Journal of Medicinal Chemistry
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compound i5b (450 mg, 1.5 mmol) was exposed to 3 M HCl/MeOH
(3.0 mL) for 2 h. A 340 mg amount of compound j5b was obtained
after purification with reverse phase HPLC (96%). 1H NMR (DMSO-
d6) δ 0.80−0.9 (m, 9H), 1.18−1.30 (m, 4H), 1.36−1.46 (m, 2H),
1.49−1.63 (m, 3H), 2.97−3.06 (m, 1H), 3.09−3.18 (m, 1H), 3.67 (t,
1H), 8.26 (brs, 3H), 8.59 (t, J = 5.2 Hz, 1H); m/z (LCMS, ESI):
found 201.1 [M + H]+, [C11H24N2O + H]+ requires 201.2.
13C NMR (D2O) δ 21.19, 21.55, 22.54, 22.69, 24.20, and 24.79 (6C,
2CH(CH3)2), 40.04 (d, JC−P = 543 Hz, 1C, CH2P), 41.22, 43.64, and
43.71 (2C, 2CH2CH(CH3)2), 54.00 and 54.32 (2C, 2CHCO), 67.32
(1C, PhCH2O−), 128.01, 128.60, 129.02, and 136.66 (6C, Ph), 158.41
(1C, Cbz CO), 177.44 and 179.96 (2C, 2CO), 31P NMR (D2O)
δ 17.45; m/z (LCMS, ESI): found 470.3 [M − 2Li + H]−;
[C21H32Li2N3O7P − 2Li + H]− requires 470.2.
(S)-2-Amino-N-isopentyl-4-methylpentanamide Hydrochloride
(j9b). Following the general procedure for Boc deprotection,
compound i9b (450 mg, 1.5 mmol) was exposed to 3 M HCl/
MeOH (3.0 mL) for 3 h. A 340 mg amount of compound j9b was
obtained after purification with reverse phase HPLC (96%). 1H NMR
(DMSO-d6) δ 0.82−0.92 (m, 12H), 1.28−1.38 (m, 2H), 1.53−1.66
(m, 4H), 3.02−3.10 (m, 1H), 3.12−3.20 (m, 1H), 3.68−3.76 (m, 1H),
8.38 (brs, 3H), 8.75 (t, J = 5.5 Hz, 1H); m/z (LCMS, ESI): found
223.1 [M + Na]+, [C11H24N2O + Na]+ requires 223.2.
General Procedure for the Synthesis of Compounds 1a−12a and
1b−12b. To a cooled solution of compound 4 (1 equiv), any of
compounds j1a−j12a or j1b−j12b (1.2−1.5 equiv), and PyBop (1.2
equiv) in anhydrous DCM was added diisopropylethylamine (4 equiv)
gradually. The reaction mixture was stirred at rt for 6 h to overnight.
The reaction mixture was then diluted with DCM up to 25 mL;
extracted with 5% citric acid (2 × 12 mL), saturated sodium
bicarbonate (2 × 12 mL), and brine (2 × 10 mL); and dried over
anhydrous sodium sulfate. The solvent was then evaporated under
reduced pressure, and the residue was purified by semipreparative
HPLC to give the intermediate that corresponds to the starting j1a−
j12a or j1b−j12b compound among k1a−k12a and k1b−k12b. This
intermediate was then hydrolyzed in the following manner: 0.2 mmol
of this intermediate was vigorously shaken at room temperature with
1−2 mL of 0.4 M LiOH aqueous solution until all the solid dissolves
(acetonitrile was used as a cosolvent whenever needed). The solution
was then stirred for 2− 24 h and concentrated under vacuum. The
final compound was then separated as a pure lithium or dilithium salt
using semipreparative reverse phase HPLC.
