Study on synthesis and inhibitory activities of NR2B ca 2 flux of isoindoline derivatives and study on chemical constituents of marsdenia tenacissima and calotropis gigantea

Study on synthesis and inhibitory activities of NR2B Ca2+-flux of isoindoline derivatives, and study on chemical constituents of Marsdenia tenacissima and Calotropis gigantea of 2-cyan

Introduction

Isoindole derivatives are one of the most important classes of N-heterocyclic biological active compounds [1-7] They have been received considerable attention from synthetic pharmacists and chemists due to their potent therapeutic and pharmacological activities [1, 3-7] Isoindolin-1- imine series have exhibited typical pharmacological activities, such as NR2B-selective NMDA (N-Methyl-D-aspartate) receptor antagonists [4] , the thrombin receptor (PAR-1) inhibitors [5-6] , and antiproliferative effect [7]

Fig 1.1 Several ioindolin-1-imine analogues [4]

Since the first multicomponent reaction (MCR) was accomplished in 1850 by Strecker [8] , MCRs show especial significance to the organic synthesis due to their operational simplicity, high productivity, short reaction time, and high yield without isolating the intermediates from simple and popular starting materials [9-12] Up to date, many well-known MCRs such as Alkynes trimerisation, Kabachnik–Fields reaction, Biginelli reaction, Asinger reaction, Mannich reaction, Passerini reaction, and Ugi reaction have been developed [12-14] The important aspects of MCRs is widely recognized and applied as a powerful tool for the general synthesis of important biologically active compounds, particularly the synthesis of N- heterocyclic compounds [12]

Up to date, several methods for the formation of isoindolin-1-imine structures have been

2 reported, but most of methods include some kind of drawbacks such like multi-step strategies, required tough reaction conditions, prolonged reaction time, low yields, and so on [4-6, 9-11]

Scheme 1.1 Synthesis of isoindolin-1-imine derivatives 1-4 [4]

Recently, our research group reported new procedures for 2,3-disubstituted isoindolin-1- imines via a three-component reaction of 2-cyanobenzaldehyde, primary amine, and alcohol in the presence of acetic acid [15] (5, Scheme 1.2) or 3-methyl-1H-pyrazol-5(4H)-one [16] (6, Scheme 1.3) with good yields and wide scope Dueing to the reactive activity of the adjacent formyl and cyano groups of 2-cyanobenzaldehyde, cascade condesation reactions can be carried out with different nucleophilic reagents via Knoevenagel condensation and Michael addition.For further development, we turned our attention towards other substrates

4-Hydroxycoumarin scaffolds are abundant in natural products [17] , and offen exhibit remarkable pharmacological properties [18-29] In addition, due to the active methylene group, 4-hydroxycoumarin is served as starting scaffold for the synthesis of many different structures with various functional groups [20-28] In an effort to exploit the potential of 4- hydroxycoumarin for the synthesis of isoindolin-1-imine analogues, we considered it worthwhile to investigate its multi-component condensations with 2-cyanobenzaldehyde and an amine

Fig 1.2 The biologically active 4-hydroxycoumarin derivatives

Design for synthesis of isoindolin-1-imine derivatives

According to the summary of the existing literatures, the formation of isoindolin-1-imine skeleton most needed benzonitrile as a substrate fragment, and the reaction of a halogen substituent compound combined with a primary amine to give the target product Otherwise, benzaldehyde could react with a primary amine to give unstable imines, and could be formed isoindolin-1-imine structure by cyclization In our previous work, isoindolin-1-imine skeletons can be afforded via condensation from 2-cyanobenzaldehyde, primary amine, and alcohol [15] or 3-methyl-1H-pyrazol-5(4H)-one [16] in high yields Based on the previous results, we choose 2-cyanobenzaldehyde, ammonium acetate or primary amine, and 4- hydroxycoumarin derivatives as reaction substrates to attempt whether isoindolin-1-imine structure can be constructed

Initially, we screened different solvents for condensation reaction of 2- cyanobenzaldehyde and 4-hydroxycoumarin with ammonium acetate or benzyl amine Notably, when ethanol was selected as a reaction solvent, these reactions yielded new structures as 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a, Scheme 1.4) under reflux condition, and 3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4- one (11a, Scheme 1.9) without catalyst under room temperature Thus, we decided to study the optimization of reaction conditions, the scope for further systematic study and accurately identify their structures.

Experiment

Synthesis of 3-substituted isoindolin-1-imine derivatives

1.3.1.1 Synthesis of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)

Based on our previous results [15-16] , we continued to explore the three-component condensation reaction of 2-cyanobenzaldehyde 1a (2 mmol), ammonium acetate 2 (2 mmol), 4-hydroxycoumarin (4-hydroxy-2H-chromen-2-one) 3a (2 mmol) in dry ethanol under reflux for 20 minutes (Scheme 1.4) The condensation reaction was carried out smoothly, quickly, and a white precipitate was afforded When the reaction completed, the white solid was filtered and washed with acetone to give 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen- 4-one 4a in excellent yield (95%) Structure of 4a was determined by using NMR data and

Scheme 1.4 Synthesis of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)

1.3.1.2 The optimization of reaction conditions

Initially, the influence of different solvents on this condensation was investigated The results summarized in Table 1.1 showed that the target product 4a was afforded in low to moderate yields in DCM (entries 1, 2), toluen (entry 3), DCE (entry 4), CH 3 CN (entry 5), THF (entry 6), and H 2 O (entries 11-13), and good yield in MeOH (entry 10) Particularly, product 4a was obtained in excellent yield in EtOH under reflux condition (entry 8), but the product 4a was only given in about 30% yield under room temperature (entry 7) Thus, dry ethanol was selected as an ideal solution for this three-component condensation To further investigate the influence of reaction time in yield, the multi-component reaction was carried out in EtOH under reflux condition with different times When the reaction was proceeded for 120 minutes, the product was obtained in 96% yield (entry 9) Consequently, the optimized reaction condition was obtained in ethanol solution under reflux for 20 minutes without any catalyst, and this condition was chosen to further synthesis of isoindolin-1-imine derivatives

Table 1.1 Solvent screening for the synthesis of 4a

Entry Solvent Time (min) Yield 4a b (%)

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), ammonium acetate (2 mmol),

4-hydroxycoumarin 3a (2 mmol), solvent (4 mL), reflux b Isolated yields d Room temperature

To further demonstration the scope of this condensation reaction, we continued to screen the significant effects of reagent 2 to this three-component reaction on the yields (Table 1.2) When reagent 2 was ammonium salts of strong acids, such as HCl and H2SO4 (Table 1.2, entries 5,

6), only a trace amount of compound 4a was observed in reaction solution When NH3,

NH 3 /AcOH (1/1, eq/eq) or NH 4 OAc was used as reagent 2 under similar conditions (Table 1.2, entries 1-4, 7-11), compound 4a was obtained in moderate to excellent isolated yields (40-95%) When ammonia solution or ammonia/AcOH solution was used, the yield of this condensation reached 78% (Table 1.2, entries 10, 11), whereas ammonium acetate 2 was the most effective reagent in term of yields of product 4a (Table 1.2, entries 1-4) When the amount of ammonium acetate increased from 1.0 (eq.) to 2.0 (eq.), no significant impact had on the overall yields of product 4a Therefore, ammonium acetate was chosen as the ideal reagent 2 for this condensation

Table 1.2 The effect of reagent 2 for the synthesis of 4a

Entry Reagent 2 Ratio (eq.) Yield 4a b (%)

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), 4-hydroxycoumarin 3a (2 mmol), solvent (4 mL), reflux, 20 minutes b Isolated yields

1.3.1.3 The scope and limitations of reaction substrates

To demonstrate the scope and limitations of this reaction, some series of isoindolin-1-imine derivatives 4, 8, and 10 were synthesized from different starting materials 1 and 3 (Table 1.3- 1.5, Scheme 1.5-1.7)

Scheme 1.5 Synthesis of 3-substituted isoindolin-1-imine derivatives 4

Firstly, a series of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one derivatives 4 were prepared via condensations of 2-cyanobenzaldehyde derivatives 1, ammonium acetate 2 and 4-hydroxycoumarin derivatives 3 in dry ethanol in excellent yields (Table 1.3) 4-Hydroxycoumarin 3 carrying different substituent groups on the aromatic ring had no significant impact on the overall yields of the corresponding products 4 (entries 1-8) To further investigate the effects of substituted 2-cyanobenzaldehydes for this condensation, the reactions of 2-cyanobenzaldehydes bearing either electron-donating groups such as methoxy

6 or n-propoxy group (entries 9-14) or electron-withdrawing groups such as nitro group with ammonium acetate 2 and 4-hydroxycoumarin derivatives 3 were also investigated The results showed that the reaction of 2-cyano-4-propoxy-5-methoxybenzaldehyde (entries 9-14) gave the corresponding products 4 in excellent yields, but 2-cyano-4-nitrobenzaldehyde (entry 15) or 2-cyano-5-nitrobenzaldehyde (entry 16) did not give the corresponding product under similar reaction conditions

Table 1.3 Synthesis of isoindole-1-imine analogs products 4

Conditions: 1 (2 mmol), ammonium acetate 2 (2 mmol), 3 (2 mmol), solvent (4 mL), reflux, 20 minutes b Isolated yields

In continuing to demonstrate the scope and limitations of this reaction, the condensation of 2-cyanobenzaldehyde 1, ammonium acetate 2, and 1,3-dicarbonyl compounds 7 were carried out (Scheme 1.6) In this case, the condensation could also be accomplished quickly and smoothly to give formation of products 8 with lower yields and longer reaction time as described in Table 1.4

Scheme 1.6 Synthesis of compounds 8 Table 1.4 Synthesis of products 8

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), ammonium acetate 2 (2 mmol), 4 (2 mmol), solvent (4 mL), reflux, 60 minutes b Isolated yields

On the other hand, this three-component condensation was also extended to other nucleophilic reagents such as 4-hydroxyquinolin-2(1H)-one derivative, which exhibited useful biological benefits [33-37] In previous reports, we showed that 4-hydroxyquinolin-2(1H)-one was one of the common starting substances in the synthetic medicinal chemistry [34-35] Thus, the three-component condensation reactions of 2-cyanobenzaldehyde 1, ammonium acetate 2, and 4-hydroxyquinolin-2(1H)-one derivatives 9 were also explored (Scheme 1.7)

Interestingly, the condensation of 4-hydroxyquinolin-2(1H)-one derivatives 9 with 1 and 2 gave the corresponding 10 quickly and smoothly in high yields (entries 1-3, Table 1.5)

However, 2-cyano-4-nitrobenzaldehyde (entry 4, Table 1.5) only gave a trace amount of product 10 under similar reaction conditions

Scheme 1.7 Synthesis of compounds 10 Table 1.5 Synthesis of products 10

Conditions: 2-cyanobenzaldehyde 1 (1 mmol), ammonium acetate 2 (1 mmol), 9 (1 mmol), solvent (4 mL), reflux, 20 minutes b Isolated yields

The structures of isoindolin-1-imine derivatives 4a-4o, 8a-8b, and 10a-10c were determined by NMR spectral data and LC-MS data, as illustrated by the representative example 4b (Figure 1.3) In its 1 H NMR spectrum in DMSO-d 6 , a signal of H-3 proton occurred as a singlet at δ 6.50 that showed HMBC correlation with C-1 (δ 3.0), C-3 (δ = 89.5), C-2’ (δ = 162.8),

10 and C-4’ (δ = 173.8), respectively H-2 proton (s, δ = 10.39) also had correlation with C-1 (δ 163.0) and C-3 (δ = 60.2) H-5’ proton showed important HMBC correlations with C-4’ (Figure 1.4)

Fig 1.3 Important 1 H and 13 C NMR assignments for compound 4b

Fig 1.4 Key HMBC correlations for compound 4b

1.3.1.5 The plausible mechanism for synthesis of isoindolin-1-imine

Thus, a plausible mechanism can reasonably be proposed for the series of products 4 and 10 (Scheme 1.8) [15-16] In the initial step, 2-cyanobenzaldehyde 1 reacted with ammonia by decomposition of ammonium acetate under reflux condition to give adduct 5 Then the adduct

5 gave unstable imine intermediate 6 by protonation and dehydration under acidic condition

Subsequent nucleophilic addition of 3 to 6 afforded 7, which afforded the title isoindolin-1- imine 4 or 10 via intramolecular cyclization

Scheme1.8 Possible mechanism for the formation of target products

Synthesis of 2,3-disubstituted isoindolin-1-imine derivatives

As part of a continuing effort in our laboratory toward the synthesis of isoindolin-1-imine derivatives, we herein describe a novel one-pot procedure for the synthesis of 3-(2- substituted-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one derivatives 11 via cascade three-component condensation This reaction was carried out by condensation of 2- cyanobenzaldehyde 1, primary amine 2 (NH 4 OAc replaced by RNH 2 ), and 4-hydrocoumarin derivatives 3 with catalyst-free under room temperature in dry dichloromethane with excellent yields, fast reactive time, and wide scope (Scheme 1.9)

1.3.2.1 The optimization of reaction conditions

Due to our previously results [15-16] , we continued to explore a three-component condensation reaction of 2-cyanobenzaldehyde 1 (2 mmol), benzylamine 2a (2 mmol), 4-hydroxy-2H- chromen-2-one 3a (2 mmol) (Scheme 1.9) It noted that the reaction processed smoothly in dry dichloromethane at room temperature for 5 minutes and obtained a new structure white solid product 11a, its structure was determined by NMR and LC-MS data.

