Design and synthesis of novel distamycin-modified nucleoside analogues as HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.antiviral.2013.12.002

Design and synthesis of nucleoside analogues have persistently attracted extensive interest because of their potential application in the field of antiviral therapy, and its study also receives additional impetus for improvement in the ProTide technology. Previous studies have made great strides in the design and discovery of monophosphorylated nucleoside analogues as potential kinase-independent antiretrovirals. In this work, a series of nucleoside phosphoramidates modified by distamycin analogues was synthesized and evaluated as nucleoside reverse transcriptase inhibitors (NRTIs) in HIV-1-infected MT-4 and CEM cells, including variations in nucleoside, alkyl moiety, and the structure of distamycin analogues. These compounds exhibited modest potency with the EC₅₀ value in the range of 1.3- to 6.5-fold lower than their corresponding parent drugs in MT-4 cells, which may be attributed to increasing intracellular availability due to the existence of distamycin analogue with favorable hydrophilic-lipophilic equilibrium. Meanwhile, the length of distamycin analogue was considered and assessed as an important factor that could affect antiviral activity and cytotoxicity. Enzymatic and metabolic stability studies have been performed in order to better understand the antiviral behavior of these compounds. The present work revealed the compounds to have a favorable and selective anti-HIV-1 activity in MT-4 and CEM cells, and helped to develop strategies for design and synthesis of effective monophosphorylated nucleoside analogues, which may be applied to antiretroviral research as NRTIs.

Full text of DOI:10.1016/j.antiviral.2013.12.002

Antiviral Research 102 (2014) 54–60
Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Design and synthesis of novel distamycin-modified nucleoside
analogues as HIV-1 reverse transcriptase inhibitors
c
Chao Li a, Chunying Ma a, Jin Zhang a, Ning Qian a, Jingjing Ding a, Renzhong Qiao a,b, , Yufen Zhao
⇑
a State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China
b State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, PR China
c Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Design and synthesis of nucleoside analogues have persistently attracted extensive interest because of
their potential application in the field of antiviral therapy, and its study also receives additional impetus
for improvement in the ProTide technology. Previous studies have made great strides in the design and
discovery of monophosphorylated nucleoside analogues as potential kinase-independent antiretrovirals.
In this work, a series of nucleoside phosphoramidates modified by distamycin analogues was synthesized
and evaluated as nucleoside reverse transcriptase inhibitors (NRTIs) in HIV-1-infected MT-4 and CEM
cells, including variations in nucleoside, alkyl moiety, and the structure of distamycin analogues. These
compounds exhibited modest potency with the EC50 value in the range of 1.3- to 6.5-fold lower than their
corresponding parent drugs in MT-4 cells, which may be attributed to increasing intracellular availability
due to the existence of distamycin analogue with favorable hydrophilic–lipophilic equilibrium. Mean-
while, the length of distamycin analogue was considered and assessed as an important factor that could
affect antiviral activity and cytotoxicity. Enzymatic and metabolic stability studies have been performed
in order to better understand the antiviral behavior of these compounds. The present work revealed the
compounds to have a favorable and selective anti-HIV-1 activity in MT-4 and CEM cells, and helped to
develop strategies for design and synthesis of effective monophosphorylated nucleoside analogues,
which may be applied to antiretroviral research as NRTIs.
Received 24 August 2013
Revised 9 November 2013
Accepted 8 December 2013
Available online 14 December 2013
Keywords:
Nucleoside analogue
Anti-HIV
Antiretrovirals
Distamycin
ProTide
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
(2009), successfully circumvents the first step of kinase-mediated
activation of nucleosides and thus confers significant antiviral
Nucleoside analogues continue to dominate antiviral therapy
and also make a significant contribution to the chemotherapy of
cancer, HBV (Hepatitis B Virus), and particularly HIV (Human
Immunodeficiency Virus) (Hostetler, 2009; Cihlar and Ray, 2010).
Without exception, nucleoside analogues with such activity re-
quire phosphorylation in vivo to their active nucleotide forms.
