Discovery of 3-(Indol-5-yl)-indazole Derivatives as Novel Myeloid Differentiation Protein 2/Toll-like Receptor 4 Antagonists for Treatment of Acute Lung Injury

Article abstract of DOI:10.1021/acs.jmedchem.9b00316

Acute lung injury (ALI) is often caused by systemic inflammatory responses. Targeting the myeloid differentiation protein 2/toll-like receptor 4 (MD2-TLR4) complex may be a promising way to treat Gram-negative bacterial-induced inflammatory disorders. In this study, we report the design and synthesis of a new series of 3-(indol-5-yl)-indazoles, which were evaluated for their anti-inflammatory activities in macrophages. Among the analogues generated, the promising 3-(indol-5-yl)-indazole analogue 22m inhibited lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in macrophages with IC₅₀ values of 0.89 and 0.53 μM, respectively. Compound 22m was then identified as an MD2-TLR4 antagonist in suppressing LPS-induced inflammatory responses. In vivo administration of 22m significantly inhibited macrophage infiltration and ameliorated histopathological changes in lung tissues of LPS-challenged mice. Our studies have identified a new 3-(indol-5-yl)-indazole, 22m, as a potent MD2-TLR4 inhibitor and lay the groundwork for future drug development of anti-inflammatory agents for the treatment of ALI.

Full text of DOI:10.1021/acs.jmedchem.9b00316

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Article
Discovery of 3-(indol-5-yl)-indazole derivatives as
novel myeloid differentiation protein 2/toll-like receptor
4
antagonists for treatment of acute lung injury
Zhiguo Liu, Lingfeng Chen, Pengtian Yu, Yali Zhang, Bo Fang,
Chao Wu, Wu Luo, Xianxin Chen, Chenglong Li, and Guang Liang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00316 • Publication Date (Web): 18 Apr 2019
Downloaded from http://pubs.acs.org on April 18, 2019
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Discovery of 3-(indol-5-yl)-indazole derivatives as novel myeloid
differentiation protein 2/toll-like receptor 4 antagonists for treatment of
acute lung injury
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a,#,
b,#
a,c
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Zhiguo Liu *, Lingfeng Chen , Pengtian Yu , Yali Zhang , Bo Fang , Chao Wu , Wu
a
a
a
a,b,
Luo , Xianxin Chen , Chenglong Li , Guang Liang *
a
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
b
School of Chemical Engineering, Nanjing University of Science and Technology,
Nanjing, Jiangsu, 210094, China
c
Department of Pharmacy, The First Affiliated Hospital, College of Medicine,
Zhejiang University, Zhejiang Hangzhou 310000, China
#
These authors contributed equally to this work.
*
Corresponding author:
Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical
University. 1210 University Town, Wenzhou, Zhejiang 325035, China
Tel: (+86)-577-86699892; Fax: (+86)-577-86699892.
E-mail: lzgcnu@163.com
Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical
University. 1210 University Town, Wenzhou, Zhejiang 325035, China
Tel: (+86)-577-86699396; Fax: (+86)-577-86699396.
E-mail: wzmcliangguang@163.com
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ABSTRACT
Acute lung injury (ALI) is often caused by systemic inflammatory responses. Targeting
myeloid differentiation protein 2/toll-like receptor 4 (MD2-TLR4) complex may be a
promising way to treat Gram-negative bacterial-induced inflammatory disorders. In this study,
we report the design and synthesis of a new series of 3-(indol-5-yl)-indazoles, which were
evaluated for their anti-inflammatory activities in macrophages. Among the analogues
generated, the promising 3-(indol-5-yl)-indazole analogue 22m inhibited lipopolysaccharides
(LPS)-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in
macrophages with IC values of 0.89 and 0.53 μM, respectively. Compound 22m was then
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identified as MD2-TLR4 antagonist in suppressing LPS-induced inflammatory responses. In
vivo administration of 22m significantly inhibited macrophage infiltration and ameliorated
histopathological changes in lung tissues of LPS-challenged mice. Our studies have identified
a new 3-(indol-5-yl)-indazole, 22m, as potent MD2-TLR4 inhibitor and lay the groundwork
for the future drug development of anti-inflammatory agents for the treatment of ALI.
Keywords: 3-(indol-5-yl)-indazole, acute lung injury, inflammation, myeloid differentiation
protein 2, toll-like receptor 4
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INTRODUCTION
Acute lung injury (ALI) and acute respiratory distress syndrome are characterized by
pulmonary infiltrates, hypoxemia, and injury to both the vascular endothelium and lung
alveolar epithelium.1, 2 ALI and acute respiratory distress syndrome commonly result from
sepsis, trauma, and acute pancreatitis. While systemic and direct lung injury may initiate the
process, expansion of inflammatory response is responsible for injury progression.
Macrophages account for approximately 95% of airspace leukocytes and are critically
involved in the development of ALI following infection and non-infectious stimuli by
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producing proinflammatory cytokines. Among these cytokines, interleukin-6 (IL-6) and
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tumor necrosis factor-α (TNF-α) contribute most to the deterioration seen in ALI. Elevated
levels of both IL-6 and TNF-α have been found in human and experimental ALI.5, 6
Experimentally, ALI has been produced in preclinical models through administration of
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bacterial endotoxin. Lipopolysaccharide (LPS), the main element of Gram-negative bacteria,
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plays an important role in acute inflammatory diseases such ALI and sepsis. Toll-like
receptor 4 (TLR4) is one of the pattern recognition receptors that recognizes
pathogen-associated molecular patterns, including LPS, lipooligosaccharide (LOS), and lipid
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A from bacteria. Studies have shown that LPS activates the TLR4 signaling pathway through
an indispensable accessory protein called the myeloid differentiation protein 2 (MD2). MD2
associates with TLR4 to form (LPS-MD2-TLR4) complex on cell membrane, leading to the
2
recruitment of adaptor protein, myeloid differentiating primary response gene 88 (MyD88).
