Studies onRubia akane(RA) derivatives. Part 8. Design, syntheses and antitumour activity of cyclic hexapeptide RA analogues possessing an alkyl substituent on the Tyr-3 aromatic ring

摘要

Studies on Rubia akane (RA) derivatives. Part 8.l Design, syntheses rnand antitumour activity of cyclic hexapeptide RA analogues possessing an alkyl substituent on the Tyr-3 aromatic ring Yukio Hitotsuyanagi,"Suguru Lee,"Izumi Ito," Kazuyuki Kondo," Koichi Takeya," Takehiro Yamagishi,b Takatoshi Nagate and Hideji Itokawa *ic,a a Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-03, Japan Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., Yoshino-cho, Ohmiya-shi, Saitama 330, Japan The effective conversion of RA-VII 1 into the naturally less-accessible RA-I14 has been devised through boron tribromide bis-0-demethylation and successive selective partial 0-methylation using diazo(trimethylsily1)methane. The O-triflate 11 prepared from RA-I14 was subjected to cross-coupling reaction with alkylstannanes to produce analogues 12,13 and 15, while compounds 13 and 15 were later converted into analogues 14 and 16, respectively.Analogues 12-16 showed antitumour activity against P-388 leukaemia both in vitro and in vivo. Introduction RA-VII 1 and bouvardin (NSC 259968) 2 are a class of antitumour bicyclic hexapeptides isolated from Rubiaceae plants. Owing to their promising antitumour activity in addition to their unique mode of action-inhibition of protein synthesis through interaction with eukaryotic 80 S ribosomes 4-peptide 1 is currently undergoing clinical trials in Japan as an anticancer agent. Their unique cycloisodityrosine structure has attracted much attention from synthetic chemists,6 and two total syntheses of compound 1 have been accomplished.7*8 A previous structure-activity relation study ' and biotransformations lo*ll of these peptides revealed that the methoxy group of the Tyr-3 residue is very important for such activity.When this methoxy group is substituted by a hydrogen atom (e.g. compound 3) or a hydroxy group (e.g. RA-I1 4), their cytotoxicity is reduced by -100 to 1000 times.' Also, this 0-demethylation has been identified as a metabolic pathway for these pep tide^.^"^'^*' In spite of such importance, no effort has been made thus far to modify the substituents of Tyr-3 because of the difficulties associated with the selective manipulation of this residue. We proposed that substitution of this methoxy group by an alkyl group, especially a sterically very similar ethyl group, would produce an analogue which possesses metabolic stability and might express more pronounced in uiuo antitumour activity. We report here on the syntheses of these analogues-and their in uitro and in uivo antitumour activities.Results and discussion We considered that RA-I1 4,12 possessing a hydroxy group at the 4 position of the Tyr-3 residue, would be a suitable precursor for these transformations. However, because of the very small amount (0.000025 of the dry roots of Rubia cordifolia) of compound 4 in the plant, an alternative access route from the more available RA-VII 1 or RA-V 5 (total -0.01) had to be devised.We first examined the selective 0-demethylation at the Tyr-3 residue of RA-VII 1under various conditions. However, the use of Lewis acid reagents (e.g.AlCl,, BCl,, BBr, or BI,) under selected conditions gave mainly RA-V 5, and an excess of these reagents resulted in di-0- demethylated product 6. The addition of nucleophiles such as A'a-2 Me9y=-:-3 HN Me Me--Fo >Me Ala-4 D-Ma-1 0 Ty-5Tyr-6 0 1; R' = R3 =OMe. R2 =H 2 R' =OMe, R2 =R3 =OH 3; R'=R2=H,R3=OMe 4; R' =OH, R2 =H. R3 = OMe 5; R' =OMe, R2 =H, R3 =OH 6; R'=R3=OH,R2=H 11; R' =OSOzCF3,R2 =H, R3 =OMe 