Studies on the inhibition of moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds

Article abstract of DOI:10.1080/15257770802271698

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5′- phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-amino

Full text of DOI:10.1080/15257770802271698

Nucleosides, Nucleotides and Nucleic Acids, 27:1011–1023, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802271698
STUDIES ON THE INHIBITION OF MOLONEY MURINE LEUKEMIA
VIRUS REVERSE TRANSCRIPTASE BY N-TRITYLAMINO ACIDS
AND N-TRITYLAMINO ACID-NUCLEOTIDE COMPOUNDS
Arthur Hawtrey,1 Anton Pieterse,1 Johann van Zyl,1 Pieter Van der Bijl,1
Marichen Van der Merwe,1 William Nel,2 and Mario Ariatti3
1Division of Pharmacology, Department of Medicine, Faculty of Health Sciences, Stellenbosch
University, Tygerberg, South Africa
2Department of Biochemistry, University of Zululand, KwaDlangezwa, South Africa
3Department of Biochemistry, Westville Campus, University of KwaZula-Natal, Durban,
South Africa
N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 ꢁ-phosphate were prepared and
2
studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse
transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5ꢁ-AMP
inhibited the enzyme partially using poly (rA).oligo d(pT)16-18 as template-primer with [3H]dTTP.
In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino
group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m,
and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above
template-primer system. The compounds all inhibited the reverse transcriptase with IC50 values of
approximately 60−80 µM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the
nucleotide 8-(6-aminohexyl) amino-adenosine-5ꢁ-phosphate, the inhibitory activity of this compound
increased significantly (IC50 = 5 µM).
Keywords N-tritylamino acid-nucleotide; reverse transcriptase; inhibitor
INTRODUCTION
Reverse transcriptase is a key enzyme involved in the replication of
retroviruses. It is used in the conversion of genomic viral RNA into proviral
DNA. Reverse transcriptase enzymes purified from a number of retroviruses
including Moloney murine leukemia virus (M-MuLV) and the AIDS virus
Received 5 September 2007; accepted 22 May 2008.
We thank Dr H. Seifart for mass-spectra.
Address correspondence to Johann van Zyl, Division of Pharmacology, Department of Medicine,
Faculty of Health Sciences, Stellenbosch University, P O Box 19063, Tygerberg 7505, South Africa. E-mail:
jmvzyl@sun.ac.za
1011

1012
A. Hawtrey et al.
(HIV-1) all possess (i) a DNA polymerase activity using either RNA or
DNA templates and (ii) a RNase activity capable of degrading RNA from
RNA-DNA hybrids.
Inhibitors of the polymerase (chemicals/drugs) fall into a number
of categories–(i) nucleoside analogues and derivatives,[1–3] (ii) naturally
occurring compounds,[4,5] and (iii) non-nucleoside inhibitors (NNI).[6–18]
Nucleoside inhibitors and nucleoside-like inhibitors act as chain elongation
terminators. The non-nucleoside inhibitors (structurally varied group of
compounds) on the other hand, all bind to a common hydrophobic al-
losteric site near the polymerase active centre. The classical non-nucleoside
inhibitor is Nevirapine (dipyridodiazepinone), discovered during a random
screening of various chemical compounds.
In our studies, we envisaged the preparation of compounds consisting
of a nucleotide component covalently linked to a hydrophobic grouping
through a spacer of approximitaley 10–15 A which is the distance of the
hydrophobic binding pocket area from the active centre of the reverse tran-
scriptase polymerase enzyme. The resulting compound would thus have a
ribonucleotide component which could presumably go to the active centre
of the enzyme while the hydrophobic section could conceivably interact
or attach at the hydrophobic pocket of the enzyme. The compounds
mentioned above can thus be thought of as chimeras of a nucleotide and
a non-nucleotide inhibitor (NNI).
In the present experiments, compounds consisting of 8-(6-aminohexyl)
amino adenosine-5-phosphate coupled to N-trityl-amino acids via a spacer,
were therefore investigated for their possible effect on the activity of
M-MuLV reverse transcriptase. These compounds in essence consist of a
hydrophobic grouping (NN) linked covalently to a nucleotide residue via
a spacer. These particular chimera structures are illustrated in Figures 1
and 5 of the manuscript. Chimeras of deoxynucleosides and non-nucleoside
reverse transcriptase inhibitors previously prepared by a number of groups
and tested for inhibition of the reverse transcriptase are mentioned in the
Discussion section. Results of our experiments are reported in the present
communication.
MATERIALS AND METHODS
+
[3H]dTTP (126 Ci/mmol) as the NH4 salt was obtained from Amer-
sham, U.K. Poly (rA), d(pT)16–18, tritylchloride, tritanol, 8-(6-aminohexyl)
amino adenosine-5ꢁ-phosphate (Li+ salt) and N-hydroxysuccinimide (NHS)
were supplied by Sigma (St. Louis, MO, USA). Oligo d(A)10 and d(A)20
were prepared by Metabion (Germany). These substances and reverse
transcriptase (M-MuLV) were supplied by Roche (Germany). All other
chemical reagents were of analytical grade.

