Discovery, Optimization, and Biological Evaluation of Sulfonamidoacetamides as an Inducer of Axon Regeneration

Article abstract of DOI:10.1021/acs.jmedchem.6b00015

Axon regeneration after injury in the central nervous system is hampered in part because if an age-dependent decline in the intrinsic axon growth potential, and one of the strategies to stimulate axon growth in injured neurons involves pharmacological manipulation of implicated signaling pathways. Here we report phenotypic cell-based screen of chemical libraries and structure-activity-guided optimization that resulted in the identification of compound 7p which promotes neurite outgrowth of cultured primary neurons derived from the hippocampus, cerebral cortex, and retina. In an animal model of optic nerve injury, compound 7p was shown to induce growth of GAP-43 positive axons, indicating that the in vitro neurite outgrowth activity of compound 7p translates into stimulation of axon regeneration in vivo. Further optimization of compound 7p and elucidation of the mechanisms by which it elicits axon regeneration in vivo will provide a rational basis for future efforts to enhance treatment strategies.

Full text of DOI:10.1021/acs.jmedchem.6b00015

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Article
Discovery, Optimization and Biological Evaluation of
Sulfonamidoacetamides as an Inducer of Axon Regeneration
Jin-Mo Ku, Kyuhee Park, Jung Hun Lee, Kyong Jin Cho, Yeon-Ju Nam,
Dae-Youn Jeong, Yu-Han Kim, SoonJung Kwon, Ju-Young Park, Jungeun
Yang, Tae-gyu Nam, Sung-Hwa Yoon, Sangmee Ahn, and Yongmun Choi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00015 • Publication Date (Web): 23 Mar 2016
Downloaded from http://pubs.acs.org on March 24, 2016
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Discovery, Optimization and Biological Evaluation of Sulfonamidoacetamides as an
Inducer of Axon Regeneration
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JinꢀMo Ku1, Kyuhee Park1, Jung Hun Lee1, Kyong Jin Cho2, YeonꢀJu Nam1, DaeꢀYoun
Jeong1, YuꢀHan Kim3, SoonJung Kwon4, JuꢀYoung Park4, Jungeun Yang1, Taeꢀgyu Nam5,
SungꢀHwa Yoon4, Sangmee Ahn3*, and Yongmun Choi1*
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1BioꢀCenter, Gyeonggi Institute of Science and Technology Promotion, 147 Gwanggyoꢀro,
Suwon, Korea 16229
2Department of Ophthalmology, Medical College, Dankook University, 119 Dandaeꢀro,
Cheonan, Korea 31116
3Department of Pharmacy, College of Pharmacy and Nanobiomedical Science & BK21 PLUS
NBM Global Research Center for Regenerative Medicine, Dankook University, 119 Dandaeꢀ
ro, Cheonan, Korea 31116
4Department of Applied Chemistry, Ajou University, 206 Worldcupꢀro, Suwon, Korea 16499
5Department of Pharmacy, Hanyang University, 55 Hanyangdaehakꢀro, Ansan, Korea 15588
* To whom correspondence should be addressed. For S. A.: phone, 82ꢀ41ꢀ550ꢀ1433; fax, 82ꢀ
41ꢀ559ꢀ7899; eꢀmail, smahn@dankook.ac.kr. For Y.C.: phone, 82ꢀ31ꢀ888ꢀ6972; fax, 82ꢀ31ꢀ
888ꢀ6979; eꢀmail, ychoi@gstep.re.kr.
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Abstract
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The axon regeneration after injury in the central nervous system is hampered due in part to an
ageꢀdependent decline in the intrinsic axon growth potential, and one of the strategies to
stimulate axon growth in injured neurons involves pharmacological manipulation of
implicated signaling pathways. Here we report phenotypic cellꢀbased screen of chemical
libraries and structureꢀactivity guided optimization that resulted in the identification of
compound 7p, which promotes neurite outgrowth of cultured primary neurons derived from
the hippocampus, cerebral cortex, and retina. In an animal model of optic nerve injury,
compound 7p was shown to induce growth of GAPꢀ43 positive axons, indicating that the in
vitro neurite outgrowth activity of compound 7p translates into stimulation of axon
regeneration in vivo. Further optimization of compound 7p and elucidating the mechanisms
by which it elicits axon regeneration in vivo will provide a rational basis for future efforts to
enhance treatment strategies.
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Introduction
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The neurons in the adult central nervous system (CNS) fail to regenerate axons after injury,
which accounts in part for the poor functional recovery in patients with traumatic and
neurodegenerative diseases.1 The failure of axon regeneration is attributable to a growthꢀ
inhibiting environment involving myelinꢀassociated inhibitors (NogoꢀA, myelin associated
glycoprotein, and oligodendrocyte/myelin glycoprotein) and glial scar at the injury site.2
However, neutralizing and/or removing such inhibitory factors are insufficient to promote
longꢀdistance axon regeneration.3 Adding to the complexity of inhibitory extrinsic factors is
an ageꢀdependent decline in the intrinsic axon growth potential; while the capacity of axon to
elongate during development is robust, cell intrinsic mechanisms regulating axon growth are
suppressed after the formation of functional synapses in the adult CNS and remain inactive
even following traumatic injury.4, 5
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Recent years have seen some progress in understanding the mechanisms that lead to
reactivation of cell intrinsic axon growth programs. The mechanisms involve an epigenetic
regulation of gene transcription, developmentꢀdependent transcription factors, mTOR
(mammalian target of rapamycin) and STAT3 (signal transducer and activator of transcription
3) signaling pathways, and manipulation of these cellular signaling pathways in conjunction
with providing a permissive environment have been considered as therapeutic approaches to
elicit axon growth in injured CNS neurons.6ꢀ10
Signaling pathways relevant to human diseases are likely to be amenable to small
molecule intervention, and bioactive small molecules have proven to be valuable tools for
exploring complex cellular processes.11, 12 Discovering such small molecules using unbiased
screening approaches and identification of protein targets of the compounds should provide a
rational basis for elucidating new drug targets and signaling pathways relevant to human
diseases.13, 14 In this regards, phenotypeꢀbased small molecule screens involving axon
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elongation can provide an opportunity for pharmacological manipulation of previously
unknown signaling factors which are implicated in cell intrinsic control of axon growth.15
These endeavors could bring forth novel therapeutic agents for the treatment of clinical
conditions associated with a loss of axon.
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In this study, in an effort to discover new pharmacological modalities to aid in axon
regeneration, we employed phenotypic cellꢀbased screens that allow visual assessment and
quantitative measurement of neurite outgrowth in vitro. The phenotypic screening campaign
and chemical modification efforts led to identification of compound 7p that enhances neurite
outgrowth in cultured primary neurons derived from the hippocampus, cerebral cortex, and
retina and that induce optic nerve regeneration in an animal model of optic nerve injury.
While it need to be determined how the compound stimulates axon growth in vivo, our results
should provide further insight into the treatment strategies for clinical conditions associated
with a loss of axon.
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Results and Discussion
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Phenotypic screens for small molecules that stimulate neurite outgrowth
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In an effort to discover synthetic small molecules that stimulate axon growth by targeting cell
intrinsic mechanisms of axon elongation, we made use of neuronal differentiation of P19
embryonic carcinoma cells by neurogenic transcription factor NeuroD2 in the primary
screen.16, 17 Upon expression of NeuroD2, P19 cells begin to sprout neurites which can be
visually detected by indirect immunofluorescence with an antibody against neuronꢀspecific
class III βꢀtubulin.18 We envisioned that the intrinsic mechanisms of axon growth in primary
neurons can be recapitulated in the neurite outgrowth of P19ꢀderived neuronꢀlike cells.
