Anti-influenza active and low toxic N-phenyl-substituted β-amidoamidines structurally related to natural antibiotic amidinomycin

Article abstract of DOI:10.1016/j.bmcl.2013.02.053

A set of racemic N-phenyl-substituted β-amidoamidines hydrochlorides 4, which are structurally related to natural antiviral agent amidinomycin (1), was synthesized in four steps starting from methacryloyl anilide (5). In the final step of the synthetic route, an uncommon monoacylation of β-aminoamidine 8 at the less reactive β-phenylamino-group took place. To rationalize this result, a mechanism which involves initial acylation at the more active amidine-function followed by intramolecular acyl-group transfer to β-phenylamino-group was suggested. All three β-amidoamidines 4d-f bearing long linear aliphatic chain (from n-C₈H₁₇ to n-C₁₂H₂₅) revealed significant in vitro activity against influenza A virus (H3N2) and modest cytotoxicity. The in vitro antiviral potency of 4d,e is 6-20 times greater than that of commercial rimantadine with lower EC₅₀ values and higher therapeutic index. The non-toxic in vivo compounds 4d-f showed a beneficial protective effect in influenza A (H3N2) infected mice.

Full text of DOI:10.1016/j.bmcl.2013.02.053

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2357–2361
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-influenza active and low toxic N-phenyl-substituted
b-amidoamidines structurally related to natural antibiotic
amidinomycin
b
b
c
b,c
Edward E. Korshin a,b, , Lyubov G. Zakharova , Yakov A. Levin , Marina P. Shulaeva , Oskar K. Pozdeev
⇑
a Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
b Arbuzov Institute of Organic and Physical Chemistry, Russian Academy of Sciences, 8 Arbuzov Str., Kazan 420088, Russia
c Department of Microbiology, Kazan State Medical University, 49 Butlerov Str., Kazan 420012, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of racemic N-phenyl-substituted b-amidoamidines hydrochlorides 4, which are structurally related
to natural antiviral agent amidinomycin (1), was synthesized in four steps starting from methacryloyl
anilide (5). In the final step of the synthetic route, an uncommon monoacylation of b-aminoamidine 8
at the less reactive b-phenylamino-group took place. To rationalize this result, a mechanism which
involves initial acylation at the more active amidine-function followed by intramolecular acyl-group
transfer to b-phenylamino-group was suggested. All three b-amidoamidines 4d–f bearing long linear ali-
phatic chain (from n-C8H17 to n-C12H25) revealed significant in vitro activity against influenza A virus
(H3N2) and modest cytotoxicity. The in vitro antiviral potency of 4d,e is 6–20 times greater than that
of commercial rimantadine with lower EC50 values and higher therapeutic index. The non-toxic in vivo
compounds 4d–f showed a beneficial protective effect in influenza A (H3N2) infected mice.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 24 December 2012
Revised 6 February 2013
Accepted 12 February 2013
Available online 22 February 2013
Keywords:
Influenza A
Antiviral activity
Imidoyl chloride
Amidine
Amide
Influenza (flu) is the cause serious public health and economic
problems. Worldwide, influenza epidemics result in several million
cases of severe illness and hundreds of thousands deaths per year.
