Influence of 4′-Substitution on the Activity of Gemcitabine and Its ProTide against VZV and SARS-CoV-2

Article abstract of DOI:10.1021/acsmedchemlett.0c00485

In addition to its therapeutic value as a chemotherapy drug, gemcitabine is of ongoing interest to the scientific community for its broad-spectrum antiviral activity. Herein the synthesis of 4′-methoxy- and 4′-fluoro-substituted gemcitabine analogues along with their phosphoramidate prodrugs is described. Among these derivatives, 4′-fluorogemcitabine proved to be active against varicella zoster virus (VZV, TK+ strain) with an EC50 of 0.042 μM and produced significant cytotoxicity (CC50 = 0.11 μM). Upon derivatization of this trifluoro nucleoside as its prodrug, decreased anti-VZV activity was observed, but with a concomitantly improved selectivity index (SI = 36). When this prodrug was tested against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its antiviral activity (EC50 = 0.73 μM) was comparable to or slightly lower than its cytotoxic concentration in measurements of cell growth and cell morphology, respectively.

Full text of DOI:10.1021/acsmedchemlett.0c00485

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Letter
Influence of 4′-Substitution on the Activity of Gemcitabine and Its
ProTide Against VZV and SARS-CoV‑2
Zihua Zheng, Elisabetta Groaz, Robert Snoeck, Steven De Jonghe, Piet Herdewijn,§
and Graciela Andrei*,§
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ABSTRACT: In addition to its therapeutic value as a chemotherapy drug,
gemcitabine is of ongoing interest to the scientific community for its broad-
spectrum antiviral activity. Herein the synthesis of 4′-methoxy- and 4′-fluoro-
substituted gemcitabine analogues along with their phosphoramidate prodrugs is
described. Among these derivatives, 4′-fluorogemcitabine proved to be active
against varicella zoster virus (VZV, TK+ strain) with an EC50 of 0.042 μM and
produced significant cytotoxicity (CC50 = 0.11 μM). Upon derivatization of this
trifluoro nucleoside as its prodrug, decreased anti-VZV activity was observed, but
with a concomitantly improved selectivity index (SI = 36). When this prodrug was
tested against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its
antiviral activity (EC50 = 0.73 μM) was comparable to or slightly lower than its
cytotoxic concentration in measurements of cell growth and cell morphology,
respectively.
KEYWORDS: Gemcitabine analogues, phosphoramidate prodrug, varicella zoster virus, severe acute respiratory syndrome coronavirus 2,
antiviral activity
emcitabine (2′-deoxy-2′,2′-difluorocytidine, dFdC; Fig-
Furthermore, gemcitabine was also demonstrated to exert
broad-spectrum in vitro activity against a range of RNA viruses.
In particular, its antiviral inhibitory effect was assessed against
Zika virus (ZIKV) (EC50 = 0.01 μM),4 hepatitis C virus
(HCV) (EC50 = 12 nM),5 human immunodeficiency virus 1
(HIV-1) (EC50 = 16.3 nM),6 and poliovirus (IC50 = 0.3 μM)7
as well as respiratory viruses such as influenza A virus (IAV)
(EC50 = 0.068 μM),8 human rhinovirus (HRV) (EC50 = 0.81−
1.92 μM),9 Middle East respiratory syndrome coronavirus
(MERS-CoV) (EC50 = 1.22 μM),10 and severe acute
respiratory syndrome coronavirus (SARS-CoV) (EC50 = 4.95
μM).10 In addition, a recent report described the activity of
this modified nucleoside against the emerging coronavirus
SARS-CoV-2 (EC50 = 1.24 μM) that is responsible for the
current worldwide viral pneumonia outbreak.11
G
ure 1) is a pyrimidine nucleoside analogue that is in
Figure 1. Gemcitabine and its 4′-substituted derivatives investigated
in this study.
clinical use for the treatment of various solid tumors, including
nonsmall cell lung, pancreatic, bladder, and breast cancer.1 By
acting as a potent antimetabolite, it inhibits two cellular
processes that are both required for DNA biosynthesis, i.e.,
nucleotide reduction and replicative DNA chain elongation.
