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ACS Medicinal Chemistry Letters
ACS Med Chem Lett. Jan 13, 2011; 2(1): 63–67.
Published online Oct 22, 2010. doi:  10.1021/ml1002162
PMCID: PMC3074239

N-3 Hydroxylation of Pyrimidine-2,4-diones Yields Dual Inhibitors of HIV Reverse Transcriptase and Integrase


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A new molecular scaffold featuring an N-hydroxyimide functionality and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of human immunodeficiency virus (HIV) was rationally designed based on 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) non-nucleoside RT inhibitors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine ring of HEPT compound to yield a chelating triad which, along with the existing benzyl group, appeared to satisfy major structural requirements for IN binding. In the mean time, this chemical modification did not severely compromise the compound’s ability to inhibit RT. A preliminary structure−activity relationship (SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl group on N-1 side chain is more important for IN binding than the one on C-6.

Keywords: HIV, integrase, reverse transcriptase, dual inhibitor, rational design

HEPT (1)1 and its analogues constitute an important class of HIV NNRTIs,2 with the best ones being MKC-442 (emivirine, 2)35 and TNK-651 (3)6 (Figure (Figure1).1). The clinical development of these compounds was halted as they were found to stimulate liver enzyme CYP 3A4 and cause prohibitively low bioavailability of protease inhibitors used in combination therapy.7 Drug−drug interactions of this type pose a major barrier to successful highly active antiretroviral therapy (HAART) against HIV/AIDS.8,9 Simplifying regimens by reducing the number of drugs used in HAART could alleviate this issue. Toward this end, we have long been interested in identifying molecular scaffolds with dually inhibitory activities, particularly against HIV RT and IN.1012

Figure 1
Representative structures of HEPT type NNRTIs.

The design of inhibitors that can accommodate binding to two distinct biological targets remains an intriguing yet challenging scientific endeavor.1315 Our previous work has demonstrated the viability of constructing RT/IN dual inhibitors from NNRTIs using a design-in strategy. Significantly, IN binding requires minimally a two metal ion chelating functionality and a hydrophobic benzyl group (Figure (Figure2).2). Certain NNRTI scaffolds, including HEPT16 and BHAP17 (e.g., delavirdine, 9), have structural moieties that are sitting on the P236 loop of the NNRTI binding pocket and are not directly involved in RT binding. These moieties provide valuable handles (highlighted, Figure Figure3)3) for incorporating a relatively hydrophilic chelating functionality (boxed) to satisfy binding to the IN (Figure (Figure33).10,12 Most other NNRTIs are deeply buried in the highly hydrophobic binding pocket, thus are less amenable to hydrophilic modifications. In these cases, designing in structural components for IN binding requires careful considerations of all binding interactions of NNRTIs.

Figure 2
Structures of important IN inhibitors: the chelating functionality (red) and hydrophobic benzyl group (blue) represent the minimal pharmacophore.
Figure 3
Previously designed RT/IN dual inhibitors based on NNRTIs.

Notably the NNRTI binding pocket is largely hydrophobic and few H-bonds are accommodated. For the HEPT type of NNRTIs, the H-bond between the N(3)H of the inhibitor (e.g., inhibitor 3, Figure Figure4)4) and the backbone carbonyl group of K101 represents the major hydrophilic interaction.18 This H-bond contributes to the NNRTI binding affinity and, more importantly, serves as an anchor for the inhibitor to adopt optimized conformation so that hydrophobic interactions can be maximized throughout the binding pocket. Additional water mediated H-bonds may be present, albeit of much less significance.3 Therefore, the N(3)H group is generally considered too important to be modified. In our design, we hypothesize that hydroxylation of the N-3 of compound 3 would not significantly compromise its binding affinity to RT. This hypothesis is based on two observations: (1) the newly incorporated OH would retain H-bonding ability; and (2) the NNRTI binding pocket has four consecutive lysine residues (K101−K104) that could potentially H-bond to the newly introduced OH. Since this minimal modification only results in an insertion of an oxygen atom to NH, we expect that the key H-bond can be formed between the OH and one of these lysine residues, and the overall conformation of the inhibitor should be largely preserved. On the other hand, such a simple hydroxylation yields a C(2)O−N(3)OH−C(4)O chelating triad (11, Figure Figure4)4) capable of chelating two magnesium ions, which along with an existing hydrophobic benzyl group at N-1 or C-6 will essentially provide the minimal pharmacophore for IN binding (Figure (Figure4).4). Therefore, through a simple N-3 hydroxylation we could convert these HEPT type NNRTIs into RT/IN dual inhibitors.

