Polyfluoroaromatic stavudine (d4T) ProTides exhibit enhanced anti-HIV activity

Article abstract of DOI:10.1016/j.bmcl.2019.126721

Human Immunodeficiency Virus (HIV) damages the immune system and leads to the life-threatening acquired immunodeficiency syndrome (AIDS). Despite the advances in the field of antiretroviral treatment, HIV remains a major public health challenge. Nucleosides represent a prominent chemotherapeutic class for treating viruses, however their cellular uptake, kinase-mediated activation and catabolism are limiting factors. Herein, we report the synthesis and in vitro evaluation of stavudine (d4T) ProTides containing polyfluorinated aryl groups against two strains; HIV-1 (IIIB) and HIV-2 (ROD). ProTide 5d containing a meta-substituted pentafluorosulfanyl (3-SF₅) aryl group showed superior antiviral activity over the parent d4T and the nonfluorinated analogue 5a. ProTide 5d has low nanomolar antiviral activity; (IC₅₀ = 30 nM, HIV-1) and (IC₅₀ = 36 nM, HIV-2) which is over tenfold more potent than d4T. Interestingly, ProTide 5d showed a significantly high selectivity indices with SI = 1753 (HIV-1) and 1461 (HIV-2) which is more than twice that of the d4T. All ProTides were screened in wild type as well as thymidine kinase deficient (TK^?) cells. Enzymatic activation of ProTide 5d using carboxypeptidase Y enzyme and monitored using both ³¹P and ¹⁹F NMR is presented.

Full text of DOI:10.1016/j.bmcl.2019.126721

Journal Pre-proofs
Polyfluoroaromatic Stavudine (d4T) ProTides Exhibit Enhanced Anti-HIV Ac-
tivity
Sahar Kandil, Christophe Pannecouque, Fiona M. Chapman, Andrew D.
Westwell, Christopher McGuigan
PII:
S0960-894X(19)30679-1
DOI:
https://doi.org/10.1016/j.bmcl.2019.126721
BMCL 126721
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Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
12 September 2019
26 September 2019
27 September 2019
Please cite this article as: Kandil, S., Pannecouque, C., Chapman, F.M., Westwell, A.D., McGuigan, C.,
Polyfluoroaromatic Stavudine (d4T) ProTides Exhibit Enhanced Anti-HIV Activity, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126721
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Polyfluoroaromatic Stavudine (d4T) ProTides Exhibit Enhanced
Anti-HIV Activity
Sahar Kandila, Christophe Pannecouqueb, Fiona M. Chapmana, Andrew D. Westwella and Christopher
McGuigana
aSchool of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10
3NB, b Rega Institute for Medical Research - Laboratory of Virology and Chemotherapy, K.U. Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Keywords:
Polyfluoroaromatic, pentafluorosulfanyl, SF5, Stavudine, d4T, ProTide, Phosphoramidate, Antiviral, Nucleoside,
HIV.
Graphical abstract
Highlights



Synthesis of polyfluoroaromatic Stavudine (d4T) ProTides.
Anti-retroviral activity against two strains; HIV-1, (IIIB) and HIV-2, (ROD) in MT4 cell line.
Evaluation of anti-HIV activity against wild type (C8166) as well as thymidine kinase
deficient (C8166-TK-) cells.

Nanomolar antiviral activity of novel ProTide 5d containing 3-SF5 aromatic ring; IC50 = 30
nM, HIV-1 and IC50 = 36 nM, HIV-2, more than tenfold that of d4T.
High selectivity index of 5d, SI = 1753 (HIV-1) and 1461 (HIV-2) twice that of d4T.
Enzymatic activation of 5d is monitored by 31P and 19F NMR.



Polyfluoroaromatic ProTides have high in vitro aqueous solubility.