We screened commercially available libraries of 170,000 synthetic small molecules
from the DIVERSet libraries, kinase directed libraries, PremiumSet library, and GPCR library
(ChemBridge). The compounds were tested at a concentration of 10 ꢁM in 384ꢀwell format
for their ability to promote neurite outgrowth in P19 cells which were transiently transfected
with a NeuroD2 cDNA construct. The compounds that enhance neurite outgrowth greater
than 2 fold as compared with neurite outgrowth in DMSOꢀtreated cells were selected, and we
were able to confirm the activity of 185 compounds (0.11% hit rate). Then, the hit
compounds were subject to a secondary screen which involves axon elongation in rat primary
hippocampal neurons. The primary hippocampal neurons have been extensively used as a
cellular system to study neuritogenesis,19, 20 and we tested the hit compounds that enhance
neurite outgrowth in P19 cells to see whether they can stimulate neurite elongation in the
neuronal context. Interestingly, the secondary screen yielded a set of compounds that share a
common chemical scaffold containing a core substructure of sulfonamido acetamide, and
compound 1 was most potent among the analogs (Figure 1). The neurite elongation activity
of compound 1 reached a plateau at concentrations between 10 and 20 ꢁM (~ 3 fold higher
than control), and it lost neurite elongation activity below 1 ꢁM. Similarly, compound 1 was
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able to enhance neurite elongation in assay with primary cortical neurons with a similar
potency as shown in primary hippocampus neurons (data not shown).
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Chemistry
Compound 1 exhibited limited water solubility and poor metabolic stability (halfꢀlife in liver
S9 fraction) in vitro, indicating that compound 1 is not an appropriate candidate for
evaluation in vivo. In order to address these deficiencies, we initiated structureꢀactivity
guided optimization using the neurite elongation of primary cortical neurons.
We sectioned the chemical structure of the compound for chemical modification as
shown in Figure 2 and planned three synthetic routes in order to synthesize the derivatives of
compound 1 as shown in Figure 3. In route A, compound A bearing R1 and R2 was used to
introduce an aromatic ring or a heteroaromatic ring which include various substituents in
section 3 of compound 1. Compound B was obtained through hydrolysis of compound A and
compound B reacted with diverse amine (C) to synthesize compound D. Synthetic route B
was advantageous for fixing R1 and R3 and changing the structure of R2. Compound E was
synthesized by substitution reaction, and then reacted with various sulfonyl chloride
compounds (F) along with the diversity of R2. There is a limitation that secondary amine (ꢀ
NHꢀ) and nitrogen (ꢀNHCOꢀ) of amide in compound E competitively reacted with alkyl
halide or acyl halide to produce mixtures. Alternatively, we chose route C to minimize
production of the mixtures (tertiary amine and secondary amide) as mentioned above. Amine
compounds bearing various substituents (R1) reacted with arylsulfonyl chloride, acyl chloride
and alkyl chloride for compound G, which was substituted with compound H to afford the
compound D. As mentioned above, we could avoid competitive reactions of secondary amine
(–NHꢀ) and amide (–NHCOꢀ) of compound E because R2 was introduced in advance.
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The synthesis of compounds 7a-x through synthetic route A is described in Scheme 1.
Compounds 2a-g were selected as starting materials and reacted with toluenesufonyl chloride
to synthesize Nꢀtoluenesulfonamides (compounds 3a-g). Substitutions of compounds 3a-g
with ethyl bromoacetate afforded compounds 4a-g, which was followed base catalyzed
hydrolysis to obtain compounds 5a-g. HATU (1ꢀ[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀ
triazolo[4,5ꢀb]pyridinium 3ꢀoxid hexafluorophosphate) was chosen to produce active ester
to conduct amide coupling of various amines (compounds 6a-t) and carboxylic acid
(compound 5a-g) for compounds 7a-x.
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Compounds 12a-g bearing 2ꢀmethoxy in R3 and 2,4ꢀdimethoxyphenyl or 4ꢀmethoxyꢀ
pyridinꢀ3ꢀyl in R6 were synthesized by reaction of compounds 10a and 10b with various
benzenesulfonylchloride through synthetic route B. Substitution reaction of compound 2a
with compounds 8 and 9 were carried out for compounds 10a and 10b, which was followed
by reaction with arylsufonyl chlorides (11a-e) to synthesize compounds 12a-g having diverse
substituents in the C ring (Scheme 2).
Compounds 17 and 18 were synthesized for substitution of sulfonamide into amide (ꢀ
CONHꢀ) or alkyl (ꢀCH2ꢀ) as linkers in section 2 via synthetic route C. Compound 2a was
reacted with chlorobenzoyl chloride (13) and 4ꢀmethylbenzyl chloride (14), respectively,
afforded Nꢀacylated compound 15 and Nꢀalkylated compound 16, which were reacted with
compound 8 to synthesize compounds 17 and 18. At the beginning, we tried to synthesize
compounds 17 and 18 through synthetic route B, however the chemical yields were low due
to a competitive reaction of two nucleophilic nitrogens of secondary amine and amide in
compound E. Alternatively, the synthetic route C were required and showed no undesired
products (Scheme 3).
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The neurite outgrowth activities of the compounds bearing the various substituents of
ring C and heteroaromatic system in section 3 were summarized in Table 1. First, we
modified section 3 to find the effective functional group on ring C. The directing effect of the
substituents on ring C was mostly shown in the ortho position (Table 1, entries 1 and 2) and
the ethoxy group, an electron donating group, confer better outgrowth activity, compared to
an electron withdrawing group, such as halogen and methoxycarbonyl (Table 1, entries 1ꢀ8).
Replacement of the 2ꢀethoxyphenyl on ring C of compound 1 with 2ꢀethoxybenzyl yielded
compound 7i where ring C was placed far away from the basic frame due to insertion of
methylene. However replacement of the 2ꢀethoxyphenyl with imidazole or thiazole yielded
compounds which exhibited cytotoxicity in neurite outgrowth assay with primary cortical
neurons (Table 1, entries 9, 10 and 11).
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We next investigated the structureꢀactivity relationship between substituents in R4 of
ring A and R5 of ring B along with 2ꢀethoxy or 2,4ꢀdimethoxy in R6 of ring C (Table 2). First,
the activity of the compounds which adopted a methoxy group in ortho or meta position in R4
was compared, and it was revealed that the meta substituted compounds exhibited less
activity as compared to compound 1 (Table 2, entries 1ꢀ2). There was no significant
difference in neurite outgrowth activity depending on electron donating/withdrawing groups
on ring A (Table 2, entries 1 and 3ꢀ6). Because 2,4ꢀdimthoxy group in R6 presented similar
activity with compound 1, chemical modification was shifted to R5 in ring B. The activities of
halogen substituted compounds were higher than that of compound 12d in that 4ꢀmethoxy
was substituted for methyl of R5 (Table 2, entries 8ꢀ11). Among them, the neurite outgrowth
activity of 4ꢀbromo substituted compound 12c was fiveꢀfold higher than that of DMSO
(Table 2, entry 10). We synthesized compounds 17 and 18 bearing 4ꢀchlorobenzoyl and
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methylbenzyl in order to test whether the sulfonamide in section 2 is a structural feature
necessary to impart activity; however the results showed that replacements of the
sulfonamide residue led to a complete loss of neurite outgrowth activity or yielded
compounds that exhibits cytotoxicity (Table 2, entries 14 and 15).
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We measured the metabolic stability in mouse liver S9 fraction of compounds 1, 7g,
12b and 12d whose neurite outgrowth activity was higher than that of DMSO. Unfortunately,
when the selected compounds were incubated in the presence of isolated mouse liver S9
fraction for 30 minutes, the residual rates were 0.3 ~ 0.7%, suggesting very low metabolic
stability. One of strategies for improving the metabolic stability of compound is to reduce the
overall lipophilicity (e.g. logP and logD). Because the binding site of metabolizing enzyme is
lipophilic, lipophilic molecules could be accepted more readily to the binding site of enzymes.
21 Thus, we designed compounds with higher metabolic stability and synthesized compounds
introducing pyridine or quinoline, as shown in Table 3. The neurite outgrowth activities were
different depending on the substituted positions of quinoline; in particular, compound 7l
showed the highest activity (Table 3, entries 1ꢀ3). Replacement of R3 with pyridine
derivatives resulted in compounds with varied potency depending on the position of nitrogen;
the most potent one was compound 7p (Table 3, entries 4ꢀ6). As expected, the lipophilicity of
compound 7p (logP = 2.43) bearing the pyridyl amide was reduced when compared to that of
compound 1 (logP = 3.76), and hence the metabolic stability of the compound 7p (remaining
compound, 61.2%) was remarkably enhanced relative to compound 1 (remaining compound,
0.7%) (Table 4). In addition, it was assumed that the small alkoxy group on pyridine was
more adequate with regards to the neurite outgrowth activity (Table 3, entries 6ꢀ8). Further
modification of compound 7p by introducing 4ꢀmethylthio or 4ꢀbromo substituent in section
2 did not adversely affect its ability to enhance neurite outgrowth of primary cortical neurons
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(Table 3, entries 9ꢀ10).
