Design, synthesis and anti-mycobacterial activity evaluation of benzofuran-isatin hybrids

Article abstract of DOI:10.1016/j.ejmech.2018.09.049

Herein we report the design and synthesis of a series of novel benzofuran-isatin hybrids, and in vitro evaluation of their anti-mycobacterial activity against both drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (MTB) strains. I

Full text of DOI:10.1016/j.ejmech.2018.09.049

Accepted Manuscript
Design, synthesis and anti-mycobacterial activity evaluation of benzofuran-isatin
hybrids
Feng Gao, Hua Yang, Tianyu Lu, Zijian Chen, Long Ma, Zhi Xu, Paul Schaffer,
Guangming Lu
PII:
S0223-5234(18)30821-3
10.1016/j.ejmech.2018.09.049
EJMECH 10760
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2018
Revised Date: 11 September 2018
Accepted Date: 16 September 2018
Please cite this article as: F. Gao, H. Yang, T. Lu, Z. Chen, L. Ma, Z. Xu, P. Schaffer, G. Lu, Design,
synthesis and anti-mycobacterial activity evaluation of benzofuran-isatin hybrids, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.049.
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ACCEPTED MANUSCRIPT
Design, synthesis and anti-mycobacterial activity evaluation of
benzofuran-isatin hybrids
1
2
3
1
1
3
2
1*
Feng Gao , Hua Yang , Tianyu Lu , Zijian Chen , Long Ma , Zhi Xu , Paul Schaffer , Guangming Lu
Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing, P. R. China;
1
2
Life Sciences, TRIUMF, Vancouver, Canada;
3
Department of Neurosurgery, NanJing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical
School, Nanjing, P. R. China
A series of novel benzofuran-isatin hybrids were designed, synthesized and evaluated for their in
vitro anti-mycobacterial activities as well as cytotoxicity. Preliminary results indicated that all
hybrids with acceptable cytotoxicity in VERO cells exhibited considerable anti-mycobacterial
activities against MTB H Rv and MDR-TB strains. It is worthy to note that hybrid 8f with no
37
cytotoxicity towards VERO cells, was found to be the most active compound against MTB H Rv
3
7
and MDR-TB. Comparing to the first-line anti-TB agents (rifampicin and isoniazid), hybrid 8f has
shown over two magnitude more active against MDR-TB. However, both the metabolic stability
and in vivo pharmacokinetic profiles of 8f were inferior to inhibitor TAM16. Further modification
based on hybrid 8f is needed to improve metabolic stability and pharmacokinetic profiles.

ACCEPTED MANUSCRIPT
Design, synthesis and anti-mycobacterial activity evaluation of
benzofuran-isatin hybrids
1
2
3
1
1
3
1*
Feng Gao , Hua Yang , Tianyu Lu , Zijian Chen , Long Ma , Zhi Xu , Guangming Lu
Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing, P. R. China;
1
2
Life Sciences, TRIUMF, Vancouver, Canada;
3
Department of Neurosurgery, NanJing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical
School, Nanjing, P. R. China
Abstract: Herein we report the design and synthesis of a series of novel
benzofuran-isatin hybrids, and in vitro evaluation of their anti-mycobacterial activity
against both drug-susceptible and multi-drug resistant (MDR) Mycobacterium
tuberculosis (MTB) strains. In parallel, cytotoxicity of these hybrids was also tested in
VERO cells. Preliminary results indicated that all hybrids with acceptable cytotoxicity
in VERO cells (CC : 128->1,024 µg/mL) exhibited considerable anti-mycobacterial
5
0
activities against MTB H Rv and MDR-TB with MIC ranging from 0.25 to 8 µg/mL.
3
7
It is worth noting that hybrid 8f with no cytotoxicity towards VERO cells
CC : >1,024 µg/mL) was found to be the most active compound (MIC: 0.25 and 0.5
(
50
µg/mL) against MTB H Rv and MDR-TB strains. Comparing to the first-line
3
7
anti-tuberculosis agents rifampicin and isoniazid, hybrid 8f has shown over two
magnitude more active against MDR-TB. The hybrid 8f was further evaluated for its
metabolic stability and in vivo pharmacokinetic profiles, with the results showing that
hybrid 8f exhibited lower metabolic stability compared to inhibitor TAM16. Further
modification based on hybrid 8f is needed to improve the metabolic stability and
pharmacokinetic profiles.
Keywords: benzofuran-isatin hybrids; anti-mycobacterial; anti-tubercular; multi-drug
resistant; structure-activity relationship
1
. Introduction
Tuberculosis (TB) is an infectious disease predominantly caused by the bacterium
Mycobacterium tuberculosis (MTB), which mainly makes the lung infection [1].
*
Corresponding author: cjr.luguangming@vip.163.com

