Bisphosphonate inhibitors of ATP-mediated HIV-1 reverse transcriptase catalyzed excision of chain-terminating 3′-azido, 3′-deoxythymidine: A QSAR investigation

Article abstract of DOI:10.1016/j.bmc.2008.08.047

We report the results of an investigation of the inhibition of the ATP-mediated HIV-1 reverse transcriptase catalyzed phosphorolysis in vitro of AZT from AZT-terminated DNA primers by a series of 42 bisphosphonates. The four most active compounds possess neutral, halogen-substituted phenyl or biphenyl sidechains and have IC₅₀ values <1 μM in excision inhibition assays. Use of two comparative molecular similarity analysis methods to analyze these inhibition results yielded a classification model with an overall accuracy of 94%, and a regression model having good accord with experiment (q² = 0.63, r² = 0.91), with the experimental activities being predicted within, on average, a factor of 2. The most active species had little or no toxicity against three human cell lines (IC₅₀^avg > 200 μM). These results are of general interest since they suggest that it may be possible to develop potent bisphosphonate-based AZT-excision inhibitors with little cellular toxicity, opening up a new route to restoring AZT sensitivity in otherwise resistant HIV-1 strains.

Full text of DOI:10.1016/j.bmc.2008.08.047

Bioorganic & Medicinal Chemistry 16 (2008) 8959–8967
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bisphosphonate inhibitors of ATP-mediated HIV-1 reverse transcriptase
catalyzed excision of chain-terminating 30-azido, 30-deoxythymidine:
A QSAR investigation
Yongcheng Song a, Julian M. W. Chan a, Zev Tovian a, Aaron Secrest b, Eva Nagy b, Kilannin Krysiak a,
Kyle Bergan a, Michael A. Parniak b, Eric Oldfield a,
*
a Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA
b Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the results of an investigation of the inhibition of the ATP-mediated HIV-1 reverse transcrip-
tase catalyzed phosphorolysis in vitro of AZT from AZT-terminated DNA primers by a series of 42 bisphos-
phonates. The four most active compounds possess neutral, halogen-substituted phenyl or biphenyl
Received 13 May 2008
Revised 11 August 2008
Accepted 22 August 2008
Available online 27 August 2008
sidechains and have IC50 values <1 lM in excision inhibition assays. Use of two comparative molecular
similarity analysis methods to analyze these inhibition results yielded a classification model with an
overall accuracy of 94%, and a regression model having good accord with experiment (q2 = 0.63,
Keywords:
Reverse transcriptase
Nucleoside reverse transcriptase inhibitor
(NRTI)
Bisphosphonate
Quantitative structure–activity relationship
(QSAR)
r
2 = 0.91), with the experimental activities being predicted within, on average, a factor of 2. The most
active species had little or no toxicity against three human cell lines (IC50avg > 200
lM). These results
are of general interest since they suggest that it may be possible to develop potent bisphosphonate-based
AZT-excision inhibitors with little cellular toxicity, opening up a new route to restoring AZT sensitivity in
otherwise resistant HIV-1 strains.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
that contains a terminal azide (–N3) rather than an –OH group.
The terminal OH ? –N3 substitution prevents DNA elongation
HIV-1 is a major cause of AIDS with ꢀ40,000,000 individuals
infected.1 The first drug approved for the clinical treatment of
HIV-1 infection was 30-azido, 30-deoxythymidine (AZT, RetrovirÒ,
RetrovisÒ, and ZidovudineÒ). When taken up into cells AZT is con-
verted to AZT-triphosphate, which can then be incorporated into
nascent viral DNA,2 resulting in a so-called ‘‘blocked DNA primer”
and, consequently, viral DNA replication is terminated. However,
shortly after the introduction of AZT, cases of resistance were
found. This resistance originates in the phosphorolytic excision
of AZT by ATP (and, to a lesser extent, PPi) catalyzed by viral re-
verse transcriptase3–5 (RT), as illustrated below:
In most cases, resistance originates from specific RT mutations
called thymidine analog mutations (TAMs).6–10 Specific inhibitors
* Corresponding author. Tel.: +1 217 333 3374; fax: +1 217 244 0997.
E-mail address: eo@chad.scs.uiuc.edu (E. Oldfield).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.047

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Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
of AZT-excision, and in principle the excision of other nucleoside
reverse transcriptase inhibitors (NRTIs), could have therapeutic
utility, since they might restore the activity of the NRTI against
TAM-containing HIV-1. Optimally, such inhibitors should not re-
duce incorporation of an NRTI, since this would clearly antagonize
their anti-retroviral activity. We demonstrate here that selective
inhibitors of RT-catalyzed phosphorolytic excision of NRTIs can
be identified.