12; R' =Me, R2 =H, R3 =OMe 13; R' =vinyl, R2 =H. R3 =OMe 14; R' =Et,R2 =H, R3 =OMe 15; R' =allyl, R2 =H, R3 =OMe 16; R' =R, Rz =H. R3 =OMe OR' QJR2 Me 7; R~=R~=H 8; R' =H, R2 =OMe 9; R'=Me,R2=H 10; R' =Me, R2 =OMe ethanethiol did not yield favourable selectivity.Other 0-demethylating reagents such as iodotrimethylsilane resulted in decomposition of the substrate. We next attempted the selective Tyr-6 0-methylation of the di-0-nor derivative 6 which was effectively obtained after slightly modifying previous condi- tions '(7 mole equivalents of boron tribromide in dichlorometh- J. Chem. SOC., Perkin Trans. 1 213 ane) in 74 yield. Initial attempts using iodomethane or dimethyl sulfate with potassium carbonate or sodium hydride under various conditions yielded little or non-selective 0-methylation. However, we found that diazo(trimethylsily1)meth-ane under modified Shioiri conditions l3 afforded the desired product RA-I1 4 predominantly, with optimised conditions.The best result was obtained when compound 6 was treated with 1.7 rnol equiv. of diazo(trimethylsily1)methane in the presence of 4-(dimethy1amino)pyridine (DMAP) (1.7 rnol equiv.) in acetonitrile-methanol (10: 1) at room temperature for 21 h which produced RA-114, RA-VII 1 and RA-V 5 in yields of 68, 19 and 4, respectively.? Although this selectivity of 0-methylation is rather unexpected, the reaction appears to have some generalities. When equimolar amounts of 4-methylphenol 7 and 2-methoxy-4-methylphenol 8 were treated with 1.7 mol equiv. of diazo(trimethylsily1)methane under the same condi- tions, yields of 38 for 4-methylanisole 9 and 60 of 3,4- dimethoxytoluene 10 were obtained, and 51 of substrate 7 and 31 of substrate 8 was recovered.The more pronounced selectivity observed for compound 6 might be attributed to the strained 14-membered ring structure which deforms the Tyr-6 aromatic ring and would enhance the reactivity of the hydroxy group attached to this ring. Introduction of the alkyl group was conducted through cross-coupling reactions of various alkylstannanes with RA- I1 0-triflate 11 which was prepared by the reaction of RA-I1 4 with N-phenyltrifluoromethanesulfonimide in quantitative yield. Compound 11 was first treated with tetramethyltin in the presence of dichlorobis(triphenylphosphine)palladium(rI) and lithium chloride to produce the methyl analogue 12 in 82 yield.However, attempted direct introduction of an ethyl group using tetraethyltin under the same conditions was unsuccessful. Thus, the more reactive tributylvinyltin was used to produce the vinyl analogue 13 (87), which was hydrogenated over palladium on carbon to furnish the ethyl analogue 14 in 92 yield. Similarly, the ally1 analogue 15 was prepared (78) from compound 11 using allyltributyltin, and was then converted into the propyl analogue 16 in 82 yield. Cytotoxicity and in uiuo antitumour activity of the prepared compounds including resynthesized analogue 3 for comparison were evaluated using P-388 leukaemia cells, and the results are summarised in Table I. The prepared alkyl analogues 12-16 are less cytotoxic than RA-VII 1, but more than ten times as toxic as the unsubstituted analogue 3.in uiuo Antitumour activity (P-388 cells) almost parallels the cytotoxicity. Although all alkyl analogues 12-16 showed antitumour activity, none of them exceeded the activity of RA-VII 1. The unsubstituted analogue 3 showed no activity. These results suggest that the alkyl group at position 6on the Tyr-3 residue produces a similar effect on the activity as a methoxy group, and this appendage site is responsible for the potentiation of the activity. In spite of the close structural similarities in steric bulkiness