Discovery and Characterization of 2-Nitro-5-(4-(phenylsulfonyl)piperazin-1-yl)- N-(pyridin-4-ylmethyl)anilines as Novel Inhibitors of the Aedes aegypti Kir1 (AeKir1) Channel

Article abstract of DOI:10.1021/acsinfecdis.8b00368

Mosquito-borne arboviral diseases such as Zika, dengue fever, and chikungunya are transmitted to humans by infected adult female Aedes aegypti mosquitoes and affect a large portion of the world's population. The Kir1 channel in Ae. aegypti (AeKir1) is an important ion channel in the functioning of mosquito Malpighian (renal) tubules and one that can be manipulated in order to disrupt excretory functions in mosquitoes. We have previously reported the discovery of various scaffolds that are active against the AeKir1 channel. Herein we report the synthesis and biological characterization of a new 2-nitro-5-(4-(phenylsulfonyl) piperazin-1-yl)-N-(pyridin-4-ylmethyl)anilines scaffold as inhibitors of AeKir1. This new scaffold is more potent in vitro compared to the previously reported scaffolds, and the molecules kill mosquito larvae.

Full text of DOI:10.1021/acsinfecdis.8b00368

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Article
Discovery and characterization of 2-nitro-5-(4-
phenylsulfonyl)piperazin-1-yl)-N-(pyridin-4-ylmethyl)anilines
(
as novel inhibitors of the Aedes aegypti Kir1 (AeKir1) channel
Christopher Aretz, M Jane Morwitzer, Austin Sanford, Alicia M Hogan,
Madalene Portillo, Sujay V Kharade, Meghan Kramer, James Mcarthey, Renata
R Trigueros, Peter M. Piermarini, Jerod S Denton, and Corey R. Hopkins
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00368 • Publication Date (Web): 04 Mar 2019
Downloaded from http://pubs.acs.org on March 6, 2019
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Discovery and characterization of 2-nitro-5-(4-
(phenylsulfonyl)piperazin-1-yl)-N-(pyridin-4-
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ylmethyl)anilines as novel inhibitors of the Aedes aegypti
Kir1 (AeKir1) channel
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Christopher D. Aretz , M. Jane Morwitzer , Austin G. Sanford , Alicia M. Hogan , Madelene V.
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Portillo , Sujay V. Kharade , Meghan Kramer , James B. McCarthey , Renata Rusconi
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1,*
Trigueros , Peter M. Piermarini , Jerod S. Denton , Corey R. Hopkins
1
University of Nebraska Medical Center, Department of Pharmaceutical Sciences, College of Pharmacy,
Omaha, NE
2
University of Nebraska Medical Center, High School Alliance, Omaha, NE
3
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN
4
Department of Entomology, Ohio State University, Wooster, OH
Email: corey.hopkins@unmc.edu
Corresponding author:
Corey R. Hopkins, Ph.D.
College of Pharmacy
Department of Pharmaceutical Sciences
986125 Nebraska Medical Center, PDD3015
nd
42 and Emile St.
Omaha, NE 68198-6125
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Omaha, NE 68198-6125
Sujay V. Kharade, Meghan Kramer, James B. McCarthey, Jerod S. Denton
T4208 Medical Center North
st
1161 21 Ave. South
Nashville, TN 37232
Renata Rusconi Trigueros, Peter M. Piermarini
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24 Thorne Hall
680 Madison Ave.
Wooster, OH 44691
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Mosquito-borne arboviral diseases such as Zika, dengue fever and chikungunya are transmitted to
humans by infected adult female Aedes aegypti mosquitoes and affect a large portion of the world’s
population. The Kir1 channel in Ae. aegypti (AeKir1) is an important ion channel in the
functioning of mosquito Malpighian (renal) tubules and one that can be manipulated in order to
disrupt excretory functions in mosquitoes. We have previously reported the discovery of various
scaffolds that are active against the AeKir1 channel. Herein we report the synthesis and biological
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characterization of
a
new 2-nitro-5-(4-(phenylsulfonyl) piperazin-1-yl)-N-(pyridin-4-
ylmethyl)anilines scaffold as inhibitors of AeKir1. This new scaffold is more potent in vitro
compared to the previously reported scaffolds, and the molecules kill mosquito larvae.
Keywords: Kir channels, Aedes aegypti, Zika, vector-borne diseases, larvae
Zika virus (ZIKV) is an arbovirus originating in Africa and Asia that was introduced to
South America in 2015 and has since spread throughout Latin America and the Caribbean. This
has coincided with the increasingly high number of reported cases of microcephaly and Guillain-
Barré syndrome and led the World Health Organization (WHO) to declare the emerging Zika
situation a “public health emergency of international concern”. The virus is spread primarily
through mosquitoes of the genus Aedes, with the urban-dwelling, anthropophilic Aedes aegypti
being suspected as the most important species contributing to the spread of ZIKV throughout Latin
America and the Caribbean. In addition to ZIKV, Ae. aegypti is the primary vector of the
arboviruses that cause chikungunya (CHIKV) and dengue (DENV) fevers, which are diseases that
are emerging and reemerging worldwide. Annually, DENV infects hundreds of millions of people,
1
causes hundreds of thousands of hospitalizations, and kills tens of thousands of people. The
economic burden is also substantial with estimates over $2 billion in the Americas and over $1
billion in southeast Asia.2,3 CHIKV had been contained to Africa and Southeast Asia; however,
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over the past decade it has spread to Europe and then to the Americas where over 2 million
suspected cases and autochthonous transmission have been reported.4-6
Unfortunately, there are no effective treatments for ZIKV, CHIKV, or DENV, either as
vaccines or therapeutics; thus, the principal strategy of controlling these diseases is to block the
vector from biting humans. This is generally accomplished using insecticides or insecticide-
treated materials (clothing, nets, etc.). Unfortunately, mosquitoes have evolved resistance to the
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commonly used classes of insecticides (e.g., pyrethroids) , and, the use of similar insecticides has
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also been implicated in the decline of beneficial insects, e.g., the honey bee (Apis mellifera). Thus,
the confluence of these two issues (resistance and decline of pollinators) has made the
identification of novel insecticides with novel mechanisms of action that are selective for
mosquitoes an urgent need.
We have previously identified an inwardly-rectifying potassium (Kir1) channel as a novel
target for the development of new mosquitocides10,11; Kir1 is abundantly expressed in mosquito
Malpighian (renal) tubules and ovaries and plays a crucial role in mosquito renal excretory
capacity, hemolymph potassium homeostasis, and fecundicity.11-15 In other insects, Kir1 channels
play important roles in renal excretions, salivary gland function, and nervous system function.16,17
We have published regarding molecules (1-3) that inhibit the Kir1 channel of Ae. aegypti (AeKir1)
in vitro, and after hemolymph injection are toxic to adult female mosquitoes (Figure 1).11-13 More
recently, we have identified a new compound, 4, that was toxic to mosquitoes in vivo after topical
application to adult females or addition to the rearing water of larvae.18,19 Excitingly, 4 was
similarly toxic to pyrethroid-susceptible and pyrethroid-resistant lab strains of adult female
mosquitoes, showed no apparent toxicity to adult honey bees, Apis mellifera, and exhibited toxic
1
8
efficacy against mosquitoes in semi-field conditions. Although 4, and the other compounds 1-3,
are important analog milestones in the development of AeKir1 inhibitors as novel mosquitocides,
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these compounds show only micromolar activity against the AeKir1 channel in vitro, and low
potency in vivo compared to conventional insecticides (e.g., pyrethroids). Thus, the need to
develop more potent compounds remains a goal of our laboratories. Herein, we report the
discovery and characterization of a new scaffold of AeKir inhibitors and their activity in a larval
toxicity assay.
