PMPA and PMEA prodrugs for the treatment of HIV infections and human papillomavirus (HPV) associated neoplasia and cancer

Article abstract of DOI:10.1016/j.ejmech.2014.03.051

The synthesis and in vitro biological evaluation of novel phosphonamidate and phosphonodiamidate prodrugs of adefovir and tenofovir are reported. The selected synthetic approach from free phosphonic acid via bis-trimethylsilyl ester intermediates affords (l)-alanine ester derivatives in 10-70% yields. When assessed for their anti-HIV activity, all the prodrugs showed submicromolar activity. Noteworthy, the most potent derivative in the adefovir series contained a 5,6,7,8-tetrahydronaphtyl group, herein reported for the first time as an aryl moiety in a ProTide. A pronounced cytostatic activity of the above prodrugs is also reported. Selected compounds were tested for their antiproliferative activity against HPV-transformed cells and they were found significantly more active in comparison to their parent compounds. In this study a slightly improved activity of the adefovir derivatives over those of tenofovir was also noticed. However, no specificity for naturally HPV-transformed cell lines was observed.

Full text of DOI:10.1016/j.ejmech.2014.03.051

European Journal of Medicinal Chemistry 78 (2014) 259e268
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
PMPA and PMEA prodrugs for the treatment of HIV infections and
human papillomavirus (HPV) associated neoplasia and cancer
Fabrizio Pertusati a, Karen Hinsinger a, Áine Sinéad Flynn b, Ned Powell b,
,
Amanda Tristram b, Jan Balzarini c, Christopher McGuigan a
*
a School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK
b HPV Oncology Research Group, Institute of Cancer and Genetics, School of Medicine, Cardiff University, UK
c Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and in vitro biological evaluation of novel phosphonamidate and phosphonodiamidate
prodrugs of adefovir and tenofovir are reported. The selected synthetic approach from free phosphonic
acid via bis-trimethylsilyl ester intermediates affords (L)-alanine ester derivatives in 10e70% yields.
Received 14 August 2013
Received in revised form
13 March 2014
Accepted 15 March 2014
Available online 17 March 2014
When assessed for their anti-HIV activity, all the prodrugs showed submicromolar activity. Noteworthy,
the most potent derivative in the adefovir series contained a 5,6,7,8-tetrahydronaphtyl group, herein
reported for the first time as an aryl moiety in a ProTide. A pronounced cytostatic activity of the above
prodrugs is also reported. Selected compounds were tested for their antiproliferative activity against
HPV-transformed cells and they were found significantly more active in comparison to their parent
compounds. In this study a slightly improved activity of the adefovir derivatives over those of tenofovir
was also noticed. However, no specificity for naturally HPV-transformed cell lines was observed.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
5,6,7,8-Tetrahydro-1-naphthol
HIV
Antiproliferative activity
HPV
PMPA/PMEA
1. Introduction
charged free phosphonate. Therefore, several prodrug approaches
are needed to improve the bioavailability of ANPs. The importance
Acyclic nucleoside phosphonates (ANPs) are broad spectrum
antiviral agents active against DNA viruses and retroviruses. The
common structural attribute of ANPs is a nucleobase attached to an
aliphatic side chain containing a phosphonomethyl residue [1e5].
The methylene bridge between the phosphonate moiety and the
rest of the molecule excludes the possibility of enzymatic
dephosphorylation. Three ANP compounds, namely cidofovir
(HPMPC), adefovir (PMEA) 1 and tenofovir ((R)-PMPA) 2 are in
current clinical use. Cidofovir is approved for the treatment of
human cytomegalovirus (HCMV) retinitis in AIDS patients. Adefovir
in its oral prodrug form, adefovir dipivoxil (Hepsera) 3, is approved
for the treatment of Hepatitis B Virus (HBV) infections, and teno-
fovir disoproxil fumarate (TDF, Viread) 4 has been approved for the
treatment of HIV and HBV infections [6] (see Fig. 1).
of ANP prodrugs has been recently reviewed [7,8].
Our ProTide technology was successfully applied to PMEA and
(R)-PMPA by Ballatore et al. [9]. Mirroring this work, Gilead Sci-
ences reported an extensive study on the application of the ProTide
approach to (R)-PMPA. In these studies, the phenyloxy isopropyl-
(L)-alaninyl phosphonamidate prodrug of (R)-PMPA (GS-7340)
emerged as a lead compound and is now in a phase III clinical trial
for the treatment of HIV (Fig. 2). Similarly, several ANP phospho-
nodiamidates containing (
L)-alanine esters were reported to exhibit
potent antiviral activities, notably bis(isobutyl-(
L)-phenylalaninyl)
phosphonodiamidate-PME-N6-(cyclopropyl)DAP (GS-9191) the
most active of the series against human papillomavirus (HPV). This
compound has currently completed Phase I clinical trials as a
topical prodrug for the treatment of HPV lesions [1].
Since the discovery of these compounds, intensive drug dis-
covery programs have led to the development of other ANPs with
diverse nucleobases, aglyco modifications and other structural
variations. The major limitation of ANPs relates to their poor oral
bioavailability, most likely due to the presence of a negatively
Some ANPs have also demonstrated an interesting cytostatic
potential, a little explored therapeutic opportunity in the case of
ANPs. In particular, PMEA has been shown to be a potent inhibitor
of highly aggressive choriocarcinoma tumors which may be of
particular interest [10,11], given the occurrence of several types of
cancer, including Kaposi sarcoma, in patients with AIDS.
Noteworthy, cidofovir also displays antineoplastic activity,
increasingly showing positive efficacy in HPV associated lesions
* Corresponding author.
E-mail address: mcguigan@cf.ac.uk (C. McGuigan).
http://dx.doi.org/10.1016/j.ejmech.2014.03.051
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

260
F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
NH2
NH2
N
N
N
N
N
N
O
N
O
P
OH
N
O
P
OH
O
OH
OH
Adefovir
1
Tenofovir 2
NH2
N
N
N
NH2
N
N
N
O
O
P
O
O
O
N
O
O
N
O
O
O
O
O
HO
O
P
OH
O
O
O
O
O
Adefovir dipivoxil 3
Tenofovir disoproxil
fumarate
4
Fig. 1. Structure of Adefovir 1, Tenofovir 2 and their prodrugs 3 and 4.
Fig. 2. Structures of GS-7340 and GS-9191.
[12]. HPV is the primary causative agent of cervical cancer, and also
gynaecological and anogenital epithelial lesions such as Vulval
Intraepithelial Neoplasia (VIN). VIN is an uncommon chronic skin
disorder, which has a high risk of recurrence post treatment and a
significant risk of progression to invasive vulval carcinoma. The
current treatment options including interferon, 5-FU and several
forms of surgery, all have considerable disadvantages [13]. One
pilot study has shown promise for the treatment of VIN with topical
cidofovir [14]. With this in mind, there is a considerable need for a
treatment option that specifically targets the cause of the lesion
with minimal side effects.
Due to the high success of the ProTide approach applied to
ANPs and other nucleosides, with the aim to expand the SAR
study of PMEA and (R)-PMPA derivatives as anti-HIV agents, we
present here a series of novel phosphonamidate and phospho-
nodiamidate prodrugs of both adefovir and tenofovir. Consid-
ering the therapeutic potential of PMEA for the treatment of
neoplastic diseases and the positive effect of cidofovir in the
treatment of the HPV-associated VIN, we assessed the anti-
proliferative activity of these derivatives in the treatment of HPV
and related agents.
2. Results and discussion
2.1. Chemistry
In 2001, we reported the synthesis of a number of adefovir and
tenofovir amidate prodrugs. However, the yields achieved with
established chemical methodologies were very modest [9]. More
recently, an efficient one-pot procedure for the synthesis of ANP
diamidate prodrugs from free phosphonic acid has been reported
[15].
We thus endeavored to apply this novel methodology. First, we
prepared new diamidate derivatives according to the approach
highlighted in Scheme 1. (L)-Alanine neopentyl and benzyl ester
amino acids were selected as designated pro-moieties on the basis
of the excellent results obtained during our anti-HCV synthesis
program [16]. Thus, overnight reaction of free ANPs with trime-
thylsilyl bromide in acetonitrile afforded the intermediate bis(-
trimethylsilyl) phosphonate esters, which without purification
were reacted with an excess of the desired (L)-alanine amino acid
ester in pyridine in the presence of triethylamine, aldrithiol-2 and
triphenylphosphine. This two-step reaction gave the corresponding

F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
261
NH2
N
NH2
N
N
2 AAOR or AAOR/ArOH
N
N
N
N
Aldrithiol-2, Ph3P
O
OH
OH
TMSBr, CH3CN,
rt, 18 h
OTMS
OTMS
O
P
O
P
N
NEt3, Py, 50 °C, 3h
10-70%
O
R
R
R = H PMEA 1
R =CH3 PMPA 2
5
6
7
8
9
R = H, R' = (L)-AlaOBn, R'' = ONaph
R = H, R' = R'' = (L)-AlaOBn
R = H, R' = R'' = (L)-AlaOCH2tBu
R = H, R' = (L)-AlaOCH2tBu, R'' = OPh
R = H, R' = (L)-AlaOCH2tBu, R'' = ONaph
NH2
10 R = H, R' = (L)-AlaOCH2tBu, R'' = O-5,6,7,8-Tetrahydronaph
11 R = CH3, R' = R'' = (L)-AlaOBn
N
N
N
N
R'
O
12 R = CH3, R' = R'' = (L)-AlaOCH2tBu
P
13 R = CH3, R' = (L)-AlaOBn, R'' = ONaph
O
R''
14 R = CH3 R' = (L)-AlaOCH2tBu, R'' = Ph
15 R = CH3 R' = (L)-AlaOCH2tBu, R'' = Naph; (d.r. 19:1)
16 R = CH3 R' = (L)-AlaOCH2tBu, R'' = Naph; (d.r. 1:5)
17 R = CH3, R' = (L)-AlaOCH2tBu, R'' = O-5,6,7,8-Tetrahydronaph
R
Scheme 1. General procedure for the synthesis of amidate ANP prodrugs 5-17.
