Novel phthalide derivatives: Synthesis and anti-inflammatory activity in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2020.112722

Phthalide is a promising chemical scaffold and has been proved to show potent anti-inflammatory efficacy. In this study, three series, total of 31 novel phthalide derivatives were designed and synthesized, their anti-inflammatory activities were screened

Full text of DOI:10.1016/j.ejmech.2020.112722

Journal Pre-proof
Novel phthalide derivatives: synthesis and anti-inflammatory activity in vitro and in
vivo
Liu Zeng Chen, Jing Wu, Kang Li, Qian Qian Wu, Rui Chen, Xin Hua Liu, Ban Feng
Ruan
PII:
S0223-5234(20)30694-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112722
EJMECH 112722
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2020
Revised Date: 31 July 2020
Accepted Date: 2 August 2020
Please cite this article as: L.Z. Chen, J. Wu, K. Li, Q.Q. Wu, R. Chen, X.H. Liu, B.F. Ruan, Novel
phthalide derivatives: synthesis and anti-inflammatory activity in vitro and in vivo, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112722.
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Novel phthalide derivatives: synthesis and anti-inflammatory activity in vitro and
in vivo
Thirty-one novel phthalide derivatives were synthesized and evaluated to discover
novel agents with anti-inflammatory activity. Among them, compound 9o was the
most one with potent NO inhibitory activity and without obvious cytotoxicity. The
preliminary mechanism of anti-inflammatory activity was revealed. The further in
vivo anti-inflammatory studies showed that this title compound had a good therapeutic
effect against AIA rats.

Novel phthalide derivatives: synthesis and anti-inflammatory activity in vitro and
in vivo
Liu Zeng Chena, Jing Wua, Kang Lia, Qian Qian Wua, Rui Chena, Xin Hua Liua,*, Ban
Feng Ruan*,b
a School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
b Key Lab of Biofabrication of Anhui Higher Education, Hefei University, Hefei
230601, P. R. China
Abstract: Phthalide is a promising chemical scaffold and has been proved to show
potent anti-inflammatory efficacy. In this study, three series, total of 31 novel
phthalide derivatives were designed and synthesized, their anti-inflammatory
activities were screened in vitro and in vivo. The anti-inflammatory activity of all the
compounds were screened on LPS induced NO production to evaluating their
inhibitory effects. Structure-activity relationship has been concluded, and finally
3-((4-((4-fluorobenzyl)oxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1
(3H)-one (compound 9o) was found to be the active one with low toxicity, which
showed 95.23% inhibitory rate at 10 μM with IC50 value of 0.76 μM against
LPS-induced NO over expression. Preliminary mechanism studies indicated that
compound 9o activated Nrf2/HO-1 signalling pathway via accumulation ROS and
blocks the NF-κB/MAPK signaling pathway. The in vivo anti-inflammatory activity
shown that compound 9o had obvious therapeutic effect against the adjuvant-induced
rat arthritis model.
Keywords: Phthalide derivatives; design; synthesis; anti-inflammatory
________________
*Corresponding author, E-mail: xhliuhx@163.com (Xin Hua Liu).
ruanbf@hfuu.edu.cn (Ban Feng Ruan).

1. Introduction
Inflammation is a series of complex defense-related reactions caused by invading
microbes or physical injuries [1]. In the innate immune response, macrophages
mediate most of the inflammatory cellular and molecular pathophysiology by
producing various pro-inflammatory mediators, and play an important role in
promoting cellular repair and protection [2-4]. Unregulated production of these
pro-inflammatory mediators such as nitric oxide (NO), interleukin-6 (IL-6), tumor
necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-12 which can lead
to wide range of diseases, including rheumatoid arthritis, sepsis and inflammatory
bowel disease[5,6]. Therefore, there is an urgent need to find effective drugs or
therapies that can inhibit the production and release of pro-inflammatory cytokines.
Toll-like receptors are transmembrane proteins of pattern recognition receptors
of lipopolysaccharide (LPS) [7]. It controls the initial inflammatory response and
plays an important role in innate immune system[8]. The complex formed by LPS and
TLR4 leads to the activation of intracellular signaling pathway [9]. The pathway
mediates the activation of IκB kinases, p38, c-Jun NH2-terminal kinase (JNK) and
(ERK)-1/2 mitogen-activated protein kinases (MAPKs), leading to activation of
transcription factors nuclear factor-κB (NF-κB) and AP-1 enter the nucleus[10, 11].
The nuclear factor erythroid 2-related factor 2 (Nrf2) is a key and important
transcription factor, which contributes to anti-inflammatory processes and regulates
the expressions of a series of antioxidant enzymes [12, 13]. In macrophages, Nrf2 can
inhibit the inflammatory response by blocking the transcription of pro-inflammatory
cytokines [14]. The activation of Nrf2 activation and regulation of HO-1 are
considered to be the main anti-inflammatory and cytoprotective enzymes [15, 16].
Therefore, targeting Nrf2/HO-1 pathway may be a promising strategy for the
treatment of inflammatory diseases [17].
Butylphthalide (NBP) is a phthalide extracted from Angelicae Sinensis Radix,
which is one of the most important traditional Chinese medicines (TCM). Due to its
effects on protecting nerve cells, antioxidation and improving brain circulation, the
racemic 3-n-butylphthalide has been approved by the SFDA of China as a new drug

mainly for the treatment of ischemic stroke since 2002. It is well-known that NBP can
inhibit platelet aggregation and thrombosis, improve microcirculation, and reduce
brain infarct volume, thus benefiting patients with ischemic stroke[18]. Besides NBP,
some other natural phthalides have been shown to active against ischemic
stroke-induced brain injury.
Previous studies show that NBP and its derivatives can suppress the inflammatory
response caused by cerebral ischemia, which is related to the inhibition of NF-κB
signaling pathway (Figure 1). Zou et al have investigated the effects of a phthalide
derivative CD21 on ischemic brain injury. The results suggest that the anti-stroke of
CD21 appear to be mediated partially via the induction of MSR1-promoted DAMP
(PRX1) clearance, TLR4/nuclear factor-κB pathway inhibition, and the resolution of
inflammation [19]. Several groups have determined that (Z)-Ligustilide, another
natural compound with phthalide skeleton, can exert anti-inflammatory effects by
regulating the NF-κB, MAPK pathways, or by regulating the AP-1 and HO-1 [20-24].
Among them, Guo et al found that Levistilide A, a phthalide dimer isolated from
Ligusticum chuanxiong and Angelica sinensis could suppress NLRP3 gene expression
by blocking the Syk-p38/JNK pathway [25]. Lee et al also found that Cnidilide, an
alkylphthalide isolated the roots of Cnidium officinale, could suppress LPS-induced
NO, PGE2, IL-1β, IL-6 and TNF-α production by AP-1 and MF-κB inactivation in
RAW 264.7 macrophages [26]. Therefore, phthalide is a promising chemical scaffold
to discovery new anti-inflammatory drugs.
Figure 1
Herein three series of phthalide derivatives were designed and synthesized
through above finding. The first one is hybrids of phthalide and 1,2,4-oxadiazole core,
inspired by the anti-inflammatory activity of some compounds with 1,2,4-oxadiazole
moiety (Series A). In order to investigate the effects of hydrophobic interaction and
steric configuration, the second series of 3-hydroxy-benzhydrolphthalides (Series B)
were synthesized. Based on the activity of (Z)-Ligustilide, the third one, a series of

