J. Org. Chem. 1984,49,1013-1021 2 X 1H, J = 7 Hz, 11.5 Hz, CCHZOH), 3.54, 3.76 (2 d, 2 X 1 H, J = 8.5 HZ, CCH~O). Anal. Calcd for CBH3604:C, 73.37; H, 9.64. Found C, 73.12; H, 9.82. (*)-16~,17-(Isopropylidenedioxy)aphidicolin. To a stirred solution of 18 mg (0.05 mmol) of the above keto alcohol in 1mL of dry THF at -78 "C under argon was added 0.2 mL (0.2 mmol) of a 1M solution of L-Selectride (Aldrich) in THF. After stirring for 2 h, cooling was discontinued. The reaction mixture was treated with 0.1 mL of ethanol, 0.15 mL of 15% aqueous NaOH, and 0.15 mL of 30% aqueous HzOZ.After stirring at room temperature for 3 h, it was diluted with 30 mL of dichloromethane. The organic phase was washed with 20 mL of saturated aqueous NaHC03 and 15 mL of saturated aqueous NaCl. The combined aqueous phase was extracted with two 2-mL portions of dichloromethane. The combined organic phase was dried (MgSO,) and then concentrated under reduced pressure. Chromatography of the residue on 15 g of silica gel with 2% methanol:lO% ethyl acetate in dichloromethane gave 12 mg (67%) of the expected diol. Analytically pure material was obtained by recrystallization of this material from ether:hexane: mp 192-195 OC; IR (CHC13) 3440 (OH) cm-'; 'H NMR (CDC13,500 MHz) 6 0.70,0.98 (2 s , 2 X 3 H, C4 and ClO-CHis), 1.34,1.42 (2 s , 2 X 3 H, CH3CCH3), 3.37, 3.47 (2 d, 2 X 1 H, J = 11 Hz, CCHZOH), 3.54, 3.76 (2 d, 2 X 1H, J = 8 Hz, CCHzO), 3.67 (dd, 1H, J = 2 , 3 Hz, C3-H). Anal. Calcd for CBHB04: C, 72.98; H, 10.12. Found C, 72.95; H, 10.21. (*)-Aphidicolin (34). To a stirred solution of 21.2 mg (0.056 mmol) of the above diol in 4 mL of methanol was added 0.2 mL of 10% aqueous HC1. After 24 h, excess solid NaHC03and KzCO3 were added. The resulting mixture was stirred for 1h and then filtered. Removal of solvent under reduced pressure and chromatography of the residue on 10 g of silica gel with 8% methanol in dichloromethane afforded 17.2 mg (91%) of (*)-aphidicolin (34).
1013
The analytically pure sample was obtained after crystallization of this material from ethyl acetate: mp 220-225 "C; IR (Nujol mull) 3500,3300 (OH) cm-'; 'H NMR (CDC13,500 MHz) 6 0.70, 1.0 (2 s, 2 X 3 H, C4 and ClO-CHis), 3.36, 3.46 (2 d, 2 X 1 H, J = 11 Hz, CH3CCHZOH), 3.37,3.45 (2 d, 2 X 1 H, J = 11 Hz, COCCHZOH), 3.67 (dd, 1 H, J = 2, 3 Hz, C3-H). Anal. Calcd for C&€%04:C, 70.97; H, 10.12. Found C, 70.81; H, 9.87. Registry No. (*)-l, 78310-39-1; 2,88802-63-5;3,88802-64-6; 4,3419-66-7;5,88802-65-7;(*)-6a, 78310-40-4; (*)-6b, 78310-41-5; (&)-6c,78310-42-6; (*)-7a, ~3391-97-6;(*)-7b, 88852-71-5;( A ) - ~ c , 88852-72-6; (*)-sa, 88802-66-8; (f)-8b, 88802-67-9; ( A ) - ~ c , 88802-68-0; (A)-9,78310-43-7; (*)-io, aaa25-34-7; (*)-ii,8880269-1; (*)-12,78310-44-a; (~)-13,aaao2-70-4; (*)-i4, 78310-54-0; (A)-15,88802-71-5; 16, mi3i-38-1; (A)-17a, 88802-72-6; (*)-in, aam-73-7; is,aaao2-73-7; 19,aaa02-74-a; (*I-20, 78310-45-9; (*)-2ia, 88802-75-9; 21b, aam-76-0; (*)-22, 15401-86-2; (k1-23, 88852-74-8; (*)-24, 78310-56-2; 88852-75-9; ~ 2 6 , 78310-46-0; (f1-27, 88852-76-0; (*I-28, 78310-57-3; (h1-29, ~331-40-5; (A)-30, m3io-48-2; (A)-31, 88802-77-1; (A)-32, 88802-78-2; (A)-33, 88802-79-3; (A)-34, 69926-98-3; (+1,2,2trimethylcycloheptanol, 88802-80-6;2,2-dimethylcycloheptanone, 7228-52-6; ll-(hydroxymethyl)-3,7,7-trimethylspiro[5.5]undec2-ene, 88802-81-7; ll-(o-nitrophenylselenomethyl)-3,7,7-trimethylspiro[5.5]undec-2-ene, 88802-82-8;o-nitrophenyl selenocyanate, 51694-22-5; methyl (*)-3-(dimethylamin0)-2-[(trimethylsilyl)methyl)]propionate, 88802-83-9; methyl 3-(dimethylamino)propionate, 3853-06-3;methyl CY-[ (trimethysily1)methyllacrylate, 78310-52-8; (A)-13a-[(tert-butyldimethylsily1)oxy]-16~,17-dihydroxy-3,3-(ethylenedioxy)-5-epi-l8,19-dinoraphidicolane, 78331-39-2; (f)-13a-[(tert-butyldimethylsilyl)oxy]-3,3-(ethylenedioxy)-l6~,17-(isopropylidenedioxy)-5-epi18,19-dinoraphidicolane, 78310-47-1; (*)-16/3,17-(isopropylidenedioxy)aphidicolan-3-one,78310-59-5; (A)-160,17(isopropylidenedioxy)aphidicolin,78310-60-8.
Synthesis of Dihydromauritine A, a Reduced Cyclopeptide Alkaloid Ruth F. Nutt, Kau-Ming Chen, and Madeleine M. JoulliB* Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received November 8, 1983
The synthesis of the strained 14-membered cyclopeptide alkaloid derivative dihydromauritine A has been accomplished from L-proline via an SN2displacement and three amide bond forming steps. The nucleophilic displacement involved a 8-bromopyrroline methyl ester and the thallium salt of Boc-phenylalanyltyramide to generate an aryloxy ether. Reduction of the pyrroline double bond with dimethylamine-borane resulted in a 6040 ratio of trans:cis &substituted proline derivatives. The critical cyclization step was accomplished by amide bond formation using an active ester derivative. Macrocyclic ring formation proceeded in similar yields when carried out at 90 "C in pyridine or at 25 OC with the acylation catalyst 1-hydroxybenzotriazole. Surprisingly, no stereoselectivity was observed in the cyclization step, and both proline diastereomers were formed in equal amounts. The introduction of the dipeptide side chain Nfl-dimethylalanylvaline was carried out most easily by using dicyclohexylcarbodiide/hydroxybenzotri activation to afford dihydromauritine A with configuration 8S,9S,5S and its stereoisomer with configuration 8R,9R,5S. This is the first synthesis of a 14-membered dihydrocyclopeptide alkaloid containing a non-proline internal amino acid. Cyclopeptide alkaloids are macrocyclic molecules of various ring sizes including 13-, 14-, or 15-membered rings, 14-membered rings being t h e most c0mmon.l Since the discovery of the first cyclopeptide alkaloid by Goutarel and Pais in 1963,2t h e field of cyclopeptide al(1) Tschesche, R.; Kaussman, E. U. "The Alkaloids"; Manske, R. H. F., Ed.; Academic Press: New York, 1975. (2) Pais, M.; Mainil, J.; Goutarel, R. Ann. Pharm. Fr. 1963,21, 139.
