Virtual Screening of Acyclovir Derivatives as Potential Antiviral Agents: Design, Synthesis, and Biological Evaluation of New Acyclic Nucleoside ProTides

Article abstract of DOI:10.1021/acs.jmedchem.7b01009

Following our findings on the anti-human immunodeficiency virus (HIV) activity of acyclovir (ACV) phosphate prodrugs, we herein report the ProTide approach applied to a series of acyclic nucleosides aimed at the identification of novel and selective antiv

Full text of DOI:10.1021/acs.jmedchem.7b01009

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Article
Virtual screening of acyclovir derivatives as potential antiviral agents: design,
synthesis and biological evaluation of new acyclic nucleoside ProTides
Marco Derudas, Christophe Vanpouille, Davide Carta, Sonia Zicari, Graciela Andrei, Robert
Snoeck, Andrea Brancale, Leonid Margolis, Jan Balzarini, and Christopher McGuigan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01009 • Publication Date (Web): 22 Aug 2017
Downloaded from http://pubs.acs.org on August 22, 2017
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Virtual screening of acyclovir derivatives as potential antiviral agents: design,
synthesis and biological evaluation of new acyclic nucleoside ProTides
Marco Derudas,+, * Christophe Vanpouille, Davide Carta, Sonia Zicari, Graciela Andrei,
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Robert Snoeck,+++ Andrea Brancale, Leonid Margolis, Jan Balzarini and Christopher
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McGuigan 
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§This work is dedicated to the memory of Prof. Chris McGuigan, a great colleague and scientist,
invaluable source of inspiration and love for research
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Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, CF10
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3NB, UK
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Eunice Kennedy Shriver National Institute of Child Health and Human Development, National
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Institutes of Health, Bethesda, MD 20892, USA
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Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
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Abstract
Following our findings on the anti-human immunodeficiency virus (HIV) activity of acyclovir
ACV) phosphate prodrugs, we herein report the ProTide approach applied to a series of acyclic
nucleosides aimed at the identification of novel and selective antiviral, in particular anti-HIV
(
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agents. Acyclic nucleoside analogues used in this study were identified through a virtual screening
using HIV-reverse transcriptase (RT), adenylate/guanylate kinase, and human DNA polymerase.
A total of thirty-nine new phosphate prodrugs were synthesised and evaluated against HIV-1 (in in
vitro and in ex-vivo human tonsillar tissue system) and human herpes viruses. Several ProTide
compounds showed substantial potency against HIV-1 at low micromolar range while the parent
nucleosides were not effective. Also, pronounced inhibition of herpesvirus replication was
observed. A carboxypeptidase-mediated hydrolysis study was performed for a selection of
compounds to assess the formation of putative metabolites and support the biological activity
observed.
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Introduction
HIV infection currently affects over 36 million people worldwide and caused about 35 million
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deaths. The rate of new HIV infections is still increasing, especially in third-world countries.
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Twenty-six anti-HIV drugs are currently available and they act by interacting with different targets
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essential for the viral replication, such as RT, protease, integrase, or the fusion/entry step. The
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current antiretroviral therapy significantly improved the life expectancy of HIV-infected
individuals by reducing the viral load and postponing the insurgence of AIDS. Despite their
success these drugs carry some limitations, such as the onset of drug resistance and adverse side-
effects. Whilst the first problem has been addressed using a cocktail of different antiretroviral
drugs, the second one still dramatically impacts the patients’ compliance.
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Several years ago we discovered that ACV, a gold-standard drug against herpes simplex virus
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(HSV) and varicella zoster virus (VZV) infections, can act as a new potential anti-HIV agent. ACV
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inhibited HIV in the presence of human herpesviruses (HHV) and this activity was attributed to the
conversion of ACV to its monophosphate form by the HHV-encoded kinases; further
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phosphorylation steps provided the active triphosphate form of ACV able to inhibit HIV-RT.3
To circumvent the required first phosphorylation step of ACV, that requires HHV kinase activity, a
series of phosphate prodrugs, so called ProTides, were synthesised. These compounds showed
anti-HIV activity in the absence of HHV infection, demonstrating their nucleoside kinase
independence.4, 5 These findings were also supported in a different study, where ACV phosphate
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prodrugs showed a full retention of antiviral activity against HSV-1 and VZV thymidine kinase
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TK)-deficient strains. Unfortunately, these compounds proved to be somewhat cytotoxic in some
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assays. To overcome this issue and to better understand the full potential of acyclic nucleoside
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analogues as new anti-HIV agent, we performed a virtual screening on a library of ACV
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derivatives. We then synthesised and biologically evaluated a series of different acyclic
nucleosides and their corresponding ProTides and investigated their mechanism of bioactivation
using a well-established enzymatic method.