Upon phosphorylation in cells, it is converted to its biologically
active metabolites, namely, gemcitabine diphosphate
(dFdCDP) and triphosphate (dFdCTP).2 While dFdCDP
acts as an inhibitor of ribonucleotide reductase (RNR), thus
preventing the biosynthesis of deoxynucleotide building blocks
that are required for DNA synthesis, dFdCTP is competitively
incorporated into DNA in place of natural 2′-deoxycytosine-5′-
triphosphate (dCTP).3
The modification of gemcitabine as a means to convert a
life-saving antitumoral drug into an active compound for the
treatment of severe viral infections represents an attractive
research endeavor. In this context, a variety of gemcitabine
analogues have been synthesized to increase its selectivity as
Received: September 4, 2020
Accepted: December 7, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00485
© XXXX American Chemical Society
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A

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a
Scheme 1. Synthetic Routes for the Preparation of 4′-Substituted Gemcitabine Analogues
a
Reagents and conditions: (a) BzCl, pyr, 93%; (b) 80% AcOH, reflux, 90%; (c) 7 M NH3 in MeOH, 85%; (d) imidazole, PPh3, I2, THF, 84%; (e)
DBU, THF, 80 °C, 65%; (f) I2, PbCO3, anhydrous MeOH for 8a, 61%; (g) I2, AgF, anhydrous THF for 8b, 35%; (h) BzCl, anhydrous pyr, 0 °C,
94 and 87% for 9a and 9b, respectively; (i) m-CPBA, CH2Cl2/H2O, 40 °C, 72 and 59% for 10a and 10b, respectively; (j) BzCl, triethylamine,
DMAP, anhydrous THF, 65 and 75% for 11a and 11b, respectively; (k) POCl3, 1,2,4-triazole, anhydrous MeCN; (l) 25% NH4OH(aq), MeCN;
(m) 7 M NH3 in MeOH, 38 and 56% for 1a and 1b, respectively, over three steps; (n) 7 M NH3 in MeOH, 68 and 51% for 12a and 12b,
respectively.
antiviral agent.12,13 The rationale behind this strategy aims at
reducing the inhibitory activity of dFdCDP and dFdCTP
against the cellular RNR and polymerase, respectively, while
maintaining the viral polymerase inhibitory capacity of the
dFdCTP metabolite.
monophosphate) was expected to provide useful information
for the design of new gemcitabine analogues.
Herein, varicella zoster virus (VZV) and human cytomega-
lovirus (HCMV) were chosen as the two most important
herpes viruses. Although there are anti-VZV and anti-HCMV
drugs on the market, toxicity is an issue for some of these
drugs, and the emergence of drug resistance has been
described for all of them among immunocompromised
patients. Novel nontoxic antiviral chemotherapeutics that are
more potent and effective than the currently available drugs are
therefore required for the treatment of these viral infections in
at-risk populations. At the same time, SARS-CoV-2 was
privileged among RNA viruses because of the extremely urgent
need to develop new antiviral treatments for this infection. As
shown in Scheme 1, commercially available gemcitabine served
as a convenient starting point for the introduction of fluoro
and methoxy substituents at the 4′-position.
First, uridine congener 5 was obtained in an overall yield of
71% via a three-step sequence including complete functional
group protection, nucleobase deamination, and O-debenzoy-
lation using standard literature procedures.14 Next, the primary
hydroxyl group of compound 5 was substituted with an iodine
atom upon treatment with iodine and triphenylphosphine to
yield 6 in good yield (84%). When 6 was subjected to an
elimination reaction under strongly basic conditions, methyl-
ene derivative 7 was obtained in 65% yield and served as a key
It is well-known that subtle structural modifications of
nucleosides can have a profound impact on their biological
profiles. In this study, we selected a methoxy group as an
electron-withdrawing substituent via the inductive effect and a
fluorine atom as a strongly inductive electron-withdrawing
substituent to modify the 4′-position of gemcitabine in order
to identify compounds that could lead to reduced cell toxicity
while retaining activity against selected viruses. Sterically, both
substituents are small, as larger groups might be detrimental
for an effective interaction with the target enzymes. In
addition, the presence of substituents with different electronic
properties might influence the reactivity of the primary
hydroxyl group, which needs to undergo intracellular
phosphorylation to deliver the active metabolite. The synthesis
of 4′-methoxy- and 4′-fluorogemcitabine analogues (1a and
1b, respectively; Figure 1) was complemented by that of the
corresponding prodrugs (2a and 2b; Figure 1). A comparison
of the antiviral activity of a nucleoside itself (which still needs
to undergo three consecutive phosphorylations) and its
prodrug (bypassing the first phosphorylation to the nucleoside
B
https://dx.doi.org/10.1021/acsmedchemlett.0c00485
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Letter
a
Scheme 2. Conversion of Gemcitabine and Its 4′-Substituted Analogues to Their Phosphoramidate Prodrugs
a
Reagents and conditions: (a) DBDC, Na2CO3, dioxane/H2O, 35% for 13a, 23% for 13b, and 59% for 13c; (b) phenyl dichlorophosphate,
triethylamine, CH2Cl2, −78 °C, quantitative yield; (c) tert-butylmagnesium chloride, anhydrous THF; (d) trifluoroacetic acid/CH2Cl2, 0 °C, 49%
for 16a, 54% for 16b, and 32% for 16c.
