344
Y. Jiang et al. / European Journal of Medicinal Chemistry 143 (2018) 334e347
DMSO-d6):
d
172.8, 171.2, 169.7, 157.9, 149.6, 141.1, 136.8, 129.9,
HRMS (AP-ESI) m/z calcd for C27H38FN5O7 [M þ H]þ 564.2828,
found 564.2830. Retention time: 15.6 min, eluted with 25% acetoni-
trile/75% water (containing 0.1% triethylamine and 0.15% tri-
fluoroacetic acid).
129.6, 129.1, 127.4, 71.3, 68.7, 60.2, 54.6, 51.5, 41.5, 35.1, 24.5, 23.3,
22.4. HRMS (AP-ESI) m/z calcd for C23H30FN5O7 [M þ H]þ 508.2202,
found 508.2200. Retention time: 6.3 min, eluted with 25% acetoni-
trile/75% water (containing 0.1% triethylamine and 0.15% tri-
fluoroacetic acid).
5.1.7. (6R,7S,10S)-ethyl-6-benzyl-10-isobutyl-2,2-dimethyl-4,8,11-
trioxo-7-((tetrahy-dro-2H-pyran-2-yl)oxy)-3-oxa-5,9,12-
triazatetradecan-14-oate(13)
Compounds 12b-12e were synthesized using the similar
method to 12a.
10 (1.21 g, 2.45 mmol) and HOBt (0.46 g, 3.4 mmol) were dis-
solved in 100 mL of dry DCM under ice bath followed by the
addition of EDCI (0.65 g, 3.4 mmol) and stirring for 30 min at room
temperature. Glycine ethyl ester hydrochloride (0.47 g, 3.4 mmol)
and TEA (0.48 mL) dissolved in 30 mL of dry acetonitrile were
added into the solution directly. The mixture was stirred for 3 h at
room temperature. DCM was evaporated with the residue being
taken up in EtOAc (40 mL). The EtOAc solution was washed with 1 N
aqueous citric acid, saturated NaHCO3 and brine for 3 times, dried
with anhydrous sodium sulfate and evaporated under vacumm. The
residue was purified by chromatography using EtOAcePE (1:2) as
mobile phase to obtain 0.69 g of colorless oil 13 (0.69 g, yield: 49%).
ESI-MS m/z:578.6 (M þ H)þ.
5.1.6.2. (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
m e t h y l - 2 - ( ( S ) - 2 - ( ( 2 S , 3 R ) - 3 - a m i n o - 2 - h y d r o x y - 4 -
phenylbutanamido)-4-methylpentanamido)propanoate hydrochlo-
ride (12b). White solid, 0.8 g, 88% yield. Mp: 111e112 ꢀC. 1H
NMR(400 MHz DMSO-d6):
d 0.85e0.91 (m, 6H), 1.25e1.28 (m, 3H),
1.43e1.51 (m, 2H),1.60e1.63 (m, 1H), 2.86e2.96 (m, 2H), 3.53 (s,
1H), 3.99e4.19 (m, 1H), 4.24e4.37 (m, 2H), 5.54e5.64 (m, 2H), 6.70
(s,1H), 7.25e7.36 (m, 5H), 7.99e8.08 (m, 4H), 8.15 (d, J ¼ 6.8 Hz,1H),
8.63 (d, J ¼ 6.7 Hz, 1H), 12.02 (d, J ¼ 5.0 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6):
d 172.3, 172.2, 171.0, 157.9, 149.5, 141.1, 136.8, 129.9,
129.6, 129.1, 127.4, 71.4, 68.6, 60.2, 54.7, 51.2, 48.0, 35.1, 24.5, 23.3,
22.4, 16.9. HRMS (AP-ESI) m/z calcd for C24H32FN5O7 [M þ H]þ
522.2359, found 522.2354. Retention time: 7.1 min, eluted with 25%
acetoni-trile/75% water (containing 0.1% triethylamine and 0.15%
trifluoroacetic acid).
5.1.8. (6R,7S,10S)-6-benzyl-10-isobutyl-2,2-dimethyl-4,8,11-trioxo-
7-((tetrahydro-2H -pyran-2-yl)oxy)-3-oxa-5,9,12-triazatetr-
adecan-14-oic acid(14)
5.1.6.3. (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-
3-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-
methylpentanamido)propanoate hydr-ochloride (12c). White solid,
0.6 g, 85% yield. Mp: 119e120 ꢀC. 1H NMR(400 MHz DMSO-d6):
13 (0.69 g, 1.2 mmol) was dissolved in 20 mL of methanol fol-
lowed by the addition of 3 N NaOH (10 mL) and stirring for 2 h at
35 ꢀC. After removing methanol under vacuum, the solution was
acidified to pH 3 with 1 N HCl solution. The solution was washed
with 15 mL of acetic ether for 3 times. The organic layer was
collected and dried with anhydrous sodium sulfate over night. After
evaporating the solvent, the residue was dried under vacuum to get
colorless oil 14 (0.51 g, yield: 78%), ESI-MS m/z: 550.7 (M þ H)þ.
d
0.84e0.88 (m, 6H), 1.40e1.60 (m, 3H), 2.47e2.49 (m, 2H),
2.87e2.97 (m, 2H), 3.21e3.29 (m, 2H), 4.00e4.05 (m, 2H),
4.18e4.27 (m, 1H), 5.55e5.58 (m, 2H), 6.72 (s, 1H), 7.25e7.36 (m,
5H), 7.92e8.14 (m, 5H), 8.27e8.29 (m, 1H), 11.98 (d, J ¼ 4.9 Hz, 1H).
