Discovery of Small Molecules as Multi-Toll-like Receptor Agonists with Proinflammatory and Anticancer Activities

Article abstract of DOI:10.1021/acs.jmedchem.7b00419

Therapies based on activation of multiple Toll-like receptors (TLRs) may offer superior therapeutic profiles than that of single TLR activation. To discover new small molecules that could activate multiple TLRs, we performed a cell-based high-Throughput screening of a small-molecule library based on TLR3-mediated NF-B activation. Subsequent structural optimization and counterscreening of other TLRs produced the first small molecule 17e (CU-CPT17e) capable of simultaneously activating TLRs 3, 8, and 9. Biochemical studies demonstrated that 17e could induce a strong immune response via the production of various cytokines in human monocytic THP-1 cells. Furthermore, 17e inhibited the proliferation of HeLa cancer cells by triggering apoptosis and arresting the cell cycle at the S phase. These results showcase potential therapeutic applications of 17e in both vaccine adjuvants and anticancer therapies based on multi-TLR activation.

Full text of DOI:10.1021/acs.jmedchem.7b00419

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Article
Discovery of Small Molecules as Multi-Toll-like Receptor
Agonists with Proinflammatory and Anticancer Activities
Lei Zhang, Varun Dewan, and HANG Hubert YIN
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 24 May 2017
Downloaded from http://pubs.acs.org on May 24, 2017
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Discovery of Small Molecules as MultiꢀTollꢀlike
Receptor Agonists with Proinflammatory and
Anticancer Activities
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Lei Zhang, Varun Dewan, and Hang Yin
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Department of Chemistry and Biochemistry and the BioFrontiers Institute, University of
Colorado, Boulder, CO 80309, USA
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Correspondence: hubert.yin@colorado.edu
KEYWORDS. Tollꢀlike receptor, agonist, highꢀthroughput screening, NFꢀκB, proinflammatory,
anticancer
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Abstract. Therapies based on activation of multiple Tollꢀlike receptors (TLRs) may offer
superior therapeutic profiles than that of single TLR activation. To discover new small molecules
that could activate multiple TLRs, we performed a cellꢀbased highꢀthroughput screening of a
smallꢀmolecule library based on TLR3ꢀmediated NFꢀκB activation. Subsequent structural
optimization and counter screening of other TLRs produced the first small molecule 17e (CUꢀ
CPT17e) capable of simultaneously activating TLRs 3, 8, and 9. Biochemical studies
demonstrated that 17e could induce a strong immune response via the production of various
cytokines in human monocytic THPꢀ1 cells. Furthermore, 17e inhibited the proliferation of HeLa
cancer cells by triggering apoptosis and arresting the cell cycle at the S phase. These results
showcase potential therapeutic applications of 17e in both vaccine adjuvants and anticancer
therapies based on multiꢀTLR activation.
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Introduction
Tollꢀlike receptors (TLRs) are a family of pattern recognition receptors (PRRs) that play an
important role in orchestrating the innate and adaptive immunity to protect the host from
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invading bacteria, viruses, and other pathogens. The activation of different TLRs has also been
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found to be promising in anticancer therapies. With significant therapeutic potential, TLR
agonists have been included in the National Cancer Institute's list of immunotherapeutic agents
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with the highest potential to treat cancer.
Therapies based on multiꢀTLR activation have considerable benefit over single TLR
activation in clinical applications for several reasons. Firstly, multiꢀTLR activation could
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augment the immune response by triggering synergism between different TLR pathways.
Invading pathogens usually release different components which can trigger several TLRs
simultaneously in distinct cellular compartments. Instead of working alone, TLRs network with
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each other to recognize these diverse components of invading pathogens.
Secondly,
polymicrobial infection also occurs when two or more invading microbes are involved at an
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infection site.
In these cases, invading microbes are more likely to trigger the activation of
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several TLRs simultaneously, sometimes even involving other PRRs. The sum of these signals
enables the host to mount an effective immune response to clear the invading pathogens.
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Moreover, the expression of TLRs varies with different tissues and cell types. Even for the
same cell type, TLR expression patterns are significantly different from one subpopulation to
another. For example, human myeloid dendritic cells (DCs) express TLRs 2 and 4, while
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plasmacytoid DCs express TLRs 7 and 9. Due to the heterogeneous distribution of TLRs, the
efficacy of single TLR activation could diminish or disappear if the target cells fail to express
that specific TLR, whereas engagement of multiple TLRs may supplement each other to reach a
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critical activation threshold to exert effective immune responses. Therefore, the potential
applications of multiꢀTLRꢀbased therapy in infectious diseases, autoimmune diseases, and cancer
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have motivated enthusiastic research interest.
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Numerous reports have shown the promising potential of multiꢀTLR activation in the
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development of adjuvant vaccines against infectious and autoimmune diseases.
For example,
the yellow fever vaccine YFꢀ17D, one of the most successful vaccines ever developed, activates
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TLRs 2, 7, 8, and 9 in dendritic cells to stimulate proinflammatory cytokines.
In another
study of the Tꢀcell response in mice, Berzofsky and coworkers found that a mixture of three TLR
ligands (TLR2/6, TLR3, and TLR9) provided more protection against viral infections compared
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to ligands for only any two of these TLRs.
Different combinations of multiple TLR agonists have also been reported to be more effective
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in the treatment of various cancers.
One of the most successful examples is the Bacillus
CalmetteꢀGuérin (BCG) vaccine which was originally used against tuberculosis. The BCG
vaccine containing components that activate TLRs 2, 4, and 9 has been further successfully
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developed to treat nonꢀmuscleꢀinvasive bladder cancer. In a separate study, the synergistic
activity between the TLR3 and TLR9 agonists resulted in an enhanced antiꢀtumor response,
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while treatment with either agonist alone failed to control tumor growth.
Combination
therapy by the coꢀdelivery of agonists targeting TLRs 7, 8, and 9 was also reported to eradicate
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large primary tumors and establish longꢀterm antitumor immunity.
Despite the potential of multiꢀTLR targeting drugs, most previous reports have been limited to
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either simply combining several TLR agonists
or covalently tethering different TLR
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agonists.
Moreover, most known TLR agonists are mimics or modifications of microbial
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components that have suboptimal pharmacokinetic properties. Small molecules provide an
alternative for TLRꢀbased therapeutics. Nonetheless, no smallꢀmolecule agonist has been
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identified for human TLRs 3, 5, or 9. To address these issues, we envisioned that an effective
strategy would be to perform a cellꢀbased highꢀthroughput screening (HTS) of smallꢀmolecule
libraries. In the present study, we chose TLR3 activation as the starting point of our HTS
campaign in the hope to extend the activation to other TLRs by further studies. Upon cellꢀbased
HTS of a smallꢀmolecule library, several hits were identified as TLR3 agonists, among which hit
6a was chosen in this study based on its potency. Satisfactorily, subsequent structureꢀactivity
relationship (SAR) studies and counter screening other TLRs found compound 17e as the first
smallꢀmolecule multiꢀTLR agonist that activates TLRs 3, 8, and 9. The therapeutic potential of
17e was also investigated in terms of its effect on induction of immune responses and anticancer
activities in vitro.
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Results and discussion
Highꢀthroughput screening. The Maybridge HitFinder v11 library comprised of 14,400
compounds was utilized for the cellꢀbased HTS. The human embryonic kidney 293 (HEK293)
cell line has been extensively used as a benchꢀmark expression system for studies involving
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TLRs. Activation of TLRs upon ligand binding initiates a downstream signaling cascade and
results in the activation of transcription factors, such as nuclear factorꢀκB (NFꢀκB), eventually
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leading to the secretion of proinflammatory cytokines and chemokines. Thus, the HEK293 cell
line was utilized for the screening system, and was coꢀtransfected with the human TLR3
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hTLR3) gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene
placed under the control of an NFꢀκBꢀinducible promoter. The screening layout (Figure S1A)
was conducted in 384ꢀwell plates in duplicate to include controls (basal, positive, and negative)
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and library compounds at a final concentration of 30 ꢁM. The efficiency (minimum false
positives) and robustness (reproducibility) of the screening procedure was calculated based on
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the Z' factor, which takes into account the standard deviation and the average values of the
positive and the negative controls (Figure S1B). The obtained Z' value of our cellꢀbased HTS
was 0.77 which was validated to be robust and reliable. TLR3 agonist
polyribosinic:polyribocytidic acid (poly I:C) was used as the positive control. SEAP secretion in
the cell supernatant was measured to indicate levels of TLR3ꢀmediated NFꢀκB activation by
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QUANTIꢀBlue detection.
Upon successful completion of the HTS, the obtained data was statistically distributed based
on the root mean square (RMS) values as depicted in Figure 1A. A total of 59 compounds were
picked based on activation of the SEAP reporter gene which overlaid most closely to the positive
controls (see green highlighted box, Figure 1A). To identify exclusive TLR3 activators from the
overall hits (NFꢀκB activators), all identified compounds were then subjected to a secondꢀround
manual screening with HEKꢀnull cells which express the NFꢀκB based SEAP reporter but no
TLRs to eliminate generic NFꢀκB activators. Further rounds of refinement eliminated cytotoxic,
nonꢀreproducible and least potent compounds based on EC calculations from SEAP reporter
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doseꢀresponse studies. From this extensive filtering process, several hits were identified as TLR3
agonists, among which hit compound 6a (EC = 22.3 ± 5.3 ꢁM) was picked in this study based
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on its potency (Figure 1B).