0022-326318411949-1013$01.50/0
kaloids has grown rapidly, and several reviews have been published on this subject.'^^-^ Although they occur in many different parts of a plant, difficulties in isolation have resulted in limited supplies of these products. Several (3) Warnhoff, E. W. Fortschr. Chem. Org. Naturst. 1970, 28, 162. Pais,M.; Jarreau, F.-X. In "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins"; Weinstein, B., Ed.; Marcel Dekker: New York, 1971; p 127. (5)Tschesche, R.Heterocycles 1976, 4, 107. (4)
0 1984 American Chemical Society
1014 J,Org. Chem., Vol. 49, No. 6, 1984 research groups have been involved in developing synthetic routes for these molecules.e-s Most synthetic studies have focused on the dihydro derivatives of the natural products. We first became interested in cyclopeptide alkaloids of the amphibine B family, a subgroup of the most commonly found 14-membered macrocycles.' These compounds appeared of interest because their structures represented the first known example of a @-hydroxyproline in a peptide of plant origin, although this amino acid was known to be present in some antibiotics, collagen, and sponges. Furthermore, a member of this family, mauritine A (la) was the only cyclopeptide alkaloid for which there was a published X-ray s t r u ~ t u r e . ~ J The " X-ray structure of this
Nutt, Chen, and Joulli6 Scheme I I-\
h
6
l a , X-Y = -CH=CHl b , X-Y = -CH,CH,-
compound presents some unique features: a highly strained 14-membered ring which exhibits a nonplanar conjugated aromatic system and a distorted @-substituted proline ring with trans stereochemistry. As previous investigators, we focused our initial efforts on the synthesis of dihydromauritine A (lb). When we began our investigations, no cyclopeptide alkaloid or dihydro derivative had been synthesized, although recently the first syntheses of 14-membered-ring dihydrocyclopeptide alkaloids were published."ab As part of our efforts directed toward the synthesis of la, we investigated two independent synthetic routes. Our model studies using a four-component condensation approach were published recently.12 We now describe the total synthesis of dihydromauritine A using the second route. The conformation of the proline moiety in l a was determined by an analysis of the splitting patterns of the a-, @-, and y-protons. The conformation in which the two substituents assume an axial-quasiaxial relationship results in an a,P-proton dihedral angle of 90° and a corresponding coupling constant of 0. This conformation is most commonly found in unstrained /3-substituted prolines and in the synthetic linear intermediates of l b that will be described later. However, in the 14-membered-cyclopeptide alkaloid mauritine A, the a,@-couplingconstant is 5.5 Hz. This value is supportive evidence for an unusual conformation brought about by the strained macrocyclic ring system. Dihydromauritine A (1b) was prepared from this sample by hydrogenation of the styryl double bond. The 'H NMR spectrum of l b shows many of the features (6) Rocchiccioli, F.;Jarreau, F.-X.; Sierra, M. G.; Mascarretti, 0. A.; Ruveda, E. A,; Chang, C.-J.; Hagaman, E. W.; Wenkert, E. Phytochem. 1979, 18, 1869. (7) Goff, D.; Lagarias, J. C.; Shih, W. C.; Klein, M. R.; Rapaport, H. J . Org. Chem. 1980, 45, 4813. (8) (a) Schmidt, U.;Griesser, H.; Lieberknecht, A,; Talbiersky, J. Angew. Chem. Int. Ed. Engl. 1981,20,280. (b) Schmidt, U.; Lieberknecht, A.; Griesser, H.; Hausler, J. Ibid. 1981, 20, 281. (c) Schmidt, U.; Liebernecht, A.; Bokens, H.; Griesser, H. J. Org. Chem. 1983, 48, 2680. (9) Kirfel, A.; Will, G.; 2. Kristallogr. 1975, 142, 368. (IO) Kirfel, A.; Will, G., Tschesche, R.; Wilhelm, H. 2. Naturforsch., B: Anorg. Chem., Org. Chem. 1976, 31B, 279. (11) Nutt, R. F. Doctoral Dissertation, University of Pennsylvania, 1981. (12) Nutt, R. F.; Joulli6, M. M. J . Am. Chem. SOC.1982, 104, 5852.
7
present in the spectrum of la. The splitting pattern of the a-and @-protonsof proline was similar to that in la, showing a,@-couplingconstants of 6.5 Hz. The retention of the distorted proline conformation after reduction of the double bond is evidence that dihydrocyclopeptide alkaloids also exhibit considerable macrocyclic ring strain. Our strategy toward the synthesis of dihydromauritine A involved the formation of three amide bonds and an aryl ether bond. A retrosynthetic analysis (Scheme I) shows that lb can be disconnected at the acylproline bond to give the macrocyclic ring 2 and the peptide side chain 3. Connection may be visualized by amide bond formation using standard methodology for carboxylic acid activation. Suitable dipeptide intermediates for acylation of the macrocyclic system would require the conversion of a valine dipeptide ester to either a hydrazide or a carboxylic acid. The synthesis of this peptide chain derivative posed a potential synthetic problem as difficulties had been reported during the preparation of carboxy terminal valine acids or hydrazides from the corresponding carboxylic esters.13-15 The macrocyclic intermediate could be disconnected at either one of two amide functionalities. We chose disconnection a t proline because there was literature precedent for higher cyclization yields in corresponding 14membered rings.16 The cyclization of 4 presents two major potential problems: (1)undesirable stereoselectivity during cyclization and (2) poor reactivity at the terminal phenylalanine amino group. The most likely linear precursor 4 would be racemic at proline. The difficult cyclization step might occur more easily for one of the two resulting stereoisomers and therefore afford only one diastereomeric form of 2. Stereoselectivity during cyclization would be expected but should favor the desired absolute configuration according to literature Low reactivity of the terminal phenylalanine amino group could decrease cyclization yield (13) Klausner, Y.; Bodansky, M. Synthesis 1974, 549. (14) Shankman, S.;Schvo, Y. J. Am. Chem. SOC.1958, 80, 1154. (15) "The Peptides", Part I; Schroder, E., Lubke, K., Eds.; Academic Press: New York, 1966; p 138. (16) Lagarias, J. C.;Houghten, R. A,; Rapoport, H. J. Am. Chem. SOC. 1978, 100,8302.
J. Org. Chem., Vol. 49, No. 6, 1984 1015
Synthesis of Dihydromauritine A Scheme I1
%
C6H5
X
OCH3
HN
DMF
loa, Y = H lob, Y = TI
8,X= H 9,x=Br I--\
OR
NHBoc
lla,R=CH, l l b , R = Na'
+ OH
0
NHbc
R
12a, R 12b, R 14a, R 14b, R
= = = =
H (Pro aS,pS; Phe a s ) H (ProaR,pR; Phe aS) Cbz (Pro aS,pS; Phe a s ) Cbz (Pro aR,pR; Phe a s )
R
13a, R 13b, R 15a, R 15b, R
= H (Pro aS,PR; Phe a s ) = H (Pro aR,pS; Phe a s )
= Cbz (Pro aS,pR;Phe a s ) = Cbz (Pro aR,pS; Phe a s )
even more than in proline-containing alkaloids.' Further retrosynthetic analysis of the trans-B(ary1oxy)proline 4 afforded a methyl &(aryloxy)pyrrolinecarboxylate (5). This intermediate can in turn be prepared from a phenolate (7) derived from tyramine and previously N-acylated with L-phenylalanine and a methyl &halogenated pyrrolinecarboxylate (6). The synthesis of trans-0-phenoxyproline had been previously described.lB As the preparation of the 0-(ary1oxy)pyrrolinecarboxylic acid ester 5 involved the reaction of a racemic allylic halide with a n optically active phenoxide, a possibility existed that the S N 2 displacement would proceed stereoselectively and reaction conditions might have to be devised to obtain the desired diastereomer (a-S,p-Sabsolute configuration at proline). The separation of diastereomeric mixtures was not expected to present any problems.
Discussion Synthesis of cis-and trans-(Ary1oxy)prolines. Our synthetic studies began with the preparation of methyl pyrrolinecarboxylate 8 described by Hausler in 1979l' (Scheme 11). Interestingly, intermediates 8 and 9 exhibited split ester carbonyl bands. This phenomenon is due to the two possible alignments of the ester carbonyl with (17) Hausler, J.; Schmidt, U. Liebigs Ann. Chem. 1979, 1881.
