Reaction of naphthoquinones with substituted nitromethanes. Facile synthesis and antifungal activity of naphtho[2,3-d]isoxazole-4,9-diones

Article abstract of DOI:10.1016/j.bmcl.2009.10.137

We report here a simple entry into naphtho[2,3-d]isoxazole-4,9-dione system containing a EWG in position 3 using the readily available 2,3-dichloro-1,4-naphthoquinone and nitromethyl derivatives in the presence of base. Antifungal activity of synthesised naphthoquinones was evaluated against ATCC and PYCC reference strains of Candida. The results suggest that the naphtho[2,3-d]isoxazole-4,9-dione scaffold has the potential to be developed into novel and safe therapeutic antifungal agents.

Full text of DOI:10.1016/j.bmcl.2009.10.137

Bioorganic & Medicinal Chemistry Letters 20 (2010) 193–195
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Reaction of naphthoquinones with substituted nitromethanes. Facile
synthesis and antifungal activity of naphtho[2,3-d]isoxazole-4,9-diones
b
c
d
d
b
Maria M. M. Santos a, , Natália Faria , Jim Iley , Simon J. Coles , Michael B. Hursthouse , M. Luz Martins ,
*
Rui Moreira a
a i-Med.UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Forças Armadas, Lisboa, Portugal
b Mycology Laboratory, IHMT/CREM, New University of Lisbon, Lisboa, Portugal
c Chemistry Department, The Open University, Milton Keynes, UK
d EPSRC National Crystallography Service, School of Chemistry, University of Southampton, Southampton, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here a simple entry into naphtho[2,3-d]isoxazole-4,9-dione system containing a EWG in posi-
tion 3 using the readily available 2,3-dichloro-1,4-naphthoquinone and nitromethyl derivatives in the
presence of base. Antifungal activity of synthesised naphthoquinones was evaluated against ATCC and
PYCC reference strains of Candida. The results suggest that the naphtho[2,3-d]isoxazole-4,9-dione scaf-
fold has the potential to be developed into novel and safe therapeutic antifungal agents.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 30 September 2009
Revised 29 October 2009
Accepted 30 October 2009
Available online 4 November 2009
Keywords:
Quinones
Isoxazoles
Heterocycles
Antifungal agents
Compounds containing the quinone group represent an impor-
tant class of biologicallyactive molecules that are widespread in nat-
ure.1 Isoxazoles also have a significant number of biological
applications, displaying hypoglycemic, analgesic, anti-inflamma-
tory, antifungal, anti-bacterial and HIV-inhibitory activity.2
Naphtho[2,3-d]isoxazole-4,9-diones 1 (Fig. 1) combine both func-
tionalities and are an attractive target. Importantly, compounds 1
also display agricultural applications.3
O
O
Cl
O
N
OR
EWG
O
4a-b
R=Et; R=Me
O
1
a
b
Figure 1. Structure of compounds 1 and 4.
To date, most of the routes to naphtho[2,3-d]isoxazole-4,9-
diones described in the literature use in situ generation of nitrile
oxides, require the synthesis of the necessary starting materials
and do not allow the introduction of electron-withdrawing group
(EWG) in position 3.4–6 To the best of our knowledge, there is only
one route that introduces an ethyl ester in position 3 of the isoxaz-
ole moiety that involves a manganese(III) acetate initiated reaction
between readily available 1,4-naphthoquinones and ethyl nitroac-
etate. This method is highly wasteful, requiring 8 equiv of ethyl
nitroacetate and 12 equiv of manganese(III) acetate.7,8
nitromethyl derivative 3a–b, in the presence of base (Scheme 1).