Design and Chemistry. With hit 6a in hand, the structural optimization was performed to
target three regions (Figure 1B): (a) the substituent effect on the diꢀbenzyl aromatic ring system
as well as the difference between diꢀbenzylation and monoꢀbenzylation; (b) replacement of the
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ether linkages in the central region; and (c) exploration of other substituted skeletons with a
preference for privileged heterocyclic motifs.
Compounds 6aꢀh, 9, and 12 were designed to investigate the SAR of the two phenyl rings at
the 6 and 7 positions of the chromene ring (Scheme 1). Intermediate 2 was prepared by
condensation of 1ꢀ(2ꢀhydroxyꢀ4,5ꢀdimethoxyphenyl)ethanꢀ1ꢀone 1 with acetone, which afforded
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6,7ꢀdihydroxyꢀ2,2ꢀdimethylchromanꢀ4ꢀone 3 by demethylation with boron tribromide. Due to the
electronꢀwithdrawing effect by the carbonyl group, the 7ꢀOH group of compound 3 is more
easily ionized. Therefore, methylation or benzylation of compound 3 will selectively occur at the
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ꢀOH group. The intermediate 3 underwent a diꢀbenzylation or methylation/benzylation or
monoꢀbenzylation to generate 2,2ꢀdimethylchromanꢀ4ꢀone intermediates 4aꢀh, 7, and 10
respectively, which were then directly reduced by sodium borohydride to give 2,2ꢀ
dimethylchromanꢀ4ꢀols 5aꢀh, 8, and 11. The regioselectivity was further confirmed by the 2D
NMR of compound 10. Target compounds 6aꢀh, 9, and 12 were then obtained through oneꢀstep
elimination under acidic condition at room temperature.
Looking to replace the dimethyl group on the 2Hꢀchromene moiety of 6a with various
substituents, compounds 17aꢀe were synthesized using a different route from that of compounds
6aꢀh (Scheme 2). The synthesis started from condensation of compound 1 with various ketones
to afford 13aꢀf, which were then demethylated by boron tribromide to give 6,7ꢀdihydroxyꢀ
chromanꢀ4ꢀones 14aꢀf. Subsequent reduction of intermediates 14aꢀf by lithium aluminium
hydride afforded chromaneꢀ4,6,7ꢀtriols 15aꢀf, which were subject to elimination of the hydroxyl
group under acidic conditions to give compounds 16aꢀf. The final target compounds 17aꢀe were
conveniently accessed via the diꢀbenzylation of 16aꢀe with 4ꢀnitrobenzyl bromide. Compound
18, a derivative with a different linker than 6a, was obtained from 2,2ꢀdimethylꢀ2Hꢀchromeneꢀ
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,7ꢀdiol 16f via a similar synthetic route, except that a diꢀbenzoylation by 4ꢀnitrobenzoyl
chloride was conducted instead of a diꢀbenzylation.
SAR studies. Based on its doseꢀresponse results, compound 6a showed significant NFꢀκB
activation at 25 ꢁM while displaying no cytotoxicity. Therefore, the activation of all the
compounds were tested at a concentration of 25 ꢁM in hTLR3 HEK293 cells. Untreated cell
samples were used as the baseline control. NFꢀκB activation of the treated samples was
quantified by normalizing to that of the control and expressed as fold changes. Those with NFꢀ
κB activation increases of more than 2.5 fold were further tested for their EC values.
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To investigate the role of the two 4ꢀnitrobenzyl groups of 6a, compounds 12 and 9 were
synthesized. There was no TLR3ꢀmediated NFꢀκB activation observed for any monoꢀbenzylated
compounds, highlighting the importance of both 4ꢀnitrobenzyl groups (Table 1). Replacing 4ꢀ
nitrobenzyl groups (6a) with 4ꢀnitrobenzoyl groups (18) abrogated the activity. Therefore, we
maintained the diꢀbenzylated groups on the chromene scaffold of 6a in the following SAR
studies.
Next, we tried to change the substituents on the two symmetric benzyl rings. Absence of the
two nitro substituents led to loss of the NFꢀκB activation (6a vs 6f, Table 2). No obvious
electronic effects on the benzyl rings were observed based on compounds 6bꢀe. However, the
position of nitro groups played an important role as the metaꢀ and orthoꢀsubstituents (6g and 6h)
showed no activity compared to their paraꢀsubstituted counterpart 6a. Meanwhile, intermediates
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aꢀe (chromones) and 5aꢀe (chromanols) were also tested. They exhibited similar substituent
effects on the benzyl groups as that of compounds 6aꢀe. On the other hand, the chromene
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scaffold was the most potent activator of TLR3ꢀmediated NFꢀκB signaling as compared to
chromone and chromanol scaffolds, exemplified by compounds 6a, 4a, and 5a.
Optimization studies were then directed to examine different substituted chromene scaffolds
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(
Table 3). Chromenes with 2,2ꢀdiethyl (17a), cyclohexane (17b), cyclopentane (17c),
cycloheptane (17d) substituents displayed no NFꢀκB activation. However, tetrahydroꢀ2Hꢀpyran
substitution (17e) significantly improved the activity with 13.9 ± 0.9 fold of NFꢀκB activation
and an EC value of 4.8 ± 0.7 ꢁM. Therefore, compound 17e (CUꢀCPT17e) was identified as
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the best derivative for subsequent biological evaluations.
Compound 17e is a multiꢀTLR agonist that activates TLRs 3, 8, and 9. We were
interested to see whether 17e could activate NFꢀκB by tapping other hTLR signaling pathways.
To this end, the best derivative 17e was tested by the SEAP assay using HEK293 cells
transfected with various hTLRs. As shown in Figure 2, compound 17e showed negligible or no
NFꢀκB activation in hTLRs 2, 4, 5, and 7 cells compared with the activation by the cognate
agonists of these TLRs. Nonetheless, compound 17e showed strong NFꢀκB activation in TLR8
and TLR9 cells with EC values of 13.5 ± 0.6 ꢁM and 5.7 ± 0.2 ꢁM respectively (Table 4).
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These results indicated that 17e is a novel smallꢀmolecule TLR3/8/9 agonist.
The therapeutic potential of 17e was further explored in a bifurcate manner: its effect on
induction of immune response and anticancer activities in vitro.
Compound 17e stimulates cytokine responses in THPꢀ1 cells. As a response to harmful
stimuli, immune cells stimulated via TLR activation release various cytokines to activate other
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immune cells to cope with invading pathogens or deleterious tissue damages. Human
monocytic THPꢀ1 cells express TLRs 1ꢀ10 and are widely used as a model system to study Tollꢀ
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like receptors. To characterize its immunostimulatory activity, compound 17e was tested in
THPꢀ1 cells along with several standards such as poly I:C (TLR3 agonist), R848 (TLR7/8
agonist), and CpG ODN2006 (TLR9 agonist). Levels of cytokine tumor necrosis factor α (TNFꢀ
α) from cell culture supernatants were analyzed by the enzymeꢀlinked immunosorbent assay
(ELISA). As shown in Figure 3A, compound 17e stimulated a strong production of TNFꢀα in
THPꢀ1 cells in a doseꢀdependent manner. Additionally, the effect of 17e on mRNA expression of
various proinflammatory cytokines was also examined by qRTꢀPCR. The results showed that the
mRNA levels of TNFꢀα (Figure 3B), interleukin 6 (ILꢀ6) (Figure 3C), and ILꢀ8 (Figure 3D)
significantly increased after 12 hours in a doseꢀdependent manner. Collectively, our data
suggested that 17e could induce strong proinflammatory signals at both transcriptional and
translational levels in human monocytic THPꢀ1 cells.
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Compound 17e inhibits the proliferation of cancer cells in a doseꢀdependent manner.
The antitumor role of TLRꢀbased therapies has been attributed to the enhancement of innate and
adaptive immunity, interference with cell cycle regulation, or a direct proapoptotic effect on
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tumor cells.
To investigate its effects on tumor cells, compound 17e was evaluated for the
antiproliferative property in several human cancer cell lines including HeLa, MDAꢀMBꢀ231,
VAꢀ13, Uꢀ2 OS, SCaBER, and MCFꢀ7 cells. Among those, the HeLa cell line was found
particularly sensitive to 17e, which may be caused by varied expression patterns and amounts of
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TLRs in different cancer cell lines.
Therefore, HeLa cells were selected for further
investigation. The antiproliferative effect of 17e on human HeLa cells was examined by the
WSTꢀ1 assay. As shown in Figure 4A, following exposure to 17e for 24, 48, and 72 hours, the
proliferation of the HeLa cell line was strongly inhibited in a doseꢀdependent manner. To
determine the degree of toxicity of 17e towards normal cells, parallel experiments were
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conducted in normal human mammary epithelial cells (HuMEC). Compound 17e showed only
mild cytotoxicity to HuMEC cells (Figure 4B and Table 5).
