3172 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Mavunkel et al.
The crude material, obtained from lyophilization, was
purified by reverse phase high-performance liquid chromatog-
raphy, using a linear gradient of 5-60% water/acetonitrile
(both containing 0.2% acetic acid), on a C18 Vydac (22.5 mm
i.d. × 25 cm, 10 µm, 300 Å) column, at room temperature. The
homogeneous pooled pure fractions were lyophilized and the
purity of the white fluffy powders determined by analytical
reverse phase HPLC, using a linear gradient of 5-80% water/
acetonitrile (both containing 0.1% TFA), on a C18 Vydac (4.6
mm i.d. × 25 cm, 10 µm, 300 Å) column, at room temperature,
and fast atom bombardment mass spectroscopy.
wt %, 0.54 g, 14.42 mmol) was washed with hexane (3 × 5
mL), suspended in DMF (20 mL), and cooled to 0 °C.
A
solution of 5f (4.20 g, 11.68 mmol) in DMF (20 mL) was added
dropwise. After the mixture was stirred at 0 °C for 30 min,
methyl bromoacetate (2.24 g, 14.42 mmol) was added and the
mixture stirred at room temperature 3 h. After diluting with
water, the mixture was extracted with EtOAc (5 × 50 mL).
The organic extracts were washed with water (3 × 50 mL) and
saturated aqueous sodium chloride and dried over Na2SO4.
After filtration and removal of the solvent in vacuo, the residue
was chromatographed (10-50% EtOAc/hexane, gradient) to
give 4.42 g (88%) of 6f as a colorless oil: 1H NMR (CDCl3) δ
1.44 (s, 9H), 1.52-1.75 (m, 4H), 2.68-2.85 (m, 4H), 3.35-3.55
(m, 2H), 3.76 (s, 3H), 3.80-4.04 (m, 2H), 4.10 (s, 2H), 4.28 (s,
2H), 7.16-7.35 (m, 5H). Anal. (C23H33N3O5) C, H, N.
f. 1-(2-P h en yleth yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e-3-a cetic Acid (7f). A mixture of 6f (4.27 g, 9.78 mmol),
Na2CO3 (4.50 g, excess), MeOH (75 mL), and water (25 mL)
was refluxed for 3 h. After the MeOH was removed in vacuo,
the resulting aqueous solution was cooled to 0 °C and carefully
acidified (pH 4) with concentrated HCl. The product was
extracted with EtOAc (3 × 100 mL); these organic extracts
were washed with saturated aqueous sodium chloride, dried
over Na2SO4, and filtered; and the solvent was removed in
vacuo to give 3.80 g (93%) of 7f as a white foam: 1H NMR
(CDCl3) δ 1.44 (s, 9H), 1.64-1.80 (m, 2H), 1.80-1.92 (m, 2H),
2.82-2.96 (m, 4H), 3.27-3.62 (m, 2H), 3.80-4.22 (m, 6H), 4.46
(br s, 2H), 7.15-7.35 (m, 5H). Anal. (C22H31N3O5‚0.75H2O)
C, H, N.
P r ep a r a t ion of 8-Boc-1,3,8-t r ia za sp ir o[4.5]d eca n -4-
on e-3-a cetic Acid s (7). a . 1-Ben zyl-4-cya n o-4-[(2-p h en -
yleth yl)a m in o]p ip er id in e (2f). To a stirred mixture of
1-benzyl-4-piperidone (1) (37.87 g, 200 mmol), 2-phenylethyl-
amine (27.16 g, 224 mmol), and acetic acid (100 mL) at room
temperature was added dropwise a solution of potassium
cyanide (16.10 g, 247 mmol) in water (35 mL). After stirring
at room temperature for 48 h, the reaction mixture was poured
into a mixture of concentrated NH4OH (200 mL) and crushed
ice (200 g). The mixture was extracted with CHCl3 (5 × 100
mL). The organic extracts were washed with saturated sodium
chloride, dried over Na2SO4, and filtered, and the solvent was
removed in vacuo. Recrystallization from diisopropyl ether
1
gave 32.37 g (51%) of 2f as white crystals: mp 65-68 °C; H
NMR (CDCl3) δ 1.25 (br s, 1H), 1.73 (t, 2H, J ) 13.5 Hz), 1.98
(d, 2H, J ) 12.6 Hz), 2.34 (t, 2H, J ) 11.0 Hz), 2.70-2.90 (m,
2H), 2.82 (t, 2H, J ) 6.9 Hz), 3.01 (q, 2H, J ) 6.9 Hz), 3.53 (s,
2H), 7.18-7.35 (m, 10H). Anal. (C21H25N3) C, H, N.
