Synthesis, cytotoxic activity, and 2D- and 3D-QSAR studies of 19-carboxyl-modified novel isosteviol derivatives as potential anticancer agents

Received Date : 18-Jul-2016

Revised Date : 12-Oct-2016

Accepted Date : 25-Oct-2016

Article type

: Research Article

Synthesis, cytotoxic activity, 2D- and 3D-QSAR studies of

19-carboxyl modified novel isosteviol derivatives as potential

anticancer agents

Cong-Jun Liu ^a, Tao Zhang ^b, Shu-Ling Yu ^c, Xing-Jie Dai ^a, Ya Wu ^a, Jing-Chao Tao* ^a

a

College of Chemistry and Molecular Engineering, New Drug Research & Development Center,

Zhengzhou University, 75 Daxue Road, Zhengzhou, Henan 450052, China

^bSchool of Pharmacy, Xinxiang Medical University, Xinxiang 453003, Henan, China

c

Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province, Henan University,

Kaifeng, Henan 475004, China

Abstract: Two series of novel acylthiosemicarbazide and oxadiazole fused-isosteviol derivatives were

synthesized based on the 19-carboxyl modification. The target compounds were evaluated for their

cytotoxicities against 3 cancer cell lines (HCT-116, HGC-27 and JEKO-1) using an MTT assay. Lead

compounds from the acylthiosemicarbazides (4) showed IC₅₀values in the lower μmolar range. For

example, compounds (4i, 4l, 4m, 4r and 4s) exhibited significant inhibitory activities against the 3 cell

lines with IC₅₀values of 0.95-3.36 μM. Furthermore, 2D-HQSAR and 3D-topomer CoMFA analyses were

established, which could be used to develop second generation of isosteviol derivatives as anticancer

agents.

Key words: Isosteviol derivatives; Synthesis; Cytotoxicity; 2D-HQSAR; 3D-topomer CoMFA.

* Corresponding author. Tel/Fax: +86-371-67767200. Email address: jctao@zzu.edu.cn.

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/cbdd.12910

1. Introduction

Cancer, the second leading cause of death after cardiovascular disorders, represents one of the most serious

clinical problems and its incidence is rapidly rising throughout the world (1-3). Despite recent advances in

cancer therapy, numerous anticancer drugs have been reported which are having severe side effects.

Development of novel compounds as chemotherapeutic agents having a limited toxicity profile is needed.

Natural products and their derivatives, such as doxorubicin, paclitaxel, vinblastine, are a rich source of

treatments for cancers, and the unique metabolites produced by diterpenoids have increasingly become

major players in anticancer drug discovery (4-8).

Isosteviol (ent-16-oxobeyeran-19-oic acid), a metabolite of stevioside isolated from the leaves of the

natural stevia plant, is a tetracyclic diterpenoid with a beyerane skeleton which becomes one of the most

popular platforms for the design of novel pharmacological agents (9, 10). Previous studies have found that

isosteviol exhibits a wide range of biological activities, including antihypertension (11, 12),

antihyperglycemic (13, 14), antioxidative (15), antidiarrheal (16), cardio- and neuroprotective effects (17,

18). To go deep into the research on isosteviol, a large number of isosteviol derivatives have been obtained

by microbial transformation and chemical modification. The typical properties of isosteviol derivatives,

such as cytotoxic activities (19-23), catalytic performance (24-27), as well as supramolecular

self-assembly properties (28-30), were further investigated in recent years. Notably, some of the synthetic

isosteviol derivatives exhibited remarkable inhibitory activity against cancer cells that captured scientific

attention with possible applications in anticancer drugs design and development. Indeed, recently

published novel cytotoxic isosteviol derivatives with D-ring modifications have shown promising results.

Notable example is the exo-methylene cyclopentanone tetracyclic diterpenoids A, which exhibited similar

anticancer activities to adriamycin with IC₅₀value of 1.58 µM (31). Moreover, some bioactive subunits

containing isosteviol derivatives, such as 1,3-amino alcohol fragment B (32) or triazole ring C (33), were

proved to possess antiproliferative activities on several human malignant cell lines. And the heterocycle

bridged carbothioamide type isosteviol derivatives D, E (34) showed remarkable cytotoxic activities (Fig.

1). However, few reports have been focused on introduction of bioactive structural fragments to

19-position of isosteviol. In addition, to the best of our knowledge, two-dimensional hologram quantitative

structure–activity relationship (2D-HQSAR) and three-dimensional topomer comparative molecular field

analyses (3D-topomer CoMFA) were scarcely employed to investigate the structure–activity relationship

of 19-carboxyl modified isosteviol derivatives.

In light of these facts, two series of novel isosteviol derivatives containing acylthiosemicarbazide and

oxadiazole moieties based on 19-carboxyl modification were synthesized in the present work, and their

cytotoxicities against HCT-116, HGC-27 and JEKO-1 cancer cell lines in vitro were evaluated.

Furthermore, two- and three-dimensional quantitative structure–activity relationships were also studied for

the validation of the observed pharmacological properties of the investigated bioactive compounds and for

the confirmation of the most important parameters controlling these properties. The contribution/contour

maps, displaying activity information, will be further taken advantage of designing new anticancer

molecules with improved potency.

2. Results and discussion

2.1. Chemistry

Synthesis of isosteviol-fused acylthiosemicarbazide subunit or oxadiazole ring was carried out as the route

shown in Scheme 1. The initial isosteviol 1 was obtained through the sulfuric acid hydrolysis of stevioside.

Its acid chloride 2 was prepared from compound 1 and oxalyl chloride by adding drops of DMF as catalyst,

which then reacted without purification with hydrazine in dichloromethane at -10 ^oC to furnish compound

3 in 89% yield. Compounds 4a-4u, the acylthiosemicarbazide subunit containing isosteviol derivatives,

were readily synthesized through condensation of compound 3 with appropriately substituted phenyl

isothiocyanates in dichloromethane with the final yields of 37–89%. The IR spectrum of compound 4s, as a

representative example of the synthesized compounds, shows absorption bands of N-H group at 3312 cm^-1

and of C=O, C=S moieties at 1735, 1330 cm^-1, respectively. In its NMR spectra, resonances are distinctly

observed at δ_H10.54, 9.61, 9.19 ppm and δ_C178.86, 171.27ppm, demonstrating the formation of

acylthiosemicarbazide substructure. Subsequently, cyclization of compounds 4a-4u took place readily in

refluxing ethanol in the presence of mercuric acetate to afford the corresponding oxadiazole derivatives

5a-5u.

In order to further investigate the effects of thiourea subunit and carbonyl group on anticancer activities,

and simultaneously to provide diverse molecule structures convenient for the subsequent QSAR studies,

compounds 6, 7 and 8 were also designed and synthesized. The synthetic approaches employed to prepare

the target derivatives were outlined in Scheme 2. Treatment of compound 3 with phenyl isocyanate

afforded the corresponding target compound 6 in good yield. Meantime, the C(16)=O group of compound

5a was reduced with sodium borohydride to give the compound 7, which was further acylated with acetic

anhydride in pyridine at room temperature to generate the compound 8 with the yield of 97%.

2.2. Evaluation of cytotoxic activity

The in vitro cytotoxic activity of the novel isosteviol derivatives obtained was examined in three human

cancer cell lines: colon carcinoma (HCT-116), gastric carcinoma (HGC-27), mantle cell lymphoma

(JEKO-1) using the MTT assay. Cisplatin was included in the experiments as a positive control. The IC₅₀

values were summarized in Table 1.

From the obtained results we can see that all the investigated isosteviol derivatives exhibited much better

cytotoxic activities than their precursor isosteviol 1. Acylthiosemicarbazides-substituted isosteviol

derivatives 4a-4u displayed much higher cytotoxicities against 3 cancer cell lines than those of the

oxadiazoles 5a-5u. Investigations of the cytotoxic activity against HCT-116 demonstrated that it was the

most sensitive cell line to the influence of the tested compounds.

The inhibitory activities against HCT-116 and HGC-27 cell lines were remarkablely improved as the

introduction of the substituted acylthiosemicarbazides to the isosteviol skeleton (4a-4u vs 3). Notably,

compounds (4i, 4l, 4m, 4r and 4s) exhibited IC₅₀values in the lower μmolar range (0.95-3.36 μM). Going

even further, substituted groups and positions on aromatic ring had a significant effect on cytotoxic

activities. Compounds with strong electron-withdrawing substituents (4e-4g, 4r and 4s) exhibited good to

excellent cytotoxic activities. Except for the compounds (4q-4s), the meta-substituted compounds were

more favorable to the cytotoxicity than the ortho and para-substituted compounds. However, replacement

of the sulfur with oxygen greatly decreased the cytotoxic activities (4a vs 6), which indicated that the

thiocarbamide moiety may play an important role in the influence to cytotoxic activity. What’s more, most

of cyclized oxadiazole compounds revealed the moderate cytotoxic activity. Only compound 5g showed

better inhibitory activity against JEKO-1 cells than the positive control cisplatin. But to our disappointment,

when the carbonyl group was reduced to hydroxyl or further acylated, the compounds (7, 8) showed poor

inhibitory activities against JEKO-1 cell with IC₅₀values of 57.24±2.5 μM and 52.53±2.4 μM, respectively.

In order to further understand the observed cytotoxic activities and determine the main controlling factors

governing these activities, quantitative structure–activity relationship (QSAR) studies were developed.

2.3. QSAR studies

2.3.1. Data sets

Forty six isosteviol derivatives (3-8) were used for the QSAR analyses, of which thirty seven compounds

were selected as training set to construct model and the other nine (4a, 4b, 4j, 4r, 4u, 5e, 5l, 5p, 7) were

assigned into test set for external model validation. The division of the dataset was done randomly, but

taking into account the diversity of the bioactivities simutaneously. The IC₅₀values against HCT-116 cells,

which was the most sensitive to the influence of the tested compounds, were converted into the

corresponding pIC₅₀(–logIC₅₀) and subsequently used as dependent variables for the 2D-HQSAR and

3D-topomer CoMFA analyses.

2.3.2. 2D- and 3D-QSAR model development

Three distinct parameters, such as hologram length, fragment distinction and fragment size, were used to

generate the 2D-HQSAR model. For the hologram length, default values of 53, 59, 61, 71, 83, 97, 151, 199,

257, 307, 353 and 401 were implemented in SYBYL. The fragment distinction for displaying molecule

information was described in terms of atoms (A), bonds (B), connections (C), hydrogen (H), chirality (Ch),

donor and acceptor (DA). The minimum (M) and maximum (N) number of atoms of the broken molecule

was defined as fragment size which was provided as default value of 4–7. Three distinct parameters as

independent variables were linearly correlated with the pIC₅₀values as dependent variables by the full

cross validated (q²) partial least square (PLS) leave-one-out (LOO) method to develop the 2D-HQSAR

model.

Initially, the influence of the fragment distinction parameters on the statistical values of models was

investigated. A series of 2D-HQSAR models (Table 2) were developed by the combination of more than

one fragment distinction parameters, while fragment size (4–7) and hologram length (53 to 401) were

respectively set. As shown in Table 2, q²value of 2D-HQSAR model based on the combination of A and B

(model 1) is 0.413. An introduction of the factor H into the combination of A and B could improve the q²

value (model 3, q²= 0.498), whereas the introduction of factor C resulted in the decreasing of q²value

(model 2, q²= 0.368), indicating that connections (C) did not represent their intrinsic structural

characteristics. Introducing Ch or DA into the combination of A/B/H, respectively, could further improve

the q²value. Especially, the combination of A/B/H/DA could generate the highest q²value of 0.589 (model

9). However, simultaneously introduction of Ch and DA into the combination of A/B/H could not further

increase the q²value (q²= 0.577). Therefore, the A/B/H/DA combination was proved to be the most

effective fragment distinction.

Based on the best 2D-HQSAR model (model 9, Table 2), the influences of distinct fragment sizes on the

statistical parameters were further investigated. The statistical results, summarized in Table 3,

demonstrated that the variation of fragment size led to the generation of a better 2D-HQSAR model on the

basis of the fragment size of 7–8, affording the highest q²value of 0.631 (model 13). Therefore, the final

optimal 2D-HQSAR model, demonstrating the q², SEcv, r²and SEE values of 0.631, 0.249, 0.882 and

0.140, respectively, was developed by using A/B/H/DA as fragment distinction parameters, 7–8 as

fragment size, and 6 as optimum number of component.

In the 3D-topomer CoMFA analysis, C-4 and C-19 were initially picked respectively from the two most

active compounds of their respective series, 4s and 5f，which were taken as the two template molecules.

All the training set compounds were automatically divided into R₁(blue) and R₂(red) fragments (Fig. 2).

Two fragments were applied with topomer alignment to make a 3D invariant representation. Steric and

electrostatic interaction energies were calculated using carbon sp³probe. These steric and electrostatic field

descriptor values and the cytotoxicities as independent and dependent variables, respectively, were

correlated to finally generate the 3D-topomer CoMFA model, which provided a cross-validated q²value of

0.572, an optimized component of 3, and a noncross-validated r²value of 0.853 with cross-validated

standard error SE_CV= 0.25 and noncross validated standard error SEE = 0.15. These data demonstrate that

the model has a predictive ability of (q²>0.2).

