Diastereoselective synthesis of new psi[(E)-CH=CMe]- and psi[(Z)-CH=CMe]-type alkene dipeptide isosteres by organocopper reagents and application to conformationally restricted cyclic RGD peptidomimetics.

Article abstract of DOI:10.1021/jo025923m

Diastereoselective synthesis of new psi[(E)-CH=CMe]- and psi[(Z)-CH=CMe]-type alkene dipeptide isosteres corresponding to dipeptides having one N-methylamino acid, and application to bioactive peptides, are described. In a key reaction introducing the chiral alpha-alkyl group of the isosteres, organocopper-mediated alkylation of syn-beta-methylated gamma-mesyloxy-alpha,beta-enoate 26a afforded E- and Z-isomers of anti-S(N)2' products in a solvent-dependent manner. The resulting two isosteres, D-Phe-psi[(E)-CH=CMe]-L-Val 27a and D-Phe-psi[(Z)-CH=CMe]-L-Val 28b, which corresponded to trans- and cis-conformers of D-Phe-L-MeVal, respectively, were utilized in a structure-activity relationship study on cyclic RGD peptides 1 and 2, in company with a psi[(E)-CH=CH]-type alkene dipeptide isostere, D-Phe-psi[(E)-CH=CH]-L-Val. The cyclic isostere-containing pseudopeptides 3, 4, and 40 were synthesized and biological activity against integrin alpha(V)beta(3) and alpha(IIb)beta(3) receptors were also evaluated.

Full text of DOI:10.1021/jo025923m

Dia ster eoselective Syn th esis of New ψ[(E)-CHdCMe]- a n d
ψ[(Z)-CHdCMe]-typ e Alk en e Dip ep tid e Isoster es by Or ga n ocop p er
Rea gen ts a n d Ap p lica tion to Con for m a tion a lly Restr icted Cyclic
RGD P ep tid om im etics
Shinya Oishi,† Takae Kamano,† Ayumu Niida,† Yoshihiko Odagaki,‡ Nobuyuki Hamanaka,‡
Mikio Yamamoto,§ Keiichi Ajito,§ Hirokazu Tamamura,† Akira Otaka,† and Nobutaka Fujii†,*
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, J apan,
Minase Research Institute, Ono Pharmaceutical Co. Ltd., Mishima-gun, Osaka, 618-8585, J apan, and
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Kohoku-ku, Yokohama, 222-0002, J apan
nfujii@pharm.kyoto-u.ac.jp
Received May 9, 2002
Diastereoselective synthesis of new ψ[(E)-CHdCMe]- and ψ[(Z)-CHdCMe]-type alkene dipeptide
isosteres corresponding to dipeptides having one N-methylamino acid, and application to bioactive
peptides, are described. In a key reaction introducing the chiral R-alkyl group of the isosteres,
organocopper-mediated alkylation of syn-â-methylated γ-mesyloxy-R,â-enoate 26a afforded E- and
Z-isomers of anti-SN2′ products in a solvent-dependent manner. The resulting two isosteres, D-Phe-
ψ[(E)-CHdCMe]-L-Val 27a and D-Phe-ψ[(Z)-CHdCMe]-L-Val 28b, which corresponded to trans-
and cis-conformers of D-Phe-L-MeVal, respectively, were utilized in a structure-activity relationship
study on cyclic RGD peptides 1 and 2, in company with a ψ[(E)-CHdCH]-type alkene dipeptide
isostere, D-Phe-ψ[(E)-CHdCH]-L-Val. The cyclic isostere-containing pseudopeptides 3, 4, and 40
were synthesized and biological activity against integrin RVâ3 and RIIbâ3 receptors were also
evaluated.
In tr od u ction
N-methylamino acid scanning of 1 demonstrated that
substitution of Val with N-methylvaline remarkably
increased antagonistic activity against RVâ3 integrin,
resulting in improved selectivity relative to RIIbâ3 inte-
grin.6 The selective RVâ3 antagonist, cyclo(-Arg-Gly-Asp-
D-Phe-MeVal-) 2, is now in phase III clinical trial. In
general, imidic peptide bonds N-terminal to N-alkylamino
acids such as proline possess similar energy barriers for
Integrins are cell membrane receptors consisting of R
and â subunits, which participate in cell-cell and cell-
matrix adhesive interactions.1 Among these receptors,
RVâ3 integrin receptors are involved in a number of
biological processes, including tumor-induced angiogen-
esis2 and adhesion of osteoclasts to bone matrix.3 Thus,
RVâ3 integrin antagonists are being developed as potential
therapeutic agents for tumor metastasis, osteoporosis,
and other diseases. A significant number of drug candi-
dates have been designed based on the RGD (Arg-Gly-
Asp) sequence, which is key recognition motif of integrin
receptors.4 In 1991, Kessler et al. reported that a cyclic
pentapeptide, cyclo(-Arg-Gly-Asp-D-Phe-Val-) 1, was a
highly active integrin RVâ3 antagonist.5 Their extensive
conformational research also revealed that peptide 1
adopted a representative type II′ â/γ structure, in which
D-Phe and Gly were at the i + 1 positions of â- and
γ-turns in dimethyl sulfoxide, respectively. In addition,
(4) (a) Kerr, J . S.; Wexler, R. S.; Mousa, S. A.; Robinson, C. S.;
Wexler, E. J .; Mohamed, S.; Voss, M. E.; Devenny, J . J .; Czerniak, P.
M.; Gudzelak, A., J r; Slee, A. M. Anticancer Res. 1999, 19, 959. (b)
Miller, W. H; Alberts, D. P.; Bhatnagar, P. K.; Bondinell, W. E.;
Callahan, J . F.; Calvo, R. R.; Cousins, R. D.; Erhard, K. F.; Heerding,
D. A.; Keenan, R. M.; Kwon, C.; Manley, P. J .; Newlander, K. A.; Ross,
S. T.; Samanen, J . M.; Uzinskas, I. N.; Venslavsky, J . W.; Yuan, C.
C.-K.; Haltiwanger, R. C.; Gowen, M.; Hwang, S.-M.; J ames, I. E.; Lark,
M. W.; Rieman, D. J .; Stroup, G. B.; Azzarano, L. M.; Salyers, K. L.;
Smith, B. R.; Ward, K. W.; J ohanson, K. O.; Huffman, W. F. J . Med.
Chem. 2000, 43, 22. (c) Kumar, C. C.; Malkowski, M.; Yin, Z.;
Tanghetti, E.; Yaremko, B.; Nechuta, T.; Varner, J .; Liu, M.; Smith,
E. M.; Neustadt, B.; Presta, M.; Armstrong, L. Cancer Res. 2001, 61,
2232. (d) Coleman, P. J .; Brashear, K. M.; Hunt, C. A.; Hoffman, W.
F.; Hutchinson, J . H.; Breslin, M. J .; McVean, C. A.; Askew, B. C.;
Hartman, G. D.; Rodan, S. B.; Rodan, G. A.; Leu, C. T.; Prueksari-
tanont, T.; Fernandez-Metzler, C.; Ma, B.; Libby, L. A.; Merkle, K. M.;
Stump, G. L.; Wallace, A. A.; Lynch, J . J .; Lynch, R.; Duggan, M. E.
Bioorg. Med. Chem. Lett. 2002, 12, 31. (e) Goodman, S. L.; Ho¨lzemann,
G.; Sulyok, G. A. G.; Kessler, H. J . Med. Chem. 2002, 45, 1045.
(5) (a) Aumailley, M.; Gurrath, M.; Muller, G.; Calvete, J .; Timpl,
R.; Kessler, H. FEBS Lett. 1991, 291, 50. (b) Haubner, R.; Gratias, R.;
Diefenbach, B.; Goodman, S. L.; J onczyk, A.; Kessler, H. J . Am. Chem.
Soc. 1996, 118, 7461. (c) Haubner, R.; Finsinger, D.; Kessler, H. Angew.
Chem. Int. Ed. Engl. 1997, 36, 1374.
* To whom correspondence should be addressed. Tel: +81-75-753-
4551; Fax: +81-75-753-4570.
† Kyoto University.
‡ Ono Pharmaceutical Co. Ltd.
§ Meiji Seika Kaisha, Ltd.
(1) (a) Hynes, R. O. Cell 1992, 69, 11. (b) Humphries, M. J . Biochem.
Soc. Trans. 2000, 28, 311.
(2) (a) Brooks, P. C.; Clark, R. A.; Cheresh, D. A. Science 1994, 264,
569. (b) Varner, J . A.; Cheresh, D. A. Curr. Opin. Cell Biol. 1996, 8,
724.
(3) Ross, F. P.; Chappel, J .; Alvarez, J . I.; Sander, D.; Butler, W. T.;
Farach-Carson, M. C.; Mintz, K. A.; Robey, P. G.; Teitelbaum, S. L.;
Cheresh, D. A. J . Biol. Chem. 1993, 268, 9901.
(6) Dechantsreiter, M. A.; Planker, E.; Matha¨, B.; Lohof, E.; Ho¨lze-
mann, G.; J onczyk, A.; Goodman, S. L.; Kessler, H. J . Med. Chem. 1999,
42, 3033.
10.1021/jo025923m CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/01/2002
6162
J . Org. Chem. 2002, 67, 6162-6173

