ProTides of BVdU as potential anticancer agents upon efficient intracellular delivery of their activated metabolites

Article abstract of DOI:10.1016/j.bmcl.2016.10.077

Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented. The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated against three different cancer cell lines; murine leukemia (L1210), human CD₄⁺T-lymphocyte (CEM) and human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable toxicity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoisomeric mixtures and comparison of their spectral properties, biological activities and enzymatic activation rate is reported.

Full text of DOI:10.1016/j.bmcl.2016.10.077

Bioorganic & Medicinal Chemistry Letters 26 (2016) 5618–5623
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
ProTides of BVdU as potential anticancer agents upon efficient
intracellular delivery of their activated metabolites
b
a
a
a
Sahar Kandil a, , Jan Balzarini , Stephanie Rat , Andrea Brancale , Andrew D. Westwell ,
⇑
Christopher McGuigan a
a School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in
terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented.
The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing
the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the
structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated
against three different cancer cell lines; murine leukemia (L1210), human CD+4 T-lymphocyte (CEM) and
human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved
against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM
and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable tox-
icity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoiso-
meric mixtures and comparison of their spectral properties, biological activities and enzymatic activation
rate is reported.
Received 31 August 2016
Revised 24 October 2016
Accepted 25 October 2016
Available online 27 October 2016
This work is dedicated to the memory of
Professor Christopher McGuigan.
Keywords:
BVdU
ProTide
Phosphoramidate
Anticancer
Nucleoside
Prodrug
Ó 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Brivudine
Over the past 40 years, nucleoside analogues have been estab-
lished as first-line antiviral and anticancer agents. In most cases
phosphorylation is required to convert the nucleoside analogues
to the corresponding 5’-monophosphates and subsequently to
the triphosphates [1–3]. However, this three-step intracellular
conversion is often inefficient in the intact cells being rate-limited
by the initial phosphorylation step; furthermore, lower, or lack of
expression of nucleoside kinases often leads to emergence of resis-
tance to the nucleoside analogue treatment [1,2]. Unfortunately,
nucleotides cannot be considered as therapeutic agents because
of their polar nature, their impermeability through the cell mem-
brane and their dephosphorylation in extracellular fluids. There-
fore, considerable efforts have focused on monophosphate
prodrugs that carry little or no charge to mask the negative charge
of the phosphate group of the nucleotides, with various chemically
or enzymatically cleavable moieties once the compound is trans-
ported into the cell [2,3]. Among all reported pro-nucleotide
approaches, the class of aryl phosphoramidate nucleosides known
as ProTides, pioneered by McGuigan and co-workers, has been pro-
ven to enhance the activity of parent nucleosides by improving
intracellular transport and/or by bypassing the rate-limiting
monophosphorylation step. These improvements serve to eventu-
ally increase the formation rate of intracellular nucleoside triphos-
phate [4]. Proof-of-principle in patients is demonstrated with two
FDA-approved therapeutic antiviral agents, Sofosbuvir used for
hepatitis C virus treatment and tenofovir alafenamide used for
human immunodeficiency virus treatment. Additionally, there
are more ProTides in the pipelines of several academic groups
and pharmaceutical companies undergoing preclinical and clinical
studies for the treatment of viral infections and cancer [2]. The
gemcitabine ProTide known as NUC-1031 represents an example
of an anticancer ProTide currently in Phase 2 clinical trial [5].
Brivudine, (E)-5-(2-bromovinyl)-20-deoxyuridine, (BVdU) Fig. 1
is a potent inhibitor of herpes simplex virus type 1 (HSV-1) and
varicella–zoster virus (VZV). The application of ProTide technology
on the BVdU scaffold led to less potent derivatives compared to the
parent agent against VZV in cell culture models [6]. This was inter-
preted as corresponding to poor intracellular delivery of BVdU
monophosphate (BVdUMP), rapid degradation to BVdU or poor
⇑
Corresponding author.
