Discovery of 2-substituted-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide as potent and selective protein arginine methyltransferases 5 inhibitors: Design, synthesis and biological evaluation

Jingwei Shao, Kongkai Zhu, Daohai Du, Yuanyuan Zhang, Hongrui Tao, Zhifeng Chen, Hualiang Jiang, Kaixian Chen, Cheng Luo, Wenhu Duan

PII: S0223-5234(18)31112-7
DOI: https://doi.org/10.1016/j.ejmech.2018.12.065 Reference: EJMECH 10999

To appear in: European Journal of Medicinal Chemistry

Received Date: 17 September 2018
Revised Date: 22 December 2018
Accepted Date: 25 December 2018

Please cite this article as: J. Shao, K. Zhu, D. Du, Y. Zhang, H. Tao, Z. Chen, H. Jiang, K. Chen, C. Luo,
W. Duan, Discovery of 2-substituted-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4- tetrahydroisoquinoline-6-carboxamide as potent and selective protein arginine methyltransferases 5 inhibitors: Design, synthesis and biological evaluation, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.065.

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Discovery of
2-Substituted-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydr oxypropyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide as Potent and Selective Protein Arginine Methyltransferases 5 Inhibitors: Design, Synthesis and Biological Evaluation

Jingwei Shao$,|, Kongkai Zhu#,|, Daohai Du†,|, Yuanyuan Zhang†, Hongrui Tao#,†, Zhifeng Chen†, Hualiang Jiang†, Kaixian Chen†,±, Cheng Luo†,±,* and Wenhu Duan$,*
§Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China;
#School of Biological Science and Technology, University of Jinan, Jinan 250022,

P.R. China;

†Drug Discovery and Design Center, CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China;
±Open Studio for Drugability Research of Marine Natural Products, Qingdao National Laboratory for Marine Science and Technology, Wenhai Road, Aoshanwei, Jimo, Qingdao, Shangdong 266237, China

| These authors contributed equally.

*Correspondence: Wenhu Duan, Email: [email protected] or Cheng Luo, E-mail: [email protected]

Abstract

Protein arginine methyltransferases 5 (PRMT5) represents an attractive drug target in epigenetic field for the treatment of leukemia and lymphoma. Here, a series of N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)amide derivatives targeting PRMT5 were designed with structure-based approach and synthesized. Among them, compound 46 showed potent and selective PRMT5 inhibition activity with an IC50 of
8.5 nM, which was approximately equivalent with the phase I clinical trial PRMT5 inhibitor GSK-3326595 (IC50 = 5.5 nM). Compound 46 also displayed pronounced anti-proliferative activity in MV4-11 cells (GI50 = 18 nM) and antitumor activity in MV4-11 mouse xenografts model. This molecule can serve as an excellent tool compound for probing the biological function of PRMT5.

Keywords: PRMT5 inhibitors; N-Substituted 1,2,3,4-tetrahydroisoquinoline derivatives; Anti-tumor

1. Introduction

Protein arginine methyltransferases (PRMTs) are S-adenosylmethionine (SAM)-dependent methyltransferase enzymes and are capable of transferring methyl groups from SAM to guanidine nitrogen of arginine residue on histone and non-histone substrate proteins [1, 2] Based on their product specificity, nine mammalian PRMTs can be classified into three groups termed type I (PRMT1, 2, 3, 4, 6, 8), type II (PRMT5, 9), and type III (PRMT7) [3], of which PRMT5 is currently the prominent

type II PRMT and catalyzes the mono- and symmetric dimethylation of arginine residues [4]. PRMT5 has several important binding partners [5-7] including RIOK1, MEP50 and pICIn, which can stimulate PRMT5 activity. Substrates [8] for PRMT5 include the histones H4 residue Arg3 (H4R3), H3 residue Arg8 (H3R8), H2A residue Arg3 (H2AR3) [9,10] and non-histone protein such as Sm proteins [11], p53 [12], p65 [13], HOXA9, E2F-1, SPT5, MBD2, PDCD4 [14], EGFR [15], CRAF [16], FGF-2
[17]and SREBP1a. PRMT5 regulates the transcription of target genes and posttranslational modifications of signaling proteins, therefore plays a significant role in diverse cellular processes such as cell cycle progression, apoptosis [18], and DNA-damage response. Overexpression or dysregulation of PRTM5 is found in a variety of cancers [19] and was suggested to have a close relationship with tumorigenesis and development [8,20]. In addition, knockdown of PRMT5 in cancer cells is associated with cell-cycle arrest, p53-independent apoptosis, and reduced migration capability. All of these indicate that PRMT5 is a promising target for cancer therapy.
In the past few years, a few small-molecule PRMT5 inhibitors [21-31] have been reported. Of these inhibitors, only two small-molecule inhibitors showed nanomolar IC50 values (Figure 1) at enzymatic level assay. Compound 1 (EPZ015666) [29] was a first-in-class, highly potent and selective PRMT5 inhibitor and it displayed effective anti-tumor activity in MCL xenograft animal models. The crystal structure of compound 1 complexed with human PRMT5 has been reported and 1 occupied the substrate binding pocket. An analog of compound 1 has progressed into phase I

clinical trials for treatment of solid tumors and non-Hodgkin’s lymphoma. Recently, Janssen reported the novel PRTM5 inhibitor 2 [32] which was predicted to occupy the SAM-binding pocket and exhibited excellent activity with an IC50 value of 0.14 nM. Compound 2 also showed good tumor growth inhibition in a small-cell lung cancer
xenograft model.

Figure 1. Structures of representative PRMT5 inhibitors.

Considering the importance of PRMT5 in tumorigenesis and the limited availability of high-quality tool compounds at the beginning of our program, we embarked on a campaign to search for potent PRMT5 inhibitors. In this study, we reported the structure-based design, synthesis and biological evaluation of a class of potent PRMT5 inhibitors with high selectivity over other PRMTs.
2. Result and Discussion

2.1. Structure-based design of novel PRMT5 inhibitors

An X-ray crystal structure analysis of human PRMT5 complexed with a highly selective inhibitor 1 (EPZ015666), showed that 1 bond to the substrate binding site with hydrogen bond and hydrophobic interactions (Figure 2A-2B). To obtain novel and potent PRMT5 inhibitors, We tried to replace the substituted pyrimidine ring with the 4,5,6,7-tetrahydrothieno[3,2-c]pyridine core (Figure 3, compound 3) by utilizing the scaffold hopping strategy. Unfortunately, it showed weak activity against PRMT5 tested by the radioactive methylation assay method that we previously reported

[26].Further analysis indicated that this site was a hydrophobic site and there was untapped chemical space could be utilized. Aiming to occupy the identified chemical space meanwhile to retain the mainly interactions, compound 4 substituted with benzyl group was synthesized. To our delight, 4 (IC50 = 240 nM) showed a two-fold increased inhibitory potency compared to 3 (IC50 = 475 nM). This result suggested the feasibility of our structure-based design strategy and prompted us to begin a structural
modification effort toward the identification of more potent PRMT5 inhibitors.

Figure 2. Crystal structure of compound 1 with the PRMT5-MEP50 protein (PDB code: 4X61). (A) Substrate binding site shown as surface with 1 and SAM represented by cyan and yellow sticks. (B) The interacting residues were displayed by lightblue sticks.
O
NH
scaffold hopping
N H H
N N N
O O 3
1 (EPZ015666)

add aromatic substituent

N

H
S N
O
4

Figure 3. Design of novel PRMT5 inhibitors.

2.2. Optimization of compound 4

The optimization of 4 was systematically performed according to three main parts of the molecule, namely the methylene group linker, the benzene ring and the 4,5,6,7-tetrahydrothieno[3,2-c]pyridine core. Initially we have focused our attention on the enzymatic potency of synthesized compounds.
Table 1. Activity of compounds 3-32.

PRMT5 MV4-11 cell (GI50, µM)
Compound R
(IC50, nM) Day 8 Day 12
3 475 NTa NTa
4 240 NTa NTa

5

260
NTa
NTa

6 60 0.436 0.172

7 39 0.184 0.154

8

91

0.375

0.209

9 50 0.256 0.147

22 23 0.147 0.162

23 18 0.181 0.066

24 19 0.193 0.089

25 49 0.204 0.134

26 19 0.415 0.186

27 19 0.201 0.141

28 25 0.258 0.135

29 23 0.197 0.143

30 44 NTa NTa

31 31.5 0.314 0.166

32 45 0.261 0.124

1
–
47
0.212
0.106
a Not tested

First, we tried to optimize the methylene group linker region. As shown in Table

1. Removal of the methylene group resulted in compound 5, which retained enzymatic potency despite its rigid structure. Introduction of an α-methyl into 4 led to a 4-fold enhancement of enzymatic potency (compound 6). Replacing the methylene group with formyl led to compound 7, which exhibited potent PRMT5 inhibitory activity with IC50 = 39 nM. It was supposed that carbonyl oxygen atom might be a hydrogen bond acceptor. Compound 8 with bulky sulfonyl was less potent than 7. Interestingly, lengthening the linker (compound 9-11) was well tolerated.
Next we explored the SAR of benzene ring region. Replacement of the benzene

ring by straight-chain group (12) dramatically deteriorated the enzymatic potency. Cyclohexane analog (13) exhibited modest potency and more hydrophilic tetrahydropyran analog (14) displayed a reduced PRMT5 activity. Derivatives (15-18) with other aromatic rings were as potent as compound 7. While exploring alternative substitutions on the benzene ring, we found that these modifications have equivalent effects on potency. The derivative substituted at position 4 of the benzene ring (compound 21) exerted slightly higher potency in comparison with compounds substituted at 2 or 3, compound 32 and 31, respectively.
Table 2. Activity of compounds 33-41.

PRMT5 MV4-11 cell (GI50, µM)

Compound R A

(IC50,

nM)

Day 8 Day 12

33 Me

1900 NTa NTa

34 Me

1900 NTa NTa

35 Me

1200 NTa NTa

36 23 0.169 0.101

37 40 NTa NTa

O

38 17 0.128 0.092
N

O

39 17 0.070 0.036
N

40 23 0.035 0.032

41 11 0.045 0.034

1 – – 47 0.212 0.106

a Not tested

Then we assessed the importance of the 4,5,6,7-tetrahydrothieno[3,2-c]pyridine core (Table 2). Replacement of the thiophene ring with furan (33), pyrrole (34), and thiazole (35) greatly reduced the activity. In contrast, benzene ring bioisosteric compound 39 exerted extremely high activity against PRMT5 with IC50 of 17 nM. Movement of the nitrogen to produce 37 was less potent (IC50 = 40 nM). Ring-contraction derivative 38 retained a good level of enzyme inhibition.
So far we have obtained a series of novel compounds which showed excellent activity in the enzymatic assay. Then the MV4-11 cell line was used to assess the proliferation effects upon treatment with PRMT5 inhibitors, allowing for the

measurement of cell growth over 12 days. The IC50 at day 8 and day 12 was shown in table 1 and 2. Most of these compounds displayed modest cellular activities with IC50 values ranging from 0.1 to 0.2 µM. Only of five compounds showed potent antiproliferative activities with IC50 values below 0.1 µM. Based on the results, we combined the optimal structures of three parts and synthesized compound 40 and 41. Unsurprisingly both of them exerted potent enzymatic and cellular activity.
All the compounds mentioned above were racemates and the enantiomers of 39-41 were prepared to evaluate the relative PRMT5 activity. As illustrated in Table 3, the S enantiomers were significantly more potent; the result was consistent with the finding reported by Duncan [28]. Thus, we obtained the most potent compound 46 which demonstrated 6-fold increases compared with compound 1 in enzymatic and cellular potencies.
Table 3. Activity of compounds 39, 42-46.

