Design and structural optimization of novel 2H-benzo[h]chromene derivatives that target AcrB and reverse bacterial multidrug resistance

Article abstract of DOI:10.1016/j.ejmech.2020.113049

Drug efflux pumps have emerged as a new drug targets for the treatment of bacterial infections in view of its critical role in promoting multidrug resistance. Herein, novel chromanone and 2H-benzo[h]chromene derivatives were designed by means of integrated molecular design and structure-based pharmacophore modeling in an attempt to identify improved efflux pump inhibitors that target Escherichia coli AcrB. The compounds were tested for their efflux inhibitory activity, ability to inhibit efflux, and the effect on bacterial outer and inner membranes. Twenty-three novel structures were identified that synergized with antibacterials tested, inhibited Nile Red efflux, and acted specifically on the AcrB. Among them, WK2, WL7 and WL10 exhibiting broad-spectrum and high-efficiency efflux inhibitory activity were identified as potential ideal AcrB inhibitors. Molecular modeling further revealed that the strong π-π stacking interactions and hydrogen bond networks were the major contributors to tight binding of AcrB.

Full text of DOI:10.1016/j.ejmech.2020.113049

Journal Pre-proof
Design and structural optimization of novel 2H-benzo[h]chromene derivatives that
target AcrB and reverse bacterial multidrug resistance
Yinhu Wang, Rawaf Alenazy, Xinjie Gu, Steven W. Polyak, Panpan Zhang, Matthew
J. Sykes, Na Zhang, Henrietta Venter, Shutao Ma
PII:
S0223-5234(20)31021-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.113049
EJMECH 113049
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 August 2020
Revised Date: 21 October 2020
Accepted Date: 23 November 2020
Please cite this article as: Y. Wang, R. Alenazy, X. Gu, S.W Polyak, P. Zhang, M.J. Sykes, N. Zhang,
H. Venter, S. Ma, Design and structural optimization of novel 2H-benzo[h]chromene derivatives that
target AcrB and reverse bacterial multidrug resistance, European Journal of Medicinal Chemistry, https://
doi.org/10.1016/j.ejmech.2020.113049.
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Graphical Abstract：
Design and structural optimization of novel 2H-benzo[h]chromene
derivatives that target AcrB and reverse bacterial multidrug
resistance
Yinhu Wanga,b,1, Rawaf Alenazyc,d,1, Xinjie Gua, Steven W Polyakc, Panpan Zhanga, Matthew J. Sykesc,
Na Zhanga, Henrietta Venterc,*, Shutao Maa,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West
Wenhua Road, Jinan 250012, China
bSchool of Pharmacy, Liaocheng University, Liaocheng, China
c Clinical and Health Sciences, University of South Australia, Adelaide, SA 5000, Australia
dDepartment of Medical Laboratory, College of Applied Medical Sciences-Shaqra, Shaqra University,
11961, Saudi Arabia
Novel chromanone and benzo[h]chromene derivatives were designed, synthesized and evaluated as
AcrB inhibitors.

Design and structural optimization of novel 2H-benzo[h]chromene derivatives
that target AcrB and reverse bacterial multidrug resistance
Yinhu Wanga,b,1, Rawaf Alenazyc,d,1, Xinjie Gua, Steven W Polyakc, Panpan Zhanga, Matthew J. Sykesc, Na Zhanga,
Henrietta Venterc,*, Shutao Maa,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 West Wenhua Road, Jinan 250012,
China
bSchool of Pharmacy, Liaocheng University, Liaocheng, China
c Clinical and Health Sciences, University of South Australia, Adelaide, SA 5000, Australia
dDepartment of Medical Laboratory, College of Applied Medical Sciences-Shaqra, Shaqra University, 11961, Saudi
Arabia
*Corresponding authors.
E-mail addresses: mashutao@sdu.edu.cn (S. Ma); rietie.venter@unisa.edu.au (H. Venter).
1These authors contributed equally
Running title: 2H-Benzo[h]chromenes derivatives as AcrB inhibitors
1

Abstract: Drug efflux pumps have emerged as a new drug targets for the treatment of bacterial infections in view of its
critical role in promoting multidrug resistance. Herein, novel chromanone and 2H-benzo[h]chromene derivatives were
designed by means of integrated molecular design and structure-based pharmacophore modeling in an attempt to identify
improved efflux pump inhibitors that target Escherichia coli AcrB. The compounds were tested for their efflux inhibitory
activity, ability to inhibit efflux, and the effect on bacterial outer and inner membranes. Twenty-three novel structures
were identified that synergized with antibacterials tested, inhibit Nile red efflux, and acted specifically on the AcrB.
Among them, WK2, WL7 and WL10 exhibiting broad-spectrum and high-efficiency efflux inhibitory activity were
identified as potential ideal AcrB inhibitors. Molecular modeling further revealed that the strong π-π stacking interactions
and hydrogen bond networks were the major contributors to tight binding of AcrB.
Keywords: 2H-Benzo[h]chromene; Multidrug resistance; AcrB inhibitors; Efflux inhibitory activity
2

1. Introduction
The decreasing efficacy of existing antibiotics for the treatment of infectious diseases caused by bacteria has accelerated
worldwide in recent years [1, 2]. In particular, multidrug resistance (MDR) in pathogenic Gram-negative bacteria,
including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter spp., poses a serious
threat to global health as bacteria have acquired resistance mechanisms to one or more classes of clinically important
antibiotics [3-6]. At present, approximately 700,000 people die of bacterial infections every year. It is estimated that by
2050 bacterial infections will cause 10 million deaths, and kill more people than cancer, if no action is taken to address
MDR now [7]. Therefore, the discovery of novel antibacterials, or development of safe adjuvants that potentiate the
activity of existing antibiotics, is urgently needed to avoid a return to the pre-antibiotic era [8].
Gram-negative bacteria are intrinsically resistant to many antibiotic classes due to the presence of a lipopolysaccharide
rich outer membrane and multidrug efflux pumps [9, 10]. The highly impermeable outer membrane effectively restricts
antibiotics from penetrating into bacteria, whilst efflux pumps recognize structurally distinct chemicals and extrude them
from the cell before they can reach their intracellular drug targets and exert their antibacterial activity [11-14]. As a result,
the development of therapeutic adjuvants that can inhibit the action of efflux pumps is a promising strategy to
re-empower those antibacterials that are subject to efflux mechanisms [15-18]. Pharmacological efflux pump inhibitors
(EPIs) have emerged as promising, alternative therapeutics that have the potential to improve antibacterial potency and
reverse MDR [19, 20].
Over-expression of intrinsic efflux pump complexes is a primordial resistance mechanism that permits bacteria to
survive when challenged with toxic chemicals [12, 21, 22]. One of the most widely studied and common efflux systems
contributing to MDR in Gram negative bacteria is the tripartite AcrAB-TolC pump, which comprises an inner membrane
transporter (AcrB), an outer membrane channel protein (TolC), and a periplasmic protein (AcrA) that connects AcrB and
TolC [23-25]. AcrB catalyses drug/H+ antiport and is the subunit responsible for selective substrate binding and expulsion,
thus playing an essential role in the efflux mechanism [26, 27]. Crystal structures of AcrB in complex with natural
substrates or small molecule EPIs have provided molecular details into the mechanisms of action and inhibition,
respectively, that help to establish AcrB as a potential therapeutic target. Structural studies have revealed that AcrB
assembles as a homotrimer, and substrates are transported through the protein using a rotational mechanism that requires
co-operation between all three subunits. Compounds that inhibit one AcrB subunit consequentially impede the entire
efflux machinery [28]. Structural studies have also identified an important inhibitor binding site, known as the
hydrophobic trap, which is voluminous, flexible, and rich in aromatic amino-acid residues, such as Phe136, Phe178,
Phe610, Phe615, Phe617 and Phe628. The phenylalanine rich pocket facilitates binding to a wide variety of hydrophobic
substrates and EPIsvia hydrophobic bonding and π-π interactions [15, 19, 29-32]. In addition, certain polar residues, such
as Asn274 and Gln176, provide further opportunities for hydrogen bonding, as do water molecules present in the
substrate binding channel [29, 31]. Over the past decade, a number of AcrB inhibitors from different chemical classes
have been discovered, including PAβN, NMP, plumbagin and MBX3135 (Fig. 1). Recent crystal structures of AcrB in
complex with MBX3135 and its derivatives have highlighted the citical role of Phe628 and Phe178 in the inhibition
mechanism and provides valuable information for the design of new and more powerful EPIs [15, 19, 31]. To date, no
EPIs have entered the clinic for various reasons, such as low chemical stability, poor selectivity for the bacterial drug
target or high cytotoxicity. New EPIs with improved pharmacology or biological activity would be a welcome addition in
the fight against MDR.
3

<Insert Fig. 1>
We have previously reported two novel classes of AcrB inhibitors, 2-naphthamide derivatives (A3 and 7g) [7, 33] and
nordihydroguaiaretic acid (NDGA) derivatives (NDGA and WD6) (Fig. 1) [34, 35]. These representative compounds
displayed promising activity as antibiotic potentiators against highly-drug resistant E. coli. Consequently, these EPIs
have emerged as lead compounds due to their efflux inhibitory activity, novel drug-like scaffolds and favourable
biological properties. In the current study we applied an integrated molecular design strategy to devise the synthesis of
novel chromanone and 2H-benzo[h]chromene derivatives by fusing the structures of A3, 7g and WD6. The ability of
these compounds to potentiate the activity of antibiotics, to inhibit AcrB-mediated substrate efflux and to target AcrB
specifically was systematically evaluated. Moreover, the structure-activity relationships (SARs) and molecular modeling
were further investigated to explore the possible mechanisms of binding and inhibition against AcrB.
2. Results and discussion
2.1 Molecular design
Our previous docking experiments demonstrated that the naphthalene moiety of A3 (Fig. 2a) and one benzene moiety of
WD6 (Fig. 2b) were both oriented parallel to the Phe628 side chain, resulting in extensive π–π stacking interactions. The
second benzene ring of WD6 was also accommodated in the hydrophobic pocket through aπ–π stacking interaction with
Phe178 [33-35]. Thus, we proposed that our newly designed compounds should contain at least two appropriately spaced
aromatic groups to force the necessary interactions with Phe628 and Phe178 simultaneously, resulting in tight binding
with AcrB. In view of the above structural constraints, and to build upon our previous findings, two hypothetic
pharmacophores for new AcrB EPIs were proposed based upon an integrated molecular design and scaffold hopping
strategy (outlined in Fig. 2). We first replaced the naphthalene moiety of A3 with a 2,2-dimethylchroman-4-one fragment,
and integrated the benzene moiety of WD6 to produce novel-structure chromanone derivatives (Fig. 2b). The benzene
moiety was then derivatized for further bonding interactions. To verify the possible binding mode of the new derivatives,
a molecular docking study was performed. As depicted in Fig. 2b, the structure of novel chromanone derivative retained
the critical binding interactions observed in A3 and WD6, namely (1) two aromatic rings for interaction with Phe628 and
Phe178 and (2) a gemdimethyl group for additional hydrophobic interactions with Met573 and Tyr327.
Subsequently, we substituted the 2,2-dimethylchroman-4-one moiety with the 2H-benzo[h]chromene core with larger
aromatic volume in order to obtain stronger π–π interactions with Phe628. Inspection of the binding pocket revealed that
a water molecule bound to Gln176 by a hydrogen bond was located near the C-5 position of the 2H-benzo[h]chromene
core. Additionally, there was still sufficient space between the 2H-benzo[h]chromene core and the water molecule to
accommodate larger groups. Therefore, further structural modification could be elaborated by introduction of certain
polar substituents at the C-5 position of the 2H-benzo[h]chromene core to serve as hydrogen bond acceptors or donors
and form additional hydrogen bonds with AcrB via water mediated interactions. Additionally, the large and flexible
pocket consisting of Gln151, Ser155, Phe178, Gly179, Ala286, and Ser287, surrounded the benzene ring at the side chain
end of the chromanone or 2H-benzo[h]chromene core, provided suitable space for introducing a variety of groups to the
terminal benzene ring.
4

On the basis of the proposed binding modes described above, the 2H-benzo[h]chromene could be divided into two
functional parts, as shown in Fig. 2c, R section as hydrogen-bond-forming“hydrophilic groups” and the Rʹ section as
“multi-functional groups”. In the R positions, polar groups (e.g. morpholinyl, oxadiazolyl, tetrazolyl, carboxyl, and
hydrazide, etc.) that could form hydrogen bonds with resident water were preserved to improve the binding affinity for
AcrB, whilst a variety of functional groups that could form hydrophobic contacts or hydrogen bonds with amino acid
residues or water molecules were introduced at the Rʹ positions. To identify more potent AcrB inhibitors for further
pharmacological evaluation, as well as to verify the SARs, structural optimization was carried out through three chemical
modifications: (1) hydrophilic modification on the 2H-benzo[h]chromene ring (R section), and (2) multi-functional
modification on the benzene ring (Rʹ section), and (3) scaffold hopping from chromanone core to 2H-benzo[h]chromene
core.
<Insert Fig. 2>
2.2. Chemistry
The synthesis of a series of the chromanone derivatives (WH series) is shown in Scheme 1. Firstly resorcinol (1) reacted
with 3-methyl-2-butenoic acid (2) in the presence of zinc chloride in phosphorus oxychloride to afford
7-hydroxy-2,2-dimethylchroman-4-one
(3).
Reaction
of
3
with
dibromoethane
provided
7-(2-bromoethoxy)-2,2-dimethylchroman-4-one (4), which was followed by substitution reaction with various
alkylamines to give chromanone derivatives WH1-WH6. Amide 6 was efficiently synthesized from substituted aniline 5
in the presence of Et3N through an acylation reaction of chloroacetyl chloride, which was subsequently reacted with 3 to
yield chromanone derivatives WH7-WH10.
<Insert Scheme 1>
The synthetic route of 2H-benzo[h]chromene-5-carboxylate 13 as key intermediate for the 2H-benzo[h]chromene
derivatives is presented in Scheme 2. Salicylaldehyde (7) as starting material was reacted with benzyl chloride to afford
benzyl protected product 8. Stobbe condensation reaction of 8 with diethyl succinate gave condensed product 9, and then
further cyclization in acetic anhydride under reflux conditions produced 4-acetoxynaphthoate 10. Subsequently, the
acetyl group of 10 was deprotected with K2CO3 in methanol, providing 4-hydroxynaphthoate 11, which was then
subjected to the [3+3] cycloaddition reaction with 3-methyl-2-butenal (12) to obtain a key intermediate 13.
<Insert Scheme 2>
The synthetic routes of the 2H-benzo[h]chromene derivatives, encompassing the WI, WJ, WK and WL series, are
outlined in Scheme 3. The key intermediate 13 was hydrolyzed with NaOH to afford 2H-benzo[h]chromene-5-carboxylic
acid (14), which was then subjected to amidation reaction with corresponding amines in the presence of TBTU to give
amide intermediates 15a-c. Deprotection and double bond reduction of 14 and 15a-c were carried out with hydrogen
under the catalysis of Pd/C, providing hydroxyl intermediates 16a-d, which were then reacted with corresponding
substituted benzyl chloride (or bromide) to obtain 5-(2,6-dimethylmorpholinoyl)-2H-benzo[h]chromene derivatives
5