respect to the pyrroline double bond. A similar occurrence was observed in a-carbonyl-substituted heterocycles, and a detailed study of this phenomenon was carried out by GUm.18 Condensation of the allylic bromide 9 with the thallium salt of the N-acylated tyramine derivative 10b in DMF afforded the 0-aryloxy-substituted pyrrolinecarboxylic acid ester 1la as a mixture of starting material and product in a 25:75 ratio. Because of the instability of the allylic bromide, the condensation step was carried out with both distilled and crude bromide. Yields for comparable reaction steps were 52% and 40% on the basis of distilled and nondistilled bromide, respectively. The N-acylated tyramine derivatives 10a and 10b used in the previous step were prepared by using three different routes. The preferred method involved the condensation of tyramine with the hydroxysuccinimide esterlg of (tertbutoxycarbony1)-L-phenylalanineto give product 10b in 98% yield. The desired 10a was also prepared by directly acylating tyramine with (tert-butoxycarbony1)phenylalanine by either the mixed anhydride methodz0 or dicyclohexylcarbodiimide (DCCI)/N-hydroxybenzotriazole (HOBt) activation.21 Although these two later methods involved a one-step procedure, isolation of pure product was more difficult. As shown in Scheme 11, crude methyl ester l l a was saponified with sodium hydroxide and the sodium salt of the carboxylic acid 1l b was isolated by precipitation with acetone and ether. Reduction of 11b with dimethylamine-borane in acetic acid generated the trans- and cis-6-(ary1oxy)proline derivatives 12 and 13 in a 6040 ratio. The overall yield of the two isomers was 52% for the three reaction steps starting with the allylic bromide 9. The two isomers 12 and 13 were easily separated by silica gel chromatography. The more mobile isomer, 12, was later determined to be the trans isomer. The trans and cis isomers 12 and 13 were converted separately, and in nearly quantitative yields, to their respective N-carbobenzoxy derivatives 14 and 15 by treatment with [ (benzyloxycarbonyl)oxy] -5-norbornene-2,3dicarboximide (CBz-HONB). These N-carbobenzoxy derivatives were suitable for making configurational assignments by NMR. A characteristic singlet for the proline a-proton of the carbobenzoxy derivative 14 derived from the more mobile free amine 12 was indicative of trans configuration. A high-field 'H NMR study showed cistrans amide bond rotational isomers, as determined by temperature studies and solvent-dependent variability of the isomer ratio. The carbobenzoxy derivative 15 of the more polar isomer 13 had a coupling constant of 6 Hz for the a-proton of proline. This value is consistent with an a,p-cis configuration. Examination by high-field NMR resolved the a-proton signal into two separate doublets, which were present in a 1:l ratio in D3COD and Me,SO. Again, acylproline bond rotamers were observed by NMR. The reduction of the imine double bond in l l b theoretically generated four possible diastereomers in equal amounts if no stereoselective reaction occurred during any of the preceding reaction steps. The two diastereomers with proline aS,@S(12a) and aR,@R(12b) absolute configuration have the a,@-transrelative stereochemistry. The (18) Gum, W.F.;Joulli6, M. M. J. Org. Chem. 1965, 30, 3982. (19) Anderson, G. W.;Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. SOC.1964,86, 1839. (20) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. SOC.1967,89, 5012. (21) Konig, W.;Geiger, R. Chem. Ber. 1970, 103, 788.
1016 J. Org. Chem., Vol. 49, No. 6,1984
Nutt, Chen, and Joulli6 Scheme IV
Scheme I11 I
+
-
yo+)
-
NHBoc
-
21
OR
1 7 a , R = Me 1 7 b , R = Na'
16a,Y=H 16b, Y = "I
..
\
0
1 8 , R = H; R , = H 20
-
22a (Pro aS,pS; Phe a s ) 22b (Pro &R,pR;Phe a s )
1 9 , R = H; R , = H 2 0 , R = H ; R , = Cbz 14a
+
NHBoc
Scheme V
14b
other two diastereomers with aS,PR (13a) and &,PS (13b) configurations exist as a,@-cis-prolineisomers. Derivatives 14 and 15, therefore, were each composed of two diastereomers. Attempts to separate the trans and cis isomers 12 and 13 into their diastereomeric components by using thin-layer chromatography and high-pressure liquid chromatography were unsuccessful. The presence of the desired diastereomer with mauritine stereochemistry (Pro a-S,PS; Phe as)in the trans isomer 12 was highly probable but could not be proven unequivocally by HPLC, TLC, or NMR. Since we had no analytical methods for determining the diastereomeric composition of the trans- and cis-P-(aryloxy)prolines, we decided to carry out additional studies to resolve this important question. The first study involved the synthesis of the carbobenzoxy derivative 14 of the trans-P-(ary1oxy)prolineby an independent synthetic route which would guarantee the formation of both diastereomers 14a and 14b. The reaction sequence shown in Scheme I11 was carried out. The allylic bromide 9 was reacted with the nonchiral thallium salt of Boc-tyramine 16b, which was prepared from tyramine and tert-butoxycarbonyl anhydride. After alkaline hydrolysis, the p-(ary1oxy)pyrrolinecarboxylate 17b was reduced to give the cis- and trans-P-(ary1oxy)prolines18 and 19 as racemic pairs. Conversion of the racemic trans isomer 19 to the carbobenzoxy derivative 20, removal of the tert-butoxycarbonyl group by trifluoroacetic acid, and complete acylation of the tyramine amino group with (tert-butoxycarbony1)-L-phenylalanine by using the N hydroxysuccinimide ester resulted in the two diastereomers 14a and 14b of Pro aS,PS; Phe aS and Pro aR,PR; Phe as absolute stereochemistry. This product was found to be identical with 14 obtained by the initially described route as measured by HPLC, TLC, and NMR. Again, no separation of diastereomers could be obtained by TLC or HPLC. The described experiments showed definitively that the two diastereomers 14a and 14b had very similar physical properties and were inseparable by analytical techniques. Our second approach to prove that the diastereomeric composition of 12 was more successful and involved the conversion of this derivative into separable diastereomers (Scheme IV). We selected (dimethylamino)-L-alanyl-Lvalyl azide (21) (the synthesis of which will be discussed later) as our acylating agent because we had already prepared the hydrazide as our side-chain intermediate to be attached later to form dihydromauritine A. Acylation via the azide method resulted in the formation of the two new diastereomers 22a and 22b, which could be separated by
1
l4
C
F
$
0
2
G
N
O
2 TFA
-
~
1
HCI, EtOAc -25 'C
23
DMF, HOBt, 2 5
'C
Cbz
23, R = H; R , = H (HCl salt) 24, R = ONp; R , = BOC 2 5 , R = ONp; R , = H (TFA salt)
I
Cbz
L C 6 H 5
26a ( 8 S , 9 S , 5 S ) 26b ( 8 R , 9 R , 5 S )
silica gel chromatography and were identified by NMR and amino acid analysis. I t is noteworthy that compound 22a is a suitable intermediate for the synthesis of dihydromauritine A. Removal of the tert-butoxycarbonyl group and activation of the proline carboxyl would give dihydromauritine A after amide bond formation. We decided, however, to use the longer approach of initially blocking the proline nitrogen with the carbobenzoxy group, the rationale being that the carbobenzoxy group would be less likely cause additional problems during the macrocyclic ring formation than the dipeptide acyl group. Problems derived from the steric bulk of the acyl side chain and racemization at the proline a-carbon would be considerably less in the case of the carbobenzoxy group than the dipeptide acyl group. Cyclization. The key intermediate 14 was converted into two suitable linear precursors for cyclization studies (Scheme V). Selective removal of the tert-butoxycarbonyl group in the presence of the carbobenzoxy group was carried out by using hydrogen chloride in EtOAc22at -30 "C to give product 23, which was a single spot on TLC. (22) Nutt, R. F.; Veber, D. F.; Saperstein, R. J. Am. Chem. SOC.1980, 102, 6539.
Synthesis of Dihydromauritine A When this intermediate was subjected to cyclization procedures using carboxyl activation with diphenylphosphoroazidate (DPPA) or DCCI and HOBt in dilute solution, only polymeric products were obtained. The second linear precursor for cyclization was prepared by reacting our key intermediate 14 with p-nitrophenyl trifluoroacetateZ3 in pyridine to form the p-nitrophenyl ester 24 of the proline carboxylic acid. The transesterification to give the p-nitrophenyl ester 24 proceeded without epimerization a t the a-position of proline. The desired active ester 24 was purified to homogeneity by chromatography using ethyl acetate-petroleum ether as eluent. Examination of the NMR spectrum of this product showed it to be two acylproline bond rotamers in a 1:l ratio. For cyclization studies, the tert-butoxycarbonyl group of the phenylalanine was removed with TFA. The resultant solid (25) was then subjected to two different cyclization procedures using (1)a dilute pyridine solution (c 0.2 mM) at 90 OC' and (2) a dilute dimethylformamide (DMF) solution (c 0.2 mM) a t 25 OC with the addition of HOBt. The latter reagent is known to drastically enhance the acylation rates of active esters in DMF and was also found to facilitate cyclizations of conformationally unfavorable macrocyclic systems.21v22Although dimerization still prevailed, the 14-membered-ring monomeric product 26 was formed and could be isolated by gel filtration using Sephadex LH20 and MeOH as solvent. Yields of monomeric product, as calculated for the three-step conversion starting from 14, was 8% for the pyridine/90 "C and 10% for the DMF/HOBt method. The results of the DMF/HOBt reaction clearly point out that for cyclization to the 1Cmembered ring, elevated temperatures show no clear advantage as was reported by Rapoport and Schmidt.Is8 Our cyclization product 26 was thoroughly characterized for cyclic, monomeric, and diastereomeric composition. The cyclic nature of our product was confirmed by the absence of an amino group &s shown by the ninhydrin test, and the absence of the carboxy-activating p-nitrophenyl ester group, as shown by UV. The decreased polarity of the product, as indicated by higher R p in several solvent systems on TLC, was also consistent with a cyclic product. The monomeric nature of our product was shown by gel filtration in 50% aqueous acetic acid.24 Using this analytical tool, our product was eluted a t a volume that corresponded to a smaller molecular weight than the linear precursor. Field desorption mass spectroscopy also confirmed our monomeric structure by giving the correct molecular ion of m / z 513. The 'H NMR spectrum of our product indicated that the two possible diastereomers 26a and 26b (racemic at proline) had formed in equal amounts. This result was quite surprising since cyclizations to macrocyclic structures, especially in strained systems, usually proceed with stereoselectivity. Extensive stereoselectivity during the macrocyclic ring formation was observed by Rapoportl and Schmidt.s The Nh4R spectra of the two diastereomers 26a and 26b showed that the proline ring had undergone a conformational change. Both proline a-protons exhibited a coupling constant of 6.6 Hz, whereas in all the trans linear intermediates singlets were observed for the a-proton of proline. A similar conformation of proline was observed in natural dihydromauritine A and mauritine A. The diastereomers 26a and 26b had identical R p in several solvent systems. (23) Sakakibara, S.;Inukai, N. Bull. Chem. SOC.Jpn. 1964,37,1231. (24) Brady, S. F.;Varga, S. L.;Freidinger, R. M.; Schwenk, D. A.; Mendlowski, M.; Holly, F. W.; Veber, D. F. J. Org. Chem. 1979,44,3101.
