Disoxaril-related 3-(diethylamino)-5-phenylisoxazoles

Article abstract of DOI:10.1016/S0014-827X(00)00002-1

Previous research has shown that 3-(dialkylamino)-5-phenylisoxazoles possessing a compact structure were active against HRV-2 and, consequently, presented a type B activity. In this paper, 3-(diethylamino)-5-phenylisoxazoles, which are structurally more elongated and related to Disoxaril, were synthesized in view to attempt type A activity against HRV-14. Unfortunately, all tested compounds were devoid of activity against HRV-14 (and HIV-1) or exhibited great toxicity. Copyright (C) 2000 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(00)00002-1

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 119–124
Disoxaril-related 3-(diethylamino)-5-phenylisoxazoles
Mauro Mazzei a,*, Ramona Dondero a, Bernardetta Ledda a, Francesca Demontis b,
Laura Vargiu b
a Department of Pharmaceutical Sciences, Uni6ersity of Geno6a, Viale Benedetto XV, 3-16132 Genoa, Italy
b Department of Experimental Biology, Uni6ersity of Cagliari, 09042 Monserrato, Cagliari, Italy
Received 19 July 1999; accepted 30 December 1999
Abstract
Previous research has shown that 3-(dialkylamino)-5-phenylisoxazoles possessing a compact structure were active against
HRV-2 and, consequently, presented a type B activity. In this paper, 3-(diethylamino)-5-phenylisoxazoles, which are structurally
more elongated and related to Disoxaril, were synthesized in view to attempt type A activity against HRV-14. Unfortunately, all
tested compounds were devoid of activity against HRV-14 (and HIV-1) or exhibited great toxicity. © 2000 Elsevier Science S.A.
All rights reserved.
Keywords: Isoxazole derivatives; Antiviral agents; Antirhinovirus activity
elongated structure were synthesized. All new deriva-
tives carry a similar design in the 4%-position of the
phenyl ring concerning a five carbon atom linker be-
tween oxygen and nitrogen, this latter having different
basic properties. In particular, when R% was a butoxy
chain ending with an oxazoline ring, as in compounds 9
and 10, the substances obtained were related to WIN
compounds with the etherocyclic rings mutually ex-
changed (see Fig. 2).
1. Introduction
In previous works we showed that the 3-(dialkyl-
amino)-5-phenylisoxazoles A (see Fig. 1) were endowed
with significant antirhinovirus activity [1,2]. Because the
structure of many A derivatives was short and compact,
in accordance with the observations of Andries et al.
[3], these compounds exerted type B activity. More
generally, the isoxazole moiety is known to possess
potential antirhinoviral properties as it is widely repre-
sented in the class of the WIN compounds (see Figs. 1
and 2). The leader of this class (WIN 51711 or Disox-
aril) inhibits picornaviral uncoating by binding to the
hydrophobic pocket on the virion surface and, as it
possesses an elongated structure, shows type A activity
[3]. Although the Disoxaril was a very active antirhi-
novirus agent, it was withdrawn from clinical studies
due to the appearance of crystallurea at high dosages in
healthy volunteers [4].
2. Chemistry
With the goal of obtaining a suitable chain in the
4%-position of phenylisoxazole A, resorcin was treated
with 5-chlorovaleronitrile in the presence of dimethyl-
formamide (DMF) and anhydrous potassium carbonate
to yield 3-(4-cyanobutoxy)phenol (1).
In turn, 2-(diethylamino)chromone (2) was prepared
with a cyclocondensation reaction between 1 and 3-(di-
ethylamino)-3-oxo-propanoic acid ethyl ester in the
presence of phosphorus oxychloride using 1,2-
dichloroethane (DCE) as solvent, as previously re-
ported for similar compounds [5] (see Scheme 1).
As depicted in Scheme 1, the treatment of the 2-(di-
ethylamino)chromone (2) with hydroxylamine hy-
drochloride in ethanol in the presence of pyridine gave
Following our interest in the class of 3-(dialkyl-
amino)-5-phenylisoxazoles, our attention was aimed at
amplifying the pattern of activity of A compounds and,
consequently, some new isoxazole derivatives with an
* Corresponding author. Fax: +39-010-573 7320.
E-mail address: mazzei@ermes.cba.unige.it (M. Mazzei)
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00002-1

120
M. Mazzei et al. / Il Farmaco 55 (2000) 119–124
Fig. 1.
shows a signal at about 8.10 ppm in the 1H NMR
spectra because of the deshielding effect of the carbonyl
in the 4-position as described for similar structures [5].