However, it appears that the first phosphorylation to the 50-mono-
phosphate is the rate-limiting step, since in many cases the ddN
(20,30-dideoxynucleoside) derivatives have a poor affinity for nucle-
oside kinases (Balzarini et al., 1987; Johnson et al., 1988). Nega-
tively charged nucleoside monophosphates cannot be used as
therapeutic agents because they are unable to cross cell mem-
branes efficiently, and they are readily dephosphorylated on cell
surfaces and in extracellular fluids by non-specific enzymes
(Ahmadibeni et al., 2011). ProTide, as a pronucleotide technology
proposed by McGuigan et al. (2005, 2006) and Mehellou et al.
activity on inactive parent nucleosides. During the past two dec-
ades, various modifications on aryl/alkyl, ester, and amino acid re-
gions of the ProTide and how these changes affect antiviral activity
and metabolic stability were described in many reports to achieve
this goal (McGuigan et al., 1996; Congiatu et al., 2005; Derudas
et al., 2009; Meneghesso et al., 2012). However, research focusing
on how to increase hydrophilic–lipophilic equilibrium of nucleo-
side monophosphorylated analogues with a consequent increase
of intracellular availability could be another key point, and not fre-
quent (Perrone et al., 2007).
Distamycin and its analogues containing three 4-amimo-1-
methyl-pyrrole-2-carboxylate residues have been proved to pos-
sess natural antiviral and antibiotic activity (Grehn et al., 1983,
1986; Pang, 2004). Also, multiple hydrogen-bond donors inter-
acted with the solvent as well as the existence of C-terminal ami-
dine potentially increase polyamide solubility in water and alter
cell permeability (Bremer et al., 2000). Conceivably, it will be a no-
vel and challenging design that distamycin as an important mask-
ing group was introduced into monophosphorylated nucleoside to
complete ProTide approach, and then the intrinsic molecular
⇑
Corresponding author Address: Mailbox 53, 15 Beisanhuan East Road, Chaoyang
District, Beijing 100029, PR China. Tel.: +86 010 64413899; fax: +86 010 82728926.
E-mail addresses: lichao@mail.buct.edu.cn (C. Li), qiao_group@163.com (R. Qiao).
0166-3542/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.antiviral.2013.12.002

C. Li et al. / Antiviral Research 102 (2014) 54–60
55
character of distamycin could endow parent nucleoside with favor-
able intracellular availability with a consequent further increase of
antiviral activity. Fortunately, recent efforts in our laboratory have
been inspired by the natural distamycin composed of N-methyl-
pyrrole carboxamides, and focused on modification of small mole-
cules with the introduction of distamycin analogues to successfully
interfere with gene expression (Li et al., 2008, 2010, 2012). Thus, it
could provide substantial experiences in reasonably constructing
the distamycin nucleoside conjugates as potential anti-HIV
prodrugs.
We here presented the synthesis and biological activity of a
series of distamycin-modified nucleoside analogues, including
variations in nucleoside, alkyl moiety, length of distamycin
analogues, and nitrile/amidine terminus (Fig. 1). All these combi-
nations allowed us to extensively study what extent this difference
might reflect the relative efficiency of phosphorylation, which
might contribute to the progress of antiviral activity, hydro-
philic–lipophilic balance and metabolic stability. This paper de-
scribed our initial attempts in this regard.
firstly generated and then converted to amidine with the introduc-
tion of dry HCl(g) and NH3(g) in proper order in absolute ethanol.
Three main side reactions in the process could occur under differ-
ent conditions, as shown in Scheme 2. Excess ethanol can cause
alcoholysis of Pinner salt to successively get compounds 4 and 5
(orthoester). In the presence of HCl(g) and another equivalent
amount of ethanol, the orthoester could be further converted into
ethyl ester 6 which also directly obtained through Pinner salt
hydrolysis. Temperature is also one of the main parameters that af-
fected the reaction. At higher than 0 °C, heat decomposition easily
occurred in the mixture and resulted in undesired product amide 3.
In addition, strictly controlling gas velocity in low flow was found
to be preferable, since excess gas turned the reaction mixture to be
complex and more than two products detected by TLC.
2.3. In vitro assays of anti-HIV activity and cytotoxicity
MT-4 cells, a human T4-positive cell line were infected with
HIV-1 at the multiplicity of infection (MOI) of 0.1, and HIV-infected
MT4 cells were incubated for 1.5 h at 37 °C in a 5% CO2 incubator.