ACCEPTED MANUSCRIPT
According to the reports, TB has been the ninth leading cause of death around the
world, ranking above HIV/AIDS [1,2]. The latest global TB report from the World
Health Organization (WHO) showed that approximately one-third of the population
worldwide infected MTB, with around 10.4 million incident cases and 1.67 million
deaths in the year 2016[1]. Drug-resistant TB (DR-TB), especially multi-drug
resistant TB (MDR-TB), is a persistent problem for the treatment of the global TB
epidemic [3,4]. It is estimated 600,000 incident cases of the first-line anti-TB agent
rifampicin (RIF)-resistant TB happened in 2016, of which 490,000 cases were
MDR-TB [1]. Therefore, it is urgent to develop novel agents to treat both
drug-susceptible and MDR-TB.
Figure 1 Schematic showing the design strategy for the novel benzofuran-isatin
hybrids
Benzofuran-based derivatives have been attracted great interest in medicinal
chemistry, due to their various biological properties, such as antibacterial [5,6],
anticancer [7], antimalarial [8] and anti-TB activities [9-13]. The benzofuran-based
lead compound TAM16 (Figure 1) showed therapeutic potential in MDR-TB patients.
TAM16 could inhibit the C-terminal thioesterase domain activity of polyketide
synthase 13, and display great activity against 38 clinical MTB strains, including
MDR-TB in vitro. Notably, resistant mutants emerged at frequencies two magnitude
lower than that for the first-line anti-TB agent isoniazid (INH) [13]. In vivo, TAM16
also exhibited excellent physiochemical, toxicological and pharmacological properties,
with the similar treatment efficiency as INH in multiple TB infected mouse models.

ACCEPTED MANUSCRIPT
Moreover, the combined application of TAM16 and RIF in the chronic infection
model was as effective as the gold-standard combination of INH and RIF at reducing
the bacterial load, but more active than RIF alone. Meanwhile, isatin
(
indoline-2,3-dione) derivatives (Figure 1) has shown potential anti-TB activities
according to the previous reports [14-18]. In this case, the combination of TAM16 and
isatin together lead to novel hybrids that may possess higher anti-TB activity than
themselves alone. Herein we report the design and synthesis of various novel
triazole-tethered benzofuran-isatin hybrids, in vitro evaluation of anti-mycobacterial
activities, and cytotoxicity test in VERO cells as well. In this study, both
drug-sensitive and drug-resistant MTB strains are expected to be effectively treated
with the novel hybrids. Preliminary structure-activity relationship (SAR) studies are
also performed to facilitate the further development of these hybrids.
2
Results and discussion
The desired benzofuran-isatin hybrids 7a-c and 8a-f were synthesized following the
methods described in Scheme 1. Isatin/5-methoxyisatin/5-bromoisatin alkylated with
1
,2-dibromoethane, producing the N-(2-bromoethyl)isatin derivatives 2a-c with yields
of 49-77% [15,16]. Subsequently, treatment of N-(2-bromoethyl)isatins 2a-c with
sodium azide at 60 °C provided the desired azido precursors 3a-c (yield: 57-78%).
The benzofuran intermediate 5 was synthesized via copper-catalyzed cyclization of
ethyl 3-(4-methoxyphenyl)-3-oxopropanoate 4 and benzoquinone, and the propargyl
benzofuran intermediate 6 was obtained by treatment of intermediate 5 with propargyl
bromide according to the method reported in the literature [12, 15]. The precursors
3
a-c and 6 were utilized for the synthesis of desired 1H-1,2,3-triazole-tethered
benzofuran-isatin hybrids 7a-c via copper-promoted azide-alkyne cycloaddition
reaction in the presence of Cu(OAc) (yield: 38-47%) [15]. Finally, condensation of
2
7
a-c with the corresponding amine hydrochlorides in the presence of sodium
bicarbonate yielded target compounds 8a-f (21-72%)