(
L
)-Alanine diamidate prodrugs 6, 7, 11 and 12 in 22e70% overall
respective benzyl derivatives (6 vs 7 and 11 vs 12, Table 1). These
results may suggest that steric factors in the amino acid moiety may
have a defined influence on the antiviral activity. The increased
antiviral potency was more pronounced in the tenofovir than the
adefovir prodrug series. It is also evident that an increased anti-HIV
activity is observed, especially in the neopentyl ester Protide for
both adefovir and tenofovir series going from phenol towards the
presumably more lipophilic and sterically encumbered tetrahydro-
1-naphthol.
yields, after purification by column chromatography. The reaction
can be monitored by 31P NMR spectroscopy analysis to ensure the
completion of each step.
When we slightly modified this procedure, adding in the second
step a 1:1 mixture of the amino acid ester and the aryl alcohol
instead of solely the amino acid ester, the desired phosphonoami-
date 5, 8e10 and 13e17 were obtained, in 10e30% yields after
column chromatography. All products were isolated as a roughly
1:1 mixture of two diastereoisomers. It is worthy to note that in the
In case of the tenofovir L-Ala-Oneopentyl-naphthyl derivative,
case of the neopentyloxy-(
L
)-alanyl naphtyloxyphosphonoamidate
we were able to obtain two fractions 15 and 16 each of one enriched
in one of the diastereoisomers. They showed a difference in activity
between the two fractions. This suggests that prodrug stereo-
chemistry may be important for the eventual metabolic conver-
sion/activation of these prodrugs.
derivatives of tenofovir we succeeded, after column chromatog-
raphy, to partially separate the two diastereoisomers obtaining two
fractions enriched respectively with one of the two di-
astereoisomers (15 d.r. 19:1 and 16 d.r. 1:5).
3. Antiviral activity
Table 1
Anti-HIV activity (EC50) and cytotoxicity (CC50) data of the test compounds.
3.1. Anti-HIV activity
Compounds
EC50
(
m
M)a
CC50 (m
M)a
(CEM)
HIV-1 (IIIB)
HIV-2 (ROD)
All compounds were tested against HIV-1 and HIV-2 to deter-
mine their antiviral activity in vitro. All prodrugs showed marked
anti-HIV activity when compared to the parent ANPs, likely due to
improved cellular permeability (Table 1).
1
5
6
7
8
9
10
2
11
12
13
14
8.4 ꢀ 5.4
8.2 ꢀ 4.0
ꢁ50
0.030 ꢀ 0.009
0.040 ꢀ 0.003
0.024 ꢀ 0.003
0.056 ꢀ 0.004
0.032 ꢀ 0.002
0.017 ꢀ 0.011
3.9 ꢀ 0.98
0.050 ꢀ 0.005
0.025 ꢀ 0.012
0.019 ꢀ 0.004
0.044 ꢀ 0.003
0.019 ꢀ 0.004
0.007 ꢀ 0.0002
3.7 ꢀ 1.2
0.12 ꢀ 0.04
0.14 ꢀ 0.09
0.20 ꢀ 0.04
0.40 ꢀ 0.04
0.045 ꢀ 0.045
0.019 ꢀ 0.009
>500
From the data collected in Table 1, it can be appreciated that
adefovir and tenofovir derivatives display low micromolar activity
against HIV-1 and HIV-2. The EC50 of tenofovir value is slightly
(w3-fold) lower compared to that of adefovir. However, their pro-
drugs consistently showed nanomolar anti-HIV activities, up to
600- to 1000-fold stronger for the adefovir prodrugs or even up to
1000- to 2000-fold stronger for the tenofovir prodrugs depending
the nature of the R1 and R2 groups on the phosphonate moiety.
Interestingly, in the adefovir series, the derivative bearing the novel
5,6,7,8-tetrahydro-1-naphthol entry as a hydrolysable aryl moiety
(10) showed the most increased antiviral activity compared to all
other adefovir protides. This is the first time this aryl unit has been
reported in ProTide. When the amino acid ester is taken into ac-
count, in general, neopentyl diamidates were more active than the
0.030 ꢀ 0.001
0.004 ꢀ 0.003
0.019 ꢀ 0.013
0.018 ꢀ 0.009
0.009 ꢀ 0.002
0.014 ꢀ 0.004
0.002 ꢀ 0.0006
0.011 ꢀ 0.001
0.012 ꢀ 0.006
0.007 ꢀ 0.005
6.9 ꢀ 6.4
1.2 ꢀ 0.1
4.4 ꢀ 0.6
2.3 ꢀ 1.1
15
6.9 ꢀ 1.7
(ratio 19:1)
16
(ratio 1:5)
17
0.003 ꢀ 0.0006
0.004 ꢀ 0.002
3.9 ꢀ 1.7
0.011 ꢀ 0.0
0.009 ꢀ 0.002
2.0 ꢀ 0.7
GS 7340
0.005b
e
40b
a
In CEM cells.
b
In MT-2 cells [17].

262
F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
Table 2
Anti-DNA virus activity of the test compounds.
MICb
(mM)
a
Compounds
EC50
(
m
M)
HSV-1 (KOS)
HSV-2 (G)
HSV-1 TKꢂ (ACVR KOS)
VV
1
5
6
7
8
9
10
2
11
13
12
14
15
16
17
4.7
6.3
7.0
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.02 ꢀ 0.01
0.7 ꢀ 0.2
0.3 ꢀ 0.07
0.6 ꢀ 0.3
0.25 ꢀ 0.07
0.13 ꢀ 0.09
105
0.008 ꢀ 0.003
0.25 ꢀ 0.07
0.2 ꢀ 0.1
0.15 ꢀ 0.07
0.1 ꢀ 0.1
0.05 ꢀ 0.03
122
0.7 ꢀ 0.2
0.6 ꢀ 0.3
0.3 ꢀ 0.1
0.55 ꢀ 0.4
0.2 ꢀ 0
0.12 ꢀ 0.1
151
7 ꢀ 4.2
0.8 ꢀ 0
2 ꢀ 1.4
1 ꢀ 0
9.5 ꢀ 3.5
5.0 ꢀ 1.7
2.7 ꢀ 1.5
3.7 ꢀ 0.6
1.6 ꢀ 0.7
1.3 ꢀ 0.6
>100
10 ꢀ 3.5
1.3 ꢀ 0.7
1.5 ꢀ 0.7
1.3 ꢀ 0.7
1.5 ꢀ 0.7
2 ꢀ 1.4
3 ꢀ 1.4
20 ꢀ 0
0.7 ꢀ 0.1
0.7 ꢀ 0.1
0.7 ꢀ 0.1
0.7 ꢀ 0.1
0.8 ꢀ 0
5.5 ꢀ 2.1
6 ꢀ 2.1
8 ꢀ 1.4
4 ꢀ 0
2 ꢀ 1.4
2 ꢀ 0
4 ꢀ 0
1.4 ꢀ 0
1.4 ꢀ 0
5.5 ꢀ 2.1
10.5 ꢀ 2.1
a
50% Effective concentration or compound concentration required to inhibit virus-induced cytopathicity by 50%.
Minimal inhibitory concentration or compound concentration required to afford a microscopically visible alteration of HEL cell morphology.
b
The compounds have also been evaluated against DNA virus
values (between 1.2 and 17
contrary, adefovir prodrugs showed submicromolar cytostatic ac-
tivity (IC50 between 0.019 and 1.6 M) (Table 3). An increased
cytostatic activity in the adefovir and tenofovir series is observed
on replacing the phenyl by either the naphthyl or tetrahy-
dronaphthyl groups, with the latter showing a >100-fold increase
in antiproliferative activity when compared to the parent nucleo-
side phosphonate.
mM, Table 3) were observed. On the
replication in HEL cell cultures such as herpes simplex virus type 1
(HSV-1), HSV-2, thymidine kinase-deficient HSV-1 and vaccinia
virus. The prodrugs of PMEA (1) but also of PMPA (2) were invari-
ably more antivirally active than the parent compounds (Table 2).
The PMEA prodrugs showed more antiviral efficacy than those
of PMPA but given the much poorer antiherpetic activity of PMPA
versus PMEA, the antiherpetic improvement for the PMPA prodrugs
was much more pronounced than observed for the PMEA prodrugs.
m
In fact, PMPA as such proved poorly active (EC50: 105e151
its prodrugs (except for 13) were inhibitory in the higher nano-
molar/lower micromolar range for the herpesviruses (0.7e2 M),
and in the lower micromolar range for VV (4e5.5 M). In the PMEA
prodrug series, the EC50 values for antiherpes activity ranged be-
tween 0.12 M and 0.7 M and for VV between 1.3 and 5 M. In
none of the cases, cellular toxicity against the HEL cell monolayers
was noticed at 100 M.
mM), but
3.3. Antiproliferative activity against naturally HPV-transformed
cell lines
m
m
To assess the antiproliferative activity of the phosphonodiami-
dates and protides of adefovir and tenofovir for the potential
treatment of HPV-induced lesions, the prodrugs 12e15, 16, 9 and 7
and the parent ANPs were initially examined in the SiHa (HPV 16þ),
HeLa (HPV 18þ) and C33A (HPVꢂ) cell cultures (Table 4).
m
m
m
m
From the data in Table 4, it can be seen that the adefovir protide
compounds are significantly more cytostatic in comparison to the
parent compounds (1 and 2). Compounds 7 and 9 and the tenofovir
protide compounds 12, 15 and 16 were further examined in cells
derived from normal, neoplastic, and tumour tissue: SiHa cells,
3.2. Cytostatic activity
The in vitro inhibitory effect of all compounds on the prolifera-
tion of murine leukemia cells (L1210), human CD4þ T-lymphocyte
cells (CEM) and human cervix carcinoma cells (HeLa) was also
evaluated. In the tenofovir prodrug series, low micromolar IC50
Table 4
IC50 values of the compounds against HPVþ and HPVꢂ cells.
Table 3
Entry
Compound
IC50 (mM)
Antiproliferative activity (IC50) of adefovir derivatives in different cell lines.
