Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza purine nucleoside analogs as anti-influenza A agents

Article abstract of DOI:10.1016/j.antiviral.2016.01.005

Among many subtypes of influenza A viruses, influenza A(H1N1) and A(H3N2) subtypes are currently circulating among humans (WHO report 2014-15). Therapeutically, the emergence of viral resistance to currently available drugs (adamantanes and neuraminidase inhibitors) has heightened alarms for developing novel drugs that could address diverse targets in the viral replication cycle in order to improve treatment outcomes. To this regard, the design and synthesis of nucleoside analog inhibitors as potential anti-influenza A agents is a very active field of research nowadays. In this study, we designed and synthesized a series of hitherto unknown 6-methyl-7-substituted-7-deaza purine nucleoside analogs, and evaluated for their biological activities against influenza A virus strains, H1N1 and H3N2. From the viral inhibition assay, we identified some effective compounds, among which, compounds 5x (IC₅₀ = 5.88 μM and 6.95 μM for H1N1 and H3N2, respectively) and 5z (IC₅₀ = 3.95 μM and 3.61 μM for H1N1 and H3N2, respectively) demonstrated potent anti-influenza A activity. On the basis of selectivity index, we conceive that compound 5x may serve as a chemical probe of interest for further lead optimization studies with a general aim of developing novel and effective anti-influenza A virus agents.

Full text of DOI:10.1016/j.antiviral.2016.01.005

Accepted Manuscript
Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-
deaza purine nucleoside analogs as anti-influenza A agents
Cai Lin, Chenghai Sun, Xiao Liu, Yiqian Zhou, Muzammal Hussain, Junting Wan,
Minke Li, Xue Li, Ruiliang Jin, Zhengchao Tu, Jiancun Zhang
PII:
S0166-3542(16)30007-9
10.1016/j.antiviral.2016.01.005
AVR 3753
DOI:
Reference:
To appear in: Antiviral Research
Received Date: 9 October 2015
Revised Date: 12 January 2016
Accepted Date: 13 January 2016
Please cite this article as: Lin, C., Sun, C., Liu, X., Zhou, Y., Hussain, M., Wan, J., Li, M., Li, X., Jin, R.,
Tu, Z., Zhang, J., Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-
deaza purine nucleoside analogs as anti-influenza A agents, Antiviral Research (2016), doi: 10.1016/
j.antiviral.2016.01.005.
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ACCEPTED MANUSCRIPT
Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza
purine nucleoside analogs as anti-influenza A agents
Cai Lin b†, Chenghai Sun a†, Xiao Liu a, Yiqian Zhoub, Muzammal Hussain b, c, Junting Wan b, Minke Li
b, Xue Li a, Ruiliang Jin e, Zhengchao Tu b*, Jiancun Zhang b, d
*
a Institute of Pharmaceutical Research, South China Normal University, Guangzhou, Guangdong,
510631, PR China
b Guangzhou Institutes of Biomedicine and Heath, Chinese Academy of Sciences, 190 Kaiyuan
Road, Guangzhou, 510530, PR China
c University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing, 100049, PR China
d State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou, PR
China
e Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University
School of Medicine, Shanghai, PR China
Address correspondence and reprint request to:
Jiancun Zhang and Zhengchao Tu, Guangzhou Institutes of Biomedicine and Health, Chinese
Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou, 510530, PR China
Tel.: +86 020 32015323; email address: zhang_jiancun@gibh.ac.cn (J. Zhang)
Tel.: +86 020 32015323; email address: tu_zhengchao@gibh.ac.cn (Z.-C. Tu)
† These authors equally contributed to this work.
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Abstract
Among many subtypes of influenza A viruses, influenza A(H1N1) and A(H3N2) subtypes are
currently circulating among humans (WHO report 2014-15). Therapeutically, the emergence of
viral resistance to currently available drugs (adamantanes and neuraminidase inhibitors) has
heightened alarms for developing novel drugs that could address diverse targets in the viral
replication cycle in order to improve treatment outcomes. To this regard, the design and
synthesis of nucleoside analog inhibitors as potential anti-influenza A agents is a very active
field of research nowadays. In this study, we designed and synthesized a series of hitherto
unknown 6-methyl-7-substituted-7-deaza purine nucleoside analogs, and evaluated for their
biological activities against influenza A virus strains, H1N1 and H3N2. From the viral inhibition
assay, we identified some effective compounds, among which, compounds 5x (IC50 = 5.88 ꢀM
and 6.95 ꢀM for H1N1 and H3N2, respectively) and 5z (IC50 = 3.95 ꢀM and 3.61 ꢀM for H1N1
and H3N2, respectively) demonstrated potent anti-influenza A activity. On the basis of
selectivity index, we conceive that compound 5x may serve as a chemical probe of interest for
further lead optimization studies with a general aim of developing novel and effective anti-
influenza A virus agents.
