Metal-Binding Pharmacophore Library Yields the Discovery of a Glyoxalase 1 Inhibitor

Article

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Cite This: J. Med. Chem. 2019, 62, 1609 −1625

Metal-Binding Pharmacophore Library Yields the Discovery of a

Glyoxalase 1 Inhibitor

Christian Perez,^†Amanda M. Barkley-Levenson,^‡Benjamin L. Dick,^†Peter F. Glatt,^†Yadira Martinez,^§

Dionicio Siegel,^§Jeremiah D. Momper,^§Abraham A. Palmer,*^,‡,∥and Seth M. Cohen*^,†

^†Department of Chemistry and Biochemistry, ^‡Department of Psychiatry, ^§Skaggs School of Pharmacy and Pharmaceutical Sciences,

∥

and Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, United States

S

* Supporting Information

ABSTRACT: Anxiety and depression are common, highly comorbid psychiatric diseases that account for a large proportion of

worldwide medical disability. Glyoxalase 1 (GLO1) has been identiﬁed as a possible target for the treatment of anxiety and

depression. GLO1 is a Zn²⁺-dependent enzyme that isomerizes a hemithioacetal, formed from glutathione and methylglyoxal, to

a lactic acid thioester. To develop active inhibitors of GLO1, fragment-based drug discovery was used to identify fragments that

could serve as core scaﬀolds for lead development. After screening a focused library of metal-binding pharmacophores, 8-

(methylsulfonylamino)quinoline (8-MSQ) was identiﬁed as a hit. Through computational modeling and synthetic elaboration,

a potent GLO1 inhibitor was developed with a novel sulfonamide core pharmacophore. A lead compound was demonstrated to

penetrate the blood−brain barrier, elevate levels of methylglyoxal in the brain, and reduce depression-like behavior in mice.

These ﬁndings provide the basis for GLO1 inhibitors to treat depression and related psychiatric illnesses.

reaction between glutathione (GSH) and MG. The resulting

thioester is then converted by glyoxalase 2 (GLO2) to a less

reactive metabolite, ^D-lactate, and in this capacity helps deplete

MG levels.^4,10Because of the role in AGE formation and

cytotoxicity, GLO1 inhibitors have been investigated as

potential oncogenic therapeutics but have not yet found

clinical success.^11,12

Recent studies using both genetic and pharmacological

techniques have implicated GLO1 in numerous behaviors,

including several that are relevant to anxiety and depres-

sion.¹³⁻¹⁷MG has been shown to be a competitive partial

GABA-A receptor agonist.¹⁵Taken together, these data suggest

that inhibition of GLO1 can cause an accumulation of MG,

leading to activation of GABA-A receptors, and thus producing

potentially therapeutic changes in behavior.

INTRODUCTION

■

Anxiety and depression are the two most common psychiatric

disorders in the U.S. and aﬀect approximately one in ﬁve adults

at some point in their lifetime. Depression is the leading cause

of worldwide disability and anxiety disorders, which are highly

comorbid with depression and are the sixth leading cause of

worldwide disability.¹There are already several drugs approved

by the U.S. Food and Drug Administration for the treatment of

both anxiety and depression; however, these drugs have

limitations. Anxiolytic drugs produce sedating side eﬀects and

have signiﬁcant abuse liability. Antidepressant drugs are not

eﬀective in all patients, take weeks to produce therapeutic

eﬀects, and produce side eﬀects that limit their use.²Therefore,

there is an urgent need for developing better therapeutics.

Glyoxalase 1 (GLO1) is a cytosolic Zn²⁺-dependent

isomerase that is part of the glyoxalase system, a pathway

involved in the detoxiﬁcation of α-oxoaldehydes.³⁻⁶Methyl-

glyoxal (MG) is a reactive metabolite generated via the

degradation of glycolytic intermediates and is capable of

forming covalent adducts with proteins and nucleotides that

result in advanced glycation end (AGE) products, reactive-

oxygen species, and apoptosis.⁶⁻⁹GLO1 catalyzes the

isomerization of a hemithioacetal formed from a spontaneous

Several natural products and natural product derivatives that

inhibit GLO1 have been described.¹⁸GSH-based inhibitors

were among the ﬁrst GLO1 inhibitors reported, with a

compound containing a hydroxamic acid metal-binding

pharmacophore (MBP) (AHC-GSH, Figure 1) being the

Received: November 28, 2018

Published: January 10, 2019

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Figure 1. Molecular structures of previously reported GLO1 inhibitors with corresponding K_ior IC₅₀values shown against human GLO1. Metal

binding atoms of the MBP are highlighted in bold red.

a

Table 1. 8-MSQ Derivatives Used To Obtain a Rudimentary SAR

a

All IC₅₀values listed are in μM.

ﬁrst tight binding inhibitor to be reported.¹⁹These GSH

mimics have poor membrane permeability and need to be

administered as prodrugs (i.e., esters), in order to achieve

membrane penetration.¹¹AHC-GSH prodrug analogs have

demonstrated some eﬃcacy in vivo, but suﬀer from rapid

clearance (in esterase deﬁcient mice) and have a narrow

therapeutic window.²⁰Both the hydroxamic acid MBP and

GSH origins of AHC-GSH, and its analogs, make these

molecules unsuitable as viable drug candidates. A second class

of reported inhibitors are ﬂavonoids and ﬂavonoid analogs

which tend to coordinate the catalytic metal of GLO1 via a

catechol moiety.²¹⁻²⁷Methyl gerfelin (M-GFN) was reported

as a potent inhibitor of GLO1 related to the ﬂavonoid class

(Figure 1).²⁶Lastly, a N-hydroxypyridinone-based inhibitor

(Chugai-3d, Figure 1) reported by Chugai Pharmaceuticals

shows an excellent IC₅₀value of 11 nM.²⁸However, to the best

of our knowledge, in vivo studies or clinical trials have not

been reported with Chugai-3d. The majority of these

inhibitors have been designed and investigated for oncogenic

indications and may not be suitable candidates for targets

within the central nervous system. Therefore, we sought to

develop a novel GLO1 inhibitor that could penetrate the

blood−brain barrier (BBB) and inhibit GLO1.

A fragment-based drug discovery (FBDD) approach was

used to identify a novel GLO1 inhibitor. The use of FBDD for

the discovery of biologically active compounds has become

increasingly important.²⁹This strategy consists of generating

small libraries of molecular fragments and screening them

against the target of interest. For metalloenzyme targets such as

GLO1, FBDD libraries have been designed consisting of

MBPs, where “hits” from these libraries can be elaborated into

lead compounds. MBPs are small molecules that have a set of

donor atoms that are capable of coordinating to the active site

metal ion of metalloenzymes.³⁰Here, we report the discovery,

design, synthesis, and evaluation of a novel class of GLO1

inhibitors obtained from metalloenzyme-targeted FBDD. An in

vitro inhibition assay and computational molecular modeling

were utilized to develop structure−activity relationship (SAR).

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Figure 2. Top: Structure of Chugai-3d bound to the Zn²⁺ion in the active the GLO1 active site (PDB 3VW9). Bottom: Superposition of 8-MSQ

using the structure of the donor atoms of Chugai-3d as a guide. For both compounds, an illustration of the interactions is provided (right) along

with an image of the structure (left) with the active site surface in gray, the Zn²⁺ion as an orange sphere, and water molecules as red spheres.

Further biological in vivo assessment of a lead compound in

mice demonstrated that it penetrates the BBB, increases MG

levels in the brain (indicating target engagement of GLO1 in

vivo), and results in changes in behavioral phenotypes in mice

comparable to other GLO1 inhibitors and prototypical tricyclic

and serotonin reuptake inhibitor (SRI) antidepressants. Our

ﬁndings lend further support to the potential of GLO1

inhibitors to provide a novel therapeutic strategy for the

treatment of depression and anxiety.

substituted or replaced (Table 1). Compounds 2 and 3 place

the coordinating endocyclic nitrogen at diﬀerent positions

within the quinoline ring. Additionally, 4 replaces the

sulfonamide with a sulfonate ester, while 5 replaces the

sulfonamide with an amide. Lastly, compound 6 incorporates a

reverse sulfonamide functional group. As highlighted by the

data in Table 1, compounds 2−6 all showed a complete loss of

activity (IC₅₀> 200 μM) against GLO1, further suggesting the

importance of the speciﬁc bidentate binding of 8-MSQ

through the nitrogen pair of donor atoms.

8-MSQ Derivatives and Rudimentary SAR. The core

quinoline scaﬀold of compound 8-MSQ has several positions

amenable for derivatization. In this study, the optimal point(s)

of attachment were determined based on computational

modeling and structural studies. Modeling studies were

performed using the Molecular Operating Environment

(MOE) software suite (see the Supporting Information for

details), which can account for ﬂexible protein domains and

can assist in pharmacophore modeling and SAR studies. Using

a crystal structure of GLO1 (PDB 3VW9),²⁸an inhibitor

pharmacophore model was generated. The crystal structure

selected has a resolution of 1.47 Å and has the Chugai-3d

inhibitor bound (Figure 1) to the active site of GLO1.

Structural analysis of this crystal structure revealed a Zn²⁺

metal center and two distinct pockets within the GLO1 active

site: a hydrophobic and a GSH-binding pocket. The hydro-

phobic pocket consists of residues Cys60, Phe62, Ile88,

Met179, Leu182, and Met183. The GSH-binding pocket

consists of Phe67, Leu69, Met157, Phe162, as well as the

conserved water network (Figure 2). The catalytic Zn²⁺has an

octahedral geometry and is coordinated by Gln33, Glu99,

Glu172, and His126, with the ﬁfth and sixth coordination sites

occupied by the O,O donor atoms of the Chugai-3d

compound (Figure 2). The Chugai-3d binding aﬃnity is

attributed to signiﬁcant contacts with both the hydrophobic

and GSH-binding pockets, as well as the water network. The 6-

phenyl moiety of Chugai-3d occupies the hydrophobic pocket

and engages in edge-to-face interactions with Phe62 and

RESULTS

■

Screening of the MBP Library. For the development of

metalloenzyme inhibitors, an MBP library has been reported

for producing hits.³¹⁻³⁴To identify MBPs that could serve as

scaﬀolds for GLO1 inhibitors, a chemical library containing

∼240 MBP fragments was screened against GLO1 utilizing a

previously reported enzyme-based assay adapted for a

multiwell plate format.³⁵The assay was performed in a 96-

well plate format using a colorimetric output that measures the

isomerization of a hemithioacetal to a lactic acid thioester. The

MBP library was initially screened at a fragment concentration

of 200 μM yielding >50 unique hits displaying >50%

inhibition. A second screening at a lower concentration of 50

μM produced 25 distinct hits. From this second screening, 8-

(methylsulfonylamino)quinoline (8-MSQ) showed essentially

complete inhibition and was subsequently shown to have an

IC₅₀value of 18.5

particularly strong activity of 8-MSQ, the fragment was chosen

for lead development.

0.5 μM (Table 1). Because of the

The lead fragment 8-MSQ has been previously reported as

an MBP, as well as a receptor unit in molecular sensors for

Zn²⁺.³⁶⁻³⁹On the basis of these previous reports, we predicted

that 8-MSQ would bind the catalytic Zn²⁺ion of GLO1 in a

bidentate fashion through the endocyclic nitrogen and

exocyclic sulfonamide-nitrogen donor atoms (Figure 1). To

validate this hypothesis, a series of compounds were

synthesized wherein the metal-coordinating atoms were

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a

Table 2. 8-MSQ Fragment Derivatives at the 8-Position

a

All IC₅₀values listed are in μM.

additional hydrophobic contacts with Met179 and Met183

(Figure 2). The 7-azaindole forms an edge-to-face interaction

with Phe67 and a hydrophobic contact with Leu69 (Figure 2).

Additionally, the 7-azaindole nitrogen atom is directly facing a

water molecule and engaging in a hydrogen bond. The binding

conformation and the critical contacts made by Chugai-3d

with GLO1 allowed for the development of a crude

pharmacophore model to guide inhibitor design derived from

8-MSQ.

the coordinating atoms (N,N) of 8-MSQ with those of the

bound Chugai-3d inhibitor. The superposition of 8-MSQ

generated a probable binding mode in which the endocyclic

nitrogen of the quinoline ring is bound at the equatorial site of

the metal center and the exocyclic nitrogen of the sulfonamide

functional group is bound axial of the metal center (Figure 2).

The docked pose was well tolerated with no major clash

predicted between the fragment and the receptor. Another

mode of binding can be modeled by alternating the position of

the donor set; however, modeling suggested that this alternate

binding pose is likely to cause signiﬁcant steric clashes with the

A binding model for 8-MSQ was obtained through docking

of the fragment into the GLO1 active site and superimposing

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a

Scheme 1. Synthesis Disubstituted Sulfonamide Quinoline Derivatives

a

(a) KNO₃, H₂SO₄, 0−25 °C; (b) R-B(OH)₂, Pd(OAc)₂, DIPA, H₂O, 100 °C; (c) R-B(OH)₂, Pd(PPh₃)₄, CH₃COOK, 1,4-dioxane 100 °C; (d)

R-B(OH)₂, Pd(dppf)Cl₂, S-Phos, K₂CO₃, H₂O, 1,4-dioxane, 100 °C; (e) R-amine, Pd₂(dba)₃, X-Phos, K₂CO₃, 1,4-dioxane 100 °C; (f) R-ethynyl,

CuI, Pd(dppf)Cl₂, X-Phos, Et₃N, 1,4-dioxane 90 °C; (g) Pd/C, H₂, MeOH, 25 °C; (h) Na₂S₂O₄, K₂CO₃, H₂O, MeOH, acetone 25 °C; (i) R-

SO₂Cl, pyridine, 25 °C.

