Glucose-containing sulfonic acid-catalyzed efficient synthesis of spiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3’-indoline] derivatives

doi:10.21203/rs.3.rs-3111679/v1

β-1-N-methyl-imidazole-2,3,4,6-tetrasulfonic-D-glucopyranosyl bisulfate ([Bmim-G-(SO3H)4]+[HSO4]-, NMI-GSA) was used as the efficient catalyst to promote the three-component reaction of 2-hydroxy-1,4-naphthoquinone, isatins and aminopyrazoles to afford a series of spiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3’-indoline] derivatives (4). Six bonds were cleaved while six new bonds (two C-N bonds and four C-C bonds) were formed to constructed a new dihydropyridine ring and a spiro skeleton in one-pot with water as the only one by-product in this highly atom-economic reaction. Two non-toxic products to normal cells have been screened out because of their special high inhibition rate of on MCF-7 cells (> 90%).

glucose-containing sulfonic acid

spiro[benzo[g]pyrazolo[3

4-b]quinoline-4

3’-indoline]

Compounds with spiro skeletons not only constitute subunits in numerous alkaloids, but are also templates for drug discovery and have been used as scaffolds for combinatorial libraries [1]. Indole and dihydroindol are important moieties of a large number of natural products and medicinal agents [2]. Especially, indoles or dihydroindoles with a spiro carbon atom at C-3 position (known as spiro-oxindole) have higher biological activities [3-5] and are the core structure of many pharmacological agents and natural alkaloids [6-9]. For example, the spiro tetrahydro-b-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration [10-13].

Multicomponent reaction is a classical method to synthesize the spirohydroindoles, and catalyst is the key factor [14]. Compared with indole, naphthoquinone is rarely used as a substrate to give spiroindolones due to its larger steric hindrance and weaker nucleophilicity. Naphthoquinone is an important framework, compounds containing naphthoquinone fragments have varied biological activities such as mitochondrial dysfunction improvement [15], antimicrobial [16], anti-trypanosomatids [17], antimalarial [18] and so on. Therefore, it is of great significance to prepare spiroindolones containing naphthoquinone skeleton. By far, several weak organic acids such as L-proline[19], p-toluenesulfonic acid[20] and melamine chloride [21] were used to promote the preparation of naphthoquinone-containing spiroindolones. But only aminopyrazole with small steric hindrance could participate in the reaction, and only four products were obtained up to now. Therefore, it's still valuable to develop more efficient catalysts for the important synthetic procedure.

Glucose-containing sulfonic acids (GSA) derived from natural glucose have four sulfonic groups and therefore show strong acidity. Moreover, the unique chair conformation of sugar ring enables the four sulfonic groups of GSA to cooperate with each other while perform their own duties. The versatile structure can therefore gather different raw materials together very efficiently and enhance their effective collision probability to make the reaction proceed smoothly under more mild conditions. Therefore, using GSA as catalyst can generally increase the yield, broaden the range of substrates and obtain products with complex, novel and diverse structures [22-24].

In view of this, we used the synthesized β-1-3’-methylmidazole-2,3,4,6-tetrasulfonic-D-glucopyranosyl bisulfate ([Bmim-G-(SO₃H)₄]⁺[HSO₄]^-, NMI-GSA) [21] as catalyst to promote the three-component reaction of 2-hydroxy-1,4-naphthoquinone, isatin derivatives and substituted aminopyrazole to afford a series of spiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3’-indoline] derivatives (Scheme 1). The anti-tumor activity of all products was evaluated preliminarily.

Materials

All the reagents were purchased commercially without further purified. Human three positive breast cancer cells (MCF-7), human three negative breast cancer cells (MDA-MB-231), human hepatoma cells (HepG2), human hepatoma cells (MHCC-97H) and Human hepatocyte (LO2) cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China).

NMR spectra were measured using a Bruker DPX 400 MHz spectrometer using TMS as internal standard. Mass spectra were recorded on a Micro TOF-Q (ESI) instrument. The Alamar Blue was performed on a microplate reader (SpectraMax M2).

Synthesis of product 4

A mixture of 2-hydroxy-1,4-naphthoquinone 1 (1 mmol), isatin derivative 2 (1 mmol), aminopyrazole 3 (1 mmol), NMI-GSA (15 mol%) and EtOH (3 mL) was stirred at 50 °C for given time until complete consumption of the starting material as monitored by TLC. After the completion, the mixture was cooled to room temperature, filtered by suction and washed with water and ethanol to give red powdered solid crude products. The pure product 4 was obtained by column chromatography (V_PE: V_EA = 4:1).

We started our investigation by employing 2-hydroxy-1,4-naphthoquinone, isatin, 5-amino-3-methyl-1-phenylpyrazole as the model substrates (Table 1).