Inhibition of Moloney Murine Leukemia Virus
1013
FIGURE 1 Preparation of N-acyl derivatives of 8-(6-aminohexyl) amino-5ꢁ-AMP. 8-(6-aminohexyl)
amino-5ꢁ-AMP (1); palmitoyl derivative (2); nicotinyl derivative (3) and bis-nucleotide with glutaryl
spacer (4).
N-Tritylglycine (9) and the N-trityl derivatives of o-(10), m-(11) and
p-(12) fluorophenylalanine were prepared by Nel and Ariatti[19] using
the method of Zervas and Theodoropoulos.[21] The N-hydroxysuccinimide
derivative of N-trityl-m-fluorophenylalanine (6) was prepared from 11 by the
procedure of Nel and Ariatti.[19]
Assay for RNA-Dependent DNA Polymerase Activity of M-MuLV
Reverse Transcriptase
The incubation system contained 50 mM Tris-HCl (pH 8.3), 20 mM KCl,
10 mM dithiothreitol, 5 mM MgCl2, reverse transcriptase (10 units), poly
rA (1.3 µg), d(pT)16 primer (0.15 µg), [3H]dTTP (1.5 µCi), and dTTP
(0.45 µg) in a final volume of 20 µl. Reactions were carried out at 37◦C.
Freshly prepared reagents were stored in crushed ice-water. Preincubated
poly rA-d(pT)16 was added to the enzyme-[3H]dTTP mixture at zero
time. At regular intervals thereafter, aliquots (2–6 µl) were removed and
mixed into 18–30 µl 0.1 M EDTA (pH 7.6). From these solutions, aliquots
(5–10 µl) were streaked on discs of DE-81 paper, dried at 37◦C and

