Synthesis and biological evaluation in vitro and in mammalian cells of new heteroaryl carboxyamides as HIV-protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.07.014

New heteroaryl HIV-protease inhibitors bearing a carboxyamide spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core modifying the type of heteroarene and the central core, with the presence of either H or benzyl group. Their in vitro inhibition activity against recombinant protease showed a general beneficial effect of carboxyamide moiety, the IC₅₀ values ranging between 1 and 15nM. In particular benzofuryl derivatives showed IC₅₀ values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such benzofuryl derivatives in terms of number of interactions in the active site, supporting the experimental results on activity.The inhibition activity of such molecules has been also evaluated in HEK293 cells expressing the protease fused to green fluorescent protein, by western blotting analysis, fluorescence microscopy and cytofluorimetry.

Full text of DOI:10.1016/j.bmc.2017.07.014

Accepted Manuscript
Synthesis and biological evaluation in vitro and in mammalian cells of new
heteroaryl carboxyamides as HIV-Protease inhibitors
M. Funicello, L. Chiummiento, F. Tramutola, M.F. Armentano, F. Bisaccia, R.
Miglionico, L. Milella, F. Benedetti, F. Berti, P. Lupattelli
PII:
S0968-0896(17)31036-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.014
BMC 13850
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 May 2017
30 June 2017
6 July 2017
Please cite this article as: Funicello, M., Chiummiento, L., Tramutola, F., Armentano, M.F., Bisaccia, F., Miglionico,
R., Milella, L., Benedetti, F., Berti, F., Lupattelli, P., Synthesis and biological evaluation in vitro and in mammalian
cells of new heteroaryl carboxyamides as HIV-Protease inhibitors, Bioorganic & Medicinal Chemistry (2017), doi:
http://dx.doi.org/10.1016/j.bmc.2017.07.014
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Synthesis and biological evaluation in vitro and in mammalian cells of new heteroaryl
carboxyamides as HIV-Protease inhibitors
a*
a
a
a
a
a
M. Funicello, L. Chiummiento, F. Tramutola, M. F. Armentano, F. Bisaccia, R. Miglionico , L.
a
b
b
a*
Milella, F. Benedetti, F. Berti and P. Lupattelli.
a
Dipartimento di Scienze, Università della Basilicata, Via Ateneo Lucano 10, 85100, Potenza-Italy
b
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Via Giorgieri 1, 34127
Trieste-Italy
Abstract:
New heteroaryl HIV-Protease inhibitors bearing a carboxyamide spacer were synthesized in few
steps and high yield, from commercially available homochiral epoxides. Different substitution
patterns were introduced onto a given isopropanoyl-sulfonamide core modifying the type of
heteroarene and the central core, with the presence of either H or benzyl group. Their in vitro
inhibition activity against recombinant protease showed a general beneficial effect of carboxyamide
moiety, the IC50 values ranging between 1 and 15 nM. In particular benzofuryl derivatives showed
IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to
identify the favorable situation of such benzofuryl derivatives in terms of number of interactions in
the active site, supporting the experimental results on activity.
The inhibition activity of such molecules has been also evaluated in HEK293 cells expressing the
protease fused to green fluorescent protein, by western blotting analysis, fluorescence microscopy
and cytofluorimetry.
Introduction
During the past two decades, great efforts have been devoted to the discovery of new drugs for the
treatment of human immunodeficiency virus (HIV) infection. The development of multiple
therapeutic agents targeting various steps of the HIV life cycle has helped transform HIV infection
1
from an inevitably fatal disease into a manageable chronic pathology.
Among the different biochemical targets for antiviral therapy development, HIV-1 protease remains
the most addressed, and its inhibitors continue to play an important role in the treatment of the
2
infection.
Detailed knowledge of the structure of HIV protease and its substrate has led to the preparation of
specific PIs, whose arrival was a pivotal moment in the development of antiretroviral therapy and
1

3
made possible the dual class triple combination therapy. Despite the already marketed PIs have an
4
evident crucial role into the highly active antiretroviral therapy (HAART) regimen and the major
advances in HIV/AIDS therapies, there are many drawbacks to current treatments, in terms of drug
toxicity, drug-drug interactions and systemic complications involving heart, kidney, bone and other
5
6
organs. Central nervous system (CNS) penetration seems challenging in current HIV therapies and
the emergence of multiple drug resistance mutations reduces long-term viral inhibition. In this
respect we had already demonstrated the beneficial effect of a heteroaromatic group in a series of
new thienyl ring containing analogues of first generation PIs as nelfinavir and saquinavir, which
7
showed to maintain or even increase their activity against either wild type or mutant HIV protease.
The concept of targeting the protein backbone in structure-based drug design prompted the
preparation of new non-peptidic templates, which can maximize interactions in the HIV-protease
8
active site, particularly with the enzyme backbone atoms. Both extensive hydrogen bonding and
hydrophobic interactions with enzyme subsites can limit the drug resistance as the geometry of the
9
catalytic site must be conserved to maintain functionality. Such approach resulted in many potent
1
0
inhibitors such FDA approved darunavir, and related compounds.
During our investigation on new peptidomimetics and non peptidic inhibitors, we found beneficial
1
1
effect of heteroaryl rings as P2 ligand. In particular, the systematic study on derivatives of simple
substituted stereodefined isopropanolamine core showed the high effect of both the functionality
between the heteroaryl group and the core and the type of heteroaryl group. Promising IC50 values
were obtained for compounds 1 and 2 (Fig. 1), bearing 3,4-dimethoxyphenyl sulfonamide group.
Figure 1
This is in line with the reported data for derivatives of Darunavir in which the amino group on the
1
2
phenyl ring is replaced by 1,3-dioxolane group. 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.
Thus, with the aim of finding new easily accessible non-peptidic PIs, we run a systematic study on
the synthesis and inhibition activity of new simple derivatives with general structure A (Fig. 2), in
which the heterocycle is spaced from the core by a carboxamide group, to enhance the H-bonding
2

