3-substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: Structure-based design, synthesis, SAR, and biological evaluation

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Open Access on 04/21/2015

3‑Substituted‑N‑(4-Hydroxynaphthalen-1-yl)arylsulfonamides as a

Novel Class of Selective Mcl‑1 Inhibitors: Structure-Based Design,

Synthesis, SAR, and Biological Evaluation

Fardokht A. Abulwerdi,^†,‡Chenzhong Liao,^†,#,⊥Ahmed S. Mady,^†,‡,#Jordan Gavin,^†Chenxi Shen,^†

Tomasz Cierpicki,^†Jeanne A. Stuckey,^§H. D. Hollis Showalter,^∥and Zaneta Nikolovska-Coleska*^,†,‡

^†Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States

§

∥

^‡Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, Life Sciences Institute, and Vahlteich Medicinal

Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States

S

* Supporting Information

ABSTRACT: Mcl-1, an antiapoptotic member of the Bcl-2

family of proteins, is a validated and attractive target for cancer

therapy. Overexpression of Mcl-1 in many cancers results in

disease progression and resistance to current chemotherapeu-

tics. Utilizing high-throughput screening, compound 1 was

identiﬁed as a selective Mcl-1 inhibitor and its binding to the

BH3 binding groove of Mcl-1 was conﬁrmed by several

diﬀerent, but complementary, biochemical and biophysical

assays. Guided by structure-based drug design and supported

by NMR experiments, comprehensive SAR studies were

undertaken and a potent and selective inhibitor, compound

21, was designed which binds to Mcl-1 with a K_iof 180 nM.

Biological characterization of 21 showed that it disrupts the interaction of endogenous Mcl-1 and biotinylated Noxa-BH3

peptide, causes cell death through a Bak/Bax-dependent mechanism, and selectively sensitizes Eμ-myc lymphomas

overexpressing Mcl-1, but not Eμ-myc lymphoma cells overexpressing Bcl-2. Treatment of human leukemic cell lines with

compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis.

Overexpression of Bcl-2 survival members is observed in

diﬀerent types of human tumor samples and cancer cell lines,

and much eﬀort has been focused on developing therapeutics

against this family of proteins for the treatment of cancers.⁵⁻⁷

Selective and potent small-molecule inhibitors have been

INTRODUCTION

■

Evasion of apoptosis or programmed cell death, a key regulator

of physiological growth control and regulation of tissue

homeostasis, is a hallmark of cancer and a contributor to the

emergence of resistance to current therapies.¹⁻³The B-cell

lymphoma-2 (Bcl-2) family of proteins regulate the intrinsic

(mitochondrial) pathway of apoptosis through a network of

protein−protein interactions between pro- and antiapoptotic

members.⁴Twenty-ﬁve known members of the family can be

grouped functionally according to their pro- and antiapoptotic

eﬀects as well as structurally according to their Bcl-2 homology

(BH) regions. The Bcl-2 antiapoptotic proteins, consisting of

Bcl-2, Bcl-X_L, Bcl-b, Bcl-w, Mcl-1, and A1, share up to four BH

domains which form the hydrophobic BH3-binding groove for

binding their cognate partners. The pro-apoptotic proteins are

divided into two groups: (a) multidomain proteins including

Bax and Bak with BH1−BH4 domains and (b) BH3-only

proteins including Bad, Bid, Bim, Noxa, and Puma, which share

homology only in the BH3 α-helical domain. The BH3 domain

possesses four conserved hydrophobic residues that are

involved in the interaction with BH3-binding groove of the

pro-survival Bcl-2 family members, which results in the

sequestering and blocking of the function of pro-death

members.

successfully developed against Bcl-2,⁸Bcl-X_L,⁹and Bcl-2/Bcl-

10−13

X_L

with Navitoclax (ABT-263), currently in phase I/II

clinical trials. The eﬃcacy of ABT-263 as a single agent has

been demonstrated in tumors with low levels of the pro-survival

Mcl-1. Several studies¹⁴⁻¹⁸have shown that resistance to ABT-

263 and its analogue ABT-737 is linked to high expression

levels of Mcl-1, and in many instances this resistance can be

overcome by treatment with agents that downregulate,

destabilize, or inactivate Mcl-1. The biological signiﬁcance of

Mcl-1 protein expression in support of cell survival has been

well documented in a number of cell systems, including human

myeloblastic leukemia,¹⁹myeloma,²⁰⁻²²B-lymphoma,²³non-

small cell lung,²⁴melanoma,²⁵pancreatic,^26,27and prostate.²⁸

Furthermore, Mcl-1 is overexpressed in many human tumor

specimens^26,29−32and metastatic tissue.³³This overexpression

contributes to chemoresistance and disease relapse.³⁴⁻³⁶It has

Received: January 3, 2014

Published: April 21, 2014

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Figure 1. (A) Structure of the HTS lead compound 1. (B) Putative binding mode of 1 to Mcl-1(PDB ID: 2NLA). Side chains residues of mNoxa

peptide are shown in blue sticks. The surface of Mcl-1 protein is colored according to the chemical shift intensity. Signiﬁcant shift (>0.09 ppm) is

represented with purple, moderate shift (≥0.03 and ≤0.09 ppm) represented with pink. (C) Overlaid ¹⁵N−¹H HSQC spectra of Mcl-1 (red) and in

the presence of 1 (Mcl-1:1 ratio of 1:2) (black), (Mcl-1:1 ratio of 1:1) (purple). Arrows show the direction of chemical shift changes upon binding

of 1. (D) Plot of chemical shift changes of Mcl-1 amide upon addition of 1 (Mcl-1:1 ratio of 1:2) as a function of Mcl-1 residue numbers.

been shown that Mcl-1 down-regulation is important toward

making multiple myeloma cells susceptible to BH3-only

proteins and therefore to mitochondrial disruption.^37,38Such

down-regulation can increase the sensitivity to rituximab-

mediated killing of chronic and acute lymphoid leukemia (CLL

and ALL).³⁹Antisense strategies targeting Mcl-1 in vitro and in

vivo have provided promising results in sensitizing human

melanoma and pancreatic cancer.⁴⁰⁻⁴²These data suggest that

therapies speciﬁcally targeting Mcl-1, either as a single agent or

in combination, can be eﬀective in the treatment of diﬀerent

human cancers.

Recently, several groups including us have reported on small

molecules⁴³⁻⁵⁰and stapled peptides^51,52as Mcl-1 inhibitors.

Herein we disclose a novel series of small-molecule Mcl-1

inhibitors discovered through high-throughput screening

(HTS) followed by the utilization of structure-based design

to develop structure−activity relationships (SAR) around lead

compound 1 (Figure 1A). Docking studies and two-dimen-

sional ¹H− ¹⁵N heteronuclear single quantum coherence

spectroscopy (HSQC) NMR studies were employed to provide

information about its binding mode which was used for the

design and synthesis of additional analogues. A SAR was

developed which resulted in the identiﬁcation of several

selective small-molecule Mcl-1 inhibitors with improved

binding. Selected analogues were then tested in a series of

complementary biochemical, biophysical, functional, and

cellular assays to evaluate their potency, speciﬁcity, and

mechanism of action.

RESULTS AND DISCUSSION

■

Lead Discovery. HTS of a 53.3K small-molecule library

was performed using a ﬂuorescence polarization (FP) binding

assay based on the interaction between recombinant human

Mcl-1 and ﬂuorescently labeled Bid BH3 peptide (Flu-Bid).

Compound 1 (Figure 1A) was one of the validated hits, which

was also previously identiﬁed as a proteasome inhibitor.⁵³

Compound 1 was resynthesized and its binding to Mcl-1 was

conﬁrmed with a K_iof 1.55 0.18 μM. Compound 1 exhibits

similar potency as MIM1 (IC₅₀= 4.72 μM), Mcl-1 small-

molecule inhibitor with a thiazolyl substituted core, which was

also discovered with high-throughput competitive FP screen

approach.⁴⁷It is known that the binding eﬃciency index (BEI)

is important in identifying leads that exhibit good potency

relative to their size.⁵⁴The BEI of compound 1, calculated as a

ratio between pK_iand molecular weight, is 14.7, which

encouraged us to further pursue with modiﬁcations of this

scaﬀold. To explore the binding mode of 1 with Mcl-1, in silico

induced ﬁt docking (IFD)⁵⁵studies were performed using the

crystal structure of Mcl-1 in complex with mNOXA BH3

peptide (PDB ID: 2NLA).⁵⁶The predicted binding model of 1

in complex with Mcl-1 revealed that the thiophene and the

naphthalene rings of 1 occupy two hydrophobic pockets, h2

and h3, in the Mcl-1 protein, mimicking two conserved

hydrophobic residues in the BH3 binding motif represented by

Leu 78 and Ile 81, respectively, in mNoxaB (Figure 1B). The

importance of the two conserved hydrophobic residues, Leu

and Ile, in the BH3 binding motif for the high aﬃnity

interaction and selective binding to Mcl-1 has been

demonstrated with structural studies of Bims2A,⁵⁷a highly

selective peptide derived from Bim BH3-only protein, as well as

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a

Scheme 1. Synthetic Route for 1 and Analogues

a

Reagents and conditions: (a) NIS, TFA, reﬂux, 24 h; (b) HS(CH₂)_nCOOCH₃(n = 1, 2), Pd(OAc)₂, Xantphos, Cs₂CO₃, LiI, ZnCl₂, THF, 60 °C,

overnight, or HS(CH₂)₃CH₃, Pd₂(dba)₃, Dppf, Et₃N, NMP, 80 °C, 2 h, or HCC(CH₂)_nOH (n = 1, 2), Pd(PPh₃)₂Cl₂, CuI, Et₃N/THF, 60 °C, 2 h

(4:1), 60 °C, 2 h; (c) Fe, AcOH, 70 °C, 1 h, or Pd/C, H₂30 psi, EtOH/EtOAc (6:1), rt, overnight; (d) RSO₂Cl, pyridine, CH₂Cl₂, rt, overnight, or

RCOCl, Et₃N, CH₂Cl₂, rt, overnight, or

, CH₃CN, 80 °C, 15 h; (e) BBr₃, CH₂Cl₂, 0 °C to rt, 1 h, or BBr₃, CH₂Cl₂, 0 °C to rt, 1

h, quench with MeOH at 0 °C; (g) phenyl boronic acid, Pd(PPh₃)₄, Na₂CO₃, THF/H₂O, 60 °C, 2 h; (h) NH₄OH, rt, 1 h; (i) NaN₃, SiCl₄, CH₃CN,

80 °C, 15 h; (j) LiOH, THF, rt, 1 h; (k) H₃CCOCl, Et₃N, 0 °C, rt, 30 min.

from recently reported selective small-molecule inhibitor in

complex with Mcl-1.⁴⁵The carboxylic acid group of 1 forms a

network of hydrogen bonds with Arg 263 and Asn 260 of Mcl-

1, mimicking the conserved Asp in BH3 peptides (Asp 83 in

mNoxa). The predicted binding model also suggests that the

phenolic group forms a hydrogen bond with His 224, which is

one of the residues composing the h3 pocket of Mcl-1. A recent

report⁵⁸on the conformational ﬂexibility of Mcl-1 and its

binding hotspots identiﬁed His 224 as an acidic hotspot in the

h3 site of Mcl-1, supporting the predicted hydrogen bonding in

this region of Mcl-1.

To validate the computationally predicted binding site and

conﬁrm the binding of 1 to the BH3 groove of Mcl-1 protein,

HSQC NMR spectroscopy studies were performed. For this

purpose, the assignment of the backbone amides of apo human

Mcl-1 was based on the work by Liu et al.,⁵⁹and a series of

¹H,¹⁵N-HSQC studies were carried out. The HSQC spectra

were of good quality with well-dispersed peaks, and

concentration-dependent perturbations of residues were

observed (Figure 1C), indicating that 1 binds Mcl-1 speciﬁcally

and causes dose-dependent perturbations of backbone amides.

The chemical shift changes in the presence of a 2-fold excess of

1 were mapped and plotted against Mcl-1 residues (Figure 1D).

Compound 1 caused moderate to signiﬁcant chemical shift

perturbations for residues of Met 231, Met 250, Leu 267, and

Phe 270, which constitute the h2 and h3 pockets and which are

predicted to be occupied by the thiophene and naphthalene

rings of 1, respectively. Moderate chemical shift perturbations

of Arg 263 and His 224 were also observed, which are predicted

to form hydrogen bonds with the thioacidic acid and phenolic

moieties of 1, respectively. Additionally, the residues in the

vicinity of the predicted binding pose (Leu 232, Val 243, Arg

248) and the ones located on an unstructured loop connecting

α-helix 3 to α-helix 4 (Lys 234, Leu 235, Lys 238, Asn 239)

were also perturbed. Overall analysis of the chemical shift

changes of the compound 1 in complex with Mcl-1 showed that

1 aﬀects the residues forming the BH3-binding groove and

provided conclusive evidence that 1 binds Mcl-1 protein at the

same site that the conserved BH3 peptides interact with Mcl-1

protein. Therefore, on the basis of our modeling and NMR

results, the substituted-N-(4-hydroxynaphthalen-1-yl)-

arylsulfonamide represents a promising class for further

optimization. A structure-based design approach was under-

taken, and a focused library of analogues of 1 was designed and

synthesized to improve the potency of this series.

Synthesis. Except for analogues 4−8, 15, and 39, which

were commercially available, all analogues were synthesized

through a novel, modular route (Scheme 1), which diﬀers from

that recently reported by Ge et al.⁵³Our route allows for facile

access to a variety of analogues with variations at R₁, R₂, and R₃

and the linker region (X). It starts with an electrophilic

aromatic substitution of 1-methoxy-4-nitronaphthalene with N-

iodosuccinamide to provide aryl iodide (47).⁶⁰This was

subjected to Pd-catalyzed C−S or C−C cross-coupling using

conditions previously reported,^61,62or developed in our lab

based on recent literature,⁶³⁻⁶⁵to provide several desired

intermediates (48). The nitro function was reduced with iron⁶⁶

or via catalytic hydrogenation⁶⁷to provide the corresponding

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amines. Reaction of the amines with appropriate sulfonyl or

acyl chlorides or 3-methyl-1-((4-phenylpiperazin-1-yl)-

sulfonyl)-1H-imidazol-3-ium⁶⁸provided the penultimate com-

pounds (49), which were demethylated with BBr₃followed by

puriﬁcation by trituration or reverse-phase HPLC to aﬀord the

target compounds (1−3, 9−14, 16−23, 25−26, 28−29, 31−

38) of >95% purity (Tables 1−3). In the case of analogues with

an ester side chain, the BBr₃step provided a convenient way to

concomitantly hydrolyze the ester in a single pot. To preserve

the methyl ester of analogue 32 (Table 3), analogue 41 (Table

4) was subjected to BBr₃conditions followed by a quench with

MeOH. For the synthesis of 49o, intermediate 49k underwent

Suzuki−Miyaura coupling^69,70with phenyl boronic acid to

provide the desired compound which was subjected to BBr₃to

provide 17 (Table 1). Aminolysis of 41 and 49s with NH₄OH⁷¹

provided carboxamides 42 (Table 4) and 49aa, respectively. 42

was then subjected to BBr₃to give 33 (Table 3). The terminal

amide of 42 and 49aa were converted by a known procedure⁷²

to tetrazoles 49cc and 49dd, which after BBr₃demethylation

provided 36 and 37 (Table 3), respectively. The thioacidic acid

analogues with a methoxy at R₃(40, 43−44) (Table 4) were

obtained via LiOH hydrolysis of esters 41, 49n, and 49o,

respectively. Acetylation of 10 (Table 1) with acetyl chloride⁷³

provided 46 (Table 4). Analogue 24 (Table 1) with a phenyl

scaﬀold in place of naphthalene was synthesized using

conditions similar to those described for 10 starting from 2-

iodoanisole (Supporting Information Scheme S1). The

syntheses of compounds 27 (Table 2) and 30 (Table 3)

were completed starting from 4-methoxy-1-naphthaldehyde

and 1-nitronaphthalene, respectively (Supporting Information

Schemes S2−S3).

Table 1. Binding Aﬃnities of Sulfonamide Analogues with

Variations at R₁

Structure−Activity Relationships. Structure-based design

of analogues based on 1 yielded a focused library of compounds

leading to clear SAR for this series. The binding aﬃnities of our

Mcl-1 inhibitors were determined by using competitive

ﬂuorescence polarization (FP) and surface plasmon resonance

(SPR) binding assays, which test the ability of inhibitors to

disrupt interaction between Mcl-1 and two diﬀerent BH3

peptides, ﬂuorescently labeled Bid and biotin-labeled Bim,

respectively. Concurrently, HSQC NMR experiments were

performed to provide structural insights of protein-bound

ligand and experimental validation for the modeling studies.

The predicted binding model showed that the thiophene ring

at R₁of 1 projects into the h2 pocket (Figure 1B), which is the

biggest and deepest pocket among the four hydrophobic

pockets of Mcl-1.⁵⁶To investigate the importance of hydro-

phobic interaction at this site and increase the binding aﬃnity

of 1, a series of analogues with variation at R₁was synthesized

and evaluated (Table 1). When R₁is changed to a methyl group

in 2, the binding aﬃnity is signiﬁcantly reduced, conﬁrmed by

SPR (IC₅₀> 100 μM) and NMR experiments which showed

lack of chemical shift perturbation of backbone residues in the

Mcl-1 BH3 binding site after adding 2 (Supporting Information

Figure S1). As was expected, isosteric replacement of the

thiophene in 1 to a phenyl in 3 maintained binding aﬃnity with

K_iof 3.56 0.45 μM. To probe the hydrophobic interactions in

the h2 pocket, analogues with alkyl and halogen substituents of

various sizes at the para-position of the phenyl ring were

prepared. From this set of compounds, a trend emerged

showing improved binding with increasing size and hydro-

phobicity of substituents. tert-Butyl phenyl (7) exhibited 4.5-

fold enhancement in FP assay (K_i= 0.81 0.04 μM) and 1.5-

fold in SPR assay (IC₅₀= 8.73 0.95 μM), and bromo phenyl

(10) 7-fold in FP assay (K_i= 0.49 0.06 μM) and 5-fold in

SPR assay (IC₅₀= 2.40 0.17 μM) improved binding over 3.

Introduction of a more polar methoxy group in 11 was

accommodated but resulted in a 3-fold decreased binding (K_i=

9.31 3.1 μM) compared to 3. Bromine substitution at the

meta-(12) and ortho-positions (13) of the phenyl ring was also

explored, and the obtained binding results from both assays, FP

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Figure 2. Putative binding modes of (A) 16, (B) 17, (C) 18 to Mcl-1 (PDB ID: 2NLA). Surface of Mcl-1 protein is colored according to the

chemical shift intensity. Signiﬁcant shift (>0.09 ppm) is represented with purple, moderate shift (≥0.03 and ≤0.09 ppm) represented with pink. Plots

of chemical shift changes of Mcl-1 amide upon addition of (D) 16 (Mcl-1:16 ratio of 1:2), (E) 17 (Mcl-1:17 ratio of 1:2), (F), and 18 (Mcl-1:18

ratio of 1:2) as a function of Mcl-1 residue numbers.