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Results and Discussion
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Molecular modelling: virtual screening
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The objective of this study was to increase the specificity of the compounds for HIV-1 RT and
reduce the affinity for the cellular DNA polymerase, while still retaining substrate action for
adenylate and guanylate kinases. A virtual screening using docking techniques was performed on
HIV-1 RT, adenylate or guanylate kinase, and a model of DNA polymerase . The docking using
nucleoside kinases involved in the first step of drug phosphorylation were not considered since the
phosphoramidate ProTide approach will be applied to the selected compounds.
Firstly, a database of 3600 compounds was built considering modifications at the base and at the
acyclic sugar moieties (Figure 1), including: simple and branched acyclic sugars (brown); oxygen,
methylene or sulphur at the 2’-position (yellow); substitution at the C-8-position of the base
including: hydrogen, methyl, fluorine, bromine or chlorine (red); substitution at the C-7-position of
the base including: nitrogen or carbon (hydrogen or methyl substituted) (blue); substitution at the
C-6-position of the base including: carbonyl, amine, chlorine, methoxide, ethoxide, phenoxy,
methylamine, or cyclopropylamine (magenta); substitution at the 2-NH position of the base
including: hydrogen, methyl, ethyl, phenyl or benzyl (green).
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Figure 1. Acyclic nucleoside derivatives.
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Based on the modification at the 6-position of the nucleobase, the database was divided in two
groups: 6-carbonyl derivatives (450 compounds), which showed base-pairing with cytidine; and 6-
O-modified and 6-N-modified derivatives (3150 compounds) which showed better base pairing
with thymidine (Figure 2).
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Figure 2. Base-pairing of the 6-carbonyl and 6-modified guanine derivatives.
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The first screening was performed on HIV-RT using a crystal structure of the enzyme co-
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crystallised with tenofovir diphosphate (PDB: 1T05). The original structure was used for the 6-
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modified ACV derivatives, while the substitution of a thymine by a cytidine was necessary for the
6-carbonyl derivatives. All the compounds were docked in their triphosphate form and the
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stereochemistry, where present, was taken into account. For this screening, a root mean square
deviation (RMSD) < 1 Å between the base of the compounds docked and the base of co-
crystallised tenofovir diphosphate was considered leading to a total of 984 compounds.
Interestingly, it was observed that branched sugar derivatives were well-tolerated due to the
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presence of a pocket in the proximity of amino acid residues Tyr115 and Phe116, which allowed
the accommodation of a substitution in the acyclic sugar moiety (Figure 3). Moreover, the
presence of a cleft that allows a potential substitution at the 2-amino position of the base was
observed. In addition, substitutions at the 8-position of the base, especially with halogens, were
well-tolerated.
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Interestingly, the compound that showed the best interactions (RMSD < 0.18 Å) was ganciclovir
(GCV)-TP (Figure 3), drug of choice for human cytomegalovirus (HCMV) infections.
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Figure 3. Docking pose of GCV triphosphate overlapped with tenofovir diphosphate (green) in the
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catalytic site of HIV-RT.
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For the second screening human adenylate kinase (PDB: 2C95) or mouse guanylate kinase (PDB:
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LVG, 88% human homology) were considered for the 6-modified and for the 6-carbonyl
analogues respectively. For these docking experiments, two different RMSDs were considered: the
first one regarding the position of the base (<1 Å) and the second one considering the position of
the phosphate moiety (<3 Å). These two values allowed a good position of the base and a
reasonable position for the phosphate moiety, which, however, in some cases, was slightly moved
compared to the crystallized compound. Similar to the results obtained for the HIV-RT docking,
branched sugar derivatives showed the best docking results in both series. In addition, the halogen
substitution at the 8-position for the 6-carbonyl series was well-tolerated. This second screening
led to a total of 218 compounds, with the 6-carbonyl derivatives being preferred over the 6-
modified ones.
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The last part of the virtual screening included the use of human DNA polymerases. It is known that
the toxicity of nucleoside analogues, especially HIV-1 RT inhibitors, is usually due to interactions
with these enzymes.10-13 The subtypes of human DNA polymerase mainly involved in the
cytotoxicity are  and , with the latter being able to incorporate antiviral nucleotide analogues,
which then act as a DNA chain terminators and consequently show cytotoxicity.14-17 As the crystal
structures for both human DNA polymerases have not yet been solved, a model for the human
polymerase  that covers the polymerase active site with the highest degree of homology with T7
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DNA polymerase has been used for the final step of our virtual screening.
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In this case, the results were analysed visually, excluding all the compounds that showed good
interactions with the catalytic site of the enzyme. A total of twenty-five compounds were
identified, and amongst these, five compounds were selected which displayed the best scoring for
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all the three docking experiments and that were easily accessible from a synthetic point of view. A
summary of the virtual screening is depicted in Figure 4.
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Figure 4. Summary of the virtual screening using HIV-RT, adenylate or guanylate kinases, and
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Figure 5 shows the five compounds selected from the virtual screening, which are: ganciclovir (1),
penciclovir (2), 2’-thio derivative of ganciclovir (3), 6-chloropenciclovir (4), and 6-
methoxypenciclovir (5).