(Z)-3-benzylidene-phthalides by eliminating the chiral hydroxy group of series B
(Series C) were constructed at last (Figure 2). On the basis of balancing
anti-inflammatory activity and toxicity, the title phthalide derivative was used to
explore ameliorate inflammation in a CFA-induced arthritis (AIA) mouse model.
Figure 2
2. Results and discussion
2.1 Chemistry
The synthesis of the first series of derivatives with 1,2,4- oxadiazole moiety
(5a~5e) was outlined in Scheme 1. Compounds (3a~3e) were obtained by reacting
different nitriles with hydroxylamine hydrochloride in the presence of triethylamine in
methanol under reflux. Amide coupling of the respective oximes with
phthalide-3-acetic acid (4), following by subsequent cyclization and afforded the title
compounds 5a~5e.
Scheme 1
The synthesis of the second series of phthalide derivatives was exhibited in
Scheme 2. Actually, this series of compounds are by-products of Pinnick oxidation. At
first, we wanted to convert the aldehyde group of 8b, a derivative of resveratrol, to the
corresponding carboxyl group. When the reaction was carried out at 0 °C, the main
product was the carboxylic acid [27]. However, when the reaction temperature went
up to 50 °C, a new main product was obtained with good yield. After characterized by
NMR, MS and the X-ray single crystal diffraction, we found that a new phthalide
derivative was synthesized (9b) in the single-step reaction. The possible mechanism
of this reaction was proposed and outlined in Figure 3.
Scheme 2
Figure 3

To understand the influence of the chirality of the second series of compounds, the
hydroxy groups of compounds 9b, 9n and 9o were eliminated to afford the
corresponding 3-benzylidenephthalides (10b, 10n and 10o) Scheme 3 [28]. The
double bond of 10b, 10n and 10o were found to be (Z)-configuration. This was
indicated by δH of the vinyl protons of 10b, 10n and 10o (δ 6.31, 6.32 and 6.29 ppm,
respectively) because the chemical shifts of the vinyl protons of unstable (E)-isomers
of 3-benzylidenephtha-lides are shifted downfield from the corresponding stable
(Z)-isomers [29].
Scheme 3
2.2 Crystal structure of compound 9b
The structure of compound 9b was determined by X-ray crystallography (Figure
4). Crystallographical data: Crystallographic data: C18H18O6, Triclinic, space group
P-1; a = 9.635(2), b = 9.956(2), c = 10.257(2) (Å);
α = 65.318(2), β = 68.283(2), γ =
68.615(2) (°), V = 804.4(3) nm3, T = 298 (2) K, Z = 2, Dc = 1.364 g/cm3, F(000) =
348, Reflections collected/unique = 5506/2223, Data/restraints/parameters =
2808/0/221, Goodness of fit on F2 = 1.082, Fine, R1 = 0.053, wR(F2) = 0.1448.
Crystallographic data (excluding structure factors) for the structures have been
deposited into the Cambridge Crystallographic Data Center (CCDC no: 844477).
Figure 4
2.3 Anti-inflammatory activity of compounds
Firstly, we determined the intrinsic cytotoxicity of the title compounds against
RAW264.7 cells by MTT assay, and found that the target compounds displayed low
intrinsic cytotoxicity. As shown in Figure 5 (A and B). All compounds showed no
significant intrinsic cytotoxicity at 20 μM. Subsequently, the anti-inflammatory
activity of all compounds were determined according to the previous method [30],
and LPS stimulated RAW264.7 cells as an evaluation model. Excessive NO

production has been found to play an important role in many inflammatory
diseases[31]. Here, all compounds were tested for their ability to inhibit NO
overexpression. Griess reagent was used to determine the concentration of NO in cell
supernatant. As shown in Figure 5 (C and D) the screening results showed that most
of the compounds could reduce the LPS-induced NO secretion at a dose of 10 µM. It
is noteworthy that most compounds exhibited stronger inhibition of NO production
compared with the references Celecoxib and Indomethacin. Therefore, these
compounds are valuable for further evaluation.
Figure 5
2.4 Inhibitory effect of NO production and expression of pro-inflammatory mediators
Different concentrations (0.625, 1.25, 2.5, 5, 10 μM) of compounds were
selected for further study. Compounds dose-dependently inhibited NO production in
LPS-stimulated RAW264.7 cells (Table 1). On the whole, the toxicity of all
compounds, lower than 40 μM, which were acceptable. The structure-activity
relationships of the three series against NO inhibitory activity were clear. Compounds
5a~5e (Series A) had no obvious inhibitory activity against NO, IR is less than 25%
and with IC50 ﹥ 10 μM. After hydroxymethylation optimization (Series B,
compounds 9a~9v), which significantly improve the inhibitory activity against NO,
among them the IR values of compounds 9e, 9f, 9i, 9o, 9p, 9q, 9R, 9t and 9u
(62.24~95.23%) are better than that of Indomethacin (61.59%); the IC50 (NO) values
of above compounds ranging from 0.76 to 10 μM), which also surpassing of
Indomethacin. What's more, the IC50s of compounds 9e, 9i, 9m and 9o is lower than
2.0 μM, which show strong activity against NO, among this series, benzyl substitution
is the best, such as compound 9o.
Unfortunately, the design based on (Z)-ligustilide failed. Compounds 10b, 10n
and 10o (Series C) exhibited inhibitory activity against NO ranging of 36%~48%,
which was significantly worse than that of Series B.

It is interesting, title compound 9o could significantly decreased production of NO
with IC50 values of 0.76 µM. At the same time, the IC50 value of compound 9o
cytotoxicity exceeds 40 µM. In addition, the inhibitory effect of compound 9o on
pro-inflammatory mediators iNOS and COX-2 expression were analyzed by Western
blot. Treatment of RAW264.7 cells with compound 9o decreased the LPS-induced
expression levels of iNOS and COX-2 in a concentration-dependent manner (Figure
6). These results prove that compound 9o prevents LPS-induced expression of
inflammatory mediators. So, it is worthy of further study.
Table 1
Figure 6
2.5 Effect of compound 9o on the intracellular ROS generation
Previous studies have shown that the increase of ROS is related to the activation
of inflammation[32]. On this basis, we investigated the effect of compound 9o on
ROS production by analyzing the fluorescence intensity of DCF-DA. As expected,
LPS stimulated ROS accumulation compared with untreated cells. Interestingly, ROS
production was significantly increased in a concentration dependent manner in the
compound 9o (0.5, 1 and 2 μM). The results showed that compound 9o could not
inhibit ROS production in LPS-pretreated RAW264.7 cells (Figure 7).
Figure 7
2.6 Compound 9o activates Nrf2/HO-1 pathway via ROS
ROS are products of normal cell metabolism [33]. Accumulation ROS may
destroy the balance between antioxidant and pro-oxidant systems, and then activate
various defense mechanisms in cells [33, 34]. Cumulative evidence suggests that ROS
has an important effect on Nrf2 activation [35]. Nrf2 is considered to be a key
transcription factor for cell homeostasis, and it has been confirmed that Nrf2 has
antioxidant and anti-inflammatory effects by interacting with various signaling

