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Design, Synthesis and Pharmacological Evaluation of 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-aryl Propanamides as Novel Smoothened (Smo) Antagonists Gang Liu, Jun Yang, Juan Wang, Xiaohua Liu, Wenjing Huang, Jie Li, Wenfu Tan, and Ao Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01247 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

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Journal of Medicinal Chemistry

Design, Synthesis and Pharmacological Evaluation of 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-aryl Propanamides as Novel Smoothened (Smo) Antagonists Gang Liu,†,ǁ,§ Jun Yang,‡,§ Juan Wang,‡,§ Xiaohua Liu,†,ǁ Wenjing Huang‡, Jie Li,ǁ Wenfu Tan,*,‡ and Ao Zhang*,†,ǁ

†

CAS Key Laboratory of Receptor Research, and Synthetic Organic & Medicinal

Chemistry Laboratory (SOMCL), Shanghai Institute of Materia Medica (SIMM), University of Chinese Academy of Sciences, Shanghai 201203, China ‡

Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai

201203, China ǁ

School of Life Science and Technology, ShanghaiTech University, Shanghai 201210,

China §

These authors contributed equally to this work.

*

To whom correspondence should be addressed. For W.T.: phone: +86-21-51980039;

fax: 86-21-51980039; E-mail: [email protected]. For

A.Z.:

phone:

+86-21-50806035;

fax:

86-21-50806035;

[email protected].

ACS Paragon Plus Environment

E-mail:


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Abstract. A series of novel Smo antagonists were developed either by directly incorporating the basic skeleton of the natural product artemisinin or by first breaking artemisinin into structurally simpler and stable intermediates and then reconstructing into diversified heterocyclic derivatives, equipped with a Smo-targeting bullet. 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-arylpropanamide

65

was

identified as the most potent with an IC50 value of 9.53 nM against the Hh signaling pathway. Complementary mechanism studies confirmed that 65 inhibits Hh signaling pathway by targeting Smo and shares the same binding site as that of the tool drug cyclopamine. Meanwhile, 65 has a good plasma exposure and an acceptable oral bioavailability.

Dose-dependent

antiproliferative

effects

were

observed

in

ptch+/-;p53-/- medulloblastoma cells, and significant tumor growth inhibitions were achieved for 65 in the ptch+/-;p53-/- medulloblastoma allograft model.


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INTRODUCTION The hedgehog (Hh) signaling pathway plays a pivotal role in the embryogenesis and tissue maintenance by controlling cell proliferation and differentiation.1 In adults, the Hh pathway is significantly downregulated, but reactivation usually occurs during tissue repair.2 The Hh ligands (Sonic, Desert, and Indian), the transmembrane receptor Patched (Ptch), the signal transducer Smoothened (Smo), and the transcription factors Gli1-3 are the major components that regulate the transcription of Hh target genes.3-5 Ptch is a 12-pass transmembrane receptor that negatively regulates the seven-pass transmembrane receptor Smo. Upon Ptch binding to the Hh ligand, the repression of Smo by Ptch is disarmed, and Smo is consequently released to initiate a downstream signaling cascade leading to the activation of transcription factors of the Gli family. It has been reported that aberrant Hh pathway activation is involved in tumorigenesis of various cancers, especially basal cell carcinoma (BCC) and medulloblastoma (MB) as the most commonly identified Hh-dependent tumors.6-10 Aberrant activation of the Hh pathway can result from abnormal up-regulation of the Hh ligands, loss of Ptch, Smo mutations, and gene amplification/chromosomal translocation of Gli1 or Gli2.7,9,11 Consequently, inhibition of Hh pathway signaling has become an attractive anticancer chemotherapeutic strategy.3,5,7,12,13 Since the natural steroidal alkaloid cyclopamine was identified as the first Smo antagonist to block Hh signaling by directly binding to Smo,14 a number of Smo-targeting small molecules have been developed in recent years.12,13,15,16 Benzamide 117-19 (GDC-0449, vismodegib) from Genentech is the first-in-class Smo antagonist that was approved by the FDA in 2012 for the treatment of locally advanced or metastatic BCC. Three years later, sonidegib20,21 (NVP-LDE225, 2), a structurally similar analogue of 1 from Novartis, was also approved by FDA in 2015



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as the second clinically prescribed Smo antagonist. In spite of the significant achievement in the development of Smo-targeted antagonists or other Hh signaling pathway inhibitors, the clinical application of 1 and 2 is strictly limited to adult BCC patients, due to many side effects including diarrhoea, constipation, decreased appetite, hair loss, muscle spasms, and tiredness.7,16,19 Such side effects can be severe in children, especially in the skeletal system. Meanwhile, development of drug resistance and patient relapse have also been reported after the clinical use of 1 for three months, possibly due to Smo mutations.7,22-24 Therefore, new Hh signaling pathway inhibitors, especially new Smo antagonists with structures distinct from benzamides 1 and 2, are highly needed.

The natural product artemisinin (3) is the active component of the sweet wormwood plant Artemisia annua L.25 Its unique 1,2,4-trioxane structural feature and significant antimalarial activity have opened a new paradigm for antimalarial drug discovery, having resulted in a number of artemisinin analogues (artemalogs) such as dihydroartemisinin, artemether, arteether and artesunate that have been clinically used


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for decades as the antimalarial treatment.25,26 To fully take advantage of the unusual endoperoxide and multiple cyclic structure of 3, an increasing number of artemalogs have been explored as treatment for many other diseases, especially cancer.27 Among which, benzyl ether 428 and dimer 529 were reported to be among the most potent showing two-digit nM potency against mouse lymphocytic leukemia L1210 and human erythroleukemic K-562 cell lines, respectively (Figure 1). Unfortunately, such high cellular antitumor potency failed to translate into tumor growth inhibition in vivo.27 For example, dimer 5, one of the most potent artemalogs in vitro, only shows moderate tumor growth inhibition in the HL-60 human leukemia xenografts.27,29 In view of the reported disappointing outcomes in the development of artemalogs as cytotoxic anticancer agents, we recently proposed to develop molecularly targeted anticancer therapies derived from 3 by combining the typical skeleton of 3 or its novel fragmented intermediates with a molecularly targeted drug bullet.30,31 Among several approaches we tried, we were delighted to find that incorporation of a Smo antagonistic structural motif into the structure of 3 led to highly potent Smo-targeting antagonists. As shown in Figure 2, two strategies were used in our design, including 1) direct merging the basic skeleton of 3 with 4-chloro-3-(pyridin-2-yl)aniline - the well-established Smo-targeting bullet17,20 as in 1 and 2; 2) first breaking endoperoxide 3 into stable key intermediates and then reconstructing with the Smo-targeting bullet (Figure 2). Compounds from strategy one are direct analogues of 3 (series I and II), whereas compounds from strategy two belong to derivatives of 3 lacking the basic skeleton (endoperoxide and polyoxycycle).



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RESULTS AND DISCUSSION Chemistry. The synthesis of artemalogs 6 and 7 was shown in Scheme 1. Starting from commercial substrate 3, acid 832 and ring-contracted acid 1033 were prepared via several steps according to literature procedures. Condensation of 8 with 4-chloro-3-(pyridin-2-yl)aniline

in

the

presence

of

1-[bis(dimethylamino)

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAT) delivered the hybrid product 6 in 62% yield. Reduction of 6 by Zn/HOAc34 led to the deoxygenated derivative 9 in 54% yield. Synthesis of the ring-contracted analog 7 was accomplished by following a similar reaction condition as that for preparation of 6 in 56% yield.


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Scheme 1. Synthesis of Artemisinin Analogues 6, 7 and 9a H

H ref 43 3 (artemisinin)

O O O

a

O O O

H

O

H

O

N

H N

ref 44

COOH

O

8

H

Cl

6 b

O O O

H

H

O 10

H COOH

O

O O O

O H

O

a

H N

N

H N

O 7

H

O

N

O 9

Cl

Cl

a

Reagents and conditions: (a) 4-chloro-3-(pyridin-2-yl)aniline, HATU, HOAT, DIPEA, CH2Cl2, rt, 4 h, 56-62%; (b) Zn, HOAc, rt, 12 h, 54%.

Acid degradation of 3 was conducted by treating with concentrated sulfuric acid (con. H2SO4) in methanol to afford keto-ester 1135 in 64% yield, which was then converted to the decalenone acid 1235 by treating with Ba(OH)2 in 77% yield (Scheme 2). The target compound 13 was obtained by condensation of 12 with 4-chloro-3-(pyridin-2-yl)aniline

in

the

presence

of

1-ethyl-3-(3-dimethyl

aminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in 34% yield. Hydrogenation36 of 12 with Pd/C under H2 afforded cis-decalone

acid

14

in

91%

yield,

which

was

then

coupled

with

4-chloro-3-(pyridin-2-yl)aniline, leading to product 15 in 56% yield. Subsequent reduction36 of 15 in the presence of NaBH4 provided decalin amide 18 as a pair of inseparable diastereoisomers in 76% yield (9:1). Bromination of 15 with bromine in the presence of acetic acid (HOAc) led to the key intermediate 16 in 94%. Without further isolation, treating 16 under microwave condition at 160 oC resulted in the



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isomerized decalenone derivative 17 in 53% yield. Thiazole derivatives 19 and 20 were synthesized by heterocyclization37 of 16 with thiourea or thioacetamide in EtOH in 71% and 37% yield, respectively. Meanwhile, treatment of 20 with acetyl chloride in dichloromethane gave acetamidothiazole 21 in 92% yield.


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As outlined in Scheme 3, compound 22 bearing a totally new pyridine-fused bicyclic framework was prepared38 by treating 11 with NH4OAc and Cu(OAc)2 in 71% yield. Hydrolysis of the intermediate 22 followed by amidation with 4-chloro-3-(pyridin-2-yl)aniline under the standard condensation conditions (EDCI, DMAP, CH2Cl2) provided pyridine-fused compound 24 in 43% overall yield. Meanwhile, quinolinyl aldehyde 25 was obtained in 51% yield by treating 22 with SeO2,39 which was further converted to acid 26 through Pinnick oxidation in 79% yield.40 Subsequent condensation of 26 with 4-chloro-3-(pyridin-2-yl)aniline was



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found quite sluggish and compound 27 was obtained in 27% yield. In addition, indole 28 was obtained in 73% yield by treating 14 with phenylhydrazine hydrochloride. N-Methylation of 28 with CH3I provided 29, which was further hydrolyzed and amidated to afford the indole-fused compound 30 in 25% overall yield (three steps). Compound 31 was prepared by methyl esterification of 14 with MeI in 91% yield. Claisen condensation41 of 31 with ethyl formate followed by treating with hydrazine hydrochloride produced 32 in 47% overall yield. The final compound 33 was prepared from 32 in 30% overall yield in a similar manner as that for 30. Scheme 4. Synthesis of Compounds 38a,ba

22

a

b AcO 68%

N+ O-

81%

c,d 64%

N

COOMe

Cl

N

COOMe 35

34

36

COOMe

O O e

N

N

92%

37a

38a CF3 O g

H N O

N

Cl

O

N

H N

61%

CF3

O

COOH 23

N

COOMe

+ H2N

N

N

f,g 36%

37b

38b

a

Reagents and conditions: (a) m-CPBA, CH2Cl2, 1 h; (b) (Ac)2O, reflux, 5 h; (c) LiOH.H2O, EtOH-H2O (1:1), rt, 2 h; (d) SOCl2, CH2Cl2, 30 min, rt; (e) morpholine, MeCN, K2CO3, rt, 3 h; (f) LiOH.H2O, EtOH-H2O (1:1), 50 oC, 12 h; (g) EDCI, DMAP, CH2Cl2, 1 h, then 4-chloro3-(pyridin-2-yl)aniline, rt, 12 h.

Substituted pyridine-fused derivative 38a were synthesized as described in Scheme 4. Oxidation of methylpyridine 22 with 3-chloroperbenzoic acid (m-CPBA) gave pyridine N-oxide 34 in 81% yield. Subsequent acetylation of 34 in refluxing


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acetic anhydride provided the 2-acetoxymethyl intermediate 35 in 68% yield. Hydrolysis of 35 followed by chlorination with SOCl2 and subsequent amination with morpholine delivered the corresponding products 37a in 58% overall yield. Amide 38a was obtained in 45% yield through hydrolysis of 37a and subsequent condensation with 4-chloro-3-(pyridin-2-yl)aniline. Meanwhile, N-biphenyl product 38b was obtained similarly by condensation of acid 23 and aniline 37b in 61% yield.



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As shown in Scheme 5, modification or replacement of the pyridine moiety in 24 by a wide variety of heteroaromatic or aliphatic rings was conducted. First, treatment of 39 with N-bromosuccinimide (NBS) at -20 oC followed by Suzuki coupling with 2-chloro-5-nitrophenyl boronic acid and reduction of the resulting intermediate by Fe powder in ethanol furnished the required aniline 41 in 16% overall yield (three steps). Two 1,2,4-oxadiazoles 42 and 44 were prepared42 from 2-chloro-5-nitrobenzoic acid and 2-chloro-5-nitrobenzonitrile respectively by following literature procedures, which were in turn reduced with Fe powder to yield precursors 43 and 45 in 82% and 84% yields, respectively. Syntheses of 1,3,4-oxadiazole 49a and l,3,4-thiadiazole 49b were accomplished43 over four steps starting from commercial available methyl 1-methyl-1H-pyrazole-5-carboxylate (46), which was reacted with hydrazine first, followed by treating with 2-chloro-5-nitrobenzoic acid to yield 47 in 50% overall yield. Subsequent cyclization of 47 mediated by POCl3,43 followed by reduction with Fe-powder afforded the 1,3,4-oxadiazole compound 49a in 73% overall yield. The 1,3,4-thiadiazole 49b was synthesized in 40% overall yield by cyclization of the intermediate 47 in the presence of Lawesson’s reagent36 in THF and subsequent reduction with Fe powder in ethanol. The final products 50-54 were obtained in 41-53% yields by condensation of acid 23 with corresponding anilines under aforementioned standard conditions. Substituted benzoimidazole derivatives 62-69 were synthesized as described in Scheme 6. Intermediates 55 and 56 were obtained over two steps from 2-chloro-5-nitrobenzoic acid following literature procedures.44 Conversion of 2-chloro-5-nitrobenzoic acid to the corresponding acid chloride and subsequent reaction with substituted 1,2-diaminobenzene followed by cyclization of the resulting intermediate in reflux acetic acid provided 58a-d in moderate yields.45 Reduction of


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58a with Fe powder in ethanol resulted in the corresponding aniline 59 in 82% yield. Treatment of 58a-d with MeI provided the corresponding methylated products 60a-e in 65-81% yields. It should be noted that 60b, 60c and 60d were generated as a pair of 5- and 6-isomers in about 1:1 ratio, which were inseparable by silica gel column.

