Synthesis, Structure-Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease

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Synthesis, Structure−Activity Relationships, and Antiviral Activity of

Allosteric Inhibitors of Flavivirus NS2B−NS3 Protease

Shenyou Nie,^∥Yuan Yao,^∥Fangrui Wu,^∥Xiaowei Wu, Jidong Zhao, Yuanda Hua, Jingyu Wu, Tong Huo,

Yi-Lun Lin, Alexander R. Kneubehl, Megan B. Vogt, Josephine Ferreon, Rebecca Rico-Hesse,

and Yongcheng Song *

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ABSTRACT: Flaviviruses, including Zika, dengue, and West Nile

viruses, are important human pathogens. The highly conserved

NS2B−NS3 protease of Flavivirus is essential for viral replication

and therefore a promising drug target. Through compound

screening, followed by medicinal chemistry studies, a novel series

of 2,5,6-trisubstituted pyrazine compounds are found to be potent,

allosteric inhibitors of Zika virus protease (ZVpro) with IC₅₀values

as low as 130 nM. Their structure−activity relationships are

discussed. The ZVpro inhibitors also inhibit homologous proteases

of dengue and West Nile viruses, and their inhibitory activities are

correlated. The most potent compounds 47 and 103 potently

inhibited Zika virus replication in cells with EC₆₈values of 300−600 nM and in a mouse model of Zika infection. These compounds

represent novel pharmacological leads for drug development against Flavivirus infections.

but complexation with NS2B is required for the catalytic

activity.¹²In the active (or known as “closed”) conformation of

ZVpro, NS2B is fully wrapped around NS3 and constitutes

part of the active site of the enzyme.^11,13−16In the “open”

conformation, NS2B is partially associated with NS3, and the

enzyme is inactive. Due to its pivotal role in viral replication,

ZVpro is a promising drug target for ZIKV infection.^9,12,17

Peptide-based covalent inhibitors of ZVpro and other

Flavivirus proteases have been reported,^12,18−21but they did

not show signiﬁcant antiviral activity in cell or animal models,

presumably due to low cell permeability or metabolic stability.

Non-peptidic inhibitors have also been disclosed, whose

activities are relatively weak, and interactions with the protease

are less characterized.^12−14,22In a recent communication,¹⁵we

reported that several 2,5,6-trisubstituted pyrazine compounds

are novel potent inhibitors of NS2B−NS3 proteases of Zika

and the related dengue and West Nile viruses. These

compounds inhibited in vitro and in vivo replication of ZIKV.

X-ray crystallographic studies show that these inhibitors bind

to an allosteric pocket of dengue NS2B−NS3 protease. In this

full article, we report the synthesis, comprehensive structure−

INTRODUCTION

■

Zika virus (ZIKV) is a species of the arthropod-borne

Flavivirus of the Flaviviridae family of RNA viruses, which

includes other important human pathogens such as yellow

fever, dengue, and West Nile viruses. ZIKV was ﬁrst discovered

and isolated from a sentinel Rhesus monkey in the Zika Forest

of Uganda in 1947.¹The virus is transmitted among humans

through Aedes mosquitoes. About 20% people infected with

ZIKV show ﬂu-like symptoms and can recover naturally.

However, ZIKV infections have been found to cause a 20-fold

increased incidence of severe neurological disorders, including

birth defects of the central nervous system (mostly micro-

cephaly)^2,3and the Guillain−Barre syndrome. An outbreak

4,5

́

of ZIKV in Brazil and in other 48 American countries in 2015-

16 aﬄicted more than 2 million people.⁶During mid-2015

through Jan-2016, >4000 cases of microcephaly with suspected

ZIKV involvement were reported, as compared to <200 cases/

year previously in Brazil.^7,8The WHO has announced that

ZIKV is a “Public Health Emergency of International

Concern”. Except for mosquito control, there have been no

antiviral drugs or vaccines for the prevention and treatment of

ZIKV infection.^9,10

Received: November 28, 2020

Published: February 17, 2021

ZIKV contains ∼10.8 kb RNA genome encoding a

polyprotein,¹¹which is cleaved by the viral NS2B−NS3

protease (ZVpro) as well as host proteases into three structural

(C, prM/M, and E) proteins and seven non-structural proteins,

including NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

NS3 contains a N-terminal serine protease domain (1−170),

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a

Scheme 1. Synthesis of Pyrazine Compounds 1−18, 25−33, 35−55, 59−65, and 72−104

a

Reagents and conditions: (a) NaOH, MeOH, reﬂux; (b) N-Boc-4-piperidinemethanol, PPh₃, diisopropyl azodicarboxylate, THF; (c) HCl (4 M in

1,4-dioxane), CH₂Cl₂, 0 °C; (d) NIS, DMSO, 72 h; (e) NaNO₂, H₂SO₄(conc.), 1 h; (f) alcohols, PPh₃, diisopropyl azodicarboxylate, THF; (g)

aryl boronic acid or aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane-H₂O, 110 °C; (h) for the synthesis of 10 and

11, diisopropylethylamine, CH₂Cl₂, 0 °C, acetyl chloride, and pivaloyl chloride; (i) for the synthesis of 33, 35, 36, and 37, K₂CO₃, DMSO, 110 °C,

overnight; for other compounds: R⁶-boronic acid or R⁶-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane-H₂O, 110 °C.

activity relationships (SAR), and biological activities of 104

pyrazine and related compounds targeting ZVpro, which have

led to the discovery of compound 47 with potent anti-ZIKV

activity. Moreover, selected ZVpro inhibitors were also found

to inhibit NS2B−NS3 proteases of dengue and West Nile

viruses, showing that these compounds are broad-spectrum

inhibitors of Flaviviruses.

Suzuki reactions on the 5-iodo and 6-chloro groups, followed

by deprotection produced compounds 5−18. Synthesis of

compounds with diﬀerent R⁵and R⁶substituents was also

started from compound 108. Two selective Suzuki reactions

were performed to introduce the R⁵and R⁶substituents,

followed by deprotection to produce compound 47 and its

analogues. For compounds 33, 35, 36, and 37, a nucleophilic

aromatic substitution reaction was used to replace the 6-Cl of

the pyrazine ring to generate the corresponding intermediates.

For the synthesis of compounds with a 2-amino substituent

(Scheme 2), mono-substitution of 1,6-dibromo-pyridine or

-pyrazine (111) with (N-Boc-piperidin-4-yl)methylamine

produced compound 112, which was iodized to give a 5-

iodo product 113. The target compounds can then be obtained

following the reactions described above.

Scheme 2 also shows the synthesis of compounds 56, 57, 58,

and 70. Methyl-3-amino-6-iodopyrazine-2-carboxylate (114)

was successively subjected to a Sandmeyer reaction to convert

its 3-amino group to a −Cl, a nucleophilic substitution reaction

on 6-I, and a Suzuki reaction on 3-Cl to give compound 117.

Upon hydrolysis of the methyl ester, the resulting acid was

converted to an amide, followed by deprotection to give the

target compounds.

RESULTS AND DISCUSSION

■

Chemical Synthesis. Compounds 1−18 with the same R⁵

and R⁶substituents were synthesized using general methods

shown in Scheme 1. Synthesis of compounds 1−4 was started

with a cyclization reaction of 4,4′-dibromo- or diiodo-benzil

(105) with glycine amide (106) in the presence of sodium

hydroxide to give 2-hydroxypyrazine 107. The hydroxy group

was alkylated with tert-butyloxycarbonyl (Boc)-protected 4-

piperidinemethanol using a Mitsunobu reaction, and the

product was deprotected to give compounds 1−4.

For the synthesis of compounds 5−18, 6-chloro-2-amino-

pyrazine (108) was selectively iodized with N-iodosuccinimide

(NIS) to give 6-chloro-5-iodo-2-aminopyrazine (109), whose

amino group was converted to a hydroxy group by treatment

with NaNO₂in H₂SO₄. A Mitsunobu reaction between the

hydroxy group and a Boc-protected piperidine- or piperazine-

containing alcohol gave compound 110. Palladium-catalyzed

The general methods for the synthesis of compounds 20−24

and 34 are depicted in Scheme 3. The aldehyde group of 4,5-

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Scheme 2. Synthesis of Compounds 19, 56−58, and 66−71

a

Reagents and conditions: (a) (N-Boc-piperidin-4-yl)methylamine, K₂CO₃, DMF, 100 °C, 12 h; (b) NIS, CH₃CN-DMSO, 24 h; (c) aryl boronic

acid or aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane-H₂O, 80 °C; (d) 2-(4-(furan-3-yl)phenyl)-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane-H₂O, 100 °C; (e) HCl (4 M in 1,4-dioxane), CH₂Cl₂, 0 °C; (f) NaNO₂,

CuCl, HCl (12 M), −10 °C; (g) (N-Boc-piperidin-4-yl)methylamine, K₂CO₃, DMSO, 50 °C; (h) LiOH, H₂O−MeOH, rt; (i) aniline, HATU (1-

[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate), diisopropylethylamine, DMF.

a

Scheme 3. Synthesis of Compounds 20−24 and 34

a

Reagents and conditions: (a) NaBH₄, MeOH, 0 °C; (b) cyanuric chloride, DMF; (c) 4-(aminomethyl)-1-Boc-piperidine, K₂CO₃, DMF; (d) aryl

boronic acid, Pd(PPh₃)₄, Na₂CO₃, 100 °C; (e) HCl (4 M in 1,4-dioxane); (f) N-bromosuccinimide, CH₃CN, 0 °C to rt, 18 h; (g) N-Boc-4-

piperidinemethanol, NaOH, 1,4-dioxane, 75 °C, 4 h; (h) aryl boronic acid, (A-^taPhos)₂PdCl₂, Na₂CO₃, 1,4-dioxane-H₂O, 100 °C, 16 h; (i)

chloroacetaldehyde, isopropyl alcohol, 105 °C, 18 h; (j) an alcohol, NaOH, 1,4-dioxane, 4 h, 75 °C; or an amine, diisopropylethylamine, CH₃CN, 4

h, 75 °C; (k) potassium vinyltriﬂuoroborate, Cs₂CO₃, THF-H₂O, 100 °C; (l) morpholine, DBU, 120 °C.

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Table 1. Structures and Inhibitory Activities of Compounds 1−18

a

b

Standard errors of all IC₅₀values are less than 30%. Unless indicated, X = CH and n = 1.

dibromothiophene-2-carbaldehyde (127) was reduced and

converted to a −Cl to give 128, which was reacted with (N-

Boc-piperidin-4-yl)methylamine to produce compound 129.

Compound 130 was obtained using Suzuki coupling reactions,

which was then deprotected to give tri-substituted thiophene

20. Starting from 2-amino-6-chloropyrazine (108), compound

21 was synthesized through bromination, nucleophilic

substitution, Suzuki coupling, and deprotection. The reaction

between 3,5-dibromo-6-chloropyrazin-2-amine (121) and

chloroacetaldehyde produced the imidazo[1,2-a]pyrazine

core in compound 124, from which compounds 22−24 were

obtained using similar methods, as described for compound

21. The reaction between pyrazine compound 118 with a 6-Cl

and potassium vinyltriﬂuoroborate gave compound 119 with a

6-vinyl group, which was subjected to an amination reaction to

give, after removing the Boc groups, compound 34.

was used as the substrate. Upon ZVpro-mediated hydrolysis,

there is a signiﬁcant increase of ﬂuorescence (Ex/Em: 360/460

nm). We screened our proprietary library of ∼1200

compounds synthesized targeting histone-modifying enzymes

such as lysine-speciﬁc demethylase 1 (LSD1).²³5,6-Di(4-

bromophenyl)-2-(piperidin-4-ylmethoxy)pyrazine (compound

1, Table 1) was found to be an inhibitor of ZVpro with an IC₅₀

value of 21.7 μM. SAR studies based on compound 1 were

performed to ﬁnd more potent inhibitors.

SAR Studies. First, a series of pyrazine compounds with

identical R⁵and R⁶substituents were synthesized, and their

inhibitory activities are shown in Table 1. Replacing the bromo

groups at the para-position of the phenyl ring in compound 1

with tert-butyl groups in compound 7 (IC₅₀: 3.14 μM),

thiophene-3-yl groups in 5 (IC₅₀: 1.42 μM), furan-3-yl groups

in 6 (IC₅₀: 1.0 μM), and iodo groups in 2 (IC₅₀: 0.52 μM)

increases the activity against ZVpro by ∼7-, 15-, 20-, and 40-

fold, respectively. This suggests that increased bulk and/or

hydrophobicity are favorable. Interestingly, introduction of

more polar hydroxymethyl groups at these positions as found

in compound 8 (IC₅₀: 0.52 μM) also resulted in a 40-fold

increase of the activity, while masking the hydroxy with an

Biochemical Assay and Initial Inhibitor Discovery. We

applied a biochemical assay for recombinant ZVpro, containing

NS2B (47−95) and NS3 (1−170) covalently connected with a

ﬂexible Gly₄-Ser-Gly₄linker. This strategy has been commonly

used for the Flavivirus protease assay.^12,18,19Benzoyl-

norleucine-lysine-lysine-arginine-(7-amino-4-methylcoumarin)

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acetyl in compound 11 (IC₅₀> 10 μM) or a pivaloyl group in

10 (IC₅₀: 9.8 μM) signiﬁcantly reduced the activity. Similarly,

compound 12 (IC₅₀: 0.62 μM) with aminomethyl groups

exhibits a comparable activity (to compounds 2 and 8). An

additional ortho-methyl group as found in compound 16

(IC₅₀: 2.14 μM) leads to a 3-fold activity reduction, suggesting

that the substitution is disfavored. While acetylation of the

primary amine groups in 12 yielded the inactive compound 15

(IC₅₀> 10 μM), masking the amine with N,N-dimethyl groups

as found in compound 18 (IC₅₀: 0.39 μM) increased the

inhibitory activity.

pyrindin-4-yl group in compound 28 (IC₅₀: 0.32 μM) increases

the inhibitory potency, while its analogue 29 (IC₅₀: 1.7 μM)

with a longer R2 side chain is less potent. Similar to compound

16, adding an ortho-methoxy group to the 5-substituent of 28

resulted in signiﬁcant activity reduction in compound 30

(IC₅₀: 3.5 μM). Compound 31 with a pyridin-3-yl group at the

R5 position is a weak inhibitor (IC₅₀: 9.9 μM).

We next wanted to optimize the R⁶substituent in potent

compounds 8 and 12, and the results are summarized in Table

4. Surprisingly, adding a meta-F group to the phenyl ring for

compound 32 (IC₅₀> 50 μM) signiﬁcantly reduced the

activity, which might indicate a strict electronic and/or steric

tolerance to the 6-phenyl ring. A series of smaller 6-

substituents were introduced. Compounds 33 (IC₅₀: 7.3

μM) with a 2-aminoethylamino group and 34 (IC₅₀> 50

μM) with a 2-morpholinyl-ethyl group are weak inhibitors.

Similarly, compounds 35 (IC₅₀> 50 μM), 36 (IC₅₀> 10 μM),

37 (IC₅₀: 6.6 μM), 38 (IC₅₀: 24.6 μM), 39 (IC₅₀: 5.8 μM),

and 40 (IC₅₀: 11.1 μM) containing ﬁve-membered hetero-

cycles are signiﬁcantly less active than 12. Compounds 41−47

with a terminal aromatic group were evaluated. While

compounds 41−45 show reduced activities, compound 46

(IC₅₀: 0.71 μM) with a pyrazol-4-yl group retains the activity

and compound 47 with a furan-3-yl group has a signiﬁcantly

enhanced activity with an IC₅₀value of 200 nM. More variety

of the 4-substituent at the 6-phenyl ring were evaluated. As

compared to 12, an acetyl group in compound 48 (IC₅₀: 3.16

μM) and an isopropyl in 49 (IC₅₀: 2.02 μM) reduced the

inhibitory activity by 3- and 5-fold, while compound 50 with a

hydroxy group (IC₅₀: 0.76 μM) shows a comparable activity.

Compounds 51−55 with a fused bicyclic aromatic ring at the

R⁶position were found to give reduced activities with IC₅₀

values of 1.1−21 μM. Three aromatic amide 6-substituents in

compounds 56−58 were found to be disfavored (IC₅₀: 12−17

μM).

Next, the length of R²side chain was evaluated. Generally, a

longer R²linker leads to a signiﬁcant reduction in activity.

Compounds 3 (IC₅₀: 4.65 μM), 17 (IC₅₀: 1.62 μM), 4 (IC₅₀

10 μM), 9 (IC₅₀: 2.1 μM), 13 (IC₅₀> 10 μM), and 14 (IC₅₀

>

10 μM) are less active than their analogues with a piperidin-4-

ylmethoxy substituent.

Several analogues of compound 12 with diﬀerent central

aromatic rings were synthesized to evaluate their SARs (Table

2). Switching to a pyridine in compound 19, thiophene in 20,

Table 2. Structures and Inhibitory Activities of Compounds

a

19−24

With the discovery of compound 47 showing an IC₅₀of 200

nM against ZVpro (as well as potent cellular anti-ZIKV activity

described below), it became the focus of the SAR studies.

Switching the positions of the R⁵and R⁶substituents produced

compound 59 (Table 5) with reduced inhibitory activity (IC₅₀:

0.68 μM). Masking the primary amino group of 59 with one

and two methyl groups for compounds 60 (IC₅₀: 3.3 μM) and

61 (IC₅₀: 1.8 μM) further reduces the potency. Similarly,

mono- and di-methylation of 47 led to activity reduction for

compounds 62 (IC₅₀: 0.53 μM) and 63 (IC₅₀: 6.1 μM).

Compounds 64 and 65 with a longer R2 side chain are

considerably less active than their corresponding analogues 59

and 47. In addition, analogous compounds 66−71 with an

−NH-containing R²substituent were synthesized. A simple

change from −O− in 47 to −NH− in 66 (IC₅₀: 0.40 μM)

resulted in a 2-fold activity decrease. Adding an −F to either

the 6- (in 67) or 5-substituent (in 68) led to signiﬁcant activity

reduction. Changing the central pyrazine ring in 66 to a

pyridine in 69 (IC₅₀: 0.79 μM) also reduced the potency.

Insertion of an amide linker into the R⁶group for compound

70 is disfavored (IC₅₀: 1.1 μM). Compounds 71 and 72 with a

thiophene-3-yl terminal group for R6 were found to have

moderate activities, suggesting that a thiophene is less favored

than a furan-3-yl group.

a

Standard errors of all IC₅₀values are less than 30%.

3-aminopyrazine in 21, and imidazo[1,2-a]pyrazine in

compounds 22−24 resulted in considerable (3−30×) activity

reduction.

Based on the SARs described above, the pyrazine core with a

2-piperidin-4-yl-methoxy group is favorable for ZVpro

inhibition. We next synthesized compounds to optimize the

R⁵substituent of compounds 12 and 18. Compound 25 (IC₅₀:

8.1 μM, Table 3) containing a 4-hydroxyphenyl group at the

R5 position exhibits more than 10-fold activity reduction, as

compared to 12 (IC₅₀: 0.62 μM). The dimethyl analogue 26 is

also a weak inhibitor (IC₅₀: 20.5 μM). Acetylation of the 5-

substituent (of 12) is disfavored in 27 (IC₅₀: 30 μM). A

Compounds 73−78 (Table 6) were synthesized to ﬁnd how

modiﬁcations of the R²substituent aﬀect ZVpro inhibition.

Changing the piperidine group to a cyclohexyl ring in 73 (IC₅₀:

3.2 μM) or a phenyl ring in 74 (IC₅₀: 3.2 μM) caused >15-fold

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Table 3. Structures and Inhibitory Activities of Compounds 25−31

a

Standard errors of all IC₅₀values are less than 30%.

activity loss. Shortening the R²length resulted in a 7-fold

reduction of potency in 75 (IC₅₀: 1.6 μM). Changing to linear

amines in 76 (IC₅₀: 1.3 μM) and 77 (IC₅₀: 2.1 μM) was also

disfavored. Compound 78 (IC₅₀: 240 nM) bearing an

additional (racemic) methyl group exhibits a comparable

activity to that of compound 47.

Compounds 79−83 (Table 7) were synthesized to ﬁne-tune

the terminal amino group of the R⁵substituent of compound

47. Changing it to an amide group in 79 (IC₅₀: 1.1 μM)

considerably reduced the activity by ∼5×, while further activity

reduction was observed upon addition of an ortho-F group for

compound 80 (IC₅₀: 2.3 μM). A hydroxyamino group in 81

(IC₅₀: 1.2 μM) is less favored than a primary amine. Adding a

methyl group to the R⁵phenyl ring in 82 (IC₅₀: 2.6 μM), or

moving the aminomethyl group to the meta-position in

compound 83 (IC₅₀: 2.9 μM) considerably reduced the

activity.

Compounds 84−103 (Table 8) were synthesized to

optimize the R⁶substituent of compound 47. The substituent

at the ortho-position for compounds 85, 86, 87, and 88, or at

the meta-position for 89, 90, and 91 of the phenyl ring

dramatically decreased the inhibitory activity, showing a strict

steric and/or electronic requirement for the pocket. Next, the

furan-3-yl group is optimized. Changing it to an amide in

compounds 91−93 (IC₅₀: 3.5−13 μM) was disfavored.

Switching to a saturated tetrahydrofuran-3-yl substituent for

compound 94 (IC₅₀: 0.59 μM) resulted in 3-fold activity drop,

while moving the tetrahydrofuran-3-yl group to the meta-

position for 95 further reduced the potency. Moreover, a

variety of analogous cyclic groups with diﬀerent steric,

electronic, or hydrogen bond-forming properties were

synthesized and evaluated, including a pyrrolin-1-yl group in

compound 96 (IC₅₀: 2.5 μM), piperidin-1-yl group in 97 (IC₅₀

> 10 μM), cyclohexyl group in 98 (IC₅₀: 16.2 μM), piperdin-4-

yl group in 99 (IC₅₀: 3.1 μM) and 100 (IC₅₀: 2.9 μM),

piperazin-1-yl group in 101 (IC₅₀: 0.65 μM), and O-containing

groups in compounds 102 (IC₅₀: 1.0 μM), 103 (IC₅₀: 130

nM), and 104 (IC₅₀> 10 μM). It is of interest that compound

103 with a ﬂexible tetrahydropyran-3-yl ring exhibited

improved inhibitory activity as compared to 47, while others

are less active.

Activity against Proteases of Dengue and West Nile

Viruses. Dengue and West Nile viruses also belong to the

Flavivirus family with a high homology to ZIKV, particularly

for their NS2B−NS3 proteases. The proteases of the four

serotypes of dengue viruses show 49−54% sequence identity

and 71−73% similarity to ZVpro, while those of West Nile

virus has 66% sequence identity and 78% similarity. 18

compounds with a wide range of inhibitory activities against

ZVpro were tested for their activity against the protease of

dengue serotype-2 and West Nile virus (DV2pro and WVpro).

