Synthesis and biological evaluation of new simple indolic non peptidic HIV Protease inhibitors: The effect of different substitution patterns Dedicated to CINMPIS on the occasion of its 20th anniversary.

Article abstract of DOI:10.1016/j.bmc.2014.06.055

New structurally simple indolic non peptidic HIV Protease inhibitors were synthesized from (S)-glycidol by regioselective methods. Following the concept of targeting the protein backbone, different substitution patterns were introduced onto the common stereodefined isopropanolamine core modifying the type of functional group on the indole, the position of the functional group on the indole and the type of the nitrogen containing group (sulfonamides or perhydroisoquinoline), alternatively. The systematic study on in vitro inhibition activity of such compounds confirmed the general beneficial effect of the 5-indolyl substituents in presence of arylsulfonamide moieties, which furnished activities in the micromolar range. Preliminary docking analysis allowed to identify several key features of the binding mode of such compounds to the protease.

Full text of DOI:10.1016/j.bmc.2014.06.055

Bioorganic & Medicinal Chemistry 22 (2014) 4792–4802
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new simple indolic non
peptidic HIV Protease inhibitors: The effect of different substitution
patterns
a
a
a
a
a
b
C. Bonini a, , L. Chiummiento , N. Di Blasio , M. Funicello , P. Lupattelli , F. Tramutola , F. Berti ,
⇑
A. Ostric b, S. Miertus c,d, V. Frecer c,e, D.-X. Kong f,g
a Dipartimento di Scienze, Università degli studi della Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy
b Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, via Giorgieri 1, 34127 Trieste, Italy
c International Centre for Applied Research and Sustainable Technology, Bratislava SK-8410, Slovakia
d University of SS. Cyril & Methodius, Faculty of Natural Sciences, Trnava, Slovakia
e Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, Bratislava SK-83232, Slovakia
f ICS-UNIDO, Area Science Park, Trieste, Italy
g State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e i n f o
a b s t r a c t
Article history:
New structurally simple indolic non peptidic HIV Protease inhibitors were synthesized from (S)-glycidol
by regioselective methods. Following the concept of targeting the protein backbone, different substitu-
tion patterns were introduced onto the common stereodefined isopropanolamine core modifying the type
of functional group on the indole, the position of the functional group on the indole and the type of the
nitrogen containing group (sulfonamides or perhydroisoquinoline), alternatively. The systematic study
on in vitro inhibition activity of such compounds confirmed the general beneficial effect of the 5-indolyl
substituents in presence of arylsulfonamide moieties, which furnished activities in the micromolar range.
Preliminary docking analysis allowed to identify several key features of the binding mode of such com-
pounds to the protease.
Received 29 April 2014
Revised 25 June 2014
Accepted 30 June 2014
Available online 4 July 2014
Dedicated to CINMPIS on the occasion of its
20th anniversary.
Keywords:
Ó 2014 Published by Elsevier Ltd.
HIV PR inhibitors
Non-peptidic inhibitors
Indoles
1. Introduction
led to the preparation of specific PIs, whose arrival was a pivotal
moment in the development of antiretroviral therapy and made
The devastating effect of the AIDS epidemic is still a reality.
However, since the highly active antiretroviral therapy (HAART)
has been employed to combat the illness, HIV infection has defi-
nitely become more manageable.1 During the last 20 years an
unprecedented success has been achieved in discovering anti-HIV
drugs as reflected by the fact that there are now more drugs
approved for the treatment of HIV than for all other viral infections
taken together.
The currently FDA approved anti-HIV drugs can be divided into
seven groups: nucleoside reverse transcriptase inhibitors, nucleo-
tide reverse transcriptase inhibitors, non-nucleoside reverse trans-
criptase inhibitors, protease inhibitors (PIs), fusion inhibitors,
co-receptor inhibitors, and integrase inhibitors.2 Detailed
knowledge of the structure of HIV protease and its substrate has
possible the dual class triple combination therapy.3 Despite the
already marketed PIs have an evident crucial role into HAART reg-
imen, their clinical utility can be limited by low bioavailability and
reduced long-term viral inhibition, with multiple protease resis-
tance mutations being observed. Thus, novel PIs with high potency
against the known HIV protease variants have been designed. We
recently demonstrated the beneficial effect of a heteroaromatic
group in a series of new thienyl ring containing analogues of nelfi-
navir and saquinavir, which showed to maintain or even increase
their activity against either wild type or mutant HIV protease.4
Recently the concept of targeting the protein backbone in struc-
ture-based drug design was introduced. Thus new non-peptidic
templates, which can maximize interactions in the HIV-protease
active site, particularly with the enzyme backbone atoms, were
developed.5 Both extensive hydrogen bonding and hydrophobic
interactions with enzyme subsites can limit the protease ability
to acquire drug resistance as the geometry of the catalytic site
must be conserved to maintain functionality.6 This new concept
⇑
Corresponding author. Tel.: +39 0971205495; fax: +39 0971205503.
E-mail address: carloc.bonini@libero.it (C. Bonini).
http://dx.doi.org/10.1016/j.bmc.2014.06.055
0968-0896/Ó 2014 Published by Elsevier Ltd.

C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
4793
R1
N
electrophile (S)-glycidol 1 for the generation of the core (Scheme 1).
The glycidol was first activated by the reaction with m-nosyl chlo-
ride to obtain compound 2. The subsequent nucleophilic displace-
ment of the nosyl group was performed by the commercially
available 4-, 5-, or 6-hydroxyindoles affording the corresponding
oxyindoles 3, 4 and 5 in good yields and mild reaction conditions,
without any competitive epoxide ring opening.8 Each oxyindole
represents the common precursor for the preparation of either per-
hydroisoquinyl- or sulfonamidyl derivatives. Indeed, perhydroiso-
quinoline (PHIQ) was introduced by direct regioselective oxiranyl
ring opening, obtaining the corresponding 6, 7 and 8 in good yield
and as single diastereoisomers (>57% overall yield in 3 steps).
For the preparation of arylsulfonamidyl compounds, the oxira-
nyl-oxyindoles 3, 4 and 5 were reacted with isobutylamine, afford-
ing the amino-derivatives 9, 10 and 11 in excellent yield. Starting
from this triad, arylsulfonyl fragments with different electronic
properties were introduced by employing suitable chlorides,
affording compounds 12a,b, 13a,b and 14a,b (>46% overall yield
in 4 steps). In particular, electron releasing 3,4-dimethoxyphenyl-
and electron withdrawing 4-nitrophenyl groups were introduced
to evaluate their potentially different effect on inhibitory activity.
Compound 13a was also transformed into 4-aminophenyl deriva-
tive 13c by Pd-catalyzed hydrogenation.
OH
Y
R2
N
H
A
Figure 1.
allowed to design different compounds of very simple structure
and to focus the interest more toward their easy synthetic avail-
ability and less to structural similarity. Our preliminary investiga-
tion showed the beneficial effect of indolyl ring on a simple
substituted stereodefined isopropanolamine core.7 In this respect,
with the aim of finding new easily accessible non-peptidic PIs,
we started a systematic study on the synthesis and inhibition
activity of new indolyl derivatives with general structure A
(Fig. 1), modifying the following parameters:
(1) Type of functional group on the indole.
(2) Position of the functional group on the indole.
(3) Type of the nitrogen containing group R1 and R2 (sulfona-
mides or PHIQ).
All these oxyindoles were tested in vitro for anti HIV-PR activity
and the results are reported in Table 1.
2. Results and discussion
This systematic study confirms the trend observed in our preli-
minary results on oxyindoles.7b In general 5-oxyindoles show bet-
ter activity than the 4- and 6-substituted ones and this difference is
Although the synthetic work can appear tedious at a first sight,
we took advantage of the commercially available bidentate
NO
2
O
K CO Het-OH
NsCl, Et N
2
3,
3
HO
O
O
S
CH Cl -10°C
DMF, rt
2
2,
80%
1
O
2
2
H
N
O
O
PHIQ
i-PrOH, rt
OH
H
O
N
H
O
NH
N
H
3; 4-oxyindole 63%
4; 5-oxyindole 72%
5; 6-oxyindole 61%
6; 4-oxyindole 91%
7; 5-oxyindole 87%
8; 6-oxyindole 93%
i-BuNH
2
i-PrOH, rt
OH
OH
O
O
NH
N
ArSO Cl, Et N
2
3
SO Ar
2
N
H
CH Cl rt
2
2,
N
H
76-94%
9; 4-oxyindole 99%
10; 5-oxyindole 99%
11; 6-oxyindole
12; 4-oxyindole;
a; Ar=4-NO -C H ; b; Ar=3,4-diMeO-C H
2
6
4
6
4
4
4
13; 5-oxyindole;
a; Ar=4-NO -C H ; b; Ar=3,4-diMeO-C H
2
6
4
6
14; 6-oxyindole;
a; Ar=4-NO -C H ; b; Ar=3,4-diMeO-C H
2
6
4
6
NH
2
OH
H
Pd/C 10%
O
N
2,
S
13a
O
O
EtOAc
74%
N
H
13c
Scheme 1.

4794
C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
Table 1
Regioisomer
PHIQ (IC50
,
lM)
4-NO2C6H4SO2NR (4-NH2C6H4SO2NR) (IC50
,
lM)
3,4-diMeOC6H4SO2NR (IC50, lM)
1
OH
R
N
O
2
R
HN
6 (24)
7 (14)
12a (346)
12b (336)
13b (1)
4-oxyindole
OH R1
N
13a (10)
13c (12)
O
R2
N
H
5-oxyindole
OH
O
R1
N
R2
8 (138)
14a (46)
14b (346)
NH
6-oxyindole
IC50 values were obtained by measuring the initial rates of hydrolysis of the fluorogenic substrate Abz-Thr-Ile-Nle-Phe(NO2)-Gln-Arg. Results are the mean of at least three
independent experiments.
derivatives 15 and 16 were recently prepared starting from avail-
H
N
O
H
N
O
able 4- and 5-aminoindoles and (S)-glycidol, by a slight modifica-
OH
OH
tion of our synthetic approach (Fig. 2).7b Their potency was
H
N
H
H
H
N
HN
disappointing, the IC50 values ranging between 150 and 180 lM.
Thus, we wanted to prepare and test the corresponding 4-nitro-
and 3,4-dimethoxyphenyl-sulphonamidyl derivatives. (S)-glycidol
was regioselectively opened with i-BuNH2, affording the aminodiol
17 (Scheme 2).
