Fluconazole analogues containing 2H-1,4-benzothiazin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moieties, a novel class of anti-Candida agents

Article abstract of DOI:10.1016/j.bmcl.2009.11.071

As a part of our program to develop new antifungal agents, a series of fluconazole analogues was designed and synthesized wherein one of the triazole moieties in fluconazole was replaced with 2H-1,4-benzothiazin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moiety. The new chemical entities thus synthesized were screened against various fungi and it was observed that the compounds 4a and 4i are potent inhibitors of Candida strains. The structure-activity relationship for these compounds is discussed.

Full text of DOI:10.1016/j.bmcl.2009.11.071

Bioorganic & Medicinal Chemistry Letters 20 (2010) 722–725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluconazole analogues containing 2H-1,4-benzothiazin-3(4H)-one or
2H-1,4-benzoxazin-3(4H)-one moieties, a novel class of anti-Candida agents
a
a
b
Hanumant B. Borate a, , Suleman R. Maujan , Sangmeshwer P. Sawargave , Mohan A. Chandavarkar ,
Sharangi R. Vaiude b, Vinay A. Joshi b, Radhika D. Wakharkar a, Ramki Iyer b, Ramesh G. Kelkar a,
Subhash P. Chavan a, Sunita S. Kunte a
*
a Division of Organic Chemistry, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, India
b FDC Ltd, 142-148, S. V. Road, Jogeshwari (W), Mumbai 400 102, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2009
Revised 10 October 2009
Accepted 13 November 2009
Available online 4 December 2009
As a part of our program to develop new antifungal agents, a series of fluconazole analogues was designed
and synthesized wherein one of the triazole moieties in fluconazole was replaced with 2H-1,4-benzothia-
zin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moiety. The new chemical entities thus synthesized were
screened against various fungi and it was observed that the compounds 4a and 4i are potent inhibitors of
Candida strains. The structure–activity relationship for these compounds is discussed.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal
Fluconazole
Benzothiazinone
Benzoxazinone
Complications in the fungal infections have increased in the
number and severity in recent years as a result of an increasing
number of immunocompromised hosts, such as patients suffering
from tuberculosis, infected with HIV and undergoing organ trans-
plantations and cancer chemotherapy, due to the use of immuno-
suppressant agents. Though there are effective antifungal agents
available in the market, they have quite a few shortcomings such
as toxicity, limited range of activity for the fungal strains, high
price and limited penetration through central nervous system.
Fluconazole (1)1,2 is an important antifungal agent used against
various fungal strains, but its extensive use has increased the num-
ber of fluconazole-resistant fungi due to mutations (see Fig. 1).
Amphotericin B (2) is the antifungal agent of choice used in fluco-
nazole-resistant fungal infections but it has a higher degree of tox-
icity than that of fluconazole.3 The immunocompromised patients
have to take long term antifungal therapy to prevent relapses and
this causes the development of resistance in fungal strains. This
compels the synthetic chemists to design and synthesize new
chemical entities in an effort to come up with new drugs which
can be used in place of current drugs like fluconazole or amphoter-
icin B against the mutated fungal strains.
fungal strains. This included derivatisation at tertiary alcohol of
fluconazole as ethers, esters, phosphates and so on or the substitu-
tion of one of the triazole unit by different moieties.4 In some of the
cases, difluorophenyl ring in fluconazole was replaced with other
moieties such as 1,4-benzothiazinones to obtain compounds 3a.5
The analogues having general structure 3b were also synthesized
and screened against Candida albicans.6 In all the above 1,4-benzo-
thiazinones and 1,4-benzoxazinones, amide nitrogen was pro-
tected as alkyl chain such as methyl or ethyl (see Fig. 2).
As a part of our efforts7 to develop new antifungal drugs, we
synthesized fluconazole analogues wherein one of the triazole
moieties in fluconazole was replaced with 2H-1,4-benzothiazin-
3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moiety and we report
herein the synthesis and in vitro antifungal activity of a series of
fluconazole derivatives against various fungi.