SiHa
HeLa
C33A
a
Compounds
IC50
(m
M)
1
2
3
4
5
6
7
2
12
e
e
e
L1210
CEM
24 ꢀ 11
HeLa
0.73
e
0.54
0.53
6.2
e
0.7
7.6
6
7.8
0.54
0.84
15 (ratio 19:1)
16 (ratio 1:5)
1
9
7
1
2
5
6
7
8
9
10
11
12
13
14
15
16
17
3.0 ꢀ 1.0
11 ꢀ 12
181 ꢀ 166
17 ꢀ 10
7.8
e
6.9 ꢀ 6.4
0.12 ꢀ 0.04
0.14 ꢀ 0.09
0.20 ꢀ 0.04
0.40 ꢀ 0.04
0.045 ꢀ 0.045
0.019 ꢀ 0.009
>500
0.041 ꢀ 0.018
0.21 ꢀ 0.08
0.24 ꢀ 0.01
0.15 ꢀ 0.07
0.068 ꢀ 0.016
0.026 ꢀ 0.009
120 ꢀ 28
1.6 ꢀ 0.7
0.67 ꢀ 0.26
1.6 ꢀ 0.8
2.2 ꢀ 1.5
0.92 ꢀ 0.22
0.45 ꢀ 0.17
>500
0.6
0.66
0.5
0.6
Table 5
IC50 values of 9 and 7 against normal, neoplastic and tumor cell lines.
1.5 ꢀ 0.9
1.2 ꢀ 0.1
4.0 ꢀ 0.1
11 ꢀ 1.0
8.0 ꢀ 3.0
17 ꢀ 1.0
12 ꢀ 1.0
5.3 ꢀ 3.9
Cell line (HPV status)
Cell origin
IC50
9
(mM)
3.7 ꢀ 1.8
4.4 ꢀ 0.6
4.0 ꢀ 0.2
2.3 ꢀ 1.1
7
5.6 ꢀ 2.3
6.9 ꢀ 1.7
2.2 ꢀ 1.3
3.9 ꢀ 1.7
SiHa (HPV16)
M08 (HPV16)
HEK (HPV negative)
Malignant
Neoplastic
Normal
0.09
0.048
0.076
0.83
0.82
0.3
1.3 ꢀ 0.5
2.0 ꢀ 0.7
a
50% Inhibitory concentration.

F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
263
significantly more active in comparison to the parent compounds
from which they were derived. A slightly better activity of the
adefovir derivative was also noticed. However, no preferential
specificity to HPV þ cell lines or transformed cell lines was
observed in this study.
The compounds of the present study warrant further investi-
gation in antiviral and cytotoxic settings.
5. Experimental section
5.1. Chemistry
Tenofovir was obtained from Ningbo Haishu Hobid Imp & Exp
Co., Ltd, Ningbo, China and Adefovir was obtained from Hubei
Maxsource chemical Co., Ltd, Wuhan, China.
The numbering of compounds follows the recommended IUPAC
nomenclature guidelines. The naming of the compounds follows
IUPAC nomenclature and/or standard accepted nomenclature for
nucleoside chemistry.
Fig. 3. Carboxypeptidase Y assay applied on ProTide 10 and monitored by 31P NMR,
25 ꢃC.
All solvents used were anhydrous and used as supplied by
Aldrich. All nucleosides and solid reagents were dried for several
hours under high vacuum over potassium hydroxide. All glassware
was oven-dried at 130 ꢃC for several hours or overnight and
allowed to cool in a desiccator or under a stream of dry nitrogen.
For thin-layer chromatography (TLC), precoated aluminium-backed
plates (60 F-54, 0.2 mm thickness; supplied by E. Merck AG,
Darmstadt, Germany) were used and developed by an ascending
elution method. After solvent evaporation, compounds were
detected by quenching of the fluorescence, at 254 or 336 nm
depending on the compound, upon irradiation with a UV lamp. For
column chromatography: Glass columns were slurry-packed in the
appropriate eluent or pre-adsorbed onto silica gel. Fractions con-
taining the product were identified by TLC and pooled, and the
solvent was removed in vacuo.
M08 vulval keratinocytes and human epidermal keratinocytes
(Table 5). Unfortunately, no specificity to naturally HPV-
transformed cell lines was observed in this study.
3.4. Mechanism of activation of the phosphonamidate derivatives
The metabolic activation of the phosphonamidates is generally
assumed to follow the same two enzymatic steps involved in the
activation of the phosphoramidates [18]. In order to prove that the
tetrahydronaphthyl derivative 10 is activated in a similar manner, a
31P NMR study has been performed to investigate the interaction of
this compound with a carboxypeptidase-type enzyme. Compound
10 was therefore incubated in an NMR tube with carboxypeptidase
Y in acetone-d6 buffered at pH ¼ 7.6 with Trizma. The reaction was
monitored by 31P NMR. The spectra in Fig. 3 show conversion of the
starting material (dP ¼ 23.2, 23.1 ppm) to the corresponding ami-
noacyl phosphonamidate ester (dP ¼ 15.0 ppm). In vivo, the ester is
then believed to undergo PeN cleavage, mediated by a phospho-
namidase or resulting from simple hydrolysis in a more acidic
subcellular compartment, to eventually release the parent drug.
1H, 31P and 13C NMR spectra were recorded in a Bruker Avance
500 spectrometer at 500 MHz, 202 MHz and 125 MHz, respectively
and auto-calibrated to the deuterated solvent reference peak in
case of 1H and 13C NMR and 85% H3PO4 for 31P NMR experiments.
All 31P and 13C NMR spectra were proton-decoupled. Coupling
constants (J) are measured in Hertz. The following abbreviations
are used in the assignment of NMR signals: s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), bs (broad singlet), dd (doublet
of doublet), ddd (doublet of doublet of doublet), dt (doublet of
triplet).
4. Conclusion
Low resolution mass analysis were performed by the service at
the Department of Chemistry, University of Wales, Cardiff, UK, us-
ing electrospray ionization technique (ES).
All analytical high-performance liquid chromatography (HPLC)
experiments were done on a Thermo Fisher Scientific Spectra
System SCM1000 provided with a System Controller SN4000, a
pump Spectra System P4000 and a Spectra UV2000 detector set
In conclusion, we have successfully prepared a family of adefovir
and tenofovir phosphonodiamidates and protides with improved
in vitro anti-HIV activity compared to previously reported data for
the parent ANP. For the first time, a 5,6,7,8-tetrahydro-1-naphthol
unit was introduced as hydrolysable aryl unit in the proTide motif
of ANPs. Phosphonoamidates bearing this structurally innovative
modification displayed an improved antiviral activity compared
with the “common” naphthyl and phenol protide units. This
modification appears to be an excellent candidate for future
improvement of the ProTide motif. The prodrugs obtained were
evaluated in vitro for antiviral (HIV type 1 and 2) and cytostatic
(murine leukemia cells and human T-lymphocyte, human cervix
carcinoma) activity. These data showed that the 5,6,7,8-tetrahydro-
1-naphthol derivative has improved inhibitory activity compared to
the phenol and naphthyl moieties in both a cytostatic and an anti-
HIV context. An enzyme study shows processing by a parallel
mechanism to known aryl entities.
and using a 5
m
M Hypersil GOLD (150 ꢄ 4.6 mm) reverse phase
column, eluting with the indicated mobile phase. The software
used was ChromQuest 5.0. All final compounds were isolated with
purity ꢁ95%.
5.1.1. Procedure A: synthesis of phosphonodiamidates [15]
To a solution of the acyclic nucleoside in dry ACN (10 ml/mmol),
TMSBr (5 eq) was added under argon and the reaction was stirred at
RT overnight. The solvents were removed under reduced pressure
without any contact with air. The residue was dissolved in anhy-
drous Et3N (2 ml/mmol) and pyridine (8 ml/mmol) and some amino
acid ester (4 eq) was added. In a separated flask, Aldrithiol-2 (6 eq)
and Ph3P (6 eq) were dissolved in anhydrous pyridine (10 ml/
mmol) and this solution was immediately added to the reaction.
Prodrug compounds 7 and 9 (adefovir) and compounds 15, 16
and 12 (tenofovir) were also tested for their antiproliferative ac-
tivity against HPV-infected neoplastic cells and were found to be

264
F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
3
The resulting mixture (green solution) was stirred at 50 ꢃC for 3e
5 h, then cooled to room temperature and the solvent completely
evaporated on a rotary evaporator (temp. water bath 40 ꢃC). The
residue was taken up in a mixture of methanol, water, toluene and
hexane (15 mL each) and then transferred into a separating funnel.
The upper phase (toluene and hexane) was removed and the lower
phase (water/methanol) was extracted again with a 1:1 mixture of
toluene/hexane (3 ꢄ 10 mL). The product was then extracted from
the water/methanol phase with 3 dichloromethane washing (30 mL
each). The dichloromethane washing were combined and dried
over MgSO4, filtered and evaporated to dryness.
aa), 20.32 (d, JC-P ¼ 6.2 Hz, CH3-aa) ppm. 31P NMR (202 MHz,
MeOH-d4):
d
24.26, 23.13 ppm. MS (ESþ) m/z: 561 [M þ Hþ].
Reverse-phase HPLC, eluting with H2O/MeOH from 90/100 to 0/100
in 30 min, Flow ¼ 1 mL/min,
l
¼ 263 nm, tR ¼ 15.14 min.
5.1.4. 9-[2-(Bis-(benzyloxy-
L-alaninyl)-phosphonomethoxy)ethyl]
adenine (6)
Following procedure A with adefovir (210 mg, 0.77 mmol) and
benzyloxy- -alaninyl ester p-TSA salt (1.04 g, 3.07 mmol), the
desired product (FP137 ¼ Cf3557) was obtained as an off-white
solid (22%, 82 mg). 1H NMR (500 MHz, MeOH-d4):
8.24 (s, 1H,
L
d
The residue was purified by flash chromatography on silica gel
eluted with DCM/MeOH 95:5 to obtain the desired products.