N
H
NH
NH
16; IC50 = 176 µM
15; IC50 = 157 µM
Figure 2.
Then the suitable arylsulfonyl moiety was introduced, namely
4-nitro- and 3,4-dimethoxyphenylsulfonyl groups, in high yield
giving 18a and 18b. After regioselective tosylation of primary
hydroxyl group, the two epoxides 19a and 19b were alternatively
prepared, and they were finally opened with 4- and 5-aminoin-
doles, affording the four new derivatives 22a,b and 23a,b (>41%
overall yield in 5 steps). In vitro tests of these aminoindolyl com-
pounds against wild type HIV-1 PR are reported in Table 2.
These results pointed out the general negative effect of amino
group on the inhibition activity of indole derivatives, whatever
the position of the nitrogen on the heterocycle. The introduction
of sulfonamidyl groups instead of PHIQ even reduced the activity
of the resulting compounds.
highly amplified by the presence of sulfonamidyl groups, no matter
which substituent is placed on the aryl ring. Promising IC50 value of
1.04 lM was obtained for compound 13b, suggesting a beneficial
effect of the two methoxy groups on the aryl ring. This is in line
with the reported data for derivatives of Darunavir in which the
aniline group is replaced by 1,3-dioxolane group.9 The two oxygen
atoms are thought to interact with the protease by two direct
hydrogen bonds and a water-mediated interaction with Gly 48,
which may stabilize the flexible flap region.
Next step of our study was to change the functionality on the
indole ring, so we focused our attention on amino indoles. PHIQ
OH
ArSO Cl
i-BuNH
O
2
2
OH
HO
HO
NH
HO
N
Ar
S
i-PrOH
Et N
3
1
O
17
2
18a; Ar = 4-NO -C H ; 74%
NH2
2
6 5
18b; Ar = 3,4-diOMe-C H ; 78%
6
5
N
H
20; 4-NH
21; 5-NH
1) TosCl
2) K CO
2
2
OH
O
H
N
N
N
Ar
2
Ar
S
S
2
3
O
O
67-77%
2
N
H
19a; Ar = 4-NO -C H ; 84%
2
6 5
19b; Ar = 3,4-diOMe-C H ; 81%
6
5
22; 4-aminoindole;
a; Ar = 4-NO -C H b; Ar = 3,4-diOMe-C H
2
6
5,
6
5
5
23; 5-aminoindole;
a; Ar = 4-NO -C H b; Ar = 3,4-diOMe-C H
2
6
5,
6
Scheme 2.

C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
4795
Table 2
Finally, we wanted to investigate the effect of carbamoyl moi-
ety, instead of amino group, considering its beneficial effect in cer-
tain non peptidic inhibitors, as Darunavir.10
The preparation of the corresponding carbamoyl derivatives of
4- and 5-indoles was straightforward. 4- and 5-aminoindoles 20
and 21 were first reacted with p-nitrophenylchlorocarbonate, to
afford the activated carbamates 24 and 25 (Scheme 3). Glycidol
was then introduced by substitution reaction, and the common
intermediates oxiranyl carbamates 26 and 27 were obtained in
good yield.
These intermediates were alternatively transformed into PHIQ
derivatives 28, 29 (>67% overall yield in 3 steps) and sulfonamides
32a,b, 33a,b,c (>63% overall yield in 4 steps) by oxiranyl ring open-
ing with PHIQ and iso-butylamine followed by sulfonylation,
respectively.
Regioisomer
4-NO2C6H4SO2NR
(IC50 M)
3,4-diMeOC6H4SO2NR
(IC50 M)
,
l
, l
1
OH
R
H
N
N
2
R
HN
22a (2630)
22b (336)
4-aminoindole
1
OH
R
H
N
N
2
R
23a (inactive)
23b (4400)
N
H
All these carbamates were tested and their inhibition activities
are listed in Table 3.
5-aminoindole
H
NH
2
N
O
ClOCO
+
Et N
3
O
CH Cl rt
2
2,
NO
NO
N
H
2
2
N
H
24; 4-carbamoyl
25; 5-carbamoyl
20; 4-NH
2
2
21; 5-NH
glycidol,
Et N
3
CH Cl rt
2
2,
H
N
O
90%
OH
O
H
N
O
H
H
O
N
PHIQ
O
O
NH
N
H
i-PrOH, rt
75%
N
H
28; 4-carbamoyl
29; 5-carbamoyl
26; 4-carbamoyl
27; 5-carbamoyl
i-BuNH
2
i-PrOH,
OH
rt, 90%
R
H
N
O
N
S
OH
H
N
ArSO Cl
2
O
2
O
NH
O
N
H
Et N, CH Cl rt
3
2
2,
O
N
H
32; 4-amino; a) R = 4-NO ; 87%
2
30; 4-carbamoyl
31; 5-carbamoyl
b) R = 3,4-diOMe; 85%
33; 5-amino; a) R = 4-NO ; 90%
2
b) R = 3,4-diOMe; 82%
c) R = 4-OMe; 77%
Scheme 3.
Table 3
Regioisomer
PHIQ (IC50
,
l
M)
4-NO2C6H4SO2NR (IC50
,
l
M)
3,4-diMeOC6H4SO2NR (IC50, lM)
1
OH
R
H
N
HN
O
N
2
R
28 (50)
32a (72)
32b (615)
O
OH R1
N
H
N
O
R2
33b (1400)
33c (4-OMe) (8)
O
29 (606)
33a (2)
N
H

4796
C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
Figure 3. 11Binding poses of some active compounds; HIV PR were presented with red/green ribbons or sticks. Darunavir was used as the reference and is presented as yellow
sticks. Ligands are shown as ball and sticks, coloured by atom types. (a) Crystal structure of darunavir with HIV PR. (b) Binding mode of 13b. (c) Binding mode of 33b. (d)
Binding mode of 33c.
Although the general positive effect on the activity of the sub-
stitution of amino group with carbamoyl one, usually ranging in
the micromolar values, the results appear difficult to rationalize.
PHIQ moiety contributes better on 4-indole derivative, whether
arylsulfonamide group matches with 5-indole one. Such a struc-
ture is very sensitive to the overall length of the molecule, depend-
ing on the linkage between the indole ring and the core of the
inhibitor, and requires a careful selection of the substituents on
the aryl system. Thus, the 3,4-dimethoxy aryl system is favourable
in compounds as 13b, but fails entirely in the corresponding carba-
mate 33b, with an extremely high IC50 of 1.4 mM. Conversely, the
nitroaryl system is effective in both the series of inhibitors. To
address this point, we have carried out a preliminary computa-
tional analysis by docking inhibitors 13, 23 and 33 inside the
proteases. Analysis of the docked poses shows that the activities
of the inhibitors are determined by the distance between the
hydrophobic part of the indole ring and the central carbon atom.
For one atom linkers, –O– or –N–, the indole ring lies mostly in
sub-site S1, rather than in sub-site S2. For a longer linker as the
carbamate one, the indole ring will lie in S2 but this forces the
arylsulfonamide side of the molecules to a deeper extent inside
S20 subsite (Fig. 3).
The nitro-(13a) or one of the two methoxy-groups (13b, Fig. 3b)
could establish favourable hydrogen bonding with the backbone
amide proton of Asp300. However, the second methoxy-group in
the larger 33b structure, clearly leads to unfavourable steric clash
(Fig. 3c). This prompted us to remove such a group as in 33c
(Fig. 3c), and this led to a three orders of magnitude gain in activity
(see Table 3).
3. Conclusion
In conclusion, our systematic study confirmed the possibility of
introducing indole ring in anti-HIV-1 PR inhibitors which are active
in the micromolar range. Although the net contribution of the indi-
vidual substituents cannot be fully rationalized, the following gen-
eral insights may be found:
(a) oxyindoles and carbamoyl indoles showed general good
activity, whereas simple aminoindoles are much less active,

C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
4797
(b) 5-substituted indoles appear to be the best regioisomers,
apparently they fit better into the S1 or S2 site of the enzyme,
depending on the linker length and distance from the core,
(c) arylsulfonamidyl moieties are in general beneficial for the
inhibition activity, but their effect is compound type-depen-
dent: 5-oxyindole matches with 3,4-diOMe-phenylsulfona-
mide group at best, whilst 5-carbamoyl indole matches
well with both 4-NO2- and 4-OMe-phenylsulfonamide moi-
ety, with a slight preference for the former,
(d) a preliminary docking analysis allowed to identify several
key features of the binding mode of our compounds to the
protease, prompting further developments of inhibitors with
improved activity.
4.04 (1H, dd, J = 6 and 11.0 Hz), 3.43–3.42 (1H, m), 2.94 (1H, t,
J = 4.5 Hz), 2.81 (1H, dd, J 3.0 and 5.5 Hz); dC (125 MHz, CDCl3)
153.0, 131.2, 128.1, 125.0, 112.8, 111.7, 103.8, 102.3, 69.6, 50.4,
44.9; MS (EI) m/z: 189 (M+) (100), 132 (80), 104 (54). Anal. Calcd
for C11H11NO2: C, 69.83; H, 5.86, N, 7.40. Found: C, 69.82; H,
5.87; N, 7.35.
4.2.3. (ꢀ)-(R)-6-Oxiranylmethoxy-1H-indole (5)
Compound 5 was isolated as a yellow solid (0.036 g, 61%). Mp
20
105 °C; [
a
]
ꢀ3.4 (c 1, CHCl3); Rf 0.4 (CH2Cl2/EtOAc 99:1); dH
D
(500 MHz, CDCl3) 8.15 (1H, s), 7.54 (1H, d, J = 11.0 Hz), 7.09 (1H,
s), 6.85 (2H, d, J = 6.5 Hz), 6.50 (1H, d, J = 1.0 Hz), 4.24 (1H, dd,
J = 1.5 and 13.5 Hz), 3.96 (1H, dd, J = 7.0 and 14.0 Hz), 3.39 (1H,
m), 2.93–2.78 (2H, m); dC (125 MHz, CDCl3) 155.1, 136.3, 123.4,
122.5, 121.2, 110.2, 102.2, 95.9, 69.3, 50.3, 44.7; MS (EI) m/z: 189
(M+) (100), 132 (80), 104 (54). Anal. Calcd for C11H11NO2: C,
69.83; H, 5.86; N, 7.40. Found: C, 69.82; H, 5.87; N, 7.38.
