Discovery of Novel Small-Molecule Inhibitors of NF-κB Signaling with Antiinflammatory and Anticancer Properties

Article abstract of DOI:10.1021/acs.jmedchem.7b01557

Excessive NF-κB activation contributes to the pathogenesis of numerous diseases. Small-molecule inhibitors of NF-κB signaling have significant therapeutic potential especially in treating inflammatory diseases and cancers. In this study, we performed a ce

Full text of DOI:10.1021/acs.jmedchem.7b01557

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Article
Discovery of Novel Small-molecule Inhibitors of Nuclear Factor-
#B Signaling with Anti-inflammatory and Anti-cancer Properties
Lei Zhang, Lei Shi, Shafer Soars, Joshua Kamps, and Hang Hubert Yin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01557 • Publication Date (Web): 05 Jun 2018
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Discovery of Novel Smallꢀmolecule Inhibitors of
Nuclear FactorꢀκB Signaling with Antiꢀ
inflammatory and Antiꢀcancer Properties
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1,
‡
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‡
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1,*
Lei Zhang, Lei Shi, Shafer Myers Soars, Joshua Kamps, and Hang Yin
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Department of Chemistry and Biochemistry and the BioFrontiers Institute, University of
Colorado, Boulder, CO 80309, USA
2
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry,
China Pharmaceutical University, Nanjing 210009, PR China
‡
Coꢀfirst author
*
Correspondence: hubert.yin@colorado.edu
KEYWORDS. NFꢀκB, highꢀthroughput screening (HTS), drug discovery, antiꢀinflammatory,
antiꢀcancer
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ABSTRACT. Excessive NFꢀκB activation contributes to the pathogenesis of numerous diseases.
Smallꢀmolecule inhibitors of NFꢀκB signaling have significant therapeutic potential especially in
treating inflammatory diseases and cancers. In this study, we performed a cellꢀbased highꢀ
throughput screening to discover novel agents capable of inhibiting NFꢀκB signaling. Based on
two hit scaffolds from the screening, we synthesized 69 derivatives to optimize the potency for
inhibition of NFꢀκB activation, leading to successful discovery of the most potent compound Z9j
with over 170ꢀfold enhancement of inhibitory activity. Preliminary mechanistic studies revealed
that Z9j inhibited NFꢀκB signaling via suppression of Src/Syk, PI3K/Akt and IKK/IκB
pathways. This novel compound also demonstrated antiꢀinflammatory and antiꢀcancer activities,
warranting its further development as a potential multifunctional agent to treat inflammatory
diseases and cancers.
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INTRODUCTION
Nuclear factorꢀκB (NFꢀκB) is a master transcription factor that regulates a number of cellular
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activities such as immune responses, inflammation, and cell survival. The mammalian NFꢀκB
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family encompasses five members, including p50, p52, p65 (RelA), RelB, and cꢀRel, which form
various homoꢀ or heterodimeric complexes. In unstimulated cells, NFꢀκB dimer is sequestered in
the cytoplasm by the inhibitory family protein IκB in a latent form. Upon activation, different
upstream pathways converge on the IKK complex which consists of two catalytic components,
IKKα and IKKβ, and a regulatory component named NFꢀκB essential modulator (NEMO) or
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IKKγ. IKK complex phosphorylates IκB, leading to its subsequent ubiquitination and
degradation by the proteasome (Figure 1A). This releases NFꢀκB dimer and allows it to
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translocate into the nucleus to activate specific target genes.
A broad range of stimuli can activate NFꢀκB signaling including pathogens, inflammatory
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cytokines, growth factors, ultraviolet, and oxidative stress. Upon activation of distinct stimuli,
many membrane receptors can initiate various signaling pathways that culminate in the
activation of NFꢀκB, for example Tollꢀlike receptors (TLRs), tumor necrosis factor receptor
(TNFR), interleukinꢀ1 receptor (ILꢀ1R), T cell receptor (TCR), and B cell receptor (BCR)
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ꢀ8
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(
Figure 1A). Among these, TLRs are the best studied receptors.
The human TLR family to
date consists of ten members which recognize a wide range of pathogenꢀassociated molecular
patterns (PAMPs) or dangerꢀassociated molecular patterns (DAMPs), such as bacterial
lipoproteins (TLR2 ligand), viral doubleꢀstranded RNA (TLR3 ligand), lipopolysaccharide (LPS;
TLR4 ligand), flagellin (TLR5 ligand), viral singleꢀstranded RNA (TLR7 and 8 ligand), and
bacterial CpG DNA (TLR9 ligand). In response to PAMPs or DAMPs, all TLR signaling
pathways initiate inflammatory cascades, resulting in NFꢀκB activation. The activation of NFꢀκB
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in turn controls the expression of hundreds of genes involved in immune responses, cell cycle,
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apoptosis, cell adhesion, tissue differentiation, and DNA repair.
Due to its key role as a master switch for many genes, it is not surprising that aberrant
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activation NFꢀκB is associated with a plethora of disorders, especially inflammatory diseases and
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cancers.
Inflammation is an important defense system by which the body protects us from
exogenous infections and endogenous damage. NFꢀκB has been recognized as a pivotal regulator
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of inflammation. Upon activation of NFꢀκB signaling, various proꢀinflammatory cytokines,
chemokines, and other mediators such as TNFꢀα and nitric oxide (NO) are expressed to remove
pathogens or damage. However, excessive or chronic activation of NFꢀκB causes detrimental
inflammation and results in high levels of inflammatory mediators, contributing to pathogenesis
of many inflammatory diseases, such as systemic lupus erythematosus, sepsis, atherosclerosis,
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rheumatoid arthritis, asthma, diabetes, and Alzheimer’s disease.
Importantly, inhibition of
NFꢀκB signaling by pharmacological interventions can potentially prevent or alleviate all these
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chronic disorders.
Constitutive activation of NFꢀκB has also been found in many cancer cells
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and causes drug resistance by increasing cell proliferation and inhibiting apoptosis.
Therefore, inhibition of NFꢀκB overactivation by smallꢀmolecule inhibitors has been recognized
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as an advantageous strategy for cancer therapy.
In this regard, NFꢀκB is an exciting
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therapeutic target for drug discovery and development.
The dynamic NFꢀκB regulatory network offers a multitude of promising options for
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developing therapeutic interventions. Generally, NFꢀκB inhibition can be achieved by targeting
any step of the signaling cascades, such as blocking the binding of stimuli to membrane receptors
at an early stage, interfering with an intracellular component of the cascade to stop the signal
transduction (e.g. kinases, phosphatases, ubiquitination, IKK complex, nuclear translocation), or
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suppressing the NFꢀκB nuclear activity (e.g. DNA binding, acetylation or methylation of NFꢀ
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κB).
However, currently there are few NFꢀκB signaling inhibitors that are entering clinical
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trials, suggesting an urgent need to develop novel agents to modulate NFꢀκB signaling.
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To discover novel agents that could inhibit NFꢀκB signaling, we herein conducted a cellꢀ
based highꢀthroughput screening (HTS) using TLRꢀmediated NFꢀκB activation as a phenotypic
platform. Our efforts led to the discovery of twelve hits, two of which were selected in this study
for extensive structureꢀactivity relationship (SAR) studies, affording the most potent compound
Z9j with over 170ꢀfold enhancement of inhibitory activity. Furthermore, we carried out
preliminary studies to understand possible inhibitory mechanism of Z9j by both cellular and
molecular approaches. Meanwhile, to investigate whether the newly discovered modulator has
therapeutic potential, we explored its antiꢀinflammatory and antiꢀcancer activities.
RESULTS AND DISCUSSION
Cellꢀbased highꢀthroughput screening. HTS has been proven to be highly efficient in
identifying biologically active small molecules as candidates for further drug discovery and
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development. HTS assays typically can be divided into two categories: biochemical and cellꢀ
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based assays. Hits discovered in a biochemical HTS cannot always translate into the same
response in a cellular context due to several limitations such as lack of cellular cofactors and
issues of membrane permeability. In contrast, a cellꢀbased HTS can reveal multiple targets in cell
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signaling, some of which cannot be captured in biochemical screening. Given the multiple
pathways that can activate NFꢀκB signaling, a cellꢀbased HTS is particularly advantageous for
the discovery of novel NFꢀκB inhibitors. Therefore, we conducted a cellꢀbased HTS using TLRꢀ
mediated NFꢀκB activation as a phenotypic platform to discover smallꢀmolecule modulators that
can inhibit any step of NFꢀκB signaling.
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Our group has successfully established a robust HTS platform based on a TLRꢀ
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overexpression system. We modified the protocol in this study to accommodate for screening
inhibitors of TLRꢀmediated NFꢀκB activation. Because of their low cost, ease of culture, and
good reliability, human embryonic kidney 293 (HEK293) cells have been extensively used as a
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benchꢀmark expression system for TLR signaling studies. The chemical library utilized in this
study was the Maybridge HitFinder v11 library which encompasses 14,400 diverse small
molecules. Since there is still a lack of smallꢀmolecule TLR7 inhibitors, the HEK293 cell line
was coꢀtransfected with the human TLR7 gene and a secreted embryonic alkaline phosphatase
(
SEAP) reporter gene placed under the control of an NFꢀκBꢀinducible promoter. Therefore,
through the HTS, we could expect either inhibitors of TLR7 itself or inhibitors of TLR7ꢀ
mediated NFꢀκB activation, both of which are of interest to drug discovery.