J. Org. Chem., Vol. 49, No. 6, 1984 1017 Scheme VI
27, R = H,Cbz; R, = OMe 28, R = H,H; R , = OMe 29, R = Me, Me; R , = OMe 30, R = Me, Me; R , = NHNH, 3 1 , R = Me, Me; R , = OH 21, R = Me, Me; R , = N,
- NH~NHz
CH2O
28
29
Ph 121
31
30
1
I - A ~ O N O H'
21
Side Chain. The dipeptide side-chain derivatives 30 and 31 of N~-dimethyl-L-alanyl-L-valinewere synthesized as shown in Scheme VI. L-Valine methyl ester was acylated by carbobenzoxy+ alanine according to the standard mixed anhydride method20 to give the crystalline protected dipeptide methyl ester 27. The carbobenzoxy blocking group was removed by transfer hydrogenation in the presence of formic acid and 10% Pd/C to give 28. The free amino group of 28 was then dimethylated by reductive alkylation with formaldehyde and NaCNBH3. Use of an excess of formaldehyde and NaCNBH3 gave the dimethylated product 29 in good yield, whereas limited amount of reagents gave 30% of an unidentified byproduct. The methyl ester of 29 was converted to intermediates 30 and 31 which were suitable for acylation of the deblocked macrocyclic system by either the azide method or by DCCI activation. The conversion to the dipeptide hydrazide 30 was carried out by using a 33% hydrazine-MeOH solution, whereas the carboxylic acid derivative 31 was prepared in 25% THFH20 a t pH 12-12.9. Both derivatives were purified by silica gel chromatography and were noncrystalline. Conversion of sterically hindered valyl esters to hydrazides or acids has been reported in the literature to be quite difficult. In our case, however, these conversions proceeded smoothly and without racemization. The products were analyzed for possible racemization at valine by literature procedures.26 Less than 0.1 % of racemization occurred during the preparation of 30 and 31. Synthesis of Dihydromauritine A. After the successful synthesis of two key components, the side chain and the macrocyclic portion of dihydromauritine A, the remaining problem was to connect these two fragments by amide bond formation after deprotection of the cyclic intermediate 26 and activation of the side-chain carboxy group. Initial attempts to remove the carbobenzoxy group were by transfer hydrogenation using formic acid and 10% P d / C (Scheme VII). The deblocked product 32 was subjected to acylation studies with N,N-dimethyl-L-alanyl-L-valyl azide (21), which had been synthesized from the dipeptide hydrazide 30 according to literature procedures.26,n This acylation attempt, however, yielded as the major reaction product, a diastereomeric mixture of the N-formylated macrocyclic ring 33 and only trace amounts of dipeptidyl acylation products. The structure of 33 was determined by 'H NMR. (25) Lam,S.;Chow, F.; Karmen, A. J. Chromatogr. 1980, 199, 295. (26) H o d , J.;Rudinger, J. Collect. Czech. Chem. Commun. 1961,26, 2333. (27) Mazur, R. H.; Schlatter, J. M. J. Org. Chem. 1964, 29, 3212.
1018 J . Org. Chem., VoE. 49, No. 6, 1984
Nutt, Chen, and Joulli6 Scheme VI1
N ''
1
HC02 H Pd/C
-.'K
32
OA
1 32
H N
l b (8S,9S,5S) IC( 8 R , 9 R , 5 S ) (formic
acid
Salt1
t
21
H
33
Since deblocking by transfer hydrogenation resulted in undesirable products in the succeeding steps we carried out the deblocking by hydrogenolysis in aqueous EtOH in the presence of a small amount of HCl. This procedure resulted in the free amine 32, which was a single spot on
TLC. T h e first attempts t o attach the side chain were carried out via the azide method. This procedure had been used very successfully in acylating the P-(aryloxy)proline of the linear intermediate 12 during the determination of the diastereomeric composition of this intermediate. For the acylation of our macrocyclic system 32, however, product yields were minimal, and therefore isolation was not attempted. Successful acylation of 32 with the side-chain component was finally accomplished by using an excess of dipeptide acid 31 and activation with DCCI in the presence of HOBt. A diastereomeric mixture of products was obtained, with one of the diastereomers having identical mobility by TLC and retention time during HPLC to dihydromauritine A, lb, which had been prepared from natural mauritine A by hydrogenation. Separation of the two diastereomers was accomplished by multiple silica gel chromatography. NMR analysis of the two isomers showed one of them to be identical with dihydromauritine A obtained from natural sources.
Experimental Section Melting points were determined on a Thomas-Hoover Unimelt capillary melting point apparatus and are corrected. Microanalyses were carried out at Merck & Co., Inc. High-pressure liquid chromatography (HPLC) analyses were carried out on Hewlett-Packard 1084B or Spectrophysics SP8000 instruments using a Waters C-18 reverse-phase column. Amino acid analyses were performed on a Beckmann Model 121MB amino acid analyzer after hydrolysis of the designated peptide in 6 N HCl for 20 h at 110 "C. A sodium citrate buffer was used for elution, and ninhydrin was used for quantitative amino acid detection. Field-desorption maw spectrometric determinations were obtained on a Perkin-Elmer 137 spectrometer. Absorptions are reported in wave numbers (cm-I). Proton magnetic resonance spectra were obtained on Varian EM 360 (60 MHz), Varian EM 390 (90 MHz), Varian XL 200 (200 MHz), Varian FC 300 (300 MHz), and Nicolet NT 360 (360 MHz) spectrometers. Chemical shifts are expressed in parts per million (6) and are relative to Me&. When deuterium oxide (D20)was used as the solvent, sodium 3-(trimethylsilyl)tetradeuteriopropionate (TTP) was used as internal standard. Multiplicities are designated as singlet (s),doublet (d),triplet (t), quartet (q), quintet (quin), and multiplet (m). The peaks are integrated in units of protons (H). Analytical thin-layer chromatography (TLC) was performed on precoated silica gel plates (250 pm) with fluorescent indicator, supplied by Analtech. Visualization was effected with ultraviolet light, ninhydrin (390 w/v) in 95% ethanol containing 2% HOAc, or t-BuOC1-KI-