3. Experimental
Melting points were determined using an Electrother-
mal apparatus and are uncorrected. Microanalyses were
carried out on a Carlo Erba 1106 elemental analyzer.
The results of elemental analysis were within 90.3%
for C and 90.1 for H and N of the theoretical value.
1H NMR spectra were performed on a Hitachi Perkin–
Elmer R 600 (60 MHz) spectrometer using TMS as
internal standard (l=0). IR spectra were recorded on
a Perkin–Elmer 398 spectrophotometer.
Fig. 2.
a good yield of the 3-(diethylamino)-5-phenylisoxazole
derivative (3). The reaction mechanism, based on the
nucleophilic attack of the hydroxylamine at the 2-posi-
tion of the chromone ring, was already described [1].
The isoxazole 3 was transformed into the ester 4 by
reaction with p-nitrobenzoyl chloride and into the ether
5 by reaction with dimethylsulfate (see Scheme 2).
Comments on these reactions are given in Section 4.
The cyanodervative 3 was then hydrogenated into the
corresponding aminoderivative 6 using platinum oxide
as catalyst in chloroform–methanol [6]. The latter com-
pound was in turn acetylated yielding 7 (see Scheme 2).
From the chromone 2, the oxazoline 8 was obtained
by means of ethanolamine in the presence of anhydrous
zinc chloride [7]. Then the benzopyran ring was opened
as above with hydroxylamine yielding the isoxazole 9;
this latter was easily methylated with dimethylsulfate to
give the oxazolinylisoxazole 10 (see Scheme 3).
3.1. 3-(4-Cyanobutoxy)phenol (1)
To 4.0 g (0.036 mol) of resorcinol dissolved in 10 ml
of DMF, 8.0 g of anhydrous potassium carbonate and
4.3 g (0.036 mol) of 5-chlorovaleronitrile were added.
The mixture was heated at 110–115°C for 5 h under a
stream of nitrogen. The final mixture was cooled,
poured onto crushed ice (adjusting the pH to 7) and
stirred for 30 min. The aqueous solution was extracted
several times with chloroform. The pooled organic ex-
tracts were counter-extracted with 2 N NaOH. The
alkaline solution was acidified with 6 M HCl and the
white solid obtained was filtered off. The crude product
was crystallized from ethyl ether obtaining 1; m.p.
69–70°C; 68% yield.
Compounds 1–10 are white crystals whose structures
are in agreement with elemental analyses and spectral
data. In particular, the H-5 of chromones 2 and 8
Scheme 1.

M. Mazzei et al. / Il Farmaco 55 (2000) 119–124
121
Scheme 2.
1H NMR (CDCl3): l 1.71–2.18 (m, 4H, OCH2-
(CH2)2), 2.45 (t, 2H, CH2CN), 3.99 (t, 2H, OCH2), 5.83
(s, 1H, OH), 6.32–7.41 (m, 4H, arom. H). IR (KBr)
cm−1: 3340, 2260. Anal. C11H13NO2: C, H, N.
added slowly with stirring. The reaction mixture was
then heated for 5 h at reflux. After cooling, a solution
of 68 g of sodium acetate trihydrate in 200 ml of water
was added and the mixture was then heated for 1.5 h at
70°C. After cooling, the organic phase was discarded
and the aqueous one was extracted three times with
chloroform. The pooled organic extracts were washed
with water, dried and evaporated under reduced pres-
sure giving a dark-red oil. The oil was stirred at r.t. for
2 h with 200 ml of 2 N NaOH and 50 ml of light
petroleum ether. The obtained solid was filtered off and
washed with water. The crude product was crystallized
from ethyl acetate obtaining 2; m.p. 85–86°C; 77.8%
yield.
3.2. 2-(Diethylamino)-7-(4-cyanobutoxy)-
4H-1-benzopyran-4-one (2)
In an ice-cooled flask, protected from moisture with
a calcium chloride drying tube, 7.0 ml (78.0 mmol) of
phosphorus oxychloride were added dropwise with stir-
ring to 10.30 g (55.0 mmol) of 3-(diethylamino)-3-oxo-
propanoic acid ethyl ester [8]. After the addition, the
mixture was removed from the ice bath and maintained
at room temperature (r.t.) for 30 min. To the resulting
yellow mixture, a solution of 9.56 g (50.0 mmol) of
3-(4-cyanobutoxy)phenol (1) in 40 ml of DCE was
1H NMR (CDCl3): l 1.25 (t, 6H, NCH2ꢀCH3), 1.68–
2.31 (m, 4H, OCH2ꢀ(CH2)2ꢀCH2CN), 2.48 (t, 2H,
CH2CN), 3.47 (q, 4H, NCH2ꢀCH3), 4.09 (t, 2H,
Scheme 3.

