Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5869
2.76-2.83 (m, 1H), 1.27 (d, 3H). 13C NMR (75 MHz, CDCl3):
δ 173.7, 52.0, 42.1, 18.1, 6.8.
131.6, 127.1, 79.1, 32.6, 19.9, 16.6. HRMS (ESI) m/e calcd for
C13H17O3NNa [M + Na]+ 258.1106; obsd, 258.1088.
A solution of (R)-methyl 3-iodo-2-methylpropanoate (6.60 g,
28.9 mmol) in anhydrous benzene (96 mL) and DMA (6.4 mL)
was added to a dry nitrogen-purged flask charged with
zinc-copper couple (3.47 g). The resulting mixture was soni-
cated under nitrogen for 40 min. Bis(tri-o-tolylphosphine)
palladium dichloride (1.12 g, 1.28 mmol) was added followed
by 11 (8.82 g, 24.1mmol). The resulting mixture was stirred
under nitrogen at 55 °C for 2 h and then allowed to cool down
to room temperature. Ethyl acetate (400 mL) was added and the
mixture filtered into a separating funnel. The mixture was washed
with aqueous hydrochloric acid (0.1 N) (400 mL) and distilled
water (3 × 100 mL), and dried, filtered, and concentrated. The
crude product was purified by flash chromatography to afford
Peptide Synthesis. (2S)-Mdcp-c[D-Cys-Gly-Phe(pNO2)-D-
Cys]NH2 (1). The cyclic tetrapeptide H-c[D-Cys-Gly-Phe(pNO2)-
D-Cys]NH2 was prepared by the manual solid-phase technique
as described elsewhere.7 To a solution of (2S)-Mdcp (15) (37.7
mg, 0.16 mmol), HBTU (62.7 mg, 0.16 mmol), and N,N-
diisopropylethylamine (DIEA) (91.0 µL, 0.82 mmol) in 5 mL
DMF were added H-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 × TFA
(90.0 mg, 0.15 mmol) and NMM (16.5 µL, 0.15 mmol). After
stirring for 30 min, the solvent was evaporated to dryness in
vacuo, and the residue was extracted with 20 mL of AcOEt.
After washing with 5% KHSO4, saturated NaHCO3, and brine,
the organic phase was dried (MgSO4), filtered, and evaporated
to dryness in vacuo. The peptide was purified by reversed-phase
HPLC. HPLC K′ 3.75; TLC Rf 0.65 (I), Rf 0.86 (II), Rf 0.16
(III); MS [M + H]+ 688.
(2S)-Mdcp-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH (3). The linear
precursor peptide was prepared by the manual solid-phase technique
using Boc-protection of the R-amino group and Mob protection of
the Pen residues and DIC/HOBt as coupling agents. The peptide was
assembled on a polystyrene-divinylbenzene (1%) resin (200-400
mesh) (Boc-Phe-resin, 0.65 equiv/g, Bachem Bioscience, King of
Prussia, PA) according to a published protocol.3 The peptide was
cleaved from the resin and deprotected by HF/anisole treatment in the
usual manner. After evaporation of the HF, the resin was extracted
three times with Et2O and subsequently three times with glacial AcOH.
The peptide was obtained in solid form through lyophilization of the
acetic acid extract. For disulfide bond formation, the peptide (150 mg)
dissolved in 20 mL MeOH was slowly added to a solution of
K3Fe(CN)6 (293 mg) in 750 mL ammonium acetate buffer (0.05N,
pH 8.5) over a period of 20 h. After lowering the pH to 4.5 by addition
of AcOH, the solution was treated with Amberlite IRA-400 (Cl). After
subsequent filtration, solvent evaporation, and dissolution of the residue
in AcOH, the product was obtained in solid form through lyophilization
and was purified by preparative reversed-phase HPLC. HPLC K′ 5.83;
TLC Rf 0.88 (I), Rf 0.81 (II), Rf 0.30 (III), MS [M + H]+ 865.
[(2S)-Mdcp1]Dyn A(1-11)-NH2 (5). The peptide was prepared
by the manual solid-phase technique by using the protocol described
above for the synthesis of the linear precursor peptide of 3. Side
chain protection was as follows: tosyl (Arg) and 2-chlorobenzy-
loxycarbonyl (Lys). HPLC K′ 3.78; TLC Rf 0.38 (II), Rf 0.32 (IV);
MS [M + H]+ 1414.
20
the desired product 12 as a light-yellow oil (4.27 g, 52%). [R]D
+39.5° (c 0.22, CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.71 (s,
2H), 7.36-7.43 (m, 7H), 5.34 (s, 2H), 3.63 (s, 3H), 3.05-3.12
(a, 1H), 2.71-2.86 (m, 2H), 2.36 (s, 6H), 1.14 (d, 3H, J ) 6.9
Hz). 13C NMR (75 MHz, CDCl3) 176.5, 166.6, 141.9, 137.1,
136.2, 129.4, 128.5, 128.19, 129.17, 127.7, 66.5, 51.7, 39.1, 33.2,
20.2, 16.5. HRMS (ESI) m/e calcd for C21H24O4Na [M + Na]+
363.1573; obsd, 363.1573.