starch.28 Preparative chromatography was carried out by using silica gel 60, 70-230 mesh, supplied by E. Merck. The following solvent systems were used for chromatographic purposes: A, 9O:lO:l (CHC1,:MeOH:concentrated NH40H); B, 9O:lO (CHC1,:MeOH); C, 9O:lO:l (CHC13:MeOH:H20);D, 60:30:4:6 (CHCl3:MeOH:HzO:concentrated NH40H); E, 60:40:10 (CHC1,:MeOH:HZ0);F, 80:202 (CHC13:MeOH:H20);G, 70:30:3 (CHC13:MeOH:H20);H, 95:5:0.5 (CHC1,:MeOH:concentrated NH,OH); I, 80:20:2 (CHC1,:MeOH:concentrated NH40H); J, 95:5:0.5 (CHC13:MeOHHzO);K, 70302.5 (CHC13:i-PrOH:H20); L, 60:30:5 (CHCl3:MeOH:H20); M, 10:5:1:3 (Et0Ac:pyr: HOAc:H20); N, 15:5:1:3 (EtOAc:pyr:HOAc:H20); 0, 70:30:3 (CHC1,:MeOH:concentrated NH40H). Methyl 1-Pyrroline-2-carboxylate(8). This compound was prepared in 72% yield: bp 55 "C (0.55 mm); R, 0.6 (B), 0.7 (A) (lit.17bp 43 OC (0.13 mm)); IR (neat) 2900, 1750, 1780, 1660, 1430, 1340, 1320, 1200, 1130,945,790,750 cm-'; 'H NMR (CDCl,, 60 MHz) 6 3.9 (s, 3 H), 2.85 (m, 2 H), 2.0 (m, 2 H), 4.2 (m, 2 H). Methyl 3-Bromo-1-pyrroline-2-carboxylate (9). This compound was prepared17in 62.5% yield: IR (neat) 2900,1730,1740, 1620,1440,1350,1320,1290,1240,1200,1150,1130,786cm-'; 'H NMR (CDCl,, 60 MHz) 6 3.93 (s,3 H), 5.1 (m, 1H), 2.5 (m, 2 H), 4.2 (m, 2 H). N-( tert -Butoxycarbonyl)-L-phenylalanyltyramide (loa). A suspension of tyramine (1.45 g, 10.5 mmol) and (tert-butoxycarbonyl)-L-phenylalanineN-hydroxysuccinimide ester'$ (3.62 g, 10 mmol) in EtOAc (50 mL) was stirred magnetically at 25 "C for 20 h. During this period, diisopropylethylamine (DIPEA) (1.7 mL) was added portionwise to keep the apparent pH of the solution at 7.2 (measured by application to moist pH paper, range 6-8). The EtOAc solution was successively washed with H20,5% citric acid solution, 10% NaHCO, solution, and brine. After being dried (MgSO,), the solution was filtered and concentrated in vacuo. Recrystallization of the residue from ether-petroleum ether gave 3.77 g (98% yield) of product mp 137-138.5 "C; R 0.5 (C); IR (CHCl,) 3500-2950,1710,1675,1520,1490,1360 cm-I; fH NMR (CDC13, 90 MHz) 6 1.4 (5, 9 H), 2.56 (t, 2 H), 3.0 (d, 2 H), 3.36 (q, 2 H), 4.26 (4, 1 H), 5.1 (d, 1 H), 5.9 (t, 1 H), 6.5 (br, 1 H), 6.75 (d, 2 H), 6.9 (d, 2 H), 7.25 (m, 5 H). Anal. Calcd for Cz2HBN204:C, 68.72; H, 7.34; N, 7.29. Found: C, 68.47; H, 7.65; N, 7.30. Methyl 3 - [ p424[N-( tert -Butoxycarbonyl)-L-phenylalanyl]amino]ethyl]phenoxy]- 1-pyrroline-2-carboxylate (lla). A solution of 10a (1.95 g, 5.07 mmol) in benzene was evaporated to a semicrystalline residue. Anhydrous ether (30 mL)
and thallium ethoxide (0.332 mL, 4.69 mmol) were added at 25 "C to give a yellow precipitate, and the volume of the reaction solution was reduced in vacuo to give a residual oil. To this were added DMF (3.5mL, stored over 4-A molecular sieves) and methyl 3-bromo-1-pyrroline-2-carboxylate (967 mg, 4.69 mmol). An additional 1.5 mL of DMF was used to transfer the allylic bromide 9. The reaction solution turned immediately into a thick paste. After 1 h at 25 "C and 20 h at -15 "C the solvent was evaporated in vacuo, ether was added, and the resultant suspension was (28) Nutt, R. F.; Holly, F. W.; Hommick, C.; Hirschmann, R.; Veber, D. F. J . Med. Chem. 1981,24,692.
Synthesis of Dihydromauritine A filtered twice through a Celite pad. The ether filtrate was extracted with four portions of water and dried with Na2S04. Filtration and evaporation of solvent gave 2.48 g of residue, which consisted of a 2:l mixture of product lla and starting material (loa). Contamination with 10a was estimated from inspection of the NMR spectrum. Diagnostic signals for loa are the aromatic protons at 6 6.75. The most diagnostic signal for product lla is the @-protonof the pyrroline ring with 6 5.6. Product lla was not purified at this stage because of its instability. Product lla had Rr 0.7 (C) and R, 0.85 (A). Sodium 3-[p-[2-[[N-(tert -Butoxycarbonyl)-L-phenylalanyl]amino]ethyl]phenoxy]- 1-pyrroline-2-carboxylate (llb). To a solution of crude lla (2.1 g) in THF (60 mL) was added 0.1 N NaOH (34 mL). After 1h at 25 "C the solvents were reduced in vacuo, and the moist residue was precipitated by the addition of acetone and ether. Most of the contaminant loa remained in the acetone-ether filtrate after isolation of product by filtration. Product (1.44 g, 71% yield based on two reaction steps) was obtained as an amorphous solid, which had Rp of 0.5 (D) and 0.5-0.8 (E); IR (Nujol) 3330,1680,1650,1620,1520,1230, 1170 cm-'; 'H NMR (Me2SO-ds,90 MHz) 6 1.4 (s,9 H), 2.2-2.6 (m, 2 H), 2.7 (t, 2 H), 2.9 (m, 2 H), 4.0 (m, 2 H), 4.25 (m, 1 H), 5.65 (m, 1 H), 6.9 (d, 2 H), 7.11 (d, 2 H), 7.25 (9, 5 H). trans - & [ p -[2-[[ N - (tert -Butoxycarbonyl)-L-phenylalanyl]amino]ethyl]phenoxy]proline (12)and the Cis Isomer 13. A solution of the carboxylate salt llb (1.34 g, 2.6 mmol) in glacial HOAc (12 mL) was treated with dimethylamine borane (230 mg, 3.9 mmol) for 15 min at 25 "C. The solvent was evaporated in vacuo, and the residue was chromatographed by using a silica gel 60 column (Merck, 70-230 mesh) and elution with solvents F and G. Chromatography yielded 366 mg of the more mobile trans isomer 12 (Rf0.4, G),230 mg of the more polar cis isomer 13 (Rr0.25, G),and 395 mg of product, which was a mixture of the two isomers. m e trans:cis product isomer ratio was 62:38. Total product yield calculated for the three reaction steps starting was 56%. The product with 3-bromo-1-pyrroline-2-carboxylate isomers were analyzed by high-pressure liquid chromatography (HPLC) using a trimethylamine phosphate buffer at pH 3.2. The trans isomer 12 had an elution time of 21.79 and was 99.46% isomerically pure. The cis isomer 13 had an elution time of 20.36 and was found to be 98.93% isomerically pure. 12: IR (Nujol) 3330,1680,1660,1630,1515,1230,1170cm-'. 