D2 receptor ligands

Accepted Manuscript Chemical synthesis, microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands Haixia Ge, Yan Zhang, Zhuo Yang, Kun Qiang, Chao Chen, Laiyu Sun, Ming Chen, Jian Zhang PII: DOI: Reference:

S0968-0896(19)30053-7 https://doi.org/10.1016/j.bmc.2019.04.014 BMC 14868

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

10 January 2019 20 March 2019 6 April 2019

Please cite this article as: Ge, H., Zhang, Y., Yang, Z., Qiang, K., Chen, C., Sun, L., Chen, M., Zhang, J., Chemical synthesis, microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.014

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Chemical synthesis, microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands Haixia Ge a*, Yan Zhang b, Zhuo Yang c, Kun Qiang a, Chao Chen a, Laiyu Sun a, Ming Chen c, Jian Zhang b*

a

School of Life Sciences, Huzhou University, Huzhou 313000, China

b

State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China

Pharmaceutical University, Nanjing, 210009, China. c

Chemical Biology Core Facility, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes

for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.

* Haixia Ge: Tel.: +86-572-2321166. Fax: +86-572-2321016. E-mail: [email protected] * Jian Zhang: Tel.: +86-25-86185157. Fax: +86-25-86185158. E-mail: [email protected]

Notes: The authors declare no competing financial interest.

1

Abstract: Dopamine D1/D2 receptors are important targets for drug discovery in the treatment of central nervous system diseases. To discover new and potential D1/D2 ligands, 17 derivatives of tetrahydroprotoberberine (THPB) with various substituents were prepared by chemical synthesis or microbial transformation using Streptomyces griseus ATCC 13273. Their functional activities on D1 and D2 receptors were determined by cAMP assay and calcium flux assay. Seven compounds showed high activity on D1/D2 receptor with low IC50 values less than 1 µM. Especially, top compound 5 showed strong antagonistic activity on both D1 and D2 receptor with an IC50 of 0.391 and 0.0757 µM, respectively. Five compounds displayed selective antagonistic activity on D1 and D2 receptor. The SAR studies revealed that (1) the hydroxyl group at C-9 position plays an important role in keeping a good activity and small or fewer substituents on ring D of THPBs may also stimulate their effects, (2) the absence of substituents at C-9 position tends to be more selective for D2 receptor, and (3) hydroxyl substitution at C-2 position and the substitution at C-9 position may facilitate the conversion of D1 receptor from antagonist to agonist. Molecular docking simulations found that Asp 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386 of D1/ D2 receptors are the key residues, which have strong interactions with the active D1/D2 compounds and may influence their functional profiles.

Keywords: Tetrahydroprotoberberines; Dopamine D1/D2 receptors; Antagonistic activities; Microbial transformation; Molecular simulations.

2

1. Introduction G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly as they involve numerous diverse physiological processes and have druggable sites at the cell surface.1 Among the GPCR superfamily, the dopamine receptors are associated with schizophrenia, depression, Parkinson’s disease, attention-deficit hyperactivity disorder, and substance abuse, etc.2 According to their pharmacological and functional characteristics, dopamine receptors can be classified into D1-like (D1 and D5) and D2-like (D2, D3 and D4) types. The dopamine receptor D1 is the most abundant of the five subtypes, and the D1 antagonists were found to be effective in the treatment of pathological gambling,3 Tourette’s syndrome,4 Lesch-Nyhan disease5 and other CNS conditions. The D2 receptor is the primary target for both typical and atypical antipsychotic drugs, and for drugs used to treat Parkinson’s disease.6, 7 But current dopaminergic drugs in the market mainly target D2/D3 receptors, while no drug is designed to target on D1 receptor.8 Therefore, discovery and development of novel D1R / D2R ligands with safer and more effective medications represents an important field of research interests for certain diseases of unmet medical need. Importantly, structural studies have uncovered two potential allosteric binding sites, which will supply valuable information for the rational design of new D1R / D2R ligands.9 Tetrahydroprotoberberines (THPBs), a class of isoquinoline alkaloids, are originally isolated from the Chinese herb Corydalis yanhusuo and various species of Stephania. THPBs and their derivatives, such as l-tetrahydropalmatine (l-THP, Fig.1), l-stepholidine (l-SPD, Fig.1), l-tetrahydroberberrubine (l-TU, Fig.1), have been demonstrated to be active at D1 and D2 receptors and become lead compounds for the development of novel drugs to treat central nervous system disorders. l-THP, a D1/D2 antagonist, has been used as a pain-killer for more than 40 years in China and has the potential for treating cocaine addiction.10, 11 l-SPD has been proved to be a very rare D1 agonist, D2 antagonist and D3 antagonist and show 3

antipsychotic, anti-addiction and memory enhancing properties.12-15 As an antagonist of D1/D2 receptors and a 5-HT1A agonist, l-TU is a potential candidate for the treatment of anxiety and depression.16, 17 In general, the multi-pharmacological activity of THPBs is mainly related to dopamine receptor.

Fig.1. Structures of l-THP, l-SPD and l-TU

Many structure-activity relationships studies indicated that the position and characteristics of the substituents on aryl rings A and D can affect the size of the A-D dihedral angle of THPBs, and then significantly influence the binding affinity and activity of ligands at dopamine D1 and D2 receptors.18-23 However, most of the reported compounds have only binding affinity data and no functional activity test, and it is impossible and difficult to perform a comprehensive analysis of the relationship between functional activity and structures, which is critical to design the novel selective and functional compounds. We are particularly interested in exploring the effect of various substituents on the selectivity of dopamine D1 and D2 receptors and structure-functional activity relationship of THPBs on D1 and D2 receptors as such compounds can be used as research tools to detect the effects of multiple receptor modulation in the treatment of related diseases. In the present work, a series of THPBs derivatives with various substituents were prepared by chemical semi- or total synthesis or microbial transformation. Biotransformation has become one of the promising and effective methods for the structural modification of natural products, and some THPBs were subjected to microbial transformation by Streptomyces griseus ATCC 13273 to determine the substrate specificity of demethylation. 17 compounds’ functional activities (agonistic or antagonistic activity) on D1 and D2 receptors were determined by cAMP assay and calcium flux assay, respectively. In addition, molecular docking simulations were performed to explore the receptor-ligand interactions and used 4

to predict the key residues influencing the functional profiles of these compounds.

2. Results and discussion 2.1

Chemistry As the previous report,24 compound 1 and tetrahydroberberrubine (TU) were synthesized through

pyrolysis monodemethylation and reduction reactions of palmatine hydrochloride and berberine hydrochloride, respectively (Scheme 1). Compounds 2 and 3 were synthesized using acetyl chloride or acetylferuloyl chloride and tetrahydroberberrubine (TU), respectively (Scheme 2). Compound 4 was synthesized from berberine hydrochloride, which first reacted with phloroglucinol in H2SO4 (60%) at 90-95 °C and then was reduced by NaBH4 (Scheme 3).24, 25 For the synthesis of compound 5, berberine hydrochloride was used as the starting material and first conducted pyrolysis monodemethylation, reacted with phloroglucinol in H2SO4 (60%) at 90-95 °C and then was reduced by NaBH4 (Scheme 3). As shown in Scheme 4, compounds 6-12 were synthesized through a seven-step process including dehydration, reduction, acidification, cyclization, alkalization, acidification, and reduction,26, 27 in which cyclization was the key step and greatly affected the total yield. The starting materials were 3, 4-(methylenedioxy) phenylethylamine or 3, 4-dimethoxyphenyl ethylamine and the substituted benzaldehydes, all of which were commercially available. Dehydration of the two starting materials gave the intermediate M1, which was directly reduced with NaBH4 in methanol to give the intermediate M2. M2 was acidified with HCl in ethanol to give M3. The mixture of M3 and glyoxal was directly treated with CuSO4 and HCl in formic acid to obtain the key intermediate M4, then M4 was dissolved in the methanol and waster, and treated with CaO to give the intermediate M5. The acidification of M5 with HCl in ethanol afforded the crude products of M6, which were purified with silica gel column chromatography. Finally, the reduction of M6 with NaBH4 in methanol obtained compounds 6-12. Among these compounds 6-12, compounds 10 and 11 are a pair of isomers, 5

which were simultaneously produced in the total synthesis and compound 11 was the predominant product compared to compound 10. Compound 13 was obtained from alkylation reaction between benzyl bromide and compound 10 (Scheme 5).

