Discovery and Optimization of 2 H-1λ2-Pyridin-2-one Inhibitors of Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer

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Discovery and Optimization of 2H‑1λ²‑Pyridin-2-one Inhibitors of

Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer

Jason M. Rohde, Surendra Karavadhi, Rajan Pragani, Li Liu, Yuhong Fang, Weihe Zhang,

Andrew McIver, Hongchao Zheng, Qingyang Liu, Mindy I. Davis, Daniel J. Urban, Tobie D. Lee,

Dorian M. Che ﬀ , Melinda Hollingshead, Mark J. Henderson, Natalia J. Martinez, Kyle R. Brimacombe,

Adam Yasgar, Wei Zhao, Carleen Klumpp-Thomas, Sam Michael, Joseph Covey, William J. Moore,

Gordon M. Stott, Zhuyin Li, Anton Simeonov, Ajit Jadhav, Stephen Frye, Matthew D. Hall,* Min Shen,*

Xiaodong Wang,* Samarjit Patnaik,* and Matthew B. Boxer

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ABSTRACT: Neomorphic mutations in isocitrate dehydrogenase

1 (IDH1) are oncogenic for a number of malignancies, primarily

low-grade gliomas and acute myeloid leukemia. We report a

medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-

2H-1λ²-quinoline-2,5(6H)-dione screening hit against the R132H

and R132C mutant forms of isocitrate dehydrogenase (IDH1).

Systematic SAR eﬀorts produced a series of potent pyrid-2-one

mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-

SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft

mouse model, after a single oral dose of 30 mg/kg, 16 h post dose,

between 16 and 48 h, (+)-119 showed higher tumoral

concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).

cellular behavior.¹¹⁻¹³For example, 2-HG inhibits α-KG-

INTRODUCTION

■

dependent enzymes,^14,15and direct evidence exists for

Over the past decade, mutant isocitrate dehydrogenases 1

inhibition of α-KG-dependent histone and DNA demethylases

(mIDH1) and 2 (mIDH2) have emerged as important targets

by 2-HG leading to elevated histone methylation, with

for treating a range of malignancies.^1,2Normal (wild-type,

consequent impact on gene expression and cell diﬀer-

WT) IDH1 or IDH2 protein catalyzes the conversion of

entiation.¹⁶

isocitrate to α-ketoglutarate (α-KG, also called 2-oxoglutarate,

Both mIDH1 and mIDH2 are attractive therapeutic targets

2-OG), using NADP⁺as a cofactor.³Critical work has

due to their genetic gain-of-function, which confers an

demonstrated that some acute myelogenous leukemias

oncogenic role that is amenable to detection via both tumor

(AMLs) and gliomas possess heterozygous somatic mutations

gene sequencing and metabolite (2-HG) measurement.¹⁷

of IDH1 at position R132 and IDH2 at R140 or R172.⁴⁻⁶

Moreover, speciﬁc inhibition of mIDH1/2 should not produce

Analysis of clinical samples has shown that the majority of low-

target-related clinical side eﬀects as WT IDH’s function would

grade gliomas and ∼20% of AML have IDH1 or IDH2

be unaﬀected. In 2017, only after ten years since the ﬁrst

mutations, and a range of other solid tumors such as

reports of the role of IDH1/2 mutations in cancer, the FDA

chondrosarcoma, cholangiocarcinoma, colon, pancreatic, and

approved the mIDH2 inhibitor enasidenib from Agios/

prostate cancer have also been found to carry IDH1/2

Celgene for treatment of relapsed or refractory AML (R/R

mutations, although to a lesser degree.^1,7

AML).¹⁸In 2018, the FDA granted priority approval to Agios’

The canonical mutations to IDH1 and IDH2 confer a

mIDH1 inhibitor AG-120 (ivosidenib) for patients with R/R

neomorphic (gain-of-function) activity. While WT IDH1

produces α-KG, α-KG is the substrate of mIDH1, which in a

Received: January 6, 2021

Published: April 6, 2021

pseudo-reverse reaction using NADPH produces R-2-hydrox-

yglutarate (2-HG).^8,9An analysis of serum 2HG levels in AML

patients bearing IDH1 or IDH2 mutants showed a clear

elevation (median 2HG of 3004 ng/mL) compared to patients

with WT IDH (median 2HG of 61 ng/mL).¹⁰This 2-HG

“oncometabolite” has been shown to play a role in modifying

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Figure 1. (A) Schematic for primary biochemical qHTS assay; (B) assay performance as measured by Z′ factor and S/B ratio; (C) workﬂow for the

identiﬁcation of small-molecule inhibitors of mutant IDH1; and (D) dose-dependent activity of hit 1 against R132H mIDH1, IDH-WT, and in a

readout interference counterscreen.

AML harboring IDH1 mutations as measured by the Abbott

RealTime IDH1 assay.¹⁹In May 2019, ivosidenib was

approved as ﬁrst-line treatment for AML with IDH1 mutation

in patients who are at least 75 years old or who have

comorbidities that preclude the use of intensive induction

chemotherapy.¹⁹Agios continues to take ivosidenib through

multiple clinical trials, notably, toward AML in combination

with the chemotherapeutic drug azacytidine and toward glioma

and cholangiocarcinoma in patients harboring mIDH1.²⁰Agios

is also advancing its pan mIDH oral inhibitor AG-881

(vorasidenib) in trials with patients with advanced solid

tumors (including gliomas) and low-grade glioma.^20,21In

addition to this, Novartis and Bayer have also advanced IDH

inhibitors IDH305 and BAY1436032 mIDH1 to clinical

trials.^22,23In 2019, an updated succinct review of all reported

classes of preclinical and clinical small-molecule mIDH

inhibitors was published.²⁴It included Agios’ tool mIDH1

inhibitor AG-5198,²⁵probe molecules from Sanoﬁ and

GlaxoSmithKline,^26,27and CNS penetrant FT-2102 from

Forma Therapeutics, Inc.²⁸

We had been involved in an early assay development and

screening program that produced the phenyl-glycine analogue

ML309 as a potent inhibitor of R132H mIDH1.^29,30We also

developed a panel of biochemical and cell-based assays to

enable inhibitor discovery campaigns and aid comparison to

known mIDH1 probe and experimental therapeutic inhib-

itors.³¹Here, we fully describe the hit-to-lead medical

chemistry campaign based on a 7,7-dimethyl-7,8-dihydro-2H-

1λ²-quinoline-2,5(6H)-dione mIDH1 inhibitor chemical series

that also emerged as a screening hit besides the chemotype that

was optimized to ML309. Optimization led to the discovery,

separation, and biological characterization of stable atro-

pisomers from a 1-arylpyridin-2(1H)-one subseries. Exemplary

analogue (+)-119 is used to demonstrate 2-HG reduction in a

pharmacokinetic/pharmacodynamic (PK/PD) model with

ivosidenib as a comparator molecule.

Our project was supported by the National Cancer

Institute’s (NCI) Experimental Therapeutics (NExT) Program

facilitated within the NCI Chemical Biology Consortium

(CBC). The goal of CBC’s NExT Program is to bring together

government, industry, and academic groups to enable a team-

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Scheme 1. General Analogue Synthesis

Table 1. Core Modiﬁcations and SAR

biochemical

IDH1 R132H

% inhibition SD at 38 μM

IDH1 R132C

% inhibition SD at 38 μM

a

compound

IC₅₀(μM) SD

1

2

1.6 0.2

>38

84

7

1.0 0.3

>38

85

9

3

>38

4

5

6

7

8

9

10

3.6 0.5

3.0 0.3

87

96

62

38

35

3

4

1

0

64

76

43

29

36

32

65

5

2

1

4

2

3

4

1.7 0.2

8.0 2.4

2.3 0.0

75 15

84

1

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled R132H and R132C mIDH1 assays (average of N = 3). IC₅₀values are

reported only for compounds where ≥75% inhibition of enzyme activity was observed at the highest concentration tested (38 μM).

science approach toward the discovery and development of

novel therapeutics (https://next.cancer.gov).

from the endpoint and normalized against control wells [no

enzyme as the positive control and dimethyl sulfoxide

(DMSO) as the negative control]. The percent inhibition at

each of the concentrations of inhibitor tested was plotted and

modeled using a four-parameter logistic ﬁt using in-house

software to determine the compound IC₅₀values. There were

nearly 600 1536-well plates in the primary qHTS, which

included library plate sets and a couple of DMSO plates

inserted in the beginning and end of the screening for data

correction purposes. The assay was performed well with an

RESULTS AND DISCUSSION

■

A diaphorase/resazurin-coupled assay for the IDH1 R132H

mutation was developed and utilized to screen our in-house

collection of nearly 390,000 small molecules (Figure 1A). In

this quantitative high-throughput screen (qHTS), the assay

was performed at six concentrations ranging from 76 μM to 2

nM. The inhibition associated with each well was computed

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Table 2. Thiazole Substitution SAR

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled R132H and R132C mIDH1 assays (average of N = 3). IC₅₀values are

reported only for compounds where ≥75% inhibition of enzyme activity was observed at the highest concentration tested (38 μM).

average Z′ factor of 0.60 and an S/B ratio around 3.2 (Figure

1B). Analysis of these dose−response curves resulted in 2155

testing concentration. Among these 2155 inhibitors, 769 were

identiﬁed as high-quality inhibitors with single or dual

compounds that showed inhibition over 30% at the highest

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Table 3. Pyridone Aryl Substituent SAR

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled R132H and R132C mIDH1 assays (average of N = 3). IC₅₀values are

reported only for compounds where ≥75% inhibition of enzyme activity was observed at the highest concentration tested (38 μM).

asymptote curves and greater than 50% inhibition (Figure 1C),

representing 0.2% of the chemical library.

1D, compound 1 displayed inhibition with an IC₅₀of 1.6 μM

against R132H mIDH1, with no readout interference and oﬀ-

target activity against WT IDH1. In addition to the R132H

mutant,^32,33we also developed the analogous assay for the

R132C mIDH1, a frequent mutation in AML and intracranial

chondrosarcoma,^34,35and we sought to develop a class of

inhibitors capable of targeting both mutations (1 had an IC₅₀

of 1.0 μM against R132C mIDH1).

Despite the moderate structural complexity of hit 1, it was

readily accessible via an eﬃcient one-pot, multicomponent

coupling (Scheme 1). Combining dione I and acetal II well

resulted in quick conversion to reactive vinylogous amide III.

The addition of a base (piperidine or KOtBu) to nitrile IV

generated an anion that when added to intermediate III,

aﬀorded intermediate V by a Michael addition−elimination

sequence. Finally, incorporation of aniline Ar₂NH₂VI provided

the pyridinone ring system through cyclodehydration. This last

two-step sequence was suﬃciently tolerant of diverse

substitution on both nitrile IV and aniline VI and was

The 2155 hit compounds identiﬁed from the screen were

thoroughly evaluated for both potency and percent inhibition;

the autoﬂuorescent compounds and promiscuous inhibitors

(compounds that show activity in more than 20% of assays

within historical screening data at NCATS) were eliminated

from follow-up examination. A total of 761 prioritized hits

were retested and screened in 12-pt dose−response in the

primary screening assay to reconﬁrm the activity. The

compounds were further tested in a readout interference

assay and IDH1-WT selectivity assay to reﬁne and triage initial

screening hits (assay hit triage workﬂow shown in Figure 1C).

After structural clustering analysis and assessment of synthetic

tractability to eliminate compounds that did not oﬀer good

starting points for hit-to-lead optimization, a 7,7-dimethyl-7,8-

dihydro-2H-1λ²-quinoline-2,5(6H)-dione chemotype (repre-

sentative hit 1, Figure 1) with good starting potency and

selectivity over WT IDH1 was identiﬁed. As shown in Figure

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Table 4. Pyridone Aryl Substituent SAR Continued

biochemical

R⁴

IDH1 R132H

IDH1 R132C

a

compd

R⁵

IC₅₀(μM) SD

% inhibition SD at 38 μM

84

64 17

IC₅₀(μM) SD

% inhibition SD at 38 μM

1

OMe

OEt

5-OMe

3-OMe

4-OMe

6-OMe

3-CO₂H

4-CO₂H

5-CO₂H

6-CO₂H

6-Me

3-N (Py)

5-N (Py)

5-OEt

6-OEt

6-Me

1.6 0.2

7

1.0 0.3

85

35

9

3

53

54

55

56

57

58

59

60

61

62

63

64

65

66

>38

2.4 0.1

>38

1.6 0.2

87

82

2

75

65

43

86

108

72

80

7

2

6

4

1

14

1

>38

12

1

108

102

81

2

3

14

1

0.91 0.06

2.1 0.1

1.0 0.1

>38

0.16 0.01

0.39 0.03

0.79 0.18

0.69 0.05

2.0 0.1

2

1

97

1.3 0.2

>38

0.064 0.004

0.16 0.02

0.50 0.09

0.24 0.05

85

76

89

1

4

1

2

75

86

82

1

7

3

4

OEt

Et

6-Et

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled R132H and R132C mIDH1 assays (average of N = 3). IC₅₀values are

reported only for compounds where ≥75% inhibition of enzyme activity was observed at the highest concentration tested (38 μM).

conducted in one pot, requiring only a single solvent exchange

from isopropanol to acetic acid.

was essentially equipotent to 1 against R132H mIDH1 but lost

eﬃcacy toward the R132C mutant. Substitution at the 4-

position of the thiazole in the form of a biaryl linkage seemed

optimal in contrast to either substitution at the 5-position or

modiﬁcation to a fused ring system, with analogues 12−14

displaying signiﬁcant losses in activity. Replacement of the

thiazole with a variety of diazoles 15−22 had a dichotomic

eﬀect on mIDH1 potency with imidazoles 17 and 18, pyrazoles

19 and 20, maintaining activity, and the remainder of the

analogues (15, 16, and 21−26) experiencing signiﬁcant losses

in biochemical potency. This SAR led us to believe that

maintaining a nitrogen positioned akin to the thiazole nitrogen

(3-position highlighted in 1, Table 2) was important, while a

nitrogen at the 2-position abrogated activity (16, 20, and 21).

Further supporting this observation, expansion of the ring to a

benzene (23−24; meta- and para-substitution) proved

unfavorable with complete loss in mIDH1 inhibitory activity,

while substitution with a 2,6-disubstituted pyridine retained

some activity (25). In an attempt to open the thiazole ring, we

synthesized amide 26, but this proved to be a detrimental

variation of the thiazole. Thus, the thiazole segment, much like

the cyclohexanone moiety, was not amenable to signiﬁcant

modiﬁcation.

Tables 3 and 4 showcase the SAR exploration of the aryl

group attached to the pyridone nitrogen (R¹). Synthetically,

this region was readily modiﬁable since it was made from the

last step in Scheme 1; thus, it was extensively investigated.

Given the steric congestion about the pyridinone aryl linkage,

we posited that the orientation of the pyridinone orthogonal or

perpendicular to the arene may be optimal, especially given the

2-methoxy substituent present in the hit molecule. Along these

lines, removal of the two methoxy groups in the form of

analogue 27 resulted in a loss of activity against both mutant

forms of IDH1 (Table 3). Installation of a variety of ortho-

We initiated the optimization of the 7,8-dihydroquinoline-

2,5(1H,6H)-dione ring system by ﬁrst establishing the essential

components necessary for activity in this region. The

cyclohexanone ring proved was necessary as monocyclic

analogues 2 and 3 were completely inactive. The carbonyl

functionality appeared to play a lesser role as des-carbonyl

analogues 4 and 5 were only slightly less potent and/or

eﬃcacious against the R132H and the R132C IDH1 mutant

enzymes (Table 1). The removal of the methyl groups (6),

contraction of the ring (7), and aromatization (8) all resulted

in signiﬁcant losses in activity. We also incorporated a

heteroatom into this ring system with analogues 9 and 10,

and while amine 9 was less potent, lactam 10 displayed

comparable activity to the hit, showing that the amide

functional group was well-tolerated. In addition to focusing

primarily on target potency, we closely monitored the activity

of our molecules against WT IDH1. Fortunately, none of the

molecules in this series showed activity against WT IDH1

(IC₅₀> 57 μM) nor did most of the compounds in the

remainder of the hit-to-lead campaign described below. This

brief survey of the core structure−activity relationships (SARs)

highlighted the importance of the geminal methyl groups, and

the six-membered ring as requirements for biochemical

potency.

The next structural element investigated was the thiazole

ring (Table 2). Given its proximity to the carbon center that

acts as a nucleophile during the conversion of III to V, as

shown above in Scheme 1, many of the modiﬁcations to the

thiazole ring required developing new synthetic sequences

which resulted in synthetic challenges and poor throughput; as

a result, Table 2 has analogues that lack the para-chloro

substituent present in the hit. The des-chloro compound 11

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Table 5. Ring-Opened Core Modiﬁcations

biochemical

core

IDH1 R132H

% inhibition SD at 38 μM

IDH1 R132C

% inhibition SD at 38 μM

a

IC₅₀(μM) SD

1

1.6 0.2

2.3 0.0

0.69 0.05

>38

7.2 0.5

>38

84

89

7

1

2

1.0 0.3

85

65

82

4

10

66

67

68

69

70

0.24 0.05

>38

102

1

72

3

>38

1.5 0.0

87

3

64

5

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled R132H and R132C mIDH1 assays. IC₅₀is reported only for

compounds with >75% inhibition at 38 μM, the highest concentration tested.

Scheme 2. General Amide Synthesis

substituents regained target potency in the instance of small

alkoxy substituents (28−30) and an ethyl substituent (32).

The ortho-methyl-substituted compound 31 was active but did

not show inhibition beyond 75% at the highest concentration.

Analogues with larger R¹groups, such as 2-iPr (33) and 2-Ph

(34), or with free hydroxyl (35) and larger alkoxy groups (36

and 37), or nitrogen-containing substituents (38−40)

provided only partial inhibition of the mutant enzymes even

at the top concentration tested (38 μM). Interestingly, the only

polar substituent to maintain activity was the 2-carboxylic acid

analogue 41. With 2-halo substitutions, a smaller 2-F (42) lost

activity against both IDH1 mutants, but a 2-Cl (43) was

equipotent to 1 for R132H, emphasizing the need of an ortho-

substitution large enough to force the pyridone ring to be

orthogonal to the central bicyclic core and restrict rotation.

Notably, both 2-triﬂuoromethyl (44) and 2-triﬂuoromethoxy

(45) analogues exhibited improved mIDH1 activity over 1,

reiterating that varying electronic character was tolerated at the

2-position. Further modiﬁcations by extension with a carbon

spacer (48) or conversion to alkyl groups (49 and 50) were

not tolerated. A handful of heterocyclic replacements such as a

2-pyridyl ring (46) were also evaluated; most heterocycles

(analogues not shown) showed diminished activities, whereas

2,3-disubstituted thiophenes, such as compounds 51 and 52,

served as viable replacements.

Probing further substitution of the pyridinone arene, we

aimed to develop a more complete understanding of this

region of the pharmacophore (Table 4). By keeping the 2-

OMe group constant and walking the second methoxy group

around the remaining positions of the ring (53−55), the

importance of the relative 2,5- and 2,6-substituent arrange-

ments became apparent. A similar scan was conducted with a

polar carboxylic acid group (56−59). The 2-OMe,6-CO₂H-

disubstituted compound 59 showed potent inhibition of both

R132H and R132C mutant isoforms. Within this subset (1 and

53−59), we also noticed that the placement of a substituent at

the 4-position led to a profound reduction of activity (54 and

57). A 2-OMe,6-Me compound (60) was also potent

indicating that an alkyl group could also be tolerated at the

6-position. Despite the synthetic challenges associated with

substituting the phenyl for a pyridine given the diminished

nucleophilic reactivity of amino pyridines in the conversion of

V to VII (Scheme 1), we synthesized analogues 61 and 62.

These analogues demonstrated that this change was only

tolerated if the pyridine nitrogen was in the 3-position, next to

the 2-OMe substituent (61). We then moved on to change the

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Table 6. Amide Substitution SAR

biochemical

IDH1 R132H

IDH1 R132C

a

compd

R⁴

R⁵

IC₅₀(μM) SD

% inhibition SD at 38 μM

IC₅₀(μM) SD

% inhibition SD at 38 μM

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

Me

iPr

tBu

iBu

H

Me

H

1.5 0.0

1.2 0.1

2.8 0.2

87

98

47

65

3

1

5

64

86

54

49

56

5

1

2

4

1

2

5

4

1

2

2.0 0.1

>38

0.81 0.05

2.0 0.1

0.73 0.10

CH₂CH₂OH

CH₂CH₂OMe

CH₂CO₂H

CH₂CH₂NH₂

CH₂CH₂NHMe

CH₂CONH₂

CH₂CH₂CH₂NHMe

Ph

CH₂Ph

cyclohexyl

N-piperidine

N-morpholine

N-piperazine

N-4-Me-piperazine

0.42 0.00

2.1 0.0

1.2 0.0

0.38 0.00

0.87 0.06

1.6 0.1

1.0 0.1

104

92

106

97

101

105

104

3

2

1

3

96

102

87

89

11

1

2.0 0.1

0.32 0.02

3.8 0.0

96

77 14

95

69 11

41

31

65

71

93

86

0.91 0.06

0

40 21

>38

1

7

3

2

3

51

82

103

88

0

4

3

1

4.2 0.0

0.21 0.00

2.9 0.2

4.1 0.3

1.5 0.0

1.2 0.1

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled mIDH1 R132H and R132C assays. IC₅₀is reported for compounds

with >75% inhibition at 38 μM, the highest concentration tested.

2-methoxy substituent to a 2-ethoxy substituent and evaluated

the 2,5- and 2,6-diOEt compounds 63 and 64. Both

compounds showed sub-micromolar IC₅₀s. This remarkable

breakthrough in IC₅₀< 1.0 μM was also observed with a 2-

ethyl group when it was combined with a 6-methyl or ethyl in

65 and 66, respectively.

dimethyl acetal II was carried out by heating the two

components without any solvent to provide intermediate III

which was then condensed with functionalized nitrile IVa

under basic conditions. After the addition of aniline VIa to

ketonitrile Va and stirring with acetic acid, we discovered that

further heating was required to eﬃciently drive pyridinone

formation in VIIa. Finally, ester hydrolysis and HATU-

mediated amide couplings were utilized to eﬃciently access a

diversity of ring-opened amide analogues (VIII).

Based on this synthetic route, we surveyed a structurally

diverse set of amides, as depicted in Table 6. The tertiary

dimethylamide 71 displayed a similar activity to that of

compound 70, but larger alkyl substituents at the R⁴position

provided analogues (72−74) with decreased mIDH1 activity.

Incorporation of either a nitrogen or oxygen atom at or near

the termini of an alkyl chain at the R⁴position (75−81) either

maintained or even provided a boost in potency. Most

noteworthy among these were ethanaloamide 75 and 2-

aminoethyl carboxamide 78, which exhibited threefold

improvements in potency compared to analogue 70. Secondary

amides such as 82, 83, and 84 with large ring substituents such

as phenyl, benzyl, and cyclohexyl, respectively, lost activity. We

also evaluated cyclic tertiary amides (85−88) and found that

piperidine analogue 85’s activity was attenuated with ∼50−

65% inhibition at the highest concentration tested. The

placement of distal heteroatoms (N and O) in morpholine

86 and N-Me-piperazine 88 maintained good potency, while

NH-piperazine 87 was sevenfold more potent in mIDH1

R132H inhibition compared to analogue 70. Importantly,

piperazine 87 also was more stable in a single point rat liver

Our earlier SAR study of the bicyclic dihydroquinoline-

dione core in hit molecule 1 had revealed that most changes

reduced potency, but lactam 10 maintained a similar activity

against the R132H IDH1 mutant (Table 1). This observation

provided an attractive opportunity to both substitute the amide

and open the ring to reveal new opportunities for SAR

exploration in the northwestern region of the chemotype. To

that end, we evaluated the ring-opened variants of the lactam,

as shown in Table 5; this was done with the symmetrical 2,6-

diethylphenyl substituent at the R¹position from lead 66. This

was a strategic decision as we realized that the possibility of

restricted rotation around N-R¹could lead to atropisomers

(vide infra), adding one more variable to our SAR analysis.

Compared to ketone 66, ester (67 and 69) and acid (68)

variants lost much, if not all mIDH1 target potency. However,

amide 70 only experienced a small drop in R132H activity and

did not show >75% inhibition at 38 μM against R132C (Table

5). This analogue proved pivotal in providing an additional

SAR handle for a new amide-substituted pyridone series.

With only moderate variations to our initial multicomponent

coupling, we were able to develop a modular synthesis tolerant

of signiﬁcant modiﬁcation to both the amide and the

pyridinone substituents (Scheme 2). The initial condensation

between keto ester Ia and N,N-dimethylformamide (DMF)

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Table 7. Pyridinone 6-Position SAR

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled mIDH1 R132H and R132C assays. IC₅₀is reported for compounds

with >75% inhibition at 38 μM, the highest concentration tested.

Table 8. In Vitro Drug-like Properties of Analogues with >10-fold Increase in R132C mIDH1 Potency over Compound 87

microsomal stability assay (70: t_1/2= 1.5 min; 87: t_1/2> 30

min).³⁶Additionally, 87 had better aqueous kinetic solubility

than methyl amide 70 (70: [M] < 1 μM; 87: [M] = 6.7 μM).

These results led us to maintain this substitution while we

examined the SAR of the pyridinone 6-substituent.

and cyclopropylmethyl 93 had benchmark potencies with IC₅₀

< 100 nM for both IDH1 mutants. The replacement of the

methyl group on the n-propyl side chain in 89 with an

electron-donating methoxy (94) or electron-withdrawing

triﬂuoromethyl group (95) led to a comparative reduction in

potency. Within cyclic ethers, the racemic tetrahydrofuran

(THF) 96 was more potent than the larger tetrahydropyran

97. Analogous pyrrolidine 98 (racemic) proved to be much

less potent with IC₅₀∼ 10 μM. We postulated that perhaps,

steric congestion to this region of the pharmacophore helps

position the piperazine away from R³(as suggestively drawn in

Our evaluation of the pyridinone 6-substituent R³is

presented in Table 7. The n-propyl analogue 89 was ∼3-fold

more potent toward R132H mIDH1 but with a marked 15-fold

improvement toward mR132C. While this trend did not carry

to the α-branched isopropyl 90, it emerged again with

analogues 91−93 with β-branching. Indeed, isobutylene 92

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Table 9. Piperazine SAR

a

b

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled mIDH1 R132H and R132C assays. Compounds showed >97%

c

inhibition at 38 μM, the highest concentration tested. Compounds showed >85% inhibition at 38 μM, the highest concentration tested.

d

Compounds are racemic.

