544
M. Y. H. Lai et al. / Bioorg. Med. Chem. 13 (2005) 533–548
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl,
97%) (0.13g, 0.50mmol) was added and the solution
stirred for 19h, then washed successively with 10% aque-
ous hydrochloric acid (20mL) and saturated aqueous
sodium hydrogen carbonate (20mL). The organic
extract was dried (MgSO4), filtered and evaporated to
dryness in vacuo. Purification of the resultant residue
by flash column chromatography (60% ethyl acetate–
hexane) yielded fully protected tripeptide (0.20g, 75%)
as a colourless oil that existed exclusively as the trans
conformer with 6% epimerization as observed by 13C
NMR analysis: [a]D +1.14 (c 4.38, CH2Cl2); mmax
(film)/cmÀ1 3317, 3033, 2944, 2858, 1737, 1699, 1648,
1535, 1498, 1454, 1391, 1279, 1255, 1199, 1168, 1093,
1034, 973, 736 and 698; dH (400MHz; CDCl3) 1.21-
1.35 (3H, m, 3 · cyclohexyl-H), 1.60–1.76 (6H, m,
Proc-H2 and 4 · cyclohexyl-H), 1.82–1.89 (2H, m,
Prob-HAHB and 1 · cyclohexyl-H), 1.98–2.07 (3H,
m, Prob-HAHB and 2 · cyclohexyl-H), 2.12–2.32 (2H,
m, Glub-H2), 2.40–2.56 (2H, m, Gluc-H2), 3.15–3.21
(1H, m, Prod-HAHB), 3.52–3.57 (1H, m, Prod-HAHB),
4.43–4.49 (1H, m, Glua-H), 4.58 (1H, dd, J 8.2 and
5.7, Proa-H), 4.90–5.20 (7H, m, NH and 3 · OCH2Ph),
7.27–7.38 (15H, m, 3 · Ph) and 7.57 (1H, d, J 7.2, NH);
dC (100MHz; CDCl3) 21.1 (CH2, cyclohexyl-C), 21.3
(CH2, cyclohexyl-C), 24.8 (CH2, cyclohexyl-C), 25.8
(CH2, Proc-C), 26.3 (CH2, Glub-C), 26.4ꢂ (CH2, Glub-
C), 28.0 (CH2, Prob-C), 28.6ꢂ (CH2, Prob-C), 29.7ꢂ
(CH2, Gluc-C), 30.1 (CH2, Gluc-C), 30.6ꢂ (CH2, cyclo-
hexyl-C), 31.1ꢂ (CH2, cyclohexyl-C), 31.4 (CH2, cyclo-
hexyl-C), 31.8 (CH2, cyclohexyl-C), 47.9 (CH2, Prod-
C), 48.1ꢂ (CH2, Prod-C), 51.6ꢂ (CH, Glua-C), 51.8
(CH, Glua-C), 59.3 (quat., Glya-C), 62.4 (CH, Proa-
C), 62.8ꢂ (CH, Proa-C), 66.0ꢂ (CH2, OCH2Ph), 66.2
(CH2, OCH2Ph), 66.7ꢂ (CH2, OCH2Ph), 66.9 (CH2,
OCH2Ph), 67.1 (CH2, OCH2Ph), 67.4ꢂ (CH2, OCH2Ph),
127.9ꢂ (CH, Ph), 128.1 (CH, Ph), 128.3 (CH, Ph), 128.5
(CH, Ph), 128.6 (CH, Ph), 128.7 (CH, Ph), 128.9ꢂ (CH,
Ph), 135.8 (quat., Ph), 135.9 (quat., Ph), 136.0 (quat.,
Ph), 154.8 (quat., NCO2), 171.1 (quat., Glua-CO),
172.0 (quat., Gly-CO), 172.2 (quat., Pro-CO) and
172.8 (quat., Gluc-CO); m/z (FAB+) 684.3291 (MH+,
C39H46N3O8 requires 684.3285).
(1:1)]; dH (400MHz; D2O) 1.35–1.58 (3H, m, 3 · cyclo-
hexyl-H), 1.80–1.94 (6H, m, Prob-HAHB and 5 · cyclo-
hexyl-H) 2.00–2.11 (3H, m, Proc-H2 and Glub-HAHB),
2.21–2.37 (4H, m, Prob-HAHB, Glub-HAHB and
2 · cyclohexyl-H), 2.57 (2H, t, J 7.2, Gluc-H2), 3.83–
4.05 (2H, m, Prod-H2), 4.44 (1H, dd, J 9.0 and 5.3,
Glua-H) and 4.52–4.56 (1H, m Proa-H); dC (100MHz;
D2O) 22.3 (2 · CH2, cyclohexyl-C), 25.7 (CH2, cyclo-
hexyl-C), 28.0 (CH2, Proc-C), 28.2 (CH2, Glub-C),
30.5 (CH2, Prob-C), 31.0 (CH2, cyclohexyl-C), 31.2
(CH2, cyclohexyl-C), 32.3 (CH2, Gluc-C), 52.2 (CH2,
Prod-C), 54.4 (CH, Glua-C), 64.7 (quat., Glya-C), 65.3
(CH, Proa-C), 118.8 (quat., q, J 290.0, CF3CO2H),
165.3 (quat., q, J 35.0, CF3CO2H), 172.6 (quat.,
Gly-CO), 176.9 (quat., Pro-CO), 177.3 (quat.,
Glua-CO) and 179.5 (quat., Gluc-CO); m/z
(FAB+) 370.1983 [M(freebase)H+, C17H28N3O6 requires
370.1978].