(K_iof 2.35 0.19 μM and 4.42 0.86 μM, respectively) and

SPR (IC₅₀= 9.68 0.88 μM and IC₅₀= 6.18 1.24 μM,

To further extend into the h2 pocket and gain additional

interaction, analogues with biphenyl substituents were synthe-

sized because biphenyl is considered a privileged moiety in

targeting protein−protein interactions.⁷⁴Analogues 16 and 17,

with para- and meta-biphenyl substituents, respectively, showed

a similar 10-fold improvement in FP-based binding assay

compared to 3 with K_ivalues of 0.37 0.10 and 0.38 0.03

μM, respectively, further conﬁrmed with SPR assay showing 6-

fold improvement. On the other hand, the ortho-biphenyl

analogue 18 showed almost the same binding aﬃnity as 3 with

K_iof 2.03 0.41 μM and IC₅₀= 9.90 3.50 μM, in FP and

SPR assays, respectively. The predicted binding models of these

compounds showed that the distal phenyl ring of 16 and 17

inserts deeper into the h2 pocket (parts A and B of Figure 2,

respectively). For 18, this phenyl ring is partially solvent

exposed (Figure 2C), which might explain its lower aﬃnity

compared to 16 and 17. In agreement with their binding

aﬃnities, chemical shift perturbation plots of 16 and 17 show

stronger perturbation of residues involved in the binding site

compared to 18 (Figure 2D−F).

respectively), indicated that para-bromo substitution in 10 is

the best, exhibiting the highest binding aﬃnity to Mcl-1 among

these three isomers. Because of peak overlap and possibility of

ambiguous assignment of the peaks in the presence of a ligand

and to improve the quality and accuracy of ligand-induced Mcl-

1 chemical shift perturbation, resonance assignments were also

determined for 10 in complex with Mcl-1 using ¹³C,¹⁵N double-

labeled Mcl-1 protein in the presence of a 2-fold excess of 10.

The HSQC spectrum of 10 showed a similar pattern of

chemical shift perturbation as with 1, which we attribute to the

structural similarity of the two analogues. Consistent with the

7-fold improved binding of 10 in comparison with 1, the

chemical shift perturbations were larger for 10 (Supporting

Information Figure S2A). In particular, signiﬁcant perturbation

of residues Ser 247, Arg 248, and Val 253 on α-helix 4, which

forms the upper rim of the h2 pocket, and residues Val 216, Val

220, and Gln 221 located toward the C-terminus of α-helix 2,

which are at the border between the h3 and h4 pockets, were

observed for 10, suggesting that the Mcl-1 conformational

ﬂexibility accommodates larger R₁substituent into the h2

pocket. Chemical shift perturbation plots derived from ¹H,¹⁵N-

HSQC experiments of the other two bromophenyl isomers

showed a similar perturbation pattern as 10 with chemical shift

changes that correlate well with the binding data in which the

strongest perturbation is observed for 10, followed by 12 and

13 (Supporting Information Figure S2A−C).

Analysis of the HSQC spectra of analogues 16−18 resulted

in an important ﬁnding that strongly supports their predicted

binding mode. One long-standing question for us was the

possibility of a ﬂipped binding mode for this class of analogues

where R₁would occupy the h4 pocket instead of h2. The

possibility of a ﬂipped conformation has been previously

documented for a diﬀerent class of dual Mcl-1 and Bcl-xL

inhibitors.⁴⁹The diﬀerential chemical shift mapping meth-

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Figure 3. Overlaid ¹⁵N−¹H HSQC spectra of Mcl-1 (red) and in the presence of 16 (Mcl-1:16 ratio of 1:2) (purple), 17 (Mcl-1:17 ratio of 1:2)

(blue), 18 (Mcl-1:18 ratio of 1:2) (green) for (A) Phe 228, (B) Met 250, and (C) Val 249 and Val 253. Arrows show the direction of chemical shift

changes upon binding of compounds. (D) Overlay of putative binding modes of 16 (purple), 17 (blue), and 18 (green) to Mcl-1 (PDB ID: 2NLA)

highlighting in red Val 249, Met 250, and Val 253 on helix 4, Phe 228 on helix 3 of Mcl-1.

Figure 4. Putative binding mode of 21. (A) Surface of the Mcl-1 protein (PDB ID: 2NLA) is colored according to the chemical shift intensity.

Signiﬁcant shift (>0.09 ppm) is represented with purple, moderate shift (≥0.03 and ≤0.09 ppm) represented with pink. (B) Plot of chemical shift

changes of Mcl-1 amide upon addition of 21 (Mcl-1:21 ratio of 1:2) as a function of Mcl-1 residue numbers. (C) Overlaid ¹⁵N−¹H HSQC spectra of

Mcl-1 (red) and in the presence of 21 (Mcl-1:21 ratio of 1:2) (black), (Mcl-1:21 ratio of 1:1) (purple). Arrows show the direction of chemical shift

changes upon binding of 21. (D) Mcl-1 residues shown to be perturbed in HSQC NMR in the presence of 21 (Mcl-1:21 ratio of 1:2) (green).

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od^75,76was applied, using the three biphenyl analogues (16, 17,

and 18) diﬀering only in the orientation of distal phenyl ring.

Comparison of the spectra of the Mcl-1 bound to these three

analogues shows distinct changes in chemical shifts exclusively

for the resonances of the residues that are in direct contact with

the modiﬁed structure of the inhibitors, facilitating binding site

mapping. Careful analysis of the HSQC spectra of these

analogues reveals a diﬀerent direction of the chemical shift

perturbations of the residues forming the h2 pocket (Met 250)

or in its vicinity (Phe 228) as well as residues located on α-helix

4 (Val 249, Val 253) (Figures 3A−C). On the other hand,

residues which are not part of the h2 pocket, such as Leu267,

Val265, His224, and Arg263, show very similar chemical shift

perturbations in the presence of the biphenyl analogues

(Supporting Information Figure S6). This ﬁnding provides

conclusive evidence for biphenyl occupation of the h2 pocket

and further supports that α-helix 4 of Mcl-1 is ﬂexible.⁵⁸

Because of encouraging binding data with 16, analogues 19

and 20 with halogenated biphenyl rings at R₁were synthesized

to further increase potency. Satisfyingly, 19 with para-

chlorobiphenyl showed a 21-fold improved binding compared

to 3, becoming the most potent analogue in our series, with a K_i

= 0.17 0.04 μM determined by the FP binding assay using

ﬂuorescent labeled Bid BH3 peptide and IC₅₀of 0.88 0.15

μM in displacement of biotin labeled Bim BH3 peptide in the

SPR-based assay (14-fold improvement). The chemical shift

perturbation plot of 19 (Supporting Information Figure S3)

showed a similar pattern of perturbations to 16, suggesting a

similar binding conformation but with a lower perturbation

magnitude, likely due to the lower solubility of 19 in our NMR

studies. To improve the physicochemical properties of the

biphenyl analogues, we synthesized and evaluated analogues 21

with para-phenoxyphenyl at R₁and its ﬂuorine substituted

congener, 22. Analogue 21 showed similar binding aﬃnity as

19 (K_i= 0.18 0.05 μM in FP, and IC₅₀= 1.45 0.29 μM in

SPR) but stronger chemical shift perturbation (Figure 4A−C),

probably due to its improved solubility. Computational docking

of 21 predicted a π−π stacking of the distal phenyl of the para-

phenoxyphenyl moiety with the phenyl of Phe270 of Mc-1

(Figure 4D), which could account for its improved aﬃnity.

Compounds 19 and 21 became the most potent analogues of

our series and exhibited BIEs of 13.5 and 14.0, respectively,

maintaining 1’s BIE. In comparison with compound 53 (K_iof

0.055 μM), recently reported Mcl-1 selective inhbitor with an

indole core structure, discovered and optimized by fragment-

based screening strategy,⁴⁵19 and 21 have binding aﬃnity in a

similar nanomolar range, being 3-fold less potent than 53. To

further increase the aqueous solubility of this class of analogues,

a para-phenylpiperazine group at R₁was introduced in 23

which led to a 6-fold decrease in binding based on FP assay (K_i

= 2.22 0.36 μM) and 8-fold decrease based on SPR assay

naphthalene core, supporting the importance of hydrophobic

interactions at h3 to the overall Mcl-1 binding.

The next focus of our SAR studies was the sulfonamide linker

for which docking results suggested lack of any speciﬁc

interactions with Mcl-1. To investigate the importance of the

sulfonamide linker, several analogues were synthesized (Table

2). Replacement of the sulfonamide with a carboxamide in 25

Table 2. Binding Aﬃnities of Analogues with Linker (X)

Variations

led to signiﬁcant 83-fold decrease of the binding aﬃnity with K_i

value of 40.8 8.50 μM compared to 10, possibly due to the

unfavorable orientation of R₁by the carboxamide linker. The

decreased binding potency was conﬁrmed by SPR and NMR

(Supporting Information Figure S4) binding studies. To

explore the impact of the ﬂexibility of the linker, a methylene

linker distal (26) or proximal (27) to the naphthalene core was

inserted and both analogues showed decreased binding aﬃnity

with K_i= 5.23 0.36 μM and K_i= 64.75 12.61 μM, relative

to 9 (6-fold) and 3 (18-fold), respectively. As expected, change

of the sulfonamide linker in 26 to a carboxamide in 28

decreased the potency, but only by 2-fold (K_i= 12.53 1.24

μM), suggesting that the insertion of the methylene linker

between the carboxamide and the pendant aryl ring provids a

degree of freedom to better orient R₁into h2 pocket of Mcl-1.

Modeling studies showed that the thioacetic acid moiety at

R₂mimics the conserved Asp of BH3-only peptides and that

the carboxylate is involved in electrostatic interaction with Asn

260 and Arg 263 of Mcl-1. Therefore, to further explore this

site, analogues with diﬀerent substituents at R₂were

synthesized (Table 3). Removal of the acid side chain (30)

or its replacement with thiobutyl (31) did not show binding up

to 100 μM in both FP and SPR assays. Introducing a methyl

ester (32) or a primary carboxamide (33) resulted in decreased

binding by 10-and 3-fold in FP assay respectively compared to

10 (6- and 2-fold decrease in SPR assay respectively),

consistent with each of these functional groups, forming a

weaker interaction with Arg 263 relative to the carboxylic acid

moiety. When the carboxylic acid was changed to an alcohol

(IC₅₀= 16.20

5.80 μM) in comparison with the biphenyl

substituent in 16. This can be attributed to a relatively polar

substitutent projecting into the hydrophobic h2 pocket and to a

less optimal conformational preference of phenylpiperazine

versus biphenyl for the h2 pocket.

To brieﬂy explore the importance of the naphthalene core to

the binding potency, which occupies the h3 hydrophobic

pocket, analogue 24 with a phenyl core was synthesized.

Binding studies showed a signiﬁcant drop in potency (K_i=

283.36

81.50 μM in FP and IC₅₀> 100 μM in SPR)

compared to the corresponding compound 10 with the

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Table 3. Binding Aﬃnities of Sulfonamide Analogues with

Variations at R₁and R₂

Table 4. Binding Aﬃnities of Sulfonamide Analogues with

Variations at R₁, R₂, and R₃

a

*Compounds were tested up to 100 μM.

(Supporting Information Figure S5). Similar signiﬁcant loss of

binding is also apparent in compounds 42 to 45 compared to

their corresponding phenolic analogues, clearly indicating the

importance of the phenolic group to the overall binding to Mcl-

1. Compound 46, where the hydroxyl group was acetylated

exhibited a K_iof 33.97 15.96 μM, which is a 69-fold decrease

in binding compared to 10. However, 46 showed improved

binding relative to 40, which might be attributed to the

formation of a hydrogen bond between the carbonyl of the

acetyl group and the protonated His 224, supported by

computational prediction.

We determined the selectivity of this class of compounds

against four other Bcl-2 antiapoptotic proteins (Bcl-2, Bcl-xL,

Bcl-w, Bﬂ-1/A1). The most potent analogues were tested in

competitive FP-based assays that were optimized for each

protein, and K_ivalues were calculated using equations

developed previously⁷⁷(Table 5). In general, all the analogues

inhibit Mcl-1 most potently with the following order of

selectively: Bﬂ-1/A1 > Bcl-w > Bcl-2 > Bcl-xL. As the BH3

domain binding proﬁle of Bﬂ-1/A1, as well as its BH3 binding

groove, is most similar to that of Mcl-1,⁷⁸it is not surprising

that the tested compounds showed less selective inhibition of

A1. The most potent analogues in this series, 19 and 21, show a

proﬁle for selectively inhibiting Mcl-1 with 7-and 19-fold versus

Bﬂ-1/A1, 8-and 9-fold versus Bcl-w, 36- and 42-fold versus Bcl-

2, and 56- and 59-fold versus Bcl-x_L, respectively. Other

reported selective Mcl-1 inhibitors, MIM1 and 53, also show

high selectivity against Bcl-xL (IC₅₀> 50 μM and K_i> 15 μM,

respectively), while 19 and 21 show better selectivity against

Bcl-2 in comparison with 53 which has 16-fold selectivity with

K_ivalue of 0.87 μM.

a

^#ND: The IC₅₀was not determined because the compound showed

nonspeciﬁc binding to the control surface; *Compounds were tested

up to 100 μM.

and the sulfur to methylene (34) or ethylene (35), both

analogues showed decreased binding compared to the parent

congener 10 with K_ivalues of 3.79 0.62 and 2.18 0.91 μM,

respectively. As was expected, bioisosteric replacement of the

carboxylic acid group with a tetrazole in analogues 36 and 37

maintained the binding aﬃnity in comparison with their parent

congeners, 10 and 21. Homologation of the thioacetic acid in

38 had no detrimental eﬀect on binding and showed a similar

K_iof 1.13 0.34 μM in FP assay and IC₅₀= 5.03 1.88 μM in

SPR assay as 15, which can be explained with the ﬂexibility of

both the thiopropanoic acid moiety and Arg 263 side chain.

However, changing the point of fusion of the naphthalene ring

in 38 substantially aﬀected the binding and 39 with 1-naphthyl

substituent showed a signiﬁcant reduction in binding to Mcl-1.

Our docking studies suggest that this might be attributed to a

clash with the residues in the h2 pocket of Mcl-1.

Modeling showed that the phenolic group of the core

naphthalene scaﬀold forms a hydrogen bond with His 224 of

Mcl-1, consistent with the reported acidic hotspot in the h3 site

of Mcl-1 close to His 224.⁵⁸Several analogues were synthesized

to probe the contribution of the phenolic group to binding to

Mcl-1 (Table 4). When the hydroxyl group is changed to a

methoxy in analogue 40, the potency decreased by 170 fold (K_i

= 86.98 15.35 μM) compared to the phenolic congener 10.

The analogue 41, where R₂is methyl thioacetate and R₃is

methoxy, did not show binding up to 100 μM, and the loss of

the binding was conﬁrmed with SPR and HSQC experiment

Biological Characterization of Mcl-1 Inhibitors. To

verify the speciﬁc binding of novel inhibitors to Mcl-1, we

employed a pull-down assay using biotin-labeled Noxa (BL-

Noxa) and whole cell lysate from the human breast cancer cell

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Table 5. Selectivity of Selected Analogues against Bcl-2 Antiapoptotic Proteins

compound

Mcl-1 K_iSD (μM)

A1/Bﬂ-1 K_iSD (μM)

Bcl-2 K_iSD (μM)

Bcl-w K_iSD (μM)

Bcl-X_LK_iSD (μM)

1

1.55 0.18

0.49 0.06

0.37 0.10

0.38 0.03

2.03 0.41

0.17 0.04

0.18 0.05

6.14 1.0

5.33 1.01

2.34 0.37

3.18 0.41

17.26 0.56

1.11 0.19

3.36 0.56

54.65 9.56

23.83 1.81

8.82 0.65

7.85 0.65

23.84 3.16

6.11 0.65

7.56 1.08

37.53 7.96

8.19 1.91

1.92 0.37

2.19 0.45

4.41 0.39

1.36 0.51

1.58 0.35

99.00 22.63

32.99 4.33

8.05 0.50

15.14 1.11

48.15 3.28

9.59 1.28

10.58 1.53

10

16

17

18

19

21

line 2LMP. As shown in Figure 5, Mcl-1 was pulled down by

BL-Noxa and, as was expected, the Bim BH3 peptide disrupted

Figure 5. Interaction of Mcl-1 inhibitors with endogenous Mcl-1

protein and Noxa. Biotin-labeled Noxa (BL-Noxa, 0.1 μM) was

incubated with whole cell lysates of 2LMP cells with or without tested

Mcl-1 inhibitors and Bim BH3 peptide as a positive control, followed

by incubation with precleared streptavidin agarose beads. Eluted beads

were subjected to Western blot analysis with anti-Mcl-1 antibody.

Figure 6. Cell death induced by Mcl-1 inhibitor 21 is Bax/Bak-

dependent. MEFs deﬁcient in Bax and Bak (gray bars) along with their

wild-type counterpart (black bars) were exposed for 15 h to diﬀerent

concentrations of 21, and the cell viability was assessed with PI

staining. Error bars represent the mean SEM. The signiﬁcance was

calculated using unpaired t test, and the number of data is shown for

each tested concentration with corresponding signiﬁcance: (**) p <

0.01 and (***) p < 0.001.

the interaction between BL-Noxa and Mcl-1. Preincubation

with several Mcl-1 inhibitors, 10, 19, and 21, completely

blocked the binding of BL-Noxa to Mcl-1, similar to Bim

peptide, demonstrating that these inhibitors can recognize and

speciﬁcally bind to the BH3 binding groove of endogenous

Mcl-1 protein.

It is well established that Bax and Bak are required for the

initiation of intrinsic, mitochondrial, apoptotic cell death, and

they are maintained in an inactive state through interaction

with the antiapoptotic proteins.^14,79Therefore, cells subjected

to inhibitors of antiapoptotic proteins are expected to undergo

cell death in a Bax/Bak-dependent manner.⁸⁰To determine the

contribution of Bak and Bax in the cell death induced by our

Mcl-1 inhibitors, we employed murine embryonic ﬁbroblasts

(MEFs) wild-type (wt) and deﬁcient in both Bax and Bak

(double knock out, DKO). Exposure of the wt MEFs to 21

resulted in a concentration-dependent cell death (assessed by

PI staining), while Bax/Bak deletion signiﬁcantly rescued cells

from 21 induced cell death (Figure 6). Although 21 initiates

cell death also in DKO MEFs (23% positive PI at 16 μM), it is

clear that 21 is more potent in wt MEFs (61% positive PI at 16

μM), indicating that Bax and/or Bak are involved in cell death

induction. Taken together, our data suggest that 21 induces cell

death in a Bax/Bak-dependent manner due to its Mcl-1

inhibitory function.

To further conﬁrm the speciﬁcity of our novel Mcl-1

inhibitors and to determine whether diﬀerent prosurvival Bcl-2

proteins could suppress the apoptotic activities of novel Mcl-1

inhibitors, we used reported cell lines developed by retroviral

transduction of lymphoma cells isolated from Eμ-myc trans-

genic mice which diﬀer only in their expression of prosurvival

Bcl-2 family proteins.⁸¹Lymphoma cells overexpressing Mcl-1

and Bcl-2 were treated with varying concentrations of tested

compounds for 15−18 h, and then cell viability was determined

by ﬂow cytometry using a ﬂuorescent reactive dye (LIVE/

DEAD ﬁxable violet stain kit). ABT-263 (navitoclax), a

selective inhibitor of Bcl-2, Bcl-xL, and Bcl-w, was used as a

positive control. As predicted, lymphoma cells overexpressing

Mcl-1 were signiﬁcantly sensitive to 19 and 21 as assessed by an

increased percentage of cell death in a concentration-dependent

manner. In contrast, 19 and 21 were ineﬀective against Eμ-

myc/Bcl-2 lymphomas (Figure 7). Importantly, 41 did not

show any activity against both cell lines overexpressing Mcl-1 or

Bcl-2, consistent with our binding studies which showed that 41

does not bind to Mcl-1. As expected, lymphoma cells

overexpressing Bcl-2 were sensitive to cell death induced by

ABT-263, while cells overexpressing Mcl-1 were insensitive to

ABT-263, consistent with its binding speciﬁcity. Collectively,

these results, demonstrate that 19 and 21 speciﬁcally bind and

inhibit Mcl-1 and have no eﬀect on Bcl-2, which is consistent

with our biochemical data for their selectivity proﬁles.