Human Immunodeficiency Virus (HIV) damages the immune system and leads to the life-
threatening acquired immunodeficiency syndrome (AIDS). Despite the advances in the field
of antiretroviral treatment, HIV remains a major public health challenge.
Nucleosides represent a prominent chemotherapeutic class for treating viral infections,
however their cellular uptake, kinase-mediated activation and catabolism are limiting
factors. Herein, we report the synthesis and in vitro evaluation of stavudine (d4T) ProTides
containing polyfluorinated aryl groups against two strains; HIV-1 (strain IIIB) and HIV-2
(strain ROD). ProTide 5d containing a meta-substituted pentafluorosulfanyl (3-SF5) aryl
group17 showed superior antiviral activity over the parent d4T and the non-fluorinated
analogue 5a. ProTide 5d has low nanomolar antiviral activity; (IC50 = 30 nM, HIV-1) and (IC50
= 36 nM, HIV-2) which is over tenfold more potent than d4T. Interestingly, ProTide 5d
showed significantly high selectivity indices (SI) = 1753 (HIV-1) and 1461 (HIV-2) which is
more than twice that of the d4T. All ProTides were screened in wild type as well as
thymidine kinase deficient (TK-) cells. Enzymatic activation of ProTide 5d using
carboxypeptidase Y enzyme and monitored using both 31P and 19F NMR is presented.
Human Immunodeficiency Virus (HIV) is a retrovirus, of the family Retroviridae, which is
characterised by the presence of the reverse transcriptase (RT) enzyme1. HIV damages the
immune system and causes higher susceptibility to life-threatening opportunistic infections,
known as acquired immunodeficiency syndrome (AIDS). Many advances have been made in
antiretroviral therapy that have turned AIDS into a chronic condition rather than a fatal
illness2. However, HIV remains a major public health challenge because of the high mutation
rate of HIV-RT, the adverse side effects of the antiretroviral treatment and the poor patient
compliance in clinical applications3. Nucleoside / nucleotide reverse-transcriptase inhibitors
(NRTIs/NtRTIs) represent the first major class of antiretroviral drugs developed 4.
Nucleoside analogues are a well-established class of antiviral and anticancer therapeutic
agents. Enzymatic phosphorylation is required to convert the nucleoside analogues to the
5’-monophosphates and subsequently to the triphosphate derivatives 5-7. Often the three-
step intracellular phosphorylation is inefficient and rate-limited by the initial
monophosphorylation step. Further, reduced expression of nucleoside kinases leads to
resistance to the nucleoside analogue treatment 5,6. Therefore, notable efforts have focused
on the delivery of monophosphate prodrugs that are capable of masking the negative

charge of the phosphate group. The monophosphate phosphoramidate pro-nucleotides,
known as ProTides, represent a successful strategy for bypassing the dependence on active
transport and nucleoside kinase-mediated activation 8,9. This is demonstrated clinically by
sofosbuvir (anti-HCV, FDA approved) 6 and acelarin (anticancer, Phase III) 9, shown in Figure
1.
Figure 1: Chemical structures of sofosbuvir (anti-HCV, FDA approved), acelarin (anticancer, Phase III) ProTides
and stavudine (d4T) nucleoside.
Stavudine (2’,3’-didehydro-2’,3’-dideoxythymidine, d4T) is an approved antiviral nucleoside
for the prevention or treatment of HIV infection, usually as part of antiretroviral
combination therapy10. In an attempt to overcome the first intracellular thymidine kinase-
directed phosphorylation step of d4T, phosphoramidate derivatives were prepared and
evaluated against HIV11.
Generally, introduction of fluorinated substituents can provide a unique combination of
electronegativity, size and lipophilicity and can also greatly affect physicochemical
properties12-14. In this work we synthesised phosphoramidate derivatives of d4T (Scheme 1)
and compared the effect of introducing different polyfluorinated variants of the aromatic
ring (4-CF3 / 3-CF3 / 3-SF5) and investigated whether the lipophilic and electronegative
properties of the corresponding ProTides would affect their anti-HIV activity and toxicity
profiles compared to the non-fluorinated ProTide.
Stavudine (d4T) ProTides were prepared using previously described phosphorochloridate
chemistry15. The phenyl phosphorodichloridates 2a-d were prepared by the reaction of
substituted phenols 1a-d with POCl3. The subsequent reaction with methyl alanine 3 to form
the corresponding phosphorochloridates 4a-d was monitored by 31P NMR. Next the
arylaminoacyl phosphorochloridates 4a-d were reacted with d4T in the presence of t-
BuMgCl as a hindered base (Scheme 1). Stirring for 24h at ambient temperature generated
the crude ProTides 5a-d that were purified by column chromatography to provide the