13C NMR (100 MHz, DMSO-d6):
d 172.2, 171.4, 171.1, 157.9, 149.7,
141.0, 136.8,129.9,129.7, 129.1, 127.4, 70.9, 68.7, 60.2, 54.7, 51.8, 41.3,
35.1, 33.9, 24.7, 23.3, 22.4. HRMS (AP-ESI) m/z calcd for C24H32FN5O7
[M þ H]þ 522.2359, found 522.2359. Retention time: 6.6 min,
eluted with 25% acetoni-trile/75% water (containing 0.1% triethyl-
amine and 0.15% trifluoroacetic acid).
5.1.9. 2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-
phenylbutanamido)-4-methylpentan-amido)-acetic acid (15)
Compound 14 (0.51 g, 0.94 mmol) was dissolved in a solution of
EtOAc (20 mL) saturated by dry HCl gas. The solution was stirred at
room temperature for 3 h. The filtered precipitate was washed by
diethyl ether to give compound 15, a white solid powder (0.33 g,
yield: 87%). Mp: 84e86 ꢀC. 1H NMR(400 MHz DMSO-d6):
5.1.6.4. (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-
4-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-
methylpentanamido)butanoate hydr-ochloride (12d). White solid,
0.7 g, 87% yield. Mp: 117e118 ꢀC. 1H NMR(400 MHz DMSO-d6):
d
0.84e0.90 (m, 6H), 1.51e1.58 (m, 2H), 1.62e1.67 (m, 1H),
2.86e2.98 (m, 2H), 3.56 (s, 1H), 3.68e3.77 (m, 2H), 4.01e4.04 (m,
1H), 4.30e4.36 (m, 1H), 6.71 (S, 1H), 7.27e7.37 (m, 5H), 8.03e8.08
(m, 4H), 8.42 (t, J ¼ 5.7 Hz, 1H), 12.52 (s, 1H). 13C NMR (100 MHz,
d
0.85e0.90 (m, 6H), 1.46e1.65 (m, 5H), 2.32e2.36 (m, 2H),
2.88e2.97 (m, 2H), 3.02e3.07 (m, 2H), 4.00e4.06 (m, 2H),
4.19e4.25 (m, 1H), 5.56 (s, 2H), 6.71 (s, 1H), 7.25e7.36 (m, 5H),
DMSO-d6): d 172.5, 171.4, 171.1, 136.8, 129.9, 129.6, 129.1, 127.4, 68.7,
60.2, 54.7, 51.5, 41.5, 35.1, 24.6, 23.4, 22.4. HRMS (AP-ESI) m/z calcd
for C23H30FN5O7 [M þ H]þ 366.2023, found 366.2022. Retention
time: 4.6 min, eluted with 25% acetoni-trile/75% water (containing
0.1% triethylamine and 0.15% trifluoroacetic acid).
8.00e8.08 (m, 4H), 8.12e8.18 (m, 2H), 11.98 (d, J ¼ 5.0 Hz, 1H). 13
C
NMR (100 MHz, DMSO-d6):
d 172.8, 172.1, 171.1, 157.9, 149.7, 141.1,
136.8, 129.9, 129.8, 129.1, 127.4, 70.9, 68.8, 60.2, 54.7, 51.8, 41.4, 38.1,
35.1, 30.9, 24.7, 23.3, 22.4. HRMS (AP-ESI) m/z calcd for C25H34FN5O7
[M þ H]þ 536.2515, found 536.2515. Retention time: 8.1 min, eluted
with 25% acetoni-trile/75% water (containing 0.1% triethylamine
and 0.15% trifluoroacetic acid).
5.2. CD13 inhibition assay [23]
The inhibitory activity of synthetic compounds towards CD13
was determined using -leucine-p-nitroanilide as substrate and
L
5.1.6.5. (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-
6-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-
methylpentanamido)hexanoate hydr-ochloride (12e). White solid,
0.7 g, 82% yield. Mp: 98e99 ꢀC. 1H NMR(400 MHz DMSO-d6):
microsomal aminopeptidase from porcine kidney microsomes
(Sigma) as the enzyme. In brief, the assay was performed in 96-well
plates in 50 mM PBS, pH 7.2 as the assay buffer, at 37 ꢀC. At the
beginning, the CD13 solution was added to compounds solutions at
various concentrations and incubated at 37 ꢀC for 5 min. Subse-
quently, the substrate was added and the mixture was incubated for
30 min at 37 ꢀC. Finally, the hydrolysis of the substrate was
measured by following the change in the absorbance measured at
405 nm with a Micro-plate Reader (Thermo Fisher, Shanghai,
China).
d
0.86e0.89 (m, 6H),1.16e1.24 (m, 2H),1.34e1.38 (m, 2H),1.44e1.58
(m, 5H), 2.28e2.34 (m, 2H), 2.87e3.09 (m, 4H), 4.00e4.06 (m, 2H),
4.20e4.26 (m, 1H), 6.67 (s, 1H), 7.25e7.41 (m, 5H), 7.97e8.19 (m,
6H), 12.01 (d, J ¼ 5.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d 172.9,
171.9, 170.9, 157.9, 149.7, 141.1, 136.8, 129.9, 129.8, 129.1, 127.4, 70.9,
68.7, 60.2, 54.7, 51.9, 41.5, 38.8, 35.1, 33.5, 28.9, 26.1, 24.7, 23.3, 22.5.