Compound 17e triggers apoptosis in HeLa cells. To gain insights into the underlying
mechanism for cell growth inhibition by 17e, we further investigated the induction of apoptosis
by 17e in HeLa cells. HeLa cells were cultured with increasing concentrations of 17e or poly I:C
or blank control (DMSO) for 24 hours. Induction of apoptosis was monitored by flow cytometry
using annexin V and propidium iodide (PI) double staining, which detects the externalization of
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phosphatidylserine (PS), a characteristic feature of cells entering apoptosis. The phospholipidꢀ
binding protein, annexin V, binds to cells with externally exposed PS, while PI staining occurs
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only after loss of membrane integrity. This dual staining distinguishes between unaffected cells
ꢀ
ꢀ
+
ꢀ
(
Q4, Annexin V /PI ), early apoptotic cells (Q3, Annexin V /PI ), late apoptotic cells (Q2,
+
+
ꢀ
+
Annexin V /PI ), and necrotic cells (Q1, Annexin V /PI ). As shown in Figure 5, treatment with
17e for 24 hours at different concentrations (10 to 40 ꢁM) resulted in an elevation of apoptotic
cell population ranging from 10% to 17% (Figures 5CꢀF), which is more effective than poly I:C
at 5 µg/mL (Figure 5G). These results suggested that the antiproliferative activity of 17e against
HeLa cells might result from its ability to directly induce apoptosis.
Compound 17e arrests the cell cycle at the S phase in HeLa cells. To delineate the
mechanism behind apoptosis, the effect of 17e on the cell cycle distribution of HeLa cells was
also examined by staining cells with PI and analyzing the percentages of G0/G1, S, and G2/M
cell populations with flow cytometry. As shown in Figure 6G, treatment with 17e for 24 hours
caused a doseꢀdependent accumulation of cells at the S phase accompanied with a reduction of
G0/G1 cells. Specifically, the percentage of cells at the G0/G1 phase was about 52% in the
control group, but gradually decreased to 40% after treatment with different concentrations of
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7e. In addition, the percentage of cells at the S phase increased from 31% in the control group
to 41% after treatment with 17e. The arrest of the cell cycle at the S phase could block DNA
synthesis and cause DNA damage. Cells are prone to undergo apoptosis when damage repair is
impeded or incomplete. Taken together, these results indicate that 17e arrested HeLa cells at the
S phase, leading to the apoptosis.
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Modulation of several targets by a single agent can provide a superior safety and efficacy
5
4ꢀ56
profile due to lower dosages and thus less side effects and drug resistance.
This is especially
beneficial to complex diseases including many infectious and autoimmune diseases as well as
57ꢀ59
35ꢀ39
cancer.
However, studies of single agents that activate multiple TLRs are still rare.
In this
study, we successfully identified the first small molecule TLR3/8/9 agonist 17e from a cellꢀbased
HTS campaign and subsequent structural optimizations. Although the molecular mechanisms by
which 17e activates TLRs 3, 8, and 9 remains elusive, some speculations can be drawn based on
the structural and functional similarities between them. TLRs 3, 8, and 9 are highly homologous
6
0,61
and all confined to the membranes of endosomes to recognize nucleic acids (RNA or DNA).
It is also interesting that 17e selectively activates TLR8 over TLR7, both of which recognize
ssRNA within the endosomes. This may be due to its different binding affinity or even distinct
6
2
binding pockets to TLR7 and TLR8. Some reports have found that activation of TLRs 7 and/or
6
3ꢀ65
8
is very sensitive to structural modifications of the agonists.
For example,
imidazoquinolines with a butyl group activated TLR7, but simply replacing with a pentyl group
66
caused specific TLR8 activation. Understanding the molecular basis of the multiꢀTLR agonist
17e will facilitate its further development in multiꢀTLR based therapies as well as help the
studies of TLR biology.
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Proinflammatory assays showed that 17e induced large amounts of cytokines such as TNFꢀα,
ILꢀ6, and ILꢀ8 in THPꢀ1 cells at both transcriptional and translational levels. A strong
inflammatory response might suggest a long duration of immune response and full deployment
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of immune cells. This brings more clinical benefit particularly to newborns and the elderly
who generally suffer from poor immunogenicity. Moreover, compound 17e can inhibit the
growth of human cervical HeLa cancer cells by inducing apoptosis and arresting the cell cycle at
the S phase. Regulation of the cell cycle and apoptosis can be important targets for cancer
6
9
chemotherapy. The mechanism underlying the antiꢀtumor property of 17e will be examined
latter by analyzing the changes of cell cycle markers, death receptors, caspases and alike. These
results suggest promising therapeutic applications of 17e in the development of vaccine
adjuvants and anticancer therapies.
Conclusion
In summary, we utilized a cellꢀbased highꢀthroughput screening to assist in the discovery of
smallꢀmolecule multiꢀTLR agonists. The strategy was to initially focus on TLR3 activation in the
HTS and SAR studies, then extend to other TLRs, eventually leading to the discovery of
compound 17e as a potent multiꢀTLR agonist which activates TLRs 3, 8, and 9. Proinflammatory
studies proved that 17e can induce a robust immune response via the production of various
cytokines in human monocytic THPꢀ1 cells at both mRNA and protein levels. 17e can also cause
growth inhibition, apoptosis induction, and cell cycle arrest in human cervical cancer cells. This
work expands the limited repertoire of available smallꢀmolecule TLR agonists and warrants inꢀ
depth investigations to explore the potential therapeutic applications in vaccine adjuvants and
anticancer therapies.
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Experimental
General Chemistry Methods. Reagents were purchased from commercial sources and used
1
as received. H NMR spectra were recorded at 400 MHz in CDCl or CD OD using residual
3
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solvent peaks (CDCl : δ 7.28; CD OD: δ 3.30) as the internal standard. C NMR spectra were
3
3
recorded at 101 MHz in CDCl using residual CHCl (77.16 ppm) as the internal reference.
3
3
Compounds were purified using flash chromatography (ACI systems, Biotage). The purity of
1
tested compounds was evaluated via H NMR (>95% sample 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 high resolution mass spectrometer.
Synthesis of 6,7ꢀDimethoxyꢀ2,2ꢀdimethylꢀ3Hꢀ1ꢀbenzopyranꢀ4ꢀone (2). To a solution of 1ꢀ
(2ꢀhydroxyꢀ4,5ꢀdimethoxyphenyl)ethanꢀ1ꢀone 1 (5 g, 25.48 mmol) in methanol, pyrrolidine (4.2
mL, 50.97 mmol) and acetone (20 ml, 272.04 mmol) were added sequentially and the reaction
mixture was heated to reflux overnight before concentrating and purifying by flash column
1
chromatography to give the title compound 2 (5.12 g, 85%) as a white solid. H NMR (400 MHz,
CDCl ) δ 7.28 (s, 1H), 6.42 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 2.68 (s, 2H), 1.47 (s, 6H); MS
3
+
+
(ESI ) m/z 237.1 [M+H] .
Synthesis of 6,7ꢀDihydroxyꢀ2,2ꢀdimethylchromanꢀ4ꢀone (3). To a solution of 2 (5 g, 21.16
mmol) in dichloromethane (20 mL) was slowly added boron tribromide (1M in dichloromethane,
o
63.49 mL, 63.49 mmol) at 0 C under nitrogen and the reaction was stirred at room temperature
overnight. The reaction mixture was poured into ice water, extracted with ethyl acetate for three
times. The combined organic layers were dried with Na SO , filtered, concentrated, and purified
2
4
1
by flash column chromatography to give the title compound 3 (3.53 g, 80%) as a white solid. H
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NMR (400 MHz, CD OD) δ 7.14 (s, 1H), 6.30 (s, 1H), 4.95 (br s, 2H), 2.61 (s, 2H), 1.39 (s, 6H);
3
+
+
MS (ESI ) m/z 209.1 [M+H] .
General Procedure for the Preparation of Compounds 4aꢀe Exemplified by 2,2ꢀ
Dimethylꢀ6,7ꢀbis((4ꢀnitrobenzyl)oxy)chromanꢀ4ꢀone (4a). A mixture of 3 (300 mg, 1.44
mmol) and potassium carbonate (1.19 g, 8.64 mmol) in acetone (20 mL) was treated with 4ꢀ
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60
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nitrobenzyl bromide (685 mg, 3.17 mmol) and stirred at 60 C under nitrogen overnight. The
reaction mixture was filtered, concentrated, and purified by flash column chromatography to give
1
the title compound 4a (648 mg, 94%) as a yellow solid. H NMR (400 MHz, CDCl ) δ 8.28 (dd,
3
J = 11.1, 8.8 Hz, 4H), 7.64 (dd, J = 8.9, 2.2 Hz, 4H), 7.39 (s, 1H), 6.46 (s, 1H), 5.29 (s, 2H), 5.24
1
3
(
s, 2H), 2.68 (s, 2H), 1.46 (s, 6H); C NMR (101 MHz, CDCl ) δ 190.83, 156.64, 155.16,
3
147.81, 147.63, 144.18, 143.15, 142.73, 127.50, 127.39, 124.00, 123.84, 113.12, 109.91, 102.53,
+
+
7
9.90, 70.05, 69.43, 48.34, 26.60; HRMS (ESI ), calcd C H LiN O (M + Li ) = 485.1536,
25 22 2 8
found = 485.1544.