Furthermore, this supports the concept that tumor cells

“addicted” to Mcl-1 protein will be the most sensitive target

cell population for selective small-molecule Mcl-1 inhibitors.

We next evaluated our most potent compounds for their

ability to inhibit cell growth in the leukemia cell lines HL-60,

MV4,11, and K-562 (Figure 8). It has been shown that AML-

derived cell lines, HL-60 and MV4,11, are sensitive to inhibition

of the antiapoptotic protein Mcl-1, while CML-derived K-562

cell line is less sensitive to Mcl-1 inhibition.⁸²Tested

compounds from our series showed inhibition of the cell

growth in a dose-dependent manner with similar potencies,

IC₅₀values ranging from 2.06 to 11.72 μM against the HL-60

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Figure 7. Sensitivity of Eμ-myc lymphoma cells overexpressing Mcl-1 and Bcl-2 antiapoptotic proteins to inhibitor-induced cell death. Eμ-myc/Mcl-

1 and Eμ-myc/Bcl-2 lymphomas were treated for 15−18 h with increasing concentrations of 19, 21, 41, and ABT-263. Dead cells were assessed by

LIVE/DEAD ﬁxable dead cell stain kit (ViVID). The data shown represents means SEM from 3−7 independent experiments. The signiﬁcance was

calculated using unpaired t test, and the number of data is shown for each tested concentration with corresponding signiﬁcance: (*) is p < 0.05, (**)

p < 0.01, and (***) p < 0.001.

and MV4,11 cell lines (Figure 8A). Interestingly, these

compounds showed decreased ability to inhibit the cell growth

of K-562, with IC₅₀values of 11.76 to 29.89 μM. Compound

41, which does not bind to Mcl-1, did not show inhibition up to

50 μM. It is important to be pointed out that 36 and 37, where

the carboxylic acid was replaced with a bioisostere tetrazole

group, show similar cellular activity in all tested cell lines in

comparison with their parent compounds, 10 and 21,

respectively. These results demonstrate that the tetrazole

group can eﬀectively replace the acid group, achieving not

only the same binding aﬃnity to Mcl-1 but also comparable

cellular activity. Furthermore, compound 32, in which the acid

group has been replaced with methyl ester and showed 10-fold

less binding to Mcl-1 as compared with 10, has also similar IC₅₀

values in tested cell lines as 10. This is probably due to

enzymatic hydrolysis of the methyl ester group in the cells and

releasing the corresponding compound with free acid group,

thus functioning as a pro-drug. These data also demonstrate

that the compounds with free acidic group in this series are cell

permeable, although the mechanism of their cell permeability

need to be further studied. Compounds 21 and 37 were

evaluated for their ability to induce apoptosis in the HL-60 cell

line using annexin-V and propidium iodide (PI) double staining

by ﬂow cytometry (Figure 8B). Both compounds eﬀectively

induced apoptosis in a dose-dependent manner. Treatment of

the HL-60 cells by 2.5, 5.0, and 10 μM of 21 and 37 for 20 h

results in 28.9% and 23.5%, 37.8% and 51.6%, and 78.4% and

74.2% of apoptotic cells (early + late), respectively, as

compared to 7.6% and 7.3% of apoptotic cells in the DMSO

controls. These results further conﬁrmed that these two

analogues have similar cellular activity, and tetrazole can

eﬀectively replace the acid group. To determine if the induction

of apoptosis by compound 37 in the HL-60 cell line depends

upon caspases, we treated the cells with compound 37 alone or

in the presence of Z-VED-FMK, a pan-caspase inhibitor (Figure

8B and Supporting Information Figure S7). The obtained

results showed that the induction of apoptosis was signiﬁcantly

inhibited in the presence of Z-VAD-FMK, clearly indicating

that the apoptotic activity of compound 37 is mediated by

caspases. Of note, Z-VAD-FMK alone has no eﬀect on cells

(Supporting Information Figure S7). Compound 41 at 40 μM

has no eﬀect on apoptosis induction just like untreated control,

which is consistent with its lack of binding to Mcl-1 and

inhibition of cell growth. We further tested compounds 19 and

21 in HL-60 cell line for their ability to induce caspase-3

activity, one of the important biochemical markers of apoptosis

(Figure 8C). Both compounds induce activation of caspase-3

activity in a dose-dependent manner, with 21 eﬀectively

inducing activation over a 24 h period starting at 2.5 μM.

Importantly, these results correlate with the ability of 21 to

induce apoposis and inhibit HL-60 cell growth.

CONCLUSIONS

■

Applying a HTS approach we have identiﬁed a novel class of

small molecules as selective Mcl-1 inhibitors. Employing

structure-based design supported by NMR studies, we

synthesized a focused library of analogues and established a

SAR for binding to Mcl-1. Careful HSQC analysis of analogues

16−18 provided strong evidence for the predicted binding

model of these compounds and further conﬁrmation that R₁

substituent of this class of inhibitors binds to one well-deﬁned

pocket of Mcl-1 known as h2 pocket. Analogues 19 and 21,

with K_ivalues of 170 and 180 nM, respectively, were developed

as the most potent compounds in this series with an overall 9-

fold increase in binding compared to 1. Binding studies showed

that 19 and 21 maintained the selectivity proﬁle of 1. Using

wild-type and Bax/Bak double knockout MEFs cells, the

contribution of these two multidomain pro-apoptotic proteins

in 21-induced cell death was determined and demonstrated that

21 primarily causes cell death in wild-type MEFs in a Bax/Bak-

dependent manner. Furthermore, 19 and 21 led to sensitization

of Eμ-myc lymphomas overexpressing Mcl-1 but did not show

eﬀect on cells overexpressing Bcl-2 antiapoptotic protein,

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Figure 8. Cell death and apoptosis induction by Mcl-1 inhibitors in human leukemic cell lines. (A) Inhibition of cell growth by designed Mcl-1

inhibitors in the HL-60, MV4,11, and K-562 leukemia cell lines. Cells were treated for 3 days, and cell growth was determined using CellTiter Glo

luminescent cell viability assay. (B) Analysis of apoptosis induced by 21 and 37 in the HL-60 leukemia cell line. Cells were treated with 21, 37, and

41 for 20 h using indicated concentrations, and apoptosis was analyzed with annexin-V and propidium iodide (PI) double staining by ﬂow cytometry.

Early apoptotic cells were deﬁned as annexin-V positive/PI-negative, and late apoptotic cells as annexin-V/PI-double positive. Induction of the

apoptosis by 37 was tested also in the presence of Z-VAD-FMK. (C) Induction of caspase-3 by 19 and 21 in the HL-60 cell line. Cells were treated

for 20 h, and caspase-3 was detected with ﬂuorometric-based assay. Results shown are the mean and SEM from at least three separate experiments.

mL/min. Semipreparative reverse-phase HPLC was performed on a

Shimadzu system with a Restek Ultra C18 (21.2 mm × 150 mm, 5 μm

particle size) column. All NMR spectra were obtained in DMSO-d₆or

CDCl₃, and results were recorded at 400 or 500 MHz on a Varian 400

or 500 instrument. Mass spectra were recorded on a Micromass LCT

time-of-ﬂight instrument utilizing electrospray ionization operating in

positive-ion (ESI+) or negative-ion (ESI) modes where indicated.

High resolution mass spectrometry (HRMS) analysis was performed

on an Agilent Q-TOF system. All ﬁnal compounds were puriﬁed to

>95% purity. Analogues 4−8, 15, and 39 were purchased from

commercial vendors.

2-Iodo-1-methoxy-4-nitronaphthalene (47). Synthesized using

reported procedure with modiﬁcation.⁶⁰A mixture of 1-methoxy-4-

nitronaphthalene (2.1 g, 10.4 mmol) and N-iodosuccinimide (2.7 g, 12

mmol) in TFA (40 mL) was heated to reﬂux and stirred for 20 h under

nitrogen. The reaction mixture was diluted with EtOAc (40 mL),

washed with saturated aqueous Na₂S₂O₃solution (30 mL), saturated

aqueous NaHCO₃(30 mL × 2), and brine (30 mL). The organic layer

was dried (MgSO₄), ﬁltered, and silica was added to ﬁltrate and the

solvent was removed under reduced pressure. The adsorbed crude

residue was puriﬁed by ﬂash column chromatography (100% hexane)

on silica gel to give 47 (2.4 g, 70%) as a light-yellow solid. Note: R_fof

starting material and product were very close and a good separation

was achieved with a relatively long silica gel column and 100% hexane

gradient. >95% pure product was needed for pd-catalyzed coupling

conﬁrming their selective targeting of Mcl-1. Most potent

developed 3-substituted-N-(4-Hydroxynaphthalen-1-yl)-

arylsulfonamide Mcl-1 inhibitors inhibited the cell growth of

AML-derived cell lines, HL-60 and MV4,11. Compounds 19

and 21 induced activation of caspase-3 in HL-60 cell line and

21 eﬀectively induced apoptosis starting at 2.5 μM. Future

studies will be directed toward optimization of this class of

compounds and in vivo evaluation.

EXPERIMENTAL SECTION

■

Chemistry Materials and Methods. All reactions were

performed under anhydrous conditions. Reagents were used as

supplied without further puriﬁcation. Reactions were monitored by

TLC using precoated silica gel 60 F254 plates. Silica gel

chromatography was performed with silica gel (220−240 mesh)

obtained from Silicycle. Purities of ﬁnal compounds were assessed by

analytical reverse-phase HPLC performed with one of the two

methods. Method A: Agilent 1100 series with an Agilent Zorbax

Eclipse Plus C18 (4.6 mm × 75 mm, 3.5 μm particle size) column with

the gradient 10% ACN/water (1 min), 10−90% ACN/water (6 min),

and 90% ACN/water (2 min) ﬂow = 1 mL/min. Method B: Shimadzu

system with a Restek Ultra C18 (4.6 mm × 150 mm, 5 μm particle

size) column with the gradient 50% ACN/water (5 min), 50−70%

ACN/water (2 min), and 70%−90% ACN/water (8 min) ﬂow = 1

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1

step. H NMR (400 MHz, CDCl₃) δ 8.59 (s, 1H), 8.58 (s, 1H), 8.21

mg, 97%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.59 (d, J =

8.66 Hz, 1H), 8.29 (s, 1H), 8.26 (d, J = 8.66 Hz, 1H), 7.74−7.66 (m,

1H), 7.64−7.55 (m, 1H), 4.58 (s, 2H), 4.29 (s, 3H). ¹³C NMR (100

MHz, CDCl₃) δ 162.40, 141.04, 130.49, 129.78, 128.26, 127.56,

126.29, 123.44, 123.24, 107.40, 93.96, 80.83, 61.95, 51.67. ESI MS: m/

z 258.1 (M + H)⁺.

(d, J = 8.42 Hz, 1H), 7.74 (t, J = 7.53 Hz, 1H), 7.65 (t, J = 7.53 Hz,

1H), 4.03 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 161.69, 142.85,

134.07, 129.99, 128.55, 128.08, 126.55, 123.92, 123.02, 83.35, 62.20.

ESI MS: m/z 330.0 (M + H)⁺.

Methyl 2-((1-Methoxy-4-nitronaphthalen-2-yl)thio)acetate

(48a). Synthesized using reported procedures with modiﬁcation.⁶³⁻⁶⁵

To a solution of Cs₂CO₃(1.5 g, 4.5 mmol) in dry THF (7 mL) under

nitrogen was added methylthioglycolate (277 μL, 2.9 mmol). The

mixture was stirred at room temperature for 10 min. At this time, a

solution of ZnCl₂(288 mg, 2.1 mmol) in dry THF (3 mL) was added

and the mixture was stirred at room temperature for an additional 10

min. Meanwhile, in a separate ﬂask, Pd(OAc)₂(36 mg, 0.16 mmol)

and xantphos (90 mg, 0.15 mmol) were premixed in dry THF (5 mL)

under nitrogen and stirred at room temperature for about 20 min. To

the solution of thiol, Cs₂CO₃, and ZnCl₂was added 47 (1.0 g, 3.1

mmol), LiI (200 mg, 1.5 mmol), and premixed solution of the catalyst

and ligand. The mixture was stirred at 60 °C under nitrogen for 20 h.

The reaction mixture was ﬁltered to remove Cs₂CO₃and silica was

added to the mixture and the solvent was removed under reduced

pressure. The adsorbed crude residue was puriﬁed by column

chromatography (hexane/EtOAc 4:1) on silica gel to give 48a (606

mg, 66%) as a yellow oil which solidiﬁed. ¹H NMR (400 MHz,

CDCl₃) δ 8.59 (d, J = 8.50 Hz, 1H), 8.37 (s, 1H), 8.19 (d, J = 8.50 Hz,

1H), 7.70 (t, J = 7.57 Hz, 1H), 7.64 (t, J = 7.57 Hz, 1H), 4.07 (s, 3H),

3.77 (s, 2H), 3.70 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.48,

159.95, 142.50, 129.73, 128.88, 127.85, 127.22, 125.94, 123.71, 122.93,

122.68, 61.92, 52.72, 35.10. ESI MS: m/z 308.1 (M + H)⁺.

Methyl 3-((1-Methoxy-4-nitronaphthalen-2-yl)thio)-

propanoate (48b). Synthesized using a similar procedure used to

prepare 48a except using methyl 3-mercaptopropionate. The mixture

was stirred at 60 °C under nitrogen for 5 h. Crude was puriﬁed using

ﬂash column chromatography (hexane/EtOAc 4:1) on silica gel with

dry loading to give 48b (194 mg, 66%) as a yellow oil. ¹H NMR (400

MHz, CDCl₃) δ 8.56 (d, J = 8.48 Hz, 1H), 8.27 (s, 1H), 8.16 (d, J =

8.48 Hz, 1H), 7.70−7.64 (m, 1H), 7.64−7.58 (m, 1H), 4.03 (s, 3H),

3.65 (s, 3H), 3.28 (t, J = 7.24 Hz, 2H), 2.65 (t, J = 7.24 Hz, 2H). ¹³C

NMR (100 MHz, CDCl₃) δ 171.64, 159.79, 142.48, 129.51, 128.93,

127.81, 126.94, 125.60, 123.62, 123.45, 122.57, 61.59, 51.90, 34.11,

28.11. ESI MS: m/z 322.0 (M + H)⁺, 343.9 (M + Na)⁺.

4-(1-Methoxy-4-nitronaphthalen-2-yl)but-3-yn-1-ol (48e).

Synthesized using a similar procedure used to prepare 48d except

using 3-butyn-1-ol as the alkyne. The crude was puriﬁed by ﬂash

column chromatography (hexane/EtOAc 3:2) on silica gel to give 48e

(338 mg, 83%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.53

(d, J = 8.56 Hz, 1H), 8.23 (s, 1H), 8.19 (d, J = 8.56 Hz, 1H), 7.63 (t, J

= 7.59 Hz, 1H), 7.53 (t, J = 7.59 Hz, 1H), 4.23 (s, 3H), 3.87 (t, J =

6.14 Hz, 2H), 2.76 (t, J = 6.14 Hz, 2H), 2.45 (s, 1H). ¹³C NMR (100

MHz, CDCl₃) δ 162.29, 141.11, 130.19, 129.91, 128.36, 127.48,

126.03, 123.43, 123.10, 109.99, 108.73, 93.90, 61.77, 60.96, 24.07. ESI

MS: m/z 272.1 (M + H)⁺.

A Representative Procedure for Iron Reduction of Nitro to

Amine. Methyl 2-((4-Amino-1-methoxynaphthalen-2-yl)thio)-

acetate (49a). Synthesized using a reported procedure.⁶⁶To a

suspension of iron powder (538 mg, 9.6 mmol) in glacial acetic acid (5

mL) was added 48a (195 mg, 0.63 mmol) dissolved in glacial acetic

acid (5 mL). The mixture was stirred at 70 °C under nitrogen for 1 h,

when the mixture turned milky. The mixture was then diluted with

EtOAc (15 mL) and washed with saturated aqueous NaHCO₃(20 mL

× 2) and brine (20 mL). Organic layer was dried (MgSO₄), ﬁltered,

and the solvent removed under reduced pressure to give 49a as a crude

as a purple oil. The crude was used in the next step without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 8.29 Hz, 1H),

7.76 (d, J = 8.29 Hz, 1H), 7.50 (t, J = 7.58 Hz, 1H), 7.43 (t, J = 7.58

Hz, 1H), 6.78 (s, 1H), 3.91 (s, 3H), 3.73 (s, 2H), 3.68 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃) δ 170.48, 147.81, 138.68, 128.54, 126.54,

125.33, 124.24, 123.36, 122.45, 121.35, 111.08, 61.41, 52.53, 35.37.

ESI MS: m/z 278.1 (M + H)⁺, 300.1 (M + Na)⁺.

Methyl 3-((4-Amino-1-methoxynaphthalen-2-yl)thio)propanoate

(49b). Synthesized using the procedure for 49a except using 48b as

the starting material. Crude was used in the next step without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 8.02 (d, J = 8.36 Hz, 1H),

7.75 (d, J = 8.36 Hz, 1H), 7.49 (t, J = 7.56 Hz, 1H), 7.42 (t, J = 7.56

Hz, 1H), 6.72 (s, 1H), 6.26 (s, 2H), 3.89 (s, 3H), 3.65 (s, 3H), 3.22 (t,

J = 7.46 Hz, 2H), 2.64 (t, J = 7.46 Hz, 2H);. ¹³C NMR (100 MHz,

CDCl₃) δ 172.38, 147.73, 138.77, 128.63, 126.51, 125.12, 123.92,

123.56, 122.36, 121.35, 110.85, 61.21, 51.78, 34.48, 27.90. ESI MS: m/

z 292.0 (M + H)⁺, 314.0 (M + Na)⁺.

3-(Butylthio)-4-methoxynaphthalen-1-amine (49c). Synthesized

using the procedure for 49a except using 48c as the starting material.

Crude was used in the next step without further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃) δ 8.03 (d, J = 8.30 Hz, 1H), 7.75 (d, J = 8.30 Hz,

1H), 7.49 (t, J = 7.42 Hz, 1H), 7.40 (t, J = 7.42 Hz, 1H), 6.72 (s, 1H),

6.30 (s, 2H), 3.92 (s, 3H), 2.96 (t, J = 7.33 Hz, 2H), 1.65 (p, J = 7.33

Hz, 2H), 1.47 (h, J = 7.33 Hz, 2H), 0.92 (t, J = 7.33 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃) δ 146.85, 138.46, 128.54, 126.42, 125.50,

124.73, 123.45, 122.20, 121.30, 110.24, 61.02, 32.29, 31.50, 22.02,

13.67. ESI MS: m/z 262.0 (M + H)⁺.

Butyl(1-methoxy-4-nitronaphthalen-2-yl)sulfane (48c). Syn-

thesized using a reported procedure.⁶¹A stirred mixture of 47 (300

mg, 0.91 mmol), Pd₂(dba)₃(42 mg, 0.05 mmol), Dppf (104 mg, 0.18

mmol), and Et₃N (0.2 mL) in dry NMP (7 mL) was ﬂushed with

nitrogen for 15 min at room temperature. Butanethiol (83 μL, 0.77

mmol) was then added, and the reaction mixture was heated to 80 °C

and stirred for 2 h. The mixture was diluted with EtOAc (10 mL) and

washed with H₂O (10 mL × 4) and brine (10 mL). The organic layer

was dried (MgSO₄), ﬁltered, and silica added to the ﬁltrate, and the

solvent was removed under reduced pressure. The adsorbed crude

residue was puriﬁed by ﬂash column chromatography (hexane to

hexane/EtOAc 99:1) on silica gel to give 48c (189 mg, 71%) as a

1

yellow oil. H NMR (400 MHz, CDCl₃) δ 8.58 (ddd, J = 0.72, 1.50,

8.36 Hz, 1H), 8.26 (s, 1H), 8.16 (ddd, J = 0.72, 1.50, 8.36 Hz, 1H),

7.68−7.59 (m, 2H), 4.03 (s, 3H), 3.03 (t, J = 7.36 Hz, 2H), 1.67 (p, J

= 7.36 Hz, 2H), 1.48 (h, J = 7.36 Hz, 2H), 0.93 (t, J = 7.36 Hz, 3H).