interactions. The type of heteroaryl group (X = O, NH, S) and the core were modified, with the
presence of either H or benzyl as R group.
Figure 2
Results and Discussion
Chemistry
For the synthesis of simple unsubstituted isopropanolamine core (R = H) we took advantage of the
established route, starting from the commercially available bidentate electrophile (S)-glycidol 1
(
Scheme 1).
Scheme 1. Synthesis of 7a-c: (a) i-BuNH
2
, i-PrOH, rt, 24h (quantit. yield); (b) 2 eq. 3,4-dimethoxyphenylsulfonyl
, Pd/C 10%, EtOH, rt (95% yield); (e) 5-
chloride, Et N, CH Cl , rt; (c) NaN , DMF, reflux (80% yield from 2); (d) H
3
2
2
3
2
heteroaryl carboxylic acid 6a-c, N,N-carbonyldiimidazole, THF, rt, 1h; then 5, rt, 24h (7a, 78%; 7b, 76%; 7c, 74%
yield).
The glycidol was first reacted with iso-butylamine affording the aminodiol 2 in excellent yield.
Treatment of 2 with 2 eq. of 3,5-dimethoxyphenylsulfonyl chloride afforded to the sulfamido-
sulfonate 3, which underwent nucleophilic displacement with NaN
3
, giving the sulfamidyl-azide 4
3

in good yield. Final catalytic hydrogenation of 4 allowed the amino-derivative 5, common precursor
of different heterocyclic amides. Thus easily available 5-heteroarylcarboxylic acids 6a-c were
alternatively coupled with the amine 5, after in situ activation with N,N’-carbonyldiimidazole,
affording amides 7a-c in good overall yield.
Since most of the commercially available inhibitors bear a benzyl moiety in the core which is
important for non-covalent interactions with the protease, we decided to prepare new benzyl
analogues to compare them with the formers in terms of inhibition activity.
The synthesis of such derivatives 12a-c was even shorter (Scheme 2) and started from the
commercially available homochiral N-Boc protected amino-epoxide 8.
Scheme 2. Synthesis of 12a-c.(a) i-BuNH
CH Cl , rt; (c) TFA/CH Cl 30%, rt, then Et
carbonyldiimidazole, Et N, THF, 1h; then 11, Et
2
, i-PrOH, 60 °C (quantit. yield); (b) 3,4-dimethoxysulfonyl chloride, Et
N (84% yield from 9); (d) 5-heteroaryl carboxylic acid 6a-c, N,N-
N, THF, rt (12a, 72%; 12b, 74%; 12c, 77% yield).
3
N,
2
2
2
2
3
3
3
Epoxide 8 was firstly opened with i-BuNH affording to the mono protected diaminoalcohol 9 in
2
good yield. The arylsulfonyl group was then introduced and the N-Boc group was efficiently
displaced by treatment with trifluoroacetic acid in dichloromethane. The ammonium trifluoroacetate
was treated with NEt , affording amine 11, which was reacted with 5-heteroarylcarboxylic acids 6,
3
previously activated with N,N’-carbonyldiimidazole. Thus, final products 12a, 12b and 12c were
obtained in four steps and excellent overall yield.
Preliminary biological essays have been conducted on all the obtained compounds: the results are
reported in Table 1.
Table 1. In vitro inhibition activity of compounds 7a-c and 12a-c.
4

Compound
IC50 (nM)
1.0
Std. error (nM)
7
7
a
0.1
315
3
b
4000
15
7
c
1
2a
2b
2c
1.1
0.2
0.2
2
1
2.0
1
13
IC50 values are obtained by measuring the initial rates of hydrolysis of the fluorogenic substrate Abz-Thr-Ile-Nle-
Phe(NO )-Gln-Arg. Results are the mean of three independent experiments
2
A general high activity (in the nanomolar range) in vitro is evident for all compounds, with notable
exception of indole derivative 7b. In the case of indole derivatives the introduction of benzyl
moiety on P ’ restored the activity of 12b, reaching those of the most active benzofurane and
1
benzothiophene derivatives. Little, if any, effect of the presence of benzyl fragment is apparent
comparing the activities of 7a and 7c with the corresponding 12a and 12c, the benzofurans 7a and
1
2a being the most active with a notable IC50 = 1 nM value.
In order to attempt a rationalization of the in vitro activity measures, we carried out a series of
1
3
docking runs on the six amides. The calculation was carried out with Autdock Vina. The best
1
4
poses were further refined by MD runs, which were carried out with the Gromacs package.
The number of favorable contacts at the S1’ and S2’ subsites for the best docking solutions is
reported in Table 2 and in Fig. 3.
5

Table 2. Number of favorable contacts established by the inhibitors at S1’ and S2’ subsites of the protease.
Inhibitor
S1’ S2’
NH2
OH
N
H
N
O
4
11
S
O
O
O
O
Ph
Amprenavir
7
a
11
7
7
b
7
c
9
1
2a
2b
2
2
2
9
7
8
1
1
2c
Figure 3. A: overlay of the best docking poses of 12a (red) and 7a (green); B,C: contacts established by 12a (B) and 7a
C) in their complexes with the protease.
(
6

In comparison with amprenavir (chosen as reference as the known inhibitor with the most
significant structural similarity with our compounds) which establishes 15 interactions on this side
of the active site, both 7a and 12a lost only four contacts, and the favorable interactions are actually
the same number in the two compounds. The benzofuryl ring in 7a and 12a is placed in a very
different way inside the site. In 12a (Fig. 3A and 3B), the S1’ site is in fact occupied by the benzyl
moiety at P1’, as expected and as in amprenavir. This sends the benzofuryl system completely
inside the S2’ site, thus allowing to keep many of the interactions observed in amprenavir. For
instance, the oxygen atom can accept hydrogen bonds from Asp129 and Asp130, as the furyl
oxygen of amprenavir. In 7a (Fig. 3A and 3C) there is lacking a canonical P1’ group, and the
conformation of the inhibitor becomes very different. The benzofuryl system is in an intermediate
position between S1’ and S2’, and can establish favorable interactions with both sites, including
hydrogen bonding at the oxygen level, with trapped water molecules. Conversely, the indole moiety
of 7b and 12b is unable to keep the same number of contacts, mostly due to its lack of ability in
accepting hydrogen bonds. No clean evidence of donation of hydrogen bond from the indole NH
group could be observed in the docking results. In 12b, the benzyl group has therefore a
compensating effect, which is much more important than those for the other compounds. As to the
benzothiophenyl derivatives 7c and 12c, the docking results are similar to those obtained for the
corresponding benzofuryl inhibitors, and the lack of two hydrogen bonding interactions can explain
the 10-fold decrease in activity in comparison with 7a and 12a. Nevertheless, 7c is by far more
active than its indole analogue 7b. An extra effect could be operating here, namely a most favorable
desolvation contribution upon binding for the more hydrophobic sulphur derivative. A similar
behavior has been already observed by us on a series of similar compounds in which the
1
1d
heterocyclic moiety was directly linked to the pseudopeptide chain by an ether linkage.
These considerations prompted us to a better analysis so we decided to test some of such inhibitors
also by a different approach in cells.
Cell-based assays for HIV-1 protease activity detection
Over the years, because of HIV-1 protease cytotoxicity in mammalian cells, the assays mainly used
1
5
16
to evaluate the HIV-1 protease inhibitors activity have been performed in vitro in bacterial, or in
1
7
yeast cells. However, to monitor the protease activity under physiological conditions, it has been
1
8
significant the contribution of Lindsten et al., which have developed a reliable and efficient assay
to screen novel drugs able to inhibit HIV-protease in mammalian cells. This method is based on the
expression of a precursor harboring the green fluorescent protein (GFP) that becomes toxic upon
autocatalytic cleavage, determining the disappearance of the fluorescence signal. The intact chimera
7