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Figure 5. Selected acyclic nucleoside analogues from the virtual screening.
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Chemistry
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The desired phosphoramidates were synthesised using tert-butylmagnesium chloride ( BuMgCl) as
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a coupling reagent, following the procedure reported by Uchiyama, which is widely used for the
synthesis of ProTides. The desired phosphorochloridates were prepared according to our
established method by coupling the appropriate aryl dichlorophosphate with the appropriate amino
acid ester to yield the desired compound as an oil.4
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Synthesis of ProTides of nucleoside analogue 1.
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For the synthesis of ganciclovir 1 ProTides (Scheme 1), the 2-amino functionality of the guanine
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base was protected using N,N-dimethylamino formamide according to the literature. The coupling
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with the appropriate phosphorochloridate (2-8 eq) was performed using an excess of BuMgCl (2-3
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mol/eq) in a mixture of THF and pyridine. The presence of the extra hydroxyl group in the
molecule played a crucial role in the synthesis of the desired compounds resulting in some cases
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only in the isolation of the bisphosphate prodrug (9 and 10). The dimethylformamidine group was
removed by refluxing in 2-propanol for 24-80 h yielding the desired compounds 15-22.
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Scheme 1
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To overcome the issue related to the bis-phosphorylated side-product, the monomethoxytrityl
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(MMT) of 1 was synthetized using monomethoxytrityl chloride (2.2 eq), triethylamine (3 eq) and
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DMAP (cat.) in DMF. The reaction held two different products: 23 bearing two protecting groups
at the 2-amino position and at the 3’-hydroxyl position and 24 bearing the monomethoxytrityl
group only at the 2-amino position.
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Scheme 2
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Compound 23 was then reacted with phenyl(benzoxy-L-alaninyl)-phosphorochloridate (2 eq) in
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the presence of BuMgCl (2 eq) to yield the desired compound 25; compound 24 was reacted with
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-naphthyl(benzoxy-L-alaninyl)-phosphorochloridate under the same condition giving the desired
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mono-phosphorylated derivative 26. The deprotection of the monomethoxytrityl group was
performed using an excess of para-toluene sulfonic acid in DCM/MeOH yielding the desired
compounds 27 and 28.
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Synthesis of ProTides of nucleoside analogue 2
The same synthetic approach was applied for the synthesis of compound 2 ProTides. Initially, the
-amino functionality of the guanine base was protected under the same conditions seen previously
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to give compound 29 in excellent yield. Unfortunately, the coupling with the appropriate
phosphorochloridate resulted in the isolation of only the bis-phosphorylated compounds 30 and 31
for L-alaninyl derivatives; while both bis- (32 and 34) and mono-phosphorylated (33 and 35)
derivatives were obtained when dimethylglycine phosphorochloridates were used. The protecting
group was removed by refluxing in 2-propanol to give the final compounds (36-41) in low yield
due to degradation of the products.
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Scheme 3
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To overcome the issue related to the deprotection step, 2 was directly reacted with
phenyl(benzoxy-L-alaninyl)-phosphorochloridate yielding compound 42 in very low yield (5%).
Alternatively, compound 2 was converted to the bis-MMT derivative 43 and subsequently reacted
with the appropriate phosphorochloridate to give 44. The deprotection step was carried out using
para-toluene sulfonic acid affording the desired compound 45.
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Scheme 4
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The protection of both 1 and 2 with the para-methoxytrityl group proved to be an effective
approach for the synthesis of monophosphorylated derivatives, having a milder deprotection step,
which led to a higher overall yield.
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Synthesis of compound 3 and its ProTides.
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The synthesis of 3 was performed adapting a reported procedure. Compound 46, easily made by
reacting epichlorohydrin with benzyl alcohol in the presence of sodium hydride and tetra-n-butyl
ammonium iodide, was reacted with tosyl chloride to give derivative 47, which was then converted
into its thio-acetyl derivative 48 by heating with potassium thioacetate in anhydrous DMF. The
deprotection of the acetyl group was performed either in methanolic ammonia or using sodium
methoxide in methanol affording compound 49 in comparable yield. A three-steps coupling
reaction between diacetyl-guanine and 48 gave the desired compound 50; the N-7 isomer was also
isolated during the purification step. The benzyl protecting groups were then converted into acetyl
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groups, using acetic anhydride and BF
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to yield 51, which was then fully deprotected using
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methanolic ammonia to obtain the desired compound 3.
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Scheme 5
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Compound 3 was protected with N,N-dimethylformamide to give 52 in 88% yield followed by the
coupling with the appropriate phosphorochloridate affording only the bis-phosphorylated
derivatives 53 and 54. The deprotection was only successful for compound 53 leading the desired
compound 55; whilst in the case of 54, degradation of the starting material was observed (Scheme
6).
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Scheme 6
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Synthesis of nucleosides analogues 4 and 5, and their ProTides.