The dimerization of MyD88 activates pro-inflammatory signal transduction through the
downstream intercellular kinase cascades and transcriptional factors, which up-regulate the
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expression of pro-inflammatory cytokines.10, 11 Therefore, targeting MD2-TLR4 complex may
be a promising way to treat ALI, sepsis, and other acute inflammatory diseases.12, 13
Several compounds with anti-inflammatory activities have been reported to target MD2 or
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TLR4 (Figure 1). Natural products such as 1 (Curcumin), 2 (Xanthohumol) and 3 (Caffeic
acid phenethyl ester) are reported as potential modulators of TLR4 signaling owing to their
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ability to interact with MD2. The triterpenoids celastrol and asiatic acid are also active in
disrupting TLR4 signaling, experimental binding studies showed these two compounds
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compete with LPS for MD-2 binding. However, these natural compounds may interact with
multiple targets in addition to MD2-TLR4. In addition, E. coli lipid A mimetics were
developed to modulate the interaction of LPS with MD2/TLR4 complex. A synthetic
phospholipid analogue 5 (PE-DTPA)18 improved the survival rate of LPS-challenged
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C57BL/6 mice in a dose-dependent manner. Cationic amphiphile (6, IAXO-102) based on
monosaccharide scaffolds efficiently inhibited TLR4 signaling by competition from LPS for
binding to MD-2. The most famous lipid A mimetic is tetra-acylated lipid A (7, Eritoran),
which directly binding to the hydrophobic pocket of MD2.20, 21 Compound 8 (TAK-242)
selectively inhibits TLR4 signal by irreversibly binding to the Cys thiol of TLR4, thereby
forming a covalent adduct with TLR4.22 Yin and co-workers23 also developed β-amino
alcohol derivative 4 targeting TLR4 as a potential antiseptic compound. However, among all
MD2-TLR4 antagonists so far developed, only antagonists 7 and 8 reached clinical trials, but
unfortunately, both failed to reduce mortality among patients with severe sepsis in Phase 3
studies.24, 25 Therefore, development of an effective MD2/TLR4 inhibitor with new structural
scaffold is highly desirable.
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Figure 1. Structures of natural and synthetic MD2/TLR4 antagonists 1-8.
Indole and indazole have wide biological applications and they are potentially valuable
as building-blocks in medicinal chemistry.26, 27 In recent years, some of the indazole and
indole derivatives, such as 9 (5-aminoindazole), 10 (benzidamine), and 11 (indomethacin),
have been applied to the treatment of acute or chronic inflammation as well as a variety of
inflammatory diseases (Figure 2).28-30 However, to the best of our knowledge, these drugs
share many of the potential adverse effects, including burning sensation, headache, itching,
3
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erythema, upset stomach, skin rash, or renal function disorders. As part of our ongoing
anti-inflammatory drug discovery activities, we are engaging to find lead compounds with
new structural skeleton for the treatment of ALI. Here, we envisioned that the introduction of
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an indole moiety to the 5-aminoindazole scaffold might yield a novel series of potent
anti-inflammatory agents. In addition, we found that a large number of natural and synthetic
compounds, such as 12 (LY2874455),33 13 (1,3,5-tris(2-hydroxyethyl)isocyanurate)34 and
FDA approved 14 (Dasatinib),35 with therapeutic effects on cancer and related diseases,
contain one N-hydroxyethyl group (Figure 2). Evidences suggest that the hydroxyethyl group
may contribute to the biological activity of these compounds, since it is the attachment point
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of the pyrophosphate radicle in cocarboxylase. We further try to see if linking hydroxyethyl
group at indole N-atom enhances the anti-inflammatory ability.
Hence, we report here the design, synthesis and anti-inflammatory evaluation of a series
of novel 3-(indol-5-yl)-indazole derivatives with or without hydroxyethyl group. Among the
tested compounds, compound 22m showed the highest anti-inflammatory activity in
macrophages. Further, we explored the potential mechanism and molecular target of 22m
through a series of biochemical assays. Interestingly, our data indicated that 22m directly
bound to MD2-TLR4 complex and disrupted MD2-TLR4 activation. 22m also showed potent
therapeutic effects in LPS-induced model of ALI. Our results provided a new lead compound,
2
2m, as potent MD2-TLR4 inhibitor for the treatment of ALI.
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Figure 2. Structures of 9-14 and drug design conception.
RESULTS AND DISCUSSION
The syntheses of 3-(indol-5-yl)-indazole derivatives (22a-22q) reported in this study was
performed as outlined in Schemes 1 and 2. Iodination of the commercially available
5-nitroindazole (15) with iodine in basic condition produced the 3-iodo-5-nitro-1H-indazole
(16). The subsequent nucleophilic substitutions placed 2-(trimethylsilyl) ethoxymethyl (SEM)
or benzyl substituent at N-1 on the indazole skeleton of 16, which were easily performed
under basic conditions due to its relatively acidic character, thus affording N-1 substituted
products 17a-17b. Furthermore, Suzuki coupling of 17a and 17b with (1H-indol-5-yl) boronic
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acid gave the key intermediates 18a and 18b, respectively.