The screening was conducted in 384ꢀwell plates in duplicate with a Z' value of 0.74, an
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indicator of robustness of the screening procedure. Transfected HEK293 cells were stimulated
with TLR7 agonist R848 before treating with library compounds at 30 ꢁM. Triptolide, a known
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NFꢀκB inhibitor,
and blank (w/ 4% DMSO) were used as positive and negative controls,
respectively (Figure S1). SEAP secretion in the cell supernatant was measured to indicate levels
of TLR7ꢀmediated NFꢀκB activation by colorimetric QUANTIꢀBlue detection. After the
screening, the data obtained were statistically distributed based on the root mean square (RMS)
values as depicted in Figure 1B. From the HTS, twelve hits were identified, among which hit
compounds S3a and Z3a were selected in this study for structural optimization and SAR studies.
The two hits were selected based on two criteria. First, they are among the top list of the twelve
identified hits based on potency. Second, both of these compounds are structurally similar with
two aromatic rings attached to a hydrazide linker (Figure 1C).
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SAR Studies. To explore SAR and identify more potent inhibitors of the TLR7ꢀmediated NFꢀ
κB activation, we designed and synthesized 31 derivatives of S3a and 38 derivatives of Z3a. All
the compounds were synthesized in a straightforward fashion following the routes outlined in
Schemes 1ꢀ7. The SAR studies were mainly focused on the bilateral aromatic rings of hit
compounds S3a and Z3a while maintaining the central hydrazide scaffold. The NFꢀκB inhibitory
activity of all the compounds were examined in HEK hTLR7 cells by the SEAP assay. The
inhibition percentages of all the compounds were initially tested at 40 ꢁM and those with more
than 50% inhibition were further tested for halfꢀmaximal inhibitory concentration (IC ) values.
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For derivatives of hit S3a, we first examined the position effect of the trifluoromethyl groups
on the phenyl ring (Table 1). It turned out that the paraꢀ substitution (S3c) was more favorable
than metaꢀ substitution (S3a), whereas orthoꢀ substitution (S3b) led to diminished activity. We
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also investigated the role of the heteroaromatic ring, particularly the necessity of the two sp ꢀ
hybridized nitrogen atoms and the amino group of S3a. The results showed that the N1 of the
pyrazine system of S3a is indispensable, as indicated by the loss of activity of S3f (Table 1). The
other two nitrogen atoms are inessential as compound S3i lacking both still showed strong NFꢀ
κB inhibitory activity. Absence of the amino group on the pyrazine ring abolished the activity
(Table 1, S3a and S3e) whereas attaching an amino or hydroxyl group (Table 1, S3d and S3h)
to the aromatic system recovered the activity, suggesting that a hydrogenꢀbond donor or electronꢀ
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donating group could offset the electron deficiency caused by two sp ꢀhybridized nitrogen atoms
compared to those with only one. Halogen substituents (Table 1, S3j and S3k) resulted in
significant decrease of NFꢀκB inhibitory activity, indicating intolerance of substituents on the
pyrazine ring. The importance of the flexible hydrazide linker was evidenced by compounds S4
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which displayed no inhibition possibly due to their more rigid structures (Table 1). Therefore,
the following SAR studies were dedicated to the bilateral aromatic rings only.
Maintaining the aminopyrazine ring, we studied various substituted phenyl rings with a
preference to paraꢀ substitution (Table 2). There appeared no obvious electronic effect of
substituents on the phenyl ring, since either electronꢀwithdrawing (e.g. S10f) or electronꢀ
donating (e.g. S10g and S10h) groups did not result in any significant difference in the activity.
Replacing the phenyl ring with pyridine proved to be unfavorable (S10k). In addition, longꢀ
chained substitution was not tolerated exemplified by compound S10l.
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Based on the two best heteroaromatic rings and four best substituted phenyl rings from above
SAR studies, we explored their different combinations. As expected, the best heteroaromatic ring
and the best substituted phenyl ring produced the most potent compound S11e among the
derivatives of hit S3a, with an IC value of 7.9 ± 0.5 ꢁM (Table 3).
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The structural optimization of hit Z3a was also focused on the bilateral aromatic rings (Table
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). Investigation of different heteroaromatic rings found that an sp ꢀhybridized nitrogen atom
adjacent to the hydrazide linker is vital to maintain the NFꢀκB inhibitory activity (Table 4, Z3a
vs Z3b and Z3dꢀZ3f). Appropriate substituents on the thiazole ring improved the activity (Table
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, Z3a vs Z3c).
Investigation of different substituted phenyl rings revealed a position effect of substituents
with a preference for the paraꢀ position (Table 5, Z3a vs Z5a and Z5b). No obvious electronic or
hydrogenꢀbonding effects were observed based on compounds Z5cꢀZ5k. However, replacing the
phenyl ring with 2ꢀpyridyl ring yielded a more potent compound Z5l compared with hit
compound Z3a.
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Maintaining the 2ꢀpyridyl ring on the right side of the molecule, we then examined the
aliphatic 2ꢀisopropyl (Z7a), phenyl (Z7b), and heteroaromatic (Z7c and Z7d) substituents on the
thiazole (Table 6). It turned out that the phenyl substituted compound Z7b resulted in significant
improvement of TLR7ꢀmediated NFꢀκB inhibitory activity.
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From Z7b, we further explored whether installing appropriate substituents on its phenyl ring
would improve the inhibitory activity (Table 7). A trend for electronic effect of substituents on
the phenyl ring was not identified, both electronꢀwithdrawing (Z9c) and electronꢀdonating (Z9dꢀ
Z9h) groups gave comparable activities. However, diꢀchloro substituted derivatives Z9iꢀZ9k
displayed significant increase of inhibitory activities. Among them, compound Z9j (CUꢀCPTꢀ
Z9j) was the most potent compound with an IC value of 0.26 ± 0.05 ꢁM. In this structure, the
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thiazole nitrogen is essential to maintain the activity, hydrophobic 2,4ꢀdichlorophenyl
substitution on the thiazole ring significantly improved the potency, and pyridineꢀ2ꢀ
carbohydrazide structure of Z9j is beneficial to the NFꢀκB inhibitory activity. In comparison to
its original hit Z3a (44.8 ± 0.9 ꢁM), compound Z9j showed over 170ꢀfold enhancement of
inhibitory activity to TLR7ꢀmediated NFꢀκB activation. Therefore, compound Z9j was selected
for further biological studies.
Mechanism of Action Studies. We began the biological investigations of Z9j by examining
its NFꢀκB signaling inhibitory mechanism using cellular approaches. To determine whether Z9j
could specifically target TLR7, we tested its TLR selectivity. HEK293 cells were coꢀtransfected
with different human TLR genes and a SEAP reporter gene under the control of NFꢀκB
promoter. The inhibitory activities of Z9j were tested using the SEAP assay. These HEK hTLR
cells were stimulated with distinct TLR ligands, including Pam3CSK4 for TLR1/2, Pam2CSK4
for TLR2/6, poly I:C for TLR3, LPS for TLR4, flagellin for TLR5, R848 for TLR7 and TLR8,
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and CpGꢀODN2006 for TLR9. As shown in Figure 2A, Z9j inhibited the SEAP signals
triggered by different TLR ligands in a similar manner. To rule out toxicity, the cell viability was
evaluated with a colorimetric WSTꢀ1 assay. Compound Z9j showed negligible cytotoxicity
within the concentrations tested (Figure 2B). Cumulatively, the results suggested that Z9j may
target signaling molecules downstream of all these homologous TLRs.
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To confirm our hypothesis, we examined whether Z9j could inhibit NFꢀκB signaling through
other membrane receptors, such as TNFR and ILꢀ1R (Figure 2C). HEKꢀBlue Null cells were
transfected with the SEAP reporter gene under the control of the NFꢀκB promoter in the absence
of TLRs. Results showed that compound Z9j doseꢀdependently inhibited the SEAP signals
stimulated by TNFꢀα (Figure 2D) and ILꢀ1β (Figure 2E) in the transfected HEKꢀBlue Null
cells. Moreover, Z9j showed similar IC50 values in both cases: 0.77 ± 0.04 µM for TNFꢀα
stimulation and 0.72 ± 0.03 µM for ILꢀ1β stimulation. These results clearly pointed to the
possibility that Z9j targets common downstream pathways shared by all these membrane
receptors.
NFꢀκB signaling can also be activated through upstream cytoplasmic kinases including
35
protein kinase C (PKC). To exclude the possibility that membrane receptors are not the target
of compound Z9j, we examined its effects on NFꢀκB signaling activated by phorbol 12ꢀmyristate
1
3ꢀacetate (PMA), a known PKC agonist (Figure 2C). Indeed, PMA treatment induced strong
NFꢀκB activation in either HEK hTLR7 (Figures 2F) or HEK Null cells (Figures 2G) as shown
by the SEAP assay. When treated with compound Z9j, the PMAꢀinduced NFꢀκB activation was
doseꢀdependently inhibited in a similar manner in both HEK hTLR7 (IC = 0.59 ± 0.02 µM) and
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HEK Null (IC = 0.37 ± 0.03 µM) cells. These data further confirmed that Z9j did not target
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membrane receptors.
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Additionally, we examined the inhibitory effect of Z9j on NFꢀκB signaling in mouse cells.