¹³C NMR (100 MHz, CDCl₃) δ 158.48, 142.53, 129.05, 128.81,

127.69, 125.69, 125.57, 125.03, 123.62, 122.34, 61.31, 32.23, 31.10,

21.92, 13.60. ESI MS: m/z 292.0 (M + H)⁺.

3-(1-Methoxy-4-nitronaphthalen-2-yl)prop-2-yn-1-ol (48d).

Synthesized using a reported procedure.⁶²A mixture of 47 (453 mg,

1.4 mmol), Pd(PPh₃)₂Cl₂(48 mg, 0.07 mmol), and CuI (28 mg, 0.15

mmol) in Et₃N (8 mL) and dry THF (3 mL) was added dropwise to a

solution of 2-propyn-1-ol (0.15 mL, 2.6 mmol) in Et₃N (3 mL) under

nitrogen at room temperature. Reaction mixture was heated to 60 °C

and stirred for 2 h then diluted with EtOAc (10 mL) and washed with

saturated aqueous NH₄Cl (15 mL × 2) and brine (15 mL). The

organic layer was dried (MgSO₄), ﬁltered, and concentrated under

reduced pressure. The crude was puriﬁed by ﬂash column

chromatography (hexane/EtOAc 3:2) on silica gel to give 48d (342

A Representative Procedure for Hydrogenation Reaction. 3-

(4-Amino-1-methoxynaphthalen-2-yl)propan-1-ol (49d). Synthes-

zied using a reported procedure.⁶⁷A stirred solution of 48d (325

mg, 1 mmol) in a mixture of EtOH (12 mL) and EtOAc (2 mL) was

hydrogenated in the presence of 10% Pd/C (80 mg) at room

temperature and under 30 psi of H₂overnight. The suspension was

ﬁltered through a pad of Celite, and the ﬁltrate was concentrated

under reduced pressure. The crude was used in the next reaction

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 8.02 (d, J

= 8.35 Hz, 1H), 7.76 (d, J = 8.35 Hz, 1H), 7.51−7.43 (m, 1H), 7.43−

7.35 (m, 1H), 6.54 (s, 1H), 3.85 (s, 3H), 3.54 (t, J = 6.08 Hz, 2H),

2.80 (t, J = 7.27 Hz, 2H), 1.85 (p, J = 6.69 Hz, 2H). ¹³C NMR (100

MHz, CDCl₃) δ 146.20, 138.66, 129.80, 128.24, 126.01, 124.51,

123.79, 122.41, 121.33, 111.39, 62.18, 61.32, 33.18, 25.51. ESI MS: m/

z 232.1 (M + H)⁺.

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4-(4-Amino-1-methoxynaphthalen-2-yl)butan-1-ol (49e). Synthe-

sized using the procedure for 49d except using 48e as the starting

material. Crude was used in the next step without further puriﬁcation.

¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 8.39 Hz, 1H), 7.72 (d, J =

8.39 Hz, 1H), 7.43 (t, J = 7.33 Hz, 1H), 7.34 (t, J = 7.33 Hz, 1H), 6.52

(s, 1H), 3.79 (s, 3H), 3.56 (t, J = 6.43 Hz, 2H), 2.67 (t, J = 6.43 Hz,

2H), 1.65 (dt, J = 6.60, 14.36 Hz, 2H), 1.56 (dt, J = 6.60, 14.36 Hz,

2H). ¹³C NMR (100 MHz, CDCl₃) δ 145.98, 138.36, 130.77, 128.39,

125.85, 124.33, 123.72, 122.34, 121.36, 111.52, 62.32, 61.97, 32.45,

29.21, 26.89.

3.64 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.83, 154.25, 138.21,

132.29, 129.91, 128.89, 128.71, 128.07, 127.41, 127.12, 125.92, 123.47,

122.48, 122.17, 122.09, 61.56, 52.70, 35.03. ESI HRMS: m/z 493.9724

(M − H)⁻.

Methyl 2-((1-Methoxy-4-(4-methoxyphenylsulfonamido)-

naphthalen-2-yl)thio)acetate (49j). Synthesized using the procedure

for 49f except using 4-methoxybenzenesulfonyl chloride, which

aﬀorded the title compound (190 mg, 77% over two steps) as a

purple oil which solidiﬁed upon standing. ¹H NMR (400 MHz,

CDCl₃) δ 8.02 (d, J = 8.42 Hz, 1H), 7.80 (d, J = 8.42 Hz, 1H), 7.69−

7.63 (m, 2H), 7.48 (t, J = 7.64 Hz, 1H), 7.40 (t, J = 7.64 Hz, 1H), 7.29

(s, 1H), 6.86−6.80 (m, 3H), 3.95 (s, 3H), 3.79 (s, 3H), 3.70 (s, 3H),

3.63 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.82, 163.12, 153.79,

130.64, 129.85, 129.52, 128.61, 128.11, 126.95, 126.90, 125.20, 123.36,

122.31, 114.12, 61.50, 55.56, 52.65, 35.09. ESI MS: m/z 447.8 (M +

H)⁺, 469.8 (M + Na)⁺.

A Representative Procedure for Sulfonamide/Amide Cou-

pling Reaction of Aryl Amines with Sulfonyl/Acyl Chlorides.

Methyl 2-((1-Methoxy-4-(thiophene-2-sulfonamido)naphthalen-2-

yl)thio)acetate (49f). A solution of the crude amine 49a dissolved

in dry CH₂Cl₂(4 mL) was added to 2-thiophenesulfonyl chloride (119

mg, 0.65 mmol). Addition of pyridine (0.08 mL, 0.99 mmol) was

followed, and the mixture was stirred at room temperature under

nitrogen overnight. The mixture was diluted with EtOAc (10 mL) and

washed with H₂O (10 mL × 3) and brine (10 mL). The organic layer

was dried (MgSO₄), ﬁltered, and the solvent removed under reduced

pressure. Crude was puriﬁed by ﬂash column chromatography

(hexane/EtOAc 7:3) on silica gel to give 49f (176 mg, 66% over

Methyl 2-((4-(3-Bromophenylsulfonamido)-1-methoxynaphtha-

len-2-yl)thio)acetate (49k). Synthesized using the procedure for 49f

except using 3-bromobenzenesulfonyl chloride, which aﬀorded the title

1

compound (331 mg, 74%) as a purple/pink solid. H NMR (400

MHz, CDCl₃) δ 8.04 (d, J = 8.40 Hz, 1H), 7.88 (s, 1H), 7.73 (d, J =

8.40 Hz, 1H), 7.62 (t, J = 9.41 Hz, 2H), 7.49 (t, J = 7.59 Hz, 1H), 7.40

(t, J = 7.59 Hz, 1H), 7.34 (s, 1H), 7.26−7.21 (m, 1H), 6.82 (s, 1H),

3.98 (s, 3H), 3.70 (s, 3H), 3.65 (s, 2H). ¹³C NMR (100 MHz, CDCl₃)

δ 169.72, 154.55, 141.06, 135.89, 130.41, 130.24, 130.03, 128.75,

127.31, 127.04, 127.02, 126.33, 125.89, 123.45, 122.90, 122.48, 122.09,

61.48, 52.55, 35.16. ESI MS: m/z 495.8, 497.8 (M + H)⁺, 517.8, 519.8

(M + Na)⁺.

1

two steps) as a purple oil which solidiﬁed upon standing. H NMR

(400 MHz, CDCl₃) δ 8.01 (d, J = 8.40 Hz, 1H), 7.82 (d, J = 8.40 Hz,

1H), 7.49−7.43 (m, 2H), 7.42−7.39 (m, 1H), 7.39−7.34 (m, 2H),

6.92−6.88 (m, 1H), 3.95 (s, 3H), 3.68 (s, 3H), 3.65 (s, 2H). ¹³C NMR

(100 MHz, CDCl₃) δ 169.91, 154.12, 139.48, 133.09, 132.51, 130.06,

128.60, 127.74, 127.39, 126.96, 125.74, 123.39, 122.29, 61.49, 52.68,

35.12. ESI MS: m/z 423.9 (M + H)⁺, 445.8 (M + Na)⁺.

Methyl 2-((4-(2-Bromophenylsulfonamido)-1-methoxynaphtha-

len-2-yl)thio)acetate (49l). Synthesized using the procedure for 49f

except using 2-bromobenzenesulfonyl chloride, which aﬀorded the title

Methyl 2-((1-Methoxy-4-(methylsulfonamido)naphthalen-2-yl)-

thio)acetate (49g). Synthesized using the procedure for 49f except

using methanesulfonyl chloride, which aﬀorded the title compound

1

compound (314 mg, 74% over two steps) as a purple oil. H NMR

1

(69 mg, 39% over two steps) as a light-pink solid. H NMR (400

(400 MHz, CDCl₃) δ 8.16−8.11 (m, 1H), 8.05−8.00 (m, 1H), 7.93

(d, J = 7.72 Hz, 1H), 7.78 (d, J = 7.72 Hz, 1H), 7.54−7.48 (m, 3H),

7.42−7.32 (m, 2H), 7.19 (s, 1H), 7.13 (s, 1H), 3.94 (s, 3H), 3.68 (s,

3H), 3.49 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.52, 154.35,

138.58, 135.17, 133.92, 132.04, 130.52, 128.75, 127.83, 127.55, 127.11,

127.07, 125.00, 123.16, 122.90, 122.26, 119.94, 61.40, 52.50, 35.13.

ESI MS: m/z 495.8, 597.8 (M + H)⁺, 517.8, 519.8 (M + Na)⁺.

Methyl 2-((4-(Benzo[b]thiophene-2-sulfonamido)-1-methoxy-

naphthalen-2-yl)thio)acetate (49m). Synthesized using the proce-

dure for 49f except using 1-benzothiophene-2-sulfonyl chloride, which

aﬀorded the title compound (132 mg, 57% over two steps) as a purple

MHz, CDCl₃) δ 8.13−8.08 (m, 1H), 8.05−8.00 (m, 1H), 7.65 (s, 1H),

7.60−7.54 (m, 2H), 6.80 (s, 1H), 3.99 (s, 3H), 3.75 (s, 2H), 3.69 (s,

3H), 3.04 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.98, 154.06,

129.61, 128.85, 127.84, 127.36, 127.19, 125.15, 123.67, 122.67, 122.07,

61.50, 52.64, 39.91, 35.00. ESI MS: m/z 355.9 (M + H)⁺, 377.9 (M +

Na)⁺.

Methyl 2-((1-Methoxy-4-(phenylsulfonamido)naphthalen-2-yl)-

thio)acetate (49h). Synthesized using the procedure for 49g except

using benzenesulfonyl chloride, which aﬀorded the title compound

(119 mg, 64% over two steps) as a purple oil which solidiﬁed upon

1

standing. H NMR (400 MHz, CDCl₃) δ 8.01 (d, J = 8.40 Hz, 1H),

oil which solidiﬁed upon standing. H NMR (400 MHz, CDCl₃) δ

7.74 (t, J = 9.06 Hz, 3H), 7.47 (q, J = 8.19, 8.80 Hz, 2H), 7.36 (t, J =

7.61 Hz, 3H), 7.29 (s, 1H), 6.93 (s, 1H), 3.95 (s, 3H), 3.68 (s, 3H),

3.61 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.80, 154.02, 139.11,

132.95, 129.95, 128.98, 128.63, 127.82, 127.33, 126.95, 126.90, 125.67,

123.36, 122.31, 122.28, 61.49, 52.63, 35.06. ESI MS: m/z 417.9 (M +

H)⁺, 439.9 (M + Na)⁺.

8.02 (d, J = 8.40 Hz, 1H), 7.90 (d, J = 8.40 Hz, 1H), 7.79 (d, J = 7.99

Hz, 1H), 7.74 (d, J = 7.99 Hz, 1H), 7.69 (s, 1H), 7.48−7.42 (m, 2H),

7.41 (s, 1H), 7.40−7.35 (m, 2H), 7.01 (s, 1H), 3.96 (s, 3H), 3.64 (s,

3H), 3.51 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.77, 154.23,

141.85, 139.53, 137.38, 130.61, 130.05, 128.66, 127.47, 127.37, 127.06,

125.72, 125.47, 125.44, 123.47, 122.62, 122.34, 122.25, 61.51, 52.58,

34.95. ESI MS: m/z 473.9 (M + H)⁺, 495.8 (M + Na)⁺.

Methyl 2-((4-(4-Chlorophenylsulfonamido)-1-methoxynaphtha-

len-2-yl)thio)acetate (49i). Synthesized using the procedure for 49f

except using 4-chlorobenzenesulfonyl chloride, which aﬀorded the title

Methyl 2-((4-([1,1′-Biphenyl]-4-ylsulfonamido)-1-methoxynaph-

thalen-2-yl)thio)acetate (49n). Synthesized using the procedure for

49f except using 4-biphenylsulfonyl chloride, which aﬀorded the title

compound (152 mg, 71% over two steps) as a purple oil which

1

compound (280 mg, 67% over two steps) as a light-pink solid. H

NMR (400 MHz, CDCl₃) δ 8.02 (d, J = 8.40 Hz, 1H), 7.74 (d, J =

8.40 Hz, 1H), 7.66 (t, J = 1.96 Hz, 1H), 7.65 (t, J = 1.96 Hz, 1H), 7.47

(ddd, J = 1.17, 6.86, 8.24 Hz, 1H), 7.38 (ddd, J = 1.17, 6.86, 8.24 Hz,

1H), 7.35−7.33 (m, 2H), 7.32 (t, J = 1.94 Hz, 1H), 7.07 (s, 1H), 3.95

(s, 3H), 3.69 (s, 3H), 3.63 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ

169.84, 154.18, 139.50, 137.67, 129.86, 129.25, 128.78, 128.66, 127.49,

127.05, 125.81, 123.42, 122.41, 122.14, 61.50, 52.64, 35.02. ESI MS:

m/z 451.9 (M + H)⁺, 473.8 (M + Na)⁺.

1

solidiﬁed upon standing. H NMR (400 MHz, CDCl₃) δ 8.02 (d, J =

8.34 Hz, 1H), 7.83−7.76 (m, 3H), 7.58 (d, J = 8.31 Hz, 2H), 7.51 (d, J

= 7.14 Hz, 2H), 7.49−7.42 (m, 2H), 7.42−7.35 (m, 3H), 7.33 (s, 1H),

6.90 (s, 1H), 3.96 (s, 3H), 3.65 (s, 3H), 3.60 (s, 2H). ¹³C NMR (100

MHz, CDCl₃) δ 169.75, 153.99, 145.89, 139.09, 137.68, 129.97,

128.98, 128.66, 128.50, 127.90, 127.86, 127.56, 127.23, 126.98, 126.93,

125.58, 123.43, 122.35, 122.33, 61.49, 52.58, 35.02. ESI MS: m/z 494.1

(M + H)⁺, 516.1 (M + Na)⁺.

Methyl 2-((4-([1,1′-Biphenyl]-3-ylsulfonamido)-1-methoxynaph-

thalen-2-yl)thio)acetate (49o). Synthesized using reported proce-

dures with modiﬁcation.^69,70A stirred mixture of 49k (307 mg, 0.62

mmol), phenylboronic acid (113 mg, 0.91 mmol), 2 M aqueous

Na₂CO₃(0.93 mL), and Pd(PPh₃)₄(72 mg, 0.06 mmol) in THF (6

mL) /H₂O (1 mL) was heated at 60 °C under nitrogen for 2 h.

Mixture was diluted with EtOAc (10 mL) and washed with H₂O (15

Methyl 2-((4-(4-Bromophenylsulfonamido)-1-methoxynaphtha-

len-2-yl)thio)acetate (41). Synthesized using the procedure for 49f

except using 4-bromobenzenesulfonyl chloride, which aﬀorded the title

compound (185 mg, 61% over two steps) as a pink/purple solid.

1

HPLC (method A, t_R= 7.66 min), purity 99%. H NMR (400 MHz,

CDCl₃) δ 8.04 (d, J = 8.38 Hz, 1H), 7.72 (d, J = 8.38 Hz, 1H), 7.61−

7.59 (m, 1H), 7.58 (t, J = 2.04 Hz, 1H), 7.54−7.47 (m, 3H), 7.44−

7.38 (m, 1H), 7.34 (s, 1H), 6.76 (s, 1H), 3.97 (s, 3H), 3.71 (s, 3H),

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mL × 2) and brine (15 mL). Organic layer was dried (MgSO₄),

ﬁltered, and concentrated under reduced pressure. The crude was

puriﬁed by ﬂash column chromatography on silica to give 49o (284

mg, 92%) as a light-brown oil. ¹H NMR (400 MHz, CDCl₃/ couple of

drops of D₂O) δ 8.00 (d, J = 8.39 Hz, 1H), 7.83 (s, 1H), 7.79−7.73

(m, 2H), 7.69 (dd, J = 7.58, 14.09 Hz, 2H), 7.47−7.40 (m, 2H), 7.37−

7.31 (m, 6H), 3.92 (s, 3H), 3.62 (s, 3H), 3.58 (s, 2H). ¹³C NMR (100

MHz, CDCl₃/ couple of drops of D₂O) δ 169.86, 154.00, 142.22,

139.49, 138.99, 133.60, 131.46, 131.02, 130.00, 129.44, 128.89, 128.63,

128.14, 127.89, 127.03, 126.98, 126.88, 125.92, 125.87, 125.80, 123.46,

122.38, 122.27, 61.43, 52.60, 34.97. ESI MS: m/z 494.1 (M + H)⁺.

Methyl 2-((4-([1,1′-Biphenyl]-2-ylsulfonamido)-1-methoxynaph-

thalen-2-yl)thio)acetate (49p). Synthesized using the procedure for

49f except using 2-biphenylsulfonyl chloride which was synthesized as

previously reported.⁸³The title compound (57 mg, 58% over two

steps) was obtained as a light-orange oil. ¹H NMR (400 MHz, CDCl₃)

δ 8.16 (d, J = 8.20 Hz, 1H), 8.00 (d, J = 8.20 Hz, 1H), 7.62 (t, J = 7.54

Hz, 1H), 7.51 (q, J = 7.13, 7.54 Hz, 3H), 7.44−7.37 (m, 2H), 7.37−

7.29 (m, 5H), 6.93 (s, 1H), 5.86 (s, 1H), 3.91 (s, 3H), 3.67 (s, 3H),

3.44 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.66, 152.79, 140.89,

138.72, 138.15, 132.77, 132.73, 129.66, 129.43, 128.59, 128.52, 128.39,

128.20, 128.11, 127.94, 127.07, 126.69, 123.16, 122.20, 122.04, 120.40,

61.44, 52.62, 34.96. ESI MS: m/z 494.0 (M + H)⁺.