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a
Scheme 1. General synthetic route of 3-(indol-5-yl)-indazole derivatives 22a-22l. Reagents
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and conditions: (a) KOH, I , DMF, rt; (b) NaH, SEMCl, DMF, 0 C to rt; or BnCl, K CO ,
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acetonitrile, reflux; (c) (1H-indol-5-yl)boronic acid, Na CO , PdCl(dppf), THF, reflux; (d)
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NaH, (2-bromoethoxy)-tert-butyldimethylsilane, DMF, 0 C to rt; (e) Fe, NH Cl,
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oC to rt ; (g) TBAF, EDA, THF, reflux.
N-alkylation of 18a with (2-bromoethoxy)-tert-butyldimethylsilane afforded
t-butyldimethylsilyl (TBS) protected product 19, which was reduced by catalytic iron and
ammonia chloride in ethanol (EtOH), yielding the corresponding 6-aminoindazole derivative
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0. Condensation of 20 with appropriate aliphatic or aromatic chloride (21a-21l), followed by
removing of SEM and TBS groups, afforded target compounds 22a-22l.
Scheme 2. General synthetic route of 3-(indol-5-yl)-indazole derivatives 22m, 22n, 22o and
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2p. Reagents and conditions: (a) Fe, NH Cl, C H OH/H O, reflux; (b) 2,6-dichlorobenzoyl
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chloride, DIPEA, CH Cl , 0 C to rt; (c) TBAF, EDA, THF, reflux.
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For the syntheses of compound 22m, key intermediate 18a was directly reduced to amine
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and then deprotection of SEM gave 22m (Scheme 2). 22n was also achieved with the same
synthetic procedure. Alternatively, hydrolysis of the SEM and TBS groups of intermediates
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9 and 20 yielding target compounds 22o and 22p, respectively. All title compounds
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(22a-22p) were fully characterized by proton nuclear magnetic resonance ( H NMR), carbon
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nuclear magnetic resonance ( C NMR), and electrospray ionization high-resolution mass
spectrometry (ESI-HRMS). The purity of chemicals was identified by HPLC method.
LPS activates MD2-TLR4 on cell membranes and transduces signal to produce
pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6.38, 39 Clinical evidence has shown
that pro-inflammatory cytokines play a significant role in the pathogenesis of
inflammatory-induced sepsis and ALI.40,
41
Hence, we established an enzyme-linked
immunosorbent assay (ELISA)42 to screen for the inhibitory activity of synthesized
compounds (22a-22p) against LPS-induced TNF-α and IL-6 release in mouse primary
macrophages
(MPMs).
Previously
reported
anti-inflammatory
compound
5-(4-methylpiperazin-1-yl)-3-propyl-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-3H-imidazo[4,5-b]-py
4
3
ridine (X12) was used as positive control, and DMSO was used as the vehicle control. The
cytokine inhibitory activity of the compounds is summarized in Figure 3. Our screening
results show that majority of the compounds tested exhibit greater inhibition of LPS-induced
TNF-α and IL-6 release compared to X12. We also evaluated their cytotoxicity in MPMs
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using the MTT method. As shown in Supplementary Figure S1A, all compounds at 10 μM
showed no cytotoxicity. Among these compounds, 22c, 22d, 22g, 22m, 22n and 22o inhibited
IL-6 release with rates greater than 50%. Meanwhile, 22c, 22g, 22m, 22o and 22p showed
high inhibitory rates against TNF-α release. Notably, 22m inhibited both TNF-α and IL-6
release with inhibitory rates reaching 83% and 91%, respectively, compared to the LPS
control.
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Figure 3. Effect of 3-(indol-5-yl)-indazole derivatives on LPS-induced cytokine production in
macrophages. The effect of compounds on LPS-induced TNF-α (A) and IL-6 (B) release in
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mouse primary peritoneal macrophages (MPMs) was measured by ELISA. Cells were
pretreated with vehicle, X12 and compounds 22a-22p (10 μM) for 2 h. Cells were then treated
with LPS (0.5 μg/mL) for 22 h. Each bar represents mean ± SEM of 3 independent
experiments. Statistical significance relative to LPS group is indicated (*p < 0.05, **p < 0.01,
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**p < 0.001 compared to LPS).
To our knowledge, there are few studies that have reported on the cytokine-inhibitory
effects of 3-(indol-5-yl)-indazole derivatives and their structure-activity relationship (SAR)
analysis. Based on the results shown in Figure 3, compound 22o with electron-withdrawing
group (-NO ) at the 5-position of indazol core shows relatively potent inhibitory effect against
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TNF-α and IL-6 compared to X12. However, after the reduction to amine group (22p), the
inhibitory activity against IL-6 dramatically declines. Next, we investigated the importance of
the substitutions at the 5-position of indazol core. After modification of acetamide group,
resulting compound 22a showed increased inhibitory activity against IL-6 but not TNF-α.
When the acetamide group was replaced with a propanamide group (22b), the potencies
against both IL-6 and TNF-α were almost completely abolished. However, further
investigation revealed that the potency loss of compound 22b was partially restored by
introducing a 3-chloropropanamide (22c) at the 5-position of indazol core.
We next examined substituted N-aryl formamide moieties by synthesizing 22d-22k.
