Design, synthesis and biological evaluation of N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide derivatives as novel xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.09.051

A series of N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide derivatives was designed, synthesized and evaluated for inhibitory potency in vitro against xanthine oxidase. The isonicotinamide series was considerably more effective than the nicotinamide series. SARs analysis revealed that the isonicotinoyl moiety played a significant role on the inhibition and that a benzyl ether tail (e.g., ortho-cyanobenzoxy) linked to the benzonitrile moiety benefits the inhibitory potency. Among these compounds, 10q (IC₅₀ = 0.3 μM) was identified to be the most potent in this work and was observed to be 28.3-fold more potent than allopurinol but 20-fold less potent than topiroxostat. The Lineweaver-Burk plot showed that 10q acted as a mixed-type inhibitor on xanthine oxidase. Molecular modeling provided a reasonable explanation for the SARs observed in this study.

Full text of DOI:10.1016/j.ejmech.2017.09.051

Accepted Manuscript
Design, synthesis and biological evaluation of N-(4-alkoxy-3-
cyanophenyl)isonicotinamide/nicotinamide derivatives as novel xanthine oxidase
inhibitors
Ting-jian Zhang, Song-ye Li, Lin Wang, Qi Sun, Qing-xia Wu, Yi Zhang, Fan-hao
Meng
PII:
S0223-5234(17)30766-3
10.1016/j.ejmech.2017.09.051
EJMECH 9766
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 April 2017
Revised Date: 24 June 2017
Accepted Date: 24 September 2017
Please cite this article as: T.-j. Zhang, S.-y. Li, L. Wang, Q. Sun, Q.-x. Wu, Y. Zhang, F.-h. Meng,
Design, synthesis and biological evaluation of N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide
derivatives as novel xanthine oxidase inhibitors, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.09.051.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design,
synthesis
and
biological
evaluation
of
N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide derivatives as novel xanthine oxidase
inhibitors
Ting-jian Zhang, Song-ye Li, Lin Wang, Qi Sun, Qing-xia Wu, Yi Zhang, Fan-hao Meng*
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang
110122, China
* Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F-h. Meng).
Abstract
A series of N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide derivatives was
designed, synthesized and evaluated for inhibitory potency in vitro against xanthine oxidase. The
isonicotinamide series was considerably more effective than the nicotinamide series. SARs
analysis revealed that the isonicotinoyl moiety played a significant role on the inhibition and that a
benzyl ether tail (e.g., ortho-cyanobenzoxy) linked to the benzonitrile moiety benefits the
inhibitory potency. Among these compounds, 10q (IC50 = 0.3 µM) was identified to be the most
potent in this work and was observed to be 28.3-fold more potent than allopurinol but 20-fold less
potent than topiroxostat. The Lineweaver-Burk plot showed that 10q acted as a mixed-type
inhibitor on xanthine oxidase. Molecular modeling provided a reasonable explanation for the
SARs observed in this study.
Keywords: Isonicotinamide; Xanthine oxidase; Hyperuricemia
1. Introduction
Uric acid is the final product of purine metabolism in humans. High serum uric acid
concentration (hyperuricemia) has been linked to a number of diseases, such as gout,
cardiovascular diseases, hypertension and renal diseases [1, 2]. In particular, gout is a common
form of inflammatory arthritis attributed to the deposition of monosodium urate crystals in and
around the joints. Gout affects millions of people worldwide and is increasing in prevalence [3].
Xanthine oxidase (XO) is a critical, rate-limiting enzyme in uric acid biosynthesis in humans that
catalyzes oxidative hydroxylation of hypoxanthine to xanthine and, finally, to uric acid. Therefore,

ACCEPTED MANUSCRIPT
XO is considered to be the most promising target for controlling uric acid levels and for the
treatment of hyperuricemia and gout [4]. Allopurinol (1) (Fig. 1), a prototype XO inhibitor and
hypoxanthine isomer, has been widely prescribed for the treatment of hyperuricemia and gout for
several decades. However, in certain cases, severe and life-threatening side effects have been
reported, including fulminant hepatitis, renal failure, and Stevens–Johnson syndrome, since its
backbone is similar to that of purine [5]. Under these limitations, there is a considerable need for
developing non-purine XO inhibitors with potent XO inhibitory activity and with fewer side
effects. In recent years, many non-purine XO inhibitors with excellent potency have been reported,
such as febuxostat (2, approved in USA, 2009) [6], Y-700 (3) [7], selenazoles (4) [8], imidazoles
(5) [9], 2-(indol-5-yl)thiazoles (6) [10], topiroxostat (7, approved in Japan, 2013) [11],
phenyl-1,2,4- triazoles (8) [12] and isocytosines (9) (Fig. 1) [13-16]. Additionally, other XO
inhibitors with various chemotypes have also been published in the literature, including isoxazoles
[17], N-(1,3-diaryl-3-oxo-propyl)amides [18], N-acetyl pyrazolines [19], chalcones [20],
9-deazaguanine [21], fraxamoside [22], pyrano[3,2-d]pyrimidine [23], 2-arylbenzo[b]furan [24]
and benzaldehydes [25].
Fig. 1 Chemical structures of selected XO inhibitors.
To the best of our knowledge, the structures of classic XO inhibitors (such as 2-9) could be
split into four fragments, marked as S1-S4, as shown in fig. 2. S1 is a functional group, which can
be further divided into two exclusive subgroups as S1a and S1b. S1a and S1b occupy two
independent regions in the XO active pocket, respectively. The most frequent member of S1a is a
carboxyl group (e.g., compound 2-6) that goes deep into the active cavity of XO and strongly

ACCEPTED MANUSCRIPT
interacts with Arg880 and Thr1010, helping ligands anchor tightly to the binding site [6, 26-28];
the S1b fragment usually bears a pyridine moiety (e.g., compounds 7-8), which occupies an
adjacent pocket towards Glu1261 and the molybdenum-pterin center and may be oxidized by XO
and covalently bound with molybdenum-pterin (e.g., compound 7) [29]. S2 usually exists as a
nitrogen-containing five-membered ring and acts as a linker between S1 and S3. S3, an aromatic
nitrile moiety, is the most permanent unit in the structure of classic XO inhibitors, which is often
located at the outer part of the active site and acts as a doorkeeper for the cavity; moreover, the
cyano group accepts a hydrogen bond from the amino of ASN768 [6, 11, 26, 28] and locks the
ligand to the XO. S4, a lipophilic tail located at the outside of the cavity, could significantly affect
the XO inhibitory potency. When S3 employs a pyridine moiety, the lipophilic tail can be omitted
(e.g., compound 7).
In our previous studies, we identified a new class of non-purine XO inhibitors with an
imidazole [9] replacing the thiazole in febuxostat. In this study, to continue our efforts in searching
for novel non-purine XO inhibitors, we used an amide fragment as an opened-ring isostere of
1,2,4-triazole to perform the role of the S2 linker and designed a series of XO inhibitors, 10a-s and
11a-o, as shown in Fig. 2.
Fig. 2 Design of the compounds 10a-s and 11a-o as XO inhibitors.

ACCEPTED MANUSCRIPT
2. Results and Discussion
2.1. Chemistry
The
synthesis
of
N-(4-alkoxy-3-cyanophenyl)isonicotinamides
10a-s
and
N-(4-alkoxy-3-cyanophenyl)nicotinamides 11a-o were performed as outlined in Scheme 1. As
shown in Scheme 1, 2-alkoxy-5-aminobenzonitriles 14a-s were considered to be the key
intermediates to synthesize the target compounds 10a-s and 11a-o. To acquire these intermediates,
we first performed a convenient method using 5-nitrosalicylaldehyde as the starting material via a
three-step procedure, involving the conversion of the aldehyde into an oxime, dehydration of the
oxime to a nitrile and, finally, the alkylation of the nitrile into 14 [30]. In this procedure, the
transformation of the oxime to the nitrile had less than a ten percent yield, even in a microwave.
Thus, we had to adopt an alternative method, as shown in Scheme 1. Commercially available
2-hydroxybenzonitrile was nitrated by concentrated HNO3 in an acetic acid solution to provide
2-hydroxy-5-nitrobenzonitrile (12). In this reaction, the nitro group could also be introduced at the
3-position to generate the by-product 2-hydroxy-3-nitrobenzonitrile, which are difficult to separate
out at large scale due to their similar polarities; therefore, we used the mixture directly. A
continuous three-step alkylation procedure with the corresponding alkyl bromide or alkyl chloride
and reduction by using iron powder and isolation with column chromatography provided the
desired 2-alkoxy-5-aminobenzonitriles 14a-s effortlessly. Compounds 14a-s were acylated by
isonicotinoyl chloride/nicotinoyl chloride prepared from isonicotinic acid/nicotinic acid to provide
the compounds 10a-s and 11a-o with good yields. A similar method was used to synthesize 12b,
12i, 12j, 12m, 12n and 13.

