Synthesis of new compounds with promising antiviral properties against group A and B Human Rhinoviruses

Article abstract of DOI:10.1016/j.bmc.2014.05.066

The human common cold, which is a benign disease caused by the Rhinoviruses, generally receives palliative symptomatic treatments, since no specific therapy against any of these viruses currently exists. In this work, some original synthetic compounds wer

Full text of DOI:10.1016/j.bmc.2014.05.066

Bioorganic & Medicinal Chemistry 22 (2014) 4061–4066
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new compounds with promising antiviral properties
against group A and B Human Rhinoviruses
a
a
b,
Angela M. Bernard a, Maria G. Cabiddu a, , Stefania De Montis , Roberto Mura , Raffaello Pompei
⇑
⇑
a Dipartimento di Scienze Chimiche e Geologiche, Università di Cagliari, Cittadella Universitaria di Monserrato, S.S. 554, Bivio per Sestu, I-09042 Monserrato (CA), Italy
b Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Università di Cagliari, Cittadella Universitaria di Monserrato, S.S. 554, Bivio per Sestu, I-09042
Monserrato (CA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The human common cold, which is a benign disease caused by the Rhinoviruses, generally receives pal-
liative symptomatic treatments, since no specific therapy against any of these viruses currently exists. In
this work, some original synthetic compounds were produced and tested, in order to find non-toxic sub-
stances with an improved protection index (PI) for infected cells, as compared to reference drugs such as
Pirodavir. We designed a series of novel molecules with a double oxygen in the central hydrocarbon chain
and some modifications of the lateral methylisoxazole and propoxybenzoate moieties of lead compound
6602 (ethyl 4-{3-[2-(3-methyl-1,2-isoxazol-5-yl)ethoxy]propoxy}benzoate). It was found that most of
these substances were actually less toxic than Pirodavir; in addition, the new molecule indicated as 8c
was more than 30 times less toxic than Pirodavir, about twice as active on the group A strain of
Rhinovirus HRV14, and even four times more effective on the group B strain HRV39, as compared to
Pirodavir’s PI.
Received 20 February 2014
Revised 23 May 2014
Accepted 29 May 2014
Available online 12 June 2014
Keywords:
Rhinoviruses
Antivirals
Cell receptors
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Rhinovirus strains, for example, bind to the intracellular adhesion
molecule 1 (ICAM-1), an immunoglobulin-like molecule, and con-
Several groups of viruses that cause an extraordinarily wide
range of illnesses belong to the Picornaviridae family.1,2 The Rhino-
virus genus contains the common cold agents and is formed from a
great number of serotypes, making it extremely difficult to prepare
an effective vaccine.3 Due to the lack of specific antiviral therapy,
most Rhinovirus illnesses are managed symptomatically. Although
effective antiviral therapy is not yet clinically available, some
promising anti-picornavirus drugs are able to reduce the duration
of illness in adults. Capsid-binding compounds such as Disoxaril
and Pirodavir have proved to be partially effective in the intranasal
treatment of patients with a common cold caused by some strains
of Human Rhinoviruses.4–10
The Rhinovirus capsid consists of 4 polypeptides VP 1, 2, 3, and
4, which all derive from the cleavage of a larger protein. The capsid
proteins determine host and tissue tropism by recognition of
cell-specific cell-membrane receptors and the virus uses these
membrane receptors to enter the target cells.11–13 Most human
sequently, capsid proteins are a potential target for the action of
specific anti-Rhinovirus drugs.14–19
In a previous study20 we tested several novel anti-Rhinovirus
compounds and demonstrated that some modifications of the cen-
tral hydrocarbon molecule chain could lead to new interesting
drugs with improved activity for certain Rhinovirus strains and
decreased cytotoxicity on cell cultures. Thus, considering that the
VP1 viral canyons of different species of Picornaviruses have a
peculiar structure in terms of size, depth and shape, we designed
novel SAR studies targeted at modifying the central chain and
lateral moieties of lead compound 6602 (1a), in order to find origi-
nal molecules whose antiviral and cytotoxic properties could prove
more effective against group A and B Rhinovirus strains.
In the present work we describe some original molecules with
very promising properties, one of which presents improved cyto-
toxic and anti-viral activity, as compared to lead drug 6602 and
also to the reference drug Pirodavir.
2. Drug design
⇑
Corresponding authors. Tel.: +39 070 675 4406; fax: +39 070 675 4388 (M.G.C.);
The molecules described in this study were derived from lead
compound 6602 (1a) described by Laconi et al.20 and represented
in Figure 1. Pirodavir (1b) was also used as a reference compound.
tel.: +39 070 675 8483; fax: +39 070 675 8482 (R.P.).
E-mail addresses: mgcabidd@unica.it (M.G. Cabiddu), rpompei@unica.it
(R. Pompei).
http://dx.doi.org/10.1016/j.bmc.2014.05.066
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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A. M. Bernard et al. / Bioorg. Med. Chem. 22 (2014) 4061–4066
Figure 1. Compound 1a: The chemical structure of lead compound 6602 (ethyl 4-
{3-[2-(3-methyl-1,2-isoxazol-5-yl)ethoxy]propoxy}benzoate). Compound 1b: The
general reference compound Pirodavir.