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Taken together, the structureꢀactivity guided optimization efforts led to identification
of a subset of compounds that exhibit improved neurite outgrowth activity and water
solubility as compared to compound 1; compound 7p was best among the derivatives as it
also exhibited improved metabolic stability relative to compound 1.
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The effect of compound 7p on optic nerve regeneration
We discovered compound 1 and its derivatives that demonstrated the ability to stimulate
neurite outgrowth of cultured primary neurons derived from hippocampus and cerebral cortex
in the brain of rat E18. The in vitro results raised the question of whether such compounds
can stimulate axon outgrowth and produce desired biological outcome such as axon
regeneration in CNS injury site. To address the question, we first tested compound 7p for its
ability to stimulate axon growth of retina neuronal cells in vitro, and then employed an
animal model of optic nerve injury to test compound 7p in vivo.
The retina neuronal cells prepared as described in Experimental Section were treated
with compound 7p at concentrations between 2 and 20 ꢁM. After a 72 hr incubation, the cells
were fixed with paraformaldehyde and stained with an antiꢀneurofilament antibody (SMIꢀ312)
to detect axons (Figure 4A and 4B). Axonal outgrowth was quantified by counting the
number of neuronal cells with axon length less than either 50 ꢀm, 50 ~ 100 ꢀm, 100 ~ 200
ꢀm, or longer than 200 ꢀm. Although the cultures were purified partially, we were able to
select retinal ganglion cells (RGCs) by cell size and morphology, and the cells bigger than 10
ꢀm in cell body diameter were counted. As shown in Figure 4C, the axonal growth
stimulatory effect of compound 7p was evident in all range of length, and was most
prominent at 10 ꢁM.
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In order to examine whether compound 7p was able to stimulate axonal outgrowth of
RGCs in an animal model of optic nerve injury, the crushed optic nerves in the injury model
were treated with either PBS or compound 7p (0.22 ꢀg per eye) for three weeks. Then the
animals were sacrificed, and optic nerve sections were stained with an antiꢀGAP43 antibody
to visualize growing axons. As shown in Figure 5A, the axons of optic nerves from animals
treated with compound 7p were significantly extended beyond the lesion epicenter as
compared to those from the shamꢀcontrol animals treated with PBS. The differences in the
number of GAPꢀ43 positive axons were evident in all cases of measurements at 500, 1000
and 1500 ꢀm distal to the crush center (Figure 5B) but was statistically significant beyond the
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1500 ꢀm (injured, 0.22 ± 0.081; injured + 7p, 0.70 ± 0.20) as determined by
quantification of GAPꢀ43 positive axons.
Conclusion
An approach to activate cell intrinsic mechanisms regulating axon growth in the adult CNS is
likely to contribute to functional recovery following traumatic injury. Upon screening
chemical libraries of 170,000 synthetic small molecules for those that stimulate neurite
outgrowth of P19ꢀderived neuronꢀlike cells, we discovered a promising hit (compound 1) that
is also capable of stimulating neurite outgrowth of cultured primary neurons. The structureꢀ
activity guided optimization and the efforts to improve metabolic stability of compound 1
resulted in the identification of compound 7p with properties more suitable for use in in vivo
studies. It is of note that the in vitro neurite outgrowth activity of compound 7p translates into
stimulation of axon regeneration in an animal model of optic nerve injury. Further
optimization of compound 7p and elucidating the mechanisms by which compound 7p elicits
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axon regeneration in vivo will provide a rational basis for future efforts to enhance treatment
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Experimental Section
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P19 cell culture and chemical treatment
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P19 embryonic carcinoma cells (6 x 106/10ꢀcm dish) were transfected with 10 µg of DNA
construct encoding NeuroD2 by using FuGENE HD. Twelve hr after transfection, the cells
were trypsinized and replated at a density of 8,000 cells/well to a 384ꢀwell plate in MEM
(minimum essential medium) containing 5% FBS, 1 mM sodium pyruvate, and 2 mM
glutamate. After a 24 hr incubation, the cells were treated with compounds for 48 hr, and then
beta III tubulin was visualized by indirect immunofluorescence. The chemical libraries used
in this work were purchased from ChemBridge Corporation (San Diego, CA, USA).
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Animal Housing and Ethics
The animal study was approved by the Animal Ethical Committee of Gyeonggi Institute of
Science & Technology Promotion and Dankook University, and carried out in accordance
with guidelines from KFDA (Korea Food and Drug Administration) and Animal and Plant
Quarantine Agency in Korea.
Cell culture of primary neurons
Entire forebrains were dissected from embryonic day 18 (E18) embryos of SpragueꢀDawley
(SD) rats as described with minor modification.22 In brief, skull covering the brain was cut
and opened with microscissors, and the brain was transferred to a clean dish with iceꢀcold
Hank’s buffered salt solution (HBSS). The meninges were teased off with microforceps, and
hippocampus and cerebral cortex were collected separately in HBSS. After being centrifuged
at 100 x g for 1 min, they were resuspended in Neurobasal medium containing serumꢀfree
supplement (B27) and 2 mM glutamate and then were triturated approximately 30 times with
a 1 mL micropipette tip to achieve a singleꢀcell suspension.The cells were washed twice with
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Neurobasal medium containing B27 and 2 mM glutamate, passed through a 40ꢀµm cell
strainer, and then seeded onto 384ꢀwell plates at a density of 2,000 cells/well. After a 24 hr
incubation, the cells were treated with compounds for 48 hr, and then beta III tubulin was
visualized by indirect immunofluorescence.
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RGCs were obtained from the postnatal day 7 (P7) SD rats and maintained in
Dulbecco’s PhosphateꢀBuffered Saline (DPBS) with 0.5% Bovine Serum Albumin (MP,
CellectTM BSA). The RGCs separation was done by using a RGC isolation kit (Miltenyi
Biotec; Auburn, CA). The RGCs were seeded on 18 mm cover glass (Deckglaser) in 12ꢀ well
plates (Costar, Corning, NY) which had been preꢀcoated with polyꢀDꢀlysine (20 ꢀg/mL)
followed by laminin (10 μg/mL) in Dulbecco's Modified Eagle's Medium (DMEM) at a
density of 200,000 cells/well. Two hour after plating, RGCs were treated with various doses
(0, 2, 10, 20 ꢀM) of compound 7p and maintained for 3 days.
Indirect immunofluorescence
The fixed cells were washed with PBS and permeabilized with 0.25% Triton Xꢀ100 in PBS
for 8 min. Primary and secondary antibodies were diluted in PBS containing 5% bovine
serum album and used for staining. The antibodies are as follows: 0.5 µg/mL of mouse antiꢀβ
III tubulin (Covance); 2 µg/mL Alexa Fluor® 488 goat antiꢀmouse IgG (Invitrogen). 4’,6ꢀ
Diamidinoꢀ2ꢀphenylindole (DAPI) was used to stain the nucleus. Confocal fluorescence
images were acquired using a LSM700 with Zen software (Carl Zeiss).
The cultured retina neuronal cells were washed with 0.1 M PBS, fix with 4%
paraformaldehyde (PFA) prepared in 0.1 M PBS for 15 min at room temperature and washed
again with 0.1 M PBS 2 times for 5 min. Nonꢀspecific protein binding was blocked by
incubation with 1% BSA and 3% Goat Serum (Vector Laboratories, Burlingame, CA)
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prepared in 0.1 M PBS containing 0.1% Triton Xꢀ100 (Sigma) for 60 min. Then cells were
incubated with primary antiꢀneurofilament antibody (SMI312 Monoclonal, 1:400, Covance)
antibody for overnight at 4℃ and then with secondary antiꢀrabbit IgG conjugated with Alexa
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594 (diluted 1:500 in PBS, Life Technologies) for 90 min at room temperature. Cells were
mounted with Vectashield kit (Vector Laboratories) and the images were photographed using
a Zeiss microscope (Zeiss, Immersol® 518F, ZEN 2012, blue edition). Using Image J
program (NIH Image, Bethesda, MD), the numbers of SMI312 positive neurons were counted.