GLO1 active site (Figure S1). Therefore, initial assessment of

these two poses pointed toward the pose shown in Figure 2 as

the most likely mode of binding.

group and the phenyl substituent (34−36) did not seem to

change inhibition signiﬁcantly. Interestingly, compounds 17

and 20 that incorporate a methoxy or nitrile at the ortho

position (relative to sulfonamide) were not tolerated;

modeling of these compounds within the active site predicted

a signiﬁcant clash with a W1 and limited intramolecular

rotation. Ultimately, compound 23 (Table 2, IC₅₀value 1.3

μM) was the most active derivative from this group by making

the best interactions within the GSH-binding pocket. Similar

to compound 8, the 2-pyridinyl group forms an edge-to-face

interaction with Phe67 and a hydrophobic contact with Leu69

(Figure S3). Additionally, the 2-pyridinyl nitrogen is directly

facing W1 and engaging in a hydrogen bond.

5-Aryl/Cyclic-8-(Methylsulfonylamino)quinoline De-

rivatives. A second set of compounds was pursued to identify

substituents that interact within the hydrophobic pocket

(Figure 2). This series of compounds complements the study

exploring the GSH-binding pocket and was later merged to

ﬁnd a lead compound with favorable substituents at each

position (vide infra). For the synthesis of this series, the

procedure shown in Scheme 1 was used. First, 5-bromoquino-

line was selectively nitrated at the 8-position, yielding 5-

bromo-8-nitroquinoline. Then, this intermediate was utilized

to attach aryl or amine substituents at the 5-position via Suzuki

or Buchwald−Hartwig coupling reactions. The products of the

coupling reaction were then reduced via palladium on carbon

under a hydrogen atmosphere or through the use of sodium

dithionite resulting in the free amine compound. Lastly, the

amine was coupled with methylsulfonyl chloride under basic

conditions. A set of 10 compounds was synthesized, with

diverse functional groups at the 5-position. Evaluation of these

compounds against GLO1 showed that polar (38, 39) or

nonaromatic (45, 46) groups were not well tolerated (Table

3). Some compounds containing hydrophobic phenyl sub-

stituents resulted in modest (∼3-fold) improvements in

inhibition relative to 8-MSQ (37, 40−44, Table 3).

Merged Compounds. Using the best performing func-

tional groups identiﬁed for both the 5- and 8-positions, a

fragment merging strategy was employed to produce a series of

compounds. The functionality at the 8-position (2-pyridinyl-

sulfonamide) was maintained throughout this series, with

structural variation at the 5-position. Additionally, a handful of

compounds were synthesized with substituents at the 6-

position, instead of the 5-position, to provide more SAR

information. Synthesis of these compounds generally followed

Scheme 1 where the intermediate, 5-bromo-8-nitroquinoline or

6-bromo-8-nitroquinoline (Scheme S2), was coupled to aryl

groups, amines, or alkynes via Suzuki, Buchwald, or

Sonogashira methods. Compounds containing alkynes were

reduced to alkanes in a one-pot reduction of the alkyne and

nitro with palladium on carbon and hydrogen. Compounds

To determine whether the computational model developed

in Figure 2 was predictive, a small set of compounds was

synthesized to evaluate the model. Compounds 7−9 were

synthesized in a straightforward, one-step manner starting with

8-aminoquinoline or 2-methyl-8-aminoquinoline and combin-

ing with the corresponding sulfonyl chloride (Scheme S1).

Enzyme-based assay evaluation of compound 7 yielded a

poorer IC₅₀value (∼2-fold loss of aﬃnity compared to 8-

MSQ) because of the introduction of a methyl substituent

(Table 1). As demonstrated in Figure 2, the introduction of a

methyl group would generate a steric clash with Gln33;

therefore, this loss of activity is consistent with the computa-

tional modeling. Compound 8 incorporates a phenyl at the 8-

sulfonamide position and results in a 2-fold improvement in

inhibition, which also validates the computational model,

where the added phenyl group can be accommodated in the

GSH binding pocket (Figure S2). From this docked pose,

compound 8 forms a hydrogen bond between the sulfone and

a water molecule (W1). An additional edge to face interaction

between the phenyl moiety and Phe67 (∼4.5 Å) and a

hydrophobic interaction between the phenyl group and Leu69

(∼4.2 Å) are predicted. Compound 9 incorporates both a 2-

methyl and an 8-phenylsulfonamide which results in a slight

increase in inhibition relative to 8-MSQ, but worse activity

when compared with 8 (Table 1). Taken together, these results

support the predicted mode of binding for 8-MSQ (Figure 2)

and indicate that functional groups at the sulfonamide moiety

are well tolerated to ﬁt in the GSH-binding pocket. Using a

ﬁxed Zn²⁺-coordination geometry for 8-MSQ, the SAR around

the fragment was explored by making independent sub-

stitutions at the sulfonamide 8-position.

8-Sulfonamidequinoline Analogs. Compound 8 dem-

onstrated that a phenyl group was well tolerated within the

GSH-binding pocket (Table 1). Therefore, diﬀerent sub-

stituents were explored to ﬁnd a more optimal group to

enhance interactions with this pocket. A diverse range of

aliphatic, functionalized phenyl, and heterocyclic functional

groups were probed at the 8-position. Preparation and

screening of several derivatives (Table 2) demonstrated that

aliphatic substituents (10−13) were not tolerated and resulted

in poorer inhibition than 8-MSQ. However, compounds that

incorporated heterocyclic or aromatic rings, such as thiophene,

furan, phenyl, and pyridine (14−15, 18−19, 21−24, and 27−

30) gave modest improvements in activity, with compound 23

showing the largest (∼14-fold) improvement. Three com-

pounds that incorporated fused-ring systems (31−33) also

showed modest improvements in activity. Alternatively,

extending the linker between the sulfonamide functional

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a

a reduction in percent immobility as compared to vehicle-

treated animals (Figure 3). This is consistent with an

antidepressant eﬀect for 60 and is similar to previously

reported results for other GLO1 inhibitors.⁴¹

Table 3. 8-MSQ Fragment Derivatives at the 5-Position

Brain Exposure and Eﬀect on MG Brain Levels. Given

the behavioral results, we used a separate cohort of mice to

examine brain levels of 60 and to establish target engagement

by measuring brain MG concentrations. Following 12.5 mg/kg

i.p. doses of 60, median brain levels of the compound at 30, 60,

120, and 240 min were 2.1, 14.4, 6.0, and 2.2 ng/mg of wet

weight brain tissue (n = 3 per time point). At 25 mg/kg i.p.,

median brain levels at 30, 60, 120, and 240 min were 0.4, 6.7,

18.9, and 8.3 ng/mg of wet weight brain tissue (n = 3 per time

point). MG levels in the brain between 30 and 240 min post-

dose were increased approximately 11-fold and 13-fold in mice

treated with 12.5 and 25 mg/kg of 60, respectively, as

compared to vehicle-treated mice (MG per mg brain tissue in

60 12.5 and 25 mg/kg groups 11.8

4.1 and 14.1

3.1,

respectively, vs 1.1 0.3 in vehicle-treated mice). Collectively,

these data suggest on-target engagement of GLO1 by 60,

resulting in accumulation of MG, and the observed behavioral

eﬀects in mice (vide supra).

DISCUSSION

■

a

All IC₅₀values listed are in μM.

In order to discover a GLO1 inhibitor, an FBDD approach

using a ∼240-component library of MBPs was utilized in two

independent screens at two diﬀerent concentrations, 200 and

50 μM. Screening led to the identiﬁcation of the highly active

8-MSQ fragment that was determined to have an IC₅₀value of

18.5 μM. Upon identifying 8-MSQ as the lead fragment,

several analogs were evaluated to determine whether metal

binding was the mechanism of GLO1 inhibition. The SAR

obtained from compounds 2−6 (Table 1) strongly suggests

that bidentate metal coordination is necessary for the observed

inhibitory activity of 8-MSQ. The identiﬁcation of 8-MSQ

prompted the development of a computational model that

identiﬁed the 5- and 8-positions as suitable positions for

derivatization, fragment growth, and lead inhibitor develop-

ment. A strategy was then implemented in which the

substituent oﬀ each position was explored independently.

Initial synthetic eﬀorts were conﬁned to substituents

exploring modiﬁcations to the 8-position, which is situated

within the GSH-binding pocket of the GLO1 active site. A

variety of substituents were explored via a sulfonamide linkage

as demonstrated in Table 2. The introduction of nonaromatic

alkane chains led to loss of inhibition (10−13). However,

compounds that incorporated heterocyclic or aromatic rings,

such as thiophene, furan, phenyl, and pyridine (14−15, 18−

19, 21−22, 24, and 27−30) all improved activity modestly.

Three compounds incorporated fused-ring systems (31−33)

and were among the best inhibitors of this subset of molecules.

Alternatively, extending the linker between the sulfonamide

functional group and the phenyl substituent (34−36) did not

change inhibition signiﬁcantly. Introduction of a 2-pyridyl

moiety (23) yielded the best inhibition against GLO1, and

computational modeling suggested that the nitrogen of the 2-

pyridyl moiety is engaged in a hydrogen bond with an active

site water (Figure S3).

containing phenyl, chlorophenyl, and ﬂuorophenyl substituents

(47−50, 53−54, 57, and 59) were primarily explored (Table

4). Additionally, compounds 51−52, 55, and 60 were inspired

by previously reported GLO1 inhibitors.²¹⁻²⁷These eﬀorts

yielded a rather ﬂat SAR, but with several inhibitors showing

IC₅₀values of <1 μM, with 49 and 60 demonstrating the best

inhibition with an IC₅₀value of 370 and 460 nM, respectively

(Table 4). A model for the binding of these compounds

(speciﬁcally compound 47) shows how the 5- and 8-

substituents occupy the hydrophobic and GSH binding

pockets in GLO1 (Figure S4).

Behavioral Testing. Preliminary behavioral testing in mice

used compound 48, which was found to be visibly toxic at

intraperitoneal (i.p.) doses of 50 mg/kg and higher.

Compound 60 was chosen as an alternative, and observational

assessment of toxicity showed no adverse responses with doses

up to 100 mg/kg i.p. To evaluate whether 60 showed an

antidepressant-like behavioral proﬁle, we employed the forced

swim test (FST), which is an assay that is widely used to screen

compounds for antidepressant eﬃcacy.⁴⁰We also used the

open ﬁeld test in an eﬀort to identify unexpected changes in

locomotor behavior following drug treatment, which might

confound our interpretation of the FST. Mice were weighed

and given an i.p. injection with either 12.5 mg/kg of the GLO1

inhibitor 60 or vehicle [8% dimethylsulfoxide (DMSO), 18%

Tween, 80% H₂O] and were returned to the home cage.

Ninety minutes after injection, mice were placed in the open

ﬁeld apparatus for 30 min. Immediately after open ﬁeld testing,

mice were transported to a diﬀerent procedure room and given

a 6 min FST. At the end of testing, mice were returned to the

colony. Approximately 3 weeks later, mice were again weighed

and given an i.p. injection with either 12.5 mg/kg of the GLO1

inhibitor 60 or vehicle. After 2 h, mice were given another 6

min FST. Drug treatment order was counterbalanced relative

to the ﬁrst day of testing, such that each mouse receive vehicle

on one day and 60 on the other. Drug-treated animals showed

Additional synthetic eﬀorts were made to explore the GLO1

active site hydrophobic pocket. A small library of inhibitors

with functionalization at the 5-position were prepared.

Generally, only hydrophobic aromatic functional groups were

tolerated (37, 40−44), while compounds containing polar

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a

Table 4. 8-MSQ Derivatives with Two Substituents

a

All IC₅₀values listed are in μM.

heterocyclic or nonaromatic substituents (38−39 and 45−46)

demonstrated signiﬁcant loss of inhibition (Table 3).

Combining preferred substituents on the 5- and 8-positions

using a fragment-merge strategy gave a series of lead

compounds (Table 4). Although the SAR of these lead

compounds was somewhat ﬂat, several compounds were

identiﬁed with IC₅₀values in the range of ∼370−800 nM,

representing a ∼40-fold improvement in activity when

compared to the parent fragment 8-MSQ. These lead

compounds are comparably active to the GLO1 inhibitor

methyl gerfelin, but are substantially more druglike (e.g.,

lacking PAINS such as the catechol group in methyl

gerfelin).⁴²

In an eﬀort to evaluate behavioral eﬀects of 60, in vivo

studies were performed in mice. We conﬁrmed that 60 reduced

immobility in the FST (Figure 3), which is consistent with

prior observations for two other GLO1 inhibitors: S-

bromobenzylglutathione cyclopentyl diester (pBBG) and

methyl gerfelin.⁴¹Importantly, the eﬀects on the FST were

not accompanied by changes in general locomotor activity,

which can confound the interpretation of the FST.⁴⁰We also

assessed levels of 60 in the brain over the time course relevant

to our behavioral studies. Brain exposure of 60 was similar to

that observed with ﬂuoxetine (a prototypical selective SRI) in

rodents⁴³and was associated with a >10-fold increase in brain

MG levels (ng/mg of brain tissue) compared to vehicle-treated

mice, indicating target engagement. Taken together, these

results show that 60 elevated brain MG levels and produced

behavioral changes that are consistent with antidepressant

eﬀects. Although not directly tested in these studies, our prior

studies showed that the behavioral eﬀects of GLO1 inhibitors

occurred more rapidly than conventional antidepressants (e.g.,

ﬂuoxetine) and could be observed using both acute and

chronic models; it is likely that the same would be true of 60.⁴¹

Our working model suggests that the eﬀects of MG are

mediated by its action as an endogenously produced

competitive partial agonist at GABA-A receptors;^14,15,44

however, a recent study has suggested an alternative

mechanism, namely, that MG may also exert antidepressant

eﬀects by direct activation of TrkB receptors.⁴⁵

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8.12 (m, 2H), 7.93 (d, J = 6.8 Hz, 1H), 3.19 (s, 3H). ESI-MS(+): m/z

223.08 [M + H]⁺.

N-(Quinolin-5-yl)methanesulfonamide (3). To a solution of

quinoli-5-amine (0.2 g, 1.38 mmol) in pyridine (3 mL),

methanesulfonyl chloride (0.161 mL, 2.08 mmol) was added, and

the mixture was allowed to stir at room temperature for 12−15 h.