between an ethyl group and a methoxy group, the inferior activity of compound 15 compared with RA-VII 1 might be explained by the differences between their electric field potential around this region and/or different metabolic pathways in uiuo.1 Experimenta1 Organic solutions, dried over Na,S04, were evaporated under an aspirator vacuum with a rotary evaporator. Medium-pressure liquid chromatography (MPLC) was performed with t Although we have disclosed the predominant formation of RA-I1 4 using diazomethane in a previous communication,' the results are inconsistent and less reproducible due to the difficulty in controlling the stoichiometry of the reagent. $ Although the 14-membered cycloisodityrosine moiety (Tyr-5 and Tyr-6) has been proposed to be the pharmacophore for this class of antitumour agent~,*~.l~ this work suggests that the Tyr-3 residue is also very important for the activity both in uitro and in viuo.214 J, Chem. SOC.,Perkin Trans. 1 Table 1 Cytotoxicity and in uiuo antitumour activity of RA analogues against P-388 leukaemia Antitumour activity (t/c) Dose Compound Cytotoxicity IC,," 0.4 1.6 6.25 1' 0.0013 144 152 Toxic 3 0.22' 92 100 101 12 0.018 105 121 149 13 0.0 13 100 110 127 14 0.0072 108 120 151 15 0.0039 I02 130 130 16 0.020 109 121 149 pg ~m-~.mg kg-'. 'Ref. 9. a Kusano C.I.G. system. Dichloromethane was distilled from calcium hydride. Mps were taken on a Yanagimoto melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-360 digital polarimeter and are recorded in units of lo-' deg cm2 g-'. IR spectra were recorded on a JASCO A-302 spectrophotometer.NMR spectra were measured on a Bruker AM 400 spectrometer. 'H Chemical shifts are referenced in CDC1, to residual CHC1, (7.26 ppm); 13C chemical shifts are referenced to the solvent (CDCl,, 77.03). J Values are given in Hz. Mass spectra were taken using a VG AutoSpecE spectrometer. 0-Methylation of di-0-norRA-VII 6 with diazo(trimethylsily1)- methane To a solution of compound 6 (65.3 mg, 0.088 mmol) and DMAP (18.3 mg, 0.15 mmol) in acetonitrile-MeOH (9: 1; 1 cm3) was added a 2 rnol dmP3 solution of diazo(trimethylsily1)- methane in hexane (0.075 cm3, 0.15 mmol), and the mixture was stirred at room temperature for 21 h. Acetic acid (0.5 cm3) was added to the mixture, which was then concentrated under reduced pressure.The residue was chromatographed on a silica gel column with CH,Cl,-MeOH (10: 1) as eluent and then separated by MPLC with CH,Cl,-EtOAc-MeOH (12:2: 1) to give RA-VII 1 (13.2 mg, 19), RA-V 5 (2.6 mg, 4) and RA-I1 4 (45.3 mg, 68). Compounds 1, 4 and 5 were confirmed by direct comparison with authentic samples. 0-Methylation of the phenols 7 and 8 using diazo(trimethy1- si1yl)methane To a solution of 4-methylphenol 7 (108 mg, 1.0 mmol), 2- methoxy-4-methylphenol 8 (138 mg, 1.0 mmol) and DMAP (208 mg, 1.7 mmol) in acetonitrile-MeOH (9: 1; 20 cm3) was added a 2 rnol dm-, solution of diazo(trimethylsily1)methane in hexane (0.85 cm3, 1.7 mmol), and the mixture was stirred at room temperature for 24 h. Acetic acid (2 cm3) was added to the mixture, which was then concentrated under slightly reduced pressure.The residue was chromatographed on silica gel (MPLC) with CH,Cl,-hexane (1:l) as eluent and then with hexane-CH,Cl,-tetrahydrofuran (THF) (1 5 :2 : l), to provide 4-methylanisole 9 (46.4mg, 38) and 3,4-dimethoxytoluene 10 (91.6 mg, 60), and 51 (54.7 mg) of initial substrate 7 and 31 (43.2 mg) of initial substrate 8 were recovered. The structures of the obtained materials were confirmed by comparison of their 'H NMR (400 MHz) spectra and TLC mobility with those of authentic samples. RA-I1 0-triflate 11 A mixture of RA-I1 4 (80.5 mg, 0.11 mmol), N-phenyltri- fluoromethanesulfonimide (1 89.6 mg, 0.53 mmol), Et,N (0.074 cm3, 0.53 mmol) and CH,C1, (4 cm3) was stirred at room temperature for 26 h.The mixture was diluted with CH,Cl,, washed successively with water, 1 mol dm-, HCl and brine, and dried over Na,SO,. The solvent was evaporated off under reduced pressure to leave a residue, which was purified using MPLC (SiO,) with CH,Cl,-AcOEt-MeOH (15 :2 :1) as eluent to give the trijate 11 as an amorphous powder (94.0 mg, 99); vmaX(CHCl,)/cm-' 3425, 3040, 1680, 1640, 1510, 1425, 1270 and 1150; 6,(400 MHz; CDCl,; major conformer) 1.10 (3 H, d, J6.7, Ala-4 P-H3), 1.31 (3 H, d, J7.0, D-Ala-1 P-H3), 1.32 (3 H, d, J 7.0, Ala-2 B-H,), 2.64 (1 H, dd, J 11.3 and 3.1, Tyr-5 P-Ha), 2.69 (3 H, s, Tyr-6 NMe), 2.92 (3 H, s, Tyr-3 NMe), 2.95 (1 H, dd, J 18.0 and 3.8, Tyr-6 8-Hb), 3.10 (1 H, dd, J 18.0 and 1 1.8, Tyr-6 8-Ha), 3.12 (3 H, s, Tyr-5 NMe), 3.45 (1 H, dd, J 13.9 and 10.6, Tyr-3 8-Ha), 3.50 (1 H, dd, J 13.9 and 5.0, Tyr-3 p-Hb), 3.62 (1 H, dd, J 10.6 and 5.0, Tyr-3 a-H), 3.67 (1 H, dd, J 11.3 and 1 1.3, Tyr-5 p-Hb), 3.93 (3 H, s, Tyr-6 OMe), 4.34 (1 H, d, J 2.0, Tyr-6 6-Hb),4.37 (1 H, qd, J 7.0 and 6.8, D-Ala- 1 a-H), 4.54(1 H,dd, J11.8and3.8,Tyr-6a-H),4.75(1H,dq, J7.6and 6.7, Ala-4 a-H), 4.84 (1 H, dq, J8.2 and 7.0, Ala-2 a-H), 5.41 (1 H, dd, J 11.3 and 3.1, Tyr-5 a-H), 6.45 (1 H, d, J6.8, D-Ala-1 NH),6.56(1 H,d, J8.2,Ala-2NH),6.57(1 H,dd, J8.4and2.0, Tyr-66-Ha),6.74(1 H,d, J7.6,Ala-4NH),6.80(1 H,d, J8.4, Tyr-6 E-H), 6.87 (1 H, dd, J 8.4 and 2.4, Tyr-5 -Ha), 7.18-7.28 (6 H, m, Tyr-3 6-and E-H,, and Tyr-5 6-Ha and -Hb) and 7.41 (1 H, dd, J 8.4 and 2.2, Tyr-5 6-Hb); 6,(100 MHz; major conformer) D-Ala-1 (20.598, 47.53~, 170.63 CO), Ala-2 (16.308, 44.36a, 172.78 CO), Tyr-3 (33.088, 39.69 NMe, 67.70a, 118.59 CF,, 121.36~, 130.976, 139.57y, 148.196, 167.52 CO), Ala-4 (18.258,46.32~, 172.15 CO), Tyr-5 (30.42 NMe, 36.798, 54.22a, 124.08", 125.80~~, 130.826b, 132.626", 135.05y, 158.096, 169.20 CO) and Tyr-6 (29.18 NMe, 35.378, 56.02 OMe, 57.22a, 112.24~", 113.27tib, 120.846", 128.00y, 146.385, 152.96cb, 171.41 CO); m/z 889 (M + l', 10) and 121 (100); HR-FAB-MS Found: (M + H), 889.3066.C,,H,,F,N,O,,S (M + H) requires m/z,889.30541. N, 300 "C (from MeOH); .ID -209.8 (c 0.49, CHCI,); v,,,(KBr)/cm-' 3390, 3320, 2940, 1670, 1620, 1500, 1445, 1410, 1265, 1210, 1130 and 1100; 6,(400 MHz; CDCl,; major conformer) 1.11 (3 H, d, J 6.7, Ala-4 D-H,), 1.30 (3 H, d, J 6.9, D-Ala-1 P-H,), 1.35 (3 H, d, J 6.9, Ala-2 0-H,), 2.32 (3 H, s, Tyr-3 q-H3), 2.64 (1 H, dd, J 11.3, 3.0, Tyr-5 8-Ha), 2.69 (3 H, s, Tyr-6 NMe), 2.84 (3 H, s, Tyr-3 NMe), 2.95 (1 H, dd, J 18.6 and 3.9, Tyr-6 8-Hb), 3.09 (1 H, dd, J 18.6 and 11.9, Tyr-6 8-Ha), 3.12 (3 H, s, Tyr-5 NMe), 3.35 (1 H, dd, J 12.9 and 10.8, Tyr-3 8-Ha), 3.39 (1 H, dd, J 12.9 and 4.9, Tyr-3 p-Hb), 3.60 (1 H dd, J 10.8 and 4.9, Tyr-3 a-H), 3.67 (1 H, dd, J 11.3 and 11.3, Tyr-5 8-H), 3.93 (3 H, s, Tyr-6 OMe), 4.34 (1 H, d, J 2.0, Tyr-6 6-Hb), 4.36 (1 H, dq, J 7.1 and 6.9, D-Ala-1 a-H), 4.54 (1 H,dd, J11.9and3.9,Tyr-6a-H),4.75(1H,dq, J7.6and6.7, Ala-4 a-H), 4.84 (1 H, dq, J 8.4 and 6.9, Ala-2 a-H), 5.42 (1 H, dd, J 11.3 and 3.0, Tyr-5 a-H), 6.40 (1 H, d, J 8.4, Ala-2 NH), 6.44 (1 H, d, J7.1, D-Ala-1 NH), 6.57 (1 H, dd, J8.4 and 2.0, Tyr-66-Ha),6.71 (1 H,d, J7.6,Ala-4NH),6.80(1 H,d, J8.4, Tyr-6 E-H), 6.87 (1 H, dd, J8.4 and 2.4, Tyr-5 -Ha), 7.01 (2 H, d-like, J 7.8, Tyr-3 E- or 6-H,), 7.09 (2 H, d-like, J 7.8, Tyr-3 6-or E-H,), 7.20 (1 H, dd, J8.4 and 2.4, Tyr-5 -Hb), 7.26 (1 H, dd, J 8.4 and 2.2, Tyr-5 Ha) and 7.42 (I H, dd, J 8.4 and 2.2, Tyr-5 6-Hb); 6,( 100 MHz; CDCI,; major conformer) D-Ala-1 (20.628,47.67~, 170.61 CO), Ala-2(16.410,44.43~, 172.51 CO), Tyr-3 (20.95q, 33.00p, 39.72 NMe, 68.20a, 