pathways[36]. To investigate the anti-inflammatory mechanisms of compound 9o, we
further determined whether compound 9o activated Nrf2/HO-1 signaling pathway in
RAW264.7 cells. The effect of compound 9o on Nrf2 / HO-1 signal pathway was
studied by Western blot. As we expected, compound 9o treatment significantly
increased Nrf2 protein expression in the nucleus and HO-1 protein expression in
RAW264.7 cells (Figure 8A). At the same time, the localization of nuclear factor Nrf2
in RAW264.7 cells was examined by immunofluorescence. As shown in the Figure
8B, Nrf2 was obviously transferred to the nucleus after compound 9o treatment, and
the higher compound 9o concentration, the more obvious Nrf2 nuclear transfer. These
results indicated that compound 9o-mediated Nrf2/HO-1 pathway activation. To
verify whether ROS is involved in the activation of the Nrf2/HO-1 pathway by
compound 9o. NO production in RAW264.7 cells co-treated with compound 9o and
NAC (ROS scavenger) were examined. Moreover, NAC significantly reversed
compound 9o-mediated suppression of NO production in LPS-stimulated RAW264.7
cells (Figure 8C), suggesting that compound 9o may exert anti-inflammatory effects
by activates Nrf2/HO-1 pathway via accumulation ROS. From Figure 8C, we found
that although NAC reversed the inhibitory effect of compound 9o on NO production,
compound 9o still had a certain ability to inhibit NO secretion. So we think that this
compound can also exert anti-inflammatory activity through other pathways.
Figure 8
2.7 Compound 9o inhibits LPS-induced activation of NF-κB and MAPKs
Activating Nrf2 can suppress activation of NF-κB signaling pathway [37, 38]. In
addition, inhibition of NF-κB and MAPKs signaling activation is considered to be an
important target for the development of anti-inflammatory drugs [39]. Thus, we
examined whether compound 9o suppressed LPS-induced activation of NF-κB and
MAPKs. Here, we analyzed it by Western blotting. The results showed compound 9o
inhibited LPS-induced degradation and resynthesis of IκB protein. Meanwhile it could
effect of p65 phosphorylation and inhibit NF-κB p65 translocate into the nucleus

(Figure 9A). We examined the effect of compound 9o on LPS-mediated MAPK signal
activation with phosphorylated antibodies. As we expected, compound 9o
time-dependently blocked LPS-induced ERK phosphorylation, JNK phosphorylation
and p38 phosphorylation (Figure 9B). These findings demonstrated that this
compound blocked NF-κB and MAPKs signaling activation.
Figure 9
2.8 In vivo activity of compound 9o
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease
characterized by joint swelling, synovitis, and progressive joint injury of cartilage and
bone [40]. The complete Freund's adjuvant (CFA)-induced arthritis (AA) is widely
used model to study RA because of its similar pathogenesis [41]. Male SD rats,
weighing 140-160 g, were randomly divided into groups with 10 rats in each group.
CFA consists of in-complete Freund's adjuvant and inactivated BCG vaccine (10
mg/mL). Rat was induced by a single intradermal injection of 0.1 mL CFA and saline
(0.9% sodium chloride, as normal) into the right hind paw. After 2 weeks, we
observed that 70-80% of the rats showed obvious swelling of the left hind paw,
pathological nodules in the tail and ears, and the weight gain of the rats was slowed
down and part of the weight was lost. From the 14th day to the 28th day, all
experimental group AIA rats were given intragastric administration of different
concentrations compound 9o (20 mg/kg, 40 mg/kg) and Sinomenine (60 mg/kg) once
a day. All rats measured the left hind swelling level and weight change every three
days. The results were shown in Figure 10A and B), We observed that compound 9o
attenuate paw swelling and weight loss in rats. Histopathological analysis of the ankle
joints was performed at the end of the experiment to assess the level of inflammation
and tissue changes. The representative histological photos of the tissue sections were
shown in the Figure 10C. Compared with normal rats, AIA rats showed extensive
inflammatory cell infiltration, synovial hyperplasia and cartilage destruction (Figure
10C normal). AA rats treated with compound 9o (20 mg/kg) showed moderate

synovial hyperplasia, inflammatory cell infiltration and cartilage destruction (Figure
10 C, 9o L). Compound 9o (40 mg/kg) ameliorated cartilage destruction and
inflammatory cell infiltration (Figure 10C, 9o H). Sinomenine (60 mg/kg) showed
moderate synovial hyperplasia and keep intact joint cavity. In conclusion, the results
indicate that compound 9o has an anti-inflammatory effect on AA rats.
Figure 10
3. Conclusion
In summary, thirty-one novel phthalide derivatives were synthesized and
evaluated to discover novel agents with anti-inflammatory activity. The preliminary
screening results indicated that most of the title compounds exhibited potent
anti-inflammatory activity. Among them, compound 9o was the active one with potent
NO inhibitory activity and without obvious cytotoxicity. Taken together, compound
9o showed anti-inflammatory activity through the regulation of three signalling
pathways (Figure 11). First, compound 9o activated Nrf2/HO-1 signalling pathway
via accumulation ROS. Second, compound 9o inhibited NF-κB signalling activation
through blocking IκB-α degradation and subsequent p65 nuclear accumulation.
Finally, compound 9o inhibited MAPKs signalling activation through blocking the
phosphorylation of ERK1/2, JNK and p38. The further in vivo anti-inflammatory
studies showed that this compound had a good therapeutic effect on AIA rats. These
findings suggest that this title compound may has great potential in the development
of anti AA drug.
Figure 11
4. Experimental section
4.1. Chemistry general
All the reagents, solvent, and other chemicals were commercially available from

Aladdin, Adamas, Tan soole et al. without further purification. Reactions were
monitored by TLC using Silica Gel GF254 precoated plates, and compounds were
visualized under UV light (λ = 254 or 365 nm). Column chromatography was
performed using silica gel 60 (40–60 µm). 1H and 13C NMR spectra were recorded on
a Bruker Avance III 400 MHz spectrometer or an Agilent 600 MHz spectrometer. The
NMR solvents were purchased from Sigma-Aldrich (China). Chemical shifts (δ, ppm)
are reported relative to the solvent peak (CDCl3): 7.26 [1H] or 77.16 [13C]; DMSO d6:
2.50 [1H] or 39.52 [13C]). Proton resonances are annotated as: chemical shift (δ),
multiplicity (br, broad; s, singlet; d, doublet; m, multiplet), coupling constant (J, Hz),
and the number of protons. The ESI-MS spectra were recorded on a Mariner System
5304 Mass spectrometer. Melting points were determined on a XT4MP apparatus
(Taike Corp., Beijing, China), and are uncorrected. All characterized compounds had
a purity of >95% as measured by analytical reversed-phase high-performance liquid
chromatography (HPLC).
4.2. Crystallographic studies
Compound 9b was chosen for X-ray diffraction analysis performed on a
BRUCKER SMART APEX-CCD diffractometer equipped with a graphite
monochromatic MoKa radiation (λ = 0.71073 A) radiation at 293(2) K. A total
reflection was collected in the range of 0.97<θ<26.1o by using a ψ-ω scan mode with
independent ones, of which I>2σ(I) were observed and used in the succeeding
refinements. The data set were corrected by SADABS program and the structure were
solved by direct methods with SHELXS-97 and refined by full-matrix least-squares
method on F2 with SHELXL-97[30]. The non-hydrogen atoms were refined
anisotropically, and the hydrogen atoms were added according to theoretical models.
The structures were refined by full-matrix least-squares method on F2 with
SHELXT-97.
4.3. General procedure A: Synthesis of compounds 5a~5e
A solution of appropriate nitriles (1a~1e, 5.0 mmol, 1.0 equiv), hydroxylamine
hydrochloride (2, 6.0 mmol, 1.2 equiv) and Et3N (6.0 mmol, 1.2 equiv) in methanol