Although vaccination plays a primary role in the prevention of
influenza, its efficacy mislaid frequently because of mismatch be-
tween the seasonal influenza vaccine and the circulating influenza
virus.1 Therefore, pharmacotherapy is crucially important for the
control and cure of influenza. However, for the time being only 4
small molecule drugs are available for the cure of influenza infec-
tion in the developed countries.2 This short list of approved anti-
influenza agents includes relatively novel neuraminidase (NA)
inhibitors like zanamivir and oseltamivir, which are sialic acid
derivatives or mimetics, and the older adamantane-derived M2
ion channel blockers like amantadine and rimantadine.2 The
emerged high level of influenza A virus resistance resulted in lim-
ited applicability of the adamantane-type M2 ion channel blocker
drugs for a single treatment of influenza.2,3 Moreover, from the
2007 to 2008 season, oseltamivir-resistant variants of influenza A
viruses became widespread, and a number of zanamivir-resistant
influenza virus strains were also detected.1,2,4
ing drugs.2a,4c,5 The drawbacks of the currently available anti-
influenza chemotherapy catalyze intensive studies to broaden an
assortment of the potent antiviral leads, including the entities
essentially dissimilar in structure and/or in the mode of action
from the currently prescribed antiviral agents.5,6 Thus, a variety
of natural products of different structures were found to act as po-
tent influenza viral NA-inhibitors.7 Particularly, a number of natu-
ral and synthetic flavonoids and polyphenols,8 chalcones,9
diarylheptanoids,10 and sesquitherpenes revealed a high influenza
antiviral activity,11 mostly through inhibition of the viral NA. A
very promising approach for treatment of influenza is associated
with recently discovered highly potent synthetic peptides, which
inhibit influenza virus replication through a strong interaction
with virus haemagglutinin of different subtypes, thus blocking vir-
al attachment to cells.12 Besides, even simple dipeptide–rimanta-
dine conjugates showed significant antiviral activity against
rimantadine-resistant H1N1 and H3N2 influenza A virus strains.13
In the search of potent antiviral agents, we revisited the struc-
tures of natural oligopeptide antibiotics 1–3, which are specifically
characterized by the acylated at the amino-group b-aminoamidine
framework (Fig. 1).14 Being the simplest natural b-amidoamidine,
(1R,3S)-amidinomycin (myxoviromycin) (1) was isolated from fer-
mentation medium of various Streptomyces species.15 Its structure
was originally resolved by chemical degradation methods,16a and
the absolute configuration was determined by X-ray analysis.16b
Subsequently, preparation of racemic 117 and 3 enantioselective
Such a rapid appearance of worldwide resistance toward re-
cently launched NA-inhibitors highlights an urgent need in novel
anti-influenza pharmaceuticals to supplement or replace the exist-
⇑
Corresponding author. Tel.: +972 8934 2558; fax: +972 8934 4142.
E-mail address: edward.korshin@weizmann.ac.il (E.E. Korshin).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.053

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E. E. Korshin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2357–2361
H
N
H
N
At the first look for potent and non-toxic antivirals within
NH2
NH
NH2
NH
b-amidoamidines we decided to modify the structure of amidino-
mycin (1) as follows: (i) To replace the chiral hydrophilic
(3-aminocyclopentyl)-fragment in the head amido-group with a
linear alkyl of variable chain-length, and thus, of different lipophil-
icity; (ii) To attach a planar phenyl ring at the nitrogen atom of the
amido-group; (iii) To substitute a highly basic parent N-unsubsti-
tuted amidine tail by essentially less basic N1,N2-diphenylamidine
moiety.28
Considering recent achievements on N-(het)arylations of
amide29 and amidine functionalities,30 the target N-phenyl substi-
tuted b-amidoamidines 4 might be prepared from the correspond-
ing N-unsubstituted b-amidoamidines. However, our attempts to
get 4 through Pd(II)- or Cu(I)-mediated phenylation of N-unsubsti-
tuted precursors failed. In contrast to that, hydrochlorides of
N-phenyl-substituted b-amidoamidines 4a–g were successfully
synthesized in four steps from methacryloyl anilide (5) according
to the optimized version of the previously reported approach
(Scheme 1).31
H2N
H2N
N
O
O
Amidinomycin (1)
Noformicin (2)
HCO NH
H
N
NH2
NH
Distamycin A (3)
N
(n = 3)
O
Me
n
Figure 1. Structural formulas of natural b-amidoamidine-type oligopeptide
antibiotics.
