Design and synthesis of stereochemically defined novel spirocyclic p2-ligands for hiv-1 protease inhibitors

Article abstract of DOI:10.1021/ol8020308

(Chemical Equation Presented) The synthesis of a series of stereochemically defined spirocyclic compounds and their use as novel P2-ligands for HIV-1 protease inhibitors are described. The bicyclic core of the ligands was synthesized by an efficient nBu₃SnH-promoted radical cyclization of a 1,6-enyne followed by oxidative cleavage. Structure-based design, synthesis of ligands, and biological evaluations of the resulting inhibitors are reported.

Full text of DOI:10.1021/ol8020308

ORGANIC
LETTERS
2008
Vol. 10, No. 22
5135-5138
Design and Synthesis of
Stereochemically Defined Novel
Spirocyclic P2-Ligands for HIV-1
Protease Inhibitors
Arun K. Ghosh,*,† Bruno D. Chapsal,† Abigail Baldridge,† Kazuhiko Ide,‡
Yashiro Koh,‡ and Hiroaki Mitsuya‡,§
Departments of Chemistry and Medicinal Chemistry, Purdue UniVersity,
West Lafayette, Indiana 47907, Department of Hematology and Infectious Diseases,
Kumamoto UniVersity School of Medicine, Kumamoto 860-8556, Japan, and
Experimental RetroVirology Section, HIV and AIDS Malignancy Branch,
Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892
akghosh@purdue.edu
Received August 29, 2008
ABSTRACT
The synthesis of a series of stereochemically defined spirocyclic compounds and their use as novel P2-ligands for HIV-1 protease inhibitors
are described. The bicyclic core of the ligands was synthesized by an efficient nBu3SnH-promoted radical cyclization of a 1,6-enyne followed
by oxidative cleavage. Structure-based design, synthesis of ligands, and biological evaluations of the resulting inhibitors are reported.
The introduction of highly active antiretroviral therapy
(HAART) in 1996, in combination with HIV-1 protease
inhibitors and reverse transcriptase inhibitors, has dramati-
cally changed the management of HIV/AIDS.1 The advent
of HAART has significantly reduced morbidity and mortality
and has improved the quality of life for HIV-infected patients,
particularly in developed nations.2 Despite this important
breakthrough, current and future management of HIV/AIDS
is being challenged by the rapid emergence of multi-drug-
resistant HIV-1 strains and drug-related side effects.3 Con-
sequently, development of novel and effective treatment
regimens are critically important.
In our continuing effort to design a new generation of
HIV-1 protease inhibitors (PIs) that combat drug resistance,
we developed a series of exceedingly potent PIs. A number
of these nonpeptidyl PIs have shown superb antiviral activity
and drug-resistance profiles. In our structure-based design
strategies, we introduced the “backbone binding concept”
with the presumption that an inhibitor that makes maximum
interactions in the protease active site, particularly hydrogen
bonding with the backbone atoms, may retain its potency
against mutant strains.4 Darunavir (1, Figure 1), which has
been approved by the FDA for the treatment of patients
harboring multi-drug-resistant HIV-1 strains, has emerged
from this approach.5,6 Our detailed X-ray structural analysis
of protein-ligand complexes revealed an extensive hydrogen
† Purdue University.
‡ Kumamoto University School of Medicine.
§ National Cancer Institute.
(1) Sepkowitz, K. A. N. Engl. J. Med. 2001, 344, 1764.
(2) Palella, F. J.; Delaney, K. M.; Moorman, A. C.; Loveless, M. O.;
Fuhrer, J.; Satten, G. A.; Aschman, D. J.; Homberg, S. D. N. Engl. J. Med.
1998, 338, 853.
(3) Boden, D.; Markowitz, M. Antimicrob. Agents Chemother. 1998,
42, 2775.
(4) Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Acc. Chem.
Res. 2008, 41, 78.
10.1021/ol8020308 CCC: $40.75
Published on Web 10/18/2008
2008 American Chemical Society