characteristic low to moderate yields associated with ProTide chemistry, for the final
products (20–34%)16. Each of the phosphoramidates was generated as a pair of
diastereoisomers at the phosphorus centre, in a roughly 1:1 ratio, as revealed by the two
closely spaced peaks in the 31P NMR spectrum (see Supporting Information). It is worth
mentioning that due to the instability of the intermediates it was not feasible to prepare the
4-SF5 analogue.
OH
+
POCl3
1 (a-d)
R
O
N
i
NH
O
O
N
O
O
O
O
O
O
P
O
P
NH
O
Cl
P
Cl
N
Cl
N
O
O
H
O
H
O
O
O
O
ii
iii
+
+
HO
O
NH3Cl
R
R
R
2 (a-d)
4 (a-d)
5 (a-d)
3
d4T
1a, 2a, 4a, 5a, R = H
1b, 2b, 4b, 5b, R = 4-CF3
1c, 2c, 4c, 5c, R = 3-CF3
1d, 2d, 4d, 5d, R = 3-SF5
Scheme 1. Synthesis of d4T phosphoramidate analogues; Reagents and conditions: i) Et3N, anhydrous Et2O, -78
C to rt, 2 h, ii) Et3N, anhydrous DCM, -78 C, 3 h, iii) t-BuMgCl, anhydrous THF, -78 C to rt, 24 h.
ProTides need to be metabolised to release the monophosphate form, which will then
undergo further intracellular phosphorylation steps to generate di- and triphosphate active
form. The proposed intracellular activation pathway of the most active ProTide 5d is
described in Scheme 2, in accordance with previous literature15. Metabolic breakdown
starts with the hydrolysis of the ProTide carboxylate ester, mediated by a carboxyesterase-
type enzyme to form intermediate A, which is followed by a spontaneous cyclisation
displacing the aryl moiety via an internal nucleophilic attack of the carboxylate residue on
the phosphorus centre to yield intermediate B. In the third step, the unstable cyclic mixed
anhydride is thought to be hydrolysed to release the intermediate C. The final step then
involves a phosphoramidase-type enzyme, which would cleave off the amino acid to
generate the corresponding monophosphate D, trapped within the cell due to the polar
nature of the monophosphate.

Scheme 2. Proposed activation pathway of d4T ProTide 5d initiated by esterase or carboxypeptidase enzyme.
To probe the activation process of ProTide 5d, Scheme 2, an enzymatic study using
carboxypeptidase Y enzyme was carried out. ProTide 5d was incubated with
carboxypeptidase Y to verify that the enzymatic cleavage of the ester motif is sufficient to
initiate the activation process and generate intermediate C. Compound 5d was dissolved in
acetone-d6 in the presence of Trizma buffer (pH 7.6), treated with carboxypeptidase Y and
31
monitored by P NMR chemical shift changes over time, Figure 2. Two peaks at δ 3.61 and
3.27 ppm in the blank spectrum recorded at 25 °C correspond to the diastereoisomers of
the parent compound 5d. Within 7 mins of incubation with carboxypeptidase Y, a single
peak at around δ 6 ppm started appearing, which is consistent with the single chemical shift
expected for the formation of achiral intermediate C via the putative intermediate B. The
peak height increased over time suggesting the accumulation of a stable product. The
spectra recorded during the enzymatic reaction suggest that within 2 h of the start of the
assay 5d was almost fully converted into metabolite C.
Figure 2. 31P NMR spectra of d4T ProTide 5d over time after carboxypeptidase Y treatment showing the signals
of the metabolite C over time.