To investigate the influence of solvents, the condensation reaction was carried out in different solvents The results were summarized in Table 1.6

Entry Solvent Time (min) Yield 11a b (%)

12 a Conditions: 2-cyanobenzaldehyde 1 (2 mmol), benzyl amine 2a (2 mmol), 4-hydroxycoumarin 3a (2 mmol), solvent (5 mL), room temperature b Isolated yields c Conditions: Reflux

The results showed that the product 11a was afforded with excellent yield in DCM for 5 and 30 min (entries 1, 2) The multi-component reaction was also carried out in Benzene (entry 3), DCE (entry 4), CH3CN (entry 5), and THF (entry 6), MeOH (entry 10) at room temperature to yield 11a in low to good yields Then, this reaction was also carried out in ethanol and in water for 5 and 180 minutes under room temperature and reflux condition The yields of 11a in ethanol were 65%, 70%, and 90% (entries 7-9), and in water were 10%, 30%, and 80% (entries 11-13), respectively Thus, the optimized reaction condition was in dry dichloromethane under room temperature with catalyst-free for 5 minutes This condition was chosen for further synthesis of isoindolin-1-imine derivatives

1.3.2.2 The scopes and limitations of reaction substrates

Table 1.7 Synthesis of isoindole-1-imine analogs products 11 a

24 11y n-Butyl 3c 90 c a Conditions: 2-cyanobenzaldehyde 1a (2 mmol), amine 2 (2 mmol), 3 (2 mmol), solvent (5 mL), room temperature, 5 minutes b Isolated yields c Conditions: 2-cyanobenzaldehyde 1a (1 mmol), amine 2 (1 mmol), 3c (1 mmol), solvent (5 mL), room temperature for 30 minutes

To further demonstrate the scope and limitations of this procedure, we prepared the condensation reaction of 2-cyanobenzaldehyde 1, alkyl amine 2, and 4-hydroxycoumarin (or

4-hydroxy-2H-chromen-2-one) 3a or 4-hydroxy-6-methylcoumarin (or 4-hydroxy-6-methyl-

2H-chromen-2-one) 3b or 4-hydroxy-2H-benzo[h]chromen-2-one 3c under room temperature in dry dichloromethane for 5 minutes for synthesis of the series of isoindolin-1-imines 11.The structure of compound 11 was determined by using NMR and LC-MS data analysis The results were summarized in Table 1.7 The reaction scope and limitations were explored according to amine and 4-hydroxycoumarin derivatives

As shown in the Table 1.7, we showed that the electronic effect from the substrate had no significant impact on the overall yields of the products 11 For example, arylalkylamines carrying either electron-withdrawing or electron-donating substituents (entries 2-6, 16; entries 17-20, 23) reacted quickly to afford the desired products in excellent yields Alkyl amines such as n-propyl (entry 7), n-butyl (entries 8, 21, 24), i-butyl (entry 9), N,N- dimethylaminoethyl (entry 15), cyclopropylmethyl (entry 10), cyclohexyl (entry 11), and hydroxyalkyl amines as ethanol-2-yl (entries 12, 22), propan-1-ol-3-yl (entry 13) or 3- methylbutan-1-ol-2-yl (entry 14) could also react quickly and smoothly to give formation of products 4 in excellent yields

1.3.2.3 The plausible mechanism for synthesis of isoindolin-1-imines 11

A plausible mechanism can reasonably be proposed for the series of products 11 (Scheme 1.10)

In the initial step, 2-cyanobenzaldehyde 1 reacted with amine to give adduct 5 Then the adduct 5 gave unstable imine intermediate 6 by protonation and dehydration Subsequent nucleophilic addition of 3 to 6 afforded 7, which afforded the title isoindolin-1-imine 11 via intramolecular cyclization

Scheme 1.10 Possible mechanism for the formation of compounds 11

Activity of isoindolin-1-imine derivatives

N-methyl-D-aspartate receptors (NMDARs) are well known as ligandgated cation-selective channels that are highly expressed in the central nervous system crucial to brain functions such as circuit development, learning, and memory [38-42] NMDARs containing different NR2 (A, B, C or D) subunits have different pharmacological and kinetic properties [40-42] NMDARs are unusual ligandgated ion channels because their activation requires the relief of

Mg 2+ block by membrane depolarization and the concomitant binding of two agonists: glycine and L-glutamate The opening of NMDARs might lead to an influx of cations, including Ca 2+ , which initiates the signal transduction cascade [38-42]

In order to development of NR2B subtype selective NMDA antagonist drugs, many NMDA receptor antagonists such as ifenprodil, besonprodil CI-104, Ro-25-6981, traxoprodil, and carbamates, 4-substituted-3-phenylquinolin-2(1H)-ones, have been reported [4-6, 42-49]

Particularly, ioindolin-1-imines exhibited strong inhibition activities of NR2B Ca 2+- flux [4]

1.3.3.2 Effect of isoindolin-1-imine derivatives as NR2B Ca 2+ flux inhibitor

The inhibitory activities of isoindolin-1-imines 4 and 11 were evaluated against NR2B Ca2+- flux as compared to ifenprodil as a positive control This assay is depending on the fluorescence measurement of free concentrations of intracellular calcium of L(tk-) cells expressing NR1a/NR2B receptor by fluorometric imaging plate reader (FLIPR) method From obtained results, several structure activities relationships were discussed as follow

The inhibitory effects of NR2B Ca 2+ -flux by compounds 4 are summarized in the Table 1.8 According to the assay results, we showed that all the tested compounds 4a-4n exhibited inhibitory activities against NR2B calcium ion-flux with IC 50 values range from97.2±10.1 to 1925.3±42.5 nM, but lower than that of ifenprodil (IC 50 = 75.3±7.3 nM)

Compounds 4a-h with R 1 =R 2 =H (entries 1-8) displayed less potent activities compared to compounds 4i-n (entries 9-13) with R 1 =methoxyl and R 2 =n-propoxyl These results suggested that the introduction of the 5- and 6-alkoxyl group into the aromatic ring was beneficial to inhibitory activity

Compound 4d (IC 50 = 250.3±20.1 nM) and 4l (IC 50 = 97.2±10.1 nM) with methoxyl substituent on aromatic ring of coumarin moiety showed better inhibitory activities than methyl, tert-butyl, and iso-propyl substituent compounds Otherwise, the presence of methyl at position 6’ (4b, entry 2) or 7’ (4c, entry 3) of the aromatic ring of coumarin moiety had no obvious contribution to activities

Replacing the methyl group by tert-butyl (4e, entry 5), iso-propyl (4f, entry 6) did not lead to signiﬁcant effect on the inhibitory activities All alkyl substituent groups on coumarin moiety derivatives had one to two-fold potent activities reduction compared to unsubstituted compounds (4a, entry 1; 4i, entry 10) These activity results showed that the presence of the alkyl group at aromatic ring of coumarin is critical

Among the compounds above, compound 4l exhibited the most effective inhibition activity, and could be a hit compound for further SAR study as a potential NR2B calcium ion flux antagonists

Table 1.8 Inhibition of NR2B Ca 2+ -flux by isoindolin-1-imine analogs

R 2 Reagent 3 Product 4 NR2B Ca 2+ -flux

The isoindolin-1-imine products 11 were prepared by one-pot procedure via three-component condensation of 2-cyanobenzaldehyde, primary amines, and 4-hydrocoumarin derivatives in dry DCM The inhibitory evaluation results of isoindolin-1-imine analogs 11 against NR2B calcium ion-flux are summarized in Table 1.9

Table 1.9 Inhibition of NR2B Ca 2+ -flux by products 11

Entry Product 11 R 1 Reagent 3 NR2B Ca 2+ -flux

IC50 (nM) Positive control Ifenprodil 75.3±7.3

As shown in Table 1.9, all the tested compounds 11 are potent NR2B calcium ion-flux inhibitors with IC50 values range from 84.2±7.5 to 2500.7±68.7 nM, but lower than that of ifenprodil (IC50 = 75.3±7.3 nM)

In the case of compounds were substituted by various substituent at 2-position of isoindolin-1-imine core (as shown in entries 1-16 of Table 1.9), all benzyl substituent compounds (11a-f) showed better inhibitory effects than compounds containing alkyl group (R 1 -) such as n-butyl (11h), i-butyl (11i) and cyclohexyl (11l ) These results suggested that the substitution of benzyl by alkyl group at 2-position had no obvious contribution to activities

However, compound 11m containing ethanol-2-yl (IC50 = 352.8±29.5 nM, entry 12) and

11n containing propan-1-ol-3-yl (IC50 = 385.4±34.2 nM, entry 13) showed enhanced effects than compounds containing alkyl group They also exhibited nearly equivalent activity of p- chlorobenzyl compound 11b (IC50 = 345.4±28.5 nM, entry 2), but revealed about 3 to 4-fold lower potential activity than that of benzyl 11a (IC 50 = 106.3±11.2 nM, entry 1) and compounds 11e-f with methoxyl group at the phenyl ring (IC 50 = 84.2±7.5, 89.1±11.5 nM, respectively)

Comparing the IC 50 values of compounds containing various substituent group at the phenyl ring at 2-position of isoindolin-1-imine core, showed that p-halogenobenzyl derivatives (11b-d, 11s) displayed reduced effects compared to benzyl group (11a, 11r) in their inhibition activity p-Methoxyl substituent derivatives (11e, 11t) had better activity than that of p-halogen substituent (11b, 11s) and benzyl group (11a, 11r, 11x) Otherwise, p- chloro- 11b (IC 50 = 345.4±28.5, entry 2) exhibited better inhibitory activity than p-bromo- 11c

(IC 50 = 642.2±23.5 nM, entry 3) and p-floro- 11d (IC 50 = 501.2±34.2 nM, entry 4) substituent compounds

Compounds 11r with 3b (entry 17) and 11x with 3c (entry 23) at position 3 of isoindolin- 1-imine core had weaker inhibitory potency as compared to 11a with 3a (entry 1) and a clear order of the introduction of 3 at position 3 for this activity was observed as the following: 3a > 3b > 3c (11a > 11r > 11x)

Finally, all above isoindolin-1-imine derivatives revealed inhibitory activities of Ca 2+ flux in cells expressing the NR2B subunit (NR2B Ca 2+ -flux) Among them, compounds 11e,

11f, 11t exhibited stronger potent inhibitory activities Thus, the substitution at 2-postion on isoindolin-1-imine core by substituted benzyl structures was potential for further synthesis of derivatives as Ca 2+ -flux inhibitors.

Preparation section

1.3.4.1 Preparation of isoindolin-1-imines 4, 8, and 10

To a stirred dry ethanol solution of 2-cyanobenzaldehyde (1, 2 mmol), and 4- hydroxycoumarin derivatives (3, 2 mmol) was added ammonium acetate (2, 2 mmol) The resulting mixture was stirred under reflux untill precipitate was afforded After completion of the reaction, solid was filtered off, washed with dichloromethane and acetone, respectively, to give the corresponding product 4a-o (89-95%)

2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)

White solid, yield 95%, mp 185-186 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.38 (s, 1H), 9.30 (s, 1H), 8.79 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.80 (dd, J = 7.7, 1.7 Hz, 1H), 7.61 (m,

1H), 7.51 (t, J = 7.5 Hz, 1H), 7.42-7.33 (m, 2H), 7.12 (td, J = 7.7, 1.0 Hz, 1H), 7.08 (dd, J 8.2, 1.0 Hz, 1H), 6.51 (s, 1H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.6, 162.9, 162.8, 154.3, 151.0, 133.4, 130.9, 130.8, 129.1, 127.6, 125.1, 123.3, 122.6, 122.4, 116.1, 89.5, 60.2; MS (ESI): m/z 293.35 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C17H13N2O3: 293.2802; Found: 293.2801

2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methyl-4H-chromen-4-one (4b)

White solid, yield 95%, mp 188-189 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.39 (s, 1H), 9.32 (s, 1H), 8.81 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.64-7.57 (m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.18 (dd, J = 8.2, 2.3 Hz, 1H), 7.00-6.94 (d, J = 8.2 Hz, 1H), 6.50 (s, 1H), 2.31 (s, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.8, 163.0, 162.8, 152.4, 151.0, 133.4, 131.5, 131.1, 129.1, 127.6, 125.0, 123.3, 123.0, 122.6, 115.9, 89.5, 60.2, 21.0 MS (ESI): m/z 307.57 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C18H15N2O3: 307.1023; Found: 307.1025

2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methyl-4H-chromen-4-one (4c)

White solid, yield 94%, mp 189-190 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.16 (s, 1H), 9.52 (s, 1H), 8.96 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.60 (td, J = 7.7, 1.1 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.33 (dd, J = 7.6, 0.9 Hz, 1H), 6.93 (d, J = 7.9 Hz, 1H), 6.89 (s, 1H), 6.46 (s, 1H), 2.32 (s, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.8, 163.1, 162.5, 152.4, 151.0, 133.4, 131.5, 131.3, 129.1, 127.6, 125.0, 123.1, 122.9, 122.6, 115.9, 89.6, 60.2, 21.1; MS (ESI): m/z 307.57 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C18H15N2O3: 307.1023; Found: 307.1025

2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methoxy-4H-chromen-4-one (4d)

White solid, yield 92%, mp 179-180 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.36 (s, 1H), 9.27 (s, 1H), 8.78 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.61 (td, J = 7.7, 1.1 Hz, 1H), 7.51 (t, J 7.6 Hz, 1H), 7.34 (dd, J = 7.6, 1.1 Hz, 1H), 7.27 (d, J = 3.0 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 6.97 (dd, J = 8.8, 3.0 Hz, 1H), 6.46 (s, 1H), 3.75 (s, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.8, 162.9, 162.6, 155.8, 154.3, 151.0, 133.3, 130.8, 129.1, 127.6, 124.3, 123.3, 122.6, 122.4, 117.5, 89.0, 60.2, 55.9; MS (ESI): m/z 323.44 [M+H] + ; HRMS (ESI): m/z [M+H] +

6-tert-butyl-2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4e)

White solid, yield 92%, mp 181-182 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.36 (s, 1H), 9.33 (s, 1H), 8.80 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 2.6 Hz, 1H), 7.61 (td, J = 7.8, 1.1 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.43 (dd, J = 8.6, 2.6 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.6 Hz, 1H), 6.46 (s, 1H), 1.28 (s, 9H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.8,

163.3, 162.8, 152.3, 151.1, 144.5, 133.3, 133.1, 129.1, 127.9, 127.6, 123.3, 122.6, 122.5, 121.1, 115.7, 89.3, 60.4, 34.5, 31.8 (3C); MS (ESI): m/z 349.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C21H21N2O3:349.1524; Found: 349.1517

2-hydroxy-3-(3-iminoisoindolin-1-yl)-8-isopropyl-4H-chromen-4-one (4f)

White solid, yield 93%, mp 183-184 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.35 (s, 1H), 9.30 (s, 1H), 8.89 – 8.70 (m, 1H), 8.12 (d, J = 7.8 Hz, 1H), 7.65 (dd, J = 7.5, 1.7 Hz, 1H), 7.62 (td, J = 7.8, 1.1 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.31 (dd, J = 7.5, 1.7 Hz, 1H), 7.07 (t, J = 7.7 Hz, 1H), 6.48 (s, 1H), 3.33-3.41 (m, 1H), 1.20-1.21 (d, J = 5.6 Hz, 6H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.8, 163.0, 162.9, 154.4, 151.0, 140.7, 133.3, 129.1, 127.5, 125.1, 123.7, 123.4, 122.5, 120.9, 116.1, 89.0, 60.1, 25.6, 21.4 (2C); MS (ESI): m/z

335.44 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C20H19N2O3: 335.1321; Found: 335.1317

3-hydroxy-2-(3-iminoisoindolin-1-yl)-1H-benzo[f]chromen-1-one (4g)

Yellow solid, yield 92%, mp 201-202 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.42 (s, 1H), 10.22 (d, J = 8.8 Hz, 1H), 9.32 (s, 1H), 8.82 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 8.8

Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.56-7.48 (m, 2H), 7.40-7.45 (m, 2H), 7.31 (d, J = 8.8 Hz, 1H), 6.60 (s, 1H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 177.3, 162.8, 162.4, 154.4, 151.2, 133.4, 131.8 (2C), 130.1, 129.2, 128.3, 127.6, 127.2, 126.9, 124.7, 123.4, 122.6, 118.1, 114.3, 91.1, 60.4; MS (ESI): m/z 343.45 [M+H] + ; HRMS (ESI): m/z [M+H] +

2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-benzo[h]chromen-4-one (4h)

Yellow solid, yield 91%, mp 200-201 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.46 (s, 1H), 9.36 (s, 1H), 8.87 (s, 1H), 8.28-8.20 (m, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.9-1.94 (m, 2H), 7.65- 7.56 (m, 4H), 7.53 (t, J = 7.5 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 6.54 (s, 1H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 174.3, 162.9, 162.8, 150.9, 150.4, 134.8, 133.4, 129.2, 128.2, 127.7 (2C), 126.5, 123.5, 123.4, 122.7, 122.3, 122.1, 121.8, 118.2, 89.7, 60.3; MS (ESI): m/z 343.45

