Novel N-3 substituted TSAO-T derivatives: Synthesis and anti-HIV-evaluation

Article abstract of DOI:10.1080/15257770801943990

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds with an alcohol functional group connected to the N-3 position through a cis or trans double bond have been prepared. These compounds have been useful to study how the conformational restriction of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme. Copyright Taylor & Francis Group, LLC.

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Novel N-3 Substituted TSAO-T
Derivatives: Synthesis and Anti-HIV-
Evaluation
María-Cruz Bonache a , Ernesto Quesada a , Chih-Wei Sheen b , Jan
Balzarini c , Nicolas Sluis-Cremer b , María Jesús Pérez-Pérez a ,
María-José Camarasa a & Ana San-Félix a
a Instituto de Química Médica (C.S.I.C.) , Madrid, Spain
b Division of Infectious Diseases , University of Pittsburgh School of
Medicine , Pittsburgh, Pennsylvania, USA
c Rega Institute for Medical Research , K. U. Leuven, Leuven,
Belgium
Published online: 10 Apr 2008.
To cite this article: María-Cruz Bonache , Ernesto Quesada , Chih-Wei Sheen , Jan Balzarini , Nicolas
Sluis-Cremer , María Jesús Pérez-Pérez , María-José Camarasa & Ana San-Félix (2008) Novel N-3
Substituted TSAO-T Derivatives: Synthesis and Anti-HIV-Evaluation, Nucleosides, Nucleotides and
Nucleic Acids, 27:4, 351-367, DOI: 10.1080/15257770801943990
To link to this article: http://dx.doi.org/10.1080/15257770801943990
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Nucleosides, Nucleotides and Nucleic Acids, 27:351–367, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770801943990
NOVEL N-3 SUBSTITUTED TSAO-T DERIVATIVES: SYNTHESIS
AND ANTI-HIV-EVALUATION
Mar´ıa-Cruz Bonache,1 Ernesto Quesada,1 Chih-Wei Sheen,2 Jan Balzarini,3
Nicolas Sluis-Cremer,2 Mar´ıa Jesu´ s Pe´rez-Pe´rez,1 Mar´ıa-Jose´ Camarasa,1
1
and Ana San-Fe´lix
1Instituto de Qu´ımica Me´dica (C.S.I.C.), Madrid, Spain
2Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania, USA
3Rega Institute for Medical Research, K. U. Leuven, Leuven, Belgium
Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating
2
groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these
compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we
were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds
with an alcohol functional group connected to the N-3 position through a cis or trans double bond
have been prepared. These compounds have been useful to study how the conformational restriction
of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme.
Keywords AIDS; HIV-1 reverse transcriptase; TSAO-T
INTRODUCTION
Human immunodeficiency virus (HIV) reverse transcriptase (RT) is an
attractive target for the development of anti-AIDS drugs.[1–3] This enzyme
plays a multifunctional role in the conversion of the ssRNA viral genome to
dsDNA.
HIV-1 RT is an asymmetric heterodimer which contains two subunits
of 66 and 51 kDa designated as p66 and p51, respectively.[4–6] The larger
Received 8 May 2007; accepted 15 November 2007.
We thank Dr. Francisco Amat for his help and advise with the photochemical experiments. We
thank the Ministery of Education of Spain for a grant to Mar´ıa-Cruz Bonache. The Spanish MEC
(project SAF2006–12713-C02) is also acknowledged for financial support. NSC was supported by grants
GM068406 and R21 AI060393 from the National Institutes of Health (USA).
Address correspondence to Ana San-Fe´lix, Instituto de Qu´ımica Me´dica (C.S.I.C.), c/Juan de la
Cierva, 3-28006 Madrid, Spain. E-mail: anarosa@iqm.csic.es
351

352
M.-C. Bonache
FIGURE 1 Structure of TSAO-T, TSAO-m3T, and TSAO-hp3T.
subunit (p66) contains both polymerase and RNase H activities, while the
smaller subunit (p51) lacks these functions.[4–6] The catalytic activity of the
RT is only observed when it is in its (hetero)dimeric form.[7–9]
TSAO-T (Figure 1) is the prototype of a unique class of non-nucleoside
reverse transcriptase inhibitors (NNRTIs).[10–13] TSAO derivatives are
highly functionalized nucleosides, which have a structure and a mechanism
of action quite distinct from conventional NNRTIs.[12] Experimental data
on TSAO derivatives strongly suggest a specific interaction of the amino
group of the 3ꢁ-spiro moiety with the carboxylic acid group of a glutamic acid
residue at position 138 of the p51 subunit of HIV-1 RT.[14–17] This residue is
located at the β7-β8 loop of the p51 subunit.[18,19] Moreover, other amino
acids of the p66 subunit also are needed for an optimal interaction of TSAO
derivatives with HIV-1 RT.[20] So, TSAO compounds are among the very few
RT inhibitors for which amino acids at both HIV-1 RT subunits (p51 and
p66) are needed for optimal interaction with the enzyme.
Since the first TSAO derivatives were synthesized many efforts to
co-crystallize these molecules with their target enzyme (HIV-1 RT) were
pursued. Unfortunately, they have been unsuccessful so far. In 2001, com-
putational docking studies led us to propose a putative binding mode of the
N -3 methyl derivative of TSAO-T (TSAO-m3T) with HIV-1 RT.[21]
The molecular model was in agreement with known structure-activity
and mutagenesis data for this unique class of inhibitors, and also with
biochemical evidence indicating that TSAO molecules can affect enzyme
dimerization.[22] In fact, the model provided strong evidence that TSAO-
m3T straddles between both subunits at the p66/p51 interface but in a
manner that does not make use of the NNRTI binding pocket.
On the other hand, our docking model indicates that the N -3 sub-
stituent at the thymine base of TSAO-m3T was found running parallel to
the subunit interface and that the presence of functional groups at this
position might provide additional interactions with amino acids at or near
the p51/p66 interface.[23]

