Microwave prompted multigram synthesis, structural determination, and photo-antiproliferative activity of fluorinated 4-hydroxyquinolinones

Article abstract of DOI:10.1016/j.bmcl.2006.09.082

3-Unsubstituted 4-hydroxyquinolin-2(1H)-one containing F and CF₃ substituent in ring is important pharmacological and synthetic target and basic synthones for a number of antibacterial fluoroquinolones and is promising potent and selective glycine site NMDA receptors. A simple facile one-step microwave enhanced multigram synthesis of such fluorinated quinolones in reasonable purity has been developed in excellent yield (85-94%) in 3-5 min, whereas conventional synthesis required the harsh conditions, long reaction period with use of environmentally unacceptable regents giving the required product in lower yield. The phototoxicity as well as the cytotoxic activities of the title compounds are evaluated against leukemia- and adenocarcinoma-derived cell lines in comparison to the normal human keratinocytes. Structure-activity relationships between the chemical structures and the antimycobacterial, antifungal activity of the evaluated compounds are also discussed.

Full text of DOI:10.1016/j.bmcl.2006.09.082

Bioorganic & Medicinal Chemistry Letters 17 (2007) 86–93
Microwave prompted multigram synthesis, structural
determination, and photo-antiproliferative activity of fluorinated
4
-hydroxyquinolinones
*
Kapil Arya and Manish Agarwal
Indian Oil Corporation Limited, Catalyst Division, R&D Centre, Faridabad, Haryana 121007, India
Received 25 August 2006; revised 23 September 2006; accepted 28 September 2006
Available online 30 September 2006
3
Abstract—3-Unsubstituted 4-hydroxyquinolin-2(1H)-one containing F and CF substituent in ring is important pharmacological
and synthetic target and basic synthones for a number of antibacterial fluoroquinolones and is promising potent and selective gly-
cine site NMDA receptors. A simple facile one-step microwave enhanced multigram synthesis of such fluorinated quinolones in rea-
sonable purity has been developed in excellent yield (85–94%) in 3–5min, whereas conventional synthesis required the harsh
conditions, long reaction period with use of environmentally unacceptable regents giving the required product in lower yield.
The phototoxicity as well as the cytotoxic activities of the title compounds are evaluated against leukemia- and adenocarcinoma-
derived cell lines in comparison to the normal human keratinocytes. Structure–activity relationships between the chemical structures
and the antimycobacterial, antifungal activity of the evaluated compounds are also discussed.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
5
16
17
The synthesis of compounds belonging to 4-hydroxyqu-
inolones constitutes an important area of research due
cycles, multiazahetrocycles, isoxazolo quinolines,
1
8
19
pyrano quinolines, piperazinyl carbomides, pyrazolo
20
1
2
3
to their use as analgesic, dye-stuffs, herbicides, orally
4
active antagonists, anti-inflammatory, antiallergenic,
quinolines, oxazino quinolines, etc. These derivatives
are used as potent antiemetic, migraine suppressing,
5
6
7
8
21
22
antitubercular, and cardiovascular agents. They have
also attracted considerable interest for various pharma-
cological targets including glycine NMDA receptor
antibacterial, fungicidal, antipyretic, anticoagulant,
2
3
CNS, memory enhancing agents, antibrain tumor
2
activity in vivo, etc.
4
antagonist and glycine (5-HT ) site antagonists related
3
2
5
to several central nervous system disorders including
stroke, epilepsy, schizophrenia, Parkinson’s disease,
Fluorinated quinolones are also extensively used in
medicinal chemistry, notable clinical examples are
worldwide patented drugs, for example, norfloxacin,
ciprofloxacin, ofloxacin, enoxacin, pefloxacin, grepa-
9
–12
and Alzheimer’s disease.
In addition 4-hydroxyqui-
nolones have recently been found to serve as key inter-
mediates in the synthesis of non-peptide GnRH
2
6
floxacin, etc., as antibacterial, antimicrobial, anticon-
2
7
19
(
nists. Such compounds are of interest for the treatment
gonadotropin releasing hormones) receptor antago-
vulsant, CNS depressing, hypotensive agents, and
some references are patented as high oral absorbents
and used for the treatment of autoimmune and rheumat-
ic diseases Further literature survey that presence of F
or CF substituent at various positions in the quinoline
1
3
of sex hormone related conditions.