Quantification of neurite outgrowth
In order to measure the neurite outgrowth of P19, primary hippocampal neurons, and primary
cortical neurons, wide field fluorescence images from four fields in each well of 384ꢀwell
plate were acquired on the ArrayScan® VTI HCS reader (Thermo Fisher Scientific) using a 10
x objective lens, and the images were analyzed with the Neuronal Profiling BioApplication
software (Thermo Fisher Scientific).
For the measurement of retina neuronal cells, the number and axonal length of neurons in
nine consecutive nonꢀoverlapping microscopic fields with areas of 13.55 mm2 (total area
40.65 mm2) were counted. Both the total number of cells and the numbers of cells which
contains axon length less than 50 ꢀm, 50 ~ 100 ꢀm, 100 ~ 200 ꢀm and longer than 200 ꢀm
were counted.
Optic nerve crush
Male Sprague Dawley (SD) rats (8 weeks of age, weighing 200 ~ 250 g) were purchased
from Samtako Bio Korea, Inc. (Osan, GyeongGiꢀDo, Korea). Rats were housed in a
temperature (22 ~ 24℃) and humidityꢀcontrolled environment under a 12 hour light/dark
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cycle (light on at 08:00 and off at 20:00). Rats were given ordinary laboratory food and water
ad libitum. Rats were randomly divided into two groups: shamꢀoperated normal control (n=3)
and compound 7p after optic nerve crush (n=4). The animals were anesthetized by
intraperitoneal injection of 50 mg/kg tiletamine plus zolazepam (Zoletil; Virbac, Carros,
France) and 15 mg/kg xylazine hydrochloride (Rompun; Bayer, Leuverkeusen, Germany).
The pupils were dilated with 1% tropicamide eye drops. Dissection of the conjunctiva was
made with forcep towards the back of the eye to expose the retrobulbar optic nerve. The optic
nerve was crushed with sharp forceps for 15 seconds at 2 mm behind connected optic head. A
shamꢀoperated normal control group was injected with saline using 30G needle intraꢀ
vitreously once in 4 days. The crushed group was injected with compound 7p (0.22 ꢀg in 5
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ꢀL per eye) prepared in 100 mM potassium phosphate buffer with 0.01 N HCl once in four
days for 3 weeks.
Preparation of optic nerve sections and immunohistochemistry
The anterior segments of the eyeballs with optic nerve and the vitreous humor were removed.
The eyeballs with optic nerve were isolated and fixed by soaking in 4% paraformaldehyde for
2 h. The fixed retinas were soaked in 30% sucrose solution prepared in PBS, pH 7.4 at 4 ℃
overnight for cryoprotection and embedded in O.C.T. compound (Tissueꢀtek) before freezing
at ꢀ80 ℃. Leica cryostat was used to section the retina (12 ꢀm). The sections were washed
with PBS three times and were incubated with 1% BSA (MP, CellectTM BSA) and 3% Goat
Serum (Vector) in PBS (1% BSA) (blocking solution) for 1 hour at room temperature to
block nonspecific protein binding. Then the sections were incubated with primary antiꢀGAPꢀ
43 BRN3α (Polyclonal, 1:200, Novus Biologicals) antibody for overnight at 4℃ and then
with secondary antiꢀrabbit IgG conjugated with Vector 488 (diluted 1:500 in PBS, Life
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technologies) for 90 min at room temperature. Sections were mounted with a Vecta shield kit
(Vector Laboratories, Inc. Burlingame, CA) and the images were photographed usinga Zeiss
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microscope (Zeiss, Immersol® 518F, ZEN 2012, blue edition). Using Image J program (NIH
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Image, Bethesda, MD), the numbers of GAPꢀ43 positive axons extending 0.5 and 1 mm from
the end of the crush site were counted.
Quantification of axon growth
For analysis of axon length, the number of GAPꢀ43 positive axon was counted in the 0.5, 1
and 1.5 mm from the end of the crush site in sections. The axons were measured by using
formula at which the counts were used to calculate and group average number of axons per
millimeter of nerve width (100 ꢀm box size). Then neuronal axon length was measured by
formula as described.23
Σαԁ=πr2X[average axon/mm]/t
Σαԁ: The total number of axon extending distance
t: thickness, r: radius of nerve
Statistics
Significant differences among groups were determined using a program, Graphpad prism
(version 5, GraphPad Software, Inc, La Jolla, California). All values are reported as mean ±
standard error of the mean. Statistical significance was set at p < 0.05.
General Methods
All substances were purchased from Sigma–Aldrich and used as received. Purchased
anhydrous MeOH was used without further purification. Analytical thinꢀlayer
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chromatography was conducted on E. Merck TLC plates (silica gel 60 F254, aluminum back).
Silica gel 60 (230–400 mesh) for column chromatography was purchased from E. Merck.
Melting points were measured with a Kofler block or Büchi Bꢀ545 melting point apparatus.
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1
The H and 13C NMR spectroscopic data were recorded in CDCl3, DMSOꢀd6, CD3OD
solutions at ambient temperature with Avance II 400 [Bruker Biospin, Germany] (400 MHz
for 1H NMR and 100 MHz for 13C NMR) and Ascend III 700 [Bruker Biospin] (700 MHz for
1H NMR and 175 MHz for 13C NMR). Chemical shifts were recorded as δ values in parts per
million (ppm) and were indirectly referenced to tetramethylsilane by the solvent signal.
Coupling constants (J) are reported in Hertz. Infrared spectra were recorded on a Nicolet
6700 FTIR spectrophotometer. Low resolution mass spectra were measured on a Micromass
Quattro Micro API (Waters, USA), whereas high resolution mass spectra were recorded on
LTQ Orbitrap XL (Thermo Scientific, USA) spectrometer. Characterization data for known
compounds were consistent with literature values.
Representative compounds and their synthetic procedures
All of the compounds were synthesized via route A (7i-k, 7l-p and 7r-g), route B (l, 7c-f and
7t-x), and route C (7q, 12e, 17 and 18) except commercially available compounds (7b, 7g
and 7h).
2-(N-(2-Methoxyphenyl)-4-methylphenylsulfonamido)-N-(4-methoxypyridin-3-
yl)acetamide (7p) via route A
To a stirred compound 2a (10.0 g, 81.2 mmol) and K2CO3 (16.8 g, 122 mmol) in DMF (150
mL) was added tosyl chloride (16.3 g, 85.3 mmol). After being stirred at rt for 10 min, the
reaction solution was quenched with water and extracted with EtOAc. The combined organic
phases were washed with water and brine, and dried over Na2SO4, concentrated. The
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resulting solid was tritured with hexane, providing 15.0 g (67%) of Nꢀ(2ꢀmethoxyphenyl)ꢀ4ꢀ
methylbenzenesulfonamide (3a) as a white solid. To a stirred compound 3a (15.0 g, 54.1
mmol) and K2CO3 (18.6 g, 135 mmol) was added dropwise ethyl bromoacetate (5.98 mL,
85.3 mmol). After stirring at rt for 2 h, the reaction solution was quenched with water and
extracted with EtOAc. The organic layer was washed with water and brine three times, dried
over Na2SO4, filtered, and concentrated to give 19.0 g (96%) of ethyl 2ꢀ(Nꢀ(2ꢀ
methoxyphenyl)ꢀ4ꢀmethylphenylsulfonamido)acetate (4a) as a white solid.
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To a solution of compound 4a (19.0 g, 52.3 mmol) was added 2.5 N NaOH (63 ꢀL, 157
mmol) at rt. The reaction mixture was heated to reflux for 6 h. After cooled to rt, the mixture
was concentrated and diluted with EtOAc and H2O. The aqueous layer was adjusted to pH 2
with 1N HCl (aq), then the product was extracted with EtOAc. The combined organic layers
were dried over Na2SO4, concentrated. The resulting residue was recrystallized from EtOAc
and
hexane
to
obtain
15.0
g
(86%)
of
2ꢀ(Nꢀ(2ꢀmethoxyphenyl)ꢀ4ꢀ
methylphenylsulfonamido)acetic acid (5a) as a white solid, To a mixture of compound 5a
(0.5 g, 1.49 mmol), compound 6p (185 mg, 1.49 mmol) and HATU (1ꢀ
[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium
3ꢀoxid
hexafluorophosphate) in DMF (20 mL) was added triethylamine (416 ꢀL, 2.98 mmol) at 0 oC.