Synthesis of Alkene Dipeptide Isosteres
and to suppress peptide bond cis/trans-isomerization,11
as well as other uses.12 To characterize bioactive confor-
mations of cyclic RGD peptides 1 and 2, particularly in
D-Phe-L-Val and D-Phe-L-MeVal moieties, we designed
two cyclic pseudopeptides 3 and 4, which contain ψ[(E)-
CHdCH]- A and ψ[(E)-CHdCMe]- B type (E)-alkene
dipeptide isosteres (EADIs), respectively. It was assumed
that the D-Phe-ψ[(E)-CHdCH]-L-Val-type EADI in 3 can
be a mimetic of the i + 1 and i + 2 â-turn sites in 1. The
methylated analogue, D-Phe-ψ[(E)-CHdCMe]-L-Val-type
EADI, in 4 can be viewed in the same way as an
equivalent of the D-Phe-L-MeVal moiety in 2. In pseudo-
peptides 3 and 4, EADI-mediated restriction of ω-angles
between D-Phe and L-Val/MeVal may permit rational
evaluation of the N-methyl group’s effect on the confor-
mation of peptide 2.
To realize the structure-activity relationship objec-
tives outlined above, we required the synthesis of un-
precedented alkene dipeptide isosteres B, which corre-
spond to dipeptides containing an N-methylamino acid
such as D-Phe-ψ[(E)-CHdCMe]-L-Val-type EADI. A num-
ber of synthetic approaches toward ψ[(E)-CHdCH]- A
and ψ[(Z)-CHdCH]-type C alkene dipeptide isosteres
have been reported to date.11-17 We attempted to syn-
F IGURE 1.
cis-trans imide isomerization. This results in greater
flexibility, which provides for a higher population of cis-
peptide bonds (ω ) 0°), as compared to usual amidic
peptide bonds.7 It is noteworthy that in water and
dimethyl sulfoxide the N-methyl group of L-MeVal in the
peptide 2 induces a more flexible γi/γ/γi arrangement,
which consists of two inverse γ-turns (γi-turns) having
Arg and Asp at the i + 1 positions and one γ-turn having
Gly at the i + 1 position.6 Although the peptide backbone
secondary structure was discussed in detail, no sugges-
tion was offered whether the flexible conformation in 2
was derived from rotation of the peptide bond between
D-Phe and L-MeVal induced by N-methylation or from
altered distribution of side chains proximal to the N-
methyl group with the maintenance of trans-amide
conformer of D-Phe-L-MeVal (ω ) 180°).
(11) (a) Bol, K. M.; Liskamp, R. M. J . Tetrahedron 1992, 48, 6425.
(b) Bohnstedt, A. C.; Prasad, J .V.N. V.; Rich, D. H. Tetrahedron Lett.
1993, 34, 5217. (c) Andres, C. J .; Macdonald, T. L.; Ocain, T. D.; Longhi,
D. J . Org. Chem. 1993, 58, 6609. (d) Boros, L. G.; De Corte, B.; Gimi,
R. H.; Welch, J . T.; Wu, Y.; Handschumacher, R. E. Tetrahedron Lett.
1994, 35, 6033. (e) Welch, J . T.; Lin, F. Tetrahedron 1996, 52, 291. (f)
Hart, S. A.; Sabat M.; Etzkorn, F. A. J . Org. Chem. 1998, 63, 7580. (g)
Hart S. A.; Etzkorn, F. A. J . Org. Chem. 1999, 64, 2998. (h) Hart, S.
A.; Trindle, C. O.; Etzkorn, F. A. Org. Lett. 2001, 3, 1789.
(12) (a) Shue, Y.-K.; Tufano, M. D.; Carrera, G. M., J r,; Kopecka,
H.; Kuyper, S. L.; Holladay, M. W.; Lin, C. W.; Witte, D. G.; Miller, T.
R.; Stashko, M.; Nadzan, A. M. Bioorg. Med. Chem. 1993, 1, 161. (b)
Christos, T. E.; Arvanitis, A.; Cain, G. A.; J ohnson, A. L.; Pottorf, R.
S.; Tam, S. W.; Schmidt, W. K. Bioorg Med. Chem. Lett. 1993, 3, 1035.
(c) Wai, J . S.; Bamberger, D. L.; Fisher, T. E.; Graham, S. L.; Smith,
R. L.; Gibbs, J . B.; Mosser, S. D.; Oliff, A. I.; Pompliano, D. L.; Rands,
E.; Kohl, N. E. Bioorg. Med. Chem. 1994, 2, 939. (d) Wada, M.; Doi,
R.; Hosotani, R.; Higashide, S.; Ibuka, T.; Habashita, H.; Nakai, K.;
Fujii, N.; Imamura, M. Pancreas 1995, 10, 301. (e) Bartlett, P. A.;
Otake, A. J . Org. Chem. 1995, 60, 3107. (f) Fujimoto, K.; Doi, R.;
Hosotani, R.; Wada, M.; Lee, J .-U.; Koshiba, T.; Ibuka, T.; Habashita,
H.; Nakai, K.; Fujii, N.; Imamura, M. Life Sci. 1997, 60, 29. (g)
Miyasaka, K.; Kanai, S.; Masuda, M.; Ibuka, T.; Nakai, K.; Fujii, N.;
Funakoshi, A. J . Auton. Nerv. Syst. 1997, 63, 179. (h) Kawaguchi, M.;
Hosotani, R.; Ohishi, S.; Fujii, N.; Tulachan, S. S.; Koizumi, M.; Toyoda,
E.; Masui, T.; Nakajima, S.; Tsuji, S.; Ida, J .; Fujimoto, K.; Wada, M.;
Doi R.; Imamura, M. Biochem. Biophys. Res. Commun. 2001, 288, 711.
(i) Tamamura, H.; Hiramatsu, K.; Miyamoto, K.; Omagari, A.; Oishi,
S.; Nakashima, H.; Yamamoto, N.; Kuroda, Y.; Nakagawa, T.; Otaka
A.; Fujii, N. Bioorg. Med. Chem. Lett. 2002, 12, 923.
(13) (a) Ibuka, T.; Habashita, H.; Funakoshi, S.; Fujii, N.; Oguchi,
Y.; Uyehara, T.; Yamamoto, Y. Angew. Chem., Int. Ed. Engl. 1990,
29, 801. (b) Ibuka, T.; Habashita, H.; Otaka, A.; Fujii, N.; Oguchi, Y.;
Uyehara T.; Yamamoto, Y. J . Org. Chem. 1991, 56, 4370. (c) Ibuka,
T.; Yoshizawa, H.; Habashita, H.; Fujii, N.; Chounan, Y.; Tanaka, M.;
Yamamoto, Y. Tetrahedron Lett. 1992, 33, 3783. (d) Noda, M.; Ibuka,
T.; Habashita, H.; Fujii, N. Chem. Pharm. Bull. 1997, 45, 1259.
(14) (a) Ibuka, T.; Nakai, K.; Habashita, H.; Hotta, Y.; Fujii, N.;
Mimura, N.; Miwa, Y.; Taga T.; Yamamoto, Y. Angew. Chem., Int. Ed.
Engl. 1994, 33, 652. (b) Fujii, N.; Nakai, K.; Tamamura, H.; Otaka,
A.; Mimura, N.; Miwa, Y.; Taga, T.; Yamamoto Y.; Ibuka, T. J . Chem.
Soc., Perkin Trans. 1 1995, 1359. (c) Ibuka, T.; Mimura, N.; Ohno, H.;
Nakai, K.; Akaji, M.; Habashita, H.; Tamamura, H.; Miwa, Y.; Taga,
T.; Fujii N.; Yamamoto, Y. J . Org. Chem. 1997, 62, 2982.
Peptidomimetics are valuable tools for conformational
investigation of bioactive peptides and proteins, and for
development of peptide-leads for pharmaceuticals.8 Alk-
ene dipeptide isosteres restrict peptide ω-angle rotations
to cis- or trans-conformers (Figure 1). They have been
widely utilized for elucidating the importance of amide
bond polarity,9 as surrogates of â-turn substructures10
(7) Fischer, G. Chem. Soc. Rev. 2000, 29, 119.
(8) For recent reviews of peptidomimetics, see: (a) Peptide Second-
ary Structure Mimetics. Tetrahedron (Symposia-in-print; No. 50, Kahn,
M., Ed.), 1993, 49, 3433. (b) Hanessian, S.; McNaughton-Smith, G.;
Lombart H.-G.; Lubell, W. D. Tetrahedron, 1997, 53, 12789. (c)
Peptidomimetics Protocols; Kazmiersk, W. M., Ed.; Humana Press,
Totowa, New J ersey, 1999. (d) Hruby, V. J .; Balse, P. M. Curr. Med.
Chem., 2000, 7, 945. (e) Kim H.-O.; Kahn, M. Comb. Chem. High
Throughput Screening, 2000, 3, 167. (f) Bursavich, M. G.; Rich, D. H.
J . Med. Chem., 2002, 45, 541.
(15) (a) Wipf, P.; Fritch, P. C. J . Org. Chem. 1994, 59, 4875. (b) Wipf,
P.; Henninger, T. C. J . Org. Chem. 1997, 62, 1586.
(9) Vasbinder, M. M.; J arvo, E. R.; Miller, S. J . Angew. Chem., Int.
Ed. 2001, 40, 2824.
(10) (a) Gardner, R. R.; Liang G.-B.; Gellman, S. H. J . Am. Chem.
Soc. 1995, 117, 3280. (b) Wipf, P.; Henninger, T. C.; Geib, S. J . J . Org.
Chem. 1998, 63, 6088. (c) Gardner, R. R.; Liang G.-B.; Gellman, S. H.
J . Am. Chem. Soc. 1999, 121, 1806.
(16) (a) Tamamura, H.; Yamashita, M.; Muramatsu, H.; Ohno, H.;
Ibuka, T.; Otaka, A.; Fujii, N. Chem. Commun. 1997, 2327. (b)
Tamamura, H.; Yamashita, M.; Nakajima, Y.; Sakano, K.; Otaka, A.;
Ohno, H.; Ibuka T.; Fujii, N. J . Chem. Soc., Perkin Trans. 1 1999, 2983.
(c) Oishi, S.; Tamamura, H.; Yamashita, M.; Odagaki, Y.; Hamanaka,
N.; Otaka A.; Fujii, N. J . Chem. Soc. Perkin Trans. 1 2001, 2445.
J . Org. Chem, Vol. 67, No. 17, 2002 6163

Oishi et al.
SCHEME 1a
SCHEME 2a ,b
a
Reagents: (a) Ac2O, pyridine, DMAP; (b) O3 gas; (c) Me2S; (d)
(EtO)2P(O)CH2CO2t-Bu, (i-Pr)2NEt, LiCl; (e) Na2CO3, MeOH; (f)
MsCl, pyridine; (g) i-PrCu(CN)MgCl‚BF3; (h) TFA; (i) (Boc)2O,
Et3N.
thesize ψ[(E)-CHdCMe]-type EADIs B using organocop-
per-mediated anti-SN2′ alkylation of â-methylated R,â-
enoates having a leaving group at the γ-position. This
was based on well-established stereochemical precedence
as in the synthesis of ψ[(E)-CHdCH]-type EADIs A.13,18
In the current paper, we describe the diastereoselective
synthesis of new ψ[(E)-CHdCMe]-type alkene dipeptide
isosteres B as well as unexpected ψ[(Z)-CHdCMe]-type
ones D. We also present an exploration of bioactive
conformation of Kessler’s cyclic RGD peptides using
pseudopeptides containing these isosteres and a ψ[(E)-
CHdCH]-type isostere A.19
a
Reagents: (a) SOCl2, MeOH; (b) Boc-Arg(Mts)-OH, DCC,
HOBt, (i-Pr)2NEt; (c) 4 M HCl-dioxane, anisole; (d) 11, DCC,
HOBt, (i-Pr)2NEt; (e) LiOH; (f) TFA, anisole; (g) Boc-Asp(OBn)-
ONB, (i-Pr)2NEt; (h) DCC, HONB; (i) 4 M HCl-dioxane; (j)
N-methylmorpholine; (k) 1M TMSBr-thioanisole/TFA, m-cresol,
b
1,2-ethanedithiol. Abbreviations: HOBt ) 1-hydroxybenzotriaz-
ole; HONB ) N-hydroxy-5-norbornene-2,3-dicarboximide; Mts )
2,4,6-trimethylphenylsulfonyl.
give γ-acetoxy-R,â-enoate 7 E-selectively in 63% yield,
which was readily isolated by flash chromatography over
silica gel. Alcoholysis of the acetyl group followed by
mesylation yielded γ-mesyloxy-R,â-enoate 9, which is a
key substrate for organocopper-mediated alkylation.
Treatment of R,â-enoate 9 with i-PrCu(CN)MgCl‚BF3
afforded an R-alkylated product, Boc-D-Phe-ψ[(E)-CHd
CH]-L-Val-Ot-Bu 10, as a single diastereomer in 84%
yield. Successive TFA treatment and N-reprotection of
10 provided N-Boc-protected EADI 11, which was utilized
for Boc-based solution-phase peptide synthesis of pseudo-
peptide 3 (Scheme 2). EADI 11 was converted to pro-
tected pseudotetrapeptide 13 by DCC condensation with
a HCl-treated sample of Boc-Arg(Mts)-Gly-OMe 12,
which was prepared by coupling of Boc-Arg(Mts)-OH and
glycine methyl ester. With the intention of avoiding
succinimide formation of an aspartic acid moiety by basic
treatment, the methyl ester of protected peptide 13 was
saponified at this stage to give the corresponding acid,
Boc-D-Phe-ψ[(E)-CHdCH]-Val-Arg(Mts)-Gly-OH 14. TFA
treatment of protected peptide 14 and coupling with Boc-
Asp(OBn)-ONB provided the linear pseudopentapeptide,
Boc-Asp(OBn)-D-Phe-ψ[(E)-CHdCH]-Val-Arg(Mts)-Gly-
OH 15. To facilitate efficient intramolecular peptide bond
formation, acid 15 was quantitatively converted to the
activated ester, Boc-Asp(OBn)-D-Phe-ψ[(E)-CHdCH]-Val-
Arg(Mts)-Gly-ONB 16. This was subjected to Boc group
deprotection using HCl in 1,4-dioxane followed by cy-
clization in highly dilute solution. The resulting protected
cyclic peptide 18 was treated with 1 M TMSBr-thioani-
sole in TFA in the presence of m-cresol and 1,2-
ethanedithiol21 to afford expected cyclic peptide 3, which
Resu lts a n d Discu ssion
Syn th esis of a Cyclic RGD P seu d op ep tid e Con -
ta in in g D-P h e-ψ[(E)-CHdCH]-L-Va l-typ e Alk en e Di-
p ep tid e Isoster e. Initially, we undertook the synthesis
of pseudopeptide 3, which contains a D-Phe-ψ[(E)-CHd
CH]-L-Val-type EADI, that is a potential equivalent of
the cyclic peptide 1. Stereoselective synthesis from chiral
amino acid derivatives utilizing 1,3-chirality transfer of
ψ[(E)-CHdCH]-type EADIs A having a disubstituted
alkene has been fully documented by us and others. Here
reaction of γ-mesyloxy-R,â-enoates using organocopper
reagents gave R-alkylated products regio- and stereo-
selectively by an anti-SN2′ mechanism.13 A protected
D-Phe-ψ[(E)-CHdCH]-L-Val-type EADI 11 was synthe-
sized according to established procedures (Scheme 1).
After protection with an acetyl group of the known allyl
alcohol 5,16b the resulting acetate 6 was subjected to a
sequence of reactions consisting of ozonolysis followed by
modified Horner-Wadsworth-Emmons olefination20 to
(17) (a) Fujii, N.; Habashita, H.; Shigemori, N.; Otaka, A.; Ibuka,
T.; Tanaka, M.; Yamamoto, Y. Tetrahedron Lett. 1991, 32, 4969. (b)
Otaka, A.; Watanabe, H.; Mitsuyama, E.; Yukimasa, A.; Tamamura,
H.; Fujii, N. Tetrahedron Lett. 2001, 42, 285. (c) Otaka, A.; Watanabe,
H.; Yukimasa, A.; Oishi, S.; Tamamura, H.; Fujii, N. Tetrahedron Lett.
2001, 42, 5443. (d) Oishi, S.; Niida, A.; Kamano, T.; Odagaki, Y.;
Tamamura, H.; Otaka, A.; Hamanaka, N.; Fujii, N. Org. Lett. 2002, 4,
1055.
(18) For
a review of regio- and stereoselective syntheses with
organocopper reagents, see: Krause, N.; Gerold, A. Angew. Chem., Int.
Ed. Engl. 1997, 36, 186.
(19) A portion of this work has appeared in a preliminary com-
munication: Oishi, S.; Kamano, T.; Niida, A.; Odagaki, Y.; Tamamura,
H.; Otaka, A.; Hamanaka, N.; Fujii, N. Org. Lett. 2002, 4, 1051.
(20) Blanchette, M. A.; Choy, W.; Davis, J . T.; Essenfeld, A. P.;
Masamune, S.; Roush, W. R.; Sakai, T. Tetrahedron Lett. 1984, 25,
2183.
1
was fully characterized by H NMR and mass spectra.
(21) Yajima, H.; Fujii, N.; Funakoshi, S.; Watanabe, T.; Murayama,
E.; Otaka, A. Tetrahedron 1988, 44, 805.
6164 J . Org. Chem., Vol. 67, No. 17, 2002