129.09~, 129.206, 135.536, 136.07y, 168.08 CO), Ala-4 (18.368, 46.31a, 172.19 CO), Tyr-5 (30.44 NMe, 36.858, 54.21~, 124.11~", 125.82~~, 130.906b, 132.696", 135.13y, 158.126, 169.25 CO) and Tyr-6 (29.23 NMe, 35.438, 56.08 OMe, 57.27~, 112.26~", 113.33?jb, 120.866", 128.12~, 146.416, 153.02cb, 171.65 CO); HR-FAB- MS Found: (M + H), 755.3807.C,,H,,N,O, (M + H) requires m/z,755.37681. N-Methyl-6-vinylphenylalanine-31RA-VII 13 To a solution of triflate 11 (62.3 mg, 0.070 mmol) in DMF (1.6 cm3) were added lithium chloride (30.6 mg, 0.72 mmol), dichlorobis(triphenylphosphine)palladium(rr) (49.3 mg, 0.070 mmol) and tributyl(viny1)tin (0.154 cm3, 0.53 mmol), and the mixture was stirred at 80 "C for 49 h. The mixture was then diluted with CH,Cl,, passed through a Celite pad, washed with saturated aq. NaCl, and dried over Na,SO,. The solvent was evaporated off under reduced pressure to leave a residue, which was purified using MPLC (SiO,) with CH,Cl,-AcOEt-MeOH (15:2 :1) as eluent to give compound 13as a crystalline powder (47.0 mg, 87); mp 257-261 "C (from MeOH); .ID -215.3 (c 0.71, CHCl,); v,,,(KBr)/cm-' 3390, 3310, 2940, 1670, 1625, 1510, 1445, 1410, 1265, 1210, 1135 and 1100; 6,(400 MHz; CDCl,; major conformer) 1.08 (3 H, d, J 6.7, Ala-4 8-H,), 1.28 (3 H, d, J 6.7, D-Ala-1 8-H,), 1.33 (3 H, d, J 6.8, Ala-2 8-H3), 2.63 (1 H, dd, J 11.3 and 2.5, Tyr-5 8-Ha), 2.68 (3 H, d, Tyr-6 NMe), 2.85 (3 H, s, Tyr-3 NMe), 2.94 (1 H, m, Tyr-6 p-Hb), 3.07 (1 H, m, Tyr-6 8-Ha), 3.10 (3 H, s, Tyr-5 NMe), 3.31-3.43 (2 H, m, Tyr-3 P-H,), 3.6 1 (1 H, dd, J 11.1 and 5.3, Tyr-3 a-H), 3.66 (1 H, dd, J 11.3 and 11.3, Tyr-5 p-Hb), 3.91 (3 H, s, Tyr-6 OMe), 4.32 (1 H, s-like, Tyr-6 6-Hb), 4.39 (1 H, dq, J 6.7 and 6.7, D-Ala-1 a-H), 4.54 (1 H, dd, J 11.8 and 3.6, Tyr-6 a-H), 4.75 (1 H, dq, J 7.7 and 6.7, Ala-4 a-H),4.79 (1 H, m, Ala-2 a-H), 5.2 1 (1 H, dd, J 11.0 and 1.4, Tyr-3 Ha), 5.40 (1 H, dd, J 11.3 and 2.5, Tyr-5a-H),5.70(1H,dd,J17.6and1.4,Tyr-38-Hb),6.49(1H, d, J 6.7, D-Ala-1 NH), 6.56 (2 H, d-like, J 8.3, Tyr-6 E-H and Ala-2 NH), 6.67 (1 H, ddd, J 17.6, 11.0 and 1.4, Tyr-3 q-H), 6.73 (1 H, d, J 7.7, Ala-4 NH), 6.78 (1 H, dd, J 8.4 and 1.8, Tyr-6 Ha), 6.85 (1 H, m, Tyr-5 -Ha), 7.08 (2 H, d-like, J 8.0, Tyr-3 6-H2), 7.19 (1 H, m, Tyr-5 E-H~), 7.24 (1 H, m, Tyr-5 6-Ha), 7.32 (2 H, d-like, J 8.0, Tyr-3 E-H,) and 7.40 (1 H, m, Tyr-5 6-Hb); 6,(100 MHz, major conformer) D-Ala-1 (20.628, 47.76a, 170.67 CO), Ala-2 (16.498, 44.50a, 172.64 CO), Tyr-3 (33.328,39.80NMe,68.16a, 113.538,126.41~, 129.446,136.07~, 136.38q, 138.506, 167.94 CO), Ala-4 (18.408, 46.39~, 172.24 CO), Tyr-5 (30.48 NMe, 36.928, 54.27a, 124.18~", 125.87cb, 130.92tjb, 132.73tia, 135.19, 158.226, 169.30 CO) and Tyr-6 (29.26 NMe, 35.498, 56.16 OMe, 57.36~1, 112.40~", 113.446b, 120.916", 128.21y, 146.516, 153.13~~, 171.68 CO); HR-FAB-MS Found: (M + H), 767.3807.C,,H,,N,O, (M + H) requires m/z,767.37681. 6-Ethyl-N-methylphenylalanine-3RA-VII14 Palladium (10) on activated carbon (61 mg) was added to a solution of the styrene 13 (60.3 mg, 0.079 mmol) in EtOH (15 cm3), and the mixture was stirred vigorously at room temperature for 1 h under hydrogen.The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was chromatographed on silica gel (MPLC) with CH,Cl,-AcOEt-MeOH (12 :2 :1) to provide compound 14 as a crystalline powder (55.8 mg, 92); mp 234234°C (from MeOH); alD -208.1 (c 0.54, CHCl,); v,,,(KBr)/cm-' 3400, 3330,2950,1670, 1620, 1510,1445, 1410, 1265, 1210, 1130and 1100; 6,(400 MHz; CDCl,; major conformer) 1.10 (3 H, d, J 6.6, Ala-4 P-H,), 1.21 (3 H, t, J7.6, Tyr-3 8-H,), 1.30 (3 H, d, J 6.9, D-Ala-1 8-H3), 1.35 (3 H, d, J6.9, Ala-2 P-H,), 2.59 (2 H, q, J7.6, Tyr-3 q-H,), 2.66 (1 H, dd, J 11.3 and 2.9, Tyr-5 