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NO2
O
N
N
O
N
NH
OH
N
O N
2
O
O
1
2
F3C
O
O
N
N
O
S
O
N
H
N
N
O
3
4, VU041
Figure 1. Previously identified AeKir1 inhibitors.
In order to further develop potent AeKir1 inhibitors, we performed a high-throughput
screening campaign against the Vanderbilt Institute of Chemical Biology compound library and
identified a compound, 5, with a unique and interesting molecular scaffold (Figure 2). This
compound, 5, is characterized by four distinct regions of the molecule that we will exploit in our
structure-activity relationship (SAR) campaign. First, the left-hand aminomethyl pyridine region
(red) will be investigated to determine the optimal moiety, second, the aryl nitro (green) will be
eliminated and replaced, third, the piperazine (black) ring system will be expanded and replaced
with spriocycles, and finally, the arylsulfonamide (blue) will be evaluated. Detailed below is the
work from our laboratory on the medicinal chemistry campaign, the in vitro pharmacology and
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lastly, the in vivo toxicology which has led to the identification of a new and more efficacious
mosquitocide for further evaluation and development.
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N
HN
N
Cl
O N
2
5
Figure 2. Newly identified scaffold from a high-throughput screen and highlighted areas for
SAR diversification.
The synthesis of the first analogs to be evaluated is shown in Scheme 1. The 2,4-
difluoronitrobenzene, 6, was reacted with the appropriate amine under basic conditions (Et
3
N,
DMSO) to give the ortho-substituted product exclusively.20 Next, either piperazine or
homopiperazine were reacted via S Ar with the 4-fluoronitrobenzene analog, 7, to yield the
N
diamino substituted 8. The final targets were realized after reaction with an appropriately chosen
sulfonyl chloride or acid chloride to yield the desired compounds (5, 9a-k, 10a-v, 11a-b, e-f). This
reaction sequence is robust and allows for scale-up of the final targets (>1 g).
Scheme 1. Synthesis of initial sulfonamide analogs.a
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R
R
NH
F
F
HN
F
HN
N
a
b
n
NH
O N
O N
2
2
O N
2
HN
6
7
n
8
1
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O
O
O
S
R
N
R1
R
N
R1
HN
N
HN
N
c
n
+
n
O N
O N
2
2
5, 9a-k,
0a-v, 11a-b
11e,f
1
a
Reagents and conditions. (a) R-NH , Et N, DMSO; (b) Et N, DMSO; (c) R SO Cl or
2
3
3
1
2
R COCl, Et N, CH Cl
1
3
2
2
The initial SAR centered around the sulfonamide (right-hand) portion of the molecule (R,
Table 1). The original hit molecule, 5, was resynthesized and tested. This compound was active
as an AeKir1 channel inhibitor (IC50 = 1.47 µM) utilizing a thallium flux assay. Addition of a
methylene linker between the sulfonyl group and the phenyl resulted in a less active compound,
9
a. Addition of a bromine, 9b, brought the activity back to the same as 5; but removing the phenyl
group led to a decrease in activity (9c, IC50 = 21 µM and 9d, IC50 = 44 µM). Further substitution
around the phenyl (or no substitution) led to the identification of more potent compounds,
highlighted by the phenyl, 9f (IC50 = 0.55 µM), and the 4-fluorophenyl, 9j (IC50 = 0.47 µM) as the
most potent compounds. Addition of a large 4-tert-butyl group led to a loss of activity (9k).
Table 1. Evaluation of the sulfonamide analogs.
N
R
N
HN
N
O N
2
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Thallium Flux
IC50 ± SEM
%
b
b
Cmpd
5
R
cLogPa
( M)
µ
Inhibition
O
O
S
3.37
1.47 ± 0.06
100
Cl
0
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0
O
O
9a
3.55
3.45
5.66 ± 0.86
1.56 ± 0.20
67
86
S
O
O
S
9b
Br
O
O
9c
S
2.29
1.65
20.8 ± 1.3
43.7 ± 19
35
O
O
9d
S
10.8
O
O
S
9e
3.24
2.88
1.11 ± 0.016
0.55 ± 0.04
100
95
O
O
S
9f
O
O
S
Cl
Cl
9g
4.03
0.98 ± 0.046
97
O
O
S
9h
3.88
4.05
3.12
1.24 ± 0.01
6.57 ± 0.35
0.47 ± 0.018
99
CF3
O
O
S
9i
95.3
100
O
O
S
9j
F
O
O
S
9k
4.29
22.6 ± 6.6
44.8
a
Small-Molecule Drug Discovery Suite 2018-2, Schrödinger, LLC, New
b
York, NY, 2018. Thallium flux AeKir1 assay; IC50 values represent the
average (mean ± SEM) of values obtained from at least three individual
experiments.
The next round of analog evaluation moved from the sulfonamide portion to the 3-amino
group (R , Table 2) while maintaining the sulfonamide as either the 4-fluorophenyl or 4-
1
chlorophenyl. The 4-pyridyl group was well tolerated or even produced a slightly more potent
analog (10a, IC50 = 0.39 µM; 10b, IC50 = 0.75 µM), when compared to the 3-pyridyl counterparts.
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Substituting the 2-furyl group was also tolerated; however, there was an interesting change of
potency where the 4-Cl-phenyl derivative was more potent, although the potencies are in a similar
range (4-Cl-phenyl, 10d, IC50 = 0.84 µM; 4-F-phenyl, 10c, IC50 = 1.29 µM). Other replacements
led to compounds that were less active than the best compound, 9j; however, all of the compounds
were within 2-fold of each other (10e-o). Replacing the methylamino linker with a methylether
was not a productive change, but again, there was a divergence between the 4-Cl-phenyl and 4-F-
phenyl moieties (4-F-phenyl, 10p, IC50 = 63 µM; 4-Cl-phenyl, 10q, IC50 = 4.3 µM). Finally,
extending the linker by a methylene group (10r,s) produced equipotent compounds; whereas,
reducing the linker (i.e., direct NH linked) was not productive (10t,u).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
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
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2
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9
0
1
2
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5
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7
8
9
0
Table 2. Evaluation of the left-hand amine moiety.
O
O
S
N
R1
N
R2
O N
2
Thallium Flux
IC50 ± SD
%
cLogPa
(
µ
M)b
Inhibition
b
Cmpd
0a
R
1
R
2
1
N
F
3.39
0.39 ± 0.24
0.75 ± 0.23
103
99
HN
1
0b
0c
0d
Cl
3.64
1
F
3.57
3.82
1.29 ± 0.10
0.84 ± 0.07
96
93
O
HN
1
Cl
10e
Cl
F
4.60
3.72
1.23 ± 0.26
0.80 ± 0.25
19
HN
1
0f
0g
0h
0i
101
N
HN
1
Cl
F
3.98
3.38
3.63
1.1 ± 0.004
1.82 ± 0.40
1.92 ± 0.03
101
95
O
1
HN
1
Cl
96
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8
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
F
F
1
0j
0k
0l
F
3.89
4.14
1.77 ± 0.60
2.17 ± 0.90
31
14
HN
1
Cl
1
Cl
3.42
3.94 ± 0.06
76.13
HN
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
O
10m
Cl
Cl
Cl
3.42
3.32
3.94
4.49 ± 0.31
3.63 ± 0.11
2.61 ± 0.66
94.4
50.58
13.12
HN
10n
N
10o
HN
N
10p
F
3.19
3.46
63 ± 13
77
96
O
101
Cl
4.33 ± 0.14
N
10r
F
3.54
3.80
0.84 ± 0.07
1.27 ± 0.05
103
101
1
0s
0t
0u
HN
Cl
N
1
F
3.21
3.46
42.6 ± 15.0
N/A
69
5
HN
1
Cl
a
Small-Molecule Drug Discovery Suite 2018-2, Schrödinger, LLC, New York,
NY, 2018. Thallium flux AeKir1 assay; IC50 values represent the average
mean ± SEM) of values obtained from at least three individual experiments.
b
(
After evaluating the left- and right-side of the molecule, we investigated chemical
substitutions at the piperazine moiety. In order to achieve this, we varied the ring size (6- to 7-
membered), introduced spirocyclic analogs, and evaluated both amide and methylene linked
compounds. The seven-membered ring systems were synthesized as previously shown; however,
the spirocyclic analogs and the methylene linked compounds’ synthesis is shown in Scheme 2.