Synthesis of Alkene Dipeptide Isosteres
SCHEME 3
SCHEME 4a
Dia ster eoselective Syn th esis of D-P h e-ψ[(E)-CHd
CMe]-L-Va l- a n d D-P h e-ψ[(Z)-CHdCMe]-L-Va l-t yp e
Alk en e Dip ep tid e Isoster es by Or ga n ocop p er -Me-
d ia ted Alk yla tion . Next, we engaged in the synthesis
of a D-Phe-ψ[(E)-CHdCMe]-L-Val-type EADI as a D-Phe-
L-MeVal dipeptide equivalent. The stereoselective con-
struction of two chiral centers adjacent to a trisubstituted
alkene with concomitant control of olefinic geometry were
required for the synthesis of ψ[(E)-CHdCMe]-type EADIs
B′.22 It was assumed that this isostere could be synthe-
sized by organocopper-mediated alkylation of â-methy-
lated γ-mesyloxy-R,â-enoates F by essentially the same
strategy as that utilized in the synthesis of ψ[(E)-CHd
CH]-type EADIs A′ from nonmethylated γ-mesyloxy-R,â-
enoates E, as shown in Scheme 3.13 In this synthetic
scheme, chirality of the amino group in EADIs B′ is
derived from the amino acid used as starting material,
while chirality of the alkyl group at the R-position
depends on the γ-mesyloxy group in substrate F . Ad-
ditionally, it was expected that alkylated products should
be obtained only as E-isomers based on previous investi-
gations.13-15
a
Reagents: (a) DIBAL-H; (b) CH2dCMeMgBr‚ZnCl2‚LiCl; (c)
(COCl)2, DMSO, (i-Pr)2NEt; (d) Zn(BH4)2; (e) Ac2O, pyridine,
DMAP; (f) O3 gas; (g) Me2S; (h) Ph3PdCHCO2t-Bu; (i) Na2CO3,
MeOH.
SCHEME 5a
Initially, efficient synthesis of EADIs B′ precursors,
γ-mesyloxy-â-methyl-R,â-enoates F , was intensively in-
vestigated (Scheme 4). For diastereoselective construction
of hydroxy groups corresponding to the chiral γ-mesyloxy
groups of F , syn- and anti-amino alcohols 20a ,b were
synthesized, respectively in selective fashion.23 Ester 19
was converted predominantly into syn-amino alcohol 20a
by reduction with DIBAL-H followed by addition of an
isopropenyl group using a Grignard reagent.24 On the
other hand, anti-amino alcohol 20b was obtained pref-
erentially by reduction of the enone with Zn(BH4)2,25
which was prepared by Swern oxidation of the syn/anti-
mixture of the allyl alcohols 20a ,b derived from 21.
Diastereomerically pure syn- and anti-amino alcohols
20a ,b could be readily separated by flash chromatogra-
phy over silica gel. After O-protection of amino alcohol
20a , ozonolysis of acetate 22a followed by reduction using
dimethyl sulfide yielded the crude ketone. Wittig reaction
using Ph3PdCHCO2t-Bu gave the corresponding γ-ac-
etoxy-â-methyl-R,â-enoates, which were deacetylated to
provide two isomeric γ-hydroxy-â-methyl-R,â-enoates.
a
Reagents: (a) 2,2-dimethoxypropane, BF3‚Et2O; (b) Ac2O,
pyridine, DMAP.
The major product was the expected syn-E-γ-hydroxy-â-
methyl-R,â-enoate 23a , while the minor isomer proved
to be the unexpected anti-E-γ-hydroxy-â-methyl-R,â-
enoate 23b, which resulted from epimerization at the
γ-hydroxy group. The anti-isomer of acetate 22b was
converted similarly into a mixture of hydroxy esters
23a ,b, which could be purified by flash chromatography
to give the respective hydroxy esters as single diastereo-
mers. Relative stereochemistries of the hydroxy groups
of 23a ,b were established by NOESY spectra of the
corresponding acetonides 24a ,b, which were prepared by
exposure to 2,2-dimethoxypropane (Scheme 5).26 In the
spectrum of 24a , a cross-peak between the 5-H proton of
the 1,3-oxazolidine ring and the benzyl protons proved
it to be the trans-isomer. On the other hand, the corre-
sponding cross-peak in the spectrum of the cis-acetonide
24b was not observed. NOE experiments of acetates
25a ,b, which were obtained by acetylation of 23a ,b,
established E-olefinic geometries of the hydroxy esters
23a ,b. NOE enhancement of the olefinic proton reso-
(22) Mitchell, H. J .; Nelson, A.; Warren, S. J . Chem. Soc., Perkin
Trans. 1 1999, 1899.
(23) Hoffman, R. V.; Maslouh, N.; Cervantes-Lee, F. J . Org. Chem.
2002, 67, 1045.
(24) Syn-selective formation of 1,2-amino alcohols by the one-pot
reaction, which included reduction with DIBAL-H and alkylation with
an alkenyl metal reagent, were previously reported: Tohdo, K.;
Hamada, Y.; Shoiri, T. Tetrahedron Lett. 1992, 33, 2031.
(25) (a) Wasserman, H. H.; Xia, M.; Petersen, A. K.; J orgensen, M.
R.; Curtis, E. A. Tetrahedron Lett. 1999, 40, 6163. (b) Travins, J . M.;
Bursavich, M. G.; Veber, D. F.; Rich, D. H. Org. Lett. 2001, 3, 2725.
(26) Benedetti, F.; Miertus, S.; Norbedo, S.; Tossi, A.; Zlatoidzky,
P. J . Org. Chem. 1997, 62, 9348.
J . Org. Chem, Vol. 67, No. 17, 2002 6165

Oishi et al.
TABLE 1. Alk yla tion of a syn -γ-Mesyloxy-â-m eth yl-r,â-u n sa tu r a ted Ester 26a a n d th e γ-Acetoxy Der iva tive 25a w ith
Or ga n ocya n ocu p r a tes
product ratioe
run
substrate
reagenta
additiveb
solvent
THF
THF
THF
THF
THF
Et2O
Et2O
Et2O
Et2O
Et2O
Et2O
Et2O-THFc
THF
conditiond
27a :28a :27b+28bf:29
yieldh (%)
1
2
3
4
5
6
7
8
26a
26a
26a
26a
26a
26a
26a
26a
26a
26a
26a
26a
25a
25a
A
B
B
B
C
A
B
B
B
C
B
B
B
B
-
-
A
B
B
B
B
A
B
C
B
B
B
B
D
D
40:51:-g:9
35:64:-g:1
28:71:-g:1
27:72:-g:1
25:73:-g:1
36:63:-g:1
46:27:26:1
70:27:-g:3
44:25:28:3
14:84:-g:2
43:42:12:3
37:62:-g:1
21:79:-g: -g
73:27:-g: -g
82
94
91
92
86
15i
90
93
81
93
97
87
39i
57i
HMPA
TMEDA
18-crown-6
-
-
HMPA
TMEDA
18-crown-6
THF
9
10
11
12
13
14
-
-
-
Et2O
a
All reactions were carried out with 4 mol equiv of reagent, A: i-PrCu(CN)MgCl‚BF3; B: i-Pr2Cu(CN)(MgCl)2‚BF3; C: i-Pr2Cu(CN)(MgCl)2.
4 mol equiv. c Et2O:THF ) 7:3. A: 0 °C, 0.5 h; B: - 78 °C, 0.5 h; C: - 78 °C, 0.5 h, then 0 °C, 0.5 h; D: - 78 °C, 0.5 h, then 0 °C, 3
b
d
h. e Product ratios were determined by HPLC and 1H NMR. f Containing small amount of an uncharacterized product. Although we
g
h
i
cannot conclusively rule out its presence, we failed to isolate the corresponding product. Combined yield. The starting material was
recovered (77, 60, and 41% for runs 6, 13, and 14, respectively).
SCHEME 6
SCHEME 7
nances of 25a ,b were observed by irradiation of the 4-H
proton (10.0% and 9.9%, respectively). Mesylation of
hydroxy esters 23a ,b gave the expected â-methylated
γ-mesyloxy-R,â-enoates 26a ,b, respectively, which were
used for organocopper-mediated alkylation (Scheme 6).
It was initially envisioned that regio- and stereoselec-
tive alkylation of â-methylated γ-mesyloxy-R,â-enoates
26a ,b by organocopper reagents would proceed without
significant difficulty based on precedence of the unm-
ethylated analogue 9. However, obvious differences in
reactivity and in stereoselectivity of alkylation products
were observed (Scheme 7 and Table 1). Treatment of the
syn-R,â-enoate 26a with a “lower-order” cyanocuprate-
BF3 complex, i-PrCu(CN)MgCl‚BF3, at -78 °C, which is
generally utilized for the synthesis of ψ[(E)-CHdCH]-type
EADIs A,13 failed to provide product. However, the same
reaction proceeded at elevated temperature (0 °C) to yield
R-alkylated products 27a and 28a with a small amount
of a reductive product, Boc-D-Phe-ψ[(E)-CHdCMe]-Gly-
Ot-Bu 29 (run 1). Unexpectedly, the major product was
an unprecedented Z-isomer of an anti-SN2′ product, Boc-
D-Phe-ψ[(Z)-CHdCMe]-D-Val-Ot-Bu 28a .27 This result
suggested that â-methylated analogue 26a has less
reactivity toward organocopper reagents than 9. Employ-
ment of a more reactive “higher-order” reagent, i-Pr2Cu-
(CN)(MgCl)2‚BF3, which tends to reduce aziridinyl
substrates,14b also provided R-alkylated products 27a and
28a with Z-selectivity even at -78 °C in excellent
combined yields (run 2). It was supposed that Z-isomer
28a was predominantly formed as a result of preferential
reaction from a more favorable conformer 26a -B in THF
(Figure 2).28 Therefore, introduction of additives such as
HMPA, TMEDA, and 18-crown-6 was attempted to
improve E-selectivity; however, increased E-selectivity
was not observed (runs 3-5). Although Et2O has been
thought to be an inappropriate solvent for alkylation
owing to sluggishness,29 its use in the present case
resulted a remarkable improvement of the E-selectivity.
Reaction of R,â-enoate 26a with i-PrCu(CN)MgCl‚BF3 in
Et2O proceeded with low efficiency (run 6), while use of
i-Pr2Cu(CN)(MgCl)2‚BF3 improved the E-selectivity with
significant production of syn-SN2′ products 27b and 28b
(run 7). Among additives investigated, HMPA most
efficiently decreased production of syn-SN2′ products to
afford the expected EADI, Boc-D-Phe-ψ[(E)-CHdCMe]-
L-Val-Ot-Bu, 27a , which could be purified by flash
(28) It could be also assumed that the formation of copper-π-allyl
complexes as reactive intermediates were involved in the alkylation.
In this case, product ratios should depend on stabilities of intermedi-
ates.
(29) (a) Ibuka, T.; Tanaka, M.; Nishii, S.; Yamamoto, Y. J . Am.
Chem. Soc. 1989, 111, 4864. (b) Ibuka, T.; Akimoto, N.; Tanaka, M.;
Nishii, S.; Yamamoto, Y. J . Org. Chem. 1989, 54, 4055.
(27) Yang, H.; Sheng, X. C.; Harrington, E. M.; Ackermann, K.;
Garcia, A. M.; Lewis, M. D. J . Org Chem. 1999, 64, 242.
6166 J . Org. Chem., Vol. 67, No. 17, 2002

