Reduction of peptide character of HIV protease inhibitors that exhibit nanomolar potency against multidrug resistant HIV-1 strains

Article abstract of DOI:10.1021/jm020537i

Novel HIV protease inhibitors containing a hydroxyethylamine dipeptide isostere as a transition state-mimic king structure were synthesized by combining substructures of known HIV protease inhibitors. Among them, TYA5 and TYB5 were proven to be not only p

Full text of DOI:10.1021/jm020537i

1764
J . Med. Chem. 2003, 46, 1764-1768
Brief Articles
Red u ction of P ep tid e Ch a r a cter of HIV P r otea se In h ibitor s Th a t Exh ibit
Na n om ola r P oten cy a ga in st Mu ltid r u g Resista n t HIV-1 Str a in s
Hirokazu Tamamura,* Yasuhiro Koh,† Satoshi Ueda, Yoshikazu Sasaki, Tomonori Yamasaki, Manabu Aoki,†
Kenji Maeda,† Yoriko Watai,‡ Hisashi Arikuni,‡ Akira Otaka, Hiroaki Mitsuya,†,§ and Nobutaka Fujii*
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, J apan; Department of
Internal Medicine II, Kumamoto University School of Medicine, Kumamoto 860-8556, J apan; Yokohama Laboratory,
SC BioSciences Corporation, Hodogaya-ku, Yokohama 240-0005, J apan; and Experimental Retrovirology Section, Medicine
Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received November 26, 2002
Novel HIV protease inhibitors containing a hydroxyethylamine dipeptide isostere as a transition
state-mimic king structure were synthesized by combining substructures of known HIV protease
inhibitors. Among them, TYA5 and TYB5 were proven to be not only potent enzyme inhibitors
(Ki ) 0.12 nM and 0.10 nM, respectively) but also strong anti-HIV agents (IC50 ) 9.5 nM and
66 nM, respectively), even against viral strains with multidrug resistance. Furthermore,
insertion of an (E)-alkene dipeptide isostere at the P1-P2 position of TYB5 led to development
of a purely nonpeptidic protease inhibitor, TYB1 (Ki ) 0.38 nM, IC50 ) 160 nM).
In tr od u ction
Ch em istr y
Disclosure of the molecular mechanism relevant to
each step of the HIV-1 life cycle has led to development
of many types of anti-HIV agents. The multiple drug-
combination chemotherapy, “highly active anti-retrovi-
ral therapy (HAART)”, which involves a combination of
reverse transcriptase inhibitors and protease inhibi-
tors,1-9 has dramatically improved the clinical treat-
ment of individuals with HIV-infection or AIDS. How-
ever, there still remain several serious problems includ-
ing the emergence of viral strains with multidrug
resistance, significant adverse effects and high costs.10
Many HIV protease inhibitors also have the disadvan-
tage of low bioavailability due to their peptide character.
All HIV protease inhibitors that have been clinically
used to date, contain one or more amide bonds. Discov-
ery and development of novel potent HIV protease
inhibitors that are active against multidrug resistant
viral strains and have excellent biostability and bio-
availability, are required for HAART. By combining
substructure units of known HIV protease inhibitors,
we have derived novel potent inhibitors that are effec-
tive even against multidrug resistant viruses. Subse-
quently, in an effort to improve bioavailability by
developing inhibitors that contain no amide bonds, an
(E)-alkene dipeptide isostere (EADI)11 was introduced
into the above inhibitors. The 3D structure of this (E)-
alkene peptide mimic (bond length, bond angle, and
rigidity) closely resembles that of the parent amide.
The efficacy of hydroxyethylamine dipeptide isosteres
(HDIs)12 as backbone replacements of amide bonds in
the position P1-P1′ of aspartyl protease inhibitors has
been well documented. Several target compounds con-
taining an HDI transition state-mimic were designed
based on structural information derived from reported
HIV protease inhibitors. Among them, TYA5 (7) has a
decahydroisoquinoline unit, which was used in saquina-
vir2,3 and nelfinavir,7 at the P1′-P2′ position, and a D-S-
(2-naphthyl)cysteine unit, which was used in Lilly’s
compounds,13 at the P2-P3 position (Figure 1). TYB5
(14) has a sulfonamide unit, which was used in am-
prenavir,8 at the P1′-P2′ position, and a D-S-(2-naphth-
yl)cysteine unit at the P2-P3 position. Starting with a
known, optically active epoxy amine 1,3,14-16 TYA5 (7)
and TYB5 (14) were synthesized as diastereomeric
sufoxides using general procedures illustrated in Scheme
1 (see Supporting Information). Target compounds
23a ,b (TYA1 and TYA2), in which the amide bonds at
the P1-P2 position of compound 7 were replaced by (E)-
alkenes, were synthesized using the (D-Ser, L-Phe)-type
EADI [Cbz-D-Ser-ψ[(E)-CHdCH]-L-Phe-OBut] 15 (see
Supporting Information)17 according to Scheme 2 in
company with their sulfone derivatives 24a ,b (TYA3
and TYA4). Stereochemistry at the C2-carbon of dia-
stereomers 23a ,b and 24a ,b has yet to be determined.
Compound 29a (TYB1), in which the amide bond at the
P1-P2 position of compound 14 was replaced by an (E)-
alkene, and its 2R isomer 29b (TYB2) were synthesized
using epoxy compounds 20a ,b according to Scheme 3.
Stereochemistry at the C2-carbon of diastereomers
29a ,b is based on X-ray analysis of 27b. 23a ,b and
29a ,b are all comprised of sufoxide diastereomixtures.
* Corresponding authors. Tel: +81 75 753 4551, Fax: +81 75 753
4570, e-mail: tamamura@pharm.kyoto-u.ac.jp/nfujii@pharm.kyoto-
u.ac.jp.
† Kumamoto University School of Medicine.
‡ SC BioSciences Corp.
§ National Cancer Institute.
10.1021/jm020537i CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/18/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1765
F igu r e 1. Structures of saquinavir, nelfinavir, amprenavir, Lilly’s compound and our initial target compounds, TYA5 and TYB5.
Sch em e 1a
a
Reagents: (i) N-(tert-butyl)-decahydroisoquinolinecarboxamide; (ii) 1 M TMSBr-thioanisole/TFA; (iii) N-Boc-D-S-(2-naphthyl)cysteine,
BOP, DIPEA, HOBt; (iv) 4 M HCl in 1,4-dioxane; (v) MsCl, DIPEA; (vi) NaIO4; (vii) isobutylamine; (viii) p-nitrobenzenesulfonyl chloride,
Et3N; (ix) Zn, AcOH.
Sch em e 2a
a
Reagents: (i) TsCl, pyridine; (ii) 2-naphthalenethiol, NaH; (iii) 4 M HCl in 1,4-dioxane; (iv) isobutylchloroformate, DIPEA; (v) CH2N2;
(vi) NaBH4, H2O; (vii) 0.5 M KOH in EtOH; (viii) N-(tert-butyl)-decahydroisoquinolinehydroxamide; (ix) TFA, thioanisole; (x) MsCl, DIPEA;
(xi) NaIO4.
Biologica l Resu lts a n d Discu ssion
Supporting Information).18 TYA5 showed potent anti-
HIV activity (Table 1). This potency was greater than
those of saquinavir and amprenavir, which have been
clinically used. TYB5 also showed potent activity. It was
The anti-HIV activity of compounds, TYA5 and TYB5,
was determined based on the inhibition of HIV-1-
induced cytopathogenicity in MT-2 cells (described in