Since compounds 1a and 1b undergo easy in vivo hydrolysis of
the ester functionality to the corresponding acid derivatives which
are almost devoid of anti-HRV activity, we disclosed some new
compounds lacking such a function in the aryloxy group.
With this aim, we substituted the above-mentioned group with
either a 1,3-benzodioxol-5-yl (6b, 7b, 8b), a 4-acetylphenyl (6c, 8c,
9c, 10c), a 4-cyanophenyl (8a), or a 4-fluorophenyl group (9d)
(Fig. 2). In some derivatives the methylisoxazolyl moiety was
substituted by either a 2-ethoxy-1,3-benzoxazol-6-yl (8a–c) or a
1,3-benzodioxol-5-yl group (9c–d, 10c). The central aliphatic chain
was shortened in molecules 7b, 8a–c, 9c–d and 10c.
Figure 2. Study of compound 8c antiviral activity when added to the cells at
different times after infection. Compound 8c and Pirodavir were used at
concentration of 5 g/mL. Both compounds were 100% effective when added to
a
l
the cells from h À1 until the 2nd h post infection; after this their activity decreased
rapidly.
Two different strains of Rhinoviruses were used in these tests, rep-
resenting the 2 major groups of this genus, namely groups A and B.
Each compound was tested for the maximum non-toxic dose
(CD50), the inhibitory dose (ID50
(PI = CD/ID).
) and the protection index
3. Results and discussion
3.1. Chemistry
3.2.1. Chemical modification and structure–activity relationship
(SAR)
All the new compounds tested showed a CD equal or even lower
than 6602, but all of them resulted far less toxic than Pirodavir on
HeLa cells; moreover, the insertion of an ethoxybenzoxazol-6-yl
group coupled with either a benzodioxolyl or a 4-acetylphenyl
group produced two compounds (8b and 8c) that were more than
60 times less toxic than pirodavir and 4 times less toxic than 6602.
The introduction of the 1,3-benzodioxol-5-yl group in 6b, 7b, 8b,
9c, 9d, 10c led to substances with poor antiviral activity. Although
their cytotoxicity was low, the PIs were far less significant than
those of 6602 and Pirodavir. The insertion of a cyano substituent
into the aryloxy group produced a compound (8a) with a PI of 16
and 33, respectively, still worse than 6602. It should also be noted
that shortening the central aliphatic chain as in 7b till 10c did not
seem to have a relevant effect on either the cytotoxicity or the
antiviral effect. Finally, when the aryloxy group was replaced by
a 4-acetylphenyl group as in 6c and 8c, the results were extremely
The general synthetic pathways for the preparation of the
reported compounds are shown in Scheme 1. Compounds 6–10
were obtained through nucleophilic substitution between
derivatives 3–5 and the aryloxypropyl bromides 2a–d, obtained
from the corresponding phenols by treatment with excess of 1,
3-dibromopropane. The primary alcohol
3 [2-(3-methyl-1,2-
isoxazol-5-yl)ethanol, n = 2] was prepared following previously
described methods.21 Compound 4 was prepared by the reaction
of 4-aminobenzene-1,3-diol with an excess of tetraethyl orthocar-
bonate. Compounds 3 (n = 0) and 5 are commercially available.
3.2. Antiviral activity
The cytotoxicity and the anti-Rhinovirus activity of 9 novel ori-
ginal compounds derived from lead 6602 are indicated in Table 1.
Scheme 1. Synthesis of compounds 6–10. Reagents and conditions: (i) n = 2, NaH, THF, À10 °C; n = 0, K2CO3, CH3CN, 80 °C; (ii) K2CO3, CH3CN, 80 °C; (iii) n = 1, NaH, THF,
À10 °C; n = 0, K2CO3, CH3CN, 80 °C.

A. M. Bernard et al. / Bioorg. Med. Chem. 22 (2014) 4061–4066
4063
Table 1
Screening of cytotoxic activity and anti-Rhinovirus properties of the novel compounds and reference molecules 6602 and Pirodavir
Compounds
Cytotoxic activity
Anti-Rhinovirus activity
a
b
CD50
(lg/mL)
HRV14 ID50
(lg/mL)
PIc
HRV39 ID50
(
lg/mL)
PI
6602
6b
50
50
0.07
50
714
1
0.07
50
714
1
6c
50
25
2
6
8
7b
50
25
2
12
4
8a
8b
8c
9c
50
25
25
0.5
25
2
8
400
1
1.5
12
0.1
25
33
16
2000
1
200
200
25
9d
25
25
1
25
1
10c
Pirodavir
50
3.1
6
8
258
6
8
516
0.012
0.006
a
b
c
CD: Cytotoxic dose on HeLa cells.
ID: Inhibitory dose.