ACCEPTED MANUSCRIPT
NO2
NO2
a, R = n-Propyl
b, R = n-Butyl
c, R = n-Pentyl
d, R = n-Hexyl
ⅰ
ⅱ
HO
RO
HO
CN
12
CN
13
CN
e, R = n-Heptyl
f, R = n-Octyl
g, R = iso-Propyl
h, R = sec-Butyl
i, R = iso-Butyl
j, R = iso-Pentyl
k, R = Cyclopentyl
l, R = Methoxyethyl
m, R = Benzyl
n, R = 4-Chlorobenzyl
o, R = 4-Cyanobenzyl
p, R = 3-Cyanobenzyl
q, R = 2-Cyanobenzyl
NH2
ⅲ
RO
X1
X2
H
CN
14
N
ⅴ
O
R O
O
O
CN
ⅳ
Cl
OH
10a-s X1 = N, X2 = CH
11a-o X2 = N, X1 = CH
r
, R = 4-Methoxybenzyl
s, R = 2-Methoxybenzyl
X
X
1X2
1X2
Scheme 1. Reagents and conditions: ( ) HNO3, AcOH, 50°C, 4 h; ( ) RCl or RBr, K2CO3, KI,
DMF, overnight; ( ) 1) Fe, NH4Cl, EtOH, H2O, reflux, 3 h; 2) isolation in column
chromatography (ethyl acetate: petroleum ether = 1: 5 to 1: 1); ( ) SOCl2, DMF, 50°C, 2 h; ( )
Et3N, DCM, -5°C, 1 h.
2.2. Xanthine oxidase inhibitory activity
Based on the in vitro inhibitory activities shown in Table 1, it was observed that the
isonicotinamides were considerably more effective than the nicotinamides. Almost all of the
compounds in the isonicotinamide series (10a-s) were effective with IC50 values ranging from 0.3
µM to 19.2 µM, whereas the majority of the compounds in the nicotinamide series were totally
inactive, except 11j and 11m. To extend the investigation range, we synthesized several
picolinamide analogs (12b, 12i, 12j, 12m and 12n) and a benzamide analog (13) as shown in Fig.
3. The evaluated results indicated that only compound 12m (IC50 = 22.6 µM) exhibited a weak
effect, whereas the rest of compounds were totally ineffective. Therefore, the isonicotinoyl moiety
was extremely important for the inhibition. Thus, our efforts for searching for potent XO
inhibitors were primarily focused on the isonicotinamide series.
Table 1 In vitro XO inhibitory potency of target compounds
Compounds
IC50 (µM) a
Compounds
IC50 (µM)
3.9 ± 0.23 #
n.a. b
10a
11a

ACCEPTED MANUSCRIPT
n.a. b
n.a.
2.3± 0.15
10b
10c
11b
3.0± 0.20 #
11c
2.7± 0.10 #
n.a.
10d
11d
5.0± 0.38 #
n.a.
10e
11e
19.2± 1.2 #
n.a.
10f
11f
9.5± 0.13 #
n.a.
10g
11g
6.1± 0.07 #
n.a.
10h
11h
1.6± 0.07 #
n.a.
10i
11i
2.0± 0.09
35.0± 1.49
n.a.
10j
11j
5.7± 0.17
1k
11k
n.a.
n.a.
10l
11l
0.9± 0.01
32.8± 1.13
n.a.
10m
10n
11m
0.8± 0.02
11n
2.3± 0.23 *
n.a.
10o
11o
2.0± 0.24 *
n.a.
10p
12b
0.3± 0.03 *
n.a.
10q
12i
0.6± 0.04 *
n.a.
10r
12j
1.8± 0.05 *
22.6± 0.81
n.a.
10s
12m
8.5± 0.09
Allopurinol
Topiroxostat
12n
0.015± 0.002
n.a.
13
a Values are means of three independent experiments.
b n.a.: not active (<60% inhibition at 50 µM).
# P＜0.05, versus compound 10b.
* P＜0.05, versus compound 10m.

ACCEPTED MANUSCRIPT
Fig. 3 Chemical structures of compounds 10b, 12b, 12i, 12j, 12m, 12n and 13.
A set of isonicotinamide derivatives with a linear R group (10a-f) has been initially
synthesized and evaluated for XO inhibitory activities in vitro. Among these derivatives,
compound 10b, bearing an n-butyl as R group, exhibited a relatively high potency with an IC50
value of 2.3 µM (Fig. 3). Shortening (10b versus 10a) or lengthening (10b versus 10c-f) the
carbon chains both damaged the potency. When the carbon chain was prolonged to n-octyl (10f,
IC50 = 19.2 µM), the inhibitory potency was greatly reduced. When the number of carbon atoms
was held constant and a methyl substituent occupied the α-position of the R group, the inhibitory
potency was obviously decreased (10a versus 10g, 10b versus 10h). However, if a methyl group
was installed at the β- or γ-position, the potency was slightly improved (10b versus 10i, 10c
versus 10j).
A cyclopentyl-substituted compound, 10k (IC50 = 5.7 µM), was involved in the series, and its
activity was nearly halved compared with its straight-chain analog 10c (IC50 = 3.0 µM). Replacing
n-butyl (10b) with methoxyethyl (10l) resulted in vanished potency, which demonstrated that a
polar atom may not be tolerated in the R group. In other words, the most potent compound in the
subseries of 10a-l possessing the aliphatic alkyl R group was 10i (IC50 = 1.6 µM), which was
5.3-fold more effective than allopurinol.
Surprisingly, employment of a benzyl for R group led to 10m (IC50 = 0.9 µM), the effect of
which was double that of with 10i, which greatly encouraged us. For the purpose of identifying
more effective XO inhibitors, some substituents were fixed on the benzyl moiety. When a chloro
atom was linked at the para-position (10n, IC50 = 0.8 µM), the potency was maintained.
Installation of an ortho-cyano (10q, IC50 = 0.3 µM) or a para-methoxy (10r, IC50 = 0.6 µM)
resulted in a 3-fold or 1.5-fold gain in potency, respectively; however, the other substituents
introduced on the benzyl moiety could reduce the potency, such as para-cyano (10o, IC50 = 2.3
µM), meta-cyano (10p, IC50 = 2.0 µM) and ortho-methoxy (10s, IC50 = 1.8 µM).

ACCEPTED MANUSCRIPT
In summary, compound 10q (IC50 = 0.3 µM), bearing an ortho-cyanobenzyl R group, was
determined to be the most potent compound in this work, which was 28.3-fold more effective than
allopurinol, although was still 20-fold less effective than topiroxostat. Enzyme kinetics studies
were performed for the representative compound 10q. The Lineweaver-Burk plot (fig. 4) revealed
that compound 10q acted as a mixed-type inhibitor of XO. This inhibition type could be explained
by potent inhibition of both the oxidized and reduced forms of XO [31]. Similar inhibitory
behavior was reported with 2-hydroxy-topiroxosat [11], a metabolite of topiroxostat catalyzed by
XO.
Fig. 4 Lineweaver-Burk plot analysis of xanthine oxidase by compound 10q.
2.3. Molecular docking
To rationalize the structure activity relationships (SARs) observed in this study and to foresee
the possible interactions of the synthesized compounds with XO, molecular modeling simulations
of topiroxostat and 10q in the binding pocket of XO were performed with MOE (Molecular
Operating Environment, version 2016.08, Chemical Computing Group Inc., Canada) software.

ACCEPTED MANUSCRIPT
The crystal structure of XO in complex with topiroxostat (PDB code: 1V97) [29] was adopted in
the docking calculations. First, a self-docking of topiroxostat into the binding pocket was
performed to validate the docking protocol [9]. The docking results demonstrated that topiroxostat
could stack well with the crystallographic ligand, and the H-bonds predicted by MOE were similar
to those found in the crystal structure (Fig. 5 A). According to the co-crystal structure, topiroxostat
was oxidized by XO and covalently bond with molybdenum-pterin [11]. Although the covalent
bonding was not simulated in the molecular stimulations, this protocol well reproduced the crystal
conformation and other interactions.
The docking pose of 10q in the binding pocket as shown in Fig. 5 B indicated that the
isonicotinoyl moiety could overlap well with the pyridin-1,2,4-triazole moiety of the original
ligand [29]. The para-N was adjacent to Glu1261 and formed an H-bond with it. This H-bond
would be essential for the inhibitory potency [29] because changing (e.g., 10m versus 11m and
12m) or removing (e.g., 10m versus 13) the para-N position could damage the potency by
breaking the H-bond. The carbonyl group exercised a similar role of the N-2 atom on
1,2,4-triazole, and linked to the amino acid residues Arg880 and Thr1010 via a water bridge [29].
In addition, the amide NH formed an extra H-bond with Glu802 carboxyl and the para-benzoxy
tail strongly interacted with some lipophilic amino acid residues, such as Leu648, Phe649 and
Phe1013. These interactions explained the relatively high activities of the isonicotinamides and
further implied that isonicotinamides stood a good chance of forming a covalent modification with
XO, similar to that of topiroxostat.
However, due to the shorter distance of the amide-S2 linker compared with 1,2,4-triazole of
topiroxostat, the cyano group was too distant from Asn768 to form an H-bond. This H-bond
definitely plays a significant role for the inhibition [6, 11, 26], thus its absence very likely
contributes to the decreased potency of the isonicotinamides compared with topiroxostat.