Novel N-3 Substituted TSAO-T Derivatives
353
Herein we decided to prepare novel series of N-3 substituted TSAO
derivatives that may give additional interactions with one or more amino
acids of the dimer interface, and thus may destabilize the RT by disrupting
crucial interface interactions.
As some of the amino acids found at the dimer interface near the
TSAO binding site have side chains with nucleophilic functional groups
(Glu-A169, Thr-A165, Lys-B49), we decided to prepare a first series of
TSAO derivatives bearing at the N -3 position of thymine strong electrophilic
moieties such as an epoxide or a nitrogen mustard with a reactive N,N -
bis-(2-chloroethyl)amine group. The alkylating properties of these groups
might lead to the formation of a covalent bond between the novel TSAO
derivatives and the amino acids of the HIV-1 RT that might persistently
disturb the enzyme.
We have previously seen that attachment of a hydroxypropyl substituent
at the N -3 position of TSAO-T resulted in a two-fold better inhibitor (TSAO-
hp3T) than the prototype compound TSAO-T (Figure 1).[21,23] This result
is consistent with the prediction that an additional hydrogen bond could be
formed with the protein backbone (Pro-B140). However, the improvement
in activity was lower than expected and we reasoned that the putative extra
hydrogen bond may be partially offset by the inherent flexibility of the
methylene linker.[21] With the aim to enhance the interaction of the N -3
substituent with HIV-1 RT we prepared a second series of compounds in
which an unsaturated bond in the linker connecting the distal functional
group (alcohol) with the N -3 position of the thymine has been introduced.
The nature of the unsaturation may play an essential role in the relative
orientation of the TSAO molecule and the terminal functional group.
Therefore, compounds containing a cis- or trans- double bond into the
linker were prepared.
On the other hand, we prepared a third series of compounds that in-
corporate at the N -3 position a photoreactive moiety, such as aryl azide and
benzophenone. These compounds would first selectively interact with the
HIV-1 RT and then, upon irradiation with an appropriate light source, the
photoactive group might react with the enzyme, via different intermediates,
forming a covalent linkage with the amino acids proximal to the TSAO
binding site. These compounds may, in addition, help to identify the amino
acids at the interface that are involved in the interaction of TSAO derivatives
with HIV-1 RT.
RESULTS AND DISCUSSION
Chemical Results
In order to interact covalently with the amino acids of the HIV-1 RT
close to the TSAO binding site, derivatives 2 and 3 bearing a bis-alkylating

354
M.-C. Bonache
SCHEME 1 Synthesis of N -3 substituted TSAO derivatives 2 and 3.
and epoxide moiety, respectively at the N -3 position (Scheme 1) have been
prepared. Compound 2 was obtained in a 68% yield by coupling the N -3 car-
boxylic acid TSAO derivative 1[23] with N,N -bis(2-chloro-ethyl)amine in the
presence of BOP and triethylamine (Scheme 1). The N -3 epoxi derivative
3 could be readily prepared in one step following our previously described
method for the selective N -3 alkylation of TSAO derivatives).[13,23] Thus,
treatment of TSAO-T (Scheme 1) with epibromohydrin in the presence of
anhydrous potassium carbonate afforded the N-3-derivative 3 in 60% yield.
In addition, compounds with rigid side chains connecting a distal func-
tional group (alcohol) with the N -3 position of TSAO-T by incorporating E
or Z double bonds into the linker were prepared as follows.
Synthesis of the ethoxycarbonylvinyl E isomer 4 was carried out fol-
lowing the procedure developed by Villarrasa et al.[24] that consisted on
addition of the NH of a pyrimidine ring to the triple bond of an activated
alkyne. Thus, when TSAO-T was treated with ethyl propiolate and DMAP,
in acetonitrile at room temperature, a quick reaction occurred to afford 4
in very good yield (82%) (Scheme 2). Only one isomer, of E configuration,
was obtained, in the light of the 1H spectrum (olefinic protons at δ 6.99 and
8.26 ppm, with J CH = CH 14.7 Hz).[24–26]
For comparative purposes we were also interested in the synthesis of
the corresponding Z isomer. Photochemical isomerization of an alkene
of E configuration is a specially convenient route for the synthesis of
the thermodynamically less stable Z isomer.[27–29] This method has been
applied with success for the trans-cis isomerization of an ethoxycarbonylethyl

Novel N-3 Substituted TSAO-T Derivatives
355
SCHEME 2 Synthesis of N -3 substituted TSAO derivatives 4–7.
moiety attached at the N -3 position of a thymine.[30] Based on these
precedents we decided to carry out the photochemical isomerization of the
E-isomer 4 to the corresponding Z-isomer 5. Due to the presence of other
light-absorbing groups in the TSAO molecule, the reaction conditions were
carefully adjusted to give a preponderance of the Z isomer without affecting
to the other light-sensitive groups.
Thus, by performing the UV difference spectra of TSAO-T and com-
pound 4 one absorption maximum at 289 nm was detected that should
correspond to the ethoxycarbonyl vinyl moiety (data not shown). This was
the wavelength used for the UV irradiation in order to ensure that only
the vinyl moiety was affected and not the rest of the molecule. Thus, we
irradiated with a medium-pressure mercury lamp a methanolic solution
of the E isomer 4, and monitored the progress of the reaction by HPLC.
After 30 minutes of irradiation with 290 nm light an equilibrium (1:1.5)
mixture of E and Z isomers was observed. Prolonged reaction times (6
hours) did not affect the rate of isomerisation. It is interesting to note
that no degradation and/or side reactions occurred in this photochemical
trans-cis isomerisation.
1
The HNMR spectrum of the mayor component of the mixture (com-
pound 5) revealed that the olefinic protons appeared at δ 6.12 and 6.70
ppm, with a vecinal coupling constant of J CH = CH 9.0 Hz as corresponds
to a Z isomer.[25,26] In addition, the stereochemistry of the double bond
of 5 was unequivocally established as Z by NOE difference experiments.
Thus, irradiation of one of the vinylic protons (δ 6.12 ppm) caused the
enhancement of the signal for the other vinylic proton (δ 6.70 ppm)
indicating that was the Z isomer.
Next, DIBAL-H (1M in THF) reduction of the carboxylic acid ester of 4
afforded the corresponding hydroxy methyl vinyl derivative 6 in 61% yield
(Scheme 2). A similar reaction with compound 5 gave 7 in 52% yield.