Scheme 1. Semi-synthesis of compounds 1 and TU. Reagents and conditions: (a) 195-205 °C, 20 min; EtOH - HCl (95:5); (b) NaBH4, MeOH, rt, 2 h.

Scheme 2. Semi-synthesis of compounds 2 and 3. Reagents and conditions: (a) 4-dimethylamiopryidine, triethylamine, CH2Cl2, rt, 3 h.

Scheme 3. Semi-synthesis of compounds 4 and 5. Reagents and conditions: (a) 60% H2SO4, Phloroglucin, 95 °C, 20 min; (b) NaBH4, MeOH, rt, 2 h; (c) 195-205 °C, 20 min; EtOH - HCl (95:5).

6

Scheme 4. Total synthesis of compounds 6-12. Reagents and conditions: (a) 100 °C, 2 h; (b) NaBH4, MeOH, rt, 2 h; (c) EtOH - HCl (95:5), rt; (d) anhydrous CuSO4, anhydrous formic acid, 40% glyoxal solution, conc. HCl, 100 °C, 4 h; (e) CaO, MeOH, H2O, rt, 2 h.

Scheme 5. Semi-synthesis of compound 13. Reagents and conditions: (a) Benzyl bromide, K2CO3, DMF, 60 °C, 4 h.

The glycosidic products 14 and 15 are a pair of diastereoisomer, which were prepared by microbial transformation with Bacillus subtilis ATCC 6633 from racemic tetrahydroberberrubine as our previous discovery (scheme 6).28

Scheme 6. Microbial semi-synthesis of compounds 14 and 15 by Bacillus subtilis ATCC 6633 Streptomyces griseus ATCC 13273 has been proven as a potent strain with astonishing demethylation ability on natural products.29, 30 In addition to TU, compounds 1, 6, 8, 9, 12, a series of THPBs (Fig. 2) with 7

methoxy and hydroxyl groups at different carbon positions on D aromatic ring (compounds A-F), were also used as substrates in microbial transformation screening to generate the new products of THPBs using Streptomyces griseus ATCC 13273. The results show that only substrate E and F could be transformed to metabolites 16 and 17, respectively (Scheme 7). In our previous report, l-tetrahydropalmatine (l-THP) could be converted to l-corydalmine.24 So we could draw a preliminary conclusions: (1) the substrates with a methylenedioxy group on aromatic ring A could not be metabolized to the demethylation products; (2) the substrates with hydroxyl groups at any positions on aromatic ring D could not be converted to the demethylation products; and (3) the site of demethylation has no regularity and selectivity, and it could be demethylated on ring A as well as on ring D.

Fig. 2. Chemical structures of the substrates in the microbial transformation screening.

8

Scheme 7. Microbial transformation of substrates E and F by Streptomyces griseus ATCC 13273. Compound 16 was white amorphous powder and its structure was characterized by HR-ESI-MS, 1

H-NMR, 13C-NMR, HSQC, HMBC and NOESY. HR-ESI-MS of compound 16 revealed the molecular ion

at m/z 342.1761 [M+H]+, which indicated a 14 amu mass of methylene group might be removed from substrate E (M.W. = 355.43, Scheme 7). The strongest fragment ions of compound 16 was at m/z 178.0911. According to the major MS characteristic fragmentation pathways of THPBs,31 we could deduce that there was one hydroxyl group on ring A. Meanwhile, the 1H-NMR spectrum exhibited three characteristic methoxymethyl signals that appeared at δH 3.71, 3.72, and 3.74 ppm with the absence of a methoxymethyl signal of the substrate E, and one hydroxyl signal at δH 8.69 ppm. Compared with the 13C-NMR spectrum of substrate E (Table 1), a carbon signal at δC 55.62 of a methoxy group is lost, and the chemical shifts of C-1, C-2 and C-3 show obvious changes , while those of C-9, C-10, C-11 and C-12 has imperceptible changes. In the HSQC spectrum, the carton signals of 3-OCH3, 11-OCH3, 10 -OCH3 at δC 55.62, 55.62, 55.50 ppm showed correlations with their proton signals at δH 3.74, 3.71, 3.72 ppm, respectively. In the HMBC spectrum, the long-bond correlations between δC 145.98 (C3) and δH 3.74 (3-OCH3), 6.64 (4-ArH), δC 147.01 (C11) and δH 3.71 (11-OCH3), 6.73 (12-ArH), δC 147.14 (C10) and δH 3.72 (10-OCH3), 6.66(9-ArH) were observed (Fig. 3a). In addition, in the nuclear overhauser effect spectroscopy (NOESY) spectrum, the proton-proton correlations between 2-OH at δH 8.69 ppm and 1-ArH at δH 6.70 ppm, 4-ArH at δH 6.64 ppm 9

and 3-OCH3 at δH 3.74 ppm, 9-ArH at δH 6.66 ppm and 10-OCH3 at δH 3.72 ppm, 12-ArH at δH 6.73 ppm and 11-OCH3 at δH 3.71 ppm, were observed obviously (Fig. 4a). Based on all the evidences, compound 16 was characterized as 2-demethyl product of substrate E. Similar to compound 16, compound 17 was the 11-demethyl product of substrate F (Scheme 7) and its structure was also characterized by multiple spectra. The molecular ion of compound 17 (C21H25NO5) was at m/z 372.2018 [M+H]+ indicating that a 14 amu mass of methylene group might be removed from substrate F (M.W.=385.46, C22H27NO5). The strongest fragment ions was at m/z 192.1070 that indicated there was one hydroxyl group on ring D.31 The 1H-NMR spectrum exhibited four characteristic methoxymethyl signals that appeared at δH 3.74, 3.72, 3.75 and 3.71 ppm with the absence of a methoxymethyl signal of the substrate F, and one hydroxyl signal at δH 8.36 ppm. Compared with the 13C-NMR spectrum of substrate F (Table 2), a carbon signal at δC 60.03 of a methoxy group is lost, and the chemical shifts of C-9, C-10, C-11 and C-12 show obvious changes , while those of C-1, C-2, C-3 and C-4 has imperceptible changes. In the HSQC spectrum, the carton signals of 2-OCH3, 3-OCH3, 10-OCH3, 12-OCH3 at δC 55.84, 55.89, 55.41, 59.24 ppm showed correlations with their proton signals at δH 3.74, 3.72, 3.75, 3.71 ppm respectively. In the HMBC spectrum, the long-bond correlations between δC 147.14 (C2) and δH 3.74 (2-OCH3), 6.88 (1-ArH) respectively, δC 147.24 (C3) and δH 3.72 (3-OCH3), 6.69 (4-ArH) respectively, δC 147.05 (C10) and δH 3.75 (10-OCH3), 6.47 (9-ArH) respectively, δC 145.44 (C12) and δH 3.71 (12-OCH3), 2.34, 3.37 (13-CH2) respectively, δC 137.15 (C11) and δH 6.47 (9-ArH) were observed (Fig. 3b). In addition, in the rotating frame overhauser enhancement spectroscopy (ROESY) spectrum, the proton-proton correlations between 1-ArH at δH 6.88 ppm and 2-OCH3 at δH 3.74 ppm, 4-ArH at δH 6.69 ppm and 3-OCH3 at δH 3.72 ppm, 9-ArH at δH 6.47 ppm and 10-OCH3 at δH 3.75 ppm, 12-OCH3 at δH 3.71 ppm and 13-CH2 at δH 3.37 ppm, were observed obviously (Fig. 4b). Finally, based on all the evidences, compound 17 was characterized as 11-demethyl product of substrate F. 10

Table 1 13C-NMR and 1H-NMR spectral data of substrate E and the product 16 Carbon E 13C-NMR 16 13C-NMR E 1H-NMR 16 1H-NMR 1 2 3 4 4a 5