The spirocyclic analogs, 11c,d, were made by reacting 7 with the tert-butyl 2,7-
diazaspiro[3.5]nonane-2-carboxylate, 12 under basic conditions (Et
Boc-protected amine, 13, was treated with acid in order to remove the protecting group (4 M HCl,
dioxane), followed by sulfonamide formation (ClSO , DMAP, pyridine) to yield the desired
3
N, DMSO). The resulting
2
R
1
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compounds, 11c,d. The methylene linked compounds were made by S
N
Ar reaction of the
commercially available reagents, 14, and 7 as described above.
Scheme 2. Synthesis of spirocyclic and methylene linked analogs.a
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
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
A
Boc
N
R
R
HN
F
HN
N
a
Boc
O N
O N
2
N
2
7
13
HN
12
O
O
S
N
R1
R
b,c
HN
N
O N
2
11c,d
B
R
R
N
HN
F
a
HN
N
X
O N
2
N
O N
2
7
HN
11g,h
1
4
X
a
Reagents and conditions. (a) Et N, DMSO; (b) 4 M HCl, dioxane; (c) R SO Cl, DMAP, pyridine.
3
1
2
The piperazine moiety was replaced with a seven-membered ring system (homopiperazine)
and the spriocyclic [5.3.0] systems in order to assess the importance of the spatial orientation of
the ring system. Either the 3-pyridyl or 4-pyridyl left-hand substituent were used in this study as
both were potent. Unfortunately, both the homopiperazine (11a,b) and the spirocyclic (11c,d)
systems were much less potent than the corresponding piperazine. To further evaluate the spatial
orientation of the terminal phenyl group (right-hand side) amide bonds were synthesized replacing
the sulfonamide systems. The amide bonds were suspected to be in a planar orientation whereas
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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
the sulfonamide group imparts a distinct “kink” in the system. The amide derivatives were also
less active (or inactive) (11e,f). Lastly, in order to introduce less planarity into the system, the
sulfonamide/amide portion was replaced with a simple methylene linker (11g,h). Both of these
analogs were more active than the amide compounds, and were similar in potency to the
sulfonamide analogs, with a slight loss in potency for the 4-fluorophenyl compound (11g).
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
Table 3. SAR of the cyclic sulfonamide derivatives.
Th Flux
IC50 ± SEM
%
cLogPa
b
Inhibition
b
Cmpd
Structure
( M)
µ
N
O
O
N
S
1
1a
HN
N
3.76
3.48 ± 0.20
2.74 ± 0.17
91
98
O2N
Cl
N
O
O
N
S
11b
HN
N
3.52
O
2
N
F
O
N
O
N
S
1
1c
Cl
4.65
4.16
3.90
4.29
6.18 ± 0.08
8.66 ± 3.50
9.33 ± 1.89
>100
56
67
23
27
HN
N
O
2
N
O
N
O
N
S
11d
F
HN
N
O
2
N
N
O
N
11e
HN
O2N
N
F
N
O
N
1
1f
HN
N
O2N
N
N
1
1g
4.15
4.41
1.92 ± 0.019
1.95 ± 0.011
101
101
HN
N
F
O2N
N
N
11h
HN
N
Cl
2
O N
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a
Small-Molecule Drug Discovery Suite 2018-2, Schrödinger, LLC, New York, NY,
b
2018. Thallium flux AeKir1 assay; IC50 values represent the average (mean ± SEM)
of values obtained from at least three individual experiments.
Having evaluated the methylpyridine, piperazine and sulfonamide portions of the
molecule, we next turned our attention to the nitrophenyl core of the molecule. First, the position
of the nitro group was studied. To this end, the compounds 12a-d were synthesized as outlined in
Scheme 3A-B. The 3-(4-(sulfonyl)piperazin-1-yl)-4-nitro-N-(pyridin-4-ylmethyl)aniline, 12a,b,
were synthesized starting from the 2,4-difluoro-1-nitrobenzene, 15, which was reacted with the
appropriately substituted piperazinesulfonamide, 16, to yield 17. The final targets were realized
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
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
by reaction of pyridine-4-ylmethanamine, 18, with 17 under basic conditions (Et N, DMSO). In
3
a similar fashion, 3-(4-(sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-4-ylmethyl)aniline, 12c,d,
were synthesized by a slight change in the ordering of the reactions. Thus, 2,6-difluoro-1-
nitrobenzene, 19, was reacted with pyridine-4-ylmethanamine, 18, to yield 20 which was then
subjected to the piperazinesulfonamide, 21, yielding the desired compounds, 12c,d.
Scheme 3. Synthesis of nitro regioisomers, 12a-d.a
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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
A
O
O
S
O
O
N
R1
16
S
N
R1
HN
F
F
F
N
a
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
NO2
17
1
5
NO2
N
N
O
O
S
NH2
N
R1
1
8
HN
N
a
1
2a,b
NO2
B
N
N
NO2
NH2
NO2
F
F
18
HN
F
a
1
9
2
0
N
O
O
O
O
S
S
N
R1
NO2
N
R1
HN
21
HN
N
a
1
2c,d
a
Reagents and conditions. (a) Et N, DMSO
3
Replacement of the nitro group with other electron withdrawing groups, or hydrogen, were
also evaluated and the synthesis of these analogs is shown in Scheme 4. The hydrogen analog was
synthesized by starting with 3-chloro-bromobenzene, 22, and reaction with the amine, 18, under
o
cross-coupling conditions (Pd
2
(dba)
3
, CyJohnPhos, Cs
2
CO
3
, 120 C, µW) to yield 23 (selective
bromo reaction). The electron withdrawing groups could be synthesized by reaction of 25 with 18
under the normal S Ar reaction to yield 26. The next reactions merged the two scaffolds (23 and
6) and these could be carried forward under similar conditions. Namely, 23 or 26 was reacted
N
2
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o
with N-Bocpiperazine under cross-coupling conditions (Pd
2
(dba)
3
, CyJohnPhos, Cs
2
CO , 120 C,
3
µW) to yield 27. Next, the Boc protecting group was removed (4 M HCl, dioxane) and then the
appropriate sulfonamide was formed (ClSO , DMAP, pyridine) yielding the desired
2
R
1
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
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
compounds, 12e-l. Final compound 12m, was completed by saponification of the ester moiety.
Scheme 4. Synthesis of non-nitro containing analogs, 12e-m.a
N
N
NH2
18
Cl
Br
HN
Cl
Boc
N
a
HN
2
2
24
2
3
N
N
a
NH2
F
Br
HN
Br
1
8
X
X
b
26
2
5
X = N, CCF , CCN,
3
CCO Me
2
N
O
O
Boc
S
N
R
N
R1
c,d
HN
X
N
HN
N
X
2
7
e
1
2m
1
2e-l
a
o
Reagents and conditions. (a) Pd (dba) , CyJohnPhos, Cs CO , dioxane, 120 C, µW; (b) Et N, DMSO;
2
3
2
3
3
(
c) 4 M HCl, dioxane; (d) R SO Cl, DMAP, pyridine; (e) LiOH, dioxane
1 2
Moving the nitro group ortho to the piperazine group was detrimental to the activity on the
4
-fluorophenylsulfonamide, 12a, but was equipotent with the 4-chlorophenylsulfonamide, 12b.