As shown in Table 9 and Figure 1, these ZVpro inhibitors also

inhibit the activity of DV2pro and WVpro, and their activities

correlate with those against ZVpro, showing R²values of 0.76

and 0.77, respectively. These results suggest that these three

Flavivirus proteases exhibit similar susceptibility to this series

of compounds. Moreover, compound 47 did not inhibit ﬁve

selected human proteases at 10 μM¹⁵showing high selectivity.

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Table 4. Structures and Inhibitory Activities of Compounds 32−58

a

Standard errors of all IC₅₀values are less than 30%.

The crystal structures of compounds 46, 47, and 66 in

complex with DV2pro have been published in our previous

communication.¹⁵Given the high homology between DV2pro

and ZVpro, particularly in the active site, the binding of these

molecules to Zika protease is expected to be similar, which has

been conﬁrmed by our enzyme kinetic studies. Other

analogues described above should adopt a similar binding

pose in the protein. In addition, it could explain many of the

SARs described above. For example, the crystal structures show

that there are strong hydrogen bonding and electrostatic

interactions between their −NH₂group and Asp75, which

explains, as compared to 47 (IC₅₀= 0.20 μM), that

compounds 62 (IC₅₀= 0.53 μM) and 63 (IC₅₀= 6.1 μM)

with −NHMe and −N(Me)₂exhibited signiﬁcantly reduced

activities. Moreover, the steric and hydrophobic nature of the

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Table 5. Structures and Inhibitory Activities of Compounds 59−72

a

Standard errors of all IC₅₀values are less than 30%.

R⁶-binding pocket may explain many of the observed SARs for

compounds in Table 8.

multiplicity of infection (MOI, the number of infectious viral

particles per cell) of ZIKV was added to a monolayer of cells to

initiate viral infection. After 1 h of virus attachment, cells were

washed and incubated with fresh medium containing

increasing concentrations of a compound for 48 h. The viral

titer of the supernatant containing newly generated ZIKV was

determined using an end-point dilution assay, and the anti-

ZIKV activity of the compound can be evaluated.¹⁵

First, selected ZVpro inhibitors were tested for their

cytotoxicity against U87 and Vero cells, using the MTT

assay. Compounds in Table 8 did not inhibit the proliferation

of these cells at 10 μM. These compounds were next evaluated

Anti-ZIKV Activity. The antiviral activity of selected ZVpro

inhibitors was evaluated in human U87 glioma cells and

monkey Vero cells,¹⁵in which ZIKV replicates rapidly. ZIKV

does not cause cytopathic eﬀects (CPE) in U87 cells,²⁴but it

causes signiﬁcant CPE and, eventually, cell death and lysis in

∼5−7 days in Vero cells lacking interferon-mediated defense.²⁵

This feature can be conveniently used to detect ZIKV in Vero

cells. The passage-3 stock of the FLR strain of ZIKV, which

was isolated from the serum of a patient infected in Colombia

in 2015,²⁶was used for the antiviral experiments. 0.01

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that ZVpro is the cellular target. Compound 50, a potent

inhibitor of ZVpro, did not inhibit ZIKV replication at 10 μM.

In addition to its 5-aminomethylphenyl substituent, compound

50 contains a polar 4-hydroxyphenyl (phenol) group at the 6-

position, which might signiﬁcantly reduce its cell permeability.

The most active compound 47 was found to potently inhibit

the replication of ZIKV strain HN16 and dengue virus

(serotype-2, strain K0049) with comparable activities in our

previous communication.¹⁵Compound 47 exhibited strong in

vivo antiviral activity in a mouse model of ZIKV infection.

Treatment with compound 47 (30 mg/kg) for 3 days inhibited

ZIKV viral loads in plasma and brain by 96 and 98% (at 24 h),

respectively, and signiﬁcantly prolonged the survival rates of

the experimental mice.¹⁵

Table 6. Structures and Inhibitory Activities of Compounds

73−78

a

CONCLUSIONS

■

Compound screening found that 2,5,6-trisubstituted pyrazine

compound 1 is a novel inhibitor of ZVpro. Iterative SAR and

medicinal chemistry studies were performed to ﬁnd more

potent inhibitors. The initial series of compounds with

identical R5 and R6 groups led to the discovery of compound

12 with an IC₅₀of 620 nM. Subsequent scaﬀold hopping did

not yield a better core, suggesting the importance of the central

pyrazine ring. Next, optimization of the R6 group of

compound 12 gave rise to a potent inhibitor 47 having a

furanylphenyl substituent showing an IC₅₀of 200 nM. Further

modiﬁcation based on 47 produced compound 103 with an

IC₅₀of 130 nM against ZVpro. Activity optimization for the R5

or R2 group did not yield more potent compounds. ZVpro

inhibitors also inhibit the activity of homologous proteases of

dengue and West Nile viruses, and their inhibitory activities

correlated well with a R²of 0.77, showing that these

compounds are broad-spectrum inhibitors of Flavivirus

proteases. Cell-based assays showed that compounds 47 and

103 exhibited the most potent antiviral activity against ZIKV

with EC₆₈values of 300−600 nM. Compound 47 also showed

strong in vivo antiviral activity in a mouse model of ZIKV

infection. Compound 47 and related compounds are potent,

broadly active inhibitors of Flavivirus proteases and therefore

represent novel pharmacological leads for developing antiviral

drugs against Zika, dengue, or West Nile virus infections.

a

Standard errors of all IC₅₀values are less than 30%.

Table 7. Structures and Inhibitory Activities of Compounds

79−83

a

EXPERIMENTAL SECTION

■

All chemicals for synthesis were purchased from Alfa Aesar (Ward

Hill, MA) or Aldrich (Milwaukee, WI). Unless otherwise stated, all

solvents and reagents were used as received. All reactions were

performed using a Teﬂon-coated magnetic stir bar at the indicated

temperature and were conducted under an inert atmosphere when

stated. The identity of the synthesized compounds was characterized

by H and ¹³C NMR spectroscopy on a Varian (Palo Alto, CA) 400-

1

MR spectrometer and a mass spectrometer (Shimadzu LCMS-2020).

Chemical shifts were reported in parts per million (ppm, δ) downﬁeld

from tetramethylsilane. Proton coupling patterns are described as

singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and

broad (br). The identity of potent inhibitors was conﬁrmed by high-

resolution mass spectroscopy (HRMS) using an Agilent 6550 iFunnel

quadrupole-time-of-ﬂight (Q-TOF) mass spectrometer with electro-

spray ionization (ESI). The purities of the ﬁnal compounds were

determined to be >95% with a Shimadzu Prominence HPLC using a

Zorbax C18 (or C8) column (4.6 × 250 mm) and monitored with a

UV detector at 254 nm.

a

Standard errors of all IC₅₀values are less than 30%.

for their anti-ZIKV activity using the method described above.

As shown in Table 8, except for compounds 1 and 50, other

compounds inhibited ZIKV replication with EC₆₈[concen-

tration at which the number of infectious ZIKV in the

supernatant is reduced by 68% (half-log)] values of 0.3−5 μM.

In addition, their anti-ZIKV activities generally correlated with

the inhibitory activities against ZVpro (Table 10), suggesting

General Synthetic Procedure for Compounds 1−4. Sodium

hydroxide solution (12.5 M, 3.2 mL, 40 mmol) was added over 30

min to a reﬂuxing mixture of 4,4′-dibromobenzil (105, 7.36 g, 20

mmol), glycine amide hydrogen chloride (106, 2.21 g, 20 mmol), and

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a

Table 8. Structures and Inhibitory Activities of Compounds 84−104

a

Standard errors of all IC₅₀values are less than 30%.

50 mL methanol. After reﬂuxing for another 30 min, the mixture was

treated with HCl (12 N, 2.5 mL), followed by KHCO₃(2 g). The

To a solution of the above 1 precursor (1.5 g, 2.49 mmol) in

CH₂Cl₂(25 mL) was dropwise added hydrogen chloride (4 N in p-

dioxane, 12.5 mL) at 0 °C. The solution was stirred overnight and the

precipitated solid was ﬁltered oﬀ to give 1.1 g of compound 1 as a pale

yellow powder. 2,3-Bis(4-bromophenyl)-5-(piperidin-4-ylmethoxy)-

pyrazine hydrochloride (1). ¹H NMR (400 MHz, DMSO-d₆): δ 8.92

(br, 2H), 8.33 (s, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz,

2H), 7.32 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 4.26 (d, J =

5.6 Hz, 2H), 3.26 (d, J = 12.0 Hz, 2H), 2.88 (t, J = 12.4 Hz, 2H), 2.11

(br, 1H), 1.90 (d, J = 12.8 Hz, 2H), 1.57−1.47 (m, 2H); ¹³C NMR

(100 MHz, DMSO-d₆): δ 158.2, 147.1, 143.4, 137.8, 137.4, 133.3,

132.1, 131.9, 131.79, 131.70, 122.9, 122.0, 70.1, 43.1, 33.3, 25.5; MS

(ESI): [M + H]⁺504.2.

subsequently formed yellow solid was ﬁltered oﬀ, washed well with

t

water, and recrystallized from BuOH. Yellow needles of 5,6-bis(4-

bromophenyl)pyrazin-2-ol (107, 5.94 g) were obtained after ﬁltration.

¹H NMR (400 MHz, CDCl₃): δ 8.12 (s, 1H), 7.54 (d, J = 8.4 Hz,

2H), 7.44 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), and 7.15 (d, J

= 8.4 Hz, 2H).

To a solution of 2-hydroxypyrazine 107 (1.12 g, 2.76 mmol) and

N-Boc-4-piperidinemethanol (594 mg, 2.76 mmol) in anhydrous

tetrahydrofuran (THF) (27 mL) were added triphenylphosphine

(1.16 g, 4.42 mmol) and DIAD (894 mg, 4.42 mmol). The reaction

mixture was stirred at room temperature for 17 h. The solution was

then concentrated and puriﬁed to give the product tert-butyl 4-(((5,6-

bis(4-bromophenyl)pyrazin-2-yl)oxy)methyl)piperidine-1-carboxylate

2,3-Bis(4-iodophenyl)-5-(piperidin-4-ylmethoxy)pyrazine hydro-

chloride (2) was prepared from 4,4′-iodobenzil, following the same

procedure as compound 1, as a hydrochloric acid salt (pale yellow

1

1 precursor (1.5 g, 90%). H NMR (400 MHz, CDCl₃): δ 8.22 (s,

powder). H NMR (400 MHz, DMSO-d₆): δ 8.93 (s, 1H), 8.59 (s,

1H), 7.47−7.41 (m, 4H), 7.30−7.24 (m, 4H), 4.26 (d, J = 6.8 Hz,

2H), 4.15 (br, 2H), 2.75 (t, J = 12.8 Hz, 2H), 2.03 (br, 1H), 1.81 (d, J

= 13.2 Hz, 2H), 1.46 (s, 9H), and 1.35−1.25 (m, 2H).

1H), 8.33 (s, 1H), 7.72−7.60 (m, 4H), 7.16−7.08 (m, 4H), 4.5 (s,

2H), 3.26 (s, 2H), 2.85 (brs, 2H), 2.10 (s, 1H), 1.87 (d, J = 10.4 Hz,

2H), 1.46 (s, 1H); MS (ESI): [M + H]⁺598.0.

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Table 9. Inhibitory Activity IC₅₀(μM) against Flavivirus

Proteases ZVpro, DV2pro, and WVpro

Table 10. Antiviral EC₆₈(μM) against ZIKV-FLR in U87

Cells

a

ZVpro

DV2pro

WVpro

ZVpro IC₅₀(μM)

ZIKV-FLR EC₆₈(μM)

44

45

1

20

10

3

32.6

26.5

21.7

10.7

9.8

4.6

3.1

2.1

1.6

1.4

1.1

0.79

0.71

0.53

0.40

0.20

0.13

>50

35

>50

13.4

10.2

8.4

6.3

3.8

3.6

0.64

0.98

0.86

0.21

0.73

0.29

0.59

2.4

>50

20.0

1.8

9.5

3.6

3.2

6.6

0.93

1.34

1.3

0.12

0.87

0.51

0.78

0.82

1

50

7

21.7

0.76

3.1

1.0

1.1

>10

5.0

3

2.5

1.2

0.6

102

79

55

46

69

59

62

66

78

103

47

1.1

7

0.71

0.79

0.68

0.53

0.40

0.24

0.13

0.20

16

17

5

55

79

69

46

62

66

47

103

0.3−0.6

J = 19.2, 10.4 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.8,

146.8, 142.9, 137.8, 137.7, 137.4, 137.2, 137.1, 132.9, 131.6, 131.5,

130.8, 63.8, 43.0, 34.3, 30.2, 28.2; MS (ESI): [M + H]⁺612.0.

2,3-Bis(4-iodophenyl)-5-(3-(piperidin-4-yl)propoxy)pyrazine hy-

drochloride (4) was prepared from 4,4′-iodobenzil, following the

same procedure as compound 1, as a hydrochloric acid salt (pale

a

Standard errors of all IC₅₀values are less than 30%.

1

yellow powder). H NMR (400 MHz, DMSO-d₆): δ 8.90 (s, 1H),

8.67 (s, 1H), 8.36 (s, 1H), 7.72 (dd, J = 15.3, 8.2 Hz, 4H), 7.16 (dd, J

= 26.0, 8.1 Hz, 4H), 4.38 (s, 2H), 3.21 (d, J = 10.8 Hz, 2H), 2.80 (d, J

= 9.9 Hz, 2H), 1.79 (s, 4H), 1.56 (s, 1H), 1.41−1.27 (m, 4H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 157.9, 146.8, 142.9, 137.7, 137.4,

137.2, 137.1, 133.0, 131.6, 131.5, 95.8, 94.8, 66.4, 43.1, 32.6, 31.8,

28.3, 25.2; MS (ESI): [M + H]⁺626.0.

General Synthetic Procedure for Compounds (5)−(18). To a

solution of 2-amino-6-chloropyrazine (108, 8 g, 62 mmol) in

dimethyl sulfoxide (DMSO) (50 mL) was added NIS (15.3 g, 68

mmol) in portions. After stirring at room temperature for 72 h, the

reaction was quenched with the sodium thiosulfate aqueous solution

(50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL),

and the combined organic layers were washed with water and brine

and dried over Na₂SO₄. The volatiles were removed in vacuo to aﬀord

a crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 5:1 to 2:1) to aﬀord 6-chloro-5-

1

iodopyrazin-2-amine (109, 12.7 g, 80%) as a yellow solid. H NMR

(400 MHz, CDCl₃): δ 7.72 (s, 1H), and 4.73 (br, 2H).

To a suspension of 109 (3.78 g, 14.9 mmol) in sulfuric acid (18

mL) at 0 °C was added sodium nitrite (1.09 g, 15.8 mmol) in three

portions. The resulting reaction mixture was stirred at 0 °C for 1 h.

The mixture was then poured into a beaker with ice while stirring.

The resulting precipitate was collected by ﬁltration, washed with

water, and dried under vacuum to aﬀord 6-chloro-5-iodopyrazin-2-ol

(3.6 g) as a yellowish solid, which is used directly for the next step. ¹H

NMR (400 MHz, DMSO-d₆): δ 7.98 (s, 1H). The crude product 6-

chloro-5-iodopyrazin-2-ol (3.6 g, 14 mmol), N-Boc-4-piperidineme-

thanol (3.1 g, 14.5 mmol), and triphenylphosphine (5.9 g, 22.5

mmol) were dissolved in THF (40 mL) and cooled to 0 °C.

Diisopropyl azodicarboxylate (4.55 g, 22.5 mmol) was added

dropwise under a nitrogen atmosphere. The mixture was warmed to

room temperature and stirred for 12 h. The volatiles were removed in

vacuo to aﬀord a crude oil, which was puriﬁed by column

chromatography (silica gel, hexanes/ethyl acetate from 10:1 to 5:1)

to aﬀord compound 110 (5.2 g, 77% for 2 steps) as an oﬀ-white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.96 (s, 1H), 4.16 (d, J = 6.4 Hz,

4H), 2.73 (t, J = 12.0 Hz, 2H), 1.96 (s, 1H), 1.77 (d, J = 12.8 Hz,

2H), 1.46 (s, 9H), and 1.33−1.19 (m, 2H).

Figure 1. Activities of ZVpro inhibitors correlated with those of (A)

DV2pro and (B) WVpro with R²values of 0.76 and 0.77, respectively.

2,3-Bis(4-iodophenyl)-5-(2-(piperidin-4-yl)ethoxy)pyrazine hydro-

chloride (3) was prepared from 4,4′-iodobenzil, following the same

procedure as compound 1, as a hydrochloric acid salt (pale yellow

1

powder). H NMR (400 MHz, DMSO-d₆): δ 8.93 (s, 1H), 8.70 (s,

Compound 110 (1.94 mmol), arylboronic acid or aryl-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane (2.51 mmol), tetrakis-

(triphenylphosphine)palladium (110 mg, 0.095 mmol), and sodium

carbonate (610 mg, 5.75 mmol) in p-dioxane/H₂O (15/3 mL) were

1H), 8.35 (s, 1H), 7.72 (dd, J = 15.6, 7.2 Hz, 4H), 7.16 (dd, J = 28.0,

7.2 Hz, 4H), 4.44 (s, 2H), 3.22 (d, J = 12.0 Hz, 2H), 2.82 (dd, J =

22.8, 11.6 Hz, 2H), 1.86 (d, J = 13.2 Hz, 2H), 1.75 (s, 3H), 1.40 (dd,

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placed in a sealed tube. The mixture was degassed and heated to 110

°C for 24 h. The reaction was then cooled and quenched with brine

(20 mL). The product was extracted with ethyl acetate (3 × 20 mL),

and the combined organic layers were washed with water and brine

and dried over Na₂SO₄. The volatiles were removed in vacuo to give a

crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 5:1 to 1:2) to aﬀord the R⁵and R⁶

identically substituted product. To a solution of this intermediate (1.5

mmol) in dichloromethane (DCM) (5 mL) was added HCl dropwise

(1.2 mL, 4 N in p-dioxane) at 0 °C. The reaction mixture was warmed

to room temperature and stirred for 12 h. The volatiles were removed

in vacuo to aﬀord an oil, which was triturated in diethyl ether and

solidiﬁed to give the ﬁnal product as a hydrochloric acid salt.

2H), 7.36 (s, 2H), 7.26 (br, 6H), 4.49 (br, 4H), 3.56 (s, 2H), 3.28 (s,

2H), 2.89 (s, 2H), 2.14 (s, 1H), 1.92 (s, 2H), 1.52 (s, 2H); δ 158.0,

148.0, 144.6, 143.5, 142.6, 137.3, 137.0, 132.4, 132.0, 129.8, 129.5,

126.6, 69.8, 63.0, 62.9, 43.2, 33.4, 25.6; MS (ESI): [M + H]⁺406.2.

((5-(2-(Piperidin-4-yl)ethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))dimethanol hydrochloride (9) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 8.90 (s, 1H), 8.67 (s, 1H), 8.36 (s, 1H), 7.72 (dd, J =

15.3, 8.2 Hz, 4H), 7.16 (dd, J = 26.0, 8.1 Hz, 4H), 4.38 (s, 2H), 3.21

(d, J = 10.8 Hz, 2H), 2.80 (d, J = 9.9 Hz, 2H), 1.79 (s, 4H), 1.56 (s,

1H), 1.41−1.27 (m, 4H); ¹H NMR (400 MHz, DMSO-d₆): δ 8.98 (s,

1H), 8.74 (s, 1H), 8.31 (s, 1H), 7.36 (d, J = 7.7 Hz, 2H), 7.31−7.21

(m, 6H), 4.49 (s, 4H), 4.45 (s, 2H), 3.21 (d, J = 11.4 Hz, 2H), 2.82

(dd, J = 22.4, 11.0 Hz, 2H), 1.87 (d, J = 13.8 Hz, 2H), 1.76 (s, 3H),

1.41 (dd, J = 22.8, 12.5 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ

157.5, 147.6, 144.0, 143.0, 142.2, 137.1, 136.9, 136.6, 132.1, 129.3,

129.1, 126.1, 63.6, 62.5, 43.0, 34.4, 30.2, 28.2; MS (ESI): [M + H]⁺

420.2.

((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-phenylene))-

bis(methylene) bis(2,2-dimethylpropanoate) hydrochloride (10) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, DMSO-d₆): δ 9.04 (s, 1H), 8.69 (s, 1H), 8.36 (s,

1H), 7.41 (d, J = 7.6 Hz, 2H), 7.36−7.19 (m, 6H), 5.08 (s, 4H), 4.30

(d, J = 6.0 Hz, 2H), 3.27 (s, 2H), 2.90 (d, J = 11.2 Hz, 2H), 2.14 (s,

1H), 1.93 (d, J = 14.4 Hz, 2H), 1.61−1.45 (m, 2H), 1.16 (s, 18H);

¹³C NMR (100 MHz, DMSO-d₆): δ 177.6, 158.1, 147.8, 144.3, 138.3,

138.0, 137.4, 136.6, 132.9, 130.1, 129.9, 127.6, 127.6, 69.9, 65.4, 43.1,

38.7, 33.4, 27.3, 25.6; MS (ESI): [M + H]⁺574.3.

N,N′-(((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))bis(methylene))diacetamide hydrochloride (11) was

prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, DMSO-d₆): δ 9.13 (d, J = 8.4 Hz, 1H), 8.78 (d,

J = 9.6 Hz, 1H), 8.36 (s, 1H), 7.42 (d, J = 7.6 Hz, 2H), 7.37−7.27 (m,

6H), 5.07 (d, J = 4.4 Hz, 4H), 4.30 (d, J = 6.0 Hz, 2H), 3.29 (d, J =

12.4 Hz, 2H), 2.90 (q, J = 11.6 Hz, 2H), 2.15 (s, 1H), 2.07 (s, 6H),

1.93 (d, J = 14.0 Hz, 2H), 1.54 (q, J = 11.6 Hz, 2H); ¹³C NMR (100

MHz, DMSO-d₆): δ 170.2, 157.6, 147.3, 143.8, 138.0, 137.7, 136.7,

135.8, 132.5, 129.6, 129.4, 127.7, 69.5, 65.02, 64.97, 42.6, 32.9, 25.1,

20.7; MS (ESI): [M + H]⁺488.3.

For the synthesis precursors of 10 and 11 only: to a solution of the

above R⁵and R⁶identically substituted product (R = 4-(OHCH₂)−

Ph, 0.2 mmol) and N-diisopropylethylamine (87 μL, 0.5 mmol) in

anhydrous DCM (3 mL) was added acetyl chloride (18 μL, 0.25

mmol) or pivaloyl chloride (31 μL, 0.25 mmol) at °C, respectively.

