Exploration of 2-((Pyridin-4-ylmethyl)amino)nicotinamide Derivatives as Potent Reversal Agents against P-Glycoprotein-Mediated Multidrug Resistance

Article abstract of DOI:10.1021/acs.jmedchem.6b01879

Overexpression of the ATP-binding cassette (ABC) transport proteins, like ABCB1, commonly referred to as P-glycoprotein (P-gp), initiates active efflux of a broad spectrum of unrelated chemotherapeutic drugs in structure and function, leading to chemotherapy failure. A series of 2-((pyridin-4-ylmethyl)amino)nicotinamide derivatives as potent reversal agents against P-glycoprotein-mediated multidrug resistance (MDR) were designed and synthesized. The majority of target compounds displayed great reversal potency, especially 9n. In-depth studies demonstrated 9n has high potency (EC₅₀ = 119.6 ± 6.9 nM), low cytotoxicity, and long duration (>24 h) in reversing adriamycin (ADM) resistance in K562/A02 cells. 9n also improved the effects of other cytotoxic agents related to MDR, increased accumulation of ADM, interrupted P-gp-mediated Rh123 efflux function, and suppressed P-gp ATPase activity in K562/A02 MDR cells. The Western blot analysis indicated that the MDR reversal by 9n was not due to a decrease in protein expression. Besides, the effect of CYP3A4 was not influenced by 9n, avoiding the toxicity caused by drug interactions. The study yielded 9n with superior properties compared to the classical inhibitor verapamil (VRP) and leading compound apatinib.

Full text of DOI:10.1021/acs.jmedchem.6b01879

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Article
Exploration of 2-((pyridin-4-ylmethyl)amino)nicotinamide derivatives as
potent reversal agents against P-glycoprotein-mediated multidrug resistance
Qianqian Qiu, Wei Shi, Zheng Li, Bo Zhang, Miaobo Pan, Jian Cui, Yuxuan Dai, Wenlong Huang, and Hai Qian
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01879 • Publication Date (Web): 16 Mar 2017
Downloaded from http://pubs.acs.org on March 16, 2017
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Exploration
of
2-((pyridin-4-ylmethyl)amino)nicotinamide
derivatives as potent reversal agents against P-glycoprotein-mediated
multidrug resistance
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a
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a
Qianqian Qiu , Wei Shi , Zheng Li , Bo Zhang , Miaobo Pan , Jian Cui , Yuxuan Dai , Wenlong
a,b,
a,b,
Huang * and Hai Qian
*
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing, PR China
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing, PR China
ABSTRACT
Overexpression of the ATPꢀbinding cassette (ABC) transport proteins, like ABCB1,
commonly referred to as Pꢀglycoprotein (Pꢀgp), initiates active efflux of a broad spectrum of
unrelated chemotherapeutic drugs in structure and function, leading to chemotherapy failure. A
series of 2ꢀ((pyridinꢀ4ꢀylmethyl)amino)nicotinamide derivatives as potent reversal agents against
Pꢀglycoproteinꢀmediated multidrug resistance (MDR) were designed and synthesized. The
of target compounds displayed great reversal potency, especially 9n. Inꢀdepth studies
9n with high potency (EC50 = 119.6 ± 6.9 nM), low cytotoxicity and long duration (> 24 h) in
reversing adriamycin (ADM)ꢀresistance in K562/A02 cells. 9n also improved the effects of other
cytotoxic agents related to MDR, increased accumulation of ADM, interrupted Pꢀgpꢀmediated
Rh123 efflux function, and suppressed Pꢀgp ATPase activity in K562/A02 MDR cells. The
blot analysis indicated the MDR reversal by 9n was not due to a decrease in protein expression.
Besides, the effect of CYP3A4 was not influenced by 9n, avoiding the toxicity caused by drug
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interactions. The study yielded 9n with superior properties compared to the classical inhibitor
Verapamil (VRP) and leading compound Apatinib.
KEYWORDS: Pꢀglycoprotein; Nicotinamide; Multidrug resistance; Reversal agents
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INTRODUCTION
Multidrug resistance (MDR), which is characterized by crossꢀresistance to a broad spectrum
of unrelated chemotherapeutic drugs in structure and function, is a pathophysiological
1
phenomenon that limits the chemotherapeutic outcomes in cancer treatment in clinic.
Overexpression of Pꢀglycoprotein (Pꢀgp, ABCB1), a main member of ATPꢀbinding cassette (ABC)
transport proteins, plays a key role in the progress of MDR in cancer. Pꢀgp is an integral
membrane protein comprising of 1280 amino acids located at the cytoplasmatic face of the
membrane. It has two homologous halves, each one containing a transmembrane domain (TMD)
2ꢀ4
and a hydrophilic nucleotide binding domain (NBD). The development of agents to evade the
5
ꢀ7
Pꢀgp efflux seems to be a practical approach to circumvent MDR. Various mechanisms are
responsible for the inhibition of anticancer drug efflux in cancer cells overexpressing Pꢀgp,
consisting in blocking drug binding sites competitively, noncompetitively, allosterically, the
8ꢀ12
inhibition of ATP hydrolysis, and the alteration of the integrity of cell membrane lipids.
In the
last three decades, great efforts have been made to study a wide series of Pꢀgp inhibitors, including
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verapamil and cyclosporin A of first generation,
dexverapamil and valspodar of second
1
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generation,
also tariquidar and zosuquidar of third generation.
Some specific physico–
chemical characteristics, such as positive charge at neutral pH, lipophilicity, and presence of
aromatic rings, are extensively used in the design of Pꢀgp modulators to conquer the limitations of
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1, 19, 20
previous generation compounds.
Some of the current Pꢀgp inhibitors have stopped clinic
studies due to inherent side effects, such as high cytotoxicity, low inhibitory potencies, and drug
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interactions.
Some others are ongoing,
therefore the pharmacotherapeutic significance of
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ABCB1 in cancer treatment emerged always. Discovering novel and potent Pꢀgp inhibitors to
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reverse drug resistance is in a strong need for development.
At present, 2ꢀ((pyridinꢀ4ꢀylmethyl) amino) nicotinamide based tyrosine kinase inhibitors
(TKIs), for example, Apatinib (YN968D1, 1) and Motesanib (AMG706, 2) have been reported to
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be potent inhibitors of Pꢀgp (Figure 1).
On the basis of the docking analysis of Motesanib
33
with human ABCB1 homology previously reported by Wang et.al., the molecule may bind to all
transmembrane drugꢀbinding sites of ABCB1 due to exceptional flexibility and large drugꢀbinding
site present in ABCB1 as shown in Figure 1A: the pyridineꢀ4ꢀylꢀmethyl amino moiety and
nicotinamide moiety of Motesanib were involved in hydrophobic contacts with the hydrophobic
pockets lined with several residues, respectively; besides, a hydrogen bond was formed with the
N1 atom of nicotinamide moiety, and large hydrophobic pocket encompassed the
3,3ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ6ꢀyl moiety. In this study, extensive efforts have been devoted
to the development of Pꢀgp inhibitors. By introducing different hydrophobic building blocks to the
nicotinamide domain, a series of novel Pꢀgp reversal agents with strong bioactivity, high safety
profile, and outstanding affinity to Pꢀgp target have been developed, which affords new insights
into the structure−activity relationship (SAR) to further exploit novel Pꢀgp reversal agents (Figure
1B).
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Figure 1. A) A two dimensional diagram with important interactions of Motesanib with the
drugꢀbinding site residues of human ABCB1 homology, cyan bubbles: polar; green bubbles:
hydrophobic residues; purple dotted arrow: hydrogen bond; B) Design of target compounds.