Key words: H1N1, H3N2, Influenza A, Nucleoside analogs
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1. Introduction
Influenza is one of the most burdensome and widespread human infectious diseases the impact of
which is felt globally each year, affecting approximately 20% of world’s population with an
estimated annual epidemics of 3 to 5 million cases of severe illness, and about 250, 000 to 500,
000 deaths (WHO, 2014). Influenza A virus represents the most significant public health
problem as estimated to have claimed 18,500 lives (laboratory-confirmed cases) in 2009 H1N1
global pandemic, and still consistently contributing to the death toll in pandemic countries across
the globe (Dawood et al., 2012; WHO, 2014; WHO, 2015). According to World Health
Organization (WHO) reports of 2014-15, H1N1 and H3N2 strains have spread quickly among
humans.
Therapeutically, the two classes of anti-influenza drugs: M2 ion channel inhibitors, also called
adamantanes (amantadine and rimantadine); and neuraminidase inhibitors (oseltamivir and
zanamivir), remain the mainstay of combating influenza A spread and treating incident
infections. But unfortunately, most of influenza A strains, including the currently circulating
H1N1 and H3N2, have become resistant to adamantanes (Deyde et al., 2007; Sheu et al., 2011).
Especially in recent years, the emergence of H1N1 resistance against oseltamivir and zanamivir
has complicated the treatment outcomes (Correia et al., 2015; Han et al., 2012; Orozovic et al.,
2014; Pizzorno et al., 2013). Nevertheless, the drug development pipeline to tackle this viral
resistance remains woefully thin, including the recently developed pyrimido-
pyrroloquinoxalinedione analogs targeting nucleoproteins (Lin et al., 2015), nucleoside analogs
(Gao et al., 2015), daidzein analogs (Chung et al., 2015), and the hydrazide derivatives targeting
non-structural protein 1 (NS1) (Barman et al., 2014). Clearly, there is a pressing need for
developing novel chemical-based anti-influenza A drugs that could improve treatment outcomes.
To this regard, the design and synthesis of nucleoside analog inhibitors as potential anti-
influenza A agents is very active field of research (Al-Saad et al., 2014; Elayadi et al., 2014; Gao
et al., 2015).
Recently, β-D-6-methyl-7-deaza-adenine ribonucleoside (compound A in Fig. 1) has been shown
to possess good inhibitory activity against tuberculosis and dengue virus. However, this
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compound causes significant cytotoxicity (Naus et al., 2014; Wu et al., 2010) and, thus, has
limited clinical use as an antiviral agent. It is highly desirable to improve the toxicity profile of
this compound. On the other hand, the introduction of a methyl group at the 2’-β-position on the
ribose ring of some nucleoside analogs has been demonstrated to be an important sugar
modification necessary to achieve excellent potency against various viruses without interfering
with the function of host cell polymerases (Eldrup et al., 2004a; Eldrup et al., 2004b; Olsen et al.,
2004). In addition, 2’-α-fluoro ribonucleosides have been shown to exhibit potent antiviral
activity to suppress influenza A virus infections in Madin-Darby canine kidney (MDCK) cells
(Tuttle et al., 1993). Collectively, the introduction of 2’-deoxy-2’-fluoro-2’-C-methyl
ribofuranosyl moiety on the ribose ring may improve the antiviral activity and reduce toxicity of
nucleoside analogs. One particular example is Sofosbuvir (Sovaldi®), shown in Fig. 1, which is
a newly improved nucleoside inhibitor that possesses the same moiety in its structure, and has
been found potent against hepatitis C virus. Based on these preliminary and intriguing findings
from literature, we devised a rationale that combining the two moieties: 2’-deoxy-2’-fluoro-2’-C-
methyl ribosyl sugars and 6-methyl-7-deazapurine bases, together in a single scaffold and then
performing structure-activity relationship (SAR) studies may reveal a novel class of anti-
influenza A virus nucleoside analogs that will be expected to possess less cytotoxicity. The
biological evaluation of the designed compounds validated our rationale as we found a
compound with more potent anti-influenza A activity and less cytotoxicity in comparison to the
reference drug Ribavirin. Here, we report the synthesis and biological evaluation of a novel
series of 6-methyl-7-substituted-7-deaza purine nucleoside analogs against influenza A virus
with the reasonable SAR revealed.