Subsequent reduction of 60a-e by Fe-powder generated the key building blocks 61a-d in 82-93% yields. The final products 62-69 were obtained in 41-53% yields by coupling of the appropriate aniline with the key intermediate 23 under the standard condensation conditions. The isomeric 68 and 69 were successfully separated by chromatography and corresponding absolute configuration was deduced by comparison of their nuclear overhauser effect (NOE) correlation analysis (see Supporting Information).



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Structure-Activity Relationship (SAR) Study. All new compounds were evaluated for their ability to inhibit the Hh signaling pathway by dual luciferase reporter assays using light II cells, which were NIH-3T3 cells stably transfected with a Gli-responsive firefly luciferase reporter and Renilla-luciferase expression vector.46 As shown in Table 1, we first evaluated artemisinin analogues 6 and 7 based on our strategy one (series I and II, Figure 2), and found that both compounds showed good inhibitory potency against the Hh signaling pathway with IC50 values of 241 nM and 180 nM, respectively, which were only 3- to 6-fold less potent than the clinically approved drug 1 (39 nM). Compared to 6, the ring-contracted derivatives 7 was slightly more potent. Interestingly, the deoxygenated compound 9 showing slightly improved potency, compared to the endoperoxide 6 (184 nM vs 241 nM). These results indicated that neither the polyoxycyclic nor the endoperoxide component is necessary for these compounds to block the Hh signaling pathway. As mentioned earlier, artemisinin framework is generally attributed to the poor PK and cytotoxicity of many reported direct artemisinin analogues, therefore, the rest of our SAR was directed to simpler derivatives lacking the polyoxycyclic and endoperoxide motifs through

major

structural

operations

of

3,

but

containing

the

4-chloro-3-(pyridin-2-yl)aniline motif - the Smo-targeting bullet, based on our strategy two (series III and IV, Figure 2).

Table 1. Inhibition of compounds to the Hh pathway activity tested by dual luciferase reporter assays in light II cells.a

Gli-luc reporter, Compound

Gli-luc reporter, Compound

IC50 (nM)


IC50 (nM)

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241 ± 14.1 6

180.0 ± 10.4 7

184 ± 39.3

39.2 ± 2.16 1

9 a

IC50 values are shown as the mean ±

SD (nM) from three separate experiments.

Since 3 is highly sensitive to both acidic and basic conditions,35 we treated 3 with H2SO4 followed by Ba(OH)2, leading to a stable bicyclic acid 12. With this structurally stable and non-peroxide acid 12 as the key intermediate, compounds 13, 15, 17 and 18 were obtained and evaluated. Unfortunately, only compound 15 containing a naphthalen-2(1H)-one motif retained moderate potency with an IC50 value of 290 nM (Table 2). Enones 13 and 17 as well as the alcoholic 18 were inactive (> 1 µM) in the Gli-luc reporter assay. Further functionalization of ketone 15 led to a small series of heterocyclic derivatives, amongst which thiazole 21 was inactive, but pyridine 24, 1-phenyl-pyrazole 33, N-methylindole 30, as well as quinoline 27 retained moderate potency with IC50 values ranging between 212–476 nM. Since pyridine 24 showed the highest potency (212 nM) and its synthesis only needs four steps from the ample commercial source of 3, it served as our lead for further structure optimization.



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Gli-luc reporter, X=

Gli-luc reporter, X=

IC50 (nM)

IC50 (nM)

>1000

>1000

13

21

290 ± 80.3 15

212 ± 110 24

>1000 17

327 ± 33.6 33

>1000 18

476 ± 56.3 30

>1000

270 ± 23.4 27


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19 a

IC50 values are shown as the mean (nM) from three separate experiments.

As shown in Table 3, incorporating a morpholinyl moiety to compound 24 lead to 38a, which showed slightly improved potency with an IC50 value of 155 nM. Replacement the Smo-targeting bullet - 4-chloro-3-(pyridin-2-yl)aniline with a biphenyl bioisostere led to compound 38b, which only showed modest inhibitory potency. Meanwhile, optimization of the pyridinyl component within the 4-chloro-3-(pyridin-2-yl)aniline motif was conducted and a series of heterocyclic bioisosteres17 were introduced (50-55 and 62-64). Quite disappointingly, introducing a 2,6-dimethylmorpholinyl (50) on the pyridinyl moiety abolished the inhibitory activity. Significant difference was observed by replacing the pyridinyl with 3-cyclopropyl 1,2,4-oxadiazole (51) and 5-methyl 1,2,4-oxadiazole (52), of which only the former compound retained good potency whereas the latter one was inactive (220 nM vs 1 µM). Moderate potency was observed on compounds 53 and 54 containing

the

bioisosetric

5-(1-methyl-1H-pyrazol-5-yl)-1,3,4-oxadiazole

and

5-(1-methyl-1H-pyrazol-5-yl)-1,3,4-thiadiazole, respectively. However, oxadiazole 53 was 2-fold less potent than thiadiazole 54 (588 nM vs 265 nM). Compounds 62 and 63 containing benzo[d]oxazole and benzo[d]thiazole, respectively, retained similar potency (243 nM vs 282 nM). To our delight, compound 64 containing the benzo[d]imidazole component showed much improved potency with an IC50 value of 46 nM. It was nearly 5-fold more potent than pyridine 24 (212 nM) and was nearly as potent as reference compound 1 (39 nM).

Table 3. Inhibition of compounds to the Hh pathway activity tested by dual



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Page 18 of 72

luciferase reporter assays in light II cells.a

Gli-luc reporter, Structure IC50 (nM)

155 ± 43.2

38a

38b

O

N

H N

CF3

~1000

O

Gli-luc reporter,

Gli-luc reporter, Y=

Y= IC50 (nM)

IC50 (nM)

>1000

265 ± 48.0 54

50

220 ± 20.0 51

243 ± 39.1 62

>1000 52

282 ± 48.1 63

588 ± 62.4


45.9 ± 7.79

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64

53 a

The IC50 values are shown as the mean ± SD (nM) from two separate experiments.

Since compound 64 showed much improved potency, we finely tuned this compound by introducing a small substituent on the benzo[d]imidazole moiety (Table 4). To our delight, the N-methyl substituted benzo[d]imidazole 65 displayed a high potency of 9.53 nM that is 4- to 5-fold more potent than both the non-substituted benzo[d]imidazole 65 and the reference compound 1. Further introduction of a 5- or 6-methoxy (66) on the benzo[d]imidazole network led to reduced potency (335 nM), however introducing a 5- or 6-fluoro substituent afforded compound 67 retaining high potency of 63 nM. Meanwhile, the 5- or 6-trifluotomethoxy substituted analogues 68 and 69 also retained good potency with IC50 values of 84 nM and 53 nM, respectively.




IC50 (nM)

IC50 (nM)

45.9 ± 7.79

63.2 ± 31.1

64

67 9.53 ± 1.19

84.5 ± 21.2 68



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65

335 ± 91.1 69

66 a

52.6 ± 27.3


Cytotoxicity and hERG Inhibition of Potent Compounds. Based on the SAR above, several compounds were identified showing significant inhibitory potency against the Hh signaling pathway with IC50 values higher than or compatible to the reference compound 1. To exclude the chemical structure related untargeted cell toxicity and cardiac toxicity, compounds 6, 24, 38a, and 65 from different structure series were selected for evaluation of their inhibitory effects against proliferation of squamous carcinoma KB cells and against hERG channel. As shown in Table 5, all these compounds showed no significant antiproliferative effects against the KB cell (IC50 > 5 µM), indicating that off-target related toxicity may not be an issue of concerns for current compounds. However, compound 38a displayed an alarming hERG blocking effect (3.3 µM) indicating its cardiac toxicity potential, otherwise the other compounds (6, 24, 65) showed IC50 values greater than 15 µM against the hERG channel. Since compound 65 showed highest potency against the Hh signaling pathway and no significant toxicity to the cell growth and hERG channel, it was selected for further profiling.

Table 5. Cytotoxicity and hERG Inhibition of Potent Compounds

Compound

IC50, nM or µM Gli-luc reporter

squamous carcinoma


hERG

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KB cells

a

6

241 ± 14.1 nM

6.61 µM

> 15.0 µM

24

212 ±110 nM

15.0 µM

> 15.0 µM

38a

155 ± 43.2 nM

>20 µM

3.31 µM

65

9.53 ± 1.19 nM

>20 µM

> 15.0 µM


Compound 65 Inhibited the mRNA Expression of Gli Target Gene Gli1. To exclude that the high potency of compound 65 observed in the dual luciferase reporter assay was due to inhibition of the luciferase enzyme itself, we evaluated the effect of 65 on the mRNA expression of Gli1, a transcriptional target gene of Gli that was frequently used as readout of the Hh signaling pathway activity.47 As shown in Figure 3, the expression of Gli1 mRNA in light II cells was robustly elevated in response to Shh conditioned medium (Shh CM), whereas treatment with compounds 65 (0.1 µM) led to significantly suppression on the mRNA expression of Gli1, equal to that of compound 1. Therefore, these data further confirm that compound 65 is a potent inhibitor against the Hh signaling pathway.



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Figure 3. Compound 65 inhibited the mRNA expression of Gli target gene Gli1. Light II cells were subjected to various treatments as indicated for 36 h, and then were collected for RT-qPCR analysis of the Gli1 mRNA expression. The results were expressed as the mean ± SD.

Compound 65 Had no Inhibitory Effect on Transcriptional Factor Activity Provoked by Tumor Necrosis Factor-α α (TNF-α) and Prostaglandin E2 (PGE2), respectively. To exclude the possibility that compound 65 may non-specifically inhibit the Hh pathway activity, we further tested the effect of compound 65 on two other unrelated transcriptional factors activity, such as NF-κB in response to TNF-α, and T-cell factor (TCF)/lymphoid enhancer factor (LEF) activity stimulated by PGE2.48 As shown in Figure 4, compound 65 had no effects on either the TCF/LEF activity in response to PGE2 (Figure 4A), or the NF-κB activity provoked by TNF-α (Figure 4B); however, significant inhibitions were observed from their respective inhibitors,

(E)-3-tosylacrylonitrile

(BAY11-7082,

(N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide


NF-κB)

and

dihydrochloride

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salt (H89, TCF/LEF), which served as positive controls (Figure 4A,B). Hence, these data suggest that compound 65 possessed selectivity on suppressing the Hh pathway activity.

Figure 4. Compound 65 had no effect on inhibiting transcriptional factors activity provoked by TNF-α and PGE2, respectively. A. The HEK293 cells after various treatments as indicated for 24 h were collected for dual luciferase reporter analysis of the NF-κB activity. B. The LS174T cells after various treatments as indicated for 6 h were collected for dual luciferase reporter analysis of TCF/LEF activity. The results were expressed as the mean ± SD.

Compound 65 Inhibited the Hh Signaling Pathway by Targeting Smo. To test whether the inhibitory effect of 65 on the Hh signaling pathway was due to targeting Smo, a central regulator of the Hh signaling pathway,49,50 we then conducted several control experiments as shown in Figure 5. First, we artificially expressed the Smo wild type plasmid to activate the Gli-luciferase activity in light II cells. Compound 65 was found to dose-dependently abolish the Gli-luciferase activity provoked by artificially forced expression of Smo wild type plasmid, similar to compound 1 (Figure 5A), suggesting that 65 inhibited the Hh signaling pathway activity by



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targeting Smo or molecules downstream of Smo. SmoW539L and SmoD473H were two Smo mutants which may cause resistance to the clinically approved Smo antagonists (e.g. 1 and 2).7,23,24 When compared to the obvious inhibitory effect on the Gli-luciferase activity stimulated by Smo wild type plasmids (Figure 5A), compound 65 had minimal inhibitory effect on the Gli-luciferase activity stimulated by ectopic expression of either SmoW539L or SmoD473H (Figure 5B, 5C), indicating that compound 65 was insensitive to SmoW539L and SmoD473H. Collectively, the capacity of 65 to inhibit the Gli-luciferase activity provoked by ectopic expression of Smo but not by ectopic expression of SmoW539L and Smo D473H suggested that compound 65 functioned by binding to Smo as an antagonist.51,52


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Figure 5. Distinct responses of compound 65 to the Gli-luciferase activity stimulated by Smo (wild type), SmoW539L, and SmoD473H plasmids, respectively. A, compound 65 dose-dependently inhibited the Gli luciferase activity initiated by ectopic expression of wild type Smo plasmid. B and C, compound 65 exhibited minimal inhibitory effect on Gli luciferase activity provoked by ectopic expression of SmoW539L or SmoD473H mutants. Light II cells transfected with Smo, SmoW539L or SmoD473H plasmids were exposed to compound 1 or 65 for 36 h, and then were harvested for dual luciferase reporter assays. The results were expressed as the mean ± SD.