356
M.-C. Bonache
SCHEME 3 Synthesis of N -3 substituted TSAO derivatives 9 and 10.
Finally, a third series of compounds was prepared in which a photoactive
group was attached at the N -3 position of TSAO-T (Scheme 3). Two
different photoactive moieties, arylazide and benzophenone were chosen as
they have been described as being among the most successful for labeling re-
ceptors and enzymes.[31–33] Upon irradiation, these photoactivable groups
produce extremely reactive species namely, a diradical, or a (singlet) nitrene
for the benzophenone and arylazide, respectively[31] that bind covalently to
receptors and enzymes. Because it is essentially imposible to predict which
group will be most readily reactive with the receptor, the use of different
groups increases the likelihood of a successful covalent incorporation.
As shown in Scheme 3, compound 9 which possesses an arylazide
photophore, was synthesized in 85% yield by a method that consisted of
coupling the previously described N -3 aminopropyl TSAO derivative 8[23]
with 4-azidosalicylic acid[34] in the presence of BOP and trietylamine. In a
similar way, coupling of 8 with commercially available 4-benzoylbenzoic acid
afforded 10 in 76% yield.
Structures of the novel N -3 TSAO derivatives were assigned on the basis
of their analytical and spectroscopic data.
Biological Results
All the synthesized compounds showed pronounced inhibitory activity,
in cell culture, against HIV-1 replication (EC50 = 0.04–0.75 µM) while
being invariably inactive against HIV-2 (Table 1). There is a good agree-
ment with the anti-HIV activities obtained in both MT-4 and CEM cell
cultures.

Novel N-3 Substituted TSAO-T Derivatives
357
TABLE 1 Inhibitory effects of test compounds on HIV-1 and HIV-2 replication in MT-4 and CEM cell
culture and recombinant HIV-1 RT
EC50 (µM)a
MT-4
HIV-1
CEM
CC50 (µM)b IC50 (µM)c
Comp.
HIV-2
HIV-1
HIV-2 HIV1/138K
CEM
HIV-1 RT
2
3
6
7
9
10
0.08 0.02 >125 0.04 0.02 ≥125
>25
>10
>2
86 30
47 16
3.4 1.9
44 7.8
≥250
8.2 1.3
2.4 0.5
0.5 0.1
1.6 0.2
0.4 0.1
0.5 0.1
1.0 0.2
0.7 0.1
0.11 0.11
0.04 0.0
>50 0.10 0.02
>2 0.05 0.03
>25 0.12 0.0
>50 0.37 0.25
>50 0.16 0.09
>20 0.06 0.01
>2 0.01 0.01
>50
>2
0.32 0.02
0.68 0.33
0.29 0.12
0.06 0.03
0.03 0.03
>25
>50
>50
>20
>2
>5
>50
>50
>10
>10
≥250
TSAO-Td
TSAO-hp3Te
12
3
3.9 0.29
a50% effective concentration, or the compound concentration required to inhibit HIV-induced
cytopathicity by 50%.
b50% cytostatic concentration, or the compound concentration required to inhibit cell proliferation
(CEM) or to reduce cell viability (MT-4) by 50%.
c50% inhibitory concentration, or the compound concentration required to inhibit recombinant
HIV-1 RT activity by 50%.
dData taken from ref. [13]
e Data taken from ref. [23].
TSAO derivative 3 contained an epoxide moiety attached to the N -3
position showed, in cell culture, an anti-HIV-1 activity comparable to that
of compound 2 with a bis-N -chloroethyl moiety at this position (Table 1).
These groups could be introduced without apreciable loss of antiviral
activity against the HIV-1 RT but did not improve activity, suggesting that
although they were well-tolerated, they were not able to provide additional
interactions with the enzyme.
The E-hydroxymethylvinyl derivative 6 was 10-fold more active in cell
culture than the corresponding Z-isomer 7, and almost equally active than
the prototype TSAO-hp3T (Table 1). With respect to the recombinant HIV-
1 RT (Table 1), the E-isomer 6 (IC50 = 0.5 µM) proved to be three-fold
more active than the Z-isomer 7 (IC50 = 1.6 µM) and equally active than the
prototype TSAO-hp3T (IC50 = 0.7 µM). These data are in agreement with
what was observed in cell culture. Taken together, these data point to the
impact of the relative orientation of the TSAO molecule and the terminal
hydroxy group for an optimal interaction with the enzyme. Thus, the propyl
linker, present in TSAO-hp3T, as well as the E-methylvinyl linker (present in
6) gave the most potent inhibitors while compound 7 with the Z-methylvinyl
linker was 3-fold less active. These results indicated that the restricted Z-
conformation may not allow the best interaction of the terminal OH with
the HIV-1 RT.
The photoaffinity-labeled TSAO derivatives 9 and 10 were 3- to 10-fold
less effective in comparison with TSAO-T (Table 1). However, they were at

358
M.-C. Bonache
least 20-fold less cytostatic in cell culture than TSAO-T. Interestingly, the
compounds showed marked activity against purified HIV-1 RT (IC50 ∼2-fold
lower than TSAO-T) (Table 1), which suggests that the loss of antiviral
activity in cell culture may be due to pharmacokinetic considerations
(uptake and/or metabolism). The potent inhibitory activity of 9 and 10
against recombinant HIV-1 RT indicated that these compounds might be
promising candidates for photoaffinity labeling experiments.
Finally, the novel compounds also were evaluated for their inhibitory
activity against a mutant HIV-1 strain that contains the E138K mutation in its
RT which is known to afford pronounced resistance to TSAO-T.[15] None of
the compounds showed inhibitory activity against such mutant HIV-1/138K
strain in CEM cell culture (Table 1) pointing to a similar mechanism of
interaction of the compounds with HIV-1 RT as the prototype TSAO-T.
Effect of UV Irradiation on the Interaction of Arylazide 9 and
Benzophenone 10 Derivatives with HIV-1 RT
A preliminary study of the interaction of the photoaffinity analogues
9 and 10 with HIV-1 RT in the presence of UV light was carried out.
Wildtype HIV-1 RT (200 nM) was incubated at room temperature with
varying concentrations of 9 and 10 and then exposed to UV irradiation
(312 nm) for varying times (30 seconds to 30 minutes). Following ir-
radiation, RT was separated from unbound drug by passage through a
Sephadex G-25 Quick Spin Protein Column (Roche, Basel, Switzerland).
Thereafter, the RNA-dependent DNA polymerase activity of the enzyme
was assessed and compared with the control reactions. These included
a no drug control that was exposed to all experimental steps including
irradiation, and a drug control which did not undergo UV exposure. Initial
experiments demonstrated a marked loss of RT activity (5–10%) after 4
minutes. Complete loss of RT activity was observed at prolonged exposure
times (20–30 minutes), however this was attributable to UV damage and not
to cross-linking of the TSAO derivative to RT. Analysis of the cross-linking
experiments that were carried out for shorter time periods demonstrated
no discernable difference with the control reactions, which suggested that
9 and 10 were not effectively cross-linked with HIV-1 RT. Consistent with
this result, the UV and CD spectra of enzymes that were subjected to UV
irradiation were essentially identical to the control enzymes, confirming that
compounds 9 and 10 did not cross-link to RT.
CONCLUSION
As shown above, all the compounds showed pronounced activity against
HIV-1 replication in cell culture. The most antivirally active compounds in