Scheme 1. Synthesis of Bicyclic Ketone 8
Figure 1. Structure of HIV protease inhibitors.
We initially set out to synthesize a series of spirocyclic
Cp-THF-derived P2-ligands and their corresponding HIV-
protease inhibitors (3a-e). A synthetic strategy was devised
so that all analogs could be synthesized from a common
precursor that gives rapid access to new polycyclic molecular
probes. The general synthesis of the bicyclic core of our new
P2-ligands was accomplished in enantiomerically pure form
as shown in Scheme 1. Optically active monoacetate 4 was
obtained in 95% ee by desymmetrization of the correspond-
ing meso-diacetate with acetyl cholinesterase.11 Protection
of alcohol 4 as a TBS ether followed by methanolysis of
the acetyl group furnished compound 5. Propargylation of 5
using propargyl bromide in the presence of NaH provided
alkyne 6 in excellent yield.
bonding network with HIV-1 protease backbone atoms and
most notably with the designed bis-THF P2-ligand.7
More recently, we reported another novel PI, GRL-06579
(2), which features a stereochemically defined bicyclic
hexahydrocyclopentylfuran (Cp-THF) P2-ligand in the hy-
droxyethylsulfonamide isostere core.8 The X-ray crystal-
lographic analysis of 2-bound HIV-1 protease documented
extensive hydrogen bonding interactions including the Cp-
THF oxygen with the backbone atoms in the S2-subsite.8
The favorable drug-resistance profile of this PI containing
the Cp-THF ligand logically prompted us to design several
structural analogs. We set out to introduce new functionalities
on this bicyclic core that could create additional interactions
within the enzyme catalytic site. The 3-position of the Cp-
THF ligand appeared particularly suitable for this purpose,
because of its proximity to the flap region and the S2-subsite
of the protease. Based upon our analysis of the X-ray crystal
structure of 2-bound protease, we planned to investigate the
effect of a structurally constrained spirocyclic motif at the
3-position of the Cp-THF ring. We speculated that a cyclic
ether oxygen or an oxazolidinone carbonyl oxygen may be
positioned in this cyclic motif to accept a hydrogen bond
from the enzyme active site residues or a backbone NH. Such
a functionality would fill in the hydrophobic pocket in the
S2-subsite as well. Furthermore, this structural feature may
improve the pharmacological profile of these inhibitors.9,10
The construction of the bicyclic core was accomplished
by an intramolecular radical cyclization of alkyne 6 using
nBu3SnH and AIBN in benzene at reflux. This provided vinyl
stannane 7 as a mixture of cis/trans diastereoisomers (2:1),
Scheme 2
.
Synthesis of Spirocyclic Ketal and Ether Ligands
(5) Ghosh, A. K.; Kincaid, J. F.; Cho, W.; Walters, D. E.; Krishnan,
K.; Hussain, K. A.; Koo, Y.; Cho, H.; Rudall, C.; Holland, L.; Buthod, J.
Bioorg. Med. Chem. Lett. 1998, 8, 687.
(6) Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasam-
udram, 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.
(7) Kovalevsky, A. Y.; Liu, F.; Leshchenko, S.; Ghosh, A. K.; Louis,
J. M.; Harrison, R. W.; Weber, I. T. J. Mol. Biol. 2006, 363, 161.
(8) Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.; Li,
J.; Kovalevsky, A. Y.; Walters, D. E.; Wedekind, J. E.; Grum-Tokars, V.;
Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.; Weber, I. T.; Mitsuya, H.
J. Med. Chem. 2006, 49, 5252.
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Org. Lett., Vol. 10, No. 22, 2008

along with trace amounts of the olefin, which presumably
formed during purification on silica gel. The mixture of
isomers was directly oxidized with a catalytic amount of
OsO4 in the presence of NaIO4 and 2,6-lutidine to afford
the key intermediate, ketone 8 in 77% yield.
Scheme 4. Synthesis of Spirooxazolidinone Ligand 18
We first turned our attention to the synthesis of spirocyclic
ketal 9 and ether 12. Molecular modeling of the correspond-
ing inhibitors suggested that the ligand oxygens could be
within hydrogen bonding distance to the NH amide bonds
of both Asp30 and Asp29 residues.
Spirocyclic dioxolane ligand 9 was obtained in 86% yield
by treatment of ketone 8 with ethylene glycol in benzene with
a catalytic amount of p-TsOH. Synthesis of ether 12 was
achieved in four consecutive steps starting from ketone 8.
Reaction of 8 with homoallyl magnesium bromide furnished
compound 10 in 76% yield. Ozonolysis of the terminal alkene
and refluxing the resulting crude aldehyde in benzene/methanol
followed by azeotropic distillation of the excess methanol
afforded methyl acetal 11 as a mixture (1:0.85) of diastereoi-
somers. Reduction of this acetal intermediate 11 furnished the
desired alcohol 12 by applying a one-pot procedure involving
(1) TMS-protection of the alcohol with hexamethyldisilazane
and (2) subsequent reduction of the acetal with triethylsilane.12
We have designed spirocyclic oxazolidinone ligands that
could potentially exploit polar interactions with the backbone
atoms and residues in the HIV-1 protease active site. Their
respective syntheses are highlighted in Scheme 3 and 4.
gave amino alcohol 17 in 67% yield. The carbonyl was
installed using triphosgene and Et3N in CH2Cl2. Deprotection
of the TBS-group provided the desired oxazolidinone 18.
The synthesis of polycyclic PIs is shown in Scheme 5.
Various synthetic ligands were reacted with 4-nitrophenyl-
chloroformate and pyridine to form the corresponding
activated carbonates, 19a-e. Reaction of the respective
Scheme 5. Synthesis of Inhibitors 3a-e
Scheme 3. Synthesis of Spirooxazolidinone Ligand 15
Cyanohydrin 13 was synthesized in 82% yield from ketone 8.
LiAlH4-reduction of the cyanide provided the corresponding
amine, which exhibited partial TMS-deprotection. Therefore,
the crude mixture was directly submitted to the next steps with
(1) formation of the methyl carbamate derivative and (2)
removal of the silyl ethers with TBAF in THF. The resulting
diol 14 was then treated with NaH in THF to give oxazolidinone
ligand 15 in 51% yield over four steps (from 13).
Synthesis of oxazolidinone 18 started with vinylstannane
7 (Scheme 4). Proto-destannylation of 7 was carried out with
CSA in CH2Cl2. Epoxidation of the resulting olefin with
m-CPBA gave epoxide 16 as a major diastereomer (93:7
ratio). Opening of the epoxide with p-methoxybenzylamine
Org. Lett., Vol. 10, No. 22, 2008
5137