2.3.3. Validation of the 2D- and 3D-QSAR models

The statistical parameters of the optimal models selected by the 2D- and 3D-QSAR methods were

summarized in Table 4. In order to investigate the predictive ability of the constructed QSAR models based

on the training set, an external test set including nine compounds was used for validation. The

cytotoxicities against HCT-116 cells of the 9 test set compounds were predicted using the two optimal

models. The 2D-HQSAR and 3D-topomer CoMFA models afforded the satisfactory r²_predvalues of 0.832

and 0.643 (Table 4), respectively. Comparing with the r²_predvalue of 3D-topomer CoMFA model, the

2D-HQSAR model had the better predictive r²value and showed good predictions for the external test set,

indicating that the 2D-HQSAR method are more suited to this kind of datasets than 3D-topomer CoMFA

method.

The detailed experimental and predicted cytotoxicities (pIC₅₀) with residual values for the test set

compounds from the two models were listed in Table 5. The predicted pIC₅₀residuals of 2D-HQSAR and

3D-topomer CoMFA models were less than 0.3 and 0.4 log unit, respectively, indicating that high

predictive QSAR models were obtained. The histogram of residual values (Fig. 3) visually displayed that

the 2D-HQSAR model had smaller residuals than 3D-topomer CoMFA model.

The correlation between the experimental and predicted activities of both the training set and the test set

was also displayed graphically in Fig. 4. It was shown that the predicted values of all compounds fall

closely to the experimental pIC₅₀values almost in a line, deviating by no more than 0.3 log units except

compounds 4b, 4u and 5t. The good agreement between experimental and predicted pIC₅₀values for the

training set and test set compounds indicates the internal robustness and external high prediction of the

QSAR models.

2.3.4. Atomic contribution maps and contour maps analyses

A significant role of QSAR model, besides predicting the activities of untested molecules, is to provide

straightforward clues about the relationship between molecular fragments and bioactivities (35). In

2D-HQSAR analysis, the results can be graphically displayed in the form of contribution maps, where the

color coding of each atom reflects its contribution to the overall activity of the molecule. The color (red,

red orange and orange) at one end of the spectrum denotes the negative contribution (NC) to the activity,

whereas the color (yellow, green blue and green) of the other end reflects positive contribution (PC) to the

activity. Additionally, the intermediate contribution (IC) is indicated by white color.

The individual atomic contribution maps of compounds 4s, 4u, 5f and 5t, the highest and the lowest active

compounds in their respective series, were depicted in Fig. 5. It can be seen from the map, the

4'-nitrophenyl, ring A and ring B of compound 4s are colored yellow and green, respectively, indicating

favorable contribution to the cytotoxic activity. The 2, 6-dimethylphenyl group was heavily colored red in

compound 4u showing significant negative effect on bioactivity. Moreover, ring A and ring B of compound

4u was colored white and red orange, demonstrating their intermediate or negative contribution to the

cytotoxic activity. In the representations of compounds 5f and 5t, the particular atoms in the oxadiazole

ring were colored yellow, suggesting that the heterocycle subunit may be the pharmacologically important

group. In addition, the facts that there are more yellow areas and less red areas in compound 5f than in

compound 5t can reasonably explain the more potent cytotoxicity of compound 5f. Furthermore, in any

molecule of the dataset, regions with intermediate or unfavorable contributions can be identified as the

potential targets for the structural modification as well as the SAR studies.

In the 3D-topomer CoMFA analysis, the contour maps around R₁and R₂(Fig. 6) were obtained by plotting

the coefficients from the 3D-topomer CoMFA model. The most active compound 4s was chosen as a

reference structure to explain the contour map. In the steric contour map, green color represents that

sterically bulky group is in favor of activity, whereas yellow color is unfavoured for activity. In the

electrostatics contour map, red and blue colors denote respectively electronegative or electropositive

groups favored for activity.

As shown in Fig. 6, a yellow region (Fig. 6, A) near the C-16 of the R₁fragment indicates that the bulk

groups are unfavorable to the anticancer activity. This can well explain the fact that compound 8

containing large group at this position has relative lower anticancer activity (IC₅₀= 15.06±1.3 μM) than

compound 5a (IC₅₀= 9.56±0.9 μM). The skeleton of R₁fragment lacks of distinct substituent so that the

electrostatics contour maps are not found around it (Fig. 6, B). In Fig. 6 (C), a green contour near the meta-

and para-position of the phenyl group in R₂fragment suggests that a bulky group would be in favor of

activities (e.g. compound 4l having meta-Br group with IC₅₀= 1.35±0.3 μM, whereas compound 4i having

meta-Cl group with IC₅₀= 1.62±0.1 μM). While the yellow contour near ortho-position of the phenyl

group, unfavoured result was observed (compound 4b having ortho-CH₃group with IC₅₀= 11.61±0.9 μM).

Furthermore, a large red region surrounding para-position of the phenyl group (Fig. 6, D) demonstrates

that introduction of electronegative groups can improve the inhibitory activity (e.g. compound 4g having

para-F group with IC₅₀= 2.40±0.5 μM, whereas compound 4s having para-NO₂group with IC₅₀

=

0.95±0.1 μM). Based on the above useful clues and informations from atomic contribution and contour

maps analyses, a conclusion can be derived that the introduction of small group(s) onto the C-16 position

of the isosteviol fragment and electron-withdrawing and bulky group(s) onto the para-position of the

phenyl group may be favorable for the inhibitory activity against HCT-116 cells.

3. Conclusions

In this work, two series of 19-carboxyl modified novel isosteviol derivatives, containing respectively

acylthiosemicarbazide and oxadiazole subunits, were rationally designed and successfully synthesized.

Their cytotoxic activities in vitro against three human cancer cell lines (HCT-116, HGC-27 and JEKO-1)

were evaluated. The results obtained revealed that the HCT-116 cell line was more sensitive to the novel

compounds than the HGC-27 and JEKO-1 cell lines. It was noteworthy that the acylthiosemicarbazide

group containing isosteviol derivatives exhibited prominent cytotoxicities against all the three cell lines.

Especially, compounds (4i, 4l, 4m, 4r and 4s) exhibited the significant inhibitory activities against the 3

cell lines with IC₅₀values of 0.95-3.36 μM, which may be exploitable as lead compounds for the

development of potent antitumor agents. In addition, 2D- and 3D-QSAR techniques were implemented on

the SAR of these compounds. The optimal 2D-HQSAR and 3D-topomer CoMFA models with good

predictive capabilities were obtained. Based on the useful clues and informations from atomic contribution

maps and contour maps generated by 2D-HQSAR and 3D-topomer CoMFA models, a conclusion can be

derived that introduction of small group(s) onto the C-16 position of isosteviol and electron-withdrawing

and bulky group(s) onto the para-position of the phenyl group may be favorable for the inhibitory activity

against HCT-116 cells. The results provided a practical method for the development of second generation

of isosteviol derivatives as anticancer agents.

4. Experimental protocols

4.1. General procedures

All chemicals were used as received unless otherwise noted. Reagent grade solvents were redistilled prior

to use. Isosteviol 1 was synthesized as described by Wu et al (36). Chromatography was performed on

silica gel (100-200 mesh). All melting points were measured with a Beijing Keyi XT5 apparatus and the

temperature was not corrected. The IR spectra were recorded as KBr pellets on a Thermo Nicolet (IR200)

1

spectrometer. H and ¹³C NMR spectra were collected with a Bruker DPX-400 spectrometer at 400 and

100 MHz, respectively, with TMS as internal standard. High-resolution mass spectra (HRMS) were

obtained with a Waters Micromass Q-Tof Micro™ instrument by the ESI technique. X-ray analysis was

taken on a Rigaku Saturn 724 CCD diffractomer (Mo-Kα, λ = 0.71073 Å).

4.2. The preparation of isosteviol derivatives

4.2.1. Compound 2

To a solution of compound 1 (0.318 g, 1 mmol) in CH₂Cl₂(10 mL), oxalyl chloride (0.191 g, 1.5 mmol)

was added and the resulting mixture was stirred for 30 min. To this was added the DMF (0.1 ml) and

stirring was continued at room temperature for 8 h. Upon completion, the solvent was evaporated in vacuo

to obtain the crude product 2 as a gray solid, which was used directly in the next step without further

purification.

4.2.2. Compound 3

o

The crude product 2 (0.366 g, 1 mmol) was dissolved in CH₂Cl₂(20 mL) at -10 C, and then 80%

hydrazine hydrate (0.1 mL, 2 mmol) was slowly added. After stirring for 2 h, the mixture was washed with

saturated NaCl aqueous solution, dried with anhydrous sodium sulfate, filtered and concentrated under

vacuum. The residue was purified by column chromatography on silica (dichloromethane / methanol = 5:1,

v/v) to give the compound 3 as a white powder. Yield 89%; Mp 82.3–84.1 °C; IR (KBr): 3323, 2925, 2846,

1737, 1633, 1503, 1454, 1402, 1372, 1321, 1249, 1199, 1151, 1110, 1087, 1028, 977, 916, 734, 695, 590,

508cm^-1; ¹H NMR (400MHz, CDCl_3,ppm): δ 7.02 (s, 1H), 3.48 (s, 2H), 2.63 (d, J = 18.64Hz, 1H), 2.03 (d,

J = 14.32Hz, 1H), 1.95 (d, J = 13.52Hz, 1H), 1.83–1.67 (m, 6H), 1.62–1.48 (m, 4H), 1.42–1.33 (m, 2H),

1.27–1.21 (m, 1H), 1.19 (s, 3H), 1.15–1.03 (m, 2H), 0.97 (s, 3H), 0.94–0.79 (m, 2H), 0.74 (s, 3H); ¹³C

NMR (100MHz, CDCl_3,ppm): δ 222.11, 177.52, 57.56, 54.67, 54.22, 48.68, 48.37, 43.04, 41.56, 40.00,

39.44, 38.01, 37.44, 37.24, 29.99, 22.11, 20.31, 19.83, 19.11, 13.44; HRMS (ESI, m/z) calcd for

C₂₀H₃₃N₂O₂[M+H]⁺333.2542. Found: 333.2539.

4.2.3. General procedure for synthesis of compounds 4a-4u

Compound 3 (0.332 g, 1 mmol) was dissolved in CH₂Cl₂(10 mL). Appropriately substituted phenyl

isothiocyanates (1.1 mmol) was added. The reaction mixture was stirred at room temperature. After

completion of the reaction monitored by TLC, the solvent was removed under reduced pressure. The

residue was purified by column chromatography on silica to give the target compound 4a-4u.

4.2.3.1. Compound 4a. White powder; Yield 64%; Mp 87.5–88.4 °C; IR (KBr): 3300, 3056, 2928, 2847,

1734, 1653, 1598, 1534, 1497, 1447, 1353, 1319, 1235, 1155, 1110, 1088, 1032, 973, 900, 854, 694, 590,

499 cm^-1; ¹H NMR (400MHz, CDCl_3,ppm): δ 10.08 (s, 1H), 9.83 (s, 1H), 8.68 (s, 1H), 7.43–7.26 (m, 5H),

2.59 (d, J = 18.44Hz, 1H), 2.16 (d, J = 14.24Hz, 1H), 1.99–1.87 (m, 3H), 1.80–1.67 (m, 4H), 1.62–1.50 (m,

5H), 1.44–1.33 (m, 3H), 1.28 (s, 3H), 1.27–1.18 (m, 2H), 0.97 (s, 3H), 0.92–0.89 (m, 1H), 0.79 (s, 3H);

¹³C NMR (100MHz, CDCl_3,ppm): δ 222.10, 176.22, 172.05, 136.99, 129.46, 129.46, 126.91, 125.30,

125.30, 57.51, 54.58, 54.09, 48.67, 48.24, 43.11, 41.31, 39.87, 39.37, 38.08, 37.23, 37.18, 30.18, 22.20,

20.31, 19.81, 18.89, 13.75; HRMS (ESI, m/z) calcd for C₂₇H₃₈N₃O₂S [M+H]⁺468.2685. Found: 468.2683.

4.2.3.2. Compound 4b. White powder. Yield 60%; Mp 112.1–114.9 °C; IR (KBr): 3284, 3058, 2929, 2847,

1736, 1643, 1590, 1527, 1458, 1370, 1344, 1246, 1154, 1109, 1045, 973, 855, 728, 698, 632, 510cm^-1; ¹H

NMR (400MHz, CDCl_3,ppm): δ 10.36 (s, 1H), 9.85 (s, 1H), 8.53 (s, 1H), 7.30–7.24 (m, 4H), 2.55 (dd, J =

18.60, 3.48Hz, 1H), 2.31 (s, 3H), 2.11 (d, J = 14.60Hz, 1H), 1.98–1.45 (m, 11H), 1.36–1.30 (m, 4H), 1.25

(s, 3H), 1.21–1.12 (m, 2H), 0.95 (s, 3H), 0.92–0.88 (m, 1H), 0.77 (s, 3H); ¹³C NMR (100MHz, CDCl_3,

ppm): δ 222.13, 176.61, 171.48, 135.74, 135.26, 131.28, 128.33, 127.88, 127.15, 57.50, 54.50, 54.04,

48.63, 48.18, 42.97, 41.22, 39.84, 39.29, 38.00, 37.14, 37.06, 30.16, 22.14, 20.27, 19.78, 18.82, 17.91,

13.69; HRMS (ESI, m/z) calcd for C₂₈H₄₀N₃O₂S [M+H]⁺482.2841. Found: 482.2839.