Novel N-3 Substituted TSAO-T Derivatives
359
cell culture were the bis-N -chloroethyl 2 and the E-hydroxymethylvinyl 6
derivatives.
The alkylating moieties (epoxide and nitrogen mustard) introduced at
the N -3 position of TSAO-T were tolerated without loss of activity but they
did not improve activity.
On the other hand, presence of an alcohol functional group linked to
the N -3 position of thymine has a significant impact on the activity provided
that this moiety was linked to this position through a flexible polymethylene
linker or a trans double bond.
Benzophenone and arylazide containing TSAO analogues derivatives
were synthetically accesible. Before attempting to use these analogues for
photoaffinity labeling, we have first established that the presence of the
photophore in the side chain is well tolerated without a significant loss of
activity. A preliminary study of the interaction of the photoaffinity analogs
9 and 10 with HIV-1 RT in the presence of UV light was carried out,
however successful cross-linking to the target protein was not obtained.
Although we were unable to cross-link the current compounds to HIV-1
RT, our studies demonstrated that the cross-linking approach is feasible in
that photoaffinity labels can be introduced on the N -3 of TSAO without
apreciable loss of inhibitory activity. The future challenge is to alter the
orientation and/or length of the spacer or to include different moieties at
the molecule to identify a TSAO derivative that can cross-link to RT.
EXPERIMENTAL
Synthesis
Microanalyses were obtained with a Heraeus CHN-O-RAPID instrument.
Electrospray mass spectra were measured on a quadropole mass spectrome-
ter equippped with an electrospray source (Hewlett Packard, LC/MS HP
1
1100). H NMR spectra were recorded with a Varian Gemini, a Varian
XL-300 (Varian, Palo Alto, CA, USA) and a Bruker AM-200 spectrometer
(Bruker, Madison, WI, USA) operating at 300 and 200 MHz with Me4Si as
internal standard. 13C NMR spectra were recorded with a Varian XL-300 and
a Bruker AM-200 spectrometer operating at 75 MHz and at 50 MHz with
Me4Si as internal standard. Analytical thin-layer chromatography (TLC) was
performed on silica gel 60 F254 (Merck). Separations on silica gel were
performed by preparative centrifugal circular thin-layer chromatography
(CCTLC) on a Chromatotron (Kiesegel 60 PF254 gipshaltig; Merck), layer
thickness (1 mm), flow rate (5 mL min-1). Preparative layer chromatog-
raphy was performed on 20 × 20 cm glass plates coated with a 2 mm
layer of silica gel PF254 (Merck). Analytical HPLC was carried out on a
Waters 484 System using a µBondapak C18 (3.9 × 300mm: 10mm). Isocratic
conditions were used: Mobile phase CH3CN/H2O (0.05%TFA). Flow rate:

