Discovery of N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2- c]quinoline-8-sulfonamide as a Potent Dual MDM2/XIAP Inhibitor

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Discovery of N‑(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-

2,3,3a,4,5,9b-hexahydrofuro[3,2‑c]quinoline-8-sulfonamide as a

Potent Dual MDM2/XIAP Inhibitor

Zhongzhi Wu, Lubing Gu, Sicheng Zhang, Tao Liu, Pradeep B. Lukka, Bernd Meibohm,

John C. Bollinger, Muxiang Zhou,* and Wei Li *

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ABSTRACT: Murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) are important cell survival

proteins in tumor cells. As a dual MDM2/XIAP inhibitor reported previously, compound MX69 has low potency with an IC₅₀value

of 7.5 μM against an acute lymphoblastic leukemia cell line EU-1. Herein, we report the structural optimization based on the MX69

scaﬀold, leading to the discovery of a 25-fold more potent analogue 14 (IC₅₀= 0.3 μM against EU-1). We demonstrate that 14

maintains its mode of action by dual targeting of MDM2 and XIAP through inducing MDM2 protein degradation and inhibiting

XIAP mRNA translation, respectively, which resulted in cancer cell growth inhibition and cell death. The results strongly suggest that

the scaﬀold based on 14 is promising for further optimization to develop a new therapeutic agent for leukemia and possibly other

cancers where MDM2 and XIAP are dysregulated.

new gene)-ﬁnger-type family of E3 ubiquitin ligases, is also a

substrate of its own RING domain E3 ligase.^18,19Under certain

conditions, such as upon binding nucleic acids or RNA, the

capacity of the MDM2 RING domain E3 ligase to target itself

for ubiquitination becomes inhibited.^20,21

X-linked inhibitor of apoptosis protein (XIAP) is an

important member of the inhibitors of apoptosis protein

(IAP) family. It speciﬁcally binds to and inhibits the activated

forms of caspases 3, 7, and 9; these enzymes induce the

intrinsic (mitochondrial) apoptotic pathway, the major cell

death mechanism activated by many chemotherapy drugs.²²

Upregulated XIAP is detected in tumor cells from a number of

cancer types, and a high level of XIAP expression is associated

with treatment resistance and a poor prognosis.²³⁻²⁵XIAP

expression is mainly regulated by an internal ribosome entry

INTRODUCTION

■

The human murine double minute 2 (MDM2) is ampliﬁed/

overexpressed in various human cancers, including childhood

acute leukemia lymphoma (ALL) and neuroblastoma (NB).¹⁻⁴

In cancer patients, MDM2 overexpression is associated with

disease progression and poor treatment outcomes.⁵⁻⁸The

main oncogenic function of MDM2 is to inhibit the tumor

suppressor p53;^9,10thus, the p53 function becomes inactivated

in MDM2-overexpressing cells, resulting in cancer cell

proliferation and growth. In addition, MDM2 plays p53-

independent roles in oncogenesis, as reported in p53-deﬁcient

cancer patients, in which MDM2 overexpression is also

involved in cancer progression and treatment resistance.¹¹⁻¹³

This is because in addition to interacting with and regulating

p53, MDM2 also interacts with other molecules involved in

oncogenesis. For instance, MDM2 can bind to nucleic acids

and speciﬁc small RNA molecules,¹⁴⁻¹⁷playing many p53-

independent roles in cancer pathogenesis. Changes in MDM2

expression level occur in multiple ways, including via gene

ampliﬁcation, transcriptional induction by p53, and regulation

at the post-translational level by a self-ubiquitination

mechanism. MDM2, a member of the RING (really interesting

Received: June 18, 2020

Published: February 8, 2021

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Figure 1. Structural modiﬁcation of the A-ring in the MX69 scaﬀold.

site (IRES)-mediated mechanism at the translational level.²⁶

The IRES-mediated translation of XIAP is speciﬁcally activated

when cells undergo cellular stress, such as exposure to radiation

or treatment with chemotherapeutic drugs.²⁷

on-target inhibition for MDM2 and XIAP and shows more

than 25-fold improvements in potency compared with MX69.

Although its unfavorable pharmacokinetic (PK) properties

prevented its further development, the current study paves a

foundation to further optimize this scaﬀold to ultimately

develop a more metabolically stable analogue for future

potential clinical development.

Our recently published studies revealed that expression of

XIAP and MDM2 in tumors is mutually regulated.^15,21We

found that the C-terminal RING domain of MDM2 binds to

the XIAP IRES, increasing the IRES-mediated XIAP trans-

lation; this in turn results in increased expression of XIAP and

drug resistance.¹⁵Conversely, binding of XIAP IRES to the

MDM2 RING domain decreases its E3 ubiquitin-ligase activity

for MDM2 self-ubiquitination, which leads to MDM2

stabilization, resulting in p53 inhibition and cancer pro-

gression.²¹Thus, the interaction between the MDM2 RING

protein and the XIAP IRES causes simultaneously increased

expression of both MDM2 and XIAP, contributing to cancer

progression and drug resistance. These mechanistic studies

suggest that simultaneous inhibition of MDM2 and XIAP

could be a promising strategy in developing targeted agents for

cancer treatment.

Toward this direction, we have recently published the

discovery of MX69 as a dual MDM2/XIAP inhibitor by high-

throughput screening compound libraries available at Emory

University using ﬂuorescence polarization assays, followed by

extensive target validation studies.²⁸MX69 binds to the C-

terminal RING domain of MDM2, which is critical for its E3-

ligase function, and is capable of blocking or disrupting the

interaction between the MDM2 RING domain and the XIAP

IRES, leading to simultaneous inhibition of both MDM2 and

XIAP. This dual inhibition results in the activation of p53 in

wild-type p53 tumor cells, similar to that of nutlin-3, a known

MDM2 inhibitor that works by disrupting the binding of

MDM2 and p53,²⁹but has the added advantage of inducing

caspases 3, 7, and 9 that is independent of the p53 status.²⁸

Such an agent should be capable of inducing apoptosis in p53-

deﬁcient cancer cells that are dependent on the expression of

high levels of both MDM2 and XIAP.

RESULTS AND DISCUSSION

■

Structural Modiﬁcation of the A-ring. For convenient

discussion on the synthesis of MX69 analogues and the

subsequent structure−activity relationship (SAR) analysis, we

designated the four rings in MX69 as rings A to D as shown in

Figure 1. The MX69 scaﬀold is generally assembled through

the Povarov reaction, a three-component Aza−Diels−Alder

reaction via in situ imine formation and subsequent formal [4 +

2]-cycloaddition to form the tetrahydroquinoline scaﬀold by

the coupling of amines, aldehydes, and alkenes. The amines

could be obtained by palladium-catalyzed hydrogenation of the

corresponding nitro compounds, which were synthesized by

the reaction of substituted anilines with 4-nitrobenzenesulfonyl

chlorides in the presence of pyridine. All the new MX69

analogues were tested for their antiproliferative activities, as

stereoisomeric mixtures as synthesized, against the human

leukemia EU-1 cell line, and MX69 was used as the reference

for comparison (Table 1).

Since there are no crystal structures of the MDM2 RING

domain available to guide our structural optimization, to get an

initial idea about the key functional groups in the MX69

scaﬀold for the antiproliferative activity, we ﬁrst probed

whether the p-carboxylic acid in MX69 is required by

converting it to a more hydrophobic moiety. Compound 1

(IC₅₀= 4.7 μM, Figure 1) formed by a simple esteriﬁcation of

the acid group shows moderately improved potency,

suggesting that the acid moiety is not absolutely required.

Next, we bioisosterically replaced the double bond in the A-

ring with an oxygen atom to form 2 (IC₅₀= 2.2 μM) or a

nitrogen atom to form 3 (IC₅₀= 3.5 μM), with the goal of

adding polarity to the molecule and thus providing enhanced

aqueous solubility from 1. Both analogues also showed slightly

increased potency, with the oxygen analogue 2 being the more

potent one. Further expansion of the A-ring from a ﬁve- to six-

membered ring to form 4 (IC₅₀= 4.6 μM) resulted in a

decrease of antiproliferative potency. Interestingly, hydrolysis

of the ester substitution in the B-ring back to the

While MX69 is well tolerated in mice, its potency is low

(IC₅₀= 7.5 μM against cancer cells) and thus requires a high

dose (up to 100 mg/kg) in order to achieve good in vivo

eﬃcacy.²⁸We report here our medicinal chemistry eﬀorts to

increase the potency of MX69, which led to the discovery of its

more potent analogue 14, N-(3,4-dimethylphenyl)-4-(4-

isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]-

quinoline-8-sulfonamide. We demonstrate that 14 maintains

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of B-ring with the ketone group demonstrated a signiﬁcant

improvement in potency. However, cyclization of the isopropyl

group to form 22 (IC₅₀= 5.2 μM) showed a 10-fold reduced

potency compared to 14. Replacement of the isopropyl group

with linear chains such as ethylene (23, IC₅₀= 30.0 μM) and

n-propyl (24, IC₅₀= 2.9 μM) failed to further increase the

anticancer activity either. Compounds 25 (IC₅₀= 3.1 μM) and

26 (IC₅₀= 3.2 μM) with a larger group, cyclohexyl and phenyl,

respectively, were approximately 6-fold less potent than 14.

Since compound 13 does not bind to the MDM2 target based

on subsequent mechanistic studies, the isobutyryl group was

considered the optimal substituent at the para position of the

B-ring.

Having the currently most potent compound 14 in hand, we

went back to investigate the eﬀect of A-ring on the

antiproliferative activity (Scheme 2D). Similarly, incorporation

of a carbon double bond (27, IC₅₀= 2.7 μM) or nitrogen (30,

IC₅₀= 3.8 μM) into the A-ring decreased the potencies by 5-

or 6-fold, respectively, compared to 14. Further saturation of

the double bond of the cyclopentene ring of 27 to form 28

(IC₅₀= 7.0 μM) also exhibited an approximately 3-fold drop in

potency. All these results are consistent with the previous

ﬁnding that a tetrahydrofuran moiety displayed higher activity

than other modiﬁcations on the A-ring.

Optimization of the C-ring Moiety. Having optimized

the A- and B-rings in the MX69 scaﬀold, next we investigated

the substitutions on the C-ring by removing either of the two

methyl substituents in parent compound 14 or by bioisosteri-

cally replacing the methyl group(s) with halogens (Scheme 3).

Removing either (31, IC₅₀= 3.3 μM; 32, IC₅₀= 1.4 μM) or

both of the methyl substituents (33, IC₅₀= 3.2 μM) in 14 all

led to reduced antiproliferative potency. Replacing one (34,

IC₅₀= 1.6 μM; 35, IC₅₀= 6.3 μM) or both methyl

substitutions (36, IC₅₀= 5.6 μM) with a chlorine atom also

resulted in lower potency. Substitutions using other halogen

atoms that are larger (37, IC₅₀= 5.2 μM) or smaller (38, IC₅₀

= 2.9 μM) than chlorine did not help, nor did replace a methyl

with a triﬂuoromethyl group (39, IC₅₀= 7.2 μM). Further

analyses of this SAR indicated that when keeping the meta-

methyl substitution intact, increasing the size of the para

substitution decreases the antiproliferative potency with the

order H (32) > F (38) > Cl (35) > CF₃(39). Thus, bulkier

substituents at either position seem to reduce the activity.

However, since compound 14 is more potent than compounds

32, 35, and 38 despite the former’s larger substituents, these

results suggest that the activity also correlates with the C-ring

substituents’ electronic eﬀects, with the two electron-donating

methyl groups of 14 striking the best balance for antiprolifer-

ative activity at this stage of the study.