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Compounds 4 and 5 were prepared according to a procedure reported by Toori et al. (Scheme 7).
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Compound 2 was acetylated using acetic anhydride and DMAP (cat.) to obtain 57 in good yield.
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Chlorination at the 6-position was achieved using POCl in the presence of
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benzyltriethylammonium chloride and N,N-dimethylaniline to yield 58. The deprotection of the
acetyl group using methanolic ammonia at room temperature led only to the deprotection of the
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acetyl group, giving compound 4; while the use of sodium methoxide in methanol at 40 C
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afforded compound 5.
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Scheme 7
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Compounds 4 and 5 were coupled with the appropriate phosphorochloridate under the same
conditions described above. The presence of the 6-chloro or 6-methoxy group greatly improved
their solubility compared to the previous analogues with the subsequent improvement of their
reactivity.
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Scheme 8
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In the case of compound 4, the monophosphorylated derivative 59 and the bisphosphorylated 60
were obtained in 9% and 24% yield respectively (Scheme 8).
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In the case of compound 5, given the previous success of 6-methoxy purine analogues in another
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antiviral program, a wider range of ProTides was considered (Scheme 9). Monophosphorylated
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derivatives (66-72) were obtained most of the time together with the bisphosphorylated ProTides
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(61-65).
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5
5
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59
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Scheme 9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
The monophosphorylated ProTides of all the parent nucleosides considered were obtained as an
inseparable mixture of isomers due to the formation of a new chiral centre at the acyclic sugar
side-chain.
39
40
41
42
Scheme 10
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
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To overcome this issue, the cyclic ProTides derivatives of 2 and 4 were prepared. Compound 6
t
was reacted with ethoxy-L-alaninyl dichlorophosphate using BuMgCl in THF/pyridine yielding
compound 73 followed by deprotection in 2-propanol to afford compound 74 (Scheme 10).
1
1
12
1
14
Scheme 11
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
Similarly, 4 was directly coupled with benzoxy-L-alaninyl phosphorochloridate providing the
desired compound 75 (Scheme 11).
37
3
In an attempt to identify the formation of a metabolite in an enzymatic assay which will be
39
40
41
discussed later, the monoacetylated derivative of 76 and its ProTide 77 were synthesised (Scheme
42
4
o
1
2). Treating 58 with ammonia gas generated in situ at 0 C gave the desired compound 76 in
4
4
46
moderate yield. The synthesis of ProTide 77 was then performed using a standard procedure
47
48
obtaining the desired compound as a mixture of four diasteroisomers, as clearly depicted in the
49
50
3
1
corresponding P-NMR spectra.
51
52
53
5
5
56
57
58
59
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Scheme 12
1
1
12
13
14
15
16
17
18
19
20
2
2
Biological Results
23
2
25
26
27
28
29
30
31
Anti-HIV activity. The synthesised compounds were first tested for HIV-1 inhibition in MT-4 cell
cultures (Table 1). While the parent nucleosides 1-5 were inactive against HIV-1, several of the
ProTides showed anti-HIV activity, with the most potent being in the low M range. Mono-
ProTides (16, 20, 22, 27, 28) of compound 1 showed anti-HIV activity between 8 and >150 M
3
3
3
35
and an antiproliferative effect between 20 and >150 M. In the case of bis-ProTides (15, 17, 18,
36
37
1
9, and 21) the anti-HIV activity varied between 8.5 and >150 M and the antiproliferative effect
38
3
40
41
42
4
4
45
between 8 and 150 µM. However, for the most active ones (i.e. 15, 17, 19 and 22) the antiviral
activity can be related to the relatively high cytotoxicity observed for these compounds.
Penciclovir (2) mono-ProTides (39, 41, 42, and 45) showed an anti-HIV activity in a range
between 10-95 M and cytotoxicity at 100 M. The phenyl-L-alaninyl-O-benzyl derivative 42
46
47
48
49
50
51
52
53
5
was the most active in this series, with an EC50= 10 M and IC50= >150 M. Its therapeutic
selectivity index (i.e. ratio IC50/EC50) was > 15. The substitution of the natural amino acid L-
alanine with the non-natural amino acid dimethylglycine led to a >4-fold reduction of anti-HIV
5
56
activity. As observed in the previous series, the bis-ProTides of 2 (36-38) and the 2-NH -DMF-
2
57
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59
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protected ProTides 30 and 31 were generally fairly cytotoxic (4-30 M) and the antiviral activity
observed may be due to the underlying cytotoxicity.
Similarly, the bis-ProTides 54 and 55 of nucleoside analogues 3 were cytotoxic (21 and 4 M,
respectively) which, as previously observed, may affect the apparent anti-HIV activity. The 2-
1
1
12
13
14
NH -DMF protected ProTides (30, 31 and 54) were tested because the HIV-RT active site showed
2
15
16
17
18
the presence of a cleft in close proximity of the 2-amino position potentially allowing a better
interaction with this enzyme.