D i l i t h i u m ( ( S ) - 2 - ( ( N - ( B e n z y l o x y c a r b o n y l ) -
aminomethylphosphonyl)amino)-4-methylpentanoyl)-L-norleuci-
nate (5a). Following the general procedure for the synthesis of
compounds 5a, 9a, 5b, and 9b, compound 4 (215 mg, 0.83 mmol) was
reacted with compound j5a (295 mg, 1.0 mmol) in anhydrous DCM
(3.0 mL), using PyBop (520 mg, 1.0 mmol) as a coupling reagent and
diisopropylethylamine (428 mg, 3.3 mmol) as a base. A 281 mg
amount of compound k5a, which is the diester version of the desired
product, was obtained after purification with reverse phase HPLC
(68%). A 100 mg amount of this compound (0.2 mmol) was then
exposed to LiOH (2.0 mL of the 0.4 M solution referred to in the
general procedure; 2.0 mL of acetonitrile was used as a cosolvent)
overnight, and the final product was separated by reverse phase HPLC
as a pure white solid (91 mg, 95%). 1H NMR (D2O) δ 0.68−0.80 (m,
9H), 1.08−1.20 (m, 4H), 1.25−1.42 (m, 2H), 1.49−1.70 (m, 3H),
3.08−3.22 (m, 2H), 3.50−3.58 (m, 1H), 4.01 and 4.02 (2 d, J = 7.6
Hz, together 1H), 4.99 (s, 2H), 7.24−7.36 (m, 5H), 31P NMR (D2O)
δ 17.50; m/z (LCMS, ESI): found 478.3 [M − Li + 2H]+;
[C21H32Li2N3O7P − Li + 2H]+ requires 478.2.
(S)-2-((N-(Benzyloxycarbonyl)aminomethylphosphonyl)amino)-
4-methyl-N-pentylpentanamide Lithium (5b). Following the general
procedure for the synthesis of compounds 5a, 9a, 5b, and 9b,
compound 4 (215 mg, 0.83 mmol) was reacted with compound j5b
(237 mg, 1.0 mmol) in anhydrous DCM (3.0 mL), using PyBop (520
mg, 1.0 mmol) as a coupling reagent and diisopropylethylamine (428
mg, 3.3 mmol) as a base. A 274 mg amount of compound k5b, which
is the PO-methyl ester version of the desired product, was obtained
after purification with reverse phase HPLC (75%). A 88 mg amount of
this compound (0.2 mmol) was then exposed to LiOH (1.5 mL of the
0.4 M solution referred to in the general procedure; an additional 1.5
mL of acetonitrile was used as a cosolvent) overnight, and the final
product was separated by reverse phase HPLC as a pure white solid
(62 mg, 72%). 1H NMR (D2O) δ 0.68−0.78 (m, 9H), 1.05−1.10 (m,
4H), 1.22−1.40 (m, 4H), 1.45−1.55 (m, 1H), 2.98 (t, J = 6.4 Hz, 2H),
3.00−3.20 (m, 2H), 3.45−3.52 (m, 1H), 4.99 (s, 2H), 7.22−7.36 (m,
5H), 31P NMR (D2O) δ 17.29; m/z (LCMS, ESI): found 428.2 [M −
Li + 2H]+; [C20H33LiN3O5P − Li + 2H]+ requires 428.2.