WI1-WI9 and 5-(morpholinylalkyl)-2H-benzo[h]chromene derivatives WL1-WL10, respectively.
In addition, 13 was also treated with NaBH4 in tetrahydrofuran (THF) to provide the corresponding alcohol 17. Further
bromination of 17 by CBr4 and PPh3 in dichloromethane produced brominated intermediate 18, followed by a
substitution reaction with 1,2,3,4-tetrazole and 2,6-dimethylmorpholine in DMF using potassium carbonate as a base to
afford 1,2,3,4-tetrazolyl product 19a and dimethylmorpholinyl product 19b, respectively. Deprotection and double bond
reduction of 19a and 19b and subsequent reaction with corresponding substituted benzyl chloride (or bromide) gave
5-(1,2,3,4-tetrazolylmethyl)-2H-benzo[h]chromene
derivatives
WJ1-WJ10
and
5-(morpholinylalkyl)-2H-benzo[h]chromene derivatives WL11-WL13.
Furthermore, 13 was successfully converted to hydrazide 21 by hydrazinolysis, and subsequent treatment with triethyl
orthoformate under reflux conditions generated 1,3,4-oxadiazole product 22. Catalytic hydrogenation of 22 produced
deprotected product 23, which was transformed by treatment with the appropriate substituted benzyl halide to
5-(1,3,4-oxadiazolyl)-2H-benzo[h]chromene derivatives WK1-WK9.
<Insert Scheme 3>
2.3. Inherent antibacterial activity
The minimum inhibitory concentration (MIC) of all chromanone and 2H-benzo[h]chromene derivatives were initially
determined using an antimicrobial susceptibility assay. This was necessary so that sub-inhibitory concentrations of the
compounds could be investigated in subsequent checkerboard titration assays to measure efflux pump inhibition without
a direct antibacterial effect. For this purpose, the MDR strain E. coli BW25113, expressing AcrB, was assayed alongside
an isotypic strain with AcrB deleted. None of the target compounds showed any antibacterial activity against the wildtype
strain at 512 µg/mL above tested strains, except WK7 that was active at 64 μg/mL.
2.4 Ability to reverse bacterial resistance
The efflux pump inhibitory activity of all compounds was addressed by assaying their ability to reverse the resistance of
certain antibiotics. All chromanone and 2H-benzo[h]chromene derivatives were tested in combination with known
substrates of AcrB, namely erythromycin (ERY), chloramphenicol (CAM), tetraphenylphosphonium (TPP) and
levofloxacin (LEV). Standard checkerboard assays in which the MIC values of a panel of antibiotics were determined in
the presence of varying concentrations of the tested compounds were performed [33, 34, 36]. Known EPIs NDGA and
A3 served as reference compounds. Rifampicin (RIF) was also included as a negative control as this is not an AcrB
substrate. Hence, any synergism with RIF would indicate the compound does not act specifically upon AcrB. Only those
compounds that reduced the MIC values against wild type E. coli BW25113 expressing AcrB for at least one
antimicrobial by 2-fold or more are presented in Table 1 and Table 2. Importantly, the absence of antibacterial activity at
the high concentration of 512 µg/mL against E. coli BW25113 eliminated the possibility that the reversal of resistance in
the checkerboard assays below was due to any intrinsic antibacterial activity of the compounds.
In the first stage of optimization, we focused on the modification of the aromatic moiety and linker of chromanone
core to give the chromanone derivatives (WH series). In this series, only three compounds (WH3, WH4 and WH8)
displayed weak or moderate synergism with antibiotics (Table 1). Among them, WH3 (8 µg/mL) and WH4 (32 µg/mL)
potentiated the activity of LEV by 2-fold, while WH4 and WH8 reduced the MIC values of TPP by 2- and 4-fold at
6

lower concentration than the reference molecules. Moreover, WH8 at 64 µg/mL increased bacterial sensitivity to CAM
and TPP by 2- and 4-fold, respectively. However, none of compounds in this series showed synergism with ERY.
Although a limited SARs was provided from the WH series chromanone derivatives, we could conclude that the
chromanone core was not an optimal scaffold for AcrB inhibitory activity.
An alternative optimization strategy was carried out by substitution of the chromanone core with a large
2H-benzo[h]chromene core because the AcrB inhibition potency was particularly sensitive to the hydrophobic structure
scaffold. On this basis, we introduced 2,6-dimethylmorpholinoyl and 1,2,3,4-tetrazolylmethylene groups (R section) at
the C-5 position of the 2H-benzo[h]chromene core and varied the substituents on terminal benzene ring (Rʹ section) at the
C-7 position to obtain the 5-(2,6-dimethylmorpholinoyl)-2H-benzo[h]chromene derivatives (WI series) and the
5-(1,2,3,4-tetrazolylmethyl)-2H-benzo[h]chromene derivatives (WJ series), respectively. Those compounds that reduced
the MIC by 2-fold or more in the WI and WJ series are listed in Table 1. Only WI7 and WI8 were moderately active with
WI7 increasing sensitivity towards Lev by 2-fold at 8 µg/ml and WI8 to both ERY and TPP at the highest concentration
tested at 128 µg/mL. In stark contrast, the WJ series exhibited improved antibacterial activity, with five compounds WJ1,
WJ5, WJ6, WJ7 and WJ10 reversing resistance. Noteworthy were WJ1, WJ5, WJ7 and WJ10 that were active with
two antibiotics in the testing panel. However, the WJ series showed a narrow antibacterial spectrum and weak synergism.
The lack of biological activity in these two series was possibly due to the space constraint of the hydrophilic binding site
between the 2H-benzo[h]chromene core and the resident water molecule. Bulky substituents, such as
2,6-dimethylmorpholinoyl and 1H-tetrazolylmethylene groups, may not be well accommodated by the hydrophilic cavity,
resulting in the absence of necessary hydrogen bonding interactions mediated by water molecule.
<Insert Table 1>
Subsequently, we introduced small polar substituents (e.g. oxadiazolyl, morpholinylalkyl, carboxylic acid, and
hydrazide) in the R positions, and investigated the effects of simultaneous changes of R and Rʹ groups on the potentiating
capacity of the compounds. These structural modifications led to the 5-(1,3,4-oxadiazolyl)-2H-benzo[h]chromene
derivatives (WK) and the 5-(morpholinylalkyl)-2H-benzo[h]chromene derivatives (WL series). The data of antibacterial
synergism of the WK and WL series are summarized in Table 2. Many compounds containing 1,3,4-oxadiazolyl or
morpholinyl groups in the WK and WL series showed promising antibacterial synergism and broad-spectrum activity
with certain exemplars yielding upto 16-fold reduction of the MIC values. Noteworthy were WK2, WL1, WL7 and
WL10 that were broadly active against all antibacterials. Similarly, WK1, WK5, WK7, WL2, WL7 and WL9 were
synergistic with three out of the four antibiotics in the screening panel. Moreover, all the active compounds were
inhibitors of ERY efflux, with WK2, WL7 and WL10 displaying a desirable combination of potency and broad-spectrum
activity. WL1 increased bacterial sensitivity to CAM and TPP by 8- and 16-fold, showing greater potent synergistic
activity than reference molecule. WK2 was also an efficient rensitizer as it potentiated the antimicrobial activity of ERY,
TPP and LEV by 2-fold at the low concentration of 8 µg/mL, and synergized with all tested four antibacterials, leading
upto a 16-fold MIC reduction at 128 µg/mL. However, none of the compounds had effect on the MIC value of RIF (16
µg/mL) consistent with the hypothesis that these compounds acted as EPIs targeting AcrB.
The chromanone (WH series) and 2H-benzo[h]chromene derivatives (WI-WL series) contain three discrete structural
fragments, which provided an opportunity to estimate an impact of each feature upon bioactivity. Considering the
influence of the aromatic core fragments, the 2H-benzo[h]chromene core (WI-WL series) seem to be more profitable
7

than the chromanone core (WH series). For example, only three compounds possessing the chromanone core in the WH
series (WH3, WH4 and WH8) displayed weak or moderate synergism activity at high doses, while those compounds
with the 2H-benzo[h]chromene core in the WJ-WL series, such as WK1, WK2, WK5, WL1, WL7 and WL10, showed
desirable potency and broad-spectrum activity at low concentrations. This highlights the 2H-benzo[h]chromene core as a
promising scaffold for EPI activity. For the hydrophilic fragments (R groups) at the C-5 position of the
2H-benzo[h]chromene core, 1,3,4-oxadiazolyl and morpholinoyl substituents were optimal to improve the antibacterial
effect of all four tested antibacterials, whereas 2,6-dimethylmorpholinoyl and 1,2,3,4-tetrazolylmethylene groups were
disfavored. As exemplified by WI1 and WK2, replacement of 2,6-dimethylmorpholinoyl group with 1,3,4-oxadiazolyl
group resulted in a significant improved synergism activity, which imply that a small polar group for R is necessary for
the activity. It is possible that the smaller polar groups can be well accommodated in the hydrophilic binding site and
form additional hydrogen bonding interactions with water molecules. Thus, the most preferred substituents for EPI
activity are as follows: morpholinoyl
>
1,3,4-oxadiazolyl
>
carboxyl
>
1,2,3,4-tetrazolylmethylene
>
2,6-dimethylmorpholinoyl ≈ 2,6-dimethylmorpholinomethylene. Considering the influence of substitution at the benzene
ring (Rʹ groups) on the biological activities, it is noted that 4-methyl (WK2) and 4-acrylamido (WL10) groups were the
most favorable. Furthermore, the substituent-free phenyl group (WK1, WL1 and WL7) was more beneficial than the
3-methoxyphenyl group (WK4, WL2 and WL8). In addition, a para-substituent on the benzene ring was favored as well.
For instance, WK3, containing an o-methyl substituted phenyl group (WK3 vs WK2), showed loss of EPI activity,
possibly due to the absence of hydrophobic contacts in the para-position.
<Insert Table 2>
2.5. The effect of the compounds on substrate transport
The bioactive compounds in the checkerboard assays described above were then assayed to determine if they directly
inhibited efflux mediated by AcrB in whole cell efflux assays [36, 37]. The lipophilic fluorescent dye Nile Red was
employed as it is a known substrate of the AcrAB-TolC pump. Nile Red is weakly fluorescent in aqueous environments
but undergoes a significant increase in fluorescence once inside the cell [7, 33]. Assays were performed upon E. coli
BW255113 cells and an isogenic AcrB deletion used to help establish the specificity of AcrB-mediated substrate efflux
and EPI mechanism of action. In this efflux assay, WK5, WK7, WJ10, WL8 and WL9 were identified as the most
potent EPIs as they completely inhibited substrate efflux to the same level as the AcrB deletion strain at the low
concentration of 50 µM (Fig. 3). Similarly, WH3, WI7, WI8, WJ1, WJ5, WJ6, WK1, WK2, WK3, WK6, WL1, WL2,
WL7 and WL10 also showed complete inhibitory activity at 100 µM. In contrast, WH4, WH8, WJ7 and WK4 were
weakly active, with inhibitory activity only observed at much higher concentrations of 100-200 µM (Fig. 1S in
Supporting data).
<Insert Fig. 3>
2.6. The effect of the compounds on the bacterial outer membrane
8

The outer membrane of Gram-negative bacteria is effective at limiting permeation of antibiotics into the cell. Compounds
that permeabilise this barrier may allow the accumulation of antibiotics inside the cells and provide similar effects as
EPIs [18, 35]. To determine if the active compounds described above possessed undesirable off-target membrane
permeabilisation properties, a nitrocefin hydrolysis assay was performed on intact E. coli BW25513. Hydrolysis of the
chromogenic β-lactam nitrocefin by β-lactamase produces a red compound that can be monitored by spectroscopy. The
rapid hydrolysis of nitrocefin is indicative of a permeabilised outer membrane that facilitates increased diffusion of the
reagent into the bacterial periplasm. The known membrane disruptor, polymyxin B (PMBN), served as a positive control.
The results, shown in Fig. 4, demonstrated that none of the bioactive compounds in either the WH-WL series affected the
permeability of the outer membrane at the tested concentrations, indicating their synergism with antibacterials was not
due to membrane permeabilization.
<Insert Fig. 4>
2.7. The effect of the compounds on the bacterial inner membrane
Finally, chemical damage of the inner membrane and consequential perturbation of the proton motive force (pmf) that
drives AcrAB-TolC activity was finally evaluated. As AcrB utilizes the pmf to transport substrates, those compounds that
perturb the pmf across the inner membrane can also inhibit efflux by an indirect, off-target mechanism [38]. To determine
whether the active compounds perturbed the pmf, we assessed the ability of those compounds to depolarize the bacterial
transmembrane potential ((∆ψ)) by using the membrane potential-sensitive dye 3,3-diethyloxacarbocyanine iodide
(DiOC2(3)). DiOC2(3) undergoes a significant increase in fluorescence once the pmf is established by the addition of
glucose. Following the treatment of cells with the ionophore and proton decoupler CCCP, the ∆ψ was dissipated and the
fluorescence intensity decreased to the level before glucose stimulation (blue curves representing bacteria not treated
with active compound). If a compound disrupted the inner membrane, the fluorescence of DiOC2(3) was decreased due to
the inability of cells to establish a proton gradient. The results are shown in Fig. 5. Importantly, none of the compounds
disrupted the bacterial inner membrane, except WL1, at the low concentration of 32 µg/mL.
<Insert Fig. 5>
2.8. In vitro cytotoxicity toward mammalian cells
A crucial parameter for developing antimicrobial agents is their selectivity for bacterial cells over mammalian cells.
Mammalian cells (HepG2 cells) were used to evaluate the in vitro cytotoxic effect of the most potent compounds WK2
and WL7. Paclitaxel was used as a positive control (IC50=12.6 nM). As shown in Table 3, when treating with WK2 and
WL7 at the concentration of 10 µM, the cell viability rates toward HepG2 cells were 94% and 98%, respectively, while
the cell inhibition rates were 87% and 66% at 100 µM, respectively. The cytotoxicity results demonstrated that WK2 and
WL7 possessed low cytotoxicity toward mammalian cells, which had a safety profile much better than paclitaxel.
9