[M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C21H15N2O3:343.1014; Found: 343.1017

2-hydroxy-3-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)-4H-chromen-4-one (4i)

Yellow solid, yield 94%, mp 184-185 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.06 (s, 1H), 9.05 (s, 1H), 8.55 (s, 1H), 7.82 (dd, J = 7.7, 1.8 Hz, 1H), 7.73 (s, 1H), 7.38 (td, J = 8.6, 1.8 Hz, 1H), 7.12 (td, J = 7.7, 1.1 Hz, 1H), 7.08 (dd, J = 8.6, 1.1 Hz, 1H), 6.81 (s, 1H), 6.41 (s, 1H), 3.92 (t, J = 6.6 Hz, 2H), 3.76 (s, 3H), 1.77 (q, J = 7.0 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.7, 163.0, 162.8, 154.3, 148.4, 145.5, 130.7 (2C), 125.2, 123.3, 122.3, 120.5, 116.1, 106.6, 104.7, 90.0, 70.5, 59.7, 56.3, 22.4, 10.8; MS (ESI): m/z

2-hydroxy-3-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)-6-methyl-4H-chromen-4-one (4k)

Yellow solid, yield 94%, mp 186-187 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.04 (s, 1H), 9.01 (s, 1H), 8.51 (s, 1H), 7.72 (s, 1H), 7.61 (d, J = 2.2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.97

(dd, J = 8.3, 2.2 Hz, 1H), 6.79 (s, 1H), 6.40 (s, 1H), 3.93 (t, J = 6.7 Hz, 2H), 3.76 (s, 3H), 2.31 (s, 3H), 1.78 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13 C NMR (101 MHz, DMSO-d6) δ 173.7, 162.9 (2C), 154.3, 152.4, 148.3, 145.6, 131.4, 131.0, 125.0, 123.0, 120.5, 115.9, 106.6, 104.7, 89.8, 70.5, 59.8, 56.3, 22.4, 20.9, 10.8; MS (ESI): m/z 395.45 [M+H] + ; HRMS (ESI): m/z

2-hydroxy-3-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)-6-methoxy-4H-chromen-4- one (4l)

Yellow solid, yield 92%, mp 189-190 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.06 (s, 1H), 8.99 (s, 1H), 8.53 (s, 1H), 7.72 (s, 1H), 7.29 (d, J = 3.0 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.97 (dd, J = 8.8, 3.0 Hz, 1H), 6.80 (s, 1H), 6.38 (s, 1H), 3.94 (t, J = 6.7 Hz, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 1.79 (q, J = 7.0 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H); 13 C NMR (101 MHz, DMSO) δ 173.3, 163.1, 162.9, 154.7, 154.3, 148.6, 148.3, 145.6, 123.9, 120.5, 118.2, 117.2, 107.2, 106.6, 104.7, 89.8, 70.5, 59.9, 56.3, 55.8, 22.4, 10.8; MS (ESI): m/z 411.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 22 H 23 N 2 O 6 :411.1512; Found: 411.1511

6-tert-butyl-2-hydroxy-3-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)-4H-chromen-4- one (4m)

Yellow solid, yield 91%, mp 221-222 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.01 (s, 1H), 9.01 (s, 1H), 8.50 (s, 1H), 7.80 (d, J = 2.6 Hz, 1H), 7.73 (s, 1H), 7.42 (dd, J = 8.6, 2.6 Hz, 1H), 7.00 (d, J = 8.6 Hz, 1H), 6.79 (s, 1H), 6.37 (s, 1H), 3.94 (t, J = 6.7 Hz, 2H), 3.76 (s, 3H), 1.78 (m, 2H), 1.29 (s, 9H), 0.99 (d, J = 7.3 Hz, 2H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 173.8, 163.2, 162.9, 154.3, 152.4, 148.3, 145.7, 144.5, 127.9, 122.5, 121.1, 120.5, 115.7, 106.6, 104.7, 89.7, 70.5, 60.0, 56.3, 34.5, 31.8 (3C), 22.4, 10.8; MS (ESI): m/z 437.51 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 25 H 29 N 2 O 5 :437.2013; Found: 437.2009

3-hydroxy-2-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)-1H-benzo[f]chromen-1-one (4n)

Yellow solid, yield 93%, mp 186-187 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.24 (d, J 8.6 Hz, 1H), 10.08 (s, 1H), 9.00 (s, 1H), 8.52 (s, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.87 (dd, J 8.1, 1.6 Hz, 1H), 7.76 (s, 1H), 7.51 (t, J = 8.5 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H), 7.31 (d, J 8.5 Hz, 1H), 6.85 (s, 1H), 6.50 (s, 1H), 3.96 (t, J = 6.7 Hz, 2H), 3.76 (s, 3H), 1.80 (m, 2H),

1.01 (t, J = 7.4 Hz, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 177.3, 163.0, 162.2, 154.4, 154.3, 148.3, 145.8, 131.9, 131.8, 130.1, 128.2, 127.2, 127.0, 124.7, 120.5, 118.1, 114.4, 106.6, 104.7, 91.4, 70.5, 59.9, 56.3, 22.4, 10.8; MS (ESI): m/z 431.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 25 H 23 N 2 O 5 :431.1502; Found: 431.1501

2-hydroxy-3-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)-4H-benzo[h]chromen-4-one (4o)

Yellow solid, yield 92%, mp 183-184 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.12 (s, 1H), 9.04 (s, 1H), 8.57 (s, 1H), 8.27-8.23 (m, 1H), 7.92 (m, 2H), 7.76 (s, 1H), 7.63-7.56 (m, 3H), 6.86 (s, 1H), 6.45 (s, 1H), 3.96 (t, J = 6.7 Hz, 2H), 3.76 (s, 3H), 1.79 (m, 2H), 1.00 (t, J = 7.4

59.8, 56.3, 22.4, 10.8; MS (ESI): m/z 431.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for

To a stirred dry ethanol solution of 2-cyanobenzaldehyde (1, 2 mmol), and 1,3-dicarbonyl compounds (7, 2 mmol) was added ammonium acetate (2, 2 mmol) The resulting mixture was stirred under reflux for 60 minutes After completion of the reaction, solvent was removed in vacuo, and crude product was purified by silica gel column chromatography with gradient eluent system DCM/MeOH (50/1-20/1) to give product 8 in yields of 83-90%

6-hydroxy-5-(3-iminoisoindolin-1-yl)-2,2-dimethyl-4H-1,3-dioxin-4-one (8a)

Yellow solid, yield 90%, mp 180-181 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.24 (s, 1H), 9.23 (s, 1H), 8.69 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H), 5.98 (s, 1H), 1.51 (s, 6H); 13 C NMR (101 MHz, DMSO-d6) δ 175.0, 164.6, 162.2, 151.4, 133.4, 128.7, 127.6, 123.3, 122.7, 100.4, 67.8, 61.6, 26.4 (2C); MS (ESI): m/z 275.41 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C14H15N2O4: 275.1014; Found: 275.1013

3-hydroxy-2-(3-iminoisoindolin-1-yl)-5,5-dimethylcyclohex-2-enone (8b)

Yellow solid, yield 90%, mp 178-179 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.25 (s, 1H), 9.36 (s, 1H), 8.93 (s, 1H), 8.14 (d, J = 7.7 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 7.5

Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 6.27 (s, 1H), 1.92 (m, 4H), 0.94 (s, 6H); 13 C NMR (101 MHz, DMSO d6) δ 188.9, 179.9, 162.8, 152.6, 132.8, 129.0, 126.8, 123.5, 122.1, 106.3, 70.2, 59.7, 51.0, 31.7, 29.2 (2C); MS (ESI): m/z 271.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 16 H 19 N 2 O 2 :271.1411; Found: 271.1410

To a stirred dry ethanol solution of 2-cyanobenzaldehyde (1, 1 mmol), and 4- hydroxyquinolin-2(1H)-one (9, 1 mmol) was added ammonium acetate (2, 1 mmol) The resulting mixture was stirred under reflux for 20 minutes After completion of the reaction, solvent was removed in vacuo, and crude product was purified by silica gel column chromatography with gradient eluent system ethyl acetate/MeOH (20/1-5/1) to give product

2-hydroxy-3-(3-iminoisoindolin-1-yl) quinolin-4(1H)-one (10a)

White solid, yield 85%, mp > 280 o C; 1 H NMR (400 MHz, MeOD) δ 8.05 (d, J = 7.5 Hz, 1H), 8.00 (d, J = 6.8 Hz, 1H), 7.68 (t, J = 7.3 Hz, 1H), 7.55 (t, J = 7.5 Hz , 1H), 7.48 (d, J 7.3 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 6.8 Hz, 1H), 6.70 (s, 1H); 13 C NMR (101 MHz, MeOD) δ 173.0, 164.6, 162.9, 151.9, 139.8, 133.1, 129.4, 129.3, 127.2, 125.0, 123.4, 122.54, 122.47, 119.5, 115.0, 96.5, 60.6; MS (ESI): m/z 292.34 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C17H14N3O2:292.1033; Found: 292.1032

2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methylquinolin-4(1H)-one (10b)

Yellow solid, mp > 280 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.21 (s, 1H), 9.30-9.42

(2H), 9.03 (s, 1H), 8.11 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.64 (s, 1H), 7.58-7.53 (m, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.11 (d, J 7.8 Hz, 2H), 6.97 (d, J = 7.8 Hz, 1H), 6.58 (s, 1H), 2.25 (s, 3H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 174.7, 163.9, 163.1, 154.6, 151.4, 137.6, 133.1, 130.8, 129.4, 128.4, 127.3, 124.6, 123.3, 122.5, 115.0, 98.0, 60.3, 21.1 MS (ESI): m/z 306.41 [M+H] + ; HRMS (ESI): m/z [M+H] +

2-hydroxy-3-(3-imino-6-methoxy-5-propoxyisoindolin-1-yl)quinolin-4(1H)-one (10c)

Colour solid, mp 215-217 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.88 (s, 1H), 9.09 (s, 1H), 8.97 (s, 1H), 8.51 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.71 (s, 1H), 7.24 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 7.4 Hz, 1H), 6.88 (t, J = 7.7 Hz, 1H), 6.76 (s, 1H), 6.49 (s, 1H), 3.84 (m, 2H), 3.73 (s, 3H), 1.76 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 174.2, 164.3, 162.9, 154.1, 148.1, 146.5, 139.9, 133.3, 129.3, 124.7, 121.8, 119.3, 114.8, 106.6, 104.6, 88.0, 70.5, 60.2, 56.2, 22.4, 10.8; MS (ESI): m/z 380.43 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 21 H 22 N 3 O 4 :380.1518; Found: 380.1514

General procedure the synthesis of 11: To a stirred dichloromethan solution of 2- cyanobenzaldehyde 1 (2 mmol) and 4-hydroxycoumarin derivatives 3 (2 mmol) was added primary amines 2 (2mmol) The resulting mixture was stirred at room temperature After the reaction was completed, solid was filtered off, washed with dichloromethane and acetone, respectively, recrystallized to give the corresponding product 11a-11x (90-98%)

3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11a)

White solid, yield 98%, mp 243–244 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.74 (s, 1H), 9.22 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 7.6, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.55 (d, J

= 7.3 Hz, 1H), 7.27–7.45 (m, 7H), 7.17 (t, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.0 Hz, 1H), 6.62 (s, 1H), 4.99 (d, J = 15.5 Hz, 1H), 4.47 (d, J = 15.5 Hz, 1H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 174.9, 161.9, 161.2, 154.6, 148.9, 135.5, 133.6, 131.1, 129.1 (2C), 128.8, 128.2 (2C), 128.1, 125.5, 123.5, 123.2, 122.8, 122.7, 122.5, 116.3, 87.9, 64.0, 45.5; MS (ESI): m/z 383.42

[M+H] + ; HRMS (ESI) Calcd for C24H19N2O3 [M+H] + :383.1267; Found: 383.1270

3-(2-(4-chlorobenzyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11b)

White solid, yield 98%, mp 246-247 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.76 (s, 1H), 9.23 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.3 Hz, 1H), 7.51- 7.57 (m, 3H), 7.30-7.45 (m, 4H), 7.16 (t, J = 7.6 Hz, 1H), 7.08 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.93 (d, J = 15.5 Hz, 1H), 4.48 (d, J = 15.6 Hz, 1H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 175.0, 161.9, 161.2, 154.6, 149.0, 134.9, 133.7, 131.9 (2C), 131.1, 130.4 (2C), 128.7, 128.0, 125.5, 123.2, 122.8, 122.7, 122.5, 121.4, 116.3, 87.8, 64.1, 45.0; MS (ESI): m/z 417.37

[M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C24H18ClN2O3:417.0934; Found: 417.0930

3-(2-(4-bromobenzyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11c)

White solid, yield 97%, mp 253-254 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.75 (s, 1H), 9.21 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.3 Hz, 1H), 7.55

(t, J = 7.5, 1.0 Hz, 1H), 7.35-7.45 (m, 6H) , 7.16 (t, J = 7.6 Hz, 1H), 7.09 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.96 (d, J = 15.7 Hz, 1H), 4.51 (d, J = 15.7 Hz, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 161.8, 161.2, 154.6, 149.0, 134.5, 133.7, 132.8, 131.1, 130.1 (2C), 129.0 (2C), 128.7, 128.0, 125.5, 123.3, 122.8, 122.7, 122.5, 116.3, 87.7, 64.1, 45.1; MS (ESI): m/z 462.33 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C24H18BrN2O3:462.3121; Found: 462.3120

3-(2-(4-fluorobenzyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11d)

White solid, yield 95%, mp 249-250 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.74 (s, 1H), 9.23 (s, 1H), 8.15 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.55 (t, J = 7.3 Hz, 1H), 7.35-7.50 (m, 4H) , 7.13-7.20 (m, 3H), 7.09 (dd, J = 8.0, 1.0 Hz, 1H), 6.60 (s, 1H), 4.95 (d, J = 15.7 Hz, 1H), 4.47 (d, J = 15.7 Hz, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 175.0, 161.9, 161.1, 161.0, 154.6, 149.0, 133.6, 131.7, 131.1, 130.5 ( 3 J CF = 7.0 Hz) (2C), 128.8, 128.0, 125.5, 123.2, 122.8, 122.7, 122.5, 116.3, 115.9 ( 2 J CF = 21.3 Hz) (2C), 87.8, 63.9, 44.9; MS (ESI): m/z 401.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C24H18FN2O3: 401.4123; Found: 401.4112

2-hydroxy-3-(3-imino-2-(4-methoxybenzyl) isoindolin-1-yl)-4H-chromen-4-one (11e)

White solid, yield 95%, mp 244-245 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.72 (s, 1H), 9.20 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.54 (t, J = 7.3 Hz, 1H), 7.38-7.46 (m, 3H), 7.35(d, J = 7.4 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.92 (d, J = 15.5 Hz, 1H ), 4.37 (d, J = 15.5 Hz, 1H), 3.72 (s, 3H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 174.9, 161.9, 160.7, 159.3, 154.6, 148.9, 133.5, 131.7, 131.1, 130.0 (2C), 129.9, 128.8, 128.0, 127.4, 125.6, 122.9, 122.6, 116.3, 114.5 (2C), 87.9, 63.7, 55.5, 44.9; MS (ESI): m/z 413.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 25 H 21 N 2 O 4 :413.4212; Found: 413.4208