(10 mL) was stirred for 6 h at 60 °C. After completion, the solvent was evaporated
under reduced pressure and the crude product (3a~3e) was used for the next step
without purification.
The appropriate oximes (3a~3e, 5.0 mmol, 1.0 equiv), phthalide-3-acetic acid (4,
0.96 g, 5.0 mmol, 1.0 equiv) and CDI (0.81 g, 5.0 mmol, 1.0 equiv) were dissolved in
DMF (15 mL). The reaction mixture was stirred at 90 °C overnight. After completion,
the solvent was evaporated under reduced pressure. The residue was dissolved in
EtOAc, washed twice with water and once with brine. The combined organic layer
was dried over Na2SO4 and then evaporated under reduced pressure. The product was
separated using silica gel column chromatography.
((3-methyl-1,2,4-oxadiazol-5-yl)methyl)isobenzofuran-1(3H)-one (5a). Yellow waxy
solid, Yield: 70.5%. 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 8.0 Hz, 1H), 7.71 (t, J
= 8.0 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 5.91 (t, J = 8.0 Hz,
13
1H), 3.78 (s, 3H), 2.99-2.87 (m, 2H). C NMR (101 MHz, CDCl3) δ 169.8, 148.7,
134.3, 129.6, 125.9, 122.1, 52.2, 39.4. ESI MS (m/z) 230 (M+H+); HRMS (ESI) (m/z)
calcd for C12H10N2O3, 230.2230.
3-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)isobenzofuran-1(3H)-one (5b). Yellow
1
waxy solid, Yield: 65.5%. H NMR (600 MHz, CDCl3) δ 8.00 (d, J = 6.0 Hz, 2H),
7.88 (d, J = 6.0 Hz, 1H), 7.64 (t, J = 6.0 Hz, 1H), 7.52 (t, J = 6.0 Hz, 1H), 7.47-7.41
13
(m, 4H), 6.03 (t, J = 6.0 Hz, 1H), 3.56-3.48 (m, 2H). C NMR (101 MHz, CDCl3) δ
174.7, 169.5, 168.5, 147.7, 134.6, 131.5, 130.0, 129.0, 127.5, 126.3, 126.1, 126.0,
122.1, 32.4. ESI MS (m/z) 293 (M+H+); HRMS (ESI) (m/z) calcd for C17H13N2O3,
293.0921, found 293.0920.
3-((3-benzyl-1,2,4-oxadiazol-5-yl)methyl)isobenzofuran-1(3H)-one (5c). White waxy
solid, Yield: 60.5%. 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.0 Hz, 1H), 7.63 (t, J
= 8.0, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.367.26 (m, 6H), 5.94 (t, J = 8.0 Hz, 1H),
13
4.09-4.01 (m, 2H), 3.49-3.39 (m, 2H). C NMR (101 MHz, CDCl3) δ 174.8, 169.7,

169.4, 147.6, 135.2, 134.4, 129.9, 129.0, 128.8, 127.2, 126.0, 125.9, 122.1, 32.2. ESI
MS (m/z) 306 (M+H+); HRMS (ESI) (m/z) calcd for C18H14N2O3, 306.3210.
3-((3-(4-phenoxyphenyl)-1,2,4-oxadiazol-5-yl)methyl)isobenzofuran-1(3H)-one (5d).
1
White waxy solid, Yield: 75.8%. H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 6.0, 2.0
Hz, 2H), 7.93 (d, J = 6.0 Hz, 1H), 7.68 (t, J = 6.0 Hz, 1H), 7.58 (t, J = 6.0 Hz, 1H),
7.44 (d, 1H), 7.37 (t, J = 6.0 Hz, 2H), 7.17 (t, J = 6.0 Hz,1H), 7.05 (t, J = 6.0 Hz, 4H),
13
6.05 (t, J = 6.0 Hz, 1H), 3.62-3.52 (m, 2H). C NMR (101 MHz, CDCl3) δ 174.6,
169.4, 168.0, 160.4, 156.0, 147.8, 134.5, 130.0, 129.3, 126.1, 124.3, 122.1, 120.9,
119.8, 118.3, 32.3. ESI MS (m/z) 385 (M+H+); HRMS (ESI) (m/z) calcd for
C23H16N2O4, 385.1183, found, 385.1181.
3-((3-([1,1'-biphenyl]-4-yl)-1,2,4-oxadiazol-5-yl)methyl)isobenzofuran-1(3H)-one
(5e). White waxy solid, Yield: 73.6%. 1H NMR (600 MHz, CDCl3) δ 8.13 (d, J = 6.0
Hz, 2H), 7.94 (d, J = 6.0 Hz, 1H), 7.71 (t, J = 6.0 Hz, 3H), 7.63 (d, J = 6.0 Hz, 2H),
7.59 (t, J = 6.0, 1H), 7.47 (t, J = 6.0, 3H), 7.39 (t, J = 6.0 Hz, 1H), 6.08 (t, J = 6.0 Hz,
1H), 3.57 (dt, J = 16.0, 9.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 175.0, 174.1,
169.9, 169.1, 148.0, 147.2, 144.5, 144.0, 143.8, 143.5, 134.4, 134.0, 131.3, 130.1,
129.6, 129.0, 128.33 (s), 128.1, 128.0, 127.7, 127.6, 127.2, 127.1, 126.6, 126.1, 125.9,
33.4. ESI MS (m/z) 368 (M+H+); HRMS (ESI) (m/z) calcd for C23H16N2O3, 368.3920.
4.4. General procedure B: Synthesis of compounds 9a-9v
Compounds 8a-8v were obtained according to the procedure as described. The
appropriate aldehyde compound (8a-8v, 2.0 mmol, 1.0 equiv) was dissolved in
DMSO and saturated sodium dihydrogenphosphate solution (8.0 mmol, 4.0 equiv) at
0 °C. Saturated sodium chlorite solution (30.0 mmol, 15.0 equiv) was then added
dropwise with vigorous stirring, and the reaction mixture was stirred at 50 °C for 6 h.
When TLC indicated complete consumption of the starting material, the resulting
mixture was extracted with EtOAc (×3), and the organic layers were combined dried
over Na2SO4. After the solvent was evaporated, the product was separated using silica