19
20
2
Compound 60, mono-ProTide of 4, showed an anti-HIV activity in the low micromolar range (1
2
23
M) and an IC50 = 20 M, while bis-ProTide 59 showed a 7-fold loss in antiviral activity while
24
25
26
retaining cytotoxicity.
27
28
29
30
31
3
3
34
35
36
37
38
Mono-ProTides (66-72) of nucleoside 5 showed an EC50 in a range between 1.1->150 M, and an
IC50 in between 12-92 M. It must be stated that the majority of the compounds showed a
substantial antiproliferative effect and the observed anti-HIV activity may result from it, with the
exception of compound 66, which showed the best inhibitory activity (EC50= 1.1 M) as well as
the best selectivity index = 21. This compound has similar biological profile as the best ACV
39
4
4
ProTide reported in our previous study. Interestingly, the substitution of the phenyl moiety with
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
the naphthol led to a dramatic loss of activity (>20 fold), which was not observed in the previous
series. Regarding the bis-ProTides (61-65), these compounds were found to be fairly cytotoxic.
The cyclic ProTides 74 and 75 were also evaluated in this study, showing an EC50= 22 M and
IC50= >150 M (selectivity index > 7) for compound 74, and EC50= 85 M and IC50 of 150 M for
75. Acetyl protected nucleoside 76 did not show any anti-HIV activity or cytotoxicity (EC50 and
IC50 > 150 M), ProTide 77 showed an EC50= 2 M for HIV and an IC50= 10 M, data similar to
its related compound 60.
58
59
60
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Table 1. Anti-HIV activity for synthesized ProTides and their related parent nucleosides in cell
1
1
lines.
12
13
Antiviral activity
Cytostatic/toxic
1
a
EC50 (µM)
activity (µM)
15
16
17
18
1
20
2
2
23
2
25
26
27
28
2
30
31
3
3
34
35
36
3
38
39
40
41
4
4
4
45
46
b
c
Amino
Acid
HIV-1
MT-4
8.5
CC50
CEM
>100
IC50
Cpds
15
Nucl
ProTide
Aryl
Ester
cLogP
MT-4
6
i
1
1
Bis
Naph
Naph
L-Ala
L-Ala
Pr
4.03
0.41
i
1
1
6
7
Mono
Pr
40
354.2
150
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
4
4
Bis
Bis
Ph
Naph
Ph
L-Ala
L-Ala
DMG
DMG
DMG
DMG
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
DMG
DMG
DMG
DMG
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
3.42
5.77
4.04
0.42
6.39
1.59
0.11
1.28
4.04
6.38
3.83
6.18
4.45
0.87
6.80
2.04
0.56
1.73
6.14
3.59
5.54
2.27
30
ND
10
>150
>150
10
17
8
15982
ND
>150
>150
13
18
1
2
9
0
Bis
47
Mono
Bis
Ph
>100
>20
>150
>150
26
21
Naph
Naph
Ph
2
2
2
2
7
8
Mono
Mono
Mono
Bis
>100
ND
20
Naph
Ph
ND
100
10
d
3
0
9
21±0
19±1
76±2
>10
d
31
Bis
Naph
Ph
3.2
8
4
3
3
6
Bis
10
7
Bis
Naph
Ph
ND
19
68
>150
95
10
21
9
ND
30
38
Bis
100
3
4
4
4
9
0
1
2
Mono
Bis
Ph
252±32
>250
96±2
>250
138±6
17±2
77±21
14±3
33±8
>150
>150
100
>150
100
4
Naph
Naph
Ph
Mono
Mono
Mono
Bis
47
48
49
5
51
52
53
5
45
Naph
Naph
Ph
d
5
4
5
5
Bis
24
7
21
5
56
57
59
Bis
Ph
20
6
0
Mono
Ph
1
20
58
59
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1
1
12
1
14
15
1
17
18
19
20
2
2
23
24
25
2
27
28
2
30
31
3
3
3
35
36
6
6
6
1
2
3
5
5
5
5
5
5
5
5
5
5
5
5
1
4
4
4
-
Bis
Bis
Ph
Naph
Naph
Naph
Ph
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
-
Bn
5.79
8.43
6.83
9.49
6.43
2.51
3.69
3.83
3.03
1.98
4.36
2.83
-0.69
1.55
3.17
-0.60
-2.54
-2.72
-2.38
-1.01
-0.76
12
8.2
19±7
4.6±0.6
6.8±1.5
7.2±1.9
7.2±0.5
32±4
20
5
t
CH
2
Bu
n
Bis
Pr
9.7
20
64
Bis
2,2-diMe-But
Cyclohexyl
Bn
5.6
9
6
6
5
6
Bis
10
11
Mono
Mono
Mono
Mono
Mono
Mono
Mono
Cyclic
Cyclic
monoacetyl
monoacetyl
-
Ph
1.1
23
67
Naph
Naph
Naph
Naph
Naph
Ph
Bn
23
27±2
12
t
6
6
7
7
8
9
0
1
CH
2
Bu
41
17±2
35
n
Pr
11
17±5
13
Me
16
15±0
10
2,2-diMe-But
24
18±2
17
72
Cyclohexyl
>150
22
15±1
92
7
7
4
5
-
Et
Bn
Bn
-
ND
>150
150
10
85
165±90
26±9
77
Ph
-
2.25
>150
>50
>150
>150
>150
>150
7
6
≥250
>150
>50
>150
>150
>150
>150
1
-
-
-
>100
2
3
4
5
-
-
-
-
-
ND
-
-
-
-
-
>500
-
-
-
-
-
400±141
246±0
-
-
-
-
-
3
38
a50% Effective concentration, or compound concentration required to inhibit virus-induced cytopathicity by 50%. 50%
b
39
40
Cytostatic concentration, or compound concentration required to decrease the viability of the cell cultures by 50%.