NFꢀκB activation leads to production of NO by inducible nitric oxide synthase (iNOS) (Figure
2C). The levels of NO production can be measured by the 2,3ꢀdiaminonaphthalene (DAN) assay.
Results showed that Z9j inhibited the NFꢀκBꢀmediated NO production triggered by either
interferon (IFN)ꢀγ (IC = 0.079 ± 0.005 µM, Figure 2H) or LPS (0.08 ± 0.01 ꢁM, Figure 4B) in
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a similar manner in mouse macrophage RAW 264.7 cells. These data indicated that the inhibition
of Z9j on NFꢀκB signaling worked in both human and mouse cell lines, though their potency
may differ to a certain extent.
On the basis of these cellular results, we moved on to explore the molecular mechanism by
which Z9j inhibited NFꢀκB signaling. After LPS stimulation in RAW 264.7 cells, three key
regulatory pathways are activated including IKK/IκB pathway, phosphatidylinositolꢀ3ꢀkinase
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(
PI3K)/Akt pathway, and mitogenꢀactivated protein kinase (MAPK) signaling pathway. The
changes of the key regulators in these pathways were investigated by Western blotting.
Since the phosphorylation of IκB and its upstream regulator IKK complex are the critical
steps for activation of NFꢀκB signaling, we first measured the effects of Z9j on the LPSꢀinitiated
IKK/IκB pathway. As shown in Figure 3A, LPS significantly stimulated the phosphorylation of
both IKKαβ and IκBα at 15 and 30 minutes. Upon preꢀtreatment of Z9j, the phosphorylation
levels of IKKαβ and subsequent IκBα were decreased at both time intervals. These results
showed that Z9j could inhibit NFꢀκB activation via the classical IKK/IκB cascade.
We then looked into the upstream signaling events for IKK/IκB activation. PI3K and its
37ꢀ39
downstream target Akt are upstream regulators of IKK/IκB signaling in various cells.
Thus,
the effects of Z9j on the LPSꢀinduced phosphorylation of PI3K and Akt were examined. LPS
induced the phosphorylation of both PI3K and Akt in macrophages (Figure 3B) and the
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induction was markedly attenuated by Z9j, indicating that Z9j can inhibit NFꢀκB activation via
the PI3K/Akt pathway.
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MAPKs are wellꢀknown signaling adaptors involved in the LPSꢀinduced macrophages. To
determine whether Z9j could affect the MAPK pathways, phosphorylation levels of cꢀJun Nꢀ
terminal kinase (JNK), p38, and extracellular signal regulated protein kinase (ERK) were
assessed. LPS treatment alone induced phosphorylation of the three major families of MAPKs at
both 15ꢂand 30 min (Figure 3C). However, Z9j showed no or negligible inhibition on the
phosphorylation levels of these MAPK isoforms. It has been reported that, once activated, the
MAPKs translocate to the nucleus to phosphorylate and thereby transcriptionally activate
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1, 42
activator protein 1 (APꢀ1).
Our results suggested that Z9j did not inhibit NFꢀκB activation
via the MAPK signaling pathway.
Then we turned to search for signaling enzymes upstream of PI3K/Akt. Activation of Src and
subsequent spleen tyrosine kinase (Syk) is an early event required for activation of PI3K
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3, 44
signaling.
We thus investigated the effect of Z9j on levels of phosphorylated Syk and Src in
LPSꢀinduced RAW 264.7 cells. As shown in Figure 3D, LPS induced increased levels of
phosphorylation of both Src and Syk in RAW 264.7 cells. More importantly, Z9j could block the
phosphorylation of both Src and subsequent Syk in a doseꢀdependent manner at 30 minutes. We
further performed the Src kinase assay in vitro and found Z9j could only suppress less than 50%
of the Src kinase activity at 5 µM (Figure 3E). The inhibition of the Src kinase activity by Z9j is
not comparable to the cellꢀbased activity, suggesting that there are probably other novel
mechanisms of actions involved.
To further examine whether Z9j could directly bind to any of the Src family kinases, we
sought to kinase profiling using KINOMEscan™ service (Eurofins DiscoverX) which tested
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compoundꢀkinase interactions for dissociation constants (K s) based on DNA labeled kinases and
d
qPCR readout. As the first attempt, we screened for 15 Src family kinases (BLK, BMX, BTK,
FGR, FYN, HCK, ITK, LCK, LYN, SRC, SYK, TEC, TXK, YES1, ZAP70) with Z9j at 10 ꢁM.
Results showed that Z9j only slightly inhibited Src (75%) and there was no inhibition against
Syk (Figure S2). Therefore, we increased the concentration of Z9j to 20 ꢁM and expanded the
screen to all the kinases involved in the above three regulatory pathways of NFꢀκB signaling.
The second round of kinase profiling comprised 40 targets, including Src family kinases, PI3Ks,
AKTs, MAPKs, and IKKs. It turned out that Z9j exhibited only mild inhibition against Erk8
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(44%) at 20 ꢁM (Figure S3). Given that Z9j did not significantly inhibit most of these kinases in
the kinaseꢀprofiling assays, there are probably other novel mechanisms of actions of Z9j
involved, such interfering proteinꢀprotein interactions.
Based on these results, we proposed a putative mechanism for Z9j inhibition on NFꢀκB
signaling (Figure 4). Briefly, Z9j might inhibit NFꢀκB signaling by suppressing the Src/Syk,
PI3K/Akt and IKK/IκB pathways, eventually leading to downregulation of inflammatory
mediators, whereas with negligible impact on the MAPK pathways. Due to the complexity of
NFꢀκB signaling and its crosstalk with other signaling pathways, further studies are needed to
determine if Z9j may also modulate other enzyme molecules or directly target certain proteinꢀ
45, 46
protein interactions thereby influencing NFꢀκB signaling pathways.
Therapeutic Potential of Z9j. Aside from looking at the possible mechanism of action of
Z9j, we further investigated its therapeutic potential. Excessive activation NFꢀκB signalinghas
11ꢀ14
been implicated in many diseases.
Particularly, inhibitors of NFꢀκB signaling have been
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recognized as potential therapeutic agents against many inflammatory diseases and cancers.
We thus studied the antiꢀinflammatory and antiꢀcancer properties of Z9j.
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The antiꢀinflammatory potential of compound Z9j was investigated in LPSꢀstimulated RAW
64.7 cells, an extensively used model to study inflammation. The effect of Z9j on the
2
production of TNFꢀα, a wellꢀknown inflammatory marker, was tested by enzymeꢀlinked
immunosorbent assay (ELISA). As shown in Figure 5A, LPS stimulation led to significant
production of TNFꢀα, an indication of a strong inflammatory response. The TNFꢀα production
was efficiently suppressed by Z9j in a doseꢀdependent manner. Another wellꢀrecognized
inflammatory marker is NO produced by iNOS after activation of NFꢀκB (Figure 2C). As shown
in Figure 5B, compound Z9j also diminished the NO levels doseꢀdependently in LPSꢀstimulated
RAW 264.7 cells with an IC value of 0.08 ± 0.01 ꢁM. These experiments demonstrated that
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Z9j could effectively reduce or prevent NFꢀκB mediated inflammation, suggesting its potential
to be further developed as an antiꢀinflammatory agent against many inflammatory diseases.
We also investigated whether the inhibition of NFꢀκB by Z9j has antiꢀcancer effects in human
cancer cells. Cervical cancer HeLa cells were selected as a test system in this study. The
antiproliferative effect of Z9j on HeLa cells was first examined by the WSTꢀ1 assay. As shown
in Figure 5C, Z9j drastically inhibited the growth of HeLa cells in a concentrationꢀ and timeꢀ
dependent manner, with a GI50 (growth inhibition of 50%) value around 1.35 ± 0.08 ꢁM
following exposure to Z9j for 48 hours. As a comparison, Z9j only showed mild cytotoxicity to
the normal human mammary epithelial cells (HuMECs). To further explore the mechanism
behind its antiproliferative effect, Z9j was subjected to an apoptosis induction assay. The HeLa
cancer cells were treated with different concentrations of Z9j for 24 hours before flow cytometry
analysis by double staining with Annexin VꢀFITC and propidium iodide (PI). The cells in
quadrant Q4 represent living cells, Q3 early apoptotic cells, Q2 late apoptotic cells, and Q1
necrotic cells (Figure 6A). Results showed that Z9j displayed evident induction of apoptosis in
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HeLa cells in a doseꢀdependent manner compared to the control group (Figure 6B), suggesting
that the antiproliferative activity of Z9j against HeLa cells may result from its ability to induce
apoptosis. The effect of Z9j on the cell cycle distribution of HeLa cells was also examined by
staining cells with propidium iodide and analyzing the percentages of G0/G1, S, and G2/M cell
populations with flow cytometry. As shown in Figures 6C and 6D, treatment with Z9j caused a
doseꢀdependent accumulation of HeLa cells at the G0/G1 phase, along with a decreased
population of G2/M phase cells. Collectively, these results suggested that compound Z9j may
also be developed as an antiꢀcancer agent.
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Taken together, these data suggested that Z9j may be further developed as a multifunctional
therapeutic agent to treat inflammatory diseases as well as cancers.