ACCEPTED MANUSCRIPT
O
O
O
Br
R1
R1
R1
Br
NaN3
O
O
O
N
N
N
K2CO3, DMF
H
N3
1
a-c
2a-c
2a: R1 = Br;
Br
3
1
1
1
a: R1 = Br;
b: R1 = OMe;
c: R1 = H.
3a: R1 = Br;
3b: R1 = OMe;
3c: R1 = H.
2b: R1 = OMe;
2c: R1 = H.
Cu(OAc) , DMF
2
O
O
O
O
O
OEt
OEt
Br
O
HO
O
OEt
O
O
O
O
Cu(OTf)2, Tol.
O
K2CO3, DMF
6
5
4
R1
R1
O
O
N
N
N
N
OEt
N
N
N
OEt
RONH2•HCl
NaHCO3
N
O
O
R2
O
O
O
O
O
O
O
7
a-c
8
a-f
7
7
7
a: R1 = Br;
b: R1 = OMe;
c: R1 = H.
8
8
8
8
8
8
a: R1 = Br, R2 = NOH;
b: R1 = Br, R2 = NOEt;
c: R1 = H, R2 = NOMe;
d: R1 = OMe, R2 = NOH;
e: R1 = OMe, R2 = NOMe;
f: R1 = OMe, R2 = NNHCSNH2.
Scheme 1 Synthesis of benzofuran-isatin hybrids 7a-c and 8a-f
The anti-mycobacterial activities of benzofuran-isatin hybrids 7a,b and 8a-f against
MTB H Rv and MDR-TB (resistant to ethambutol, INH and RIF) strains were
3
7
investigated. The minimum inhibitory concentration (MIC) is defined as the lowest
concentration that inhibits the visible bacterial growth. The MIC values for the
synthesized hybrids, together with TAM16, RIF and INH, were measured (Table 1).
All hybrids exhibited promising anti-mycobacterial activities against MTB H Rv and
3
7
MDR-TB strains, with MIC ranging from 0.25 to 8 µg/mL. However, all hybrids were
less active than the parent compound TAM16 (MIC: <0.06 µg/mL), indicating that
benzofuran backbone is crucial for the binding with polyketide synthase 13.
The SAR results indicated that substitutents on both C-3 and C-5 position of isatin
moiety significantly influenced their anti-mycobacterial activities. Hybrids with
electron-donating -OMe group at C-5 position were found more active than the
corresponding unsubstituted or -Br substituted hybrids. At C-3 position,
hydrogen-bond donor such as hydrazinecarbothioamide or oximine could also enhance
the anti-mycobacterial activity. The relative contribution of the substituents at C-3