E-mail address: kandils1@cf.ac.uk (S. Kandil).
http://dx.doi.org/10.1016/j.bmcl.2016.10.077
0960-894X/Ó 2016 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

S. Kandil et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5618–5623
5619
O
N
the amino acid ester 5, Scheme 2. The formation of the key phos-
phorochloridate was monitored by 31P NMR. Next, the arylaminoa-
cyl phosphorochloridate 6 was reacted with either BVdU or its
carboxy methyl ester precursor 2 in the presence of 1-methylimi-
dazole (NMI), Scheme 2. Overnight reactions at ambient tempera-
ture generated crude materials that were purified by column
chromatography to provide low to moderate yields typical of pre-
vious phosphoramidate ProTide syntheses (3.4–28%) [15]. Each of
the phosphoramidate compounds was generated as a pair of
diastereoisomers at the phosphate center, in roughly 1:1 ratio, as
revealed by the two closely spaced peaks in the 31P NMR spectrum.
It has been reported before that the biological activity of phos-
phoramidates can be dependent on the configuration of the phos-
phorus centre [16,17]. Single phosphorus centre diastereoisomers
were separated from their corresponding racemic mixtures of com-
pounds (35, 36, 47 and 49) using a combination of gradient column
chromatography and preparative thin liquid chromatography.
Obtaining four pairs of fast eluting (f) and slow eluting (s)
diastereoisomers; (35f/35s, 36f/36s, 47f/47s and 49f/49s, respec-
tively), offered the opportunity to compare their spectroscopic,
anti-proliferative and enzymatic activation rate profiles. Fig. 2A,
shows the 31P NMR of the diastereoisomeric mixture of 36 and that
of its single diastereoisomer components separated (36s and 36f),
Fig. 2B and C, respectively. It is noteworthy that in the case of pro-
line-based ProTides (39 and 51), we obtained single P diastereoiso-
mers, however the reasons behind this observed stereoselectivity
still need to be investigated.
Br
O
N
NH
O
Br
NH
O
O
P
O
N
H
O
O
O
HO
O
O
OH
OH
BVdU (Brivudine)
NB1011 (Thymectacin)
Fig. 1. Chemical structure of the BVdU and its ProTide derivative NB1011.
onward phosphorylation of BVdUMP to the bioactive triphosphate
[6]. However, NewBiotics Inc. had independently prepared NB1011
(Thymectacin) which has recently entered Phase I/II clinical trials
for the treatment of colon cancer [7,8]. Further studies on
NB1011 revealed that it is selectively toxic to tumour cells express-
ing elevated levels of thymidylate synthase (TS), a key enzyme in
DNA synthesis [9].
Some SAR optimisation of NB1011 was previously reported
against human breast (MCF-7, MDA MB 231), prostate (PC3), colon
(HT115) and bladder (T24) cancer cell lines [10]. In this work we
report the synthesis and biological evaluation of an extensive ser-
ies of BVdU phosphoramidate derivatives. The tuning of the parent
structure involved combined modifications of the amino acid ester,
the aromatic masking group on the phosphate moiety and the 5-
position of the BVdU nucleoside base. We have synthesized 46
BVdU phosphoramidate derivatives 8–53 possessing improved
cytotoxic activity against three tumour cell lines; murine leukemia
(L1210), human CD+4 T-lymphocyte (CEM) and human cervical car-
cinoma (HeLa). Moreover, separation of four phosphorus centre
diastereoisomeric pairs was successfully achieved and provided
useful comparative insights. Applying the previously reported
computational and NMR studies, the absolute stereochemistry of
the phosphorus centre of some of these diastereoisomers has been
predicted.