The reaction solution was allowed to stir at rt for 2 h. The reaction was quenched with H2O,
diluted with EtOAc. The organic layer was washed with water and brine, dried over Na2SO4,
concentrated. The residue was purified by flash chromatography (EtOAc/Hexane, 1:1, v:v) to
o
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give 250 mg (38%) of compound 7p as a white solid, mp 153.0 C; H NMR (700 MHz,
CDCl3) δ 9.45 (s, 1H), 9.32 (s, 1H), 8.36 (d, J = 6.3 Hz, 1 H), 7.58 (d, J = 7.7 Hz, 2 H), 7.35
(t, J = 7.0 Hz, 1 H), 7.31ꢀ7.29 (m, 3H), 6.99ꢀ6.95 (m, 2H), 6.86 (d, J = 8.4 Hz, 1 H), 4.36 (s,
13
2H), 4.12 (s, 3H), 3.47 (s, 3H), 2.46 (s, 3H); C NMR (175 MHz, CDCl3) δ 166.59, 155.45,
154.21, 146.37, 143.78, 141.43, 134.96, 131.46, 130.34, 129.10, 127.74, 126.89, 124.23,
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120.75, 111.92, 105.55, 55.95, 55.02, 54.70, 21.61; LRMS (ESI) m/z: 442.1 [M+H]+; HRMS
(ESI) m/z: Calcd. for [M+H]+ C22H24N3O5S: 442.1431; found: 442.1435.
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N-(2,5-Dimethoxyphenyl)-2-(4-fluoro-N-(2-
methoxyphenyl)phenylsulfonamido)acetamide (12a) via route B
A mixture of compound 2a (0.98 mL, 8.7 mmol), compound 8 (2.0 g, 8.7 mmol) and K2CO3
o
(1.8 g, 13.1 mmol) in DMF (17.4 mL) was heated at 80 C for 2 h. The reaction was
quenched with H2O and the solution was diluted with EA and washed with H2O and brine.
The organic layer was dried over Na2SO4, filtered and concentrated. The resulting residue
was purified by flash chromatography (EtOAc/Hexane, 1:2, v:v) to give compound 1a as a
brown solid. To a solution of compound 10a (20 mg, 0.06 mmol) and compound 11a (13 mg,
0.07 mmol) in DCM (0.3 mL) was added pyridine (25.0 ꢀL, 0.32 mmol). After 3 h at rt, the
reaction was quenched with H2O, and then the solution was diluted with DCM. The organic
layer was washed with CuSO4 (aq), H2O and brine, dried over Na2SO4, filtered, concentrated.
The residue was purified by flash chromatography (EtOAc/Hexane, 1:4, v:v) to obtain 22 mg
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(78%) of compound 12a as a yellow solid, mp 148.4 C; H NMR (400 MHz, DMSOꢀd6) δ
9.25 (s, 1 H), 7.75ꢀ7.72 (m, 3 H), 7.46ꢀ7.33 (m, 4 H), 7.01ꢀ6.98 (m, 3 H), 6.64 (dd, J = 8.8,
13
2.8 Hz, 1 H), 4.42 (s, 2 H), 3.84 (s, 3 H), 3.68 (s, 3 H), 3.41 (s, 3 H); C NMR (175 MHz,
DMSOꢀd6) δ 167.01, 165.79, 164.36, 156.03, 153.52, 143.29, 135.89, 135.89, 132.56, 130.93,
128.15, 127.15, 120.91, 116.61, 112.97, 112.44, 108.55, 107.34, 56.95, 56.18, 55.86; LRMS
(ESI) m/z: 475.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C23H24FN2O6S: 475.1334;
found: 475.1336.
4-Chloro-N-(2-((2,5-dimethoxyphenyl)amino)-2-oxoethyl)-N-(2-
methoxyphenyl)benzamide (17) via route C
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To a mixture of compound 2a (246 mg, 2.0 mmol) and K2CO3 (345 mg, 2.5 mmol) in DMF
(10 mL) was added compound 13 (417 mg, 2.4 mmol) at rt. The solution was allowed to stir
at rt for 2 h. The reaction was quenched with H2O and the solution was diluted with EA and
washed with H2O and brine. The organic layer was dried over Na2SO4, filtered and
concentrated. The resulting residue was purified by flash chromatography (EtOAc/Hexane,
1:2, v:v) to give 497 mg (95%) of intermediate 15 as a white solid. After LC/MS analysis of
intermediate 15, to a solution of compound 15 (26.1 mg, 0.1 mmol) and compound 8 (25.7
mg, 0.1 mmol) in DMF (5 mL) was added K2CO3 (20.7 mg, 0.15 mmol). The solution was
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o
heated to 80 C for 2 h. The reaction was quenched with H2O, and then the solution was
diluted with DCM. The organic layer was washed with H2O and brine, dried over Na2SO4,
filtered, concentrated. The residue was purified by flash chromatography (EtOAc/Hexane, 1:4,
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v:v) to obtain 40 mg (83%) of compound 17 as a white solid, mp 161.8 C; H NMR (400
MHz, DMSOꢀd6) δ 9.38 (s, 1 H), 7.81 (s, 1 H), 7.33ꢀ7.21 (m, 6 H), 7.02ꢀ6.96 (m, 2 H), 6.85 (t,
J = 7.6 Hz, 1 H), 6.64 (dd, J = 8.8, 2.8 Hz, 1 H), 4.77ꢀ4.74 (m, 1 H), 4.25ꢀ4.20 (m, 1 H), 3.81
(s, 3 H), 3.73 (s, 3 H), 3.69 (s, 3 H); 13C NMR (175 MHz, CDCl3) δ 166.43, 155.50, 153.99,
142.77, 139.52, 137.16, 132.45, 130.70, 129.31, 128.88, 128.04, 126.62, 121.08, 111.98,
111.53, 109.04, 106.20, 56.85, 55.82, 54.93, 54.82; LRMS (ESI) m/z: 455.1 [M+H]+; HRMS
(ESI) m/z: Calcd. for [M+H]+ C24H24ClN2O5: 455.1368; found: 455.1369.
N-(2-Ethoxyphenyl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide (1)
mp 178.3 oC; 1H NMR (400MHz, CDCl3): δ 9.56 (S, 1 H), 8.36 (d, J = 8.0 Hz, 1 H), 7.57 (d,
J = 8.0 Hz, 1 H), 7.51 (d, J = 7.6 Hz, 2 H), 7.34ꢀ7.27 (m, 3 H), 7.09 (t, J = 6.4 Hz, 1 H),
6.99ꢀ6.97 (m, 3 H), 6.79 (d, J = 1.2 Hz, 1 H), 4.35 (s, 2 H), 4.26 (q, J = 8.8, 6.8 Hz, 2 H),
3.36 (s, 3 H), 2.45 (s, 3 H), 1.69 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, DMSOꢀd6) δ
166.36, 155.54, 147.60, 143.62, 135.66, 132.08, 130.35, 129.36, 127.41, 126.96, 126.74,
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124.15, 120.41, 120.32, 119.40, 112.56, 111.96, 64.22, 55.10, 54.12, 20.97, 14.63; LRMS
(ESI) m/z: 455.2 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C24H27N2O5S: 455.1635;
found: 455.1635.
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N-(3-Methoxyphenyl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide
(7b)
o
mp 206.7 C; 1H NMR (700MHz, CD3OD): δ 7.60 (d, J = 7.7 Hz, 2 H), 7.39ꢀ7.35 (m, 3 H),
7.31 (dd, J = 7.7, 1.4 Hz, 1 H), 7.24ꢀ7.22 (m, 2 H), 7.02 (t, J = 7.7 Hz, 2 H), 6.97 (t, J = 7.7
Hz, 1 H), 6.71 (dd, J = 8.4, 2.8 Hz, 1 H), 4.38 (s, 2 H), 3.80 (s, 3 H), 3.60 (s, 3 H), 2.46 (s, 3
H); 13C NMR (175 MHz, CD3OD) δ 167.71, 160.21, 156.30, 144.11, 138.80, 136.02, 131.63,
130.15, 129.23, 129.12, 127.60, 120.63, 112.08, 112.03, 109.81, 105.71, 54.73, 54.33. 54.13,
20.05; LRMS (ESI) m/z: 441.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C23H25N2O5S:
441.1479; found: 441.1482.