8-Ha), 2.68 (3 H, s, Tyr-6 NMe), 2.83 (3 H, s, Tyr-3, NMe), 2.94 (I H, dd, J 18.3 and 3.8, Tyr-6 p-Hb), 3.09 (1 H, dd, J 18.3 and 11.9, Tyr-6 8-Ha), 3.12 (3 H, s, Tyr-5 NMe), 3.34 (1 H, dd, J 13.8 and 10.9, Tyr-3 8-Ha), 3.39 (1 H, dd, J 13.8 and 4.7, Tyr-3 p-Hb), J.Chem. Soc., Perkin Trans. I 215 3.60 (1 H, dd, J 10.9 and 4.7, Tyr-3 a-H), 3.67 (1 H, dd, J 11.3 and 11.3, Tyr-5 8-Hb), 3.92 (3 H, s, Tyr-6 OMe), 4.33 (1 H, J 1.8, Tyr-6 6-Hb),4.37 (1 H, qd, J 6.9 and 6.7, D-Ala-1 a-H), 4.54 (1 H, dd, J 11.9 and 3.8, Tyr-6 a-H), 4.75 (1 H, J7.6 and 6.6, Ala-4 a-H), 4.83 (1 H, dq, J 8.0 and 6.9 Ala-2 a-H), 5.41 (1 H, dd, J 11.3 and 2.9, Tyr-5 a-H), 6.45 (1 H, d, J 6.7, D-Ala-1 NH), 6.52 (1 H, d, J 8.0 Ala-2 NH), 6.57 (1 H, dd, J 8.4 and 1.8, Tyr-6 -Ha), 6.71 (1 H, d, J7.6, Ala-4NH), 6.79 (1 H, d, J8.4, Tyr-6 E-H), 6.86 (1 H, dd, J 8.4 and 2.3, Tyr-5 -Ha), 7.04 (2 H, d-like, J 7.8, Tyr-3 6-or E-H,), 7.1 1 (2 H, d-like, J 7.8, Tyr-3 E-or 6-H,), 7.20 (I H, dd, J 8.4 and 2.3, Tyr-5 E-H~), 7.26 (1 H, dd, J 8.4 and 2.1, Tyr-5 Ha) and 7.42 (1 H, dd, J 8.4 and 2.1, Tyr-5 6-Hb); 6, (100 MHz; CDC1,; major conformer) D-Ala-1 (20.688,47.83a, 170.69 CO), Ala-2 (16.588,44.53a, 172.54CO), Tyr-3 (15.628, 28.45q, 33.12p, 39.73 NMe, 68.33a, 128.088, 129.236, 135.92y, 142.676, 168.04 CO), Ala-4 (18.473, 46.41a, 172.25 CO), Tyr-5 (30.51 NMe, 36.97p, 54.25a, 124.22~", 125.90~~,130.986b, 132.786", 135.17y, 158.234, 169.33 CO) and Tyr-6 (29.28 NMe, 35.508, 56.16 OMe, 57.37a, 112.32~", 113.406b, 120.926", 128.18y, 146.526, 153.13cb, 171.77 CO); HR-FAB-MS Found: (M + H), 769.3947. C42H53N608 (M + H) requires m/z,769.39251.l,-Allyl-N-methylphenylalanine-3RA-VII 15 To a solution of triflate 11 (48.3 mg, 0.054 mmol) in DMF (1 cm3) were added lithium chloride (24.0 mg, 0.57 mmol), dichlorobis(triphenylphosphine)palladium(II) (39.0 mg, 0.056 mmol) and allyltributyltin (0.17 cm3, 0.55 mmol), and the mixture was stirred at 80 "C for 55 h. The mixture was diluted with CH,Cl,, passed through a Celite pad, washed with saturated aq. NaC1, and dried over Na,S04. The solvent was evaporated off under reduced pressure to leave a residue, which was purified using MPLC (SiO,) with CH,Cl,-AcOEt-MeOH (12 :2 :1) as eluent to give compound 15 as a crystalline powder (33.2 mg, 78), mp 224-227 "c(from MeOH); aD -159.7 (c 0.50, CHCl,); v,,,(KBr)/cm-' 3390, 3310, 2940, 1665, 1625, 1510, 1500, 1440, 1410, 1265, 1200, 1160, 1135 and 1100; 6,(400 MHz; CDCl,; major conformer) 1.09 (3 H, d, J 6.9, Ala-4 8-H3), 1.29 (3 H, d, J6.9, D-Ala-1 P-H,), 1.34 (3 H, d, J 6.9, Ala-2 8-H3), 2.65 (1 H, dd, J 11.3 and 3.0, Tyr-5 8-Ha), 2.68 (3 H, s, Tyr-6 NMe), 2.83 (3 H, s, Tyr-3 NMe), 2.95 (1 H, dd, J 18.0 and 3.7, Tyr-6 p-Hb), 3.08 (1 H, dd, J 18.0 and 11.9, Tyr-6 8-Ha), 3.1 1 (3 H, s, Tyr-5 NMe), 3.30-3.41 (4 H, m, Tyr-3 p-and q-H,), 3.60 (1 H, dd, J 10.8 and 4.7, Tyr-3 a-H), 3.66 (1 H, dd, J 11.3 and 11.3, Tyr-5 p-Hb), 3.92 (3 H, s, Tyr-6 OMe), 4.33 (1 H, d, J 1.7, Tyr-6 6-Hb), 4.38 (1 H, qd, J6.9 and 6.7, D-Ala-1 a-H), 4.54 (1 H, dd, J 11.9 and 3.7, Tyr-6 a-H), 4.76 (1 H, dq, J 7.6 and 6.9, Ala-4 a-H), 4.82 (1 H, dq, J 7.9 and 6.9, Ala-2 a-H), 4.99-5.07(2H,rn,Tyr-31-H2),5.41(1 H,dd,Jl1.4and3.0,Tyr-5 a-H), 5.94 (1 H, m, Tyr-3 0-H), 6.44 (1 H, d, J 6.7, D-Ala-1 NH), 6.51 (1 H,d, J7.9,Ala-2NH), 6.57(1 H,dd, J8.4and 1.7,Tyr-6 Ha), 6.71 (1 H,d, J7.6,Ala-4NH), 6.79(1 H,d, J8.4, Tyr-6 E-H), 6.86 (1 H, dd, J 8.4 and 2.3, Tyr-5 -Ha), 7.04 (2 H, d-like, J 7.8, Tyr-3 E-or 6-H,), 7.09 (2 H, d-like, J 7.8, Tyr-3 6-or E-H,), 7.20(1 H,dd, J8.4and2.3,Tyr-5 -Hb),7.25(l H,dd, J8.4and 2.1, Tyr-5 Ha) and 7.41 (1 H, dd, J8.4 and 2.1, Tyr-5 6-Hb);6, (100 MHz; major conformer) D-Ala-1 (20.688, 47.77a, 170.66 CO), Ala-2 (16.578, 