4. Experimental section
4.1. Chemistry
4.3. Ring opening of the epoxides with PHIQ: general procedure
Preparative chromatography was carried out on Merck silica gel
(0.063–0.200 mm particle size) by progressive elution with oppor-
tune solvent mixtures. 1H and 13C NMR spectra were normally car-
ried out in CDCl3 solutions on a VARIAN INOVA 500 MHz or Bruker
400 MHz and referenced to Me4Si. Mass spectra were obtained
with a Hewlett–Packard 5971 mass-selective detector on a Hew-
lett–Packard 5890 gas chromatograph [(OV-1 capillary column
between 70 and 250 °C (20 °C minꢀ1)]. The optical purity was eval-
uated by using a polarimeter JASCO Mod Dip-370. Dichlorometh-
ane was dried by distillation over anhydrous CaCl2 in inert
atmosphere. Dry dimethylformamide was commercially available.
Compound 2 was prepared according to the literature8a starting
from the commercially available (S)-(ꢀ)-glycidol 1.
PHIQ (0.043 g, 0.18 mmol) was added to a stirred solution of
suitable epoxide (0.15 mmol) in i-PrOH (2 mL) at room tempera-
ture. After 20 h the solvent was removed under reduced pressure
and the crude purified by column chromatography on silica gel
(CHCl3/CH3OH 95:5).
4.3.1. (ꢀ)-(3S,4aS,8aS)-2-[(2R)-Hydroxy-3-(1H-indol-4-yloxy)-
propyl]-decahydro-isoquinoline-3-carboxylic acid tert-
butylamide (6)
Compound 6 was isolated as a pink solid (0.061 g, 91%); mp
20
85 °C; [
a
]
ꢀ61.2 (c 1.5, CHCl3); Rf 0.4 (CHCl3/CH3OH 95:5); dH
D
(500 MHz, CDCl3) 8.51 (1H, s), 7.14–7.06 (3H, m), 6.65 (1H, d,
J = 2.5 Hz), 6.53 (1H, d, J = 7.5 Hz), 6.25 (1H, s, NH), 4.42
(1H, d, J = 9.0 Hz), 4.07 (1H, t, J = 8.0 Hz), 3.12 (1H, s, OH), 2.99
(1H, d, J = 7.5 Hz), 2.78 (1H, d, J = 8.0 Hz), 2.66 (1H, d, J = 8.0 Hz),
2.44 (1H, d, J = 7.5 Hz), 2.31 (1H, d, J = 8.0 Hz), 1.92–1.39 (9H, m),
1.34 (9H, s), 1.28–1.16 (4H, m); dC (125 MHz, CDCl3) 171.5, 152.1,
137.3, 122.8, 122.5, 118.6, 105.1, 100.7, 99.5, 70.4, 68.2, 59.5,
4.2. Nosyl displacement with hydroxyindoles: general
procedure
K2CO3 (0.1433 g, 1.04 mmol) was added to a stirred solution of
hydroxyindole (0.0461 g, 0.35 mmol) in dry DMF (4 mL) at room
temperature under argon atmosphere; after 1 h a DMF solution
(3 mL) of compound 2 (0.0815 g, 0.31 mmol) was added and the
mixture was stirred overnight. After 14 h (TLC control, CHCl3/CH3OH
99:1) the reaction mixture was quenched by adding ammonium
chloride (saturated aqueous solution), then was extracted with
diethyl ether and the organic layer washed with brine. After drying
over Na2SO4, the organic layer was concentrated in vacuo and the
crude was purified by column chromatography on silica gel (eluent:
CH2Cl2/EtOAc 99:1).
58.4, 50.6, 35.7, 33.1, 30.8, 30.6, 28.6, 26.1, 25.7, 20.4. IR (cmꢀ1
)
3311, 2925, 1652, 1365. Anal. Calcd for C25H37N3O3: C, 70.22; H,
8.72; N, 9.83. Found: C, 70.20; H, 8.73; N, 9.80.
4.3.2. (ꢀ)-(3S,4aS,8aS)-2-[(2R)-Hydroxy-3-(1H-indol-5-yloxy)-
propyl]-decahydro-isoquinoline-3-carboxylic acid tert-
butylamide (7)
Compound 7 was isolated as a yellow thick oil (0.063 g, 87%);
20
[a
]
ꢀ70.3 (c 2, CHCl3); Rf 0.4 (CHCl3/CH3OH 95:5); dH (500 MHz,
D
CDCl3) 8.45 (1H, s), 7.29 (1H, t, J = 9.0 Hz), 7.21 (1H, t, J = 2.5 Hz),
7.11 (1H, d, J = 2.5 Hz), 6.87 (1H, dd, J = 2.5 and 9.0 Hz), 6.47 (1H,
t, J = 2.5 Hz), 6.29 (1H, s, NH), 4.19 (1H, s, OH), 4.11 (1H, dd,
J = 4.0 and 9.5 Hz), 3.95 (1H, dd, J = 7.0 and 9.0 Hz), 3.01–2.22
(5H, m), 1.91–139 (10H, m), 1.35 (9H, s), 1.22–1.18 (3H, m); dC
(125 MHz, CDCl3) 173.9, 152.9, 131.3, 128.2, 125.1, 112.6, 111.8,
103.7, 102.7, 71.2, 70.3, 68.3, 59.4, 58.6, 50.7, 35.7, 35.7, 33.1,
30.8, 30.6, 29.6, 28.6, 26.1, 25.7, 20.5. IR (cmꢀ1) 3311, 2925,
1652, 1365. Anal. Calcd for C25H37N3O3: C, 70.22; H, 8.72; N,
9.83. Found: C, 70.19; H, 8.76; N, 9.85.
4.2.1. (ꢀ)-(R)-4-Oxiranylmethoxy-1H-indole (3)
Compound 3 was isolated as a brown thick oil (0.038 g, 63%).
20
[
a]
ꢀ6.4 (c 1.6, CHCl3); Rf 0.5 (CH2Cl2/EtOAc 99:1); dH
D
(500 MHz, CDCl3) 8.25 (1H, s), 7.14–7.05 (3H, m), 6.71 (1H, t,
J = 2.5 Hz), 6.55 (1H, d, J = 8.0 Hz), 4.38 (1H, dd, J = 3.0 and
11.0 Hz), 4.17 (1H, dd, J = 6.0 and 11.0 Hz), 3.49–3.47 (1H, m),
2.96 (1H, t, J = 5.0 Hz), 2.85 (1H, dd, J = 2.5 and 5.0 Hz); dC
(125 MHz, CDCl3) 152.1, 133.2, 127.1, 120.5, 117.6, 112.3, 103.4,
102.7, 70.5, 50.9, 44.3. MS (EI) m/z: 189 (M+) (100), 132 (63), 104
(50). Anal. Calcd for C11H11NO2: C, 69.83; H, 5.86; N, 7.40. Found:
C, 69.85; H, 5.84; N, 7.45.
4.3.3. (ꢀ)-(3S,4aS,8aS)-2-[(2R)-Hydroxy-3-(1H-indol-6-yloxy)-
propyl]-decahydro-isoquinoline-3-carboxylic acid tert-
butylamide (8)
4.2.2. (ꢀ)-(R)-5-Oxiranylmethoxy-1H-indole (4)
Compound 8 was isolated as a brown thick oil (0.063 g, 93%);
20
Compound 4 was isolated as a brown thick oil (0.045 g, 72%).
[a
]
D
ꢀ66.0 (c 1.3, CH3OH); Rf 0.6 (CHCl3/CH3OH 95:5); dH
20
[
a]
D
ꢀ2.2 (c 1.2, CHCl3); Rf 0.5 (CH2Cl2/EtOAc 99:1); dH
(500 MHz, CDCl3) 8.53 (1H, s), 7.51 (1H, d, J = 8.5 Hz), 7.21 (1H, t,
J = 2.5 Hz), 6.90 (1H, d, J = 2.0 Hz), 6.79 (1H, dd, J = 2.5 and
9.0 Hz), 6.48 (1H, t, J = 2.0 Hz), 6.24 (1H, s, NH), 4.16 (1H, t,
(500 MHz, CDCl3) 8.16 (1H, s), 7.31–7.14 (3H, m), 6.92 (1H, dd,
J = 1.5 and 8.5 Hz), 6.5 (1H, s), 4.27 (1H, dd, J = 3.5 and 12.0 Hz),

4798
C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
J = 5.0 Hz), 4.08 (1H, dd, J = 4.5 and 9.0 Hz), 3.95 (1H, dd, J 6.5 and
9.5 Hz), 2.95 (1H, dd, J = 2.0 and 11.5 Hz), 2.76–2.64 (2H, m), 2.4
(1H, dd, J = 5.0 and 13.5 Hz), 2.28 (1H, dd, J = 3.0 and 12.0 Hz),
1.92–1.40 (11H, m), 1.37 (9H, s), 1.34–1.21 (3H, m); dC (125 MHz,
CDCl3) 174.0, 155.0, 136.4, 123.4, 122.5, 121.1, 110.1, 102.1, 95.9,
70.9, 70.2, 68.3, 59.5, 58.5, 50.7, 35.7, 33.1, 30.8, 30.6, 28.6, 26.1,
25.7, 20.5. IR (cmꢀ1) 3311, 2925, 1652, 1365. Anal. Calcd for
4.4.2. (+)-(R)-1-(1H-Indol-5-yloxy)-3-isobutylamino-propan-2-
ol (10)
Compound 10 was obtained as a colourless thick oil (0.358 g,
20
99%). [
a
]
+6.5 (c 1.3, CH3OH); Rf 0.5 (CHCl3/CH3OH 9:1); dH
D
(500 MHz, CDCl3) 8.66 (1H, s), 7.25–7.10 (3H, m), 6.85 (1H, dd,
J = 1.5 and 8.5 Hz), 6.45 (1H, s), 4.22–4.18 (1H, m), 4.02–3.97 (2H,
m), 3.72–3.68 (2H, m), 2.87 (2H, d, J = 7.0 Hz), 1.85–1.80 (1H, m),
0.94 (3H, d, J = 3.5 Hz), 0.93 (3H, d, J = 3.5 Hz); dC (125 MHz, CDCl3)
152.9, 131.2, 128.2, 125.1, 112.4, 111.7, 103.6, 101.9, 71.3, 67.9,
57.4, 51.8, 27.7, 20.4. IR (cmꢀ1) 3402, 3313, 3050, 2958, 1455,
1159. Anal. Calcd for C15H22N2O2: C, 68.67; H, 8.45; N, 10.68.
Found: C, 68.69; H, 8.43; N, 10.64.