H-2), 8.19 (s, 1H, H-8), 7.38e7.30 (m, 10H, Ph),5.16, 5.13 (ABq, 2H,
JAB ¼ 12.2 Hz, CH2Ph), 5.13, 5.08 (ABq, 2H, JAB ¼ 2.2 Hz, CH2Ph),
4.37 (t, J ¼ 4.8 Hz, 2H, NeCH2), 3.97 (m, 2H, 2 ꢄ NeCH), 3.82 (t,
J ¼ 5.0 Hz, 2H, OCH2CH2N), 3.72 (dd, JHP ¼ 8.2 Hz, J ¼ 10.2 Hz, 1H,
CH2eP), 3.67 (dd, JHP ¼ 8.9, J ¼ 13.2 Hz, 1H, CH2eP), 1.34 (d,
J ¼ 7.1 Hz, 3H, CH3-aa), 1,28 (d, J ¼ 7.1 Hz, 3H, CH3-aa); 13C NMR
5.1.2. Procedure B: synthesis of phosphonoamidates [15]
To a solution of the acyclic nucleoside in dry ACN (10 ml/mmol),
TMSBr (5 eq) was added under argon and the reaction was stirred at
RT overnight. Then the solvents were removed under reduced
pressure without any contact with air. The residue was dissolved in
anhydrous Et3N (2 ml/mmol) and pyridine (8 ml/mmol) and some
amino acid ester (1.5 eq) and aryl alcohol (1.5 eq) were added. In a
separated flask, Aldrithiol-2 (6 eq) and Ph3P (6 eq) were dissolved
in anhydrous pyridine (10 ml/mmol) and this solution was imme-
diately added to the reaction. The resulting mixture (green solu-
tion) was stirred at 50 ꢃC for 3 h.
The residue was taken up in a mixture of methanol, water,
toluene and hexane (15 mL each) and then transferred into a
separating funnel. The upper phase (toluene and hexane) was
removed and the lower phase (water/methanol) was extracted
again with a 1:1 mixture of toluene/hexane (3 ꢄ 10 mL). The
product was then extracted from the water/methanol phase with 3
dichloromethane washing (30 mL each). The dichloromethane
washing were combined and dried over MgSO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography on silica gel eluted with DCM/MeOH 95:5 to obtain the
desired products.
(125 MHz, MeOH-d4):
d
175.41 (d, 3JC-P ¼ 3.9 Hz, C]O), 175.37 (d,
3JC-P ¼ 2.6 Hz, C]O), 156.82 (C-6), 153.02 (CH-2), 150.71 (C-4),
143.71 (CH-8), 137.35 (C-ipso Ph), 137.33 (C-ipso Ph), 129.64, 129.61,
129.42, 129.40, 129.36 (Ph), 119.92 (C-5), 72.22 (d, 2JC-P ¼ 13.2 Hz,
OCH2CH2N), 68.55 (d, 1JC-P ¼ 135.1 Hz, PeCH2), 67.99, 67.93
(OCH2Ph), 50.21, 49.88 (NeCH), 44.65 (NeCH2), 21.14 (d, 3JC-
P ¼ 4.9 Hz, CH3-aa), 20.95 (d, 3JC-P ¼ 6.2 Hz, CH3-aa) ppm; 31P
NMR (202 MHz, MeOH-d4):
[M þ H]. Reverse-phase HPLC, eluting with H2O/MeOH from 90/10
to 0/100 in 30 min, Flow ¼ 1 mL/min, ¼ 263 nm, tR ¼ 19.44 min.
d
23.10 ppm. MS (ESþ) m/z: 596
d
5.1.5. 9-[2-(Bis-(2,2-dimethylpropoxy-L-alaninyl)-
phosphonomethoxy)ethyl]adenine (7)
Following procedure A with adefovir (200 mg, 0.73 mmol) and
2,2-dimethylpropoxy- -alaninyl ester p-TSA salt (970 mg,
2.93 mmol), the desired product was obtained as an off-white solid
(64%, 273 mg). 1H NMR (500 MHz, MeOH-d4):
8.21 (s, 2H, H-2, H-
L
d
8), 4.44 (m, 2H, NeCH2), 3.97 (m, 2H, 2 ꢄ NeCH), 3.94 (m, 2H,
OCH2CH2N), 3.90, 3.79 (AB, 2H, JAB ¼ 11.0 Hz, OCH2C(CH3)3), 3.86,
3.73 (AB, 2H, JAB ¼ 11.0 Hz, OCH2C(CH3)3), 3.78 (dd, JHP ¼ 8.2 Hz,
J ¼ 13.7 Hz, 1H, CH2eP), 3.74 (dd, JHP ¼ 9.3 Hz, J ¼ 13.9 Hz, 1H, CH2e
P), 1.36 (d, J ¼ 7.2 Hz, 3H, CH3-aa), 1.34 (d, J ¼ 7.3 Hz, 3H, CH3-aa),
0.95 (s, 9H, C(CH3)3), 0.94 (s, 9H, C(CH3)3) ppm; 13C NMR (125 MHz,
5.1.3. 9-[2-(Naphthyloxy-(benzyloxy-
phosphonomethoxy)ethyl]adenine (5)
Following procedure B with adefovir (400 mg, 1.46 mmol), 2,2-
dimethylpropoxy- -alaninyl ester p-TSA salt (790.42 mg,
L-alaninyl)-
3
3
L
MeOH-d4):
d
175.69 (d, JC-P ¼ 5.2 Hz, C]O), 175.62 (d, JC-
2.34 mmol) and phenol (337.75 mg, 2.34 mmol), the title com-
¼ 3.9 Hz, C]O), 157.26 (C-6), 153.70 (CH-2), 150.73 (C-4), 143.47
P
pound was obtained as an off-white solid (55%, 450 mg). 1H NMR
(CH-8), 119.91 (C-5), 75.42, 75.34 (OCH2C(CH3)3), 72.26 (d,
3J ¼ 13.2 Hz, OCH2CH2N), 68.51 (d, 1JC-P ¼ 136.3 Hz, CH2eP), 50.20,
49.78 (NeCH), 44.62 (NeCH2), 32.31, 32.29 (C(CH3)33), 26.76, 26.74
(C(CH3)3), 21.39 (d, 3JC-P ¼ 4.8 Hz, CH3-aa), 21.26 (d, JC-P ¼ 5.8 Hz,
(500 MHz, MeOH-d4): d 8.18 (s,1H, H-2), 8.16 (s,1H, H-2), 8.10 (s,1H,
H-8), 8.07 (s, 1H, H-8), 8.01 (d, J ¼ 10.0 Hz, 1H, Naph), 7.98 (d,
J ¼ 10.0 Hz, 1H, Naph), 7.87 (d J ¼ 10.0 Hz, 2H, Naph), 7.69 (d,
J ¼ 5.0 Hz, 1H, Naph), 7.68 (d, J ¼ 5.0 Hz, 2H, Naph), 7.53e7.50 (m,
2H, Naph), 7.47e7.45 (m, 2H, Naph), 7.39e7.31 (m, 4H, Naph), 7.30e
7.27 (m,10H, CH2Ph), 5.08, 5.02 (ABq, 2H, JAB ¼ 12.2 Hz, CH2Ph), 5.03
(s, 2H, CH2Ph), 4.40e4.35 (m, 4H, NeCH2), 4.10e3.97 (m, 6H, CH2e
P, NeCH), 3.92e3.88 (m, 4H, OCH2CH2N), 1.23 (d, J ¼ 7.0 Hz, 3H,
CH3-aa), 1.19 (d, J ¼ 7.0 Hz, 3H, CH3-aa) ppm. 13C NMR (125 MHz,
CH3-aa) ppm. 31P NMR (202 MHz, MeOH-d4): 23.19 ppm. MS (ESþ)
d
m/z: 578.28 [M þ Na]. Reverse-phase HPLC, eluting with H2O/
MeOH from 90/10 to 0/100 in 30 min, Flow ¼ 1 mL/min,
tR ¼ 15.44 min.
l
¼ 263 nm,
5.1.6. 9-[2-(Phenyloxy-(2,2-dimethylpropoxy-L-alaninyl)-
phosphonomethoxy)ethyl]adenine (8)
MeOH-d4):
d
175.31 (d, 3JC-P ¼ 3.7 Hz, C]O),174.97 (d, 3JC-P ¼ 4.2 Hz,
C]O), 157.27, 157.25 (C-6), 153.73, 153.69 (CH-2), 150.65 (C-4),
147.45 (d, 2JC-P ¼ 8.7 Hz, C-ipso ONaph), 147.40 (d, 2JC-P ¼ 8.7 Hz, C-
ipso ONaph), 143.19 (CH-8), 137.57 (C-ipso CH2Ph), 137.33 (C-ipso
CH2Ph), 136.36 (C-Naph), 129.56, 129.33, 129.31 (CH-Ph), 128.83,
128.77 (CH-Naph), 128.08 (d, 3JC-P ¼ 4.2 Hz, C-Naph), 127.98 (d, 3JC-
Following procedure B with adefovir (218 mg, 0.80 mmol), 2,2-
dimethylpropoxy- -alaninyl ester p-TSA salt (423.16 mg,
L
1.28 mmol) and phenol (120.16 mg, 1.28 mmol), the title compound
was obtained as an off-white solid (25%, 100 mg). 1H NMR
(500 MHz, MeOH-d4):
d 8.21 (s, 1H, H-2), 8.20 (s, 1H, H-2), 8.18 (s,
¼
5.0 Hz, C-Naph), 127.47, 127.39, 126.45, 126.42, 125.98,
1H, H-8), 8.16 (s, 1H, H-8), 7.30e7.22 (m, 4H, Ph), 7.14 (t, J ¼ 8.2 Hz,
2H, Ph), 7.12 (t, J ¼ 8.1 Hz, 2H, Ph), 7.06 (d, J ¼ 8.1 Hz, 2H, Ph), 4.43
(m, 4H, NeCH2), 4.07e3.85 (m, 10H, CH2eP, NeCH, OCH2CH2N),
3.81, 3.71 (AB, JAB ¼ 10.0 Hz, 2H, OCH2C(CH3)3), 3.80, 3.70 (AB,
JAB ¼ 10.0 Hz, 2H, OCH2C(CH3)3), 1.29 (d, J ¼ 7.3 Hz, 3H, CH3-aa), 1.28
(d, J ¼ 7.3 Hz, 3H, CH3-aa), 0.95 (s, 9H, C(CH3)3), 0.94 (s, 9H, C(CH3)3)
P
125.92 (CH-Naph), 122.84, 122.57 (CH-Naph), 119.98, 119.92 (C-5),
3
3