PI: Protection index (CD/ID).
different depending on the concomitant modification of the isoxaz-
olyl moiety. 6c showed a modest PI of 8, whilst 8c, where the
methylisoxazolyl group was substituted by a 2-ethoxy-1,3-benz-
oxazolyl group, presented a dramatic improvement of its activity
and cytotoxicity on cultured cells. Its PI for the group A Rhinovirus
was 400 (about half of that shown by 6602, but almost twice that
of Pirodavir = 258), but for group B it was 2000, far better than
6602 (714) and Pirodavir (516). Although the overall specific anti-
viral activity of 8c was lower than that of 6602 and Pirodavir, it is
important to consider that in all cases, compound 8c was less toxic
on cell cultures than all the other lead or reference compounds
tested, so that in most cases its PI was highly improved with
respect to both 6602 and pirodavir.
some properties that render it less toxic and more active on Rhino-
viruses than the reference compounds. Since Pirodavir, the most
studied reference compound, has already shown some sort of
anti-common cold activity at least as an in vivo topical treatment,
but has yet to be launched onto the drug market because of its
toxicity, we believe that substance 8c has shown a significant
improvement in terms of cell toxicity and anti-viral activity and
merits possible development for in vivo studies and for preclinical
testing.
5. Experimental
5.1. Synthesis and characterization
3.2.2. Action mechanism of the new compounds
5.1.1. General
Figure 2 shows the antiviral activity of compound 8c on HRV14
when it is added to the infected cell culture at different times after
infection. Pirodavir was used as the reference molecule. 8c exerts
its inhibition effect on the Rhinovirus when added to the cells
within the 3rd h after infection. From the 4th h onwards the activ-
ity rapidly decreases to non-significant levels. This behaviour is
quite similar to that expressed by Pirodavir, and means that 8c is
an antiviral molecule which exerts its activity in the early phases
of viral replication, such as during virus-to-cell adhesion and virus
entry into the cells. In this context, compound 8c seems to behave
exactly like the reference Pirodavir, which alters virion adhesion to
the cells by blocking the interaction between the Rhinovirus capsid
canyon and the cell receptor ICAM 1. Moreover, it is important to
point out that substance 8c, just like Pirodavir, has a reversible
effect on Rhinovirus inhibition and does not show any kind of
direct virucidal activity.
NMR spectra were recorded in CDCl3 on 500 Varian spectrome-
ter at room temperature and TMS as internal standard. Chemical
shifts are reported as follows: chemical shifts, multiplicity and cou-
pling constants. Mass spectra were recorded on Agilent 5973 N
(Cpsil 32 m) and Nermag R10-10 (quartz-Cpsil 5.25 m). (E.I.
70 eV) Microanalysis was performed with a Perkin Elmer 2400 ser-
ies 2. Infrared spectra were obtained using a Bruker-FT-IR.
Analytical thin layer chromatography was performed using
0.25 mm silica gel 60-F plates. Flash chromatography was per-
formed using 200–400 mesh silica gel (Merk KGaA). Unless other-
wise stated, yields refer to chromatography and spectroscopically
pure materials. THF was freshly distilled from sodium/benzophe-
none. Reactions requiring anhydrous conditions were performed
in oven dried glassware under Argon atmosphere.
5.1.2. Preparation of 4-(3-bromopropoxy)benzonitrile (2a)
A solution of 1,3-dibromopropane (4.24 g, 21.2 mmol, 2.13 mL) in
CH3CN (8 mL), was added dropwise to a stirred mixture of 4-hydrox-
ybenzonitrile (0.50 g, 4.2 mmol) and K2CO3 (0.87 g, 6.3 mmol) in CH3
CN (10 mL) at room temperature and then stirred at 80 °C for 7 h. The
reaction mixture was cooled, the solvent was removed under reduced
pressure and the residue was dissolved in CH2Cl2 (30 mL), washed
with 10% NaOH (2 Â 20 mL), and dried (Na2SO4). The solvent was
evaporated under reduced pressure and the residue was purified by
silica gel column chromatography with petroleum ether (40–60)/
diethyl ether (4:1) as eluent to give 2a (0.68 g, 68%).
4. Conclusions
The aim of this study was to induce some modifications on lead
compound 6602 in order to obtain a number of derivatives with
improved cytotoxic and anti-Rhinovirus activities. In fact, by
substituting the isoxazolyl moiety with a benzoxazolyl group,
and the ethyl ester with a 4-acetylphenyl group, we obtained a
new compound, indicated by the label 8c, which was 4 times less
toxic than molecule 6602 and more than 60 times less toxic than
the reference compound Pirodavir. Furthermore, anti-Rhinovirus
activity resulted highly improved, since the protection index of
8c was about 3 times superior to that of compound 6602 on
HRV39, about twice as high as that of Pirodavir on HRV14 and
up to 4 times better that that shown by Pirodavir on HRV39. We
conclude that the novel original molecule named 8c has acquired
Colourless oil; IR (neat): 2226 cmÀ1 (CN). 1H NMR (CDCl3,
500 MHz): d 2.29 (m, CH2CH2Br), 3.55 (t, CH2Br, J = 6.5 Hz), 4.12
(t, OCH2, J = 6.0 Hz), 6.92 (d, 2 ArH, J = 9.0 Hz), 7.53 (d, 2 ArH,
J = 9.0 Hz); 13C NMR (CDCl3, 125,8 MHz): d 29.6 (CH2), 31.9 (CH2),
65.7 (CH2), 104.1 (C), 115.2 (2CH), 119.1 (CN), 133.9 (2CH), 161.9
(C); MS (EI): m/z (%) 241 (29), 239 (27), 160 (4), 119 (100), 102

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A. M. Bernard et al. / Bioorg. Med. Chem. 22 (2014) 4061–4066
(12), 64 (10), 41 (55). Anal. Calcd for C10H10BrNO: C, 50.02; H, 4.20;
N, 5.83. Found: 50.16; H, 4.12; N, 5.76.