122
M. Mazzei et al. / Il Farmaco 55 (2000) 119–124
OCH2), 5.47 (s, 1H, H-3), 6.70–7.05 (m, 2H, H-6, H-8),
8.10 (d, 1H, H-5). IR (KBr) cm−1: 2240, 1610, 1540.
Anal. C18H22N2O3: C, H, N.
1H NMR (CDCl3): l 1.20 (t, 6H, NCH2ꢀCH3), 1.78–
2.16 (m, 4H, OCH2ꢀ(CH2)2ꢀCH2CN), 2.45 (t, 2H,
CH2CN), 3.38 (q, 4H, NCH2ꢀCH3,), 3.88–4.23 (m, 5H,
OCH3, OCH2), 6.26 (s, 1H, H-4), 6.45–6.73 (m, 2H,
H-3%, H-5%), 7.88 (d, 1H, H-6%). IR (KBr) cm−1: 1615,
1565, 1545. Anal. C19H25N3O3: C, H, N.
3.3. 3-(Diethylamino)-5-[2%-hydroxy-4%-
(4-cyanobutoxy)phenyl]isoxazole (3)
To a solution of 1.2 g (3.82 mmol) of 2 in 40 ml of
ethanol, 1 g of hydroxylamine hydrochloride and 1.5 ml
of pyridine were added. The mixture was refluxed for
24 h. The final solution was evaporated under reduced
pressure to leave a pale yellow solid. The solid was
dissolved in a small amount of 2 N NaOH, filtering off
any impurity of the unreacted starting product. Then
the alkaline solution was acidified with 6 M HCl ob-
taining a white precipitate, which was filtered off and
washed with water. The crude product was crystallized
from ethanol obtaining 3; m.p. 188–189°C; 68.2%
yield.
3.6. 3-(Diethylamino)-5-[2%-hydroxy-4%-
(5-aminopentoxy)phenyl]isoxazole (6)
A solution of 1 g (3.04 mmol) of 3 in 20 ml of
chloroform and 20 ml of methanol was hydrogenated at
60 psi in a Parr apparatus with 0.25 g of platinum oxide
until gas uptake ceased. The catalyst was filtered off
and the solvent was distilled under reduced pressure.
The residue was treated with a solution of sodium
hydrogencarbonate and the free base was crystallized
from ethyl acetate–cyclohexane obtaining 6; m.p. 128–
129°C; 60.5% yield.
1H NMR (CDCl3): l 1.10 (t, 6H, NCH2ꢀCH3), 1.55–
2.12 (m, 4H, OCH2(CH2)2), 2.51 (t, 2H, CH2CN), 3.38
(q, 4H, NCH2ꢀCH3), 3.65–4.20 (m, 3H, OCH2, OH),
6.42 (s, 1H, H-4), 6.52–6.70 (m, 2H, H-3%, H-5%), 7.73
(d, 1H, H-6%). IR (KBr) cm−1: 3000 (broad), 2240,
1620, 1590. Anal. C18H23N3O3: C, H, N.
1H NMR (DMSO-d6): l 1.21 (t, 6H, NCH2ꢀCH3),
1.35–1.90 (m, 6H, OCH2ꢀ(CH2)3ꢀCH2), 2.65–2.98 (m,
2H, CH2NH2), 3.05–3.57 (m, 6H, NCH2ꢀCH3, NH2),
3.99 (t, 2H, OCH2), 6.25–6.46 (m, 2H, H-4, OH),
6.50–6.85 (m, 2H, H-3%, H-5%), 7.62 (d, 1H, H-6%). IR
(KBr) cm−1: 3350, 3310, 3100 (broad), 1620, 1555.
Anal. C18H27N3O3: C, H, N.
3.4. 3-(Diethylamino)-5-[2%-p-nitrobenzoyloxy-4%-
(4-cyanobutoxy)phenyl]isoxazole (4)
3.7. 3-(Diethylamino)-5-[2%-hydroxy-4%-
(5-acetylaminopentoxy)phenyl]isoxazole (7)
To 1 g (3.04 mmol) of 3 in 5 ml of pyridine, 0.98 g
(5.3 mmol) of p-nitrobenzoyl chloride was added and
the solution was heated at 110°C for 8 min. At the end
the mixture was cooled and 15 ml of water was added
obtaining a solid. The solid was filtered off and washed
with water. The crude product was crystallized from
ethyl acetate–cyclohexane obtaining 4; m.p. 80–81°C;
51% yield.