are in the range 500 nM to 51.3
second set of compounds having low activity (IC50 values
P100 M) are shown in Figure 1B. What is immediately apparent
from these results is that none of the conventional, nitrogen-con-
taining bisphosphonates, such as incadronate (35), zoledronate
(36), or minodronate (44), Figure 1B, used to treat bone-resorption
diseases, have appreciable activity in inhibiting AZT-excision, since
lM (Table 1). The structures of a
l
their IC50 values are all P100 lM. Several of the other inactive
While screening large libraries of compounds for activity in
inhibiting AZT-excision might be one approach to discovering
NRTI-excision inhibitors, current assay methods are not well suited
for high-throughput screening. An alternative approach might,
however, be to screen smaller libraries of compounds containing
hydrolytically stable analogs of diphosphate or triphosphate,
which could block AZT (or PPi) binding to RT. Conceptually, the
simplest analogs of PPi to consider are the so-called bisphospho-
nate class of drugs (used to treat bone-resorption diseases) in
which the central, bridging oxygen atom is replaced by a methy-
lene (2) or substituted methylene group, such as 3, 4:
compounds (31, 38, and 39) are also known to be active in inhibit-
ing bone resorption,15,16 and in each case their target is the enzyme
farnesyl diphosphate synthase (FPPS). However, targeting FPPS is
an undesirable feature for an AZT-excision inhibitor since it is
likely to lead to toxicity in host cells.
In the case of the bisphosphonates that are far more active in
inhibiting AZT-excision, we find a clearly different pattern of
sidechain substitution to that seen in the nitrogen-containing
bisphosphonates. All of the nitrogen-containing bisphosphonates
active in bone resorption have a basic nitrogen atom that is pro-
tonated when bound to the FPPS enzyme,17 whereas in the case
the potent excision inhibitors, the sidechain is expected (based
on pKa values) to be neutral. The four most active compounds
have IC50 values 61 lM and in each case, these compounds con-
tain halogen substituents on a phenyl or biphenyl ring. In con-
trast, the two least ‘‘active” compounds (27 and 28, with IC50
values of 38, 51.3 lM, respectively), each contain electron-
donating ring substituents. Of all of the active compounds, none
were potent inhibitors of RT-catalyzed DNA synthesis, as shown
in Figure 2, a desirable feature for an excision inhibitor. We also
found that there was little or no activity of these bisphospho-
nates against the human cell lines tested: IC50 values are given
in Table 1. For example, 3 had an IC50 (averaged over the three
and in previous work11 we reported that the bisphosphonate 3 had
some activity in this context. More recently, Cruchaga et al.12 have
followed up on this work, finding that the bisphosphonate pamidr-
onate (4) competitively inhibited the phosphorolytic activity of RT,
and synergistically inhibited HIV-1 RT in combination with AZT-
triphosphate, in the presence of either ATP or PPi. And in more re-
cent work, this group found similar results with the anti-viral com-
pound 2-O-galloylpunicalin (2GP13). Here, we report the results of
an investigation of the activity of 3 and 41 other bisphosphonates
in inhibiting RT-catalyzed AZT-excision by ATP. The most active
compounds are far more potent than are 3 or pamidronate (4),
and we use here a variety of structure–activity relationship meth-
ods to relate their structure with function. We also investigate the
direct inhibition of RT-catalyzed DNA synthesis by these bisphos-
phonates, finding in most cases that, unlike 2GP, they have little
or no activity, an observation of potential importance for their fur-
ther development as inhibitors of NRTI-excision during HIV-1
replication.
cell lines investigated, MCF-7, SF-268, NCI-H460) of ꢀ229
while the most active species (5) had an IC50 of ꢀ234 M. In
contrast, species such as zoledronate and minodronate (36, 44)
had IC50 values of ꢀ15, 5 M, due to potent inhibition of the far-
nesyl diphosphate synthase enzyme. As expected, 3 and 5 had
very weak activity against this target (44, 8.5 M vs 0.14,
0.12 M for minodronate, zoledronate: data not shown). The
lM,
l
l
l
l
presence of both phosphonate groups is critical for activity, as
can be seen in the series 5, 29, 30. Both of the latter compounds
have P–C (phosphinate) groups, replacing 1 or 2 –OH groups
with CH3 groups, and neither compound has any appreciable
activity in inhibiting AZT-excision.
2.2. Quantitative structure–activity relationships (QSAR)
We next sought to see to what extent it might be possible to
make a more detailed analysis of the activity results shown in
Table 1. We first carried out a pharmacophore modeling investi-
gation and obtained the common feature hypothesis shown in
Figure 3A. However, this approach did not yield any highly pre-
dictive models. We thus next moved to using CoMSIA modeling
methods, which, in previous work on bisphosphonate inhibi-
tors,18–20 have been shown to be highly predictive. These meth-
ods require an alignment of all molecules within a common
frame of reference (Fig. 3B), which in this case was constructed
using the flexible alignment algorithm in the program MOE.21
We then used the default settings in Sybyl 7.3 to generate steric,
hydrophobic, electrostatic, donor, and acceptor CoMSIA descrip-
tors at grid points surrounding the alignment. Given the discrete
distribution of the RT-enzyme inhibition data, we first investi-
gated the possible utility of a classification approach, rather than
regression methods, to analyze the CoMSIA results. For such
classification approaches, compound activity can be assigned
using an ordered scale (i.e., active, moderately active, inactive)
rather than specific numeric values. The soft independent mod-
2. Results and discussion
2.1. Structure–activity relationships (SAR)
We investigated 42 bisphosphonates for activity in RT-cata-
lyzed ATP-mediated AZT-excision reactions in vitro. The structures
of 25 compounds (3, 5–28) having activity in the range 0.5–50 lM
are shown (rank ordered in terms of decreasing activity) in Figure
1A and the structures of 17 additional compounds (29–45) having
low (P100 lM) activity are shown in Figure 1B. The synthesis and
characterization of all compounds except 29 and 30 have been re-
ported elsewhere14 and the samples used here were from the same
batches. The synthesis of 29 and 30 were shown in Section 4, using
our previous methods.
