Heterocyclic HIV-1 protease inhibitors.

Article abstract of DOI:10.1021/ol990586y

[formula: see text] A series of simple heterocyclic HIV-1 protease inhibitors were developed on the basis of size, shape, and electronic complementarity to the active site of the enzyme. The C2-symmetric heterocycles do not contain a transition-state isos

Full text of DOI:10.1021/ol990586y

ORGANIC
LETTERS
1999
Vol. 1, No. 2
249-252
Heterocyclic HIV-1 Protease Inhibitors
Paul W. Baures
111 Willard Hall, Department of Chemistry, Kansas State UniVersity,
Manhattan, Kansas 66506
baures@ksu.edu
Received April 13, 1999
ABSTRACT
A series of simple heterocyclic HIV-1 protease inhibitors were developed on the basis of size, shape, and electronic complementarity to the
active site of the enzyme. The C -symmetric heterocycles do not contain a transition-state isostere nor are they active site directed irreversible
2
inhibitors; thus, they represent the success of a new design strategy. The first generation heterocycles inhibit the protease in the micromolar
range, whereas control compounds show no bioactivity at the same concentrations.
The development of the HIV-1 protease inhibitors now
clinically available has brought hope to thousands of people
infected with the HIV virus.1 These inhibitors were developed
around transition-state design elements previously used
against other aspartic proteases such as renin.2 Clinical
candidates having suitable potency and specificity against
HIV-1 protease were identified by researchers rapidly.
However, modifications of the drug candidates were required
to improve the pharmacokinetics of these early compounds.
This paper details the development of a simple class of
HIV-1 protease inhibitors designed on the basis of the size,
shape, and electronic complementarity of the core structures
to the active site of the enzyme. The heterocyclic core
structures exclude the use of transition-state mimics, elimi-
nating the complications of stereochemistry which often
accompany these mimics. Moreover, the heterocycles offer
potential benefits in the final pharmacokinetic properties of
the compounds due to their amphoteric nature.
protease inhibitors which would reasonably be assumed to
offer potential benefits in the ease of synthesis, aqueous
solubility, and oral bioavailability of the compounds. The
choice of our core heterocycles was made on the basis of
three important pieces of information described in the
following paragraphs.
Heterocycles were first considered as possible core
structures after examination of imidazolium hydrogen male-
ate (Figure 1), which forms an extended hydrogen bonded
salt in the solid state.3 The molecules are organized inter-
molecularly by the formation of NH‚‚‚O hydrogen bonds as
well as short CH‚‚‚O contacts. While the carboxylate
distances in this structure (carbon-to-carbon distance of 6.5
Å) are significantly greater than a typical separation observed
for HIV-1 protease active site residues (carbon-to-carbon
distance of 5.1 Å),4 some degree of active site flexibility is
considered possible given the substrate diversity recognized
by the protease.2c
Inhibitor Design. Our goal at the outset of this project
was to consider an alternative design strategy for HIV-1
Development of novel C2-symmetric inhibitors has led to
potent compounds for which X-ray cocrystal structures are
available.5 Most of the symmetric compounds do not bind
(1) Havlir, D. V.; Lange, J. M. A. AIDS 1998, 12, S165.
(2) Selected reviews of HIV-1 protease inhibitor development: (a) West,
M. L.; Fairlie, D. P. Trends Pharmacol. Sci. 1995, 16, 67. (b) Kempf, D.
J.; Sham, H. L. Curr. Pharm. Design 1996, 2, 225. (c) Wlodawer, A.;
Vondrasek, J. Annu. ReV. Biophys. Biomol. Struct. 1998, 27, 249.
(3) Hsu, B.; Schlemper, E. O. Acta Crystallogr. 1980, B36, 3017.
(4) This is the average distance in the HIV-1 protease structures with
the PDB codes 1HVI, 1HVJ, 1HVK, and 1HVL.
10.1021/ol990586y CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/29/1999