Subsequently, cells were cultured in 96-well microtiter plates in
the presence of various concentrations of compounds and aliquots
of culture supernatants were removed from the wells on the fifth
day after infection for p24 antigen assays. The applied p24 enzyme
immunoassay (EIA) was the unmodified kinetic which utilizes a
murine mAb to HIV core protein coated onto microwell strips to
which the antigen present in the test culture supernatant samples
binds. Percent viral inhibition was calculated by comparing the
p24 values from untreated infected cells. EC50 (representing the
concentration of compounds reducing HIV-1 replication by 50%)
was determined from the concentration–response curve using
the median effect method.
2. Materials and methods
2.1. Experimental chemistry
Unless otherwise noted, all materials were obtained from com-
mercial suppliers and dried and purified by standard procedures.
1H NMR and 13C NMR spectra were measured on a Bruker AV600
spectrometer, and chemical shifts in ppm are reported relative to
internal Me4Si (CDCl3 and DMSO-d6). MS (ESI) mass spectral data
were recorded on a Finnigan LCQDECA mass spectrometer. HRMS
data were measured using Bruker APEX IV Fourier Transform Ion
Cyclotron Resonance Mass spectrometer. Purities of all target com-
pounds (P98%) were determined by an Agilent 1200 HPLC system
with a Agilent Zorbax SB-Aq C-18 column (4.6 ꢀ 150 mm), UV
detector at 295 nm, mobile CH3CN/H2O (0.6%trifluoroacetic
acid) = 32–67% and flow rate of 1 mL/min. Polyamides with differ-
ent lengths were prepared according to our previous work (Li et al.,
2010). General procedure and data for the synthesis of target com-
pounds were shown in Supporting Information.
CEM/0 or CEM/TK-cells were suspended at 250,000 cells/mL cell
culture medium and infected with approximately 100 CCID50 (1
CCID50 is the 50% cell culture infective dose) of HIV-1. Then,
100
lL of the infected cell suspension was added to the wells of
a 96-well microtiter plate containing 100
lL of an appropriate
dilution of the test compounds. After 4 days, giant cell formation
was recorded microscopically in the HIV-infected CEM cell
cultures.
2.2. By-products in Pinner reaction
Cytotoxicity of the conjugate was evaluated in parallel with
antiviral activity using the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) method. It was based on the viabil-
ity of mock-infected cells, as monitored by the MTT method. CC50
(representing the concentration of compounds reducing cell viabil-
In order to improve the product yield, it should be pointed out
that, effective inhibition of by-products in Pinner reaction must be
not neglected. Pinner salt, an important reactive intermediate,
Fig. 1. Structural features of nucleosides and distamycin-modified nucleoside analogues 1a–1d and 2a–2d.

56
C. Li et al. / Antiviral Research 102 (2014) 54–60
Scheme 1. Reagents and conditions: (i) PCl3, CH2Cl2; (ii) R1-OH; (iii) Et3N; (iv) Et3N/CCl4, THF/DMF; (v) HCl/EtOH, NH3(q), EtOH.
Scheme 2. The side reactions in Pinner reaction: hydrolysis, alcoholysis, and heat decomposition.
ity by 50%) was determined from the concentration–response
curve using the median effect method.
solubility assay. The partition coefficient (log P) was calculated
according to the following equation:
½drugꢂo
logP ¼ log
2.4. Solubility and permeability
½drugꢂaq
where [drug]o is the drug concentration in the organic phase and
[drug]aq is the drug concentration in the aqueous phase. In addition,
Clog P values were calculated with a software package the Molecu-
lar Operating Environment (MOE).
Solubility was measured at pH 7.4 by using an HPLC–UV meth-
od. Test compounds were initially dissolved in DMSO at
10 mg mLꢁ1. 10
lL of this stock solution was spiked into pH 7.4
phosphate buffer (1.0 mL) with the final DMSO concentration
being 1%. The mixture was stirred for 4 h at room temperature
and then concentrated at 3000 rpm for 10 min. The saturated
supernatants were transferred to other vials for analysis by
HPLC–UV. Each sample was performed in triplicate. For quantifica-
tion, analysis was carried out on Waters 2695 system equipped
with a Diode Array Detector. A SB C18 250 ꢀ 4.5 mm column was
used with an isocratic elution at a flow rate of 1.0 mL minꢁ1. The
elution condition was acetonitrile (B) in water (A, with 0.6% triflu-
oroacetic acid) at 40%. Aqueous concentration was determined by
comparison of the peak area of the saturated solution with a stan-
dard curve plotted peak area versus known concentrations.