2. Materials and methods
2.1. Experimental chemistry
Reagents were purchased from commercial suppliers and used without further purification unless
otherwise noted. NMR spectra were recorded on Bruker Avance 400 MHz (1H at 400MHz) and
13
500 MHz (1H at 500 MHz, C at 125 MHz) spectrometer. Chemical shifts values are given in
ppm and referred as the internal standard to TMS (tetramethylsilane). The peak patterns are
indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet and dd, doublet
of doublets. The coupling constants (J) are reported in hertz (Hz). LC-MS data were recorded on
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Agilent HPLC/MCD instrument. Silica gel column chromatography was performed over silica
gel 200−300 mesh, and the eluent was a mixture of ethyl acetate and petroleum ether, or, a
mixture of isopropanol and dichloromethane.
2.1.1. Inhibitor design and synthesis
The sugar and base moieties were synthesized by using the inexpensive and easily available
material. Diisopropylidine derivative of D-mannitol was used as the starting material for the
sugar moiety synthesis, which underwent symmetrical oxidative cleavage to afford the
isopropylidine protected D-glyceraldehyde. Compound 1 was then synthesized by following the
procedures described by Wang et al. (Wang et al., 2009). Detail procedures described by Kim et
al. (Kim et al., 2012) were followed to obtain the base moiety, 4-Chloro-7H-pyrrolo[2,3-
d]pyrimidine (13.2% overall yield) through a five steps process using ethyl cyanoacetate as the
starting material. Coupling of the sugar moiety and base moiety gave the compound 3. Various
modifications were performed at 7-position to obtain the desired nucleoside analogs. Details
regarding the syntheses and characterizations of the compounds can be found in the
Supplementary data.
2.2. Biological experiments
2.2.1. Cells and virus
MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco)
supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 ꢀg/ml streptomycin at
37°C in 5% CO2. The influenza A virus strains H1N1 (A/WSN/33) and H3N2 (A/Hong
Kong/8/68) were propagated in the allantoic cavity of 9-day-old embryonated chicken eggs. The
harvested viruses were then inoculated in MDCK cells for adaptation. The culture suspensions
were collected and stored in multiple single-use aliquots at -80 °C for further use.
2.2.2. Cytotoxicity assay
MDCK cells were grown as monolayers in 96-well plates after seeding for 18–24 h. The medium
was removed and rinsed twice with Hanks’ solution and the compounds were serially diluted in
100 ꢀl medium containing 0.3% BSA. After incubating for 72 h at 37 °C in a humidified 5%
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CO2 incubator, 50ul diluted CCK-8 reagent was added to each well and incubated for 2 h. The
absorbance was measured at 450 nm using Envision Plate Reader (PerKin Elmer). The CC50
values were calculated by analyzing the data using Graph Pad Prism 5.
2.2.3. Viral inhibition assay
MDCK cells were grown to confluence in 96-well microtiter plates. The medium was removed,
and fifty microliters, equal to approximately 0.01 MOI of H1N1 virus or H3N2 virus were added
to the plates and incubated at 37 °C for 2 h. The cells were then covered with 50 ꢀl of medium
containing various amounts of Ribavirin or one of the synthesized compounds in the presence of
1 ꢀg/ml TPCK trypsin and 0.3% BSA. After incubation in 5% CO2 at 37 °C for 72 h, 50 ꢀl of
diluted CCK-8 reagent were added to each well, and the mixture was incubated for 2 h. The
A450 was then measured using Envision Plate Reader (PerKin Elmer). Data were analyzed using
Graph Pad Prism 5.