2
8
4-Hydroxyquinolines are important synthones and they
are used as synthetic precursors of many naturally
3
nucleus plays an important role in modifying the biolog-
19,26e
ical activities.
1
4
occurring alkaloids,
polycyclic condensed hetero-
4-Hydroxyquinolin-2(1H)-one derivatives have been
synthesized by cyclization of N-acetylanthranilic acid
Keywords: Fluorinated 4-hydroxyquinolinones; Antiproliferative activ-
ity; MW irradiation.
2
9
derivatives,
acid with anilines using ZnCl2 and POCl ,
condensation of malonates/malonic
7
,30
*
Corresponding author. Tel.: +91 9911317131; fax: +91 129 2294381;
e-mail: aryakapil2001@yahoo.com
3
and
32
cyclization of malanodianilides using AlCl3, PPA,
3
1
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.082

K. Arya, M. Agarwal / Bioorg. Med. Chem. Lett. 17 (2007) 86–93
87
3
3
CH SO H/P O , etc. Recently, they have been synthe-
3
new possible antitumoral, photochemically reactive,
antimycobacterial or antifungal compounds.
3
2
5
sized by the reaction of 2-aminoacetophenone and
acylating reagent such as phosgene, dimethylcarbonate
or diethylcarbonate in the presence of basic catalyst
sodium ethoxide, sodium hydride, 3-aminopropylamin-
de (NAPA), potassium 3-aminopropylamide (KAPA),
Literature survey reveals that 3-unsubstituted 4-
hydroxyquinolin-2-(1H)-one quinolines have been pre-
pared conventionally by environmentally unacceptable
procedure using hazardous reagents and volatile solvent,
3
4
and refluxing in anhydrous toluene.
3
0,32
for example, PPA/ZnCl + POCl /P O , etc.
2
and 3-
unsubstituted fluoroquinolones are patented as pharma-
cological agents, the synthetic details of which are not
3
2
5
All these methods suffer from many disadvantages like
long reaction period, use of strong acids (PPA, H SO ,
etc.)/bases (NaOEt, sodium hydride, potassium 3-
aminopropylamide (KAPA), sodium 3-aminopropyla-
mide (NAPA), etc.), dehydrating agents (ZnCl , AlCl ),
and hazardous solvents/reagents like toluene, POCl3,
CH SO H, and P O . Further, method involving use
of diethyl malonate was found unsuitable for synthesis
2
4
4
1,42
available.
2
3
In view of these observations we have successfully syn-
thesized fluorinated 4-hydroxyquinolin-2-(1H)-one (hav-
ing F and CF substitution at various positions) by
3
condensation of fluorinated anilines (X = F, CF ) with
3
malonic acid in unexpensive multimode domestic micro-
wave oven in reasonable purity (TLC) in 85–94% yield in
3
4
2
5
3
2
of 3-unsubstituted 4-hydroxyquinolone.
The exploitation of microwaves for assisting different
3–5 min using 4–5 drops of DMF, which act as energy
transfer agent and homogenizer to increase the reaction
3
5
organic reactions has blossomed into an important
tool in synthetic organic chemistry with large horizon
of applications due to the timelessness, ease of
workability, and ecofriendliness, so microwave-assisted
organic reactions are the best alternatives to an envi-
ronmentally unacceptable procedure. However, recently
more interest has been focused on dry-media synthesis
and particularly on solvent-free procedure using
various mineral oxides and solventless reactions with
4
3
temperature. Nonreproducibility of results observed
by some workers in domestic microwave oven can be
overcome by previous cartography of energy distribu-
3
8b
tion
and performing the reactions under continuous
microwave power emission without on–off cycles. The
reactions carried out with similar weight of load always
located in the same place as determined above in the
oven are highly reproducible in present studies.
3
6
neat reactants in the absence of any support or
solvent.