The octanol–water partition coefficient was determined by the
traditional shake-flask technique. To 2 mL of a buffer solution
(pH 7.4) containing 100 lM of each compound, 2 mL of n-octanol,
previously saturated with buffer solution, were added. The mixture
was shaken for 40 min, centrifuged and the aqueous and organic
2.5. In vitro metabolic stability
Pooled human liver microsomes (HLM) were purchased from
Sigma–Aldrich. An incubation mixture with a final volume of
0.1 mL consisted of human liver microsomes in 50 mM KH2PO4–
K2HPO4 phosphate buffer (pH 7.4) and 0.5
The concentration of human liver microsomes was 0.4 mg mLꢁ1
Reactions were started by adding 80 L of NADPH (final concentra-
lM test compound.
.
l
tion of 1.0 mM). The mixture was incubated at 37 °C for 0 and
30 min. The reaction was terminated by the addition of acetonitrile
equivalent to the volume of the reaction mixture. All incubations
were made in triplicate. Analysis was carried out on Waters 2695
equipped with a Diode Array Detector. A SB C18 250 ꢀ 4.5 mm col-
umn was used with a gradient elution at a flow rate of
1.0 mL minꢁ1. The initial elution condition was acetonitrile (B) in
water (A, with 0.6% trifluoroacetic acid) at 30%. The concentration
of B increased linearly to 90% over 30 min. The mobile phase was
phases were separated. Samples (10
lL) were injected directly into
HPLC (Waters 2695), and test conditions were the same as

C. Li et al. / Antiviral Research 102 (2014) 54–60
57
then returned to the initial condition and re-equilibrated for 3 min.
The column effluent was analyzed using a Bruker ESQUIRE-LCTM
ion trap spectrometer equipped with a gas nebulizer probe. The
ion source was operated at 300 °C with the capillary voltage at
11 V. Nitrogen was used as drying gas at a flow rate of 4 L/min.
For metabolic stability determinations, chromatograms were
analyzed for parent compound disappearance from the reaction
mixtures. The parent compound peak area in the 0 min incubation
sample was considered to be the 100% value and parent compound
levels were expressed as percent (%) parent remaining.
cessfully achieved conversion from nitrile to amidine group, and
afforded 1c and 1d with moderate yields. Following the same pro-
cedure, treatment of nucleoside AZT gave the corresponding com-
pounds 2a–2d.
3.2. Anti-HIV activity and cytotoxicity
All compounds were evaluated for anti-HIV-1 activity and cyto-
toxicity in human lymphocyte MT-4 and CEM cells, and tests were
also conducted in thymidine kinase-deficient CEM cells (Table 1).
i
Both d4T (1) and its monophosphorylated derivatives pr-1 and
chex-1 as positive controls exhibited proper potencies and toxici-
2.6. Enzymatic assays
ties against HIV-1, with EC50 values of 0.27–4.3
lM and CC50 values
5 mg of the appropriate nucleoside analogue was first dissolved
of 74–245 M in MT-4 and CEM/0 cells, which were consistent
l
in 200 lL of DMSO-d6 and then 400 lL of Tris buffer solution
with the data previously reported (Pokrovsky et al., 2001). The iso-
propyl distamycin phosphoramidate (1b) with a EC50 value of
(pH = 7.4) was added. A 31P NMR was conducted at this stage to
see the peaks of the compound in DMSO-d6 and Tris buffer, so that
it acted as a reference (t = 0 h).To this mixture was added 0.3 mg of
0.08
MT-4 cell than its parent compounds 1 and ipr-1 respectively,
accompanied with a increasing cytotoxicity (CC50 = 98 M, vs
M for pr-1) due to the presence of distamycin moiety. So,
lM showed 3.3- and 3.1-fold inhibitory against HIV-1 in
carboxypeptidase
units mgꢁ1, EC number 3.4.17.1) that had already been dissolved
in 200
L of Tris buffer. 31P NMR of the reaction mixture was then
A
(purchased from Sigma–Aldrich, >50
l
i
>245
l
l
a similar selectivity index (SI) of 1225 was obtained compared to
i
carried out every 1 h for 24 h at 25 °C.
that of pr-1 (1448), but significantly higher than that of 1 (274).