1014
A. Hawtrey et al.
then immersed in 0.5 M sodium phosphate (pH 6.8) for 5 minutes. This
procedure was repeated with fresh 0.5 M phosphate. Discs were dried at
60◦C and counted for radioactivity by liquid scintillation.
Test compounds were dissolved either in H2O, EtOH: H2O (1:1, v/v) or
EtOH. Final concentration of EtOH in test runs or controls was 10% (v/v).
The nucleotide-palmitate compound (2, Figure 1) was also solubilized in
an aqueous solution as follows: An EtOH solution of nucleotide-palmitate
(400 µg/40 µl) was taken to dryness and the residue suspended in 500
µl H2O. This suspension was adjusted to pH 7.5 by careful addition of
0.05 M Tris (free base). Water was then added to bring the volume to 850 µl
(only partial solubilization). Sodium chloride (1 M, 150 µl) was then rapidly
added with stirring to produce a clear solution. This was filtered through a
0.22 µM sterile filter. Spectral analysis (UV) revealed a 60% solubilization of
the compound.
8-(6-Aminohexyl) Amino-adenosine-5ꢁ-phosphate-palmitate (2)
To palmitoyl-N-hydroxysuccinimide (7.2 mg, 0.02 mmol) in 3.2 ml
pyridine:H2O (9:1, v/v) was added a slightly warm solution of 8-(6-
aminohexyl) amino-adenosine-5ꢁ-phosphate (9.2 mg, 0.02 mmole of I) in
3.2 ml pyridine: H2O (9:1,v/v). The clear solution was kept at room
temperature for 20 hours in the dark. Thereafter, the reaction mixture was
taken to dryness under N2 at 40◦C. The residue was treated with toluene
(5 ml) and again taken to dryness under N2. This was repeated once more.
The resulting dry residue was dissolved in ethanol (40◦C) and applied
to silica gel 60F254 plates (20 × 20 cm) which were then developed with
n-butanol : AcOH : H2O (12:3:5, v/v/v, solvent A). The title compound (Rf,
0.48) was extracted into EtOH:AcOH (95:5) at 40◦C, which was filtered and
taken to dryness under a stream of N2 at 40◦C to give a clear white crystalline
residue. UV (EtOH): λmax 278 nm and λmin 245 nm, λmax 278 nm (log ε
4.36). Silica gel TLC on 60F254 plates gave a Rf = 0.48 (solvent A). Mass
spectrum: m/z 698 (M-H)− (sample in 50% acetonitrile).
8-(6-Aminohexyl) Amino-adenosine-5ꢁ-phosphate-nicotinamide (3)
To a solution of nicotinic acid-N-hydroxysuccinimide (6 mg 0.02 mmol)
in 2.6 ml pyridine: H2O (9:1, v/v) was added a warm solution (40◦C) of 8-(6-
aminohexyl) amino-AMP (9.2 mg, 0.02 mmol) in 2.4 ml pyridine : H2O (9
:1, v/v). After mixing, the solution was kept at room temperature (20◦C) in
the dark for 24 h. The solution was then taken to dryness under a stream of
N2 at 40◦C. The residue was treated with 4 ml of toluene and again taken to
dryness. The final residue was dissolved in 2.0 ml of ethanol (96%) at 40◦C
and purified on silica gel 60F254 plates developed in solvent A. The required
band (Rf 0.18) was extracted from the silica gel into a solution containing
ethanol: H2O:conc. NH4OH (5:4:2, v/v/v) at 37◦C. The extract was filtered

Inhibition of Moloney Murine Leukemia Virus
1015
and dried in vacuo at 37◦C. The residue was dissolved in EtOH:H2O (1:2,
v/v, 3 ml) at 37◦C and centrifuged (10 000 × g, 4 min). Portions of the
supernatant were used for analysis and assays with reverse transcriptase.
The sample was chromatographically pure (Rf 0.18, solvent A), UV: λmax
278 nm, (278/260 ratio, 1.28). Mass spectrum: m/z 567 (molecular mass,
566.5) (sample in 50% acetonitrile containing 0.1% formic acid).
Bis-8-(6-aminohexyl) Amino-5ꢁ-AMP-glutarate (4)
This was prepared in the same manner as the palmitoyl (2) and nicotinyl
(3) derivatives. A solution of 8-(6-aminohexyl) amino-5ꢁ-AMP (9.2 mg,
0.02 mmol) and bis-N-hydroxysuccimimide ester of glutaric acid (3.0 mg,
0.01 mmol) in 7.0 ml pyridine: H2O (9:1, v/v) was kept at 20◦C for 22
hours. Thereafter, the reaction mixture was worked up as described for
compound 3. Purification was effected on silica gel 60F254 plates (solvent
A). Compound 4 was extracted into EtOH: H2O (4:1, v/v) containing 5%
acetic acid. The product was freely soluble in H2O. UV (H2O, pH 7.5): λmax
278 nm and λmin 242 nm. Mass spectrum: m/z, 1017 (M-H)− (sample in
50% acetonitrile). Molecular mass, 1018.
Preparation of Nicotinyl-trismethylolamide (5)
To a solution of the NHS ester of nicotinic acid (8.8 mg, 0.04 mmol)
in DMF:pyridine (3:1, v/v, 0.25 ml) was added a solution of tris (hydrox-
ymethyl) aminomethane (4,84 mg, 0.04 mmol) in 0.45 ml of the same
solvent. The clear solution was stored in the dark for 20 hours at 20◦C. It
was then taken to dryness at 35◦C under a stream of N2. Traces of pyridine
in the product were removed by repeated co-evaporation with toluene. The
final residue was dissolved in EtOH (0.3 ml) and applied to a silica gel 60F254
TLC plate (20 × 20 cm) which was developed in solvent A. Compound 5 (Rf
0.44) was extracted into EtOH (25 ml) at 30◦C. The extract was filtered and
concentrated to dryness under N2 (30◦C). The residue was dissolved in H2O
(2 ml) and stored at −20◦C. UV (H2O, pH 5.5): λmax 260 and 219 nm, λmax
260 nm (log ε 3.48). Mass spectrum: m/z 227 (M+).
Rf values (silica gel 60F254 plates in chloroform:methanol, 4:1 v/v)
for tris (hydroxymethyl) amino-methane, N-hydroxysuccinimide ester of
nicotinic acid, nicotinic acid and nicotinyl-tris-methylolamide were 0.06,
0.46, 0.27, and 0.15, respectively.
8-(6-Aminohexyl) Amino-adenosine-5ꢁ-monophosphate-N-trityl-m-fluoro-DL-
phenylalanine (7)
To a solution of 8-(6-aminohexyl) amino-adenosine-5ꢁ-monophosphate
(Li+ salt, 4.6mg, 0.01 mmol) in 0.4 ml pyridine:H2O (7:3, v/v) was added the
N-hydroxysuccinimide ester of N-trityl-m-fluoro-DL-phenylalanine (5.3 mg,
0.01 mmole) in 0.2 ml pyridine:H2O (7:3, v/v). The reaction mixture was