gel column chromatography.
3-(hydroxy(4-hydroxyphenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one
(9a).
White waxy solid, Yield: 62.6%. 1H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 7.13
(d, J = 8.0 Hz, 2H), 6.69 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 6.23 (s, 1H), 5.79 (d, J =
4.0 Hz, 1H), 5.51 (d, J = 4.0 Hz, 1H), 4.92(t, J = 4.0 Hz, 1H), 3.82 (s, 3H), 3.78 (s,
3H).13C NMR (101 MHz, DMSO-d6) δ 167.6, 165.9, 159.2, 157.1, 152.3, 130.6,
128.6, 115.0, 107.1, 100.3, 99.2, 82.7, 73.0, 56.3, 56.2, 42.6.
3-(hydroxy(4-methoxyphenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one
(9b).
White solid, Yield: 73.3%, MP:202.3-203.5oC. 1H NMR (400 MHz, DMSO-d6) δ 7.25
(d, J = 8.0 Hz, 2H), 6.87 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 6.24 (s, 1H), 5.89 (d, J =
4.0 Hz, 1H), 5.54 (d, J = 4.0 Hz, 1H), 4.97 (t, J = 4.0 Hz, 1H), 3.82 (s, 3H), 3.77 (s,
3H), 3.73 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 166.1, 159.5, 151.2, 130.4, 127.8,
113.8, 99.4, 99.3, 82.5, 73.4, 71.48 (s), 55.9, 55.8, 55.3, 42.6. ESI MS (m/z) 353
(M+Na); HRMS (ESI) (m/z) calcd for C18H18O6Na, 353.0996, found, 353.1004.
3-((4-ethoxyphenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one
(9c).
White solid, Yield: 64.4%, MP:154.1-154.8 oC.1H NMR (600 MHz, DMSO-d6) δ 7.21
(d, J = 12.0 Hz, 2H), 6.83 (d, J = 12.0 Hz, 2H), 6.51 (s, 1H), 6.21 (s, 1H), 5.85 (d, J =
6.0 Hz, 1H), 5.50 (s, 1H), 4.94 (t, J = 6.0 Hz, 1H), 3.98-3.95 (q, J = 6.0, 2H), 3.79 (s,
3H), 3.75 (s, 3H), 1.28 (t, J = 6.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 168.1,
166.1, 159.4, 159.0 (s), 151.1, 130.0, 127.7, 114. 5, 107.6, 99.3, 82.4, 73.6, 63.5, 55.9,
55.8, 14.8. ESI MS (m/z) 345 (M+H+); HRMS (ESI) (m/z) calcd for C19H21O6,
345.1333, found, 345.1361.
3-(hydroxy(4-propoxyphenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one
(9d).
o
1
White solid, Yield: 62.6%, MP:148.6-149.5 C. H NMR (600 MHz, DMSO-d6) δ
7.22 (d, J = 6.0 Hz, 2H), 6.84 (d, J = 12.0 Hz, 2H), 6.52 (s, 1H), 6.23 (s, 1H), 5.83 (d,
J = 6.0 Hz, 1H), 5.51 (d, J = 6.0 Hz, 1H), 4.93 (t, J = 6.0 Hz, 1H), 3.88 (t, J = 6.0 Hz,
2H), 3.80 (s, 3H), 3.76 (s, 3H), 1.72-1.66 (m, 2H), 0.95 (t, J = 6.0 Hz, 3H). 13C NMR

(101 MHz, CDCl3) δ 168.2, 166.1, 159.4, 159.2, 151.1, 130.0, 127.7, 114.5, 107.6,
99.3, 82.5, 73.6, 69.61 (s), 55.9, 55.8, 22.6, 10.5. ESI MS (m/z) 381 (M+H+); HRMS
(ESI) (m/z) calcd for C21H23O6, 381.1309, found, 381.1337.
3-((4-butoxyphenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one
(9e).
o
1
White solid, Yield: 58.9%, MP:205.8-206.4 C. H NMR (600 MHz, DMSO-d6) δ
7.21 (d, J = 6.0 Hz, 2H), 6.84 (d, J = 6.0 Hz, 2H), 6.51 (s, 1H), 6.21 (s, 1H), 5.85 (d, J
= 6.0 Hz, 1H), 5.50 (d, J = 6.0 Hz, 1H), 4.94 (t, J = 6.0 Hz, 1H), 3.91 (t, J = 6.0 Hz,
2H), 3.79 (s, 3H), 3.75 (s, 3H), 1.67-1.62 (m, 2H), 1.42-1.36 (m, 2H), 0.90 (t, J = 6.0
Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 168.3, 166.1, 159.4, 159.2, 151.1, 130.0,
127.7, 114.4, 107.6, 99.4, 99.3, 82.5, 73.6, 67.8, 55.9, 55.8, 31.3, 19.2, 13.8. ESI MS
(m/z) 395 (M+Na); HRMS (ESI) (m/z) calcd for C21H23O6, 395.1465., found,
395.1494.
3-((4-(hexyloxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one (9f).
o
1
White solid, Yield: 67.1%, MP:157.6-158.5 C. H NMR (600 MHz, DMSO-d6) δ
7.21 (d, J = 12.0 Hz, 2H), 6.84 (d, J = 6.0 Hz, 2H), 6.52 (s, 1H), 6.22 (s, 1H), 5.83 (d,
J = 6.0 Hz, 1H), 5.50 (s, 1H), 4.93 (t, J = 6.0 Hz, 1H), 3.91 (t, J = 6.0, 2H), 3.80 (s,
3H), 3.76 (s, 3H), 1.69-1.64 (m, 2H), 1.39-1.37 (m, 2H), 1.29-1.28 (m, 4H), 0.86 (t, J
= 6.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 168.3, 166.1, 159.4, 159.2, 151.2,
130.0, 127.7, 114.4, 107.6, 99.4, 99.3, 82.5, 73.6, 68.1, 55.9, 55.8, 31.6, 29.2, 25.7,
22.6, 14.0. ESI MS (m/z) 401 (M+H+); HRMS (ESI) (m/z) calcd for C23H29O6,
401.1959., found, 401.1990.
3-(hydroxy(4-isobutoxyphenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one (9g).
o
1
White solid, Yield: 53.8%, MP:155.5-156.7 C. H NMR (400 MHz, DMSO-d6) δ
7.24 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 6.20 (s, 1H), 5.86 (s,
1H), 5.53 (d, J = 4.0 Hz, 1H), 4.95 (s, 1H), 4.39-4.34 (m, 1H), 3.82 (s, 3H), 3.76 (s,
3H), 1.67-.62 (m, 2H), 1.21 (t, J = 4.0 Hz, 3H), 0.91 (t, J = 8.0 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 167.1, 165.4, 158.7, 157.1, 151.8, 131.7, 128.2, 114.9, 106.6,
99.6, 98.8, 82.1, 74.0, 72.3, 55.7, 28.5, 19.0, 9.5. ESI MS (m/z) 373 (M+
H+); HRMS

