 H, m, CH,CH,CO,), 3.32 and 3.42 (2 H, ABq, J 17, CH,CO,), 3.51, 3.54, 3.55, 3.58, 3.60 (each 3 H, s, OMe), 3.68 and 3.79 (2 H, ABq,J 18,CH,CO,), 3.69 (2 H, s, CH,CO,),4.33 and 4.53 (2 H, ABq, J 13, lactone-6-H2), 4.55 (1 H, d, J 8, lactone-5-H), 4.81 (1 H, dd, J 8 and 3,lactone-4-H), 5.00 and 5.06 (2 H, ABq, J 12, CH,CBr,), 5.12 and 5.19 (2 H, ABq, J 12, CH,Ph), 5.61 (1 H, d, J 3,lactone-3-H), 5.74 (1 H, s, CHPh), 7.25-7.43 (1 0 H, m, 2 x Ph) 7.48 (1 H, br s, lactam-NH) and 9.69 and 10.04 (each 1 H, br s, pyrrole-NH); G,(CDCl,, 100 MHz) 19.18, 19.79 and 20.53 (3 x CH,CH,CO,), 29.28, 29.70, 30.28, 30.69, 31.02, 31.44, 34.64, 34.90 and 35.97 (3 x CH,CH,CO,, 3 x CH,CO,, CH,CCH,, CBr,), 51.50, 51.63, 51.80, 51.85 and 52.31 (5 x OMe), 65.81, 66.18, 67.62, 70.16, 73.38 and 74.10 (ribonic lactone, C-4 and CH,Ph), 76.75 (CH,CBr,), 104.82 (CHPh), 116.53, 117.00, 119.25, 122.40 (2 C), 127.32 (2 C), 128.14(2C), 128.48(2C), 128.61 (2C), 130.46(2C), 130.57, 131.41, 135.54, 138.08, 138.41 and 148.56 (C=C) and 158.81, 160.63, 165.59, 170.66, 171.83, 172.02, 173.00, 173.34, 173.55 and 173.94 (10 x C=O); m/z (FD) 1359, 1361, 1363 and 1365 (1:3:3:1, M', 100%).(ii) The ester 38a (peak X in Fig. 1) as an oil (25.4 mg, 22%) (Found: MH+, 1360.1229); &,,,(MeCN)/nm 283; G,(CDCl,, 400 MHz) 2.39-2.54 (8 H, m) and 2.69 (2 H, t, 7, CH,CH,CO,), 2.78 and 3.15 (2 H, ABq, J 16, CH,CCH,), 2.95-3.06 (4 H, m, CH2CH,C02 and CH,CCH,), 3.15 and 3.48 (2 H, ABq, J 16, CH,CO,), 3.55, 3.56, 3.56, 3.57, 3.58 (each 3 H, s, OMe), 3.68 and 3.87 (2 H,ABq,J18, CH,CO,), 3.71 and 3.81 (2 H, ABq, J 18, CH,CO,), 4.40 and 4.61 (2 H, ABq, J 13, lactone-6-H2), 4.73 (1 H, d, J 8,lactone-5-H), 4.94 (1 H, dd, J 8 and 3, lactone-4-H), 4.99 and 5.08 (2 H, ABq, J 12, CH,CBr,), 5.17 and 5.25 (2 H, ABq, J 12, CH,Ph), 5.64 (1 H, d, J 3, lactone-3-H), 5.83 (1 H, s, CHPh), 7.28-7.47 (10 H, m, 2 x Ph), 7.69 (1 H, br s, lactam-NH) and 9.24 and 9.89 (each 1 H, br s, pyrrole-NH); G,(CDCl,, 100 MHz) 19.28, 20.00 and 20.45 (3 x CH,CH,CO,), 29.10, 30.12, 30.49, 30.61, 31.03, 32.94, 34.80, 35.05 and 36.05 (3 x CH,CH,CO,, 3 x CH,CO,, CH,CCH,, CBr,), 51.49, 51.64, 51.70, 51.85 and 52.37 (5 x OMe), 65.82, 66.02, 67.74, 70.22, 73.34 and 74.12 (ribonic lactone, C-4 and CH,Ph), 76.71 (CH,CBr,), 105.07 (CHPh), 116.19, 116.72, 119.37, 122.52, 122.63, 127.32 (2 C), 127.88, 128.20, 128.39 (2 C), 128.53 (2 C), 128.63 (2 C), 130.48, 130.57, 131.10, 134.40, 136.03, 138.83 and 148.31 (GC) and 158.82, 160.30, 165.22, 171.15, 171.75, 171.83, 173.30, 173.47and 173.53(2C)(lO x C=O);m/z(FD) 1359,1361,1363 and 1365(1:3:3:1,M+, 100%).