Table 7); we suspect that the amide is also not coplanar to the

pyridine pi-system. It could be that such a conformation is

more desirable toward R132C activity and results in the >10-

fold increase in potency observed in analogues 89, 91−93, and

96 compared to the methyl analogue 87. We assessed in vitro

drug-like properties in this set (Table 8); isobutylene 92 had a

desirable balance between potency, rat liver microsome

stability, and permeability. Thus, it was held at the R³position

for further SAR studies. We also noticed that all compounds

had low aqueous kinetic solubility.

(PAMPA), rat and mouse microsome stability, and solubility]

nor in vivo PK proﬁles (data not shown). Another noteworthy

observation was that the analogues described in Tables 7−10

were at least 1000-fold selective for mIDH1s over WT IDH1.

Finally, our SAR studies shifted back to the pyridinone 1-

arene (Table 10). Analogous to our earlier studies, an ortho

substituent proved essential. The complete removal of all

substituents in 113 led to a 7.5-fold drop in R132H potency

and a more signiﬁcant 55-fold drop in R132C potency. The

presence of one ortho ethyl in 114 was 1−3-fold less potent

compared to its diethyl counterpart 92. While the combination

of ethyl,methyl-di-ortho substitutions in 115 led to a potency

drop, especially toward the R132C mutant, the ethyl-chloro

combination in 116 was equipotent to 92. Keeping the ortho-

ethyl group constant, we shifted to the meta position (R¹⁰) and

found that methyl 117, methoxy 118, and chloro 119

maintained their potency across both mutants. While it was

apparent that steric congestion around the pyridone seemed

critical for activity, we anticipated that hindered rotation

around the pyridone N-aryl bond likely led to the existence of

atropisomers that could have dissimilar activities. As two

representative examples, we separated the atropisomers in 118

and 119 and discovered that they had a ∼3- and 3−8-fold

Upon optimizing the R³substituent, the NR⁴R⁵site (see

VIII in Scheme 2) was further explored (Table 9). A subset of

analogues with diverse groups at the piperazine 3-position such

as methyl 99, ethyl 101, propyl 102, cyclopropyl 103,

hydroxymethyl 104, ketone 105, triﬂuoromethyl 106, cyano

107, acid 108, and amide 109 maintained their potency against

both IDH mutants, showcasing that electron-donating and

-withdrawing groups were tolerated at this position. The 2-

methyl piperazine 100 was also potent. Furthermore, hetero-

bicyclic systems also produced analogues 110−112 with

nanomolar activity. Although these substitutions were tolerated

for enzymatic inhibition, these additions typically did not

provide additional beneﬁts with respect to their in vitro drug-

like properties [parallel artiﬁcial membrane permeability assay

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Table 10. SAR at the Pyridinone N1-Aryl Ring

biochemical

IDH1 R132H

IDH1 R132C

a

compd

92

113

R⁹

R¹⁰

R¹¹

IC₅₀(μM) SD

Et

H

Et

H

0.044 0.003

0.33 0.00

0.058 0.010

3.2 0.6

114

115

116

117

Et

H

Me

OMe

H

0.056 0.004

0.27 0.00

0.044 0.003

0.114 0.007

0.12 0.02

0.16 0.01

2.2 0.2

Me

Cl

H

0.055 0.004

0.11 0.02

0.075 0.000

0.13 0.03

0.67 0.04

0.10 0.01

0.072 0.005

0.54 0.10

0.063 0.015

118

H

(+)-118

(−)-118

119

(+)-119

(−)-119

AG-120

0.42 0.05

Et

Cl

H

0.114 0.007

0.081 0.005

0.25 0.04

NCATS-SM5637, NSC 791985

0.065 0.009

a

IC₅₀values were determined utilizing the diaphorase- and resazurin-coupled R132H and R132C mIDH1 assays. All compounds showed >80%

inhibition at 38 μM, the highest concentration tested.

Table 11. 2-HG Levels (% of Vehicle Control) in U87-mIDH1 (R132H) Cells after 48 h Incubation with the Compound

Compared to DMSO and Drug-like Properties

a

b

% 2-HG

IDH1 R132H

single point

PAMPA

solubility

compd

91

92

99

100

103

105

108

111

112

118

(−)-119

(+)-119

100 nM

500 nM

IC₅₀(μM) SD

RLM (t_1/2, min)

(10⁻⁶cm/s)

(μg/mL)

33 25

13 3.1

11 5.5

14 5.1

17 3.8

23 4.2

29 16

11 3.1

32 8.4

40 1.7

ND

12 6.4

16 1.1

0.11 0.01

0.044 0.003

0.039 0.003

0.078 0.005

0.035 0.002

0.055 0.004

0.094 0.000

0.062 0.004

0.114 0.007

0.25 0.04

17

14

25

17

12

2

23

>30

9

16

<1

16

<1

225

213

40

48

275

<1

89

99

549

255

434

8

2.3

11 5.3

14 0.81

13 3.1

10 8.6

10 3.4

18 2.8

19 3.7

ND

>30

5.9 0.16

4.1 0.17

0.081 0.005

<1

a

b

Values represent % 2-HG after 48 h incubation with an inhibitor, where 100% is normalized to 2-HG levels in the DMSO-treated control. The

enzymatic R132H IC₅₀has been reintroduced in the table to visualize the entire data set for all compounds in this table. ND: Not Determined.

diﬀerence in the R132H and R132C biochemical potencies,

respectively.

biochemical IC₅₀s toward R132H mIDH1 were similar (114

and 81 nM). Notably, in this cellular assay, (+)-119 appears

100 times more potent (<0.7 nM) than in the biochemical

enzyme inhibition assay and more potent than AG-120 which

had a comparable biochemical potency.

For the compounds in Table 11, an analysis of drug-like

properties using our in-house high throughput assays showed

that analogues 111, 118, (−)-119, and (+)-119 had promising

stability in rat liver microsomes with t_1/2> 30 min, high passive

permeability, and low aqueous kinetic solubility. From this

subset, based on its potent cellular activity, we chose (+)-119

to showcase the chemical series in PK/PD studies.

Having developed several biochemically potent mIDH1

inhibitors, for a representative set of compounds, we next

assessed cellular activity in U87 cells, a cell line of glioblastoma

origin engineered to express the IDH1 R132H mutation.

Inhibition of 2-HG production, secreted from cells into media,

was measured by mass spectrometry 48 h after the addition of

compounds at 100 and 500 nM (Table 11). Analogue (+)-119

lowered 2-HG levels by more than 94% at 100 nM. Dose−

response curves for atropisomer (+)-119, the corresponding

unseparated mixture of atropisomers 119, and the approved

drug AG-120 are shown in Figure 2. (+)-119 was more potent

at reducing 2-HG levels than 119, even though their

To directly assess target engagement in cells, we used a high-

throughput CETSA platform that uses a split NanoLuciferase

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lactone hydrolysis and in situ elimination of the tertiary alcohol

provided the sensitive intermediate VIIc. We observed

proximity-promoted deleterious cyclization back to lactone

VIIb under a variety of neutralization conditions as well during

the subsequent amide coupling. Careful neutralization with

acetic acid to neutral pH and subsequent removal via wash

steps was necessary following lactone hydrolysis. The amide

bond formation also had to adhere to a strict order of addition

of HATU, iPr₂NEt, and then the Boc-protected piperazine to

furnish VIIIa in 95% yield. Subsequently, separation of

atropisomers proved optimal at the stage of the protected

piperazine VIIIa with the use of a CHIRAL OD-H column.

Final deprotection, which necessitated short reaction times

with a signiﬁcant excess (35 equiv) of triﬂuoroacetic acid in

order to minimize relactonization, followed by neutralization of

the TFA salt by aqueous sodium bicarbonate provided the

pure atropisomer (+)-119. The stereochemical conﬁguration

of the atropisomer was proved by X-ray analysis of a single

crystal of the intermediate VIIb (Supporting Information) that

was formed during attempts to form a HCl salt of (+)-119

(NCATS-SM5637, NSC 791985).

Figure 2. Dose-dependent reduction of 2-HG in U87-mIDH1

(R132H) cells (N = 3).

A single dose of 30 mg/kg (+)-119 administered orally to

CD1 mice led to good exposure in the plasma with a C_max

∼3.34 μM and AUC_lastof 24,371 h·ng/g. It also crossed the

blood brain barrier with an AUC of 6996 h·ng/g and a brain/

plasma ratio of 28% (Figure 4).

(SplitNanoLuc) reporter to detect soluble protein in cells.³⁷

After tagging the 15-amino acid sequence 86b from NanoLuc

to the C-terminus of R132H mIDH1 (termed IDH1(R132H)-

86b) and transient expression in HEK293T cells, soluble

protein was measured by complementation of the larger 11S

fragment to 86b. This reconstitutes NanoLuciferase activity

and allows its quantiﬁcation using the substrate furimazine.

Dose−response testing measured the ability of compounds to

increase the thermal stability of IDH1(R132H)-86b when cells

were heated to 56 °C for 3.5 min. (+)-119 and AG-120 were

equipotent in this assay, whereas the corresponding mixture

119 had a 10-fold greater EC₅₀(Figure 3).

We devised a scalable route to (+)-119 and related

analogues to enable in vivo testing (Scheme 3). Commencing

with a similar multicomponent coupling with dicarbonyl 6,6-

dimethyldihydro-2H-pyran-2,4(3H)-dione Ic, a multicompo-

nent coupling reaction eﬃciently assembled VIIb which upon

We compared the activities of (+)-119 and AG-120 in vivo,

using the U87 mIDH1 (R132H) xenograft to assess PK/PD

responses (Figure 5). After a single 30 mg/kg p.o. dose, AG-

120 reached a higher plasma C_maxthan (+)-119 but after 8 h,

(+)-119 displayed higher plasma concentrations. Similarly, 4 h

after dosing, concentrations of (+)-119 in tumor were higher

than AG-120. (+)-119 had very slow clearance from tumor

with sustained average concentrations of 9.5, 7.2, and 6.8 μM

at 24, 36, and 48 h, respectively, compared to 1.6, 1.4, and 0.77

μM for AG-120. These higher sustained tumoral concen-

trations of (+)-119 appear to be responsible for the greater

reduction in tumor 2-HG, as compared to AG-120, from the

16 h time point onward. If this promising activity translates to

the clinical setting, it could provide a therapeutic advantage

over AG-120 (see the Supporting Information for proﬁling

data on (+)-119).

CONCLUSIONS

■

An extended medicinal chemistry campaign starting with a 7,8-

dihydro-2H-1λ²-quinoline-2,5(6H)-dione chemotype led to

the discovery of new amide-substituted pyridones as potent

R132H and R132C mIDH1 inhibitors. The lead compound

119 had nanomolar activities against both IDH1 mutants and

lowered the mIDH1-catalyzed product 2-HG in cells. The

single atropisomer (+)-119 (NCATS-SM5637, NSC 791985)

was shown to engage mIDH1 in cells and reduce 2-HG

production more potently that 119 (13-fold in HEK293

engagement assay and 30-fold in U87 2-HG assay). In a PK/

PD model, after a single oral dose at 30 mg/kg, (+)-119

showed sustained tumor concentrations (7 μM after 48 h) and

greater reduction in 2-HG levels at later time points (16−48 h)

than the approved mIDH1 drug AG-120. Further head-to-head

comparative animal studies in various mIDH1-driven cancer

models with key compounds from this chemical series, such as

NCATS-SM5637 (NSC 791985), and AG-120 are ongoing

and will be reported in due course.

Figure 3. Dose-dependent stabilization of NanoLuc-tagged R132H

mIDH1 in HEK293 cells (N = 3).

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Scheme 3. (A) Synthesis of Isobutylene Analogue (+)-119 Including Separation of Atropisomers; (B) Formation of VIIb from

(+)-119 and Its X-ray Coordinates of Its Single Crystal

Figure 4. PK parameters and conc vs time proﬁles in brain and plasma of CD1 mice (n = 3) after a single 30 mpk oral dose of (+)-119 formulated

as a solution in 20% PEG300, 40% of Solutol solution (30% w/w in water), and 40% DI water.

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Figure 5. PK/PD in a U87 R132H mIDH-based xenograft mouse model: (A) Concentrations of (+)-119 or AG-120 in plasma and tumor after

administration of a single 30 mpk PO dose. (B) Concentrations of 2-HG in tumor.

mL penicillin, and 100 μg/mL streptomycin. 8000 cells/well/100 μL

EXPERIMENTAL SECTION

■

were seeded into clear, tissue culture-treated 96-well plates (Corning)

Materials and Methods: Biology. mIDH1-R132H and mIDH-

in 100 μL culture medium excluding wells along the edge (which

R132C Enzyme Assay. Pubchem AID 624002. Enzyme buﬀer was

instead were ﬁlled with culture medium only) and incubated

dispensed into black, solid 1536-well plates at 3 μL/well in 20 mM

overnight at 37 °C, 5% CO₂, 90% RH. Culture medium was aspirated

Tris buﬀer, pH 7.5, containing ﬁnal concentrations of 10 mM MgCl₂,

and replaced with 100 μL of culture medium containing either a

20 mM NaCl, 0.001% Tween 20, 0.05% BSA, 2 mM β-ME, and 5.2

vehicle (DMSO) or mIDH1 inhibitor. Assays were established with

nM IDH1 R132H. Then, 23 nL of compounds or DMSO was

an inhibitor tested at two concentrations 500 and 100 nM or as seven-

delivered to each well using a pin tool. The inherent compound

point dose response. After 48 h, 75 μL of the culture medium was

ﬂuorescence was measured at this point at 590 nm on a ViewLux plate

collected and snap-frozen on dry ice. Samples were analyzed by LC−

reader (Ex 525, Em 598, bodipy ﬁlter); this is test for assay

MS for 2-HG concentrations using Rapidﬁre/Mass Spectrometry

interference as we measure resoruﬁn (em 590 nm in the end). The

(Quintara). Remaining cells were assessed for viability by adding 100

substrate buﬀer (3 μL; 20 mM Tris buﬀer, pH 7.5, containing ﬁnal

concentrations of 10 mM MgCl₂, 20 mM NaCl, 0.001% Tween 20,

0.05% BSA, 0.008 mM NADPH, and 1 mM α-ketoglutarate) was

added to start the enzymatic reaction, and the plate was brieﬂy spun at

μL of fresh media and 50 μL of CellTiter-Glo reagent (Promega).

Luminescence was read using a ViewLux High-throughput CCD

imager (PerkinElmer).

SplitLuc CETSA. The mIDH1(R132H) open reading frame was

270g. After the reaction was allowed to progress for 80 min at room

cloned into a pcDNA3.1(+) backbone using the NheI and EcoRI

temperature, the remaining NADPH was detected with the

restriction sites. A 15 amino acid tag (86b, Gly-Ser-HiBiT-Gly-Ser)

diaphorase/resazurin-coupled system. A total of 3 μL of detection

buﬀer (20 mM Tris buﬀer, pH 7.5, containing ﬁnal concentrations of

10 mM MgCl₂, 20 mM NaCl, 0.001% Tween 20, 0.05% BSA, 0.53

μM diaphorase and 0.012 mM resazurin) was added, and after 5 min,

was fused in-frame to the carboxy terminus. HEK293T cells (obtained

from ATCC; CRL-1573) were cultured in DMEM (4.5 g/L glucose)

with 10% fetal bovine serum, 6 mM ^L-glutamine, 1 mM sodium

pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin. Cells were

the ﬂuorescence at 590 nm was measured on an Envision plate reader

grown at 37 °C in a humidiﬁed incubator maintained at 5% CO₂.

Cells were transfected in T75 ﬂasks using a reverse transfection

procedure, where 9 mL of complexes (45 μL Lipofectamine 2000 and

22.5 μg DNA) was combined with 10 mL of HEK293T cell

suspension (1 × 10⁶cells/mL, 10 million cells total). After 24 h, cells

were harvested by trypsinization, resuspended at 1 × 10⁶cells/mL

(DPBS with CaCl₂and MgCl₂plus 1 g/L glucose), and dispensed (15

μL cells/well) into 384-well PCR plates (Roche) using a Multidrop

Combi (Thermo Fisher). Compounds (63 nL) or DMSO vehicle

control (63 nL) were subsequently pinned using a pin tool (GNF)

and incubated for 1 h at 37 °C. Plates were sealed and heated at 56 °C

for 3.5 min and cooled to 25 °C using an AB qPCR machine (Roche)

with a ramp speed of 1.5 °C/s for the heating phase and max ramp

rate for the cooling phase. Three microliters of 6% NP40 was added

per well and incubated for 30 min to allow cell lysis followed by the

addition of 11S (puriﬁed from Escherichia coli) and furimazine

(Promega) substrate (at ﬁnal concentrations of 100 nM and 0.5×,

respectively). Samples were analyzed for luminescence intensity using

a ViewLux reader.

(Ex 544, Em 590, bodipy ﬁlter, 10 ﬂashes). The % activity was

determined from the corrected ﬂuorescence values. The activity was

normalized with 5.2 nM R132H IDH1 (no inhibitor) as 0% inhibition

and the activity with no enzyme as 100% inhibition.

We also measured for assay interference by measuring dose-

dependent activity against the diaphorase/resazurin-coupled system

by repeating the assay mentioned above in the absence of mIDH1.

This will ﬂag false positives that may inhibit the NADPH to NADP⁺

conversion.

Concentration−response curves were ﬁtted to the signals arising

from the resulting ﬂuorescence. The concentration−response curves

were then classiﬁed based on curve quality (r2), response magnitude,

and degree of measured activity, and compounds were subsequently

categorized based on their curve class. Active inhibitors showed

concentration-dependent increases in ﬂuorescence, concordant with a

decrease in IDH1 R132H activity and less substrate NADPH

utilization. Inactive compounds showed no eﬀect on the ﬂuorescence

signal relative to the DMSO control.

Cellular 2-HG Production Assay. 2-HG secreted from cells was

quantiﬁed as previously described with modiﬁcations. U87-R132H

cells were cultured in Dulbecco’s modiﬁed Eagle medium (DMEM)

high glucose with glutamine supplemented with 10% FBS, 100 units/

Kinetic Solubility Assay. Pion’s patented μSOL assay was used for

kinetic solubility determination. In this assay, the classical saturation

shake-ﬂask solubility method was adapted as previously described.³⁸

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Test compounds were prepared in 10 mM DMSO stock and diluted

to a ﬁnal drug concentration of 150 μM in the aqueous solution (pH

7.4, 100 mM phosphate buﬀer). Samples were incubated at room

temperature for 6 h and vacuum-ﬁltered using Tecan Te-Vac to

remove any precipitates. The concentration of the compound in the

ﬁltrate was measured via UV absorbance (λ: 250−498 nm). The

unknown drug concentration was determined by comparing the fully

solubilized reference plate which contained 17 μM of the compound

dissolved in spectroscopically pure n-propanol. All compounds were

tested in duplicates. The kinetic solubility (μg/mL) of compounds

was calculated using the μSOL Evolution software. The three controls

used were albendazole (low solubility), phenazolpyridine (moderate

solubility), and furosemide (high solubility).³⁹

Rat Liver Microsome Stability Assay. Single time point micro-

somal stability was determined in a 96-well HTS format. Sample

preparation was automated using a Tecan EVO 200 robot. A high-

resolution LC/MS (Thermo Q Exactive) instrument was used to

measure the percentage of compound remaining after incubation

using a previously described method.⁴⁰Six standard controls were

tested in each run: buspirone and propranolol (for short half-life),

loperamide and diclofenac (for short to medium half-life), and

carbamazepine and antipyrine (for long half-life). 10 mM DMSO

stock solutions of the drugs were ﬁrst diluted to 10 μM in 1:2 MeCN/

DI H₂O and then further diluted to 1 μM in assay buﬀer. Brieﬂy, the

incubate consisted of 0.5 mg/mL microsomal protein, 1.0 μM drug

concentration, and NADPH regeneration system (containing 0.650

mM NADP⁺, 1.65 mM glucose 6-phosphate, 1.65 mM MgCl₂, and 0.2

unit/mL G6PDH) in 100 mM phosphate buﬀer at pH 7.4. The

incubation was carried out at 37 °C for 15 min.³⁶The reaction was

quenched by adding 555 μL of acetonitrile (∼1:2 ratio) containing

0.28 μM albendazole (internal standard). Sample acquisition and data

analysis were done using a previously described method.⁴⁰

Parallel Artiﬁcial Membrane Permeability Assay. The stirring

double-sink PAMPA method (patented by pION Inc.) was employed

to determine the permeability of compounds via PAMPA as published

before.⁴¹The PAMPA lipid membrane consisted of an artiﬁcial

membrane of a proprietary lipid mixture and dodecane (pION Inc.),

optimized to predict gastrointestinal tract passive permeability. The

lipid was immobilized on a plastic matrix of a 96-well “donor” ﬁlter

plate placed below a 96-well “acceptor” plate. pH 7.4 solution was

used in both donor and acceptor wells. The test articles, stocked in 10

mM DMSO solutions, were diluted to 0.05 mM in aqueous buﬀer

(pH 7.4), and the concentration of DMSO was 0.5% in the ﬁnal

solution. During the 30 min permeation period at room temperature,

the test samples in the donor compartment were stirred using the

Gutbox Technology (pION Inc.) to reduce the aqueous boundary

layer. The test article concentrations in the donor and acceptor

compartments were measured using a UV plate reader (Nano Quant,

calculated for plasma and brain: T_max, C_max, and AUC_24h. Animals were

also monitored during the in-life phase by once daily cageside

observations; no adverse clinical signs were noted as part of the PK

report.

PK/PD. A PK/PD study was performed in 6-week old female

athymic nude mice (nu/nu NCr). Mice were housed in microisolator

cages with food and water provided ad libitum. All work was

performed on an approved Institutional Animal Care and Use

Committee protocol in AAALACi accredited facilities with automated

12 on−12 oﬀ light cycles. Mice were implanted subcutaneously with

the U87MG human glioblastoma cell line (1 × 107 cells/mouse),

containing the IDH1 R132H mutation. On the day of tumor staging

(∼200 mg), mice were randomized into 3 groups of 32 mice each.

These groups were treated with a single oral dose of either vehicle

(10% NMP, 40% PEG300, 25% [30% Solutol in water], 25%

deionized water), compound (+)-119 at 30 mg/kg, or AG120 at 30

mg/kg. Mice were anesthetized with isoﬂurane by inhalation and the

tumors and K₂EDTA anticoagulated blood were collected from

cohorts of 4 mice per group at 1, 2, 4, 8, 16, 24, 36, and 48 h post

dose. Plasma was obtained by centrifugation and all samples were

ﬂash-frozen until analysis. (+)-119 and AG120 in plasma and tumor

tissue and 2-HG in tumor were quantiﬁed using validated LC−MS/

MS methods.

Use of Animal Subjects. All animal studies included as part of this

manuscript were performed in accordance with institutional guide-

lines as deﬁned by Institutional Animal Care and Use Committee

(IACUC).

EXPERIMENTAL SECTION CHEMISTRY

■

General Methods for Chemistry. All air- or moisture-sensitive

reactions were performed under positive pressure of nitrogen with

oven-dried glassware. Anhydrous solvents or reagents such as

dichloromethane, DMF, acetonitrile, methanol, and triethylamine

were purchased from Sigma-Aldrich. To follow most chemical

reactions, LC/MS of reaction aliquots was analyzed using a gradient

of 4−100% acetonitrile (containing 0.025% triﬂuoroacetic acid) and

water (containing 0.05% triﬂuoroacetic acid) with a 4.5 min run time

at a ﬂow rate of 1 mL/min in an Agilent Extend-C18 column (3.5 μm,

4.6 × 100 mm) at a temperature of 50 °C using an Agilent diode array

detector. Conﬁrmation of molecular formulae was accomplished using

electrospray ionization in the positive mode with the Agilent

Masshunter software (version B.02).

Preparative puriﬁcation was performed on a Waters semi-

preparative HPLC system. The column used was a Phenomenex

Luna C18 (5 μm, 30 × 75 mm) at a ﬂow rate of 45 mL/min. The

mobile phase consisted of acetonitrile and water (each containing

0.1% triﬂuoroacetic acid). A gradient of 10−50% acetonitrile over 8

min was used during the puriﬁcation. Fraction collection was

triggered by UV detection (220 nM). This puriﬁcation method is

referred to as Standard Acidic Gradient Method.

Purity of all ﬁnal compounds was ≥95% as determined on an

Agilent LC/MS (Agilent Technologies, Santa Clara, CA) using a 7

min gradient of 4−100% acetonitrile (containing 0.025% triﬂuoro-

acetic acid) and water (containing 0.05% triﬂuoroacetic acid) with an

8 min run time at a ﬂow rate of 1 mL/min. A Phenomenex Luna C18

column (3 μm, 3 × 75 mm) was used at a temperature of 50 °C using

an Agilent diode array detector. Mass determination was performed

using an Agilent 6130 mass spectrometer with electrospray ionization

in the positive mode.

̈

Inﬁnite 200 PRO, Tecan Inc., Mannedorf, Switzerland). Permeability

calculations were performed using pION Inc. software and were

expressed in units of 10⁻⁶cm/s. Compounds with a low or weak UV

signal were analyzed using high-resolution LC/MS (Thermo

QExactive). The three controls used were ranitidine (low

permeability), dexamethasone (moderate permeability), and verapa-

mil (high permeability).

Mouse PK Studies. Studies were conducted by Pharmaron. Fed

male CD1 mice (sourced from Si Bei Fu LaboratoryAnimal

Technology Co. Ltd.), approximately 6−8 weeks of age and weight

of approximately 25−30 g, were dosed with (+)-119 at 30 mpk dose

PO. The formulation was a solution at 3 mg/mL in 20% PEG300,

40% of Solutol solution (30% w/w in water), and 40% DI water. This

was prepared prior to dosing a cohort of N = 24 mice. Plasma and

whole brain were collected from N = 3 mice at 15 min, 30 min, 1 h, 2

h, 4 h, 6 h, 84, and 24 h postdose. Approximately, 0.200 mL of blood

was collected via the heart puncture at each time point. Blood samples

were then transferred into plastic microcentrifuge tubes containing

heparin−Na as an anticoagulant. Samples were then centrifuged at

4000g for 5 min at 4 °C to obtain plasma. Plasma samples were then

stored in polypropylene tubes, quickly frozen, and kept at −75 °C

until analyzed by LC/MS/MS. The following PK parameters were

¹H NMR spectra were recorded on Varian 400 MHz

spectrometers. Chemical shifts are reported in ppm with a

nondeuterated solvent (DMSO-h₆at 2.50 ppm) as an internal

standard for DMSO-d₆solutions. High-resolution mass spectra

(HRMS) were recorded on an Agilent 6210 Time-of-Flight LC/MS

system.

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Synthetic Procedures. Synthesis of Nitrile Intermediates

(Scheme 1, Intermediate IV).

EtOH and AcOH. The reaction mixture was concentrated under a

stream of air, and 2-(6-chlorobenzo[d]thiazol-2-yl)acetonitrile was

used without further puriﬁcation.