By this method, the isoxazole 1a was obtained in 22% yield (35%
based on recovered 2) after 3 d at reflux in the presence of NEt3
in ethanol. Using methyl nitroacetate 3b the isoxazole 1b was ob-
tained in 19% yield, along with the isoxazole 1a (7% yield) due to
ester exchange, and 2-chloro-3-ethoxy-1,4-naphthoquinone 4a
(13% yield). To avoid the problem of ester exchange, we changed
the solvent to MeOH but did not observe any formation of isoxaz-
ole after 3 d at reflux (Table 1). In this case, we only isolated
Recently we described naphtho[2,3-d]isoxazole-4,9-dione-3-
carboxylates 1a–b as potent, non-cytotoxic, antiapoptotic agents,
through modulation of apoptotic pathways.9 The two compounds
were synthesized from 1 equiv of each commercially available
O
O
Cl
Cl
O
base
+
O2N
N
EWG
EWG
O
2
O
1a-d
starting material, 2,3-dichloro-1,4-naphthoquinone
2 and a
3a-d
a
b
c
d
EWG=CO2Et; EWG=CO2Me; EWG=SO2Ph; EWG=COPh
* Corresponding author.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
Scheme 1. Synthesis of naphtho[2,3-d]isoxazole-4,9-diones 1a–d.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.137

194
M. M. M. Santos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 193–195
Table 1
When 2-bromo-1,4-naphthoquinone 5 was used as starting
material, naphthoquinone 6 (78% yield) was obtained from reac-
tion with 3a, after 6.5 h at reflux in the presence of NEt3 in ethanol
(Scheme 2). Only after 29 h at reflux, was the isoxazole 1a obtained
(13% yield), along with the naphthoquinone 6 (26% yield).12,13 The
latter obviously arises from Michael addition of the nitronate anion
at the least substituted quinone carbon atom followed by loss of
the nitrite ion. Attack at the C–H carbon is presumably preferred
because (i) this centre is less sterically hindered, and (ii) bromine
stabilizes an adjacent anion better than does an H atom.
When we used 2,3-dichloro-5,8-dimethoxy-1,4-naphthoqui-
none 7 and 3a as starting materials, after 1.5 h at reflux in ethanol
in the presence of 2 equiv of DIEA and molecular sieves, we iso-
lated compound 8 in 56% yield (Scheme 2).14 In this case, no forma-
tion of isoxazole was observed, even after 30 h at reflux in the
presence of NEt3 (Table 1).
Reaction conditions used for the synthesis of compounds 1a–d, 6 and 8
Starting materials
Solvent
EtOH
Base
DIEA
Reflux (h)
6.5
Product (yield %)
2
3a
1a (33)
4a (11)
5
EtOH
EtOH
NEt3
NEt3
6.5
29.0
6 (78)
1a (13)
6 (26)
7
2
EtOH
EtOH
DIEA
DIEA
1.5
30.0
8 (56)
8 (37)
3b
3c
EtOH
DIEA
6.5
1b (33)
4a (12)
MeOH
EtOH
CH3CN
CH3CN
CH3CN
NEt3
DIEA
NEt3
DIEA
K2CO3
72.0
96.0
4.0
2.0
48.0
4b (33)
4a (15)
1c (10)
—
2
2
—
This method seems to be general for other nitromethyl
derivatives, as it was also possible to obtain naphtho[2,3-d]isoxaz-
ole-4,9-diones with a sulfone 1c and a ketone 1d in position 3
3d
EtOH
DIEA
DABCO
NEt3
DIEA
K2CO3
24.0
6.0
5.0
4.5
5.0
4a (41)
1d (12)
1d (32)
1d (32)
1d (23)
CH3CN
CH3CN
CH3CN
CH3CN
(Scheme 1). When reaction of
2
was attempted with
phenylsulfonylnitromethane 3c or benzoylnitromethane 3d in the
presence of DIEA and ethanol, only 2-chloro-3-ethoxy-1,4-naphtho-
quinone 4a, from reaction of the quinone with the solvent, could be
isolated (Table 1).10a However, when we changed the solvent to
acetonitrile, it was then possible to obtain the isoxazoles 1c and
1d, respectively (Table 1).10b Isoxazole 1c was only formed in the
presence of NEt3 (10% yield). In the presence of DIEA or K2CO3 a com-
plex mixture of products was obtained. Isoxazole 1d however was
obtained using as bases DABCO (12% yield), NEt3 (32% yield), DIEA
(32% yield) or K2CO3 (23% yield). To try to improve the yield of 1d,
we decided to study the effect of using 2 equiv of nitroacetate 3d,
however in the presence of NEt3 in CH3CN, we obtained a more com-
plex mixture, and we could only isolate isoxazole 1d in 14% yield.