For the cyclohexyl ester derivatives, it is worth pointing out that
3. Results and discussion
the parent compound chex-1 having the bulky cyclohexyl group
as ester moiety did not show ideal activity (EC50 = 1.1
4 cell), however, 1d via modification of distamycin showed a 6.5-
fold increase in anti-HIV potency, being active at 0.17 M. As ex-
lM, in MT-
3.1. Synthesis
l
Depicted in Scheme 1 is our synthetic route developed to allow
for efficient introduction of distamycin moiety on the monopho-
sphorylated d4T through Atherton-Todd reaction under mild con-
ditions. The synthesis of d4T monophosphate started with
treatment of d4T with phosphorus trichloride in CH2Cl2, to give
nucleotide dichloro-phosphate in excellent yield, which was trea-
ted in the mixed solution of the corresponding alcoholysis reagent
and triethylamine to provide the key intermediate. Another inter-
mediate NH2Py3-b-CN containing three monomers was synthe-
sized according to our previous similar work (Li et al., 2010).
Subsequently, the active P-H from monophosphorylated d4T and
NH2 from distamycin backbone were linked through Atherton-
Todd reaction in the presence of tetrachloromethane as catalyst
to obtain distamycin-modified d4T with nitrile-terminal, 1a and
1b. Direct Pinner reaction of these nitrile-terminal products suc-
pected, favorable permeability of distamycin derivative could
weaken steric-hindrance influence and contribute to intracellular
availability, resulting in the increasing of antiviral activity. Addi-
tionally, to better demonstrate function of distamycin moiety,
the anti-HIV-1 activity of nitrile-terminal 1a and 1c were evaluated
and results displayed ꢃ2.0-fold lower activity than their corre-
sponding amidine derivatives, suggesting that positively charged
amidine terminus may influence the realization of intact mem-
brane permeability. This correlated with their CC50 values of
137 lM for 1a and 113 lM for 1c recovered from high cytotoxicity.
Striking differences were found for the antiviral activity of
tested compounds in HIV-1-infected CEM/TK- cells. Compound 1
proved feebly active against HIV-1 replication in CEM/TK- cells at
a concentration as high as ꢃ60
lM. In contrast, the analogues
1a–d showed markedly inhibitory to HIV-1 in CEM/TK- cells. Their
Table 1
Anti-HIV-1 activity and cytotoxicity of distamycin-modified nucleoside conjugates.
R1
R2
EC50
(
lM)
CC50 (lM)
SIc (MT-4)
a
b
Compds.
Nucleoside
MT-4
CEM/0
CEM/TK-
MT-4
CEM/0
1
d4T
d4T
d4T
d4T
d4T
d4T
d4T
AZT
AZT
AZT
AZT
AZT
AZT
AZT
–
H
i-pr
Cyclohex
i-pr
i-pr
Cyclohex
Cyclohex
H
i-pr
Cyclohex
i-pr
–
–
–
-CN
-CNHNH2
-CN
-CNHNH2
–
–
–
ACN
ACNHNH2
ACN
ACNHNH2
ACN
ACNHNH2
0.27
0.25
1.1
0.16
0.08
0.40
0.17
0.022
0.026
0.005
0.013
0.007
0.004
0.002
>100
>100
0.28
0.33
4.3
1.12
0.65
1.39
0.43
0.031
0.043
0.032
0.065
0.078
0.011
0.011
>100
>100
57.8
5.7
74
184
>200
>200
188
133
192
120
>200
>200
>200
177
89
274
ipr-1
>287
>245
137
98
113
76
>1148
>223
856
1225
282
chex-1
1a
1b2
1c
34.9
0.88
1.07
1.31
0.77
>100
3.25
2.13
1.15
1.32
2.19
0.97
NDd
ND
1d
447
2
46
2091
>7000
>32,200
6308
7571
18,250
23,500
ND
ipr-2
>182
>161
82
53
73
42
124
55
chex-2
2a
2b
2c
2d
i-pr
Cyclohex
Cyclohex
–
121
156
145
112
NO2Py3-b-CN
NO2Py3-b-CNHNH2
–
–
ND
a
b
c
Representing the concentration of compounds reducing HIV-1 replication by 50%.