Table 1. Antiviral Activities and Cytotoxicities of 4′-Substituted Gemcitabine Analogues and Their Prodrugs against VZV and
HCMV
a
antiviral activity, EC50 (μM)
e
VZV
HCMV
AD-169 strain
HEL cytotoxicity (μM)
VZV SI
TK+ strain
b
c
compound
TK+ strain
TK− strain
Davis strain
MCC
20
CC50
TK− strain
gemcitabine
1a
1b
12a
12b
2a
2b
2c
acyclovir
brivudin
ganciclovir
cidofovir
0.028 0.020
>100
0.042 0.014
>100
>100
>100
2.32 0.43
0.074 0.006
1.6
0.054 0.012
>100
0.166 0.13
>100
>100
>100
0.074 0.004
>100
0.053 0.049
>100
0.0036 0.0003
<1
−
2
−
−
−
36
4
265
>8333
−
−
b
<1
−
<1
−
−
−
29
3
d
>100
20
ND
0.815 0.64
>100
0.93 0.75
>100
0.11
d
>100
>100
>100
>100
>100
>440
>300
350
ND
ND
ND
d
>100
>100
d
>100
>100
2.84 0.021
0.089 0.011
22.15 11.6
6.04
15.47 6.40
0.066 0.007
4.00 0.00
0.032 0.00
ND
84.35 22.1
0.34 0.25
424 21.7
>300
231.22 49.2
150.02 29.1
d
d
ND
19
>49
−
d
d
0.036 0.004
ND
ND
d
d
ND
ND
ND
2.77 0.53
0.65 0.38
1.67 0.48
0.34 0.056
d
d
ND
300
−
a
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). Minimum cytotoxic
concentration that causes a microscopically detectable alteration of cell morphology. Cytotoxic concentration required to reduce the cell growth by
c
d
e
50%. Not determined. SI = CC50/EC50.
synthon for the further introduction of a methoxy or fluorine
substituent at the 4′-position to afford intermediate com-
pounds 8a and 8b in 61 and 35% yield, respectively. The
stereoselectivity of this step was most likely due to the
participation of the C2 carbonyl of the uracil nucleobase via
formation of a 4′-anhydro intermediate, as previously
postulated.14−16 Subsequent protection of the 3′-hydroxy
group of 8a and 8b as a benzoyl moiety furnished compounds
9a and 9b in 94 and 87% yield, respectively; these were further
converted to 10a and 10b by transfer of the 3′-benzoyl group
to the 5′-position upon treatment with m-CPBA in a solvent
mixture (CH2Cl2/H2O, 4/1). Then the benzoyl group of 10a
and 10b was deprotected to afford 4′-methoxy- and 4′-
fluorouridine analogues 12a and 12b in 68 and 51% yield,
respectively. Alternatively, the 3′-hydroxy group of compounds
10a and 10b was reprotected using benzoyl chloride in
pyridine followed by nucleobase conversion via a triazole
intermediate and final deprotection under basic conditions to
afford compounds 1a and 1b in 38 and 56% yield, respectively,
over three steps.15,17
bypassing the first rate-limiting phosphorylation step in the
nucleoside activation cascade to their 5′-triphosphate forms
and facilitate intracellular delivery.18 For instance, the
application of this approach to gemcitabine led to enhanced
biological activities and decreased drug resistance.19 Herein,
phosphoramidate prodrugs of compounds 1a and 1b along
with that of gemcitabine were synthesized as shown in Scheme
2. First, 1a, 1b, and gemcitabine were selectively protected at
the 3′-position by treatment with di-tert-butyl dicarbonate
(DBDC) and sodium carbonate in a 4:1 mixture of dioxane
and water as the solvent to afford compounds 13a−c,
respectively. In parallel, L-alanine benzyl ester hydrochloride
(14) was converted to its phenyl aminoacyl phosphorochlor-
idate 15 upon reaction with phenyl phosphorochloridate. Then
compounds 13a−c were reacted with compound 15 to furnish
compounds 16a−c in moderate yields. Finally, the Boc
protecting group was cleaved under acidic conditions to afford
the desired compounds 2a−c.