We show inFigure 1A the structures of 25 bisphosphonates hav-
ing high-to-moderate activity in inhibiting RT-catalyzed AZT-exci-
sion by ATP, ordered in terms of decreasing activity. The IC50 values

Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
8961
Figure 1. Structures of bisphosphonate inhibitors. (A) Twenty-five active species investigated, rank ordered by decreasing activity. (B) Additional, inactive species (IC50
P100 M).
l
eling of class analogy22 (SIMCA) module in Sybyl 7.3 provides
one approach with which to create such classification models,
based on the CoMSIA fields of the aligned structures. Using this
approach we find that the resulting cross-validated model has an
overall classification accuracy of 94%, with 100% accuracy among
inactive and moderately active groups and 90% accuracy among
actives, Figure 4A (see the Supplementary Material Table S1, for
more details).
Given confidence in the results of the classification approach,
which can be applied to all of the compounds investigated, we
then investigated use of a standard regression method. Due to
the limited range of the numeric data, we assigned arbitrarily
large (300 lM) activities to the inactive compounds, in order to
increase the range, basically the same approach we used in a pre-
vious study.20 We then performed a CoMSIA analysis on all com-
pounds, using a partial least squares (PLS) regression approach.

8962
Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
Table 1
shown in Figure 3C–E. The (green) favored steric features
(Fig. 3C) are coincident with the meta-position of the phenyl ring,
while the unfavorable steric feature (yellow, Fig. 3C) correlate
with the occurrence of para-biphenyl substituted and inactive
n-alkyl species. The (yellow) favored hydrophobic feature
(Fig. 3E) is basically coincident with the (red) negative charge-
favorable field feature (Fig. 3D), and can be attributed to the
importance of having either a halogen or aromatic ring feature
in the meta-position of the phenyl ring. Experimental and pre-
dicted pIC50’s including statistical parameters for the final model
are given in Table 2, with training and test set predictions shown
graphically in Figure 4B, there being on average a factor of 2ꢁ er-
ror in the IC50 predictions.
We next considered what features might be missing from the
CoMSIA fields, or equivalently, why is the dichlorophenyl species
5 so active? One possibility is that these halogenated species might
be involved in charge-transfer interactions with an electron-rich
amino-acid sidechain in the protein, such as a tyrosine or phenyl-
alanine. Indeed, a TAM mutation found in many HIV-1 mutants and
present in our HIV-1 involves T215, and in previous work24 it has
been proposed that a major contact between ATP and RT involves
aromatic ring stacking interactions between the ATP adenine base
and an aromatic ring at position 215.24 In the case of a highly elec-
tron deficient bisphosphonate sidechain, this interaction could be
quite substantial. Of course, in the absence of concrete structural
Inhibition of ATP-mediated AZT-excision from HIV-1 reverse transcriptase
Compounda
Excision inhibition
MCF-7
SF-268
NCI-H460
IC50 (lM)
IC50
(l
M)
IC50
(l
M)
IC50
(lM)
5
6
7
8
9
0.5
0.9
1.0
1.0
1.3
1.5
1.9
2.1
2.2
2.4
2.9
3.2
3.3
3.5
3.9
3.9
4.6
5.0
5.2
5.4
7.9
9.5
11.2
38.0
51.3
213
41
>300
225
259
165
208
48
>300
236
225
122
241
39
>300
253
248
141
10
11
12
13
14
15
3
16
17
18
19
20
21
22
23
24
25
26
27
28
173
224
175
>300
>300
>300
>100b
128
>100b
139
>100b
118
>300
289
188
>300
269
176
>300
253
212
>100b
>100b
>300
84b
>100b
>100b
>300
95b
>100b
>100b
>300
95b
296
n.d.
297
n.d.
301
n.d.
>300
>300
>300
>300
205
>300
>300
>300
>300
186
>300
>300
>300
>300
187
>300
>300
>300
a
information, we cannot rule out other possibilities, such as a
p–p
Structures shown in Figure 1.
