The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor

Article

pubs.acs.org/jmc

The Discovery of I‑BET726 (GSK1324726A), a Potent

Tetrahydroquinoline ApoA1 Up-Regulator and Selective BET

Bromodomain Inhibitor

Romain Gosmini,^†,¶Van Loc Nguyen,^†Jerome Toum,^†Christophe Simon,^†,+Jean-Marie G. Brusq,^‡

́

̂

Gael Krysa,^‡Olivier Mirguet,^†,▲Alizon M. Riou-Eymard,^‡Eric V. Boursier,^§Lionel Trottet,^§

Paul Bamborough,*^,⊥Hugh Clark,^○Chun-wa Chung,^⊥Leanne Cutler,^#Emmanuel H. Demont,^∥

Rejbinder Kaur,^∥Antonia J. Lewis,^∇Mark B. Schilling,^○Peter E. Soden,^∥Simon Taylor,^#Ann L. Walker,^◆

Matthew D. Walker,^○Rab K. Prinjha,*^,∥and Edwige Nicodeme^‡

̀

‡

§

^†Candidate Discovery, Discovery Biology, DMPK, GlaxoSmithKline Les Ulis, Centre de Recherches Franco

̧

is Hyaﬁl,

́

GlaxoSmithKline R&D, 25 Avenue du Quebec, 91140 Villebon-sur-Yvette, France

^∥Epinova Discovery Performance Unit, ^⊥Computational & Structural Chemistry, ^#Experimental Medicine Unit, ^∇Biological Sciences,

^○Exploratory Development Sciences, ^◆Discovery Partnerships Medicinal Chemistry, GlaxoSmithKline Stevenage, Medicines

Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

S

* Supporting Information

ABSTRACT: Through their function as epigenetic readers of the

histone code, the BET family of bromodomain-containing proteins

regulate expression of multiple genes of therapeutic relevance, including

those involved in tumor cell growth and inﬂammation. BET

bromodomain inhibitors have profound antiproliferative and anti-

inﬂammatory eﬀects which translate into eﬃcacy in oncology and

inﬂammation models, and the ﬁrst compounds have now progressed

into clinical trials. The exciting biology of the BETs has led to great

interest in the discovery of novel inhibitor classes. Here we describe the

identiﬁcation of a novel tetrahydroquinoline series through up-

regulation of apolipoprotein A1 and the optimization into potent

compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these eﬀects are produced by

inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure−activity relationships of

binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET

inhibitors.

four members of this family, BRD2, BRD3, and BRD4

(together with BRDT, whose expression is restricted to testis

and ovary), activate transcription via recognition of the

acetylated lysine state of histone tails and other proteins and

recruitment of polymerases and transcription factors.^8,9Each

BET protein contains two bromodomains, modules which

speciﬁcally recognize the acetylated state of their partners,

including the histone protein tails whose acetylation forms part

of the histone code of epigenetic gene regulation. Small

molecule inhibitors of bromodomain binding to histones have

emerged over recent years and are now of great current interest

for wide-ranging applications including atherosclerosis, inﬂam-

mation, and oncology.¹⁰⁻¹³

INTRODUCTION

■

Atherosclerosis is a signiﬁcant cause of mortality, and although

currently treated by lipid-modifying drugs such as statins, a

need remains for novel treatments. Decreased risk of

atherosclerosis is associated with higher levels of high density

lipoprotein (HDL) cholesterol. The apolipoprotein A1 protein

(ApoA1) is a major component of HDL, and its overexpression

is correlated with increased HDL cholesterol levels.^1,2ApoA1

up-regulation therefore oﬀers a novel alternative to existing

treatments for atherosclerosis, although until quite recently no

molecular mechanism to bring that about was known.³

We recently reported the use of a cellular reporter assay to

discover novel ApoA1 up-regulators, such as the benzodiaze-

pine (BZD) compounds exempliﬁed by 1 (GSK525762A/I-

BET762, Figure 1), which is currently in clinical develop-

ment.⁴⁻⁶These were shown via chemoproteomics and other

approaches to bind to the tandem bromodomains of the

bromodomain and extra terminal domain (BET) family

proteins and also have potent anti-inﬂammatory activity.⁷The

A thienotriazolodiazepine with structural similarity to 1,

discovered by a group at Mitsubishi Tanabe as an inhibitor of

CD28-dependent T-cell growth and inﬂammation, was also

Received: July 11, 2014

A

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Figure 1. Some examples of BET bromodomain inhibitors, 1−8.