Placing the nitro group ortho to both amino groups was beneficial. Both 12c and 12d were
submicromolar in activity. Next, removing the nitro group (12e,f) or replacing the nitrophenyl
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1
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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
with a pyridine (12g,h) or CF
3
(12k,l) were not productive changes as each of these compounds
were micromolar (IC50’s = 4 – 9 µM). However, replacing the nitro group with a cyano did
produce potent compounds (12i, IC50 = 0.738 µM and 12j, IC50 = 0.361 µM). Lastly, introduction
of the carboxylic acid (12m) moiety had no effect on the potency compared to the nitro group.
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
Table 4. Evaluation of nitro replacements and regioisomers.
Thallium
Flux
IC50 ± SEM
%
cLogPa
( M)
µ
b
Inhibition
b
Cmpd
Structure
N
O
O
S
N
12a
3.41
ND
8
HN
N
F
NO
2
N
O
O
S
N
12b
3.66
1.20 ± 0.09
101
HN
N
Cl
NO
2
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
NO2
N
12c
12d
12e
3.51
3.76
3.95
4.21
3.59
3.85
3.56
0.833 ± 0.06
0.246 ± 0.01
9.63 ± 0.66
4.01 ± 0.19
4.96 ± 0.22
3.69 ± 0.08
0.738 ± 0.045
105
107
81
HN
HN
HN
HN
HN
HN
N
F
Cl
F
O
NO
2
N
N
N
N
N
N
N
O
N
O
N
12f
62
Cl
O
N
12g
93
F
N
O
N
12h
96
Cl
Cl
N
O
N
12i
99
HN
NC
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N
O
O
S
N
1
2j
3.31
5.13
4.87
0.361 ± 0.016
2.70 ± 0.028
3.24 ± 0.13
101
95
HN
NC
N
F
N
O
O
S
N
12k
HN
N
Cl
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
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
3
F C
N
O
O
S
N
1
2l
97
HN
N
F
3
F C
N
O
O
S
N
HN
N
12m
3.87
2.02 ± 0.11
101
Cl
O
OH
a
Small-Molecule Drug Discovery Suite 2018-2, Schrödinger, LLC, New York,
b
NY, 2018. Thallium flux AeKir1 assay; IC50 values represent the average (mean
SEM) of values obtained from at least three individual experiments.
±
Having identified a novel scaffold as AeKir1 inhibitors, we next evaluated these
compounds in a manual patch clamp assay and compared them to our previously disclosed
compound, 4.18 4 was the first AeKir inhibitor that was shown to be active after topical
administration to the adult female mosquito or addition to the larval rearing water; however, the
in vitro potency for this compound was moderate (Thallium Flux = 1.7 µM; Patch clamp, IC50
=
238 nM). We have discovered a number of compounds that have significantly improved potency
versus 4. The 3- to 4-fold increase in potency seen in the thallium flux assay translated well to the
manual patch clamp assay (Table 5). As we have seen in the past, the compounds are more potent
in the patch clamp assay (left-shifted potency) and our best compound, 12j, is ~9-fold more potent
than 4. In addition, we performed selectivity screening against hKir1.1 and hKir2.1 in thallium
flux assays and found that these compounds were inactive, or weakly active (Table 5).
Table 5. Patch clamp and selectivity data for select compounds.
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1
1
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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
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5
5
5
5
5
5
5
5
5
6
Cmpd
9j
Thallium flux IC50 Patch Clamp IC50
95% CI (nM)
hKir1.1,
M (%
inhibition)
1.90 (27)
NA
NA
NA
NA
7.0 (22)
hKir2.1,
µ
M
a
b
µ
( M)
(nM)
µ
b
0.47 ± 0.018
0.39 ± 0.24
0.84 ± 0.07
57.0
49.7
34.2
35.5
39.0
27.6
238
49.9-64.8
36.2-67.6
23.6-44
29.2-42.7
22.2-64.3
18.3-39.5
192-294
NA
NA
NA
NA
NA
NA
1
1
1
1
1
0a
0d
0f
2i
0.80 ± 0.25
0.738 ± 0.045
0.361 ± 0.016
1.7 (0.6 – 2.8)
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
2j
4
a
Manual patch clamp IC50 values represent the average (mean ± SEM) of values obtained from at least three
b
individual experiments. NA = not active
We next screened a selection of the newly developed compounds for larval toxicity at 100
μM and compared their efficacies to 4 and a small molecule insecticide that has been shown to
inhibit insect Kir1 channels (flonicamid).16 As shown in Figure 3A, all of the compounds had
limited toxicity (<50% mortality) within 24 h, but 12j was the most effective in inducing ~42%
mortality. 4, flonicamid, and the parent compound 5 only produced ~6%, 13%, and ~2% mortality,
1
9
respectively, within 24 h. As we have seen previously with 4 , the toxicity of nearly all the
compounds increased dramatically between 24 and 48 h, with the exceptions of flonicamid and 5.
Notably, 9j, 12i, 12j, and 12k all elicited >80% mortality by 48 h, which was significantly greater
than flonicamid (12% mortality) and 5 (22% mortality) and statistically similar to 4 (67.5%
mortality). In addition to larval toxicity, we compared the 24 h topical toxicity of 9j, flonicamid,
and 4 against adult female mosquitoes using a dose of 12.5 nmol/mosquito. As shown in Figure
3
C, the efficacy of 9j (49%) was statistically similar to that of flonicamid (58.7%) and significantly
greater than that of 4 (17.5%). Thus, this new scaffold appears to not only improve the in vitro
potency but also the in vivo efficacy against both larval and adult female mosquitoes.
A
B
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b
b
b
100
75
100
75
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b
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ab
ab
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a
25
25
a
a
a
a
0
0
Flo
4
5
9j
12i
12j
12k
Flo
4
5
9j
12i
12j
12k
C
80
60
40
a
a
b
20
0
Flo
4
9j
st
Figure 3. The 24 h (A) and 48 h (B) mortality of 1 instar Ae. aegypti after addition of small
molecules (100 µM) to the rearing water. Values are means ± SEM based on 6-18 replicates of 6
larvae each. C) 24 h topical efficacy of small molecules (12.5 nmol/mosquito) against adult female
Ae. aegypti. Values are means ± SEM based on 5-24 replicates of 10 adult females each. In all
2
1
panels, Abbott’s correction was applied to the percent mortalities to account for the negative
control mortality, and the lower-case letters indicate statistical categorization of the means as
determined by a one-way ANOVA with a Tukey’s multiple comparisons test (P < 0.05).
In conclusion, we report a novel scaffold as potent and efficacious AeKir1 channel
inhibitors. The new scaffold is superior to the previously reported 4 in both in vitro potency and
in vivo efficacy. Structure-activity relationship studies confirmed that the sulfonamide moiety was
critical for activity. In addition, the nitro group was not required and the pyridylmethyl amine
could be exchanged for other heterocyclic moieties. Further evaluation in patch clamp assay
identified compounds that were ~10-fold more potent than our previously reported inhibitor and
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with no activity against the closely related human Kir channels. Lastly, we have shown these
compounds to be active against both mosquito larval and adult female mosquitoes, which expands
the potential application of these molecules as novel insecticides. However, future studies will be
needed to evaluate other chemical and toxicological properties of the molecules to determine their
potential suitably as insecticides for field use, such as stability, biodegradability, cuticular
penetration, and safety to non-target organisms (e.g,. mammals, beneficial insects, aquatic
organisms).
0
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EXPERIMENTAL SECTION
1
13
All H & C NMR spectra were recorded on Bruker AV-400 (500 MHz) instrument.
