A. Stefanucci et al.
were added dropwise to a solution of the Boc-amino acid
(200 mg) or Lonidamine (200 mg, 0.62 mmol) in THF
(10 mL) stirring at −20 °C for 30 min. NH4OH solution
(2.1 eq., 0.37 mL) was added to the reaction stirring at
−20 °C for 30 min, then the reaction was maintained at r.t.
overnight. The reaction mixture was evaporated to dryness
and the residue extracted with EtOAc. The organic phase
was washed with 5% citric acid, NaHCO3 s.s. and NaCl s.s.,
dried on Na2SO4 anhydrous and evaporated to obtain the
desired amide derivative as a crude white product. This pro-
cedure was also applied to the synthesis of LONI B deriva-
tive previously characterized in the literature (Angapelly
Boc-L-tert-Leu-OH has been converted to its amide deriva-
tive following the general procedure D. The Boc protec-
tion has been removed with a mixture of TFA/DCM =1:1
at r.t. for 1 h, then the intermediate TFA salt was coupled
with Lonidamine, following the general procedure B. The
crude product was chromatographed on silica gel column
using EtOAc/n-hexane from 20:80 to 40:60 as eluent, to
give LONI 3 as sole product. Rf = 0.11 (EtOAc/n-hexane
1
3:7), overall yield 90%, HPLC tR = 19.95 min. H NMR
(300 MHz, DMSO-d6) δ 8.18 (d, 1H, H5′), 7.73 (d, 1H,
H4), 7.71, 7.26 (brs, 2H, NH2),7.69 (s, 1H, H3′), 7.54 (d,
1H, NH), 7.46 (t, 1H, H5), 7.30–7.36 (m, 2H, H6 and H6′),
5.84 (s, 2H, H1), 4.41–4.45 (m, 1H, CHα L-tert-Leu), 0.95
(s, 9H, 3×CH3 L-tert-Leu). 13C NMR (300 MHz, DMSO-d6)
δ 172.12, 161.24, 141.55, 137.90, 133.76, 133.52, 130.82,
129.57, 128.24, 127.72, 123.38, 122.52, 122.28, 110.99,
59.15, 50.16, 35.00, 27.06. LRMS calcd. for C21H22Cl2N4O2:
432.1: found: 455.2 [M+Na]+.
( S ) - 2 - ( 1 - ( 2 , 4 - d i ch l o ro b e n z y l ) - 1 H - i n d a z o l e -
3-carboxamido)-3,3-dimethylbutanoic acid (LONI 1).
Boc-tert-Leu-OH was transformed in its methyl ester deriv-
ative following the general procedure A. The so-obtained
product was coupled with Lonidamine following the gen-
eral procedure B, to give an intermediate product (LONI
2), which was converted in LONI 1, following the general
procedure C; overall yield 30%, HPLC tR=20.69 min. 1H
NMR (300 MHz, DMSO-d6) δ 8.17 (d, 1H, H5’), 7.74 (d,
1H, H4), 7.70 (s, 1H, H3’), 7.59 (d, 1H, NH), 7.47 (t, 1H,
H5), 7.28–7.37 (m, 2H, H6 and H6’), 6.84 (d, 1H, H7), 5.85
(s, 2H, H1), 4.29–4.32 (m, 1H, CHα L-tert-Leu), 0.98 (s,
9H, 3×CH3 3 L-tert-Leu). 13C NMR (300 MHz, DMSO-d6)
δ 174.04, 161.52, 141.51, 137.50, 133.55, 133.43, 130.90,
129.57, 128.26, 127.75, 123.42, 122.51, 122.22, 111.02,
108.87, 101.85, 60.25, 50.16, 34.69, 27.11. LRMS calcd.
for C21H21Cl2N3O3: 433.1; found: 456.1 [M+Na]+.