3-(2-(3,4-dimethoxyphenethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11f)

White solid, yield 95%, mp 237-238 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.48 (s, 1H), 8.95 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.3 Hz ,1H), 7.53 (t, J = 7.3 Hz, 1H), 7.36-7.47 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.09 (dd, J = 8.0, 1.0 Hz, 1H), 6.82 (d, J = 7.3 Hz, 1H), 6.79 (s, 1H), 6.71 (d, J = 7.3 Hz, 1H), 6.66 (s, 1H), 3.99 (m, 1H ), 3.68 (s, 3H), 3.67 (s, 3H), 3.43 (m, 1H), 2.99(m, 1H), 2.87 (m, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 175.0, 161.9, 160.4, 158.4, 154.6, 149.1, 147.9, 133.3, 131.1, 130.8, 128.8, 127.9, 125.6, 123.4, 123.0, 122.8, 122.5, 121.0, 116.3, 112.6, 112.1, 87.8, 63.5, 55.9, 55.5, 43.8, 32.7; MS (ESI): m/z 457.44 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 27 H 24 N 2 O 5 : 457.1707; Found: 457.1692

2-hydroxy-3-(3-imino-2-propylisoindolin-1-yl)-4H-chromen-4-one (11g)

White solid, yield 97%, mp 247-248 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.62 (s, 1H), 9.06 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 7.6, Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.37-7.53 (m, 3H), 7.19 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.0 Hz, 1H), 6.65 (s, 1H), 3.69 (m, 1H), 3.26 (m, 1H), 1.96 (m, 2H), 0.85 (m, 3H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 174.9, 161.9, 160.7, 154.5, 148.9, 133.3, 131.1, 129.0, 127.9, 125.5, 123.4, 123.0, 122.8, 122.5, 116.3, 88.3, 63.5, 43.9, 20.4, 19.8; MS (ESI): m/z 333.45 [M-H] - ; HRMS (ESI) [M-H] - Calcd for C 20 H 17 N 2 O 3 :333.1367; Found: 333.1364

3-(2-butyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11h)

White solid, yield 95%, mp 230-231 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.59 (s, 1H), 9.02 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.57-7.64 (m, 2H), 7.50 (t, J 7.3, 1H), 7.36-7.45 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1.0, 1H), 6.64 (s, 1H), 3.72 (m, 1H), 3.26 (m, 1H), 1.64 (m, 1H), 1.27 (m, 1H), 0.82 (m, 3H); 13 C NMR (100 MHz, DMSO) δ 174.9, 161.9, 160.6, 154.5, 148.8, 133.3, 131.1, 129.1, 127.9, 125.5, 123.3, 123.0, 122.7, 122.5, 116.3, 88.2, 63.5, 42.2, 29.5, 19.9, 14.0; MS (ESI): m/z 349.5 [M+H] + ; HRMS (ESI) Calcd for C 21 H 21 N 2 O 3 [M+H] + :349.1547; Found: 349.1543

2-hydroxy-3-(3-imino-2-isobutylisoindolin-1-yl)-4H-chromen-4-one (11i)

White solid, yield 94%, mp 255-256 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.55 (s, 1H), 8.98 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.55– 7.52 (m, 2H), 7.35–7.45 (m, 2H), 7.18 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 8.0, 1.0 Hz, 1H), 6.61 (s, 1H), 3.53 (m, 1H), 3.07 (m, 1H), 2.17 (m, 1H), 0.87 (m, 6H); 13 C NMR (100 MHz DMSO- d 6 ) δ 174.7, 161.8, 161.1, 154.5, 148.9, 133.4, 131.1, 128.9, 127.9, 125.6, 123.4, 123.0, 122.8, 122.5, 116.3, 88.0, 63.8, 49.2, 27.2, 20.4, 19.8; MS (ESI): m/z 347.40 [M-H] - ; HRMS (ESI) [M-H] - Calcd for C 21 H 19 N 2 O 3 :347.1545; Found: 347.1543

3-(2-(cyclopropylmethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11k)

White solid, yield 97%, mp 251-252 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.57 (s, 1H), 9.02 (s, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.53 (t, J = 7.3 Hz, 1H), 7.36-7.45 (m, 2H), 6.75 (s, 1H), 3.74 (m, 1H), 2.99 (m, 1H), 1.22 (m, 1H), 0.42-0.58 (m, 3H), 0.20-0.29 (m, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 174.8, 161.8, 160.3, 154.5, 149.0, 133.4, 131.1, 129.0, 127.9, 125.5, 123.4 123.0, 122.8, 122.5, 116.2, 88.0, 63.7, 46.6, 9.8, 4.1, 3.4; MS (ESI): m/z 347.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for

3-(2-cyclohexyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11l)

White solid, yield 91%, mp 252-253 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.45 (s, 1H), 8.88 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.95 (dd, J = 7.6, 1.0 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.26 (dd, J = 7.3, 1.0 Hz, 1H), 7.17 (t, J 7.5 Hz, 1H), 7.06 (dd, J = 8.0, 1.0 Hz, 1H), 6.66 (s, 1H), 3.84 (m, 1H), 1.74-2.00 (m, 4H),

1.40-1.62 (m, 3H), 1.19-1.36 (m, 2H), 0.86 (m, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 173.4, 161.7, 160.3, 154.4, 149.1, 133.1, 130.9, 129.2, 127.7, 125.6, 123.5, 122.6, 122.5, 122.0, 116.2, 90.1, 62.7, 55.4, 29.8, 29.5, 29.3, 25.9, 25.2; MS (ESI): m/z 373.45 [M-H] - ; HRMS (ESI): m/z [M-H] - Calcd for C23H21N2O3:373.1542; Found: 373.1645

2-hydroxy-3-(2-(2-hydroxyethyl)-3-iminoisoindolin-1-yl)-4H-chromen-4-one (11m)

White solid, yield 94%, mp 243-244 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.57 (s, 1H), 8.98 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.53 (t,

123.4, 122.9, 122.8, 122.6, 116.3, 88.2, 63.9, 58.5, 45.0; MS (ESI): m/z 347.41 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C19H17N2O4:347.1143; Found: 337.1132

2-hydroxy-3-(2-(3-hydroxypropyl)-3-iminoisoindolin-1-yl)-4H-chromen-4-one (11n)

6.63 (s, 1H), 3.75 (m, 1H), 3.45-3.47 (m, 2H), 3.30 (m, 1H), 1.82 (m, 2H) 13 C NMR (100 MHz, DMSO-d 6 ) δ 175.0, 161.8, 160.7, 154.5, 148.9, 133.2, 131.1, 129.0, 127.9, 125.5, 123.4, 122.8, 122.6, 122.5, 116.3, 88.2, 63.9, 58.5, 45.7, 30.6; MS (ESI): m/z 351.44 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C20H19N2O4:351.1303; Found: 351.1302

2-hydroxy-3-(2-(2-hydroxy-3-methylbutyl)-3-iminoisoindolin-1-yl)-4H-chromen-4-one (11o)

J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.28 (dd, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H),

Hz, 1H), 2.09 (m, 1H), 0.83-0.92 (m, 6H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 174.2, 162.5, 162.2, 154.5, 149.5, 133.3, 131.0, 128.9, 127.6, 125.6, 123.4, 122.8, 122.5, 122.0, 116.2, 89.5, 65.4, 60.2, 49.1, 27.9, 20.5 20.4; MS (ESI): m/z 379.41 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C22H23N2O4:379.1632; Found: 379.1630

3-(2-(2-(1H-indol-3-yl) ethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11p)

White solid, yield 93%, mp 238-239 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.89 (s, 1H), 9.59 (s, 1H), 9.10 (s, 1H), 8.09-8.14 (m, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.40-7.52 (m, 4H),

7.25-7.35 (m, 2H), 7.17-7.19 (m, 2H), 7.02 (dd, J = 8.0, 1.0 Hz, 1H), 6.86 (s, 1H), 6.73 (t, J 7.3 Hz, 1H), 4.05 (m, 1H), 3.60 (m, 1H), 3.24 (m, 1H), 3,09 (m, 1H); 13 C NMR (100 MHz, DMSO-d 6 ) δ 175.2, 162.2, 160.6, 154.6, 148.9, 136.5, 133.3, 131.3, 129.1, 127.9, 127.4, 125.7, 123.6, 123.0, 122.8, 122.7, 122.5, 121.4, 118.8, 118.7, 116.4, 111.8, 110.8, 88.4, 63.5, 43.4, 23.3; MS (ESI): m/z 436.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C27H22N3O3: 436.1612; Found: 436.1610

3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4-one (11q)

White solid, yield 98%, mp 248–249 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.73 (s, 1H), 9.21 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.71 (s, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.54 (m, 1H),

7.33–7.45 (m, 5H), 7.30(d, J = 7.6 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.01 (dd, J = 8.0, 1.0 Hz, 1H), 6.60 (s, 1H), 4.98 (d, J = 15.5 Hz, 1H), 4.46 (d, J = 15.5 Hz, 1H), 2.35 (s, 3H); 13 C NMR

(101 MHz, DMSO-d 6 ) δ 175.0, 162.0, 161.0, 152.6, 149.0, 135.5, 133.6, 131.0, 129.1 (2C), 128.8, 128.2 (2C), 128.1, 128.0, 125.4, 123.2, 123.1, 122.7, 122.5, 116.1, 87.8, 64.0, 45.5, 20.9; MS (ESI): m/z 397.45 [M+H] + ; HRMS (ESI) Calcd for C25H21N2O3 [M+H] + :397.1527; Found: 397.1520

3-(2-(4-chlorobenzyl)-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4-one (11r)

White solid, yield 97%, mp 250-251 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.77 (s, 1H),

Hz, 1H), 7.33-7.44 (m, 5H), 7.22 (d, J = 7.3 Hz, 1H), 7.01 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.95 (dd, J = 15.5 Hz, 1H), 4.48 (d, J = 15.5 Hz, 1H), 2.25-2.35 (3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 175.1, 161.3, 161.1, 152.6, 149.1, 134.5, 133.6, 132.8, 131.8, 131.4, 130.1 (2C), 129.0 (2C), 128.7, 128.0, 125.4, 123.2, 122.7, 122.4, 116.1, 87.8, 64.1, 45.0, 20.9; MS (ESI): m/z 431.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 25 H 20 ClN 2 O 3 :431.1117; Found: 431.1110

2-hydroxy-3-(3-imino-2-(4-methoxybenzyl)isoindolin-1-yl)-6-methyl-4H-chromen-4-one (11s)

White solid, yield 96%, mp 251-252 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.72 (s, 1H), 9.22 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.32-7.41 (m, 3H), 7.23 (t, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.92 (dd, J = 15.7 Hz, 1H), 4.48 (d, J = 15.7 Hz, 1H), 3.71 (s, 3H), 2.25-2.36 (3H); 13 C NMR

(101 MHz, DMSO-d 6 ) δ 175.0, 162.1, 160.3, 159.3, 152.6, 149.0, 135.5, 133.6, 131.8, 131.4, 130.1 (2C), 128.8, 128.0, 127.4, 125.4, 123.2, 122.6, 122.5, 116.1, 114.5 (2C), 87.9, 63.7, 55.5, 44.9, 20.9; MS (ESI): m/z 427.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C 26 H 22 N 2 O 4 : 427.1621; Found: 427.1612

3-(2-(3,4-dimethoxyphenethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4- one (11t)

White solid, yield 97%, mp 227-228 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.49 (s, 1H), 8.69 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.78(s, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 7.36 (m, 3H), 7.23 (t, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.0, 1.0 Hz, 1H), 6.81(d, J = 7.9 Hz, 1H), 6.78 (s, 1H), 6.72 (d, J = 7.9 Hz, 1H), 3.99 (m, 1H), 3.57-3.67 (6H), 3.48 (m, 1H), 2.98 (m, 1H), 2.86 (m, 1H), 2.24-2.37 (m, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 175.1, 162.1, 160.4, 152.6, 149.1, 149.0, 147.9, 133.3, 131.8, 131.4, 130.8, 128.9, 127.8, 125.4, 123.0, 122.5, 121.0, 116.1, 112.1, 112.2, 87.9, 63.5, 55.8, 55.5, 43.8, 32.7, 20.9; MS (ESI): m/z

3-(2-butyl-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4-one (11u)

White solid, yield 95%, mp 235-236 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.49 (s, 1H), 8.92 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.34 (m, 1H), 7.22 (dd, J = 7.8, 1.0 Hz, 1H), 7.02 (dd, J = 8.0, 1.0 Hz, 1H), 6.60 (s, 1H), 3.68 (m, 1H), 3.23 (m, 1H), 2.24-2.36 (3H), 1.63 (m, 2H), 1.27 (m, 2H), 0.84 (m, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 174.9, 161.9, 160.5, 152.6, 149.0, 133.2, 131.2, 131.7, 131.3, 129.0, 127.8, 125.4, 123.1, 122.9, 122.5, 116.1, 87.9, 63.7, 42.2, 29.4, 20.9, 19.9, 14.0; MS (ESI): m/z 363.45 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C22H23N2O3: 363.1623; Found: 363.1614

2-hydroxy-3-(2-(2-hydroxyethyl)-3-iminoisoindolin-1-yl)-6-methyl-4H-chromen-4-one (11v)

White solid, yield 91%, mp 233-234 o C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.50 (s, 1H), 8.91 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.53 (d, J = 7.5

Hz, 1H), 7.34 (m, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.00 (dd, J = 8.0, 1.0 Hz, 1H), 6.60 (s, 1H), 5.09 (1H), 3.72-3.91 (m, 2H), 3.18-3.29 (m, 1H), 2.24-2.35 (3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 174.9, 162.0, 161.3, 152.6, 149.1, 133.2, 131.8, 131.4, 131.3, 129.1, 127.8, 125.3, 122.9, 122.5, 116.0, 88.1, 63.6, 58.4, 44.9, 20.9; MS (ESI): m/z 351.41 [M+H] + ; HRMS (ESI): m/z [M+H] + Calcd for C20H19N2O4:351.1318; Found: 363.1310

3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-benzo[h]chromen-4-one (11w)

White solid, yield 92%, mp 255–256 o C; 1 H NMR (400 MHz, DMSO-d 6 ): δ 9.83 (s, 1H), 9.31 (s, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.53- 7.69 (m, 5H), 7.40–7.45 (m, 3H), 7.33-7.37 (m, 2H), 7.28 (dd, J = 7.3, 1.0 Hz, 1H), 7.10 (dd, 8.0, 1.0 Hz, 1H), 6.68 (s, 1H), 4.03 (d, J = 15.7 Hz, 1H), 4.54 (d, J = 15.7 Hz, 1H); 13 C NMR

(101 MHz, DMSO-d 6 ) δ 175.5, 161.7, 161.1, 150.8, 148.9, 135.5, 134.9, 133.6, 131.1, 129.1 (2C), 128.9, 128.2 (2C), 128.1, 127.8, 126.6, 123.6, 123.3, 122.8, 122.6, 122.1, 122.0, 121.7, 118.4, 117.8, 88.2, 64.0, 45.6; MS (ESI): m/z 433.45 [M+H] + ; HRMS (ESI) Calcd for