360
M.-C. Bonache
1 ml/min. Detection: UV (254 nm). All retention times are quoted in
minutes. Irradiations were carried out in a Pyrex cell (inmersion well)
with light of 290 nm wavelenth using a medium-pressure mercury lamp.
IR spectra were recorded with a Schimadzu IR-435 spectrometer.UV spectra
were recorded with a Perkin Elmer Lambda 16 Spectrophotometer.
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[[N,N-
bis(2-chloroethyl)carbamoyl]methyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-1ꢁꢁ,2ꢁꢁ-
oxathiole-2ꢁꢁ,2ꢁꢁ-dioxide) (2). To a cooled (-20◦C) solution of compound
1[23] (0.1 g, 0.16 mmol) and triethylamine (TEA) (0.04 mL, 0.32 mmol) in
dry dichloromethane (4 mL) was added N,N -bis(2-chloro-ethyl)amine (0.03
g, 0.16 mmol). After 15 minutes, BOP (0.07 g, 0.16 mmol) was added. The
mixture was stirred at room temperature overnight and then evaporated
to dryness. The residue was dissolved in ethyl acetate (2 mL) and washed
successively with 10% citric acid (5 mL), 10% NaHCO3 (5 mL), and brine
(5 ml). The organic phase was dried (Na2SO4), filtered, and evaporated to
dryness. The residue was purified by CCTLC on the chromatotron (hexane:
1
ethyl acetate, 2:1) to give 2 (0.07 g, 68%) as a white foam; H NMR [300
MHz, (CD3)2CO] δ 0.78, 0.93 (2s, 18H, 2t-Bu), 1.95 (s, 3H, CH3-5), 3.71
(m, 4H, CH2Cl), 3.94 (m, 4H, CH2N), 4.05 (dd, 1H, H-5ꢁa, J4 ,5 a = 3.8 Hz,
ꢁ
ꢁ
J5 a,5 b = 13.2 Hz), 4.13 (dd, 1H, H-5ꢁb, J4 ,5 b = 3.5 Hz), 4.34 (t, 1H, H-4ꢁ),
4.80 (d, 1H, H-2ꢁ), 4.89 (d, 2H, CH2N, J = 10.7 Hz), 5.74 (s, 1H, H-3ꢁꢁ),
ꢁ
ꢁ
ꢁ
ꢁ
6.07 (d, 1H, H-1ꢁ, J1 ,2 = 8.0 Hz), 6.50 (bs, 2H, NH2−4ꢁꢁ), 7.63 (s, 1H, H-6);
ꢁ
ꢁ
13
C NMR [75 MHz, (CD3)2CO] δ 13.6 (CH3−5), 18.3, 18.9 [(CH3)3-C-Si],
25.5, 26.6 [(CH3)3-C-Si], 44.2 (2 CH2Cl), 45.6 (NCH2), 49.5 (2 CH2N), 47.0
(CH2N), 49.1 (CH), 62.9 (C-5ꢁ), 75.3 (C-2ꢁ), 83.6 (C-4ꢁ), 88.7 (C-3ꢁꢁ), 93.4
(C-3ꢁ), 92.7 (C-1ꢁ), 111.1 (C-5), 134.9 (C-6), 151.5 (C-4ꢁꢁ), 151.9 (C-2), 163.1
(C-4), 163.9 (CON). Anal. calcd. for C30H52Cl2N4O9SSi2: C, 46.68; H, 6.79;
N, 7.26. Found: C, 46.36; H, 6.28; N, 7.51. MS (ES+) m/z 771.4 (M+1)+.
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[2,3-
epoxypropyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-1ꢁꢁ;,2ꢁꢁ-oxathiole-2ꢁ,2ꢁꢁ-dioxide)
(3). To a solution of TSAO-T[13] (0.10 g, 0.17 mmol) in dry acetone
(5 mL) was added dried and powdered K2CO3 (0.026 g, 0.19 mmol)
and epibromohydrin (0.032 mL, 0.51 mmol). The reaction mixture was
refluxed for 8 hours and then concentrated to dryness. The residue was
dissolved in ethyl acetate (20 mL), washed with brine (2 × 20 mL), dried
(Na2SO4), filtered, evaporated to dryness, and purified by CCTLC on the
chromatotron (dichloromethane/ethyl acetate, 20:1) to give 3 (0.075 g,
1
60%) as a white, amorphous solid; H NMR [300 MHz, (CD3)2CO] δ 0.81,
0.96 (2s, 18H, 2t-Bu), 1.95 (s, 3H, CH3−5), 2.60 (m, 1H, OCHa), 2.69 (m,
1H, OCHb), 3.12 (m, 1H, CH), 3.81 (m, 1H, NCHa), 4.06 (dd, 1H, H-5ꢁa,
J4 ,5 a = 4.1 Hz, J5 a,5 b = 12.0 Hz), 4.11 (dd, 1H, H-5ꢁb, J4 ,5 b = 6.7 Hz), 4.26
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
(m, 1H, NCHb), 4.35 (t, 1H, H-4ꢁ), 4.69 (d, 1H, H-2ꢁ), 5.78 (s, 1H, H-3ꢁꢁ),
6.11 (d, 1H, H-1ꢁ, J1 ,2 = 6.4 Hz), 6.57 (bs, 1H, NH2−4ꢁꢁ), 7.56 (s, 1H, H-6);
ꢁ
ꢁ

Novel N-3 Substituted TSAO-T Derivatives
361
13C NMR [75 MHz, (CD3)2CO] δ 13.3 (CH3-5), 18.2, 18.8 [(CH3)3-C-Si],
25.6, 26.4 [(CH3)3-C-Si], 43.5 (OCH2), 47.0 (CH2N), 49.1 (CH), 62.5
(C-5ꢁ), 75.2 (C-2ꢁ), 83.9 (C-4ꢁ), 88.5 (C-3ꢁꢁ), 93.5 (C-3ꢁ), 92.5 (C-1ꢁ), 111.5
(C-5), 134.2 (C-6), 151.6 (C-4ꢁꢁ), 151.7 (C-2), 163.4 (C-4). Anal. calcd. for
C27H47N3O9SSi2: C, 50.21; H, 7.33; N, 6.51. Found: C, 50.08; H, 7.49; N,
6.10. MS (ES+) m/z 646.2 (M+1)+.
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[(E)-2-
ethoxycarbonylvinyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-1ꢁꢁ,2ꢁꢁ-oxathiole-2ꢁꢁ,2ꢁꢁ-
dioxide) (4). A mixture of TSAO-T[13] (0.4 g, 0.68 mmol), ethyl propiolate
(0.082 mL, 0.84 mmol) and 4-(dimethylamino)pyridine (DMAP) (0.102
g, 0.84 mmol) was stirred at room temperature for 1.5 hours. Solvent was
evaporated to dryness and the residue was dissolved in ethyl acetate (20
mL) and washed successively with HCl 0.1 N (2 × 15 mL), 10% NaHCO3
(2 × 15 mL), and brine (2 × 15 mL). The organic phase was dried
(Na2SO4), filtered, evaporated to dryness, and purified by CCTLC on the
chromatotron (hexane:ethyl acetate, 2:1) to give 4 (0.39 g, 82%) as a white
foam; 1H NMR [300 MHz, (CD3)2CO] δ 0.80, 0.97 (2s, 18H, 2t-Bu), 1.27 (t,
3H, OCH2CH3, J = 7.0 Hz), 1.98 (s, 3H, CH3-5), 4.06 (dd, 1H, H-5ꢁa, J4 ,5 a
=
ꢁ
ꢁ
3.4 Hz, J5 a,5 b = 12.2 Hz), 4.12 (dd, 1H, H-5ꢁb, J4 ,5 b = 3.6 Hz), 4.20 (q,
ꢁ
ꢁ
ꢁ
ꢁ
2H, OCH2CH3), 4.39 (t, 1H, H-4ꢁ), 4.65 (d, 1H, H-2ꢁ), 5.78 (s, 1H, H-3ꢁꢁ),
6.13 (d, 1H, H-1ꢁ, J1 ,2 = 8.1 Hz), 6.45 (bs, 2H, NH2−4ꢁꢁ), 6.99 (d, 1H,
NCH = CH), 7.56 (s, 1H, H-6), 8.26 (d, 1H, NCH = CH, J = 14.7 Hz);
13C NMR [75 MHz, (CD3)2CO] δ 13.2 (CH3-5), 14.5 (OCH2CH3), 18.1,
18.6 [(CH3)3-C-Si], 25.0, 26.3 [(CH3)3-C-Si], 61.0 (OCH2), 63.1 (C-5ꢁ), 75.5
(C-2ꢁ), 85.5 (C-4ꢁ), 87.6 (C-3ꢁꢁ), 92.7 (C-1ꢁ, C-3ꢁ), 111.4 (C-5), 113.8 (NCH =
CH), 134.8 (C-6), 135.3 (NCH = CH), 151.7 (C-4ꢁꢁ), 150.9 (C-2), 162.3
(C-4), 167.4 (CO). Anal. calcd. for C29H49N3O10 SSi2: C, 50.63; H, 7.18;
N, 6.11. Found: C, 50.30; H, 7.45; N, 6.85. MS (ES+) m/z 688.4 (M+1)+.
HPLC (CH3CN:H2O, 70:30) retention time: 10.58 minutes.
ꢁ
ꢁ
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[(Z)-2-
ethoxycarbonylvinyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-1ꢁꢁ,2ꢁꢁ-oxathiole-2ꢁꢁ,2ꢁꢁ-
dioxide) (5). A solution of the E isomer 4 (0.100 g, 0.14 mmol) in
methanol (100 mL) in a Pyrex cell (inmersion well) was irradiated
under argon atmosphere at room temperature with light of 290 nm
wavelenth using a medium-pressure mercury lamp. The progress of the
reaction was monitored by HPLC. After 30 minutes of irradiation an
equilibrium (1:1.5) mixture of E and Z isomers were obtained. Prolonged
reaction times (6 hours) did not affect to the composition of the mixture.
Solvent was evaporated under reduced pressure to give a residue which
were purified by preparative thin layer chromatography using as eluent
(dichloromethane:ethyl acetate, 40:1). After several developments two
different moving bands were separated.
From the fastest moving band E isomer 4 (0.040 g, 40%) was isolated.

