Open Journal of Medicinal Chemistry, 2013, 3, 128-135
Published Online December 2013 (http://www.scirp.org/journal/ojmc)
http://dx.doi.org/10.4236/ojmc.2013.34015
Open Access OJMC
Synthesis and Evaluation of
2-Amino-4H-Pyran-3-Carbonitrile
Derivatives as Antitubercular Agents
Chunxia Chen1*, Minghui Lu2*, Zhihui Liu3, Junting Wan2, Zhengchao Tu2,
Tianyu Zhang2#, Ming Yan1#
1Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences,
Sun Yat-sen University, Guangzhou, China
2State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Science, Guangzhou, China
3Guangzhou Chest Hospital, Guangzhou, China
Received March 2, 2013; revised April 2, 2013; accepted April 9, 2013
Copyright © 2013 Chunxia Chen et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
A series of 2-amino-4H-pyran-3-carbonitrile derivatives were designed and synthesized. Their antitubercular activities
were evaluated against autoluminescent M. tuberculosis H37Ra and standard strain M. tuberculosis H37Rv. No obvious
antitubercular activities could be observed (MIC > 10 g/mL). The results are in sharp contrast with the previously re-
ported data.
Keywords: 2-Amino-4H-Pyran-3-Carbonitrile; Synthesis; Antitubercular Activity
1. Introduction
Tuberculosis (TB) is a chronic disease caused by My-
cobacterium tuberculosis. It continues to be a serious
threat for human health [1,2]. Every year about two mil-
lion people die of this disease and almost eight million
people get tuberculosis. The present antitubercular treat-
ment typically requires the combination of at least two
first-line drugs (rifampicin, isoniazid, ethambutol and
pyrazinaimde) for an extended period (6 - 12 months).
The poor compliance to the rigid implementation of the-
rapy leads to the emergence of multi-drug-resistant (MDR)
and extensively drug-resistant (XDR) strains of Myco-
bacterium tuberculosis, which have brought new chal-
lenges for clinical treatment [3-6]. In addition, the rising
incidence of TB and HIV co-infection makes the treat-
ment more difficult.
The development of antitubercular agents with new
action mechanism is an urgent task [7,8]. In recent ten
years, a number of candidates have appeared with prom-
ising activities against sensitive and resistant Mycobacte-
rium tuberculosis strains. In 2007, Perumal and co-
workers reported 2-aminopyranopyridine-3-carbonitriles
as a new type of antitubercular agents (Scheme 1) [9].
Several compounds showed excellent antitubercular ac-
tivity comparable with isoniazid. Recently Perumal, Sri-
ram and co-workers also found that 1,2,4-oxadiazoles de-
rived from 2-aminopyranopyridine-3-carbonitriles show-
ed enhanced antitubercular activity (Scheme 1) [10]. These
results strongly suggest that 2-amino-4H-pyran-3-carbo-
nitrile is a new pharmacophore of antitubercular agents.
Recently we have developed efficient methods for the
synthesis of homochiral 2-amino-4H-pyran-3-carboni-
triles [11,12]. We are interested in the effect of chiral
center of these compounds on the antitubercular activity.
We are also interested in the further improvement of the
antitubercular activity of 2-amino-4H-pyran-3-carboni-
triles by structural modifications. In this paper, we report
the synthesis of racemic and homochiral 2-amino-pyra-
nopyridine-3-carbonitriles as well as their structural ana-
logs. The antitubercular activity was evaluated in vitro
against autoluminescent Mycobacterium tuberculosis
H37Ra and standard strain Mycobacterium tuberculosis
H37Rv.
*These authors contribute equally to this study.
#Corresponding authors.

C. X. CHEN ET AL.
Open Access OJMC
129
Ar
O
NH
2
CN
Ar
N
Ar = 4-Cl-C
6
H
4
, MIC0.92 (TB), 1.84 (MDRTB)
Ar = 4-F-C
6
H
4
, MIC 0.97 (TB),0.97 (MDRTB)
Ar = 4-Me
2
N-C
6
H
4
, MIC0.43 (TB),0.43 (MDRTB)
Ar
O
NH
2
Ar
N
Ar = 4-Cl-C
6
H
4
-, MIC 0.35(TB)
Ar= 2,4-diCl-C
6
H
3
, MIC 0.31 (TB)
N
N
OCl
Scheme 1. 2-Amino-4H-pyran-3-carbonitrile and their derivatives with potent antitubercular activity.
2. Results and Discussion
2.1. Chemistry
Racemic 2-aminopyranopyridine-3-carbonitriles 2a-2g were
prepared from dienones 1a-1g and malononitrile in the
presence of piperidine. Generally excellent yields (92% -
99%) were obtained (Scheme 2).
