Discovery of potential dual-target prodrugs of HIV-1 reverse transcriptase and nucleocapsid protein 7

Article abstract of DOI:10.1016/j.bmcl.2020.127287

In the present work, we described the design, synthesis and biological evaluation of a novel series of potential dual-target prodrugs targeting the HIV-1 reverse transcriptase (RT) and nucleocapsid protein 7 (NCp7) simultaneously. Among them, the most effective compound 7c was found to inhibit HIV-1 wild-type (WT) strain at double-digit nanomolar concentration (EC₅₀ = 42 nM) in MT-4 cells, and sub-micromole (EC₅₀ = 0.308 μM) to inhibit HIV-1 NL4-3 strain in TZM-bl cells. This is a significant improvement over the parent drug MT. In addition, it showed moderate inhibitory potency (EC₅₀ = 1.329 μM) against the HIV-1 K103N/Y181C double mutant strain (MT-4 cells). The metabolic stability in human plasma of compound 7c indicated that it can release the active forms of the parent drugs MT and AZT in a linear time-independent manner and turn out to be a potential prodrug.

Full text of DOI:10.1016/j.bmcl.2020.127287

Journal Pre-proofs
Discovery of potential dual-target prodrugs of HIV-1 reverse transcriptase and
nucleocapsid protein 7
Songkai Sun, Boshi Huang, Zhuo Li, Zhao Wang, Lin Sun, Ping Gao,
Dongwei Kang, Chin-Ho Chen, Kuo-Hsiung Lee, Dirk Daelemans, Erik De
Clercq, Christophe Pannecouque, Peng Zhan, Xinyong Liu
PII:
S0960-894X(20)30397-8
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127287
BMCL 127287
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 February 2020
15 May 2020
23 May 2020
Please cite this article as: Sun, S., Huang, B., Li, Z., Wang, Z., Sun, L., Gao, P., Kang, D., Chen, C-H., Lee, K-
H., Daelemans, D., De Clercq, E., Pannecouque, C., Zhan, P., Liu, X., Discovery of potential dual-target
prodrugs of HIV-1 reverse transcriptase and nucleocapsid protein 7, Bioorganic & Medicinal Chemistry Letters
(2020), doi: https://doi.org/10.1016/j.bmcl.2020.127287
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Discovery of potential dual-target prodrugs of HIV-1 reverse transcriptase
and nucleocapsid protein 7
Songkai Suna, Boshi Huanga, Zhuo Lia, Zhao Wanga,Lin Suna, Ping Gaoa, Dongwei Kanga, Chin-
Ho Chenb, Kuo-Hsiung Leec,d, Dirk Daelemanse, Erik De Clercqe, Christophe Pannecouquee,
Peng Zhana,*, Xinyong Liua,*
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology, Ministry of Education,
School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Ji’nan, 250012
b Duke University Medical Center, Box 2926, SORF, Durham, North Carolina 27710, United States.
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North
Carolina, Chapel Hill, North Carolina 27599-7568, United States.
d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung
40402, Taiwan.
e Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U.Leuven,
Herestraat 49 Postbus 1043 (09.A097), B-3000 Leuven, Belgium.
Abstract
In the present work, we described the design, synthesis and biological evaluation of a novel
series of potential dual-target prodrugs targeting the HIV-1 reverse transcriptase (RT) and
nucleocapsid protein 7 (NCp7) simultaneously. Among them, the most effective compound 7c
was found to inhibit HIV-1 wild-type (WT) strain at double-digit nanomolar concentration (EC50
= 42 nM) in MT-4 cells, and sub-micromole (EC50 = 0.308 μM) to inhibit HIV-1 NL4-3 strain in
TZM-bl cells. This is a significant improvement over the parent drug MT. In addition, it showed
moderate inhibitory potency (EC50 = 1.329 μM) against the HIV-1 K103N/Y181C double mutant
strain (MT-4 cells). The metabolic stability in human plasma of compound 7c indicated that it
can release the active forms of the parent drugs MT and AZT in a linear time-independent
manner and turn out to be a potential prodrug.
Keywords: HIV-1, NCp7, RT, Dual-target prodrug, Anti-HIV activity, Metabolic stability
Acquired immune deficiency syndrome (AIDS), primarily caused by human
immunodeficiency virus type 1 (HIV-1), remains an urgent problem in the world. Currently,
combination antiretroviral therapy (cART) targeting different proteins/steps in the HIV-1 life