Incorporation of a chlorine atom at the meta-position of the phenyl ring provided 22d, which
displayed significant increases in potency against IL-6 release. Moreover, introduction of a
chlorine (22e) or fluoro (22f) atom at para-position of the phenyl ring obviously decreased the
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anti-inflammatory potency. When 2,6-dichlorobenzamide was introduced (22g), the ability
against TNF-α remarkably increased compared to 22d with an approximately 47% inhibitory
rate. Comparatively, modification of electron-donating groups like 3-methyl (22h),
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,5-dimethoxy group (22i) or heterocycles (22j and 22k), led to a dramatic loss in inhibitory
activity against both IL-6 and TNF-α. Interestingly, the introduction of cinnamamide moiety
yielding 22l, which contains a Michael receptor, exhibited very weak anti-inflammatory
activity. These results implied that halogen group substituted benzamide was crucial for the
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anti-inflammatory effect. Compared to 22g, the removal of ethanol group at R position
yielding 22m was found to dramatically improve the activities against TNF-α and IL-6 (83%
and 91% inhibitory rates, respectively), indicating that the hydroxyethyl group may not
contribute to the anti-inflammatory benefits. Further introduction of a bulky group like benzyl
into indazole core of 22m afforded 22n, which demonstrated decreased anti-inflammatory
potency. Taken together, the preliminary SAR analysis for the TNF-α and IL-6 inhibitory
activities of the novel 3-(indol-5-yl)-indazole derivatives may provide valuable information
for the further development of anti-inflammatory compounds.
Among the screened compounds, 22c, 22m and 22o showed most promising effects
against IL-6 and TNF-α release. Thus, we selected these compounds for further investigation
of potential dose-dependent cytokine inhibitory effects. We also included compound 22g,
which significantly inhibited IL-6 and appeared to show inhibitory effect on TNF-α as well.
MPMs were pretreated with the three active compounds in increasing concentrations (0.5, 1,
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.5, 5.0, 10, and 20 μM) for 30 min. Cells were then challenged with 0.5 μg/mL LPS for 24 h.
The release of TNF-α and IL-6 in the culture medium was determined by ELISA. As shown
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in Figure 4, all four active compounds exhibited a dose-dependent inhibition of LPS-induced
cytokines. In particular, compounds 22g, 22m and 22o suppressed LPS-induced IL-6 with
IC values in the 0.5-1.0 micromole range (IC values = 0.73 μM, 0.53 μM and 0.84 μM,
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respectively). Interestingly, 22m showed suppressed TNF-α release with an IC value of 0.89
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μM, which is obviously smaller compared to values obtained with compounds 22c, 22g and
2o (3.47, 6.31 and 6.99 μM). As expected, 22m did not show cytotoxicity in MPMs in the
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concentration range from 0.5 to 20 μM (Supplementary Figure S1B). These results suggest
that 22m may be a promising anti-inflammatory candidate for the treatment of ALI and
sepsis.
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Figure 4. 3-(indol-5-yl)-indazole derivatives inhibited LPS-induced inflammatory cytokines
in a dose-dependent manner. MPMs were pretreated with vehicle or compounds at increasing
concentrations for 2 h. Subsequently, cells were incubated with LPS (0.5 μg/mL) for 22 h. (A)
IL-6 and (B) TNF-α levels as measured by ELISA. Each bar represents mean ± SEM of 3
independent. Statistical significance relative to the LPS group is indicated (*p < 0.05, **p <
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.01 compared to LPS).
As mentioned earlier, upon LPS recognition, the MD2-TLR4 mediates cytokine gene
expression through the activation of the nuclear factor (NF)-κB. Hence, we examined whether
compound 22m altered LPS-induced NF-κB signaling. We prepared nuclear fractions from
MPMs challenged with LPS. As shown in Figure 5A, the NF-κB p65 subunit nuclear levels
were increased in MPMs exposed to LPS. Pretreatment of cells with 22m suppressed the
levels indicating inhibition of NF-κB activation. We confirmed these results by infecting
RAW 264.7 macrophages with NFκB-EGFP reporter. As shown in Figure 5B and 5C, flow
cytometric analysis indicated that 22m significantly reduced the transcriptional activity of
NF-κB as detected by green fluorescent protein (GFP) fluorescence. These data show that
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2m exerts its anti-inflammatory activity by inhibiting NF-κB.
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Figure 5. Compound 22m attenuated LPS-induced activation of NF-κB signaling. (A)
Western blot analysis of NF-κB p65 subunit levels in nuclear fractions. Macrophages were
challenged with LPS, with or without pretreatment with compound 22m. Lamin B was used
as loading control. (B, C) NF-κB reporter assay showing suppressed activation of NF-κB
following 22m treatment. RAW264.7 cells stably expressing NF-κB-EGFP reporter were
subjected to flow cytometry (B) to detect NF-κB activation. Quantification of fluorescence is
shown in panel C (**p < 0.01 compared to Ctrl, ##p < 0.01 compared to LPS). (D) Schematic
illustration showing MD2-TLR4 signaling initiated by LPS. (E) Kinase selectivity profile of
compound 22m against 98 kinases.
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A schematic diagram showing mechanisms of LPS trafficking and TLR4-mediated
inflammatory signaling is presented in Figure 5D. The activated receptor dimer of
(LPS-MD-2-TLR4) recruits the intracellular adaptor MyD88 to activate NF-κB through a
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cascade of multiple kinase phosphorylation. The proteins involved between MyD88 and
NF-κB are complex and include kinases such as IL-1 receptor-associated kinase 4 (IRAK4),
IRAK1, transforming growth factor-β-activated kinase 1 (TAK1), inhibitory κB kinase α and
β (IKKα, IKKβ), and mitogen-activated protein kinases (MAPK). To identify the possible
molecular target of 22m in this cascade, we profiled the inhibitory activity of 22m against a
panel of 98 kinases using DiscoverX’s KINOMEscan platform. This panel comprises
majority of proteins invovled in the MyD88-NF-κB cascade as well as other proteins
distributed across the whole kinome (Supplementary Table S1). Our results show that 22m
did not inhibit any of these 98 kinases (Figure 5E and Supplementary Table S1). These results
indicate that kinases in MyD88-NF-κB cascade may not be targeted by 22m.