To explore the effect of chiral centers in 2a-2g, ho-
mochiral (S)-2a, (S)-2d, (R)-2a, and (R)-2d were pre-
pared. Excellent yields and enantioselectivities were achiev-
ed using chiral thiourea-tertiary amines 3a and 3b as the
catalysts (Scheme 3) [11].
2-Aminopyranopyridine-3-carbonitriles 2h-2n derived
from monoenones 1h-1n were also prepared (Scheme 4)
[12]. Cyclic enones 1h-1j reacted with malononitrile in
the presence of triethylamine. The reaction of acyclic
enones 1k-1n was achieved using piperidine as the cata-
lyst. Generally products 2h-2n were obtained in good
yields.
For a further understanding the effect of C (sp3) chiral
structure in 2-aminopyranopyridine-3-carbonitriles, the
compounds 4a and 4j with achiral pyridine structure
were designed. The treatment of 2-amino-pyran 2a and
2j with ammonium acetate provided 4a and 4j in good
yields (Scheme 5) [13].
2.2. Evaluation of Antitubercular Activity
The antitubercular activity of racemic 2-amino-4H-pyran-
3-carbonitriles 2a-2e, homochiral 2-amino-4H-pyran-3-
carbonitriles (S)-2a, (S)-2d, (R)-2a, and (R)-2d were eva-
luated against autoluminescent M. tuberculosis H37Ra
[14]. This screen model is fast and cost-efficient for the
preliminary evaluation of antitubercular activity. Isoni-
azid and rifampicin were used as the positive control and
the results are listed in Figure 1. The bacteria growth
was conveniently monitored by the bioluminescence in-
tensity. Unexpectedly all compounds including 2a and
2d did not showed obvious antitubercualr activity.
We further examined the inhibitive activity of the
compounds against standard strain M. tuberculosis H37Rv
and the results are summarized in Table 1. Perumal and
co-workers reported that compound 2a and 2d possess
excellent antitubercular activities (MIC 0.97 and 0.92
g/mL against H37Rv respectively) [9]. Our present
study led to significantly different results. Racemic 2a,
2d and their homochiral enantiomers did not show obvi-
ous antitubercular activities (MIC > 10 g/mL). Other
structural analogs 2b, 2c, 2e-2g also appeared to be in-
efficient. The compounds 2h-2n and 4a, 4j with further
structural diversities still showed disappointed antituber-
cular activities. These results brought the question about
the reported antitubercular activity of 2-amino-4H-pyran-
3-carbonitriles by Perumal and co-workers. Although a
clear conclusion could not be achieved so far, the further
examination of the reported data is highly desirable.
3. Conclusion
In conclusion, we designed and synthesized a series of 2-
amino-4H-pyran-3-carbonitriles and their structural ana-
logs. Homochiral 2-amino-4H-pyran-3-carbonitriles were
also prepared via the organocatalytic enantioselctive re-
action. The antitubercualr activities of these compounds
were determined against autoluminescent M. tuberculosis
H37Ra and standard strain M. tuberculosis H37Rv, how-
ever, no obvious inhibitive activities could be observed.
The results are in sharp contrast with the previously re-
ported data. Before the further attempt to develop 2-
amino-4H-pyran-3-carbonitriles as potential antituber-
cular agents, the clarification of the contradictive activity
data is required.
4. Experimentals
1H and 13C NMR spectra were recorded on a Bruker Ad-
vance 400 MHz spectrometer as solutions in CDCl3.
Chemical shifts in 1H NMR spectra are reported in parts
per million (ppm, δ) downfield from the internal standard
Me4Si (TMS, δ = 0 ppm). Chemical shifts in 13C NMR
spectra are reported relative to the central line of the
chloroform signal (δ = 77.0 ppm). The following abbre-
viations are used to designate chemical shift mutiplicities:
s = singlet, d = doublet, m = multiplet. High-resolution
mass spectra were obtained with Shimadazu LCMS-IT-
TOF mass spectrometer. Infrared (IR) spectra were re-
corded on a Bruker Tensor 37 spectrophotometer. Data
are represented as follows: frequency of absorption
(cm−1), intensity of absorption (s = strong, m = medium,
w = weak). The flash column chromatography was car-

C. X. CHEN ET AL.
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130
Ar
O
Ar NCCN piperdine
EtOH, rtAr
O
NH
2
CN
Ar
1a-1g 2a-2g
XX
92-99% yields
2a:Ar=4-F-C
6
H
4
,X=CH
3
N; 2b:Ar=3-F-C
6
H
4
,X=CH
3
N;
2c: Ar = 3,4-diF-C
6
H
3
,X=CH
3
N; 2d:Ar=4-Cl-C
6
H
4
,X=CH
3
N;
2e:Ar=4-F-C
6
H
4
,X=O;2f:Ar=4-F-C
6
H
4
,X=S;2g:Ar=4-F-C
6
H
4
,X=CH
2
Scheme 2. Synthesis of 2-amino-4H-pyran-3-carbonitriles 2a-2g.