4.2.3.3. Compound 4c. White powder. Yield 37%; Mp 104.6–107.2 °C; IR (KBr): 3296, 3046, 2927, 2847,

1

1735, 1652, 1611, 1537, 1451, 1348, 1298, 1249, 1152, 1043, 975, 900, 781, 718, 693, 623, 571cm^-1; H

NMR (400MHz, CDCl_3,ppm): δ 9.94 (s, 1H), 9.78 (s, 1H), 8.48 (s, 1H), 7.28 (t, J = 7.98Hz, 1H), 7.15 (d,

J = 9.00Hz, 2H), 7.08 (d, J = 7.52Hz, 1H), 2.57 (dd, J = 18.60, 3.56Hz, 1H), 2.36 (s, 3H), 2.14 (d, J =

14.48Hz, 1H), 1.97–1.31 (m, 14H), 1.25 (s, 3H), 1.24–1.17 (m, 3H), 0.96 (s, 3H), 0.93–0.87 (m, 1H), 0.77

(s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.14, 175.97, 171.89, 139.60, 136.62, 129.34, 127.88,

125.91, 122.31, 57.48, 54.57, 54.09, 48.66, 48.23, 43.05, 41.33, 39.86, 39.35, 38.05, 37.20, 37.16, 30.15,

22.15, 21.39, 20.29, 19.80, 18.89, 13.71; HRMS (ESI, m/z) calcd for C₂₈H₄₀N₃O₂S [M+H]⁺482.2841.

Found: 482.2840.

4.2.3.4. Compound 4d. White powder. Yield 41%; Mp 102.3–104.7 °C; IR (KBr): 3293, 3029, 2926, 2847,

1735, 1652, 1606, 1517, 1449, 1344, 1290, 1236, 1153, 1111, 1044, 974, 818, 727, 694, 586, 500cm^-1; ¹H

NMR (400MHz, CDCl₃, ppm): δ 9.94 (s, 1H), 9.80 (s, 1H), 8.59 (s, 1H), 7.22 (d, J = 8.56Hz, 2H), 7.19 (d,

J = 8.48Hz, 2H), 2.58 (dd, J = 18.64, 3.48Hz, 1H), 2.34 (s, 3H), 2.13 (d, J = 14.44Hz, 1H), 1.96–1.31 (m,

15H), 1.25 (s, 3H), 1.24–1.14 (m, 2H), 0.96 (s, 3H), 0.92–0.89 (m, 1H), 0.77 (s, 3H); ¹³C NMR (100MHz,

CDCl₃, ppm): δ 222.15, 176.21, 171.84, 137.02, 134.12, 130.12, 130.12, 125.43, 125.43, 57.48, 54.56,

54.08, 48.65, 48.23, 43.03, 41.33, 39.85, 39.35, 38.04, 37.18, 37.16, 30.15, 22.12, 21.08, 20.29, 19.80,

18.87, 13.71; HRMS (ESI, m/z) calcd for C₂₈H₄₀N₃O₂S [M+H]⁺482.2841. Found: 482.2839.

4.2.3.5. Compound 4e. White powder. Yield 73%; Mp 102.1–102.3 °C; IR (KBr): 3290, 3041, 2928, 2847,

1734, 1656, 1621, 1542, 1494, 1456, 1342, 1249, 1152, 1107, 1033, 974, 856, 807, 752, 699, 622, 570cm^-1;

¹H NMR (400MHz, CDCl₃, ppm): δ 10.59 (s, 1H), 10.00 (s, 1H), 8.69 (d, J = 14.84Hz, 1H),, 7.67 (t, J =

7.22Hz, 1H), 7.26–7.13 (m, 3H), 2.56 (dd, J = 18.64, 3.12Hz, 1H), 2.13 (d, J = 14.76Hz, 1H), 2.00–1.31

(m, 14H), 1.28 (s, 3H), 1.25–1.12 (m, 3H), 0.95 (s, 3H), 0.93–0.87 (m, 1H), 0.79 (s, 3H); ¹³C NMR

(100MHz, CDCl₃, ppm): δ 222.08, 176.61, 171.62, 156.66 (d, J = 246.06Hz), 128.40 (d, J = 2.75Hz),

128.21 (d, J = 7.67Hz), 125.50 (d, J = 16.44Hz), 124.38, 116.17 (d, J = 19.94Hz), 57.52, 54.56, 54.04,

48.67, 48.16, 43.08, 41.26, 39.86, 39.30, 38.06, 37.23, 37.16, 30.14, 22.08, 20.31, 19.80, 18.77, 13.72;

HRMS (ESI, m/z) calcd for C₂₇H₃₇FN₃O₂S [M+H]⁺486.2591. Found: 486.2588.

4.2.3.6. Compound 4f. White powder. Yield 80%; Mp 119.3–121.5 °C; IR (KBr): 3299, 3079, 2928, 2847,

1734, 1655, 1611, 1543, 1490, 1444, 1349, 1283, 1253, 1148, 1045, 970, 861, 776, 721, 682, 571, 519cm^-1;

¹H NMR (400MHz, CDCl₃, ppm): δ 10.47 (s, 1H), 9.89 (s, 1H), 8.88 (s, 1H), 7.33 (q, J = 7.54Hz, 1H),

7.25 (d, J = 10.00Hz, 1H), 7.16 (d, J = 8.00Hz, 1H), 6.94 (td, J = 8.16, 1.76Hz, 1H), 2.58 (dd, J = 18.60,

3.20Hz, 1H), 2.15 (d, J = 14.64Hz, 1H), 1.99–1.32 (m, 14H), 1.27 (s, 3H), 1.25–1.19 (m, 3H), 0.96 (s, 3H),

0.92–0.86 (m, 1H), 0.78 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.08, 176.30, 172.25, 162.71 (d, J

= 245.24Hz), 139.05 (d, J = 10.15Hz), 130.20 (d, J = 9.28Hz), 120.53, 113.30 (d, J = 21.10Hz), 112.44 (d,

J = 24.04Hz), 57.43, 54.53, 54.01, 48.66, 48.20, 43.13, 41.23, 39.80, 39.35, 38.07, 37.23, 37.13, 30.19,

22.24, 20.29, 19.78, 18.84, 13.70; HRMS (ESI, m/z) calcd for C₂₇H₃₇FN₃O₂S [M+H]⁺486.2591. Found:

486.2587.

4.2.3.7. Compound 4g. White powder. Yield 89%; Mp 112.5–115.1 °C; IR (KBr): 3294, 3062, 2929, 2847,

1

1734, 1654, 1509, 1465, 1343, 1220, 1153, 1046, 974, 835, 798, 731, 586, 507cm^-1; H NMR (400MHz,

CDCl₃, ppm): δ 10.38 (s, 1H), 9.93 (s, 1H), 8.77 (s, 1H), 7.35–7.31 (m, 2H), 7.10–7.06 (m, 2H), 2.58 (dd, J

= 18.56, 3.52Hz, 1H), 2.12 (d, J = 15.12Hz, 1H), 1.99–1.32 (m, 14H), 1.27 (s, 3H), 1.24–1.17 (m, 3H),

0.96 (s, 3H), 0.94–0.87 (m, 1H), 0.78 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.00, 176.87, 171.78,

161.12 (d, J = 245.39Hz), 133.28, 127.85 (d, J = 7.41Hz), 127.85 (d, J = 7.41Hz), 116.11 (d, J = 22.64Hz),

116.11 (d, J = 22.64Hz), 57.42, 54.51, 54.00, 48.66, 48.22, 43.04, 41.25, 39.80, 39.34, 38.05, 37.19, 37.13,

30.20, 22.24, 20.29, 19.77, 18.81, 13.71; HRMS (ESI, m/z) calcd for C₂₇H₃₇FN₃O₂S [M+H]⁺486.2591.

Found: 486.2586.

4.2.3.8. Compound 4h. White powder. Yield 74%; Mp 114.3–117.1 °C; IR (KBr): 3311, 3062, 2927, 2847,

1735, 1657, 1593, 1537, 1470, 1443, 1340, 1229, 1155, 1129, 1055, 1034, 974, 856, 755, 732, 684, 621,

1

572, 452 cm^-1; H NMR (400MHz, CDCl₃, ppm): δ 10.57 (s, 1H), 9.99 (s, 1H), 8.78 (s, 1H), 7.67 (d, J =

7.24Hz, 1H), 7.47 (dd, J = 7.96, 1.36Hz, 1H), 7.33 (td, J = 7.66, 1.36Hz, 1H), 7.24 (td, J = 7.68, 1.44Hz,

1H), 2.55 (dd, J = 18.64, 3.48Hz, 1H), 2.13 (d, J = 14.84Hz, 1H), 2.01–1.30 (m, 13H), 1.28 (s, 3H),

1.25–1.11 (m, 4H), 0.95 (s, 3H), 0.93–0.86 (m, 1H), 0.78 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ

222.09, 176.59, 171.63, 134.83, 130.64, 130.00, 128.97, 128.19, 127.45, 57.54, 54.51, 54.01, 48.64, 48.16,

43.09, 41.18, 39.83, 39.26, 38.03, 37.12, 30.17, 29.68, 22.15, 20.28, 19.77, 18.77, 13.73; HRMS (ESI, m/z)

calcd for C₂₇H₃₇ClN₃O₂S [M+H]⁺502.2295. Found: 502.2291.

4.2.3.9. Compound 4i. White powder. Yield 80%; Mp 114.0–115.5 °C; IR (KBr): 3294, 3070, 2927, 2848,

1

1735, 1655, 1594, 1537, 1474, 1343, 1232, 1155, 1044, 972, 922, 865, 778, 728, 680, 621, 573cm^-1; H

NMR (400MHz, CDCl₃, ppm): δ 10.56 (s, 1H), 9.94 (s, 1H), 8.90 (s, 1H), 7.43 (s, 1H), 7.35–7.32 (m, 1H),

7.30 (t, J = 7.80Hz, 1H), 7.21 (d, J = 7.48Hz, 1H), 2.58 (dd, J = 18.56, 3.40Hz, 1H), 2.14 (d, J = 14.52Hz,

1H), 2.00–1.32 (m, 14H), 1.27 (s, 3H), 1.25–1.18 (m, 3H), 0.96 (s, 3H), 0.93–0.86 (m, 1H), 0.78 (s, 3H);

¹³C NMR (100MHz, CDCl₃, ppm): δ 222.07, 176.40, 172.10, 138.79, 134.50, 130.00, 126.56, 125.42,

123.50, 57.46, 54.55, 54.03, 48.68, 48.20, 43.14, 41.22, 39.82, 39.37, 38.09, 37.23, 37.16, 30.24, 22.33,

20.32, 19.80, 18.86, 13.74; HRMS (ESI, m/z) calcd for C₂₇H₃₇ClN₃O₂S [M+H]⁺502.2295. Found:

502.2293.

4.2.3.10. Compound 4j. White powder. Yield 88%; Mp 118.2–120.3 °C; IR (KBr): 3293, 2928, 2848, 1736,

1

1655, 1595, 1533, 1491, 1338, 1235, 1154, 1090, 1045, 1012, 973, 828, 732, 572, 503cm^-1; H NMR

(400MHz, CDCl₃, ppm): δ 10.48 (s, 1H), 9.91 (s, 1H), 8.87 (s, 1H), 7.33 (s, 4H), 2.58 (dd, J = 18.56,

3.24Hz, 1H), 2.13 (d, J = 14.48Hz, 1H), 1.97–1.33 (m, 14H), 1.27 (s, 3H), 1.25–1.15 (m, 3H), 0.96 (s, 3H),

0.94–0.86 (m, 1H), 0.78 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.05, 176.61, 172.09, 136.07,

132.07, 129.25, 129.25, 126.81, 126.81, 57.41, 54.53, 54.01, 48.69, 48.24, 43.11, 41.27, 39.80, 39.37,

38.09, 37.26, 37.15, 30.22, 22.26, 20.31, 19.80, 18.84, 13.73; HRMS (ESI, m/z) calcd for C₂₇H₃₇ClN₃O₂S

[M+H]⁺502.2295. Found: 502.2293.