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Figure 6. Identification of MD2-TLR4 as the molecular target of compound 22m. (A) SPR
analysis of 22m to binding to recombinant MD2 protein; (B) SPR analysis of 22m to binding
to TLR4 protein; (C) SPR analysis of 22m to binding to MyD88 protein. (D-E)
Co-immunoprecipitation showing effect of 22m pretreatment on MD2-TLR4 complex
formation in macrophages exposed to LPS. Panel E showing quantification of
immunoreactivity (*p < 0.05 compared to control; #p < 0.05 compared to LPS). (F) TLR4
dimerization in HEK293T co-expressing HA and Flag-tagged TLR4. (G) Flow cytometric
analysis of the effect of 22m on binding of FITC-labeled LPS on macrophage cell surface.
(H) Molecular docking analysis of 22m with MD2-TLR4 complex.
Based on our findings, we postulated that proteins upstream of MyD88-NF-κB cascade
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might be the target of 22m. To test this, we examined the direct interaction of 22m with
recombinant MD2, TLR4, and MyD88 proteins by surface plasmon resonance (SPR) analysis.
As shown in Figure 6A-C, 22m binds to recombinant MD2 and TLR4 protein with a KD
value of 185 and 146 μM, respectively, while it did not interact with intracellular adaptor
protein MyD88. These data suggest that MD2-TLR4 may be the direct target of 22m in
producing its anti-inflammatory activity.
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To confirm MD2-TLR4 as the target of 22m, we performed co-immunoprecipitation to
investigate MD2-TLR4 complex formation. Our results show that 22m significantly inhibited
dimerization of the MD2-TLR4 complex in LPS-activated MPMs (Figure 6D-E). We then
constructed Flag-TLR4 and HA-TLR4 plasmids to investigate the dimerization of TLR4. In
Flag-TLR4 and HA-TLR4 co-expressing HEK293 cells, 22m inhibited the homodimerization
of TLR4 as determined by co-immunoprecipitation analysis (Figure 6F). We then investigated
the ability of 22m to compete with LPS binding to MD2-TLR4 complex at the cellular level.
MPMs were challenged with fluorescein isothiocyanate (FITC) -labeled LPS (FITC-LPS)
with or without 22m pretreatment and fluorescence was analyzed by flow cytometry. Here we
show that pretreatment with 22m significantly inhibited FITC-LPS binding to the cell surface
(Figure 6G). These data validate the interaction of 22m with its molecular target MD2/TLR4.
By the way, although we excluded the kinases using kinome assay, we do not know if 22m
can target other TLRs. There are 10 human TLR members in the TLR family. This is a
limitation of this study. The measurement of the interactions between 22m and recombinant
proteins of other TLRs to clarify the specificity of 22m to TLR4 will be needed in the future.
We then performed molecular docking analysis to investigate the binding mode between
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2m and MD2-TLR4 complex (PDB: 3FXI ). In our simulations, the 2,6-dichlorobenzamide
part of 22m inserted deep into the MD2 hydrophobic pocket and interacted with the inside
hydrophobic residues such as Ile-52, Phe-121, Phe-126 and Ile-153 (Figure 6H). The indole
fragment of 22m stretches to the rim of MD2 pocket and formed a key hydrogen bond with
Lys-125 residue of MD2. The indazole group locates to the MD2-TLR4 interface, further
forming a hydrogen bond with Glu-439 residue of TLR4.
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Excessive production of inflammatory cytokines induced by LPS leads to coagulation,
endothelial damage and microvascular leakage, resulting in lung organ dysfunction and
injury.45, 46 We, therefore, investigated the potential effect of 22m on ALI in LPS-challenged
mice. Histological analysis showed significant lung edema, pulmonary congestion,
neutrophilic infiltration and thickening of the alveolar wall in lung tissues of mice challenged
with LPS (Figure 7A). Pretreatment of mice with compound 22m effectively reduced airspace
inflammation and amended the tissue structure of pulmonary lobules. Scoring of lung injury
showed that 22m significantly reduces injury score to levels comparable to X12 (Figure 7B).
These results indicate that 22m is protective against LPS-induced ALI.
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Figure 7. Effect of 22m on histopathological changes in lung tissues of mice challenged with
LPS. (A) H&E staining of lung sections. (B) Lung injury scores (n=8 per group; ns: no
significance; *p < 0.05, **p < 0.01 compared to LPS).
Myeloperoxidase (MPO) is a neutrophil enzyme that promotes oxidative stress in
numerous inflammatory pathologies, and serves as a marker of neutrophil accumulation
within tissues.47, 48 To examine the effect of 22m on neutrophil lung infiltration, we measured
MPO activity in lung tissues harvested from LPS-challenged mice. Figure 8A shows that the
treatment of mice with 22m at 5 and 10 mg/kg significantly decreases LPS-induced MPO
activity. Under the same experimental conditions, 22m reduced LPS-induced serum TNF-α
levels (Figure 8B).
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Figure 8. 22m attenuate LPS-induced lung inflammation and pulmonary macrophage
infiltration in mice. Male C57BL/6 mice were treated with compounds by tail vein injection
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sacrificed 6 h after LPS injection. Serum and lung tissues were collected. (A) MPO activity in
lung tissue lysates (**p < 0.01 compared to LPS). (B) Serum levels of TNF-α (**p < 0.01
compared to LPS). (C) F4/80 immunohistochemical staining of lung tissues showing
increased macrophage infiltration following LPS challenge. Compound 22m prevented
macrophage infiltration as seen by F4/80 staining (brown). (D) Quantification of
F4/80-positive cells (**p < 0.01 compared to LPS).