Hence, we have developed a facile, safe, and economic
method for 3-unsubstituted 4-hydroxyquinolin-2-(1H)-
one with no need of paraphernalia requiring the stream
Recently microwave-enhanced synthesis of some
7
3
3
Lange et al.
-substituted hydroxyquinolines has been studied.
3
of N , since in present case instead of ethanol, water is
2
7a
reported the synthesis of 3-substituted
-hydroxyquinoline under focused microwaves using
formed as byproduct which is removed easily. Further
workup procedure is simple involving the washing from
water to give product in reasonable purity (TLC) with
no need of further use of solvent for purification proce-
dure Scheme 1).
4
substituted aniline with excess amount of substituted
malonic ester under the stream of N to remove the eth-
2
anol which is formed as byproduct. However, these
microwave-mediated methods have disadvantages like
3
2,37
(
i) use of volatile solvents (diethyl ether, hexane, and
Earlier report mentioned
that the yield of the reac-
acetone) in workup process, (ii) it was found unsuitable
for the synthesis of trifluoromethyl substituted quino-
lones, reaction remaining at the intermediate malanodi-
anilide stage. More interesting is that one can obtain
excellent results even using inexpensive domestic micro-
wave oven which is available elsewhere rather than to
tion strongly depends on the relative amount of the
starting material (malonates/malonic acid and anilines)
used, prompting to study in detail this effect using differ-
ent molar ratio of the malonic acid and anilines, and re-
sults are summarized in Table 1.
3
5c,36a–36d,38
use systematically monomode oven.
In all these cases it has been found that reactions proceed
with higher yields with shorter reaction time when 1:1
molar ratio of malonic acid and anilines was used in open
vessel. It is essential to work in an open vessel to enable
the formed water to escape from reaction mixture, which
is polar microwave active product, while the 1.5:1/2:1
molar ratio of starting materials gave lower yield with
higher time which makes it unsuitable for synthesis of re-
quired fluoroquinolines. These optimal conditions were
then applied for the synthesis of a range of 3-unsubstitut-
ed 4-hydroxyquinolones depicted in Table 2.
Recently, 4-hydroxyquinolin-2(1H)-one derivatives
were prepared in a new profitable way these com-
pounds show strong photo-antiproliferative activity,
higher than that of 8-methoxypsoralen (8-MOP), the
most widely employed drug for photochemotherapy.
Moreover, their activity is devoid of mutagenicity
3
9
and skin phototoxicity. For these features, fluorinat-
ed quinolinones and other analogues appeared to be
very promising photochemotherapeutic agents. The
great importance of this category of heterocycles
and continuation to our earlier interest on environeco-
nomic synthesis of biodynamic heterocycles under
Further multigram synthesis (18–22 g) of representa-
tive quinolines (4d, e, h; X = 8-CF , 7-CF , H) is also
3
3
4
0
microwaves oriented our attention to the synthesis
of fluorinated 3-unsubstituted 4-hydroxyquinolin-
carried out safely in open tall beaker (4 cm radius) in
reasonable purity (TLC) in 10–15min (Table 3) under
microwaves.
2
(1H)-one with F and CF3 at various positions as

88
K. Arya, M. Agarwal / Bioorg. Med. Chem. Lett. 17 (2007) 86–93
OH
4
H
O
O
N
X
5
C
COOH
COOH
6
7
3
MW
X
+
CH2
C
X
H C
2
2
Neat + DMF
1
NH2
N
O
8
H
N
H
X
4
a, c, d
1
2
1a; X= 2-F; 1c; X= 4-F
1d; X= 2-CF3
3
a, c, d
OH
5
X
6
4
3
.
H
1
N
H
2
O
O
N
7
X
O
C
8
X
NH2
1
b, e-g
X
MW
CH2
C
4b, e-g
+
Neat + DMF
X
5
OH
COOH
COOH
N
H
H C
2
6
4
3
2
3
b, e-g
2
1
7
N
O
1
1
b; X= 3-F; 1e; X= 3-CF3
f; X= 3-CH3; 1g;X= 3-Cl
8
H
5
Scheme 1.