CONCLUSION
NFꢀκB plays an essential role in regulating a number of cellular activities, thus its aberrant
activation contributes to pathogenesis of numerous diseases, such as arthritis, asthma,
Alzheimer’s disease, sclerosis, sepsis, cardiovascular diseases, diabetes, inflammatory diseases,
and many types of cancers. Developing novel smallꢀmolecule agents that inhibit NFꢀκB
signaling would provide potential therapeutics for the treatment of many diseases as well as
valuable tools for biological studies. We in this study utilized the strategy of cellꢀbased highꢀ
throughput screening to discover novel inhibitors of NFꢀκB signaling via the TLR7 pathway.
Structural modification and SAR studies of two hit compounds led to successful discovery of the
best compound Z9j with an over 170ꢀfold of enhanced NFꢀκB inhibitory activity. Mechanistic
studies using both cellular and molecular approaches revealed that Z9j inhibited NFꢀκB
signaling by suppressing Src/Syk, PI3K/Akt and IKK/IκB pathways. Moreover, preliminary
therapeutic studies showed that Z9j effectively inhibited NFꢀκBꢀassociated inflammatory
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responses in macrophage cells, as well as caused cell growth inhibition, apoptosis induction, and
cell cycle arrest in human cervical cancer cells. We obtained herein a novel NFꢀκB inhibitor with
great potential for further development as multifunctional therapeutic agent to treat inflammatory
diseases as well as cancer. Our work has also showcased the efficiency of the cellꢀbased HTS
strategy to discover novel therapeutic agents.
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EXPERIMENTAL SECTION
General Chemistry Methods. Reagents were purchased from commercial sources and used as
1
received. H NMR spectra were recorded at 400 MHz in DMSOꢀd using a residual solvent peak
6
1
3
(
2.50 ppm) as the internal standard. C NMR spectra were recorded at 101 MHz in DMSOꢀd
6
using residual DMSO (39.52 ppm) as the internal reference. Compounds were purified using
flash chromatography (ACI systems, Biotage). Purities of all tested compounds were tested using
1
13
H NMR, C NMR, and highꢀperformance liquid chromatography (HPLC) analysis. NMR and
HPLC analysis showed that all tested compounds have >95% purity. Mass spectrometry was
performed at the mass spectrometry facility of the Department of Chemistry at the University of
Colorado at Boulder on a double focusing highꢀresolution mass spectrometer.
General Procedure for the Preparation of Compounds S3aꢀk, S10aꢀl, and S11aꢀh
Exemplified by 3ꢀAminoꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide (S3a).
Oꢀbenzotriazolꢀ1ꢀylꢀN,N,N',N'ꢀtetramethyluronium tetrafluoroborate (TBTU, 346 mg, 1.08
mmol) and triethylamine (0.30 mL, 2.16 mmol) were added to a solution of 3ꢀaminopyrazineꢀ2ꢀ
carboxylic acid (100 mg, 0.72 mmol) and 3ꢀ(trifluoromethyl)benzeneꢀ1ꢀcarbohydrazide (176 mg,
0
.86 mmol) in dimethylformamide (DMF, 10 mL) and the resulting solution stirred at room
temperature overnight. The reaction mixture was poured into water and extracted with ethyl
acetate. The combined organic layers were dried with Na SO , filtered, concentrated, and
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purified by flash column chromatography to give the title compound S3a (213 mg, 91%) as a
1
lightꢀyellow solid. H NMR (400 MHz, DMSOꢀd ) δ 10.77 (s, 1H), 10.68 (s, 1H), 8.30 (s, 1H),
6
8.26 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.91 (s, 1H), 7.80 (t, J = 7.6 Hz,
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H), 7.54 (br s, 2H); C NMR (101 MHz, DMSOꢀd ) δ 165.71, 164.63, 155.59, 147.94, 133.84,
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1
32.06, 131.67, 130.42, 129.76 (q, J = 32.1 Hz), 128.94 (q, J = 3.9 Hz), 125.01, 124.50 (q, J =
+
+
3
.9 Hz), 124.35 (q, J = 272.4 Hz); HRMS (ESI ), calcd C H F N O (M + H ) = 326.0865,
13 11 3 5 2
found = 326.0862.
3
ꢀAminoꢀN'ꢀ(2ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide (S3b). Lightꢀyellow
1
solid, yield 92%. H NMR (400 MHz, DMSOꢀd ) δ 10.60 (s, 1H), 10.49 (s, 1H), 8.29 (d, J = 2.3
6
1
3
Hz, 1H), 7.90 (d, J = 2.3 Hz, 1H), 7.86 – 7.79 (m, 2H), 7.74 – 7.69 (m, 2H), 7.53 (br s, 2H); C
NMR (101 MHz, DMSOꢀd ) δ 166.57, 165.52, 155.55, 147.89, 134.54 (d, J = 2.3 Hz), 132.90,
6
1
31.64, 130.96, 129.72, 127.34 (q, J = 31.7 Hz), 126.89 (d, J = 4.6 Hz), 125.06, 121.28 (d, J =
+
+
273.8 Hz); HRMS (ESI ), calcd C H F N O (M + H ) = 326.0865, found = 326.0861.
1
3
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3
5
2
3
ꢀAminoꢀN'ꢀ(4ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide (S3c). Yellow solid,
1
yield 89%. H NMR (400 MHz, DMSOꢀd ) δ 10.69 (br s, 2H), 8.29 (d, J = 1.8 Hz, 1H), 8.11 (d,
6
1
3
J = 8.1 Hz, 2H), 7.93 (s, 1H), 7.90 (d, J = 2.3 Hz, 2H), 7.53 (br s, 2H); C NMR (101 MHz,
DMSOꢀd ) δ 165.46, 164.86, 155.60, 147.86, 137.09, 131.99 (d, J = 31.5 Hz), 131.67, 128.84,
6
+
1
25.99 (q, J = 3.6 Hz), 125.72, 125.17, 121.66 (d, J = 272.3 Hz); HRMS (ESI ), calcd
+
C H F N O (M + H ) = 326.0865, found = 326.0867.
13
11
3
5
2
3ꢀAminoꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)picolinohydrazide (S3d). White solid, yield 90%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.70 (s, 1H), 10.39 (s, 1H), 8.26 (s, 0H), 8.22 (d, J = 7.7 Hz,
6
0
H), 7.99 (d, J = 7.7 Hz, 0H), 7.86 (dd, J = 4.2, 1.4 Hz, 1H), 7.79 (t, J = 7.8 Hz, 0H), 7.31 (dd, J
1
3
= 8.5, 4.2 Hz, 1H), 7.22 (dd, J = 8.5, 1.5 Hz, 1H), 6.83 (s, 2H). C NMR (101 MHz, DMSOꢀd )
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δ 166.98, 164.59, 147.06, 136.30, 134.01, 132.06, 130.37, 129.72 (q, J = 32.2 Hz), 128.84 (q, J =
+
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.7 Hz), 128.23, 128.05, 125.06, 124.51 (q, J = 3.8 Hz), 124.39 (q, J = 272.5 Hz); HRMS (ESI ),
+
calcd C H F N O (M + H ) = 325.0912, found = 325.0912.
1
4
12
3
4
2
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
N'ꢀ(3ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide (S3e). Lightꢀyellow solid, yield
1
9
5%. H NMR (400 MHz, DMSOꢀd ) δ 10.97 (s, 2H), 9.23 (s, 1H), 8.95 (s, 1H), 8.82 (s, 1H),
6
1
3
8
.27 – 8.23 (m, 2H), 8.00 (d, J = 7.3 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H); C NMR (101 MHz,
DMSOꢀd ) δ 164.53, 162.71, 148.55, 144.59, 144.21, 144.12, 133.81, 132.08, 130.44, 129.62 (q,
6
J = 32.2 Hz), 128.97 (q, J = 3.7 Hz), 124.51 (q, J = 4.0 Hz), 124.36 (q, J = 272.5 Hz); HRMS
+
+
(
ESI ), calcd C H F N O (M + H ) = 311.0756, found = 311.0755.
13 10 3 4 2
2ꢀAminoꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)nicotinohydrazide (S3f).White solid, yield 98%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.76 (s, 1H), 10.53 (s, 1H), 8.26 – 8.23 (m, 2H), 8.16 (d, J =
6
3
.5 Hz, 1H), 8.04 – 8.00 (m, 2H), 7.81 (t, J = 7.8 Hz, 1H), 7.08 (s, 2H), 6.65 (dd, J = 7.7, 4.8 Hz,
1
3
1H); C NMR (101 MHz, DMSOꢀd ) δ 167.57, 165.04, 159.26, 152.61, 137.12, 133.77, 132.04,
6
1
2
3
30.48, 129.82 (q, J = 32.1 Hz), 129.02 (q, J = 3.6 Hz), 124.48 (q, J = 3.8 Hz), 124.35 (q, J =
+
+
72.5 Hz), 111.87, 107.82; HRMS (ESI ), calcd C H F N O (M + H ) = 325.0912, found =
14
12
3
4
2
25.0916.
3
ꢀChloroꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide (S3g). Yellow solid,
1
yield 97%. H NMR (400 MHz, DMSOꢀd ) δ 11.00 (s, 2H), 8.80 (d, J = 2.4 Hz, 1H), 8.73 (d, J =
6
2
.4 Hz, 1H), 8.29 (s, 1H), 8.25 (d, J = 7.9 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.81 (t, J = 7.8 Hz,
1
3
1H); C NMR (101 MHz, DMSOꢀd ) δ 164.34, 162.90, 146.78, 146.47, 146.03, 143.06, 133.59,
6
1
32.14, 130.45, 129.79 (d, J = 32.2 Hz), 129.05 (q, J = 3.4 Hz), 124.57 (q, J = 3.6 Hz), 124.36
+
+
(
d, J = 272.6 Hz); HRMS (ESI ), calcd C H ClF N O (M + H ) = 345.0366, found = 345.0363.