RESULTS AND DISCUSSION
Chemistry
The synthetic route for target compounds 8a-s and 9a-n is depicted in Scheme 1. The structures of
all target compounds are presented in Table 1. The key intermediate 5 was prepared through a
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copper oxide catalyzed nucleophilic aromatic substitution.
Then the reaction of compound 5
with the corresponding amines in CH Cl gave target aryl amides 8a-s and 9a-n. The chemical
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structures of the target compounds were confirmed by H NMR, C NMR and MS. Besides,
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highꢀperformance liquid chromatography (HPLC) was employed to ascertain the purity of target
compounds. Results indicated that all target compounds had a purity superior to 95%
(
Experimental Section).
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Scheme 1. Synthesis of 2ꢀ((pyridinꢀ4ꢀylmethyl) amino) nicotinamides 8a-s and 9a-n. Reagents
2 3
and conditions: (i) CuO (0.1 equiv), K CO , 110 °C, 2 h; (ii) EDCI/HOBT, DMF, 16 h at room
temperature.
Table 1. Structures of the synthesized compounds 8a-s and 9a-n.
Compound
8a
R
Compound
8h
R
Compound
8o
R
8b
8i
8p
8
c
8j
8k
8l
8q
8r
8s
8d
8
e
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Biological evaluation
Intrinsic toxicity of target compounds and the ability to reverse ADM-resistance
Looking forward an optimal template to replace the hydrophobic amine domain of 1, the
cytotoxicity and reversal activity of the first synthesized 19 compounds 8a-s were investigated in
K562 and its ADMꢀselected derivative Pꢀgp overexpressing K562/A02 cells by MTT method. It
was obvious that resistant K562/A02 cells (IC50 for ADM of 53.95 ± 2.11 ꢁM) displayed about
1
05.8ꢀfold resistance than sensitive K562 cells (IC50 for ADM of 0.51 ± 0.05 ꢁM) as indicated in
Table 2. Most of the target compounds displayed low intrinsic cytotoxicity against both K562 and
K562/A02 cells, because their IC50 values were at a high micromolar level (IC50s > 100 ꢁM) in the
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two cell lines. In contrast, compound 8b, 8e and 8n are found to exhibit higher toxicity (IC50 < 30
ꢁM) in both cell lines, as well as the positive control 1 (IC50s of 15.87 ± 0.36 ꢁM and 19.76 ± 0.89
ꢁM in K562 and K562/A02 respectively). VRP has lower cytotoxic effect toward the two cell
lines with IC50 values of 37.3 and 31.2 ꢁM. Based on the IC50s and related data, the survival rates
of 8a-s at the concentration of 5.0 ꢁM were all above 90%, suitable for testing its inhibitory effect
on Pꢀgp in the MDR cell line. Furthermore, the majority of target compounds had almost equal
IC50 values of cytotoxicity against both K562 and K562/A02 cells, suggesting that unlike ADM,
they were not possible to be substrates of Pꢀgp.
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a
Table 2 Cytotoxicity of the target compounds 8a-s against K562 and K562/A02 cell lines
IC50 (µM)
K562/A02
IC50 (µM)
Compound
K562
>100
Compound
K562
K562/A02
>100
8a
>100
28.69 ± 1.13
>100
8l
8m
8n
66.12 ± 0.55
>100
8b
24.24 ± 0.86
>100
>100
8
c
16.97 ± 1.41
39.52 ± 0.38
>100
27.93 ± 0.59
>100
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>100
>100
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8e
78.92 ± 3.63
>100
27.93 ± 0.59
>100
8p
>100
8f
8q
>100
>100
8g
>100
>100
8r
>100
>100
8h
>100
>100
8s
51.36 ± 1.55
37.30 ± 1.88
15.87 ± 0.36
0.51 ± 0.05
>100
8
i
>100
>100
VRP
1
31.28 ± 2.19
19.76 ± 0.89
53.95 ± 2.11
8j
>100
>100
8k
>100
>100
ADM
a
MTT method was used for determination of the IC50s for the target compounds. Data was
analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three independent
tests.
Then the reversal activity of 8a-s in ADMꢀresistance were evaluated preliminarily in
K562/A02 cells at nonꢀtoxic concentration of 5.0 ꢁM by MTT. When the hydrophobic region was
replaced with the monocycle series, we got the target compounds 8a-j. As depicted in Table 3,
aniline substituted with electronꢀdonating, electronꢀwithdrawing and hindered group (8a-f)
excreted no promoting activity, just similar to VRP (RF = 5.42). Once extending carbon chain
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length of arylalkylamines (8g-j), this modification increased the RF value considerably from 8.63
of 1 to 16.60 of 8i, the activity of 8g was slightly inferior to 1, a minor improvement of the
inhibitory activity could be achieved in 8h, while the substitution of
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ꢀ(thiophenꢀ3ꢀyl)ethanꢀ1ꢀamine almost deprived the reversal activity of 8j, suggesting the
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importance of a suitable chain length and the hydrogenꢀbond interaction of dimethoxy group
contribution to the potency of 8i. However, compounds containing fusedꢀrings in the domain
(8k-m) which may possess limited hydrophobic pocket space and less flexibility, showed no
positive effect on reversing ADMꢀresistance and exhibited lower RF values than 1. Surprisingly,
in biaryl series, aniline substituted with Nꢀcontaining alicyclic series and 1,4'ꢀbipiperidine series
(8n-s), most compounds retained the reversal activity as VRP, even 8n and 8q exhibited much
higher RF value than 1, of 12.69 and 18.80 respectively. A major contribution to this considerable
activity is likely due to flexible hydrophobic rings and polar oxygen atom. Hydrophilic
heterocyclic such as thiamethoxam azole would have the opposite effect, such as 8o. The
bialicyclic compound 8s reduced the πꢀπ stocking interactions, resulting in a significant drop of
activity. The biphenyl domain associated with the larger hydrophobic effect promoted the potency
of 8n, simultaneously the oxygen atom in morpholine perhaps interacted with the hydrogen bond
acceptor of Pꢀgp, resulting in approximately 2ꢀfold increase in potency of 8q over 1. All the
preliminary results demonstrated appropriate length of carbon chain could reach the extra
hydrophobic pockets and interactions of πꢀπ stocking and hydrogen bond were necessary.
a
Table 3 ADMꢀresistance reversal activity of 8a-s at 5.0 ꢀM concentration in K562/A02 cells .
Compound
8a
IC50/ADM (µM)
14.33 ± 2.37
6.41± 0.39
RF
Compound
IC50/ADM (µM)
12.73 ± 1.67
32.91 ± 1.91
4.25 ± 0.28
RF
4.24
1.69
12.69
4.60
5.21
3.76
8.42
4.27
5.29
6.60
8l
8m
8n
8o
8
b
8c
12.63 ± 0.56
10.20 ± 1.25
8.17 ± 0.70
8
d
11.72 ± 1.34
10.34 ± 1.27
8e
8p
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f
15.39 ± 1.07
6.81 ± 0.82
5.63 ± 0.96
3.25 ± 0.76
39.35 ± 2.80
6.97 ± 0.28
3.51
7.92
9.58
16.60
1.37
7.74
8q
8r
2.87 ± 0.16
10.94 ± 1.23
45.10 ± 4.89
9.95 ± 0.98
6.25 ± 1 .23
53.95 ± 2.11
18.80
4.93
1.20
5.42
8.63
1.00
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8
i
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Control
a
The IC50 value was determined after exposure to a series of ADM concentrations with different
compounds at 5.0 ꢀM in K562/A02 cells; Reversal fold (RF, foldꢀchange in drug sensitivity) =
b
(IC50 without inhibitor / (IC50 with inhibitor). 0.1% DMSO was added as solvent control. Data
were analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three
independent tests.