(S)-2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-
3-methylbutanoic acid (LONI 4). Boc-Val-OH was trans-
formed in its methyl ester derivative following the general
procedure A. The so-obtained product was coupled with
Lonidamine following the general procedure B, to give an
intermediate product (LONI 5), which was converted in
LONI 4 following the general procedure C; overall yield
1
88%, HPLC tR = 20.17 min. H NMR (300 MHz, DMSO-
d6) δ 8.17 (d, 1H, H5′), 7.86 (d, 1H, H4), 7.75 (d, 1H, NH),
7.70 (s, 1H, H3′), 7.47 (t, 1H, H5), 7.28–7.36 (m, 2H, H6
and H6′), 6.79 (d, 1H, H7) 5.85 (s, 2H, H1), 4.35–4.39
(m, 1H, CHα Val), 2.20–2.22 (m, 1H, CH(CH3)2), 0.94
(t, 6H, 2×CH3 2 Val). 13C NMR (300 MHz, DMSO-d6) δ
173.25, 161.94, 141.50, 137.90, 133.83, 133.72, 133.40,
130.68, 129.54, 128.27, 127.74, 123.38, 122.61, 122.25,
110.94, 57.39, 50.12, 30.50, 19.60, 18.72. LRMS calcd. for
C20H19Cl2N3O3: 419.0; found: 442.1 [M+Na]+.
(S)-methyl 2-(1-(2,4-dichlorobenzyl)-1H-indazole-
3-carboxamido)-3,3-dimethylbutanoate (LONI 2): Boc-
tert-Leu-OH was transformed in its methyl ester derivative
following the general procedure A. The so-obtained product
was coupled with Lonidamine following the general pro-
cedure B, to give LONI 2 as crude material. The residue
was chromatographed on silica gel column using EtOAc/n-
hexane = 20:80 as eluent. Rf = 0.68 (EtOAc/n-hexane
30:70), overall yield 25%, HPLC tR =22.24 min. 1H-NMR
(300 MHz, DMSO-d6) δ 8.13 (d, 1H, H5’), 7.76 (d, 1H, H4),
7.70 (s, 1H, H3′), 7.65 (d, 1H, NH), 7.31–7.37 (m, 2H, H6
and H6′), 6.87 (d, 1H, H7), 5.85 (s, 2H, H1), 4.43–4.47 (m,
1H, CHα L-tert-Leu), 3.66 (s, 3H, OCH3 L-tert Leu), 0.98
(s, 9H, 3×CH3 L-tert-Leu). 13C NMR (300 MHz, DMSO-d6)
δ 171.71, 161.76, 141.50, 137.50, 133.81, 133.70, 133.58,
130.99, 129.57, 128.26, 127.80, 123.51, 122.49, 122.09,
111.06, 59.94, 52.28, 50.18, 34.66, 26.85. LRMS calcd. for
C22H23Cl2N3O3: 447.1; found: 470.2 [M+Na]+.
(S)-methyl 2-(1-(2,4-dichlorobenzyl)-1H-indazole-
3-carboxamido)-3-methylbutanoate (LONI 5). Boc-Val-OH
was transformed in its methyl ester derivative following the
general procedure A. The so-obtained product was coupled
with Lonidamine following the general procedure B to give
the crude product which was chromatographed on silica gel
column using EtOAc/n-hexane from 20:80 to 30:70 as elu-
ent. LONI 5 was obtained in 76% overall yield, Rf = 0.67
1
(EtOAc/n-hexane 30:70), HPLC tR = 21.64 min. H NMR
(300 MHz, DMSO-d6) δ 8.14-8.18 (m, 2H, H5′ and H4),
7.74 (d, 1H, NH), 7.70 (s, 1H, H3′), 7.47 (t, 1H, H5), 7.28–
7.37 (m, 2H, H6 and H6′), 6.77 (d, 1H, H7), 5.84 (s, 2H,
H1), 4.36–4.40 (m, 1H, CHα Val), 3.65 (s, 3H, OCH3 Val),
2.21-2.25 (m, 1H, CH(CH3)2), 0.90 (dd, 6H, 2×CH3 2 Val).
13C NMR (300 MHz, DMSO-d6) δ 172.36, 162.16, 141.48,
137.87, 133.78, 133.73, 133.37, 130.65, 129.49, 128.24,
(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(2,4-d
ichlorobenzyl)-1H-indazole-3-carboxamide (LONI 3).
1 3