13: IR (Nujol) 3320, 1660,1520,1235,1170cm-'. The trans isomer was triturated with MeOH to give an amorphous solid, which gave a good elemental analysis for the monohydrate. Anal. Calcd for C27H35N306-H20: C, 62.91; H, 7.19; N, 8.16. Found: C, 62.88; H, 7.09; N, 8.19. trans -N-Carbobenzoxy-8-[p -[2-[[ N - (tert -butoxycarbonyl)-~-phenylalanyl]amino]ethyl]phenoxy]proline (14). To a suspension of 12 (325.5 mg, 0.655 mmol) in THF-H20 (2:1, 15 mL) was added with stirring EbN (0.251 mL, 1.73 mmol) and CBZ-HONB (226.6 mg, 0.723 mmol). After 30 min at 25 "C, the solvents were evaporated in vacuo, water (10mL) was added, and the solution was extracted with ether (20 mL). The ether extract was backwashed with two portions of water (10 mL each). The combined water extracts were evaporated to a volume of 10 mL and acidified with citric acid. The precipitated product was filtered and washed with water to give 410 mg (99% yield) of product 14: R, 0.4 (F); IR (CHCl,) 3430,2920,1700,1680,1505, 1410 cm-'; 'H NMR (CDC13,90 MHz) 6 1.37 (8, 9 H), 2.2 (m, 2 H), 2.57 (t, 2 H), 3.0 (d, 2 H), 3.35 (q, 2 H), 3.75 (t, 2 H), 6.15 (t, 1 H), 6.85 (d, 2 H), 7.0 (d, 2 H), 7.15-7.4 (m, 10 H), 9.0 (s, 1 H). A 300-MHz NMR spectrum of 14 in CD30D showed two singlets for the proline a-proton at 6 4.6 and 4.62. In MeaO-d,, a 360-MHz NMR spectrum of 14 showed two singlets for the same proton at 6 4.27 and 4.3. These two singlets coalesced at 70 "C. cis -N-Carbobenzoxy-fi-[p -[2-[[N-(t e r t -butoxyca~bonyl)-~-phenylalanyl]amino]ethyl]phenoxy]proline ( 15). The carbobenzoxy derivative 15 of the minor reduction product 13 was prepared by the method described for the synthesis of 14. A quantitative yield of product 15 was obtained: Rr 0.5 (F); IR (CHC13)3330,2920,1700,1680,1500,1470,1350 cm-'; 'H NMR (CDC13, 90 MHz) 6 1.35 (9, 9 H), 2.15 (t, 2 H), 2.5 (t, 2 H), 2.9 (t, 2 H), 3.25 (q, 2 H), 3.65 (t, 2 H), 4.25 (q, 1 H), 4.68 (d, 1 H, J = 6 Hz), 4.9-5.1 (m, 3 H), 5.4 (d, 1H), 6.22 (t, 1H), 6.6-6.9 (m, 4 H), 7.0-7.4 (m, 10 H). A 360-MHz NMR spectrum of 15 in
J. Org. Chem., Vol. 49, No. 6,1984 1019 CD30D showed two doublets for the proline a-proton which had a similq chemical shift at 6 4.65 and 4.70 and, therefore, appear as a triplet. A 200-MHz spectrum in MezSO-d6 showed two doublets at 6 4.58 and 4.64. These signals coalesced at 58 "C and showed one doublet at 6 4.60 at 83 "C. N-( tert-Butoxycarbony1)tyramine(16a). To a suspension of tyramine (5.37 g, 39.1 mmol) in EtOAc (50 mL) was added with magnetic stirring, and occasional cooling, tert-butoxycarbonyl anhydride (9 g, 41.2 mmol). After 30 min at 25 "C, EtOAc (100 mL) was added and the reaction solution was extracted with H20 (1X 50 mL), dilute citric acid (1 X 50 mL), NaHC03 solution (1 X 50 mL), and saturated NaCl solution (1 X 30 mL). The EtOAc layer was dried with MgS04 and evaporated in vacuo to give an oil as residue. Crystallization from EtOAc-petroleum ether gave a first crop of 3.5 g, mp 75-77 "C. A second crop of 5.0 g, mp 73-75 "C (total yield 92%),was obtained by evaporation of the first crop fiitrate and storage of the residue at 5 "C for 24 h. Both crops were a single spot by TLC with Rp 0.2 (H) and 0.6 (B): IR (CHC13)3480 (w), 3330 (m), 3230 (m), 2900 (m), 1680 (s), 1610 (m), 1590 (w), 1500 (s),1360 (s), 1250 (s), 1183 (s), 1060 (m) cm-'; 'H NMR (CDCl,, 60 MHz) 6 1.45 (s, 9 H), 2.65 (t, 2 H), 3.3 (q, 2 H), 4.5 (m, 1 H), 5.9 (br, 1 H), 6.7 (d, 2 H), 6.95 (d, 2 H). Methyl 3-[p42-[(tert -Butoxycarbonyl)amino]ethyl]phenoxy]-l-pyrroline-2-carboxylate(17a). To a solution of 16a (0.70 g, 2.95 mmol) in ether (20 mL) was added thallium ethoxide (0.209 mL, 2.96 mmol). A yellow precipitate formed immediately. The solid was treated with benzene (2 X 10 mL) and benzene was removed in vacuo. The thallium salt was dissolved in DMF (3.5 mL) and the solution treated with 0.60 g of crude bromide 9 in DMF (1.5 mL). A precipitate formed immediately. After 1 1 / 2 h, at room temperature, the solvent was removed in vacuo and the residue triturated with ether. The mixture was filtered through Celite, and the fiitrate wai extracted with 3 X 30 mL of water. The organic layer was dried (Na2S04) to give 1.0 g of crude product. NMR analysis showed the product to be contaminated by an equal amount of 16a: 'H NMR (CDCl,, 250 MHz) 6 1.42 (s, 9 H), 2.1-2.4 (m, 2 H), 2.7 (m, 2 H), 3.3 (m, 2 H), 3.9 (s, 3 H), 4.2 (m, 2 H), 5.65 (m, 1H), 6.35 (m, 1 H),6.87 (d, 2 H), 7.12 (d, 2 H). trans -194~424 (tert-Butoxycarbonyl)amino]ethyl]phenoxylproline (19)and the Cis Isomer 18. A solution of 17a (970 mg) in THF (10 mL) and H20 (2 mL) was adjusted to pH 11with NaOH. After 90 min at 25 "C, the flocculent precipitate was filtered and washed with THF to give 420 mg of the sodium carboxylate salt 17b Rf0.3 (A) and 0.1 (B);IR (Nujol) 1690,1660, 1600,1520,1280,1230,1170 cm-'; 'H NMR (CD30D,360 MHz) 6 1.42 (s, 9 H), 1.87 (m, 1 H), 2.46 (m, 1 H), 2.69 (t, 2 H), 3.2 (t, 2 H), 7.10 (d, 2 H). A solution of 17b (400 mg,1.08 "01) in acetic acid (15 mL) was treated with dimethylamine-borane (121 mg, 2.05 mmol) for 30 min at 25 "C. The solvent was evaporated in vacuo, and the residual solid was chromatographed on a silica gel column (50 g). Elution with solvent systems F and G gave 143 mg of pure trans isomer 19 and 107 mg of pure cis isomer 18. 18 IR (Nujol) 3330,3150,2100-2900,1680,1625,1600,1525, 1330, 1290,1220,1170cm-'. 19 IR (Nujol) 3350,3100,2100-2800,1700, 1610,1515, 1350, 1280, 1175 cm-l. trans -N-Carbobenzoxy-&[p4 2 4(tert-butoxycarbonyl)amino]ethyl]phenoxy]proline (20). Intermediate 20 was prepared by the procedure described for the synthesis of 14 using of 19,119.7mg (0.38 "01) of CBZ-HNOB, 127.5 mg (0.364 "01) and 0.16 mL (1.1mmol) of Et3N. Product 20 was isolated (166 mg, 94% yield): R, 0.8 (G); IR (CHClJ 3400, 2930, 1720, 1510, 1420,1340,1150,1120,990 cm-'; 'H NMR (CDC13,360 MHz) 6 1.45 (s, 9 H), 2.2 (br, 2 H), 2.75 (t, 2 H), 3.35 (br, 2 H), 3.75 (m, 2 H), 4.57 and 5.0 (br, 1 H), 4.62 (s, 1 H), 5.15 (m, 1 H), 5.2 (s, 2 H), 6.9-7.4 (m). Preparation of 14 by the N-( tert -Butoxycarbonyl)tyramine Route. The tert-butoxycarbonyl group of derivative 20 was removed by treating 20 (150 mg, 0.31 mmol) with TFA (1mL) for 2 min at 25 "C. The TFA was evaporated to give a residual oil, which was precipitated with ether and petroleum ether to give a gummy hygroscopic solid. The deblocked product had an R, of 0.05 (G). After drying the deblocked product in vacuo for 2 h, it was suspended in 5 mL of ethyl acetate, and (tert-butoxycarbonyl)-L-phenylalanineN-hydroxysuccinimideester (118 mg, 0.326 mmol) and DIPEA (0.054 mL, 0.314 mmol) were added.