109.43 146.96 147.13 111.67 126.34 28.51

112.33 144.60 145.98 111.82 130.00 28.46

6

50.83

51.00

8

57.54

57.55

8a 9 10 11 12 12a 13

126.25 109.23 146.96 147.13 111.78 126.28 35.77

126.41 109.45 147.14 147.01 111.99 126.17 36.86

14 14a 2-OCH3/OH 3-OCH3 10-OCH3 11-OCH3

59.15 129.80 55.62 55.48 55.37 55.40

58.92 124.65 55.62 55.50 55.62

6.73 (s, 1H) 6.87 (s, 1H) 2.53 (m, 1H) 2.93 (m, 1H) 2.44 (m, 1H) 3.05 (m, 1H) 3.48 (d, J = 15 Hz, 1H) 3.87 (d, J = 15 Hz, 1H) 6.66 (s, 1H) 6.67 (s, 1H) 2.59 (m, 1H) 3.36 (m, 1H) 3.41 (m, 1H) 3.75 (s, 3H) 3.72 (s, 3H) 3.72 (s, 3H) 3.71 (s, 3H)

6.70 (s, 1H) 6.64 (s, 1H) 2.57 (m,1H) 2.90 (m,1H) 2.43 (m,1H) 3.03 (m,1H) 3.47 (d, J=15Hz, 1H) 3.86 (d, J=15Hz, 1H) 6.66 (s, 1H) 6.73 (s, 1H) 2.50 (m, 1H) 3.17 (m, 1H) 3.36 (m, 1H) 8.69 (s, 1H) 3.74 (s, 3H) 3.72 (s, 3H) 3.71 (s, 3H)

Fig. 3. a Key HMBC correlation of compound 16; b Key HMBC correlation of compound 17.

11

Fig. 4. a Key NOESY correlation of compound 16; b Key ROESY correlation of compound 17.

Table 2 13C-NMR and 1H-NMR spectral data of substrate F and the product 17 Carbon F 13C-NMR 17 13C-NMR F 1H-NMR 17 1H-NMR 1 2 3 4 4a 5

109.63 147.17 147.23 111.79 126.43 28.48

109.67 147.14 147.24 111.81 126.51 28.55

6

50.44

50.60

8

57.77

57.72

8a 9 10 11 12 12a 13

129.87 105.16 150.57 139.72 151.30 120.20 30.63

124.70 104.72 147.05 137.15 145.44 120.11 30.74

14 14a 2-OCH3 3-OCH3 10-OCH3 11-OCH3/OH 12- OCH3

58.67 130.12 55.68 55.83 55.37 60.03 60.23

58.89 130.66 55.84 55.89 55.41 59.24

2.2

6.88 (s, 1H) 6.69 (s, 1H) 2.61 (m, 1H) 2.90 (m, 1H) 2.43 (m, 1H) 3.04 (m, 1H) 3.50 (d, J=15Hz, 1H) 3.85 (d, J=15Hz, 1H) 6.54 (s, 1H) 2.34 (m, 1H) 3.38 (m, 1H) 3.32 (m, 1H) 3.77 (s, 3H) 3.76 (s, 3H) 3.76 (s, 3H) 3.73 (s, 3H) 3.72 (s, 3H)

6.88 (s, 1H) 6.69 (s, 1H) 2.62 (m, 1H) 2.91 (m, 1H) 2.43 (m, 1H) 3.03 (m, 1H) 3.49 (d, J=15Hz, 1H) 3.82 (d, J=15Hz, 1H) 6.47 (s, 1H) 2.34 (m, 1H) 3.37 (m, 1H) 3.37 (m, 1H) 3.74 (s, 3H) 3.72 (s, 3H) 3.75 (s, 3H) 8.36 (s, 1H) 3.71 (s, 3H)

In vitro evaluations of biological activity

2.2.1 Antagonistic activity assay on CHO-K1 cells expressing D1 and D2 receptors Since most of reported THPBs compounds exhibited antagonistic activity on dopamine D1 and D2 receptors, we first evaluated the antagonistic activity in dose-response studies using the cAMP assay on D1 receptor and calcium flux assay on D2 receptor, respectively. Compounds l-THP and the known D1/D2 receptor antagonist SCH23390 are used as the reference compounds. Furthermore, their half maximal 12

inhibitory concentrations (IC50) were calculated. The structures and bioactive results of all compounds are listed in Table 3, and the sigmoidal dose-response curves of the representative compounds with significant antagonistic activity are illustrated in Fig. 5.

Fig. 5 a. Antagonist effect of representative compounds 1, 4, 5, 7, 11, and 12 on D1/CHO-K1 cells； b. Antagonist effect of representative compounds 2, 5, 6, 7, 10, 11, and 12 on D2/CHO-K1 cells. The reference compound l-THP showed good antagonistic activity on the D1 receptor with an IC50 value of 1.63 µM. Compared with l-THP, compound 1 has a hydroxyl group at R3 (C-9) position, which showed greatly improved activity with an IC50 of 0.41 µM. Further moving the hydroxyl group from the R3 position to R5 (C-11) position got compound 6, which exhibited slightly decreased activity with an IC50 value of 0.67 µM on the D1 receptor. Compared with compound 1, keeping a hydrogen atom at R3 position and introducing different groups to ring D gave compounds 7, 8 and 17, but all three compounds showed dramatically decreased activity on the D1 receptor. These results indicated that a hydroxyl group at R3 position is a better option to keep a good activity, while adding hydroxyl or methoxyl groups at R5 and R6 (C-12) position on ring D might attenuate the activity on D1 receptor. Replacing R1 (C-2) and R2 (C-3) methoxyl groups of reference compound l-THP to hydroxyl groups offered compound 4, which exhibited strong antagonistic activity with an IC50 value of 0.63 µM on the D1 receptor. This result indicated that methoxyl groups at R1 and R2 positions may not be essential to keep a 13

good activity. Further replacing the methoxyl groups at R3 position with a hydroxyl group, which has been proven to be a good option at R3 position in compound 1, offered the most potential compound 5, which showed the best activity with an IC50 value of 0.391 µM on the D1 receptor. Similarly, deleting the hydroxyl group from R3 position and modifying the substituents at R2 and R5 positions obtained compound 16, which showed a very low activity (IC50 > 10 µM). These results also confirmed the importance of a hydroxyl group at R3 position. To further extend the SAR studies, we also synthesized a series of compounds with a dioxymethylene ring at R1 and R2 positions, including compounds 2, 3, 9-15. Compound 2 bearing an acetyl group at R3 position on ring D exhibited antagonistic activity on D1 receptor with an IC50 value of 1.08 µM, while compound 3 bearing an acetylferuloyl group showed lower activity (IC50 > 10 µM). So we deduce that a bulky group on R3 position might decrease the activity for these dioxymethylene compounds on D1 receptor. Furthermore, compounds 14 and 15 bearing a big glycosidic group on R3 position were demonstrated to be very low activity on D1 receptor. These results are consistent with compounds 2 and 3, which confirmed our deduction that a bulky group at R3 position can abolish the bioactivity of THPBs on D1 receptor. Interestingly, compounds 10 and 11 with only one hydroxyl and no other substituent on ring D showed similar antagonistic effect to the reference compound l-THP with an IC50 value of 1.03 and 1.42 µM, respectively on the D1 receptor. Especially, compounds 10 and 11, a pair of isomers bearing a hydroxyl group on different positions on D ring, possessed similar potent bioactivity. While, compounds 9, 12 and 13, which has more or bigger substituents on ring D besides a hydroxyl group, were examined to show decreased activity. These results are suggested that THPBs compounds prefer small substituents on ring D to get a good antagonistic activity on the D1 receptor. Besides D1 receptor, the antagonistic effect of THPBs compounds on D2 receptor was also evaluated by calcium flux assay. As shown in Tables 3, the reference compound l-THP showed strong antagonistic 14