heterocycles and the use outlined in this paper is that the
heterocycles are proposed to interact directly within the active
site in this work rather than their previous peripheral
inclusion.
Inhibitor Synthesis. The compounds 1-6 synthesized for
this study were prepared from the commercially available
dicarboxylic acids (Chart 1). The conformationally con-
Chart 1. Compounds Synthesized for This Study
Figure 1. Small molecule X-ray structure of imidazolium hydrogen
maleate6 organized intermolecularly by short NH‚‚‚O and CH‚‚‚O
contacts. The hydrogen bond distances are (a) 2.39 Å, (b) 3.03 Å,
and (c) 2.78 Å. The CH‚‚‚O angle in b is 117°.
symmetrically to the active site of HIV-1 protease; however,
A-76928 does bind symmetrically with the two hydroxyl
groups of the dihydroxyethylene core of this compound
gauche to one another (Figure 2).5 A conformational
strained control compound, 6, was synthesized to support
the hypothesis that the heterocyclic rings in 1-5 would be
necessary for enzyme inhibition. The compounds were
synthesized by standard coupling reactions7 in CH2Cl2
employing dicyclohexylcarbodiimide, 1-hydroxybenzotri-
azole, and triethylamine, giving poor yields (2-25%).
Compounds 1, 3, 5, and 6 were prepared by coupling the
appropriate dicarboxylic acid with L-phenylalanine tert-butyl
ester hydrochloride. The syntheses of 2 and 4, which also
contain two valine residues, were done from 1 and 3,
respectively, by deprotecting the tert-butyl ester with 4 N
HCl in dioxane before coupling the product of the reaction
with L-valine tert-butyl ester hydrochloride. The synthesized
Figure 2. Design rationale for the heterocyclic core structure on
the basis of the gauche hydroxyl groups in A-76928 and the small
molecule crystal structure of imidazolium hydrogen maleate.
1
compounds were characterized by H and 13C NMR spec-
constraint involving these gauche hydroxyl groups (Figure
2) was considered on the basis of this information. A trans
imidazoline would place the appended R groups in the same
general location as found in A-76928. Yet, the imidazole
was chosen for its simplicity and its ready availability for
the testing of this hypothesis.
There was one final consideration in choosing the imid-
azole and the other aromatic heterocycles as a component
of our design: namely, that these heterocycles have already
been utilized in HIV-1 protease inhibitors to improve the
water solubility and oral bioavailability of the final com-
pound.6 The major difference between the prior use of the
troscopy as well as mass spectrometry.8
Enzyme Inhibition Studies. The ability of these com-
pounds to inhibit HIV-1 protease was tested by using the
previously reported colorimetric peptide substrate (H-Lys-
Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2) with some
modifications.9
In addition to testing the synthesized compounds, a known
HIV-1 protease inhibitor, acetylpepstatin (7), was included
in the study.10 Three heterocycles, imidazole (8), imidazole-
4,5-dicarboxylic acid (9), and 4,5-diphenylimidazole (10),
were included to verify the necessity of both the heterocyclic
ring and the appended amino acids for bioactivity against
the enzyme.
(5) Hosur, M. V.; Bhat, T. N.; Kempf, D. J.; Baldwin, E. T.; Liu, B.;
Gulnik, S.; Wideburg, N. E.; Norbeck, D. W.; Appelt, K.; Erickson, J. W.
J. Am. Chem. Soc. 1994, 116, 847.
Lam, P. Y. S. J. Med. Chem. 1998, 41, 2019. (e) De Lucca, G. V.; Kim, U.
T.; Liang, J.; Cordova, B.; Klabe, R. M.; Garber, S.; Bacheler, L. T.; Lam,
G. N.; Wright, M. R.; Logue, K. A.; Erickson-Viitanen, S.; Ko, S. S.;
Trainer, G. L. J. Med. Chem. 1998, 41, 2411.
(7) Klausner, Y. S.; Bodansky, M. Synthesis 1972, 453.
(8) Spectra are included with the Supporting Information.
(9) Richards, A. D.; Phylip, L. H.; Farmerie, W. G.; Scarborough, P. E.;
Alvarez, A.; Dunn, B. M.; Hirel, P.-H.; Konvalinka, J.; Strop, P.; Pavlickova,
L.; Kostka, V.; Kay, J. J. Biol. Chem. 1990, 265, 7733. Our inhibitor assays
were done at 320 nm instead of 300 nm because this wavelength minimized
background variations.
(6) (a) Thompson, S. K.; Murthy, K. H.; Zhao, B.; Winborne, E.; Green,
D. W.; Fisher, S. M.; DesJarlais, R. L.; Tomaszek, T. A., Jr.; Meek, T. D.;
Gleason, J. G.; Abdel-Meguid, S. S. J. Med. Chem. 1994, 37, 3100. (b)
Abdel-Meguid, S. S.; Metcalf, B. W.; Carr, T. J.; Demarsh, P.; DesJarlais,
R. L.; Fisher, S.; Green, D. W.; Ivanoff, L.; Lambert, D. M.; Murthy, K.
H. M.; Petteway, S. R., Jr.; Pitts, W. J.; Tomaszek, T. A., Jr.; Winborne,
E.; Zhao, B.; Dreyer, G. B.; Meek, T. D. Biochemistry 1994, 33, 11671.
(c) Turner, S. R.; Strohbach, J. W.; Tommasi, R. A.; Aristoff, P. A.; Johnson,
P. D.; Skulnick, H. I.; Dolak, L. A.; Seest, E. P.; Tomich, P. K.; Bohanon,
M. J.; Horng, M.-M.; Lynn, J. C.; Chong, K.-T.; Hinshaw, R. R.;
Watenpaugh, K. D.; Janakiraman, M. N.; Thaisrivongs, S. J. Med. Chem.
1998, 41, 3467. (d) Han, Q.; Chang, C.-H.; Li, R.; Ru, Y.; Jadhav, P. K.;
(10) Richards, A. D.; Broadhurst, A.; Ritchie, A. J.; Dunn, B. M.; Kav,
J. FEBS Lett. 1989, 253, 214.
250
Org. Lett., Vol. 1, No. 2, 1999