C25H37N3O3: C, 70.22; H, 8.72; N, 9.83. Found: C, 70.20; H, 8.71;
N, 9.86.
4.3.4. (ꢀ)-(3S,4aS,8aS)-(1H-Indol-4-yl)-carbamic acid 3-(3-tert-
butylcarbamoyl-octahydro-isoquinolin-2-yl)-(2R)-hydroxy-
propyl ester (28)
Compound 28 was isolated as a brown powder (0.050 g, 80%)
4.4.3. (+)-(1H-Indol-5-yl)-carbamic acid (2R)-hydroxy-3-
isobutylamino-propyl ester (31)
starting from 27. Mp 130 °C; [a]
20 +36.0 (c 1, CH3OH); Rf 0.8 (CHCl3/
D
CH3OH 9:1); dH (500 MHz, CDCl3) 8.68 (1H, s), 7.11 (1H, t,
J = 2.5 Hz), 6.85 (1H, d, J 8.0 Hz), 6.48–6.35 (3H, m), 4.37 (1H, d,
J = 8.0 Hz), 4.06–4.11 (2H, m), 2.90 (1H, d, J = 11.5 Hz), 2.65 (1H,
t, J = 5.0 Hz), 2.26 (2H, t, J = 12.0 Hz), 2.23–1.38 (11H, m), 1.36
(9H, s), 1.21–1.18 (3H, m); dC (125 MHz, CDCl3) 173.7, 138.6,
136.7, 129.7, 128.1, 125.2, 115.7, 111.3, 106.4, 102.6, 70.1, 68.1,
59.4, 58.8, 55.7, 50.8, 35.7, 33.0, 31.9, 30.7, 30.6, 29.7, 29.3, 25.7.
IR (cmꢀ1) 3411, 3282, 3042, 2937, 1731, 1642, 1312, 1238, 1124.
Anal. Calcd for C26H38N4O4: C, 66.36; H, 8.14; N, 11.91 Found: C,
66.33; H, 8.18; N, 11.88.
Compound 31 was obtained as yellow thick oil (0.270 g, 92%)
from 21 (0.24 g, 0.96 mmol), i-BuNH2 (0.3 mL, 2.9 mmol), after
20
34 h. [a]
+4.9 (c 1.4, CH3OH); Rf 0.6 (CHCl3/CH3OH 7:3); dH
D
(500 MHz, CDCl3) 8.45 (1H, s), 7.66 (1H, s), 7.27–7.04 (3H, m),
6.46 (1H, s), 4.28–4.25 (1H, m), 4.13–3.94 (2H, m), 2.75–2.41
(4H, m), 1.75–1.70 (1H, m), 0.91 (3H, d, J = 3.0 Hz), 0.89 (3H, d,
J = 3.0 Hz); dC (125 MHz, CDCl3) 154.4, 133.0, 130.1, 128.0, 125.3,
125.1, 115.5, 111.3, 102.6, 67.9, 67.2, 57.6, 51.3, 28.3, 20.5. IR
(cmꢀ1) 3316, 2958, 1704, 1557, 1481, 1237, 1063. Anal. Calcd for
C
16H23N3O3: C, 62.93; H, 7.59; N, 13.76. Found: C, 62.89; H, 7.55;
N, 13.68.
4.3.5. (ꢀ)-(3S,4aS,8aS)-(1H-Indol-5-yl)-carbamic acid 3-(3-tert-
butylcarbamoyl-octahydro-isoquinolin-2-yl)-(2R)-hydroxy-
propyl ester (29)
4.5. Synthesis of arylsulfonamides: general procedure
Compound 29 was isolated as a brown powder (0.052 g, 75%)
Dry triethylamine (0.14 mL, 1.01 mmol) and the suitable aryl-
sulfonyl chloride (0.93 mmol) were added to a stirred solution of
the substrate (0.78 mmol) in dry CH2Cl2 (40 mL) at room temperature
under argon atmosphere. After 24 h (TLC control in CHCl3/CH3OH
99:1) the reaction mixture was quenched by adding a 5% solution
of H2SO4 and extracted by CH2Cl2. The organic layer was washed
with a NaHCO3 (saturated aqueous solution) and brine, then it was
dried over Na2SO4 and concentrated under reduced pressure.
The crude was purified by column chromatography on silica gel
(CHCl3/CH3OH 99:1).
20
starting from 27. Mp 139 °C; [
a
]
D
+42.0 (c 1.2, CH3OH); Rf 0.6
(CHCl3/CH3OH 9:1); dH (500 MHz, CDCl3) 8.31 (1H, s), 7.68 (1H,
s), 7.31 (1H, d, J = 9.0 Hz), 7.21 (1H, t, J = 2.5 Hz), 7.11 (1H, d,
J = 8.0 Hz), 6.85 (1H, br s), 6.47 (1H, t, J = 2.5 Hz), 6.50 (1H, s),
6.16 (1H, s), 4.39 (1H, d, J = 8.0 Hz), 4.08–4.12 (2H, m), 2.92 (1H,
d, J = 11.5 Hz), 2.63 (1H, t, J = 5.0 Hz), 2.28 (2H, t, J = 12.0 Hz),
2.22–1.39 (11H, m), 1.35 (9H, s), 1.22–1.18 (3H, m); dC (125 MHz,
CDCl3) 173.7, 154.4, 133.1, 130.0, 129.7, 128.1, 125.2, 115.7,
111.3, 102.6, 70.1, 68.1, 59.4, 58.8, 50.8, 35.7, 33.0, 31.9, 30.7,
30.6, 29.7, 29.3, 28.7, 26.1, 25.7, 20.6. IR (cmꢀ1) 3410, 3280,
3046, 2939, 1730, 1642, 1308, 1245, 1128. Anal. Calcd for
4.5.1. (+)-N-[(2R)-Hydroxy-3-(1H-indol-4-yloxy)-propyl]-N-
C26H38N4O4: C, 66.36; H, 8.14; N, 11.91. Found: C, 66.39; H, 7.15;
isobutyl-4-nitro-benzenesulfonamide (12a)
N, 11.93.
Compound 12a was isolated as a yellow solid (0.285 g, 82%). Mp
20
142 °C; [
a
]
D
+24.0 (c 1.0, CHCl3); Rf 0.6 (CHCl3/CH3OH 99:1); dH
4.4. Ring opening of the epoxides with iso-butylamine: general
procedure
(500 MHz, CDCl3) 8.25 (1H, s), 8.18 (2H, d, J = 8.5 Hz), 7.90 (2H, d,
J = 8.5 Hz), 7.06 (1H, s), 6.98–7.01 (2H, m),), 6.42 (1H, s), 6.39
(1H, d, J = 8.0 Hz), 4.18–4.09 (1H, m), 3.41–3.37 (1H, m), 3.13–
2.95 (5H, m), 1.98–1.94 (1H, m), 0.96–0.84 (6H, m); dC (125 MHz,
CDCl3) 151.3, 149.1, 145.8, 133.3, 128.4, 128.2, 124.5, 124.2,
119.3, 107.8, 102.6, 98.5, 74.2, 65.3, 57.7, 52.2, 25.7, 20.9. IR
(cmꢀ1) 3408, 3123, 2898, 1489, 1353, 1224. Anal. Calcd for
i-ButNH2 (1.12 g, 1.48 mmol) was added to a stirred solution of
suitable epoxide (1.38 mmol) in i-PrOH (30 mL) at room tempera-
ture for 26 h. Then the solvent was removed under reduced pres-
sure. The crudes of compounds 11, 17 and 30 were used in the
subsequent reaction without any purification. Compounds 9, 10
and 31 were purified by column chromatography on silica gel.
C21H25N3O6S: C, 56.36; H, 5.63; N, 9.39; S, 7.17. Found: C, 56.38;
H, 5.61; N, 9.35; S, 7.20.
4.4.1. (+)-(R)-1-(1H-Indol-4-yloxy)-3-isobutylamino-propan-2-
4.5.2. (+)-N-[(2R)-Hydroxy-3-(1H-indol-4-yloxy)-propyl]-N-
ol (9)
isobutyl-3,4-dimethoxy-benzenesulfonamide (12b)
Compound 9 was obtained as a colourless thick oil (0.400 g,
Compound 12b was isolated as a white solid (0.340 g, 94%). Mp
20
20
99%). [
a]
+6.8 (c 1.2, CHCl3); Rf 0.5 (CHCl3/CH3OH 9:1); dH
118 °C; [
a]
D
+4.1 (c 1.6, CHCl3); Rf 0.6 (CHCl3/CH3OH 99:1); dH
D
(500 MHz, CDCl3) 8.30 (1H, s), 7.14–7.05 (3H, m), 6.68 (1H, d,
J = 2.5 Hz), 6.55 (1H, d, J = 7.5 Hz), 4.20–4.15 (1H, m), 2.95–2.85
(2H, m), 3.72–3.68 (2H, m), 2.87 (2H, d, J = 7.0 Hz), 1.85–1.80
(1H, m), 0.94 (3H, d, J = 3.5 Hz), 0.93 (3H, d, J = 3.5 Hz); dC
(125 MHz, CDCl3) 152.9, 131.2, 128.2, 125.1, 112.4, 111.7, 103.6,
101.9, 71.3, 67.9, 57.4, 51.8, 27.7, 20.4. IR (cmꢀ1) 3402, 3313,
3050, 2958, 1455, 1159. Anal. Calcd for C15H22N2O2: C, 68.67; H,
8.45; N, 10.68. Found: C, 68.69; H, 8.43; N, 10.72.