cycle contributes significantly to the success in treating HIV-1 infections [1,2]. Although new
antiretroviral therapy for HIV has been successfully applied, infections caused by drug-resistant
strains are also observed in treatment-intensive locations at the same time [3]. Therefore, there is
an urgent need to exploit novel anti-HIV drugs with new scaffolds and mechanisms of action.
HIV-1 nucleocapsid protein 7 (NCp7), a 55-amino acid portion of the HIV-1 Gag
polyprotein, has attracted significant attention as a next-generation target due to its highly
conservative feature and pivotal role at many steps of the viral replication cycle [4]. For HIV-1
replication, NCp7 must bind to viral RNA via two zinc knuckle motifs (C-X2-C-X4-H-X4-C
pattern), templating the assembly of multiple Gag polyproteins at the membrane surface to direct
formation of a new viral particle [5]. NCp7 also plays an important role in HIV-1 reverse
transcription. Interactions between RT and NCp7 are required for the formation of the HIV-1 RT
initiation complex. In the presence of NCp7, HIV-1 reverse transcriptase (RT) changes
conformation after the binding of the protein and primer-template. NCp7-faciliated annealing
changes to a high-efficiency conformation after the formation of the double stranded primer-
template [6]. At the completion of proviral DNA synthesis, interference in these steps leads to the
improper HIV-1 maturation and loss of viral infectivity.
The compound S-acyl-2-mercaptobenzamide thioester (SAMT) only binds to the C-
terminus of the zinc finger of the NCp7 protein and has a high selectivity. Inhibitors first
acetylate Cys39 in zinc knuckle motifs of the protein through nucleophilic interaction, and then
transfer to Lys33 and Lys38 through intramolecular acyl groups. Eventually, the zinc finger
structure is changed and the zinc ion is expelled to inactivate the protein. Subsequently, SAMT is
transferred to intramer acyl group to obtain 2-mercaptobenzamide thioester (MT). MT is
acetylated with intracellular acetyl-CoA to obtain SAMT, which plays its role again (Fig. 1).
Through recycling, the half-life of the drug is increased and the dosage of the drug is reduced.
Therefore, SAMT and MT are good anti-HIV drug candidates [7]. However, compared with most
drugs used to treat HIV-1 infection, the antiviral activity of NCp7 inhibitors SAMT and MT is
significantly weaker. At the same time, the thioester bond in SAMT is highly susceptible to
hydrolysis, making them difficult to formulate as a therapeutic product. Therefore, it is generally
believed that prodrug modification of MT can improve its antiviral activity and stability [8].

ZF~Cys-SCOCH3
ZF~Cys-SH
O
S
SH
H
H
N
NH2
O
N
NH2
O
O
O
SAMT
MT
AcCoA
Fig. 1. In vivo transformation of inhibitors SAMT and MT [7]
.
Nucleoside HIV-1 reverse transcriptase inhibitors (NRTIs), the earliest anti-HIV drug
applied in clinical practice, still plays an important role nowadays. Zidovudine (AZT) is the first
anti-AIDS drug in the world approved by the US FDA because of its good antiviral activity and
is still the most basic component of combination antiretroviral therapy [9]. However, with the
appearance of drug-resistant strains, there is an urgent need to modify their structural resistance.
The design idea of dual-target prodrugs is that a single chemical entity molecule with two
pharmacophores related to activity can simultaneously act on two targets of the same disease or
two domains of a single target [10]. Dual-target prodrugs are composed of four moieties: two
parent drugs, a spacer, and a specifier. The specifier, as a substrate for a specific enzyme, is often
connected to the parent drug via a selfimmolative spacer. The spacer is incorporated to promote
enzymatic cleavage of the specifier, and must spontaneously eliminate to release the desired
active agent after specifier removal [11-13]. Single-component multi-target drugs of the same
disease have an obvious advantage over single-target drugs in the treatment of dysfunctional
diseases [14-16]. There have been many articles reporting dual-target prodrugs of HIV-1 inhibitors
[17-19]. Since NCp7 cooperates with reverse transcriptase, we designed to link NCp7 inhibitor
(MT) and reverse transcriptase inhibitor (AZT) through an ester bond linker to form a dual-target
prodrug. Hydrolyzed by esterases, these prodrugs can release the original drugs to play a role
(Fig. 2). We expect these compounds to have the advantages of their parent drugs, including
activity against HIV-1 wild strain and resistant strain [20]. And we intend to explore how different
lengths and types of linkers affect the antiviral activity of compounds.

