Synthesis of biologically active dicarba analogues of the peptide hormone oxytocin using ring-closing metathesis.

Article abstract of DOI:10.1021/ol027160v

Facile synthesis of cis and trans olefinic analogues of oxytocin 1 that have carbon in place of sulfur is achieved via ring-closing metathesis (RCM) on a resin-bound linear precursor peptide. Hydrogenation of the cis olefin, 3, proceeds selectively to gen

Full text of DOI:10.1021/ol027160v

ORGANIC
LETTERS
2003
Vol. 5, No. 1
47-49
Synthesis of Biologically Active Dicarba
Analogues of the Peptide Hormone
Oxytocin Using Ring-Closing Metathesis
,†
Jake L. Stymiest,† Bryan F. Mitchell,‡ Susan Wong,‡ and John C. Vederas*
Department of Chemistry, UniVersity of Alberta, Edmonton,
Alberta, Canada, T6G 2G2, and Perinatal Research Centre,
Department of Obstetrics and Gynecology, UniVersity of Alberta, Canada, TH5 3V9
john.Vederas@ualberta.ca
Received October 23, 2002
ABSTRACT
Facile synthesis of cis and trans olefinic analogues of oxytocin 1 that have carbon in place of sulfur is achieved via ring-closing metathesis
(RCM) on a resin-bound linear precursor peptide. Hydrogenation of the cis olefin, 3, proceeds selectively to generate the previously reported
saturated derivative 5. Biological testing on rat uterus strips shows that cis compound 3 has an EC50 value of 38 ng/mL (EC50 for oxytocin
is 2.7 ng/mL) whereas 5 and trans olefin 4 are less active.
The synthesis of diamino acids and cyclic peptide derivatives
using ring-closing metathesis (RCM) affords the opportunity
to substitute a metabolically less stable disulfide bridge with
two methylene groups, a structural change that results in
replacement of LL-cystine with LL-diaminosuberic acid.1
Although such replacement should in principle impose
negligible change on the overall peptide structure, the polarity
of the sulfurs and their preference for a close to 90° dihedral
angle is clearly different from the two methylenes in the
“dicarba” analogue. Oxytocin 1 is a mammalian nonapeptide
hormone that controls mammary and uterine smooth muscle
contraction,2 has neurotransmitting properties in the central
nervous system, and displays autocrine and/or paracrine
functions in the ovaries and testes.2,3 The synthesis of a fully
saturated dicarba analogue of oxytocin has been described,4
but it involves a cumbersome multistep procedure to
incorporate a selectively protected diaminosuberic acid
moiety. The compound was reported as being “less active”
(2) For a review, see: (a) Lehninger, A. L.; Nelson, D. L.; Cox, M.
Principles of Biochemistry, 2nd ed.; Worth Publishers, Inc.: New York,
1993; pp 750-752. For examples of oxytocin analogues see: (b) Be´lec,
L.; Slaninova, J.; Lubell, W. D. J. Med. Chem. 2000, 43, 1448-1445. (c)
ZhongQing, Y.; Blomberg, D.; Sethson, I.; Brickman, K.; Ekholm, K.;
Johansson, B.; Nilsson, A.; Kihlberg, J. J. Med. Chem. 2002, 45, 2512-
2519.
† Department of Chemistry.
‡ Perinatal Research Centre.
(1) For some early reports, see: (a) Miller, S. J.; Blackwell, H. E.;
Grubbs, R. H. J. Am. Chem. Soc. 1996, 118, 9606-9614. (b) Williams, R.
M.; Liu, J. J. Org. Chem. 1998, 63, 2130-2132. (c) Gao, Y.; Lane-Bell,
P.; Vederas, J. C. J. Org. Chem. 1998, 63, 2133-2143.
(3) (a) Nicholson, H. D. ReV. Reproduction 1996, 1, 69-72. (b) Harris,
G. C.; Nicholson, H. D. J. Endocrinol. 1998, 156, 35-42.
(4) Keller, O.; Rudinger, J. HelV. Chim. Acta. 1974, 57, 5, 1253-1259.
10.1021/ol027160v CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/10/2002