3-(2-butyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-benzo[h]chromen-4-one (11x)

White solid, yield 90%, mp 237–238 o C; 1 H NMR (400 MHz, DMSO-d 6 ): δ 9.58 (s, 1H), 9.00 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.93 (dd, J = 7.6 Hz, 1H), 7.50-7.69 (m, 5H), 7.41 (m, 1H), 6.67 (s, 1H), 3.74 (m, 1H), 3.30 (m,

1H), 1.66 (m, 2H), 1.28 (m, 2H), 0.83 (m, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 175.4, 161.6, 160.7, 150.7, 148.8, 134.9, 133.3, 129.1, 128.2, 127.8, 126.6, 123.6, 123.0, 122.5, 122.1, 122.0, 121.1, 118.3, 117.7, 88.3, 63.5, 42.3, 29.5, 19.9, 14.0; MS (ESI): m/z 399.40

[M+H] + ; HRMS (ESI) Calcd for C 25 H 23 N 2 O 3 [M+H] + :399.1607; Found: 399.1601

1.3.4.3 Preparation of Calcium flux functional assay

NR1a/NR2B receptor transfected L(tk-) cells were plated in 96-well format at 3 x 10 4 cells/well and grown for 1 day in normal growth medium (Dulbeccos MEM with Na pyruvate,

4500 mg glucose, pen/strep, glutamine, 10% FCS and 0.5 mg/mL geneticin) Receptor expression was induced by the addition of 10 nM dexamethasone in the presence of 500 àM ketamine for 15–24 h Solutions of NR2B antagonists were prepared in DMSO and serially diluted with DMSO to yield 10 solutions differing by 3-fold in concentration 96-well drug plate was prepared by diluting the DMSO solution 250-fold into assay buffer (Hanks balanced salt solution (HBSS) Mg 2+ free containing 20 mM HEPES, 2 mM CaCl 2 , 0.1% BSA, and 250 àM probenecid After induction, the cells were washed twice with assay buffer and loaded with 4 àM of the calciumfluorescence indicator fluo-3 AM in assay buffer containing pluronic F-127 and 100 àM ketamine at 37 o C for 1 h The cells were then washed five times with assay buffer leaving 40 àL of buffer in each well Fluorescence intensity was measured in a fluorometric imaging plate reader (FLIPR), using an excitation of 488 nm, and emission at

530 nm Five seconds after starting the recording of fluorescence intensity, 50 àL of agonist solution (40 àM glutamate/glycine, the final concentration 10 àM) was added and after 1 min, when fluorescence signal was stable, 50 àL of NR2B antagonists and control solutions from the drug plate were added, and then the fluorescence intensity recorded for an other 30 min The IC50 values were determined by non-linear least squares fitting of the end fluorescence values [46-47]

Conclusions

In conclusion, a novel one-pot method for the synthesis of isoindolin-1-imine derivatives (4, 8, and 10) was devoloped This method is a simple three-component condensation of 2- cyanobenzaldhyde, ammonium acetate, and 4-hyroxycoumarin derivatives or 1,3-dicarbonyl compounds or 4-hydroxyquinolin-2(1H)-one in ethanol under reflux for 20-60 minutes with excellent yields In addition, we have also developed a new protocol, simple, efficient procedure for the preparation of 3-(2-substituted-3-iminoisoindolin-1-yl)-2-hydroxy-4H- chromen-4-ones 11 via three-component condensation reaction of 2-cyanobenzaldehyde, primary amine, and 4-hydroxycoumarin derivatives in dry dichloromethane with catalyst-free condition at room temperature These methodologies offer significant efficient for the synthesis of isoindolin-1-imine derivatives with regard to operational simplicity, excellent yields, fast reactive time, without catalyst, and little environmental impact, and are good valid contribution to the synthesis of N-heterocyclic compounds

The inhibitory activities of the synthesized isoindolin-1-imine analogs against NR2B

Ca 2+ flux were investigated The evalution results showed that all tested isoindolin-1-imine derivatives exhibited acceptable inhibitory effects against Ca 2+ flux in cells expressing the NR2B subunit (NR2B Ca 2+ -flux) Noteably, compounds 4l, 11e, 11f, and 11t exhibited stronger potent inhibitory activities

Study on synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-one derivatives via one-pot reaction

Introduction

Isoindolin-1-ones are one of the most important classes of N-heterocyclic biological active substances [50-54] They have been attracted considerable attention from synthetic pharmacists and chemists due to their potent therapeutic and pharmacological activities [50-56] A series of isoindolin-1-one bridged to 4-(1,2-benzisothiazol-3-yl)-1-piperazinyl moiety were revealed acceptable dopamine D2, serotonin 5-HT1a, and 5-HT2 receptor antagonistic actions in vitro, and could antagonize the apomorphine-induced climbing ability in mice [51] In addition, isoindolin-1-ones also exhibited as inhibitory agents of the MDM2-p53 interaction [53-54] , enzymatic and potent cellular inhibitory of KDR kinase activities [55] Otherwise, a series of 2- piperazino-isoindolin-1-one derivatives as nonpeptide urotensin-II (U-II) receptor antagonists in vitro, in vivo were reported [56] (Fig 2.1)

Nonpeptide Urotensin-II Receptor Antagonists Sedative and anxiolytic drug

Fig 2.1 Structure of isoindolin-1-one activity analogs

Due to the potential of isoindolin-1-one derivatives, several methods have been developed and published for the synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-ones [57-59]

In 1999, Nitya G K et al carried out the synthesis of isoindolin-1-ones by a mixture of 2- iodobenzamide or its N-substituted derivatives and a selected acetylenic carbinol in the presence of bis(triphenylphosphine)palladium(II) dichloride and copper(I) iodide as co- catalyst at 80 o C for 24 h (Method A) to give target products in good yields Otherwise, when the palladium and copper (I) iodide as co-catalyst, synthetic reactions were carried out at room temperature only to give disubstituted alkynol mediate products The target products isoindolin-1-ones could be obtain by cyclation of disubstituted alkynols in the presence of sodium ethoxide in ethanol under reflux condition (Method B) (Scheme 2.1) [57]

Scheme 2.1 Synthesis of isoindolin-1-one derivatives by palladium and copper (I) iodide co-catalyzed

In 2009, Wu et al [58] provided a method for synthesis of isoindolin-1-ones through the coupling reaction of carbonyl compounds and 2-alkyl-3-hydroxyisoindol-1-ones catalyzed by boron trifluoride-diethyl ether complex in dichloromethane at room temperature in good yields (Scheme 2.2) [58]

Scheme 2.2 Synthesis of isoindolin-1-ones by boron trifluoridediethyl ether-catalyzed [58]

In 2010, Alice Devineau et al [59] reported a new two-step method for the synthesis of isoindolin-1-ones under efficient DBU-catalyzed trichloroacetimidation of an alcohol compound and following by ditriflylamine (Tf 2 NH) condition in excellent yields (55-100%) The trichloroacetimidate method exhibited useful applications for carbon-carbon bond building procedure (Scheme 2.3) [59]

Scheme 2.3 Synthesis of isoindolin-1-one derivatives by DBU-catalysed [59]

Due to results above, several methods for synthesis of 2-substituted-3-(2- oxoalkyl)isoindolin-1-ones have been reported, but had more drawbacks such as multi-step protocols, and only observed in low to moderate isolated yields, usage of uncommon starting materials, the usage of catalyst agents and reaction solvents which have adverse effect on environment Thus, the development of a green, efficient, and convenient method for the synthesis of this analog is required

Thiamine hydrochloride (VB 1 ), which is salt contains a pyrimidine ring and a thiazole ring linked by a methylene bridge, is a cheap, stable, and a non-toxic agent VB 1 is one of the important nutrient agents of the animals and people, which must obtain from their diet In body, the deficiency of VB 1 could be effect in Korsakoff’s syndrome, optic neuropathy, and affected the peripheral nervous and cardiovascular system In synthetic chemistry, VB1 exhibited as a powerful catalyst to be application in various organic reactions More recently, our group reported VB1-catalyzed reactions for the synthesis of heterocyclic compounds, such as dihydropyridines [60] , 1,2-dihydro-naphth[1,2-e][1,3]oxazine-3-one [61] , and 2,3- dihydroquinazolin-4(1H)-ones [62] in high yields, and short reaction time

Fig 2.2 Structure of thiamine hydrochloride (VB 1 )

In continuation of our group for the development methods for synthesis of N-heterocyclic products, hence we report a green, facile, efficient, and environmentally friendly procedure for the synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-ones in one-pot via three-component condensation reaction of phthalaldehydic acid, primary amine, and ketone in the presence of

VB1 as catalyst (as Scheme 2.4)

Scheme 2.4 Synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-one derivatives

Results and Discussion

2.2.1 The optimization of reaction catalyst

In the initial step, for screening the influence of catalysts on the yields of condensation, a reaction of phthalaldehydic acid 1 (1 mmol), benzylamine 2a (1 mmol), and acetophenone 3a

(1 mmol) in the presence of different catalysts in 4 mL H 2 O was carried at reflux condition The results are summarized in Table 2.1

As shown in Table 2.1, in the presence 10% mol of Et3N (entry 1), NaHCO3 (entry 2), AlCl3 (entry 3), NH4Cl (entry 4), and AcOH (entry 5) as catalysts, product 4a [58-59] was only observed in low to moderate yields

Particularly, when the reaction was carried out in the presence of VB1 as catalyst, yield of product 4a increased to 81% (10% mol VB1, entry 6) and 80% (15% mol VB1, entry 7) for 3 h

In addition, the effect of time on product of yields was obvious from the Table 1 When the reaction time was extended from 1 h to 10 h, the yield of 4a increased from 60 to 89% (entries

6, 9-11) In the absence of catalyst, the reaction only obtained a trace amount of target product (entry 12)

Considering efficient and eco-friendly point of view, VB1 was selected as optimized catalyst for further experiments

Table 2.1 Catalyst screening for the synthesis of 4a

Entry Catalyst Catalyst (mol %) Time (h) Yields 4a a, b (%)

12 None 5 Trace a The reaction carried out in H 2 O at reflux temperature b Isolated yields

2.2.2 The optimization of reaction solvent

In continuation to demonstrate effect of solvents on this procedure, the condensation reaction of phthalaldehydic acid 1 (1 mmol), benzylamine 2a (1 mmol), and acetophenone 3a (1 mmol) in the presence of 10 mol% of VB1 as catalyst was carried out in various solvents (4 mL) under reflux temperature to yield target product 4a The results are summarized in Table 2.2

As shown in Table 2.2, the results suggested that the target product 4a was afforded in low to moderate yields in DCM, toluene, CH3CN and THF (entries 1-4) Using EtOH as solvent reaction, product 4a was observed in high yield (entry 5) for 5 h Noteably, when reaction solvent is water, the yield of 4a increased to 81% for 3 h (entry 6), 88% for 5 h (entry

7), and 89% for 10 h (entry 8) after stirring under reflux In addition, the yields of 4a were improved when the reaction temperature was rised from 20 o C to reflux (entries 7-11)

Therefore, from an efficient and eco-friendly point of view, water was chosen as reaction solvent for further synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-ones 4

Entry Solvent Time (h) Temp ( o C) Yield 4a b (%)

2.2.3 The scope and limitations of reaction substrates

To demonstrate the scope and limitations of this procedure, a series of isoindolin-1-one derivatives 4 were synthesized from different starting materials (Table 2.3; Scheme 2.4)

Table 2.3 Screening scope and limitations of reaction substrates for the synthesis of 4

Initially, series of 2-substituted-3-(2-oxoalkyl)isoindolin-1-ones 4 were synthesized via the reaction condensation of phthalaldehydic acid 1 (1 mmol), amine 2 (1 mmol), and ketone

3 (1 mmol) in the presence of 10 mol% VB1 as catalyst in 4 mL water under reflux to give target products 4 (in the Table 2.3) When compound 3 was acetophenone, this reaction was carried out with various amines 2 (entries 1-12) These results showed that the reactions of primary amines 2 were obtained target products 4 in good yields (entries 1-12) Among them, 4- methoxybenzylamine (entry 2) had the most effect on yield of product 4 Otherwise, the reaction was also prepared with amine 2 as aniline (entry 13) but did not obtain the corresponding product under similar reaction conditions On the other hand, this condensation reaction was carried out with 3 was acetone (entry 14), but product 4 was only observed in very low yield.

The plausible mechanism

A plausible mechanism can reasonably be proposed for the series of products 4 (Scheme 2.6)

In the initial step, the condensation between phthalaldehydic acid 1 and amine 2 gave adduct 5 Then the adduct 5 gave unstable imine intermediate 6 by protonation and dehydration At the same time, ketone 3 isomerizes to 3’ under VB1-catalyzed, subsequent nucleophilic addition of 3’ to 6 afforded 7, which afforded the title isoindolin-1-one 4 via intramolecular cyclization Due to results above, the three-component condensation reaction could not be proceeded smoothly when using the alkyl acetones as the starting materials The probable reason was that it was difficult for these alkyl acetones to form the enol intermediate as 3’, which meant that the reaction could not proceed smoothly to afford the target products 4

Scheme 2.6 Plausible mechanism can reasonably be proposed for the series of products 4

Conclusions

In conclusion, a green, simple, and efficient three-component procedure via one-pot reaction has been developed for the synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-ones from phthalaldehydic acid, primary amine, and ketone in the presence of VB1 as catalyst in the aqueous solution under reflux Prominent advantages of this method for synthesis of isoindolin-1-one derivatives including simplicity of operation, high yields, and using of environmental friendly catalyst Therefore, this procedure is a good valid contribution to the synthesis of isoindolin-1-ones

Experiment section

To a stirred aqueous solution of phthalaldehydic acid (1, 1 mmol), and ketone (3, 1 mmol) was added primary amine (2, 1 mmol) and 10%mol of VB 1 The resulting mixure was stirred under reflux condition for 5 h After completion of the reaction, 30 mL water was added, and then the reaction solution was extracted with DCM (3*30 mL) The combined organic layers were washed with water, brine, and dried over anhydrous sodium sulfate and concentrated under reduced pressure The crude products were purified by chromatography on silica gel column by AcOEt/PE = 1/30 to 1/5 (v/v) to afford the corresponding products 4 (78-90%)

2-benzyl-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4a)

1H NMR (400 MHz, CDCl 3 ) δ 7.93 (m, 1H), 7.81 (d, J = 7.4 Hz, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.53-7.34 (m, 5H), 7.15-7.30 (m, 5H), 5.32-5.22 (m, 1H), 5.09 (d, J = 15.4 Hz, 1H), 4.56 (d,

J = 15.4 Hz, 1H), 3.51 (dd, J = 17.6, 5.3 Hz, 1H), 3.17 (dd, J = 17.6, 7.2 Hz, 1H) [58-59]

MS (ESI): m/z 342.33 [M+H] + ; HR-MS (ESI) Calcd for C23H20NO2 [M+H] + :342.1403; Found: 342.1412

2-(4-methoxybenzyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4b)