13 9 3 4 2
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ꢀHydroxyꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)picolinohydrazide (S3h). White solid, yield
1
9
3%. H NMR (400 MHz, DMSOꢀd ) δ 11.85 (br s, 1H), 11.15 (br s, 2H), 8.26 – 8.22 (m, 3H),
6
8.00 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.61 (dd, J = 8.5, 4.3 Hz, 1H), 7.49 (dd, J =
1
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
8
.5, 1.2 Hz, 1H); C NMR (101 MHz, DMSOꢀd ) δ 168.23, 164.47, 157.68, 140.67, 133.67,
6
1
32.09, 131.04, 130.47, 130.04, 129.80 (q, J = 32.2 Hz), 129.04 (d, J = 3.5 Hz), 126.57, 124.51
+
+
(
q, J = 3.7 Hz), 124.34 (q, J = 272.5 Hz); HRMS (ESI ), calcd C H F N O (M + H ) =
14 11 3 3 3
3
26.0753, found = 326.0748.
N'ꢀ(3ꢀ(trifluoromethyl)benzoyl)picolinohydrazide (S3i). White solid, yield 99%. H NMR
400 MHz, DMSOꢀd ) δ 10.87 (s, 1H), 10.79 (s, 1H), 8.72 (ddd, J = 4.8, 1.6, 1.0 Hz, 1H), 8.27
1
(
6
(s, 1H), 8.24 (d, J = 7.9 Hz, 1H), 8.10 – 8.03 (m, 2H), 8.00 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.8
13
Hz, 1H), 7.68 (ddd, J = 6.9, 4.8, 1.9 Hz, 1H); C NMR (101 MHz, DMSOꢀd ) δ 164.49, 163.69,
6
1
49.57, 149.18, 138.36, 133.84, 132.08, 130.42, 129.76 (q, J = 32.1 Hz), 128.94 (q, J = 3.4 Hz),
+
127.56, 124.52 (q, J = 3.8 Hz), 124.37 (q, J = 272.5 Hz), 122.90; HRMS (ESI ), calcd
+
C H F N O (M + H ) = 310.0803, found = 310.0802.
14
11
3
3
2
3
ꢀAminoꢀ6ꢀbromoꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide
(S3j).
1
Yellow solid, yield 99%. H NMR (400 MHz, DMSOꢀd ) δ 10.77 (s, 1H), 10.63 (s, 1H), 8.44 (s,
6
13
1
H), 8.24 – 8.21 (m, 2H), 8.00 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.69 (s, 2H); C
NMR (101 MHz, DMSOꢀd ) δ 164.73, 164.71, 154.64, 150.05, 133.80, 132.08, 130.46, 129.78
6
(
q, J = 32.2 Hz), 128.99 (d, J = 3.7 Hz), 124.48 (q, J = 4.2 Hz), 124.35 (q, J = 272.5 Hz), 122.24;
+
+
HRMS (ESI ), calcd C H BrF N O (M + H ) = 403.9970, found = 403.9966.
1
3
10
3
5
2
3
ꢀAminoꢀ6ꢀchloroꢀN'ꢀ(3ꢀ(trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide
(S3k).
1
Lightꢀyellow solid, yield 94%. H NMR (400 MHz, DMSOꢀd ) δ 10.78 (s, 1H), 10.64 (s, 1H),
6
8.41 (s, 1H), 8.25 – 8.21 (m, 2H), 8.00 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.69 (s, 2H);
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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
13
C NMR (101 MHz, DMSOꢀd ) δ 164.73, 164.68, 154.47, 147.50, 133.80, 132.38, 132.08,
6
1
30.45, 129.78 (q, J = 32.1 Hz), 128.98 (q, J = 3.5 Hz), 124.48 (q, J = 3.8 Hz), 124.35 (q, J =
+
+
272.5 Hz), 123.09; HRMS (ESI ), calcd C H ClF N O (M + H ) = 360.0475, found =
1
3
10
3
5
2
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
60.0475.
Synthesis
of
Nꢀ(4ꢀoxoꢀ2ꢀpropylꢀ1,4ꢀdihydropteridinꢀ3(2H)ꢀyl)ꢀ3ꢀ
mixture of 3ꢀaminoꢀN'ꢀ(3ꢀ
trifluoromethyl)benzoyl)pyrazineꢀ2ꢀcarbohydrazide S3a (100 mg, 0.31 mmol), butyraldehyde
(
trifluoromethyl)benzamide
(S4).
A
(
S5 (0.054 mL, 0.61 mmol) and a catalytic amount of pꢀtoluenesulfonic acid monohydrate in
dioxane (10 mL) was refluxed for 2 h. The reaction mixture was concentrated and extracted with
ethyl acetate. The combined organic layers were dried with Na SO , filtered, concentrated, and
2
4
purified by flash column chromatography to give the title compound S4 (57 mg, 49%) as a lightꢀ
1
yellow solid. H NMR (400 MHz, DMSOꢀd ) δ 11.19 (s, 1H), 8.41 (d, J = 2.6 Hz, 1H), 8.27 –
6
8.22 (m, 3H), 8.04 – 8.02 (m, 2H), 7.82 (t, J = 7.8 Hz, 1H), 5.13 (q, J = 5.0, 4.2 Hz, 1H), 1.82 –
1
3
1
.77 (m, 2H), 1.54ꢀ1.30 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H); C NMR (101 MHz, DMSOꢀd ) δ
6
1
64.60, 160.16, 154.40, 147.70, 135.20, 133.30, 132.35, 130.50, 129.78 (q, J = 32.2 Hz), 129.30
(q, J = 3.3 Hz), 126.65, 124.68 (q, J = 3.8 Hz), 124.32 (q, J = 272.5 Hz), 70.99, 36.61, 16.71,
+
+
1
4.15; HRMS (ESI ), calcd C H F N O (M + H ) = 380.1334, found = 380.1333.
17 17 3 5 2
Preparation of Nꢀhexylꢀ4ꢀ(hydrazinecarbonyl)benzamide (S7l). A mixture of 4ꢀ
hexylcarbamoyl)benzoic acid S9l (300 mg, 1.20 mmol), hydrazine (0.38 mL, 12.03 mmol),
(
HOBt (163 mg, 1.20 mmol), and EDCI (277 mg, 1.44 mmol) in DMF (20 mL) and stirred at
room temperature overnight. The reaction mixture was poured into water and extracted with
ethyl acetate. The combined organic layers were dried with Na SO , filtered, concentrated, and
2
4
purified by flash column chromatography to give the title compound S7l (291 mg, 92%) as a
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white solid. H NMR (400 MHz, DMSOꢀd ) δ 9.91 (s, 1H), 8.56 (s, 1H), 7.88 (s, 4H), 4.56 (br s,
6
+
2
H), 3.31 – 3.19 (m, 2H), 1.52 (m, 2H), 1.36 – 1.22 (m, 6H), 0.87 (t, J = 8.0 Hz, 3H); MS (ESI )
+
m/z 264.2 [M+H] .
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
Preparation of 4ꢀ(hexylcarbamoyl)benzoic acid (S9l). 1ꢀ(3ꢀDimethylaminopropyl)ꢀ3ꢀ
ethylcarbodiimide hydrochloride (EDCI, 415 mg, 2.17 mmol) and N,Nꢀdiisopropylethylamine
(
DIPEA, 0.75 mL, 4.51 mmol) were added to a solution of terephthalic acid S8 (300 mg, 1.81
mmol), hexylamine (0.24 mL, 1.81 mmol) and 1ꢀhydroxybenzotriazole (HOBt, 293 mg, 2.17
mmol) in DMF (20 mL) and stirred at room temperature overnight. The reaction mixture was
poured into water and extracted with ethyl acetate. The combined organic layers were dried with
Na SO , filtered, concentrated, and purified by flash column chromatography to give the title
2
4
1
compound S9l (427 mg, 95%) as a white solid. H NMR (400 MHz, DMSOꢀd6) δ 13.46 (br s,
1
H), 8.62 (t, J = 5.6 Hz, 1H), 8.03 – 7.98 (m, 2H), 7.95 – 7.90 (m, 2H), 3.26 (td, J = 7.1, 5.6 Hz,
+
2H), 1.57 – 1.45 (m, 2H), 1.35 – 1.22 (m, 6H), 0.86 (t, J = 8.0 Hz, 3H); MS (ESI ) m/z 250.1
+
[
M+H] .
3
ꢀAminoꢀN'ꢀ(4ꢀchlorobenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10a). Yellow solid, yield 87%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.58 – 10.56 (m, 2H), 8.29 (d, J = 1.9 Hz, 1H), 7.95 – 7.90
6
13
(
m, 3H), 7.61 (d, J = 8.4 Hz, 2H), 7.51 (br s, 2H); C NMR (101 MHz, DMSO) δ 165.72,
+
1
65.01, 155.57, 147.88, 137.14, 131.74, 131.65, 129.85, 129.10, 125.10; HRMS (ESI ), calcd
+
C H ClN O (M + H ) = 292.0601, found = 292.0608.