ACCEPTED MANUSCRIPT
Fig. 5 Docking poses of topiroxostat (A, purple) and compound 10q (B, yellow) within the protein
binding pocket and overlaid with the original ligand topiroxostat (green).
3. Conclusions
We
designed,
synthesized
and
identified
a
series
of
N-(4-alkoxy-3-cyanophenyl)isonicotinamide/nicotinamide derivatives as novel XO inhibitors.
Among them, the most promising compound obtained was 10q (IC50 = 0.3 µM), which was
28.3-fold more effective than allopurinol and was 20-fold less effective than topiroxostat. The
Lineweaver-Burk plot showed that compound 10q acted as a mixed-type XO inhibitor. SARs
analysis revealed that the isonicotinoyl moiety played a significant role on the inhibition and that a
benzyl ether tail (e.g., ortho-cyanobenzoxy) linked to the benzonitrile moiety will benefit the
inhibitory potency. Molecular modeling provided a reasonable explanation for the SARs observed
in this study. In a word, it is worthwhile to explore more promising XO inhibitors based on
isonicotinamide scaffold, as these compounds, especially compound 10q, constitute plausible lead
candidates for further investigations.
4. Experimental Protocols
4.1. Chemical synthesis
Unless otherwise indicated, reagents and solvents were purchased from commercial sources
and used without further purification. All reactions were monitored by TLC using silica gel
aluminum cards (0.2 mm thickness) with a fluorescent indicator at 254 nm. The column
chromatography was performed using silica gel (200-300 mesh) from Qingdao Ocean Chemicals
(Qingdao, Shandong, China). Melting points were recorded on an RD-1 melting apparatus and
1
were uncorrected. H NMR and 13C NMR spectra were recorded on a Bruker 600 MHz

ACCEPTED MANUSCRIPT
spectrometer. Chemical shifts were expressed in parts per million using tetrametrylsilane as an
internal reference and DMSO-d6 as the solvent. ESI-HRMS data were gathered using a Bruker
microTOF-Q instrument.
4.1.1. Synthesis of 2-hydroxy-5-nitrobenzonitrile (12)
Concentrated nitric acid (74.4 mL, 1.1 mol) was added slowly and dropwise to a solution of
2-hydroxybenzonitrile (100 g, 0.84 mol) in acetic acid (200 mL) at 50 to maintain the reactive
temperature between 50 and 70 . The mixture was stirred for another 4 h after the addition was
complete, and then poured into ice water. The resulting precipitate was collected by filtration,
washed with water (100 mL× 3) and dried to give a pale yellow solid of 82 g with a yield of
59.5%. A small amount of the sample (0.2 g) was further purified by column chromatography to
give a slight yellow solid. Mp 195.2-196.8°C (Mp > 200°C [30]). 1H NMR (600 MHz, DMSO-d6)
δ
12.29 (s, 1H), 8.60 (d, J = 2.9 Hz, 1H), 8.36 (dd, J = 9.3, 2.9 Hz, 1H), 7.17 (d, J = 9.3 Hz, 1H). 13C
NMR (150 MHz, DMSO-d6) δ 165.63, 139.32, 130.28, 130.09, 116.79, 115.04, 99.65.
4.1.2. General procedure for the synthesis of 2-alkoxy-5-aminobenzonitriles (14)
A mixture of nitration product 12 (110 mmol), alkyl bromide/chloride (110 mmol), anhydrous
potassium carbonate (165 mmol) and potassium iodide (11 mmol) in DMF (80 mL) was stirred
overnight at 60 . After completion of the reaction as determined by TLC, the mixture was cooled
to room temperature, poured into water (300 mL) and extracted with ethyl acetate (100 mL × 3).
The combined organic layers were washed with brine, dried over magnesium sulfate anhydrous
and evaporated in a vacuum to afford alkylated products 13, as a yellow to brown oil, which were
used directly in the next step.
Reduced iron powder (223.8 mmol) and ammonium chloride (29.1 mmol) was added to a
solution of 13 (58.2 mmol) in ethanol (15 mL) and water (15 mL). The reactive solution was
refluxed for 3 h. The completion of the reaction was proved by TLC. The mixture was cooled to
room temperature and poured into water (50 mL) before extracting with ethyl acetate (50 mL × 3).
The combined organic layers were washed with brine, dried over sodium sulfate anhydrous, and
concentrated under reduced pressure to give crude products, which were then purified using
column chromatography (ethyl acetate: petroleum ether = 1: 5 to 1: 1) to obtain the pure
2-alkoxy-5-aminobenzonitriles 14a-s.

ACCEPTED MANUSCRIPT
4.1.2.1. 5-Amino-2-propoxybenzonitrile (14a)
A yellow solid, yield 45.2%. Mp 69.8-71.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 6.96 (d, J = 9.0
Hz, 1H), 6.83 (dd, J = 9.0, 2.8 Hz, 1H), 6.76 (d, J = 2.8 Hz, 1H), 5.10 (s, 2H), 4.08 (t, J = 6.4 Hz,
2H), 1.81 – 1.67 (m, 2H), 0.96 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 151.54,
142.90, 120.52, 117.00, 116.69, 114.83, 100.72, 70.46, 22.01, 10.26.
4.1.2.2. 5-Amino-2-butoxybenzonitrile (14b)
A yellow solid, yield 42.0%. Mp 49.5-51.4 °C. 1H NMR (600 MHz, DMSO-d6) δ 6.95 (d, J = 9.0
Hz, 1H), 6.84 (dd, J = 8.9, 2.8 Hz, 1H), 6.77 (d, J = 2.8 Hz, 1H), 5.05 (s, 2H), 3.96 (t, J = 6.4 Hz,
2H), 1.66 (m, 2H), 1.50 – 1.37 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO-d6)
δ 151.54, 142.90, 120.50, 116.99, 116.66, 114.82, 100.69, 68.70, 30.68, 18.61, 13.65.
4.1.2.3. 5-Amino-2-(pentyloxy)benzonitrile (14c)
A yellow solid, yield 36.4%. Mp 71.7-73.42 °C. 1H NMR (600 MHz, DMSO-d6) δ 6.94 (d, J = 9.0
Hz, 1H), 6.84 (dd, J = 9.0, 2.8 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 5.05 (s, 2H), 3.95 (t, J = 6.4 Hz,
2H), 1.75 – 1.57 (m, 2H), 1.45 – 1.27 (m, 4H), 0.88 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 151.55, 142.90, 120.50, 116.99, 116.69, 114.81, 100.73, 68.99, 28.29, 27.55, 21.82,
13.88.
4.1.2.4. 5-Amino-2-(hexyloxy)benzonitrile (14d)
A yellow solid, yield 35.7%. Mp 84.5-86.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 6.94 (d, J = 9.0
Hz, 1H), 6.84 (dd, J = 8.9, 2.8 Hz, 1H), 6.77 (d, J = 2.8 Hz, 1H), 5.05 (s, 2H), 3.95 (t, J = 6.4 Hz,
2H), 1.66 (m, 2H), 1.45 – 1.36 (m, 2H), 1.33 – 1.24 (m, 4H), 0.91 – 0.82 (m, 3H). 13C NMR (150
MHz, DMSO-d6) δ 151.54, 142.90, 120.50, 116.99, 116.67, 114.84, 100.72, 69.00, 30.90, 28.54,
25.01, 22.05, 13.85.
4.1.2.5. 5-Amino-2-(heptyloxy)benzonitrile (14e)
A yellow oil, yield 42.4%. 1H NMR (600 MHz, DMSO-d6) δ 6.95 (d, J = 9.0 Hz, 1H), 6.84 (dd, J

ACCEPTED MANUSCRIPT
= 8.9, 2.8 Hz, 1H), 6.76 (d, J = 2.8 Hz, 1H), 5.05 (s, 2H), 4.11 (t, J = 6.4 Hz, 2H), 1.77 – 1.66 (m,
2H), 1.46 – 1.37 (m, 2H), 1.37 – 1.21 (m, 6H), 0.85 (t, J = 6.9 Hz, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 151.53, 142.89, 120.49, 116.98, 116.66, 114.83, 100.71, 68.98, 31.19, 28.64, 28.57,
25.34, 22.07, 13.94.
4.1.2.6. 5-Amino-2-(octyloxy)benzonitrile (14f)
A yellow oil, yield 34.8%. 1H NMR (600 MHz, DMSO-d6) δ 6.94 (d, J = 9.0 Hz, 1H), 6.84 (dd, J
= 9.0, 2.8 Hz, 1H), 6.77 (d, J = 2.8 Hz, 1H), 5.05 (s, 2H), 3.95 (t, J = 6.4 Hz, 2H), 1.72 – 1.60 (m,
2H), 1.45 – 1.33 (m, 2H), 1.34 – 1.21 (m, 8H), 0.85 (t, J = 7.0 Hz, 3H). δ 151.54, 142.90, 120.50,
116.98, 116.67, 114.83, 100.72, 68.99, 31.18, 28.65, 28.61, 28.34, 25.35, 22.08, 13.96.
4.1.2.7. 5-Amino--2-isopropoxybenzonitrile (14g)
A yellow oil, yield 32.6%. 1H NMR (600 MHz, DMSO-d6) δ 6.96 (d, J = 9.0 Hz, 1H), 6.83 (dd, J
= 8.9, 2.8 Hz, 1H), 6.76 (d, J = 2.8 Hz, 1H), 5.09 (s, 2H), 4.46 (m, 1H), 1.23 (d, J = 6.1 Hz, 6H).
13C NMR (150 MHz, DMSO-d6) δ 150.28, 143.26, 120.51, 117.53, 117.21, 116.44, 102.51, 72.27,
21.85.
4.1.2.8. 5-Amino-2-(sec-butoxy)benzonitrile (14h)
A yellow oil, yield 42.3%. 1H NMR (600 MHz, DMSO-d6) δ 6.95 (d, J = 9.0 Hz, 1H), 6.83 (dd, J
= 8.9, 2.8 Hz, 1H), 6.76 (d, J = 2.8 Hz, 1H), 5.09 (s, 2H), 4.27 (m, 1H), 1.59 (m, 2H), 1.19 (d, J =
6.1 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 150.59, 143.15, 120.56,
117.25, 117.18, 116.45, 102.31, 76.93, 28.51, 19.03, 9.30.
4.1.2.9. 5-Amino-2-isobutoxybenzonitrile (14i)
A yellow solid, yield 23.4%. Mp 44.2-45.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 6.93 (d, J = 9.0
Hz, 1H), 6.84 (dd, J = 8.9, 2.8 Hz, 1H), 6.80 – 6.76 (m, 1H), 5.05 (s, 2H), 3.73 (d, J = 6.5 Hz, 2H),
1.98 (m, 1H), 0.97 (d, J = 6.7 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 151.67, 142.89, 120.50,
116.93, 116.64, 114.79, 100.69, 75.13, 27.73, 18.84.
4.1.2.10. 5-Amino-2-(isopentyloxy)benzonitrile (14j)