Compounds dissolved in dimethyl sulfoxide (DMSO).
b
interaction with an electron-rich DNA base, although this would
probably be expected to have an effect on DNA polymerase activ-
ity, which is generally not seen. In either case, however, the pres-
ence of halogen substituents in the most potent inhibitors
The optimal number of components in the final PLS model was
determined to be 4 by using q2 values, as obtained from the
SAMPLS23 leave-one-out cross-validation technique implemented
in Sybyl. This four component CoMSIA model (n = 35) gave
q2 = 0.63, r2 = 0.91, and F = 73, as shown graphically in Figure 4B
(full details shown in Supplementary Material Table S2). Five iter-
ations of a leave-two-out analysis were performed and 10 itera-
tions of activity data randomization were then applied to
confirm the predictive ability of the model, and that the final
model was not the result of a chance correlation. The hydropho-
bic, electrostatic and steric fields generated from the model are
suggests the possibility that
p/p or charge-transfer interactions
might be involved. To test this hypothesis, we computed semi-
empirical electron affinities (EA) as well as the ab initio vertical elec-
tron affinities (VEA) for each of the bisphosphonate sidechains to
see: (1) if there was any correlation between electron affinity and
excision activity and (2), whether electron affinity might be a useful
additional descriptor. The computed EA and VEA values are shownin
Table S3 in the Supplementary Material, and we observe good agree-
ment between ab initio and semi-empirical calculations, r2 = 0.81,
F = 99, p < 0.0001 (Table S3 and Fig. S2, Supplementary Material).
Figure 2. Inhibition of RT-catalyzed ATP-mediated phosphorolysis (green) and DNA synthesis (orange) at 5 lM inhibitor. Foscarnet, used as a control.

Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
8963
Figure 3. Structure–activity schematics. (A) Common feature pharmacophore: yellow spheres, combined feature of acceptor and metal ligator representing a phosphonate
group; purple sphere, donor or hydrophobic; green sphere, aromatic; cyan sphere, aromatic or hydrophobic. (B–E) CoMSIA alignment and results; (B) flexible common feature
alignment used for CoMSIA, showing heavy atoms only; (C) steric fields; green favored, yellow disfavored; (D) electrostatic fields; blue positive charge favored, red, positive
charge-disfavored; (E) hydrophobic fields; yellow favorable, gray disfavored.
When the experimental pIC50 = (ꢂlog10 [IC50 (M)]) values for AZT-
3. Conclusion
excision by the active compounds are plotted against the EA and
VEA values, we obtain the result shown in Figure 4C, in which
we see a modest correlation: r2 = 0.55, F = 28 and p < 0.0001 for
EA and r2 = 0.37, F = 14, p = 0.0012 for the VEA. These results
are of interest since they suggest that it might be possible to im-
prove upon the CoMSIA results by incorporating the EA or VEA
values as additional descriptors. This indeed turns out to be
the case (Supplemental Information Tables S4–S6) with incorpo-
ration of the VEA term resulting in significant improvements in
r2 (0.91 ? 0.97), F (73 ? 144) and the rms error (0.32 ? 0.10).
Clearly then, using the CoMSIA and VEA results lead to good
activity predictions, and should be of help in the future design
of improved excision inhibitors.
The bisphosphonates we have investigated here have IC50 val-
ues as low as 500 nM in AZT-excision. This is ꢀ6ꢁ higher than that
found with 2GP, however, unlike 2GP, the bisphosphonates tested
here have essentially no activity on DNA polymerase activity. This
is of importance since our purpose is to block, exclusively, AZT-
excision (not AZT incorporation). We also find that the most potent
inhibitors, in general, have IC50 values for the inhibition of human
The results we have presented above are of interest for a num-
ber of reasons. First, we have tested a library of 42 bisphospho-
nates for their ability to inhibit the HIV-1 reverse transcriptase
catalyzed phosphorolysis of AZT from AZT-terminated primers by
ATP. The four most active compounds are all halogen containing
aromatic species having IC50 values of 61 lM. Second, we have
used 3D-QSAR methods to investigate structure–activity relation-
ships. A classification method was found to have an accuracy of
94% in categorizing compounds into three discrete classes: active,
moderately active and inactive. In addition, a CoMSIA partial least
square regression model was found to yield a predictive model
(q2 = 0.63, r2 = 0.90, F = 73, n = 35) for AZT-excision. Interpretation
of the resulting fields showed a preference for large, hydropho-
bic/steric substituents at the meta- rather than para-position of
the first ring. Additionally, electron-withdrawing ring substituents
(negative charge favored) enhanced activity. Third, we find that
there is no significant correlation (r2 = 0.10) between RT-catalyzed
AZT-excision inhibition and the growth inhibition of three human
cell lines, with the most potent excision inhibitors having essen-
cell growth >250 lM, since they do not inhibit the human FPPS (or
GGPPS) enzymes, again making them of interest for further
tially no activity (>250 lM) in cell growth inhibition. Fourth, we
find that there is no inhibition of RT-catalyzed DNA synthesis by
development.