To better explore the hydrophobic template for nicotinamide Pꢀgp inhibitors, we therefore
selected 8i, 8n and 8q as our starting points for further modification. Our optimized efforts were
directed to chain elongation and increase of rings to find more hydrophobic pockets of Pꢀgp.
Firstly, the cytotoxicity of compounds 9a-n was determined (Table 4). All the compounds had
high IC50 values towards both K562 and K562/A02 cell lines (IC50s > 50 ꢁM). 9a-n at the
concentration of 5.0 ꢁM had no toxic effect on the cells and was used to investigate reversal of
resistance to ADM in K562/A02 cells.
a
Table 4 Cytotoxicity of the target compounds 9a-n against K562 and K562/A02 cell lines
IC50 (µM)
K562/A02
>100
>100
IC50 (µM)
K562/A02
>100
>100
Compound
K562
Compound
K562
9
9
a
>100
69.97 ± 5.24
9j
>100
>100
b
9k
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>100
>100
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>100
82.15 ± 2.85
86.03 ± 3.93
>100
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>100
>100
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>100
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4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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2
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9
0
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9
0
1
2
3
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7
8
9
0
h
72.55± 2.95
>100
85.73 ± 1.74
>100
ADM
9i
a
MTT method was used for determination of the IC s for the target compounds. Data was
50
analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three independent
tests.
The reversal potency of 9a-n was presented in Table 5. In comparison with the parent 8n, the
triꢀring derivative 9b and the chainꢀextended flexible compound 9d kept excellent reversal activity,
due to the distance and the bulk of the relevant deep hydrophobic pockets. While the activity of 9a
and 9c was in a sharp decline, it is possible that their improper conformation prevented stretching
into the pockets, and so 9e is. Prolongation of the chain in 8q brought unpleasant response, the
compounds 9f and 9g exhibited markedly reduced reversal activity, even vanished, indicating the
hydrophobic structure of 8q represented the most suitable length for one of the pockets.
Gratifyingly, based on 8i, the activity of 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline derivatives
were proved. 9h, the cyclization derivative of 8i, showed a slight improvement in potency, while
presence of more hindered rings at the αꢀposition caused steric hindrance to generate a sharp drop
in activity (9i - m). Fortunately, adopting 7n as the amine source contributed to the optimized
compound 9n and resulted in the RF value up to 32.26, which was almost 4ꢀfold higher than that
of
1.
The
results
indicated
that
modifications
at
αꢀposition
of
6
,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline played a sharp declining role in the reversal activity,
and the sufficient chain length and benzene rings prompted 9n to stretch into deep pockets and
interact with more hydrophobic regions. After the completion of evaluation for reversal activity of
all compounds, clear SARs had been developed and summarized in Figure 2.
a
Table 5 ADMꢀresistance reversal activity of 9a-n at 5.0 ꢀM concentration in K562/A02 cells .
Compound
IC50/ADM (µM)
RF
Compound
IC50/ADM (µM)
RF
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a
14.40 ± 1.47
4.16 ± 0.76
44.35 ± 3.21
5.60 ± 0.64
7.14 ± 1.30
10.07 ± 1.02
27.83 ± 2.31
3.16 ± 0.95
21.34 ± 2.58
3.94
13.65
1.28
9j
37.35 ± 2.57
19.18 ± 1.39
22.62 ± 1.85
16.50 ± 1.42
1.76 ± 0.21
10.59 ± 0.69
6.52 ± 1 .13
56.77 ± 3.62
1.52
2.96
2.51
3.44
32.26
5.36
8.71
1.00
9k
b
9l
9
c
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9
m
9d
10.14
7.95
9
n
9
e
9
f
5.64
VRP
1
9
9
g
2.04
b
h
17.97
2.66
Control
9
i
a
The IC50 value was determined after exposure to a series of ADM concentrations with different
compounds at 5.0 ꢀM in K562/A02 cells; Reversal fold (RF, foldꢀchange in drug sensitivity) =
b
(
IC50 without inhibitor / (IC50 with inhibitor). 0.1% DMSO was added as solvent control. Data
were analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three
independent tests.
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0
Figure 2. SARs summary of 2ꢀ((pyridinꢀ4ꢀylmethyl)amino)nicotinamide derivatives.
To sum up, when coꢀadministered with ADM at the same condition, most target compounds
exerted stronger reversal activity than the classical Pꢀgp inhibitor VRP. Among the active
compounds, compounds 8q, 9h and 9n showed the most promising reversal potency with RF
values of 18.80, 17.97 and 32.26 at 5.0 ꢁM concentration, respectively, displayed superior reversal
activity to 1. In terms of the capacity against Pꢀgp, compound 9n holds the most potential.
Dose–response relationship and selective index (SI) of 9n
To afford a quantitative measurement of the efficacy of reversal to ADMꢀresistance, we have
determined the EC50 values of the superior compounds 8q and 9n in reversing ADM resistance in
36, 37
K562/A02 cells as previously reported.
As illustrated in Figure 3, 9n was so potent in
reversing ADM resistance with EC50 in nanomolar range (119.6 ± 6.9 nM) and 8q had a higher
EC50 value of 329.0 ± 12.9 nM. As the most promising compound, the safety of 9n was evaluated
with the intrinsic cytotoxicity against normal human gastric epithelial cell strainꢀ1 (GESꢀ1). The
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result manifested that 9n showed no cytotoxicity toward GESꢀ1 cell line (IC50 > 100 ꢁM). As a
result, the selective index (SI, ratio of IC50 toward GESꢀ1 to EC50 for reversing ADM resistance)
37
for 9n was very high (> 836.1), suggesting 9n as a MDR modulator may be safe for normal
human cells when coꢀadministrated with anticancer agents.
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Figure 3. A) EC50 values of 8q and 9n in reversing ADMꢀresistance in K562/A02 cells. The
percent of IC50 of ADM = [(IC50 of ADM at each modulator concentration / IC50 of ADM without
modulator) × 100%)]. EC50 refers to the concentration of modulator that can reduce the % of IC50
of ADM by half. Each data point is presented as mean ± SD for three independent tests; B) The
survival rate of different concentrations of 9n was determined in GESꢀ1 cells. Data were analyzed
with GraphPad Prism 5.0 software and presented as mean ± SD for three independent tests.
Effect of 9n on reversing P-gp-mediated resistance to other anticancer drugs
MDR is accompanied by resistance to other chemotherapeutic drugs that may have different
structures and mechanisms of action. Hence, we wanted to evaluate if 9n can also lower the
Pꢀgpꢀmediated resistance to other drugs like paclitaxel (PTX), vinblastine (VLB), daunorubicin
(DNR) and cyclophosphamide (CTX). Figure 4 illustrates clearly that 9n enhanced cytotoxic
effects of various agents (PTX, VLB and DNR) related to the Pꢀgpꢀmediated MDR in the resistant
cells, but had no effect on the potency of nonꢀMDR cytotoxic (as CTX). The results indicate that
inhibition of Pꢀgp function contributes to the reversal of resistance.
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0
Figure 4. Reversing potency of 9n on Pꢀgpꢀmediated resistance to other anticancer agents. DNR,
PTX, and VLB are Pꢀgp substrates, whereas CTX is not. Each data point is presented as mean ±
SD for three independent tests.
Duration of drug effect
In further evaluation for the duration of action of compound 9n, the assay was performed as
3
6, 38
previously reported.
The duration time shown in Table 6 demonstrated the reversal activity of
5
.0 ꢁM VRP almost disappeared right after its removal from the medium, indicating that the
action of VRP continued to exist no more than 6 h. In sharp contrast, 5.0 ꢁM 9n had significant
reversal potency even at 24 h after its removal. Another positive control, Apatinib, displayed
sustaining reversal activity inferior to 9n. Consequently, it is can be drawn that 9n possessed
potent reversal activity, which persisted for much longer duration (>24 h) compared with control 1
and VRP (<6 h) after removal of the agent from the culture.