1016
A. Hawtrey et al.
left in the dark at room temperature for 20 hours. It was then taken to
dryness under a stream of N2 at 30◦C and traces of pyridine removed by
co-evaporation with toluene using N2. The crude product was taken up in
dioxane:EtOH: H2O (2:2:1, v/v/v, 0.6 ml) and chromatographed on silica
gel 60F254 plates (20 × 20 cm) in CHCl3:CH3OH (4:1, v/v). The product,
which remained at the origin with unreacted 8-(6-aminohexyl) amino-AMP)
was selectively extracted into ethanol (5 ml) at 30◦C. Chromatography
(silica gel 60F254 in n-propanol: NH4OH:H2O, 55:10:35, v/v/v) gave a single
UV254 absorbing spot (Rf, 0.74) which was also positive for the trityl group
(2% aqueous HClO4, 100◦C), while 8-(6-aminohexyl) amino-AMP and N-
hydroxysuccinimide gave Rf values of 0.55 and 0.66 respectively. UV (EtOH):
λmax 278 nm, 278/240 ratio 2.19, λmax 278 nm (log ε 4.25). Mass spectrum:
m/z 243.1 (trityl), 299.1, 353.3, 381.4, 409.3, 537.4, 627.3, 869.5 (molecular
mass 869.5) (sample in methanol containing 0.1% formic acid).
The compounds 8-(6-aminohexyl) amino-AMP-N-trityl-m-fluoro-DL-
phenylalanine and 8-(6-aminohexyl) amino-AMP gave Rf values on silica gel
60F254 (EtOH:H2O, 5:1, v/v) of 0.60 and 0.09, respectively.
RESULTS AND DISCUSSION
Our studies commenced originally with the idea of coupling various
chemical groups to the terminal primary amino group of the aminohexyl
chain at position 8 in 8-(6-aminohexyl) amino-adenosine-5ꢁ-phosphate (I in
Figure 1). These derivatives could then be examined to explore possible
inhibitory or stimulatory activity against the reverse transcriptase enzyme of
Moloney mouse leukemia virus. In the first phase of this work we attached
(i) palmitic acid, (ii) nicotinic acid and (iii) a further 8-(-6-aminohexyl)
amino-adenosine-5ꢁ-phosphate to the aminohexyl moiety with glutaric acid
spacer (Figure 1). All couplings were by N-hydroxy-succinimide activation of
carboxyl groups. Synthesized compounds were purified by TLC chromatog-
raphy on silica gel 60F254 plates.
With poly (rA)-oligo d(pT)16 as template-primer, incorporation of
[3H]dTTP into poly dT was inhibited to varying extents with the com-
pounds mentioned above. Figure 2 shows time curves for incorporation
of [3H]dTTP where the palmitoyl derivative (2) gave approximately 35%
inhibition at 10−4 M and the bis-nucleotide with glutaryl spacer (4), 30%
inhibition at 10−4 M. It is noteworthy that 8-(6-aminohexyl)-amino-AMP (1)
stimulated incorporation at the early phase of the time curve (up to 10
minutes).
In Figure 3, it is seen that 8-(6-aminohexyl)-amino-AMP-nicotinamide
(3) at 10−4 M inhibited incorporation by some 60%. At 10−5 M, incorpora-
tion was inhibited by approximately 35%. Nicotinic acid, nicotinamide and