A solution of 1 g (3.00 mmol) of 6 was treated with
5 ml of acetic anhydride at 70°C for 1 h. At the end, the
solution was poured onto crushed ice and the suspen-
sion was stirred for 1 h. The precipitate was filtered off
and the solid was crystallized from ethyl acetate–
ethanol obtaining 7; m.p. 169–170°C; 84% yield.
1H NMR (DMSO-d6): l 1.12 (t, 6H, NCH2ꢀCH3),
1.38–1.74 (m, 6H, OCH2ꢀ(CH2)3ꢀCH2), 1.85 (s, 3H,
COCH3), 2.85–3.54 (m, 6H, NCH2ꢀCH3, CH2NH),
4.00 (t, 2H, OCH2), 6.38 (s, 1H, H-4), 6.51–6.73 (m,
2H, H-3%, H-5%), 7.50–8.12 (m, 2H, H-6%, NH), 10.52 (s,
1H, OH). IR (KBr) cm−1: 3295 (sharp), 3100 (broad),
1640, 1610, 1565. Anal. C20H29N3O4: C, H, N.
1H NMR (CDCl3): l 1.07 (t, 6H, NCH2ꢀCH3), 1.72–
2.16 (m, 4H, OCH2ꢀ(CH2)2ꢀCH2CN), 2.50 (t, 2H,
CH2CN), 3.22 (q, 4H, NCH2ꢀCH3), 4.13 (t, 2H,
OCH2), 5.94 (s, 1H, H-4), 6.80–7.13 (m, 2H, H-3%,
H-5%), 7.87 (d, 1H, H-6%), 8.46 (near s, 4H, arom. H). IR
(KBr) cm−1
:
2245, 1745, 1625, 1610. Anal.
C25H26N4O6: C, H, N.
3.8. 2-(Diethylamino)-7-[4-(2%-oxazolinyl)-
butoxy]-4H-1-benzopyran-4-one (8)
3.5. 3-(Diethylamino)-5-[2%-methoxy-4%-
(4-cyanobutoxy)phenyl]isoxazole (5)
To 2 g (6.36 mmol) of 2 dissolved in 10 ml of
ethanolamine, 0.9 g of anhydrous zinc chloride was
added and the mixture was heated at 120°C for 2 h. At
the end the mixture was cooled and poured onto
crushed ice (adjusting the pH to 7). The suspension was
extracted three times with chloroform; then the pooled
organic extracts were dried and evaporated under re-
duced pressure. To the resulting oil, 30 ml of 2 N
NaOH and 5 ml of cyclohexane were added and the
To 1 g (3.04 mmol) of 3 dissolved in 5 ml of 2 N
NaOH, 0.2 ml of dimethylsulfate was added dropwise.
The mixture was heated at 70°C for 2 h and then, after
cooling, extracted three times with diethyl ether. The
pooled organic extracts were washed with water, dried
and evaporated under reduced pressure to give a white
solid which was crystallized from ethyl acetate–cyclo-
hexane obtaining 5; m.p. 100–101°C; 46% yield.

M. Mazzei et al. / Il Farmaco 55 (2000) 119–124
123
4. Results and discussion
mixture was allowed to stir for 1 h on an ice bath. The
solid that separated out was filtered off and washed
with water. The crude product was crystallized from
ethanol obtaining 8; m.p. 90–91°C; 52.1% yield.
1H NMR (CDCl3): l 1.28 (t, 6H, NCH2ꢀCH3), 1.65–
3.00 (m, 6H, OCH2ꢀ(CH2)3), 3.21–3.90 (m, 8H,
NCH2ꢀCH3, CH2 oxazoline), 4.02 (t, 2H, OCH2), 5.40
(s, 1H, H-3), 6.79–7.08 (m, 2H, H-6, 8), 8.06 (d, 1H,
H-5). IR cm−1: 1630, 1605, 1545. Anal. C20H26N2O4: C,
H, N.
Regarding the chemistry, it is interesting to note that
the methylation of the hydroxy group in 2% of the
isoxazole 3, probably due to the presence of the
cyanobutoxy substituent in 4%, is more difficult with
respect to the previous series having a methoxy group
in 4%; in the present case, there is the formation of many
by-products and, consequently, the yield is low. In this
context, the use of methyl iodide, instead of dimethyl
sulfate, does not achieve any significant improvement in
yield (data not shown). When the cyano group is
replaced with the oxazoline ring, as in the isoxazole 9,
the methylation occurs in a more favorable yield: in this
case methyl iodide gives a slightly better yield with
respect to dimethyl sulfate.