44.49a, 172.47 CO), Tyr-3 (33.12P, 39.73q and NMe, 68.26a, 115.751, 128.82~, 129.326, 136.48y, 137.368, 138.395, 167.98 CO), Ala-4 (18.458, 46.37a, 172.21 CO), Tyr-5 (30.48 NMe, 36.948, 54.22a, 124.18~", 125.86~~, 130.96Zib, 132.756", 135.16y, 158.185, 169.29 CO) and Tyr-6 (29.25 NMe, 35.488, 56.13 OMe, 57.33a, 112.29~", 113.37lib, 120.896", 128.14y, 146.481;, 153.09~~, 171.71 CO); HR-FAB-MS Found: (M + H), 781.3937.C4,H5,N6O8 (M + H) requires m/z, 781.39251. N-Methyl-~-propylphenylalanine-3R A-VII 16 Palladium (10) on activated carbon (31 mg) was added to a solution of compound 15 (30.3 mg, 0.039 mmol) in EtOH 216 J. Chem. SOC.,Perkin Trans. 1 (8 cm3), and the mixture was stirred vigorously at room temperature for 1.5 h under hydrogen. The mixture was filtered, and the filtrate was concentrated under reduced pressure.The residue was chromatographed on silica gel (MPLC) with CH,CI,-AcOEt-MeOH (12 :2 :1) as eluent to provide compound 16 as a crystalline powder (25.0 mg, 8279, mp 235-238°C (from MeOH); aD -200.3 (c 0.51, CHCl,); v,,,(KBr)/cm-l 3390, 3320,2940, 1670, 1620, 1510, 1440, 1405, 1265, 1130 and 1100; 6,(400 MHz; CDCl,; major conformer) 0.90 (3 H, t, J7.3, Tyr-3 1-H3),1.09 (3 H, d, J6.7, Ala-4 P-H,), 1.29 (3 H, d, J 6.9, D-Ala-1 P-H3), 1.34 (3 H, d, J 6.9, Ala-2 8-H3), 1.61 (2 H, m, Tyr-3 0-H,), 2.55 (2 H, t-like, J7.5, Tyr-3 q-H,), 2.64 (1 H, dd, J 1 1.3 and 3.0, Tyr-5 P-Ha), 2.68 (3 H, s, Tyr-6NMe), 2.83 (3 H, s, Tyr-3NMe), 2.95 (1 H, dd, J 17.8 and 3.7, Tyr-6 8-Hb), 3.08 (1 H, dd, J 17.8 and 12.0, Tyr-6 P-Ha),3.1 1 (3 H, s, Tyr-5 NMe), 3.34 (1 H, dd, J 13.9 and 10.7, Tyr-3 P-Ha), 3.38 (1 H, dd, J 13.9 and 5.0, Tyr-3 P-Hb), 3.61 (1 H, dd, J 10.7 and 5.0, Tyr-3 a-H), 3.66 (1 H, dd, J 11.3 and 11.3, Tyr-5 P-Hb), 3.92(3 H,s,Tyr-6OMe), 4.34(1 H,d, J2.0,Tyr-66-Hb),4.39(1 H, dq, J6.9 and 6.9, D-Ala-1 a-H), 4.55 (1 H, dd, J 12.0 and 3.7, Tyr-6 a-H), 4.75 (1 H, dq, J 7.6 and 6.7, Ala-4 a-H), 4.82 (1 H, dq, J8.1 and6.9,Ala-2a-H), 5.41 (1 H,dd, J11.3and3.0,Tyr-5 a-H), 6.52 (1 H, d, J 6.9, D-Ala-1 NH), 6.56 (1 H, d, J 8.1, Ala-2 NH), 6.57 (1 H, dd, J8.3 and 2.0, Tyr-6 -Ha), 6.72 (1 H, d, J 7.6, Ala-4 NH), 6.79 (1 H, d, J 8.3, Tyr-6 E-H), 6.86 (1 H, dd, J 8.4 and 2.4, Tyr-5 -Ha), 7.03 (2 H, d-like, J 7.9, Tyr-3 E-or 6-H,), 7.08 (2 H, d-like, J7.9, Tyr-3 6-or E-H,), 7.20 (1 H, dd, J 8.4 and 2.4, Tyr-5 8-Hb), 7.25 (1 H, dd, J 8.4 and 2.2, Tyr-5 Ha)and 7.41 (1 H, dd, J8.4 and 2.2, Tyr-5 6-Hb); 6,(100 MHz; CDCl,; major conformer) D-Ala-1 (20.67p, 47.75a, 170.71 CO), Ala-2 (16.398, 44.50a, 172.86 CO), Tyr-3 (13.691, 33.088, 37.57q, 39.77NMe,68.37a, 128.708, 129.116, 135.797, 141.071;, 167.97 CO), Ala-4 (18.368, 46.41a, 172.35 CO), Tyr-5 (30.51 NMe, 36.91p, 54.2501, 124.20~", 125.88~~, 130.926b, 132.776", 135.13y, 158.216, 169.30 CO) and Tyr-6 (29.29 NMe, 35.488, 56.14 OMe, 57.33a, 112.29~", 113.42Fb, 120.916", 128.20y, 146.49(, 153.08~~, 171.71 CO); HR-FAB-MS Found: (M + H), 783.4090. C43H55N60, (M + H) requires m/z,783.40811.Cell survival by MTT assay MTT colorimetric assay was performed in a 96-well plate.I6 The assay is dependent on the reduction of MTT by the mito- chondrial dehydrogenase of viable cells to give a blue formazan product which can be measured spectrophotometrically. Mouse P-388 leukaemia cells (2 x lo4 cells cm-,) were inoculated in each well with 0.1 cm3 of RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10fetal calf serum (Flow Lab- oratories, UK), 100 units of penicillin and 100 pg cm-3 of streptomycin.After overnight incubation (37 "C; 5 CO,), sample solution (0.1 cm3) was added to each well and the plates were incubated for 2 days. Then MTT (0.05 cm3) (200 pg cm-, PBS) was added to each well and the plates were incubated