After stirring, H₂O (50 mL) was added to the reaction mixture and

the solution was extracted with EtOAc (3 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated via

rotary evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting a gradient of 0−16% MeOH in CH₂Cl₂. Yield

0.11 g (38%). ¹H NMR (400 MHz, CDCl₃): δ 8.41 (dd, J₁= 8.4 Hz,

J₂= 1.6 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 7.81 (t, J = 8.4 Hz, 1H),

7.64 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.59 (dd, J₁= 8.4 Hz, J₂= 4.0

Hz, 1H), 3.51 (s, 3H). ESI-MS(+): m/z 223.11 [M + H]⁺.

Quinolin-8-yl Methanesulfonate (4). To a solution of 8-

hydroxyquinoline (0.2 g, 1.38 mmol) in pyridine (3 mL),

methanesulfonyl chloride (0.322 mL, 4.13 mmol) was added, and

the mixture was allowed to stir at room temperature for 12−15 h.

After stirring, H₂O (50 mL) was added to the reaction mixture and

the solution was extracted with EtOAc (3 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated via

rotary evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting a gradient of 0−70% EtOAc in hexanes. Yield

0.085 g (34%). ¹H NMR (400 MHz, CDCl₃): δ 8.91 (dd, J₁= 4.0 Hz,

J₂= 1.6 Hz, 1H), 8.15 (dd, J₁= 8.4 Hz, J₂= 1.2 Hz, 1H), 7.73 (d, J =

8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.43

(dd, J₁= 8.4 Hz, J₂= 4.4 Hz, 1H), 3.40 (s, 3H). ESI-MS(+): m/z

224.25 [M + H]⁺.

Figure 3. Administration of compound 60 at 120 min before

behavioral testing in the FST produced a signiﬁcant decrease in the

time spent immobile compared to vehicle-treated controls (ANOVA;

F_1,14= 5.06, p = 0.041). There were no main eﬀects of sex or test

order, and no signiﬁcant interactions (F_1,14≤ 3.48, p > 0.05). There

was no eﬀect of drug treatment on locomotor activity (F_1,21= 0.052, p

> 0.05; data not shown), indicating that our results were not

confounded by hypo- or hyper-locomotion.

EXPERIMENTAL SECTION

■

N-(Quinolin-8-yl)acetamide (5). To a solution of 8-amino-

quinoline (0.2 g, 1.38 mmol) in pyridine (3 mL), acetyl chloride

(0.148 mL, 2.08 mmol) was added, and the mixture was allowed to

stir at room temperature for 12−15 h. After stirring, H₂O (50 mL)

was added to the reaction mixture and the solution was extracted with

EtOAc (3 × 50 mL). The combined organic layers were dried over

MgSO₄, ﬁltered, and concentrated via rotary evaporation. The

resulting crude oil was puriﬁed via silica gel chromatography eluting

General. All reagents and solvents were obtained from commercial

sources and used without further puriﬁcation. Microwave reactions

were performed in 10 or 35 mL microwave reaction vials using a CEM

Discover S reactor. Column chromatography was performed using a

Teledyne ISCO CombiFlash Rf system with prepacked silica

cartridges or High Performance Gold C18 columns. H/¹³C NMR

1

spectra were recorded at ambient temperature on a 400 or 500 Varian

FT-NMR instrument located in the Department of Chemistry and

Biochemistry at the U.C. San Diego. Mass spectra were obtained from

the Molecular Mass Spectrometry Facility (MMSF) in the Depart-

ment of Chemistry and Biochemistry at the University of California,

San Diego. The purity of all compounds used in assays was

determined to be ≥95% by high-performance liquid chromatography

(HPLC) analysis (Table S1 and Figure S5) using an Agilent 6230

Accurate-Mass LC-TOFMS from the MMSF at U.C. San Diego.

N-(Quinolin-8-yl)methanesulfonamide (8-MSQ). To a sol-

ution of 8-aminoquinoline (0.1 g, 0.7 mmol) in pyridine (3 mL),

methanesulfonyl chloride (0.16 mL, 2.1 mmol) was added. The

mixture was stirred at room temperature for 12−15 h. After stirring,

H₂O (50 mL) was added to the reaction mixture and the solution was

extracted with EtOAc (3 × 50 mL). The combined organic layers

were dried over MgSO₄, ﬁltered, and concentrated via rotary

evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting a gradient of 0−80% EtOAc in hexanes. The

1

a gradient of 0−60% EtOAc in hexanes. Yield 0.076 g (32%). H

NMR (400 MHz, CDCl₃): δ 9.78 (br, 1H), 8.81 (dd, J₁= 4.0 Hz, J₂=

1.6 Hz, 1H), 8.77 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 8.17 (dd, J₁= 8.0

Hz, J₂= 1.6 Hz, 1H), 7.55−7.48 (m, 2H), 7.47 (dd, J₁= 8 Hz, J₂= 5

Hz, 1H), 2.35 (s, 3H). ESI-MS(+): m/z 187.23 [M + H]⁺.

N-Methylquinoline-8-sulfonamide (6). To a solution of

quinoline-8-sulfonyl chloride (0.2 g, 0.88 mmol) in pyridine (3

mL), methylamine (2 M tetrahydrofuran solution, 1.31 mL, 0.082

mmol) was added and allowed to stir at room temperature for 12−15

h. After stirring, H₂O (50 mL) was added to the reaction mixture and

the solution was extracted with EtOAc (3 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated via

rotary evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting a gradient of 0−55% EtOAc in hexanes. Yield

1

0.110 g (43%). H NMR (400 MHz, CDCl₃): δ 9.03 (d, J = 4.0 Hz,

1H), 8.47 (d, J = 8.0 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 8.09 (d, J =

8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.59 (dd, J₁= 8.0 Hz, J₂= 4.0

Hz, 1H), 6.26 (br, 1H), 2.59 (d, J = 5.6 Hz, 3H). ESI-MS(+): m/z

223.15 [M + H]⁺.

1

product was isolated as an oﬀ-white solid. Yield 0.085 g (55%). H

NMR (400 MHz, CDCl₃): δ 8.93 (br, 1H), 8.83 (dd, J₁= 4.4 Hz, J₂=

2 Hz, 1H), 8.20 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.87 (dd, J₁= 7.2

Hz, J₂= 1.6 Hz, 1H), 7.58−7.48 (m, 3H), 3.03 (s, 3H). ESI-MS(+):

m/z 223.08 [M + H]⁺, 245.04 [M + Na]⁺.

N-(2-Methylquinolin-8-yl)methanesulfonamide (7). To a

solution of 2-methylquinolin-8-amine (0.2 g, 1.26 mmol) in pyridine

(3 mL), methanesulfonyl chloride (0.296 mL, 3.79 mmol) was added,

and the mixture was allowed to stir at room temperature for 15 h.

After stirring, H₂O (50 mL) was added to the reaction mixture and

the solution was extracted with EtOAc (3 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated via

rotary evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting 0−10% MeOH in CH₂Cl₂. The desired

N-(Isoquinolin-8-yl)methanesulfonamide (2). To a solution of

isoquinolin-8-amine (0.2 g, 1.38 mmol) in pyridine (3 mL),

methanesulfonyl chloride (0.161 mL, 2.08 mmol) was added, and

the mixture was allowed to stir at room temperature for 12−15 h.

After stirring, H₂O (50 mL) was added to the reaction mixture and

the solution was extracted with EtOAc (3 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated via

rotary evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting a gradient of 0−20% MeOH in CH₂Cl₂. Yield

1

product was isolated as a dark brown solid. Yield 0.191 g (64%). H

NMR (DMSO-d₆, 400 MHz): δ 9.17 (br, 1H), 8.29 (d, J = 8.4 Hz,

1H), 7.67 (m, 2H), 7.51 (m, 2H), 3.15 (s, 3H), 2.70 (s, 3H). ESI-

MS(+): m/z 237.07 [M + H]⁺, 258.98 [M + Na]⁺.

1

0.161 g (52%). H NMR (400 MHz, DMSO-d₆): δ 10.75 (br, 1H),

9.92 (s, 1H), 8.70 (d, J = 6.4 Hz, 1H), 8.47 (d, J = 6.4 Hz, 1H), 8.18−

1616

DOI: 10.1021/acs.jmedchem.8b01868

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Journal of Medicinal Chemistry

Article

Protocol for sulfonamide coupling method Ato a solution of 8-

aminoquinoline (0.1 g, 0.69 mmol) in pyridine (3 mL) the

corresponding sulfonyl chloride was added, and the mixture was

allowed to stir at room temperature for 12−15 h. To the resulting

mixture was added H₂O (50 mL), and then the solution was extracted

with EtOAc (3 × 50 mL). The combined organic layers were dried

over MgSO₄, then ﬁltered, and concentrated via rotary evaporation.

The resulting crude oil was puriﬁed via silica gel chromatography

eluting a gradient of EtOAc in hexanes.

Protocol for sulfonamide coupling method Bto a solution of 8-

aminoquinoline (0.1 g, 0.69 mmol) in pyridine (3 mL), the

corresponding sulfonyl chloride (1.04 mmol) was added. The clear

solution was heated in a microwave reactor at 130 °C for 3 min

(power = 300 W). The solution was then poured into H₂O (12 mL)

causing the product to precipitate, which was then isolated via

ﬁltration, and rinsed with water. If no precipitate was formed, the

aqueous phase was extracted CH₂Cl₂(2 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and evaporated in

vacuo to obtain a solid when co-evaporated with Et₂O.

3.6 Hz, 1H), 2.18−1.14 (m, 10H). ESI-MS(+): m/z 291.17 [M +

H]⁺, 313.13 [M + Na]⁺.

N-(Quinolin-8-yl)thiophene-2-sulfonamide (14). It is synthe-

sized utilizing sulfonamide coupling method B and isolated as a dark

1

brown solid. Yield 0.179 g (89%). H NMR (400 MHz, CDCl₃): δ

9.30 (br, 1H), 8.76 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.12 (dd, J₁=

8.4 Hz, J₂= 1.6 Hz, 1H), 7.91 (dd, J₁= 6.4 Hz, J₂= 2.4 Hz, 1H), 7.62

(dd, J₁= 3.6 Hz, J₂= 1.2 Hz, 1H), 7.51 (m, 2H), 7.44 (d, J = 4.4 Hz,

1H), 7.42 (d, J = 4.0 Hz, 1H), 6.92 (t, J = 3.6 Hz, 1H). ESI-MS(+):

m/z 291.02 [M + H]⁺, 312.97 [M + Na]⁺.

1-Methyl-N-(quinolin-8-yl)-1H-imidazole-4-sulfonamide

(15). It is synthesized utilizing sulfonamide coupling method A using

1.5 equiv of sulfonyl chloride. The product precipitated as H₂O was

added to reaction mixture. The solid was isolated via ﬁltration and not

1

further puriﬁed and isolated as a pink solid. Yield 0.089 g (48%). H

NMR (CDCl₃, 400 MHz): δ 9.41 (br, 1H), 8.79 (dd, J₁= 4.0 Hz, J₂=

1.6 Hz, 1H), 8.12 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.89 (t, J = 4.4

Hz, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.46−7.41 (m, 3H), 7.36 (s, 1H),

7.25 (s, 1H), 3.65 (s, 3H). ESI-MS(+): m/z 289.22 [M + H]⁺, 311.16

[M + Na]⁺.

4-(1H-Pyrazol-1-yl)-N-(quinolin-8-yl)benzenesulfonamide

(16). It is synthesized utilizing sulfonamide coupling method B and

isolated as a white solid. Yield 0.330 g (99%). ¹H NMR (CDCl₃, 400

MHz): δ 9.26 (br, 1H), 8.76 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.10

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.98 (d, J = 9.2 Hz, 2H), 7.88 (d, J

= 2.8 Hz, 1H), 7.86 (dd, J₁= 6.8 Hz, J₂= 2.0 Hz, 1H), 7.68 (d, J = 2.0

Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.43 (m, 3H), 6.45 (dd, J₁= 2.8

Hz, J₂= 2.0 Hz, 1H). ESI-MS(+): m/z 351.08 [M + H]⁺, 373.06 [M

+ Na]⁺.

2-Methoxy-N-(quinolin-8-yl)benzenesulfonamide (17). It is

synthesized utilizing sulfonamide coupling method A, puriﬁed with a

gradient of 0−25% EtOAc in hexanes, and isolated as a white solid.

Yield 0.123 g (56%). ¹H NMR (CDCl₃, 400 MHz): δ 9.70 (br, 1H),

8.79 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.07 (dd, J₁= 8.4 Hz, J₂= 1.6

Hz, 1H), 7.98 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.81 (dd, J₁= 8.4

Hz, J₂= 1.6 Hz, 1H), 7.42−7.32 (m, 3H), 6.94 (t, J = 8.4 Hz, 1H),

6.78 (t, J = 8.4 Hz, 1H), 3.81 (s, 3H). ESI-MS(+): m/z 315.09 [M +

H]⁺.

N-(Quinolin-8-yl)benzenesulfonamide (8). It is synthesized

utilizing sulfonamide coupling method B and isolated as a white solid.

Yield 0.179 g (91%). ¹H NMR (CDCl₃, 400 MHz): δ 9.25 (br, 1H),

8.76 (d, J = 4.4 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 8.0 Hz,

2H), 7.82 (dd, J₁= 6.4 Hz, J₂= 2.4 Hz, 1H), 7.41 (m, 6H). ESI-

MS(+): m/z 285.03 [M + H]⁺.

N-(2-Methylquinolin-8-yl)benzenesulfonamide (9). To a

solution of 2-methylquinolin-8-amine (0.2 g, 1.26 mmol) in pyridine

(3 mL), benzenesulfonyl chloride (0.486 mL, 3.79 mmol) was added,

and the mixture was allowed to stir at room temperature for 15 h.