found to target the BET bromodomains.^14,15An exemplar of

this chemotype, 2 (JQ1), has been released to the worldwide

academic community by the Chemical Probe Consortium.¹⁶

This molecule shows strong eﬀects in a range of cancer cell

types. For example, NUT midline carcinoma (NMC) is an

aggressive tumor in which the BRD4−NUT fusion oncoprotein

prevents normal cell diﬀerentiation.¹⁷Treatment of NMC cell

lines with 2 halted proliferation and restored normal diﬀer-

entiation.¹⁸2 and other BET inhibitors also show eﬀects in

models of hematologic cancers (including acute myeloid

leukemia, Burkitt’s lymphoma, MLL-fusion leukemia, multiple

myeloma, and B-cell lymphoblastic leukemia) and neuro-

blastoma.¹⁹⁻²³

Other classes of BET bromodomain inhibitors have been

reported. We recently described an unrelated series of

quinoline isoxazole ApoA1 up-regulators (e.g., 3, I-BET151),

which also exert their eﬀects through binding to the BET

bromodomains.^24,253 has been investigated for indications

including acute leukemia, including MLL-related acute myeloid

leukemia, and JAK2-driven myeloproliferative neoplasms.^20,26

Compound 4 (RVX-208) was also discovered as an ApoA1 up-

regulator and has entered clinical trials for atherosclerosis.²⁷It

has since been found that its molecular binding partners include

the BET tandem bromodomains.²⁸

binding to the BET family of bromodomain-containing

proteins.⁴We therefore screened representative compounds

from the tetrahydroquinoline series in a ﬂuorescence anisotropy

(FA) binding assay by measuring the displacement of a ligand

from the acetyl-lysine site of recombinant BET bromodomains.

As with previously published series of BET inhibitors, BET

binding and ApoA1 reporter activation correlate reasonably

well, although this is limited by the tight binding limit of the FA

assay and factors such as cell permeability.^4,25In this article, we

show the ApoA1 reporter activation data, which was used to

make the decisions that drove lead optimization, alongside the

BET bromodomain activity which was gathered later. The

Results and Discussion section concludes with X-ray structures

showing the binding modes of representative molecules in

BRD2 and BRD4, which will be used to rationalize the

structure−activity relationships found.

Identiﬁcation of Tetrahydroquinolines as ApoA1 Up-

Regulators. Tetrahydroquinoline 9 (Table 1) was identiﬁed

from the output of a high-throughput screening campaign as an

Table 1. ApoA1 Luciferase Activation (Fold) of Initial Hits

and Analogues after 16 h in HepG2 Cells

Beyond cell-based screening, the BET bromodomains are

highly tractable to structure-based and fragment-based drug

discovery, taking advantage of increasing understanding of the

molecular architecture and binding preferences of the acetyl-

lysine site.²⁹Examples of molecules developed from fragments

binding in the AcK site and growing outward to exploit

additional binding interactions include the tetrahydroquinazo-

linone 5 (PFI-1), isoxazoles such as 6 and 7, and compounds

incorporating elements of these such as 8.³⁰⁻³⁵In this

manuscript we describe the discovery of a new series of

tetrahydroquinoline-based ApoA1 up-regulators that function

through BET inhibition and their optimization to a potent and

selective compound active in models of acute inﬂammation.

ApoA1 reporter activation

relative

a

compd

stereochemistry

R₁

R₄

1.25 μM

10 μM

9

mixture

cis

Me

Et

H

1.7

2.3−3.3

10

11

12

13

14

15

Me

H

trans

1.1

cis

1.7−2.0

1.6

RESULTS AND DISCUSSION

■

mixture

H

1.4−2.9

≤1.1

Molecular Targets of the Tetrahydroquinolines.

Throughout the optimization work reported here, the

molecular mode of action of the tetrahydroquinolines was

unknown. In due course, we discovered that other families of

ApoA1 up-regulators operate by speciﬁc, histone-competitive

Pr

H

Bu

H

≤1.1

a

All compounds are racemates. “Mixture” indicates a mixture of

diastereoisomers.