Method 1: 2-(4-(4-Chlorophenyl)thiazol-2-yl)acetonitrile (N1 or

IVa in Schemes 2 and 3 ). To a solution of 2-bromo-1-(4-

chlorophenyl)ethanone (2.33 g, 10 mmol) in ethanol (25 mL) was

added 2-cyanoethanethioamide (1 g, 10 mmol). The reaction mixture

was heated at re ﬂ ux for 15.5 h. The reaction mixture was cooled to 0

°C. A precipitate formed and was removed by ﬁ ltration, washed with

hexanes, and subsequently dried under vacuum. The product, 2-(4-(4-

chlorophenyl)thiazol-2-yl)acetonitrile is a brown powder; LCMS: m /

z (M + H)⁺= 235.0; ¹H NMR (400 MHz, CDCl₃): δ 7.88 −7.77 (m,

2H), 7.48 (s, 1H), 7.44 −7.35 (m, 2H), 4.17 (s, 2H).

2-(5-Chlorobenzo[d]thiazol-2-yl)acetonitrile (N6, Intermediate

for Compound 14). 2-(5-Chlorobenzo[d]thiazol-2-yl)acetonitrile

(N6, intermediate for compound 14) was prepared according to

Method 4, however reﬂuxing was conducted overnight followed by

heating in a microwave at 120 °C for 1 h and at 150 °C for 1 h. The

reaction mixture was concentrated under a stream of air, and 2-(5-

chlorobenzo[d]thiazol-2-yl)acetonitrile was used without further

puriﬁcation; LCMS: m/z (M + H)⁺= 209.0.

2-(4-Phenylthiazol-2-yl)acetonitrile (N2, Intermediate for Com-

pound 11). Synthesized by Method 1 substituting 2-bromo-1-

phenylethanone as a starting material. Following the reaction, the

mixture was concentrated and puriﬁed via silica gel chromatography

(0−30% EtOAc/hexanes). The product is a red-orange solid (1.53 g,

77%); LCMS: m/z (M + H)⁺= 201.1.

Method 5: 2-(1-(4-Chlorophenyl)-1H-imidazol-4-yl)acetonitrile

(N7, Intermediate for Compound 15). A mixture of 2-(1H-

imidazol-4-yl)acetonitrile (150 mg, 1.4 mmol), 1-chloro-4-iodoben-

zene (467 mg, 1.96 mmol), 4,7-dimethoxy-1,10-phenanthroline (101

mg, 0.42 mmol), copper(I) oxide (20 mg, 0.14 mmol), cesium

carbonate (776 mg, 2.38 mmol), PEG (250 mg), and DMSO (1.5

mL) was heated with stirring at 110 °C for 24 h. The reaction mixture

was diluted with water, 0.1 N HCl, and EtOAc and extracted (2×).

The organic layers were combined, dried with magnesium sulfate,

concentrated, and puriﬁed via reverse-phase chromatography (C18)

(5−100% acetonitrile/water [0.1% TFA]) to aﬀord 2-(1-(4-

chlorophenyl)-1H-imidazol-4-yl)acetonitrile (35 mg, 12%) as a yellow

oil; LCMS: m/z (M + H)⁺= 218.0.

Method 2: 2-(4′-Chloro-[1,1′-biphenyl]-3-yl)acetonitrile (N3,

Intermediate for Compound 23). In a microwave vial, 2-(3-

bromophenyl)acetonitrile (300 mg, 1.53 mmol), (4-chlorophenyl)-

boronic acid (287 mg, 1.84 mmol), tetrakis(triphenylphosphine)-

palladium(0) (88 mg, 0.077 mmol), 2 M aqueous sodium carbonate

solution (2.3 mL), and dimethoxyethane (10 mL) were combined.

The reaction mixture was heated in a microwave with stirring at 140

°C for 1 h. The reaction mixture was diluted with water and extracted

with CH₂Cl₂(2 × 10 mL); the organic layers were combined, dried

with magnesium sulfate, concentrated, and puriﬁed via silica gel

chromatography (0−25% EtOAc/hexanes) to aﬀord 2-(4′-chloro-

[1,1′-biphenyl]-3-yl)acetonitrile (298 mg, 86%); ¹H NMR (400

MHz, CDCl₃): δ 7.62−7.38 (m, 7H), 7.37−7.28 (m, 1H), 3.82 (t, J =

0.7 Hz, 2H).

Method 6: (2-(1-Phenyl-1H)-pyrazol-3-yl)acetonitrile (N8, Inter-

mediate for Compound 19). A mixture of copper(II) acetate (382

mg, 2.1 mmol), 2-(1H-pyrazol-3-yl)acetonitrile (150 mg, 1.4 mmol),

phenylboronic acid (341 mg, 2.8 mmol), triethylamine (0.390 mL, 2.8

mmol), pyridine (0.227 mL, 2.8 mmol), 4 Å molecular sieves (500

mg), and dichloromethane (10 mL) was heated at 55 °C overnight.

The reaction mixture was ﬁltered, extracted (DCM/1 N HCl), dried

with magnesium sulfate, and concentrated, and 2-(1-phenyl-1H)-

pyrazol-3-yl)acetonitrile was used without further puriﬁcation;

LCMS: m/z (M + H)⁺= 184.1.

2-(6-(4-Chlorophenyl)pyridin-2-yl)acetonitrile (N4, Intermediate

for Compound 25). Method 3: 2-(6-Bromopyridin-2-yl)acetonitrile:

A solution of n-butyllithium in hexanes (1.6 M, 17.4 mL, 27.9 mmol)

was added slowly to a solution of acetonitrile (1.5 mL, 28.7 mmol) in

THF (40 mL) at −78 °C. A precipitate formed. The slurry was stirred

at this temperature for 30 min. A solution of 2,6-dibromopyridine (2

g, 8.4 mmol) in THF (10 mL) was added slowly to the slurry. The

reaction mixture was stirred at −78 °C for 45 min. The mixture was

allowed to warm slowly to rt over 30 min. The reaction mixture was

diluted with water and extracted with EtOAc (2 × 10 mL); the

organic layers were combined, dried with magnesium sulfate,

concentrated, and puriﬁed via silica gel chromatography (0−40%

EtOAc/hexanes) to aﬀord 2-(6-bromopyridin-2-yl)acetonitrile (1.65

g, 99%) as a yellow oil that solidiﬁed upon cooling; LCMS: m/z (M +

H)⁺= 197.0. Method 2 was used to aﬀord 2-(6-(4-chlorophenyl)-

pyridin-2-yl)acetonitrile N4; LCMS: m/z (M + H)⁺= 229.1.

Method 7: 2-(2-Phenyl-1H-imidazol-5-yl)acetonitrile. A mixture

of 5-(chloromethyl)-2-phenyl-1H-imidazole hydrochloride (197 mg,

0.86 mmol) and sodium cyanide (127 mg, 2.58 mmol) in DMSO (3

mL) was stirred at rt overnight. The reaction mixture was diluted with

water and saturated aqueous sodium bicarbonate solution, extracted

(EtOAc × 2), dried with magnesium sulfate, and concentrated, and 2-

(2-phenyl-1H-imidazol-5-yl)acetonitrile was used without further

puriﬁcation.

Method 8: tert-Butyl 5-(Cyanomethyl)-2-phenyl-1H-imidazole-1-

carboxylate (N9, Intermediate for Compound 17). A mixture of 2-

(2-phenyl-1H-imidazol-5-yl)acetonitrile (50 mg, 0.27 mmol), Boc₂O

(0.070 mL, 0.3 mmol), and DMAP (trace) in acetonitrile (3 mL) and

sodium cyanide (127 mg, 2.58 mmol) in DMSO (3 mL) was stirred at

rt for 40 min and concentrated under a stream of air. tert-Butyl 5-

(cyanomethyl)-2-phenyl-1H-imidazole-1-carboxylate was used with-

out further puriﬁcation; LCMS: m/z (M + H)⁺= 284.1 (weak).

Method 4: 2-(6-Chlorobenzo[d]thiazol-2-yl)acetonitrile (N5,

Intermediate for Compound 13). Malononitrile (65 mg, 0.98

mmol) and 2-amino-5-chlorobenzenethiol (157 mg, 0.98 mmol)

were heated at 50 °C for 4 h and at reﬂux for 1 h in a mixture of

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3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-

pyridin-2(1H)-one (2). Step 1: 2-Oxo-1,2-dihydropyridine-3-carbo-

thioamide: Ammonium sulﬁde (0.509 mL, 2.99 mmol) was added to

a solution of 2-oxo-1,2-dihydropyridine-3-carbonitrile (211 mg, 1.757

mmol) in methanol (14 mL). The reaction mixture was heated in a

microwave at 130 °C for 2 h. The mixture stood overnight at rt and

crystals formed. The mixture was further cooled to 0 °C for 4 h. The

methanol was poured oﬀ and the solid was triturated with methanol

and used as is in the following step. ¹H NMR (400 MHz, DMSO-d₆):

δ 12.60 (s, 1H), 11.31 (s, 1H), 9.98 (s, 1H), 8.93 (dd, J = 7.4, 2.2 Hz,

1H), 7.77 (dd, J = 6.2, 2.3 Hz, 1H), 6.52 (dd, J = 7.4, 6.2 Hz, 1H).

Step 2: 3-(4-(4-Chlorophenyl)thiazol-2-yl)pyridin-2(1H)-one: To

2-oxo-1,2-dihydropyridine-3-carbothioamide (124 mg, 0.804 mmol)

in ethanol (2 mL) was added 2-bromo-1-(4-chlorophenyl)ethanone

(188 mg, 0.804 mmol). The reaction mixture was heated at reﬂux for

17.5 h. The reaction mixture was cooled to rt and diluted with

hexanes. The solid was removed by ﬁltration, washed with hexanes,

and dried on high vacuum. The product (213 mg, 65%) is a red-

Method 9: 2-(4-Phenyl-1H-imidazol-1-yl)acetonitrile (N10, Inter-

mediate for Compound 16). A mixture of 4-phenyl-1H-imidazole

(250 mg, 1.7 mmol), chloroacetonitrile (0.22 mL, 3.5 mmol), and

potassium carbonate (1.2 g, 8.7 mmol) in DMF (8 mL) was stirred at

rt for 22 h. The reaction mixture was diluted with water, extracted

(EtOAc × 2), dried with magnesium sulfate, and concentrated to

aﬀord 2-(4-phenyl-1H-imidazol-1-yl)acetonitrile as a brown solid

which was used without further puriﬁcation; LCMS: m/z (M + H)⁺=

184.1.

1

brown powder. H NMR (400 MHz, DMSO-d₆): δ 12.10 (s, 1H),

2-(3-Phenyl-1H-pyrazol-1-yl)acetonitrile (N11, Intermediate for

Compound 20). 2-(3-Phenyl-1H-pyrazol-1-yl)acetonitrile (N11,

intermediate for compound 20) was synthesized by Method 9;

LCMS: m/z (M + H)⁺= 184.1 (weak).

8.28 (dd, J = 7.2, 2.1 Hz, 1H), 7.80 (s, 1H), 7.76−7.68 (m, 2H), 7.30

(s, 1H), 7.20−7.11 (m, 2H), 6.15 (dd, J = 7.2, 6.3 Hz, 1H).

Step 3: 3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-

dimethoxyphenyl)pyridin-2(1H)-one (Table 1; compound 2): To a

mixture of 3-(4-(4-chlorophenyl)thiazol-2-yl)pyridin-2(1H)-one (60

mg, 0.208 mmol), copper(II) acetate (56.6 mg, 0.312 mmol), and 2,5-

dimethoxyphenylboronic acid (76 mg, 0.416 mmol) were added 1,4-

dioxane (2 mL) and pyridine (0.2 mL). The reaction mixture was

sealed and heated at 80 °C for 60 h. The mixture was ﬁltered with

thiol resin, washed with EtOAc, and concentrated. The crude mixture

was diluted with DMSO and puriﬁed by reverse-phase chromatog-

raphy (Standard Acidic Gradient Method) to aﬀord as a TFA salt, 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione. LCMS: m/z (M +

H)⁺= 425.1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.70 (dd, J = 7.3, 2.0

Hz, 1H), 8.16 (s, 1H), 8.07 (d, J = 8.6 Hz, 2H), 7.78 (dd, J = 6.6, 2.0

Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.17 (dd, J = 8.5, 1.3 Hz, 1H),

7.10−7.02 (m, 2H), 6.59 (t, J = 6.9 Hz, 1H), 3.73 (s, 3H), 3.68 (s,

3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₂H₁₈ClN₂O₃S,

425.0721; found, 425.0723. Retention time = 3.787 min.

2-(4-Phenyl-1H-pyrazol-1-yl)acetonitrile (N12, Intermediate for

Compound 21). 2-(4-Phenyl-1H-pyrazol-1-yl)acetonitrile (N12,

intermediate for compound 21) was synthesized by Method 9;

LCMS: m/z (M + H)⁺= 184.1 (weak).

Method 10: 3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (1).

Step 1: In a vial, 5,5-dimethylcyclohexane-1,3-dione (0.100 g, 0.713

mmol) and DMF-DMA (0.096 mL, 0.713 mmol) were mixed and

stirred well (no solvent) for 5 min. The reaction mixture became

yellow oil.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-6-

methylpyridin-2(1H)-one (3).

Step 2: To the mixture were added i-PrOH (2.55 mL), 2-(4-(4-

chlorophenyl)thiazol-2-yl)acetonitrile (167 mg, 0.713 mmol), and

piperidine (0.071 mL, 0.713 mmol). The reaction mixture was

allowed to stir at rt for 3 h. The solid turned into solution. After 3 h, a

precipitate formed at which point the solvent was removed by blowing

down under a stream of air with mild heating at 30 °C.

Step 1: 2-(4-(4-Chlorophenyl)thiazol-2-yl)-N -(2,5-

dimethoxyphenyl)acetamide: To a mixture of 2-(4-(4-chlorophenyl)-

thiazol-2-yl)acetic acid (155 mg, 0.611 mmol) in CH₂Cl₂(3 mL)

were added HATU (348 mg, 0.916 mmol), DIPEA (320 μ L, 1.833

mmol), and 2,5-dimethoxyaniline (103 mg, 0.672 mmol). The

reaction mixture was stirred for 1 h at room temperature. The

reaction mixture was diluted with water and CH₂Cl₂and extracted (2

× 15 mL); the organic layers were combined, dried with magnesium

sulfate, concentrated, and puri ﬁ ed via silica gel chromatography to

a ﬀ ord 2-(4-(4-chlorophenyl)thiazol-2-yl)-N -(2,5-dimethoxyphenyl)-

acetamide as a solid; LCMS: m /z (M + H)⁺= 389.

Step 3: To the resulting residue were added acetic acid (1 mL) and

2,5-dimethoxyaniline (109 mg, 0.713 mmol). The reaction mixture

was stirred for 15 min at rt, a precipitate formed almost immediately.

The solvent was removed by blowing down under a stream of air with

mild heating at 30 °C. The crude mixture was diluted with DMSO

and puriﬁed by reverse-phase chromatography (Standard Acidic

Gradient Method) to aﬀord as a TFA salt, 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydroqui-

1

noline-2,5(1H,6H)-dione LCMS: m/z (M + H)⁺= 521.1; H NMR

(400 MHz, DMSO-d₆): δ 9.04 (s, 1H), 8.22 (s, 1H), 8.12−8.05 (m,

2H), 7.58−7.50 (m, 2H), 7.23 (d, J = 9.2 Hz, 1H), 7.13 (dd, J = 9.1,

3.1 Hz, 1H), 7.05 (d, J = 3.0 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 2.62

(d, J = 17.7 Hz, 1H), 2.46 (d, J = 2.9 Hz, 2H), 2.22 (d, J = 17.7 Hz,

1H), 0.99 (s, 3H), 0.94 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd

for C₂₈H₂₆ClN₂O₄S, 521.1296; found, 521.1321. Retention time =

3.858 min.

Step 2: 3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphen-

yl)-6-methylpyridin-2(1H)-one (Table 1; Compound 3): A mixture

of 2-(4-(4-chlorophenyl)thiazol-2-yl)-N-(2,5-dimethoxyphenyl)-

acetamide (74 mg, 0.190 mmol), (E)-4-methoxybut-3-en-2-one

(38.8 μL, 0.381 mmol), and potassium 2-methylpropan-2-olate

(21.35 mg, 0.190 mmol) was heated at 80 °C for 3.5 h. The reaction

mixture was diluted with methanol/CH₂Cl₂/water and to alleviate the

emulsion, it is acidiﬁed with 1 N HCl, extracted with EtOAc, and

concentrated. The crude mixture was diluted with DMSO and

puriﬁed by reverse-phase chromatography (Standard Acidic Gra-

dient Method) to aﬀord as a TFA salt, 3-(4-(4-chlorophenyl)thiazol-

2-yl)-1-(2,5-dimethoxyphenyl)-6-methylpyridin-2(1H)-one. LCMS:

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m/z (M + H)⁺= 521.1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.61 (d, J

= 7.4 Hz, 1H), 8.15−8.03 (m, 3H), 7.55−7.47 (m, 2H), 7.18 (d, J =

9.1 Hz, 1H), 7.07 (dd, J = 9.1, 3.1 Hz, 1H), 6.98 (d, J = 3.1 Hz, 1H),

6.59 (dd, J = 7.5, 0.9 Hz, 1H), 3.74 (s, 3H), 3.69 (s, 3H), 2.03 (d, J =

0.8 Hz, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₃H₂₀ClN₂O₃S,

439.0878; found, 439.0897. Retention time = 3.822 min.

(ESI): m/z (M + H)⁺calcd for C₂₆H₂₂ClN₂O₄S, 493.0983; found,

493.0973. Retention time = 3.741 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-6,7-

dihydro-1H-cyclopenta[b]pyridine-2,5-dione (7). This compound

was prepared using Method 10 using cyclopentane-1,3-dione in step 1

to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphen-

yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS:

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-5,6,7,8-tetrahydroquinolin-2(1H)-one (4). To a solution of

3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (35 mg, 0.067 mmol)

compound 1 in THF (2 mL) was added lithium aluminum hydride

(0.101 mL, 0.101 mmol). The reaction mixture was stirred at rt for 1

h and then heated at 60 °C for 12 h. The reaction mixture was cooled

to 0 °C and quenched with methanol and a bit of water, diluted with

EtOAc, and extracted (2 × 25 mL); the organic layers were

combined, dried with magnesium sulfate, and concentrated. The

crude mixture was diluted with DMSO and puriﬁed by reverse-phase

chromatography (Standard Acidic Gradient Method) to aﬀord as a

TFA salt, 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphen-

yl)-7,7-dimethyl-5,6,7,8-tetrahydroquinolin-2(1H)-one. LCMS: m/z

(M + H)⁺= 507.2; ¹H NMR (400 MHz, DMSO-d₆): δ 8.48 (s, 1H),

8.13−8.00 (m, 3H), 7.54−7.45 (m, 2H), 7.16 (d, J = 9.1 Hz, 1H),

7.05 (dd, J = 9.1, 3.0 Hz, 1H), 6.87 (d, J = 3.0 Hz, 1H), 3.73 (d, J =

1.4 Hz, 3H), 3.66 (s, 3H), 2.73 (t, J = 6.5 Hz, 2H), 2.09 (d, J = 17.8

Hz, 1H), 1.78 (d, J = 17.8 Hz, 1H), 1.47 (t, J = 6.7 Hz, 2H), 0.88 (s,

3H), 0.84 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₈H₂₈ClN₂O₃S, 507.1504; found, 507.1509. Retention time = 4.196

min.

1

m/z (M + H)⁺= 479.1; H NMR (400 MHz, DMSO-d₆): δ 8.77 (s,

1H), 8.27 (s, 1H), 8.14 (d, J = 8.61 Hz, 2H), 7.54 (d, J = 8.61 Hz,

2H), 7.27 (d, J = 9.00 Hz, 1H), 7.07−7.22 (m, 2H), 3.76 (d, J = 12.91

Hz, 5H), 2.77−2.92 (m, 1H), 2.55−2.77 (m, 3H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₅H₂₀ClN₂O₄S, 479.0827; found, 479.0840.

Retention time = 3.672 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-5-

hydroxyquinolin-2(1H)-one (8). To a solution of 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,8-dihydroqui-

noline-2,5(1H,6H)-dione (25 mg, 0.051 mmol) compound 6 in 1,4-

dioxane (3 mL) was added DDQ (17.27 mg, 0.076 mmol). The

reaction mixture was stirred at 70 °C for 12 h. The organic solvent

was concentrated. The crude mixture was diluted with DMSO and

puriﬁed by reverse-phase chromatography (Standard Acidic Gra-

dient Method) to aﬀord as a TFA salt, 3-(4-(4-chlorophenyl)thiazol-

2-yl)-1-(2,5-dimethoxyphenyl)-5-hydroxyquinolin-2(1H)-one.

LCMS: m/z (M + H)⁺= 491.2; HRMS (ESI): m/z (M + H)⁺calcd

for C₂₆H₂₀ClN₂O₄S, 491.0827; found, 491.0817. Retention time =

3.619 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,8-

dihydro-1,7-naphthyridine-2,5(1H,6H)-dione (9). This compound

was prepared using Method 10 using tert-butyl 3,5-dioxopiperidine-1-

carboxylate in step 1 followed by Boc deprotection after step 3 using

TFA to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxy-

phenyl)-7,8-dihydro-1,7-naphthyridine-2,5(1H,6H)-dione as a TFA

1

salt. LCMS: m/z (M + H)⁺= 494.1; H NMR (400 MHz, DMSO-

d₆): δ 9.03 (s, 1H), 8.30 (s, 1H), 8.14−8.07 (m, 2H), 7.59−7.52 (m,

2H), 7.29 (d, J = 9.2 Hz, 1H), 7.19 (dd, J = 9.2, 3.1 Hz, 1H), 7.06 (d,

J = 3.1 Hz, 1H), 3.98−3.79 (m, 2H), 3.76 (s, 3H), 3.74 (s, 3H);

HRMS (ESI): m/z (M + 2H)²⁺calcd for C₂₅H₂₂ClN₃O₄S, 493.0821;

found, 493.0863. Retention time = 3.148 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinolin-2(1H)-one (5). Step 1: To a solution

of 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (200 mg, 0.384

mmol) compound 1 in EtOH (10 mL) was added sodium

borohydride (29.0 mg, 0.768 mmol). Gas evolution was observed.

The reaction mixture was stirred at 50 °C for 12 h. The reaction

mixture was cooled to rt, diluted with water and EtOAc, and extracted

(2 × 25 mL); the organic layers were combined, dried with

magnesium sulfate, and concentrated. The crude mixture was taken to

the next step without further puriﬁcation.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,8-

dihydro-1,6-naphthyridine-2,5(1H,6H)-dione (10). This compound

was prepared using Method 10 using tert-butyl 2,4-dioxopiperidine-1-

carboxylate in step 1 followed by Boc deprotection using TFA to

aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-

7,8-dihydro-1,6-naphthyridine-2,5(1H,6H)-dione as a TFA salt.

1

LCMS: m/z (M + H)⁺= 494.2; H NMR (400 MHz, DMSO-d₆):

δ 9.06 (s, 1H), 8.20 (s, 1H), 8.13−8.04 (m, 2H), 7.92 (s, 1H), 7.57−

7.47 (m, 2H), 7.21 (dd, J = 8.8, 0.8 Hz, 1H), 7.15−7.06 (m, 2H), 3.74

(s, 3H), 3.71 (s, 3H), 3.42−3.30 (m, 2H), 2.73−2.60 (m, 1H), 2.40

(dt, J = 17.6, 6.5 Hz, 1H); HRMS (ESI): m/z (M + Na)⁺calcd for

C₂₅H₂₀ClN₃NaO₄S, 516.0755; found, 516.0768. Retention time =

3.822 min.

Step 2: To a solution of 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-

dimethoxyphenyl)-5-hydroxy-7,7-dimethyl-5,6,7,8-tetrahydroquino-

lin-2(1H)-one (38 mg, 0.073 mmol) in 1,4-dioxane (10 mL) was

added pTSA (1.38 mg, 0.1 equiv). The reaction mixture was stirred at

rt for 4 h. The organic solvent was concentrated. The crude mixture

was diluted with DMSO and puriﬁed by reverse-phase chromatog-

raphy (Standard Acidic Gradient Method) to aﬀord as a TFA salt, 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-di-

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(4-phenylthiazol-2-yl)-

7,8-dihydroquinoline-2,5(1H,6H)-dione (11). This compound was

prepared using Method 10 using 2-(4-phenylthiazol-2-yl)acetonitrile

N2 in step 2 to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-dimethyl-3-(4-

phenylthiazol-2-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA

methyl-7,8-dihydroquinolin-2(1H)-one. LCMS: m/z (M + H)⁺

=

1

505.2; H NMR (400 MHz, DMSO-d₆): δ 8.60 (s, 2H), 8.01−8.23

(m, 6H), 7.54 (m, 2H), 7.18 (d, J = 8.61 Hz, 3H), 7.18−7.30 (m,

2H), 7.06−7.18 (m, 2H), 7.00 (d, J = 3.13 Hz, 2H), 6.47 (d, J = 9.78

Hz, 2H), 5.63 (d, J = 9.78 Hz, 1H), 3.73 (d, J = 19.96 Hz, 7H), 2.37−

2.48 (m, 2H), 2.14 (s, 1H), 0.84−1.14 (m, 7H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₈H₂₆ClN₂O₃S, 505.1347; found, 505.1351.

Retention time = 4.152 min.

1

salt. LCMS: m/z (M + H)⁺= 487.2; H NMR (400 MHz, DMSO-

d₆): δ 9.14 (s, 1H), 8.25 (s, 1H), 8.18−8.11 (m, 2H), 7.57 (dd, J =

8.3, 7.0 Hz, 2H), 7.50−7.41 (m, 1H), 7.33 (d, J = 9.1 Hz, 1H), 7.23

(dd, J = 9.1, 3.1 Hz, 1H), 7.14 (d, J = 3.0 Hz, 1H), 3.85 (s, 3H), 3.81

(s, 3H), 2.72 (d, J = 17.8 Hz, 1H), 2.64−2.49 (m, 2H), 2.31 (d, J =

17.7 Hz, 1H), 1.09 (s, 3H), 1.03 (s, 3H); HRMS (ESI): m/z (M +

Na)⁺calcd for C₂₈H₂₆N₂NaO₄S, 509.1505; found, 509.1529.