than the parent hormone 1, which may be due to greater
conformational flexibility at the bis-methylene unit. In the
present study, we show that substitution of the cysteines with
L-allylglycine residues allows RCM for facile generation of
more rigid olefinic analogues of 1 and provides access to
the saturated dicarba derivative (Figure 1).
of oxytocin on Rink amide NovaGel resin in conjunction
with standard Fmoc chemistry for amino acid coupling
(Scheme 1).11 This resin conveniently provides the C-terminal
Scheme 1. Synthesis of the Linear Peptide Backbonea
a Conditions: (a) Fmoc-Leu-OH, (b) Fmoc-Pro-OH, (c) Fmoc-
allylglycine-OH, (d) Fmoc-Asn(N-Trt)-OH, (e) Fmoc-Gln(N-Trt)-
OH, (f) Fmoc-Ile-OH, (g) Fmoc-Tyr(O-tBu)-OH, (h) Fmoc-allyl-
glycine-OH.
Figure 1. RCM Strategy for Peptidic Dicarba Analogues
Ruthenium-catalyzed RCM has been previously achieved5,6
on an assortment of peptidic diene systems1,7,8 with varying
yields. To date, the cyclization of bis-allylglycine-containing
peptides to make hormone analogues has not been reported,
nor have such olefinic analogues been investigated for
biological activity. It is interesting that the X-ray crystal
structure of free oxytocin 1 does not allow exact determi-
nation of the conformation at the disulfide,9 although this is
clearly fixed in a single orientation upon binding to a
neurophyseal carrier protein.10
amide functionality upon cleavage. Protection of the side
chains of tyrosine (O-tBu), asparagine (N-Trt), and glutamine
(N-Trt) with acid-labile groups is essential. The trityl (Trt)
groups ensure optimal coupling by preventing tandem
cyclization and dehydration of the primary amide side
chains,12,13 whereas the tert-butyl group is necessary to avoid
interference of the phenol in the RCM reaction.
Resin-bound linear peptide 2 could then be cyclized using
10 mol % Grubbs (benzylidene-bis(tricyclohexyl-phosphine)-
dichlororuthenium) catalyst6,7 to give a mixture of olefinic
products. Upon completion of this reaction, it is essential to
add DMSO (50 equiv relative to the catalyst loading) to the
resin-bound peptide. Failure to do so results in the production
of a mixture of products and ruthenium-containing contami-
nants that is exceedingly difficult to separate, even by
reverse-phase HPLC. This technique is an adaptation of a
literature procedure14 wherein DMSO was added to a
solution-phase RCM reaction unrelated to peptide synthesis.15
Removal of the remaining Fmoc group followed by acidic
cleavage from the resin with concomitant side chain depro-
tection affords a 4:1 mixture of cis and trans isomers 3 and
4, respectively (Scheme 2).
The synthesis is initiated by first building the linear peptide
backbone 2 using allyglycine in place of cysteine residues
(5) For recent reviews, see: (a) Furstner, A. Angew. Chem., Int. Ed. 2000,
39, 3012-3043. (b) Grubbs, R. H.; Chang, S. Tetrahedron 1998, 54, 4413-
4450. (c) Schuster, M.; Bechert, S. Angew. Chem., Int. Ed. Engl. 1997, 36,
2036-2056. (d) Grubbs, R. H.; Miller, S. J.; Fu, G. C. Acc. Chem. Res.
1995, 28, 446-452.
(6) Second generation imidazole/imidazoline Grubbs catalyst: (a) Mor-
gan, J. P.; Grubbs, R. H. Org. Lett. 2000, 2, 3153-3155. (b) Briot, A.;
Bujard, M.; Gouverneur, V.; Nolan, S. P.; Mioskowski, C. Org. Lett. 2000,
2, 1517-1519. (c) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org.
Lett. 1999, 1, 953-956. (d) Huang, J.; Stevens, E. D.; Nolan, S. P.; Petersen,
J. L. J. Am. Chem. Soc. 1999, 121, 2674-2678.
(7) Examples of RCM on peptides in solution: (a) Reichwein, J. F.;
Liskamp, R. M. J. Eur. J. Org. Chem. 2000, 2335-2344. (b) Reichwein, J.
F.; Versluis, C.; Liskamp, R. M. J. J. Org. Chem. 2000, 65, 6187-6195.
(c) Reichwein, J. F.; Versluis, C.; Liskamp, R. M. J. J. Org. Chem. 2000,
65, 6187-6195. (d) Kasmaier, U.; Maier, S. Org. Lett. 1999, 1 (11), 1763-
1766. (e) Gao, Y.; Wei, C.; Burke, T. R., Jr. Org. Lett. 2001, 3 (11), 1617-
1620.
(8) Examples of RCM on resin-bound peptides. (a) Schmiedeberg, N.;
Kessler, H. Org. Lett. 2002, 4 (1), 59-62. (b) Jarvo, E. R.; Copeland, G.
T.; Papaioannou, N.; Bonitatebus, P. J., Jr.; Miller, S. J. J. Am. Chem. Soc.
1999, 121, 11638-11643. (c) Schafmeister, C. E.; Po, J.; Verdine, G. L. J.
Am. Chem. Soc. 2000, 122, 5891-5892.
(11) (a) Adams, J. H.; Cook, R. M.; Hudson, D.; Jammalamadaka, V.;
Lyttle, M. H.; Songster, M. F. J. Org. Chem. 1998, 63, 3706-3716. (b)
Sieber, P.; Rinker, B. Tetrahedron Lett. 1991, 32, 739-742.
(12) Novabiochem Catalog 2002-2003; Calbiochem-Novabiochem Inc.,
San Diego, CA.
(13) Several peptide syntheses were done using asparagine and glutamine
subunits without side chain protection in the presence of an extra 1 equiv
of HOBt.10 In all attempts, a substantial amount of dehydrated product was
obtained, as shown by ES/MS as a (M-18) peak.
(14) Ahn, Y. M.; Yang, K.; Georg, G. I. Org. Lett. 2001, 3, 9, 1411-
1413.
(15) To our knowledge, this is the first time ruthenium byproducts have
been removed using simple DMSO injection and filtration when working
with resin-bound peptides.
(9) Wood, S. P.; Tickle, I. J.; Treharne, A. M.; Pitts, J. E.; Mascarenhas,
Y.; Li, J. Y.; Husain, J.; Cooper, S.; Blundell, T. L.; Hruby, V. J.; Buku,
A.; Fischman, A. J.; Wyssbrod, H. R. Science 1986, 232 (4750), 633-636.
(10) Rose, J. P.; Wu, C.; Hsiao, C.; Breslow, E.; Wang, B. Nat. Struct.
Biol. 1996, 3 (2), 163-169.
48
Org. Lett., Vol. 5, No. 1, 2003