The acylation of hydroxy group in 2% also gives poor
results: thus, the benzoylation reaction to form the
derivative 4 occurs under strong conditions and low
yield (with respect to those acylations reported in [2])
while in the acetylation of 6, following the reaction
conditions used by us, the hydroxy group does not
react.
In Table 1, the effect of the synthesized chromones (2
and 8) and isoxazoles (3, 4, 5, 6, 7, 9 and 10) on the
multiplication of HRV-14 is reported, this effect being a
marker of a type A antirhinovirus activity [1,3]. The
biological tests were performed as described in [2]. With
regard to antirhinovirus properties, chromones are not
toxic but are void of activity. Isoxazoles are ineffective
and also very toxic. In this context, the Disoxaril-re-
lated isoxazole 10 is the most toxic. Hence, the aim of
finding derivatives capable of covering both type A and
type B antirhinovirus activity, utilizing a unique 3-(di-
ethylamino)-5-phenylisoxazole moiety was unsuccessful.
Furthermore, the synthesized compounds do not
present significant activity against HIV-1 (data not
shown).
3.9. 3-(Diethylamino)-5-[2%-hydroxy-4%-(4-
[2%-oxazolinyl]butoxy)phenyl]isoxazole (9)
To a solution of 1 g (2.79 mmol) of 8 in 20 ml of
ethanol, 1 g of hydroxylamine hydrochloride and 1.5 ml
of pyridine were added. The mixture was refluxed for
24 h. The final solution was evaporated under reduced
pressure to yield a pale yellow solid. The solid was
dissolved in a small amount of 2 N NaOH, filtering off
any impurity of unreacted starting product. Then the
alkaline solution was acidified with 6 M HCl obtaining
a white precipitate, which was filtered off and washed
with water. The crude product was crystallized from
ethanol obtaining 9; m.p. 151–152°C; 74.8% yield.
1H NMR (CDCl3): l 1.19 (t, 6H, NCH2ꢀCH3), 1.60–
2.65 (m, 6H, OCH2ꢀ(CH2)3), 3.12–3.80 (m, 8H,
NCH2ꢀCH3, CH2 oxazoline), 4.00 (t, 2H, OCH2), 6.40
(s, 1H, H-4), 6.48–6.70 (m, 2H, H-3% 5%), 7.70 (d, 1H,
H-6%). IR cm−1: 1620, 1545, 1450. Anal. C20H27N3O4:
C, H, N.
3.10. 3-(Diethylamino)-5-{2%-methoxy-4%-[4-
(2%-oxazolinyl)butoxy]phenyl}isoxazole (10)
To a solution of 1 g (2.68 mmol) of 9 in 20 ml of
acetone, 0.5 ml of methyl iodide and 1.4 g of anhydrous
potassium carbonate were added and the resulting mix-
ture was heated at 50°C for 24 h. After 12 h of heating,
another 0.5 ml of methyl iodide was added. At the end,
the solvents were eliminated under reduced pressure
and 30 ml of 2 N NaOH were added to the residue,
with stirring. The alkaline suspension was extracted
many times with chloroform. Then the pooled organic
extracts were dried and evaporated under reduced pres-
sure. The crude product was crystallized from ethyl
acetate–cyclohexane obtaining 10; m.p. 85–86°C;
91.3% yield.
Table 1
Effect of compounds 2–10 on the multiplication of HRV-14
a
b
Comp.
CC50 (mM)
EC50 (mM)
MT-4
HRV-14
2
3
4
\200
5.6
5
\200
\5.6
\5
5
6
7
8
9
10
5.8
35
22
\5.8
\35
\22
\200
\34
\4.4
\200
34
1H NMR (CDCl3): l 1.12 (t, 6H, NCH2ꢀCH3), 1.52–
2.56 (m, 6H, OCH2ꢀ(CH2)3), 3.09–3.70 (m, 8H,
NCH2ꢀCH3, CH2 oxazoline), 3.90–4.23 (m, 5H, OCH3,
OCH2), 6.19 (s, 1H, H-4), 6.42–6.70 (m, 2H, H-3% 5%),
7.80 (d, 1H, H-6%). IR (KBr) cm−1: 1620, 1575, 1545.
Anal. C21H29N3O4: C, H, N.
4.4
a CC50, compound concentration required to reduce the viability of
mock-infected MT-4 by 50% (at 37°C).
b EC50, compound concentration required to achieve 50% protec-
tion of MT-4 cells against the cytopathic effect of HRV-14 (at 33°C).

124
M. Mazzei et al. / Il Farmaco 55 (2000) 119–124
broekx, P.J. Lewi, A comparative test of 15 compounds against
Acknowledgements
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