The results of the bioassay are shown in Figure 3 following
the normalization of the absorbances at 5 min and against
the observed substrate hydrolysis over the course of the
experiment. Compounds 1-6 and 8-10 were tested at 1 µM,
pharmacokinetics of the compounds. While this work does
not attempt to mimic the transition-state geometry, recent
reports on the development of achiral, potent, and specific
serine protease inhibitors support the hypothesis that this
could be attained.15 For example, the active site chemical
functionality in HIV-1 protease has been reported to form a
copper(II) chelatesdesigned by comparison with small
molecule organometallics.16 In addition, researchers at Phar-
macia & Upjohn, Inc. are developing a class of HIV-1
protease inhibitors which do contain a hydroxyl group to
hydrogen bond to the active site, but which is not part of a
stereogenic center.6d
Incorporation of heterocyclic rings into existing HIV-1
protease inhibitors had previously been done on the premise
of improving water solubility or oral bioavailability of the
compound.6 The results of these modifications have thus far
been mixed, with both successes and failures being reported.
Dupont-Merck’s cyclic ureas were reported to have poor
aqueous solubilities despite the presence of two substituted
heterocyclic rings (pyrazole, imidazole, 1,2,3-triazole, 1,2,4-
triazole, or tetrazole) or benzo-fused analogues.6a These
researchers hypothesized that the symmetric structures had
greater crystal packing energies, thereby making them less
soluble.6d,e The preparation of asymmetric ureas containing
one benzo-fused pyrazole ring did result in a series of
inhibitors with improved oral bioavailability.17
Figure 3. Bioassay of compounds 1-6 as well as 8 (imidazole),
9 (imidazole-4,5-dicarboxylic acid), and 10 (4,5-diphenylimidazole)
all tested at 1 µM in DMSO alongside the known HIV-1 protease
inhibitor acetylpepstatin (7) at 3.5 µM.11
It appears the compounds reported herein bind to HIV-1
protease with occupation of the active site by the heterocyclic
ring, since the observed enzyme inhibition data indicates a
dependence on both the presence of the ring as well as the
presence of the linked amino acids. On this basis, the
proposed binding with the HIV-1 protease active site is
shown schematically in Figure 4 for the imidazole and
pyrazole inhibitors. The utilization of the C-H bond of the
2-position of the imidazole-containing inhibitors may provide
some additional stabilization. This position is known to be
acidic enough to exchange with D2O at room temperature18
and to form short CH‚‚‚O contacts in small molecule X-ray
structures.3,19
The triazole inhibitors could potentially interact in either
version of the proposed interactions. The similarity of the
observed in Vitro enzyme inhibition of 1-5 precludes a
current judgment regarding the possible contributions of pKa
versus hydrogen bonding for the heterocycle core struc-
tures.
while acetylpepstatin (7) was used at 3.5 µM.11 The error
bars are calculated standard deviations obtained by averaging
three separate experiments except for compounds 6 and 8-10
which were done in duplicate. The increase in absorbance
for 5 and 7 is likely due to some precipitation (turbidity)
which occurs with these inhibitors over the course of the
experiment.
Discussion. Although an oversimplification of ligand/
inhibitor design, the important features include appropriately
matching the size and shape of the small molecule to the
macromolecular binding site as well as presenting an
electronic and hydrophobic surface which complements the
macromolecular surface.12
Traditional aspartic protease inhibition has generally
utilized two design approaches, either the use of transition-
state isosteres13 or active-site directed irreversible inhibitors.14
Transition-state isosteres have been used with the greatest
success in the development of clinical drugs against HIV-1
protease.2
Conclusions. HIV-1 protease inhibitors have been devel-
oped which contain heterocyclic rings complementary to the
The outcome of this project was the development of
noncovalent inhibitors of HIV-1 protease with achiral
heterocyclic core structures that might logically improve the
(15) Katz, B. A.; Clark, J. M.; Finer-Moore, J. S.; Jenkins, T. E.; Johnson,
C. R.; Ross, M. J.; Luong, C.; Moore, W. M.; Stroud, R. M. Nature 1998,
391, 608.
(16) Florence, L.; Ledecq, M.; Ben atallah, Z.; Sicsic, S.; Rosny, E. de;
Arnaud, G.; Miche`le, R.-R.; Rene´, L.; Olivier, K.; Franc¸ois, D. “Copper(II)
Chelates as a New Generation of Non-Peptide Inhibitors of the Human
Immunodeficiency Virus”, presented at the 26th National Medicinal
Chemistry Symposium, Richmond, VA, June 14-18, 1998; Abstract A-21.
(17) Rodgers, J. D.; Lam, P. Y. S.; Johnson, B. L.; Wang, H.; Ko, S. S.;
Seitz, S. P.; Trainor, G. L.; Anderson, P. S.; Klabe, R. M.; Bacheler, L. T.;
Cordova, B.; Garber, S.; Reid, C.; Wright, M. R.; Chang, C.-H.; Erickson-
Viitanen, S. Chem. Biol. 1998, 5, 597.
(11) Inhibition by 1 and 5 drops off quickly below 1 µM. Determination
of the compounds Ki values will be published at a later date.
(12) (a) Babine, R. E.; Bender, S. L. Chem. ReV. 1997, 97, 1359. (b)
Bo¨hm, H. J.; Klebe, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 2588.
(13) Dreyer. G. B.; Metcalf, B. W.; Tomaszek, T. A., Jr.; Carr, T. J.;
Chandler, A. C., III; Hyland, L.; Fakhoury, S. A.; Magaard, V. W.; Moore,
M. L.; Strickler, J. E.; Debouck, C.; Meek, T. D. Proc. Natl. Acad. Sci.
U.S.A. 1989, 86, 9752.
(14) Rose, R. B.; Rose, J. R.; Salto, R.; Craik, C. S.; Stroud, R. M.
Biochemistry 1993, 32, 12498.
(18) Harris, T. M.; Randall, J. C. Chem. Ind. 1965, 1728.
(19) Gorbitz, C. H.; Husdal, J. Acta Chem. Scand. 1998, 52, 218.
Org. Lett., Vol. 1, No. 2, 1999
251