<Insert Table 3>
2.9. Molecular docking
Molecular docking studies were next performed to predict the possible binding mode, to explain the SARs and to guide
future structural optimisation experiments. The crystal structure of AcrB (PDB code: 5eno) served as the receptor protein,
and WK2 and WL10 were selected as representative EPIs for molecular docking. As illustrated in Fig. 6a, WK2 was
well accommodated in the well-defined hydrophobic trap of AcrB. The 2H-benzo[h]chromene core and phenyl groups
were oriented parallel to the aromatic side chains of Phe628 and Phe178 respectively, resulting in extensive π-π stacking
interactions. The gemdimethyl group attached at the C-2 position of the 2H-benzo[h]chromene core was encased by
Try327 and Met573 through hydrophobic interactions. Moreover, the nitrogen atom of 1,3,4-oxadiazolyl group at the C-5
position of the 2H-benzo[h]chromene core formed a hydrogen bond with a water molecule, which, in turn, bound to
Gln176 by hydrogen bonds. The flexible methyl ether linker at the C-7 position of the 2H-benzo[h]chromene core
induced a conformational flexibility in the molecule that allowed optimal π-stacking interactions between the two
aromatic rings and side chains of Phe628 and Phe178. Notably, WL10 adopted similar binding mode to WK2, forming
strong π-π stacking interactions with Phe628 and Phe178, and hydrogen bond networks with Gln176 and water molecule.
Additionally, a critical water molecule bound to the nitrogen atom of 4-acrylamide group was involved in a hydrogen
bond network with Gln151, Ser155 and Ala286, effectively anchoring the EPI to these three amino acid residues (Fig. 6b).
We propose that this extended hydrogen bonding network contributes to the potent bioactivity of of the optimal WK and
WL series.
<Insert Fig. 6>
Overall, on the basis of an integrated molecular design strategy and molecular modeling, fifty-one benzochromene and
2H-benzo[h]chromene derivatives were designed, synthesized and evaluated for their EPI activity. Among them,
twenty-three compounds (Table 4) were found to synergize with at least one antibacterial. Preliminary SARs derived
from this study suggest: (1) the compounds containing the 2H-benzo[h]chromene core were favorable for enhanced the
EPI activity, whereas those containing the chromanone core were less active; (2) the efflux inhibitory activity was
dependent upon substituents at the C-5 position of 2H-benzo[h]chromene core with 1,3,4-oxadiazolyl and
morpholinoyl groups preferred; (3) introduction of a hydrophilic or lipophilic group at the para position of the terminal
benzene ring are important contributors for activity, and the introduction of methyl and acylamido groups were optimal.
In comparison to the lead compounds A3 and NDGA, the 2H-benzo[h]chromene derivatives described here seemed to be
more favorable as they displayed superior efficacy, and possessed significant and desirable EPI activity, indicating a
successful outcome for this structure-guided drug designprogram. More importantly, the above SARs provides new
insight into the discovery of novel AcrB inhibitors.
<Insert Table 4>
10

Noteworthy are compounds in the WK and WL series containing 1,3,4-oxadiazolyl and morpholinoyl groups at the
C-5 position of the 2H-benzo[h]chromene core, respectively, that generally display better efficacy than the other
compounds in the above two series as well as those in the WH, WI and WJ series, which is probably due to form an
additional hydrogen bond networks, resulting in tighter binding with AcrB. In all five series, exemplars WK2, WL1,
WL7 and WL10 all exhibited broad-spectrum, and high-efficiency, EPI activity. Especially, WK2 was the most efficient
potential EPI as it potentiated the activity of ERY, TPP and LEV even at the low concentration of 8 µg/mL, and
synergized with all four antibacterials tested, leading upto a 16-fold MIC reduction at 128 µg/mL. Moreover, WL1 had
the greatest synergism with CAM and increased its sensitivity by 8-fold at 128 µg/mL, whereas WK2, WL7 and WL10
at 128 µg/mL markedly decreased the MIC values of ERY, TPP and LEV by 8-, 16- and 6-fold, respectively. In contrast,
the compounds in the WH, WI and WJ series only showed a narrow antibacterial spectrum and weak synergism.
Subsequently, twenty-four compounds with synergetic activity were selected to further assess their ability to inhibit Nile
Red efflux and their effect on inner- and outer membrane permeabilisation. The results showed that all the active
compounds inhibited AcrB-mediated substrate efflux at a reasonable concentration, and none of them permeabilised the
inner and outer membrane, except WL1 that was a strong disruptor of the inner membrane. Consequently, WH3, WH4,
WH8, WI8, WI9, WJ1, WJ5-WJ7, WJ10, WK1-WK7, WL2 and WL7-WL10 were all identified as ideal AcrB
inhibitors using the selection criteria described by Lomovskaya et al [1, 39].
3. Conclusions
This study illustrates the successful application of an integrated molecular design approach followed by chemical
optimizations proposed by in silico analyses to confirm 2H-benzo[h]chromene nucleus as an optimal scaffold to obtain
novel and potent AcrB inhibitors. Fifty-one compounds were designed, synthesized and evaluated for their EPI activity.
Twenty-three compounds were identified as ideal AcrB inhibitors that synergized with at least one antibacterials tested,
leading to 2- to 16-fold MIC reduction. They were also able to completely inhibit Nile red efflux at 50-200 µM, and
targeted specifically on the AcrB. Among the above ideal EPIs, WK2, WL7 and WL10 exhibited the most
broad-spectrum, and high-efficiency EPI activity. In conclusion, these newly 2H-benzo[h]chromene derivatives display
potent biological activities as a novel class of antibacterial adjuvants and have potential for further development.
4. Experimental
4.1. Chemistry
All reagents and chemicals were commercially available and used without further purification. All reactions were
monitored by analytical thin-layer chromatography (TLC) using silica gel GF254 plates. Purification of crude products
1
was carried out by flash column chromatography using silica gel 60 (particle size 0.040-0.063 mm). H NMR and 13C
NMR spectra were recorded by using Bruker instrument at 600 and 100 MHz spectrometer with TMS as an internal
standard. The following abbreviations are used to denote peak patterns in NMR spectra: singlet (s), doublet (d), triplet (t),
multiplet (m), as well as doublet of doublets (dd). Low-resolution mass spectra (ESI-MS) data were recorded on an API
4000 instrument (Applied Biosystems, Connecticut, USA). The purity of all target compounds was performed on an
Agilent 1200 Series HPLC system ((Agilent, Santa Clara County, USA) equipped with a diamonil C18 column (150 ×
11

4.6 mm, 5 μm). HPLC conditions: solvent A = H2O, solvent B = CH3OH; flow rate = 1.0 mL/min. All tested compounds
have a purity ≥ 95%.
4.1.1. 7-Hydroxy-2,2-dimethylchroman-4-one (3)
A mixture of resorcinol 1 (10.00 g, 90.82 mmol), 3-methyl-2-butenoic acid (2) (9.09 g, 90.82 mmol), zinc chloride (13.74
g, 136.22 mmol) and phosphorus oxychloride (139.24 g, 0.91 mol) was heated at 50 °C for 3 h. the reaction mixture was
then poured into ice water (80 mL), and the resulting precipitate was filtered off. The residue was purified by silica
column chromatography to afford 3 (10.27 g, 59%) as brown solid. mp 172-174 °C, ESI-MS m/z calcd for C11H13N3 [M
+ H]+ 193.1, found 193.4.
4.1.2. 7-(2-Bromoethoxy)-2,2-dimethylchroman-4-one (4)
To a solution of 3 (5.00 g, 26.00 mmol) in acetonitrile (50 mL) were added K2CO3 (7.19 g, 52.00 mmol) and
dibromoethane (19.55 g, 104.00 mmol). The reaction mixture was heated at 75 °C for 8 h. The mixture was concentrated
under reduced pressure and purified by flash column chromatography on silica gel to give yellow oil 4 (4.53 g, 58%).
ESI-MS m/z calcd for C13H16BrO3 [M + H]+ 299.0, found 299.1.
4.1.3. General methods for the preparation of the chromanone derivatives WH1-WH6
A solution of 4 (150 mg, 0.50 mmol) in DMF (10 mL), K2CO3 (140 mg, 1.00 mmol) and corresponding alkylamine (0.75
mmol) were added. The solution was stirred at 78 °C for 6 h. Upon completion, the reaction mixture was poured in water
(40 mL) and extracted with dichloromethane, washed with brine, and the solvent was removed under vacuum. The
residue was purified by flash column chromatography (DCM/MeOH) to afford the desired compounds.
4.1.3.1. 7-(2-(2,6-Dimethylmorpholino)ethoxy)-2,2-dimethylchroman-4-one (WH1)
Colorless oil, yield 45%, Rf = 0.33 (petroleum ether/EtOAc = 30:1). HPLC purity100.00% (A%/B% = 25:75, Rt = 4.183
min). 1H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 8.7 Hz, 1H), 6.52 (dd, J = 8.8, 2.3 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H),
4.06 (t, J = 5.7 Hz, 2H), 3.46 (dtd, J = 12.3, 6.0, 2.0 Hz, 2H), 2.78–2.69 (m, 2H), 2.63 (s, 2H), 2.58 (t, J = 5.7 Hz, 2H),
1.68–1.58 (m, 2H), 1.31 (s, 6H), 0.96 (d, J = 6.3 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 191.03, 165.30, 161.89, 128.26,
114.21, 109.59, 101.78, 79.60, 71.57 (2C), 66.03, 59.78 (2C), 56.89, 48.58, 26.71 (2C), 19.15 (2C). ESI-HRMS m/z
calcd for C19H28NO4 [M + H]+ 334.2018, found 334.2014.
4.1.3.2. 2,2-Dimethyl-7-(2-((3-morpholinopropyl)amino)ethoxy)chroman-4-one (WH2)
Colorless oil, yield 59%, Rf = 0.27 (petroleum ether/EtOAc = 30:1). HPLC purity 98.30% (A%/B% = 15:85, Rt = 4.619
min). 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 8.8 Hz, 1H), 6.52 (dd, J = 8.8, 2.3 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H),
4.01 (t, J = 5.5 Hz, 2H), 3.47 (t, J = 4.6 Hz, 4H), 2.81 (t, J = 5.4 Hz, 2H), 2.63 (s, 2H), 2.53 (t, J = 6.9 Hz, 2H), 2.23 (q, J
= 7.4, 5.7 Hz, 6H), 1.50 (p, J = 6.9 Hz, 2H), 1.31 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 190.99, 165.28, 161.89, 128.26,
114.25, 109.47, 101.71, 79.60, 67.45, 66.93 (2C), 57.42, 53.75 (2C), 48.59, 48.54, 48.44, 26.68, 26.22 (2C). ESI-MS m/z
calcd for C19H28NO4 [M + H]+ 334.2, found 334.3. ESI-HRMS m/z calcd for C20H31N2O4 [M + H]+ 363.2284, found 363.
2287.
4.1.3.3. 7-(2-(Isopropylamino)ethoxy)-2,2-dimethylchroman-4-one (WH3)
12