ACCEPTED MANUSCRIPT
1
A yellow oil, yield 39.1%. H NMR (600 MHz, DMSO-d6) δ 6.98 – 6.95 (m, 1H), 6.84 (dd, J =
9.0, 2.8 Hz, 1H), 6.77 (d, J = 2.8 Hz, 1H), 5.04 (s, 2H), 4.01 – 3.93 (m, 2H), 1.82 – 1.73 (m, 1H),
1.58 (q, J = 6.7 Hz, 2H), 0.92 (t, J = 6.3 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 151.54,
142.91, 120.50, 117.00, 116.67, 114.81, 100.68, 67.48, 37.36, 24.53, 22.41.
4.1.2.11. 5-Amino-2-((cyclopentyl)oxy)benzonitrile (14k)
A yellow oil, yield 36.1%. 1H NMR (600 MHz, DMSO-d6) δ 6.95 (d, J = 9.0 Hz, 1H), 6.84 (dd, J
= 9.0, 2.8 Hz, 1H), 6.75 (d, J = 2.8 Hz, 1H), 5.06 (s, 2H), 4.77 (td, J = 5.7, 3.0 Hz, 1H), 1.88 –
1.77 (m, 2H), 1.77 – 1.64 (m, 4H), 1.62 – 1.51 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 150.43,
142.98, 120.51, 117.10, 116.66, 116.54, 101.87, 80.90, 32.10, 23.33.
4.1.2.12. 5-Amino-2-(2-methoxyethoxy)benzonitrile (14l)
An off-white solid, yield 22.1%. Mp 75.3-76.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 6.97 (d, J =
9.0 Hz, 1H), 6.85 (dd, J = 8.9, 2.7 Hz, 1H), 6.79 (d, J = 2.7 Hz, 1H), 5.13 (s, 2H), 4.18 – 4.00 (m,
2H), 3.72 – 3.56 (m, 2H), 3.32 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 151.43, 142.91, 120.51,
117.00, 116.86, 114.89, 100.75, 70.32, 68.66, 58.33.
4.1.2.13. 5-Amino-2-(benzyloxy)benzonitrile (14m)
A pale yellow powder, yield 42.7%. Mp 89.6-91.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.44 (d, J
= 7.3 Hz, 2H), 7.40 (d, J = 7.3 Hz, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 6.84 (dd,
J = 8.9, 2.8 Hz, 1H), 6.80 (d, J = 2.8 Hz, 1H), 5.10 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ
151.02, 143.23, 136.71, 128.45, 127.92, 127.52, 120.32, 117.00, 116.68, 115.35, 101.05, 70.48.
4.1.2.14. 5-Amino-2-((4-chlorobenzyl)oxy)benzonitrile (14n)
1
An off-white solid, yield 29.9%. Mp 121.4-123.6 °C. H NMR (600 MHz, DMSO-d6) δ 7.50 –
7.43 (m, 4H), 7.03 (d, J = 9.0 Hz, 1H), 6.84 (dd, J = 9.0, 2.8 Hz, 1H), 6.79 (d, J = 2.8 Hz, 1H),
5.11 (s, 4H). 13C NMR (150 MHz, DMSO-d6) δ 150.75, 143.35, 135.76, 132.51, 129.35, 128.48,
120.27, 116.92, 116.66, 115.40, 101.09, 69.68.
4.1.2.15. 5-Amino-2-((4-cyanobenzyl)oxy)benzonitrile (14o)

ACCEPTED MANUSCRIPT
An off-white solid, yield 30.6%. Mp 167.1-168.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.89 (d, J
= 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 9.0 Hz, 1H), 6.84 (dd, J = 8.9, 2.8 Hz, 1H),
6.81 (d, J = 2.8 Hz, 1H), 5.18 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 150.54, 143.49, 142.54,
132.46, 127.86, 120.25, 118.72, 116.68, 115.25, 110.57, 101.05, 69.49.
4.1.2.16. 5-Amino-2-((3-cyanobenzyl)oxy)benzonitrile (14p)
A grey powder, yield 31.6%. Mp 108.0-110.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.89 (s, 1H),
7.80 (m, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 6.85 (dd, J = 8.9, 2.8 Hz, 1H),
6.81 (d, J = 2.8 Hz, 1H), 5.17 (s, 2H), 5.14 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ150.57,
143.52, 138.46, 132.24, 131.74, 130.91, 129.79, 120.27, 118.65, 116.88, 116.67, 115.43, 111.44,
101.18, 69.41.
4.1.2.17. 5-Amino-2-((2-cyanobenzyl)oxy)benzonitrile (14q)
An off-white powder, yield 38.6%. Mp 130.0-131.3 °C. 1H NMR (600 MHz, CDCl3) δ 7.82 (d, J
= 7.7 Hz, 1H), 7.67 (m, 2H), 7.44 (t, J = 7.6 Hz, 1H), 6.88 (m, 3H), 5.29 (s, 2H), 3.66 (s, 2H). 13C
NMR (150 MHz, DMSO-d6) δ 150.57, 143.66, 139.63, 133.45, 133.24, 129.37, 129.18, 120.22,
117.02, 116.79, 116.70, 115.40, 111.27, 101.22, 68.90.
4.1.2.18. 5-Amino-2-((4-methoxybenzyl)oxy)benzonitrile (14r)
1
A yellow solid, yield 42.8%. Mp 127.3-128.9 °C . H NMR (600 MHz, DMSO-d6) δ 7.36 (d, J =
8.6 Hz, 2H), 7.05 (d, J = 9.0 Hz, 1H), 6.98 – 6.92 (m, 2H), 6.84 (dd, J = 9.0, 2.8 Hz, 1H), 6.78 (d,
J = 2.8 Hz, 1H), 5.12 (s, 2H), 5.02 (s, 2H), 3.75 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 159.06,
151.09, 143.12, 129.42, 128.52, 120.33, 117.03, 116.65, 115.52, 113.83, 101.12, 70.37, 55.08.
4.1.2.19. 5-Amino-2-((2-methoxybenzyl)oxy)benzonitrile (14s)
An off-white powder, yield 36.0%. Mp 87.0-88.6 °C. 1H NMR (600 MHz, CDCl3) δ 7.51 (dd, J =
6.9, 0.6 Hz, 1H), 7.29 (td, J = 8.2, 1.6 Hz, 1H), 6.98 (td, J = 7.5, 0.7 Hz, 1H), 6.92 – 6.83 (m, 3H),
6.81 (dd, J = 8.8, 2.9 Hz, 1H), 5.15 (s, 2H), 3.86 (s, 3H), 3.56 (s, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 156.81, 151.27, 143.15, 129.39, 128.68, 124.38, 120.41, 120.30, 117.03, 116.71,
115.19, 110.91, 100.94, 66.00, 55.44.

ACCEPTED MANUSCRIPT
4.1.3. General procedure for the synthesis of N-(4-alkoxy-3-cyanophenyl)isonicotinamides
(10a-s)
Isonicotinoyl chloride hydrochloride (0.72 mmol) in dichloromethane (15 mL) was added
dropwise to a mixture of 2-alkoxy-5-aminobenzonitrile (0.6 mmol), trimethylamine (1.5 mmol) in
dichloromethane (15 mL) at -5°C. After the addition was completed, the mixture was stirred for 1
h at the same temperature, followed by the addition of water (30 mL). The reaction mixture was
extracted with dichloromethane (60 mL × 3). The combined organic layers were washed with
brine, dried over sodium sulfate anhydrous and concentrated under reduced pressure to provide the
crude products, which were then purified using column chromatography (ethyl acetate: petroleum
ether = 1: 1) to give the pure products 10a-s.
4.1.3.1. N-(3-cyano-4-propoxyphenyl)isonicotinamide (10a)
An off-white crystal, yield 75.1%. Mp > 230 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1H),
8.80 (dd, J = 4.4, 1.6 Hz, 2H), 8.10 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.85 (dd, J
= 4.4, 1.6 Hz, 2H), 7.29 (d, J = 9.2 Hz, 1H), 4.09 (t, J = 6.4 Hz, 2H), 1.87 – 1.69 (m, 2H), 1.01 (t,
J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 163.89, 156.83, 150.34, 141.42, 131.80,
127.40, 124.90, 121.48, 116.14, 113.62, 100.18, 70.32, 21.81, 10.19. ESI-HRMS calcd. for
C16H16N3O2 [M + H]+ 282.1237, found: 282.1229.
4.1.3.2. N-(4-butoxy-3-cyanophenyl)isonicotinamide (10b)
1
A yellow powder, yield 89.4%. Mp 199.8-202.5 °C. H NMR (600 MHz, DMSO-d6) δ 10.61 (s,
1H), 8.80 (d, J = 5.9 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.85 (dd, J
= 4.4, 1.6 Hz, 2H), 7.30 (d, J = 9.2 Hz, 1H), 4.14 (t, J = 6.4 Hz, 2H), 1.74 (m, 2H), 1.57 – 1.38 (m,
2H), 0.95 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 163.90, 156.86, 150.35, 141.43,
131.79, 127.42, 124.91, 121.49, 116.15, 113.61, 100.17, 68.65, 30.43, 18.59, 13.64. ESI-HRMS
calcd. for C17H18N3O2 [M + H]+ 296.1394, found: 296.1403.
4.1.3.3. N-(3-cyano-4-(pentyloxy)phenyl)isonicotinamide (10c)
An off-white powder, yield 86.8%. Mp 175.1-176.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61