Table 1. Comparative results obtained for the synthesis of 4d and 4e by different relative amounts of the starting material
a
Compound
X
Molar ratio
malonic acid:amine)
Temp (ꢁC)
Time (min)
Isolated yield (%)
(
4d
4d
4d
4e
4e
4e
4h
4h
4h
8-CF
8-CF
8-CF
7-CF
7-CF
7-CF
H
3
3
3
3
3
3
1:1
145
142
141
142
138
138
135
133
128
5
6
7
4
7
8
3
6
9
85
80
78
88
81
84
94
80
89
1.5:1
2:1
1:1
1.5:1
2:1
1:1
H
1.5:1
2:1
H
a
Final temperature is measured at the end of microwave irradiation by introducing a glass thermometer in the reaction mixture in the beaker.
Table 2. Physical data of synthesized compounds 4a–g
Compound
X
Reaction time
Molecular formula
Mp (ꢁC)
Classical method
Microwave method
a
yield (%)
a
yield (%)
(
min)
(min)
[
[
[
[
33]
34]
34]
34]
4
4
4
4
4
4
4
4
a
b
c
d
e
f
8-F
7-F
600
480
960
75
67
78
62
71
80
76
81
3
4
3
5
4
4
4
3
90
89
85
85
88
88
85
94
C
C
C
C
C
C
C
C
9
9
9
H
H
H
6
FNO
FNO
FNO
2
2
2
310
318
245
295
290
305
340
360
6
6-F
6
8-CF
7-CF
3
1080
960
840
860
560
10
10
10
9
H
6
6
9
F
F
3
NO
NO
2
3
H
H
3
2
7-CH
7-Cl
H
3
NO
ClNO
FNO
2
g
h
H
6
2
9
H
7
2
a
Isolated yield.
To study the range of this electrophilic attack of the
amide toward aromatic ring, some meta-substituted ani-
lines (1b, e–g) with fluorine, trifluoromethyl, methyl, and
chlorine as the substituent were treated with malonic
acid under microwaves to yield 4b, e–g in one step.
Although in these cases two isomers (4 and 5) could
be expected, only one isomer was obtained (4b, e–g),
the structure of which could be unequivocally assigned
as 7-substituted 4-hydroxyquinolin-2-ones on the basis
of PMR spectra.

K. Arya, M. Agarwal / Bioorg. Med. Chem. Lett. 17 (2007) 86–93
Table 3. Multigram synthesis (50 g) of compounds 4d, 4e, and 4h
89
a
Compound
X
Temp (ꢁC)
Time (min)
Isolated yield
(
%)
(g)
4d
4e
4h
8-CF
7-CF
H
3
138
135
137
10
13
12
81
86
89
18
20
22
3
The condensation of fluorinated anilines with malonic
acid afforded the title compound instead of the interme-
diate malanodianilides, which is confirmed by spectral
studies.
meric compound 4c is only slightly active and substitu-
tion for a methyl group leads to an inactive derivative
4b. From this preliminary screening the most active
compounds were also evaluated on a human intestinal
adenocarcinoma cell line (LoVo) and one line of
immortalized, not tumorigenic, human keratinocytes
(NCTC 2544). From Table 6 it appears that the photo-
toxicity of the most active compounds, in particular 4e,
is higher in the tumor cell lines in comparison to the
normal ones (NCTC 2544). In preliminary experiments
devoted to the search for a possible molecular target,
compound 4e was evaluated for its potential capability
to induce single strand breaks in a plasmid DNA, as a
model.
IR spectra of the products (4a–h) displayed characteris-
tic absorption in the region 3390–2980 (br, OH, NH,
and CH stretching), 1670–1640 (s, NH–C@O), 1610–
1
1
600 (s, C@C), and 760–740 (C–F). The H NMR spec-
tra of 4a (X = 8-F) showed singlet at d 6.38 (s, 1H, H-3),
7
.32–7.65 (m, 2H, H-6 and H-7), 7.81 (dd, J = 7.2Hz, H-
5
pearance of CO (acidic) at 1740 cm
amino group absorption at 3450–3330 cm
confirmed the formation of 4.