B

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Table 2. Mean Potency (ApoA1 Luciferase and BET FA) and Solubility of 16−29 (* Denotes Single Enantiomer, Otherwise

Racemic Mixtures with cis Stereochemistry)

a

See Supporting Information, Table S1, for standard deviations and other notes.

up-regulator of ApoA1 in the HepG2 cell luciferase reporter

assay. At 10 μM, 9 increased luciferase luminescence by 2.2−

3.3-fold after 16 h. Because of this promising level of activity,

this compound and analogues, which were originally purchased

from a commercial vendor as mixtures of undeﬁned stereo-

chemistry, were investigated further. The para-methyl analogue

was prepared (see Scheme 1, Synthetic Chemistry) and

separated into its cis and trans stereoisomers 10 and 11. The

ApoA1 activation ability of the cis isomer 10 was similar to 9,

but its trans isomer 11 was inactive (Table 1). The cis isomer of

9 was also synthesized, giving 12, which retained ApoA1

activity. These results strongly suggested that the activity

resided in the cis stereochemistry.

Initial Exploration at the N1 Position. To understand the

eﬀects of modiﬁcations at the acetamide position, analogues

were ﬁrst sought within the GSK compound collection. Like 9,

compounds 13−15 originated from a commercial supplier. The

propionamide analogue 13 retained levels of ApoA1 activity

comparable to those of the acetamide 9, but the butyramide

and pentanamide analogues 14 and 15 were inactive. This

suggested that further modiﬁcation of this position was unlikely

to be productive. However, the knowledge that SAR existed in

modiﬁcations at the N1 position as well as in the stereo-

chemistry of the tetrahydroquinolinone core led us to

investigate some of the other positions of this template.

Optimization of the 6-Position. The key 6-bromo

tetrahydroquinoline intermediate N-((2S*,4R*)-1-acetyl-6-

bromo-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)formamide

(56) was used to prepare an initial set of aryl substituted

racemic cis-stereochemistry analogues through Suzuki chem-

istry (see Scheme 2, Synthetic Chemistry). The choice of this

substituent position was inspired by a pharmacophore model

involving the overlay of THQs such as 10 with members of

another series of ApoA1 up-regulators, which will be reported

in due course. Introduction of the unsubstituted phenyl ring at

the 6-position (R₆= H, Table 2) yielded 16, which was found

to be a highly potent ApoA1 activator. This encouraged us to

make additional substituted analogues. Para substitution of the

6-phenyl ring was well tolerated (17−22), with polar groups

oﬀering some improvement both in activity and solubility. The

ring was particularly tolerant of highly polar functionality, for

example the carboxylic acid (19). Interestingly, the acid

substituent at this position did not negatively impact the

cellular activity of the molecule (compare the acid 19 to its

methyl ester 18).

Because of the tolerance of charged functionality on this ring,

a range of sizable substituents with varying degrees of basicity

(morpholine, piperazine, and piperidine) were prepared, for

example 23−25. These substituents were very well tolerated at

both 3- and 4- positions of the 6-phenyl ring, giving enhanced

solubility and similar small improvements in potency (Table 2).

Because of the excellent activity of the piperidine 25, it was

subjected to chiral separation to yield a pair of enantiomers,

compounds 28 and 29. Almost all of the ApoA1 reporter

activity resided in one enantiomer, 28. Although no sample of

the racemate (25) remained for testing by the time the

bromodomain assays were developed, the relative activity of the

pair of separated enantiomers in the BET assays mirrored that

in the ApoA1 reporter assay. The absolute stereochemistry of

the active enantiomer was later shown by X-ray crystallography

to be the (2S,4R) stereoisomer.

Two intermediates (26 and 27) prepared en route to 25

(Scheme 4, Synthetic Chemistry) were tested to determine the

eﬀect of removing the THQ 4-position aryl substituent. Both

were very much weaker in the ApoA1 and BET assays than 25

C

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or its active enantiomer 28 (Table 2), showing the importance

of the 4-anilino substituent for maintaining potency.

Additional properties of 28 are shown in Table 3. Its

solubility was good and in vitro clearance in hepatocytes from

comparable to the equivalent tertiary butyl 30, it seems likely

that the polarity of 32 negatively impacts its cell permeability.

However, the greatest ApoA1 activity was obtained with simple

4-methyl, 4-chloro, or 4-ﬂuoro substituents (33−35).

Ortho and meta substitution of the 4-anilino ring was also

well tolerated. For example, the 2-OMe analogue (39) was

comparable to the 3-OMe analogue 38.