After stirring, H₂O (50 mL) was added to the reaction mixture and

the solution was extracted with EtOAc (3 × 50 mL). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated via

rotary evaporation. The resulting crude oil was puriﬁed via silica gel

chromatography eluting 0−10% MeOH in CH₂Cl₂. The desired

1

product was isolated as a light brown solid. Yield 0.247 g (66%). H

NMR (DMSO-d₆, 400 MHz): δ 9.76 (br, 1H), 8.18 (d, J = 8.4 Hz,

1H), 7.89 (m, 2H), 7.62−7.39 (m, 7H), 2.63 (s, 3H). ESI-MS(+): m/

z 299.11 [M + H]⁺, 321.05 [M + Na]⁺.

N-(Quinolin-8-yl)cyclopropanesulfonamide (10). It is synthe-

sized utilizing sulfonamide coupling method A using 1.5 equiv of

sulfonyl chloride, puriﬁed with a gradient of 0−30% EtOAc in

hexanes, and isolated as a white solid. Yield 0.080 g (45%). ¹H NMR

(CDCl₃, 400 MHz): δ 8.92 (br, 1H), 8.83 (dd, J₁= 4.0 Hz, J₂= 1.6

Hz, 1H), 8.19 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.92 (dd, J₁= 7.2

Hz, J₂= 1.6 Hz, 1H), 7.57−7.47 (m, 3H), 2.57 (m, 1H), 1.29 (m,

2H), 0.90 (m, 2H). ESI-MS(+): m/z 249.14 [M + H]⁺.

3-Methoxy-N-(quinolin-8-yl)benzenesulfonamide (18). It is

synthesized utilizing sulfonamide coupling method A using 1.5 equiv

of sulfonyl chloride, puriﬁed with a gradient of 0−25% EtOAc in

1

hexanes, and isolated as a white solid. Yield 0.20 g (92%). H NMR

(CDCl₃, 400 MHz): δ 9.25 (br, 1H), 8.67 (dd, J₁= 4.0 Hz, J₂= 1.6

Hz, 1H), 8.00 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.84 (m, 1H), 7.48

(m, 1H), 7.39 (m, 3H), 7.33 (dd, J₁= 8.4 Hz, J₂= 4.4 Hz, 1H), 7.20

(t, J₁= 8.0 Hz, 1H), 6.88 (m, 1H), 3.63 (s, 3H). ESI-MS(+): m/z

315.13 [M + H]⁺.

4-Methoxy-N-(quinolin-8-yl)benzenesulfonamide (19). It is

synthesized utilizing sulfonamide coupling method B and isolated as

an oﬀ-white solid. Yield 0.117 g (54%). ¹H NMR (400 MHz, CDCl₃):

δ 9.20 (br, 1H), 8.76 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.09 (dd, J₁=

8.0 Hz, J₂= 1.6 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.80 (dd, J₁= 6.0

Hz, J₂= 2.4 Hz, 1H), 7.43 (m, 3H), 6.81 (d, J = 8.8 Hz, 2H), 3.75 (s,

3H). ESI-MS(+): m/z 315.02 [M + H]⁺.

N-(Quinolin-8-yl)propane-2-sulfonamide (11). It is synthe-

sized utilizing sulfonamide coupling method A using 3.3 equiv of

sulfonyl chloride, puriﬁed with a gradient of 0−40% EtOAc in

1

hexanes, and isolated as a tan solid. Yield 0.040 g (23%). H NMR

(400 MHz, CDCl₃): δ 8.86 (br, 1H), 8.82 (dd, J₁= 4.4 Hz, J₂= 2.0

Hz, 1H), 8.18 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.90 (dd, J₁= 7.6

Hz, J₂= 2.0 Hz, 1H), 7.54 (m, 3H), 3.37 (sept, J = 6.8 Hz, 1H), 1.38

(s, 1H), 1.37 (s, 3H). ESI-MS(+): m/z 251.09 [M + H]⁺.

N-(Quinolin-8-yl)butane-1-sulfonamide (12). It is synthesized

utilizing sulfonamide coupling method A using 1.5 equiv of sulfonyl

chloride, puriﬁed with a gradient of 0−15% EtOAc in hexanes, and

2-Cyano-N-(quinolin-8-yl)benzenesulfonamide (20). It is

synthesized utilizing sulfonamide coupling method A, puriﬁed with

a gradient of 0−70% EtOAc in hexanes, and isolated as a white solid.

1

isolated as a dark oil. Yield 0.090 g (49%). H NMR (CDCl₃, 400

1

Yield 0.070 g (33%). H NMR (400 MHz, CDCl₃): δ 8.80 (dd, J₁=

MHz): δ 8.89 (br, 1H), 8.80 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.17

(dd, J₁= 8.0 Hz, J₂= 1.4 Hz, 1H), 7.84 (dd, J₁= 7.2 Hz, J₂= 1.6 Hz,

1H), 7.54−7.45 (m, 3H), 3.12 (m, 2H), 1.81 (m, 2H), 1.35 (sex, J =

7.2 Hz, 2H), 0.79 (t, J = 7.2 Hz, 3H). ESI-MS(+): m/z 265.12 [M +

H]⁺.

4.0 Hz, J₂= 1.6 Hz, 1H), 8.18 (dd, J₁= 8.0 Hz, J₂= 1.2 Hz, 1H), 8.09

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.73−7.39 (m, 7H). ESI-MS(+):

m/z 310.11 [M + H]⁺.

3-Cyano-N-(quinolin-8-yl)benzenesulfonamide (21). It is

synthesized utilizing sulfonamide coupling method A using 1.5

equiv of sulfonyl chloride, puriﬁed with a gradient of 0−70% EtOAc

N-(Quinolin-8-yl)cyclohexanesulfonamide (13). It is synthe-

sized utilizing sulfonamide coupling method A using 3 equiv of

sulfonyl chloride, puriﬁed with a gradient of 0−15% EtOAc in

1

in hexanes, and isolated as a white solid. Yield 0.070 g (32%). H

1

NMR (CDCl₃, 400 MHz): δ 9.25 (br, 1H), 8.77 (dd, J₁= 4.4 Hz, J₂=

1.6 Hz, 1H), 8.17−8.09 (m, 3H), 7.86 (dd, J₁= 7.2 Hz, J₂= 1.2 Hz,

1H), 7.70 (dd, J₁= 8.0 Hz, J₂= 1.2 Hz, 1H), 7.54−7.42 (m, 4H). ESI-

MS(+): m/z 310.11 [M + H]⁺.

hexanes, and isolated as a clear oil. Yield 0.034 g (17%). H NMR

(CDCl₃, 400 MHz): δ 8.85 (br, 1H), 8.81 (dd, J₁= 4.4 Hz, J₂= 1.6

Hz, 1H), 8.18 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.88 (dd, J₁= 7.2

Hz, J₂= 1.6 Hz, 1H), 7.53−7.46 (m, 3H), 3.09 (tt, J₁= 7.2 Hz, J₂=

1617

DOI: 10.1021/acs.jmedchem.8b01868

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Article

4-Cyano-N-(quinolin-8-yl)benzenesulfonamide (22). It is

synthesized utilizing sulfonamide coupling method B and isolated as

an orange solid. Yield 0.106 g (99%). ¹H NMR (CDCl₃, 400 MHz): δ

9.27 (br, 1H), 8.76 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.12 (dd, J₁=

8.4 Hz, J₂= 1.6 Hz, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.85 (dd, J₁= 7.2

Hz, J₂= 1.2 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.53 (dd, J₁= 8.4 Hz,

J₂= 1.2 Hz, 1H), 7.46 (m, 2H). ESI-MS(+): m/z 310.07 [M + H]⁺.

N-(Quinolin-8-yl)pyridine-2-sulfonamide (23). It is synthe-

sized utilizing sulfonamide coupling method A using 1.5 equiv of

sulfonyl chloride, puriﬁed with a gradient of 0−50% EtOAc in

hexanes, and isolated as a brown solid. Yield 0.112 g (57%). ¹H NMR

(CDCl₃, 400 MHz): δ 9.94 (br, 1H), 8.85 (m, 1H), 8.59 (m, 1H),

8.37 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H), 8.09 (m, 2H), 7.76−7.48 (m,

5H). ESI-MS(+): m/z 286.12 [M + H]⁺.

N-(Quinolin-8-yl)naphthalene-2-sulfonamide (31). It is syn-

thesized utilizing sulfonamide coupling method B and isolated as a

white solid. Yield 0.20 g (87%). ¹H NMR (CDCl₃, 400 MHz): δ 9.35

(br, 1H), 8.76 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.51 (d, J = 2.0 Hz,

1H), 8.06 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.88 (m, 3H), 7.78 (m,

2H), 7.55 (m, 2H), 7.41 (m, 3H). ESI-MS(+): m/z 335.04 [M + H]⁺.

N-(Quinolin-8-yl)benzo[d]thiazole-6-sulfonamide (32). It is

synthesized utilizing sulfonamide coupling method A using 1.1 equiv

of sulfonyl chloride, puriﬁed with a gradient of 0−40% EtOAc in

1

hexanes, and isolated as an oﬀ-white solid. Yield 0.11 g (45%). H

NMR (400 MHz, CDCl₃): δ 9.33 (br, 1H), 9.07 (s, 1H), 8.72 (d, J =

4.4 Hz, 1H), 8.58 (s, 1H), 8.05 (m, 3H), 7.88 (m, 1H), 7.43 (m, 2H),

7.38 (m, 1H). ESI-MS(+): m/z 342.03 [M + H]⁺, 363.99 [M + Na]⁺.

2-(Methylthio)-N-(quinolin-8-yl)benzo[d]thiazole-6-sulfona-

mide (33). It is synthesized utilizing sulfonamide coupling method B

N-(Quinolin-8-yl)pyridine-3-sulfonamide (24). It is synthe-

sized utilizing sulfonamide coupling method A using 1.1 equiv of

sulfonyl chloride. The product precipitated as H₂O was added to

reaction mixture. The solid was isolated via ﬁltration and not further

1

and isolated as a light brown solid. Yield 0.20 g (81%). H NMR

(CDCl₃, 400 MHz): δ 9.28 (br, 1H), 8.74 (dd, J₁= 4.4 Hz, J₂= 1.6

Hz, 1H), 8.41 (dd, J₁= 1.6 Hz, J₂= 0.4 Hz, 1H), 8.08 (dd, J₁= 8.4

Hz, J₂= 1.6 Hz, 1H), 7.90 (dd, J₁= 8.8 Hz, J₂= 1.6 Hz, 1H), 7.90

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.85 (dd, J₁= 6.4 Hz, J₂= 2.8 Hz,

1H), 7.75 (dd, J₁= 8.4 Hz, J₂= 0.4 Hz, 1H), 7.42 (m, 3H). ESI-

MS(+): m/z 388.05 [M + H]⁺.

1

puriﬁed and isolated as a white solid. Yield 0.198 g (99%). H NMR

(CDCl₃, 400 MHz): δ 9.33 (br, 1H), 9.07 (dd, J₁= 2.4 Hz, J₂= 0.8

Hz, 1H), 8.70 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.60 (dd, J₁= 4.8

Hz, J₂= 1.6 Hz, 1H), 8.14 (m, 1H), 8.06 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz,

1H), 7.85 (dd, J₁= 7.2 Hz, J₂= 1.6 Hz, 1H), 7.47−7.39 (m, 2H), 7.38

(dd, J₁= 8.4 Hz, J₂= 4.4 Hz, 1H), 7.25 (m, 1H). ESI-MS(+): m/z

286.10 [M + H]⁺, 307.99 [M + Na]⁺.

6-Morpholino-N-(quinolin-8-yl)pyridine-3-sulfonamide

(25). It is synthesized utilizing sulfonamide coupling method B and

isolated as a white solid. Yield 0.100 g (78%). ¹H NMR (CDCl₃, 400

MHz): δ 9.20 (br, 1H), 8.76 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.65

(m, 1H), 8.10 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.87 (dd, J₁= 9.2

Hz, J₂= 2.8 Hz, 1H), 7.82 (m, 1H), 7.45 (m, 3H), 6.45 (d, J = 9.2 Hz,

1H), 3.72 (m, 4H), 3.54 (m, 4H). ESI-MS(+): m/z 371.10 [M + H]⁺,

393.17 [M + Na]⁺.

1-Phenyl-N-(quinolin-8-yl)methanesulfonamide (34). It is

synthesized utilizing sulfonamide coupling method B and isolated as

1

a yellow solid. Yield 0.20 g (97%). H NMR (CDCl₃, 400 MHz): δ

8.87 (br, 1H), 8.71 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.18 (dd, J₁=

8.4 Hz, J₂= 1.6 Hz, 1H), 7.80 (dd, J₁= 7.6 Hz, J₂= 1.6 Hz, 1H), 7.54

(dd, J₁= 8.8 Hz, J₂= 1.2 Hz, 1H), 7.48 (m, 2H), 7.25 (m, 1H), 7.15

(t, J = 8.0 Hz, 2H), 7.08 (d, J = 7.2 Hz, 2H). ESI-MS(+): m/z 299.06

[M + H]⁺, 321.08 [M + Na]⁺.

2-Phenyl-N-(quinolin-8-yl)ethane-1-sulfonamide (35). It is

synthesized utilizing sulfonamide coupling method A using 1.5 equiv

of sulfonyl chloride, puriﬁed with a gradient of 0−20% EtOAc in

1

hexanes, and isolated as a tan solid. Yield 0.143 g (66%). H NMR

4-(tert-Butyl)-N-(quinolin-8-yl)benzenesulfonamide (26). It

(CDCl₃, 400 MHz): δ 8.96 (br, 1H), 8.81 (d, J = 4.0 Hz, 1H), 8.19

(d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.58−7.47 (m, 3H),

7.18−7.11 (m, 3H), 6.97 (d, J = 8.4 Hz, 3H), 3.40 (t, J = 8.0 Hz, 2H),

3.40 (t, J = 8.0 Hz, 2H), 3.16 (t, J = 8.0 Hz, 2H). ESI-MS(+): m/z

313.18 [M + H]⁺.

is synthesized utilizing method sulfonamide coupling B and isolated as

1

a white solid. Yield 0.216 g (91%). H NMR (CDCl₃, 400 MHz): δ

9.23 (br, 1H), 8.75 (dd, J₁= 4.4 Hz, J₂= 2.0 Hz, 1H), 8.10 (dd, J₁=

8.4 Hz, J₂= 1.6 Hz, 1H), 7.83 (m, 3H), 7.43 (m, 3H), 7.37 (d, J = 8.8

Hz, 2H), 1.24 (s, 9H). ESI-MS(+): m/z 341.08 [M + H]⁺.