(ESI) (m/z) calcd for C21H25O6, 373.1646, found, 373.1675.
3-(hydroxy(4-(2-methoxyethoxy)phenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-o
o
1
ne (9h). White solid, Yield: 66.0%, MP:102.6-103.4 C. H NMR (600 MHz,
DMSO-d6) δ 7.22 (d, J = 6.0 Hz, 2H), 6.85 (d, J = 12.0 Hz, 2H), 6.51 (s, 1H), 6.21 (s,
1H), 5.86 (d, J = 6.0 Hz, 1H), 5.51 (d, J = 6.0 Hz, 1H), 4.94 (t, J = 6.0, 1H), 4.03 (t, J
= 6.0, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.61 (t, J = 6.0, 2H), 3.27 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 168.3, 166.1, 159.4, 158.8, 151.1, 132.7, 130.5, 127.7, 114.7,
114.5, 107.6, 99.3, 99.2, 82.5, 73.5, 71.0, 67.3, 59.2, 55.9, 55.8, 40.8, 29.7. ESI MS
(m/z) 375 (M+H+); HRMS (ESI) (m/z) calcd for C20H23O7, 375.1438, found,
375.1471.
3-(hydroxy(4-(3-methoxypropoxy)phenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-
o
1
one (9i). White solid, Yield: 66.2%, MP:100.8-103.9 C. H NMR (600 MHz,
DMSO-d6) δ 7.22 (d, J = 6.0 Hz, 2H), 6.85 (d, J = 6.0 Hz, 2H), 6.52 (s, 1H), 6.22 (s,
1H), 5.83 (d, J = 6.0 Hz, 1H), 5.51 (d, J = 6.0 Hz, 1H), 4.94 (t, J = 6.0 Hz, 1H), 3.97
(t, J = 6.0 Hz, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.44 (t, J = 6.0 Hz, 2H), 3.22 (s, 3H),
1.92-1.89 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 168.3, 166.1, 159.4, 158.9, 151.2,
130.4, 127.8, 114.4, 107.6, 99.4, 99.2, 82.6, 73.5, 69.2, 64.9, 58.7, 55.9, 55.8, 29.6.
ESI MS (m/z) 411 (M+Na); HRMS (ESI) (m/z) calcd for C21H25O6, 411.1414, found,
411.1426.
3-(hydroxy(4-(4-methoxybutoxy)phenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-o
o
1
ne (9j). White solid, Yield: 58.8%, MP:179.5-182.4 C. H NMR (400 MHz,
DMSO-d6) δ 7.23 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 6.24 (s,
1H), 5.89 (d, J = 4.0 Hz, 1H), 5.54 (d, J = 4.0, 1H), 4.97 (t, J = 4.0 Hz, 1H), 3.95 (t, J
= 6.0, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 3.35 (t, J = 4.0, 2H), 3.23 (s, 3H), 1.74-1.69 (m,
2H), 1.66-1.59 (m, 2H). 13C NMR (101 MHz, DMSO) δ 167.1, 165.5, 158.7, 157.9,
151.7, 131.7 , 128.1, 113.6, 106.6, 99.7, 98.7, 82.2, 72.4, 71.5, 67.1, 57.8, 55.8, 55.7,
25.6, 25.5. ESI MS (m/z) 425 (M+H+); HRMS (ESI) (m/z) calcd for C21H23O6,
425.1571, found, 425.1567.

3-((4-(2-ethoxyethoxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-o
o
1
ne (9k). White solid, Yield: 48.5%, MP:192.0-192.7 C. H NMR (400 MHz,
DMSO-d6) δ 7.24 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 6.23 (s,
1H), 5.87 (d, J = 4.0 Hz, 1H), 5.53 (d, J = 4.0 Hz, 1H), 4.96 (t, J = 4.0 Hz, 1H), 4.05
(t, J = 4.0, 2H), 3.82 (s, 3H), 3.77 (s, 3H), 3.67 (t, J = 4.0, 2H), 3.51-3.46 (q, J = 4.0
Hz, 2H), 1.12 (t, J = 8.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.1, 165.5,
158.7, 157.8, 151.7, 131.9, 128.1, 113.6, 106.6, 99.7, 98.7, 82.2, 72.4, 68.3, 67.0, 65.7,
55.8, 55.7, 15.1. ESI MS (m/z) 389 (M
389.1595, found, 389.1625.
+
H+); HRMS (ESI) (m/z) calcd for C21H25O7,
3-(hydroxy(4-(2-phenoxyethoxy)phenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-o
o
1
ne (9l). White solid, Yield: 57.4%, MP:96.9-97.3 C. H NMR (400 MHz, CDCl3) δ
7.34-7.29 (m, 4H), 6.96 (d, J = 8.0 Hz, 5H), 6.40 (s, 1H), 6.03 (s, 1H), 5.48 (d, J = 4.0
Hz, 1H), 5.13 (d, J = 4.0 Hz, 1H), 4.34 (s, 4H), 3.91 (s, 3H), 3.74 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 166.1, 159.3, 158.6, 151.2, 131.0, 129.5, 127.9, 121.2, 114.7,
114.6, 107.50 (s), 99.4, 99.2, 82.6, 73.4, 66.7, 66.4, 55.9, 55.8. ESI MS (m/z) 459
(M+Na); HRMS (ESI) (m/z) calcd for C25H24O7Na, 459.1414, found, 459.1419.
3-((4-(but-3-en-1-yloxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-
o
1
one (9m). White solid, Yield: 54.7%, MP:95.2-96.9 C. H NMR (400 MHz,
DMSO-d6) δ 7.23 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 6.23 (s,
1H), 5.89-5.82 (m, 2H), 5.53 (d, J = 4.0 Hz, 1H), 5.06-4.94 (m, 3H), 3.94 (t, J = 4.0
Hz, 2H), 3.82 (s, 3H), 3.77 (s, 3H), 2.19-2.14 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
168.3, 166.1, 159.4, 159.0, 151.2, 137.8, 130.3, 127.7, 115.2, 114.4, 107.6, 99.4, 99.2,
82.6, 73.5, 67.2, 55.9, 55.8, 30.1, 28.4. ESI MS (m/z) 371 (M+
H+); HRMS (ESI) (m/z)
calcd for C21H23O6, 371.1495, found, 371.1501.
3-((4-(benzyloxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H)-one
1
(9n). White solid, Yield: 56.8%, H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.0 Hz,
2H), 7.47-7.34 (m, 5H), 7.02 (d, J = 8.0 Hz, 2H), 6.73 (s, 1H), 6.45 (d, J = 4.0 Hz,
1H), 6.32 (s, 1H), 5.12 (s, 2H), 3.99 (s, 3H), 3.97 (s, 3H). 13C NMR (101 MHz,