Ar
O
Ar NC CN
3a or 3b
toluene, rt
(10 mol%)Ar
O
NH
2
CN
Ar
1a,1d (R)-2a,2dand (S)-2a, 2d
CF
3
F
3
C N
HN H
S
N
CF
3
F
3
C N
HN H
S
N
3a 3b
NN
Me Me
94-96% yields
98-99% ee
Scheme 3. Synthesis of homochiral 2-amino-4H-pyran-3-carbonitriles.
X
O
FNC CN
X
O
F
NH
2
CN
triethylamine
ethanol, rt
1h-1j
2h-2j
75-78% yields
2h:X=CH
2
;2i:X= O;2j:X=S
O
NH
2
CN
R
1
R
2
R
3
R
1
O
R
2
R
3
NC CN piperdine
ethanol, rt
1k-1n 2k-2n
78-86% yields
2k:R
1
=Ph,R
2
=CN,R
3
=4-F-C
6
H
4
;2l:R
1
=Me,R
2
=Ph,R
3
=Ph;
2m:
R
1
=Ph,
R
2
=Ph,
R
3
=4-Cl- C
6
H
4
;2n:
R
1
=Ph,
R
2
=Ph,
R
3
=2-thiophen
y
l
Scheme 4. Synthesis of 2-amino-4H-pyran-3-carbonitriles 2h-2n derived from monoenones.
ried out over silica gel (230 - 400 mesh), purchased from
Qingdao Haiyang Chemical Co. Ltd. Melting points were
recorded on an electrothermal digital melting point ap-
paratus and were uncorrected. TLC analysis was per-
formed on precoated silica gel GF254 slides, and visual-
ised by either UV irradiation. Unless otherwise stated, all
reagents were obtained from commercial sources and
used as received. The solvents were used as commercial
anhydrous grade without further purification. Enanti-
omeric excesses were determined by HPLC using a Dai-
cel Chiralpak AD-H column (4.6 mm × 25 cm) and
eluting with hexane/2-PrOH solution.

C. X. CHEN ET AL.
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131
N
O
NH2CN
FF EtOAC, refluxN
N
NH2CN
F F
2a 4a
S
O
NH2CN
FS
N
NH2
CN
F
2j 4j
80%yield
85% yield
NH4OAc, AcOH
EtOAC, reflux
NH4OAc, AcOH
EtOAc
EtOAc
Scheme 5. Synthesis of pyridine analogs 4a and 4j.
100
1000
10000
100000
1000000
Day0Day1 Day2 Day3
Biolum inescence intensity
(RLU/0.05mL)
I n cu b atio n ti me
compounds
DMS
O
INH
RIF
2a
R-2a
S-2a
2d
R
-2d
S-2d
2b
2c
2e
Figure 1. Inhibitive activity against autoluminescent M.
tuberculosis H37Ra, (INH = isoniazid, RIF = rifampicin. All
the compounds were tested at the concentration of 10 g/
mL).
4.1. Typical Procedure for the Synthesis of
Compounds 2a-2g
A mixture of (3E, 5E)-3,5-bis(4-fluorobenzylidene)-1-
methylpiperidin-4-one 1a (66.1 mg, 0.2 mmol), malononi-
trile (19.8 mg, 0.3 mmol) and piperidine (17.0 mg, 0.2
mmol) in ethanol (2 mL) were stirred for 12 h at room
temperature. The precipitate was filtered to provide 2a as
a white solid.
4.1.1. (E)-2 - A mi no- 8-(4-Fluorobenzylidene)- 4-
(4-Fluorophenyl)-6-Methyl-5,6,7,8-
Tetrahydro-4H-Pyrano-[3,2-c]Pyridine-3-
Carbonitrile (2a) [11]
White solid, yield 94%, mp 197˚C - 198˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.24 - 7.17 (4H, m, ArH), 7.08-
7.02 (4H, m, ArH), 6.86 (1H, s, HC=C), 4.55 (2H, s,
NH2), 4.03 (1H, s, CH), 3.52 (1H, d, J = 13.4 Hz, CH2),
3.36 (1H, d, J = 13.0 Hz, CH2), 2.95 (1H, d, J = 15.9 Hz,
CH2), 2.72 (1H, d, J = 15.4 Hz, CH2), 2.28 (3H, s, CH3).
4.1.2. (E)-2 - A mi no- 8-(3-Fluorobenzylidene)- 4-
(3-Fluorophenyl)-6-Methyl-5,6,7,8-
Tetrahydro-4H-Pyrano-[3,2-c]Pyridine-3-
Carbonitrile (2b) [9]
White solid, yield 92%, mp 169˚C - 172˚C; 1H NMR
(400 MHz, CDCl3): δCDCl3 = 7.33 (2H, d, J = 5.9 Hz,
ArH), 7.08 - 7.04 (1H, d, J = 7.6 Hz, ArH), 7.01 - 6.96
(4H, t, J = 19.2 Hz, ArH), 6.93 - 6.91 (1H, d, J = 9.6 Hz,
ArH), 6.85 (1H, s, HC=C), 4.59 (2H, s, NH2), 4.04 (1H, s,
CH), 3.55 (1H, d, J = 14.4 Hz, CH2), 3.36 (1H, d, J =
14.6 Hz, CH2), 2.97 (1H, d, J = 15.8 Hz, CH2), 2.74 (1H,
d, J = 16.0 Hz, CH2), 2.29 (3H, s, CH3).