Table 1. Optimization of the Reaction Conditions^a

Entry	Cat. (pH / mol%)	Solvent	Time (h)	Temp. (^oC)	Yields (%)^b
1	None	EtOH	0.75	50	Nr^c
2	SSA (1.87 / 15)^d	EtOH	0.75	50	52
3	AcOH (2.29 / 15)^e	EtOH	0.75	50	20
4	T_SOH (1.84 / 15)	EtOH	0.75	50	60
5	[Bmim]⁺HSO₄^-(1.85 / 15)	EtOH	0.75	50	58
6	L-Proline (6.40 / 15)	EtOH	0.75	50	40
7	H₂SO₄(1.75 / 15)^f	EtOH	0.75	50	35
8	N-methylimidazole (8.54 / 15)	EtOH	0.75	50	20
9	NMI-GSA (1.24 / 15)	EtOH	0.75	50	90
10	NMI-GSA (1.24 / 5)	EtOH	0.75	50	60
11	NMI-GSA (1.24/ 10)	EtOH	0.75	50	75
12	NMI-GSA (1.24/ 20)	EtOH	0.75	50	81
13	NMI-GSA (1.24 / 15)	H₂O	0.75	50	75
14	NMI-GSA (1.24/ 15)	DCM	0.75	50	65
15	NMI-GSA (1.24 / 15)	CH₃CN	0.75	50	70
16	NMI-GSA (1.24 / 15)	THF	0.75	50	40
17	NMI-GSA (1.24 / 15)	EtOH	0.75	0	Nr^c
18	NMI-GSA (1.24 / 15)	EtOH	0.75	r.t.	50
19	NMI-GSA (1.24 / 15)	EtOH	0.75	80	80
20	NMI-GSA (1.24/ 15)	EtOH	2	50	85
21	NMI-GSA (1.24 / 15)	EtOH	5	50	85
22	NMI-GSA (1.24 / 15)	EtOH	10	50	83

^aReaction conditions: 2-hydroxy-1,4-naphthoquinone (1 mmol), isatin(1 mmol), 5-amino-3-methyl-1-phenyl pyrazole(1 mmol). ^bisolated yields. ^c No reaction. ^d Synthesized in our lab [24]. ^e 1 mol/L, aq. ^f 1 mol/L, aq.

At first, a range of acids were screened. Common organic acids (entries 2-6) and sulfuric acid (entry 7) can promote the reaction but give low yield (20-60%). The stronger the acidity of the catalyst, the higher the yield, which indicates that strong acid condition is the key factor of this reaction. It also shows that acid-drove electrophilic addition may be the first step of the reaction. In the case of similar acidity, organic acids give a higher yield of 4a than inorganic acids, which may be due to the higher solubility. Gratifyingly, the exciting result is afforded by using NMI-GSA as the organocatalyst (entry 9). N-methylimidazole (entry 8) can also promote the reaction, but the yields are very low (20%), which indicate the indispensability of the sugar ring. The amount of catalyst was screened subsequently (entries 9-12). While increasing the loading of NMI-GSA from 5 mol % to 15 mol %, the yield of 4a is increased from 60 % - 90 % (entries 9-11), which shows the important role of catalyst concentration. However, the yield decreased unexpectedly when the molar amount of catalyst is over 15 mol % (entry 12). A possible reason is that the starting material or the products have been destroyed when excess amount of NMI-GSA was added. Ethanol is the best solvent (entry 9 vsentries 13-16) maybe due to its appropriate solubility to both of raw materials and catalyst. It was also realized that the process is efficiently facilitated at 50 ^oC (entry 9 vsentries 17-19). Finally, we found that the yield can’t be raised by prolonging the reaction time (entry 9 vsentries 20-22).

Overall, the best result is obtained by using 15 mol % of NMI-GSA as the catalyst, EtOH as solvent at 25 ^oC for 0.75 h.

With these optimized reaction conditions (Table 1, entry 10), the generality and limitation of this protocol was then systematically evaluated (Table 2).

Table 2. Substrate Scope for the Synthesis of Products 4^a

Entry	Product	R₁	R₂	R₃	Time (h)	Yield (%)
1	4a	H	CH₃	H	0.75 (12)^[19]	90 (70)^[19]
2	4b	5-F	CH₃	H	0.75 (5)^[20]	86 (86) ^[20]
3	4c	5-Cl	CH₃	H	0.75 (7)^[20]	81 (86) ^[20]
4	4d	5-NO₂	CH₃	H	0.75	84
5	4e	5-CH₃	CH₃	H	0.75 (7)^[20]	94 (86) ^[20]
6	4f	5-OCH₃	CH₃	H	0.75	88
7	4g	H	4-Me-C₆H₄	H	2	63
8	4h	5-F	4-Me-C₆H₄	H	2	76
9	4i	5-Cl	4-Me-C₆H₄	H	2	80
10	4j	5-NO₂	4-Me-C₆H₄	H	2	72
11	4k	5-CH₃	4-Me-C₆H₄	H	2	88
12	4l	5-OCH₃	4-Me-C₆H₄	H	2	91
13	4m	H	4-OMe-C₆H₃	H	2.5	71
14	4n	5-F	4-OMe-C₆H₃	H	2.5	82
15	4o	5-Cl	4-OMe-C₆H₃	H	2.5	85
16	4p	5-NO₂	4-OMe-C₆H₃	H	2.5	81
17	4q	5-CH₃	4-OMe-C₆H₃	H	2.5	93
18	4r	5-OCH₃	4-OMe-C₆H₃	H	2.5	87
19	4s	H	CH₃	4-CH₃	4	80
20	4t	5-F	CH₃	4-CH₃	4	82
21	4u	5-Cl	CH₃	4-CH₃	4	87
22	4v	5-NO₂	CH₃	4-CH₃	4	83
23	4w	5-CH₃	CH₃	4-CH₃	4	90
24	4x	5-OCH₃	CH₃	4-CH₃	4	92