activity on D2 receptor with an IC50 value of 0.446 µM. Among the tested THPBs, compounds 4 and 5 exhibited the best antagonistic activity on D2 receptor with an IC50 value of 0.040 and 0.076 µM, respectively (Table 3). Similar as the reference compound l-THP, compounds 2, 10 and 11 also possessed good antagonistic effect on D2 receptor with a nanomolar IC50 of 0.42, 0.19 and 0.50 µM, respectively. While, other compounds 1, 3, 6, 7, 8, 9, 12, 16 and 17 display weak effects with the IC50 values between 1 to 9 µM. And compounds 13-15 have very low activity on D2 receptor (IC50 > 10 µM). From these data about D2 receptor, we can get similar SAR conclusion as D1 receptor: a) the hydroxyl group at R3 position is important to get good antagonistic activity on D2 receptor, while a bulky group at R3 position can abolish the bioactivity; b) THPBs compounds bearing small or less substituents on ring D exhibit better antagonistic activity. Meanwhile, we noticed that the substituents at R3 position also play an important role on the selectivity of D1 and D2 receptors, such as compounds 8, 9, 16, 17 without substituents of R3. These compounds display some antagonistic effect on D2 receptor with an IC50 value of 8.81, 4.94, 3.58 and 6.36 µM, respectively, while have relatively low activity on D1 receptor (IC50 > 10 µM). For other compounds without R3 substituents, compounds 6, 7, 10, 11, 12 have antagonistic activity on both D1 and D2 receptors. Except compound 6, all of them show antagonistic activity on D2 receptor, which is greater than on D1 receptor.

Table 3 Structures and antagonistic effect of compounds on D1 and D2 receptors

No.

C14

R1

R2

R3

R4 15

R5

R6

D1R

D2R

IC50, µM

IC50, µM

l-THP 1 2 3

-

OCH3

OCH3

OCH3

OCH3

H

H

1.63

0.45

±

OCH3

OCH3

OH

OCH3

H

H

0.41

3.11

OCOCH3

OCH3

H

H

1.08

0.42

OCH3

H

H

>10

7.96

±

-CH2-

±

-CH2-

4 5 6 7 8 9 10 11 12 13

±

OH

OH

OCH3

OCH3

H

H

0.63

0.040

±

OH

OH

OH

OCH3

H

H

0.39

0.076

±

OCH3

OCH3

H

OCH3

OH

H

0.67

2.59

±

OCH3

OCH3

H

OH

H

H

6.51

1.53

±

OCH3

OCH3

H

OH

OCH3

OCH3

>10

8.81

±

-CH2-

H

OCH3

OH

H

>10

4.94

±

-CH2-

H

H

H

OH

1.03

0.19

±

-CH2-

H

OH

H

H

1.42

0.50

±

-CH2-

H

OCH3

OH

OCH3

7.42

1.08

±

-CH2-

H

H

H

>10

>10

14

-

-CH2-

OCH3

H

H

>10

>10

15

+

-CH2-

OCH3

H

H

>10

>10

16 17

±

OH

OCH3

H

OCH3

OCH3

H

>10

3.58

±

OCH3

OCH3

H

OCH3

OH

OCH3

>10

6.36

0.0034

2.05

SCH 23390

2.2.2 Agonistic activity assay on CHO-K1 cells expressing D1 and D2 receptors The agonistic activity of the THPBs compounds were determined on CHO-K1 cells expressing D1 and D2 receptors by cAMP assay and calcium flux assay, respectively. We screened the agonist effects of all tested compounds at the concentration of 10 µM toward the D1 and D2 receptors. The results are summarized in Table 4, as shown in the table, none of the THPBs compounds displays significant agonistic activity on D1 and D2 receptors at a concentration up to 10 µM (Table 4), except that the D1 receptor agonist SKF38393 and the D2 agonist dopamine showed their normal agonistic activity on the corresponding receptor with EC50 of 665.9 nM and 0.99 nM, respectively. However, compound 16 display a 16

trend of agonistic activity with 24.38% stimulation on D1 receptor. Considering the D1 antagonistic activity of compounds 4, 5 and the currently known D1 agonists of THPBs compounds, such as l-SPD (Fig. 1), l-isocorypalmine (Fig. 6),32 12-chloroscoulerine (Fig. 6),33 we can speculate that only one hydroxyl substitution at R1 position on aryl ring A and the substitution at R3 position may facilitate the conversion of D1 receptor from antagonist to agonist. The result deserves further exploration and research.

Fig.6. Structures of l-isocorypalmine and l-12-chloroscoulerine

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 l-THP SKF38393 Dopamine

Table 4 Agonist effect of compounds on D1 and D2 receptors Test Concentration (µM) D1 Receptor D2 Receptor Stimulation% Stimulation% (Mean±SD) (Mean±SD) -4.32±2.34 5.52±0.83 10 0.00±11.10 4.50±0.21 10 -3.69±2.77 2.97±0.61 10 -3.17±1.71 2.53±0.19 10 -5.41±0.56 4.27±0.36 10 -5.16±7.41 3.35±1.08 10 -3.91±0.40 2.39±0.12 10 5.95±0.12 6.88±1.94 10 -3.70±1.29 3.81±0.46 10 1.80±5.27 2.22±0.20 10 -4.41±3.77 2.17±0.74 10 -5.75±0.65 1.29±0.14 10 4.48±3.42 2.50±1.05 10 -1.05±1.16 2.96±0.33 10 -7.46±2.30 3.17±1.29 10 3.67±0.96 24.38±5.99 10 -4.17±1.36 3.74±0.28 10 -7.43±4.86 3.51±0.34 10 EC50: 665.9 nM EC50: 0.99 nM

17

2.3 Interactions between compounds and D1 / D2 receptors Using flexible docking in Discovery Studio 3.0, docking simulations were carried out to explore the binding mode of these THPBs analogues with the D1 receptor homology model as our newly published data34 and D2 crystal structure. The antagonist-bound conformation of the D1 receptor was constructed and the inactive-state structure of D3 receptor (PDB entry: 3PBL) was selected as the template. Although most of newly synthesized compounds are racemic, it is well known that the absolute configuration of THPBs has significant impact on their binding properties towards dopamine receptors. The S-enantiomer has high binding affinity, while the R-enantiomer exhibits low or absent affinity.16,

23, 34

Thus, we chose the

S-enantiomer of the tested compounds to study their binding mode. For D1 receptor, compound 5 with the strongest antagonistic activity was chosen as the delegate. As shown in Fig. 6a, the protonated and positive charged NH+ of compound 5 generated a salt bridge with residue Asp 103, which anchored the molecule into the binding site. And two hydroxyl groups on ring A, facing down towards the intracellular space, formed strong hydrogen bonds with residues Ile 104, Ser 107, Ser 202, and Ser 198. Especially, the central toggle switch, Trp 2856.48, had strong hydrophobic interactions with compound 5. This hydrophobic interaction further limited the free rotation of the Trp 2856.48, which was critical for the class A GPCR activation.35 In addition, other active compounds, such as 1, 4, 6, 7, 10, 11, were used to compare their simulated D1-ligand complexes with compound 5, (Fig. 6b). All these compounds displayed quite similar binding poses and formed similar interactions with surrounding key residues in flexible docking. In contrast, inactive compounds 3, 13, 14, and 15 had different docking mode with significantly lower scores of -CDOCKER_ENERGY, which were consistent with the experimental test results.