Table 3. Some Properties of Compound 28

To summarize the above results, the THQ 4-position phenyl

ring showed only small substituent eﬀects on BET activity,

although more signiﬁcant diﬀerences were seen in cellular

ApoA1 up-regulation. Compounds with the 4-chlorophenyl

aniline at the THQ 4-position (such as 34) seemed to oﬀer the

best combination of properties and potency. Incorporating this

feature, the 6-position was then revisited to explore more

diversely substituted phenyls.

Combining the piperidine-containing 6-aryl group of 25 with

the chlorophenyl aniline 4-position of 34 led to 40, a molecule

which, although potent, did not aﬀord an advantage over 25 or

34. Moving the acid group of 34 from the para to the meta

position of the 6-aryl substitient, or replacing it with an ester at

the meta position, was slightly detrimental (41, 42).

Replacement of the acid by a primary carboxamide (43) had

little eﬀect. Other positions were rather more sensitive to

change, however. For instance, the addition of a small methyl

substituent at the ortho position of the THQ 6-phenyl

substituent led to a marked reduction of ApoA1 activity (44),

an observation that was also found later in the BET binding

assay.

28

peptide-based FRET (pIC₅₀)

LPS-stimulated IL6 pIC₅₀

BRD4

6.7 0.03 (2)

PBMC

HWB

8.5 0.2 (2)

a

7.2 0.6 (670)

FASSIF solubility

μg/mL

125

1.8

hepatocyte clearance (mL/min/g

tissue)

human

rat

0.8

0.9

6.0

dog

b

primate

P450 proﬁle IC₅₀(μM)

1A2 (ER)

>33

14

20

8

The THQ 2-methyl group was suspected to be important for

activity because of the preference for 28 over its enantiomer 29.

To further assess its signiﬁcance, an analogue of 21 lacking the

2-methyl substituent (45) was prepared and tested. This was

weaker than 21 in all assays, conﬁrming that the THQ 2-

position methyl substituent does play a role in both ApoA1

induction and BET binding.

2C9 (FCA)

2C19 (BMC)

2D6 (MMMC)

3A4 (7BQ)

3A4 (DEF)

10

2

a

This compound was used as a standard in the HWB assay: the mean

pIC₅₀excludes 3 occasions when ﬁtted values could not be obtained.

b

After this exploration, the most promising compounds were

33 and 34, which showed potency comparable to that of 25.

Pure enantiomers of these two compounds (46, 47) were

prepared according to Scheme 6, Synthetic Chemistry. These

molecules had very similar properties, so 47 (GSK1324726A, I-

BET726) was chosen as our leading molecule from this series.

Synthetic Chemistry. Compounds 9 and 13−15 were

purchased. All others were synthesized using the general routes

shown in Schemes 1−6.⁴⁰Compounds 10−12 were prepared

according to Scheme 1. Aniline was condensed with crotonic

acid, giving the Michael addition adduct, which was then

cyclized to 2-methyl-2,3-dihydroquinolin-4(1H)-one (50) by

heating in polyphosphoric acid (PPA) at 110 °C. Following

acetylation under standard conditions, reductive amination in

the presence of 4-methylaniline and titanium tetrachloride

followed by reduction with sodium borohydride gave

compounds 10 and 11 as a mixture of cis- and trans-

diastereoisomers. The two isomers were easily separated by

ﬂash chromatography.

Compounds 16−22, 36, 37, 41, 42, and 44 were synthesized

according to Scheme 2. 4-Bromoaniline was reacted with

acetaldehyde in the presence of benzotriazole in toluene at

room temperature. N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-

ethyl)-4-bromoaniline (52) was obtained in modest yield.

Reaction with N-vinyl formamide in the presence of a Lewis

acid such as BF₃·Et₂O in dry THF gave the hetero-Diels−Alder

adduct 53 as a racemic cis-mixture. The tetrahydroquinoline

nitrogen atom was then substituted with an acyl chloride in the

Cynomolgous monkey.