From Table 2, it can be seen that 2-hydroxy-1,4-naphthoquinone can successfully participate in the reaction. Aminopyrazole derivatives with large steric hindrance substituents at 1- or 3- position can also react (63-94%). However, because it is proximity to the spiro ring, aminopyrazole with large group on the 3-position can only give relative low yield (4g-4r, R₂ = Ar, 63-93%). But in general, the greater the steric hindrance, the longer the reaction time, for example, the synthesis of 4s-4x takes 4 hours (entries 19-24). It is worth mentioning that the reaction gives higher yield in shorter time (entries 1-3 and 5, 4a-4c and 4e) when there is small group on the 3-position of aminopyrazole compared with the literature value [19, 20]. The electronic effect of the group on isatin has slight influence on the yield. Generally, isatins containing electron-withdrawing groups (F, Cl and NO₂) give slightly lower yield. Due to the limited commercial availability of raw materials, the steric hindrance of isatin and the electronic effect of aminopyrazole could not be further explored.

According to the previous literature [22-24], a possible mechanism was proposed (Scheme 2). Initially, intermediate I is derived from isatin in the presence of strong acid catalyst NMI-GSA. Subsequently, I is attacked by aminopyrazole to form intermediate II. Intermediate II is then reacted with 2-hydroxy-1,4-naphthoquinone to form intermediate III. The target product 4 is afforded from III via cyclization and dehydration in turn. As mentioned above, the strong acidity of the sulfonic group, the sugar ring’s chair conformation and the suitable solubility of the catalyst in EtOH make it efficiently.

3-Methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4a)

Red powder, m.p. 246.3-248.1 °C; IR (KBr) (v_max , cm^-1): 3151, 3082, 2962, 2910, 1710, 1675, 1646; ¹H NMR (400 MHz, DMSO-d₆): δ 1.63 (3H, s, CH₃), 6.87-8.08 (13H, m, Ar-H), 9.83 (1H, s, NH), 10.70 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.6, 179.3, 178.8, 145.0, 141.3, 141.0, 138.2, 136.7, 136.1, 135.2, 133.3, 131.7, 129.8, 129.5, 128.1, 127.2, 125.9, 123.9, 122.9, 121.8, 115.2, 109.0, 101.3, 49.8, 18.5. HRMS (ESI) m/z: cacld for C₂₈H₁₈N₄O₃ (M+H)⁺ found (expected): 459.1812 (459.1894).

5'-Fluoro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4b)

Red powder, m.p. 257.5-259.4 °C; IR (KBr) (v_max , cm^-1): 3592, 3442, 2930, 2911, 1711, 1679, 1642; ¹H NMR (400 MHz, DMSO-d₆): δ 1.66 (3H, s, CH₃), 6.90-8.07 (12H, m, Ar-H), 9.88 (1H, s, NH), 10.73 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.7, 179.3, 178.8, 159.4, 157.1, 144.9, 141.2, 138.1, 137.6, 136.2, 135.2, 133.3, 131.7, 129.9, 129.5, 127.3, 126.0, 125.8, 123.0, 114.6, 114.4, 114.2, 112.0, 111.7, 109.7, 109.6, 100.8, 50.3, 11.3. HRMS (ESI) m/z: cacld for C₂₈H₁₇N₄O₃F (M+H)⁺ found (expected): 477.2986 (477.2957).

5'-Chloro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4c)

Red powder, m.p. 255.6-257.0 °C; IR (KBr) (v_max , cm^-1): 3564, 3394, 3028, 2921, 1715, 1674, 1645; ¹H NMR (400 MHz, DMSO-d₆): δ 1.67 (3H, s, CH₃), 6.93-8.08 (12H, m, Ar-H), 9.89 (1H, s, NH), 10.84 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.7, 179.3, 178.8, 159.4, 157.1, 144.9, 138.1, 137.5, 136.2, 135.2, 133.3, 131.7, 129.9, 129.5, 127.3, 126.0, 125.8, 123.0, 114.6, 114.4, 114.2, 112.0, 111.7, 109.7, 109.6, 100.8, 50.3, 11.3. HRMS (ESI) m/z: cacld for C₂₈H₁₇N₄O₃Cl (M+H)⁺ found (expected): 493.1452 (493.1462).