1H NMR (400 MHz, CDCl3) δ 7.98-7.86 (m, 1H), 7.81 (m, 2H), 7.58 (t, J = 7.4 Hz, 1H), 7.38-7.56 (m, 4H), 7.15-7.33 (m, 3H), 6.92 (d, J = 8.2 Hz, 2 H), 5.33-5.21 (m, 1H), 5.07 (d, J

= 15.3 Hz, 1H), 4.56 (d, J = 15.3 Hz, 1H), 3.52 (dd, J = 17.6, 5.3 Hz, 1H), 3.18 (dd, J = 17.6, 7.2 Hz, 1H)

MS (ESI): m/z 372.40 [M+H] + ; HR-MS (ESI) Calcd for C 24 H 22 NO 3 [M+H] + :372.1508; Found: 372.1502

2-(4-chlorobenzyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4c)

1H NMR (400 MHz, CDCl3) δ 7.92 (m, 1H), 7.83 (m, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.38- 7.56 (m, 6H), 7.15-7.30 (m, 3H), 5.32-5.21 (m, 1H), 5.07 (d, J = 15.4 Hz, 1H), 4.56 (d, J 15.4 Hz, 1H), 3.53 (dd, J = 17.6, 5.3 Hz, 1H), 3.19 (dd, J = 17.6, 7.3 Hz, 1H)

MS (ESI): m/z 375.50 [M+H] + ; HR-MS (ESI) Calcd for C 23 H 19 ClNO 2 [M+H] + :375.1038; Found: 375.1030

2-(4-bromobenzyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4d)

1H NMR (400 MHz, CDCl3) δ 7.93 (m, 1H), 7.83 (m, 2H), 7.57 (t, J = 7.3 Hz, 1H), 7.38- 7.56 (m, 4H), 7.15–7.30 (m, 4H), 5.33-5.22 (m, 1H), 5.08 (d, J = 15.4 Hz, 1H), 4.56 (d, J 15.4 Hz, 1H), 3.52 (dd, J = 17.6, 5.3 Hz, 1H), 3.18 (dd, J = 17.6, 7.3 Hz, 1H)

MS (ESI): m/z 420.45 [M+H] + ; HR-MS (ESI) Calcd for C23H19BrNO2 [M+H] + :420.0512; Found:420.0501

2-(4-fluorobenzyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4e)

1H NMR (400 MHz, CDCl 3 ) δ 7.91 (m, 1H), 7.80 (m, 2H), 7.55 (t, J = 7.3 Hz, 1H), 7.38- 7.56 (m, 4H), 7.10-7.25 (m, 4H), 5.32-5.21 (m, 1H), 5.05 (d, J = 15.3 Hz, 1H), 4.54 (d, J 15.3 Hz, 1H), 3.52 (dd, J = 17.4, 5.2 Hz, 1H), 3.18 (dd, J = 17.4, 7.3 Hz, 1H)

MS (ESI): m/z 360.44 [M+H] + ; HR-MS (ESI) Calcd for C23H19FNO2 [M+H] + : 360.1319; Found:360.1324

MS (ESI): m/z 356.35 [M+H] + ; HR-MS (ESI) Calcd for C24H22NO2 [M+H] + : 356.1611; Found:356.1605

1H NMR (400 MHz, CDCl 3 ) δ 7.93-7.95 (m, 2H), 7.83 (m, 1H), 7.57 (m, 1H), 7.40-7.50 (m, 5H), 5.31-5.20 (dd, J = 7.2, 5.3 Hz, 1H), 3.68 (m, 1H), 3.51 (dd, J = 17.6, 5.3 Hz, 1H), 3.27 (m, 1H), 3.17 (dd, J = 17.6, 7.2 Hz, 1H), 1.73 (m, 2H), 0.84 (m, 3H)

MS (ESI): m/z 294.55 [M+H] + ; HR-MS (ESI) Calcd for C19H20NO2 [M+H] + : 294.1425; Found: 294.1415

2-butyl-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4h)

1H NMR (400 MHz, CDCl3) δ 7.92-7.91 (m, 2H), 7.80 (m, 1H), 7.54 (m, 1H), 7.42-7.50 (m, 5H), 5.28-5.20 (dd, J = 7.3, 5.2 Hz, 1H), 3.73 (m, 1H), 3.51 (dd, J = 17.8, 5.2 Hz, 1H), 3.25 (m, 1H), 3.17 (dd, J = 17.8, 7.3 Hz, 1H), 1.70 (m, 2H), 1.25 (m, 2H), 0.83 (m, 3H) [59]

MS (ESI): m/z 308.45 [M+H] + ; HR-MS (ESI) Calcd for C 20 H 22 NO 2 [M+H] + : 308.1413; Found: 308.1403

2-isobutyl-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4i)

1H NMR (400 MHz, CDCl3) δ 7.94-7.91 (m, 2H), 7.83 (m, 1H), 7.52 (m, 1H), 7.40-7.48 (m, 5H), 5.26-5.18 (dd, J = 7.2, 5.3 Hz, 1H), 3.60 (m, 1H), 3.51 (dd, J = 17.8, 5.3 Hz, 1H), 3.17 (dd, J

MS (ESI): m/z 308.40 [M+H] + ; HR-MS (ESI) Calcd for C 20 H 22 NO 2 [M+H] + : 308.1413; Found: 308.1403

2-(cyclopropylmethyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4k)

1H NMR (400 MHz, CDCl 3 ) δ 7.95-7.93 (m, 2H), 7.84 (m, 1H), 7.53 (m, 1H), 7.40-7.49 (m, 5H), 5.23-5.17 (dd, J = 7.2, 5.3 Hz, 1H), 3.72 (m, 1H), 3.54 (dd, J = 17.6, 5.3 Hz, 1H), 3.16 (dd, J

2-(2-hydroxyethyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4l)

1H NMR (400 MHz, CDCl 3 ) δ 7.95-7.92 (m, 2H), 7.85 (m, 1H), 7.55 (m, 1H), 7.41-7.48 (m, 5H), 5.25-5.18 (dd, J = 7.3, 5.3 Hz, 1H), 3.70-3.85 (m, 2H), 3.68 (m, 1H), 3.53 (dd, J = 17.6, 5.3

2-(2-hydroxy-3-methylbutyl)-3-(2-oxo-2-phenylethyl)isoindolin-1-one (4m)

1H NMR (400 MHz, CDCl3) δ 7.94-7.91 (m, 2H), 7.85 (m, 1H), 7.54 (m, 1H), 7.41-7.47 (m, 5H), 5.25-5.18 (dd, J = 7.3, 5.2 Hz, 1H), 4.17 (m, 1H) , 3.62-3.73 (m, 2H), 3.53 (dd, J = 17.7, 5.2

Hz, 1H), 3.15 (dd, J = 17.7, 7.3 Hz, 1H), 2.00-2.06 (m, 1H), 0.81-0.91 (m, 6H); MS (ESI): m/z 338.40 [M+H] + ; HR-MS (ESI) Calcd for C 21 H 23 NO 3 [M+H] + : 338.1713; Found: 338.1714

Study on C 21 steroidal glycosides from the stems of Marsdenia tenacissima

Introduction

Marsdenia tenacissima (Roxb.) (M tenacissima), is one of important species of Marsdenia genus of plant in family Asclepiadaceae [63-65] (Fig 3.1) It is widely grown in the southwestern provinces of China, and tropical regions [63-71]

Fig 3.1 Marsdenia tenacissima (Roxb.) plant

M tenacissima is used as herbal medicine for the treatment of asthma, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia [64-66] In particular, the extract of M tenacissima has been shown to have anticancer activity [64-68] There are major active constituents in M tenacissima such as C21 steroidal glycosides, alkaloids, flavonoids, and other components [64-

In the clinical trials, M tenacissima extract has been used for treating patients with cancers such as esophageal cancer, gastric cancer and lung cancer [63-65] It exhibited growth inhibitory activity of cancer cells However, whether M tenacissima inhibits tumor angiogenesis and its underlying mechanism(s) in endothelial cells are unclear [63-71] Otherwise, it also has effects in immune system such as lymphocytes and concanavalin LPS-induced proliferation ability of human lymphocytes and other activities [74-75]

Previous studies on its chemical compositions, pharmacological effects are detailed summarized as follow

To date, ninety one C21 skeleton compounds, which are the most important chemical compositions, were isolated from stems of M tenacissima, such as tenacigenins derivatives, diester derivatives of tenacigenin B, and glycosides of tenacigenin B derivatives, marsdenoside, tenacissosides, and marsdekoiside, tenacigenoside derivatives, etc [76-103] All isolated compounds from this plant were summarized as below:

11α-O-Tigloyl-12β-O-acetyltenacigenin B H Tig Ac [77] 11α-O-2-Methylbutyryl-12β-O- acetyltenacigenin B

11α,12β-Di-O-tigloyl-tenacigenin B H Tig Tig [90]

Marsdenoside A Allo-Ole Bu Tig [91]

Marsdenoside B Allo-Ole Tig Tig [91]

Marsdenoside C Allo-Ole Bu Bz [91]

Marsdenoside E Allo-Ole Pro Ac [91]

Marsdenoside F Allo-Ole Ac Ac [91]

Marsdenoside H Glc-Allo-Ole Bu Ac [91]

Marsdenoside J Allo-Ole HPA Ac [82]

Marsdenoside K Glc-Allo-Cym Bz Ac [82]

Tenacissoside A Glc-Allo-Ole Tig Ac [85]

Tenacissoside B Glc-Allo-Ole Tig Tig [85]

Tenacissoside C Glc-Allo-Ole Bz Tig [85]

Tenacissoside D Glc-Allo-Ole Bu Tig [85]

Tenacissoside E Glc-Allo-Ole Bz Bu [85]

Tenacissoside G Allo-Ole Tig Ac [92]

Tenacissoside H Allo-Ole Bu Ac [92]

Tenacissoside I Allo-Ole Bz Ac [92]

Tenacissoside N Glc-Glc-Allo-Ole Ac Tig [93]

Tenacigenoside E Glc-Glc-Allo-Ole Tig Tig [90]

3-O-6-Deoxy-3-O-methyl-β-D- allopyranosyl-(1-4)-β-D-oleandropyranosyl tenacigenin C

Marsdekoiside B Allo-Ole-Cym Bz H [78]

Marsdeoreophiside B Glc-Allo-Ole-Cym Cin H [78]

Tenacissoside J Glc-Glc-Allo-Ole-Cym Bz H [78]

Tenacissoside K Glc-Allo-Ole-Cym Bz H [96]

Tenacissoside M Thv-Cym-Cym H Ac [97]

Marstenacisside A Glc-Thv-Ole-Cym-Dig H H [98]

Marstenacisside B Glc-Thv-Ole-Cym H H [98]

Marstenacisside C Glc-Glc-Thv-Ole-Cym H H [98]

Marstenacisside D Glc-Glc-Thv-Ole-Cym-Cym H H [98]

Marstenacisside E Glc-Thv-Ole-Cym Bz H [88]

Marstenacisside F Glc-Thv-Ole-Cym-Dig Bz H [88]

Marstenacisside G Glc-Glc-Thv-Ole-Cym H H [88]

Marstenacisside H Glc-Thv-Ole-Cym-Dig H - [88]

Marstenacisside I Glc-Thv-Ole-Cym-Cym H - [88]

Marstenacisside J Glc-Glc-Thv-Ole-Cym Ac - [88]

Thevetopyranosyl-(1→4)-β- oleandropyranosyl-(1→4)-β- cymaropyranosyl-(1→4)-β- digitoxopyranoxy-8,14,17,20- tetrahydroxypregnan-20-yl cinnamate

Thv-Ole-Cym-Dig H Cin [80]

Thevetopyranosyl-(1→4)-β- oleandropyranosyl-(1→4)-β- digitoxopyranoxyl-(1→4)-β- digitoxopyranoxy-8,14,17,20- tetrahydroxypregnan-12-ylbenzoate

Thv-Ole-Dig-Dig Bz H [80]

Thv-Ole-Cym-Dig Bz H [80]

Compound R 1 R 2 R 3 Reference digitoxopyranoxy-8,14,17,20- tetrahydroxypregnan-12-yl benzoate

Thevetopyranosyl-(1→4)-β- oleandropyranosyl-(1→4)-β- cymaropyranosyl-(1→4)-β- digitoxopyranoxy-8,14,17,20- tetrahydroxypregnan-12-yl cinnamate

Thv-Ole-Cym-Dig Cin H [80]

S3 Allo-Ole-Dig-Dig Bz H [99]

S4 Allo-Ole-Cym-Dig Bz H [99]

S5 Allo-Ole-Cym-Dig Cin H [99]

Tri-O-acetylsogenin Ac Tig Ac Ac [101]

Mono-O-ocetyldrevo Q (17β) Ac Isoval Ac [102]

3,12-Di-O-acetyl iso-drevogenin P (17α) Ac H Ac [102]

Tri-O-acetyl iso-drevogenin P (17α) Ac Ac Ac [102]

Otherwise, some flavonoids, alkaloids, sterols, etc were isolated from this plant [63-65, 103]

3.1.2 Biological activities of of M tenacissima

3.1.2.1 Biological activities of the total extract of M tenacissima

In the early 1970s, M tenacissima extract was firstly applied into treatment of malignant tumors Up to date, M tenacissima extract has been reported in in vitro and in vivo anti-tumor activities The results were summarized in Table 3.1

Table 3.1 Anti-cancer activity of total extract of M tenacissima

In vitro, growth inhibition activity: malignant lymphoma, esophageal cancer, lung cancer, stomach, liver, and cervical

M tenacissima extract exhibit inhibition activity of cancer cell lines (% inhibition rate): malignant lymphoma (40.0%), esophageal cancer (26.3%), lung cancer

Growth inhibition activity in mice:

- Cell lines: Transplant stomach cancer S180 cells, mice transplant H22 cells, transplant stomach cancer P388 cells

- Dose: 18, 9, and 4.5 mg/kg treatment in 10 days

- Mice transplant H22 cells (18, 9, and 4.5 mg/kg): 49.32, 32.04, and 22.3 (%)

Antitumor effect of adenoviral- mediated p53 gene transfer in an ascitic tumor-bearing mouse model:

- Dose: 4 mL/mouse/day (0.1M) in 7 days

Anti-cancer mutation and anti- tumor in mouse:

- Dose: 0.1, 0.2, and 0.4 mg/mL, 1 mL/time, 2 times/day in 24 days

- S180 cells (Dose: 0.1, 0.2, and 0.4 mg/mL): 1.84, 1.61, and 1.52 (g), and control (3.42 g)

- H22 cells (Dose: 0.1, 0.2, and 0.4 mg/mL): 1.89, 1.68, and 1.54 (g), and control (3.58 g)

In addition, “Ai-xiao-ping”, made from M tenacissima extract, was proven by SFDA in

1987 It was used for the treatment of digestive malignancies tumors, especially in primary liver cancer, esophageal and gastric cancer, and lung cancer in clinic It also had synergistic anti-tumor activity when combined with other treatments such as chemotherapy or radiation

Otherwise, M tenacissima extract exhibited effects on the immune system such as lymphocytes M tenacissima extract could promote T and B cell proliferation in normal immune cells with the non-cytotoxic concentration [117-118] It was also used for the treatment of chronic bronchitis, pneumonia, laryngitis, gastritis, stomach ulcers, nephritis, etc [119]

3.1.2.2 Biological activities of M tenacissima steroids

The cytotoxicities of C 21 steroidal glycosides from M tenacissima were summarized in the Table 3.1

Table 3.1 Activity of steroid glycosides extract from M tenacissima

In vitro: growth inhibition activity Bel-7402 cells

All compounds exhibit inhibition activity

Tenacissoside L-P showed the strongest inhibition activity of SGC -7901 cells; And on mouse colon C-

26 cells inhibited the weakest inhibition activity

P-Glycoprotein- Mediated multidrug resistance in HepG2/Dox cells

The sensitivity of HepG2/Dox cells to the antitumor drugs doxorubicin, vinblastine, puromycin, and paclitexel was increased by 18-, 10- , 11-, and 6-fold by 20 àg/mL (or

25 àM) of 1, and 16-, 53-, 16-, and 326-fold by 20 àg/mL (or 39 àM) of 2

Tenacissoside I In vitro: growth inhibition activity of SGC-7901, K562, Bel-7402, SMMC-

IC 50 (μg/mL): 12.27, 53.34, 18.39, 10.92, 6.35, 8.04, 49.51, 12.89, 14.22, and 72.23, respectively DDP is as a positive control IC50 values of Tenacissoside I > DDP in BGC-

Growth inhibition activity in S180 cells at concentration of 300, 150, and 75 mg/mL, inhibition rates as 54.36, 32.29, and 9.45% It exhibits weaker activity than DDP (79.52%)

Because M tenacissima extract has low in vitro cytotoxicity, and potent in vivo anti- tumor activity, its anti-tumor activity may be from enhancing the immune system

Due to the activities of M tenacissima, we are interested to conduct systematic study C21 steroidal glycosides of M tenacissima.