Colorless oil, yield 95%, Rf = 0.38 (petroleum ether/EtOAc = 30:1). HPLC purity 96.50% (A%/B% = 15:85, Rt = 7.804
min). 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 8.7 Hz, 1H), 6.53 (dd, J = 8.8, 2.4 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H),
3.98 (t, J = 5.7 Hz, 2H), 2.78 (t, J = 5.7 Hz, 2H), 2.72–2.66 (m, 1H), 2.64 (s, 2H), 1.32 (s, 6H), 0.92 (d, J = 6.2 Hz, 6H).
13C NMR (100 MHz, CDCl3) δ 191.07, 165.47, 161.90, 128.24, 114.21, 109.58, 101.73, 79.59, 68.01, 48.58 (2C), 46.04,
26.70 (2C), 22.86 (2C). ESI-HRMS m/z calcd for C16H24NO3 [M + H]+ 278.1756, found 278.1757.
4.1.3.4. 7-(2-(Cyclopentylamino)ethoxy)-2,2-dimethylchroman-4-one (WH4)
Brown oil, yield 85%, Rf = 0.37 (petroleum ether/EtOAc = 30:1). HPLC purity 100.00% (A%/B% = 15:85, Rt = 10.628
min). 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 8.8 Hz, 1H), 6.52 (dd, J = 8.8, 2.3 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H),
3.98 (t, J = 5.7 Hz, 2H), 2.96 (p, J = 6.3 Hz, 1H), 2.77 (t, J = 5.7 Hz, 2H), 2.63 (s, 2H), 1.70–1.61 (m, 2H), 1.58–1.48 (m,
2H), 1.39 (qd, J = 7.5, 5.4, 2.6 Hz, 2H), 1.31 (s, 6H), 1.21 (qt, J = 8.0, 6.0, 2.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
191.04, 165.48, 161.89, 128.21, 114.17, 109.57, 101.71, 79.56, 68.01, 59.67, 48.57, 47.27, 33.15 (2C), 26.69 (2C), 24.07
(2C). ESI-HRMS m/z calcd for C18H26NO3 [M + H]+ 304.1912, found 304.1899.
4.1.3.5. 7-(2-((Furan-2-ylmethyl)amino)ethoxy)-2,2-dimethylchroman-4-one (WH5)
Brown oil, yield 64%, Rf = 0.40 (petroleum ether/EtOAc = 30:1). HPLC purity 95.28% (A%/B% = 15:85, Rt = 6.082
min). 1H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 1.8 Hz, 1H), 6.52 (dd, J = 8.8, 2.4 Hz, 1H),
6.41 (d, J = 2.3 Hz, 1H), 6.31 (dd, J = 3.1, 1.9 Hz, 1H), 6.18 (d, J = 3.1 Hz, 1H), 3.99 (t, J = 5.6 Hz, 2H), 3.66 (s, 2H),
2.78 (t, J = 5.6 Hz, 2H), 2.43 (p, J = 1.8 Hz, 2H), 1.31 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 191.05, 165.38, 161.89,
153.41, 142.00, 128.25, 114.24, 110.15, 109.55, 107.18, 101.72, 79.59, 67.74, 48.58, 47.60, 46.07, 26.70 (2C).
ESI-HRMS m/z calcd for C18H22NO4 [M + H]+ 316.1549, found 316.1530.
4.1.3.6. 7-(2-((1H-Benzo[d]imidazol-2-yl)thio)ethoxy)-2,2-dimethylchroman-4-one (WH6)
White solid, yield 50%, mp 68-70 °C, Rf = 0.32 (petroleum ether/EtOAc = 30:1). HPLC purity 98.78% (A%/B% = 15:85,
1
Rt = 7.514 min). H NMR (400 MHz, DMSO-d6) δ 8.26 – 8.19 (m, 1H), 8.12 (d, J = 6.2 Hz, 2H), 8.00–7.93 (m, 1H),
7.62 (td, J = 6.2, 5.6, 3.4 Hz, 2H), 7.46 (s, 1H), 7.40 (s, 1H), 4.57 (t, J = 5.4 Hz, 2H), 3.99 (t, J = 5.3 Hz, 2H), 2.50 (q, J =
1.9 Hz, 6H). 13C NMR (100 MHz, DMSO) δ 190.80, 165.01, 161.97, 150.07, 144.05, 132.71, 128.09, 122.75, 122.13,
121.59, 117.82, 114.33, 110.82, 109.92, 102.44, 80.11, 67.29, 48.19, 30.20, 26.60 (2C). ESI-RHMS m/z calcd for
C21H22NO3S [M + H]+ 369.1273, found 369.1262.
4.1.4. General methods for the preparation of amide 6
To a solution of substituted aniline 5 (10.00 mmol) in DCM (20 mL) were added Et3N (20.00 mmol), and chloroacetic
chloride (12.00 mmol) was added at 0 °C slowly. The solvent was removed in vacuo and the crude residue was purified
by flash column chromatography on silica gel to give amide 6.
4.1.5. General methods for the preparation of the chromanone derivatives WH7-WH10
The preparation method of WH7-WH10 is similar to that of WH1-WH6.
4.1.5.1. 2-((2,2-Dimethyl-4-oxochroman-7-yl)oxy)-N-(4-nitrophenyl)acetamide (WH7)
13

Yellow solid, yield 46%, mp 196-198 °C, Rf = 0.78 (petroleum ether/EtOAc = 10:1). HPLC purity 95.11% (A%/B% =
1
15:85, Rt = 2.532 min). H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 8.25 (dd, J = 9.1, 4.5 Hz, 2H), 7.90 (d, J = 8.8
Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 6.70 (dd, J = 8.8, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 4.89 (s, 2H), 2.73 (d, J = 6.0
Hz, 2H), 1.39 (s, 6H). 13C NMR (100 MHz, DMSO) δ 190.82, 167.37, 164.59, 161.69, 144.96, 143.03, 128.14, 126.28,
125.45, 119.74, 115.76, 114.67, 109.84, 102.61, 80.23, 67.50, 48.18, 26.57 (2C). ESI-MS m/z calcd for C19H19N2O6 [M +
H]+ 371.1, found 371.3.
4.1.5.2. N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-((2,2-dimethyl-4-oxochroman-7-yl)oxy)acetamide (WH8)
White solid, yield 56%, mp 176-178 °C, Rf = 0.75 (petroleum ether/EtOAc = 10:1). HPLC purity 99.72% (A%/B% =
1
15:85, Rt = 3.973 min). H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.31 (d, J = 1.9 Hz, 1H), 8.13 (d, J = 8.6 Hz,
1H), 8.07 (dd, J = 8.7, 1.9 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 6.70 (dd, J = 8.8, 2.3 Hz, 1H), 6.55 (d, J = 2.3 Hz, 1H), 4.89
(s, 2H), 2.72 (s, 2H), 1.38 (s, 6H). 13C NMR (100 MHz, DMSO) δ 190.81, 167.84, 164.46, 161.68, 143.40, 137.03,
132.35, 128.15, 124.22, 122.90, 121.50, 117.36, 116.13, 114.73, 109.82, 102.69, 80.23, 67.43, 48.18, 26.56 (2C).
ESI-HRMS m/z calcd for C21H18F3N2O4 [M + H]+ 419.1218, found 419.1213.
4.1.5.3. N-(3,4-Dicyanophenyl)-2-((2,2-dimethyl-4-oxochroman-7-yl)oxy)acetamide (WH9)
White solid, yield 67%, mp 212-214 °C, Rf = 0.75 (petroleum ether/EtOAc = 10:1). HPLC purity 100.00% (A%/B% =
1
15:85, Rt = 5.073 min). H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.14–8.01 (m, 2H),
7.69 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 8.8, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 4.89 (s, 2H), 2.72 (s, 2H), 1.39 (s, 6H).
13C NMR (100 MHz, DMSO) δ 190.83, 167.83, 164.46, 161.68, 143.32, 135.61, 128.15, 123.94, 116.52, 115.99, 114.73,
110.26, 109.83, 108.81, 103.29, 102.70, 80.25, 67.43, 48.18, 26.58 (2C). ESI-HRMS m/z calcd for C21H19N3O5 [M + H]+
376.1297, found 393.1292.
4.1.5.4. N-(3-Acetamido-4-fluorophenyl)-2-((2,2-dimethyl-4-oxochroman-7-yl)oxy)acetamide (WH10)
White solid, yield 68%, mp 190-192 °C, Rf = 0.40 (petroleum ether/EtOAc = 8:1). HPLC purity 99.51% (A%/B% =
1
15:85, Rt = 1.835 min). H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.07 (p, J = 4.5 Hz, 1H), 7.72–7.62 (m, 3H),
7.42 (dd, J = 8.5, 1.9 Hz, 1H), 6.69 (dd, J = 8.7, 2.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 4.83 (s, 2H), 2.77 (d, J = 4.5 Hz,
3H), 2.72 (s, 2H), 1.39 (s, 6H). 13C NMR (100 MHz, DMSO) δ 190.83, 167.05, 164.60, 163.87, 161.69, 161.05, 158.59,
142.26, 131.35, 128.14, 115.45, 114.66, 109.86, 106.99, 102.62, 80.24, 67.50, 48.18, 26.76, 26.58 (2C). ESI-HRMS m/z
calcd for C21H21FN2O5 [M + H]+ 401.1512, found 423.1512.
4.1.6. 2-(Benzyloxy)benzaldehyde (8)
To a suspension of salicylaldehyde 7 (0.00 g, 81.88 mmol) in DMF (40 mL) was added benzyl chloride (15.55 g, 122.83
mmol) and K2CO3 (22.63 g, 163.77 mmol), and the mixture was kept stirring at 70 °C for 6 h. After the completion of the
reaction, water (30 mL) was added, extracted with EtOAc (3 × 20 mL), washed with brine, and dried over anhydrous
Na2SO4. The solvent was removed in vacuo. The residue was purified by column chromatography to furnish 8 as white
solid (13.52g, 78%). mp 48-50 °C, ESI-MS m/z calcd for C14H13O2 [M + H]+ 213.1, found 213.3.
4.1.7. (E)-4-(2-(Benzyloxy)phenyl)-3-(ethoxycarbonyl)but-3-enoic acid (9)
14

Sodium (2.25 g, 98.00 mmol) was dissolved in EtOH (100 mL), which was added portionwise to a stirred solution of 8
(13.00 g, 61.25 mmol) and diethyl succinate (13.87 g, 79.62 mmol) in EtOH (50 mL). The reaction mixture was then
stirred and refluxed for 4 h. After the reaction finished, the resulting solution was concentrated in vacuo. The residue was
poured into water (300 mL), extracted three times with EtOAc, washed with saturated brine and dried with anhydrous
Na2SO4. The organic phase was concentrated and purified by silica flash chromatography to afford 9 as brown oil
(15.72g, 75%). ESI-MS m/z calcd for C20H21O5 [M + H]+ 341.1, found 341.3.
4.1.8. Ethyl 4-acetoxy-8-(benzyloxy)-2-naphthoate (10)
The above product 9 (15.00 g, 44.07 mmol) and sodium acetate (3.62 g, 44.07 mmol) were dissolved in acetic anhydride
(118.18 g, 881.38 mmol). The reaction mixture was refluxed for 6 h. Upon completion of the reaction, the solution was
evaporated to dryness. Then water (300 mL) was added, and extracted with DCM (3 × 60 mL), washed with saturated
Na2CO3 and brine. The combined organic layers were concentrated to dryness under vacuum, and recrystallized from
EtOAc and Et2O to afford 10 as light yellow solid (14.57 g, 91%). mp 96-98 °C, ESI-MS m/z calcd for C22H24NO5 [M +
NH4]+ 382.1, found 382.4.
4.1.9. Ethyl 8-(benzyloxy)-4-hydroxy-2-naphthoate (11)
To a solution of 10 (10.00 g, 27.40 mmol) in MeOH (100 mL) at room temperature was added K2CO3 (5.70 g, 41.20
mmol) slowly. After stirring for 2 h, the reaction solution was concentrated to dryness under vacuum. Water (100 mL)
was added, and then acidified with concentrated hydrochloric acid to pH = 6. The resulting precipitate was filtered off,
and recrystallized from EtOAc and hexane to obtain 11 as a white solid (13.52g, 78%). mp 181-183 °C, ESI-MS m/z
calcd for C20H22NO4 [M + NH4]+ 340.1, found 340.4.
4.1.10. Ethyl 7-(benzyloxy)-2,2-dimethyl-2H-benzo[h]chromene-5-carboxylate (13)
A mixture of 11 (5.00 g, 15.51 mmol), 3-methyl-2-butenal 12 (1.57 g, 18.61 mmol), PhB(OH)2 (2.08 g, 17.06 mmol),
and acetic acid (10 mL) in toluene (120 mL) was refluxed using Dean-Stark trap for 34 h. The resultant reaction solution
was diluted with EtOAc (200 mL), and washed with saturated Na2CO3 and brine in turn. The organic layer was dried, and
evaporated in vacuo. The resultant residue was purified by silica column chromatography to give 13 as yellow solid (3.50
g, 58%). mp 100-102 °C, ESI-MS m/z calcd for C25H28NO4 [M + NH4]+ 406.2, found 406.5.
4.1.11. 7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromene-5-carboxylic acid (14)
To a stirred suspension of NaOH (2.06 g, 51.50 mmol) in mixed solution (MeOH:H2O =1:1, 40 mL) was added 13 (4.00
g, 10.30 mmol). The mixture was stirred at 70 °C for 6 h. After the reaction came to end, MeOH was removed in vacuo.
The resultant mixture was acidified with concentrated hydrochloric acid to pH = 1, the resulting precipitate was filtered
off, and dried to give 14 as yellow solid (3.56 g, 96%). mp 161-163 °C, ESI-MS m/z calcd for C22H22O6 [M - H]- 359.1,
found 359.3
4.1.12. (7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromen-5-yl)(2,6-dimethylmorpholino)methanone (15a)
To a stirred suspension of 14 (3.50 g, 9.71 mmol) in CH3CN (50 mL) was added TBTU (3.12 g, 9.71 mmol). The
mixture was stirred at rt. for 1 h and then 2,6-dimethylmorpholine (cis:tran = 1:1) and N, N-diisopropylethylamine (1.12g,
9.71 mmol) were added. The mixture was still stirred at rt. for another 2 h. The resulting suspension was evaporated
15

under reduced pressure and the residue was treated with 1N HCl, 5% NaHCO3 and saturated brine, and dried over
anhydrous Na2SO4. The organic extracts were concentrated under reduced pressure, and was purified by column
chromatography (petroleum ether/EtOAc) to afford 15a as yellow solid (3.23g, 77%). mp 160-162 °C, ESI-MS m/z calcd
for C29H32NO4 [M + H]+ 458.2, found 458.4.
4.1.13. 7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromene-5-carbohydrazide (15b)
Compound 15b was prepared according to the procedure depicted for 15a. Yellow solid, ESI-MS m/z calcd for
C23H23N2O3 [M + H]+ 375.2, found 375.3.
4.1.14. (7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromen-5-yl)(morpholino)methanone (15c)
Compound 15c was prepared according to the procedure depicted for 15a. Yellow solid, ESI-MS m/z calcd for
C27H28NO4 [M + H]+ 430.2, found 430.5.
4.1.15.
(2,6-Dimethyltetrahydro-2H-pyran-4-yl)(7-hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)methanone
(16a)
To a solution of 15a (2.50 g, 5.47 mmol) in MeOH (20 mL) was added Pd/C (100 mg) under an atmosphere of hydrogen.
The mixture was stirred overnight. The resulting solution was filtered off to remove palladium carbon. The filtrate was
concentrated in vacuo to give 16a (2.21g) as white solid, which was used for the next step without further purification.
ESI-MS m/z calcd for C22H28NO4 [M + H]+ 370.2, found 370.5.
4.1.16. 7-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carbohydrazide (16b)
Compound 16b was prepared according to the procedure depicted for 16a. white solid, ESI-MS m/z calcd for
C16H19N2O3 [M + H]+ 287.1, found 287.3.
4.1.17. (7-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(morpholino)methanone (16c)
Compound 16c was prepared according to the procedure depicted for 16a. Yellow solid, ESI-MS m/z calcd for
C20H24NO4 [M + H]+ 342.2, found 342.1.
4.1.18. 7-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carboxylic acid (16d)
Compound 16d was prepared according to the procedure depicted for 16a. Yellow solid, ESI-MS m/z calcd for C16H15O4
[M - H]- 271.1, found 271.5.
4.1.19. General procedure for the preparation of the 5-(2,6-dimethylmorpholinoyl)-2H-benzo[h]chromene derivatives
WI1-WI9
To a solution of 15 (0.44 mmol, 1.0 eq) in CH3CN (20 mL) was slowly added K2CO3 (0.88 mmol, 2.0 eq) and substituted
benzyl halide (0.66 mmol, 1.5 eq). The reaction mixture was stirred at 55 °C for 4-8 h and concentrated in vacuo. The
residue was redissolved in EtOAc (30 mL), washed with brine, dried with Na2SO4 and concentrated. The residue was
purified by silica gel column chromatography (petroleum ether/EtOAc) to yield the target compounds WI1-WI 9 that
were the mixture (cis : tran = 1:1).
16