3-Methyl-5'-nitro-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4d)

Red powder, m.p. 243.9-245.7 °C; IR (KBr) (v_max , cm^-1): 3422, 3191, 2948, 2213, 1734, 1680, 1620, 1530, 1392; ¹H NMR (400 MHz, DMSO-d₆): δ 1.68 (3H, s, CH₃), 7.16-8.21 (12H, m, Ar-H), 10.03 (1H, s, NH), 11.46 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.8, 179.3, 179.2, 147.8, 144.7, 142.5, 141.6, 138.1, 137.2, 136.4, 135.1, 133.4, 131.6, 130.0, 129.5, 127.5, 126.1, 125.9, 125.7, 123.2, 119.8, 113.9, 109.2, 100.1, 49.8, 11.4. HRMS (ESI) m/z: cacld for C₂₈H₁₇N₅O₅ (M+H)⁺ found (expected): 504.1461 (504.1402).

3,5'-Dimethyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4e)

Red powder, m.p. 255.4-256.3 °C; IR (KBr) (v_max , cm^-1): 3420, 3062, 2946, 2836, 2216, 1755, 1727, 1621; ¹H NMR (400 MHz, DMSO-d₆): δ 1.64 (3H, s, CH₃), 2.09 (3H, s, CH₃), 6.80-8.07 (12H, m, Ar-H), 9.81 (1H, s, NH), 10.59 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.6, 179.4, 178.8, 145.0, 141.0, 138.8, 138.2, 136.8, 136.0, 135.2, 133.3, 131.7, 130.7, 129.8, 129.5, 128.6, 128.3, 127.2, 126.0, 125.8, 124.5, 122.9, 115.4, 101.4, 49.8, 30.6, 20.5, 11.3. HRMS (ESI) m/z: cacld for C₂₉H₁₉N₄O₃ (M+H)⁺ found (expected): 473.2749 (473.2708).

5'-Methoxy-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4f)

Red powder, m.p. 240.3-242.1 °C; IR (KBr) (v_max , cm^-1): 3426, 3343, 2938, 2360, 2342, 2209, 1755, 1727, 1678; ¹H NMR (400 MHz, DMSO-d₆): δ 1.65 (3H, s, CH₃), 3.61 (3H, s, OCH₃), 6.69-8.08 (12H, m, Ar-H), 9.78 (1H, s, NH), 10.51 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.6, 179.4, 178.7, 155.1, 145.0, 141.0, 138.2, 138.1, 137.9, 137.8, 136.0, 135.2, 134.7, 133.2, 131.8, 129.9, 129.5, 127.2, 126.0, 125.8, 122.9, 115.2, 112.5, 111.1, 109.3, 101.3, 55.3, 50.3, 11.3. HRMS (ESI) m/z: cacld for C₂₉H₁₉N₄O₄ (M+H)⁺ found (expected): 489.1266 (489.1257).

3-Methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4g)

Red powder, m.p. 243.1-244.6 °C; IR (KBr) (v_max , cm^-1): 3151, 3082, 2940, 2202, 1710, 1640, 1593; ¹H NMR (400 MHz, DMSO-d₆): δ 2.27 (3H, s, CH₃), 6.57-8.07 (17H, m, Ar-H), 9.79 (1H, s, NH), 10.17 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.4, 179.4, 178.7, 149.3, 142.0, 140.6, 138.1, 137.9, 137.2, 136.4, 135.2, 133.3, 131.8, 129.7, 129.6, 129.5, 128.4, 128.2, 127.9, 127.6, 125.9, 125.9, 124.0, 123.1, 121.5, 115.5, 109.2, 101.1, 50.0, 20.8. HRMS (ESI) m/z: cacld for C₃₄H₂₂N₄O₃ (M+H)⁺ found (expected): 535.3030 (535.3065).

5'-Fluoro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4h)

Red powder, m.p. 254.2-256.1 °C; IR (KBr) (v_max , cm^-1): 3592, 3342, 2951, 2212, 1711, 1679, 1649; ¹H NMR (400 MHz, DMSO-d₆): δ 2.28 (3H, s, CH₃), 6.55-8.08 (16H, m, Ar-H), 9.93 (1H, s, NH), 10.20 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.7, 179.3, 178.8, 159.4, 157.1, 144.9, 141.2, 138.1, 137.6, 136.2, 135.2, 133.3, 131.7, 129.9, 129.5, 127.3, 126.0, 125.8, 123.0, 119.9, 118.6, 116.2, 116.1, 114.6, 114.4, 114.2, 112.0, 111.7, 109.7, 109.6, 100.8, 50.3, 11.3. HRMS (ESI) m/z: cacld for C₃₄H₂₁N₄O₃F (M+H)⁺ found (expected): 553.1266 (553.1270).