Retention time = 3.718 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,8-

dihydroquinoline-2,5(1H,6H)-dione (6). This compound was pre-

pared using Method 10 using cyclohexane-1,3-dione in step 1 to

aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-

7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z

3-(5-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (12). This com-

pound was prepared using Method 10 using 2-(5-(4-chlorophenyl)-

thiazol-2-yl)acetonitrile in step 2 to aﬀord 3-(5-(4-chlorophenyl)-

thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydroqui-

1

(M + H)⁺= 493; H NMR (400 MHz, DMSO-d₆): δ 9.04 (s, 1H),

8.21 (d, J = 1.2 Hz, 1H), 8.11−8.02 (m, 2H), 7.57−7.46 (m, 2H),

7.21 (d, J = 9.1 Hz, 1H), 7.14−7.02 (m, 2H), 3.74 (d, J = 1.1 Hz,

3H), 3.70 (s, 3H), 2.51 (d, J = 1.2 Hz, 2H), 2.47 (m, 4H); HRMS

noline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺

=

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1

521.2; H NMR (400 MHz, DMSO-d₆): δ 9.03 (d, J = 4.7 Hz, 1H),

(ESI): m/z (M + H)⁺calcd for C₂₈H₂₈N₃O₄, 470.2074; found,

8.49 (s, 1H), 7.84−7.75 (m, 2H), 7.64−7.54 (m, 2H), 7.32 (d, J = 9.2

Hz, 1H), 7.26−7.17 (m, 1H), 7.13 (d, J = 3.1 Hz, 1H), 3.84 (s, 3H),

3.79 (s, 3H), 2.71 (d, J = 17.7 Hz, 1H), 2.54 (d, J = 3.1 Hz, 2H), 2.30

(d, J = 17.8 Hz, 1H), 1.08 (s, 3H), 1.02 (s, 3H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₆H₂₄ClN₂O₄S, 495.114; found, 495.1159.

Retention time = 3.787 min.

470.2054. Retention time = 2.59 min.

3-(6-Chlorobenzo[d]thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (13). This com-

pound was prepared using Method 10 using 2-(6-chlorobenzo[d]-

thiazol-2-yl)acetonitrile N5 in step 2 to aﬀord 3-(6-chlorobenzo[d]-

thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydroqui-

noline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺

=

1

495.1; H NMR (400 MHz, DMSO-d₆): δ 9.14 (s, 1H), 8.27 (d, J =

2.1 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.56 (dd, J = 8.7, 2.2 Hz, 1H),

7.25 (d, J = 9.1 Hz, 1H), 7.18−7.05 (m, 2H), 3.75 (s, 3H), 3.72 (s,

3H), 2.64−2.60 (m, 2H), 2.35−2.17 (m, 2H), 1.00 (s, 3H), 0.94 (s,

3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₆H₂₄ClN₂O₄S,

495.114; found, 495.1138. Retention time = 3.795 min.

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-2,5-dioxo-1,2,5,6,7,8-

hexahydro-quinoline-3-carbonitrile dione (18). Step 1: Similar to

Method 10; Step 2: Similar to Method 10 with stirring only for 1 h.

Also, aniline was added prior to concentration; Step 3: Acetic acid was

added and stirred overnight at rt. The reaction mixture was diluted

with water and DCM and extracted (2×); the organic layers were

combined, dried with magnesium sulfate, concentrated, and puriﬁed

via silica gel chromatography (10−100% EtOAc/hexanes) to aﬀord 1-

(2,5-dimethoxyphenyl)-7,7-dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydro-

quinoline-3-carbonitrile (70% on 2.85 mmol scale); LCMS: m/z (M

+ H)⁺= 353.1. Step 4: 1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-2,5-

dioxo-1,2,5,6,7,8-hexahydroquinoline-3-carboximidamide: To a mix-

ture of 1-(2,5-dimethoxyphenyl)-7,7-dimethyl-2,5-dioxo-1,2,5,6,7,8-

hexahydroquinoline-3-carbonitrile (60 mg, 0.170 mmol) in MeOH

(1 mL) was added sodium methanolate (398 μL, 1.703 mmol) in

MeOH. The reaction mixture was stirred at 45 °C for 45 min and

then ammonia hydrochloride (91 mg, 1.703 mmol) and AcOH (1.5

mL) were added. The reaction mixture was stirred at 60 °C for 12 h.

The reaction mixture was diluted with water and DCM and extracted

(2×); the organic layers were combined, dried with magnesium

sulfate, concentrated, and puriﬁed via silica gel chromatography (10−

100% EtOAc/hexanes) to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydroquinoline-3-carboximida-

mide; LCMS: m/z (M + H)⁺= 370.1. Step 5: To a mixture of 1-(2,5-

dimethoxyphenyl)-7,7-dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydroqui-

noline-3-carboximidamide (50 mg, 0.135 mmol) in THF (1 mL) were

added 2-bromo-1-(4-chlorophenyl)ethan-1-one (135 μL, 0.135

mmol) and sodium bicarbonate (11.37 mg, 0.135 mmol). The

reaction mixture was stirred at 70 °C for 2 h and then AcOH (7.75

μL, 0.135 mmol) was added. The reaction mixture was stirred at 70

°C for 2 h. The solvent was removed by blowing down under a stream

of air with mild heating at 30 °C. The crude mixture was diluted with

DMSO and puriﬁed by reverse-phase chromatography (Standard

Acidic Gradient Method) to aﬀord as a TFA salt, 3-(5-(4-

chlorophenyl)-1H-imidazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione. LCMS: m/z (M +

3-(5-Chlorobenzo[d]thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (14). This com-

pound was prepared using Method 10 using 2-(5-chlorobenzo[d]-

thiazol-2-yl)acetonitrile N6 in step 2 to aﬀord 3-(5-chlorobenzo[d]-

thiazol-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydroqui-

noline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺

=

1

495.1; H NMR (400 MHz, DMSO-d₆): δ 9.15 (s, 1H), 8.20−8.12

(m, 2H), 7.46 (dd, J = 8.5, 2.0 Hz, 1H), 7.25 (d, J = 9.1 Hz, 1H),

7.18−7.05 (m, 2H), 3.75 (s, 3H), 3.72 (s, 3H), 2.64 (d, J = 17.8 Hz,

1H), 2.24 (d, J = 17.8 Hz, 1H), 1.00 (s, 3H), 0.94 (s, 3H); HRMS

(ESI): m/z (M + Na)⁺calcd for C₂₈H₂₅ClN₂NaO₄S, 543.1116; found,

543.113. Retention time = 3.811 min.

3-(1-(4-Chlorophenyl)-1H-imidazol-4-yl)-1-(2,5-dimethoxyphen-

yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (15). This

compound was prepared using Method 10 using 2-(1-(4-chloro-

phenyl)-1H-imidazol-4-yl)acetonitrile N7 in step 2 to aﬀord 3-(1-(4-

chlorophenyl)-1H-imidazol-4-yl)-1-(2,5-dimethoxyphenyl)-7,7-di-

methyl-7,8-dihydro-quinoline-2,5(1H,6H)-dione as a TFA salt.

1

LCMS: m/z (M + H)⁺= 504.1; H NMR (400 MHz, DMSO-d₆):

δ 8.65 (s, 1H), 8.38 (d, J = 1.4 Hz, 1H), 8.20 (d, J = 1.4 Hz, 1H),

7.75−7.62 (m, 2H), 7.59−7.48 (m, 1H), 7.18 (d, J = 9.1 Hz, 1H),

7.07 (dd, J = 9.1, 3.0 Hz, 1H), 6.94 (d, J = 3.0 Hz, 1H), 3.72 (s, 3H),

3.67 (s, 3H), 2.63 (dd, J = 3.7, 1.9 Hz, 1H), 2.38 (d, J = 8.1 Hz, 1H),

2.31−2.26 (m, 1H), 0.96 (s, 3H), 0.91 (s, 3H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₈H₂₇ClN₃O₄, 504.1685; found, 504.1678.

Retention time = 3.343 min.

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(4-phenyl-1H-imida-

zol-1-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (16). This com-

pound was prepared using Method 10 using 2-(4-phenyl-1H-

imidazol-1-yl)acetonitrile N10 in step 2 to aﬀord 1-(2,5-dimethox-

yphenyl)-7,7-dimethyl-3-(4-phenyl-1H-imidazol-1-yl)-7,8-dihydroqui-

1

H)⁺= 504.1; H NMR (400 MHz, DMSO-d₆): δ 8.78 (s, 1H), 7.88

noline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺

=

(d, J = 8.2 Hz, 2H), 7.63 (s, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.20 (d, J =

9.2 Hz, 1H), 7.15−6.95 (m, 2H), 3.71 (d, J = 13.8 Hz, 6H), 2.66−

2.54 (m, 2H), 2.42 (d, J = 4.5 Hz, 2H), 0.98 (s, 3H), 0.92 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₇ClN₃O₄, 504.1685;

found, 504.1709. Retention time = 3.171 min.

470.2; ¹H NMR (400 MHz, DMSO-d₆): δ 8.71 (s, 1H), 8.35 (s, 1H),

8.27 (s, 1H), 7.88−7.80 (m, 2H), 7.41 (dd, J = 8.3, 7.1 Hz, 2H),

7.33−7.19 (m, 2H), 7.12 (dd, J = 9.1, 3.1 Hz, 1H), 7.02 (d, J = 3.1

Hz, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 2.60 (d, J = 17.6 Hz, 1H), 2.44

(d, J = 4.1 Hz, 2H), 2.19 (d, J = 17.5 Hz, 1H), 0.99 (s, 3H), 0.94 (s,

3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₈N₃O₄, 470.2074;

found, 470.2095. Retention time = 3.02 min.

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(1-phenyl-1H-pyrazol-

3-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (19). This compound

was prepared using Method 10 using 2-(1-phenyl-1H-pyrazol-3-

yl)acetonitrile N8 in step 2 to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-3-(1-phenyl-1H-pyrazol-3-yl)-7,8-dihydroquinoline-2,5-

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(2-phenyl-1H-imida-

zol-4-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (17). This com-

pound was prepared using Method 10 using tert-butyl 5-

(cyanomethyl)-2-phenyl-1H-imidazole-1-carboxylate N9 in step 2

followed by treatment with TFA to aﬀord 1-(2,5-dimethoxyphenyl)-

7,7-dimethyl-3-(2-phenyl-1H-imidazol-4-yl)-7,8-dihydroquinoline-

2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺= 470.2; ¹H

NMR (400 MHz, DMSO-d₆): δ 8.71 (s, 1H), 8.01 (d, J = 7.6 Hz,

2H), 7.45 (d, J = 31.3 Hz, 3H), 7.18 (d, J = 9.1 Hz, 1H), 7.08 (dd, J =

9.1, 3.0 Hz, 2H), 6.94 (d, J = 3.0 Hz, 1H), 3.73 (s, 3H), 3.68 (s, 3H),

2.66−2.55 (m, 1H), 2.40 (d, J = 8.2 Hz, 1H), 2.29 (p, J = 1.9 Hz,

1H), 2.13 (d, J = 17.5 Hz, 1H), 0.97 (s, 3H), 0.92 (s, 3H); HRMS

1

(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺= 470.2; H

NMR (400 MHz, DMSO-d₆): δ 8.68 (s, 1H), 8.51 (d, J = 2.5 Hz,

1H), 7.94−7.86 (m, 2H), 7.56−7.47 (m, 2H), 7.37−7.28 (m, 1H),

7.20 (d, J = 9.1 Hz, 1H), 7.16−7.05 (m, 2H), 6.98 (d, J = 3.0 Hz,

1H), 3.75 (s, 3H), 3.71 (s, 3H), 2.56 (d, J = 17.6 Hz, 1H), 2.41 (d, J =

7.3 Hz, 2H), 2.14 (d, J = 17.6 Hz, 1H), 0.98 (s, 3H), 0.93 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₈N₃O₄, 470.2074; found,

470.2094. Retention time = 3.539 min.

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(3-phenyl-1H-pyrazol-

1-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (20). This compound

4931

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was prepared using Method 10 using 2-(3-phenyl-1H-pyrazol-1-

yl)acetonitrile N11 in step 2 to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-3-(3-phenyl-1H-pyrazol-1-yl)-7,8-dihydroquinoline-2,5-

(dd, J = 11.1, 6.7 Hz, 2H), 7.19 (d, J = 8.9 Hz, 2H), 7.08 (d, J = 9.4

Hz, 1H), 7.01−6.93 (m, 1H), 6.47 (s, 0H), 3.73 (s, 3H), 3.69 (s, 3H),

2.51 (s, 3H), 2.14 (d, J = 17.8 Hz, 1H), 0.97 (s, 3H), 0.92 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₈N₃O₄, 470.2074; found,

470.2073. Retention time = 3.428 min.

1

(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺= 470.2; H

NMR (400 MHz, DMSO-d₆): δ 8.64 (d, J = 2.6 Hz, 1H), 8.51 (s,

1H), 7.96−7.88 (m, 2H), 7.50−7.41 (m, 2H), 7.41−7.32 (m, 1H),

7.22 (d, J = 9.1 Hz, 1H), 7.12 (dd, J = 9.1, 3.1 Hz, 1H), 7.04 (d, J =

3.0 Hz, 1H), 6.96 (d, J = 2.6 Hz, 1H), 3.75 (s, 3H), 3.72 (s, 3H), 2.57

(d, J = 17.5 Hz, 1H), 2.44 (d, J = 4.2 Hz, 2H), 2.16 (d, J = 17.6 Hz,

1H), 0.99 (s, 3H), 0.94 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd

for C₂₈H₂₈N₃O₄, 470.2074; found, 470.2074. Retention time = 3.66

min.

3-(4′-Chloro-[1,1′-biphenyl]-3-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (23). This com-

pound was prepared using Method 10 using 2-(4′-chloro-[1,1′-

biphenyl]-3-yl)acetonitrile N3 in step 2 to aﬀord 3-(4′-chloro-[1,1′-

biphenyl]-3-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydro-

quinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺=

1

514.2; H NMR (400 MHz, DMSO-d₆): δ 8.12 (s, 1H), 7.99−7.93

(m, 1H), 7.79−7.60 (m, 4H), 7.56−7.45 (m, 3H), 7.19 (d, J = 9.1 Hz,

1H), 7.08 (dd, J = 9.1, 3.1 Hz, 1H), 6.97 (d, J = 3.0 Hz, 1H), 3.74 (s,

3H), 3.71 (s, 3H), 2.97 (s, 3H), 2.60−2.49 (m, 1H), 2.47−2.32 (m,

2H), 2.14 (d, J = 17.5 Hz, 1H), 0.98 (s, 3H), 0.94 (s, 3H); HRMS

(ESI): m/z (M + H)⁺calcd for C₃₁H₂₉ClNO₄, 514.178; found,

514.1777. Retention time = 3.835 min.

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(4-phenyl-1H-pyrazol-

1-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (21). This compound

was prepared using Method 10 using 2-(4-phenyl-1H-pyrazol-1-

yl)acetonitrile N12 in step 2 to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-3-(4-phenyl-1H-pyrazol-1-yl)-7,8-dihydroquinoline-2,5-

1

(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺= 470.2; H

NMR (400 MHz, DMSO-d₆): δ 8.95 (d, J = 0.8 Hz, 1H), 8.44 (s,

1H), 8.25 (d, J = 0.8 Hz, 1H), 7.67−7.59 (m, 2H), 7.41−7.31 (m,

2H), 7.26−7.16 (m, 2H), 7.12 (dd, J = 9.1, 3.1 Hz, 1H), 7.04 (d, J =

3.1 Hz, 1H), 3.75 (s, 3H), 3.72 (s, 3H), 2.62−2.50 (m, 1H), 2.43 (d, J

= 4.1 Hz, 2H), 2.17 (d, J = 17.6 Hz, 1H), 0.99 (s, 3H), 0.94 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₈N₃O₄, 470.2074; found,

470.208. Retention time = 3.608 min.

3-([1,1′-Biphenyl]-4-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-

7,8-dihydroquinoline-2,5(1H,6H)-dione (24). This compound was

prepared using Method 10 using 2-([1,1′-biphenyl]-4-yl)acetonitrile

in step 2 to aﬀord 3-([1,1′-biphenyl]-4-yl)-1-(2,5-dimethoxyphenyl)-

7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt.

1

LCMS: m/z (M + H)⁺= 480.2; H NMR (400 MHz, DMSO-d₆): δ

8.09 (s, 1H), 7.83−7.75 (m, 2H), 7.74−7.65 (m, 4H), 7.51−7.42 (m,

2H), 7.40−7.31 (m, 1H), 7.20 (d, J = 9.2 Hz, 1H), 7.08 (dd, J = 9.1,

3.1 Hz, 1H), 6.98 (d, J = 3.0 Hz, 1H), 3.74 (s, 3H), 3.72 (s, 3H),

2.45−2.33 (m, 2H), 2.14−2.10 (m, 2H), 0.99 (s, 3H), 0.94 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₃₁H₃₀NO₄, 480.2169; found,

480.2176. Retention time = 3.719 min.

3-(6-(4-Chlorophenyl)pyridin-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (25). This com-

pound was prepared using Method 10 using 2-(6-(4-chlorophenyl)-

pyridin-2-yl)acetonitrile N4 in step 2 to aﬀord 3-(6-(4-chlorophenyl)-

pyridin-2-yl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydroqui-

1-(2,5-Dimethoxyphenyl)-7,7-dimethyl-3-(5-phenyl-1H-pyrazol-

3-yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (22). Step 1: A mixture

of 1-(2,5-dimethoxyphenyl)-7,7-dimethyl-2,5-dioxo-1,2,5,6,7,8-hexa-

hydroquinoline-3-carbonitrile (60 mg, 0.17 mmol) (intermediate

after step 3 in the synthesis of compound 18) in concentrated HCl (3

mL) was heated at 80 °C for 22 h. The reaction mixture was diluted

with water, extracted (DCM/MeOH × 3), dried with magnesium

sulfate, and concentrated to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydroquinoline-3-carboxylic acid

which was used without further puriﬁcation; LCMS: m/z (M + H)⁺

= 372.1. Step 2: To the acid (28 mg, 0.075 mmol) in DCM (3 mL)

were added a drop of DMF and oxalyl chloride (0.033 mL, 0.38

mmol). The reaction mixture was stirred at rt for 1.2 h. The reaction

mixture was concentrated under a stream of argon, rediluted with

DCM, and reconcentrated to aﬀord 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydroquinoline-3-carbonyl chlor-

ide; this was veriﬁed by LCMS that showed the formation of the

methyl ester on the addition of MeOH to an aliquot from the

reaction.

noline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺

=

515.1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.94 (s, 1H), 8.27 (dd, J =

7.2, 1.6 Hz, 1H), 8.16 (d, J = 8.6 Hz, 2H), 7.95−7.85 (m, 2H), 7.59

(d, J = 8.6 Hz, 2H), 7.19 (d, J = 9.1 Hz, 1H), 7.08 (dd, J = 9.1, 3.0 Hz,

1H), 6.99 (d, J = 3.0 Hz, 1H), 3.73 (s, 3H), 3.70 (s, 3H), 2.58 (d, J =

17.6 Hz, 1H), 2.41 (d, J = 7.2 Hz, 2H), 2.15 (d, J = 17.6 Hz, 1H),

0.98 (s, 3H), 0.93 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₀H₂₈ClN₂O₄, 515.1732; found, 515.175. Retention time = 3.8 min.

N-(4-Chlorobenzyl)-1-(2,5-dimethoxyphenyl)-7,7-dimethyl-2,5-

dioxo-1,2,5,6,7,8-hexahydroquinoline-3-carboxamide (26). This

compound was prepared using Method 10 using N-(4-chloroben-

zyl)-2-cyanoacetamide in step 2 to aﬀord N-(4-chlorobenzyl)-1-(2,5-

dimethoxyphenyl)-7,7-dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydroqui-

noline-3-carboxamide as a TFA salt. LCMS: m/z (M + H)⁺= 495.2;

¹H NMR (400 MHz, DMSO-d₆): δ 9.55 (t, J = 6.0 Hz, 1H), 8.79 (s,

Step 3: To a solution of acetophenone (0.026 mL, 0.23 mmol) in

THF (1 mL) that had been cooled to −78 °C was added a solution of

LiHMDS (1 M THF, 0.225 mL, 0.225 mmol) slowly. The reaction

mixture was continued to stir at this temperature for 1 h (faint yellow

solution) at which point a solution of 1-(2,5-dimethoxyphenyl)-7,7-

dimethyl-2,5-dioxo-1,2,5,6,7,8-hexahydroquinoline-3-carbonyl chlor-

ide (VI) (0.075 mmol) in THF (1.5 mL) was added. The reaction

mixture became more yellow and was allowed to warm slowly for 1.5

h. The reaction mixture turned from yellow to red (likely red is

doubly deprotonated trione). Step 4: Hydrazine (3 equiv) in ethanol

was added and stirring resumed for 1 h. Acetic acid (3 drops) was

added, and the reaction mixture turned from red to yellow along with

the formation of a precipitate. The reaction mixture was heated at 50

°C for 1 h and stood at rt for 1 week. The crude mixture was diluted

with DMSO and puriﬁed by reverse-phase chromatography (Stand-

ard Acidic Gradient Method) to aﬀord as a TFA salt, 1-(2,5-

dimethoxyphenyl)-7,7-dimethyl-3-(5-phenyl-1H-pyrazol-3-yl)-7,8-di-

hydroquinoline-2,5(1H,6H)-dione. LCMS: m/z (M + H)⁺= 470.2;

¹H NMR (400 MHz, DMSO-d₆): δ 13.14 (s, 1H), 8.48 (s, 1H), 7.81

1H), 7.40−7.29 (m, 2H), 7.30 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 9.1

Hz, 1H), 7.09 (dd, J = 9.1, 3.1 Hz, 1H), 6.99 (d, J = 3.0 Hz, 1H),

4.54−4.38 (m, 2H), 3.72 (s, 3H), 3.69 (s, 3H), 2.59 (d, J = 17.8 Hz,

1H), 2.42 (d, J = 2.2 Hz, 2H), 2.17 (d, J = 17.8 Hz, 1H), 0.96 (s, 3H),

0.90 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₇H₂₈ClN₂O₅,

495.1681; found, 495.1691. Retention time = 3.467 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-phenyl-7,8-di-

hydroquinoline-2,5 (1H,6H)-dione (27). This compound was

prepared using Method 10 using aniline in step 3 to aﬀord N4-(4-

((1H-tetrazol-5-yl)methoxy)-3-chlorophenyl)-N6-([1,1′-biphenyl]-4-

yl)pyrimidine-4,6-diamine as a TFA salt. LCMS: m/z (M + H)⁺=

1

461; H NMR (400 MHz, DMSO-d₆): δ 9.06 (s, 1H), 8.22 (s, 1H),

8.13−8.05 (m, 2H), 7.67−7.51 (m, 5H), 7.47−7.40 (m, 2H), 2.45 (s,

2H), 2.42 (s, 2H), 0.96 (s, 6H); HRMS (ESI): m/z (M + H)⁺calcd

for C₂₆H₂₂ClN₂O₂S, 461.1085; found, 461.1086. Retention time =

3.853 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-methoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (28). This com-

pound was prepared using Method 10 using 2-methoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-methoxy-

(d, J = 7.4 Hz, 1H), 7.74−7.66 (m, 1H), 7.45−7.31 (m, 1H), 7.27

4932

https://doi.org/10.1021/acs.jmedchem.1c00019

J. Med. Chem. 2021, 64, 4913−4946

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

phenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

TFA salt. LCMS: m/z (M + H)⁺= 491; ¹H NMR (400 MHz,

DMSO-d₆): δ 9.05 (s, 1H), 8.22 (s, 1H), 8.13−8.04 (m, 2H), 7.61−

7.49 (m, 3H), 7.37 (dd, J = 7.7, 1.6 Hz, 1H), 7.31 (dd, J = 8.5, 1.2 Hz,

1H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 3.77 (s, 3H), 2.61−2.51 (m, 2H),

2.250−2.20 (m, 2H), 0.98 (s, 3H), 0.94 (s, 3H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₇H₂₄ClN₂O₃S, 491.1191; found, 491.1203.

Retention time = 3.86 min.

1-([1,1′-Biphenyl]-2-yl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (34). This com-

pound was prepared using Method 10 using [1,1′-biphenyl]-2-

amine in step 3 to aﬀord 1-([1,1′-biphenyl]-2-yl)-3-(4-(4-

chlorophenyl)thiazol-2-yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5-

1

(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺= 537.2; H

NMR (400 MHz, DMSO-d₆): δ 8.92 (s, 1H), 8.22 (s, 1H), 8.13−7.99

(m, 2H), 7.75−7.58 (m, 2H), 7.58−7.44 (m, 2H), 7.24 (qq, J = 7.1,

3.8, 3.0 Hz, 7H), 2.56 (d, J = 18.0 Hz, 1H), 2.40 (s, 1H), 2.18 (d, J =

16.2 Hz, 1H), 2.00 (d, J = 18.0 Hz, 1H), 0.91 (s, 3H), 0.48 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₃₂H₂₆ClN₂O₂S, 537.139;

found, 537.1398. Retention time = 4.037 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ethoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (29). This com-

pound was prepared using Method 10 using 2-ethoxyaniline in step

3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-ethoxyphenyl)-

7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-hydroxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (35). This com-

pound was prepared using Method 10 using 2-aminophenol in step

3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-hydroxyphenyl)-

7,7-dimethyl-7,8-dihydro-quinoline-2,5(1H,6H)-dione as a TFA salt.

1

LCMS: m/z (M + H)⁺= 505.1; H NMR (400 MHz, DMSO-d₆): δ

9.04 (s, 1H), 8.20 (s, 1H), 8.07 (d, J = 8.7 Hz, 2H), 7.57−7.48 (m,

3H), 7.34 (dd, J = 7.7, 1.7 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.14 (t, J

= 7.6 Hz, 1H), 4.06 (q, J = 6.9 Hz, 2H), 2.60 (d, J = 17.8 Hz, 1H),

2.50 (d, J = 16.2 Hz, 1H), 2.40 (d, J = 16.3 Hz, 1H), 2.16 (d, J = 17.7

Hz, 1H), 1.11 (t, J = 6.9 Hz, 3H), 0.97 (s, 3H), 0.93 (s, 3H); HRMS

(ESI): m/z (M + H)⁺calcd for C₂₈H₂₆ClN₂O₃S, 505.1347; found,

505.1354. Retention time = 3.927 min.

1

LCMS: m/z (M + H)⁺= 477.1; H NMR (400 MHz, DMSO-d₆): δ

10.14 (s, 1H), 9.03 (s, 1H), 8.20 (s, 1H), 8.07 (d, J = 8.4 Hz, 2H),

7.52 (d, J = 8.3 Hz, 2H), 7.41−7.33 (m, 1H), 7.24 (dd, J = 7.9, 1.6

Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 2.57 (d, J

= 17.8 Hz, 1H), 2.45 (s, 2H), 2.29 (d, J = 17.7 Hz, 1H), 0.97 (s, 3H),

0.94 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₆H₂₂ClN₂O₃S,

477.1034; found, 477.1046. Retention time = 3.642 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-isopropoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (30). This com-

pound was prepared using Method 10 using 2-isopropoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-isopropox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(2-phenoxy-

phenyl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (36). This com-

pound was prepared using Method 10 using 2-phenoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(2-

phenoxyphenyl)-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA

1

TFA salt. LCMS: m/z (M + H)⁺= 519.2; H NMR (400 MHz,

DMSO-d₆): δ 9.03 (d, J = 1.3 Hz, 1H), 8.19 (d, J = 1.4 Hz, 1H), 8.07

(d, J = 8.6 Hz, 2H), 7.56−7.46 (m, 3H), 7.34 (d, J = 7.7 Hz, 1H),

7.29 (d, J = 8.4 Hz, 1H), 7.12 (t, J = 7.0 Hz, 1H), 4.63 (h, J = 6.0 Hz,

1H), 2.62 (d, J = 17.7 Hz, 1H), 2.50 (d, J = 16.1 Hz, 1H), 2.40 (d, J =

16.3 Hz, 1H), 2.15 (d, J = 17.7 Hz, 1H), 1.13 (d, J = 5.9 Hz, 3H),

1.04 (d, J = 6.0 Hz, 3H), 0.97 (s, 3H), 0.93 (s, 3H); HRMS (ESI): m/

z (M + H)⁺calcd for C₂₉H₂₈ClN₂O₃S, 519.1504; found, 519.1492.