Compound R

Configur-a tion

PRMT5 (IC50, nM)

MV4-11 cell (GI50, µM)

Day 8 Day 12

O

39 N racemic 17 0.070 0.036

O

42 N S 11 0.075 0.053

43 R 33 0.333 0.213

44 S 13 0.037 0.023

45 R 59 0.213 0.187
46 S 8.5 0.025 0.018
1 – – 47 0.212 0.106

2.3. Methyltransferase enzymatic selectivity of 46

Compound 46 with good potencies in both enzyme and leukemia cell was selected as represent compound to evaluate its selectivity over other methyltransferases enzymes. Not unexpectedly compound 46 showed no activity against these tested methyltransferases enzymes at a 100 µM concentration, indicating that 46 was a selective PRMT5 inhibitor.
Table 4. Selectivity Profile of 46

Protein IC50 (µΜ) Protein IC50 (µΜ)
PRMT1 > 100 PRMT6 > 100
PRMT3 > 100 PRMT7 > 100
PRMT4 > 100 PRMT8 > 100

2.4. Pharmacokinetic Study

With the potent and selective PRMT5 inhibitors in hand, we tried to evaluate the

pharmacokinetic properties of compound 42, 44 and 46 at a dose of 10 mg/kg（i.g., intragastric）in rats. As shown in Table 5, among these three compounds, 46 showed the longest half-time and highest oral exposure. However it had a poor aqueous
solubility. We improved the solubility by forming the corresponding hydrochloride

salt.

Table 5. Pharmacokinetic Profile of 42, 44 and 46 in Ratsa

Cmax AUC
T T
Comp
ound (ng/m max

(h) 1/2

(h) (ng·h/m
L) L)
42 14.3 0.333 0.945 15.8
44 102.0 0.417 2.14 94.6
46 32.0 0.583 4.60 168
aCmax, maximum concentration; Tmax, time of maximum concentration; T1/2,

elimination half-life; AUC, area under the plasma concentration time curve. Data are the mean values.

2.5. Characterization of Cell Methylation

To further evaluate the cellular activity of 46 targeting PRMT5, we analyzed its effects on methylation of the previously reported PRMT5 substrate SmD3 in MV4-11 cell. SmD3, a protein that could be used to track the cell biochemical activity of PRMT5 [29,33-35]. As shown in figure 4, compound 46 significantly inhibited the methylation of SmD3 in a dose dependent manner, suggesting that 46 strongly suppressed MV4-11 cell proliferation via inhibiting PRMT5 activity.

Figure 4. Effects of 46 on cellular target inhibition as determined by sMDA Western blot.
2.6. In Vivo Antitumor Activity of Compound 46

To assess the in vivo antitumor efficacy of 46, MV4-11 xenograft mouse model was used. Compound 46 was administrated orally as hydrochloride salt at doses of 100 mg/kg once daily for 30 days. The results showed that, compared with the vehicle group, 46 could suppress tumor growth with the tumor growth inhibition rate (TGI) of 54.0%.

Figure 5. The inhibitory effect of 46 on tumor growth in MV4-11 xenograft model. Compound 46 was administered orally once daily for 30 days. Results are expressed as the mean ± SEM (n = 7 for inhibitor-treated group, n = 7 for vehicle control group). The percentage of tumor growth inhibition values (TGI) was measured on the final day of the study for the drug-treated mice compared with the vehicle control mice: (∗) P< 0.05, (∗∗) P< 0.01, (∗∗∗) P< 0.001, versus vehicle group, using Student’s t test.
2.7. Predicted binding mode with PRMT5 of 46

Although we tried to obtain the crystal structure of PRMT5 complexed with compound 46 to understand the binding mode of 46, we failed. Therefore, molecular docking was used to probe the binding mode of compound 46 with PRMT5. As shown in Figure 6, 46 showed similar binding mode with EPZ015666. The entire scaffold located in the hydrophobic pocket composed of residues F300, A301, Y304, L312, L319, Y324, V326, F327, Y334, L437, V503, F577, W579, and F580. The Quinoline
part in both 46 and EPZ015666 was well overlapped, and the hydroxyl in 46 formed a hydrogen bond with residue E444. All of the three benzene rings in 46 showed π-π

interactions with PRMT5. Hydrophobic, hydrogen bond, cation-π, and π-π interactions were observed and contributed to the binding affinity by further analysis (Figure 7).

Figure 6. The binding mode of 46 simulated by molecular docking. (A) Binding mode analysis of 46 with PRMT5. PRMT5 is shown in cartoon diagram in white, while 46 and EPZ015666 are represented as cyan and magenta sticks, respectively. (B) A close-up view of the binding mode of 46. The interacting residues are shown as green sticks.

Figure 7. The detailed interactions of 46 with PRMT5 obtained by molecular docking.

2.8. Chemistry

As outlined in scheme 1, N-(tert-Butoxycarbonyl)-4-piperidone was selected as the starting material and was subjected to Vilsmeier-Haack formylation reaction to produce tert-butyl 4-chloro-3-formyl-5,6-dihydropyridine-1(2H)-carboxylate (48). Compound 48 was reacted with ethyl mercaptoacetate or ethyl glycolate in the presence of base to yield the intermediate 49a or 49b, followed by hydrolysis to obtain intermediates 50a or 50b. Carboxylic acid 50c was synthesized according to the reported literature [36]. Condensation of 50a-c with 51 [29] followed by deprotection led to the key intermediates 53a-c, which were converted to the final compound 3, 4, 6-9, 11-34 by reductive amination with corresponding aldehydes or ketones, nucleophilic substitution with halohydrocarbons or condensation reaction with a series of acids.
Compounds 37 and 39-46 were prepared by analogous methods from intermediate 61 or 57. Compound 61 was commercial available; while 57 was synthetized from 6-bromoisoquinoline. The bromine atom of 6-bromoisoquinoline was replaced by cyano group using Zn(CN)2 in the presence of Pd(PPh3)4 to form the intermediate 54, followed by hydrolysis and esterify to produce compound 55. The reduction of 55 by catalytic hydrogenation of PtO2 yielded the key intermediate 56, which was treated with (Boc)2O to achieve compound 57.
Scheme 1. Synthesis of Compounds 3, 4, 6-9 and 11-34a

aReagents and conditions: (a) POCl3, DMF, DCM, 0 °C to rt; (b) methyl 2-mercaptoacetate, Et3N, DCM, rt; or NaH, methyl 2-hydroxyacetate, THF, 0 °C to reflux; (c) NaOH, EtOH or MeOH, reflux; (d) 51, EDCI, HOBt, Et3N, DCM, rt; (e) TFA or HCl in dioxane, DCM, rt; (f) aldehyde, NaBH3CN, MeOH; or ketone, NaBH3CN, AcOH, MeOH; or acid, EDCI, HOBt, Et3N, DCM, rt; (g) Zn(CN)2,
Pd(PPh3)4, DMF, 90 °C; (h) i) NaOH, EtOH, reflux; ii) con.H2SO4, MeOH, reflux; (i) PtO2, AcOH, MeOH; (j) (Boc)2O, Et3N, DCM, rt.
Compound 5, 10, 35, 36 and 38 were prepared following the route illustrated in

scheme 2. Deprotection of compound 49b provided compound 54, which was converted to 55a upon reaction with phenyl carbonochloridate or 55b through Buchwald-Hartwig cross-coupling reaction. Hydrolysis of esters 55a and 55b under basic conditions provided the corresponding acids 56a and 56b. Acids 56 were finally reacted with 51 to give target compounds. Compound 67, 71 and 74 were synthesized according to the reported literature [36-38].
Scheme 2. Synthesis of Compound 5 and 10a

aReagents and conditions: (a) TFA, DCM, rt; (b) iodobenzene, Cs2CO3, X-PHOS, Pd2(dba)3; or phenyl carbonochloridate, Et3N, DCM, rt; (c) NaOH, EtOH or MeOH, reflux; (d) PtO2, AcOH, EtOH; (e) 4-(dimethylamino)benzoic acid, EDCI, HOBt, Et3N, DCM, rt; (f) 51, EDCI, HOBt, Et3N, DCM, rt.
3. Conclusion

In summary, we rationally designed and prepared a novel series of

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl) amide derivatives as potent PRMT5 inhibitors. Systematic SAR explorations led to the identification of 46, which exhibited the most potent PRMT5 inhibitory activity with IC50 value of 8.5 nM and excellent selectivity over other methyltransferases enzymes. Moreover, the compound 46 showed a robust antiproliferative activity against MV4-11 cells (GI50 = 18 nM) and was approximately 6-fold more potent than EPZ015666 (1). Besides, Western blot analysis revealed that 46 suppressed PRMT5 activity at nanomolar concentrations. Furthermore, significant antitumor efficacy was observed for 46 in the MV4-11 xenograft model. Overall, 46 could be used as an effective chemical probe to investigate new functions of PRMT5 in biology and also served as a good lead compound for the development of new PRMT5-targeting therapeutic agents.
4. Experimental section

4.1. Chemistry

All purchased chemicals and solvents were used without further purification unless otherwise noted. Flash chromatography was performed using silica gel (300−400 mesh). All reactions were monitored by thin-layer chromatography (TLC), using silica gel plates with fluorescence F254 and visualized under UV light. 1H NMR spectral data were recorded on Varian Mercury 300 or 400 NMR spectrometer. Chemicals shifts (δ) were reported in parts per million, coupling constants (J) values were in hertz, and the splitting patterns were abbreviated as follows: s for singlet; d for doublet; t for triplet; q for quartet and m for multiplet. The high resolution of ESI-MS was recorded on a Finnigan/MAT95 spectrometer.