2.2.4. Plaque reduction assay
The plaque reduction assay was performed in 6 well tissue culture plates. The MDCK cells were
seeded at 6 × 105 cells/well and incubated in 5% CO2 at 37 °C for 18-24 h. The monolayer of
MDCK cells was then infected with H1N1 or H3N2 virus (100 PFU/well) for 1h at 37 °C. The
inoculums were removed, and the cells were washed twice with Hanks’ solution. The cells were
then overlaid with 1% agar MEM-containing Ribavirin or the synthesized compound 5x in the
presence of 1 ꢀg/ml trypsin and 0.3% BSA. Three days after infection, the monolayers were
fixed and stained with 0.1% crystal violet solution, and the plaques counted.
3. Results and discussion
3.1. Chemistry
The synthesis was focused to combine 2’-deoxy-2’-fluoro-2’-C-methyl ribosyl sugar and 6-
methyl-7-deazapurine base to yield a novel nucleoside scaffold (4), and further evaluating the
antiviral activities.
The novel scaffold (4) was synthesized by using a route described in Scheme 1. Initially, the
coupling of 2’-C-methyl-2’-fluoro sugar with 6-chloro-7-deazapurine suffered from low yield
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and poor anomeric stereoselectivity under Mitsunobu condition. Moreover, the desired product
failed to be obtained via the Vorbruggen reaction. Therefore, we employed a previously reported
method that was effective to couple 2’-C-methyl-2’-fluoro sugar part with purine bases through
double stereoselective inversion of β-lactol (Reddy et al., 2011). Treatment of the known
compound 1 with carbon tetra bromide and triphenylphosphine (CBr4/PPh3, Appel reaction) in
anhydrous DCM gave the α-bromo ribofuranose 2 as a major product. The SN2 coupling between
2 and 4-chloro-7H-pyrrolo [2, 3-d] pyrimidine with KOtBu was accomplished to give the key
intermediate 3 in an acceptable yield (50%). The compound 4 was then obtained readily through
a palladium-catalyzed reaction in 96% yield (Scheme 1).
Next, the halogenation of 4 with N-Iodosuccinimide (NIS), N-Bromosuccinimide (NBS) and N-
Chlorosuccinimide (NCS) in DMF afforded the halogenated nucleoside intermediates 6a-c,
respectively, and then subsequent deprotection yielded 5a-d (Scheme 2).
In the following steps, 7-iodo-7-deazapurine nucleoside (5b) served as a key intermediate for the
synthesis of 7-substituted 7-deazapurine ribonucleosides via various palladium catalysts. Under
the catalytic effect of Pd(PPh3)4, 5b was first reacted with Zn(CN)2 in dry DMF to obtain 7-
cyano derivative 5e, and then the subsequent oxidation reaction provided 7-formamido derivative
5f. Next, using the same catalyst, the Stille reaction (Baillargeon and Stille, 1986) of 5b with
tributyl(vinyl)tin and 2-(tributylstannyl)thiophene, respectively, provided 7-ethyl and 7-
thiophen-2’-yl substituted nucleosides 5h and 5j in acceptable yields. Further, the coupling of
compound 5b with corresponding substituted alkynyl reagents was performed by using
Pd(PPh3)4 and CuI (Sonogashira reaction) (Sonogashira, 2002) at mild temperature to obtain the
products 5g and 5q in 98% and 51% yields, respectively. Moreover, 5b was also used as building
block for the synthesis of the 7-(het)aryl 7-deazapurine nucleosides via the Suzuki cross-
coupling reaction (Miyaura and Suzuki, 1995) with various heteroaryl boronic acids. The
reactions were conducted in a water/acetonitrile 2:1 mixture with Pd(OAc)2, PPh3 and sodium
carbonate to provide 7-heteroaryl derivatives 5k-p in moderate yields. Finally, compound 5b was
converted to 7-carbomethoxyvinyl and 7-aromatic vinyl derivatives 5i and 5r-z (moderate to
high yields) in the presence of Pd(OAc)2 and PPh3 by treatment with corresponding substituted
vinyl reagents, called Heck reaction (Scheme 3).
3.2. Biological results
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3.2.1. Evaluation of anti-influenza virus activity and cytotoxicity
All the synthesized nucleoside analogs were evaluated for their in vitro anti-influenza A activity
against two viral strains, A/WSN/33 (H1N1) and A/Hong Kong/8/68 (H3N2), while using
Ribavirin as a positive control. In addition, the cytotoxicity was also evaluated in parallel using
MDCK cells. The considered measurements of the in vitro screening were IC50 (compound
concentration that inhibits viral replication by 50%) and cytotoxic concentration CC50
(compound concentration that reduces cell viability by 50%). The selectivity index (SI = ratio of
CC50 to IC50) was also calculated where needed as an estimate of therapeutic window.