MHz, CDCl₃) δ 169.87, 161.82, 160.79, 158.36, 153.87, 150.83 (d, J =

2.72 Hz), 132.65, 129.83, 129.66, 128.61, 127.99, 126.88 (d, J = 4.95

Hz), 125.47, 123.39, 122.35 (d, J = 6.41 Hz), 121.67 (d, J = 8.41 Hz),

117.23, 116.86, 116.63, 61.50, 52.69, 35.06. ESI MS: m/z 528.1 (M +

H)⁺, 550.1 (M + Na)⁺.

Methyl 2-((1-Methoxy-4-(4-phenylpiperazine-1-sulfonamido)-

naphthalen-2-yl)thio)acetate (49u). As reported previously,⁶⁸to a

solution of 1,1′-sulfonyldiimidazole (1.98 g, 10 mmol) in CH₂Cl₂(40

mL) was added methyl triﬂuoromethanesulfonate (1.64 g, 10 mmol)

at 0 °C. The solvent was removed after 3 h stirring. To the resulting

residue in CH₃CN (40 mL) was added 1-phenylpiperazine (1.08 g,

6.67 mmol). After being stirred at room temperature overnight, the

reaction mixture was concentrated under reduced pressure, and the

crude was puriﬁed using ﬂash column chromatography on silica to give

1-((1H-imidazol-1-yl)sulfonyl)-4-phenylpiperazine (1.17 g, 60% yield

over 2 steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s,

1H), 7.30−7.23 (m, 3H), 7.16 (s, 1H), 6.93 (t, J = 7.33 Hz, 1H),

6.89−6.84 (m, 2H), 3.37−3.30 (m, 4H), 3.26−3.20 (m, 4H). ¹³C

NMR (100 MHz, CDCl₃) δ 150.21, 136.62, 130.80, 129.34, 121.43,

117.64, 117.20, 48.89, 46.42. ESI MS: m/z 293.0 (M + H)⁺.

To a solution of 1-((1H-imidazol-1-yl)sulfonyl)-4-phenylpiperazine

(85 mg, 0.29 mmol) in CH₂Cl₂(3 mL) cooled at 0 °C was added

methyl triﬂuoromethanesulfonate (0.035 mL, 0.32 mmol). After being

stirred for 2 h at 0 °C, the reaction mixture was concentrated under

reduced pressure to give 3-methyl-1-((4-phenylpiperazin-1-yl)-

sulfonyl)-1H-imidazol-3-ium as a beige solid which was used in the

next step without further puriﬁcation.

Methyl 2-((4-(4′-Chloro-[1,1′-biphenyl]-4-ylsulfonamido)-1-me-

thoxynaphthalen-2-yl)thio)acetate (49q). Synthesized using the

procedure for 49f except using 4′-chlorobiphenyl-4-sulfonyl chloride,

which aﬀorded the title compound (284 mg, 80% over two steps) as a

pink oil which solidiﬁed upon standing. ¹H NMR (400 MHz, CDCl₃)

δ 8.02 (d, J = 7.14 Hz, 1H), 7.80 (d, J = 7.14 Hz, 1H), 7.78 (d, J = 7.54

Hz, 2H), 7.54 (d, J = 7.54 Hz, 2H), 7.49−7.44 (m, 2H), 7.44−7.40

(m, 3H), 7.40−7.35 (m, 1H), 7.33 (s, 1H), 6.97 (s, 1H), 3.96 (s, 3H),

3.65 (s, 3H), 3.61 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.73,

154.05, 144.56, 138.08, 137.53, 134.79, 129.97, 129.19, 128.67, 128.48,

128.01, 127.80, 127.40, 126.98, 126.93, 125.64, 123.40, 122.35, 122.33,

61.50, 52.60, 35.03. ESI MS: m/z 528.1 (M + H)⁺, 550.1 (M + Na)⁺.

Methyl 2-((4-(2′,4′-Diﬂuoro-[1,1′-biphenyl]-4-ylsulfonamido)-1-

methoxynaphthalen-2-yl)thio)acetate (49r). Synthesized using the

procedure for 49f except using 2′,4′-diﬂuoro-biphenyl-4-sulfonyl

chloride, which aﬀorded the title compound (75 mg, 58% over two

A solution of 3-methyl-1-((4-phenylpiperazin-1-yl)sulfonyl)-1H-

imidazol-3-ium (0.29 mmol) and aniline 49a (0.29 mmol) in

CH₃CN (3 mL) was stirred at 80 °C for 15 h. The reaction mixture

was diluted with EtOAc (10 mL), washed with 1 N HCl, 1 N NaOH,

H₂O, and brine, dried (MgSO₄), and ﬁltered. The solvent was

removed under reduced pressure, and the crude was puriﬁed using

ﬂash column chromatography on silica to give 49u (36 mg, 25% over

1

three steps) as a purple solid. H NMR (400 MHz, CDCl₃) δ 8.12−

8.06 (m, 1H), 8.06−8.00 (m, 1H), 7.71 (s, 1H), 7.59−7.52 (m, 2H),

7.22 (t, J = 7.94 Hz, 2H), 6.88−6.81 (m, 3H), 6.75 (s, 1H), 3.98 (s,

3H), 3.74 (s, 2H), 3.68 (s, 3H), 3.43−3.37 (m, 4H), 3.15−3.08 (m,

4H). ¹³C NMR (100 MHz, CDCl₃) δ 169.86, 153.45, 150.61, 129.16,

128.93, 128.69, 128.57, 127.15, 127.07, 123.77, 123.51, 122.71, 121.71,

120.67, 116.73, 61.50, 52.59, 49.15, 46.48, 35.20. ESI MS: m/z 502.1

(M + H)⁺, 524.1 (M + Na)⁺.

1

steps) as a purple oil which solidiﬁed upon standing. H NMR (400

MHz, CDCl₃) δ 8.02 (d, J = 8.41 Hz, 1H), 7.83−7.76 (m, 3H), 7.50

(d, J = 8.16 Hz, 2H), 7.46 (d, J = 7.94 Hz, 1H), 7.40 (d, J = 7.94 Hz,

1H), 7.37−7.29 (m, 2H), 6.98 (s, 1H), 6.96−6.86 (m, 2H), 3.96 (s,

3H), 3.66 (s, 3H), 3.61 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ

169.84, 154.01, 139.74, 138.18, 131.40 (dd, J = 4.57, 9.75 Hz), 130.03,

129.43 (d, J = 3.03 Hz), 128.65, 127.76, 127.61, 127.02, 126.96,

125.70, 123.44, 122.37, 122.32, 111.98 (dd, J = 3.71, 21.44 Hz),

105.88−103.01 (m), 61.51, 52.63, 34.95. ESI MS: m/z 530.1 (M +

H)⁺.

Methyl 2-((4-(4-Bromobenzamido)-1-methoxynaphthalen-2-yl)-

thio)acetate (49v). Synthesized using the procedure for 49f except

using 4-bromobenzoyl chloride and Et₃N as the base, which aﬀorded

1

the title compound (245 mg, 66%) as a pink solid. H NMR (400

MHz, CDCl₃) δ 8.14 (s, 1H), 8.09 (d, J = 8.38 Hz, 1H), 7.88 (s, 1H),

7.82−7.73 (m, 3H), 7.63−7.56 (m, 2H), 7.56−7.43 (m, 2H), 3.98 (s,

3H), 3.74 (s, 2H), 3.68 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

170.08, 165.42, 153.22, 133.17, 132.01, 131.74, 131.53, 128.82, 128.59,

128.43, 126.80, 126.77, 123.92, 123.50, 122.67, 121.57, 61.45, 52.57,

35.18. ESI MS: m/z 460.0, 462.0 (M + H)⁺, 482.0, 484 (M + Na)⁺.

Methyl 2-((4-((4-Chlorophenyl)methylsulfonamido)-1-methoxy-

naphthalen-2-yl)thio)acetate (49w). Synthesized using the proce-

dure for 49f except using 4-chlorobenzylsulfonyl chloride, which

aﬀorded the title compound (110 mg, 41% over two steps) as a light-

pink solid. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 8.31 Hz, 1H),

7.75 (d, J = 8.31 Hz, 1H), 7.65 (s, 1H), 7.57 (t, J = 7.54 Hz, 1H), 7.50

(t, J = 7.54 Hz, 1H), 7.22−7.16 (m, 4H), 6.73 (s, 1H), 4.37 (s, 2H),

4.00 (s, 3H), 3.75 (s, 2H), 3.70 (s, 3H). ¹³C NMR (100 MHz, CDCl₃)

δ 170.00, 153.34, 135.04, 132.09, 128.91, 128.74, 128.31, 127.98,

127.18, 127.08, 126.88, 123.66, 122.67, 122.59, 121.65, 61.55, 57.41,

52.66, 34.96. ESI MS: m/z 465.8 (M + H)⁺, 487.8 (M + Na)⁺.

Methyl 2-((4-(2-(4-Chlorophenyl)acetamido)-1-methoxynaph-

thalen-2-yl)thio)acetate (49x). Synthesized using the procedure for

49f except using 4-chlorophenylacetyl chloride and Et₃N as the base,

which aﬀorded the title compound (208 mg, 69% over two steps) as a

light-yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.34 Hz,

1H), 7.82 (s, 1H), 7.49 (m, 1H), 7.45−7.38 (m, 4H), 7.38−7.32 (m,

3H), 3.94 (s, 3H), 3.82 (s, 2H), 3.73 (s, 2H), 3.67 (s, 3H). ¹³C NMR

Methyl 2-((1-Methoxy-4-(4-phenoxyphenylsulfonamido)-

naphthalen-2-yl)thio)acetate (49s). Synthesized using the procedure

for 49f except using 4-phenoxybenzenesulfonyl chloride, which

aﬀorded the title compound (244 mg, 72% over two steps) as a

1

light-brown oil. H NMR (400 MHz, CDCl₃) δ 8.02 (d, J = 8.43 Hz,

1H), 7.80 (d, J = 8.43 Hz, 1H), 7.66 (d, J = 8.14 Hz, 2H), 7.52−7.46

(m, 1H), 7.42−7.32 (m, 4H), 7.23 (s, 1H), 7.17 (t, J = 7.38 Hz, 1H),

6.94 (d, J = 8.52 Hz, 2H), 6.87 (d, J = 8.52 Hz, 2H), 3.95 (s, 3H), 3.69

(s, 3H), 3.66 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.85, 161.65,

155.11, 153.85, 132.59, 130.09, 129.85, 129.59, 128.60, 128.03, 126.90,

126.85, 125.50, 124.81, 123.42, 122.38, 122.31, 120.02, 117.64, 61.50,

52.67, 35.05. ESI MS: m/z 509.8 (M + H)⁺.

Methyl 2-((4-(4-(4-Fluorophenoxy)phenylsulfonamido)-1-me-

thoxynaphthalen-2-yl)thio)acetate (49t). Synthesized using the

procedure for 49f except using 4-(4-ﬂuoro-phenoxy)-benzenesulfonyl

chloride, which aﬀorded the title compound (93 mg, 72% over two

1

steps) as a yellow oil which solidiﬁed upon standing. H NMR (400

MHz, CDCl₃) δ 8.02 (d, J = 8.42 Hz, 1H), 7.80 (d, J = 8.42 Hz, 1H),

7.66 (d, J = 8.72 Hz, 2H), 7.51−7.45 (m, 1H), 7.42−7.36 (m, 1H),

7.34 (s, 1H), 7.07−7.00 (m, 2H), 6.94−6.88 (m, 2H), 6.84 (d, J = 8.72

Hz, 2H), 3.95 (s, 3H), 3.69 (s, 3H), 3.66 (s, 2H). ¹³C NMR (100

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(100 MHz, CDCl₃) δ 170.16, 169.79, 153.07, 133.95, 132.72, 130.93,

129.52, 128.47, 128.24, 127.90, 126.80, 126.72, 123.43, 123.32, 122.67,

120.80, 61.41, 52.59, 43.65, 35.14. ESI MS: m/z 429.9 (M + H)⁺,

451.8 (M + Na)⁺.

except using 49e and 4-bromobenzenesulfonyl chloride, which

aﬀorded the title compound (226 mg, 49% over two steps) as a

light-pink solid. HPLC (method A, t_R= 6.94 min), purity 96%. H

1

NMR (400 MHz, DMSO-d₆) δ 10.09 (s, 1H), 7.99−7.91 (m, 2H),

7.70 (d, J = 8.43 Hz, 2H), 7.54 (d, J = 8.43 Hz, 2H), 7.49 (t, J = 7.34

Hz, 1H), 7.40 (t, J = 7.34 Hz, 1H), 6.81 (s, 1H), 4.33 (t, J = 5.42 Hz,

1H), 3.78 (s, 3H), 3.37 (q, J = 5.42 Hz, 2H), 2.59 (t, J = 7.01 Hz, 2H),

1.49−1.28 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 152.35,

139.45, 132.62, 130.46, 130.42, 129.33, 128.54, 128.24, 126.97, 126.82,

126.78, 126.02, 124.16, 122.21, 62.36, 60.92, 32.66, 28.85, 26.90. ESI

MS: m/z 464.1, 466.1 (M + H)⁺, 486.1, 488.1 (M + Na)⁺. ESI HRMS:

m/z 462.0378 (M − H)⁻.

Methyl 2-((1-Methoxy-4-(2-phenylacetamido)naphthalen-2-yl)-

thio)acetate (49y). Synthesized using the procedure for 49f except

using phenylacetyl chloride. Crude was triturated with cold methylene

chloride to yield the title compound (154 mg, 49% over two steps) as

1

a white solid. H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 8.38 Hz,

1H), 7.94 (s, 1H), 7.53−7.44 (m, 4H), 7.40 (q, J = 7.27 Hz, 2H), 7.33

(s, 1H), 7.29−7.24 (m, 2H), 3.96 (s, 3H), 3.89 (s, 2H), 3.78 (s, 2H),

3.71 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.10, 169.71, 152.55,

134.54, 129.69, 129.46, 128.70, 128.40, 127.92, 127.64, 126.59, 123.48,

122.57, 122.48, 120.78, 61.40, 52.61, 44.64, 35.16. ESI MS: m/z 396.1

(M + H)⁺, 418.1 (M + Na)⁺.

4-Bromo-N-(3-(butylthio)-4-methoxynaphthalen-1-yl)-

benzenesulfonamide (49z). Synthesized using the procedure for 49f

except using 49c and 4-bromobenzenesulfonyl chloride, which

aﬀorded the title compound (55 mg, 57% over two steps) as a light-

brown oil which solidiﬁed. ¹H NMR (400 MHz, CDCl₃) δ 8.01 (d, J =

8.44 Hz, 1H), 7.77 (d, J = 8.44 Hz, 1H), 7.59 (d, J = 8.36 Hz, 2H),

7.49 (d, J = 8.36 Hz, 2H), 7.46 (d, J = 7.85 Hz, 1H), 7.35 (t, J = 7.85

Hz, 1H), 7.21 (s, 1H), 7.16 (s, 1H), 3.94 (s, 3H), 2.81 (t, J = 7.24 Hz,

2H), 1.58 (p, J = 7.24 Hz, 2H), 1.45 (h, J = 7.24 Hz, 2H), 0.92 (t, J =

7.24 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 152.71, 138.26, 132.23,

128.92, 128.88, 128.65, 127.99, 127.25, 126.93, 126.37, 125.79, 124.49,

122.19, 122.04, 61.01, 31.92, 31.20, 21.95, 13.67. ESI MS: m/z 479.8,

481.8 (M + H)⁺.

N-(3-(((2H-Tetrazol-5-yl)methyl)thio)-4-methoxynaphthalen-1-

yl)-4-bromobenzenesulfonamide (49cc). Synthesized using a re-

ported procdure.⁷²To a suspension of 42 (127 mg, 0.26 mmol) and

sodium azide (255 mg, 3.9 mmol) in dry CH₃CN (5 mL) in a glass

tube was added SiCl₄(0.15 mL, 1.3 mmol) via syringe. The tube was

sealed, and the stirring reaction mixture was heated to 80 °C for 15 h.

The reaction was quenched with 2 M Na₂CO₃. The solution was then

washed with EtOAc (10 mL × 2). The aqueous phase was acidiﬁed

with 1 N HCl, and the mixture was extracted with EtOAc (15 mL ×

5). The combined organic extracts were washed with brine (20 mL),

dried (MgSO₄), ﬁltered, and concentrated under reduced pressure to

give 49cc (66 mg, 50%) as a tan solid. The crude was used in the next

1

step without further puriﬁcation. H NMR (400 MHz, DMSO-d₆) δ

10.31 (s, 1H), 7.93 (d, J = 8.40 Hz, 1H), 7.83 (d, J = 8.40 Hz, 1H),

7.65 (d, J = 8.04 Hz, 2H), 7.55−7.48 (m, 3H), 7.44−7.38 (m, 1H),

7.14 (s, 1H), 6.53 (s, 1H), 4.42 (s, 2H), 3.78 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ 153.77, 139.46, 132.64, 130.46, 129.17, 129.07,

128.36, 127.58, 127.06, 126.23, 123.97, 122.53, 122.22, 61.68, 25.09.

ESI MS: m/z 505.8, 507.8 (M + H)⁺, 527.8, 529.8 (M + Na)⁺.

N-(3-(((2H-Tetrazol-5-yl)methyl)thio)-4-methoxynaphthalen-1-

yl)-4-phenoxybenzenesulfonamide (49dd). Synthesized using the

procedure for 49cc except using 49aa as the starting material which

2-((4-(4-Bromophenylsulfonamido)-1-methoxynaphthalen-2-yl)-

thio)acetamide (42). Synthesized using a reported procedure.⁷¹

Compound 41 (82 mg, 0.16 mmol) was added to aqueous NH₄OH

(29%, 3 mL). The mixture was stirred at room temperature for 1 h.

Workup included diluting the mixture with EtOAc (10 mL) and

washing with H₂O (10 mL × 2) and brine (10 mL). Organic layer was

dried (MgSO₄), ﬁltered, and concentrated under reduced pressure to

give 42 (69 mg, 90%) as a pink solid. Crude was used in the next step

without further puriﬁcation. HPLC (method A, t_R= 6.50 min), purity

1

aﬀorded the title compound (128 mg, 95%) as a tan solid. H NMR

(500 MHz, DMSO-d₆) δ 10.14 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.87

(d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.56 (t, J = 7.5 Hz, 1H),

7.48−7.43 (m, 1H), 7.40 (t, J = 7.4 Hz, 2H), 7.20 (t, J = 7.3 Hz, 1H),

7.17 (s, 1H), 7.01−6.94 (m, 4H), 4.44 (s, 2H), 3.79 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ 160.90, 155.45, 153.57, 134.18, 130.75,

130.55, 129.76, 129.50, 128.35, 127.49, 126.94, 126.14, 125.17, 124.15,

122.62, 122.16, 120.13, 118.27, 61.66, 25.13. ESI MS: m/z 517.9 (M −

H)⁻.