Eﬀects of Substitutions on the D-ring and on the NH

Moieties. In the next phase of our SAR analysis, modiﬁcations

in the D-ring were designed by a simple introduction of a

chlorine substituent to diﬀerent positions. Compound 2 was

selected as a parent scaﬀold, and incorporation of chlorine into

the 6- or 7-position aﬀorded compounds 40 and 41 (Scheme

4A). An alternative pathway yielded 7-chlorinated compound

43 and 9-chlorinated compound 44 based on the scaﬀold

bearing a methanesulfonyl group in the B-ring and a carbon

double bond in the A-ring (Scheme 4B). These compounds all

exhibited abolishment or major reduction in anticancer

activity. The best, 44 (IC₅₀= 23.4 μM), is approximately 6-

fold less potent than its parent compound 42 (IC₅₀= 6.2 μM),

and the others all have IC₅₀values larger than 50 μM. These

Table 1. Antiproliferative Activities for New MX69

Analogues against the Leukemia EU-1 Cell Line (Data

Represent the Mean SD of Three Independent

Experiments)

compound

IC₅₀(μM)

compound

IC₅₀(μM)

1

2

2a

3

3a

4

4.7 0.6

2.2 0.3

>50

3.5 0.2

>50

26

27

28

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

MX69

3.2 0.3

2.7 0.2

7.0 0.9

3.8 0.4

3.3 0.2

1.4 0.2

3.2 0.4

1.6 0.2

6.3 0.8

5.6 0.5

5.2 0.7

2.9 0.3

7.2 0.8

>50

6.2 0.7

>50

23.4 3.0

1.2 0.2

1.6 0.2

7.5 0.5

4.6 0.3

>50

4a

5

18.3 2.6

20.5 2.9

4.2 0.4

3.6 0.3

4.1 0.4

0.5 0.1

0.3 0.1

2.0 0.2

3.6 0.3

6.1 0.5

5.2 0.5

30 3.3

2.9 0.3

3.1 0.4

5a

10

11

12

13

14

15

16

17

18

22

23

24

25

corresponding acids 2a, 3a, or 4a (IC₅₀> 50 μM) all resulted

in a signiﬁcant loss of activity. Attempts to add another ring to

the A-ring for either the ester 5 (IC₅₀= 18.3 μM) or the acid

5a (IC₅₀= 20.5 μM) also resulted in signiﬁcant loss of potency.

Collectively, these results are consistent with the trend in the

transformation from the original MX69 to compound 1,

further suggesting that a hydrophobic substitution in this B-

ring is beneﬁcial. The activities for these compounds are

summarized in Table 1 and their syntheses are shown in

Scheme 1.

Structural Modiﬁcation of the B-ring Substitution.

Since compound 2 bearing the oxygen atom in the A-ring

displayed the highest potency, this scaﬀold was used for

subsequent SAR exploration. The ester substitution on the B-

ring is not metabolically stable in vivo where esterase is

abundant. In order to increase the metabolic stability of

compound 2, analogues were designed with other more stable

substitutions in the B-ring (Scheme 2B). Unfortunately,

replacement of the ester group by isopropyl (10, IC₅₀= 4.2

μM), bromo (11, IC₅₀= 3.6 μM), or methanesulfonyl moieties

(12, IC₅₀= 4.1 μM) did not exhibit improved potency

compared to 2. Replacing the substituted phenyl group with a

naphthyl group (18, IC₅₀= 6.1 μM) did not help either.

However, compounds 13 and 14 displayed an approximately 4-

fold increased potency after replacing the para-ester moiety by

acetyl or isobutyryl groups, with IC₅₀values of 0.5 and 0.3 μM,

respectively. Moving the acetyl group and isobutyryl group

from para to meta position aﬀorded 16 (IC₅₀= 2.0 μM) and

17 (IC₅₀= 3.6 μM), leading to 4-fold and 7-fold drops in

potency, respectively. Reduction of the carbonyl group of

isobutyryl moiety to the hydroxyl group to form 15 (IC₅₀= 2.0

μM) caused an approximately 4-fold potency loss compared to

14. Analogues with diﬀerent ketone moieties were further

synthesized (Scheme 2C) based on the results that substitution

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a

Scheme 1. Synthesis of 1−5 and 2a−5a through Modiﬁcation of the A-ring

a

(a) Pyridine, CH₂Cl₂, and r.t.; (b) Pd/C, H₂, MeOH, and r.t.; (c) Sc(OTf)₃, 4 Å molecular sieves, CH₃CN, and r.t.; (d) KOH, MeOH/H₂O, and

r.t.; and (e) TFA, CH₂Cl₂, and r.t.

outcomes suggest that it is not appropriate to occupy the

position of the D-ring with substituents. The incorporation of

methyl group(s) on the nitrogen(s) of 14 was investigated as

well. The resulting 45 (IC₅₀= 1.2 μM) and 46 (IC₅₀= 1.6

μM) have 2-fold and 3-fold decreased potency, respectively, so

the NH groups are important for antiproliferative activity

(Scheme 4C).

Biological Characterization of Compound 14. Com-

pound 14 Attenuates the Proliferation in Cancer Cells but

Shows a Negligible Inhibitory Eﬀect on Normal Hema-

topoiesis. Based on the screening on the 40 new MX69

analogues using the water-soluble tetrazolium salt (WST) assay

in the EU-1 cell line, compound 14 exhibited signiﬁcantly

enhanced anticancer activity with an IC₅₀value of 0.3 μM,

representing a 25-fold increase in activity compared to MX69

(IC₅₀= 7.5 μM). Furthermore, we tested four additional

cancer cell lines including one ALL (EU-3) and three NB (NB-

1643, SHEP1, and LA1-55N). WST cytotoxicity assays showed

that 14 consistently exhibited potent cytotoxicity against these

tested cell lines with more sensitivity of ALL (IC₅₀= 0.3−0.4

μM) than NB (IC₅₀= 0.5−1.2 μM) to 14 (Figure 2A).

Furthermore, the results of colony formation assays showed

that 14 potently inhibits cell growth in all the three NB cell

lines. We observed a signiﬁcant reduction in both colony

number and size in 14-treated cells as compared with controls

(Figure 2B).

To evaluate the possible inhibitory eﬀect of 14 on normal

hematopoiesis, we performed Clonogenic assays for CFU-GM

and BFU-E using human normal bone marrow mononuclear

(NBMM) cells, with doxorubicin (Dox) as the reference

control. The CFU-GM and BFU-E colony numbers and size in

14-treated samples were similar to the control, whereas both

the colony number and size were signiﬁcantly reduced in Dox-

treated samples (Figure 2C,D).

Compound 14 Inhibits Expression of MDM2 through

Post-translational Modiﬁcation. To determine whether the

more potent compound 14 maintains its mechanism of action

as a dual MDM2/XIAP inhibitor, similar to its parental

compound MX69, we performed Western blot assays for the

eﬀects of 14 on MDM2 and XIAP expression. Results showed

that 14 induced a remarkable downregulation of MDM2 and

XIAP in a dose-dependent manner and occurred at

approximately 2−4 h after treatment, followed by steady-

state suppression (Figure 3A). Downregulation of MDM2 was

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Scheme 2. (A) Synthesis of Intermediates 9a and 9b; Reagents and Conditions: (a) N-Butyllithium, CuCN, LiCl, THF, and

−78 °C; (b) Isobutyryl Chloride, THF, and −78 °C; (c) HCl (aq, 3 M), CH₂Cl₂/H₂O, and r.t; (B) Synthesis of 10−18 through

Modiﬁcation of the B-ring; Reagents and Conditions: (d) Sc(OTf)₃, 4 Å Molecular Sieves, CH₃CN, and r.t.; (e) Pd/C, H₂,

MeOH, and r.t.; (C) Synthesis of 22−26 with Diﬀerent Ketone Moieties in the B-ring; Reagents and Conditions: (f) Oxalyl

Chloride, DMF, CH₂Cl₂, and 0 °C−r.t; (g) N,O-Dimethylhydroxylamine Hydrochloride, NEt₃, CH₂Cl₂, and 0 °C−r.t; (h)

THF, Corresponding Lithium Reagents, −78 °C or Grignard Reagents, and r.t.; (D) Synthesis of 27−30 through the A-ring

Modiﬁcation on Parent Compound 14; Reagents and Conditions: (i) Pd/C, H₂, MeOH, and r.t.; (j) Dess−Martin Reagent,

CH₂Cl₂, and r.t.; and (k) TFA, CH₂Cl₂, and r.t.

accompanied by increased expression of p53. To further

evaluate the mechanism by which 14 inhibits MDM2, we

performed MG132 (protein degradation inhibitor) treatment

and cycloheximide (CHX, protein synthesis inhibitor) chase

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a

Scheme 3. Synthesis of 31−39 through Modiﬁcation of the C-ring

a

(a) Pyridine, CH₂Cl₂, r.t. (b) Pd/C, H₂, MeOH, r.t. (c) Sc(OTf)₃, 4 Å molecular sieves, CH₃CN, r.t.

Scheme 4. (A,B) Synthesis of 40−44 through Modiﬁcation of the D-ring and (C) Synthesis of 45 and 46 by Methylation of the

a

NH Group.

a

Reagents and conditions: (a) pyridine, CH₂Cl₂, and r.t.; (b) Pd/C, H₂, MeOH, and r.t.; (c) Sc(OTf)₃, 4 Å molecular sieves, CH₃CN, and r.t; (d)

NaH, MeI, THF, and r.t.

assays in EU-1 cells treated with 14; results showed that 14

induced increased MDM2 protein degradation. As can be seen

in Figure 3B, the observed downregulation of MDM2 by 14

was blocked by MG132. The CHX chase assay results showed

that the half-life of MDM2 in untreated EU-1 cells was >120

min, whereas 14 treatment decreased the MDM2 half-life to

<60 min (Figure 3C). In contrast, the half-life of p53 in

untreated cells was <30 min, and the time was increased to

>120 min by treatment with 14. Since the MDM2 protein

stability is regulated by a self-ubiquitination mechanism, we

tested whether 14-induced MDM2 protein degradation is

mediated through this mechanism. Immunoprecipitation−

Western blot assay results showed that 14 induced

ubiquitination of endogenous MDM2 in EU-1 cells (Figure

3D). These results suggest that 14 downregulates MDM2

through induction of MDM2 self-ubiquitination and degrada-

tion. Furthermore, activation of p53 following 14-mediated

MDM2 ubiquitination and degradation led to activation of the

p53 downstream targets p21 and PUMA that induce cell cycle

arrest and apoptosis (Figure 3E).

Compound 14 Inhibits XIAP Translation and Activity. We

investigated whether compound 14-mediated inhibition of

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Figure 2. Eﬀects of 14 on cancer cell viability and growth as well as on normal human hematopoiesis. (A) WST assay for cytotoxic eﬀects of 14 on

two ALL cell lines (EU-1 and EU-3) and three NB cell lines (NB-1643, SHEP1, and LA1-55N). The cells were treated at the doses indicated for 48

h. Data represent the mean SD of three independent experiments. (B) Representative colony formation of NB cell lines as indicated treated with

or without 14 for 2 weeks. (C,D) Comparison of inhibitory eﬀects of 14 and Dox on CFU-GM and BFU-E in NBMM cells using in vitro colony

formation analysis. NBMM cells (1 × 10⁵) were incubated with GM-CSF or Epo in the absence or presence of 1 μM either 14 or Dox. The

colonies were counted after 14 days of culture. Comparison of colony numbers, *p < 0.01.