GFP-PR is detectable only in the presence of an efficient inhibitor. Thus, the GFP fluorescence
quantification provides a useful method to monitor the inhibition of protease activity in vivo.
We have evaluated the cytotoxicity of inhibitors to HEK293 cells by MTT assay. We first observed
that cell proliferation was just weakly inhibited (CC50 > 200 μM) after 24 h exposure to 12a, 12b,
1
9
and 12c inhibitors, in a dose-dependent manner. Then, all subsequent assays were performed
using concentrations which did not affect cell viability.
The presence of the intact chimeric GFP-PR protein in transiently transfected cells, cultured with or
without specific protease inhibitors, was investigated by western blotting, using a polyclonal
antibody to GFP. As shown in Fig. 4, in absence of any inhibitor, the GFP-PR precursor was not
detectable, confirming that the chimera was processed by autocatalytic cleavage. Conversely, the
use of 10 μM of darunavir (DRV), a commercial protease inhibitor used as positive controls, has
allowed the accumulation of the precursor and a strong band corresponding to the full GFP-PR
protein was observed. In cells treated with 10 μM of 12a, 12b, and 12c, the expression of the
chimera, even though lower respect to the positive controls, indicates that its processing is partially
blocked by the inhibitors.
Figure 4. Western blotting analysis of GFP-PR chimera. The transfected HEK293 cells were incubated with 10 M of
each inhibitor for 24 h and the chimera was detected using anti GFP primary antibody. The band corresponding to the
GFP-PR is not visible in the absence of protease inhibitors (CTRL). Tubulin was used as a loading control.
The presence of the intact HIV-1 protease in pcDNA3-GFP-PR transfected cells was also
monitored measuring the accumulation of GFP fluorescence using both fluorescence microscopy
and fluorescence-activated cell sorting (FACS) analysis. As shown in Fig. 5A, only a weak
background was detected in absence of any inhibitor (CTRL), whereas a strong increase in
fluorescence intensity was detected in cells treated with DRV. A lower fluorescence intensity was
observed in cells treated with 12a, 12b and 12c. These results have been confirmed by quantitative
analysis of fluorescence intensity performed with FACS (Fig. 5B). Also using higher concentrations
of 12a, 12b and 12c, the inhibitory activity is not improved.
8

Figure 5. (A) Fluorescence analysis. HEK293 cells expressing GFP-PR fusion protein were visualized by
fluorescence microscopy after incubation with each inhibitor for 24 h. Images (40X magnification) are representative of
three independent experiments. (B) Flow cytometry analysis of GFP accumulation. HEK293 cells were transiently
transfected with pcDNA3/GFP-PR and treated for 24 h with HIV-1 protease inhibitors at indicated concentrations.
Values are means ± SD of three replicates from three independent experiments. Unpaired data were assessed for
statistical significance using the Student’s t test. **p < 0.01.
Taken together, these results demonstrate that our molecules are able to inhibit PR activity in
mammalian cells, although less efficiently than Darunavir, and encourage us to pursue further
studies. Indeed, because of the side effects frequently associated with HIV protease inhibitors and
the antiviral drug-resistance, the development of new inhibitors, safer and increasingly specific, it is
needed. In the near future, the assays used in this work will allow us to easily screen, with extreme
sensitivity, our inhibitors also against HIV protease mutants and in others cell lines, to better assess
their potential.
Conclusion
We synthesized in few steps and high yield new simple benzofuryl, indolyl and benzothienyl HIV
Protease inhibitors bearing a carboxyamide spacer, which in general showed in vitro activity against
native protease in the range of 1-15 nM. Docking analysis supported the experimental data showing
the number of favorable contacts established by the inhibitors at S1’ and S2’ subsites of the
9

protease, matching with the different activities observed. Such derivatives also showed inhibition
activity in mammalian cells, demonstrating their promising potential.
Experimental section
Chemistry
Preparative chromatography was carried out on Merck silica gel (0.063–0.200 mm particle size) by
1
13
progressive elution with opportune solvent mixtures. H and C NMR spectra were normally
carried out in CDCl solutions on a VARIAN INOVA 500 MHz or Bruker 400 MHz and referenced
to Me Si. Mass spectra were obtained with a Hewlett–Packard 5971 mass-selective detector on a
3
4
Hewlett–Packard 5890 gas chromatograph [(OV-1 capillary column between 70 and 250 °C (20 °C
-
min 1)]. The optical purity was evaluated by using a polarimeter JASCO Mod Dip-370.
Dichloromethane was dried by distillation over anhydrous CaCl
2
in inert atmosphere. Dry THF and
DMF were commercially available.
1
1a
(
2R)-3-isobutylamino-propane-1,2-diol (2)
i-BuNH
2
(5.851 g, 80 mmol) was added to a stirred solution of (S)-glycidol (1) (0.308 g, 4.0 mmol)
were
in i-PrOH (20 mL) at room temperature. After 24 h the solvent and the excess of i-BuNH
2
removed under reduced pressure and the aminodiol (2) was obtained as oil in quantitative yield
1
(
0.589 g, 4.0 mmol). Rf 0.1 (EtOAc); H NMR (500 MHz, CDCl
3
)  (ppm) 3.75 (m, 3H), 3.23 (d,
H
J = 6.5 Hz, 1H), 3.01 (m, 3H), 1.92 (m, 1H), 0.90 (d, J = 6.0 Hz, 3H), 0.89 (d, J = 6.0 Hz, 3H). MS
+
(
EI) m/z 148 (M+1) . Anal. Calcd for C
7
H
17NO : C, 57.11; H, 11.64; N, 9.51. Found C, 57.0; H,
2
1
1.7; N, 9.4.
(
2R)-2-hydroxy-3-[N-isobutyl-N-(3,4-dimethoxyphenylsulfonyl)]propyl-3,4-
dimethoxybenzenesulfonate (3)
To a stirred solution of aminodiol (2) (0.115 g, 0.78 mmol) in anhydrous CH
2
Cl
2
(40 mL), Et N
3
(
0.28 mL, 2.02 mmol) and 3,4-dimethoxyphenylsulfonyl chloride (0.400 g, 1.86 mmol) were added
at room temperature and under Ar atmosphere. After 24 h the reaction was quenched by adding
saturated aqueous NaHCO solution and brine. The organic layer was extracted, dried over Na SO
filtered and concentrated in vacuo. The crude containing compound (3) was used in the subsequent
3
2
4
,
2
0
reaction without any purification.
2S)-N-(3-azido-2-hydroxypropyl)-N-isobutyl-3’,4’-dimethoxybenzenesulfonamide (4)
The product (3) (0.78 mmol) was dissolved in anhydrous DMF (10 mL) and NaN (0.104 g, 1.60
mmol) was added at room temperature. The reaction mixture was warmed at reflux and kept stirring
(
3
1
0