(500 MHz, CDCl3) 8.31 (1H, s), 7.47 (1H, dd, J = 2.0 and 8.5 Hz),
7.29 (1H, d, J = 2.0 Hz), 7.14–7.06 (3H, m), 6.95 (1H, d, J = 8.5 Hz),
6.58–6.53 (2H, m), 4.39 (1H, br s), 4.22–4.00 (2H, m), 3.85 (3H,
s), 3.82 (3H, s), 3.29 (2H, d, J = 5.5 Hz), 3.02–2.97 (2H, m), 2.87–
2.83 (1H, m), 1.95–1.90 (1H, m), 1.32–1.29 (2H, m), 0.87 (3H, d,
J = 2.5 Hz), 0.81 (3H, d, J = 2.5 Hz); dC (125 MHz, CDCl3) 153.0,
150.1, 148.5, 132.9, 129.1, 128.4, 124.1, 118.5, 118.2, 115.8,
112.4, 107.5, 102.5, 99.0, 74.1, 66.7, 57.1, 56.2, 55.3, 26.5, 20.9. IR

C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
4799
20
(cmꢀ1) 3421, 3118, 2954, 1565, 1320, 1148. Anal. Calcd for
23H30N2O6S: C, 59.72; H, 6.54; N, 6.06; S, 6.93. Found: C, 59.68;
4.5.7. (+)-N-(2R,3-Dihydroxy-propyl)-N-isobutyl-4-nitro-
benzenesulfonamide (18a)
C
H, 6.52; N, 6.11; S, 6.90.
Compound 18a was isolated as a thick oil (0.192 g, 74%). [
a
]
D
+2.8 (c 0.8, CHCl3); Rf 0.4 (CHCl3/CH3OH 95:5); dH (500 MHz, CDCl3)
8.38 (2H, d, J = 7.5 Hz), 8.03 (2H, d, J = 7.0 Hz), 3.93–3.89 (1H, m),
3.73–3.65 (1H, m), 3.23 (2H, d, J = 6.5 Hz), 3.04–3.00 (3H, m),
1.98–1.92 (1H, m), 0.91 (3H, d, J = 2.5 Hz), 0.90 (3H, d, J = 2.5 Hz);
dC (125 MHz, CDCl3) 150.1, 144.6, 128.5, 124.4, 70.1, 58.2, 51.8,
27.1, 19.9. IR (cmꢀ1) 3410, 3071, 2957, 1709, 1535, 1346, 1161.
Anal. Calcd for C13H20N2O6S: C, 46.98; H, 6.07; N, 8.43; S, 9.65.
Found: C, 46,95; H, 6.09; N, 8.46; S, 9.62.
4.5.3. (+)-N-[(2R)-Hydroxy-3-(1H-indol-5-yloxy)-propyl]-N-
isobutyl-4-nitro-benzenesulfonamide (13a)
Compound 13a was isolated as a yellow solid (0.279 g, 80%). Mp
20
135 °C; [
a]
+18.0 (c 0.8, CHCl3); Rf 0.7 (CHCl3/CH3OH 99:1); dH
D
(500 MHz, CDCl3) 8.29 (1H, d, J = 8.5 Hz), 8.16 (1H, s), 7.99 (2H, d,
J = 8.5 Hz), 7.27 (1H, d, J = 7.0 Hz), 7.20 (1H, d, J = 2.5 Hz), 7.07
(1H, s), 6.80 (1H, dd, J = 1.5 and 8.5 Hz), 6.47 (1H, s), 4.22–4.18
(1H, m), 4.00–3.99 (2H, m), 3.42–3.38 (2H, m), 3.09–3.05 (2H, m),
2.86 (2H, d, J = 4.5 Hz), 2.01–1.98 (1H,m), 0.92 (3H, d, J = 2.5 Hz),
0.90 (3H, d, J = 2.5 Hz); dC (125 MHz, CDCl3) 152.6, 149.9, 145.1,
131.3, 128.5, 128.3, 124.0, 112.3, 111.8, 103.8, 102.4, 70.2, 68.8,
57.4, 51.8, 26.7, 19.9. IR (cmꢀ1) 3417, 3102, 2961, 1529, 1349,
1159. Anal. Calcd for C21H25N3O6S: C, 56.36; H, 5.63; N, 9.39; S,
7.17. Found: C, 56.38; H, 5.61; N, 9.34; S, 7.15.
4.5.8. (+)-N-(2R,3-Dihydroxy-propyl)-N-isobutyl-3,4-dimethoxy-
benzenesulfonamide (18b)
20
Compound 18b was isolated as a thick oil (0.211 g, 78%). [a]
D
+1.5 (c 1.5, CHCl3); Rf 0.3 (CHCl3/CH3OH 97:3); dH (500 MHz, CDCl3)
7.44 (1H, d, J = 8.5 Hz), 7.23 (1H, s), 6.96 (1H, d, J = 8.5 Hz), 3.93
(3H, s), 3.91 (3H, s), 3.70–3.51 (3H, m), 2.73–2.58 (2H, m), 2.40–
2.31 (2H, m), 1.96–1.89 (1H, m), 0.90 (3H, d, J = 2.5 Hz), 0.89 (3H,
d, J = 2.5 Hz); dC (125 MHz, CDCl3) 153.0, 150.1, 129.2, 118.4,
4.5.4. (+)-N-[(2R)-Hydroxy-3-(1H-indol-5-yloxy)-propyl]-N-
isobutyl-3,4-dimethoxy-benzenesulfonamide (13b)
115.6, 112.2, 68.0, 64.9, 57.3, 56.2, 51.8, 25.3, 20.2. IR (cmꢀ1
)
Compound 13b was isolated as a white solid (0.339 g, 94%). Mp
3391, 3172, 2808, 1523, 1354, 1187. Anal. Calcd for C15H25NO6S:
C, 51.86; H, 7.25; N, 4.03; S, 9.23. Found: C, 51.84; H, 7.22; N,
4.06; S, 9.22.
20
118 °C; [
a]
+4.1 (c 1.6, CHCl3); Rf 0.6 (CHCl3/CH3OH 99:1); dH
D
(500 MHz, CDCl3) 8.14 (1H, s), 7.45 (1H, dd, J 1.5 and 8.0 Hz),
7.29–7.26 (2H, m), 7.19 (1H, d, J = 2.5 Hz), 7.09 (1H, s), 6.93 (1H,
d, J = 8.5 Hz), 6.83 (1H,dd, J 2.0 and 8.5 Hz), 6.47 (1H, t, J 1.0 Hz),
4.26–4.22 (1H, m), 4.06–3.99 (2H, m), 3.93 (3H, s), 3.91 (3H, s),
3.33–3.21 (2H, m), 3.04–2.92 (2H,m), 1.98–1.96 (1H, m), 0.94
(3H, d, J = 8.5 Hz), 0.90 (3H, d, J 8.5 Hz); dC (125 MHz, CDCl3)
152.8, 152.6, 149.1, 131.3, 130.5, 128.3, 125.1, 121.3, 112.4,
111.7, 110.6, 109.9, 103.7, 102.4, 70.3, 69.2, 58.3, 56.2, 56.0, 52.7,
27.0, 20.0. IR (cmꢀ1) 3390, 2954, 1509, 1262, 1137. Anal. Calcd
for C23H30N2O6S: C, 59.72; H, 6.54; N, 6.06; S, 6.93. Found: C,
59.70; H, 6.51; N, 6.03; S, 6.90.
4.5.9. (+)-(1H-indol-4-yl)-carbamic acid (2R)-hydroxy-3-
[isobutyl-(4-nitro-benzenesulfonyl)-amino]-propyl ester (32a)
Compound 32a was isolated as a yellow thick oil (0.332 g, 87%).
[a]
20 +10.2 (c 1.2, CHCl3); Rf 0.6 (CHCl3/CH3OH 99:1); dH (500 MHz,
D
CDCl3) 8.36 (br s, 1H), 8.16 (2H, d, J = 8.5 Hz), 7.87 (2H, d,
J = 8.5 Hz), 7.18–7.20 (m, 4H), 6.96 (br s, 1H), 6.50–6.51 (m, 1H),
4.26 (1H, dd, J = 5.0 and 11.5 Hz), 4.16–4.08 (2H, m), 3.25–3.02
(4H, m), 1.95–1.90 (1H, m), 0.91 (3H, d, J = 3.0 Hz), 0.89 (3H, d,
J = 3.0 Hz); dC (125 MHz, CDCl3) 153.7, 151.2, 145.9, 136.1, 130.3,
128.4, 127.3, 124.4, 124.1, 114.9, 112.5, 102.6, 66.3, 64.5, 57.3,
52.2, 25.8, 19.9. IR (cmꢀ1) 3420, 3073, 2952, 1721, 1513, 1354,
1168. Anal. Calcd for C22H26N4O7S: C, 53.87; H, 5.34; N, 11.42; S,
6.54. Found: C, 53.83; H, 5.38; N, 11.46; S, 6.52.
4.5.5. (+)-N-[(2R)-Hydroxy-3-(1H-indol-6-yloxy)-propyl]-N-
isobutyl-4-nitro-benzenesulfonamide (14a)
Compound 14a was isolated as a yellow solid (0.265 g, 76%). Mp
20
131 °C; [
a]
+10.8 (c 0.5, CHCl3); Rf 0.7 (CHCl3/CH3OH 99:1); dH
D
(500 MHz, CDCl3) 8.35 (2H, dd, J = 2.0 and 7.0 Hz), 8.10 (1H, s),
8.03 (2H, dd, J = 2.0 and 7.0 Hz), 7.52 (1H, d, J = 8.5 Hz), 7.15 (1H,
m), 7.15 (1H, d, J = 2.0 Hz), 6.77 (1H, dd, J = 2.0 and 8.5 Hz), 6.51
(1H, m), 4.25 (1H, br s), 4.03 (2H, d, J = 5.0 Hz), 3.43–3.38 (2H,
m), 3.09–3.06 (2H, m), 2.88 (1H, br s), 2.03–2.00 (1H, m), 0.94
(3H, d, J = 2.5 Hz), 0.91 (3H, d, J = 2.5 Hz); dC (125 MHz, CDCl3)
154.8, 150.0, 144.9, 136.3, 128.5, 124.3, 123.4, 122.9, 121.5,
4.5.10. (+)-(1H-indol-4-yl)-carbamic acid (2R)-hydroxy-3-
[isobutyl-(3,4-dimethoxy-benzenesulfonyl)-amino]-propyl
ester (32b)
20
Compound 32b was isolated as a thick oil (0.335 g, 85%). [a]
D
+2.4 (c 1.0, CHCl3); Rf 0.7 (CH2Cl2/EtOAc 98:2); dH (500 MHz, CDCl3)
8.36 (1H, br s), 7.47 (1H, m), 7.33 (1H, d, J = 8.5 Hz), 7.18 (1H, d,
J = 5.5 Hz), 7.10–7.06 (3H, m), 6.9 (1H, m), 6.80 (1H, d, J = 8.5 Hz),
6.4 (1H, s), 4.26–4.17 (2H, m), 4.08 (1H, br s), 3.82 (3H, s), 3.79
(3H, s), 3.41 (1H, br s), 3.18–3.05 (2H, m), 2.93–2.80 (2H, m),
1.85–1.78 (1H, m), 0.85 (3H, d, J = 2.5 Hz), 0.82 (3H, d, J = 2.5 Hz);
dC (125 MHz, CDCl3) 152.7, 149.1, 136.5, 130.0, 129.5, 123.9,
122.6, 121.3, 110.6, 109.9, 107.8, 98.4, 69.2, 66.9, 58.4, 56.2, 56.1,
52.5, 27.1, 20.1, 19.9. IR (cmꢀ1) 3392, 3106, 2955, 1548, 1193,
1178. Anal. Calcd for C24H31N3O7S: C, 57.01; H, 6.18; N, 8.31; S,
6.34. Found: C, 57.04; H, 6.15; N, 8.33; S, 6.37.
110.0, 102.4, 95.4, 69.9, 68.8, 57.7, 51.9, 29.5, 19.9. IR (cmꢀ1
)
3430, 3156, 2889, 1565, 1332, 1104. Anal. Calcd for C21H25N3O6S:
C, 56.36; H, 5.63; N, 9.39; S, 7.17. Found: C, 56.40; H, 5.66; N,
9.40; S, 7.20.