4.1.19.1.
(2,2-Dimethyl-7-((4-methylbenzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)(2,6-dimethyl-morpholino)methanon
e (WI1)
White solid, yield 73%, mp 212-214 °C, Rf = 0.74 (petroleum ether/EtOAc = 10:1). HPLC purity100.00% (A%/B% =
5:95, Rt = 3.749 min). 1H NMR (600 MHz, CDCl3) δ 7.83 (d, J = 8.5 Hz, 1H), 7.70 (d, 1H), 7.45–7.37 (m, 3H), 7.25 (dd,
J = 8.0, 3.0 Hz, 2H), 6.92 (d, J = 7.6 Hz, 1H), 5.26–5.16 (t, 2H), 4.70 (dd, 1H), 3.78–3.63 (m, 1H), 3.63–3.45 (m, 1H),
3.43–3.29 (m, 1H), 3.01 (ddt, 1H), 2.75 (ddd, 1H), 2.70–2.58 (m, 1H), 2.58–2.51 (m, 1H), 2.41 (s, 3H), 1.98–1.86 (m,
2H), 1.45 (d, J = 2.3 Hz, 6H), 1.29 (dd, J = 6.4, 2.6 Hz, 3H), 1.05 (d, J = 6.2 Hz, 3H). ESI-HRMS m/z calcd for
C30H36NO4 [M + H]+ 474.2644, found 474.2851.
4.1.19.2.
(7-((4-(Tert-butyl)benzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(2,6-dimethyl-morpholino)methanone
(WI2 )
White solid, yield 49%, mp 200-202 °C, Rf = 0.79 (petroleum ether/EtOAc = 10:1). HPLC purity 95.61% (A%/B% =
1
5:95, Rt = 5.052 min). H NMR (600 MHz, CDCl3) δ 7.84 (d, J = 8.5 Hz, 1H), 7.73 (d, 1H), 7.46 (t, J = 2.3 Hz, 4H),
7.42–7.37 (m, 1H), 6.96 – 6.91 (m, 1H), 5.21 (t, J = 5.4 Hz, 2H), 4.71 (dd, 1H), 3.79–3.64 (m, 1H), 3.64–3.46 (m, 1H),
3.36 (dd, 1H), 3.01 (ddt, 1H), 2.86–2.69 (m, 1H), 2.68–2.58 (m, 1H), 2.56 (d, 1H), 1.92 (m, 2H), 1.45 (d, J = 2.0 Hz, 6H),
1.38 (s, 9H), 1.29 (t, J = 5.6 Hz, 3H), 1.05 (dd, J = 6.5, 3.1 Hz, 3H). ESI-HRMS m/z calcd for C33H42NO4 [M + H]+
516.3114, found 516.3363.
4.1.19.3.
(2,6-Dimethylmorpholino)(7-((3-methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]-chromen-5-yl)methano
ne (WI3)
White solid, yield 64%, mp 176-178 °C, Rf = 0.71 (petroleum ether/EtOAc = 10:1). HPLC purity 95.04% (A%/B% =
1
5:95, Rt = 3.216 min). H NMR (600 MHz, CDCl3) δ 7.84 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 7.37 (dt, 2H), 7.10 (d, J =
7.6 Hz, 1H), 7.05 (t, J = 1.9 Hz, 1H), 6.93–6.88 (m, 2H), 5.22 (t, J = 5.2 Hz, 2H), 4.71 (dd, 1H), 3.85 (s, 3H), 3.77–3.46
(m, 2H), 3.38 (dd, 1H), 3.02 (ddt, 1H), 2.77 (ddd, 1H), 2.69–2.52 (m, 2H), 1.94 (ddd, 2H), 1.45 (d, J = 2.6 Hz, 6H), 1.30
(dd, J = 6.6, 3.1 Hz, 3H), 1.05 (dd, J = 6.3, 3.8 Hz, 3H). ESI-HRMS m/z calcd for C30H36NO5 [M + H]+ 490.2590, found
490.3004.
4.1.19.
4.4-(((5-(2,6-Dimethylmorpholine-4-carbonyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)benzonit
rile (WI4)
White solid, yield 68%, mp 186-188 °C, Rf = 0.63 (petroleum ether/EtOAc = 10:1). HPLC purity 98.94% (A%/B% =
1
5:95, Rt = 2.407 min). H NMR (600 MHz, CDCl3) δ 7.87 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.68 (d, 1H),
7.62 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 5.35–5.27 (m, 2H), 4.74–4.66 (m, 1H), 3.77–
3.63 (m, 1H), 3.60–3.50 (m, 1H), 3.34 (dd, 1H), 3.01 (ddt, 1H), 2.85–2.70 (m, 1H), 2.70–2.60 (m, 1H), 2.57 (dq, 1H),
1.93 (dt, 2H), 1.45 (s, 6H), 1.30 (dd, J = 6.3, 3.1 Hz, 3H), 1.05 (t, J = 6.1 Hz, 3H). ESI-RHMS m/z calcd for C30H32N2O4
[M + H]+ 485.2440, found 485.2796.
17

4.1.19.5.
(2,6-Dimethylmorpholino)(7-((4-fluorobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]-chromen-5-yl)-methanone
(WI5)
White solid, yield 75%, mp 194-196 °C, Rf = 0.67 (petroleum ether/EtOAc = 10:1). HPLC purity 99.68% (A%/B% =
1
5:95, Rt = 9.416 min). H NMR (600 MHz, CDCl3) δ 7.85 (d, J = 8.4 Hz, 1H), 7.68 (d, 1H), 7.48 (t, J = 6.6 Hz, 2H),
7.42–7.36 (m, 1H), 7.13 (t, J = 8.3 Hz, 2H), 6.91 (d, J = 2.7 Hz, 1H), 5.20 (t, J = 4.6 Hz, 2H), 4.70 (dd, 1H), 3.70 (dt, 1H),
3.60–3.48 (m, 1H), 3.35 (dd, 1H), 3.01 (ddt, 1H), 2.76 (dt, 1H), 2.69–2.53 (m, 2H), 1.94 (qd, 2H), 1.45 (s, 6H), 1.29 (t, J
= 5.3 Hz, 3H), 1.05 (t, J = 5.4 Hz, 3H). ESI-MS m/z calcd for C29H33FNO4 [M + H]+ 478.2, found 478.5.
4.1.19.6.
(7-((2,4-Dichlorobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(2,6-dimethyl-morpholino)methanone
(WI6)
White solid, yield 68%, mp 92-94 °C, Rf = 0.64 (petroleum ether/EtOAc = 10:1). HPLC purity 98.31% (A%/B% = 5:95,
Rt = 8.184 min). 1H NMR (600 MHz, CDCl3) δ 7.86 (d, J = 8.5 Hz, 1H), 7.69 (d, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.47 (t, J
= 1.4 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.32 (dd, J = 8.3, 2.0 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 5.30 (d, J = 1.8 Hz, 2H),
4.76–4.67 (m, 1H), 3.78–3.63 (m, 1H), 3.62–3.50 (m, 1H), 3.37 (ddt, 1H), 3.02 (ddt, 1H), 2.78 (ddd, 1H), 2.69–2.60 (m,
1H), 2.60–2.52 (m, 1H), 1.99–1.86 (m, 2H), 1.45 (d, J = 2.2 Hz, 6H), 1.30 (dd, J = 6.3, 2.7 Hz, 3H), 1.05 (t, J = 5.9 Hz,
3H). ESI-HRMS m/z calcd for C29H32Cl2NO4 [M + H]+ 528.1708, found 528.1889.
4.1.19.7.
(7-((2,6-Dichlorobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(2,6-dimethyl-morpholino)methanone
(WI7)
White solid, yield 45%, mp 112-114 °C, Rf = 0.68 (petroleum ether/EtOAc = 10:1). HPLC purity 97.57% (A%/B% =
1
5:95, Rt = 3.824 min). H NMR (600 MHz, CDCl3) δ 7.90–7.84 (m, 1H), 7.63–7.53 (m, 1H), 7.47–7.38 (m, 3H), 7.31
(dd, J = 8.6, 7.6 Hz, 1H), 7.06 (dd, J = 7.7, 0.9 Hz, 1H), 5.58–5.36 (m, 2H), 4.67 (dd, 1H), 3.66 (s, 1H), 3.50 (d, 1H),
3.37 (dd, 1H), 3.16–2.88 (m, 1H), 2.82–2.50 (m, 3H), 1.91 (dt, 2H), 1.47–1.43 (m, 6H), 1.27 (dd, J = 7.1, 4.2 Hz, 3H),
1.10–1.01 (m, 3H). ESI-HRMS m/z calcd for C29H32Cl2NO4 [M + H]+ 528.1708, found 528.1939.
4.1.19.8.
(7-((4-Bromobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(2,6-dimethyl-morpholino)methanone
(WI8)
White solid, yield 54%, mp 198-199 °C, Rf = 0.64 (petroleum ether/EtOAc = 10:1). HPLC purity 97.71% (A%/B% =
1
5:95, Rt = 3.694 min). H NMR (600 MHz, CDCl3) δ 7.85 (d, J = 8.5 Hz, 1H), 7.67 (d, 1H), 7.55 (d, J = 8.3 Hz, 2H),
7.42–7.36 (m, 3H), 6.87 (d, J = 7.6 Hz, 1H), 5.19 (d, J = 5.8 Hz, 2H), 4.71 (dd, 1H), 3.68 (dd, 1H), 3.62–3.46 (m, 1H),
3.36 (dd, 1H), 3.14–2.90 (m, 1H), 2.86–2.70 (m, 1H), 2.70–2.53 (m, 2H), 1.92 (h, J = 7.7 Hz, 2H), 1.45 (s, 6H), 1.30 (d,
J = 6.3 Hz, 3H), 1.05 (d, J = 6.3 Hz, 3H). ESI-HRMS m/z calcd for C29H33BrNO4 [M + H]+ 538.1593, found 538.1797.
4.1.19.9.
(2,2-Dimethyl-7-((4-nitrobenzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)(2,6-dimethyl-morpholino)methanone (WI9)
18

Yellow solid, yield 71%, mp 182-184 °C, Rf = 0.62 (petroleum ether/EtOAc = 10:1). HPLC purity 98.19% (A%/B% =
10:90, Rt = 3.694 min). 1H NMR (600 MHz, CDCl3) δ 8.30 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.4 Hz, 1H), 7.74–7.63 (m,
3H), 7.37 (t, J = 8.1 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 5.36 (s, 2H), 4.71 (dd, 1H), 3.77–3.63 (m, 1H), 3.61–3.48 (m, 1H),
3.36 (dd, 1H), 3.01 (ddt, 1H), 2.85–2.70 (m, 1H), 2.70–2.53 (m, 2H), 1.94 (qd, 2H), 1.46 (s, 6H), 1.30 (d, J = 6.1 Hz, 3H),
1.05 (t, J = 5.1 Hz, 3H). ESI-HRMS m/z calcd for C29H33N2O6 [M + H]+ 505.2338, found 505.2597.
4.1.20. General procedure for the preparation of the 5-(morpholinylalkyl)-2H-benzo[h]chromene derivatives WL1-WL10
The preparation method of WL1-WL10 is similar to that of WI1-WI9.
4.1.20.1. 7-(Benzyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carboxylic acid (WL1)
White solid, yield 87%, mp 189-191 °C, Rf = 0.29 (petroleum ether/EtOAc = 5:1). HPLC purity 97.88% (A%/B% =
25:75, Rt = 1.603 min). 1H NMR (600 MHz, CDCl3) δ 8.76 (d, J = 0.9 Hz, 1H), 7.86 (dt, J = 8.5, 0.9 Hz, 1H), 7.57–7.53
(m, 2H), 7.48–7.43 (m, 3H), 7.39–7.36 (m, 1H), 6.93–6.90 (m, 1H), 5.31 (s, 2H), 3.29 (t, J = 6.8 Hz, 2H), 1.93 (t, J = 6.8
Hz, 2H), 1.47 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 173.01, 155.14, 149.41, 136.94, 129.20, 128.66 (2C), 127.96,
127.92, 127.29 (2C), 125.84, 123.93, 119.10, 115.39, 114.34, 106.01, 74.29, 70.19, 32.73, 26.73 (2C), 21.81. ESI-HRMS
m/z calcd for C23H26NO4 [M - H]- 361.1440, found 361.1437.
4.1.20.2. 7-((3-Methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carboxylic acid (WL2)
White solid, yield 76%, mp 184-186 °C, Rf = 0.26 (petroleum ether/EtOAc = 5:1). HPLC purity 99.36% (A%/B% =15:85,
1
Rt = 1.606 min). H NMR (600 MHz, CDCl3) δ 8.79 (d, J = 0.8 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.49–7.43 (m, 1H),
7.36 (t, J = 7.8 Hz, 1H), 7.14–7.10 (m, 2H), 6.94–6.89 (m, 2H), 5.29 (s, 2H), 3.87 (s, 3H), 3.30 (t, J = 6.8 Hz, 2H), 1.94 (t,
J = 6.8 Hz, 2H), 1.47 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 173.25, 159.88, 155.11, 149.42, 138.59, 129.69, 129.22,
128.00, 125.82, 123.94, 119.38, 119.19, 115.43, 114.39, 113.71, 112.39, 106.09, 74.29, 70.09, 55.26, 32.73, 26.73 (2C),
21.82. ESI-HRMS m/z calcd for C24H27NO5 [M - H]- 391.1546, found 391.1544.
4.1.20.3. 7-((4-Acetamidobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carboxylic acid (WL3)
White solid, yield 77%, mp 150-152 °C, Rf = 0.41 (petroleum ether/EtOAc = 10:1). HPLC purity 95.28% (A%/B% =
10:90, Rt = 3.501 min).1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.78 (s, 1H), 8.29 (s, 1H), 7.39 (d, J = 8.2 Hz,
2H), 7.08 (d, J = 7.8 Hz, 1H), 6.80 (t, J = 7.4 Hz, 3H), 4.51 (s, 2H), 3.01 (t, J = 6.8 Hz, 2H), 1.98 (s, 3H), 1.67 (t, J = 6.8
Hz, 2H), 0.97 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 169.30, 168.36, 153.15, 151.03, 138.80, 136.97, 132.71, 128.13
(2C), 127.92, 127.44, 125.64, 124.71, 119.06 (2C), 117.76, 115.17, 108.76, 74.26, 41.89, 32.12, 26.04 (2C), 24.38, 21.81.
ESI-HRMS m/z calcd for C25H29N2O5 [M -H]- 418.1655, found 418.1650.
4.1.20.4. 7-(Benzyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carbohydrazide (WL4)
White solid, yield 32%。mp 170-172 °C, Rf = 0.48 (petroleum ether/EtOAc = 10:1). HPLC purity 98.56% (A%/B% =
1
12:88, Rt = 2.534 min). H NMR (600 MHz, CDCl3) δ 7.89 (s, 1H), 7.85–7.82 (m, 1H), 7.51 (dd, J = 7.3, 1.8 Hz, 2H),
7.47–7.42 (m, 2H), 7.43–7.37 (m, 2H), 7.28 (s, 1H), 6.91 (d, J = 7.7 Hz, 1H), 5.24 (s, 2H), 4.23–4.11 (s, 2H), 3.01 (t, J =
6.7 Hz, 2H), 1.88 (t, J = 6.7 Hz, 2H), 1.45 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 170.72, 154.52, 149.48, 136.84,
131.75, 128.68 (2C), 128.09, 127.78, 127.57 (2C), 126.62, 124.16, 114.41, 113.14, 112.38, 106.01, 74.68, 70.31, 32.47,
26.87 (2C), 20.62. ESI-MS m/z calcd for C23H25N2O3 [M + H]+ 377.2, found 377.4.
19