Scheme 2 Cyclization of 2a
Scheme 3. Reduction of 3
Testing for biological activity of 3-5 utilized freshly
excised rat uterus strips according to established literature
procedures.18 Preliminary results show that cis isomer 3 is
the most active dicarba analogue with an EC50 value of 38
ng/mL, which is approximately 14-fold less than that of
oxytocin 1, whose EC50 is 2.7 ng/mL. Trans isomer 4 and
saturated dicarba analogue 5 are about an order of magnitude
less potent than 3 with EC50 values of 242 and 338 ng/mL,
respectively.
In summary, RCM on resin-bound allylglycine-containing
peptides provides an effective and rapid method for the
synthesis of cyclic dicarba analogues of biologically active
peptide hormones such as oxytocin 1. The ability of RCM
to introduce more rigid ethylene bridges in place of
metabolically less stable disulfide moieties may prove to be
useful for generation of analogues of a host of biologically
active peptides and proteins. The yields of resin-bound olefin
metathesis are acceptable when compared with other peptide
RCM cyclizations7,19 and may potentially be improved by
use of second generation catalysts.6 The synthesis of other
analogues of oxytocin using carbon bridges of varying
lengths is currently under investigation, as is application of
the methodology to other biologically active peptides.
a Conditions: (a) 10 mol % (PCy3)2Cl2Ru(CHPh) (Grubbs
catalyst), DCM, reflux, 24h. (b) 50 equiv of DMSO, rt, 12 h. (c)
20% Pip/DMF, rt, 5 min. (d) 90% TFA/5% DCM/5% anisole, 1 h.
(e) RP-HPLC.
Separation of 3 and 4 is facile using reverse-phase HPLC.
The combined yield of these pure olefinic products is ca.
45% from resin-bound material 2.
The geometry of the double bond could be determined by
NMR spectrometry using multiple decoupling experiments
in which all methylene protons adjacent to the olefin protons
in 3 or 4 (assigned by gCOSY) are simultaneously irradiated
using two frequencies. This allows the coupling constants
of the AB quartet formed by the two olefin protons in 3 and
4 to be determined. The JAB for 3 (cis isomer) is 10.9 Hz,
whereas the corresponding JAB value for 4 (trans isomer) is
15.9 Hz.
Hydrogenation of a mixture of 3 and 4 using a variety of
conditions selectively reduces 3 to bis-methylene analogue
5 without altering 4. Initial attempts at reducing the cis/trans
mixture of free cyclic peptides after acidic cleavage but
without Fmoc removal using N,N-dimethylacetamide (DMAc)
with H2 at 1 atm gave only starting material. Attempted
reduction using in situ diimide production (triisopropyl
benzenesulfonyl hydrazide (TPSH) and base)7,16 yielded fully
deprotected peptides with selective hydrogenation of 3 to
5.17 Purified cis isomer 3 is soluble in ethanol and easily
hydrogenated in quantitative yield (Scheme 3).
Acknowledgment. We thank Dr. T. Nakashima for
assistance with simultaneous double-decoupling experiments.
These investigations were supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC),
Boehringer Ingelheim Canada, Ltd., Bio ChemPharma Shire,
Ltd., and the Canada Research Chair in Bioorganic and
Medicinal Chemistry (J.C.V.).
Supporting Information Available: Characterization and
biological data for compounds 3-5. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL027160V
(18) Mitchell, B. F.; Fang, Xin; Wong, S. Biology of Reproduction 1998,
59 (6), 1321-1327.
(19) Blackwell, H. E.; Sadowsky, J. D.; Howard, R. J.; Sampson, J. N.;
Chao, J. A.; Steinmetz, W. E.; O’Leary, D. J.; Grubbs, R. H. J. Org. Chem.
2001, 66, 5291-5302.
(16) Cusack, N. J. Tetrahedron 1976, 32, 2157-2162.
(17) No “Fmoc” group was detectable by 1H NMR, and only the olefin
protons of trans olefinic isomer 6 were detected.
Org. Lett., Vol. 5, No. 1, 2003
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