4.2.3.11. Compound 4k. White powder. Yield 79%; Mp 116.7–118.1 °C; IR (KBr): 3302, 3058, 2928, 2847,

1735, 1655, 1589, 1534, 1465, 1439, 1340, 1294, 1234, 1155, 1027, 974, 855, 754, 732, 663, 622, 572,

516cm^-1; ¹H NMR (400MHz, CDCl₃, ppm): δ 10.55 (s, 1H), 9.98 (s, 1H), 8.80 (s, 1H), 7.65 (d, J = 8.04Hz,

1H), 7.62 (d, J = 7.96Hz, 1H), 7.38 (t, J = 7.66Hz, 1H), 7.18 (t, J = 7.72Hz, 1H), 2.55 (dd, J = 18.64,

2.92Hz, 1H), 2.14 (d, J = 14.76Hz, 1H), 2.02–1.88 (m, 1H), 1.78–1.42 (m, 9H), 1.34–1.31 (m, 2H), 1.29 (s,

3H), 1.25–1.14 (m, 5H), 0.95 (s, 3H), 0.92–0.91 (m, 1H), 0.79 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm):

δ 222.10, 176.62, 171.65, 136.29, 133.23, 129.38, 128.69, 128.21, 121.55, 57.58, 54.53, 54.02, 48.66,

48.21, 43.12, 41.18, 39.85, 39.28, 38.05, 37.14, 37.14, 30.25, 22.26, 20.30, 19.79, 18.82, 13.78; HRMS

(ESI, m/z) calcd for C₂₇H₃₇BrN₃O₂S [M+H]⁺546.1790. Found: 546.1785.

4.2.3.12. Compound 4l. White powder. Yield 67%; Mp 120.2–121.7 °C; IR (KBr): 3289, 2928, 2847, 1733,

1

1653, 1590, 1535, 1472, 1344, 1231, 1155, 1044, 974, 863, 776, 733, 678, 621, 573cm^-1; H NMR

(400MHz, CDCl₃, ppm): δ 10.50 (s, 1H), 9.95 (s, 1H), 8.91 (s, 1H), 7.56 (t, J = 1.70Hz, 1H), 7.38 (d, J =

7.96Hz, 2H), 7.25 (t, J = 7.24Hz, 1H), 2.58 (dd, J = 18.56, 3.24Hz, 1H), 2.14 (d, J = 14.56Hz, 1H),

1.99–1.32 (m, 14H), 1.28 (s, 3H), 1.26–1.18 (m, 3H), 0.97 (s, 3H), 0.94–0.90 (m, 1H), 0.78 (s, 3H); ¹³C

NMR (100MHz, CDCl₃, ppm): δ 222.05, 176.54, 172.15, 138.89, 130.29, 129.58, 128.43, 124.19, 122.39,

57.45, 54.55, 54.02, 48.68, 48.19, 43.15, 41.19, 39.82, 39.37, 38.09, 37.23, 37.16, 30.24, 22.35, 20.32,

19.80, 18.86, 13.74; HRMS (ESI, m/z) calcd for C₂₇H₃₇BrN₃O₂S [M+H]⁺546.1790. Found: 546.1789.

4.2.3.13. Compound 4m. White powder. Yield 70%; Mp 124.8–126.6 °C; IR (KBr): 3297, 2929, 2847,

1734, 1655, 1591, 1532, 1488, 1461, 1338, 1280, 1235, 1154, 1072, 1044, 1010, 974, 825, 731, 571,

499cm^-1; ¹H NMR (400MHz, CDCl₃, ppm): δ 10.42 (s, 1H), 9.89 (s, 1H), 8.82 (s, 1H), 7.49 (d, J = 8.52Hz,

2H), 7.27 (d, J = 8.64Hz, 2H), 2.58 (dd, J = 18.56, 3.82Hz, 1H), 2.13 (d, J = 14.52Hz, 1H), 1.96–1.33 (m,

14H), 1.27 (s, 3H), 1.25–1.19 (m, 3H), 0.97 (s, 3H), 0.94–0.90 (m, 1H), 0.77 (s, 3H); ¹³C NMR (100MHz,

CDCl₃, ppm): δ 222.03, 176.49, 172.16, 136.52, 132.25, 132.25, 127.07, 127.07, 119.98, 57.40, 54.54,

54.01, 48.69, 48.24, 43.13, 41.27, 39.80, 39.38, 38.09, 37.27, 37.15, 30.20, 22.25, 20.32, 19.80, 18.85,

13.73; HRMS (ESI, m/z) calcd for C₂₇H₃₇BrN₃O₂S [M+H]⁺546.1790. Found: 546.1787.

4.2.3.14. Compound 4n. White powder. Yield 77%; Mp 178.6–179.4 °C; IR (KBr): 3334, 3075, 2930,

2846, 1735, 1603, 1543, 1460, 1357, 1313, 1245, 1154, 1112, 1046, 1025, 974, 927, 857, 789, 747, 698,

1

574, 503cm^-1; H NMR (400MHz, CDCl₃, ppm): δ 9.56 (s, 2H), 8.01 (s, 1H), 7.65 (s, 1H), 7.21 (t, J =

7.70Hz, 1H), 7.00 (t, J = 7.18Hz, 1H), 6.95 (d, J = 8.12Hz, 1H), 3.85 (s, 3H), 2.60 (d, J = 18.64Hz, 1H),

2.15 (d, J = 14.88Hz, 1H), 2.00–1.89 (m, 2H), 1.81–1.50 (m, 9H), 1.45–1.32 (m, 3H), 1.26 (s, 3H),

1.24–1.15 (m, 4H), 0.96 (s, 3H), 0.76 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.29, 176.94, 171.21,

144.53, 127.41, 125.73, 124.30, 121.16, 111.56, 57.55, 55.73, 54.61, 54.14, 48.69, 48.31, 43.12, 41.45,

39.91, 39.41, 38.07, 37.20, 37.20, 30.01, 21.98, 20.31, 19.83, 18.94, 13.72; HRMS (ESI, m/z) calcd for

C₂₈H₄₀N₃O₃S [M+H]⁺498.2790. Found: 498.2804.

4.2.3.15. Compound 4o. White powder. Yield 89%; Mp 98.4–100.2 °C; IR (KBr): 3298, 3054, 2930, 2846,

1735, 1654, 1602, 1538, 1461, 1351, 1229, 1156, 1087, 1044, 973, 900, 860, 775, 690, 627, 571, 455cm^-1;

¹H NMR (400MHz, CDCl₃, ppm): δ 10.02 (s, 1H), 9.74 (s, 1H), 8.62 (s, 1H), 7.29 (t, J = 7.82Hz, 1H), 6.96

(s, 1H), 6.93 (d, J = 7.80Hz, 1H), 6.79 (d, J = 8.20Hz, 1H), 3.79 (s, 3H), 2.58 (dd, J = 18.64, 3.00Hz, 1H),

2.15 (d, J = 17.48Hz, 1H), 2.00–1.32 (m, 13H), 1.27 (s, 3H), 1.24–1.17 (m, 4H), 0.96 (s, 3H), 0.94–0.87

(m, 1H), 0.77 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.22, 175.95, 172.24, 160.34, 138.02,

130.20, 117.03, 112.63, 110.72, 57.49, 55.44, 54.58, 54.09, 48.68, 48.25, 43.11, 41.34, 39.87, 39.38, 38.08,

37.25, 37.18, 30.16, 22.15, 20.31, 19.82, 18.91, 13.74; HRMS (ESI, m/z) calcd for C₂₈H₄₀N₃O₃S [M+H]⁺

498.2790. Found: 498.2830.

4.2.3.16. Compound 4p. White powder. Yield 72%; Mp 112.2–112.8 °C; IR (KBr): 3291, 3050, 2930, 2846,

1736, 1653, 1602, 1512, 1463, 1347, 1297, 1246, 1172, 1110, 1033, 973, 830, 733, 695, 586, 517cm^-1; ¹H

NMR (400MHz, CDCl₃, ppm): δ 9.81 (s, 2H), 8.52 (s, 1H), 7.25 (d, J = 8.84Hz, 2H), 6.92 (d, J = 8.40Hz,

2H), 3.81 (s, 3H), 2.68 (dd, J = 18.64, 2.52Hz, 1H), 2.12 (d, J = 14.52Hz, 1H), 1.96–1.35 (m, 13H), 1.25 (s,

3H), 1.24–1.16 (m, 4H), 0.96 (s, 3H), 0.92–0.90 (m, 1H), 0.77 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm):

δ 222.15, 176.63, 171.74, 158.61, 129.41, 127.53, 127.53, 114.74, 114.74, 57.49, 55.49, 54.46, 54.08,

48.67, 48.25, 43.02, 41.34, 39.86, 39.37, 38.06, 37.18, 37.18, 30.17, 22.16, 20.30, 19.82, 18.87, 13.73;

HRMS (ESI, m/z) calcd for C₂₈H₄₀N₃O₃S [M+H]⁺498.2790. Found: 498.2787.

4.2.3.17. Compound 4q. Yellow powder. Yield 70%; Mp 111.4–115.6 °C; IR (KBr): 3297, 2930, 2849,

1

1735, 1665, 1608, 1586, 1510, 1457, 1347, 1267, 1165, 1110, 1086, 976, 857, 741, 511cm^-1; H NMR

(400MHz, CDCl₃, ppm): δ 10.10 (s, 1H), 8.43 (s, 1H), 8.02 (d, J = 8.32Hz, 1H), 7.54 (t, J = 7.76Hz, 1H),

7.18 (t, J = 7.24Hz, 1H), 2.87 (d, J = 32.84Hz, 1H), 2.51 (dd, J = 18.60, 3.24Hz, 1H), 2.14 (d, J = 14.12Hz,

1H), 1.87–1.28 (m, 14H), 1.25 (s, 3H), 1.21–1.11 (m, 4H), 0.89 (s, 3H), 0.69 (s, 3H); ¹³C NMR (100MHz,

CDCl₃, ppm): δ 222.47, 179.82, 173.19, 139.99, 134.29, 133.90, 126.01, 125.46, 124.88, 57.56, 54.61,

54.10, 48.72, 48.30, 41.42, 39.84, 39.42, 38.09, 37.34, 37.19, 29.86, 22.05, 20.32, 19.80, 19.00, 13.82;

HRMS (ESI, m/z) calcd for C₂₇H₃₇N₄O₄S [M+H]⁺513.2536. Found:513.2532.

4.2.3.18. Compound 4r. Yellow powder. Yield 72%; Mp 124.0–126.4 °C; IR (KBr): 3309, 3095, 2930,

2848, 1736, 1659, 1616, 1529, 1467, 1347, 1247, 1155, 1090, 1045, 974, 888, 830, 799, 735, 672, 587,

509cm^-1; ¹H NMR (400MHz, CDCl₃, ppm): δ 10.72 (s, 1H), 9.84 (s, 1H), 9.21 (s, 1H), 8.31 (t, J = 4.00Hz,

1H), 8.06 (dd, J = 8.24, 1.36Hz, 1H), 7.87 (dd, J = 8.04, 1.20Hz, 1H), 7.52 (t, J = 8.16Hz, 1H), 2.58 (dd, J

= 18.56, 3.40Hz, 1H), 2.17 (d, J = 14.36Hz, 1H), 1.94–1.36 (m, 13H), 1.33 (s, 3H), 1.29–1.20 (m, 4H),

0.97 (s, 3H), 0.94–0.93 (m, 1H), 0.80 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.17, 177.00, 173.18,

148.28, 139.15, 130.93, 129.51, 120.71, 119.71, 57.38, 54.50, 53.93, 48.71, 48.23, 43.31, 41.19, 39.75,

39.39, 38.13, 37.33, 37.13, 30.22, 22.34, 20.32, 19.78, 18.86, 13.77; HRMS (ESI, m/z) calcd for

C₂₇H₃₇N₄O₄S [M+H]⁺513.2536. Found:513.2532.

4.2.3.19. Compound 4s. Yellow powder. Yield 50%; Mp 137.7–140.1 °C; IR (KBr): 3312, 3112, 2929,

2848, 1735, 1659, 1595, 1548, 1512, 1465, 1330, 1246, 1179, 1111, 1044, 973, 852, 731, 698, 587, 530,

493cm^-1; ¹H NMR (400MHz, CDCl₃, ppm): δ 10.54 (s, 1H), 9.61 (s, 1H), 9.19 (s, 1H), 8.19 (d, J = 9.08Hz,

2H), 7.71 (d, J = 9.04Hz, 2H), 2.58 (dd, J = 18.56, 3.28Hz, 1H), 2.18 (d, J = 14.28Hz, 1H), 1.95–1.38 (m,

12H), 1.33 (s, 3H), 1.29–1.21 (m, 5H), 0.97 (s, 3H), 0.94–0.92 (m, 1H), 0.77 (s, 3H); ¹³C NMR (100MHz,

CDCl₃, ppm): δ 222.45, 178.86, 171.27, 134.27, 134.01, 125.46, 125.46, 124.75, 124.75, 57.53, 54.57,

54.06, 48.70, 48.28, 43.49, 41.39, 39.80, 39.39, 38.07, 37.28, 37.16, 29.89, 22.01, 20.29, 19.79, 18.95,

13.77; HRMS (ESI, m/z) calcd for C₂₇H₃₇N₄O₄S [M+H]⁺513.2536. Found:513.2532.