ACCEPTED MANUSCRIPT
position to the activity in the hybrids was: NNHCSNH > NOH > NOMe > NOEt >
2
O.
Among the synthesized benzofuran-isatin hybrids, compound 8f (MICMTB H37Rv: 0.25
µg/mL and MICMDR-TB: 0.5 µg/mL) was the most active against MTB H Rv and
37
MDR-TB strains, with 128 and over 256 folds more effective than the RIF (MIC: 64
µg/mL) and INH (MIC: >128 µg/mL) against MDR-TB, and 2 times more active than
RIF (MIC: 0.5 µg/mL) against MTB H Rv. It is worth mentioning that the values of
3
7
the resistance index (RI, MIC(MDR-TB)/MIC(MTB H37Rv)) for the benzofuran-isatin
hybrids were around 1, suggesting that these hybrids may have novel mechanism of
action against TB.
Table 1 Structure, anti-mycobacterial activity and cytotoxicity of benzofuran-isatin
hybrids 7a,b and 8a-f
MIC (µg/mL)
a
Compounds
R1
R2
CC (µΜ)
SI
50
MTB H Rv
MDR-TB
3
7
7
7
8
8
a
b
a
b
Br
OMe
Br
O
4
2
8
2
512
128
128
64
O
NOH
NOEt
NOMe
NOH
NOMe
1
0.5
4
512
512
Br
2
256
128
8
c
H
1
2
>1,024
>1,024
>1,024
>1,024
>2,048
>1,024
8
d
OMe
OMe
0.5
1
1
8
e
0.5

ACCEPTED MANUSCRIPT
8
f
OMe
NNHCSNH
2
0.25
<0.06
0.06
0.5
0.5
<0.06
>128
64
>1,024
>1,024
256
>4,096
>16,384
4,096
TAM16
INH
-
-
-
-
-
-
RIF
512
1,024
a
SI: selectivity index, CC /MIC
MTB H37Rv
5
0
Cytotoxicity (CC ) of hybrids 7a,b and 8a-f was sequentially tested in VERO cells,
5
0
(green monkey kidney epithelial cells) [16]. All hybrids displayed excellent
toxicological profiles with CC50 ranging from 128 to >1,024 µg/mL. The
structure-cytotoxicity relationship study indicated that substitution of isatin moiety on
C-3 position for hybrids 8a-f generally reduced the cytotoxicity compared to the
corresponding ketones, 7a,b. Interestingly, the most active hybrid 8f (CC : >1,024
50
µg/mL) showed no discernible cytotoxicity against VERO cells.
To evaluate the selectivity profiles of these hybrids, the selectivity index (SI) values
from the ratio of CC /MIC were calculated (Table 1). All benzofuran-isatin
5
0
MTB H37Rv
hybrids possessed moderate to high SI values, and half of the derivatives have SI values
even greater than 1,024. The SI of the most active hybrid 8f was over 4,096, higher than
RIF and INH (SI values: 1,024 and 4,096, respectively), suggesting that hybrid 8f
holds a profile of excellent selectivity.
Table 2 The metabolic stability and in vivo pharmacokinetic values of hybrid 8f and
TAM16 in mice
Pharmacokinetics (p.o.)
Compd.
Metabolic stability
(
t , min)
1
/2
AUC0-inf
ng•h/mL)
Cmax (ng/mL)
t1/2 (h)
Tmax (h)
(
8
f
28.9
>60
389
4.26
9.88
1.24
3.46
2,564
TAM16
5,064
39,329
t : half-life;
1/2
Cmax: the peak concentration;