18

Fig. 6. a. Binding pose and interactions of compound 5 (gray) in D1 receptor. Critical residues are marked in pink sticks. b. Binding poses of compounds 1, 4, 5, 6, 7, 10, 11 in the respective simulated D1-ligand complexes. Hydrogen bonds and hydrophobic interactions are indicated as red and blue dashed lines. Compound 5 also exhibited best antagonistic activity on D2 receptor and was chosen as the representative. As illustrated in Fig. 7a, the protonated and positive charged NH+ of compound 5 could generate a salt bridge with residues Asp 114 and Tyr 416, and two hydroxyl groups on ring A formed hydrogen bonds with residues Cys 118, Ser 193 and Ser 197, correspondingly. Similarly, Trp 3866.48, the toggle switch, generated strong hydrophobic interactions with compound 5, which can stabilize Trp 3866.48 and limit its free rotation. Active compounds 1, 2, 4, 6, 7 and 8 also displayed quite similar binding poses as compound 5 and could form critical interactions respectively with surrounding key residues (Fig. 7b). But inactive compounds 13, 14 and 15 had the lowest scores of –CDOCKER_ENERGY, which were consistent with their experimental data on D2 receptor. As shown in the representative D2-14 complex (Fig. 7c), compound 14 could not form hydrogen bonds and hydrophobic interactions with the key residues Asp 114, Cys 118, Ser 197 and Trp 386, Instead, it could form weak hydrogen bonds and hydrophobic interactions with the residues Ser 193, Tyr 416 and Trp 100. This docking result may also indicate to a certain extent that compound 14 is inactive on the D2 receptor. 19

Fig. 7. a. Binding pose and interactions of compound 5 (gray) in D2 receptor. Critical residues are marked in pink sticks. b. Binding poses of compounds 1, 2, 4, 5, 6, 7, 8 in the respective simulated D1-ligand complexes. c. Binding poses of compound 14 (cyan) in D2 receptor. Critical residues are marked in pink sticks. Hydrogen bonds and hydrophobic interactions are indicated through red and blue dashed lines.

3. Conclusions In this work, a series of THPBs derivatives with various substituents were prepared by chemical seimor total synthesis method, and some of them were subjected to microbial transformation by Streptomyces griseus ATCC 13273 to determine the substrate specificity of demethylation. Compounds 16 and 17 were microbial region-selective demethylation products of substrates E and F by Streptomyces griseus ATCC 13273, and their structure were characterized by multiple spectra including HR-ESI-MS, 1H-NMR, 13

C-NMR, HSQC, HMBC and NOESY. The functional activities (agonistic or antagonistic activity) of synthesized compounds on D1 and D2

receptors were determined by cAMP assay and calcium flux assay. The activity results indicated that these THPBs compounds behave as antagonists of D1 and D2 receptors. Our preliminary SAR studies revealed that the hydroxyl group at R3 (C-9) position plays an important role to keep a good activity and active THPBs compounds prefer small or less substituents on ring D. Especially, the chemical modification offered us many active compounds, including top compounds 4 and 5 with high activity on both D1 and D2 receptors. At the same time, we found that the absence of substituents at R3 (C-9) position of THPBs tend to be more selective for D2 receptor, and speculated that only one C-2 hydroxyl substitution on aryl ring A and 20

the substitution at R3 (C-9) position may facilitate the conversion of D1 receptor from antagonist to agonist. Further molecular docking simulations well explained the interactions between THPBs compounds and protein key residues including 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386. These studies provide good leading compounds and valuable SAR information for druggable D1/D1 ligand development.

4. Experimental section 4.1 Chemistry Thin-layer chromatography (TLC) was performed on silica gel GF254 plates of 0.5 mm of thickness for analysis. Layers were air dried and activated at 110 °C for 0.5 h before use. NMR spectra (1H–NMR, 13

C-NMR, 1H-1H NOESY, 1H-1H ROESY, HMBC, and HSQC) were recorded on a Bruker Avance 500 or

300 MHz spectrometer with tetramethylsilane as the internal standard, and chemical shifts were expressed in δ (parts per million). HR-ESI-TOF-MS experiments were performed on a G1969A time of flight (TOF)-MS (Agilent Technologies, Santa Clara, CA, USA). ESI-MS experiments were performed on an Agilent 1100 Series MSD Trap mass spectrometer. Streptomyces griseus ATCC 13273 was obtained from a courtesy of Prof. J. P. N. Rosazza of University of Iowa, USA. 4.1.1 10-Methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,3,9-triol (5) Berberine hydrochloride (8.0 g, 0.0208 mol) was heated in an oil-bath at 205 °C for 15 min. The crude product was dissolved in EtOH / HCl (95:5) and stirred at the room temperature for 1 h, filtered to obtain berberrubine hydrochloride. Berberrubine hydrochloride (6.0 g, 0.017 mol) and phloroglucin (6.0 g, 0.048 mol) were mixed with 60% H2SO4 (50 mL) and stirred at 95 °C in an oil bath for 20 min. The mixture was cooled, concentrated, and resuspended in H2O/acetone (1:1), and then the intermediate was purified by silica gel chromatography (CHCl2 / MeOH, 20:1). A mixture of the intermediate and 50 mL of MeOH was refluxed for 20 min, then NaBH4 (1.9 g, 0.05 mol) was added in small portions with stirring at room temperature. Three hours later, the resulting solids were filtered, washed with MeOH and purified by recrystallization from ethanol to obtain 5 with a yield of 16%. White solid; 1H NMR (DMSO-d6, 500 MHz) δ: 2.67 (m, 2H, 6-H, 13-H), 2.44 (m, 1H, 5-H), 2.53 (m, 1H, 21

5-H), 2.83 (m, 1H, 6-H), 3.04 (brs, 1H, 13-H), 3.12 (d, J = 15Hz, 1H, 8-H), 3.25 (m, 1H, 14-H), 3.79 (s, 3H, -OCH3), 3.99 (d, J = 15Hz, 1H, 8-H), 6.45 (s, 1H, 4-H), 6.57(d, J = 8Hz, 1H, 12-H), 6.63 (s, 1H, 1-H), 6.77 (d, J = 8Hz, 1H, 11-H), 8.53, 8.50 ( brs, 2H, -OH), 8.66 ( brs, 1H, -OH). 13CNMR (DMSO-d6, 125 MHz) δ: 107.68 (C1), 144.92 (C2), 143.57 (C3), 110.21 (C4), 118.50 (C4a), 27.93 (C5), 51.13 (C6), 53.41 (C8), 121.78 (C8a), 141.93 (C9), 143.66 (C10), 112.43 (C11), 114.94 (C12), 124.46 (C12a), 35.69 (C13), 58.63 (C14), 127.31 (C14a), 55.96 (-OCH3; ESI-MS: m/z 314.2 [M+H]+. 4.1.2 2,3,10-Trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3, 2-a]isoquinolin-11-ol (6) A mixture of 3,4-dimethoxyphenethylamine (14.9 g, 0.082 mol) and 3-methoxy-4-hydroxybenzaldehyde (9.1 g, 0.082 mol) was heated in an oil-bath at 100 °C for 2 h. The product (M1) was dissolved in ethanol (200 mL) by reflux for 20 min, and NaBH4 (5.0 equiv) was added portionwise at the room temperature. The mixture was stirred for 3 h, then concentrated in vacuo. The residue was dissolved in water (600 mL). The resulting aqueous phase was extracted with ethyl acetate (3×200 mL) and the combined organic layers were rinsed with saturated brines (200 mL). The EtOAC phase was dried over Na2SO4 and concentrated in vacuo to obtain M2. M2 was dissolved in EtOH / HCl (95:5) and removed solution to give the hydrochlorate (M3), which was used for next step without further purification. To a suspension of anhydrous CuSO4 (4 equiv) and the above hydrochlorate (M3, 1 equiv) in anhydrous formic acid (75 mL) was added 40% glyoxal solution (2 equiv) at 100 °C. The reaction mixture was stirred at 100 °C for 4 h, concentrated hydrochloric acid (0.8, 0.8, 0.8, 0.6 and 0.6 equiv) was added respectively at 30 min, 50 min, 80 min, 150 min, and 210 min through this process. The mixture was filtered after the reaction completed. The filtrate was concentrated in vacuo. The residue (M4) was dissolved in MeOH (1400 mL) and water (90 mL), CaO was added portionwise to adjust pH to 9-10. The suspension was stirred at the room temperature for 2 h, filtered and concentrated in vacuo. The resulting residue (M5) was dissolved in EtOH / HCl (95:5) and purified via silica gel chromatography (CH2Cl2 / MeOH, 30:1-10:1) to give the product M6. A mixture of M6 and ethanol (100 mL) was refluxed for 20 min to dissolve, then NaBH4 (5.0 equiv) was added portionwise at the room temperature. Two hours later, the resulting solids 22