Compound 65 Shares the Same Binding Site on Smo with Cyclopamine. Having characterized Smo as the cellular target of compound 65, we further mapped its binding site on Smo with complementary methods. Given the fact that the vast majority of currently reported Smo antagonists or agonists were identified to function by binding the common site in the transmembrane domain of Smo as that of the tool drug cyclopamine47,49 — the first reported archetypical Smo antagonist,53 we set out to determine whether compound 65 binds Smo in a similar mode. As shown in Figure 6, the Smo biding site of 65 was tested by using BODIPY-cyclopamine, a fluorescent derivative of cyclopamine.46 It was found that compound 65 decreased the binding of



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BODIPY-cyclopamine to Smo, analogous to the action of cyclopamine (Figure 6A). However, itraconazole,54 another Smo atagonist with binding site dictinct from that of cyclopamine, had no effect on the binding of BODIPY-cyclopamine to Smo (Figure 6A). These results were further recapitulated by FACS analysis (Figure 6B), thus suggesting that compound 65 acted on the common binding site similar to that of cyclopamine. To further confirm this argument, we tested the influence of 65 on the stimulation of the Hh pathway activity by a specific Smo agonistic tool drug 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]-be nzo[b]thiophene-2-carboxamide (SAG), whose binding site on Smo overlaps with that of cyclopamine.55 As shown in Figure 6C, the SAG-induced Gli luciferase activity was counteracted by increasing concentrations of 65, accompanied with enhancement of SAG EC50 values (Figure 6D), thus suggesting that compound 65 acted as a competitive inhibitor of SAG. In conclusion, these data clearly suggested that compound 65 is a Smo antagonist and shares a same binding mode as that of cyclopamine.


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Figure 6. Compound 65 shares the same binding site on Smo with cyclopamine. A. BODIPY-cyclopamine competition analysis tested by fluorescent microscope. Photographs were representatives from three distinct experiments with identical results; B. Cells after various treatments as indicated were subjected to BODIPY-cyclopamine binding analysis by FACS. C-D: Compound 65 is a competitive inhibitor of Smo agonist SAG. Light II cells were exposed to SAG with or without increasing concentrations of compound 65 for 36 h, and Gli-luciferase activity of light II cells were measured by dual luciferase reporter assays. C. Compound 65 counteracted the Gli-luciferase activity provoked by SAG; D. SAG EC50 values obtained by increasing concentrations of compound 65.



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Figure 7. Compound 65 suppressed proliferation of ptch+/-;p53-/- medulloblastoma cells. A. Compound 65 inhibited the proliferation of ptch+/-;p53-/- medulloblastoma cells. The results were expressed as the mean ± SD. B. Compound 65 inhibited the expression of Gli1 at mRNA level in ptch+/-;p53-/- medulloblastoma cells. The results were expressed as the mean ± SD. C. Compound 65 treatment caused apoptosis of ptch+/-;p53-/- medulloblastoma cells.

Compound 65 Inhibited the Proliferation of Medulloblastoma Cells Isolated from ptch+/-;p53-/- Mice. Medulloblastoma spontaneously aroused in ptch+/-;p53-/-


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mice has been widely used to evaluate the anticancer effect of Smo antagonists, as the growth of medulloblastoma from these mice is addicted to the constitutive Hh activity caused by loss of one allele of ptch.56,57 In this regard, medulloblastoma cells from nude mice allografted with a primary medulloblastoma from a ptch+/-;p53-/-mouse were isolated for testing the antiproliferative effect of compound 65. As revealed by MTS analysis in Figure 7A, compound 65 dose-dependently inhibited the proliferation of ptch+/-;p53-/-medulloblastoma cells in a fashion similar to that of compound 1 (Figure 7A). Considering that Hh signaling pathway may transcriptionally up-regulate the anti-apoptotic gene Bcl-2 to prevent the cellular apoptosis,58 we then tested whether the inhibitory effect of compound 65 on the proliferation of ptch+/-;p53-/- medulloblastoma cells was caused by inducing apoptosis. Using annexin V-fluorescein isothiocyanate (FITC) and propidiumiodide (PI) double staining meathod,59 we observed that exposure of compound 65 for 24 h obviously caused apoptosis of ptch+/-;p53-/- medulloblastoma cells, with the apoptotic rate of 26.3% compared to 0.9% of the control cohort. Meanwhile, no cytotoxic effect of compound 65 was observed, as revealed by no cells were stained by PI only (Figure 7C). Pharmacokinetic (PK) Study of Compound 65. Since the most potent compound 65 displayed favorable profiles in vitro as a novel Smo antagonist, we further investigated its pharmacokinetic properties in SD rats. After intravenous (i.v.) injection of 1 mg/kg of the new inhibitor 65, low systemic plasma clearance (CL = 9.31 mL/min/kg) and a good volume of distribution (Vss = 2229 mL/kg) were obtained. The mean residence time (MRT) was 3.97 h. The oral exposure of compound 65 at the dose of 3.0 mg/kg was 1836 h•ng/mL, resulting in an acceptable estimated bioavailability of 27.4% (Table 7). The overall PK parameters of compound



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Page 30 of 72

65 encouraged its further evaluation in vivo.

Table 7. Pharmacokinetics parameters of 65 after p.o. and i.v. administrationa-c T1/2

Tmax

Cmax

AUClast

AUCINF_obs

CL_obs

MRTINF_obs

Vss_obs

(h)

(h)

(ng/mL)

(h*ng/mL)

(h*ng/mL)

(mL/min/kg)

(h)

(mL/kg)

p.o.

1.40

0.25

799

1464

1477

--

1.53

--

i.v.

5.95

-

-

1783

1836

9.31

3.97

2229

F(%)

27.4%

a

Values are the average of three runs. Vehicle: DMSO, Tween 80, normal saline. CL, clearance; Vss,

volume of distribution; T1/2, half-life; Cmax, maximum concentration; Tmax, time of maximum concentration; AUC0-∞, area under the plasma concentration time curve; F, oral bioavailability. bDose: p.o. at 3.0 mg/kg; cDose: i.v. at 1.0 mg/kg

Compound 65 Significantly Inhibited the Tumor Growth in the ptch+/-;p53-/Medulloblastoma Allograft Mice Model. To test whether the high in vitro potency of the new Smo inhibitor 65 can be translated into in vivo antitumor efficacy, we next examined the tumor growth inhibition (TGI) of 65 on nude mice subcutaneously engrafted with a primary medulloblastoma from a ptch+/-;p53-/- mouse.46,60 Compound 65 was administered via intraperitoneal injection (i.p.) at a dose of 25 or 50 mg/kg twice a day for 13 consecutive days. Compared to the vehicle control, compound 65 obviously inhibited the growth of ptch+/-;p53-/- mededulloblastoma at either dose (Figure 8A), with TGI of 81.7%, and 91.5%, respectively, and no significant body weight loss was observed at both doses (Figure 8B). Meanwhile, we observed that the growth inhibition of medulloblastoma by compound 65 paralleled the repression of Hh pathway activity, as reflected by reductions of Gli1 expression at the mRNA level in respective cohorts (Figure 8C).


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Figure 8. Antitumor activity of compound 65 in ptch+/-;p53-/- medulloblastoma allograft model. Mice bearing in medulloblastoma were administered compound 1 or 65 twice a day for 13 days. A. Compound 65 inhibited the growth of ptch+/-;p53-/medulloblastoma. The results were expressed as the mean ± SEM. B. Administration of compound 65 had no influence on the body weight of mice. The results were expressed as the mean ± SD. C. Administration of compound 65 suppressed the Gli1 expression at mRNA level of medulloblastoma. After 4 h of last dosage, tumor samples were collected for examining the expression of Gli1 mRNA. The results were expressed as the mean ± SD.

CONCLUSIONS In summary, we have developed a series of novel Smo antagonists either by directly incorporating the basic skeleton of the natural product artemisinin containing



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the typical polyoxycycle and endoperoxide elements or by first breaking artemisinin into structurally simpler and stable intermediates and then reconstructing into diversified heterocyclic derivatives, equipped with a Smo-targeting bullet. The inhibitory activity of these compounds against the Hh signaling pathway was determined by dual luciferase reporter assays that guided further structural optimization. 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-arylpropanamide 65 bearing

artemisinin-derived

propanoyl

motif

and

the

2-(2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)

Smo-targeting

bullet

- 4-chloro-3-(1-methyl-1H-

benzo[d]imidazol-2-yl)aniline moiety showed the highest inhibitory potency with an IC50 value of 9.53 nM, which was 4-fold more potent than the clinically prescribed Smo antagonist 1. Further mechanism studies confirmed that compound 65 is a Smo antagonist and shares the same binding site on Smo as that of the tool drug cyclopamine. In ptch+/-;p53-/- medulloblastoma cells and allograft model, compound 65 dose-dependently inhibited the cell proliferation and showed significant tumor growth inhibition. Although compound 65 is somewhat less efficacious in vivo than 1 and failed to serve as a next generation Smo antagonist to suppress the SmoW539L and Smo D473H mutants, it represents a new strategy to develop new Smo antagonists absent of the traditional benzamide profile, and more importantly provides an alternative practice to turn natural products (e.g. artemisinin) from non-targeted to targeted anticancer therapies.


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EXPERIMENTAL SECTION Chemistry. All reactions were performed in glassware containing a Tefloncoated stir bar. Solvents and chemical reagents were obtained from commercial sources and used without further purifications. 1H NMR spectral data were recorded in CDCl3 or CDCl3 + CD3OD on Varian Mercury 300 or 400 NMR spectrometer and

13

C NMR

was recorded in CDCl3 or CDCl3 + CD3OD on Varian Mercury 400 or 500 NMR spectrometer. Chemical shifts (δ) are reported in ppm downfield from an internal TMS standard. Low and high-resolution mass spectra were obtained in the ESI mode. Flash column chromatography on silica gel (200-300 mesh) was used for the routine purification of reaction products. The column output was monitored by TLC on silica gel (200-300 mesh) precoated on glass plates (15 x 50 mm), and spots were visualized by UV light at 254 or 365 nM. NOE were used in the structural assignment. Compounds 843, 1044, 1239, 1446, 4253, 4453, 5555, 5655, 58a56 and 5956 were prepared according to corresponding literature procedures. HPLC analysis was conducted for all bioassayed compounds on an Agilent Technologies 1260 series LC system (Agilent ChemStation Rev.A.10.02; ZORBAX-C18, 4.6 mm × 150 mm, 5 µM, MeOH (0.1% DEA)/H2O, rt) with two ultraviolet wavelengths (uv 254 and 214 nM). All the assayed compounds displayed a chemical purity of 95%-99% in both wavelengths. General Procedure for Synthesis of Compounds 6 and 7. To a solution of the acid intermediate

(8

and

10)

(0.2

mmol)

in

CH2Cl2

(3

mL)

was

added

4-chloro-3-(pyridin-2-yl)aniline (0.2 mmol), HATU (0.3 mmol), HOAt (0.2 mol) and DIPEA (0.9 mmol). The mixture was stirred at rt for 4 h, and then diluted with water



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(8 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined organic phase was dried over Na2SO4 and concentrated. The residue was subjected to column chromatography on silica gel (CH2Cl2/EtOAc 3:1) to give the corresponding target compound 6 or 7. 10-(4-Chloro-3-(pyridin-2-yl)phenylamino) derivative 6. White solid (64 mg, 62%). 1

H NMR (300 MHz, CDCl3) δ 9.24 (s, 1H), 8.70 (d, J = 4.8 Hz, 1H), 7.86 – 7.56 (m,

4H), 7.38 (d, J = 8.7 Hz, 1H), 7.30 – 7.26 (m, 1H), 5.47 (s, 1H), 5.03 – 4.97 (m, 1H), 2.70 – 2.24 (m, 4H), 2.06 – 1.98 (m, 2H), 1.86 – 1.77 (m, 2H), 1.71 – 1.67 (m, 1H), 1.39 – 1.15 (m, 7H), 0.99 – 0.89 m, 7H).

13

C NMR (126 MHz, CDCl3) δ 170.09,

156.68, 149.25, 139.16, 137.53, 136.08, 130.30, 126.41, 124.87, 122.55, 122.49, 121.01, 102.98, 90.78, 80.82, 68.83, 51.62, 43.15, 38.75, 37.57, 36.54, 34.18, 30.63, 25.74, 24.87, 19.96, 11.66. MS (ESI, [M + H]＋) m/z 513.2. HRMS (ESI) calcd for C28H34ClN2O5, 513.2151; found, 513.2161. 9-(4-Chloro-3-(pyridin-2-yl)phenylamino) ring-contracted derivative 7. White solid (54 mg, 56%). 1H NMR (300 MHz, CDCl3) δ 8.94 (s, 1H), 8.72 (d, J = 4.8 Hz, 1H), 7.80 – 7.67 (m, 4H), 7.43 (d, J = 8.7 Hz, 1H), 7.32 – 7.28 (m, 1H), 5.81 (s, 1H), 2.41 – 2.30 (m, 2H), 2.16 – 1.99 (m, 3H), 1.85 (s, 3H), 1.58 – 1.45 (m, 5H), 1.41 – 0.96 (m, 7H). 13C NMR (126 MHz, CDCl3) δ 172.37, 156.98, 150.26, 140.27, 136.95, 136.61, 131.38, 127.92, 125.57, 123.29, 123.19, 121.44, 104.80, 98.33, 89.47, 87.36, 52.24, 48.96, 37.69, 37.58, 32.96, 26.71, 26.53, 25.91, 25.15, 20.49. MS (ESI, [M + H]＋) m/z 485.1. HRMS (ESI) calcd for C26H30ClN2O5, 485.1838; found, 485.1834. Synthesis of 10-(4-chloro-3-(pyridin-2-yl)phenylamino) deoxyartemisinin derivative 9. To a solution of 6 (51 mg, 0.1 mmol) in HOAc (2 mL) was added Zn powder (131 mg, 2 mmol). The mixture was stirred at rt for 12 h, and then filtered over a pad of celite. The residue was slowly treated with saturated NaHCO3 until pH >8 and extracted with CH2Cl2 (3 × 5 mL). The organic phase was dried over Na2SO4, filtered,


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concentrated under reduced pressure and purified by silica gel chromatography (CH2Cl2/EtOAc 3:1) to give 9 as a white solid (27 mg, 54%). 1H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 8.70 (d, J = 4.8 Hz, 1H), 7.87 (d, J = 2.7 Hz, 1H), 7.78 – 7.73 (m, 1H), 7.64 – 7.70 (m, 2H), 7.38 (d, J = 8.7 Hz, 1H), 7.30 – 7.26 (m, 1H), 5.38 (s, 1H), 4.69 – 4.63 (m, 1H), 2.63 – 2.41 (m, 2H), 2.35 – 2.22 (m, 1H), 2.06 – 1.56 (m, 6H), 1.50 (s, 3H), 1.30 – 1.17 (m, 5H), 1.00 – 0.88 (m, 6H).