12
11
5
2
3ꢀAminoꢀN'ꢀ(4ꢀbromobenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10b). Yellow solid, yield 86%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.59 – 10.57 (m, 2H), 8.28 (d, J = 2.1 Hz, 1H), 7.90 – 7.85
6
13
(
m, 3H), 7.75 (d, J = 8.4 Hz, 2H), 7.51 (br s, 2H); C NMR (101 MHz, DMSO) δ 165.71,
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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
+
1
65.16, 155.57, 147.89, 132.09, 132.04, 131.66, 130.03, 126.11, 125.08; HRMS (ESI ), calcd
+
C H BrN O (M + H ) = 336.0096, found = 336.0101.
12
11
5
2
3ꢀAminoꢀN'ꢀ(2,6ꢀdichlorobenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10c). Lightꢀyellow solid,
1
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
yield 93%. H NMR (400 MHz, DMSOꢀd ) δ 10.78 (s, 1H), 10.64 (s, 1H), 8.29 (d, J = 2.3 Hz,
6
13
1
H), 7.90 (d, J = 2.3 Hz, 1H), 7.56 – 7.47 (m, 5H); C NMR (101 MHz, DMSO) δ 165.03,
+
1
62.72, 155.53, 147.89, 134.99, 132.33, 132.08, 131.61, 128.65, 125.03; HRMS (ESI ), calcd
+
C H Cl N O (M + H ) = 326.0212, found = 326.0212.
12
10
2
5
2
3
ꢀAminoꢀN'ꢀ(2ꢀchloroꢀ4ꢀnitrobenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10d). Yellow solid,
1
yield 82%. H NMR (400 MHz, DMSOꢀd ) δ 10.72 – 10.69 (m, 2H), 8.40 – 8.29 (m, 3H), 7.90
6
1
3
(d, J = 2.2 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.53 (br s, 2H); C NMR (101 MHz, DMSO) δ
1
65.46, 164.50, 155.57, 149.12, 148.01, 140.76, 132.11, 131.70, 131.04, 125.27, 124.85, 122.90;
+
+
HRMS (ESI ), calcd C H ClN O (M + H ) = 337.0452, found = 337.0450.
1
2
10
6
4
3ꢀAminoꢀN'ꢀ(2,4ꢀdifluorobenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10e). Yellow solid, yield
1
9
7
1
9%. H NMR (400 MHz, DMSOꢀd ) δ 10.61 (s, 1H), 10.32 (s, 1H), 8.28 (d, J = 2.3 Hz, 1H),
6
.89 (d, J = 2.3 Hz, 1H), 7.74 (q, J = 8.2 Hz, 1H), 7.52 – 7.39 (m, 3H), 7.24 (td, J = 8.5, 2.0 Hz,
1
3
H); C NMR (101 MHz, DMSOꢀd ) δ 165.48 , 164.19 (d, J = 238.6 Hz), 162.85 (d, J = 2.1
6
Hz), 160.42 (dd, J = 253.8, 12.8 Hz), 155.57 , 147.90 , 132.44 (d, J = 10.5 Hz), 131.66 , 125.02 ,
+
1
19.37 (d, J = 18.4 Hz), 112.39 (d, J = 25.2 Hz), 105.31 (t, J = 26.2 Hz); HRMS (ESI ), calcd
+
C H F N O (M + H ) = 294.0803, found = 294.0803.
12
10
2
5
2
3ꢀAminoꢀN'ꢀ(4ꢀcyanobenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10f). Lightꢀyellow solid, yield
1
9
0%. H NMR (400 MHz, DMSOꢀd ) δ 10.75 (s, 1H), 10.67 (s, 1H), 8.29 (d, J = 2.3 Hz, 1H),
6
1
3
8
.07 – 8.04 (m, 4H), 7.90 (d, J = 2.3 Hz, 1H), 7.53 (br s, 2H); C NMR (101 MHz, DMSO) δ
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65.70, 164.70, 155.58, 147.96, 136.94, 133.12, 131.68, 128.75, 124.97, 118.69, 114.69; HRMS
+
+
(ESI ), calcd C H N O (M + H ) = 283.0943, found = 283.0943.
13 11 6 2
3ꢀAminoꢀN'ꢀ(4ꢀmethoxybenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10g). Yellow solid, yield
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
9
9%. H NMR (400 MHz, DMSOꢀd ) δ 10.48 (s, 1H), 10.32 (s, 1H), 8.28 (d, J = 2.3 Hz, 1H),
6
13
8
.00 – 7.75 (m, 3H), 7.50 (br s, 2H), 7.05 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H); C NMR (101 MHz,
DMSO) δ 165.78, 165.53, 162.45, 155.55, 147.78, 131.64, 129.84, 125.28, 125.13, 114.15,
+
+
5
5.86; HRMS (ESI ), calcd C H N O (M + H ) = 288.1097, found = 288.1095.
13 14 5 3
3
ꢀAminoꢀN'ꢀ(4ꢀhydroxybenzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10h). Yellow solid, yield
1
7
2%. H NMR (400 MHz, DMSOꢀd ) δ 10.44 (s, 1H), 10.20 (s, 1H), 10.13 (s, 1H), 8.28 (s, 1H),
6
1
3
7.89 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.51 (br s, 2H), 6.85 (d, J = 8.4 Hz, 2H); C NMR (101
MHz, DMSO) δ 165.77, 165.70, 161.08, 155.54, 147.75, 131.61, 129.96, 125.33, 123.59,
+
+
1
15.43; HRMS (ESI ), calcd C H N O (M + H ) = 274.0940, found = 274.0943.
12 12 5 3
3ꢀAminoꢀN'ꢀ(4ꢀ(trifluoromethoxy)benzoyl)pyrazineꢀ2ꢀcarbohydrazide (S10i). Yellow
1
solid, yield 95%. H NMR (400 MHz, DMSOꢀd ) δ 10.62 (s, 2H), 8.29 (d, J = 2.3 Hz, 1H), 8.04
6
13
(d, J = 8.8 Hz, 2H), 7.90 (d, J = 2.3 Hz, 1H), 7.55ꢀ7.53 (m, 4H); C NMR (101 MHz, DMSOꢀ
d6) δ 165.72, 164.85, 155.58, 151.12 (q, J = 1.7 Hz), 147.90, 132.07, 131.66, 130.32, 125.07,
+
+
1
21.28, 120.42 (d, J = 257.3 Hz); HRMS (ESI ), calcd C H F N O (M + H ) = 342.0814,
13 11 3 5 3
found = 342.0814.
Methyl 4ꢀ(2ꢀ(3ꢀaminopyrazineꢀ2ꢀcarbonyl)hydrazineꢀ1ꢀcarbonyl)benzoate (S10j). Yellow
1
solid, yield 70%. H NMR (400 MHz, DMSOꢀd ) δ 10.67 (s, 1H), 10.64 (s, 1H), 8.29 (d, J = 2.2
6
Hz, 1H), 8.10 (d, J = 8.3 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 2.2 Hz, 1H), 7.53 (br s,
1
3
2
H), 3.90 (s, 3H); C NMR (101 MHz, DMSO) δ 166.10, 165.70, 165.27, 155.58, 147.92,
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
5
5
5
6
+
1
37.10, 132.77, 131.66, 129.77, 128.37, 125.05, 52.91; HRMS (ESI ), calcd C H N O (M +
14 14 5 4
+
H ) = 316.1046, found = 316.1046.
1
3ꢀAminoꢀN'ꢀnicotinoylpyrazineꢀ2ꢀcarbohydrazide (S10k). White solid, yield 90%. H NMR
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
(
400 MHz, DMSOꢀd ) δ 10.70ꢀ10.66 (m, 2H), 9.07 (dd, J = 2.2, 0.8 Hz, 1H), 8.78 (dd, J = 4.8,
6
1
1
1
.7 Hz, 1H), 8.29 (d, J = 2.3 Hz, 1H), 8.26 (ddd, J = 8.0, 2.3, 1.7 Hz, 1H), 7.90 (d, J = 2.3 Hz,
1
3
H), 7.60 – 7.53 (m, 3H); C NMR (101 MHz, DMSO) δ 165.73, 164.65, 155.58, 152.98,
+
48.90, 147.95, 135.69, 131.68, 128.65, 125.00, 124.16; HRMS (ESI ), calcd C H N O (M +
1
1
11
6
2
+
H ) = 259.0943, found = 259.0939.
4
ꢀ(2ꢀ(3ꢀAminopyrazineꢀ2ꢀcarbonyl)hydrazineꢀ1ꢀcarbonyl)ꢀNꢀhexylbenzamide
(S10l).
1
Lightꢀyellow solid, yield 78% from compound S8. H NMR (400 MHz, DMSOꢀd ) δ 10.61ꢀ
6
1
0.58 (m, 2H), 8.62 (t, J = 5.6 Hz, 1H), 8.29 (d, J = 2.3 Hz, 1H), 8.00 – 7.94 (m, 4H), 7.90 (d, J
2.2 Hz, 1H), 7.52 (br s, 2H), 3.27 (q, J = 8.2, 7.4 Hz, 2H), 1.55 – 1.52 (m, 2H), 1.30 (s, 6H),
=
1
3
0.88 (t, J = 6.5 Hz, 3H); C NMR (101 MHz, DMSO) δ 165.78, 165.72, 165.43, 155.58, 147.89,
37.97, 135.04, 131.66, 127.90, 127.73, 125.11, 31.49, 29.47, 26.64, 22.53, 14.40; HRMS
1
+
+
(ESI ), calcd C H N O (M + H ) = 385.1988, found = 385.1989.