these potent bisphosphonate excision inhibitors. When taken to-

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Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
Table 2
CoMSIA results for inhibition of AZT-excision by bisphosphonates
Compounda
CoMSIA pIC50 predictions
2
Experimental
Test setsb
4
IC50
(
lM)
pIC50 (M)
Training Set
1
3
5
Pred
Residual
5
6
7
8
9
0.5
0.9
1.0
1.0
1.3
1.5
1.9
2.1
2.2
2.4
2.9
3.2
3.3
3.5
3.9
3.9
4.6
5.0
5.2
5.4
7.9
9.5
6.3
6.1
6.0
6.0
5.9
5.8
5.7
5.7
5.7
5.6
5.5
5.5
5.5
5.5
5.4
5.4
5.3
5.3
5.3
5.3
5.1
5.0
5.0
4.4
4.3
6.1
5.9
5.6
5.9
5.9
5.9
5.9
5.5
5.5
5.8
5.4
5.5
5.6
5.0
5.4
5.2
4.7
5.5
5.8
5.4
4.9
4.9
5.0
4.4
4.9
6.1
5.9
5.8
5.8
5.8
5.9
5.8
5.6
5.7
5.6
5.4
5.7
5.5
5.1
5.5
5.2
4.4
5.6
5.8
5.3
5.1
5.0
4.8
4.2
4.4
6.1
5.9
5.8
5.8
5.8
5.8
5.7
5.5
5.7
5.6
5.4
5.7
5.6
5.1
5.4
5.2
5.5
5.6
5.8
5.2
5.1
5.0
5.8
4.2
4.6
6.1
5.8
5.8
5.8
5.7
5.8
5.7
5.6
5.7
5.6
5.5
5.6
5.6
5.2
5.4
5.2
5.3
5.5
5.8
5.1
5.1
5.0
5.2
4.2
4.5
6.2
5.9
5.8
5.9
5.8
5.8
5.8
5.6
5.6
5.9
5.7
5.6
5.6
5.2
5.4
5.5
5.3
5.5
5.8
5.2
5.1
4.9
5.1
4.2
4.5
6.1
5.9
5.8
5.8
5.6
5.8
5.7
5.6
5.7
5.6
5.5
5.6
5.6
5.2
5.4
5.2
5.3
5.5
5.8
5.2
5.0
4.7
5.2
4.2
4.5
6.1
5.8
5.8
5.8
5.6
5.8
5.8
5.5
5.7
5.9
5.5
5.7
5.6
5.2
5.4
5.2
4.4
5.5
5.8
5.1
5.1
4.7
5.8
4.2
4.5
0.2
0.2
0.2
0.1
0.3
0.0
0.0
0.1
0.0
10
11
12
13
14
15
3
16
17
18
19
20
21
22
23
24
25
26
27
28
ꢂ0.3
0.0
ꢂ0.2
ꢂ0.1
0.3
0.0
0.2
0.9
ꢂ0.2
ꢂ0.5
0.2
0.0
0.3
11
38
51
ꢂ0.9
0.2
ꢂ0.2
32
38
41
42
40
33
43
34
31
45
302
302
302
302
302
302
302
302
302
302
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.8
3.1
3.9
3.7
4.2
3.2
3.9
3.6
3.3
3.4
3.3
3.3
3.5
3.3
4.1
3.4
3.9
3.6
3.6
3.7
3.4
3.3
3.5
3.3
4.0
3.3
3.9
3.6
3.6
3.6
3.3
3.3
3.5
3.3
4.1
3.3
3.9
3.7
3.6
3.6
3.2
3.4
3.6
3.4
4.1
3.2
3.9
3.6
3.6
3.4
3.3
3.3
3.5
3.3
4.0
3.3
3.9
3.6
3.6
3.6
3.3
3.3
3.5
3.3
4.1
3.3
3.9
3.6
3.6
3.6
0.2
0.2
0.0
0.2
ꢂ0.5
0.2
ꢂ0.4
ꢂ0.1
ꢂ0.1
0.0
q2
0.63
0.91
73
35
4
0.70
0.96
115
33
5
0.68
0.95
108
33
5
0.66
0.95
107
33
5
0.67
0.97
151
33
0.61
0.95
110
33
5
r2
F test
N
n
5
Steric (%)
0.19
0.32
0.28
0.20
0.19
0.19
0.19
0.18
0.34
0.29
0.21
0.18
Electrostatic (%)
Hydrophobic (%)
Donor (%)
0.33
0.27
0.20
0.35
0.27
0.20
0.35
0.27
0.20
0.35
0.27
0.20
a
Structures shown in Figure 1.
Bold values represent predicted activities of compounds that were not included in the training set.
b
gether, these results strongly support the idea that bisphospho-
nates may have utility as inhibitors of AZT-excision catalyzed by
HIV-1 RT, provided suitable formulations or delivery vehicles are
available to facilitate uptake of the charged bisphosphonates into
HIV-infectable target cells such as T-lymphocytes. Nonetheless, gi-
ven the low cytotoxicity of the novel bisphosphomnates described
in the present work, because they do not target the FPPS enzyme,
such compounds are of interest in the context of the future devel-
opment of novel anti-infectives for HIV-1.
gel F254 plates (Merck) and UV light (254 nm) and 1% KMnO4 solu-
tion were used for visualization.