Results and discussion

The 80% (v/v) ethanolic extract of the stems of M tenacissima was prepared to repeat column chromatography (silica gel and reversed phase silica gel) with various solvent systems to yield twenty C21 steroidal glycosides including 3 new compounds and 17 known ones

The structures of all isolated compounds were confirmed by NMR and MS data The structures of compounds were summarized in Fig 3.2

Glc-Glc-Allo-Ole Ac Tig

Glc-Allo-Ole Tig Ac

Bu Glu-Allo-Cym Bz Ac

Glc-Thv-Ole-Cym Bz H Allo-Ole-Cym Bz H

Fig 3.2 Structure of isolated compounds

Compound 10 was isolated as a white amorphous powder, the molecular formula was established to be C45H70O14 by EIS-MS (m/z 857.50, [M+Na] + ) In the 1D and 2D NMR spectra of 10, we showed that the patterns suggested to a C21 steroid skeleton [88-91] In the 1 H and 13 C NMR spectra of compound 10, two anomeric proton signals at δ H 4.82 (d, J = 8.2 Hz, 1H) and 4.61 (d, J = 9.5 Hz, 1H), with two corresponding carbon signals at δ C 99.1 and 95.6, respectively (Table 3.2) indicated that structure of 10 consists two sugars

Then, compound 10 carried out hydrolysis by HCl 0.5 % in methanol In the acid hydrolysis solution of 10, two sugars as cymarose and 6-deoxy-3-O-methyl-β-allopyranose were identified by TLC procedure in comparison with authentic samples The structures of the two sugars were determined by NMR analysis and in comparison with data in reported literatures [88-91]

NMR spectroscopic data of 10 displayed proton and carbon signals at δ H 2.24 (s, 3H) and δ C 30.0 to assume the presence of acetyl group at C-21 position [83, 92] The proton signals at δ H 0.80 (t, J = 7.4 Hz, 3H), 0.99 (d, J = 5.7 Hz, 3H), a ester carbonyl carbon at δ C 175.6 suggested to 2-methylbutyryl moiety [81, 83, 92]

Methyl protons at δ H 1.78 (3H, s), 1.77 (d, J 5.4 Hz, 3H), a ester carbonyl carbon at 167.4, two carbon signals at δC 128.0, 138.6 of double bond indicated the presence of tigloyl moiety [85, 90, 91]

Tigloyl moiety was connected to the C-

11 hydroxyl group according to key HMBC correlation from proton at δH 5.42 at C-11 to C-1’ (δ C 167.4), and 2-methylbutyryl moiety was assigned to attached to the C-12 hydroxyl group of aglycone by important correlation between proton at δ H 5.06 at 12-position and C-1” (δ C 175.6) Thus, the aglycone of 10 was determined as 11α-O-tigloyl-12β-O-2-methylbutyryl tenacigenin B

In addition, in its HMBC spectrum, we also showed that proton at δ H 4.61 (d, J = 9.5 Hz, 1H) at C-1 of cymarose should be assigned to C-3 of aglycone and proton at δH 4.82 (d, J 8.2 Hz, 1H) at C-1 of 6-deoxy-3-O-methyl-β-allopyranose sugar was assigned to be C-4 of cymarose Thus, structure of 10 was determined as 3-O-6-deoxy-3-O-methyl-β-D- allopyranosyl-(1→4)-β-D-cymmanopyranosyl-11α-O-tigloyl-12β-O-2-methylbutyryl tenacigenin B (Fig 3.3)

Table 3.2 1 H and 13 C NMR (400 Hz) spectral data for compound 10 (CDCl 3 , δ in ppm)

Compound 13 was obtained as a white solid, displayed a molecular formula C42H66O14Na from the quasi-molecular ion peak at m/z 817.50 [M+Na] + in its ESI-MS spectrum The 13 C NMR spectrum of 13 showed 42 carbon signals (Table 3.3)

Table 3.3 1 H and 13 C NMR (400 Hz) spectral data for compound 13 (CDCl 3 , δ in ppm)

In the NMR spectroscopic data of 13, a simplet proton signal at δ H 2.24 (s, 3H) and two carbons at δC 29.8 and 210.7 indicated the presence of acetyl moiety The proton signals at δH

0.90 (t, J = 7.4 Hz, 3H), 1.04 (d, J = 5.7 Hz, 3H), 1.99 (3H, s) and two ester carbonyl carbons at δC 170.7 and 175.6 suggested the presence of characteristic of acetyl [82-83, 92-93] and 2- methylbutyryl group [81, 83, 92]

, respectively In addition, HMBC spectrum of 13 displayed key correlation between proton H-11 at δ H 5.37 (t, J = 10.1 Hz) and carbon C-1’ of acetyl unit, and key correlation between the proton H-12 at δ H 5.00 (d, J = 10.1 Hz) and C-1” of 2- methylbutyryl moiety Thus, the aglycone of 13 was determined as 11α-O-acetyl-12β-O-2- methylbutyryl tenacigenin B The acidic hydrolysis solution of 13 consist two sugars, which were identified as olenadrose and 6-deoxy-3-O-methyl-β-allopyranose by TLC in comparison with authentic sugars The structures of two sugars were also confirmed based on NMR analysis and in comparison with previously reported data [81, 92]

In addition, the HMBC spectrum of 13 showed that the location of 6-deoxy-3-O- methyl-β-allopyranose moiety was assigned to be C-4 of oleandrose sugar by key HMBC correlation signal from proton at δ H 4.82 (d, J = 8.2 Hz) at 1-position to C-4 of oleandrose sugar Oleandrose moiety was linked to C-3 of aglycone according to key HMBC correlations of 13 Thus, structure of 13 was established as 3-O-6-deoxy-3-O- methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-acetyl-12β-O-2- methylbutyryl tenacigenin B (Fig 3.4)

Compound 14 was isolated as a white amorphous powder, the molecular formula was established to be C 44 H 62 O 14 by EIS-MS (m/z 837.51, [M+Na] + ) and the NMR spectroscopic analysis suggesting the presence of C 21 steroid skeleton The NMR spectra of compound 14 exhibited two anomeric proton signals at δ H 4.79 (d, J = 8.2 Hz, 1H) and 4.60 (d, J = 9.5 Hz,

1H), with corresponding carbon signals at δ C 99.1 and δ C 97.0, respectively (Table 3.4), thus structure of 14 consists two sugar moiety units Acidic hydrolysis solution of 14 consists two sugars were identified as olenadrose and 6-deoxy-3-O-methyl-β-allopyranose by TLC and NMR analysis in comparison with authentic sugars and NMR data in literatures [78-91, 92]

Proton signal at δ H 2.21 (s, 3H) and two carbon signals at δ C 30.0 and 210.7 suggested the presence of acetyl group [81, 91] The proton signals at δ H 7.43 (t, J = 7.7 Hz, 1H), 7.56 (t, J 6.8 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H) of aromatic ring, and one carbon at δ C 166.0 were assigned to benzoyl group [77, 91] Three proton signals at δ H 1.60 and one carbon at δ C 170.7 indicated the presence of acetate group [77, 82, 92]

Furthermore, the location of benzoyl and acetyl units was assigned by key HMBC correlations The location of acetyl group was assigned to C-11 hydroxyl group of aglycone by key correlation of proton H-11 at δ H 5.60 (t, J = 10.1 Hz) and C-1’ (δ C 170.7) of acetyl moiety Benzoyl part was linked to C-12 hydroxyl group according to important HMBC correlation of proton H-12 at δ H 5.13 (d, J = 10.1 Hz) to carbon at δ C 166.0 of benzoyl group Thus, the aglycone of 14 was determined as 11α-O-acetyl-12β-O-benzoyl tenacigenin B In the HMBC spectrum of 14, we showed that one proton at δ H 4.70 at 1-position of olenadrose was assigned to the C-3 of aglycone and a proton at 1-position of 6-deoxy-3-O-methyl-β- allopyranose should be assigned to C-4 of olenadrose sugar

Thus, structure of 14 was determined as 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-

(1→4)-β-D-olenadropyranosyl-11α-O-acetyl-12β-O-benzoyl tenacigenin B (Fig 3.5)

Fig 3.5 Structure of compound 14 Table 3.4 1 H and 13 C NMR (400 Hz) spectral data for compound 14 (CDCl 3 , δ in ppm)

Compound 1 as Tenacissoside F [98] Compound 1 was obtained as a white, amorphous powder; 1

H NMR (400 MHz, CDCl3) δ 1.03 (3H, s, 19-CH3), 1.08 (3H, s, 18-CH3), 1.23 (3H, d, J = 6.2 Hz, Allo-6-CH3), 1.37 (3H, d, J = 4.9 Hz, Ole-6-CH3), 1.50 (1H, m, Ole-H-2), 1.71 (1H, d, J = 10.1 Hz, H-9), 2.22 (3H, s, 21-CH3), 2.33 (1H, ddd, J = 11.5, 4.5, 2.0 Hz, Ole-H-2), 2.93 (1H, d, J = 7.3 Hz, H-17), 3.18 (1H, dd, J = 9.6, 2.5 Hz, Allo-H-4), 3.30 (1H, t, J = 10.1

Hz, H-11), 3.37 (2H, m, Ole-H-4, 5), 3.38 (3H, s, Ole-3-OCH3), 3.41 (1H, m, Ole-H-3), 3.46 (1H, d, J = 9.0 Hz, Allo-H-2), 3.57 (1H, m, Allo-H-5), 3.57 (1H, d, J = 10.1 Hz, H-12), 3.62 (1H, m, H-3), 3.67 (3H, s, Allo-3-OCH3), 3.80 (1H, t, J = 2.5 Hz, Allo-H-3), 4.61 (1H, dd, J 9.6, 1.6 Hz, Ole-H-1), 4.81 (1H, d, J = 8.0 Hz, Allo-H-1); ESI-MS: m/z 691.50 [M+Na] + ; HR- ESI-MS: m/z 691.3811 [M+Na] + (calcd for C35H56O12Na, m/z 691.3807)

Compound 2 as Marsdenoside D [91] Compound 2 was isolated as a white amorphous powder; 1 H NMR (400 MHz, CDCl3)δ 0.91 (3H, t, J = 7.4 Hz, 4’-CH3), 1.05 (3H, s, 19-CH3), 1.06 (3H, s, 18-CH3), 1.16 (3H, d, J = 7.0 Hz, 5’-CH3), 1.26 (3H, d, J = 6.2 Hz, Allo-6-CH3), 1.37 (3H, d, J = 4.9 Hz, Ole-6-CH 3 ), 1.50 (1H, m, Ole-H-2), 1.71 (1H, d, J = 10.0 Hz, H-9),

2.21 (3H, s, 21-CH 3 ), 2.33 (1H, ddd, J = 11.8, 4.5, 2.0 Hz, Ole-H-2), 2.92 (1H, d, J = 7.3 Hz, H-17), 3.18 (1H, dd, J = 9.6, 2.5 Hz, Allo-H-4), 3.37 (2H, m, Ole-H-4, 5), 3.38 (3H, s, Ole-3- OCH3), 3.40 (1H, m, Ole-H-3), 3.48 (1H, d, J = 9.0 Hz, Allo-H-2), 3.57 (1H, m, Allo-H-5), 3.64 (1H, m, H-3), 3.67 (3H, s, Allo-3-OCH3), 3.80 (1H, t, J = 2.5 Hz, Allo-H-3), 3.88 (1H, t,

J = 10.0 Hz, H-11), 4.60 (1H, dd, J = 9.6, 1.7 Hz, Ole-H-1), 4.80 (1H, d, J = 8.3 Hz, Allo-H-1),

4.83 (1H, d, J = 10.0 Hz, H-12); 13 C NMR (CDCl3) δ 210.9 (C-20, C=O), 177.2 (-COO-), 99.6 (Allo-C-1), 96.8 (Ole-C-1), 81.3 (Allo-C-3), 79.4 (Ole-C-4), 78.9 (Ole-C-3), 77.9 (C-12), 76.4 (C-3), 72.7 (Allo-C-4), 71.8 (C-14), 71.6 (Allo-C-2), 71.4 (Ole-C-5), 71.3 (Allo-C-5), 69.8 (C-11), 66.9 (C-8), 61.7 (Allo-3-OCH 3 ), 60.3 (C-17), 55.8 (Ole-3-OCH 3 ), 52.7 (C-9), 45.9 (C-13), 44.5 (C-5), 41.5 (C-2’), 39.6 (C-10), 38.3 (C-1), 36.2 (Ole-C-2), 34.9 (C-4), 31.9 (C-7), 30.1 (C-21), 29.4 (C-2), 26.8 (C-15), 26.9 (C-3’), 26.6 (C-6), 25.2 (C-16), 18.8 (Ole-C-

[M+Na] + ; HR-ESI-MS: m/z 775.4312 [M+Na] + (calcd for C40H64O13Na, m/z 775.4307)

Compound 3 as Tenacissoside N [93] Compound 3 was obtained as a white, amorphous powder; 1 H NMR (400 MHz, C5D5N) δ 1.16 (s, 3H, 19-CH3), 1.26 (3H, 18-CH3), 1.53 (s, 3H, 5'-CH3)，1.60 (3H, d, J = 5.9 Hz, Allo-6-CH 3 ), 1.60 (3H, d, J = 6.0 Hz, Ole-6-CH3), 1.78 (3H, d, J = 6.8 Hz, 4’-CH 3 ), 2.10 (s, 3H, 2”-CH 3 ), 2.26 (3H, s, 21-CH 3 ), 2.92 (1H, d, J = 6.4 Hz, H-