2
,2ꢀDimethylꢀ6,7ꢀbis((4ꢀ(trifluoromethyl)benzyl)oxy)chromanꢀ4ꢀone (4b). White solid,
1
yield 92%. H NMR (400 MHz, CDCl ) δ 7.65 (dd, J = 12.0, 8.2 Hz, 4H), 7.57 (dd, J = 8.1, 4.2
3
Hz, 4H), 7.41 (s, 1H), 6.47 (s, 1H), 5.22 (s, 2H), 5.17 (s, 2H), 2.67 (s, 2H), 1.46 (s, 6H); 13C
NMR (101 MHz, CDCl3) δ 190.88, 156.63, 155.64, 143.04, 140.95, 139.99, 127.27, 127.09,
125.69, 125.66, 125.62, 125.58, 125.51, 125.47, 125.44, 125.40, 125.36, 112.98, 110.08, 102.45,
+
+
79.76, 70.58, 69.81, 48.35, 26.56; HRMS (ESI ), calcd C H F LiO (M + Li ) = 531.1582,
2
7
22
6
4
found = 531.1577.
6
,7ꢀBis((3ꢀfluoroꢀ4ꢀnitrobenzyl)oxy)ꢀ2,2ꢀdimethylchromanꢀ4ꢀone (4c). Yellow solid, yield
1
8
7%. H NMR (400 MHz, CDCl ) δ 8.08–8.00 (m, 2H), 7.40–7.33 (m, 2H), 7.31–7.28 (m, 2H),
3
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7
.19 (s, 1H), 6.35 (s, 1H), 5.16 (s, 2H), 5.12 (s, 2H), 2.60 (s, 2H), 1.37 (s, 6H); C NMR (101
MHz, CDCl ) δ 190.77, 157.11, 156.70, 154.71, 154.47, 146.04, 145.96, 144.93, 144.85, 142.42,
3
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26.70, 126.68, 126.51, 126.48, 122.27, 122.23, 122.19, 122.15, 116.50, 116.28, 113.29, 109.84,
0
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+
1
02.59, 80.02, 69.30, 68.74, 48.32, 26.64, 26.59; HRMS (ESI ), calcd C H F LiN O (M +
25
20
2
2
8
+
Li ) = 521.1348, found = 521.1359.
6
,7ꢀBis((3,4ꢀdifluorobenzyl)oxy)ꢀ2,2ꢀdimethylchromanꢀ4ꢀone (4d). White solid, yield 85%.
1
H NMR (400 MHz, CDCl ) δ 7.35 (s, 1H), 7.30–7.24 (m, 2H), 7.19–7.11 (m, 4H), 6.43 (s, 1H),
3
1
3
5.07 (s, 2H), 5.02 (s, 2H), 2.65 (s, 2H), 1.44 (s, 6H); C NMR (101 MHz, CDCl ) δ 190.85,
3
1
1
1
1
2
56.63, 155.58, 151.74, 151.66, 151.62, 151.53, 151.38, 151.26, 151.21, 151.09, 149.27, 149.19,
49.14, 149.06, 148.91, 148.79, 148.75, 148.62, 142.90, 134.01, 133.96, 133.92, 133.02, 132.96,
32.92, 123.20, 123.17, 123.14, 123.12, 123.10, 123.09, 123.06, 123.02, 117.59, 117.42, 117.34,
17.16, 116.36, 116.29, 116.18, 116.11, 112.93, 110.15, 102.41, 79.73, 70.17, 69.37, 48.32,
+
6.53; HRMS (ESI+), calcd C H F LiO (M + Li ) = 467.1458, found = 467.1453.
25 20
4
4
6
,7ꢀBis((3ꢀfluoroꢀ4ꢀmethoxybenzyl)oxy)ꢀ2,2ꢀdimethylchromanꢀ4ꢀone (4e). White solid,
1
yield 83%. H NMR (400 MHz, CDCl ) δ 7.38 (s, 1H), 7.21–7.13 (m, 4H), 7.01–6.93 (m, 2H),
3
1
3
6.44 (s, 1H), 5.07 (s, 2H), 5.02 (s, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 2.66 (s, 2H), 1.45 (s, 6H); C
NMR (101 MHz, CDCl ) δ 190.95, 156.57, 156.05, 153.60, 153.57, 151.15, 151.12, 147.62,
3
1
47.51, 147.39, 147.28, 143.15, 129.94, 129.88, 128.87, 128.81, 123.40, 123.37, 123.25, 123.22,
115.53, 115.38, 115.34, 115.19, 113.40, 113.20, 112.78, 110.42, 102.41, 79.63, 70.76, 69.91,
+
5
6.29, 56.27, 48.41, 26.63; HRMS (ESI+), calcd C H F LiO (M + Li ) = 491.1857, found =
2
7
26
2
6
4
91.1855.
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General Procedure for the Preparation of Compounds 5aꢀe Exemplified by 2,2ꢀ
Dimethylꢀ6,7ꢀbis((4ꢀnitrobenzyl)oxy)chromanꢀ4ꢀol (5a). A mixture of 4a (400 mg, 0.84
mmol) and sodium borohydride (64 mg, 1.67 mmol) in absolute methanol (20 mL) was stirred at
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0 C. After 3 h, the solution was poured into water and extracted with ethyl acetate. The organic
phase was dried with Na SO , filtered, concentrated, and purified by flash column
2
4
1
chromatography to afford the title compound 5a (325 mg, 81%) as a yellow solid. H NMR (400
MHz, CDCl ) δ 8.27–8.19 (m, 4H), 7.66–7.59 (m, 4H), 7.08 (s, 1H), 6.40 (s, 1H), 5.21 (s, 2H),
3
5.21 (s, 2H), 4.77 (dd, J = 14.3, 7.8 Hz, 1H), 2.17 (dd, J = 13.4, 6.1 Hz, 1H), 1.83 (dd, J = 13.4,
13
8
.7 Hz, 1H), 1.70 (d, J = 7.4 Hz, 1H), 1.43 (s, 3H), 1.30 (s, 3H); C NMR (101 MHz, CDCl ) δ
3
1
49.36, 148.48, 147.59, 147.52, 144.92, 144.25, 142.09, 127.57, 127.40, 123.85, 123.75, 116.55,
+
114.69, 103.30, 75.66, 71.06, 69.58, 63.49, 42.91, 28.80, 25.71; HRMS (ESI ), calcd
+
C H LiN O (M + Li ) = 487.1693, found = 487.1692.
2
5
24
2
8
2
,2ꢀDimethylꢀ6,7ꢀbis((4ꢀ(trifluoromethyl)benzyl)oxy)chromanꢀ4ꢀol (5b). White solid, yield
1
9
8%. H NMR (400 MHz, CDCl ) δ 7.65–7.60 (m, 4H), 7.55 (dd, J = 8.1, 2.9 Hz, 4H), 7.09 (s,
3
1
H), 6.43 (s, 1H), 5.13 (s, 2H), 5.10 (s, 2H), 4.74 (dd, J = 14.1, 7.1 Hz, 1H), 2.15 (d, J = 4.8 Hz,
1H), 2.12 (dd, J = 12.5, 5.2 Hz, 1H), 1.79 (dd, J = 13.4, 8.8 Hz, 1H), 1.42 (s, 3H), 1.30 (s, 3H);
1
3
C NMR (101 MHz, CDCl ) δ 149.79, 148.43, 142.36, 141.57, 140.95, 127.42, 127.18, 125.53,
3
1
25.49, 125.45, 125.41, 125.37, 125.33, 125.30, 116.42, 115.04, 103.28, 75.60, 71.67, 69.97,
+
+
63.44, 42.83, 28.81, 25.55; HRMS (ESI ), calcd C H F LiO (M + Li ) = 533.1739, found =
27
24
6
4
5
33.1743.
6
,7ꢀBis((3ꢀfluoroꢀ4ꢀnitrobenzyl)oxy)ꢀ2,2ꢀdimethylchromanꢀ4ꢀol (5c). Yellow solid, yield
1
8
8%. H NMR (400 MHz, CDCl ) δ 8.12–8.08 (m, 2H), 7.47–7.35 (m, 4H), 7.08 (s, 1H), 6.37 (s,
3
1
H), 5.18 (s, 4H), 4.81–4.76 (m, 1H), 2.18 (dd, J = 13.4, 6.1 Hz, 1H), 1.86–1.80 (m, 1H), 1.71
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(
d, J = 7.6 Hz, 1H), 1.44 (s, 3H), 1.31 (s, 3H); C NMR (101 MHz, CDCl ) δ 157.09, 154.45,
3
1
48.97, 148.63, 146.86, 146.78, 146.14, 146.06, 141.79, 126.51, 126.49, 126.38, 126.36, 122.33,
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0
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+
+
6
8.93, 63.43, 42.88, 28.82, 25.67; HRMS (ESI ), calcd C H F LiN O (M + Li ) = 523.1504,
25 22 2 2 8
found = 523.1508.