1020 J . Org. Chem., Vol. 49, No. 6, 1984 After 30 min at 25 "C, the solvent was evaporated in vacuo, and the residue was redissolved in ether and extracted with three portions of water. The combined water extracts were acidified with dilute citric acid solution, and the precipitated product was collected to give 184 mg (94% yield) of 14. This sample was identical with 14 prepared by the previously described route. Comparisons of the two products were made by thin-layer chromatography in systems A, I, F, and G and also by 90- and 250-MHz NMR in CDC13 and CD30D. p -Nitrophenyl N-Carbobenzoxy-trans-#?-[p-[2-[[N(tert-butoxycarbonyl)-L-phenylalanyl]amino]et hyllphenoxylprolinate (24). To a solution of 14 (750 mg, 1.18 mmol) in 3 mL of pyridine was added p-nitrophenyl trifluoroacetateB (900 mg, 4.0 mmol). After 4 h dt 25 "C, the solvent was evaporated and the dark residue was chromatographed by using silica gel (150 g) and EtOAe-petroleum ether eluents with the following EtOAc concentrations: 25% (100 mL), 33% (500 mL), and 50%. Fractions containing product (Rf 0.4, 50% EtOAc-petroleum ether) were combined to give 900mg (100% yield) of product 24. In the NMR spectrum of 24 some protons appeared as two peaks of equal intensity, which was attributed to the two acylproline bond rotamers: IR (CHC13)3500,1770, 1700, 1680, 1620,1600, 1490, 1340, 1160, 1120 cm-'; 'H NMR (CDC13,360 MHz) 6 1.4 (s), 2.35 (m, 2 H), 2.5-2.75 (m, 2 H), 3.0 (m, 2 H), 3.25-3.5 (m, 2 H), 3.75-4.0 (m, 2 H), 4.25 (4, 1H), 4.75, 4.85 (s, 1H), 4.95, 5.37 (d, 1H), 5.05 (m, 1 H), 5.2 (s, 2 H), 8.15, 8.25 (d, 2 H), 6.8-7.4 (Ar H). (5S,8S,9S)-Nu,9-Ethylene-5-benzyl-N8-carbobenzoxyl&dihydro-p-phencyclopeptine(26a)and the Diasteredmer with 5S,8R,9R Configuration (26b). Deprotection of 24 To Give 25. To 870 mg (1.15 mmol) of p-nitrophenyl ester 24 was added TFA (9 mL), and the reaction solution was kept at 25 "C for 3 min. The trifluoroacetate salt 25 was precipitated with ether and petroleum ether, and the resultant amorphous solid was dried in vacuo for 30 min. The product was a single spot on TLC with R, 0.2 (J). Product 25 was dissolved in 12 mL of degassed DMF. Cyclization in Pyridine. One-third (4 mL) of the DMF solution containing 25 was added dropwise over a period of 1 h to pyridine (1900 mL) at 92 "C. The reaction was kept at 92 "C for 20 h. The monomeric product 26 was isolated as described below. This cyclization procedure yielded 24 mg of methanol insoluble material, 56 mg of trimer and polymeric material, 77 mg of dimeric material, and 15 mg (8% yield) of monomeric product 26. Cyclization in DMF. Two-thirds (8 mL) of the freshly prepared DMF solution containing 25 was added to degassed DMF (4200 mL) which contained (1.5 g, 10 mmol) and DIPEA (1mL, 5.8 mmol). The reaction solution was kept at 25 "C for 5 days. This reaction yielded 78 mg of MeOH-insoluble material, 134 mg of trimer and polymer, 99 mg of dimers, and 35 mg (10% yield) of monomeric product 26. Both cyclization reactions were worked up in the same manner. The solvents were evaporated in vacuo, the residue was disaolved in EtOAc, and the solution was extracted with water (lx), NaHC03 solution (3X), dilute citric acid solution (2x), and saturated NaCl solution (lx). After being dried with Na2SO4,the EtOAc solution was evaporated in vacuo and partially dissolved in MeOH. The insoluble material was centrifuged and the supernatant liquid chromatographed on a Sephadex LH20 column (2.5 X 100 cm) using methanol for elution. The pyridine reaction product was chromatographed in two equal batches and the dimethylformamide reaction product in three batches. Elution of product was monitored by ultraviolet absorption at 254 nm. Three major peptide-containing fractions were eluted before elution of p-nitrophenol. Fractions that made up the third peptide containing peak, as monitored by UV detection and TLC evaluation, were combined, the solvent was evaporated, and the residue was purified further by chromatography on silica gel using ethyl acetate-petroleum ether (8:2). Further characterization of the monomeric product was carried out by molecular weight determination using gel filtration through Sephadex G-25F (2.5 X 100 cm column) in 50% aqueous HOAC.~,Product was eluted in fractions corresponding to the elution volume of 275-290 mL; the elution volume for the linear precursor 14 was 250-265 mL. Monomeric product 26 was a single spot on TLC with R, 0.55 (H) and Rf 0.2 (ether). Product 26 gave the correct molecular
Nutt, Chen, and Joulli6 ion peak of m/z 513 by field-desorption mass spectroscopy: IR (CHC13) 3380,2900,3000,1690,1675,1500,1420,1350,1170,1130 cm-'; 'H NMR (CDC13,360 MHz) 6 3.68 (d, 6.6 Hz), 3.75 (d, 6.6 Hz), 5.3 (m, 1 H), 5.6 (d, 8.1 Hz), 5.9 (d, 8.3 Hz), 4.5 (d, 12 Hz), 4.7 (d, 11Hz), 5.G5.2 (four d). Additional absorption signals were observed at 6 2.75-3.0, 3.5-4.2, 6.8-7.4. N-Carbobenzoxy-L-alanyl-L-valine Methyl Ester (27). To a solution containing N-carbobenzoxy-L-alanine (7.0 g, 31.5 mmol) and N-methylmorpholine (3.46 mL, 31.5 mmol) in EtOAc (120 mL) at 0 "C was added with magnetic stirring isobutyl chloroformate (4.09 mL, 1.5 mmol). After stirring at 0 "C for 15 min, valine methyl ester hydrochloride (5.03 g, 30 mmol) was added, followed by addition of N-methylmorpholine (4.5 mL, 41 mmol) over a period of 20 min to keep the apoarent pH at 7.0 (measured with moist pH paper, range 6-8). After being stirred at 0 "C for an additional 20 min, the reaction mixture was extracted with NaHC03solution (3 X 80 ml), dilute citric acid (2 X 50 mL), and HzO (1X 50 mL). The EtOAc layer was dried with MgSO,, and the solvent was evaporated in vacuo to give 10.1 g (100% yield) of crystalline product 27. Recrystallization from ether and petroleum ether gave 8.4 g of product, mp 83.5-84 "C. The product was a single spot by TLC with Rp 0.4 (H) and 0.75 (C);IR (CHCl,) 3410,2970,1725,1695,1500em-'; 'H NMR (CDC13,90 MHz) 6 0.82 (d, 3 H), 0.9 (d, 3 H), 1.4 (d, 3 H), 2.12 (m, 1 H), 3.7 (s,3 H), 4.27 (quin, 1 H), 4.5 (dd, 1H), 5.1 (s,2 H), 5.37 (d, 1 H), 6.54 (d, 1 H), 7.3 (s, 5H). Anal. Calcd for C17H24N205: C, 60.70; H, 7.19; N, 8.33. Found: C, 61.07; H, 7.30; N, 8.08. L-Alanyl-L-valineMethyl Ester (28). A suspension of 27 (3.0 g, 8.9 mmol) in methanol (120 mL) was treated with 97% formic acid (12 mL) and 10% Pd/C (1g) for 10 min and filtered through a Celite pad, and the filtrate was evaporated in vacuo to give an oil (3.5 9). The product was ninhydrin positive and was a single spot on TLC with R p 0.2 (H) and 0.1 (C). N,N-Dimethyl-t-alanyl-L-valine Methyl Ester (29). To formate salt 28 (prepared from 8.9 mmol of 27) in MeOH (30 mL) was added 30% aqueous formaldehyde solution (3.5 mL) and NaCNBH3 (660 mg). The reaction solution was kept at 25 "C for 1h, the solvent was evaporated in vacuo, and the semisolid residue was chromatographed on a 300-g silica gel 60 (E. Merck) column by eluting with solvents C and F. Elution of a less polar byproduct (730 mg, Rf 0.9, F) was followed by elution of product 29 formate salt (1.5 g, 63% yield) as an oil: 'H NMR (CDC13, 90 MHz) 6 0.9 (d, 3 H), 1.0 (d, 3 H), 1.4 (d, 3 H), 2.2 (m, 1 H), 2.65 (s, 6 H), 3.75 (9, 1 H), 3.7 (s,3 H), 4.45 (dd, 1H). A sample (500 mg) of the formate salt of 29 was converted to the free base by dissolving the salt in NaHC03 and extracting the base into CHpC12.After the organic extracts were dried with Na2S04,the solvent was evaporated in vacuo to give 340 mg (83% yield) of free base 99: R, 0.6 (C) as an oil; 'H NMR (CDCl,, 90 MHz) 6 0.87 (d, 3 H), 0.95 (d, 3 H), 1.2 (d, 3 H), 2.15 (m, 1 H), 2.28 (s, 6 H), 2.95 (q, 1 H), 3.72 (s, 3 H), 4.5 (dd, 1 H), 7.6 (d, 1 H). N,N-Dimethyl-L-alanyl-L-valine Hydrazide (30). To a solution of formate salt 29 (900 mg, 3.26 mmol) in MeOH (6.6 mL) ww added hydrazine (3.3 mL). After 2 h at 25 "C the solvents were evaporated to a residual solid, which was chromatographed by using a silica gel 60 column (75 g) and eluting with solvent K. Fractions containing product 30 (R, 0.65, F, 0.5, A) were combined to give a main crop of 460 mg (61% yield) and a second crop of product from side fractions (320 mg). The main crop of product was triturated with ether to give 376 mg of 30 as an amorphous solid, which was a single spot by TLC: IR (Nujol) 90 MHz) 3250,3100,1635,1530,1225,1200 em-'; 'H NMR (DzO, 6 0.95 (dd, 6 H), 1.22 (d, 3 H), 2.02 (m, 1 H), 2.26 (s, 6 H), 3.12 ( q , 1 H), 4.0 (d, 1H). In CD30D the 6 H of the isopropyl group of valine appeared as a doublet. Anal. Calcd for Cl$-IZN4O2:C, 52.15; H, 9.63; N, 24.33. Found: C, 52.05; H, 9.98; N, 24.40. N,N-Dimethyl-L-alanyl-L-valine (31). A solution of 29 (300 mg, 1.25 mmol) in THF-HpO (1:3, 8 mL) was kept at pH 12 for 30 min and pH 12.9 for 40 min by periodic addition of 1.25 N NaOH. The solution was adjusted to pH 7.2 with 10% HCl and lyophilized. The product was chromatographed by using a silica gel column (60 g) and eluted with solvents L and E. Fractions containing product (which appeared as a single spot by TLC and had Rf 0.4 (E)] were combined to give 202 mg (72% yield) of 31.