total syntheses of (1R,3S)-amidinomycin (1) have been reported.18
The natural amidinomycin (1) exhibited a wide range of promising
antibacterial,15,19 antiprotozoal activities,20 high in vitro and
in vivo antiviral potencies.21 Similarly to 1, another simple b-
amidoamidine-type natural antibiotic (4S)-(ꢀ)-noformicin (2), iso-
lated from a culture of Nocardia formica,22 and its synthetic racemic
analogue were also reported to reveal high and diverse antiviral
potency.23 Nevertheless, further studies of both amidinomycin
(1) and noformicin (2) have been interrupted, probably because
of high toxicity of these natural products.15,21a–c,23a,b An entire
mode of the antiviral action of 1 and 2 was not elucidated. How-
ever, the biological activity of oligopeptide antibiotics, especially
of distamycin A (3) and its derivatives, is generally associated with
blocking the template function of DNA by binding to AT sequences
in the DNA minor groove.24 Ab initio calculations evaluated strong
bindings of different isomers of amidinomycin (1) and noformicin
(2) to the minor groove of DNA at both AT and GC sequences.25
Extensive SAR analysis within a wide set of minor groove binders
led to clarification of some general factors, which could favor a
development of more biologically potent and less toxic agents.26,27
In particular, taking advantage of hydrophobic interaction with the
minor groove of DNA, introduction of the planar aromatic rings and
lipophilic alkyl substituents to the head-group was suggested to fa-
vor the selective antibacterial and antitumor activity.27 On the
other hand, while an elevated toxicity of natural oligopeptide-type
antibiotics 1–3 was associated with a high basicity of the N-unsub-
stituted amidine tail group (pKa 12–12.5),27,28 application of less
basic (within pKa gap 5–9) tail groups could lead to therapeutically
relevant level of toxicity.27 In this Letter we demonstrate, that the
same structural and physico-chemical factors favor anti-influenza
Reaction of the anilide 5 with phosphorus pentachloride under
properly controlled conditions afforded pure metacrylimidoyl
chloride 6 in up to 95% yield. Being rather stable to vacuum
distillation, the compound 6 represents a rare example of pure
a
a
,b-unsaturated imidoyl chlorides,32 and differs essentially from
sporadically explosive even at room temperature N-phenyl
acrylimidoyl chloride.31 Upon treatment of imidoyl chloride 6 with
aniline (2 equiv), a one-pot exchange of chlorine atom to anilino-
group and unusually rapid Michael addition of aniline to b-carbon
of unsaturated system occurred,33 leading to formation of
N-phenyl-substituted b-aminoamidine hydrochloride 7 in good yield.
The direct acetylation of 7 was unsuccessful, but after conversion of
the salt 7 into free base 8, the latter was smoothly acylated with a
set of acyl chlorides (1 equiv) to give the target monoacylated
b-amidoamidines 4a–g as hydrochlorides (Scheme 1, Table 1).
Although reactions of N-aryl-substituted
a-aminoamidines
with Ac2O also afforded monoacylated at the -amino-group deriv-
a
atives,34 the mechanism of monoacylation of b-aminoamidine 8 to
form b-amidoamidines 4 is not obvious in view of apparently less
nucleophilic character of b-phenylamino-group in 8 compared to
N1,N2-diphenylamidine moiety. Indeed, competitive acetylation of
(2-phenylamino)isobutironitrile (9) and N1,N2-diphenylacetami-
dine (10), which modeled the b-phenylamino- and N1,N2-dipheny-
lamidine-functionalities in b-aminoamidine 8 respectively, led to
isolation of N1-acetylated amidine 11 (92%) and amidine hydro-
chloride 12 (97%), whereas 9 was recovered practically unchanged
(Scheme 2).
A
activity and low toxicity of racemic b-amidoamidines 4
(Scheme 1) structurally related to natural antiviral agent amidino-
Thus, monoacylation of 8 leading to 4 proceeds probably
through initial acylation at the more nucleophilic N1,N2-diphenyl-
amidine fragment with formation of the N1-acylated intermediate
13 (Scheme 3). The latter is subjected to HCl-mediated rearrange-
ment to more thermodynamically stable b-amido-amidinium salt
4, perhaps via six-membered cyclic hemidiaminal intermediate
14. A possibility of a facile acyl group migration from amidine-
function to b-amino-group in N-unsubstituted b-aminoamidines
have been proved experimentally.35 A potential viability of the
intermediate 14 is deduced from a high-yielding isolation of
1,3-diaryl-4-arylaminoimidazolium chlorides as the dehydration
products of five-membered analogs of 14 upon treatment of
mycin (1).
a
b
O
NPh
PhNH
NHPh
NHPh
NHPh
Cl
Cl
6
5
7
8
c
Ph
N
d
NHPh
PhNH
NHPh
NPh
RC(O)
N-aryl-substituted a
-amidoamidines with acyl chlorides.36
Cl
NHPh
The in vitro antiviral activity against influenza A virus strain St.
Petersburg/34/72 (H3N2) and cytotoxicity toward Madin–Darby
canine kidney (MDCK) cells of b-amidoamidine hydrochlorides
4a–g and b-aminoamidine hydrochloride 7 were tested according
to the reported methods.37 In brief, MDCK cells in the maintenance
medium were infected with 1 infective dose or 100 doses of influ-
enza A virus (1 virus dose induced 50 3% cells death under the
4a-g
Scheme 1. Synthesis of N1,N2-diphenyl-2-(N-phenylacylamido)isobutyramidines
hydrochlorides 4 (amidine hydrochlorides 4 and 7 are shown in the real amidinium
chloride form). Reagents and conditions: (a) PCl5, benzene, 40–50 °C, 6 h, 95%; (b)
PhNH2 (2.05 equiv), ether, from 5 °C to rt, overnight, 86%; (c) aqueous NH3
(50 equiv), CH2Cl2, 98%; (d) RC(O)Cl (1.02 equiv), ether, from 5 °C to rt, overnight,
79–94%.