1766 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Brief Articles
Sch em e 3a
a
Reagents: (i) isobutylamine; (ii) p-nitrobenzenesulfonylchloride, Et3N; (iii) TFA, thioanisole; (iv) MsCl, DIPEA; (v) NaIO4; (vi) Zn,
AcOH.
Ta ble 1. Anti-HIV Activity, Cytotoxicity, and HIV Protease
Inhibitory Activity of the Synthetic Compounds
Ta ble 2. Anti-HIV Activity of TYA5, TYB5, and TYB1 against
HIV-1 Clinical Isolates
IC50 (nM) (fold change)a MDRb
compd (no.)
IC50 (nM)a
CC50 (µM)b
SIc
Ki (nM)e
wild type
compd (no.) HIV104pre
TYA5 (7) 15 ( 2
HIVTM
HIVMM
HIVJ SL
TYA1 (23a )
TYA2 (23b)
TYA3 (24a )
TYA4 (24b)
TYA5 (7)
TYB1 (29a )
TYB2 (29b)
TYB5 (14)
AZT
>1000
>1000
>1000
>1000
9.5 ( 0.3
160 ( 40
>1000
66 ( 2
27 ( 9
17 ( 3
36 ( 11
NTd
NT
NT
NT
NT
3.9 ( 0.19
8.0 ( 1.1
NT
33 ( 12
>100
11 ( 3
>100
NT
NT
NT
NT
NT
0.12
0.38
NT
0.10
NT
0.19
NT
0.28
34 ( 4 (2×)
43 ( 6 (1×)
32 ( 4 (2×)
36 ( 8 (1×)
220( 40 (15×)
25 ( 6 (1×)
TYB5 (14) 31 ( 3
TYB1 (29a ) 180 ( 30 >1000 (>6×) 520 ( 120 (3×) 340 ( 30 (2×)
410
58
AZT
2.5 ( 0.7 43 (17×)
37(15×)
230 ( 20 (12×) 320 ( 2 (17×) 550 (29×)
480 (24×) 530 (27×) 800 (40×)
64 (26×)
saquinavir 19 ( 4
amprenavir 20 ( 3
500
650
a
IC50 values are based on the inhibition of HIV p24 antigen
expression in PBMC. All data with standard deviation are the
mean values for at least three experiments. Data without standard
saquinavir
amprenavir
indinavir
b
deviation are derived from the value for one experiment. Amino
acid substitutions in the protease-encoding region are shown in
Supporting Information.
a
IC50 values are based on the inhibition of HIV-induced
b
cytopathogenicity in MT-2 cells. CC50 values are based on the
reduction of the viability of mock-infected MT-2 cells. All data with
standard deviation are the mean values for at least three
independent experiments. Data without standard deviation are
tors, protease inhibitors with high anti-HIV activity
including against MDR HIV-1 have resulted.
Finally, work was undertaken to reduce the peptide
character of TYA5 and TYB5. TYB1, an amide bond
mimic-containing compound based on TYB5, having syn
geometry as in TYB5, showed moderate anti-HIV activ-
ity. Alternatively, TYA1-4, based on TYA5, did not
show significant activity (Table 1). (Either TYA1 or
TYA2 and either TYA3 or TYA4 are the same syn
isomers as TYA5.) It is reasonable that the anti isomer,
TYB2, did not show significant activity, whereas it was
not readily explainable why the anti-HIV activities of
compounds having amide bond mimics are less than
those of the corresponding amide-containing compounds.
Nonetheless, the anti-HIV activity of TYB1 was noted.
As such, the HIV protease inhibitory activities of TYA5,
TYB5, and TYB1 were evaluated. HIV protease activity
(the rate of enzyme reaction) was determined by use of
a surface-enhanced laser desorption/ionization (SELDI)-
MS method for quantitation of the parent peptide
substrate (see Supporting Information).19,20 As shown
in Table 1 (Ki), the protease inhibitory activities of TYA5
and TYB5 were stronger than those of saquinavir and
crixivan.4 TYA5 and TYB5 showed almost the same
protease inhibitory activity, while TYA5 showed 7-fold
higher anti-HIV activity in cells than TYB5. A suitable
correlation between protease inhibitory activity and
anti-HIV activity for these compounds was not appar-
ent. TYB1 also exhibited strong protease inhibitory
activity, although this activity was weaker than that of
derived from the value for one experiment. c Selectivity index (SI)
d
is shown as CC50/IC50
.
NT: not tested. e Ki values are based on
HIV-1 protease inhibitory activity.
noted that both TYA5 and TYB5 exhibited high selec-
tivity indexes (SIs) as comparable to that of saquinavir.
Next, the anti-HIV activities of TYA5 and TYB5 were
determined against multidrug resistant (MDR) strains
as measured by the inhibition of HIV p24 antigen
expression in peripheral blood mononuclear cells (PBMC)
(described in Supporting Information).18 TYB5 showed
nearly equivalent activity against three MDR strains
as against a wild-type strain, HIV104pre. A reverse
transcriptase inhibitor, AZT, and the reported protease
inhibitors, saquinavir and amprenavir, showed 12-40-
fold reduced potency against the three MDR strains as
compared to against HIV104pre (Table 2). TYA5 also
exhibited effective activity (half potency) against MDR
strains, except for HIVJ SL. The structure of TYA5 was
based on the combination of a decahydroisoquinoline
unit derived from saquinavir and a D-S-(2-naphthyl)-
cysteine unit used in Lilly’s compounds. The TYB5
structure was derived by combining a sulfonamide unit
from amprenavir with a D-S-(2-naphthyl)cysteine unit.
TYA5 and TYB5 are effective against MDR strains,
while saquinavir and amprenavir exhibited low efficacy.
By combining structural subunits from known inhibi-