A comparison between the 1H NMR spectra of the diastereoiso-
meric pair (49s, 49f) revealed a characteristic pattern difference of
the benzylic methylene protons, Fig. 3. For the 49f diastereoisomer,
the two protons display a singlet signal, Fig. 3A, while the 49s
diastereoisomer shows a double doublet signal, Fig. 3B. Similar
findings were reported before for a partially separated racemic
mixture of another BVdU ProTide analogue using preparative HPLC
and were previously explained through conformational studies
[10]. The three aromatic rings (nucleoside base, benzyl ester and
naphthyl) are stacked in
p-p interactions in the case of the Sp
diastereoisomer. This imparts relative rigidity of this conformation
and justifies the observed non-equivalent double of doublet split-
ting NMR pattern of the benzylic methylene hydrogens. On the
other hand, the Rp counterpart does not show such interaction
among the aromatic rings, resulting in the greater flexibility of
the benzylic methylene group reducing the magnetic differences
between the two protons and hence, they appear as a singlet sig-
nal. Therefore, by combining the NMR and the conformational data,
we can propose the Rp configuration to the fast-eluting
diastereoisomer 49f and, consequently, the Sp absolute configura-
tion to the slow-eluting diastereoisomer 49s.
ProTides 8–53 described above were evaluated for their cyto-
static activity against a panel of three established tumour cell lines
in vitro: L1210 (murine leukemia), CEM (human CD+4 T-lympho-
cyte), and HeLa (human cervix). In each case a thymidine kinase-
deficient (TKꢀ) mutant of the parent cell line is included to probe
the effect of TK deficiency on the cytostatic activity of the test com-
BVdU is prepared from 5-iodo-20-deoxyuridine 1, which was
used to prepare the carboxymethyl ester derivative, 2 via Heck
reaction with methyl acrylate in the presence of palladium acetate.
Hydrolysis of 2 was carried out using NaOH followed by acidifica-
tion to get the carboxylic acid derivative 3, which was treated with
N-bromosuccinimide (NBS) to give BVdU [11], Scheme 1.
The target BVdU ProTides were all prepared using the exten-
sively described phosphorochloridate chemistry [12,13]. The phe-
nyl phosphorodichloridate 4a was used to introduce the phenolic
aromatic masking unit of the ProTide. For the naphthyl analogues,
1-naphthol was reacted with phosphoryl chloride to give the
required dichloridate 4b, Scheme 2. The second component of
the ProTide motif is an amino acid ester (general formula, 5), if
not commercially available, was prepared by esterification of the
appropriate amino acids using standard methods [14]. The ary-
laminoacyl phosphorochloridate (general formula, 6), were pre-
pared by reacting the aryl phosphorodichloridate 4a or 4b with
1
2
3
BVdU
Scheme 1. Synthesis of BVdU; Reagents and conditions; i) Pd (OAc)2, PPh3, Me acrylate, ii) NaOH, HCl, iii) NBS, K2CO3.

5620
S. Kandil et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5618–5623
O
O
N
R2
O
R1
R1
O
R2
O
NH
O
NH
O
R2
O
O
O
O
P
O
N
O
P
O
P
R3
HO
N
O
c
O
R3
R4
H
b
Cl
Cl
R3
NH2X
+
+
N
Cl
R4
O
Ar
H
R4
O
Ar
OH
O
OH
Ar
Scheme 2. Synthesis of BVdU phosphoramidate analogues; Reagents and conditions: a) Et3N, anhydrous DCM, ꢀ78 °C, 2–5 h, b) NMI, anhydrous THF, ꢀ78 °C to r.t., 16–18 h.
Fig. 2. A) 31P NMR of the racemic mixture 36, B) slow eluting diastereoisomer, 36s, C) fast eluting diastereoisomer 36f.