116.82 (d, JC-P ¼ 3.7 Hz, CH-Naph), 116.68 (d, JC-P ¼ 3.7 Hz, CH-
3
3
Naph), 72.46 (d, JC-P ¼ 13.7 Hz, OCH2CH2N), 72.35 (d, JC-
1
¼ 13.7 Hz, OCH2CH2N), 67.97, 67.87 (OCH2Ph), 67.78 (d, JC-
P
1
¼ 155.0 Hz, CH2eP), 68.29 (d, JC-P ¼ 153.7 Hz, CH2eP), 50.98,
P
50.92 (NeCH), 44.64, 44.58 (NeCH2), 20.81 (d, 3JC-P ¼ 5.0 Hz, CH3-
ppm. 13C NMR (125 MHz, MeOH-d4):
d
175.33 (d, 3JC-P ¼ 4.0 Hz, C]

F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
265
3
O), 175.09 (d, JC-P ¼ 4.0 Hz, C]O), 157.32,157.30 (C-6), 153.76,
OTetrahydronaph), 146.43, 146.41 (CH-2), 145.71, 145.67 (CH-8),
2
2
3
153.72 (CH-2), 151.50 (d, JC-P ¼ 9.0 Hz, C-ipso OPh), 151.41 (d, JC-
140.70 (C-Tetrahydronaph), 129.90 (d, JC-P ¼ 6.2 Hz, C-Tetrahy-
3
¼ 9.0 Hz, C-ipso OPh), 150.75, 150.73 (C-4), 143.41, 143.38 (CH-8),
dronaph), 129.76 (d, JC-P ¼ 6.2 Hz, C-Tetrahydronaph), 126.86,
P
3
130.76, 130.74, 126.17, 126.11 (Ph), 121.94 (d, JC-P ¼ 4.5 Hz, Ph),
126.82, 126.79, 126.76 (CH-Tetrahydronaph), 119.68, 119.66 (C-5),
3
3
121.76 (d, JC-P ¼ 4.5 Hz, Ph), 119.99, 119.95 (C-5), 75.40, 75.37
118.26 (d, JC-P
¼
3.4 Hz, CH-Tetrahydronaph), 75.42, 75.40
3
3
(OCH2C(CH3)3), 72.37 (d, JC-P ¼ 11.9 Hz, OCH2CH2N), 72.26 (d, JC-
(OCH2C(CH3)3)), 72.22 (d, 3JC-P ¼ 12.5 Hz, OCH2CH2N), 72.20 (d, 3JC-
¼ 12.9 Hz, OCH2CH2N), 67.51 (d, 1JC-P ¼ 155.3 Hz, CH2eP), 67.48 (d,
¼ 12.5 Hz, OCH2CH2N), 67.70 (d, 1JC-P ¼ 153.7 Hz, CH2eP), 50.95,
P
P
1JC-P ¼ 155.3 Hz, CH2eP), 51.02, 50.92 (NeCH), 44.69, 44.65 (Ne
CH2), 32.34 (C(CH3)3), 26.77, 26.74 (C(CH3)3), 21.26 (d, 3JC-P ¼ 5.3 Hz,
CH3-aa), 20.74 (d, 3JC-P ¼ 5.3 Hz, CH3-aa) ppm. 31P NMR (202 MHz,
50.93 (NeCH), 45.15, 45.16 (NeCH2), 32.37, 32.36 (C(CH3)3), 30.52
(CH2-Tetrahydronaph), 26.81 (C(CH3)3), 24.60, 24.58, 23.88, 23.85,
23.76 (CH2-Tetrahdronaph), 21.40 (d, 3JC-P ¼ 5.0 Hz, CH3-aa), 20.81
3
MeOH-d4):
d
23.71, 22.68 ppm. MS (ESþ) m/z: 513.49 [M þ Naþ].
(d, JC-P ¼ 6.2 Hz, CH3-aa) ppm. 31P NMR (202 MHz, MeOH-d4):
Reverse-phase HPLC, eluting with H2O/MeOH from 90/100 to 0/100
d
23.41, 22.47 ppm. MS (ESþ) m/z: 545.2 [M þ Hþ]. Reverse-phase
in 30 min, Flow ¼ 1 mL/min,
l
¼ 263 nm, tR ¼ 15.74 min.
HPLC, eluting with H2O/MeCN from 90/10 to 40/60 in 15 min,
then 40/60 isocratic for 15 min then 40/60 to 0/100 in 10 min,
5.1.7. 9-[2-( -Napthyloxy-(2,2-dimethylpropoxy-
d
L
-alaninyl)-
Flow ¼ 1 mL/min,
l
¼ 263 nm, tR ¼ 26.7 min; 27.6 min.
phosphonomethoxy)ethyl]adenine. (9)
Following procedure B with adefovir (210 mg, 0.76 mmol), 2,2-
dimethylpropoxy- -alaninyl ester p-TSA salt (384 mg, 1.16 mmol),
5.1.9. 9-[(R)-2-(bis-(benzyloxy-L-alaninyl)-phosphonomethoxy)
propyl]adenine (11)
L
and naphthol (167 mg, 1.16 mmol), the desired product (9) was
Following procedure A with tenofovir (200 mg, 0.69 mmol) and
benzyloxy- -alaninyl ester p-TSA salt (970 mg, 2.76 mmol), the
desired product 11 was obtained as an off-white solid (75%,
0.320 g). 1H NMR (500 MHz, MeOH-d4):
8.22 (s, 1H, H-2), 8.17 (s,
obtained as an off-white solid (45%, 189 mg). 1H NMR (500 MHz,
L
MeOH-d4): d 8.19 (s, 1H, H-2), 8.16 (s, 1H, H-8), 8.13 (s, 1H, H-8), 8.11
(s, 1H, H-8), 8.03 (d, J ¼ 10.0 Hz, 1H, Naph), 8.00 (d, J ¼ 10.0 Hz, 1H,
Naph), 7.87 (d, J ¼ 10 Hz, 2H, Naph), 7.69 (d, J ¼ 5.0 Hz, 1H, Naph),
7.68 (d, J ¼ 5.0 Hz, 1H, Naph), 7.54e7.51 (m, 2H, Naph), 7.48e7.35
(m, 2H, Naph) 7.32e7.34 (m, 4H, Naph), 4.43 (m, 4H, NeCH2), 4.16e
4.02 (m, 10H, CH2eP, NeCH, OCH2CH2N), 3.80, 3.66 (AB,
JAB ¼ 10.5 Hz, 2H, OCH2C(CH3)3), 3.76, 3.68 (AB, JAB ¼ 10.5 Hz, 2H,
OCH2C(CH3)3), 1.25 (d, J ¼ 6.9 Hz, 3H, CH3-aa), 1.23 (d, J ¼ 7.1 Hz, 3H,
d
1H, H-8), 7.37e7.29 (m, 10H, 2 ꢄ OeCH2Ph), 5.19, 5.15 (AB,
JAB ¼ 12.0 Hz, 1H, OCH2Ph), 5.08, 5.02 (AB, JAB ¼ 12.5 Hz, 1H,
OCH2Ph), 4.32 (dd, 1H, J ¼ 14.5, 3.1 Hz, 1H, NeCH2), 4.14 (dd, 1H,
J ¼ 14.5, 7.1 Hz, NeCH2), 4.02e3.98 (m, 1H, NeCH), 3.97e3.92 (m,
1H, NeCH), 3.86e3.80 (m, 1H, OeCH), 3.77 (dd, J ¼ 13.0 Hz,
JHP ¼ 7.7 Hz, 1H, PeCH2), 3.48 (dd, J ¼ 13.0 Hz, JHP ¼ 10.9 Hz, 1H, Pe
CH2), 1.37 (d, J ¼ 7.2 Hz, 3H, CH3-aa), 1.25 (d, J ¼ 7.1 Hz, 3H, CH3-aa),
1.12 (d, J ¼ 6.2 Hz, 3H, CH3-tenofovir) ppm. 13C NMR (125 MHz,
CH3-aa), 0.90 (s, 9H, C(CH3)3), 0.88 (s, 9H, C(CH3)3) ppm. 13C NMR
3
(125 MHz, MeOH-d4):
d
175.31 (d, JC-P ¼ 3.7 Hz, C]O), 174.97 (d,
3JC-P ¼ 4.2 Hz, C]O), 157.27, 157.25 (C-6), 153.74, 153.68 (CH-2),
150.74, 150.71 (C-4), 147.45, 147.40 (d, 2JC-P ¼ 8.7 Hz, C-ipso ONaph),
143.23, 143.20 (CH-8), 136.29 (C-Naph), 128.84, 128.80 (CH-Naph),
128.08 (d, 3JC-P ¼ 4.2 Hz, C-Naph), 127.98 (d, 3JC-P ¼ 5.0 Hz, C-Naph),
127.76, 127.45, 127.41, 126.48, 126.45, 125.99, 125.94 (CH-Naph),
3
3
MeOH-d4):
d
175.42 (d, JC-P ¼ 5.3 Hz, C]O), 175.35 (d, JC-
¼ 2.9 Hz, C]O), 157.25 (C-6), 153.70 (C-2), 150.99 (C-4), 143.92 (C-
P
8), 137.33 (C-ipso Ph), 137.24 (C-ipso Ph), 129.54, 129.36, 129.32,
3
129.30 (Ph), 119.79 (C-5), 77.52 (d, JC-P ¼ 13.0 Hz, OeCH), 67.97,
67.89 (2 ꢄ OeCH2Ph), 66.07 (d, 1JC-P ¼ 137.1 Hz, PeCH2), 50.18, 49.71
(2 ꢄ NeCH), 49.19 (NeCH2), 21.08 (d, 3JC-P ¼ 4.7 Hz, CH3-aa), 20.81
3
122.82, 122.62 (CH-Naph), 119.96, 119.94 (C-5), 116.84 (d, JC-
3
¼ 3.9 Hz, CH-Naph), 116.70 (d, JC-P ¼ 3.7 Hz, CH-Naph), 75.39,
P
3
(d, JC-P ¼ 6.3 Hz, CH3-aa), 16.61 (CH3-tenofovir) ppm. 31P NMR
3
75.35 (OCH2C(CH3)3), 72.22 (d, JC-P ¼ 12.5 Hz, OCH2CH2N), 72.20
(202 MHz, MeOH-d4):
d
23.75 ppm. MS (ESþ) m/z: 632 (M þ Naþ,
(d, JC-P ¼ 12.5 Hz, OCH2CH2N), 67.78 (d, 1JC-P ¼ 155.3 Hz, CH2eP),
3
100%), 610 (M þ Hþ, 46%). Reverse-phase HPLC, eluting with H2O/
67.72 (d, 1JC-P ¼ 154.5 Hz, CH2eP), 50.95, 49.90 (NeCH), 44.69, 44.61
ACN from 90/100 to 0/100 in 25 min, Flow ¼ 1 mL/min,
tR ¼ 13.37 min.
l
¼ 254 nm,
3
(NeCH2), 32.37, 32.36 (C(CH3)3), 26.81 (C(CH3)3), 21.18 (d, JC-
¼ 5.2 Hz, CH3-aa), 20.66 (d, 3JC-P ¼ 6.2 Hz, CH3-aa), ppm. 31P NMR
P
(202 MHz, MeOH-d4):
d
24.29, 23.34 ppm. MS (ESþ) m/z: 541
[M þ Hþ]. Reverse-phase HPLC, eluting with H2O/MeOH from 90/
5.1.10. 9-[(R)-2-(Bis-(2,2-dimethylpropoxy-L-alaninyl)-
phosphonomethoxy)propyl]adenine (12)
100 to 0/100 in 30 min, Flow ¼ 1 mL/min,
l
¼ 263 nm, tR ¼ 15.8 min.