ACCEPTED MANUSCRIPT
(s, 1H), 8.80 (dd, J = 4.4, 1.6 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H),
7.85 (dd, J = 4.4, 1.6 Hz, 2H), 7.29 (d, J = 9.2 Hz, 1H), 4.12 (t, J = 6.5 Hz, 2H), 1.82 – 1.68 (m,
2H), 1.50 – 1.29 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 163.90,
156.85, 150.35, 141.43, 131.79, 127.41, 124.91, 121.48, 116.15, 113.62, 100.17, 68.92, 28.05,
27.50, 21.79, 13.90. ESI-HRMS calcd. for C18H20N3O2 [M + H]+ 310.1550, found: 310.1564.
4.1.3.4. N-(3-cyano-4-(hexyloxy)phenyl)isonicotinamide (10d)
A pink crystal, yield 70.7%. Mp 138.3-139.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H),
8.79 (dd, J = 4.4, 1.6 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.85 (dd, J
= 4.4, 1.6 Hz, 2H), 7.28 (d, J = 9.2 Hz, 1H), 4.12 (t, J = 6.5 Hz, 2H), 1.81 – 1.68 (m, 2H), 1.50 –
1.37 (m, 2H), 1.36 – 1.27 (m, 4H), 0.88 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ
163.89, 156.85, 150.35, 141.43, 131.80, 127.40, 124.90, 121.48, 116.14, 113.61, 100.18, 68.93,
30.87, 28.31, 24.96, 22.05, 13.86. ESI-HRMS calcd. for C19H22N3O2 [M + H]+ 324.1707, found:
324.1715.
4.1.3.5. N-(3-cyano-4-(heptyloxy)phenyl)isonicotinamide (10e)
A gray powder, yield 66.7%. Mp 118.8-119.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1H),
8.80 (d, J = 5.8 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.88 – 7.81 (m,
2H), 7.29 (d, J = 9.2 Hz, 1H), 4.13 (t, J = 6.4 Hz, 2H), 1.83 – 1.65 (m, 2H), 1.51 – 1.38 (m, 2H),
1.38 – 1.19 (m, 6H), 0.87 (t, J = 6.9 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 166.17, 163.86,
156.83, 150.60, 150.31, 141.43, 131.79, 127.37, 124.89, 122.74, 121.49, 116.12, 113.59, 100.18,
68.92, 31.21, 28.35, 28.32, 25.26, 22.01, 13.92. ESI-HRMS calcd. for C20H24N3O2 [M + H]+
338.1869, found: 338.1865.
4.1.3.6. N-(3-cyano-4-(octyloxy)phenyl)isonicotinamide (10f)
A brown powder, yield 56.2%. Mp 102.9-104.4 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61 (s,
1H), 8.80 (dd, J = 4.5, 1.5 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.85
(dd, J = 4.4, 1.6 Hz, 2H), 7.29 (d, J = 9.2 Hz, 1H), 4.12 (t, J = 6.4 Hz, 2H), 1.83 – 1.66 (m, 2H),
1.48 – 1.40 (m, 2H), 1.36 – 1.21 (m, 8H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO-d6)
δ 163.87, 156.83, 150.34, 141.41, 131.80, 127.37, 124.88, 121.47, 116.13, 113.59, 100.18, 68.92,

ACCEPTED MANUSCRIPT
31.20, 28.65, 28.62, 28.34, 25.29, 22.08, 13.95. ESI-HRMS calcd. for C21H26N3O2 [M + H]+
352.2020, found: 352.2028.
4.1.3.7. N-(3-cyano-4-isopropoxyphenyl)isonicotinamide (10g)
1
A brown powder, yield 74.0%. Mp 220.5-222.4 °C. H NMR (600 MHz, DMSO-d6) δ 10.61 (s,
1H), 8.80 (d, J = 5.9 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.94 (dd, J = 9.2, 2.6 Hz, 1H), 7.85 (dd, J
= 4.5, 1.4 Hz, 2H), 7.33 (d, J = 9.3 Hz, 1H), 4.76 (m, 1H), 1.32 (d, J = 6.0 Hz, 6H). 13C NMR (150
MHz, DMSO-d6) δ 163.92, 155.87, 150.35, 141.44, 131.78, 127.35, 124.97, 121.49, 116.33,
115.06, 101.19, 71.70, 21.65. ESI-HRMS calcd. for C16H16N3O2 [M + H]+ 282.1237, found:
282.1232.
4.1.3.8. N-(4-(sec-butoxy)-3-cyanophenyl)isonicotinamide (10h)
An off-white powder, yield 74.9%. Mp 165.8-166.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61
(s, 1H), 8.80 (dd, J = 4.4, 1.6 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.94 (dd, J = 9.2, 2.7 Hz, 1H),
7.85 (dd, J = 4.4, 1.6 Hz, 2H), 7.32 (d, J = 9.3 Hz, 1H), 4.56 (h, J = 6.0 Hz, 1H), 1.70 – 1.63 (m,
2H), 1.28 (d, J = 6.1 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 163.89,
156.18, 150.34, 141.43, 131.75, 127.35, 124.95, 121.48, 116.28, 114.98, 101.15, 76.40, 28.40,
18.85, 9.22. ESI-HRMS calcd. for C17H18N3O2 [M + H]+ 296.1394, found: 296.1386.
4.1.3.9. N-(3-cyano-4-isobutoxyphenyl)isonicotinamide (10i)
An off-white powder, yield 71.6%. Mp 174.1-175.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61
(s, 1H), 8.80 (d, J = 5.5 Hz, 2H), 8.09 (d, J = 2.3 Hz, 1H), 7.95 (dd, J = 9.1, 2.3 Hz, 1H), 7.85 (d, J
= 5.5 Hz, 2H), 7.30 (d, J = 9.2 Hz, 1H), 3.92 (d, J = 6.4 Hz, 2H), 2.06 (m, 1H), 1.01 (d, J = 6.7 Hz,
6H). 13C NMR (150 MHz, DMSO-d6) δ 163.90, 155.87, 150.34, 141.44, 131.78, 127.33, 124.96,
121.48, 116.33, 115.04, 101.20, 71.69. ESI-HRMS calcd. for C17H18N3O2 [M + H]+ 296.1394,
found: 296.1398.
4.1.3.10. N-(3-cyano-4-(isopentyloxy)phenyl)isonicotinamide (10j)
An off-white powder, yield 75.0%. Mp 159.1-161.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61
(s, 1H), 8.80 (dd, J = 4.4, 1.6 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H),

ACCEPTED MANUSCRIPT
7.85 (dd, J = 4.4, 1.6 Hz, 2H), 7.32 (d, J = 9.2 Hz, 1H), 4.16 (t, J = 6.6 Hz, 2H), 1.81 (m, 1H),
1.66 (q, J = 6.6 Hz, 2H), 0.95 (d, J = 6.7 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 163.89,
156.84, 150.35, 141.42, 131.79, 127.40, 124.91, 121.48, 116.14, 113.60, 100.16, 67.47, 37.06,
24.60, 22.38. ESI-HRMS calcd. for C18H20N3O2 [M + H]+ 310.1550, found: 310.1558.
4.1.3.11. N-(3-cyano-4-(cyclopentyloxy)phenyl)isonicotinamide (10k)
An off-white powder, yield 71.8%. Mp 218.2-219.7 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.61
(s, 1H), 8.80 (dd, J = 4.5, 1.5 Hz, 2H), 8.09 (d, J = 2.6 Hz, 1H), 7.94 (dd, J = 9.2, 2.6 Hz, 1H),
7.85 (dd, J = 4.4, 1.6 Hz, 2H), 7.30 (d, J = 9.2 Hz, 1H), 4.99 (t, J = 5.7 Hz, 1H), 1.95 (m, 2H),
1.80 – 1.65 (m, 4H), 1.67 – 1.51 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 163.90, 155.93,
150.34, 141.44, 131.73, 127.28, 124.96, 121.49, 116.22, 114.91, 100.98, 80.77, 32.16, 23.47.
ESI-HRMS calcd. for C18H18N3O2 [M + H]+ 308.1394, found: 308.1399.
4.1.3.12. N-(3-cyano-4-(2-methoxyethoxy)phenyl)isonicotinamide (10l)
A yellow crystal, yield 62.4%. Mp 228.0-229.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.62 (s,
1H), 8.80 (dd, J = 4.5, 1.5 Hz, 2H), 8.10 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.85
(dd, J = 4.5, 1.6 Hz, 2H), 7.32 (d, J = 9.2 Hz, 1H), 4.34 – 4.21 (m, 2H), 3.77 – 3.63 (m, 2H), 3.34
(s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 163.94, 156.70, 150.36, 141.43, 131.98, 127.36,
124.99, 121.49, 116.18, 113.72, 100.24, 70.09, 68.59, 58.41. ESI-HRMS calcd. for C16H16N3O3
[M + H]+ 298.1186, found: 298.1192.
4.1.3.13. N-(4-(benzyloxy)-3-cyanophenyl)isonicotinamide (10m)
A yellow powder, yield 77.5%. Mp 223.1-224.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.63 (s,
1H), 8.80 (d, J = 5.4 Hz, 2H), 8.12 (d, J = 2.6 Hz, 1H), 7.96 (dd, J = 9.2, 2.6 Hz, 1H), 7.85 (dd, J
= 4.5, 1.6 Hz, 2H), 7.49 (d, J = 7.4 Hz, 2H), 7.45 – 7.31 (m, 4H), 5.29 (s, 2H). 13C NMR (150
MHz, DMSO-d6) δ 163.94, 156.40, 150.36, 141.40, 136.04, 132.13, 128.58, 128.16, 127.61,
127.32, 124.99, 121.50, 116.15, 114.18, 100.54, 70.31. ESI-HRMS calcd. for C20H16N3O2 [M +
H]+ 330.1237, found: 330.1242.
4.1.3.14. N-(4-((4-chlorobenzyl)oxy)-3-cyanophenyl)isonicotinamide (10n)