(S)-2-((N-(Benzyloxycarbonyl)aminomethylphosphonyl)amino)-
N-isopentyl-4-methylpentanamide Lithium (9b). Following the
general procedure for the synthesis of compounds 5a, 9a, 5b, and
9b, compound 4 (215 mg, 0.83 mmol) was reacted with compound
j9b (237 mg, 1.0 mmol) in anhydrous DCM (3.0 mL), using PyBop
(520 mg, 1.0 mmol) as a coupling reagent and diisopropylethylamine
(428 mg, 3.3 mmol) as a base. A 245 mg amount of compound k9b,
which is the PO-methyl ester version of the desired product, was
obtained after purification with reverse phase HPLC (67%). A 88 mg
amount of this compound (0.2 mmol) was then exposed to LiOH (1.5
mL of the 0.4 M solution referred to in the general procedure; an
additional 1.5 mL of acetonitrile was used as a cosolvent) overnight,
and the final product was separated by reverse phase HPLC as a pure
white solid (78 mg, 90%). 1H NMR (CD3OD) δ 0.88−0.98 (m, 12H),
1.38−1.46 (m, 3H), 1.54−1.68 (m, 2H), 1.75−1.83 (m, 1H), 3.18−
3.32 (m, 4H), 3.68−3.74 (m, 1H), 5.10 (s, 2H), 7.28−7.42 (m, 5H),
13C NMR (CD3OD) δ 21.53, 21.86, 21.88, 22.54, 24.68, and 25.95
(6C, 2CH(CH3)2), 37.72 and 38.29 (2C, 2CH2CH(CH3)2), 40.73 (d,
JC−P = 543 Hz, 1C, CH2P), 44.22 and 44.29 (1C, HNCH2CH2), 54.51
(1C, CHCONH), 66.69 (1C, PhCH2O), 127.89, 128.01, 128.47, and
137.28 (6C, Ph), 158.0 (1C, Cbz CO), 177.05 (1C, CO), 31P
NMR (CD3OD) δ 15.75; m/z (LCMS, ESI): found 440.3 and 873.5
[M + Li]+ and [2 M + Li]+; [C20H33LiN3O5P + Li]+ requires 440.2.
ITC, X-ray Crystallography, and Molecular Modeling. Details
of the experimental procedures of ITC are provided in the Supporting
Information. The crystal structures of the complexes of thermolysin
with 1a, 2a, 3a, 4a, 6a, 8a, 9a, 11a, 1b, and 2b were previously
reported under PDB IDs 3T8G, 3T74, 3T87, 3T8C, 3T8H, 3T8D,
4H57, 4D9W, 3T73, and 3T8F. Molecular modeling experiments were
carried out on some of the ligands that do not have crystal structures
(e.g., 3b, 4b, 6b, 7a, and 7b). The purpose of these experiments was to
predict the potential binding modes of the P2′ side chains of these
ligands in the ligand−protein complexes. The steric compatibility, as
well as the interactions between a modeled P2′ side chain and the
hydration water structures observed in the crystallographic complexes
of ligands with smaller P2′ side chains, could then be investigated (e.g.,
the position of the side chain of 3b relative to the hydration waters
observed in the crystal structure of the 2b−TLN complex). All the
modeling experiments described herein were performed using SYBYL-
X software; Tripos Inc. Modeling experiments were performed
according to the following protocol:
D i l i t h i u m ( ( S ) - 2 - ( ( N - ( B e n z y l o x y c a r b o n y l ) -
aminomethylphosphonyl)amino)-4-methylpentanoyl)-L-leucinate
(9a). Following the general procedure for the synthesis of compounds
5a, 9a, 5b, and 9b, compound 4 (130 mg, 0.5 mmol) was reacted with
compound j9a (221 mg, 0.75 mmol) in anhydrous DCM (3.0 mL),
using PyBop (312 mg, 0.6 mmol) as a coupling reagent and
diisopropylethylamine (258 mg, 2.0 mmol) as a base. A 177 mg
amount of compound k9a, which is the diester version of the desired
product, was obtained after purification with reverse phase HPLC
(71%). A 100 mg amount of this compound (0.2 mmol) was then
exposed to LiOH (2.0 mL of the 0.4 M solution referred to in the
general procedure; an additional 2.0 mL of acetonitrile was used as a
cosolvent) for 10 h, and the final product was separated by reverse
Either the crystal structure, for which the relative position of the
modeled side chain was to be compared with (e.g., that of ligand 2b;
PDB ID: 3T8F), or a model system of the active site constructed
based on this crystal structure was used for the modeling experiment.
1
phase HPLC as a pure white solid (78 mg, 81%). H NMR (D2O) δ
0.66−0.78 (m, 12H), 1.23−1.58 (m, 6H), 3.04−3.20 (m, 2H), 3.49−
3.57 (m, 1H), 4.02−4.08 (m, 1H), 4.97 (s, 2H), 7.20−7.34 (m, 5H);
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dx.doi.org/10.1021/jm401609a | J. Med. Chem. 2014, 57, 2315−2333