DMSO-d6) δ 167.6, 165.9, 159.2, 158.1, 152.2, 137.6, 132.5, 128.9, 128.6, 128.3,
128.1, 114.5, 107.1, 100.1, 99.2, 82.6, 72.8, 69.5, 56.3, 56.2, 49.1.
3-((4-((4-fluorobenzyl)oxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3
o
1
H)-one (9o). White solid, Yield: 69.9%, MP:190.5-191.0 C. H NMR (600 MHz,
DMSO-d6) δ 7.47 (t, J = 6.0, 2H), 7.24 (d, J = 6.0 Hz, 2H), 7.19 (t, J = 6.0 Hz, 2H),
6.93 (d, J = 12.0 Hz, 2H), 6.52 (s, 1H), 6.21 (s, 1H), 5.85 (s, 1H), 5.73(s, 1H), 5.51 (d,
13
J = 6.0, 1H), 5.05 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H). C NMR (101 MHz, CDCl3) δ
166.1, 159.5, 158.6, 151.1, 13.07, 129.3, 127.8, 115.6, 115.4, 114.9, 107.6, 99.3, 82.3,
73.7, 69.4, 56.0, 55.7. ESI MS (m/z) 425 (M+H+); HRMS (ESI) (m/z) calcd for
C24H22FO6, 425.1395, found, 425.1426.
3-((4-((4-chlorobenzyl)oxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3
1
H)-one (9p). White solid, Yield: 63.5%, H NMR (400 MHz, CDCl3) δ 7.38 (s, 4H),
7.33 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 6.41 (s, 1H), 6.00 (s, 1H), 5.48 (d, J
13
= 4.0 Hz, 1H), 5.14 (s, 1H), 5.07 (s, 2H), 3.93 (s, 3H), 3.73 (s, 3H). C NMR (101
MHz, DMSO-d6) δ 167.5, 166.0, 159.2, 157.9, 152.2, 136.7, 132.8, 129.9, 128.9,
128.7, 114.6, 100.2, 99.3, 82.6, 72.9, 68.7, 56.3, 56.2.
3-((4-((4-bromobenzyl)oxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3
1
H)-one (9q). White solid, Yield: 65.5%, H NMR (400 MHz, CDCl3) δ 7.54 (d, J =
8.0 Hz, 2H), 7.34-7.33 (m, 4H), 6.98 (d, J = 8.0 Hz, 2H), 6.41 (s, 1H), 6.01 (s, 1H),
5.49 (d, J = 4.0 Hz, 1H), 5.14 (d, J = 4.0 Hz, 1H), 5.06 (s, 2H), 3.94(s, 3H), 3.73 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 167.5, 166.0, 159.2, 157.9, 152.2, 137.1,
132.7, 131.8, 130.2, 128.7, 121.3, 115.7, 115.0, 114.6, 107.1, 100.2, 99.3, 82.6, 72.9,
68.7, 56.3.
3-(hydroxy(4-((4-methylbenzyl)oxy)phenyl)methyl)-5,7-dimethoxyisobenzofuran-1(3H
1
)-one (9r). White solid, Yield: 58.5%, H NMR (400 MHz, CDCl3) δ 7.34-7.31 (m,
4H), 7.21 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 6.40(s, 1H), 5.96 (s, 1H), 5.47
(d, J = 4.0 Hz, 1H), 5.15 (d, J = 4.0 Hz, 1H), 5.05 (s, 2H), 3.92 (s, 3H), 3.70 (s, 3H),

2.38 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 164.5, 163.4, 159.2, 157.7, 152.2,
143.5 137.5, 134.5, 129.4, 128.2, 119.2, 114.5, 97.2, 69.5, 56.2, 21.2.
4-((4-((4,6-dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)(hydroxy)methyl)phenoxy
1
)methyl)benzonitrile (9s). White solid, Yield: 56.6%, H NMR (400 MHz, CDCl3) δ
7.70 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 6.96 (d, J
= 8.0 Hz, 2H), 6.41 (d, J = 4.0 Hz, 1H), 6.10 (s, 1H), 5.46 (d, J = 8.0 Hz, 1H), 5.15 (s,
2H), 5.09 (d, J = 4.0, 1H), 3.92 (s, 3H), 3.76 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 167.5, 166.0, 159.2, 157.7, 152.2, 143.5, 132.9, 128.7, 128.5, 119.2, 114.6, 110.9,
107.1, 100.2, 99.3, 82.6, 72.9, 68.6, 56.3, 56.2, 40.97-40.40 (m), 40.11 (d, J = 21.2
Hz), 39.72 (d, J = 15.5 Hz), 39.38 (s).
3-(hydroxy(4-((4-(trifluoromethoxy)benzyl)oxy)phenyl)methyl)-5,7-dimethoxyisobenzo
furan-1(3H)-one (9t). White solid, Yield: 54.3%, 1H NMR (400 MHz, CDCl3) δ 7.49
(d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0
Hz, 2H), 6.41 (s, 1H), 6.04 (s, 1H), 5.49 (d, J = 4.0 Hz, 1H), 5.14 (d, J = 4.0 Hz, 1H),
5.09 (s, 2H), 3.93 (s, 3H), 3.74 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.5,
166.0, 159.2, 157.9, 152.2, 148.3, 137.1, 132.8, 130.0, 128.7, 121.5, 114.5, 107.1,
100.2, 99.3, 82.6, 72.9, 68.6, 56.3, 56.2, 49.1.
3-((4-((4-(difluoromethoxy)benzyl)oxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenz
1
ofuran-1(3H)-one (9u). White solid, Yield: 59.3%, H NMR (400 MHz, CDCl3) δ
7.35-7.31 (m, 5H), 7.22 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.40 (s, 1H),
5.96 (s, 1H), 5.49 (d, J = 4.0 Hz, 1H), 5.16 (d, J = 8.0, 1H), 5.06 (s, 2H), 3.93 (s, 3H),
3.71 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.6, 1656.0 (s), 159.2, 158.0, 152.2,
150.9, 134.6, 132.7, 129.9, 128.7, 119.2, 116.8, 114.5, 107.1, 100.2, 99.3, 82.6, 72.9,
68.8, 60.2, 56.5, 56.3, 5.2.
3-((4-(cyclohexylmethoxy)phenyl)(hydroxy)methyl)-5,7-dimethoxyisobenzofuran-1(3H
)-one (9v). White solid, Yield: 52.6%, 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 8.0
Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 6.38 (s, 1H), 6.00 (d, J = 11.0 Hz, 1H), 5.46-5.42

(m, 1H), 5.12-5.09 (m, 1H), 3.89 (s, 3H), 3.86 (s, 1H), 3.76-3.71 (m, 4H), 1.88-1.70
(m, 5H), 1.32-1.16 (m, 4H), 1.09-1.01 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ
162.7, 158.0, 157.8, 140.0, 134.9, 131.3, 130.7, 128.8, 114.7, 113.5, 107.7, 98.2, 73.6,
73.0, 56.3, 55.9, 49.1, 44.9, 37.5, 29.7, 26.5, 25.9, 25.7.
4.5. General procedure C: Synthesis of compounds 10b, 10n and 10o
The appropriate compound (9b, 9n and 9o, 1.0 mmol, 1.0 equiv), p-TsOH (2.0
mmol, 2.0 equiv) was dissolved in benzene (5 mL). The reaction mixture was then
stirred at 60 °C for 6 h. After completion, the solvent was evaporated under reduced
pressure. The residue was dissolved in EtOAc, washed twice with water and once
with brine. The combined organic layer was dried over Na2SO4 and then evaporated
under reduced pressure. The product was separated using silica gel column
chromatography.
(Z)-5,7-dimethoxy-3-(4-methoxybenzylidene)isobenzofuran-1(3H)-one (10b). White
solid, Yield: 60.5%, 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.0 Hz, 2H), 6.94 (d,
J = 8.0 Hz, 2H), 6.72 (s, 1H), 6.43 (s, 1H), 6.31 (s, 1H), 3.98 (s, 3H), 3.96 (s, 3H),
3.87 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.5, 164.1, 159.6, 145.1, 142.9,
131.7, 126.4, 114.9, 106.9, 103.3, 100.7, 96.4, 56.8, 56.5, 55.7.
(Z)-3-(4-(benzyloxy)benzylidene)-5,7-dimethoxyisobenzofuran-1(3H)-one
(10n).
1
White solid, Yield: 66.5%, H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.0 Hz, 2H),
7.47 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 8.0, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.02 (d, J =
8.0 Hz, 2H), 6.73 (s, 1H), 6.44 (s, 1H), 6.32 (s, 1H), 5.12 (s, 2H), 3.99 (s, 3H), 3.97 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 167.5, 164.1, 159.5, 158.8, 145.1, 143.0,
137.3, 131.7, 128.9, 128.4, 128.4, 126.6, 115.8, 106.9, 103.3, 100.7, 96.4, 69.7, 56.8,
56.6.
(Z)-3-(4-((4-fluorobenzyl)oxy)benzylidene)-5,7-dimethoxyisobenzofuran-1(3H)-one
1
(10o). White solid, Yield: 62.8%, H NMR (600 MHz, CDCl3) δ 7.79 (d, J = 6.0 Hz,
2H), 7.38 – 7.34 (m, 4H), 6.97 (d, J = 6.0 Hz, 2H), 6.70 (s, 1H), 6.42 (s, 1H), 6.29 (s,