The mixture was stirred for 2 h before it was quenched with saturated

NaHCO₃. The mixture was extracted with ethyl acetate (3 × 80 mL),

and the combined organic layers were washed with water and brine

and dried over Na₂SO₄. The volatiles were removed in vacuo to aﬀord

a crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 1.5:1 to 1:1) to aﬀord precursors of 10

(112 mg, 95%) or 11 (124 mg, 92%) as a colorless oil. For the

1

precursor of 10: H NMR (400 MHz, CDCl₃): δ 8.22 (s, 1H), 7.41

(dd, J = 25.6, 7.4 Hz, 4H), 7.29−7.18 (m, 4H), 5.09 (s, 4H), 4.27 (d,

J = 6.0 Hz, 2H), 4.15 (s, 2H), 2.82−2.65 (m, 2H), 2.15−1.91 (m,

7H), 1.82 (d, J = 11.7 Hz, 2H), 1.51−1.34 (m, 9H), 1.29 (dd, J =

1

25.3, 13.6 Hz, 2H); for the precursor of 11: H NMR (400 MHz,

CDCl₃): δ 8.24 (s, 1H), 7.44 (d, J = 6.9 Hz, 2H), 7.37 (d, J = 8.3 Hz,

2H), 7.26 (s, 4H), 5.10 (s, 4H), 4.29 (d, J = 6.0 Hz, 2H), 4.16 (s,

2H), 2.76 (t, J = 14.2 Hz, 2H), 2.01 (s, 1H), 1.83 (d, J = 13.0 Hz,

2H), 1.55 (s, 2H), 1.47 (s, 9H), 1.28−1.18 (m, 18H).

5-(Piperidin-4-ylmethoxy)-2,3-bis(4-(thiophen-3-yl)phenyl)-

pyrazine hydrochloride (5) was prepared following the same

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, DMSO-

d₆): δ 9.13 (s, 1H), 8.79 (s, 1H), 8.34 (s, 1H), 7.94−7.85 (m, 2H),

7.68 (t, J = 7.7 Hz, 4H), 7.60 (dd, J = 5.5, 2.4 Hz, 2H), 7.55 (dd, J =

3.0, 1.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H),

4.29 (d, J = 6.3 Hz, 2H), 3.27 (d, J = 11.5 Hz, 2H), 2.88 (dd, J = 22.6,

11.5 Hz, 2H), 2.13 (s, 1H), 1.91 (d, J = 13.2 Hz, 2H), 1.54 (dd, J =

22.8, 11.1 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.6, 147.3,

143.8, 140.7, 140.6, 137.1, 136.7, 135.2, 134.5, 132.3, 130.2, 129.9,

127.3, 127.2, 126.1, 126.1, 125.8, 125.8, 121.7, 121.4, 69.5, 42.7, 33.0,

25.2; MS (ESI): [M + H]⁺510.2.

((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-phenylene))-

dimethanamine hydrochloride (12) was prepared following the same

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, D₂O): δ

8.02 (s, 1H), 7.20 (d, J = 7.6 Hz, 2H), 7.11 (s, 6H), 4.10 (s, 2H), 3.89

(s, 4H), 3.22 (d, J = 10.0 Hz, 2H), 2.79 (t, J = 10.4 Hz, 2H), 1.99 (s,

1H), 1.84 (d, J = 11.6 Hz, 2H), 1.41−1.32 (m, 2H); MS (ESI): [M +

H]⁺404.2.

2,3-Bis(4-(furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazine

hydrochloride (6) was prepared following the same procedure as a

hydrochloric acid salt. H NMR (400 MHz, DMSO-d₆): δ 8.93 (s,

((5-(2-(Piperidin-4-yl)ethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))dimethanamine hydrochloride (13) was prepared follow-

ing the same procedure as a hydrochloric acid salt. H NMR (400

1

1H), 8.57 (s, 1H), 8.33 (s, 1H), 8.19 (d, J = 6.0 Hz, 2H), 7.75−7.67

(m, 2H), 7.61−7.49 (m, 4H), 7.44 (d, J = 8.0 Hz, 2H), 7.36 (d, J =

8.0 Hz, 2H), 6.95 (s, 2H), 4.30 (d, J = 6.0 Hz, 2H), 3.30 (d, J = 11.6

Hz, 2H), 2.91 (t, J = 12.4 Hz, 2H), 2.14 (s, 1H), 1.94 (d, J = 13.6 Hz,

2H), 1.51 (dd, J = 24.4, 12.0 Hz, 2H); ¹³C NMR (100 MHz, DMSO-

d₆): δ 158.0, 147.7, 144.9, 144.8, 144.2, 140.3, 140.1, 137.3, 136.9,

132.6, 131.8, 130.5, 130.2, 125.7, 109.0; MS (ESI): [M + H]⁺429.2.

2,3-Bis(4-(tert-butyl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazine

hydrochloride (7) was prepared following the same procedure as a

hydrochloric acid salt. ¹H NMR (400 MHz, DMSO-d₆): δ 9.00−8.27

(br, 4H), 8.30 (s, 1H), 7.38−7.26 (m, 8H), 4.27 (d, J = 6.2 Hz, 2H),

3.33−3.24 (m, 2H), 2.90 (t, J = 11.8 Hz, 2H), 2.13 (br, 1H), 1.93 (d,

J = 13.0 Hz, 2H), 1.58−1.48 (m, 2H), 1.26 (s, 18H); ¹³C NMR (100

MHz, DMSO-d₆): δ 157.4, 151.2, 150.3, 147.4, 144.0, 135.7, 135.4,

131.9, 129.2, 129.0, 125.0, 124.9, 69.4, 42.7, 34.4, 34.3, 33.0, 31.1,

31.0, 25.2; HRMS (ESI⁺): calcd for C₃₀H₄₀N₃O [M + H]⁺, 458.3166;

found, 458.3179.

MHz, D₂O): δ 8.28 (s, 1H), 7.51−7.43 (m, 2H), 7.44−7.30 (m, 6H),

4.57−4.44 (m, 2H), 4.20 (s, 4H), 3.44 (d, J = 12.8 Hz, 2H), 3.00 (t, J

= 12.6 Hz, 2H), 2.05 (d, J = 14.3 Hz, 2H), 1.93−1.78 (m, 3H), 1.50

(dd, J = 24.7, 13.4 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.8,

149.2, 143.4, 138.0, 137.8, 133.4, 132.8, 131.7, 130.4, 130.3, 128.7,

128.6, 64.9, 43.9, 42.6, 34.0, 30.2, 28.2; MS (ESI): [M + H]⁺418.3.

((5-(2-(Piperazin-1-yl)ethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))dimethanamine hydrochloride (14) was prepared follow-

1

ing the same procedure as a hydrochloric acid salt. H NMR (400

MHz, D₂O): δ 8.39 (s, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.43 (dd, J =

7.8, 5.0 Hz, 6H), 4.90 (d, J = 4.7 Hz, 2H), 4.21 (s, 4H), 3.88−3.80

(m, 6H), 3.67 (d, J = 4.6 Hz, 4H); ¹³C NMR (100 MHz, D₂O): δ

157.5, 148.8, 144.8, 138.2, 138.1, 133.4, 132.8, 132.5, 130.4, 130.3,

128.7, 128.7, 60.1, 56.0, 48.8, 42.6, 42.6, 40.6; MS (ESI): [M + H]⁺

419.3.

N,N′-(((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))bis(methylene))diacetamide hydrochloride (15) was

prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, DMSO-d₆): δ 9.17 (d, J = 10.1 Hz, 1H), 8.82

(d, J = 10.0 Hz, 1H), 8.45 (dt, J = 11.2, 5.7 Hz, 2H), 8.32 (d, J = 1.4

Hz, 1H), 7.36 (d, J = 7.3 Hz, 2H), 7.28 (d, J = 7.2 Hz, 2H), 7.19 (t, J

((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-phenylene))-

dimethanol hydrochloride (8) was prepared following the same

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, DMSO-

d₆): δ 9.00 (s, 1H), 8.65 (s, 1H), 8.33 (s, 1H), 7.58 (d, J = 19.6 Hz,

2788

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= 9.0 Hz, 4H), 4.28 (d, J = 6.3 Hz, 2H), 4.25 (d, J = 5.7 Hz, 4H), 3.28

(d, J = 12.1 Hz, 2H), 2.89 (q, J = 11.7 Hz, 2H), 2.14 (s, 1H), 1.92 (d,

J = 12.7 Hz, 2H), 1.88 (s, 6H), 1.54 (dd, J = 23.1, 11.2 Hz, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 169.3, 169.3, 157.5, 147.4, 143.9,

140.2, 139.3, 137.0, 136.7, 132.2, 129.5, 129.3, 126.9, 126.9, 69.4,

54.9, 42.6, 41.7, 41.7, 33.0, 25.2, 22.6; MS (ESI): [M + H]⁺488.3.

((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(3-methyl-4,1-

phenylene))dimethanamine hydrochloride (16) was prepared follow-

The reaction mixture was warmed to room temperature and stirred

for 12 h. The volatiles were removed in vacuo to aﬀord an oil, which

was triturated in diether ether and solidiﬁed to give the ﬁnal product

as a hydrochloric acid salt.

4-(3-(4-(Aminomethyl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-

2-yl)phenol hydrochloride (25) was prepared following the same

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, DMSO-

d₆): δ 9.03 (bs, 1H), 8.76 (bs, 1H), 8.43 (bs, 3H), 8.28 (s, 1H), 7.42

(s, 4H), 7.10 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 4.25 (d, J =

6 Hz, 2H), 3.99 (d, J = 6 Hz, 2H), 3.26 (d, J = 13.2 Hz, 2H), 2.87

(dd, J = 12.4 Hz, 24 Hz, 2H), 2.11 (bs, 1H), 1.89 (d, J = 12.4 Hz,

2H), 1.51 (dd, J = 12 Hz, 22.8 Hz, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 162.8, 158.2, 147.7, 144.1, 139.3, 138.9, 133.0, 132.2,

130.2, 130.2, 130.10, 130.06, 70.0, 51.1, 43.0, 33.4, 25.6; MS (ESI):

[M + H]⁺391.1.

1

ing the same procedure as a hydrochloric acid salt. H NMR (400

MHz, D₂O): δ 8.26 (s, 1H), 7.17 (d, J = 9.6 Hz, 2H), 7.02 (s, 4H),

4.20 (d, J = 5.6 Hz, 2H), 3.99 (s, 2H), 3.39 (d, J = 12.4 Hz, 2H), 2.96

(t, J = 11.2 Hz, 2H), 2.14 (br, 1H), 2.09 (br, 3H), 2.00 (br, 3H), 1.52

(dd, J = 23.6, 11.6 Hz, 2H); MS (ESI): [M + H]⁺432.3.

N,N′-(((5-(2-(Piperidin-4-yl)ethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))bis(methylene))bis(Hydroxylamine) hydrochloride (17)

was prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, D₂O): δ 8.16 (s, 1H), 7.37 (d, J = 7.7 Hz, 2H),

7.30 (d, J = 7.7 Hz, 6H), 4.39 (s, 2H), 4.31 (s, 4H), 3.29 (d, J = 12.3

Hz, 2H), 2.85 (t, J = 12.2 Hz, 2H), 1.91 (d, J = 13.5 Hz, 2H), 1.82−

1.71 (m, 3H), 1.41−1.31 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ

158.8, 148.8, 143.8, 139.0, 138.9, 132.3, 130.52, 130.48, 130.3, 130.2,

129.2, 128.5, 64.8, 54.4, 43.9, 34.0, 30.2, 28.2; MS (ESI): [M + H]⁺

450.2.

1,1′-((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))bis(N,N-dimethylmethanamine) hydrochloride (18) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, D₂O): δ 8.20 (s, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.30

(s, 6H), 4.26 (d, J = 4.0 Hz, 2H), 4.18 (s, 4H), 3.37 (d, J = 11.6 Hz,

2H), 2.94 (t, J = 12.4 Hz, 2H), 2.71 (s, 12H), 2.15 (s, 1H), 2.00 (d, J

= 12.8 Hz, 2H), 1.51 (dd, J = 26.4, 12.8 Hz, 2H); ¹³C NMR (100

MHz, DMSO-d₆): δ 157.9, 147.4, 143.7, 139.2, 138.8, 132.9, 131.0,

130.8, 130.8, 130.2, 129.9, 129.8, 69.6, 58.9, 58.8, 42.6, 41.5, 41.4,

32.9, 25.1; MS (ESI): [M + H]⁺406.3.

General Synthetic Procedure for Compounds 25−32, 38−

55, and 72−104. Compound 110 (1.2 g, 2.66 mmol), 4-[(tert-

butoxycarbonylamino)methyl]phenylboronic acid pinacol ester (2.66

mmol), tetrakis(triphenylphosphine)palladium (154 mg, 0.13 mmol),

and sodium carbonate (564 mg, 5.32 mmol) in p-dioxane/H₂O (15/3

mL) were placed in a sealed tube. The mixture was degassed and

heated to 80 °C for 12 h. The reaction was cooled and quenched with

brine (20 mL). The product was extracted with ethyl acetate (3 × 20

mL), and the combined organic layers were washed with water and

brine and dried over Na₂SO₄. The volatiles were removed in vacuo to

aﬀord a crude oil, which was puriﬁed by column chromatography

(silica gel, hexanes/ethyl acetate from 5:1 to 1:1) to aﬀord the

corresponding R⁵substituted product. For the intermediate of 47: ¹H

NMR (400 MHz, CDCl₃): δ 8.19 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H),

7.34 (d, J = 8.0 Hz, 2H), 5.04 (s, 1H), 4.34 (d, J = 5.2 Hz, 2H), 4.21

(d, J = 6.4 Hz, 2H), 4.15 (s, 2H), 2.73 (t, J = 11.2 Hz, 2H), 1.99−1.92

(m, 1H), 1.79 (d, J = 12.4 Hz, 2H), 1.44 (s, 18H), and 1.30−1.20 (m,

2H).

4-(3-(4-((Dimethylamino)methyl)phenyl)-5-(piperidin-4-

ylmethoxy)pyrazin-2-yl)phenol hydrochloride (26) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.23 (s, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.41 (d, J

= 8.0 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.3 Hz, 2H), 4.31

(s, 4H), 3.51 (d, J = 12.6 Hz, 2H), 3.06 (t, J = 12.1 Hz, 2H), 2.23 (s,

1H), 2.10 (d, J = 13.8 Hz, 2H), 1.63 (dd, J = 23.6, 11.5 Hz, 2H); ¹³C

NMR (100 MHz, D₂O): δ 158.3, 155.9, 148.3, 144.3, 139.3, 131.5,

131.2, 130.6, 130.4, 129.7, 129.1, 115.0, 70.2, 60.5, 43.6, 42.0, 32.9,

25.0; MS (ESI): [M + H]⁺419.2.

N-(4-(3-(4-(Aminomethyl)phenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)benzyl)acetamide hydrochloride (27) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.15 (s, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.25 (d, J

= 8.1 Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 4.23

(s, 4H), 4.06 (s, 2H), 3.38 (d, J = 12.8 Hz, 2H), 2.94 (t, J = 12.8 Hz,

2H), 2.13 (s, 1H), 1.99 (d, J = 13.6 Hz, 2H), 1.92 (s, 3H), 1.51 (dd, J

= 24.0, 11.5 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 174.0, 158.6,

148.7, 144.4, 138.1, 136.4, 133.2, 131.9, 130.3, 129.8, 128.6, 126.9,

110.0, 70.2, 43.6, 42.65, 42.63, 32.9, 25.0, 21.8; MS (ESI): [M + H]⁺

446.2.

(4-(6-(Piperidin-4-ylmethoxy)-3-(pyridin-4-yl)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (28) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.59 (d, J = 5.6 Hz, 2H), 8.40 (s, 1H), 7.81 (d, J = 6.0 Hz,

2H), 7.51 (dd, J = 21.2, 8.0 Hz, 4H), 4.42 (d, J = 6.0 Hz, 2H), 4.24 (s,

2H), 3.53 (d, J = 12.4 Hz, 2H), 3.09 (t, J = 12.4 Hz, 2H), 2.29 (s,

1H), 2.14 (d, J = 13.2 Hz, 2H), 1.66 (dd, J = 24.0, 11.6 Hz, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 158.3, 152.7, 149.3, 142.3, 138.7,

136.1, 135.1, 133.3, 131.7, 129.6, 128.8, 126.2, 69.6, 42.2, 41.4, 32.4,

24.6; MS (ESI): [M + H]⁺476.2.

(4-(6-(2-(Piperidin-4-yl)ethoxy)-3-(pyridin-4-yl)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (29) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.66 (s, 2H), 8.42 (s, 1H), 8.01 (s, 2H), 7.57 (br, 2H), 7.52

(br, 2H), 4.61 (s, 2H), 4.26 (s, 2H), 3.45 (d, J = 12.4 Hz, 2H), 3.01

(t, J = 12.4 Hz, 2H), 2.07 (d, J = 13.6 Hz, 2H), 1.92 (br, 3H), 1.51

(dd, J = 23.2, 11.2 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 159.9,

155.7, 151.0, 140.9, 138.3, 136.8, 134.5, 134.2, 130.6, 129.2, 127.3,

65.2, 43.9, 42.6, 33.9, 30.2, 28.2 MS (ESI): [M + H]⁺390.2.

(3-Methoxy-4-(6-(piperidin-4-ylmethoxy)-3-(pyridin-4-yl)pyrazin-

2-yl)phenyl)methanamine hydrochloride (30) was prepared following

The above R⁵-substituted compound (1.94 mmol), arylboronic

acid or aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.51 mmol),

tetrakis(triphenylphosphine)palladium (110 mg, 0.095 mmol), and

sodium carbonate (610 mg, 5.75 mmol) in p-dioxane/H₂O (15/3

mL) were placed in a sealed tube. The mixture was degassed and

heated to 110 °C for 24 h. The reaction was then cooled and

quenched with brine (20 mL). The product was extracted with ethyl

acetate (3 × 20 mL), and the combined organic layers were washed

with water and brine and dried over Na₂SO₄. The volatiles were

removed in vacuo to give a crude oil, which was puriﬁed by column

chromatography (silica gel, hexanes/ethyl acetate from 5:1 to 1:2) to

aﬀord the corresponding R⁶-substituted product. For the precursor of

47: ¹H NMR (400 MHz, CDCl₃): δ 8.22 (s, 1H), 7.75 (s, 1H), 7.50−

7.34 (m, 7H), 7.20 (d, J = 7.8 Hz, 2H), 6.70 (s, 1H), 4.85 (s, 1H),

4.36−4.24 (m, 4H), 4.20−4.07 (m, 2H), 2.76 (t, J = 12.4 Hz, 2H),

2.03−1.97 (m, 1H), 1.84 (d, J = 12.4 Hz, 3H), 1.54−1.40 (m, 18H),

and 1.37−1.27 (m, 2H).

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 9.18 (bs, 1H), 8.88 (bs, 1H), 8.73 (bs, 2H), 8.65 (bs,

3H), 8.46 (s, 1H), 7.70 (bs, 2H), 7.26 (d, J = 7.2 Hz, 1H), 7.21 (s,

1H), 4.28 (d, J = 5.6 Hz, 2H), 4.04 (d, J = 4.4 Hz, 2H), 3.26 (s, 3H),

2.87 (d, J = 11.2 Hz, 2H), 2.12 (bs, 1H), 1.89 (d, J = 13.2 Hz, 2H),

1.52 (d, J = 12.8 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 159.7,

155.6, 147.5, 143.6, 141.6, 138.2, 133.8, 133.6, 126.1, 124.8, 122.2,

113.0, 70.4, 55.2, 43.0, 33.3, 25.5; MS (ESI): [M + H]⁺406.2.

N,N-Dimethyl-1-(4-(6-(piperidin-4-ylmethoxy)-3-(pyridin-3-yl)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (31) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

To a solution of R⁶-substituted compound (1.5 mmol) in DCM (5

mL) was dropwise added HCl (1.2 mL, 4 N in p-dioxane) at 0 °C.

(400 MHz, D₂O): δ 8.70 (s, 1H), 8.61 (d, J = 5.4 Hz, 1H), 8.38 (d, J

= 7.5 Hz, 1H), 8.31 (s, 1H), 7.85 (t, J = 6.8 Hz, 1H), 7.44 (dd, J =

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19.5, 7.6 Hz, 4H), 4.31 (d, J = 6.0 Hz, 2H), 4.25 (s, 2H), 3.39 (d, J =

11.1 Hz, 2H), 2.96 (t, J = 12.8 Hz, 2H), 2.17 (s, 1H), 2.01 (d, J = 13.9

Hz, 2H), 1.53 (q, J = 12.7 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ

159.7, 149.7, 147.1, 141.4, 140.2, 137.8, 137.7, 137.4, 134.2, 131.3,

130.8, 130.7, 126.9, 70.4, 60.5, 43.6, 42.2, 42.0, 32.8, 24.9; MS (ESI):

[M + H]⁺404.2.

(4-(3-(4-(Aminomethyl)-3-ﬂuorophenyl)-5-(piperidin-4-

ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride (32) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, D₂O): δ 8.32 (s, 1H), 7.40 (d, J = 14.0 Hz, 5H),

7.28 (t, J = 8.4 Hz, 2H), 4.38 (d, J = 6.0 Hz, 2H), 4.25 (s, 2H), 4.19

(s, 2H), 3.51 (d, J = 12.4 Hz, 2H), 3.07 (t, J = 12.8 Hz, 2H), 2.27 (s,

1H), 2.12 (d, J = 14.0 Hz, 2H), 1.64 (dd, J = 23.6, 11.6 Hz, 2H); ¹³C

NMR (100 MHz, D₂O): δ 161.7, 159.2, 158.8, 147.6, 143.7, 140.9,

140.8, 137.7, 132.9, 132.6, 131.1, 131.0, 130.2, 128.8, 126.2, 126.2,

120.4, 120.2, 117.1, 116.8, 110.0, 70.3, 43.6, 42.6, 36.9, 36.8, 32.8,

25.0; MS (ESI): [M + H]⁺422.2.

(43) was prepared following the same procedure as a hydrochloric

acid salt. H NMR (400 MHz, DMSO-d₆): δ 9.18 (s, 1H), 8.87 (s,

1

1H), 8.73 (s, 1H), 8.49 (br, 3H), 8.42 (s, 1H), 7.90 (d, J = 8.0 Hz,

2H), 7.61 (d, J = 8.0 Hz, 2H), 7.43 (dd, J = 20.0, 8.0 Hz, 4H), 4.61 (s,

2H), 4.32 (d, J = 5.6 Hz, 2H), 4.04 (s, 2H), 3.29 (d, J = 10.4 Hz, 2H),

2.90 (dd, J = 22.4, 10.8 Hz, 2H), 1.94 (d, J = 13.2 Hz, 2H), 1.63−1.49

(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.8, 149.3, 146.7,

143.8, 138.2, 138.0, 136.6, 133.8, 132.9, 131.1, 129.6, 128.8, 120.9,

119.6, 69.7, 54.9, 42.6, 41.8, 32.9, 25.2; MS (ESI): [M + H]⁺472.2.