ACCEPTED MANUSCRIPT
1
An off-white powder, yield 79.6%. Mp 225.9-227.1 °C. H NMR (600 MHz, DMSO-d6) δ 10.64
(s, 1H), 8.80 (d, J = 5.7 Hz, 2H), 8.12 (d, J = 2.5 Hz, 1H), 7.96 (dd, J = 9.2, 2.5 Hz, 1H), 7.85 (d, J
= 5.9 Hz, 2H), 7.51 (s, 4H), 7.38 (d, J = 9.2 Hz, 1H), 5.29 (s, 2H). 13C NMR (150 MHz, DMSO-d6)
δ 163.96, 156.19, 150.36, 141.39, 135.08, 132.79, 132.25, 129.45, 128.61, 127.31, 125.00, 121.50,
116.09, 114.18, 100.58, 69.50. ESI-HRMS calcd. for C20H15ClN3O2 [M + H]+ 364.0847, found:
364.0841.
4.1.3.15. N-(3-cyano-4-((4-cyanobenzyl)oxy)phenyl)isonicotinamide (10o)
An off-white powder, yield 84.2%. Mp > 240 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.65 (s, 1H),
8.80 (d, J = 5.8 Hz, 2H), 8.14 (d, J = 2.4 Hz, 1H), 7.96 (dd, J = 9.2, 2.5 Hz, 1H), 7.92 (d, J = 8.2
Hz, 2H), 7.85 (d, J = 5.8 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 9.2 Hz, 1H), 5.41 (s, 2H).
13C NMR (150 MHz, DMSO-d6) δ 163.98, 155.98, 150.37, 141.81, 141.38, 132.58, 132.38,
127.94, 127.35, 125.05, 121.48, 118.68, 116.02, 114.12, 110.80, 100.59, 69.31. ESI-HRMS calcd.
for C21H15N4O2 [M + H]+ 355.1190, found: 355.1198.
4.1.3.16. N-(3-cyano-4-((3-cyanobenzyl)oxy)phenyl)isonicotinamide (10p)
An off-white powder, yield 87.2%. Mp > 240 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.65 (s, 1H),
8.80 (d, J = 4.9 Hz, 2H), 8.14 (d, J = 2.3 Hz, 1H), 7.98 (dd, J = 9.1, 2.3 Hz, 1H), 7.95 (s, 1H), 7.84
(m, 4H), 7.66 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 9.2 Hz, 1H), 5.35 (s, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 163.96, 156.01, 150.36, 141.37, 137.78, 132.39, 132.32, 131.97, 131.02, 129.91,
127.34, 125.01, 121.48, 118.60, 116.03, 114.16, 111.56, 100.63, 69.17. ESI-HRMS calcd. for
C21H15N4O2 [M + H]+ 355.1190, found: 355.1186.
4.1.3.17. N-(3-cyano-4-((2-cyanobenzyl)oxy)phenyl)isonicotinamide (10q)
A light yellow crystal, yield 81.7%. Mp 221.5-222.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.66
(s, 1H), 8.80 (dd, J = 4.5, 1.5 Hz, 2H), 8.15 (d, J = 2.6 Hz, 1H), 8.01 (dd, J = 9.1, 2.6 Hz, 1H),
7.96 (d, J = 7.5 Hz, 1H), 7.86 (dd, J = 4.5, 1.6 Hz, 2H), 7.80 (m, 2H), 7.61 (td, J = 7.5, 1.6 Hz,
1H), 7.47 (d, J = 9.2 Hz, 1H), 5.43 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 163.98, 156.00,
150.36, 141.37, 139.01, 133.56, 133.40, 132.54, 129.42, 129.40, 127.35, 125.10, 121.49, 116.97,

ACCEPTED MANUSCRIPT
115.87, 114.09, 111.33, 100.60, 68.64. ESI-HRMS calcd. for C21H15N4O2 [M + H]+ 355.1190,
found: 355.1180.
4.1.3.18. N-(3-cyano-4-((4-methoxybenzyl)oxy)phenyl)isonicotinamide (10r)
An off-white powder, yield 85.6%. Mp > 240 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.62 (s, 1H),
8.80 (s, 2H), 8.10 (d, J = 2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.85 (d, J = 5.9 Hz, 2H), 7.41
(t, J = 8.5 Hz, 3H), 7.05 – 6.83 (m, 2H), 5.20 (s, 2H), 3.77 (s, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 163.93, 159.24, 156.49, 150.36, 141.41, 132.01, 129.59, 127.82, 127.29, 124.96,
121.49, 116.18, 114.27, 113.96, 100.55, 70.21, 55.11. ESI-HRMS calcd. for C21H18N3O3 [M + H]+
36031343, found: 360.1355.
4.1.3.19. N-(3-cyano-4-((2-methoxybenzyl)oxy)phenyl)isonicotinamide (10s)
A cyan crystal, yield 78.2%. Mp 208.6-209.7 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.63 (s, 1H),
8.80 (d, J = 5.0 Hz, 2H), 8.12 (s, 1H), 8.01 – 7.92 (m, 1H), 7.86 (d, J = 5.4 Hz, 2H), 7.45 (d, J =
7.3 Hz, 1H), 7.37 (m, 2H), 7.08 (d, J = 8.2 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 5.23 (s, 2H), 3.84 (s,
3H). 13C NMR (150 MHz, DMSO)-d6) δ 163.92, 156.95, 156.62, 150.35, 141.41, 132.05, 129.72,
128.88, 127.38, 124.97, 123.67, 121.48, 120.39, 116.19, 114.07, 111.07, 100.44, 66.01, 55.52.
ESI-HRMS calcd. for C21H18N3O3 [M + H]+ 360.1343, found: 360.1351.
4.1.4. synthesis of N-(4-alkoxy-3-cyanophenyl)nicotinamides (11a-o)
Compounds 11a-o were synthesized by the similar method for the preparation of compounds
10a-s with nicotinoyl chloride instead of isonicotinoyl chloride.
4.1.4.1. N-(3-cyano-4-propoxyphenyl)nicotinamide (11a)
1
An off-white powder, yield 86.3%. Mp 156.4-157.9 °C. H NMR (600 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.10 (d, J = 1.7 Hz, 1H), 8.77 (dd, J = 4.8, 1.6 Hz, 1H), 8.36 – 8.24 (m, 1H), 8.10 (d, J =
2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.58 (ddd, J = 8.0, 4.8, 0.7 Hz, 1H), 7.29 (d, J = 9.2
Hz, 1H), 4.10 (t, J = 6.4 Hz, 2H), 1.85 – 1.71 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). 13C NMR (150
MHz, DMSO-d6) δ 163.98, 156.69, 152.29, 148.64, 135.41, 132.07, 130.11, 127.27, 124.74,
123.55, 116.19, 113.59, 100.16, 70.31, 21.82, 10.20. ESI-HRMS calcd. for C16H16N3O2 [M + H]+

ACCEPTED MANUSCRIPT
282.1237, found: 282.1233.
4.1.4.2. N-(4-butoxy-3-cyanophenyl)nicotinamide (11b)
An off-white powder, yield 74.2%. Mp 82.0-83.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.27 (s,
1H), 9.14 (d, J = 1.8 Hz, 1H), 8.79 (dd, J = 4.8, 1.6 Hz, 1H), 8.37 – 8.28 (m, 1H), 7.93 (dd, J = 8.0,
1.4 Hz, 1H), 7.70 (dd, J = 7.8, 1.6 Hz, 1H), 7.64 – 7.54 (m, 1H), 7.33 (t, J = 7.9 Hz, 1H), 4.10 (t, J
= 6.3 Hz, 2H), 1.67 (m, 2H), 1.40 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 164.31, 155.21, 152.48, 148.72, 135.46, 132.58, 131.47, 131.03, 129.51, 124.51,
123.63, 116.21, 106.38, 74.36, 31.57, 18.48, 13.50. ESI-HRMS calcd. for C17H18N3O2 [M + H]+
296.1394, found: 296.1406.
4.1.4.3. N-(3-cyano-4-(pentyloxy)phenyl)nicotinamide (11c)
1
An off-white powder, yield 73.2%. Mp 124.0-124.6 °C. H NMR (600 MHz, DMSO-d6) δ 10.61
(s, 1H), 9.13 (d, J = 1.7 Hz, 1H), 8.79 (dd, J = 4.9, 1.6 Hz, 1H), 8.44 – 8.26 (m, 1H), 8.10 (d, J =
2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.69 – 7.55 (m, 1H), 7.29 (d, J = 9.2 Hz, 1H), 4.12 (t,
J = 6.5 Hz, 2H), 1.83 – 1.68 (m, 2H), 1.49 – 1.27 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (150
MHz, DMSO-d6) δ 163.78, 156.73, 151.76, 148.20, 136.05, 132.03, 130.30, 127.32, 124.78,
123.79, 116.20, 113.62, 100.15, 68.92, 28.06, 27.51, 21.80, 13.91. ESI-HRMS calcd. for
C18H20N3O2 [M + H]+ 310.1550, found: 310.1567.
4.1.4.4. N-(3-cyano-4-(hexyloxy)phenyl)nicotinamide (11d)
1
An off-white powder, yield 78.1%. Mp 121.0-122.6 °C. H NMR (600 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.10 (s, 1H), 8.77 (d, J = 4.0 Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.09 (d, J = 1.9 Hz, 1H),
7.94 (d, J = 9.1 Hz, 1H), 7.58 (dd, J = 7.7, 4.8 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 4.13 (t, J = 6.3
Hz, 2H), 1.85 – 1.64 (m, 2H), 1.44 (m, 2H), 1.32 (m, 4H), 0.89 (t, J = 6.6 Hz, 3H). 13C NMR (150
MHz, DMSO-d6) δ 163.99, 156.71, 152.30, 148.63, 135.41, 132.04, 130.11, 127.31, 124.77,
123.57, 116.18, 113.63, 100.15, 68.92, 30.86, 28.30, 24.96, 22.04, 13.87. ESI-HRMS calcd. for
C19H22N3O2 [M + H]+ 324.1707, found: 324.1712.
4.1.4.5. N-(3-cyano-4-(heptyloxy)phenyl)nicotinamide (11e)

