Design, synthesis, and biological evaluation of HIV/FIV protease inhibitors incorporating a conformationally constrained macrocycle with a small P3′ residue

Article abstract of DOI:10.1016/S0960-894X(00)00641-7

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tripeptide (Ala-Val-Phe), a motif with a small P3′ residue effective against the FIV protease and the drug-resistant HIV proteases, has been

Full text of DOI:10.1016/S0960-894X(00)00641-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 219±222
Design, Synthesis, and Biological Evaluation of HIV/FIV
Protease Inhibitors Incorporating a Conformationally Constrained
Macrocycle with a Small P30 Residue
Chi Ching Mak,a Van-Duc Le,a Ying-Chuan Lin,b John H. Elderb
and Chi-Huey Wonga,*
aDepartment of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 19 September 2000; accepted 3 November 2000
AbstractÐA series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tri-
peptide (Ala-Val-Phe), a motif with a small P30 residue e€ective against the FIV protease and the drug-resistant HIV proteases, has
been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were
developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also e€ective against some
drug-resistant as well as TL3-resistant HIV proteases. # 2001 Elsevier Science Ltd. All rights reserved.
Despite the approval of several inhibitors of the HIV
protease,1,2 many drug-resistant variants of HIV have
been identi®ed.2 4 Feline immunode®ciency virus (FIV)
causes an immunode®ciency syndrome in cats compar-
able to AIDS in humans. Although the active site struc-
tures of HIV and FIV proteases are superimposable and
have an identical mechanism of catalysis,5,6 all the
approved inhibitors, with Ki values in the low nanomolar
range for HIV protease, only bind to the FIV protease in
the micromolar range. FIV protease has a more restric-
ted P3 binding subsite than does the wild-type HIV pro-
tease and resembles many drug-resistant HIV proteases
that were found to have a shrunk P3 binding subsite;7
good inhibitors of the FIV protease with small P3 resi-
due, especially Ala, were thus shown to be better inhibi-
tors of the wild-type and drug-resistant HIV proteases.7
For example, compound 1 (TL3) with Ala residue in P3
positions demonstrated nanomolar inhibitions in wild-
type FIV/HIV proteases and some drug-resistant HIV
mutants. Also 1 showed strong ability to inhibit replica-
tion of FIV, HIV, and SIV with virtually the same degree
of e€ectiveness (ꢀ1 mM) in tissue culture.7 FIV protease
therefore has been considered as the drug-resistant
phenotype of HIV protease and used as a model for the
development of new inhibitors to tackle the problem of
drug resistance.
It has been demonstrated that the introduction of
macrocyclic structures in HIV protease inhibitors can
improve their binding anity and resistance toward
proteolytic enzymes.8 14 In addition, HIV protease inhi-
bitors containing the norstatine analogue (3-amino-2-
hydroxy-4-phenylbutyric acid),15 18 particularly 2 (JE-
2147) (Fig. 1), have very potent antiviral activities in
vitro and exhibit good oral bioavailability and plasma
pharmacokinetic pro®les. These reports have prompted
us to disclose our approach to the design, synthesis,
and in vitro evaluation of a series of norstatine-based
macrocyclic inhibitors 3±7 (Fig. 1). The macrocycle was
designed to mimic the conformationally constrained
Ala-Val-Phe motif which, with Ala in the P3 position,
was used in the development of FIV and drug-resistant
HIV protease inhibitors. We have found that several of
these structures inhibit the FIV and drug-resistant HIV
enzymes at low nanomolar concentrations.
Results and Discussion
The X-ray structure of FIV protease complexed with 119
reveals that the P1 and P3 residues are positioned very
*Corresponding author. Tel.: +1-858-784-2487; fax: +1-858-784-2409;
e-mail: wong@scripps.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00641-7