(iii) The 2,2-dibromoethenyl ester 39a (peak P in Fig. 1) as an oil (7.6 mg, 7%); iE,,,(CH,Cl,)/nm 289; G,(CDCl,, 400 MHz) 2.41-2.69 and 2.963.01 (12 H, m, 3 x CH,CH,CO,), 2.82 and 3.11 (2 H, ABq, J 15) and 2.85 and 3.14 (2 H, ABq, J 15, CH,CCH,), 3.23 and 3.48 (2 H, ABq, J 17, CH,C02), 3.54, 3.56, 3.57,3.61 and 3.62 (each 3 H, s, OMe), 3.68 and 3.87 (2 H, ABq,J18, CH,CO,), 3.75(2 H, s, CH,CO,),4.37 and 4.61 (2 H, ABq, J 13, lactone-6-H2), 4.70 (1 H, d, J 8, lactone-5-H), 4.89 (1 H, dd, J 8 and 3,lactone-4-H), 5.18 and 5.24 (2 H, ABq, J 12, CH,Ph), 5.57 (1 H, d, J 3, lactone-3-H), 5.80 (1 H, s, CHPh), 7.28-7.45 (11 H, m, 2 x Ph and lactam-NH), 8.02 (1 H, s, CHCBr,) and 9.38 and 9.80 (each 1 H, br s, pyrrole-NH); m/z (FD) 1279,1281 and 1283 (1 :2: 1, M', 100%).(iv) The 2,2-dibromoethenyl ester 39b (peak Q in Fig. 1) as an oil (4.7 mg, 4%); Amax(CH2Cl,)/nm 289; G,(CDCl,, 400 MHz) 2.39-2.58,2.67-2.73 and 2.95-3.05 (1 5H, m, 3 x CH,CH,CO, and CH~CCHAHB), 3.12 (1 H, d, J 17, CHAHBCO,), 3.16 (1 H, d, J 15, CH~CCHAH,), 3.46-3.98 (5 H, m, CHAHBCO, and 2 x CH2C02), 3.53, 3.55, 3.56, 3.58 and 3.59 (each 3 H, s, OMe), 4.39 and 4.63 (2 H, ABq, J 13,1actone-6-H2), 4.77 (1 H, d, J 7,lactone-5-H), 4.98 (1 H, dd, J 8 and 3,lactone-4-H), 5.18 and 5.27 (2 H, ABq, J 12, CH,Ph), 5.60 (1 H, d, J 3,lactone-3-H), 5.85 (1 H, s, CHPh), 7.27-7.65 (10 H, m, 2 x Ph), 7.65 (1 H, br s, lactam-NH), 8.01 (1 H, s, CH=CBr,) and 8.99 and 10.1 1 (each 1 H, br s, pyrrole-NH); m/z (FD) 1279, 1281 and 1283 (1 :2: 1, M+, 100%).4-[5-Benzyloxycarbonyl-3-(2-methox ycar bonylethyl)-4-(methoxycarbonylmethyl)pyrrol-2-ylmethyl]-2,&bis(2-methoxycarbonylethyl)-3,7-bis(methoxycar~nylmethyl)-9-(2,2,2-tribromoethoxycarbonyl)-4,5-dihydrodipyrrin-1(10H)-one 40 A solution of the resolved lactam 38b (75.4 mg, 55 pmol) in methanol (6.5 cm3) and tetrahydrofuran (1.2 cm3) was stirred with a solution of sodium methoxide (1.19 mg, 22 pmol) in methanol (0.1 cm3) under argon at room temperature for 25 min. Water (5 cm3) was added, the pH was adjusted to ca. 4 with glacial acetic acid and the solution was extracted with dichloromethane (4 x 4 cm3).The combined extracts were washed with water (1 x 2.5 cm3), dried and evaporated. The residue was purified by preparative TLC, eluting with diethyl ether-methanol (19 :l), to give the lactam 40b (61.9 mg, 97%) as a glass (Found: M', 11 55.0782. C,,H,,Br,N,O, requires M, 1 155.0847). The resolved lactam 38a (94.0 mg, 69 pmol) was similarly converted into the enantiomeric lactam 40a (70.4 mg, 88%) (Found: M', 1155.0782). Both enantiomers 40 were identical (apart from optical properties) to authentic racemic material by TLC [run separately and in admixture, Rf0.32, diethyl ether-methanol (19: l)], 'H and I3C NMR and UV spectro-scopy. 4,19-Methylene-2,8,13,18-tetrakis(2-methoxycarbonylethyl)-3,7,12,17-tetrakis(methoxycarbonylmethyl)bilan-l(4H)-one 54 The conversion of the resolved lactams 40 into the respective enantiomers of the spiro lactam 54was accomplished using the chemistry and procedures developed for the racemic ~eries.