The antiviral activity data, as summarized in Table 1, revealed the significant resistance of both
strains against most of the synthesized compounds (IC50 values > 100 ꢀM) compared to the
reference Ribavirin. However, among the twenty-six compounds (Table 1), ten compounds in
case of H1N1, while eleven compounds in case of H3N2 showed certain level of antiviral
activity with IC50 values < 100 ꢀM. Especially, the compounds 5r (IC50 = 7.11 ꢀM and 10.53
ꢀM for H1N1 and H3N2, respectively), 5x (IC50 = 5.88 ꢀM and 6.95 ꢀM for H1N1 and H3N2,
respectively) and 5z (IC50 = 3.95 ꢀM and 3.61 ꢀM for H1N1 and H3N2, respectively) were more
potent and active than Ribavirin (IC50 = 10.92 ꢀM and 11.2 ꢀM for H1N1 and H3N2,
respectively). The inhibition data for these three compounds has been shown in Fig. 2.
As far as the cytotoxicity data is concerned, most of the novel nucleoside analogs with 2’-deoxy-
2’-fluoro-2’-C-methyl ribosyl sugars demonstrated no significant inhibitory effect (CC50 > 100
ꢀM) on the growth of MDCK cells, which indicates they possess low cytotoxic potential.
Surprisingly, the cytotoxic evaluation of most active compounds revealed that 5r and 5z possess
a certain level of cytotoxic potential having CC50 values 53.2 ꢀM and 28.3 ꢀM, respectively, in
comparison to the compound 5x and the reference Ribavirin (both having CC50 values > 100
ꢀM). However, despite the lower CC50 values in comparison to the reference Ribavirin, the
differential activity exhibited by these compounds in terms of SI values (SIs of 5r = 7.48 and
5.05 for H1N1 and H3N2, respectively; SIs of 5z = 7.16 and 7.83 for H1N1 and H3N2,
respectively) is quite significant and remains an open question for further investigations.
Interestingly, 5x possesses SI values (> 17 in case of H1N1 and > 14 in case of H3N2) that are
much better in comparison to 5r and 5z. The SI value demonstrates the differential
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activity/selectivity of a compound towards its target. The greater the SI value of the compound is,
the more selective it is. Usually, an SI value below 2 indicates general toxicity of a pure
compound (Koch et al., 2005). Based on this assumption, the SI values of 5r, 5x, and 5z exhibit a
high degree of viral selectivity. Since 5x has an IC50 very close to 5z and SI values two times that
of 5z, we conceived 5x has a higher potency than 5z and thus selected it for further evaluation in
plaque reduction assay.
Inspired by design strategy of target compounds and their anti-influenza inhibitory activity, we
also performed anti-tuberculosis (anti-T.B) and anti-dengue virus assays. The results showed that
all the compounds demonstrated IC50 values > 100 ꢀM in anti-T.B assay. Whereas, some of the
compounds demonstrated considerable IC50 values against dengue virus (detail will be reported
in the future).
3.2.2. Structure-activity relationship (SAR) studies
A comparison of the effect of different C7-substituents on antiviral activity was made with
Ribavirin. For the first SAR, to begin with, compounds 5j-r bearing bulky substituents, including
substituted and unsubstituted benzenes (5k-n), (hetero)aromatics (5j, 5o and 5p), styrene (5r)
and phenylacetylene (5q), at the C7-position of the 7-deazapurine base demonstrated better
inhibitory activities than the compounds with smaller substituents, such as halogen atoms (5b-d)
and other electron-withdrawing groups (5e-i). The antiviral activity was further improved to be
more potent when the phenyl group (5k, IC50 = 92.3 ꢀM for H1N1) was replaced by substituted
benzenes, such as 4-fluoro benzene (5l, IC50 = 72.2 ꢀM and 65. 3 ꢀM for H1N1 and H3N2,
respectively), 4-trifluoromethyl benzene (5m, IC50 = 55.1 ꢀM and 48.15 ꢀM for H1N1 and
H3N2, respectively) and 4-methoxyl benzene (5n, IC50 = 72.6 ꢀM and 86 ꢀM for H1N1 and
H3N2, respectively). Whereas, the antiviral activity declined in the order of less to no when the
phenyl group was changed to thienyl (5j, IC50 = 64.8 ꢀM for H3N2), 4-pyridyl (5o, IC50 > 100
ꢀM) and naphthyl (5p, IC50 > 100 ꢀM) groups, respectively. It is noteworthy that the (E)-7-styryl
derivative exhibited slightly stronger activity (5r, IC50 = 7.11 ꢀM and 10.53 ꢀM for H1N1 and
H3N2, respectively) in comparison to Ribavirin, while 7-phenylethynyl analog (5q, IC50 = 50.6
ꢀM and 47 ꢀM for H1N1 and H3N2, respectively) demonstrated decreased activity.