), 10.21 (br, 1H, NH), and 11.94 (br, 1H, OH). Disap-
and primary
À1
À1
further
The obtained results (data not shown) indicate that 4e,
after irradiation in the presence of DNA, is not able
to induce any significant damage to DNA thus suggest-
ing that another target at cellular level may be involved
in its phototoxic effect. In parallel to the cytotoxic eval-
uation, flow cytometry was employed to study cell cycle
variations upon irradiation. The effects of the most ac-
tive compound 4e were evaluated after 24, 48, and
72 h from irradiation in the leukemic cell line. The per-
centage of the cells in the different phases of the cell cy-
cle is shown in Table 7.
In case of meta-substituted anilines (1b, e–g) (X = F,
CF , Cl, and CH ) two isomers (4 and 5) could be
expected, only one isomer was obtained showing H
3
3
1
NMR signals at d 6.21–6.38 (s, 1H, H-3), 7.26–7.40
(
d, J = 2Hz, H-8), 7.35–7.60 (dd, J = 2 + 7 Hz, H-6),
7.65–7.92 (d, J = 7Hz, H-5), 10.22–10.32 (br, 1H,
NH), and 11.76–11.94 (br, 1H, OH). On the basis
1
of H NMR spectra it is unequivocally identified as
1
3
7
-substituted quinoline derivatives (4b, e–g).
C
NMR of 4 showed a sharp signal at d 85.6–86.5
(
(
CH), 119.2–138.5 (aromatic carbons), 164.6–165.3
C@O), and 172.3–173.4 (C–OH). The presence of
It can be observed that treatment with 4e in combina-
tion with UVA induces a reduction of the S phase at
48 and 72 h after irradiation especially for the highest
dose utilized. This is accompanied by a concomitant
block in G1 phase. This event is followed at 48 and
72 h after the irradiation by massive induction of apop-
tosis, as observed by the appearance of a sub G1 peak
(apoptotic cells) that refers to cells with DNA content
1
9
fluorine was confirmed by F NMR spectra, where
the C–F signal was observed at d À118.05–120.42
(
6
compounds 4a–c) and CF3 signal at d À63.25/
4.07 ppm in case of compounds 4d–e. In the mass
spectra of 4a and 4b molecular ion peaks were ob-
served at 179 corresponding to their molecular weight
along with base peak at 109 (C H O ) and 4d, e
molecular ion peaks were observed at 229 corre-
sponding to their molecular weight along with base
peak at 43(C H OF ), other characteristic peaks
3
2
2
4
4,45
lower than G1.
In fact, apoptosis induces the activa-
tion of endogenous nucleases, which are responsible for
nucleic acid degradation.
9
5
3
observed are described in Table 4.
All compounds prepared were evaluated for their in vitro
antimycobacterial activity. The highest activity (72%
inhibition) against Mycobacterium tuberculosis was
found for 7-trifluoromethyl4-hydroxyquinolin-2-(1H)-
one (4e). Two other compounds, (4d and 4f) with the
identical substitution on the aromatic part of the mole-
cule, exert a comparable activity. The majority of com-
pounds exhibited only modest antimycobacterial
activity (see Table 8).
The phototoxicity of title compounds was investigated
first on a cell line of human tumor HL-60 (human
promyelocytic leukemia). Table 5 shows the extent of
cell survival expressed as GI50, which is the concentra-
tion, expressed in mM, which induces 50% of inhibi-
tion of cell growth, after irradiation at different UVA
doses.
Control experiments with UVA light or compounds
alone were carried out without significant cytotoxic
effects (data not shown). The results, shown in
Table 5, indicate that compound 4a is not active, instead
The evaluation of in vitro antifungal activity of the syn-
thesized compounds was performed against eight fungal
strains. The results revealed no interesting activity
against the majority of strains tested.