5'-Chloro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4i)

Red powder, m.p. 261.9-263.7 °C; IR (KBr) (v_max , cm^-1): 3564, 3394, 2931, 2212, 1715, 1674, 1646; ¹H NMR (400 MHz, DMSO-d₆): δ 2.29 (3H, s, CH₃), 6.93-8.08 (16H, m, Ar-H), 9.93 (1H, s, NH), 10.31 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): 180.4, 179.4, 178.7, 149.3, 142.0, 140.6, 138.1, 137.9, 137.2, 136.4, 135.2, 133.3, 131.8, 129.7, 129.6, 129.5, 128.4, 128.2, 127.9, 127.6, 125.9, 124.0, 123.1, 121.5, 115.5, 109.2, 101.1, 50.0, 20.8. HRMS (ESI) m/z: cacld for C₃₄H₂₁N₄O₃Cl (M+H)⁺ found (expected): 569.2077 (569.2075).

3-Methyl-5'-nitro-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4j)

Red powder, m.p. 239.2-241.1 °C; IR (KBr) (v_max , cm^-1): 3422, 3191, 2947, 2207, 1734, 1680, 1620, 1536, 1352; ¹H NMR (400 MHz, DMSO-d₆): δ 2.29 (3H, s, CH₃), 6.62-8.12 (16H, m, Ar-H), 10.08 (1H, s, NH), 10.88 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): 180.4, 179.8, 179.2, 159.4, 149.7, 142.4, 141.1, 138.5, 138.3, 136.7, 135.7, 133.8, 132.3, 130.2, 130.2, 130.1, 128.7, 128.1, 126.4, 126.4, 125.0, 124.5, 123.6, 122.0, 116.0, 113.3, 109.7, 101.7, 55.5, 20.7. HRMS (ESI) m/z: cacld for C₃₄H₂₁N₅O₅ (M+H)⁺ found (expected): 580.1858 (580.1815).

3,5'-Dimethyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4k)

Red powder, m.p. 241.6-243.3 °C; IR (KBr) (v_max, cm^-1): 3420, 3062, 2985, 2361, 1755, 1727, 1621; ¹H NMR (400 MHz, DMSO-d₆): δ 2.19 (3H, s, CH₃), 2.28 (3H, s, CH₃), 6.54-8.06 (16H, m, Ar-H), 9.83 (1H, s, NH), 10.07 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): 180.4, 179.4, 178.6, 155.0, 149.5, 140.5, 139.0, 137.9, 137.5, 136.3, 135.7, 133.7, 132.3, 129.8, 129.3, 129.2, 129.1, 127.9, 127.0, 125.4, 124.1, 122.6, 115.2, 112.3, 108.5, 100.8, 55.5, 20.0. HRMS (ESI) m/z: cacld for C₃₅H₂₃N₄O₃ (M+H)⁺ found (expected): 549.2937 (549.2921).

5'-Methoxy-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4l)

Red powder, m.p. 212.3-214.2 °C; IR (KBr) (v_max , cm^-1): 3426, 3343, 2921, 2842, 2363, 2218, 1755, 1727, 1678; ¹H NMR (400 MHz, DMSO-d₆): δ 2.28 (3H, s, CH₃), 3.64 (3H, s, OCH₃), 6.54-8.08 (12H, m, Ar-H), 9.81 (1H, s, NH), 10.00 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.4, 179.4, 178.6, 155.0, 149.4, 140.5, 139.0, 138.0, 137.2, 136.3, 135.4, 135.2, 133.2, 131.8, 129.7, 129.6, 129.4, 128.4, 127.9, 127.6, 125.9, 123.1, 115.5, 112.8, 111.2, 109.5, 101.0, 55.4, 50.5, 20.8. HRMS (ESI) m/z: cacld for C₃₅H₂₃N₄O₄ (M+H)⁺ found (expected): 565.1708 (565.1770).

3-Methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4m)

Red powder, m.p. 222.8-224.6 °C; IR (KBr) (v_max , cm^-1): 3151, 3082, 2936, 2210, 1716, 1660, 1607; ¹H NMR (400 MHz, DMSO-d₆): δ 3.74 (3H, s, OCH₃), 6.58-8.07 (17H, m, Ar-H), 9.86 (1H, s, NH), 10.16 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.0, 178.8, 178.7, 158.9, 149.1, 142.0, 138.2, 138.2, 138.1, 137.9, 136.2, 135.2, 131.9, 129.7, 129.5, 128.6, 128.3, 127.5, 125.9, 125.8, 124.0, 123.1, 112.8, 109.2, 109.2, 102.6, 101.3, 100.2, 55.0, 50.0. HRMS (ESI) m/z: cacld for C₃₄H₂₂N₄O₄ (M+H)⁺ found (expected): 511.1729 (511.1714).