2-(N-(2-Methoxyphenyl)-4-methylphenylsulfonamido)-N-phenylacetamide (7c)
o
mp 167.9 C; 1H NMR (400 MHz, CDCl3) δ 9.35 (S, 1 H), 7.61 (dd, J = 21.2, 8.4 Hz, 4 H),
7.38ꢀ7.33 (m, 5 H), 7.15 (t, J = 7.2 Hz, 1 H), 7.02 (d, J = 8.4 Hz, 1 H), 6.95ꢀ6.89 (m, 2 H),
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4.25 (s, 2 H), 3.87 (s, 3 H), 2.48 (s, 3 H); C NMR (100 MHz, DMSOꢀd6) δ 166.36, 155.74,
143.10, 138.54, 136.84, 132.93, 129.91, 129.22, 128.72, 127.17, 126.91, 123.36, 123.36,
120.18, 119.03, 112.24, 55.21, 52.84, 20.96; LRMS (ESI) m/z: 411.1 [M+H]+; HRMS (ESI)
m/z: Calcd. for [M+H]+ C22H23N2O4S: 411.1373; found: 411.1386.
N-(2-Ethylphenyl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide (7d)
mp 152.2 oC; 1H NMR (400 MHz, DMSOꢀd6) δ 9.27 (S, 1 H), 7.57 (d, J = 8.0 Hz, 2 H), 7.40
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(d, J = 8.0 Hz, 2 H), 7.35ꢀ7.28 (m, 3 H), 7.22ꢀ7.13 (m, 3 H), 7.01 (d, J = 8.0 Hz, 1 H), 6.93 (t,
J = 7.6 Hz, 1 H), 4.38 (s, 2 H), 3.32 (s, 3 H), 2.50ꢀ2.44 (m, 2 H), 2.41 (s, 3 H), 1.03 (t, J =
7.6 Hz, 3 H); 13C NMR (100 MHz, CD3OD) δ 170.19, 157.62, 145.59, 139.98, 137.14,
135.61, 133.10, 131.66, 130.57, 130.02, 129.07, 128.89, 127.92, 127.43, 127.00, 121.99,
113.50, 55.97, 55.51, 25.30, 21.50, 15.00; LRMS (ESI) m/z: 439.2 [M+H]+; HRMS (ESI) m/z:
Calcd. for [M+H]+ C24H27N2O4S: 439.1686; found: 439.1695.
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N-(2-Fluorophenyl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide (7e)
mp 141.3 oC; 1H NMR (400 MHz, DMSOꢀd6) δ 9.69 (S, 1 H), 7.90ꢀ7.86 (m, 1 H), 7.56 (d, J
= 8.4 Hz, 2 H), 7.40 (d, J = 8.0 Hz, 2 H), 7.34ꢀ7.23 (m, 3 H), 7.17ꢀ7.14 (m, 2 H), 7.00 (d, J =
13
7.6 Hz, 1 H), 6.95 (t, J = 7.6 Hz, 1 H), 4.43 (s, 2 H), 3.43 (s, 3 H), 2.41 (s, 3 H); C NMR
(175 MHz, CDCl3) δ 166.98, 156.42, 153.49, 144.17, 135.03, 130.48, 130.27, 129.46, 128.23,
126.05, 124.77, 124.44, 122.11, 121.22, 115.02, 114.91, 112.36, 55.57, 55.55, 21.54; LRMS
(ESI) m/z: 429.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C22H22FN2O4S: 429.1279;
found: 429.1283.
N-(2-Bromophenyl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide (7f)
mp 186.7 oC; 1H NMR (400 MHz, DMSOꢀd6) δ 9.45 (S, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 7.68
(dd, J = 8.0, 1.2 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 2 H), 7.40ꢀ7.31 (m, 5 H), 7.49ꢀ7.10 (m, 1 H),
7.01ꢀ6.93 (m, 2 H), 4.40 (s, 2 H), 3.43 (s, 3 H), 2.41 (s, 3 H); 13C NMR (100 MHz, CD3OD) δ
166.85, 155.69, 143.41, 136.09, 135.54, 132.64, 132.40, 130.12, 129.32, 128.13, 127.38,
126.90, 126.67, 125.01, 120.31, 112.40, 55.20, 53.33, 20.98; LRMS (ESI) m/z: 489.0, 491.0
[M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C22H22BrN2O4S: 489.0478; found: 489.0480.
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Methyl 2-(2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamido)benzoate (7g)
mp 156.7 oC, 1H NMR (700 MHz, CDCl3) δ 11.88 (s, 1H), 8.73 (d, J = 8.3 Hz, 1H), 8.08 (dd,
J = 8.0, 1.3 Hz, 1H), 7.92 (dd, J = 7.8, 1.5 Hz, 1H), 7.54 (m, 3H), 7.31 (td, J = 8.3, 1.5 Hz,
1H), 7.26 (d, J = 8.0 Hz, 2H), 7.13 (t, J = 7.3 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.75 (d, J =
8.0 Hz, 1H), 4.42 (s, 2H), 4.08 (s, 3H), 3.27 (s, 3H), 2.43 (s, 3H); 13C NMR (175 MHz,
CDCl3) δ 168.27, 168.06, 155.42, 143.44, 140.52, 136.05, 134.22, 133.54, 130.92, 130.11,
129.06, 128.01, 127.32, 122.94, 120.95, 120.70, 116.29, 111.47, 55.14, 54.63, 52.61, 21.48;
LRMS (ESI) m/z: 469.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C24H25N2O6S:
469.1428; found: 469.1437.
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N-(2,4-Dimethoxyphenyl)-2-(N-(2-methoxyphenyl)-4-
methylphenylsulfonamido)acetamide (7h)
mp 163.3 oC; 1H NMR (CDCl3, 400 MHz) δ 9.19 (s, 1 H), 8.15 (d, J = 8.8 Hz, 1 H), 7.59 (d,
J = 10.4 Hz, 2 H), 7.38 (dd, J = 8.0, 1.2 Hz, 1 H), 7.35ꢀ7.27 (m, 3 H), 6.97 (t, J = 8.0 Hz, 1
H), 6.83 (dd, J = 8.4, 1.2 Hz, 1 H), 6.54 (d, J = 2.4 Hz, 1 H), 6.48 (dd, J = 8.8, 2.8 Hz, 1 H),
4.34 (s, 2 H), 3.99 (s, 3 H), 3.81 (s, 3 H), 3.43 (s, 3 H), 2.45 (s, 3 H); 13C NMR (100 MHz,
CDCl3) δ 166.37, 156.77, 155.86, 150.06, 143.75, 135.66, 132.07, 130.36, 129.24, 128.01,
127.25, 121.07, 120.89, 120.82, 111.93, 103.81, 98.91, 56.05, 55.55, 54.96, 54.71, 21.55;
LRMS (ESI) m/z: 471.2 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C24H27N2O6S:
471.1585; found: 471.1590.
N-(2-Ethoxybenzyl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide (7i)
mp 143.6 oC; 1H NMR (400 MHz, DMSOꢀd6) δ 8.12 (t, J = 4.0 Hz, 1 H), 7.54 (d, J = 8.4 Hz,
2 H), 7.38ꢀ7.27 (m, 4 H), 7.20 (t, J = 6.4 Hz, 1 H), 6.98ꢀ6.88 (m, 4 H), 6.82 (t, J = 7.2 Hz, 1
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H), 4.23ꢀ4.20 (m, 4 H), 4.03ꢀ3.98 (m, 2 H), 3.36 (s, 3 H), 2.40 (s, 3 H), 1.31 (t, J = 6.8 Hz, 3
H); 13C NMR (100 MHz, CDCl3) δ 168.07, 157.03, 156.29, 143.91, 135.29, 135.29, 130.44,
130.23, 130.14, 128.75, 128.23, 127.99, 125.94, 121.30, 120.96, 120.33, 112.10, 111.07,
63.58, 54.97, 54.69, 54.46, 39.32, 31.59, 21.56, 14.13; LRMS (ESI) m/z: 469.1 [M+H]+;
HRMS (ESI) m/z: Calcd. for [M+H]+ C25H29N2O5S: 469.1792; found: 469.1796.