O
NH
N
O
O
O
SH
Drug design based on
dual-target prodrug strateg
S
O
O
linker
NH
HO
OH
linker
O
y
+
+
N
N3
O
O
O
O
O
O
O
HN
O
HN
HO
O
N3
NH2
7a-7e
Dual-target prodrug
OH
Zidovudine(NRTI)
MT (NCp7 inhibitor)
SH
O
S
O
OH
HCHO
HN
HN
O
spontaneously
O
O
NH
N
NH2
NH2
HO
OH
linker
O
O
O
O
2-mercaptomethanol benzamide thioester
MT
S
O
O
linker
N3
O
O
O
O
Esterase hydrolysis
HN
O
7a-7e
NH
N
OH
O
linker=
linker=
7a,7b,7c
7d,7e
O
n=1,2,3
n
HO
R1=H, R2=CH3
R1=R2=CH3
N3
R
1 R2
Zidovudine (AZT)
Fig. 2. Design and metabolism of NCp7 and RT targeted dual prodrugs
The synthetic routes for the newly designed dual-target prodrugs are outlined in Scheme 1.
We synthesize the compound MT based on previous reports [8]. And then we combined MT,
AZT and a linker containing ester bonds to form the target compound. The target compounds 7a-
7e were fully characterized by mass spectra (MS), proton nuclear magnetic resonance (1H NMR)
and carbon nuclear magnetic resonance (13C NMR). The purity of compounds 7a-7e was
checked by high performance liquid chromatography (HPLC).
SH
O
O
N
H
NH2
NH2
O
SH
S
S
i
ii
HN
COOH
O
H
N
O
NH
NH2
O
O
N
O
1
O
3
2
S
O
linker
O
v
N3
O
O
O
HN
O
O
O
NH2
NH
NH
NH
N
N
N
O
7a-7e
O
iii
O
iv
O
O
O
Cl
O
O
linker
HO
O
linker
HO
N3
O
O
N3
O
O
N3
5a-5e
6a-6e
4

Scheme 1. Reagents and conditions: (i) β-alaninamide·HCL, HBTU, DIEA, DMF, r.t., overnight;(ii)
TCEP·HCL, DIEA, DCM/water=9/1, r.t.,4h; (iii) Corresponding acid anhydride, DMAP, DMF, r.t.,12h;
-
(iv) NaHCO3, Bu4N+ •HSO4 , DCM, r.t., 4 h; (v) K2CO3, DMF,70℃ reflow, overnight;
The antiviral activity of compounds 7a-7e, was evaluated in MT-4 cell cultures infected
with HIV-1 WT strain (IIIB), K103N/Y181C (RES056), one of the reverse transcriptase inhibitor
resistant double-mutant strain observed in clinic. Zidovudine (AZT) and 2-mercaptobenzamide
thioester (MT) were chosen as reference drugs. The results, including EC50, CC50, SI (selectivity
index, CC50/EC50 ratio) and FR (fold resistance, EC50mutant/EC50wt ratio), are summarized in
Table 1.
Table 1.
Antiviral potency against HIV-1 IIIB and RES056 strains and cytotoxicity in MT-4 cells.
Compd.
Linker
EC50 (μM)a
RES056
CC50 (μM)b
SIc
FRd
IIIB
IIIB
959
RES056
51.77
7a
7b
0.129 ± 0.099 2.402 ± 0.36
0.068 ± 0.011 1.619 ± 0.06
0.042 ± 0.025 1.329 ± 0.39
0.044 ± 0.017 1.963 ± 0.47
0.805 ± 0.511 104.4 ± 21.3
5.261 ± 2.319 7.936 ± 0.13
0.011 ± 0.001 1.197 ± 0.07
124.35
>202.39
> 197.89
> 197.89
> 193.59
332.44
18.6
23.8
31.6
44.6
129.7
1.5
> 2994
> 4660
> 4500
> 240
63
> 125.0
> 148.9
> 100.8
> 1.854
41.89
7c
7d
7e
MT
AZT
> 7.48
> 645
> 6.249
108.8
a EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-
1-induced cytotoxicity, as determined by the MTT method.
b CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
determined by the MTT method.
c SI: selectivity index, the ratio of CC50/EC50.
d FR: fold resistance, ratio of EC50 value against K103N/Y181C double mutant type HIV-1 over EC50
value against WT HIV-1 (EC50mutant/EC50wt).

