Lysine sulfonamides as novel HIV-protease inhibitors: Nε-Acyl aromatic α-amino acids

Article abstract of DOI:10.1016/j.bmcl.2006.04.011

A series of lysine sulfonamide analogues bearing Nε-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.

Full text of DOI:10.1016/j.bmcl.2006.04.011

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3459–3462
Lysine sulfonamides as novel HIV-protease inhibitors:
Ne-Acyl aromatic a-amino acids
à
*
´
´
Brent R. Stranix, Jean-Franc¸ois Lavallee, Guy Sevigny, Jocelyn Yelle,
§
´
Valerie Perron, Nicholas LeBerre, Dominik Herbart and Jinzi J. Wu
Ambrilia Biopharma Inc., 1000 chemin du Golf, Verdun, QC, Canada H3E 1H4
Received 16 February 2006; revised 31 March 2006; accepted 3 April 2006
Available online 27 April 2006
Abstract—A series of lysine sulfonamide analogues bearing Ne-acyl aromatic amino acids were synthesized using an efficient
synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and
multiple-protease inhibitor-resistant HIV viruses.
Ó 2006 Elsevier Ltd. All rights reserved.
Protease inhibitors (PIs) along with reverse transcriptase
inhibitors (RTIs) are the basis of the highly active
anti-retroviral therapy (HAART), which has led to a
significant reduction in HIV-associated morbidity and
mortality since its widespread availability in 1996.1
HIV-1 protease is an excellent therapeutic target since
its inhibition prevents proteolytic processing of the
Gag and Gag-Pol polyproteins, thereby blocking viral
maturation.2–4 Unfortunately, long-term use of
HAART often leads to the development of a high
proportion of drug resistance in AIDS patients.5,6 One
of the highest priorities in anti-retroviral drug research
today is the development of new HIV inhibitors for
the treatment of patients infected by multi-resistant viral
strains through combination therapy.
studies of one such compound suggested that this class
of molecules was promising.8 We had noted from our
previous studies that potent inhibitors could be obtained
through the acylation of the e-terminal amine of the ly-
sine scaffold with a second amino acid moiety, in partic-
ular those with aromatic side chains. We further
ventured to develop more potent compounds based on
this scaffold through a rational approach consisting of
modulating the e-terminal acylated aromatic amino
acid9 (Fig. 1).
Using (S)-a-amino-caprolactam 1 as starting material
(Scheme 1), reductive alkylation followed by sulfonami-
dation of the free amine gave excellent yields of enantio-
merically pure crystalline intermediate lactam. A novel
and mild procedure was developed in order to effect a
reductive cleavage of the lactam quantitatively and with-
out any detectable racemization. Reaction of the inter-
mediate diamide with Boc2O/DMAP in acetonitrile10
gave a quantitative conversion to the bis–boc intermedi-
ate 2. Reduction of the resulting activated lactam was
effected by NaBH4 in EtOH, yielding a bis–boc-protect-
ed lysinol derivative 3. This procedure concomitantly
During the course of our research, we established that a
novel scaffold base on lysine sulfonamides could
generate potent compounds for both enzyme inhibition
and anti-viral effects.7 Furthermore, cross-resistance
Keywords: HIV protease inhibitor; Lysine; Amino acids; Resistant
viral strains.
*
Corresponding author. Tel.: +1 514 732 3218; fax: +1 514 751
2709; e-mail: bstranix@ambrilia.com
O
Present address: Gemin-X Biotechnologies Inc., 3576 Avenue du
Parc, Montreal, QC, Canada H2X 2H7.
N
Ar
H2N
S
CH2
N
H
4
O2
NH
à
Present address: Chronogen Inc., 521 Rachel E Suite 22, Montreal,
QC, Canada H1W 4A4.
R
OH
§
Figure 1. General Structure of lysine sulfonamide HIV protease
inhibitors.
Present address: INRS-Institut Armand-Frappier, 531, des Prairies
Blvd, Building 27, Laval, Que., Canada H7V 1B7.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.011