1

98%. H NMR (400 MHz, DMSO-d₆) δ 10.32 (s, 1H), 7.95 (d, J =

8.50 Hz, 1H), 7.85 (d, J = 8.50 Hz, 1H), 7.72 (d, J = 8.20 Hz, 2H),

7.60 (d, J = 8.20 Hz, 2H), 7.56−7.50 (m, 2H), 7.43−7.37 (m, 1H),

7.21 (s, 1H), 7.15 (s, 1H), 3.88 (s, 3H), 3.53 (s, 2H). ¹³C NMR (100

MHz, DMSO-d₆) δ 169.76, 151.88, 139.67, 132.70, 129.69, 129.18,

129.05, 128.23, 127.46, 127.03, 126.41, 125.24, 124.89, 123.95, 121.82,

61.27, 35.91. ESI MS: m/z 480.7, 482.7 (M + H)⁺, 502.7, 504.7 (M +

Na)⁺. ESI HRMS: m/z 478.9742 (M − H)⁻.

Methyl 3-((1-Methoxy-4-(naphthalene-2-sulfonamido)-

naphthalen-2-yl)thio)propanoate (49ee). Synthesized using the

procedure for 49f except using 49b and 2-naphthalenesulfonyl

chloride, which aﬀorded the title compound (180 mg, 59% over two

2-((1-Methoxy-4-((4-phenoxyphenyl)sulfonamido)naphthalen-2-

yl)thio)acetamide (49aa). Synthesized using the procedure for 42

except using 49s as the starting material, which aﬀorded the title

1

steps) as a light-pink solid. H NMR (400 MHz, CDCl₃) δ 8.30 (s,

1

1H), 7.98 (d, J = 8.39 Hz, 1H), 7.90−7.85 (m, 2H), 7.85−7.80 (m,

2H), 7.80−7.76 (m, 1H), 7.59 (t, J = 7.36 Hz, 1H), 7.53 (t, J = 7.36

Hz, 1H), 7.43 (t, J = 7.39 Hz, 1H), 7.34 (t, J = 7.39 Hz, 1H), 7.19 (s,

1H), 6.98 (s, 1H), 3.89 (s, 3H), 3.64 (s, 3H), 2.94 (t, J = 7.23 Hz, 2H),

2.40 (t, J = 7.23 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 171.84,

153.85, 135.99, 134.83, 131.94, 129.66, 129.43, 129.20, 129.02, 128.91,

128.75, 127.82, 127.78, 127.54, 126.95, 126.72, 125.21, 123.73, 122.39,

122.19, 109.99, 61.20, 51.79, 34.05, 27.65. ESI MS: m/z 481.9 (M +

H)⁺, 503.9 (M + Na)⁺.

compound (179 mg, quantitative) as a light-purple solid. H NMR

(500 MHz, DMSO-d₆) δ 10.14 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.87

(d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.56−7.46 (m, 2H), 7.41

(t, J = 7.0 Hz, 2H), 7.39−7.33 (m, 1H), 7.23−7.14 (m, 2H), 7.11 (s,

1H), 7.04−6.94 (m, 4H), 3.82 (s, 3H), 3.50 (s, 2H). ¹³C NMR (125

MHz, DMSO-d₆) δ 169.84, 155.74, 130.72, 129.77, 129.55, 128.23,

127.07, 125.57, 125.09, 124.94, 124.55, 121.45, 120.02, 118.17, 109.99,

61.17, 36.13. ESI MS: m/z 495.0 (M + H)⁺, 517.0 (M + Na)⁺.

4-Bromo-N-(3-(3-hydroxypropyl)-4-methoxynaphthalen-1-yl)-

benzenesulfonamide (49bb). Synthesized using the procedure for 49f

except using 49d and 4-bromobenzenesulfonyl chloride, which

aﬀorded the title compound (163 mg, 33% over two steps) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.11 (s, 1H), 7.94 (t, J

= 7.53 Hz, 2H), 7.70 (d, J = 8.53 Hz, 2H), 7.53 (d, J = 8.53 Hz, 2H),

7.51−7.45 (m, 1H), 7.42−7.35 (m, 1H), 6.86 (s, 1H), 4.47 (s, 1H),

3.79 (s, 3H), 3.37 (t, J = 5.87 Hz, 2H), 2.69−2.60 (m, 2H), 1.59−1.49

(m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 152.33, 139.44, 132.62,

130.38, 130.34, 129.28, 128.55, 128.23, 127.00, 126.88, 126.78, 126.01,

124.09, 122.21, 62.33, 60.67, 33.79, 25.69. ESI MS: m/z 449.9, 451.9

(M + H)⁺, 471.9, 473.9 (M + Na)⁺.

2-((4-(4-Bromophenylsulfonamido)-1-methoxynaphthalen-2-yl)-

thio)acetic Acid (40). To a solution of 41 (425 mg, 0.85 mmol) in dry

THF (2 mL) was added 1N aqueous LiOH (4 mL). The mixture was

stirred at room temperature under nitrogen for 1 h. Reaction mixture

was diluted with water (10 mL) and washed with EtOAc (10 mL × 2).

Aqueous phase was acidiﬁed with 1 N HCl and extracted with EtOAc

(10 mL × 3). Combined organic extracts were washed with brine,

dried (MgSO₄), and ﬁltered. The solvent was removed under reduced

pressure. Crude was triturated with cold CH₂Cl₂to give 40 (305 mg,

74%) as a white/tan solid. HPLC (method A, t_R= 6.85 min), purity

1

>99%. H NMR (400 MHz, DMSO-d₆) δ 10.32 (s, 1H), 7.92 (d, J =

8.38 Hz, 1H), 7.87 (d, J = 8.38 Hz, 1H), 7.68 (d, J = 8.08 Hz, 2H),

7.57 (d, J = 8.08 Hz, 2H), 7.51 (t, J = 7.56 Hz, 1H), 7.40 (t, J = 7.56

4-Bromo-N-(3-(4-hydroxybutyl)-4-methoxynaphthalen-1-yl)-

benzenesulfonamide (45). Synthesized using the procedure for 49f

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Journal of Medicinal Chemistry

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Hz, 1H), 7.09 (s, 1H), 3.84 (s, 3H), 3.65 (s, 2H). ¹³C NMR (100

MHz, DMSO-d₆) δ 170.59, 152.07, 139.60, 132.69, 129.83, 129.18,

129.13, 128.28, 127.54, 127.09, 126.57, 124.60, 124.56, 124.01, 121.86,

61.30, 34.63. ESI HRMS: m/z 479.9578 (M − H)⁻.

DMSO-d₆) δ 171.46, 152.56, 140.16, 133.03, 131.56, 129.53, 129.44,

127.25, 127.05, 126.17, 125.46, 124.56, 123.76, 122.75, 113.34, 37.16.

ESI HRMS: m/z 388.0319 (M − H)⁻.

2-((4-(4-Chlorophenylsulfonamido)-1-hydroxynaphthalen-2-yl)-

thio)acetic Acid (9). Synthesized using the procedure for 1 except 49i

was used as the starting material. The title compound (48 mg, 20%)

was obtained as a white solid after trituration with a mixture of

CH₃CN:H₂O 1:1 and cold CH₂Cl₂and without a need for HPLC

2-((4-([1,1′-Biphenyl]-4-ylsulfonamido)-1-methoxynaphthalen-2-

yl)thio)acetic Acid (43). Synthesized using the procedure for 40

except 49n was used as the starting material. Crude was triturated with

cold CH₂Cl₂to give 43 (88 mg, 70%) as an white solid. HPLC

(method A, t_R= 7.22 min), purity 98%. ¹H NMR (400 MHz, DMSO-

d₆) δ 12.74 (s, 1H), 10.21 (s, 1H), 7.95 (d, J = 8.44 Hz, 1H), 7.91 (d, J

= 8.44 Hz, 1H), 7.79−7.70 (m, 4H), 7.68−7.61 (m, 2H), 7.53−7.48

(m, 1H), 7.45 (t, J = 7.28 Hz, 2H), 7.39 (t, J = 7.28 Hz, 2H), 7.08 (s,

1H), 3.83 (s, 3H), 3.61 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ

170.52, 151.84, 144.73, 139.09, 138.87, 129.90, 129.50, 128.93, 128.26,

127.89, 127.78, 127.49, 126.47, 124.56, 124.21, 124.18, 121.77, 61.26,

34.56. ESI HRMS: m/z 478.0787 (M − H)⁻.

1

puriﬁcation. HPLC (method B, t_R= 6.72 min), purity 95%. H NMR

(400 MHz, DMSO-d₆) δ 12.77 (s, 1H), 10.03 (s, 1H), 9.84 (s, 1H),

8.15 (d, J = 8.12 Hz, 1H), 7.85 (d, J = 8.12 Hz, 1H), 7.66−7.59 (m,

2H), 7.59−7.52 (m, 2H), 7.46 (dt, J = 7.28, 14.85 Hz, 2H), 7.05 (s,

1H), 3.54 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.48, 152.72,

139.06, 137.95, 131.47, 129.66, 129.60, 129.19, 127.15, 126.25, 125.48,

124.24, 123.63, 122.84, 113.38, 37.11. ESI HRMS: m/z 421.9930 (M

− H)⁻.

2-((4-([1,1‴-Biphenyl]-3-ylsulfonamido)-1-methoxynaphthalen-

2-yl)thio)acetic Acid (44). Synthesized using the procedure for 40

except 49o was used as the starting material. Crude was triturated with

cold CH₂Cl₂to give 44 (152 mg, 59%) as a white solid. HPLC

(method A, t_R= 7.20 min), purity 98%. ¹H NMR (400 MHz, DMSO-

d₆) δ 12.69 (s, 1H), 10.20 (s, 1H), 7.90 (d, J = 8.46 Hz, 1H), 7.86 (d, J

= 8.46 Hz, 1H), 7.82 (d, J = 7.81 Hz, 1H), 7.72 (s, 1H), 7.63 (d, J =

7.81 Hz, 1H), 7.55 (t, J = 7.74 Hz, 1H), 7.50−7.44 (m, 1H), 7.43−

7.38 (m, 4H), 7.38−7.30 (m, 2H), 7.11 (s, 1H), 3.82 (s, 3H), 3.61 (s,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.46, 151.97, 141.49,

140.71, 138.92, 131.42, 130.33, 129.80, 129.49, 129.43, 128.66, 128.24,

127.47, 127.14, 126.43, 125.88, 125.21, 124.64, 124.59, 124.04, 121.77,

61.23, 34.56. ESI HRMS: m/z 478.0788 (M − H)⁻.

2-((4-(4-Bromophenylsulfonamido)-1-hydroxynaphthalen-2-yl)-

thio)acetic Acid (10). Synthesized using the procedure for 1 except 41

was used as the starting material. The title compound (134 mg, 45%)

was obtained as a white solid after trituration with a mixture of

CH₃CN:H₂O 1:1 and cold CH₂Cl₂and without a need for HPLC

1

puriﬁcation. HPLC (method A, t_R= 6.53 min), purity 99%. H NMR

(400 MHz, DMSO-d₆) δ 12.80 (s, 1H), 10.05 (s, 1H), 9.87 (s, 1H),

8.14 (d, J = 8.17 Hz, 1H), 7.84 (d, J = 8.17 Hz, 1H), 7.71 (d, J = 8.48

Hz, 2H), 7.54 (d, J = 8.48 Hz, 2H), 7.48 (t, J = 7.03 Hz, 1H), 7.43 (t, J

= 7.03 Hz, 1H), 7.04 (s, 1H), 3.53 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 171.52, 152.74, 139.45, 132.56, 131.45, 129.66, 129.28,

127.18, 126.92, 126.27, 125.48, 124.21, 123.63, 122.86, 113.38, 37.10.

ESI HRMS: m/z 465.9418 (M − H)⁻.

2-((1-Hydroxy-4-(4-methoxyphenylsulfonamido)naphthalen-2-

yl)thio)acetic Acid (11). Synthesized using the procedure for 1 except

49j was used as the starting material. The title compound (25 mg,

29%) was obtained as a white solid after HPLC puriﬁcation. HPLC

(method A, t_R= 6.20 min), purity 95%. ¹H NMR (400 MHz, DMSO-

d₆) δ 9.70 (s, 1H), 8.10 (d, J = 8.07 Hz, 1H), 7.89 (d, J = 7.78 Hz,

1H), 7.52 (d, J = 8.63 Hz, 2H), 7.49−7.36 (m, 2H), 7.00−6.93 (m,

3H), 3.75 (s, 3H), 3.47 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ

171.47, 162.75, 152.44, 131.81, 131.63, 129.49, 129.37, 127.05, 126.18,

125.48, 124.92, 123.93, 122.75, 114.57, 113.35, 56.02, 37.24. ESI

HRMS: m/z 418.0424 (M − H)⁻.

A Representative Procedure for a Single-Pot Ester Hydrol-

ysis and Demethylation with BBr₃. 2-((1-Hydroxy-4-(thiophene-

2-sulfonamido)naphthalen-2-yl)thio)acetic Acid (1). To a stirred

solution of 49f (48 mg, 0.12 mmol) suspended in dry CH₂Cl₂(1.5

mL) was added BBr₃(1 M in CH₂Cl₂, 0.5 mL, 0.5 mmol) dropwise at

0 °C under nitrogen. The mixture was allowed to warm up to room

temperature. After 1 h of stirring, the starting material was entirely

consumed and the product formed as determined by TLC and MS

(ESI⁻). The mixture was slowly added to a stirred solution of saturated

aqueous NH₄Cl (20 mL) at 0 °C. The solution was extracted with

EtOAc (15 mL × 2). The combined organic extracts were washed with

brine (15 mL), dried (MgSO₄), and ﬁltered, and the solvent was

removed under reduced pressure. The crude was puriﬁed using a C₁₈

reverse phase semipreparative HPLC column with solvent A (0.1% of

TFA in water) and solvent B (0.1% of TFA in CH₃CN) as eluents to

give 1 (59) (28 mg, 59%) as a white/tan solid. HPLC (method A, t_R=

2-((4-(3-Bromophenylsulfonamido)-1-hydroxynaphthalen-2-yl)-

thio)acetic Acid (12). Synthesized using the procedure for 1 except

49k was used as the starting material. The title compound (21 mg,

35%) was obtained as a white/tan solid after trituration with a mixture

of CH₃CN:H₂O 1:1 and cold CH₂Cl₂and without a need for HPLC

1

6.05 min), purity 92%. H NMR (400 MHz, DMSO-d₆) δ 10.10 (s,

puriﬁcation. HPLC (method A, t_R= 6.69 min), purity 94%. H NMR

1H), 8.17−8.14 (m, 1H), 7.89−7.84 (m, 2H), 7.52−7.40 (m, 2H),

7.36 (dd, J = 1.32, 3.74 Hz, 1H), 7.09 (s, 1H), 7.07 (dd, J = 3.74, 4.97

Hz, 1H), 3.56 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.54,

152.87, 140.58, 133.57, 132.80, 131.71, 129.80, 128.03, 127.20, 126.22,

125.46, 124.33, 123.61, 122.83, 113.36, 37.25. ESI HRMS: m/z

393.9870 (M − H)⁻.

2-((1-Hydroxy-4-(methylsulfonamido)naphthalen-2-yl)thio)-

acetic Acid (2). Synthesized using the procedure for 1 except 49t was

used as the starting material. The title compound (27 mg, 48%) as a

white solid after HPLC puriﬁcation. HPLC (method A, t_R= 5.14 min),

purity 97%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.40 (s, 1H), 8.20 (d, J

= 8.09 Hz, 1H), 8.14 (d, J = 8.09 Hz, 1H), 7.56 (p, J = 6.86 Hz, 2H),

7.47 (s, 1H), 3.69 (s, 2H), 2.99 (s, 3H). ¹³C NMR (100 MHz, DMSO-

d₆) δ 171.73, 152.58, 131.89, 129.38, 127.31, 126.30, 125.65, 125.18,

124.32, 122.82, 113.55, 39.79, 37.03. ESI HRMS: m/z 326.0164 (M −

H)⁻.

(400 MHz, DMSO-d₆) δ 10.08 (s, 1H), 8.15 (d, J = 7.56 Hz, 1H),

7.87−7.78 (m, 2H), 7.75 (t, J = 1.78 Hz, 1H), 7.62−7.56 (m, 1H),

7.52−7.39 (m, 3H), 7.01 (s, 1H), 3.52 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 171.52, 152.92, 142.07, 135.93, 131.78, 131.49, 129.77,

129.59, 127.18, 126.34, 126.28, 125.53, 124.02, 123.58, 122.89, 122.35,

113.43, 37.24. ESI HRMS: m/z 465.9414 (M − H)⁻.

2-((4-(2-Bromophenylsulfonamido)-1-hydroxynaphthalen-2-yl)-

thio)acetic Acid (13). Synthesized using the procedure for 1 except

49l was used as the starting material. The title compound (34 mg,

40%) was obtained as a white solid after HPLC puriﬁcation. HPLC

(method A, t_R= 6.41 min), purity 85%. ¹H NMR (400 MHz, DMSO-

d₆) δ 12.80 (s, 1H), 10.23 (s, 1H), 9.88 (s, 1H), 8.16−8.10 (m, 1H),

8.07−8.02 (m, 1H), 7.86 (dd, J = 1.52, 7.83 Hz, 1H), 7.78 (dd, J =

1.52, 7.83 Hz, 1H), 7.52−7.46 (m, 3H), 7.42 (td, J = 1.33, 7.60 Hz,

1H), 7.05 (s, 1H), 3.46 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ

171.42, 152.60, 139.23, 135.82, 134.64, 131.92, 131.77, 129.49, 128.52,

127.18, 126.32, 125.44, 123.98, 123.78, 122.78, 119.92, 113.54, 37.03.

ESI HRMS: m/z 465.9408 (M − H)⁻.

2-((1-Hydroxy-4-(phenylsulfonamido)naphthalen-2-yl)thio)acetic

Acid (3). Synthesized using the procedure for 1 except 49h was used as

the starting material. The title compound (21 mg, 22%) was obtained

as a white solid after HPLC puriﬁcation. HPLC (method A, t_R= 6.15

2-((4-(Benzo[b]thiophene-2-sulfonamido)-1-hydroxynaphthalen-

2-yl)thio)acetic Acid (14). Synthesized using the procedure for 1

except 49m was used as the starting material. The title compound (13

mg, 22%) was obtained as an orange solid after HPLC puriﬁcation.

1

min), purity 97%. H NMR (400 MHz, DMSO-d₆) δ 12.76 (s, 1H),

9.91 (s, 1H), 9.79 (s, 1H), 8.14 (d, J = 8.22 Hz, 1H), 7.88 (d, J = 8.22

Hz, 1H), 7.67−7.61 (m, 2H), 7.61−7.56 (m, 1H), 7.54−7.47 (m, 3H),

7.46−7.39 (m, 1H), 7.01 (s, 1H), 3.51 (s, 2H). ¹³C NMR (100 MHz,

1

HPLC (method B, t_R= 7.52 min), purity 99%. H NMR (400 MHz,

DMSO-d₆) δ 10.32 (s, 1H), 8.11 (d, J = 8.14 Hz, 1H), 8.02 (d, J = 8.14

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dx.doi.org/10.1021/jm500010b | J. Med. Chem. 2014, 57, 4111−4133

Journal of Medicinal Chemistry

Article

Hz, 1H), 7.89 (t, J = 8.09 Hz, 2H), 7.72 (s, 1H), 7.47 (t, J = 7.56 Hz,

1H), 7.41 (t, J = 7.35 Hz, 2H), 7.38−7.32 (m, 1H), 7.14 (s, 1H), 3.40

(s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.57, 153.29, 141.31,

141.04, 137.80, 131.73, 129.99, 129.94, 127.65, 127.22, 126.21, 125.86,

125.54, 123.96, 123.54, 123.41, 122.92, 113.33, 37.41. ESI HRMS: m/z

444.0041 (M − H)⁻.