4
e we observe the highest activity. Interestingly, the iso-

Table 4. Spectral data of synthesized compounds 4a-e
À1
13
19
Compound IR (cm
)
1H NMR (d, ppm)
NH/OH
C NMR (d, ppm)
F NMR (d, PPM) Mass m/z (%)
a
CH
Ar-H
+
4
4
4
4
4
4
4
4
a
b
c
d
e
f
3380–3020 (br), 1665
6.25 (s, 1H)
7.32–7.65 (m, 2H, H-6
and H-7), 7.81 (dd,
J = 7.28 Hz H-5)
10.21 (br, s, NH), 11.85 (br, s,
OH)
85.6 (CH), 120.8–138.2
(aromatic carbons),
164.8 (C@O), 172.3
À120.42 (s, 8-F)
À119.20 (s, 7-F)
À118.05 (s, 6-F)
179 ([M ] 8.8), 160
(40.2), 151 (56), 123 (25),
109 (100), 43 (15)
(
(
s, C@O), 1605 (s), 740
s, C–F)
(
10.25 (br s, NH), 12.90 (br s, OH) 85.9 (CH), 119.2–135.2
C–OH)
3390–2980 (br), 1640
6.38 (s, 1H)
6.35 (s, 1H)
6.50 (s, 1H)
6.21 (s, 1H)
2.54 (s, 1H,
7.43 (d, J = 2 Hz, H-8),
7.56 (dd, J = 2 + 7 Hz,
H-6), 8.19 (d, J = 7 Hz,
H-5)
—
(
(
s, C@O), 1610 (s), 750
s, C–F)
(aromatic carbons),
164.9 (C@O), 172.8
(C–OH)
10.21 (br s, NH), 11.74 (br s, OH) 86.2(CH), 121.2–137.2
(aromatic carbons),
3360–3090 (br), 1665
7.34 (d, J = 8 Hz, H-8),
7.50 (dd, J = 2 + 8 Hz,
H-7), 8.26 (d, J = 2 Hz,
H-5)
—
(
(
s, C@O), 1605 (s), 760
s, C–F)
165.3 (C@O), 173.4
(C–OH)
+
229 ([M ]12.5), 221 (4.5),
3380–3090 (br), 1665
7.38 (t, J = 7.5 Hz, H-6), 10.28 (br s, NH), 10.94 (br s, OH) 86.4 (CH), 121.8–138.2
À64.02 (s, 8-CF
3
)
(
(
s, C@O), 1602 (s), 750
s, C–F)
7.65–7.43 (m, 2H, H-5
and H-7)
(aromatic carbons),
130.5 (C–CF ), 165.8
C@O), 173.4 (C–OH)
160(32.2), 120 (28.3),
105(27), 92 (54.3), 64
(23.1), 51 (48.5), 43 (100)
3
(
+
3390–3010 (br), 1660
7.26 (d, J = 2 Hz, H-8),
7.35 (dd, J = 2 + 7 Hz
H-6), 7.65 (d, J = 7 Hz,
H-5)
10.32(br s, NH), 11.76 (br s, OH) 86.3(CH), 120.3–135.7
(aromatic carbons),
À63.25 (s,7-CF
3
)
229 ([M ]11.8), 221 (5.6),
(
(
s, C@O), 1605 (s), 760
s, C–F)
190 (13.8), 170 (5.6), 160
(30.5), 120 (28.3), 105
(23.6), 64 (25.3), 43 (100)
—
131.2 (C–CF
(C@O), 173.1 (C–OH)
10.32 (br s, NH), 11.76 (br s, OH) 28.3 (CH ), 85.3 (CH),
3
), 165.4
320–2930 (br), 1645
s, C@O), 1605 (w),
7.19 (d, J = 2 Hz, H-8),
7.29 (dd, J = 2 + 7 Hz,
H-6), 7.82 (d, J = 7 Hz,
H-5)
3
—
—
—
(
CH
(
3
), 6.2
121.3–134.8 (aromatic
carbons), 164.8 (C@O),
172.6 (C–OH)
s, 1H)
g
h
3380–3020 (br), 1670
6.32 (s, 1H)
6.33 (s, 1H)
7.12 (d, J = 2 Hz, H-8),
7.25 (dd, J = 2 + 7 Hz,
H-6), 7.65 (d, J = 7 Hz,
H-5)
11.32(br s, NH), 11.76 (br s, OH) 87.6 (CH), 119.6–132.6
(aromatic carbons),
—
(
(
s, C@O), 1605 (s), 780
s, C–Cl)
164.9 (C@O), 172.9
(C–OH)
+
161 (M ), 133 (200), 105
3370–3030 (br), 1650
s, C@O), 1600 (s)
7.32–7.76 (m, 3H), 8.26
(d, 1H, J = 8 Hz)
10.47 (br s, NH), 11.42 (br s, OH) 85.3 (CH), 118.6–138.4
(aromatic carbons),
(
(45), 91 (56), 43 (100)
1
65.4(C@O), 178.6
C–OH)
(
a
Exchangeable with deuterium.