Table 6. Duration of MDR reversal effect in K562/A02 cells after incubation and washout of VRP,
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1
and 9n.
a
IC50/ADM [
ꢀ
M] (RF)
5.0 µM 1
Treatment
No wash
Control
5.0 µM VRP
5.0 µM 9n
55.69 ± 2.72 (1.00)
10.22 ± 1.46 (5.45)
6.31 ± 1.02 (8.83)
7.58 ± 0.63 (7.35)
9.10 ± 0.49 (6.12)
14.03 ± 1.74 (3.97)
22.64 ± 2.05 (2.46)
1.74 ± 0.25 (32.01)
1.99 ± 0.40 (27.98)
2.78 ± 0.72 (20.03)
3.83 ± 0.69 (14.54)
7.10 ± 1.36 (7.84)
b
Wash, 0 h
Wash, 6 h
Wash, 12 h
Wash, 24 h
nd
40.36 ± 3.87 (1.38)
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nd
nd
nd
nd
nd
nd
a
Reversal fold (RF) = (IC50 without inhibitor)/(IC50 with inhibitor).The values were presented as
b
the mean ± SD for three independent tests. nd: not determined.
Effect of 9n on ADM accumulation
The defect in the accumulation of MDR cytotoxics is one property of the MDR phenotypes.
Hence, whether the reversal effect of 9n is associated with a concomitant increase in ADM
accumulation should be investigated. The autoꢀfluorescence property of ADM allows an
accumulation assay by spectrofluorometry to be carried out. The results were depicted in Figure 5.
In the absence of inhibitors, ADM accumulation in K562 cells was about 4.4ꢀfold higher than that
of K562/A02 cells (P < 0.001), probably accounting for Pꢀgpꢀmediated ADM efflux. At modulator
concentrations down to 1.0 ꢁM, 9n and 1 significantly contributed the significant enhancement of
the 2.5ꢀh accumulation of ADM into K562/A02 cells. However, VRP increased ADM
accumulation only when at the highest tested concentration of 5.0 ꢁM. These results indicated that
9n was a potent Pꢀgp inhibitor, which exhibited greater potency in increasing accumulation of
ADM into K562/A02 cells than VRP and 1.
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9
0
Figure 5. Effect of 9n on intracellular ADM accumulation in K562/A02 cells. The results are
presented as the mean ± SD for three independent experiments; (***) P < 0.001, (**) P < 0.01, (*)
P < 0.05 relative to the negative control (K562/A02).
Inhibitory effect of P-gp-mediated Rhodamine123 efflux
To assess the potency of MDR reversal agents on Pꢀgp efflux function, Pꢀgpꢀdependent fluorescent
39, 40
rhodamine 123 (Rh123) efflux has been widely used.
Intracellular Rh123 accumulation of 9n
was determined via flow cytometry. The intracellular quantity of Rh123ꢀassociated mean
fluorescence intensity (MFI) at 5, 10, 25, 30, 60 and 90min in 5.0 ꢁM 9nꢀtreated cells as
illustrated in Figure 6 was significantly more intense than that in negative control, in 5.0 ꢁM
VRPꢀtreated cells, and in 5.0 ꢁM 1ꢀtreated cells, indicating the reversal activity of 9n was superior
to VRP and 1. The results suggested that 9n can reverse MDR by inhibiting Pꢀgpꢀmediated drug
efflux significantly.
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Figure 6. Effect of 9n on efflux of Rh123 from K562/A02. Each data point is presented as mean ±
SD for three independent experiments.
Effect of 9n on P-gp-ATPase Activity
In further evaluation, the effect of 9n on PꢀgpꢀATPase activity was determined. VRP, being an
41, 42
inhibitor as well as a substrate of Pꢀgp, is one of the stimulators of PꢀgpꢀATPase.
At 50 ꢁM,
VRP strongly increased the PꢀgpꢀATPase activity over the basal level by 2.56ꢀfold (Figure 7).
Also, 1 had emitted stimulated activity on PꢀgpꢀATPase of 2.68ꢀfold at 5.0 ꢁM, even at low
concentration of 0.5 ꢁM, the lumiescence level was 1.41ꢀfold higher than the basal level (Figure
7
). However, 9n triggered adverse inhibitory effect on PꢀgpꢀATPase at 0.5 ꢁM even, which was
obviously lower than the basal level (Figure 7), suggesting that 9n, different from 1 and VRP,
could suppress Pꢀgp ATPase activity.
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0
Figure 7. Effect of 9n on PꢀgpꢀATPase activity. Sodium vanadate inhibitable ATPase (Pꢀgp
ATPase) was studied as described in Experimental Section. Pꢀgp ATPase was measured in the
absence (basal activity) or presence of Pꢀgp modulators (VRP, 9n and 1). Results are presented as
the mean ± SD for three independent experiments. (***) P < 0.001, (**) P < 0.01, (*) P < 0.05
relative to the basal activity.
Effect of 9n on P-gp expression
Western blot analysis indicated a band with a molecular weight of approximately 170ꢀkDa in
the K562/A02 cell lysates, suggesting the presence of ABCB1 protein. In contrast, this band was
not present in sensitive K562, indicating the absence of ABCB1 protein (Figure 8A and B). The
reversal of ABCB1 ꢀmediated MDR can be achieved either by inhibiting their function or by
lowering their expression. Therefore, we determined the effect of incubating cells with 9n at 0.1,
1
.0, or 5.0 ꢁmol/L, to confirm that the reversal of ABCB1ꢀ mediated MDR by 9n was not due to a
decrease in the protein expression levels. Subsequently, the expression levels of ABCB1 in the
respective cell lysates were measured. There was no significant change in the expression levels of
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the ABCB1 protein in the K562/A02 cells (Figure 8A and B). These findings revealed that the
MDR reversal by 9n was not due to a decrease in protein expression, but most likely obtained by
direct inhibition of the efflux function of ABCB1.
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Figure 8. Western blot analysis showing the expression of ABCB1 after exposure to 0.1, 1.0, and
5
.0 ꢁmol/L 9n. (A) Effect of 9n on expression level of ABCB1 in K562/A02 cells for 72 h. (B)
Band intensity was analyzed by Quantity One software and protein expression was presented as
the ratio of target protein's band intensity to that of βꢀTubulin. Representative result is shown here
and similar results were obtained in two other independent trials.
Effect of 9n on cytochrome P3A4 (CYP3A4)
There is a lot of overlap about the characteristics of substrate and tissue distribution between Pꢀgp
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and CYP3A4. Indeed, as the substrates of CYP3A4, a few secondꢀgeneration Pꢀgp inhibitors
revealed unpredictable pharmacokinetic properties when combining with chemotherapeutic agents.
Therefore, the new generation Pꢀgp inhibitors are expected to be safer agents without effect on
CYP3A4 at effective dose. In order to evaluate the potential of compound 9n as a safe Pꢀgp
modulator, an in vitro inhibition assay for rat liver CYP3A4 were performed. As shown in Figure
0
1
2
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9
0
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0
9
, ketoconazole, a widely used specific inhibitor of CYP3A4, significantly suppressed the potency
of rat liver CYP3A4 in a concentrationꢀdependent manner, whereas 9n did not inhibit the potency
of CYP3A4 even at the concentration of 40.0 ꢀM, which is much higher than the EC50 value.
These results demonstrated that compound 9n might be a relatively safe Pꢀgp inhibitor suitable for
coꢀadministering with chemotherapy agents metabolized by liver CYP3A4.