4.5.6. (+)-N-[(2R)-Hydroxy-3-(1H-indol-6-yloxy)-propyl]-N-
isobutyl-3,4-dimethoxy-benzenesulfonamide (14b)
Compound 14b was isolated as a white solid (0.339 g, 94%). Mp
20
126 °C; [
a]
+2.2 (c 1.0, CHCl3); Rf 0.6 (CHCl3/CH3OH 99:1); dH
D
(500 MHz, CDCl3) 8.23 (1H, br s), 7.51 (1H, d, J = 8.5 Hz), 7.46
(1H, dd, J = 2.0 and 8.5 Hz), 7.30 (1H, d, J = 2.0 Hz), 7.14–7.13 (1H,
m), 6.94 (1H, d, J = 8.5 Hz), 6.83 (1H, s), 6.76 (1H, dd, J = 2.0 and
8.5 Hz), 6.49 (1H, s), 4.25 (1H, br s), 4.03–3.96 (2H, m), 3.94 (3H,
s), 3.93 (3H, s), 3.38–3.29 (2H, m), 3.22 (1H, d, J = 4.0 Hz), 3.09–
2.93 (2H, m), 2.40–1.96 (1H, m), 0.95 (3H, d, J = 2.5 Hz), 0.92 (3H,
d, J = 2.5 Hz); dC (125 MHz, CDCl3) 155.2, 152.8, 149.3, 136.6,
130.7, 123.6, 122.7, 121.5, 110.9, 110.5, 110.0, 102.9, 102.5, 95.5,
70.2, 69.3, 58.4, 56.6, 56.4, 52.6, 29.9, 20.3. IR (cmꢀ1) 3402, 3098,
2922, 1560, 1280, 1142. Anal. Calcd for C23H30N2O6S: C, 59.72; H,
6.54; N, 6.06; S, 6.93. Found: C, 59.68; H, 6.52; N, 6.10; S, 6.90.
4.5.11. (+)-(1H-indol-5-yl)-carbamic acid (2R)-hydroxy-3-
[isobutyl-(4-nitro-benzenesulfonyl)-amino]-propyl ester (33a)
Compound 33a was isolated as a yellow thick oil (0.344 g, 90%).
20
[a
]
+8.8 (c 0.8, CHCl3); Rf 0.6 (CHCl3/CH3OH 99:1); dH (500 MHz,
D
CDCl3) 8.38 (1H, s), 8.24 (2H, br s), 7.95 (2H, br s), 7.63 (1H, s),
7.28–7.00 (4H, m), 6.46 (1H, s), 4.27 (1H, dd, J = 5.0 and 11.5 Hz),
4.18–4.09 (2H, m), 3.27–3.01 (4H, m), 1.95–1.91 (1H, m), 0.92
(3H, d, J = 3.0 Hz), 0.89 (3H, d, J = 3.0 Hz); dC (125 MHz, CDCl3)
154.2, 149.9, 144.8, 133.1, 129.8, 128.6, 128.4, 128.1, 125.5,
124.3, 115.6, 111.5, 102.4, 68.8, 66.8, 57.4, 51.6, 26.8, 19.9. IR
(cmꢀ1) 3410, 3071, 2957, 1709, 1535, 1346, 1161. Anal. Calcd for

4800
C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
C22H26N4O7S: C, 53.87; H, 5.34; N, 11.42; S, 6.54. Found: C, 53.82;
H, 5.36; N, 11.45; S, 6.51.
and finally with brine. The organic layer was dried and evaporated
under reduced pressure. Toluene-4-sulfonic acid (2R)-hydroxy-3-
[isobutyl-(4-nitro-benzenesulfonyl)-amino]-propyl ester and
toluene-4-sulfonic acid (2R)-hydroxy-3-[isobutyl-(3,4-dimethoxy-
benzenesulfonyl)-amino]-propyl ester were obtained as yellow oil
and used in the subsequent reaction without any purification.
4.5.12. (+)-(1H-indol-5-yl)-carbamic acid (2R)-hydroxy-3-
[isobutyl-(3,4-dimethoxy-benzenesulfonyl)-amino]-propyl
ester (33b)
20
Compound 33b was isolated as a thick oil (0.323 g, 82%). [a]
D
+1.8 (c 1.0, CHCl3); Rf 0.7 (CH2Cl2/EtOAc 98:2); dH (500 MHz, CDCl3)
8.38 (1H, br s), 7.72–7.68 (1H, m), 7.43–7.39 (1H, m), 7.32–7.24
(2H, m), 7.19 (1H, s), 7.11–7.07 (1H, m), 6.95–6.85 (1H, m), 6.44
(1H, s), 4.28–4.14 (2H, m), 4.07 (1H, br s), 3.84 (3H, s), 3.83 (3H,
s), 3.58 (1H, br s), 3.25–3.12 (2H, m), 3.03–2.85 (2H, m), 1.91–
1.82 (1H, m), 0.88 (3H, d, J = 2.5 Hz), 0.83 (3H, d, J = 2.5 Hz); dC
(125 MHz, CDCl3) 154.3, 152.7, 149.1, 133.0, 130.1, 129.9, 128.0,
125.3, 121.3, 115.6, 111.6, 111.3, 110.6, 109.9, 102.6, 69.2, 66.8,
58.3, 56.2, 56.1, 52.5, 27.1, 20.1, 19.9. IR (cmꢀ1) 3445, 3108,
2918, 1498, 1265, 1123. Anal. Calcd for C24H31N3O7S: C, 57.01; H,
6.18; N, 8.31; S, 6.34. Found: C, 57.04; H, 6.22; N, 8.28; S, 6.32.
4.5.16. N-Isobutyl-4-nitro-N-(R)-oxiranylmethyl-
benzenesulfonamide (19a) and N-Isobutyl-3,4-dimethoxy-N-
(R)-oxiranylmethyl-benzenesulfonamide (19b)
Potassium carbonate was added to a stirred solution of the
crude tosyl derivative of 18a (or 18b) in methanol (26 mL) at room
temperature.
After disappearance of the starting material, the reaction was
quenched by adding ammonium chloride (saturated aqueous solu-
tion), then extracted with diethyl ether and washed with brine. The
combined extracts were dried over Na2SO4 and evaporated under
reduced pressure to give the crude epoxide.
N-Isobutyl-4-nitro-N-(R)-oxiranylmethyl-benzenesulfonamide
4.5.13. (+)-(1H-indol-5-yl)-carbamic acid (2R)-hydroxy-3-
[isobutyl-(4-methoxy-benzenesulfonyl)-amino]-propyl ester
(33c)
(19a) was isolated as thick oil (0.280 g, 84% in two steps) [a]
20 +2.4
D
(c 1.2, CHCl3); Rf 0.6 (EP/EtOAc 7:3); dH (500 MHz, CDCl3) 8.38 (2H,
d, J = 8.5 Hz), 8.04 (2H, d, J = 8.5 Hz), 3.70 (1H, dd, J = 15.0 and
3.0 Hz), 3.13 (1H, dd, J = 14.0 and 8.0 Hz), 3.06–2.90 (3H, m),
2.79–2.81 (1H, dd, J = 5.0 and 3.0 Hz), 2.53 (1H, dd, J = 5.0 and
3.0 Hz), 2.06–1.97 (1 H, m), 0.97 (3H, d, J = 7.0 Hz), 0.92 (3H, d,
J = 6.5 Hz); dC (125 MHz, CDCl3) 151.3, 145.9, 128.4, 124.3, 58.1,
51.6, 50.2, 45.4, 25.7, 20.2. IR (cmꢀ1) 3389, 3135, 2885, 1537,
1328, 1137. Anal. Calcd for C13H18N2O5S: C, 49.67; H, 5.77; N,
8.91; S, 10.20. Found: C, 49.64; H, 5.79; N, 8.88; S, 10.23.
Compound 33c was isolated as a brown solid (0.285 g, 77%). Mp
20
96 °C; [a]
+3.2 (c 1.0, CHCl3); Rf 0.6 (CHCl3/MeOH 95:5); dH
D
(500 MHz, CDCl3) 8.44 (1H, s), 7.69–7.50 (3H, m), 7.23 (1H, d,
J = 9.5 Hz), 7.13 (1H, s), 7.04 (1H, d, J = 9.0 Hz), 6.96–6.88 (3H, m),
6.42 (1H, s), 4.24–4.09 (3H, m), 3.77 (3H, s), 3.60 (1H, s), 3.46
(1H, d, J = 8.5 Hz), 3.13 (2H, br s), 2.91 (2H, d, J = 8.5 Hz), 1.24–
1.17 (6H, m); dC (125 MHz, CDCl3) 162.9, 154.4, 133.0, 130.9,
129.9, 129.5, 128.7, 128.0, 125.3, 115.6, 114.3, 111.3, 102.4, 69.1,
66.7, 58.3, 55.5, 52.4, 27.1, 19.9. IR (cmꢀ1) 3486, 3122, 2866,
1423, 1196, 1141. Anal. Calcd for C23H29N3O6S: C, 58.09; H, 6.15;
N, 8.84; S, 6.74. Found: C, 58.11; H, 6.18; N, 8.82; S, 6.75.