Moreover, due to the presence of the 3-SF5 group in 5d, we considered that the activation
process of ProTide 5d using carboxypeptidase Y enzyme can be also monitored using 19F
NMR chemical shift changes over time, Figure 3. The appearance of two doublet peaks at δ
62.15 and 62.48 ppm in the blank spectrum corresponds to the diastereoisomers of the
parent compound 5d. The new doublet signal appearing at δ 61.86 and 62.21
(corresponding to the four fluorine atoms of the SF5 group) indicating the release of 3-SF5
phenol (1d).
Figure 3. 19F NMR spectra of ProTide 5d over time after carboxypeptidase Y treatment showing the signals of
the metabolites over time.
Both 31P NMR and 19F NMR monitoring experiments, Figure 2 and 3, indicate that ProTide 5d
activation is initiated by the carboxpeptidase enzyme Y and that after 1.25h of incubation
most of the ProTide is metabolised. Quantification of intracellular drug levels would be the
focus of future investigation.
Nucleoside reverse transcriptase inhibitors (NRTIs) and nucleotide analogue reverse-
transcriptase inhibitors (NtRTIs) act as chain terminators for both viral and host DNA
synthesis4. The former describes their antiviral activity, while the latter describes their drug
toxicity/side effects. Hence, d4T ProTides 5a-d were screened for both antiviral activity and
cytotoxicity.
The antiviral activity profile of compounds 5a-d was evaluated in MT4 cells against two HIV
strains; HIV-1 (IIIB) and HIV-2 (ROD) in parallel with stavudine (d4T) as a positive control. The
biological results are expressed as IC50, CC50 and SI (selectivity index; CC50/IC50 ratio).

All ProTides showed markedly enhanced antiviral activity compared to the parent d4T
except the non-fluorinated ProTide 5a which is slightly more potent than d4T. The best
activity was observed across both HIV types in ProTide 5d with the meta-
pentafluorosulfanyl (3-SF5) substituent attached to its aromatic ring. ProTide 5d has low
nanomolar antiviral activity; (IC50 = 30 nM, HIV-1) and (IC50 = 36 nM, HIV-2) which is over
tenfold more potent than d4T, Table 1. Additionally, ProTide 5d showed significantly high
selectivity indices (SI) = 1753 (HIV-1) and 1461 (HIV-2) which is more than twice that of the
d4T. This could be attributed to its enhanced lipophilic and/or electronegative properties
imparted by the presence of five fluorine atoms. Also, the position of the CF3 substituent in
the phenyl ring of 5b and 5c has little influence on the anti-HIV activity but more effect on
the toxicity profile which was in favour of the meta-CF3 (5c) over the para-CF3 (5b) as
observed in the selectivity index values; SI (5c) = 1612 (HIV-1) and 1349 (HIV-2) compared to
SI (5b) = 1159 (HIV-1) and 921 (HIV-2), Table 1.
HIV-1 (IIIB)
CC50 (µM)
HIV-2 (ROD)
CC50 (µM)
comp
d4T
5a
IC50 (µM)
0.40±0.09
SI
IC50 (µM)
0.45±0.09
SI
313±7
251±7
782
313±7
251±7
695
930
912
1349
1461
0.21±0.09
1195
1159
1612
1753
0.27±0.11
5b
0.037±0.002
0.041±0.006
0.030±0.012
42.9±12.6
66.1±24.8
52.6±4.9
0.047±0.022
0.049±0.017
0.036±0.020
42.9±12.6
66.1±24.8
52.6±4.9
5c
5d
Table 1. Anti-HIV-1 and 2 activity and cytotoxicity of stavudine (d4T) and ProTide analogues 5a-d.
Further, we tested our compounds in the C8166 cell line and a variant thymidine kinase
deficient variant (C8166-TK-) to probe the effect of TK deficiency on the activity of the test
compounds and the degree to which the ProTides could bypass this dependence.
As shown in Table 2, the fluorinated ProTides 5b-d displayed greatly enhanced antiviral
potency than the non-fluorinated analogue 5a or the parent d4T across. ProTide 5b (4-CF3)
has around 180-fold higher activity than d4T, while ProTide 5d (3-SF5) is around 75-fold
more active than d4T. It is interesting to observe that compounds 5a, 5b and 5d can retain
their activity in TK deficient cells while d4T has completely lost its activity. While ProTide 5c
(IC50 = 0.039 µM) shows better activity than 5a (IC50 = 0.30 µM) and d4T (IC50 = 4.06 µM), it is