E. E. Korshin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2357–2361
2359
Table 1
Anti-influenza virus A activity and cytotoxicity of b-amidoamidines hydrochlorides 4a–g and b-aminoamidine hydrochloride 7 in MDCK cellsa
Ph
N
NHPh
PhNH
NHPh
RC(O)
Cl
Cl
NHPh
NHPh
4a-g
7
Therapeutic index, TId
1 dose 100 doses
b
c
Compound
R
Antiviral activity, EC50
(
l
M)
Cytotoxicity, CC50
(l
M)
1 dose
100 doses
4a
4b
4c
4d
4e
4f
4g
7
C6H5
CH3
>100
68
24
0.95
1.82
6.95
15.6
ND
ND
ND
98
1.98
7.3
27.8
72
ND
43.5
286
153
216
247
936
890
1240
85
<2.85
2.25
9
260
514
128
80
—
—
n-C3H7
n-C8H17
n-C10H21
n-C12H25
n-C17H35
—
2.2
125
128
32
17
—
—
256
Rimantadine
5.45
1395
32
a
b
c
Virus strain and abbreviations used: influenza virus A: St. Petersburg/34/72 (H3N2); MDCK cells: Madin–Darby canine kidney cells; ND: not detected (EC50 P200
Effective concentration (EC50) that required to reduce virus-induced cytopathogenicity by 50% in 48 h experiment.
Cytotoxic concentration (CC50) that caused 50% reduction of MDCK cell viability after 48 h incubation.
lM).
d
TI represents the ratio of CC50 to EC50
.
In contrast to inactive parent b-aminoamidine 7, even the small-
est N-phenyl-substituted b-amidoamidines 4a–c exhibited consid-
erable antiviral activity and moderate cytotoxicity (Table 1).
Essential increase of the head-group hydrophobic properties as in
C8–C12 alkylamido-derivatives 4d–f led to significant in vitro activ-
ity against the applied influenza A virus, whereas further elongation
of the n-alkyl chain to C17 as in 4g hampered the antiviral proper-
ties. A similar positive influence of the unbranched C6–C10 aliphatic
2 PhNH
CN
2 PhNH
Ac
CN
AcCl (1.0 equiv)
9
9 (96% recovery)
+
ether, r.t.,
overnight
2
NHPh
N
NHPh
+
Ph
Cl
12 (97%)
NPh
NPh
NHPh
10
11 (92%)
Scheme 2. Competitive acylation of 2-(phenylamino)isobutyronitrile (9) and N1,N2-
diphenylacetamidine (10).
chain on the anti-influenza A (H3N2 and H1N1) activity of
x-ami-
noalkyl rupestonates was recently reported.11a In the same vein,
rather subtle changes of length from 5 to 7 atoms in the linear
hydrophobic ether chain of 3-(alkoxymethyl)-4-pyrrolidinobenzoic
acid derivatives yielded up to 10,000-times enhancement in inhibi-
tion of the influenza A viral NA of both H3N2 and H1N1 types.38
Among the tested substances, the most potent pelargonoyl- 4d
and undecanoyl-derivative 4e were found to be considerably more
active against influenza A virus compared to commercial drug
rimantadine, while the tridecanoyl-analog 4f revealed similar to
rimantadine antiviral properties (Table 1). The antiviral effects of
b-amidoamidines 4d–f were more pronounced in experiments
with 100 virus doses infection. Notable, that even the simplest
benzoylation or acetylation at the b-amino-group as in 4a,b
reduced the cytotoxicity 2–4 times compared to the parent
b-aminoamidine 7. Further elongation of the alkyl chain in
b-amidoamidines 4 provided gradual lack of toxicity, and the
highest members of this set 4e–g exhibited low cytotoxicity.
Preliminary in vivo sub-acute toxicity studies of the pelargo-
noyl-derivative 4d in rodents showed no toxicity at high dosage.