5.1.4.1. 5-{[3-(1,3-Benzodioxol-5-yloxy)propoxy]-3-methyl-1,2-
iso-xazole (7b). (0.17 g, 41%), yellow crystals; mp: 100–
5-(3-Bromopropoxy)-1,3-benzodioxole (2b)22 (0.73 g, 68%),
1-[4-(3-bromopropoxy)phenyl]ethanone (2c)23 (0.74 g, 69%) and
1-(3-bromopro-poxy)-4-fluorobenzene (2d)24 (0.69 g, 70%) were
prepared in a manner similar to that described above for 2a, using
1,3-benzodioxol-5-ol (0.58 g, 4.2 mmol),or 1-(4-hydroxy-phenyl)eth-
anone (0.57 g, 4,2 mmol), or 4-fluorophenol (0.47 g, 4.2 mmol), in
place of 4-hydroxybenzonitrile, respectively, and the 1H NMR, 13C
NMR and MS data were in accordance with literature values.
102 °C; 1H NMR (CDCl3, 500 MHz): d 2.16 (s, CH3), 2.23 (qnt, OCH2
CH2CH2O, J = 6.0 Hz), 4.04 (t, Isoxazole-OCH2, J = 6.0 Hz), 4.33 (t,
CH2OAr, J = 6.0 Hz), 5.06 (s, Isoxazole-H), 5.91 (s, OCH2O), 6.31
(dd, Ar-H, J = 2.0 Hz, J = 8.5 Hz), 6.48 (d, ArH, J = 2.0 Hz), 6.69 (d,
Ar-H, J = 8.5 Hz); 13C NMR (CDCl3, 125,8 MHz): d 12.3 (CH3), 28.9
(CH2), 64.5 (CH2), 68.7 (CH2), 86.7 (CH), 98.1 (CH, Isoxazole),
101.1 (OCH2O), 105.7 (CH), 107.9 (CH), 141.8 (C), 148.3 (C), 154.1
(C, Isoxazole), 162.2 (C), 173.2 (C, Isoxazole); MS (EI): m/z (%) 277
(42), 149 (52), 140 (100), 138 (46), 82 (56). Anal. Calcd for
5.1.3. Preparation of 6b and 6c
C14H15NO5: C, 60.64; H, 5.45; N, 5.05. Found: 60.48; H, 5.52; N,
A
solution of 2-(3-methyl-1,2-isoxazol-5-yl)ethanol (0.20 g,
4.94.
1.6 mmol)21 in anhydrous THF (1 mL) was added at room temper-
ature to a stirred suspension of pentane-washed sodium hydride
(60% w/v in mineral oil, 0.09 g, 2,3 mmol) in THF (5 mL) in an argon
atmosphere. After 1 h the appropriate halides 2b (0,41 g,
1.6 mmol) or 2c (0,41 g, 1.6 mmol) in THF (5 mL) were added at
-10 °C and the resulting mixture was stirred and warmed to a room
temperature over 18 h and then treated with brine (20 mL). The
organic layer was extracted with diethyl ether (3 Â 20 mL), dried
(Na2SO4) and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography with petroleum ether
(40–60)/diethyl ether (4:1) as eluent to give the final products.
5.1.5. 2-Ethoxy-1,3-benzoxazol-6-ol (4)25
To a mixture of of 4-aminobenzene-1,3-diol hydrochloride
(1.00 g, 6.2 mmol) and anhydrous sodium acetate (0.52 g,
6.2 mmol) in anhydrous CHCl3 (30 mL), a slight excess of tetraethyl
orthocarbonate (2.50 g, 13.0 mmol) was added and then stirred for
16 h at 60 °C. The reaction was worked up by adding saturated
NaCl solution (20 mL) and the aqueous phase was extracted with
CH2Cl2 (3 Â 20 mL). The organic extracts were dried (NaSO4) and
evaporated under reduced pressure, and the residue purified by sil-
ica gel column chromatography with petroleum ether (40–60)/
ethyl acetate (3:1) as eluent to give 4 (0.78 g, 70%).
5.1.3.1. 5-{2-[3-(1,3-Benzodioxol-5-yloxy)propoxy]ethyl}-3-methyl-
The 1H-NMR and 13C NMR data were in agreement with the lit-
erature values. MS (EI): m/z (%) 179 (34), 151 (100), 122 (39), 107
(12), 95 (88), 80 (16), 67 (30), 52 (31), 39 (41). Anal. Calcd for
C9H9NO3: C, 60.33; H, 5.06; N, 7.82; O, 26.79. Found: 60.28; H,
5.11; N, 7.76; O, 26.85.
1,2-isoxazole (6b).