Figure 3. Eﬀects of 14 on the expression of MDM2 and XIAP and activation of p53. (A) Western blot assays showed the dose−response and time

course of MDM2 and XIAP inhibition as well as p53 induction by 14 in the EU-1 cell line treated with doses and times as indicated. (B) The EU-1

cells with or without 14 treatment (1 μM for 8 h) were treated with 10 μM of MG132 for an additional 4 h and then Western blot assays were

performed for the expression of proteins as indicated. (C) CHX chase assay for the detection of protein turnover in EU-1 cells treated with or

without (control) 1 μM of 14 for 4 h. Numerical labels under each band of Western blots represent the expression levels after normalization for

GAPDH, compared with untreated samples (deﬁned as 1 unit). (D) IP and Western blot assay using anti-MDM2 and anti-ubiquitin antibodies,

respectively, to detect the eﬀects of 14 on the ubiquitination of endogenous MDM2 in EU-1 cells. (E) Western blot for the expression of p53 and

its targets p21 and PUMA in EU-1 cells treated with 14.

XIAP, such as MX69-mediated, occurs at the translational

level. We performed linear sucrose gradient fractionation to

assess the state of polyribosome association of XIAP mRNA in

EU-1 cells subjected to 14 treatment. We found that 14

induced a downregulation in polyribosome association. This

was shown by a shift in XIAP mRNA from fractions containing

enriched translating polyribosomes to fractions containing

translation-inactive complexes, monoribosomes (Figure 4A).

We also tested the eﬀects of 14-mediated inhibition of XIAP

on the activation of caspases 3 and 9, as well as the cleavage of

the death substrate PARP. As shown in Figure 4B, the cleavage

of caspases 3 and 9 and PARP can be detected 8 h after 14

treatment in EU-1 cells. Simultaneously, we treated EU-1 cells

with MX69 for comparison, and results showed that 14

induced a stronger cleavage of caspases 3 and 9 and PARP at a

much lower concentration (1 μM) than MX69 (5 μM). These

results suggest that the increased potency of 14 relative to

MX69 in inhibiting EU-1 cells is closely associated with the

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Figure 4. (A) EU-1 cells were treated with or without 1 μM of 14 for 4 h and their cytoplasmic lysates were fractionated on a sucrose gradient.

RNA was extracted from each of the fractions and subjected to qRT-PCR for the analysis of the distribution of XIAP and GAPDH mRNAs. Data

show the percentage of the total amount of the corresponding mRNA in each fraction and represent the mean

SD of three independent

experiments. (B) Western blot showed the activation of caspases 3 and 9 as well as the cleavage of the death substrate PARP in EU-1 cells following

treatment with 5 μM of MX69 and 1 μM of 14 for times indicated.

1

enhanced inhibition of XIAP function as well as the activation

of p53.

cis-isomers). A comparative depiction of the H NMR spectra

of the stereoisomers of 14 is presented in Figure S2, panel A.

To elucidate the absolute conﬁguration of these stereoisomers,

single-crystal X-ray diﬀraction (SCXRD) studies were under-

taken. Isomer 14a provided crystals suitable for SCXRD

analysis, and its absolute conﬁguration was determined as

3aR,4S,9bR (Figure 5C). Multiple attempts to crystallize 14b

and 14d under various conditions failed to yield crystals of

suﬃcient quality for X-ray analysis. Several alternatives were

attempted until ultimately, Boc-14b and Boc-14d, Boc

derivatives prepared by N-protection of 14b and 14d,

respectively, yielded suitable crystals. As shown in Figure

5D,E, the absolute conﬁgurations 3aR,4R,9bR and 3aS,4S,9bS

were established for 14b and 14d, respectively, based on the

SCXRD of the corresponding Boc derivatives. By elimination,

the absolute conﬁguration of the remaining isomer, 14c, is

unambiguously established as 3aS,4R,9bS. The absolute

structures of 14a−d are shown in Figure 5B. The

antiproliferative activity of the four isomers was subsequently

PK Study Exhibits Limited In Vivo Metabolic Stability of

14. Prior to embarking on the eﬃcacy studies in murine cancer

models to further assess the potential in vivo eﬃcacy of

compound 14, we investigated its PK behavior after intra-

venous (IV) and oral administration in rats to ensure that

potentially eﬃcacious drug exposures could be reached and

maintained in vivo. The resulting average concentration−time

proﬁles are shown in Figure S1. After an IV administration of

10 mg/kg, plasma concentrations followed a reproducible

biexponential proﬁle with peak concentrations of 8.20 2.47

μg/mL (mean SD) and an area under the concentration−

time curve (AUC) of 4.75 0.51 μg h/mL. The clearance was

with 2.12 0.21 L/h/kg relatively fast, indicating limited in

vivo metabolic stability. Consequently, despite a relatively large

volume of distribution of 2.88 0.51 L/kg as expected for a

lipophilic compound such as 14, the elimination half-life

remained very short at 0.94 0.11 h. After oral administration

of 25 mg/kg of 14, the exposure remained relatively limited

with an AUC of 0.71 0.31 μg h/mL. The resulting low oral

bioavailability of 5.9% was likely the consequence of the

limited in vivo stability and the related high hepatic ﬁrst-pass

inactivation after oral absorption. These unfavorable PK

properties prevented a further comprehensive in vivo

evaluation of compound 14, and eﬀorts are underway to

create more viable analogues with increased metabolic stability.

Separation of Stereoisomers 14 and Identiﬁcation of

Absolute Conﬁgurations for Individual Isomers. All the

current MX69 analogues contain three chiral centers, with two

chiral centers in the A-ring restricted to the same geometry

(either both α- or both β-conﬁgurations) due to the structural

constriction during the synthesis. Thus, the most potent

compound 14 is a mixture of four stereoisomers. Since

diﬀerent stereoisomers often have diﬀerent biological activities,

the separation was undertaken by chiral HPLC and several

analytical conditions were tested. Utilization of a cellulose-4

column with methanol as the mobile phase was found to

provide the best separation for the four isomers (14a−d shown

in Figure 5A). These conditions were applied to a preparative

method to give the individual isomers with good purities

tested by the WST cytotoxicity assays. Compound 14d (IC₅₀

=

0.3 μM) exhibited an outstanding antiproliferative activity,

with 5- to 8-fold higher potency compared to the other three

isomers (IC₅₀values of 14a−c are 1.9, 1.6, and 2.4 μM,

respectively). It is interesting that 14d exhibited the same

activity as 14, implying a complicated interaction among the

isomers or between the isomers with a site of action.

Molecular Modeling Studies to Preliminarily Under-

stand the SAR of the MX69 Scaﬀold. Since no crystal

structure of the MDM2 RING domain is presently available

and we are actively working to generate this protein and to

determine its complexes with potent MX69 analogues, as a

preliminary step to understand the observed SAR described

above, we employed the published NMR structure of the

MDM2 RING domain protein (PDB: 2HDP) for modeling.³⁰

We examined the MDM2 RING domain and identiﬁed the

most likely binding site for MX69 (Figure 6, top panels) that

also fulﬁlled two important requirements: (1) the size of the

site must be large enough to accommodate MX69 or its larger

analogues such as compound 14 and (2) the site must be close

to the critical residue Cys464 because it is well known that the

point mutation C464A will abolish the E3-ligase activity of

MDM2.³¹Consistent with these requirements, this site has a

“7” shape, which is highly compatible with the overall

1

(>98%). Preliminary identiﬁcation by H NMR indicated that

14a/14c and 14b/14d are two pairs of racemates (trans- and

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Figure 5. (A) Chiral HPLC separation of the stereoisomeric mixture 14. (B) Stereostructure of isomers 14a−d. ORTEP plot (thermal ellipsoids

set at 50% probability) of the structures obtained from the SCXRD analyses of (C) 14a, (D) Boc-14b, and (E) Boc-14d. Hydrogen atoms are

removed for clarity. The reliability of the absolute stereochemistry assignments is established by Flack parameters of −0.017(9) for 14a,

−0.020(11) for Boc-14b, and −0.005(5) for Boc-14d.

geometry of MX69 and its analogues. Subsequent docking

studies generated reasonable binding conformations for MX69

analogues, as exempliﬁed for MX69 and 14d shown in Figure

6, since isomer 14d with absolute conﬁguration 3aS,4S,9bS

exhibited the highest potency.

This model revealed several interesting features that are

consistent with the SAR results described above for the MX69

scaﬀold. First, the B-ring moiety of MX69 occupies the deeper

pocket 2. Serendipitously, this pocket is large and is formed

mostly by the nearby hydrophobic residues. This may explain

why compound 14 with a less polar but a larger B-ring

substitution is signiﬁcantly better than MX69 for MDM2

binding. Second, the sulfonamide NH in the head moiety is

very close to the MDM2 surface due to the strong interactions

between K473 and both the SO₂moiety (hydrogen bond) and

the dimethylphenyl ring (cation−π interaction). Thus, the

sulfonamide NH between the C/D rings has limited tolerance

for large substitutions, as we found with reduced activities in

compounds 45 and 46. The other NH in the central moiety of

MX69 also has limited surrounding space. This is consistent

with our SAR data that a substitution at either NH is highly

detrimental to MDM2 binding and thus the antiproliferative

potency. Third, substitutions on the D-ring, especially to the

K473 side of the MDM2 RING domain surface, are predicted

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Figure 6. A molecular model of MX69 and compound 14d binding the MDM2 RING domain (PDB: 2HDP). The binding site is identiﬁed using

SiteMap in Schrodinger suites. Top panels show best docking poses on the MDM2 electrostatic surface (blue: positive; red: negative). The deep,

hydrophobic pocket 2 may explain the improved binding of compound 14d over MX69 at the B-ring substitution and the untolerated substitutions

at the sulfonamide. Bottom panels show 2D ligand interactions (green: hydrophobic residues).

to reduce the potency because of the tight space between the

D-ring and the MDM2 RING domain. This is consistent with

the signiﬁcant loss of potency for compounds 40, 41, 43, and,

to a lesser extent, 44. Fourth, the ﬁve-member A-ring in the

central body of MX69 is partially exposed to solvents and does

not seem to signiﬁcantly contribute to MDM2 binding except

for maintaining the proper geometry for the B-ring moiety to

enter pocket 2. This is consistent with the marginal

improvement observed when we bioisosterically replaced the

CC double bond of this ring with O or NH and the

detrimental eﬀects when we added a hydrophobic fused ring.

Last, both methyl substitutions in the C-ring ﬁt very well to a

hydrophobic region in pocket 1, consistent with the optimal

potency observed for compound 14 over compounds 31−39.

Although very preliminary and hypothetical at this stage, this

binding model does explain many of our observed SAR. When

an X-ray structure of the MDM2 RING domain becomes

available, we will revisit this model and reﬁne it, so as to better

guide our ongoing eﬀorts to optimize the MX69 scaﬀold.

CONCLUSIONS

■

In this report, we performed a preliminary, systemic SAR

investigation based on the MX69 scaﬀold as a dual MDM2/

XIAP inhibitor and discovered compound 14 that achieves a

25-fold increase in potency over the original MX69 hit. We

also reported a preliminary molecular binding model that

could explain most of the observed SAR for the MX69 scaﬀold.

We demonstrated that 14 sustains the on-target of MDM2 and

XIAP activity by inducing MDM2 protein degradation and

inhibiting XIAP mRNA translation, which leads to cancer

inhibition and death.

However, PK studies revealed that the in vivo stability of 14

is not ideal. We are currently working to design new analogues

with improved PK properties. Nevertheless, the presented data

strongly suggest that this scaﬀold is promising for further

optimization to develop a new therapeutic agent for leukemia

and possibly other cancers where MDM2 and XIAP are

dysregulated.