Indeed, all the four Balb/c male mice, subcutaneously (SC) single
dosed with 1000 mg/kg of 4d (solution in saline/DMSO 4:1), sur-
vived for at least 10 days after administration. Additionally, the
separate group of four mice (two of both sexes) was well tolerated
300 mg/kg daily SC treatment with 4d for 6 days with no dramatic
change in the behavior and in the body weight at the day 10. The
acute toxicities (a median lethal doses LD50) of the most in vitro ac-
tive compounds 4d–f were roughly estimated in the groups of
male mice (four in each group) upon SC administration in increas-
ing single doses from 1000 mg/kg up to 4000 mg/kg (500 mg/kg
step) (Table 2).
NPh
NPh
RC(O)Cl
- HCl
PhNH
NHPh
NPh
Ph
H
N
R
8
O
13
NPh
NPh
OH
Ph
HCl
N
NHPh
PhN
RC(O)
Cl
NHPh
R
14
4
Scheme 3. A plausible mechanism for monoacylation of 8 leading to 4.
standard experimental conditions), and incubated with each com-
pound at various concentrations for 48 h at 37 °C under a 5% CO2
atmosphere. Typical cytopathic effects (CPE) of the cells were ob-
served for each compound. The inhibition of the virus-induced
CPE for each sample was recorded relative to the cell control and
the virus control. In the case of the most potent compounds 4d,e,
the hemadsorption in chicken erythrocytes method was addition-
ally used for evaluation of antiviral activity, and similar results of
the both assays (5–8% deviation) were averaged. The measured
effective concentrations for 50%-inhibition of virus-induced cyto-
pathogenicity (EC50
) and the cytotoxic concentrations (CC50),
which caused 50% damage of MDCK cells, are summarized in Ta-
ble 1. All the data represent mean values for 2–4 separate
experiments.
In vivo anti-influenza protective activity was tested in the influ-
enza virus A/Aichi/2/68 (H3N2)-infected Balb/c mice of both sexes.
To the groups of mice (10 animals in each group) the tested

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E. E. Korshin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2357–2361
Table 2
Protective activity against experimental influenza A infection and acute toxicity of b-amidoamidines hydrochlorides 4 in micea
c
Compound
R
No. of infected survivors/total no. of infected miceb
14 days
Acute toxicity, LD50 (mg/kg)
7 days
Control (saline/DMSO)
4d
4e
4f
3/10
6/10
7/10
6/10
9/10
2/10
5/10
6/10
4/10
8/10
ND
n-C8H17
n-C10H21
n-C12H25
3000
3500
4000
175
Rimantadine
a
b
c
Mice were infected intranasally by mouse-adapted influenza virus A/Aichi/2/68 (H3N2).
Mice were dosed daily, totally 5 times, each time with 10% of LD50. The compounds were administered subcutaneously (SC) in saline/DMSO (4:1, total 100
Lethal dose (LD50) corresponds to 50% mortality of the animals upon a single dose SC administration in saline/DMSO (4:1, total 200 L). The toxicity was estimated in the
lL).
l
5 days test.
2. (a) Das, K. J. Med. Chem. 2012, 55, 6263; (b) De Clercq, E. Nat. Rev. Drug Disc.
2006, 5, 1015.
compounds 4 were SC administered. Each mouse was 5 times
dosed with 10% of LD50/dose of the tested compounds in saline–
DMSO solution (4:1, total 100 ll). Administration of the tested
compounds was provided in prophylaxis 24 h and 1 h before
infecting, followed by treatment 24, 48 and 72 h after infecting.
For comparison, the reference drug rimantadine was examined un-
der the same conditions. Control group of animals was similarly
3. (a) Deyde, V. M.; Xu, X.; Bright, R. A.; Shaw, M.; Smith, C. B.; Zhang, Y.; Shu, Y.;
Gubareva, L. V.; Cox, N. J.; Klimov, A. I. J. Infect. Dis. 2007, 196, 249; (b)
Gubareva, L. V.; Trujillo, A. A.; Okomo-Adhiambo, M.; Mishin, V. P.; Deyde, V.
M.; Sleeman, K.; Nguyen, H. T.; Sheu, T. G.; Garten, R. J.; Shaw, M. W.; Fry, A. M.;
Klimov, A. I. Antivir. Ther. 2010, 15, 1151.
4. (a) Meijer, A.; Lackenby, A.; Hungnes, O.; Lina, B.; van der Werf, S.; Schweiger,
B.; Opp, M.; Paget, J.; van de Kassteele, J.; Hay, A.; Zambon, M. Emerg. Infect. Dis.