for 4 h. The reaction was then quenched by adding H
with CH Cl (3 x 30 mL) and the organic layer was dried with anhydrous Na
concentrated in vacuo. The crude was purified by column chromatography on silica gel (CH
2
O (20 mL). The mixture was then extracted
SO , filtered and
Cl
-12.0 (c
= 8.8
2
2
2
4
2
2
/
2
D
0
EtOAc 9:1) and compound (4) was isolated as brown oil (0.232 g, 80% from (2)). []
1
1
.0, CH
2
Cl
2
); Rf 0.5 (CH
2
Cl
2
/ EtOAc 9:1); H NMR (400 MHz, CDCl
3
) 
H
(ppm) 7.41 (dd, J
1
Hz, J
2
= 1.6 Hz, 1H); 7.24 (d, J = 1.6 Hz, 1H); 6.95 (d, J = 8.8 Hz, 1H); 4.00 (m, 1H); 3.94 (s, 3H),
.92 (s, 3H), 3.37 (dd, J = 14.0 Hz, J = 4.8 Hz, 2H), 3.25 (brs, 1H), 3.17 (dd, J = 15.0 Hz, J
.0 Hz, 1H), 2.97 (m, 2H), 2.85 (dd, J = 15.0 Hz, J = 8.0 Hz, 1H), 1.85 (m, 1H); 0.93 (d, J = 5.6
3
8
1
2
1
2
=
1
2
1
3
Hz, 3H); 0.87 (d, J = 5.6 Hz, 3H). C NMR (100 MHz, CDCl
3
) 
C
(ppm) 152.7, 149.1, 129.8,
+
1
21.3, 110.6, 109.9, 69.6, 58.6, 56.3, 56.1, 54.2, 53.2, 27.1, 20.0, 19.5. MS (EI) m/z 373 (M+1) .
Anal. Calcd for C15
S, 8.7.
H
24
N
4
5
O S: C, 48.37; H, 6.50; N, 15.04; S, 8.61. Found C, 48.2; H, 6.7; N, 15.0;
(
2S)-N-(3-amino-2-hydroxypropyl)-N-isobutyl-3’,4’-dimethoxybenzenesulfonamide (5)
Compound (4) (0.186 g, 0.50 mmol) was dissolved in EtOH (25 mL) and Pd/C 10% (0.026 g) was
added. The reaction mixture was stirred under H atmosphere at room temperature. After 4 h the
2
reaction mixture was fluxed with Ar, filtered on a celite path and concentrated in vacuo. (2S)-N-(3-
amino-2-hydroxypropyl)-N-isobutyl-3’,4’-dimethoxybenzenesulfonamide (5) was obtained as
2
0
brown solid (0.164 g, 95%). Mp = 88-90 °C []
D
+12.4 (c 1.0, CH
3
OH); Rf 0.6 (CH
2
Cl / EtOH
2
1
9
7
5:5); H NMR (400 MHz, DMSOd
6
)  (ppm) 7.96 (s, 2H), 7.39 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H),
H
.13 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 5.2 Hz, 1H), 3.83 (brs, 6H), 3.34 (brs, 1H), 3.20 (m, 1H), 2.94
1
3
(
(
m, 4H), 2.70 (m, 2H), 1.88 (m, 1H), 0.83 (d, J = 6.4 Hz, 3H), 0.79 (d, J = 6.4 Hz, 3H). C NMR
100 MHz, DMSOd
6
) 
C
(ppm) 152.2, 148.7, 129.9, 120.9, 111.3, 109.8, 66.2, 57.4, 55.9, 55.8,
+
5
2.6, 26.3, 19.9, 19.8. MS (EI) m/z 347 (M+1) . Anal. Calcd for C15
H
26
N
2
5
O S: C, 52.00; H, 7.56;
N, 8.09; S, 9.26. Found C, 52.1; H, 7.4; N, 8.2; S, 9.3
Condensation of compound (5) with 5-heteroaryl carboxylic acids: general procedure
-Heteroaryl carboxylic acid (0.40 mmol) was dissolved in anhydrous THF (10 mL) and N,N’-
5
carbonyldiimidazole (0.065 g, 0.40 mmol) was added while stirring. The mixture was kept stirring
for 1 h at room temperature. Then amine (5) (0.155 g, 0.33 mmol) was added and the mixture was
kept stirring for 24 h. The solvent was evaporated and the crude was purified by column
chromatography on silica gel (CH
2
Cl / EtOAc 4:1).
2
Benzofuran-5-carboxylic acid {(2S)-3-[(3,4-dimethoxy-benzenesulfonyl)-isobutyl-amino]-2-
hydroxy-propyl}-amide (7a)
2
D
0
The product (7a) was isolated as white solid (0.126 g, 78% yield). []
+7.5 (c, 0.5, CH
2
Cl ); Rf
2
1
0
.4 (CH
2
Cl
2
/ EtOAc 3:2); H NMR (500 MHz, CDCl
3
)  (ppm) 8.18 (s, 1H), 7.79 (d, J = 8.0 Hz,
H
1
1