6
,7ꢀBis((3,4ꢀdifluorobenzyl)oxy)ꢀ2,2ꢀdimethylchromanꢀ4ꢀol (5d). White solid, yield 75%.
1
H NMR (400 MHz, CDCl ) δ 7.28–7.23 (m, 2H), 7.16–7.08 (m, 4H), 7.04 (s, 1H), 6.39 (s, 1H),
3
4.98 (s, 2H), 4.94 (s, 2H), 4.70 (dd, J = 14.1, 7.0 Hz, 1H), 2.47 (d, J = 6.8 Hz, 1H), 2.09 (dd, J =
13
1
3.4, 6.2 Hz, 1H), 1.76 (dd, J = 13.4, 8.9 Hz, 1H), 1.41 (s, 3H), 1.28 (s, 3H); C NMR (101
MHz, CDCl ) δ 151.67, 151.59, 151.54, 151.46, 151.23, 151.16, 151.10, 151.04, 149.68, 149.20,
3
1
1
1
6
4
49.12, 149.07, 148.99, 148.76, 148.70, 148.63, 148.58, 148.43, 142.19, 134.61, 134.56, 134.52,
33.98, 133.93, 133.89, 123.39, 123.36, 123.33, 123.30, 123.19, 123.15, 123.13, 123.09, 117.39,
17.22, 117.04, 116.56, 116.46, 116.39, 116.34, 116.16, 115.16, 103.24, 75.62, 71.28, 69.54,
+
+
3.34, 42.75, 28.84, 25.48; HRMS (ESI ), calcd C H F LiO (M + Li ) = 469.1614, found =
25
22
4
4
69.1612.
6,7ꢀBis((3ꢀfluoroꢀ4ꢀmethoxybenzyl)oxy)ꢀ2,2ꢀdimethylchromanꢀ4ꢀol (5e). White solid, yield
1
7
1%. H NMR (400 MHz, CDCl ) δ 7.20–7.10 (m, 4H), 7.03 (s, 1H), 6.96–6.89 (m, 2H), 6.40 (s,
3
1
H), 4.97 (s, 2H), 4.93 (s, 2H), 4.74–4.67 (m, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 2.14 (d, J = 5.7 Hz,
1H), 2.09 (dd, J = 13.4, 6.1 Hz, 1H), 1.77 (dd, J = 13.4, 8.7 Hz, 1H), 1.40 (s, 3H), 1.28 (s, 3H);
13
C NMR (101 MHz, CDCl ) δ 153.53, 153.46, 151.08, 151.02, 150.02, 148.24, 147.34, 147.26,
3
1
47.24, 147.15, 142.47, 130.59, 130.53, 129.95, 129.89, 123.57, 123.54, 123.33, 123.30, 116.17,
15.71, 115.52, 115.46, 115.27, 115.25, 113.29, 113.13, 103.33, 75.45, 71.75, 70.02, 63.43,
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+
+
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6.26, 56.23, 42.80, 28.81, 25.69; HRMS (ESI ), calcd C H F LiO (M + Li ) = 493.2014,
2
7
28
2
6
found = 493.2016.
General Procedure for the Preparation of Compounds 6aꢀh Exemplified by 2,2ꢀ
Dimethylꢀ6,7ꢀbis((4ꢀnitrobenzyl)oxy)ꢀ2Hꢀchromene (6a). Compound 5a (200 mg, 0.42 mmol)
was dissolved in acetone (10 mL) and then 4 N HCl (0.8 mL) was added. The reaction mixture
was stirred overnight at room temperature. The reaction was basified with saturated NaHCO3
solution and extracted with ethyl acetate. The combined organic layers were dried with Na SO ,
10
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56
57
58
59
60
2
4
filtered, concentrated, and purified by flash column chromatography to afford the title compound
1
6
4
=
a (184 mg, 96%) as a yellow solid. H NMR (400 MHz, CDCl ) δ 8.25 (dd, J = 8.3, 3.1 Hz,
3
H), 7.62 (dd, J = 8.3, 3.1 Hz, 4H), 6.62 (s, 1H), 6.45 (s, 1H), 6.21 (d, J = 9.7 Hz, 1H), 5.53 (d, J
1
3
9.7 Hz, 1H), 5.22 (s, 2H), 5.19 (s, 2H), 1.42 (s, 6H); C NMR (101 MHz, CDCl ) δ 149.12,
3
1
48.44, 147.61, 147.54, 144.93, 144.23, 141.98, 129.19, 127.59, 127.44, 123.85, 123.75, 121.48,
+
1
14.61, 114.35, 103.50, 76.35, 71.41, 69.76, 29.72, 27.76; HRMS (ESI ), calcd C H LiN O
25 22
2
7
+
(M + Li ) = 469.1587, found = 469.1594.
2
,2ꢀDimethylꢀ6,7ꢀbis((4ꢀ(trifluoromethyl)benzyl)oxy)ꢀ2Hꢀchromene (6b). White solid,
1
yield 97%. H NMR (400 MHz, CDCl ) δ 7.67–7.61 (m, 4H), 7.55 (d, J = 8.1 Hz, 4H), 6.65 (s,
3
1
H), 6.48 (s, 1H), 6.22 (d, J = 9.7 Hz, 1H), 5.53 (d, J = 9.7 Hz, 1H), 5.17 (s, 2H), 5.13 (s, 2H),
1
3
1
.43 (s, 6H); C NMR (101 MHz, CDCl ) δ 149.58, 148.34, 142.33, 141.63, 140.95, 128.92,
3
127.38, 127.22, 125.43 (dq, J = 10.9, 3.8 Hz), 121.65, 114.60, 114.40, 103.56, 76.24, 71.98,
+
+
7
0.20, 27.74; HRMS (ESI ), calcd C H F LiO (M + Li ) = 515.1633, found = 515.1635.
27 22 6 3
6
,7ꢀBis((3ꢀfluoroꢀ4ꢀnitrobenzyl)oxy)ꢀ2,2ꢀdimethylꢀ2Hꢀchromene (6c). Yellow solid, yield
1
9
0%. H NMR (400 MHz, CDCl ) δ 8.13–8.08 (m, 2H), 7.47–7.39 (m, 2H), 7.37–7.34 (m, 2H),
3
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.62 (s, 1H), 6.43 (s, 1H), 6.21 (d, J = 9.7 Hz, 1H), 5.55 (d, J = 9.7 Hz, 1H), 5.19 (s, 2H), 5.15 (s,
1
3
H), 1.42 (s, 6H); C NMR (101 MHz, CDCl ) δ 157.08, 154.39, 148.75, 148.61, 146.84,
3
46.76, 146.10, 146.02, 141.74, 129.43, 126.50, 126.47, 126.38, 126.35, 122.32, 122.28, 122.25,
22.21, 121.35, 116.59, 116.50, 116.37, 116.29, 114.88, 114.24, 103.51, 76.44, 70.73, 69.15,
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
+
+
27.78; HRMS (ESI ), calcd C H F LiN O (M + Li ) = 505.1399, found = 505.1407.
2
5
20
2
2
7
6
,7ꢀBis((3,4ꢀdifluorobenzyl)oxy)ꢀ2,2ꢀdimethylꢀ2Hꢀchromene (6d). White solid, yield 90%.
1
H NMR (400 MHz, CDCl ) δ 7.29 (dt, J = 16.5, 5.3 Hz, 2H), 7.19–7.11 (m, 4H), 6.64 (s, 1H),
3
6.48 (s, 1H), 6.23 (d, J = 9.7 Hz, 1H), 5.53 (d, J = 9.7 Hz, 1H), 5.03 (s, 2H), 4.99 (s, 2H), 1.44 (s,
13
6H); C NMR (101 MHz, CDCl ) δ 151.61 (dd, J = 12.7, 6.2 Hz), 151.18 (dd, J = 12.6, 7.4 Hz),
3
149.56, 149.14 (dd, J = 12.7, 6.0 Hz), 148.71 (dd, J = 12.6, 7.1 Hz), 148.39, 142.25, 134.66 (dd,
J = 5.3, 4.0 Hz), 133.98 (dd, J = 5.4, 4.0 Hz), 128.93, 123.25 (ddd, J = 12.3, 6.4, 3.7 Hz), 121.66,
117.48–117.00 (m), 116.40 (dd, J = 17.7, 15.5 Hz), 114.70, 114.43, 103.59, 76.23, 71.57, 69.79,
+
+
27.73; HRMS (ESI ), calcd C H F LiO (M + Li ) = 451.1509, found = 451.1508.
25 20
4
3
6
,7ꢀBis((3ꢀfluoroꢀ4ꢀmethoxybenzyl)oxy)ꢀ2,2ꢀdimethylꢀ2Hꢀchromene (6e). White solid,
1
yield 88%. H NMR (400 MHz, CDCl ) δ 7.24–7.07 (m, 4H), 6.94 (dt, J = 10.9, 8.5 Hz, 2H),
3
6.62 (s, 1H), 6.47 (s, 1H), 6.21 (d, J = 9.7 Hz, 1H), 5.50 (d, J = 9.7 Hz, 1H), 5.01 (s, 2H), 4.96 (s,
13
2H), 3.90 (s, 3H), 3.89 (s, 3H), 1.42 (s, 6H); C NMR (101 MHz, CDCl ) δ 153.56, 153.50,
3
151.12, 151.06, 149.93, 148.22, 147.40, 147.30, 147.19, 142.46, 130.68, 130.62, 129.97, 129.91,
128.66, 123.54, 123.51, 123.39, 123.36, 121.81, 115.71, 115.52, 115.34, 114.96, 114.16, 113.30,
113.16, 103.65, 76.12, 72.07, 70.24, 56.27, 56.25, 27.76; HRMS (ESI+), calcd C H F LiO (M
27 26 2 5
+
+
Li ) = 475.1908, found = 475.1909.