3-Phenyl-N-(quinolin-8-yl)propane-1-sulfonamide (36). It is

synthesized utilizing sulfonamide coupling method A using 1.1 equiv

of sulfonyl chloride, puriﬁed with a gradient of 0−30% EtOAc in

4-Fluoro-N-(quinolin-8-yl)benzenesulfonamide (27). It is

synthesized utilizing sulfonamide coupling method B and isolated as

an amber solid. Yield 0.150 g (72%). ¹H NMR (CDCl₃, 400 MHz): δ

8.77 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.21 (dd, J₁= 8.4 Hz, J₂= 1.6

Hz, 1H), 7.88−7.92 (m, 2H), 7.83 (dd, J₁= 7.6 Hz, J₂= 1.2 Hz, 1H),

7.58 (dd, J₁= 8.0 Hz, J₂= 1.2 Hz, 1H), 7.46−7.50 (m, 2H), 7.09 (t, J

= 8.4 Hz, 2H). ESI-MS(+): m/z 303.05 [M + H]⁺, 324.99 [M + Na]⁺.

3,4-Diﬂuoro-N-(quinolin-8-yl)benzenesulfonamide (28). It is

synthesized utilizing sulfonamide coupling method A using 1.1 equiv

of sulfonyl chloride, puriﬁed with a gradient of 0−20% EtOAc in

1

hexanes, and isolated as a tan solid. Yield 0.185 g (82%). H NMR

(CDCl₃, 400 MHz): δ 9.23 (br, 1H), 8.69 (dd, J₁= 4.4 Hz, J₂= 1.6

Hz, 1H), 8.01 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.83 (m, 3H), 7.39

(d, J = 4.8 Hz, 2H), 7.34 (dd, J₁= 8.0 Hz, J₂= 4.0 Hz, 1H), 7.12 (d, J

= 8.4 Hz, 2H), 2.48 (t, J = 7.6 Hz, 2H), 1.53 (sex, J = 7.6 Hz, 2H),

0.81 (t, J = 7.2 Hz, 3H). ESI-MS(+): m/z 327.09 [M + H]⁺, 349.03

[M + Na]⁺.

5-Bromo-8-nitroquinoline. A solution of 5-bromoquinoline (10

g, 48.1 mmol) in concentrated H₂SO₄(40 mL) was cooled to 0 °C

under a nitrogen atmosphere. To this was added potassium nitrate

(7.77 g, 77 mmol) slowly and portion-wise. The resulting reaction

mixture was then allowed to warm up to room temperature slowly and

then stirred for an additional 15 h at room temperature. The resulting

mixture was then poured onto ice, which caused the formation of a

precipitate. The precipitate was then ﬁltered and rinsed with excess

amount of H₂O. The resulting product was not further puriﬁed and

1

hexanes, and isolated as a brown solid. Yield 0.10 g (46%). H NMR

(CDCl₃, 400 MHz): δ 8.75 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.11

(dd, J₁= 8.0 Hz, J₂= 1.2 Hz, 1H), 7.84 (dd, J₁= 7.2 Hz, J₂= 1.2 Hz,

1H), 7.76−7.66 (m, 2H), 7.51−7.40 (m, 3H), 7.13 (m, 1H). ESI-

MS(+): m/z 321.11 [M + H]⁺.

N-(Quinolin-8-yl)benzo[d][1,3]dioxole-5-sulfonamide (29).

It is synthesized utilizing sulfonamide coupling method B and isolated

1

as a dark brown solid. Yield 0.08 g (70%). H NMR (CDCl₃, 400

1

MHz): δ 9.20 (br, 1H), 8.77 (dd, J₁= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.11

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.80 (dd, J₁= 6.4 Hz, J₂= 2.4 Hz,

1H), 7.50 (dd, J₁= 8.0 Hz, J₂= 2.0 Hz, 1H), 7.45 (m, 2H), 7.43 (t, J

= 4.0 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 5.96

(s, 2H). ESI-MS(+): m/z 329.09 [M + H]⁺.

was isolated as a light yellow solid. Yield 10.63 g (87%). H NMR

(CDCl₃, 400 MHz): δ 9.08 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.62

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 7.90 (d, J₁= 0.8 Hz, 2H), 7.67 (dd,

J₁= 8.4 Hz, J₂= 4.0 Hz, 1H). ESI-MS(+): m/z 253.21, 255.17 [M +

H]⁺.

2-Oxo-N-(quinolin-8-yl)indoline-5-sulfonamide (30). It is

synthesized utilizing sulfonamide coupling method B and isolated as

an orange solid. Yield 0.09 g (91%). ¹H NMR (CDCl₃, 400 MHz): δ

10.72 (br, 1H), 9.80 (br, 1H), 8.86 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H),

8.34 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.76 (m, 2H), 7.60 (m, 3H),

7.50 (t, J = 8.0 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 3.47 (s, 2H). ESI-

MS(+): m/z 340.09 [M + H]⁺.

The protocol for Suzuki coupling method A is as follows: to a

solution of 5-bromo-8-nitroquinoline (0.3 g, 1.18 mmol) in H₂O (10

mL) were added boronic acid (1.78 mmol), diisopropylamine (0.498

mL, 3.56 mmol), and palladium acetate (0.018 g, 0.08 mmol), and the

solution was heated to reﬂux for 16 h. To the reaction mixture was

added H₂O (50 mL), and the solution was extracted with EtOAc (3 ×

50 mL). The reaction mixture was ﬁltered through celite and

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concentrated and then the product was puriﬁed via silica gel

chromatography utilizing a gradient of EtOAc in hexanes.

(m, 2H), 7.39−7.28 (m, 2H), 7.20−7.17 (m, 1H). ESI-MS(+): m/z

287.32 [M + H]⁺.

The protocol for Suzuki coupling method B is as follows: to a

solution of 5-bromo-8-nitroquinoline (0.3 g, 1.18 mmol) in 1,4-

dioxane (10 mL) were added boronic acid (1.78 mmol), potassium

acetate (0.233 g, 2.37 mmol), and Pd(PPh₃)₄(0.068 g, 0.06 mmol),

and the solution was degassed for 5−10 min. The reaction mixture

was then heated to reﬂux for 16 h under nitrogen atmosphere. The

reaction mixture was ﬁltered through celite and concentrated, and

then the product was puriﬁed via silica gel chromatography utilizing a

gradient of EtOAc in hexanes.

5-(Furan-2-yl)-8-nitroquinoline. It is synthesized utilizing

Suzuki coupling method B. The product was puriﬁed via silica gel

chromatography eluting a gradient of 0−30% EtOAc in hexanes. Yield

1

0.181 g (64%). H NMR (CDCl₃, 400 MHz): δ 9.04 (d, J = 4.0 Hz,

1H), 8.88 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.80 (d, J =

8.0 Hz, 1H), 7.68 (m, 1H), 7.57 (dd, J₁= 8.0 Hz, J₂= 4.0 Hz, 1H),

6.84 (d, J = 2.4 Hz, 1H), 6.63 (m, 1H). ESI-MS(+): m/z 241.31 [M +

H]⁺.

8-Nitro-5-(thiophen-2-yl)quinoline. It is synthesized utilizing

Suzuki coupling method B. The product was puriﬁed via silica gel

chromatography eluting a gradient of 0−40% EtOAc in hexanes. Yield

The protocol for Suzuki coupling method C is as follows: to a

solution of 5-bromo-8-nitroquinoline (0.3 g, 1.18 mmol) in H₂O (5

mL) and 1,4-dioxane (5 mL) were added boronic acid (1.78 mmol),

potassium carbonate (0.328 g, 2.37 mmol), S-Phos (0.05 g, 0.12

1

0.300 g (99%). H NMR (CDCl₃, 400 MHz): δ 9.08 (m, 1H), 8.65

(d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H),

7.55 (m, 2H), 7.27−7.24 (m, 2H). ESI-MS(+): m/z 257.27 [M +

H]⁺.

.

mmol), and Pd(dppf)Cl₂CH₂Cl₂(0.097 g, 0.12 mmol), and the

solution was degassed for 5−10 min. The reaction mixture was then

heated to reﬂux for 16 h under nitrogen atmosphere. The reaction

mixture was ﬁltered through celite and concentrated, and then the

product was puriﬁed via silica gel chromatography utilizing a gradient

of EtOAc in hexanes or MeOH in CH₂Cl₂.

5-(Benzo[d][1,3]dioxol-5-yl)-8-nitroquinoline. It is synthe-

sized utilizing Suzuki coupling method A. The product was puriﬁed

via silica gel chromatography eluting 0−40% EtOAc in hexanes. Yield

1

0.167 g (48%). H NMR (CDCl₃, 400 MHz): δ 9.03 (d, J = 4.0 Hz,

The protocol for Buchwald coupling is as follows: to a solution of

5-bromo-8-nitroquinoline (0.3 g, 1.18 mmol) in dimethylformamide

(10 mL) were added an amine (3.56 mmol), potassium carbonate

(0.49 g, 3.6 mmol), X-Phos (0.11 g, 0.24 mmol), and Pd₂(dba)₃(0.22

g, 0.24 mmol), and the solution was degassed for 5−10 min. The

reaction mixture was then irradiated in a microwave reactor at 140 °C

for 30 min. The resulting mixture was ﬁltered through celite and

concentrated, and then the product was puriﬁed via silica gel

chromatography utilizing a gradient of EtOAc in hexanes.

The protocol for Sonogashira coupling is as follows: to a solution of

5-bromo-8-nitroquinoline (0.3 g, 1.18 mmol) in 1,4-dioxane (10 mL)

and Et₃N (10 mL) were added an alkyne (1.78 mmol), X-Phos (0.057

g, 0.12 mmol), Pd(dppf)Cl₂·CH₂Cl₂(0.097 g, 0.12 mmol), and CuI

(0.023 g, 0.12 mmol), and the solution was degassed for 5−10 min.

The reaction mixture was then heated to reﬂux for 16 h under

nitrogen atmosphere. The reaction mixture was ﬁltered through celite

and concentrated, and then the product was puriﬁed via silica gel

chromatography utilizing a gradient of EtOAc in hexanes.

8-Nitro-5-phenylquinoline. It is synthesized utilizing Suzuki

coupling method A. The product was puriﬁed with a gradient of 0−

30% EtOAc in hexanes. The desired product was isolated as a light tan

solid. Yield 0.297 g (75%). ¹H NMR (CDCl₃, 400 MHz): δ 9.03 (d, J

= 4.0 Hz, 1H), 8.30 (d, J = 4.0 Hz, 1H), 8.06 (d, J = 4.0 Hz, 1H),

7.56−7.43 (m, 7H). ESI-MS(+): m/z 251.34 [M + H]⁺.

8-Nitro-5-(pyridin-4-yl)quinoline. It is synthesized utilizing

Suzuki coupling method C. The product was puriﬁed with a gradient

of 0−15% MeOH in CH₂Cl₂. Yield 0.132 g (44%). ¹H NMR (CDCl₃,

400 MHz): δ 9.10 (dd, J₁= 4 Hz, J₂= 1.6 Hz, 1H), 8.81−8.80 (m,

2H), 8.23 (dd, J₁= 8.8 Hz, J₂= 1.6 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H),

7.59 (d, J = 8.0 Hz, 1H), 7.56 (dd, J₁= 8.8 Hz, J₂= 4.4 Hz, 1H),

7.40−7.39 (m, 2H). ESI-MS(+): m/z 252.25 [M + H]⁺.

1H), 8.34 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.52−7.47

(m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 6.88−6.87 (m, 2H), 6.06 (s, 2H).

ESI-MS(+): m/z 295.36 [M + H]⁺.

8-Nitro-5-(piperidin-1-yl)quinoline. It is synthesized utilizing

the Buchwald coupling method. The product was puriﬁed with a

gradient of 0−25% EtOAc in hexanes. Yield 0.182 g (60%). ¹H NMR

(CDCl₃, 400 MHz): δ 9.04 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.47

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.49 (dd,

J₁= 8.4 Hz, J₂= 4.0 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 3.14 (t, J = 5.6

Hz, 4H), 1.88 (pent, J = 5.6 Hz, 4H), 1.72 (m, 2H). ESI-MS(+): m/z

258.34 [M + H]⁺.

4-(8-Nitroquinolin-5-yl)morpholine. It is synthesized utilizing

the Buchwald coupling method. The product was puriﬁed with a

gradient of 0−25% EtOAc in hexanes. Yield 0.202 g (68%). ¹H NMR

(CDCl₃, 400 MHz): δ 9.00 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.49

(dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.50 (dd,

J₁= 8.4 Hz, J₂= 4.0 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 3.97 (t, J = 4.4

Hz, 4H), 3.16 (t, J = 4.4 Hz, 4H). ESI-MS(+): m/z 260.11 [M + H]⁺.

N-(5-Phenylquinolin-8-yl)methanesulfonamide (37). It is

synthesized utilizing sulfonamide coupling method A. The product

was puriﬁed with a gradient of 0−25% EtOAc in hexanes and isolated

1

as an oﬀ-white solid. Yield 0.033 g (24%). H NMR (CDCl₃, 400

MHz): δ 9.03 (br, 1H), 8.84 (d, J = 3.6 Hz, 1H), 8.31 (d, J = 8.4 Hz,

1H), 7.92 (d, J = 8.4 Hz, 1H), 7.51−7.43 (m, 7H), 3.08 (s, 3H). ESI-

MS(+): m/z 299.10 [M + H]⁺.