4
42
c50% Inhibitory concentration, or compound concentration required to inhibit cell proliferation by 50%. ND = not
4
4
d
determined. N
2
-DMF-protected.
45
46
47
48
Next we tested a selection of ProTides and their respective parent nucleosides using ex-vivo human
49
50
51
3
tonsillar tissues infected with HIV and HHV. This system of histoculture offers major advantages
52
53
5
5
56
57
58
over single cell cultures as it retains tissue cytoarchitecture and many important functional aspects
of cell–cell interactions in vivo. In particular, cultures of human lymphoid tissue allow the testing
of anti-HIV drugs upon HIV inoculation ex vivo. Lymphoid tissue explants support productive
59
60
2
3
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1
1
12
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16
17
18
19
20
2
HIV infection without exogenous cell activation, and retain the pattern of expression of key cell
2
3
surface molecules relevant to HIV infection. In this system, 1 proved to be fairly effective in
inhibiting HIV at an EC50= 5 μM. Compounds 28 and 74, respectively the mono-ProTide and the
cyclic-ProTide of 1, showed retention of antiviral activity (EC50= 11 and 4.6 M, respectively)
demonstrating that the delivery of the monophosphate form of 1 through a ProTide is as efficient
as the phosphorylation mediated by the HHV-TK present in the tissue cultures. 2 displayed an
EC50= 11 M versus HIV-1, result expected since this compound is a well-known anti-HSV drug
and as such it is an efficient substrate for the HHV-thymidine kinase. This activity was also in
accordance with the results obtained for its ProTide 42 in the MT4 assay (EC50= 10 M). An
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
important result was obtained for compound 3 which lacked any anti-HIV activity (EC50> 30 M)
in this assay, although it functions as a submicromolar anti-HSV compound (as reported in Table
3). Similar results were obtained for its bis-ProTide 55, which did not inhibit HIV either. We can
speculate that 3, despite being a very close analogue of 1, is a poor substrate for HIV-RT and the
inhibitory activity found in the MT-4 cell assay for 54 and 55 was just due to the underlying
toxicity of these compounds.
39
40
41
42
4
4
45
Both compounds 4 and 5 were not effective in this assay (EC50> 60 M and > 30 M,
respectively), while their phosphate prodrugs were able to inhibit HIV in a low micromolar range:
EC50= 4 M for mono-ProTide 60 and EC50= 9 M for cyclic ProTide 74 (both derivatives of
46
4
48
49
50
51
52
nucleoside analogue 4); and EC50= 0.9 M for 66 (mono-ProTide of 5). The lack of activity
reported for 4 and 5 is probably related to their poor conversion to the monophosphate form by
TK-HHV. This assumption is supported by the lack of anti-HSV activity of these compounds
53
5
5
(Table 3). Similar result was obtained for the monoacetylated derivative 76 (EC50>10 M), while
56
57
58
its ProTide 77 showed an EC50= 4 M.
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Table 2. Inhibitory Activity of ProTides against HIV-1 in human tonsillar tissues ex vivo.
1
1
12
13
1
15
Anti-HIV Activity
Amino
Cps
Nucl
ProTide
Aryl
Ester
50a
Acid
EC (M)
16
17
1
19
20
2
2
23
24
25
2
27
28
29
30
3
3
3
3
35
36
37
38
39
2
8
1
Mono
Naph
L-Ala
Bn
Bn
Bn
Bn
Bn
Et
Bn
Bn
-
11
>20
>3
4
42
2
Mono
Ph
Ph
Ph
Ph
-
L-Ala
5
6
6
7
5
0
6
4
3
Bis
L-Ala
4
Mono
L-Ala
5
Mono
L-Ala
0.9
4.6
9
1
Cyclic
L-Ala
75
4
Cyclic
-
L-Ala
7
7
MonoAcetyl 4
Mono
Ph
-
L-Ala
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
5
2
3
4
5
-
-
-
11
-
-
-
>30
>60
>30
>10
-
-
-
-
-
-
7
6
MonoAcetyl 4
-
-
a50% Effective concentration, or compound concentration required to inhibit virus-induced cytopathicity by 50%.