In all the above reactions, a complex mixture of products was
obtained, and we could only isolate as the major products the
products described in Table 1.
O
O
Br
CO2Et
Br
NEt3
+
O2N
CO2Et
CO2Et
O
6
O
5
3a
MeO
MeO
O
MeO
O
Cl
Cl
Cl
Cl
DIEA
EtO2C
EtO2C
+
O2N
O
MeO
O
7
3a
8
Scheme 2. Reactions of 2-bromo-1,4-naphthoquinone
dimetoxi-1,4-naphthoquinone 7 with 3a.
5 and 2,3-dichloro-5,8-
Structural assignment of products 1 was based on spectroscopic
data,15,16 which accorded with the literature.7 Confirmation of the
isoxazole structure 1 came from the X-ray crystallographicanalysis.17
Naphtho[2,3-d]isoxazole-4,9-diones 1a–d and naphthoqui-
nones 4a–b, 6 and 8 were evaluated for their antifungal activity
against various ATCC and PYCC reference strains of Candida.18
The MIC50 values were calculated using probit analysis based on
triplicate assays and are presented in Table 2. All compounds
2-chloro-3-methoxy-1,4-naphthoquinone 4b (33% yield)—presum-
ably due to the lower temperature of reflux.
We then decided to continue working on this new methodology
in order to improve the low yields of naphtho[2,3-d]isoxazole-4,9-
dione-3-carboxylates 1a–b. An improved synthesis was accom-
plished, using diisopropylethylamine (2 equiv) as base, in the pres-
ence of molecular sieves.10a In this case, the reaction proceeded
faster with no starting material being recovered; after 6.5 h we iso-
lated isoxazole 1a (33% yield), along with 2-chloro-3-ethoxy-1,4-
naphthoquinone 4a11 (11% yield) formed by substitution of a Cl
atom by the solvent. Using methyl nitroacetate 3b the isoxazole
1b was obtained in 33% yield, along with 2-chloro-3-ethoxy-1,4-
naphthoquinone 4a (12% yield).10a
showed a reasonable level of antifungal activity at the
ranging from the lowest MIC50 value of 0.2 g/mL for compound 1a
against PYCC 2545 (Candida parapsilosis) and PYCC 2418T (Candida
lg/mL level,
l
glabrata) to the highest of 47.9
ATCC 90028 (Candida albicans).
lg/mL for compound 4a against
Inspection of the data in Table 2, allows the following observa-
tions to be made:
Table 2
Determination of MIC50
lg/mL (lM) for compounds 1a–d, 4a–b, 6 and 8
Compound
C. albicans
ATCC 90028
C. albicans
C. krusei
PYCC 3341
C. parapsilosis
PYCC 2545
C. parapsilosis
ATCC 22019
C. glabrata
PYCC 3436T
PYCC 2418T
1a
1b
1c
1d
4a
4b
6
3.1 (11.5)
3.1 (12.2)
19.2 (56.7)
21.6 (71.2)
47.9 (202.4)
16.5 (73.9)
1.5 (4.5)
0.5 (1.8)
0.2 (0.7)
0.8 (2.5)
0.5 (1.6)
2.3 (9.5)
2.3 (10.4)
0.3 (0.9)
5.6 (12.2)
0.5 (0.5)
0.6 (2.3)
0.6 (2.4)
3.3 (9.7)
6.4 (21.2)
0.9 (3.9)
1.0 (4.6)
nda
0.2 (0.6)
0.3 (1.3)
0.5 (1.6)
4.0 (13.2)
11.4 (48.3)
1.2 (5.3)
0.4 (1.1)
7.0 (15.2)
0.4 (0.5)
0.5 (1.7)
0.5 (1.8)
2.1 (6.1)
1.0 (3.3)
9.6 (40.4)
1.0 (4.7)
1.0 (3.2)
8.4 (18.4)
0.4 (0.5)
0.2 (0.6)
0.7 (2.6)
0.5 (1.5)
14.4 (47.6)
0.9 (3.8)
3.7 (16.7)
3.9 (12.0)
2.3 (5.1)
0.3 (0.4)
8
25.7 (56.0)
1.1 (1.2)
3.0 (6.5)
0.9 (1.0)
AmpB
a
Not determinate due to absence of yeast growth in all bioassays.