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B. R. Stranix et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3459–3462
O
Boc
d
O
N
HN
N
N
a, b, c
O2S
Boc
NH
O2S
Boc
H2N
OH
3
2
1
HN Boc
e, f
AcN
R1
R1
R1
N
HN
R2
O2S
g
N
R2
HO
R2
HO
N
N
H
O
COR3
OH
O
COR3
O
5-40
NH2
4
Scheme 1. Reagents and conditions: (a) i-PrCHO, STAB, DCM 89%; (b) 4-(AcNH)PhSO2Cl, TEA, EtOAc 88%; (c) Boc2O, MeCN, DMAPcat.
80–99%; (d) NaBH4, EtOH 6 h; (e) HCl, EtOH, rt 95%; (f) EDAC HOBt, DMF, 35–95%; (g) R3 COX, acetone:1 M, K2CO3 1:1.
Table 1. Inhibition constants of compounds for HIV aspartyl protease
and whole-cell anti-viral inhibition
removed the acetyl group protecting the aniline moiety.
An acidic deprotection of the Boc groups liberated the
free amines. The amino alcohol obtained could be
selectively acylated with a variety of activated substituted
amino acids. Acylation of the pendant aniline was not
observed. The N-substituted amino acids 4 were
obtained by Schotten–Baumann type acylations of com-
mercially available amino acids with commercial anhy-
drides, acid chlorides or active esters. The biphenyl
derivatives were obtained by the reaction of 2-Br Phe
derivatives with phenyl boronic acids through Suzuki-
type coupling. The final products 5–40 were then sub-
jected to a preparative RPHPLC purification followed
by LC/MS and NMR characterization.9 The products
were then evaluated as protease inhibitors in an in vitro
enzyme assay11 and in cell based assays.12 The effect of
spacer length between the e-amide and a phenyl group
has been discussed previously,7 along with the effect of
an a-amino group. The encouraging results prompted
us to examine more closely the effects of the phenyl
group’s distance and geometry using non-natural amino
acids. Table 1 describes the inhibition constants (IC50)
of the compounds 5–13 on the activity of purified HIV
protease, and their anti-viral activity (EC50) determined
in whole-cell cytopathic assay.
O
R1
N
CH2OH
(CH2)
O
O
O
S
CH3
4 N
H
N
R2
O
H2N
Compound R1
R2
a
IC50 (nM) EC50
a,b
(nM)
5
6
H
H
H
C6H5CH2
Ph
C6H5CH2CH2
12
>300
40
2000
NR
9000
NR
1000
120
258
200
16
7
8
CH2– C6H4CH (TIC)
130
9
CH3
H
C6H5CH2
6.5
1.2
10
11
12
13
Naphthyl-1-CH2
Naphthyl-2-CH2
Biphenyl-2-CH2
(C6H5)2CH
H
1.2
0.521
0.500
H
H
a Mean of at least two experiments.
b Anti-viral activity using the molecular clone HIV-1 NL4-3 in MT-4
cells.
the bulk of the R2 group in the terminal amino acid also
gave significant increase in IC50 potency. Both 1 and 2
naphthylalanines (10 and 11) gave similar results in
IC50, 1.2 nM and a very potent 100–200 nM in the
whole-cell anti-viral assay. Our strategy then focused
on combining both the geometric aspect and the hydro-
phobic bulk by using an o-biphenyl alanine 12 and a
diphenylalanine 13. This approach, using the steric bulk
of the second phenyl group to enrich the trans confor-
mation population, yielded highly potent compounds
with an IC50 of 0.5 nM, and anti-viral EC50 values of
200 and 16 nM for both compounds, respectively.
Encouraged by an impressive activity of 16 nM for
compound 13 we addressed the stereochemistry of the
diastereomeric compound. We synthesized the stereoiso-
mers through similar chemistry as discussed using the
commercially available D-amino acids, and tested the
purified compounds 13SR, 13RR and 13RS (>97% puri-
ty, de >95%) against the purified enzyme and in whole-
cell anti-viral assay. We confirmed the S,S stereoisomer
13 to be the most active compound as shown in Table 2.
The Moc-Phe derivative 5, closely related to
a
compound from a previous article, gave a respectable
IC50 of 12 nM and some moderate anti-viral activity in
whole-cell assays of 2 lM. As in our previous approach
the spacer length between the phenyl and e-amide was
varied in the amino acid series using Moc-Phg 6, placing
the phenyl directly on the carbon and Moc-HomoPhe, 7
increasing the distance by one methylene unit. The effect
of decreasing or increasing the distance was similar in
that a marked decrease in anti-protease and anti-viral
activity was noted. In order to explore the conforma-
tional preference of the phenyl moiety, we synthesized
a conformationaly locked phenylalanine analogue using
a Moc-Tic 8, which places the phenyl in a (-g) gauche
position.13 This resulted in a >10-fold decrease in
enzyme inhibition potency. Conversely, favouring the
trans conformation by placing a methyl group onto
the a0-amino group (Moc-N-Me-Phe) 9 increased the
potency ꢀ2-fold with respect to the Moc-Phe. Increasing