All of the novel compounds (1a, 1b, and 2a−c) were
evaluated for their antiviral activity (expressed as EC50) against
VZV (strains TK+, thymidine kinase-competent, and TK−,
thymidine kinase-deficient) and HCMV (strains AD-169 and
The aryloxy triester phosphoramidate prodrug or ProTide
technology is a strategy that has proven to be very effective in
C
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Table 2. Antiviral Activities and Cytotoxicities of 4′-Substituted Gemcitabine Analogues and Their Prodrugs against SARS-
CoV-2 in Vero Cells
a
antiviral activity, EC50 (μM)
cytotoxicity (μM)
b
c
compound
gemcitabine
1a
1b
2a
UC-1074 strain
UC-1075 strain
>0.0016
≥86.2 20.4
0.096 0.034
≥55.1 42.8
0.73 0.15
MCC
0.008
>100
0.16
CC50
0.0043 0.0008
>100
>0.0032
ND
0
0
d
0
0.36
>100
≥2.4 2.3
0.26 0.13
≥93.6 11.1
1.44 0.62
0.020 0.009
≥100
2b
2c
4
0
>0.032
0
>0.0128
0
0.048 0.018
>40
remdesivir
hydroxychloroquine
5.8 3.1
8.1 2.4
1.52 1.60
1.74 0.68
>40
0
100
36.9 7.5
a
b
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 PFU. Minimum cytotoxic concentration that
c
d
causes a microscopically detectable alteration of cell morphology. Cytotoxic concentration required to reduce cell growth by 50%. Not
determined.
Davis), with concomitant determination of cytotoxicity and
cytostatic effects (CC50) (Table 1). Gemcitabine was included
as a reference compound, and acyclovir, brivudine, ganciclovir,
and cidofovir were included as positive controls.
Interestingly, in this cellular test system, compounds 1b and 2b
were 20 and 10 times more active than remdesivir, respectively.
In summary, the synthesis of 4′-methoxy- and 4′-
fluorogemcitabine analogues and their phosphoramidate
prodrugs and an evaluation of their antiviral activities against
VZV, HCMV, and SARS-CoV-2 have been described. The
introduction of a fluorine atom at the 4′-position led to a
trifluorinated gemcitabine analogue that exhibited potent
activity but no selectivity against these three viruses. A
phosphoramidate prodrug of the 4′-fluoro congener displayed
reduced activity against VZV but less pronounced cytotoxicity
with an enhanced selectivity index. A similar although
somewhat reduced effect was observed against HCMV.
Unfortunately, this effect was not determined upon testing
against SARS-CoV-2.
The potent cytostatic activity of gemcitabine in human
embryonic lung (HEL) fibroblasts (CC50 = 0.0036 μM) leads
to a complete absence of selective antiviral activity. The
phosphoramidate prodrug of gemcitabine (compound 2c) was
100-fold less inhibitory toward HEL cell growth (CC50 = 0.34
μM), which is in agreement with what was previously observed
for other ProTides of gemcitabine. The introduction of a
fluorine at the 4′-position of gemcitabine yielded compound
1b, which was in general less active against VZV and HCMV.
Remarkably, an EC50 of 0.042 μM was observed against the
TK+ strain of VZV, making it only 2-fold less active than
gemcitabine. Moreover, 1b displayed a 30-fold lower cytostatic
effect than gemcitabine (CC50 = 0.11 μM vs 0.0036 μM for
gemcitabine).
Interestingly, phosphoramidate prodrug 2b is less active than
the parent nucleoside 1b, showing moderate activity against
the TK+ and TK− strains of VZV with EC50 values in the 2−3
μM range. However, a 700-fold lower cytostatic activity was
observed for ProTide 2b in comparison with the parent
compound 1b. Consequently, compound 2b showed a
selectivity index (SI) of 36, which was higher than those of
1b (SI = 2) and 2c (SI = 4). On the other hand, the 4′-
methoxy gemcitabine analogue 1a and the 4′-substituted
uridine analogues 12a and 12b were completely devoid of
activity against VZV and HCMV.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00485.
■
sı
Experimental details and characterization data for the
reported compounds, NMR spectra, and biological
assays (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Graciela Andrei − Laboratory of Virology and Chemotherapy,
Rega Institute for Medical Research, KU Leuven, 3000
Leuven, Belgium; Email: graciela.andrei@kuleuven.be
In a second screening assay, the cytosine-containing
compounds were tested against two different clinical isolates
of SARS-CoV-2 in Vero cells (Table 2). Remdesivir and
hydroxychloroquine were included as positive controls. As
expected, the high cytotoxicity of gemcitabine in Vero cells
precluded selective antiviral activity. The application of the
ProTide technology to gemcitabine (affording compound 2c)
led to decreased cytotoxicity but also lack of activity against
SARS-CoV-2. The introduction of a 4′-methoxy group
afforded compound 1a and the corresponding prodrug 2a,
both of which were devoid of antiviral activity and cytotoxicity.