three species was low, giving grounds for optimism that the

series would have good pharmacokinetic properties. Com-

pound 28 was potent in an alternative BET bromodomain TR-

FRET assay measuring displacement of a labeled histone tail

peptide. Furthermore, it showed potent anti-inﬂammatory

activity in vitro, as measured by inhibition of the pro-

inﬂammatory cytokine IL6 in LPS-stimulated peripheral

blood mononuclear cells (PBMCs) and human whole blood

(HWB). However, moderate inhibition of multiple cytochrome

P450 isoforms was also found. Because P450 enzymes are

involved in the oxidative metabolism of statins, which are

widely used for treatment of atherosclerosis, an additional

consideration was to minimize any risk of drug−drug

interactions by reducing the P450 inhibition of compounds

which might be coadministered.³⁶

Our next step was to explore greater diversity at the THQ 4-

position. On the basis of the results described above for

compounds 18−19, it was decided to do this ﬁrst in

compounds substituted at the THQ 6-position by p-CO₂H

phenyl and p-CO₂Me phenyl. Para substitution of the THQ 4-

anilino ring was well tolerated (Table 4), with even quite

sterically bulky groups such as t-butyl retaining acceptable

activity (30). Introduction of groups of similar size but greater

polarity such as the methyl sulfone (31, 32) led to some loss of

ApoA1 potency. Because the BET activity of 32 was

D

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Table 4. Mean Potency of 30−47 in ApoA1 Reporter and BET Bromodomain Assays (*Denotes Single Enantiomer, Otherwise

a

Racemic Mixtures with cis Stereochemistry)

a

See Supporting Information Table S2 for standard deviation.

a

Scheme 1. Synthesis of Tetrahydroquinoline Compounds 10−11

a

Reagents and conditions: (a) toluene, reﬂux, 43%; (b) PPA, 110 °C, 29%; (c) AcCl, DMAP, 0 °C to RT, 86%; (d) 4-methylaniline, TiCl₄, TEA,

THF/DCM, 0 °C to RT then NaBH₄, RT, 15%.

presence of pyridine, giving 54. The formamide was

deprotected using 6 N HCl in reﬂuxing methanol to aﬀord

55, which reacted with phenylboronic acid under Chan−Lam−

Evans conditions. Finally, R₆was introduced using Suzuki

coupling. Tetrahydroquinoline compounds could also be

obtained from 54 using the same reagents and conditions in

a diﬀerent order, beginning with the installation of the R₆

substituent, as depicted in Scheme 3.

When the R₆group bears a hydrophilic basic heterocycle

such as piperidine or morpholine, compounds such as 23−26

and 40 could be obtained from intermediate 54 as shown in

Scheme 4, beginning with Suzuki coupling with 3- or 4-

formylphenylboronic acid. Reductive amination in the presence

of the desired cyclic amine and sodium triacetoxyborohydride

aﬀorded intermediates of general form 59 which, after

deprotection and copper-mediated Chan−Lam−Evans cou-

pling, gave products 23−25.

Compound 45, lacking the tetrahydroquinoline 2-position

methyl substituent, was prepared according to Scheme 5. 2,3-

Dihydroquinolin-4(1H)-one was brominated at position 6 in

the presence of NBS in DCM. The tetrahydroquinoline

nitrogen atom was then substituted using an acetic anhydride,

giving 61. A Suzuki coupling reaction was used to introduce the

4-methoxyophenyl substituent at position 6, aﬀording the 6-aryl

tetrahydroquinolinon-4-one. Finally, the substituent at position

4 was introduced by reductive amination with aniline in the

presence of titanium tetrachloride before reduction with

sodium triacetoxyborohydride, giving compound 45.

47 can also be prepared in an eight-step synthesis starting

from isopropyl carbamate. Reaction with crotonyl chloride at

−20 °C formed the but-2-enoyl carbamate 63. An aza-Michael

E

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a

Scheme 2. Representative Synthesis of Tetrahydroquinoline Compounds 16−22, 36−37, 41−42, and 44

a

Reagents and conditions: (a) MeCHO, toluene, RT, 82%; (b) N-vinylformamide, BF₃·Et₂O, THF, −5 °C, 56%; (c) MeCOCl, DCM, pyridine, 0

°C, 100%; (d) MeOH, 6 N HCl, reﬂux, 91%; (e) PhB(OH)₂, Cu(OAc)₂, TEA, DCM, RT; (f) R₆B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, DME, H₂O, reﬂux.