B. R. Stranix et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3459–3462
3461
Table 2. The effect of stereoisomers on the inhibition constants of
compounds for HIV aspartyl protease and whole-cell anti-viral assays
mentally in order to evaluate the impact of the
changes at the enzyme inhibition level. IC50 values were
determined from a dose–response curve, whereas Ki val-
ues were obtained by fitting initial rates to a tight-bind-
ing inhibition equation.14 Also, EC50 were determined
for laboratory adapted wild-type strain NL4-3, multi-
PI-resistant strain 4596 and Saquinavir-resistant strain
(Saqr strain)15–19 to assess their potential against
resistant viral strains.
O
H
(R,S) N
CH2OH
O
O
O
S
(CH2)4
N
H
CH3
N
(R,S)
O
Ph
Ph
H2N
a
a,b
Compound
Absolute configuration
IC50
EC50
16
13
S,S
S,R
R,R
R,S
0.500
25
The non-acylated compound 14 (obtained by acid catal-
ysed Boc deprotection of 18) gave an IC50 of 0.5 nM and
an excellent anti-viral activity of 44 nM against NL4-3
strain. Small alkyl amides such as acetyl (15) and cyclo-
propyl (16) were first examined. The acetyl gave a reduc-
tion in enzyme inhibition but retained a potent 66 nM
on the wt whole-cell assay. Cyclopropyl amide was more
potent with a Ki of 0.45 nM and EC50 of 25 and 46 nM
for wt and 4596 strains, respectively. A more bulky
amide bearing a cyclohexyl ring (17) showed a decrease
in potency for both enzymatic and whole-cell assays. A
13SR
13RR
13RS
3,000
> 100,000
850
> 300
13
a Mean of at least two experiments.
b Anti-viral activity using the molecular clone HIV-1 NL4-3 in MT-4
cells.
We also explored the effect of various acyl groups on the
a0-amino of the L-diphenylalanine portion of the mole-
cule (Table 3). Both IC50 and Ki were assessed experi-
Table 3. Inhibition constants of compounds 13–40 for HIV aspartyl protease and anti-viral assays
O
H
N
CH OH
2
O
O
1
S
(CH )
2 4
N
H
R
N
Ph
Ph
13-40
H N
2
R1
Kia,c (nM))
EC50 (nM) Saqr
a,e
a
IC50 (nM)
a,b
EC50 (nM) NL4-3
a,d
EC50 (nM) 4596
Compound
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
CH3O–CO
0.50
0.50
1.5
0.04
0.56
16
44
66
25
93
41
62
78
19
34
68
39
32
42
24
153
84
>
38
43
19
H
Ac
66
46
Cyclopropyl-CO
Cyclohexyl-CO
(CH3)3CO–CO
(CH3)2N–CO
0.43
<3.8
0.40
0.44
0.51
0.52
0.43
0.46
0.68
0.65
0.40
0.40
0.90
0.40
0.50
0.24
0.12
0.48
0.39
0.63
0.40
0.53
0.33
0.56
0.5
0.45
17
186
290
56
0.50
0.58
0.55
0.60
0.47
0.44
0.55
0.66
0.40
0.50
0.70
0.50
0.40
0.34
0.40
0.36
0.02
0.58
0.40
0.47
0.33
0.48
0.45
4-Morpholine-CO
Pyrazine-CO
Pyrrole-2-CO
46
51
2-Pyridyl-CO
3-Pyridyl-CO
4-Pyridyl-CO
161
25
26
36
2-CH3-3-pyridyl-CO
6-CH3-3-pyridyl-CO
3-HO-2-pyridyl-CO
6-H2N-3-pyridyl-CO
6-HO-3-pyridyl-CO
3-Picolyl-CO
34
186
72
>
194
17
18
20
64
64
88
16
55
14
128
18
2-Picolyl-O-CO
3-Picolyl-O-CO
4-Picolyl-O-CO
2-HO-phenyl-CO
3-HO-phenyl-CO
4-HO-phenyl-CO
3-HO-4-NO2-phenyl-CO
4-HO-3-NO2-phenyl-CO
4-HO-3-CH3O-phenyl-CO
14
4
18
13
134
30
34
10
35
23
a Mean of at least three experiments.
b Anti-viral activity using the molecular clone HIV-1 NL4-3 in MT-4 cells.
c The Ki is determined using XLfit3 software (Version: 3.0.3 Build: 09) according to the following equation (Inhibition)14: Y = Vo/(2 E)
*
{((K + x ꢁ E)2 + 4
K
E)0.5 ꢁ (K + x ꢁ E)}; where Y is the enzyme rate (RFU/min), x is the concentration of the compound (gM), Vo is the
*
*
maximum rate (initial rate measured from 200 to 600 s) (RFU/min), E is the protease concentration (gM) and K is the Ki of the compound.
d 4596; 46I, 82T, 84V, 10R, 63P.
e SaqR; 48V, 90M.