were filtered, washed with MeOH to crude which recrystallized from EtOAC, to provide 6. Yield: 25%. Light yellow solid; 1H NMR (500 MHz, DMSO-d6) δ: 2.43 (m, 2H, 5-H, 6-H), 2.59 (m, 1H, 13-H), 2.92 (m, 1H, 6-H), 3.03 (m, 1H, 5-H), 3.27 (m, 1H, 13-H), 3.39 (m, 1H, 14-H), 3.46 (d, 1H, J = 15 Hz, 8-H), 3.72 (s, 3H, -OCH3), 3.72 (s, 3H, -OCH3), 3.75 (s, 3H, -OCH3), 3.82 (d, 1H, J = 15Hz, 8-H), 6.56 (s, 1H, 12-ArH), 6.62 (s, 1H, 9-ArH), 6.67 (s, 1H, 1-ArH), 6.86 (s, 1H, 4-ArH), 8.69 (s, 1H, -OH); 13C NMR (125 MHz, DMSO-d6) δ: 109.49 (C1), 147.12 (C2), 147.15 (C3), 109.70 (C4), 126.31 (C4a), 28.51 (C5), 50.82 (C6), 57.63 (C8), 124.83 (C8a), 115.07 (C9), 144.68 (C10), 145.88 (C11), 111.71 (C12), 124.45 (C12a), 35.42 (C13), 59.20 (C14), 129.91 (C14a), 55.38 (-OCH3), 55.62 (-OCH3), 55.73 (-OCH3); ESI-MS: m/z 342.39 [M+H] +. 4.1.3 2, 3-Dimethoxy-5, 8, 13, 13a-tetrahydro-6H-isoquinolino[3, 2-a]isoquinolin-10-ol (7) Preparation and purification of compound 7 was carried out according to the procedure of compound 6. Yield: 21%. Light yellow solid; 1H NMR (500 MHz, DMSO-d6) δ: 2.41 (m, 1H, 6-H), 2.48 (m, 1H, 5-H), 2.60 (m, 1H, 13-H), 2.92 (m, 1H, 6-H), 3.04 (m, 1H, 5-H), 3.31 (m, 1H, 14-H), 3.40 (m, 1H, 13-H), 3.49 (d, 1H, J = 15Hz, 8-H), 3.72 (s, 3H, -OCH3), 3.74 (s, 3H, -OCH3), 3.86 (d, 1H, J = 15Hz, 8-H), 6.47 (s, 1H, 9-ArH), 6.56 (d, 1H, J = 8Hz, 12-ArH), 6.67 (s, 1H, 1-ArH), 6.86 (s, 1H, 4-ArH), 6.94 (d, 1H, J = 8Hz, 11-ArH), 9.10 (s, 1H, -OH); 13C NMR (75 MHz, DMSO-d6) δ: 106.36 (C1), 147.09 (C2), 147.13 (C3), 111.68 (C4), 126.29 (C4a), 28.48 (C5), 50.72 (C6), 57.93 (C8), 129.21 (C8a), 111.90 (C9), 155.50 (C10), 113.48 (C11), 135.38 (C12), 124.61 (C12a), 35.35 (C13), 59.37 (C14), 129.87 (C14a), 55.68 (-OCH3), 55.36 (-OCH3); ESI-MS: m/z 312.22 [M+H] +. 4.1.4 2, 3, 11, 12-Tetramethoxy-5, 8, 13, 13a-tetrahydro-6H-isoquinolino[3, 2-a]isoquinolin-10-ol (8) Preparation and purification of compound 8 was carried out according to the procedure of compound 6. Yield: 18%. 23

Light yellow solid; 1H NMR (300 MHz, DMSO-d6) δ: 2.39 (m, 2H, 13-H, 6-H), 2.60 (m, 1H, 5-H), 3.01 (m, 2H, 5-H, 6-H), 3.30 (m, 1H, 13-H), 3.37 (m, 1H, 14-H), 3.47 (d, 1H, J = 15Hz, 8-H), 3.71 (s, 3H, -OCH3), 3.73 (s, 3H, -OCH3), 3.74 (s, 3H, -OCH3), 3.75 (s, 3H, -OCH3), 3.82 (d, 1H, J = 15Hz, 8-H), 6.46 (s, 1H, 9-ArH), 6.68 (s, 1H, 4-ArH), 6.87 (s, 1H, 1-ArH), 8.31 (s, 1H, -OH); 13C NMR (75 MHz, DMSO-d6) δ: 109.67 (C1), 147.18 (C2), 147.25 (C3), 111.81 (C4), 126.52 (C4a), 28.57 (C5), 50.62 (C6), 58.91 (C8), 124.72 (C8a), 104.72 (C9), 147.05 (C10), 137.15 (C11), 145.44 (C12), 120.13 (C12a), 30.76 (C13), 59.26 (C14), 130.07 (C14a), 55.41 (-OCH3), 55.85 (-OCH3), 55.89 (-OCH3), 57.74 (-OCH3); ESI-MS: m/z 372.06 [M+H] +. 4.1.5 10-Methoxy-5, 8, 13, 13a-tetrahydro-6H-[1, 3]dioxolo[4, 5-g]isoquinolino[3, 2-a]isoquinolin-11-ol (9) Preparation and purification of compound 9 was carried out according to the procedure of compound 6. Yield: 15%. White solid; 1H NMR (500 MHz, DMSO-d6) δ: 2.44 (m, 2H, 5-H, 6-H), 2.59 (m, 1H, 13-H), 2.88 (m, 1H, 6-H), 3.01 (m, 1H, 5-H), 3.20 (m, 1H, 13-H), 3.38 (m, 1H, 14-H), 3.46 (d, 1H, J = 15Hz, 8-H), 3.72 (s, 3H, -OCH3), 3.82 (d, 1H, J=15Hz, 8-H), 5.94 (d, 2H, J = 4.5Hz, -OCH2O-), 6.54 (s, 1H, 12-ArH), 6.62 (s, 1H, 9-ArH), 6.65 (s, 1H, 1-ArH), 6.90 (s, 1H, 4-ArH), 8.67 (s, 1H, -OH);

13

C NMR (125 MHz, DMSO-d6) δ:

105.65 (C1), 145.64 (C2), 145.90 (C3), 107.96 (C4), 127.38 (C4a), 28.93 (C5), 50.64 (C6), 57.50 (C8), 126.30 (C8a), 109.65 (C9), 144.69 (C10), 145.32 (C11), 115.10 (C12), 124.71 (C12a), 35.51 (C13), 59.45 (C14), 131.07 (C14a), 100.44 (-OCH2O-), 55.62 (-OCH3); ESI-MS: m/z 326.32 [M+H] +. 4.1.6 5,8,13,13a-Tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-12-ol (10) and 5,8,13, 13a-tetrahydro-6H-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-10-ol (11) Preparation and purification of compound 10 and 11 which are isomers and co-products of the cyclization reaction were carried out according to the procedure of compound 6. Yield: 12% (10), 26% (11). 24