N-(5-(Pyridin-4-yl)quinolin-8-yl)methanesulfonamide (38).

It is synthesized utilizing sulfonamide coupling method A. The

product was puriﬁed with a gradient of 0−10% MeOH in CH₂Cl₂and

isolated as an oﬀ-white solid. Yield 0.054 g (35%). ¹H NMR (CDCl₃,

400 MHz): δ 9.09 (br, 1H), 8.86 (d, J = 4.0 Hz, 1H), 8.74 (d, J = 5.2

Hz, 2H), 8.25 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.51−

7.47 (m, 2H), 7.39 (m, 2H), 3.09 (s, 3H). ESI-MS(+): m/z 300.14

[M + H]⁺.

N-(5-(Pyrimidin-5-yl)quinolin-8-yl)methanesulfonamide

(39). It is synthesized utilizing sulfonamide coupling method A. The

product was puriﬁed with a gradient of 0−10% MeOH in CH₂Cl₂and

isolated as an oﬀ-white solid. Yield 0.048 g (32%). ¹H NMR (CDCl₃,

400 MHz): δ 9.29 (s, 1H), 9.12 (br, 1H), 8.88 (d, J = 4.0 Hz, 1H),

8.85 (s, 2H), 8.14 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.55

(dd, J₁= 8.4 Hz, J₂= 4.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 3.09 (s,

3H). ESI-MS(+): m/z 301.10 [M + H]⁺.

N-(5-(4-Chlorophenyl)quinolin-8-yl)methanesulfonamide

(40). It is synthesized utilizing sulfonamide coupling method A. The

product was puriﬁed with a gradient of 0−40% EtOAc in hexanes and

isolated as an oﬀ-white solid. Yield 0.042 g (24%). ¹H NMR (CDCl₃,

400 MHz): δ 9.03 (br, 1H), 8.85 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H),

8.24 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H),

7.49−7.38 (m, 6H), 3.07 (s, 3H). ESI-MS(+): m/z 333.23 [M + H]⁺.

8-Nitro-5-(pyrimidin-5-yl)quinoline. It is synthesized utilizing

Suzuki coupling method C. The product was puriﬁed with a gradient

of 0−15% MeOH in CH₂Cl₂. Yield 0.138 g (46%). ¹H NMR (CDCl₃,

400 MHz): δ 9.39 (s, 1H), 9.14 (d, J = 4.0 Hz, 1H), 8.90 (s, 2H),

8.17 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.63−7.60 (m,

2H). ESI-MS(+): m/z 253.43 [M + H]⁺.

5-(4-Chlorophenyl)-8-nitroquinoline. It is synthesized utilizing

Suzuki coupling method B. The product was puriﬁed via silica gel

chromatography eluting a gradient of 0−30% EtOAc in hexanes. Yield

1

0.182 g (54%). H NMR (CDCl₃, 400 MHz): δ 9.03 (d, J = 4.0 Hz,

1H), 8.30 (d, J = 4.0 Hz, 1H), 8.06 (d, J = 4.0 Hz, 1H), 7.56−7.43

(m, 7H). ESI-MS(+): m/z 285.43 [M + H]⁺.

5-(3,4-Diﬂuorophenyl)-8-nitroquinoline. It is synthesized

utilizing Suzuki coupling method A. The product was puriﬁed via

silica gel chromatography eluting 0−40% EtOAc in hexanes. Yield

1

0.311 g (92%). H NMR (CDCl₃, 400 MHz): δ 9.09 (d, J = 4.0 Hz,

1H), 8.25 (d, J = 8.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.56−7.53

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N - ( 5 - ( 3 , 4 - D i ﬂ u o r o p h e n y l ) q u i n o l i n - 8 - y l ) -

methanesulfonamide (41). It is synthesized utilizing sulfonamide

coupling method A. The product was puriﬁed with a gradient of 0−

35% EtOAc in hexanes and isolated as a tan solid. Yield 0.068 g

(39%). ¹H NMR (CDCl₃, 400 MHz): δ 9.04 (br, 1H), 8.85 (dd, J₁=

4.0 Hz, J₂= 1.6 Hz, 1H), 8.23 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 7.89

(d, J = 8.0 Hz, 1H), 7.51 (dd, J₁= 8.0 Hz, J₂= 4.0 Hz, 1H), 7.46 (d, J

= 8.0 Hz, 1H), 7.33−7.14 (m, 3H), 3.08 (s, 3H). ESI-MS(+): m/z

335.41 [M + H]⁺.

N-(5-(Furan-2-yl)quinolin-8-yl)methanesulfonamide (42). It

is synthesized utilizing sulfonamide coupling method A. The product

was puriﬁed with a gradient of 0−25% EtOAc in hexanes and isolated

as a red-brown oil. Yield 0.091 g (42%). ¹H NMR (CDCl₃, 400

MHz): δ 9.03 (br, 1H), 8.81−8.77 (m, 2H), 7.86 (d, J = 8.0 Hz, 1H),

7.73 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 1.6 Hz, 1H), 7.53 (dd, J₁= 8.0

Hz, J₂= 4.0 Hz, 1H), 6.66 (d, J = 3.2 Hz, 1H), 6.58 (dd, J₁= 3.2 Hz,

J₂= 1.6 Hz, 1H), 3.04 (s, 3H). ESI-MS(+): m/z 289.13 [M + H]⁺.

N-(5-(Thiophen-2-yl)quinolin-8-yl)methanesulfonamide

(43). It is synthesized utilizing sulfonamide coupling method A. The

product was puriﬁed with a gradient of 0−55% EtOAc in hexanes and

isolated as faint yellow crystals. Yield 0.128 g (68%). ¹H NMR

(CDCl₃, 400 MHz): δ 9.05 (br, 1H), 8.84 (dd, J₁= 4.0 Hz, J₂= 1.6

Hz, 1H), 8.58 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.88 (d, J = 8.0 Hz,

1H), 7.62 (d, J = 8.0 Hz, 1H), 7.52 (dd, J₁= 8.0 Hz, J₂= 4.0 Hz, 1H),

7.45 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 7.20−7.18 (m, 2H), 3.07 (s,

3H). ESI-MS(+): m/z 305.33 [M + H]⁺.

N-(5-(Benzo[d][1, 3]dioxol-5-yl)quinolin-8-yl)-

methanesulfonamide (44). It is synthesized utilizing sulfonamide

coupling method A. The product was puriﬁed with a gradient of 0−

50% EtOAc in hexanes and isolated as a faint yellow solid. Yield 0.086

g (46%). ¹H NMR (CDCl₃, 400 MHz): δ 9.00 (br, 1H), 8.83 (dd, J₁

= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.33 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H),

7.88 (d, J = 8.0 Hz, 1H), 7.47−7.44 (m, 2H), 6.95−6.87 (m, 3H),

6.05 (s, 2H), 3.06 (s, 3H). ESI-MS(+): m/z 343.17 [M + H]⁺.

N-(5-(Piperidin-1-yl)quinolin-8-yl)methanesulfonamide

(45). It is synthesized utilizing sulfonamide coupling method A. The

product was puriﬁed with a gradient of 0−75% EtOAc in hexanes and

isolated as a yellow solid. Yield 0.091 g (48%). ¹H NMR (CDCl₃, 400

MHz): δ 8.80 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.67 (br, 1H), 8.54

(dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.48 (dd,

J₁= 8.0 Hz, J₂= 4.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 3.03−2.95 (m,

4H), 2.94 (s, 3H), 1.85 (pent, 4H), 1.66 (br, 2H). ESI-MS(+): m/z

306.11 [M + H]⁺.

N-(5-Morpholinoquinolin-8-yl)methanesulfonamide (46). It

is synthesized utilizing sulfonamide coupling method A. The product

was puriﬁed with a gradient of 0−75% EtOAc in hexanes and isolated

as a yellow solid. Yield 0.101 g (51%). ¹H NMR (CDCl₃, 400 MHz):

δ 8.82 (dd, J₁= 4.0 Hz, J₂= 2.0 Hz, 1H), 8.72 (br, 1H), 8.56 (dd, J₁=

8.4 Hz, J₂= 2.0 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.50 (dd, J₁= 8.4

Hz, J₂= 4.0 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 3.97 (t, J = 4.4 Hz,

4H), 3.07 (t, J = 4.4 Hz, 4H), 2.97 (s, 3H). ESI-MS(+): m/z 308.54

[M + H]⁺.

J₂= 1.6 Hz, 1H), 8.33 (dd, J₁= 8.8 Hz, J₂= 2.0 Hz, 1H), 8.06 (d, J =

8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.51 (dd, J₁= 8.8 Hz, J₂= 4.0

Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.06−6.97 (m, 3H), 3.86 (s, 3H).

ESI-MS(+): m/z 281.31 [M + H]⁺.

5-(3-Methoxyphenyl)-8-nitroquinoline. It is synthesized utiliz-

ing Suzuki coupling method C. The product was puriﬁed with a

gradient of 0−50% EtOAc in hexanes and isolated as a tan solid. Yield

0.249 g (75%). ¹H NMR (CDCl₃, 400 MHz): δ 9.03 (dd, J₁= 4.0 Hz,

J₂= 1.6 Hz, 1H), 8.33 (dd, J₁= 8.8 Hz, J₂= 1.6 Hz, 1H), 8.05 (d, J =

8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.49 (dd, J₁= 8.8 Hz, J₂= 4.0

Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 3.89 (s,

3H). ESI-MS(+): m/z 281.19 [M + H]⁺.

5-(3-Chloro-4-methoxyphenyl)-8-nitroquinoline. It is synthe-

sized utilizing Suzuki coupling method B. The product was puriﬁed

with a gradient of 0−60% EtOAc in hexanes and isolated as a tan

solid. Yield 0.30 g (68%). ¹H NMR (CDCl₃, 400 MHz): δ 9.06 (dd, J₁

= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.30 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H),

8.06 (d, J = 8.0 Hz, 1H), 7.54−7.46 (m, 3H), 7.33 (dd, J₁= 8.4 Hz, J₂

= 2.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 4.00 (s, 3H). ESI-MS(+): m/

z 315.15 [M + H]⁺.

5-(3,4-Dichlorophenyl)-8-nitroquinoline. It is synthesized

utilizing Suzuki coupling method B. The product was puriﬁed with

a gradient of 0−50% EtOAc in hexanes and isolated as a tan solid.

Yield 0.30 g (75%). ¹H NMR (CDCl₃, 400 MHz): δ 9.05 (dd, J₁= 4.0

Hz, J₂= 1.6 Hz, 1H), 8.30−8.05 (m, 2H), 7.54−7.46 (m, 3H), 7.32

(dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 4.00 (s,

3H). ESI-MS(+): m/z 319.23 [M + H]⁺.

5-(3,5-Dimethoxyphenyl)-8-nitroquinoline. It is synthesized

utilizing Suzuki coupling method C. The product was puriﬁed with a

gradient of 0−35% EtOAc in hexanes and isolated as a brown solid.

Yield 0.19 g (53%). ¹H NMR (CDCl₃, 400 MHz): δ 9.07 (dd, J₁= 4.0

Hz, J₂= 1.6 Hz, 1H), 8.36 (dd, J₁= 8.8 Hz, J₂= 1.6 Hz, 1H), 8.06 (d,

J = 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.51 (dd, J₁= 8.8 Hz, J₂=

4.0 Hz, 1H), 6.60−6.55 (m, 3H), 3.84 (s, 6H). ESI-MS(+): m/z

311.26 [M + H]⁺.

8-Nitro-5-(phenylethynyl)quinoline. It is synthesized utilizing

the Sonogashira coupling method. The product was puriﬁed with a

gradient of 0−35% EtOAc in hexanes and isolated as a brown solid.

1

Yield 0.206 g (63%). H NMR (CDCl₃, 400 MHz): δ 9.11 (m, 1H),

8.79 (dd, J₁= 8.8 Hz, J₂= 1.6 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.83

(d, J = 8.0 Hz, 1H), 7.66−7.64 (m, 3H), 7.45−7.42 (m, 3H). ESI-

MS(+): m/z 275.21 [M + H]⁺.

5-((4-Fluorophenyl)ethynyl)-8-nitroquinoline. It is synthe-

sized utilizing the Sonogashira coupling method. The product was

puriﬁed with a gradient of 0−25% EtOAc in hexanes and isolated as

an oﬀ-white solid. Yield 0.17 g (49%). ¹H NMR (CDCl₃, 400 MHz):

δ 9.11 (dd, J₁= 4 Hz, J₂= 1.6 Hz, 1H), 8.77 (dd, J₁= 8.0 Hz, J₂= 1.6

Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.66−

7.62 (m, 3H), 7.15−7.11 (m, 2H). ESI-MS(+): m/z 293.21 [M +

H]⁺.

The protocol for reduction A is as follows: this method was utilized

to reduce corresponding nitro compounds into amines, which were

utilized in the synthesis of compounds 37−39, 41−47, 50−52, and

55−60. A solution of 5-aryl/cyclic/ethynyl-8-nitroquinoline in

MeOH (25 mL) and Pd/C (10% loading, 0.05 equiv) was degassed

for 5−10 min. The solution mixture was then purged with hydrogen

gas (1 atm) and stirred at room temperature for 1−2 h. The resulting

mixture was then ﬁltered through celite and concentrated. The

product identity was conﬁrmed via thin layer chromatography (TLC)

and MS and used in the next step without further puriﬁcation.