3462
B. R. Stranix et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3459–3462
similar pattern was observed for terminal carbamates
and ureas, where smaller moieties were more potent
than bulky groups. This is exemplified in the case of car-
bamate compounds Moc (13) and Boc (18), Ki 0.04 and
0.5 nM, EC50 wt 16 and 41 nM, respectively. Similar Kis
were obtained for both dimethyl and morpholyl ureas 19
and 20. However, a slight increase is noted for the
dimethyl urea whole-cell EC50 value. Heterocyclic
amides and phenolic amides were introduced as R1 in
order to assess the influence of H-bond acceptors and
donors at this position. 2-pyrrole carboxamide 22, 2-
pyridyl and pyrazinoyl amides (23 and 21) gave very po-
tent compounds with the pyrazinoyl compound showing
EC50s for NL4-3 and 4596 strains of 19 and 46 nM,
respectively. The nicotinoyl and 4-picoloyl derivatives
24 and 25 were added and a small regiomeric effect
was observed, especially significant on the 2-pyridyl
derivative 23, where a significant drop in potency was
observed in the whole-cell assays. The addition of sub-
stituents CH3, NH2 and OH to the nicotinamide and
picoloyl amide moieties (26–30) showed some influence
on EC50. Increasing the distance between the terminal
amine and the heterocycles was assessed by compounds
3-picolyl-CO 31 (2 atoms) and carbamates 32–34 (3
atoms). Of these, the three carbamates 32–34 gave high-
ly potent compounds with EC50s of 17–20 nM for NL4-
3 and 13–18 nM for the multi-resistant strain 4596. The
Ki of compound 34 gave an excellent 0.02 nM on the
purified enzyme. Phenolic amides 35–37 were synthe-
sized and very similar Kis were obtained for the 2, 3
and 4 OH regiomers, with the 30-OH derivative 36 show-
ing the best activity in the whole-cell assays. Increasing
the acidity by addition of a nitro group to the phenyl
ring had the effect of lowering both the Ki and EC50
for the 30-phenol 39. Addition of an electron-withdraw-
ing group or an electron-donating group to the 40-OH
(38 and 40) did not affect the Ki however a marked
improvement was noted on the whole-cell assay with
an excellent 14 nM being recorded for compound
OCH3. Also tabulated in Table 3 are results for whole-
cell assays for strain Saqr. Only 4 compounds (13, 16,
32 and 34) were evaluated which in all cases showed a
similar or greater potency on this strain than on the
wild-type strain.
Acknowledgments
We gratefully acknowledge A. Dubois, A. M. Lemieux,
Y. Zhao and B. Tian for their participation in the bio-
logical analyses. The following reagents were obtained
through the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH: HIV-1
mutated strains, SaqR (catalog no. 2948) from Dr. N.
Roberts and P. Tomlinson, and 4596 (catalog no.
2840) from Dr. E. Emini and Dr. W. Schleif.
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The results shown in Table 3 suggest that the acylation
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In summary, the acylation of the e-amine of N-isobu-
tyl-N-aminophenylsulfonamido lysinol with substituted
aromatic amino acids yields potent HIV protease
inhibitors. In particular, compounds bearing the vari-
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