To investigate whether 22m prevented macrophage infiltration in the LPS-induced ALI
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model, we utilized F4/80, a widely used marker of macrophages.49 Immunohistochemical
analysis revealed increased F4/80 immunoreactivity in lung tissues harvested from
LPS-challenged mice, while F4/80 positive macrophages decreased significantly with 22m
and X12 treatment (Figure 8C and 8D). This indicates that 22m prevented macrophage
infiltration following LPS challenge. We next investigated the inhibitory effect of compound
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2m on the mRNA levels of a panel of proinflammatory factors in lung tissues. Real-time
qPCR analysis revealed that treatment of LPS-challenged mice with 22m decreased the
mRNA levels of TNF-α, IL-6, IL-1β, IL-12 and IL-33 in lung tissues (Figure 9). In addition,
cyclooxygenase-2 (COX-2) mRNA levels were also reduced upon 22m treatment. These
results suggest that 22m can inhibit macrophage infiltration and reduce proinflammatory
cytokine production in lung tissues of ALI mice.
Figure 9. 22m reduced mRNA levels of inflammatory genes induced by LPS in mouse lung
tissues. (A-F) mRNA levels of (A) TNF-α, (B) IL-6, (C) IL-1β, (D) COX-2, (E) IL-12, and
(F) IL-33 as assessed by quantitative RT-PCR (data normalized to β-actin; n=8 per group; *p
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<
0.05, **p < 0.01 compared to LPS).
CONCLUSIONS
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Acute inflammation is the leading cause of ALI, and the rate of hospitalization of
patients with ALI is increasing.50 Thus, there is a pressing need to identify new
anti-inflammatory molecules and drug targets to prevent or mitigate inflammatory responses
in these patients. Numerous studies demonstrated that inhibiting MD2-TLR4 complex
suppresses LPS-induced inflammatory responses and the development of ALI in experimental
models. Therefore, antagonists directly targeting MD2-TLR4 have emerged as attractive
pharmacological targets for ALI and other inflammatory disorders.51
In the present study, we have designed and synthesized a novel series of
3
-(indol-5-yl)-indazole derivatives and evaluated their anti-inflammatory activity. Active
compound 22m inhibited LPS-induced TNF-α and IL-6 production in macrophages. We also
showed that 22m leads to reduced activity of NF-κB. However, kinase phosphorylation
profiling showed that 22m may not directly inhibit kinases involved in the MD2-TLR4
pathway. Then, we found that 22m directly bound MD2 and TLR4 complex through a series
of molecular studies including SPR, co-immunoprecipitation assays, flow cytometric
determination of LPS binding, and molecular docking. In vivo investigation in
LPS-challenged mice showed that treatment with 22m significantly decreases macrophage
infiltration, MPO activity, and inflammatory cytokines. The outcome of 22m treatment was
normalization of pulmonary histopathological changes. Although the Kd values of 22m-MD2
and 22m-TLR4 interactions are about 150μM, higher than that of the previously reported
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MD2 inhibitor 2i (Kd=34μM) , 22m represents a new lead structure targeting MD2/TLR4
complex. Taken together, our studies show that 3-(indol-5-yl)-indazole derivative 22m is a
new lead for the design and discovery of MD2-TLR4 antagonists for the treatment of ALI and
potentially other inflammatory diseases.
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EXPERIMENTAL SECTION
General Synthetic Chemistry Methods.All reagents and solvents were purchased from Alfa
Aesar and Sigma-Aldrich, respectively. Other chemicals were obtained from local suppliers
and were used without further purification. All reactions were monitored by thin-layer
chromatography (250 silica gel 60 F254 glass plates). Melting points were determined on a
Fisher-Johns melting apparatus and were uncorrected. Flash chromatography was performed
1
using Silica Gel 60 (E. Merck, 70-230 mesh). H NMR spectra was recorded on a Bruker 400,
500 and 600 MHz instruments, and the chemical shifts were expressed in terms of parts per
million with TMS as the internal reference. High-resolution mass spectrometry (HRMS) was
measured with an Agilent G6520 quadrupole time-of-flight mass spectrometer in electrospray
mode. Analytical HPLC analyses were performed at ambient temperature on Agilent 1260
liquid chromatograph fitted with Inertex C18 column (4.6 mm × 150 mm, 5 μm particle size).
All general chemicals were the highest available grade. The purity of all synthetic compounds
was determined by HPLC analysis and was higher than 95%.
3
-Iodo-5-nitro-1H-indazole (16). I (15.66 g, 61.6 mmol) and KOH pellets (3.58 g, 63.7
2
mmol) were successively added to a stirred solution of 5-nitro-1H-indazole (15, 5.0 g, 30.8
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6
o
mmol) in DMF (60 mL) at 65 C. After 3 h, the result mixture was poured into 1M HCl (60
mL) and 10% Na S O (100 mL). The mixture was stirred for 20 min, and the precipitate was
2
2
3
collected. The solid was dissolved in EtOAc, and the insoluble residue was filtered. The
filtrate was evaporated to give compound 16 (7.99 g, 90%) as a yellow solid. m.p 157.5-160.6
0
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o
1
C. H NMR (500 MHz, CD OD) δ (ppm) 8.42 (s, 1H, H-4), 8.30 (d, J = 7.5 Hz, 1H, H-7),
3
1
3
7
.66 (d, J = 5.0 Hz, 1H, H-6). C NMR (125 MHz, CD OD) δ (ppm) 144.26, 144.18, 128.18,
3
-
1
23.44, 119.59, 112.35, 96.39. ESI-MS m/z: 287.6 (M-H) , calcd for C H IN O : 288.93.