favorable electrostatic interactions with the catalytic aspartic
acid side chains. This may imply that other new classes of
inhibitors remain to be discovered and that some of these
classes may offer advantages in the synthesis and develop-
ment of the drug candidate(s) or both.
In addition, this work addresses electronic complementarity
with functionality not traditionally considered in drug design,
such as CH groups as hydrogen bond donors. In an era of
data mining, the importance of subtleties can often go
unnoticed in the wealth of data. The energy of association
of the imidazole CH group is yet undetermined, but it is
apparent that its inclusion did not preclude bioactivity. This
suggests that medicinal chemists may be able to design
alternative structures for a variety of known drugs, with each
alternative structure having potential differences in potency,
specificity, toxicity, pharmacokinetics, or other important
properties.
Future efforts are directed at discovering more potent
compounds through analogue synthesis. Specificity is also
being determined by measuring the bioactivity of these
inhibitors against other aspartic proteases.
Figure 4. (a) The proposed interaction of an imidazole-based
inhibitor with the two catalytic aspartic acid side chains. The
diagram indicates contributors resulting from symmetry-equivalent
hydrogen-bonded structures plus interactions resulting from proton
transfer by an aspartic acid side chain (partial bonds). In addition,
the potential interaction of the C-H group of the 2-position in the
imidazole ring is indicated by dashes. (b) The proposed interaction
of a pyrazole-based inhibitor with the two catalytic aspartic acid
side chains. The partial bonds indicate symmetry-related structures
in the absence of proton transfer from the aspartic acid side chain,
whereas the dashes represent hydrogen-bonding interactions only.
The emboldened hydrogens serve to indicate the pairwise relation-
ship of the hydrogens in this diagram.
Acknowledgment. The author thanks Kansas State
University and the Kansas State University Department of
Chemistry for the funds, space, and opportunity to pursue
this research.
Supporting Information Available: Experimental de-
1
tails, H and 13C NMR spectra of 1-6, and a larger Figure
3. This material is available free of charge via the Internet
at http://pubs.acs.org.
size and shape of the active site. Furthermore, despite
differences in the structure of the imidazole, pyrazole, and
triazole rings, all of the heterocycles could plausibly form
OL990586Y
252
Org. Lett., Vol. 1, No. 2, 1999