4.2.3.20. Compound 4t. White powder. Yield 75%; Mp 126.4–127.9 °C; IR (KBr): 3309, 3087, 2929, 2847,

1735, 1659, 1581, 1530, 1468, 1370, 1336, 1295, 1231, 1152, 1103, 1050, 974, 861, 813, 780, 735,

573cm^-1; ¹H NMR (400MHz, CDCl₃, ppm): δ 10.75 (s, 1H), 9.95 (s, 1H), 8.82 (s, 1H), 7.65 (d, J = 7.88Hz,

1H), 7.47 (d, J = 2.28Hz, 1H), 7.30 (dd, J = 8.64, 2.36Hz, 1H), 2.57 (dd, J = 18.56, 3.40Hz, 1H), 2.12 (d, J

= 14.56Hz, 1H), 1.97–1.87 (m, 1H), 1.78–1.46 (m, 9H), 1.37–1.35 (m, 3H), 1.27 (s, 3H), 1.26–1.16 (m,

4H), 0.96 (s, 3H), 0.93–0.90 (m, 1H), 0.78 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.09, 176.70,

171.94, 133.74, 132.98, 131.25, 129.89, 129.65, 127.62, 57.44, 54.47, 53.95, 48.66, 48.20, 43.12, 41.22,

39.77, 39.29, 38.04, 37.16, 37.11, 30.13, 22.13, 20.28, 19.76, 18.74, 13.72; HRMS (ESI, m/z) calcd for

C₂₇H₃₆Cl₂N₃O₂S[M+H]⁺536.1905. Found: 536.1902.

4.2.3.21. Compound 4u. White powder. Yield 58%; Mp 126.2–127.3 °C; IR (KBr): 3279, 3037, 2926,

2847, 1738, 1632, 1523, 1469, 1372, 1337, 1236, 1163, 1111, 1090, 1036, 973, 922, 772, 735, 676, 584,

509cm^-1; ¹H NMR (400MHz, CDCl₃, ppm): δ 10.09 (s, 1H), 8.31 (s, 1H), 8.11 (dd, J = 8.32, 1.16Hz, 1H),

8.03 (s, 1H), 7.62 (t, J = 7.80Hz, 1H), 7.25 (d, J = 7.00Hz, 1H), 2.99 (s, 3H), 2.91 (s, 3H), 2.58 (dd, J =

18.64, 3.28Hz, 1H), 2.20 (d, J = 13.92Hz, 1H), 1.95–1.92 (m, 2H), 1.85–1.45 (m, 9H), 1.41–1.35 (m, 2H),

1.32 (s, 3H), 1.29–1.19 (m, 5H), 0.96 (s, 3H), 0.77 (s, 3H); ¹³C NMR (100MHz, CDCl₃, ppm): δ 222.57,

176.51, 174.15, 144.25, 144.05, 124.58, 124.58, 122.87, 122.87, 58.48, 57.38, 54.48, 53.92, 48.76, 48.30,

43.42, 41.27, 39.73, 39.43, 38.15, 37.35, 37.13, 30.12, 22.21, 20.33, 19.77, 18.92, 18.40, 13.74; HRMS

(ESI, m/z) calcd for C₂₉H₄₂N₃O₂S[M+H]⁺496.2998. Found: 496.2994.

4.2.4. General procedure for synthesis of compounds 5a-5u

Compound 4a-4u (1 mmol) was dissolved in EtOH (20 mL), and Hg(OAc)₂(0.351 g, 1.1 mmol) was

added.The mixture was stirred at reflux for 8 h. The reaction mixture was filtered and concentrated under

reduced pressure. The residue was diluted with saturated NaCl aqueous solution and extracted with

CH₃CO₂C₂H₅. The combined organic layer was dried over anhydrous sodium sulfate, filtered and

concentrated. The residue was purified on silica gel column to give the corresponding product 5a-5u.

4.2.4.1. Compound 5a. White solid. Yield 97%; Mp 115.2–116.3 °C; IR (KBr): 3298, 3137, 3087, 2925,

2849, 1735, 1626, 1599, 1559, 1499, 1451, 1373, 1319, 1243, 1172, 1132, 1082, 1049, 972, 856, 751, 692,

1

505cm^-1; H NMR (400MHz, CDCl_3,ppm): δ 8.27 (s, 1H), 7.46 (d, J = 7.68Hz, 2H), 7.32 (t, J = 7.74Hz,

2H), 7.04 (t, J = 7.30Hz, 1H), 2.58 (dd, J = 18.60, 3.56Hz, 1H), 2.51 (d, J = 13.40Hz, 1H), 2.12–1.97 (m,

2H), 1.78–1.51 (m, 9H), 1.44–1.34 (m, 3H), 1.31 (s, 3H), 1.27–1.01 (m, 4H), 0.97 (s, 3H), 0.52 (s, 3H);

¹³C NMR (100MHz, CDCl_3,ppm): δ 222.25, 165.43, 159.88, 138.17, 129.32, 129.32, 122.75, 117.38,

117.38, 57.06, 54.69, 54.27, 48.72, 48.44, 41.26, 39.58, 39.35, 38.26, 37.85, 37.51, 37.18, 30.01, 21.47,

20.16, 19.83, 18.51, 13.05; HRMS (ESI, m/z) calcd for C₂₇H₃₆N₃O₂[M+H]⁺434.2808. Found: 434.2804.

4.2.4.2. Compound 5b. White solid. Yield 70%; Mp 198.4–200.5 °C; IR (KBr): 3444, 3058, 2924, 2850,

1

1736, 1626, 1598, 1558, 1458, 1374, 1249, 1090, 975, 857, 750, 546cm^-1; H NMR (400MHz, CDCl_3,

ppm): δ 7.77 (d, J = 7.92Hz, 1H), 7.30 (s, 1H), 7.26 (d, J = 7.64Hz, 1H), 7.21 (t, J = 7.12Hz, 1H), 7.02 (td,

J = 7.40, 0.76Hz, 1H), 2.59 (dd, J = 18.60, 7.68Hz, 1H), 2.50 (d, J = 13.52Hz, 1H), 2.36 (s, 3H), 2.13–1.94

(m, 2H), 1.80–1.52 (m, 9H), 1.41–1.34 (m, 3H), 1.30 (s, 3H), 1.27–1.13 (m, 4H), 0.97 (s, 3H), 0.54 (s, 3H);

¹³C NMR (100MHz, CDCl_3,ppm): δ 222.22, 165.57, 160.28, 136.41, 130.70, 127.09, 126.52, 123.51,

118.68, 57.01, 54.67, 54.27, 48.71, 48.44, 41.23, 39.59, 39.35, 38.25, 37.85, 37.51, 37.19, 30.03, 21.42,

20.16, 19.85, 18.52, 17.83, 13.10; HRMS (ESI, m/z) calcd for C₂₈H₃₈N₃O₂[M+H]⁺448.2964. Found:

448.2960.

4.2.4.3. Compound 5c. White solid. Yield 37%; Mp 108.6–109.1 °C; IR (KBr):3295, 3090, 2929, 2850,

1737, 1629, 1601, 1561, 1493, 1454, 1374, 1318, 1256, 1229, 1131, 1052, 975, 932, 866, 778, 691cm^-1; ¹H

NMR (400MHz, CDCl_3,ppm): δ 7.33 (s, 1H), 7.23–7.17 (m, 3H), 6.88 (d, J = 6.80Hz, 1H), 2.58 (dd, J =

18.56, 3.56Hz, 1H), 2.50 (d, J = 13.48Hz, 1H), 2.34 (s, 3H), 2.11–1.98 (m, 2H), 1.79–1.53 (m, 9H),

1.48–1.35 (m, 3H), 1.32 (s, 3H), 1.22–1.13 (m, 4H), 0.97 (s, 3H), 0.53 (s, 3H); ¹³C NMR (100MHz, CDCl_3,

ppm): δ 222.16, 165.31, 159.61, 139.31, 137.65, 129.15, 123.92, 118.29, 114.78, 57.01, 54.66, 54.27,

48.70, 48.43, 41.24, 39.53, 39.32, 38.19, 37.82, 37.52, 37.14, 29.97, 21.58, 21.48, 20.14, 19.81, 18.43,

13.04; HRMS (ESI, m/z) calcd for C₂₈H₃₈N₃O₂[M+H]⁺448.2964. Found: 448.2960.

4.2.4.4. Compound 5d. White solid. Yield 25%; Mp 101.2–103.4 °C; IR (KBr): 3291, 3063, 2928, 2850,

1

1737, 1629, 1560, 1516, 1455, 1374, 1248, 1173, 1130, 1051, 974, 816, 506cm^-1; H NMR (400MHz,

CDCl_3,ppm): δ 7.88 (s, 1H), 7.32 (d, J = 8.44Hz, 2H), 7.13 (d, J = 8.32Hz, 2H), 2.58 (dd, J = 18.60,

3.64Hz, 1H), 2.50 (d, J = 13.48Hz, 1H), 2.31 (s, 3H), 2.04–1.96 (m, 2H), 1.79–1.52 (m, 9H), 1.45–1.34 (m,

3H), 1.31 (s, 3H), 1.28–1.19 (m, 4H), 0.97 (s, 3H), 0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ

222.26, 165.29, 159.97, 135.49, 132.39, 129.82, 129.82, 117.51, 117.51, 57.02, 54.66, 54.25, 48.70, 48.42,

41.24, 39.56, 39.33, 38.23, 37.82, 37.47, 37.16, 29.98, 21.44, 20.70, 20.14, 19.82, 18.49, 13.04; HRMS

(ESI, m/z) calcd for C₂₈H₃₈N₃O₂[M+H]⁺448.2964. Found: 448.2961.

4.2.4.5. Compound 5e. White solid. Yield 38%; Mp 91.7–93.6 °C; IR (KBr): 3286, 3070, 2929, 2848, 1735,

1625, 1591, 1559, 1495, 1458, 1373, 1337, 1255, 1131, 1049, 974, 928, 873, 794, 752, 572cm^-1; ¹H NMR

(400MHz, CDCl_3,ppm): δ 8.22 (td, J = 8.36, 1.32Hz, 1H), 7.16 (t, J = 7.92Hz, 1H), 7.14–7.09 (m, 1H),

7.02–6.97 (m, 1H), 2.60 (dd, J = 18.60, 3.68Hz, 1H), 2.51 (d, J = 13.56Hz, 1H), 2.17–1.98 (m, 2H),

1.82–1.53 (m, 9H), 1.45–1.36 (m, 3H), 1.32 (s, 3H), 1.30–1.20 (m, 4H), 0.97 (s, 3H), 0.53 (s, 3H); ¹³C

NMR (100MHz, CDCl_3,ppm): δ 222.16, 165.84, 158.89, 151.58 (d, J = 241.00Hz), 126.32 (d, J =

10.35Hz), 124.90 (d, J = 3.68Hz), 122.89 (d, J = 7.24Hz), 118.77, 114.85 (d, J = 18.34Hz), 57.01, 54.70,

54.27, 48.70, 48.47, 41.20, 39.54, 39.34, 38.24, 37.85, 37.49, 37.17, 29.99, 21.51, 20.15, 19.83, 18.49,

13.03; HRMS (ESI, m/z) calcd for C₂₇H₃₅FN₃O₂[M+H]⁺452.2713. Found: 452.2709.

4.2.4.6. Compound 5f. White solid. Yield 22%; Mp 118.7–121.5 °C; IR (KBr): 3280, 3152, 3094, 2927,

2849, 1735, 1614, 1559, 1493, 1454, 1373, 1256, 1173, 1149, 1051, 951, 862, 775, 682, 573cm^-1; ¹H NMR

(400MHz, CDCl_3,ppm): δ 8.70 (s, 1H), 7.33–7.28 (m, 2H), 7.14 (dd, J = 8.12, 1.40Hz, 1H), 6.74 (td, J =

8.32, 2.12Hz, 1H), 2.58 (dd, J = 18.60, 3.64Hz, 1H), 2.52 (d, J = 13.52Hz, 1H), 2.10–2.00 (m, 2H),

1.80–1.53 (m, 9H), 1.45–1.36 (m, 3H), 1.33 (s, 3H), 1.25–1.16 (m, 4H), 0.97 (s, 3H), 0.52 (s, 3H); ¹³C

NMR (100MHz, CDCl_3,ppm): δ 222.32, 165.69, 163.39 (d, J = 243.59Hz), 159.53, 139.62 (d, J =

10.89Hz), 130.55 (d, J = 9.45Hz), 112.91, 109.43 (d, J = 21.21Hz), 104.66 (d, J = 26.76Hz), 57.01, 54.64,

54.21, 48.72, 48.40, 41.20, 39.52, 39.34, 38.21, 37.84, 37.55, 37.15, 29.99, 21.50, 20.14, 19.81, 18.46,

13.04; HRMS (ESI, m/z) calcd for C₂₇H₃₅FN₃O₂[M+H]⁺452.2713. Found: 452.2710.

4.2.4.7. Compound 5g. White solid. Yield 58%; Mp 185.5–187.2 °C; IR (KBr): 3293, 3159, 3087, 2929,

2849, 1735, 1627, 1563, 1509, 1454, 1419, 1374, 1222, 1131, 1051, 973, 833, 807, 619, 555, 511cm^-1; ¹H

NMR (400MHz, CDCl_3,ppm): δ 8.29 (s, 1H), 7.41 (d, J = 9.04Hz, 1H), 7.40 (d, J = 9.08Hz, 1H), 7.04 (d,

J = 8.56Hz, 1H), 7.02 (d, J = 8.72Hz, 1H), 2.58 (dd, J = 18.56, 3.64Hz, 1H), 2.48 (d, J = 13.52Hz, 1H),

2.10–1.96 (m, 2H), 1.79–1.52 (m, 9H), 1.45–1.35 (m, 3H), 1.31 (s, 3H), 1.25–1.17 (m, 4H), 0.97 (s, 3H),

0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.30, 165.39, 159.91, 158.62 (d, J = 240.55Hz),

134.17, 119.12 (d, J = 7.83Hz), 119.12 (d, J = 7.83Hz), 115.99 (d, J = 22.61Hz), 115.99 (d, J = 22.61Hz),

57.01, 54.64, 54.23, 48.73, 48.42, 41.23, 39.53, 39.34, 38.21, 37.83, 37.51, 37.16, 29.99, 21.45, 20.15,

19.83, 18.48, 13.05; HRMS (ESI, m/z) calcd for C₂₇H₃₅FN₃O₂[M+H]⁺452.2713. Found: 452.2710.