Retention time = 4.026 min.

1

salt. LCMS: m/z (M + H)⁺= 553.1; H NMR (400 MHz, DMSO-

d₆): δ 8.99 (s, 1H), 8.20 (s, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.60−7.48

(m, 4H), 7.34 (dt, J = 18.2, 7.6 Hz, 3H), 7.08 (dt, J = 7.6, 3.3 Hz,

2H), 6.98 (d, J = 8.0 Hz, 2H), 2.68 (d, J = 17.8 Hz, 1H), 2.53−2.35

(m, 3H), 1.01 (s, 3H), 0.89 (s, 3H); HRMS (ESI): m/z (M + H)⁺

calcd for C₃₂H₂₆ClN₂O₃S, 553.1347; found, 553.1347. Retention time

= 4.048 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(o-tolyl)-7,8-

dihydroquinoline-2,5(1H,6H)-dione (31). This compound was

prepared using Method 10 using o-toluidine in step 3 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(o-tolyl)-7,8-dihy-

droquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺

1-(2-(Benzyloxy)phenyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (37). This com-

pound was prepared using Method 10 using 2-(benzyloxy)aniline in

step 3 to aﬀord 1-(2-(benzyloxy)phenyl)-3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as

1

= 475.1; H NMR (400 MHz, DMSO-d₆): δ 9.07 (s, 1H), 8.21 (s,

1

a TFA salt. LCMS: m/z (M + H)⁺= 567.1; H NMR (400 MHz,

1H), 8.07 (d, J = 8.2 Hz, 2H), 7.60−7.39 (m, 5H), 7.32 (d, J = 7.6

Hz, 1H), 2.56 (d, J = 17.8 Hz, 1H), 2.46 (d, J = 8.1 Hz, 2H), 2.12 (d,

J = 17.9 Hz, 1H), 2.00 (s, 3H), 0.97 (s, 3H), 0.94 (s, 3H); HRMS

(ESI): m/z (M + H)⁺calcd for C₂₇H₂₄ClN₂O₂S, 475.1242; found,

475.1249. Retention time = 3.934 min.

DMSO-d₆): δ 9.02 (s, 1H), 8.21 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H),

7.58−7.49 (m, 3H), 7.43−7.34 (m, 2H), 7.19 (dd, J = 7.4, 3.7 Hz,

6H), 5.22−5.09 (m, 2H), 2.58 (d, J = 17.8 Hz, 1H), 2.46 (d, J = 16.1

Hz, 1H), 2.35 (d, J = 16.2 Hz, 1H), 2.17 (d, J = 17.9 Hz, 1H), 0.95 (s,

3H), 0.82 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₃H₂₈ClN₂O₃S, 567.1504; found, 567.1516. Retention time = 3.99

min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ethylphenyl)-7,7-dimeth-

yl-7,8-dihydroquinoline-2,5(1H,6H)-dione (32). This compound was

prepared using Method 10 using 2-ethylaniline in step 3 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-ethylphenyl)-7,7-dimethyl-7,8-

dihydroquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M +

1-(2-Aminophenyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (38). This com-

pound was prepared using Method 10 using benzene-1,2-diamine in

step 3 to aﬀord 1-(2-aminophenyl)-3-(4-(4-chlorophenyl)thiazol-2-

yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA

1

H)⁺= 489.2; H NMR (400 MHz, DMSO-d₆): δ 9.08 (s, 1H), 8.22

(s, 1H), 8.13−8.05 (m, 2H), 7.58−7.49 (m, 4H), 7.49−7.41 (m, 1H),

7.32 (d, J = 7.7 Hz, 1H), 2.63−2.50 (m, 1H), 2.49−2.40 (m, 2H),

2.34 (dd, J = 15.1, 7.5 Hz, 1H), 2.32−2.19 (m, 1H), 2.14 (d, J = 17.9

Hz, 1H), 1.08 (t, J = 7.6 Hz, 3H), 0.98 (s, 3H), 0.94 (s, 3H); HRMS

(ESI): m/z (M + H)⁺calcd for C₂₈H₂₆ClN₂O₂S, 489.1398; found,

489.1417. Retention time = 4.023 min.

1

salt. LCMS: m/z (M + H)⁺= 476.1; H NMR (400 MHz, DMSO-

d₆): δ 9.00 (s, 1H), 8.18 (s, 1H), 8.07 (d, J = 8.2 Hz, 2H), 7.52 (d, J =

8.2 Hz, 2H), 7.18 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.1 Hz, 1H), 6.83

(d, J = 8.1 Hz, 1H), 6.66 (t, J = 7.5 Hz, 1H), 5.30 (s, 2H), 2.57 (d, J =

17.7 Hz, 1H), 2.47 (s, 1H), 2.35 (d, J = 16.3 Hz, 1H), 2.25 (d, J =

17.7 Hz, 1H), 0.96 (s, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₆H₂₃ClN₃O₂S, 476.1194; found, 476.1207. Retention time = 3.721

min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-isopropylphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (33). This com-

pound was prepared using Method 10 using 2-isopropylaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-isopropyl-

phenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(2-

(methylamino)phenyl)-7,8-dihydroquinoline-2,5(1H,6H)-dione

(39). This compound was prepared using Method 10 using N1-

methylbenzene-1,2-diamine in step 3 to aﬀord 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-1-(2-(methylamino)phenyl)-7,8-dihydro-

quinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺=

1

TFA salt. LCMS: m/z (M + H)⁺= 503.1; H NMR (400 MHz,

DMSO-d₆): δ 9.07 (s, 1H), 8.20 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H),

7.64−7.49 (m, 4H), 7.46−7.37 (m, 1H), 7.28 (d, J = 7.4 Hz, 1H),

2.63 (d, J = 17.9 Hz, 1H), 2.55−2.38 (m, 3H), 2.08 (d, J = 17.9 Hz,

1H), 1.14 (d, J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 0.97 (s, 3H),

0.92 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₉H₂₈ClN₂O₂S,

503.1555; found, 503.1558. Retention time = 4.121 min.

1

490.1; H NMR (400 MHz, DMSO-d₆): δ 9.01 (d, J = 1.5 Hz, 1H),

8.19 (d, J = 1.4 Hz, 1H), 8.12−8.02 (m, 2H), 7.59−7.47 (m, 2H),

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7.37−7.27 (m, 1H), 7.01 (dd, J = 7.7, 1.6 Hz, 1H), 6.80−6.64 (m,

2H), 5.51 (q, J = 4.7 Hz, 1H), 2.69−2.51 (m, 5H), 2.40−2.30 (m,

1H), 2.13 (d, J = 17.7 Hz, 1H), 0.94 (d, J = 7.9 Hz, 6H); HRMS

(ESI): m/z (M + H)⁺calcd for C₂₇H₂₅ClN₃O₂S, 490.1351; found,

490.136. Retention time = 3.845 min.

droquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M +

Na)⁺= 545.1; HRMS (ESI): m/z (M + H)⁺calcd for

C₂₇H₂₀ClF₃N₂NaO₃S, 567.0727; found, 567.0755. Retention time =

3.96 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(pyridin-2-yl)-

7,8-dihydroquinoline-2,5(1H,6H)-dione (46). This compound was

prepared using Method 10 using pyridin-2-amine in step 3 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(pyridin-2-yl)-7,8-di-

hydroquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M +

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-(dimethylamino)-

phenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (40).

This compound was prepared using Method 10 using N1,N1-

dimethylbenzene-1,2-diamine in step 3 to aﬀord 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2-(dimethylamino)phenyl)-7,7-dimeth-

yl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z

1

H)⁺= 462.1; H NMR (400 MHz, DMSO-d₆): δ 9.07 (s, 1H), 8.72

(ddd, J = 4.9, 1.9, 0.8 Hz, 1H), 8.24 (s, 1H), 8.16 (td, J = 7.7, 1.9 Hz,

1H), 8.12−8.06 (m, 2H), 7.73−7.62 (m, 2H), 7.58−7.50 (m, 2H),

2.60 (d, J = 17.8 Hz, 1H), 2.47 (s, 2H), 2.12 (d, J = 17.8 Hz, 1H),

0.98 (s, 3H), 0.96 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₅H₂₁ClN₃O₂S, 462.1038; found, 462.1057. Retention time = 3.723

min.

1

(M + H)⁺= 504.2; H NMR (400 MHz, DMSO-d₆): δ 9.06 (d, J =

1.4 Hz, 1H), 8.21 (d, J = 1.5 Hz, 1H), 8.13−8.02 (m, 2H), 7.57−7.42

(m, 2H), 7.24 (ddd, J = 7.7, 5.0, 1.5 Hz, 3H), 7.15 (td, J = 7.5, 1.4 Hz,

1H), 2.52 (d, J = 1.4 Hz, 7H), 2.44 (s, 1H), 2.33−2.26 (m, 2H), 0.94

(d, J = 3.8 Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₈H₂₇ClN₃O₂S, 504.1507; found, 504.1516. Retention time = 4.019

min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-methoxybenzyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (47). This com-

pound was prepared using Method 10 using (2-methoxyphenyl)-

methanamine in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-

(2-methoxybenzyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-

2-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-

5,6,7,8-tetrahydro-quinolin-1(2H)-yl)benzoic Acid (41). This com-

pound was prepared using Method 10 using 2-aminobenzoic acid in

step 3 to aﬀord 2-(3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-dimethyl-

2,5-dioxo-5,6,7,8-tetrahydroquinolin-1(2H)-yl)benzoic acid as a TFA

1

dione as a TFA salt. LCMS: m/z (M + H)⁺= 505.1; H NMR (400

MHz, DMSO-d₆): δ 9.03 (s, 1H), 8.20 (s, 1H), 8.07 (d, J = 8.6 Hz,

2H), 7.52 (d, J = 8.6 Hz, 2H), 7.27 (t, J = 7.9 Hz, 1H), 7.08 (d, J = 8.3

Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 5.39 (s,

2H), 3.88 (s, 3H), 2.85 (s, 2H), 2.46 (d, J = 11.6 Hz, 2H), 0.94 (s,

6H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₆ClN₂O₃S,

505.1347; found, 505.1334. Retention time = 3.997 min.

1

salt. LCMS: m/z (M + H)⁺= 505.1; H NMR (400 MHz, DMSO-

d₆): δ 13.21 (s, 1H), 9.04 (s, 1H), 8.19 (s, 1H), 8.13 (d, J = 7.5 Hz,

1H), 8.07 (d, J = 8.6 Hz, 2H), 7.80 (s, 1H), 7.68 (t, J = 7.5 Hz, 1H),

7.52 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 7.6 Hz, 1H), 2.66−2.55 (m,

1H), 2.41−2.27 (m, 2H), 2.14 (d, J = 17.7 Hz, 1H), 0.97 (s, 3H),

0.89 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₇H₂₂ClN₂O₄S,

505.0983; found, 505.1001. Retention time = 3.523 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ﬂuorophenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (42). This com-

pound was prepared using Method 10 using 2-ﬂuoroaniline in step

3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-ﬂuorophenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt.

LCMS: m/z (M + H)⁺= 479.1; H NMR (400 MHz, DMSO-d₆):

1

δ 9.06 (s, 1H), 8.23 (s, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.71−7.49 (m,

5H), 7.49−7.42 (m, 1H), 2.61 (d, J = 17.8 Hz, 1H), 2.48 (d, J = 2.0

Hz, 2H), 2.31 (d, J = 17.7 Hz, 1H), 0.99 (s, 3H), 0.94 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₆H₂₁ClFN₂O₂S, 479.0991;

found, 479.0991. Retention time = 3.885 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(1-phenyleth-

yl)-7,8-dihydroquinoline-2,5(1H,6H)-dione (48). Step 1 1-((4,4-

Dimethyl-2,6-dioxocyclohexylidene)methyl)urea: To a solution of

triethyl orthoformate (0.89 mL, 5.35 mmol) and urea (214 mg, 3.57

mmol) in DMF (1.5 mL) was added isopropanol (10 mL). The

resulting solution was heated at 80 °C for 2 h and cooled to 0 °C. A

white precipitate formed and was removed by ﬁltration (washed with

water and hexanes). 1-((4,4-Dimethyl-2,6-dioxocyclohexylidene)-

methyl)urea was isolated as a white solid (480 mg, 64%); LCMS:

m/z (M + H)⁺= 211.1.

1-(2-Chlorophenyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (43). This com-

pound was prepared using Method 10 using 2-chloroaniline in step

3 to aﬀord 1-(2-chlorophenyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-

7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt.

1

LCMS: m/z (M + H)⁺= 495.1; H NMR (400 MHz, DMSO-d₆): δ

9.07 (s, 1H), 8.23 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.83−7.76 (m,

1H), 7.63 (dd, J = 3.4, 1.9 Hz, 3H), 7.53 (d, J = 8.3 Hz, 2H), 2.60 (d,

J = 17.8 Hz, 1H), 2.47 (s, 2H), 2.12 (d, J = 17.8 Hz, 1H), 0.98 (s,

3H), 0.95 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₆H₂₁Cl₂N₂O₂S, 495.0695; found, 495.0716. Retention time = 3.956

min.

Step 2 3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-7,8-dihy-

dro-2H-chromene-2,5(6H)-dione: A mixture of 1-((4,4-dimethyl-2,6-

dioxocyclohexylidene)methyl)urea (40 mg, 0.19 mmol), nitrile 1 (54

mg, 0.23 mmol), and a solution of benzyltrimethylammonium

hydroxide (40% in MeOH, 0.113 mL, 0.285 mmol) in DMF/

MeOH (1:1−1 mL) was heated at 140 °C for 1 h 20 min. Upon

cooling, the mixture was diluted with water, acidiﬁed at 0 °C with 1 N

HCl, stirred overnight, and ﬁltered to aﬀord a brown solid (3-(4-(4-

chlorophenyl)thiazol-2-yl)-7,7-dimethyl-7,8-dihydro-2H-chromene-

2,5(6H)-dione, 61 mg, 91%); LCMS: m/z (M + H)⁺= 386.0.

Step 3 (48): A solution of 3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-

dimethyl-7,8-dihydro-2H-chromene-2,5(6H)-dione (34 mg, 0.088

mmol) and 1-phenylethan-1-amine (20.6 mg, 0.17 mmol) in DMF

(1.0 mL) was heated at 150 °C for 2 h. The crude mixture was diluted

with DMSO and puriﬁed by reverse-phase chromatography (Stand-

ard Acidic Gradient Method) to aﬀord as a TFA salt, 3-(4-(4-

chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(1-phenylethyl)-7,8-dihy-

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(2-

(triﬂuoromethyl)phenyl)-7,8-dihydroquinoline-2,5(1H,6H)-dione

(44). This compound was prepared using Method 10 using 2-

(triﬂuoromethyl)aniline in step 3 to aﬀord 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-1-(2-(triﬂuoromethyl)phenyl)-7,8-dihydro-

quinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺=

1

529.1; H NMR (400 MHz, DMSO-d₆): δ 9.05 (d, J = 1.5 Hz, 1H),

8.23 (d, J = 1.6 Hz, 1H), 8.10−8.01 (m, 3H), 7.97 (t, J = 7.8 Hz, 1H),

7.84 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.57−7.48 (m, 2H),

2.70−2.55 (m, 2H), 2.42−2.27 (m, 1H), 2.10 (d, J = 17.9 Hz, 1H),

0.98 (s, 3H), 0.90 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₇H₂₁ClF₃N₂O₂S, 529.0959; found, 529.0976. Retention time =

3.936 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-1-(2-

(triﬂuoromethoxy)phenyl)-7,8-dihydroquinoline-2,5(1H,6H)-dione

(45). This compound was prepared using Method 10 using 2-

(triﬂuoromethoxy)aniline in step 3 to aﬀord 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-1-(2-(triﬂuoromethoxy)phenyl)-7,8-dihy-

1

droquinoline-2,5(1H,6H)-dione. LCMS: m/z (M + H)⁺= 489.1; H

NMR (400 MHz, DMSO-d₆): δ 9.00 (s, 1H), 8.19 (s, 1H), 8.09−8.02

(m, 2H), 7.55−7.48 (m, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.30−7.19 (m,

3H), 3.06 (m, 1H), 2.69−2.51 (m, 2H), 2.13 (d, J = 17.7 Hz,

2H),1.93 (d, J = 6.9 Hz, 3H), 0.95 (s, 3, H), 0.94 (s, 3H); HRMS

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(ESI): m/z (M + H)⁺calcd for C₂₈H₂₆ClN₂O₂S, 489.1398; found,

489.1375. Retention time = 4.042 min.

pound was prepared using Method 10 using 2,4-dimethoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,4-dimethox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

1

TFA salt. LCMS: m/z (M + H)⁺= 521.13; H NMR (400 MHz,

CDCl₃): δ 9.33 (s, 1H), 8.03−7.96 (m, 2H), 7.56 (s, 1H), 7.42 (dd, J

= 8.8, 2.2 Hz, 2H), 7.11 (d, J = 9.1 Hz, 1H), 6.70−6.64 (m, 2H), 3.90

(s, 3H), 3.78 (s, 3H), 2.55−2.45 (m, 3H), 2.34 (d, J = 17.9 Hz, 1H),

1.06 (d, J = 6.4 Hz, 6H). Aliphatic region complicated signiﬁcantly by

amide rotamers; HRMS (ESI): m/z (M + H)⁺calcd for

C₂₈H₂₆ClN₂O₄S, 521.1296; found, 521.1308. Retention time =

3.882 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-isopropyl-7,7-dimethyl-7,8-

dihydroquinoline-2,5(1H,6H)-dione (49). This compound was

prepared according to the method used to make compound 48

using isopropylamine in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-

2-yl)-1-isopropyl-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (55). This com-

pound was prepared using Method 10 using 2,6-dimethoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-dimethox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

1

dione as a TFA salt. LCMS: m/z (M + H)⁺= 427.1; H NMR (400

MHz, DMSO-d₆): δ 8.94 (s, 1H), 8.21 (s, 1H), 8.12−8.03 (m, 2H),

7.57−7.48 (m, 2H), 4.81 (br s, 1H), 3.08 (s, 2H), 2.45 (s, 2H), 1.60

(d, J = 6.7 Hz, 6H), 1.08 (s, 6H); HRMS (ESI): m/z (M + H)⁺calcd

for C₂₃H₂₄ClN₂O₂S, 427.1242; found, 427.1242. Retention time =

3.952 min.

1

TFA salt. LCMS: m/z (M + H)⁺= 521.13; H NMR (400 MHz,

DMSO-d₆): δ 9.05 (s, 1H), 8.22 (s, 1H), 8.13−8.04 (m, 2H), 7.58−

7.49 (m, 3H), 6.91 (d, J = 8.6 Hz, 2H), 3.75 (s, 6H), 2.52−2.46 (m,

3H), 2.35 (d, J = 17.9 Hz, 1H), 0.96 (s, 6H); HRMS (ESI): m/z (M

+ H)⁺calcd for C₂₈H₂₆ClN₂O₄S, 521.1296; found, 521.1318.

Retention time = 3.864 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-cyclohexyl-7,7-dimethyl-

7,8-dihydroquinoline-2,5(1H,6H)-dione (50). This compound was

prepared using Method 10 using cyclohexanamine in step 3 to aﬀord

3-(4-(4-chlorophenyl)thiazol-2-yl)-1-cyclohexyl-7,7-dimethyl-7,8-di-

hydroquinoline-2,5(1H,6H)-dione as a TFA salt. LCMS: m/z (M +

3-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-

5,6,7,8-tetrahydroquinolin-1(2H)-yl)-2-methoxybenzoic Acid (56).

This compound was prepared using Method 10 using 3-amino-2-

methoxybenzoic acid in step 3 to aﬀord 3-(3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydroquinolin-

1(2H)-yl)-2-methoxybenzoic acid as a TFA salt. LCMS: m/z (M +

H)⁺= 535.1; ¹H NMR (400 MHz, DMSO-d₆): δ 13.33 (s, 1H), 9.08

(s, 1H), 8.24 (s, 1H), 8.13−8.04 (m, 2H), 7.93 (dd, J = 7.8, 1.8 Hz,

1H), 7.64 (d, J = 7.4 Hz, 1H), 7.58−7.49 (m, 2H), 7.42 (t, J = 7.8 Hz,

1H), 3.62 (s, 3H), 2.57 (d, J = 17.8 Hz, 1H), 2.53 (s, 1H), 2.46−2.40

(m, 1H), 2.32 (d, J = 17.8 Hz, 1H), 0.99 (s, 3H), 0.96 (s, 3H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₄ClN₂O₅S, 535.1089;

found, 535.1078. Retention time = 3.604 min.

4-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-

5,6,7,8-tetrahydroquinolin-1(2H)-yl)-3-methoxybenzoic Acid (57).

This compound was prepared using Method 10 using 4-amino-3-

methoxybenzoic acid in step 3 to aﬀord 4-(3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydroquinolin-

1(2H)-yl)-3-methoxybenzoic acid as a TFA salt. LCMS: m/z (M +

H)⁺= 535.1; ¹H NMR (400 MHz, DMSO-d₆): δ 13.37 (s, 1H), 9.06

(s, 1H), 8.23 (s, 1H), 8.12−8.05 (m, 2H), 7.75 (d, J = 7.8 Hz, 2H),

7.53 (dd, J = 8.2, 6.3 Hz, 3H), 3.84 (s, 3H), 2.62−2.50 (m, 2H),

2.49−2.40 (m, 1H), 2.22 (d, J = 17.7 Hz, 1H), 0.99 (s, 3H), 0.94 (s,

3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₄ClN₂O₅S,

535.1089; found, 535.1099. Retention time = 3.643 min.

3-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-

5,6,7,8-tetrahydroquinolin-1(2H)-yl)-4-methoxybenzoic Acid (58).

This compound was prepared using Method 10 using 3-amino-4-

methoxybenzoic acid in step 3 to aﬀord 3-(3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydroquinolin-

1(2H)-yl)-4-methoxybenzoic acid as a TFA salt. LCMS: m/z (M +

H)⁺= 535.1; ¹H NMR (400 MHz, DMSO-d₆): δ 13.01 (s, 1H), 9.05

(s, 1H), 8.23 (s, 1H), 8.15 (dd, J = 8.7, 2.2 Hz, 1H), 8.12−8.05 (m,

2H), 7.94 (d, J = 2.2 Hz, 1H), 7.57−7.51 (m, 2H), 7.41 (d, J = 8.8

Hz, 1H), 3.86 (s, 3H), 2.62 (d, J = 17.9 Hz, 1H), 2.55−2.49 (m, 1H),

2.47−2.39 (m, 1H), 2.19 (d, J = 17.8 Hz, 1H), 0.99 (s, 3H), 0.94 (s,

3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₄ClN₂O₅S,

535.1089; found, 535.1112. Retention time = 3.596 min.

2-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-

5,6,7,8-tetrahydroquinolin-1(2H)-yl)-3-methoxybenzoic Acid (59).

This compound was prepared using Method 10 using 2-amino-3-

methoxybenzoic acid in step 3 to aﬀord 2-(3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydroquinolin-

1(2H)-yl)-3-methoxybenzoic acid as a TFA salt. LCMS: m/z (M +

H)⁺= 535.1; ¹H NMR (400 MHz, DMSO-d₆): δ 13.19 (s, 1H), 9.05

(s, 1H), 8.21 (s, 1H), 8.12−8.04 (m, 2H), 7.76−7.65 (m, 2H), 7.61−

7.50 (m, 3H), 3.81 (s, 3H), 2.58−2.49 (m, 2H), 2.37 (d, J = 2.8 Hz,

2H), 0.97 (s, 3H), 0.94 (s, 3H); HRMS (ESI): m/z (M + H)⁺calcd

1

H)⁺= 467.2; H NMR (400 MHz, DMSO-d₆): δ 8.93 (s, 1H), 8.19

(s, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 4.35−4.20

(m, 1H), 2.73−2.66 (m, 3H), 2.54−2.39 (m, 2H), 1.81 (d, J = 12.8

Hz, 2H), 1.68 (t, J = 14.3 Hz, 3H), 1.43 (d, J = 13.2 Hz, 2H), 1.22 (d,

J = 9.9 Hz, 2H), 1.07 (s, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₆H₂₈ClN₂O₂S, 467.1555; found, 467.1555. Retention time = 4.193

min.

Methyl 2-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-

dioxo-5,6,7,8-tetrahydro-quinolin-1(2H)-yl)thiophene-3-carboxy-

late (51). This compound was prepared using Method 10 using

methyl 2-aminothiophene-3-carboxylate in step 3 to aﬀord methyl 2-

(3-(4-(4-chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-5,6,7,8-

tetrahydroquinolin-1(2H)-yl)thiophene-3-carboxylate as a TFA salt.

1

LCMS: m/z (M + H)⁺= 525.07; H NMR (400 MHz, CDCl₃): δ

9.34 (s, 1H), 8.02−7.95 (m, 2H), 7.61 (d, J = 5.7 Hz, 1H), 7.58 (s,

1H), 7.50 (t, J = 6.0 Hz, 1H), 7.45−7.39 (m, 2H), 3.71 (s, 3H),

2.61−2.45 (m, 4H), 1.08 (d, J = 9.4 Hz, 6H). Aliphatic region

complicated signiﬁcantly by amide rotamers; HRMS (ESI): m/z (M +

H)⁺calcd for C₂₆H₂₂ClN₂O₄S₂, 525.0704; found, 525.071. Retention

time = 3.812 min.