362
M.-C. Bonache
From the slowest moving band Z isomer 5 (0.060 g, 60%) was isolated;
1H NMR [300 MHz, (CD3)2CO] δ 0.83, 0.96 (2s, 18H, 2t-Bu), 1.17 (t, 3H,
OCH2CH3, J = 5.2 Hz), 1.93 (s, 3H, CH3-5), 4.05 (dd, 1H, H-5ꢁa, J4 ,5 a = 4.1
ꢁ
ꢁ
Hz, J5 a,5 b = 11.4 Hz), 4.09 (q, 2H, OCH2CH3), 4.10 (dd, 1H, H-5ꢁb, J4 ,5 b
=
ꢁ
ꢁ
ꢁ
ꢁ
3.5 Hz), 4.31 (t, 1H, H-4ꢁ), 4.70 (d, 1H, H-2ꢁ), 5.75 (s, 1H, H-3ꢁꢁ), 6.01 (d,
1H, H-1ꢁ, J1 ,2 = 7.9 Hz), 6.12 (d, 1H, NCH = CH), 6.47 (bs, 1H, NH2−4ꢁꢁ),
6.70 (d, 1H, NCH = CH, J = 9.0 Hz), 7.55 (s, 1H, H-6). Anal. calcd. for
C29H49N3O10 SSi2: C, 50.63; H, 7.18; N, 6.11. Found: C, 50.30; H, 7.45; N,
6.85. MS (ES+) m/z 688.4 (M+1)+. HPLC (CH3CN:H2O, 70:30) retention
time : 5.46 minutes.
ꢁ
ꢁ
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[(E)-3-
hydroxy-1-propenyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-1ꢁꢁ,2ꢁꢁ-oxathiole-2ꢁꢁ,2ꢁꢁ-
dioxide) (6). DIBAL-H (1 M solution in THF, 0.28 mL, 0.28 mmol) was
added dropwise into a solution of 4 (0.2 g, 0.29 mmol) in dry THF (0.5 mL)
at 0◦C over 10 minutes with stirring under argon. After the reaction mixture
was allowed to reach room temperature, stirring was continued for 3 hours.
Saturated aqueous NH4Cl (0.5 mL) was then added, and the gel-like solid
was filtered off using a Celite bed. Evaporation of the solvent gave a residue
that was purified by CCTLC on the chromatotron using hexane/ethyl
1
acetate (2:1) to give 6 (0.11 g, 61%) as a white foam; H NMR [300 MHz,
(CD3)2CO] δ 0.82, 0.98 (2s, 18H, 2t-Bu), 1.96 (s, 3H, CH3-5), 4.05 (dd, 1H,
H-5ꢁa, J4 ,5 a = 3.7 Hz, J5 a,5 b = 12.2 Hz), 4.13 (dd, 1H, H-5ꢁb, J4 ,5 b = 3.5 Hz),
4.16 (t, 1H, OH, J = 5.8 Hz), 4.23 (m, 2H, CH2OH), 4.36 (t, 1H, H-4ꢁ), 4.65
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
(d, 1H, H-2ꢁ), 5.78 (s, 1H, H-3ꢁꢁ), 6.12 (d, 1H, H-1ꢁ, J1 ,2 = 8.2 Hz), 6.50 (bs,
1H, NH2−4ꢁꢁ), 6.56 (m, 1H, NCH = CH), 6.82 (m, 1H, NCH = CH, J = 14.4
Hz), 7.52 (s, 1H, H-6); 13C NMR [75 MHz, (CD3)2CO] δ 13.8 (CH3-5), 18.1,
18.6 [(CH3)3-C-Si], 25.0, 26.3 [(CH3)3-C-Si], 61.7 (CH2OH), 63.5 (C-5ꢁ),
75.1 (C-2ꢁ), 85.9 (C-4ꢁ), 87.2 (C-3ꢁꢁ), 92.8 (C-1ꢁ, C-3ꢁ), 111.6 (C-5), 123.4
(CH = CH), 130.3 (NCH = CH), 134.6 (C-6), 151.4 (C-4ꢁꢁ), 150.6 (C-2),
162.5 (C-4). Anal. calcd. for C27H47N3O9SSi2: C, 50.21; H, 7.33; N, 6.51.
Found: C, 50.50; H, 7.85; N, 6.75. MS (ES+) m/z 646.4 (M+1)+.
ꢁ
ꢁ
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[(Z)-3-
hydroxy-1-propenyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-1ꢁꢁ,2ꢁꢁ-oxathiole-2ꢁꢁ,2ꢁꢁ-
dioxide) (7). Following the previosly described method for the synthesis of
6, a solution of 5 (0.050 g, 0.07 mmol) in dry THF (0.25 mL) was treated
with DIBAL-H (1 M solution in THF, 0.14 mL, 0.14 mmol). Purification of
the residue by CCTLC on the chromatotron using hexane/ethyl acetate
1
(2:1) afforded 7 (0.058 g, 52%) as a white foam; H NMR [300 MHz,
(CD3)2CO] δ 0.82, 0.96 (2s, 18H, 2t-Bu), 1.94 (s, 3H, CH3-5), 3.64 (t, 1H,
OH, J = 5.8 Hz), 3.92 (t, 2H, CH2OH), 4.04 (dd, 1H, H-5ꢁa, J4 ,5 a = 3.8 Hz,
ꢁ
ꢁ
J5 a,5 b = 12.3 Hz), 4.12 (dd, 1H, H-5ꢁb, J4 ,5 b = 3.4 Hz), 4.32 (t, 1H, H-4ꢁ),
ꢁ
ꢁ
ꢁ
ꢁ
4.69 (d, 1H, H-2ꢁ), 5.74 (s, 1H, H-3ꢁꢁ), 5.89 (m, 1H, NCH = CH), 6.05 (d,
1H, H-1ꢁ, J1 ,2 = 8.0 Hz), 6.09 (m, 1H, NCH = CH, J = 9.5 Hz), 6.45 (bs,
ꢁ
ꢁ