4.2. Synthesis
The synthesis and characterization of all compounds except 29
and 30 have been reported elsewhere14 and the samples used here
were from the same batches. The synthesis of 29 and 30 were
shown below:
4. Experimental
4.1. General
4.2.1. 2-(3,4-Dichlorophenyl)ethyl-1,1-bis(methylphosphinic
acid) (29)
NaH (44 mg, 1.1 mmol, 60% in oil) was added at 0° C to diethyl
methylenebis(methylphosphinate)25 (228 mg, 1 mmol) in dry THF.
3,4-Dichlorobenzyl bromide (240 mg, 1 mmol) was added to the
resulting solution and after 1 h, the reaction was quenched with an
NH4Cl solution and extracted with diethyl ether (3 ꢁ 20 mL). The or-
ganic layer was dried and evaporated and the residue purified using
column chromatography (silica gel, ethyl acetate:MeOH/10:1). The
All reagents used were purchased from Aldrich (Milwaukee,
WI). The purities of all compounds were routinely monitored by
using 1H and 31P NMR spectroscopy at 400 or 500 MHz on Varian
(Palo Alto, CA) Unity spectrometers using, in some instances, abso-
lute spin-count quantitative analyses. TLC was performed on silica

Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
8965
Figure 4. CoMSIA modeling results; (A) SIMCA results, the overall classification accuracy is 94%. Actual and predicted classes shown, yellow box indicating classification
error; (B) PLS regression results, q2 = 0.63, r2 = 0.90, F = 73, n = 35. Test and training set results shown: training = , test = s; (C) activity–electron affinity correlations. d = EA,
r
2 = 0.55, F = 28;
= VEA, r2 = 0.37, F = 14.
diethyl ester obtained was treated with 3 equiv. of TMSBr in CH2Cl2
for 6 h. The mixture was then evaporated to dryness, dissolved in
water/MeOH, and neutralized to pH ꢀ6. Compound 29 was obtained
by filtration as a white powder. 1H NMR (400 MHz, D2O): d 1.21 (d,
J = 14.4 Hz, 6H, CH3), 2.3–2.45 (m, 1H, CH), 2.96 (td, J = 15.2 Hz,
6.8 Hz, 2H, CH2), 7.1–7.3 (m, 3H, aromatic); 31P NMR (162 MHz,
D2O): d 47.4; Anal. Calcd for C10H13Cl2O4P2ꢃ1.5H2Oꢃ0.6NaBr: C,
27.19; H, 3.65. Found: C, 27.14; H, 3.47.
inhibitor was determined using 0.2 U/mL of poly(rA)-oligo(dT)12–18
2 nM RT, 5 L of 50 mM Tris–
M [3H]-TTP in a final volume of 50
HCl, pH 8.0 containing 60 mM KCl, and 10 mM MgCl2. Samples were
incubated for 20 min at 37 °C and then quenched by adding 200
of ice-cold 10% trichloroacetic acid containing 20 mM sodium pyro-
phosphate. After a 20 min incubation on ice, samples were filtered
,
l
l
l
L
through 1.2 lm glass fiber type-C filters in 96-well microplates (Mil-
lipore, Bedford MA). The filters were washed sequentially with 10%
TCA and ethanol, and the extent of [3H]-TMP incorporation into the
precipitated nucleic acid was quantified by liquid scintillation
spectrometry.
4.2.2. 2-(3,4-Dichlorophenyl)-1-(methylphosphino)
ethylphosphonic acid (30)
NaH (44 mg, 1.1 mmol, 60% in oil) was added at 0 °C to triethyl
methylphosphinomethylenephosphonate26 (258 mg, 1 mmol) in
dry THF. 3,4-Dichlorobenzyl bromide (240 mg, 1 mmol) was added
to the resulting solution and after 1 h, the reaction was quenched
with an NH4Cl solution and extracted with diethyl ether
(3 ꢁ 20 mL). The organic layer was dried and evaporated and the
residue purified using column chromatography (silica gel, ethyl
acetate:MeOH/10:1). The diethyl ester obtained was treated with
4.5 equiv. of TMSBr in CH2Cl2 for 6 h. The mixture was then evap-
orated to dryness, dissolved in water/MeOH, and evaporated again
to give compound 30 as a white powder. 1H NMR (400 MHz, D2O):
d 1.23 (d, J = 14.4 Hz, 3H, CH3), 2.2–2.35 (m, 1H, CH), 2.85–3.05 (m,
2H, CH2), 7.1–7.3 (m, 3H, aromatic); 31P NMR (162 MHz, D2O): d
18.3 (s, 1P), 49.6 (s, 1P). Anal. Calcd for C9H12Cl2O5P2: C, 26.75;
H, 3.24. Found: C, 26.90; H, 3.00.