The synthesis of compounds 4 was achieved from epoxide 5 and
(un)substituted benzothiazinone or benzoxazinone moieties 6 as
shown in Schemes 1 and 2.
Epoxide 5a8,9 was prepared from ketone 9 by Corey–Chaykovsky
epoxidation method. Ketone 9 was in turn obtained from 1,2-difluo-
robenzene via its acylated intermediate 8 as shown in Scheme 1.
Other epoxides such as 5b, 5c and 5d were synthesized10 using same
strategy from their respective starting materials.
Various fluconazole derivatives and analogues have been syn-
thesized and checked for the antifungal activity against different
The synthesis of benzoxazinone 6a11 and benzothiazinone 6b11
was achieved from commercially available 2-aminophenol 10a and
2-aminothiophenol 10b, respectively, as shown in Scheme 1. Stir-
* Corresponding author. Tel.: +91 20 25902546; fax: +91 20 25902629.
E-mail address: hb.borate@ncl.res.in (H.B. Borate).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.071

H. B. Borate et al. / Bioorg. Med. Chem. Lett. 20 (2010) 722–725
723
OH
OH
O
OH
F
Me
HO
Me
O
OH
N
N
N
N
N
OH OH
HO HO
N
O
Me
COOH
F
O
O
Me
Amphotericin B (2)
Fluconazole (1)
HO
OH
NH2
Figure 1. Structures of fluconazole and amphotericin B.
ring 2-aminophenol 10a with chloroacetyl chloride in DCM at
room temperature for 2 h yielded N-acylated intermediate 11a
which on treatment with potassium carbonate in ethyl acetate at
refluxing temperature yielded benzoxazinone 6a. The same reac-
tion sequence using 2-aminothiophenol 10b afforded benzothiazi-
none 6b. Rest of the benzoxazinones and benzothiazinones were
either prepared by the synthetic sequence given in Scheme 1 or
were commercially available.
Benzoxazinone and benzothiazinone so prepared were reacted
with epoxides 5 in presence of potassium carbonate and tetra-
butylammonium bromide (TBAB) in ethyl acetate at refluxing tem-
perature for 10–12 h to yield desired compounds 412 in good yields
as shown in Scheme 2.
OH
N
H
N
N
N
Y
N
N
N
O
N
N
Y
Cl
R
S
X
R1
X= S, O
Y= N, CH
Me
3b
O
O
R= Cl, H
3a
R1=Me, Et
Figure 2. Structures of azole analogues containing benzothiazinone and benzox-
azinone moieties.
O
N
N
O
O
N
N
Cl
F
N
N
i
F
F
iii
F
ii
F
F
5
F
8
F
9
7
Reagents i) Chloroacetyl chloride, AlCl3, 0ºC to RT, 10 hr. ii) Triazole, K2CO3, DMF, 85ºC, 8 hr
iii) Trimethylsulfoxonium iodide, cetrimide, aq. KOH, DCM, 45ºC, 12 hr
H
N
H
N
O
O
NH2
i
ii
Cl
XH
X
XH
10
X = O for 10a,11a and 6a ; X= S for 10b, 11b and 6b respectively
Reagents i) Chloroacetyl chloride, DCM, RT, 2 hr ii) K2CO3, EtOAc, RT, 12 hr
11
6
Scheme 1. Synthesis of epoxide intermediate (5) and benzothiazinone (6b) and benzoxazinone (6a) moieties.
O
N
O
O
OH
R2
N
N
K2CO3, TBAB,
EtOAc
N
N
HN
X
X
N
N
R3
R3
+
Reflux, 8-10 hr
R1
R1
R2
4
5
6
5a: R2=R3=F
2
3
5b
: R =R =Cl
2
3
5c
: R =Br, R =H
5d: R2=F, R3=H
Scheme 2. Synthesis of compounds 4 from epoxide 5 and benzothiazinone or benzoxazinone moieties 6.

724
H. B. Borate et al. / Bioorg. Med. Chem. Lett. 20 (2010) 722–725
Table 1
MIC obtained for compounds 4 by broth macro-dilution method
Sr. no.