4.1.3. (E)-2 - Amino-8-( 3,4-Difluorobenzylidene)-4-
(3,4-Difluorophenyl)-6-methyl-5,6,7,8-
Tetrahydro-4H-Pyrano[3,2-c]Pyridine-3-
Carbonitrile (2c)
White solid, yield 92%, mp 207˚C - 209˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.19 - 6.94 (6H, m, ArH), 6.80
(1H, s, HC=C), 4.63 (2H, s, NH2), 4.01 (1H, s, CH), 3.50
(1H, d, J = 13.8 Hz, CH2), 3.34 (1H, d, J = 14.0 Hz, CH2),
2.96 (1H, d, J = 16.1 Hz, CH2), 2.72 (1H, d, J = 15.8 Hz,
CH2), 2.30 (3H, s, CH3); 13C NMR (100 MHz, CDCl3): δ
= 158.77, 140.11, 139.09, 133.02, 127.70, 125.36, 123.78,
121.27, 119.07, 117.92, 117.75, 117.74, 117.60, 117.42,
117.25, 116.72, 112.66, 60.09, 55.13, 54.47, 44.90, 41.09;
IR (KBr) v/cm−1: 3484 (m), 2194 (s), 1681 (m), 1644 (m),
1595 (m), 1517 (s), 1396 (w), 1264 (w), 1110 (m), 910
(w), 785 (w); HRMS (ESI) calcd for C23H17N3OF4
+ [M +
H]+: 428.1381, found: 428.1389.
4.1.4. (E)-2 - A mi no- 8-(4-Chloro benzylidene)-4-
(4-Chlorophe-nyl)-6-Methyl-5,6,7,8-
Tetrahydro-4H-Pyrano[3,2-c]Pyridine-3-
Carbonitrile (2d) [11]
White solid, yield 96%, mp 205˚C - 207˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.35 - 7.32 (4H, m, ArH), 7.21 -

C. X. CHEN ET AL.
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Table 1. Evaluation of antitubercular activity against M.
tuberculosis H37Rv.
Compounds MIC (g/mL)
2a >10
2b >10
2c >10
2d >10
2e >10
2f >10
2g >10
(R)-2a >10
(R)-2d >10
(S)-2a >10
(S)-2d >10
2h >10
2i >10
2j >10
2k >10
2l >10
2m >10
2n >10
4a >10
4j >10
Isoniazid 0.03
Rifampicin <0.25
7.13 (4H, m, ArH), 6.84 (1H, s, HC=C), 4.57 (2H, s,
NH2), 4.02 (1H, s, CH), 3.51 (1H, d, J = 13.8 Hz, CH2),
3.35 (1H, d, J = 13.6 Hz, CH2), 2.94 (1H, d, J = 16.0 Hz,
CH2), 2.72 (1H, d, J = 16.0 Hz, CH2), 2.27(3H, s, CH3).
4.1.5. (E)2-Amino-8-(4 -F luorobenzylidene)-4-
(4-Fluorophenyl)-4,5,7,8-Tetrahydropyrano
[4,3-b]Pyran-3-Carbonitrile (2e)
While solid, yield 99%, mp 221˚C - 223˚C; 1H NMR
(400 MHz, DMSO-d6): δ = 7.44 - 7.05 (8H, m, ArH),
6.92 (1H, s, HC=C), 6.90 (2H, s, NH2), 4.59 (1H, d, J =
14.0 Hz, CH2), 4.48 (1H, d, J = 13.9 Hz, CH2), 4.17 (1H,
d, J = 15.5, CH2), 4.15 (1H, s, CH), 3.72 (1H, d, J = 15.6
Hz, CH2); 13C NMR (100 MHz, DMSO): δ = 162.50,
160.08, 159.70, 139.17, 138.07, 131.72, 131.02, 130.99,
129.42, 129.30, 126.09, 120.38, 120.15, 115.61, 115.55,
115.40, 115.34, 112.96, 65.12, 64.90, 55.77; IR (KBr)
v/cm−1: 3472 (m), 3312 (w), 2220 (s), 1685 (m), 1660
(m), 1603 (s), 1508 (s), 1414 (w), 1391 (w), 1264 (w),
1228 (s), 1155 (w), 1098 (m), 883 (w), 838 (w), 780 (w),
744 (m); HRMS (ESI) calcd for C22H16N2O2F2
+ [M +
Na]+: 401.1072, found: 401.1065.