Following procedure A with tenofovir (200 mg, 0.69 mmol) and
neopentyloxy-L-alaninyl ester p-TSA salt (928 mg, 2.76 mmol). The
desired product 12 was obtained as an off-white solid (76%,
5.1.8. 9-[2-(5,6,7,8-Tetrahydro-1-naphthyloxy-(2,2-
dimethylpropoxy-L-alaninyl)-Phosphonomethoxy)ethyl]
adenine (10)
0.298 g). 1H NMR (500 MHz, MeOH-d4):
d 8.27 (s, 1H, H-2), 8.24 (s,
Following procedure B with adefovir (230 mg, 0.84 mmol), 2,2-
dimethylpropoxy- -alaninyl ester p-TSA salt (421 mg, 1.27 mmol),
1H, H-8), 4.43 (dd, J ¼ 14.5, 3.1 Hz, 1H, NeCH2), 4.25 (dd, J ¼ 14.6,
7.3 Hz, 1H, NeCH2), 4.05e3.93 (m, 3H, 2 ꢄ NeCH, OeCH), 3.94, 3.82
(AB, JAB ¼ 10.0 Hz, 2H, OCH2C(CH3)3), 3.86 (dd, J ¼ 12.5 Hz,
JHP ¼ 7.5 Hz, 2H, PeCH2), 3.78, 3.67 (AB, JAB ¼ 10.0 Hz, 2H,
OCH2C(CH3)3), 3.58 (dd, J ¼ 12.8 Hz, JHP ¼ 11.0 Hz, 2H, PeCH2), 1.43
(d, J ¼ 7.2 Hz, 3H, CH3-aa), 1.36 (d, J ¼ 7.2 Hz, 3H, CH3-aa), 1.23 (d,
L
and tetrahydronaphthol (188 mg, 1.27 mmol), the desired product
(FP137 ¼ Cf3476) was obtained as an off-white solid (8%, 35 mg). 1H
NMR (500 MHz, MeOH-d4): d 8.22 (s, 1H, H-2), 8.20 (s, 1H, H-8), 8.17
(s, 1H, H-2), 8.15 (s, 1H, H-8), 7.06e6.94 (m, 4H, CH-tetrahy-
dronaph), 6.86 (t, J ¼ 6.4 Hz, 2H, CH-tetrahydronaph), 4.45 (m, 4H,
NeCH2), 4.09e3.92 (m, 10H, CH2eP, NeCH, OCH2CH2N), 3.83, 3.73
(AB, JAB ¼ 11.0 Hz, 2H, OCH2C(CH3)3), 3.81, 3.71 (AB, JAB ¼ 9.0 Hz, 2H,
OCH2C(CH3)3), 2.76e2.71 (m, 4H, CH2-tetrahydronaph), 2.57e2.52
(m, 4H, CH2-tetrahydronaph), 1.76e1.75 (m, 8H, CH2-tetrahy-
dronaph),1.31 (d, J ¼ 6.9 Hz, 3H, CH3-aa),1.30 (d, J ¼ 7.1 Hz, 3H, CH3-
J ¼ 6.3 Hz, 3H, CH3-tenofovir), 0.99 (s, 9H, C(CH3)3), 0.94 (s, 9H,
3
C(CH3)3) ppm. 13C NMR (125 MHz, MeOH-d4):
d
175.77 (d, JC-
3
¼ 4.6 Hz, C]O), 175.35 (d, JC-P ¼ 2.7 Hz, C]O), 157.28 (C-6),
P
3
153.70 (C-2), 151.07 (C-4), 144.04 (C-8), 119.82 (C-5), 77.69 (d, JC-
1
¼ 13.3 Hz, OeCH), 75.50, 75.36 (2 ꢄ OeCH2), 66.12 (d, JC-
P
¼ 136.2 Hz, PeCH2), 50.26, 49.69 (2 ꢄ NeCH), 48.53 (NeCH2),
P
aa), 0.93 (s, 9H, C(CH3)3), 0.92 (s, 9H, C(CH3)3) ppm. 13C NMR
32.37, 32.27 (C(CH3)3)), 26.79, 26.76 (C(CH3)3)), 21.37 (d, JC-
3
3
3
(125 MHz, MeOH-d4):
d
175.40 (d, JC-P ¼ 3.8 Hz, C]O), 175.05 (d,
¼ 5.0 Hz, CH3-aa), 21.15 (d, JC-P ¼ 6.2 Hz, CH3-aa), 16.70 (CH3-
P
3JC-P ¼ 4.2 Hz, C]O), 152.44 (C-6), 150.5 (C-4), 149.80 (d, JC-
tenofovir) ppm. 31P NMR (202 MHz, MeOH-d4):
d
23.91 ppm. MS
2
¼ 6.2 Hz, C-ipso OTetrahydronaph), 149.70 (d, 2JC-P ¼ 6.2 Hz, C-ipso
(ESþ) m/z: 570 (M þ Naþ). Reverse-phase HPLC, eluting with H2O/
P

266
F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
MeOH from 90/100 to 0/100 in 30 min, Flow ¼ 1 mL/min,
¼ 254 nm, tR ¼ 26.04 min.
Reverse-phase HPLC, eluting with H2O/ACN from 90/100 to 0/100 in
30 min, Flow ¼ 1 mL/min, ¼ 254 nm, tR ¼ 15.15 min.
l
l
5.1.13. 9-[(R)-2-(naphtyloxy-(2,2-dimethylpropoxy-L-alaninyl))-
phosphonomethoxy)propyl]adenine (15)
5.1.11. 9-[(R)-2-(naphtyloxy-(benzyloxy-L-alaninyl))-
phosphonomethoxy)propyl]adenine (13)
Reaction was carried out following procedure B with tenofovir
(200 mg, 0.69 mmol), neopentyloxy- -alaninyl ester p-TSA salt
Following procedure B with tenofovir (200 mg, 0.69 mmol),
benzyloxy- -alaninyl ester p-TSA salt (364 mg, 1.03 mmol) and 1-
L
L
(228 mg, 0.69 mmol) and 1-naphthol (100 mg, 0.69 mmol). Puri-
fication by column chromatography allows isolation of compound
15 (27 mg, ratio SP : Rp : 19:1, 7%) as off-white solid: 1H NMR
(500 MHz, MeOH-d4, only the major diastereoisomer is described):
naphthol (149 mg, 1.03 mmol), the title compound 13 was obtained
as an off-white solid (120 mg, ratio: 1:1, yield: 30%). 1H NMR
(500 MHz, MeOH-d4): d 8.17 (s,1H, H-2), 8.13 (s,1H, H-2), 8.10 (s,1H,
H-8), 8.06 (s, 1H, H-8), 8.03 (d, 1H, J ¼ 8.0 Hz, Naph), 7.99 (d, 1H,
J ¼ 8.0 Hz, Naph), 7.88 (d, 2H, J ¼ 8.0 Hz, Naph), 7.71e7.68 (m, 2H,
Naph), 7.55e7.51 (m, 2H, Naph), 7.50e7.40 (m, 5H, Naph), 7.33e7.29
(m, 11H, Naph, Ph), 5.07, 5.00 (AB, JAB ¼ 12.0 Hz, 2H, OCH2Ph), 5.04
(s, 2H, OCH2Ph), 4.34 (dd, 1H, J ¼ 9.5 Hz, 3.0 Hz, NeCH2), 4.31 (dd,
1H, J ¼ 10 Hz, 3.0 Hz, NeCH2), 4.19 (dd, 1H, J ¼ 9.0 Hz, 7.0 Hz, Ne
CH2), 4.16 (dd, 1H, J ¼ 7.5 Hz, 5.5 Hz, NeCH2), 4.12e4.06 (m, 4H, Pe
CH2, OeCH), 4.02e3.99 (m, 1H, NeCH), 3.95 (dd, 1H, J ¼ 13.0 Hz,
JHP ¼ 9.5 Hz, PeCH2), 3.94e3.91 (m, 1H, NeCH), 3.85 (dd, 1H,
J ¼ 13.5 Hz, JHP ¼ 9.5 Hz, PeCH2),1.25 (d, 3H, J ¼ 7.0 Hz, CH3-aa),1.23
(d, 3H, J ¼ 7.0 Hz, CH3-aa),1.18 (d, 3H, J ¼ 6.5 Hz, CH3-tenofovir),1.08
(d, 3H, J ¼ 6.5 Hz, CH3-tenofovir) ppm. 13C NMR (125 MHz, MeOH-
d
8.16 (s, 1H, H-2), 8.11 (s, 1H, H-8), 8.05 (d, 1H, J ¼ 8.5 Hz, Naph),
7.87 (d, 1H, J ¼ 8.0 Hz, Naph) 7.70e7.69 (m,1H, Naph), 7.54e7.46 (m,
2H, Naph), 7.42e7.41 (m, 2H, Naph), 4.39 (dd, J ¼ 14.6, 3.2 Hz, 1H,
NeCH2), 4.23 (dd, J ¼ 14.6, 6.9 Hz, 1H, NeCH2), 4.14 (dd, 1H,
J ¼ 13.0 Hz, J HP ¼ 9.0 Hz, PeCH2), 4.10, 4.07 (m, 1H, OeCH), 4.05e
4.01 (m, 1H, NeCH), 3.94 (dd, 1H, J ¼ 13.0 Hz, J HP ¼ 9.0 Hz, PeCH2),
3.79, 3.67 (AB, 2H, JAB ¼ 10.5 Hz, OeCH2), 1.24 (d, J ¼ 7.2 Hz, 3H,
CH3-aa),1.12 (d, J ¼ 6.3 Hz, 3H, CH3-tenofovir), 0.90 (s, 9H, -C(CH3)3)
ppm. 13C NMR (125 MHz, MeOH-d4, only the major diastereoisomer
is described):
d
175.34 (d, 3JC-P ¼ 3.5 Hz, C]O), 157.20 (C-6), 153.65
(C-8), 150.98 (C-4), 147.48 (d, 2JC-P ¼ 5.5 Hz, C-ipso Naph), 143.69 (C-
2), 136.30 (C-Naph), 128.80 (CeH Naph), 128.07 (d, J ¼ 5.4 Hz, C-
Naph), 127.78, 127.33, 126.47, 125.98, 122.98 (CeH Naph), 119.77 (C-
5), 116.80 (d, 3JC-P ¼ 3.7 Hz, CH-Naph), 78.98 (d, 3JC-P ¼ 13.3 Hz, Oe
CH), 75.43 (OeCH2), 65.44 (d, 1JC-P ¼ 156.3 Hz, PeCH2), 50.96 (Ne
CH), 48.89 (NeCH2), 32.30 (C(CH3)3), 26.75 (C(CH3)3), 20.57 (d, 3JC-
d4):
d
174.99 (d, 3JC-P ¼ 3.6 Hz, C]O), 174.70 (d, 3JC-P ¼ 3.6 Hz, C]O),
2
157.19 (C-6), 153.70, 153.63 (C-8), 150.95 (C-4), 147.50 (d, JC-
¼ 3.6 Hz, C-ipso ONaph), 147.43 (d, 2JC-P ¼ 3.6 Hz, C-ipso ONaph),
P
143.67, 143.47 (C-2), 137.19, 137.18 (C-ipso CH2Ph), 136.27, 129.55,
129.33, 129.32, 128.78, 128.05, 127.73, 127.41, 127.28, 126.43, 126.42,
125.94, 125.88,122.95,122.67 (Naph, Ph),119.73 (C-5), 116.74 (d, 3JC-
¼ 6.3 Hz, CH3-aa), 16.74 (CH3-tenofovir) ppm. 31P NMR (202 MHz,
P
MeOH-d4, only the major diastereoisomer is described): d 24.90 ppm.