K. Arya, M. Agarwal / Bioorg. Med. Chem. Lett. 17 (2007) 86–93
91
Table 5. Photocytotoxicity of test compounds against HL-60 cell line
a
compounds GI50 (mM)
Table 8. Calculated antimycobaterial evaluation (% of inhibition) and
antifungal susceptibility (MIC) comparison with standards: pyrazin-
a
amide (PZA) and fluconazole
Compound
GI50 (lm)
À1
b
.25 J cm
À2
À2
Compound
X
% inhibition at
À1
MIC (lmol mL
)
1
2.5 J cm
6
.25 lg mL
4
4
4
4
4
4
a
b
c
d
e
f
>10
>10
10
10
4
4
4
4
4
4
4
4
a
b
c
d
e
f
8-F
7-F
50
52
35
69
72
54
2
31.25/62.5
31.25/31.25
125/250
>10
>10
7.8 ± 2.2
7.3 ± 1.9
0.9 ± 0.1
6-F
8-CF
7-CF
3
>500/>500
125/125
6.2 ± 0.7
>10
3
10
7-CH
7-Cl
H
3
>500/>500
>500/>500
>500/>500
—
a
Concentration of compound required to inhibit the cell growth by
50% after 72 h of exposure as determined by MTT assay.
g
h
0
a
b
À2
UVA dose expressed in J cm as measured at 365 nm with a Cole–
Parmer radiometer.
PZA
Fluconazole
—
—
100
—
1.95/3.91
a
À1
MIC = 12.5 lg mL , data from Ref. 46.
Table 6. Photocytotoxicity of test compounds against NCTC 2544 and
a
LoVo cell lines
À1
dard (MIC = 3.91 lmol mL after 120 h, see Table 8).
The negative antifungal screening results do not allow
us to draw detailed conclusions on potential structure–
activity relationships.
b
Compound
Cell line GI50 (lm)
NCTC 2544
LoVo
c
.25 J cm
À2
À2
b
1.25 J cm
À2
À2
2.5 J cm
1
2.5 J cm
4
4
4
e
c
d
7.0 ± 1.3
>20
>20
2.5 ± 0.7
>20
7.8 ± 2.2
3.7 ± 0.8
>20
18.2 ± 2.1
1.8 ± 0.2
12.1 ± 1.9
10.1 ± 1.3
In summary, the synthesis and biological evaluation of
eight new substituted 4-hydroxyquinolin-2-(1H)-one
are described. In the first series, among the compounds
with substituted fluoro, the highest antifungal effect was
found for 7-fluoromethyl4-hydroxyquinolin-2-(1H)-one
a
b
c
Human cell lines: NCTC 2544 Human keratinocytes; LoVo intestinal
adenocarcinoma.
Concentration of compound required to inhibit the cell growth by
(
4b). In the second series, among the compounds bear-
5
0% after 72 h of exposure as determined by MTT assay.
À1 À2
ing trifluoromethyl (4d–f) have shown the highest bio-
logical activity, that is, against M. tuberculosis H37Rv.
UVA dose expressed in J cm
Cole–Parmer radiometer.