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1H), 8.40 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 8.8 Hz, 2H), 7.56 (d, J =

8.8 Hz, 1H), 7.14 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H), 2.24 (s, 3H).

3-Chloro-N-(3,4-dimethylphenyl)-4-nitrobenzenesulfonamide

EXPERIMENTAL SECTION

■

General Chemistry. All solvents and chemical reagents were

purchased from commercial sources and used directly without further

puriﬁcation. Glassware was oven-dried before use. All reactions were

performed under an argon atmosphere. Analytical thin-layer

chromatography was performed on silica gel 60 GF254 and

monitored under UV light. Flash chromatography was performed

on 230−400 mesh silica gel (Fisher Scientiﬁc). NMR spectra were

obtained on a Bruker Ascend 400 (Billerica, MA) spectrometer. The

purity of all ﬁnal compounds (as stereoisomers not separable on the

UPLC column used) was ≥95% as determined by UPLC and further

conﬁrmed by proton NMR (details given in the Supporting

Information). High-resolution mass spectrometry data (HRMS)

were obtained on a Waters Acquity UPLC linked to a Waters

Acquity Photodiode Array Detector and a Waters qTof mass detector.

UPLC analyses were performed using a BEH C18 (2.1 × 50 mm, 1.7

μm) column and a mixture of solvent methanol/water at a ﬂow rate of

0.3 mL/min, monitored by UV absorption at the appropriate

wavelength. Chemical shifts are given in ppm. Tetramethylsilane is

used as an internal reference for NMR spectra taken in chloroform-d.

All coupling constants (J) are given in hertz (Hz).

1

(6k). 87.5% yield; H NMR (400 MHz, CDCl₃): δ 7.88−7.86 (m,

2H), 7.65 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H), 7.21 (s, 1H), 7.05 (d, J =

8.0 Hz, 1H), 6.98 (1, 1H), 6.93 (d, J = 8.0 Hz, 1H), 2.23 (s, 6H).

2-Chloro-N-(3,4-dimethylphenyl)-4-nitrobenzenesulfonamide

1

(6l). 84% yield; H NMR (400 MHz, DMSO-d₆): δ 10.74 (s, 1H),

8.43 (d, J = 2.0 Hz, 1H), 8.30−8.22 (m, 2H), 6.98 (d, J = 8.0 Hz,

1H), 6.89 (d, J = 1.6 Hz, 1H), 6.82 (dd, J₁= 8.0 Hz, J₂= 2.4 Hz, 1H),

2.10 (s, 3H), 2.08 (s, 3H).

General Procedure for Hydrogenation to form Anilines 7a−

7l. Sulfonamide (16.32 mmol) and Pd/C (10% Pd base) were mixed

in MeOH (50 mL) at room temperature. Hydrogen gas was

introduced via a H₂balloon. The reaction mixture was stirred

under a H₂atmosphere overnight at room temperature. The reaction

mixture was ﬁltered to remove the solid. The solution was

concentrated under reduced pressure and the resulting residues

were puriﬁed by column chromatography (silica gel, CH₂Cl₂/MeOH

= 9/1 v/v) to aﬀord compounds 7a−7l.

4-Amino-N-(3,4-dimethylphenyl)benzenesulfonamide (7a).

1

87.6% yield; H NMR (400 MHz, DMSO-d₆): δ 9.60 (s, 1H); 7.37

General Procedure for the Synthesis of Sulfonamides 6a−

6l. Substituted benzenesulfonyl chloride (26.58 mmol) and 3,4-

dimethylaniline (29.23 mmol) were dissolved in anhydrous methylene

chloride (100 mL) at room temperature under argon. Pyridine (2.31

g, 29.23 mmol) was added dropwise via a syringe at room

temperature. The reaction mixture was stirred at room temperature

overnight under an argon atmosphere. Then, the volatile was removed

under reduced pressure. The yellow solid residues were subjected to

ﬂash column chromatography (silica gel, CH₂Cl₂) to aﬀord

compounds 6a−6l.

(d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 6.80 (d, J =

8.0 Hz, 1H), 6.52 (d, J = 8.0 Hz, 2H), 5.91 (s, 2H), 2.09 (s, 3H), 2.08

(s, 3H).

4-Amino-N-(p-tolyl)benzenesulfonamide (7b). Yield 59.4%. ¹H

NMR (400 MHz, DMSO-d₆): δ 9.69 (s, 1H), 7.34 (d, J = 8.8 Hz,

2H), 6.99 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 6.50 (d, J =

8.8 Hz, 2H), 5.95 (s, 2H), 2.17 (s, 3H).

4-Amino-N-(m-tolyl)benzenesulfonamide (7c). 87.2% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 9.80 (s, 1H), 7.38 (d, J = 8.4 Hz,

2H), 7.09−7.05 (m, 1H), 6.86 (s, 2H), 6.77 (d, J = 7.2 Hz, 1H), 6.52

(d, J = 8.4 Hz, 2H), 5.97 (s, 2H), 2.18 (s, 3H).

N-(3,4-Dimethylphenyl)-4-nitrobenzenesulfonamide (6a). 87%

1

yield; H NMR (400 MHz, DMSO-d₆): δ 10.37 (s, 1H); 8.36 (d, J

4-Amino-N-phenylbenzenesulfonamide (7d). 96.3% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 9.86 (s, 1H), 7.38 (d, J = 8.0 Hz,

2H), 7.21−7.17 (m, 2H), 7.05 (d, J = 8.0 Hz, 2H), 6.98−6.94 (m,

1H), 6.52 (s, 2H), 5.97 (s, 2H).

= 8.0 Hz, 2H), 7.96 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 1H), 6.88

(s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 2.11 (s, 3H), 2.09 (s, 3H).

N-(3-Bromo-4-methylphenyl)-4-nitrobenzenesulfonamide (6b).

94.3% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 10.75 (s, 1H),

8.39 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H), 7.30 (s, 1H), 7.24

(d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 2.23 (s, 3H).

4-Nitro-N-(m-tolyl)benzenesulfonamide (6c). 99% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 10.56 (s, 1H), 8.38 (d, J = 8.0 Hz,

2H), 8.00 (d, J = 8.0 Hz, 2H), 7.15−7.11 (m, 1H), 6.93−6.88 (m,

3H), 2.20 (s, 3H).

4-Amino-N-(3-chloro-4-methylphenyl)benzenesulfonamide (7e).

1

90.3% yield; H NMR (400 MHz, DMSO-d₆): δ 9.98 (s, 1H), 7.38

(d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 6.94 (d, J =

8.0 Hz, 1H), 6.54 (d, J = 8.4 Hz, 2H), 6.01 (s, 2H), 2.19 (s, 3H).

4-Amino-N-(4-chloro-3-methylphenyl)benzenesulfonamide (7f).

1

94.1% yield; H NMR (400 MHz, DMSO-d₆): δ 9.98 (s, 1H), 7.38

4-Nitro-N-phenylbenzenesulfonamide (7d). 92.7% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 10.63 (s, 1H), 8.37 (d, J = 8.4 Hz,

2H), 7.99 (d, J = 8.4 Hz, 2H), 7.28−7.24 (m, 1H), 7.11−7.06 (m,

1H).

N-(3-Chloro-4-methylphenyl)-4-nitrobenzenesulfonamide (6e).

86.1% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 10.74 (s, 1H),

8.38 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.4 Hz,

1H), 7.14 (d, J = 2.0 Hz, 1H), 6.99 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H).

N-(4-Chloro-3-methylphenyl)-4-nitrobenzenesulfonamide (6f).

97.5% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 10.74 (s, 1H),

8.39 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.4 Hz,

1H), 7.14 (d, J = 2.0 Hz, 1H), 6.99 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H),

2.21 (s, 3H).

(d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 6.95 (d, J =

8.0 Hz, 1H), 6.54 (d, J = 8.4 Hz, 2H), 6.01 (s, 2H), 2.19 (s, 3H).

4-Amino-N-(3,4-dichlorophenyl)benzenesulfonamide (7g).

99.2% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 10.28 (s, 1H),

7.47 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 2.0 Hz,

1H), 7.05 (dd, J₁= 8.8 Hz, J₂= 2.4 Hz, 1H), 6.55 (d, J = 8.4 Hz, 2H),

6.07 (s, 2H).

4-Amino-N-(3,4-dibromophenyl)benzenesulfonamide (7h).

1

60.3% yield; H NMR (400 MHz, DMSO-d₆): δ 9.98 (s, 1H), 7.38

(d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 6.95 (d, J =

8.0 Hz, 1H), 6.54 (d, J = 8.4 Hz, 2H), 6.01 (s, 2H), 2.19 (s, 3H).

4-Amino-N-(4-ﬂuoro-3-methylphenyl)benzenesulfonamide (7i).

1

97.5% yield; H NMR (400 MHz, DMSO-d₆): δ 9.74 (s, 1H), 7.35

N-(3,4-Dichlorophenyl)-4-nitrobenzenesulfonamide (6g). 99.6%

yield; ¹H NMR (400 MHz, DMSO-d₆): δ 11.03 (s, 1H), 8.40 (d, J =

8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.32

(d, J = 2.4 Hz, 1H), 7.11 (dd, J₁= 8.8 Hz, J₂= 2.4 Hz, 1H).

N-(3,4-Dibromophenyl)-4-nitrobenzenesulfonamide (6h). 94.9%

yield; ¹H NMR (400 MHz, DMSO-d₆): δ 11.03 (s, 1H), 8.39 (d, J =

8.8 Hz, 2H), 8.03 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.8 Hz, 1H), 7.44

(d, J = 2.4 Hz, 1H), 7.08 (dd, J₁= 8.8 Hz, J₂= 2.4 Hz, 1H).

N-(4-Fluoro-3-methylphenyl)-4-nitrobenzenesulfonamide (6i).

96.6% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 10.51 (s, 1H),

8.38 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.8 Hz, 2H), 7.06−6.99 (m,

2H), 6.91−6.87 (m, 1H), 2.14 (s, 3H).

(d, J = 8.0 Hz, 2H), 7.00−6.92 (m, 2H), 6.88−6.86 (m, 1H), 6.52 (d,

J = 8.0 Hz, 2H), 5.98 (s, 2H), 2.12 (s, 3H).

4-Amino-N-(3-methyl-4-(triﬂuoromethyl)phenyl)-

benzenesulfonamide (7j). 93.8% yield; ¹H NMR (400 MHz, DMSO-

d₆): δ 10.42 (s, 1H), 7.51−7.46 (m, 3H), 7.07−7.04 (m, 2H), 6.55 (d,

J = 8.8 Hz, 2H), 6.07 (s, 2H), 2.32 (s, 3H).

4-Amino-3-chloro-N-(3,4-dimethylphenyl)benzenesulfonamide

1

(7k). 84.2% yield; H NMR (400 MHz, DMSO-d₆): δ 7.44 (dd, J₁=

8.0 Hz, J₂= 1.6 Hz, 1H), 7.20−7.16 (m, 1H), 6.93 (d, J = 8.4 Hz,

1H), 6.80 (s, 1H), 6.76 (dd, J₁= 8.0 Hz, J₂= 2.0 Hz, 1H), 6.71 (d, J =

7.6 Hz, 1H), 6.53−6.49 (m, 1H), 5.98 (s, 2H), 2.07 (s, 3H), 2.06 (s,

3H).

4-Amino-2-chloro-N-(3,4-dimethylphenyl)benzenesulfonamide

N-(3-Methyl-4-(triﬂuoromethyl)phenyl)-4-nitrobenzenesulfona-

1

mide (6j). 89.8% yield; H NMR (400 MHz, DMSO-d₆): δ 11.15 (s,

(7l). 87% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 9.90 (s, 1H); 7.61

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(d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H),

6.77 (dd, J₁= 8.0 Hz, J₂= 2.4 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 6.45

(dd, J₁= 8.8 Hz, J₂= 2.4 Hz, 1H), 6.23 (s, 2H), 2.08 (s, 3H), 2.07 (s,

3H).