19 25 6 3
1
N'ꢀ(4ꢀ(trifluoromethoxy)benzoyl)picolinohydrazide (S11a). White solid, yield 99%. H
NMR (400 MHz, DMSOꢀd ) δ 10.73 (s, 1H), 10.70 (s, 1H), 8.72 (d, J = 4.6 Hz, 1H), 8.08 – 8.03
6
13
(
m, 4H), 7.70 – 7.66 (m, 1H), 7.54 (d, J = 8.2 Hz, 2H); C NMR (101 MHz, DMSOꢀd ) δ
6
1
64.72 , 163.71 , 151.12 (d, J = 1.9 Hz), 149.63 , 149.17 , 138.35 , 132.08 , 130.34 , 127.53 ,
+
+
122.88 , 121.27 , 120.42 (q, J = 257.2 Hz); HRMS (ESI ), calcd C H F N O (M + H ) =
14
11
3
3
3
3
26.0753, found = 326.0753.
N'ꢀ(4ꢀ(trifluoromethyl)benzoyl)picolinohydrazide (S11b). White solid, yield 97%. H NMR
1
(400 MHz, DMSOꢀd ) δ 10.84 (s, 1H), 10.78 (s, 1H), 8.72 (d, J = 4.5 Hz, 1H), 8.14 – 8.03 (m,
6
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H), 7.93 (d, J = 8.1 Hz, 2H), 7.68 (ddd, J = 6.9, 4.8, 1.8 Hz, 1H); C NMR (101 MHz, DMSOꢀ
d6) δ 164.83, 163.69, 149.59, 149.17, 138.34, 136.80 (d, J = 1.5 Hz), 132.13 (q, J = 31.9 Hz),
+
128.89, 127.54, 126.02 (q, J = 3.7 Hz), 124.32 (q, J = 272.6 Hz), 122.89; HRMS (ESI ), calcd
+
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
C H F N O (M + H ) = 310.0803, found = 310.0806.
14
11
3
3
2
1
N'ꢀ(4ꢀbromobenzoyl)picolinohydrazide (S11c). White solid, yield 97%. H NMR (400 MHz,
DMSOꢀd ) δ 10.71 (s, 1H), 10.67 (s, 1H), 8.72 – 8.71 (m, 1H), 8.09 – 8.02 (m, 2H), 7.88 (d, J =
6
1
3
8
.5 Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H), 7.67 (ddd, J = 6.9, 4.8, 2.1 Hz, 1H); C NMR (101 MHz,
DMSO) δ 165.03, 163.69, 149.64, 149.16, 138.35, 132.10, 132.04, 130.05, 127.52, 126.12,
+
+
1
22.87; HRMS (ESI ), calcd C H BrN O (M + H ) = 320.0035, found = 320.0038.
13 11 3 2
1
N'ꢀ(4ꢀchlorobenzoyl)picolinohydrazide (S11d). White solid, yield 91%. H NMR (400 MHz,
DMSOꢀd ) δ 10.69 (s, 1H), 10.66 (s, 1H), 8.72 – 8.71 (m, 1H), 8.09 – 8.03 (m, 2H), 7.95 (d, J =
6
1
3
8
.6 Hz, 2H), 7.67 (ddd, J = 6.9, 4.8, 2.2 Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H); C NMR (101 MHz,
DMSO) δ 164.91, 163.71, 149.64, 149.16, 138.34, 137.15, 131.74, 129.88, 129.09, 127.51,
+
+
1
9
22.87; HRMS (ESI ), calcd C H ClN O (M + H ) = 276.0540, found = 276.0544.
13 11 3 2
3
ꢀHydroxyꢀN'ꢀ(4ꢀ(trifluoromethoxy)benzoyl)picolinohydrazide (S11e). White solid, yield
1
9%. H NMR (400 MHz, DMSOꢀd ) δ 11.90 – 10.75 (m, 3H), 8.24 (d, J = 4.2 Hz, 1H), 8.06 (d,
6
1
3
J = 8.3 Hz, 2H), 7.62 – 7.48 (m, 4H); C NMR (101 MHz, DMSOꢀd ) δ 168.31, 164.69, 157.69,
6
1
51.20, 140.64, 131.85, 131.07, 130.37, 130.01, 126.55, 121.30, 120.41 (q, J = 257.3 Hz);
+
+
HRMS (ESI ), calcd C H F N O (M + H ) = 342.0702, found = 342.0704.
1
4
11
3
3
4
3ꢀHydroxyꢀN'ꢀ(4ꢀ(trifluoromethyl)benzoyl)picolinohydrazide (S11f). Lightꢀyellow solid,
1
yield 83%. H NMR (400 MHz, DMSOꢀd ) δ 11.90 – 11.15 (m, 3H), 8.25 (s, 1H), 8.13 (d, J =
6
1
3
7
.3 Hz, 2H), 7.94 (d, J = 7.8 Hz, 2H), 7.60 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H); C NMR (101
MHz, DMSOꢀd ) δ 168.22, 164.75, 157.69, 140.65, 136.61, 132.20 (q, J = 31.0 Hz), 131.07,
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3
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5
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6
+
1
30.02, 128.90, 126.57, 126.08 (d, J = 3.5 Hz), 124.32 (q, J = 272.5 Hz); HRMS (ESI ), calcd
+
C H F N O (M + H ) = 326.0753, found = 326.0760.
14
11
3
3
3
N'ꢀ(4ꢀbromobenzoyl)ꢀ3ꢀhydroxypicolinohydrazide (S11g). Lightꢀyellow solid, yield 87%.
1
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
H NMR (400 MHz, DMSOꢀd ) δ 11.14 (br s, 3H), 8.23 (d, J = 3.4 Hz, 1H), 7.87 (d, J = 8.2 Hz,
6
13
2
H), 7.76 (d, J = 8.1 Hz, 2H), 7.59 (dd, J = 7.8, 4.1 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H); C NMR
(
101 MHz, DMSO) δ 168.20, 164.94, 157.74, 140.57, 132.70, 132.08, 131.97, 130.05, 127.51,
+
+
1
26.55, 126.20; HRMS (ESI ), calcd C H BrN O (M + H ) = 335.9984, found = 335.9985.
13 11 3 3
N'ꢀ(4ꢀchlorobenzoyl)ꢀ3ꢀhydroxypicolinohydrazide (S11h). Lightꢀyellow solid, yield 82%.
1
H NMR (400 MHz, DMSOꢀd ) δ 11.87 – 11.06 (m, 3H), 8.24 (dd, J = 4.4, 1.4 Hz, 1H), 7.95 (d,
6
1
3
J = 8.6 Hz, 2H), 7.64 – 7.59 (m, 3H), 7.49 (dd, J = 8.5, 1.4 Hz, 1H); C NMR (101 MHz,
DMSO) δ 168.29, 164.85, 157.69, 140.64, 137.27, 131.53, 131.08, 130.01, 129.89, 129.16,
+
+
1
26.55; HRMS (ESI ), calcd C H ClN O (M + H ) = 292.0489, found = 292.0490.
13 11 3 3
General Procedure for the Preparation of Compounds Z3aꢀf, Z5aꢀn, Z7aꢀd, and Z9aꢀo
Exemplified by N'ꢀ(4ꢀchlorobenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z3a). Oꢀ
benzotriazolꢀ1ꢀylꢀN,N,N',N'ꢀtetramethyluronium tetrafluoroborate (TBTU, 336 mg, 1.05 mmol)
and triethylamine (0.30 mL, 2.10 mmol) were added to a solution of 2ꢀmethylꢀ1,3ꢀthiazoleꢀ4ꢀ
carboxylic acid (100 mg, 0.70 mmol) and 4ꢀchlorobenzeneꢀ1ꢀcarbohydrazide (143 mg, 0.84
mmol) in DMF (10 mL) and the resulting solution stirred at room temperature overnight. The
reaction mixture was poured into water and extracted with ethyl acetate. The combined organic
layers were dried with Na SO , filtered, concentrated, and purified by flash column
2
4
1
chromatography to give the title compound Z3a (196 mg, 95%) as a white solid. H NMR (400
MHz, DMSOꢀd ) δ 10.61 (s, 1H), 10.33 (s, 1H), 8.25 (s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.61 (d, J
6
1
3
= 8.6 Hz, 2H), 2.75 (s, 3H); C NMR (101 MHz, DMSO) δ 166.97, 164.90, 160.33, 148.22,
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+
+
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37.13, 131.73, 129.88, 129.07, 125.55, 19.28; HRMS (ESI ), calcd C H ClN O S (M + H ) =
1
2
11
3
2
2
96.0261, found = 296.0268.
N'ꢀ(4ꢀchlorobenzoyl)ꢀ2ꢀmethylthiazoleꢀ5ꢀcarbohydrazide (Z3b). White solid, yield 96%. H
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
NMR (400 MHz, DMSOꢀd ) δ 10.69 (s, 1H), 10.67 (s, 1H), 8.33 (s, 1H), 7.93 (d, J = 8.5 Hz,
6
13
2
1
H), 7.62 (d, J = 8.5 Hz, 2H), 2.72 (s, 3H); C NMR (101 MHz, DMSO) δ 170.93, 165.34,
+
60.09, 144.13, 137.32, 132.78, 131.49, 129.83, 129.18, 19.67; HRMS (ESI ), calcd
+
C H ClN O S (M + H ) = 296.0261, found = 296.0267.