220
C. C. Mak et al. / Bioorg. Med. Chem. Lett. 11 (2001) 219±222
the S isomer 10 (Fig. 2). In addition, replacement of
Cbz in TL3 with para-toluenesulfonyl protecting groups
increases the activity (ꢀ5-fold) toward FIV protease.20
As a result, extending the main chain in the N-terminus
of 9 with Z-Ala-Val-OH and p-CH3-C6H4SO2-Ala-Val-
OH to produce 3 and 4, respectively, is our initial
approach. Dunn et al.21 have also reported that nano-
molar inhibition for FIV protease can be achieved by
capping the N-terminus with the small acetyl protecting
group together with a relatively large side-chain residue
such as naphthalene methyl in the P3 and P30 positions.
Modeling of these inhibitors complexed with the
enzyme revealed the naphthalene group has moved
from the P3 or P30 binding subsites toward the outside
of the cleft. Based on this observation, we also have
designed compounds 5±7 containing the dipeptides Ac-
Trp-Val-OH, Ac-Phe-Val-OH and Ac-Tyr-Val-OH as
the P3 and P2 residues.
Figure 1.
Our most ecient synthetic route toward our inhibitors
3±7 is illustrated in Scheme 1, as described for cyclic
inhibitor 5. The synthesis involves the preparation of the
hydroxyl acid 12 derived from 11,22 followed by incor-
poration of the macrocycle 8 after Boc removal. Macro-
cyclization of 8 was achieved eciently by the treatment
of Cs2CO3 in CH3CN (10 mM) in the presence of 1 equiv
of tetrabutylammonium iodide at room temperature.
Despite introduction of the ¯exible trimethylene ether
linker, the NMR spectrum indicates four unequivalent
aromatic protons in the cycle, suggesting that the 15-
membered ring system is highly constrained and the
aromatic ring is not able to rotate freely.
closely in the neighboring hydrophobic pockets. Con-
necting the P1 and P3 residues with the aliphatic chain in a
macrocycle may render the solution conformation similar
to the bound conformation in the active site of the pro-
tease. With this speculation, we decided to choose the
macrocycle 8 (Scheme 1) as a mimic of the tripeptide
(Ala-Val-Phe)11 13 in our macrocyclic protease inhibitors.
The con®guration of the C-OH stereogenic center in the
norstatine-based protease inhibitors is known to be
important for the inhibitory activity.15 18 Speci®cally,
compound 9 with R stereochemistry around its essential
carbinol function was found to have better inhibitory
activity (ꢀ6-fold) for HIV/FIV proteases than that of
The inhibitory activities of each compound against FIV,
HIV, drug-resistant mutant HIV, and TL3-resistant
mutant HIV proteases were determined as described
previously,7 and the results are summarized in Table 1.
The results revealed that 3 and 4 have similar potency
against HIV/FIV proteases though they are slightly less
potent than TL3. However the inhibitors 5±7 showed a
comparable activity to TL3 against HIV protease.
Compounds 5±7 also exhibited remarkable activities
toward FIV protease with IC50 values about 2- to 3-fold
lower than TL3. In particular, 7 was found to be the
most potent cyclic inhibitor of FIV protease reported to
date, with an IC50 value of only 27 nM.
Although TL3 displayed a signi®cant inhibitory activity
against HIV/FIV and some drug-resistant HIV pro-
teases, its development of resistance has been studied
Scheme 1. Reagents and conditions: (a) Boc-Val-OH, HBTU,
DIPEA, THF; (b) TFA, CH2Cl2; (c) Boc-Tyr-OH, HBTU, DIPEA,
THF; (d) CsCO3, TBAI, CH3CN; (e) TFA, CH2Cl2; (f) Ac-Trp-Val-
OH, HBTU, DIPEA, THF; (g) LiOH, MeOH/H2O; (h) TFA,
CH2Cl2; (i) HBTU, DIPEA, DMF.
Figure 2.

C. C. Mak et al. / Bioorg. Med. Chem. Lett. 11 (2001) 219±222
221
Table 1. Biological activity (IC50 nM) of TL3 and 3±7 against FIV-WT, HIV-WT, drug-resistant HIV and TL3-resistant HIV proteases
Drug-resistant HIV mutants
TL3-resistant HIV mutants
FIV PR
HIV PR
HIV
(G48V)
HIV
(V82F)
HIV
(V82A)
HIV (L24I, M46I, F53L,
L63P, V77I, V82A)
Inhibitors
IC50 (nM)
TL3
72
95
147
49
35
27
4
10
13
10
7
20.5 (5Â)a
ndb
nd
71 (7Â)
94 (13Â)
57 (9.5Â)
14.9 (4Â)
nd
nd
30 (3Â)
32 (4Â)
26 (4Â)
16 (4Â)
nd
nd
10 (1Â)
12 (2Â)
11 (2Â)
144 (36Â)
nd
nd
49 (5Â)
79 (11Â)
70 (12Â)
3
4
5
6
7
6
aNumber in parentheses denotes number of fold higher IC50 values in inhibition compared to that of the wild-type HIV PR.
bNot determined.
under forcing conditions and TL3-resistant HIV proteases
with up to six mutations have been identi®ed. Since inhi-
bitors 5±7 showed better activity toward FIV protease as
compared to TL3, the inhibitory activities of 5±7 were also
evaluated against several drug-resistant and TL3-resistant
HIV proteases. All these mutant enzymes have their
mutated residues a€ect the S3 and S30 subsites, which
have been identi®ed as the important areas associated
with the development of drug resistance.7
Figure 4.
Compared to TL3, which is 5-fold weaker toward the
drug-resistant mutant G48V, compounds 5±7 are less
e€ective against this mutant, and similarly e€ective
against the V82F mutant. In the case for TL3-resistant
mutants, the results were also noteworthy. The cyclic
inhibitors 5±7, in general, have higher potency than that
of TL3. In particular, 5±7 showed an improved inhibi-
tory activity against the hexa-mutant induced by TL3.
These results indicate that inhibitors with the designed
cyclic motif such as 5±7 may also be e€ective against a
broad range of HIV variants.
attributed to the macrocycle better pre-organized in a
favorable enzyme-bound conformation, resulting in an
entropy advantage gained over the acyclic compound.11 14
It is also interesting to note that the keto amide 17 (Fig. 4)
is about 10-fold more active than the corresponding
hydroxyl amide 9 against HIV/FIV proteases.5,23 Oxida-
tion of 5±7 to the corresponding keto amide analogues
may generate a better series of protease inhibitors.
The macrocycle was found to be important for the over-
all activity of the inhibitors, as the hydroxyl methyl esters
13±15 (Fig. 3) were found to be less potent than that of 9
against HIV/FIV proteases. Also, hexapeptide 16, which
is the acyclic analogue of 7, displayed about 20- or 35-
fold higher IC50 values for wild-type FIV and HIV PRs,
respectively, as compared to 7. Such phenomena may be
In summary, we have described a general strategy to
introduce a structural and functional mimic of the tri-
peptide (Ala-Val-Phe) in the form of a 15-membered
ring into our protease inhibitors. Certain inhibitors were
developed expressing low nanomolar HIV/FIV protease
inhibitory activity. Bicyclic inhibitors incorporating two
similar macrocycles are currently under investigation
and will be reported in due course.
Acknowledgements
This research was supported by the N.I.H. (GM48870).
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