¼ 4.5 Hz, C-2 Naph), 116.66 (d, 3JC-P ¼ 5.0 Hz, C-2 Naph), 77.88 (d,
P
MS (ESþ) m/z: 577 (M þ Naþ). Reverse-phase HPLC, eluting with
H2O/MeOH from 90/100 to 0/100 in 30 min, Flow ¼ 1 mL/min,
3JC-P ¼ 13.7 Hz, OeCH), 77.63 (d,3JC-P ¼ 13.1 Hz, OeCH), 68.00, 67.89
1
1
(OeCH2Ph), 65.51 (d, JC-P ¼ 157.2 Hz, PeCH2), 65.35 (d, JC-
l
¼ 254 nm, tR ¼ 22.45 min.
¼ 156.7 Hz, PeCH2), 50.97 (d, J ¼ 4.5 Hz, NeCH), 49.18 (NeCH2),
P
Further elution afforded 16 (16 mg, ratio SP : Rp 1:5, 4%) as an off-
3
3
20.88 (d, JC-P ¼ 5.3 Hz, CH3-aa), 20.22 (d, JC-P ¼ 6.2 Hz, CH3-aa),
white solid: 1H NMR (500 MHz, MeOH-d4, only the major diaste-
reoisomer is described):
16.79, 16.60 (CH3-tenofovir) ppm. 31P NMR (202 MHz, MeOH-d4):
d 8.17 (s, 1H, H-2), 8.12 (s, 1H, H-8), 7.96 (d,
d
24.89, 23.60 ppm. MS (ESþ) m/z: 575 (M þ Hþ). Reverse-phase
J ¼ 8.1 Hz, 1H, Naph), 7.86 (d, J ¼ 8.0 Hz, 1H, Naph), 7.68e7.66 (m,
1H, Naph), 7.53e7.44 (m, 2H, Naph), 7.36e7.34 (m, 2H, Naph), 4.36
(dd, J ¼ 14.6, 3.2 Hz, 1H, NeCH2), 4.22 (dd, J ¼ 14.6, 7.2 Hz, 1H, Ne
CH2), 4.13e4.03 (m, 3H, PeCH2, NeCH, OeCH), 4.06 (dd,
JHP ¼ 10.0 Hz, J ¼ 12.5 Hz, 1H, PeCH2) 3.76, 3.68 (AB, JAB ¼ 10.5 Hz,
2H, OeCH2), 1.26 (d, J ¼ 7.2 Hz, 3H, CH3-aa), 1.22 (d, J ¼ 6.2 Hz, 3H,
CH3-tenofovir), 0.88 (s, 9H, C(CH3)3) ppm. 13C NMR (125 MHz,
HPLC, eluting with H2O/MeOH from 90/100 to 0/100 in 30 min,
Flow ¼ 1 mL/min,
l
¼ 254 nm, tR ¼ 16.29 min.
5.1.12. 9-[(R)-2-(Phenyloxy-(2,2-dimethylpropoxy-L-alaninyl))-
phosphonomethoxy)propyl]adenine (14)
Following procedure B with tenofovir (200 mg, 0.69 mmol),
neopentyloxy- -alaninyl ester p-TSA salt (343 mg, 1.03 mmol) and
L
MeOH-d4, only the major diastereoisomer is described): d 174.95 (d,
phenol (97 mg, 1.03 mmol), the title compound 14 was obtained as
3JC-P ¼ 3.9 Hz, C]O), 155.34 (C-6), 150.94 (C-8), 150.79 (C-4), 147.39
an off-white solid (37 mg, ratio: 2:1, yield: 10%). 1H NMR (500 MHz,
(d, JC-P ¼ 9.7 Hz, C-ipso Naph), 144.35 (C-2), 136.27 (C-Naph),
2
MeOH-d4):
d
8.23 (s, 1H, H-2), 8.22 (s, 1H, H-8), 8.20 (s, 1H, H-2),
128.82 (CH-Naph), 128.00 (d, JCP ¼ 6.2 Hz, C-Naph), 127.74, 127.38,
3
8.19 (m, 1H, H-8), 7.35e7.32 (m, 2H, Ph), 7.29e7.25 (m, 2H, Ph),
7.20e7.12 (m, 4H, Ph), 7.00 (d, 2H, J ¼ 8.5 Hz, Ph), 4.42 (dd, 1H,
J ¼ 14.5, 3.5 Hz, 1H, NeCH2), 4.38 (dd, 1H, J ¼ 15.0, 3.5 Hz, 1H, Ne
CH2), 4.27 (dd, 1H, J ¼ 14.5, 7.0 Hz, NeCH2), 4.23 (dd, 1H, J ¼ 14.5,
8.0 Hz, NeCH2), 4.06e3.97 (m, 4H, OeCH, OeCH2), 3.87e3.71 (m,
4H, PeCH2, OeCH2), 1.31 (d, 3H, J ¼ 7.5 Hz, CH3-aa), 1.27 (d, 3H,
J ¼ 7.0 Hz, CH3-aa), 1.24 (d, 3H, J ¼ 5.5 Hz, CH3-tenofovir), 1.21 (d,
126.46, 125.87, 122.62 (CH-Naph), 119.59 (C-5), 116.57 (d, JC-
3
¼ 3.6 Hz, CH-Naph), 77.60 (d, JC-P ¼ 13.4 Hz, OeCH), 75.35 (Oe
P
CH2), 65.51 (d, 1JC-P ¼ 157.1 Hz, PeCH2), 50.90 (NeCH), 49.00 (Ne
CH2), 32.30 (C(CH3)3), 26.67 (C(CH3)3), 21.18 (d, 3JC-P ¼ 5.3 Hz, CH3-
aa), 16.86 (CH3-tenofovir) ppm. 31P NMR (202 MHz, MeOH-d4, only
the major diastereoisomer is described):
d
23.64 ppm. MS (ESþ) m/z:
577 (M þ Naþ). Reverse-phase HPLC, eluting with H2O/MeOH from
3H, J ¼ 6.0 Hz, CH3-tenofovir), 0.94 (s, 9H, C(CH3)3), 0.93 (s, 9H,
90/100 to 0/100 in 30 min, Flow ¼ 1 mL/min,
tR ¼ 14.63 min.
l
¼ 254 nm,
3
C(CH3)3) ppm. 13C NMR (125 MHz, MeOH-d4):
d
175.27 (d, JC-
¼ 3.7 Hz, C]O), 175.07 (d, 3JC-P ¼ 3.8 Hz, C]O), 157.28, 157.22 (C-
P
6), 153.71, 153.61 (C-8), 151.53 (d, J ¼ 7.1 Hz, C-ipso OPh), 151.50,
5.1.14. 9-[2-(5,6,7,8-Tetrahydro-1-naphthyloxy-(2,2-
dimethylpropoxy-L-alaninyl)-phosphonomethoxy)propyl]adenine
(17)
Reaction was carried out following procedure B with tenofovir
(500 mg, 1.74 mmol), neopentyloxy- -alaninyl ester p-TSA salt
(881 mg, 2.61 mmol) and 5,6,7,8-tetrahydro-1-naphthol (387 mg,
151.43 (C-4), 143.83, 143.63 (C-2), 130.68, 126.12, 126.02, 121.94,
3
121.91, 121.68 (d, JCP ¼ 7 Hz, Ph), 119.95 (C-5), 77.76 (d, JC-
¼ 12.7 Hz, OeCH), 77.63 (d, 3JC-P ¼ 13.7 Hz, OeCH), 75.38 (OeCH2),
P
65.27 (d, 1JC-P ¼ 156.1 Hz, PeCH2), 65.17 (d, 1JC-P ¼ 156.0 Hz, PeCH2),
51.02, 50.85 (NeCH), 49.20 (NeCH2), 32.33(C(CH3)3), 26.74, 26.70
(C(CH3)3), 21.26 (d, 3JC-P ¼ 5.25 Hz, CH3-aa), 20.58 (d, 3JC-P ¼ 6.0 Hz,
CH3-aa), 16.77, 16.72 (CH3-tenofovir) ppm. 31P NMR (202 MHz,
L
2.61 mmol). The desired product (17) was obtained as an off-white
solid (8%, 35 mg). 1H NMR (500 MHz, MeOH-d4):
d
8.20 (s, 1H, H-2),
MeOH-d4):
d
24.34, 22.99 ppm. MS (ESþ) m/z: 505 (M þ Hþ).