(4-(3-(4-Phenoxyphenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (44) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.11 (s, 1H), 7.25 (d, J = 7.7 Hz, 2H), 7.13 (dd, J = 15.2, 7.6

Hz, 4H), 7.03−6.93 (m, 3H), 6.64 (d, J = 7.6 Hz, 2H), 6.46 (d, J =

7.6 Hz, 2H), 4.01 (s, 4H), 3.42 (d, J = 12.0 Hz, 2H), 2.93 (t, J = 12.6

Hz, 2H), 1.98 (s, 1H), 1.87 (d, J = 13.6 Hz, 2H), 1.49 (dd, J = 24.8,

12.8 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.2, 157.3, 155.5,

148.1, 142.9, 138.3, 132.5, 131.9, 131.3, 129.9, 129.4, 128.9, 127.0,

124.1, 118.9, 117.4, 69.7, 43.4, 42.5, 32.8, 24.9; MS (ESI): [M + H]⁺

46.2.7.

(4-(3-(1-Methyl-1H-pyrazol-4-yl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (38) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.28 (s, 1H), 9.06 (d, J = 9.6 Hz, 1H), 8.67

(s, 3H), 8.13 (s, 1H), 7.74 (s, 1H), 7.59 (d, J = 7.6 Hz, 2H), 7.43 (d, J

= 7.6 Hz, 2H), 7.06 (s, 1H), 4.29 (d, J = 6.0 Hz, 2H), 4.09 (d, J = 5.6

Hz, 2H), 3.79 (s, 3H), 3.27 (d, J = 11.6 Hz, 2H), 2.89 (dd, J = 22.4,

11.2 Hz, 2H), 2.13 (s, 1H), 1.92 (d, J = 13.2 Hz, 2H), 1.58 (dd, J =

23.6, 11.6 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.2, 142.9,

141.7, 139.4, 138.8, 134.7, 131.9, 130.8, 129.65, 129.60, 120.0, 69.7,

43.0, 42.3, 33.4, 25.6; MS (ESI): [M + H]⁺379.2.

(4-(5-(Piperidin-4-ylmethoxy)-3-(4-(pyridin-4-yl)phenyl)pyrazin-

2-yl)phenyl)methanamine hydrochloride (45) was prepared following

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

1

D₂O): δ 8.77 (d, J = 6.0 Hz, 2H), 8.29 (d, J = 7.2 Hz, 3H), 7.85 (d, J

= 7.6 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.41 (dd, J = 14.8, 7.6 Hz,

4H), 4.38 (d, J = 5.6 Hz, 2H), 4.18 (s, 2H), 3.51 (d, J = 12.0 Hz, 2H),

3.07 (t, J = 12.0 Hz, 2H), 2.27 (s, 1H), 2.13 (d, J = 13.2 Hz, 2H), 1.65

(dd, J = 23.2, 10.0 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.7,

157.1, 148.1, 144.0, 141.0, 140.7, 138.1, 134.6, 132.8, 132.5, 130.8,

130.2, 128.7, 127.8, 124.3, 70.2, 43.5, 42.6, 32.8, 24.9; MS (ESI): [M

+ H]⁺452.2.

(4-(5-(Piperidin-4-ylmethoxy)-3-(1H-pyrazol-4-yl)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (39) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.06 (s, 1H), 7.54 (d, J = 5.3 Hz, 4H), 7.43 (d, J = 7.6 Hz,

2H), 4.29 (d, J = 11.6 Hz, 4H), 3.51 (d, J = 12.4 Hz, 2H), 3.07 (t, J =

12.6 Hz, 2H), 2.21 (s, 1H), 2.10 (d, J = 14.0 Hz, 2H), 1.62 (q, J =

11.6 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.6, 142.3, 141.9,

137.6, 134.4, 133.6, 129.7, 129.7, 129.3, 119.2, 70.0, 43.6, 42.7, 32.8,

25.0; MS (ESI): [M + H]⁺365.2.

(4-(3-(4-(1H-Pyrazol-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methan-amine hydrochloride (46) was prepared

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.18 (s, 2H), 8.15 (s, 1H), 7.41 (s, 2H), 7.34−

7.27 (m, 6H), 4.20 (s, 2H), 4.14 (s, 2H), 3.49 (d, J = 12.9 Hz, 2H),

3.02 (t, J = 13.2 Hz, 2H), 2.12 (br, 1H), 2.03 (d, J = 15.3 Hz, 2H),

1.64−1.54 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.5, 148.8,

143.2, 138.1, 135.9, 132.7, 131.11, 131.04, 131.02, 131.00, 130.95,

130.4, 130.1, 128.8, 125.1, 70.1, 43.5, 42.6, 32.9, 25.0; MS (ESI): [M

+ H]⁺441.2.

1

(4-(3-(3,5-Dimethylisoxazol-4-yl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (40) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.37 (s, 1H), 7.48 (s, 4H), 4.34 (d, J = 5.6 Hz,

2H), 4.21 (s, 2H), 3.52 (d, J = 12.4 Hz, 2H), 3.08 (t, J = 12.8 Hz,

2H), 2.27 (s, 1H), 2.13 (d, J = 14.0 Hz, 2H), 2.03 (s, 3H), 1.99 (s,

3H), 1.69−1.59 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 169.2,

160.4, 158.8, 145.1, 139.6, 137.5, 133.9, 132.9, 129.6, 129.0, 113.9,

70.3, 43.6, 42.6, 32.8, 24.9, 10.6, 9.5; MS (ESI): [M + H]⁺394.2.

(4-(3-(4-(3,5-Dimethylisoxazol-4-yl)phenyl)-5-(piperidin-4-

ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride (41) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, DMSO-d₆): δ 9.12 (s, 1H), 8.81 (s, 1H), 8.47 (s,

3H), 8.39 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.47−7.32 (m, 6H), 4.31

(d, J = 6.0 Hz, 2H), 4.02 (d, J = 5.2 Hz, 2H), 3.29 (d, J = 11.2 Hz,

2H), 2.91 (dd, J = 22.8, 12.4 Hz, 2H), 2.41 (s, 3H), 2.23 (s, 3H), 2.16

(s, 1H), 1.94 (d, J = 13.6 Hz, 2H), 1.56 (dd, J = 24.4, 11.2 Hz, 2H);

¹³C NMR (100 MHz, D₂O): δ 165.4, 158.0, 157.7, 147.3, 143.6,

138.4, 137.0, 133.8, 132.6, 130.3, 130.0, 129.5, 128.7, 128.6, 115.3,

69.6, 42.6, 41.8, 32.9, 25.2, 11.6, 10.6; MS (ESI): [M + H]⁺470.2.

(4-(3-(4-(1H-1,2,3-Triazol-1-yl)phenyl)-5-(piperidin-4-

ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride (42) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, D₂O): δ 8.38 (s, 1H), 8.19 (s, 1H), 7.93 (s, 1H),

7.59 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.34 (dd, J = 21.2,

8.0 Hz, 4H), 4.24 (d, J = 6.0 Hz, 2H), 4.16 (s, 2H), 3.50 (d, J = 12.4

Hz, 2H), 3.05 (t, J = 12.4 Hz, 2H), 2.16 (s, 1H), 2.06 (d, J = 13.6 Hz,

2H), 1.60 (dd, J = 24.2, 11.2 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ

158.5, 147.7, 143.5, 137.9, 136.3, 134.5, 132.8, 132.0, 131.2, 130.1,

128.8, 123.8, 120.3, 116.0, 70.1, 43.6, 42.6, 32.9, 25.0; MS (ESI): [M

+ H]⁺442.2.

(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

yl)phenyl)methanamine hydrochloride (47) was prepared following

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

1

D₂O): δ 8.24 (s, 1H), 7.94 (s, 1H), 7.61 (s, 1H), 7.51 (dd, J = 8.4, 1.8

Hz, 2H), 7.41 (br, 6H), 6.85 (s, 1H), 4.35 (br, 2H), 4.17 (s, 2H),

3.50 (d, J = 13.3 Hz, 2H), 3.06 (t, J = 13.3 Hz, 2H), 2.12 (br, 1H),

2.11 (d, J = 14.6 Hz, 2H), 1.68−1.58 (m, 2H); ¹³C NMR (100 MHz,

D₂O): δ 158.4, 148.8, 144.5, 143.3, 139.4 (2), 138.3, 135.6, 132.6,

132.4, 131.1, 130.3, 130.0, 128.7, 125.0, 108.1, 70.0, 43.5, 42.6, 32.9,

24.9; HRMS (ESI⁺) calcd for C₂₇H₂₉N₄O₂[M + H]⁺, 441.2291;

found, 441.2285.

1-(4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)ethan-1-one hydrochloride (48) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.08 (br, 1H), 8.78 (br, 1H), 8.41 (br, 4H),

7.89 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz,

2H), 7.35 (d, J = 8.4 Hz, 2H), 4.29 (d, J = 6 Hz, 2H), 3.99 (d, J = 5.2

Hz, 2H), 3.27 (d, J = 12.0 Hz, 2H), 2.88 (d, J = 10.4 Hz, 2H), 2.57 (s,

3H), 2.15 (br, 1H), 1.91 (d, J = 11.6 Hz, 2H), 1.53 (d, J = 12 Hz,

2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 198.0, 158.2, 147.2, 144.4,

143.0, 138.5, 136.9, 134.3, 133.6, 130.4, 130.0, 129.2, 128.6, 70.1,

55.4, 43.0, 42.2, 33.4, 25.6; MS (ESI): [M + H]⁺417.2.

(4-(3-(4-Isopropylphenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (49) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 8.65 (s, 4H), 8.34 (s, 1H), 7.44 (d, J = 7.6 Hz, 2H),

7.41−7.29 (m, 4H), 7.21 (d, J = 7.2 Hz, 2H), 4.29 (d, J = 6.0 Hz,

2H), 4.01 (s, 2H), 3.28 (d, J = 12.4 Hz, 2H), 2.90 (t, J = 10.8 Hz,

3H), 2.14 (s, 1H), 1.93 (d, J = 13.2 Hz, 2H), 1.55 (dd, J = 24.2, 11.8

(1-(4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)methanol hydrochloride

2790

https://dx.doi.org/10.1021/acs.jmedchem.0c02070

J. Med. Chem. 2021, 64, 2777−2800

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Hz, 2H), 1.20 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆):

δ 157.6, 149.0, 147.8, 143.4, 138.6, 135.6, 133.6, 132.1, 129.5, 129.4,

128.6, 126.2, 69.5, 42.6, 41.8, 33.1, 33.0, 25.2, 23.7; MS (ESI): [M +

H]⁺417.3.

(4-(3-(4-(Furan-3-yl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-2-

yl)phenyl)methanamine hydrochloride (59) was prepared following

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.01 (s, 1H), 7.69 (s, 1H), 7.41 (s, 1H), 7.21 (dd, J = 20.0,

7.6 Hz, 6H), 7.04 (d, J = 8.1 Hz, 2H), 6.61 (s, 1H), 4.09 (d, J = 6.0

Hz, 2H), 3.98 (s, 2H), 3.34 (d, J = 12.4 Hz, 3H), 2.88 (t, J = 12.0 Hz,

2H), 2.00 (s, 1H), 1.91 (d, J = 14.0 Hz, 2H), 1.44 (dd, J = 23.6, 10.8

Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.3, 148.5, 144.4, 143.8,

139.3, 138.2, 135.6, 133.3, 131.8, 131.6, 130.7, 130.2, 130.1, 128.7,

125.2, 108.2, 70.2, 43.5, 42.6, 32.9, 24.9; MS (ESI): [M + H]⁺441.2.

1-(4-(3-(4-(Furan-3-yl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-

2-yl)phenyl)-N-methylmethanamine hydrochloride (60) was pre-

4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-

2-yl)phenol hydrochloride (50) was prepared following the same

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, DMSO-

d₆): δ 9.79 (s, 1H), 8.25−9.82 (m, 5H), 7.34−7.40 (m, 4H), 7.20 (d,

J = 8.8 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.27 (d, J = 6.4 Hz, 2H),

4.00 (d, J = 5.2 Hz, 2H), 3.27 (br, 2H), 2.89 (d, J = 11.2 Hz, 2H),

2.11 (br, 1H), 1.91 (d, J = 13.2 Hz, 2H), 1.48−1.51 (m, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 158.5, 158.0, 148.4, 143.5, 139.5,

133.8, 131.8, 131.4, 129.8, 129.0, 128.8, 115.5, 69.8, 43.2, 42.3, 33.4,

25.6; MS (ESI): [M + H]⁺391.2.

1

pared following the same procedure as a hydrochloric acid salt. H

NMR (400 MHz, D₂O): δ 8.17 (s, 1H), 7.85 (s, 1H), 7.57 (s, 1H),

7.48−7.24 (m, 6H), 7.18 (d, J = 7.4 Hz, 2H), 6.76 (s, 1H), 4.24 (d, J

= 4.8 Hz, 2H), 4.16 (s, 2H), 3.49 (d, J = 10.4 Hz, 2H), 3.03 (t, J =

11.6 Hz, 2H), 2.68 (s, 3H), 2.17 (s, 1H), 2.06 (d, J = 12.4 Hz, 2H),

1.60 (dd, J = 24.4, 12.8 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ

158.3, 148.2, 144.4, 144.0, 139.3, 138.7, 135.8, 131.9, 131.8, 131.3,

130.3, 130.1, 129.6, 125.2, 125.1, 108.2, 70.2, 51.7, 43.6, 32.9, 32.0,

25.0; MS (ESI): [M + H]⁺455.2.

1-(4-(3-(4-(Furan-3-yl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-

2-yl)phenyl)-N,N-dimethylmethanamine hydrochloride (61) was

prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, D₂O): δ 8.07 (s, 1H), 7.73 (s, 1H), 7.44 (s,

1H), 7.28 (t, J = 7.3 Hz, 4H), 7.23 (d, J = 7.7 Hz, 2H), 7.07 (d, J = 7.8

Hz, 2H), 6.64 (s, 1H), 4.16−4.11 (m, 4H), 3.36 (d, J = 12.6 Hz, 2H),

2.89 (d, J = 12.8 Hz, 2H), 2.67 (s, 6H), 2.05 (s, 1H), 1.94 (d, J = 13.4

Hz, 2H), 1.47 (dd, J = 24.5, 11.9 Hz, 2H); ¹³C NMR (100 MHz,

D₂O): δ 158.4, 148.2, 144.4, 144.1, 139.3, 135.8, 132.0, 131.8, 130.6,

130.4, 130.4, 130.1, 129.8, 125.2, 125.1, 108.2, 70.2, 60.5, 43.6, 42.0,

32.9, 24.9; MS (ESI): [M + H]⁺469.2.

(4-(3-(1H-Benzo[d]imidazol-6-yl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (51) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.59 (s, 1H), 8.82 (s, 1H), 9.07 (s, 1H),

8.43 (bs, 4H), 7.95 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.4

Hz, 1H), 7.34−7.40 (m, 4H), 4.29 (d, J = 6.4 Hz, 2H), 3.98 (d, J =

6.4 Hz, 2H), 3.28 (d, J = 12 Hz, 2H), 2.90 (d, J = 11.6 Hz, 2H), 2.15

(bs, 1H), 1.92 (d, J = 12.8 Hz, 2H), 1.52−1.60 (m, 2H); ¹³C NMR

(100 MHz, DMSO-d₆): δ 158.19, 147.32, 144.34, 141.85, 138.63,

136.45, 134.25, 131.12, 130.05, 129.21, 128.01, 116.12, 114.61, 65.35,

43.07, 42.20, 31.75, 15.61; MS (ESI): [M + H]⁺414.2.

(4-(3-(1H-Benzo[d]imidazole-4-yl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (52) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.43 (s, 1H), 9.06 (bs, 1H), 8.78 (bs, 1H),

8.50 (s, 1H), 8.39 (bs, 3H), 7.83 (d, J = 7.6 Hz, 2H), 7.38−7.40 (m,

2H), 7.23 (d, J = 7.2 Hz, 2H), 4.25 (d, J = 6.4 Hz, 2H), 3.93 (d, J =

4.8 Hz, 2H), 2.87 (d, J = 11.6 Hz, 4H), 2.11 (bs, 1H), 1.89 (d, J =

12.4 Hz, 2H), 1.51 (d, J = 11.2 Hz, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 170.8, 158.2, 144.9, 144.2, 142.0, 137.9, 134.6, 134.3,

129.8, 129.0, 127.0, 126.5, 125.8, 115.6, 114.9, 70.3, 65.4, 60.2, 34.6,

21.2; MS (ESI): [M + H]⁺415.2.

1-(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-

2-yl)phenyl)-N-methylmethanamine hydrochloride (62) was pre-

1

pared following the same procedure as a hydrochloric acid salt. H

NMR (400 MHz, D₂O): δ 8.07 (s, 1H), 7.76 (s, 1H), 7.54 (s, 1H),

7.28 (d, J = 7.9 Hz, 2H), 7.24−7.16 (m, 4H), 7.12 (d, J = 7.8 Hz,

2H), 6.67 (s, 1H), 4.1−4.00 (m, 4H), 3.43 (d, J = 6.1 Hz, 2H), 2.97

(t, J = 12.6 Hz, 2H), 2.64 (s, 3H), 2.06 (s, 2H), 1.95 (d, J = 13.2 Hz,

2H), 1.59−1.48 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.5,

148.8, 144.7, 143.2, 139.5, 138.9, 135.6, 132.6, 131.3, 130.8, 130.4,

130.2, 129.9, 125.1, 116.0, 108.3, 70.1, 52.3, 43.7, 33.07, 32.11, 25.0;

MS (ESI): [M + H]⁺455.2.

1-(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-

2-yl)phenyl)-N,N-dimethylmethanamine hydrochloride (63) was

prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, D₂O): δ 8.25 (s, 1H), 7.94 (s, 1H), 7.61 (s,

1H), 7.50 (d, J = 7.2 Hz, 2H), 7.45−7.35 (m, 6H), 6.84 (s, 1H), 4.34

(d, J = 6.0 Hz, 2H), 4.29 (s, 2H), 3.50 (d, J = 11.6 Hz, 2H), 3.06 (t, J

= 13.2 Hz, 2H), 2.24 (s, 1H), 2.11 (d, J = 14.4 Hz, 2H), 1.63 (dd, J =

24.0, 11.6 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.7, 149.1,

144.5, 143.6, 139.5, 135.8, 132.6, 131.6, 130.6, 130.4, 130.3, 129.2,

125.2, 125.1, 108.2, 70.1, 60.6, 43.5, 42.0, 32.9, 24.9; MS (ESI): [M +

H]⁺429.2.

(4-(3-(1H-Indol-2-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (53) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 11.51 (s, 1H), 9.11 (s, 1H), 8.88 (s, 1H), 8.53 (s, 3H),

8.24 (s, 1H), 7.64−7.47 (m, 5H), 7.32 (d, J = 7.5 Hz, 1H), 7.19−7.05

(m, 1H), 7.00−6.89 (m, 1H), 4.51 (d, J = 4.6 Hz, 2H), 4.13 (d, J =

4.4 Hz, 2H), 3.31 (d, J = 8.9 Hz, 2H), 2.92 (dd, J = 21.7, 11.0 Hz,

2H), 2.17 (s, 1H), 1.99 (d, J = 11.7 Hz, 2H), 1.61 (dd, J = 22.3, 10.1

Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.6, 143.7, 140.1,

139.2, 136.6, 134.3, 133.7, 131.2, 129.3, 129.1, 127.8, 122.9, 120.8,

119.6, 112.0, 104.2, 69.7, 42.6, 42.0, 33.1, 25.2; MS (ESI): [M + H]⁺

414.2.

(4-(3-(1H-Indol-4-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (54) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.27 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 9.2 Hz,

3H), 7.24 (d, J = 7.2 Hz, 2H), 7.12 (t, J = 7.2 Hz, 1H), 6.97 (d, J = 7.2

Hz, 1H), 4.26 (d, J = 4.8 Hz, 2H), 4.09 (s, 2H), 3.49 (d, J = 12.4 Hz,

2H), 3.03 (t, J = 12.4 Hz, 2H), 2.18 (s, 1H), 2.05 (d, J = 14.0 Hz,

2H), 1.59 (dd, J = 25.6, 13.2 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ

158.5, 149.8, 143.9, 138.4, 135.9, 132.3, 131.5, 129.6, 129.0, 128.4,

126.3, 126.1, 121.5, 121.2, 112.4, 70.2, 43.6, 42.6, 32.8, 24.9; MS

(ESI): [M + H]⁺414.2.

(4-(3-(4-(Furan-3-yl)phenyl)-6-(2-(piperazin-1-yl)ethoxy)pyrazin-

2-yl)phenyl)methanamine hydrochloride (64) was prepared following

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.36 (s, 1H), 7.93 (s, 1H), 7.60 (s, 1H), 7.53 (d, J = 8.0 Hz,

4H), 7.41−7.34 (m, 4H), 6.85 (s, 1H), 4.18 (s, 2H), 3.85 (s, 7H),

3.66 (s, 6H); ¹³C NMR (100 MHz, D₂O): δ 157.3, 148.6, 145.1,

144.5, 139.4, 138.2, 135.8, 133.4, 132.3, 132.1, 130.3, 130.2, 128.7,

125.4, 125.2, 108.3, 60.0, 56.0, 48.8, 42.6, 40.5; MS (ESI): [M + H]⁺

456.2.

(4-(3-(1H-Indol-5-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (55) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.27 (s, 1H), 7.71 (s, 1H), 7.47−7.35 (m, 6H), 7.32 (d, J =

7.4 Hz, 1H), 7.21 (d, J = 7.3 Hz, 1H), 4.36 (s, 2H), 4.17 (s, 2H), 3.51

(d, J = 11.5 Hz, 2H), 3.06 (t, J = 13.1 Hz, 2H), 2.22 (s, 1H), 2.10 (d, J

= 13.5 Hz, 2H), 1.65 (dd, J = 24.7, 12.0 Hz, 2H); ¹³C NMR (100

MHz, D₂O): δ 158.6, 151.1, 143.4, 139.3, 136.1, 132.8, 130.6, 130.3,

129.1, 128.8, 127.6, 126.6, 123.6, 122.5, 111.6, 70.2, 43.7, 42.7, 33.2,

25.3; MS (ESI): [M + H]⁺414.2.

(4-(3-(4-(Furan-3-yl)phenyl)-5-(2-(piperidin-4-yl)ethoxy)pyrazin-

2-yl)phenyl)methanamine hydrochloride (65) was prepared following

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.00 (s, 1H), 7.71 (s, 1H), 7.44 (s, 1H), 7.28−7.04 (m, 6H),

6.61 (s, 1H), 4.19 (s, 1H), 3.99 (s, 2H), 3.25 (d, J = 11.2 Hz, 2H),

2.77 (t, J = 12.4 Hz, 1H), 1.80 (d, J = 13.6 Hz, 2H), 1.62 (s, 2H), 1.29

(dd, J = 21.2, 10.4 Hz, 1H); ¹³C NMR (100 MHz, D₂O): δ 158.5,

1

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149.0, 144.5, 143.2, 139.4, 138.3, 135.8, 132.6, 132.5, 131.2, 130.3,

130.1, 128.8, 125.1, 108.2, 64.7, 43.8, 42.6, 34.0, 30.2, 28.2; MS

(ESI): [M + H]⁺455.2.