General Procedure A: Synthesis of compounds 3, 4 and 12. Intermediates 53a-c (1.0 mmol) and corresponding aldehydes (2.0 mmol) were dissolved in in 5 mL methanol and stirred for 30 minutes, then sodium cyanoborohydride (2.0 mmol) was added and the mixture was stirred for 4 hours. Water was added and the reaction mixture was partitioned between ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography (dichloromethane/ methanol, v/v, 90:10) to give the desired product.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-methyl-4,5,6,7-tetrahyd rothieno[3,2-c]pyridine-2-carboxamide (3). The title compound was prepared from 53a and formaldehyde following general procedure A. Purity: 98.6%. Yellow solid (56%).1H NMR (300 MHz, CD3OD) δ 7.18 (s, 1H), 7.13 (m, 3H), 7.05 (s, 1H), 4.08 (m,
1H), 3.80 (s, 2H), 3.45 (d, J = 5.2 Hz, 2H), 3.40 (m, 2H), 2.93 (m, 6H), 2.79 (m, 2H),

2.72 (m, 2H), 2.46 (s, 3H). HRMS (ESI+) m/z calcd for C21H28N3O2S 386.1902; found 386.1883.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-benzyl-4,5,6,7-tetrahyd rothieno[3,2-c]pyridine-2-carboxamide (4). The title compound was prepared from 53a and benzaldehyde following general procedure A. Purity: 97.2%. Yellow solid (45%). 1H NMR (300 MHz, CD3OD) δ 7.43 – 7.26 (m, 5H), 7.16 (s, 1H), 7.12 (m, 4H),
4.14 (m, 1H), 4.03 (s, 2H), 3.74 (s, 2H), 3.45 (d, J = 5.0 Hz, 4H), 3.16 (t, J = 5.9 Hz, 2H),

3.00 (m, 2H), 2.96 – 2.78 (m, 6H). HRMS (ESI+) m/z calcd for C27H32N3O2S 462.2215;

found 462.2204.

General Procedure B: Synthesis of compounds 5, 7, 9-11, 15-33 and 52. To a solution of corresponding acids (1.0 mmol) in DCM (6 mL) was added EDCI (1.5 mmol), HOBt (1.5 mmol), Et3N (3.0 mmol) and corresponding amines (1.0 mmol). The mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (10 mL) and extracted with DCM (3x10 mL). The combined organic layers were then dried and concentrated. The residue was purified by silica gel chromatography (dichloromethane/ methanol, v/v, 90:10) to give the desired product.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-phenyl-4,5,6,7-tetrahyd rothieno[3,2-c]pyridine-2-carboxamide (5). The title compound was prepared from 66a and 51 following general procedure B. Purity: 96.2%. Yellow solid (60%).1H NMR (300 MHz, CD3OD) δ 7.31 – 7.20 (m, 3H), 7.14 (m, 3H), 7.07 (m, 1H), 7.01 (d, J
= 8.2 Hz, 2H), 6.84 (t, J = 7.4 Hz, 1H), 4.16 – 4.03 (m, 3H), 3.85 (s, 2H), 3.57 (t, J = 5.5

Hz, 2H), 3.51 – 3.41 (m, 2H), 2.95 (m, 6H), 2.76 (m, 2H). HRMS (ESI+) m/z calcd for C26H30N3O2S 448.2053; found 448.2064.
General Procedure C: Synthesis of compounds 6, 13 and 14. Intermediate 53a (1.0 mmol), corresponding ketone (2.0 mmol) and acetic acid (0.2 mmol) were dissolved in 5 mL methanol and stirred for 30 minutes, then sodium cyanoborohydride (2.0 mmol) was added and the mixture was heated to 65 °C for 12 hours. Water was added and the reaction mixture was partitioned between ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, and concentrated in vacuum. The resulting residue was purified by silica gel

chromatography (dichloromethane/ methanol, v/v, 90:10) to give the desired product.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(1-phenylethyl)-4,5,6,7

-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (6). The title compound was prepared from 53a and acetophenone following general procedure C. Purity: 95.0%. White solid (43%). 1H NMR (300 MHz, CD3OD) δ 7.42 – 7.20 (m, 5H), 7.16 – 6.98 (m,
5H), 4.06 (m, 1H), 3.79 (s, 2H), 3.62 (m, 1H), 3.51 (m, 1H), 3.47 – 3.36 (m, 2H), 3.28

(d, J = 5.9 Hz, 1H), 2.92 (m, 4H), 2.81 (m, 3H), 2.77 – 2.63 (m, 3H), 1.46 (d, J = 6.7 Hz, 3H). HRMS (ESI+) m/z calcd for C28H34N3O2S 476.2372; found 476.2359.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-benzoyl-4,5,6,7-tetrahy drothieno[3,2-c]pyridine-2-carboxamide (7). The title compound was prepared from 53a and benzoic acid following general procedure B. Purity: 97.8%. Yellow solid (73%) 1H NMR (300 MHz, CD3OD) δ 7.46 (m, 6H), 7.14 (m, 4H), 4.69 (s, 1H), 4.41 (s,
1H), 4.25 – 3.94 (m, 4H), 3.69 (s, 1H), 3.46 (s, 2H), 3.20 (m, 2H), 2.93 (m, 6H). HRMS

(ESI+) m/z calcd for C27H30N3O3S 476.2008; found 476.2013.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(phenylsulfonyl)-4,5,6, 7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (8). To a mixture of 53a (100 mg,
0.27 mmol) and Et3N (112 µL, 0.81 mmol) in dichloromethane (6 mL) was added benzenesulfonyl chloride (62 mg, 0.32 mmol ) and the mixture was stirred for 4 hours at room temperature. Dichloromethane and water were added. The organic layer was separated and washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuum. The resulting residue was purified by silica gel chromatography (dichloromethane/ methanol, v/v, 90:10) to give 8 as yellow solid(70

mg, 51%). Purity: 95.8%. 1H NMR (300 MHz, CD3OD) δ 7.84 (d, J = 7.4 Hz, 2H), 7.70 – 7.54 (m, 3H), 7.12 (m, 4H), 7.04 (d, J = 7.4 Hz, 1H), 4.11 (m, 1H), 4.03 (s, 2H),
3.87 (s, 2H), 3.43 (m, 4H), 2.98 (m, 4H), 2.81 (m, 4H). HRMS (ESI+) m/z calcd for C26H30N3O4S2 512.1678; found 512.1670.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(2-phenylacetyl)-4,5,6, 7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (9). The title compound was prepared from 53a and 2-phenylacetic acid following general procedure B. Purity: 99.0%. White solid (57%). 1H NMR (300 MHz, CD3OD) δ 7.23 (m, 6H), 7.14 (m, 3H),
7.06 (s, 1H), 4.50 (m, 2H), 4.12 (m, 1H), 3.85 (m, 6H), 3.45 (d, J = 5.5 Hz, 2H), 3.01 (m,

4H), 2.84 (m, 3H), 2.61 (m, 1H). HRMS (ESI+) m/z calcd for C28H32N3O3S 490.2164;

found 490.2167.

Phenyl-2-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-6,7-di hydrothieno[3,2-c]pyridine-5(4H)-carboxylate (10). The title compound was prepared from 66b and 51 following general procedure B. White solid (61%). Purity: 99.6%. 1H NMR (300 MHz, CD3OD) δ 7.38 (t, J = 7.6 Hz, 2H), 7.23 (m, 2H), 7.12 (m, 5H),
7.03 (d, J = 7.6 Hz, 1H), 4.58 (s, 1H), 4.46 (s, 1H), 4.20 – 4.02 (m, 1H), 3.93 (m, 1H),

3.86 (s, 2H), 3.79 (s, 1H), 3.46 (d, J = 5.5 Hz, 2H), 3.05 – 2.70 (m, 8H). HRMS (ESI+)

m/z calcd for C27H30N3O4S 492.1957; found 492.1962.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(3-phenylpropanoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (11). The title compound was prepared from 53a and 3-phenylpropanoic acid following general procedure B. Purity: 99.5%. White solid (70%). 1H NMR (300 MHz, CD3OD) δ 7.48 – 6.86 (m, 10H), 4.49

(s, 1H), 4.26 (s, 1H), 4.10 (m, 1H), 3.91 – 3.77 (m, 3H), 3.70 (m, 1H), 3.47 (t, J = 7.2 Hz, 2H), 2.94 (m, 6H), 2.76 (m, 6H). HRMS (ESI+) m/z calcd for C29H34N3O3S 504.2321;
found 504.2320.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-propyl-4,5,6,7-tetrah ydrothieno[3,2-c]pyridine-2-carboxamide (12). The title compound was prepared from 53a and propionaldehyde following general procedure A. Purity: 97.3%. Yellow solid (59%). 1H NMR (300 MHz, CD3OD) δ 7.10 (m, 5H), 4.14 – 3.98 (m, 1H), 3.75 (s,
2H), 3.45 (d, J = 5.8 Hz, 2H), 3.40 (s, 2H), 2.89 (d, J = 6.0 Hz, 6H), 2.81 (d, J = 5.1 Hz,

2H), 2.75 – 2.59 (m, 2H), 2.58 – 2.43 (m, 2H), 1.61 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H). HRMS (ESI+) m/z calcd for C23H32N3O2S 414.2210; found 414.2199.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-cyclohexyl-4,5,6,7-te trahydrothieno[3,2-c]pyridine-2-carboxamide (13). The title compound was prepared from 53a and cyclohexanone following general procedure C. Purity: 96.2%. Yellow solid (64%). 1H NMR (300 MHz, CD3OD) δ 7.13 (m, 4H), 7.04 (d, J = 6.3 Hz, 1H), 4.08 (p, J = 6.0 Hz, 1H), 3.78 (s, 2H), 3.59 (s, 2H), 3.51 – 3.42 (m, 2H), 3.00 – 2.84 (m,
8H), 2.79 – 2.52 (m, 3H), 1.92 (m, 4H), 1.69 (m, 1H), 1.45 – 1.12 (m, 5H). HRMS

(ESI+) m/z calcd for C26H36N3O2S 454.2523; found 454.2522.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(tetrahydro-2H-pyra n-4-yl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (14). The title compound was prepared from 53a and dihydro-2H-pyran-4(3H)-one following general procedure C. Purity: 98.6%. Yellow solid (44%). 1H NMR (300 MHz, CD3OD) δ 7.14 (m, 4H), 7.05 (m, 1H), 4.15 – 3.95 (m, 3H), 3.79 (s, 2H), 3.54 (s, 2H), 3.44 (m,

4H), 3.00 – 2.82 (m, 8H), 2.79 – 2.62 (m, 2H), 1.87 (m, 2H), 1.63 (m, 2H). HRMS

(ESI+) m/z calcd for C25H34N3O3S 456.2321; found 456.2327.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-isonicotinoyl-4,5,6,7