ACCEPTED MANUSCRIPT
Tmax: the time to reach peak concentration;
AUC: the area under the curve.
Hybrid 8f was selected for the further investigation on metabolic stability and in vivo
pharmacokinetics in mice (25 mg/kg, single oral administration) due to its excellent
selectivity. The results were listed in Table 2. Comparing to TAM16 (t : over 60
1
/2
min), hybrid 8f showed much lower metabolic stability (t : 28.9 min).
1
/2
Pharmacokinetic investigation displayed that hybrid 8f was inferior to TAM16 in
term of Cmax (389 ng/mL vs 5,064 ng/mL), t (4.26 h vs 9.88 h), T (1.24 h vs 3.46
1/2
max
h) and AUC0-inf (2,564 ng•h/mL vs 39,329 ng•h/mL). Therefore, further modification
based on hybrid 8f is needed to improve its metabolic stability and pharmacokinetic
profiles.
3
Conclusions
In summary, a series of novel triazole-tethered benzofuran-isatin hybrids were
designed and prepared via concise organic synthesis. All synthetic benzofuran-isatin
compounds exhibited decent in vitro anti-mycobacterial activities against both
susceptible and multidrug-resistant MTB strains. These hybrids showed minimal
cytotoxicity, and the most active hybrid 8f had comparable anti-mycobacterial activity
to RIF and INH against both drug-sensitive MTB H Rv and MDR-TB strains.
3
7
However, both the metabolic stability and pharmacokinetic profiles for 8f could be
improved through the further modification of its functional groups.
4
4
. Experimental section
.1. Synthesis
N-(2-azidoethyl)isatins
3a-c
and
(1.0
ethyl
were
2
-(4-methoxyphenyl)-5-(prop-2-yn-1-yloxy)benzofuran-3-carboxylate
6
prepared via the methods reported in the literature [13, 18].
A
mixture of N-(2-azidoethyl)isatins 3a-c
mmol),
ethyl
2
-(4-methoxyphenyl)-5-(prop-2-yn-1-yloxy)benzofuran-3-carboxylate 6 (1.0 mmol)

ACCEPTED MANUSCRIPT
and Cu(OAc) (100 mg) in DMF (50 mL) was stirred at room temperature for 48 h
2
under N . After filtration, the filtrate was concentrated under reduced pressure. The
2
residue was purified by silica gel column chromatography with PE: EA=3:1 to give
benzofuran-isatin hybrids 7a-c.
To 7a-c (1 mmol), sodium bicarbonate (2 mmol), hydroxylamine/
methoxyamine/ethoxyamine hydrochloride (1.8 mmol), water (10 mL) and
o
tetrahydrofuran (THF, 30 mL) were added and the mixture was stirred at 40 C for 12 h.
After cooling to room temperature, the mixture was extracted with EA (20 mL×3). The
organic phases were washed with water (50 mL×2) and brine (50 mL), and then dried
with Na SO . After filtration, the filtrate was concentrated under reduced pressure to
2
4
give a residue, which was further purified by silica gel chromatography with PE:
EA=3:1 to give benzofuran-isatin hybrids 8a-f.
4
.1.1.
ethyl
5
-((1-(2-(5-bromo-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-(4-
methoxyphenyl)-3a,7a-dihydrobenzofuran-3-carboxylate (7a)
1
Yellow solid, yield: 38%. H NMR (400 MHz, DMSO-d ) δ 1.37 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.88 (3H, s, OMe), 4.17 (2H, s, -CH -), 4.36 (2H, q, J = 5.6 Hz,
2
2
3
2
CO CH CH ), 4.69 (2H, t, J = 1.2 Hz, -CH -), 5.22 (2H, t, J = 1.2 Hz, -CH -), 6.84 (1H,
2
2
3
2
2
d, J = 6.4 Hz, Ar-H), 7.06 (1H, d, J = 7.2 Hz, Ar-H), 7.12 (2H, d, J = 7.2 Hz, Ar-H),
1
3
7
.58-7.71 (4H, m, Ar-H), 8.00 (1H, d, J = 7.2 Hz, Ar-H), 8.34 (1H, s, triazole-H). C
NMR (101 MHz, DMSO-d ) δ 163.612, 162.715, 161.461, 157.336, 155.592, 148.452,
6
1
1
5
43.663, 143.321, 133.237, 131.917, 131.414, 127.929, 125.515, 123.219, 121.680,
15.307, 114.505, 112.318, 109.487, 107.776, 106.485, 64.947, 62.191, 60.856,
+
+
5.876, 47.469, 14.520. ESI-MS m/z: 667 [M+Na] , 669 [M+2+Na] .
4
5
4
.1.2.
ethyl
-((1-(2-(5-methoxy-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-(
-methoxyphenyl)benzofuran-3-carboxylate (7b)
1
Yellow solid, yield: 47%. H NMR (400 MHz, DMSO-d ) δ 1.36 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.75 (3H, s, OMe), 3.88 (3H, s, OMe), 4.15 (2H, s, -CH -), 4.36 (2H, q,
2
2
3
2
J = 6.0 Hz, CO CH CH ), 4.69 (2H, t, J = 1.2 Hz, -CH -), 5.18 (2H, t, J = 1.2 Hz,
2
2
3
2