(0.19 g, 40%), colourless oil; 1H NMR (CDCl3,
500 MHz): d 2.01 (qnt, OCH2CH2CH2O, J = 6.0 Hz), 2.23 (s, CH3), 2.98
(t, Isoxazole-CH2, J = 6.5 Hz), 3.63 (t, OCH2CH2CH2O, J = 6.0 Hz), 3.73 (t,
Isoxazole-CH2CH2O, J = 6.5 Hz), 3.96 (t, CH2O-Benzodioxole, J = 6.0 Hz),
5.88 (s, Isoxazole-H), 5.92 (s, OCH2O), 6.32 (dd, Ar-H, J = 2.5 Hz,
J = 8,0 Hz), 6.50 (dd, Ar-H, J = 2.5 Hz, J = 6.0 Hz), 6.71 (dd, Ar-H,
J = 3.0 Hz); 13C NMR (CDCl3, 125.8 MHz): d 11.3 (CH3), 27.5 (CH2),
29.3 (CH2), 65.5 (CH2), 67.4 (CH2), 67.9 (CH2), 98.0 (CH), 101.1 (OCH2O),
102.4(CH, Isoxazole), 105.7 (CH), 107.9 (CH), 141.6 (C), 149.9 (C), 159.6
(C, Isoxazole), 159.8 (C), 170.4 (C, Isoxazole); MS (EI): m/z (%) 305 (51),
138 (100), 110 (37), 68 (85), 41 (55). Anal. Calcd for C16H19NO5: C,
62.94; H, 6.27; N, 4.59. Found: 62.86; H, 6.12; N, 4.76.
5.1.6. 4-{3-[(2-Ethoxy-1,3-benzoxazol-6-yl)oxy]propoxy}
benzonitrile (8a)
A solution of 2-ethoxy-1,3-benzoxazol-6-ol 4 (0.30 g, 1.7 mmol)
in CH3CN (2 mL), was added dropwise to a stirred mixture of 4-(3-
bromopropoxy)benzonitrile 2a (0.40 g, 1.7 mmol) and K2CO3
(0.35 g, 2.5 mmol) in CH3CN (10 mL) at room temperature and then
stirred at 80 °C for 7 h. The reaction mixture was cooled, the sol-
vent was removed under reduced pressure and the residue was
dissolved in CH2Cl2 (30 mL), washed with 10% NaOH (2 Â 20 mL),
and dried (Na2SO4). The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column chroma-
tography with petroleum ether (40–60)/diethyl ether) (4:1) as elu-
ent to give 8a (0.51 g, 89%).
5.1.3.2. 1-(4-{3-[2-(3-Methyl-1,2-isoxazol-5-yl)ethoxy]propoxy}
phe-nyl)ethanone (6c).
(0.23 g, 47%), colourless oil; IR
(neat): 1669 cmÀ1 (C@O). 1H NMR (CDCl3, 500 MHz): d 2.03 (qnt,
OCH2CH2CH2O, J = 6.0 Hz), 2.18 (s, CH3), 2.52 (s, CH3CO), 2.95 (t,
Isoxazole-CH2, J = 6.5 Hz), 3.60 (t, OCH2CH2CH2O, J = 6.0 Hz), 3.70
(t, Isoxazole-CH2CH2O, J = 6.0 Hz), 4.06 (t, CH2OAr, J = 6.0 Hz),
5.83 (s, 1H, Isoxazole-H), 6.90 (d, 2 Ar-H, 8.5 Hz), 7.90 (d, 2 Ar-H,
9 Hz); 13C NMR (CDCl3, 125.8 MHz): d 11.3 (CH3), 26.3 (CH3CO),
27.5 (CH2), 29.4 (CH2), 64.9 (CH2), 67.1 (CH2), 67.9 (CH2), 102.4
(CH, Isoxazole), 114.1 (2CH), 130.4 (C), 130.6 (2CH), 159.8 (C, Isox-
azole), 162.9 (C), 170.3 (C, Isoxazole), 196.7 (C@O); MS (EI): m/z (%)
303 (22), 288 (16), 161 (25), 149 (22), 121 (45), 110 (41), 68 (87),
43 (100). Anal. Calcd for C17H21NO4: C, 67.31; H, 6.98; N, 4.62.
Found: 67.46; H, 6.72; N, 4.74.