The protocol for reduction B is as follows: this method was utilized

to reduce corresponding nitro compounds into amines, which were

utilized in the synthesis of compounds 40, 48−49, and 53−54. To a

solution of chlorophenyl-8-nitroquinoline in MeOH/acetone/H₂O

(1:1:1, 15 mL) was added K₂CO₃(4 equiv) and sodium dithionate (4

equiv) and then allowed to stir at room temperature under nitrogen

overnight. The resulting solution was concentrated to remove organic

solvents. The resulting aqueous solution was diluted with water (25

mL) and then extracted with EtOAc (3 × 50 mL). The combined

organic fractions were concentrated to yield product. The product

5-(3-Chlorophenyl)-8-nitroquinoline. It is synthesized utilizing

Suzuki coupling method B. The product was puriﬁed with a gradient

of 0−35% EtOAc in hexanes and isolated as a tan solid. Yield 0.222 g

(65%). ¹H NMR (CDCl₃, 400 MHz): δ 9.01 (m, 1H), 8.24 (m, 1H),

8.03 (d, J = 8.4 Hz, 1H), 7.54−7.19 (m, 6H). ESI-MS(+): m/z 285.24

[M + H]⁺.

5-(4-Fluorophenyl)-8-nitroquinoline. It is synthesized utilizing

Suzuki coupling method C. The product was puriﬁed with a gradient

of 0−35% EtOAc in hexanes and isolated as a brown solid. Yield

0.167 g (53%). ¹H NMR (CDCl₃, 400 MHz): δ 9.02 (dd, J₁= 4.0 Hz,

J₂= 1.6 Hz, 1H), 8.25 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 8.04 (d, J =

8.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 8.25 (dd, J₁= 8.4 Hz, J₂= 4.0

Hz, 1H), 7.43−7.40 (m, 2H), 7.24−7.20 (m, 2H). ESI-MS(+): m/z

269.19 [M + H]⁺.

5-(4-Methoxyphenyl)-8-nitroquinoline. It is synthesized utiliz-

ing Suzuki coupling method C. The product was puriﬁed with a

gradient of 0−50% EtOAc in hexanes and isolated as a tan solid. Yield

0.110 g (33%). ¹H NMR (CDCl₃, 400 MHz): δ 9.05 (dd, J₁= 4.0 Hz,

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identity was conﬁrmed via TLC and MS and used in the next step

without further puriﬁcation.

N-(5-(3-Chloro-4-methoxyphenyl)quinolin-8-yl)pyridine-2-

sulfonamide (53). 5-(3-Chloro-4-methoxyphenyl)-8-nitroquinoline

was reduced to an amine utilizing reduction method B. The

corresponding amine was then coupled to pyridine-2-sulfonyl chloride

utilizing sulfonamide coupling method A. The product was puriﬁed

with a gradient of 0−100% EtOAc in hexanes and isolated as a white

N-(5-Phenylquinolin-8-yl)pyridine-2-sulfonamide (47). 8-

Nitro-5-phenylquinoline was reduced to an amine utilizing reduction

method A. The corresponding amine was then coupled to pyridine-2-

sulfonyl chloride utilizing sulfonamide coupling method A. The

product was puriﬁed with a gradient of 0−40% EtOAc in hexanes and

isolated as a white solid. Yield 0.115 g (70%). ¹H NMR (CDCl₃, 400

MHz): δ 9.60 (br, 1H), 8.81 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.62−

8.60 (m, 1H), 8.22 (dd, J₁= 8.4 Hz, J₂= 1.6 Hz, 1H), 8.14 (d, J = 8.0

Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.87 (td, J₁= 8.0 Hz, J₂= 1.6 Hz,

1H), 7.49−7.36 (m, 8H). HR-ESI-MS calcd for [C₂₀H₁₆N₃O₂S]⁺,

362.0958; found, 362.0951.

1

solid. Yield 0.15 g (51%). H NMR (CDCl₃, 400 MHz): δ 9.59 (br,

1H), 8.82 (dd, J₁= 4.4 Hz, J₂= 2.0 Hz, 1H), 8.61−8.60 (m, 1H), 8.19

(dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.96 (d, J

= 8.0 Hz, 1H), 7.88 (td, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.43−7.39 (m,

3H), 7.36 (d, J = 8.0 Hz, 1H), 7.26 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H),

7.04 (d, J = 8.4 Hz, 1H), 3.97 (s, 3H). HR-ESI-MS calcd for

[C₂₁H₁₆ClN₃NaO₃S]⁺, 448.0493; found, 448.0491.

N-(5-(3,4-Dichlorophenyl)quinolin-8-yl)pyridine-2-sulfona-

mide (54). 5-(3,4-Dichlorophenyl)-8-nitroquinoline was reduced to

an amine utilizing reduction method B. The corresponding amine was

then coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide

coupling method A. The product was puriﬁed with a gradient of 0−

100% EtOAc in hexanes and isolated as a white solid. Yield 0.17 g

(58%). ¹H NMR (CDCl₃, 400 MHz): δ 9.61 (br, 1H), 8.85 (dd, J₁=

4.4 Hz, J₂= 2.0 Hz, 1H), 8.62−8.60 (m, 1H), 8.15−8.12 (m, 2H),

7.99 (d, J = 8.0 Hz, 1H), 7.89 (td, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.56−

7.35 (m, 5H), 7.23 (m, 1H). HR-ESI-MS calcd for

[C₂₀H₁₄Cl₂N₃O₂S]⁺, 430.0178; found, 430.0180.

N-(5-(4-Chlorophenyl)quinolin-8-yl)pyridine-2-sulfonamide

(48). 5-(4-Chlorophenyl)-8-nitroquinoline was reduced to an amine

utilizing reduction method B. The corresponding amine was then

coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide coupling

method A. The product was puriﬁed with a gradient of 0−40% EtOAc

1

in hexanes and isolated as a white solid. Yield 0.061 g (79%). H

NMR (CDCl₃, 400 MHz): δ 9.60 (br, 1H), 8.80 (dd, J₁= 4.0 Hz, J₂=

1.6 Hz, 1H), 8.60 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 8.15 (m, 2H),

7.96 (d, J = 8.0 Hz, 1H), 7.87 (td, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.44−

7.29 (m, 7H). HR-ESI-MS calcd for [C₂₀H₁₅ClN₃O₂S]⁺, 396.0568;

found, 396.0563.

N-(5-(3,5-Dimethoxyphenyl)quinolin-8-yl)pyridine-2-sulfo-

namide (55). 5-(3,5-Dimethoxy phenyl)-8-nitroquinoline was

reduced to an amine utilizing reduction method A. The corresponding

amine was then coupled to pyridine-2-sulfonyl chloride utilizing

sulfonamide coupling method A. The product was puriﬁed with a

gradient of 0−100% EtOAc in hexanes and isolated as an oﬀ-white

solid. Yield 0.145 g (56%). ¹H NMR (CDCl₃, 400 MHz): δ 9.58 (br,

1H), 8.77−8.75 (m, 1H), 8.58−8.57 (m, 1H), 8.26 (dd, J₁= 8.0 Hz,

J₂= 1.6 Hz, 1H), 8.12 (dd, J₁= 8.0 Hz, J₂= 0.8 Hz, 1H), 7.94 (dd, J₁

= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.86 (td, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H),

7.41−7.34 (m, 3H), 6.50 (m, 3H), 3.79 (s, 6H). HR-ESI-MS calcd for

[C₂₂H₂₀N₃O₄S]⁺, 422.1169; found, 422.1165.

N-(5-Phenethylquinolin-8-yl)pyridine-2-sulfonamide (56).

8-Nitro-5-(phenylethynyl) quinoline was reduced to an amine

utilizing reduction method A. The corresponding amine was then

coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide coupling

method A. The product was puriﬁed with a gradient of 0−100%

EtOAc in hexanes and isolated as an oﬀ-white solid. Yield 0.045 g

(15%). ¹H NMR (CDCl₃, 400 MHz): δ 9.51 (br, 1H), 8.77 (d, J = 2.4

Hz, 1H), 8.57 (d, J = 3.6 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.07 (d, J

= 8.4 Hz, 1H), 7.82−7.81 (m, 2H), 7.42−7.11 (m, 8H), 3.23 (t, J =

8.4 Hz, 2H), 2.95 (t, J = 8.4 Hz, 2H). HR-ESI-MS calcd for

[C₂₂H₂₀N₃O₂S]⁺, 390.1271; found, 390.1265.

N-(5-(3-Chlorophenyl)quinolin-8-yl)pyridine-2-sulfonamide

(49). 5-(3-Chlorophenyl)-8-nitroquinoline was reduced to an amine

utilizing reduction method B. The corresponding amine was then

coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide coupling

method A. The product was puriﬁed with a gradient of 0−50% EtOAc

1

in hexanes and isolated as a light pink solid. Yield 0.05 g (16%). H

NMR (CDCl₃, 400 MHz): δ 9.60 (br, 1H), 8.82−8.80 (m, 1H), 8.61

(dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 8.17−8.12 (m, 2H), 7.97 (d, J = 8.0

Hz, 1H), 7.87 (td, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.43−7.32 (m, 6H),

7.18 (t, J = 8.0 Hz, 1H). HR-ESI-MS calcd for [C₂₀H₁₅ClN₃O₂S]⁺,

396.0568; found, 396.0565.

N-(5-(4-Fluorophenyl)quinolin-8-yl)pyridine-2-sulfonamide

(50). 5-(4-Fluorophenyl)-8-nitroquinoline was reduced to an amine

utilizing reduction method A. The corresponding amine was then

coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide coupling

method A. The product was puriﬁed with a gradient of 0−45% EtOAc

in hexanes and isolated as a light yellow solid. Yield 0.091 g (36%). ¹H

NMR (CDCl₃, 400 MHz): δ 9.59 (br, 1H), 8.80−8.79 (m, 1H), 8.60

(dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 8.15−8.11 (m, 2H), 7.97 (d, J = 8.0

Hz, 1H), 7.88 (td, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.44−7.31 (m, 5H),

7.17−7.13 (m, 2H). HR-ESI-MS calcd for [C₂₀H₁₅FN₃O₂S]⁺,

380.0864; found, 380.0866.

N-(5-(4-Methoxyphenyl)quinolin-8-yl)pyridine-2-sulfona-

mide (51). 5-(4-Methoxyphenyl)-8-nitroquinoline was reduced to an

amine utilizing reduction method A. The corresponding amine was

then coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide

coupling method A. The product was puriﬁed with a gradient of 0−

50% EtOAc in hexanes and isolated as a white solid. Yield 0.12 g

(43%). ¹H NMR (CDCl₃, 400 MHz): δ 9.60 (br, 1H), 8.80−8.79 (m,

1H), 8.61−8.60 (m, 1H), 8.25 (dd, J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 8.14

(dd, J₁= 4.0 Hz, J₂= 0.8 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.88 (td,

J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.42−7.35 (m, 4H), 6.97−6.91 (m,

3H). HR-ESI-MS calcd for [C₂₁H₁₈N₃O₃S]⁺, 392.1063; found,

392.1061.

N-(5-(3-Methoxyphenyl)quinolin-8-yl)pyridine-2-sulfona-

mide (52). 5-(3-Methoxyphenyl)-8-nitroquinoline was reduced to an

amine utilizing reduction method A. The corresponding amine was

then coupled to pyridine-2-sulfonyl chloride utilizing sulfonamide

coupling method A. The product was puriﬁed with a gradient of 0−

100% EtOAc in hexanes and isolated as a white solid. Yield 0.074 g

(21%). ¹H NMR (CDCl₃, 400 MHz): δ 9.59 (br, 1H), 8.79 (dd, J₁=

4.0 Hz, J₂= 1.6 Hz, 1H), 8.61−8.59 (m, 1H), 8.22 (dd, J₁= 8.0 Hz, J₂

= 1.6 Hz, 1H), 8.13−8.11 (m, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.86 (td,

J₁= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.41−7.29 (m, 5H), 7.01−6.00 (m,

2H). HR-ESI-MS calcd for [C₂₁H₁₈N₃O₃S]⁺, 392.1063; found,

392.1065.

N-(5-(4-Fluorophenethyl)quinolin-8-yl)pyridine-2-sulfona-

mide (57). 5-((4-Fluorophenyl)ethynyl)-8-nitroquinoline was re-

duced to an amine utilizing reduction method A. The corresponding

amine was then coupled to pyridine-2-sulfonyl chloride utilizing

sulfonamide coupling method A. The product was puriﬁed with a

gradient of 0−100% EtOAc in hexanes and isolated as an oﬀ-white

1

solid. Yield 0.07 g (31%). H NMR (CDCl₃, 400 MHz): δ 9.50 (br,

1H), 8.75 (d, J = 2.4 Hz, 1H), 8.56 (d, J = 3.6 Hz, 1H), 8.24 (d, J =

8.4 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.83−7.78 (m, 2H), 7.43−7.35

(m, 2H), 7.17 (d, J = 8.4 Hz, 1H), 7.03−7.00 (m, 2H), 6.92 (m, 2H),

3.21 (t, J = 8.4 Hz, 2H), 2.92 (t, J = 8.4 Hz, 2H). HR-ESI-MS calcd

for [C₂₂H₁₈FN₃NaO₂S]⁺, 430.0996; found: 430.0994.

6-Bromo-8-nitroquinoline. A solution of 4-bromo-2-nitroaniline

(5 g, 23.04 mmol) in toluene (30 mL) and 6 M HCl (30 mL) was

heated to reﬂux for ∼15 min. To this was added acrolein (1.93 g, 34.6

mmol) and allowed to heat at reﬂux for an additional 2 h. The

resulting solution was then partitioned, and the organic layer was

discarded. The aqueous layer was made strongly basic (pH 10−11)

with 6 M NaOH. The resulting basic mixture was then extracted with

EtOAc (3 × 50 mL). The combined organic layers were dried over

MgSO₄, ﬁltered, concentrated then puriﬁed via silica gel chromatog-

raphy eluting 0−40% EtOAc in hexanes. The desired product was

isolated as a pink-orange solid. Yield 1.21 g (21%). ¹H NMR (CDCl₃,

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400 MHz): δ 9.08 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.20−8.18 (m,

2H), 8.13 (d, J₁= 2.4 Hz, 1H), 7.60 (dd, J₁= 8.4 Hz, J₂= 4 Hz, 1H).

ESI-MS(+): m/z 253.19 [M + H]⁺.