= 2.0 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H), 6.28 (s, 1H), 5.64−5.62 (m,

2H), 4.75 (d, J = 3.2 Hz, 1H), 4.20 (s, 1H), 4.05 (d, J = 8.8 Hz, 1H),

3.95 (s, 3H), 3.05−2.98 (m, 1H), 2.54−2.47 (m, 1H), 2.21 (s, 6H),

1.80−1.74 (m, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 166.8, 149.2,

146.8, 137.6, 134.4, 133.8, 133.4, 130.4, 130.2, 130.0, 129.5, 129.0,

128.2, 126.3, 125.9, 125.5, 123.6, 119.7, 115.5, 57.2, 52.2, 45.6, 45.3,

31.4, 19.8, 19.2. HRMS (ESI): calcd for C₂₈H₂₈N₂NaO₄S, 511.1662

[M + Na]⁺; found, 511.1650. Purity: 97.6% by UPLC (R_t= 3.44

min).

Methyl 4-(8-(N-(3,4-Dimethylphenyl)sulfomoyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinolin-4-yl)benzoate (2). 68.5% yield; ¹H

NMR (400 MHz, CDCl₃): δ 8.09 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 2.0

Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.45 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz,

1H), 7.01 (d, J = 8.0 Hz, 1H), 6.90 (d, J = 2 Hz, 1H), 6.81 (dd, J₁=

8.0 Hz, J₂= 2.0 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 6.27 (s, 1H), 5.20

(d, J = 7.6 Hz, 1H), 4.88 (d, J = 2.8 Hz, 1H), 4.30 (s, 1H), 3.95 (s,

3H), 3.77−3.72 (m, 1H), 3.69−3.67 (m, 1H), 2.80−2.74 (m, 1H),

2.21 (s, 3H), 2.20 (s, 3H), 2.09−1.97 (m, 1H), 1.51−1.47 (m, 1H).

¹³C NMR (100 MHz, CDCl₃): δ 166.7, 148.7, 148.0, 146.3, 145.9,

137.4, 134.6, 134.5, 133.3, 133.2, 131.3, 130.1, 129.9, 129.6, 128.6,

128.2, 128.0, 127.9, 127.2, 126.4, 123.2, 123.0, 121.4, 119.1, 119.0,

118.7, 114.5, 114.3, 75.2, 74.8, 66.3, 65.0, 56.6, 56.0, 52.3, 52.2, 44.4,

42.4, 28.3, 24.1, 19.8, 19.1. HRMS (ESI): calcd for C₂₇H₂₈N₂NaO₅S,

515.1611 [M + Na]⁺; found, 515.1586. Purity: 98.9% by UPLC (R_t=

3.28 min).

tert-Butyl 8-(N-(3,4-Dimethylphenyl)sulfamoyl)-4-(4-

(methoxycarbonyl)phenyl)-2,3,3a,4,5,9b-hexahydro-1H-pyrrolo-

[3,2-c]quinoline-1-carboxylate (3b). The crude product was used

directly for the next step without further puriﬁcation.

Methyl 4-(9-(N-(3,4-Dimethylphenyl)sulfamoyl)-3,4,4a,5,6,10b-

hexahydro-2H-pyrano[3,2-c]quinolin-5-yl)benzoate (4). ¹H NMR

(400 MHz, CDCl₃): δ 8.07 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 2.0 Hz,

0.3H), 7.72 (d, J = 2.0 Hz, 0.7H), 7.53 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz,

0.3H), 7.44 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 0.7H), 7.01−6.98

(m, 1H), 6.90 (s, 1H), 6.84−6.80 (m, 1H), 6.59 (d, J = 8.8 Hz, 0.3H),

6.48 (d, J = 8.8 Hz, 0.7H), 6.41 (s, 0.3H), 6.35 (s, 0.7H), 5.23 (d, J =

5.6 Hz, 0.3H), 4.80 (s, 0.7H), 4.78 (s, 0.3H), 4.57 (s, 0.7H), 4.37−

4.36 (m, 1H), 4.04−4.02 (m, 0.7H), 3.95 (s, 3H), 3.71−3.69 (m,

1H), 3.45−3.40 (m, 0.7H), 3.07−3.02 (m, 0.3H), 2.20 (s, 3H), 2.19

(s, 3H), 2.08−2.05 (m, 0.7H), 1.85−1.82 (m, 1H), 1.74−1.62 (m,

1H), 1.57−1.41 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 166.7,

148.0, 146.5, 137.5, 134.5, 133.5, 130.9, 130.2, 128.9, 127.6, 126.5,

123.3, 119.5, 119.3, 113.6, 54.7, 52.3, 38.3, 34.7, 31.6, 25.3, 23.7, 22.7,

22.0, 19.8, 19.2, 14.1. HRMS (ESI): calcd for C₂₈H₃₁N₂O₅S, 507.1948

[M + H]⁺; found, 507.1957. Purity: 95.4% by UPLC (R_t= 1.19 min).

Methyl 4-(2-(N-(3,4-Dimethylphenyl)sulfamoyl)-6,6a,7,11b-tet-

rahydro-5H-indeno[2,1-c]quinolin-6-yl)benzoate (5). ¹H NMR

(400 MHz, CDCl₃): δ 8.10 (d, J = 7.6 Hz, 2H), 7.74 (s, 1H), 7.53

(d, J = 7.6 Hz, 2H), 7.42 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.15−7.14

(m, 2H), 7.05 (s, 1H), 6.91 (d, J = 7.2 Hz, 1H), 6.77 (s, 1H), 6.72 (d,

J = 8.0 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H), 6.21 (s, 1H), 4.87 (s, 1H),

4.50 (d, J = 4.8 Hz, 1H), 4.36 (s, 1H), 3.97 (s, 3H), 3.16−3.11 (m,

2H), 2.38−2.30 (m, 1H), 2.19 (s, 3H), 2.13 (s, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 166.8, 157.9, 151.0, 149.2, 146.4, 144.3, 140.6,

137.85, 137.81, 136.9, 134.2, 134.1, 131.6, 130.6, 130.4, 130.0, 129.4,

128.7, 127.8, 126.4, 125.6, 124.64, 124.57, 124.2, 123.6, 122.7, 119.7,

52.4, 42.3, 34.6, 31.6, 29.7, 25.2, 22.6, 19.8, 19.1, 14.1. HRMS (ESI):

calcd for C₃₂H₃₁N₂O₄S, 539.1999 [M + H]⁺; found, 539.1990. Purity:

98.4% by UPLC (R_t= 3.46 min).

General Procedure for the Synthesis of Aldehydes 9a and

9b. To a solution of 1-bromo-4-(diethoxymethyl)benzene or 1-

bromo-3-(diethoxymethyl)benzene (45.30 g, 174.81 mmol) in 200

mL of anhydrous tetrahydrofuran under an argon atmosphere was

added dropwise n-butyllithium (76.88 mL of 2.5 N hexanes solution,

192.2 mmol) at −78 °C in a dry ice/acetone bath. After stirring at

−78 °C for 2 h, a solution of CuCN (15.66 g, 184.81 mmol) and LiCl

(14.82 g, 349.62 mmol) in 200 mL of anhydrous tetrahydrofuran was

added dropwise with stirring at −78 °C under argon. The resulting

solution was stirred at −78 °C for 30 min, then slowly warmed to

room temperature, and stirred for another hour. Isobutyryl chloride

(18.08 mL, 174.81 mmol) was added dropwise into the solution at

−78 °C within 1 h. The reaction mixture was allowed to warm up to

room temperature and stirred for another 4 h. The reaction was

quenched by the addition of 100 mL of water at room temperature

with vigorous stirring. THF solvent was removed under reduced

pressure. The residue 8a or 8b was extracted with methylene chloride

(3 × 100 mL). HCl (100 mL, aq, 3 M) was then added to the

solution and the mixture was stirred for 3 h. The organic layer was

separated and dried over anhydrous MgSO₄, ﬁltered, and concen-

trated under reduced pressure. The crude liquid was puriﬁed by

column chromatography (silica gel, CH₂Cl₂) to aﬀord pure

compound 9a or 9b.

4-Isobutyrylbenzaldehyde (9a). 91.5% yield; ¹H NMR (400 MHz,

DMSO-d₆): δ 10.11 (s, 1H); 8.14 (d, J = 8.0 Hz, 2H), 8.05 (d, J = 8.0

Hz, 2H), 3.70 (m, 1H), 1.12 (d, J = 6.8 Hz, 6H).

3-Isobutyrylbenzaldehyde (9b). 94.1% yield; ¹H NMR (400 MHz,

DMSO-d₆): δ 10.12 (s, 1H); 8.48 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H),

8.15 (d, J = 7.6 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 3.77−3.70 (m, 1H),

1.14 (d, J = 6.8 Hz, 6H).

Synthesis of 4-Formyl-N-methoxy-N-methylbenzamide (20). 4-

Formylbenzoic acid (10.00 g, 66.61 mmol) was dissolved in

anhydrous methylene chloride (100 mL) at room temperature

under argon. Oxalyl chloride (10.15 g, 79.93 mmol) was added

dropwise via a syringe at room temperature. The reaction mixture was

cooled to 0 °C and 0.5 mL of anhydrous DMF was added. The

resulted mixture was stirred at room temperature for 4 h. Then, N,O-

dimethylhydroxylamine hydrochloride (9.75 g, 99.92 mmol) was

added at room temperature. Triethylamine (20.22 g, 199.83 mmol)

was added to the reaction mixture at 0 °C. After stirring at room

temperature overnight, the reaction was quenched by adding water.

The organic layer was separated, dried over anhydrous MgSO₄,

ﬁltered, and concentrated under reduced pressure. The yellow oil

residue was subjected to ﬂash column chromatography (silica gel,

CH₂Cl₂/MeOH = 19/1 v/v) to aﬀord a pale-yellow oil product

1

(10.58 g, 82.2% yield). H NMR (400 MHz, CDCl₃): δ 10.07 (s,

1H), 7.94 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 3.54 (s, 3H),

3.39 (s, 3H).

General Procedure for the Synthesis of MX69 Analogues.

Anilines (0.27 g, 0.87 mmol), aldehydes (0.87 mmol), and Sc(OTf)₃

(0.17 mmol) were mixed together and dissolved in anhydrous

CH₃CN (10 mL) at room temperature under argon. The reaction

mixture was stirred for 1 h at room temperature. Alkene (1.74 mmol)

was then added via a syringe. The resulting mixture was stirred at

room temperature under argon overnight. The reaction was quenched

by adding 50 mL of water at room temperature and neutralized by

adding NaHCO₃. The solution was extracted with ethyl acetate (3 ×

50 mL). The extracts were dried over anhydrous MgSO₄, ﬁltered, and

concentrated under reduced pressure to dryness. The solid residues

were puriﬁed by column chromatography to aﬀord the desired

compounds.