4.26. N-Benzyloxycarbonyl-N-methylglycine53 (31)
Following procedure A, protection of sarcosine in
7:2 H2O–dioxane afforded crude carbamate 31
(5.00g, 100%) as a light green oil: dH (200MHz;
CD3OD): 3.01 (3H, s, CH3N), 4.09 (2H, s, Glya-H2),
5.16 (2H, s, OCH2Ph), 5.75 (1H, br s, CO2H) and 7.36
(5H, s, Ph); dC (50MHz; CD3OD) 35.8ꢁ (CH3, CH3N),
36.4 (CH3, CH3N), 51.1ꢁ (CH2, CH2CO2H), 51.3
(CH2, Glya-C), 68.5 (CH2, OCH2Ph), 68.6 (CH2,
OCH2Ph), 128.6 (CH, Ph), 128.8 (CH, Ph) 128.9 (CH,
Ph), 129.5 (CH, Ph), 137.9 (quat., Ph) 158.5 (quat.,
NCO2) and 172.9 (quat., CO2H); m/z (EI+) 223 (M+,
5%).
4.27. Dibenzyl-N-benzyloxycarbonyl-N-methylglycyl-L-
prolyl-L-glutamate (33)
Following procedure B, coupling of amine31 2 with car-
bamate 31 using triethylamine as the base yielded fully
protected tripeptide 33 (0.174g, 47%) as a clear gum fol-
lowing purification by flash column chromatography
(ethyl acetate): [a]D À47.2 (c 0.05, MeOH); dH
(200MHz; CDCl3; Me4Si) 1.90–2.44 (8H, m, Prob-H2,
Proc-H2, Glub-H2 and Gluc-H2,), 2.95 (3H, s, CH3N),
3.40–3.47 (2H, m, Prod-H2), 3.76–3.86 (1H, m, Gly-
NCHAHB), 4.15–4.23 (1H, m, Gly-NCHAHB), 4.54–
4.57 (1H, m, Proa-H), 4.57–4.59 (1H, m, Glua-H),
5.06 (2H, s, OCH2Ph), 5.09 (2H, s, OCH2Ph), 5.27
(2H, s, OCH2Ph) and 7.25–7.33 (15H, m, 3 · Ph); dC
(50MHz; CDCl3) 25.0 (CH2, Proc-C), 27.0 (CH2,
Glub-C), 27.4 (CH2, Prob-C), 30.0 (CH2, Gluc-C),
35.6 (CH3, CH3N), 36.1ꢁ (CH3, CH3N), 46.4 (CH2,
Prod-C), 51.3 (CH2, Glya-C), 51.7 (CH, Glua-C),
60.0 (CH, Proa-C), 66.3 (CH2, OCH2Ph), 67.0 (CH2,
OCH2Ph), 67.3 (CH2, OCH2Ph), 127.7 (CH, Ph),
127.9 (CH, Ph), 128.1 (CH, Ph), 128.4 (CH, Ph),
128.5 (CH, Ph), 135.0 (quat., Ph), 156.9 (quat.,
NCO2), 168.3 (quat., Gly-CO), 171.0 (quat., Glu-CO),
171.3 (quat., Glu-CO) and 172.4 (quat., Pro-CON);
m/z (EI+) 629.2726 (M+, C35H39N3O8 requires
629.2737).
4.25. Cyclohexylglycyl-L-prolyl-L-glutamic acid trifluoro-
acetate salt (14)
Following procedure C, hydrogenation of the above
amide in 3:2 ethyl acetate–trifluoroacetic acid yielded
analogue 14 (23mg, 35%) as a colourless hygroscopic
gummy oil following purification by C18 RP HPLC
[90% water (0.05% trifluoroacetic acid)–10% acetonit-
rile, 13mLminÀ1]. Compound 14 existed exclusively as
the trans conformer with 6% epimerization as observed
by HPLC analysis:§ [a]D À34.1 [c 0.18, MeOH–H2O
ꢂ Denotes resonances assigned to epimer.
§ Analytical reverse-phase HPLC studies on the mixture [Altech
Econosphere C18 Si column, 150 · 4.6mm, 5mm; 5min flush with
H2O (0.05% TFA) then steady gradient over 25min to MeCN as
eluent at flow rate of 1cm3/min; detection using diode array] indicated
it was a 94:6 mixture of two eluting peaks with retention times of
13.53 and 14.13min at 210nm wavelength, respectively.