Fig. 3. 1H NMR spectra of the benzylic methylene protons (CH2) of the isolated diastereoisomers; (a) 49f, (b) 49s.
pounds and the degree to which the ProTides could bypass this
dependence. The murine L1210 cell line was included because
these tumour cells can be used in a mouse tumour model. Also, a
normal human non-tumourigenic primary monolayer cell line
(lung fibroblast HEL cells) is included for comparative purposes,
and BVdU as a positive parental drug control, Table 1. For the mur-
ine leukemia cell line (L1210), alanine-based ProTides were
IC50 = 4.8
static against the human cervical carcinoma (HeLa) (IC50 = 160 -
M), compounds 33 -valinyl cyclohexyl ester naphthoxy
phosphoramidate, IC50 = 7.2 M) and 36 ( -tryptophanyl ethyl
ester naphthoxy phosphoramidate, IC50 = 7.8 M) were found to
boost the antiproliferative activity significantly. Interestingly,
BVdU and its phosphoramidate ProTide derivatives generally
showed poor, if any cytotoxicity against the human lung fibroblast
cell cultures displaying minimal cytotoxic concentration (MCC)
lM) being the most active. While BVdU is poorly cyto-
l
(L
l
L
l
amongst the most active compounds with compound 23 (
nyl benzyl ester naphthoxy phosphoramidate, IC50 = 1.8
showing 20-fold increase in potency compared to BVdU (IC50 = 38 -
M). Interestingly, while BVdU itself does not show any notewor-
thy anti-proliferative activity against the human T-lymphocyte cell
line (CEM) (IC50 > 100 M), most of its phosphoramidate deriva-
tives showed a greatly enhanced activity with compound 37 (
phenylalaninyl ethyl ester naphthoxy phosphoramidate,
L
-alani-
lM)
values of P100 lM in the vast majority of the test compounds,
l
Table 1. These findings point to a considerable extent of selectivity
of the synthesised ProTides, which is potentially beneficial from a
drug development viewpoint.
It has been noticed before that BVdU, in contrast to most other
thymine-based nucleoside analogues, often shows an increased
l
L
-

S. Kandil et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5618–5623
5621
Table 1
Inhibitory effects of BVdU ProTides on the proliferation of murine leukemia (L1210, L1210/TK-), human CD4+ T-lymphocyte (CEM, CEM/TK-) and human cervical carcinoma (HeLa,
HeLa/TKꢀ) cells, and their toxicity against human non-tumourigenic lung fibroblast (HEL) cultures.
No.
R
Ar
Ester
AA
IC50
(
l
M)
MCCa
HEL
L1210/0
L1210/TKꢀ
Cem/0
Cem/TKꢀ
HeLa
HeLa/TKꢀ
8
9
Br
Br
Br
Br
Br
Br
Br
Br
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Bn
Et
Me
Et
Me
Et
cHex
Et
cHex
Me
Et
Me
cHex
Bn
NeoPnt
Bn
cHex
Et
iPr
Bn
Bn
Bn
2-Bu
Bn
Ala
Ala
Trp
Trp
Phe
Phe
Val
Tyr
Ala
Ala
Phe
Phe
Val
Ala
Ala
Ala
Ala
Ala
Ala
Met
Val
Pro
Ala
Gly
2.8 0.3