ACCEPTED MANUSCRIPT
-
CH -), 6.86 (1H, d, J = 7.2 Hz, Ar-H), 7.05 (1H, d, J = 6.8 Hz, Ar-H), 7.12-7.13 (4H,
2
m, Ar-H), 7.57 (1H, s, Ar-H), 7.61 (1H, d, J = 6.8 Hz, Ar-H), 8.01 (1H, d, J = 6.8 Hz,
+
+
Ar-H), 8.34 (1H, s, triazole-H). ESI-MS m/z: 597 [M+H] , 619 [M+Na] .
4
5
.1.3.
ethyl
-((1-(2-(5-bromo-3-(hydroxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-(4-methoxyphenyl)benzofuran-3-carboxylate (8a)
1
Yellow solid, yield: 54%. H NMR (400 MHz, DMSO-d ) δ 1.36 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.88 (3H, s, OMe), 4.20 (2H, s, -CH -), 4.36 (2H, q, J = 5.6 Hz,
2
2
3
2
CO CH CH ), 4.70 (2H, t, J = 1.2 Hz, -CH -), 5.18 (2H, t, J = 1.2 Hz, -CH -), 6.81 (1H,
2
2
3
2
2
d, J = 6.8 Hz, Ar-H), 7.06 (1H, d, J = 7.2 Hz, Ar-H), 7.12 (2H, d, J = 6.8 Hz, Ar-H),
.44 (1H, d, J = 6.8 Hz, Ar-H), 7.58-7.62 (2H, m, Ar-H), 8.00 (1H, d, J = 7.2 Hz,
7
1
3
Ar-H), 8.06 (1H, s, Ar-H), 8.31 (1H, s, triazole-H), 13.78 (1H, s, NOH). C NMR (101
MHz, DMSO-d ) δ 183.627, 163.619, 161.461, 158.661, 156.146, 155.606, 148.454,
6
1
1
5
44.524, 143.379, 131.414, 127.929, 125.719, 124.436, 121.680, 118.275. 114.484,
14.192, 112.311, 111.830, 109.511, 107.768, 106.434, 62.191, 60.856, 56.306,
+
+
5.876, 47.440, 14.513. ESI-MS m/z: 660 [M+H] , 662 [M+2+H] .
4
5
.1.4.
ethyl
-((1-(2-(5-bromo-3-(ethoxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)me
thoxy)-2-(4-methoxyphenyl)benzofuran-3-carboxylate (8b)
1
Yellow solid, yield: 49%. H NMR (400 MHz, DMSO-d ) δ 1.36-1.41 (6H, m,
6
NOCH CH and CO CH CH ), 3.89 (3H, s, OMe), 4.16-4.20 (2H, m, -CH -), 4.35-4.51
2
3
2
2
3
2
(
4H, m, NOCH CH and CO CH CH ), 4.70 (2H, t, J = 1.2 Hz, -CH -), 5.16 (2H, t, J =
2 3 2 2 3 2
1
.2 Hz, -CH -), 6.82 (1H, d, J = 7.2 Hz, Ar-H), 7.06 (1H, d, J = 7.2 Hz, Ar-H), 7.13
2
(2H, d, J = 6.8 Hz, Ar-H), 7.44-7.48 (1H, m, Ar-H), 7.51-7.57 (2H, m, Ar-H), 7.85 (1H,
d, J = 7.2 Hz, Ar-H), 8.00 (2H, d, J = 7.2 Hz, Ar-H), 8.30 (1H, s, triazole-H). ESI-MS
+
+
m/z: 710 [M+H] , 712 [M+2+H] .
4
5
2
.1.5.
ethyl
-((1-(2-(3-(methoxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
-(4-methoxyphenyl)benzofuran-3-carboxylate (8c)
1
Yellow solid, yield: 72%. H NMR (400 MHz, DMSO-d ) δ 1.37 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.85 (3H, s, OMe), 4.15-4.22 (5H, m, -CH - and NOMe), 4.36 (2H, q, J
2
2
3
2