Chemical shifts are reported in ppm relative to residual solvent peaks as an internal standard set to
δH 7.26 or δC 77.0 (CDCl ). Data are reported as follows: chemical shift, multiplicity (br = broad,
3
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (Hz), and
integration. Low resolution mass spectra were obtained on an Agilent 1260 LCMS with
electrospray ionization, with a gradient of 5-95% MeCN in 0.1% formic acid water over 4 min.
Analytical thin layer chromatography was performed on LuxPlate silica gel 60 F254 plates.
Visualization was accomplished with UV light, and/or the use of ninhydrin, anisaldehyde and ceric
ammonium molybdate solutions followed by charring on a hot-plate. Chromatography on silica
gel was performed using Silica Gel 60 Å (230-400 mesh) from Sorbent Technologies. Solvents for
extraction, washing and chromatography were HPLC grade. All reagents were purchased from
Aldrich Chemical Co. (or similar) and were used without purification. All reagents and solvents
were commercial grade and purified prior to use when necessary. Microwave synthesis was
performed on an Anton Paar Monowave 400 equipped with an autosampler.
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Final compounds were purified on a Gilson preparative reversed-phase HPLC system
comprised of a 322 aqueous pump with solvent-selection valve, 334 organic pump, GX-271 liquid
hander, two column switching valves, and a 159 UV detector. UV wavelength for fraction
collection was user-defined, with absorbance at 254 nm always monitored. Column: Phenomenex
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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Axia-packed Luna C18, 50 x 21.2 mm, 5 µm. For Acidic Method: Mobile phase: CH
3
CN in H
0.1% formic acid). Gradient conditions: 2.0 min equilibration, followed by user-defined gradient
starting organic percentage, ending organic percentage, duration, typically 15 mins), hold at 95%
2
O
(
(
CH
3
3
CN in H
CN in H
2
O (0.1% TFA) for 2 min, 20 mL/min, 23 °C. For Basic Method: Mobile phase:
CH
2
O (0.05% v/v NH OH). Gradient conditions: 0.75 min equilibration, followed by
4
user-defined gradient (starting organic percentage, ending organic percentage, duration), hold at
5% CH CN in H O (0.05% v/v NH OH) for 1 min, 50 mL/min, 23 °C. The purity of all
9
3
2
4
compounds was determined by LCMS to be >95% (or as stated).
General Procedure for the Synthesis of 5, 9a-k, 10a-v, 11a,b,e,f, 12a-d. To a two-dram
vial was added 2,4-difluoronitrobenzene, 6, (10.8 µL, 0.100 mmol), dimethyl sulfoxide (1.00 mL),
triethylamine (20.8 µL, 0.150 mmol), followed by the amine (0.100 mmol) of interest. After
stirring at room temperature for 16 h, triethylamine (20.8 µL, 0.150 mmol) and the cyclic diamine
(0.150 mmol) of interest were added. The reaction was stirred for an additional 16 h at room
temperature. The reaction was diluted with ethyl acetate (15 mL) and washed with 1 M HCl (15
mL). The aqueous layer was basified with 1 M NaOH to pH 9 and extracted with ethyl acetate (15
mL) twice. Organic layers were combined and washed with saturated brine (15 mL), dried over
anhydrous Na
2
SO , filtered, and concentrated under reduced pressure. The crude product, 8, was
4
used without further purification.
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To a two-dram vial was added the starting compound, 8, (0.100 mmol), dichloromethane
(
3.00 mL), triethylamine (20.8 µL, 0.150 mmol), and the electrophile (R
1
SO
2
Cl or R COCl) (0.150
1
mmol) of interest. After stirring at room temperature for 16 h, the reaction was diluted with
dichloromethane (15 mL) and washed with water (15 mL). The aqueous layer was extracted with
0
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8
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9
0
dichloromethane (15 mL) twice. Organic layers were combined, dried over anhydrous Na
2
SO ,
4
filtered, and concentrated. The crude products were purified by flash column chromatography on
silica gel (dry loaded using silica/DCM) with a gradient of 0-20% methanol in dichloromethane to
yield the final compounds (5, 9a-k, 10a-v, 11a-b).
5-(4-((4-chlorophenyl)sulfonyl)piperazine-1-yl)-2-nitro-N-(pyridine-3-
ylmethyl)aniline (5). Yield = 12 mg; 23%. LCMS: R
T
= 2.474 min., >98% @ 215 and 254 nm,
) δ 8.78 (d, J = 5.9 Hz, 1H), 8.56 (s, 1H),
.43 (d, J = 4.0 Hz, 1H), 7.83 (d, J = 9.7 Hz, 1H), 7.78 – 7.60 (m, 5H), 7.30 (dd, J = 7.6, 4.9 Hz,
H), 6.28 (dd, J = 9.7, 1.9 Hz, 1H), 5.88 (s, 1H), 4.56 (d, J = 5.9 Hz, 2H), 3.42 (s, 4H), 2.87 (s,
+
1
m/z = 488.1 [M + H] . H NMR (499 MHz, DMSO-d
6
8
1
1
3
4H). C NMR (126 MHz, DMSO-d
6
) δ 155.37, 149.83, 149.34, 147.53, 139.36, 136.05, 135.14,
1
34.57, 130.61, 130.33, 129.26, 124.55, 124.38, 105.60, 95.30, 46.63, 46.01, 44.01.
5
-(4-(benzylsulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-ylmethyl)aniline
(9a).
Compound was purchased from ChemDiv. LCMS: R
T
= 2.297 min., >98% @ 215 and 254 nm,
+
1
m/z = 468.1 [M + H] . H NMR (499 MHz, CDCl ) δ 8.75 (t, J = 5.0 Hz, 1H), 8.63 (s, 1H), 8.57
3
(d, J = 3.7 Hz, 1H), 8.09 (d, J = 9.6 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.38 (s, 5H), 7.31 (dd, J =
7.7, 4.9 Hz, 1H), 6.18 (dd, J = 9.7, 2.3 Hz, 1H), 5.76 (d, J = 2.1 Hz, 1H), 4.53 (d, J = 5.4 Hz, 2H),
13
4
1
9
.25 (s, 2H), 3.18 (dd, J = 22.3, 5.3 Hz, 8H). C NMR (126 MHz, CDCl ) δ 155.47, 149.31,
3
48.80, 146.85, 134.72, 133.03, 130.67, 129.18, 129.02, 128.94, 128.46, 125.26, 123.86, 104.90,
5.18, 57.49, 47.36, 45.36, 44.63.
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-(4-((4-bromophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-ylmethyl)aniline
(
9b). Compound was purchased from ChemDiv. LCMS: R
T
= 2.521 min., >98% @ 215 and 254
+
1
nm, m/z = 532.0 [M + H] . H NMR (499 MHz, CDCl
3
) δ 8.74 (t, J = 5.1 Hz, 1H), 8.62 (s, 1H),
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
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
8.57 (d, J = 3.8 Hz, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.68 (dd, J = 15.0, 8.2 Hz, 3H), 7.61 (d, J = 8.5
Hz, 2H), 7.30 (dd, J = 7.7, 4.9 Hz, 1H), 6.17 (dd, J = 9.7, 2.2 Hz, 1H), 5.77 (d, J = 1.9 Hz, 1H),
13
4
.52 (d, J = 5.4 Hz, 2H), 3.40 – 3.32 (m, 4H), 3.14 – 3.04 (m, 4H). C NMR (126 MHz, CDCl )
3
δ 155.18, 149.32, 148.79, 146.80, 134.69, 134.40, 133.80, 133.02, 132.62, 129.25, 129.21, 128.46,
1
23.87, 104.75, 95.28, 46.67, 45.38, 44.63.
5-(4-(ethylsulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-ylmethyl)aniline
(9c).