Figure 4
The design of novel RT/IN dual inhibitor 11 from NNRTI 3. The H-bond crucial to RT binding is maintained (red) and the conformation of the molecule remains unchanged. The N-3 hydroxylation yields a chelating triad (blue), a critical structural component ...

This design is verified in silico by molecular modeling. All modeling was carried out using the Schrödinger modeling suite package.19 Docking of all compounds was carried out using Glide v2.5 at Standard Precision.20 For docking into HIV-1 IN, both Mg2+ ions and the interfacial hydrophobic pocket between the HIV-1 IN and DNA were defined as required constraints. Docking of 3 and 11 reveals their mode of binding in the NNRTI binding pocket almost identically (Figure (Figure5A).5A). Interestingly the OH of 11 and NH of 3 are both situated in close proximity to the K101 backbone (1.7 Å and 1.6 Å respectively). Accordingly, the H-bonding allows both inhibitors to adopt a nearly identical conformation in the binding pocket, suggesting that N-3 hydroxylation should not significantly compromise RT binding. Docking of 11 into our recent homology model21 of HIV-1 IN catalytic core domain (CCD) in complex with Mg2+ and DNA indicated the newly designed inhibitor 11 also showed reasonable IN binding (Figure (Figure5B).5B). The 3-N hydroxylation of the pyrimidine ring yields a chelating triad for Mg2+ binding while allowing the placement of the benzyl group into the protein−DNA interfacial hydrophobic pocket, a crucial pharmacophore requirement for potent HIV-1 ST inhibition.22,23

Figure 5
(A) Mode of binding of 3 (gray) and 11 (green) in the HIV NNRTI binding pocket. Both compounds hydrogen bonded to the backbone carbonyl oxygen atom of K101. (B) Mode of binding of 11 (green) in HIV-1 IN CCD in complex with Mg2+ and DNA. The 3-N hydroxyl ...

The designed inhibitors are synthetically accessed through a route outlined in Scheme 1. The synthesis of advanced intermediates 2, 3 and 2933 is well-established in the literature24 whereas the key transformation, the N-3 hydroxylation, is rarely known. This was achieved through deprotonation of HEPT intermediates and the subsequent treatment with an oxidizing agent. After screening of a wide array of oxidants it was found that m-CPBA can effect this N-3 hydroxylation efficiently (Scheme 1). Adaptation of this method also allowed us to successfully prepare an N-3 amino analogue (16), wherein mesityl sulfonyl-O-hydroxyl amine (MSH)25 was employed as the aminating agent.

Testing of these compounds against recombinant HIV IN provided the first validation of our design. In this assay both the 3′ processing (3′P) and the strand transfer (ST) activities, the two primary functions of IN, were assessed. As shown in Table 1, all N-3 hydroxylated compounds (1115, 17, 18) selectively inhibit ST over 3′P at low micromolar concentrations, whereas the unmodified compound (3) did not show any inhibitory activity against HIV IN. This observation strongly suggests that the N-3 hydroxyl group, partially forming the chelating triad (Figure (Figure4,4, 11), is crucial to IN binding. Interestingly, the N-3 amino compound (16) also turned out inactive in IN assay. It is not surprising that the N-3 hydroxylation yields a better chelating triad than the N-3 amination as O tends to have a higher affinity for Mg2+ than N. In addition, compounds 17 and 18 demonstrate significantly reduced (ca. 10-fold) inhibition against ST when compared with compounds 1115, indicating that the benzyl group at the N-1 side chain is far more important for IN binding than the one at the C-6 position of the pyrimidine-2,4-dione ring (Figure (Figure4,4, compound 11).