b. 1-Ben zyl-4-[(2-p h en yleth yl)a m in o]p ip er id in e-4-ca r -
boxa m id e (3f). To 90% H2SO4 (200 mL) was added 2f (32.00
g, 100 mmol) and the mixture stirred for 20 h. After this
mixture was cautiously poured into a mixture of concentrated
NH4OH (750 mL) and crushed ice (750 g), the resulting
alkaline mixture was extracted with CHCl3 (5 × 100 mL). The
organic extracts were washed with saturated sodium chloride,
dried over Na2SO4, and filtered, and the solvent was removed
in vacuo. Recrystallization from 2-propanol gave 27.03 g (80%)
of 3f as an off-white powder: mp 89-92 °C; 1H NMR (CDCl3)
δ 1.57 (d, 2H, J ) 11.3 Hz), 1.95-2.06 (m, 2H), 2.06-2.17 (m,
2H), 2.62-2.80 (m, 6H), 3.42 (s, 2H), 5.29 (s, 1H), 6.85 (s, 1H),
7.16-7.35 (m, 10H). Anal. (C21H27N3O) C, H, N.
1-P h en yl-8-Boc-1,3,8-tr iazaspir o[4.5]decan -4-on e-3-ace-
tic a cid (7a ): mp 200-201 °C; 1H NMR (CDCl3) δ 1.50 (s, 9H),
1.63-1.83 (m, 2H), 2.38-2.56 (m, 2H), 3.43-3.70 (m, 2H),
3.90-4.15 (m, 2H), 4.23 (s, 2H), 4.79 (s, 2H), 6.68-7.05 (m,
3H), 7.20-7.35 (m, 2H). Anal. (C20H27N3O5) C, H, N.
1-(4-Meth ylp h en yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
1
4-on e-3-a cetic a cid (7b): mp 173-175 °C; H NMR (CDCl3)
δ 1.45 (s, 9H), 1.64-1.84 (m, 2H), 2.20-2.40 (m, 5H), 3.42-
3.60 (m, 2H), 3.88-4.10 (m, 2H), 4.20 (s, 2H), 4.75 (s, 2H), 6.78
(d, 2H, J ) 11.4 Hz), 7.08 (d, 2H, J ) 11.4 Hz). Anal.
(C21H29N3O5) C, H, N.
1-(Cycloh exylm eth yl)-8-Boc-1,3,8-tr iazaspir o[4.5]decan -
1
4-on e-3-a cetic a cid (7c): mp 148-149 °C; H NMR (CDCl3)
c. 1-(2-P h en yleth yl)-8-ben zyl-1,3,8-tr ia za sp ir o[4.5]d ec-
2-en -4-on e (4f). A mixture of 3f (11.80 g, 34.97 mmol),
triethyl orthoformate (23 mL, excess), acetic acid (5 mL), and
toluene (75 mL) was refluxed for 20 h. The mixture was cooled
to room temperature and diluted with water (100 mL) and
CHCl3 (100 mL). The mixture was made basic with aqueous
sodium hydroxide, the layers were separated, and the aqueous
layer was extracted with CHCl3 (3 × 50 mL). The combined
organic extracts were dried over Na2SO4 and filtered, and the
solvent was removed in vacuo. Chromatography (1-15%
MeOH/CH2Cl2, gradient) gave 8.79 g (73%) of 4f as a pale
yellow oil: 1H NMR (CDCl3) δ 1.57 (d, 2H, J ) 12.5 Hz), 1.90-
2.22 (m, 2H), 2.79 (br s, 2H), 3.07 (br s, 2H), 3.61 (t, 2H, J )
7.1 Hz), 3.68 (br s, 2H), 7.15 (d, 2H, J ) 6.9 Hz), 7.20-7.42
(m, 8H), 7.80 (s, 1H). Anal. (C22H25N3O‚0.5H2O) C, H, N.