4.1.6. 2-Amin o-8-(4-Flu orobenzyli dene)- 4-
(4-Fluorophenyl)-4,5,7,8-Tetrahydrothiopyrano
[4,3-b]Pyran-3-Carbonitrile (2f)
White solid, yield 98%, mp 205˚C - 207˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.25 - 7.21 (4H, m, ArH), 7.10 -
7.03 (4H, m, ArH), 6.93 (1H, s, HC=C), 4.53 (2H, s,
NH2), 4.03 (1H, s, CH), 3.58 (2H, q, J = 9.0 Hz, CH2),
3.04 (2H, q, J = 12.0 Hz, CH2); 13C NMR (100 MHz,
DMSO-d6): δ = 159.65, 143.42, 141.39, 135.84, 129.13,
128.74, 128.51, 127.51, 127.39, 127.16, 126.35, 124.39,
120.23, 114.04, 55.97, 43.21, 27.20, 27.15; IR (KBr)
v/cm−1: 3452 (m), 3355 (m), 2360 (s), 2192 (m), 2024
(w), 1673 (s), 1627 (w), 1599 (w), 1506 (s), 1410 (m),
1233 (m), 1126 (m), 881 (w), 850 (w); HRMS (ESI)
calcd for C22H16N2OF2S+ [M + Na]+: 417.0844, found:
417.0843.
4.1.7. (E)-2 - A mi no- 8-(4-Fluorobenzylidene)- 4-
(4-Fluorophenyl)-5,6,7,8-Tetrahydro-4H-
Chromene-3-Carbonitrile (2g) [10]
White solid, yield 99%, mp 211˚C - 213˚C: 1H NMR
(400 MHz, CDCl3): δ = 7.25 - 7.20 (4H, m, ArH), 7.06 -
7.02 (4H, m, ArH), 6.83 (1H, s, HC=C), 4.56 (2H, s,
NH2), 3.97 (1H, s, CH), 2.74 - 2.70 (1H, m, CH2), 2.60 -
2.56 (1H, m, CH2), 2.04 - 1.92 (2H, m, CH2), 1.74 - 1.52
(2H, m, CH2); HRMS (ESI) calcd for C23H18N2OF2
+ [M
+ Na]+: 399.1279, found: 399.1261..
4.2. Typical Procedure for the Synthesis of
Homochiral Compounds (S)-2a, (S)-2d,
(R)-2a, and (R)-2d
A mixture of (3E, 5E)-3,5-bis(4-fluorobenzylidene)-1-
methylpiperidin-4-one 1a (66.1 mg, 0.2 mmol), malononi-
trile (19.8 mg, 0.3 mmol) and 3a (9.0 mg, 0.02 mmol) in
toluene (2 mL) were stirred for 28 h at room temperature.
The white precipitate (S)-2a was collected by the centri-
fugalization.
4.2.1. (S,E)- 2 -Amino-8- (4 -Fluorobenz yli dene)-4-
(4-Fluorophenyl)-6-Methyl-5,6,7,8-Tetrahydro-
4H-Pyrano-[3,2-c]Pyridine-3-Carbonitrile
(S-2a) [11]
White solid, yield 94%, mp 197˚C - 198˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.24 - 7.17 (4H, m, ArH), 7.08-
7.02 (4H, m, ArH), 6.86 (1H, s, HC=C), 4.55 (2H, s,
NH2), 4.03 (1H, s, CH), 3.52 (1H, d, J = 13.4 Hz, CH2),
3.36 (1H, d, J = 13.0 Hz, CH2), 2.95 (1H, d, J = 15.9 Hz,
CH2), 2.72 (1H, d, J = 15.4 Hz, CH2), 2.28 (3H, s, CH3).
Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (i-PrOH/hexane = 30:70,

C. X. CHEN ET AL.
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133
254 nm, 0.8 mL/min), tr (major) = 10.6 min, tr (minor) =
7.5 min, 99%ee.
4.2.2. (R,E) -2 -Amino-8- (4 -Fluorobenz yli dene)-4-
(4-Fluorophenyl)-6-Methyl-5,6,7,8-Tetrahydro-
4H-Pyrano-[3,2-c]Pyridine-3-Carbonitrile
(R-2a) [11]
White solid, yield 94%, mp 197˚C - 198˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.24 - 7.17 (4H, m, ArH), 7.08 -
7.02 (4H, m, ArH), 6.86 (1H, s, HC=C), 4.55 (2H, s,
NH2), 4.03 (1H, s, CH), 3.52 (1H, d, J = 13.4 Hz, CH2),
3.36 (1H, d, J = 13.0 Hz, CH2), 2.95 (1H, d, J = 15.9 Hz,
CH2), 2.72 (1H, d, J = 15.4 Hz, CH2), 2.28 (3H, s, CH3).
Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (i-PrOH/hexane = 30:70,
254 nm, 0.8 mL/min), tr(major) = 7.5 min, tr (minor) =
10.6 min. 98%ee.
4.2.3. (S,E)-2- Amino-8- (4-Chlorob enzylidene)-4-
(4-Chlorophe-nyl)-6-Methyl-5,6,7,8-
Tetrahydro-4H-Pyrano[3,2-c]Pyridine-3-
Carbonitrile (S-2d) [11]
White solid, yield 96%, mp 205˚C - 207˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.35 - 7.32 (4H, m, ArH), 7.21-
7.13 (4H, m, ArH), 6.84 (1H, s, HC=C), 4.57 (2H, s,
NH2), 4.02 (1H, s, CH), 3.51 (1H, d, J = 13.8 Hz, CH2),
3.35 (1H, d, J = 13.6 Hz, CH2), 2.94 (1H, d, J = 16.0 Hz,
CH2), 2.72 (1H, d, J = 16.0 Hz, CH2), 2.27 (3H, s, CH3).
Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (i-PrOH/hexane = 30:70,
254 nm, 0.8 mL/min), tr (major) = 9.7 min, tr (minor) =
7.6 min, 98%ee.
4.2.4. (R,E) -2 - Ami no - 8- (4 -C hl oro b enz yl i dene)- 4 -
(4-Chlorophe-nyl)-6-Methyl-5,6,7,8-
Tetrahydro-4H-Pyr-ano[3,2-c]Pyridine-3-
Carbonitrile (R-2d) [11]
White solid, yield 96%, mp 205˚C - 207˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.35 - 7.32 (4H, m, ArH), 7.21 -
7.13 (4H, m, ArH), 6.84 (1H, s, HC=C), 4.57 (2H, s,
NH2), 4.02 (1H, s, CH), 3.51 (1H, d, J = 13.8 Hz, CH2),
3.35 (1H, d, J = 13.6 Hz, CH2), 2.94 (1H, d, J = 16.0 Hz,
CH2), 2.72 (1H, d, J = 16.0 Hz, CH2), 2.27 (3H, s, CH3).
Enantiomeric excess was determined by HPLC with a
CHIRALPAK AD-H column (i-PrOH/hexane = 30:70,
254 nm, 0.8 mL/min), tr (major) = 7.6 min, tr (minor) =
9.7 min, 99%ee.
4.3. Typical Procedure for the Synthesis of
Compounds 2h-2j
A mixture of (E)-2-(4-fluorobenzylidene)-3,4-dihy-dro-
naphthalen -1(2H)-one 1h (50.5 mg, 0.2 mmol), malono-
nitrile (19.8 mg, 0.3 mmol) and triethylamine (20.2 mg,
0.2 mmol) in ethanol (2 mL) were stirred for 12 h at
room temperature. The precipitate was filtered to provide
2h as a yellow solid.
4.3.1. 2-Amino-4-(4-Fl u orophenyl)- 5, 6-Dihydro-
4H-Benzo[h]Chrom ene-3-Ca rbonitrile (2h) [12]
Yellow solid, yield 94%, mp 184˚C - 186˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.46 (1H, d, J = 7.2 Hz, ArH),
7.26 - 7.19 (4H, m, ArH), 7.11 (1H, d, J = 6.8 Hz, ArH),
7.01 (2H, t, J = 8.2 Hz, ArH), 4.58 (2H, s, NH2), 4.07
(1H, s, CH), 2.81 (1H, dd, J = 15.9, 8.1 Hz, CH2), 2.69
(1H, dt, J = 15.7, 7.8 Hz, CH2), 2.16 (1H, dt, J = 15.9,
7.9 Hz, CH2), 2.09 - 1.96 (1H, m, CH2); HRMS (ESI)
calcd for C20H15N2OF+ [M + Na]+: 341.1061, found:
341.1054..
4.3.2. 2-Amino-4-(4-Fl u orophenyl)- 4, 5-
Dihydropyrano[3,2-c]Chromene-3-Carbonitrile
(2i)
Yellow solid, yield 75%, mp 175˚C - 177˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.34 (1H, d, J = 7.6 Hz, ArH),
7.30 - 7.14 (3H, m, ArH), 7.05 (2H, dd, J = 12.0, 5.0 Hz,
ArH), 6.96 (1H, t, J = 7.5 Hz, ArH), 6.80 (1H, d, J = 8.1
Hz, ArH), 4.67 (2H, s, NH2), 4.62 - 4.55 (1H, d, J = 13.6
Hz, CH2), 4.41 (1H, d, J = 13.7 Hz, CH2), 4.03 (1H, s,
CH); 13C NMR (100 MHz, CDCl3): δ = 163.61, 161.17,
158.86, 154.14, 138.16, 136.81, 130.46, 129.55, 129.48,
121.36, 121.13, 116.58, 116.08, 116.00, 115.76, 104.75,
66.37, 60.87, 39.07; IR (KBr) v/cm−1: 3327 (m), 2195
(m), 1709 (s), 1656 (s), 1600 (s), 1506 (m), 1356 (m),
1230 (s), 1157 (m), 1101 (w), 1036 (w), 836 (m), 755
(w); HRMS (ESI) calcd for C19H13N2O2F+ [M + Na]+:
343.0853, found: 343.0857..