ACCEPTED MANUSCRIPT
A brown crystal, yield 66.4%. Mp 119.0-120.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.55 (s,
1H), 9.10 (d, J = 1.8 Hz, 1H), 8.77 (dd, J = 4.8, 1.6 Hz, 1H), 8.34 – 8.23 (m, 1H), 8.09 (d, J = 2.6
Hz, 1H), 7.94 (dd, J = 9.2, 2.6 Hz, 1H), 7.62 – 7.53 (m, 1H), 7.29 (d, J = 9.2 Hz, 1H), 4.13 (t, J =
6.4 Hz, 2H), 1.82 – 1.68 (m, 2H), 1.47 – 1.38 (m, 2H), 1.30 (m, 6H), 0.87 (t, J = 6.9 Hz, 3H). 13C
NMR (150 MHz, DMSO-d6) δ 164.00, 156.71, 152.30, 148.62, 135.41, 132.03, 130.12, 127.32,
124.78, 123.57, 116.18, 113.64, 100.15, 68.92, 31.21, 28.35, 28.31, 25.27, 22.01, 13.94.
ESI-HRMS calcd. for C20H24N3O2 [M + H]+ 338.1863, found: 338.1869.
4.1.4.6. N-(3-cyano-4-(octyloxy)phenyl)nicotinamide (11f)
A brown crystal, yield 57.5%. Mp 117.3-118.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.55 (s, 1H),
9.10 (d, J = 1.9 Hz, 1H), 8.77 (dd, J = 4.8, 1.5 Hz, 1H), 8.28 (dt, J = 7.9, 1.9 Hz, 1H), 8.09 (d, J =
2.6 Hz, 1H), 7.94 (dd, J = 9.2, 2.6 Hz, 1H), 7.58 (dd, J = 7.8, 4.9 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H),
4.13 (t, J = 6.4 Hz, 2H), 1.81 – 1.69 (m, 2H), 1.50 – 1.40 (m, 2H), 1.37 – 1.20 (m, 8H), 0.86 (t, J =
6.9 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 164.00, 156.71, 152.30, 148.62, 135.41, 132.03,
130.11, 127.31, 124.77, 123.57, 116.18, 113.64, 100.15, 68.91, 31.18, 28.63, 28.60, 28.33, 25.29,
22.07, 13.96. ESI-HRMS calcd. for C21H26N3O2 [M + H]+ 352.2020, found: 352.2029.
4.1.4.7. N-(3-cyano-4-isopropoxyphenyl)nicotinamide (11g)
1
An off-white powder, yield 77.2%. Mp 191.6-192.4 °C. H NMR (600 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.10 (s, 1H), 8.77 (d, J = 3.7 Hz, 1H), 8.33 – 8.22 (m, 1H), 8.09 (d, J = 2.6 Hz, 1H), 7.93
(dd, J = 9.2, 2.7 Hz, 1H), 7.58 (dd, J = 7.6, 4.8 Hz, 1H), 7.32 (d, J = 9.3 Hz, 1H), 4.76 (m, 1H),
1.32 (d, J = 6.0 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 164.00, 155.72, 152.29, 148.63,
135.41, 132.04, 130.13, 127.22, 124.80, 123.56, 116.37, 115.08, 101.19, 71.68, 21.65. ESI-HRMS
calcd. for C16H16N3O2 [M + H]+ 282.1237, found: 282.1239.
4.1.4.8. N-(4-(sec-butoxy)-3-cyanophenyl)nicotinamide (11h)
An off-white powder, yield 57.4%. Mp 101.8-109.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.11 (s, 1H), 8.77 (d, J = 3.8 Hz, 1H), 8.37 – 8.22 (m, 1H), 8.09 (d, J = 2.6 Hz, 1H), 7.93
(dd, J = 9.2, 2.7 Hz, 1H), 7.65 – 7.54 (m, 1H), 7.31 (d, J = 9.3 Hz, 1H), 4.55 (h, J = 6.0 Hz, 1H),
1.75 – 1.54 (m, 2H), 1.28 (d, J = 6.1 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6) δ 163.99, 156.04, 152.29, 148.64, 135.41, 132.01, 130.12, 127.26, 124.80, 123.56,
116.33, 115.01, 101.14, 76.39, 28.41, 18.87, 9.23. ESI-HRMS calcd. for C17H18N3O2 [M + H]+
296.1394, found: 296.1384.
4.1.4.9. N-(3-cyano-4-isobutoxyphenyl)nicotinamide (11i)
An off-white powder, yield 64.5%. Mp 158.1-160.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.11 (s, 1H), 8.77 (s, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.10 (d, J = 2.4 Hz, 1H), 7.94 (dd, J =
9.1, 2.4 Hz, 1H), 7.58 (dd, J = 7.6, 4.9 Hz, 1H), 7.29 (d, J = 9.1 Hz, 1H), 3.91 (d, J = 6.4 Hz, 2H),
2.06 (m, 1H), 1.01 (d, J = 6.7 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 163.97, 156.77, 152.29,
148.63, 135.40, 132.07, 130.10, 127.27, 124.70, 123.55, 116.12, 113.64, 100.17, 74.84, 27.62, 18.77.
ESI-HRMS calcd. for C17H18N3O2 [M + H]+ 296.1394, found: 296.1397.
4.1.4.10. N-(3-cyano-4-(isopentyloxy)phenyl)nicotinamide (11j)
1
An off-white powder, yield 68.5%. Mp 130.2-132.2 °C. H NMR (600 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.10 (d, J = 1.5 Hz, 1H), 8.77 (dd, J = 4.7, 1.3 Hz, 1H), 8.40 – 8.20 (m, 1H), 8.09 (d, J =
2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.58 (dd, J = 7.8, 4.9 Hz, 1H), 7.31 (d, J = 9.2 Hz, 1H),
4.15 (t, J = 6.6 Hz, 2H), 1.82 (m, 1H), 1.66 (q, J = 6.7 Hz, 2H), 0.95 (d, J = 6.7 Hz, 6H). 13C NMR
(150 MHz, DMSO-d6) δ 163.96, 156.69, 152.28, 148.64, 135.40, 132.06, 130.10, 127.25, 124.74,
123.54, 116.19, 113.55, 100.15, 67.45, 37.08, 24.61, 22.38. ESI-HRMS calcd. for C18H20N3O2 [M
+ H]+ 310.1550, found: 310.1546.
4.1.4.11. N-(3-cyano-4-(cyclopentyloxy)phenyl)nicotinamide (11k)
1
An off-white powder, yield 70.2%. Mp 165.0-166.7 °C. H NMR (600 MHz, DMSO-d6) δ 10.54
(s, 1H), 9.10 (d, J = 1.7 Hz, 1H), 8.77 (dd, J = 4.8, 1.6 Hz, 1H), 8.37 – 8.22 (m, 1H), 8.09 (d, J =
2.6 Hz, 1H), 7.93 (dd, J = 9.2, 2.7 Hz, 1H), 7.58 (m, 1H), 7.29 (d, J = 9.2 Hz, 1H), 4.98 (t, J = 5.7
Hz, 1H), 2.02 – 1.83 (m, 2H), 1.81 – 1.67 (m, 4H), 1.67 – 1.54 (m, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 163.99, 155.79, 152.29, 148.63, 135.41, 131.98, 130.13, 127.18, 124.81, 123.56,
116.27, 114.93, 100.97, 80.76, 32.17, 23.47. ESI-HRMS calcd. for C18H18N3O2 [M + H]+
308.1394, found: 308.1390.

ACCEPTED MANUSCRIPT
4.1.4.12. N-(3-cyano-4-(2-methoxyethoxy)phenyl)nicotinamide (11l)
An off-white crystal, yield 86.4%. Mp 125.0-126.4 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.56 (s,
1H), 9.10 (d, J = 1.8 Hz, 1H), 8.77 (dd, J = 4.8, 1.6 Hz, 1H), 8.38 – 8.20 (m, 1H), 8.10 (d, J = 2.6
Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.58 (m, 1H), 7.31 (d, J = 9.2 Hz, 1H), 4.27 (dd, J = 5.3,
3.7 Hz, 2H), 3.79 – 3.65 (m, 2H), 3.34 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 164.03, 156.56,
152.32, 148.63, 135.43, 132.23, 130.12, 127.26, 124.84, 123.58, 116.23, 113.73, 100.21, 70.10,
68.58, 58.41. ESI-HRMS calcd. for C16H16N3O3 [M + H]+ 298.1186, found: 298.1196.
4.1.4.13. N-(4-(benzyloxy)-3-cyanophenyl)nicotinamide (11m)
A light yellow crystal, yield 90.2%. Mp 181.8-183.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.57
(s, 1H), 9.10 (s, 1H), 8.12 (d, J = 2.6 Hz, 1H), 8.77 (d, J = 3.6 Hz, 1H), 8.33 – 8.22 (m, 1H), 7.95
(dd, J = 9.2, 2.6 Hz, 1H), 7.58 (m, 1H), 7.49 (d, J = 7.0 Hz, 2H), 7.46 – 7.34 (m, 4H), 5.29 (s, 2H).
13C NMR (150 MHz, DMSO-d6) δ 164.04, 156.26, 152.32, 148.64, 136.06, 135.43, 132.39,
130.10, 128.58, 128.16, 127.61, 127.20, 124.84, 123.58, 116.20, 114.18, 100.53, 70.31.
ESI-HRMS calcd. for C20H16N3O2 [M + H]+ 330.1237, found: 330.1235.
4.1.4.14. N-(4-((4-chlorobenzyl)oxy)-3-cyanophenyl)nicotinamide (11n)
1
An off-white powder, yield 88.7%. Mp 230.2-231.7 °C. H NMR (600 MHz, DMSO-d6) δ 10.58
(s, 1H), 9.10 (d, J = 1.6 Hz, 1H), 8.77 (dd, J = 4.8, 1.6 Hz, 1H), 8.43 – 8.19 (m, 1H), 8.12 (d, J =
2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.58 (m, 1H), 7.55 – 7.44 (m, 4H), 7.38 (d, J = 9.2 Hz,
1H), 5.29 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 164.04, 156.04, 152.33, 148.64, 135.43,
135.11, 132.78, 132.50, 130.08, 129.45, 128.61, 127.18, 124.84, 123.57, 116.13, 114.18, 100.56,
69.49. ESI-HRMS calcd. for C20H15ClN3O2 [M + H]+ 364.0847, found: 364.0843.
4.1.4.15. N-(3-cyano-4-((4-cyanobenzyl)oxy)phenyl)nicotinamide (11o)
An off-white powder, yield 75.1%. Mp > 230 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.59 (s, 1H),
9.10 (s, 1H), 8.78 (d, J = 3.8 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.96 (dd,
J = 9.2, 2.5 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H), 7.59 (dd, J = 7.8, 4.8 Hz,
1H), 7.37 (d, J = 9.2 Hz, 1H), 5.41 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 164.05, 155.83,
152.34, 148.64, 141.83, 135.42, 132.65, 132.57, 130.06, 127.92, 127.19, 124.87, 123.57, 118.68,