As shown in Table 1, it can be observed that some newly synthesized dual-target prodrugs
are more active than the parent drug MT, and are comparable or slightly weaker than AZT. At
the same time, the toxicity and selectivity index of these prodrugs on MT4 cells are superior to
the two parent drugs. This series of prodrugs showed moderate to excellent activity against the
WT HIV-1 strain with EC50 values ranging from 0.042 μM to 0.805 μM. Among them,
compound 7c exhibited highest potency against WT HIV-1 strain with an EC50 value of 0.042
μM, which was up to 125 times more active than that of parent drug MT (EC50 = 5.261 μM) and
was comparable to AZT (EC50 = 0.011 μM). Moreover, 7c was found to be a potent inhibitor
against the HIV-1 double mutant strain RES056 (EC50 = 1.329 μM). It can be seen that 7c is a
potential prodrug based on HIV-1 RT with micromolar inhibitory activity against the most
common HIV-1 RT inhibitor-resistant strains. Both anti-HIV activity and cytotoxicity are
determined after 5-day incubation in cell-based assays using MTT method [21], the prodrug
molecule should possibly transform to its parent drugs entirely and function as HIV-1 inhibitor.
In brief, compound 7c is active against the WT and mutant HIV-1 strains (FR value = 31.6).
Next, in order to verify the rationality of our dual-target prodrug strategy, we sought to test
the newly synthesized compounds in a multicycle assay using fully infectious HIV-1 wild-type
(NL4-3) virus and TZM-bl target cells for invitro anti-HIV-1 activity. Parent drugs MT and AZT
were included as an in-line control to allow for direct comparison with the new compounds.
Besides, the toxicity of MT, AZT and the new compounds towards the TZM-bl cells was also
assessed. Table 2 shows the anti- HIV potency (EC50, as measured by a luciferase gene
expression assay [22,23]), cytotoxicity (CC50) as well as selectivity index (SI, the ratio of
CC50/EC50) for each of the compounds and control. As shown in Table 2, it is obvious that
prodrug 7c exhibited the best activity against HIV-1 NL4-3 virus with the lowest EC50 values
(EC50 = 0.308 μM). Compared with the parent drug MT (EC50 = 2.363 μM), the activity has been
greatly improved, which proved the rationality of the dual-target prodrug strategy again.
However, the activity of this series of compounds has not exceeded AZT, and further
modification is needed. In addition, the antiviral activity results suggested that the linker length
and the number of branches showed significant impact on the antiviral activity of this series of
dual-target prodrugs. To be specific, prodrugs which bear a longer linker exhibited more potent
antiviral activity (EC50: 7a>7b>7c), and the activity of the prodrug is similar when there is only
one branch in the linker, but the activity decreases significantly when the linker contains two

branches(EC50: 7b≈7d<<7e). We speculate that the steric effect may affects the decomposition of
prodrug molecules, thereby affecting the release of the parent drug and reducing the activity.
Table 2.
Anti-HIV-1 activity and cytotoxicity of the novel phenylalanine derivatives in TZM-bl cells infected with
the HIV-1 NL4-3 virus.
Compounds
Linker
EC50 (μM)a
CC50 (μM)b
SIc
7a
0.414 ± 0.084
> 1.656
> 4.00
7b
7c
0.332 ± 0.086
0.308 ± 0.085
> 1.619
> 1.583
> 5.26
> 4.76
7d
7e
0.522 ± 0.131
> 1.55
> 1.583
> 1.548
> 3.03
> 1.00
MT
2.363 ± 0.758
0.016 ± 0.005
82.486 ± 6.911
> 0.374
> 34.91
> 23.37
AZT
a EC50: the concentration of the compound required to achieve 50% protection of TZM-bl cells against
HIV-1-induced cytopathic effect, determined in at least triplicate against HIV-1 in TZM-bl cells; values
are the mean ± SD of at least two parallel tests.
b CC50: the concentration of the compound required to reduce the viability of uninfected cells by 50%,
determined in at least triplicate against HIV-1 in TZM-bl cells; values were averaged from at least four
independent experiments.
c SI: selectivity index, the ratio of CC50/EC50.
Most importantly, to verify our hypothesis, 7c was further assessed for its ability of
conversion to the parent drugs MT and AZT in vitro. Incubate 7c in human plasma (pH 7.4 ±
0.1) at 37 °C for 1 hour, and analyze the content of prodrug and parent drugs in plasma by LC-
MS/MS. The dual-target prodrug 7c was completely hydrolyzed by enzyme after 10 minutes of
incubation. Accordingly, the content of the parent drugs MT and AZT increased over time (Fig.
3). To evaluate whether compound 7c indeed acts as a potential prodrug, we further checked if
the formation amount of MT was linearly with decrease amount of 7c (within 10min). As
revealed in Fig. 4, the linear curve fitted well (R2 = 0.99), indicating that compound 7c possibly