Inhibition of Moloney Murine Leukemia Virus
1017
20
18
16
14
12
10
8
6
4
2
0
0
5
10
15
20
25
30
35
Time (min)
FIGURE 2 Time course of incorporation of [3H]dTTP into DNA using poly(rA).d(pT)16 as polymer
template and M-MuLV reverse transcriptase in the absence (∇) and presence of different compounds;
(ꢀ), 8-(6-aminohexyl) amino-5ꢁ-AMP (10−4M); (O), palmitoyl derivative (10−4M); (ꢁ), bis-AMP-
glutarate (10−4M).
sodium palmitate (all at 10−4 M) had no effect on the activity of the reverse
transcriptase (results not shown).
In addition to 8-(6-aminohexyl) amino-5ꢁ-AMP (1, Figure 1) nicotinyl-
trismethylolamide (5, Figure 4) was prepared as a control in this study.
This non-nucleotide compound displayed an initial stimulation of
20
18
16
14
12
10
8
6
4
2
0
0
5
10
15
20
25
30
35
Time (min)
FIGURE 3 Time course of incorporation of [3H]dTTP into DNA using poly(rA).d(pT)16 as polymer
template and the reverse transriptase in the absence (ꢁ) and presence of the nicotinyl derivative of
8-(6-aminohexyl) amino-5ꢁ-AMP at 10−5M (◦), and 10−4M (•).

1018
A. Hawtrey et al.
CH2OH
O
C
NH
C
CH2OH
CH2OH
N
(5)
FIGURE 4 Structure of the non-nucleoside derivative nicotinyl-trismethylolamide used as a control in
the incorporation studies with the nucleotidyl compounds illustrated in Figure 1.
incorporation although this effect was less marked at 20 minutes and
thereafter. The result paralleled the time curve of incorporation in the
presence of 8-(6-aminohexyl) amino-5ꢁ-AMP (Figure 2). Results with
nicotinyl-trismethylolamide are not shown.
Having shown that attachment of certain groups (palmitoyl, nicotinyl,
glutaryl-AMP) to the aminohexyl side chain of nucleotide I, enabled the
resulting modified nucleotides to inhibit partially the DNA polymerase
activity of MuLV reverse transcriptase (Figures 1 through 3), we were
encouraged to attempt further modification of nucleotide I.
In view of the very detailed research carried out over the last few years
on non-nucleoside inhibitors of reverse transcriptase, it was considered of
interest to attach more hydrophobic groups (molecules) to the aminohexyl
side chain of nucleotide I. It was therefore decided to attach N-tritylamino
acids to the side chain amino group of the nucleotide (Figure 5). In previous
work in our laboratory,[19,20] N-trityl derivatives of different substituted
phenylalanines, glycine and histidine were prepared by the method of Zer-
vas and Theodoropoulos[21] and attached to puromycin amino nucleoside
(PANS) for studies on protein synthesis. Couplings of N-trityl amino acids to
PANS were facilitated by NHS activation of carboxyl groups. This approach
has been employed in the present study to prepare 7 (Figure 5).
Using poly (rA)-d(pT)16 as template-primer, M-MuLV reverse
transcriptase-catalyzed incorporation of [3H]dTTP into DNA was assayed
in the absence or presence of various N-trityl amino acids and the 5ꢁ-AMP
derivative 7. The results are shown in Table 1 and are reported as IC50 values.
It is seen that at the N-trityl amino acid level, m-fluoro- DL-phenyl-alanine
is the most active (IC50 = 65 µM). However, if this particular N-trityl amino
acid is coupled to the nucleotide 8-(6-aminohexyl) amino-5ꢁ-AMP, the
resulting compound (7) showed increased inhibitory activity (IC50 = 5
µM). The IC50 value varied between 2 and 5 for different preparations
of compound (7). These results are interesting in that they demonstrate
that linkage of a hydrophobic aromatic structure such as N-trityl-m-fluoro-
DL-phenylalanine to the C-8 position of the purine nucleotide 5ꢁ-AMP
via a 1,6-diaminohexyl spacer brings about increased inhibitory activity
over the starting N-trityl amino acid. The distance between the purine C-8
position and the methane carbon of the N-trityl group is approximately
10 A. Detailed x-ray studies by a number of groups on co-crystals of HIV-1