ACCEPTED MANUSCRIPT
=
5.6 Hz, CO CH CH ), 4.70 (2H, t, J = 1.6 Hz, -CH -), 5.17 (2H, t, J = 1.2 Hz, -CH -),
2
2
3
2
2
6
.92 (1H, d, J = 6.8 Hz, Ar-H), 7.06-7.14 (4H, m, Ar-H), 7.35-7.38 (1H, m, Ar-H), 7.56
(1H, s, Ar-H), 7.62 (1H, d, J = 6.8 Hz, Ar-H), 7.88 (1H, d, J = 6.0 Hz, Ar-H), 8.01 (2H,
1
3
d, J = 6.8 Hz, Ar-H), 8.30 (1H, s, triazole-H). C NMR (101 MHz, DMSO-d ) δ
6
+
ESI-MS m/z: 618 [M+Na] .
4
.1.6.
ethyl
5
-((1-(2-(3-(hydroxyimino)-5-methoxy-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl
)methoxy)-2-(4-methoxyphenyl)benzofuran-3-carboxylate (8d)
1
Yellow solid, yield: 53%. H NMR (400 MHz, DMSO-d ) δ 1.36 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.73 (3H, s, OMe), 3.88 (3H, s, OMe), 4.18 (2H, s, -CH -), 4.36 (2H, q,
2
2
3
2
J = 5.6 Hz, CO CH CH ), 4.69 (2H, t, J = 1.2 Hz, -CH -), 5.18 (2H, t, J = 1.2 Hz,
2
2
3
2
-
CH -), 6.81 (1H, d, J = 6.8 Hz, Ar-H), 6.88 (1H, d, J = 6.8 Hz, Ar-H), 7.06 (1H, d, J =
2
7
.2 Hz, Ar-H), 7.12 (2H, d, J = 6.8 Hz, Ar-H), 7.58-7.62 (3H, m, Ar-H), 8.00 (2H, d, J
1
3
=
7.2 Hz, Ar-H), 8.30 (1H, s, triazole-H). C NMR (101 MHz, DMSO-d ) δ 163.612,
6
1
1
1
1
62.599, 161.454, 161.345, 156.664, 155.606, 148.446, 143.321, 137.283, 131.414,
27.921, 125.501, 121.695, 118.049, 115.847, 114.498, 114.185, 114.024, 112.303,
10.116, 107.768, 106.463, 65.020, 62.205, 60.858, 56.139, 56.037, 55.876, 47.484,
+
4.513. ESI-MS m/z: 634 [M+Na] .
4
.1.7.
ethyl
5
-((1-(2-(5-methoxy-3-(methoxyimino)-2-oxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-y
l)methoxy)-2-(4-methoxyphenyl)benzofuran-3-carboxylate (8e)
1
Yellow solid, yield: 57%. H NMR (400 MHz, DMSO-d ) δ 1.37 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.74 (3H, s, OMe), 3.88 (3H, s, OMe), 4.15-4.22 (5H, m, -CH - and
2
2
3
2
NOMe), 4.35 (2H, q, J = 5.6 Hz, CO CH CH ), 4.68 (2H, t, J = 4.0 Hz, -CH -), 5.18
2
2
3
2
(
2H, t, J = 1.2 Hz, -CH -), 6.84-6.96 (2H, m, Ar-H), 7.06 (1H, d, J = 7.2 Hz, Ar-H),
2
7
7
6
.13 (2H, d, J = 7.2 Hz, Ar-H), 7.45 (1H, s, Ar-H), 7.57 (1H, s, Ar-H), 7.62 (1H, d, J =
.2 Hz, Ar-H), 8.01 (2H, d, J = 7.2 Hz, Ar-H), 8.29 (1H, s, triazole-H). ESI-MS m/z:
+
48 [M+H] .
4
5
3
.1.8.
ethyl
-((1-(2-(3-(2-carbamothioylhydrazono)-5-methoxy-2-oxoindolin-1-yl)ethyl)-1H-1,2,
-triazol-4-yl)methoxy)-2-(4-methoxyphenyl)benzofuran-3-carboxylate (8f)