13

C NMR (126 MHz,

CDCl3) δ 169.49, 156.25, 148.86, 138.86, 137.04, 135.37, 129.84, 126.05, 124.32, 122.11, 121.92, 120.48, 107.53, 96.75, 82.19, 64.80, 44.72, 39.16, 38.54, 35.13, 33.92, 33.87, 29.21, 24.85, 23.07, 21.68, 18.25, 11.22. MS (ESI, [M + H]＋) m/z 497.2. HRMS (ESI) calcd for C28H34ClN2O4, 497.2202; found, 497.2214. General Procedure for Synthesis of Compounds 13 and 15. To a solution of the acid 12 or 14 (0.2 mmol) in CH2Cl2 (2 mL) was added EDCI (0.4 mmol) and DMAP (0.02

mmol).

The

mixture

was

stirred

at

rt

for

1

h,

and

then

4-chloro-3-(pyridin-2-yl)aniline (41 mg, 0.2 mmol) was added. The reaction was stirred at rt for 12 h and then diluted with water (10 mL). The mixture was extracted with CH2Cl2 (3 × 5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/EtOAc 4:1) to give corresponding target compound 13 or 15. (R)-N-(4-Chloro-3-(pyridin-2-yl)phenyl)-2-((1S,4R,4aS)-4-methyl-7-oxo-1,2,3,4,4a, 5,6,7-octahydronaphthalen-1-yl)propanamide (13). White solid (29 mg, 34%). 1H NMR (300 MHz, CDCl3) δ 9.39 (s, 1H), 8.66 (d, J = 4.8 Hz, 1H), 7.88 – 7.76 (m, 3H), 7.67 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.33 – 7.29 (m, 1H), 5.56 (s, 1H), 2.54 – 2.26 (m, 4H), 2.09 – 1.98 (m, 3H), 1.88 – 1.83 (m, 1H), 1.75 – 1.60 (m, 2H), 1.32 – 1.26 (m, 1H), 1.16 (d, J = 6.3 Hz, 3H), 1.07 – 0.94 (m, 4H).

13

C NMR (126

MHz, CDCl3) δ 200.81, 175.13, 171.41, 157.23, 149.85, 139.71, 138.27, 137.07,



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Page 36 of 72

131.22, 127.31, 125.85, 123.52, 123.27, 121.88, 121.16, 50.06, 46.96, 43.23, 39.29, ＋

35.62, 35.39, 34.27, 24.64, 20.90, 17.30. MS (ESI, [M + H] ) m/z 423.2. HRMS (ESI) calcd for C25H28ClN2O2, 423.1834; found, 423.1834. (R)-N-(4-Chloro-3-(pyridin-2-yl)phenyl)-2-((1R,4R,4aS,8aS)-4-methyl-7-oxodecahy dronaphthalen-1-yl)propanamide (15). White solid (48 mg, 56%). 1H NMR (300 MHz, CDCl3) δ 8.97 (m, 1H), 8.65 (d, J = 4.8 Hz, 1H), 7.98 (dd, J = 8.7, 2.7 Hz, 1H), 7.83 – 7.78 (m, 1H), 7.72 – 7.68 (m, 2H), 7.40 (d, J = 8.7 Hz, 1H), 7.35 – 7.31 (m, 1H), 2.37 – 2.10 (m, 5H), 1.93 – 1.58 (m, 6H), 1.43 – 1.19 (m, 3H), 1.05 – 0.98 (m, 4H), 0.92 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 212.52, 175.27, 156.41, 149.18, 138.84, 137.67, 136.40, 130.61, 126.40, 125.33, 122.92, 122.37, 121.21, 44.24, 43.21, 42.45, 38.42, 37.54, 36.98, 35.02, 27.81, 27.15, 25.48, 19.51, 15.13. MS (ESI, [M + H]＋) m/z 425.3. HRMS (ESI) calcd for C25H30ClN2O2, 425.1990; found, 425.1995. Synthesis

of

(2R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((1R,4R,4aS,8aS)-7-

hydroxy-4-methyldecahydronaphthalen-1-yl)propanamide (18). To a solution of 15 (42 mg, 0.1 mmol) in EtOH (2 mL) was added NaBH4 (8 mg, 0.2 mmol), The reaction mixture was stirred for 1 h at 0 °C. The mixture was quenched with water (10 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/MeOH 20:1) to give 18 as white solid (32 mg, 76%). 1H NMR (300 MHz, CDCl3/CD3OD) δ 8.51 (d, J = 2.4 Hz, 1H), 7.76 – 7.68 m, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.46 and 7.38 (s, 1H), 7.30 – 7.20 (m, 2H), 4.01 – 3.96 and 3.51 – 3.40 (m, 1H), 2.17 – 2.07 (m, 1H), 1.83 – 1.01 (m, 16H), 0.92 – 0.83 (m, 1H), 0.70 (d, J = 5.7 Hz, 3H).

13

C NMR (126 MHz,

CDCl3/CD3OD) δ 176.35 and 176.27 (1C), 156.17 and 156.07 (1C), 148.89, 138.37,


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137.59 and 137.50 (1C), 136.53, 130.53, 126.40, 125.46, 122.83, 122.66 and 122.56 (1C), 121.76 and 121.67 (1C), 71.59, 44.38 and 44.33 (1C), 43.55, 42.83, 35.97, ＋

35.59, 30.12, 29.82, 27.34, 26.33, 26.25, 19.52, 15.20. MS (ESI, [M + H] ) m/z 427.2. HRMS (ESI) calcd for C25H32ClN2O2, 427.2147; found, 427.2147. Synthesis

of

(R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((1R,4R,4aR,8aR)-4-

methyl-7-oxo-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl)propanamide (17). 15 (170 mg, 0.4 mmol) was dissolved in HOAc (5 mL), and then Br2 (23 µL, 0.44 mmol) in HOAc (1 mL) was added dropwise to the reaction. The mixture was stirred for 6 h at 50 °C and then slowly treated with saturated NaHCO3 until pH >8 and extracted with CH2Cl2 (2 × 10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to afford compound 16 as white solid. The crude product 16 was used without further purification. A microwave tube was charged with 16 (50 mg, 0.1 mmol) and DMF (2 mL) and heated to 160 °C under microwave for 10 min. The reaction mixture was diluted with water (20 mL) and washed with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/EtOAc 4:1) to give compound 17 as white solid (25 mg, 53%). 1H NMR (300 MHz, CDCl3) δ 8.79 (s, 1H), 8.68 (d, J = 5.1 Hz, 1H), 7.91 (dd, J = 8.7, 2.7 Hz, 1H), 7.84 – 7.78 (m, 1H), 7.73 – 7.70 (m, 2H), 7.40 (d, J = 8.7 Hz, 1H), 7.35 – 7.31 (m, 1H), 7.26 – 7.21 (m, 1H), 6.01 (d, J = 10.2 Hz, 1H), 2.45 – 2.23 (m, 2H), 2.08 – 1.83 (m, 4H), 1.67 – 1.48 (m, 3H), 1.33 – 1.26 (m, 1H), 1.14 – 1.04 (m, 4H), 0.99 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 199.94, 175.04, 156.48, 156.09, 149.24, 138.96, 137.55, 136.39, 130.62, 128.83, 126.57, 125.26, 122.87, 122.48, 121.17, 45.09, 44.15, 42.55, 35.05,



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＋

34.58, 34.06, 33.48, 25.57, 19.92, 15.26. MS (ESI, [M + H] ) m/z 423.3. HRMS (ESI) calcd for C25H28ClN2O2, 423.1834; found, 423.1845. Synthesis

of

(R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((4aS,5R,8R,8aS)-2,8-

dimethyl-4,4a,5,6,7,8,8a,9-octahydronaphtho[2,3-d]thiazol-5-yl)propanamide

(19).

To a solution of 16 (50 mg, 0.1 mmol) in EtOH (3 mL) was added thioacetamide (15 mg, 0.2 mmol). The reaction mixture was stirred under reflux overnight and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/MeOH 40:1) to give compound 19 as white solid (34 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 4.8 Hz, 1H), 8.04 (s, 1H), 7.80 – 7.65 (m, 4H), 7.41 (d, J = 8.7 Hz, 1H), 7.32 – 7.28 (m, 1H), 2.94 (d, J = 16.8 Hz, 1H), 2.74 – 2.44 (m, 6H), 2.31 – 2.25 (m, 1H), 2.10 – 1.94 (m, 3H), 1.66 – 1.57 (m, 2H), 1.32 – 1.27 (m, 2H), 1.17 (d, J = 6.6 Hz, 3H), 1.10 – 1.02 (m, 1H), 0.83 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.23, 163.05, 156.49, 149.42, 147.75, 139.29, 136.14, 130.60, 126.87, 126.85, 124.98, 122.70, 121.21, 45.26, 43.82 , 43.62, 35.32, 34.14, 27.79, 25.92, 25.53, 22.85, 19.97, 19.25, 15.47. MS (ESI, [M + H]＋) m/z 480.3. HRMS (ESI) calcd for C27H31ClN3OS, 480.1871; found, 480.1884. Synthesis

of

(R)-2-((4aS,5R,8R,8aS)-2-acetamido-8-methyl-4,4a,5,6,7,8,8a,9-

octahydronaphtho[2,3-d]thiazol-5-yl)-N-(4-chloro-3-(pyridin-2-yl)phenyl)propanami de (21). To a solution of 16 (50 mg, 0.1 mmol) in EtOH (3 mL) was added thiourea (15 mg, 0.2 mmol). The reaction mixture was stirred under reflux overnight and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/MeOH 10:1) to give compound 20 as white solid. The crude product 20 was used without further purification. To a solution of 20 (24 mg, 0.05 mmol) in CH2Cl2 (1 mL) at 0 °C was added acetyl chloride (7 µL, 0.1 mmol). The reaction mixture was stirred for 30 min at 0 °C,


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and diluted with water (10 mL). The mixture was extracted with CH2Cl2 (3 × 5 mL) and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/MeOH 20:1) to give compound 21 as white solid (24 mg, 92%). 1H NMR (300 MHz, CDCl3) δ 10.33 (s, 1H), 8.68 (d, J = 4.8 Hz, 1H), 8.17 (s, 1H), 7.79 – 7.75 (m, 3H), 7.66 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 9.3 Hz, 1H), 7.32 – 7.27 (m, 1H), 2.90 (d, J = 16.8 Hz, 1H), 2.72 – 2.66 (m, 1H), 2.48 – 2.21 (m, 6H), 2.06 – 1.92 (m, 3H), 1.64 – 1.53 (m, 2H), 1.27 – 1.01 (m, 6H), 0.83 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.22, 167.65, 156.42, 155.98, 149.38, 141.94, 139.25, 137.18, 136.16, 130.63, 126.86, 125.05, 122.71, 122.65, 121.51, 121.17, 45.15, 43.63, 35.28, 34.23, 27.82, 25.84, 25.03, 23.26, 22.58, 19.95, 15.55. MS (ESI, [M + H]＋) m/z 523.3. HRMS (ESI) calcd for C28H32ClN4O2S, 523.1929; found, 523.1935. Synthesis of (R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((5R,8S)-2,5-dimethyl5,6,7,8-tetrahydroquinolin-8-yl)propanamide (24). To a solution of 11 (282 mg, 1.0 mmol) in EtOH (3 mL) was added Cu(OAc)2 (499 mg, 2.5 mmol) and NH4OAc (231 mg, 3.0 mmol). The reaction mixture was stirred for 4 h at rt and then filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (petroleum ether/EtOAc 10:1) to give compound 22 as colorless oil (175 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 7.39 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 3.62 (s, 3H), 3.45 – 3.38 (m, 1H), 3.15 – 3.06 (m, 1H), 2.85 – 2.78 (m, 1H), 2.42 (s, 3H), 2.05 – 1.94 (m, 2H), 1.80 – 1.67 (m, 1H), 1.49 – 1.36 (m, 1H), 1.25 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 7.2 Hz, 3H).



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To a solution of compound 22 (117 mg, 0.5 mmol) in EtOH (3 mL) and H2O (3 mL) was added lithium hydroxide monohydrate (42 mg, 1.0 mmol). The reaction was stirred for 12 h at 50 °C. The aqueous layer was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 10 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 23 as colorless oil. The crude product 23 was used to the next step without further purification. To a solution of the acid 23 (47 mg, 0.2 mmol) in CH2Cl2 (2 mL) was added EDCI (77 mg, 0.4 mmol) and DMAP (2.5 mg, 0.02 mmol). The mixture was stirred at rt for 1 h, and then 4-chloro-3-(pyridin-2-yl)aniline (41 mg, 0.2 mmol) was added. The reaction was stirred at rt for 12 h and then diluted with water (10 mL). The mixture was extracted with CH2Cl2 (3 × 5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/EtOAc 2:1) to give 24 as white foam (43 mg, 51%). 1H NMR (300 MHz, CDCl3) δ 10.49 (s, 1H), 8.69 (d, J = 4.9 Hz, 1H), 7.74 – 7.69 (m, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.34 (d, J = 8.7 Hz, 1H), 7.29 – 7.24 (m, 1H), 7.02 (d, J = 8.0 Hz, 1H), 3.38 – 3.25 (m, 2H), 2.84 – 2.76 (m, 1H), 2.59 (s, 3H), 2.18 – 2.13 (m, 1H), 2.06 – 1.97 (m, 1H), 1.66 – 1.58 (m, 1H), 1.45 – 1.37 (m, 1H), 1.26 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 7.2 Hz, 3H).

13

C NMR (126 MHz, CDCl3) δ 174.81, 156.73,

156.36, 153.92, 149.43, 139.31, 137.94, 136.53, 136.09, 135.85, 130.43, 126.07, 124.77, 122.51, 122.41, 121.50, 120.61, 46.11, 44.14, 32.43, 31.60, 27.25, 24.05, 21.47, 14.51. MS (ESI, [M + H]＋) m/z 420.2. HRMS (ESI) calcd for C25H27ClN3O, 420.1837; found, 420.1840. Synthesis of (R)-methyl 2-(2-(4-chloro-3-(pyridin-2-yl)phenylcarbamoyl)-5-methyl quinolin-8-yl) propanoate (27). To a solution of 22 (247 mg, 1.0 mmol) in dioxane (5


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mL) was added SeO2 (220 mg, 2.0 mmol). The reaction mixture was heated under reflux for 4 h. After cooling, the mixture was filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (petroleum ether/EtOAc 8:1) to give compound 25 as colorless oil (131 mg, 51%). 1H NMR (300 MHz, CDCl3) δ 10.12 (s, 1H), 8.40 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 4.94 (q, J = 7.2 Hz, 1H), 3.59 (s, 3H), 2.63 (s, 3H), 1.59 (d, J = 7.2 Hz, 3H). To a solution of 25 (51 mg, 0.2 mmol) in t-BuOH (3 mL) and H2O (1 mL) was added 2-methyl-2-butene (140 mg, 2.0 mmol), then NaH2PO4.2H2O (156 mg, 1.0 mmol), NaClO2 (54 mg, 0.6 mmol) in H2O (1 mL) was added. The reaction mixture was stirred at rt for 3 h, and then diluted with water (20 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/MeOH 10:1) to give compound 26 as pale oil. The crude product 26 was used without further purification. To a solution of 26 (27 mg, 0.1 mmol) in CH2Cl2 (2 mL) was added EDCI (38 mg, 0.2 mmol) and DMAP (1.2 mg, 0.01 mmol). The mixture was stirred at rt for 1 h, and then 4-chloro-3-(pyridin-2-yl)aniline (20 mg, 0.1 mmol) was added. The reaction was stirred at rt for 12 h and then diluted with water (10 mL). The mixture was extracted with CH2Cl2 (3 × 5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to column chromatography on silica gel (petroleum ether/EtOAc 2:1) to give compound 27 as white solid (12 mg, 27%). 1H NMR (300



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MHz, CDCl3) δ 10.29 (s, 1H), 8.76 (d, J = 4.8 Hz, 1H), 8.53 (d, J = 8.7 Hz, 1H), 8.41 – 8.36 (m, 2H), 8.07 (dd, J = 8.7, 2.7 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.33 – 7.29 (m, 1H), 4.21 (q, J = 6.9 Hz, 1H), 3.55 (s, 3H), 2.71 (s, 3H), 1.69 (d, J = 6.9 Hz, 3H).

13

C NMR (126 MHz, CDCl3) δ 177.23, 162.57, 157.04, 149.53,

147.78, 143.85, 139.77, 138.18, 137.51, 135.94, 135.03, 134.16, 130.64, 129.26, 129.19, 128.65, 127.03, 124.81, 122.48, 122.45, 120.92, 118.48, 52.25, 43.63, 18.58, 18.01. MS (ESI, [M + H] ＋ ) m/z 460.2. HRMS (ESI) calcd for C26H23ClN3O3, 460.1422; found, 460.1433. Synthesis of (R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((6aS,7R,10R,10aS)-5,10dimethyl-6,6a,7,8,9,10,10a,11-octahydro-5H-benzo[b]carbazol-7-yl)propanamide (30). To a solution of 14 (95 mg, 0.4 mmol) in HOAc (5 mL) was added phenylhydrazine hydrochloride (69 mg, 0.48 mmol). The reaction mixture was heated under reflux for 2 h and concentrated in vacuo. Water (10 mL) was added and the mixture treated with Saturated NaHCO3 until pH = 5 and then extracted with CH2Cl2 (3 × 10 mL). The combined organic phase was dried over Na2SO4 and concentrated to give compound 28 as pale yellow oil. The crude product 28 was used without further purification. To a solution of 28 (62 mg, 0.2 mmol) in DMSO (2 mL) was added CH3I (114 mg, 0.8 mmol) and KOH (156 mg, 0.3 mmol). The reaction was stirred at 60 °C for 3 h, and then diluted with water (10 mL). The aqueous layer was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (petroleum ether/EtOAc 10:1) to give compound 29 as colorless oil (56 mg, 82%). 1H


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NMR (300 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 7.03 (t, J = 7.8 Hz, 1H), 3.67 (s, 3H), 3.58 (s, 3H), 2.99 (d, J = 16.2 Hz, 1H), 2.68 – 2.55 (m, 2H), 2.45 – 2.35 (m, 3H), 1.91 – 1.82 (m, 1H), 1.62 – 1.58 (d, J = 13.2 Hz, 1H), 1.46 – 1.32 (m, 3H), 1.22 – 1.01 (m, 5H), 0.80 (d, J = 6.3 Hz, 3H). To a solution of compound 29 (68 mg, 0.2 mmol) in EtOH (2 mL) and H2O (2 mL) was added lithium hydroxide monohydrate (17 mg, 0.4 mmol). The reaction was stirred for 12 h at 50 °C. The aqueous layer was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the acid intermediate as colorless oil. The crude acid was used to the next step without further purification. To a solution of the acid in CH2Cl2 (2 mL) was added EDCI (77 mg, 0.4 mmol) and DMAP (2.5 mg, 0.02 mmol). The mixture was stirred at rt for 1 h, and then 4-chloro-3-(pyridin-2-yl)aniline (41 mg, 0.2 mmol) was added. The reaction was stirred at rt for 12 h and then diluted with water (10 mL). The mixture was extracted with CH2Cl2 (3 × 5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/EtOAc 2:1) to give 30 as white solid (31 mg, 30%). 1H NMR (300 MHz, CDCl3) δ 8.73 (d, J = 5.1 Hz, 1H), 8.14 (s, 1H), 7.99 (dd, J = 8.7, 2.7 Hz, 1H), 7.85 – 7.74 (m, 2H), 7.67 (d, J = 2.7 Hz, 1H), 7.48 – 7.42 (m, 2H), 7.37 – 7.33 (m, 1H), 7.26 – 7.22 (m, 1H), 7.17 – 7.04 (m, 2H), 3.52 (s, 3H), 3.01 (d, J = 16.3 Hz, 1H), 2.69 – 2.63 (m, 1H), 2.51 – 2.28 (m, 3H), 2.16 – 2.10 (m, 1H), 2.01 – 1.93 (m, 1H), 1.66 – 1.59 (m, 3H), 1.38 – 1.30 (m, 2H), 1.18 (d, J = 6.6 Hz, 3H), 1.10 – 1.05 (m, 1H), 0.83 (d, J = 6.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.30, 156.60, 149.48, 138.98, 137.52, 137.19, 136.18, 133.46, 130.78, 127.22, 126.51, 125.34, 122.82, 122.24, 121.18, 120.59, 118.62, 117.84,



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108.43, 107.53, 45.31, 44.05, 43.71, 35.53, 34.53, 29.00, 27.83, 26.07, 23.28, 20.20, ＋

18.24, 15.61. MS (ESI, [M + H] ) m/z 512.3. HRMS (ESI) calcd for C32H35ClN3O, 512.2463; found, 512.2469. Synthesis of (R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((4aS,5R,8R,8aS)-5-methyl1-phenyl-4,4a,5,6,7,8,8a,9-octahydro-1H-benzo[f]indazol-8-yl)propanamide (33). A solution of 14 (238 mg, 1.0 mmol) in THF (10 mL) was cooled to 0 °C. NaH (600 mg, 15 mmol) and CH3I (2130 mg, 15 mmol) was added to the solution. The reaction was stirred for 1 h at rt, and then quenched with ice water and extracted with CH2Cl2 (3 × 10 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give 31 as colorless oil (229 mg, 91%). 1

H NMR (300 MHz, CDCl3) δ 3.67 (s, 3H), 2.41 – 2.14 (m, 6H), 2.08 – 2.03 (m, 1H),

1.85 – 1.62 (m, 4H), 1.46 – 1.20 (m, 3H), 1.17 – 1.07 (m, 4H), 0.96 (d, J = 6.3 Hz, 3H). A solution of 31 (51 mg, 0.2 mmol) in THF (5 mL) was cooled to 0 °C. NaH (160mg, 4 mmol) and ethyl formate (296 mg, 4 mmol) was added to the solution. The reaction was slowly warmed to rt and stirred for another 10 min. The reaction was quenched with ice water, and then the mixture was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 10 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product as colorless oil. To a solution of the crude product in EtOH (5 mL) was added phenylhydrazine hydrochloride (34 mg, 0.24 mmol). The reaction mixture was heated under reflux for 1 h and concentrated in vacuo. The residue was purified by silica column chromatography (petroleum ether/EtOAc 5:1) to give 32 as colorless oil (33 mg, 47%). 1

H NMR (300 MHz, CDCl3) δ 7.49 – 7.44 (m, 5H), 7.36 – 7.31 (m, 1H), 3.68 (s, 3H),


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2.85 (d, J = 16.2 Hz, 1H), 2.75 – 2.53 (m, 2H), 2.44 – 2.25 (m, 3H), 1.91 – 1.86 (m, 1H), 1.76 – 1.70 (m, 1H), 1.51 – 1.40 (m, 3H), 1.16 – 0.95 (m, 5H), 0.88 (d, J = 6.0 Hz, 3H). To a solution of compound 32 (70 mg, 0.2 mmol) in EtOH (2 mL) and H2O (2 mL) was added lithium hydroxide monohydrate (17 mg, 0.4 mmol). The reaction was stirred for 12 h at 50 °C. The aqueous layer was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the acid intermediate as colorless oil. The crude acid was used to the next step without further purification. To a solution of the acid in CH2Cl2 (2 mL) was added EDCI (77 mg, 0.4 mmol) and DMAP (2.5 mg, 0.02 mmol). The mixture was stirred at rt for 1 h, and then 4-chloro-3-(pyridin-2-yl)aniline (41 mg, 0.2 mmol) was added. The reaction was stirred at rt for 12 h and then diluted with water (10 mL). The mixture was extracted with CH2Cl2 (3 × 5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/EtOAc 2:1) to give 33 as white solid (31 mg, 30%). 1H NMR (300 MHz, CDCl3) δ 8.67 (d, J = 4.2 Hz, 1H), 7.88 (s, 1H), 7.77 – 7.60 (m, 4H), 7.48 – 7.26 (m, 8H), 2.84 (d, J = 15.9 Hz, 1H), 2.68 – 2.54 (m, 2H), 2.40 – 2.27 (m, 2H), 2.07 – 1.96 (m, 2H), 1.70 – 1.55 (m, 3H), 1.47 – 1.26 (m, 2H), 1.14 – 1.01 (m, 4H), 0.87 (d, J = 6.6 Hz, 3H).

13

C NMR (126 MHz,

CDCl3) δ 175.02, 156.43, 149.25, 140.21, 139.01, 138.99, 137.13, 136.51, 136.24, 130.60, 129.22, 126.78, 126.76, 125.17, 123.28, 122.76, 122.51, 121.26, 116.37, 45.10, 43.81, 43.39, 35.40, 34.64, 27.63, 25.85, 23.07, 19.97, 19.24, 15.64. MS (ESI, [M + H] ＋ ) m/z 525.4. HRMS (ESI) calcd for C32H34ClN4O, 525.2416; found, 525.2426.



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Synthesis

of

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(R)-N-(4-chloro-3-(pyridin-2-yl)phenyl)-2-((5R,8S)-5-methyl-2-

(morpholinomethyl)-5,6,7,8-tetrahydroquinolin-8-yl)propanamide

(38a).

To

a

solution of compound 22 (124 mg, 0.5 mmol) in CH2Cl2 (10 mL) was added m-CPBA (138 mg, 0.8 mmol). The reaction was stirred for 1 h at rt, and then washed with saturated NaHCO3. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/EtOAc 1:1) to give 34 as white solid (107 mg, 81%). 1H NMR (300 MHz, CDCl3) δ 7.18 – 7.04 (m, 2H), 3.87 – 3.78 (m, 1H), 3.65 – 3.59 (m, 1H), 3.45 (s, 3H), 2.85 – 2.74 (m, 1H), 2.51 (s, 3H), 2.08 – 1.99 (m, 1H), 1.93 – 1.70 (m, 2H), 1.37 – 1.23 (m, 7H). A solution of compound 34 (53 mg, 0.2 mmol) in (Ac)2O (5 mL) was heated under reflux for 5 h. After removal of the solvent, the residue was diluted with water (10 mL). The mixture was treated with Saturated NaHCO3 until pH>8 and washed with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (petroleum ether/EtOAc 3:1) to give 35 as pale yellow oil (41 mg, 68%). 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 5.15 – 5.04 (m, 2H), 3.62 (s, 3H), 3.47 – 3.42 (m, 1H), 3.15 – 3.09 (m, 1H), 2.89 – 2.82 (m, 1H), 2.14 (s, 3H), 2.07 – 1.99 (m, 2H), 1.81 – 1.70 (m, 1H), 1.51 – 1.40 (m, 1H), 1.26 (d, J = 6.8 Hz, 3H), 1.10 (d, J = 7.2 Hz, 3H). To a solution of compound 35 (61 mg, 0.2 mmol) in EtOH (2 mL) and H2O (2 mL) was added lithium hydroxide monohydrate (17 mg, 0.4 mmol). The reaction was stirred for 2 h at rt. The aqueous layer was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under


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reduced pressure to give the intermediate as colorless oil. The crude product was dissolved in CH2Cl2 (5 mL) at rt, SOCl2 (36 mg, 0.3 mmol) in CH2Cl2 (1 mL) was added. The reaction was stirred for 30 min at rt and then diluted with water (5 mL). The mixture was treated with Saturated NaHCO3 until pH>8 and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the 36 as pale yellow oil. The crude product 36 was used without further purification. To a solution of crude product 36 (36 mg, 0.2 mmol) in MeCN (5 mL) was added morpholine (26 mg, 0.3 mmol) and K2CO3 (56 mg, 0.4 mmol). The reaction was stirred for 3 h at rt, and then diluted with water (10 mL) and washed with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give 37a as pale yellow oil. To a solution of the crude compound 37a (66 mg, 0.2 mmol) in EtOH (2 mL) and H2O (2 mL) was added lithium hydroxide monohydrate (17 mg, 0.4 mmol). The reaction was stirred for 12 h at 50 °C. The aqueous layer was adjusted to pH 5 by adding hydrochloride acid (0.5 M) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the acid intermediate as colorless oil. The crude acid was used to the next step without further purification. To a solution of the acid in CH2Cl2 (2 mL) was added EDCI (77 mg, 0.4 mmol) and DMAP (2.5 mg, 0.02 mmol). The mixture was stirred at rt for 1 h, and then 4-chloro-3-(pyridin-2-yl)aniline (41 mg, 0.2 mmol) was added. The reaction was stirred at rt for 12 h and then diluted with water (10 mL). The mixture was extracted



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with CH2Cl2 (3 × 5 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica column chromatography (CH2Cl2/MeOH 20:1) to give 38a as white solid (45 mg, 45%). 1H NMR (300 MHz, CDCl3) δ 10.59 (s, 1H), 8.69 (d, J = 4.9 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.68 – 7.57 (m, 4H), 7.35 (d, J = 8.7 Hz, 1H), 7.29 – 7.22 (m, 2H), 3.73 (d, J = 13.9 Hz, 1H), 3.62 – 3.57 (m, 5H), 3.32 – 3.28 (m, 2H), 2.87 – 2.81 (m, 1H), 2.48 – 2.39 (m, 4H), 2.21 – 2.15 (m, 1H), 2.04 – 2.00 (m, 1H), 1.73 – 1.60 (m, 1H), 1.47 – 1.35 (m, 1H), 1.28 (d, J = 6.9 Hz, 3H), 1.15 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 174.81, 156.64, 156.59, 153.92, 149.44, 139.09, 137.96, 137.50, 136.45, 135.78, 130.30, 126.03, 124.79, 122.75, 122.41, 121.17, 121.10, 66.78, 64.31, 53.84, 46.25, 44.05, 32.55, 31.52, 27.49, 21.41, 14.48. MS (ESI, [M + H]＋) m/z 505.3. HRMS (ESI) calcd for C29H34ClN4O2, 505.2365; found, 505.2358. (R)-2-((5R,8S)-2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-(2-methyl-4'-(triflu oromethoxy)biphenyl-3-yl)propanamide

(38b).

Condensation

of

2-methyl-4'-(trifluoromethoxy)biphenyl-3-amine (0.1 mmol) and acid 23 (0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound 38b was obtained as white solid (29 mg, 61%). 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.22 – 7.15 (m, 5H), 7.00 – 6.93 (m, 2H), 3.93 – 3.85 (m, 1H), 3.16 – 3.11 (m, 1H), 2.85 – 2.76 (m, 1H), 2.51 (s, 3H), 2.25 – 2.20 (m, 1H), 2.08 – 2.03 (m, 1H), 1.75 – 1.65 (m, 1H), 1.46 – 1.39 (m, 1H), 1.32 (d, J = 7.2 Hz, 4H), 1.27 (d, J = 6.8 Hz, 3H). 13

C NMR (126 MHz, CDCl3) δ 174.68, 157.45, 155.26, 148.90, 142.04, 141.31,

137.46, 136.98, 136.48, 131.36, 127.86, 126.97, 126.62, 123.81, 122.09, 122.28 (q, J = 257 Hz), 121.23, 46.55, 43.43, 33.20, 32.51, 25.83, 24.88, 22.01, 15.81, 15.37. MS (ESI, [M + H]＋) m/z 483.3. HRMS (ESI) calcd for C28H30F3N2O2, 483.2254; found,


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483.2246. Synthesis of (R)-N-(4-chloro-3-(5-((2S,6R)-2,6-dimethylmorpholino)pyridin-2-yl) phenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide (50). A solution of compound 39 (192 mg, 1.0 mmol) in MeCN (10 mL) was cooled to -20 °C, NBS (196 mg, 1.1 mmol) was added and the reaction mixture was stirred for 30 min at -20 °C. After removal of the solvent, the residue was purified by silica column chromatography (CH2Cl2/EtOAc 4:1) to give 40 as colorless oil (122 mg, 45%). 1H NMR (300 MHz, CDCl3) δ 7.92 (d, J = 3.3 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.00 (dd, J = 8.7, 3.3 Hz, 1H), 3.81 – 3.66 (m, 2H), 3.35 – 3.31 (m, 2H), 2.42 – 2.34 (m, 2H), 1.20 (d, J = 6.3 Hz, 6H). To a solution of compound 40 (121 mg, 0.5 mmol) in DME (8 mL) was added 2-chloro-5-nitrophenylboronic acid (121 mg, 0.6 mmol), Pd(PPh3)4 (35 mg, 0.03 mmol) and 2N Na2CO3 (1 mL). The reaction mixture was heated under reflux for 3 h. After removal of the solvent, the residue was purified by silica column chromatography (petroleum ether/EtOAc 5:1) to give the intermediate as off-white foam in 40% yield. The intermediate was dissolved in EtOH (5 mL) and H2O (2 mL), Fe powder (78 mg, 1.4 mmol) and NH4Cl (106 mg, 2.0 mmol) was added. The reaction mixture was heated under reflux for 4 h, and then filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 41 as pale yellow solid. Condensation of amine 41 (32 mg, 0.1 mmol) and acid 23 (24 mg, 0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound 50 was obtained as white foam (22 mg, 42%). 1H NMR (300



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MHz, CDCl3) δ 10.41 (s, 1H), 8.34 (d, J = 3.0 Hz, 1H), 7.65 – 7.63 (m, 1H), 7.56 – 7.44 (m, 3H), 7.34 – 7.30 (m, 1H), 7.20 (dd, J = 8.7, 3.0 Hz, 1H), 7.03 – 6.99 (m, 1H), 3.86 – 3.80 (m, 2H), 3.55 – 3.50 (m, 2H), 3.39 – 3.24 (m, 2H), 2.84 – 2.74 (m, 1H), 2.60 (s, 3H), 2.56 – 2.48 (m, 2H), 2.21 – 2.11 (m, 1H), 2.06 – 1.98 (m, 1H), 1.71 – 1.58 (m, 1H), 1.45 – 1.36 (m, 1H), 1.30 – 1.16 (m, 12H). 13C NMR (151 MHz, CDCl3) δ 174.69, 156.35, 153.91, 147.30, 145.34, 139.06, 137.85, 137.28, 136.48, 136.02, 130.38, 126.08, 124.62, 122.28, 121.52, 121.46, 120.03, 71.51, 53.69, 46.04, 44.16, 32.40, 31.59, 27.18, 24.09, 21.46, 19.08, 14.52. MS (ESI, [M + H]＋) m/z 533.3. HRMS (ESI) calcd for C31H38ClN4O2, 533.2678; found, 533.2677. General Procedure for Synthesis of Compounds 51 and 52. To a solution of nitrobenzene 42 or 44 (0.2 mmol) in EtOH (5 mL) and H2O (2 mL) was added Fe powder (1.4 mmol) and NH4Cl (2.0 mmol). The reaction mixture was heated under reflux for 4 h, and then filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give corresponding aniline (43 and 45) as pale yellow solid. Condensation of aniline 43 or 45 (0.1 mmol) and acid 23 (0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound 51 or 52 was obtained as white foam. (R)-N-(4-Chloro-3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)phenyl)-2-((5R,8S)-2,5-dim ethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide (51). (21 mg, 47%). 1H NMR (400 MHz, CDCl3) δ 11.22 (s, 1H), 7.97 (d, J = 2.8 Hz, 1H), 7.87 (dd, J = 8.8, 2.8 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 3.36 – 3.22 (m, 2H), 2.84 – 2.78 (m, 1H), 2.65 (s, 3H), 2.21 – 2.13 (m, 2H), 2.04 – 1.98 (m,


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1H), 1.69 – 1.59 (m, 1H), 1.45 – 1.35 (m, 1H), 1.27 (d, J = 6.8 Hz, 3H), 1.16 – 1.09 (m, 7H).

13

C NMR (151 MHz, CDCl3) δ 175.25, 173.70, 172.67, 156.15, 153.79,

138.24, 136.73, 136.35, 131.83, 127.13, 123.88, 123.59, 121.90, 121.72, 46.97, 43.54, ＋

32.42, 31.49, 27.94, 23.96, 21.42, 14.35, 7.95, 7.93, 6.90. MS (ESI, [M + H] ) m/z 451.2. HRMS (ESI) calcd for C25H28ClN4O2, 451.1895; found, 451.1898. (R)-N-(4-Chloro-3-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl)-2-((5R,8S)-2,5-dimethyl5,6,7,8-tetrahydroquinolin-8-yl)propanamide (52). (18 mg, 43%). 1H NMR (300 MHz, CDCl3) δ 10.97 (s, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.71 (dd, J = 8.7, 2.7 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 3.35 – 3.24 (m, 2H), 2.84 – 2.77 (m, 1H), 2.66 (s, 3H), 2.63 (s, 3H), 2.19 – 2.12 (m, 1H), 2.05 – 1.99 (m, 1H), 1.66 – 1.58 (m, 1H), 1.46 – 1.37 (m, 1H), 1.27 (d, J = 6.9 Hz, 3H), 1.16 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.93, 175.01, 167.14, 156.18, 153.82, 138.03, 136.69, 136.24, 131.29, 126.93, 126.11, 122.50, 122.14, 121.63, 46.63, 43.72, ＋

32.40, 31.50, 27.59, 23.95, 21.43, 14.40, 12.35. MS (ESI, [M + H] ) m/z 425.1. HRMS (ESI) calcd for C23H26ClN4O2, 425.1739; found, 425.1743. Synthesis of (R)-N-(4-Chloro-3-(5-(1-methyl-1H-pyrazol-5-yl)-1,3,4-oxadiazol-2-yl) phenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide

(53).

To a solution of methyl 1-methyl-1H-pyrazole-5-carboxylate (700 mg, 5.0 mmol) in MeOH (20 mL) was added N2H4·H2O (1.94 mL, 40.0 mmol), and the reaction was under reflux for 4 h. After removal of the solvent, the solid was washed with petroleum ether/ CH2Cl2 (1:1) and dried in vacuo. The crude product was dissolved in THF (20 mL) at 0 °C, 2-chloro-5-nitrobenzoyl chloride (1100 mg, 5.0 mmol) and Et3N (1.38 mL, 10.0 mmol) was added. The reaction was warmed to rt and stirred overnight. After removal of the solvent, the residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed



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with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 47 as pale yellow solid. The crude compound 47 (324 mg, 1.0 mmol) was dissolved in POCl3 (5 mL) and heated under reflux for 2 h. After cooling, the mixture was poured into ice water, basified by means of 2N aqueous NaOH and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were dried over Na2SO4 and evaporated. The residue was purified by silica column chromatography (CH2Cl2/ EtOAc 20:1) to give 48a as white solid (250 mg, 82%).1H NMR (300 MHz, CDCl3) δ 8.96 (d, J = 2.7 Hz, 1H), 8.36 (dd, J = 8.7, 2.7 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H), 7.01 (d, J = 2.1 Hz, 1H), 4.40 (s, 3H). To a solution of 48a (61 mg, 0.2 mmol) in EtOH (5 mL) and H2O (2 mL) was added Fe powder (78 mg, 1.4 mmol) and NH4Cl (106 mg, 2.0 mmol). The reaction mixture was heated under reflux for 4 h, and then filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 49a as white solid. Condensation of amine 49a (28 mg, 0.1 mmol) and acid 23 (24 mg, 0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound 53 was obtained as white solid (25 mg, 53%). 1H NMR (300 MHz, CDCl3) δ 11.34 (s, 1H), 8.12 – 8.10 (m, 1H), 7.84 (dd, J = 8.7, 2.7 Hz, 1H), 7.60 – 7.55 (m, 2H), 7.47 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 6.96 – 6.95 (m, 1H), 4.37 (s, 3H), 3.39 – 3.22 (m, 2H), 2.86 – 2.79 (m, 1H), 2.68 (s, 3H), 2.20 – 2.15 (m, 1H), 2.05 – 2.00 (m, 1H), 1.71 – 1.59 (m, 1H), 1.48 – 1.35 (m, 1H), 1.28 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 7.3 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.36, 162.45,


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157.76, 156.09, 153.73, 138.91, 138.45, 136.71, 136.31, 131.82, 127.00, 126.68, 123.66, 122.44, 121.70, 121.24, 109.10, 47.03, 43.52, 39.85, 32.42, 31.51, 27.98, ＋

24.06, 21.40, 14.32. MS (ESI, [M + H] ) m/z 491.3. HRMS (ESI) calcd for C26H28ClN6O2, 491.1957; found, 491.1957. Synthesis of (R)-N-(4-Chloro-3-(5-(1-methyl-1H-pyrazol-5-yl)-1,3,4-thiadiazol-2-yl) phenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide

(54).

To a solution of 47 (324 mg, 1.0 mmol) in THF (5 mL) was added lawesson’s reagent (606 mg, 1.5 mmol). The reaction was heated under reflux for 4 h. The mixture was basified by means of 2N aqueous NaOH, and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were dried over Na2SO4 and evaporated. The residue was purified by silica column chromatography (CH2Cl2/ EtOAc 20:1) to give 48b as white solid. (164 mg, 51%). 1H NMR (300 MHz, CDCl3) δ 9.27 (d, J = 2.7 Hz, 1H), 8.26 (dd, J = 8.7, 2.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 4.34 (s, 3H). To a solution of 48b (64 mg, 0.2 mmol) in EtOH (5 mL) and H2O (2 mL) was added Fe powder (78 mg, 1.4 mmol) and NH4Cl (106 mg, 2.0 mmol). The reaction mixture was heated under reflux for 4 h, and then filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 49b as white solid. Condensation of amine 49b (29 mg, 0.1 mmol) and acid 23 (24 mg, 0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound 54 was obtained as white solid (21 mg, 41%). 1H NMR (300 MHz, CDCl3) δ 11.37 (s, 1H), 8.24 (d, J = 2.7 Hz, 1H), 7.98 (dd, J = 8.7, 2.7 Hz,



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1H), 7.56 – 7.54 (m, 2H), 7.46 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.75 (d, J = 2.1 Hz, 1H), 4.37 (s, 3H), 3.39 – 3.33 (m, 1H), 3.29 – 3.22 (m, 1H), 2.84 – 2.80 (m, 1H), 2.69 (s, 3H), 2.20 – 2.14 (m, 1H), 2.05 – 2.00 (m, 1H), 1.72 – 1.60 (m, 1H), 1.47 – 1.35 (m, 1H), 1.28 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 7.2 Hz, 3H).

13

C NMR (126

MHz, CDCl3) δ 175.35, 163.36, 159.22, 156.03, 153.94, 138.83, 138.60, 136.59, 136.15, 131.60, 131.14, 128.41, 126.22, 123.34, 121.70, 121.13, 109.98, 47.04, 43.56, 39.98, 32.41, 31.58, 27.95, 24.10, 21.39, 14.32. MS (ESI, [M + H]＋) m/z 507.2. HRMS (ESI) calcd for C26H28ClN6OS, 507.1728; found, 507.1723. General Procedure for Synthesis of Compounds 62-64. Condensation of aniline 55, 56, or 59 (0.1 mmol) and acid 23 (0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound (62-64) was obtained as white solid. (R)-N-(3-(Benzo[d]oxazol-2-yl)-4-chlorophenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-te trahydroquinolin-8-yl)propanamide (62). (19 mg, 41%). 1H NMR (300 MHz, CDCl3) δ 11.07 (s, 1H), 8.27 (d, J = 2.7 Hz, 1H), 7.86 – 7.82 (m, 1H), 7.69 (dd, J = 8.7, 2.7 Hz, 1H), 7.61 – 7.53 (m, 2H), 7.44 (d, J = 8.7 Hz, 1H), 7.40 – 7.36 (m, 2H), 7.07 (d, J = 8.1 Hz, 1H), 3.38 – 3.27 (m, 2H), 2.85 – 2.78 (m, 1H), 2.68 (s, 3H), 2.20 – 2.13 (m, 1H), 2.06 – 1.98 (m, 1H), 1.72 – 1.59 (m, 1H), 1.47 – 1.38 (m, 1H), 1.27 (d, J = 6.9 Hz, 3H), 1.18 (d, J = 7.2 Hz, 3H).

13

C NMR (126 MHz, CDCl3) δ 175.13, 160.63,

156.18, 153.80, 150.42, 141.81, 138.09, 136.68, 136.26, 131.79, 127.08, 126.07, 125.55, 124.57, 122.82, 122.08, 121.65, 120.57, 110.65, 46.71, 43.70, 32.40, 31.50, 27.68, 23.99, 21.41, 14.40. MS (ESI, [M + H]＋) m/z 460.2. HRMS (ESI) calcd for C27H27ClN3O2, 460.1786; found, 460.1789. (R)-N-(3-(Benzo[d]thiazol-2-yl)-4-chlorophenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-te trahydroquinolin-8-yl)propanamide (63). (22 mg, 48%). 1H NMR (300 MHz, CDCl3)


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δ 10.98 (s, 1H), 8.18 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.85 (dd, J = 8.7, 2.4 Hz, 1H), 7.55 – 7.52 (m, 2H), 7.45 – 7.40 (m, 2H), 7.07 (d, J = 8.0 Hz, 1H), 3.37 – 3.27 (m, 2H), 2.85 – 2.77 (m, 1H), 2.71 (s, 3H), 2.20 – 2.15 (m, 1H), 2.04 – 1.97 (m, 1H), 1.64 – 1.59 (m, 1H), 1.46 – 1.35 (m, 1H), 1.27 (d, J = 6.9 Hz, 3H), 1.18 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.14, 163.90, 156.27, 154.05, 152.46, 138.25, 136.62, 136.20, 136.18, 132.15, 131.30, 126.36, 126.26, 125.40, 123.43, 122.32, 121.88, 121.67, 121.44, 46.72, 43.85, 32.45, 31.57, 27.70, 24.14, 21.47, 14.44. MS (ESI, [M + H]＋) m/z 476.2. HRMS (ESI) calcd for C27H27ClN3OS, 476.1558; found, 476.1556. (R)-N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-2-((5R,8S)-2,5-dimethyl-5, 6,7,8-tetrahydroquinolin-8-yl)propanamide (64). (24 mg, 53%). 1H NMR (300 MHz, CDCl3) δ 11.15 (s, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 8.7, 2.4 Hz, 1H), 7.71 – 7.62 (m, 2H), 7.53 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.30 – 7.26 (m, 2H), 7.04 (d, J = 8.0 Hz, 1H), 3.37 – 3.21 (m, 2H), 2.84 – 2.76 (m, 1H), 2.66 (s, 3H), 2.17 – 2.10 (m, 1H), 2.03 – 1.99 (m, 1H), 1.69 – 1.61 (m, 1H), 1.47 – 1.35 (m, 1H), 1.26 (d, J = 6.9 Hz, 3H), 1.16 (d, J = 7.2 Hz, 3H).

13

C NMR (126 MHz, CDCl3) δ 175.37,

155.97, 154.01, 148.90, 138.43, 136.63, 136.14, 131.13, 128.51, 124.93, 122.99, 122.97, 122.55, 122.28, 121.72, 46.94, 43.57, 32.40, 31.53, 29.72, 27.80, 23.99, 21.42, 14.39. MS (ESI, [M + H] ＋ ) m/z 459.2. HRMS (ESI) calcd for C27H28ClN4O, 459.1946; found, 459.1940. Synthesis

of

(R)-N-(4-chloro-3-(1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)-2-

((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide (65) A solution of 58a (273 mg, 1.0 mmol) in THF (10 mL) was cooled to 5 °C, NaH (80 mg, 2.0 mmol) was added and stirred for 2 h, then CH3I (284 mg, 2.0 mmol) was added to the solution. The reaction was stirred overnight at rt, and then quenched with



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ice water and extracted with CH2Cl2 (3 × 10 mL). The combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give 60a as colorless oil (218 mg, 76%). 1H NMR (300 MHz, CDCl3) δ 8.51 (d, J = 2.4 Hz, 1H), 8.35 (dd, J = 8.7, 2.4 Hz, 1H), 7.88 – 7.85 (m, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.48 – 7.35 (m, 3H), 3.73 (s, 3H). To a solution of 60a (58 mg, 0.2 mmol) in EtOH (5 mL) and H2O (2 mL) was added Fe powder (78 mg, 1.4 mmol) and NH4Cl (106 mg, 2.0 mmol). The reaction mixture was heated under reflux for 4 h, and then filtered over a pad of celite and concentrated in vacuo. The residue was diluted with water (20 mL) and extracted with CH2Cl2 (3 × 10 mL), and the combined organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 61a as pale yellow solid. Condensation of amine 61a (26 mg, 0.1 mmol) and acid 23 (24 mg, 0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound 65 was obtained as white solid (23 mg, 49%). 1H NMR (300 MHz, CDCl3) δ 10.80 (s, 1H), 7.86 – 7.77 (m, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.61 (dd, J = 8.7, 2.4 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.42 – 7.31 (m, 4H), 7.01 (d, J = 8.1 Hz, 1H), 3.68 (s, 3H), 3.34 – 3.26 (m, 2H), 2.85 – 2.77 (m, 1H), 2.56 (s, 3H), 2.17 – 2.13 (m, 1H), 2.03 – 1.99 (m, 1H), 1.69 – 1.57 (m, 1H), 1.45 – 1.37 (m, 1H), 1.26 (d, J = 6.9 Hz, 3H), 1.15 (d, J = 7.2 Hz, 3H).

13

C NMR (126 MHz, CDCl3) δ

175.11, 156.17, 153.82, 151.29, 142.88, 138.00, 136.57, 136.11, 135.66, 130.09, 130.06, 128.02, 123.36, 122.93, 122.37, 122.33, 121.57, 120.15, 109.64, 46.41, 43.90, 32.42, 31.57, 30.89, 27.52, 24.11, 21.44, 14.44. MS (ESI, [M + H]＋) m/z 473.2. HRMS (ESI) calcd for C28H30ClN4O, 473.2103; found, 473.2114. General Procedure for Synthesis of Compounds 66-69. The synthetic method for


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compounds 60b-60d was similar to that of compound 60a and the target compounds was obtained as a mixture of isomers, which were inseparable by silica gel column. Condensation of aniline 61b or 61c (0.1 mmol) and acid 23 (0.1 mmol) was conducted by following a procedure similar to that of preparation of compound 24, and the target compound (66 or 67) was obtained as inseparable isomer. Whereas, the isomers 68 and 69 (from 61d) can be separated by silica gel column. (R)-N-(4-Chloro-3-(5-methoxy- and 6-methoxy-1-methyl-1H-benzo[d]imidazole -2-yl)phenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide (66). White solid (21 mg, 43%). 1H NMR (300 MHz, CDCl3) δ 10.79 (s, 1H), 7.70 – 7.66 (m, 1.5H), 7.63 – 7.59 (m, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.30 – 7.27 (m, 1H), 7.03 – 6.93 (m, 2H), 6.85 (d, J = 2.4 Hz, 0.5H), 3.91 and 3.88 (s, 3H), 3.65 and 3.63 (s, 3H), 3.35 – 3.27 (m, 2H), 2.85 – 2.77 (m, 1H), 2.56 (s, 3H), 2.18 – 2.13 (m, 1H), 2.05 – 1.99 (m, 1H), 1.69 – 1.57 (m, 1H), 1.45 – 1.32 (m, 1H), 1.27 (d, J = 6.7 Hz, 3H), 1.16 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.02, 156.83, 156.29, 156.19, 153.82, 151.42, 150.48, 143.65, 137.97, 137.47, 136.55, 136.28, 136.08, 130.38, 130.22, 130.18, 130.03, 130.00,

128.09, 128.01,

123.40, 123.31, 122.25, 122.21, 121.56, 120.67, 113.08, 111.64, 109.99, 102.21, 93.12, 55.95, 55.89, 46.35, 43.94, 32.42, 31.60, 30.97, 30.89, 27.50, 24.12, 21.44, 14.44. MS (ESI, [M + H]＋) m/z 503.3. HRMS (ESI) calcd for C29H32ClN4O2, 503.2208; found, 503.2215. (R)-N-(4-Chloro-3-(5-fluoro- and 6-fluoro-1-methyl-1H-benzo[d]imidazol-2-yl) phenyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide (67). White solid (22 mg, 44%). 1H NMR (400 MHz, CDCl3) δ 10.98 and 10.96 (s, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.72 (dd, J = 8.8, 4.8 Hz, 0.5H), 7.57 – 7.51 (m, 2H), 7.47 (dd, J = 9.2, 2.4 Hz, 0.5H), 7.40 (d, J = 8.8 Hz, 1H), 7.32 (dd, J = 8.8, 4.8 Hz, 0.5H),



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7.11 – 7.01 (m, 2.5H), 3.67 and 3.64 (s, 3H), 3.31 – 3.25 (m, 2H), 2.85 – 2.76 (m, 1H), 2.56 (s, 3H), 2.17 – 2.11 (m, 1H), 2.03 – 1.97 (m, 1H), 1.68 – 1.58 (m, 1H), 1.44 – 1.34 (m, 1H), 1.26 (d, J = 6.8 Hz, 3H), 1.15 (d, J = 7.2 Hz, 3H).

13

C NMR

(126 MHz, CDCl3) δ 175.13, 159.73 (d, J = 241 Hz), 159.38 (d, J = 238 Hz), 156.03, 153.67, 152.58, 151.88, 151.86, 143.01 (d, J = 12 Hz), 139.06, 138.00, 136.49, 136.00, 135.67 (d, J = 12 Hz), 132.10, 129.96, 129.62, 127.79, 127.71, 123.25, 123.22, 122.34, 122.29, 121.49, 120.72 (d, J = 10 Hz), 111.21 (d, J = 26 Hz) , 110.61 (d, J = 26 Hz), 109.95 (d, J = 10 Hz), 105.60 (d, J = 24 Hz), 96.27 (d, J = 26 Hz), 46.42, 43.68, 32.28, 31.39, 30.98, 30.93, 27.44, 23.96, 21.35, 14.36. MS (ESI, [M + H]＋) m/z 491.3. HRMS (ESI) calcd for C28H29ClFN4O, 491.2008; found, 491.2015. (R)-N-(4-Chloro-3-(1-methyl-5-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)ph enyl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide

(68).

White solid (13 mg, 23%). 1H NMR (400 MHz, CDCl3) δ 11.03 (s, 1H), 7.81 – 7.80 (m, 1H), 7.69 (s, 1H), 7.61 – 7.55 (m, 2H), 7.43 – 7.38 (m, 2H), 7.24 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 3.69 (s, 3H), 3.35 – 3.28 (m, 2H), 2.83 – 2.79 (m, 1H), 2.58 (s, 3H), 2.18 – 2.12 (m, 1H), 2.05 – 1.99 (m, 1H), 1.71 – 1.61 (m, 1H), 1.45 – 1.35 (m, 1H), 1.27 (d, J = 6.8 Hz, 3H), 1.16 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.15, 155.96, 153.61, 153.09, 144.86, 142.92, 138.13, 136.89, 136.34, 134.19, 130.08, 129.50, 127.79, 123.23, 122.53, 121.73, 120.73 (q, J = 257 Hz), 116.94, 112.95, 110.08, 46.42, 43.54, 32.32, 31.35, 31.07, 27.37, 23.79, 21.38, 14.40. MS (ESI, [M + H]＋) m/z 557.2. HRMS (ESI) calcd for C29H29ClF3N4O2, 557.1926; found, 557.1921. (R)-N-(4-Chloro-3-(1-methyl-6-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)phen yl)-2-((5R,8S)-2,5-dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)propanamide (69). White solid (12 mg, 21%). 1H NMR (400 MHz, CDCl3) δ 11.00 (s, 1H), 7.81 – 7.79 (m, 2H),


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7.61 – 7.54 (m, 2H), 7.42 (d, J = 8.8 Hz, 1H), 7.29 (m, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 3.68 (m, 3H), 3.34 – 3.27 (m, 2H), 2.84 – 2.79 (m, 1H), 2.58 (s, 3H), 2.20 – 2.12 (m, 1H), 2.05 – 1.99 (m, 1H), 1.71 – 1.61 (m, 1H), 1.44 – 1.33 (m, 1H), 1.27 (d, J = 6.8 Hz, 3H), 1.15 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 175.12, 156.03, 153.65, 152.85, 145.22, 141.28, 138.13, 136.79, 136.27, 135.56, 130.07, 129.48, 127.82, 123.20, 122.49, 121.67, 120.80, 120.68 (q, J = 257 Hz), 116.31, 102.98, 46.43, 43.63, 32.34, 31.41, 31.07, 27.40, 23.87, 21.39, 14.41. MS (ESI, [M + H]＋) m/z 557.2. HRMS (ESI) calcd for C29H29ClF3N4O2, 557.1926; found, 557.1932. ShhN Conditioned Medium (CM) Preparation. ShhN CM was prepared as previously described.57,68 In brief, HEK293 cells were transfected with GFP or plasmid containing the N-terminal signaling domain of the Shh (ShhN). The CM was collected for stimulating the Hh pathway activity after transfection of 48 h. Dual Luciferase Reporter Assay. Cells were seeded in 96-well plates, followed by various treatments as indicated. The luciferase activity in the cell lysates was measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions in a luminometer (Molecular Devices; Sunnyvale, CA). The firefly luciferase values were normalized to Renilla values. Reverse

Transcription and

Quantitative

Polymerase

Chain

Reaction

(RT-qPCR). After various indicated treatment, the cells were subjected for extraction of total RNA using an RNAiso Plus Kit (TaKaRa; Dalian, China) according to the manufacturer’s instructions. The obtained total RNA was further reversely transcribed to cDNA with a SuperScript III Kit (TaKaRa). The quantitative PCR analyses were



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conducted in triplicate with a SYBR Green Kit (TaKaRa) in an iCycler iQ system (Bio-Rad; Hercules, CA) using the following primers: mGUSB: 5’-CTGCCACGGCGATGGA-3’; 5’-ACTGCATAATAATGGGCACTGTTG- 3’; mGli1: 5’-GCAGTGGGTAACATGAGTGTCT-3’; 5′-AGGCACTAGAGTTGAGGAATTGT-3’. The mRNA level of the Gli1 was normalized to that of GUSB. Medulloblastoma Cells Culture and the 3-(4, 5-Dimethylthiazol-2-yl)-5 (3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium

(MTS)

Assay.

Medulloblastoma cells were obtained from Ptch+/-;p53-/- mice as previously reported.57,68 Briefly, medulloblastoma allografts were mechanically minced and digested by collagenase. The cells were cultured in Neurobasal A medium (Invitrogen) containing B-27 supplement (Invitrogen), EGF 20 ng/ml (Invitrogen), bFGF 20 ng/ml (Invitrogen), nonessential amino acids, N-acetyl cysteine 60 µg/ml. Medulloblastoma cells were seeded into 96-well plates and subjected to various treatments as indicated for 72 h. The MTS assays were performed using the MTS Cell Proliferation Kit (Merck Millipore; Bedford, MA) according to manufacturer’s instructions. Annexin V-fluorescein isothiocyanate (FITC) and propidiumiodide (PI) double staining:

Medulloblastoma cells after treated with compound 65 for 24 h were

collected for Annexin V-FITC and PI double staining using a kit from Beoytime (Suzhou, China) according to manufacturer’s instructions. After that, the cells were


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subjected to fluorescence-activated cell sorting (FACS; Becton Dickinson; San Jose, CA) analysis. Fluorescent BODIPY-Cyclopamine Competition Assay. The HEK293 cells seeded onto coverslips were transfected with hSmo expression constructs, followed by incubation with BODIPY-cyclopamine (Selleck Chemicals) and tested drugs as indicated for 10 h. After washed with PBS, fixed with paraformaldehyde, incubated with Triton X-100 solution, the cells were then subjected to FACS analysis or were mounted with DAPI and visualized using a fluorescence microscope (Leica; Wetzlar, Germany). Medulloblastoma Allograft Model. Mouse medulloblastoma model was established as previously described using Ptch+/-; p53-/- mouse.57,68 The primary intracranial medullblastomas spontaneously developed in Ptch+/-; p53-/- mice were harvested and subcutaneously allografted into athymic nude mice (Beijing HFK Bio-Technology; Beijing, China). The tumors in nude mice were further collected, cut into 1 mm3 fragments and inoculated subcutaneously into the right flank of athymic nude mice. When the tumor volume reached 100–150 mm3, the mice were administered with vehicle, compound 60, or compound 1. Both compound 60 and compound 1 were formulated as a suspension in 0.5% methylcellulose, 0.2% Tween-80. The volume of the tumors was measured twice per week using microcaliper and calculated as ‘Volume = [length (mm) ×width2 (mm2)]/2’. The tumor growth inhibition (TGI) was calculated on the last day of the study by comparing the tumor volume of treated mice with that of the vehicle control mice with the following



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formula: 100 × {1 − [(tumor volume of treated mice at last day – tumor volume of treated mice at day 0) / (tumor volume of control mice at final day − tumor volume of control mice at day 0)]}.

ASSOCIATED CONTENT Supporting Information Available. The Supporting information is available free of charge via the Internet at http://pubs.acs.org. Copies of the 1H and

13

C spectra of all

new compounds. AUTHOR INFORMATION Corresponding Author For

W.T.:

phone:

+86-21-51980039;

fax:

86-21-51980039;

E-mail:

+86-21-50806035;

fax:

86-21-50806035;

E-mail:

[email protected]. For

A.Z.:

phone:

[email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by grants from Chinese NSF (81430080, 81373277). Supporting from the National Program on Key Basic Research Project of China (2015CB910603), the International Cooperative Program (GJHZ1622) and Key Program of the Frontier Science (160621) of the Chinese Academy of Sciences, the Shanghai

Commission

of

Science

and

Technology

(16XD1404600,

14431905300, 14431900400), as well as grant from CAS Key Laboratory of


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Receptor Research of SIMM (SIMM1606YKF-08, SIMM1606YZZ-06) are also highly appreciated. ABBREVIATIONS USED Hh, hedgehog; Ptch, Patched; Smo, Smoothened; BCC, basal cell carcinoma; MB, medulloblastoma; Artemalogs, artemisinin analogues; DIPEA, diisopropyl ethylamine; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HOAT, 1-hydroxy-7-azabenzotriazole; EDCI, 1-ethyl-3-(3dimethylaminopropyl)carbodimide;

DMAP,

dimethylaminopyridine;

PK,

pharmacokinetic; MCPBA, 3-chloroperbenzoic acid; NOE, nuclear overhauser effect; SAR, structure-activity relationship; hERG, human Ether-a-go-go Related Gene; Shh CM, the Shh conditioned medium; TNF-α, tumor necrosis factor-α; PGE2, prostaglandin E2; BODIPY, boron-dipyrromethene; FACS, fluorescence-activated cell sorting; SAG, 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl) phenyl]methyl]-benzo[b]thiophene-2-carboxamide; TNF-α, tumor necrosis factor-α; PGE2, prostaglandin E2; TCF, T-cell factor; LEF, lymphoid enhancer factor; FITC, V-fluorescein isothiocyanate; PI, propidiumiodide; MRT, the mean residence time; Vss, volume of distribution; MRT, the mean residence time; CL, clearance; T1/2, half-life; Cmax, maximum concentration; Tmax, time of maximum concentration; AUC0-∞, area under the plasma concentration time curve; F, oral bioavailability; TGI, tumor growth inhibition; i.p., intraperitoneal; i.v., intravenous; RT-qPCR, Reverse transcription and quantitative polymerase chain reaction. References: (1) Ingham, P. W.; McMahon, A. P. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001, 15, 3059– 3087.



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(2) Beachy, P. A.; Karhadkar, S. S.; Berman, D. M. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004, 432 (7015): 324–331. (3) Ng, J. M.; Curran, T. The Hedgehog’s tale: developing strategies for targeting cancer. Nat. Rev. Cancer 2011, 11, 493-501. (4) Corbit, K. C.; Aanstad, P.; Singla, V.; Norman, A. R.; Stainier, D. Y.; Reiter, J. F. Vertebrate Smoothened functions at the primary cilium. Nature 2005, 437(7061): 1018–1021. (5) Ruat, M.; Hoch, L.; Faure, H.; Rognan, D. Targeting of Smoothened for therapeutic gain. Trends Pharmacol. Sci. 2014, 35, 237-246. (6) Jiang, J. Hui; C. C. Hedgehog signalling in development and cancer. Dev. Cell 2008, 15, 801–812. (7) Pak, E.; Segal, R. A. Hedgehog signal transduction: key players, oncogenic drivers and cancer therapy. Dev. Cell 2016, 38, 333-344. (8) Gibson, P.; Tong, Y.; Robinson, G.; Thompson, M. C.; Currle, D. S.; Eden, C.; Kranenburg, T. A.; Hogg, T.; Poppleton, H.; Martin, J.; Finkelstein, D.; Pounds, S.; Weiss, A.; Patay, Z.; Scoggins, M.; Ogg, R.; Pei, Y.; Yang, Z. J.; Brun, S.; Lee, Y.; Zindy, F.; Lindsey, J. C.; Taketo, M. M.; Boop, F. A.; Sanford, R. A.; Gajjar, A.; Clifford, S. C.; Roussel, M. F.; McKinnon, P. J.; Gutmann, D. H.; Ellison, D. W.; Wechsler-Reya, R.; Gilbertson, R. J. Subtypes of medulloblastoma have distinct developmental origins. Nature 2010, 468, 1095−1099. (9) Reifenberger, J.; Wolter, M.; Knobbe, C. B.; Köhler; Schönicke, A.; Scharwächter, C.; Kumar, K.; Blaschke, B.; Ruzicka, T.; Reifenberger, G. Somatic mutations in the PTCH, SMOH, SUFUH, and TP53 genes in sporadic basal cell carcinoma. Br. J. Dermatol. 2005, 152, 43−51.


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Table of Graphic:

Design, Synthesis and Pharmacological Evaluation of 2-(2,5-Dimethyl-5,6,7,8-tetrahydroquinolin-8-yl)-N-aryl Propanamides as Novel Smoothened (Smo) Antagonists Gang Liu,†,ǁ,§ Jun Yang,‡,§ Juan Wang,‡,§ Xiaohua Liu,†,ǁ Wenjing Huang‡, Jie Li,ǁ Wenfu Tan,*,‡ and Ao Zhang*,†,ǁ

H O O O

retain the poly oxycycle and peroxide of 3 couple with a Smotargeting bullet

H

H

H

O O O

O O O

H

O

1 tegy Stra

N

gy te

destroy the instable skeleton of 3 reconstruct into new heterocycles

Ar

couple with a Smotargeting bullet

N

O

N

Cl

H N

2

COOH

H N

Cl

O

O 3 (artemisinin)

H

O

H N

O

ra St

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 72 of 72

65 O

N N Cl


IC50 = 9 nM F%: 27% > 80% TGI

Design, Synthesis, and Pharmacological ... - ACS Publications

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