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
2
5
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Anti-HSV activity. The synthesized compounds were also evaluated for their ability to
-
inhibit HSV-1, HSV-2, and a TK-deficient (TK ) HSV-1 strain (ACV-resistant) in
confluent virus-infected human embryonic lung fibroblasts (HEL cultures) (Table 3).
1
1
Parent compound 1 showed antiviral activity (EC50) of 0.03 M, 0.04 M and 4.3 M
12
1
14
-
against HSV-1, HSV-2 and HSV-1 TK , respectively. Similar to the anti-HIV activity in
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
tissues ex vivo, its ProTides were found to be slightly less active compared to the parent
nucleoside (with an activity against the HSV-1 KOS strain in the range of 0.09-2 M for
the mono-ProTides 16, 20, 22, 27 and 28, and in the range of 0.3-3 M for the bis-
ProTides 15, 17, 19 and 21). Similar activity was obtained against HSV-2 with an EC50 in
the range of 0.3-3 M for the mono-ProTides, and 0.9-2.5 M for the bis-ProTides. As
expected, while the parent nucleoside 1 showed a loss of activity of 140-fold against
-
HSV-1 TK , some of the ProTides (mono- and bis-) showed a good retention of the
31
3
3
antiviral activity against this strain. In particular, compounds 18, 19, 20 and 22 showed a
34
3
36
full retention of antiviral activity (<2.5 fold difference in EC50 values between the HSV-1
3
38
+
-
TK and TK strains). While compounds 17, 21 and 28 still show considerable activity
39
40
against these two HSV-1 strains, compounds 15 and 16 displayed marginal activity
41
4
4
-
against the TK mutant virus. No cytotoxicity was observed for any of these compounds
4
45
(MCC> 100 M).
46
4
48
49
50
51
52
In spite of being at least 15-fold (for HSV-1) and 30-fold (for HSV-2) less potent than the
parent nucleoside, the majority of these compounds were found as active as ACV, the
current drug of choice for HSV infections.
53
5
5
56
57
Mono-ProTides 39, 41, 42, and 45 showed a loss of antiherpes activity of 16- to 150-
fold against wild-type HSV-1, and 27- to 132-fold against HSV-2 compared to the
58
59
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1
1
12
13
14
15
16
17
18
19
-
parent nucleoside 2 (EC50= 0.5, 0.6 and 4 M against HSV-1, HSV-2 and HSV-1 TK
strain, respectively). In addition, no activity was found for the mono-ProTides of 2
-
against the HSV-1 TK strain. Contrary to the results observed for derivatives of 1, bis-
ProTides of 2 (compounds 30, 31, 37, 38, and 40) were found to be inactive against the
three HSV strains. Similarly, compounds 54 and 55 (i.e. bis-ProTides of 3) were found
inactive (EC50= >100 M) against HSV-1 and HSV-2, while the parent compound 3
showed a sub-micromolar activity.
20
2
2
23
24
25
26
Compound 5 did not show any anti-HSV activity (EC50>100 M). Its mono-ProTide
derivatives, with the exception of compound 70, which showed a good to moderate
inhibitory activity (EC50= 9-38 M and EC50= 9-20 M against HSV-1 and HSV-2,
27
28
-
respectively), with retention of some activity against the TK strain for compounds 66,
29
3
31
67, 68, and 72. Compound 68, bearing the naphthyl as an aryl group, L-alanine as an
3
3
amino acid and neopentyl as an ester, showed the best antiviral profile with an EC50 of 9
34
3
36
-
M, 9 M and 15 M against HSV-1, HSV-2 and HSV-1 TK , respectively. In this
37
38
series, as already observed for derivatives of 2 and 3, the bis-ProTides 61, 62, 63, 64, and
39
40
41
6
5 were found totally inactive (EC50>100 M).
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
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Table 3. Anti-HSV activity of the synthesized ProTides and their corresponding parent
nucleosides.