2-((1-Hydroxy-4-(4-phenoxyphenylsulfonamido)naphthalen-2-

yl)thio)acetic Acid (21). Synthesized using the procedure for 1 except

49s was used as the starting material. The title compound (18 mg,

13%) was obtained as a white solid after HPLC puriﬁcation. HPLC

(method B, t_R= 10.24 min), purity 99%. ¹H NMR (400 MHz, DMSO-

d₆) δ 12.81 (s, 1H), 9.87 (s, 1H), 9.83 (s, 1H), 8.14 (d, J = 8.31 Hz,

1H), 7.86 (d, J = 8.31 Hz, 1H), 7.59 (d, J = 8.75 Hz, 2H), 7.50 (t, J =

7.09 Hz, 1H), 7.44 (t, J = 7.81 Hz, 3H), 7.23 (t, J = 7.09 Hz, 1H),

7.05−6.98 (m, 5H), 3.55 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ

171.53, 160.76, 155.60, 152.58, 134.17, 131.57, 130.75, 129.84, 129.76,

127.06, 126.16, 125.48, 125.07, 124.60, 123.78, 122.80, 120.03, 118.33,

113.38, 37.15. ESI HRMS: m/z 480.0579 (M − H)⁻.

2-((4-([1,1′-Biphenyl]-4-ylsulfonamido)-1-hydroxynaphthalen-2-

yl)thio)acetic Acid (16). Synthesized using the procedure for 1 except

49n was used as the starting material. The title compound (29 mg,

45%) was obtained as a white/tan solid after HPLC puriﬁcation.

1

HPLC (method B, t_R= 8.81 min), purity 99%. H NMR (400 MHz,

DMSO-d₆) δ 9.92 (s, 1H), 8.11 (d, J = 7.93 Hz, 1H), 7.90 (d, J = 7.93

Hz, 1H), 7.76 (d, J = 7.47 Hz, 2H), 7.72−7.62 (m, 4H), 7.50−7.42

(m, 3H), 7.43−7.36 (m, 2H), 7.02 (s, 1H), 3.46 (s, 2H). ¹³C NMR

(100 MHz, DMSO-d₆) δ 171.52, 152.74, 144.57, 139.00, 138.90,

131.65, 129.57, 129.52, 128.92, 128.00, 127.63, 127.46, 127.08, 126.18,

125.55, 124.53, 123.85, 122.81, 113.42, 37.27. ESI HRMS: m/z

464.0630 (M − H)⁻.

2-((4-(4-(4-Fluorophenoxy)phenylsulfonamido)-1-hydroxynaph-

thalen-2-yl)thio)acetic Acid (22). Synthesized using the procedure for

1 except 49t was used as the starting material. The title compound (36

mg, 44%) as a white solid after HPLC puriﬁcation. HPLC (method A,

1

t_R= 7.17 min), purity 95%. H NMR (400 MHz, DMSO-d₆) δ 12.78

(s, 1H), 9.83 (s, 1H), 9.79 (s, 1H), 8.11 (d, J = 8.21 Hz, 1H), 7.83 (d, J

= 8.21 Hz, 1H), 7.55 (d, J = 8.46 Hz, 2H), 7.44 (dt, J = 6.92, 21.86 Hz,

2H), 7.25 (t, J = 8.52 Hz, 2H), 7.11−7.02 (m, 2H), 6.99−6.96 (m,

2H), 6.95 (s, 1H), 3.51 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ

171.53, 161.09, 152.57, 151.54 (d, J = 2.57 Hz), 134.14, 131.59,

129.86, 129.68, 127.08, 126.19, 125.48, 124.61, 123.80, 122.80, 122.17

(d, J = 8.70 Hz), 117.90, 117.45, 117.22, 113.38, 37.17. ESI HRMS:

m/z 498.0485 (M − H)⁻.

2-((1-Hydroxy-4-(4-phenylpiperazine-1-sulfonamido)-

naphthalen-2-yl)thio)acetic Acid (23). Synthesized using the

procedure for 1 except 49u was used as the starting material. The

title compound (14.5 mg, 48%) was obtained as a white solid after

HPLC puriﬁcation. HPLC (method A, t_R= 6.55 min), purity 92%. ¹H

NMR (400 MHz, DMSO-d₆) δ 9.77 (s, 1H), 9.59 (s, 1H), 8.15 (t, J =

7.91 Hz, 2H), 7.59−7.53 (m, 1H), 7.53−7.48 (m, 2H), 7.20−7.14 (m,

2H), 6.89 (d, J = 7.91 Hz, 2H), 6.77 (t, J = 7.26 Hz, 1H), 3.64 (s, 2H),

3.24−3.16 (m, 4H), 3.14−3.04 (m, 4H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 171.57, 152.02, 150.84, 131.08, 129.40, 128.56, 127.32,

126.28, 125.52, 125.43, 123.76, 122.95, 120.04, 116.48, 113.63, 48.54,

46.36, 37.06. ESI HRMS: m/z 472.1002 (M − H)⁻.

2-((4-([1,1′-Biphenyl]-3-ylsulfonamido)-1-hydroxynaphthalen-2-

yl)thio)acetic Acid (17). Synthesized using the procedure for 1 except

49o was used as the starting material. The title compound (48 mg,

34%) was obtained as a white/tan solid after HPLC puriﬁcation.

1

HPLC (method A, t_R= 6.99 min), purity 95%. H NMR (400 MHz,

DMSO-d₆) δ 12.73 (s, 1H), 9.93 (s, 1H), 9.79 (s, 1H), 8.10 (d, J =

8.25 Hz, 1H), 7.88−7.81 (m, 2H), 7.79 (s, 1H), 7.60−7.50 (m, 2H),

7.50−7.41 (m, 4H), 7.41−7.32 (m, 3H), 7.04 (s, 1H), 3.45 (s, 2H).

¹³C NMR (100 MHz, DMSO-d₆) δ 171.44, 152.66, 141.46, 140.82,

139.01, 131.50, 131.30, 130.18, 129.60, 129.55, 128.66, 127.17, 127.07,

126.23, 126.07, 125.51, 125.26, 124.59, 123.73, 122.81, 113.44, 37.16.

ESI HRMS: m/z 464.0632 (M − H)⁻.

2-((4-([1,1′-Biphenyl]-2-ylsulfonamido)-1-hydroxynaphthalen-2-

yl)thio)acetic Acid (18). Synthesized using the procedure for 1 except

49p was used as the starting material. The title compound (20 mg,

43%) was obtained as a white/tan solid after HPLC puriﬁcation.

1

HPLC (method B, t_R= 8.95 min), purity 84%. H NMR (400 MHz,

DMSO-d₆) δ 12.77 (s, 1H), 9.76 (s, 1H), 9.70 (s, 1H), 8.12 (d, J =

8.03 Hz, 1H), 7.95 (d, J = 8.03 Hz, 1H), 7.74 (d, J = 8.30 Hz, 1H),

7.58 (t, J = 7.46 Hz, 1H), 7.52 (t, J = 7.46 Hz, 1H), 7.48−7.41 (m,

1H), 7.41−7.34 (m, 1H), 7.23 (q, J = 4.82, 5.81 Hz, 1H), 7.17 (t, J =

7.46 Hz, 3H), 7.01−6.95 (m, 1H), 6.93 (s, 1H), 6.91 (s, 1H), 3.43 (s,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.57, 152.24, 141.36,

139.92, 138.75, 133.21, 132.68, 131.42, 129.62, 129.37, 128.85, 128.36,

127.55, 127.48, 126.97, 126.21, 125.49, 124.57, 123.79, 122.69, 113.65,

37.07. ESI HRMS: m/z 464.0636 (M − H)⁻.

2-((4-(4′-Chloro-[1,1′-biphenyl]-4-ylsulfonamido)-1-hydroxy-

naphthalen-2-yl)thio)acetic Acid (19). Synthesized using the

procedure for 1 except 49q was used as the starting material. The

title compound (36 mg, 25%) was obtained as a white solid after

HPLC puriﬁcation. HPLC (method B, t_R= 10.23 min), purity >99%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.75 (s, 1H), 9.96 (s, 1H), 9.81 (s,

2-((4-(4-Bromobenzamido)-1-hydroxynaphthalen-2-yl)thio)-

acetic Acid (25). Synthesized using the procedure for 1 except 49v was

used as the starting material. The title compound was obtained (8.5

mg, 30%) as a white solid after HPLC puriﬁcation. HPLC (method A,

1

t_R= 6.72 min), purity 99%. H NMR (400 MHz, DMSO-d₆) δ 10.36

(s, 1H), 8.23 (d, J = 5.36 Hz, 1H), 8.02 (d, J = 8.05 Hz, 2H), 7.83 (d, J

= 5.36 Hz, 1H), 7.78 (d, J = 8.05 Hz, 2H), 7.57−7.50 (m, 3H), 3.72 (s,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.74, 165.71, 151.94,

133.90, 131.89, 130.66, 130.30, 128.64, 127.11, 126.17, 126.13, 125.80,

125.47, 123.80, 122.97, 113.65, 37.15. ESI HRMS: m/z 429.9747 (M

− H)⁻.

2-((4-((4-Chlorophenyl)methylsulfonamido)-1-hydroxynaphtha-

len-2-yl)thio)acetic Acid (26). Synthesized using the procedure for 1

except 49w was used as the starting material. The title compound

(15.5 mg, 20%) was obtained as a white solid after HPLC puriﬁcation.

1H), 8.10 (d, J = 8.09 Hz, 1H), 7.88 (d, J = 8.09 Hz, 1H), 7.77 (d, J =

8.24 Hz, 2H), 7.72−7.64 (m, 4H), 7.52 (d, J = 8.24 Hz, 2H), 7.47−

7.36 (m, 2H), 6.99 (s, 1H), 3.45 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 171.45, 152.51, 143.18, 139.25, 137.67, 133.88, 131.56,

129.50, 129.39, 129.27, 128.05, 127.65, 127.09, 126.24, 125.50, 124.54,

123.84, 122.78, 113.44, 37.04. ESI HRMS: m/z 498.0247 (M − H)⁻.

2-((4-(2′,4′-Diﬂuoro-[1,1′-biphenyl]-4-ylsulfonamido)-1-hydroxy-

naphthalen-2-yl)thio)acetic Acid (20). Synthesized using the

procedure for 1 except 49r was used as the starting material. The

title compound (31 mg, 48%) as a white solid after HPLC puriﬁcation.

1

HPLC (method B, t_R= 6.22 min), purity 98%. H NMR (400 MHz,

DMSO-d₆) δ 12.86 (s, 1H), 9.84 (s, 1H), 9.55 (s, 1H), 8.20 (d, J =

8.36 Hz, 1H), 8.07 (d, J = 8.36 Hz, 1H), 7.60−7.51 (m, 2H), 7.44 (s,

1H), 7.43−7.38 (m, 4H), 4.49 (s, 2H), 3.71 (s, 2H). ¹³C NMR (100

MHz, DMSO-d₆) δ 171.81, 152.35, 133.45, 133.18, 131.79, 129.37,

128.93, 128.72, 127.23, 126.30, 125.63, 124.99, 124.28, 122.79, 113.67,

57.16, 36.96. ESI HRMS: m/z 436.0086 (M − H)⁻.

1

2-((4-(2-(4-Chlorophenyl)acetamido)-1-hydroxynaphthalen-2-

yl)thio)acetic Acid (28). Synthesized using the procedure for 1 except

49x was used as the starting material. The title compound (19 mg,

10%) was obtained as a white solid after trituration with a mixture of

CH₃CN:H₂O 1:1 and cold CH₂Cl₂and without a need for HPLC

HPLC (method A, t_R= 7.12 min), purity 97%. H NMR (400 MHz,

DMSO-d₆) δ 12.71 (s, 1H), 9.97 (s, 1H), 9.80 (s, 1H), 8.10 (d, J =

8.16 Hz, 1H), 7.86 (d, J = 8.16 Hz, 1H), 7.69 (d, J = 7.72 Hz, 2H),

7.62 (d, J = 7.72 Hz, 2H), 7.59−7.53 (m, 1H), 7.47−7.34 (m, 3H),

7.19 (t, J = 8.31 Hz, 1H), 7.00 (s, 1H), 3.46 (s, 2H). ¹³C NMR (100

MHz, DMSO-d₆) δ 171.46, 152.55, 139.44, 138.77, 132.51 (dd, J =

4.88, 9.80 Hz), 131.58, 129.82 (d, J = 2.86 Hz), 129.50, 127.66,

127.06, 126.23, 125.50, 124.50, 123.81, 122.78, 113.47, 112.73 (dd, J =

3.78, 21.26 Hz), 105.15 (t, J = 26.72 Hz), 37.04. ESI HRMS: m/z

500.0441 (M − H)⁻.

1

puriﬁcation. HPLC (method B, t_R= 5.77 min), purity 93%. H NMR

(400 MHz, DMSO-d₆) δ 9.96 (s, 1H), 8.23−8.17 (m, 1H), 7.89−7.83

(m, 1H), 7.57−7.50 (m, 3H), 7.47−7.39 (m, 4H), 3.78 (s, 2H), 3.66

(s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.72, 169.89, 151.36,

135.73, 131.66, 131.49, 129.70, 128.69, 127.15, 127.00, 126.11, 126.07,

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125.40, 123.21, 123.02, 113.51, 42.24, 37.21. ESI HRMS: m/z

400.0417 (M − H)⁻.

Hz, 2H), 7.38 (t, J = 7.23 Hz, 1H), 7.31 (t, J = 7.23 Hz, 1H), 6.73 (s,

1H), 3.34 (t, J = 6.35 Hz, 2H), 2.61 (t, J = 7.48 Hz, 2H), 1.54 (p, J =

6.68 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 149.29, 139.59,

132.49, 130.41, 129.31, 128.21, 126.80, 126.16, 125.66, 125.40, 123.69,

123.54, 122.46, 122.39, 60.51, 33.15, 26.18. ESI HRMS: m/z 434.0063

(M − H)⁻.

2-((1-Hydroxy-4-(2-phenylacetamido)naphthalen-2-yl)thio)-

acetic Acid (29). Synthesized using the procedure for 1 except 49y was

used as the starting material. The title compound (50 mg, 61%) was

obtained as a white solid after trituration with a mixture of

CH₃CN:H₂O 1:1 and cold CH₂Cl₂and without a need for HPLC

4-Bromo-N-(4-hydroxy-3-(4-hydroxybutyl)naphthalen-1-yl)-

benzenesulfonamide (35). Synthesized using the procedure for 1

except 45 was used as the starting material. The title compound (49

mg, 34%) was obtained as a white solid after HPLC puriﬁcation.

1

puriﬁcation. HPLC (method A, t_R= 6.07 min), purity 94%. H NMR

(400 MHz, DMSO-d₆) δ 12.78 (s, 1H), 9.97 (s, 1H), 9.66 (s, 1H),

8.26−8.12 (m, 1H), 7.92−7.77 (m, 1H), 7.59−7.47 (m, 3H), 7.42 (d, J

= 7.64 Hz, 2H), 7.37 (t, J = 7.40 Hz, 2H), 7.31−7.24 (m, 1H), 3.77 (s,

2H), 3.67 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.73, 170.25,

151.26, 136.74, 129.70, 129.58, 128.76, 127.10, 126.97, 126.92, 126.19,

126.06, 125.38, 123.22, 122.99, 113.49, 43.09, 37.13. ESI HRMS: m/z

366.0802 (M − H)⁻.

4-Bromo-N-(3-(butylthio)-4-hydroxynaphthalen-1-yl)-

benzenesulfonamide (31). Synthesized using the procedure for 1

except 49z was used as the starting material. The title compound (10

mg, 8%) was obtained as a white solid after HPLC puriﬁcation. HPLC

(method A, t_R= 8.44 min), purity 97%. ¹H NMR (400 MHz, CDCl₃)

δ 8.23 (d, J = 7.40 Hz, 1H), 7.79 (d, J = 7.40 Hz, 1H), 7.59−7.50 (m,

4H), 7.50−7.45 (m, 1H), 7.37 (s, 1H), 7.26 (s, 1H), 6.60 (s, 1H), 3.49

(s, 1H), 2.65 (t, J = 7.24 Hz, 2H), 1.52−1.45 (m, 2H), 1.45−1.35 (m,

2H), 0.90 (t, J = 7.24 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

154.04, 138.33, 132.18, 131.86, 131.23, 128.98, 128.09, 127.90, 126.29,

123.80, 123.47, 122.92, 122.16, 111.25, 36.61, 31.70, 21.74, 13.60. ESI

HRMS: m/z 463.9998 (M − H)⁻.

Methyl 2-((4-(4-Bromophenylsulfonamido)-1-hydroxynaphtha-

len-2-yl)thio)acetate (32). To a suspension of 41 (75 mg, 0.15

mmol) in dry CH₂Cl₂(1.5 mL) was added BBr₃(1 M in CH₂Cl₂, 0.4

mL, 0.4 mmol) dropwise at 0 °C. The mixture was allowed to warm up

to room temperature and stirred under nitrogen. The starting material

was entirely consumed as determined by TLC and analytical HPLC

after 30 min. The mixture was again cooled down to 0 °C and MeOH

(2 mL) was added. After addition, the mixture was allowed to warm up

to room temperature and stirred for 1 h when a new spot formed as

monitored by TLC. The mixture was slowly added to a stirring

solution of saturated aqueous NH₄Cl (15 mL) at 0 °C. The solution

was extracted with EtOAc (15 mL × 2). The combined organic

extracts were washed with brine (15 mL), dried (MgSO₄), and ﬁltered.

The solvent was removed under reduced pressure. The crude was

puriﬁed using a C₁₈reverse phase semipreparative HPLC column with

solvent A (0.1% of TFA in water) and solvent B (0.1% of TFA in

CH₃CN) as eluents to give 32 (31 mg, 43%) as a white solid. HPLC

(method A, t_R= 7.45 min), purity 98%. ¹H NMR (400 MHz, DMSO-

d₆) δ 10.08 (s, 1H), 9.90 (s, 1H), 8.15 (d, J = 8.22 Hz, 1H), 7.85 (d, J

= 8.22 Hz, 1H), 7.73 (d, J = 8.58 Hz, 2H), 7.55 (d, J = 8.58 Hz, 2H),

7.52−7.41 (m, 2H), 6.99 (s, 1H), 3.60 (s, 5H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 170.21, 152.67, 139.44, 132.58, 131.45, 129.39, 129.23,

127.20, 126.93, 126.30, 125.51, 124.25, 123.67, 122.85, 113.07, 52.64,

36.18. ESI HRMS: m/z 479.9586 (M − H)⁻.

2-((4-(4-Bromophenylsulfonamido)-1-hydroxynaphthalen-2-yl)-

thio)acetamide (33). Synthesized using the procedure for 1 except 41

was used as the starting material. The title compound (24 mg, 42%) as

a yellow solid after trituration with a mixture of CH₃CN:H₂O 1:1 and

cold CH₂Cl₂and without a need for HPLC puriﬁcation. HPLC

(method B, t_R= 9.37 min), purity 97%. ¹H NMR (400 MHz, DMSO-

d₆) δ 11.14 (s, 1H), 9.98 (s, 1H), 8.14 (d, J = 8.15 Hz, 1H), 7.87−7.78

(m, 2H), 7.69 (d, J = 8.16 Hz, 2H), 7.56−7.48 (m, 3H), 7.44 (p, J =

6.86 Hz, 2H), 6.98 (s, 1H), 3.47 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 173.00, 154.82, 139.41, 132.55, 132.15, 131.51, 129.34,

127.54, 126.92, 126.20, 125.68, 123.58, 123.23, 112.74. ESI HRMS:

m/z 464.9591 (M − H)⁻.