Novel N-3 Substituted TSAO-T Derivatives
363
1H, NH2−4ꢁꢁ), 7.55 (s, 1H, H-6). Anal. calcd. for C27H47N3O9SSi2: C, 50.21;
H, 7.33; N, 6.51. Found: C, 50.50; H, 7.85; N, 6.75. MS (ES+) m/z 646.4
(M+1)+.
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[3-Nꢁ-(4-
azidosalicylyl)aminopropyl]thymine]-3ꢁ-spiro-5”-(4”-amino-1”,2”-oxathiole-
2”,2”-dioxide) (9). To a solution of compound 8[23] (0.10 g, 0.16 mmol) in
dry dichloromethane (10 mL) 4-azido salicylic acid[34] (0.034 g, 0.19 mmol)
and BOP (0.070 g, 0.16 mmol) was added. After 15 minutes, triethylamine
(TEA) (0.032 mL, 0.24 mmol) was added. The mixture was stirred at room
temperature overnight and then evaporated to dryness. The residue was
dissolved in ethyl acetate (2 mL) and washed successively with 10% citric
acid (10 mL), 10% NaHCO3 (10 mL) and brine (10 ml). The organic phase
was dried (Na2SO4), filtered and evaporated to dryness. The residue was
purified by CCTLC on the chromatotron (dichloromethane: methanol,
50:1) to give 9 (0.11 g, 85%) as a white foam; UV/Vis (MeOH) λmax (ε,
1
M−1cm−1) = 268 (60600), 309 (22000) nm. IR (KBr) ν = 2100 cm−1. H
NMR [300 MHz, (CD3)2CO] δ 0.79, 0.97 (2s, 18H, 2 (CH3)3-C-Si), 1.92
(m, 2H, CH2CH2N), 1.94 (s, 3H, CH3-5), 3.42 (m, 2H, CH2NHCO), 4.04
(m, 2H, H-5ꢁa, CH2N), 4.10 (dd, 1H, H-5ꢁb, J4 ,5 b = 3.6 Hz, J5 a,5 b = 12.3
ꢁ
ꢁ
ꢁ
ꢁ
Hz), 4.33 (t, 1H, H-4ꢁ), 4.64 (d, 1H, H-2ꢁ), 5.76 (s, 1H, H-3ꢁꢁ), 6.10 (d, 1H,
H-1ꢁ, J1 ,2 = 8.1 Hz), 6.43 (bs, 1H, NH2−4ꢁꢁ), 6.54 (s, 1H, Ph), 6.60 (d, 1H,
Ph), 7.51 (s, 1H, H-6), 7.77 (d, 1H, Ph), 8.20 (t, 1H, NHCO); 13C RMN
[75 MHz, (CD3)2CO] δ 12.8 (CH3-5), 18.0, 18.6 [(CH3)3-C-Si], 25.3, 26.0
[(CH3)3-C-Si], 28.9 (CH2), 37.0 (CH2NHCO), 39.4 (CH2N), 62.8 (C-5ꢁ),
75.1 (C-2ꢁ), 84.5 (C-4ꢁ), 87.1 (C-3ꢁꢁ), 92.2 (C-3ꢁ), 92.3 (C-1ꢁ), 108.0 (CH-Ph),
110.1 (CH-Ph), 111.2 (C-5), 128.7 (CH-Ph), 134.2 (C-6), 145.9 (C-N3),
151.6 (C-4ꢁꢁ), 151.6 (C-2), 163.3 (C-4), 163.3 (CONH). Anal. calcd. for
C34H53N7O10 SSi2: C, 50.54; H, 6.61; N, 12.13. Found: C, 50.26; H, 6.48; N,
12.45. MS (ES+) m/z 808.3 (M+1)+.
ꢁ
ꢁ
[1-[2ꢁ,5ꢁ-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3-N-[3-Nꢁ[(4-
(benzoyl)-carbonylphenyl]aminopropyl]thymine]-3ꢁ-spiro-5ꢁꢁ-(4ꢁꢁ-amino-
1ꢁꢁ,2ꢁꢁ-oxathiole-2ꢁꢁ,2ꢁꢁ-dioxide) (10). Following the previously described
method, a solution of 8[23] (0.1 g. O.15 mmol), BOP (0.066 g, 0.15 mmol),
4-benzoylbenzoic acid (0.040 g, 0.18 mmol) and TEA (0.030 mL, 0.23
mmol) in dry dichloromethane was stirred at room temperature overnight
and then evaporated to dryness. The residue was purified by CCTLC on
the chromatotron (hexane:ethyl acetate, 1:1) to give 10 (0.097 g, 76%) as
1
a white foam; UV/Vis (MeOH) λmax (ε, M−1cm−1) = 260 (28000) nm. H
NMR [300 MHz, (CD3)2CO] δ 0.80, 0.96 (2s, 18H, 2t-Bu), 1.93 (m, 2H,
CH2CH2N), 1.95 (s, 3H, CH3-5), 3.46 (m, 2H, CH2NHCO), 4.10 (m, 4H,
H-5ꢁa, CH2N), 4.34 (t, 1H, H-4ꢁ), 4.66 (d, 1H, H-2ꢁ), 5.77 (s, 1H, H-3ꢁꢁ),
6.12 (d, 1H, H-1ꢁ, J1 ,2 = 8.0 Hz), 6.47 (bs, 1H, NH2-4ꢁꢁ), 7.51 (m, 9H,
ꢁ
ꢁ
13
Ph), H-6), 8.22 (t, 1H, NHCO);
C RMN [75 MHz, (CD3)2CO] δ 12.8