K
as measured at 365 nm with a
In conclusion, we have presented a new economical,
safe, environmentally benign solvent free synthesis of
fluorinated 4-hydroxyquinolin-2(1H)-one under micro-
wave irradiation. The operational simplicity, avoiding
the use of solvent and solid support, and high yield in
significantly very short reaction time, can impose this
procedure as a useful and attractive alternative to the
currently available methods.
a
Table 7. Percentage of HL-60 in the different phases of the cell cycle
b
Treatment
G1
G2
9.0
S
Apoptotic cells
0.8
Nonirradiated cells
39.0
51.7
À2
UVA irradiated cells without drug (2.5 J cm
)
2
4
7
4 h
8 h
2 h
36.7
48.2
35.0
13.0
10.9
10.5
50.3
8.6
11.7
9.4
40.9
54.4
On the basis of the biological evaluation, compound 4e
seems to be very attractive as a potential drug for pho-
tochemotherapy and antimicrobial agents, while, among
the compounds bearing trifluoromethyl (4d–f) have
shown the highest antituberculosis activity, that is,
against M. tuberculosis H37Rv. Hence, experiments
aimed at defining the target(s) at cellular level and the
phototoxicity mechanism are in progress.
À2
4
4
c 2.5 mM CUVA (2.5 J cm
)
)
2
4
7
4 h
8 h
2 h
30.8
44.7
44.2
10.8
10.3
12.0
58.4
45.0
43.8
13.2
27.0
40.5
À2
c 5.0 mM CUVA (2.5 J cm
2
4
7
4 h
8 h
2 h
34.6
49.6
56.0
11.1
11.7
6.9
54.3
38.6
37.1
21.0
26.0
46.0
a
The percentage of each phase of the cell cycle (G1, S, and G2/M) were
calculated on living cells.
Human promyelocytic leukemia cells (HL-60) were
grown in RPMI-1640 medium (Sigma Co., MO,
USA), human keratinocytes (NCTC 2544) were grown
in DMEM (Sigma Co., MO, USA), and intestinal ade-
nocarcinoma cells (LoVo) were grown in Ham’s F12
medium (Sigma Co., MO, USA) all supplemented
b
The percentage of apoptotic cells is referred to cells population
characterized by the appearance of a sub-G1 peak.
À1
Only the 8-fluoromethyl4-hydroxyquinolin-2-(1H)-one
(
with 115 U mL of penicillin G (Invitrogen, Milano,
Italy), 115 lg mL streptomycin (Invitrogen, Milano
À1
4a) and especially 7-fluoromethyl4-hydroxyquinolin-2-
1H)-one (4b) showed some promising in vitro anti-
(
fungal activity against Trichophyton mentagrophytes,
the most susceptible fungal strain evaluated
(
is only modest in comparison with fluconazole, the stan-
Italy) and 10% fetal bovine serum (Invitrogen, Milan-
o, Italy). Individual wells of a 96-well tissue culture
microtiter plate (Falcon BD) were inoculated with
À1
3
MIC = 31.25–62.5 lmol mL ), although this activity
100 mL of complete medium containing 8 · 10 HL-
3
60 cells or 5 · 10 NCTC 2544 and LoVo cells. The

92
K. Arya, M. Agarwal / Bioorg. Med. Chem. Lett. 17 (2007) 86–93
plates were incubated at 37 ꢁC in a humidified 5%
incubator for 18 h prior to the experiments. After
medium removal, 100 mL of the drug solution, dis-
solved in DMSO and diluted with Hanks’ balanced
salt solution (HBSS, pH 7.2), was added to each well
and incubated at 37 ꢁC for 30 min and then irradiated.
France, for his generous support and valuable
suggestions. We are also thankful to Indian Oil
Corporation, R&D Center, for spectral and financial
supports and Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (TAACF) for antimycobac-
terial data.
After irradiation, the solution was replaced with the
medium, and the plates were incubated for 72 h. Cell
viability was assayed by the MTT [(3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide)] test, as
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The authors wish to express their appreciation to Dr.
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53. IR spectra (KBr) were recorded on a Magna FT IR–550
1
19
13
spectrophotometer, H and F and C NMR spectra
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3
3
)
2
SO] were taken on a Bruker À300DX
spectrometer at 300, 84.25, and 200MHz, respectively,
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1
92; Dandia, A.; Arya, K.; Sati, M.; Sarawgi, P.;
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(Niles, IL), equipped with a 365-CX sensor.
4
6
71; Suarez, M.; Loupy, A.; Perez, E.; Moran, L.; Gerona,