Synthesis of Alkene Dipeptide Isosteres
ment of E-selectivity in the alkylation of anti-isomer 26b
was not observed, even following treatment with i-Pr2-
Cu(CN)(MgCl)2‚BF3 in Et2O. This resulted in preferential
production of Z-isomer 28b along with syn-SN2′products
27a and 28a and a reductive product 29 (run 4). In
contrast, alkylation of 26b with i-PrCu(CN)MgCl‚BF3 in
Et2O failed to proceed (run 3). The unexpected Z-isomer,
Boc-D-Phe-ψ[(Z)-CHdCMe]-L-Val-Ot-Bu, 28b represents
a potential mimetic of the cis-peptide bond between D-Phe
and L-MeVal (Figure 1, D).11 We therefore utilized the
Z-isomer in a conformational study on cyclic RGD peptide
2.
Regiochemical assignments of alkylated products 27a ,b
and 28a ,b and the reduced product 29 were performed
by 1H NMR (NOE experiments, 1H-1H COSY and
NOESY spectra). The observed enhancement of the 2-H
resonance by irradiation of the olefinic proton indicated
27a ,b to be E-isomers (13.2% and 12.0%). The olefinic
resonance following irradiation of the 3-methyl protons
indicated that 28a ,b and 29 were Z-isomers (14.0%,
13.3%, and 3.1%).30 Stereochemistries of the resulting
alkyl groups of 27a ,b and 28a ,b were established by
circular dichroism (CD) measurements based on an
empirical method used for determination of R-alkyl group
configuration of R-alkyl-â,γ-enoates.31 Negative Cotton
effects around 220 nm in CD spectra of 27a and 28b
indicated 2R-isomers, while positive Cotton effects of 27b
and 28a indicated 2S-isomers. The crystal structure of
28b also supported these assignments.32
F IGURE 2.
TABLE 2. Alk yla tion of a n
a n ti-γ-Mesyloxy-â-m eth yl-r,â-u n sa tu r a ted Ester 26b w ith
Or ga n ocya n ocu p r a tes
product ratioc
run reagenta solvent conditionb 27b:28b:27a +28a :29 yielde (%)
1
2
3
4
A
B
A
B
THF
THF
Et2O
Et2O
A
B
A
A
27:69:-d:4
41:54:-d:5
-
65
87
-
68
f
7: 41:26:26
a
All reactions were carried out with 4 mol equiv of reagent, A:
b
i-PrCu(CN)MgCl‚BF3; B: i-Pr2Cu(CN)(MgCl)2‚BF3. A: - 78 °C,
0.5 h then 0 °C, 3 h; B: - 78 °C, 0.5 h. c Product ratios were
determined by HPLC and 1H NMR. Although we cannot conclu-
d
sively rule out its presence, we failed to isolate the corresponding
product. e Combined yield. f The starting material was recovered.
Syn th esis of Cyclic RGD P seu d op ep tid es Con -
ta in in g D-P h e-ψ[(E)-CHdCMe]-L-Va l- a n d D-P h e-ψ-
[(Z)-CHdCMe]-L-Va l-typ e Alk en e Dip ep tid e Isos-
ter es. The resulting Boc-D-Phe-ψ[(E)-CHdCMe]-L-Val-
Ot-Bu 27a and Boc-D-Phe-ψ[(Z)-CHdCMe]-L-Val-Ot-Bu
28b, were utilized for the synthesis of cyclic RGD
pseudopeptides (Schemes 8 and 9). Fmoc-based solid-
phase peptide synthesis (SPPS) was employed in order
to prevent trisubstituted alkene moieties from isomer-
izing during strong acid treatments needed for final
deprotection in Boc-based synthesis. Isosteres 27a and
28b were converted to the corresponding Fmoc-amino
acids 30 and 35 beforehand. For the synthesis of 4,
cyclization of linear peptide precursor was attempted
without protection of side chain functional group. A
hydrazino linker was constructed on a solid-support prior
to peptide synthesis since the C-terminal peptide hy-
drazide 34 should be selectively activated by the azide
method33 without modification of a carboxylic acid func-
tionality of Asp (Scheme 8). Accordingly, p-nitrophenyl
carbonate Wang resin 31 was treated with hydrazine
hydrate in DMF to afford a resin possessing a hydrazino
linker 32, which could be easily cleaved by TFA treat-
chromatography over silica gel (run 8). In contrast, 18-
crown-6 increased Z-selectivity of alkylation (run 10). We
also examined mixed solvents consisting of THF and Et2O
to quantitatively estimate solvent effects on E-selectivity.
Alkylation of 26a with i-Pr2Cu(CN)(MgCl)2‚BF3 in Et2O
in the presence of 4 equiv of THF gave nearly equal
amounts of E- and Z-isomers of anti-SN2′ products 27a
and 28a (run 11). The product ratio of 27a and 28a
following alkylation in Et2O-THF (7:3) was identical to
that in THF alone (run 12). As such, Z-selectivity seemed
dependent on addition of cyclic ethers such as THF or
18-crown-6, although the reason was unclear. Alkylation
of â-methylated γ-acetoxy-R,â-enoate 25a with i-Pr2Cu-
(CN)(MgCl)2‚BF3 in THF or Et2O afforded R-alkylated
products 27a and 28a with Z- or E-selectivity, respec-
tively, in similar solvent-dependent fashion as observed
in the reactions of mesylate 26a . In this case, the overall
yields were decreased in comparison with that from 26a ,
probably due to the deficient leaving-group ability of the
acetoxy group of 25a (runs 13 and 14).
Organocopper-mediated alkylation of the anti-γ-mesyl-
oxy-R,â-enoate 26b, which was expected to provide Boc-
D-Phe-ψ[(E)-CHdCMe]-D-Val-Ot-Bu 27b by an anti-SN2′
mechanism, was also investigated in similar fashion
(Scheme 7 and Table 2). Treatment of 26b with the
“lower-order” cyanocuprate-BF3 complex, i-PrCu(CN)-
MgCl‚BF3, in THF preferentially yielded a Z-isomer of
an anti-SN2′ product, Boc-D-Phe-ψ[(Z)-CHdCMe]-L-Val-
Ot-Bu, 28b in a moderate yield (run 1). The “higher-
order” cyanocuprate-BF3 complex, i-Pr2Cu(CN)(MgCl)2‚
BF3, at -78 °C also provided R-alkylated products 27b
and 28b with similar Z-selectivity (run 2). In sharp
contrast to the alkylation of syn-isomer 26a , improve-
(30) An E-isomer of the reductive products, D-Phe-ψ[(E)-CHdCMe]-
Gly 41, resulted from treatment of the mesylate 26a using Gilman-
type reagent, Me2CuLi‚LiI‚2LiBr, as shown below.29
(31) Ibuka, T.; Habashita, H.; Funakoshi, S.; Fujii, N.; Baba, K.;
Kozawa, M.; Oguchi, Y.; Uyehara, T.; Yamamoto, Y. Tetrahedron:
Asymmetry 1990, 1, 379.
(32) The crystal structure of 28b was presented in the preliminary
communication.19
(33) Honzl, J .; Rudinger, J . Collect. Czech. Chem. Commun. 1961,
26, 2333.
J . Org. Chem, Vol. 67, No. 17, 2002 6167

Oishi et al.
SCHEME 8a
TABLE 3. Biologica l Effects of Cyclic RGD P ep tid es
a n d P seu d op ep tid es a ga in st In tegr in Recep tor s
RVâ3
IC50 (nM)
98
6.8
R
IIbâ3
peptide
Qb
IC50 (nM)
Qb
1
2.9
1.0
0.53
0.37
RGDSa
1
2
3
1
270
770
280
140
100
0.069
0.014
0.037
0.034
1.4
3.6
3.3
4
40
18000
180
370000
1400
a
A linear peptide RGDS (H-Arg-Gly-Asp-Ser-OH) was used as
a standard peptide. b Q values were calculated as Q ) IC50(peptide)/
IC50(RGDS).
38, which was subjected to cyclization using DPPA and
NaHCO3 in DMF.36 Deprotection of protected cyclic
peptide 39 using 95% TFA, followed by HPLC purifica-
tion yielded the expected cyclic pseudopeptide 40 having
a D-Phe-ψ[(Z)-CHdCMe]-L-Val-type isostere, in 20% yield
from the isostere 35.
a
Reagents: (a) TFA; (b) Fmoc-OSu, Et3N; (c) NH2NH2‚H2O; (d)
Fmoc-based SPPS; (e) TFA; (f) HCl, isoamyl nitrite; (g) (i-Pr)2NEt.
Biologica l Activities of Cyclic RGD P ep tid es a n d
P seu d op ep tid es Con ta in in g Alk en e Dip ep tid e Isos-
ter es. Alkene moieties of pseudopeptides 3 and 4, which
include ψ[(E)-CHdCH]- and ψ[(E)-CHdCMe]-type EA-
DIs, respectively, were assumed to exhibit the similar
structures (ω ) 180°), corresponding to the peptide bonds
between D-Phe and L-Val/MeVal in 1 and 2. It was
presumed that a higher potency of methylated analogue
4 as compared to 3 should enable an interpretation of
the contribution of conformation to the increased bioac-
tivity of 2. On the other hand, if bioactivities of pseudopep-
tides 3 and 4 were equivalent, this showed that com-
paratively free rotation about the D-Phe-L-MeVal peptide
bond might result in increased bioactivity of 2. Evalua-
tion of the biological activities of the cyclic pseudopeptides
3, 4 and 40 against integrin receptors (RVâ3 and RIIbâ3)
was performed using a competitive binding assay with
immobilized RVâ3 and RIIbâ3 integrins, in comparison with
Kessler’s cyclic RGD peptides 1 and 2 (Table 3). More
potent antagonistic activities of pseudopeptides 3 and 4
were observed in comparison with 1 against on RVâ3 and
SCHEME 9a ,b
R
IIbâ3 integrins. In contrast, pseudopeptides 40, which
a
Reagents: (a) TFA; (b) Fmoc-OSu, Et3N; (c) Fmoc-based SPPS;
contained a ψ[(Z)-CHdCMe]-type isostere, showed ex-
ceedingly low potency against these receptors. Further-
more, the difference between the activities of the
pseudopeptides 3 and 4 was minimal. These results
suggest that a cis-conformation for the peptide bond in
D-Phe-L-Val/MeVal is inappropriate for interaction of
cyclic peptides 1 and 2 with integrins. Taken together,
it can be concluded that the N-methyl group in 2
influences conformation not by simple addition of a
methyl group followed by redistribution of close side
chains, but by a subtle rotation of the ω-angle of D-Phe-
MeVal away from an exact trans-conformation.7 From the
viewpoint of selectivity against the two integrin receptors,
peptide 2 was the most potent agent against RVâ3 integrin
despite being comparatively less potent against RIIbâ3
integrin. Overall, it exhibited the best profile. Limited
(d) AcOH-TFE-CH2Cl2 (1:1:3); (e) DPPA, NaHCO3; (f) 95% TFA-
H2O. bAbbreviations: Clt resin ) 2-chlorotrityl resin; Pbf )
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; DPPA ) diphen-
ylphosphoryl azide.
ment to yield a peptide hydrazide. Fmoc-amino acid-
containing 30 was successively coupled onto the linker
using normal Fmoc-based SPPS. Following 95% TFA
treatment of the protected peptidyl resin 33, the resulting
peptide hydrazide 34 was cyclized by the azide method
in highly diluted DMF solution to yield the cyclic
pseudopeptide 4,34 which contains a D-Phe-ψ[(E)-CHd
CMe]-L-Val-type isostere, in 14% yield from 30.
In contrast, cyclic pseudopeptide 40 was synthesized
by well-established cyclic peptide synthesis protocols
(Scheme 9). A protected peptide resin 37 was constructed
by Fmoc-based SPPS on glycinyl 2-chlorotrityl (Clt) resin
36, which can provide side chain-protected peptides
following mild acidic treatment.35 Exposing resin 37 to
AcOH-TFE-CH2Cl2 (1:1:3) provided protected peptide
(35) (a) Barlos, K.; Gatos, D.; Kallitsis, J .; Papaphotiu, G.; Sotiriu,
P.; Wenqing, Y.; Scha¨fer, W. Tetrahedron Lett. 1989, 30, 3943. (b)
Barlos, K.; Gatos, D.; Kapolos, S.; Papaphotiu, G.; Scha¨fer, W.;
Wenqing, Y. Tetrahedron Lett. 1989, 30, 3947. (c) Barlos, K.; Gatos,
D.; Kapolos, S.; Poulos, C.; Scha¨fer, W.; Wenqing, Y. Int. J . Pept. Protein
Res. 1991, 38, 555.
(34) The pseudopeptide 4 contained about 6% of an uncharacterized
impurity by 1H NMR analysis, which failed to be removed by HPLC.
(36) Shioiri, T.; Yamada, S. Chem. Pharm. Bull. 1974, 22, 855.
6168 J . Org. Chem., Vol. 67, No. 17, 2002