2-(3-(4-(4-Chlorophenyl)thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-

5,6,7,8-tetrahydroquinolin-1(2H)-yl)thiophene-3-carbonitrile (52).

This compound was prepared using Method 10 using 2-amino-

thiophene-3-carbonitrile in step 3 to aﬀord 2-(3-(4-(4-chlorophenyl)-

thiazol-2-yl)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydroquinolin-

1(2H)-yl)thiophene-3-carbonitrile as a TFA salt. LCMS: m/z (M +

1

H)⁺= 492.1; H NMR (400 MHz, DMSO-d₆): δ 9.04 (s, 1H), 8.27

(s, 1H), 8.08 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 5.7 Hz, 1H), 7.65 (d, J =

5.7 Hz, 1H), 7.53 (d, J = 8.6 Hz, 2H), 2.68 (d, J = 17.8 Hz, 2H), 2.51

(m, 2H), 1.02 (s, 3H), 1.00 (s, 3H); HRMS (ESI): m/z (M + H)⁺

calcd for C₂₅H₁₉ClN₃O₂S₂, 492.0602; found, 492.0606. Retention

time = 3.776 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,3-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (53). This com-

pound was prepared using Method 10 using 2,3-dimethoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,3-dimethox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

1

TFA salt. LCMS: m/z (M + H)⁺= 521.13; H NMR (400 MHz,

CDCl₃): δ 9.36 (s, 1H), 8.00 (d, J = 7.8 Hz, 2H), 7.57 (d, J = 0.9 Hz,

1H), 7.42 (d, J = 7.7 Hz, 2H), 7.26 (s, 1H), 7.12 (d, J = 8.4 Hz, 1H),

6.81 (d, J = 7.9 Hz, 1H), 3.97 (d, J = 0.6 Hz, 3H), 3.76 (d, J = 0.9 Hz,

3H), 2.53−2.36 (m, 4H), 1.07 (t, J = 9.8 Hz, 6H). Aliphatic region

complicated signiﬁcantly by amide rotamers; HRMS (ESI): m/z (M +

H)⁺calcd for C₂₈H₂₆ClN₂O₄S, 521.1296; found, 521.1272. Retention

time = 3.908 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,4-dimethoxyphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (54). This com-

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1

for C₂₈H₂₄ClN₂O₅S, 535.1089; found, 535.1099. Retention time =

3.547 min.

dione as a TFA salt. LCMS: m/z (M + H)⁺= 503.2; H NMR (400

MHz, DMSO-d₆): δ 9.10 (s, 1H), 8.22 (s, 1H), 8.08 (d, J = 8.6 Hz,

2H), 7.53 (d, J = 8.6 Hz, 2H), 7.44 (t, J = 7.6 Hz, 1H), 7.34 (t, J = 8.0

Hz, 2H), 2.50 (d, J = 2.8 Hz, 2H), 2.32 (d, J = 14.0 Hz, 3H), 2.21−

2.10 (m, 1H), 1.96 (s, 3H), 1.16−1.01 (m, 3H), 0.95 (s, 6H); HRMS

(ESI): m/z (M + H)⁺calcd for C₂₉H₂₈ClN₂O₂S, 503.1555; found,

503.1562. Retention time = 4.101 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-methoxy-6-methylphen-

yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (60). This

compound was prepared using Method 10 using 2-methoxy-6-

methylaniline in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-

1-(2,3-dimethoxyphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5-

1

(1H,6H)-dione as a TFA salt. LCMS: m/z (M + H)⁺= 505.1; H

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (66). This com-

pound was prepared using Method 10 using 2-(4-(4-chlorophenyl)-

thiazol-2-yl)acetonitrile in step 2 and 2,6-diethylaniline in step 3 to

aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-7,7-

dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a TFA salt.

NMR (400 MHz, DMSO-d₆): δ 9.08 (s, 1H), 8.23 (s, 1H), 8.13−8.02

(m, 2H), 7.58−7.42 (m, 3H), 7.16−7.03 (m, 2H), 3.74 (s, 3H),

2.48−2.37 (m, 2H), 2.269 (m, 2H), 1.99 (s, 3H), 0.96 (s, 3H), 0.95

(s, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₆ClN₂O₃S,

505.1347; found, 505.135. Retention time = 3.963 min.

LCMS: m/z (M + H)⁺= 517.2; H NMR (400 MHz, DMSO-d₆):

1

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-methoxypyridin-3-yl)-

7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (61). This

compound was prepared using Method 10 using 2-methoxypyridin-

3-amine in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-

methoxypyridin-3-yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-

dione as a TFA salt. LCMS: m/z (M + H)⁺= 492.11; ¹H NMR (400

MHz, CD₃OD): δ 9.04 (s, 1H), 8.39−8.38 (m, 1H), 8.22 (s, 1H),

8.07 (d, J = 8.4 Hz, 2H), 7.91−7.89 (m, 1H), 7.52 (d, J = 8.4 Hz,

2H), 7.27 (d, J = 8.4 Hz, 1H), 3.86 (s, 3H), 3.66−3.53 (m, 1H),

3.47−3.32 (m, 1H), 2.57 (d, J = 20 Hz, 1H), 2.23 (d, J = 20 Hz, 1H),

1.19−0.94 (m, 6H). Aliphatic region complicated signiﬁcantly by

amide rotamers; HRMS (ESI): m/z (M + H)⁺calcd for

C₂₆H₂₃ClN₃O₃S, 492.1143; found, 492.116. Retention time = 3.836

min.

δ 9.11 (s, 1H), 8.23 (s, 1H), 8.13−8.05 (m, 2H), 7.59−7.47 (m, 3H),

7.38 (d, J = 7.7 Hz, 2H), 2.51 (s, 2H), 2.40−2.26 (m, 4H), 2.16 (dq, J

= 15.0, 7.5 Hz, 2H), 1.07 (t, J = 7.5 Hz, 6H), 0.95 (s, 6H); HRMS

(ESI): m/z (M + H)⁺calcd for C₃₀H₃₀ClN₂O₂S, 517.1711; found,

517.1699. Retention time = 4.187 min.

Method 11: Methyl 5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-

diethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate

(67). Step 1: In a vial, methyl 3-oxobutanoate (0.385 mL, 3.57 mmol)

and DMF−DMA (0.474 mL, 3.57 mmol) were mixed and heated well

at 100 °C for 15 min. The reaction mixture became red oil.

Step 2: To the mixture were added i-PrOH (40 mL), 2-(4-(4-

chlorophenyl)thiazol-2-yl)acetonitrile N1/IVa (837 mg, 3.57 mmol),

and potassium tert-butoxide (400 mg, 3.57 mmol). The reaction

mixture was allowed to stir at rt for 2 h at which point the solvent was

removed.

Step 3: To the resulting residue were added acetic acid (30 mL)

and 2,6-dimethylaniline (646 μL, 3.9 mmol). The reaction mixture

was stirred for 15 min and the mixture was diluted with water and

extracted (EtOAc × 2). The organic layers were combined (not dried

with magnesium sulfate) and concentrated.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(4-methoxypyridin-3-yl)-

7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (62). This

compound was prepared using Method 10 using 4-methoxypyridin-

3-amine in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(4-

methoxypyridin-3-yl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-

dione as a TFA salt. LCMS: m/z (M + H)⁺= 492.11; ¹H NMR (400

MHz, CD₃OD): δ 9.01 (s, 1H), 8.21−8.19 (m, 2H), 8.08−8.06 (m,

2H), 7.88−7.86 (m, 1H), 7.52 (d, J = 8.4 Hz, 2H), 6.48 (d, J = 8.4

Hz, 1H), 3.76 (s, 3H), 3.68−3.64 (m, 1H), 2.77−2.73 (m, 2H),

2.41−2.41 (m, 1H), 1.20−0.96 (m, 6H). Aliphatic region complicated

signiﬁcantly by amide rotamers; HRMS (ESI): m/z (M + H)⁺calcd

for C₂₆H₂₃ClN₃O₃S, 492.1143; found, 492.1148. Retention time =

3.133 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,5-diethoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (63). This com-

pound was prepared using Method 10 using 2,5-diethoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,5-diethox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

Step 4: The residue was taken up in DMF (40 mL) and heated at

125 °C for 1.5 h. The reaction mixture was diluted with water and

EtOAc and extracted (2×); the organic layers were combined, dried

with Na₂SO₄, and concentrated. The crude mixture was diluted with

DMSO and puriﬁed by reverse-phase chromatography (Standard

Acidic Gradient Method) to aﬀord as a TFA salt, methyl 5-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-6-oxo-1,6-

dihydropyridine-3-carboxylate (1.05 g, 60%); LCMS: m/z (M + H)⁺

1

TFA salt. LCMS: m/z (M + H)⁺= 549.16; H NMR (400 MHz,

CDCl₃): δ 9.34 (s, 1H), 8.03−7.97 (m, 2H), 7.57 (s, 1H), 7.42 (d, J =

8.5 Hz, 2H), 7.04 (d, J = 1.6 Hz, 2H), 6.78−6.75 (m, 1H), 4.10−3.95

(m, 4H), 2.50 (d, J = 18.4 Hz, 3H), 2.36 (d, J = 18.0 Hz, 1H), 1.42 (t,

J = 7.0 Hz, 3H), 1.19 (t, J = 7.0 Hz, 3H), 1.07 (t, J = 7.3 Hz, 6H).

Aliphatic region complicated signiﬁcantly by amide rotamers; HRMS

(ESI): m/z (M + H)⁺calcd for C₃₀H₃₀ClN₂O₄S, 549.1603; found,

549.1609. Retention time = 4.023 min.

1

= 493.1; H NMR (400 MHz, DMSO-d₆): δ 9.14 (s, 1H), 8.22 (s,

1H), 8.12−8.03 (m, 2H), 7.59−7.44 (m, 3H), 7.36 (d, J = 7.7 Hz,

2H), 3.89 (s, 3H), 3.31 (s, 7H), 2.61−2.50 (m, 1H), 2.47−2.34 (m,

3H), 2.33−2.07 (m, 8H), 1.05 (t, J = 7.5 Hz, 6H), 1.00 (t, J = 7.6 Hz,

0H), 0.82 (s, 1H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₇H₂₆ClN₂O₃S, 493.1347; found, 493.1338. Retention time = 4.247

min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-meth-

yl-6-oxo-1,6-dihydropyridine-3-carboxylic Acid (68). To a solution

of methyl 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-

methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (1.0 g, 2.03 mmol,

compound 67) in THF (10 mL) and MeOH (10 mL) was added

lithium hydroxide (0.168 g, 14.22 mmol) and the mixture became

yellow. The mixture was stirred for 1 h at 70 °C, concentrated with a

stream of air, and diluted with DCM. The pH of the aqueous layer

was adjusted to pH 7 using 1 N HCl, the mixture was extracted with 2

× 25 mL DCM, and the organic layers were dried with Na₂SO₄and

concentrated. The crude mixture was diluted with DMSO and

puriﬁed by reverse-phase chromatography (Standard Acidic Gra-

dient Method) to aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethoxyphenyl)-7,7-di-

methyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (64). This com-

pound was prepared using Method 10 using 2,6-diethoxyaniline in

step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethox-

yphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione as a

1

TFA salt. LCMS: m/z (M + H)⁺= 549.16; H NMR (400 MHz,

CDCl₃): δ 9.34 (s, 1H), 8.03−7.97 (m, 2H), 7.56 (s, 1H), 7.45−7.34

(m, 3H), 6.70 (dd, J = 8.5, 3.8 Hz, 2H), 4.06 (hd, J = 9.8, 7.2 Hz,

4H), 2.49 (s, 2H), 2.40 (s, 2H), 1.22 (t, J = 7.0 Hz, 6H), 1.07 (d, J =

11.2 Hz, 6H). Aliphatic region complicated signiﬁcantly by amide

rotamers; HRMS (ESI): m/z (M + H)⁺calcd for C₃₀H₃₀ClN₂O₄S,

549.161; found, 549.1609. Retention time = 4.023 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ethyl-6-methylphenyl)-

7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-dione (65). This

compound was prepared using Method 10 using 2-ethyl-6-methylani-

line in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2-ethyl-

6-methylphenyl)-7,7-dimethyl-7,8-dihydroquinoline-2,5(1H,6H)-

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diethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid;

1.20 (d, J = 6.6 Hz, 6H), 1.08 (t, J = 7.5 Hz, 6H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₉H₃₁ClN₃O₂S, 520.182; found, 520.1817.

Retention time = 3.916 min.

1

LCMS: m/z (M + H)⁺= 478.7; H NMR (400 MHz, DMSO-d₆): δ

13.33 (s, 1H), 9.17 (s, 1H), 8.19 (s, 1H), 8.11−8.02 (m, 2H), 7.57−

7.42 (m, 3H), 7.35 (d, J = 7.7 Hz, 2H), 2.34−2.19 (m, 5H), 2.14 (dq,

J = 15.1, 7.5 Hz, 2H), 1.04 (t, J = 7.6 Hz, 6H); HRMS (ESI): m/z (M

+ H)⁺calcd for C₂₆H₂₄ClN₂O₃S, 479.1191; found, 479.1186.

Retention time = 3.835 min.

N-(tert-Butyl)-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (73).

This compound was prepared using Method 12 using 2-

methylpropan-2-amine to aﬀord N-(tert-butyl)-5-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-6-oxo-1,6-

Ethyl 5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-

oxo-2-phenyl-1,6-dihydro-pyridine-3-carboxylate (69). This com-

pound was prepared using Method 11 using ethyl 3-oxo-3-

phenylpropanoate in step 1 to aﬀord ethyl 5-(4-(4-chlorophenyl)-

thiazol-2-yl)-1-(2,6-diethylphenyl)-6-oxo-2-phenyl-1,6-dihydropyri-

1

dihydropyridine-3-carboxamide. LCMS: m/z (M + H)⁺= 534.1; H

NMR (400 MHz, DMSO-d₆): δ 8.68 (s, 1H), 8.49 (d, J = 7.5 Hz,

1H), 8.20 (s, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H),

7.48 (dd, J = 8.2, 7.1 Hz, 1H), 7.37 (s, 1H), 7.35 (s, 1H), 4.06 (dq, J

= 13.4, 6.7 Hz, 1H), 2.32 (dq, J = 15.2, 7.6 Hz, 2H), 2.18 (dq, J =

15.1, 7.5 Hz, 2H), 2.00 (s, 3H), 1.20 (d, J = 6.6 Hz, 6H), 1.08 (t, J =

7.5 Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for C₃₀H₃₃ClN₃O₂S,

534.1977; found, 534.1983. Retention time = 4.032 min.

1

dine-3-carboxylate. LCMS: m/z (M + H)⁺= 569.2; H NMR (400

MHz, DMSO-d₆): δ 9.16 (s, 1H), 8.28 (s, 1H), 8.15−8.02 (m, 2H),

7.62−7.46 (m, 2H), 7.26−7.01 (m, 8H), 3.91 (q, J = 7.1 Hz, 2H),

2.60−2.48 (m, 1H), 2.47−2.15 (m, 6H), 1.04 (t, J = 7.5 Hz, 6H),

0.78 (t, J = 7.1 Hz, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₃H₃₀ClN₂O₃S, 569.166; found, 569.1643. Retention time = 4.366

min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-N-iso-

butyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (74).

This compound was prepared using Method 12 using 2-

methylpropan-1-amine to aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-

1-(2,6-diethylphenyl)-N-isobutyl-2-methyl-6-oxo-1,6-dihydropyri-

1

dine-3-carboxamide. LCMS: m/z (M + H)⁺= 534.2; H NMR (400

MHz, DMSO-d₆): δ 8.70 (s, 1H), 8.61 (t, J = 5.8 Hz, 1H), 8.18 (d, J =

0.6 Hz, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.46

(dd, J = 8.2, 7.1 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 3.08 (dd, J = 6.8,

5.8 Hz, 2H), 2.29 (dq, J = 15.2, 7.6 Hz, 2H), 2.15 (dq, J = 15.1, 7.5

Hz, 2H), 2.00 (s, 3H), 1.83 (dt, J = 13.4, 6.7 Hz, 1H), 1.08 (t, J = 7.5

Hz, 6H), 0.91 (d, J = 6.6 Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd

for C₃₀H₃₃ClN₃O₂S, 534.1977; found, 534.1974. Retention time =

4.012 min.

Method 12: 5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-N,2-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide

(70). To a solution of 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-

diethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid,

compound 68 (250 mg, 0.522 mmol), in DMF (volume: 5 mL) were

added 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyli-

souronium hexaﬂuorophosphate(V) (397 mg, 1.044 mmol), N-

ethyl-N-isopropylpropan-2-amine (365 μL, 2.088 mmol), and

methanamine (1044 μL, 2.088 mmol). The mixture became yellow

and the reaction mixture was stirred for 6 h at 60 °C, diluted with

water, extracted with 3 × 10 mL DCM, and washed with brine. The

organic layer was dried and concentrated. The crude mixture was

diluted with DMSO and puriﬁed by reverse-phase chromatography

(Standard Acidic Gradient Method) to aﬀord 5-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-N,2-dimethyl-6-oxo-

1,6-dihydropyridine-3-carboxamide; LCMS: m/z (M + H)⁺= 492.1;

¹H NMR (400 MHz, DMSO-d₆): δ 8.73 (s, 1H), 8.54 (d, J = 4.7 Hz,

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-N-(2-hy-

droxyethyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide

(75). This compound was prepared using Method 12 using 2-

aminoethan-1-ol to aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-

diethylphenyl)-N-(2-hydroxyethyl)-2-methyl-6-oxo-1,6-dihydropyri-

1

dine-3-carboxamide. LCMS: m/z (M + H)⁺= 522.1; H NMR (400

MHz, DMSO-d₆): δ 8.71 (s, 1H), 8.61 (t, J = 5.6 Hz, 1H), 8.18 (d, J =

0.6 Hz, 1H), 8.15−8.00 (m, 2H), 7.56−7.49 (m, 2H), 7.46 (dd, J =

8.2, 7.1 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 3.53 (t, J = 6.1 Hz, 2H),

3.35−3.30 (m, 2H), 3.14 (s, 1H), 2.29 (dd, J = 15.1, 7.6 Hz, 2H),

2.15 (dq, J = 15.1, 7.5 Hz, 2H), 2.00 (s, 3H), 1.08−1.02 (m, 6H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₂₉ClN₃O₃S, 522.1613;

found, 522.1627. Retention time = 3.505 min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-N-(2-

methoxyethyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxa-

mide (76). This compound was prepared using Method 12 using 2-

methoxyethan-1-amine to aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-

1-(2,6-diethylphenyl)-N-(2-methoxyethyl)-2-methyl-6-oxo-1,6-dihy-

dropyridine-3-carboxamide. LCMS: m/z (M + H)⁺= 536.1; ¹H NMR

(400 MHz, DMSO-d₆): δ 8.71 (d, J = 4.7 Hz, 2H), 8.21 (s, 1H), 8.11

(d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.48 (dd, J = 8.2, 7.1 Hz,

1H), 7.37 (s, 1H), 7.35 (s, 1H), 3.50 (td, J = 5.4, 1.3 Hz, 2H), 3.47−

3.39 (m, 2H), 3.31 (s, 3H), 2.39−2.26 (m, 2H), 2.18 (dq, J = 15.1,

7.5 Hz, 2H), 2.02 (s, 3H), 1.08 (t, J = 7.5 Hz, 6H); HRMS (ESI): m/

z (M + H)⁺calcd for C₂₉H₃₁ClN₃O₃S, 536.1769; found, 536.1774.

Retention time = 3.789 min.

1H), 8.18 (s, 1H), 8.13−8.05 (m, 2H), 7.56−7.48 (m, 2H), 7.46 (dd,

J = 8.3, 7.1 Hz, 1H), 7.34 (d, J = 7.7 Hz, 2H), 2.96 (s, 1H), 2.78 (d, J

= 4.5 Hz, 3H), 2.27 (dt, J = 15.2, 7.6 Hz, 2H), 2.14 (dq, J = 15.1, 7.5

Hz, 2H), 2.01 (s, 3H), 1.05 (t, J = 7.6 Hz, 6H); HRMS (ESI): m/z

(M + H)⁺calcd for C₂₇H₂₇ClN₃O₂S, 492.1507; found, 492.1506.

Retention time = 3.75 min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-N,N,2-

trimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (71). This

compound was prepared using Method 12 using dimethylamine to

aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-

N,N,2-trimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide. LCMS:

1

m/z (M + H)⁺= 506.1; H NMR (400 MHz, DMSO-d₆): δ 8.58

(s, 1H), 8.19 (s, 1H), 8.10 (d, J = 8.6 Hz, 2H), 7.56−7.47 (m, 2H),

7.49−7.41 (m, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 3.01 (s, 3H), 2.96 (s,

3H), 2.29 (dq, J = 15.2, 7.6 Hz, 2H), 2.16 (dq, J = 15.0, 7.5 Hz, 2H),

1.84 (s, 3H), 1.06 (t, J = 7.6 Hz, 6H); HRMS (ESI): m/z (M + H)⁺

calcd for C₂₈H₂₉ClN₃O₂S, 506.1664; found, 506.1659. Retention time

= 3.819 min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-N-iso-

propyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (72).

This compound was prepared using Method 12 using propan-2-

amine to aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-N-isopropyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxa-

mide. LCMS: m/z (M + H)⁺= 520.1; ¹H NMR (400 MHz, DMSO-

d₆): δ 8.68 (s, 1H), 8.49 (d, J = 7.5 Hz, 1H), 8.20 (s, 1H), 8.10 (d, J =

8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.48 (dd, J = 8.2, 7.1 Hz, 1H),

7.37 (s, 1H), 7.35 (s, 1H), 4.06 (dq, J = 13.4, 6.7 Hz, 1H), 2.32 (dq, J

= 15.2, 7.6 Hz, 2H), 2.18 (dq, J = 15.1, 7.5 Hz, 2H), 2.00 (s, 3H),

(5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-

methyl-6-oxo-1,6-dihydro-pyridine-3-carbonyl)glycine (77). Step 1:

Followed Method 12 using methyl glycinate.

Step 2: To a solution of the crude acid from step 1 in 1:1 THF/

MeOH (∼0.3 mM) was added lithium hydroxide (∼10 equiv) and

stirred for 1 h at 70 °C. The mixture was concentrated with a stream

of air and diluted with DCM. The pH of the aqueous layer was

adjusted to pH 7 using 1 N HCl and extracted with 2× DCM; the

organic layers were dried with Na₂SO₄and concentrated. The crude

mixture was diluted with DMSO and puriﬁed by reverse-phase

chromatography (Standard Acidic Gradient Method) to aﬀord

compound 77. LCMS: m/z (M + H)⁺= 536.1; ¹H NMR (400 MHz,

DMSO-d₆): δ 12.69 (s, 1H), 9.00 (t, J = 5.9 Hz, 1H), 8.77 (s, 1H),

8.21 (d, J = 0.6 Hz, 1H), 8.14−8.07 (m, 2H), 7.57−7.52 (m, 2H),

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7.49 (dd, J = 8.2, 7.2 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 3.96 (d, J =

5.9 Hz, 2H), 2.32 (dq, J = 15.2, 7.6 Hz, 2H), 2.18 (dq, J = 15.1, 7.5

Hz, 2H), 2.08 (s, 3H), 1.08 (td, J = 7.6, 0.6 Hz, 6H); HRMS (ESI):

m/z (M + H)⁺calcd for C₂₈H₂₇ClN₃O₄S, 536.1405; found, 536.1417.

Retention time = 3.491 min.

2H), 7.57−7.45 (m, 3H), 7.43−7.32 (m, 4H), 7.18−7.09 (m, 1H),

2.42−2.28 (m, 2H), 2.21 (dq, J = 15.1, 7.5 Hz, 2H), 2.09 (s, 3H),

1.10 (t, J = 7.5 Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₂H₂₉ClN₃O₂S, 554.1664; found, 554.1682. Retention time = 4.043

min.

N-(2-Aminoethyl)-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-di-

ethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide

(78). This compound was prepared using Method 12 using ethane-

1,2-diamine to aﬀord N-(2-aminoethyl)-5-(4-(4-chlorophenyl)thiazol-

2-yl)-1-(2,6-diethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-

carboxamide as a TFA salt. LCMS: m/z (M + H)⁺= 521.1; ¹H NMR

(400 MHz, DMSO-d₆): δ 8.86 (s, 1H), 8.77 (t, J = 5.6 Hz, 1H), 8.23

(d, J = 0.6 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 7.81 (s, 2H), 7.55 (d, J =

8.4 Hz, 2H), 7.53−7.45 (m, 1H), 7.38 (s, 1H), 7.36 (s, 1H), 3.51 (q, J

= 6.1 Hz, 2H), 3.04 (q, J = 5.9 Hz, 2H), 2.31 (dd, J = 15.2, 7.5 Hz,

2H), 2.17 (dq, J = 15.1, 7.5 Hz, 2H), 2.08 (s, 3H), 1.13−1.04 (m,

6H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₈H₃₀ClN₄O₂S,

517.1711; found, 521.1773. Retention time = 2.669 min.

N-Benzyl-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphen-

yl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (83). This

compound was prepared using Method 12 using phenylmethanamine

to aﬀord N-benzyl-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide. LCMS:

1

m/z (M + H)⁺= 567.7; H NMR (400 MHz, DMSO-d₆): δ 9.13

(t, J = 5.9 Hz, 1H), 8.77 (s, 1H), 8.18 (s, 1H), 8.12−8.04 (m, 2H),

7.57−7.41 (m, 3H), 7.41−7.21 (m, 7H), 4.48 (d, J = 5.9 Hz, 2H),

2.22 (ddq, J = 56.8, 15.1, 7.5 Hz, 4H), 2.02 (s, 3H), 1.05 (t, J = 7.5

Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for C₃₃H₃₁ClN₃O₂S,

568.182; found, 568.1842. Retention time = 3.966 min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-N-cyclohexyl-1-(2,6-diethyl-

phenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (84).