5'-Fluoro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4n)

Red powder, m.p. 240.5-242.0 °C; IR (KBr) (v_max , cm^-1): 3592, 3442, 2996, 2215, 1711, 1679, 1642; ¹H NMR (400 MHz, DMSO-d₆): δ 3.75 (3H, s, OCH₃), 6.55-8.08 (16H, m, Ar-H), 9.93 (1H, s, NH), 10.18 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 181.0, 179.8, 179.3, 159.7, 159.5, 149.7, 141.3, 139.8, 139.5, 138.6, 138.6, 136.7, 135.7, 133.8, 132.3, 130.2, 130.1, 128.1, 126.4, 124.9, 123.7, 115.4, 113.3, 112.1, 110.2, 101.4, 55.5, 51.0. HRMS (ESI) m/z: cacld for C₃₄H₂₁N₄O₄F (M+H)⁺ found (expected): 553.3108 (553.3170).

5'-Chloro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4o)

Red powder, m.p. 237.4-238.6 °C; IR (KBr) (v_max , cm^-1): 3564, 3394, 2985, 2209, 1715, 1674, 1645; ¹H NMR (400 MHz, DMSO-d₆): δ 3.75 (3H, s, OCH₃), 6.60-8.08 (16H, m, Ar-H), 9.91 (1H, s, NH), 10.29 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 181.0, 179.8, 179.0, 159.5, 149.6, 141.4, 139.9, 138.5, 136.8, 135.7, 133.8, 132.3, 130.3, 130.2, 130.0, 128.4, 128.1, 126.4, 125.8, 124.9, 124.6, 123.7, 115.2, 113.4, 111.0, 101.3, 55.5, 50.7. HRMS (ESI) m/z: cacld for C₃₄H₂₁N₄O₄Cl (M+H)⁺ found (expected): 585.1864 (585.1824).

3-Methyl-5'-nitro-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4p)

Red powder, m.p. 217.3-219.2 °C; IR (KBr) (v_max , cm^-1): 3422, 3191, 2990, 2212, 1734, 1680, 1620, 1558, 1354; ¹H NMR (400 MHz, DMSO-d₆): δ 3.73 (3H, s, CH₃), 6.57-8.07 (16H, m, Ar-H), 9.87 (1H, s, NH), 10.16 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.9, 179.8, 179.2, 159.4, 149.7, 142.4, 141.1, 138.5, 138.3, 136.7, 135.7, 133.8, 132.3, 130.2, 130.2, 130.1, 128.7, 128.1, 126.4, 126.4, 125.0, 124.5, 123.6, 122.0, 116.0, 113.3, 109.7, 101.7, 55.5, 50.5. HRMS (ESI) m/z: cacld for C₃₄H₂₁N₅O₆ (M+H)⁺ found (expected): 596.1878 (596.1865).

3,5'-Dimethyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4q)

Red powder, m.p. 254.3-256.3 °C; IR (KBr) (v_max , cm^-1): 3420, 3062, 3002, 2987, 2360, 2209, 1755, 1727, 1621; ¹H NMR (400 MHz, DMSO-d₆): δ 2.19 (3H, s, CH₃), 3.74 (3H, s, OCH₃), 6.55-8.07 (16H, m, Ar-H), 9.83 (1H, s, NH), 10.05 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.9, 179.9, 179.1, 159.4, 149.6, 141.0, 140.0, 138.6, 138.5, 136.7, 135.7, 133.7, 132.3, 130.8, 130.3, 130.2, 130.1, 128.9, 128.0, 126.4, 125.1, 123.6, 116.2, 113.3, 109.5, 101.8, 55.5, 50.6, 21.0. HRMS (ESI) m/z: cacld for C₃₅H₂₃N₄O₄ (M+H)⁺ found (expected): 565.1526 (565.1570).

5'-Methoxy-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4r)

Red powder, m.p. 216.4-218.1 °C; IR (KBr) (v_max , cm^-1): 3426, 3343, 2998, 2945, 2836, 2202, 1755, 1727, 1678; ¹H NMR (400 MHz, DMSO-d₆): δ 1.65 (3H, s, CH₃), 3.61 (3H, s, OCH₃), 6.69-8.08 (16H, m, Ar-H), 9.78 (1H, s, NH), 10.51 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.9, 179.9, 179.0, 159.4, 155.4, 149.7, 141.1, 139.4, 138.6, 136.7, 135.9, 135.7, 133.7, 132.4, 130.3, 130.2, 130.1, 128.0, 126.4, 125.1, 123.6, 116.0, 113.2, 111.6, 110.0, 101.7, 55.9, 55.5, 51.0. HRMS (ESI) m/z: cacld for C₃₅H₂₃N₄O₅ (M+H)⁺ found (expected): 581.1878 (581.1819).