Figure 9. The effect of 9n and ketoconazole on CYP3A4. Ketoconazole, a specific inhibitor of
CYP3A4, was used as the positive control. 6βꢀOH testosterone (6βꢀOHT) is the specific product
of testosterone metabolized by CYP3A4. The data represent as the mean ± SD for three
independent experiments.
CONCLUSIONS
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In summary, a series of Apatinib analogues incorporating 2ꢀ((pyridinꢀ4ꢀylmethyl) amino)
nicotinamide scaffold as MDR modulators with low cytotoxity were designed and synthesized.
This investigation contributed to the identification of 9n as a potent Pꢀgp inhibitor, which was
superior to 1 and VRP in reversing Pꢀgpꢀmediated ADMꢀresistance in K562/A02 cells and possess
outstanding reversal potency with EC50 in namomolar range (EC50 = 119.6 ± 6.9 nM).
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Moreover, the MDR reversal potency of 9n continued to exist more than 24 h. 9n at a
nonꢀtoxic concentration of 5.0 ꢁM (cell viability > 99.0%) enhanced cytotoxic effect of other
MDRꢀrelated anticancer agents (PTX, VLB and DNR) toward K562/A02 cells. Further research
was conducted to explore the action mechanism preliminarily. 9n was proved to significantly
inhibit the transport activity of Pꢀgp by intracellular accumulation of ADM and efflux of Rh123 in
resistant K562/A02 cells. Unlike VRP, 9n acted as the inhibitor of PꢀgpꢀATPase, which partly
accounting for the mechanism action of the reversal activity. Except the expressing level in K562
cell line, the other lanes representing K562/A02 in different incubator conditions had no
significant difference, suggesting the MDR reversal by 9n was not due to a decrease in protein
expression, but most likely obtained by direct inhibition of the efflux function of ABCB1.
More importantly, 9n did not affect the activity of CYP3A4 even at high concentration to
avoid the pharmacokinetic interactions in vitro assay. Further intensive investigations including
docking analysis and in vivo assays are still in progress for 9n which holds promising potential in
the development of Pꢀgpꢀmediated MDR reversal modulator in cancer chemotherapy.
EXPERIMENTAL SECTION
General chemistry
All starting materials, reagents and solvents were obtained from commercial sources and used
without further purification unless otherwise indicated. Purifications by column chromatography
were carried out over silica gel (200ꢀ300 mesh) and monitored by thin layer chromatography
(TLC) performed on GF/UV 254 plates and were visualized using UV light at 254 and 365 nm.
Melting points were taken on a RYꢀ1 meltingꢀpoint apparatus and were uncorrected. NMR spectra
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were recorded in DMSOꢀd on a Bruker ACFꢀ300Q instrument (300 MHz for H, 75 MHz for C;
6
Bruker Instruments, Inc., Billerica, MA, USA), chemical shifts are expressed as values (ppm)
relative to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
tetramethylsilane as internal standard, and coupling constants (J values) were given in hertz (Hz).
Abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
dd = doublets of doublet, br = broad. ESIꢀMS were recorded with Waters ACQUITY UPLC
Systems with Mass (Waters, Milford, MA).
Purities of Target Compounds
The purities of all target compounds was established by HPLC with LCꢀ20A chromatograph
system (SHIMADZU, Japan). The analytical column was 5020ꢀ02745 INERTSIL ODSꢀSP
4
.6*150 nm, 5 ꢁM (GL Sciences, Japan). UV signals were recorded at 254 nm at column
temperature of 40 °C. The content results of compounds 8a-s and 9a-n were intelligently
calculated with the LCꢀsolution. The purities of all target compounds were >95% for biological
evaluation.
General procedure for the synthesis of compound 5
The starting material 2ꢀchloronicotinic acid (3, 25.3 mmol) was treated with copper oxide (2.53
mmol) and potassium carbonate (4, 25.3 mmol) in a roundꢀbottom flask for stirring. After adding
pyridinꢀ4ꢀylmethanamine (126.5 mmol), the mixtures were heated for 2 h in 110℃. Then the
mixtures were diluted with ethyl acetate (40 mL), and filtered. The filter cake was dissolved in 20
ml water for adjusting pH to 5ꢀ6 with 4 N hydrochloric acid. The obtained white crude was
recrystallized in ethanol (15 mL) to afford compound 5 with high purity.
2-((pyridin-4-ylmethyl)amino)nicotinic acid (5)
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5
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7
8
9
1
Yield: 89.2%; grayꢀwhite solid; H NMR (300 MHz, DMSOꢀd ) δ: 13.22 (s, 1H), 8.60 (t, J = 5.9
6
Hz, 1H), 8.48 (s, 2H), 8.19 (dd, J = 4.7, 1.9 Hz, 1H), 8.11 (dd, J = 7.7, 1.9 Hz, 1H), 7.29 (d, J =
1
3
4
.7 Hz, 2H), 6.64 (dd, J = 7.7, 4.7 Hz, 1H), 4.73 (d, J = 5.8 Hz, 2H); C NMR (75 MHz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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33
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35
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DMSOꢀd
6
) δ: 170.78, 159.02, 151.33 , 150.46, 149.05, 136.60, 122.78, 114.11, 106.62, 43.63;
+
ESIꢀMS m/z: 230.1 [M+H] .
General procedure for the synthesis of 8a-s and 9a-n
The crudes of compounds 8a-s and 9a-n were prepared by compound 5 and diverse amines in the
presence of condensation agents EDCI and HOBT in DMF. Compound 5 (0.23 g, 1.1 mmol),
EDCI (0.23 g, 1.2 mmol) and HOBT (0.16 g, 1.2 mmol) were dissolved and stirred in DMF (5 mL,
0
.2 M) with ice bath for 30min, then the corresponding amine 6a-s or 7a-n (1 mmol) was added to
the reaction solution. The reaction mixture was removed to room temperature for 16 h and
monitored by TLC. After reaction completion, 50 mL water was added to the mixture solution,
which was next extracted with ethyl acetate (30 mL × 3). The combined organic layers were
2 3 2 4
washed with saturated Na CO (50 mL × 2), brine (50 mL × 2) in sequence, dried over Na SO ,
and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel
using dichloromethane / methanol (50:1) as eluent to obtain desire product compound 8a-s and
9a-n with high purity.
N-(3-methoxyphenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8a)
1
Yield: 83.1%; white solid; m.p. 113ꢀ115 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 10.59 (s, 1H),
9
.23 (s, 1H), 8.87 (d, J = 5.4 Hz, 2H), 8.41 (d, J = 7.0 Hz, 1H), 8.12 (d, J = 4.8 Hz, 1H), 8.02 (d, J
=
5.4 Hz, 2H), 7.45 (s, 1H), 7.45ꢀ7.28 (m, 2H), 6.96 (t, J = 5.7 Hz, 1H), 6.74 (d, J = 6.9 Hz, 1H),
13
5
6
.03 (s, 2H), 3.76 (s, 3H); C NMR (75 MHz, DMSOꢀd ) δ: 162.25, 160.12, 158.75, 151.41,
150.46, 149.05, 141.35, 134.99, 130.08, 122.79, 116.75, 111.08, 110.71, 109.81, 107.01, 56.08,
+
43.63; ESIꢀMS m/z: 335.2 [M+H] . Purity 98%; t 4.59 min; eluent CH CN/H O 55/45 0.1% FA;
r
3
2
fl 1.2 mL/min.