10: Light yellow solid; 1H NMR (500 MHz, DMSO-d6) δ: 2.26 (m, 1H, 6-H), 2.41 (m, 1H, 5-H), 2.88 (m, 1H, 6-H), 2.99 (m, 1H, 13-H), 3.04 (m, 1H, 5-H), 3.31 (m, 1H, 13-H), 3.39 (m, 1H, 14-H), 3.51 (d, 1H, J = 15Hz, 8-H), 3.86 (d, 1H, J = 15Hz, 8-H), 5.95 (s, 2H, -OCH2O-), 6.53 (d, 1H, J = 7.5Hz, 11-ArH), 6.62 (d, 1H, J = 7.5Hz, 9-ArH), 6.57 (s, 1H, 1-ArH), 6.89 (s, 1H, 4-ArH), 6.94 (t, 1H, J = 7.5Hz, 10-ArH); 13C NMR (125 MHz, DMSO-d6) δ: 106.08 (C1), 145.88 (C2), 146.19 (C3), 108.53 (C4), 127.91 (C4a), 29.45 (C5), 51.09 (C6), 58.39 (C8), 126.54 (C8a), 116.83 (C9), 136.29 (C10), 112.45 (C11), 155.21 (C12), 121.76 (C12a), 31.75 (C13), 59.76 (C14), 131.82 (C14a), 100.99 (-OCH2O-); ESI-MS: m/z 296.02[M+H] +. 11: Light yellow solid; 1H NMR (500 MHz, DMSO-d6) δ: 2.47 (m, 2H, 6-H, 5-H), 2.59 (d, 1H, J = 16Hz, 13-H), 2.91 (m, 1H, 6-H), 3.04 (br, 1H, 5-H), 3.27 (d, 1H, J = 16Hz, 13-H), 3.41 (br, 1H, 14-H), 3.49 (d, 1H, J = 14Hz, 8-H), 3.85 (d, 1H, J = 14Hz, 8-H), 5.94 (d, 2H, J = 2.5Hz, -OCH2O-), 6.47 (s, 1H, 1-ArH) 6.56 (d, 1H, J = 8Hz, 11-ArH), 6.66 (s, 1H, 4-ArH), 6.90 (s, 1H, 9-ArH), 6.92 (d, 1H, J =8Hz, 12-ArH), 9.09 (s, 1H, -OH);

13

C NMR (125 MHz, DMSO-d6) δ: 105.59 (C1), 145.36 (C2), 145.71 (C3), 107.97 (C4), 129.27

(C4a), 28.88 (C5), 50.51 (C6), 57.76 (C8), 127.36 (C8a), 111.88 (C9), 155.09 (C10), 113.53 (C11), 135.28 (C12), 124.50 (C12a), 35.36 (C13), 59.61 (C14), 131.08 (C14a), 100.45 (-OCH2O-); ESI-MS: m/z 296.18 [M+H] +. 4.1.7 10, 12-Dimethoxy-5, 8, 13, 13a-tetrahydro-6H-[1, 3]dioxolo[4, 5-g]isoquinolino[3, 2-a]isoquinolin11-ol (12) Preparation and purification of compound 12 was carried out according to the procedure of compound 6. Yield: 28%. Light yellow solid; 1H NMR (300 MHz, DMSO-d6) δ: 2.33 (m, 2H, 5-H, 6-H), 2.60 (m, 1H, 13-H), 2.89 (m, 1H, 6-H), 3.02 (m, 1H, 5-H), 3.29 (m, 2H, 13-H, 14-H), 3.47 (d, 1H, J = 15Hz, 8-H), 3.70 (s, 3H, -OCH3), 3.74 (s, 3H, -OCH3), 3.81 (d, 1H, J = 15Hz, 8-H), 5.94 (m, 2H, -OCH2O-), 6.46 (s, 1H, 9-ArH-), 6.66 (s, 1H, 1-ArH-), 6.93 (s, 1H, 4-ArH), 8.34 (s, 1H, -OH); 25

13

C NMR (75 MHz, DMSO-d6) δ: 105.74

(C1), 145.41 (C2), 145.41 (C3), 108.03 (C4), 124.53 (C4a), 28.92 (C5), 50.49 (C6), 59.20 (C8), 127.42 (C8a), 104.70 (C9), 145.70 (C10), 137.22 (C11), 147.10 (C12), 120.02 (C12a), 30.92 (C13), 59.26 (C14), 131.14 (C14a), 100.53 (-OCH2O-), 55.86 (-OCH3), 57.58 (-OCH3); ESI-MS: m/z 356.14 [M+H] +. 4.1.8 12-(Benzyloxy)-5, 8, 13, 13a-tetrahydro-6H-[1, 3]dioxolo[4, 5-g]isoquinolino[3, 2-a]isoquinoline (13) Compound 10 (0.2 g, 0.68 mmol) was diluted with dry dimethylformamide (DMF, 15 mL), and benzyl bromide (2 equiv) was added slowly, followed by anhydrous K2CO3 (2 equiv). The mixture was stirred at 80 °C for 4h. The mixture was partitioned between water and EtOAC. The organic layers were washed with water and saturated aqueous NaCl. The extract were dried over anhydrous Na2SO4 and concentrated to obtain the crude product. The crude was purified by silica gel chromatography (CH2Cl2 / MeOH, 200:1- 60:1) to obtain the product 13. Yield: 75%. Light yellow solid; 1H NMR (300 MHz, DMSO-d6) δ: 2.39 (m, 2H, 5-H, 6-H), 2.61 (m, 1H, 13-H), 2.90 (m, 1H, 5-H), 3.06 (m, 1H, 6-H), 3.42 (m, 2H, 13-H, 14-H), 3.56 (d, 1H, J = 15Hz, 8-H), 3.91 (d, 1H, J = 15Hz, 8-H), 5.14 (s, 2H, -CH2-Ar), 5.94 (d, 2H, J = 6.9Hz, -OCH2O-), 6.67 (s, 1H, 1-ArH), 6.7 (d, 1H, J = 8Hz, 9-Ar-H), 6.85 (d, 1H, J = 8Hz, 11-Ar-H), 6.90 (s, 1H, 4-ArH), 7.09 (t, 1H, J = 8Hz, 10-Ar-H), 7.36 (m, 3H, 3’ ,4’, 5’-ArH), 7.47 (d, 2H, J = 7Hz, 2’, 6’-Ar-H);

13

C NMR (75 MHz, DMSO-d6) δ: 105.56 (C1),

145.38 (C2), 145.63 (C3), 108.04 (C4), 127.59 (C4a), 28.92 (C5), 50.33 (C6), 57.63 (C8), 135.87 (C8a), 118.13 (C9), 126.21 (C10), 109.14 (C11), 155.58 (C12), 123.12 (C12a), 31.18 (C13), 59.00 (C14), 131.12 (C14a), 137.38 (C1’), 127.20 (C2’, C6’), 128.35 (C3’, C5’), 127.41 (C4’); ESI-MS: m/z 386.30 [M+H] +. 4.1.9 2, 3, 10, 11-Tetramethoxy-5, 8, 13, 13a-tetrahydro-6H-isoquinolino[3, 2-a]isoquinoline (E) Preparation and purification of compound E was carried out according to the procedure of compound 6. Yield: 35%. Light yellow solid; ESI-MS: m/z 356.25 [M+H]+; 1H NMR (500 MHz, DMSO-d6) and MHz, DMSO-d6) see Table 1. 26

13

C NMR (125

4.1.10 2, 3, 10, 11, 12-Pentamethoxy-5, 8, 13, 13a-tetrahydro-6H-isoquinolino[3, 2-a]isoquinoline (F) Preparation and purification of compound F was carried out according to the procedure of compound 6. Yield: 33%. Light yellow solid; ESI-MS: (m/z) 386.37 [M+H]+; 1HNMR (500 MHz, DMSO-d6) and

13

CNMR (125

MHz, DMSO-d6) see Table 2.

4.2 Analytical and preparative scale biotransformation, isolation and identification of products 29 Cultures were grown by a two-stage procedure in 50 mL of soybean meal glucose medium held in 250 mL culture flasks. The soybean meal glucose medium contained (in g/L) 20 glucose, 5 yeast extract, 5 soybean meal, 5 NaCl, and 5 K2HPO4 in distilled water and was adjusted to pH 7.0 with 6 N HCl before being autoclaved at 121 °C for 15 min. Cultures were incubated with shaking at 180 rpm at 28 °C. A 10% inoculum derived from 48-h-old stage I cultures was used to initiate stage II cultures, which were incubated for 24 h before receiving 10 mg of substrates in 1 mL of ethanol and incubations were conducted as before. Substrate controls consisted of sterile medium and substrates incubated under the same conditions but without microorganism. Cultures were incubated for 5 days and extracted with equal volume of EtOAc. The organic phase was concentrated and spotted on silica gel TLC plate, which was developed by CHCl 2 / MeOH (30:1, v/v). The results were visualized by spraying developed plated with KBiI4 reagent. Using 24-h-old stage II cultures, substrate (300 mg) was distributed evenly among thirty 150 mL culture flasks. Substrate-containing cultures were incubated for 5 days and then extracted with equal EtOAc for three times. The organic solvent layer was evaporated to dryness. The crude extracts of substrates E and F were subjected to silica gel column chromatography eluted with solvent system of CH2Cl2 / MeOH (90:1 to 20:1) to afford the products 16 and 17, respectively, and the structures were identified based on mass spectrometry, nuclear magnetic resonance and 2D-NMR spectra data. 27

4.2.1 3, 10, 11–Trimethoxy-5, 8, 13, 13a–tetrahydro-6H–isoquinolino[3, 2-a]isoquinolin-2-ol (16) White solid; Yield: 19%; HR-ESI-MS: (m/z) 342.1761 [M+H]+; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) see Table 1. 4.2.2 2, 3, 10, 12-Tetramethoxy-5, 8, 13, 13a-tetrahydro-6H-isoquinolino[3, 2-a]isoquinolin-11-ol (17) Light yellow solid; Yield: 28%; HR-ESI-MS: (m/z) 372.2018 [M+H] +; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) see Table 2.

