Design, synthesis, and X-ray structure of substituted bis-tetrahydrofuran (bis-THF)-derived potent HIV-1 protease inhibitors

Article abstract of DOI:10.1021/ml100289m

We investigated substituted bis-THF-derived HIV-1 protease inhibitors in order to enhance ligand-binding site interactions in the HIV-1 protease active site. In this context, we have carried out convenient syntheses of optically active bis-THF and C4-substituted bis-THF ligands using a [2,3]-sigmatropic rearrangement as the key step. The synthesis provided convenient access to a number of substituted bis-THF derivatives. Incorporation of these ligands led to a series of potent HIV-1 protease inhibitors. Inhibitor 23c turned out to be the most potent (K_i = 2.9 pM; IC₅₀ = 2.4 nM) among the inhibitors. An X-ray structure of 23c-bound HIV-1 protease showed extensive interactions of the inhibitor with the protease active site, including a unique water-mediated hydrogen bond to the Gly-48 amide NH in the S2 site.

Full text of DOI:10.1021/ml100289m

LETTER
pubs.acs.org/acsmedchemlett
Design, Synthesis, and X-ray Structure of Substituted
Bis-tetrahydrofuran (Bis-THF)-Derived Potent HIV-1 Protease Inhibitors
Arun K. Ghosh,*,† Cuthbert D. Martyr,† Melinda Steffey,† Yuan-Fang Wang,‡ Johnson Agniswamy,‡
||
Masayuki Amano,§ Irene T. Weber,‡ and Hiroaki Mitsuya§,
†Departments of Chemistry and Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, United States
‡Department of Biology, Molecular Basis of Disease, Georgia State University, Atlanta, Georgia 30303, United States
§Departments of Hematology and Infectious Diseases, Kumamoto University Graduate School of Medical and Pharmaceutical Sciences,
Kumamoto 860-8556, Japan;
Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, United States
S Supporting Information
b
ABSTRACT: We investigated substituted bis-THF-derived HIV-1 protease inhibitors in
order to enhance ligand-binding site interactions in the HIV-1 protease active site. In this
context, we have carried out convenient syntheses of optically active bis-THF and C4-
substituted bis-THF ligands using a [2,3]-sigmatropic rearrangement as the key step. The
synthesis provided convenient access to a number of substituted bis-THF derivatives.
Incorporation of these ligands led to a series of potent HIV-1 protease inhibitors. Inhibitor
23c turned out to be the most potent (Ki = 2.9 pM; IC50 = 2.4 nM) among the inhibitors. An X-ray structure of 23c-bound HIV-1
protease showed extensive interactions of the inhibitor with the protease active site, including a unique water-mediated hydrogen
bond to the Gly-48 amide NH in the S2 site.
KEYWORDS: HIV-1 protease inhibitors, Darunavir, bis-THF, drug-resistant, design, synthesis, X-ray structure
n our continuing studies aimed at the design and synthesis of
novel HIV-1 protease inhibitors to combat drug resistance, we
number of syntheses of the bis-THF ligand have been reported,
including several optically active routes from our research
group.8-12 Quaedflieg and co-workers reported a chiral synthesis
that involved a conjugate addition of nitromethane.13 Other
syntheses have been attempted with the use of various catalysts to
obtain the desired product.14 Interestingly, many of these
syntheses require late-stage resolution to obtain the enantio-
merically pure ligand. Furthermore, the reported syntheses are
not convenient for the preparation of C4-substituted derivatives.
In view of this shortcoming, we investigated a new optically active
synthesis utilizing an efficient [2,3]-sigmatropic rearrangement
as the key step and an inexpensive chiral precursor as the starting
material. The synthetic route provided convenient access to
optically active bis-THF and functionalized bis-THF ligands.
We incorporated these ligands in the hydroxyethylsulfonamide
isostere, and the resulting HIV-1 protease inhibitors were
evaluated in enzyme inhibitory and antiviral assays.