4.1.20.5. 7-((3-Methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5-carbohydrazide (WL5)
White solid, yield 73%, mp 139-141 °C, Rf = 0.38 (petroleum ether/EtOAc = 10:1). HPLC purity 99.58% (A%/B% =
18:82, Rt = 3.244 min). 1H NMR (600 MHz, CDCl3) δ 7.91–7.88 (m, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.43–7.32 (m, 3H),
7.09 (dd, J = 7.7, 1.3 Hz, 1H), 7.05 (t, J = 2.0 Hz, 1H), 6.93–6.87 (m, 2H), 5.21 (s, 2H), 4.02 (d, 2H), 3.84 (s, 3H), 3.00 (t,
J = 6.7 Hz, 2H), 1.87 (t, J = 6.7 Hz, 2H), 1.44 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 170.70, 159.84, 154.49, 149.47,
138.44, 131.70, 129.75, 127.78, 126.61, 124.15, 119.75, 114.43, 113.37, 113.21, 113.16, 112.43, 106.06, 74.68, 70.22,
55.28, 32.46, 26.86 (2C), 20.62. ESI-MS m/z calcd for C24H27N2O4 [M + H]+ 407.2, found 407.4.
4.1.20.6.
N-(4-(((5-(Hydrazinecarbonyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)phenyl)acetamide
(WL6)
White solid, yield 53%, mp 225-227 °C, Rf = 0.27 (petroleum ether/EtOAc = 5:1). HPLC purity 97.38% (A%/B% =
30:70, Rt = 8.856 min). 1H NMR (600 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.77 (s, 1H), 9.49 (s, 1H), 7.70 (s, 1H), 7.39 (d,
J = 8.3 Hz, 2H), 7.03 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 8.0 Hz, 3H), 4.51 (s, 2H), 4.47 (s, 2H), 2.78 (t, J = 6.9 Hz, 2H),
1.99 (s, 3H), 1.66 (t, J = 6.8 Hz, 2H), 0.98 (s, 6H). 13C NMR (150 MHz, DMSO-d6) δ 168.96, 168.36, 152.65, 150.64,
138.98, 136.92, 133.36, 131.37, 128.10 (2C), 126.22, 125.56, 125.16, 119.07 (2C), 113.57, 113.43, 108.56, 74.47, 41.93,
31.95, 26.14 (2C), 24.38, 20.77. ESI-MS: calcd for C25H31N4O4 [M + NH4]+ 451.2, found 451.4.
4.1.20.7. (7-(Benzyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(morpholino)methanone (WL7)
White solid, yield 85%, mp 171-172 °C, Rf = 0.70 (petroleum ether/EtOAc = 10:1). HPLC purity 100.00% (A%/B% =
15:85, Rt = 6.633 min). 1H NMR (400 MHz, DMSO-d6) δ 7.66 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 7.1 Hz, 2H), 7.48 (s, 1H),
7.45 – 7.33 (m, 4H), 7.08 (d, J = 7.7 Hz, 1H), 5.29 (s, 2H), 3.67 (s, 4H), 3.48 (s, 2H), 3.18 (d, J = 5.7 Hz, 2H), 2.70 (d,
2H), 1.88 (s, 2H), 1.38 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 169.92, 154.45, 149.38, 137.00, 133.17, 128.62 (2C),
127.99, 127.42 (2C), 126.88, 125.86, 124.82, 114.31, 111.56, 110.79, 106.00, 74.70, 70.19, 66.97 (2C), 47.46, 41.97,
32.33, 26.93, 26.76, 20.21. ESI-HRMS m/z calcd for C27H30NO4 [M + H]+432.2175, found 432.2135.
4.1.20.8. (7-((3-Methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)(morpholino)methanone (WL8)
White solid, yield 64%, mp 138-140 °C, Rf = 0.68 (petroleum ether/EtOAc = 10:1). HPLC purity 96.10% (A%/B% =
15:85, Rt = 5.142 min). 1H NMR (600 MHz, CDCl3) δ 7.83 (d, J = 8.5 Hz, 1H), 7.73 (s, 1H), 7.36 (dt, J = 12.8, 7.9 Hz,
2H), 7.10 (d, J = 7.5 Hz, 1H), 7.05 (t, J = 2.0 Hz, 1H), 6.94–6.88 (m, 2H), 5.22 (d, J = 4.1 Hz, 2H), 3.93 – 3.86 (m, 2H),
3.85 (s, 3H), 3.84–3.77 (m, 2H), 3.60 (d, J = 33.7 Hz, 2H), 3.34 (d, J = 45.6 Hz, 2H), 3.10 – 3.00 (m, 1H), 2.63 (d, J =
17.1 Hz, 1H), 1.93 (q, J = 8.4, 7.4 Hz, 2H), 1.45 (s, 6H). 113C NMR (150 MHz, CDCl3) δ 169.89, 159.83, 154.44, 149.39,
138.62, 133.17, 129.69, 126.88, 125.86, 124.82, 119.63, 114.34, 113.36, 113.00, 111.59, 110.82, 106.07, 74.70, 70.15,
66.98, 55.28, 47.48, 41.98, 32.34, 32.34, 26.94, 26.72, 20.22. ESI-MS m/z calcd for C28H32NO5 M + H]+ 462.2, found
462.4.
4.1.20.9.
(2,2-Dimethyl-7-((4-(trifluoromethyl)benzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)(morpholino)methanone (WL9)
20

White solid, yield 44%, mp 242-244 °C, Rf = 0.62 (petroleum ether/EtOAc = 10:1). HPLC purity 100.00% (A%/B% =
15:85, Rt = 5.555 min). 1H NMR (600 MHz, CDCl3) δ 7.86 (d, J = 8.5 Hz, 1H), 7.73–7.67 (m, 3H), 7.63 (d, J = 8.0 Hz,
2H), 7.37 (t, J = 8.1 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 5.30 (s, 2H), 3.89 (q, J = 5.1 Hz, 2H), 3.86–3.79 (m, 2H), 3.66–
3.53 (m, 2H), 3.41–3.26 (m, 2H), 3.04 (dt, J = 15.3, 6.8 Hz, 1H), 2.70–2.59 (m, 1H), 1.94 (q, J = 7.1 Hz, 2H), 1.46 (s,
6H). 13C NMR (150 MHz, CDCl3) δ 169.84, 153.99, 149.47, 141.04, 133.49, 127.34 (2C), 126.92, 125.75, 125.64,
125.62, 125.59, 125.57, 124.99, 124.73, 114.74, 111.70, 110.47, 106.02, 74.79, 69.31, 66.97, 47.45, 41.98, 32.30, 26.87,
26.77, 20.21. ESI-MS m/z calcd for C28H29F3NO4 [M + H]+ 500.2, found 500.3.
4.1.20.10.
N-(4-(((2,2-Dimethyl-5-(morpholine-4-carbonyl)-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)phenyl)acrylamide
(WL10)
White solid, yield 38%, mp 128-130 °C, Rf = 0.27 (petroleum ether/EtOAc = 10:1). HPLC purity 98.82% (A%/B% =
1
15:85, Rt = 3.294 min). H NMR (600 MHz, DMSO-d6) δ 10.22 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.5 Hz,
1H), 7.50–7.45 (m, 3H), 7.39 (t, J = 8.1 Hz, 1H), 7.09 (d, J = 7.7 Hz, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.28 (dd, J =
17.0, 1.9 Hz, 1H), 5.78–5.75 (m, 1H), 5.23 (s, 2H), 3.69 (d, J = 19.8 Hz, 4H), 3.54–3.42 (m, 2H), 3.19 (d, J = 13.3 Hz,
2H), 2.83–2.55 (m, 2H), 1.87 (q, J = 6.6 Hz, 2H), 1.38 (s, 6H). 13C NMR (150 MHz, DMSO-d6) δ 168.65, 163.62, 154.27,
149.02, 139.24, 134.39, 132.38, 132.29, 128.90 (2C), 127.42, 126.47, 126.31, 124.50, 119.80 (2C), 114.03, 112.30,
110.09, 107.02, 75.15, 69.87, 66.73, 55.38, 47.37, 41.84, 31.99, 27.02, 26.81, 20.04. ESI-HRMS m/z calcd for
C30H33N2O5 [M - H]- 499.2233, found 499.2243.
4.1.21. (7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromen-5-yl)methanol (17)
The key intermediate 13 (3.00 g, 7.72 mmol) was dissolved in THF (20 mL) at 0 °C. LiAlH4 (0.59 g, 15.44 mmol) was
slowly added to the above solution. The mixture was stirred for another 2 h. After the reaction completed, the mixture
was quenched with water (30 mL), extracted with DCM (3 × 35 mL), washed with saturated brine, dried with sodium
sulphate and concentrated in vacuo. The residue was purified via silica gel column chromatography (petroleum
ether/EtOAc) to give 17 as yellow solid (2.07 g, 77%). mp 96-98 °C, ESI-MS m/z calcd for C23H26NO3 [M + NH4]+ 364.2,
found 364.4.
4.1.22. 7-(Benzyloxy)-5-(bromomethyl)-2,2-dimethyl-2H-benzo[h]chromene (18)
To a solution of 17 (2.00 g, 5.77 mmol) and CBr4 (2.11 g, 6.35 mmol) in DCM (15 mL) at 0 °C was added PPh3 (1.67 g,
6.35 mmol) slowly. The mixture was stirred for 3 h, and the reaction solvents were concentrated to dryness. The residue
was purified by flash column chromatography to afford 18 as yellow solid (1.63 g, 69%). mp 89-91 °C, ESI-MS m/z
calcd for C23H22BrO2 [M + H]+ 409.1, found 409.4.
4.1.23. 1-((7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromen-5-yl)methyl)-1H-tetrazole (19a)
A solution of 18 (3.80 g, 9.28 mmol), K2CO3 (2.57 g, 18.56 mmol) and 1H-tetrazole (0.78 g, 11.14 mmol) in DMF (15
mL) was heated at 75 °C for 5 h. The reaction mixture was then cooled to room temperature, diluted with EtOAc (30 mL),
and washed with water (30 mL). The organic extract was dried, and the residue was purified by flash column
chromatography (petroleum ether/EtOAc) to afford 19a as white solid (2.22 g, 47%). mp 100-102 °C, ESI-MS m/z calcd
for C24H24N5O2 [M + NH4]+ 416.2, found 416.5.
21