1
6
H), 7.70 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.31 (m, 1H), 7.17 (s, 1H),
.85 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H), 4.42 (bs, 1H), 4.05 (m, 2H), 3.91 (s, 3H), 3.77 (s, 3H), 3,50
1
3
(
(
m, 2H), 2.90 (m, 3H), 1.90 (m, 1H), 0.97 (d, J = 7.0 Hz, 3H), 0.92 (d, J = 7.0 Hz, 3H). C NMR
125 MHz, CDCl )  (ppm) 169.7, 156.8, 152.8, 149.1, 146.4, 129.7, 128.6, 127.7, 123.6, 121.1,
20.9, 111.6, 110.7, 109.8, 109.7, 107.1, 70.5, 59.3, 56.2, 52.8, 42.9, 27.5, 20.1, 20.0. MS (EI) m/z
3
C
1
4
6
1
+
91 (M+1) Anal. Calcd for C24
H
30
N
2
O S: C, 58.76; H, 6.16; N, 5.71; S, 6.54. Found C, 58.7; H,
7
.2; N, 5.6; S, 6.5.
H-Indole-5-carboxylic acid {(2S)-3-[(3,4-dimethoxy-benzenesulfonyl)-isobutyl-amino]-2-
hydroxy-propyl}-amide (7b)
2
0
The product (7b) was isolated as white solid (0.123 g, 76% yield). []
D
+41.3 (c, 1.0, CH
2
Cl ); Rf
2
1
0
.5 (CH
2
Cl
2
/ EtOAc 55:45); H NMR (400 MHz, CDCl
3
)  (ppm) 8.59 (s, 1H), 8.22 (s, 1H), 7.71
H
(
d, J = 8.4 Hz, 1H), 7.37 (m, 4H), 7.15 (d, J = 1.6 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.64 (s, 1H),
4
1
.74 (d, J = 2.4 Hz, 1H), 4.05 (d, J = 5.6 Hz, 2H), 3.89 (s, 3H), 3.70 (s, 3H), 3.45 (m, 2H), 3.05 (m,
1
3
H), 2.85 (m, 2H), 1.90 (m, 1H), 0.94 (d, J = 5.6 Hz, 3H), 0.88 (d, J = 5.6 Hz, 3H). C NMR (100
MHz, CDCl
3
) 
C
(ppm) 163.1, 157.1, 144.6, 133.6, 129.7, 129.4, 128.1, 125.5, 116.2, 114.4, 112.7,
+
1
11.3, 102.8, 70.0, 59.0, 55.6, 53.1, 44.2, 27.3, 20.0. MS (EI) m/z 490 (M+1) . Anal. Calcd for
C
24
H
31
N
3
6
O S: C, 58.88; H, 6.38; N, 8.58; S, 6.55. Found C, 58.9; H, 6.3; N, 8.7; S, 6.5.
Benzo[b]thiophene-5-carboxylic acid {(2S)-3-[(3,4-dimethoxy-benzenesulfonyl)-isobutyl-
amino]-2-hydroxy-propyl}-amide (7c)
2
0
The product (7c) was isolated as an oil (0.123 g, 74% yield). []
D
+2.3 (c, 1.0, CH
2
Cl ); Rf 0.3
2
1
(
CH
2
Cl
2
/ EtOAc 7:3); H NMR (500 MHz, CDCl
3
)  (ppm) 8.38 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H),
H
7
7
3
=
.80 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 5.5 Hz, 1H), 7.44 (d, J = 5.5 Hz, 1H), 7.36 (d, J = 7.0 Hz, 2H),
.18 (s, 1H), 6.90 (d, J = 8.0 Hz, 1H), 4.41 (d, J = 5.5 Hz, 1H), 4.06 (m, 2H), 3.92 (s, 3H), 3.76 (s,
H), 3.65 (m, 1H), 3.40 (m, 1H), 3.05 (m, 1H), 2.90 (m, 1H), 2.80 (m, 1H), 1.92 (m, 1H), 0.96 (d, J
13
7.0 Hz, 3H), 0.91 (d, J = 7.0 Hz, 3H). C NMR (125 MHz, CDCl
3
)  (ppm) 169.7, 152.8, 149.1,
C
1
5
5
43.1, 139.6, 129.8, 129.7, 128.0, 124.4, 122.9, 122.8, 122.5, 121.2, 110.7, 109.8, 70.5, 59.3, 56.2,
+
6.1, 52.8, 42.9, 27.5, 20.2, 20.0. MS (EI) m/z 507 (M+1) . Anal. Calcd for C24
H
30
N
2
O
6
2
S : C,
6.90; H, 5.97; N, 5.53; S, 12.66. Found C, 56.8; H, 6.0; N, 5.6; S, 12.6.
2
1
(
1S)(2R)-(1-Benzyl-2-hydroxy-3-iso-butylamino-propyl)-carbamic acid tert-butyl ester (9)
i-BuNH (5.851 g, 80 mmol) was added to a stirred solution of (2S,3S)-1,2-Epoxy-3-(Boc-amino)-
2
4
-phenylbutane (8) (1.053 g, 4.0 mmol) in i-PrOH (20 mL) and the mixture was warmed at 60 °C..
After 6 h the solvent and the excess of i-BuNH were removed under reduced pressure and the
2
2
0
product (9) was obtained as white solid in quantitative yield (1.340 g, 4.0 mmol). []
D
+7.0 (c,
1
1
.0, CHCl
3
); Rf 0.35 (CHCl
3
/ CH
3
OH 9:1); H NMR (500 MHz, CDCl
3
)  (ppm) 7.25 (m, 5H),
H
1
2