Scheme 3. Alternative Synthesis of Tetrahydroquinoline

Compounds, e.g., 30−35, 38−39, and 43

Scheme 5. Synthesis of 2-H Tetrahydroquinoline Compound

45

a

Reagents and conditions: (a) R₆B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, DME,

H₂O, reﬂux; (b) MeOH, 6 N HCl, reﬂux; (c) R₆PhB(OH)₂,

Cu(OAc)₂, TEA, DCM, RT.

a

Reagents and conditions: (a) NBS, DCM, 0 °C to RT, 86%; (b)

Ac₂O, 80 °C, 69%; (c) 4-MeOPhB(OH)₂, Pd(PPh₃)₄, Cs₂CO₃, DME,

H₂O, reﬂux, 67%; (d) aniline, TiCl₄, TEA, THF/DCM, 0 °C to RT

then NaBH(OAc)₃, AcOH, RT, 15%.

reaction with 4-bromoaniline was achieved stereoselectively

under palladium catalyzed conditions, then cyclized to give the

syn tetrahydroquinoline 65 using sodium borohydride and

magnesium chloride.⁴¹N-Acylation of the tetrahydroquinoline

was followed by a Suzuki coupling of the 6-bromo group with

{4-[(ethoxy)carbonyl]phenyl}boronic acid to give 67. Depro-

tection of the 4-aminotetrahydroquinoline was achieved using

aluminum chloride and triethylamine to give 68. The

mechanism of this deprotection is not fully understood but

requires between 3 and 4 equiv of aluminum chloride; typically,

we used 3.6 equiv to achieve full conversion. Fewer than 3

equiv of aluminum chloride gave no reaction, while 4 or more

equiv of aluminum chloride, by contrast, resulted in the very

a

Scheme 4. Synthesis of Basic Tetrahydroquinoline Compounds, e.g., 23−26 and 40

a

Reagents and conditions: (a) 3- or 4-formylPhB(OH)₂, Pd(PPh₃)₄, Na₂CO₃, DME, H₂O, reﬂux; (b) NaBH(OAc)₃, AcOH, cyclic II^aryamines,

solvent, RT; (c) MeOH, 6M HCl, reﬂux; (d) R₄PhB(OH)₂, Cu(OAc)₂, TEA, DCM, RT.

F

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a

Scheme 6. Enantioselective Route to Compound 47

a

Reagents and conditions: (a) crotonyl chloride, lithium tert-butoxide, THF, −20 °C, 29%; (b) 4-bromoaniline, (R-BINAP)Pd(MeCN)₂(OTf)₂,

toluene, THF, 20 °C, 67%, ee = 92%; (c) NaBH₄, MgCl₂·6H₂O, EtOH, H₂O, −15 °C, 78%, 1 cis-isomer, ee = 92%; (d) AcCl, pyridine, CH₂Cl₂, 0°C,

78%, puriﬁcation by chiral chromatography, ee >99%; (e) {4-[(ethyloxy)carbonyl]phenyl}boronic acid, Pd(PPh₃)₄, NaHCO₃, EtOH, H₂O, 80 °C,

92%; (f) AlCl₃, Et₃N, MeOH, CH₂Cl₂, 0 °C, 98%; (g) 1-bromo-4-chlorobenzene, DavePhos, Bis(dibenzylideneacetone)palladium, sodium tert-

butoxide, toluene, 70 °C, 37% (94% based on recovered starting material); (h) 2M NaOH, EtOH, RT, 77%.

rapid degradation of the starting material to a mixture of

unidentiﬁed products. The Hartwig−Buchwald reaction with 1-

bromo-2-chlorobenzene is sensitive to the quality of the sodium

tert-butoxide as any acid formed by ester hydrolysis of 68 is

unreactive and remains as a byproduct following the reaction.

Basic ester hydrolysis completed the synthesis of 47.

Table 5. Properties of Compound 47

Potency and Selectivity. Knowing that compound 47 is a

BET bromodomain inhibitor, we proﬁled it in additional

binding and selectivity assays. Besides having activity in the FA

assay, 47 is a potent competitor of labeled histone peptide

binding to all four BET isoforms in TR-FRET format, with IC₅₀

at or below 100 nM against each (Table 5). To conﬁrm this,

the K_dwas measured by surface plasmon resonance (SPR) and

determined to be 20−25 nM (Supporting Information, Figure

S2). Isothermal titration calorimetry (ITC) results suggested

that 47 binds to the tandem bromodomain of His-tagged-

BRD4 (1−477) with 2:1 stoichiometry, consistent with the

binding of one molecule at each bromodomain site, with high

aﬃnity (K_D4.4 0.9 nM).³⁷

47

peptide-based FRET (pIC₅₀)

BRD2

BRD3

BRD4

BRDT

7.4 0.06 (6)

7.1 0.03 (5)

7.0 0.20 (39)