Representing the concentration of compounds reducing cell viability by 50%.
Selectivity index, SI = CC50/EC50
Not detection.
.
d

58
C. Li et al. / Antiviral Research 102 (2014) 54–60
EC50 values varied between 0.77 and 1.31
l
M. Thus, these ana-
played unusual results far below the expected, with EC50 values
of 1.63 M for 1b4 and 0.07 M for 2b4, respectively. The suitable
size of masking molecules as an important factor should be consid-
ered, since bulky backbone may inhibit the original penetrating
ability of distamycin analogues and thus reduce intracellular
availability.
logues were at least 44- to 75-fold more effective than 1 in inhib-
iting HIV-1 replication in CEM/TK- cells. This indicated that a
successful bypass of the first phosphorylation step can turn d4T
from an inactive compound to a moderately active anti-HIV-1
agent.
l
l
As for AZT, antiviral activity and cytotoxicity of three control
compounds against HIV-1 were tested, presenting EC50 values of
3.4. Permeability and aqueous solubility
i
0.022, 0.026 and 0.005
l
M for 2, pr-2 and chex-2, respectively.
Compared to d4T assay, the similar trend in antiviral potency of
2a–2d was observed via the modification of distamycin analogues,
namely slightly increasing activity and cytotoxicity. Although
cyclohexyl phosphoramidate 2d exhibited more favorable inhibi-
Modification at phosphonate of nucleotide with distamycin
analogues expected to improve intracellular availability of the
drugs. It can increase the lipophilicity that may lead to potential
improvement of intracellular transportation of the compounds.
Accordingly, the log P values obtained in the present study (Table 3)
indicated the modification by distamycin analogues facilitated
lipophilic property compared to their parent nucleosides. For in-
tory activity against HIV-1 (EC50 = 0.002
pared to chex-2, the considerable cytotoxicity with CC50 value of
42 M drove the selectivity index (23500) down remarkably. In
lM, in MT-4 cell) com-
l
addition, AZT was >3200-fold less active in CEM/TK- than CEM/0
cells, while 2a and 2b were only moderately (ꢃ17-fold) less inhib-
itory to HIV-1 replication in CEM/TK- cells than CEM/0 cells, thus
confirming the ability of phosphoramidates to deliver AZT mono-
phosphate into cells. As to the influence of distamycin terminus,
no striking distinction in activity data was observed between ni-
trile- and amidine-terminal compounds.
stance, compound 1c (log P 1.01; EC50 0.40
log P and a 3-fold increase in anti-HIV-1 activity in comparison
to parent compound chex-1 (log P 0.72; EC50 1.1
M). Compound
lM) exhibited larger
l
2a (log P ꢁ1.09; EC50 0.013
lM) showed a 2-fold enhancement in
anti-HIV-1 activity compared to ipr-2 (log P ꢁ1.12; EC50
0.026 lM). Interestingly, change at distamycin terminal with ami-
dine instead of nitrile group enhanced hydrophilic property, and
also further improved anti-HIV activity. For example, amidine-ter-
As promoties controls, distamycin analogues NO2Py3-b-CN and
NO2Py3-b-CNHNH2 were devoid of anti-HIV-1 activity at 100
in MT-4 and CEM cells, whereas they were cytostatic at CC50 of
145 and 112 M, respectively.
lM
minal compounds 1d (log P 0.67; EC50 0.17
lM) and 2b (log P
ꢁ1.58; EC50 0.007 M) presented lower log P and increased anti-
l
l
HIV-1 activity than their nitrile-terminal compounds 1c and 2a.
From these data, it can be concluded that antiviral activity of the
phosphoramidate prodrugs was strongly dependent on proper
hydrophilic–lipophilic equilibrium by promoieties, rather than
enhancement of single factor.