2009, 15, 552; (b) Dharan, N. J.; Gubareva, L. V.; Meyer, J. J.; Okomo-Adhiambo,
M.; McClinton, R. C.; Marshall, S. A.; St. George, K.; Epperson, S.; Brammer, L.;
Klimov, A. I.; Bresee, J. S.; Fry, A. M. JAMA 2009, 301, 1034; (c) Moscona, A. N.
Eng. J. Med. 2009, 360, 953.
5. Kirchmair, J.; Rollinger, J. M.; Liedl, K. R.; Seidel, N.; Krumbholz, A.; Schmidtke,
M. Future Med. Chem. 2011, 3, 437.
6. (a) Müller, K. H.; Kakkola, L.; Nagaraj, A. S.; Cheltsov, A. V.; Anastasina, M.;
Kainov, D. E. Trends Pharmacol. Sci. 2012, 33, 89; (b) Muratore, G.; Goracci, L.;
Mercorelli, B.; Foeglein, A.; Digard, P.; Crusiani, G.; Palù, G.; Loregian, A. PNAS
2012, 109, 6247.
treated 5 times with the vehicle (100 ll of saline–DMSO 4:1 v/v
each dose). The efficacies of b-amidoamidine hydrochlorides 4d–f
in mice were evaluated on the basis of the number of survived ro-
dents at the day 7 and 14 post infecting with experimental influ-
enza A infection (Table 2).
Dissimilarly to very toxic amidinomycin
1 (LD50 10–20
mg/kg),15,21a–c the tested N-phenyl-substituted b-amidoamidines
4d–f are non-toxic compounds (LD50 3000–4000 mg/kg) (Table 2).
The compounds 4d–f revealed a considerable anti-influenza A pro-
tective effect, albeit upon high dosage (10% of LD50) only. In fact,
the control mice began to die from day 2 after the virus infection,
and by the day 7 only three from the control ten mice survived. In
contrast to that, in the group of mice treated with undecanoyl-
derivative 4e, a mortality of animals started from the day 5, and
by the day 7 seven from ten animals were still alive. A similar,
but less pronounced protective effects were observed for com-
pounds 4d and 4f. However, none of the tested b-amidoamidines
4d–f exhibited a considerable protective activity upon a dosage
6100 mg/kg, and all these compounds were less active in vivo
compared to commercial drug rimantadine.
7. Grienke, U.; Schmidtke, M.; von Grafenstein, S.; Kirchmair, J.; Liedl, K. R.;
Rollinger, J. M. Nat. Prod. Rep. 2012, 29, 11.
8. (a) Thapa, M.; Kim, Y.; Desper, J.; Chang, K.-O.; Hua, D. H. Bioorg. Med. Chem.
Lett. 2012, 22, 353; (b) Gao, L.; Zu, M.; Wu, S.; Liu, A.-L.; Du, G.-H. Bioorg. Med.
Chem. Lett. 2011, 21, 5964; (c) Liu, A.-L.; Wang, H.-D.; Lee, S. M. Y.; Wang, Y.-T.;
Du, G.-H. Bioorg. Med. Chem. 2008, 16, 7141; (d) Miki, K.; Nagai, T.; Suzuki, K.;
Tsujimura, R.; Koyama, K.; Kinoshita, K.; Furuhata, K.; Yamada, H.; Takahashi, K.
Bioorg. Med. Chem. Lett. 2007, 17, 772.
9. Dao, T.-T.; Tung, B.-T.; Nguyen, P.-H.; Thuong, P.-T.; Yoo, S.-S.; Kim, E.-H.; Kim,
S.-K.; Oh, W.-K. J. Nat. Prod. 2010, 73, 1636.
10. Grienke, U.; Schmidtke, M.; Kirchmair, J.; Pfarr, K.; Wutzler, P.; Dürrwald, R.;
Wolber, G.; Liedl, K. R.; Stuppner, H.; Rollinger, J. M. J. Med. Chem. 2010, 53, 778.
11. (a) Zhao, J.; Aisa, H. A. Bioorg. Med. Chem. Lett. 2012, 22, 2321; (b) Kiyohara, H.;
Ichino, C.; Kawamura, Y.; Nagai, T.; Sato, N.; Yamada, H. J. Nat. Med. 2012, 66,
55.
12. (a) Nicol, M. Q.; Ligertwood, Y.; Bacon, M. N.; Dutia, B. M.; Nash, A. A. J. Gen.