4.2.4.8. Compound 5h. White solid. Yield 26%; Mp 186.4–188.4 °C; IR (KBr): 3360, 3074, 2920, 2849,

1731, 1616, 1552, 1523, 1474, 1444, 1399, 1373, 1309, 1269, 1229, 1170, 1131, 1110, 1052, 974, 927, 857,

744, 711, 465cm^-1; ¹H NMR (400MHz, CDCl_3,ppm): δ 8.33 (dd, J = 8.32, 1.36Hz, 1H), 7.40 (dd, J = 8.00,

1.40Hz, 1H), 7.32 (td, J = 8.72, 1.44Hz, 1H), 7.00 (td, J = 7.70, 1.44Hz, 1H), 2.61 (dd, J = 18.56, 3.72Hz,

1H), 2.51 (d, J = 13.56Hz, 1H), 2.17–1.99 (m, 2H), 1.83–1.53 (m, 9H), 1.45–1.35 (m, 3H), 1.33 (s, 3H),

1.31–1.15 (m, 4H), 0.97 (s, 3H), 0.54 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.09, 165.94, 158.73,

134.38, 129.14, 128.13, 123.17, 120.84, 118.20, 56.97, 54.70, 54.26, 48.68, 48.47, 41.16, 39.50, 39.31,

38.27, 37.84, 37.50, 37.15, 30.01, 21.53, 20.13, 19.82, 18.50, 13.06; HRMS (ESI, m/z) calcd for

C₂₇H₃₅ClN₃O₂[M+H]⁺468.2418. Found: 468.2416.

4.2.4.9. Compound 5i. White solid. Yield 19%; Mp 116.8–118.6 °C; IR (KBr): 3282, 3088, 2926, 2849,

1735, 1626, 1597, 1557, 1481, 1452, 1372, 1317, 1253, 1131, 1051, 974, 909, 871, 776, 733, 683, 619,

572cm^-1; ¹H NMR (400MHz, CDCl_3,ppm): δ 8.96 (s, 1H), 7.55 (s, 1H), 7.26–7.24 (m, 2H), 7.03–7.00 (m,

1H), 2.56 (dd, J = 18.60, 3.64Hz, 1H), 2.52 (d, J = 13.56Hz, 1H), 2.13–2.01 (m, 2H), 1.82–1.53 (m, 9H),

1.45–1.34 (m, 3H), 1.33 (s, 3H), 1.26–1.13 (m, 4H), 0.97 (s, 3H), 0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,

ppm): δ 222.36, 165.70, 159.64, 139.25, 135.04, 130.37, 122.73, 117.18, 115.63, 57.02, 54.64, 54.18,

48.72, 48.43, 41.21, 39.53, 39.36, 38.18, 37.83, 37.57, 37.14, 29.98, 21.57, 20.14, 19.81, 18.43, 13.02;

HRMS (ESI, m/z) calcd for C₂₇H₃₅ClN₃O₂[M+H]⁺468.2418. Found: 468.2416.

4.2.4.10. Compound 5j. White solid. Yield 10%; Mp 126.7–129.2 °C; IR (KBr): 3286, 3077, 2923, 2849,

1735, 1624, 1558, 1493, 1454, 1409, 1373, 1320, 1245, 1173, 1130, 1093, 1051, 972, 925, 828, 732,

504cm^-1; ¹H NMR (400MHz, CDCl_3,ppm): δ 8.72 (s, 1H), 7.41 (d, J = 8.88Hz, 2H), 7.29 (d, J = 8.88Hz,

2H), 2.57 (dd, J = 18.56, 3.56Hz, 1H), 2.48 (d, J = 13.52Hz, 1H), 2.09–1.97 (m, 2H), 1.79–1.52 (m, 9H),

1.43–1.36 (m, 3H), 1.31 (s, 3H), 1.26–1.12 (m, 4H), 0.97 (s, 3H), 0.51 (s, 3H); ¹³C NMR (100MHz, CDCl_3,

ppm): δ 222.36, 165.55, 159.60, 136.69, 129.29, 129.29, 127.70, 118.57, 118.57, 56.97, 54.59, 54.18,

48.71, 48.40, 41.18, 39.48, 39.32, 38.19, 37.80, 37.50, 37.13, 29.97, 21.43, 20.12, 19.80, 18.45, 13.03;

HRMS (ESI, m/z) calcd for C₂₇H₃₅ClN₃O₂[M+H]⁺468.2418. Found: 468.2413.

4.2.4.11. Compound 5k. White solid. Yield 91%; Mp 196.8–198.5 °C; IR (KBr): 3389, 3289, 3066, 2929,

2847, 1735, 1618, 1555, 1443, 1401, 1371, 1314, 1268, 1129, 1049, 1024, 972, 928, 750, 702, 572cm^-1; ¹H

NMR (400MHz, CDCl_3,ppm): δ 8.32 (dd, J = 8.32, 1.40Hz, 1H), 7.56 (dd, J = 8.00, 1.36Hz, 1H), 7.36 (td,

J = 7.86, 1.44Hz, 1H), 6.93 (td, J = 7.68, 1.48Hz, 1H), 2.61 (dd, J = 18.60, 3.72Hz, 1H), 2.50 (d, J =

13.56Hz, 1H), 2.15–1.99 (m, 2H), 1.83–1.53 (m, 9H), 1.41–1.35 (m, 3H), 1.33 (s, 3H), 1.31–1.13 (m, 4H),

0.97 (s, 3H), 0.54 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.16, 165.98, 158.79, 135.50, 132.43,

128.82, 123.70, 118.41, 111.31, 56.98, 54.70, 54.27, 48.69, 48.48, 41.17, 39.51, 39.33, 38.28, 37.85, 37.52,

37.17, 30.04, 21.53, 20.15, 19.84, 18.53, 13.09; HRMS (ESI, m/z) calcd for C₂₇H₃₅BrN₃O₂[M+H]⁺

512.1913. Found: 512.1908.

4.2.4.12. Compound 5l. White solid. Yield 88%; Mp 126.2–128.4 °C; IR (KBr): 3282, 3090, 2934, 2849,

1

1735, 1625, 1595, 1556, 1480, 1454, 1373, 1319, 1251, 1172, 1051, 975, 894, 774, 681cm^-1; H NMR

(400MHz, CDCl_3,ppm): δ 7.69 (s, 1H), 7.28–7.25 (m, 1H), 7.20–7.19 (m, 2H), 2.56 (dd, J = 18.60, 3.56Hz,

1H), 2.50 (d, J = 13.52Hz, 1H), 2.06–2.01 (m, 2H), 1.84–1.51 (m, 10H), 1.41–1.35 (m, 3H), 1.33 (s, 3H),

1.26–1.12 (m, 3H), 0.97 (s, 3H), 0.51 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.32, 165.65, 159.83,

138.99, 130.71, 126.00, 123.04, 120.16, 116.46, 56.99, 54.60, 54.12, 48.70, 48.40, 41.15, 39.47, 39.35,

38.06, 37.81, 37.60, 37.11, 29.91, 21.58, 20.12, 19.80, 18.38, 13.02; HRMS (ESI, m/z) calcd for

C₂₇H₃₅BrN₃O₂[M+H]⁺512.1913. Found: 512.1907.

4.2.4.13. Compound 5m. White solid. Yield 95%; Mp 133.1–135.2 °C; IR (KBr): 3284, 3186, 3124, 3074,

2935, 2849, 1735, 1624, 1594, 1558, 1491, 1454, 1407, 1373, 1319, 1245, 1175, 1131, 1074, 1051, 1009,

974, 825, 501cm^-1; ¹H NMR (400MHz, CDCl_3,ppm): δ 8.45 (s, 1H), 7.44 (d, J = 8.88Hz, 2H), 7.36 (d, J =

8.88Hz, 2H), 2.58 (dd, J = 18.52, 3.56Hz, 1H), 2.49 (d, J = 13.52Hz, 1H), 2.12–1.97 (m, 2H), 1.79–1.52

(m, 9H), 1.45–1.36 (m, 3H), 1.31 (s, 3H), 1.26–1.12 (m, 4H), 0.97 (s, 3H), 0.51 (s, 3H); ¹³C NMR

(100MHz, CDCl_3,ppm): δ 222.33, 165.60, 159.49, 137.16, 132.22, 132.22, 118.90, 118.90, 115.13, 56.97,

54.60, 54.19, 48.71, 48.40, 41.18, 39.48, 39.32, 38.19, 37.81, 37.49, 37.13, 29.98, 21.44, 20.12, 19.81,

18.45, 13.03; HRMS (ESI, m/z) calcd for C₂₇H₃₅BrN₃O₂[M+H]⁺512.1913. Found: 512.1908.

4.2.4.14. Compound 5n. White solid. Yield 44%; Mp 162.3–164.1 °C; IR (KBr): 3400, 3082, 2957, 2909,

2842, 1738, 1624, 1592, 1559, 1528, 1488, 1462, 1345, 1249, 1216, 1176, 1120, 974, 928, 748, 475cm^-1;

¹H NMR (400MHz, CDCl_3,ppm): δ 8.10 (t, J = 4.62Hz, 1H), 7.52 (s, 1H), 7.02–6.98 (m, 2H), 6.93–6.89

(m, 1H), 3.94 (s, 3H), 2.60 (dd, J = 18.60, 3.68Hz, 1H), 2.51 (d, J = 13.52Hz, 1H), 2.17–1.99 (m, 2H),

1.81–1.52 (m, 9H), 1.42–1.35 (m, 3H), 1.31 (s, 3H), 1.24–1.13 (m, 4H), 0.97 (s, 3H), 0.53 (s, 3H); ¹³C

NMR (100MHz, CDCl_3,ppm): δ 222.27, 165.36, 159.40, 146.99, 127.35, 124.41, 121.27, 116.78, 109.98,

57.00, 55.76, 54.67, 54.25, 48.69, 48.45, 41.21, 39.54, 39.31, 38.24, 37.82, 37.43, 37.15, 29.97, 21.49,

20.12, 19.82, 18.50, 13.01; HRMS (ESI, m/z) calcd for C₂₈H₃₈N₃O₃[M+H]⁺464.2913. Found: 464.2910.

4.2.4.15. Compound 5o. White solid. Yield 57%; Mp 110.5–113.1 °C; IR (KBr): 3292, 3091, 2937, 2849,

1

1735, 1629, 1599, 1559, 1497, 1456, 1285, 1161, 975, 852, 771, 689cm^-1; H NMR (400MHz, CDCl_3,

ppm): δ 8.76 (s, 1H), 7.21 (t, J = 8.18Hz, 1H), 7.18 (s, 1H), 6.92 (d, J = 8.00Hz, 1H), 6.60 (dd, J = 8.12,

1.48Hz, 1H), 3.82 (s, 3H), 2.58 (dd, J = 18.56, 3.28Hz, 1H), 2.50 (d, J = 13.44Hz, 1H), 2.14–1.99 (m, 2H),

1.79–1.52 (m, 9H), 1.48–1.35 (m, 3H), 1.32 (s, 3H), 1.26–1.13 (m, 4H), 0.97 (s, 3H), 0.53 (s, 3H); ¹³C

NMR (100MHz, CDCl_3,ppm): δ 222.39, 165.47, 160.58, 159.91, 139.38, 130.07, 109.51, 108.68, 102.94,

57.01, 55.29, 54.65, 54.23, 48.73, 48.45, 41.24, 39.55, 39.34, 38.25, 37.84, 37.52, 37.16, 30.02, 21.52,

20.15, 19.84, 18.50, 13.05; HRMS (ESI, m/z) calcd for C₂₈H₃₈N₃O₃[M+H]⁺464.2913. Found: 464.2910.

4.2.4.16. Compound 5p. White solid. Yield 64%; Mp 71.8–74.3 °C; IR (KBr): 3284, 3072, 2934, 2849,

1

1735, 1628, 1563, 1512, 1456, 1373, 1242, 1176, 1035, 974, 829, 592, 518cm^-1; H NMR (400MHz,

CDCl_3,ppm): δ 7.89 (s, 1H), 7.36 (d, J = 8.96Hz, 2H), 6.88 (d, J = 9.00Hz, 2H), 3.79 (s, 3H), 2.57 (dd, J =

18.56, 3.60Hz, 1H), 2.48 (d, J = 13.48Hz, 1H), 2.12–1.94 (m, 2H), 1.78–1.50 (m, 9H), 1.44–1.33 (m, 3H),

1.29 (s, 3H), 1.24–1.13 (m, 4H), 0.97 (s, 3H), 0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.36,

165.17, 160.26, 155.57, 131.31, 119.50, 119.50, 114.50, 114.50, 57.00, 55.55, 54.62, 54.21, 48.71, 48.41,

41.22, 39.55, 39.32, 38.19, 37.81, 37.44, 37.15, 29.97, 21.40, 20.13, 19.82, 18.47, 13.03; HRMS (ESI, m/z)

calcd for C₂₈H₃₈N₃O₃[M+H]⁺464.2913. Found: 464.2913.