N-(3,4-Dimethylphenyl)-4-(4-isopropylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (10). 75.6% yield; ¹H

NMR (400 MHz, CDCl₃): δ 7.89 (s, 0.6H), 7.80 (s, 0.4H), 7.44−

7.39 (m, 1H), 7.36−7.32 (m, 2H), 7.28−7.26 (m, 3H), 7.01−6.97

(m, 1H), 6.99 (d, J = 1.6 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.50 (d, J

= 8.8 Hz, 1H), 6.48 (d, J = 4.4 Hz, 0.4H), 6.42 (s, 0.6H), 5.19 (d, J =

7.2 Hz, 0.4H), 4.78 (d, J = 2.4 Hz, 0.4H), 4.62 (s, 0.6H), 4.54 (d, J =

4.8 Hz, 0.6H), 4.31 (s, 0.4H), 4.06−4.02 (m, 0.6H), 3.89−3.87 (m,

0.4H), 3.82 (d, J = 11.2 Hz, 0.6H), 3.76−3.69 (m, 1H), 2.97−2.92

(m, 1H), 2.76−2.71 (m, 0.4H), 2.41−2.35 (m, 0.6H), 2.19 (s, 6H),

2.09−2.04 (m, 1H), 1.78−1.72 (m, 0.6H), 1.65−1.57 (m, 0.4H),

Methyl 4-(8-(N-(3,4-Dimethylphenyl)sulfamoyl)-3a,4,5,9b-tetra-

1

hydro-3H-cyclopenta[c]quinolin-4-yl)benzoate (1). 79.2% yield; H

NMR (400 MHz, CDCl₃): δ 8.07 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.0

Hz, 2H), 7.40 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 1H), 7.36 (s, 1H), 7.02

(d, J = 8.0 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 6.81 (dd, J₁= 8.0 Hz, J₂

1941

https://dx.doi.org/10.1021/acs.jmedchem.0c00932

J. Med. Chem. 2021, 64, 1930−1950

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1.29−1.27 (m, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 149.3, 148.9,

148.8, 148.2, 138.3, 137.9, 137.5, 134.6, 134.5, 133.4, 133.3, 131.4,

130.12, 130.11, 130.09, 128.6, 128.1, 127.8, 127.7, 127.0, 126.9, 126.8,

126.4, 123.2, 123.1, 121.7, 119.2, 119.1, 118.9, 114.2, 114.0, 75.5,

75.0, 66.5, 65.0, 56.6, 56.1, 44.7, 42.3, 33.83, 33.78, 28.6, 24.3, 24.0,

19.8, 19.8, 19.2. HRMS (ESI): calcd for C₂₈H₃₂N₂NaO₃S, 499.2026

[M + Na]⁺; found, 499.1997. Purity: 99.5% by UPLC (R_t= 3.49

min).

4-(4-Bromophenyl)-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (11). 79.2%; ¹H

NMR (400 MHz, CDCl₃): δ 7.80 (d, J = 2.0 Hz, 1H), 7.53 (d, J =

8.4 Hz, 2H), 7.44 (dd, J₁= 8.4 Hz, J₂= 2.4 Hz, 1H), 7.31 (d, J = 8.0

Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.80 (d, J

= 8.0 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 5.18 (d, J = 7.6 Hz, 1H), 4.77

(d, J = 3.2 Hz, 1H), 4.31 (s, 1H), 3.95 (s, 3H), 3.75−3.71 (m, 1H),

3.69−3.65 (m, 1H), 2.72−2.70 (m, 1H), 2.20 (s, 3H), 2.19 (s, 3H),

2.03−1.97 (m, 1H), 1.55−1.51 (m, 1H). ¹³C NMR (100 MHz,

CDCl₃): δ 148.7, 147.8, 140.1, 139.6, 137.6, 134.4, 134.3, 133.6,

133.5, 132.0, 131.9, 131.7, 131.4, 130.5, 130.2, 130.14, 130.06, 129.8,

128.6, 128.2, 128.0, 127.9, 127.5, 123.3, 123.2, 122.4, 121.8, 121.7,

119.24, 119.15, 119.0, 114.4, 114.2, 75.3, 74.8, 66.4, 65.0, 56.4, 55.8,

44.6, 42.5, 29.7, 28.4, 24.2, 19.8, 19.2. HRMS (ESI): calcd for

C₂₅H₂₅BrN₂NaO₃S, 535.0661 [M + Na]⁺; found, 535.0659. Purity:

95.3% by UPLC (R_t= 3.41 min).

MHz, DMSO-d₆): δ 204.0, 203.9, 149.6, 149.1, 147.3, 137.2, 137.1,

136.4, 135.7, 135.3, 131.9, 131.8, 131.0, 130.32, 130.28, 129.3, 129.2,

128.9, 128.8, 128.0, 127.4, 127.3, 127.1, 126.2, 121.7, 121.5, 121.0,

118.4, 117.9, 117.6, 114.6, 114.3, 75.0, 74.5, 66.1, 64.6, 55.8, 55.0,

44.0, 42.0, 35.0, 28.5, 24.5, 20.0, 19.5, 19.4, 19.1. HRMS (ESI): calcd

for C₂₉H₃₃N₂O₄S, 505.2161 [M + H]⁺; found, 505.2151. Purity:

99.3% by UPLC (R_t= 3.29 min).

4-(3-Acetylphenyl)-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (16). 67.4% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 9.77 (s, 0.5H), 9.73 (s, 0.5H), 8.00

(d, J = 3.6 Hz, 1H), 7.96−7.91 (m, 1H), 7.73−7.70 (m, 1H), 7.60−

7.55 (m, 2H), 7.39 (d, J = 8.8 Hz, 0.5H), 7.33 (d, J = 8.4 Hz, 0.5H),

7.16 (s, 0.5H), 6.96 (d, J = 8.0 Hz, 1H), 6.89−6.80 (m, 2.5H), 6.74−

6.71 (m, 1H), 5.11 (d, J = 7.2 Hz, 0.5H), 4.86 (s, 0.5H), 4.43 (d, J =

4.8 Hz, 0.5H), 3.94−3.88 (m, 0.5H), 3.80 (d, J = 10.8 Hz, 0.5H),

3.74−3.68 (m, 0.5H), 3.63−3.57 (m, 0.5H), 3.52−2.50 (m, 0.5H),

2.70−2.65 (m, 0.5H), 2.59 (s, 3H), 2.34−2.29 (m, 0.5H), 2.11 (s,

3H), 2.09 (s, 3H), 1.98−1.92 (m, 0.5H),1.81−1.76 (m, 0.5H), 1.56−

1.50 (m, 0.5H), 1.31−1.29 (m, 0.5H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 198.4, 149.7, 149.2, 142.7, 142.5, 137.5, 137.3, 137.2, 137.1,

136.39, 136.36, 133.7, 131.89, 131.86, 131.8, 131.0, 130.32, 130.28,

129.4, 129.3, 128.3, 128.03, 127.96, 127.3, 127.1, 126.5, 126.2, 121.7,

121.5, 121.0, 118.4, 117.9, 117.6, 114.6, 114.3, 75.1, 74.6, 66.0, 64.6,

55.8, 55.0, 44.2, 42.0, 28.6, 27.30, 27.27, 24.4, 20.03, 20.02, 19.1.

HRMS (ESI): calcd for C₂₇H₂₈N₂NaO₄, 499.1662 [M + Na]⁺; found,

499.1648. Purity: 98.7% by UPLC (R_t= 3.19 min).

Methyl N-(3,4-Dimethylphenyl)-4-(4-(methylsulfonyl)phenyl)-

2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12).

1

73% yield; H NMR (400 MHz, DMSO-d₆): δ 9.75 (s, 0.5H), 9.71

N-(3,4-Dimethylphenyl)-4-(3-isobutyrylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (17). 59.4% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 9.77 (s, 0.6H), 9.74 (s, 0.4H), 8.03

(d, J = 3.6 Hz, 1H), 7.96−7.91 (m, 1H), 7.73−7.71 (m, 1H), 7.68−

7.55 (m, 2H), 7.39 (d, J = 8.8 Hz, 0.6H), 7.18 (s, 0.6H), 6.96 (d, J =

8.0 Hz, 1H), 6.89−6.80 (m, 2.4H), 6.74−6.71 (m, 1H), 5.10 (d, J =

7.2 Hz, 0.4H), 4.86 (s, 0.4H), 4.43 (d, J = 4.8 Hz, 0.6H), 3.94−3.88

(m, 0.6H), 3.80 (d, J = 10.8 Hz, 0.6H), 3.71−3.68 (m, 2H), 3.63−

3.57 (m, 0.8H), 3.52−2.50 (m, 0.6H), 2.70−2.65 (m, 0.4H), 2.34−

2.29 (m, 0.6H), 2.09−2.11 (m, 9H), 1.98−1.92 (m, 1H),1.87−1.76

(m, 1H), 1.51−1.50 (m, 1H), 1.31−1.29 (m, 0.4H), 1.19−1.05 (m,

6.6H). ¹³C NMR (100 MHz, DMSO-d₆): δ 204.25, 204.22, 149.7,

149.2, 142.8, 142.6, 137.2, 137.1, 136.40, 136.36, 136.3, 136.2, 133.5,

131.9, 131.8, 131.0, 130.32, 130.27, 129.5, 129.4, 128.4, 128.3, 128.0,

127.8, 127.3, 127.1, 126.6, 126.2, 121.7, 121.5, 121.0, 118.4, 117.9,

117.6, 114.6, 114.3, 75.1, 74.6, 66.1, 64.6, 55.8, 55.0, 44.2, 42.0, 35.11,

35.08, 30.0, 28.5, 24.4, 20.0, 19.6, 19.5, 19.3, 19.1. HRMS (ESI): calcd

for C₂₉H₃₂N₂NaO₄S, 527.1975 [M + Na]⁺; found, 527.1965. Purity:

95.5% by UPLC (R_t= 3.33 min).

N-(3,4-Dimethylphenyl)-4-(naphthalene-1-yl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (18). 76.4% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 9.76 (s, 0.6H), 9.73 (s, 0.4H), 8.27−

8.25 (m, 1H), 8.00−7.88 (m, 2H), 7.78 (d, J = 7.2 Hz, 0.4H), 7.66−

7.64 (m, 1H), 7.58−7.53 (m, 3.6H), 7.41−7.29 (m, 1.6H), 6.99−6.96

(m, 1H), 6.89−6.72 (m, 3.4H), 5.62 (d, J = 2.0 Hz, 0.4H), 5.29 (d, J

= 7.2 Hz, 0.4H), 4.50 (d, J = 4.8 Hz, 0.6H), 4.48 (s, 0.4H), 3.92−3.88

(m, 0.6H), 3.74−3.69 (m, 0.6H), 3.56−3.51 (m, 1H), 2.85−2.67 (m,

0.4H), 2.70−2.65 (m, 0.6H), 2.11−2.10 (m, 6H), 2.02−1.92 (m,

0.6H), 1.84−1.78 (m, 0.4H), 1.48−1.43 (m, 0.6H), 1.19−1.10 (m,

0.6H). ¹³C NMR (100 MHz, DMSO-d₆): δ 149.8, 137.4, 137.2, 137.1,

136.5, 134.1, 133.8, 132.0, 131.84, 131.77, 131.1, 130.4, 130.3, 129.3,

128.8, 128.2, 128.0, 127.2, 127.0, 126.84, 126.76, 126.2, 126.0, 125.9,

123.7, 123.1, 121.6, 121.4, 118.2, 117.8, 117.5, 114.8, 114.2, 75.3,

74.5, 66.0, 64.8, 60.2, 51.0, 42.5, 31.2, 29.0, 25.0, 20.0, 19.1. HRMS

(ESI): calcd for C₂₉H₂₉N₂O₃S, 485.1893 [M + H]⁺; found, 485.1888.

Purity: 97.3% by UPLC (R_t= 1.29 min).

(s, 0.5H), 7.96−7.94 (m, 2H), 7.75−7.70 (m, 2H), 7.60 (s, 0.5H),

7.55 (s, 0.5H), 7.40 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 0.5H), 7.33 (dd, J₁=

8.8 Hz, J₂= 2.0 Hz, 1H), 7.19 (s, 0.5H), 6.96 (d, J = 8.0 Hz, 1H),

6.89−6.80 (m 3H), 6.73−6.70 (m, 1H), 5.12 (d, J = 7.2 Hz, 0.5H),

4.89 (d, J = 2.8 Hz, 0.5H), 4.44 (d, J = 4.4 Hz, 0.5H), 3.92−3.84 (m,

1H), 3.75−3.50 (m, 2.5H), 2.72−2.67 (m, 0.5H), 2.33−2.29 (m,

0.5H), 2.11 (s, 3H), 2.10 (s, 3H), 1.99−1.92 (m, 0.5H), 1.81−1.76

(m, 1H), 1.56−1.53 (m, 0.5H), 1.48−1.28 (m, 1H). ¹³C NMR (100

MHz, DMSO-d₆): δ 148.9, 148.1, 140.2, 137.1, 136.3, 131.9, 130.3,

129.3, 128.0, 127.6, 127.3, 127.3, 121.7, 121.0, 118.0, 114.6, 74.5,

66.0, 54.9, 44.0, 43.8, 24.4, 20.0, 19.1. HRMS (ESI): calcd for

C₂₆H₂₈N₂NaO₅S₂, 535.1332 [M + Na]⁺; found, 535.1329. Purity:

99.5% by UPLC (R_t= 3.03 min).