not active in TK- cells. This is most probably because 5c uptake is enhanced but it falls back
to its free nucleoside form and cannot be re-phosphorylated in these cells, Table 2.
HIV-1 (IIIB)
ID
d4T
5a
IC50 WT (µM)
4.06±0.18
IC50 TK- (µM)
NA
0.30±0.04
0.32±0.15
0.075±0.000
NA
5b
5c
0.022±0.006
0.039±0.007
0.054±0.039
5d
0.335±0.004
Table 2. Antiviral activity of stavudine (d4T) and ProTide analogues 5a-d against HIV-1 strain IIIB in the C8166
cell line and the thymidine kinase deficient (C8166-TK-) cell line. NA: not active
Introduction of fluorine atoms into molecular structures increases their hydrophobicity.
Thus, we investigated the in vitro turbidimetric aqueous solubility of the polyfluorinated
ProTides 5b-d using five concentrations (1, 3, 10, 30 and 100 μM) in phosphate buffered
saline (PBS; pH 7.4) solution (1% DMSO), which were incubated at 37°C for 2 hr. The
estimated precipitation range (lower and upper bound) and a mid-range value were
calculated for compounds 5b-d. Nicardipine and pyrene were used as control compounds.
The results show that ProTides with polyfluoroaromatic substituents 5b-d retain high
aqueous solubility with estimated precipitation range 100 μM (lower bound) and >100 μM
(upper bound), Table 3, confirming their potential advantage for intestinal absorption.
Estimated Precipitation range (µM)
Estimated
solubility
(µM)
Compound
MW
ClogPa
Lower
Upper
bound
100
100
100
Calculated mid-range
bound
100
100
100
100
10
d4T
5b
5c
224.22 -0.4875
533.40 1.9788
533.40 1.9788
591.44 2.3288
100
100
100
100
20
> 100
> 100
> 100
> 100
10-30
3-10
5d
100
30
Nicardipine
pyrene
-
-
-
-
3
10
6.5
Table 3. Aqueous solubility of 5b-d versus control compounds (nicardipine and pyrene) in PBS (pH=7.4)
buffered aqueous solution. aCalculated by PerkinElmer ChemDraw 16.0 software.

In this study, a series of polyfluoroaromatic ProTides of d4T were synthesised. The antiviral
activity was evaluated in vitro against the two HIV types; HIV-1 (strain IIIB) and HIV-2 (strain
ROD). Our findings identified compound 5d featuring a meta-pentafluorosulfanyl (SF5) in its
aromatic ring as promising lead. ProTide 5d has low nanomolar antiviral activity; (IC50 = 30
nM, HIV-1) and (IC50 = 36 nM, HIV-2) which is over tenfold more potent than d4T. Moreover,
5d has the best activity / toxicity profile with a selectivity index (SI) = 1753 (HIV-1) and 1461
(HIV-2), which is more than twice that of the parent d4T (SI) = 782 (HIV-1) and 695 (HIV-2).
Additionally, ProTides 5a, 5c and 5d retained some activity in the thymidine kinase deficient
(C8166-TK-) cell line. Furthermore, the polyfluorinated ProTides 5b-d retained aqueous
solubility comparable to that of the parent d4T. Considering the above findings, it is
concluded that 5d represents a promising basis for further development of NtRTI anti-HIV
therapies.
Acknowledgment
The authors would like to thank the Welsh Government (A4B-Academic Expertise for
Business). The technical assistance of Mrs. Kristien Erven, Mr. Kris Uyttersprot and Mrs.
Cindy Heens for the HIV experiments is gratefully acknowledged. The authors also would
like to dedicate this article to the memory of Prof. Christopher McGuigan.
Supplementary data
Supplementary data including chemistry and carboxypeptidase Y enzymatic assay
procedures. 1H, 31P, 19F and 13C NMR spectra of Protides 5a-d as well as HPLC analysis
reports associated with this article can be found, in the online version.
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2(1992) 701–704.
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Thompson, E. Ghazaly, C. McGuigan, J. Med. Chem. 57 (4) (2014) 1531.
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Kaul, C. McLaren, G. Skowron, N. M. Kouttab, T. A. Kennedy, A. B. Weitberg, G. A.
Curt, The Journal of Infectious Diseases, 167, 1 (1993) 21–29.
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16. General experimental procedure for the preparation of ProTides (5a-d)
d4T phosphoramidates (5a-d) were prepared applying the previously described
phosphochloridate chemistry. To a stirring solution of d4T (1.0 eq.) in THF, tert-
butylmagnesium chloride (1.0 M in THF) (1.1 eq) was added dropwise over 1 min, and
the appropriate phosphochloridate (3.0 eq.) in anhydrous THF was added at room
temperature and stirred for 16 h. The solvent was removed under reduced pressure and
the yellow oil obtained was dissolved in DCM and washed with 0.5 M HCl and water. The
organic layer was dried over MgSO4, filtered, reduced to dryness and purified by flash
chromatography.
17. Spectral data of ProTide 2′,3′-Didehydro-2′,3′-dideoxythymidine-5′-(3-(penta
fluorosulfanyl)
phenyl
methoxy
alaninyl phosphate) (5d);
1H
NMR
(CDCl3): δ 9.11 (bs, 1H, NH), 7.54-7.49 (m, 2H, ArH), 7.41-7.31 (m, 2H, ArH), 7.21-
7.11 (2m, 1H, H-6), 6.97-6.93 (2m, 1H, H-1′), 6.28-6.20 (2m, 1H, H-2′), 5.88 -5.82 (2m,
1H, H-3′), 5.00-4.92 (2m, 1H, H-4′), 4.35-4.17 (2m, 2H, H-5′), 3.98-3.88 (m, 1H,
Ala CH), 3.64, 3.64 (2s, 3H, OCH3), 1.82 (bs, 1H, NH), 1.78, 1.74 (2d, J = 1 Hz, 3H, CH3-