Synthesis of Dihydromauritine A
J. Org. Chem., Vol. 49, No. 6, 1984 1021
11Hz), 6.8-7.3,4.56 (d, 1 H), 5.81 (d, 1H), 7.7 (d, 1 H); 'H NMR Combination of side fractions gave additional product (280 mg), (CD,OD, 360 MHz) 6 lb, 0.95 (dd, 6 H), 1.23 (d, 3 H), 2.05 (m, which was contaminated with NaC1; IR (Nujol) 3230,2150-3000, 1 H), 2.25 (m, 1 H), 2.52 (m, 1 H), 2.53 (m, 1 H), 2.85 (m, 1 H), 1675,1585,1310,1250,1150,1090,1025 cm-'; 'H NMR (90MHz) 2.9 (m, 1 H), 3.98 (m, 1 H), 3.0 (9, 1 H), 2.28 ( 5 , 6 H), 2.75 (m, 6 (DzO)0.92 (dd, 6 HI, 1.46 (d, 3 HI, 2.12 (m, 1 HI, 2.69 (s,6 HI, 2 H), 3.68 (m, 1 H), 4.25 (t, 1H), 4.0 (m, 1 H), 4.05 (d, 1 H, J = 3.72 (4, 1H), 4.07 (d, 1H); (DCl) 0.95 (d), 1.55 (d), 2.22 (m), 2.90 6.5 Hz), 4.43 (d, 1 H), 5.25 (d, t, 1 H, J = 6, 11 Hz), 6.85-7.2 (m, (d), 4.07 (q), 4.32 (d); (NaOD) 0.92 (dd), 1.22 (d), 2.03 (m), 2.16 (q), 3.1 (q),4.05 (d). The dipeptide acid 31 was analyzedfor optical 9 H); IC, 0.96 (dd, 6 H), 1.21 (d, 3 H), 2.05 (m, 1 H), 2.25 (m), purity at the valine a-carbon by the method described in the 2.55 (m), 2.53 (m, 1 H), 2.8 (m, 1 H), 2.9 (m, 1 H), 3.63 (m, 1 H), literature.% The analysis showed the product 31 to be more than 3.1 (4,1 H), 2.25 (8, 6 H), 2.75 (m, 2 H), 3.63 (d, lH), 3.91 (dd, 99.9% optically pure at the a-carbon of valine. 1H), 3.96 (m, 1 H), 4.02 (d, 1 H, J = 6.5 Hz), 4.55 (d, 1 H), 5.17 (55,SS,9S )-5-Benzyl-1,2-dihydro-Nu-(NJV-dimethyl-L(d, t, 1 H, J = 6, 11 Hz), 6.8-7.4 (m, 9 H). alanyl-L-valyl)-N',9-et hylene-p -phencyclopeptine (DiAcylation of 12 with NJV-Dimethyl-L-alanyl-L-valyl Azide hydromauritine A, lb) and (5S,8R ,9R)-5-Benayl-1,2-dihy(21) To Give 22a and 22b. Dipeptide azide 21 (0.108 mmol) was dro-Nu-(N~-dimethyl-~-alanyl-~-valyl)-Nu,9-ethylene-p prepared from NJ-dimethyl-L-alanyl-L-valine hydrazide (30,25 phencyclopeptine (IC). A suspension of 26 (7.43 mg, 0.0144 mg, 0.108 mmol) in DMF (0.5 mL) as follows: the DMF solution mmol) and 10% Pd/C (0.43 mg) in EtOH-H20 (8:2, 4 ml) that of 30 was cooled to -25 OC and acidified with 5.5 M HC1 in THF contained 10% HCl (0.02 mL) was treated with a slow stream (0.09 mL, 0.0049 mmol). Isoamyl nitrite was added until a positive of H2at atmospheric pressure. After 2 h, the mixture was filtered test was obtained on moist KI-starch paper (0.020 mL), and the through a Celite pad and the filtrate was evaporated in vacuo to reaction mixture was allowed to stand for 30 min at -25 OC. The give 5.66 mg (95% yield) of deblocked product 32 as the hydrotrans-8-(ary1oxy)prolinederivative 12 (49.84 mg, 0.10 mmol) was chloride salt. This material was a single spot [R,0.5 (A), 0.75 (MI] added at -25 O C to an aliquot (0.010 mL, 0.002 mmol) of the nitrite by TLC and gave a positive ninhydrin test. solution, and the mixture was neutralized with DIPEA to an To a solution of 32 hydrochloride salt (5.66 mg, 0.013 mmol), apparent pH 7.2 (as measured by application to moist pH paper, dipeptide acid 31 (44 mg, 0.119 mmol), and HOBt (40.1 mg, 0.29 range 6-8). After 24 h at -25 O C and 24 h at 5 "C, the solvent mmol) in DMF (1.5 mL) was added DCCI (30.6 mg, 0.14 mmol) was evaporated and the residue was chromatographed by using and DIPEA (0.025 mL, 0.14 mmol). The reaction mixture was silica gel (10 g) and eluting with solvent H. The two isomeric stirred magnetically at 25 OC for 9 h and filtered, and the filtrate products were separated to give 12 mg of the less polar isomer, was evaporated in vacuo. The residue was purified by chroma25 mg of the polar isomer, and 27 mg of product containing both tography on several silica gel columns using solvent systems C, isomers. The two isomeric products had Rp of 0.42 and 0.44 in N, and A. Solvent C separated dicyclohexylurea and DCCI from solvent M and 0.56 and 0.63 in solvent 0. By HPLC using product. Solvent N was used to separate the two product diatrimethylamine phosphate buffer (pH 3.2):MeCN (73) as eluent, stereomers and a dipeptide byproduct impurity. Solvent A was the two isomers had elution times of 8.89 and 10.87 min. Amino most efficient in separating the product from HOBt. At an acid analysis after total amide bond hydrolysis gave valine to intermittent stage during the purification, 4.1 mg (54% yield) of phenylalanine ratios of 1.02:0.97 for the more polar isomer and a mixture of the two diastereomers l b and IC were obtained. 1.06:0.95 for the less polar isomer. NMR spectroscopy gave Further chromatography in solvent system N gave lb (1.4 mg) evidence for two acylproline bond rotamers for the less polar and IC (0.9 mg).The identity of one of the syntheticdiastereomers product and one rotamer for the more polar isomer. 'H NMR with dihydromauritine A (obtained from natural mauritine A) (CDC13,300 MHz) of more polar isomer: 6 0.95 (t), 1.3 (d), 1.4 was established by thin-layer chromatography in three solvent (s), 2.08 (m), 2.25 (m), 2.35 (m), 2.6 (br s), 3.05 (br), 3.4 (br), 3.8 systems, by HPLC, and by NMR spectroscopy: TLC (silica gel, (m, q, 2 H), 4.1 (t, 1H), 4.22 (9, 1H), 4.55 (s, 1 H), 4.65 (t, 1H), 5 X 20 cm) R lb, 0.49 (A), 0.45 (C),0.40 (N), IC, 0.42 (A), 0.40 4.97 (m, 1H), 5.1 (9, 1H), 5.85 (br, 1 H), 6.9-7.0 (dd, 4 H), 7.2-7.4 (C), 0.46 (N); kPLC (0.01% HOAc, 210 nm) lb, 9.74 min, IC, 11.57 (m), 8.35 (br, 1 H). min; synthetic and natural lb, 8.96 min (one peak); 'H NMR (CDC13,360 MHz) 6 lb, 0.93 (dd, 6 H), 1.21 (d, 3 H), 2.05 (m, 1 H), 2.2 (obscured) and 2.5 (m, 1H), 2.4 (m, 1H), 2.98 (m, 1H), Acknowledgment. We thank Dr. S. A. H. Shah in 2.95 (m, 1 H), 4.15 (m, 1H), 3.05 (q, 1H), 2.25 (8, 6 H), 2.85 (m, Professor R. Tschesche's laboratory for an authentic sam2 H), 3.7 (m, 1 H), 4.25 (m, 1 H), 3.9 (m, 1H), 3.9 (d, 1H, J = ple of mauritine A and for his interest in our work. We 6 Hz), 4.5 (t, 1 H), 5.3 (d, t, 1 H, J = 6, 10 Hz), 6.8-7.3,4.61 (d, are grateful to the National Science Foundation for gen1 H), 5.9 (d, 1 H), 7.7 (d, 1 H); IC, 0.95 (dd, 6 H), 1.15 (d, 3 H), erous financial support and to Merck Sharp & Dohme 2.05 (m, 1H), 2.2 (obscured) and 2.56 (m, 1H), 2.4 (m, 1H), 2.95 Research Laboratories for help in the determination of (m, 1H), 2.9 (m, 1 H), 4.1 (m, 1H), 3.09 (9, 1 H), 2.17 (s, 6 H), elemental analyses, HPLC, lH NMR, and mass spectral 2.75 (m, 1H), 2.85 (m, 1H), 3.65 (m, 1H), 3.9 (m, 1H), 3.8 (m, determinations. 1 H), 3.8 (d, 1H, J = 6 Hz), 4.53 (t, 1H), 5.21 (d, t, 1 H, J = 6,