I
have developed a variety of exceedingly potent inhibitors.1 One
of these inhibitors, darunavir (1, Ki = 16 pM, antiviral IC50 = 4.1
nM, Figure 1) was first approved in June 2006 for the treatment
of HIV/AIDS patients harboring drug-resistant HIV-1 variants.2
Later, darunavir received an expanded approval for the treatment
of all therapy-naïve HIV/AIDS patients, including pediatric
patients.3 Darunavir incorporates a stereochemically defined
(3R,3aS,6aR)-bis-tetrahydrofuranyl (bis-THF) urethane as the
P2 ligand in a (R)-hydroxyethyl sulfonamide isostere.4 Structural
studies of darunavir documented that the bis-THF ligand is
involved in a network of hydrogen bonding interactions with the
protein-backbone of the HIV-1 protease, as formulated in our
“backbone binding” concept.5 A number of other clinical pro-
tease inhibitors (2, Brecanavir; 3, GS-8374) have also incorpo-
rated this bis-THF ligand and demonstrated marked antiviral
potency against a panel of multidrug-resistant HIV-1 variants.6,7
In our continuing studies toward maximizing ligand-binding
site interactions, based upon the X-ray structure of 1-bound HIV-
1 protease, we further speculated that the incorporation of an
alkoxy substituent at the C4-position of bis-THF could lead to
new interactions with the backbone NH of Gly-48. We therefore
required a stereoselective synthesis that would provide access to
C4-substituted bis-THF derivatives for further optimization. The
bis-THF ligand contains three contiguous stereogenic centers. A
The synthesis of the bis-THF ligand started with known
compound 4.11 It was prepared in multigram quantities by Wittig
olefination of (S)-glyceraldehyde acetonide with (ethoxycarbonyl-
methylene) triphenylphosphorane, as described previously.11
The requisite (S)-glyceraldehyde can be obtained from ascorbic
Received: December 6, 2010
Accepted: January 13, 2011
Published: January 27, 2011
r
2011 American Chemical Society
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Scheme 2. Syntheses of Substituted Bis-THF Ligands
Figure 1. Structures of protease inhibitors 1-3.
Scheme 1. Synthesis of Bis-THF Ligand 11
yield. Only a small amount of the transesterification product
(<5%) and a small amount (<5%) of starting material were
recovered in this reaction.
We then investigated a [2,3]-sigmatropic rearrangement of 7
under a variety of conditions. Reaction of 7 with KOtBu at 23 °C
proceeded smoothly but provided 9 a 4:1 diastereoselective ratio
at the C2-chiral center. Lowering the temperature to -10 °C
resulted in a marked increase in selectivity (17:1) with a
moderate yield (63%). It should be noted that the C2-chiral
center would be eliminated en route to the bis-THF ligand;
however, stereoselectivity is important for the synthesis of C4-
derivatives. Reactions with LiHMDS in THF at -20 °C provided
9 in 55% yield and 6:1 diastereoselectivity. However, the same
reaction at -40 °C to -30 °C for 1 h provided the best yield.
Diastereomer 9 was obtained as a single product in 80% yield.
The stereochemical outcome of the [2,3]-sigmatropic rearrange-
ment can be rationalized by the proposed transition-state model
8, in which the allylic C-O bond is orthogonal to the plane of the
allylic CdC and is antiperiplanar with respect to the approaching
carbanion, as described by Bruckner and co-workers.23 For the
synthesis of the bis-THF ligand, alcohol 9 was converted to the
corresponding mesylate. LAH reduction of the mesylate pro-
vided alcohol 10 in 74% yield, over 2 steps. Oxidative cleavage of
the terminal alkene followed by acid-catalyzed cyclization, in the
presence of a catalytic amount of p-TsOH in CHCl3 at reflux,
afforded the bis-THF alcohol 11 in 80% yield.
acid,15,16 L-arabinose,17 L-serine,18 and L-tartaric acid,19 as reported.
We used the L-tartaric acid-derived procedure for our synthesis.