3.3. Structural effect of distamycin on antiviral activity
Carboxamide in each unit contributes available hydrogen-bond
donor to interact with solvent, which could provide the substantial
basis for increasing aqueous solubility and intracellular availability
with a consequent increase of antiviral activity. The problem that
influence of amount of N-methylpyrrole carboxamide on potential
biological activity can be naturally put forward. Here, polyamides
containing different amount of N-methylpyrrole unites as distamy-
cin analogues were constructed and linked to alkyl monophosph-
orylated nucleoside, and antiviral data were shown in Table 2.
Both 1b2 and 2d2 (n = 2) presented sharply decreased inhibitory
Aqueous solubility of the tested compounds was measured (Ta-
ble 3) and observed that all the distamycin-modified prodrugs also
presented favorable equilibrium between oil and water phases. The
log S ranging from ꢁ1 to ꢁ5 provided a potential possibility as
drugs (Jorgensen and Duffy, 2000). By the introduction of distamy-
cin moieties, compounds 1a–d and 2a–d (log S from ꢁ2.73 to
ꢁ3.67) showed more lipophilic than their parent compounds,
i
while pr- compounds exhibited a markedly increased log S com-
pared to chex- ones, suggesting increased aqueous solubility. These
results were consistent with the permeability assay (log P) above.
potency with EC50 values of 0.20 lM and 0.018 lM in MT-4 cells
respectively compared to those with intact distamycin, while
monocarboxamides 1b1 and 2d1 (n = 1) merely showed initial anti-
viral activity and cytotoxicity as with their parent compounds. It
could be concluded that an integrated distamycin system played
a vital role in achieving improvement of biological activity. How-
ever, two tested compounds containing four carboxamides dis-
3.5. Metabolic stability
We next turned our attention to metabolic stability of these
nucleoside analogues, which was evaluated by a human liver
microsomes incubation assay (Table 3). Compounds 1a–d were
Table 2
Anti-HIV-1 activity and cytotoxicity of 1b and 2d with different amount of N-methylpyrrole carboxamide.
Compds.
n
EC50
(
lM)
CC50
(
lM)
SI (MT-4)
MT-4
CEM/0
CEM/TK-
MT-4
CEM/0
1
2
3
4
0.22
0.20
0.08
1.63
1.87
1.89
0.65
5.33
2.13
2.01
0.17
NDa
223
179
98
>200
>200
133
1013
895
1225
112
182
>200
1
2
3
4
0.021
0.018
0.002
0.07
0.152
0.201
0.011
ND
0.133
0.122
0.009
ND
193
166
42
>200
>200
156
9190
9222
23,500
3157
221
>200
a
Not detection.

C. Li et al. / Antiviral Research 102 (2014) 54–60
59
Table 3
mycin-modified AZT series. Attempts to modulate the length of
distamycin moieties through N-methylpyrrole unit diminished
the metabolic stability to some extent (1b1–4 and 2d1–4). Com-
pound 1b1 showed lower metabolic stability and higher log P com-
pared to the other three kinds. These results indicated that
increasing lipophilicity could be correlated with decreasing stabil-
ity, and might be also due to increased steric hindrance through
distamycin moiety so that the phosphamide would be less avail-
able to metabolic enzymes.
Aqueous solubility, permeability and metabolic stability of nucleoside analogues.
Compds.
log P
log S
Stabilityb
Expt.
Calc.a
Expt.