(d, J = 8 Hz, 1H), 6.77 (d, J = 8 Hz, 1H), 5.14−5.16 (m, 1H), 3.99

(bs, 2H), 3.27 (d, J = 13.6 Hz, 2H), 2.83 (bs, 2H), 1.93 (bs, 2H),

1.81−1.84 (m, 1H), 1.52−1.56 (m, 2H), 1.32 (d, J = 6 Hz, 3H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 157.9, 154.8, 147.6, 143.9, 143.8,

138.8, 137.0, 132.9, 132.6, 130.2, 129.8, 127.3, 125.9, 125.5, 115.4,

108.6, 79.4, 75.1, 41.5, 28.4, 27.8, 16.9; MS (ESI): [M + H]⁺455.2.

4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

yl)benzamide hydrochloride (79) was prepared following the same

2-Methoxy-4-(5-(piperidin-4-ylmethoxy)-3-(4-(thiophen-3-yl)-

phenyl)pyrazin-2-yl)benzamide hydrochloride (72) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 8.91 (br, 1H), 8.57 (br, 1H), 8.39 (s, 1H),

7.96 (br, 1H), 7.74 (d, J = 7.9 Hz, 2H), 7.72 (d, J = 8.0 Hz, 1H),

7.65−7.59 (m, 2H), 7.55 (br, 1H), 7.47 (d, J = 7.9 Hz, 2H), 7.11 (s,

1H), 7.00 (d, J = 8.0 Hz, 1H), 4.33 (d, J = 6.2 Hz, 2H), 3.68 (s, 3H),

3.31 (d, J = 12.5 Hz, 2H), 2.96−2.88 (m, 2H), 2.16 (br, 1H), 1.94 (d,

J = 13.6 Hz, 2H), 1.58−1.49 (m, 2H); ¹³C NMR (100 MHz, DMSO-

d₆): δ 165.9, 157.9, 147.9, 143.2, 142.4, 140.6, 136.5, 135.4, 132.4,

132.1, 131.5, 130.7, 130.2, 128.8, 127.4, 126.2, 125.9, 121.9, 113.2,

69.6, 66.4, 42.8, 32.9, 26.3; MS (ESI): [M + H]⁺501.2.

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, DMSO-

d₆): δ 8.77 (br, 2H), 8.40 (s, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 7.99 (s,

1H), 7.81 (d, J = 7.7 Hz, 2H), 7.74 (s, 1H), 7.59 (d, J = 7.7 Hz, 2H),

7.43−7.40 (m, 4H), 6.98 (s, 1H), 4.33 (d, J = 5.6 Hz, 2H), 3.09−3.05

(m, 2H), 2.92 (br, 2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H),

1.56−1.47 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 167.6,

158.0, 147.8, 144.6, 143.4, 141.4, 140.1, 136.3, 133.4, 132.5, 130.2,

129.4, 127.53, 127.46, 125.4, 125.3, 108.7, 69.7, 42.7, 33.0, 25.3; MS

(ESI): [M + H]⁺455.5.

(4-(5-(Cyclohexylmethoxy)-3-(4-(furan-3-yl)phenyl)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (73) was prepared following the

1

2-Fluoro-4-(3-(4-(furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)benzamide hydrochloride (80) was prepared following

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 8.45 (bs, 3H), 8.33 (s, 1H), 8.21 (s, 1H), 7.73 (s, 1H),

7.57 (d, J = 7.6 Hz, 2H), 7.38−7.42 (m, 6H), 6.96 (s, 1H), 4.20 (d, J

= 5.2 Hz, 2H), 3.99 (d, J = 4.8 Hz, 2H), 1.80 (d, J = 10.8 Hz, 2H),

1.70 (d, J = 11.2 Hz, 2H), 1.64 (d, J = 10.8 Hz, 1H), 1.21 (dd, J =

12.8 Hz, 25.6 Hz, 4H), 1.04−1.07 (m, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 158.4, 147.9, 144.9, 143.7, 140.4, 139.1, 137.0, 134.0,

132.8, 132.6, 130.5, 130.0, 129.1, 125.7, 125.6, 109.0, 71.6, 42.2, 37.3,

29.7, 26.4, 25.7. MS (ESI): [M + H]⁺440.2.

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 8.80 (br, 1H), 8.48 (br, 1H), 8.39 (s, 1H), 7.77−7.75

(m, 1H), 7.74 (br, 1H), 7.66 (br, 1H), 7.63 (d, J = 8.3 Hz, 2H), 7.59

(t, J = 7.8 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.26−7.19 (m, 2H), 7.00

(s, 1H), 4.33 (d, J = 6.3 Hz, 2H), 3.33−3.30 (m, 2H), 2.96−2.87 (m,

2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H), 1.56−1.48 (m, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ 164.8, 159.0, 158.0, 147.9, 144.6,

142.9, 142.0, 140.1, 135.9, 132.6, 131.5, 130.2, 128.9, 128.7, 125.4,

125.3, 108.6, 70.0, 43.2, 35.3, 25.6; MS (ESI): [M + H]⁺473.2.

N-(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-

2-yl)benzyl)hydroxylamine hydrochloride (81) was prepared follow-

(4-(5-(Benzyloxy)-3-(4-(furan-3-yl)phenyl)pyrazin-2-yl)phenyl)-

methanamine hydrochloride (74) was prepared following the same

1

procedure as a hydrochloric acid salt. H NMR (400 MHz, DMSO-

d₆): δ 8.40−8.42 (m, 3H), 8.23 (s, 1H), 7.74 (s, 1H), 7.58 (d, J = 8.4

Hz, 1H), 7.52−7.57 (m, 3H), 7.38−7.41 (m, 8H), 7.34−7.36 (m,

2H), 6.70 (s, 1H), 5.48 (s, 2H), 3.99 (d, J = 5.6 Hz, 2H); ¹³C NMR

(100 MHz, DMSO-d₆): δ 157.9, 147.9, 145.0, 144.1, 140.4, 139.1,

137.0, 136.8, 134.0, 133.0, 132.7, 130.5, 130.0, 129.1, 128.9, 128.8,

128.6, 125.7, 125.6, 109.0, 68.1, 42.3; MS (ESI): [M + H]⁺434.2.

(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-yloxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (75) was prepared following the

1

ing the same procedure as a hydrochloric acid salt. H NMR (400

MHz, D₂O): δ 8.20 (s, 1H), 7.87 (s, 1H), 7.58 (s, 1H), 7.49−7.21

(m, 8H), 6.78 (s, 1H), 4.39 (d, J = 7.6 Hz, 2H), 4.26 (d, J = 6.4 Hz,

2H), 3.52−3.42 (m, 2H), 3.03 (s, 2H), 2.17 (s, 1H), 2.06 (d, J = 12.4

Hz, 2H), 1.59 (dd, J = 23.6, 10.4 Hz, 2H); ¹³C NMR (100 MHz,

D₂O): δ 158.6, 148.9, 144.4, 143.6, 139.4, 139.2, 135.7, 132.4, 131.5,

130.5, 130.3, 130.0, 128.4, 125.2, 115.8, 108.2, 70.1, 54.3, 43.5, 32.9,

24.9; MS (ESI): [M + H]⁺457.2.

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 8.29 (s, 1H), 8.20 (s, 1H), 7.73 (s, 1H), 7.56 (d, J = 8

Hz, 2H), 7.38−7.40 (m, 4H), 7.29 (d, J = 8 Hz, 2H), 7.15 (d, J = 8

Hz, 2H), 6.96 (s, 1H), 5.27 (bs, 1H), 4.10 (d, J = 5.6 Hz, 2H), 3.65

(bs, 2H), 3.23 (bs, 2H), 2.00 (bs, 2H), 1.64−1.66 (m, 2H); ¹³C NMR

(100 MHz, DMSO-d₆): δ 157.2, 156.3, 154.4, 147.6, 144.9, 144.3,

140.3, 137.4, 137.0, 133.1, 132.6, 130.4, 129.7, 127.0, 125.7, 109.0,

79.2, 78.3, 71.6, 43.5; MS (ESI): [M + H]⁺427.2.

(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

yl)-3-methylphenyl)methanamine hydrochloride (82) was prepared

following the same procedure as a hydrochloric acid salt. H NMR

1

(400 MHz, D₂O): δ 8.10 (s, 1H), 7.77 (s, 1H), 7.47 (s, 1H), 7.28 (d,

J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.13 (s, 3H), 6.67 (s, 1H),

4.18 (d, J = 6.0 Hz, 2H), 4.02 (s, 2H), 3.37 (d, J = 12.4 Hz, 2H), 2.92

(q, J = 11.2 Hz, 2H), 2.09 (br, 1H), 1.97 (d, J = 12.4 Hz, 2H), 1.8 (br,

3H), 1.49 (dd, J = 23.6, 11.6 Hz, 2H); MS (ESI): [M + H]⁺455.2.

(3-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

yl)phenyl)methanamine hydrochloride (83) was prepared following

3-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-

2-yl)oxy)propan-1-amine hydrochloride (76) was prepared following

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

1

DMSO-d₆): δ 8.44 (bs, 3H), 8.36 (s, 1H), 8.22 (s, 1H), 8.09 (bs,

3H), 7.74 (s, 1H), 7.57 (d, J = 8 Hz, 2H), 7.37−7.42 (m, 6H), 6.97

(s, 1H), 4.48 (t, J = 6 Hz, 2H), 3.99 (d, J = 5.6 Hz, 2H), 2.96 (d, J = 6

Hz, 2H), 2.09−2.13 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ

158.0, 147.9, 145.0, 144.0, 140.4, 139.0, 136.8, 134.1, 132.9, 132.7,

130.5, 129.9, 129.1, 125.7, 125.6, 109.0, 64.0, 42.2, 36.6, 34.6; MS

(ESI): [M + H]⁺401.2.

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 9.14 (br, 1H), 8.84 (br, 1H), 8.48 (br, 3H), 8.37 (s,

1H), 8.24 (s, 1H), 7.76 (s, 1H), 7.72 (s, 1H), 7.58 (d, J = 8.0 Hz,

2H), 7.46 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.28 (t, J = 8.0

Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 6.98 (s, 1H), 4.33 (d, J = 6.2 Hz,

2H), 4.02 (q, J = 5.1 Hz, 2H), 3.29 (d, J = 12.3 Hz, 2H), 2.91 (q, J =

12.1 Hz, 2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H), 1.60−1.52

(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.8, 147.4, 144.6,

143.6, 140.0, 138.9, 136.2, 134.4, 132.4, 132.3, 130.1, 129.9, 129.6,

128.5, 128.3, 125.3, 125.2, 108.6, 69.6, 42.7, 42.2, 33.0, 25.2; MS

(ESI): [M + H]⁺441.5.

(2-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)-5-(furan-3-yl)phenyl)methanol hydrochloride (84) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, D₂O): δ 8.34 (s, 1H), 7.97 (s, 1H), 7.70 (s, 1H),

7.64 (s, 1H), 7.43−7.29 (m, 5H), 7.10 (s, 1H), 6.88 (s, 1H), 4.54 (s,

2H), 4.27 (s, 2H), 4.14 (s, 2H), 3.51 (d, J = 12.0 Hz, 2H), 3.07 (t, J =

12.0 Hz, 2H), 2.22 (s, 1H), 2.10 (d, J = 13.2 Hz, 2H), 1.63 (dd, J =

25.6, 13.6 Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.3, 148.8,

144.5, 144.1, 139.5, 139.3, 137.8, 134.5, 132.9, 132.8, 132.5, 131.4,

129.9, 128.5, 125.2, 125.1, 124.5, 108.2, 70.2, 61.4, 43.6, 42.6, 32.8,

24.9; MS (ESI): [M + H]⁺471.2.

2-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-

2-yl)oxy)-N-methylethan-1-amine hydrochloride (77) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 8.32 (bs, 1H), 8.20 (s, 1H), 7.73 (s, 1H),

7.57 (d, J = 7.2 Hz, 2H), 7.38−7.40 (m, 3H), 7.26−7.28 (m, 2H),

7.15 (d, J = 8 Hz, 2H), 6.96 (s, 1H), 4.53 (t, J = 5.2 Hz, 2H), 4.10 (d,

J = 5.2 Hz, 2H), 2.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ

157.8, 156.3, 144.9, 144.4, 140.4, 140.3, 137.4, 137.0, 132.8, 132.6,

130.4, 129.7, 127.0, 125.7, 125.6, 109.0, 78.9, 78.3, 63.8, 43.5; MS

(ESI): [M + H]⁺401.2.

(4-(3-(4-(Furan-3-yl)phenyl)-5-(1-(piperidin-4-yl)ethoxy)pyrazin-

2-yl)phenyl)methanamine hydrochloride (78) was prepared following

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 9.09 (bs, 1H), 8.78 (bs, 1H), 8.45 (bs, 3H), 8.30 (s,

1H), 7.75 (s, 1H), 7.57 (d, J = 8 Hz, 2H), 7.39−7.41 (m, 6H), 7.26

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(4-(3-(2-Fluoro-4-(furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)-

DMSO-d₆): δ 157.8, 145.4, 144.6, 143.9, 141.9, 137.9, 137.5, 135.2,

134.9, 134.5, 134.0, 133.4, 129.6, 128.9, 128.6, 122.4, 118.6, 109.8,

108.7, 69.8, 42.6, 41.7, 32.9, 25.1; MS (ESI): [M + H]⁺466.2.

(4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)(pyrrolidin-1-yl)methanone hydrochloride (91)

was prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, DMSO-d₆): δ 9.22 (s, 1H), 8.92 (d, J = 9.2 Hz,

1H), 8.51 (br, 3H), 8.40 (s, 1H), 7.50−7.40 (m, 6H), 7.37 (d, J = 8.0

Hz, 2H), 4.30 (d, J = 6.0 Hz, 2H), 4.00 (s, 2H), 3.45 (t, J = 6.0 Hz,

2H), 3.36 (t, J = 6.0 Hz, 2H), 3.28 (d, J = 12.0 Hz, 2H), 2.90 (dd, J =

22.4, 10.4 Hz, 2H), 2.15 (s, 1H), 1.93 (d, J = 13.6 Hz, 2H), 1.83 (dt, J

= 23.6, 11.6 Hz, 4H), 1.56 (dd, J = 23.6, 12.0 Hz, 2H); ¹³C NMR

(100 MHz, DMSO-d₆): δ 168.1, 158.2, 147.6, 144.2, 139.6, 138.7,

137.6, 134.2, 133.3, 130.0, 129.9, 129.2, 127.4, 70.1, 49.3, 46.4, 43.0,

42.2, 33.4, 26.4, 25.6, 24.3; MS (ESI): [M + H]⁺472.3.

pyrazin-2-yl)phenyl)methanamine hydrochloride (85) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.40 (d, J = 8.8 Hz, 1H), 9.04 (d, J = 8.8

Hz, 1H), 8.59 (br, 3H), 8.44 (s, 1H), 8.34 (s, 1H), 7.77 (s, 1H),

7.63−7.54 (m, 2H), 7.40 (dd, J = 23.6, 8.4 Hz, 5H), 7.05 (s, 1H),

4.26 (d, J = 6.0 Hz, 2H), 3.97 (d, J = 5.2 Hz, 2H), 3.26 (d, J = 11.6

Hz, 2H), 2.88 (dd, J = 22.0, 11.2 Hz, 2H), 2.14 (s, 1H), 1.91 (d, J =

12.6 Hz, 2H), 1.56 (dd, J = 23.2, 11.2 Hz, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 160.3, 157.9, 157.8, 144.9, 144.7, 142.9, 140.8, 138.1,

135.2, 135.1, 133.8, 133.5, 132.4, 132.4, 128.6, 128.5, 124.6, 124.49,

124.47, 124.4, 121.8, 112.6, 112.4, 108.6, 69.8, 42.5, 41.7, 32.9, 25.1;

MS (ESI): [M + H]⁺459.2.

(4-(3-(4-(Furan-3-yl)-2-methoxyphenyl)-5-(piperidin-4-

ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride (86) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, D₂O): δ 8.26 (s, 1H), 7.95 (s, 1H), 7.62 (s, 1H),

7.42−7.23 (m, 6H), 7.02 (s, 1H), 6.85 (s, 1H), 4.22 (s, 2H), 4.13 (s,

2H), 3.49 (d, J = 14.0 Hz, 2H), 3.31 (s, 3H), 3.03 (t, J = 13.2 Hz,

2H), 2.15 (s, 1H), 2.05 (d, J = 12.8 Hz, 2H), 1.59 (dd, J = 24.4, 13.2

Hz, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.8, 156.0, 146.8, 145.1,

144.5, 139.6, 138.6, 134.9, 132.4, 131.9, 131.8, 128.9, 128.4, 125.3,

125.2, 118.5, 108.8, 108.3, 70.2, 55.1, 43.6, 42.6, 32.9, 24.9; MS

(ESI): [M + H]⁺471.2.

(4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)(morpholino)methanone hydrochloride (92)

was prepared following the same procedure as a hydrochloric acid

1

salt. H NMR (400 MHz, DMSO-d₆): δ 9.27 (d, J = 10.4 Hz, 1H),

8.98 (d, J = 9.6 Hz, 1H), 8.55 (s, 3H), 8.40 (s, 1H), 7.46 (dd, J = 9.6,

8.4 Hz, 4H), 7.41−7.32 (m, 4H), 4.29 (d, J = 6.0 Hz, 2H), 4.01 (d, J

= 5.6 Hz, 2H), 3.60 (s, 11H), 3.28 (d, J = 11.6 Hz, 2H), 2.90 (dd, J =

22.4, 10.8 Hz, 2H), 2.15 (s, 1H), 1.93 (d, J = 12.8 Hz, 2H), 1.57 (q, J

= 12.4 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.0, 158.1,

147.5, 144.0, 139.5, 138.6, 135.8, 133.7, 133.2, 129.97, 129.95, 129.0,

127.3, 69.9, 66.6, 66.3, 42.9, 41.9, 33.1, 25.3; MS (ESI): [M + H]⁺

488.3.

(4-(3-(4-(Furan-3-yl)-2-methylphenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (87) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.15 (s, 1H), 8.83 (s, 1H), 8.49 (s, 3H),

8.36 (s, 1H), 7.94 (s, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.45−7.38 (m,

5H), 7.31 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 6.82 (s, 1H),

4.31 (d, J = 5.6 Hz, 2H), 4.01 (d, J = 4.8 Hz, 2H), 3.29 (d, J = 12.0

Hz, 2H), 2.90 (d, J = 10.8 Hz, 2H), 2.34 (s, 3H), 2.14 (s, 1H), 1.93

(d, J = 13.2 Hz, 2H), 1.56 (d, J = 12.0 Hz, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 157.7, 147.6, 143.6, 143.4, 140.9, 138.6, 136.6, 135.2,

133.6, 132.3, 132.1, 131.9, 129.5, 128.7, 128.4, 127.3, 124.4, 111.1,

69.6, 42.6, 41.8, 33.0, 25.2, 21.4; MS (ESI): [M + H]⁺455.2.

(4-(3-(4-(Furan-3-yl)-3-methylphenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (88) was prepared

(4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)(piperidin-1-yl)methanone hydrochloride (93)

was prepared following the same procedure as a hydrochloric acid

1

salt. H NMR (400 MHz, DMSO-d₆): δ 9.17 (s, 1H, NH), 8.86 (s,

1H, NH), 8.47 (s, 3H, NH), 8.37 (s, 1H), 7.46−7.20 (m, 8H), 4.26

(s, 2H), 3.97 (s, 2H), 3.53 (s, 2H), 3.24 (s, 4H), 2.86 (s, 2H), 2.11 (s,

1H), 1.90 (d, J = 13.2 Hz, 2H), 1.60−1.32 (m, 8H); ¹³C NMR (100

MHz, DMSO-d₆): δ 168.3, 157.7, 147.2, 143.7, 138.8, 138.3, 136.5,

133.8, 132.8, 129.6, 129.5, 128.7, 126.6, 69.6, 42.6, 41.8, 32.9, 25.1,

24.0; MS (ESI): [M + H]⁺486.3.

(4-(5-(Piperidin-4-ylmethoxy)-3-(4-(tetrahydrofuran-3-yl)phenyl)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (94) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.36 (d, J = 11.0 Hz, 1H), 8.99 (d, J = 8.8

Hz, 1H), 8.55 (s, 3H), 8.42 (s, 1H), 8.23 (s, 1H), 7.75 (s, 1H), 7.51

(s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.36 (dd, J = 23.4, 8.0 Hz, 4H), 7.17

(d, J = 8.0 Hz, 1H), 6.98 (s, 1H), 4.22 (d, J = 6.0 Hz, 2H), 3.95 (d, J =

5.2 Hz, 2H), 3.26 (d, J = 11.6 Hz, 2H), 2.87 (q, J = 11.2 Hz, 2H),

2.11 (s, 1H), 2.06 (br, 3H), 1.91 (d, J = 13.2 Hz, 2H), 1.55 (dd, J =

22.8, 10.8 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.5, 148.4,

144.4, 144.0, 139.8, 138.1, 136.5, 136.1, 133.6, 133.0, 132.0, 130.6,

128.9, 128.5, 127.3, 125.3, 123.0, 108.6, 69.6, 42.5, 41.7, 33.0, 25.1,

19.5; MS (ESI): [M + H]⁺455.2.

(4-(3-(4-(Furan-3-yl)-3-methoxyphenyl)-5-(piperidin-4-

ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride (89) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, DMSO-d₆): δ 9.17 (s, 1H), 8.85 (s, 1H), 8.48 (br,

3H), 8.37 (s, 1H), 8.16 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 8.4 Hz,

1H), 7.50−7.36 (m, 4H), 7.04 (d, J = 21.6 Hz, 3H), 4.33 (d, J = 5.6

Hz, 2H), 4.01 (s, 2H), 3.67 (s, 3H), 3.29 (d, J = 10.0 Hz, 2H), 2.90

(dd, J = 21.6, 10.8 Hz, 2H), 2.16 (s, 1H), 1.94 (d, J = 12.4 Hz, 2H),

1.56 (dd, J = 22.4, 11.2 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ

157.7, 155.5, 147.5, 143.8, 143.1, 141.9, 138.8, 137.1, 133.6, 132.4,

129.5, 128.7, 127.3, 121.9, 120.9, 120.8, 112.9, 109.3, 69.6, 55.3, 42.6,

41.8, 33.0, 25.2; MS (ESI): [M + H]⁺471.2.