Inhibition of Moloney Murine Leukemia Virus
1019
NH2
N
N
NH2
(
CH2)6
NH
O
N
O
P
N
O
C
-O
O
O
CH2
Tr
CH
HN
N
O
OH
(1)
(6)
F
O
OH
OH
NH2
N
N
O
N
NH
C
CH CH2
Tr
NH
(CH2)6
HN
O
P
N
(C6H5)3 – C – OH (8)
F
-O
O
O
(9)
(C6H5)3 – C – NH – CH2 – COOH
OH
(7)
(C6H5)3 – C – NH – CH – CH2 –
COOH
Z
OH OH
X = F; Y & Z = H (10)
Y = F; X & Z = H (11)
Z = F; X & Y = H (12)
X
Y
FIGURE 5 N-tritylamino acids coupled to 8-(6-aminohexyl)-amino-5ꢁ-AMP (1). N-tritylamino acids
were activated as their N-hydroxysuccinimide derivatives. N-hydroxysuccinimide derivative of N-trityl-
m-fluoro-DL-phenylalanine (6). N-trityl-m-fluoro-DL-phenylalanine-8-(6-aminohexyl) amino-5ꢁ-AMP (7).
Tritanol (8). N-tritylglycine (9). N-trityl-(o, m, p)-fluoro-DL-phenylalanines (10, 11, 12). Tr = trityl.
reverse transcriptase and nevirapine (non-nucleoside inhibitor) give an
approximate distance of 10A between the non-nucleoside binding site and
the active center.[22]
It is of interest to note that Steitz and co-workers[22,23] suggested the
idea of linking a non-nucleoside RT inhibitor (NNRTI) to substrates of
the reverse transcriptase such as the primer-template itself or a dNTP.
The design features of the nucleotide-N-trityl-m-fluoro-DL-phenylalanine
derivative are consistent with the idea of Steitz and co-workers. At present,
we do not fully understand how 8-(6-aminohexyl) amino-AMP-N-trityl-m-
fluoro-DL-phenylalanine elicits its effect. We believe that
i The N-trityl-phenylalanine portion of the molecule may be binding to the
hydrophobic non-nucleoside area of the reverse transcriptase.
ii The 5ꢁ-AMP component of the large inhibitor may be somewhere near
the active centre of the enzyme. As mentioned above, the spacer between
the purine ring and the trityl group of the inhibitor is approximately 10
A. It should also be noted that 5ꢁ-AMP is a ribose-containing molecule.