3-Methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4s)

Red powder, m.p. 244.8-247.6 °C; IR (KBr) (v_max , cm^-1): 3151, 3082, 2921, 2859, 2363, 2218, 1710, 1675, 1632; ¹H NMR (400 MHz, DMSO-d₆): δ 2.40 (3H, s, CH₃), 3.36 (3H, s, CH₃), 6.86-8.06 (12H, m, Ar-H), 9.69 (1H, s, NH), 10.67 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.6, 179.3, 178.8, 144.7, 141.3, 140.9, 136.8, 136.7, 135.9, 135.6, 135.2, 133.3, 131.7, 129.9, 129.8, 128.1, 126.0, 125.8, 123.9, 122.9, 121.8, 115.2, 109.0, 101.1, 49.8, 20.5, 11.2. HRMS (ESI) m/z: cacld for C₂₉H₂₀N₄O₃ (M+H)⁺ found (expected): 473.1878 (473.1808).

5'-Fluoro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4t)

Red powder, m.p. 198.7-200.7 °C; IR (KBr) (v_max , cm^-1): 3592, 3442, 2947, 2361, 2343, 2207, 1711, 1679, 1642; ¹H NMR (400 MHz, DMSO-d₆): δ 1.64 (3H, s, CH₃), 2.40 (3H, s, CH₃), 6.89-8.07 (11H, m, Ar-H), 9.74 (1H, s, NH), 10.70 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.7, 179.3, 178.9, 144.6, 141.1, 138.2, 138.1, 137.5, 137.0, 136.0, 135.6, 135.2, 133.3, 131.8, 129.9, 129.8, 128.6, 126.0, 125.8, 123.0, 114.5, 114.4, 114.2, 109.7, 109.7, 100.6, 50.3, 20.5, 11.2. HRMS (ESI) m/z: cacld for C₂₉H₁₉N₄O₃F (M+H)⁺ found (expected): 491.1452 (491.1414).

5'-Chloro-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4u)

Red powder, m.p. 252.4-254.2 °C; IR (KBr) (v_max , cm^-1): 3564, 3394, 2934, 2361, 2212, 1715, 1674, 1645; ¹H NMR (400 MHz, DMSO-d₆): δ 1.65 (3H, s, CH₃), 2.40 (3H, s, CH₃), 6.92-8.07 (11H, m, Ar-H), 9.74 (1H, s, NH), 10.81 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.7, 179.3, 178.9, 144.6, 141.1, 138.2, 138.1, 137.5, 137.0, 136.0, 135.6, 135.2, 133.3, 131.8, 129.9, 129.9, 128.6, 126.0, 125.9, 123.0, 114.6, 114.4, 114.2, 109.7, 109.7, 100.6, 50.3, 20.6, 11.2. HRMS (ESI) m/z: cacld for C₂₉H₁₉N₄O₃Cl (M+H)⁺ found (expected): 507.1864 (507.1818).

3-Methyl-5'-nitro-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4v)

Red powder, m.p. 238.3-240.1 °C; IR (KBr) (v_max , cm^-1): 3422, 3191, 2996, 2211, 1734, 1680, 1620, 1545, 1352; ¹H NMR (400 MHz, DMSO-d₆): δ 1.65 (3H, s, CH₃), 2.41 (3H, s, CH₃), 7.14-8.21 (11H, m, Ar-H), 9.91 (1H, s, NH), 11.44 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.8, 179.4, 179.1, 147.8, 144.5, 142.5, 141.5, 137.2, 137.1, 136.2, 135.5, 135.2, 133.4, 131.6, 130.0, 129.9, 126.1, 125.9, 125.7, 123.2, 119.8, 113.8, 109.2, 99.9, 49.8, 20.6, 11.3. HRMS (ESI) m/z: cacld for C₂₉H₁₉N₅O₅ (M+H)⁺ found (expected): 518.2077 (518.2059).

3,5'-Dimethyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4w)

Red powder, m.p. 207.9-209.6 °C; IR (KBr) (v_max , cm^-1): 3420, 3062, 2990, 2885, 2336, 2212, 2107, 1755, 1727, 1621; ¹H NMR (400 MHz, DMSO-d₆): δ 1.63 (3H, s, CH₃), 2.14 (3H, s, CH₃), 2.40 (3H, s, CH₃), 6.79-8.06 (11H, m, Ar-H), 9.68 (1H, s, NH), 10.57 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.6, 179.4, 178.9, 144.7, 140.9, 138.8, 136.8, 136.8, 135.9, 135.6, 135.2, 133.3, 131.7, 130.7, 129.9, 129.8, 128.3, 126.0, 125.8, 124.5, 122.9, 115.3, 108.8, 101.2, 49.8, 20.5, 20.5, 11.2. HRMS (ESI) m/z: cacld for C₃₀H₂₁N₄O₃ (M+H)⁺ found (expected): 487.1095 (487.1065).