-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (15). The title compound was prepared from 53a and isonicotinic acid following general procedure B. Purity: 98.6%. White solid (65%). 1H NMR (300 MHz, CD3OD) δ 8.69 (d, J = 4.3 Hz, 2H), 7.47 (m, 2H), 7.29 (s, 1H), 7.14 (m, 2H), 7.00 (m, 2H), 4.65 (s, 1H), 4.30 (s, 1H), 4.11 (m, 1H),
4.03 (t, J = 5.6 Hz, 1H), 3.85 (s, 1H), 3.81 (s, 1H), 3.66 – 3.56 (m, 1H), 3.45 (m, 2H),

3.07 – 2.84 (m, 6H), 2.83 – 2.67 (m, 2H). HRMS (ESI+) m/z calcd for C26H29N4O3S

477.1960; found 477.1957.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(furan-2-carbonyl)-4

,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (16). The title compound was prepared from 53a and furan-2-carboxylic acid following general procedure B. Purity: 99.6%. White solid (68%). 1H NMR (300 MHz, CD3OD) δ 7.71 (s, 1H), 7.20 (s, 1H),
7.07 (m, 4H), 6.99 (d, J = 6.9 Hz, 1H), 6.60 (mz, 1H), 4.61 (s, 2H), 4.17 – 4.05 (m, 1H),

3.97 (s, 2H), 3.80 (s, 2H), 3.45 (d, J = 5.9 Hz, 2H), 2.92 (m, 6H), 2.75 (t, J = 5.5 Hz, 2H). HRMS (ESI+) m/z calcd for C25H28N3O4S 466.1801; found 466.1797.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(thiophene-2-carbon yl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (17). The title compound was prepared from 53a and thiophene-2-carboxylic acid following general procedure
B. Purity: 99.5%. White solid (70%). 1H NMR (300 MHz, CD3OD) δ 7.69 (s, 1H), 7.49 (s, 1H), 7.10 (m, 6H), 4.65 (m, 2H), 4.09 (m, 1H), 3.98 (s, 2H), 3.82 (m, 2H), 3.46

(m, 2H), 2.95 (m, 6H), 2.76 (m, 2H). HRMS (ESI+) m/z calcd for C25H28N3O3S2 482.1572; found 482.1572.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(2-naphthoyl)-4,5,6, 7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (18). The title compound was prepared from 53a and 2-naphthoic acid following general procedure B. Purity: 97.8%. White solid (63%). 1H NMR (300 MHz, CD3OD) δ 7.98 (m, 4H), 7.58 (m, 3H),
7.43 – 6.85 (m, 5H), 4.76 (s, 1H), 4.45 (s, 1H), 4.11 (m, 4H), 3.76 (m, 1H), 3.46 (m, 2H),

2.97 (m, 8H). HRMS (ESI+) m/z calcd for C31H32N3O3S 526.2164; found 526.2155.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-methylbenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (19). The title compound was prepared from 53a and 4-methylbenzoic acid following general procedure B. Purity: 99.3%. White solid (64%). 1H NMR (300 MHz, CD3OD) δ 7.45 – 7.22 (m, 5H), 7.01 (m, 4H), 4.62 (s, 1H), 4.36 (s, 1H), 4.10 (m, 2H), 3.78 (s, 2H), 3.70 (m, 1H), 3.47 (d, J = 5.9 Hz, 2H), 2.92 (m, 6H), 2.72 (m, 2H), 2.41 (s, 3H). HRMS (ESI+) m/z calcd for C28H32N3O3S 490.2164; found 490.2168.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-methoxybenzoyl)- 4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (20). The title compound was prepared from 53a and 4-methoxybenzoic acid following general procedure B. Purity: 98.4%. White solid (75%). 1H NMR (300 MHz, CD3OD) δ 7.43 (d, J = 8.8 Hz, 2H),
7.04 (m, 6H), 4.52 (s, 2H), 4.07 (m, 1H), 3.82 (m, 7H), 3.54 – 3.36 (m, 2H), 2.92 (m,

6H), 2.80 – 2.62 (m, 2H). HRMS (ESI+) m/z calcd for C28H32N3O4S 506.2114; found

506.2116.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-fluorobenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (21). The title compound was prepared from 53a and 4-fluorobenzoic acid following general procedure B. Purity: 98.9%. White solid (64%). 1H NMR (300 MHz, CD3OD) δ 7.52 (m, 2H), 7.25 (m, 3H),
7.02 (m, 4H), 4.60 (s, 1H), 4.37 (s, 1H), 4.07 (m, 2H), 3.75 (m, 3H), 3.45 (s, 2H), 2.90 (m, 6H), 2.69 (m, 2H). HRMS (ESI+) m/z calcd for C27H29FN3O3S 494.1914; found 494.1920.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-chlorobenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (22). The title compound was prepared from 53a and 4-chlorobenzoic acid following general procedure B. Purity: 99.4%. White solid (68%). 1H NMR (300 MHz, CD3OD) δ 7.53 (d, J = 8.5 Hz, 2H),
7.46 (d, J = 8.5 Hz, 2H), 7.35 – 6.78 (m, 5H), 4.62 (s, 1H), 4.34 (s, 1H), 4.07 (m, 2H),

3.77 (s, 2H), 3.67 (s, 1H), 3.47 (d, J = 5.7 Hz, 2H), 2.91 (m, 6H), 2.71 (m, 2H). HRMS

(ESI+) m/z calcd for C27H29ClN3O3S 510.1618; found 510.1621.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-bromobenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (23). The title compound was prepared from 53a and 4-bromobenzoic acid following general procedure B. Purity: 98.8%. White solid (59%). 1H NMR (300 MHz, CD3OD) δ 7.67 (d, J = 7.8 Hz, 2H),
7.38 (d, J = 7.8 Hz, 2H), 7.30 – 6.85 (m, 5H), 4.62 (s, 1H), 4.32 (s, 1H), 4.07 (m, 2H),

3.80 (s, 2H), 3.67 (s, 1H), 3.46 (d, J = 5.7 Hz, 2H), 2.92 (m, 6H), 2.73 (m, 2H). HRMS

(ESI+) m/z calcd for C27H29BrN3O3S 554.1113; found 554.1121.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-acetylbenzoyl)-4,

5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (24). The title compound was prepared from 53a and 4-acetylbenzoic acid following general procedure B. Purity: 99.3%. White solid (66%). 1H NMR (300 MHz, CD3OD) δ 8.11 (d, J = 8.0 Hz, 2H),
7.58 (m, 2H), 7.12 (m, 5H), 4.68 (s, 1H), 4.35 (s, 1H), 4.12 (m, 1H), 4.05 (s, 1H), 3.90

(m, 2H), 3.66 (s, 1H), 3.46 (m, 2H), 3.02 (m, 6H), 2.85 (m, 2H), 2.64 (s, 3H). HRMS

(ESI+) m/z calcd for C29H32N3O4S 518.2114; found 518.2112.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-(trifluoromethyl)b enzoyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (25). The title compound was prepared from 53a and 4-(trifluoromethyl)benzoic acid following general procedure B. Purity: 99.3%. White solid (60%). 1H NMR (300 MHz, CD3OD) δ 7.81 (m, 2H), 7.66 (m, 2H), 7.33 – 6.83 (m, 5H), 4.66 (s, 1H), 4.30 (s, 1H), 4.06 (m,
2H), 3.81 (s, 2H), 3.64 (s, 1H), 3.47 (s, 2H), 2.94 (m, 6H), 2.73 (m, 2H). HRMS (ESI+)

m/z calcd for C28H29F3N3O3S 544.1882; found 544.1883.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-(tert-butyl)benzoy l)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (26). The title compound was prepared from 53a and 4-(tert-butyl)benzoic acid following general procedure B. Purity: 98.8%. White solid (63%). 1H NMR (300 MHz, CD3OD) δ 7.55 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.35 – 6.80 (m, 5H), 4.64 (s, 1H), 4.37 (s, 1H), 4.11 (s,
1H), 4.04 – 3.95 (m, 1H), 3.88 (s, 2H), 3.70 (s, 1H), 3.47 (d, J = 5.7 Hz, 2H), 2.93 (m,

6H), 2.80 (m, 2H), 1.36 (s, 9H). HRMS (ESI+) m/z calcd for C31H38N3O3S 532.2634;

found 532.2640.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-nitrobenzoyl)-4,5,

6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (27). The title compound was prepared from 53a and 4-nitrobenzoic acid following general procedure B. Purity: 98.3%. White solid (69%). 1H NMR (300 MHz, CDCl3) δ 8.32 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 7.8 Hz, 2H), 7.14 (m, 3H), 7.02 (m, 2H), 4.76 (s, 1H), 4.39 (s, 1H), 4.08 (m, 2H),
3.93 (m, 1H), 3.75 (m, 3H), 3.44 (s, 1H), 2.97 (m, 4H), 2.90 (m, 2H), 2.70 (m, 2H). HRMS (ESI+) m/z calcd for C27H29N4O5S 521.1859; found 521.1855.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-cyanobenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (28). The title compound was prepared from 53a and 4-cyanobenzoic acid following general procedure B. Purity: 97.9%. White solid (69%). 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 7.8 Hz, 2H), 7.14 (m, 4H), 7.03 (m, 1H), 4.74 (s, 1H), 4.38 (s, 1H), 4.12 (m, 2H),
3.98 (m, 1H), 3.82 (m, 1H), 3.68 (m, 2H), 3.47 – 3.37 (m, 1H), 3.08 (m, 1H), 2.98 (m,

5H), 2.75 (m, 2H). HRMS (ESI+) m/z calcd for C28H29N4O3S 501.1960; found

501.1960.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-(dimethylamino)b enzoyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (29). The title compound was prepared from 53a and 4-(dimethylamino)benzoic acid following general procedure B. Purity: 96.3%. White solid (58%). 1H NMR (400 MHz, CD3OD) δ 7.35 (d, J = 8.5 Hz, 2H), 7.05 (m, 4H), 6.97 (d, J = 7.0 Hz, 1H), 6.75 (d, J = 8.5 Hz,
2H), 4.48 (s, 2H), 4.10 – 4.00 (m, 1H), 3.81 (s, 2H), 3.71 (s, 2H), 3.52 – 3.36 (m, 2H),

2.99 (s, 6H), 2.85 ( m, 6H), 2.72 – 2.61 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 171.9,

162.3, 151.8, 136.58, 133.6, 133.5, 133.1, 129.2, 128.7, 126.7, 126.6, 126.0, 122.2,

111.2, 66.0, 61.0, 56.0, 51.2, 43.7, 40.2, 28.7. HRMS (ESI+) m/z calcd for C29H35N4O3S

519.2430; found 519.2429.