4.2.4.17. Compound 5q. Pale yellow solid. Yield 50%; Mp 163.6–165.3 °C; IR (KBr): 3325, 2941, 2849,

1

1735, 1691, 1600, 1551, 1505, 1448, 1347, 1268, 1149, 1054, 979, 878, 783, 743, 584cm^-1; H NMR

(400MHz, CDCl_3,ppm): δ 10.58 (s, 1H), 8.80 (d, J = 8.60Hz, 1H), 8.31 (d, J = 8.52Hz, 1H), 7.73 (t, J =

7.88Hz, 1H), 7.14 (t, J = 7.86Hz, 1H), 2.59 (dd, J = 18.64, 2.64Hz, 1H), 2.51 (d, J = 13.84Hz, 1H),

2.12–2.01 (m, 2H), 1.85–1.58 (m, 9H), 1.44–1.38 (m, 3H), 1.35 (s, 3H), 1.32–1.12 (m, 4H), 0.98 (s, 3H),

0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.19, 166.71, 158.06, 136.80, 135.15, 134.49, 126.18,

121.81, 119.85, 56.96, 54.69, 54.23, 48.66, 48.47, 41.05, 39.43, 39.31, 38.21, 37.84, 37.55, 37.15, 30.00,

21.53, 20.13, 19.81, 18.49, 13.03; HRMS (ESI, m/z) calcd for C₂₇H₃₅N₄O₄[M+H]⁺479.2658. Found:

479.2655.

4.2.4.18. Compound 5r. Pale yellow solid. Yield 41%; Mp 117.3–118.5 °C; IR (KBr): 3270, 3099, 2935,

2851, 1735, 1615, 1560, 1531, 1454, 1352, 1251, 1173, 1092, 1052, 977, 939, 886, 799, 737cm^-1; ¹H NMR

(400MHz, CDCl_3,ppm): δ 10.18 (s, 1H), 8.40 (s, 1H), 7.90 (d, J = 8.04Hz, 1H), 7.79 (dd, J = 8.08, 1.64Hz,

1H), 7.52 (t, J = 8.16Hz, 1H), 2.60–2.54 (m, 2H), 2.14–2.11 (m, 2H), 1.86–1.57 (m, 9H), 1.46–1.39 (m,

3H), 1.37 (s, 3H), 1.28–1.02 (m, 4H), 0.98 (s, 3H), 0.54 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ

222.77, 166.23, 159.60, 148.99, 139.52, 130.32, 123.39, 117.19, 110.97, 57.06, 54.63, 54.13, 48.77, 48.40,

41.16, 39.54, 39.45, 38.22, 37.89, 37.71, 37.18, 30.00, 21.74, 20.18, 19.82, 18.45, 13.03; HRMS (ESI, m/z)

calcd for C₂₇H₃₅N₄O₄[M+H]⁺479.2658. Found: 479.2656.

4.2.4.19. Compound 5s. Pale yellow solid. Yield 79%; Mp 128.3–128.9 °C; IR (KBr): 3270, 3104, 2936,

1

2851, 1734, 1625, 1592, 1554, 1514, 1453, 1334, 1260, 1177, 1111, 1052, 975, 851, 750, 691cm^-1; H

NMR (400MHz, CDCl_3,ppm): δ 9.67 (s, 1H), 8.25 (d, J = 9.08Hz, 2H), 7.62 (d, J = 9.08Hz, 2H), 2.57 (dd,

J = 18.64, 3.20Hz, 1H), 2.52 (d, J = 13.92Hz, 1H), 2.12–2.01 (m, 2H), 1.79–1.56 (m, 9H), 1.46–1.37 (m,

3H), 1.35 (s, 3H), 1.27–1.01 (m, 4H), 0.98 (s, 3H), 0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ

222.70, 166.39, 158.89, 143.94, 142.30, 125.67, 125.67, 116.57, 116.57, 56.99, 54.55, 54.14, 48.80, 48.45,

41.16, 39.43, 39.37, 38.18, 37.84, 37.63, 37.14, 29.99, 21.44, 20.16, 19.81, 18.43, 13.08; HRMS (ESI, m/z)

calcd for C₂₇H₃₅N₄O₄[M+H]⁺479.2658. Found: 479.2655.

4.2.4.20. Compound 5t. White solid. Yield 61%; Mp 96.0–97.8 °C; IR (KBr): 3281, 3097, 2933, 2849,

1736, 1619, 1556, 1531, 1474, 1455, 1396, 1315, 1131, 1102, 1048, 976, 866, 822, 767, 696cm^-1; ¹H NMR

(400MHz, CDCl_3,ppm): δ 8.34 (d, J = 8.88Hz, 1H), 7.41 (s, 1H), 7.29 (d, J = 9.34Hz, 1H), 2.60 (dd, J =

18.64, 2.96Hz, 1H), 2.49 (d, J = 13.24Hz, 1H), 2.17–1.99 (m, 2H), 1.83–1.56 (m, 10H), 1.44–1.36 (m, 3H),

1.32 (s, 3H), 1.25–1.13 (m, 3H), 0.97 (s, 3H), 0.53 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 222.17,

166.14, 158.47, 133.17, 128.83, 128.25, 127.57, 121.33, 119.08, 56.96, 54.68, 54.25, 48.70, 48.49, 41.15,

39.48, 39.33, 38.26, 37.85, 37.54, 37.15, 30.03, 21.54, 20.15, 19.83, 18.49, 13.09; HRMS (ESI, m/z) calcd

for C₂₇H₃₄Cl₂N₃O₂[M+H]⁺502.2028. Found: 502.2024.

4.2.4.21. Compound 5u. White solid. Yield 42%; Mp 248.6–250.3 °C; IR (KBr): 3176, 3062, 2941, 2847,

1

1738, 1629, 1563, 1450, 1375, 1255, 1214, 1110, 1028, 976, 769, 694, 591cm^-1; H NMR (400MHz,

CDCl_3,ppm): δ 7.10 (s, 3H), 2.42–2.37 (m, 2H), 2.26 (s, 6H), 1.99–1.89 (m, 1H), 1.69–1.34 (m, 11H),

1.30–1.23 (m, 2H), 1.21 (s, 3H), 1.17–1.14 (m, 4H), 0.95 (s, 3H), 0.38 (s, 3H); ¹³C NMR (100MHz, CDCl_3,

ppm): δ 222.48, 165.29, 162.35, 136.03, 136.03, 134.98, 128.40, 128.40, 127.37, 57.11, 54.55, 54.20,

48.67, 48.28, 41.19, 39.60, 39.27, 37.86, 37.71, 37.29, 37.17, 29.57, 20.79, 20.11, 19.83, 18.38, 18.24,

18.23, 12.56; HRMS (ESI, m/z) calcd for C₂₉H₄₀N₃O₂[M+H]⁺462.3121. Found: 462.3116.

4.2.5. Compound 6

Phenyl isocyanate (0.131 g, 1.1 mmol) was added to a solution of compound 3 (0.332 g, 1 mmol) in

CH₂Cl₂at room temperature. After stirring for 1h, the solvent was removed under vacuo. The mixture was

extracted with CH₃CO₂C₂H₅and H₂O, and the organic layer washed with saturated NaCl aqueous solution,

dried with anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by

column chromatography on silica (petroleum ether/ethyl acetate = 5:1, v/v) to give the compound 6 as a

white powder. Yield 96%; Mp 118.6–120.3 °C; IR (KBr): 3266, 3062, 2929, 2848, 1738, 1635, 1509, 1468,

1373, 1314, 1242, 1087, 1110, 1045, 976, 929, 899, 861, 799, 711, 588, 508, 449cm^-1; ¹H NMR (400MHz,

CDCl_3,ppm): δ 10.19 (s, 1H), 8.79 (s, 1H), 7.84 (d, J = 7.28Hz, 2H), 7.49 (t, J = 7.42Hz, 1H), 7.36 (t, J =

7.70Hz, 2H), 2.59 (dd, J = 18.60, 3.64Hz, 1H), 2.16 (d, J = 14.56Hz, 1H), 1.97–1.78 (m, 3H), 1.74–1.59

(m, 5H), 1.55–1.34 (m, 5H), 1.24 (s, 3H), 1.20–1.11 (m, 4H), 0.97 (s, 3H), 0.92–0.88 (m, 1H), 0.78 (s, 3H);

¹³C NMR (100MHz, CDCl_3,ppm): δ 222.43, 174.43, 164.45, 132.20, 131.17, 128.51, 128.51, 127.33,

127.33, 57.71, 54.64, 54.16, 48.69, 48.33, 43.36, 41.57, 39.94, 39.42, 38.08, 37.28, 37.22, 30.00, 21.96,

20.29, 19.84, 19.12, 13.65; HRMS (ESI, m/z) calcd for C₂₇H₃₈N₃O₃[M+H]⁺452.2913. Found: 452.2916.

4.2.6. Compound 7

NaBH₄(0.244 g, 2 mmol) was added to a solution of compound 5a (0.433 g, 1 mmol) in EtOH at room

temperature for 3h. After completion the solvent was removed in vacuo. The residue was diluted with

saturated NaCl aqueous solution and extracted with CH₃CO₂C₂H₅. The combined organic layer was dried

over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column

chromatography on silica (petroleum ether/ethyl acetate = 2:1, v/v) to give the compound 7 as a white solid.

Yield 98%; Mp 129.5–130.7 °C; IR (KBr): 3253, 3139, 3055, 2935, 2844, 1720, 1628, 1599, 1560, 1499,

1

1451, 1373, 1321, 1246, 1171, 1147, 1118, 1052, 998, 971, 897, 749, 692, 632, 570, 502cm^-1; H NMR

(400MHz, CDCl_3,ppm): δ 7.87 (s, 1H), 7.45 (d, J = 8.52Hz, 2H), 7.33 (t, J = 7.98Hz, 2H), 7.03 (t, J =

7.36Hz, 1H), 3.85 (dd, J = 10.56, 4.64Hz, 1H), 2.49 (d, J = 13.48Hz, 1H), 2.13–2.06 (m, 1H), 1.89 (d, J =

12.40Hz, 1H), 1.78–1.66 (m, 4H), 1.55–1.32 (m, 8H), 1.25 (s, 3H), 1.24–0.93 (m, 6H), 0.90 (s, 3H), 0.55

(s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 165.79, 159.69, 138.10, 129.30, 129.30, 122.65, 117.27,

117.27, 80.51, 57.10, 55.76, 55.19, 42.87, 42.01, 41.97, 41.49, 39.68, 38.29, 37.88, 37.48, 33.58, 30.01,

24.86, 21.51, 20.27, 18.47, 12.93; HRMS (ESI, m/z) calcd for C₂₇H₃₈N₃O₂[M+H]⁺436.2964. Found:

436.2960.

4.2.7. Compound 8

A mixture of compound 7 (0.435 g, 1 mmol) and acetic anhydride (0.306 g, 3 mmol) in pyridine was

stirred at room temperature with stirring until completion of the reaction as monitored by TLC and then

concentrated under reduced pressure. Saturated NaCl aqueous solution was added and extracted with

CH₂Cl₂. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude

product was purified by column chromatography on silica (petroleum ether / ethyl acetate = 5:1, v/v) to

afford the pure product 8 as a white solid. Yield 97%; Mp 245.2–247.1 °C; IR (KBr): 3255, 3060, 2942,

2848, 1735, 1633, 1602, 1566, 1501, 1453, 1370, 1248, 1173, 1146, 1118, 1060, 1034, 974, 749, 690,

1

635cm^-1; H NMR (400MHz, CDCl_3,ppm): δ 8.00 (s, 1H), 7.47 (d, J = 7.72Hz, 2H), 7.33 (t, J = 7.98Hz,

2H), 7.04 (t, J = 7.36Hz, 1H), 4.69 (dd, J = 10.36, 4.60Hz, 1H), 2.51 (d, J = 13.40Hz, 1H), 2.12–2.08 (m,

1H), 2.02 (s, 3H), 1.90–1.72 (m, 5H), 1.62–1.32 (m, 8H), 1.28 (s, 3H), 1.25–1.20 (m, 2H), 1.08–0.92 (m,

3H), 0.90 (s, 3H), 0.52 (s, 3H); ¹³C NMR (100MHz, CDCl_3,ppm): δ 171.60, 165.71, 159.75, 138.15,

129.30, 129.30, 122.62, 117.23, 117.23, 81.67, 57.11, 55.55, 54.68, 42.23, 41.42, 41.28, 40.77, 39.72,

38.25, 37.90, 37.47, 34.43, 29.87, 24.88, 21.48, 21.21, 20.06, 18.41, 12.96; HRMS (ESI, m/z) calcd for

C₂₉H₄₀N₃O₃[M+H]⁺478.3070. Found: 478.3067.