Compound was purchased from ChemDiv. LCMS: R
T
= 1.990 min., >98% @ 215 and 254 nm,
+
1
m/z = 406.1 [M + H] . H NMR (499 MHz, CDCl
Hz, 1H), 8.13 (d, J = 9.6 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.38 (dd, J = 7.4, 5.0 Hz, 1H), 6.25
dd, J = 9.6, 2.0 Hz, 1H), 5.82 (d, J = 1.7 Hz, 1H), 4.58 (d, J = 5.3 Hz, 2H), 3.37 (s, 8H), 2.98 (q,
3
) δ 8.79 (s, 1H), 8.68 (s, 1H), 8.59 (d, J = 3.8
(
1
3
J = 7.4 Hz, 2H), 1.38 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl
3
) δ 155.47, 148.40, 147.97,
1
46.78, 135.58, 133.66, 129.30, 125.35, 124.19, 104.98, 95.23, 47.36, 45.15, 44.55, 44.42, 7.82.
5
-(4-(methylsulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-ylmethyl)aniline (9d).
= 1.899 min., >98% @ 215 and 254 nm, m/z = 392.1 [M + H]+.
Yield = 15 mg; 36%. LCMS: R
1H NMR (499 MHz, DMSO-d
T
6
) δ 8.80 (s, 1H), 8.60 (s, 1H), 8.43 (d, J = 3.8 Hz, 1H), 7.89 (d, J =
9.7 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.33 (dd, J = 7.5, 4.9 Hz, 1H), 6.38 (dd, J = 9.7, 1.8 Hz, 1H),
1
3
5.98 (s, 1H), 4.61 (d, J = 5.9 Hz, 2H), 3.44 (s, 4H), 3.09 (d, J = 4.3 Hz, 4H), 2.83 (s, 3H). C NMR
126 MHz, DMSO-d ) δ 155.75, 149.86, 149.36, 147.67, 136.08, 135.17, 129.28, 124.62, 124.39,
05.78, 95.31, 46.90, 45.64, 44.08, 34.98.
(
6
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2
-nitro-N-(pyridin-3-ylmethyl)-5-(4-tosylpiperazin-1-yl)aniline (9e). Yield = 34 mg;
+
1
7
0%. LCMS: R
T
= 2.386 min., >98% @ 215 and 254 nm, m/z = 468.1 [M + H] . H NMR (499
MHz, DMSO-d
6
) δ 8.86 (s, 1H), 8.66 (s, 1H), 8.53 (d, J = 3.7 Hz, 1H), 7.91 (d, J = 9.7 Hz, 1H),
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
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7.81 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.39 (dd, J = 7.5, 4.9
Hz, 1H), 6.35 (dd, J = 9.7, 1.8 Hz, 1H), 5.96 (d, J = 1.6 Hz, 1H), 4.64 (d, J = 5.9 Hz, 2H), 3.51 (s,
13
4
1
4
H), 2.89 (s, 4H), 2.43 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 155.30, 149.84, 149.35, 147.52,
6
44.84, 136.04, 135.13, 132.55, 130.87, 129.27, 128.48, 124.55, 124.33, 105.55, 95.27, 46.65,
6.06, 44.00, 21.93.
2-nitro-5-(4-(phenylsulfonyl)piperazin-1-yl)-N-(pyridin-3-ylmethyl)aniline
(9f).
Compound was purchased from ChemDiv. LCMS: R
T
= 2.289 min., >98% @ 215 and 254 nm,
+
1
m/z = 454.1 [M + H] . H NMR (499 MHz, CDCl
3.8 Hz, 1H), 8.07 (d, J = 9.6 Hz, 1H), 7.76 (d, J = 7.4 Hz, 2H), 7.64 (dd, J = 19.8, 7.6 Hz, 2H),
.56 (t, J = 7.6 Hz, 2H), 7.29 (dd, J = 7.7, 4.9 Hz, 1H), 6.16 (dd, J = 9.7, 2.3 Hz, 1H), 5.77 (d, J =
3
) δ 8.76 – 8.69 (m, 1H), 8.62 (s, 1H), 8.57 (d, J
=
7
1
3
1.9 Hz, 1H), 4.51 (d, J = 5.3 Hz, 2H), 3.45 – 3.30 (m, 4H), 3.12 – 3.02 (m, 4H). C NMR (126
MHz, CDCl ) δ 155.25, 149.31, 148.80, 146.82, 135.32, 134.70, 133.25, 133.02, 129.27, 129.21,
27.76, 125.28, 123.85, 104.73, 95.15, 46.66, 45.44, 44.63.
3
1
5
-(4-((3,4-dichlorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-
ylmethyl)aniline (9g). Yield = 34.1 mg; 8%. LCMS: R = 2.637 min., >98% @ 215 and 254 nm,
) δ 8.74 (s, 1H), 8.62 (s, 1H), 8.57 (s, 1H), 8.09
(d, J = 9.6 Hz, 1H), 7.84 (s, 1H), 7.65 (dd, J = 16.8, 8.0 Hz, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.35 –
T
+
1
m/z = 522.0 [M + H] . H NMR (499 MHz, CDCl
3
7
4
1
.27 (m, 1H), 6.17 (d, J = 9.5 Hz, 1H), 5.78 (s, 1H), 4.52 (d, J = 4.4 Hz, 2H), 3.37 (s, 4H), 3.11 (s,
1
3
H). C NMR (126 MHz, CDCl
3
) δ 155.11, 149.32, 148.78, 146.79, 138.33, 135.31, 134.71,
34.17, 133.02, 131.36, 129.52, 129.27, 126.68, 125.43, 123.88, 104.74, 95.36, 46.69, 45.38,
44.63.
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-nitro-N-(pyridin-3-ylmethyl)-5-(4-((4-(trifluoromethyl)phenyl)sulfonyl)piperazin-
1
-yl)aniline (9h). Yield = 89.1 mg; 21%. LCMS: R
T
= 2.551 min., >98% @ 215 and 254 nm, m/z
+
1
=
522.1 [M + H] . H NMR (499 MHz, CDCl
3
) δ 8.73 (s, 1H), 8.62 (s, 1H), 8.57 (s, 1H), 8.08 (d,
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
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
J = 9.6 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.33
–
7.26 (m, 1H), 6.16 (d, J = 9.5 Hz, 1H), 5.77 (s, 2H), 4.52 (d, J = 4.6 Hz, 2H), 3.36 (s, 4H), 3.12
13
(
s, 4H). C NMR (126 MHz, CDCl ) δ 155.17, 149.33, 148.78, 146.78, 139.18, 134.84, 134.69,
3
1
33.02, 129.24, 128.24, 126.47, 125.44, 123.88, 121.98, 104.76, 95.38, 46.73, 45.39, 44.62.
5
-(4-(naphthalen-2-ylsulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-ylmethyl)aniline
(9i). Compound was purchased from ChemDiv. LCMS: R = 2.559 min., >98% @ 215 and 254
) δ 8.71 (t, J = 4.8 Hz, 1H), 8.60 (s, 1H),
.54 (d, J = 3.9 Hz, 1H), 8.34 (s, 1H), 8.04 (d, J = 9.7 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.93 (d,
J = 7.9 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.67 (dd, J = 17.9, 7.7 Hz, 3H), 7.29 – 7.24 (m, 1H),
.14 (dd, J = 9.7, 2.1 Hz, 1H), 5.74 (d, J = 1.8 Hz, 1H), 4.49 (d, J = 5.3 Hz, 2H), 3.41 – 3.32 (m,
T
+
1
nm, m/z = 504.1 [M + H] . H NMR (499 MHz, CDCl
3
8
6
1
3
4H), 3.21 – 3.09 (m, 4H). C NMR (126 MHz, CDCl
3
) δ 155.18, 149.28, 148.79, 146.81, 135.04,
34.68, 133.01, 132.39, 132.20, 129.50, 129.25, 129.20, 129.15, 127.99, 127.82, 125.23, 123.82,
22.76, 104.69, 95.09, 46.68, 45.50, 44.61.