Compound no.
Structure 4
Activity against organisms MIC in
l
g/mL*
C. albicans ATCC 24433
A. niger ATCC 16404
F. proliferatum ATCC 10052
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Fluconazole
Amphotericin B
1
128
1
>128
2
0.25
0.25
1
1
4
2
2
2
8
0.5
8
16
32
NI
4
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
X = S, R1 = H, R2 = R3 = F
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
X = S, R1 = 7-Cl, R2 = R3 = F
X = S, R1 = 7-Br, R2 = R3 = F
X = S, R1 = 7-OMe, R2 = R3 = F
X = S, R1 = 7-Me, R2 = R3 = F
X = S, R1 = 7-Cl, R2 = F, R3 = H
X = S, R1 = 7-OMe, R2 = Br, R3 = H
X = S, R1 = 7-OMe, R2 = F, R3 = H
X = O, R1 = H, R2 = R3 = F
X = O, R1 = 6-Cl, R2 = R3 = F
X = O, R1 = 6-Br, R2 = R3 = F
X = O, R1 = 6-NO2, R2 = R3 = F
X = O, R1 = 6-Ac, R2 = R3 = F
X = O, R1 = 6-Cl, R2 = Br, R3 = H
X = O, R1 = 6-Cl, R2 = F, R3 = H
X = O, R1 = 6-Ac, R2 = Br, R3 = H
16
32
NI: no inhibition.
*
For fluconazole and the synthetic compounds, MIC is recorded as the concentration exhibiting 80% inhibition as compared to the positive control while for amphotericin B,
MIC is recorded as the concentration exhibiting complete inhibition.
All the newly synthesized compounds 4 were tested for anti-
fungal activity against various fungi including C. albicans ATCC
24433, Aspergillus niger ATCC 16404 and Fusarium proliferatum
ATCC 10052 (Table 1, Sr. no. 3–18). In vitro evaluation of anti-
fungal activity was performed by determining the minimum
inhibitory concentration (MIC) following broth dilution meth-
ods.13,14 Broth dilution testing was performed in accordance with
the Clinical and Laboratory Standards Institute (CLSI) documents
M-27 A2 and M-38 A with RPMI 1640 medium buffered to pH
7.0 with MOPS buffer. Known antifungal agents, fluconazole and
amphotericin-B, were used as positive control. End points were
determined after 48 h visually and by using spectrophotometer
wherever necessary. The activity parameters are enumerated in
Table 1.
1. The compounds containing benzothiazinone moiety are more
active than those containing benzoxazinone (compound no 4a
vs 4i).
2. In the benzothiazinone series, replacement of fluorine by
hydrogen reduces the activity (compound nos 4b v/s 4f and
4d v/s 4h). The same is true for benzoxazinone series (com-
pound no 4j v/s 4o).
3. In the benzothiazinone series, halogen substituent at C7 position
is tolerated with slight decrease in activity (compound no 4a v/s
4b or 4c) while substituents like methoxy or methyl decrease the
activity considerably (compound no 4a v/s 4d or 4e).
4. In case of benzoxazinone series, no substituent at C6 is toler-
ated and all compounds having substituents like Cl, Br, NO2 or
Ac lost the activity (compound no 4i v/s 4j, 4k, 4l or 4m).
The compounds 4 prepared in the present work exhibited anti-
fungal activity while the corresponding starting materials 5 and 6
did not exhibit any antifungal activity. The antifungal activity
exhibited by the compounds 4 was confirmed by secondary
screening of compounds 4a and 4i against various strains of Can-
dida and it was observed that the activity of 4a is excellent against
the C. albicans ATCC24433, C. albicans ATCC90028 and C. glabrata
ATCC90030 when compared with amphotericin B and fluconazole
as shown in Table 2.