The presence of a 4′-fluorine substituent (compound 1b)
reduced the cytotoxicity of gemcitabine, although no selective
antiviral activity was observed. The 4′-fluorophosphoroamidate
prodrug 2b was less cytostatic (CC50 = 1.44 μM) than the
parent nucleoside 1b and displayed minimal antiviral activity.
Authors
Zihua Zheng − Laboratory of Virology and Chemotherapy,
Rega Institute for Medical Research, KU Leuven, 3000
Leuven, Belgium
Elisabetta Groaz − Medicinal Chemistry, Rega Institute for
Medical Research, KU Leuven, 3000 Leuven, Belgium
Robert Snoeck − Laboratory of Virology and Chemotherapy,
Rega Institute for Medical Research, KU Leuven, 3000
Leuven, Belgium
Steven De Jonghe − Laboratory of Virology and
Chemotherapy, Rega Institute for Medical Research, KU
Leuven, 3000 Leuven, Belgium; orcid.org/0000-0002-
3872-6558
Piet Herdewijn − Medicinal Chemistry, Rega Institute for
Medical Research, KU Leuven, 3000 Leuven, Belgium;
orcid.org/0000-0003-3589-8503
D
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Syndrome Coronavirus Infection. Antimicrob. Agents Chemother.
2014, 58, 4885−4893.
Complete contact information is available at:
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(11) Zhang, Y.-N.; Zhang, Q.-Y.; Li, X.-D.; Xiong, J.; Xiao, S.-Q.;
Wang, Z.; Zhang, Z.-R.; Deng, C.-L.; Yang, X.-L.; Wei, H.-P.; Yuan,
Z.-M.; Ye, H.-Q.; Zhang, B. Gemcitabine, Lycorine and Oxy-
sophoridine Inhibit Novel Coronavirus (SARS-CoV-2) in Cell
Culture. Emerging Microbes Infect. 2020, 9, 1170−1173.
(12) Patent application WO 2014209979 A1.
Author Contributions
§P.H. and G.A. contributed equally to this work. All of the
authors approved the final version of the manuscript.
Notes
The authors declare no competing financial interest.
(13) Patent application WO 2015054465 A1.
(14) Wang, G. Y.; Deval, J.; Hong, J.; Dyatkina, N.; Prhavc, M.;
Taylor, J.; Fung, A.; Jin, Z. N.; Stevens, S. K.; Serebryany, V.; Liu, J.
W.; Zhang, Q. L.; Tam, Y. E.; Chanda, S. M.; Smith, D. B.; Symons, J.
A.; Blatt, L. M.; Beigelman, L. Discovery of 4′-Chloromethyl-2′-
deoxy-3′,5′-di-O-isobutyryl-2′-fluorocytidine (ALS-8176), A First-in-
Class RSV Polymerase Inhibitor for Treatment of Human Respiratory
Syncytial Virus Infection. J. Med. Chem. 2015, 58, 1862−1878.
(15) Martinez-Montero, S.; Deleavey, G. F.; Kulkarni, A.; Martin-
Pintado, N.; Lindovska, P.; Thomson, M.; Gonzalez, C.; Gotte, M.;
Damha, M. J. Rigid 2′,4′-Difluororibonucleosides: Synthesis, Con-
formational Analysis, and Incorporation into Nascent RNA by HCV
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(16) Martinez-Montero, S.; Deleavey, G. F.; Dierker-Viik, A.;
Lindovska, P.; Ilina, T.; Portella, G.; Orozco, M.; Parniak, M. A.;
Gonzalez, C.; Damha, M. J. Synthesis and Properties of 2′-Deoxy-
2′,4′-difluoroarabinose-Modified Nucleic Acids. J. Org. Chem. 2015,
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ACKNOWLEDGMENTS
■
Z.Z. thanks the China Scholarship Council (CSC) for funding
(Grant 201704910837). The authors are grateful to Mr. Brecht
Dirix for excellent technical assistance in the antiviral assays.
ABBREVIATIONS
HCMV, human cytomegalovirus; SARS-CoV-2, severe acute
respiratory syndrome coronavirus 2; VZV, varicella zoster virus
■
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