1

HPLC (method B, t_R= 9.60 min), purity 98%. H NMR (400 MHz,

DMSO-d₆) δ 9.84 (s, 1H), 9.13 (s, 1H), 8.12 (d, J = 8.17 Hz, 1H),

7.87 (d, J = 8.17 Hz, 1H), 7.69 (d, J = 8.49 Hz, 2H), 7.51 (d, J = 8.49

Hz, 2H), 7.39 (t, J = 7.31 Hz, 1H), 7.33 (t, J = 7.31 Hz, 1H), 6.67 (s,

1H), 4.36 (s, 1H), 3.42−3.33 (m, 2H), 2.58 (t, J = 5.70 Hz, 2H),

1.45−1.26 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 149.19,

139.58, 132.50, 130.54, 129.39, 128.16, 126.78, 126.20, 125.66, 125.39,

123.64, 123.61, 122.58, 122.46, 61.16, 32.47, 29.39, 26.67. ESI HRMS:

m/z 448.0223 (M − H)⁻.

N-(3-(((2H-Tetrazol-5-yl)methyl)thio)-4-hydroxynaphthalen-1-

yl)-4-bromobenzenesulfonamide (36). Synthesized using the proce-

dure for 1 except 49cc was used as the starting material. The title

compound (20 mg, 42%) was obtained as a white solid after HPLC

1

puriﬁcation. HPLC (method A, t_R= 6.45 min), purity 98%. H NMR

(400 MHz, DMSO-d₆) δ 10.00 (s, 1H), 8.13 (d, J = 8.04 Hz, 1H), 7.75

(d, J = 8.04 Hz, 1H), 7.64 (d, J = 8.62 Hz, 2H), 7.49−7.43 (m, 3H),

7.42−7.36 (m, 1H), 6.91 (s, 1H), 4.25 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 153.78, 139.47, 132.51, 131.79, 130.43, 129.21, 127.45,

126.87, 126.27, 125.59, 124.10, 123.57, 123.08, 26.54. ESI HRMS: m/z

489.9647 (M − H)⁻.

N-(3-(((2H-Tetrazol-5-yl)methyl)thio)-4-hydroxynaphthalen-1-

yl)-4-phenoxybenzenesulfonamide (37). Synthesized using the

procedure for 1 except 49dd was used as the starting material. The

title compound (32 mg, 53%) as a white solid after HPLC puriﬁcation.

1

HPLC (method A, t_R= 6.88 min), purity 96%. H NMR (500 MHz,

DMSO-d₆) δ 9.84 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4

Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.51−7.46 (m, 1H), 7.44−7.38 (m,

3H), 7.20 (t, J = 7.4 Hz, 1H), 6.99−6.94 (m, 5H), 4.28 (s, 2H). ¹³C

NMR (125 MHz, DMSO-d₆) δ 160.70, 155.63, 153.61, 134.27, 131.88,

130.73, 130.54, 129.77, 127.33, 126.16, 125.60, 125.06, 124.51, 123.71,

123.04, 119.98, 118.35, 111.64, 26.59. ESI MS: m/z 503.9 (M − H)⁻.

3-((1-Hydroxy-4-(naphthalene-2-sulfonamido)naphthalen-2-yl)-

thio)propanoic Acid (38). Synthesized using the procedure for 1

except 49ee was used as the starting material. The title compound (23

mg, 15%) was obtained as a white solid after HPLC puriﬁcation.

HPLC (method B, t_R= 7.08 min), purity >99%. ¹H NMR (400 MHz,

DMSO-d₆) δ 12.31 (s, 1H), 10.01 (s, 1H), 9.60 (s, 1H), 8.23 (s, 1H),

8.14−8.06 (m, 2H), 8.06−7.97 (m, 3H), 7.80 (d, J = 8.52 Hz, 1H),

7.68 (t, J = 7.36 Hz, 1H), 7.60 (t, J = 7.36 Hz, 1H), 7.43 (p, J = 6.84

Hz, 2H), 6.87 (s, 1H), 2.62 (t, J = 6.83 Hz, 2H), 2.17 (t, J = 6.83 Hz,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.97, 152.44, 137.07,

134.56, 131.90, 131.48, 129.68, 129.51, 129.23, 129.20, 128.33, 128.20,

128.00, 126.94, 126.22, 125.51, 124.61, 123.96, 122.93, 122.70, 113.33,

34.12, 29.59. ESI HRMS: m/z 452.0630 (M − H)⁻.

2-((1-Acetoxy-4-(4-bromophenylsulfonamido)naphthalen-2-yl)-

thio)acetic Acid (46). Synthesized using a reported procedure.⁷³

A

stirred solution of 10 (50 mg, 0.11 mmol) in dry THF (1.5 mL) was

cooled to 0 °C. Et₃N (31 μL, 0.22 mmol) was added, and the mixture

was stirred for 5 min before acetyl chloride (10 μL, 0.14 mmol) was

added at 0 °C. The mixture was stirred under nitrogen for an

additional 20 min then diluted with EtOAc (10 mL) and washed with

H₂O (10 mL × 3). The organic layer was dried (MgSO₄), ﬁltered, and

concentrated under reduced pressure. The crude was subjected to ﬂash

column chromatography (hexane/EtOAc 4:1) on silica gel to aﬀord 46

(34 mg, 61%) as a white solid. HPLC (method A, t_R= 7.87 min),

purity 99%. ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 8.10 Hz, 1H),

7.96 (d, J = 8.48 Hz, 2H), 7.76 (d, J = 8.10 Hz, 1H), 7.71 (d, J = 8.48

Hz, 2H), 7.69−7.59 (m, 2H), 7.37 (s, 1H), 3.63 (s, 2H), 1.79 (s, 3H).

¹³C NMR (100 MHz, CDCl₃) δ 170.13, 161.35, 147.04, 137.50,

132.14, 131.33, 131.18, 129.73, 129.46, 128.87, 128.46, 126.94, 125.55,

4-Bromo-N-(4-hydroxy-3-(3-hydroxypropyl)naphthalen-1-yl)-

benzenesulfonamide (34). Synthesized using the procedure for 1

except 49aa was used as the starting material. The title compound (16

mg, 32%) was obtained as a white solid after HPLC puriﬁcation.

1

HPLC (method A, t_R= 6.48 min), purity 95%. H NMR (400 MHz,

DMSO-d₆) δ 9.87 (s, 1H), 9.17 (s, 1H), 8.11 (d, J = 8.34 Hz, 1H),

7.82 (d, J = 8.34 Hz, 1H), 7.68 (d, J = 8.50 Hz, 2H), 7.49 (d, J = 8.50

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122.76, 121.64, 114.58, 28.48, 24.29. ESI HRMS: m/z 507.9533 (M −

H)⁻.

in 7% D₂O. The binding mode of the compounds has been

1

characterized by recording H,¹⁵N -HSQC experiments with a 138

Fluorescence Polarization-Based Binding Assays. Sensitive

and quantitative FP-based binding assays were developed and

optimized to determine the binding aﬃnities of small-molecule

inhibitors to the recombinant Mcl-1, A1/Bﬂ-1, Bcl-w, Bcl-2, and Bcl-

xL proteins. Protocols for expression and puriﬁcation of used

antiapoptotic proteins and determination of K_dvalues of ﬂuorescent

probes to proteins are provided in the Supporting Information. On the

basis of the K_dvalues, the concentrations of the proteins used in the

competitive binding experiments were 90 nM for Mcl-1, 40 nM for

Bcl-w, 50 nM for Bcl-xL, 60 nM for Bcl-2, and 4 nM for A1/Bﬂ-1. The

ﬂuorescent probes, Flu-BID and FAM-BID, were ﬁxed at 2 nM for all

assays except for A1/Bﬂ-1, where FAM-BID was used at 1 nM. Then 5

μL of the tested compound in DMSO and 120 μL of protein/probe

complex in the assay buﬀer (100 mM potassium phosphate, pH 7.5;

100 μg/mL bovine gamma globulin; 0.02% sodium azide, purchased

from Invitrogen, Life Technologies, supplemented with 0.01% Triton

X-100) were added to assay plates (Microﬂuor 2Black, Thermo

Scientiﬁc), incubated at room temperature for 3 h, and the polarization

values (mP) were measured at an excitation wavelength at 485 nm and

an emission wavelength at 530 nm using the plate reader Synergy H1

Hybrid, BioTek. IC₅₀values were determined by nonlinear regression

ﬁtting of the competition curves (GraphPad Prism 6.0 Software). The

K_ivalues were calculated as described previously.⁷⁷

Solution Competitive Surface Plasmon Resonance Based

Assay Using Immobilized Biotin-Labeled Bim BH3 Peptide.

The solution competitive SPR-based assay was performed on Biacore

2000. N terminal biotin-labeled Bim BH3 peptide (141−166 amino

acids) was immobilized on streptavidin (SA) chip giving density of

1400 RU (Response Units). The preincubated Mcl-1 protein (20 nM)

with tested small-molecule inhibitors for at least 30 min was injected

over the surfaces of the chip. Response units were measured at 15 s in

the dissociation phase, and the speciﬁc binding was calculated by

subtracting the control surface (Fc1) signal from the surfaces with

immobilized biotin-labeled Bim BH3. IC₅₀values were determined by

nonlinear least-squares analysis using GraphPad Prism 6.0 software.

Molecular Modeling. Crystal structure of Mcl-1 in complex with

μL solution of uniformly ¹⁵N-labeled Mcl-1 (75 μM) in the absence

and presence of added compounds with the indicated molar ratio

concentrations. All spectra were acquired at 30 °C on a Bruker 600

MHz NMR spectrometer equipped with a cryogenic probe, processed

using Bruker TopSpin and rNMR,⁸⁴and were analyzed with Sparky.⁸⁵

Plots of chemical shift changes were calculated as ((Δ¹H ppm)²+

(0.2(Δ¹⁵N ppm))²)^0.5of Mcl-1 amide upon addition of compound.

The absence of a bar in a chemical shift plot indicates no chemical shift

diﬀerence or the presence of a proline or residue that is overlapped or

not assigned.

Biotin−Streptavidin Pull-Down Experiment. Human breast

cancer 2LMP cells, a subclone of the MDA-MB-231 cell line, were

lysed in CHAPS buﬀer (10 mM HEPES (pH 7.4), 2.5 mM EDTA, 150

mM NaCl, 1.0% CHAP). Precleared cell lysates were incubated with

diﬀerent concentrations of compounds followed by incubation with

biotinylated Noxa BH3 peptide (18−43) and streptavidin−agarose

beads to pull-down Mcl-1 protein bound to Noxa peptide. Beads were

washed with CHAPS buﬀer, and Mcl-1 protein was eluted by boiling

in SDS-PAGE sample buﬀer and analyzed by Western blotting using

Mcl-1 antibody (Santa Cruz).

Cell Culture and Cell Viability Assays. MEFs cells, wild-type

and Bax/Bak double knockout, were gifts from Shaomeng Wang at the

University of Michigan and were cultured in DMEM (Life

Technologies), supplemented with 10% fetal bovine serum (FBS)

(Thermo Scientiﬁc HyClone). The retroviral transduced lymphoma

cells isolated from Eμ-myc transgenic mice were gifts from Ricky W.

Johnstone at University of Melbourne, Melbourne, Australia, and

cultured as previously described.⁸¹Human leukemia cell lines HL-60,

K-562, and MV-4−11 were obtained from American Type Culture

Collection (ATCC). The cells were cultured in RPMI 1640 medium

(Life Technologies), all supplemented with 10% FBS. MEFs cells were

seeded in 12-well plates at 0.5 × 10⁶cells/well, left to adhere, and then

treated for 15 h with increasing concentrations of the compounds. The

cells were harvested, washed with phosphate-buﬀered saline (PBS),

and stained with 0.025 mg/mL propidium iodide (MP Biomedicals).

The percentage of propodium iodide positive population was

determined by ﬂow cytometry and calculated using WinList 3.0. The

Mcl-1 and Bcl-2 retroviral transduced lymphoma Eμ-myc cells were

seeded in 24-well plates at 0.5 × 10⁶cells/well. They were treated with

diﬀerent concentrations of tested compounds for 15−18 h. The cells

were harvested and stained with violet LIVE/DEAD ﬁxable dead cell

stain kit (Invitrogen) according to manufacturer’s protocol. The

percentage of ﬂuorescent positive cells was determined by ﬂow

cytometry and calculated using WinList 3.0. The eﬀect of the

compounds on tested leukemia cells viability was evaluated by

CellTiter Glo luminescent cell viability assay (Promega). Cells were

plated in 12-well plates at 0.5 × 10⁶cells/well and treated with various

concentrations of the compounds and incubated for 3 days. Cell

viability was determined by measuring intracellular ATP levels with the

CellTiter Glo reagent and reading the luminescence with the Synergy

H1 Hybrid BioTek plate reader. Percent cell growth was calculated

relative to DMSO treated cells, and IC₅₀values were calculated by

nonlinear regression analysis using GraphPad Prism 6.0 Software.

Determination of Caspase Activity. HL-60 cells were seeded in

12-well plates at 0.5 × 10⁶cells/well. After 20 h treatment with

diﬀerent concentrations of the compounds, caspase 3 activity was

determined using the ﬂuorometric substrates DEVD-AFC following

the protocol of the Caspase-3 ﬂuorometric assay kit (BioVision).

Caspase 3 activity is reported as the fold change relative to DMSO

treated cells.

mNoxa BH3 peptide (PDB entry 2NLA) and in silico Schrodinger’s

̈

IFD were used to model the binding poses of our designed

compounds with Mcl-1. IFD is allowing incorporation of the protein

and ligand ﬂexibility in the docking protocol, which consists of the

following steps: (i) constrained minimization of the protein with an

RMSD cutoﬀ of 0.18 Å, (ii) initial Glide docking of the ligand using a

softened potential (van der Waals radii scaling), (iii) one round of

Prime side chain prediction for each protein/ligand complex, on

residues within deﬁned distance of any ligand pose, (iv) prime

minimization of the same set of residues and the ligand for each

protein/ligand complex pose, (v) Glide redocking of each protein/

ligand complex structure within a speciﬁed energy of the lowest energy

structure, (vi) estimation of the binding energy (IFDScore) for each

output pose. All docking calculations were run in the extra precision

(XP) mode of Glide. The center of the grid box of the Mcl-1 was

deﬁned by the Val 249 (in h1), Phe 270 (in h2), Val 220 (in h3/h4),

and Val 216 (in h4). The size of the grid box was set to 15 Å. Default

values were used for all other parameters. Schrodinger’s MC/SD

̈

dynamic simulation performs constant temperature calculations that

take advantage of the strengths of Monte Carlo methods for quickly

introducing large changes in a few degree of freedom and stochastic

dynamics for its eﬀective local sampling of collective motions. The

MC/SD dynamic simulation time in our study was set to 100 ps by

allowing movement of the docked ligand and the residues, which is

less than 6 Å to the ligand. The force ﬁeld used was set to

OPLS_2001. Default values were used for all other parameters.

NMR Studies. ¹⁵N-labeled or ¹⁵N, ¹³C-labeled Mcl-1 proteins for

NMR studies were prepared and puriﬁed using the same protocol as

for unlabeled protein with the exception that the bacteria were grown

on M9 minimal media supported with 3 g/L of ¹³C-glucose and/or 1

g/L of (¹⁵NH₄)₂SO₄. Protein samples were prepared in a 20 mM

sodium phosphate, 150 mM NaCl, and 1 mM DTT solution at pH 7

Analysis of Apoptosis. To determine the induction of apoptosis,

HL-60 cells were plated in 12-well plates at 0.5 × 10⁶cells/well and

treated with various concentrations of tested compounds. After 20 h,

cells were harvested, washed with PBS, and treated with annexin-V

FITC and propidium iodide using BD annexin V FITC assay kit (BD

Biosciences Pharmingen). The percentage of cells undergoing

apoptosis was assessed by ﬂow cytometry within 1 h and analyzed

using WinList 3.0. Apoptosis was also determined in the presence of

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Journal of Medicinal Chemistry

Article

Z-VAD-FMK (Bachem), pan caspase-inhibitor. For this purpose sells

were pretreated with 100 μM Z-VAD-FMK for about 1 h before

adding tested compounds.

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(3) Fulda, S.; Debatin, K. M. Extrinsic versus intrinsic apoptosis

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S

* Supporting Information

Additional experimental details for synthesis of analogues 24,

27, and 30 and intermediates, protein expression and

puriﬁcation, ﬂuorescence polarization-based binding assay,

and additional HSQC chemical shift perturbation plots and

apoptosis studies. This material is available free of charge via

the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

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inhibitors: small molecules with a big impact on cancer therapy. Cell

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Corresponding Author

*Phone: +1-734-615-9202 and +1-734-764-6683. Fax: +1-734-

763-8764. E-mail: zanetan@med.umich.edu.

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Present Address

^⊥For C.L.: School of Medical Engineering, Hefei University of

Technology, Hefei, Anhui 230009, China.

Author Contributions

^#These authors contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

This research was supported by grants from the U.S. National

Cancer Institute, National Institutes of Health R01CA149442,

R21CA0158976, and R21NS056915 to Zaneta Nikolovska-

Coleska. We are grateful to Kalle Gehring from McGill

University, Montreal, Canada, for providing us with the NMR

assignment of human Mcl-1 protein. We thank Lei Miao for

synthesizing the intermediate for analogue 23, Julie DiBernardo

for her help with growing Eμ-myc cells, Ronald Craig for his

input in ﬂow cytometric analysis, and George Lund for his help

with NMR experimental setup.

ABBREVIATIONS USED

■

Bcl-2, B-cell lymphoma-2; BH, Bcl-2 homology; DKO, double

knockout; FP, ﬂuorescence polarization; IFD, induced ﬁt

docking; MEF, murine embryonic ﬁbroblast; Mcl-1, myeloid

cell leukemia sequence 1; PI, propidium iodide; SPR, surface

plasmon resonance. Solvent and reagent abbreviations used:;

CH₃CN, acetonitrile; NH₄Cl, ammonium chloride; NH₄OH,

ammonium hydroxide; (NH₄)₂SO₄, ammonium sulfate; Dppf,

1,1′-bis(diphenylphosphino)ferrocene; Pd(PPh₃)₂Cl₂, bis-

(triphenylphosphine)palladium(II) dichloride; BBr₃, boron

tribromide; Cs₂CO₃, cesium carbonate; CuI, copper(I) iodide;

CH₂Cl₂, dichloromethane; DTT, dithiothreitol; EtOAc, ethyl

acetate; EtOH, ethanol; HCl, hydrogen chloride; LiOH, lithium

hydroxide; MgSO₄, magnesium sulfate; MeOH, methanol;

NMP, N-methyl-2-pyrrolidone; Pd(OAc)₂, palladium acetate;

NaHCO₃, sodium bicarbonate; Na₂CO₃, sodium carbonate;

NaCl, sodium chloride; NaOH, sodium hydroxide; Na₂S₂O₃,

sodium thiosulfate; Pd(PPh₃)₄, tetrakis(triphenylphosphine)-

palladium(0); THF, tetrahydrofuran; Et₃N, triethylamine; TFA,

triﬂuoroacetic acid; Pd₂(dba)₃, tris(dibenzylideneacetone)-

dipalladium(0); ZnCl₂, zinc chloride; Z-VAD-FMK, Z-Val-

Ala-^DL-Asp-ﬂuoromethylketone

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3-substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: Structure-based design, synthesis, SAR, and biological evaluation

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