4.3.3. Assay of RT-catalyzed ATP-mediated phosphorolysis of
chain-terminating AZT
In vitro ATP-dependent AZTMP excision reactions were carried
out using a direct assay, essentially as previously described.4
Briefly, a 19-nt DNA primer (30- CAG GGG GGA AAA GAA AAT T-
50) was annealed to a 57-nt DNA template (50-CGT TGT CAG TGA
ATC AGC CCT TCC AGT CCC CCC TTT TCT TTT AAA AAG TGG CTA
AGA-30) to prepare the heteropolymeric template/primer (T/P).
The 19 nt DNA primer was 50-radiolabeled with [ 32P]-ATP and
c-
T4 polynucleotide kinase (New England BioLabs, Ipswich, MA)
according to the manufacturer’s instructions. The labeled primer
was purified using denaturing polyacrylamide gel electrophoresis
on 7M urea-16% polyacrylamide gels, followed by elution from
the gel. The purified 50-32P-labeled primer was then annealed to
the 57 nt DNA template using a 1:1.2 molar ratio of template to
primer, by heating to 90 °C followed by slow cooling to ambient
temperature to form the 57 nt/19 nt template-primer (57/19 T/P)
substrate. AZTMP was added to the primer 30-terminus of the
4.3. Biological activity testing
4.3.1. Protein expression
TAM-RT with the mutations D67N/K70R/T215F/K219Q was ex-
pressed and purified as previously described.27 The protein con-
centration of the purified enzyme was determined by
spectrophotometry at 280 nm using an extinction coefficient of
57 nt/50-[32P]-19 nt T/P by incubating with wt RT and 100
lM
AZTTP for 30 min at 37 °C. The resulting [32P]-labeled, chain-termi-
nated 20 nt primer was purified by elution of the appropriate band
following electrophoretic resolution on 7M urea-16% acrylamide
denaturing gels. The purified chain-terminated primer was then
re-annealed to the 57 nt DNA template DNA primer and stored at
ꢂ20 °C for use as T/P in the phosphorolysis experiments.
260,450 Mꢂ1 cmꢂ1. [3H]-TTP and [ 32P]-ATP were products of
c-
PerkinElmer Life Sciences (Boston, MA). The homopolymeric tem-
plate-primer (T/P) poly(rA)-oligo(dT)12–18 used for steady state ki-
netic studies was obtained from GE Healthcare (Piscataway, NJ).
AZTTP as well as DNA oligonucleotides used to prepare the hetero-
polymeric T/P for excision assays were obtained from Trilink Bio-
technologies (San Diego, CA).
The phosphorolytic removal of chain-terminating 30-AZTMP in
the absence or the presence of varying concentrations of bisphos-
phonates was assayed by incubating TAM-RT with the AZTMP-ter-
minated T/P (4:1 ratio of RT:T/P) in 50 mM Tris–HCl, pH 8.0,
50 mM KCl. Reactions were initiated by addition of 10 mM MgCl2
and 3.0 mM ATP. Inorganic pyrophosphatase (0.01 U; Sigma) was
included in phosphorolysis reactions using ATP, to ensure removal
of any contaminating PPi in the ATP preparation. Aliquots were re-
moved at defined times and quenched with an equal volume of gel
4.3.2. Steady-state analysis of bisphosphonate inhibition of RT
DNA polymerase activity
TAM-RT RNA-dependent DNA polymerase activity in the ab-
sence or presence of varying concentrations of bisphosphonate

8966
Y. Song et al. / Bioorg. Med. Chem. 16 (2008) 8959–8967
loading dye (98% deionized formamide, 10 mM EDTA and 1 mg/mL
each of bromophenol blue and xylene cyanol). The 20 nt 30-AZTMP
terminated substrate and 19 nt excision products were separated
by denaturing gel electrophoresis and quantified by
phosphorimaging.
The dataset was divided into three classes: active (IC50 <10
lM),
moderately active (10–100 M), and inactive (IC50 >100 M). SIM-
l
l
CA analysis was then performed by using the assigned classes and
CoMSIA fields (see below), with default settings (CoMFA standard
scaling and column filtering at 2.0 kcal/mol). Models were auto-
matically cross-validated by the algorithm, with classification
rates, sum of squared residuals and other details being reported.