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N-(1H-Imidazol-2-yl)-2-(N-(2-methoxyphenyl)-4-methylphenylsulfonamido)acetamide
(7j)
mp 215.8 oC; 1H NMR (400 MHz, DMSOꢀd6) δ 11.75 (br, 1 H), 11.14 (br, 1 H), 7.54 (d, J =
8.4 Hz, 2 H), 7.39ꢀ7.30 (m, 4 H), 6.99ꢀ6.91 (m, 2 H), 6.72 (s, 2 H), 4.40 (s, 2 H), 3.43 (s, 3 H),
2.41 (s, 3 H); 13C NMR (100 MHz, DMSOꢀd6) δ 155.71, 143.11, 136.82, 132.86, 129.93,
129.24, 127.13, 126.99, 120.09, 112.22, 55.21, 52.14, 20.96; LRMS (ESI) m/z: 401.1 [M+H]+;
HRMS (ESI) m/z: Calcd. for [M+H]+ C19H21N4O4S: 401.1278; found: 401.1292.
2-(N-(2-Methoxyphenyl)-4-methylphenylsulfonamido)-N-(thiazol-2-yl)acetamide (7k)
o
1
mp 187.2 C; H NMR (400 MHz, DMSOꢀd6) δ 12.06 (s, 1 H), 7.54 (d, J = 8.0 Hz, 2 H),
7.47 (d, J = 7.6 Hz, 1 H), 7.39 (d, J = 8.0 Hz, 3H), 7.32 (t, J = 8.8 Hz, 1 H), 7.23 (d, J = 7.6
Hz, 1 H), 6.99ꢀ6.93 (m, 2 H), 4.49 (s, 2 H), 3.40 (s, 3 H), 2.41 (s, 3 H); 13C NMR (100 MHz,
DMSOꢀd6) δ 166.95, 155.64, 143.20, 137.62, 136.63, 132.91, 129.99, 129.24, 127.13, 126.95,
120.19, 113.61, 112.22, 55.18, 52.14, 20.96; LRMS (ESI) m/z: 418.1 [M+H]+; HRMS (ESI)
m/z: Calcd. for [M+H]+ C19H20N3O4S2: 418.0890; found: 418.0892.
2-(N-(2-Methoxyphenyl)-4-methylphenylsulfonamido)-N-(quinolin-3-yl)acetamide (7l)
mp 177.0 oC; 1H NMR (700 MHz, CDCl3) δ 10.35 (s, 1H), 9.05 (s, 1H), 8.64 (s, 1H), 7.95 (d,
J = 8.4 Hz, 1 H), 7.86 (d, J = 8.4 Hz, 1 H), 7.70 (t, J = 7.7 Hz, 1 H), 7.66 (d, J = 7.7 Hz, 2
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H), 7.54 (t, J = 7.0 Hz, 1 H), 7.34 (t, J = 7.0 Hz, 1 H), 7.32 (d, J = 7.7 Hz, 2 H), 6.97ꢀ6.93 (m,
2 H), 4.40 (s, 2H), 3.81 (s, 3H), 2.45 (s, 3H); 13C NMR (175 MHz, CDCl3) δ 167.07, 156.34,
145.04, 144.32, 143.60, 134.34, 130.95, 130.51, 129.50, 129.03, 128.80, 128.38, 128.14,
127.93, 127.61, 127.05, 123.60, 121.63, 113.05, 56.49, 55.58, 21.68; LRMS (ESI) m/z: 462.1
[M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C25H24N3O4S: 462.1482; found: 462.1491.
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2-(N-(2-Methoxyphenyl)-4-methylphenylsulfonamido)-N-(quinolin-5-yl)acetamide (7m)
mp 195.0 oC; 1H NMR (700 MHz, CDCl3) δ 9.92 (s, 1H), 9.00 (s, 1H), 8.63 (d, J = 8.4 Hz, 1
H), 8.11 (d, J = 8.4 Hz, 1 H), 7.96 (d, J = 7.7 Hz, 1 H), 7.66 (t, J = 8.4 Hz, 1 H), 7.68 (d, J =
7.7 Hz, 2 H), 7.60ꢀ7.58 (m, 1 H), 7.41ꢀ7.37 (m, 3 H), 7.00ꢀ6.98 (m, 3 H), 4.35 (br, 2H), 3.64
(s, 3H), 2.50 (s, 3H); 13C NMR (175 MHz, CDCl3) δ 167.27, 156.25, 150.28, 148.44, 144.27,
134.35, 132.03, 130.47, 130.10, 129.51, 129.41, 129.00, 128.48, 127.93, 127.18, 122.72,
121.38, 121.16, 121.04, 112.59, 55.88, 55.74, 21.68; LRMS (ESI) m/z: 462.1 [M+H]+;
HRMS (ESI) m/z: Calcd. for [M+H]+ C25H24N3O4S: 462.1482; found: 462.1493.
2-(N-(2-Methoxyphenyl)-4-methylphenylsulfonamido)-N-(quinolin-6-yl)acetamide (7n)
mp 177.0 oC; 1H NMR (700 MHz, CDCl3) δ 9.78 (s, 1H), 8.86 (s, 1H), 8.80 (s, 1H), 8.12 (d,
J = 8.4 Hz, 1 H), 7.85 (d, J = 8.4 Hz, 1 H), 7.68 (t, J = 7.0 Hz, 1 H), 7.66 (d, J = 8.4 Hz, 2
H), 7.58 (t, J = 7.0 Hz, 1 H), 7.39 (t, J = 7.0 Hz, 1 H), 7.38 (d, J = 7.7 Hz, 2 H), 7.07 (d, J =
8.4 Hz, 1 H), 6.98 (t, J = 7.7 Hz, 1 H), 6.91 (d, J = 7.7 Hz, 1 H), 4.34 (br, 2H), 3.97 (s, 3H),
2.48 (s, 3H); 13C NMR (175 MHz, CDCl3) δ 167.25, 156.35, 150.48, 148.94, 144.47, 134.35,
131.03, 130.47, 130.10, 129.51, 129.41, 129.00, 128.48, 127.93, 127.18, 122.72, 121.38,
121.16, 121.04, 112.59, 55.88, 55.74, 21.78; LRMS (ESI) m/z: 462.1 [M+H]+; HRMS (ESI)
m/z: Calcd. for [M+H]+ C25H24N3O4S: 462.1482; found: 462.1495.
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yl)acetamide (7o)
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mp 123.0 oC; 1H NMR (700 MHz, CDCl3) δ 9.70 (s, 1H), 8.12 (d, J = 4.2 Hz, 1 H), 7.60 (d, J
= 7.7 Hz, 2 H), 7.33ꢀ7.24 (m, 5H), 7.09 (dd, J = 8.4, 4.9 Hz, 1 H), 6.95 (t, J = 7.7 Hz, 1 H),
6.84 (d, J = 8.4 Hz, 1 H), 4.44 (s, 2H), 4.04 (s, 3H), 3.47 (s, 3H), 2.46 (s, 3H); 13C NMR (175
MHz, CDCl3) δ 155.50, 143.62, 141.27, 139.30, 131.57, 130.19, 129.03, 127.74, 126.97,
120.66, 119.68, 117.31, 111.77, 55.95, 55.09, 54.95, 21.60; LRMS (ESI) m/z: 442.1 [M+H]+;
HRMS (ESI) m/z: Calcd. for [M+H]+ C22H24N3O5S: 442.1431; found: 442.1429.