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 21%. H NMR (400 MHz, DMSO-d ) δ 1.36 (3H, t, J = 5.6 Hz,
6
CO CH CH ), 3.76 (3H, s, OMe), 3.88 (3H, s, OMe), 4.21 (2H, s, -CH -), 4.36 (2H, q,
2
2
3
2
J = 5.6 Hz, CO CH CH ), 4.72 (2H, t, J = 1.2 Hz, -CH -), 5.16 (2H, t, J = 1.2 Hz,
2
2
3
2
-
CH -), 6.87 (2H, s, Ar-H), 7.04 (1H, d, J = 7.6 Hz, Ar-H), 7.12 (2H, d, J = 6.8 Hz,
2
Ar-H), 7.42 (1H, s, Ar-H), 7.56-7.65 (2H, m, Ar-H), 7.99 (2H, d, J = 6.4 Hz, Ar-H),
.32 (1H, s, triazole-H), 8.74, 9.16 (2H, s, NNHCSNH ), 12.23 (1H, s, NNHCSNH ).
8
2
2
+
ESI-MS m/z: 692 [M+Na] .
4
.2. MIC determination
The in vitro activities of benzofuran-isatin derivatives 7a,b and 8a-f, along with the
references TAM16, RIF and INH against MTB H37Rv and MDR-TB were evaluated
via rapid direct susceptibility test technique [19,20]. The wells of a sterile 48-well plate
were filled with 100 mL two-fold diluted tested compounds and 100 mL MTB H Rv
3
7
-3
(
or MDR-TB) suspension containing 4×10 mg cells. The plates were incubated at
3
7 °C in a wet box. The MIC was determined by observing the quantity and state of the
cells in each test well by a continuous visual high magnification system, and
re-determined 7 days later.
4
.3. Cytotoxicity
The cytotoxicity (CC ) of the synthesized benzofuran-isatin hybrids 7a,b and 8a-f,
5
0
together with the references TAM16, RIF and INH were examined by the
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay in a
3
mammalian VERO cells [21,22]. The compounds were dissolved in DMSO with
concentrations from 1024 to 1 µg/mL. The VERO cells were maintained in culture
o
medium at 37 C under 5% CO atmosphere. Cells were seeded in 96-well plates
2
4
(
1×10 cell per well) and allowed to recover for 24 h. After 72 h of exposure, cells were
harvested and cell viability was assessed by MTT assay. The CC50 values were
calculated by Bliss analyses.
4
.4 Pharmacokinetic Profiles determination

ACCEPTED MANUSCRIPT
SPF female ICR mice (20-25 g) were used in the pharmacokinetic study, and each
treatment group had 3 mice which were dosed with hybrid 8f or TAM16 suspension at
2
5 mg/kg (oral administration/p.o.) [23]. Compounds were suspended in 0.5% CMC
for p.o., and blood was collected from the jugular vein of each mouse at the following
time points: 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after oral administration. Total area under
the concentration time curve (AUC), the elimination half-time (t ), the peak
1
/2
concentration (Cmax) and the time to reach peak concentration (Tmax) of samples were
determined directly from the experimental data using WinNonlin V6.2.1.
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899-904

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A series of novel benzofuran-isatin hybrids were assessed for their in vitro anti-mycobacterial
activities.
All hybrids exhibited considerable anti-mycobacterial activities.
The activity of hybrid 8f was no inferior to RIF and INH, but the metabolic stability and
pharmacokinetic profiles still waits for optimization.