M. M. M. Santos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 193–195
195
4. Alguacil, R.; Fariña, F.; Martín, M. V.; Paredes, M. C. Tetrahedron Lett. 1995, 36,
6773.
5. Stevens, J.; Welton, T.; Deville, J.; Behar, V. Tetrahedron Lett. 2003, 44, 8901.
6. Matsuura, T.; Bode, J. W.; Hachisu, Y.; Suzuki, K. Synlett 2003, 1746.
7. Chuang, C.; Wu, Y.; Jiang, M. Tetrahedron 1999, 55, 11229.
8. Snider, B. B.; Che, Q. Tetrahedron 2002, 58, 7821.
9. Santos, D.; Santos, M. M. M.; Viana, R. J. S.; Castro, R. E.; Moreira, R.; Rodrigues,
C. M. P. Chem. Biol. Interact. 2009, 180, 175.
(1) There was marked variability among the strains with regard
to their susceptibilities to the various test compounds. This
is particularly evident for the C. albicans ATCC 90028 and
PYCC 3436T strains (e.g., compounds 1c, 1d and 4a). Such
variability suggests there are biological factor(s) affecting
strain/species and test compound bioactivity.
10. (a) Nitromethyl derivative (2.2 mmol) and DIEA (4.4 mmol) were added to a
solution of 2,3-dichloro-1,4-naphthoquinone (2.2 mmol) in EtOH (50 mL) with
molecular sieves. After reflux, under argon atmosphere, the solution was
filtered and evaporated to dryness. The residue was submitted to flash
chromatography (n-hexane/ethyl acetate 8:2).; (b) Nitromethyl derivative
(2.2 mmol) and base (4.4 mmol) were added to a solution of 2,3-dichloro-1,4-
naphthoquinone (2.2 mmol) in CH3CN (50 mL). After reflux, under argon
atmosphere, the mixture was diluted with ethyl acetate and washed with H2O
(3Â). The organic extract was dried and concentrated. The residue was
submitted to flash chromatography (n-hexane/ethyl acetate 8:2).
11. Fieser, L. F.; Brown, R. H. J. Am. Chem. Soc. 1949, 71, 3609.
12. Compound 6: mp 122–124 °C; IR (NaCl) 1721, 1674; dH (400 MHz, CDCl3) 1.26
(3H, t, J = 8.0 Hz, CH3), 3.92 (2H, s, CH2), 4.18 (2H, q, J = 8.0 Hz, OCH2), 7.76 (2H,
t, J = 4.0 Hz, ArH), 8.10–8.13 (1H, m, ArH), 8.15–8.18 (1H, m, ArH); dC (100 MHz,
CDCl3) 14.1 (CH3), 37.1 (CH2), 61.6 (OCH2), 127.3 (CHAr), 127.7 (CHAr), 131.1
(CAr), 131.2 (CAr), 134.2 (CHAr), 134.4 (CHAr), 141.2 (CAr), 144.7 (CAr), 168.1
(CO), 177.4 (CO), 181.1 (CO).
(2) Strain ATCC 90028 appeared to be the least sensitive, to
almost all the compounds. Only compound 6, with an
MIC50 of 1.5 lg/mL demonstrated antifungal activity equiva-
lent to that of AmpB (Amphotericin B) for this strain. In addi-
tion, this compound also had notable activity against PYCC
strains 2545 and 3436T.