1
1
5
-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-ylmethyl)aniline
9j). Yield = 102.8 mg; 31%. LCMS: R = 2.326 min., >98% @ 215 and 254 nm, m/z = 472.1 [M
) δ 8.73 (s, 1H), 8.62 (s, 1H), 8.57 (d, J = 3.9 Hz, 1H), 8.08 (d,
(
T
+
1
+ H] . H NMR (499 MHz, CDCl
3
J = 9.6 Hz, 1H), 7.82 – 7.73 (m, 2H), 7.67 (d, J = 7.3 Hz, 1H), 7.35 – 7.17 (m, 3H), 6.17 (d, J =
13
9
.7 Hz, 1H), 5.77 (s, 1H), 4.52 (d, J = 5.0 Hz, 2H), 3.36 (s, 4H), 3.08 (s, 4H). C NMR (126 MHz,
CDCl
3
) δ 155.22, 149.30, 148.79, 146.81, 134.70, 133.03, 130.51, 130.43, 129.22, 125.35, 123.87,
1
16.70, 116.52, 104.75, 95.25, 46.66, 45.40, 44.62.
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-(4-((4-(tert-butyl)phenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-3-
ylmethyl)aniline (9k). Compound was purchased from ChemDiv. LCMS: R
T
= 2.725 min., >98%
) δ 8.86 (t, J = 5.8 Hz,
1H), 8.66 (s, 1H), 8.52 (d, J = 3.5 Hz, 1H), 7.91 (d, J = 9.7 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.74
+
1
@
215 and 254 nm, m/z = 510.1 [M + H] . H NMR (499 MHz, DMSO-d
6
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
–
7.61 (m, 4H), 7.38 (dd, J = 7.5, 5.0 Hz, 1H), 6.36 (dd, J = 9.8, 1.7 Hz, 1H), 5.98 (s, 1H), 4.64 (d,
13
J = 5.8 Hz, 2H), 3.51 (s, 4H), 2.91 (s, 4H), 1.30 (d, J = 34.1 Hz, 9H). C NMR (126 MHz, DMSO-
d
6
) δ 157.38, 155.38, 149.84, 149.34, 147.53, 136.04, 135.14, 132.63, 129.26, 128.43, 127.24,
1
24.53, 124.34, 105.56, 95.26, 46.62, 46.15, 44.01, 35.85, 31.64.
5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-4-ylmethyl)aniline
(10a). Yield = 90.4 mg; 27%. LCMS: R
T
= 2.192 min., >98% @ 215 and 254 nm, m/z = 472.1
) δ 8.87 (t, J = 6.0 Hz, 1H), 8.52 (d, J = 5.6 Hz, 2H),
.90 (d, J = 9.7 Hz, 1H), 7.80 (dd, J = 8.6, 5.0 Hz, 2H), 7.49 (t, J = 8.7 Hz, 2H), 7.35 (d, J = 5.3
+
1
[
M + H] . H NMR (499 MHz, DMSO-d
6
7
Hz, 2H), 6.34 (d, J = 9.8 Hz, 1H), 5.83 (s, 1H), 4.63 (d, J = 6.1 Hz, 2H), 3.43 (s, 4H), 2.88 (s, 4H).
13C NMR (126 MHz, DMSO-d
6
) δ 150.24, 150.21, 146.63, 146.21, 131.12, 130.93, 122.75,
1
17.34, 117.16, 105.20, 95.47, 95.17, 95.01, 46.15, 45.55, 44.99.
-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-4-ylmethyl)aniline
5
(
10b). Yield = 208.7 mg; 60%. LCMS: R = 2.286 min., >98% @ 215 and 254 nm, m/z = 488.1
T
+
[
M + H] . NMR (499 MHz, CDCl
3
) δ 8.81 (s, 1H), 8.59 (d, J = 4.3 Hz, 2H), 8.09 (d, J = 9.6 Hz,
1H), 7.72 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.17 (d, J = 9.6 Hz, 1H), 5.62
13
(
s, 1H), 4.53 (d, J = 5.1 Hz, 2H), 3.32 (s, 4H), 3.03 (s, 4H). C NMR (126 MHz, CDCl ) δ 155.06,
3
1
4
51.30, 150.39, 146.85, 139.98, 133.77, 129.65, 129.26, 129.12, 125.37, 121.65, 104.85, 95.34,
6.62, 45.98, 45.30.
5
-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-(furan-2-ylmethyl)-2-nitroaniline
(10c). Yield = 60 mg; 39%. LCMS: R = 2.836 min, >98% @ 215 and 254 nm, m/z = 461.1 [M
t
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+
1
+
H] . H NMR (499 MHz, CDCl
3
) δ 8.64 (s, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.87 – 7.76 (m, 2H),
7
.41 (d, J = 11.2 Hz, 2H), 7.26 (d, J = 7.7 Hz, 1H), 6.37 (s, 1H), 6.29 (s, 1H), 6.19 (d, J = 9.6 Hz,
1
3
1
H), 6.01 (s, 1H), 4.48 (d, J = 4.1 Hz, 2H), 3.48 (s, 4H), 3.16 (s, 4H). C NMR (126 MHz, CDCl )
3
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
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
δ 155.16, 150.91, 146.91, 142.29, 130.50, 130.42, 129.08, 125.30, 125.02, 116.69, 116.51, 110.60,
07.60, 104.66, 95.14, 46.77, 45.46, 40.28, 29.71.
1
5
-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-N-(furan-2-ylmethyl)-2-nitroaniline
10d). Yield = 22.2 mg; 29%. LCMS: R = 2.942 min, >98% @ 215 and 254 nm, m/z = 477.0 [M
) δ 8.62 (s, 1H), 8.06 (d, J = 9.6 Hz, 1H), 7.71 (d, J = 7.8 Hz,
(
t
+
1
+
H] . H NMR (499 MHz, CDCl
3
2H), 7.53 (d, J = 7.9 Hz, 2H), 7.37 (s, 1H), 6.35 (s, 1H), 6.26 (s, 1H), 6.16 (d, J = 9.6 Hz, 1H),
1
3
5.98 (s, 1H), 4.46 (d, J = 4.5 Hz, 2H), 3.45 (s, 4H), 3.13 (s, 4H). C NMR (126 MHz, CDCl
3
) δ
1
1
55.14, 150.91, 146.91, 142.30, 139.96, 133.91, 129.63, 129.14, 129.09, 125.32, 110.61, 107.61,
04.67, 95.16, 46.78, 45.45, 40.28.
N-benzyl-5-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-2-nitroaniline
(10e).
Compound was purchased from ChemDiv. LCMS: R
t
= 3.051 min, >98% @ 215 and 254 nm, m/z
+
1
= 487.0 [M + H] . H NMR (499 MHz, CDCl
3
) δ 8.75 (s, 1H), 8.07 (d, J = 9.6 Hz, 1H), 7.68 (d, J
=
8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.40 – 7.26 (m, 5H), 6.13 (dd, J = 9.6, 2.1 Hz, 1H), 5.79
13
(
d, J = 1.7 Hz, 1H), 4.49 (d, J = 5.2 Hz, 2H), 3.40 – 3.28 (m, 4H), 3.09 – 3.01 (m, 4H). C NMR
(
126 MHz, CDCl ) δ 154.97, 147.19, 139.94, 137.46, 133.90, 129.61, 129.11, 129.07, 128.96,
3
127.68, 126.98, 125.21, 104.53, 95.72, 47.22, 46.72, 45.32.