This compound was prepared using Method 12 using cyclohexan-

amine to aﬀord 5-(4-(4-chlorophenyl)thiazol-2-yl)-N-cyclohexyl-1-

(2,6-diethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxa-

mide. LCMS: m/z (M + H)⁺= 567.7; ¹H NMR (400 MHz, DMSO-

d₆): δ 8.64 (s, 1H), 8.46 (d, J = 7.7 Hz, 1H), 8.17 (s, 1H), 8.11−8.04

(m, 2H), 7.56−7.41 (m, 3H), 7.33 (d, J = 7.7 Hz, 2H), 3.72 (dd, J =

7.4, 3.6 Hz, 0H), 2.30 (dq, J = 15.2, 7.6 Hz, 2H), 2.15 (dq, J = 15.1,

7.5 Hz, 2H), 1.97 (s, 3H), 1.88 (d, J = 9.6 Hz, 2H), 1.71 (s, 2H), 1.58

(d, J = 13.0 Hz, 1H), 1.35−1.22 (m, 4H), 1.05 (t, J = 7.5 Hz, 6H);

HRMS (ESI): m/z (M + H)⁺calcd for C₃₂H₃₅ClN₃O₂S, 560.2133;

found, 560.2134. Retention time = 4.151 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-meth-

yl-5-(piperidine-1-carbonyl)pyridin-2(1H)-one (85). This compound

was prepared using Method 12 using piperidine to aﬀord 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-methyl-5-(piperi-

dine-1-carbonyl)pyridin-2(1H)-one. LCMS: m/z (M + H)⁺= 546.1;

¹H NMR (400 MHz, DMSO-d₆): δ 8.58 (d, J = 0.8 Hz, 1H), 8.22 (d,

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-meth-

yl-N-(2-(methylamino)-ethyl)-6-oxo-1,6-dihydropyridine-3-carbox-

amide (79). This compound was prepared using Method 12 using

tert-butyl(2-aminoethyl) (methyl)carbamate to aﬀord tert-butyl(2-(5-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-6-

oxo-1,6-dihydro-pyridine-3-carboxamido)ethyl)(methyl)carbamate

followed by Boc deprotection using TFA in DCM to aﬀord 5-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-N-(2-

(methylamino)ethyl)-6-oxo-1,6-dihydropyridine-3-carboxamide as a

1

TFA salt. LCMS: m/z (M + H)⁺= 535.1; H NMR (400 MHz,

DMSO-d₆): δ 8.86 (s, 1H), 8.83 (t, J = 5.7 Hz, 1H), 8.44 (s, 1H),

8.23 (s, 1H), 8.12 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.54−

7.45 (m, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 3.55 (q, J = 6.0 Hz, 2H),

3.14 (s, 2H), 2.70−2.63 (m, 3H), 2.31 (dq, J = 15.2, 7.6 Hz, 2H),

2.17 (dq, J = 15.1, 7.5 Hz, 2H), 2.09 (s, 3H), 1.08 (t, J = 7.5 Hz, 6H);

HRMS (ESI): m/z (M + H)⁺calcd for C₂₉H₃₂ClN₄O₂S, 535.1929;

found, 535.1938. Retention time = 2.712 min.

N-(2-Amino-2-oxoethyl)-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-

(2,6-diethylphenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carbox-

amide (80). This compound was prepared using Method 12 using 2-

aminoacetamide to aﬀord N-(2-amino-2-oxoethyl)-5-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-6-oxo-1,6-

J = 0.9 Hz, 1H), 8.17−8.06 (m, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.51−

7.46 (m, 1H), 7.38 (s, 1H), 7.36 (s, 1H), 3.65 (s, 2H), 3.45 (s, 4H),

2.39−2.15 (m, 4H), 1.88 (d, J = 0.8 Hz, 3H), 1.63 (s, 4H), 1.12−1.05

(m, 6H); HRMS (ESI): m/z (M + H)⁺calcd for C₃₁H₃₃ClN₃O₂S,

546.1977; found, 546.1998. Retention time = 4.064 min.

1

dihydropyridine-3-carboxamide. LCMS: m/z (M + H)⁺= 535.1; H

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-meth-

yl-5-(morpholine-4-carbonyl)pyridin-2(1H)-one (86). This com-

pound was prepared using Method 12 using morpholine to aﬀord

3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-methyl-5-

(morpholine-4-carbonyl)pyridin-2(1H)-one. LCMS: m/z (M + H)⁺=

548.1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.60 (s, 1H), 8.19 (s, 1H),

8.10 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.48−7.43 (m,

1H), 7.35 (s, 1H), 7.33 (s, 1H), 3.62 (m, 8H), 2.36−2.12 (m, 4H),

1.86 (d, J = 1.6 Hz, 3H), 1.11−1.01 (m, 6H); HRMS (ESI): m/z (M

+ Na)⁺calcd for C₃₀H₃₀ClN₃NaO₃S, 570.1589; found, 570.1605.

Retention time = 3.787 min.

NMR (400 MHz, DMSO-d₆): δ 8.80 (d, J = 7.6 Hz, 2H), 8.21 (s,

1H), 8.11 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.6 Hz, 1H), 7.51−7.43

(m, 2H), 7.36 (d, J = 7.7 Hz, 2H), 7.06 (s, 2H), 3.84 (d, J = 5.8 Hz,

2H), 2.32 (dq, J = 15.2, 7.6 Hz, 2H), 2.18 (dq, J = 15.2, 7.5 Hz, 2H),

2.08 (s, 3H), 1.08 (t, J = 7.5 Hz, 6H); HRMS (ESI): m/z (M + H)⁺

calcd for C₂₈H₂₈ClN₄O₃S, 535.1565; found, 535.155. Retention time

= 3.378 min.

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-meth-

yl-N-(3-(methylamino)propyl)-6-oxo-1,6-dihydropyridine-3-car-

boxamide (81). This compound was prepared using Method 12 using

tert-butyl (3-aminopropyl)(methyl)carbamate followed by boc

deprotection using TFA in DCM to aﬀord 5-(4-(4-chlorophenyl)-

thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-N-(3-(methylamino)-

propyl)-6-oxo-1,6-dihydropyridine-3-carboxamide as a TFA salt.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-meth-

yl-5-(piperazine-1-carbonyl)pyridin-2(1H)-one (87). This compound

was prepared using Method 12 using piperazine to aﬀord 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-methyl-5-(pipera-

zine-1-carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z (M +

1

LCMS: m/z (M + H)⁺= 549.1; H NMR (400 MHz, DMSO-d₆):

δ 8.76 (d, J = 2.8 Hz, 2H), 8.34 (s, 1H), 8.23 (s, 1H), 8.11 (d, J = 8.5

Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.52−7.46 (m, 1H), 7.38 (s, 1H),

7.36 (s, 1H), 3.34 (q, J = 6.5, 6.1 Hz, 2H), 2.99 (p, J = 6.6 Hz, 2H),

2.60 (t, J = 5.4 Hz, 3H), 2.31 (dq, J = 15.2, 7.6 Hz, 2H), 2.17 (dq, J =

15.1, 7.5 Hz, 2H), 2.05 (s, 3H), 1.86 (p, J = 7.0 Hz, 2H), 1.08 (t, J =

7.5 Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₀H₃₃ClN₄NaO₂S, 571.1905; found, 571.1919. Retention time =

2.708 min.

1

H)⁺= 547.1; H NMR (400 MHz, DMSO-d₆): δ 8.56 (s, 1H), 8.19

(s, 1H), 8.16−8.03 (m, 2H), 7.57−7.36 (m, 3H), 7.34 (d, J = 7.7 Hz,

2H), 3.57 (s, 2H), 3.28 (s, 4H), 2.70 (d, J = 14.8 Hz, 1H), 2.52 (p, J =

1.9 Hz, 0H), 2.26 (dt, J = 15.2, 7.4 Hz, 2H), 2.17 (s, 3H), 1.85 (s,

3H), 1.06 (t, J = 7.5 Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₀H₃₂ClN₄O₂S, 547.1929; found, 547.1942. Retention time = 2.691

min.

N-Benzyl-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphen-

yl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (88). This

compound was prepared using Method 12 using 1-methylpiperazine

to aﬀord N-benzyl-5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide as a TFA

5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-meth-

yl-6-oxo-N-phenyl-1,6-dihydropyridine-3-carboxamide (82). This

compound was prepared using Method 12 using aniline to aﬀord 5-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-methyl-6-

oxo-N-phenyl-1,6-dihydropyridine-3-carboxamide. LCMS: m/z (M +

H)⁺= 554.1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.63 (s, 1H), 8.88

(s, 1H), 8.23 (s, 1H), 8.12 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 7.9 Hz,

1

salt. LCMS: m/z (M + H)⁺= 561.2; H NMR (400 MHz, DMSO-

d₆): δ 8.70 (s, 1H), 8.23 (s, 1H), 8.12 (d, J = 8.6 Hz, 2H), 7.54 (d, J =

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8.6 Hz, 2H), 7.52−7.47 (m, 1H), 7.38 (s, 1H), 7.37 (s, 1H), 3.42 (m,

8H), 2.84 (s, 3H), 2.39−3.15 (m, 4H), 1.90 (s, 3H), 1.09 (t, J = 7.5

Hz, 6H); HRMS (ESI): m/z (M + H)⁺calcd for C₃₁H₃₄ClN₄O₂S,

561.2086; found, 561.2076. Retention time = 2.74 min.

Hz, 1H), 8.19−8.10 (m, 2H), 7.59−7.51 (m, 2H), 7.49 (t, J = 7.7 Hz,

1H), 7.37 (dd, J = 7.6, 4.5 Hz, 2H), 4.08 (d, J = 14.1 Hz, 1H), 3.78

(d, J = 14.7 Hz, 1H), 3.73−3.61 (m, 1H), 3.60−3.48 (m, 1H), 3.49−

3.38 (m, 1H), 3.26−3.13 (m, 1H), 3.10 (d, J = 6.0 Hz, 1H), 2.43−

2.29 (m, 2H), 2.25 (td, J = 15.0, 7.5 Hz, 1H), 2.11 (dd, J = 14.9, 7.4

Hz, 1H), 1.10 (q, J = 7.1 Hz, 14H); HRMS (ESI): m/z (M + H)⁺

calcd for C₃₂H₃₆ClN₄O₂S, 575.2242; found, 575.2225. Retention time

= 2.79 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-isobu-

tyl-5-(piperazine-1-carbonyl)pyridin-2(1H)-one (91). This com-

pound was prepared via Method 13 using methyl 5-methyl-3-

oxohexanoate in step 1 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-

1-(2,6-diethylphenyl)-6-isobutyl-5-(piperazine-1-carbonyl)pyridin-

1

2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺= 589.2; H NMR

(400 MHz, DMSO-d₆): δ 8.93 (s, 1H), 8.73 (d, J = 0.7 Hz, 1H), 8.24

(d, J = 0.7 Hz, 1H), 8.18−8.10 (m, 2H), 7.59−7.52 (m, 2H), 7.49 (d,

J = 7.6 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 4.06 (s, 1H), 3.81 (s, 1H),

3.64 (m, 2H), 3.16 (m, 5H), 2.45−1.91 (m, 3H), 1.34 (dq, J = 13.5,

6.7 Hz, 1H), 1.10 (d, J = 7.9 Hz, 8H), 0.63 (d, J = 6.5 Hz, 6H);

HRMS (ESI): m/z (M + H)⁺calcd for C₃₃H₃₈ClN₄O₂S, 589.2399;

found, 589.2381. Retention time = 1.982 min.

Method 13: 3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-5-(piperazine-1-carbonyl)-6-propylpyridin-2(1H)-one (89).

Steps 1−3: A mixture of ethyl 3-oxohexanoate (0.674 g, 4.26 mmol)

and 1,1-dimethoxy-N,N-dimethylmethanamine (0.566 mL, 4.26

mmol) was stirred for 1 h at 100 °C. The mixture was diluted with

IPA (10 mL). 2-(4-(4-Chlorophenyl)thiazol-2-yl)acetonitrile (1.00 g,

4.26 mmol) and K^tOBu (0.478 g, 4.26 mmol) were added and stirred

at 50 °C for 3 h. The solvent was removed. To the residue were added

2,6-diethylaniline (0.772 mL, 4.69 mmol) and AcOH (6.10 mL, 107

mmol) and sonicated to a homogeneous mixture. The reaction

mixture was stirred at 70 °C for 4 h, cooled to room temperature,

diluted with EtOAc, and washed with water. The organic layer was

dried, concentrated, and puriﬁed by column chromatography to aﬀord

ethyl 5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-oxo-

2-propyl-1,6-dihydro-pyridine-3-carboxylate; LCMS: m/z (M + H)⁺=

535.3.

Step 4−5: To a solution of ethyl 5-(4-(4-chlorophenyl)thiazol-2-

yl)-1-(2,6-diethylphenyl)-6-oxo-2-propyl-1,6-dihydropyridine-3-car-

boxylate (1.5 g, 2.8 mmol) in THF (10 mL) and MeOH (10 mL) was

added lithium hydroxide (0.470 g, 19.6 mmol). The reaction mixture

was stirred at 60 °C for 3 h. The solvent was removed by rotovap,

diluted with DCM, quenched with 1 N HCl, washed with brine (2 ×

50 mL), dried over MgSO₄, and concentrated to aﬀord the 5-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-oxo-2-propyl-1,6-

dihydropyridine-3-carboxylic acid; LCMS: m/z (M + H)⁺= 507.0.

Without further puriﬁcation, to the crude acid (1.00 g, 1.97 mmol)

were added 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetrame-

thylisouronium hexaﬂuorophosphate(V) (1.125 g, 2.96 mmol), DMF

(5 mL), N-ethyl-N-isopropylpropan-2-amine (0.689 mL, 3.94 mmol),

and piperazine (0.255 g, 2.96 mmol) (mixture became yellow). The

reaction mixture was stirred for 1 h at rt, diluted with water, extracted

with 3 × 10 mL of DCM, and washed with brine. The organic layer

was dried and concentrated. The crude mixture was diluted with

DMSO and puriﬁed by reverse-phase chromatography (Standard

Acidic Gradient Method) to aﬀord as a TFA salt, 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(piperazine-1-car-

bonyl)-6-propylpyridin-2(1H)-one; LCMS: m/z (M + H)⁺= 575.1;

¹H NMR (400 MHz, DMSO-d₆): δ 8.54 (d, J = 2.1 Hz, 1H), 8.18 (d,

Synthesis of Intermediates.

6,6-Dimethyldihydro-2H-pyran-2,4(3H)-dione (Ic ). To NaH (1.92 g,

80 mmol) in anhydrous THF (200 mL) was added, at 0 °C, methyl

acetoacetate (9.28 g, 80 mmol) dropwise. After 10 min of stirring, n -

BuLi (32 mL, 2.5 M solution in hexanes, 80 mmol) was added

dropwise, and the orange solution was stirred at 0 °C for 10 more

min. Dry acetone (7.5 mL, 82 mmol) was added at once, and the

mixture was stirred for 10 min at 0 °C. NaOH (80 mL, 2.5 M solution

in water) was then added, and the mixture was stirred for 12 h at

room temperature, whereupon it was acidi ﬁ ed (2.5 M HCl solution)

and extracted with ether (3 × 200 mL). The organic layer was washed

with brine and dried with Na₂SO₄. After ﬁ ltration, the solvent was

evaporated with rotovap. The residue was dissolved in a minimum of

CH₂Cl₂and precipitated with pentane as a brownish solid, yield

(62%) mp 126 −127 °C. ¹H NMR (400 MHz, CDCl₃): 1.48 (s, 6H);

2.66 (s, 2H); 3.40 (s, 2H).

Method D: Ethyl 3-Oxo-4-(tetrahydrofuran-3-yl)butanoate. To a

solution of 2-(tetrahydrofuran-3-yl)acetic acid (1.00 g, 7.68 mmol) in

DCM (10 mL) were added DMAP (1.408 g, 11.53 mmol) and DCC

(2.378 g, 11.53 mmol) and then 2,2-dimethyl-1,3-dioxane-4,6-dione

(1.107 g, 7.68 mmol) was added and the mixture was stirred for 12 h

at room temperature. The insoluble urea was then removed by

ﬁltration over Celite, and the solvent was evaporated under reduced

pressure. The crude product was redissolved in ethyl acetate and

washed with 1 M HCl. The organic phase was dried over Na₂SO₄,

ﬁltered, and evaporated. The crude product was used without further

puriﬁcation. The crude product was dissolved in ethanol (20 mL) and

reﬂuxed for 6 h. The organic solvent was evaporated under reduced

pressure and puriﬁed by column chromatography to aﬀord ethyl 3-

oxo-4-(tetrahydrofuran-3-yl) butanoate.

J = 2.0 Hz, 1H), 8.09 (d, J = 7.8 Hz, 2H), 7.49 (dd, J = 16.9, 7.7 Hz,

4H), 7.34 (d, J = 7.9 Hz, 2H), 3.63 (s, 1H), 3.55 (s, 1H), 3.28 (d, J =

4.1 Hz, 2H), 3.28 (s, 4H), 2.81 (s, 1H), 2.70 (s, 5H), 2.61 (s, 1H),

2.29 (dt, J = 15.3, 7.3 Hz, 4H), 2.14 (s, 4H), 1.27 (s, 2H), 1.07 (t, J =

7.4 Hz, 8H), 0.60 (t, J = 7.1 Hz, 4H); HRMS (ESI): m/z (M + H)⁺

calcd for C₃₂H₃₆ClN₄O₂S, 575.2242; found, 575.2223. Retention time

= 2.804 min.

Ethyl 3-Oxo-4-(tetrahydro-2H-pyran-4-yl)butanoate. This com-

pound was prepared using Method 14 using 2-(tetrahydro-2H-pyran-

4-yl)acetic acid to aﬀord ethyl 3-oxo-4-(tetrahydro-2H-pyran-4-

yl)butanoate.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-iso-

propyl-5-(piperazine-1-carbonyl)pyridin-2(1H)-one (90). This com-

pound was prepared via Method 13 using methyl 4-methyl-3-

oxopentanoate in step 1 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-

1-(2,6-diethylphenyl)-6-isopropyl-5-(piperazine-1-carbonyl)pyridin-

1

2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺= 575.2; H NMR

tert-Butyl 3-(4-Ethoxy-2,4-dioxobutyl)pyrrolidine-1-carboxylate.

(400 MHz, DMSO-d₆): δ 8.79 (s, 1H), 8.68 (s, 1H), 8.23 (d, J = 0.6

This compound was prepared using Method 14 using 2-(1-(tert-

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butoxycarbonyl)pyrrolidin-3-yl)acetic acid to aﬀord tert-butyl 3-(4-

ethoxy-2,4-dioxobutyl)pyrrolidine-1-carboxylate.

2H), 3.52 (d, J = 29.4 Hz, 3H), 3.24 (s, 1H), 2.31 (d, J = 61.9 Hz,

4H), 1.67 (dd, J = 21.3, 1.1 Hz, 6H), 1.31 (s, 2H), 1.26−1.06 (m,

6H). Aliphatic region complicated signiﬁcantly by amide rotamers;

HRMS (ESI): m/z (M + H)⁺calcd for C₃₃H₃₆ClN₄O₂S, 587.2242;

found, 587.2243. Retention time = 2.781 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-6-(cyclopropylmethyl)-1-(2,6-

diethylphenyl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-one (93).

This compound was prepared using Method 13 using ethyl 4-

cyclopropyl-3-oxobutanoate in step 1 to aﬀord 3-(4-(4-

chlorophenyl)thiazol-2-yl)-6-(cyclopropylmethyl)-1-(2,6-diethylphen-

yl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-one as a TFA salt.

1

LCMS: m/z (M + H)⁺= 587.2; H NMR (400 MHz, DMSO-d₆):

δ 8.79 (br s, 2H), 8.73 (s, 1H), 8.21 (s, 1H), 8.16−8.07 (m, 2H),

7.55−7.44 (m, 3H), 7.34 (d, J = 7.7 Hz, 2H), 4.07 (m, 1H), 3.74 (m,

1H), 3.51 (m, 3H), 3.12 (m, 3H), 2.35 (m, 2H), 2.12 (m, 5H), 1.07

(t, J = 7.5 Hz, 6H), 0.52−0.12 (m, 2H), −0.05 (m, 1H), −0.27 (m,

1H); HRMS (ESI): m/z (M + H)⁺calcd for C₂₆H₂₈ClN₁₄O,

587.2254; found, 587.2255. Retention time = 2.803 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-

methoxyethyl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-one (94).

This compound was prepared using Method 13 using methyl 5-

methoxy-3-oxopentanoate in step 1 to aﬀord 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-methoxyethyl)-5-(piperazine-

1-carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺=

Method 15: 3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-6-(2-methylprop-1-en-1-yl)-5-(piperazine-1-carbonyl)-

pyridin-2(1H)-one (92). Steps 1−3: A mixture of 6,6-dimethyldihy-

dro-2H-pyran-2,4(3H)-dione (3.03 g, 21.30 mmol) and 1,1-

dimethoxy-N,N-dimethylmethanamine (2.83 mL, 21.30 mmol) was

stirred for 15 min at room temperature. The reaction mixture became

a yellow solid. To the mixture were added 2-propanol (50 mL), 2-(4-

(4-chlorophenyl)thiazol-2-yl)acetonitrile (5.00 g, 21.3 mmol), and

piperidine (8.4 mL, 85 mmol) and then sonicated to get a

homogeneous mixture. The mixture was stirred at 70 °C for 4 h.

The solvent was removed by rotovap. To the residue were added 2,6-

diethylaniline (3.86 mL, 23.4 mmol) and acetic acid (61.00 mL, 1065

mmol) and the reaction mixture was sonicated to dissolve solids. The

mixture was stirred at 70 °C for 4 h and cooled to room temperature

and diluted with DCM and washed with water, sat NaHCO₃solution,

and brine solution. The organic layer was dried over Na₂SO₄and

concentrated. The crude product was puriﬁed by column chromatog-

raphy (10:90 EA/Hex to 100% EA) to aﬀord the product, 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-7,7-dimethyl-7,8-di-

hydro-2H-pyrano[4,3-b]pyridine-2,5(1H)-dione; LCMS: m/z (M +

H)⁺= 520.2.

Step 4: To a solution of 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-

diethylphenyl)-7,7-dimethyl-7,8-dihydro-1H-pyrano[4,3-b]pyridine-

2,5-dione (3.5 g, 6.74 mmol) in THF (25 mL) and MeOH (25 mL)

was added lithium hydroxide (1.13 g, 47.2 mmol) and the reaction

mixture was stirred at room temperature for 1 h. The solvent was

removed and diluted with DCM and quenched with acetic acid (2.70

mL, 47.2 mmol). The organic layer was washed several times with

brine and dried over Na₂SO₄and concentrated to aﬀord the crude 5-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-methyl-

prop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carboxylic acid; LCMS:

m/z (M + H)⁺= 519.2. Yield ∼95%. The crude acid was used in the

next step without further puriﬁcation.

1

591.2; H NMR (400 MHz, DMSO-d₆): δ 8.74 (d, J = 0.5 Hz, 2H),

8.24 (d, J = 0.5 Hz, 1H), 8.18−8.10 (m, 2H), 7.58−7.47 (m, 3H),

7.39 (s, 1H), 7.37 (s, 1H), 4.04 (s, 1H), 3.85−3.43 (m, 3H), 3.16 (d,

J = 35.4 Hz, 6H), 3.02 (d, J = 0.6 Hz, 3H), 2.85 (s, 1H), 2.33 (t, J =

1.9 Hz, 2H), 2.17 (s, 2H), 1.11 (s, 7H). Aliphatic region complicated

signiﬁcantly by amide rotamers; HRMS (ESI): m/z (M + H)⁺calcd

for C₃₂H₃₆ClN₄O₃S, 591.2191; found, 591.2195. Retention time =

3.741 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(pi-

perazine-1-carbonyl)-6-(3,3,3-triﬂuoropropyl)pyridin-2(1H)-one

(95). This compound was prepared using Method 13 using ethyl

6,6,6-triﬂuoro-3-oxohexanoate in step 1 to aﬀord 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(piperazine-1-car-

bonyl)-6-(3,3,3-triﬂuoropropyl)pyridin-2(1H)-one as a TFA salt.

1

LCMS: m/z (M + H)⁺= 629.2; H NMR (400 MHz, DMSO-d₆):

δ 8.77 (m, 3H), 8.24 (s, 1H), 8.16−8.08 (m, 2H), 7.55−7.47 (m,

3H), 7.38 (d, J = 7.7 Hz, 2H), 3.72 (br m, 5H), 3.17 (br m, 2H), 2.36

(dt, J = 15.1, 7.6 Hz, 2H), 2.02 (br m, 3H), 1.08 (t, J = 7.5 Hz, 6H);

HRMS (ESI): m/z (M + H)⁺calcd for C₃₂H₃₃ClF₃N₄O₂S, 629.1959;

found, 629.195. Retention time = 2.851 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(pi-

perazine-1-carbonyl)-6-((tetrahydrofuran-3-yl)methyl)pyridin-

2(1H)-one (96). This compound was prepared using Method 13 using

ethyl 3-oxo-4-(tetrahydrofuran-3-yl)butanoate in step 1 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(piperazine-

1-carbonyl)-6-((tetrahydrofuran-3-yl)methyl)pyridin-2(1H)-one as a

1

TFA salt. LCMS: m/z (M + H)⁺= 617.2; H NMR (400 MHz,

DMSO-d₆): δ 8.68 (s, 1H), 8.18 (s, 1H), 8.10−8.05 (m, 2H), 7.48−

7.41 (m, 3H), 7.31 (d, J = 7.7 Hz, 2H), 3.98 (s, 1H), 3.72 (s, 1H),

3.46 (d, J = 17.9 Hz, 2H), 3.07−2.95 (m, 6H), 2.57−2.49 (m, 1H),

2.16−1.90 (m, 6H), 1.71 (d, J = 21.7 Hz, 1H), 1.59 (d, J = 10.6 Hz,

2H), 1.38−135 (m, 1H), 1.02 (d, J = 9.1 Hz, 6H); HRMS (ESI): m/z

(M + H)⁺calcd for C₃₄H₃₈ClN₄O₃S, 617.2348; found, 617.2332.

Retention time = 2.722 min.

Steps 5 and 6: To a solution of 5-(4-(4-chlorophenyl)thiazol-2-yl)-

1-(2,6-diethylphenyl)-2-(2-methylprop-1-en-1-yl)-6-oxo-1,6-dihydro-

pyridine-3-carboxylic acid (2.0 g, 3.85 mmol) were added HATU

(2.198 g, 5.78 mmol), DMF (volume: 10 mL), DIPEA (1.346 mL,

7.71 mmol), and piperazine (1.077 g, 5.78 mmol). The reaction

mixture was stirred for 1 h at rt, diluted with water, extracted with 3 ×

10 mL of DCM, and washed with brine. The organic layer was dried

and concentrated. The crude product (without further puriﬁcation)

was diluted with DCM (10 mL) and TFA (10.39 mL, 135 mmol) and

stirred for 1 h at rt. The solvent was concentrated under vacuum, and

the crude mixture was diluted with DMSO and puriﬁed by reverse-

phase chromatography (Standard Acidic Gradient Method) to aﬀord

as a TFA salt, 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphen-

yl)-6-(2-methylprop-1-en-1-yl)-5-(piperazine-1-carbonyl)pyridin-

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(pi-

perazine-1-carbonyl)-6-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-

2(1H)-one (97). This compound was prepared using Method 13 using

ethyl 3-oxo-4-(tetrahydro-2H-pyran-4-yl)butanoate in step 1 to aﬀord

3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(pipera-

zine-1-carbonyl)-6-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-

1

2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺= 631.2; H NMR

(400 MHz, DMSO-d₆): δ 8.67 (s, 1H), 8.19 (s, 1H), 8.08 (d, J = 8.6

Hz, 2H), 7.51−7.41 (m, 3H), 7.33 (d, J = 7.7 Hz, 2H), 4.00 (s, 1H),

3.67−3.60 (m, 3H), 2.95 (td, J = 11.6, 2.4 Hz, 2H), 2.37−2.23 (m,

2H), 2.22−1.98 (m, 4H), 1.22−0.88 (m, 16H); HRMS (ESI): m/z

(M + Na)⁺calcd for C₃₅H₃₉ClN₄NaO₃S, 653.2324; found, 653.2336.