7
4
3
2
3
-Iodo-5-nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-indazole (17a). To a solution of
compound 16 (2.0 g, 6.92 mmol) and NaH (80%, 540 mg, 17.99 mmol) in DMF (20 mL) was
added 2-(trimethylsilyl)ethoxymethyl chloride (1.50 mL, 8.30 mmol) in an ice bath.
Subsequently, the reaction mixture was stirred at room temperature for 2.5 h and poured into
ice water (50 mL). The mixture was stirred for 30 min, and the precipitate was collected. The
residue was purified by flash chromatography to give compound 17a (2.6 g, 90%) as a yellow
o
1
solid. m.p 87.7-88.4 C. H NMR (500 MHz, CDCl ) δ (ppm) 8.72-8.27 (m, 2H, H-6, H-7),
3
7
.69 (s, 1H, H-4), 5.78 (s, 2H, NCH O), 3.62 (s , 2H, OCH CH Si(CH ) ), 0.92 (s, 2H,
2 2 2 3 3
13
OCH CH Si(CH ) ), -0.02 (s, 9H, OCH CH Si(CH ) ). C NMR (125 MHz, CDCl ) δ (ppm)
2
2
3 3
2
2
3 3
3
143.47 (C-8), 142.33 (C-5), 128.80 (C-9), 122.88 (C-6), 119.43 (C-4), 110.59 (C-7), 95.34
(
C-3), 78.57 (NCH O), 67.25 (OCH CH Si(CH ) ), 17.72 (OCH CH Si(CH ) ), -1.48 ×3
2 2 2 3 3 2 2 3 3
+
(
Si(CH ) ). ESI-MS m/z: 420.9 (M+Na) , calcd for C H IN O Si: 419.2.
3
3
13 18
3
3
1
-Benzyl-3-iodo-5-nitro-1H-indazole (17b). A mixture of 16 (0.4 g, 1.38 mmol), K CO
2
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(
0.38 g, 2.76 mmol) and benzyl bromide (164 μL, 1.38 mmol) in acetonitrile (20 mL) was
heated to reflux for 1 h. The acetonitrile was removed in vacuo, and the resulting residue was
extracted with EtOAc for three times. The organic layer was separated and dried over
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anhydrous MgSO , concentrated, and purified by flash chromatography to afford compound
4
o
1
1
7b (0.48 g, 95%) as a yellow solid. m.p 151.5-153.2 C. H NMR (500 MHz, CDCl ) δ
3
(ppm) 8.48 (s, 1H, H-4), 8.25 (d, J = 10.0 Hz, 1H, H-7), 7.37-7.23 (m, 6H, H-6, Bn-(H2-H6)),
13
5
.64 (s, 2H, Bn-CH ). C NMR (125 MHz, CDCl ) δ (ppm) 143.00 (C-5), 142.10 (C-8),
2
3
135.18 (Bn-C1), 129.06 (Bn-C3, Bn-C5), 128.50 (Bn-C4), 128.42 (C-9), 127.34 (Bn-C2,
Bn-C6), 122.56 (C-6), 119.62 (C-4), 110.07 (C-7), 94.44 (C-3), 54.26 (Bn-CH ). ESI-MS m/z:
2
+
3
80.1 (M+H) , calcd for C H IN O : 379.8.
1
4
10
3
2
3
-(1H-Indol-5-yl)-5-nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-indazole (18a).
A
mixture of compound 17a (3.48 g, 8.30 mmol), aqueous 2 M Na CO (33 mL, 66 mmol),
2
3
PdCl (dppf)·DCM (1.63 g, 2 mmol), and 5-indolylboronic acid (1.77 g, 9.96 mmol) in THF
2
(33 mL) was heated to reflux for 5 h under an atmosphere of nitrogen. The mixture was
allowed to cool to room temperature and diluted with H O and extracted with EtOAc. The
2
combined organic layer was washed with brine, dried over MgSO , filtered, and evaporated.
4
The residue was purified by flash chromatography to give compound 18a (2.74 g, 67%) as a
1
yellow oil. H NMR (500 MHz, CDCl ) δ (ppm) 9.06 (s,1H, NH), 8.42 (s, 1H, H-4), 8.34 (d, J
3
=
12.0 Hz, 1H, H-7), 8.22 (s, 1H, H-4'), 7.79 (d, J = 8.0 Hz, 1H, H-6'), 7.66 (d, J = 12.0 Hz,
H, H-6), 7.57 (d, J = 8.0 Hz, 1H, H-7'), 7.31 (s, 1H, H-2'), 6.70 (s, 1H, H-3'), 5.83 (s, 2H,
N-CH -O), 3.67 (t, J = 8.0 Hz, 2H, OCH CH Si(CH ) ), 0.93 (t, J = 8.0 Hz, 2H,
1
2
2
2
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OCH CH Si(CH ) ), -0.05 (s, 9H, OCH CH Si(CH ) ). C NMR (125 MHz, CDCl ) δ (ppm)
2
2
3 3
2
2
3 3
3
1
48.94 (C-3), 143.15 (C-8), 142.93 (C-5'), 136.24 (C-5), 128.37 (C-8'), 123.46 (C-9'), 122.26
(C-9), 121.96 (C-6), 120.41 (C-4), 119.86 (C-4'), 111.92 (C-6'), 110.23 (C-7, C-7'), 103.36
C-3'), 67.02 (OCH CH Si(CH ) ), 17.79 (OCH CH Si(CH ) ), -1.41 (OCH CH Si(CH ) ).