17 5.0
13.2 0.3
18 1.0
32 2.0
30 3.0
9.3 0.2
48 5.0
2.6 0.2
14 0.0
67 13.0
>100
75 8.0
4.0 2.3
15 2.0
1.8 0.0
9.2 3.7
8.0 5.2
18 1.0
P 250
0.35 0.1
2.0 1.4
6.7 0.6
4.2 0.2
6.2 0.7
5.6 0.7
10 0.0
26 4.0
1.5 0.0
15 2.0
57 6.0
>100
79 3.0
36 5.0
0.89 0.08
0.24 0.3
2.9 0.0
0.47 0.2
1.6 0.2
167 69.0
10 4.0
16 1.0
3.1 0.5
3.9 0.1
19 1.0
36 1.0
32 11.0
24 0.6
14 6.0
23 2.0
23 4.0
8.6 1.1
>100
15 4.0
34 6.0
55 2.0
>100
69 8.0
19 2.0
13 1.0
9.3 3.9
6.1 0.2
28 12.0
14 4.0
>250
6.0 0.1
9.9 0.9
15 2.0
18 2.0
17 0.0
32 2.0
21 0.0
16 1.0
14 2.0
6.7 3.2
9.5 2.3
10 0.0
>100
25 5.0
47 1.0
24 11.0
69 43.0
81 7.0
51 6.0
13 4.0
5.4 0.9
5.8 0.6
16 3.0
15 1.0
>250
38 2.0
48 7.0
33 6.0
18 3.0
34 2.0
40 4.0
12 0.0
47 9.0
16 0.0
10 0.0
80 10.0
>100
3.1 1.7
9.6 1.8
1.2 0.0
2.7 0.1
2.8 1.8
3.8 2.8
7.4 0.4
8.2 2.2
6.5 1.7
6.1 1.0
35 1.0
66 31.0
38 7.0
42 9.0
4.7 1.3
1.1 0.3
3.0 1.6
4.7 0.0
5.5 0.3
22.0 9.0
3.6 0.3
5.2 3.5
4.3 2.4
1.1 0.6
7.7 5.0
P100
100
100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
P100
20
>100
100
P20
10
P4
Br
COOMe
COOMe
COOMe
COOMe
COOMe
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
P 100
32 5.0
32 13.0
33 17.0
32 20.0
84 13.0
21 1.0
>250
12 5.0
23 2.0
23 1.0
36 19.0
31 8.0
7.1 2.3
15 1.0
17 2.0
18 3.0
21 3.0
5.2 0.3
12 0.0
15 3.0
19 1.0
18 3.0
P100
100
P100
Bn
D-Ala
33
34
Br
Br
Br
Nap
Nap
Nap
cHex
Et
Me
Val
Val
Trp
8.3 0.2
16 3.0
7.7 0.9
40 2.0
8.3 0.6
8.7 0.7
8.2 0.3
7.5 0.4
18 1.0
27 7.0
19 8.0
23 5.0
13 1.0
8.4 0.3
26 0.0
15 5.0
9.2 1.2
8.8 0.0
11 1.0
56 2.0
42 5.0
32 6.0
35 6.0
16 1.0
54 4.0
10 0.0
26 3.0
38 4.0
8.8 0.1
16 3.0
2.4 0.3
28 9.0
4.0 0.9
2.5 0.1
7.0 0.5
8.2 0.1
14 0.0
14 6.0
6.8 2.1
12 3.0
9.6 0.4
6.9 0.4
8.3 0.0
6.9 0.5
3.0 1.7
8.9 0.9
14 0.0
40 3.0
32 2.0
22 9.0
21 8.0
16 7.0
46 3.0
12 1.0
50 7.0
9.6 2.8
7.4 1.3
8.4 1.8
9.6 1.7
39 5.0
7.1 0.5
9.8 1.5
7.8 0.6
4.8 1.0
13 8.0
11 1.0
13 1.0
29 1.0
17 2.0
9.6 0.1
19 9.0
11 4.0
10 2.0
11 0.0
11 0.0
49 3.0
35 8.0
27 2.0
29 3.0
10 1.0
38 2.0
12 3.0
52 8.0
>100
8.4 1.5
9.2 2.3
8.0 0.4
26 2.0
6.1 1.1
7.6 1.6
5.8 1.3
4.6 1.0
6.3 1.1
9.5 1.4
6.6 2.8
12 2.0
10 0.0
8.2 0.5
12 4.0
9.5 0.2
8.6 0.0
6.2 2.3
8.9 0.8
47 1.0
46 4.0
20 10.0
32 8.0
8.1 1.7
42 1.0
8.1 1.5
58 8.0
100
7.2 0.2
27 5.0
26 14.0
43 3.0
7.8 0.7
3.9 1.3
8.2 0.1
18 3.0
73 0.0
30 3.0
32 2.0
33 6.0
38 7.0
38 1.0
36 0.0
29 10.0
25 6.0
36 4.0
34 1.0
79 7.0
57 0.0
37 1.0
37 4.0
29 2.0
P 100
5.0 0.9
7.2 1.1
3.2 1.9
17 7.0
6.0 1.4
2.3 1.2
3.1 0.4
2.8 0.4
1.8 0.8
6.4 1.0
6.1 1.2
10 0.0
9.3 0.0
1.7 0.8
11 3.0
6.3 2.1
7.4 0.3
19 13.0
32 7.0
15 10.0
26 10.0
29 9.0
11 6.0
11 2.0
40 2.0
9.7 0.2
35 9.0
0.20 0.16
P100
P100
100
100
–
P100
35
100
100
>100
100
>100
100
>100
>100
100
P20
>100
100
>100
>100
100
35f
35s
36
36 f
36s
37
38