7.3 0.25 (4)

K_dby SPR (nM)

BRD2

BRD3

BRD4

25

20

23

8

5

7

K_dby ITC (nM)³⁷

BRD4

4.4 0.9

Compound 47 was tested in a range of ﬂuorescence thermal

melting assays (Supporting Information Table S3) and showed

no signiﬁcant interaction with any bromodomain tested other

than the BET family and CREBBP. Consistent with this, in a

panel of recombinant bromodomain acetyl-lysine site directed

binding assays (Table 6, Supporting Information Figure S1),

only weak activity was found against other bromodomains apart

from CREBBP and EP300. To estimate the true selectivity for

BRD4 over CREBBP, isothermal titration calorimetry experi-

ments were carried out, which conﬁrmed that, as expected, 47

binds in a 1:1 ratio with the single bromodomain of CREBBP

and that the K_dwas 6.3 μM, >1000-fold weaker than that for

BRD4.³⁷

CREBBP

6300 500

LPS-stimulated IL6 pIC₅₀

solubility (μg/mL)

PBMC

HWB

7.3 0.25 (20)

6.4 0.2 (12)

FASSIF

106

(<25%), indicating a high degree of selectivity against those

classes of nonbromodomain targets included.

Preclinical Developability Proﬁle. Compound 47

showed an improved P450 proﬁle over 28 (Table 7), with

low potential to inhibit the major human CYP isoforms (IC₅₀

values >10 μM). In vitro clearances determined with liver

hepatocytes were low across all species, including human (CLi

<1.7 mL/min/g liver). Compound 47 had medium perme-

ability in an artiﬁcial membrane assay. However, using singly

expressed human recombinant enzymes 47 was shown to be a

To assess its selectivity against targets unrelated to

bromodomains, 47 was screened in a panel of 50 receptor,

ion channel and transporter assays (Supporting Information,

Table S4). No signiﬁcant activity was found at 1 or 10 μM

G

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a

Table 6. Selectivity of 47

Table 7. Properties of Compound 47

bromodomain

modiﬁer

pIC₅₀

47

ATAD2

<

4.5

8.5

9.2

8.7

9.4

8.5

9.4

9.6

8.9

4.5

8.4

9.0

4.5

6.5

6.2

4.5

5.1

4.5

5.0

4.8

5.0

4.8

4.5

4.7

hepatocyte clearance

(mL/min/g tissue)

mouse

<0.85

ATAD2B

rat

<0.85

<1.7

BAZ2A

dog

BAZ2B

a

primate

human

<0.85

BPTF/FALZ

BRD1

BRD2(1)

b

P450 proﬁle IC₅₀(μM)

1A2 (ER)

>33

13

BRD2(2)

2C9 (FCA)

BRD3(1)

2C19

(BMC)

12

BRD3(2)

BRD4(1)

2D6

(MMMC)

>33

BRD4(2)

BRD4(1,2)

BRD4 (full-length, short isoform)

BRD1

3A4 (7BQ)

3A4 (DEF)

32

21

<

BRD7

time-dependent P450

inhibition

3A4 (DEF)

4-fold shift in IC₅₀over 30 min

(>100 μM to 24 μM)

BRD9

BRDT(1)

permeability (nm/s)

artiﬁcial

membrane

95

BRDT(2)

BRPF1

<

BRPF3

c

free fraction (F_ub)

mouse

rat

0.013

0.009

0.065

0.017

CECR2

CREBBP

dog

human

b

EP300

GCN5

<

a

Cynomolgus monkey. Fluorometric assay with Cypex cDNA

PBRM1(2)

PBRM1(5)

PCAF

c

expressed isoforms. Measured using rapid equilibrium dialysis at a

nominal concentration of 1000 ng/mL. Assay performed using blood.

<

SMARCA2

TAF1(2)

Oral Activity. We have previously reported the activity of

compound 47 in neuroblastoma assays.³⁷Because of the links

between ApoA1 up-regulation, BET inhibition, and inﬂamma-

tory disease that emerged as this work was underway,⁷

compound 47 was assessed in an acute mouse inﬂammation

model. After a single oral administration of compound at

various concentrations followed by intraperitoneal LPS

challenge, the eﬀect on hepatic mRNA expression of the

acute phase protein PAI-1 was measured. The compound

showed dose-dependent inhibition of PAI-1 expression, with

very signiﬁcant activity already observed at the 3 mg/kg dose

(Figure 2).