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,7ꢀBis(benzyloxy)ꢀ2,2ꢀdimethylꢀ2Hꢀchromene (6f). White solid, yield 97%. H NMR (400
MHz, CDCl ) δ 7.53–7.45 (m, 4H), 7.45–7.32 (m, 6H), 6.68 (s, 1H), 6.55 (s, 1H), 6.25 (d, J = 9.7
3
1
3
Hz, 1H), 5.52 (d, J = 9.7 Hz, 1H), 5.16 (s, 2H), 5.11 (s, 2H), 1.46 (s, 6H). C NMR (101 MHz,
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11
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49
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60
CDCl ) δ 150.23, 148.15, 142.76, 137.75, 137.09, 128.51, 128.42, 127.84, 127.75, 127.61,
3
+
127.35, 121.95, 114.93, 114.01, 103.64, 76.09, 72.94, 71.05, 27.81; HRMS (ESI ), calcd
+
C H LiO (M + Li ) = 379.1885, found = 379.1885.
25
24
3
1
2
,2ꢀDimethylꢀ6,7ꢀbis((3ꢀnitrobenzyl)oxy)ꢀ2Hꢀchromene (6g). Yellow solid, yield 95%. H
NMR (400 MHz, CDCl ) δ 8.34 (d, J = 1.8 Hz, 2H), 8.21–8.13 (m, 2H), 7.85–7.76 (m, 2H), 7.57
3
(dd, J = 15.4, 7.6 Hz, 2H), 6.68 (s, 1H), 6.50 (s, 1H), 6.24 (d, J = 9.7 Hz, 1H), 5.54 (d, J = 9.7
Hz, 1H), 5.19 (s, 2H), 5.16 (s, 2H), 1.43 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 149.21,
1
48.45, 148.39, 148.33, 142.06, 139.63, 139.00, 133.14, 132.97, 129.64, 129.52, 129.13, 122.93,
22.79, 122.09, 121.92, 121.56, 114.60, 114.32, 103.47, 76.33, 71.32, 69.65, 27.76; HRMS
1
+
+
(
ESI ), calcd C H LiN O (M + Li ) = 469.1587, found = 469.1584.
25 22 2 7
1
2
,2ꢀDimethylꢀ6,7ꢀbis((2ꢀnitrobenzyl)oxy)ꢀ2Hꢀchromene (6h). Yellow solid, yield 87%. H
NMR (400 MHz, CDCl ) δ 8.23–8.15 (m, 2H), 8.05 (dd, J = 7.9, 1.0 Hz, 1H), 7.94 (dd, J = 7.9,
3
1.0 Hz, 1H), 7.73–7.64 (m, 2H), 7.53–7.47 (m, 2H), 6.69 (s, 1H), 6.51 (s, 1H), 6.25 (d, J = 9.7
13
Hz, 1H), 5.55 (s, 2H), 5.52 (d, J = 9.7 Hz, 1H), 5.51 (s, 2H), 1.43 (s, 6H); C NMR (101 MHz,
CDCl ) δ 149.06, 148.33, 146.86, 146.75, 142.06, 134.46, 134.04, 133.95, 133.86, 129.03,
3
128.75, 128.49, 128.31, 128.21, 125.01, 124.85, 121.62, 114.52, 113.89, 103.44, 76.30, 69.26,
+
+
6
7.94, 27.76; HRMS (ESI ), calcd C H LiN O (M + Li ) = 469.1587, found = 469.1582.
25 22 2 7
7
ꢀMethoxyꢀ2,2ꢀdimethylꢀ6ꢀ((4ꢀnitrobenzyl)oxy)chromanꢀ4ꢀone (7). To a solution of 3 (400
mg, 1.92 mmol) and potassium carbonate (292 mg, 2.11 mmol) in N,Nꢀdimethylformamide (20
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6
o
mL) was added methyl iodide (0.12 ml, 1.92 mmol). The reaction mixture was stirred at 80 C
for 8 h. When the reaction was completed, the mixture was cooled to room temperature, and then
4
ꢀnitrobenzyl bromide (415 mg, 1.92 mmol) and potassium carbonate (292 mg, 2.11 mmol) was
0
1
2
3
4
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9
0
1
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7
8
9
0
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7
8
9
0
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2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
added. The resultant mixture was stirred at room temperature overnight. The solution was poured
into water and extracted with ethyl acetate. The organic phase was dried with Na SO , filtered,
2
4
concentrated, and purified by flash column chromatography to afford the title compound 7 (617
1
mg, 90%) as a yellow solid. H NMR (400 MHz, CDCl ) δ 8.22 (d, J = 8.8 Hz, 2H), 7.61 (d, J =
3
1
3
8.9 Hz, 2H), 7.29 (s, 1H), 6.43 (s, 1H), 5.18 (s, 2H), 3.91 (s, 3H), 2.64 (s, 2H), 1.44 (s, 6H); C
NMR (101 MHz, CDCl ) δ 190.80, 156.94, 156.90, 147.54, 144.28, 142.47, 127.63, 123.77,
3
+
1
12.29, 109.29, 100.95, 79.71, 69.96, 56.22, 48.31, 26.60; HRMS (ESI ), calcd C H LiNO (M
19 19
6
+
+ Li ) = 364.1372, found = 364.1370.
Synthesis of 7ꢀMethoxyꢀ2,2ꢀdimethylꢀ6ꢀ((4ꢀnitrobenzyl)oxy)chromanꢀ4ꢀol (8). A mixture
of 7 (400 mg, 1.12 mmol) and sodium borohydride (85 mg, 2.24 mmol) in absolute methanol (20
o
mL) was stirred at 60 C. After 3 h, the solution was poured into water and extracted with ethyl
acetate. The organic phase was dried with Na SO , filtered, concentrated, and purified by flash
2
4
1
column chromatography to afford the title compound 8 (350 mg, 87%) as a yellow solid. H
NMR (400 MHz, CDCl ) δ 8.17 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 6.96 (s, 1H), 6.35
3
(
s, 1H), 5.09 (s, 2H), 4.68 (dd, J = 14.3, 7.1 Hz, 1H), 3.80 (s, 3H), 2.31 (d, J = 7.0 Hz, 1H), 2.07
1
3
(dd, J = 13.4, 6.1 Hz, 1H), 1.76 (dd, J = 13.4, 8.7 Hz, 1H), 1.39 (s, 3H), 1.26 (s, 3H); C NMR
(
101 MHz, CDCl ) δ 150.80, 148.48, 147.35, 145.14, 141.52, 127.67, 123.63, 115.23, 114.16,
3
+
1
01.17, 75.50, 70.88, 63.33, 55.81, 42.80, 28.83, 25.60; HRMS (ESI ), calcd C H LiNO (M +
19 21
6
+
Li ) = 366.1529, found = 366.1533.
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Page 23 of 53
Journal of Medicinal Chemistry
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Synthesis
of
7ꢀMethoxyꢀ2,2ꢀdimethylꢀ6ꢀ((4ꢀnitrobenzyl)oxy)ꢀ2Hꢀchromene
(9).
Compound 8 (200 mg, 0.56 mmol) was dissolved in acetone (10 mL) and then 4 N HCl (0.8 mL)
was added. The reaction mixture was stirred overnight at room temperature. The reaction was
10
11
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13
14
15
16
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basified with saturated NaHCO solution and extracted with ethyl acetate. The combined organic
3
layers were dried with Na SO , filtered, concentrated, and purified by flash column
2
4
1
chromatography to afford the title compound 9 (176 mg, 93%) as a yellow solid. H NMR (400
MHz, CDCl ) δ 8.22 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.9 Hz, 2H), 6.55 (s, 1H), 6.45 (s, 1H), 6.18
3
1
3
(d, J = 9.6 Hz, 1H), 5.48 (d, J = 9.7 Hz, 1H), 5.14 (s, 2H), 3.85 (s, 3H), 1.42 (s, 6H); C NMR
(
101 MHz, CDCl ) δ 150.77, 148.53, 147.44, 145.20, 141.42, 128.44, 127.66, 123.67, 121.64,
3
+
+
1
13.86, 113.26, 101.40, 76.19, 71.26, 55.94, 27.77; HRMS (ESI ), calcd C H LiNO (M + Li )
19 19 5
= 348.1423, found = 348.1422.