39
40
41
Br
Nap
Et
Trp
Br
Br
Br
Br
Br
Br
Br
Br
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Nap
Et
Me
Et
Et
n-Pnt
n-Pnt
Et
Phe
Phe
Pro
OMeTyr
Val
Phe
Tyr
Met
Gly
DMG
Trp
42
43
44
45
Et
Br
Br
COOMe
cHex
Neo-pnt
Et
46
47f
47s
48
COOMe
COOMe
Nap
Nap
Et
Val
Ala
49
49f
49s
50
51
52
Bn
100
COOMe
COOMe
COOMe
COOMe
Nap
Nap
Nap
Nap
BVdU
cHex
Et
n-Pnt
Et
Val
Pro
Phe
Met
>100
>100
>100
>100
–
33 6.0
62 11.0
160 21.0
53
a
MCC or minimal cytotoxic concentration in (lM) required to afford a microscopically visible alteration of cell morphology.
cytostatic activity against thymidine kinase-deficient tumour cell
lines. The molecular/biochemical basis for this phenomenon is still
unclear [18]. Here, a pronounced increase of cytostatic activity was
also observed for several BVdU ProTides (i.e. 8, 16, 21 for L1210
and the majority of compounds for CEM and HeLa). These observa-
tions are of particular interest since certain forms of drug resis-
tance of cancer cells have been reported to be caused by
thymidine kinase deficiency (i.e. drug resistance against 5-FdUrd-
and 5-trifluoromethyl-dUrd-treated cancers) [19,20]. It would
therefore be reasonable to suggest the use of such BVdU ProTides
to treat tumours that became refractory to FdUrd/CF3dUrd
treatment.
All compounds 8–53, were evaluated as mixtures of two phos-
phate diastereoisomers (Rp and Sp in 1:1 ratio). Interestingly, we
were able to separate four pairs of diastereoisomers, 35f/35s,
36f/36s, 47f/47s and 49f/49s, which offered the opportunity to
compare their relative biological activity, Table 1. It could be con-
cluded that in general, the fast eluting diastereoisomer 35f showed
a relatively better cytostatic activity across the three cell lines than
its slow eluting diastereoisomer 35s. Apart from 35f/35s, the other
three pairs of diastereoisomers, 36f/36s, 47f/47s and 49f/49s, dis-
played very similar activity profiles across the three cell lines. In
two cases, 36 and 49, the activity of the parent racemic mixture
(1:1 ratio) and that of its single diastereoisomeric components

5622
S. Kandil et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5618–5623
Scheme 3. Proposed activation pathway of phosphoramidate 35; i) esterase or carboxypeptidase-type enzyme, ii) and iii) spontaneous, iv) phosphoramidase-type enzyme.
(36f/36s and 49f/49s, respectively) were evaluated. In both cases
the anti-proliferative activity were very similar, Table 1.
pound without measurable toxic side-effects. Also, given the
increased activity of some of the BVdU prodrugs against TK-defi-
cient tumour cell lines, the use of BVdU ProTides may perhaps be
more efficient in suppression of TK- based resistance development.