~ At each stage of the sequence 47-48 --+49-50---51, the pure enantiomers were shown by TLC, 'H NMR spectroscopy and mass spectrometry to match authentic samples of their racemic analogues which had been rigorously +characterised. Finally, the spiro lactams 54b (Found: M , 964.3916.C4,H6,N,Ol7 requires M, 964.3954) and 54a (Found: M', 964.3905) were both identical to authentic racemic material by TLC [run separately and in admixture, R, 0.37, diethyl ether-methanol (19:1)], UV and 'H and I3C NMR spectro~copy.~ As described in the text, the first time the conversion of 48b into 50b was attempted under the published conditions,' the main product after the hydrogenation step was the bis(a-$ree) compound53b (Found: M +,952.3946.C,,H,,N,O, requires M, 952.3954); &(CDCl,, 400 MHz) 2.242.40 and 2.51-2.72 (18 H, m, 4 x CH,CH,CO, and CHAHBCCHAHB), 2.91 and 2.92 (each 1 H, d, J 15, CHAHBCCHAHB), 3.25 (1 H, d, J 16, CHAHBCO,), 3.34-3.77 (9 H, m, CHAHBCO~, 3 X CH2CO2 and 10-H,), 3.62 (6 H, s, 2 x OMe), 3.65 (9 H, s, 3 x OMe), 3.68, 3.71 and 3.74 (each 3 H, s, OMe), 6.43 (2 H, m, 2 x wH), 7.12 (s, lactam-NH) and 8.55, 8.68 and 9.22 (each 1 H, br s, pyrrole-NH); m/z (FD) 952 (100%). This undesired process could be almost completely avoided simply by carrying out the iodination in the normal way except running the reaction at room temperature for just 20 min, followed by work-up and hydrogenation.This gave the desired product 50b (86%) along with a very small amount of bis(a-free) product 53b (trace to 7% in different runs). For the conversion of the bis(a-free) product 53b into the spiro lactam 54b,paraformaldehyde (5.6 mg) was first stirred with trifluoroacetic acid (10 cm3) at room temperature for 30 min. An aliquot of this solution (0.2 cm3) and trifluoroacetic acid (0.88 cm3) were stirred with a solution of the bis(a-free) tripyrrolic lactam 53b (1.8 mg, 1.9 pmol) in methanol (83 mm3) under argon at room temperature. After 20 min, a further aliquot (0.2 cm3) of the paraformaldehyde-trifluoroacetic acid solution was added.After a further 35 min, the solution was evaporated and the residue was dissolved in dichloromethane (2 cm3), washed with water (1 cm3) followed by 5% aqueous sodium hydrogen carbonate (1 cm3), dried and evaporated. J. Chem. SOC.,Perkin Trans. 1,1996 2089 The residue was purified by preparative TLC, eluting with dichloromethane-methanol (19: l), to give the spiro lactam 54b (0.5 mg, 27%) identified by direct chromatographic and spectroscopic comparison with authentic material. 4 A. R. Battersby, C. J. R. Fookes, M. J. Meegan, E. McDonald and H. K. W. Wurziger, J. Chem. SOC.,Perkin Trans. 1, 1982,2786. 5 A. R. Battersby, G. L. Hodgson, E. Hunt, E.McDonald and J. Saunders, J. Chem. SOC.,Perkin Trans. 1, 1976,273. 