5'-Methoxy-3-methyl-1-phenylspiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3'-indoline]-2',5,10(1H,11H)-trione (4x)

Red powder, m.p. 240.3-242.1 °C; IR (KBr) (v_max , cm^-1): 3426, 3343, 3219, 2991, 2836, 2202, 1755, 1727, 1678; ¹H NMR (400 MHz, DMSO-d₆): δ 1.64 (3H, s, CH₃), 2.40 (3H, s, CH₃), 3.60 (3H, s, OCH₃), 6.68-8.07 (11H, m, Ar-H), 9.65 (1H, s, NH), 10.49 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.6, 179.4, 178.7, 155.1, 144.7, 140.9, 137.9, 136.8, 135.9, 135.7, 135.2, 134.6, 133.2, 131.8, 129.9, 129.8, 126.0, 125.8, 122.9, 115.1, 112.5, 111.1, 109.3, 101.1, 55.3, 50.3, 20.5, 11.3. HRMS (ESI) m/z: cacld for C₃₀H₂₁N₄O₄ (M+H)⁺ found (expected): 503.2880 (503.2814).

Alamar Blue assay was used to evaluate the anti-cancer activity on human three positive breast cancer cells (MCF-7), human three negative breast cancer cells (MDA-MB-231), human hepatoma cells (HepG2), human hepatoma cells (MHCC-97H) and cytotoxicity on human hepatocyte cells (LO2) of all products in vitro. The inhibitory activity of compounds on cell proliferation was measured by inhibition rate of cell proliferation which was calculated according to the following formula:

Inhibition rate (%) = (A_control- A_sample) / (A_control- A_blank)

In the formula: A_controlrepresents the fluorescence value of control group, A_samplerepresents the fluorescence value of sample group, and A_blankrepresents the fluorescence value of blank group.

The inhibition rates of different concentrations of compound 4 on each cell strain were showed in Table S1-S5 and Figure S1-S3. The result showed that: (1) Products 4h-4t have no obvious inhibition on normal LO2 cells at 12.5 µg/mL, indicating their weak binding capacity to LO2 cells due to the large steric hindrance from substituents on 1-phenyl or 3-phenyl of pyrazole ring. (2) At 12.5 µg/mL, this series of products show specific inhibition on MCF-7 cells. The inhibition rate of eight compounds (4b, 4c, 4e, 4g, 4s, 4t, 4w, 4x) exceed 50%. It is particularly noteworthy that 4w and 4x, which are non-toxic to normal cells, show high inhibition rate of on MCF-7 cells (> 90%). (3) At high concentration (25 µg/mL and 50 µg/mL), most of the products have strong inhibition on four kinds of cancer cells, but also have cytotoxicity to LO2 cells.

In conclusion, we have reported an efficient and practical protocol for the synthesis of spiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3’-indoline] derivatives (4) from the three-component reaction of 2-hydroxy-1,4-naphthoquinone, isatins and aminopyrazoles catalyzed by a Glucose-containing sulfonic acid catalyst β-1-N-methyl-imidazole-2,3,4,6-tetrasulfonic-D-glucopyranosyl bisulfate ([Bmim-G-(SO₃H)₄]⁺[HSO₄]^-, NMI-GSA). Six bonds were cleaved while six new bonds (two C-N bonds and four C-C bonds) were formed to constructed a new dihydropyridine ring and a spiro skeleton in one-pot with water as the only one by-product in this highly atom-economic reaction. The high efficiency of catalyst from its strong acidity greatly expands the scope of reaction substrates, 2-hydroxy-1,4-naphthoquinone with lower nucleophilicity and aminopyrazole derivatives with large steric hindrance substituents at 1- or 3- position can successfully participate in the reaction to give medium to high yield under mild condition. In addition to the high atom- and step-economy, this method also has the advantage of mild reaction conditions (50^oC) and convenient post-treatment, providing an efficient and rapid way for the synthesis of target products. Two non-toxic compounds to normal cells have been screened out because of their special high inhibition rate of on MCF-7 cells (> 90%), indicated their potential in new drug development.

Ethical Approval

Not applicable.

Competing interests

The authors declare no competing interests.

Authors’ contributions

Rui Yuan was contributed to investigation, methodology, data curation and writing. Xiaowei Chi was contributed to validation, investigation, editing and original draft. Lizhuo Zhang was contributed to experimental and data curation. Jinjuan Liu was contributed to bioactivity evaluation. Yu Wan was contributed to resources investigation, supervision, structure and properties analysis. Hui Wu was contributed to conceptualization, supervision, reviewing and funding acquisition.

Funding

We are grateful to the foundation of the “Aid project for PhD faculties in Jiangsu Normal University” (17XLR023) for financial support.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Schemes 1 and 2 are available in the Supplementary Files section.

The full Result and discussion section with images is available in the Supplementary Files section.

No competing interests reported.

SupportingInformation06.23.pdf
1.jpg
Scheme 1. Synthesis of spiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3’-indoline] derivatives 4
2.jpg
Scheme 2. Proposed reaction mechanism
Resultanddiscussionwithimages.docx

Glucose-containing sulfonic acid-catalyzed efficient synthesis of spiro[benzo[g]pyrazolo[3,4-b]quinoline-4,3’-indoline] derivatives

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Abstract

Introduction

Experimental

Result and discussion

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References

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Supplementary Files

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