White solid; mp: 122 °C; IR (nujol): 2224 cmÀ1 (CN). 1H NMR
(CDCl3, 500 MHz): d 1.51 (t, CH3, J = 7.0 Hz), 2.31 (qnt, OCH2CH2-
CH2O, J = 6.0 Hz), 4.17 (t, OCH2, J = 6.0 Hz), 4.23 (t, CH2O,
J = 6.0 Hz), 4.59 (q, CH3CH2, J = 7.0 Hz), 6.83 (dd, Benzoxazole-H,
J = 2.0 Hz, J = 8.5 Hz), 6.95 (d, Benzoxazole-H, J = 2.0 Hz), 6.97 (d,
2 Ar-H, J = 9.0 Hz), 7.35 (d, Benzoxazole-H, J = 8.5 Hz), 7.59 (d, 2
Ar-H, J = 8.5 Hz). 13C NMR (CDCl3, 125,8 MHz): d 14.3 (CH3), 29.1
(CH2), 64.8 (CH2), 65.0 (CH2), 68.1 (CH2), 96.8 (CH), 104.1 (C),
111.6 (CH), 115.2 (2CH), 117.8 (CH), 119.1 (CN), 134.0 (2CH),
134.9 (C), 148.8 (C), 155.3 (C), 162.1 (C, Oxazole), 163.0 (C); MS
(EI): m/z (%) 338 (44), 151 (100), 132 (52), 102 (42), 90 (12), 77
(25), 41 (39). Anal. Calcd for C19H18N2O4: C, 67.44; H, 5.36; N,
8.28. Found: 67.36; H, 5.22; N, 8.38.
5.1.4. Preparation of 7b
5-(3-Bromopropoxy)-1,3-benzodioxole (2b) (0.41 g, 1.6 mmol)
in CH3CN (2 mL), was added dropwise at room temperature to a
stirred solution of 3-methylisoxazol-5-(4H)-one morpholine salt
(0.30 g 1.6 mmol), K2CO3 (0.69 g, 5.0 mmol) in CH3CN (10 mL),
and then was stirred at 80 °C for 7 h. The reaction mixture was
cooled, the solvent was removed under reduced pressure and the
residue was dissolved in CH2Cl2 (30 mL), washed with 10% NaOH
(2 Â 20 mL), and dried (Na2SO4). The solvent was evaporated under
reduced pressure and the residue was purified by silica gel column
chromatography with petroleum ether (40–60)/diethyl ether (4:1)
as eluent to give the final product.
8b and 8c were prepared using a similar synthetic method
to that described above for 8a, using 5-(3-bromopropoxy)-1,
3-benzodioxole 2b (0.44 g, 1.7 mmol) or 1-[4-(3-bromoprop-
oxy)phenyl]ethanone 2c (0.43 g, 1.7 mmol) in place of 4-(3-bromo-
propoxy)benzonitrile, respectively.
5.1.6.1. 6-[3-(1,3-Benzodioxol-5-yloxy)propoxy]-2-ethoxy-1,3-
ben-zoxazole (8b).
(0.42 g, 69%), white solid; mp: 104–
106 °C; 1H NMR (CDCl3, 500 MHz): d 1.51 (t, CH3, J = 7.0 Hz), 2.24

A. M. Bernard et al. / Bioorg. Med. Chem. 22 (2014) 4061–4066
4065
(qnt, OCH2CH2CH2O, J = 6.0 Hz), 4.10 (t, CH2O J = 6.0 Hz), 4.16 (t,
OCH2, J = 6.0 Hz), 4.59 (q, CH3CH2, J = 7.0 Hz), 5.92 (s, OCH2O), 6.35
(dd, Benzodioxole-H, J = 2.5 Hz, J = 8.5 Hz), 6.52 (d, Benzodioxole-H,
J = 2.5 Hz), 6.71 (d, Benzodioxole-H, J = 8.5 Hz), 6.84 (dd, Benzoxaz-
ole-H, J = 2.0 Hz, J = 8.5 Hz), 6.96 (d, Benzoxazole-H, J = 2.0 Hz), 7.35
(d, Benzoxazole-H, J = 8.5 Hz); 13C NMR (CDCl3, 125,8 MHz): d 14.3
(CH3), 29.4 (CH2), 65.37 (CH2), 65.42 (CH2), 68.0 (CH2), 96.8 (CH),
98.1 (CH), 101.1 (OCH2O), 105.7 (CH), 107.9 (CH), 111.6 (CH), 117.7
(CH), 134.7 (C), 141.7 (C), 148.2 (C), 148.8 (C), 154.4 (C), 155.5 (C),
162.9 (C, Oxazole); MS (EI): m/z (%) 357 (70), 192 (61), 178 (32),
164 (23), 149 (100), 137 (62), 121 (41), 107 (32), 93 (22), 79 (24),
65 (34). Anal. Calcd for C19H19NO6: C, 63.86; H, 5.36; N, 3.92. Found:
63.76; H, 5.32; N, 3.78.
(CDCl3, 125.8 MHz): d 29.3 (CH2), 65.0 (2CH2), 98.1 (CH), 101.1
(OCH2O), 105.7 (CH), 106.9 (C), 107.9 (CH), 115.5 (2CH, d, JCF = 7.9 -
Hz), 115.8 (2CH, d, JCF = 23.0 Hz), 122.3 (C), 154.9 (C), 155.0 (C),
157.3 (CF, d, JCF = 238.1 Hz); MS (EI): m/z (%) 290 (55), 138 (100),
125 (45), 107 (14), 95 (39). Anal. Calcd for C161515FO4: C, 66.20;
H, 5.21. Found: 66.32; H, 5.30.
5.1.8. 1-{4-[3-(1,3-Benzodioxol-5-ylmethoxy)propoxy]phenyl}-
ethanone (10c)
A solution of 1,3-benzodioxol-5-ylmethanol (0.50 g, 3.3 mmol)
in anhydrous THF (5 mL) was added at room temperature to a stir-
red suspension of pentane-washed sodium hydride (60% w/v in
mineral oil, 0.17 g, 4.2 mmol) in THF (20 mL) in an argon atmo-
sphere. After 1 h the halide 2c (0.84 g, 3.3 mmol) in THF (5 mL)
was added at room temperature and the resulting mixture was
stirred at 60 °C for 16 h and then treated with brine (20 mL). The
organic layer was extracted with diethyl ether (3 Â 20 mL), dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography with petroleum
ether (40–60)/diethyl ether (1:1) as eluent to give 10c (0.52 g,
48%).