2
,2ꢀDimethylꢀ7ꢀ((4ꢀnitrobenzyl)oxy)ꢀ2Hꢀchromenꢀ6ꢀol (12) was prepared using a similar
1
procedure to compound 9. Yellow solid, yield 68% from compound 3. H NMR (400 MHz,
CDCl ) δ 8.28 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.9 Hz, 2H), 6.64 (s, 1H), 6.40 (s, 1H), 6.25 (d, J
3
13
=
9.7 Hz, 1H), 5.54 (d, J = 9.7 Hz, 1H), 5.20 (s, 2H), 1.41 (s, 6H); C NMR (101 MHz, CDCl )
3
δ 146.44, 145.15, 144.23, 143.52, 139.50, 129.31, 127.88, 123.98, 121.80, 115.18, 112.34,
+
+
1
01.55, 69.80, 27.59; HRMS (ESI ), calcd C H LiNO (M + Li ) = 334.1267, found =
18 17 5
334.1262.
2,2ꢀDiethylꢀ6,7ꢀbis((4ꢀnitrobenzyl)oxy)ꢀ2Hꢀchromene (17a). Yellow solid, total yield 28%.
1
H NMR (400 MHz, CDCl ) δ 8.25 (dd, J = 8.8, 4.5 Hz, 4H), 7.62 (dd, J = 8.9, 2.1 Hz, 4H), 6.59
3
(s, 1H), 6.44 (s, 1H), 6.30 (d, J = 10.0 Hz, 1H), 5.41 (d, J = 10.0 Hz, 1H), 5.22 (s, 2H), 5.17 (s,
1
3
2
H), 1.77–1.68 (m, 2H), 1.65–1.59 (m, 2H), 0.93 (t, J = 7.4 Hz, 6H); C NMR (101 MHz,
CDCl ) δ 149.49, 149.15, 147.59, 147.51, 145.04, 144.30, 141.61, 127.58, 127.48, 126.64,
3
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1
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2
2
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2
2
2
2
2
2
2
3
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5
5
5
6
+
1
23.81, 123.73, 122.90, 114.56, 114.35, 102.97, 82.02, 71.49, 69.77, 32.08, 7.99; HRMS (ESI ),
+
calcd C H LiN O (M + Li ) = 497.1900, found = 497.1909.
27 26
2
7
6
,7ꢀBis((4ꢀnitrobenzyl)oxy)spiro[chromeneꢀ2,1'ꢀcyclohexane] (17b). Yellow solid, total
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
yield 33%. H NMR (400 MHz, CDCl ) δ 8.25 (dd, J = 8.8, 6.1 Hz, 4H), 7.62 (dd, J = 8.9, 2.0
3
Hz, 4H), 6.62 (s, 1H), 6.51 (s, 1H), 6.22 (d, J = 9.7 Hz, 1H), 5.57 (d, J = 9.8 Hz, 1H), 5.23 (s,
1
3
2
H), 5.18 (s, 2H), 1.95–1.86 (m, 2H), 1.77–1.68 (m, 2H), 1.60–1.33 (m, 6H); C NMR (101
MHz, CDCl ) δ 149.06, 148.41, 147.58, 147.50, 145.00, 144.29, 142.02, 128.94, 127.59, 127.49,
3
123.82, 123.73, 121.90, 115.53, 114.36, 103.68, 77.00, 71.39, 69.79, 35.74, 25.29, 21.35; HRMS
+
+
(ESI ), calcd C H LiN O (M + Li ) = 509.1900, found = 509.1900.
28 26 2 7
6
,7ꢀBis((4ꢀnitrobenzyl)oxy)spiro[chromeneꢀ2,1'ꢀcyclopentane] (17c). Yellow solid, yield
1
3
0%. H NMR (400 MHz, CDCl ) δ 8.25 (dd, J = 8.8, 4.5 Hz, 4H), 7.64–7.60 (m, 4H), 6.62 (s,
3
1
H), 6.44 (s, 1H), 6.24 (d, J = 9.7 Hz, 1H), 5.58 (d, J = 9.7 Hz, 1H), 5.21 (s, 2H), 5.18 (s, 2H),
13
2.17–2.10 (m, 2H), 1.91–1.84 (m, 2H), 1.74–1.67 (m, 2H), 1.62–1.56 (m, 2H); C NMR (101
MHz, CDCl ) δ 148.97, 148.53, 147.59, 147.51, 144.97, 144.27, 142.03, 128.28, 127.59, 127.47,
3
1
23.83, 123.74, 122.24, 115.41, 114.31, 103.65, 87.30, 71.40, 69.78, 39.16, 23.54; HRMS
+
+
(ESI ), calcd C H LiN O (M + Li ) = 495.1744, found = 495.1745.
2
7
24
2
7
6
,7ꢀBis((4ꢀnitrobenzyl)oxy)spiro[chromeneꢀ2,1'ꢀcycloheptane] (17d). Yellow solid, total
1
yield 24%. H NMR (400 MHz, CDCl ) δ 8.24 (dd, J = 8.8, 2.1 Hz, 4H), 7.62 (dd, J = 8.8, 3.6
3
Hz, 4H), 6.61 (s, 1H), 6.47 (s, 1H), 6.18 (d, J = 9.7 Hz, 1H), 5.59 (d, J = 9.7 Hz, 1H), 5.21 (s,
13
2
H), 5.18 (s, 2H), 1.81–1.55 (m, 12H); C NMR (101 MHz, CDCl ) δ 149.03, 148.39, 147.94,
3
1
47.51, 145.00, 144.31, 141.97, 129.90, 127.59, 127.49, 123.81, 123.73, 120.52, 115.28, 114.27,
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9
+
+
1
03.80, 81.05, 71.40, 69.79, 39.14, 29.56, 21.59; HRMS (ESI ), calcd C H LiN O (M + Li ) =
2
9
28
2
7
523.2057, found = 523.2070.
General Procedure for the Preparation of Compounds 24aꢀe Exemplified by 6,7ꢀBis((4ꢀ
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nitrobenzyl)oxy)ꢀ2',3',5',6'ꢀtetrahydrospiro[chromeneꢀ2,4'ꢀpyran] (17e). A mixture of 1ꢀ(2ꢀ
hydroxyꢀ4,5ꢀdimethoxyphenyl)ethanꢀ1ꢀone 1 (600 mg, 3.06 mmol), pyrrolidine (0.23 mL,
4.59mmol), tetrahydroꢀ4Hꢀpyranꢀ4ꢀone (0.32 mL, 4.59 mmol) and methanol (20 mL) was stirred
at room temperature overnight. The reaction mixture was then concentrated in vacuo and the
residue
purified
by
flash
chromatography
to
give
6,7ꢀdimethoxyꢀ2',3',5',6'ꢀ
1
tetrahydrospiro[chromaneꢀ2,4'ꢀpyran]ꢀ4ꢀone 13e (749 mg, 88%) as a white solid. H NMR (400
MHz, CDCl ) δ 7.26 (s, 1H), 6.47 (s, 1H), 3.93 (s, 3H), 3.87 (s, 3H), 3.86–3.80 (m, 2H), 3.79–
3
+
3
.74 (m, 2H), 2.68 (s, 2H), 2.02–1.97 (m, 2H), 1.80–1.73 (m, 2H); MS (ESI ) m/z 279.1
+
[
M+H] . To a solution of 13e (600 mg, 2.16 mmol) in dichloromethane (20 mL) was slowly
o
added boron tribromide (1M in dichloromethane, 6.47 mL, 6.47 mmol) at 0 C under nitrogen.
Then the reaction mixture was allowed to warm to room temperature and stirred overnight. The
resultant mixture was poured into ice water, extracted with ethyl acetate for three times. The
combined organic layers were dried with Na SO , filtered, concentrated, and purified by flash
2
4
column chromatography to give 6,7ꢀdihydroxyꢀ2',3',5',6'ꢀtetrahydrospiro[chromaneꢀ2,4'ꢀpyran]ꢀ
1
4
ꢀone 14e (329 mg, 61%) as a white solid. H NMR (400 MHz, CD OD) δ 7.16 (s, 1H), 6.37 (s,
3
1H), 3.75–3.67 (m, 2H), 3.59–3.48 (m, 2H), 2.77 (s, 2H), 2.41–2.31 (m, 2H), 2.04–1.96 (m, 2H);
+
+
MS (ESI ) m/z 251.1 [M+H] . To a mixture of 14e (200 mg, 0.80 mmol) in dry THF (20 mL)
o
was added portionꢀwise lithium aluminium hydride (91 mg, 1.67 mmol) at 0 C. The reaction
mixture was stirred at room temperature for 2 h then poured into water and extracted with ethyl
acetate. The organic phase was concentrated to dryness to afford 15e, which was carried out to
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the next step without further purification. Compound 15e was dissolved in acetone (10 mL) and
then 4 N HCl (0.8 mL) was added. The reaction mixture was stirred at room temperature
overnight. The reaction was basified with saturated NaHCO solution and extracted with ethyl
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
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0
acetate. The combined organic layers were dried with Na SO , filtered, concentrated, and
2
4
purified by flash column chromatography to afford 2',3',5',6'ꢀtetrahydrospiro[chromeneꢀ2,4'ꢀ
1
pyran]ꢀ6,7ꢀdiol 16e (126 mg, 67% from compound 14e) as a white solid. H NMR (400 MHz,
CDCl ) δ 6.56 (s, 1H), 6.46 (s, 1H), 6.29 (d, J = 9.7 Hz, 1H), 6.21 (s, 1H), 5.50 (s, 1H), 5.49 (d, J
3
= 9.7 Hz, 1H), 3.98–3.91 (m, 2H), 3.81–3.76 (m, 2H), 2.00–1.95 (m, 2H), 1.81–1.75 (m, 2H);
+
+
MS (ESI ) m/z 235.1 [M+H] . A mixture of 16e (100 mg, 0.43 mmol) and potassium carbonate
(354 mg, 2.56 mmol) in acetone (20 mL) was treated with 4ꢀnitrobenzyl bromide (203 mg, 0.94
o
mmol) and stirred at 60 C under nitrogen overnight. The reaction mixture was filtered,
concentrated, and purified by flash column chromatography to give the title compound 17e (183
1
mg, 85%) as a yellow solid. H NMR (400 MHz, CDCl ) δ 8.26 (t, J = 8.6 Hz, 4H), 7.63 (dd, J =
3
8
.8, 6.4 Hz, 4H), 6.64 (s, 1H), 6.55 (s, 1H), 6.29 (d, J = 9.7 Hz, 1H), 5.54 (d, J = 9.7 Hz, 1H),
.24 (s, 2H), 5.19 (s, 2H), 3.89 (td, J = 11.2, 2.6 Hz, 2H), 3.76 (dt, J = 11.6, 4.2 Hz, 2H), 1.95 (d,
5
1
3
J = 12.7 Hz, 2H), 1.81–1.74 (m, 2H); C NMR (101 MHz, CDCl ) δ 149.31, 147.85, 147.66,
3
147.57, 144.80, 144.07, 142.37, 127.57, 127.50, 123.86, 123.77, 122.76, 115.13, 114.45, 103.71,
+
+
7
4.20, 71.35, 69.84, 63.24, 35.69; HRMS (ESI ), calcd C H LiN O (M + Li ) = 511.1693,
27 24 2 8
found = 511.1697.