6 J. H. Mathewson and A. H. Corwin, J. Am. Chem. Soc., 1961,83, 135. Enzymic studies Each enantiomer, 54a and 54b, of the spiro lactam was hydrolysed with aqueous methanolic potassium hydroxide exactly as described for racemic 54. These conditions had been shown to hydrolyse only the eight methyl ester groups. The solutions of the potassium salts of the enantiomeric octa-acids, 4a and 4b,after adjustment of the pH as earlier, were used for inhibition experiments on cosynthetase with synthetic hy- droxymethylbilane 1 as substrate. The methods and controls developed for the study of racemic 4, which have been fully de~cribed,~were followed. The data so obtained are presented as Dixon plots in Fig.3, from which the Kivalues in Table 1 are derived. Acknowledgements Grateful acknowledgement is made to Zeneca, Roche Products, F. Hoffmann-La Roche and EPSRC for financial support. We also thank the SERC for an Instant Award (to M. C. C.) and for a Studentship (to N. C.), Dr A. F. Drake (Birkbeck College, London) for measuring the CD spectra, Dr G. J. Hart for assisting with the early enzymic studies and Dr A. Capretta and Dr A. C. Spivey for their help with the manuscript. References 1 Part 43: F. Kiuchi, F. J. Leeper and A. R. Battersby, Chem. Biol., 1995, 2, 527. 2 Preliminary report of part of this work: M. A. Cassidy, N. Crockett, F. J. Leeper and A. R. Battersby, J. Chem. SOC.,Chem. Commun., 1991,384. 3 F. J. Leeper and A. R. Battersby, Chem. Rev., 1990, 90, 1261. F. J. Leeper, Nut. Prod. Rep., 1989,6, 171. 7 W. M. Stark, C. J. Hawker, G. J. Hart, A. Philippides, P. M. Petersen, J. D. Lewis, F. J. Leeper and A. R. Battersby, J. Chem. SOC.,Perkin Trans. I, 1993, 2875; W. M. Stark, G. J. Hart and A. R. Battersby, J. Chem. SOC.,Chem. Commun., 1986,465. 8 A. R. Battersby, C. J. R. Fookes, K. E. Gustafson-Potter, E. McDonald and G. W. J. Matcham, J. Chem. SOC.,Perkin Trans. I, 1982,2413. 9 A. H. Jackson, K. R. N. Rao, N. S. Ooi and E. Adelakun, Tetrahedron Lett., 1984,25,6049. 10 A. R. Battersby, E. Hunt, E. McDonald, J. B. Paine I11 and J. Saunders, J. Chem. SOC.,Perkin Trans. I, 1976, 1008. 11 B. V. Gregorovich, K. S. Y. Liang, D. M. Clugston and S. F. McDonald, Can. J. Chem., 1968,46,3291. 12 M. W. Roomi and S. F. McDonald, Can. J. Chem., 1970,48, 139. 13 B. Haveaux, A. Dekoker, M. Rens, A. R. Sidani, J. Toye and L. Ghosez, Org. Synth., 1979,59,26. 14 C. C. DukeandR. J. Wells, Aust. J. Chem., 1987,40, 1641. 15 G. J. Hart and A. R. Battersby, Biochem. J.,1985,232, 151. 16 M. Dixon and E. C. Webb, in Enzymes, 3rd edn., Longman, London, 1979, p. 62. 17 A. C. Spivey, A. Capretta, C. S. Frampton, F. J. Leeper and A. R. Battersby, J. Chem. SOC.,Perkin Trans, I, 1996,2091. 18 A. D. Miller, F. J. Leeper and A. R. Battersby, J. Chem. Soc., Perkin Trans. I, 1989, 1943. 19 G. W. Kenner, J. Rimmer, K. M. Smith and J. F. Unsworth, J. Chem. Soc.,Perkin Trans. I, 1977,332. 20 A. R. Battersby, M. H. Block, C. J. R. Fookes, P. J. Harrison, G. B. Henderson and F. J. Leeper, J. Chem. SOC.,Perkin Trans. I, 1992,2175. 21 L. Hough, J. K. N. Jones and D. L. Mitchell, Can. J. Chem., 1958,36, 1720. Paper 6/01621I Received 7th March 1996 Accepted 7th June 1996 2090 J. Chem. SOC.,Perkin Trans, I, 1996

ISSN:1472-7781    

DOI:10.1039/P19960002079

出版商:RSC    

年代:1996

数据来源: RSC