Calc.a
% remaining
ipr-1
chex-1
1a
ꢁ0.21
0.72
ꢁ0.17
1.01
ꢁ2.31
ꢁ3.30
ꢁ2.85
ꢁ3.05
ꢁ2.01
ꢁ2.37
ꢁ3.07
ꢁ3.49
ꢁ3.67
ꢁ1.86
ꢁ2.85
ꢁ2.73
ꢁ2.95
ꢁ3.52
ꢁ3.58
ꢁ2.85
ꢁ3.17
ꢁ3.82
ꢁ2.29
ꢁ3.11
ꢁ3.01
ꢁ3.12
ꢁ2.20
ꢁ2.66
ꢁ3.42
ꢁ3.83
ꢁ3.93
ꢁ2.15
ꢁ3.31
ꢁ2.88
ꢁ2.98
ꢁ3.69
ꢁ3.80
ꢁ2.87
ꢁ3.33
ꢁ4.10
52
45
72
83
44
68
85
67
77
48
31
65
71
54
69
34
63
75
ꢁ0.09
ꢁ0.24
ꢁ0.83
ꢁ0.55
ꢁ0.07
1.01
ꢁ0.06
ꢁ0.33
ꢁ0.88
ꢁ0.60
ꢁ0.06
1.12
1b
1b1
1b2
1b4
1c
3.6. Enzymatic studies
1d
0.67
0.85
ipr-2
chex-2
2a
ꢁ1.12
ꢁ0.50
ꢁ1.09
ꢁ1.58
ꢁ0.15
ꢁ0.30
ꢁ0.67
ꢁ0.55
ꢁ0.09
ꢁ1.23
ꢁ0.33
ꢁ1.12
ꢁ1.39
ꢁ0.06
ꢁ0.21
ꢁ0.76
ꢁ0.49
ꢁ0.06
The mechanism of activation of the ProTides involved a first
enzymatic activation step mediated by carboxypeptidase-type en-
zyme(s), which hydrolyzed the amide of the polyamide moiety
(Scheme 3). To probe the activation of the nucleosides ProTides
to the monophosphorylated form inside cells, we performed an
enzymatic study using carboxypeptidase A following the conver-
sion by 31P NMR. On the basis of the favorable activity against
HIV-1 and metabolic stability in human liver microsomes, com-
pounds 1b and 2d were considered for the evaluation. The assay
was carried out by dissolving the compounds in DMSO-d6 and Tris
buffer (pH = 7.4), incubating with the enzyme and recording a
blank for each sample before the addition of the enzyme.
2b
2c
2d
2d1
2d2
2d4
a
Calculated from a software package the Molecular Operating Environment
(MOE).
b
Human liver microsome incubation assay, data from at least two experiments
in parallel.
found to be metabolically stable with 67–83% of their parent com-
pounds. Further, replacing the nitrile group of (1a or 1c) with ami-
dine (1b or 1d) obtained good metabolic stability, but with poor
permeability (lower log P). A similar result was found in the dista-
In the case of compound 1b (Fig. 2), the experiment showed a
fast hydrolysis of the starting material (dp = 9.98 and 9.81, two dia-
stereoisomers) to the intermediate type i (Scheme 3) (dp = 6.25).
This intermediate was then processed to a compound of type iii
Scheme 3. Proposed metabolic activation route for d4T phosphoramidates.
Fig. 2. Carboxypeptidase-mediated cleavage of compound 1b, monitored by 31P NMR.

60
C. Li et al. / Antiviral Research 102 (2014) 54–60
Balzarini, J., Cooney, D.A., Dalal, M., Kang, G.J., Cupp, J.E., De Clercq, E., Broder, S.,
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4. Conclusion
activity of distamycin
A analogs: substitutions on the different pyrrole
In summary, we herein reported the synthesis and antiviral
activity of a series of nucleoside monophosphates modified by al-
kyl moiety and distamycin analogues. Detailed effects on inhibi-
tory activity and cytotoxicity against HIV-1 in MT-4 and CEM
cells were evaluated for variations in nucleoside, alkyl ester as well
as structure of distamycin analogues. Owing to the introduction of
distamycin skeleton, most of tested compounds exhibited favor-
able antiviral activities with the EC50 value in the range of 1.3- to
6.5-fold lower than for their corresponding controls. The increased
inhibitory activity against HIV-1 could be attributed to the proper
hydrophilic–lipophilic equilibrium of distamycin promoieties. That
was not to say that distamycin analogues with any length have the
ability to improve antiviral activity of parent compounds via mod-
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Acknowledgments
Support of this research by the National Nature Science Founda-
tion of China is gratefully acknowledged (Nos. 21372024,
21202005, 21232005, and 21172016). We thank Prof. Aixia Yan
for permeability and solubility predicting.
Appendix A. Supplementary data
Pang, Y.P., 2004. Nonbonded bivalence approach to cell-permeable molecules that
target DNA sequences. Bioorg. Med. Chem. 12, 3063–3068.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.antiviral.
2013.12.002.
Perrone, P., Luoni, G.M., Kelleher, M.R., Daverio, F., Angell, A., Mulready, S., Congiatu,
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Smith, D.B., McGuigan, C., 2007. Application of the phosphoramidate ProTide
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