5-(4-(azetidin-1-yl)benzoyl)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypr opyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (30). The title compound was prepared from 53a and 4-(azetidinyl)benzoic acid following general procedure B. Purity: 93.0%. White solid (69%).1H NMR (300 MHz, CD3OD) δ 7.34 (d, J = 8.4 Hz, 2H), 7.08 (m, 5H), 6.48 (d, J = 8.4 Hz, 2H), 4.53 (m, 2H), 4.09 (m, 1H), 3.94
(t, J = 7.2 Hz, 4H), 3.88 (m, 4H), 3.45 (s, 2H), 3.12 – 2.87 (m, 6H), 2.80 (t, J = 5.9 Hz,

2H), 2.48 – 2.31 (m, 2H).

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(3-fluorobenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (31). The title compound was prepared from 53a and 3-fluorobenzoic acid following general procedure B. Purity: 95.6%. White solid (64%). 1H NMR (300 MHz, CD3OD) δ 7.51 (m, 1H), 7.34 – 7.17
(m, 4H), 6.99 (m, 4H), 4.60 (s, 1H), 4.30 (s, 1H), 4.02 (m, 2H), 3.72 (s, 2H), 3.63 (s, 1H), 3.46 (d, J = 5.7 Hz, 2H), 2.85 (m, 6H), 2.67 (m, 2H). HRMS (ESI+) m/z calcd for C27H29FN3O3S 494.1914; found 494.1922.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(2-fluorobenzoyl)-4, 5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (32). The title compound was prepared from 53a and 2-fluorobenzoic acid following general procedure B. Purity: 96.8%. White solid (69%). 1H NMR (300 MHz, CD3OD) δ 7.60 – 7.47 (m, 1H), 7.41
(m, 1H), 7.34 – 7.19 (m, 3H), 7.19 – 7.06 (m, 2H), 7.05 – 6.85 (m, 2H), 4.65 (s, 1H),

4.22 (s, 1H), 4.07 (m, 2H), 3.76 (m, 2H), 3.59 (t, J = 5.4 Hz, 1H), 3.53 – 3.37 (m, 2H),

2.99 – 2.79 (m, 6H), 2.77 – 2.62 (m, 2H). HRMS (ESI+) m/z calcd for C27H29FN3O3S

494.1914; found 494.1915.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-methyl-4,5,6,7-tetra hydrofuro[3,2-c]pyridine-2-carboxamide (33). The title compound was prepared from 53b and formaldehyde following general procedure A. Purity: 97.7%. Yellow solid (45%).1H NMR (300 MHz, CD3OD) δ 7.16 – 7.05 (m, 3H), 7.02 (d, J = 5.4 Hz, 1H), 6.89 (s, 1H), 4.13 – 3.98 (m, 1H), 3.74 (s, 2H), 3.46 (d, J = 5.9 Hz, 2H), 3.39 (s, 2H),
2.91 (m, 2H), 2.85 (m, 2H), 2.79 (t, J = 5.8 Hz, 2H), 2.69 – 2.59 (m, 4H), 2.46 (s, 3H).

13C NMR (126 MHz, CDCl3) δ 159.7, 151.9, 146.9, 134.4, 134.4, 129.3, 127.1, 127.1,

126.4, 118.9, 113.8, 66.7, 61.5, 56.6, 52.5, 52.1, 51.7, 45.8, 43.2, 29.4, 24.6. HRMS

(ESI+) m/z calcd for C21H28N3O3 370.2131; found 370.2119.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-methyl-4,5,6,7-tetra hydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (34). The title compound was prepared from 53c and formaldehyde following general procedure A. Purity: 98.8%. Yellow solid (34%). 1H NMR (300 MHz, CD3OD) δ 7.22 – 7.04 (m, 4H), 6.51 (s, 1H), 4.20 – 4.08 (m, 1H), 4.01 (s, 2H), 3.95 (s, 2H), 3.50 – 3.38 (m, 2H), 3.33 (d, J = 7.9 Hz,
2H), 3.14 (t, J = 5.9 Hz, 2H), 3.01 (t, J = 6.0 Hz, 2H), 2.95 (t, J = 5.9 Hz, 2H), 2.88 (m,

2H), 2.83 (s, 3H). HRMS (ESI+) m/z calcd for C21H29N4O2 369.2285; found 369.2298.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-methyl-4,5,6,7-tetra hydrothiazolo[4,5-c]pyridine-2-carboxamide (35). The title compound was prepared from 74 and 51 following general procedure B. Purity: 95.4%. Yellow solid (57%).1H NMR (300 MHz, CD3OD) δ 7.18 – 7.04 (m, 3H), 7.00 (m, 1H), 4.08 (p, J = 5.9 Hz, 1H),

3.74 (s, 2H), 3.50 (dd, J = 5.7, 3.5 Hz, 2H), 3.45 (s, 2H), 2.95 (m, 4H), 2.84 (m, 2H),

2.77 (t, J = 5.7 Hz, 2H), 2.71 – 2.65 (m, 2H), 2.47 (s, 3H). HRMS (ESI+) m/z calcd for C20H27N4O2S 387.1855; found 387.1853.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-5-(4-(dimethylamino)b enzoyl)-3-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxamide (36). The title compound was prepared from 70 and 51 following general procedure B. Purity: 99.6%. Yellow solid (65%). 1H NMR (300 MHz, CD3OD) δ 7.39 (d, J = 8.8 Hz, 2H),
7.05 (m, 4H), 6.79 (d, J = 8.8 Hz, 2H), 4.53 (s, 2H), 4.12 – 4.00 (m, 1H), 3.86 (s, 2H),

3.73 (s, 2H), 3.46 (t, J = 5.6 Hz, 2H), 3.02 (s, 6H), 2.87 (m, 6H), 2.68 (m, 2H), 2.23 (s,

3H). HRMS (ESI+) m/z calcd for C30H37N4O3S 533.2581; found 533.2582.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-(dimethylamino)b enzoyl)-1,2,3,4-tetrahydroisoquinoline-7-carboxamide (37). The title compound was prepared from 63 and 4-(dimethylamino)benzoic acid following general procedure B. Purity: 99.4%. Yellow solid (67%).1H NMR (300 MHz, CD3OD) δ 7.61 (s, 1H), 7.54 (s, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.22 (d, J = 7.8 Hz, 1H), 7.09 (m, 4H), 6.76 (d, J = 7.5 Hz,
2H), 4.75 (m, 2H), 4.16 (m, 1H), 3.95 (s, 2H), 3.83 (s, 2H), 3.48 (s, 2H), 3.04 (m, 12H),

2.86 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 167.7, 151.7, 133.9, 132.8, 132.2, 131.8,

129.3, 129.1, 128.8, 127.1, 126.7, 126.4, 125.5, 125.2, 122.4, 111.2, 66.1, 60.2, 55.3,

50.9, 43.6, 40.2, 27.6. HRMS (ESI+) m/z calcd for C31H37N4O3 513.2860; found

513.2851.

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-(dimethylamino)b enzoyl)isoindoline-5-carboxamide (38). The title compound was prepared from 73

and 51 following general procedure B. Purity: 96.8%. White solid (60%). 1H NMR (300 MHz, CD3OD) δ 7.71 (s, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.25 (m, 1H), 7.06 (m, 4H),
6.76 (d, J = 6.8 Hz, 2H), 4.86 (s, 2H), 4.81 (s, 1H), 4.71 (s, 1H), 4.12 (s, 1H), 3.81 (d, J

= 5.1 Hz, 2H), 3.50 (t, J = 4.7 Hz, 2H), 3.00 (s, 6H), 2.91 (m, 4H), 2.76 (t, J = 6.5 Hz, 2H). HRMS (ESI+) m/z calcd for C30H35N4O3 499.2704; found 499.2710.
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-(dimethylamino)b enzoyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (39). The title compound was prepared from 60 and 4-(dimethylamino)benzoic acid following general procedure B. Purity: 98.5%. White solid (71%).1H NMR (300 MHz, CD3OD) δ 7.62 (s, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.13 (m, 4H), 6.77 (d, J = 8.8 Hz, 2H), 4.79 (s, 2H), 4.25 – 4.11 (m, 1H),
3.98 (s, 2H), 3.81 (s, 2H), 3.50 (d, J = 4.6 Hz, 2H), 3.11 (t, J = 5.7 Hz, 2H), 3.05 – 2.93 (m, 8H), 2.93 – 2.76 (m, 4H). HRMS (ESI+) m/z calcd for C25H28N3O3S2 482.1572;
found 482.1572. HRMS (ESI+) m/z calcd for C31H37N4O3 513.2860; found 513.2848. N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-methoxybenzoyl)- 1,2,3,4-tetrahydroisoquinoline-6-carboxamide (40). The title compound was prepared from 60 and 4-methoxybenzoic acid following general procedure B. Purity: 98.0%. White solid (63%).1H NMR (300 MHz, CDCl3) δ 7.61 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 8.7 Hz, 2H), 7.14 (m, 4H), 7.01 (d, J = 5.7 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H),
6.84 (s, 1H), 4.80 (s, 2H), 4.04 (m, 1H), 3.83 (m, 5H), 3.80 – 3.69 (m, 2H), 3.63 (m, 1H),

3.50 – 3.39 (m, 1H), 2.93 (m, 5H), 2.75 (m, 1H), 2.67 – 2.49 (m, 2H). HRMS (ESI+)

m/z calcd for C30H34N3O4 500.2544; found 500.2556.

2-(4-bromobenzoyl)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,

2,3,4-tetrahydroisoquinoline-6-carboxamide (41). The title compound was prepared from 60 and 4-bromobenzoic acid following general procedure B. Purity: 98.1%. White solid (80%).1H NMR (300 MHz, CDCl3) δ 7.69 – 7.51 (m, 4H), 7.33 (d, J = 8.2 Hz, 2H), 7.22 – 7.08 (m, 3H), 7.00 (d, J = 5.9 Hz, 1H), 6.88 (s, 1H), 4.89 (s, 1H), 4.59 (s,
1H), 4.03 (m, 2H), 3.83 (d, J = 14.9 Hz, 1H), 3.79 – 3.70 (m, 1H), 3.62 (d, J = 14.8 Hz,

2H), 3.52 – 3.38 (m, 1H), 2.94 (m, 5H), 2.81 – 2.68 (m, 1H), 2.67 – 2.49 (m, 2H).

HRMS (ESI+) m/z calcd for C29H31BrN3O3 548.1543; found 548.1535.

(S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-(dimethylami no)benzoyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (42). The title compound was prepared from (S)-60 and 4-(dimethylamino)benzoic acid following general procedure B. Purity: 98.9%. White solid (75%). 1H NMR (300 MHz, CD3OD) δ 7.62 (d, J = 6.4 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.19 – 7.11 (m, 3H), 7.08 (s, 1H), 6.78 (d,
J = 8.8 Hz, 2H), 4.81 (s, 2H), 4.17 (m, 1H), 4.00 (s, 2H), 3.83 (s, 2H), 3.51 (d, J = 5.4

Hz, 2H), 3.12 (d, J = 5.9 Hz, 2H), 3.01 (m, 8H), 2.95 – 2.80 (m, 4H). HRMS (ESI+) m/z

calcd for C31H37N4O3 513.2860; found 513.2852.