12
11
3
2
1
N'ꢀ(4ꢀchlorobenzoyl)thiazoleꢀ2ꢀcarbohydrazide (Z3c). White solid, yield 73%. H NMR
(
400 MHz, DMSOꢀd ) δ 10.86 (s, 1H), 10.73 (s, 1H), 8.15 (d, J = 3.0 Hz, 1H), 8.11 (d, J = 3.0
6
13
Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H); C NMR (101 MHz, DMSO) δ
+
1
64.98, 162.26, 159.12, 144.76, 137.28, 131.51, 129.87, 129.16, 126.68; HRMS (ESI ), calcd
+
C H ClN O S (M + H ) = 282.0104, found = 282.0109.
11
9
3
2
1
N'ꢀ(4ꢀchlorobenzoyl)ꢀ1Hꢀindoleꢀ2ꢀcarbohydrazide (Z3d). White solid, yield 80%. H NMR
(
400 MHz, DMSOꢀd ) δ 11.75 (s, 1H), 10.65 (s, 1H), 10.58 (s, 1H), 7.97 (d, J = 8.5 Hz, 2H),
6
7
.68ꢀ7.63 (m, 3H), 7.47 (d, J = 8.2 Hz, 1H), 7.28 (s, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.08 (t, J = 7.5
1
3
Hz, 1H); C NMR (101 MHz, DMSO) δ 165.37, 161.26, 137.26, 137.11, 131.68, 129.91,
+
1
29.86, 129.18, 127.44, 124.21, 122.22, 120.41, 112.85, 103.95; HRMS (ESI ), calcd
+
C H ClN O (M + H ) = 314.0696, found = 314.0705.
16
13
3
2
3
ꢀChloroꢀN'ꢀ(4ꢀchlorobenzoyl)ꢀ6ꢀmethylbenzo[b]thiopheneꢀ2ꢀcarbohydrazide
(Z3e).
1
White solid, yield 97%. H NMR (400 MHz, DMSOꢀd ) δ 10.78 (s, 1H), 10.52 (s, 1H), 7.97 –
6
7
.94 (m, 3H), 7.82 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.3 Hz, 1H), 2.49 (s,
1
3
3
H); C NMR (101 MHz, DMSO) δ 164.99, 160.56, 138.45, 137.60, 137.34, 134.28, 131.49,
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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
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5
5
5
5
5
5
5
5
6
+
1
29.88, 129.18, 128.46, 128.29, 123.38, 122.85, 121.24, 21.70; HRMS (ESI ), calcd
+
C H Cl N O S (M + H ) = 379.0075, found = 379.0075.
17
13
2
2
2
N'ꢀ(4ꢀchlorobenzoyl)ꢀ3ꢀisopropylꢀ1Hꢀpyrazoleꢀ5ꢀcarbohydrazide (Z3f). Lightꢀyellow solid,
1
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
yield 92%. H NMR (400 MHz, DMSOꢀd ) δ 13.11 (s, 1H), 10.50 (s, 1H), 10.05 (s, 1H), 7.93 (d,
6
J = 8.5 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H), 6.49 (s, 1H), 2.99 (dq, J = 11.2, 5.8, 4.6 Hz, 1H), 1.25
1
3
(
d, J = 6.9 Hz, 6H); C NMR (101 MHz, DMSO) δ 165.09, 161.86, 151.22, 145.35, 137.05,
+
+
1
3
31.86, 129.84, 129.05, 102.38, 25.54, 22.76; HRMS (ESI ), calcd C H ClN O (M + H ) =
14 16 4 2
07.0962, found = 307.0966.
N'ꢀ(2ꢀchlorobenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z5a). White solid, yield 94%. H
1
NMR (400 MHz, DMSOꢀd ) δ 10.44 (s, 1H), 10.34 (s, 1H), 8.25 (s, 1H), 7.57 – 7.43 (m, 4H),
6
13
2
.75 (s, 3H); C NMR (101 MHz, DMSO) δ 166.90, 165.76, 160.06, 148.17, 135.02, 131.93,
+
+
1
31.00, 130.36, 129.93, 127.54, 125.52, 19.28; HRMS (ESI ), calcd C H ClN O S (M + H ) =
12 11 3 2
296.0261, found = 296.0266.
1
N'ꢀ(3ꢀchlorobenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z5b). White solid, yield 90%. H
NMR (400 MHz, DMSOꢀd ) δ 10.65 (s, 1H), 10.36 (s, 1H), 8.25 (s, 1H), 7.95 (t, J = 1.9 Hz, 1H),
6
13
7
.88 (d, J = 7.8 Hz, 1H), 7.70 – 7.67 (m, 1H), 7.57 (t, J = 7.9 Hz, 1H), 2.75 (s, 3H); C NMR
(
101 MHz, DMSO) δ 166.99, 164.53, 160.29, 148.17, 134.93, 133.78, 132.17, 131.04, 127.74,
+
+
1
26.69, 125.61, 19.28; HRMS (ESI ), calcd C H ClN O S (M + H ) = 296.0261, found =
12 11 3 2
2
96.0264.
N'ꢀ(3ꢀcyanobenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z5c). White solid, yield 99%. H
NMR (400 MHz, DMSOꢀd ) δ 10.76 (s, 1H), 10.43 (s, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 8.22 (d, J
1
6
1
3
=
8.0 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 2.75 (s, 3H); C NMR (101
MHz, DMSO) δ 167.04, 164.13, 160.28, 148.08, 135.81, 133.95, 132.66, 131.54, 130.49, 125.72,
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+
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18.64, 112.20, 19.28; HRMS (ESI ), calcd C H N O S (M + H ) = 287.0603, found =
1
3
11
4
2
2
87.0605.
Methyl 3ꢀ(2ꢀ(2ꢀmethylthiazoleꢀ4ꢀcarbonyl)hydrazineꢀ1ꢀcarbonyl)benzoate (Z5d). White
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
solid, yield 70%. H NMR (400 MHz, DMSOꢀd ) δ 10.75 (s, 1H), 10.38 (s, 1H), 8.52 (t, J = 1.6
6
Hz, 1H), 8.26 (s, 1H), 8.17 (dt, J = 7.9, 1.6 Hz, 2H), 7.70 (t, J = 7.8 Hz, 1H), 3.91 (s, 3H), 2.76
1
3
(
s, 3H); C NMR (101 MHz, DMSO) δ 166.99, 166.15, 165.05, 160.29, 148.20, 133.54, 132.73,
+
1
32.56, 130.39, 129.68, 128.72, 125.62, 52.89, 19.29; HRMS (ESI ), calcd C H N O S (M +
1
4
14
3
4
+
H ) = 320.0705, found = 320.0705.
1
N'ꢀ(3ꢀfluorobenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z5e). White solid, yield 97%. H
NMR (400 MHz, DMSOꢀd ) δ 10.63 (s, 1H), 10.37 (s, 1H), 8.26 (s, 1H), 7.78 (d, J = 7.8 Hz,
6
1
H), 7.70 (d, J = 9.7 Hz, 1H), 7.59 (td, J = 7.9, 6.0 Hz, 1H), 7.47 (td, J = 8.4, 2.0 Hz, 1H), 2.75
1
3
(
s, 3H); C NMR (101 MHz, DMSOꢀd ) δ 166.99, 164.56 (d, J = 2.5 Hz), 162.39 (d, J = 244.6
6
Hz), 160.34 , 148.18, 135.22 (d, J = 6.9 Hz), 131.23 (d, J = 7.9 Hz), 125.62, 124.15 (d, J = 2.8
+
Hz), 119.27 (d, J = 21.0 Hz), 114.71 (d, J = 23.0 Hz), 19.28; HRMS (ESI ), calcd C H FN O S
1
2
11
3
2
+
(
M + H ) = 280.0556, found = 280.0558.
N'ꢀ(3ꢀbromobenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z5f). Lightꢀyellow solid, yield
1
9
9%. H NMR (400 MHz, DMSOꢀd ) δ 10.65 (s, 1H), 10.37 (s, 1H), 8.25 (s, 1H), 8.09 (t, J = 1.8
6
13
Hz, 1H), 7.93 – 7.90 (m, 1H), 7.83ꢀ7.80 (m, 1H), 7.51 (t, J = 7.9 Hz, 1H), 2.75 (s, 3H); C NMR
(
101 MHz, DMSO) δ 166.99, 164.44, 160.29, 148.16, 135.09, 135.07, 131.29, 130.61, 127.06,
+
+
125.64, 122.22, 19.29; HRMS (ESI ), calcd C H BrN O S (M + H ) = 339.9755, found =
1
2
11
3
2
3
39.9757.
N'ꢀ(3ꢀhydroxybenzoyl)ꢀ2ꢀmethylthiazoleꢀ4ꢀcarbohydrazide (Z5g). White solid, yield 75%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.40 (s, 1H), 10.25 (s, 1H), 9.76 (s, 1H), 8.24 (s, 1H), 7.93 –
6
ACS Paragon Plus Environment
29