2-((pyridin-4-ylmethyl)amino)-N-(4-(trifluoromethoxy)phenyl)nicotinamide (8b)
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Yield: 79.8%; pale yellow solid; m.p. 125ꢀ127 °C; H NMR (300 MHz,DMSOꢀd ) δ: 10.46 (s,
6
1
H), 8.47ꢀ8.40 (m, 3H), 8.16 (d, J = 3.6 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 9.0 Hz, 2H),
.38 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 5.2 Hz, 2H), 6.70 (dd, J = 7.5, 4.9 Hz, 1H), 4.68 (d, J = 5.8
7
13
Hz, 2H); C NMR (75 MHz, DMSOꢀd
6
) δ: 166.70, 156.93, 151.38, 149.52, 143.99, 138.03,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
37.29, 122.03, 121.35, 111.21, 110.73, 109.81, 42.87; ESIꢀMS m/z: 389.2 [M+H] . Purity 97%; t
r
4
.73 min; eluent CH
3
CN/H
2
O 55/45 0.1% FA; fl 1.2 mL/min.
N-(4-(tert-butyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8c)
1
Yield: 85.7%; white solid; m.p. 141ꢀ143 °C; H NMR (300 MHz,DMSOꢀd
6
) δ: 10.39 (s, 1H), 8.84
(
d, J = 5.7 Hz, 3H), 8.24 (d, J = 7.2 Hz, 1H), 8.11 (d, J = 4.5 Hz, 1H), 7.98 (d, J = 5.6 Hz, 2H),
7
.67 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 6.86ꢀ6.77 (m, 1H), 4.96 (s, 2H), 1.29 (s, 9H);
13
C NMR (75 MHz, DMSOꢀd
6
) δ: 164.83, 160.64, 154.22, 146.60, 141.27, 140.08, 135.88, 125.22,
+
1
24.81, 120.49, 112.14, 42.26, 34.07, 31.15; ESIꢀMS m/z: 361.3 [M+H] . Purity 97%; t 5.81 min;
r
eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
3
2
N-(3-chloro-4-methoxyphenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8d)
1
Yield: 73.6%; grey solid; m.p. 186ꢀ188 °C; H NMR (300 MHz, DMSOꢀd
6
) δ:10.26 (s, 1H), 8.46
(d, J = 5.8 Hz, 3H), 8.15 (d, J = 3.3 Hz, 1H), 8.09 (t, J = 1.5 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H),
7.60 (dd, J = 8.9, 2.5 Hz, 1H), 7.29 (d, J = 5.6 Hz, 2H), 7.16 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 7.6,
13
6
4.8 Hz, 1H), 4.69 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H) ; C NMR (75 MHz, DMSOꢀd ) δ: 166.34,
156.90, 151.23, 150.89, 149.80, 149.33, 137.05, 132.37, 122.20, 122.04, 120.46, 112.64, 111.20,
+
1
10.73, 56.12, 42.84; ESIꢀMS m/z: 369.2 [M+H] . Purity 98%; t
r
3 2
4.66 min; eluent CH CN/H O
55/45 0.1% FA; fl 1.2 mL/min.
N-(3-chloro-4-fluorophenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8e)
1
Yield: 71.9%; pale yellow solid; m.p. 183ꢀ185 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 10.44 (s,
1
H), 8.47ꢀ8.41 (m, 3H), 8.17 (d, J = 3.4 Hz, 1H), 8.10ꢀ8.04 (m, 2H), 7.66ꢀ7.63 (m, 1H), 7.43 (t, J
= 9.1 Hz, 1H), 7.29 (d, J = 5.4 Hz, 2H), 6.70 (dd, J = 7.6, 4.8 Hz, 1H), 4.69 (d, J = 6.0 Hz, 2H);
13
6
C NMR (75 MHz, DMSOꢀd ) δ: 166.64, 156.88, 155.04, 151.82, 151.48, 149.70, 149.35,
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137.23 , 136.04, 122.07, 120.92, 118.96, 116.86, 116.57, 111.20, 110.50, 42.86; ESIꢀMS m/z:
+
3
57.1 [M+H] . Purity 96%; t
r
3 2
4.90 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
2-((pyridin-4-ylmethyl)amino)-N-(3,4,5-trimethoxyphenyl)nicotinamide (8f)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Yield: 80.4%; white solid; m.p. 216ꢀ218 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 10.32 (s, 1H),
8
2
.83 (d, J = 6.0 Hz, 3H), 8.20 (d, J = 7.3 Hz, 1H), 8.10 (d, J = 4.1 Hz, 1H), 7.97 (d, J = 6.0 Hz,
13
H), 7.20 (s, 2H), 6.81ꢀ6.78 (m, 1H), 4.94 (s, 2H), 3.78 (s, 6H), 3.65 (s, 3H); C NMR (75 MHz,
DMSOꢀd ) δ: 164.82, 160.74, 152.31, 141.06, 134.52, 124.81, 112.10, 98.41, 60.13, 43.80;
6
+
ESIꢀMS m/z: 395.2 [M+H] . Purity 99%; t
r
4.42 min; eluent CH
3
CN/H
2
O 55/45 0.1% FA; fl 1.2
mL/min.
N-(pyridin-4-ylmethyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8g)
1
Yield: 81.5%; white solid; m.p. 101ꢀ103 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.69 (d, J = 1.8
Hz, 1H), 9.26 (s, 1H), 8.87 (d, J = 2.2 Hz, 4H), 8.35 (d, J = 7.2 Hz, 1H), 8.08ꢀ7.96 (m, 5H), 6.87
13
(d, J = 3.1 Hz, 1H), 4.95 (s, 2H), 4.78 (s, 2H); C NMR (75 MHz, DMSOꢀd
6
) δ: 166.79, 160.47,
1
3
60.23, 154.31, 145.86, 141.51, 140.08, 125.05, 124.77, 112.56, 124.77, 42.45; ESIꢀMS m/z:
+
r 3 2
20.2 [M+H] . Purity 97%; t 4.28 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
N-(3,4-dimethoxybenzyl)-N-methyl-2-((pyridin-4-ylmethyl)amino)nicotinamide (8h)
1
Yield: 76.8%; white solid; m.p. 172ꢀ174 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 8.44 (s, 2H), 8.01
(
s, 1H), 7.46ꢀ7.17 (m, 3H), 6.90ꢀ6.62 (m, 4H), 6.66 (d, J = 2.4 Hz, 1H), 4.60ꢀ4.51 (m, 2H),
13
3
1
5
5
.73ꢀ3.65 (m, 8H), 2.87 (s, 3H) ; C NMR (75 MHz, DMSOꢀd ) δ: 174.92, 157.41, 151.02,
6
50.41, 149.05, 148.71, 135.67, 129.36, 122.79, 121.65, 116.83, 113.92, 112.97, 107.92, 56.83,
+
2.33, 43.63, 35.13; ESIꢀMS m/z: 393.2 [M+H] . Purity 96%; t 4.31 min; eluent CH CN/H O
r
3
2
5/45 0.1% FA; fl 1.2 mL/min.