4
1
1
1
.71 (d, J = 8.4 Hz, 1H), 3.80 (m, 1H), 3.48 (dd, J
H), 2.99 (d, J = 4.7 Hz, 1H), 2.87 (dd, J = 13.7 Hz, J
.73 (m, 1H), 1.35 (s, 9H), 0.93 (d, J = 6.6 Hz, 6H). C NMR (125 MHz, CDCl
37.9, 129.5, 128.4, 126.3, 79.4, 70.6, 57.9, 54.1, 51.4, 36.7, 28.33, 28.27, 20.53, 20.51. Anal.
: C, 67.82; H, 9.59; N, 8.33; Found C, 67.7; H, 9.7; N, 8.3.
1S)-(2R)-{1-Benzyl-3-[(3,4-dimethoxy-benzenesulfonyl)-isobutyl-amino]-2-hydroxy-propyl}-
1
= 10.8 Hz, J
2
= 6.3 Hz, 1H), 3.02 (d, J = 4.7 Hz,
1
2
= 7.9 Hz, 1H), 2.70 (m, 2H), 2.45 (m, 2H),
13
3
): δ (ppm) 155.9,
Calcd for C19
H
32
N
2
O
3
(
carbamic acid tert-butyl ester (10)
To a stirred solution of aminoalcohol (9) (0.262 g, 0.78 mmol) in anhydrous CH
2
Cl
2
(40 mL), Et N
3
(
0.28 mL, 2.02 mmol) and 3,4-dimethoxyphenylsulfonyl chloride (0.200 g, 0.93 mmol) were added
at room temperature and under Ar atmosphere. After 24 h the reaction was quenched by adding cold
% aqueous H SO solution (15 mL). The organic layer was washed with saturated aqueous
NaHCO solution and brine, dried over Na SO , filtered and concentrated in vacuo. The crude
5
2
4
3
2
4
2
0
containing compound (10) was used in the subsequent reaction without any purification. []
D
+9.8
1
(
c, 1.0, CHCl
3
). Rf = 0.3 (EP / EtOAc 7:3). H NMR (500 MHz, CDCl
3
) 
H
(ppm) 7.41 (dd, J
1
= 8.4
Hz, J = 2.4 Hz, 1H), 7.28 (m, 6H), 6.95 (d, J = 8.4 Hz, 1H), 4.61 (bd, 1H), 3.96 (s, 3H), 3.94 (s,
2
3
0
1
H), 3.80 (bd, 2H), 3.14 (m, 2H), 2.93 (m, 4H), 1.87 (m, 1H), 1.35 (s, 9H), 0.93 (d, J = 6.5 Hz, 3H),
1
3
.89 (d, J = 6.5 Hz, 3H). C NMR (125 MHz, CDCl
28.5, 126.4, 121.3, 110.6, 109.9, 72.6, 58.5, 56.3, 56.2, 53.6, 35.5, 28.2, 27.2, 20.1, 19.9. Anal.
S: C, 60.42; H, 7.51; N, 5.22; S, 5.97. Found C, 60.2; H, 7.6; N, 5.1; S, 6.0
2R)-(3S)-N-(3-Amino-2-hydroxy-4-phenyl-butyl)-N-isobutyl-(3,4-dimethoxy)-
3
)  (ppm) 152.7, 149.1, 137.8, 130.1, 129.5,
C
Calcd for C27
H
40
N
2
O
7
(
benzenesulfonamide (11)
To a stirred solution of (10) (0.418 g, 0.78 mmol) in anhydrous CH
2
Cl (30 mL), trifluoroacetic
2
acid (13 mL) was added at room temperature. After 40 min the reaction mixture was concentrated
and treated with toluene (3 x 20 mL), evaporated under vacuum. The crude was treated with Et
and purified by chromatography on silica gel (CHCl / CH OH 9:1), affording 0.286 g of compound
). Rf = 0.5 (CHCl
= 8.5 Hz, 1H), 7.25 (m, 6H), 6.93 (d, J = 6.8 Hz, 1H),
.93 (s, 3H), 3.91 (s, 3H), 3.71 (bs, 1H), 3.23 (m, 3H), 2.95 (m, 3H), 2.60 (m, 1H), 1.85 (m, 1H),
3
N
3
3
2
0
1
(
11) (84% yield). []
D
-2.9 (c, 1.0, CH
2
Cl
2
3
/CH OH 9:1); H NMR (500 MHz,
3
CDCl )  (ppm) 7.42 (dd, J
3
H
1
= 2.0 Hz, J
2
3
0
1
4
7
1
3
.87 (m, 6H). C NMR (125 MHz, CDCl
3
)  (ppm) 152.6, 149.1, 138.4, 130.4, 129.3, 128.7,
C
26.6, 121.3, 110.6, 110.0, 72.6, 58.3, 56.3, 56.2, 55.7, 52.3, 38.6, 27.1, 20.1, 19.9. MS (EI) m/z
+
37 (M+1) . Anal. Calcd for C22
.2; N, 6.5; S, 7.3.
H
32
N
2
5
O S: C, 60.53; H, 7.39; N, 6.42; S, 7.34. Found C, 60.7; H,
Condensation of compound (11) with 5-heteroaryl carboxylic acids: general procedure
1
3

5
-Heteroaryl carboxylic acid (0.40 mmol) was dissolved in anhydrous THF (10 mL) and N,N’-
carbonyldiimidazole (0.065 g, 0.40 mmol) was added while stirring. The mixture was kept stirring
for 1 h at room temperature. Then a solution of compound (11) (0.181 g, 0.33 mmol), Et N (0.1
mL) in CH Cl (5 mL) was added and the mixture was kept stirring for 24 h. The solvent was
evaporated and the crude was purified by column chromatography on silica gel .
1S)-(2R)-Benzofuran-5-carboxylic acid {1-benzyl-3-[(3,4-dimethoxy-benzenesulfonyl)-iso-
3
2
2
(
butyl-amino]-2-hydroxy-propyl}-amide (12a)
20
The product (12a) was isolated as white amorphous solid (0.138 g, 72% yield). []
D
+19.4 (c, 0.5,
1
CH
2
Cl
2
); Rf = 0.4 (CH
2
Cl
2
/ EtOAc 95:5); H NMR (500 MHz, CDCl
3
) 
= 1.4 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H),
= 2.2 Hz, 1H), 7.20 (m, 6H), 6.81 (d, J = 8.6 Hz, 1H), 6.73 (d, J = 2.2 Hz,
H), 6.39 (d, J = 8.6 Hz, 1H), 4.33 (m, 1H), 3.95 (m, 1H), 3.85 (s, 3H), 3.78 (s, 3H), 3.22 (dd, J
H
(ppm) 7.81 (d, J = 1.5
Hz, 1H), 7.60 (d, J = 2.2 Hz, 1H), 7.48 (dd, J
1
= 8.6 Hz, J
2
7
1
1
.29 (dd, J
1
= 8.6 Hz, J
2
1
=
13
5 Hz, J
2
= 4.5 Hz, 1H), 3.10 (m, 3H), 2.82 (m, 2H), 1.80 (m, 1H), 0.80 (d, J = 6.5 Hz, 6H). C
)  (ppm) 168.3, 156.7, 152.7, 149.1, 146.4, 137.8, 130.0, 129.4, 129.0,
28.7, 127.6, 126.7, 123.3, 121.3, 120.7, 111.5, 110.6, 109.9, 107.0, 72.8, 58.8, 56.2, 56.1, 54.9,
NMR (125 MHz, CDCl
3
C
1
5
6
1
2
+
3.5, 35.1, 27.2, 20.1, 20.0. MS (EI) m/z 581 (M+1) . Anal. Calcd for C31
H
36
N
2
7
O S: C, 64.12; H,
.25; N, 4.82; S, 5.52. Found C, 64.0; H, 6.3; N, 4.9; S, 5.4.
H-Indole-5-carboxylic acid {1-benzyl-3-[(3,4-dimethoxy-benzenesulfonyl)-iso-butyl-amino]-
-hydroxy-propyl}-amide (12b)
2
0
The product (12b) was isolated as white solid (0.141 g, 74% yield). []
D
-15.7 (c, 1.0, CH OH);
3
1
Rf = 0.5 (CH
2
Cl
2
/ AcOEt 4:1); H NMR (400 MHz, CDCl
3
)  (ppm) 8.38 (s, 1H), 7.92 (s, 1H),
H
7
.30 (m, 10H), 6.85 (d, J = 8.5 Hz, 1H), 6.59 (s, 1H), 6.44 (d, J = 8.5 Hz, 1H), 4.38 (m, 1H), 4.02
1
3
(
m, 1H), 3.89 (s, 1H), 3.30 (m, 5H), 3.00 (m, 3H), 1.89 (m, 1H), 0.86 (m, 6H). C NMR (100 MHz,
CDCl )  (ppm) 167.2, 153.0, 150.2, 138.9, 138.4, 129.2, 128.6, 128.2, 128.0, 127.1, 126.3, 124.5,
19.2, 118.4, 115.8, 112.4, 111.7, 111.2, 102.6, 70.3, 57.8, 57.6, 56.4, 56.1, 50.8, 35.3, 25.3, 20.2,
3
C
1
2
+
0.0. MS (EI) m/z 580 (M+1) . Anal. Calcd for C31
H
37
N
3
O S: C, 64.23; H, 6.43; N, 7.25; S, 5.53.
6
Found C, 64.1; H, 6.5; N, 7.3; S, 5.5.
Benzo[b]thiophene-5-carboxylic acid {1-benzyl-3-[(3,4-dimethoxy-benzenesulfonyl)-isobutyl-
amino]-2-hydroxy-propyl}-amide (12c)
2
0
The product (12c) was isolated as white amorphous solid (0.151 g, 77% yield). []
D
11.7 (c, 0.5,
1
CH
2
Cl
2
); Rf = 0.4 (CH
2
Cl
2
/EtOAc = 93/7); H NMR (500 MHz, CDCl
3
)  (ppm) 8.09 (s, 1H), 7.89
H
(
d, J = 8.5 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 5.0 Hz, 1H), 7.30 (m, 8H), 6.87 (d, J = 8.5
Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 4.42 (m, 2H), 4.05 (m, 1H), 3.92 (s, 3H), 3.82 (s, 3H), 3.31 (dd,
= 15.0 Hz, J = 4.5 Hz, 1H), 3.16 (m, 3H), 2.92 (m, 2H), 1.90 (m, 1H), 0.89 (d, J = 6.5 Hz, 6H).
J
1
2
1
4