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.16 (bs, 1H), 8.86 (bs, 1H), 8.48 (bs, 3H),

8.31 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.32−7.40 (m, 4H), 7.21 (d, J

= 8.4 Hz, 2H), 4.26 (d, J = 5.6 Hz, 2H), 3.99 (d, J = 7.6 Hz, 3H),

3.88−3.93 (m, 1H), 3.76 (dd, J = 8 Hz, 15.6 Hz, 1H), 3.35 (t, J = 8.4

Hz, 1H), 3.25 (d, J = 13.2 Hz, 2H), 2.87 (d, J = 11.2 Hz, 2H), 2.26−

2.29 (m, 1H), 2.12 (bs, 1H), 1.88−1.92 (m, 3H), 1.52−1.56 (m, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ 158.1, 148.0, 143.9, 143.8, 139.0,

136.5, 134.1, 132.7, 130.1, 129.9, 129.1, 127.6, 74.1, 69.9, 68.1, 55.4,

44.3, 43.0, 34.2, 33.4, 25.6; MS (ESI): [M + H]⁺445.3.

(4-(5-(Piperidin-4-ylmethoxy)-3-(3-(tetrahydrofuran-3-yl)phenyl)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (95) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 8.30 (s, 1H), 7.14−7.33 (m, 8H), 4.23 (d, J

= 5.6 Hz, 2H), 3.85 (t, J = 6.8 Hz, 1H), 3.65−3.67 (m, 4H), 3.21−

3.27 (m, 4H), 3.08 (d, J = 12 Hz, 2H), 2.63−2.66 (m, 2H), 2.12 (bs,

1H), 1.98 (bs, 1H), 1.77 (d, J = 14 Hz, 2H), 1.60 (bs, 1H), 1.28−1.36

(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.2, 148.0, 144.6,

143.0, 138.6, 137.3, 132.7, 129.6, 128.9, 128.8, 128.2, 127.9, 127.6,

127.1, 74.0, 70.6, 67.9, 55.3, 44.6, 44.2, 34.7, 34.2, 27.8; MS (ESI):

[M + H]⁺444.6.

(4-(5-(Piperidin-4-ylmethoxy)-3-(4-(pyrrolidin-1-yl)phenyl)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (96) was prepared

5-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-

2-yl)-2-(furan-3-yl)benzonitrile hydrochloride (90) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 9.07 (s, 1H), 8.75 (s, 1H), 8.45 (s, 3H),

8.19 (s, 1H), 7.43 (q, J = 8.4 Hz, 4H), 7.26 (d, J = 8.8 Hz, 2H), 6.45

(d, J = 8.8 Hz, 2H), 4.29 (d, J = 6.4 Hz, 2H), 4.02 (d, J = 6.0 Hz, 2H),

3.29 (d, J = 12.0 Hz, 2H), 3.23 (s, 4H), 2.90 (dd, J = 23.6, 12.4 Hz,

2H), 2.14 (s, 1H), 1.95 (s, 7H), 1.54 (dd, J = 24.2, 10.8 Hz, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 157.5, 148.2, 147.7, 142.6, 139.6,

(400 MHz, DMSO-d₆): δ 9.25 (s, 1H), 8.92 (s, 1H), 8.54 (s, 3H),

8.41 (d, J = 2.1 Hz, 1H), 8.29 (s, 1H), 7.97 (s, 1H), 7.86 (s, 1H), 7.63

(dd, J = 27.6, 8.0 Hz, 2H), 7.46 (d, J = 7.2 Hz, 2H), 7.40 (d, J = 6.4

Hz, 2H), 7.02 (s, 1H), 4.30 (s, 2H), 4.00 (s, 2H), 3.26 (d, J = 11.2

Hz, 2H), 2.88 (dd, J = 19.6, 8.8 Hz, 2H), 2.13 (s, 1H), 1.91 (d, J =

13.2 Hz, 2H), 1.55 (dd, J = 22.0, 10.4 Hz, 2H); ¹³C NMR (100 MHz,

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133.3, 130.7, 130.3, 129.3, 128.6, 123.90, 111.1, 69.2, 47.2, 42.7, 41.8,

33.0, 25.2, 25.0; MS (ESI): [M + H]⁺444.3.

2H), 4.26 (d, J = 6 Hz, 2H), 3.99 (d, J = 5.2 Hz, 2H), 3.91 (d, J = 10.8

Hz, 2H), 2.88 (dd, J = 11.2 Hz, 22.8 Hz, 2H), 2.73−2.76 (m, 1H),

2.11 (bs, 1H), 1.90 (d, J = 13.2 Hz, 2H), 1.64 (bs, 4H), 1.51 (d, J =

12 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.1, 148.1, 147.0,

143.8, 139.1, 136.3, 134.0, 132.6, 130.1, 129.9, 129.0, 127.0, 69.9,

67.7, 43.0, 42.2, 40.5, 33.8, 33.3, 25.6; MS (ESI): [M + H]⁺459.3.

(4-(3-(4-Morpholinophenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

yl)phenyl)methanamine hydrochloride (104) was prepared following

(4-(3-(4-(Piperidin-1-yl)phenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (97) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.32 (s, 1H), 7.59 (dd, J = 22.2, 7.9 Hz, 4H),

7.41 (br, 4H), 4.38 (s, 2H), 4.21 (s, 2H), 3.64 (br, 4H), 3.52 (d, J =

12.4 Hz, 2H), 3.09 (t, J = 12.0 Hz, 2H), 2.27 (s, 1H), 2.13 (d, J = 12.8

Hz, 2H), 2.03 (br, 4H), 1.77 (s, 2H), 1.66 (dd, J = 25.2, 12.8 Hz,

2H); ¹³C NMR (100 MHz, D₂O): δ 158.8, 147.6, 143.8, 141.9, 139.3,

137.9, 132.8, 132.5, 131.8, 130.2, 128.8, 120.9, 70.2, 57.1, 43.6, 42.6,

32.9, 25.0, 23.3, 20.5; MS (ESI): [M + H]⁺458.3.

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 9.24 (d, J = 10.4 Hz, 1H), 8.91 (dd, J = 20.8, 10.8 Hz,

1H), 8.54 (s, 3H), 8.27 (s, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.39 (d, J =

8.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 4.29

(d, J = 6.0 Hz, 2H), 4.01 (d, J = 5.2 Hz, 2H), 3.75 (br, 4H), 3.27 (d, J

= 12.4 Hz, 2H), 3.18 (br, 4H), 2.89 (q, J = 11.6 Hz, 2H), 2.14 (s,

1H), 1.92 (d, J = 12.6 Hz, 2H), 1.55 (q, J = 11.6 Hz, 2H); ¹³C NMR

(100 MHz, DMSO-d₆): δ 157.6, 150.3, 147.6, 143.1, 139.0, 133.5,

131.3, 130.6, 129.4, 128.7, 114.6, 69.4, 65.8, 48.1, 42.6, 41.8, 40.2,

39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 33.0, 25.2; MS (ESI): [M + H]⁺

460.3.

(4-(3-(4-Cyclohexylphenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

yl)phenyl)methanamine hydrochloride (98) was prepared following

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

DMSO-d₆): δ 8.97 (bs, 1H), 8.68 (bs, 1H), 8.37 (bs, 3H), 8.30 (s,

1H), 7.29−7.39 (m, 6H), 7.15 (d, J = 8 Hz, 2H), 4.27 (s, 2H), 3.99

(s, 2H), 3.27 (d, J = 11.6 Hz, 2H), 2.88 (d, J = 11.2 Hz, 2H), 2.11 (bs,

1H), 1.90 (d, J = 13.6 Hz, 3H), 1.65−1.76 (m, 5H), 1.51 (d, J = 12

Hz, 2H), 1.35 (bs, 4H), 1.12−1.20 (m, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 158.1, 148.7, 148.2, 143.8, 139.1, 136.0, 134.0, 132.6,

130.0, 129.9, 129.0, 127.0, 110.0, 69.9, 43.9, 43.1, 42.2, 34.2, 33.3,

26.7, 26.0, 25.6; MS (ESI): [M + H]⁺457.3.

General Synthetic Procedure for Compounds 33, 35, 36,

and 37. To a solution of R⁵-substituted compound (88 mg, 0.16

mmol) in DMSO (3 mL) were added pyrazole (17 mg, 0.25 mmol)

and potassium carbonate (45 mg, 0.32 mmol). The resulting mixture

was heated at 120 °C for 24 h before it was cooled to room

temperature. The mixture was extracted with ethyl acetate (3 × 20

mL), and the combined organic layers were washed with water and

brine and dried over Na₂SO₄. The volatiles were removed in vacuo to

aﬀord a crude oil, which was puriﬁed by column chromatography

(silica gel, hexanes/ethyl acetate from 2:1 to 1.5:1) to give the

corresponding R⁶-substituted product (35 mg, 38%). For the

precursor of 35: ¹H NMR (400 MHz, CDCl₃): δ 8.32 (s, 1H),

7.69 (s, 1H), 7.65 (s, 1H), 7.21 (s, 4H), 4.86 (s, 1H), 4.29 (br, 1H),

4.25 (d, J = 6.8 Hz, 2H), 4.18 (br, 2H), 2.74 (t, J = 12.8 Hz, 2H), 2.03

(s, 1H), 1.81 (d, J = 12.8 Hz, 2H), 1.45 (br, 18H), 1.28−1.21 (m, 2).

To a solution of R⁶-substituted compound (0.08 mmol) in DCM

(5 mL) was dropwise added HCl (0.1 mL, 4 N in p-dioxane) at 0 °C.

The reaction mixture was warmed to room temperature and stirred

for 5 h. The volatiles were removed in vacuo to aﬀord an oil, which

was triturated in diether ether and solidiﬁed to give the ﬁnal product

hydrochloric acid salt.

(4-(3-(4-(Piperidin-4-yl)phenyl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (99) was prepared

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.24 (s, 1H), 7.38 (s, 6H), 7.25 (d, J = 7.6 Hz,

2H), 4.36 (d, J = 5.2 Hz, 2H), 4.18 (s, 2H), 3.51 (t, J = 12.0 Hz, 4H),

3.18−3.01 (m, 4H), 2.97−2.88 (m, 1H), 2.25 (s, 1H), 2.16−2.02 (m,

4H), 1.87 (dd, J = 26.4, 12.8 Hz, 2H), 1.63 (dd, J = 25.6, 12.0 Hz,

2H); ¹³C NMR (100 MHz, D₂O): δ 158.7, 149.2, 145.21, 143.6,

138.4, 135.7, 132.5, 131.3, 130.1, 130.0, 128.7, 126.5, 70.1, 44.2, 43.5,

42.6, 38.8, 32.8, 29.1, 24.9; MS (ESI): [M + H]⁺458.3.

(4-(5-(Piperidin-4-ylmethoxy)-3-(4-(1,2,3,6-tetrahydropyridin-4-

yl)phenyl)pyrazin-2-yl)phenyl)methanamine hydrochloride (100)

was prepared following the same procedure as a hydrochloric acid

1

salt. H NMR (400 MHz, DMSO-d₆): δ 9.52 (s, 2H, NH), 9.15 (s,

1H, NH), 8.86 (s, 1H, NH), 8.49 (s, 4H), 8.34 (s, 1H), 7.44−7.32

(m, 8H), 6.24 (s, 6H), 4.26 (d, J = 5.2 Hz, 2H), 3.97 (d, J = 4.0 Hz,

2H), 3.69.18 (s, 2H), 3.60−3.24 (m, 4H), 2.90−2.85 (m, 2H), 2.65

(s, 2H), 2.11 (s, 1H), 1.88 (d, J = 12.0 Hz, 2H), 1.54 (dd, J = 25.6,

12.0 Hz, 2H); MS (ESI): [M + H]⁺456.3.

N¹-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)ethane-1,2-diamine hydrochloride (33) was prepared

1

(4-(3-(6-(Piperazin-1-yl)pyridin-3-yl)-5-(piperidin-4-ylmethoxy)-

pyrazin-2-yl)phenyl)methanamine hydrochloride (101) was prepared

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 7.62−7.43 (m, 4H), 7.41 (s, 1H), 4.24 (d, J = 6.0

Hz, 2H), 4.16 (s, 2H), 3.62 (d, J = 5.6 Hz, 2H), 3.38 (d, J = 11.2 Hz,

2H), 3.15 (s, 2H), 2.94 (t, J = 12.0 Hz, 2H), 2.10 (s, 1H), 1.98 (d, J =

13.6 Hz, 2H), 1.56−1.42 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ

160.3, 152.8, 134.8, 131.4, 129.8, 129.4, 128.0, 112.4, 70.6, 43.6, 42.6,

38.5, 32.9, 25.0; MS (ESI): [M + H]⁺357.2.

(4-(5-(Piperidin-4-ylmethoxy)-3-(1H-pyrazol-1-yl)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (35). ¹H NMR (400 MHz,

D₂O): δ 8.26 (s, 1H), 7.76 (s, 1H), 7.57 (s, 1H), 7.30 (d, J = 7.2 Hz,

2H), 7.16 (d, J = 7.2 Hz, 2H), 4.22 (d, J = 6.0 Hz, 2H), 4.07 (s, 2H),

3.37 (d, J = 12.4 Hz, 2H), 2.94 (t, J = 12.4 Hz, 2H), 2.13 (s, 1H), 1.98

(d, J = 13.2 Hz, 2H), 1.57−1.46 (m, 2H); ¹³C NMR (100 MHz,

D₂O): δ 158.2, 142.2, 141.9, 138.6, 136.0, 134.2, 133.1, 132.1, 128.9,

128.8, 108.1, 70.5, 43.5, 42.6, 33.0, 25.0; 365.2; MS (ESI): [M + H]⁺

365.2.

1

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, D₂O): δ 8.20 (s, 1H), 7.97 (s, 1H), 7.79 (d, J = 12.0 Hz,

1H), 7.35 (s, 4H), 7.04 (d, J = 12.0 Hz, 1H), 4.27 (s, 2H), 4.09 (s,

1H), 3.83 (s, 4H), 3.35 (s, 6H), 2.94 (t, J = 12.0 Hz, 4H), 2.15 (s,

2H), 1.99 (d, J = 12.0 Hz, 2H), 1.50 (d, J = 12.0 Hz, 2H); ¹³C NMR

(100 MHz, D₂O): δ 158.9, 152.1, 144.6, 143.7, 143.3, 138.5, 137.4,

133.4, 133.2, 130.2, 129.2, 124.5, 111.8, 70.2, 43.6, 42.9, 42.6, 42.2,

32.8, 25.0; MS (ESI): [M + H]⁺460.3.

(4-(5-(Piperidin-4-ylmethoxy)-3-(6-(tetrahydro-2H-pyran-4-yl)-

pyridin-3-yl)pyrazin-2-yl)phenyl)methanamine hydrochloride (102)

was prepared following the same procedure as a hydrochloric acid salt.

¹H NMR (400 MHz, DMSO-d₆): δ 9.11 (bs, 1H), 8.82−8.85 (m,

1H), 8.60 (s, 1H), 8.49 (bs, 3H), 8.43 (s, 1H), 8.01 (d, J = 7.6 Hz,

1H), 7.56 (d, J = 8 Hz, 1H), 7.46 (d, J = 8 Hz, 2H), 7.40 (d, J = 8.4

Hz, 2H), 4.28 (d, J = 6 Hz, 2H), 4.00 (d, J = 5.2 Hz, 2H), 3.93 (d, J =

10.8, 2H), 3.32−3.62 (m, 4H), 3.26 (d, J = 11.6 Hz, 2H), 3.14 (bs,

1H), 2.88 (d, J = 12 Hz, 2H), 2.14 (bs, 1H), 1.90 (d, J = 12.8 Hz,

2H), 1.59−1.74 (m, 4H), 1.51−1.59 (m, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ 158.3, 146.2, 144.6, 144.0, 138.0, 134.6, 134.2, 133.6,

130.1, 129.4, 129.3, 129.2, 122.8, 70.3, 67.2, 65.4, 43.0, 42.2, 41.1,

34.6, 33.3, 31.8, 31.7, 25.5, 15.6; MS (ESI): [M + H]⁺460.3.

(4-(5-(Piperidin-4-ylmethoxy)-3-(4-(tetrahydro-2H-pyran-4-yl)-

phenyl)pyrazin-2-yl)phenyl)methanamine hydrochloride (103) was

prepared following the same procedure as a hydrochloric acid salt. ¹H

NMR (400 MHz, DMSO-d₆): δ 9.02 (bs, 1H), 8.70−8.72 (m, 1H),

8.40 (bs, 3H), 8.32 (s, 1H), 7.32−7.41 (m, 6H), 7.20 (d, J = 8 Hz,

(4-(5-(Piperidin-4-ylmethoxy)-3-(1H-1,2,3-triazol-1-yl)pyrazin-2-

yl)phenyl)methanamine hydrochloride (36) was prepared following

1

the same procedure as a hydrochloric acid salt. H NMR (400 MHz,

D₂O): δ 8.43 (s, 1H), 7.87 (s, 2H), 7.30 (d, J = 7.6 Hz, 2H), 7.11 (d,

J = 7.6 Hz, 2H), 4.26 (d, J = 5.6 Hz, 2H), 4.07 (s, 2H), 3.38 (d, J =

12.4 Hz, 2H), 2.95 (t, J = 12.8 Hz, 2H), 2.16 (s, 1H), 1.99 (d, J = 13.6

Hz, 2H), 1.57−1.46 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 158.4,

141.1, 139.5, 137.1, 136.6, 135.4, 133.4, 129.0, 128.7, 70.7, 43.5, 42.6,

32.6, 24.8; MS (ESI): [M + H]⁺366.2.

(4-(3-(1H-Imidazol-1-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)-

phenyl)methanamine hydrochloride (37) was prepared following the

1

same procedure as a hydrochloric acid salt. H NMR (400 MHz,

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D₂O): δ 9.46 (s, 1H), 8.57 (s, 1H), 8.13 (s, 1H), 7.86 (d, J = 7.6 Hz,

2H), 7.59 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 4.36 (d, J = 6.0 Hz, 2H),

4.16 (s, 2H), 3.34 (d, J = 12.8 Hz, 2H), 2.90 (t, J = 12.8 Hz, 2H), 2.15

(s, 1H), 1.94 (d, J = 14.4 Hz, 2H), 1.46 (dd, J = 24.8, 11.6 Hz, 2H);

¹³C NMR (100 MHz, D₂O): δ 156.6, 143.9, 139.6, 134.7, 134.4,

sodium carbonate (216 mg, 2 mmol) in p-dioxane/H₂O (8/2 mL)

were placed in a sealed tube. The mixture was degassed and heated to

100 °C for 24 h. The reaction was then cooled and quenched with

brine (10 mL). The mixture was extracted with ethyl acetate (3 × 20

mL), and the combined organic layers were washed with water and

brine and dried over Na₂SO₄. The volatiles were removed in vacuo to

aﬀord a crude oil, which was puriﬁed by column chromatography

(silica gel, hexanes/ethyl acetate from 5:1 to 1:2) to aﬀord the

corresponding R⁶-substituted a or b (78% yield for a). For compound

133.6, 129.8, 128.8, 125.1, 121.0, 119.0, 70.9, 43.5, 42.7, 32.6, 25.1;

MS (ESI): [M + H]⁺365.2.

General Synthetic Procedure for Compounds 19, 66−69,

and 71. 2,6-Dibromopyrazine (111a, X = N, 7.14 g, 30 mmol) or 2,6-

dibromopyridine (111b, X = CH, 7.14 g, 30 mmol), tert-butyl 4-

(aminomethyl)piperidine-1-carboxylate (7.07 g, 33 mmol), and

potassium carbonate (8.28 g, 60 mmol) in N,N-dimethylformamide

(DMF) (30 mL) were placed in a sealed tube. The mixture was

stirred at 100 °C for 12 h. The reaction was cooled and quenched

with saturated brine (50 mL). The mixture was extracted with ethyl

acetate (3 × 100 mL), and the combined organic layers were washed

with water and brine and dried over Na₂SO₄. The volatiles were

removed in vacuo to aﬀord 112a or 112b as a white solid, which was

used in the next step without further puriﬁcation. For compound

1

a (precursor of 66): H NMR (400 MHz, CDCl₃): δ 7.90 (s, 1H),

7.73 (s, 1H), 7.46 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.2

Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 6.69 (s,

1H), 4.89 (t, J = 5.9 Hz, 1H), 4.85 (br, 1H), 4.28 (d, J = 5.9 Hz, 2H),

4.16−4.11 (m, 2H), 3.36 (t, J = 6.1 Hz, 2H), 2.71 (t, J = 11.8 Hz,

2H), 1.79−1.76 (m, 3H), 1.45 (s, 9H), 1.44 (s, 9H), and 1.26−1.16

1

(m, 2H). For compound b (precursor of 69): H NMR (400 MHz,

CDCl₃): δ 7.70 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.45 (br, 1H), 7.34

(br, 4H), 7.13 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 6.67 (s,

1H), 6.42 (d, J = 8.4 Hz, 1H), 4.83 (t, J = 5.5 Hz, 2H), 4.28 (d, J = 5.3

Hz, 2H), 4.14−4.09 (m, 2H), 3.25 (t, J = 5.8 Hz, 2H), 2.71 (t, J =

13.4 Hz, 2H), 1.81−1.78 (m, 3H), 1.46 (s, 9H), 1.45 (s, 9H), and

1.27−1.20 (m, 2H).

1

112a: H NMR (400 MHz, CDCl₃): δ 7.85 (s, 1H), 7.76 (s, 1H),

5.00 (s, 1H), 4.14−4.11 (m, 2H), 3.25 (t, J = 5.9 Hz, 2H), 2.68 (t, J =

11.8 Hz, 2H), 1.73−1.70 (m, 3H), 1.44 (s, 9H), and 1.21−1.09 (m,

2H). For compound 112b: ¹H NMR (400 MHz, CDCl₃): δ 7.23 (t, J

= 7.8 Hz, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.26 (d, J = 7.8 Hz, 1H), 4.76

(t, J = 5.4 Hz, 1H), 4.10 (br, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.67 (t, J =

12.8 Hz, 2H), 1.77−1.67 (m, 3H), 1.44 (s, 9H), and 1.19−1.09 (m,

2H).

The above R⁶-substituted compound (0.2 mmol) was dissolved in

DCM (2 mL). HCl (0.2 mL, 4 N in p-dioxane) was slowly added to

the reaction mixture at 0 °C. After 0.5 h, the reaction was warmed to

room temperature and stirred for 12 h. The volatiles were removed in

vacuo to aﬀord an oil, which was triturated in diethyl ether and

solidiﬁed to give 66 and 69 hydrochloric salt as a white and pale-

yellow powder, respectively.