Thus, a novel class of anti-Candida agents was synthesized by a
short synthetic sequence and in good to excellent yields. Some of
the compounds exhibited good antifungal activity against C. albi-
cans and two of the compounds (4a and 4i) exhibited excellent
anti-Candida activity. These observations will be very useful in
designing newer molecules in the pursuit of more effective anti-
fungal agents.
The above results indicated following points regarding the
structure–activity relationship of the compounds studied in the
present work.
Acknowledgements
S.R.M. and S.P.S. thank CSIR, New Delhi for the award of senior
research fellowship. Financial supports from Department of Sci-
ence and Technology, New Delhi and FDC Ltd, Mumbai are grate-
fully acknowledged.
Table 2
MIC obtained for compounds 4a and 4i by broth micro-dilution method
MIC in l
g/mL*
Supplementary data
Fungus
Amphotericin B
Fluconazole
4a
4i
Experimental procedures and spectral data for all compounds
prepared for antifungal activity testing are available. Supplemen-
tary data associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2009.11.071.
C. albicans ATCC24433
C. albicans ATCC10231
C. albicans ATCC2091
C. albicans ATCC90028
C. glabrata ATCC90030
C. krusei ATCC6258
0.25
0.5
0.5
0.5
0.25
0.5
0.25
1.0
0.5
0.5
4
0.03
0.12
0.12
0.03
0.06
4
0.12
0.5
0.25
0.12
0.25
16
64
2
References and notes
C. tropicalis ATCC750
0.5
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*
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11. Huang, X.; Chan, C.-C. Synthesis 1984, 851.
12. The experimental procedures and spectral data for all compounds prepared
for antifungal activity testing in this article are given as Supplementary data.
General procedure for the synthesis of fluconazole analogues 4: To a flame
dried K2CO3 (0.18 mol), tetra-butylammonium bromide (TBAB, 0.09 mol) was
added followed by the addition of compound
6 (0.09 mol) in dry ethyl
acetate (200 mL). Reaction mixture was stirred at reflux for 30 min. Then
epoxide 5 (0.09 mol) dissolved in dry ethyl acetate (200 mL) was added to
the refluxing mixture drop wise over a period of 10 min and stirring was
continued for further 12 h at the same temperature. It was then cooled to
room temperature, diluted with water (800 mL), extracted with ethyl acetate
(3 Â 400 mL), dried over Na2SO4, concentrated and purified by column
chromatography to give pure compound. Various compounds were prepared
by using the general procedure described above and spectral data for all the
compounds prepared are given as Supplementary data. Spectral data for
representative compound are given below: 4-[2-(2,4-Difluorophenyl)-2-
hydroxy-3-[1,2,4]triazol-1-yl-propyl]-4H-benzo[1,4]thiazin-3-one
(4a):
Yield: 78%; Pale yellow solid, mp 123 °C; 1H NMR (400 MHz, CDCl3):
d
3.17 (d, J = 14 Hz, 1H), 3.24 (d, J = 14 Hz, 1H), 4.43 (d, J = 14 Hz, 1H), 4.48 (d,
J = 14 Hz, 1H), 4.60 (s, 2H), 5.52 (br s, 1H), 6.41–6.47 (m, 1H), 6.68–6.73 (m,
1H), 6.92–6.98 (m, 1H), 7.13–7.26 (m, 3H), 7.50–7.58 (m, 1H), 7.76 (s, 1H),
8.15 (s, 1H). 13C NMR (100 MHz, CDCl3,): 31.9, 52.3, 56.1, 76.4, 103.3 (t),
111.3 (d), 119.1, 122.8, 123.9, 125.2, 126.9, 128.2, 130.1, 139.4, 144.6, 151.0,
157.4 (d), 162.8 (d), 168.4. IR (Chloroform): 1647, 3332 cmÀ1. MS (ESI) m/z:
403.3074 (M+1), 425.2999 (M+Na).
13. CLSI: Reference method for broth dilution antifungal susceptibility testing of
yeasts; Approved standard, second edition M27-A2, 2002.
14. CLSI: Reference method for broth dilution antifungal susceptibility testing of
filamentous fungi; Approved standard M38-A, 2002.