Dibal-H reduction of 4 resulted in alcohol 5 (Scheme 1).20,21
We investigated the O-alkylation of 5 using tert-butyl bromo-
acetate 6 under a variety of conditions. The use of KOtBu in THF
at 23 °C for 2 h resulted in a single product, 7 in 56% yield.
O-alkylation, using Cs2CO3 in DMF also proceeded with a
moderate yield (59%). However, the use of CsOH-H2O and
activated molecular sieves, in the presence of tetrabutylammo-
nium iodide in acetonitrile, provided the best results.22 These
conditions afforded the desired O-alkylated product 7 in 85%
As shown in Scheme 2, rearrangement product 9 was utilized
for the synthesis of C4-substituted bis-THF ligands. Alcohol 9
can be conveniently functionalized into derivatives which can
further interact with the HIV-1 protease backbone atoms. Thus,
protection of the free hydroxyl group provided methyl and
benzyl ethers 12 and 13, respectively. LAH reduction, followed
by ozonolysis, and acid-catalyzed cyclization resulted in com-
pounds 14 and 15, respectively. Inversion of the C2-hydroxyl
group of 9 using Mitsunobu’s protocol24 gave 16 in 87% yield.
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Figure 2. Stereoview of the X-ray structure of inhibitor 23c (green)-bound HIV-1 protease (pdb code: 3QAA). All strong hydrogen bonding
interactions are shown as dotted lines.
Scheme 3. Syntheses of Protease Inhibitors
Table 1. Structure and Activity of Inhibitors
Selective deprotection of the nitro benzoate with potassium
carbonate in methanol at 0 °C, and subsequent protection with
methyl iodide or benzyl bromide provided methyl and benzyl
ethers 17 and 18, respectively. LAH reduction, ozonolysis, and
cyclization resulted in the desired substituted bis-THF ligands 19
and 20.
As shown in Scheme 3, alcohols 14, 15, 19, and 20 were
activated with p-nitrophenyl chloroformate to provide mixed
activated carbonates 21a-d. These carbonates were reacted with
amine 22 to afford HIV-1 protease inhibitors 23a-d.8 These
inhibitors were all evaluated for their activity in both enzyme
inhibitory and antiviral assays. The results are shown in Table 1.
The enzyme inhibitory activity (Ki) was determined according to
an assay protocol reported by Toth and Marshall.25 The inhibi-
tors displayed extremely potent enzyme inhibitory activity.
Compound 23c (Ki = 0.0029 nM) is the most potent compound
in this series. Its diastereomer 23a (Ki = 0.035 nM) displayed a
loss in enzyme inhibitory activity. The benzyl derivatives 23b and
23d have also shown reduced enzyme activity. Antiviral IC50
values were determined using the MTT assay.26,27 Consistent
with its enzyme inhibitory potency, inhibitor 23c exhibited an
impressive antiviral activity (IC50 = 2.4 nM).
To gain molecular insights into ligand-binding site interac-
tions responsible for the potent antiviral activity of inhibitor 23c,
we have determined an X-ray crystal structure of GRL-04410
(23c) complexed with the wild-type protease. The structure was
refined to an R-factor of 0.175 at a high resolution of 1.40 Å. The
structure comprises the protease dimer and inhibitor in two
orientations related by a 180° rotation, with 55/45 relative
a Values are means of at least three experiments. b Human T-lymphoid
(MT-2) cells were exposed to 100 TCID50 values of HIV-1LAI and
cultured in the presence of each PI, and IC50 values were determined
using the MTT assay.
occupancies (pdb code: 3QAA). The protease dimer structure
is essentially identical to that in the protease-darunavir complex
with a rmsd of 0.14 Å on the CR atoms.28 The inhibitor
interactions in the active site cavity consist of a series of hydrogen
bonds and weaker CH O interactions, as described previously
3 3 3
for darunavir and GRL-09865.28,29 The critical differences are
provided by the methoxy group on the bis-THF ligand in 23c.