(R)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-methoxybenzoyl

)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (43). The title compound was prepared from (R)-60 and 4-methoxybenzoic acid following general procedure B. Purity: 97.9%. White solid (65%).1H NMR (300 MHz, CDCl3) δ 7.62 (s, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.14 (m, 4H), 7.01 (d, J = 5.4 Hz, 1H), 6.94 (m,
3H), 4.81 (s, 2H), 4.07 (m, 1H), 3.87 (m, 5H), 3.76 (m, 2H), 3.67 (m, 1H), 3.52 – 3.38

(m, 2H), 2.95 (t, J = 8.3 Hz, 5H), 2.78 (m, 1H), 2.62 (m, 2H). HRMS (ESI+) m/z calcd

for C30H34N3O4 500.2544; found 500.2556.

(S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(4-methoxybenzoyl)

-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (44). The title compound was prepared from (S)-60 and 4-methoxybenzoic acid following general procedure B. Purity: 98.7%. White solid (66%). 1H NMR (300 MHz, CDCl3) δ 7.61 (s, 1H), 7.57 (m,
1H), 7.47 – 7.40 (m, 2H), 7.20 – 7.07 (m, 4H), 7.03 – 6.97 (m, 1H), 6.94 (m, 3H), 4.80

(s, 2H), 4.12 – 3.97 (m, 1H), 3.83 (m, 5H), 3.74 (m, 2H), 3.62 (m, 1H), 3.49 – 3.39 (m,

1H), 2.92 (s, 5H), 2.80 – 2.69 (m, 1H), 2.68 – 2.48 (m, 2H). 13C NMR (151 MHz,

CDCl3) δ 167.4, 161.0, 133.7, 129.0, 128.7, 127.8, 127.8, 126.5, 126.5, 125.9, 124.9,

66.1, 61.1, 56.0, 55.4, 51.2, 43.8, 28.8. HRMS (ESI+) m/z calcd for C30H34N3O4

500.2544; found 500.2553.

(R)-2-(4-bromobenzoyl)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl

)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (45). The title compound was prepared from (R)-60 and 4-bromobenzoic acid following general procedure B. Purity: 97.2%. White solid (62%). 1H NMR (300 MHz, CDCl3) δ 7.65 – 7.53 (m, 4H), 7.33 (d,
J = 8.3 Hz, 2H), 7.19 – 7.08 (m, 3H), 7.01 (m, 1H), 6.89 (s, 1H), 4.89 (s, 1H), 4.66 –

4.53 (m, 1H), 4.05 (d, J = 3.7 Hz, 2H), 3.86 (m, 1H), 3.81 – 3.72 (m, 1H), 3.66 (m, 2H),

3.46 (m, 1H), 2.96 (m, 5H), 2.82 – 2.74 (m, 1H), 2.71 – 2.52 (m, 2H). HRMS (ESI+)

m/z calcd for C29H31BrN3O3 548.1543; found 548.1535

(S)-2-(4-bromobenzoyl)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl

)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide (46). The title compound was prepared from (S)-60 and 4-bromobenzoic acid following general procedure B. Purity:

98.5%. White solid (68%). 1H NMR (300 MHz, CDCl3) δ 7.67 – 7.55 (m, 4H), 7.33

(d, J = 8.1 Hz, 2H), 7.14 (m, 3H), 7.02 (m, 1H), 6.91 (m, 1H), 4.89 (m, 1H), 4.72 – 4.54

(m, 1H), 4.04 (m, 2H), 3.85 (d, J = 15.1 Hz, 1H), 3.73 (m, 1H), 3.64 (m, 2H), 3.48 (m,

1H), 2.93 (m, 5H), 2.79 – 2.72 (m, 1H), 2.60 (m, 2H). 13C NMR (126 MHz, CDCl3) δ

167.2, 134.6, 134.1, 133.9, 131.9, 128.7, 127.8, 126.5, 126.5, 125.8, 125.0, 124.4, 66.0,

61.1, 56.1, 51.2, 43.6, 29.1. HRMS (ESI+) m/z calcd for C29H31BrN3O3 548.1543;

found 548.1535.

Synthesis of tert-butyl 4-chloro-3-formyl-5,6-dihydropyridine-1(2H)-carboxylate (48). A solution of anhydrous DMF (13.7 mL, 0.15 mol ) in anhydrous DCM (20 mL) was cooled to 0 °C. POCl3 (19.3 mL, 0.25 mol ) was added dropwise. Upon complete addition, the solution was stirred at 0 °C for 15 minutes and tert-butyl-4-oxopiperidine-1-carboxylate (10.0g, 0.05 mmol) in anhydrous DCM (30 mL)was added and the stirring was continued at room temperature for 2 hours. The reaction mixture was quenched with water and extracted with dichloromethane (2 x
200 mL). The organic layer was separated, dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford crude product (6.64 g, Yield 54%). 1H NMR (300 MHz, CDCl3) δ 10.12 (s, 1H), 4.11 (s, 2H), 3.59 (q, J = 5.8 Hz, 2H), 2.66 (s, 2H), 1.45 (s, 9H).
Synthesis of 5-tert-butyl 2-ethyl 6,7-dihydrothieno[3,2-c]pyridine-2,5(4H)-dicarboxylate (49a). The crude product 48 (6.64 g, 27.0 mmol) was dissolved in DCM. Ethylmercaptoacetae (3 mL, 27.0 mmol) and Et3N (9.4 mL, 67.5 mmol) was added slowly at room temperature. The reaction

mixture was stirred for 12 hours and quenched with water. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate, v/v, 95:5) to give the desired product (5.80 g, Yield 50%). 1H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 4.45 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 3.69 (t, J = 5.4 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H),
1.50 – 1.39 (s, 9H), 1.32 (t, J = 7.1 Hz, 3H).

Synthesis of 5-tert-butyl 2-ethyl 6,7-dihydrofuro[3,2-c]pyridine-2,5(4H)-dicarboxylate (49b). To a solution of ethyl glycolate (1.90 mL, 20.0 mmol) in THF (25 mL) was added sodium hydride (720 mg, 30 mmol) at 0 °C and stirred at room temperature for 1 hour. The solution of 48 (2.45 g, 10.0 mmol) in THF (10 mL) was added at 0 °C. Upon complete addition, the reaction mixture was heated to reflux for 2 hours and quenched with water. The mixture was extracted with ethyl acetate (2 x 100 mL). The organic layer was separated, dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford product, which was purified by silica gel chromatography (petroleum ether/ethyl acetate, v/v, 90:10) (750 mg, Yield 36 %).1H NMR (300 MHz, CDCl3) δ 7.01 (s, 1H), 4.34 (q, J = 7.2 Hz, 4H), 3.73 (s, 2H), 2.76 (s, 2H), 1.47 (s, 9H), 1.35 (t, J
= 7.2 Hz, 3H).

General Procedure D: Synthesis of compounds 50, 58, 66, 70, and 73. To a solution of ester (1.0 mmol) in ethanol or methanol (10 mL), aq. 5 N NaOH (2.0 mmol) was added. The reaction mixture was refluxed for 2 hours before the solvent was removed on a rotavapor. The remaining solution was added water, acidified with

hydrochloric acid to pH 5 and extracted with ethyl acetate (2 x 100 mL). The organic
layer was separated, dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford product.
5-(tert-butoxycarbonyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylic acid (50a). The title compound was prepared from 49a following general procedure D. Yellow solid (86%). 1H NMR (300 MHz, DMSO-d6) δ 12.97 (s, 1H), 7.52 (s, 1H), 4.42 (s, 2H), 3.62 (t, J = 5.6 Hz, 2H), 2.81 (t, J = 5.6 Hz, 2H), 1.42 (s, 9H).
5-(tert-butoxycarbonyl)-4,5,6,7-tetrahydrofuro[3,2-c]pyridine-2-carboxylic acid (50b). The title compound was prepared from 49b following general procedure D. Yellow solid (90%). 1H NMR (300 MHz, CDCl3) δ 6.98 (s, 1H), 4.29 (s, 2H), 3.68 (s,
2H), 2.69 (s, 2H), 1.46 (s, 9H).
tert-butyl

2-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-6,7-dihydrothie no[3,2-c]pyridine-5(4H)-carboxylate (52a). The title compound was prepared from 50a with 51 following general procedure B. Yellow solid (70%). 1H NMR (300 MHz, CD3OD) δ 7.21 (s, 1H), 7.14 (m, 3H), 7.05 (d, J = 8.0 Hz, 1H), 4.36 (s, 2H), 4.18 – 4.04
(m, 1H), 3.87 (s, 2H), 3.69 (m, 2H), 3.45 (d, J = 5.9 Hz, 2H), 2.98 (m, 4H), 2.80 (m, 4H), 1.49 (s, 9H).
tert-butyl

2-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-6,7-dihydrofuro [3,2-c]pyridine-5(4H)-carboxylate (52b). The title compound was prepared from 50b with 51 following general procedure B. Yellow solid (50%). 1H NMR (300 MHz, CD3OD) δ 7.15 – 7.05 (m, 3H), 7.01 (d, J = 7.9 Hz, 1H), 6.91 (s, 1H), 4.32 (s, 2H), 4.10

– 4.01 (m, 1H), 3.73 (s, 2H), 3.69 (t, J = 5.8 Hz, 2H), 3.47 (dd, J = 6.0, 2.4 Hz, 2H), 2.91

(m, 2H), 2.88 – 2.81 (m, 2H), 2.71 – 2.62 (m, 2H), 2.57 (t, J = 5.7 Hz, 2H), 1.48 (s, 9H).

di-tert-butyl

2-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-6,7-dihydro-1H

-pyrrolo[3,2-c]pyridine-1,5(4H)-dicarboxylate (52c). The title compound was prepared from 50c with 51 following general procedure B. Yellow solid (52%). 1H NMR (300 MHz, CD3OD) δ 7.26 – 7.14 (m, 3H), 7.10 (d, J = 5.7 Hz, 1H), 6.37 (s, 1H),
4.28 (s, 2H), 4.23 – 4.14 (m, 1H), 4.11 (s, 2H), 3.66 (m, 2H), 3.44 (d, J = 4.5 Hz, 2H),

3.24 (t, J = 5.9 Hz, 2H), 3.00 (m, 4H), 2.83 (s, 2H), 1.57 (s, 9H), 1.49 (d, J = 7.4 Hz,

9H).