5), 1.32, 1.27 (2d, J = 6.5 Hz, 3H, Ala CH3); 31P NMR (CDCl3): δ 3.10, 2.60; 19F NMR
(CDCl3): δ 83.21 (p, 149.9 Hz, 1F), 62.82, 62.80 (2d, 149.9,
J
=
J
=
150.4 Hz, 4F); 13C NMR (CDCl3): 173.88, 173.66 (2d, 3JC-P = 6.5, 7.4 Hz, C=O ester),
163.68, 163.63 (ArC, C-4), 154.41 (m, ArC, CSF5), 150.76 ( C=O, C-2), 150.18, 150.04
(2d, 2JC-P = 5.8 Hz, ArC ipso phenyl), 135.64, 135.44 (ArCH, C-6), 133.05, 132.77 (C-
3′), 129.91 (ArCH) ,127.73, 127.63 (C-2′), 123.80, 123.60 (2d, 2JC-P
= 3.8,
5 Hz, ArCH), 122.66 (m, ArCH), 118.55 (m, ArCH), 111.45, 111.27 (ArC, C-5), 89.96,
89.68 (C-1′), 84.48, 84.42 (2d, 3JC-P = 2.5 Hz, C-4′), 67.58, 66.86 (2d, 2JC-P = 4, 3.8 Hz, C-
5′), 52.69 (OCH3), 50.25, 50.16 (Ala CH), 20.93, 20.82 (2d, 5, 6.3 Hz, Ala CH3), 12.35,
12.25 (CH3, C-5). MS (ES+) m/z: 614.1 (M+Na+), C20H23F5N3O8PS, Reverse-phase
HPLC, eluting with H2O/CH3CN from 90/10 to 0/100 in 30 min; flow = 1 mL/min, tR =
16.01 min. yield 34%.
Highlights



Synthesis of polyfluoroaromatic Stavudine (d4T) ProTides.
Anti-retroviral activity against two strains; HIV-1, (IIIB) and HIV-2, (ROD) in MT4 cell line.
Evaluation of anti-HIV activity against wild type (C8166) as well as thymidine kinase deficient
(C8166-TK-) cells.

Nanomolar antiviral activity of novel ProTide 5d containing 3-SF5 aromatic ring; IC50 = 30 nM,
HIV-1 and IC50 = 36 nM, HIV-2, more than tenfold that of d4T.



High selectivity index of 5d, SI = 1753 (HIV-1) and 1461 (HIV-2) twice that of d4T.
Enzymatic activation of 5d is monitored by 31P and 19F NMR.
Polyfluoroaromatic ProTides have high in vitro aqueous solubility.
18.