functions as a prodrug. Definitely, we should admit that the release of the parent MT from 7c
was in a quite fast manner, which is undergoing further investigation in our lab.
110
% Remaining of 7c
100
% Formation of MT
90
% Formation of AZT
80
70
60
50
40
30
20
10
0
0
20
40
60
Incubation time (min)
Fig. 3. % Remaining of prodrug 7c and % formation of its parent drug MT and AZT during the 1 h test.
Measured in triplicate for each time point.
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.5
1
1.5
2
2.5
Concentration of decreased prodrug 7c
(μM)
Fig. 4. Linear fit of the reduced micromolar concentration of the prodrug 7c and the micromolar
concentration of the formed MT (within 10 minutes)
In summary, we have designed compounds 7a-7e as potential dual-target prodrugs of HIV-1
reverse transcriptase and NCp7 through a self-destructive spacer degradation. We evaluated
these compounds, along with the parent drug MT and AZT for their activities against WT and
the most challenging K103N/Y181C double-mutant strain of HIV-1 in MT-4 cells, as well as
NL4-3 virus in TZM-bl cells. 7c was found to inhibit the WT HIV-1 strain at nanomolar
concentrations and the NL4-3 virus strain at submicromolar concentration. Compared with the
parent drug MT, its activity is greatly improved, which is comparable to or slightly weaker than

AZT. Especially, compound 7c showed potent activity against the HIV-1 K103N/Y181C double
mutant, which is the first discovery of a potential dual-target prodrug inhibitor based on HIV-1
RT and NCp7. Metabolic stability test in human plasma demonstrated that 7c could release its
parent drugs MT and AZT in a linearly time-independent manner (during the 10min), and
therefore likely to be a potential prodrug. Based on the obtained results, compound 7c was
initially shown to be a promising dual target prodrug of RT and NCp7.
Acknowledgements
We gratefully acknowledge financial support from the National Natural Science Foundation of
China (NSFC Nos. 81573347, 81973181, 81903453), the Key Project of NSFC for International
Cooperation (No. 81420108027), Shandong Provincial Key research and development project
(Nos. 2017CXGC1401, 2019JZZY021011), the Taishan Scholar Program at Shandong Province,
KU Leuven (GOA 10/014), and NIH MERIT Award R37 AI027690 (to E.A.). The technical
assistance of Kris Uyttersprot and Kristien Erven, for the HIV experiments, is gratefully
acknowledged.
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Highlights
1. Dual-target prodrug of HIV-1 reverse transcriptase inhibitor and NCp7 inhibitor.
2. Dual target prodrug modification improves antiviral activity and selectivity index.
3. The compound releases parent drugs linearly in the plasma, with prodrug properties.
Table 1.
Antiviral potency against HIV-1 IIIB and RES056 strains and cytotoxicity in MT-4 cells.

Compd.
Linker
EC50 (μM)a
RES056
CC50 (μM)b
SIc
FRd
IIIB
IIIB
959
RES056
51.77
7a
7b
0.129 ± 0.099 2.402 ± 0.36
0.068 ± 0.011 1.619 ± 0.06
0.042 ± 0.025 1.329 ± 0.39
0.044 ± 0.017 1.963 ± 0.47
0.805 ± 0.511 104.4 ± 21.3
5.261 ± 2.319 7.936 ± 0.13
0.011 ± 0.001 1.197 ± 0.07
124.35
>202.39
> 197.89
> 197.89
> 193.59
332.44
18.6
23.8
31.6
44.6
129.7
1.5
> 2994
> 4660
> 4500
> 240
63
> 125.0
> 148.9
> 100.8
> 1.854
41.89
7c
7d
7e
MT
AZT
> 7.48
> 645
> 6.249
108.8
a EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-
1-induced cytotoxicity, as determined by the MTT method.
b CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
determined by the MTT method.
c SI: selectivity index, the ratio of CC50/EC50.
d FR: fold resistance, ratio of EC50 value against K103N/Y181C double mutant type HIV-1 over EC50
value against WT HIV-1 (EC50mutant/EC50wt).
Table 2.
Anti-HIV-1 activity and cytotoxicity of the novel phenylalanine derivatives in TZM-bl cells infected with
the HIV-1 NL4-3 virus.
Compounds
Linker
EC50 (μM)a
CC50 (μM)b
SIc
7a
0.414 ± 0.084
> 1.656
> 4.00
7b
7c
0.332 ± 0.086
0.308 ± 0.085
> 1.619
> 1.583
> 5.26
> 4.76
7d
0.522 ± 0.131
> 1.583
> 3.03