N-(3,4-dimethoxyphenethyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8i)
1
Yield: 85.2%; white solid; m.p. 185ꢀ187 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.80 (s, 1H), 9.07
(d, J = 4.8 Hz, 1H), 8.88 (d, J = 6.3 Hz, 2H), 8.30 (d, J = 6.5 Hz, 1H), 8.09 (d, J = 4.5 Hz, 1H),
7.99 (d, J = 6.4 Hz, 2H), 6.95 (dd, J = 7.2, 6.0 Hz, 1H), 6.88ꢀ6.85 (m, 2H), 6.77ꢀ6.75 (m, 1H),
1
3
5.03 (s, 2H), 3.71 (d, J = 4.6 Hz, 6H), 3.50 (dd, J = 12.9, 6.8 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H); C
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR (75 MHz, DMSOꢀd ) δ: 165.52, 159.80, 153.51, 148.57, 141.43, 140.42, 131.65, 124.79,
6
+
1
20.48, 114.36, 112.50, 112.12, 111.90, 55.48, 55.35, 44.29, 34.29; ESIꢀMS m/z: 393.3 [M+H] .
r 3 2
Purity 98%; t 4.45 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
2-((pyridin-4-ylmethyl)amino)-N-(2-(thiophen-2-yl)ethyl)nicotinamide (8j)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Yield: 74.5%; yellow solid; m.p. 130ꢀ132°C; H NMR (300 MHz, DMSOꢀd
6
) δ: 8.92ꢀ8.62 (m,
2
7
3
1
4
H), 8.47 (d, J = 5.6 Hz, 2H), 8.10 (dd, J = 4.7, 1.5 Hz, 1H), 7.93 (dd, J = 7.6, 1.5 Hz, 1H),
.49ꢀ7.14 (m, 3H), 7.05ꢀ6.80 (m, 2H), 6.62 (dd, J = 7.5, 4.9 Hz, 1H), 4.67 (d, J = 5.9 Hz, 2H),
13
.66ꢀ3.40 (m, 2H), 3.08 (t, J = 6.9 Hz, 2H); C NMR (75 MHz, DMSOꢀd
6
) δ: 167.63, 157.05,
50.88, 149.80, 149.36, 141.41, 136.30, 126.91, 125.20, 124.07, 122.05, 111.16, 110.21, 42.76,
+
0.80, 29.05; ESIꢀMS m/z: 339.2 [M+H] . Purity 98%; t
r
4.60 min; eluent CH
3
CN/H
2
O 55/45 0.1%
FA; fl 1.2 mL/min.
N-(naphthalen-1-yl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8k)
1
Yield: 81.3%; yellow solid; m.p. 197ꢀ199 °C; H NMR (300 MHz, DMSOꢀd ) δ: 10.49 (s, 1H),
6
8
.71 (s, 1H), 8.47ꢀ8.38 (m, 3H), 8.22 (s, 1H), 8.01ꢀ7.88 (m, 3H), 7.58 (d, J = 4.1 Hz, 4H), 7.30 (s,
13
6
2H), 6.75 (s, 1H), 4.70 (s, 2H); C NMR (75 MHz, DMSOꢀd ) δ: 167.52, 157.29, 151.45, 149.67,
149.39, 126.46, 126.07, 125.49, 124.14, 122.05, 111.37, 110.14, 42.87; ESIꢀMS m/z: 355.2
+
[M+H] . Purity 97%; t
r
4.72 min; eluent CH
3 2
CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
2-((pyridin-4-ylmethyl)amino)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)nicotinamide (8l)
1
Yield: 79.2%; white solid; m.p. 144ꢀ146 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 8.87ꢀ8.54 (m, 4H),
8
2
1
.11ꢀ8.03(m, 2H), 7.33 (s, 2H), 7.33ꢀ7.16 (m, 4H), 6.59 (s, 1H), 5.26 (s, 1H), 4.71 (s, 2H), 2.78 (s,
13
6
H), 1.98 (s, 2H), 1.82 (s, 2H); C NMR (75 MHz, DMSOꢀd ) δ: 167.14, 157.16, 150.81, 149.81,
49.38, 136.60, 128.74, 127.81, 126.67, 125.85, 111.11, 110.32, 46.91, 42.85, 29.75, 28.85, 20.40;
+
ESIꢀMS m/z: 359.3 [M+H] . Purity 96%; t
r
5.03 min; eluent CH
3
CN/H
2
O 55/45 0.1% FA; fl 1.2
mL/min.
N-(1H-benzo[d]imidazol-2-yl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8m)
1
Yield: 82.7%; white solid; m.p. 239ꢀ241 °C; H NMR (300 MHz, DMSOꢀd ) δ: 12.46 (s, 2H),
6
9
.75 (s, 1H), 8.50ꢀ8.40 (m, 3H), 8.12 (t, J = 2.2 Hz, 1H), 7.44 (t, J = 2.7 Hz, 2H), 7.35 (s, 2H),
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8
9
1
3
7.19 (t, J = 2.6 Hz, 2H), 6.64 (t, J = 5.2 Hz, 1H), 4.77 (d, J = 5.1 Hz, 2H); C NMR (75 MHz,
DMSOꢀd
6
) δ: 172.89, 157.81, 151.67, 149.96, 149.42, 139.17, 130.24, 122.40, 122.11, 113.46,
+
1
11.77, 111.46, 42.87; ESIꢀMS m/z: 345.2 [M+H] . Purity 97%; t
r
4.25 min; eluent CH
3 2
CN/H O
55/45 0.1% FA; fl 1.2 mL/min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-([1,1'-biphenyl]-4-yl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8n)
1
Yield: 78.2%; white solid; m.p. 190ꢀ192 °C; H NMR (300 MHz,DMSOꢀd
6
) δ: 10.40 (s, 1H), 8.51
13
(
(
s, 3H), 8.17 (s, 2H), 7.77 (m, 7H), 7.46 (s, 2H), 7.31 (s, 2H), 6.71 (s, 1H), 4.72 (s, 2H); C NMR
75 MHz, DMSOꢀd
6
) δ: 166.61, 156.95 , 151.24 , 149.73, 149.38, 139.62, 138.31, 137.26, 135.43,
+
128.88, 127.08, 126.72, 126.27, 122.07, 121.00, 111.12, 42.89; ESIꢀMS m/z: 381.2 [M+H] .
r 3 2
Purity 97%; t 4.93 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
N-(5-(3-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8o)
1
Yield: 80.3%; white solid; m.p. 221ꢀ223 °C; H NMR (300 MHz,DMSOꢀd ) δ: 8.78ꢀ8.21 (m, 5H),
6
13
7
.48ꢀ7.34 (m, 6H), 7.10 (s, 1H), 6.69 (s, 1H), 4.74 (s, 2H), 3.84 (s, 3H); C NMR (75 MHz,
DMSOꢀd6) δ: 167.00, 161.08, 159.68, 157.34, 152.71, 149.38, 138.52, 131.48, 130.53, 119.31,
+
116.63, 111.43, 111.20, 108.43, 55.27, 42.93; ESIꢀMS m/z: 419.2 [M+H] . Purity 97%; t
r
5.18 min;
3 2
eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
N-(4-(piperidin-1-yl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8p)
1
Yield: 83.8%; white solid; m.p. 217ꢀ219 °C; H NMR (300 MHz,DMSOꢀd
6
) δ: 10.09 (s, 1H),
8
7
.51ꢀ8.11 (m, 3H), 8.12 (d, J = 4.8 Hz, 1H), 8.05 (d, J = 6.6 Hz, 1H), 7.53 (d, J = 8.9 Hz, 2H),
.28 (d, J = 4.8 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 6.67 (dd, J = 7.4, 4.8 Hz, 1H), 4.67 (d, J = 5.8
13
Hz, 2H), 3.08 (d, J = 5.3 Hz, 4H), 1.61ꢀ1.51 (m, 6H); C NMR (75 MHz, DMSOꢀd
6
) δ: 166.00,
1
4
5
56.93, 150.85, 149.76, 149.36, 148.40, 136.86, 130.15, 122.02, 121.86, 115.85, 111.20, 49.92,
+
2.85, 25.23, 23.83; ESIꢀMS m/z: 388.3 [M+H] . Purity 98%; t
r
5.68 min; eluent CH
3 2
CN/H O
5/45 0.1% FA; fl 1.2 mL/min.