Synthesis of Alkene Dipeptide Isosteres
ter t-Bu tyl (4R,5R,2E)-4-Acetoxy-5-[N-(ter t-bu toxyca r -
bon yl)a m in o]-6-p h en ylh ex-2-en oa te (7). To a solution of
the acetate 6 (6.46 g, 20.2 mmol) in EtOAc (250 mL) was
bubbled O3 gas at -78 °C until a blue color persisted. To the
above solution was added Me2S (29.7 mL, 404 mmol), and the
mixture was stirred for 30 min at 0 °C. The mixture was dried
over MgSO4. Concentration under reduced pressure gave an
oily aldehyde, which was used immediately in the next step
without further purification. To a stirred suspension of LiCl
(2.14 g, 50.5 mmol) in MeCN (120 mL) under argon were added
(EtO)2P(O)CH2CO2t-Bu (12.0 mL, 50.5 mmol) and (i-Pr)2NEt
(8.79 mL, 50.5 mmol) at 0 °C. After 20 min, the above aldehyde
in MeCN (60 mL) was added to the above mixture at 0 °C,
and the mixture was stirred at this temperature for 4 h. The
mixture was concentrated under reduced pressure, and the
residue was extracted with EtOAc. The extract was washed
successively with saturated citric acid, brine, 5% NaHCO3, and
brine and dried over MgSO4. Concentration under reduced
pressure followed by flash chromatography over silica gel with
n-hexanes-EtOAc (5:1) gave the title compound 7 (5.38 g, 63%
yield) as colorless crystals: mp 101-105 °C (n-hexane/Et2O
) 3:1); [R]29D +47.8 (c 1.06, CHCl3); 1H NMR (270 MHz, CDCl3)
δ 1.38 (s, 9 H), 1.45 (s, 9 H), 2.14 (s, 3 H), 2.78 (d, J ) 7.2 Hz,
2 H,), 4.14 (m, 1 H), 4.68 (m, 1 H), 5.37 (m, 1 H), 5.80 (d, J )
15.4 Hz, 1 H), 6.70 (dd, J ) 15.4, 5.2 Hz, 1 H), 7.08-7.34 (m,
5 H). Anal. Calcd for C23H33NO6: C, 65.85; H, 7.93; N, 3.34.
Found: C, 65.79; H, 8.23; N, 3.31.
ter t-Bu tyl (4R,5R,2E)-5-[N-(ter t-Bu toxycar bon yl)am in o]-
4-h yd r oxy-6-p h en ylh ex-2-en oa te (8). Powdered Na2CO3
(5.44 g, 51.3 mmol) was added to a solution of the ester 7 (5.38
g, 12.8 mmol) in dry MeOH (30 mL) at room temperature, and
the mixture was stirred for 1 h. The mixture was filtered, and
the filtrate was concentrated under reduced pressure. The
residue was extracted with EtOAc, and the extract was washed
with water and dried over MgSO4. Concentration under
reduced pressure followed by flash chromatography over silica
gel with n-hexanes-EtOAc (3:1) gave the title compound 8
(4.35 g, 89% yield) as colorless crystals: mp 91-95 °C
(n-hexane/Et2O ) 3:1); [R]30D +59.3 (c 0.977, CHCl3); 1H NMR
(270 MHz, CDCl3) δ 1.38 (s, 9 H), 1.46 (s, 9 H), 2.94 (m, 2 H),
3.05 (m, 1 H), 3.81 (m, 1 H), 4.27 (m, 1 H), 4.82 (m, 1 H), 5.99
(dd, J ) 15.4, 1.9 Hz, 1 H), 6.81 (dd, J ) 15.4, 4.6 Hz, 1 H),
7.18-7.35 (m, 5 H). Anal. Calcd for C21H31NO5: C, 66.82; H,
8.28; N, 3.71. Found: C, 66.71; H, 8.44; N, 3.60.
ter t-Bu tyl (4R,5R,2E)-5-[N-(ter t-Bu toxycar bon yl)am in o]-
4-m eth ylsu lfon yloxy-6-ph en ylh ex-2-en oate (9). To a stirred
mixture of the alcohol 8 (4.35 g, 11.5 mmol) and pyridine (18.5
mL, 230 mmol) in CHCl3 (18 mL) was added dropwise MsCl
(8.92 mL, 115 mmol) at 0 °C, and the mixture was stirred for
1 h at this temperature. The mixture was poured into water
at 0 °C. The whole was extracted with EtOAc, and the extract
was washed successively with saturated citric acid, brine, 5%
NaHCO3, brine and dried over MgSO4. Concentration under
reduced pressure followed by flash chromatography over silica
gel with n-hexanes-EtOAc (3:1) gave the title compound 9
(4.75 g, 90% yield) as colorless crystals: mp 132-135 °C (n-
hexane/Et2O ) 3:1); [R]27D +58.6 (c 1.07, CHCl3); 1H NMR (270
MHz, CDCl3) δ 1.37 (s, 9 H), 1.47 (s, 9 H), 2.79 (dd, J ) 13.8,
7.9 Hz, 1 H), 2.96 (dd, J ) 13.8, 6.5 Hz, 1 H), 3.06 (s, 3 H),
4.14 (m, 1 H), 4.69 (d, J ) 9.2 Hz, 1 H), 5.22 (m, 1 H), 6.02 (d,
J ) 15.8 Hz, 1 H), 6.79 (dd, J ) 15.8, 6.2 Hz, 1 H), 7.20-7.35
(m, 5 H). Anal. Calcd for C22H33NO7S: C, 58.00; H, 7.30; N,
3.07. Found: C, 58.14; H, 7.22; N, 2.86.
ter t-Bu tyl (2R,5R,3E)-5-[N-(ter t-Bu toxycar bon yl)am in o]-
2-isop r op yl-6-p h en ylh ex-3-en oa te (Boc-D-P h e-ψ[(E)-CHd
CH]-Va l-Ot-Bu , 10). To a stirred slurry of CuCN (3.73 g, 41.7
mmol) in THF (50 mL) was added a solution of i-PrMgCl in
THF (1.5 M, 27.8 mL, 41.7 mmol) at -78 °C, and the mixture
was stirred for 15 min at 0 °C. BF3‚Et2O (5.28 mL, 41.7 mmol)
was added to the above mixture at -78 °C. After 5 min, a
solution of the mesylate 9 (4.75 g, 10.4 mmol) in dry THF (20
mL) was added dropwise to the above reagent at -78 °C, and
rotation or loss of polarity in the D-Phe-L-Val/MeVal
peptide bonds of pseudopeptides 3 and 4 seemed to induce
increased affinity for RIIbâ3 integrin, resulting in the lower
selectivities (RIIbâ3/RVâ3) relative to their peptidic coun-
terparts 1 and 2.
Con clu sion
In conclusion, the synthesis of new ψ[(E)-CHdCMe]-
type alkene isosteres was accomplished utilizing alkyla-
tion of â-methylated γ-mesyloxy-R,â-enoates by organo-
copper reagents. Alkylation afforded unprecedented
Z-isomers of anti-SN2′ products as well as expected
E-isomers. These are potential dipeptide mimetics of
ω-constrained cis- and trans-peptide bonds between an
amino acid and an N-methyl amino acid, respectively. In
addition, pseudopeptides 3, 4, and 40, which contain
D-Phe-ψ[(E)-CHdCH]-L-Val, D-Phe-ψ[(E)-CHdCMe]-L-
Val, and D-Phe-ψ[(Z)-CHdCMe]-L-Val-type isosteres, re-
spectively, were synthesized utilizing three separate
routes for conformational analysis of Kessler’s cyclic RGD
peptides 1 and 2, and antagonistic activities against
integrins were evaluated. The reduced difference in
activities against RVâ3 integrin between the cyclic
pseudopeptides 3 and 4, as compared to between cyclic
peptides 1 and 2, suggest that the high activity of 2 may
be derived from relatively free rotation about the D-Phe-
L-MeVal ω-angle caused by N-methylation. These dis-
tinctive alkene dipeptide isosteres may be useful tools
for exploration of bioactive conformations of peptides
containing N-methylamino acids.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are uncorrected. Chemi-
cal shifts of the compounds, of which 1H NMR spectra were
recorded in CDCl3, are reported in parts per million downfield
from internal Me4Si (s ) singlet, d ) doublet, dd ) double
doublet, ddd ) doublet of double doublet, t ) triplet, m )
multiplet). Those of the compounds measured in DMF-d7 and
DMSO-d6 are calibrated to the solvent signal (2.75 and 2.50
ppm, respectively). For flash chromatographies, silica gel 60
H
(silica gel for thin-layer chromatography, Merck) and
Wakogel C-200 (silica gel for column chromatography) were
employed. For HPLC separations, a Cosmosil 5C18-ARII
analytical (4.6 × 250 mm, flow rate 1 mL/min) column or a
Cosmosil 5C18-ARII preparative (20 × 250 mm, flow rate 11
mL/min) column was employed, and eluting products were
detected by UV at 220 nm. A solvent system consisting of 0.1%
TFA solution (v/v, solvent A) and 0.1% TFA in MeCN (v/v,
solvent B) were used for HPLC elution.
(3R,4R)-O-Acetyl-4-[N-(ter t-bu toxyca r bon yl)a m in o]-5-
p h en ylp en t-1-en -3-ol (6). To a stirred solution of the alcohol
516b (12.9 g, 46.5 mmol), pyridine (75.2 mL, 930 mmol), and
DMAP (568 mg, 4.65 mmol) in CHCl3 (50 mL) at 0 °C was
added Ac2O (43.8 mL, 465 mmol). The mixture was stirred for
3 h with warming to room temperature. The mixture was
poured into water at 0 °C, and the whole was extracted with
EtOAc. The extract was washed successively with saturated
citric acid, brine, saturated NaHCO3, and brine and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexanes-EtOAc
(3:1) gave the title compound 6 (13.8 g, 93% yield) as colorless
crystals: mp 60-63 °C (n-hexane/Et2O ) 5:1); [R]24 +55.2 (c
D
0.977, CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.38 (s, 9 H), 2.10
(s, 3 H), 2.78 (d, J ) 6.9 Hz, 2 H), 4.07 (m, 1 H), 4.67 (m, 1 H),
5.15-5.30 (m, 3 H), 5.79 (ddd, J ) 17.4, 10.5, 6.2 Hz, 1 H),
7.08-7.32 (m, 5 H). Anal. Calcd for C18H25NO4: C, 67.69; H,
7.89; N, 4.39. Found: C, 67.81; H, 8.04; N, 4.31.
J . Org. Chem, Vol. 67, No. 17, 2002 6169

Oishi et al.
the stirring was continued for 30 min followed by quenching
with 20 mL of a 1:1 saturated NH4Cl-28% NH4OH solution.
The mixture was extracted with Et2O, and the extract was
washed with brine and dried over MgSO4. Concentration under
reduced pressure followed by flash chromatography over silica
gel with n-hexanes-EtOAc (5:1) yielded the title compound
DCC (290 mg, 1.41 mmol) at 0 °C, and the mixture was stirred
overnight. The mixture was filtered, and the filtrate was
concentrated under reduced pressure. The residue was ex-
tracted with EtOAc, and the extract was washed successively
with 5% NaHCO3, brine, 0.1 N HCl, and brine and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with EtOAc gave the title
compound 13 (527 mg, 73% yield) as a colorless powder: mp
10 (3.56 g, 84% yield) as colorless crystals: mp 78-81 °C (n-
1
hexane/Et2O ) 10:1); [R]26 -43.0 (c 0.999, CHCl3); H NMR
D
(300 MHz, CDCl3) δ 0.74 (d, J ) 6.7 Hz, 3 H), 0.86 (d, J ) 6.6
Hz, 3 H), 1.40 (s, 9 H), 1.42 (s, 9 H), 1.86 (m, 1 H), 2.50 (m, 1
H), 2.77 (dd, J ) 13.5, 7.0 Hz, 1 H), 2.87 (dd, J ) 13.5, 5.8 Hz,
1 H), 4.33-4.52 (m, 2 H), 5.44-5.50 (m, 2 H), 7.13-7.30 (m, 5
H). Anal. Calcd for C24H37NO4: C, 71.43; H, 9.24; N, 3.47.
Found: C, 71.16; H, 9.06; N, 3.42.
151-154 °C; [R]22 -29.5 (c 1.01, CHCl3); 1H NMR (300 MHz,
D
CDCl3) δ 0.67 (d, J ) 6.4 Hz, 3 H), 0.79 (d, J ) 6.5 Hz, 3 H),
1.34 (s, 9 H), 1.45-1.77 (m, 3 H), 1.87-2.02 (m, 2 H), 2.26 (s,
3 H), 2.50 (m, 1 H), 2.65 (s, 6 H), 2.74 (m, 1 H), 2.84 (dd, J )
13.4, 6.7 Hz, 1 H), 3.16-3.25 (m, 2 H), 3.69 (s, 3 H), 3.90 (dd,
J ) 17.8, 5.6 Hz, 1 H), 4.03 (dd, J ) 17.8, 5.7 Hz, 1 H), 4.28
(m, 1 H), 4.50 (m, 1 H), 4.85 (m, 1 H), 5.46-5.54 (m, 2 H),
6.17 (m, 1 H), 6.34 (m, 2 H), 6.80-6.92 (m, 3 H), 7.10-7.26
(m, 5 H), 7.46 (m, 1 H). Anal. Calcd for C38H56N6O8S: C, 60.30;
H, 7.46; N, 11.10. Found: C, 60.02; H, 7.47; N, 10.99.
(2R,5R,3E)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-2-isop r o-
p yl-6-p h en ylh ex-3-en oic a cid (Boc-D-P h e-ψ[(E)-CHdCH]-
Va l-OH, 11). The ester 10 (533 mg, 1.32 mmol) was dissolved
in TFA (10 mL) at 0 °C, and the mixture was stirred overnight
at room temperature. Concentration under reduced pressure
gave an oily residue, which was dissolved in CHCl3-DMF-
H2O (50:9:1, 6 mL). To the mixture were added Et3N (0.552
mL, 3.96 mmol) and (Boc)2O (864 mg, 3.96 mmol) at 0 °C, and
the mixture was stirred for 3 h at room temperature. The
mixture was concentrated under reduced pressure to give an
oily residue, which was acidified with saturated citric acid and
extracted with EtOAc. The extract was washed with brine and
dried over MgSO4. Concentration under reduced pressure
followed by flash chlomatography over silica gel with n-hex-
anes-EtOAc (1:1) gave the title compound 11 (379 mg, 82%
Boc-D-P h e-ψ[(E)-CH dCH ]-Va l-Ar g(Mt s)-Gly-OH (14).
To a stirred solution of the protected tetrapeptide ester 13 (470
mg, 0.620 mmol) in MeOH (1 mL) was added 1 N LiOH (1.24
mL, 1.24 mmol) at room temperature. After 3 h, the mixture
was acidified with 1 N HCl and concentrated under reduced
pressure. The residue was extracted with EtOAc, and the
extract was washed with 0.1 N HCl and brine and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with CHCl3-MeOH (10:0
to 9:1) gave the title compound 14 (461 mg, 99% yield) as a
colorless amorphous semisolid: [R]18 -19.8 (c 5.79, CHCl3);
D
yield) as a colorless oil: [R]20 -28.5 (c 1.54, CHCl3); 1H NMR
1H NMR (300 MHz, CDCl3) δ 0.63 (d, J ) 6.4 Hz, 3 H), 0.75
(d, J ) 6.1 Hz, 3 H), 1.32 (s, 9 H), 1.53 (m, 2 H), 1.69 (m, 1 H),
1.89 (m, 2 H), 2.24 (s, 3 H), 2.51 (m, 1 H), 2.61 (m, 7 H), 2.76
(m, 1 H), 3.17 (m, 2 H), 3.82-4.10 (m, 2 H), 4.23 (m, 1 H),
4.52 (m, 1 H), 5.46 (m, 2 H), 6.43 (m, 2 H), 6.85 (s, 2 H), 7.04-
7.25 (m, 6 H), 7.74 (m, 1 H); LRMS (FAB), m/z 743 (MH+, base
peak), 681, 561, 414, 185, 119, 91, 70, 57; HRMS (FAB), m/z
calcd for C37H55N6O8S (MH+) 743.3802, found: 743.3774.
D
(300 MHz, CDCl3) δ 0.75 (d, J ) 6.2 Hz, 3 H), 0.90 (d, J ) 6.6
Hz, 3 H), 1.39 (s, 9 H), 1.93 (m, 1 H), 2.64 (m, 1 H), 2.76 (dd,
J ) 13.3, 6.6 Hz, 1 H), 2.88 (dd, J ) 13.3, 6.2 Hz, 1 H), 4.25-
4.70 (m, 2 H), 5.49 (m, 2 H), 7.10-7.30 (m, 5 H); LRMS (FAB),
m/z 348 (MH+, base peak), 292, 256, 248, 246, 230, 200, 185,
164, 156, 149, 129, 91, 57, 41; HRMS (FAB), m/z calcd for
C
20H30NO4 (MH+) 348.2175, found: 348.2166.
Boc-Ar g(Mts)-Gly-OMe (12). To a stirred dry MeOH (66
Boc-Asp (OBn )-D-P h e-ψ[(E)-CHdCH]-Va l-Ar g(Mts)-Gly-
OH (15). DCC (536 mg, 2.60 mmol) was added to the mixture
of Boc-Asp(OBn)-OH (646 mg, 2.00 mmol) and N-hydroxy-5-
norbornene-2,3-carboxyimide (HONB, 358 mg, 2.00 mmol) in
THF (10 mL) at 0 °C, and the mixture was stirred for 1 h at
room temperature. The solution was filtrated and the filtrate
was concentrated reduced pressure. The residue was dissolved
in EtOAc followed by addition of n-hexane to give a powder of
Boc-Asp(OBn)-ONB (866 mg), which was used in next step
without further purification. To the protected pseudotetrapep-
tide 14 (470 mg, 0.632 mmol) was added successively anisole
(0.5 mL) and TFA (5 mL) at 0 °C, and the mixture was stirred
for 2 h at room temperature. The mixture was concentrated
under reduced pressure, and the residue was dissolved in DMF
(5 mL). To the stirred solution were added successively Et3N
(0.176 mL, 1.26 mmol) and the above Boc-Asp(OBn)-ONB (353
mg, 0.728 mmol), and the stirring was continued overnight at
room temperature. Concentration under reduced pressure gave
an oily residue, which was acidified with saturated citric acid
and extracted with EtOAc. The extract was washed with
saturated citric acid, brine, and water. Concentration under
reduced pressure followed by flash chromatography over silica
gel with CHCl3-MeOH (10:0 to 9:1) gave the title compound
mL) was added dropwise SOCl2 (9.70 mL, 133 mmol) at -78
°C, and the mixture was stirred at room temperature for 1 h.
L-Glycine (5.0 g, 66.6 mmol) was added to the mixture, and
the mixture was heated under reflux for 3 h. The mixture was
concentrated under reduced pressure to give a semisolid, which
was dissolved in DMF (200 mL). To the above mixture were
added successively (i-Pr)2NEt (23.1 mL, 133 mmol), Boc-Arg-
(Mts)-OH (22.8 g, 49.9 mmol), HOBt‚H2O (7.64 g, 49.9 mmol),
and DCC (15.5 g, 74.9 mmol) at 0 °C, and the mixture was
stirred overnight. The mixture was filtered, and the filtrate
was concentrated under reduced pressure to give an oily
residue, which was extracted with EtOAc. The extract was
washed successively with saturated citric acid, brine, 5%
NaHCO3, and brine and dried over MgSO4. Concentration
under reduced pressure followed by flash chromatography over
silica gel with EtOAc gave the title compound 12 (26.1 g, 99%
yield) as a colorless amorphous semisolid: [R]24D -8.80 (c 1.02,
CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.40 (s, 9 H), 1.55-1.73
(m, 3 H), 1.80-1.92 (m, 1 H), 2.26 (s, 3 H), 2.65 (s, 6 H), 3.15-
3.35 (m, 2 H), 3.70 (s, 3 H), 3.91 (dd, J ) 17.8, 5.5 Hz, 1 H),
4.06 (dd, J ) 17.8, 5.9 Hz, 1 H), 4.25 (m, 1 H), 5.55 (d, J ) 7.7
Hz, 1 H), 6.19 (br, 1 H), 6.34 (m, 2 H), 6.88 (s, 2 H), 7.48 (t, J
) 5.7 Hz, 1 H); LRMS (FAB), m/z 528 (MH+), 472, 428, 346,
185, 119, 70, 57, 41 (base peak); HRMS (FAB), m/z calcd for
15 (466 mg, 77% yield) as a colorless powder: [R]21 -28.5 (c
D
1
0.980, CHCl3); H NMR (300 MHz, CDCl3) δ 0.63-0.92 (m, 6
C
23H38N5O7S (MH+) 528.2492, found: 528.2505.
Boc-D-P h e-ψ[(E)-CHdCH]-Va l-Ar g(Mts)-Gly-OMe (13).
H), 1.39 (s, 9 H), 1.45-2.12 (m, 5 H), 2.23 (s, 3 H), 2.55-2.87
(m, 11 H), 3.17 (m, 2 H), 3.80-4.11 (m, 2 H), 4.38-4.65 (m, 3
H), 5.00-5.09 (m, 2 H), 5.35-5.47 (m, 1 H), 5.49-5.61 (m, 1
H), 6.10 (m, 1 H), 6.83-6.94 (m, 3 H), 7.06-7.35 (m, 11 H),
7.66 (m, 1 H); LRMS (FAB), m/z 948 (MH+), 848, 202, 154 (base
peak), 119, 91, 70, 57; HRMS (FAB), m/z calcd for C48H66N7O11S
(MH+) 948.4541, found: 948.4553.
To the protected dipeptide ester 12 (936 mg, 1.77 mmol) were
added successively anisole (0.5 mL) and 4 M HCl in 1,4-dioxane
(5 mL) at 0 °C, and the mixture was stirred for 1 h at room
temperature. The mixture was concentrated under reduced
pressure to give an oily residue, which was dissolved in DMF
(5 mL). To the solution were added successively (i-Pr)2NEt
(0.618 mL, 3.54 mmol), Boc-D-Phe-ψ[(E)-CHdCH]-Val-OH 11
(327 mg, 0.941 mmol), HOBt‚H2O (144 mg, 0.941 mmol), and
Cyclo[-Asp (OBn )-D-P h e-ψ[(E)-CHdCH]-Va l-Ar g(Mt s)-
Gly-] (18). To a stirred mixture of the protected pseudopen-
tapeptide 15 (201 mg, 0.211 mmol) and HONB (45 mg, 0.254
6170 J . Org. Chem., Vol. 67, No. 17, 2002

Synthesis of Alkene Dipeptide Isosteres
mmol) in EtOAc (2 mL) was added DCC (52 mg, 0.254 mmol)
at 0 °C, the mixture was stirred for 3 h at room temperature.
The solution was filtrated, and the filtrate was concentrated
under reduced pressure to give an oily residue, which was
washed with n-hexane and Et2O. 4 M HCl-dioxane (5 mL)
was added to the residue, and the mixture was stirred for 30
min at room temperature. The mixture was concentrated
under reduced pressure, and the residue was dissolved in DMF
(400 mL). Subsequently, pH of the solution was adjusted to
8.0 with 10% N-methylmorpholine in DMF at -20 °C. After 3
h, the solution was concentrated under reduced pressure, and
the residue was extracted with EtOAc. The extract was washed
with 1 N HCl, brine, 5% NaHCO3, and brine and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with CHCl3-MeOH (98:2
to 90:10) gave the title compound 18 (145 mg, 83% yield) as
CO3 and brine and dried over MgSO4. Concentration under
reduced pressure followed by flash chromatography over silica
gel with n-hexanes-EtOAc (10:1) gave a syn-allyl alcohol 20a
(17.3 g, 66% yield) and an anti-allyl alcohol 20b (4.21 g, 16%
yield), in order of elution.
Compound 20a : colorless crystals; mp 56-58 °C (n-hexane/
Et2O ) 3:1); [R]21D +38.5 (c 0.337, CHCl3); 1H NMR (300 MHz,
CDCl3) δ 1.37 (s, 9 H), 1.68 (s, 3 H), 2.44 (m, 1 H), 2.91 (d, J
) 7.3 Hz, 2 H), 3.90 (m, 1 H), 3.94 (m, 1 H), 4.78 (d, J ) 7.5
Hz, 1 H), 4.92 (m, 1 H), 5.02 (m, 1 H), 7.17-7.33 (m, 5 H).
Anal. Calcd for C17H25NO3: C, 70.07; H, 8.65; N, 4.81. Found:
C, 70.32; H, 8.87; N, 4.64.
Compound 20b: colorless crystals; mp 134-137 °C (n-
hexane/Et2O ) 3:1); [R]25 +47.6 (c 0.713, CHCl3); 1H NMR
D
(300 MHz, CDCl3) δ 1.33 (s, 9 H), 1.81 (s, 3 H), 2.35-2.52 (br,
1 H), 2.62-2.77 (m, 1 H), 2.89 (dd, J ) 14.2, 3.8 Hz, 1 H), 3.97
(m, 1 H), 4.18 (m, 1 H), 4.64 (s, 1 H), 4.98 (m, 1 H), 5.07 (m, 1
H), 7.17-7.33 (m, 5 H). Anal. Calcd for C17H25NO3: requires
C, 70.07; H, 8.65; N, 4.81. Found: C, 69.77; H, 8.56; N, 4.68.
colorless crystals: mp 291 °C (decomp, Et2O); [R]18 -10.0 (c
D
1
0.497, DMF); H NMR (300 MHz, DMF-d7) δ 0.68 (d, J ) 6.6
Hz, 3 H), 0.82 (d, J ) 6.5 Hz, 3 H), 1.40-1.66 (m, 3 H), 1.80-
2.01 (m, 2 H), 2.25 (s, 3 H), 2.50 (t, J ) 9.4 Hz, 1 H), 2.59 (dd,
J ) 15.8, 6.1 Hz, 1 H), 2.66 (s, 6 H), 2.78 (m, 1 H), 2.87 (m, 1
H), 2.89-3.00 (m, 1 H), 3.19 (m, 2 H), 3.40 (dd, J ) 15.3, 3.6
Hz, 1 H), 4.22 (dd, J ) 15.4, 7.7 Hz, 1 H), 4.40 (m, 1 H), 4.54
(m, 1 H), 4.82 (td, J ) 8.2, 6.2 Hz, 1 H), 5.09-5.19 (m, 2 H),
5.54-5.77 (m, 2 H), 6.65-6.85 (m, 1 H), 6.85-7.08 (m, 3 H),
7.17-7.46 (m, 10 H), 7.62 (d, J ) 8.7 Hz, 1 H), 7.78 (m, 1 H),
7.85 (d, J ) 7.6 Hz, 1 H), 8.13 (d, J ) 8.5 Hz, 1 H). Anal. Calcd
for C43H55N7O8S: C, 62.22; H, 6.68; N, 11.81. Found: C, 61.98;
H, 6.70; N, 11.52.
Gen er a l P r oced u r e for An ti-Selective Syn th esis of
N-Boc-P r otected 4-Am in oa lk -1-en -3-ols. To a stirred solu-
tion of oxalyl dichloride (28.4 mL, 326 mmol) in CH2Cl2 (350
mL) was added dropwise DMSO (46.3 mL, 653 mmol) in CH2-
Cl2 (50 mL) at -78 °C under argon. After 10 min, a solution
of Boc-D-phenylalaninol 21 (41.0 g, 163 mmol) in CH2Cl2 (100
mL) was added to the above mixture at -78 °C, and the
mixture was stirred for 1 h at this temperature. (i-Pr)2NEt
(227 mL, 1306 mmol) was added to the mixture at -78 °C,
and the mixture was stirred for 15 min with warming to 0 °C.
Saturated NH4Cl was added to the mixture, and the whole
was extracted with Et2O. The extract was washed successively
with 5% citric acid, water, 5% NaHCO3, and brine and dried
over MgSO4. Concentration under reduced pressure gave an
aldehyde. To a stirred solution of CH2dCMeMgBr‚ZnCl2‚LiCl
in THF (1216 mL, 408 mmol) was added the above aldehyde
in dry THF (100 mL) dropwise at -78 °C under argon, and
the mixture was stirred for 3 h with warming to 0 °C. The
mixture was quenched with saturated citric acid at -78 °C
followed by concentration under reduced pressure. The residue
was extracted with EtOAc, and the extract was washed with
water, 5% NaHCO3, and brine and dried over MgSO4. Con-
centration under reduced pressure followed by flash chroma-
tography over silica gel with n-hexanes-EtOAc (3:1) gave the
diastereomixture of allyl alcohols (30.2 g). Next, the allyl
alcohols were converted into the corresponding enone by the
same procedure with that of the above Swern oxidation. To a
stirred solution of the enone in dry Et2O (150 mL) was added
Zn(BH4)2 in Et2O (0.25 M, 1000 mL, 248 mmol) at -78 °C
under argon, and the stirring was continued for 1 h with
warming to 0 °C. The mixture was quenched with saturated
citric acid at -78 °C. The same purification described for that
of stereoselective synthesis of syn-allyl alcohol yielded a syn-
allyl alcohol 20a (4.59 g, 10% yield) and anti-allyl alcohol 20b
(14.2 g, 29% yield), in order of elution.
Cyclo[-Ar g-Gly-Asp-D-P h e-ψ[(E)-CHdCH]-Val-]‚TFA (3).
The protected cyclic peptide 18 (145 mg, 0.174 mmol) was
treated with 1 M TMSBr-thianisole/TFA (10 mL) in the
presence of m-cresol (0.488 mL, 4.66 mmol) and 1,2-ethanedithi-
ol (0.200 mL, 2.38 mmol) at 0 °C for 3 h. After concentration
under reduced pressure, ice-cold Et2O was added. The result-
ing powder was collected by centrifugation, and the powder
was washed three times with Et2O. Purification by preparative
HPLC (23% B in A) gave the title cyclic peptide 3 as mono-
TFA salt (93 mg, 79% yield) of colorless freeze-dried powder,
1
[R]24 -45.0 (c 0.636, AcOH); tR ) 14.5 min (23% B in A); H
D
NMR (600 MHz, DMSO-d6) δ 0.64 (d, J ) 6.6 Hz, 3 H), 0.77
(d, J ) 6.6 Hz, 3 H), 1.36-1.59 (m, 3 H), 1.75-1.91 (m, 2 H),
2.36 (dd, J ) 16.0, 5.7 Hz, 1 H), 2.41 (t, J ) 9.2 Hz, 1 H),
2.67-2.70 (m, 2 H) 2.80 (dd, J ) 13.4, 6.2 Hz, 1 H), 3.09 (m,
2 H), 3.31 (m, 1 H), 4.11 (dd, J ) 15.4, 7.9 Hz, 1 H), 4.25 (m,
1 H), 4.41 (m, 1 H), 4.59 (td, J ) 8.4, 5.8 Hz), 5.45 (dd, J )
15.2, 6.2 Hz, 1 H), 5.50 (dd, J ) 15.2, 9.3 Hz, 1 H), 7.17 (m, 3
H), 7.26 (m, 2 H), 7.47 (br, 1 H), 7.62 (d, J ) 7.8 Hz, 1 H), 7.65
(d, J ) 8.6 Hz, 1 H), 7.68 (dd, J ) 7.9, 3.0 Hz, 1 H), 8.02 (d, J
) 8.6 Hz, 1 H), 12.20 (br, 1 H); LRMS (FAB), m/z 558 (MH+),
91, 87 (base peak), 70; HRMS (FAB), m/z calcd for C27H40N7O6
(MH+) 558.3040, found: 558.3052.
Gen er a l P r oced u r e for Syn -Selective Syn th esis of
N-Boc-P r otected 4-Am in oa lk -1-en -3-ols: Syn th esis of
(3R,4R)-4-[N-(ter t-Bu t oxyca r b on yl)a m in o]-2-m et h yl-5-
p h en ylp en t-1-en -3-ol (20a ) a n d Its (3S,4R)-Isom er (20b).
To a stirred solution of Boc-D-Phe-OMe 19 (25.0 g, 89.5 mmol)
in toluene-CH2Cl2 (4:1, 125 mL) was added dropwise a
solution of DIBAL-H in toluene (1.0 M, 179 mL, 179 mmol) at
-78 °C under argon, and the mixture was stirred for 1 h at
this temperature. In another flask, to a stirred solution of
ZnCl2 (36.6 g, 268 mmol) and LiCl (11.4 g, 268 mmol) in dry
THF (90 mL) was added dropwise a solution of CH2d
CMeMgBr in THF (0.5 M, 537 mL, 268 mmol) at 0 °C under
argon, and the mixture was stirred for 1 h. The resulting
solution of CH2dCMeMgBr‚ZnCl2‚LiCl in THF was added
dropwise to the above substrate mixture at -78 °C, and the
mixture was stirred for 3 h with warming to 0 °C. The mixture
was quenched with saturated citric acid at -78 °C, and the
whole was concentrated under reduced pressure. The residue
was extracted with EtOAc, and the extract was washed
successively with saturated citric acid, brine, saturated NaH-
ter t-Bu tyl (4R,5R,2E)-5-[N-(ter t-Bu toxycar bon yl)am in o]-
4-h yd r oxy-3-m eth yl-6-p h en ylh ex-2-en oa te (23a ) a n d Its
(4S,5R,2E)-Isom er (23b). By use of a procedure similar to
that described for the successive treatment of the acetate 6
with O3 gas and Me2S, the acetate 22a (5.77 g, 17.3 mmol)
was converted into the corresponding ketone. To the stirred
solution of the ketone in CHCl3 (20 mL) was added Ph3Pd
CHCO2t-Bu (17.4 g, 51.9 mmol), and the mixture was gently
refluxed for 2 d. The mixture was concentrated under reduced
pressure, and purified by flash chromatography over silica gel
to give a yellow oil (4.29 g). To a solution of the above oil in
dry MeOH (10 mL) was added powdered Na2CO3 (4.20 g, 39.5
mmol) at room temperature, and the mixture was stirred for
3 h. The mixture was filtered, and the filtrate was extracted
with Et2O. The extract was washed with water and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexanes-EtOAc
J . Org. Chem, Vol. 67, No. 17, 2002 6171

Oishi et al.
(7:1) gave the title compound 23a (2.99 g, 44% yield) and 23b
(336 mg, 4.9% yield), in order of elution.
and the stirring was continued for 30 min at -78 °C and for
30 min at 0 °C. The mixture was quenched with saturated
NH4Cl-28% NH4OH (1:1, 2 mL), and the whole was extracted
with Et2O. The extract was washed with brine and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexanes-EtOAc
(40:1) gave the mixture of the title compounds 27a , 28a , and
29 (83.4 mg, 93% yield). The product ratio was determined by
RP-HPLC and 1H NMR analyses (27a :28a :29 ) 70:27:3).
Compound 23a : colorless powder; mp 45-46 °C; [R]22
D
+45.8 (c 2.42, CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.38 (s, 9
H), 1.45 (s, 9 H), 2.00 (m, 3 H), 2.95 (m, 2 H), 3.93 (m, 2 H),
4.79 (d, J ) 9.6 Hz, 1 H), 5.89 (d, J ) 0.9 Hz, 1 H), 7.20-7.34
(m, 5 H); LRMS (FAB), m/z 392 (MH+, base peak), 336, 292,
280, 262, 236, 220, 218, 201, 164, 144, 120, 116, 99, 91, 57,
41, 29; HRMS (FAB), m/z calcd for C22H34NO5 (MH+) 392.2437,
found: 392.2451.
Compound 27a : colorless powder; [R]21 -58.8 (c 0.629,
D
Compound 23b: colorless powder; mp 80-82 °C; [R]25
CHCl3); ∆ꢀ ) -11.04 (228 nm, isooctane); tR ) 56.6 min (65%
D
1
1
+2.51 (c 0.796, CHCl3); H NMR (300 MHz, CDCl3) δ 1.34 (s,
B in A); H NMR (300 MHz, CDCl3) δ 0.59 (d, J ) 6.6 Hz, 3
9 H), 1.48 (s, 9 H), 2.14 (d, J ) 1.0 Hz, 3 H), 2.73 (m, 1 H),
2.82 (dd, J ) 14.3, 4.2 Hz, 1 H), 4.01 (m, 1 H), 4.27 (m, 1 H),
4.69 (d, J ) 8.1 Hz, 1 H), 5.95 (m, 1 H) 7.14-7.31 (m, 5 H).
Anal. Calcd for C22H33NO5: C, 67.49; H, 8.50; N, 3.58. Found:
C, 67.43; H, 8.27; N, 3.48.
H), 0.88 (d, J ) 6.4 Hz, 3 H), 1.40 (s, 9 H), 1.41 (s, 9 H), 1.55
(d, J ) 1.3 Hz, 3 H), 1.97 (m, 1 H), 2.35 (d, J ) 10.7 Hz, 1 H),
2.67 (dd, J ) 13.2, 7.8 Hz, 1 H), 2.92 (dd, J ) 13.1, 4.7 Hz, 1
H), 4.45 (m, 1 H), 4.56 (m, 1 H), 5.15 (m, 1 H), 7.14-7.27 (m,
5 H). LRMS (FAB), m/z 418 (MH+, base peak), 326, 306, 260,
245, 214, 199, 170, 164, 143, 120, 91. HRMS (FAB), m/z calcd
for C25H40NO4 (MH+) 418.2957, found: 418.2951.
Compound 28a : colorless oil; [R]25D +135.9 (c 0.103, CHCl3);
∆ꢀ ) +27.40 (226 nm, isooctane); tR ) 62.7 min (65% B in A);
1H NMR (300 MHz, CDCl3) δ 0.43 (d, J ) 6.7 Hz, 3 H), 0.88
(d, J ) 6.4 Hz, 3 H), 1.40 (s, 9 H), 1.43 (s, 9 H), 1.63 (d, J )
1.3 Hz, 3 H), 2.00 (m, 1 H), 2.74 (dd, J ) 13.1, 7.3 Hz, 1 H),
2.92 (d, J ) 10.9 Hz, 1 H), 3.02 (m, 1 H), 4.38-4.62 (m, 2 H),
5.23 (m, 1 H), 7.13-7.38 (m, 5 H); LRMS (FAB), m/z 418 (MH+,
base peak), 362, 326, 306, 270, 260, 250, 244, 214, 199, 170,
ter t-Bu tyl (2E)-3-[(4R,5R)-4-Ben zyl-N-(ter t-bu toxyca r -
bon yl)-2,2-dim eth yl-1,3-oxazolidin -5-yl]bu t-2-en oate (24a).
To a stirred solution of the hydroxy ester 23a (90.8 mg, 0.231
mmol) and 2,2-dimethoxypropane (0.0855 mL, 0.695 mmol) in
CH2Cl2 (1.0 mL) was added dropwise BF3‚Et2O (0.00171 mL,
0.0139 mmol) at 0 °C, and the mixture was stirred overnight
with warming to room temperature. Saturated NaHCO3 (1 mL)
was added to the mixture at 0 °C, and the whole was extracted
with Et2O. The extract was washed with brine and dried over
MgSO4. Concentration under reduced pressure followed by
flash chromatography over silica gel with n-hexanes-EtOAc
(3:1) gave the title compound 24a (89.2 mg, 89% yield) as a
164, 143, 120, 111, 91, 57; HRMS (FAB), m/z calcd for C25H40
-
NO4 (MH+) 418.2957, found: 418.2950.
colorless powder: mp 109-110 °C (n-hexane); [R]27 +26.9 (c
D
Compound 29: colorless oil; [R]24 -81.63 (c 0.049, CHCl3);
D
0.852, CHCl3); 1H NMR (300 MHz, CDCl3) δ 1.25 (s, 3 H), 1.46
(s, 9 H), 1.55 (s, 9 H), 1.62 (s, 3 H), 1.85 (s, 3 H), 3.01 (m, 1 H),
3.18 (dd, J ) 13.2, 2.8 Hz, 1 H), 4.05 (m, 1 H), 4.31 (dd, J )
5.6, 0.6 Hz, 1 H), 5,71 (m, 1 H), 7.19-7.33 (m, 5 H). Anal. Calcd
for C25H37NO5: C, 69.58; H, 8.64; N, 3.25. Found: C, 69.44;
H, 8.64; N, 3.28.
tR ) 49.3 min (65% B in A); 1H NMR (300 MHz, CDCl3) δ 1.39
(s, 9 H), 1.43 (s, 9 H), 1.75 (d, J ) 1.4 Hz, 3 H), 2.86 (m, 3 H),
3.04 (dd, J ) 14.9, 0.7 Hz, 1 H), 4.49 (m, 2 H), 5.19 (m, 1 H),
7.17-7.31 (m, 5 H); LRMS (FAB), m/z 376 (MH+, base peak),
264, 228, 203, 172, 143, 128, 57; HRMS (FAB), m/z calcd for
C
22H34NO4 (MH+), 376.2488, found: 376.2485.
ter t-Bu tyl (4R,5R,2E)-4-Acetoxy-5-[N-(ter t-bu toxyca r -
bon yl)a m in o]-3-m eth yl-6-p h en ylh ex-2-en oa te (25a ). To a
stirred solution of the hydroxy ester 23a (1.77 g, 4.52 mmol)
in CHCl3 (5 mL), pyridine (7.31 mL, 90.4 mmol), and DMAP
(55 mg, 0.450 mmol) was added Ac2O (4.26 mL, 45.2 mmol) at
0 °C, and the stirring was continued overnight with warming
to room temperature. The mixture was poured into ice-cooled
water, and the whole was extracted with EtOAc. The extract
was washed successively with saturated citric acid, brine,
saturated NaHCO3, and brine and dried over MgSO4. Con-
centration under reduced pressure followed by flash chroma-
tography over silica gel with n-hexanes-EtOAc (3:1) gave the
title compound 25a (1.83 g, 93% yield) as colorless crystals:
(2R,5R,3E)-5-[N-(9-Flu or en ylm eth oxycar bon yl)am in o]-
2-isop r op yl-3-m et h yl-6-p h en ylh ex-3-en oic Acid (F m oc-
D-P h e-ψ[(E)-CHdCMe]-L-Va l-OH, 30). The ester 27a (245
mg, 0.586 mmol) was dissolved in TFA (5 mL), and the mixture
was stirred for 1.5 h at room temperature. Concentration
under reduced pressure gave an oily residue, which was
dissolved in MeCN-H2O (2:1, 3.9 mL). Et3N (0.163 mL, 1.17
mmol) and a solution of Fmoc-OSu (207 mg, 0.616 mmol) in
MeCN (2.6 mL) were added to the above solution at 0 °C. After
being stirred for 3.5 h, the mixture was acidified with 0.1 N
HCl and was extracted with EtOAc. The extract was washed
with 0.1 N HCl and brine and dried over MgSO4. Concentra-
tion under reduced presssure followed by flash chromatogra-
phy over silica gel with n-hexanes-EtOAc (2:1) gave the title
mp 67-68 °C (n-hexane/Et2O ) 3:1); [R]23 +65.3 (c 1.20,
D
1
CHCl3); H NMR (300 MHz, CDCl3, at 323 K) δ 1.36 (s, 9 H),
compound 30 (198 mg, 69% yield) as a colorless oil: [R]22
D
1.44 (s, 9 H), 2.05 (d, J ) 1.1 Hz, 3 H), 2.13 (s, 3 H), 2.77 (m,
2 H), 4.24 (m, 1 H), 4.56 (m, 1 H), 5.04 (m, 1 H), 5.61 (m, 1 H),
7.12-7.32 (m, 5 H). Anal. Calcd for C24H35NO6: C, 66.49; H,
8.14; N, 3.23. Found: C, 66.19; H, 8.26; N, 3.08.
-34.0 (c 1.29, CHCl3); 1H NMR (300 MHz, CDCl3 at 328 K) δ
0.62 (d, J ) 6.5 Hz, 3 H), 0.92 (d, J ) 6.4 Hz, 3 H), 1.52 (br, 3
H), 2.01 (m, 1 H), 2.49 (d, J ) 10.6 Hz, 1 H), 2.63 (m, 1 H),
2.85 (m, 1 H), 4.16 (t, J ) 6.5 Hz, 1 H), 4.30-4.65 (m, 4 H),
5.22 (d, J ) 8.8 Hz, 1 H), 7.05 (m, 2 H), 7.13-7.30 (m, 5 H),
7.32-7.39 (m, 2 H), 7.52 (m, 2 H), 7.72 (m, 2 H); LRMS (FAB),
m/z 484 (MH+), 440 (base peak), 245, 235, 196, 179; HRMS
Gen er a l P r oced u r e for Syn th esis of ψ[(E)-CHdCMe]-
typ e Alk en e Dip ep tid e Isoster es via Or ga n ocop p er -
Med ia ted Alk yla tion : Syn th esis of ter t-Bu tyl (2R,5R,3E)-
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-2-isop r op yl-3-m eth yl-
6-p h en ylh ex-3-en oa te (Boc-D-P h e-ψ[(E)-CHdCMe]-L-Va l-
Ot-Bu , 27a ), Its (2S,5R,3Z)-Isom er (Boc-D-P h e-ψ[(Z)-CHd
CMe]-D-Va l-Ot-Bu , 28a ) a n d ter t-Bu t yl (5R,3Z)-5-[N-
(ter t-Bu t oxyca r b on yl)a m in o]-3-m et h yl-6-p h en ylh ex-3-
en oa te (Boc-D-P h e-ψ[(Z)-CHdCMe]-Gly-Ot-Bu , 29). To a
stirred suspension of CuCN (75.0 mg, 0.837 mmol) in dry Et2O
(1 mL)-HMPA (0.146 mL, 0.837 mmol) was added dropwise
a solution of i-PrMgCl in Et2O (0.87 M, 1.93 mL, 1.68 mmol)
at -78 °C under argon, and the mixture was stirred for 15
min at 0 °C. BF3‚Et2O (0.106 mL, 0.837 mmol) was added to
the above mixture, and the mixture was stirred for 3 min. A
solution of the mesylate 26a (100 mg, 0.212 mmol) in dry Et2O
(1.5 mL) was added dropwise to the above reagent at -78 °C,
(FAB), m/z calcd for
C
31H34NO4 (MH+) 484.2488, found:
484.2479.
Gen er a l P r oced u r e for Syn th esis of P r otected P ep tid e
Resin s. Protected peptide-resins were manually constructed
by Fmoc-based solid-phase peptide synthesis. t-Bu ester for
Asp, (Boc)2 or Pbf for Arg (for 33 or 37, respectively) were
employed for side-chain protection. Fmoc deprotection were
achieved by 20% piperidine in DMF (2 × 1 min, 1 × 20 min).
Fmoc-amino acids except for Fmoc-D-Phe-ψ[(E/Z)-CHdCMe]-
Val-OH were coupled by treatment with 5 equiv of reagents
[Fmoc-amino acid, N,N′-diisopropylcarbodiimide (DIPCDI),
and HOBt‚H2O] to free amino group (or hydrazino group) in
DMF for 1.5 h.
6172 J . Org. Chem., Vol. 67, No. 17, 2002

Synthesis of Alkene Dipeptide Isosteres
H -Asp (Ot-Bu )-D-P h e-ψ[(E)-CH dCMe]-Va l-Ar g(Boc)2-
Gly-NHNHCO-Wa n g Resin (33). p-Nitrophenyl carbonate
Wang resin (0.93 mmol/g, 161 mg, 0.15 mmol) was treated with
NH2NH2‚H2O (0.046 mL, 0.75 mmol) in DMF (2 mL) at room
temperature for 2 h to give a hydrazino linker. Gly and Arg-
(Boc)2 residues were coupled by general coupling protocol.
Fmoc-D-Phe-ψ[(E)-CHdCMe]-Val-OH 30 (51.0 mg, 0.105 mmol)
was incorporated by twice treatments with DIPCDI (0.019 mL,
0.126 mmol) and HOBt‚H2O (0.016 mg, 0.105 mmol) for 1.5 h
each. After capping of free amino groups with Ac2O-pyridine,
Asp(Ot-Bu) residue was coupled by general coupling protocol
to afford the title protected peptide resin 33.
to solid-phase extraction over basic alumina gel with CHCl3-
MeOH (9:1) to remove inorganic salts. The resulting cyclic
protected peptide 39 was treated with 95% TFA solution for
1.5 h at room temperature. Concentration under reduced
pressure and purification by preparative HPLC (linear gradi-
ent of B in A, 19 to 25% over 60 min) gave the cyclic
pseudopeptide 40 (15.7 mg, 20% yield from 35) as freeze-dried
powder: [R]22 -5.61 (c 0.712, H2O); tR ) 30.6 min (linear
D
gradient of B in A, 20 to 35% over 30 min); 1H NMR (600 MHz,
DMSO-d6) δ 0.08 (m, 3 H), 0.66 (d, J ) 5.8 Hz, 3 H), 1.44 (m,
2 H), 1.70-1.79 (m, 4 H), 1.86 (m, 1 H), 1.93 (m, 1 H), 2.22
(m, 1 H), 2.42-2.58 (m, 3 H), 3.00 (m, 1 H), 3.05 (m, 1 H),
3.12 (m, 1 H), 3.39 (m, 1 H), 3.49 (dd, J ) 16.6, 5.3 Hz, 1 H),
3.76 (dd, J ) 16.6, 5.8 Hz, 1 H), 4.40 (m, 1 H), 4.74 (m, 1 H),
5.32 (d, J ) 10.3 Hz, 1 H), 7.13-7.26 (m, 5 H), 7.37-7.53 (m,
3 H), 7.91 (m, 1 H), 8.55 (m, 1 H), 12.11 (br, 1 H); LRMS (FAB),
m/z 572 (MH+), 185, 154, 137, 93, 91, 70 (base peak); HRMS
(FAB), m/z calcd for C28H42N7O6 (MH+) 572.3197, found:
572.3218.
Cyclo(-Ar g-Gly-Asp -D-P h e-ψ[(E)-CH dCMe]-Va l-)‚TF A
(4). The protected peptide resin 33 was treated with TFA for
1.5 h at room temperature, and the mixture was filtered.
Concentration under reduced pressure followed by preparative
HPLC (linear gradient of B in A, 14 to 20% over 60 min) gave
a peptide hydrazide 34 as a colorless powder. To a stirred
solution of 34 in DMF (12 mL) were added a solution of 4 M
HCl in DMF (0.079 mL, 0.316 mmol) and isoamyl nitrite (0.014
mL, 0.105 mmol) at -40 °C. After being stirred for 30 min at
-20 °C, the mixture was diluted with precooled DMF (72 mL).
To the above solution was added (i-Pr)2NEt (0.183 mL, 1.05
mmol) at -40 °C, and the mixture was stirred for 24 h at -20
°C. Concentration under reduced pressure and purification by
preparative HPLC (linear gradient of B in A, 19 to 25% over
60 min) gave the cyclic pseudopeptide 434 (10.3 mg, 14% yield
In tegr in -Bin d in g Assa ys. Compounds were evaluated for
their inhibitory activities in RVâ3 and RIIbâ3-ELISA (enzyme
linked immunosorbent assay). RVâ3 was purified from human
placenta, using RGDSPK-sepharose CL-4B affinity chroma-
tography, followed by mono Q ion exchange chromatography,
according to Pytela’s protocol.37
RIIbâ3 was purified from human
platelet by RGDSPK-sepharose CL-4B as well.37 RVâ3 and RIIbâ3
binding assays were performed according to the modified
method of Kouns et al.38 EIA plates were coated with RVâ3 or
from 30) as freeze-dried powder: [R]21 -36.8 (c 0.516, H2O);
D
tR ) 31.2 min (linear gradient of B in A, 20 to 40% over 40
RIIbâ3, and blocked with bovine serum albumin. In each
1
min); H NMR (600 MHz, DMSO-d6) δ 0.43 (d, J ) 6.5 Hz, 3
reaction, a test sample in the reaction mixture (20 mM Tris-
HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4, 0.100
mL) including vitronectin or fibrinogen was added to the
receptor-coated plate and incubated for 4 h at 25 °C. Thereafter
the ligand binding was measured using anti-vitronectin rabbit
antibody and peroxidase-conjugated anti-rabbit IgG antibody
for RVâ3, or peroxidase-conjugated anti-fibrinogen antibody for
H), 0.79 (d, J ) 6.4 Hz, 3 H), 1.35-1.50 (m, 5 H), 1.81 (m, 2
H), 1.97 (m, 1 H), 2.33 (d, J ) 11.2 Hz, 1 H), 2.45 (dd, J )
16.7, 6.1 Hz, 1 H), 2.69 (dd, J ) 13.2, 9.2 Hz, 1 H), 2.78 (m, 1
H), 2.83 (dd, J ) 13.2, 4.9 Hz, 1 H), 3.08 (m, 2 H), 3.39 (dd, J
) 14.5, 2.5 Hz, 1 H), 3.85-3.90 (m, 2 H), 4.47 (m, 1 H), 4.62
(ddd, J ) 16.9, 8.5, 5.1 Hz, 1 H), 5.25 (d, J ) 8.5 Hz, 1 H),
7.13-7.17 (m, 3 H), 7.19-7.27 (m, 3 H), 7.31 (d, J ) 7.9 Hz, 1
H), 7.49 (br, 1 H), 7.78 (m, 1 H), 8.69 (d, J ) 7.9 Hz, 1 H),
12.26 (br, 1 H); LRMS (FAB), m/z 572 (MH+), 185 (base peak),
154, 137, 93; HRMS (FAB), m/z calcd for C28H42N7O6 (MH+)
572.3197, found: 572.3185.
R
IIbâ3, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
as the substrate of peroxidase. The IC50 values were deter-
mined from measurement of absorbance at 415 nm.
Ackn owledgm en t. We thank Dr. Terrence R. Burke,
J r., NCI, NIH, for reading manuscript and valuable
discussions. This work was supported by Grant-in-Aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology, J apan, and
the J apan Health Science Foundation. S.O. is grateful
for Research Fellowships of the J SPS for Young Scien-
tists.
Syn th esis of H-Asp (Ot-Bu )-D-P h e-ψ[(Z)-CHdCMe]-Va l-
Ar g(P bf)-Gly-Clt Resin (37). Arg(Pbf) residue was coupled
by general coupling protocol on H-Gly-Clt resin (0.63 mmol/g,
216 mg, 0.136 mmol). Fmoc-D-Phe-ψ[(Z)-CHdCMe]-Val-OH 35
(55.0 mg, 0.113 mmol) was incorporated by twice treatments
with DIPCDI (0.021 mL, 0.136 mmol) and HOBt‚H2O (0.017
mg, 0.113 mmol) for 1.5 h each. After capping of free amino
groups with Ac2O-pyridine, Asp(Ot-Bu) residue was coupled
by general coupling protocol to afford the title protected peptide
resin 37.
Cyclo(-Ar g-Gly-Asp -D-P h e-ψ[(Z)-CH dCMe]-Va l-)‚TF A
(40). The protected peptide resin 37 was subjected to AcOH/
TFE/CH2CH2 (1:1:3, 10 mL) treatment for 2 h at room
temperature. After filtration of the residual resin, the filtrate
was concentrated under reduced pressure to give the crude
protected peptide 38 as a colorless powder. To a stirred
suspension of 38 and NaHCO3 (57.1 mg, 0.680 mmol) in DMF
(41 mL) was added DPPA (0.0879 mL, 0.408 mmol) at -40
°C. The mixture was stirred for 36 h with warming to room
temperature and filtered. The filtrate was concentrated under
reduced pressure to give an oily residue, which was subjected
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization data for 22a ,b, 24b, 25b, 26a ,b,
27b, 28b, 35, and 41; 1H NMR spectra for all new compounds.
This material are available free of charge via the Internet at
http://pubs.acs.org.
J O025923M
(37) Pytela, R.; Pierschbacher, M. D.; Argraves, S.; Suzuki, S.;
Rouslahti, E. Methods Enzymol. 1987, 144, 475.
(38) Kouns, W. C.; Kirchhofer, D.; Hadvary, P.; Edenhofer, A.;
Weller, T.; Pfenninger, G.; Baumgartner, H. R.; J ennings, L. K.;
Steiner, B. Blood 1992, 80, 2539.
J . Org. Chem, Vol. 67, No. 17, 2002 6173