Virol. 2012, 93, 980; (b) Altman, S. E.; Jones, J. C.; Schultz-Cherry, S.; Brandt, C.
R. Virology 2009, 388, 248; (c) Jones, J. C.; Turpin, E. A.; Bultmann, H.; Brandt, C.
R.; Schultz-Cherry, S. J. Virol. 2006, 80, 11960.
13. Shibnev, V. A.; Garaev, T. M.; Finogenova, M. P.; Shevchenko, E. S.; Burtseva, E. I.
Pharm. Chem. J. 2012, 46, 1.
14. Grout, R. J. In The Chemistry of Amidines and Imidates; Patai, S., Ed.; Wiley-
Interscience: London, 1975; pp 255–281.
15. (a) Kikuchi, K. J. Antibiot. 1955, 8, 145; (b) Nakamura, S.; Karasawa, K.;
Yonehara, H.; Tanaka, N.; Umezawa, H. J. Antibiot. 1961, 14, 103.
16. (a) Nakamura, S. Chem. Pharm. Bull. 1961, 9, 641; (b) Kaneda, M.; Nakamura, S.;
Iitaka, Y. J. Antibiot. 1980, 23, 778.
17. (a) Berger, H.; Paul, H.; Hilgetag, G. Chem. Ber. 1968, 101, 1525; (b) Paul, H.;
Berger, H.; Boeden, H.; Hilgetag, G. Arch. Pharm. 1968, 301, 512.
In summary, we described an expedient synthesis of nontoxic
N-phenylated b-amidoamidines 4 and a reasonable mechanism of
their formation through intramolecular acyl fragments transfer.
The bearing long linear aliphatic chain b-amidoamidines 4d–f
(Alk = n-C8H17 ꢀ n-C12H25) exhibited significant anti-influenza A
(H3N2) activity in vitro and substantial protective effect against
experimental influenza A infection in mice.
Acknowledgments
ˇ
18. (a) Chenevert, R.; Lavoie, M.; Courchesne, G.; Martin, R. Chem. Lett. 1994, 93; (b)
This study was supported by the Israel Ministry of Absorption
through the Kamea Fellowship (to E.E.K.), and by the Arbuzov
Institute of Organic and Physical Chemistry (Research Council
Grant-in-Aid).
Sung, S.-Y.; Frahm, A. W. Arch. Pharm. 1996, 329, 291; (c) Nagata, H.; Taniguchi,
T.; Ogasawara, K. Tetrahedron: Asymmetry 1997, 8, 2679.
19. Sung, S.-Y.; Kist, M.; Frahm, A. W. Arch. Pharm. 1997, 330, 21.
20. Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; Nishihara-Tukashima,
A.; Shibahara, S.; Kondo, S.; Yamada, H.; Omura, S. J. Antibiot. 2010, 63, 381.
21. (a) Brunner, R.; Machek, G. Die Antibiotica III; Springer: Nürnberg, 1970. pp
440–441; (b) Kobayashi, N. Tohoku J. Exp. Med. 1959, 70, 95; (c) Hinuma, Y.;
Sato, S.; Chiba, M.; Kosaka, Y.; Koroya, M. Jpn. J. Microbiol. 1958, 2, 117; (d)
Hinuma, Y.; Sato, S.; Chiba, M.; Ikeda, T.; Miyakawa, T.; Mizuno, H. Jpn. J.
Microbiol. 1958, 2, 63; (e) Kuroya, M.; Kikuchi, K.; Kobayashi, N.; Matsumoto,
K.; Chiba, T.; Matsuura, S.; Hinuma, Y. Jpn. J. Microbiol. 1957, 1, 85.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.053.
22. Gray, R. A. Phytopathology 1955, 45, 281; (b) Peck, R. L.; Shafer, H. M.; Wolf, F. J.
U.S. Patent 2,804,463, 1957; Chem. Abstr. 1958, 52, 8474.
23. (a) Diana, G. D. J. Med. Chem. 1973, 16, 857; (b) Diana, G. D.; Salvador, U. J.;
Zalay, E. S.; Pancic, F. J. Med. Chem. 1973, 16, 1050; (c) Sidwell, R. W.; Dixon, G.
J.; Seller, S. M.; Frank, M., Jr. Appl. Microbiol. 1968, 16, 370.