1
3
C NMR (125 MHz, CDCl
3
)  (ppm) 168.3, 152.7, 149.2, 139.4, 137.8, 130.2, 129.9, 129.2,
C
1
5
6
28.7, 128.4, 128.0, 126.7, 124.2, 122.7, 122.6, 122.3, 121.3, 110.6, 109.9, 72.8, 58.8, 56.1, 54.9,
+
3.5, 35.1, 27.2, 20.1, 20.0. MS (EI) m/z 597 (M+1) . Anal. Calcd for C31
H
36
N
2
O
6
S : C, 62.39; H,
2
.08; N, 4.69; S, 10.75. Found C, 62.3; H, 6.1; N, 4.6; S, 10.7.
Inhibition of Recombinant HIV-Pr
IC50 values were determined at pH 5.5 using recombinant wild-type HIV-1 PR from Bachem and
the fluorogenic substrate Abz-Thr-Ile-Nle-Phe(p-NO )-Gln-Arg-NH2 (Abz-NF*-6; Bachem AG,
2
Bubendorf, CH). Atazanavir (IC50 = 20 pM) was used in this assay as reference inhibitor for
titration of the active enzyme. Full details are given in the Supporting Information.
Biology
Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose, dimethyl sulfoxide (DMSO),
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma
Aldrich (Milan, Italy). Trypsin-EDTA solution, Fetal Bovine Serum (FBS), glutamine, penicillin-
streptomycin and Phosphate Buffered Saline (PBS) were purchased from Euroclone (Milan, Italy).
Cell culture and drug treatment
Human embryonic kidney (HEK293) cells were maintained in DMEM supplemented with 10%
FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a humidified
incubator with 5% CO . HIV-protease inhibitors were dissolved in DMSO to a stock concentration
2
of 30 mM and diluted with complete DMEM to get the required concentrations. The final DMSO
concentration in the cell cultures was no greater than 0.8%: this concentration doesn’t affect cell
viability. DMSO treated cells were used as control in all the experiments.
Cell viability assay
2
2
Cell viability was determined using the MTT assay, based on the capability of living cells to
reduce the water-soluble tetrazolium salt into an insoluble purple formazan by cleavage of the
tetrazolium ring by dehydrogenase enzymes. The water insoluble product is solubilized using
organic solvents and the absorbance of the resulting colored solutions are measured, getting a direct
relation to the number of viable cells. Briefly, HEK293 cells were plated in 96-well plates at a
4
density of 1 x 10 cells/well and treated for 24 h with various concentrations of each HIV-1 protease
inhibitor (10, 25, 50, 100 and 200 M). Cells were then incubated with MTT solution (0.5 mg/ml in
1
5

DMEM) for 4 h at 37°C. The formazan crystals were finally dissolved in DMSO / i-PrOH (1:1)
solution. The absorbance was measured at a wavelength of 570 nm, with background subtraction at
6
30–690 nm, using a GLOMAX Multidetection System.
Cell Transfection and Western blot analysis
HEK293 cells, grown in complete DMEM medium, were seeded in 24-well plates at the density of
5
2
.5 x 10 cells/well. The next day, cells at
ꢀ
70% confluence were transfected with Trans-IT 2020
(
Mirus) according to the manufacturer’s instructions, using the pcDNA3-GFP-PR plasmid as GFP
reporter system (gift from Nico Dantuma, Addgene plasmid # 20253). Where indicated,
immediately after transfection, cells were treated with 10 µM of each HIV-1 protease inhibitors.
Darunavir (DRV) (Sigma), solubilized in DMSO, was used as positive controls. After 24 hours,
cells were lysed in ice-cold RIPA buffer (0.1% sodium dodecyl sulfate, 1% NP-40, 0.5% sodium
deoxycholate in PBS pH 7.4) supplemented with protease inhibitors cocktail. One hundred
micrograms of proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane
(
Amersham Bioscience). After blocking with 5% non-fat dry milk in TRIS buffered saline-Tween
0 (TBS-T), the membrane was incubated with 1:1000 anti-GFP (Thermo Scientific) primary
2
antibody and then with peroxide-conjugated anti-rabbit secondary antibody. Proteins were detected
using Enhanced Chemiluminescence reagents (Promega) and chemiluminescence was detected with
Chemidoc XRS detection system equipped with Image Lab Software for image acquisition (Bio-
Rad).
Fluorescence microscopy and flow cytometric analysis
GFP-PR expression was detected 24 hours after transfection. For fluorescence microscopy, cells
were grown on coverslips, fixed with 4% paraformaldehyde in PBS and observed under a
fluorescent microscope (Nikon 80i). To perform cytofluorimetric analysis, cells were harvested,
resuspended in PBS and GFP fluorescence was detected using a FACS Canto II flow cytometer
(
Ex/Em: 480/510 nm).
Modelling
The docking runs were carried out on the reference pdb structure 3NU3, complex of wild type HIV
protease with amprenavir. The ligand and water molecules were removed from the structure, while
hydrogens and charges were added with MGLTools. The inhibitors molecules were prepared and
optimized with the MMFF forcefield as implemented in Spartan 10 (Wave Function Inc.). The size
1
6