4.3.4. Human cell growth inhibition assay
Human tumor cell lines MCF-7 (breast adenocarcinoma), NCI-
H460 (lung large cell), and SF-268 (central nervous system glio-
blastoma) were obtained from the National Cancer Institute and
maintained at 100% humidity and 5% CO2 at 37 °C. Cell lines were
4.4.3. CoMSIA alignment
To carry out our comparative molecular similarity indices anal-
ysis (CoMSIA)30 calculations, we began by building and geometry
optimizing the bisphosphonate structures in the Molecular Operat-
ing Environment program (MOE).21 Minimized structures were
generated using the MMFF94x forcefield31 and default minimiza-
tion parameters, which apply three successive non-linear optimi-
zation methods: steepest descent, conjugate gradient and
truncated-Newton. Minimized structures were then manually
aligned to the lowest energy conformation of the most active com-
pound (5). The manual alignment was subsequently refined using
the flexible alignment module in MOE, which perceives common
features between molecules (e.g., aromaticity and volume) and at-
tempts to improve the alignment of such features. The aligned
compounds were then exported to Sybyl 7.332 for analysis: all
CoMSIA fields were computed on a rectangular grid, using default
grid spacing and probe atoms.
cultured in RPMI-1640 medium supplemented with 2 mM L-gluta-
mine and 10% fetal bovine serum (Gibco, Grand Island, NY) at 37 °C
in a 5% CO2 atmosphere with 100% humidity. 0.01 M compound
stock solutions were prepared in water or DMSO. A broth microdi-
lution method was used to determine bisphosphonate growth inhi-
bition IC50 values. Compounds were half-log serial diluted from
316
Inc., Corning, NY) using cell culture media. In cases where DMSO
was used, the range of dilution was 100 M–10 nM. Cells were pla-
ted at a density of 5000 cells/well and contained 10 L of the com-
pound of interest in a final volume of 100 L. Cells were then
lM–10 nM into 96-well TC-treated flat bottom plates (Corning
l
l
l
incubated for 4 days, at which time an MTT ((3-(4,5-dimethylthia-
zole-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation as-
say (ATCC, Manassas, VA) was performed, in order to obtain
growth inhibition dose response curves. Briefly, cells were incu-
bated for 2 h with the tetrazolium salt, lysed with detergent and
incubated O/N, protected from light and at room temperature.
The absorbance at 600 nm was read the following day using a
SpectraMax Plus 384 spectrophotometer (Molecular Devices, Sun-
nyvale, CA). The compound concentrations for 50% growth inhibi-
4.4.4. Quantum chemical calculations
Vertical electron affinities (VEA) were calculated for the side-
chains of aromatic bisphosphonates using the Gaussian 03 pro-
gram33 following an approach described previously.34 Basically,
compounds were geometry optimized using the B3LYP functional
*
tion (IC50 values) were obtained by fitting the absorbance data to
and a 6-31G basis set, and a frequency calculation was used to
ðImax
a rectangular hyperbolic function: I ¼
ÞðC0Þ, where I is the percent
confirm that the geometries did not represent saddle points. Single
point energy calculations were then carried out for the neutral,
optimized geometries, using B3LYP, a 6-311+G(2df,p) basis set
with tight convergence, no symmetry constraints, and an ultrafine
grid. UB3LYP was used to calculate the single point energy struc-
tures, after adding an additional electron, again using the
6-311+G(2df,p) basis set and the options specified for the neutral
molecules. The stability of the wave function for the charged spe-
cies was verified with the Stable = Opt keyword. The VEA was ta-
ken to be the difference between the total eigen-energies of the
neutral and charged species. For purposes of comparison, sidechain
electron affinities were also computed semi-empirically within
Maestro using the PM3 Hamiltonian.35 Structures of the sidechains
were prepared using LigPrep36 with default options. Electron affin-
ities (EA) were computed using QikProp37 specifying full, rather
than fast mode.
IC50þC
inhibition, Imax = 100% inhibition and C is the concentration of the
inhibitor, using GraphPad PRISM 3.0 software for Windows
(Graphpad Software Inc., San Diego, CA).
4.3.5. FPPS inhibition
Human FPPS was expressed and purified as described previ-
ously.28 Human FPPS assays were carried out using 96-well plates
with a 200 lL reaction mixture in each well. The condensation of
geranyl diphosphate with isopentenyl diphosphate was monitored
by a continuous spectrophotometric assay for phosphate releasing
enzymes.29 The reaction buffer contained 50 mM Tris–HCl, 1 mM
MgCl2, at pH 7.4. The compounds investigated were pre-incubated
with the enzyme for 30 min at 20 °C. The IC50 values were obtained
by fitting the inhibition data to a normal dose–response curve
using Origin 6.1 (OriginLab Corporation, Northampton, MA).
4.4. Computational methods
Acknowledgments
4.4.1. Pharmacophore model
This work was supported by the United States Public Health
Service (NIH Grants AI-060452 and AI-052010 to M.A.P., GM-
073216 to E.O.). Y.S. was supported by a Leukemia and Lymphoma
Society Special Fellowship. J.M.W.C. was a Jean Dreyfus Boissevein
Fellow.
A common feature pharmacophore was constructed in MOE21
using the consensus pharmacophore module and the polarity-
charge-hydrophobicity (PCH) scheme. The pharmacophore was
perceived from energy minimized structures of the most active
compounds using tolerances of 1.2 and a threshold of 25%. The fi-
nal model was simplified by combining the features of each indi-
vidual P–O group into two master functions, each describing the
phosphonate group as an anionic metal ligator. Tolerances (sphere
sizes) encompassing each feature was enlarged uniformly in order
to relax the mapping requirements.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.08.047.
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