4.3.3. 2-Amino-4-(4-Fl u orophenyl)- 4, 5-
Dihydrothiochromeno[4,3-b]Pyran-3-
Carbonitrile (2j)
Yellow solid, yield 76%, mp 158˚C - 160˚C; 1H NMR
(400 MHz, CDCl3): δ = 7.53 (1H, d, J = 16.8 Hz, ArH),
7.36 - 7.29 (2H, m, ArH), 7.28 - 7.14 (3H, m, ArH), 7.04
(2H, dd, J = 12.1, 4.9 Hz, ArH), 4.63 (2H, s, NH2), 4.13
(1H, s, CH), 3.29 (1H, d, J = 15.1 Hz, CH2), 3.09 (1H, d,
J = 15.1 Hz, CH2); 13C NMR (100 MHz, CDCl3): δ =
163.63, 161.18, 158.63, 142.14, 137.53, 132.76, 129.77,
129.70, 129.11, 127.15, 125.58, 123.28, 119.20, 116.03,
115.81, 107.77, 60.99, 42.39, 27.00; IR (KBr) v/cm−1:
3475 (w), 2360 (w), 2197 (s), 2024 (w), 1692 (s), 1635
(w), 1598 (s), 1506 (w), 1407 (s), 1343 (w), 1258 (w),
1219 (w), 1121 (s), 848 (w), 728 (w); HRMS (ESI) calcd
for C19H13N2OFS+ [M + Na]+: 359.0625, found: 359.0631.
4.4. Typical Procedure for the Synthesis of
Compounds 2k-2n
A mixture of (E)-2-benzoyl-3-(4-fluorophenyl)acryloni-

C. X. CHEN ET AL.
Open Access OJMC
134
itrile 1k (50.2 mg, 0.2 mmol), malononitrile (19.8 mg,
0.3 mmol), and piperidine (17.0 mg, 0.2 mmol) in tolu-
ene (2 mL) was stirred at room temperature for 24 h.
After the solvent was evaporated, the residue was puri-
fied by flash column chromatography (ethyl acetate/pe-
troleum ether = 1:5) to give 2k.
4.4.1. 2- Ami no-4-(4-Fluorophenyl) - 6-Phenyl-
4H-Pyran-3,5-Dicarbonitrile (2k)
Yellow solid, yield 85%, mp 61˚C - 63˚C; 1H NMR (400
MHz, CDCl3): δ = 7.74 (2H, dd, J = 7.0, 1.5 Hz, ArH),
7.56 - 7.40 (3H, m, ArH), 7.38 - 7.27 (2H, m, ArH), 7.10
(2H, ddd, J = 8.6, 5.0, 2.7 Hz, ArH), 4.89 (2H, s, NH2),
4.35 (1H, s, CH); 13C NMR (100 MHz, CDCl3): δ =
163.98, 161.48, 157.86, 157.70, 136.49, 131.96, 129.71 ,
129.60, 129.51, 128.81, 127.80, 117.76, 116.83, 116.34,
116.12, 90.70, 59.91, 40.11; IR (KBr) v/cm−1: 3333 (w),
2198 (w), 1673 (s), 1602 (w), 1508 (m), 1401 (m), 1341
(s), 1263 (w), 1081 (m), 743 (w); HRMS (ESI) calcd for
C19H12N3OF+ [M + Na]+: 340.0857, found: 340.0891.
4.4.2. 2-Amino-6-Me thyl-4,5- Di phenyl-4H-
Pyran-3-Carbonitrile (2l) [12]
White solid, yield 78%, 1H NMR (400 MHz, CDCl3): δ
= 7.23 - 7.13 (6H, m, ArH), 7.09 - 7.07 (2H, m, ArH),
6.90 - 6.87 (2H, m, ArH), 4.49 (2H, s, NH2), 4.18 (1H, s,
CH), 1.80 (3H, s, CH3).
4.4.3. 2-Amin o-4-(4-Chlorophenyl)- 5, 6 -Diphenyl-
4H-Pyran-3-Carbonitrile (2m) [12]
White solid, yield 80%, 1H NMR (400 MHz, CDCl3): δ
= 7.26 - 7.07 (12H, m, ArH), 6.84 - 6.82 (2H, m, ArH),
4.56 (2H, s, NH2), 4.35 (1H, s, CH).