General Procedure E: Synthesis of compounds 53, 60, 63 and 64. To a solution of 52, 59, 62 or 49b (1.0 mmol) in dichloromethane (5 mL) was added trifluoroacetate (10.0 mmol) or HCl in dioxane (4 M, 10 mmol) at room temperature and stirred for 4 hours. The reaction mixture was neutralized with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (2 x 100 mL). The organic layer was separated, dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford crude product, which was purified by silica gel chromatography
(dichloromethane / methanol, v/v, 93:7 to 90:10).
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4,5,6,7-tetrahydrothieno[ 3,2-c]pyridine-2-carboxamide(53a). The title compound was prepared from 52a following general procedure E. Yellow solid (90%). 1H NMR (300 MHz, CD3OD) δ 7.16 (s, 1H), 7.11 (s, 3H), 7.03 (s, 1H), 4.14 – 4.00 (m, 1H), 3.72 (s, 4H), 3.44 (d, J = 5.8
Hz, 2H), 3.06 (d, J = 5.5 Hz, 2H), 2.89 (s, 2H), 2.82 (d, J = 5.6 Hz, 4H), 2.72 – 2.58 (m,

2H).

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4,5,6,7-tetrahydrofuro[3, 2-c]pyridine-2-carboxamide(53b). The title compound was prepared from 52b following general procedure E. Yellow solid (78%). 1H NMR (300 MHz, CD3OD) δ 7.10 (m, 3H), 7.03 (d, J = 5.2 Hz, 1H), 6.90 (s, 1H), 4.06 (p, J = 5.9 Hz, 1H), 3.74 (d, J
= 3.6 Hz, 4H), 3.46 (d, J = 6.0 Hz, 2H), 3.11 (t, J = 5.9 Hz, 2H), 2.92 (s, 2H), 2.90 – 2.82 (m, 2H), 2.73 – 2.64 (m, 2H), 2.61 (t, J = 5.9 Hz, 2H).
N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4,5,6,7-tetrahydro-1H-py rrolo[3,2-c]pyridine-2-carboxamide (53c). The title compound was prepared from
52c following general procedure E. The residue was used for next step with purity.
Synthesis of isoquinoline-6-carbonitrile (54). 6-bromoisoquinoline (5.0 g, 24.0 mmol) was dissolved in N, N-dimethylformamide (25 mL) and zinc cyanide (1.69 g,
14.4 mmol) was added. The suspension was degassed under argon bubbling for 10 min before Pd(PPh3)4 (1.39 g, 1.2 mmol) was added. The reaction mixture was heated to 90 °C for 10 hours and then diluted with ethyl acetate. The solution was washed brine for three times, dried over anhydrous sodium sulfate, and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate, v/v, 75:25) to give the title product as white solid (2.79 g, 84%). 1H NMR (300 MHz, CDCl3) δ 9.36 (s, 1H), 8.69 (d, J = 5.7 Hz, 1H), 8.24 (s, 1H), 8.10 (d, J = 8.6 Hz, 1H), 7.74 (dd, J = 12.6, 7.1 Hz, 2H).
Synthesis of methyl isoquinoline-6-carboxylate (55). To a solution of 54 (2.76 g,

1.0 mmol) in ethanol (10 mL), aq. 5 N KOH (2.0 mmol) was added. The reaction mixture was refluxed for 12 hours before the solvent was removed on a rotavapor.

The residue was dissolved in methanol (100 mL) and was added H2SO4 (2 mL). The solution was refluxed for 4 hours and concentrated in vacuum. The mixture was added water, neutralized with saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (3 x 100 mL). The organic layer was separated, dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford product (2.62 g, 81%). 1H NMR (300 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.67 (s, 1H), 8.62 (d, J = 5.7 Hz,
1H), 8.25 (d, J = 8.5 Hz, 1H), 8.16 – 8.10 (m, 1H), 8.05 (d, J = 5.7 Hz, 1H), 3.94 (s, 3H).

General Procedure F: Synthesis of compounds 56 and 68. Compound 55 or 67 (3.0 mmol) was dissolved in methanol (10 mL), PtO2 (0.03 mmol) and acetic acid (9.0 mmol) was added. The mixture was stirred at room temperature under hydrogen atmosphere for 6 hours. The mixture was filtered, and the filtrate was concentrated to dryness, dissolved in dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel chromatography (dichloromethane / methanol, v/v, 90:10) to give the title product.
methyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate (56). The title compound was prepared from 55 following general procedure F. Gray solid (93%). 1H NMR (300 MHz, DMSO-d6) δ 7.68 (d, J = 4.7 Hz, 2H), 7.18 – 7.11 (m, 1H), 3.87 (s, 2H), 3.82 (s, 3H), 2.93 (t, J = 5.8 Hz, 2H), 2.73 (t, J = 5.8 Hz, 2H).
Synthesis of 2-tert-butyl 6-methyl 3,4-dihydroisoquinoline-2,6(1H)-dicarboxylate (57). Compound 56 (1.0 mmol) was dissolved in dichloromethane (5 mL), (Boc)2O (1.2 mmol) and Et3N (1.5 mmol) was added. The mixture was stirred at room

temperature for 6 hours. The mixture was added water and extracted with dichloromethane. The organic layer was separated, dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel chromatography (dichloromethane
/ methanol, v/v, 90:10) to give the title product as white solid (90%). 1H NMR (300 MHz, CD3OD) δ 7.82 (d, J = 6.1 Hz, 2H), 7.24 (d, J = 8.6 Hz, 1H), 4.61 (s, 2H), 3.89 (s,
3H), 3.66 (t, J = 5.7 Hz, 2H), 2.88 (t, J = 5.9 Hz, 2H), 1.53 (s, 9H).

2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (58). The title compound was prepared from 57 following general procedure D. White solid (83%). 1H NMR (300 MHz, DMSO-d6) δ 12.88 (s, 1H), 7.73 (d, J = 6.9 Hz, 2H), 7.29
(d, J = 8.6 Hz, 1H), 4.55 (s, 2H), 3.56 (t, J = 5.9 Hz, 2H), 2.83 (t, J = 5.8 Hz, 2H), 1.43 (s, 9H).
tert-butyl

6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-3,4-dihydroisoq uinoline-2(1H)-carboxylate (59). The title compound was prepared from 58 with 51 following general procedure B. Yellow solid (73%). 1H NMR (300 MHz, CD3OD) δ 7.66 (d, J = 6.8 Hz, 2H), 7.29–7.11 (m, 5H), 4.58 (s, 2H), 4.34 (s, 2H), 4.29 (m, 1H),
3.63 (t, J = 5.6 Hz, 2H), 3.55–3.41 (m, 4H), 3.24 (m, 1H), 3.14 (m, 3H), 2.84 (t, J = 5.8 Hz, 2H), 1.49 (s, 9H).
(R)-tert-butyl

6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-3,4-dihydroisoq uinoline-2(1H)-carboxylate ((R)-59). The title compound was prepared from 58 with (R)-51 following general procedure B. Yellow solid (77%). 1H NMR (300 MHz,

CD3OD) δ 7.66 (d, J = 6.8 Hz, 2H), 7.29–7.11 (m, 5H), 4.58 (s, 2H), 4.34 (s, 2H), 4.29

(m, 1H), 3.63 (t, J = 5.6 Hz, 2H), 3.55–3.41 (m, 4H), 3.24 (m, 1H), 3.14 (m, 3H), 2.84 (t,

J = 5.8 Hz, 2H), 1.49 (s, 9H).

(S)-tert-butyl

6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-3,4-dihydroisoq uinoline-2(1H)-carboxylate ((S)-59). The title compound was prepared from 58 with (S)-51 following general procedure B. Yellow solid (66%). 1H NMR (300 MHz, CD3OD) δ 7.66 (d, J = 6.8 Hz, 2H), 7.29–7.11 (m, 5H), 4.58 (s, 2H), 4.34 (s, 2H), 4.29
(m, 1H), 3.63 (t, J = 5.6 Hz, 2H), 3.55–3.41 (m, 4H), 3.24 (m, 1H), 3.14 (m, 3H), 2.84 (t,

J = 5.8 Hz, 2H), 1.49 (s, 9H).

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4-tetrahydroisoq uinoline-6-carboxamide (60). The title compound was prepared from 59 following general procedure D. Yellow solid (88%). 1H NMR (300 MHz, CD3OD) δ 7.52 (d, J = 5.1 Hz, 2H), 7.13 – 6.98 (m, 5H), 4.09 (dt, J = 11.8, 5.9 Hz, 1H), 3.95 (s, 2H), 3.73 (s,
2H), 3.52 – 3.44 (m, 2H), 3.05 (t, J = 6.0 Hz, 2H), 2.85 (m, 4H), 2.75 (dd, J = 11.8, 5.9 Hz, 2H), 2.69 – 2.58 (m, 2H).
(R)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4-tetrahydroi soquinoline-6-carboxamide ((R)-60). The title compound was prepared from (R)-59 following general procedure D. Yellow solid (92%). 1H NMR (300 MHz, CD3OD) δ 7.52 (d, J = 5.1 Hz, 2H), 7.13 – 6.98 (m, 5H), 4.09 (dt, J = 11.8, 5.9 Hz, 1H), 3.95 (s,
2H), 3.73 (s, 2H), 3.52 – 3.44 (m, 2H), 3.05 (t, J = 6.0 Hz, 2H), 2.85 (m, 4H), 2.75 (dd,

J = 11.8, 5.9 Hz, 2H), 2.69 – 2.58 (m, 2H).

(S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4-tetrahydroi soquinoline-6-carboxamide ((S)-60). The title compound was prepared from (S)-59 following general procedure D. Yellow solid (89%). 1H NMR (300 MHz, CD3OD) δ 7.52 (d, J = 5.1 Hz, 2H), 7.13 – 6.98 (m, 5H), 4.09 (dt, J = 11.8, 5.9 Hz, 1H), 3.95 (s,
2H), 3.73 (s, 2H), 3.52 – 3.44 (m, 2H), 3.05 (t, J = 6.0 Hz, 2H), 2.85 (m, 4H), 2.75 (dd,

J = 11.8, 5.9 Hz, 2H), 2.69 – 2.58 (m, 2H).

tert-butyl

7-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)-3,4-dihydroisoq uinoline-2(1H)-carboxylate (62). The title compound was prepared from 61 with 51 following general procedure B. White solid (70%). 1H NMR (300 MHz, CD3OD) δ 7.57 (d, J = 6.7 Hz, 2H), 7.16 (d, J = 8.6 Hz, 1H), 7.09 (m, 3H), 7.00 (d, J = 5.8 Hz, 1H),
4.52 (s, 2H), 4.09 (m, 1H), 3.73 (s, 2H), 3.62 (t, J = 5.9 Hz, 2H), 3.57 – 3.40 (m, 2H),

2.85 (m, 6H), 2.74 – 2.57 (m, 2H), 1.49 (s, 9H).