4.3 Functional activity assay on D1 and D2 receptors GenScript Corp (Piscataway, NJ, USA) performed the functional activity assays of all compounds against D1 and D2 receptors. The cAMP assay with a CISBIO Kit and calcium flux assay with FLIPR ® calcium-4 Kit of Molecular Device were used to get agonistic and antagonistic activities on D1 and D2 receptors, respectively. New compounds and the control articles were dissolved in DMSO to get the stock solutions, which were stored at -20 °C. HBSS buffer (with 20 mM HEPES buffer, pH 7.4) was used to dilute the stock solutions and get 5× working solution before use. The final concentrations of the tested compounds were 16 nM, 80 nM, 400 nM, 2 μM and 10 μM for the antagonistic assay. And the final concentration was 10 µM for the agonistic effects at the D1 and D2 receptors. For CHO-K1 cells expressing D1 and D2 receptors, they were cultured in the 15-cm dishes and maintained at 37 °C/ 5% CO2. When the cell confluency reached 90%, the cells were collected and inoculated into 384 microplates for experiments. For CHO-K1/D1 cells, they were cultured with Ham's F12 containing 10% fetal bovine serum and 200 μg/ml Zeocin. And CHO-K1/Gα15/D2 cells were cultured with Ham's F12 containing 10% fetal bovine serum, 200μg/ml Zeocin and 100μg/ml Hygromycin B. 4.3.1 cAMP assays on D1 receptor We followed the detailed protocols in the manual of the CISBIO Kit and conducted cAMP assays on D1 receptor. Briefly, we seeded 3000 cells/5 μL (CHO-K1 cells expressing D1 receptor) in 384-well plates 28

and then added 5 μL 2× compound solution before 0.5-hour incubation at RT. For agonistic assays, we only put test articles. While for inhibitory assays, we added a mixture of 4× test articles and 4× EC80 positive agonist volume. Then, we added 10 μL detection reagents and incubated it for another hour before we tested the signal of the plate using PheraStar and recorded the data of the 665 nM and 620 nM in each well in Excel. Then, we plotted the ratio of the two readouts as a function of the log of the cumulative doses of compounds. Finally, we used data analysis wizard written by GenScript to analyze the EC50 and IC50 and got dose response curves of agonist with four-parameter-logistic-equation by the software GraphPad Prism 6. The percentage of compound effect was calculated as: (1) (2) 4.3.2 Calcium flux assay on D2 receptor We seeded CHO-K1 cells expressing D2 receptor in a 384-well black-wall, clear-bottom plate at a density of 15,000 cell per well in 20 μl of growth medium and cultured them for 18 hours prior to the day of experiment and maintained at 37 °C/ 5% CO2. Agonistic activity assay: we first added 20 μL of dye-loading solution into the well, and placed the plate into a 37 °C incubator for 60 minutes, followed by a 15-minute incubation at room temperature. Then, we added 10 μL compounds or control agonist into respective wells of the assay plate during reading in FLIPR, and placed the plate containing 5× compound or control agonist solution in FLIPR. After that, we added solutions into the cell plate automatically at the 20 seconds and monitored the fluorescence signal for an additional period of 100 seconds (21 s to 120 s). Antagonistic activity assay: we added 20 μL of dye-loading solution and 10 μL 5× compound solution into the well and placed the plate into a 37 °C incubator for 60 minutes, followed by a 15-minute incubation at room temperature. Subsequently, we added 12.5 μL 5× EC80 control agonist into respective wells of the 29

assay plate during reading in FLIPR, and placed the plate containing 5× EC80 control agonist solution in FLIPR. After that, we added solutions into the cell plate automatically at the 20 seconds and monitored the fluorescence signal for an additional period of 100 seconds (21 sec to 120 sec.) We recorded the data using ScreenWorks (version 3.1) as FMD files with FLIPR and stored on the GenScript computer network for off-line analysis. We conducted data acquisition and analyses using ScreenWorks (version 3.1) program and exported to excel, and then calculated the average value of the first 20-second (1s to 20s) readings as the baseline and also calculated the relative fluorescent units (ΔRFU) intensity values by subtracting the average value of baseline from the maximal fluorescent units (21s to 120s) . The following are the equations used to calculate the percentage of compound effects: (3) (4)

4.4 Computational methods for molecular docking simulations We downloaded the full amino acid sequences of D1 receptor (UniProt Code: P21728) from the web site of UniProtKB (http://www.uniprot.org/uniprot/),36 and performed sequence similarity searches for D1 receptor using the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/).37 We selected the inactive-state structure of D3 receptor (PDB code: 3PBL) as the template to construct the antagonistic conformations of D1 receptor and optimized the homology modeling and loop refinement with Modeler 9.17.38,

39

We

truncated these residues before Asp 19 for the N terminal and after Cys 347 for the C terminal in the sequence of D1 receptor. Since the third extracellular loop (ECL3, between TM4 and TM5) and the third intracellular loop (ICL3, between TM5 and TM6) had long-flexible sequences, we also truncated 19 residues from Pro 168 (ECL3) to Cys 186 (ECL3) and 35 residues from Gln 222 (ICL3) to Pro 256 (ICL3). So, the sequence of D1 receptor was from ASP19 to Lys 167 (ECL3) and from Asp 187 (ECL3) to Ala 221 (ICL3) 30

and from Glu 257 (ICL3) to Cys 347. We also considered and constructed the disulfide bridge/bond between Cys 298 and Cys 307 in TM6 and TM7 of D1 receptor. Based on this truncated sequence of D1 receptor, we performed the sequence alignments and homology modeling. After we got the D1 receptor model, we evaluated with Discrete Optimized Protein Energy (DOPE) 40 measurement and Ramachandran plot,41, 42 and further validated with enrichment test. The details refer to the published computational data.34 We downloaded the X-ray crystal structure of D2 receptor (PDB ID: 6cm4) from the PDB database (http://www.rcsb.org/pdb) and used it to conduct flexible dockings of all compounds with D1/D2 receptors using flexible docking protocol in discovery studio 3.0. We first defined the active site of D1 receptor based on the key residues Asp 103, Thr 108, Trp 285 and set the radius of the sphere as 12.0 Å. Also, we selected residues Lys 81, Asp 103, Thr 108, Asp 187, Ser 188, Ser 197, Ile 201, Trp 285, Phe 313 for flexible processing. For the active site of D2 receptor, we defined it using the ligand 8NU from PDB and set the radius of the sphere as 11.4 Å. We also selected residues Trp 100, Asp 114, Thr 119, Phe 198, Phe 382, Trp 386, Phe 389, Thr 412, Tyr 416 for flexible processing. For other parameters, we kept them as default settings in flexible docking.

Supplementary Material Supplementary material available as pdf Acknowledgment This work was supported by the National Natural Science Foundation of China (NSFC NO. 21302052).

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Highlights 

THPBs derivatives were prepared by chemical synthesis or microbial transformation.

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The functional activity of compounds on D1/D2 receptors were tested.

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5 compounds displayed selective antagonistic activity on D2 receptor.

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Graphic abstract

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