4.1.24. (2S,6R)-4-((7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromen-5-yl)methyl)-2,6-dimethyl-morpholine(19b)
Compound 19b was prepared according to the procedure depicted for 19a. Colorless oil, ESI-MS m/z calcd for
C29H34NO3 [M + H]+ 444.2, found 444.5.
4.1.25. 5-((1H-Tetrazol-1-yl)methyl)-2,2-dimethyl-2H-benzo[h]chromen-7-ol (20a)
Compound 20a was prepared according to the procedure depicted for 16a. White solid, ESI-MS m/z calcd for
C17H22N5O2 [M + NH4]+ 328.1, found 328.4.
4.1.26. 5-(((2S,6R)-2,6-Dimethylmorpholino)methyl)-2,2-dimethyl-2H-benzo[h]chromen-7-ol (20b)
Compound 20b was prepared according to the procedure depicted for 16a. Yellow solid, ESI-MS m/z calcd for
C22H30NO3 [M + H]+ 356.2, found 356.4.
4.1.27. General procedure for the preparation of the 5-(1,2,3,4-tetrazolylmethyl)-2H-benzo[h]chromene derivatives
WJ1-WJ10
The preparation method of WJ1-WJ10 was similar to that of WL1-WL10.
4.1.27.1. 1-((7-(Benzyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-1H-tetrazole (WJ1)
White solid, yield 71%, mp 145-147 °C, Rf = 0.52 (petroleum ether/EtOAc = 10:1). HPLC purity 97.61% (A%/B% =
1
10:90, Rt = 2.887 min). H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.87 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.49 (d, J =
7.1 Hz, 2H), 7.45–7.39 (m, 2H), 7.39–7.31 (m, 2H), 6.86 (d, J = 7.7 Hz, 1H), 5.92 (s, 2H), 5.22 (s, 2H), 2.78 (t, J = 6.7
Hz, 2H), 1.89 (t, J = 6.7 Hz, 2H), 1.37 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 154.36, 152.96, 149.52, 137.07, 128.82,
128.60 (2C), 127.92, 127.34 (2C), 127.24, 125.98, 124.81, 115.57, 114.36, 113.82, 105.99, 74.18, 70.18, 55.44, 32.38,
26.67 (2C), 19.86. ESI-HRMS m/z calcd for C24H28N5O2 [M + H]+ 401.1899, found 418.1864.
4.1.27.2. 1-((7-((3-Methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-1H-tetrazole (WJ2)
White solid, yield 58%, mp 159-161 °C, Rf = 0.56 (petroleum ether/EtOAc = 8:1). HPLC purity 97.66% (A%/B% =
1
18:82, Rt = 6.277 min). H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.86 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.40 (t, J =
8.1 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.09 – 7.01 (m, 2H), 6.91 (dd, J = 10.4, 7.0 Hz, 2H), 5.68 (s, 2H), 5.21 (s, 2H), 3.82
(s, 3H), 2.56 (t, J = 6.7 Hz, 2H), 1.85 (d, J = 13.4 Hz, 2H), 1.36 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 159.88, 154.26,
150.10, 142.25, 138.43, 129.77, 128.11, 127.46, 126.46, 124.80, 119.70, 115.64, 114.51, 113.37, 113.32, 113.27, 106.38,
74.44, 70.29, 55.30, 51.31, 32.15, 26.64 (2C), 19.63. ESI-HRMS m/z calcd for C25H27N4O3 [M + H]+ 431.2083, found
431.1884.
4.1.27.3. 4-(((5-((1H-Tetrazol-1-yl)methyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)-methyl)benzonitrile
(WJ3)
White solid, yield 70%, mp 200-202°C, Rf = 0.50 (petroleum ether/EtOAc = 8:1). HPLC purity 97.07% (A%/B% = 18:82,
Rt = 5.070 min). 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.80 (s, 1H), 7.74 – 7.69 (m, 2H),
7.59 (d, J = 8.0 Hz, 2H), 7.33 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 5.95 (s, 2H), 5.28 (s, 2H), 2.82 (t, J = 6.7 Hz,
2H), 1.91 (t, J = 6.7 Hz, 2H), 1.39 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 153.66, 153.00, 149.63, 142.47, 132.48 (2C),
22

129.26, 127.49 (2C), 125.78, 124.63, 118.69, 115.00, 114.90, 113.99, 111.79, 106.00, 104.99, 74.31, 69.12, 55.29, 32.33,
26.66 (2C), 19.88. ESI-HRMS m/z calcd for C25H27N6O2 [M + H]+ 426.1930, found 426.1918.
4.1.27.4.
1-((2,2-Dimethyl-7-((4-(trifluoromethyl)benzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-1H-tetrazole (WJ4)
White solid, yield 65%, mp 158-160°C, Rf = 0.47 (petroleum ether/EtOAc = 8:1). HPLC purity 98.52% (A%/B% = 10:90,
Rt = 4.300 min). 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.83 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.1 Hz,
2H), 7.60 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.1 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 5.94 (s, 2H), 5.28 (s, 2H), 2.81 (t, J = 6.7
Hz, 2H), 1.90 (t, J = 6.7 Hz, 2H), 1.38 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 153.90, 152.99, 149.60, 141.11, 129.12,
127.29 (2C), 127.25, 125.86, 125.65, 125.61, 125.58, 125.54, 124.69, 115.16, 114.80, 113.95, 105.99, 74.27, 69.33,
55.34, 32.35, 26.66 (2C), 19.88. ESI-HRMS m/z calcd for C25H27F3N5O2 [M +H]+ 469.1851, found 469.1870.
4.1.27.5.
1-((2,2-Dimethyl-7-((4-(trifluoromethoxy)benzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-1H-tetrazole
(WJ5)
White solid, yield 46%, mp 154-156 °C, Rf = 0.57 (petroleum ether/EtOAc = 8:1). HPLC purity 97.66% (A%/B% =
1
10:90, Rt = 3.538 min). H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.79 (s, 1H), 7.51 (d, J =
8.5 Hz, 2H), 7.40 (t, J = 8.1 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 6.90 (d, J = 7.7 Hz, 1H), 5.70 (s, 2H), 5.23 (s, 2H), 2.58 (t,
J = 6.7 Hz, 2H), 1.86 (t, J = 6.6 Hz, 2H), 1.37 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 153.97, 150.14, 148.98, 142.29,
135.51, 129.03, 128.84 (2C), 128.34, 127.45, 126.36, 124.70, 121.20 (2C), 115.21, 114.77, 113.43, 106.24, 74.49, 69.39,
51.24, 32.14, 26.63 (2C), 19.64. ESI-HRMS m/z calcd for C25H23F3N4O3Na [M + H]+ 485.1800, found 485.1768.
4.1.27.6. 1-((2,2-Dimethyl-7-((4-nitrobenzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-1H-tetrazole (WJ6)
Yellow solid, yield 89%, mp 150-151 °C, Rf = 0.49 (petroleum ether/EtOAc = 8:1). HPLC purity 97.63% (A%/B% =
10:90, Rt = 3.375 min). 1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H), 8.31–8.24 (m, 2H), 7.87–7.78 (m, 2H), 7.65 (d, J =
8.4 Hz, 2H), 7.33 (t, J = 8.1 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 5.95 (s, 2H), 5.33 (s, 2H), 2.82 (t, J = 6.7 Hz, 2H), 1.91 (t,
J = 6.7 Hz, 2H), 1.39 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 153.60, 153.02, 149.64, 147.64, 144.45, 129.32, 127.55
(2C), 127.26, 125.77, 124.63, 123.91 (2C), 115.09, 114.88, 114.03, 106.02, 74.33, 68.91, 55.28, 32.33, 26.66 (2C), 19.89.
ESI-MS: calcd for C24H27N6O4 [M + NH4]+ 463.2, found 463.5.
4.1.27.7.
4-(((5-((1H-Tetrazol-1-yl)methyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)-methyl)-N-methylbenzamide
(WJ7)
White solid, yield 27%, mp 212-214 °C, Rf = 0.49 (petroleum ether/EtOAc = 5:1). HPLC purity 97.34% (A%/B% =
10:90, Rt = 3.207 min). 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 7.82 (dd, J = 10.1, 8.2 Hz, 3H), 7.74 (s, 1H), 7.51 (d,
J = 8.0 Hz, 2H), 7.39 (t, J = 8.1 Hz, 1H), 6.88 (d, J = 7.7 Hz, 1H), 6.33–6.24 (m, 1H), 5.70 (s, 2H), 5.26 (s, 2H), 3.03 (d,
J = 4.7 Hz, 3H), 2.61 (t, J = 6.7 Hz, 2H), 1.87 (t, J = 6.7 Hz, 2H), 1.37 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 167.83,
153.96, 150.09, 142.36, 140.24, 134.44, 128.44, 127.38, 127.32 (2C), 127.26 (2C), 126.33, 124.77, 114.89, 114.77,
113.30, 106.49, 74.47, 69.79, 51.09, 32.15, 26.87, 26.63 (2C), 19.66. ESI-HRMS m/z calcd for C26H28N5O3 [M + H]+
458.2222, found 458.2434.
23

4.1.27.8.
4-(((5-((1H-Tetrazol-1-yl)methyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)-N-isopropylbenzamid
e (WJ8)
White solid, yield 59%，mp 201-203 °C, Rf = 0.45 (petroleum ether/EtOAc = 8:1). HPLC purity 99.85% (A%/B% =
25:75, Rt = 4.149min). 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.36 (d, J = 7.6 Hz, 1H), 7.92–7.89 (m, 2H), 7.69
(d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.42 (s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 5.89 (s, 2H), 5.31 (s, 2H), 4.06 (d, J =
6.9 Hz, 1H), 2.69 (t, J = 7.0 Hz, 2H), 1.71 (t, J = 6.9 Hz, 2H), 1.18 (d, J = 6.6 Hz, 6H), 0.94 (s, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 165.64, 152.71, 150.99, 147.24, 144.99, 140.48, 134.69, 132.00, 130.64, 127.94 (2C), 127.70, 127.35,
126.99 (2C), 125.40, 115.22, 106.52, 74.51, 69.25, 49.58, 41.45, 31.67, 25.92 (2C), 22.84 (2C), 19.71. ESI-MS m/z calcd
for C28H32N5O3 [M + H]+ 486.2, found 486.5.
4.1.27.9. N-(4-(((5-((1H-Tetrazol-1-yl)methyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)phenyl)
acetamide (WJ9)
White solid, yield 39%, mp 195-197 °C, Rf = 0.38 (petroleum ether/EtOAc = 8:1). HPLC purity 96.28% (A%/B% =
1
10:90, Rt = 4.070 min). H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.77 (s, 1H), 7.55 (d, J =
8.0 Hz, 2H), 7.47–7.35 (m, 4H), 6.91 (d, J = 7.7 Hz, 1H), 5.68 (s, 2H), 5.17 (s, 2H), 2.58 (t, J = 6.5 Hz, 2H), 2.18 (s, 3H),
1.85 (t, J = 6.6 Hz, 2H), 1.36 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 168.40, 154.23, 150.06, 142.32, 137.82, 132.61,
128.42 (2C), 128.19, 127.40, 126.42, 124.82, 120.10 (2C), 115.37, 114.51, 113.30, 106.42, 74.43, 70.08, 51.19, 32.15,
26.63 (2C), 24.59, 19.63. ESI-MS m/z calcd for C26H28N5O3 [M + H]+ 458.2, found 458.5.
4.1.27.10. N-(4-(((5-((1H-Tetrazol-1-yl)methyl)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)phenyl)
acrylamide (WJ10)
43%, mp 197-199 °C, Rf = 0.37 (petroleum ether/EtOAc = 8:1). HPLC purity 95.55% (A%/B% = 10:90, Rt = 6.173 min).
1H NMR (400 MHz, CD White solid, yield Cl3) δ 8.38 (s, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.75 (s, 1H), 7.64 (d, J = 8.0 Hz,
2H), 7.44–7.36 (m, 3H), 6.90 (d, J = 7.7 Hz, 1H), 6.49–6.39 (m, 1H), 6.30 (dd, J = 16.8, 10.1 Hz, 1H), 5.76 (d, J = 10.2
Hz, 1H), 5.66 (s, 2H), 5.15 (s, 2H), 2.58 (t, J = 6.6 Hz, 2H), 1.85 (t, J = 6.6 Hz, 2H), 1.36 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 163.73, 154.20, 150.03, 142.40, 137.74, 132.82, 131.14, 128.40 (2C), 128.26, 127.92, 127.37, 126.41 (2C),
124.79, 120.26, 115.26, 114.50, 113.29, 106.37, 74.42, 70.02, 51.15, 32.15, 26.63 (2C), 19.63. ESI-MS m/z calcd for
C27H28N5O3 [M + H]+ 470.2, found 470.4.
4.1.28. General procedure for the preparation of the 5-(morpholinylalkyl)-2H-benzo[h]chromene derivatives
WL11-WL13
The preparation method of WL11-WL13 was similar to that of WL1-WL10.
4.1.28.1.
(2S,6R)-4-((7-(Benzyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-2,6-dimethylmorpholine (WL11)
25
White solid, yield 55%, mp 132-134 °C, Rf = 0.61 (petroleum ether/EtOAc = 10:1), [α]
= -153.85 (C=1, CH3OH).
D
HPLC purity 96.23% (A%/B% = 2:98, Rt = 4.665 min). 1H NMR (400 MHz, DMSO-d6) δ 7.62 (d, J = 7.6 Hz, 2H), 7.53
(d, J = 7.0 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.38–7.33 (m, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.00 (d, J = 7.7 Hz, 1H), 5.29 (s,
24

2H), 3.53 (s, 4H), 2.92 (t, J = 6.8 Hz, 2H), 2.69 (d, J = 10.8 Hz, 2H), 1.85 (t, J = 6.7 Hz, 2H), 1.70 (t, J = 10.6 Hz, 2H),
1.35 (s, 6H), 1.03 (d, J = 6.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 154.16, 148.81, 137.45, 134.07, 128.55 (2C),
127.82, 127.24 (2C), 126.34, 124.63, 124.51, 115.75, 114.37, 114.21, 105.56, 73.85, 71.96 (2C), 70.12, 61.78, 59.52
(2C), 32.73, 26.81 (2C), 19.59, 19.17 (2C). ESI-MS m/z calcd for C29H36NO3 [M + H]+ 446.3, found 446.5.
4.1.28.2.
(2S,6R)-4-((7-((3-Methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-2,6-dimethylmorphol
ine (WL12)
25
White solid, yield 67%, mp 96-98 °C, Rf = 0.63 (petroleum ether/EtOAc = 10:1), [α] D = -19.40(C=1, CH3OH). HPLC
purity 99.05% (A%/B% = 2:98, Rt = 10.171 min). 1H NMR (400 MHz, DMSO-d6) δ 7.62 (t, J = 4.2 Hz, 2H), 7.31 (dt, J
= 12.9, 7.9 Hz, 2H), 7.12–7.06 (m, 2H), 6.98 (d, J = 7.7 Hz, 1H), 6.94–6.88 (m, 1H), 5.26 (s, 2H), 3.77 (s, 3H), 3.52 (s,
4H), 2.92 (t, J = 6.7 Hz, 2H), 2.68 (d, J = 10.8 Hz, 2H), 1.84 (t, J = 6.7 Hz, 2H), 1.69 (t, J = 10.6 Hz, 2H), 1.34 (s, 6H),
1.02 (d, J = 6.2 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 159.83, 154.12, 148.82, 139.08, 134.07, 129.59, 126.36, 124.63,
124.49, 119.40, 115.79, 114.39, 114.28, 113.29, 112.72, 105.54, 73.85, 71.94 (2C), 69.98, 61.86, 59.52 (2C), 55.24,
32.73, 26.80 (2C), 19.59, 19.17 (2C). ESI-MS m/z calcd for C30H38NO4 [M + H]+ 476.3, found 476.4.
4.1.28.3.
(2S,6R)-4-((2,2-Dimethyl-7-((4-(trifluoromethyl)benzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)methyl)-2,6-dimethyl
morpholine (WL13)
25
White solid, yield 30%, mp 150-152 °C, Rf = 0.66 (petroleum ether/EtOAc = 10:1), [α]
= -25.00 (C=1, CH3OH).
D
HPLC purity 95.35% (A%/B% = 2:98, Rt = 4.355 min). 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 8.1 Hz, 2H), 7.74
(d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 7.7 Hz, 1H), 5.42 (s, 2H), 3.52 (d, J =
12.6 Hz, 4H), 2.94 (t, J = 6.7 Hz, 2H), 2.68 (d, J = 10.7 Hz, 2H), 1.85 (t, J = 6.7 Hz, 2H), 1.71 (t, J = 10.6 Hz, 2H), 1.35
(s, 6H), 1.02 (d, J = 6.2 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 153.70, 148.90, 141.50, 134.33, 128.31, 127.18 (2C),
126.39, 125.55, 125.53, 124.52, 124.38, 115.94, 114.78 (2C), 113.95, 105.52, 73.93, 71.95 (2C), 69.25, 61.84, 59.51
(2C), 32.70, 26.79 (2C), 19.60, 19.16 (2C). ESI-MS m/z calcd for C30H35F3NO3 [M + H]+ 514.2, found 514.5.
4.1.29. 7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromene-5-carbohydrazide (21)
A solution of 13 (5.00 g, 12.87 mmol) in hydrazine hydrate (30 mL) was stirred and refluxed for 8 h. The reaction
solution was concentrated to produce the crude product 21 as yellow oil (4.87g, 98%), which was used directly for the
next step without purification.
4.1.30. 2-(7-(Benzyloxy)-2,2-dimethyl-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (22)
The above crude product 21 (4.87 g, 13.01 mmol) was dissolved in triethyl orthoformate (9.64 g, 65.05 mmol), which
was refluxed for 12 h. The reaction mixture was then cooled to room temperature, the solvent was removed under
reduced pressure, and the residue was purified by flash column chromatography (petroleum ether/EtOAc) to afford 22 as
yellow solid (2.98, 60%). mp 198-200 °C, ESI-MS m/z calcd for C24H21N2O3 [M + H]+ 385.1, found 385.3.
4.1.31. 2,2-Bimethyl-5-(1,3,4-oxadiazol-2-yl)-3,4-dihydro-2H-benzo[h]chromen-7-ol (23)
The preparation method of 23 was similar to that of 16a. Yellow solid, mp 203-205 °C, ESI-MS m/z calcd for
25