4.4.4. 2- Ami no-5,6-Dipheny l -4-(Thiophen-2-yl) -
4H-Pyran-3-Carbonitrile (2n) [12]
White solid, yield 86%, 1H NMR (400 MHz, CDCl3): δ
= 7.25 - 7.10 (9H, m, ArH), 6.95 - 6.93 (2H, m, ArH),
6.87 - 6.85 (1H, m, ArH), 6.79 (1H, d, J = 2.8, Hz, ArH),
4.66 (1H, s, CH), 4.58 (2H, s, NH2).
4.5. Typical Procedure for the Synthesis of
Compounds 4a and 4j
A mixture of compound 2a (39.1 mg, 0.1 mmol), Ac-
ONH4 (92 mg, 1.2 mmol), and AcOH (1.0 mL) in EtOAc
(1.0 mL) was refluxed for 24 h. After cooled to room
temperature, EtOAc (10 mL) was added. The mixture
was washed with saturated aqueous NaHCO3 (10 mL)
and brine (5 mL), and dried over anhydrous Na2SO4.
After the solvent was evaporated under vacuum, the re-
sidue was purified by flash column chromatography
(ethyl acetate/petroleum ether = 2:5) to give the products
4a.
4.5.1. (E)-2 - A mi no- 8-(4-Fluorobenzylidene)- 4-
(4-Fluorophenyl)-6-Methyl-5,6,7,8-Tetrahydro-
1,6-Naphthyridine-3-Carbonitrile (4a)
Yellow solid, yield 80%, mp 208˚C - 210˚C; 1H NMR
(400 MHz, CDCl3): δ = 8.02 (1H, s, HC=C), 7.31 - 7.10
(8H, m, ArH), 5.15 (2H, s, NH2), 3.61 (2H, s, CH2), 3.27
(2H, s, CH2), 2.35 (3H, s, CH3); 13C NMR (100 MHz,
CDCl3): δ = 164.50, 163.38, 162.20, 161.18, 157.36,
153.20, 152.20, 132.66, 131.78, 131.48, 131.40, 130.21,
130.13, 129.39, 116.55, 116.22, 116.00, 115.58, 115.37,
90.78, 55.84, 55.27, 45.52; IR (KBr) v/cm−1: 3427 (s),
2215 (w), 2024 (w), 1627 (m), 1602 (m), 1558 (s), 1506
(s), 1423 (w), 1224 (m), 1160 (w), 1103 (s), 917 (w), 829
(w), 558 (m); HRMS (ESI) calcd for C23H18N4F2
+ [M +
H]+: 389.1572, found: 389.1569.
4.5.2. 2-Amin o- 4- ( 4-Fl uorophenyl )- 5H -Thiochromeno
[4,3-b]Pyridine-3-Carbonitrile (4j)
Yellow solid, yield 85%, mp 202˚C - 204˚C; 1H NMR
(400 MHz, CDCl3): δ = 8.32 (1H, d, J = 6.6 Hz, ArH),
7.51-7.11 (7H, m, ArH), 5.26 (2H, s, NH2), 3.65 (2H, s,
CH2); 13C NMR (100 MHz, CDCl3): δ = 164.51, 162.03,
158.00, 154.68, 151.51, 136.62, 133.51, 131.06, 130.57,
130.53, 130.50, 128.39, 127.84, 126.26, 116.94, 116.39,
116.17, 90.92, 27.21; IR (KBr) v/cm−1: 3446 (s), 2360
(w), 2213 (w), 2023 (w), 1626 (s), 1559 (m), 1427 (m),
1224 (w), 1074 (s), 843 (w), 767 (w), 546 (m); HRMS
(ESI) calcd for C19H12N3FS+ [M + H]+: 334.0809, found:
334.0804.
4.6. Evaluation of Antitubercular Activity
Autoluminescent M. tuberculosis H37Ra was constructed
as previously reported [14] and was inoculated in a 50
mL centrifuge tube containing 5 mL 7H9 with 0.1%
Tween80 and 10% ODAC, then incubated at 37˚C with
shaking. When the culture reached an OD600 nm of 0.7,
the culture was diluted. 50 L diluted H37Ra were in-
oculated in sterile 384 well plate. The RLU of each well
should be between 8000 - 12000 and was recorded as the
basic luminescence of Day 0. The test compounds and
the positive drugs were added to the 384 well plate in
triplicate by the Echo520 with the final concentration 1
or 10 g/mL. The luminescent values were detected for
the following three days. The data were analyzed with
the Excel compared to the DMSO control to estimate the
inhibition activity of the compounds.
The antitubercular activities against M. tuberculosis
H37Rv were determined by standard agar dilution me-
thod [15].
5. Acknowledgements
Financial supports from National Natural Science Foun-
dation of China (Nos. 20972195, 21172270) and Guang-

C. X. CHEN ET AL.
Open Access OJMC
135
dong Engineering Research Center of Chiral Drugs are
gratefully acknowledged.
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