5.1.6.2.
phe-nyl)ethanone (8c).
1-(4-{3-[(2-Ethoxy-1,3-benzoxazol-6-yl)oxy]propoxy}
(0.30 g, 49%), white solid; mp: 112–
114 °C; IR (nujol) 1668 cmÀ1 (C=O). 1H NMR (CDCl3, 500 MHz): d
1.52 (t, CH3CH2, J = 7.0 Hz), 2.32 (qnt, OCH2CH2CH2O, J = 6.0 Hz),
2.57 (s, CH3CO), 4.19 (t, CH2O, J = 6.0 Hz), 4.26 (t, OCH2,
J = 6.0 Hz), 4.60 (q, CH3CH2, J = 7.0 Hz), 6.85 (dd, Benzoxazole-H,
J = 2.5 Hz, J = 8.5 Hz), 6.96 (d, Benzoxazole-H, J = 2.5 Hz), 6.97 (d,
2 Ar-H, J = 9.0 Hz), 7.36 (d, Benzoxazole-H, J = 8.5 Hz), 7.95 (d, 2
Ar-H, J = 9.0 Hz); 13C NMR (CDCl3, 125,8 MHz): d 14.3 (CH3), 26.3
(COCH3), 29.2 (CH2), 64.7 (CH2), 65.1 (CH2), 68.1 (CH2), 96.8 (CH),
111.6 (CH), 114.2 (2CH), 117.7 (CH), 130.4 (C), 130.6 (2CH), 134.8
(C), 148.8 (C), 155.4 (C), 162.8 (C, Oxazole), 163.0 (C), 196.7
(C=O); MS (EI): m/z (%) 355 (86), 177 (22), 162 (26), 151 (59),
135 (15), 121 (22), 106 (23), 91 (24), 77 (25), 43 (100). Anal. Calcd
for C20H21NO5: C, 67.59; H, 5.96; N, 3.94. Found: 67.44; H, 5.82; N,
3.78.
Yellow solid; mp 42–44 °C; IR (nujol): 1669 cmÀ1 (C@O). 1H
NMR (CDCl3, 500 MHz): d 2.09 (qnt, CH2, J = 6.0 Hz), 2.55 (s, CH3
CO), 3.63 (t, 2H, OCH2, J = 6.0 Hz), 4.13 (t, ArOCH2, J = 6.0 Hz),
4.56 (s, Benzodioxole-CH2O), 5.94 (s, OCH2O), 6.76 (d, Benzodiox-
ole-H, J = 6.5 Hz), 6.79 (d, Benzodioxole-H, J = 6.5 Hz), 6.86 (s, Ben-
zodioxole-H), 6.91 (d, 2 Ar-H, J = 9.0 Hz), 7.92 (d, 2 Ar-H, J = 9.0 Hz);
13C NMR (CDCl3, 125,8 MHz): d 26.3 (CH3), 29.6 (CH2), 65.1 (CH2),
66.2 (CH2), 72.9 (CH2), 101.0 (OCH2O), 108.0 (CH), 108.4 (CH),
114.2 (2CH), 121.2 (CH), 130.4 (C), 130.6 (2CH), 134.9 (C), 147.1
(C), 147.8 (C), 163.0 (C), 196.9 (C=O); MS (EI): m/z (%) 328 (25),
135 (100), 121 (12), 105 (10), 77 (28), 43 (19). Anal. Calcd for
5.1.7. Preparation of 9c and 9d
A solution of 1,3-benzodioxol-5-ol (0.50 g, 3.6 mmol) in CH3CN
(5 mL), was added dropwise to a stirred mixture of appropriate
halide 2c (0.92 g, 3.6 mmol) or 2d (0.83 g, 3.6 mmol) and K2CO3
(0.74 g, 5.4 mmol) in CH3CN (20 mL) at room temperature and then
stirred at 80 °C for 7 h. The reaction mixture was cooled, the sol-
vent was removed under reduced pressure and the residue was
dissolved in CH2Cl2 (30 mL), washed with 10% NaOH (2 Â 20 mL),
and dried (Na2SO4). The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column chroma-
tography with petroleum ether (40–60)/diethyl ether (2:1) as elu-
ent to give the final products.
C19H20O5: C, 69.50; H, 6.14. Found: 69.42; H, 6.08.
5.2. Biological assay
5.2.1. Cells and viruses
The cytotoxic and antiviral activity of the compounds was stud-
ied on both HeLa cells (Ohio strain) and Hep-2 cells grown in
DMEM with 1% non-essential amino acids, 200 lg/mL streptomy-
cin, 200 units/mL penicillin G and 10% fetal calf serum (GIBCO Lab-
oratories INC). Cell lines were kept at 37 °C in a humidified
atmosphere with 5% CO2. Rhinoviruses HRV14 (group A) and
HRV39 (group B), were purchased from the American Type Culture
Collection (ATCC). For all the above mentioned viruses, working
stocks were prepared as cellular lysates using DMEM with 2% heat
inactivated fetal calf serum.