d . 1-(2-P h en yleth yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e (5f). A mixture of 4f (8.75 g, 25.22 mmol), MeOH (150
mL), and concentrated HCl (30 mL) was hydrogenated at 45
psi of H2 in the presence of 5% Pd/C (10 g) for 4 days. The
catalyst was filtered off and the MeOH removed in vacuo. The
remaining acidic aqueous solution was made basic by the
addition of solid Na2CO3. This alkaline mixture was diluted
with 1,4-dioxane (150 mL) and cooled to 0 °C, and di-tert-butyl
dicarbonate (10.90 g, 50.0 mmol) was added. After stirring
at room temperature for 20 h, the reaction mixture was diluted
with water (250 mL) and extracted with ether (5 × 100 mL).
The organic extracts were dried over Na2SO4 and filtered, and
the solvent was removed in vacuo. Chromatography (1-5%
MeOH/CH2Cl2, gradient) gave 8.47 g (93%) of 5f as white
crystals: mp 138-140 °C; 1H NMR (CDCl3) δ 1.44 (s, 9H),
1.53-1.70 (m, 4H), 2.68-2.84 (m, 4H), 3.30-3.55 (m, 2H),
3.78-4.05 (m, 2H), 4.21 (s, 2H), 6.68 (br s, 1H), 7.10-7.34 (m,
5H). Anal. (C20H29N3O3) C, H, N.
δ 0.75-0.90 (m, 2H), 1.10-1.30 (m, 4H), 1.50 (s, 9H), 1.60-
1.80 (m, 8H), 2.30 (d, 2H, J ) 7.2 Hz), 3.40-3.50 (m, 2H),
3.80-4.00 (m, 2H), 4.05-4.15 (m, 2H), 4.20 (s, 2H). Anal.
(C21H35N3O5) C, H, N.
1-n -P r op yl-8-Boc-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e-3-
a cetic a cid (7d ): mp 180-183 °C dec; 1H NMR (CDCl3) δ 0.93
(t, 3H, J ) 7.1 Hz), 1.46 (s, 9H), 1.50-1.90 (m, 6H), 2.55 (t,
2H, J ) 6.5 Hz), 3.30-3.67 (m, 2H), 3.95-4.20 (m, 2H), 4.08
(br d, 2H, J ) 7.2 Hz), 4.31 (brs, 2H). Anal. (C17H29N3O5) C,
H, N.
1-Cycloh exyl-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e-
3-a cetic a cid (7e): mp 172-173 °C; 1H NMR (CDCl3) δ 1.20-
1.42 (m, 8H), 1.47 (s, 9H), 1.58-1.85 (m, 6H), 2.64-2.74 (m,
1H), 3.42-3.70 (m, 2H), 3.70-3.95 (m, 2H), 3.95-4.15 (m, 2H),
4.39 (s, 2H). Anal. (C20H33N3O5) C, H, N.
B. Com p u ta tion a l P r oced u r es. All modeling work was
done using the programs CHARMm and Quanta13 on Silicon
Graphics computers. Energy minimizations were terminated
when the drop in energy after 50 steps of minimization was
less than 0.2 kcal mol-1
. The spirocyclic mimetic has no
rotatable bonds of structural consequence; hence no confor-
mational search was performed. The structure was optimized
using second-derivative minimization techniques. The com-
parative modeling entailed achieving an optimal end to end
distance wherein the N- and C-terminal heteroatoms of the
spirocyclic mimetic could closely match the corresponding
heteroatoms of Pro2 and Phe5. Moreover, the hydrophobic side
chains of Phe5 and Pro3 were accounted for in the mimetic by
a bulky substituent at the 1-position of the spirocycle. These
features are represented in Figure 1 where the thick bonds
correspond to the mimetic, the thin bonds are of the cyclic
peptide (D-Arg-Arg-Cys-Pro-Gly-Phe-Cys-D-Tic-Oic-Arg), and
the Pro3, Phe5 hydrophobic domain is shown as dark gray
spheres.
e. 1-(2-P h en yleth yl)-8-Boc-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e-3-a cetic Acid Meth yl Ester (6f). Sodium hydride (60