8.18 (s, 2H, H-2, H-8), 8.10 (s, 1H, H-8), 7.07e7.01 (m, 2H, Naph),

F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
267
6.98e6.95 (m, 2H, Naph), 6.89e6.86 (m, 2H, Naph), 4.43 (dd,
J ¼ 14.6, 3.2 Hz, 1H, NeCH2), 4.37 (dd, J ¼ 14.6, 3.1 Hz, 1H, NeCH2),
4.26 (dd, J ¼ 14.7, 6.8 Hz, 1H, NeCH2), 4.23 (dd, J ¼ 15.7, 7.5 Hz, 1H,
NeCH2), 4.08e3.97 (m, 6H, PeCH2, NeCH, OeCH), 3.86e3.80 (m,
4H, PeCH2, OeCH2), 3.75e3.72 (m, 2H, OeCH2), 2.75e2.73 (m, 4H,
CH2-Tetrahydronaph), 2.57e2.55 (m, 4H, CH2-Tetrahydronaph),
1.74e1.72 (m, 8H, CH2-Tetrahydronaph),1.35 (d J ¼ 7.15 Hz, 3H, CH3-
aa),1.30 (d J ¼ 6.9 Hz, 3H, CH3-aa),1.24 (d, J ¼ 7.2 Hz, CH3-tenofovir),
Dorset, UK), 0.1 nM Cholera Toxin (SigmaeAldrich, Dorset, UK),
4 mM Glutamine (SigmaeAldrich, Dorset, UK) and 100 units/mL
penicillin and 100
mg/mL streptomycin (SigmaeAldrich, Dorset,
UK). All cell types were cultured at 37 ꢃC in 5% CO2.
5.4. Assessment of the IC50 values
5.4.1. Initial prodrug screen
1.18 (d, J ¼ 6.26 Hz, 3H, CH3-tenofovir), 0.94 (C(CH3)3), 0.95 (9H,s,
Prior to treatment SiHa, HeLa and C33A cells were seeded at
3
C(CH3)3) ppm. 13C NMR (125 MHz, MeOH-d4):
d
175.44 (d, JC-
4 ꢄ 103 cells per well in 96 well plates and allowed to adhere for
3
¼ 2.5 Hz, C]O), 175.12 (d, JC-P ¼ 3.7 Hz, C]O), 152.20 (C-6),
24 h. After this period cells were treated with 1 mM, 10 mM and
P
157.25 (C-6), 153.68, 153.63 (CH-2), 151.03, 150.97(C-4), 149.88 (d,
2JC-P ¼ 10.0 Hz, C-ipso OTetrahydronaph), 149.80 (d, 2JC-P ¼ 10.0 Hz,
C-ipso OTetrahydronaph), 143.76, 143.65 (CH-8), 140.64 (C-Tetra-
100
were detached with trypsin/EDTA (SigmaeAldrich, Dorset, UK) and
re-suspended in a 1 g/mL 7-Aminoactinomycin D (SigmaeAldrich,
mM parent and protide compounds in triplicate. After 72 h cells
m
3
hydronaph), 129.90 (d, JC-P ¼ 5.0 Hz, C-Tetrahydronaph), 126.80,
Dorset, UK)/FACS Flow (BD Biosciences, Oxford, UK) solution and
analysed for cell viability on an Accuri C6 flow cytometer (BD
Bioscience, Oxford, UK).
126.76, 126.73, 126.69 (CH-Tetrahydronaph), 119.77 (C-5), 118.19 (d,
3JC-P ¼ 2.5 Hz, CH-Tetrahydronaph), 118.17 (d, JC-P ¼ 3.7 Hz, CH-
3
3
3
Tetrahydronaph), 77.9 (d, JC-P ¼ 13.75 Hz, OeCH), 77.7 (d, JC-
¼ 12.5 Hz, OeCH), 75.43, 75.41 (OeCH2), 65.58 (d, 1JC-P ¼ 156.3 Hz,
5.4.2. Further examination of compounds 7 and 9 in models
representing cells at different stages of neoplasia
Prior to treatment SiHa cells and M08 vulval keratinocytes were
seeded at 4 ꢄ 103 cells per well in 96 well plates and human
epidermal keratinocytes were seeded at 3.5 ꢄ 104 cells per well in
24 well plates. Cells were allowed to adhere for 24 h. After this
P
1
PeCH2), 65.44 (d, JC-P ¼ 156.3 Hz, PeCH2), 50.94, 50.96 (NeCH),
49.88, 49.65 (NeCH2), 32.34, 32.31 (C(CH3)3), 30.76, 30.49 (CH2-
Tetrahydronaph), 26.75, 26.75 (C(CH3)3), 24.56, 24.51, 23.84, 23.81,
23.71 (CH2-Tetrahydronaph), 21.38 (d, 3JC-P ¼ 5.0 Hz, CH3-aa), 20.62
(d, JC-P ¼ 6.3 Hz, CH3-aa), 16.92, 16.79 (CH3-tenofovir) ppm. 31P
3
NMR (202 MHz, MeOH-d4):
d
23.96, 22.75 ppm. MS (ESþ) m/z: 559
period cells were treated with 0.1 mM and 1 mM of compounds 9 and
(M þ Hþ). Reverse-phase HPLC, eluting with H2O/MeOH from 90/
7 in replicate. After 96 h cells were detached with trypsin/EDTA
(SigmaeAldrich, Dorset, UK) and re-suspended in pH 7.4 Phosphate
Buffered Saline (SigmaeAldrich, Dorset, UK) and manually counted
in 0.4% Trypan Blue (SigmaeAldrich, Dorset, UK) solution using a
Neubauer haemocytometer.
100 to 0/100 in 30 min, Flow ¼ 1 mL/min,
tR ¼ 17.67 min.
l
¼ 254 nm,
5.2. Carboxypeptidase Y (EC 3.4.16.1) assay
The experiment was carried out by dissolving compound 10
(3.0 mg) in acetone-d6 (0.15 mL) and by adding 0.30 mL of Trizma
buffer (pH 7.6). After the 31P NMR data were recorded at 25 ꢃC as a
control, a previously defrosted carboxypeptidase Y (0.1 mg dis-
solved in 0.15 mL of Trizma) was added to the sample. Next, the
5.5. Antiviral and cytostatic assays
The compounds were evaluated against the following viruses:
herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-
deficient (TK-) HSV-1 KOS strain resistant to ACV (ACVr), herpes
simplex virus type 2 (HSV-2) strain G, and vaccinia virus Lederle
strain. Confluent cell cultures in microtiter 96-well plates were
inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose
to infect 50% of the cell cultures) in the presence of varying con-
centrations of the test compounds. Viral cytopathicity or plaque
formation was recorded as soon as it reached completion in the
control virus-infected cell cultures that were not treated with the
test compounds. Antiviral activity was expressed as the EC50 or
compound concentration required to reduce virus-induced
cytopathogenicity.
Inhibition of HIV-1 (IIIB)- and HIV-2(ROD)-induced cytopa-
thicity in CEM cell cultures was measured in microtiter 96-well
plates containing w3 ꢄ 105 CEM cells/mL infected with 100
CCID50 of HIV per milliliter and containing appropriate dilutions of
the test compounds. After 4e5 days of incubation at 37 ꢃC in a CO2-
controlled humidified atmosphere, CEM giant (syncytium) cell
formation was examined microscopically. The EC50 (50% effective
concentration) was defined as the compound concentration
required to inhibit HIV-induced giant cell formation by 50%.
For the cytostatic experiments, all assays were performed in 96-
well microtiter plates. To each well were added (5e7.5) ꢄ 104 tumor
cells and a given amount of the test compound. The cells were
allowed to proliferate for 48 h (murine leukemia L1210 cells) or
72 h (human lymphocytic CEM and human cervix carcinoma HeLa
cells) at 37 ꢃC in a humidified CO2-controlled atmosphere. At the
end of the incubation period, the cells were counted in a Coulter
counter. The IC50 (50% inhibitory concentration) was defined as the
concentration of the compound that inhibited cell proliferation by
50%.
31
sample was submitted to P NMR experiments (at 25 ꢃC) and the
spectra were recorded every 7 min over 12 h. 31P NMR recorded
data were processed and analyzed with the Bruker Topspin 2.1
program.
5.3. HPV assay
5.3.1. Methods
Parent and daughter compounds were formulated in w1%
Dimethyl sulfoxide (Fisher Scientific, Loughborough, UK) and pH
7.4 phosphate buffered saline (SigmaeAldrich, Dorset, UK) for
in vitro assays.
5.3.2. Cell culture
SiHa (HPV16þ), HeLa (HPV18þ) and C33A (HPVꢂ) human
epithelial cervical carcinoma cell lines (American Type Culture
Collection, Manassas, VA) were grown in Dulbecco’s modified Ea-
gle’s Medium (SigmaeAldrich, Dorset, UK) supplemented with 10%
foetal bovine serum (Autogen Bioclear, Wiltshire, UK) and
100 units/mL penicillin and 100
mg/mL streptomycin (Sigmae
Aldrich, Dorset, UK). Human epidermal keratinocytes (GIBCO/Invi-
trogen, Paisley, UK) were grown in EpiLife culture medium with
supplemented calcium with Human Keratinocyte Growth Supple-
ment (GIBCO/Invitrogen, Paisley, UK). The M08 vulval keratinocytes
were a HPV16 positive short term cell line derived from a biopsy of
VIN3, and were grown in Glasgow Modified Eagles Medium
(SigmaeAldrich, Dorset, UK) supplemented with 10% foetal bovine
serum (Autogen Bioclear, Wiltshire, UK), 0.01
mg/mL EGF (Sigmae
Aldrich, Dorset, UK), 0.5 g/mL Hydrocortisone (SigmaeAldrich,
m

268
F. Pertusati et al. / European Journal of Medicinal Chemistry 78 (2014) 259e268
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The antiviral work was supported by the KU Leuven (GOA 10/
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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