C17H17N2O3 [M + H]+ 297.1, found 297.4.
4.1.32. General procedure for the preparation of the 2H-benzo[h]chromene derivatives WK1-WK9
The preparation method of WK1-WK9 was similar to that of WL1-WL10.
4.1.32.1. 2-(7-(Benzyloxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK1)
White solid, yield 66%, mp 150-152 °C, Rf = 0.57 (petroleum ether/EtOAc = 10:1). HPLC purity 97.55% (A%/B% =
1
2:98, Rt = 2.819 min). H NMR (400 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.34 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.59–7.48
(m, 3H), 7.47–7.40 (m, 2H), 7.40–7.33 (m, 1H), 7.15 (d, J = 7.7 Hz, 1H), 5.34 (s, 2H), 3.19 (t, J = 6.7 Hz, 2H), 1.92 (t, J
= 6.7 Hz, 2H), 1.41 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 165.20, 154.80, 152.22, 149.70, 136.88, 128.67 (2C), 128.23,
128.02, 127.46, 127.40 (2C), 124.38, 120.18, 115.90, 114.43, 114.00, 106.17, 74.56, 70.27, 32.58, 26.73 (2C), 22.43.
ESI-HRMS m/z calcd for C24H23N2O3 [M + H]+ 387.1708, found 387.1780.
4.1.32.2. 2-(2,2-Dimethyl-7-((4-methylbenzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK2)
White solid, yield 80%, mp 120-122 °C, Rf = 0.60 (petroleum ether/EtOAc = 10:1). HPLC purity 96.73% (A%/B% =
4:96, Rt = 3.519 min). 1H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 8.32 (s, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.50 (t, J =
8.1 Hz, 1H), 7.43 (d, J = 7.7 Hz, 2H), 7.24 (d, J = 7.7 Hz, 2H), 7.14 (d, J = 7.7 Hz, 1H), 5.28 (s, 2H), 3.19 (t, J = 6.7 Hz,
2H), 2.33 (s, 3H), 1.92 (t, J = 6.7 Hz, 2H), 1.41 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 165.20, 154.88, 152.20, 149.68,
137.79, 133.83, 129.33 (2C), 128.22, 127.53 (2C), 127.47, 124.40, 120.12, 115.96, 114.31, 113.96, 106.15, 74.54, 70.22,
32.58, 26.72 (2C), 22.43, 21.26. ESI-HRMS m/z calcd for C25H25N2O3 [M + H]+ 401.1865, found 401.1862.
4.1.32.3. 2-(2,2-Dimethyl-7-((2-methylbenzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK3)
White solid, yield 77%, mp 176-178 °C, Rf = 0.58 (petroleum ether/EtOAc = 10:1). HPLC purity 97.67% (A%/B% =
1
4:96, Rt = 3.519 min). H NMR (400 MHz, DMSO-d6) δ 9.33 (s, 1H), 8.30 (s, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.58–7.47
(m, 2H), 7.32–7.18 (m, 4H), 5.32 (s, 2H), 3.19 (t, J = 6.7 Hz, 2H), 2.39 (s, 3H), 1.92 (t, J = 6.7 Hz, 2H), 1.41 (s, 6H). 13
C
NMR (100 MHz, CDCl3) δ 165.16, 154.92, 152.21, 149.72, 136.87, 134.65, 130.49, 128.64, 128.34, 128.23, 127.47,
126.07, 124.37, 120.19, 115.83, 114.41, 114.02, 106.00, 74.56, 68.99, 32.58, 26.73 (2C), 22.43, 19.03. ESI-HRMS m/z
calcd for C25H25N2O3 [M + H]+ 401.1865, found 401.1866.
4.1.32.4. 2-(7-((3-Methoxybenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK4)
White solid, yield 81%, mp 158-160 °C, Rf = 0.63 (petroleum ether/EtOAc = 10:1). HPLC purity 96.60% (A%/B% =
4:96, Rt = 3.221 min). 1H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.36 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.51 (t, J =
8.1 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.16–7.07 (m, 3H), 6.93 (dd, J = 8.3, 2.5 Hz, 1H), 5.32 (s, 2H), 3.78 (s, 3H), 3.19 (t,
J = 6.7 Hz, 2H), 1.93 (t, J = 6.7 Hz, 2H), 1.41 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 165.20, 159.87, 154.76, 152.20,
149.71, 138.50, 129.72, 128.22, 127.46, 124.38, 120.17, 119.54, 115.89, 114.45, 114.00, 113.46, 112.84, 106.22, 74.56,
70.16, 55.26, 32.58, 26.73 (2C), 22.43. ESI-HRMS m/z calcd for C25H25N2O4 [M + H]+ 417.1814, found 417.1824.
4.1.32.5.
4-(((2,2-Dimethyl-5-(1,3,4-oxadiazol-2-yl)-3,4-dihydro-2H-benzo[h]chromen-7-yl)oxy)methyl)Benzonitrile
(WK5)
26

White solid, yield 53%, mp 195-197 °C, Rf = 0.53 (petroleum ether/EtOAc = 10:1). HPLC purity 99.49% (A%/B% =
1
4:96, Rt = 2.381 min). H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.36 (s, 1H), 7.95–7.87 (m, 2H), 7.78–7.70 (m,
3H), 7.50 (t, J = 8.1 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 5.48 (s, 2H), 3.19 (t, J = 6.7 Hz, 2H), 1.93 (t, J = 6.7 Hz, 2H), 1.41
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 165.14, 154.11, 152.28, 149.83, 142.27, 132.55 (2C), 128.27, 127.55 (2C),
127.25, 124.21, 120.51, 118.66, 115.48, 115.08, 114.23, 111.88, 106.15, 74.69, 69.19, 32.52, 26.72 (2C), 22.40.
ESI-HRMS m/z calcd for C25H22N3O3 [M + H]+ 412.1661, found 412.1661.
4.1.32.6. 2-(2,2-Dimethyl-7-((4-nitrobenzyl)oxy)-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK6)
Yellow solid, yield 58%, mp 197-199 °C, Rf = 0.50 (petroleum ether/EtOAc = 10:1). HPLC purity 97.28% (A%/B% =
10:90, Rt = 2.817 min). 1H NMR (600 MHz, CDCl3) δ 8.55 (s, 1H), 8.52 (s, 1H), 8.33–8.27 (m, 2H), 7.91 (d, J = 8.5 Hz,
1H), 7.70 (d, J = 8.7 Hz, 2H), 7.44 (t, J = 8.1 Hz, 1H), 6.87 (d, J = 7.7 Hz, 1H), 5.40 (s, 2H), 3.35 (t, J = 6.8 Hz, 2H),
1.98 (t, J = 6.7 Hz, 2H), 1.48 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 165.11, 154.06, 152.30, 149.85, 147.67, 144.25,
128.28, 127.62 (2C), 127.24, 124.20, 123.96 (2C), 120.54, 115.44, 115.16, 115.08, 114.27, 106.17, 74.70, 68.96, 32.52,
26.72 (2C), 22.40. ESI-HRMS m/z calcd for C24H22N3O5 [M + H]+ 431.1559, found 432.1585.
4.1.32.7. 2-(7-((4-Fluorobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK7)
White solid, yield 64%, mp 166-168 °C, Rf = 0.61 (petroleum ether/EtOAc = 10:1). HPLC purity 98.77% (A%/B% =
10:90, Rt = 4.564 min). 1H NMR (600 MHz, CDCl3) δ 8.53 (s, 1H), 8.50 (s, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.51 (dd, J =
8.4, 5.5 Hz, 2H), 7.46 (t, J = 8.1 Hz, 1H), 7.13 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 7.6 Hz, 1H), 5.26 (s, 2H), 3.34 (t, J = 6.8
Hz, 2H), 1.98 (t, J = 6.8 Hz, 2H), 1.48 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 165.18, 163.36, 161.73, 154.63, 152.23,
149.73, 132.60, 132.58, 129.25, 128.23, 127.39, 124.35, 120.26, 115.67, 115.53, 114.60, 114.06, 106.15, 74.59, 69.64,
32.56, 26.72 (2C), 22.41. ESI-HRMS m/z calcd for C24H22FN2O3 [M + H]+ 405.1614, found 405.1650.
4.1.32.8. 2-(7-((2,4-Dichlorobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK8)
White solid, yield 67%, mp 142-144 °C, Rf = 0.59 (petroleum ether/EtOAc = 10:1). HPLC purity 99.83% (A%/B% =
1
2:98, Rt = 10.541 min). H NMR (600 MHz, CDCl3) δ 8.55 (s, 1H), 8.53 (s, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.58 (d, J =
8.3 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.31 (dd, J = 8.3, 2.1 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H),
5.35 (s, 2H), 3.35 (t, J = 6.8 Hz, 2H), 1.98 (t, J = 6.8 Hz, 2H), 1.49 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 165.17,
154.17, 152.27, 149.80, 134.27, 133.33, 133.16, 129.58, 129.36, 128.27, 127.43, 127.36, 124.26, 120.40, 115.62, 114.95,
114.14, 106.28, 74.63, 66.92, 32.55, 26.73(2C), 22.41. ESI-HRMS m/z calcd for C24H21Cl2N2O3 [M + H]+ 455.0929,
found 455.0944.
4.1.32.9. 2-(7-((4-Bromobenzyl)oxy)-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-5-yl)-1,3,4-oxadiazole (WK9)
White solid, yield 58%, mp 162-164 °C, Rf = 0.66 (petroleum ether/EtOAc = 10:1). HPLC purity 100.00% (A%/B% =
10:90, Rt = 6.337 min).1H NMR (600 MHz, CDCl3) δ 8.53 (s, 1H), 8.50 (d, J = 0.8 Hz, 1H), 7.91–7.87 (m, 1H), 7.59–
7.55 (m, 2H), 7.45 (dd, J = 8.5, 7.7 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 6.91–6.88 (m, 1H), 5.24 (s, 2H), 3.35 (t, J = 6.8 Hz,
2H), 1.98 (t, J = 6.8 Hz, 2H), 1.48 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 165.15, 154.50, 152.24, 149.75, 135.87,
131.82 (2C), 129.07 (2C), 128.23, 127.36, 124.30, 121.96, 120.30, 115.70, 114.69, 114.10, 106.16, 74.60, 69.55, 32.56,
26.72 (2C), 22.42. ESI-HRMS m/z calcd for C24H22BrN2O3 [M + H]+ 465.0814, found 465.0829.
27

4.2. Antibacterial evaluation
4.2.1. Antimicrobial assays
Antimicrobial assays were performed applying the two-fold broth dilution method as previously described by our group
[33, 34].
4.2.2 Checkerboard titration assay
Possible synergism of the tested compounds with antimicrobials were evaluated by checkerboard titration assay as
previously described by our group [33-35].
4.2.3. Nile red efflux assay
Nile red efflux assay was carried out to assess the ability of the tested compounds to inhibit efflux as previously
described by our group [33-35].
4.2.4. Nitrocefin uptake assay
The influence of the tested compounds on outer membrane permeability of E. coli BW25513 was investigated by
nitrocefin uptake assay as previously described by our group [33, 34].
4.2.5. Measurement of the electrochemical gradient over the inner membrane
The effect of the tested compounds on pmf across the inner-membrane was evaluated by applying the DiOC2(3))
fluorescent method as previously described by our group [33, 34].
Declaration of competing interest
The authors declare that this study was carried out only with public funding. There is no funding or no agreement with
commercial for profit firms.
Acknowledgments
This research was supported financially by the National Natural Science Foundation of China (81973179, 81673284 and
81903449), the National Health and Medical Research Council of Australia (GN1147538), Key research and
development project of Shandong Province (2017CXGC1401), Major Project of Research and development of Shandong
Province (2019GSF108051), and the China-Australia Centre for Health Sciences Research (CACHSR no. 2019GJ05).
RA is the recipient of a PhD scholarship from the Government of Saudi Arabia.
28