N-Isobutyl-3,4-dimethoxy-N-(R)-oxiranylmethyl-benzenesul-
fonamide (19b) was isolated as thick oil (0.282 g, 81% in two steps)
[a]
20 +1.6 (c 0.8, CHCl3); Rf 0.7 (EP/EtOAc 6:4); dH (500 MHz, CDCl3)
D
7.42 (1H, d, J = 8.5 Hz), 7.25 (1H, s), 6.95 (1H, d, J = 8.5 Hz), 4.05–
3.97 (1H, m), 3.95 (3H, s), 3.93 (3H, s), 3.56–3.50 (3H, m), 3.20–
2-97 (3H, m), 1.98–1.92 (1H, m), 0.97–0.89 (6H, m); dC
(125 MHz, CDCl3) 153.2, 150.3, 129.1, 118.5, 115.4, 112.2, 56.6,
56.1, 51.7, 50.2, 45.6, 25.7, 20.1. IR (cmꢀ1) 3492, 3202, 2796,
1540, 1365, 1204. Anal. Calcd for C15H23NO5S: C, 54.69; H, 7.04;
N, 7.25; S, 9.73.
4.5.14. (+)-(R)-4-Amino-N-[3-(1H-indol-5-yloxy)-2-
hydroxypropyl]-N-isobutyl-benzenesulfonamide (13c)
Compound 13a (0.104 g, 0.23 mmol) was added to a suspension
of 10% Pd/C (14 mg) in ethyl acetate (20 mL) under hydrogen atmo-
sphere. After 28 h the reaction mixture was filtered on a plate of
Celite, concentrated under vacuo and purified by column chroma-
tography on silica gel (CHCl3/CH3OH 95:5) to afford compound 13c
4.6. Ring opening of epoxides with aminoindoles: general
procedure
20
as a violet thick oil (0.071 g, 74%). [
a
]
D
+12.0 (c 1, CH3OH); Rf 0.3
(CHCl3/CH3OH 95:5); dH (500 MHz, CD3OD) 7.64 (2H, d,
J = 9.0 Hz), 7.26 (1H, d, J = 8.5 Hz), 7.18 (1H, d, J = 3.0 Hz), 7.06
(1H, d, J = 2.5 Hz), 6.99 (1H, d, J = 9.0 Hz), 6.79 (1H, dd, J = 2.0 and
9.0 Hz), 6.36 (1H, d, J = 2.5 Hz), 4.18–4.14 (1H, m), 4.00 (1H, dd,
J = 4.0 and 10.0 Hz), 3.94 (1H, dd, J = 5.0 and 9.5 Hz), 3.42
(1H, dd, J = 4.5 and 15.0 Hz), 3.11 (1H, dd, J 7.0 and 14.5 Hz), 2.96
(1H, dd, J 7.5 and 13.5 Hz), 2.88 (1H, dd, J = 7.5 and 13.5 Hz),
2.01–1.97 (1H, m), 0.93 (3H, d, = 3.0 Hz), 0.91 (3H, d, J 3.0 Hz); dC
(125 MHz, CD3OD) 157.1, 154.1, 133.1, 129.8, 126.2, 114.5, 113.2,
112.9, 112.7, 104.4, 102.2, 71.9, 70.3, 59.1, 53.1, 28.0, 20.5. IR
(cmꢀ1) 3465, 3312, 3065, 2954, 1509, 1262, 1137. Anal. Calcd for
4-Aminoindole 20 (or 5-aminoindole 21) (0.145 g, 1.10 mmol)
was added to a stirred solution of the epoxide 19a (0.283 g,
0.9 mmol) (or epoxide 19b, 0.296 g, 0.9 mmol) in i-PrOH (15 mL).
The mixture was heated at reflux temperature until disappearance
of epoxide (about 10 h, TLC control, CHCl3/CH3OH 9:1). After cool-
ing, the solvent was removed under reduced pressure and the
crude was purified by column chromatography on silica gel (elu-
ent: CHCl3/CH3OH 9:1).
N-[(2R)-Hydroxy-3-(1H-indol-4-ylamino)-propyl]-N-isobutyl-
4-nitro-benzenesulfonamide (22a) was isolated as a brown thick
20
C21H27N3O4S: C, 60.41; H, 6.52; N, 10.06; S, 7.68. Found: C, 60.39;
oil (0.309 g, 77%). [
a]
+18.0 (c 0.8, CHCl3); Rf 0.6 (CHCl3/CH3OH
D
H, 6.53; N, 10.10; S, 7.65.
99:1); dH (500 MHz, CDCl3) 8.18 (2H, d, J = 8.5 Hz), 8.15 (1H, s),
7.85 (2H, d, J = 8.5 Hz), 7.06 (1H, s), 6.98–6.93 (1H, m), 6.80 (1H,
d, J = 8.0 Hz), 6.42 (1H, s), 6.19 (1H, d, J = 7.5 Hz), 4.15–4.04 (1H,
m), 3.39–3.35 (1H, m), 3.24–3.19 (3H, m), 2.96 (2H, d, J = 7.5 Hz),
1.91–1.84 (1H, m), 0.85–0.82 (6H, m); dC (125 MHz, CDCl3) 151.5,
146.1, 135.8, 135.3, 128.3, 127.4, 124.3, 124.2, 107.2, 104.3,
102.1, 99.4, 68.2, 57.3, 53.2, 51.2, 25.9, 20.1. IR (cmꢀ1) 3422,
3112, 2945, 1587, 1298, 1087. Anal. Calcd for C21H26N4O5S: C,
56.49; H, 5.87; N, 12.55; S, 7.18. Found: C, 56.51; H, 5.88; N,
12.52; S, 7.20.
4.5.15. Toluene-4-sulfonic acid (2R)-hydroxy-3-[isobutyl-(4-
nitro-benzenesulfonyl)-amino]-propyl ester and toluene-4-
sulfonic acid (2R)-hydroxy-3-[isobutyl-(3,4-dimethoxy-
benzenesulfonyl)-amino]-propyl ester
Dry pyridine (0.095 mL, 1.16 mmol) and tosyl chloride (0.222 g,
1.16 mmol) were added to a stirred solution of compound 18a
(0.351 g, 1.06 mmol) (or 18b, 0.368 g, 1.06 mmol) in dry CH2Cl2
(15 mL) at room temperature. The mixture was stirred in argon
atmosphere for 20 h, then it was washed with diluted hydrochloric
acid (0.1 M, 10 mL), with a saturated aqueous solution of NaHCO3
N-[(2R)-Hydroxy-3-(1H-indol-4-ylamino)-propyl]-N-isobutyl-3,
4-dimethoxy-benzenesulfonamide (22b) was isolated as a brown

C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
4801
thick oil (0.278 g, 67%). [
a
]
20 +3.5 (c 1.2, CHCl3); Rf 0.5 (CHCl3/CH3OH
s), 6.50–6.51 (1H, m), 4.60–4.63 (1H, m), 4.03–4.10 (1H, m),
3.32–3.45 (1H, m), 2.90–2.92 (1H, m), 2.71–2.75 (1H, m); dC
(125 MHz, CDCl3) 153.8, 134.7, 130.3, 127.8, 124.5, 114.9, 112.4,
106.9, 102.6, 64.6, 50.3, 44.2. IR (cmꢀ1) 3465, 3218, 2804, 1425,
1308, 1121. Anal. Calcd for C12H12N2O3: C, 62.06; H, 5.21; N,
12.06. Found: C, 62.09; H, 5.24; N, 12.08.
D
99:1); dH (500 MHz, CDCl3) 8.18 (1H, s), 7.35 (1H, dd, J = 2.0 and
8.5 Hz), 7.02–6.94 (2H, m), 7.83–7.78 (2H, m), 6.47–6.40 (1H, m),
6.24 (1H, d, J = 8.5 Hz), 4.18 (1H, br s), 3.85 (3H, s), 3.80 (3H, s),
3.40–3.36 (1H, m), 3.29–3.18 (2H, m), 3.15–2.82 (4H, m), 1.90–
1.81 (1H, m), 0.87 (3H, d, J = 2.5 Hz), 0.81 (3H, d, J = 2.5 Hz); dC
(125 MHz, CDCl3) 152.7, 149.2, 141.3, 136.5, 130.2, 123.4, 122.1,
121.3, 117.2, 110.7, 110.0, 101.9, 99.8, 98.8, 69.3, 58.6, 56.3, 56.2,
54.0, 47.4, 27.3, 20.1, 20.0. IR (cmꢀ1) 3408, 3145, 2814, 1505, 1290,
1121. Anal. Calcd for C23H31N3O5S: C, 59.85; H, 6.77; N, 9.10; S,
6.95. Found: C, 59.88; H, 6.80; N, 9.07; S, 6.99.
(+)-(1H-Indol-5-yl)-carbamic acid (R)-oxiranylmethyl ester (27)
was obtained as a yellow solid (0.626 g, 90% in two steps). Mp
20
109 °C; [
a
]
+7.0 (c 1, CHCl3); Rf 0.3 (CH2Cl2/EtOAc 8:2); dH
D
(500 MHz, CDCl3) 8.18 (1H s), 7.69 (1H, s), 7.33–7.13 (3H, m),
6.70 (1H, s), 6.51 (1H, d, J = 2.5 Hz), 4.56 (1H, dd, J = 3.0 and
12.5 Hz), 4.01 (1H, dd, J = 5.5 and 11.0 Hz), 3.30–2.71 (3H, m); dC
(125 MHz, CDCl3) 153.7, 133.0, 130.0, 128.1, 125.3, 115.7, 111.3,
111.1, 102.7, 65.5, 49.8, 44.7. IR (cmꢀ1) 3456, 3264, 2708, 1418,
1292, 1065. Anal. Calcd for C12H12N2O3: C, 62.06; H, 5.21; N,
12.06. Found: C, 62.09; H, 5.24; N, 12.09.
N-[(2R)-Hydroxy-3-(1H-indol-5-ylamino)-propyl]-N-isobutyl-4-
nitro-benzenesulfonamide (23a) was isolated as a brown thick oil
20
(0.297 g, 74%). [
a]
+21.0 (c 1.0, CHCl3); Rf 0.6 (CHCl3/CH3OH
D
99:1); dH (500 MHz, CDCl3) 8.18 (2H, d, J = 8.5 Hz), 8.16 (1H, s),
7.85 (2H, d, J = 8.5 Hz), 7.06 (1H, s), 6.98–6.93 (1H, m), 6.80 (1H,
d, J = 8.0 Hz), 6.42 (1H, s), 4.19 (1H, d, J = 7.5 Hz), 4.12–4.05 (1H,
m), 3.41–3.38 (1H, m), 3.25–3.21 (3H, m), 2.95 (2H, d, J = 7.5 Hz),
1.91–1.82 (1H, m), 0.85–0.82 (6H, m); dC (125 MHz, CDCl3) 151.5,
146.2, 137.1, 129.3, 128.3, 124.5, 124.2, 123.8, 113.6, 104.8,
102.5, 101.7, 68.4, 57.1, 53.7, 51.4, 26.2, 20.1. IR (cmꢀ1) 3465,
3112, 2944, 1517, 1323, 1178. Anal. Calcd for C21H25N4O5S: C,
56.49; H, 5.87; N, 12.55; S, 7.18. Found: C, 56.51; H, 5.89; N,
12.51; S, 7.15.