N-(4-morpholinophenyl)-2-((pyridin-4-ylmethyl)amino) nicotinamide (8q)
1
Yield: 79.2%; white solid; m.p. 215ꢀ217 °C; HNMR (300 MHz,DMSOꢀd ) δ: 10.10 (s, 1H),
6
8
.54ꢀ8.45 (m, 3H), 8.13 (d, J = 4.4 Hz, 1H), 8.07 (d, J = 7.3 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H),
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Journal of Medicinal Chemistry
Page 28 of 56
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7.29 (d, J = 4.7p Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 6.67 (dd, J = 7.2, 5.0 Hz, 1H), 4.68 (d, J = 5.8
13
Hz, 2H), 3.74 (s, 4H), 3.07(s, 4H); C NMR (75 MHz, DMSOꢀd
6
) δ: 166.06, 156.93, 150.91,
149.77, 149.37, 147.67, 136.90, 130.79, 122.02, 121.85, 115.11, 111.16, 66.05, 48.75, 42.83;
+
ESIꢀMS m/z: 390.3 [M+H] . Purity 99%; t
r 3 2
4.60 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2
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mL/min.
N-(4-(4-methylpiperazin-1-yl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (8r)
1
Yield: 80.5%; light yellow solid; m.p. 220ꢀ222 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 10.11 (s,
1H), 8.69ꢀ8.29 (m, 3H), 8.13ꢀ8.04 (m, 2H), 7.55 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 5.7 Hz, 2H), 6.92
(d, J = 9.0 Hz, 2H), 6.66 (dd, J = 7.6, 4.9 Hz, 1H), 4.67 (d, J = 5.9 Hz, 2H), 3.24ꢀ2.98 (m, 4H),
13
2
1
.47ꢀ2.32 (m, 4H), 2.20 (s, 3H); C NMR (75 MHz, DMSOꢀd
6
) δ: 166.03, 156.91, 150.89, 149.81,
49.34, 147.66, 136.89, 130.41, 122.02, 121.84, 115.33, 111.68, 54.55 , 48.35, 45.73, 42.81;
+
ESIꢀMS m/z: 403.3 [M+H] . Purity 98%; t 5.33 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2
r
3
2
mL/min.
[1,4'-bipiperidin]-1'-yl(2-((pyridin-4-ylmethyl)amino)pyridin-3-yl)methanone (8s)
1
Yield: 71.3%; white solid; m.p. 106ꢀ107°C; H NMR (300 MHz, CDCl
3
) δ: 8.53 (s, 2H), 8.13 (d, J
= 4.7 Hz, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.28 (d, J = 3.4 Hz, 1H), 6.60 (dd, J = 7.2, 5.1 Hz, 1H),
6.36 (d, J = 5.6 Hz, 1H), 4.68 (d, J = 5.8 Hz, 2H), 4.32 (s, 2H), 2.92 (s, 2H), 2.52 (s, 4H), 2.27 (s,
13
3
2H), 1.95ꢀ1.91 (m, 2H), 1.75ꢀ1.27 (m, 8H); C NMR (75 MHz, CDCl ) δ: 149.12, 135.39, 111.39,
+
61.86, 49.80, 43.35, 28.07, 25.74, 24.11; ESIꢀMS m/z: 380.3 [M+H] . Purity 97%; t
r
5.89 min;
eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min
3
2
N-benzhydryl-2-((pyridin-4-ylmethyl)amino)nicotinamide (9a)
1
Yield: 77.5%; white solid; m.p. 145ꢀ147 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 9.33 (d, J = 7.6
Hz, 1H), 8.68ꢀ8.46 (m, 3H), 8.16 (t, J = 7.2 Hz, 2H), 7.38ꢀ7.28 (m, 12H), 6.64 (s, 1H), 6.42 (d, J =
1
3
7
1
6
.5 Hz, 1H), 4.66 (s, 2H); C NMR (75 MHz, DMSOꢀd ) δ: 167.09, 157.08, 151.04, 150.46,
49.75, 149.36, 142.02, 137.11, 128.35, 127.61, 127.05, 122.08, 111.13, 110.08, 56.19, 42.80;
+
ESIꢀMS m/z: 395.3 [M+H] . Purity 98%; t 5.22 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2
r
3
2
mL/min
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Journal of Medicinal Chemistry
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N-(9H-fluoren-2-yl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (9b)
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Yield: 71.3 %; white solid; m.p. 180ꢀ181 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 10.38 (s, 1H),
8
.53 (s, 3H), 8.16 (s, 3H), 7.94ꢀ7.51 (m, 4H), 7.33 (s, 4H), 6.71 (s, 1H), 4.71 (s, 2H), 3.93 (s, 2H);
13
6
C NMR (75 MHz, DMSOꢀd ) δ: 166.52, 156.95, 151.14, 150.51, 149.57, 143.51, 142.87, 140.93,
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37.80, 137.03, 126.73, 126.21, 125.01, 119.94, 119.55, 117.53, 111.25, 42.89, 36.48; ESIꢀMS
+
m/z: 393.2 [M+H] . Purity 99%; t
r
3 2
5.57 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
N-(2-benzoylphenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (9c)
1
Yield: 82.9%; pale yellow solid; m.p. 146ꢀ148 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 10.59 (s,
1H), 8.45 (s, 2H), 8.26 (s, 1H), 8.09 (s, 1H), 7.67ꢀ7.62 (m, 5H), 7.58ꢀ7.50 (m, 2H), 7.47 (d, J = 7.3
Hz, 2H), 7.38 (d, J = 7.1 Hz, 1H), 7.21 (s, 2H), 6.57 (d, J = 4.9 Hz, 1H), 4.61 (d, J = 5.4 Hz, 2H); ;
13
6
C NMR (75 MHz, DMSOꢀd ) δ: 196.06, 162.50, 158.75, 151.41, 150.46, 149.05, 141.29, 136.25,
134.99, 132.50, 131.68, 129.97, 129.24, 128.60, 124.88, 124.35, 122.79, 110.71, 109.81, 43.63;
+
ESIꢀMS m/z: 409.2 [M+H] . Purity 99%; t 5.86 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2
r
3
2
mL/min.
N,N-dibenzyl-2-((pyridin-4-ylmethyl)amino)nicotinamide (9d)
1
Yield: 83.7%; white solid; m.p. 197ꢀ199 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 8.42 (s, 2H), 8.00
(d, J = 4.2 Hz, 1H), 7.50 (d, J = 6.9 Hz, 1H), 7.31ꢀ7.22 (m, 12H), 6.90 (s, 1H), 6.58 (t, J = 6.0 Hz,
13
1H), 4.60ꢀ4.58 (m, 6H); C NMR (75 MHz, DMSOꢀd
6
) δ: 169.28, 154.61, 150.05 , 149.19,
+
148.74, 136.62, 135.08, 128.57, 122.07, 114.72, 111.52, 56.56, 43.05; ESIꢀMS m/z: 409.3 [M+H] .
r 3 2
Purity 97%; t 5.35 min; eluent CH CN/H O 55/45 0.1% FA; fl 1.2 mL/min.
N-(2-chloro-6-methylphenyl)-2-(2-((pyridin-4-ylmethyl)amino)nicotinamido)thiazole-5-carboxa
mide (9e)
1
Yield: 74.1%; white solid; m.p. 169ꢀ171 °C; H NMR (300 MHz, DMSOꢀd
6
) δ: 12.71 (s, 1H),
10.07 (s, 1H), 8.79 (s, 1H), 8.47 (s, 2H), 8.39ꢀ8.35 (m, 2H), 8.21 (d, J = 3.6 Hz, 1H), 7.47ꢀ7.38 (m,
13
1H), 7.31ꢀ7.25 (m, 4H), 6.69 (dd, J = 7.6, 4.8 Hz, 1H), 4.75 (d, J = 5.5 Hz, 2H), 2.25 (s, 3H);
C
NMR (75 MHz, DMSOꢀd ) δ: 170.42, 167.55, 160.98, 158.75, 151.41, 150.46, 149.05, 136.62,
6
135.56, 134.99, 133.09, 130.11, 129.08, 128.16, 127.07, 122.79, 120.30, 110.71, 109.81, 43.63,
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