ACCEPTED MANUSCRIPT
116.06, 114.09, 110.80, 100.57, 69.30. ESI-HRMS calcd. for C21H15N4O2 [M + H]+ 355.1190,
found: 355.1195.
4.1.5. Synthesis of compounds 12b, 12i, 12j, 12m and 12n
Compounds 12b, 12i, 12j, 12m and 12n were synthesized by the similar method for the
preparation of compounds 10a-s with picolinoyl chloride instead of isonicotinoyl chloride.
4.1.5.1. N-(4-butoxy-3-cyanophenyl)picolinamide (12b)
An off-white powder, yield 77.0%. Mp 62.9-64.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.64 (s,
1H), 8.71 (d, J = 4.6 Hz, 1H), 8.67 (dd, J = 8.3, 1.4 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.10 (td, J =
7.7, 1.6 Hz, 1H), 7.72 (ddd, J = 7.5, 4.7, 1.0 Hz, 1H), 7.54 (dd, J = 7.8, 1.3 Hz, 1H), 7.33 (t, J =
8.0 Hz, 1H), 4.22 (t, J = 6.2 Hz, 2H), 1.89 – 1.77 (m, 2H), 1.65 – 1.54 (m, 2H), 0.94 (t, J = 7.4 Hz,
3H). 13C NMR (150 MHz, DMSO-d6) δ 161.69, 150.72, 148.59, 148.57, 138.57, 131.80, 128.20,
127.58, 124.92, 124.59, 122.18, 116.19, 105.07, 75.03, 31.70, 18.59, 13.62. ESI-HRMS calcd. for
C17H18N3O2 [M + H]+ 296.1394, found: 296.1412.
4.1.5.2. N-(3-cyano-4-isobutoxyphenyl)picolinamide (12i)
1
An off-white powder, yield 82.4%. Mp 94.7-96.9 °C. H NMR (600 MHz, DMSO-d6) δ 10.84 (s,
1H), 8.74 (d, J = 4.5 Hz, 1H), 8.23 (m, 1H), 8.16 – 8.11 (m, 2H), 8.07 (td, J = 7.7, 1.4 Hz, 1H),
7.75 – 7.61 (m, 1H), 7.26 (d, J = 9.2 Hz, 1H), 3.90 (d, J = 6.5 Hz, 2H), 2.05 (m, 1H), 1.00 (d, J =
6.7 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 162.62, 156.69, 149.59, 148.44, 138.12, 131.81,
127.28, 127.02, 124.70, 122.46, 116.20, 113.50, 100.13, 74.82, 27.62, 18.77. ESI-HRMS calcd.
for C17H18N3O2 [M + H]+ 296.1394, found: 296.1399.
4.1.5.3. N-(3-cyano-4-(isopentyloxy)phenyl)picolinamide (12j)
An off-white powder, yield 71.1%. Mp 90.0-92.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.85 (s,
1H), 8.74 (d, J = 4.6 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 8.17 – 8.13 (m, 2H), 8.07 (td, J = 7.7, 1.4
Hz, 1H), 7.68 (dd, J = 7.4, 4.8 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 4.15 (t, J = 6.6 Hz, 2H), 1.87 –
1.74 (m, 1H), 1.66 (m, 2H), 0.93 (m, 6H). 13C NMR (150 MHz, DMSO-d6) δ 162.62, 156.69,
149.59, 148.44, 138.12, 131.81, 127.28, 127.02, 124.70, 122.46, 116.20, 113.50, 100.13, 74.82,

ACCEPTED MANUSCRIPT
27.62, 18.77. ESI-HRMS calcd. for C18H20N3O2 [M + H]+ 310.1550, found: 310.1541.
4.1.5.4. N-(4-(benzyloxy)-3-cyanophenyl) picolinamide (12m)
1
An off-white powder, yield 77.5%. Mp 180.2-181.9 °C. H NMR (600 MHz, DMSO-d6) δ 10.57
(s, 1H), 9.10 (d, J = 1.9 Hz, 1H), 8.77 (dd, J = 4.8, 1.6 Hz, 1H), 8.33 – 8.24 (m, 1H), 8.12 (d, J =
2.6 Hz, 1H), 7.95 (dd, J = 9.2, 2.6 Hz, 1H), 7.58 (dd, J = 7.9, 4.8 Hz, 1H), 7.49 (d, J = 7.2 Hz, 2H),
7.43 (t, J = 7.6 Hz, 2H), 7.41 – 7.33 (m, 2H), 5.29 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ
164.03, 156.25, 152.32, 148.63, 136.06, 135.42, 132.38, 130.09, 128.57, 128.15, 127.60, 127.19,
124.83, 123.56, 116.19, 114.18, 100.52, 70.30. ESI-HRMS calcd. for C20H16N3O2 [M + H]+
330.1237, found: 330.1235.
4.1.5.5. N-(4-((4-chlorobenzyl)oxy)-3-cyanophenyl)picolinamide (12n)
1
An off-white powder, yield 79.4%. Mp 165.7-168.5 °C. H NMR (600 MHz, DMSO-d6) δ 10.88
(s, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.28 (d, J = 2.6 Hz, 1H), 8.20 – 8.11 (m, 2H), 8.07 (td, J = 7.7,
1.6 Hz, 1H), 7.69 (ddd, J = 7.5, 4.8, 1.0 Hz, 1H), 7.56 – 7.46 (m, 4H), 7.36 (d, J = 9.2 Hz, 1H),
5.29 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 162.68, 155.95, 149.54, 148.45, 138.15, 135.14,
132.75, 132.24, 129.42, 128.60, 127.20, 127.06, 124.84, 122.50, 116.21, 114.02, 100.48, 69.44.
ESI-HRMS calcd. for C20H15ClN3O2 [M + H]+ 364.0847, found: 364.0840.
4.1.6. synthesis of N-(4-(benzyloxy)-3-cyanophenyl)benzamide (13)
Compound 13 was synthesized by the similar procedure for the synthesis of compounds 10 with
benzoyl chloride instead of isonicotinoyl chloride.
A white crystal, yield 85.3%. Mp 194.2-195.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H),
8.13 (d, J = 2.6 Hz, 1H), 8.01 – 7.88 (m, 3H), 7.61 (t, J = 7.3 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H),
7.49 (d, J = 7.3 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.37 (m, 2H), 5.29 (s, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 165.50, 156.04, 136.10, 134.40, 132.76, 131.79, 128.56, 128.46, 128.13, 127.60,
127.13, 124.75, 116.27, 114.10, 100.45, 70.28. ESI-HRMS calcd. for C21H17N2O2 [M + H]+
329.1285, found: 329.1292.
4.2. Assay of the in vitro XO inhibitory activity

ACCEPTED MANUSCRIPT
In vitro bovine XO inhibitory potency was assayed spectrophotometrically by measuring the
uric acid formation at 294 nm using a UV-2100 spectrophotometer (UNICO) at 25°C. The testing
method was based on the procedure reported by Matsumoto et al. [11], with modification. The
assay mixture contained 0.1 M sodium pyrophosphate buffer (pH 8.3), 0.3 mM Na2EDTA, 1 mM
xanthine, 25 U/L XO (Sigma, X4875), and the test compound. The inhibition of XO by various
compounds was measured by the reduction of the uric acid concentration. The enzyme was
pre-incubated for 10 min with the test compound, and the reaction was started by an addition of
xanthine. Allopurinol and topiroxostat were used as positive controls. All tests were performed in
triplicate. Compounds presenting inhibitory effects over 60% at a concentration of 50 µM were
further tested at a wide range of concentrations to calculate their IC50 values using SPSS 20.0
software.
4.3. Molecular modeling
Molecular modeling studies were performed with MOE (Molecular Operating Environment,
version 2016.08) software [32]. The crystal structure of XO in complex with topiroxostat (PDB
code: 1V97) [29] obtained from the Protein Data Bank was adopted in docking calculations. The
structure was protonated, polar hydrogens were added and energy minimization was carried out
(RMSD gradient = 0.1 kcal/mol, AMBER10: EHT field) [33]. The docking procedure used the
standard protocol implemented in MOE and all parameters were maintained as the defaults. The
ligand–enzyme complex with the lowest S score was selected.
Acknowledgments
This work was supported by the Chinese National Science Foundation (Grant Nos. 81274182
and 81573687) and was the project of a Liaoning distinguished professor.
References
[1]
[2]
M. Gliozzi, N. Malara, S. Muscoli, V. Mollace, The treatment of hyperuricemia, Int. J. Cardiol.
213(2016) 23-27.
C. Chen, J.-M. Lü, Q. Yao, Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR)