Discovery of Conformationally Restricted Human Glutaminyl Cyclase

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Discovery of Conformationally Restricted Human Glutaminyl Cyclase Inhibitors as Potent Anti-Alzheimer’s Agents by Structure-Based Design Van-Hai Hoang, Van T.H. Ngo, Minghua Cui, Nguyen Van Manh, PhuongThao Tran, Jihyae Ann, Heejin Ha, Hee Kim, Kwanghyun Choi, Young Ho Kim, Hyerim Chang, Stephani Joy Y. Macalino, Jiyoun Lee, Sun Choi, and Jeewoo Lee J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00751 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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Journal of Medicinal Chemistry

Discovery of Conformationally Restricted Human Glutaminyl Cyclase Inhibitors as Potent Anti-Alzheimer’s Agents by Structure-Based Design

Van-Hai Hoang,†,# Van T.H. Ngo,⊥,# Minghua Cui,‡,# Nguyen Van Manh,† Phuong-Thao Tran,¶ Jihyae Ann,† Hee-Jin Ha,§ Hee Kim,§ Kwanghyun Choi,§ Young-Ho Kim,§ Hyerim Chang,‡ Stephani Joy Y. Macalino,‡ Jiyoun Lee,∥ Sun Choi,*,‡ Jeewoo Lee*,†

Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of

†

Pharmacy, Seoul National University, Seoul 08826, Republic of Korea ‡

National Leading Research Laboratory of Molecular Modeling & Drug Design, College of

Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of Korea §

Medifron DBT, Sandanro 349, Danwon-Gu, Ansan-City, Gyeonggi-Do 15426, Republic of Korea

∥

Department of Global Medical Science, Sungshin University, Seoul 01133, Republic of Korea

⊥

Laboratory of Theoretical and Computational Biophysics & Faculty of Pharmacy, Ton Duc

Thang University, Ho Chi Minh City 75307, Vietnam ¶

Department of Pharmaceutical Chemistry, Hanoi University of Pharmacy, Hanoi 10000,

Vietnam

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 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

ABSTRACT Alzheimer’s disease (AD) is an incurable, progressive neurodegenerative disease whose pathogenesis cannot be defined by one single element but consist of various factors; thus, there is a call for alternative approaches to tackle the multifaceted aspects of AD. Among the potential alternative targets, we aim to focus on glutaminyl cyclase (QC), which reduces the toxic pyroform of β-amyloid in the brains of AD patients. On the basis of a putative active conformation of the prototype inhibitor 1, a series of N-substituted thiourea, urea and α-substituted amide derivatives was developed. The structure-activity relationship analyses indicated that conformationally restrained inhibitors demonstrated much improved QC inhibition in vitro compared to nonrestricted analogs, and several selected compounds demonstrated desirable therapeutic activity in an AD mouse model. The conformational analysis of a representative inhibitor indicated that the inhibitor appeared to maintain the Z-E conformation in the active site, as is critical for its potent activity.


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■ INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with cognitive and behavioral dysfunction that ultimately leads to severe dementia. A variety of therapeutic strategies have been developed to provide symptomatic relief; however, no diseasemodifying therapies have yet emerged because the causes and pathogenesis of AD have not been completely elucidated. One of the leading theories of AD pathogenesis is “the amyloid hypothesis,” which has now been put into question because clinical trials have largely failed to confirm it.1,2 Most of the tested drugs are small molecules and antibodies targeting Aβ producing enzymes and Aβ aggregates, and they seemingly show little efficacy or unacceptable toxicity. Nevertheless, there is a general consensus that anti-amyloid therapy is still a viable approach for early stage patients, given that the accumulation of the Aβ peptides begins decades before any symptoms appear.3 Moreover, there are numerous types of Aβ fibrils and oligomers that are either neurotoxic4,5 or possibly neuroprotective6,7; thus, targeting specific forms of Aβ species may provide effective treatment options in addition to other potential alternatives. The N-truncated Aβ peptides generated from the N-terminal hydrolysis of Aβ are the main constituents of amyloid plaques, represented as Aβn-40/42, where n ranges from 2 to 11.8-10 Interestingly, more than 50% of the total Aβ plaques in the AD brain comprise pyroglutamate Aβ (pE-Aβ), AβΝ3pE3-40/42 and AβΝ3pE11-40/42, which are produced by cyclization of the N-terminal glutamates (E), E3 and E11 in Aβ.11-13 The pE-Aβs appear to be present only in AD brain, not normal brain, and aggregate quickly and irreversibly into soluble toxic oligomers that form inert amorphous fibrils over a long period of time. In addition, pE-Aβs function as key initiators of AD pathogenesis by acting as seeds for aggregation of Aβ1-40/42 and tau,14-16 suggesting that the pE-Aβs may serve as a potential target to augment current anti-amyloid strategies.17



The pE-Aβ peptides are generated by glutaminyl cyclase (QC), which is mainly found in the hippocampus and cortex of mammalian brain.18-20 Importantly, QC is overexpressed in the brains of AD patients and animal models,21,22 and the knock-out of QC rescued cognitive function in AD model mice.23 It has also been reported that small molecule inhibitors of QC efficiently reduced the brain levels of pE-Aβ and Aβ plaques24 and improved memory deficits in AD mice.22 These studies suggested that inhibition of brain QC activity may bring about beneficial effects for AD patients.25,26 Several research groups have developed QC inhibitors that are based on imidazole (1)27 and benzimidazole core structures (2, 3)28,29 (Figure 1). The most prominent candidate is PQ912 (3), developed by Probiodrug, which showed improvement in synaptic and neurological functions in AD patients without significant toxicity in clinical trials.30,31 More recently, Rubinsztein and colleagues introduced the N-methyltriazole-based inhibitor (4, SEN177), which was discovered through 3D-pharmacophore model-based in silico methods.32 Similarly, Wu and colleagues reported a diphenyl group conjugated imidazole inhibitor (5), which significantly decreased the amount of pE-Aβ3-42 in a cell-based model and improved behavior in AD model mice.33 Naturally occurring inhibitors34,35 and other small molecule inhibitors developed by the fragment-based approach36 have also been reported, demonstrating moderate in vitro activity. It should be noted that while these QC inhibitors reduced brain levels of pE-Aβ3-42 and improved cognitive functions, it is still in debate whether these inhibitors directly affected the QC activity in brain. Brooks and colleagues reported that compound 1a did not appear to cross the blood brain barrier (BBB) in mice, suggesting that the therapeutic effect of QC inhibition may be associated with peripheral mechanisms.37


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Figure 1. Reported QC inhibitors

Previously reported QC inhibitors are designed based on the N-terminal (Glu-Phe) structure of its substrate Aβ3-42, containing the three principle pharmacophores including zinc-binding group (A), hydrogen bonding donor (B) and phenyl group (C), respectively, in inhibitor 1. In addition to this basic scaffold, our research group previously reported a series of QC inhibitors with a new extended pharmacophore based on the N-terminal tripeptide (Glu-Phe-Arg) of Aβ3E-42. The additional pharmacophore mimicking Arg (D) was tethered to the phenyl group of compound 6 (Figure 1).38 The extended pharmacophore helped to improve the potency, ranging from 5 to 40fold enhancement compared to 1. In vivo experiments also indicated that compound 6 significantly reduced the brain concentrations of pE-Aβ and total Aβ while restoring cognitive functions in an AD animal model. Our molecular modeling studies revealed that the Arg mimetic 2-aminopyridine group (colored in magenta) appeared to form strong interactions with the carboxylate group of Glu327 in the QC binding site, supporting our hypothesis that the additional pharmacophore provides an extra binding interaction. Since the 2-aminopyridine group in compound 6 can freely



rotate in the active site, there may be a favorable conformation that will generate more potent inhibitors. The X-ray structure of the hQC-PBD150 (1a) complex39 showed that the ligand resided in the hQC active site with a bent conformation, the so-called Z-E conformation. We performed the docking analysis of the prototype inhibitor 1 using the same X-ray crystal structure of hQC, and compound 1 in the similar Z-E orientation appeared to form favorable interactions in the active site as shown in Figure 2.

Figure 2. The docked pose of 1 in the Z-E conformation in hQC. 1 is displayed as sticks with purple carbon atoms, and Zn2+ is the magenta ball. The interacting residues are shown as thin sticks with their carbon atoms in light blue. Hydrogen bonds are depicted as black dashed lines. Based on these observations, we hypothesized that a rigidification strategy to generate the bent conformation of 1 might improve its binding potency. Therefore, we decided to incorporate a conformational blocker to the thiourea/urea nitrogen that is proximal to the dimethoxyphenyl group to induce the formation of Z-E conformers as described in Figure 3.


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Figure 3. Strategy to facilitate Z-E conformation by N-substitution on thiourea/urea analogs

In this work, we synthesized a series of N-substituted thiourea/urea analogs of 1 and investigated their structure-activity relationships (SAR) by performing in vitro enzyme activity assays. We added alkyl groups of different sizes and added various aromatic and heteroaromatic functional groups to search for additional binding interactions. Several potent inhibitors were selected based on in vitro activity, and in vivo efficacy was further evaluated by measuring their ability to reduce the formation of Aβ3(pE)-42 in acute and transgenic AD mice models. Lastly, we performed molecular docking studies to identify the specific binding interactions of the most potent compound and examined our hypothesis.

■ RESULTS AND DISCUSSION Chemistry. The N-substituted thiourea and urea compounds were obtained by the coupling reaction between N-substituted 3,4-dimethoxyaniline fragments and the imidazole, 1-(3-azidopropyl)-5methyl-1H-imidazole or 3-(5-methyl-1H-imidazol-1-yl)propan-1-amine. The syntheses of N-substituted 3,4-dimethoxyaniline derivatives were accomplished by the two different synthetic pathways where either 3,4-dimethoxyaniline (7) or its 2-



nitrobenzenesulfonamide (33) were directly N-substituted (Scheme 1). In the first pathway, most of the N-substituted 3,4-dimethoxyaniline derivatives (8, 9 and 11-29) were synthesized by the reductive amination of 7 using corresponding aldehydes or ketones, respectively (Method A). In the case of N-cyclopropyl derivative 10, cyclopropanone was generated in situ from 1-ethoxy-1(trimethylsilyloxy)cyclopropane under reflux in acidic condition.40 The N-phenyl derivative 30 was obtained by the Buchwald-Hartwig amination using iodobenzene (Method B). N-thiazol-2-yl derivative 31 was prepared using 2-chlorothiazole through SNAr reaction under acidic condition (Method C). N-oxazol-2-yl derivative 32 was prepared by the modified Robinson-Gabriel method41 in which the phenyl carbamate of 7 was reacted with aminoacetaldehyde dimethyl acetal, and then the resulting urea was cyclized in situ under acidic condition (Method D). In the second pathway, N-substituted aniline derivatives were synthesized through 2-nitrobenzenesulfonamide derivative 33, prepared by reacting 7 with 2-nitrobenzenesulfonyl chloride.42 The N-alkylation of 33 was conducted by either direct alkylation with corresponding alkyl halides for the syntheses of 34-35 or Mitsunobu reaction with the corresponding alcohols for the syntheses of 36-43. The protected sulfonamide group was readily cleaved by a nucleophilic aromatic substitution with thiophenol in basic media.


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Scheme 1. Synthesis of N-substituted 3,4-dimethoxyaniline derivativesa

Reagents and conditions: (a) (Method A) aldehyde or ketone, NaBH3CN, AcOH, MeOH, 40 °C, 4 h; 1ethoxy-1-(trimethylsilyloxy)cyclopropane, AcOH/MeOH (1:1), NaBH3CN, reflux, overnight; (Method B) iodobenzene, Pd(OAc)2, xantphos, Cs2CO3, dioxane, reflux, overnight for 30; (Method C) 2-chlorothiazole, TsOH, EtOH, reflux, overnight for 31; (Method D) i) PhOCOCl, NEt3 then (CH3O)2CHCH2NH2, CH2Cl2, r.t., overnight; ii) c-HCl, MeOH, r.t., 2 h for 32; (b) 2-nitrobenzenesulfonyl chloride, NEt3, CH2Cl2, 0 °Cr.t., 4 h; (c) alkyl halide, Cs2CO3, DMF, r.t., 2 h; (d) hydroxyalkyl derivatives, Ph3P, DEAD, CH2Cl2, r.t., overnight; (e) thiophenol, K2CO3, MeCN, r.t., overnight. a

The syntheses of thiourea and urea compounds were accomplished by coupling Nsubstituted 3,4-dimethoxyaniline (8-43) prepared in Scheme 1 with either one of the two imidazole fragments (44, 45) (Scheme 2). 3-(5-Methyl-1H-imidazol-1-yl)propan-1-amine 44 was obtained by following the previously described method,27 and its azide surrogate 45, 1-(3-azidopropyl)-5methyl-1H-imidazole, was prepared from 44 using the diazo-transfer reagent, imidazole-1-sulfonyl azide hydrochloride, under CuSO4 catalysis in basic media.43 Aza-Wittig coupling reactions44



between the synthesized N-substituted 3,4-dimethoxyaniline fragments and 45 under carbon disulfide provided the N-substituted thiourea compounds 46-61. Meanwhile, the use of carbon dioxide instead of carbon disulfide afforded N-substituted urea compounds (62-79, 81, 87, 88, 9096 and 98) (Method E) and Boc-protected penultimate intermediates, which were hydrolyzed to final amine compounds 80, 82-85 and 97 (Method F). N-Phenyl urea derivative 86 was obtained by the coupling reaction using triphosgene between the two amines, 30 and 44 (Method G). NPiperidin-4-yl urea derivative 89 was obtained from N-benzylpiperidin-4-yl urea 94 by hydrogenation in acidic condition (Method H).

Scheme 2. Synthesis of N-substituted thiourea and urea compoundsa

Reagents and conditions: (a) 1H-imidazole-1-sulfonyl azide hydrogen chloride, CuSO4, K2CO3, MeOH, r.t., overnight; (b) CS2, Ph3P, toluene, heat, 4 h to overnight; (c) (Method E) CO2, Ph3P, toluene, heat, 4 h to overnight; (Method F) i) CO2, Ph3P, toluene, heat, 4 h to overnight, ii) CF3CO2H, CH2Cl2, r.t., overnight; (Method G) triphosgene, pyridine, CH2Cl2, r.t. then 44, reflux; (Method H) i) CO2, Ph3P, toluene, heat, 4 h to overnight, ii) Pd/C, c-HCl, H2, MeOH, r.t.

a

The syntheses of α-substituted amide derivatives were carried out by following the pathway described in Scheme 3. 2-(3,4-Dimethoxyphenyl)acetonitrile 99 was alkylated with the corresponding alkyl halides, which were then hydrolyzed to respective acids in basic aqueous


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medium at high temperature. The resulting acids were condensed with amine 44 using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) reagent to afford the final compounds, 100-108, respectively.

Scheme 3. Synthesis of α-substituted amide compoundsa

Reagents and conditions: (a) NaH, alkyl halide, THF or DMF, r.t. to 60 oC; (b) NaOH, H2O, EtOH, reflux, 24 h; (c) 44, EDC.HCl, HOBt, NEt3, r.t., overnight.

a

In vitro activity. Structure Activity Relationships of QC inhibitors The QC inhibitory activities of final compounds were determined by using a fluorogenic substrate, Gln-AMC (L-glutamine 7-amido-4-methylcoumarin) and pyroglutamyl peptidase (pGAP) as an auxiliary enzyme, as described previously.38,45 These results are summarized in Tables 1-4, together with the IC50 values of the previously reported inhibitors 1-5 for comparison. First, we investigated the SAR of the N-substituted thiourea derivatives, as shown in Table 1. The incorporation of alkyl groups, such as methyl (46), ethyl (47) and isopropyl (48) groups, increased the potencies by 2-, 3- and 10-fold, respectively, compared to 1. As the size of



the conformation-blocking alkyl group increased, the QC inhibition also improved – likely due to the formation of the Z-E conformer, the active conformation. Isobutyl (49) and cyclopentylmethyl (50) derivatives also exhibited similar degrees of potency, suggesting that the improvement in activity arises from the favorable conformational conversion, rather than from the increased lipophilicity. When cyclic alkyl groups, including cyclopropyl (51), cyclobutyl (52), cyclopentyl (53), and cyclohexyl (54), were incorporated into the same position, the cyclopropyl derivative (51) displayed a similar activity compared to 1, whereas cyclobutyl and cyclopentyl derivatives exhibited improved potencies of up to 6-fold as the size of substituent increased progressively. The incorporation of an aromatic ring generally yielded much improved potency, as demonstrated by compounds 55-61 with IC50 values in the low nanomolar range. Notably, the N-4-fluorobenzyl derivative (58) displayed excellent inhibition, with an IC50 value of 1.3 nM: much more potent than its parent compound 1.

Table 1. IC50 values for inhibition of hQC by N-substituted thiourea compounds

Compound

R

1

H

IC50 (nM)a 29.2

2

47.9

3

29.0

4

16.7

5

118

6

4.5


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46

 Me

47 48 49 50

12.4

(±1.8)



8.8

(±1.2)



2.8

(±2.4)

6.8

(±2.7)

4.1

(±0.3)





51



25.6

(±4.3)

52



8.6

(±0.8)

53



6.5

(±0.8)

54



4.7

(±0.4)

4.6

(±0.4)

3.4

(±0.9)

3.5

(±2.0)

F

1.3

(±0.8)

Cl

5.2

(±0.8)

5.4

(±3.7)

5.6

(±3.4)

55





56 F 

57 Cl

58 59

60

61





 N  N



The values indicate the mean of at least three experiments and the numbers in parentheses refer to the standard deviation a

Next, we investigated the SAR of the urea derivatives as shown in Tables 2-3. The unsubstituted urea (62) was found to be much less potent than its thiourea surrogate 1. However, we observed a similar trend in the thiourea derivatives once an alkyl group was introduced to the urea nitrogen. The QC inhibition increases upon the addition of the N-alkyl groups with increasing size as follows: acyclic (N-methyl (63) < N-ethyl (64) < N-isopropyl (65) < N-isobutyl (66) < Ncyclopentylmethyl (67)) and cyclic alkyl substituents (N-cyclopropyl (68) < N-cyclobutyl (69) < N-cyclopentyl (70) < N-cyclohexyl (71)). In particular, N-cyclopentylmethyl (67) and Ncyclohexyl (71) derivatives exhibited 26- and 36-fold enhancements in potency compared to 62, indicating that the effect of the conformational restriction was found to be more marked in the series of urea than that of thiourea. Among the N-benzylic urea derivatives (72-76), the N-4fluorobenzyl derivative (75) also showed the most potent activity in this series, having the IC50 value of 3.9 nM. N-Pyridinyl derivatives (77-82) displayed a similar extent of inhibition with a range of IC50 = 8.7-32.5 nM. Previously, we have found that the 2-aminopyridine moiety in 6 provided an additional binding interaction with the enzyme.38 Therefore, we incorporated this group on the nitrogen with various spacer lengths. Surprisingly, N-2-aminopyridinyl derivatives 84 and 85 with a 4-carbon linker displayed excellent inhibitions, having the IC50 values of 3.6 and 1.6 nM, respectively. Because both of these compounds contained a flexible linker, we believe that their potent activities may arise from a strong ionic interaction between the 2-aminopyridyl group and the carboxylate of Glu327 rather than conformational restriction based on our docking study (Figure S1). N-Phenyl and N-heterocyclic derivatives in this series (86-88) showed only modest inhibitions.


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Table 2. IC50 values for inhibition of hQC by N-alkyl/aryl substituted urea compounds

Compound

R

62

H

IC50 (nM)a 162

(±18.5)

63

 Me

78.7

(±20.6)

64



27.0

(±1.4)

65



17.5

(±0.6)

7.6

(±3.4)

6.2

(±1.9)

66 67





68



38.5

(±16.3)

69



26.5

(±2.1)

70



19.4

(±5.1)

71



4.5

(±0.1)

13.3

(±1.3)

20.5

(±0.7)

12.9

(±6.5)

72





73 F 

74 Cl



75 76

77

78

79

80

81



(±0.5)

12.5

(±2.1)

8.7

(±1.4)

F

30.6

(±13.3)

Cl

32.5

(±6.4)

NH2

23.5

(±9.1)

11.0

(±1.4)

17.6

(±7.3)

36.9

(±5.2)

3.6

(±1.1)

1.6

(±0.3)

93.0

(±18.4)

69.6

(±11.4)

98.8

(±9.3)

 Cl

 N  N  N  N  N

82



83



NH2 N

NH2 N



NH2

84

85

3.9

F

N



HN

NH2

N

86



87



88



N O N S


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Based on the finding that the N-cyclohexyl urea derivative (71) was one of the most potent inhibitors, we decided to study additional analogs containing bioisosteric piperidine surrogates, as shown in Table 3. The N-piperidin-4-yl (89) and N-(1-methylpiperidin-4-yl) (90) derivatives exhibited potent inhibitions, with IC50 values of 8.2 and 6.1 nM, respectively, comparable to that of 71. Incorporation of bulkier groups, such as ethyl (91) and acetyl (92) groups, on the nitrogen of piperidine led to marked reductions in potency. On the other hand, phenyl (93) and benzyl (9496) groups in the same position did not affect potency significantly. N-piperazinylethyl (97) and morpholinylethyl (98) derivatives showed moderate inhibitions.

Table 3. IC50 values for inhibition of hQC by N-piperidinyl urea compounds

Compound

IC50 (nM)a

R

89



NH

8.2

(±0.9)

90



N

6.1

(±1.5)

91



N

81.0

(±30.5)

92



N

53.9

(±5.2)

93



N

16.4

(±5.8)

O



94



N



N

95

7.5

(±1.9)

13.6

(±3.5)

23.3

(±6.4)

F

96

97 98



N N

 N

NH

15.0

(±3.9)

N

O

25.0

(±3.8)




Finally, we examined α-substituted amide derivatives as the bioisosteres of the N-alkyl urea moiety. The unsubstituted amide (100) showed modest activity comparable to that of the urea compound 62. However, incorporation of alkyl groups at the α-position led to a progressive improvement in potency as conformational restriction was enhanced (100 < 101 (methyl) < 102 (dimethyl) < 103 (cyclopropyl)). The ring expansion at the same position (104-106) did not improve potency any further. α-Benzyl amide analogues (107, 108) showed only moderate inhibition. In general, α-substituted amide analogs appeared to be less potent than N-substituted urea analogs.


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Table 4. IC50 values for inhibition of hQC by α-substituted amide compounds

Compound 100 101 102 103 104

105

IC50 (nM)a

X 



























106 

108

(±12.7)

112

(±34.5)

77.3

(±18.5)

24.3

(±4.5)

37.1

(±12.2)

22.9

(±4.2)

77.5

(±0.7)

39.0

(±1.4)

81.6

(±18.9)



107



183

 N




Toxicity Study On the basis of the in vitro QC inhibition, we selected the 24 most potent compounds having IC50 values less than 10 nM for further evaluation. For in vitro toxicity study as shown in Table 5, we first examined the cell viability of each compound in a hippocampal neuronal cell line (HT-22) and found that a total of 11 compounds were nontoxic at 10 μM. We also examined the human ether-a-go-go-related gene (hERG) potassium channel inhibition of each compound and found that 16 compounds exhibited less than 50% inhibition at the same concentration.

Table 5. Toxicity studies of selected QC inhibitors Cell viability at 10 μM (% of control) 88.4

hERG FP (% inhibition at 10 μM)

47

In vitro IC50 (nM) 8.8

48

2.8

100

46.3

49

6.8

100

39.9

50

4.1

100

60.0

52

8.6

86.7

11.5

53

6.5

81.7

31.8

54

4.7

78.3

59.4

55

4.6

80.4

71.5

56

3.4

91.7

77.6

57

3.5

88.2

86.0

58

1.3

100

83.5

59

5.2

100

86.8

60

5.4

89.3

34.5

61

5.6

100

32.3

66

7.6

100

23.7

67

6.2

100

29.6


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71

4.5

88.6

12.2

75

3.9

90.7

40.6

77

8.7

100

33.2

84

3.6

100

48.0

85

1.6

82.2

40.8

89

8.2

88.3

26.3

90

6.1

100

46.1

94

7.5

92.4

73.5

In vivo activity. Acute AD model To assess in vivo efficacy, we tested all 24 compounds that were selected from our in vitro activity study in the acute mouse model of AD. In this study, we injected 5 μg of human Aβ3-40, together with each compound (25 mg/kg), into ICR mice (male, six weeks old) by intracerebroventricular (icv) injection. We determined the activity of each compound on the next day by measuring the levels of human AβN3pE-40 in the brain extracts of each of the mice. As shown in Table 6, four compounds (77, 84, 89 and 90) suppressed greater than 20% of the formation of AβN3pE-40 compared to the vehicle control. While the tested compounds mostly demonstrated similar in vitro activities, their in vivo activities appear to vary. We believe that this apparent discrepancy can be attributed to the use of different substrates and enzyme species for in vitro assays (glutaminyl-AMC tested against human QC) and in vivo studies (human Aβ3-40 tested in mice), as well as compound-specific characteristics such as solubility, enzyme reaction kinetics, and metabolic rate. To examine whether these compounds exert their activity by penetrating the blood-brain barrier (BBB), we again performed the same experiment by injecting the three effective compounds (77, 84, and 90) via intraperitoneal (ip) injection while human Aβ3-40 was injected by



icv injection prior to the ip administration. As shown in Table 5, all three compounds exhibited significant AβN3pE-40 lowering effects (> 20% inhibition) that were also comparable to the activities observed in the icv administered experiments, suggesting that these compounds are likely to cross the BBB. However, since these experiments are an indirect measure comparing biological effects after the administration, possible peripheral association with QC inhibitory effect should be considered for future mechanism studies.

Table 6. QC inhibition in acute model studies in vivo.a In vitro IC50 (nM)

% inhibition of AβN3pE-40 formation (icv injected) (ip injected)

47

8.8

NE

48

2.8

6.0

49

6.8

NE

50

4.1

2.96

52

8.6

NE

53

6.5

1.17

54

4.7

4.01

55

4.6

15.28

56

3.4

6.66

57

1.3

NE

58

3.5

NE

59

5.2

0.73

60

5.4

5.31

61

5.6

11.58

66

7.6

NE

67

6.2

2.96

71

4.5

NE

75

3.9

NE


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77

8.7

25.98

22.87

84

3.6

24.38

22.74

85

1.6

13.70

89

8.2

26.26

NT

90

6.1

35.46

26.10

94

7.5

19.40

5 μL of human Aβ3−40 in PBS (1 μg/μL) was injected into the deep cortical/hippocampus of 5-week-old ICR mice (25 g, n = 4, male) using a stereotaxic frame to induce acute Aβ toxicity. Test compounds were administered via icv or ip injection. Sandwich ELISA was performed for the quantification of the brain AβN3pE-40. NE: not effective, NT: not tested a

Additionally, we determined mouse and human liver microsomal stabilities of these three compounds. The remaining percentage values of intact compounds after 30 min for 77, 84 and 90 were found to be 12.8%, 21.6% and 89% for mouse and 44%, 43.1% and 100% for human, respectively, indicating that 90 was the most stable in metabolism. We further tested compound 90 for selectivity against isoQC, an isozyme of QC with a differential substrate specificity.46 Compound 90 was found to have an IC50 value of 304 nM for isoQC, which was more than 50-fold greater than that for QC, indicating that 90 is highly specific for QC. We also determined the IC50 value of 90 in mouse QC as 28.4 nM, indicating that 90 is an hQC selective inhibitor.

AD Model To assess the desired therapeutic effects of 90 in AD, we performed long-term in vivo studies in 5XFAD transgenic mice. It has been reported that 5XFAD mice generate severe AD related pathologies with massive increases of Aβ and pyroform Aβ in the brain47; this model is thus suitable to study Aβ-induced neurodegeneration. Compound 90 was administered by ip injection to 32-week-old 5XFAD mice at a dose of 25 mg/kg every day for 4 weeks. After finishing the 4



week administration, the brain concentrations of AβN3pE-40/42 and Aβ40/42 were measured by sandwich ELISA. As shown in Figure 4, compound 90 significantly reduced the brain concentrations of AβN3pE-40 (Figure 4A) and AβN3pE-42 (Figure 4B) by 57.9 and 50.5 percent, respectively, compared to the vehicle control. Compound 90 also reduced the Aβ40 and Aβ42 levels (Figure 4C and 4D) substantially, by 36.6 and 35.1 percent without significant toxicity, indicating that the inhibition of QC not only reduced the amount of pE-Aβ in brains but also contributed to the overall reduction in Aβ levels as previously reported.48,49

Figure 4. Inhibition of the formation of human AβN3pE-40/42 and Aβ1-40/42 in 5XFAD mice. (a) Brain concentrations of AβN3pE-40; (b) brain concentrations of AβN3pE-42; (c) brain concentrations of Aβ40; (c) brain concentrations of Aβ42. Data were analyzed by unpaired t-test or one-way ANOVA. (*: p < 0.05, **: p < 0.01).


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Computational study. Our initial hypothesis of this study is that since the bent conformation of compound 1 with a Z-E orientation is the most active conformation, the N-substituted thiourea/urea analogs with a conformational blocker will favor the Z-E conformation and thus improve the overall potency. Both in vitro and in vivo activity tests showed enhanced activity compared to the unsubstituted analogs, which is consistent with our hypothesis. To better understand how our conformational restriction strategy worked to improve QC inhibition, we performed comprehensive computational analysis. We first performed conformational analysis for the prototype inhibitors 1 and 62. The unsubstituted thiourea 1 displayed the Z-E form as the dominant conformation, whereas the unsubstituted urea 62 had both Z-Z and Z-E conformations, with Z-Z as the major form. We also employed an incremental conformation search by calculating the dihedral angles of the urea/thiourea groups. The plot showing the relationship between energy and conformation revealed that lower energy was observed for 62 when its urea group was in the Z-Z form, suggesting that 62 was more stable in the Z-Z conformation (Figure 5A). On the other hand, the corresponding thiourea group of 1 was more stable in the Z-E conformation (Figure 5B). Given that the bioactive conformation of the QC inhibitor favors the Z-E form and that 62 has a mixture of Z-Z/Z-E conformations, this analysis explains why compound 62 displays 5 times less inhibitory activity than that of 1.



Figure 5. Plots of energy versus incremental conformational changes from calculated dihedral angles (A) Thiourea in 1, (B) urea in 62, and (C) N-piperidinyl urea in 90. The X axis represents the dihedral angle (C-C-N-C) found near the 5-methyl imidazole ring, while the Y axis represents the dihedral angle (N-C-N-C) near the dimethoxy phenyl group. Dihedral angle degree close to 0 denotes the E-form, and close to 180/-180 denotes the Z-form. The plots show that low energy conformations occupied mostly Z-E form in thiourea 1 and N-substituted urea 90. In contrast, diversity (both Z-Z and Z-E) of low energy conformations exist for the urea compound, 62.

Using the most stable conformers from the conformational analysis, we optimized and calculated the total free energy of both 1 (Z-E) and 62 (Z-Z). Only minimal changes were observed after optimization, as both structures maintained the stable conformations. The resulting total free energies of 1 and 62 are -1390.33 and -1067.37 kcal/mol, respectively. These values indicate that the structure of compound 1 is more stable than that of 62, whose higher energy may result from the unconstrained dihedral of its urea group leading to a mixed conformational population. Among the synthesized compounds in which different substituents were added to the proximal nitrogen of urea/thiourea, 90 exhibited excellent inhibitory activity toward hQC, with 5- and 26-fold increased activity compared to 1 and 62, respectively. The conformational analysis of 90 demonstrated that the Z-E form was its sole conformation with high stability, consistent with our design strategy (Figure 5C). The most stable conformer of 90 has a total free energy of -1357.68 kcal/mol, which


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is comparable to that of 1. This analysis supported our hypothesis that the design strategy restricted to the bioactive Z-E form provided more potent inhibitors against hQC. To investigate the binding interactions of 90, the protonated piperidine form of 90 at pH 7.4 was used for Glide QM-Polarized Ligand Docking. The results indicated that 90 was docked very well at the active site of hQC39 with Z-E conformation as anticipated. The binding interactions examined were as follows: the 5-methyl imidazole group chelated with zinc and formed H-bonding interaction with the indole NH of Trp329. Additionally, it produced hydrophobic interactions with Leu249 and Trp207. The oxygen of urea formed an H-bonding interaction with Gln304. The 3,4dimethoxy phenyl group displayed hydrophobic interactions with Ile321, Pro324, and Phe325, in which the phenyl ring presented π-π interactions with Phe325. The N-substituted piperidinyl group not only restricted the conformation but also showed hydrophobic interactions with Tyr299, Ile303, and Val302 (Figure 6).

Figure 6. Docked result of 90 in hQC. (A) Binding interactions of 90 at the active site of hQC. 90 is displayed as sticks with cyan carbon atoms, and Zn2+ is the purple ball. The interacting residues are shown as thin sticks with their carbon atoms in light blue. Hydrogen bonds are depicted as black dashed lines. (B) 2D representation of the interactions of 90 with the active site residues of the hQC. Hydrophobic interactions are marked in light brown. Hydrogen bonds are shown as



red-dotted arrows with directionality. The π-π stacking interaction is marked by a blue disc and arrow.

■ CONCLUSION In this work, we developed a series of N-substituted thiourea and urea surrogates of 1 as conformationally restricted analogs that favor a bent conformation (Z-E conformation). Our indepth SAR analyses indicated that conformational restriction by the proximal N-substituent was able to improve the potency with respect to QC inhibition by up to 20-fold for thiourea, 100-fold for urea and 8-fold for amide. Among the library of compounds, N-4-fluorobenzyl thiourea (58) and urea (75) analogs exhibited excellent QC inhibition, with the IC50 values of 1.3 and 3.9 nM, respectively. For further evaluation of the selected inhibitors with IC50 values of less than 10 nM, we measured the levels of AβN3pE-40 in an acute model and examined their cytotoxicities, hERG inhibitions and metabolic stabilities. Among the selected inhibitors, 90 showed the most promising efficacy and druggable profile and thus was evaluated in the AD model for in vivo efficacy. After 4 weeks of administration, 90 significantly reduced the brain concentrations of pyroform Aβ and total Aβ in the 5XFAD mouse model. The conformational analysis of the two prototype inhibitors 1 and 62 indicated that unsubstituted thiourea 1 displayed mostly a bioactive Z-E conformation, whereas unsubstituted urea 62 existed in a mixed conformation with a major Z-Z form, explaining why 62 is much less potent than 1. The docking analysis of 90 indicated that the Z-E conformer is the dominant form and displayed the three key interactions in the active site, supporting our hypothesis that the introduction of a conformational blocker favors the active conformation. Although recent clinical failures in anti-amyloid therapies might have disappointed researchers in the related field, amyloid-targeting drugs can still be beneficial for early-stage AD patients, and they offer advantages for combination therapy by lowering the levels of toxic Aβ species.7 We


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demonstrated that highly potent and selective QC inhibitors with promising in vitro and in vivo activity can be rationally designed, which may open a new avenue for alternative therapeutic strategies. ■ EXPERIMENTAL SECTION General. All chemical reagents were commercially available. A Buchi B-540 apparatus was used to determine melting points, which are uncorrected. Silica gel flash column chromatography was performed on silica gel 60, 230–400 mesh, Merck. The PLC plates used in the present study were PLC silica gel 60 F254, 1 mm, Merck. Proton NMR spectra were detected on a JEOL JNM-LA 300 at 300 MHz, Bruker Analytik, DE/AVANCE Digital 400 at 400 MHz, a Bruker Analytik, DE/AVANCE Digital 500 at 500 MHz, and a JEOL JNM-ECA-600 at 600 MHz. Chemical shifts are calculated in ppm units, with Me4Si as a reference standard. A VG Trio-2 GC−MS instrument and a 6460 Triple Quad LC−MS instrument were used to determine mass spectra. All final compounds were purified to > 95% purity using high-performance liquid chromatography (HPLC) on an Agilent 1120 Compact LC (G4288A) instrument using Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm) and an Agilent Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm).

General procedure for reductive amination (Procedure A). To a mixture of 3,4dimethoxyaniline 7 (1.0 eq) and ketone or aldehyde (1.0 eq) in MeOH was added acetic acid (5.0 eq), followed by NaBH3CN (3.0 eq). After stirring at 40 °C for 4 h, the reaction mixture was basified by NaHCO3 solution and then extracted with CH2Cl2. The organic layer was concentrated in vacuo and purified by silica gel column chromatography (EtOAc/ n-hexane, 1:4 to 1:1) to obtain the secondary amine 8-29. General procedure for N-alkylation (Procedure B). A mixture of sulfonamide 33 (1.0 eq), alkyl



halide derivative (1.0 eq) and excess cesium carbonate in DMF was stirred at 60 °C for 1 h or at room temperature for 2 h. The reaction mixture was quenched by water and extracted three times with EtOAc. The combined organic layer was washed with water and brine, concentrated in vacuo and used for the next step without further purification. General procedure for Mitsunobu-type reaction (Procedure C). To a mixture of sulfonamide (1.0 eq), alcohol (1.5 eq) and Ph3P (1.5 eq) in CH2Cl2 was slowly added ethyl azodicarboxylate (1.5 eq). The reaction mixture was stirred overnight at room temperature, washed by water and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/ n- hexane 1/1) to afford a desired product. General procedure for desulfurization (Procedure D). To a mixture of sulfonamide (1.0 eq) and excess potassium carbonate in CH3CN was added thiophenol (2.0 eq). The reaction mixture was stirred overnight at room temperature, added to dilute NaOH solution and extracted several times with CH2Cl2. The combined organic layer was washed with water, dried over MgSO4 and purified by silica gel column chromatography (EtOAc/ n-hexane 1/4 for 34-36, 1/1 for 37-43). (Caution: The experiment should be carried out in a fume hood. The aqueous solution should be collected and detoxified with hydrogen peroxide). General procedure for Boc-protection (Procedure E). To an ice-cooled solution of amine (1.0 eq) in CH2Cl2 was added DMAP (0.1 eq) and di-tert-butyl dicarbonate (1.2 eq for mono-protection, 2.5 eq for di-protection) in CH2Cl2. The reaction mixture was stirred at room temperature overnight, quenched by water and extracted several times with CH2Cl2. The combined organic layer was washed with 10% aqueous NaHCO3 solution, water and brine and dried over MgSO4. The solution was filtered, concentrated in vacuo and the residue was purified by silica gel column chromatography (EtOAc/ n-hexane 1/4). General procedure for Boc-deprotection reaction (Procedure F). To a stirred solution of N-Boc


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protected amine in CH2Cl2 was added trifluoroacetic acid (10 eq). The reaction mixture was stirred overnight, concentrated in vacuo and purified by ion-exchange resin (strongly basic form) to provide a free amine. General procedure for aza-Wittig coupling reaction. (Procedure G) To a stirred solution of N-substituted 3,4-dimethoxyaniline (1.0 eq) and azide 45 (1.1 eq) in toluene was added triphenylphosphine (1.2 eq). The reaction mixture was degassed and aerated with carbon dioxide. After stirring at 80 °C for 4-8 h under a carbon dioxide atmosphere, the mixture was concentrated in vacuo and purified by silica gel column chromatography (MeOH/ Methanol:Dichloromethane 1:1 (v/v) 1/13 for 62-88, 1/9 for 90-98) to yield a desired product.

(Procedure H) A solution of azide 45 (1.0 eq) and triphenylphosphine (1.2 eq) in toluene was stirred 30 min under a nitrogen atmosphere, and a solution of carbon disulfide (1.1 eq) in toluene was then added. The mixture was stirred for 1 h, and N-substituted 3,4-dimethoxyaniline (1.0 eq) was added. After stirring at 40 °C overnight, the mixture was concentrated in vacuo and purified by silica gel column chromatography (MeOH/CH2Cl2 1/19) to yield a desired product. General procedure for C-alkylation (Procedure I). To a cooled suspension of sodium hydride (1.1 eq) in anhydrous THF was added a solution of 2-(3,4-dimethoxyphenyl)acetonitrile 99 (1.0 eq) in anhydrous THF at 0 °C. After stirring for 10 minutes, a solution of alkyl halide (1.05 eq) was added to the mixture. The reaction mixture was stirred at room temperature or at 60 °C until the starting material was consumed. After completion, the reaction mixture was quenched with water and extracted twice with EtOAc. The combined organic layer was washed by water, brine and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/ n-hexane 1/3) to afford an α-substituted acetonitrile. General procedure for hydrolysis of nitrile group (Procedure J). To a solution of nitrile in MeOH was added the same amount of water and excess NaOH. The reaction mixture was refluxed



Page 32 of 63

for 24 h and then cooled to room temperature. The mixture was washed by EtOAc twice, acidified with dilute solution HCl and extracted several times with EtOAc. The combined organic layer was washed with water and concentrated in vacuo to yield α-substituted acid, which was used for the next step without further purification. General procedure for EDC coupling reaction (Procedure K). To a solution of amine 44 (1.0 eq) in CH2Cl2 was stepwise added triethylamine (3.0 eq), 1-hydroxybenzotriazole (HOBt) (1.2 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl (EDC·HCl) (1.2 eq) and α-substituted acid (1.1 eq) at room temperature. After stirring overnight, the reaction mixture was diluted with CH2Cl2 and washed several times with water. The organic layer was dried over MgSO4, concentrated in vacuo and purified by silica gel column chromatography (MeOH/CH2Cl2 1/14) to provide an amide product. 1-(3,4-Dimethoxyphenyl)-1-methyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea

(46).

From compound 34, procedure H, yield 52%, white solid, mp 80-81 oC: 1H NMR (400 MHz, CDCl3) δ 7.30 (s, 1H), 6.90 (d, J = 8.48 Hz, 1H), 6.74 (dd, J = 8.37, 2.13 Hz, 1H), 6.70 (s, 1H), 6.65 (d, J = 2.16 Hz, 1H), 5.40 (br, NH), 3.89 (s, 3H), 3.84 (s, 3H), 3.82 (t, J = 7.16 Hz, 2H), 3.61 (s, 3H), 3.56 (q, J = 6.64 Hz, 2H), 2.12 (d, J = 0.96 Hz, 3H), 1.96 (quintet, J = 7.12 Hz, 2H); HRMS (ESI) calc. for C17H25N4O2S [M + H]+ 349.1693, found: 349.1697. 1-(3,4-Dimethoxyphenyl)-1-ethyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea

(47).

From compound 35, procedure H, yield 29%, white solid, mp 51-52 oC: 1H NMR (300 MHz, CDCl3) 7.77 (s, 1H), 6.90 (d, J = 8.43 Hz, 2H), 6.77 (s, 1H), 6.69 (dd, J = 8.40, 2.37 Hz, 1H), 6.60 (d, J = 2.37 Hz, 1H), 5.39 (t, J = 6.60 Hz , 1H), 4.16 (q, J = 6.69 Hz, 2H), 3.90 (t, J = 7.68 Hz, 2H), 3.88 (s, 3H), 3.82 (s, 3H), 3.58 (q, J = 6.57 Hz, 2H), 2.15 (s, 3H), 2.02 (quintet, J = 7.14 Hz, 2H), 1.17 (t, J = 7.14 Hz, 3H); HRMS (ESI) calc. for C18H27N4O2S [M + H]+ 363.1849, found: 363.1847.


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1-(3,4-Dimethoxyphenyl)-1-isopropyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (48). From compound 8, procedure H, yield 78%, white solid, mp 59-60 oC: 1H NMR (300 MHz, CDCl3) δ 7.47 (s, 1H), 6.92 (d, J = 8.61 Hz, 1H), 6.73 (s, 1H), 6.63 (dd, J = 8.43, 2.19 Hz, 1H), 6.52 (d, J = 2.22 Hz, 1H), 5.86 (quintet, J = 6.78 Hz, 1H), 5.21 (t, J = 5.67 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H), 3.81 (t, J = 7.14 Hz, 2H), 3.58 (q, J = 6.39 Hz, 2H), 2.13 (d, J = 0.90 Hz, 3H), 1.98 (quintet, J = 7.32 Hz, 2H), 1.08 (s, 3H), 1.06 (s, 3H); HRMS (ESI) calc. for C19H29N4O2S [M + H]

+

377.2006, found: 377.2008. 1-(3,4-Dimethoxyphenyl)-1-isobutyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (49). From compound 9, procedure H, yield 65%, white solid, mp 76-77 oC: 1H NMR (300 MHz, CDCl3) δ 7.70 (s, 1H), 6.91 (d, J = 8.43 Hz, 1H), 6.78 (s, 1H), 6.72 (dd, J = 8.43, 2.37 Hz, 1H), 6.62 (d, J = 2.40 Hz, 1H), 5.47 (t, J = 6.09 Hz, NH), 4.08 (t, J = 6.66 Hz, 2H), 3.89 (s, 3H), 3.88 (d, J = 7.89 Hz, 2H), 3.84 (s, 3H), 3.58 (q, J = 6.96 Hz, 2H), 2.16 (s, 3H), 1.98 (quintet, J = 7.32 Hz, 2H), 1.871.78 (m, 1H), 0.93 (d, J = 6.60 Hz, 6H); HRMS (ESI) calc. for C20H31N4O2S [M + H]+ 391.2162, found 391.2171. 1-(Cyclopentylmethyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)thiourea (50). From compound 36, procedure H, yield 25%, white solid, mp = 87-88 oC: 1H

NMR (300 MHz, CDCl3) δ 7.48 (s, 1H), 6.87 (d, J = 8.61 Hz, 1H), 6.70 (s, 1H), 6.67 (dd, J

= 8.43, 2.37 Hz, 1H), 6.58 (d, J = 2.40 Hz, 1H), 5.36 (t, J = 5.85 Hz, 1H), 4.12 (d, J = 6.96 Hz, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.78 (t, J = 7.32 Hz, 2H), 3.54 (q, J = 7.68 Hz, 2H), 2.09 (s, 3H), 2.06-1.99 (m, 1H), 1.83 (quintet, J = 6.78 Hz, 2H), 1.57-1.42 (m, 4H), 1.21-1.18 (m, 2H), 0.800.76 (m, 2H); HRMS (ESI) calc. for C22H33N4O2S [M + H]+ 417.2319, found 417.2323. 1-Cyclopropyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (51). From compound 10, procedure H, yield 34%, white solid, 159 oC (decomposition): 1H NMR (300 MHz, CD3OD) δ 7.49 (d, J = 1.08 Hz, 1H), 6.86 (d, J = 8.43 Hz, 1H), 6.59 (d, J = 2.22 Hz,



1H), 6.57 (s, 1H), 6.52 (dd, J = 8.43, 2.40 Hz, 1H), 3.87 (t, J = 7.14 Hz, 2H), 3.75 (s, 3H), 3.71 (s, 3H), 3.58 (t, J = 6.96 Hz, 2H), 3.09-3.02 (m, 1H), 2.12 (d, J = 0.90 Hz, 3H), 1.96 (quintet, J = 6.96 Hz, 2H), 0.80-0.68 (m, 2H), 0.51-0.46 (m, 2H); HRMS (ESI) calc. for C19H27N4O2S [M + H]+ 375.1849, found 375.1846. 1-Cyclobutyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (52). From compound 11, procedure H, yield 52%, white solid, mp 126-127 oC: 1H NMR (300 MHz, CDCl3) δ 7.40 (s, 1H), 6.93 (d, J = 8.43 Hz, 1H), 6.71 (s, 1H), 6.62 (dd, J = 8.40, 2.19 Hz, 1H), 6.51 (d, J = 2.01 Hz, 1H), 5.70 (quintet, J = 7.89 Hz, 1H), 5.25 (t, J = 5.67 Hz, 1H), 3.91 (s, 3H), 3.83 (s, 3H), 3.80 (t, J = 7.32 Hz, 2H), 3.55 (q, J = 6.42 Hz, 2H), 2.20-2.16 (m, 2H), 2.12 (s, 3H), 1.93 (quintet, J = 7.14 Hz, 2H), 1.72-1.39 (m, 4H); HRMS (ESI) calc. for C20H29N4O2S [M + H]+ 389.2006, found 389.2014. 1-Cyclopentyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (53). From compound 12, procedure H, yield 68%, white solid, mp 57-59 oC: 1H NMR (300 MHz, CDCl3) δ 7.57 (s, 1H), 6.91 (d, J = 8.40 Hz, 1H), 6.75 (s, 1H), 6.62 (dd, J = 8.22, 2.19 Hz, 1H), 6.53 (d, J = 2.19 Hz, 1H), 5.76-5.64 (m, 1H), 5.24 (t, J = 5.31 Hz, 1H), 3.90 (s, 3H), 3.84 (t, J = 7.50 Hz, 2H), 3.83 (s, 3H), 3.59 (br, 2H), 2.14 (s, 3H), 1.98-1.91 (m, 4H), 1.56-1.44 (m, 4H), 1.231.94 (m, 2H); HRMS (ESI) calc. for C21H31N4O2S [M + H]+ 403.2162, found 403.2167. 1-Cyclohexyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (54). From compound 13, procedure H, yield 69%, white solid, mp 62-64 oC: 1H NMR (300 MHz, CDCl3) δ 7.56 (s, 1H), 6.98 (d, J = 8.43 Hz, 1H), 6.79 (s, 1H), 6.60 (dd, J = 8.40, 2.19 Hz, 1H, 6.51 (d, J = 2.19 Hz, 1H), 5.40-5.27 (m, 1H), 5.17 (t, J = 5.52 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.83 (t, J = 7.05 Hz, 2H), 3.59 (brm, 2H), 2.15 (s, 3H), 1.96-1.92 (m, 4H), 1.71 (d, J = 12.63 Hz, 2H), 1.56 (d, J = 13.71 Hz, 1H), 1.47-1.36 (m, 2H), 1.04-0.81 (m, 3H); HRMS (ESI) calc. for C22H33N4O2S [M + H]+ 417.2319, found 417.2327.


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1-Benzyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea

(55).

From compound 21, procedure H, yield 50%, white solid, mp 157-159 oC: 1H NMR (300 MHz, CDCl3) δ 7.70 (s, 1H), 7.28-7.22 (m, 5H), 6.79 (d, J = 8.61 Hz, 1H), 6.77 (s, 1H), 6.50 (dd, J = 8.40, 2.37 Hz, 1H), 6.29 (d, J = 2.37 Hz, 1H), 5.53 (t, J = 5.67 Hz, 1H), 5.43 (s, 2H), 3.89 (t, J = 7.14 Hz, 2H), 3.84 (s, 3H), 3.63-3.59 (m, 5H), 2.17 (s, 3H), 2.01 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C23H29N4O2S [M + H]+ 425.2006, found 425.2013. 1-(3,4-Dimethoxyphenyl)-1-(3-fluorobenzyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)thiourea (56). From compound 23, procedure H, yield 62%, white solid, mp 95-96 oC: 1H

NMR (300 MHz, CDCl3) δ 7.31 (s, 1H), 7.20 (qd, J = 8.25, 6.24 Hz, 1H), 7.08-7.03 (m, 2H),

6.92 (td, J = 8.61, 2.37 Hz, 1H), 6.80 (d, J = 8.58 Hz, 1H), 6.71 (s, 1H), 6.50 (dd, J = 8.40, 2.37 Hz, 1H), 6.35 (d, J = 2.37 Hz, 1H), 5.47 (t, J = 5.67 Hz, 1H), 5.42 (s, 2H), 3.85 (s, 3H), 3.82 (t, J = 7.14 Hz, 2H), 3.68 (s, 3H), 3.61 (q, J = 6.78 Hz, 2H), 2.13 (d, J = 0.90 Hz, 3H), 1.97 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C23H28FN4O2S [M + H]+ 443.1912, found 443.1921. 1-(3-Chlorobenzyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)thiourea (57). From compound 25, procedure H, yield 43%, white solid, mp 90-91 oC: 1H

NMR (300 MHz, CDCl3) δ 7.42 (s, 1H), 7.29 (s, 1H), 7.21-7.18 (m, 3H), 6.82 (d, J = 8.43 Hz,

1H), 6.73 (s, 1H), 6.52 (dd, J = 8.43, 2.37 Hz, 1H), 6.35 (d, J = 2.19 Hz, 1H), 5.48 (t, J = 6.06 Hz, NH), 5.40 (s, 2H), 3.85 (s, 3H), 3.84 (t, J = 7.14 Hz, 2H), 3.69 (s, 3H), 3.62 (q, J = 6.57 Hz, 2H), 2.15 (s, 3H), 2.01 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C23H28ClN4O2S [M + H]+ 459.1616, found 459.1620. 1-(3,4-Dimethoxyphenyl)-1-(4-fluorobenzyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)thiourea (58). From compound 22, procedure H, yield 55%, white solid, mp 54-56 oC: 1H

NMR (300 MHz, CDCl3) δ 7.59 (s, 1H), 7.26 (dd, J = 8.43, 5.49 Hz, 2H), 6.92 (t, J = 8.61 Hz,

2H), 6.79 (d, J = 7.89 Hz, 1H), 6.75 (s, 1H), 6.46 (dd, J = 8.43, 2.40 Hz, 1H), 6.31 (d, J = 2.22 Hz,



1H), 5.50 (t-like, 1H), 5.39 (s, 2H), 3.86 (t, J = 7.14 Hz, 2H), 3,85 (s, 3H), 3.67 (s, 3H), 3.61 (q, J = 6.96 Hz, 2H), 2.16 (s, 3H), 1.97 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C23H28FN4O3S [M + H]+ 443.1912, found 443.1909. 1-(4-Chlorobenzyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)thiourea (59). From compound 24, procedure H, yield 34%, white solid, mp 71-72 oC: 1H

NMR (300 MHz, CDCl3) δ 7.34 (s, 1H), 7.26-7.21 (m, 4H), 6.82 (d, J = 8.43 Hz, 1H), 6.73 (s,

1H), 6.50 (dd, J = 8.43, 2.37 Hz, 1H), 6.36 (d, J = 2.37 Hz, 1H), 5.51 (t, J = 5.70 Hz, NH), 5.41 (s, 2H), 3.99 (s, 3H), 3.99 (t, J = 8.25 Hz, 2H), 3.87 (s, 3H), 3.65 (q, J = 7.32 Hz, 2H), 2.17 (s, 3H), 2.08 (quintet, J = 6.60 Hz, 2H); HRMS (ESI) calc. for C23H28ClN4O2S [M + H]+ 459.1616, found 459.1615. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(pyridin-3ylmethyl)thiourea (60). From compound 26, procedure H, yield 78%, white solid, mp 58-60 oC: 1H

NMR (300 MHz, CD3OD) δ 8.41 (d, J = 2.19 Hz, 1H), 8.40 (dd, J = 4.95, 1.47 Hz, 1H), 7.88

(dt, J = 8.04, 2.19 Hz, 1H), 7.55 (d, J = 0.90 Hz, 1H), 7.36 (dd, J = 7.86, 4.38 Hz, 1H), 6.93 (d, J = 8.40 Hz, 1H), 6.66 (s, 1H), 6.59 (d, J = 2.37 Hz, 1H), 6.52 (dd, J = 8.43, 2.73 Hz, 1H), 5.52 (s, 2H), 3.90 (t, J = 7.14 Hz, 2H), 3.80 (s, 3H), 3.70 (s, 3H), 3.59 (t, J = 6.78 Hz, 2H), 2.19 (d, J = 0.93 Hz, 3H), 1.99 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C22H28N5O2S [M + H]+ 426.1958, found 426.1969. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(pyridin-4-ylmethyl)thiourea (61). From compound 29, procedure H, yield 59%, white solid, mp 61-62 oC: 1H NMR (300 MHz, CDCl3) δ 8.51 (d, J = 6.06, 1.65 Hz, 2H), 7.83 (s, 1H), 7.25 (t, J = 4.38, 1.44 Hz, 2H), 6.82 (d, J = 8.40 Hz, 1H), 6.79 (s, 1H), 6.56 (dd, J = 8.43, 2.40 Hz, 1H), 6.43 (d, J = 2.37 Hz, 1H), 5.68 (t, J = 5.31 Hz, NH), 5.42 (s, 2H), 3.94 (t, J = 6.96 Hz, 2H), 3.85 (s, 3H), 3.71 (s, 3H), 3.66 (q, J = 6.78 Hz, 2H), 2.18 (d, J = 0.90 Hz, 3H), 2.08 (quintet, J = 7.14 Hz, 2H); HRMS (ESI)


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calc. for C22H27N5O2S [M + H]+ 426.1958, found 426.1930. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (62). From compound 7, procedure G, yield 56%, white solid, mp 100-101 oC: 1H NMR (300 MHz, CDCl3) δ 7.59 (s, 1H), 7.42 (s, 1H), 7.17 (d, J = 2.40 Hz, 1H), 6.77-6.74 (m, 2H), 6.67 (dd, J = 8.61, 2.37 Hz, 1H), 5.76 (t, J = 4.95 Hz, NH), 3.94 (t, J = 6.75 Hz, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.23 (q, J = 6.21 Hz, 2H), 2.19 (s, 3H), 1.98 (quintet, J = 6.60 Hz, 2H); HRMS (ESI) calc. for C16H23N4O3 [M + H]+ 319.1765, found 319.1780. 1-(3,4-Dimethoxyphenyl)-1-methyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (63). From compound 34, procedure G, yield 32%, white solid, mp 103-104 oC: 1H NMR (300 MHz, CDCl3) δ 7.31 (s, 1H), 6.86 (d, J = 8.43 Hz, 1H), 6.77 (dd, J = 8.40, 2.37 Hz, 1H), 6.70-6.69 (m, 2H), 6.65 (d, J = 2.16 Hz, 1H), 4.34 (t, J = 5.85 Hz, NH), 3.87 (s, 3H), 3.85 (s, 3H), 3.82 (t, J = 7.32 Hz, 2H), 3.20 (s, 3H), 3.19 (q, J = 6.60 Hz, 2H), 2.12 (d, J = 0.75 Hz, 3H), 1.86 (quintet, J = 7.12 Hz, 2H); HRMS (ESI) calc. for C17H25N4O3 [M + H]+ 333.1921, found 333.1929. 1-(3,4-Dimethoxyphenyl)-1-ethyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (64). From compound 35, procedure G, yield 35%, white solid, mp 110-111 oC: 1H NMR (400 MHz, CDCl3) δ 7.29 (s, 1H), 6.83 (d, J = 8.25 Hz, 1H), 6.70-6.66 (m, 2H), 6.62 (d, J = 2.22 Hz, 1H), 4.18 (t, J = 5.88 Hz, NH), 3.84 (s, 3H), 3.80 (s, 3H), 3.78 (t, J = 7.14 Hz, 2H), 3.65 (q, J = 7.14 Hz, 2H), 3.14 (q, J = 6.60 Hz, 2H), 2.08 (d, J = 0.90 Hz, 3H), 1.83 (quintet, J = 7.14 Hz, 2H), 1.05 (t, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C18H26N4O3 [M + H]+ 347.2078, found 347.2079. 1-(3,4-Dimethoxyphenyl)-1-isopropyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea

(65).

From compound 8, procedure G, yield 73%, white solid, mp 60-61 oC: 1H NMR (300 MHz, CDCl3) δ 7.40 (s, 1H), 6.83 (d, J = 8.43 Hz, 1H), 6.68 (s, 1H), 6.65 (dd, J = 8.43, 2.40 Hz, 1H), 6.55 (d, J = 2.19 Hz, 1H), 4.81 (quintet, J = 6.78 Hz. 1H), 4.04 (t, J = 5.85 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.76 (t, J = 7.14 Hz, 2H), 3.12 (q, J = 6.39 Hz, 2H), 2.08 (d, J = 0.90 Hz, 3H), 1.87 (quintet,



Page 38 of 63

J = 7.14 Hz, 2H), 0.99 (s, 3H), 0.97 (s, 3H); HRMS (ESI) calc. for C19H29N4O3 [M + H]+ 361.2234, found 361.2237. 1-(3,4-Dimethoxyphenyl)-1-isobutyl-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (66). From compound 9, procedure G, yield 64%, white solid, mp 55-56 oC: 1H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 6.86 (d, J = 8.43 Hz, 1H), 6.76 (dd, J = 8.25, 2.37 Hz, 1H), 6.73 (s, 1H), 6.67 (d, J = 2.19 Hz, 1H), 4.29 (t, J = 6.03 Hz, 1H), 3.88 (s. 3H), 3.84 (s, 3H), 3.82 (t, J = 7.32 Hz, 2H), 3.46 (d, J = 7.50 Hz, 2H), 3.18 (q, J = 6.60 Hz, 2H), 2.13 (d, J = 0.90 Hz, 3H), 1.88 (quintet, J = 6.96 Hz, 2H), 1.74-1.62 (m, 1H), 0.89 (d, J = 6.78 Hz, 6H); HRMS (ESI) calc. for C20H31N4O3 [M + H]+ 375.2391, found 375.2426. 1-(Cyclopentylmethyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (67). From compound 36, procedure G, yield 33%, white solid, mp 55-56 oC: 1H NMR (300 MHz, CDCl3) δ 7.42 (s, 1H), 6.83 (d, J = 8.43 Hz, 1H), 6.71 (m, 2H), 6.63 (d, J = 2.37 Hz, 1H), 4.23 (t, J = 5.85 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 3.78 (t, J = 7.32 Hz, 2H), 3.54 (d, J = 7.68 Hz, 2H), 3.14 (q, J = 6.60 Hz, 2H), 2.09 (s, 3H), 1.97 (quintet, J = 6.96 Hz, 1H), 1.83 (quintet, J = 6.78 Hz, 2H), 1.57-1.42 (m, 4H), 1.21-1.18 (m, 2H), 0.80-0.76 (m, 2H); HRMS (ESI) calc. for C22H33N4O3 [M + H]+ 401.2547, found 401.2560. 1-Cyclopropyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (68). From compound 10, procedure G, yield 28%, white solid, mp 84-85 oC: 1H NMR (500 MHz, CDCl3) δ 7.93 (s, 1H), 7.62-7.60 (m, 2H), 6.67 (s, 1H), 6.66 (d, J = 6.70 Hz, 1H), 5.00 (s, 1H), 3.95 (t, J = 6.95 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.25 (q, J = 6.25 Hz, 2H), 2.96-2.93 (m, 1H), 2.19 (s, 3H), 1.97-1.94 (m, 2H), 0.82-0.78 (m, 2H). 0.55-0.52 (m, 2H); HRMS (ESI) calc. for C19H37N4O3 [M + H]+ 359.2078, found 359.2082. 1-Cyclobutyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea

(69).

From compound 11, procedure G, yield 50%, white solid, mp 77-78 oC: 1H NMR (500 MHz,


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CDCl3) δ 7.51 (s, 1H), 6.89 (d, J = 8.40 Hz, 1H), 6.74 (s, 1H), 6.67 (dd, J = 8.35, 2.15 Hz, 1H), 6.58 (d, J = 2.10 Hz, 1H), 4.94 (quintet, J = 7.90 Hz, 1H), 4.11 (t, J = 5.60 Hz, 1H), 3.90 (s, 3H), 3.85 (s, 3H), 3.82 (t, J = 7.15 Hz, 2H), 3.13 (q, J = 6.35 Hz, 2H), 2.13 (s, 3H), 2.11-2.07 (m, 2H), 1.83 (quintet, J = 6.70 Hz, 2H), 1.77-1.69 (m, 2H), 1.58-1.49 (m, 1H), 1.45-1.39 (m, 1H); HRMS (ESI) calc. for C20H29N4O3 [M + H]+ 373.2234, found 373.2236. 1-Cyclopentyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea

(70).

From compound 12, procedure G, yield 79%, white solid, mp 50-51 oC: 1H NMR (500 MHz, CDCl3) δ 7.61 (s, 1H), 6.86 (d, J = 8.40 Hz, 1H), 6.77 (s, 1H), 6.68 (dd, J = 8.30, 1.90 Hz, 1H), 6.60 (d, J = 1.85 Hz, 1H), 4.79-4.73 (m, 1H), 4.08 (t, J = 5.50 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.85 (t, J = 8.50 Hz, 2H), 3.14 (q, J = 6.35 Hz, 2H), 2.15 (s, 3H), 1.85-1.81 (m, 4H), 1.50-1.48 (m, 4H), 1.29-1.23 (m, 2H); HRMS (ESI) calc. for C21H31N4O3 [M + H]+ 387.2391, found 387.2387. 1-Cyclohexyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea

(71).

From compound 13, procedure G, yield 48%, white solid, mp 53-55 oC: 1H NMR (500 MHz, CDCl3) δ 7.66 (s, 1H), 6.85 (d, J = 8.40 Hz, 1H), 6.78 (s, 1H), 6.67 (dd, J = 8.35, 2.05 Hz, 1H), 6.58 (d, J = 2.10 Hz, 1H), 4.36 (tt, J = 11.90, 3.20 Hz, 1H), 4.05 (t, J = 5.60 Hz, 1H), 3.89 (s, 3H), 3.87-3.85 (m, 5H), 3.14 (q, J = 6.25 Hz, 2H), 2.16 (s, 3H), 1.87-1.80 (m, 4H), 1.72-1.69 (m, 2H), 1.56-1.54 (m, 1H), 1.40-1.34 (m, 2H), 1.01 (qd, J = 12.4, 3.15 Hz, 2H), 0.89 (qt, J = 13.1, 3.40 Hz, 1H); HRMS (ESI) calc. for C22H33N4O3 [M + H]+ 401.2547, found 401.2554. 1-Benzyl-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (72). From compound 21, procedure G, yield 51%, white solid, mp 112-113 oC: 1H NMR (300 MHz, CDCl3) δ 7.57 (s, 1H), 7.27-7.19 (m, 5H), 6.77 (d, J = 8.61 Hz, 1H), 6.76 (s, 1H), 6.58 (dd, J = 8.43, 2.37 Hz, 1H), 6.39 (d, J = 2.40 Hz, 1H), 4.77 (s, 2H), 4.34 (t, J = 5.85 Hz, 1H), 3.86 (t, J = 7.14 Hz, 2H), 3.84 (s, 3H), 3.67 (s, 3H), 3.19 (q, J = 6.60 Hz, 2H), 2.15 (d, J = 0.75 Hz, 3H), 1.87 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C23H29N4O3 [M + H]+ 409.2234, found 409.2237.



1-(3,4-Dimethoxyphenyl)-1-(3-fluorobenzyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (73). From compound 23, procedure G, yield 56%, white solid, mp 123-125 oC: 1H NMR (300 MHz, CDCl3) δ 7.28 (s, 1H), 7.16 (qd, J = 7.86, 5.67 Hz, 1H), 6.94-6.84 (m, 3H), 6.73 (d, J = 8.43 Hz, 1H), 6.67 (s, 1H), 6.54 (dd, J = 8.43, 2.40 Hz, 1H), 6.39 (d, J = 2.19 Hz, 1H), 4.72 (s, 2H), 4.28 (t, J = 5.88 Hz, 1H), 3.81 (s, 3H), 3.77 (t, J = 7.14 Hz, 2H), 3.67 (s, 3H), 3.15 (q, J = 6.39 Hz, 2H), 2.09 (s, 3H), 1.82 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C23H28FN4O3 [M + H]+ 427.2140, found 427.2155. 1-(3-Chlorobenzyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (74). From compound 25, procedure G, yield 54%, white solid, mp 53-54 oC: 1H NMR (300 MHz, CDCl3) δ 7.32 (s, 1H), 7.17-7.13 (m, 3H), 7.06-7.03 (m, 1H), 6.75 (d, J = 8.43 Hz, 1H), 6.67 (s, 1H), 6.55 (dd, J = 8.40, 2.37 Hz, 1H), 6.39 (d, J = 2.40 Hz, 1H), 4.70 (s, 2H), 4.29 (t, J = 5.67 Hz, NH), 3.81 (s, 3H), 3.77 (t, J = 7.32 Hz, 2H), 3.67 (s, 3H), 3.18 (q, J = 6.78 Hz, 2H), 2.09 (s, 3H), 1.86 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C23H28ClN4O3 [M + H]+ 443.1844, found 443.1899. 1-(3,4-Dimethoxyphenyl)-1-(4-fluorobenzyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (75). From compound 22, procedure G, yield 52%, white solid, mp 87-88 oC: 1H NMR (300 MHz, CDCl3) δ 7.59 (s, 1H), 7.16 (dd, J = 8.04, 5.49 Hz, 2H), 6.93 (t, J = 7.86 Hz, 2H), 6.78 (d, J = 8.43 Hz, 1H), 6.76 (s, 1H), 6.54 (dd, J = 8.43, 1.47 Hz, 1H), 6.40 (d, J = 1.83 Hz, 1H), 4.73 (s, 2H), 4.33 (t, J = 5.85 Hz, 1H), 3.85 (s, 3H), 3.85 (t, J = 7.14 Hz, 2H), 3.71 (s, 3H), 3.18 (q, J = 6.60 Hz, 2H), 2.15 (s, 3H), 1.86 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C23H28FN4O3 [M + H]+ 427.2140, found 427.2147. 1-(4-Chlorobenzyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)urea (76). From compound 24, procedure G, yield 25%, white solid, mp 54-55 oC: 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 1H), 7.24 (d, J = 8.44 Hz, 2H), 7.15 (d, J = 8.36 Hz, 2H), 6.78 (d, J = 8.52 Hz,


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1H), 6.71 (s, 1H), 6.57 (dd, J = 8.52, 2.40 Hz, 1H), 6.42 (s, 1H), 4.73 (s, 2H), 4.30 (t, J = 5.72 Hz, NH), 3.85 (s, 3H), 3.83 (t, J = 7.08 Hz, 2H), 3.72 (s, 3H), 3.21 (q, J = 6.52 Hz, 2H), 2.12 (s, 3H), 1.89 (quintet, J = 6.88 Hz, 2H); HRMS (ESI) calc. for C23H28ClN4O3 [M + H]+ 443.1844, found 443.1851. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(pyridin-3-ylmethyl)urea (77). From compound 26, procedure G, yield 76%, white solid, mp 94-95 oC: 1H NMR (300 MHz, CD3OD) δ 8.41 (dd, J = 4.95, 1.65 Hz, 1H), 8.35 (d, J = 1.65 Hz, 1H), 7.74 (dt, J = 7.89, 1.83 Hz, 1H), 7.55 (s, 1H), 7.38 (dd, J = 7.89, 4.77 Hz, 1H), 6.91 (d, J = 8.58 Hz, 1H), 6.66 (d, J = 2.40 Hz, 1H), 6.65 (s, 1H), 6.60 (dd, J = 8.61, 2.55 Hz, 1H), 4.85 (s, 2H), 3.90 (t, J = 7.14 Hz, 2H), 3.81 (s, 3H), 3.72 (s, 3H), 3.16 (t, J = 6.60 Hz, 2H), 2.18 (d, J = 1.11 Hz, 3H), 1.86 (quintet, J = 7.08 Hz, 2H); HRMS (ESI) calc. for C22H28N5O3 [M + H]+ 410.2187, found 410.2189. 1-(3,4-Dimethoxyphenyl)-1-((6-fluoropyridin-3-yl)methyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (78). From compound 27, procedure G, yield 54%, white solid, mp 59-60 oC: 1H NMR (300 MHz, CDCl3) δ 7.99 (s, 1H), 7.85 (dt, J = 8.25, 2.55 Hz, 1H), 7.36 (s, 1H), 6.91 (dd, J = 8.43, 2.94 Hz, 1H), 6.84 (d, J = 8.43 Hz, 1H), 6.74 (s, 1H), 6.57 (dd, J = 8.40, 2.37 Hz, 1H), 6.50 (d, J = 2.37 Hz, 1H), 4.80 (s, 2H), 4.36 (t, J = 6.24 Hz, NH), 3.90 (s, 3H), 3.84 (t, J = 7.14 Hz, 2H), 3.80 (s, 3H), 3.25 (q, J = 6.78 Hz, 2H), 2.16 (d, J = 0.75 Hz, 3H), 1.94 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C22H27FN5O3 [M + H]+ 428.2092, found 428.2088. 1-((6-Chloropyridin-3-yl)methyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (79). From compound 28, procedure G, yield 87%, white solid, mp 72-73 oC: 1H NMR (300 MHz, CDCl3) δ 8.14 (d, J = 2.37 Hz, 1H), 7.65 (dd, J = 8.07, 2.40 Hz, 1H), 7.31-7.24 (m, 2H), 6.80 (d, J = 8.43 Hz, 1H), 6.70 (s, 1H), 6.52-6.74 (m, 2H), 4.75 (s, 2H), 4.32 (t, J = 5.88 Hz, NH), 3.85 (s, 3H), 3.82 (t, J = 7.14 Hz, 2H), 3.76 (s, 3H), 3.20 (q, J = 6.42 Hz, 2H), 2.12 (d, J = 0.90 Hz, 3H), 1.89 (quintet, J = 6.78 Hz, 2H); HRMS (ESI) calc. for C22H27ClN5O3 [M + H]+



444.1797, found 444.1792. 1-((6-Aminopyridin-3-yl)methyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (80). From compound 39, procedure G then F, yield 67%, white solid, mp 155157 oC: 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J = 2.01 Hz, 1H), 7.36 (dd, J = 8.43, 2.40 Hz, 1H), 7.26 (d, J = 0.93 Hz, 1H), 7.72 (d, J = 8.61 Hz, 1H), 6.65 (s, 1H), 6.47 (dd, J = 8.43, 2.37 Hz, 1H), 6.40 (d, J = 2.37 Hz, 1H), 6.37 (d, J = 8.43 Hz, 1H), 4.58 (s, 2H), 4.31 (s, 2H), 4.21 (t, J = 5.85 Hz, 1H), 3.80 (s, 3H), 3.76 (t, J = 7.14 Hz, 2H), 3.69 (s, 3H), 3.12 (q, J = 6.24 Hz, 2H), 2.06 (d, J = 0.90 Hz, 3H), 1.79 (quintet, J = 7.32 Hz, 2H); HRMS (ESI) calc. for C22H29N6O3 [M + H]+ 425.2296, found 425.2296. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(pyridin-4-ylmethyl)urea (81). From compound 29, procedure G, yield 54%, white solid, mp 79-80 oC: 1H NMR (300 MHz, CDCl3) δ 8.46 (dd, J = 4.41, 1.65 Hz, 2H), 7.29 (s, 1H), 7.13 (dd, J = 4.38, 1.47 Hz, 2H), 6.75 (d, J = 8.40 Hz, 1H), 6.66 (s, 1H), 6.57 (dd, J = 8.43, 2.37 Hz, 1H), 6.45 (d, J = 2.37 Hz, 1H), 4.73 (s, 2H), 4.36 (t, J = 5.85 Hz, NH), 3.80 (s, 3H), 3.77 (t, J = 7.14 Hz, 2H), 3.69 (s, 3H), 3.19 (q, J = 6.75 Hz, 2H), 2.08 (d, J = 0.90 Hz, 3H), 1.87 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C22H28N5O3 [M + H ]+ 410.2187, found 410.2181. 1-((2-Aminopyridin-4-yl)methyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (82). From compound 40, procedure G then F, yield 52%, white solid, mp 105107 oC: 1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 5.13 Hz, 1H), 7.29 (s, 1H), 6.73 (d, J = 8.43 Hz, 1H), 6.66 (s, 1H), 6.57 (dd, J = 8.43, 2.00 Hz, 1H), 6.47 (d, J = 2.40 Hz, 1H), 6.44 (dd, J = 5.28, 1.26 Hz, 1H), 6.36 (s, 1H), 4.60 (s, 2H), 4.44 (s, 2H), 4.31 (t, J = 5.85 Hz, 1H), 3.80 (s, 3H), 3.77 (t, J = 7.14 Hz, 2H), 3.70 (s, 3H), 3.16 (q, J = 6.21 Hz, 2H), 2.08 (d, J = 0.75 Hz, 3H), 1.82 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C23H29N6O3 [M + H]+ 425.2296, found 425.2293. 1-(2-(2-Aminopyridin-4-yl)ethyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-


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yl)propyl)urea (83). From compound 41, procedure G then F, yield 83%, white solid, mp 223224 oC: 1H NMR (300 MHz, CD3OD) δ 7.74 (d, J = 5.13 Hz, 1H), 7.52 (s, 1H), 6.99 (d, J = 8.43 Hz, 1H), 6.74 (dd, J = 8.25, 2.37 Hz, 1H), 6.64 (s, 1H), 6.63 (d, J = 2.37 Hz, 1H), 6.45-6.41 (m, 2H), 3.89-3.81 (m, 7H), 3.76 (s, 3H), 3.14 (t, J = 6.78 Hz, 2H), 2.75 (t, J = 6.93 Hz, 2H), 2.17 (d, J = 0.93 Hz, 3H), 1.89 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C23H30N6O3 [M + H]+ 439.1837, found 439.1847. 1-(4-(2-Aminopyridin-4-yl)butyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (84). From compound 42, procedure G then F, yield 94%, white solid, mp 150151 oC: 1H NMR (300 MHz, CD3OD) δ 7.71 (d, J = 5.31 Hz, 1H), 7.53 (s, 1H), 6.98 (d, J = 8.43 Hz, 1H), 6.77 (d, J = 2.37 Hz, 1H), 6.72 (dd, J = 8.43, 2.40 Hz, 1H), 6.64 (s, 1H), 6.41 (d, J = 5.31 Hz, 1H), 6.38 (s, 1H), 3.87 (t, J = 7.14 Hz, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.63 (t, J = 7.32 Hz, 2H), 3.11 (t, J = 6.60 Hz, 2H), 2.48 (t, J = 7.14 Hz, 2H), 2.16 (d, J = 0.93 Hz, 3H), 1.83 (quintet, J = 7.14 Hz, 2H), 1.64-1.46 (m, 4H); HRMS (ESI) calc. for C25H35N6O3 [M + H]+ 467.2765, found 467.2767. 1-(4-(2-(2-Aminoethylamino)pyridin-4-yl)butyl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl1H-imidazol-1-yl)propyl)urea (85). From compound 43, procedure G then F, yield 89%, white solid, mp 64-65 oC: 1H NMR (300 MHz, CDCl3) δ 7.87 (d, J = 5.13 Hz, 1H), 7.28 (s, 1H), 6.79 (d, J = 8.40 Hz, 1H), 6.65 (d, J = 2.37 Hz, 1H), 6.63 (dd, J = 8.43, 2.37 Hz, 1H), 6.58 (d, J = 2.22 Hz, 1H), 6.31 (d, J = 4.02 Hz, 1H), 6.14 (s, 1H), 4.69 (br, 1H), 4.16 (t, J = 6.06 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.75 (t, J = 6.96 Hz, 2H), 3.58 (t, J = 6.93 Hz, 2H), 3.27 (q, J = 5.67 Hz, 2H), 3.10 (q, J = 6.24 Hz, 2H), 2.87 (t, J = 5.70 Hz, 2H), 2.42 (t, J = 7.14 Hz, 2H), 2.07 (s, 3H), 1.78 (quintet, J = 7.14 Hz, 2H), 1.56-1.42 (m, 4H); HRMS (ESI) calc. for C27H40N7O3 [M + H]+ 510.3187, found 510.3186. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-phenylurea (86). The



solution of 30 (1.0 eq) in toluene was cooled to 0 oC and added with pyridine (1.0 eq) followed by triphosgene (0.34 eq). The reaction mixture was stirred for 2 h at 70 oC and cooled down to 0 oC. After adding amine 44 (1.0 eq) and pyridine (1.0 eq), the reaction mixture was refluxed overnight. The mixture was washed with NaHCO3 solution, water and brine, and concentrated in vacuo. The residue was purified by silica gel chromatography to afford 86. Yield 51%, white solid, mp 117118 oC; 1H NMR (300 MHz, CDCl3) δ 7.46 (s, 1H), 7.33-7.22 (m, 4H), 7.13 (t, J = 7.14 Hz, 1H), 6.85-6.80 (m, 2H), 6.78 (s, 1H), 6.73 (s, 1H), 4.63 (t, J = 5.85 Hz, 1H), 3.87 (t, J = 7.32 Hz, 2H), 3.86 (s, 3H), 3.80 (s, 3H), 3.23 (q, J = 6.24 Hz, 2H), 2.15 (s, 3H), 1.95 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C22H27N4O3 [M + H]+ 395.2078, found 395.2080. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(oxazol-2-yl)urea (87). From compound 32, procedure G, yield 61%, white solid, mp 139-140 oC: 1H NMR (300 MHz, CDCl3) δ 8.81 (t, J = 5.44 Hz, 1H), 7.42 (s, 1H), 7.07-7.05 (m, 2H), 6.94-6.82 (m, 2H), 6.72 (s, 1H), 6.53 (d, J = 3.30 Hz, 1H), 3.91-3.82 (m, 8H), 3.37 (q, J = 6.03 Hz, 2H), 2.15 (d, J = 0.90 Hz, 3H), 1.99 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C19H24N5O4 [M + H]+ 386.1823, found 386.1839. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(thiazol-2-yl)urea (88). From compound 31, procedure G, yield 62%, white solid, mp 140-141 oC: 1H NMR (300 MHz, CD3OD) δ 7.58 (d, J = 1.08 Hz, 1H), 7.27 (d, J = 3.66 Hz, 1H), 7.11 (d, J = 8.43 Hz, 1H), 7.02 (d, J = 3.87 Hz, 1H), 6.93-6.87 (m, 2H), 6.65 (s, 1H), 4.00 (t, J = 6.96 Hz, 2H), 3.95 (s, 3H), 3.81 (s, 3H), 3.26 (t, J = 6.66 Hz, 2H), 2.21 (d, J = 1.08 Hz, 3H), 2.01 (quintet, J = 6.75 Hz, 2H); HRMS (ESI) calc. for C19H24N5O3S 402.1594, found 402.1599. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(piperidin-4-yl)urea (89). A suspension of 94, Pd/C (10 mol%) and 2 drop of conc. HCl in MeOH was hydrogenated under a balloon of hydrogen for 1 h. The mixture was filtered off Celite, washed with MeOH. The


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combined filtrate was concentrated in vacuo and the residue was purified by PLC (silica gel, MeOH/ CH2Cl2 1/9) to provide 89. Yield 93%, white solid, mp 119-121 oC; 1H NMR (300 MHz, CD3OD) δ 7.52 (s, 1H), 7.02 (d, J = 8.25 Hz, 1H), 6.73 (dd, J = 8.46, 2.37 Hz, 1H), 6.64 (s, 1H), 3.87 (t, J = 7.14 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.11 (t, J = 6.75 Hz, 2H), 3.01 (d, J = 12.27 Hz, 2H), 2.68-2.60 (m, 2H), 2.17 (d, J = 0.93 Hz, 3H), 1.84-1.79 (m, 4H), 1.29 (qd, J = 12.09, 4.32 Hz, 2H); HRMS (ESI) calc. for C21H32N5O3 [M + H]+ 402.2500, found 402.2491. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(1-methylpiperidin-4yl)urea (90). From compound 14, procedure G, yield 65%, white solid, mp 85-86 oC: 1H NMR (300 MHz, CD3OD) δ 7.54 (s, 1H), 7.02 (d, J = 9.15 Hz, 1H), 6.75-6.71 (m, 2H), 6.64 (s, 1H), 4.36-4.28 (m, 1H), 3.89-3.84 (m, 5H), 3.81 (s, 3H), 3.10 (t, J = 6.78 Hz, 2H), 2.88 (d, J = 11.73 Hz, 2H), 2.20 (s, 3H), 2.17 (d, J = 0.93 Hz, 3H), 2.14 (t, J = 5.13 Hz, 2H), 1.86-1.79 (m, 4H), 1.521.36 (m, 2H); HRMS (ESI) calc. for C22H34N5O3 416.2656, found 416.2655. 1-(3,4-Dimethoxyphenyl)-1-(1-ethylpiperidin-4-yl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (91). From compound 15, procedure G, yield 51%, white solid, mp 76-77 oC: 1H NMR (300 MHz, CDCl3) δ 7.26 (s, 1H), 6.79 (d, J = 8.43 Hz, 1H), 6.65 (s, 1H), 6.62 (dd, J = 8.40, 2.37 Hz, 1H), 6.52 (d, J = 2.37 Hz, 1H), 4.43-4.34 (m, 1H), 4.04 (t, J = 6.06 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.76 (t, J = 7.32 Hz, 2H), 3.12 (q, J = 6.39 Hz, 2H), 2.93-2.89 (br, 2H), 2.35 (q, J = 7.14 Hz, 2H), 2.07 (d, J = 0.90 Hz, 3H), 2.03-1.96 (m, 2H), 1.82-1.72 (m, 4H), 1.44-1.33 (m, 2H), 0.99 (t, J = 7.14 Hz, 3H); HRMS (ESI) calc. for C23H36N5O3 430.2813, found 430.2813. 1-(1-Acetylpiperidin-4-yl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (92). From compound 16, procedure G, yield 84%, white solid, mp 58-59 oC: 1H NMR (300 MHz, CDCl3) δ 7.27 (s, 1H), 6.81 (d, J = 8.43 Hz, 1H), 6.66 (s, 1H), 6.58 (dd, J = 8.25, 2.22 Hz, 1H), 6.48 (d, J = 2.37 Hz, 1H), 4.63-4.55 (m, 2H), 4.02 (t, J = 4.41 Hz, 1H), 3.89 (s, 3H), 3.79 (s, 3H), 3.74 (t, J = 7.14 Hz, 2H), 3.79-3.72 (m, 1H), 3.13-3.05 (m, 3H), 2.53 (t, J = 10.8 Hz,



1H), 2.07 (s, 3H), 1.94 (s, 3H), 1.82-1.76 (m, 4H), 1.20-1.12 (m, 2H); HRMS (ESI) calc. for C23H34N5O4 [M + H]+ 444.2605, found 444.2612. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(1-phenylpiperidin-4yl)urea (93). From compound 17, procedure G, yield 56%, white solid, mp 87-88 oC: 1H NMR (300 MHz, CDCl3) δ 7.55 (s, 1H), 7.22-7.16 (m, 2H), 6.87-6.84 (m, 3H), 6.82-6.77 (m, 2H), 6.71 (dd, J = 8.43, 2.37 Hz, 1H), 6.59 (d, J = 2.19 Hz, 1H), 4.55-4.50 (m, 1H), 4.11 (t, J = 5.55 Hz, NH), 3.88 (s, 3H), 3.85 (s, 3H), 3.82 (t, J = 7.14 Hz, 2H), 3.65-3.61 (br, 2H), 3.17 (q, J = 6.21 Hz, 2H), 2.84-2.76 (m, 2H), 2.15 (d, J = 0.90 Hz, 3H), 1.87-1.82 (m, 4H), 1.51-1.41 (m, 2H); HRMS (ESI) calc. for C27H36N5O3 478.2813, found 478.2823. 1-(1-Benzylpiperidin-4-yl)-1-(3,4-dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1yl)propyl)urea (94). From compound 18, procedure G, yield 78%, white solid, mp 53-55 oC: 1H NMR (300 MHz, CDCl3) δ 7.29 (d, J = 0.75 Hz, 1H), 7.25-7.16 (m, 5H), 6.84 (d, J = 8.40 Hz, 1H), 6.94 (s, 1H), 6.64 (dd, J = 8.40, 2.19 Hz, 1H), 6.55 (d, J = 2.19 Hz, 1H), 4.43 (tt, J = 12.09, 4.05 Hz, 1H), 4.03 (t, J = 5.67 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.77 (t, J = 7.14 Hz, 2H), 3.42 (s, 2H), 3.12 (q, J = 6.60 Hz, 2H), 2.85 (d, J = 11.37 Hz, 2H), 2.11 (d, J = 0.90 Hz, 3H), 2.07 (t, J = 10.26 Hz, 2H), 1.81 (quintet, J = 7.14 Hz, 2H), 1.76-1.72 (m, 2H), 1.38 (qd, J = 12.09, 3.66 Hz, 2H); HRMS (ESI) calc. for C28H38N5O3 [M + H]+ 492.2969, found 492.2973. 1-(3,4-Dimethoxyphenyl)-1-(1-(4-fluorobenzyl)piperidin-4-yl)-3-(3-(5-methyl-1H-imidazol1-yl)propyl)urea (95). From compound 19, procedure G, yield 45%, white solid, mp 48-49 oC: 1H NMR (300 MHz, CDCl3) δ 7.27 (s, 1H), 7.17 (dd, J = 8.43, 5.49 Hz, 2H), 6.91 (t, J = 8.61 Hz, 2H), 6.80 (d, J = 8.43 Hz, 1H), 6.65 (s, 1H), 6.62 (dd, J = 8.43, 2.19 Hz, 1H), 6.51 (d, J = 2.19 Hz, 1H), 4.42-4.34 (m, 1H), 4.01 (t, J = 5.31, NH), 3.83 (s, 3H), 3.78 (s, 3H), 3.75 (t, J = 7.14 Hz, 2H), 3.36 (s, 2H), 3.11 (q, J = 6.03 Hz, 2H), 2.82-2.79 (br, 2H), 2.06 (d, J = 0.90 Hz, 3H), 2.05-2.01 (m, 2H), 1.81 (quintet, J = 6.96 Hz, 2H), 1.74-1.68 (br, 2H), 1.38-1.34 (m, 2H); HRMS (ESI) calc. for


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C28H37FN5O3 510.2875, found: 510.2871. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(1-(pyridin-3ylmethyl)piperidin-4-yl)urea (96). From compound 20, procedure G, yield 74%, white solid, mp 116-117 oC: 1H NMR (300 MHz, CDCl3) δ 8.40 (d, J = 1.65 Hz, 1H), 8.39 (dd, J = 4.74, 1.65 Hz, 1H), 7.51 (dt, J = 7.68, 1.83 Hz, 1H), 7.26 (s, 1H), 7.14 (dd, J = 7.86, 4.92 Hz, 1H), 6.80 (d, J = 8.43 Hz, 1H), 6.65 (s, 1H), 6.60 (dd, J = 8.43, 2.37 Hz, 1H), 6.51 (d, J = 2.19 Hz, 1H), 4.38 (tt, J = 12.09, 3.87 Hz, 1H), 4.01 (t, J = 5.88 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.73 (t, J = 7.14 Hz, 2H), 3.37 (s, 2H), 3.08 (q, J = 6.21 Hz, 2H), 2.77 (d, J = 11.55 Hz, 2H), 2.16-2.02 (m, 2H), 2.06 (d, J = 0.72 Hz, 3H), 1.77 (quintet, J = 7.14 Hz, 2H), 1.70 (d, J = 10.80 Hz, 2H), 1.32 (qd, J = 12.09, 3.66 Hz, 2H); HRMS (ESI) calc. for C27H36N6O3 [M + H]+ 510.3187, found 510.3188. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(2-(piperazin-1yl)ethyl)urea (97). From compound 37, procedure G then F, yield 91%, white solid, mp 108-109 oC: 1H

NMR (300 MHz, CD3OD) δ 7.53 (s, 1H), 7.01 (d, J = 8.40 Hz, 1H), 6.89 (d, J = 2.37 Hz,

1H), 6.85 (dd, J = 8.43, 2.37 Hz, 1H), 6.64 (s, 1H), 3.91 (t, J = 7.32 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 3.78 (t, J = 6.75 Hz, 2H), 3.13 (t, J = 6.78 Hz, 2H), 2.80 (t, J = 4.95 Hz, 4H), 2.49-2.44 (m, 6H), 2.18 (d, J = 1.11 Hz, 3H), 1.88 (quintet, J = 6.78 Hz, 2H); HRMS (ESI) calc. for C22H35N6O3 [M + H]+ 431.27645, found 431.2773. 1-(3,4-Dimethoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1-(2-morpholinoethyl)urea (98). From compound 38, procedure G, yield 54%, white solid, mp 59-60 oC: 1H NMR (300 MHz, CDCl3) δ 7.27 (s, 1H), 6.82 (d, J = 8.43 Hz, 1H), 6.75-6.70 (m, 2H), 6.65 (s, 1H), 4.26 (t, J = 6.06 Hz, NH), 3.84 (s, 3H), 3.80 (s, 3H), 3.77-3.68 (m, 4H), 3.61 (t, J = 4.56 Hz, 4H), 3.13 (q, J = 6.57 Hz, 2H), 2.42-2.37 (m, 6H), 2.08 (d, J = 1.11 Hz, 3H), 1.83 (quintet, J = 7.14 Hz, 2H); HRMS (ESI) calc. for C22H34N5O4 [M + H]+ 432.2605, found 432.2601. 2-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)acetamide (100).



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Procedure K, yield 67%, white solid, mp 85-86 oC: 1H NMR (300 MHz, CDCl3) 7.32 (s, 1H), 6.82 (d, J = 8.61 Hz, 1H), 6.75-6.70 (m, 3H), 5.75 (br, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.79 (t, J = 7.14 Hz, 2H), 3.46 (s, 2H), 3.22 (q, J = 6.57 Hz, 2H), 2.10 (s, 3H), 1.89 (quintet, J = 6.96 Hz, 2H); HRMS (ESI) calc. for C17H24N3O3 [M + H]+ 318.1789, found 318.1801. 2-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)propanamide

(101).

Procedure K, yield 16%, white solid, mp 88-89 oC: 1H NMR (300 MHz, CDCl3) δ 7.33 (s, 1H), 6.86-6.79 (m, 3H), 6.75 (s, 1H), 5.39 (br, 1H), 3.88 (s, 6H), 3.77 (t, J = 7.14 Hz, 2H), 3.47 (q, J = 7.14 Hz, 1H), 3.22 (q, J = 8.06 Hz, 2H), 2.11 (s, 3H), 1.86 (quintet, J = 6.96 Hz, 2H), 1.50 (d, J = 7.14 Hz, 3H); HRMS (ESI) calc. for C18H26N3O3 [M + H]+ 332.1969, found 332.1962. 2-(3,4-Dimethoxyphenyl)-2-methyl-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)-propanamide (102). Procedure K, yield 59%, white solid, mp 89-91 oC: 1H NMR (300 MHz, CDCl3): δ 7.30 (s, 1H), 6.93-6.83 (m, 3H), 6.73 (s, 1H), 5.23 (br, NH), 3.89 (d, J = 6.78 Hz, 6H), 3.78 (t, J = 6.96 Hz, 2H), 3.21 (quintet, J = 6.60 Hz, 2H), 2.12 (s, 3H), 1.88 (quintet, J = 7.14 Hz, 2H), 1.55 (s, 6H); HRMS (FAB): calcd for C19H28N3O3 [M + H]+ 346.2125, found 346.2131. 1-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1yl)propyl)cyclopropanecarboxamide (103). Procedure K, yield 47%, white solid, mp 71-73 oC: 1H

NMR (300 MHz, CD3OD) δ 7.51 (d, J = 0.90 Hz, 1H), 6.96-6.94 (m, 3H), 6.65 (m, 1H), 3.85

(t, J = 7.14 Hz, 2H), 3.82 (s, 6H), 3.14 (t, J = 6.78 Hz, 2H), 2.16 (d, J = 0.90 Hz, 3H), 1.82 (quintet, J = 6.75 Hz, 2H), 1.45 (dd, J = 6.75, 3.84 Hz, 2H), 1.04 (dd, J = 6.60, 3.66 Hz, 2H); HRMS (ESI) calc. for C19H26N3O3 [M + H]+ 344.1969, found 344.1964. 1-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)cyclobutanecarboxamide (104). Procedure K, yield 22%, white solid, mp 75-77 oC: 1H NMR (300MHz, CDCl3) δ 7.30 (s, 1H), 6.87-6.82 (m, 2H), 6.73 (br, 2H), 5.14 (br, 1H), 3.90 (s, 3H), 3.87 (s, 3H), 3.71 (t, J = 7.32 Hz, 2H), 3.17 (q, J = 6.60 Hz, 2H), 2.76 (br, 2H), 2.45 (br, 2H), 2.19 (br, 1H), 2.10 (s, 3H), 1.83-


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1.79 (m, 3H); HRMS (ESI) calc. for C20H28N3O3 [M + H]+ 358.2125, found 358.2117. 1-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1yl)propyl)cyclopentanecarboxamide (105). Procedure K, yield 32%, white solid, mp 76-77 oC: 1H

NMR (300 MHz, CD3OD): δ 7.38 (d, J = 0.93 Hz, 1H), 6.97-6.94 (m, 2H), 6.91-6.88 (m, 1H),

6.61 (s, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.71 (t, J = 7.35 Hz, 2H), 3.17 (t, J = 6.60 Hz, 2H), 2.492.44 (m, 2H), 2.04 (d, J = 0.93 Hz, 3H), 2.01-1.91 (m, 2H), 1.83 (quintet, J = 6.57 Hz, 2H), 1.721.68 (m, 4H); HRMS (FAB) calcd for C21H30N3O3 [M + H]+ 372.2287, found: 372.2287. 1-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)cyclohexane-1carboxamide (106). Procedure K, yield 43%, white solid, mp 79-80 oC: 1H NMR (300 MHz, CDCl3) δ 7.22 (s, 1H), 6.89-6.74 (m, 3H), 6.65 (s, 1H), 5.34 (t, J = 5.67 Hz, NH), 3.81 (s, 3H), 3.79 (s, 3H), 3.65 (t, J = 7.14 Hz, 2H), 3.12 (q, J = 6.60 Hz, 2H), 2.19-2.11 (m, 2H), 2.02 (s, 3H), 1.901.83 (m, 2H), 1.77 (p, J = 6.96 Hz, 2H), 1.54-1.45 (m, 4H), 1.36-1.29 (m, 2H); HRMS (ESI) calc. for C22H32N3O3 [M + H]+ 386.2441, found 386.2441. 2-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)-3-phenylpropanamide (107). Procedure K, yield 57%, white solid, mp 85-86 oC: 1H NMR (300 MHz, CDCl3) δ 7.18-7.12 (m, 3H), 7.10-7.06 (m, 3H), 6.81-6.69 (m, 3H), 6.63 (s, 1H), 5.90 (t, J = 5.67 Hz, NH), 3.78 (s, 3H), 3.77 (s, 3H), 3.60-3.52 (m, 2H), 3.49-3.36 (m, 2H), 3.21-3.10 (m, 1H), 3.03-2.94 (m, 1H), 2.91 (dd, J = 5.10, 12.03 Hz, 1H), 1.99 (s, 3H), 1.73 (quintet, J = 6.78 Hz, 2H); HRMS (ESI) calc. for C24H30N3O3 [M + H]+ 408.2282, found 408.2317. 2-(3,4-Dimethoxyphenyl)-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)-3-(pyridin-3yl)propanamide (108). Procedure K, yield 51%, white solid, mp 109-110 oC: 1H NMR (300 MHz, CDCl3) δ 8.36-8.35 (m, 2H), 7.42-7.38 (m, 2H), 7.12 (dd, J = 7.68, 4.77 Hz, 1H), 6.84 (d, J = 1.44 Hz, 1H), 6.76-6.74 (m, 2H), 6.68 (s, 1H), 6.49 (br, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.66 (t, J = 7.14 Hz, 2H), 3.57 (t, J = 7.89 Hz, 1H), 3.47 (dd, J = 13.38, 8.07 Hz, 1H), 3.19-3.02 (m, 2H), 2.97 (dd,



J = 13.26, 6.78 Hz, 1H), 2.05 (s, 3H), 1.82 (quintet, J = 6.60 Hz, 2H); HRMS (ESI) calc. for C23H29N4O3 [M + H]+ 408.2160, found 408.2178.

Biological Study. In vitro QC activity assay. For testing the inhibition activities of test compounds, QC activity was fluorometrically evaluated. The used buffer consisted of 25mM HEPES (Sigma), with pH7.0, adjusted with HCl. The substrate H-Gln-AMC hydrobromide (L-glutamine 7-amido-4methylcoumarin, BACHEM, Switzerland) was use in a concentration of 0.4mM. The auxiliary enzyme pGAPase (50units, Qiagen, Germany) was prediluted 1:250 in HEPES. The human QC (10 g/ml, rhQPCT, R&D systems) was prediluted 1:250 in HEPES. A typical reaction mixture consisted of 25L of substrate, 50L of the test compounds and 25L of pGAPase. After incubation in 96-well black plates (Greiner, Austria) for 10min at 37°C the reaction was started by adding 50 L of the hQC solution. Cytotoxicity assays. HT-22 (mouse hippocampal cells) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with 10% (vol/vol) fetal bovine serum and antibiotics (100 mg/ml penicillin/streptomycin mix) in a humidified atmosphere at 37 oC with 5% CO2. 5000 HT-22 cells per well were seeded into a clear 96-well plate one day prior to assay. Medium was removed from the plate, and cells were treated with 25L solution of each compound at 10 M and incubated at 37

oC

for 18 h. 15L of 3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was added to each well and incubated for 3 h. The formazan formed was dissolved in dimethyl sulfoxide (DMSO) and absorbance at 570 nm was measured using a microplate reader (Sunrise, TECAN).OD values from each well were


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subtracted with vehicle control and cell viability was calculated by using the signals from vehicle control as 100%. hERG assay. The affinity of small molecules for the hERG channel was determined using s Predictor hERG fluorescence polarization assay kit (Invitrogen., USA). The assay is based on the principle of fluorescence polarization where a red fluorescent tracer is displaced from the hERG channel by compounds that bind to the channel. Sample triplicates were then run on fluorescence polarization following the protocol of the manufacturer. Fluorescence at 530/25-excitation and a 590/35-emission of each well were on a plate reader, Infinite F200 pro (Techan, Austria). Animals and treatment. For acute model study, ICR mice were utilized. ICR mice were obtained from SAMTACO (Korea). Five microgram in five microliter of human Aβ3−40 (62-0-99, American peptide) in PBS was injected into the deep cortical/hippocampus to 5 weeks old ICR mice (25 g, n = 4, male) using stereotaxic frame(myNeuroLab, USA). Test compounds were administrated viadeep cortical/hippocampus or i.p. injection.After mice wereanaesthetized with Zoletil50 and 2% Rumpun (1:2 ratio, 0.4 mL/kg) i.m., the animal was placed into a stereotaxic frame. A small incision (1cm) was made over the midline of the skull and exposed the landmarks of the cranium (bregma and lambda). A small hole was drilled through the skull. Human A-40was injected into the deep cortical/hippocampus by using the following coordinates : AP -2.0 mm, ML 1.2 mm, DV 1.0 mm from the bregma. Using a Hamilton 25L syringe with 27-gauge guide needle, a 5 L volume containing human Aβ3−40 was delivered. For transgenic mice model study, a total of 12 (n=6 for vehicle; n=6 for compound treatment) 5XFAD mice (Jackson lab, ME) was utilized. 5XFAD mice co-express a total of five FAD mutations, includingAPP with three FAD mutations [K670N/M671L (Swedish), I716V (Florida) and V717I (London)] and PS1 with two mutations (M146L and L286V) under the control of



neuronal specific Thy1 promoter. In this study, we followed the national guidelines inconducting animal experiments. All procedures for animal tests were approved by the Medifron Animal Care and Use Committee (approval number, Medifron 2017-1, IACUC). All surgical procedures were performed with care to minimize pain and discomfort.To evaluate the test compounds on AD model mice, the test compounds were administrated via intraperitoneal injection to mice to 32 week old male 5xFAD mice for 4weeks. Mice were housed individually with a 12:12 h light: dark cycle (07:00–19:00 hours). Aβ Sandwich ELISAs. Sandwich ELISA was performed for the quantification of N3pE-40 (for acute model mouse brain), Aβ42(for TG mouse brain), and N3pE-42 (for TG mouse brain). The mouse brains were completely lysed with RIPAbuffer using a sonicator (SONICS, USA), and ultracentrifuged at 100,000 x g, 4°C for 1 h. Protein concentrations were determined using a BCA kit (Thermo scientific). Sample duplicates were then run on Aβ42and N3pE-42 Aβ specific sandwich ELISAs following the protocol of the manufacturer (#27418 for N3pE-40, #27711 for Aβ42, #27716 for N3pE-42, IBL Japan). Optical densities at 450 nm of each well were read on a plate reader, Safire (Tecan, Switzerland). The concentration of the sample Aβ42 and N3pE-42 Aβ were determined by their standard curves. All readings were in the linear range of the assay. The concentration values were normalized to total brain weight and expressed in pg or ng of Aβper mg of total protein. After the average of the duplicates was determined, the mean and SEMfor each group were calculated. Statistical Analyses. Data was expressed as mean ± SEM. All experiments were repeated at least 3 times. Statistical analyses between groups were performed using SPSS software(SPSS, Chicago, IL). For group comparisons of variables, we analyzed data with either One-way ANOVA with Bonferroni post-hoc test or independent samples t-test in SPSS. (ns: p > 0.05, * : p < 0.05, ** : p < 0.01,*** : p < 0.001)


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Computational study. Conformational search. 1, 62 and 90 were submitted for CSearch of the MacroModel v.11.9 in Maestro v.11.5 (Schrödinger, LLC, NY, USA). Mixed torsional/Low-mode sampling was utilized and the maximum number of steps were set as 1000. Coordinate scan by dihedral angles for the two compounds were also performed using scan method in MacroModel module. During this process, the atoms involved in urea/thiourea dihedral angle calculation of both compounds were manually picked. Ab initio quantum mechanics calculations. Total energy calculation for 1 and 62 were performed using Single Point Energy and Optimization module of Jaguar v.9.9 in Maestro. Density functional theory was selected and all the options were chosen for energy calculation using 100 iterations. The whole process was operated in a solvated system. The setting was the same for both single point energy and optimization calculations. Protein structure preparation. The X-ray crystal structure of the human glutaminyl cyclase (PDB ID: 3PBB)39 was selected and prepared utilizing the Protein Preparation Wizard in Maestro. During the preparation process, bond orders were assigned, zero-order bonds to Zn2+ were created, and hydrogen atoms were added. All the hydrogen atoms were energy minimized with the optimized potential for liquid simulation (OPLS) 2005 force field until the average root mean square deviation for hydrogen atoms reached 0.30 Å. Ligand preparation. The protonation states of 90 were predicted by the pKa prediction module in ADMET PredictorTM (Simulations Plus, Lancaster, CA, USA). The three-dimensional structures of the ligand molecules were produced by LigPrep v.3.4 in Maestro. The resulting structures were energy minimized in implicit solvent with OPLS 2005 force field in Maestro.



Glide quantum mechanics polarized ligand docking (QPLD). The prepared ligand molecules were docked to hQC by Glide QM-Polarized Ligand Docking in Maestro. Firstly, the grid for the active site was generated using the centroid of the co-crystallized ligand, PBD150, and the grid box size was set as default value. Metal coordination constraint was set as tetrahedral geometry for the Zn2+. For the initial docking, Glide SP docking was performed with the maximum number of 30 poses per ligand. The partial charges of the docked ligands were calculated using Jaguar with the accurate QM level. Lastly, the ligands accompanied with the updated charges were re-docked using Glide extra precision (XP) with maximum number of 30 poses per ligand. All the molecular graphic figures were generated using PyMOL software (http://www.pymol.org). All computational studies were undertaken on an Intel Xeon Octa-Core 2.67 GHz workstation with Linux CentOS release 6.7.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Molecular formula strings (CSV) The synthetic procedure and characterization details of intermediates; the purities of all final compounds; docking study of compound 84 (PDF). ■ AUTHOR INFORMATION Corresponding Author * Phone: 82-2-880-7846. Fax: 82-2-888-0649. E-mail: [email protected] * Phone: 82-2-3277-4503. Fax: 82-2-3277-2851. E-mail: [email protected] #

These authors contributed equally


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Notes The authors declare no competing financial interest. ■ ACKNOWLEDGEMENT This work was supported by the Midcareer Researcher Program (2017R1A2B4011094) and Medical Research Center (MRC) grant (2018R1A5A2025286) funded by the National Research Foundation of Korea (NRF). ■ ABBREVIATIONS USED A -amyloid; AD, Alzheimer’s disease; APP, amyloid precursor protein; E, glutamate; pE, pyroglutamate; QC, glutamyl cyclase; SAR, structure activity relationship

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inhibitor PQ912 improves cognition in mouse models of Alzheimer’s disease-Studies on relation to effective target occupancy. J. Pharm. Exp. Ther. 2017, 362, 119-130. (30) Lues, I.; Weber, F.; Meyer, A.; Buhring, U.; Hoffmann, T.; Kuhn-Wache, K.; Manhart, S.; Heiser, U.; Pokorny, R.; Chiesa, J.; Glund, K. A phase 1 study to evaluate the safety and pharmacokinetics of PQ912, a glutaminyl cyclase inhibitor, in healthy subjects. Alzheimers Dement. 2015, 1, 182-195. (31)

New

alzheimer’s

drug

shows

safety,

hints

of

efficacy

in

phase

2,

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Table of Contents graphic O O

N

N

H N

H N O

62 IC50 = 162 nM Z-Z conformer

O

N

H N

N

N O

O

N

90 IC50 = 6.1 nM Z-E conformer "Bioactive conformation"


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Discovery of Conformationally Restricted Human Glutaminyl Cyclase

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