(S)-4-(3-Methoxy-2-methyl-3-oxopropyl)-3,5-dimethylbenzoic acid
(13). To a solution of 12 (4.1 g, 12.1 mmol) in MeOH (20 mL),
10% Pd/C (240 mg) was added, and the mixture was stirred
overnight under atmospheric pressure of H2. The reaction mixture
was then filtered through a short pad of celite, and the filter
cake was washed with EtOAC (2 × 20 mL). The filtrate and
washings were combined and concentrated in vacuo to give 13
as a white solid (2.9 g, 96%); mp 128.4-129.9 °C. [R]D20 +57.3°
(c 0.20, CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.78 (s, 2H),
3.68 (s, 3H), 3.11-3.18 (q, 1H), 2.77-2.92 (m, 2H), 2.42 (s,
6H), 1.20 (d, 3H, J ) 6.72 Hz). 13C NMR (75 MHz, CDCl3) δ
176.5, 172.1, 142.8, 137.2, 129.9, 126.9, 51.7, 39.0, 33.2, 20.2,
16.5. ΗRMS (ESI) m/e calcd for C14H17O4 [M - H]- 249.1205;
obsd, 249.1126.
(S)-Methyl 3-(4-Carbamoyl-2,6-dimethylphenyl)-2-methyl-
propanoate (14). Oxalyl chloride (2.76 mL, 31.7 mmol) was
added slowly to a solution of 13 (2.64 g, 10.6 mmol) in CH2Cl2
(20 mL) at room temperature under nitrogen atmosphere, and
slow gas formation was observed. DMF (20 µL) was then added,
accelerating gas evolution considerably. After stirring at room
temperature for 2 h, organic solvent was removed, and the brown
oily residue was dissolved in THF (20 mL). Aqueous NH4OH
(25%) (23 mL) was then added to the reaction mixture at 0 °C
and stirred at the same temperature for 30 min. The reaction
mixture was then acidified with HCl (1 N) at 0 °C, and extracted
with EtOAc (2 × 50 mL). After concentration, the residue was
purified by flash chromatography to give 14 as a white solid
(2.3 g, 87%); mp 91.8-93.2 °C; [R]D20 +58.5° (c 0.22, CHCl3).
1H NMR (300 MHz, CDCl3) δ 7.44 (s, 2H), 6.0 (br s, 1H), 5.76
(br s, 1H), 3.63 (s, 3H), 3.05-3.10 (q, 1H), 2.74-2.85 (m, 2H),
2.36 (s, 6H), 1.15 (d, 3H, J ) 6.75 Hz). 13C NMR (75 MHz,
CDCl3) δ 176.5, 169.5, 140.8, 137.3, 130.8, 127.1, 51.7, 39.1,
33.1, 20.3, 16.5. ΗRMS (ESI) m/e calcd for C14H19O3NNa [M
+ Na]+ 272.1263; obsd, 272.1256.
Acknowledgment. This work was financially supported by
the National University of Singapore (to Y.L.) and by a grant
from the U.S. National Institutes of Health (to P.W.S.).
Supporting Information Available: Experimental details and
refs 20-26. This material is available free of charge via the Internet
References
(1) Hansen, D. W., Jr.; Stapelfeld, A.; Savage, M. A.; Reichman, M.;
Hammond, D. L.; Haaseth, R. C.; Mosberg, H. I. Systemic Analgesic
Activity and δ-Opioid Selectivity in [2,6-Dimethyl-Tyr1,D-Pen2,D-
Pen5]Enkephalin. J. Med. Chem. 1992, 35, 684–687.
(2) Schiller, P. W.; Lu, Y.; Weltrowska, G.; Berezowska, I.; Wilkes, B. C.;
Nguyen, T. M.-D.; Chung, N. N.; Lemieux, C. A General New Concept
for the Development of Opioid Peptide Derived µ-, δ-and κ Antago-
nists. In Peptides: The WaVe of the Future, Proceedings of the 2nd
International Peptide Symposium/17th American Peptide Symposium;
Lebl, M., Houghten, R. A., Eds.; Kluwer Academic Publishers:
Dordrecht, The Netherlands, 2001; pp 676-678.
(3) Lu, Y.; Nguyen, T. M.-D.; Weltrowska, G.; Berezowska, I.; Lemieux,
C.; Chung, N. N.; Schiller, P. W. [2′,6′-Dimethyltyrosine]dynorphin
A(1-11)-NH2 Analogues Lacking an N-Terminal Amino Group:
Potent and Selective κ Opioid Antagonists. J. Med. Chem. 2001, 44,
3048–3053.
(4) Schiller, P. W.; Weltrowska, G.; Nguyen, T.M.-D.; Lemieux, C.;
Chung, N. N.; Lu, Y. Conversion of δ-, κ- and µ-Receptor Selective
Opioid Peptide Agonists into δ-, κ- and µ-Selective Antagonists. Life
Sci. 2003, 73, 691–698.
(2S)-2-Methyl-3-(2,6-dimethyl-4-carbamoylphenyl)propanoic Acid
[(2S)-Mdcp] (15). To a solution of 14 (2.03 g, 8.16 mmol) in
THF (50 mL) was added an aqueous solution of LiOH (1N, 50
mL) at 0 °C. After stirring at this temperature for 2.5 h, the
organic solvent was removed and the aqueous phase was
neutralized with precooled HCl (1 N) at 0 °C, and extracted
with EtOAc (2 × 75 mL). The combined EtOAc extracts were
washed with brine, dried over Na2SO4, and concentrated to yield
20
15 as a white solid (1.5 g, 78%); mp 224.1-225.9 °C. [R]D
+
64.2 ° (c 0.25, MeOH); 1H NMR (300 MHz, DMSO-d6) δ 12.17
(br s, 1H), 7.80 (s, 1H), 7.51 (s, 2H), 7.18 (s, 1H), 2.96-3.03
(q, 1H), 2.50-2.74 (m, 2H), 2.32 (s, 6H), 1.04 (d, 3H, J ) 6.72
Hz). 13C NMR (75 MHz, CDCl3) δ 177.0, 167.9, 139.8, 136.3,