of the docking grid was 65 x 50 x 40 Ǻ, comprising the whole protein. MD refinement of the
complexes was carried out at 300 °K in the NTV ensemble with 500 ps runs.
Acknowledgments
Financial support has been provided by MIUR (Italian Ministry of University) – PRIN
2
0109Z2XRJ_009: ‘Progettazione e sintesi stereoselettiva di composti attivi verso bersagli proteici
coinvolti in patologie virali e tumorali’ and Università degli Studi della Basilicata.
References and notes
1
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
2
Ghosh, A. K.; Osswald, H. L.; Prato, G. J. Med. Chem. 2016, 59, 5172-5208.
Wensing, A. M. J.; Van Maarseveen, N. M.; Nijhuis, M. Antiviral Res. 2010, 85, 59-74.
3
4
(a) Mitsuya, H.; Maeda, K.; Das, D.; Ghosh, A. K. In HIV-1: Molecular Biology and Pathogenesis: Clinical
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b) Ghosh, A. K.; Chapsal, B. D. In Aspartic Acid Proteases as Therapeutic Targets, Ghosh, A. K., Ed.; Methods and
Principles in Medicinal Chemistry, Vol. 45; Wiley-VCH: Weinheim, Germany, 2010; pp 169−204.
(
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(a) Palmisano, L.; Vella, S. Ann. Ist. Super. Sanità, 2011, 47, 44−48;
(
b) Maartens, G.; Celum, C.; Lewin, S. R. Lancet 2014, 384, 258−271.
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(a) McArthur, J. C.; Brew, B. J.; Nath, A. Lancet Neurol. 2005, 4, 543−555;
(
b) Kramer-Hämmerle, S.; Rothenaigner, I.; Wolff, H.; Bell, J. E.; Brack-Werner, R. Virus Res. 2005, 111, 194−213.
7
(a) Bonini, C.; Chiummiento, L.; De Bonis, M.; Di Blasio, N.; Funicello, M.; Lupattelli, P.; Pandolfo, R.; Tramutola,
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(
8
(
a) Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Acc. Chem. Res. 2008, 41, 78;
b) Ghosh, A. K.; Chapsal, B.; Mitsuya, H. In Aspartic Acid Proteases as Therapeutic Targets; Ghosh, A., Ed.; Wiley-
VCH: Weinheim, Germany, 2010; pp 205–243.
(
9
(
a) Ghosh, A. K.; Anderson, D. D.; Weber, I. T.; Mitsuya, H. Angew. Chem., Int. Ed. 2012, 51, 1778-1802;
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. ChemMedChem 2010, 5, 1850-1854;
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(
(
T. J. Med. Chem. 2013, 56, 1074-1083.
1
0
(a) Ghosh, A. K.; Dawson, Z. L.; Mitsuya, H. Bioorg. Med. Chem. 2007, 15, 7576−7580;
(
b) Amano, M.; Tojo, ꢁ.; Salcedo-Gꢂmez, P. M.; Campbell, J. R.; Das, D.; Aoki, M.; Xu, C. X.; Rao, K. V.; Ghosh, A.
K.; Mitsuya, Antimicrob. Agents Chemother. 2013, 57, 2036−2046;
c) Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasamudram, T.; Kincaid, J. F.; Boross, P.; Wang, Y. F.;
(
Tie, Y.; Volarath, P.; Gaddis, L.; Harrison, R. W.; Weber, I. T.; Ghosh, A. K.; Mitsuya, H. Antimicrob. Agents
Chemother. 2003, 47, 3123−3129;
(d) Ghosh, A. K.; Osswald, H. L.; Glauninger, K.; Agniswamy, J.; Yuan-Fang Wang, Y.-F.; Hayashi, H.; Aoki, M.;
Weber, I. T.; Mitsuya, H. J. Med. Chem. 2016, 59, 6826-6837.
1
1
(a) Bonini, C.; Chiummiento, L.; Di Blasio, N.; Funicello, M.; Lupattelli, P.; Tramutola, F.; Berti, F.; Ostric, A.;
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See Supplementary Material for details.
A small aliquot of the crude was purified by column chromatography on silica gel (CH
1
9
2
0
2
Cl
/ EtOAc 95:5); H NMR (400 MHz,
= 2.0 Hz, 1H), 7.24 (d, J = 2.0 Hz,
H), 6.93 (t, J = 8.0 Hz, 2H), 4.05 (m, 2H), 3.97 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H), 3.93 (s, 3H), 3.05 (m, 5H), 1.90 (m,
H), 0.89 (dd, J = 12.4 Hz, J = 6.8 Hz, 6H).
Kanemitsu, T.; Inoue, M.; Yoshimura, N.; Yoneyama, K.; Watarai, R.; Miyazaki, M.; Odanaka, Y.; Nagata, K.; Itoh,
T. Eur. J. Org. Chem. 2016, 1874-1880.
2
/ EtOAc 95:5) affording
2
0
1
(
3) (21% yield) as amorphous solid. []
)  (ppm) 7.54 (dd, J = 8.4 Hz, J
D
-6.0 (c 1.0, CH
= 2.0 Hz, 1H), 7.33 (dd, J
2
Cl
2
); Rf = 0.3 (CH
2 2
Cl
CDCl
2
3
H
1
2
1
= 8.4 Hz, J
2
1
1
2
2
1
2
2
Gerbino, A.; Schena, G.; Milano, S.; Milella, L.; Barbosa, A.F.; Armentano, M.F.; Procino, G.; Svelto, M.;
Carmosino, M. PLoS One 2016, 11(5):
e0156021.
1
8