17), 3.46 (s, 3H, Ole-3-OCH 3 ), 3.77 (s, 3H, Allo-3-OCH 3 ), 4.75 (1H, d, J = 8.6 Hz, Ole-H-1),

5.18 (1H, d, J = 7.9 Hz, Glc-H-1), 5.25 (1H, d, J = 7.8 Hz, Allo-H-1), 5.32 (1H, d, J = 10.1 Hz, H-12), 5.72 (1H, t, J = 10.1 Hz, H-11), 6.88 (1H, q, J = 7.1 Hz, H-3’); ESI-MS: m/z 1139.50

[M+Na] + ; HR-ESI-MS: m/z 1139.4212 [M+Na] + (calcd for C54H84O24Na, m/z 1139.4206) Compound 4 as Marsdenoside F [91] Compound 4 was obtained as a white, amorphous powder; 1 H NMR (400 MHz, CDCl3) δ 1.03 (3H, s, 19-CH3), 1.06 (3H, s, 18-CH3), 1.24 (3H, d, J = 6.7 Hz, Allo-6-CH 3 ), 1.37 (3H, d, J = 5.3 Hz, Ole-6-CH 3 ), 1.51 (1H, m, Ole-H-2), 1.93 (-CH 3 ), 1.97 (-CH 3 ), 1.98 (1H, d, J = 10.1 Hz, H-9), 2.19 (3H, s, 21-CH 3 ), 2.31 (1H, ddd, J 12.2, 4.6, 1.6 Hz, Ole-H-2), 2.93 (1H, d, J = 7.3 Hz, H-17), 3.19 (1H, dd, J = 9.5, 3.2 Hz,

Allo-H-4), 3.34 (1H, m, Ole-H-5), 3.35 (1H, t, J = 7.1 Hz, Ole-H-4), 3.37 (3H, s, Ole-3- OCH3), 3.41 (1H, m, Ole-H-3), 3.48 (1H, dd, J = 8.3, 2.8 Hz, Allo-H-2), 3.56 (1H, m, Allo-H-

5), 3.64 (1H, m, H-3), 3.66 (3H, s, Allo-3-OCH3 ), 3.79 (1H, t, J = 2.6 Hz, Allo-H-3), 4.57 (1H, dd, J = 9.6, 1.5 Hz, Ole-H-1), 4.79 (1H, d, J = 7.7 Hz, Allo-H-1), 4.94 (1H, d, J = 10.1

Hz, H-12), 5.31 (1H, t, J = 10.1 Hz, H-11); 13 C NMR (CDCl3) δ 211.1 (C-20, C=O), 171.1 (2C, -COO-, C-1’, C-1”), 99.5 (Allo-C-1), 96.9 (Ole-C-1), 81.0 (Allo-C-3), 79.3 (Ole-C-4), 78.8 (Ole-C-3), 76.5 (C-12), 76.6 (C-3), 72.8 (Allo-C-4), 71.9 (C-14), 71.4 (Allo-C-2), 71.2 (Ole-C-5), 71.0 (Allo-C-5), 69.8 (C-11), 66.7 (C-8), 61.8 (Allo-3-OCH 3 ), 60.2 (C-17), 55.6 (Ole-3-OCH 3 ), 52.0 (C-9), 45.9 (C-13), 44.3 (C-5), 21.6 (C-2’), 39.1 (C-10), 37.5 (C-1), 36.5 (Ole-C-2), 34.7 (C-4), 31.7 (C-7), 30.0 (C-21), 29.5 (C-2), 26.7 (C-15), 26.6 (C-6), 25.1 (C-

16), 21.0 (2C – C-2’, C-2”), 18.7 (Ole-C-6), 17.9 (Allo-C-6), 16.6 (C-18), 13.1 (C-19); ESI- MS: m/z 799.50 [M+Na] + ; HR-ESI-MS: m/z 799.4117 [M+Na] + (calcd for C40H62O14Na, m/z 799.4112)

Compound 5 as Tenacissoside A [85] Compound 5 was obtained as a white, amorphous powder; 1 H NMR (400 MHz, C 5 D 5 N) δ 1.19 (3H, s, H-19), 1.24 (3H, s, H-18), 1.53 (3H, J 6.3 Hz, Allo-6-CH 3 ), 1.61 ( 3H, J = 6.1 Hz, Ole-6-CH 3 ), 1.74 (s, 3H, H-4’), 1.75 (d, J = 6.8

Hz, 3H, H-5’), 1.89 (3H, s, H-2″), 2.22 (3H, s, H-21), 2.94 (1H, d, J = 7.3 Hz, H-17β), 3.23- 3.34 (m, 1H, Glc-H-5), 3.31 (3H, s, Ole-3-OCH3), 3.33 (1H, m, Glc-H-2), 3.43 (1H, m, Ole- H-3), 3.47 (1H, dd, J = 8.2, 2.8 Hz, Allo-H-2), 3.53 (1H, m, Glc-H-3), 3.58 (1H, m, Glc-H-4), 3.61 (1H, m, Glc-CH2), 3.62 (1H, m, H-3), 3.71 (3H, s, Allo-3-OCH3), 3.75 (1H, t, J = 3.0 Hz, Allo-H-3), 3.83 (1H, m, Glc-CH2), 4.82 (1H, dd, J = 9.7, 1.8 Hz, Ole-H-1), 4.86 (1H, d, J 7.4 Hz, Glc-H-1), 5.19 (1H, d, J = 8.0 Hz, Allo-H-1), 5.36 (1H, d, J = 10.2 Hz, H-12), 5.71 (1H, t, J = 10.2 Hz, H-11), 6.80 (1H, m, H-3’); ESI-MS: m/z 977.55 [M+Na] + ; HR-ESI-MS: m/z 977.4823 [M+Na] + (calcd for C 48 H 74 O 19 Na, m/z 977.4815)

Compound 6 as tenacissoside H [92] Compound 6 was isolated as a white, amorphous powder; 1 H NMR (400 MHz, CDCl3) δ 0.81 (3H, t, J = 7.2 Hz, 4′-CH3), 1.05 (3H, d, J = 6.7

Hz, 5’-CH3), 1.06 (3H, s, 19-CH3), 1.08 (3H, s, 18-CH3), 1.26 (3H, d, J = 5.8 H z, Allo-6-

CH3), 1.38 (3H, d, J = 5.8 Hz, Ole-6-CH3), 1.97 (3H, s, 2”-CH3), 2.22 (3H, s, 21-CH3), 2.94 (1H, d, J = 7.4 Hz, H-17), 3.18 (1H, d, J = 9.5 Hz, Allo-4-H), 3.42 (3H, s, Ole-3-OCH3), 3.66 (3H, s, Allo-3-OCH 3 ), 3.81 (1H, s, Allo-3-H), 4.59 (1H, d, J = 9.7 Hz, Ole-1-H), 4.79 (1H, d,

J = 7.9 Hz, Allo-1-H ), 5.03 (1H, d, J = 10.2 Hz, H-12), 5.43 (1H, t, J = 10.2 Hz, H-11); 13 C NMR (400 MHz, CDCl 3 ) δ 210.3 (C-20, C=O), 175.4 (-COO-), 170.4 (-COO-), 99.8 (Allo-C-

1), 96.7 (Ole-C-1), 81.4 (Allo-C-3), 79.8 (Ole-C-4), 79.3 (Ole-C-3), 75.9 (C-12), 76.6 (C-3), 72.9 (Allo-C-4), 71.7 (C-14), 71.8 (Allo-C-2), 71.3 (Ole-C-5), 71.1 (Allo-C-5), 68.5 (C-11), 66.8 (C-8), 61.7 (Allo-3-OCH3), 60.6 (C-17), 57.7 (Ole-3-OCH3), 51.9 (C-9), 46.1 (C-13), 44.4 (C-5), 39.5 (C-10), 38.3 (C-1), 36.4 (Ole-C-2), 35.0 (C-4), 31.5 (C-7), 30.0 (C-21), 29.7 (C-2), 26.9 (C-15), 26.8 (C-3’), 26.7 (C-6), 25.3 (C-16), 21.3 (C-2”), 20.5 (C-2’), 20.3 (Ole- C-6), 17.9 (Allo-C-6), 17.0 (C-5’), 16.4 (C-18), 12.8 (C-19), 11.6 (C-4’); ESI-MS: m/z 817.52 [M+Na] + ; HR-ESI-MS: m/z 817.4521 [M+Na] + (calcd for C 42 H 66 O 14 Na, m/z 817.4518)

Compound 7 as Marsdenoside A [91] Compound 7 was obtained as a white, amorphous powder; 1 H NMR (400 MHz, CDCl 3 ) δ 0.82 (3H, t, J = 7.3 Hz, 4’-CH 3 ), 1.03 (3H, d, J = 6.9

Hz, 5’-CH3), 1.05 (3H, s, 19-CH3), 1.08 (3H, s, 18-CH3), 1.26 (3H, d, J = 6.2 Hz, Allo-6-CH3), 1.37 (3H, d, J = 5.2 Hz, Ole-6-CH3), 1.49 (1H, m, Ole-H-2), 1.75 (3H, s, 5”-CH3), 1.76 (3H, d,

J = 6.5 Hz, 4”-CH3), 2.01 (1H, d, J = 10.1 Hz, H-9), 2.08 (1H, q, J = 6.9 Hz, H-20), 2.22 (3H, s, 21-CH3), 2.31 (1H, dd, J = 11.7, 4.0 Hz, Ole-H-2), 2.92 (1H, d, J = 7.2 Hz, H-17), 3.18 (1H, d, J = 9.5 Hz, Allo-H-4), 3.35 (1H, t, J = 6.7 Hz, Ole-H-4), 3.36 (1H, m, Ole-H-5), 3.38 (3H, s, Ole-3-OCH 3 ), 3.41 (1H, m, Ole-H-3), 3.48 (1H, dd, J = 7.5, 2.7 Hz, Allo-H-2), 3.56 (1H, m, Allo-H-5), 3.63 (1H, m, H-3), 3.66 (3H, s, Allo-3-OCH 3 ), 3.79 (1H, s, Allo-H-3), 4.58 (1H, dd,

J = 9.7, 1.6 Hz, Ole-H-1), 4.80 (1H, d, J = 8.1 Hz, Allo-H-1), 5.06 (1H, d, J = 10.1 Hz, H-12),

5.42 (1H, t, J = 10.1 Hz, H-11), 6.78 (1H, q, J = 6.0 Hz, H-3”); 13 C NMR (400 MHz, CDCl3) δ 210.7 (C-20, C=O), 175.2 (C-1’, -COO-), 167.3 (C-1”, -COO-), 138.3 (C-3”), 128 (C-2”), 99.6 (Allo-C-1), 96.8 (Ole-C-1), 81.3 (Allo-C-3), 79.5 (Ole-C-4), 78.9 (Ole-C-3), 76.4 (C-3), 74.6 (C-12), 72.8 (Allo-C-4), 71.8 (Allo-C-2), 71.5 (C-14), 71.3 (Ole-C-5), 71.2 (Allo-C-5), 68.9 (C-11), 66.9 (C-8), 61.9 (Allo-3-OCH 3 ), 60.0 (C-17), 56.0 (Ole-3-OCH 3 ), 51.7 (C-9), 45.8 (C-13), 44.5 (C-5), 41.3 (C-2’), 39.1 (C-10), 37.6 (C-1), 36.1 (Ole-C-2), 34.9 (C-4), 31.8 (C-7), 30.0 (C-21), 29.0 (C-2), 26.9 (C-15), 26.6 (C-6), 26.0 (C-3’), 24.9 (C-16), 18.6 (Ole-C-

6), 17.9 (Allo-C-6), 16.7 (C-18), 15.2 (C-5’), 14.5 (C-5”), 12.2 (C-19), 11.9 (C-4”), 11.8 (C- 4’); ESI-MS: m/z 857.52 [M+Na] + ; HR-ESI-MS: m/z 857.4811 [M+Na] + (calcd for

Compound 8 as Marsdenoside C [91] Compound 8 was isolated as a white powder; 1 H NMR (400 MHz, CDCl3) δ 0.79 (3H, t, J = 7.4 Hz, 4’-CH 3 ), 0.88 (3H, d, J = 7.0 Hz, 5’-CH3), 1.09 (3H, s, 19-CH 3 ), 1.16 (3H, s, 18-CH 3 ), 1.28 (3H, d, J = 6.2 Hz, Allo-6-CH 3 ), 1.42 (3H, d,

Conclusions

Marsdenia tenacissima (Roxb.) Wight et Arn., is known as a famous traditional Chinese medicine, it is widely used in the treatment of cancer, and other diseases Twenty compounds were isolated from the ethanolic extract of the stem of Marsdenia tenacissima, including three new compounds as 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-cymmanopyranosyl-11α-O-tigloyl- 12β-O-2-methylbutyryl tenacigenin B (10), 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)- β-D-oleandropyranosyl-11α-O-acetyl-12β-O-2-methylbutyryl tenacigenin B (13), 3-O-6-deoxy-3-

O-methyl-β-D-allopyranosyl-(1→4)-β-D-olenadropyranosyl-11α-O-acetyl-12β-O-benzoyl tenacigenin B (14), and seventeen known ones.

Experimental section

Column chromatography (CC) was prepared using silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., China), sephadex LH-20 (20-100 àm, Pharmacia), RP-18 (20-45 àm, Fuji Silysia Chemical Ltd., Japan) All solvents and reagents used were analytically pure grade and purchased from the Chemical plant, Shanghai, P R.China 1 H, 13 C, and 2D-NMR data spectra were recorded on a Bruker AMX 400 and 500 Hz instruments ESI-MS were recorded on a Bruker Esquire 3000 Plus Spectrometer HR-ESI-MS were measured on a Micromass Q-Tif Global mass spectrometer HPLC was run on Waters 2690 Separation module with Alltech ELSD 2000 detector and a kromacil C18 column Melting points were determined with a SGW-X4 apparatus

Dried stems of M tenacissima (5.0 kg), which was collected in Hebei, was extracted three times with EtOH (80%) under reflux for 3 h, and then isolated solution was concentrated in vacuum to give color residue (750.0 g) The residue was resuspended in H2O, and extracted with ethyl acetate and n-butanol to give three residue parts: 100 g ethyl acetate, 150 g n- butanol and water residue The ethyl acetate extract 100 g and n-butanol extract 120 g were subjected to column chromatography (silica gel, ODS) to give 20 compounds 1 to 20, including 3 new compounds and 17 known ones The extraction and isolation of 1-20 were summarized in Fig 3.6

Fig 3.6 The extraction and isolation of constituents of M tenacissima

Compound 10 was obtained as a white, amorphous powder; 1 H NMR and 13 C NMR data, see Table 3.2; ESI-MS: m/z 857.50 [M+Na] + ; HR-ESI-MS: m/z 857.4832 [M+Na] + (calcd for

Compounds (4 mg, each) were dissolved in 3 mL of 0.5 M HCl in MeOH solution for 1 hour and diluted with water (4 mL) The resulting solution was neutralised with aqueous NaOH; 10 mL water was added and extracted three times with CH 2 Cl 2 (3x10 mL) Sugars in aqueous layer were analysed by TLC, and compared with authentic sugar samples

Study on the isolation, structural determination, and cytotoxicities of cardenolides

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