N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1,2,3,4-tetrahydroisoq uinoline-6-carboxamide (63). The title compound was prepared from 62 following general procedure D. Yellow solid (90%). 1H NMR (300 MHz, CD3OD) δ 7.54 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 7.11 (m, 4H), 7.03 (m, 1H), 4.15 – 4.04 (m, 1H), 3.89 (s, 2H),
3.74 (s, 2H), 3.49 (d, J = 5.1 Hz, 2H), 3.08 (t, J = 6.0 Hz, 2H), 2.96 – 2.78 (m, 6H), 2.67 (t, J = 5.8 Hz, 2H).
ethyl 4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate (64). The title compound was prepared from 49b following general procedure D. Yellow solid (93%). 1H NMR (300 MHz, DMSO) δ 7.58 (s, 1H), 4.25 (q, J = 7.1 Hz, 2H), 4.03 (s, 2H), 3.24

(d, J = 5.7 Hz, 2H), 2.97 (s, 2H), 1.89 (s, 1H), 1.26 (t, J = 7.1 Hz, 3H).
Synthesis of ethyl 5-phenyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate (65a). Compounds 64 (180 mg, 0.85 mmol) and iodobenzene (348 mg, 1.70 mmol) were dissolved in dry toluene and cesium carbonate (555 mg, 1.70 mmol) was added. The suspension was degassed under argon bubbling for 10 min before Pd(dppf)2Cl2 (40 mg, 0.01 mmol) was added. The reaction mixture was heated to 90 °C for 5 hours and concentrated. The residue was purified by silica gel chromatography (dichloromethane / methanol, v/v, 90:10) to give the title product as white solid (100 mg, 41%).1H NMR (300 MHz, CDCl3) δ 7.55 (s, 1H), 7.33 – 7.21 (m, 2H), 6.99 (d, J = 8.3 Hz, 2H), 6.88 (t, J = 7.3 Hz,
1H), 4.34 (t, 7.2 Hz, 2H), 4.29 (s, 2H), 3.62 (t, J = 5.6 Hz, 2H), 3.00 (t, J = 5.6Hz, 2H),

1.37 (t, J = 7.2 Hz, 3H).

Synthesis of 2-ethyl 5-phenyl 6,7-dihydrothieno[3,2-c]pyridine-2,5(4H)-dicarboxylate (65b). To a mixture of 64 (1.0 mmol) and Et3N (2.5 mmol) in dry dichloromethane (6 mL) was added phenyl carbonochloridate (1.2 mmol) and the mixture was stirred for 4 hours at room temperature. Dichloromethane and water were added. The organic layer was separated and washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuum. The resulting residue was purified by silica gel chromatography (dichloromethane/ methanol, v/v, 90:10) to give 65b as white solid (80%). 1H NMR (300 MHz, DMSO-d6) δ 7.66 (s, 1H), 7.39 (t, J = 7.3 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H),
7.16 (d, J = 7.5 Hz, 2H), 4.74 (s, 1H), 4.56 (s, 1H), 4.27 (q, J = 6.8 Hz, 2H), 3.89 (s, 1H),

3.76 (s, 1H), 3.00 (s, 2H), 1.28 (t, J = 7.0 Hz, 3H).

5-phenyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylic acid (66a). The title compound was prepared from 65a following general procedure D. The residue was used for next step without purity.
5-(phenoxycarbonyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylic acid (66b). The title compound was prepared from 65b following general procedure D. White solid (85%). 1H NMR (300 MHz, CD3OD) δ 7.53 (s, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 7.13 (d, J = 7.9 Hz, 2H), 4.77 (s, 1H), 4.60 (s, 1H), 3.98 (s, 1H),
3.85 (s, 1H), 2.96 (s, 2H).

ethyl 3-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate (68). The title compound was prepared from 67 following general procedure F. Yellow solid (40%). 1H NMR (300 MHz, CD3OD) δ 4.31 (q, J = 7.2 Hz, 2H), 4.23 (s, 2H), 3.55 (t, J = 6.1 Hz,
2H), 3.16 (t, J = 6.0 Hz, 2H), 2.43 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H).

ethyl

5-(4-(dimethylamino)benzoyl)-3-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-car boxylate (69). The title compound was prepared from 68 and 4-(dimethylamino)benzoic acid following general procedure B. White solid (76%). 1H NMR (300 MHz, CD3OD) δ 7.39 (d, J = 8.9 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 4.59 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 3.88 (s, 2H), 3.02 (s, 6H), 2.95 (t, J = 5.5 Hz, 2H), 2.37 (s,
3H), 1.34 (t, J = 7.1 Hz, 3H).

5-(4-(dimethylamino)benzoyl)-3-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine- 2-carboxylic acid (70). The title compound was prepared from 69 following general procedure D. White solid (87%). 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.35

(d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H), 4.49 (s, 2H), 3.74 (m, 2H), 2.96 (s, 6H), 2.89 (m, 2H), 2.32 (s, 3H).
methyl 2-(4-(dimethylamino)benzoyl)isoindoline-5-carboxylate (72). The title compound was prepared from 71 and 4-(dimethylamino)benzoic acid following general procedure B. White solid (70%). 1H NMR (300 MHz, CD3OD) δ 7.97 (s, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.49 – 7.33 (m, 1H), 6.79 (d, J = 8.9 Hz, 2H), 4.99 (s, 4H), 3.89 (s, 3H), 3.01 (d, J = 9.9 Hz, 6H).
2-(4-(dimethylamino)benzoyl)isoindoline-5-carboxylic acid (73). The title compound was prepared from 72 following general procedure D. White solid (80%). 1H NMR (300 MHz, DMSO-d6) δ 7.87 (d, J = 7.4 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H),
7.49 – 7.31 (m, 1H), 6.73 (d, J = 8.8 Hz, 2H), 4.92 (s, 4H), 2.97 (s, 6H).

4.2. PRMT5 enzymatic activity inhibition assays

Radiometric-based scintillation proximity assay (SPA) was used to detect the PRMT5 inhibition activities of synthesized compounds. The PRMT5-MEP50 complex (purchased from BPS, with Cat. No. 51045) was used as enzyme, and the H4 peptide was used as substrate. [3H]-SAM (PerkinElmer, Lot. No. 2146246), SAM (Sigma, Cat. No. A7007-100MG), SAH (Sigma, Cat. No. A9384-25MG), and 384-well Flashplate (Perkin Elmer, Cat. No. SMP410A001PK) were purchased. Final concentrations of SAM, substrates, and enzyme were 0.25 µM, 0.1 µM, and 2.0 nM, respectively. See Supporting Information for details.
4.3. Enzymatic selectivity test assays

The methods to test the inhibition activity of compound 46 against PRMT1,

PRMT3, PRMT4, PRMT6, PRMT7, and PRMT8 were the same as that we have previously published26, see Supporting Information for details.
4.4. 8, 12-day Proliferation Assay

The methods were the same as that we have previously reported [26], see Supporting Information for details.
4.5. Western Blot Assays

After 4 days treatment, cells that were incubated with different concentrations of compounds were lysed in 100 µL of total lysis buffer. Total cell lysates were collected and boiled for 10 min in 2 × SDS sample buffer and subsequently subjected to 4–12% SDS−PAGE, then were transferred onto PVDF membrane (Millipore) and blocked in 5% nonfat dry milk in 0.1% Tween 20 PBS buffer for 1 h. Blots were incubated with primary antibodies in blocking buffer overnight at 4°C. After three times washes with TBST at room temperature, HRP-conjugated secondary antibodies (Millipore) were added for 1 h and then the membranes were washed three times with TBST for 15 min. The signal was read on membranes in Amersham Imager 600 after ECL substrate (Millipore) reagent added.
4.6. Pharmacokinetic study in mice

See Supporting Information for details.

4.7. 30-d efficacy xenograft studies

All animal studies related to handling, care and treatment were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Institute of Materia Medica. For the in vivo efficacy

xenograft studies, MV4-11 cells (5 x 106 cells/mouse) were subcutaneously inoculated at the right flanks of Balb/c nude mouse in 100 µL of a mixture of PBS and Matrigel (PBS:Matrigel, 1:1) for tumor growth. After 10 days of inoculation, the mean tumor size reached 150 mm3 and the compound treatments were initiated. Mice were grouped using a randomized block design (7 mice per group). Vehicle (0.5% methylcellulose in water) and compounds were administered orally once daily at a dose volume of 10mL/kg for 30 days. Tumor volumes were determined by measuring in two dimensions, length (L) and perpendicular width (W), with a caliper, and expressed in cubic millimeters using the formula: 0.5 x L x W2. Body weights were measured every day for 15 days, then twice weekly for the last 15 days. Results were analyzed and graphed using the software of GraphPad Prism 5.0. The percentage of tumor growth inhibition value (TGI) is calculated at the end of the efficacy study according to:
∆tumor volumetreated/∆tumor volumecontrol)*100

Where the ∆tumor volumes represent the mean tumor volume at the last day minus the mean tumor volumes at the first day of the treatment.
4.8. Molecular docking

The docking simulation was performed using Schrödinger software running under Maestro version 7.5. Glide program embedded in Maestro 7.5 was used to carry out molecular docking. During the docking process, the coordinates of the protein (PRMT5:MEP50 and H4 peptide) were first minimized using the Protein Preparation Wizard Workflow with default settings, and docking grids were then created by

defining residues within 15 Å around EPZ015666. Finally, the designed compounds that were prepared by the LigPrep panel (version 2.3, Schrödinger, LLC, New York, NY) were docked into the well-defined docking grids with the extra precision (XP) mode.
ABBREVIATIONS USED

PRMT, protein arginine methyltransferases; MBD2, Methyl-CpG-binding domain protein 2; PDCD4, Programmed cell death protein 4; EGFR, epidermal growth factor receptor; FGF-2, fibroblast growth factor; SREBF1, Sterol regulatory element-binding transcription factor 1; MCL, Mantle cell lymphoma; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; DCM, dichloromethane; X-PHOS, 2-dicyclohexylphosphorus-2,4,6-triisopropylbiphenyl; Pd2(dba)3, Tris(dibenzylideneacetone)dipalladium.
Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENTS

We gratefully acknowledge financial support from the National Science and Technology Major Project (2018ZX09711002-004-013), National Natural Science Foundation of China (81625022, 21472208, and 81803438) and the Shandong Provincial Natural Science Foundation (No. ZR2017BH038).
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 A potent and selective PRMT5 inhibitor 46 (IC50 = 8.5 nM) was identified.

 The structure-activity relationship (SAR) of this class of structures was discussed.

 Compound 46 displayed pronounced antitumor activity in MV4-11 mouse xenografts model.GSK3326595