for a further 4 h. The resulting formazan was dissolved in Me,SO (0.15 cm3).The plates were placed on a plate shaker for 5 min and read immediately at 540 nm. The IC,, (pg cmp3)-value was defined as that concentration of sample which caused 50 reduction of growth in sample-treated cells, with respect to the controls. The IC,,-value was calculated by using the probit test. in vivo Antiturnour activity P-388 murine leukaemia cells (1 x lo6 cells) were inoculated i.p. into female CDF, mice (67 weeks old, control n = 16; test n = 8) on day 0. Samples, suspended in 0.5gum arabic-saline solution, were administered i.p. on days 1-5. The antitumour activity was estimated according to the NCI tumour panel screening method.' ' References 1 Part 7, Y. Hitotsuyanagi, J.Suzuki, Y. Matsumoto, K. Takeya and H. Itokawa, J. Chem. SOC.,Perkin Trans. I, 1994, 1887. 2 H. Itokawa, K. Takeya, K. Mihara, N. Mori, T. Hamanaka, T. Sonobe and Y. Iitaka, Chem. Pharm. Bull., 1983, 31, 1424; H. Itokawa, K. Takeya, N. Mori, T. Hamanaka, T. Sonobe and K. Mihara, Chem. Pharm. Bull., 1984,32,284. 3 S. D. Jolad, J. J. Hoffmann, S. J. Torrance, R. M. Wiedhopf, J. R. Cole, S. K. Arota, R. B. Bates, R. L. Gargiulo and G. R. Kriek, J. Am. Chem. SOC., 1977,99,8040. 4 M. Zalacain, E. Zaera, D. Vhzquez and A. Jimknez, FEBS Lett., 1982, 148, 95. 5 (a) H. Majima, S. Tsukagoshi, H. Furue, M. Suminaga, K. Sakamoto, R. Wakabayashi, S. Kishino, H. Niitani, A. Murata, A. Genma, N. Nukariya, K. Uematsu, T. Furuta, M. Kurihara, F.Yoshida, S. Isomura, T. Takemoto, M. Hirashima, T. Izumi, I. Nakao, Y. Ohashi, K. Ito and R. Asai, Jpn. J. Cancer Chemother., 1993, 20, 67; (b)F. Yoshida, R. Asai, H. Majima, S. Tsukagoshi, H. Furue, M. Suminaga, K. Sakamoto, H. Niitani, A. Murata, M. Kurihara, T. Izumi, I. Nakao, Y. Ohashi and K. Ito, Jpn. J. Cancer Chemother., 1994,21, 199. 6 R. B. Bates, S. L. Gin, M. A. Hassen, V. J. Hruby, K. D. Janda, G. R. Kriek, J.-P. Michaud and D. B. Vine, Heterocycles, 1984, 22, 785; M. E. Jung, D. Jachiet and J. C. Rohloff, Tetrahedron Lett., 1989, 30, 421 1; R. K. Olsen and X. Feng, Tetrahedron Lett., 1991, 32, 5721; J. Org. Chem., 1992, 57, 5811; A. V. R. Rao, T. K. Chakraborty, K. L. Reddy and A. S. Rao, Tetrahedron Lett., 1992, 33, 4799; R. Beugelmans, A. Bigot and J. Zhu, Tetrahedron Lett., 1994, 35, 5649; M. E. Jung, D. Jachiet, S. I. Khan and C. Kim, Tetrahedron Lett., 1995,36, 361. 7 T. Inaba, I. Umezawa, M. Yuasa, T. Tnoue, S. Mihashi, H. Ttokawa and K. Ogura, J. Org. Chem., 1987,52,2957. 8 (a) D. L. Boger and D. Yohannes, J. Am. Chem. SOC., 1991, 113, 1427; (b) D. L. Boger, D. Yohannes, J. Zhou and M. A. Patane, J. Am. Chem. Soc., 1993,115, 3420. 9 H. Itokawa, K. Kondo, Y. Hitotsuyanagi, A. Nakamura, H. Morita and K. Takeya, Chem. Pharm. Bull., 1993,41,1266. 10 R. J. Petroski, R. B. Bates, G. S. Linz and J. P. Rosazza, J. Pharm. Sci., 1983,72, 1291. 11 H. Itokawa, K. Saito, H. Morita, K. Takeya and K. Yamada, Chem. Pharm. Bull., 1992,40, 2984. 12 H. Ttokawa, K. Takeya, N. Mori, T. Sonobe, S. Mihashi and T. Hamanaka, Chem. Pharm. Bull., 1986,34,3762. 13 T. Aoyama, S. Terasawa, K. Sudo and T. Shioiri, Chem. Pharm. Bull., 1984,32, 3759. 14 A. M. Echavarren and J. K. Stille, J. Am. Chem. SOC.,1987, 109, 5478. 15 D. L. Boger, D. Yohannes and J. B. Meyers, Jr., J. Org. Chem., 1992, 57, 1319; D. L. Boger, M. A. Patane, Q. Jin and P. A. Kitos, Bioorg. Med, Chem., 1994, 2, 85. 16 J. Carmichael, W. G. DeGraff, A. F. Gazdar, J. D. Minna and J. B. Mitchell, Cancer Rex, 1987,47,936. 17 J. M. Venditti, R. A. Wesley and J. Plowman, Adu. Pharmacol. Chemother., 1984, 20, 1. Paper 5/03064A Received 15th May 1995 Accepted 8th August 1995 J. Chem. SOC.,Perkin Trans. 1 217