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From the first SAR, we conceived that heteroaryl groups could increase the anti-influenza
activity, particularly, when the trans-vinyl was used as the linker at C7-position in the 7-
deazapurine base. Therefore, in an effort to find a more active and potent compound, this series
was further optimized by placing various substituents in the phenyl ring (5s-u) or replacing the
phenyl group with heteroaryl groups (5v-z). Our results showed that introduction of electron-
withdrawing groups such as fluoro and trifluoromethyl groups at the 4-position of phenyl ring
could result in loss of antiviral activity of nucleoside analogs (5s and 5t), while the addition of
electron-donating groups, such as 4-methoxystyryl group (5u, IC50 = 28 ꢀM and 26 ꢀM for
H1N1 and H3N2, respectively), could decrease the potency. Moreover, the (1, 1’-biphenyl)-4-
ylvinyl nucleoside analog (5v) also exhibited loss of antiviral activity, most probably, because of
the too bulkyl group that warrants further investigations.
Furthermore and interestingly, there was a distinct difference in the potency among nucleoside
derivatives bearing heteroaryl substituents, even with the same hetero atom, at the diverse
positions. For example, compound 5x having the nitrogen atom of vinylpyridine ring at 2-
position demonstrated improved antiviral activity (IC50 = 5.88 ꢀM and 6.95 ꢀM for H1N1 and
H3N2, respectively), but the activity was completely lost when the nitrogen atom was at 4-
position (5w). These results indicate that hetero atoms at the 2-position of vinylhetaryl-
substituted series are required for antiviral activity, but their presence at 4-position may induce
loss of activity. Nevertheless, compound 5y containing 2-Vinylpyrazine demonstrated slightly
potent activity (IC50 = 23.6 ꢀM and 54.52 ꢀM for H1N1 and H3N2, respectively), but not as
potent as that of 5x. Finally, encouraged by the improved activity and potency of heteroaryl
substitution in case of compound 5x, we decided to substitute it with 2-Vinylthiophene. As
expected, (E)-2-(thiophen-2-yl)vinyl analog exhibited the most potent antiviral activity (5z, IC50
= 3.95 ꢀM and 3.61 ꢀM for H1N1 and H3N2, respectively) in comparison to 5r and 5x, and the
reference Ribavirin. However, considering the cytotoxic potential (Table 1) and SI values, 5x
may be a promising lead in managing influenza A infections.
3.2.3. Plaque reduction assay
In order to validate the anti-influenza A activity, compound 5x was further assessed in a plaque
reduction assay using Ribavirin as reference. The images in Fig. 3 show that compound 5x and
Ribavirin inhibited plaque formation by influenza virus H1N1 and H3N2 in a concentration-
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dependent manner, ranging from 1.1 ꢀM to 30 ꢀM. The plaque formation was effectively
inhibited by 5x at a concentration of 30 ꢀM. Moreover, compared to our reference drug
Ribavirin, compound 5x showed better plaque suppressive effect, indicating that 5x was an
obviously potent inhibitor consistent with the viral inhibition results discussed in Table 1.
4. Conclusions
We have designed and synthesized a class of novel 6-methyl-7-substituted-7-deaza purine
nucleoside analogs by using a stereoselective synthetic approach accompanied by some well-
known cross-coupling reactions. We also successfully explored the SAR of synthesized
compounds in relation to anti-influenza virus activity. From viral inhibition assay, we have
identified compound 5x as the most active inhibitor against wild-type influenza A strains, H1N1
(IC50 = 5.88 ꢀM) and H3N2 (IC50 = 6.95 ꢀM), which is more potent than Ribavirin. We conceive
that compound 5x may serve as a chemical probe of interest for further SAR and lead
optimization studies with a general aim of developing novel and effective anti-influenza A virus
agents.