5.1.7.1. 1-{4-[3-(1,3-Benzodioxol-5-yloxy)propoxy]phenyl}etha-
none (9c).
(1.11 g, 98%),white solid; mp: 135–137 °C; IR
(nujol): 1669 cmÀ1 (C@O). 1H NMR (CDCl3, 500 MHz): d 2.27
(qnt, OCH2CH2CH2O, J = 6.0 Hz), 2.57 (s, CH3CO), 4.10 (t, OCH2,
J = 6.0 Hz), 4.23 (t, CH2O, J = 6.0 Hz), 5.92 (s, OCH2O), 6.35 (dd, Ben-
zodioxole-H, J = 2.5 Hz, J = 8.5 Hz), 6.51 (d, Benzodioxole-H,
J = 2.5 Hz), 6.71 (d, Benzodioxole-H, J = 8.5 Hz), 6.95 (d, 2 Ar-H,
5.2.2. Cytotoxic activity
The cytotoxicity of the test compounds was evaluated by mea-
suring the effect produced on cell morphology and cell growth
in vitro. Cell monolayers were prepared in 24-well tissue culture
plates and exposed to various concentrations of the compounds.
Plates were checked by light microscopy after 24, 48 and 72 h. Cyto-
toxicity was scored as morphological alterations (e.g., rounding up,
shrinking, detachment). The viability of the cells was determined by
a tetrazolium-based colorimetric method using 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT), as previously
described.24,26 The 50% cytotoxic dose (CD50) is the concentration
of the compound that reduced the absorbance of the control sample
by 50%.
J = 8.5 Hz), 7.94 (d,
2
Ar-H, J = 9.0 Hz); 13C NMR (CDCl3,
125.8 MHz): d 26.3 (CH3), 29.2 (CH2), 64.7 (CH2), 65.1 (CH2), 98.1
(CH), 101.1 (OCH2O), 105.7 (CH), 107.9 (CH), 114.2 (2CH), 130.4
(C), 130.6 (2CH), 141.7 (C), 148.3 (C), 154.3 (C), 162.8 (C), 196.7
(C@O); MS (EI): m/z (%) 314 (84), 149 (23), 138 (100), 121 (12),
107 (14), 91 (12), 43 (44). Anal. Calcd for C18H18O5: C, 68.78; H,
5.77. Found: 68.64; H, 5.72.
5.1.7.2.
(9d).
5-[3-(4-Fluorophenoxy)propoxy]-1,3-benzodioxole
(0.71 g, 68%), white solid; mp: 96–98 °C; 1H NMR
(CDCl3, 500 MHz): d 2.24 (qnt, OCH2CH2CH2O, J = 6.0 Hz), 4.09 (t,
OCH2, J = 6.0 Hz), 4.13 (t, CH2O, J = 6.0 Hz), 5.92 (s, OCH2O), 6.35
(dd, Benzodioxole-H, J = 2.5 Hz, J = 8.5 Hz), 6.51 (d, Benzodioxole-
H, J = 2.5 Hz), 6.71 (d, Benzodioxole-H, J = 8.5 Hz), 6.86 (dd, 2
Ar-H, J = 9.0 Hz, JHF = 4.5 Hz), 6.98 (t, 2 Ar-H, J = 9.0 Hz); 13C NMR
5.2.3. Inhibition of virus multiplication
The Rhinovirus inhibition assay was evaluated by a one-step
viral infection of cell monolayers, followed by virus yield titration
in an agar-plaque assay. HeLa cell monolayers were prepared in

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A. M. Bernard et al. / Bioorg. Med. Chem. 22 (2014) 4061–4066
24 multiwell plates and were infected by the Rhinoviruses at a MOI
of 1. Next, serial dilutions of the test compounds were added and
after 24–36 h of incubation at 33 °C and 3% CO2, when the cyto-
pathic effect in the control cells was almost total, the monolayers
were frozen and thawed and the viruses in the supernatant were
titrated by the plaque assay method. The antiviral activity assay
on the Rhinoviruses was carried out by the 50% plaque reduction
assay in Hep2 cells as previously described.20 The compound con-
centration required to inhibit virus plaque formation by 50% is
expressed as the 50% inhibitory concentration (ID50), and calcu-
lated by dose–response curves and linear regression.
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5.2.4. Time-drug addiction assay
Time-reduction studies were performed on cell monolayers
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Acknowledgment
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J. R.; Davies, Huw M. L. Tetrahedron 2008, 64, 6901.
This work was partly financed by the DiBS Department and
partly by the FBS 2012.
22. Kubota, H.; Watanabe, T.; Kakefuda, A.; Masuda, N.; Wada, K.; Ishii, N., et al.
Bioorg. Med. Chem. 2004, 12, 871.
Supplementary data
23. Al-Smadi, M.; Hanold, N.; Kalbitz, H.; Meier, H. Synthesis 2009, 15, 2539.
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Supplementary data (1H NMR, 13C NMR and MS spectra) for
new compounds 6–10 associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.05.
066. These data include MOL files and InChiKeys of the most
important compounds described in this article.
26. Enizot, F.; Lang, R. J. Immunol. Methods 1986, 89, 271.