1H), 5.06 (s, 2H), 3.96 (s, 3H), 3.94 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.5,
164.1, 159.5, 158.6, 145.0, 143.0, 136.3, 133.0, 131.7, 130.1, 128.9, 126.8, 115.8,
106.8, 103.3, 100.7, 96.4, 68.9, 56.8, 56.6.
4.6 Cell culture
Mouse peritoneal macrophages were purchased from BeNa Culture Collection
Company (BeiJing, China). RAW264.7 cells were cultured in DMEM (Hyclone, USA)
supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries,
Israel) in a humidified 5% CO2 containing atmosphere at 37°C. The cells grew until
they converged to 70-80% before treatment.
4.7 Determination of NO
RAW264.7 cells (7×104 cells/well) were seeded into 48-well plate and used for
experiments 24 h later. RAW264.7 cells were pretreated with compounds (10 μM) for
1 h, co-treated with LPS (0.5 μg/mL) for 24 h. After 24 h collect cell supernatant for
experiment. NO production was measured using Griess Reagent assay (Beyotime).
Griess reagent were mixed at a ratio of 1:1, and reacted at room temperature for
15 min. Measurement of absorbance at 450 nm with enzyme reader.
4.8 Cell viability assay (MTT)
RAW264.7 cells (1×104 cells/well) were seeded into 96-well plate and used for
experiments 24 h later. Compounds (20 μM) treated cell for 24 h. Forty microliters of
MTT solution (5 mg/mL, Sigma-Aldrich) was then added and incubated for an
additional 4 h. After 4 h, cell culture supernatants were removed and DMSO (150 μL)
was added to added into per well for dissolving the resulting crystals. Shaking about
10-15 min and the absorbance at 492 nm was measured by a microplate reader
(MQX200, Bio-Tek, USA)
4.9 Western blotting
RAW264.7 cells were seeded into 6-well plate with 2×106 cells per well and
maintained about 24 h. RAW264.7 cell were pretreated with compound 9o (0.5, 1, 2
μM) for 1 h, co-treated with LPS (0.5 μg/mL) for 0.5 h or 24h.The cells were lysed in
300 μL RIPA cell lysis buffer (Contains PMSF and phosphatase inhibitors, Beyotime
china) and incubated on ice for 30 min. Collecting supernatant by centrifugation. The

same amount of protein was separated by SDS-PAGE and transferred to PVDF
membrane (GE Healthcare, Amersham, UK). The blotted membrane incubated with
the primary antibodies and allowed to react for an additional 16 h at 4 °C. All
antibodies obtain from cell signaling Technology, USA. The membranes incubated
with a 1:5000 dilutions of HRP-conjugated secondary antibody (Beyotime Biotech,
Nantong, China) for 1h at room temperature. Signals were visualized using ECL
system (Thermo Fisher Scientific).
4.10 Determination of ROS level
RAW264.7 cells were seeded into 6-well plate with 1.5×106 cells per well and
maintained about 24 h. RAW264.7 cells were pretreated with compound 9o (0.5, 1, 2
μM) for 1 h, co-treated with LPS (0.5 μg/mL) for 6 h. The cells were treated with
DCFH-DA (BestBio, China) for 30 min at 37 °C cell incubator, and then wash the
cells 3 times with cold serum-free fresh medium or PBS. Finally, according to the
manufacturer's instructions, use flow cytometry detecting the intracellular reactive
oxygen species and analyze by flowjo software.
4.11 Immunofluorescence
RAW264.7 cells were seeded into on glass coverslip in six well plates, fixed with
4% polyformaldehyde (w/v) for 20 minutes at room temperature, and sealed in TBS
containing 0.1% Triton X-100 with 5% BSA for 1 hour. The cells were incubated with
a primary antibody. After the washing step, they are dyed with DAPI the images were
acquired by inverted fluorescence microscope (Olympus, Tokyo, Japan).
4.12 In vivo experiment
The 50 male SD rats were obtained from Animal Department of Anhui Medical
University (China). Rats were kept in room temperature of 22 ± 2°C and humidity
of around 60% and under a 12:12 h light-dark cycle. All animal protocols were
approved by the Ethics Committee in Animal Experimentation at Anhui Medical
University (Hefei, China) following the guidelines for Care and Use of Laboratory
Animals. After acclimation for one week, rats were randomly divided into groups with
10 rats in each group, including the Normal group, AA group, Sinomenine (60 mg/kg),
compound 9o L (20 mg/kg) and compound 9o H (40 mg/kg). Complete Freund's

adjuvant consists of incomplete Freund's adjuvant (sigma, USA) and inactivated BCG
vaccine (China) (10 mg/mL). Rat was induced by a single intradermal injection of 0.1
mL CFA and saline. All rats were given intragastric administration once a day. All rats
measured the left hind swelling level and weight every three days. Mice were
anesthetized and sacrificed after 29 days. The ankle joint was harvested fixed in 4%
neutral buffered polyformaldehyde solution, and then decalcified in 10% EDTA. The
ankles were cut into 4 mm thick and stained with hematoxylin and eosin (HE) for
histopathological examination. The histological image was obtained using
3DHISTECH's Slide Converter (3DHISTECH, Hungary).
4.13 Statistical analysis
Data were expressed as the mean ±SEM. Statistical significance was assessed by
one-way ANOVA and Turkish test, and differences between the two groups were
examined by SPSS (version 14.0; SPNN Inc., Chicago, IL, USA). p<0.05 was
considered to be statistically significant. All experiments dates were repeated at least
three times.
Supporting Information
The following files are available free. 1H NMR and 13C NMR spectra and HRMS
of compounds.
Acknowledgment
This work was supported by the National Natural Science Funding of China
(21977001).
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Figure Captions
Table 1. Inhibition rates and IC50 values of compounds
Figure 1. Structures of some phthalide derivatives with anti-inflammatory activity
Figure 2. Structures of Series A~C
Figure 3. Proposed mechanism for the synthesis of compound 9b
Figure 4. DIAMOND drawing of compound 9b
Figure 5. Preliminary screening cytotoxic evaluation and inhibition of NO production
Figure 6. Inhibition of the Cytokine Production production
Figure 7. Treatment with LPS induced the accumulation of ROS in RAW264.7 cells.
Figure 8. Effects of compounds 9o on Nrf2/HO-1 pathway in RAW264.7 cells.
Figure 9. Compound 9o inhibits LPS-induced activation of NF-κB and MAPKs
Figure 10. Therapeutic effect of compound 9o on AA rats
Figure 11. Proposed mechanisms for anti-inflammatory action of compound 9o
Scheme 1. Synthesis of compounds 5a~5e
Scheme 2. Synthesis of compounds 9a~9v
Scheme 3. Synthesis of compounds 10b,10n,10o