4.7. Protease inhibition assay
Biological assays were performed by measuring the increase in
the fluorescence due to the Abz-NF⁄-6 (Km = 37
8 lM;
Vmax = 690 90 nmol minꢀ1 (mg protease)ꢀ1; Kcat = 0.29 0.03 Sꢀ1
;
Kcat/Km = 7.8 0.3 mMꢀ1) substrate’s hydrolysis by a commercially
available HIV-PR, at a kexc and at a kem of 325 nm and 420 nm,
N-[(2R)-Hydroxy-3-(1H-indol-5-ylamino)-propyl]-N-isobutyl-3,
respectively; 114 lL of the fluorogenic substrate (with a concen-
4-dimethoxy-benzenesulfonamide (23b) was isolated as a brown
tration of 53 lM, obtained by diluting 10 lL of a stock solution
containing 10 mg mLꢀ1 of substrate in DMSO with 1.99 mL of pH
20
thick oil (0.286 g, 69%). [
a]
D
+4.1 (c 1.0, CHCl3); Rf 0.6 (CHCl3/
CH3OH 98:2); dH (500 MHz, CDCl3) 8.06 (1H, s), 7.42 (1H, dd,
J = 2.0 and 8.5 Hz), 7.22–7.14 (2H, m), 6.95–6.91 (2H, m), 6.71
(1H, d, J = 8.5 Hz), 6.41 (1H, s), 4.15–4.13 (1H, m), 3.94 (3H, s),
3.91 (3H, s), 3.36–3.16 (4H, m), 3.02–2.91 (2H, m), 1.94–1.89
(1H, m), 0.94 (3H, d, J = 2.5 Hz), 0.92 (3H, d, J = 2.5 Hz); dC
(125 MHz, CDCl3) 152.9, 149.4, 141.4, 131.1, 130.5, 129.0, 125.0,
121.6, 113.2, 112.0, 110.9, 110.2, 104.3, 102.2, 69.2, 58.7, 56.5,
56.4, 54.1, 50.1, 27.5, 20.4, 20.2. IR (cmꢀ1) 3422, 3202, 2774,
1445, 1288, 1091. Anal. Calcd for C23H31N3O5S: C, 59.85; H, 6.77;
N, 9.10; S, 6.95. Found: C, 59.84; H, 6.79; N, 9.09; S, 6.94.
5.5 MES buffer) were put in a thermostated cuvette (25 °C) with
75 lL of MES buffer (containing 100 mM 2-[N-morpholino]ethan-
sulfonic acid (MES)/NaOH, pH 5.5; 400 mM NaCl; 1 mM ethylen-
diaminotetracetic acid (EDTA); 1 mM dithiotreitol (DTT)),
obtaining a final concentration of 30
lM and starting measuring
the fluorescence. After 1.5 min the HIV-PR was added (11
lL of a
solution obtained diluting 1:100, with a MES/BSA buffer, a stock
solution of 0.4 mg mLꢀ1 of HIV-PR in a 10 mM sodium phosphate
pH = 6.5, 1 mM EDTA/10% glycerol/0.05% mercaptoethanol/50–
100 mM NaCl), obtaining an enzymatic concentration of 10 nm.
The increase in the fluorescence was then measured; after 1 min
4.6.1. (1H-Indol-4-yl)-carbamic acid 4-nitro-phenyl ester (24)
and (1H-Indol-5-yl)-carbamic acid 4-nitro-phenyl ester (25)
Dry triethylamine (0.54 mL, 3.9 mmol) and p-nitrophenylchlo-
rocarbonate (0.786 g, 3.9 mmol) were added to a stirred solution
of 4-aminoindole 22 (or 5-aminoindole 23) (0.396 g, 3.0 mmol) in
dry CH2Cl2 (30 mL) at room temperature and in argon atmosphere.
After 2 h a TLC control (CH2Cl2/EtOAc 99:1) showed the disappear-
ance of the aminoindole and the reaction was quenched by adding
water (15 mL). The organic layer was washed with brine, dried
over Na2SO4 and concentrated in vacuo affording compound 24
(or 25) as a yellow solid. This compound was used as crude for
the following reaction.
the inhibitor containing solution was added (2 lL) and the fluores-
cence measured for other additional 10 min. For each inhibitor, a
stock solution in DMSO was prepared by weight, then diluted it
with DMSO or MES buffer. The amount of the inhibition was eval-
uated comparing the initial rates, extrapolated from the linear
parts of the curves obtained by plotting fluorescence versus time,
of the catalyzed reaction in the presence of different inhibitor’s
concentration. IC50 values were obtained simply by plotting the
different slopes versus inhibitor’s concentrations (expressed using
a logarithmical scale) and interpolating the value corresponding to
the 50% of inhibition.
4.8. Molecular modelling
4.6.2. (+)-(1H-Indol-4-yl)-carbamic acid (R)-oxiranylmethyl
ester (26) and (+)-(1H-Indol-5-yl)-carbamic acid (R)-
oxiranylmethyl ester (27)
To inspect the binding mode of the synthesized inhibitors to the
HIV-PR, conformation searching, docking and binding pose refining
were carried out for each of the molecules using MOE program
(Chemical Computing Group, Montreal, QC, Canada. Version
2012.10). First, crystal structures of Darunavir (ligand 017 from
the Protein Data Bank entry 2IEO) and Nelfinavir (ligand 1UN from
the PDB entry 2Q63) were taken as references for the sulfonamide
derivatives and decahydro-isoquinoline derivatives, respectively.
Then, possible conformations of the ligands were generated by a
grid searching algorithm considering the linkers to be the flexible
part of the molecules. After molecular mechanics geometry optimi-
zation, each of the conformers were inserted into the binding site
by superimposing the ligand to the reference inhibitor structure.
A multi-step in situ energy minimization was carefully performed
Dry triethylamine (0.7 mL, 5 mmol) and S-glycidol 1 (0.279 g,
3.8 mmol) were added to a stirred solution of the crude compound
24 (or 25) in dry CH2Cl2 (30 mL) at room temperature and under
argon atmosphere. After 10 h and the disappearance of glycidol
(by TLC monitoring, CH2Cl2/EtOAc 8:2) the mixture was diluted
with CH2Cl2. The organic layer was washed with brine, dried over
Na2SO4 and concentrated in vacuo. The crude was purified by col-
umn chromatography (CH2Cl2/EtOAc 8:2).
(+)-(1H-Indol-4-yl)-carbamic acid (R)-oxiranylmethyl ester (26)
was obtained as a yellow solid (0.612 g, 88% in two steps). Mp
20
113 °C; [
a]
+9.2 (c 1.2, CHCl3); Rf 0.4 (CH2Cl2/EtOAc 8:2); dH
D
(500 MHz, CDCl3) 8.36 (1H, br s) 7.18–7.20 (4H, m), 6.96 (1H, br

4802
C. Bonini et al. / Bioorg. Med. Chem. 22 (2014) 4792–4802
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205–243.
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2012, 51, 1778; (b) Ghosh, A. K.; Xu, C.-X.; Rao, K. V.; Baldridge, A.; Agniswamy,
J.; Wang, Y.-F.; Weber, I. T.; Aoki, M.; Miguel, S. G. P.; Amano, M.; Mitsuya, H.
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Med. Chem. 2013, 56, 1074.
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Marino-Merlo, F. Bioorg. Med. Chem. Lett. 2012, 22, 2948; (b) Chiummiento, L.;
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and the complexes were refined with help of the MOE LigX module.
For each compound, over 30 reasonable ligand poses (out of many
generated ligand conformations) were further refined and only the
pose with the lowest energy of the enzyme-inhibitor complex was
kept.
Acknowledgments
Financial support has been provided by MIUR (Italian Ministry
of University) – PRIN 20109Z2XRJ_009: ‘Progettazione e sintesi
stereoselettiva di composti attivi verso bersagli proteici coinvolti
in patologie virali e tumorali’ and Università degli Studi della
Basilicata.
9. Wang, Y.-F.; Tie, Y.; Boross, P. I.; Tozser, J.; Ghosh, A. K.; Harrison, R. W.; Weber,
I. T. J. Med. Chem. 2007, 50, 4509.
References and notes
10. Ghosh, A. K.; Dawson, Z. L.; Mitsuya, H. Bioorg. Med. Chem. 2007, 15, 7576.
11. Crystal structures of Darunavir (ligand 017 from PDB structure 2IEO) and
Nelfinavir (ligand 1UN from structure 2Q63) were taken as references, for
sulfonamide derivatives and decahydro-isoquinoline derivatives, respectively.
In crystal structure, Darunavir can form hydrophobic interactions in P1, P2, P10
and P20 and about nine hydrogen-bonds with HIV-PR residues ASP25, ASP250,
ASP30, ASP290, ASP300, GLY270 and the crystallized water.
1. (a) Global Report: UNAIDS report on the global AIDS epidemic 2012, WHO Library
Cataloguing-in-Publication Data, Joint United Nations Programme on HIV/AIDS
(UNAIDS), 2012.; (b) Flexner, C. N. Eng. J. Med. 1998, 338, 1281.
2. Mehellou, Y.; De Clercq, E. J. Med. Chem. 2010, 53, 521.
3. Wensing, A. M. J.; Van Maarseveen, N. M.; Nijhuis, M. Antiviral Res. 2010, 85, 59.
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Lupattelli, P.; Pandolfo, R.; Tramutola, F.; Berti, F. J. Med. Chem. 2010, 53, 1451;