4-(4-Acetylphenyl)-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-

1

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (13). H NMR (400

MHz, CDCl₃): δ 8.02−7.99 (m, 2H), 7.89 (d, J = 2.0 Hz, 0.5H), 7.81

(d, J = 2.0 Hz, 0.5H), 7.54−7.51 (m, 2H), 7.47−7.43 (m, 1H), 7.00

(s, 0.5H), 6.98 (s, 0.5H), 6.90 (s, 1H), 6.82−6.80 (m, 1H), 6.57 (dd,

J₁= 8.4 Hz, J₂= 2.4 Hz, 1H), 6.39 (s, 0.5H), 6.35 (s, 0.5H), 5.20 (d, J

= 7.2 Hz, 0.5H), 4.88 (d, J = 3.2 Hz, 0.5H), 4.61 (s, 0.5H), 4.54 (d, J

= 4.8 Hz, 0.5H), 4.34 (s, 0.5H), 4.17−4.01 (m, 1H), 3.93−3.85 (m,

1H), 3.77−3.63 (m, 1H), 2.78−2.76 (m, 0.5H), 2.64 (s, 3H), 2.45−

2.39 (m, 0.5H), 2.19 (s, 6H), 2.08−2.00 (m, 0.5H),1.75−1.70 (m,

0.5H), 1.54−1.47 (m, 0.5H). ¹³C NMR (100 MHz, CDCl₃): δ

197.87, 197.85, 148.7, 148.0, 146.5, 146.1, 137.5, 137.4, 137.0, 136.6,

134.6, 134.5, 133.33, 133.26, 131.3, 130.1, 129.9, 128.9, 128.5, 128.4,

128.0, 127.8, 127.3, 126.6, 123.2, 123.0, 119.1, 119.0, 118.7, 114.6,

114.3, 75.2, 74.7, 66.3, 65.0, 56.5, 56.0, 44.4, 42.4, 28.3, 26.71, 26.67,

24.1, 19.8, 19.1. HRMS (ESI): calcd for C₂₇H₂₈N₂NaO₄S, 499.1662

[M + Na]⁺; found, 499.1631. Purity: 95.3% by UPLC (R_t= 3.19

min).

N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (14). 41.7% yield; ¹H

NMR (400 MHz, DMSO-d₆): δ 9.78,9.75 (s, 1H), 7.99 (d, J = 8.4 Hz,

2H), 7.62−7.55 (m, 3H), 7.39 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 0.55H),

7.33 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 0.45H), 7.18 (s, 0.55H), 6.96 (d, J

= 8.4 Hz, 1H), 6.89−6.86 (m, 1.45H), 6.84−6.80 (m, 1H), 6.74−6.70

(m, 1H), 5.11 (d, J = 7.2 Hz, 0.45 H), 4.84 (d, J = 2.8 Hz, 0.45H),

4.43 (d, J = 4.8 Hz, 0.55H), 3.90−3.88 (m, 0.55H), 3.79 (d, J = 10.8

Hz, 0.55H), 3.72−3.59 (m, 2H), 3.51−3.49 (m, 0.55H), 2.69−2.67

(m, 0.45H), 2.30−2.27 (m, 0.55H), 2.11 (s, 3H), 2.09 (s, 3H), 1.96−

1.93 (m, 0.55H), 1.83−1.72 (m, 0.55H), 1.56−1.52 (m, 0.55H),

1.32−1.30 (m, 0.45H), 1.11 (d, J = 6.8 Hz, 6H). ¹³C NMR (100

N-((3,4-Dimethylphenyl)-sulfonyl-2,3,3a,4,5,9b-hexahydrofuran-

[3,2-c]quinolin-4-yl)-N-methoxy-N-methylbenzamide (21). 87.3%

1

yield; H NMR (400 MHz, DMSO-d₆): δ 9.75, 9.71 (s, 1H), 7.61

(d, J = 8.0 Hz, 2H), 7.55−7.50 (m, 3H), 7.38 (dd, J₁= 8.4 Hz, J₂= 4.4

Hz, 0.38 H), 7.32 (dd, J₁= 8.4 Hz, J₂= 2.4 Hz, 1H), 7.17 (s, 0.38 H),

6.96 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.84−6.80 (m,

1H), 6.75−6.70 (m, 1H), 5.11 (d, J = 7.6 Hz, 0.67 H), 4.81 (d, J = 2.8

Hz, 0.66H), 4.33 (d, J = 2.8 Hz, 0.43H), 3.90−3.88 (m, 0.5H), 3.77−

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3.70 (m, 1H), 3.52 (s, 3H), 3.26 (s, 3H), 2.69−2.67 (s, 0.76H),

2.32−2.26 (m, 0.65H), 2.11−2.09 (m, 6H), 1.99−1.93 (m, 0.85H),

1.82−1.75 (m, 1H), 1.57−1.55 (m, 0.67H), 1.37−1.33 (m, 0.72H).

N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-3a,4,5,9b-tetra-

hydro-3H-cyclopenta[c]quinoline-8-sulfonamide (27). 72.6% yield;

¹H NMR (400 MHz, CDCl₃): δ 7.96 (d, J = 8.0 Hz, 2H), 7.47 (d, J =

8.0 Hz, 2H), 7.40−7.37 (m, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.85 (s,

1H), 6.82 (d, J = 8.0 Hz, 1H), 6.76−6.71 (m, 1H), 6.60 (d, J = 8.4

Hz, 1H), 5.60 (d, J = 10.4 Hz, 2H), 4.71 (s, 1H), 4.25 (s, 1H), 4.02

(d, J = 8.4 Hz, 1H), 3.55 (sep, J = 6.8 Hz, 1H), 3.02−2.96 (m, 1H),

2.51−2.45 (m, 1H), 2.17 (s, 3H), 1.78−1.72 (m, 2H), 1.22 (d, J = 6.4

Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 204.1, 149.2, 146.7, 137.6,

135.5, 134.4, 133.8, 133.4, 130.4, 130.2, 129.0, 128.8, 128.2, 126.5,

125.9, 125.5, 123.6, 119.7, 115.5, 57.2, 45.6, 45.3, 35.4, 31.5, 19.8,

19.2, 19.1. HRMS (ESI): calcd for C₃₀H₃₂N₂NaO₃S, 523.2026 [M +

Na]⁺; found, 523.2003. Purity: 97.9% by UPLC (R_t= 3.47 min).

tert-Butyl 8-(N-(3,4-Dimethylphenyl)sulfamoyl)-4-(4-isobutyryl-

phenyl)-2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,2-c]quinoline-1-car-

boxylate (29). The crude product was used directly for the next step

without further puriﬁcation.

4-(4-Isobutyrylphenyl)-N-(p-tolyl)-2,3,3a,4,5,9b-hexahydrofuro-

[3,2-c]quinoline-8-sulfonamide (31). 72.2% yield; ¹H NMR (400

MHz, CDCl₃): δ 7.98−7.95 (m, 2H), 7.86 (d, J = 2.0 Hz, 0.6H), 7.76

(d, J = 1.6 Hz, 0.4H), 7.52−7.48 (m, 2H), 7.42−7.38 (m, 1H), 7.03−

6.93 (m, 4H), 6.84 (s, 0.4H), 6.78 (s, 0.6H), 6.56−6.53 (m, 1H), 5.18

(d, J = 7.2 Hz, 0.4H), 4.84 (d, J = 2.8 Hz, 0.4H), 4.74 (s, 0.6H), 4.50

(d, J = 4.8 Hz, 0.6H), 4.44 (s, 0.4H), 4.05−4.01 (m, 0.6H), 3.90 (d, J

= 10.8 Hz, 0.6H), 3.87−3.83 (m, 0.6H), 3.72−3.64 (m, 0.8H), 2.56−

2.51 (m, 1H), 2.76−2.72 (m, 0.4H), 2.42−2.38 (m, 0.6H), 2.26 (s,

3H), 2.05−1.99 (m, 1H), 1.71−1.68 (m, 0.6H), 1.52−1.43 (m,

0.4H), 1.22 (d, J = 6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ

204.0, 148.7, 147.9, 146.0, 145.6, 136.3, 134.9, 134.2, 131.4, 130.0,

129.8, 128.9, 128.6, 128.4, 127.9, 126.6, 122.1, 122.0, 114.4, 114.2,

75.2, 74.8, 66.4, 65.1, 56.7, 56.1, 44.4, 42.5, 35.5, 35.4, 28.4, 24.2,

20.8, 19.14, 19.11. HRMS (ESI): calcd for C₂₈H₃₀N₂NaO₄S, 513.1818

[M + Na]⁺; found, 513.1788. Purity: 96.3% by UPLC (R_t= 3.29

min). HRMS (ESI): calcd for C₂₈H₃₀N₂NaO₄S, 513.1818 [M + Na]⁺;

found, 513.1788. Purity: 96.3% by UPLC (R_t= 3.29 min).

4-(4-Isobutyrylphenyl)-N-(m-tolyl)-2,3,3a,4,5,9b-hexahydrofuro-

[3,2-c]quinoline-8-sulfonamide (32). 58.7% yield; ¹H NMR (400

MHz, DMSO-d₆): δ 9.93 (s, 0.5H), 9.90 (s, 0.5H), 7.99 (d, J = 8.0

Hz, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.57 (s, 1H), 7.42 (d, J = 8.4 Hz,

0.5H), 7.35 (d, J = 8.8 Hz, 0.5H), 7.21 (s, 0.5H), 7.11−7.07 (m, 1H),

6.91−6.88 (m, 2.5H), 6.80 (d, J = 7.2 Hz, 1H), 6.75−6.71 (m, 1H),

5.11 (d, J = 7.2 Hz, 0.5H), 4.85 (s, 0.5H), 4.43 (d, J = 4.4 Hz, 0.5H),

3.91−3.87 (m, 0.5H), 3.80 (d, J = 10.8 Hz, 0.5H), 3.74−3.45 (m,

3H), 2.71−2.65 (m, 0.5H), 2.31−2.24 (m, 0.5H), 2.20 (s, 3H), 1.97−

1.92 (m, 0.5H), 1.80−1.75 (m, 0.5H), 1.58−1.53 (m, 0.5H), 1.32−

1.30 (m, 0.5H), 1.10 (d, J = 6.8 Hz, 6H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 203.95, 203.92, 149.7, 149.2, 147.3, 147.0, 138.8,

138.73, 138.69, 135.7, 135.3, 131.0, 129.4, 129.3, 129.2, 128.9, 128.8,

128.0, 127.4, 127.3, 127.0, 126.1, 124.6, 124.5, 121.0, 120.4, 120.2,

118.4, 117.0, 116.7, 114.6, 114.4, 75.0, 74.5, 66.1, 64.6, 55.8, 55.0,

44.0, 42.0, 35.1, 35.0, 28.5, 24.5, 21.54, 21.53, 19.5, 19.45, 19.43.

HRMS (ESI): calcd for C₂₈H₃₀N₂NaO₄S, 513.1818 [M + Na]⁺;

found, 513.1788. Purity: 98.3% by UPLC (R_t= 3.28 min).

118.4, 114.6, 114.4, 75.0, 74.5, 66.1, 64.6, 55.8, 55.0, 43.9, 41.9, 35.1,

35.0, 28.5, 24.5, 19.5, 19.45, 19.43. HRMS (ESI): calcd for

C₂₇H₂₈N₂NaO₄S, 499.1662 [M + Na]⁺; found, 499.1629. Purity:

99.2% by UPLC (R_t= 3.23 min).