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yl)acetamide (7q)
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mp 122.0 oC; H NMR (700 MHz, CDCl3) δ 9.29 (s, 1H), 8.54 (dd, J = 7.8, 1.4 Hz, 1H), 7.90
(dd, J = 5.0, 1.5 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.38 (dd, J = 7.8, 1.3 Hz, 1H), 7.33 (t, J =
7.9 Hz, 1H), 7.28 (d, J = 7.9 Hz, 2H), 6.98 (t, J = 7.3 Hz, 1H), 6.89 (dd, J = 7.7, 5.0 Hz, 1H),
6.83 (d, J = 8.2 Hz, 1H), 4.35 (s, 2H), 4.15 (s, 3H), 3.46 (s, 3H), 2.44 (s, 3H); 13C NMR (175
MHz, CDCl3) δ 167.54, 155.86, 153.53, 143.93, 140.65, 135.50, 131.86, 130.48, 129.28,
128.05, 127.30, 126.88, 122.37, 121.01, 117.12, 112.08, 55.06, 54.82, 53.93, 21.52; LRMS
(ESI) m/z: 442.2 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C22H25N3O5S: 442.1431;
found: 442.1434.
N-(2-Ethoxypyridin-3-yl)-2-(N-(2-methoxyphenyl)-4-
methylphenylsulfonamido)acetamide (7r)
mp 133.0 oC; 1H NMR (700 MHz, CDCl3) δ 9.37 (s, 1H), 8.57 (d, J = 7.7 Hz, 1 H), 7.89 (d, J
= 4.9 Hz, 1 H), 7.57 (d, J = 7.7 Hz, 2 H), 7.51 (d, J = 7.7 Hz, 1 H), 7.34 (t, J = 7.7 Hz, 1 H),
6.99 (t, J = 7.7 Hz, 1 H), 6.89 (t, J = 7.7 Hz, 1 H), 6.82 (d, J = 8.4 Hz, 1 H), 4.59 (q, J = 7.0
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Hz, 2 H), 4.36 (s, 2H), 3.39 (s, 3H), 2.45 (s, 1H), 1.64 (t, J = 7.0 Hz, 3H); 13C NMR (175
MHz, CDCl3) δ 166.233, 161.24, 147.66, 143.81, 143.72, 135.42, 135.20, 129.28, 128.16,
127.69, 127.26, 124.01, 120.81, 119.73, 111.37, 106.24, 64.73, 52.89, 21.56, 15.07; LRMS
(ESI) m/z: 456.2 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C23H26N3O5S: 456.1588;
found: 456.1591.
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methylphenylsulfonamido)acetamide (7s)
mp 124.0 oC; 1H NMR (700 MHz, CDCl3) δ 9.38 (s, 1H), 8.58 (d, J = 7.0 Hz, 1 H), 7.89 (d, J
= 9.1 Hz, 1 H), 7.57ꢀ7.55 (m, 1H), 7.34 (t, J = 7.0 Hz, 1 H), 7.29ꢀ7.28 (m, 2 H), 6.99 (t, J =
7.7 Hz, 1 H), 6.87 (dd, J = 7.7, 4.9 Hz, 1 H), 6.80 (d, J = 8.4 Hz, 1 H), 7.39 (t, J = 7.0 Hz, 1
H), 7.38 (d, J = 7.7 Hz, 2 H), 7.07 (d, J = 8.4 Hz, 1 H), 6.98 (t, J = 7.7 Hz, 1 H), 6.91 (d, J =
7.7 Hz, 1 H), 4.34 (br, 2H), 3.97 (s, 3H), 2.48 (s, 3H); 13C NMR (175 MHz, CDCl3) δ 166.97,
155.25, 152.16, 143.32, 140.30, 135.57, 132.03, 130.12, 129.11, 127.16, 126.48, 126.37,
121.90, 120.06, 116.44, 112.37, 68.61, 55.08, 53.92, 21.90, 21.00; LRMS (ESI) m/z: 470.2
[M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C24H28N3O5S: 470.1744; found:470.1753.
N-(2-Ethoxyphenyl)-2-(N-(3-methoxyphenyl)-4-methylphenylsulfonamido)acetamide (7t)
mp 175.7 oC; 1H NMR (700 MHz, CD3OD) δ 8.17 (d, J = 8.4 Hz, 1 H), 7.55 (d, J = 7.7 Hz, 2
H), 7.40 (d, J = 7.7 Hz, 2 H), 7.13ꢀ7.09 (m, 3 H), 7.06 (d, J = 8.4 Hz, 1 H), 6.92 (t, J = 7.0
Hz, 1 H), 6.89 (d, J = 9.1 Hz, 2 H), 4.38 (s, 2 H), 4.24 (q, J = 7.0 Hz, 2 H), 3.79 (s, 3 H),
2.47 (s, 3 H), 1.61 (t, J = 7.0 Hz, 3 H); 13C NMR (175 MHz, CD3OD) δ 166.84, 159.78,
148.14, 144.58, 134.25, 132.13, 129.45, 129.40, 127.74, 126.73, 124.44, 120.28, 119.47,
114.09, 111.33, 64.40, 55.41, 54.57, 20.10, 13.99; LRMS (ESI) m/z: 455.2 [M+H]+; HRMS
(ESI) m/z: Calcd. for [M+H]+ C24H28N3O5S: 455.1635; found: 455.1640.
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oC; H NMR (400 MHz, CDCl3) δ 9.31 (S, 1 H), 8.33 (d, J = 7.6 Hz, 1 H), 7.51 (d, J = 8.0
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Hz, 2 H), 7.36ꢀ7.29 (m, 5 H), 7.20ꢀ7.18 (m, 2 H), 7.06 (t, J = 9.2 Hz, 1 H), 6.94 (t, J = 9.2 Hz,
2 H), 4.34 (s, 2 H), 4.24 (q, J = 8.8, 6.8 Hz, 2 H), 2.47 (s, 3 H), 1.68 (t, J = 7.2 Hz, 3 H);
13C NMR (100 MHz, DMSOꢀd6) δ 165.64, 147.68, 143.98, 139.49, 134.36, 129.75, 129.11,
128.00, 127.78, 127.49, 126.81, 124.30, 120.38, 119.70, 111.99, 64.12, 54.38, 21.01, 14.64;
LRMS (ESI) m/z: 425.2 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+ C23H25N2O4S:
425.1530; found: 425.1544.
N-(2-Ethoxyphenyl)-2-(N-(2-fluorophenyl)-4-methylphenylsulfonamido)acetamide (7v)
o
1
mp 162.3 C; H NMR (CDCl3, 400 MHz) d 9.40 (s, 1 H), 8.36 (dd, J = 8.8, 2.0 Hz, 1 H),
7.60 (d, J = 8.4 Hz, 2 H), 7.40ꢀ7.31 (m, 4 H), 7.16 (t, J = 8.4 Hz, 1 H), 7.10ꢀ7.05 (m, 2 H),
6.97ꢀ6.93 (m, 2 H), 4.31 (s, 2 H), 4.25 (q, J = 7.2 Hz, 2 H), 2.47 (s, 3 H), 1.66 (t, J = 7.2 Hz,
3 H); 13C NMR (175 MHz, CDCl3) δ 165.71, 157.91, 147.76, 144.59, 134.46, 131.85, 130.81,
129.78, 127.87, 127.29, 124.80, 124.16, 120.79, 119.48, 117.13, 116.93, 111.04, 64.58, 55.28,
21.65, 14.93; LRMS (ESI) m/z: 443.1 [M+H]+; HRMS (ESI) m/z: Calcd. for [M+H]+
C23H24FN2O4S: 443.1436; found: 443.1444.
2-((N-(2-Chlorophenyl)-4-methylphenyl)sulfonamido)-N-(2-ethoxyphenyl)acetamide (7w)
mp 163.8 oC; 1H NMR (700 MHz, CDCl3) δ 9.36 (s, 1H), 8.33 (dd, J = 7.9, 1.3 Hz, 1H), 7.60 (d, J =
8.2 Hz, 2H), 7.44 (dd, J = 7.7, 1.7 Hz, 1H), 7.37 (dd, J = 7.8, 1.6 Hz, 1H), 7.34ꢀ7.17 (m, 4H), 7.05 (td,
J = 7.9, 1.5 Hz, 1H), 6.98 ꢀ 6.86 (m, 2H), 4.58ꢀ4.25 (m, 2H), 4.20 (s, 2H), 2.44 (s, 3H), 1.62 (t, J = 7.0
Hz, 3H); 13C NMR (175 MHz, CDCl3) δ 165.93, 147.77, 144.52, 136.62, 135.06, 133.85, 132.71,
131.01, 130.19, 129.77, 128.08, 127.71, 127.31, 124.15, 120.79, 119.67, 111.03, 64.54, 55.39, 21.65,
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