Retention time = 2.746 min.

1

2(1H)-one; LCMS: m/z (M + H)⁺= 587.2; H NMR (400 MHz,

DMSO-d₆): δ 8.91 (s, 1H), 8.08−8.02 (m, 2H), 7.95 (s, 1H), 7.53−

7.43 (m, 3H), 7.39−7.29 (m, 2H), 5.39 (s, 1H), 4.38 (s, 1H), 3.78 (s,

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3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(pi-

perazine-1-carbonyl)-6-(pyrrolidin-3-ylmethyl)pyridin-2(1H)-one

(98). This compound was prepared using Method 13 using tert-butyl

3-(4-ethoxy-2,4-dioxobutyl)pyrrolidine-1-carboxylate in step 1 fol-

lowed by Boc deprotection using TFA in DCM to aﬀord 3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(piperazine-1-car-

bonyl)-6-(pyrrolidin-3-ylmethyl)pyridin-2(1H)-one as a TFA salt.

calcd for C₃₆H₃₉ClFN₄O₃, 629.2689; found, 629.2701. Retention time

= 2.889 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-

methylprop-1-en-1-yl)-5-(4,7-diazaspiro[2.5]octane-7-carbonyl)-

pyridin-2(1H)-one (103). This compound was prepared using

Method 15 using tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate

in step 5 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-dieth-

ylphenyl)-6-(2-methylprop-1-en-1-yl)-5-(4,7-diazaspiro[2.5]octane-7-

carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺=

613.3; ¹H NMR (400 MHz, DMSO-d₆): δ 8.71 (s, 1H), 8.41 (s, 1H),

8.29 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.48−7.21 (m,

3H), 5.33 (s, 1H), 4.43−4.33 (m, 1H), 3.70−3.37 (m, 3H), 3.07−

2.90 (m, 2H), 2.71−2.59 (m, 2H), 2.44−1.90 (m, 4H), 1.58 (d, J =

10.3 Hz, 6H), 1.26−0.72 (m, 9H); HRMS (ESI): m/z (M + H)⁺

calcd for C₃₅H₃₈ClN₄O₂S, 613.2399; found, 613.2427. Retention time

= 2.85 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(3-

(hydroxymethyl)piperazine-1-carbonyl)-6-(2-methylprop-1-en-1-

yl)pyridin-2(1H)-one (104). This compound was prepared using

Method 15 using tert-butyl 2-(hydroxymethyl)piperazine-1-carbox-

ylate in step 5 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-

diethylphenyl)-5-(3-(hydroxymethyl)piperazine-1-carbonyl)-6-(2-

methylprop-1-en-1-yl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z

(M + H)⁺= 617.2; ¹H NMR (400 MHz, DMSO-d₆): δ 8.73 (s, 1H),

8.25 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.43

(t, J = 7.7 Hz, 1H), 7.29 (dd, J = 20.5, 7.7 Hz, 2H), 5.29 (s, 1H),

4.63−4.53 (m, 1H), 3.73−3.39 (m, 3H), 3.21−2.63 (m, 2H), 2.44−

2.18 (m, 4H), 2.16−1.99 (m, 2H), 1.63−1.51 (m, 7H), 1.16−1.05

(m, 3H), 0.98 (t, J = 7.5 Hz, 3H); HRMS (ESI): m/z (M + H)⁺calcd

for C₃₄H₃₈ClN₄O₃S, 617.2348; found, 617.2376. Retention time =

2.743 min.

1

LCMS: m/z (M + H)⁺= 616.2; H NMR (400 MHz, DMSO-d₆): δ

8.96 (br s, 2H), 8.74 (s, 1H), 8.54 (br s, 2H), 8.24 (s, 1H), 8.16−8.01

(m, 2H), 7.60−7.44 (m, 3H), 7.37 (d, J = 7.7 Hz, 2H), 4.19−2.56 (m,

10H), 2.44−1.94 (m, 5H), 1.63 (dd, J = 101.3, 45.9 Hz, 5H), 1.39−

1.15 (m, 1H), 1.10 (t, J = 7.5 Hz, 6H); HRMS (ESI): m/z (M + H)⁺

calcd for C₂₉H₃₄ClF₃N₉O, 616.2521; found, 616.2524. Retention time

= 2.321 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(3-

methylpiperazine-1-carbonyl)-6-(2-methylprop-1-en-1-yl)pyridin-

2(1H)-one (99). This compound was prepared using Method 15 using

tert-butyl 2-methylpiperazine-1-carboxylate in step 5 to aﬀord 3-(4-

(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(3-methylpi-

perazine-1-carbonyl)-6-(2-methylprop-1-en-1-yl)pyridin-2(1H)-one

1

as a TFA salt. LCMS: m/z (M + H)⁺= 601.1; H NMR (400 MHz,

DMSO-d₆): δ 8.73 (d, J = 9.4 Hz, 1H), 8.25 (s, 1H), 8.09 (dd, J = 8.9,

2.3 Hz, 2H), 7.53 (dd, J = 8.7, 2.3 Hz, 2H), 7.43 (t, J = 7.7 Hz, 1H),

7.29 (dd, J = 22.5, 7.7 Hz, 2H), 5.30 (s, 1H), 4.55 (dd, J = 32.5, 13.8

Hz, 1H), 4.33−4.23 (m, 1H), 3.72−3.48 (m, 1H), 3.23−2.62 (m,

3H), 2.42−2.24 (m, 2H), 2.16−1.97 (m, 2H), 1.63−1.52 (m, 6H),

1.24 (dd, J = 12.0, 6.4 Hz, 1H), 1.11 (dt, J = 11.5, 7.3 Hz, 6H), 0.98

(t, J = 7.5 Hz, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₄H₃₈ClN₄O₂S, 601.2399; found, 601.2395. Retention time = 2.805

min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(2-

methylpiperazine-1-carbonyl)-6-(2-methylprop-1-en-1-yl)pyridin-

2(1H)-one (100). This compound was prepared using Method 15

using tert-butyl 3-methylpiperazine-1-carboxylate in step 5 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(3-methyl-

piperazine-1-carbonyl)-6-(2-methylprop-1-en-1-yl)pyridin-2(1H)-one

4-(5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-

methylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)-

piperazin-2-one (105). This compound was prepared using Method

15 using tert-butyl 2-oxopiperazine-1-carboxylate in step 5 to aﬀord 4-

(5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-meth-

ylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)piperazin-2-

1

as a TFA salt. LCMS: m/z (M + H)⁺= 601.1; H NMR (400 MHz,

1

DMSO-d₆): δ 8.70 (s, 1H), 8.25 (s, 1H), 8.13−8.05 (m, 2H), 7.53

(dd, J = 8.9, 2.3 Hz, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.35−7.21 (m,

2H), 5.28 (s, 1H), 4.94 (s, 1H), 3.59 (d, J = 14.1 Hz, 1H), 3.27−2.93

(m, 5H), 2.42−2.21 (m, 2H), 2.10 (dp, J = 26.2, 7.5 Hz, 2H), 1.63−

1.47 (m, 6H), 1.38−1.19 (m, 1H), 1.17−1.06 (m, 6H), 0.99 (t, J =

7.5 Hz, 3H); HRMS (ESI): m/z (M + H)⁺calcd for C₃₄H₃₈ClN₄O₂S,

601.2399; found, 601.2388. Retention time = 2.824 min.

one as a TFA salt. LCMS: m/z (M + H)⁺= 601.1; H NMR (400

MHz, DMSO-d₆): δ 8.70 (d, J = 8.2 Hz, 1H), 8.24 (d, J = 1.8 Hz,

1H), 8.17−7.97 (m, 3H), 7.58−7.36 (m, 4H), 5.23 (s, 1H), 3.89−

3.79 (s, 2H), 3.26−2.95 (m, 4H), 2.41−2.00 (m, 4H), 1.61−1.42 (m,

7H), 1.20−0.86 (m, 6H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₃H₃₄ClN₄O₃S, 601.2035; found, 601.2049. Retention time = 3.612

min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(3-

ethylpiperazine-1-carbonyl)-6-(2-methylprop-1-en-1-yl)pyridin-

2(1H)-one (101). This compound was prepared using Method 15

using tert-butyl 2-ethylpiperazine-1-carboxylate in step 5 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-5-(3-ethylpi-

perazine-1-carbonyl)-6-(2-methylprop-1-en-1-yl)pyridin-2(1H)-one

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-

methylprop-1-en-1-yl)-5-(3-(triﬂuoromethyl)piperazine-1-

carbonyl)pyridin-2(1H)-one (106). This compound was prepared

using Method 15 using tert-butyl 2-(triﬂuoromethyl)piperazine-1-

carboxylate in step 5 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-

(2,6-diethylphenyl)-6-(2-methylprop-1-en-1-yl)-5-(3-

(triﬂuoromethyl)piperazine-1-carbonyl)pyridin-2(1H)-one as a TFA

1

as a TFA salt. LCMS: m/z (M + H)⁺= 615.1; H NMR (400 MHz,

1

DMSO-d₆): δ 8.75 (s, 1H), 8.25 (s, 1H), 8.14−8.05 (m, 2H), 7.57−

7.50 (m, 2H), 7.47−7.40 (m, 1H), 7.37−7.22 (m, 2H), 5.29 (s, 1H),

4.67−4.52 (m, 1H), 3.72−3.64 (m, 1H), 3.23−3.05 (m, 2H), 3.01−

2.61 (m, 2H), 2.45−2.20 (m, 2H), 2.14−1.99 (m, 2H), 1.66−1.51

(m, 9H), 1.16−1.06 (m, 3H), 1.02−0.94 (m, 3H), 0.87−0.79 (m,

2H); HRMS (ESI): m/z (M + H)⁺calcd for C₃₅H₄₀ClN₄O₂S,

615.2555; found, 615.2525. Retention time = 2.848 min.

salt. LCMS: m/z (M + H)⁺= 655.2; H NMR (400 MHz, DMSO-

d₆): δ 8.75 (s, 1H), 8.24 (s, 1H), 8.10 (dd, J = 9.1, 2.5 Hz, 2H), 7.57−

7.48 (m, 2H), 7.43 (td, J = 7.7, 4.6 Hz, 1H), 7.30 (dd, J = 18.6, 7.0

Hz, 2H), 5.25 (s, 1H), 4.52−4.44 (m, 1H), 4.15−3.98 (m, 1H),

3.23−2.56 (m, 5H), 2.38−2.17 (m, 2H), 2.16−1.99 (m, 2H), 1.56 (d,

J = 11.2 Hz, 6H), 1.16−0.91 (m, 6H); HRMS (ESI): m/z (M + H)⁺

calcd for C₃₄H₃₅ClF₃N₄O₂S, 655.2116; found, 655.214. Retention

time = 3.952 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-

methylprop-1-en-1-yl)-5-(3-propylpiperazine-1-carbonyl)pyridin-

2(1H)-one (102). This compound was prepared using Method 15

using tert-butyl 2-propylpiperazine-1-carboxylate in step 5 to aﬀord 3-

(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-methyl-

prop-1-en-1-yl)-5-(3-propylpiperazine-1-carbonyl)pyridin-2(1H)-one

4-(5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-

methylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)-

piperazine-2-carbonitrile (107). This compound was prepared using

Method 15 using tert-butyl 2-cyanopiperazine-1-carboxylate in step 5

to aﬀord 4-(5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-

2-(2-methylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)-

1

as a TFA salt. LCMS: m/z (M + H)⁺= 629.3; H NMR (400 MHz,

DMSO-d₆): δ 8.75 (s, 1H), 8.25 (s, 1H), 8.16−8.05 (m, 2H), 7.53

(dt, J = 9.7, 2.9 Hz, 2H), 7.49−7.38 (m, 1H), 7.29 (dd, J = 22.4, 7.9

Hz, 2H), 5.28 (s, 1H), 4.66−4.31 (m, 2H), 3.72−3.67 (m, 1H),

3.23−2.94 (m, 2H), 2.89−2.52 (m, 2H), 2.43−2.19 (m, 2H), 2.15−

1.99 (m, 2H), 1.65−1.50 (m, 6H), 1.37 (d, J = 1.2 Hz, 1H),, 1.16−

1.05 (m, 4H), 1.04−0.82 (m, 8H); HRMS (ESI): m/z (M + H)⁺

piperazine-2-carbonitrile as a TFA salt. LCMS: m/z (M + H)⁺

=

1

612.2; H NMR (400 MHz, DMSO-d₆): δ 8.67 (d, J = 8.6 Hz, 1H),

8.24 (s, 1H), 8.09 (dd, J = 8.3, 1.7 Hz, 2H), 7.58−7.48 (m, 2H), 7.42

(t, J = 7.6 Hz, 1H), 7.29 (dd, J = 17.7, 7.3 Hz, 2H), 5.24 (d, J = 8.0

Hz, 1H), 4.45−4.12 (m, 1H), 3.23−2.97 (m, 2H), 2.93−2.63 (m,

4H), 2.44−2.20 (m, 2H), 2.20−2.01 (m, 2H), 1.67−1.50 (m, 6H),

4941

https://doi.org/10.1021/acs.jmedchem.1c00019

J. Med. Chem. 2021, 64, 4913−4946

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1.11 (t, J = 12.8, 7.4, Hz, 3H), 1.00 (t, J = 7.4 Hz, 3H); HRMS (ESI):

m/z (M + H)⁺calcd for C₃₄H₃₅ClN₅O₂S, 612.2195; found, 612.22.

Retention time = 3.776 min.

octane-3-carboxylate in step 5 to aﬀord 5-(3,8-diazabicyclo[3.2.1]-

octane-8-carbonyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethyl-

phenyl)-6-(2-methylprop-1-en-1-yl)pyridin-2(1H)-one as a TFA salt.

1

4-(5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-

methylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)-

piperazine-2-carboxylic Acid (108). This compound was prepared

using Method 15 using 1-(tert-butyl) 2-methyl piperazine-1,2-

dicarboxylate in step 5 followed by ester hydrolysis using LiOH in

THF/MeOH and Boc deprotection using TFA in DCM to aﬀord 4-

(5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-meth-

ylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)piperazine-2-

LCMS: m/z (M + H)⁺= 613.2; H NMR (400 MHz, DMSO-d₆): δ

8.22 (s, 1H), 8.08 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3 Hz, 2H), 7.41

(t, J = 7.7 Hz, 2H), 7.27 (dd, J = 13.8, 7.7 Hz, 2H), 5.24 (s, 1H),

3.98−3.92 (m, 2H), 3.25−3.05 (m, 4H), 2.78−2.53 (m, 4H), 2.37−

2.02 (m, 4H),1.90 (s, 3H), 1.55 (s, 3H), 1.04−0.97 (m, 6H); HRMS

(ESI): m/z (M + H)⁺calcd for C₃₅H₃₈ClN₄O₂S, 613.2399; found,

613.2389. Retention time = 2.798 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-6-(2-methylprop-1-en-1-yl)-1-

phenyl-5-(piperazine-1-carbonyl)pyridin-2(1H)-one (113). This

compound was prepared using Method 15 using aniline in step 3

to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-6-(2-methylprop-1-en-1-

yl)-1-phenyl-5-(piperazine-1-carbonyl)pyridin-2(1H)-one as a TFA

1

carboxylic acid as a TFA salt. LCMS: m/z (M + H)⁺= 631.1; H

NMR (400 MHz, DMSO-d₆): δ 8.75 (s, 1H), 8.25 (s, 1H), 8.09 (dd, J

= 8.2, 1.3 Hz, 2H), 7.53 (dd, J = 8.6, 2.3 Hz, 2H), 7.43 (td, J = 7.7, 5.6

Hz, 1H), 7.36−7.18 (m, 2H), 5.28 (s, 1H), 4.58−4.48 (m, 1H),

4.20−4.04 (m, 1H), 3.87−3.57 (m, 1H), 3.12−2.62 (m, 2H), 2.44−

2.17 (m, 2H), 2.20−1.99 (m, 2H), 1.63−1.49 (m, 6H), 1.24−1.05

(m, 3H), 1.03−0.94 (m, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₂₇H₂₈Cl N₁₄O₃, 631.214; found, 631.2151. Retention time = 3.038

min.

4-(5-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-

methylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)-

piperazine-2-carboxamide (109). This compound was prepared

using Method 15 using piperazine-2-carboxamide in step 5 to aﬀord

4-(5-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-2-(2-

methylprop-1-en-1-yl)-6-oxo-1,6-dihydropyridine-3-carbonyl)-

piperazine-2-carboxamide as a TFA salt. LCMS: m/z (M + H)⁺=

630.3; ¹H NMR (400 MHz, DMSO-d₆): δ 8.76 (s, 1H), 8.26 (s, 1H),

8.09 (dd, J = 8.0, 6.0 Hz, 2H), 7.53 (dd, J = 8.0, 6.2 Hz, 2H), 7.43 (t, J

= 7.7 Hz, 1H), 7.37−7.20 (m, 2H), 5.29 (d, J = 9.5 Hz, 1H), 4.72−

4.55 (m, 1H), 4.02−3.72 (m, 1H), 3.66−3.54 (m, 1H), 3.17−2.99

(m, 2H), 2.82 (t, J = 13.0 Hz, 1H), 2.68−2.62 (m, 1H), 2.42−2.22

(m, 2H), 2.15−1.99 (m, 2H), 1.67−1.50 (m, 6H), 1.11 (t, J = 7.5 Hz,

3H), 1.05−0.91 (m, 3H); HRMS (ESI): m/z (M + H)⁺calcd for

C₃₄H₃₄ClF₅N₃O, 630.2305; found, 630.2311. Retention time = 2.753

min.

1

salt. LCMS: m/z (M + H)⁺= 531.2; H NMR (400 MHz, DMSO-

d₆): δ 8.82 (s, 1H), 8.06−8.01 (m, 2H), 7.93 (d, J = 1.8 Hz, 1H),

7.61−7.49 (m, 3H), 7.48−7.42 (m, 2H), 7.37 (s, 1H), 7.19 (s, 1H),

5.59 (s, 1H), 4.25 (s, 2H), 3.84 (s, 2H), 3.64 (d, J = 18.3 Hz, 2H),

3.14 (s, 2H), 1.63 (dd, J = 9.4, 1.1 Hz, 6H). Aliphatic region

complicated signiﬁcantly by amide rotamers; HRMS (ESI): m/z (M +

H)⁺calcd for C₂₉H₂₈ClN₄O₂S, 531.1616; found, 531.1631. Retention

time = 2.583 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ethylphenyl)-6-(2-meth-

ylprop-1-en-1-yl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-one

(114). This compound was prepared using Method 15 using 2-

ethylaniline in step 3 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-

(2-ethylphenyl)-6-(2-methylprop-1-en-1-yl)-5-(piperazine-1-

carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺=

1

559.2; H NMR (400 MHz, DMSO-d₆): δ 8.86 (s, 1H), 8.04 (d, J =

8.7 Hz, 2H), 7.94 (s, 1H), 7.53−7.47 (m, 2H), 7.45 (d, J = 8.6 Hz,

2H), 7.44−7.00 (m, 2H), 5.62−5.42 (m, 1H), 4.35−4.25 (m, 2H),

3.90−3.50 (m, 4H), 3.35−3.10 (m, 2H), 2.50−2.25 (m, 2H), 1.67−

1.57 (m, 6H), 1.25−1.05 (m, 3H). Aliphatic region complicated

signiﬁcantly by amide rotamers; HRMS (ESI): m/z (M + H)⁺calcd

for C₃₁H₃₂ClN₄O₂S, 559.1929; found, 559.1937. Retention time =

2.675 min.

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-

methylprop-1-en-1-yl)-5-(2,6-diazaspiro[3.3]heptane-2-carbonyl)-

pyridin-2(1H)-one (110). This compound was prepared using

Method 15 using tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate

in step 5 to aﬀord 3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-dieth-

ylphenyl)-6-(2-methylprop-1-en-1-yl)-5-(2,6-diazaspiro[3.3]heptane-

2-carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z (M + H)⁺=

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ethyl-6-methylphenyl)-6-

(2-methylprop-1-en-1-yl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-

one (115). This compound was prepared using Method 15 using 2-

ethyl-6-methylaniline in step 3 to aﬀord 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-1-(2-ethyl-6-methylphenyl)-6-(2-methylprop-1-en-1-yl)-

5-(piperazine-1-carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS:

1

m/z (M + H)⁺= 533.2; H NMR (400 MHz, DMSO-d₆): δ 8.95 (s,

599.2; H NMR (400 MHz, DMSO-d₆): δ 8.69 (d, J = 4.2 Hz, 1H),

8.23 (d, J = 8.1 Hz, 1H), 8.07 (dd, J = 8.7, 2.3 Hz, 2H), 7.56−7.49

(m, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.29 (d, J = 7.6 Hz, 2H), 5.30 (s,

1H), 4.08 (t, J = 11.4 Hz, 4H), 2.70−2.59 (m, 2H), 2.24−2.09 (m,

6H), 1.58 (s, 6H), 1.08−1.02 (m, 6H). HRMS (ESI): m/z (M + H)⁺

calcd for C₃₄H₃₆ClN₄O₂S, 599.2242; found, 599.225. Retention time

= 2.749 min.

2H), 8.71 (d, J = 6.5 Hz, 1H), 8.22 (s, 1H), 8.12 (d, J = 8.6 Hz, 2H),

7.52 (t, J = 8.4 Hz, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.33 (dd, J = 15.7,

8.1 Hz, 2H), 3.57 (d, J = 87.5 Hz, 4H), 3.16 (t, J = 47.1 Hz, 4H), 2.20

(t, J = 39.9 Hz, 1H), 1.98 (s, 3H), 1.90 (s, 3H), 1.08 (t, J = 7.5 Hz,

3H). Aliphatic region complicated signiﬁcantly by amide rotamers;

HRMS (ESI): m/z (M + H)⁺calcd for C₂₉H₃₀ClN₄O₂S, 533.1773;

found, 533.1792. Retention time = 2.6 min.

1-(2-Chloro-6-ethylphenyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-6-

(2-methylprop-1-en-1-yl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-

one (116). This compound was prepared using Method 15 using 2-

chloro-6-ethylaniline in step 3 to aﬀord 1-(2-chloro-6-ethylphenyl)-3-

(4-(4-chlorophenyl)thiazol-2-yl)-6-(2-methylprop-1-en-1-yl)-5-(pi-

perazine-1-carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS: m/z

(M + H)⁺= 593.2; ¹H NMR (400 MHz, DMSO-d₆): δ 8.88 (s, 1H),

8.03 (d, J = 8.7 Hz, 2H), 7.94 (s, 1H), 7.55−7.39 (m, 5H), 5.55−5.35

(m, 1H), 4.40−4.20 (m, 1H), 3.85−3.70 (m, 1H), 3.60−3.45 (m,

2H), 3.35−3.10 (m, 4H), 2.60−2.25 (m, 2H), 1.74−1.63 (m, 6H),

1.30−1.05 (m, 3H). Aliphatic region complicated signiﬁcantly by

amide rotamers; HRMS (ESI): m/z (M + H)⁺calcd for

C₃₁H₃₁Cl₂N₄O₂S, 593.1539; found, 593.1528. Retention time =

4.187 min.

5-((1R,5S)-3,8-Diazabicyclo[3.2.1]octane-3-carbonyl)-3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-methylprop-

1-en-1-yl)pyridin-2(1H)-one (111). This compound was prepared

using Method 15 using tert-butyl (1R,5S)-3,8-diazabicyclo[3.2.1]-

octane-8-carboxylate in step 5 to aﬀord 5-((1R,5S)-3,8-diazabicyclo-

[3.2.1]octane-3-carbonyl)-3-(4-(4-chlorophenyl)thiazol-2-yl)-1-(2,6-

diethylphenyl)-6-(2-methylprop-1-en-1-yl)pyridin-2(1H)-one as a

1

TFA salt. LCMS: m/z (M + H)⁺= 613.2; H NMR (400 MHz,

DMSO-d₆): δ 8.73 (s, 1H), 8.24 (s, 1H), 8.15−8.04 (m, 2H), 7.58−

7.48 (m, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.29 (dd, J = 26.3, 7.7 Hz,

2H), 5.23 (s, 1H), 4.44 (d, J = 14.0 Hz, 1H), 4.14−3.86 (m, 2H),

3.61−3.34 (m, 2H), 2.96 (d, J = 14.0 Hz, 1H), 2.65 (p, J = 1.9 Hz,

1H), 2.44−2.27 (m, 2H), 2.15−1.97 (m, 2H), 1.96−1.71 (m, 3H),

1.55 (dd, J = 10.4, 1.3 Hz, 6H), 1.14 (t, J = 7.7 Hz, 3H), 0.96 (t, J =

7.5 Hz, 3H); HRMS (ESI): m/z (M + Na)⁺calcd, for

C₃₅H₃₇ClN₄NaO₂S, 635.2218; found, 635.2206. Retention time =

2.823 min.

5-(3,8-Diazabicyclo[3.2.1]octane-8-carbonyl)-3-(4-(4-

chlorophenyl)thiazol-2-yl)-1-(2,6-diethylphenyl)-6-(2-methylprop-

1-en-1-yl)pyridin-2(1H)-one (112). This compound was prepared

using Method 15 using tert-butyl (1R,5S)-3,8-diazabicyclo[3.2.1]-

3-(4-(4-Chlorophenyl)thiazol-2-yl)-1-(2-ethyl-5-methylphenyl)-6-

(2-methylprop-1-en-1-yl)-5-(piperazine-1-carbonyl)pyridin-2(1H)-

one (117). This compound was prepared using Method 15 using 2-

ethyl-5-methylaniline in step 3 to aﬀord 3-(4-(4-chlorophenyl)-

thiazol-2-yl)-1-(2-ethyl-5-methylphenyl)-6-(2-methylprop-1-en-1-yl)-

5-(piperazine-1-carbonyl)pyridin-2(1H)-one as a TFA salt. LCMS:

4942

https://doi.org/10.1021/acs.jmedchem.1c00019

J. Med. Chem. 2021, 64, 4913−4946

Article Doi

Discovery and Optimization of 2 H-1λ2-Pyridin-2-one Inhibitors of Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer

DOI: 10.1021/acs.jmedchem.1c00019

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