24. a Hahn, F. E. In Antibiotics III. Mechanism of Action of Antimicrobial and Antitumor
Agents; Corcoran, J. W., Hahn, F. E., Eds.; Springer-Verlag: New York, 1975; p
References and notes
1. (a) Thorlund, K.; Awad, T.; Boivin, G.; Thabane, L. BMC Infect. Dis. 2011, 11, 134;
(b) Govorkova, E. A.; Webster, R. G. Viruses 2010, 2, 1510.

E. E. Korshin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2357–2361
79; (b) Geierstanger, B. H.; Wemmer, D. E. Annu. Rev. Biophys. Biomol. Struct. 32. For sporadic examples of
2361
a
,b-unsaturated imidoyl chlorides, see: (a) von
1995, 24, 463.
Brown, J.; Rudolph, W. Chem. Ber. 1934, 67B, 269; (b) Freudenberg, K.; Gehrke,
G. Chem. Ber. 1951, 84, 443; (c) Tanaka, M.; Kobayashi, T. Synthesis 1985, 967;
(d) Onys0ko, P. P.; Kim, T. V.; Kiseleva, E. I.; Sinitsa, A. D. Zh. Obsch. Khim. 1990,
60, 229. Chem. Abstr. 1990, 113, 59310.
25. Sapse, A.-M.; Feng, W.; Fugler-Domenico, L.; Kabir, S.; Joseph, T.; Lown, J. W. J.
Biomol. Struct. Dyn. 1993, 10, 709.
26. Cortesi, R.; Esposito, E. Mini-Rev. Med. Chem. 2010, 10, 218.
27. (a) Suckling, C. J. J. Phys. Org. Chem. 2008, 21, 575; (b) Suckling, C. Future Med.
Chem. 2012, 4, 971.
33. Michael additions of anilines to methacryloyl-derivatives usually require a
long heating and/or the presence of transition metal catalyst. See, e.g: (a)
Kawatsura, M.; Hartwig, J. F. Organometallics 2001, 20, 1960; (b) Fadini, L.;
Togni, A. Chem. Commun. 2003, 30.
34. Korshin, E. E.; Soboleva, G. I.; Levin, Y. A.; Zakharova, L. G.; Litvinov, I. A.;
Naumov, V. A.; Podval0niy, E. A.; Efremov, Y. Y. Russ. J. Org. Chem. 1992, 28, 973.
35. Barker, P. L.; Gendler, P. L.; Rapoport, H. J. Org. Chem. 1981, 46, 2455.
36. Korshin, E. E.; Soboleva, G. I.; Levin, Y. A.; Podval0niy, E. A.; Efremov, Y. Y. Russ. J.
Org. Chem. 1993, 29, 482.
28. The basicities of N-unsubstituted amidine function in 1–3 and N1,N2-
diphenylamidino-function in 4 (as a base) must be very similar to basicities
of acetamidine and N1,N2-diphenylacetamidine (10), respectively. The
measured pKa value (in H2O) for acetamidine is 12.4, and for 10 is 8.3. See:
Häfelinger, G.; Kuske, F. K. H. In The Chemistry of Amidines and Imidates; Patai,
S., Rapoport, Z., Eds.; Wiley: Chichester, 1991; Vol. 2, pp 77–78.
29. See, e.g: (a) Beletskaya, I. P.; Cheprakov, A. V. Coord. Chem. Rev. 2004, 248, 2337;
(b) Corbet, J.-P.; Mignani, G. Chem. Rev. 2006, 106, 2651; (c) Guo, X.; Rao, H.; Fu,
H.; Jiang, Y.; Zhao, Y. Adv. Synth. Catal. 2006, 348, 2197.
37. Shigeta, S.; Konno, K.; Yokota, T.; Nakamura, K.; De Clercq, E. Antimicrob. Agents
Chemother. 1988, 32, 906.
30. (a) Rauws, T. R. M.; Maes, B. U. W. Chem. Soc. Rev. 2012, 41, 2463; (b) Krasavin,
M. Tetrahedron Lett. 2012, 53, 2876.
38. Li, Y.; Silamkoti, A.; Kolavi, G.; Mou, L.; Gulati, S.; Air, G. M.; Brouillette, W. J.
Bioorg. Med. Chem. 2012, 20, 4582.
31. Korshin, E. E.; Zakharova, L. G.; Levin, Y. A.; Ashrafullina, L. K.; Podval0niy, E. A.;
Efremov, Y. Y. Russ. Chem. Bull. 1992, 41, 2082.