(3) Only compounds 1a and 1b showed a level of activity equiv-
alent to that of AmpB against strain ATCC 22019. Compound
1a appeared to have the broadest antifungal activity, show-
ing MIC50 values at or below that of AmpB for all strains,
except ATCC 90028. Compound 1b was next in breadth of
activity, with MIC50 values at or below those of AmpB for
four of the six strains. Compound 1c demonstrated reason-
13. McElvain, S. M.; Engelhardt, E. L. J. Am. Chem. Soc. 1944, 66, 1077.
14. Compound 8: mp 134–135 °C; IR (NaCl) 1723, 1661; dH (400 MHz, CDCl3) 1.25
(3H, t, J = 8.0 Hz, CH3), 3.65 (2H, s, CH2), 3.93 (3H, s, OMe), 4.15 (2H, q,
J = 8.0 Hz, OCH2); dC (100 MHz, CDCl3) 14.1 (CH3), 32.9 (CH2), 61.6 (OCH2), 62.0
(OCH3), 124.4 (CAr), 136.7 (CAr), 141.6 (CAr), 153.2 (CAr), 169.0 (CO), 182.1
(CO).
able antifungal activity, with MIC50 levels <1.0
lg/mL
against PYCC strains, 2545, 3436T and 2418T.
(4) The order of the antifungal activity in compounds 1 seems to
depend on the nature of the EWG in position 3 and varies in
the order ester > sulfone > ketone.
15. Compound 1c: mp 190–191 °C; IR (NaCl) 1716, 1675; dH (400 MHz, CDCl3) 7.67
(2H, t, J = 8.0 Hz, ArH), 7.78 (1H, t, J = 8.0 Hz, ArH), 7.83–7.92 (2H, m, ArH),
8.24–8.30 (4H, m, ArH); dC (100 MHz, CDCl3) 118.9 (CAr), 127.7 (CHAr), 128.1
(CHAr), 129.5 (2 CHAr), 129.9 (2 CHAr), 131.5 (CAr), 133.0 (CAr), 134.3 (CHAr),
135.5 (CHAr), 135.8 (CHAr), 137.3 (CAr), 162.5 (CAr), 166.6 (CN), 172.3 (CO),
175.7 (CO). HMRS C17H10NO5S (M+1) 340.02797, found 340.02847.
16. Compound 1d: mp 124–125 °C; IR (NaCl) 1689, 1589; dH (400 MHz, CDCl3) 7.56
(2H, t, J = 8.0 Hz, ArH), 7.69–7.73 (1H, m, ArH), 7.84–7.89 (2H, m, ArH), 8.08
(2H, d, J = 8.0 Hz, ArH), 8.21–8.23 (1H, m, ArH), 8.30–8.32 (1H, m, ArH); dC
(100 MHz, CDCl3) 121.2 (CAr), 127.7 (CHAr), 127.8 (CHAr), 129.0 (2 CHAr),
130.5 (2 CHAr), 132.0 (CAr), 133.3 (CAr), 134.7 (CHAr), 134.9 (CAr), 135.3
(CHAr), 135.5 (CHAr), 157.8 (CAr), 165.3 (CN), 177.0 (CO), 177.1 (CO), 188.9
(CO). HMRS C18H10NO4 (M+1) 304.06098, found 304.06211.
(5) Compounds 1d, 4a and 4b, also showed reasonable antifungal
activities equivalent to that of AmpB against one strain, each.
Compound 8, although exhibiting fair antifungal activity at
the lg/mL level against all strains, did not present any MIC50
values at or below that of AmpB against any of the strains.