Antiviral Activity
Cytotoxicity
a
EC50 (M)
(M)
1
1
1
13
14
15
1
17
18
-
Amino
Acid
HSV-1
(KOS)
HSV-2
(G)
HSV-1 TK
b
Cps
Nucl
ProTide
Aryl
Ester
MCC
(KOS)
≥73 ± 39
≥75 ± 35
14.5 ± 7.8
2.5 ± 2.1
1.5 ± 0.7
i
1
5
1
1
1
1
1
Bis
Mono
Bis
Naph
Naph
Ph
L-Ala
L-Ala
L-Ala
L-Ala
DMG
Pr
1.2 ± 1.1
2 ± 0
1.4 ± 0.8
3.0 ± 1.4
2.5 ± 2.1
2.5 ± 2.1
2.5 ± 2.1
≥100
>100
>100
>100
ND
i
16
Pr
1
1
7
8
Bn
Bn
Bn
3.0 ± 1.4
1.3 ± 1.1
1.5 ± 0.8
19
20
2
Bis
Naph
Ph
19
Bis
2
23
24
25
26
27
28
29
0
.30 ±
0.28
.90 ±
.14
0.85 ±
.07
2
2
2
0
1
2
1
1
1
Mono
Bis
Ph
DMG
DMG
DMG
Bn
Bn
Bn
0.09 ± 0.02
0.30 ± 0.28
0.60 ± 0.28
0.15 ± 0.07
1.6 ± 0.6
1.4 ± 0.8
>100
>100
>100
0
Naph
Naph
0
Mono
30
31
0
3
3
34
3
36
3
38
39
40
41
42
4
4
4
46
47
48
49
5
51
52
53
5
5
27
1
1
2
2
2
2
2
2
2
2
2
2
3
3
4
4
Mono
Mono
Bis
Ph
Naph
Ph
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
DMG
DMG
DMG
DMG
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
>100
0.6 ± 0.4
0.4 ± 0
>100
3.0 ± 1.4
0.5 ± 0.1
>100
3.0 ± 1.4
3.0 ±1.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
2
8
c
3
0
c
31
Bis
Naph
Ph
>100
>100
>100
3
3
3
3
6
Bis
>100
>100
>100
7
Bis
Naph
Ph
>100
>100
>100
8
9
Bis
>100
>100
>100
Mono
Bis
Ph
29 ± 13
>20
39 ± 27
>20
100 ± 0
>20
40
Naph
Naph
Ph
4
4
1
2
Mono
Mono
Mono
Bis
75 ± 35
8.0 ± 6.0
73 ± 39
>100
52 ± 9
16 ± 6.0
79 ± 30
>100
>100 ± 0
≥100
>100
>100
>100
≥100
≥100
>100
>100
45
Naph
Naph
Ph
>100 ± 0
>100
c
5
4
5
5
9
0
Bis
>100
>100
>100
5
6
Bis
Ph
>100
>100
>100
56
57
Mono
Ph
>100
>100
>100
58
59
60
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Page 29 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
1
14
15
1
17
18
19
20
2
2
23
24
25
2
27
28
2
30
31
3
3
34
35
36
6
6
6
1
2
3
5
5
5
5
5
5
5
5
5
5
5
5
1
4
-
Bis
Bis
Ph
Naph
Naph
Naph
Ph
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
-
Bn
100
>100
>100
>100
100
>100
>100
>100
>100
39 ± 27
20
>100
≥100
≥100
≥100
>100
>100
>100
>100
100
t
CH
2
Bu
n
Bis
Pr
>100
>100
64
Bis
2,2-diMe-But
Cyclohexyl
Bn
>100
>100
6
6
5
6
Bis
>100
>100
Mono
Mono
Mono
Mono
Mono
Mono
Mono
Cyclic
Cyclic
-
Ph
39 ± 27
38
29 ± 23
20
67
Naph
Naph
Naph
Naph
Naph
Ph
Bn
t
6
6
7
7
8
9
0
1
CH
2
Bu
9.0 ± 1.4
14 ± 2
>100
9.0 ± 0
15 ± 8
>100
15 ± 7
≥20
n
Pr
Me
>100
≥20
≥100
100
2,2-diMe-But
11 ± 2
16 ± 6
ND
10 ± 1
16 ± 6
ND
72
Cyclohexyl
40 ± 28
ND
≥100
ND
7
7
4
5
-
Et
Bn
-
-
>100
>100
>100
60 ± 82
>100
>250
ACV
1
-
0.4 ± 0.5
0.4 ± 0.3
0
.04 ±
-
-
-
-
-
0.03 ± 0.03
4.3 ± 6.4
>100
0.03
0.6 ± 0.3
0.5 ± 0.1
>100
2
3
4
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.5 ± 0.2
0.4 ± 0.1
>100
4.0
1.6 ± 1.1
>100
>100
>100
>100
>100
>100
>100
>100
3
38
a50% Effective concentration, or compound concentration required to inhibit virus-induced cytopathicity
3
40
b
by 50%. Minimum cytotoxic concentration, or compound concentration required to cause a
4
42
c
microscopically detectable alteration of normal cell morphology. N
2
-DMF-protected. ND = not
4
4
determined.
45
46
47
48
Anti-HCMV activity. The synthesized compounds were also evaluated for their ability
49
5
51
to inhibit two strains (AD-169 and Davis) of HCMV in confluent HEL cell cultures
52
53
(Table 4). Compound 1 showed an EC50 of 7.4 M (AD-169 strain) and 7.3 M (Davis
5
5
56
57
58
strain); its mono-ProTides showed an EC50 in a range between 6.6->50 M (AD-169
strain) and in a range between 4.9-24 M (Davis strain); the bis-ProTides showed a loss
59
60
2
9
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