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Acknowledgments
The research was supported in part by Foshan Municipal Funds for Scientific and Technological
Innovation Projects (2013HK100012), Guangzhou Science and Technology Project
(2014J4100222), National Natural Science Foundation of China Grant (21402205). M.H is
sponsored by CAS-TWAS President’s PhD fellowship for international students.
Conflict of interest
None
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Schemes
Scheme 1. Reagents and conditions: i) CBr4, PPh3, DCM, -20 to -15 °C; ii) 4-chloro-7H-pyrrolo
[2, 3-d] pyrimidine, tBuOK, MeCN: tBuOH = 1.5: 1, r.t. to 50 °C; iii) AlMe3, Pd(PPh3)4, THF,
80 °C.
Scheme 2. Reagents and conditions: i) for 6a: NIS, DMF, 50°C; for 6b: NBS, DMF, r.t.; for 6c:
NCS, DMF, 50°C; ii) NH3/MeOH, r.t.
Scheme 3. Reagents and conditions: i) Zn(CN)2, Pd(PPh3)4, DMF, 150 °C; ii）NH4OH, H2O2,
dioxane, 100 °C; iii) for 5h: nBu3(CHCH2)Sn, Pd(PPh3)4, DMF, 100 °C; for 5j: nBu3(2-
thienyl)Sn, Pd(PPh3)4, DMF, 100 °C; iv) for 5k-5p: R1-B(OH)2, Pd(OAc)2, PPh3, Na2CO3,
H2O/MeCN, 100 °C; v) for 5g: Pd(PPh3)4, CuI, TEA, triethylsilylacetelene, THF, 45 °C; and
then K2CO3, MeOH, r.t; for 5q: Pd(PPh3)4, CuI, TEA, arylacetelene, THF, 50 °C; vi) for 5r-5z:
vinyl
compound,
Pd(OAc)2,
PPh3,
TEA,
DMF,
100
°C.
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Figure Legends
Figure 1. Structures with potent antiviral activity. The colored parts appear to be crucial for
potent antiviral activity.
Figure 2. Dose-response curves for compounds 5r, 5x and 5z against influenza A virus strains
A/WSN/33 (H1N1) and A/Hong Kong/8/68 (H3N2).
Figure 3. Reduction of plaque formation by A/WSN/33 (H1N1) and A/Hong Kong/8/68 (H3N2)
virus in MDCK cells upon treatment with compound 5x and Ribavirin. VC, virus control
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Table 1. In vitro anti-influenza A virus activity of the test compounds in two strains, and the
cytotoxic evaluation in MDCK cells
Antiviral activity IC50 (µM)a
Cytotoxicity
Entry
Compound
R
CC50 (µM)b
H1N1
>100
H3N2
>100
1
2
3
4
5
6
7
8
H
I
>100
5a
5b
5c
5d
5e
5f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
52.6
>100
>100
>100
>100
>100
>100
>100
Br
Cl
CN
CONH2
5g
5h
9
>100
>100
>100
64.8
>100
>100
5i
5j
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11
12
13
14
15
5k
5l
92.3
72.2
55.1
72.6
>100
>100
65.3
48.15
86
>100
>100
>100
>100
>100
5m
5n
5o
>100
16
>100
100
>100
5p
17
18
50.6
7.11
47
92.8
53.2
5q
5r
10.53
19
>100
>100
90.2
5s
20
21
22
5t
5u
5v
>100
28
>100
26
28.8
>100
>100
>100
>100
23
24
>100
5.88
>100
6.95
>100
>100
5w
5x

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25
23.6
54.52
>100
5y
26
27
3.95
3.61
11.2
28.3
5z
S
-
10.92
>100
Ribavirin
a Concentration required to inhibit influenza A virus growth by 50%
b Cytotoxic concentration required to inhibit MDCK cells growth by 50%

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Figure 1

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Highlights
•
•
•
•
•
A novel nucleoside scaffold, compound 4, was preparedwith high stereoselectivity
26 6-methyl 7-substituted-7-deaza purine nucleoside analogues were synthesized
Anti-influenza A virus activity and cytotoxicity evaluation were conducted
Three compounds 5r, 5x and 5z showed more potent antiviral activity than Ribavirin
5x may serve as a potential lead compound for anti-influenza A drug development