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(
2
2
3 3
2
2
3 3
2
2
3 3
+
ESI-MS m/z: 409.1 (M+H) , calcd for C H N O Si: 408.6.
2
1
24
4
3
1
-Benzyl-3-(1H-indol-5-yl)-5-nitro-1H-indazole (18b). Following general procedure for the
synthesis of 17a, the crude residue was purified by flash chromatography to furnish
1
compound 18b (177 mg, 61%) as a yellow oil. H NMR (500 MHz, CDCl ) δ (ppm) 9.07 (s,
3
1
H, NH), 8.41 (s, 1H, H-4), 8.31-8.19 (m, 1H, H-7), 7.82 (d, J = 8.0 Hz, 1H, H-6), 7.56 (d, J
=
8.4 Hz, 1H, H-4'), 7.46-7.24 (m, 7H, Bn-(H2-H6), H2', H7'), 6.70 (s, 1H, H-3'), 5.70 (s, 2H,
13
Bn-CH ). C NMR (125 MHz, DMSO-d ) δ (ppm) 148.76 (C-3), 142.78 (C-8, C-5'), 142.51
2
6
(C-5), 136.24 (Bn-C1), 135.94 (C-8'), 128.94 (Bn-C3, Bn-C5), 128.44 (Bn-C2, Bn-C6),
1
1
3
28.17 (Bn-C4), 127.25 (C-2'), 125.26 (C-9), 123.74 (C-6), 121.99(C-4), 121.68 (C-4'),
21.58 (C-6'), 120.28 (C-7'), 120.01 (C-7), 103.39 (C-3'), 53.54 (Bn-CH ). ESI-MS m/z:
2
+
68.7 (M+H) , calcd for C H N O : 367.1.
22 16
4
2
3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-5-nitro-1-{[2-(trimethylsilyl)
ethoxy]methyl}-1H-indazol (19) . To a mixture of compound 18a (1.0 g, 2.44 mmol) and
NaH
(190
mg,
6.34
mmol)
in
DMF
(9
mL)
was
added
(
2-bromoethoxy)(tert-butyl)dimethylsilane (703 mg, 2.94 mmol) in an ice-bath. Subsequently,
the reaction mixture was stirred at room temperature for 3 h. The resulting solution was
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quenched with redistilled water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over MgSO , filtered, and evaporated. The residue was purified by
4
flash chromatography (silica gel; Hexane/EtOAc, gradient, 10-30% EtOAc) to afford
1
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1
1
1
1
1
1
2
2
2
2
2
2
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2
2
2
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3
3
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5
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compound 19 (1.11 g, 86%) as a yellow oil. H NMR (500 MHz, CDCl ) δ (ppm) 9.06 (s, 1H,
3
H-4), 8.34 (d, J = 10.0 Hz, 1H, H-7), 8.19 (s, 1H, H-4'), 7.79 (d, J = 10.0 Hz, 1H, H-6), 7.66
(
d, J = 10.0 Hz, 1H, H-6'), 7.51 (d, J = 10.0 Hz, 1H, H-7'), 5.83 (s, 2H, N-CH -O), 4.30 (t, J =
2
8
.0 Hz, 2H, OCH CH Si(CH ) ), 3.97 (t, J = 8.0 Hz, 2H, N-CH CH -O), 3.67 (t, J = 8.0 Hz,
2 2 3 3 2 2
2H, N-CH CH -O), 0.93 (t, J = 8.0 Hz, 2H, OCH CH Si(CH ) ), 0.84 (s, 9H,
2
2
2
2
3 3
O-Si(CH ) C(CH ) ), -0.05 (s, 9H, OCH CH Si(CH ) ), -0.11 (s, 6H, O-Si(CH ) C(CH ) ).
3
2
3 3
2
2
3 3
3 2
3 3
1
3
C NMR (125 MHz, CDCl ) δ (ppm) 149.04 (C-3), 143.00 (C-5', C-8), 129.77 (C-5), 129.14
3
(C-8'), 121.84 (C-9'), 121.31 (C-6, C-9), 120.54 (C-4), 119.86 (C-4'), 110.17 (C-6'), 110.13
(
C-7, C-7'), 101.93 (C-3'), 78.21 (N-CH -O), 66.98 (OCH CH Si(CH ) ), 62.43
2
2
2
3 3
(N-CH CH -O),
48.98
(N-CH CH -O),
25.81
(O-Si-(CH ) C(CH ) ,
25.65
2
2
2
2
2 2
3 3
(
O-Si-(CH ) C(CH ) ), 17.81 (OCH CH Si(CH ) ), -1.45 (OCH CH Si(CH ) ), -5.66×2
2
2
3 3
2
2
3 3
2
2
3 3
+
(
O-Si-(CH ) C(CH ) ). ESI-MS m/z: 567.2 (M+H) , calcd for C H N O Si : 566.3.
2
2
3 3
29 42
4
4
2
3
-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethoxy]m
ethyl}-1H-indazol-5-amine (20) . The compound 19 (1.15 g, 2.03 mmol) was dissolved in
ethanol (40 mL) and water (10 mL). Iron powder (0.98 g, 17.44 mmol) and ammonium
o
chloride (0.24 g, 4.46 mmol) were then added, and the resulting mixture was heated to 78 C
for 4 h. The ethanol was removed in vacuo, and the resulting residue was basified with
sodium bicarbonate and extracted with EtOAc. The organic layer was separated and dried
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