7e
> 1.55
> 1.548
> 1.00
MT
2.363 ± 0.758
0.016 ± 0.005
82.486 ± 6.911
> 0.374
> 34.91
> 23.37
AZT
a EC50: the concentration of the compound required to achieve 50% protection of TZM-bl cells against
HIV-1-induced cytopathic effect, determined in at least triplicate against HIV-1 in TZM-bl cells; values
are the mean ± SD of at least two parallel tests.
b CC50: the concentration of the compound required to reduce the viability of uninfected cells by 50%,
determined in at least triplicate against HIV-1 in TZM-bl cells; values were averaged from at least four
independent experiments.
c SI: selectivity index, the ratio of CC50/EC50.
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

ZF~Cys-SCOCH3
ZF~Cys-SH
O
S
SH
H
H
N
NH2
O
N
NH2
O
O
O
SAMT
MT
AcCoA
Fig. 1. In vivo transformation of inhibitors SAMT and MT.
O
NH
N
O
O
O
SH
Drug design based on
dual-target prodrug strateg
S
O
O
linker
NH
HO
OH
linker
O
y
+
+
N
N3
O
O
O
O
O
O
O
HN
O
HN
HO
O
N3
NH2
7a-7e
Dual-target prodrug
OH
Zidovudine(NRTI)
MT (NCp7 inhibitor)
SH
O
S
O
OH
HCHO
HN
HN
O
spontaneously
O
O
NH
N
NH2
NH2
HO
OH
linker
O
O
O
O
2-mercaptomethanol benzamide thioester
MT
S
O
O
linker
N3
O
O
O
O
Esterase hydrolysis
HN
O
7a-7e
NH
N
OH
O
linker=
linker=
7a,7b,7c
7d,7e
O
n=1,2,3
n
HO
R1=H, R2=CH3
R1=R2=CH3
N3
R
1 R2
Zidovudine (AZT)
Fig. 2. Design and metabolism of NCp7 and RT targeted dual prodrugs

110
100
90
80
70
60
50
40
30
20
10
0
% Remaining of 7c
% Formation of MT
% Formation of AZT
0
20
40
60
Incubation time (min)
Fig. 3. % Remaining of prodrug 7c and % formation of its parent drug MT and AZT during the 1 h test.
Measured in triplicate for each time point.
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.5
1
1.5
2
2.5
Concentration of decreased prodrug 7c
(μM)
Fig. 4. Linear fit of the reduced micromolar concentration of the prodrug 7c and the micromolar
concentration of the formed MT (within 10 minutes)
Graphical abstract

O
NH
N
O
O
O
SH
Drug design based on
dual-target prodrug strateg
S
O
O
linker
NH
HO
OH
linker
O
y
+
+
N
N3
O
O
O
O
O
O
O
HN
O
HN
HO
O
N3
Zidovudine(NRTI)
EC
7a-7e
Dual-target prodrug
NH2
MT (NCp7 inhibitor)
EC
OH
EC
50 (μM)
=
50 (μM)
=
50 (μM)
=
0.042 ~ 0.129 (MT4, IIIB)
1.329 ~ 104.4 (MT4,RES056)
0.308 ~ >1.55 (TZM-bl, NL4-3)
5.261 (MT4, IIIB)
7.936 (MT4,RES056)
2.363 (TZM-bl, NL4-3)
0.011 (MT4, IIIB)
1.197 (MT4,RES056)
0.016 (TZM-bl, NL4-3)
CC
CC
50 (μM) =
CC
50 (μM)
=
50 (μM)
=
124.53 ~ >202.39 (MT4)
> 1.548 (TZM-bl)
> 7.48 (MT4)
> 0.347 (TZM-bl)
332.44 (MT4)
82.49 (TZM-bl)