As shown in Figure 2, the methoxy oxygen forms a water-
mediated hydrogen bond with the amide NH of Gly-48. Furthermore,
the methyl group forms a CH O interaction with the carbonyl
3 3 3
oxygen of Gly-48 that could stabilize the conformation of the
flexible flaps. The flexibility of the flaps is important for
the binding of substrates or inhibitors and for the catalytic
activity of HIV protease. The flap conformation and flexibility
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can be altered by sequence polymorphisms and drug resistant
mutations.30-32 Consequently, inhibitors such as 23c with
strong flap interactions are expected to retain high affinity for
drug resistant variants of the protease.
(2) FDA approves Darunavir on June 23, 2006: FDA approved a
new HIV treatment for patients who do not respond to existing drugs.
Please see http://www.fda.gov/bbs/topics/NEWS/2006/NEW01395.
html.
(3) On October 21, 2008, FDA granted traditional approval to
Prezista (darunavir), coadministered with ritonavir and with other
antiretroviral agents, for the treatment of HIV-1 infection in treatment-
experienced adult patients. In addition, a new dosing regimen for treatment-
naïve adult patients was approved.
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Weber, I. T.; Mitsuya, H. A Privileged Ligand for Darunavir and a New
Generation of HIV-Protease Inhibitors That Combat Drug-Resistance.
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HIV Protease Inhibitors Targeting Protein Backbone: An Effective
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Stray, K. M.; Lee, W. A. Suppression of HIV-1 Protease Inhibitor
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In conclusion, we investigated C4-alkoxy substituted bis-
THF-derived HIV-1 protease inhibitors in order to enhance
ligand-binding site interactions in the HIV-1 protease active
site. In this context, we have developed an optically active
synthesis of the bis-THF and C4-substituted bis-THF ligands
using a [2,3]-sigmatropic rearrangement as the key step.
Incorporation of C4-substituted bis-THF ligands resulted in a
series of highly potent HIV protease inhibitors. Compound 23c
is remarkably potent (Ki = 2.9 pM; IC50 = 2.4 nM). The
stereochemical importance of the methoxy substituent is evi-
dent, as the corresponding epimer is significantly less potent. A
protein-ligand X-ray structure of 23c-bound HIV-1 protease
revealed extensive interactions of the inhibitor in the active site
of HIV-1 protease. It maintained all key backbone hydrogen
bonding interactions similar to those of darunavir. Of particular
importance, the methoxy oxygen on the bis-THF ligand is
involved in a unique water-mediated hydrogen bond to the Gly-
48 amide NH. Further design and ligand optimization involving
this interaction is in progress.
’ ASSOCIATED CONTENT
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1H- and 13C-NMR spectral data for all new compounds. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
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N. C.; Thompson, W. J.; Culberson, C.; Fitzgerald, P. M. D.; Lee, H. Y.;
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(12) Ghosh, A. K.; Li, J.; Perali, R. S. A Stereoselective Anti-aldol
Route to (3R,3aS,6aR)-Tetrahydro-2H-furo[2,3-b]furan-3-ol: A Key
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Corresponding Author
*E-mail: akghosh@purdue.edu (A.K.G.).
Funding Sources
Financial support by the National Institutes of Health (GM
53386, A.K.G.; GM 062920, I.T.W.) is gratefully acknowledged.
This work was also supported in part by the Intramural Research
Program of the Center for Cancer Research, National Cancer
Institute, National Institutes of Health, and in part by a Grant-in-
aid for Scientific Research (Priority Areas) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan
(Monbu Kagakusho) and a Grant for Promotion of AIDS
Research from the Ministry of Health, Welfare, and Labor of
Japan.
’ ACKNOWLEDGMENT
We thank the staff at the Southeast Regional-Collaborative
Access Team (SER-CAT) at the Advanced Photon Source,
Argonne National Laboratory, for assistance during X-ray data
collection. Use of the Advanced Photon Source was supported by
the U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences, under Contract No. W-31-109-Eng-38. We
thank Drs. K. V. Rao and Bruno Chapsal (Purdue University) for
helpful discussions.
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