Generally, the activity profiles suggested that our library of phos-
phoramidates were able to release the nucleoside monophosphate
within intact cells and afford pronounced cytostatic activity in
both the wild-type and TK- cancer cell lines, as shown in Table 1.
Nucleoside analogues are challenged by numerous inherent and
acquired cancer resistance mechanisms that can significantly limit
The side products from the catabolic conversion of the BVdU
ProTides to BVdU-MP are amino acids, and phenol or naphthol.
Whereas the release of naturally occurring amino acids should
not be a matter of concern in terms of potential side-effects, the
release of naphthol or phenol might be. However, it should be
noted there are currently two ProTide derivatives approved for
clinical use (i.e. TAF, or tenofovir alafenamide [21,22] and Sofosbu-
vir [2]) that release phenol upon conversion to the parent com-
Fig. 4. 31P NMR spectra of ProTide 35f (A) and ProTide 35s (B) over time (every 7 min) after treatment with carboxypeptidase Y (5 mg in acetone-D/Trizma) showing the
signals of different metabolites.

S. Kandil et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5618–5623
5623
their effectiveness. ProTides are specifically designed to overcome
Acknowledgements
some key cancer resistance pathways and thereby achieve a supe-
rior antineoplastic effect. To exert their anticancer activity, the Pro-
Tides are metabolised to release the free nucleoside
monophosphate form, which will then generate the corresponding
active forms, di- and/or triphosphates. The proposed intracellular
activation route of the ProTides has been described for other Pro-
Tide families [23] and is exemplified by compound 35 in Scheme 3.
The proposed mechanism of activation of the ProTides involves a
first enzymatic activation step (i) mediated by a carboxypepti-
dase-type enzyme that hydrolyses the ester of the aminoacyl moi-
ety to produce the intermediate A. Next, a spontaneous cyclization
(ii) displacing the aryl moiety via an internal nucleophilic attack of
the carboxylate residue on the phosphorus center to yield B. In a
third step (iii), the unstable ring is hydrolysed to release the inter-
mediate C. The last step (iv) involves a phosphoramidase-type
enzyme, which cleaves-off the amino acid to generate the corre-
sponding nucleoside monophosphate D, Scheme 3. This assay
was designed to verify whether an enzymatic cleavage of the ester
motif would be sufficient to trigger the first steps of the activation
route and generate the intermediate C [24].
We thank the MRC (SARTRE/DPFS) for the financial support of
this work. We are grateful to Mrs. Lizette van Berckelaer, Mrs.
Leentje Persoons and Mrs. Frieda De Meyer for dedicated technical
help. The biological research was supported by the KU Leuven
(GOA 15/19 TBA).
A. Supplementary data
Supplementary data (experimental procedures and spectro-
scopic characterisation data of the compounds 8–53 as well as
the computational study) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.10.
077. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
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To probe the difference between the P diastereoisomer activa-
tion rate, Scheme 3, an enzymatic study using carboxypeptidase
Y while monitoring the conversion by 31P NMR was performed.
Each diastereoisomer of compound 35; (35f and 35s) was dissolved
in acetone-d6 in the presence of Trizma buffer (pH 7.6) and treated
with carboxypeptidase Y to monitor the metabolic conversion of
the ProTide using 31P NMR spectroscopy over time, Fig. 4. In the
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against three different cancer cell lines; murine leukemia
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noma (HeLa) and for toxicity against human non-tumourigenic
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kinase-deficient cancer cell lines. Poor, if any cytotoxic activity
was observed in HEL cell cultures pointing to a considerable
degree of selectivity. Separation of four pairs of P diastereoiso-
mers and the comparison of some spectral properties, anticancer
cell activity and enzymatic activation features is described.
Molecular modeling studies were conducted in an attempt to
explain some of our findings.
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