In summary, we describe a new methodology for the synthesis of
isoxazoles 1 containing an EWG in position 3. Although the yields
are lower than those described for the synthesis of isoxazole 1a
(lit. 55% yield),7 our procedure uses cleaner chemistry and only
1 equiv of both commercial starting materials. Also, our method is
not restricted to the use of ethyl nitroacetate, but seems to be gen-
eral for other nitromethyl derivatives. In addition, all the test com-
ꢀ
17. Crystal data for C14H9NO5, Mr = 271.22, triclinic space group P1, a = 7.1485(3),
b = 8.5516(2), c = 10.1860(5) Å,
U = 594.50(4) Å3, Z = 2, Dcalcd = 1.515 g cmÀ3
pale yellow crystal 0.6 Â 0.4 Â 0.05 mm, hmax = 27.49°, 2704 unique data,
10685 measured (Rint = 0.0272), R1 = 0.0363 and wR2 = 0.0988 [I > 2 (I)].
a
= 79.315(2), b = 88.841(2),
c
= 76.382(2)°,
,
l
= 0.117 mmÀ1
, F(0 0 0) = 280,
r
pounds showed effective levels of antifungal activity at the lg/mL
Supplementary data have been deposited with the CCDC deposition number
CCDC 674974.
level. Importantly, 1a and 1b showed the broadest activity and were
equivalent to or better than the standard antifungal drug Amphoter-
icin B against five of the six strains examined. The observation that
naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates 1a–b are non-
cytotoxic in human cellular lines9 strongly suggests that the naph-
tho[2,3-d]isoxazole-4,9-dione scaffold has the potential to be devel-
oped into novel and safe therapeutic antifungal agents. The
mechanism for the formation of isoxazoles 1a–d is understudy.
18. Determination of MIC Levels: Antifungal activities of 1a–d, 4a–b, 6 and 8 and
amphotericin B (AmpB; Sigma–Aldrich, Sigma A2411), were based on Minimal
Inhibitory concentrations for 50% cell death (MIC50). Microtitre plate bioassays
for determining the MIC50 values were based on modified protocols outlined in
the Clinical and Laboratory Standards Institute (CLSI), old National Committee
for Clinical Laboratory Standards (NCCLS), document M27-A, National
Committee for Clinical Laboratory Standards., Wayne, PA, USA. The
compounds were first dissolved in dimethylsulfoxide (DMSO) (1 mg/mL)
then further diluted 10-fold in SG (100 lg/mL, stock solution) (SG minimal
medium: 6.7 mg/mL yeast nitrogen base w/o amino acids, 2% glucose). Next, 30
separate dilutions were made of each compound so that the microtitre plate
wells had final concentrations of 97.5, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 7.5,
5.0, 3.75, 2.5, 1.25, 1.0, 0.75, 0.5, 0.25, 0.125, 0.1, 0.075, 0.05, 0.0250, 0.0150,
Acknowledgments
0.0125, 0.01, 0.005 and 0.0025 lg/mL of each test compound or AmpB. The
This project was supported by Fundação para a Ciência e Tecn-
ologia (Portugal). The authors would also like to thank EPSRC for
funding the National Crystallography Service.
dilutions included respective volumes of stock solution and medium, plus
10
200
l
l
L (approx. 5 Â 103 cells) of stock yeast culture to bring the final volume to
L/ well. Control wells included 20 lL DMSO up to 200 lL SG. All bioassay
dilutions were done in triplicate. The plates were incubated at 30 °C for 48 h.
Level of yeast cell reproduction for each concentration of the test compounds
and AmpB was determined by optical density (OD) using a microtitre plate
reader (Microplate Reader Model 680, Biorad) at 655 nm. OD readings were
converted to% cell reproduction (OD concentration/OD control). The mean of
the triplicate readings was then determined. MIC50 values were determined by
probit analysis of % cell reproduction versus concentration of test compounds
or ampB using the statistical analysis program provided by SPSS (Chicago, IL,
USA).
References and notes
1. Dewick, P. M., In Medicinal Natural Products, 2nd ed., Wiley, Chichester, UK,
2002
2. Pinho e Melo, T. M. V. D. Curr. Org. Chem. 2005, 9, 925.
3. (a) Kano, H.; Ogata, M.; Yukinaga, H. U.S. Patent 3,835,168, 1974.; (b) Kano, H.;
Ogata, M.; Yukinaga, H. U.S. Patent 3,933,828, 1976.