1020
A. Hawtrey et al.
TABLE 1 The effect of different N-trityl amino acids, N-trityl-m-fluoro-DL-phenylalanyl-8-
(6-aminohexyl) amino-5ꢁ-AMP and tritanol on DNA synthesis by Moloney leukemia virus
reverse transcriptasea
N-trityl derivatives
IC50(µM)
Tritanol
N-trityl-glycine
N-trityl-L-phenylalanine
N-trityl-o-fluoro-DL-phenylalanine
N-trityl-m-fluoro-DL-phenylalanine
N-trityl-p-fluoro-DL-phenylalanine
N-trityl-m-fluoro-DL-phenylalanyl-8 (6-aminohexyl)-5ꢁ-AMP
110
95
75
80
65
74
5
aIncubations all contained poly (rA).d(pT)16 as template-primer and [3H] dTTP. IC50 = 50%
inhibition of activity. Tritanol and all the N-trityl amino acids and derivatives were dissolved
in ethanol. Final concentration in incubations was 10% (v/v). Controls contained the same
percentage of ethanol. Each compound was assayed at three concentrations.
iii There is a possibility that 8-(6-aminohexyl) amino-5ꢁ-AMP-N-trityl-m-
fluoro-DL-phenylalanine may be displacing the poly (rA) template at
the growth point. However, competition experiments using varying con-
centrations of poly (rA)-d(pT)16 at fixed concentrations of nucleotide-
N-trityl-m-fluoro-DL-phenylalanine showed the compound to act as a
non-competitive inhibitor (results not shown).
It is important to note that various groups have previously reported
on the preparation and biological evaluation of chimeras consisting of
dideoxy nucleosides (ddN) and non-nucleoside reverse transcriptase in-
hibitors (NNRTI). The Spanish-Belgian research groups[24,25] have carried
out very detailed studies on this group of compounds. These dimers have
the general formula shown below:
[ddN]-Spacer-[NNRTI]
[ddN] = AZT or Thymidine
[NNRTI] = TSAO-T or HEPT (T represents thymidine)
−
Spacer = −(CH2)n where n = 3−9
As shown above, the two classes of inhibitors were linked at the N-
3 position of the thymidine base of each component compound by an
appropriate spacer [-N-(CH2)n-N-]. The spacer is an aliphatic bridge of
varying length. Because the spacer is linked through the N-3 position of each
thymidine ring, these modified bases in the dimers are incapable of base
pairing with A residues of the template-primer. The compounds prepared
in our work on the other hand used a spacer linked at the 8 position of

Inhibition of Moloney Murine Leukemia Virus
1021
the A (purine) ring and therefore are still capable of base pairing with
a thymidine (T) residue of the template-primer. Further, our compounds
are mono-phosphorylated at the 5ꢁ-position of the ribose ring. A French
research group has also investigated various conjugates containing both
a nucleoside analog (AZT or ddC) and a non-nucleoside type inhibitor
(HepT analogues) for their ability to block HIV replication.[26] Interesting
work has also been carried out with synthetic phospholipid-AZT conjugates
and their effects on HIV replication.[27,28]
Of further interest in respect of the hydrophobic trityl group, is the
interesting work reported by Cushman and co-workers[29] on the synthesis
and biological evaluation of some alkenyl diaryl methanes (ADAMS). These
compounds behave as NNRTIs. One of the most active was 3ꢁ,3ꢁꢁ-dibromo-
4ꢁ,4ꢁꢁ-dimethoxy-5ꢁ,5ꢁꢁ-bis (methoxycarbonyl)-1,1-diphenyl-1-heptene shown
below
This molecule inhibited the reverse transcriptase-catalyzed extension of
oligo (dG) primer with poly (rC) as template (IC50, 1 µM) but was without
effect in the poly (rA).oligo (dT) template-primer system. It should be noted
that compounds presented in this study gave inconsistent results using poly
(rU).oligo (dA)10–20 as the template-primer system with and without the N-
tritylated amino acid nucleotide compounds.
As mentioned by Cushman,[29] the NNRTIs are made up of a large and
structurally diverse set of compounds. It is interesting to note, however, that
many of these compounds possess two substituted aromatic ring systems,
which may also contain hetero atoms in the rings (pyridine, pyrimidine-
dione). Examples include, HEPT derivatives (acyclouridine derivatives) and
the non-nucleoside reverse transcriptase inhibitor DABO and others.It is
noted that the ADAM shown above has two substituted benzene rings
attached to a methene carbon. Results obtained in the work presented
herein demonstrate that the strongly hydrophobic triphenyl methyl (trityl)
group plays a crucial role in potentiating the inhibitory effect on Moloney
murine leukemia virus reverse transcriptase by C-8 substituted 5ꢁ-AMP
derivatives. Moreover, findings are consistent with the notion that the purine
nucleotide component of 7 resides near the active centre of the enzyme
while the trityl component may be located in the nearby hydrophobic
pocket.

1022
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