N-(3-Chloro-4-methylphenyl)-4-(4-isobutyrylphenyl)-

2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (34).

1

56.6% yield; H NMR (400 MHz,, DMSO-d₆): δ 10.13 (s, 0.6H),

10.08 (s, 0.4H), 8.00 (d, J = 8.0 Hz, 2H), 7.62−7.58 (m, 3H), 7.41

(dd, J₁= 8.8 Hz, J₂= 2.4 Hz, 0.6H), 7.35 (dd, J₁= 8.8 Hz, J₂= 1.6 Hz,

0.4H), 7.26 (s, 0.6H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (dd, J₁= 8.0 Hz,

J₂= 1.6 Hz, 1H), 7.01−6.99 (m, 1.4H), 6.77−6.72 (m, 1H), 5.12 (d, J

= 7.2 Hz, 0.4H), 4.87 (d, J = 3.2 Hz, 0.4H), 4.44 (d, J = 4.8 Hz,

0.6H), 3.92−3.88 (m, 0.6H), 3.81 (d, J = 10.8 Hz, 0.6H), 3.74−3.60

(m, 2H), 3.52−3.49 (m, 0.4H), 2.72−2.67 (m, 0.4H), 2.33−2.27 (m,

0.6H), 2.21 (s, 3H), 1.99−1.91 (m, 0.6H), 1.82−1.72 (m, 0.6H),

1.57−1.53 (m, 0.4H), 1.34−1.31 (m, 0.4H), 1.11 (d, J = 6.8 Hz, 6H).

¹³C NMR (100 MHz, DMSO-d₆): δ 203.9, 149.9, 149.3, 147.2, 147.0,

138.0, 135.7, 135.3, 133.7, 133.6, 132.0, 132.0, 131.0, 130.7, 130.6,

129.3, 129.2, 128.9, 128.8, 128.0, 127.4, 127.3, 126.9, 126.4, 125.5,

121.1, 120.0, 119.7, 118.8, 118.5, 114.7, 114.4, 75.0, 74.4, 66.1, 64.6,

55.7, 55.0, 43.9, 41.9, 35.05, 35.02, 28.5, 24.5, 19.5, 19.4, 19.2. HRMS

(ESI): calcd for C₂₈H₂₉ClN₂NaO₄S, 547.1429 [M + Na]⁺; found,

547.1453. Purity: 96.0% by UPLC (R_t= 3.39 min).

N-(4-Chloro-3-methylphenyl)-4-(4-isobutyrylphenyl)-

2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (35).

1

67.5% yield; H NMR (400 MHz, DMSO-d₆): δ 10.10 (s, 0.6H),

10.06 (s, 0.4H), 8.00 (d, J = 7.6 Hz, 2H), 7.62−7.55 (m, 3H), 7.42

(dd, J₁= 8.8 Hz, J₂= 2.0 Hz, 0.6H), 7.36 (dd, J₁= 8.8 Hz, J₂= 2.0 Hz,

0.4H), 7.28−7.25 (m, 1.6H), 7.04 (s, 1H), 6.96−6.92 (m, 1.4H),

6.76−6.72 (m, 1H), 5.12 (d, J = 7.2 Hz, 0.4H), 4.86 (d, J = 2.8 Hz,

0.4H), 4.44 (d, J = 4.8 Hz, 0.6H), 3.92−3.89 (m, 0.6H), 3.81 (d, J =

10.8 Hz, 0.6H), 3.74−3.58 (m, 2H), 3.52−3.49 (m, 0.4H), 2.71−2.65

(m, 0.4H), 2.33−2.26 (m, 0.6H), 2.21 (s, 3H), 1.98−1.93 (m, 0.6H),

1.83−1.75 (m, 0.6H), 1.57−1.53 (m, 0.4H), 1.35−1.29 (m, 0.4H),

1.15 (d, J = 6.8 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ 203.97,

149.8, 149.3, 147.2, 147.0, 137.7, 136.5, 135.7, 135.3, 131.0, 129.8,

129.2, 128.9, 128.1, 127.5, 126.5, 125.6, 122.3, 122.1, 121.0, 119.0,

118.7, 118.4, 114.4, 105.8, 75.0, 74.5, 66.1, 66.0, 64.6, 55.7, 55.0, 43.9,

41.9, 35.05, 35.02, 28.5, 24.5, 20.2, 19.4. HRMS (ESI): calcd for

C₂₈H₂₉ClN₂NaO₄S, 547.1429 [M + Na]⁺; found, 547.1440. Purity:

96.9% by UPLC (R_t= 3.41 min).

N-(3,4-Dichlorophenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (36). 69.8% yield; ¹H

NMR (400 MHz, CDCl₃): δ 7.99 (d, J = 8.2 Hz, 2H), 7.84−7.76 (m,

1H), 7.52 (d, J = 8.2 Hz, 2H), 7.49−7.43 (m, 1H), 7.32 (d, J = 8.7

Hz, 0.7H), 7.18 (d, J = 3.6 Hz, 1H), 7.10−7.06 (m, 0.3H), 7.06−7.02

(m, 0.3H), 7.00 (dd, J₁= 8.7, J₂= 2.6 Hz, 1H), 6.64−6.60 (m, 0.7H),

6.59−6.55 (m, 1H), 5.21 (d, J = 7.5 Hz, 1H), 4.89 (d, J = 3.0 Hz,

1H), 4.38 (s, 0.7H), 4.36 (s, 0.3H), 3.80−3.64 (m, 2H), 3.60−3.50

(m, 1H), 2.82−2.72 (m, 1H), 2.18 (s, 1H), 2.09−1.94 (m, 1H),

1.56−1.46 (m, 1H), 1.24 (s, 3H), 1.22 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 204.0, 149.1, 148.2, 145.8, 145.4, 136.8, 136.7, 136.4,

135.9, 132.9, 131.5, 130.8, 130.2, 130.1, 128.9, 128.6, 128.4, 128.3,

127.9, 127.1, 126.6, 126.4, 122.3, 121.9, 120.2, 120.0, 119.1, 114.6,

114.4, 75.2, 74.7, 66.5, 65.1, 56.6, 56.1, 44.3, 42.4, 35.51, 35.46, 28.4,

24.2, 19.14, 19.10. HRMS (ESI): calcd for C₂₇H₂₆Cl₂N₂NaO₄S,

567.0883 [M + Na]⁺; found, 567.0944. Purity: 96.8% by UPLC (R_t=

3.43 min).

4-(4-Isobutyrylphenyl)-N-phenyl-2,3,3a,4,5,9b-hexahydrofuro-

[3,2-c]quinoline-8-sulfonamide (33). 56.6% yield; ¹H NMR (400

MHz, DMSO-d₆): δ 9.99, 9.97 (s, 1H), 8.00 (d, J = 8.0 Hz, 2H),

7.62−7.56 (m, 3H), 7.41 (dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 0.5H), 7.35

(dd, J₁= 8.4 Hz, J₂= 2.0 Hz, 0.5H), 7.24−7.21 (m, 2.5H), 7.11−7.08

(m, 2H), 7.01−6.98 (m, 1H), 6.92 (s, 0.5H), 6.73 (t, J = 8.4 Hz, 1H),

5.11 (d, J = 7.6 Hz, 0.5H), 4.85 (d, J = 2.8 Hz, 0.5H), 4.43 (d, J = 4.8

Hz, 0.5H), 3.91−3.88 (m, 0.5H), 3.80 (d, J = 10.4 Hz, 0.5H), 3.72−

3.59 (m, 2H), 3.52−3.49 (m, 0.5H), 2.72−2.67 (m, 0.5H), 2.33−2.27

(m, 0.5H),1.99−1.92 (m, 0.5H), 1.82−1.72 (m, 0.5H), 1.57−1.52

(m, 0.5H), 1.34−1.29 (m, 0.5H), 1.11 (d, J = 6.8 Hz, 6H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 204.0, 203.9, 149.7, 149.2, 147.3, 147.0,

138.8, 135.7, 135.3, 131.0, 129.50, 129.45, 129.3, 129.2, 128.9, 128.8,

128.0, 127.5, 127.4, 127.0, 126.0, 123.9, 123.8, 121.1, 120.0, 119.8,

N-(3,4-Dibromophenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-

hexahydrofuro[3,2-c]quinoline-8-sulfonamide (37). 56.6%; ¹H

NMR (400 MHz, DMSO-d₆): δ 10.41 (s, 0.6H), 10.36 (s, 0.4H),

8.00 (d, J = 7.6 Hz, 2H), 7.63−7.55 (m, 4H), 7.46−7.36 (m, 2H),

7.32 (s, 0.4H), 7.09−7.04 (m, 1.6H), 6.79−6.75 (m, 1H), 5.13 (d, J =

7.6 Hz, 0.4H), 4.88 (d, J = 2.8 Hz, 0.4H), 4.45 (d, J = 4.8 Hz, 0.6H),

3.92−3.88 (m, 0.6H), 3.83 (d, J = 10.8 Hz, 0.6H), 3.75−3.59 (m,

2H), 3.52−3.49 (m, 0.4H), 2.72−2.67 (m, 0.4H), 2.33−2.27 (m,

0.6H), 1.99−1.91 (m, 0.4H), 1.80−1.72 (m, 0.6H), 1.59−1.52 (m,

0.6H), 1.36−1.32 (m, 0.4H), 1.11 (d, J = 6.8 Hz, 6H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 204.0, 203.9, 150.1, 149.5, 147.1, 146.9,

139.6, 135.8, 135.3, 134.61, 134.57, 131.0, 129.3, 129.2, 128.9, 128.8,

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1

128.1, 127.5, 127.4, 125.9, 125.0, 124.51, 124.46, 123.8, 123.6, 121.1,

120.3, 119.9, 118.5, 117.9, 117.7, 114.8, 114.5, 74.9, 74.4, 66.1, 64.6,

55.7, 54.9, 43.8, 41.8, 35.1, 35.0, 28.5, 24.5, 19.5, 19.45, 19.43. HRMS

(ESI): calcd for C₂₇H₂₆Br₂N₂NaO₄S, 654.9872 [M + Na]⁺; found,

654.9943. Purity: 95.3% by UPLC (R_t= 3.46 min).

N-(4-Fluoro-3-methylphenyl)-4-(4-isobutyrylphenyl)-

2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (38).

68.4% yield; ¹H NMR (400 MHz, DMSO-d₆): δ 9.88 (s, 0.4H),

9.83 (s, 0.6H), 8.00 (d, J = 8.0 Hz, 2H), 7.62−7.58 (m, 2.4H), 7.51

(s, 0.6H), 7.37 (d, J = 8.8 Hz, 0.4H), 7.32 (d, J = 8.4 Hz, 0.6H), 7.22

(s, 0.4H), 7.03−6.89 (m, 3.6H), 6.72 (t, J = 8.4 Hz, 1H), 5.11 (d, J =

7.6 Hz, 0.6H), 4.86 (d, J = 2.8 Hz, 0.6H), 4.43 (d, J = 4.8 Hz, 0.4H),

3.92−3.88 (m, 0.4H), 3.81 (d, J = 10.4 Hz, 0.4H), 3.72−3.57 (m,

2H), 3.52−3.46 (m, 0.6H), 2.71−2.65 (m, 0.6H), 2.33−2.27 (m,

0.4H), 2.14 (s, 3H), 1.99−1.90 (m, 0.4H), 1.80−1.75 (m, 0.4H),

1.56−1.53 (m, 0.6H), 1.34−1.29 (m, 0.6H), 1.11 (d, J = 6.4 Hz, 6H).

¹³C NMR (100 MHz, DMSO-d₆): δ 203.9, 149.7, 149.2, 147.2, 147.0,