5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-2-ylmethyl)aniline
(
10f). Yield = 7.1 mg; 4.6%. LCMS: R
t
= 2.615 min, >98% @ 215 and 254 nm, m/z = 472.1 [M
+
1
+
H] . H NMR (499 MHz, DMSO-d
6
) δ 9.17 (s, 1H), 8.59 (s, 1H), 7.91 (d, J = 9.6 Hz, 1H), 7.83
(
dd, J = 15.8, 7.2 Hz, 2H), 7.51 (t, J = 8.7 Hz, 2H), 7.42 (d, J = 7.4 Hz, 1H), 7.35 (d, J = 4.8 Hz,
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H), 6.36 (d, J = 10.0 Hz, 1H), 6.07 (s, 1H), 4.65 (d, J = 4.7 Hz, 2H), 3.52 (s, 4H), 2.96 (s, 4H).
13C NMR (126 MHz, DMSO-d
29.18, 124.31, 123.45, 122.89, 117.79, 117.61, 105.50, 95.61, 48.40, 46.63, 46.16.
) δ 157.81, 155.52, 149.83, 147.65, 137.95, 131.63, 131.55,
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1
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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0
5
-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(pyridin-2-ylmethyl)aniline
(10g). Yield = 6.0 mg; 3.6%. LCMS: R = 2.728 min., >98% @ 215 and 254 nm, m/z = 488.0 [M
) δ 9.20 (t, J = 5.2 Hz, 1H), 8.61 (d, J = 4.7 Hz, 1H), 7.94
d, J = 9.7 Hz, 1H), 7.89 – 7.72 (m, 5H), 7.45 (d, J = 7.8 Hz, 1H), 7.37 (dd, J = 7.0, 5.3 Hz, 1H),
.39 (dd, J = 9.8, 2.5 Hz, 1H), 6.09 (d, J = 2.4 Hz, 1H), 4.68 (d, J = 5.2 Hz, 2H), 3.59 – 3.49 (m,
T
+
1
+
H] . H NMR (499 MHz, DMSO-d
6
(
6
1
3
4H), 3.11 – 2.96 (m, 4H). C NMR (126 MHz, DMSO-d
139.37, 137.93, 134.61, 130.62, 130.38, 129.17, 124.31, 123.45, 122.89, 105.50, 95.60, 48.40,
6.63, 46.12.
6
) δ 157.82, 155.51, 149.82, 147.65,
4
5
-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-((tetrahydro-2H-pyran-3-
yl)methyl)aniline (10h). Yield = 118.5 mg; 74%. LCMS: R = 2.812 min, >98% @ 215 and 254
) δ 8.39 (s, 1H), 8.04 (d, J = 9.6 Hz, 1H),
7.84 – 7.76 (m, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.14 (d, J = 9.7 Hz, 1H), 5.87 (s, 1H), 3.91 (d, J =
T
+
1
nm, m/z = 479.1 [M + H] . H NMR (499 MHz, CDCl
3
1
2
1.4 Hz, 1H), 3.86 – 3.77 (m, 1H), 3.57 – 3.43 (m, 5H), 3.40 – 3.29 (m, 1H), 3.28 – 3.05 (m, 6H),
13
.10 – 1.90 (m, 2H), 1.65 (d, J = 38.1 Hz, 2H), 1.44 (d, J = 9.5 Hz, 1H). C NMR (126 MHz,
CDCl ) δ 166.49, 164.45, 155.32, 147.64, 131.47, 130.52, 130.45, 129.12, 124.97, 116.70, 116.52,
3
104.39, 94.57, 70.96, 68.58, 46.78, 45.56, 44.89, 35.53, 27.46, 24.57.
5-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-((tetrahydro-2H-pyran-3-
yl) methyl)aniline (10i). Yield = 48.8 mg; 30%. LCMS: R
T
= 2.931 min, >98% @ 215 and 254
) δ 8.39 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H),
.72 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 6.14 (d, J = 9.6 Hz, 1H), 5.87 (s, 1H), 3.91 (d,
+
1
nm, m/z = 495.1 [M + H] . H NMR (499 MHz, CDCl
3
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J = 11.4 Hz, 1H), 3.86 – 3.78 (m, 1H), 3.50 (d, J = 24.2 Hz, 5H), 3.35 (s, 1H), 3.21 (dd, J = 12.6,
6
.5 Hz, 2H), 3.16 (s, 4H), 2.06 – 1.89 (m, 2H), 1.66 (d, J = 32.7 Hz, 2H), 1.44 (d, J = 9.1 Hz, 1H).
13C NMR (126 MHz, CDCl
) δ 155.29, 147.63, 139.96, 133.88, 129.62, 129.15, 129.13, 124.99,
3
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
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5
5
5
5
5
5
5
6
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
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7
8
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0
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2
3
4
5
6
7
8
9
0
104.38, 94.59, 70.96, 68.58, 46.79, 45.53, 44.89, 35.52, 27.45, 24.55.
N-(3,3-difluorocyclobutyl)-5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-
nitroaniline (10j). Yield = 29 mg; 20%. LCMS: R
T
= 2.894 min, >98% @ 215 and 254 nm, m/z
+ 1
=
471.0 [M + H] . H NMR (499 MHz, DMSO-d
6
) δ 8.33 (d, J = 5.2 Hz, 1H), 7.95 (d, J = 9.7 Hz,
1
H), 7.92 – 7.85 (m, 2H), 7.54 (t, J = 8.7 Hz, 2H), 6.44 (d, J = 9.8 Hz, 1H), 5.89 (s, 1H), 4.11 (s,
1
3
2H), 3.59 (s, 4H), 3.24 – 3.11 (m, 2H), 3.04 (s, 4H), 2.69 (d, J = 15.6 Hz, 2H). C NMR (126
MHz, DMSO-d ) δ 155.40, 146.32, 131.61, 131.59, 131.20, 131.12, 128.81, 124.03, 117.33,
17.15, 105.45, 94.79, 46.17, 45.89, 42.70, 36.95.
6
1
5
-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-N-(3,3-difluorocyclobutyl)-2-
nitroaniline (10k). Yield = 79 mg; 51%. LCMS: R = 2.983 min, >98% @ 215 and 254 nm, m/z
) δ 8.40 (s, 1H), 8.07 (d, J = 9.5 Hz, 1H), 7.72 (d, J
T
+
1
=
487.0 [M + H] . H NMR (499 MHz, CDCl
3
= 7.6 Hz, 2H), 7.54 (d, J = 7.7 Hz, 2H), 6.20 (d, J = 9.6 Hz, 1H), 5.66 (s, 1H), 3.91 (s, 1H), 3.47
13
(
s, 4H), 3.24 – 3.04 (m, 6H), 2.67 – 2.50 (m, 2H). C NMR (126 MHz, CDCl ) δ 155.29, 145.87,
3
1
40.02, 133.86, 129.65, 129.28, 129.15, 125.37, 104.88, 94.89, 46.73, 45.48, 43.57, 43.39, 43.20.
5
-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-N-cyclopropyl-2-nitroaniline
Yield = 4 mg; 7%. LCMS: R = 3.035 min, >98% @ 215 and 254 nm, m/z = 437.0 [M + H] . H
NMR (499 MHz, DMSO-d ) δ 8.25 (d, J = 1.9 Hz, 1H), 7.91 (d, J = 9.8 Hz, 1H), 7.84 – 7.78 (m,
H), 7.80 – 7.74 (m, 2H), 6.47 – 6.40 (m, 2H), 3.65 – 3.53 (m, 4H), 3.13 – 3.03 (m, 4H), 2.62 (td,
(10l).
+
1
T
6
3
13
J = 6.6, 2.9 Hz, 1H), 0.89 (dd, J = 6.7, 1.9 Hz, 2H), 0.60 (dd, J = 3.6, 2.3 Hz, 2H). C NMR (126
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