8-Nitro-6-(phenylethynyl)quinoline. It is synthesized utilizing

the Sonogashira coupling method. The product was puriﬁed with a

gradient of 0−100% EtOAc in hexanes and isolated as an oﬀ-white

solid. Yield 0.12 g (40%). ¹H NMR (CDCl₃, 400 MHz): δ 9.00 (dd, J₁

= 4.4 Hz, J₂= 1.6 Hz, 1H), 8.20−8.09 (m, 3H), 7.56−7.51 (m, 3H),

7.38−7.36 (m, 3H). ESI-MS(+): m/z 275.21 [M + H]⁺.

6-((4-Fluorophenyl)ethynyl)-8-nitroquinoline. It is synthe-

sized utilizing the Sonogashira coupling method. The product was

puriﬁed with a gradient of 0−100% EtOAc in hexanes and isolated as

an oﬀ-white solid. Yield 0.15 g (50%). ¹H NMR (CDCl₃, 400 MHz):

δ 9.11 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.33−8.03 (m, 3H), 7.80−

7.61 (m, 2H), 7.37−7.36 (m, 3H). ESI-MS(+): m/z 293.11 [M +

H]⁺.

addition of the substrate. The substrate (150 μL) was then added to

the wells yielding a maximum amount of 5% DMSO per well. The

enzyme activity was measured utilizing a Biotek Synergy HT or H4

plate reader by measuring absorbance at 240 nm every 1 min for a

duration of 8 min. The rate of absorbance increase was compared for

samples versus controls containing no inhibitor (set at 100% activity).

The absorbance reading for background wells containing DMSO,

buﬀer, and substrate (no enzyme or inhibitor) was subtracted from

the experimental wells. All IC₅₀values were benchmarked against a

positive control compound Chugai-1 (1-hydroxy-4,6-diphenylpyridin-

2(1H)-one). Chugai-1 was prepared and evaluated under the assay

conditions described above and gave an IC₅₀value of 1.9 0.2 μM,

which is comparable to the value reported in the original literature

report (1.19 μM).²⁸

Molecular Modeling. Modeling studies were performed using the

Molecular Operating Environment (MOE) 2014 software suite. The

glyoxalase crystal structure titled “Human Glyoxalase I with an N-

hydroxypyridone inhibitor” with PDB ID 3VW9 was obtained from the

Protein Data Bank.²⁸The PDB ID 3VW9 crystal structure was

utilized because the Chugai-3d inhibitor utilizes bidentate metal

binding to the GLO1 active site Zn(II) ion, which is also the

anticipated binding mode for 8-MSQ and its derivatives. In addition,

Chugai-3d has several substituents that result in tight binding to

GLO1, which can be used as a guide for designing suitable

substituents for appending to 8-MSQ. A binding model for 8-MSQ

and its analogs was obtained through visual positioning of the

fragment into the GLO1 active site and superimposing the

coordinating atoms (N,N) of 8-MSQ with those of the bound

Chugai-3d inhibitor (O,O). The bound Chugai-3d inhibitor was then

removed from the structure and replaced with 8-MSQ (or the

appropriate 8-MSQ derivative, Figures S1 −S4), which then utilized

the position of the metal-coordinating donor atoms of the Chugai-3d

inhibitor as an initial guide for the binding mode of 8-MSQ and its

derivatives. Once 8-MSQ was modeled in, the “builder” tool of MOE

was utilized to introduce substituents to the bound 8-MSQ fragment.

Substituents were built onto the bound 8-MSQ fragment and

inspected via the “contacts/clash” visualizing tool of MOE. The

substituents were freely rotated and manually moved to generate

interactions while avoiding clashes as predicted by MOE. By building

in functional groups to 8-MSQ and visually inspecting the protein

active site, it allowed for a hypothesis to be generated and predictions

for which substituents were sterically tolerated as well as which

substituents could form interactions within the protein active site. The

protein structure was not minimized or altered in any way during

these modeling exercises. PDB ﬁles for all modeled compounds have

been provided as part of the Supporting Information.

Animal Subjects. Male and female FVB/NJ mice (n = 21) were

bred in house at U.C. San Diego and were housed 2−5 per cage on

Sani-Chip bedding with food (Envigo 8604; Indianapolis, IN) and

water was given ad libitum. All animals were between 108 and 148

days old at the start of testing. Mice were housed on a 12 h/12 h

light−dark cycle with lights on at 06:00, and all behavioral testing

occurred during the light phase. All procedures were approved by the

local Institutional Animal Care and Use Committee and were

conducted in accordance with the NIH Guidelines for the Care and

Use of Laboratory Animals.

Forced Swim Test. The FST procedure was based on protocols

used previously by our laboratory and others to test GLO1 inhibitors

and classical antidepressants for their ability to reduce depression-like

behavior.^41,46Brieﬂy, mice were placed into white opaque plastic

buckets (61 cm high, 48 cm diameter) ﬁlled 48 cm high with 23−25

tap water for 6 min. Test sessions were videotaped by a camera

positioned directly above swim buckets. An experimenter blind to

treatment scored the last 4 min of each video for time spent immobile,

which is a measure of depression-like behavior and is reduced by

antidepressant treatment.

6-((3,5-Dimethoxyphenyl)ethynyl)-8-nitroquinoline. It is

synthesized utilizing the Sonogashira coupling method. The product

was puriﬁed with a gradient of 0−100% EtOAc in hexanes and

1

isolated as a tan solid. Yield 0.29 g (73%). H NMR (CDCl₃, 400

MHz): δ 9.07 (dd, J₁= 4.0 Hz, J₂= 1.6 Hz, 1H), 8.24 (dd, J₁= 4.0 Hz,

J₂= 1.6 Hz, 1H), 8.19−8.15 (m, 2H), 7.60 (dd, J₁= 8.4 Hz, J₂= 4.4

Hz, 1H), 6.73 (d, J = 2.4 Hz), 6.53 (t, J = 2.4 Hz), 3.82 (s, 6H). ESI-

MS(+): m/z 335.33 [M + H]⁺.

N-(6-Phenethylquinolin-8-yl)pyridine-2-sulfonamide (58).

8-Nitro-6-(phenylethynyl)quinoline was reduced to an amine utilizing

reduction method A. The corresponding amine was then coupled to

pyridine-2-sulfonyl chloride utilizing sulfonamide coupling method A.

The product was puriﬁed with a gradient of 0−100% EtOAc in

1

hexanes and isolated as an oﬀ-white solid. Yield 0.065 g (35%). H

NMR (CDCl₃, 400 MHz): δ 9.48 (br, 1H), 8.71 (d, J = 4.4 Hz, 1H),

8.56 (d, J = 4.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz,

1H), 7.85−7.77 (m, 2H), 7.37−7.34 (m, 2H), 7.24−7.11 (m, 7H),

3.05 (t, J = 8.4 Hz, 2H), 2.96 (t, J = 8.4 Hz, 2H). HR-ESI-MS calcd

for [C₂₂H₂₀N₃O₂S]⁺, 390.1271; found, 390.1268.

N-(6-(4-Fluorophenethyl)quinolin-8-yl)pyridine-2-sulfona-

mide (59). 6-((4-Fluorophenyl)ethynyl)-8-nitroquinoline was re-

duced to an amine utilizing reduction method A. The corresponding

amine was then coupled to pyridine-2-sulfonyl chloride utilizing

sulfonamide coupling method A. The product was puriﬁed with a

gradient of 0−100% EtOAc in hexanes and isolated as an oﬀ-white

solid. Yield 0.075 g (36%). ¹H NMR (CDCl₃, 400 MHz): δ 9.45 (br,

1H), 8.72 (d, J = 4.4 Hz, 1H), 8.56 (d, J = 4.4 Hz, 1H), 8.05 (d, J =

8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.82−7.78 (m, 2H), 7.39−7.36

(m, 2H), 7.13 (s, 1H), 7.04−7.01 (m, 2H), 6.91−6.87 (m, 2H), 3.02

(t, J = 8.4 Hz, 2H), 2.94 (t, J = 8.4 Hz, 2H). HR-ESI-MS calcd for

[C₂₂H₁₈FN₃NaO₂S]⁺, 430.0996; found, 430.0994.

N-(6-(3,5-Dimethoxyphenethyl)quinolin-8-yl)pyridine-2-

sulfonamide (60). 6-((3,5-Dimethoxyphenyl)ethynyl)-8-nitroquino-

line was reduced to an amine utilizing reduction method A. The

corresponding amine was then coupled to pyridine-2-sulfonyl chloride

utilizing sulfonamide coupling method A. The product was puriﬁed

with a gradient of 0−100% EtOAc in hexanes and isolated as an oﬀ-

1

white solid. Yield 0.165 g (45%). H NMR (CDCl₃, 400 MHz): δ

9.50 (br, 1H), 8.73 (d, J = 4.4 Hz, 1H), 8.57 (d, J = 4.4 Hz, 1H), 8.07

(d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.87 (s, 1H), 7.83 (td, J₁

= 8.0 Hz, J₂= 1.6 Hz, 1H), 7.41−7.35 (m, 2H), 7.21 (s, 1H), 6.32 (s,

3H), 3.74 (s, 6H), 3.06 (t, J = 8.4 Hz, 2H), 2.92 (t, J = 8.4 Hz, 2H).

HR-ESI-MS calcd for [C₂₄H₂₄N₃O₄S]⁺, 450.1482; found, 450.1483.

Human GLO1 Assay. Recombinant human glyoxalase I (GLO1)

was purchased from R&D Systems (Catalog #4959-GL). Assays were

carried out in 100 mM sodium phosphate, pH 7.0 buﬀer, utilizing 96-

well Clear UV Plate (Corning UV Transparent Microplates, Catalog

#3635). A fresh solution of GSH (100 mM) and MG (100 mM) was

prepared in Millipore grade water. The substrate for the assay was

prepared by adding 0.99 mL of GSH and 0.99 mL of MG to 14.5 mL

of buﬀer. The substrate mixture was vortexed vigorously for 15 s and

then allowed to sit at room temperature for 20 min. The initial well

volume was 50 μL containing GLO1 (40 ng) and inhibitor. This

protein and inhibitor mixture was incubated for 15−20 min prior to

Open Field Test. Locomotor behavior was measured using an

open ﬁeld test, which has been described previously.⁴⁷Brieﬂy, mice

were placed in the center of a square chamber (43 × 43 × 33 cm,

Accuscan, Columbus, OH) with dim overhead lighting inside of

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DOI: 10.1021/acs.jmedchem.8b01868

J. Med. Chem. 2019, 62, 1609−1625

Journal of Medicinal Chemistry

Article

sound- and light-attenuating boxes and allowed to freely explore for

30 min. A grid of infrared detection beams in each chamber and

Versamax software was used to track animal location and locomotor

activity (distance traveled) during the test.

interest, and a member of the Scientiﬁc Advisory Board for

Forge Therapeutics. Both companies may potentially beneﬁt

from the research results of certain projects in the laboratory of

S.M.C. The terms of this arrangement have been reviewed and

approved by the University of California, San Diego in

accordance with its conﬂict of interest policies.

Determination of 60 and MG in Mouse Brain Tissue. Mice

were anesthetized with isoﬂurance, the chest cavity was opened to

expose the pulsating heart, and a 25 gauge butterﬂy needle was

attached to a 25 mL syringe ﬁlled with phosphate buﬀered saline

(PBS) inserted into the left ventricle. The right atrium was then cut to

allow outﬂow of systemic blood, and then PBS was slowly infused into

the heart to purge residual systemic blood over approximately 20 min.

When liver and kidneys were pale in color, the brain was removed,

weighed, placed in a polypropylene tube, ﬂash frozen in liquid

nitrogen, and then stored at −80 °C. For quantitative analysis of each

probe compound in the brain, whole mouse brains were minced (∼20

mg per piece), then extracted with acetonitrile, homogenized using a

bead beater, and centrifuged at 5000 rpm for 5 min. The supernatant

was collected and ﬁltered through a 2 μm ﬁlter to remove any residual

particulate matter. Determination of 60 and MG in brain extracts was

performed with liquid chromatography-tandem mass spectrometry

(LC-MS/MS). Eleven-point calibration curves, ranging between 5

and 5000 ng/mL, were constructed for each analyte by spiking a

known amount of each compound into a blank matrix (untreated

mouse brain) and used as the basis to determine unknown

concentrations. Results were normalized to wet weight of brain tissue

(i.e., ng/mg of brain tissue).

ACKNOWLEDGMENTS

■

The authors acknowledge Dr. Yongxuan Su (U.C. San Diego,

Molecular Mass Spectrometry Facility) for aid with HR-MS

and HPLC compound purity analysis. This work was

supported by grants from the National Institutes of Health

(R01 GM098435 to S.M.C.; F32AA025515 to A.M.B.-L.).

B.L.D. was supported by the National Institute of Health

Molecular Biophysics Training Grant (T32GM008326-26).

ABBREVIATIONS

■

FBDD, fragment-based drug discovery; FST, forced swim test;

GLO1, glyoxalase 1; GLO2, glyoxalase 2; GSH, glutathione;

MBP, metal-binding pharmacophore; MG, methylglyoxal;

MSQ-8, 8-(methylsulfonylamino)quinoline

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ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b01868.

■

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Zn²⁺ion the GLO1 active site and chemical illustration;

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SMILES strings formula for all compounds (PDF)

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PDB ﬁle of 8-MSQ modeled in GLO1 (PDB)

PDB ﬁle of compound 8 modeled in GLO1 (PDB)

PDB ﬁle of compound 23 modeled in GLO1 (PDB)

PDB ﬁle of compound 47 modeled in GLO1 (PDB)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: aapalmer@ucsd.edu, aap@ucsd.edu. Phone: 858-534-

2093 (A.A.P.).

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Seth M. Cohen: 0000-0002-5233-2280

Notes

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and receives income as member of the Scientiﬁc Advisory

Board for Cleave Biosciences and is a co-founder, has an equity

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Metal-Binding Pharmacophore Library Yields the Discovery of a Glyoxalase 1 Inhibitor

DOI: 10.1021/acs.jmedchem.8b01868

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