To a solution of compound 112a or 112b (∼30 mmol) in

CH₃CN/DMSO (20/10 mL) was added NIS (8.1 g, 36 mmol). After

stirring at room temperature for 24 h, the reaction was quenched with

sodium thiosulfate aqueous solution (50 mL). The mixture was

extracted with ethyl acetate (3 × 100 mL), and the combined organic

layers were washed with water and brine and dried over Na₂SO₄. The

volatiles were removed in vacuo to aﬀord a crude oil, which was

puriﬁed by column chromatography (silica gel, hexanes/ethyl acetate

from 10:1 to 3:1) to give 113a or 113b as a white solid (8.95 g, 60%

over two steps). For compound 113a: ¹H NMR (400 MHz, CDCl₃):

δ 7.64 (s, 1H), 4.86 (t, J = 5.6 Hz, 1H), 4.14−4.11 (m, 2H), 3.24 (t, J

= 5.9 Hz, 2H), 2.69 (t, J = 12.2 Hz, 2H), 1.73−1.69 (m, 3H), 1.45 (s,

5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)-N-(piperi-

1

din-4-ylmethyl)pyrazin-2-amine hydrochloride (66). H NMR (400

MHz, DMSO-d₆): δ 9.00 (br, 2H), 8.67 (br, 2H), 8.38 (br, 4H), 8.21

(s, 1H), 8.02 (s, 1H), 7.75 (s, 1H), 7.54 (d, J = 7.3 Hz, 2H), 7.37−

7.30 (m, 6H), 6.96 (s, 1H), 3.98 (br, 2H), 3.28−3.24 (m, 4H), 2.83

(q, J = 10.8 Hz, 2H), 1.89−1.85 (m, 3H), 1.46−1.38 (m, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 153.0, 148.1, 144.5, 143.8, 139.8,

139.6, 137.9, 137.6, 132.5, 131.7, 130.0, 129.3, 128.5, 125.3, 125.1,

108.6, 55.0, 42.9, 41.9, 33.5, 26.4; MS (ESI): [M + H]⁺440.5.

5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)-N-(piperi-

1

9H), and 1.21−1.13 (m, 2H). For compound 113b: H NMR (400

din-4-ylmethyl)pyridin-2-amine hydrochloride (69). H NMR (400

MHz, CDCl₃): δ 7.64 (d, J = 8.5 Hz, 1H), 6.10 (d, J = 8.5 Hz, 1H),

4.74 (t, J = 5.4 Hz, 1H), 4.14−4.09 (m, 2H), 3.14 (t, J = 5.9 Hz, 2H),

2.69 (t, J = 12.2 Hz, 2H), 1.73−1.69 (m, 3H), 1.45 (s, 9H), and

1.20−1.11 (m, 2H).

MHz, DMSO-d₆): δ 9.05 (br, 1H), 8.83 (br, 2H), 8.44 (br, 3H), 8.28

(s, 1H), 7.91 (br, 2H), 7.77 (s, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.41 (d,

J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H),

7.01 (s, 1H), 3.97 (d, J = 6.3 Hz, 2H), 3.38 (br, 2H), 3.28 (d, J = 13.5

Hz, 2H), 2.85 (q, J = 12.3 Hz, 2H), 1.95−1.91 (m, 3H), 1.48−1.40

(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 153.9, 144.6, 140.4,

136.6, 133.2, 130.4, 129.5, 129.0, 125.3, 124.9, 123.1, 108.5, 46.3,

42.5, 41.6, 33.1, 25.9; MS (ESI): [M + H]⁺439.6.

Compound 113a or 113b (994 mg, 2 mmol), 4-((N-Boc-

amino)methyl)phenylboronic acid (527 mg, 2.1 mmol), tetrakis-

(triphenylphosphine)palladium (69 mg, 3 mol %), and sodium

carbonate (424 mg, 4 mmol) in p-dioxane/H₂O (15/3 mL) were

placed in a sealed tube. The mixture was degassed and heated to 80

°C for 24 h. The reaction was then cooled and quenched with brine

(20 mL). The product was extracted with ethyl acetate (3 × 20 mL),

and the combined organic layers were washed with water and brine

and dried over Na₂SO₄. The volatiles were removed in vacuo to aﬀord

a crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 5:1 to 1:1) to give the corresponding R⁵-

substituted product a and b (842 mg, 73% yield for a). For compound

a (intermediate of 66): ¹H NMR (400 MHz, CDCl₃): δ 7.88 (s, 1H),

7.63 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 4.94 (t, J = 5.9 Hz,

1H), 4.88 (br, 1H), 4.37 (d, J = 5.9 Hz, 2H), 4.16−4.11 (m, 2H),

3.31 (t, J = 5.9 Hz, 2H), 2.71 (t, J = 12.2 Hz, 2H), 1.77−1.74 (m,

3H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.25−1.15 (m, 2H). Compound

b (intermediate of 69): ¹H NMR (400 MHz, CDCl₃): δ 7.38 (d, J =

8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.36

(d, J = 8.4 Hz, 1H), 4.88 (br, 2H), 4.36 (br, 2H), 4.16−4.11 (m, 2H),

3.18 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 12.2 Hz, 2H), 2.10 (br, 1H),

1.77−1.74 (m, 2H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.22−1.15 (m,

2H).

((6-((Piperidin-4-ylmethyl)amino)pyridine-2,3-diyl)bis(4,1-

phenylene))dimethanamine hydrochloride (19) was prepared follow-

1

ing the same procedure as a hydrochloric acid salt. H NMR (400

MHz, D₂O): δ 8.09 (d, J = 9.3 Hz, 2H), 7.44 (s, 2H), 7.36 (d, J = 8.1

Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 9.3 Hz, 2H), 4.21 (s,

2H), 4.15 (s, 2H), 3.52−3.46 (m, 4H), 3.05 (t, J = 13.2 Hz, 2H),

2.13−2.08 (m, 3H), 1.59−1.49 (m, 2H); ¹³C NMR (100 MHz,

D₂O): δ 152.8, 146.4, 144.5, 136.5, 134.8, 132.4, 132.2, 130.4, 130.2,

128.9, 128.8, 124.8, 110.3, 46.6, 43.6, 42.55, 42.48, 32.8, 25.9; MS

(ESI): [M + H]⁺402.3.

5-(4-(Aminomethyl)phenyl)-6-(2-ﬂuoro-4-(furan-3-yl)phenyl)-N-

(piperidin-4-ylmethyl)pyrazin-2-amine hydrochloride (67) was pre-

1

pared following the same procedure as a hydrochloric acid salt. H

NMR (400 MHz, D₂O): δ 8.13 (s, 1H), 7.84 (br, 2H), 7.60 (d, J =

7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.33−7.26 (m, 3H), 6.72 (s,

1H), 4.21 (s, 2H), 3.72 (s, 2H), 3.44 (d, J = 13.1 Hz, 2H), 2.99−2.93

(m, 2H), 2.06−1.96 (m, 3H), 1.51−1.42 (m, 2H); ¹³C NMR (100

MHz, D₂O): δ 159.4, 153.7, 152.2, 144.3, 140.1, 136.5, 136.1, 135.6,

132.9, 132.7, 130.4, 129.7, 129.3, 128.8, 128.6, 121.5, 112.5, 108.1,

45.3, 43.6, 42.6, 32.7, 26.0; MS (ESI): [M + H]⁺458.2.

The above R⁵-substituted compound a or b (1 mmol), 2-(4-(furan-

3-yl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.1 mmol),

tetrakis(triphenylphosphine)palladium (57.7 mg, 5 mol %), and

5-(4-(Aminomethyl)-3-ﬂuorophenyl)-6-(4-(furan-3-yl)phenyl)-N-

(piperidin-4-ylmethyl)pyrazin-2-amine hydrochloride (68) was pre-

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pared following the same procedure as a hydrochloric acid salt. H

NMR (400 MHz, D₂O): δ 8.11 (s, 1H), 7.81 (br, 1H), 7.47 (br, 1H),

7.39 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.25 (br, 1H),

7.08−7.05 (m, 2H), 6.72 (s, 1H), 4.22 (d, J = 6.0 Hz, 2H), 4.10 (s,

2H), 3.36 (d, J = 13.6 Hz, 2H), 2.92 (t, J = 12.9 Hz, 2H), 2.11 (br,

1H), 1.98 (d, J = 14.8 Hz, 2H), 1.54−1.44 (m, 2H); ¹³C NMR (100

MHz, D₂O): δ 160.5, 158.6, 148.8, 144.5, 141.2, 139.4, 138.5, 135.3,

132.6, 131.5, 131.1, 130.3, 125.9, 125.1, 125.0, 119.6, 116.6, 108.2,

70.6, 43.5, 36.7, 32.9, 25.0; MS (ESI): [M + H]⁺458.6.

To a solution of crude acid (70 mg) and 4-aminomethyl-1-Boc-

piperidine (42 mg, 0.2 mmol) in DMF (3 mL) were added N,N-

diisopropylethylamine (87 μL, 0.5 mmol) and HATU (114 mg, 0.3

mmol). The mixture was stirred for 12 h before it was quenched with

H₂O. The mixture was extracted with ethyl acetate (3 × 80 mL), and

the combined organic layers were washed with water and brine and

dried over Na₂SO₄. The volatiles were removed in vacuo to aﬀord a

crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 1.5:1 to 1:1) to aﬀord the corresponding

1

5-(4-(Aminomethyl)phenyl)-N-(piperidin-4-ylmethyl)-6-(4-(thio-

phen-3-yl)phenyl)pyridin-2-amine hydrochloride (71) was prepared

following the same procedure as a hydrochloric acid salt. H NMR

(400 MHz, DMSO-d₆): δ 8.84 (br, 2H), 8.61 (br, 2H), 8.31 (br, 4H),

7.95 (s, 1H), 7.71−7.58 (m, 3H), 7.38−7.35 (m, 4H), 7.18 (d, J = 8.1

Hz, 2H), 3.97 (q, J = 5.4 Hz, 2H), 3.35 (br, 2H), 3.28 (d, J = 13.5 Hz,

2H), 2.85 (q, J = 11.6 Hz, 2H), 1.95−1.91 (m, 3H), 1.46−1.36 (m,

2H); MS (ESI): [M + H]⁺455.2.

amides (62 mg) as colorless oil. For 56 precursor: H NMR (400

MHz, CDCl₃): δ 9.92 (s, 1H), 8.74 (br, 1H), 8.66 (s, 1H), 7.80 (d, J

= 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.33

(d, J = 8.0 Hz, 2H), 4.98 (br, 1H), 4.36 (d, J = 5.2 Hz, 2H), 4.16 (br,

1H), 3.47 (t, J = 5.6 Hz, 2H), 2.70 (br, 1H), 1.90−1.78 (m, 3H), 1.91

1

(s, 1H), 1.47 (s, 9H), 1.45 (s, 9H); for 57 precursor: H NMR (400

MHz, CDCl₃): δ 9.93 (s, 1H), 8.75 (t, J = 5.6 Hz, 1H), 8.68 (s, 1H),

7.82 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.6 Hz,

2H), 7.41 (d, J = 7.9 Hz, 2H), 4.93 (br, 1H), 4.38 (d, J = 5.2 Hz, 2H),

4.12 (br, 2H), 3.48 (t, J = 6.0 Hz, 2H), 2.71 (t, J = 13.3 Hz, 2H),

1.90−1.78 (m, 3H), 1.48 (s, 9H), 1.45 (s, 9H), 1.25 (s, 2H); for 58

General Synthetic Procedure for Compounds 56, 57, 58,

and 70. To a solution of 3-amino-6-iodopyrazine-2-carboxylate (114,

1.8 g, 6.6 mmol) in 12 N HCl−H₂O (v/v = 5/1, 54 mL) at −10 °C

was added sodium nitrite solution (0.7 M solution, 18 mL) in three

portions. The resulting reaction mixture was stirred at −10 °C for 1 h

before copper(I) chloride (0.78 g, 7.88 mmol) in 12 N HCl solution

(1.8 mL) was added slowly and stirred for additional 2 h. The mixture

was extracted with ethyl acetate (3 × 50 mL), and the combined

organic layers were washed with water and brine and dried over

Na₂SO₄. The volatiles were removed in vacuo to give a crude residue,

which was puriﬁed by column chromatography (silica gel, hexanes/

ethyl acetate from 20:1 to 10:1) to aﬀord 3-chrolo-6-iodopyrazine-2-

carboxylate 115 (770 mg, 39%). ¹H NMR (400 MHz, CDCl₃): δ 8.75

(s, 1H), 4.02 (s, 3H).

3-Chrolo-6-iodopyrazine-2-carboxylate (115, 298 mg, 1 mmol),

tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (206 mg, 0.98

mmol), and potassium carbonate (300 mg, 2.2 mmol) in DMSO (6

mL) were placed in a sealed tube. The mixture was stirred at 50 °C

for 12 h. The reaction was cooled and quenched with saturated brine

(30 mL). The mixture was extracted with ethyl acetate (3 × 20 mL),

and the combined organic layers were washed with water and brine

and dried over Na₂SO₄. The volatiles were removed in vacuo to aﬀord

a residue, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 2:1 to 1.5:1) to aﬀord 116 (270 mg, 70%).

¹H NMR (400 MHz, CDCl₃): δ 8.35 (s, 1H), 8.06 (t, J = 5.3 Hz,

1

precursor: H NMR (400 MHz, CDCl₃): δ 9.94 (s, 1H), 8.75 (t, J =

5.4 Hz, 1H), 8.68 (s, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.8

Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 7.9 Hz, 2H), 4.96 (s,

1H), 4.37 (d, J = 4.8 Hz, 2H), 4.22−4.06 (m, 2H), 3.48 (t, J = 6.0 Hz,

2H), 2.71 (t, J = 12.3 Hz, 2H), 1.49−1.42 (m, 3H), 1.49−1.42 (m,

18H), 1.25−1.20 (m, 2H).

To a solution of the above amides (1.5 mmol) in DCM (5 mL) was

dropwise added HCl (1.2 mL, 4 N in p-dioxane) at 0 °C. The reaction

mixture was warmed to room temperature and stirred for 5 h. The

volatiles were removed in vacuo to aﬀord an oil, which was triturated

in diether ether and solidiﬁed to give the ﬁnal product as a

hydrochloric salt.

For the synthesis of 70 precursor: compound 57 precursor (X = I,

65 mg, 0.088 mmol), furan-3-yl boronic acid (17 mg, 0.15 mmol),

tetrakis(triphenylphosphine)palladium (10 mg, 0.009 mmol), and

sodium carbonate (43 mg, 0.4 mmol) in p-dioxane/H₂O (4/1 mL)

were placed in a sealed tube. The mixture was degassed and heated to

80 °C for 12 h. The reaction was then cooled and quenched with

brine (20 mL). The product was extracted with ethyl acetate (3 × 30

mL), and the combined organic layers were washed with water and

brine and dried over Na₂SO₄. The volatiles were removed in vacuo to

give a crude oil, which was puriﬁed by column chromatography (silica

gel, hexanes/ethyl acetate from 1.5:1 to 1:1) to aﬀord the product 70

precursor (244 mg, 63%).

3-(4-(Aminomethyl)phenyl)-N-(4-chlorophenyl)-6-((piperidin-4-

ylmethyl)amino)pyrazine-2-carboxamide hydrochloride (56). ¹H

NMR (400 MHz, DMSO-d₆): δ 10.55 (s, 1H), 9.23−9.21 (m, 2H),

8.98 (s, 1H), 8.92 (d, J = 10.0 Hz, 2H), 8.74 (s, 1H), 8.65 (br, 3H),

8.25 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.0 Hz,

2H), 7.43 (d, J = 8.8 Hz, 2H), 4.04 (d, J = 5.6 Hz, 2H), 3.45 (s, 2H),

3.21 (d, J = 11.2 Hz, 2H), 2.79 (dd, J = 23.2, 11.2 Hz, 2H), 1.91 (s,

1H), 1.81 (d, J = 12.8 Hz, 2H), 1.43 (dd, J = 25.2, 13.2 Hz, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ 164.9, 153.4, 144.2, 136.9, 136.8,

135.7, 134.0, 129.4, 128.5, 128.1, 125.7, 124.5, 123.1, 44.8, 42.8, 41.9,

33.4, 26.3; MS (ESI): [M + H]⁺451.2.

1H), 4.07 (dd, J = 13.9, 6.9 Hz, 2H), 3.91 (s, 3H), 3.36 (t, J = 6.1 Hz,

2H), 2.65 (t, J = 11.5 Hz, 2H), 1.78−1.63 (m, 3H), 1.41 (s, 9H),

1.19−1.08 (m, 2H).

Compound 116 (270 mg, 0.70 mmol), 4-[(tert-

butoxycarbonylamino)methyl]phenylboronic acid pinacol ester (300

mg, 0.9 mmol), tetrakis(triphenylphosphine)palladium (40 mg, 0.035

mmol), and sodium carbonate (220 mg, 2.07 mmol) in p-dioxane/

H₂O (10/2 mL) were placed in a sealed tube. The mixture was

degassed and heated to 105 °C for 24 h. The reaction was then cooled

and quenched with brine (20 mL). The product was extracted with

ethyl acetate (3 × 30 mL), and the combined organic layers were

washed with water and brine and dried over Na₂SO₄. The volatiles

were removed in vacuo to give a crude oil, which was puriﬁed by

column chromatography (silica gel, hexanes/ethyl acetate from 1.5:1

to 1:1) to aﬀord the product 117 (244 mg, 63%) and a small portion

3-(4-(Aminomethyl)phenyl)-N-(4-iodophenyl)-6-((piperidin-4-

ylmethyl)amino)pyrazine-2-carboxamide hydrochloride (57). ¹H

NMR (400 MHz, DMSO-d₆): δ 10.51 (s, 1H), 9.03−8.87 (m, 2H),

8.69 (ddd, J = 31.6, 19.2, 8.4 Hz, 2H), 8.49 (br, 3H), 8.26 (d, J = 8.0

Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.63 (dd, J = 29.2, 8.4 Hz, 4H),

4.06 (d, J = 5.2 Hz, 2H), 3.55 (s, 2H), 3.24 (d, J = 11.2 Hz, 2H), 2.81

(dd, J = 23.2, 11.2 Hz, 2H), 1.91 (s, 1H), 1.82 (d, J = 13.2 Hz, 2H),

1.42 (dd, J = 25.2, 13.2 Hz, 2H); ¹³C NMR (400 MHz, DMSO-d₆): δ

165.3, 153.8, 144.6, 138.0, 137.7, 137.3, 136.1, 134.3, 129.8, 126.1,

124.9, 123.9, 88.8, 45.2, 43.2, 42.3, 33.8, 26.7; MS (ESI): [M + H]⁺

543.2.

1

of crude product (38 mg). H NMR (400 MHz, CDCl₃): δ 8.34 (s,

1H), 8.02 (t, J = 5.3 Hz, 1H), 7.49 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 7.6

Hz, 2H) 4.02 (dd, J = 13.9, 6.9 Hz, 2H), 3.88 (s, 3H), 3.36 (t, J = 6.1

Hz, 2H), 2.65 (t, J = 11.5 Hz, 2H), 1.78−1.63 (m, 3H), 1.41 (s, 9H),

1.19−1.08 (m, 2H).

To a solution of compounds 117 (244 mg, 0.44 mmol) in MeOH−

H₂O (v/v = 4/1, 5 mL) was added lithium hydroxide (105 mg, 4.4

mmol) at room temperature. It was allowed to stir for 24 h before

removing the solvents to give a residue, which was acidiﬁed by 1 N

HCl and extracted with ethyl acetate (3 × 30 mL). The combined

organic layers were washed with water and brine and dried over

Na₂SO₄. The volatiles were removed in vacuo to give a crude acid,

which was used in the next step without further puriﬁcation.

3-(4-(Aminomethyl)phenyl)-N-(4-bromophenyl)-6-((piperidin-4-

ylmethyl)amino)pyrazine-2-carboxamide hydrochloride (58). ¹H

NMR (400 MHz, DMSO-d₆): δ 10.54 (s, 1H), 8.99 (s, 2H), 8.79−

8.60 (m, 2H), 8.50 (s, 3H), 8.26 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.8

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Hz, 2H), 7.63−7.48 (m, 4H), 4.05 (dd, J = 11.2, 5.2 Hz, 2H), 3.45 (t,

J = 6.0 Hz, 2H), 3.23 (d, J = 11.6 Hz, 2H), 2.86−2.72 (m, 2H), 1.91

(s, 1H), 1.81 (d, J = 13.2 Hz, 2H), 1.41 (dd, J = 23.6, 11.2 Hz, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ 164.9, 153.4, 144.2, 137.2, 136.9,

135.7, 133.9, 131.4, 129.3, 125.7, 124.5, 123.4, 116.2, 61.9, 44.8, 42.8,

41.9, 33.3, 31.5, 26.3; MS (ESI): [M + H]⁺495.1.

3-(4-(Aminomethyl)phenyl)-N-(4-(furan-3-yl)phenyl)-6-((piperi-

din-4-ylmethyl)amino)pyrazine-2-carboxamide hydrochloride (70)

was prepared by cross-coupling with 49 precursor, followed by

deprotection. ¹H NMR (400 MHz, DMSO-d₆): δ 10.51 (s, 1H), 9.01

(s, 1H), 8.90 (d, J = 8.0 Hz, 1H), 8.83 (t, J = 6.0 Hz, 1H), 8.66−8.56

(m, 1H), 8.46 (s, 3H), 8.29 (d, J = 8.0 Hz, 2H), 8.19 (s, 1H), 7.84 (d,

J = 8.4 Hz, 2H), 7.75 (d, J = 1.6 Hz, 1H), 7.63 (dd, J = 18.4, 8.4 Hz,

4H), 6.98 (s, 1H), 4.09 (q, J = 6.0 Hz, 2H), 3.44 (s, 2H), 3.26 (d, J =

12.0 Hz, 2H), 2.83 (dd, J = 24.4, 12.4 Hz, 2H), 1.95 (s, 1H), 1.85 (d,

J = 12.8 Hz, 2H), 1.50−1.37 (m, 2H); ¹³C NMR (100 MHz, DMSO-

d₆): δ 164.7, 153.5, 144.3, 144.1, 139.1, 136.8, 136.5, 135.8, 133.9,

129.3, 128.1, 125.7, 125.4, 124.7, 121.7, 108.6, 44.8, 42.9, 41.9, 33.4,

26.3; MS (ESI): [M + H]⁺483.2.

General Synthetic Procedure for Compound 20. To a

solution of 4,5-dibromothiophene-2-carboxaldehyde 127 (540 mg, 2

mmol) in MeOH (4 mL), NaBH₄(79 mg, 2.1 mmol) was added

slowly at 0 °C. The reaction mixture was stirred at room temperature

for 1 h and quenched with water (5 mL). The product was extracted

with diethyl ether (3 × 20 mL), and the combined organic layers were

washed with water and brine and dried over Na₂SO₄. Upon removal

of the solvent carefully, the residual oil was dried and used in the next

step without puriﬁcation. It was dissolved in DMF (10 mL) and the

solution was cooled to 0 °C. Cyanuric chloride (369 mg, 2 mmol) was

added slowly at 0 °C and stirred at room temperature for 10 h. The

reaction was quenched with saturated NaHCO₃(10 mL). The

product was extracted with diethyl ether (3 × 20 mL), and the

combined organic layers were washed with water and brine and dried

over Na₂SO₄. Removal of the solvents in vacuo aﬀorded compound

128 as a colorless oil, which was used without puriﬁcation.