4.3. General procedure for cytotoxicity assay

Cytotoxicity assay in vitro: HCT-116, HGC-27 and JEKO-1 cells were cultured in RPMI-1640 medium

(GIBCO：31800-022) supplemented with 10% FBS, 100 U/mL of penicillin and 100 mg/mL of

streptomycin at 37 ^oC in a 5% CO₂humidified atmosphere. Cell cytotoxicity was assayed by MTT method.

o

Briefly, cells were seeded in 96-well tissue culture plates. After 24 h incubation at 37 C, 5% CO₂,

removed the culture medium and replaced with fresh medium containing the studied compounds in

different concentrations to the wells, and the cells were incubated for another 72 h. Afterwards, the MTT

(MP: 102227) solution (0.5 mg/mL) was added and incubated for an additional 4 h. Two hundred

microliters of DMSO was added to each well to dissolve the reduced MTT crystals. Optical density of each

well was measured at 492/630 nm with enzyme immunoassay instrument (TECAN: Infinite 200 Pro). Then

the inhibitory percentage of each compound at various concentrations to the cell proliferation was

calculated, and the IC₅₀value was determined.

4.4. Computational studies

The computational studies containing 2D-HQSAR and 3D-Topomer CoMFA were carried out by using the

SYBYL (Tripos Inc., St. Louis, USA) following the standard procedures provided in the manual.

2D-HQSAR was run from SYBYL using default parameters: hologram lengths (53, 59, 61, 71, 83, 97, 151,

199, 257, 307, 353 and 401), atom count in fragments (min = 4 and max = 7) were used. The best model

was selected on the basis of best crossvalideted r². Two options dialog boxes, which are show statistics for

model ensemble and retain fragment information for preditiction statistics, were checked. For the

3D-topomer CoMFA, all the training set compounds were automatically divided into R₁and R₂fragments

and followed by automated PLS analysis, fragment alignment and activity predictions. Statistical

parameters such as q², r², number of components were generated. Standard errors of prediction were

recorded for both sets and steric and electrostatic contour maps were obtained.

Acknowledgments

We look forward to financial support from the National Natural Science Foundation of China (Project No.

20772113), Scientific Research Fund of Xinxiang Medical University (2014QN148) and the Doctoral

Foundation of Henan Institute of Engineering (D09002).

Supplementary data

Supporting information file related to this article is named as Appendix S1 which can be found at XXX

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Figure legends

Synthesis, cytotoxic activity, 2D- and 3D-QSAR studies of 19-carboxyl modified

novel isosteviol derivatives as potential anticancer agents

Cong-Jun Liu ^a, Tao Zhang ^b, Shu-Ling Yu ^c, Xing-Jie Dai ^a, Ya Wu ^a, Jing-Chao Tao* ^a

a

College of Chemistry and Molecular Engineering, New Drug Research & Development Center,

Zhengzhou University, 75 Daxue Road, Zhengzhou, Henan 450052, China

^bSchool of Pharmacy, Xinxiang Medical University, Xinxiang 453003, Henan, China

c

Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province, Henan University,

Kaifeng, Henan 475004, China

Fig. 1. Chemical structures of compounds A-E

Fig. 2. Fragmentation pattern (R₁and R₂) for all molecules of dataset in 3D-topomer CoMFA analysis

Fig. 3. The histogram of residuals between experimental values versus predicted values of pIC₅₀of the test

set in the 2D-HQSAR and 3D-topomer CoMFA model

Fig. 4. Scatter plots of experimental values versus predicted values of pIC₅₀of the training and test set

from 2D-HQSAR (A) and 3D-topomer CoMFA (B) analyses, respectively.

Fig. 5. The 2D-HQSAR contribution maps of compounds 4s, 4u, 5f and 5t

Fig. 6. Contour maps of 3D-topomer CoMFA. (A) Steric contour map depicted around R₁; (B) electrostatic

contour map depicted around R₁; (C) Steric contour map depicted around R₂; (D) electrostatic contour map

depicted around R₂.

.

Scheme 1. Reagents and conditions: (i) oxalyl chloride, DMF, DCM, rt, 8h; (ii) N₂H₄H₂O, DCM, -10 ^oC, 1

h, 89%; (iii) isothiocyanates, DCM, rt, 0.5h; (iv) Hg(OAc)₂, EtOH, reflux, 5h.

Scheme 2. Reagents and conditions: (i) phenyl isocyanate, DCM, rt, 1h, 96%; (ii) NaBH₄, EtOH, rt, 3h,

98%; (iii) Ac₂O, pyridine, rt, 6h, 97%.

Tables:

Synthesis, cytotoxic activity, 2D- and 3D-QSAR studies of 19-carboxyl modified

novel isosteviol derivatives as potential anticancer agents

Cong-Jun Liu ^a, Tao Zhang ^b, Shu-Ling Yu ^c, Xing-Jie Dai ^a, Ya Wu ^a, Jing-Chao Tao* ^a

a

College of Chemistry and Molecular Engineering, New Drug Research & Development Center,

Zhengzhou University, 75 Daxue Road, Zhengzhou, Henan 450052, China

^bSchool of Pharmacy, Xinxiang Medical University, Xinxiang 453003, Henan, China

c

Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province, Henan University,

Kaifeng, Henan 475004, China

Table 1. Cytotoxic activities of isosteviol derivatives in vitro from different cancer types ^a

Cytotoxic activities (IC_50,μM)^b,c

Cd

HCT-11

>100

HGC-27

>100

JEKO-1

>100

HCT-11

8.56±0.4

7.92±0.9

HGC-27

9.87±0.4

JEKO-1

1

5b

5c

5d

5e

5f

5g

5h

5i

16.51±1.1

3

15.20±0.5 27.42±0.9 5.17±0.3

3.43±0.2 6.72±0.7 3.66±0.3

11.61±0.9 12.04±0.3 10.18±0.5

12.47±1.1 29.34±1.1

4a

4b

4c

4d

4e

4f

4g

4h

4i

14.00±0.5 14.54±0.9 36.61±3.8

7.17±0.5

5.00±0.6

7.56±0.4

9.65±0.3

3.39±0.2

3.75±0.4

3.92±0.1

3.69±0.3

1.84±0.1

2.40±0.5

5.14±0.8

1.62±0.1

2.07±0.3

8.26±0.4

1.35±0.3

6.15±0.6

6.78±0.2

7.19±0.9

3.11±0.3

5.33±0.9

4.21±0.1

9.08±0.3

11.62±1.1

3.15±0.3

4.85±0.1

11.98±1.0 3.73±0.3

6.22±0.1

2.50±0.1

14.70±0.7 7.49±0.1

16.21±0.8

18.78±0.3 17.18±1.2 10.95±0.5

11.20±0.2 16.84±1.7 15.56±0.8

12.26±0.9 11.68±0.4 24.53±1.2

12.89±0.9 20.25±1.2 19.00±1.3

10.22±0.8 15.60±1.3 27.83±0.9

5j

11.31±0.7 21.77±1.6

5k

5l

2.89±0.3

4.95±0.1

3.36±0.3

7.77±0.8

4j

5m

5n

5o

5p

5q

5r

5s

5t

4k

4l

10.78±1.1 14.76±0.7

6.43±0.3

12.45±0.5 11.41±0.3 16.42±0.5

8.69±0.7 10.33±0.5 39.76±2.3

12.22±0.4 22.77±0.1

2.31±0.1

2.94±0.1

9.19±0.8

4.91±0.8

7.24±0.6

3.08±0.4

3.20±0.2

25.00±2.3

10.45±0.6

14.08±0.1

4m 1.60±0.1

4n

4o

4p

4q

4r

8.71±0.9

3.62±0.6

6.25±0.6

9.36±0.4

14.95±0.2 26.00±1.3 39.92±0.6

10.14±0.3 14.67±1.1 31.89±0.8

6.40±0.1

8.54±0.1

31.72±1.3

24.33±0.4 31.86±2.7

32.91±1.6 18.22±0.5 76.52±3.6

27.17±0.9 16.73±0.5 91.80±13.2

16.74±1.3 12.53±0.5 23.64±0.6

1.134±0.1 2.77±0.1

2.57±0.1

2.96±0.3

13.57±1.3

5u

6

4s

4t

0.95±0.1

3.08±0.2

2.65±0.2

9.82±0.7

7

8.39±0.7

17.51±0.8 57.24±2.5

4u

5a

13.61±0.9 17.12±1.0 27.15±1.2

9.56±0.9 11.64±0.3 32.08±2.8

8

15.06±1.3 17.50±0.8 52.53±2.4

Cisplatin 3.91±0.3 2.03±0.1 2.74±0.2

^aHTC-116 is colon-derived; HGC-27 is gastric-derived and JEKO-1 is mantle cell lymphoma-

derived

^bValues are the mean of triplicate of three independent experiments

^c±SD

Table 2. 2D-HQSAR analysis for various fragment distinction using default fragment size (4–7)

model

1

Fragment distinction

A/B

q²

SEcv

0.294

0.310

0.281

0.280

0.289

0.286

0.274

0.322

0.262

0.286

0.279

0.284

0.262

0.288

0.278

r²

SEE

HL

199

307

401

61

N

2

3

4

5

3

4

6

3

4

5

3

6

0.413

0.368

0.498

0.516

0.451

0.480

0.522

0.382

0.589

0.462

0.487

0.485

0.577

0.455

0.539

0.636

0.683

0.756

0.824

0.678

0.750

0.733

0.897

0.849

0.640

0.705

0.754

0.837

0.636

0.849

0.232

0.220

0.196

0.169

0.221

0.198

0.205

0.131

0.159

0.234

0.212

0.197

0.163

0.235

0.159

2

A/B/C

3

A/B/H

4

A/B/Ch

5

A/B/DA

401

353

53

6

A/B/C/H

7

A/B/H/Ch

A/B/C/Ch

A/B/H/DA

A/B/C/DA

A/B/Ch/DA

A/B/C/H/Ch

A/B/H/Ch/DA

A/B/C/Ch/DA

A/B/C/H/Ch/DA

8

401

83

9

10

11

12

13

14

15

401

353

83

401

q²: cross-validated correlation coefficient (LOO). SE_CV

:

cross-validated standard error. r²:

noncross-validated correlation coefficient. SEE: noncross validated standard error. HL: hologram length.

N: optimum number of component. Fragment distinction: A-atoms, B-bonds, C-connections, H-hydrogen,

Ch-chirality, DA-donor and acceptor.

Table 3. 2D-HQSAR analysis for the influence of fragment sizes using the best fragment

distinction (A/B/H/DA)

model

Fragment size

q²

SEcv

0.288

0.273

0.270

0.263

r²

SEE

0.266

0.186

0.178

0.159

HL

59

N

1

6

5

6

1

2

3

4

1–3

2–4

3–5

3–7

0.423

0.555

0.551

0.587

0.508

0.793

0.805

0.848

71

401

83

5

4–6

4–7

4–8

5–8

5–9

6–7

6–8

6–9

7–8

7–9

8–9

0.569

0.589

0.625

0.630

0.577

0.576

0.637

0.613

0.631

0.590

0.566

0.269

0.262

0.246

0.245

0.266

0.243

0.255

0.249

0.262

0.270

0.831

0.849

0.850

0.856

0.844

0.853

0.875

0.882

0.876

0.823

0.168

0.159

0.156

0.155

0.162

0.154

0.145

0.140

0.144

0.172

53

83

71

97

71

61

97

61

151

6

5

6

5

6

7

8

9

10

11

12

13

14

15

Table 4. Statistical parameters of the optimal 2D-HQSAR and 3D-topomer CoMFA models

Model method

2D-HQSAR

q²

SEcv

0.249

0.250

r²

SEE

0.140

0.150

N

6

r²

pred

0.631

0.572

0.882

0.853

0.832

0.643

3D-topomer CoMFA

3

Table 5. Experimental and predicted activities (pIC₅₀) with residual values for the nine test set

compounds investigating the predictability of 2D-HQSAR and 3D-topomer CoMFA models

2D-HQSAR model

3D-topomer CoMFA model

Cd

Exp (pIC₅₀)

Pred (pIC₅₀)

Res

Pred (pIC₅₀)

-0.465

-0.683

-0.308

-0.199

-0.737

-1.100

-1.006

-1.064

-1.082

Res

4a

4b

4j

-0.535

-1.065

-0.316

-0.057

-1.134

-0.856

-1.110

-0.939

-0.924

-0.565

-0.811

-0.375

-0.344

-1.211

-1.087

-1.054

-0.928

-0.879

0.030

-0.254

0.059

0.288

0.077

0.231

-0.056

-0.011

-0.045

-0.070

-0.382

-0.008

0.143

-0.397

0.244

-0.105

0.125

0.159

4r

4u

5e

5l

5p

7

Article Doi

Synthesis, cytotoxic activity, and 2D- and 3D-QSAR studies of 19-carboxyl-modified novel isosteviol derivatives as potential anticancer agents

DOI: 10.1111/cbdd.12910

Source and publish data:

Authors:

Article abstract of DOI:10.1111/cbdd.12910

Full text of DOI:10.1111/cbdd.12910

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