Synthesis of 2,2ꢀDimethylꢀ2Hꢀchromeneꢀ6,7ꢀdiyl bis(4ꢀnitrobenzoate) (18). To a mixture
of 2,2ꢀdimethylꢀ2Hꢀchromeneꢀ6,7ꢀdiol 16f (100 mg, 0.52 mmol) and triethylamine (0.18 mL,
1.30 mmol) in dichloromethane (20 mL) was added 4ꢀnitrobenzoyl chloride (241 mg, 1.30
mmol), and the reaction was stirred overnight. The reaction mixture was concentrated and
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purified by flash column chromatography to give the title compound 18 (189 mg, 74%) as a
1
yellow solid. H NMR (400 MHz, CDCl ) δ 8.27–8.22 (m, 8H), 7.02 (s, 1H), 6.82 (s, 1H), 6.32
3
1
3
(
d, J = 9.9 Hz, 1H), 5.71 (d, J = 9.9 Hz, 1H), 1.49 (s, 6H); C NMR (101 MHz, CDCl ) δ
3
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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59
60
1
62.61, 162.12, 151.54, 150.98, 150.95, 141.44, 135.04, 134.04, 133.91, 131.76, 131.17, 131.11,
+
123.78, 123.77, 120.87, 120.13, 120.08, 111.25, 77.07, 28.13; HRMS (ESI ), calcd
+
C H LiN O (M + Li ) = 497.1172, found = 497.1171.
25
18
2
9
Reagents. TLR ligands Pam CSK , Pam CSK , poly I:C, lipopolysaccharide, flagellin, R848,
3
4
2
4
ODN2006 were purchased from InvivoGen (San Diego, CA, USA). Phorbol 12ꢀmyristate 13ꢀ
acetate (PMA) and WSTꢀ1 proliferation reagent were purchased from SigmaꢀAldrich (St. Louis,
2
MO, USA). RNeasy Mini kit and RT First Strand kit were purchased from QIAGEN (Valencia,
CA, USA). SsoAdvanced Universal SYBR Green Supermix for qPCR was purchased from
BioRad (Hercules, CA, USA). The primers for TNFꢀα (Accession #: NM_000594), ILꢀ6
(Accession #: NM_000600), ILꢀ8 (Accession #: NM_000584), and GAPDH (Accession #:
NM_002046) were purchased from QIAGEN (Valencia, CA, USA). ELISA kit and FITC
Annexin V Apoptosis Detection kit were purchased from BD Biosciences (San Jose, CA, USA).
Cell Cycle Phase Determination kit was purchased from Cayman Chemical (Ann Arbor, MI,
USA). All the synthesized small molecules were dissolved in dimethyl sulfoxide at 10 mM as a
stock solution.
Cell culture. HEK293 cells stably transfected with human TLR2, TLR3, TLR4, TLR5,
TLR7, TLR8, or TLR9 and a SEAP reporter gene were obtained from InvivoGen. SEAP
expression is maintained by culturing the cells in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 1% PenStrep (100 U/mL Penicillin and 100
ꢁ
g/mL Streptomycin) containing selective antibiotics such as Zeocin and/or Blasticidin
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(
InvivoGen) according to the manufacturer's instructions. THPꢀ1 cells were cultured in RPMI
1
640 medium supplemented with 10% FBS and 1% PenStrep. Human cervical adenocarcinoma
HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% PenStrep. All the cells
0
1
2
3
4
5
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7
8
9
0
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2
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2
3
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9
0
o
were maintained in a humidified incubator containing 5% CO at 37 C.
2
Highꢀthroughput screening. The HTS was carried out at the HTS core facility of the
University of Colorado at Boulder. Briefly, hTLR3 HEK293 cells were seeded at 20,000
cells/well in duplicate in 384ꢀwell plates. Poly I:C was used as a positive control at 5 µg/mL.
Various instruments such as CyBiꢀWell, 96ꢀ and 384ꢀchannel simultaneous pipettor, precision
microplate pipetting system, EL406 combination washer dispenser, PlateLoc Thermal microplate
sealer and EnVision multilabel plate reader were used to carry out the screening. The library
compounds were dissolved in DMSO and used at 30 µM in the screening.
NFꢀκB activation by QUANTIꢀBlue SEAP assay. HEK293 cells seeded at a density of
4
o
5×10 cells/well in 96ꢀwell plates for 24 h at 37 C before treatment. The positive control ligands
for different TLRs are Pam CSK for TLR1/2, poly I:C for TLR3, lipopolysaccharide for TLR4,
3
4
FLAꢀBS for TLR5, R848 for TLR7 and TLR8, and ODN2006 for TLR9. After treatment for 24
h, a sample buffer (20 µL) from each well of the cell culture supernatants was collected and
transferred to a transparent 96ꢀwell plate. Each well was treated with 180 µL of QUANTIꢀBlue
o
(
InvivoGen) buffer and incubated at 37 C for 1 h. Optical density was measured using a plate
reader (Beckman Coulter, DTX 880) at an absorbance of 620 nm.
3
WSTꢀ1 assay for cell growth inhibition. Cells were seeded at a density of 5×10 cells/well
in 100 ꢁL of culture medium in 96ꢀwell plates and incubated for 24 h. A series of concentrations
of indicated compounds were added to the wells and incubated for additional 24, 48, or 72 h.
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WSTꢀ1 proliferation reagent (10 ꢁL/well) was added and incubated at 37 C until a color change
was observed (30 min to 2 h). Data were quantified on a BeckmanꢀCoulter DTX 880 Multimode
Detector using absorbance at 450 nm. Growth inhibition (%) was determined using the following
formula: Growth inhibition (%) = (1 – [Compounds (OD ) – Background (OD )]/[Control
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4
50
450
(OD ) – Background (OD )]) × 100.
4
50
450
6
qRTꢀPCR. THPꢀ1 cells were seeded at a density of 1.5×10 cells/well in RPMI 1640 growth
medium in 6‑ well plates. Cells were differentiated with PMA (20 nM) for 2 days then grown in
fresh RPMI 1640 medium for additional 24 h. Nonadherent cells and medium were removed and
replaced with fresh RPMI 1640 medium. The cells were treated with the indicated concentrations
of compounds then incubated for 12 h. Cells were collected after washing with phosphate
o
buffered saline, and frozen at ꢀ70 C until ready for assay. Total RNA was extracted with an
RNeasy Mini kit (QIAGEN) according to the manufacturer’s instruction. Reverse transcription
2
was performed on a BioRad T100 thermal cycler with a Qiagen RT First Strand kit. qPCR was
performed on a BioꢀRad CFX96 RealꢀTime PCR system using the SYBR Green method. The
data were analyzed by ∆∆C method.
t
6
Cytokineꢀspecific ELISA. THPꢀ1 cells were seeded at a density of 1.5×10 cells/well in
RPMI 1640 growth medium in 6‑ well plates. Cells were differentiated with PMA (20 nM) for 2
days then grown in fresh RPMI 1640 medium for additional 24 h. Nonadherent cells and medium
were removed and replaced with fresh RPMI 1640 medium. The cells were treated with the
indicated concentrations of compounds then incubated for 24 h. The cell culture supernatants
o
were collected and frozen at ꢀ70 C until ready for cytokine measurement with a ELISA kit (BD
Biosciences) per manufacturer’s specifications.
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