364
M.-C. Bonache
(CH3-5), 18.1, 18.6 [(CH3)3-C-Si], 25.0, 26.3 [(CH3)3-C-Si], 29.1 (CH2),
37.4 (CH2NHCO), 39.5 (CH2N), 62.8 (C-5ꢁ), 75.1 (C-2ꢁ), 84.9 (C-4ꢁ), 87.3
(C-3ꢁꢁ), 92.1 (C-1ꢁ), 92.3 (C-3ꢁ), 112.5 (C-5), 130.0, 130.2, 129.0, 127.6 (Ph),
134.3 (C-6), 137.9, 138.9, 140.3 (C-Ph), 151.7 (C-4ꢁꢁ), 151.8 (C-2), 163.3
(C-4), 166.0 (CONH), 195.8 (PhCOPh). Anal. calcd. for C41H58N4O10 SSi2:
C, 57.58; H, 6.84; N, 6.55. Found: C, 57.28; H, 6.47; N, 6.14. MS (ES+) m/z
855.5 (M+1)+.
Biological Methods
Cells and Viruses
Human immunodeficiency virus type 1 [HIV-1 (IIIB)] was obtained from
Dr. R. C. Gallo (when at the National Cancer Institute, Bethesda, MD). HIV-
2 (ROD) was provided by Dr. L. Montagnier (when at the Pasteur Institute,
Paris, France).
Activity Assay of Test Compounds against HIV-1 and HIV-2 in CEM and MT-4
Cell Cultures
A total number of 4 × 106 CEM or 3 × 105 MT-4 cells per milliliter
were infected with HIV-1 (IIIB) or HIV-2 (ROD) or HIV-1/138K (a virus
strain that was selected in the presence of TSAO-m3T and that contained the
E138K mutation in its RT) at ∼100 CCID50 (50% cell culture infective dose)
per milliliter of cell suspension. Then an amount of 100 µL of the infected
cell suspension was transferred to microtiter plate wells and mixed with 100
µL of the appropriate dilutions of the test compounds. Syncytia formation
(CEM) or HIV-induced cytopathicity (MT-4) was recorded microscopically
(CEM) or by trypan blue dye exclusion (MT-4) in the HIV-infected cell cul-
tures after 4 days (CEM) or 5 days (MT-4). The 50% effective concentration
(EC50) of the test compounds was defined as the compound concentration
required to inhibit virus-induced cytopathicity (CEM) or to reduce cell
viability (MT-4) by 50%. The 50% cytostatic or cytotoxic concentration
(CC50) was defined as the compound concentration required to inhibit
CEM cell proliferation by 50% or to reduce the number of viable MT-4 cells
in mock-infected cell cultures by 50%.
Inhibitory Effect of Test Compounds against Recombinant HIV-1 RT
HIV-1 RT was purified as described previously.[35,36] Inhibition of the
RNA-dependent DNA polymerase activity of HIV-1 RT was assessed using a
fixed time scintillation proximity assay. Briefly, reactions were carried out in
50mM Tris-HCl pH 7.8 (37◦C), 50 mM KCl, 1 mM MgCl2 and contained
700 nM of poly(rA)-oligo(dT)18 (the oligo(dT)18 was synthesized with a
biotin moiety on the 5ꢁ-end), 5µM [3H]TTP, and variable concentrations
of ligand dissolved in DMSO (3% final concentration). Reactions were

Novel N-3 Substituted TSAO-T Derivatives
365
initiated by the addition of 20 nM of wildtype or E138K HIV-1 RT and
incubated for 20 minutes at 37◦C. Reactions were quenched by addition
of 0.5 mg/ml scintillant impregnated streptavidin coated polyvinyltoluene
beads (Amersham) in a quench solution containing 100 mM EDTA. Plates
were allowed to rest for 2 hours prior to counting radioactivity using a
Hewlett-Packard plate reader. The 50% inhibitory concentration (IC50) was
defined as the concentration of the compound required to inhibit enzyme
activity by 50%.
Photoaffinity Cross-Linking Experiments
200 nM wildtype HIV-1 RT was incubated at room temperature with
compounds 9 and 10 (concentrations ranged from 1 µM to 25 µM) in
50 Tris-HCl pH 7.8, 50 mM KCl, and 1mM MgCl2. The reaction mixtures
were then irradiated with a hand-held UV lamp (Fisher Biotech) at 312 nm
wavelength with an intensity of 1.15 mW/cm2 for varying times (30 seconds
to 30 minutes).
Following irradiation, RT was separated from unbound drug by passage
through a Sephadex G-25 Quick Spin Protein Column (Roche). Thereafter,
the RNA-dependent DNA polymerase activity of the enzyme was assessed as
described above. Control reactions included no inhibitor (and in this case
they were subjected to UV irradiation) or inhibitor (without exposure to UV
irradiation).
In addition to the activity assays described above, the RT that was sub-
jected to UV irradiation was also assessed by ultraviolet (UV) absorption and
circular dichroism (CD) spectroscopy. The UV absorption spectroscopy was
carried out using a Beckman Coulter DU530 UV/VIS spectrophotometer.
CD spectra were collected at ambient room temperature (24◦C) using an
Aviv Instruments CD Spectrometer (Model 202).
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