active carbonate with known8 amine 20 in the presence of
diisopropylethylamine afforded PIs 3a-d. For the synthesis
of inhibitor 3e, amine 20 was reacted with active carbonate
19e to provide urethane 21. Removal of the PMB group from
21 by exposure to ceric ammonium nitrate (CAN) afforded
inhibitor 3e.
We examined all inhibitors for their enzymatic potency
as well as their cellular activity, and the results are displayed
in Table 1. As shown, most inhibitors exhibited excellent
stereochemical identity of each diastereomer was determined
by extensive NOESY experiments. Diastereomer 3b-(S)
showed an enzymatic Ki of 0.81 nM. The 3b-(R) isomer is
slightly more potent (Ki ) 0.38 nM). The removal of the
methoxy group from 3b resulted in inhibitor 3c, which
showed a loss of enzyme inhibitory activity. Both inhibitors
3b and 3c have shown comparable antiviral activity. We have
examined stereochemically defined oxazolidinone derivatives
as P2-ligands. Inhibitor 3d displayed a Ki of 0.29 nM.
Diastereomeric inhibitor 3e is slightly more potent than 3d
in both enzyme inhibitory as well as in antiviral assays (IC50
) 21 nM in MT-2 cells). The inhibitors in Table 1 in general
are significantly less potent than UIC-PI (TMC-126),14 the
corresponding methoxysulfonamide derivative of darunavir
or Cp-THF-containing inhibitor 2.8
Table 1. Enzymatic and Antiviral Activity of PIs
In conclusion we have designed and synthesized a series
of inhibitors containing stereochemically defined novel
spirocyclic P2-ligands. The syntheses of these ligands were
carried out from the key intermediate 8, which was efficiently
prepared in optically active form by using a radical cycliza-
tion as the key step. The spirooxazolidinone-derived inhibitor
3e is the most potent inhibitor in this series. While these
inhibitors contain novel P2-ligands, it appears that the
spirocyclic motif at the 3-position of the Cp-THF ring
resulted in a significant reduction in potency. Further design
and optimization of the ligand binding site interactions are
in progress.
Acknowledgment. Financial support by the National
Institutes of Health (GM 53386, A.K.G.) 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.
We thank Mr. David D. Anderson (Purdue University)
for his help with the HPLC and NOESY analysis.
Supporting Information Available: Experimental pro-
cedures, spectral data, and 1H NMR and 13C NMR spectra
for compounds 5-21 and 3a-e. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL8020308
a Ki determined following protocol as described by Toth and Marshall,
mean values of at least four determinations.13 b MT-2 cells (2 × 104/mL)
were exposed to 100 TCID50 of HIV-1LAI and cultured in the presence of
various concentrations of PIs, and the IC50’s were determined by using the
MTT assay on day 7 of culture.6 c Tested as a 1:0.85 mixture.
(9) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward,
K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615.
(10) Lipinski, C. A. J. Pharmacol. Toxicol. Methods 2000, 44, 235.
(11) Deardorff, D. R.; Windham, C. Q.; Craney, C. L. Org. Synth. 1996,
9, 487.
(12) Yoo, S. J.; Kim, H. O.; Lim, Y.; Kim, J.; Jeong, L. S. Bioorg.
Med. Chem. 2002, 10, 215.
enzymatic potency. Dioxolane-based analogue 3a displayed
a Ki value of 0.16 nM. Inhibitor 3b contains the spirocyclic
methyl acetal as a mixture (1:0.85 ratio) of diastereomers.
These diastereomers were separated by HPLC, and the
(13) Toth, M. V.; Marshall, G. R. Int. J. Pep. Protein Res. 1990, 36,
544.
(14) Ghosh, A. K.; Sridhar, P.; Kumaragurubaran, N.; Koh, Y.; Weber,
I. T.; Mitsuya, H. ChemMedChem 2006, 1, 937.
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