Table 1
Inhibitory Activities of Compounds 1118 against HIV IN

To further characterize the mechanism of action of our newly designed compounds, a representative compound 13 was tested head-to-head with raltegravir (7, Figure Figure2)2) against a recombinant HIV IN containing major resistant mutations G140S and Q148H (Figure (Figure66).26,27 This assay revealed that, although inhibiting IN at a higher concentration when compared with 7, compound 13 does show a similar resistance profile to 7, further proving that our new inhibitors are targeting IN ST.

Figure 6
Comparison of newly designed inhibitor 13 with raltegravir 7 in recombinant HIV IN with G140S and Q148H mutants.

Having established these new inhibitors as valid HIV IN inhibitors, we then studied their ability to inhibit HIV RT. Through a quick biochemical assay using a commercial RT assay kit it was found that N-3 hydroxylated compounds (e.g., 13) and aminated compound (16) all show inhibitory activity at low or sub-micromolar range (Table 2). Apparently an N-3 OH or NH2 allows these compound to virtually maintain the key H-bonding ability as expected, though the unmodified N(3)H group of compound 3 appears to form better H-bonding as compound 3 inhibits RT at a considerably lower concentration. Meanwhile, a dramatic fluorine effect was also observed with compounds 12, 14 and 18, where a fluorine substitution at the para position of the C-6 benzyl completely disables RT binding. By contrast, the para position of the other benzyl group in N-1 side chain appears to be highly tolerant toward fluorine substitution (compound 13).

Table 2
Anti-RT and Anti-HIV Activities of Inhibitors 1118

Finally these compounds were tested in cell-based antiviral assay against HIV-1. With the exception of compounds with a terminal fluorine atom on the C-6 benzyl group (12, 14 and 18), and the one with N-3 amino group (16), these inhibitors generally show excellent antiviral potency and safety (Table 2). The best compounds in this series, compounds 11 and 13, demonstrate low nanomolar anti-HIV activity that is comparable to or even better than that of reference compound 3.

In summary, through rational design we have generated a novel series of anti-HIV compounds that are dually active against IN and RT at low and sub-micromolar concentrations. Our design involves a minimal structural modification on a well-known NNRTI inhibitor scaffold (HEPT) to introduce inhibitory activity against IN. Major structural determinants for IN binding as well as a dramatic fluorine effect for RT binding were identified through preliminary SAR efforts. In the end, two compounds (11 and 13) were found to have low nanomolar inhibitory activities against HIV-1 in cell culture. Further studies of this newly designed chemotype as a novel IN inhibitor scaffold are currently underway and will be reported in due course.


We thank Roger Ptak at Southern Research Institute for the cell-based antiviral assay and the Minnesota Supercomputing Institute for providing all the necessary computational resources.


RT, reverse transcriptase; IN, integrase; HIV, human immunodeficiency virus; HEPT, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine; NNRTI, non-nucleoside RT inhibitor; SAR, structure−activity relationship; HAART, highly active antiretroviral therapy; CCD, catalytic core domain; MSH, mesityl sulfonyl-O-hydroxyl amine; TBAI, tetrabutylammonium iodide; 3′-P, 3′ processing; ST, strand transfer.

Funding Statement

National Institutes of Health, United States


This research was supported by the Center for Drug Design at the University of Minnesota, the Center for Cancer Research, National Cancer Institute and an IATAP grant to Y.P., NIH.

Supporting Information Available

Supporting Information Available

Synthesis of 1118, experimental details, compound characterization (1H, 13C NMR, HRMS), HPLC analysis for final products and assay methods. This material is available free of charge via the Internet at http://pubs.acs.org.

Supplementary Material


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