Synthesis of Compound 21. To a solution of 2-amino-6-

chloropyrazine 108 (2.0 g, 15.5 mmol) in acetonitrile (20 mL) was

added N-bromosuccinimide (6.9 g, 39 mmol) at 0 °C. The resulting

mixture was allowed to warm to room temperature and stirred for 18

h, and to this was added water. The mixture was extracted with ethyl

acetate (3 × 50 mL), and the combined organic layers were washed

with water and brine and dried over Na₂SO₄. The volatiles were

removed in vacuo to aﬀord a crude residue, which was puriﬁed by

column chromatography (silica gel, hexanes/ethyl acetate from 5:1 to

2:1) to give 121 as a yellow solid (3.93 g, 89%). ¹H NMR (400 MHz,

CDCl₃): δ 5.23 (s, 2H, NH).

To a solution of 121 (990 mg, 3.44 mmol) in 1,4-dioxane (10 mL)

was added N-Boc-4-piperidinemethanol (832 mg, 3.85 mmol) and

sodium hydroxide (320 mg, 8.0 mmol). The resulting mixture was

heated at 75 for 4 h °C. After cooled to room temperature, it was

extracted with ethyl acetate (3 × 30 mL), and the combined organic

layers were washed with water and brine and dried over Na₂SO₄. The

volatiles were removed in vacuo to aﬀord a crude residue, which was

puriﬁed by column chromatography (silica gel, hexanes/ethyl acetate

1

from 2:1 to 1:1) to give 122 as a yellow solid (885 mg, 61%). H

NMR (400 MHz, CDCl₃): δ 4.87 (s, 1H), 4.84 (s, 1H), 4.22 (d, J =

6.4 Hz, 2H), 4.12 (s, 2H), 2.74 (t, J = 12.4 Hz, 2H), 1.98 (s, 1H),

1.76 (d, J = 13.0 Hz, 2H), 1.46 (s, 9H), 1.27 (d, J = 12.2 Hz, 2H).

Compound 122 (90 mg, 0.214 mmol), 4-[(tert-Boc-methyl]-

phenylboronic) acid pinacol ester (166 mg, 0.5 mmol), bis[di-tert-

butyl(4-dimethylaminophenyl)phosphine] dichloropalladium(II) (15

mg, 0.021 mmol), and potassium phosphate (212 mg, 1 mmol) in p-

dioxane-H₂O (6/1.5 mL) were placed in a sealed tube. The mixture

was degassed and heated to 110 °C for 16 h. The reaction was then

cooled and quenched with brine (20 mL). The product was extracted

with ethyl acetate (3 × 30 mL), and the combined organic layers were

washed with water and brine and dried over Na₂SO₄. The volatiles

were removed in vacuo to give a crude oil, which was puriﬁed by

column chromatography (silica gel, hexanes/ethyl acetate from 2:1 to

1

1:2) to aﬀord 123 (66 mg, 43%). H NMR (400 MHz, CDCl₃): δ

8.02 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 7.8 Hz,

2H), 7.32 (d, J = 8.0 Hz, 2H), 5.12 (s, 1H), 5.01 (s, 1H), 4.35 (s,

4H), 4.23 (s, 2H), 4.16−4.10 (m, 2H), 2.81−2.70 (m, 2H), 2.02 (s,

1H), 1.82 (d, J = 12.6 Hz, 2H), 1.49 (s, 27H), 1.28 (d, J = 6.8 Hz,

2H).

To a solution of compound 123 (66 mg, 0.092 mmol) in DCM (3

mL) was dropwise added HCl (0.3 mL, 4 N in p-dioxane) at 0 °C.

The reaction mixture was warmed to room temperature and stirred

for 5 h. The volatiles were removed in vacuo to aﬀord an oil, which

was triturated in diether ether and solidiﬁed to give 21 (44 mg, 91%)

as a hydrochloric acid salt.

To a solution of 4-(aminomethyl)-1-Boc-piperidine (214 mg, 1

mmol) and potassium carbonate (166 mg, 1.2 mmol) in DMF (5

mL), a solution of 128 (145 mg, 0.5 mmol) in DMF (3 mL) was

added slowly at 0 °C, and the mixture was stirred at room

temperature for 10 h. Upon quenching with saturated NaHCO₃(10

mL), the product was extracted with diethyl ether (3 × 50 mL), and

the combined organic layers were washed with water and brine and

dried over Na₂SO₄. Column chromatography (silica gel, hexanes/

ethyl acetate from 40:1 to 2:1) of the residue oil gave compound 129

as a pale-yellow oil (69% yield for the three steps).

A

mixture of 129 (186 mg, 0.4 mmol), 4-[(tert-

((5-Amino-6-(piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1-

phenylene))dimethanamine hydrochloride (21). ¹H NMR (400

MHz, D₂O): δ 7.85 (d, J = 6.4 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H),

7.61 (dd, J = 25.6, 7.2 Hz, 4H), 4.36 (d, J = 5.2 Hz, 2H), 4.29 (s, 2H),

4.27 (s, 2H), 3.48 (d, J = 12.8 Hz, 2H), 3.03 (t, J = 12.0 Hz, 2H), 2.23

(s, 1H), 2.05 (d, J = 13.6 Hz, 2H), 1.57 (dd, J = 24.0, 11.8 Hz, 2H);

¹³C NMR (100 MHz, D₂O): δ 156.6, 151.7, 134.3, 133.9, 133.7,

133.0, 129.5, 129.42, 129.36, 128.8, 128.4, 127.6, 70.1, 43.5, 42.7,

32.7, 25.1; MS (ESI): [M + H]⁺419.2.

butoxycarbonylamino)methyl]phenylboronic acid pinacol ester (300

mg, 0.9 mmol), tetrakis(tri-phenylphosphine)palladium (46 mg, 0.04

mmol), and sodium carbonate (212 mg, 2 mmol) in 1,4-dioxane/H₂O

(5/1 mL) was heated to 100 °C for 12 h. The reaction was then

quenched with brine (10 mL). The product was extracted with diethyl

ether (3 × 20 mL), and the combined organic layers were washed

with water and brine and dried over Na₂SO₄. Upon removal of the

solvent, the product was puriﬁed by column chromatography (silica

gel, n-hexanes/ethyl acetate from 5:1 to 1:2) to give 130 as a white

solid (260 mg, 90%).

To a solution of 130 (0.1 mmol) in DCM (2 mL) at 0 °C, HCl

(0.2 mL, 4 N in 1,4-dioxane) was added slowly and then stirred at

room temperature for 12 h. Upon removal of the solvent carefully, the

residual oil was treated with anhydrous diethyl ether and vacuum-

dried to give 20 (white powder) as a hydrochloric acid salt (92%

yield).

((5-(((Piperidin-4-ylmethyl)amino)methyl)thiophene-2,3-diyl)bis-

(4,1-phenylene))dimethanamine hydrochloride (20−1226). ¹H

NMR (400 MHz, D₂O): δ 7.43 (s, 1H), 7.40 (br, 8H), 4.57 (s,

2H), 4.19 (s, 4H), 3.50 (d, J = 12.8 Hz, 2H), 3.15 (d, J = 6.8 Hz, 2H),

3.05 (t, J = 12.0 Hz, 2H), 2.18 (s, 1H), 2.08 (d, J = 14.0 Hz, 2H),

1.59−1.50 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 140.6, 137.9,

136.1, 133.9, 133.8, 132.6, 131.8, 130.5, 129.9, 129.6, 129.1, 129.0,

50.8, 45.4, 43.1, 42.64, 42.57, 30.7, 25.8; MS (ESI): [M + H]⁺421.6.

General Synthetic Procedure for Compounds 22−24. To a

solution of 121 (600 mg, 2.09 mmol) in isopropyl alcohol (10 mL)

was added chloroacetaldehyde (50% wt in water, 1.7 mL, 14 mmol).

The resulting mixture was reﬂuxed at 105 °C for 18 h, then cooled to

room temperature, and concentrated. The residue was dissolved in

DCM (30 mL) and washed sequentially with sat. NaHCO₃(10 mL),

H₂O (10 mL), and brine (10 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and removed in vacuo to give a residue, which was

puriﬁed by column chromatography (silica gel, hexanes/ethyl acetate

1

from 5:1 to 2:1) to aﬀord 124 (260 mg, 40%). H NMR (400 MHz,

CDCl₃): δ 7.94 (s, 1H), 7.90 (s, 1H).

A solution of 124 (122 mg, 0.39 mmol), alcohol nucleophiles (0.38

mmol), and sodium hydroxide (20 mg, 0.5 mmol) in 1,4-dioxane (3

mL), or a solution of amine nucleophiles (0.38 mmol) and N,N-

diisopropylethylamine (87 μL, 0.5 mmol) in acetonitrile (3 mL) was

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heated at 75 for 4 h °C. After cooled to room temperature, it was

concentrated and extracted with ethyl acetate (3 × 20 mL). The

combined organic layers were washed with water and brine and dried

over Na₂SO₄. The volatiles were removed in vacuo to aﬀord a crude

residue, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 2:1 to 1:2) to give 125 as a solid (885 mg,

crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 4:1 to 2:1) to aﬀord 119 (108 mg, 90%).

¹H NMR (400 MHz, CDCl₃): δ 8.14 (s, 1H), 7.47 (d, J = 8.0 Hz,

2H), 7.34 (d, J = 7.9 Hz, 2H), 6.83 (dd, J = 16.9, 10.6 Hz, 1H), 6.43

(dd, J = 16.9, 2.1 Hz, 1H), 5.46 (dd, J = 10.6, 2.1 Hz, 1H), 5.06 (s,

1H), 4.34 (d, J = 5.0 Hz, 2H), 4.26 (d, J = 6.4 Hz, 2H), 4.18−4.06

(m, 2H), 2.73 (t, J = 10.8 Hz, 2H), 2.00−1.94 (m, 1H), 1.81 (d, J =

12.9 Hz, 2H), 1.45 (s, 18H), 1.35−1.25 (m, 2H); ¹³C NMR (100

MHz, CDCl₃): δ 158.1, 156.0, 154.9, 144.4, 143.6, 139.2, 136.8,

133.9, 132.9, 130.0, 127.4, 120.8, 79.5, 70.3, 44.4, 35.9, 28.5, 28.5,

22.0.

To a solution of compound 119 (80 mg, 0.15 mmol) in DBU (2

mL) was added morpholine (0.1 mL). The resulting mixture was

heated at 120 °C for 24 h before it was cooled to room temperature.

The mixture was extracted with ethyl acetate (3 × 20 mL), and the

combined organic layers were washed with water and brine and dried

over Na₂SO₄. The volatiles were removed in vacuo to aﬀord a crude

oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 1:1 to 1:2) to give 120 (52 mg, 57%).¹H

NMR (400 MHz, CDCl₃): δ 8.08 (s, 1H), 7.42 (d, J = 7.8 Hz, 2H),

7.32 (d, J = 7.8 Hz, 2H), 5.00 (s, 1H), 4.33 (d, J = 5.3 Hz, 2H), 4.18

(d, J = 6.4 Hz, 2H), 4.14−4.04 (m, 2H), 3.67−3.57 (m, 4H), 2.96−

2.89 (m, 2H), 2.78−2.67 (m, 4H), 2.36 (s, 4H), 1.96 (s, 1H), 1.79 (d,

J = 12.6 Hz, 2H), 1.44 (s, 18H), 1.31−1.24 (m, 2H).

1

58%). For the intermediate of compound 24: H NMR (400 MHz,

CDCl₃): δ 7.69 (d, J = 1.1 Hz, 1H), 7.56 (s, 1H), 4.96 (s, 1H), 4.29

(s, 1H), 4.22 (s, 2H), 4.15−4.07 (m, 1H), 3.82 (s, 1H), 1.97−1.66

(m, 4H), 1.43 (s, 9H).

Compound 125 (0.16 mmol), 4-[(tert-butoxycarbonylamino)-

methyl]phenylboronic acid pinacol ester (133 mg, 0.4 mmol),

bis[di-tert-butyl(4-dimethylaminophenyl)phosphine]

dichloropalladium(II) (15 mg, 0.021 mmol), and potassium

phosphate (127 mg, 0.6 mmol) in p-dioxane−H₂O (5/1 mL) were

placed in a sealed tube. The mixture was degassed and heated to 110

°C for 16 h. The reaction was then cooled and quenched with brine

(20 mL). The product was extracted with ethyl acetate (3 × 30 mL),

and the combined organic layers were washed with water and brine

and dried over Na₂SO₄. The volatiles were removed in vacuo to give a

crude oil, which was puriﬁed by column chromatography (silica gel,

hexanes/ethyl acetate from 2:1 to 1:2) to aﬀord 126. For the

precursor of compound 24: ¹H NMR (400 MHz, CDCl₃): δ 7.48 (s,

1H), 7.32−7.25 (m, 7H), 7.08 (d, J = 7.6 Hz, 2H), 5.60 (s, 1H), 4.97

(s, 1H), 4.79 (s, 1H), 4.42−4.35 (m, 4H), 4.24 (d, J = 3.6 Hz, 2H),

4.02−3.87 (m, 3H), 1.89 (br, 2H), 1.71 (br, 2H), 1.47−1.40 (m,

27H).

To a solution of compound 120 (52 mg, 0.085 mmol) in DCM (3

mL) was dropwise added HCl (0.3 mL, 4 N in p-dioxane) at 0 °C.

The reaction mixture was warmed to room temperature and stirred

for 5 h. The volatiles were removed in vacuo to aﬀord an oil, which

was triturated in diether ether and solidiﬁed to give 34 (41 mg, 92%)

as a hydrochloric acid salt.

To a solution of compound 126 (0.08 mmol) in DCM (3 mL) was

dropwise added HCl (0.1 mL, 4 N in p-dioxane) at 0 °C. The reaction

mixture was warmed to room temperature and stirred for 5 h. The

volatiles were removed in vacuo to aﬀord an oil, which was triturated

in diether ether and solidiﬁed to give the ﬁnal product as a

hydrochloric acid salt.

(4-(3-(2-Morpholinoethyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-

1

yl)phenyl)methanamine hydrochloride (34). H NMR (400 MHz,

D₂O): δ 8.22 (s, 1H), 7.69−7.53 (m, 4H), 4.36 (d, J = 5.6 Hz, 2H),

4.28 (s, 2H), 4.08 (d, J = 13.2 Hz, 2H), 3.81−3.72 (m, 2H), 3.60 (t, J

= 7.2 Hz, 2H), 3.56−3.41 (m, 4H), 3.30 (t, J = 7.2 Hz, 2H), 3.17 (t, J

= 12.4 Hz, 2H), 3.08 (t, J = 12.8 Hz, 2H), 2.26 (s, 1H), 2.13 (d, J =

14.0 Hz, 2H), 1.65 (dd, J = 25.6, 13.2 Hz, 2H); ¹³C NMR (100 MHz,

D₂O): δ 159.0, 146.9, 144.2, 136.9, 133.4, 131.6, 129.7, 129.2, 70.2,

63.6, 55.2, 51.7, 43.6, 42.7, 32.8, 27.6, 25.0; MS (ESI): [M + H]⁺

412.3.

((8-(Piperidin-4-ylmethoxy)imidazo[1,2-a]pyrazine-5,6-diyl)bis-

(4,1-phenylene)) dimethanamine hydrochloride (22) was prepared

1

following the general procedure. H NMR (400 MHz, D₂O): δ 7.97

(s, 1H), 7.78 (s, 1H), 7.58−7.48 (m, 4H), 7.46 (d, J = 8.0 Hz, 2H),

7.34 (d, J = 8.0 Hz, 2H), 4.63 (d, J = 6.0 Hz, 2H), 4.23 (s, 2H), 4.13

(s, 2H), 3.50 (d, J = 12.4 Hz, 2H), 3.08 (t, J = 12.0 Hz, 2H), 2.37 (s,

1H), 2.16 (d, J = 13.6 Hz, 2H), 1.74−1.58 (m, 2H); ¹³C NMR (100

MHz, D₂O): δ 149.6, 138.7, 136.6, 134.9, 133.0, 131.4, 130.5, 130.0,

129.7, 128.5, 128.0, 124.9, 124.4, 116.2, 71.1, 43.5, 42.5, 32.7, 24.9;

MS (ESI): [M + H]⁺443.2.

Activity and Inhibition Assays for Flavivirus Proteases.

Expression and puriﬁcation of ﬂavivirus proteases were performed as

described previously.¹⁵The activity and inhibition assay for ZVpro

was performed using the enzyme (1 nM) and benzoyl-norleucine-

lysine-lysine-arginine 7-amino-4-methylcoumarin (Bz-Nle-Lys-Lys-

Arg-AMC, 20 μM) as the substrate in HEPES buﬀer (20 mM, pH

7.3) containing 0.05% Triton X-100. To determine IC₅₀, triplicate

samples of a compound with concentrations ranging from 1 nM to 10

μM were incubated with the enzyme for 10 min before adding the

substrate to initiate the reaction in a 96-well plate (ﬁnal volume, 100

μL). The ﬂuorescent signal (Ex: 360 nm, Em: 460 nm) of each well

was monitored every 30 s, using a Tecan microplate reader. The initial

velocity data were imported into Prism (version 5.0), and IC₅₀values

from three independent experiments with standard deviation were

obtained using a standard dose−response curve ﬁtting. Enzyme

inhibition assays for DV2pro and WVpro were performed similarly.

Cellular Antiviral Activity Testing. Anti-ZIKV activity was

(S)-((8-(3-Aminopyrrolidin-1-yl)imidazo[1,2-a]pyrazine-5,6-diyl)-

bis(4,1-phenylene))dimethanamine hydrochloride (23) was prepared

1

following the general procedure. H NMR (400 MHz, DMSO-d₆): δ

8.61 (br, 3H), 8.52 (br, 3H), 8.36 (br, 3H), 7.65−7.59 (m, 3H), 7.46

(d, J = 8.0 Hz, 2H), 7.41−7.33 (m, 4H), 7.21 (s, 1H), 5.16 (t, J = 13.2

Hz, 2H), 4.09 (s, 2H), 3.95 (s, 2H), 3.29 (s, 1H), 2.12 (s, 1H), 1.88

(s, 1H), 1.77−1.62 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ

145.6, 137.7, 135.1, 134.6, 133.4, 132.9, 132.5, 132.3, 131.6, 130.4,

130.2, 128.8, 119.7, 115.2, 110.0, 52.8, 49.5, 47.3, 42.1, 42.0, 29.1; MS

(ESI): [M + H]⁺414.2.

(R)-((8-(3-Aminopiperidin-1-yl)imidazo[1,2-a]pyrazine-5,6-diyl)-

bis(4,1-phenylene))dimethanamine hydrochloride (24) was prepared

1

following the general procedure. H NMR (400 MHz, D₂O): δ 8.61

(br, 3H), 8.51 (br, 3H), 8.36 (br, 3H), 7.62−7.59 (m, 3H), 7.44 (d, J

= 7.2 Hz, 2H), 7.35 (dd, J = 13.6, 4.4 Hz, 2H), 7.19 (s, 1H), 5.14 (t, J

= 9.8 Hz, 2H), 4.07 (d, J = 5.6 Hz, 2H), 3.93 (d, J = 5.6 Hz, 2H), 3.42

(s, 2H), 3.27 (s, 2H), 2.09 (s, 1H), 1.87 (d, J = 9.2 Hz, 2H), 1.71−

1.56 (m, 2H); MS (ESI): [M + H]⁺428.2.

Synthesis of Compound 34. Compound 118 (120 mg, 0.23

mmol), potassium vinyltriﬂuoroborate (39 mg, 0.29 mmol), tetrakis-

(triphenylphosphine)palladium (14 mg, 0.012 mmol), and cesium

carbonate (147 mg, 0.45 mmol) in THF−H₂O (5/1 mL) were placed

in a sealed tube. The mixture was degassed and heated to 110 °C for

12 h. The reaction was then cooled and quenched with brine (20

mL). The product was extracted with ethyl acetate (3 × 20 mL), and

the combined organic layers were washed with water and brine and

dried over Na₂SO₄. The volatiles were removed in vacuo to give a

evaluated in human U87 glioma following our previous methods.¹⁵

2

× 10⁴U87 cells/well were seeded in 96-well plates and cultured in

DMEM with 2% FBS to form a monolayer of cells. 0.01 MOI of ZIKV

was added. After incubation for 1 h, the supernatant was removed and

cells were washed with PBS. Fresh medium (150 μL/well) containing

various concentrations of a compound in triplicate were added. Upon

incubation at 37 °C for 48 h, aliquots of the supernatant from each

well were used to determine ZIKV titers. Half-log serial dilution of the

viral supernatant (50 μL) was added to a monolayer of Vero cells in

quadruplicate in 96-well plates and cultured for 7 days. CPE/cell lysis

was determined with a microscope, followed by the MTT assay.

TCID₅₀was calculated based on the highest dilution in which ≥50%

(i.e., ≥2 out of the 4 quadruplicate wells) of Vero cells were infected

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with ZIKV. Compared to controls, the ability of a compound to

reduce TCID₅₀can be determined. The results were from at least two

independent experiments.

Author Contributions

^∥S.N., Y.Y., and F.W. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02070.

ACKNOWLEDGMENTS

■

This work was supported by a grant (W81XWH-18-1−0368)

from USAMRAA of the US Department of Defense and a

grant (RP180177) from Cancer Prevention and Research

Institute of Texas to Y.S. and NIH grants R01AI099483 and

R01AI098715 to R.R.-H.

Molecular formula strings for all compounds (CSV)

AUTHOR INFORMATION

■

ABBREVATIONS

■

Corresponding Author

Ac-CoA, acetyl coenzyme A; AR, androgen receptor; SAR,

structure−activity relationships; DMF, N,N-dimethylforma-

mide; LSD1, lysine speciﬁc demethylase 1; ZVpro, Zika virus

protease; DV2pro, dengue (serotype 2) virus protease; WVpro,

West Nile virus protease; CPE, cytopathic eﬀects; MOI,

multiplicity of infection; NIS, N-iodosuccinimide

Yongcheng Song − Department of Pharmacology and

Chemical Biology, Baylor College of Medicine, Houston,

Texas 77030, United States; orcid.org/0000-0003-2611-

2476; Phone: 713-798-7415; Email: ysong@bcm.edu

Authors

Shenyou Nie − Department of Pharmacology and Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Yuan Yao − Department of Pharmacology and Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Fangrui Wu − Department of Pharmacology and Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Xiaowei Wu − Department of Pharmacology and Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Jidong Zhao − Department of Pharmacology and Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

Yuanda Hua − Department of Pharmacology and Chemical

Biology, Baylor College of Medicine, Houston, Texas 77030,

United States

REFERENCES

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Journal of Medicinal Chemistry

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J. Med. Chem. 2021, 64, 2777−2800

Article Doi

Synthesis, Structure-Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease

DOI: 10.1021/acs.jmedchem.0c02070

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