Brief Articles
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the corresponding peptidic compound TYB5. Replace-
ment of the amide bond by an (E)-alkene at the P1-P2
position of TYB5 caused a significant decrease both in
protease inhibitory activity and in anti-HIV activity.
This suggested either that insertion of an EADI at the
P1-P2 position or an increase in hydrophobicity is not
suitable. Significant loss of anti-HIV activity of TYB1
might underline the importance of the P1-P2 amide
bond for interaction with the enzyme. This might also
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In conclusion, novel HDI-containing HIV protease
inhibitors, TYA5 and TYB5, which are highly effective
even against MDR strains, have been found by combin-
ing substructure units of reported inhibitors. Further-
more, a purely nonpeptidic inihibitor TYB1 has been
developed based on TYB5.
Ack n ow led gm en t. This work was supported in part
by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and
Technology, J apan, and the J apan Health Science
Foundation. The authors wish to thank Drs. Yoshihiko
Odagaki and Nobuyuki Hamanaka from Minase Re-
search Institute, Ono Pharmaceutical Co., Ltd., for
X-ray analysis. We are also grateful to Dr. Kazuhisa
Yoshimura from Center for AIDS Research, Kumamoto
University, for helpful discussions. Our thanks are also
extended to Dr. Terrence R. Burke, J r., NCI-Frederick,
NIH, for proofreading the manuscript and providing
useful comments.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization data of novel synthetic com-
pounds, procedures of biological assays, synthetic scheme for
EADI 15 (Scheme S1), X-ray crystallographic data for 27b,
and HPLC charts for TYA1, TYA2, TYA5, TYB1, TYB2, and
TYB5. This material is available free of charge via the Internet
at http://pubs.acs.org.
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