Intravenous Activity in a Mouse Septic Shock Model.

Sepsis is an acute inﬂammatory condition characterized by high

mortality rates and a signiﬁcant unmet medical need despite

considerable research eﬀort.³⁸Therefore, compound 47 was

tested in a mouse septic shock model using a therapeutic mode

of treatment (dosing after induction of sepsis). An intra-

peritoneal injection of LPS (15−20 mg/kg) was ﬁrst

administered to Balb/c mice. When clinical signs were

observed, compound 47 was intravenously dosed at 10 mg/

kg. Only 25% of mice in the vehicle group survived, while 56%

of mice treated with compound survived after 5 days (Figure

3), indicating that compound 47 is able to prevent death of

diseased animals following a single administration.

TAF1L(2)

TIF1α/TRIM24 (BD)

TIF1α/TRIM24 (PHD + BD)

TIF1γ/TRIM33 (PHD + BD)

<

WDR9(2)

a

Activity in BROMOscan^SMrecombinant protein binding assays

carried out at DiscoveRx (http://www.discoverx.com).

time-dependent inhibitor of CYP3A4 based on a 4-fold shift in

IC₅₀. Its free fraction was also relatively low.

The pharmacokinetic properties of compound 47 were

investigated in four preclinical species: mouse, rat, dog, and

nonhuman primate (Table 8). It had a low-moderate clearance

across the preclinical species (<30% liver blood ﬂow).

Consistent with this, 47 was stable in vitro in hepatocytes

from preclinical species. The in vitro/in vivo clearance

correlation across preclinical species indicates that, based on

human in vitro clearance, human blood clearance could be low

to moderate. The volume of distribution was low to moderate

(0.5−1.0 L/kg), indicating some distribution into the tissues.

Elimination half-life was moderate in dog and primate (1.8 and

2.7 h, respectively) and longer in mouse (3.6 h). Compound 47

therefore has the potential to be administered once or twice

daily in human. Oral bioavailability was moderate in the rat (ca.

28%) and higher in mouse, dog, and primate (90%, 80%, and

100%, respectively). The systemic exposure in mouse was

suitable for exploration of the pharmacodynamics in vivo, and

exposures achieved in the rat and dog via the oral route allow

for progression to safety assessment evaluation in these species.

Binding Modes of Optimized Tetrahydroquinolines.

In a previous publication, we showed the structure of the

racemic tetrahydroquinoline 48 (Figure 4A) as an example of a

fragment binding to the N-terminal bromodomain of BRD2.²⁹

The amino acids of the BET family of bromodomains are

highly conserved in the acetyl-lysine pocket (Figure 4B), so it is

not surprising that 47 in BRD4 inherits the general binding

H

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Table 8. Preclinical Pharmacokinetic Parameters for Compound 47 (Values are Mean n = 3, IV Infusion at 1 mg/kg, and PO

Dose at 3 mg/kg unless Otherwise Stated)

species

CLb (mL/min/kg)

3.1 0.4

V_ss(L/kg)

t_1/2iv (h)

AUC_0−tpo (ng·h/mL)

F_po(%)

a

mouse

0.48 0.1

2.0 0.4

0.8 0.1

1.0 0.4

3.6 0.2

>8

6028

45

b

rat

26

4

1

539 96

28

1

beagle dog

6.0

1.8 0.6

2.7 0.1

5900 968

10968 3852

80 12

c

primate

3.0 0.9

103 35

a

b

c

Balb/c bolus IV dose at 10 mg/kg. Terminal t_1/2estimated from n = 1 rat. Cynomolgus monkey, IV infusion at 1 mg/kg, and PO dose at 2 mg/

kg. Parameters are from plasma analysis.

the THQ template mimic the interactions made by the acetyl-

lysine side chain found in X-ray structures of the BET

bromodomains bound to acetylated peptides (Figure 5B).^29,39

The methyl group of the N1-acetyl of the THQ ﬁlls the small

pocket between the buried water chain and the aromatic side

chain of Phe83 in the same position as the terminal methyl of

the acetyl lysine side chain.

The crystal structure of the racemic fragment 48 in BRD2

showed that the bound enantiomer had (S) absolute chirality at

the C2 position and that the 2-methyl group ﬁlled a small

lipophilic pocket near Leu110 in the ZA loop. The C2 methyl

group of 47 fulﬁls the same role and makes direct contact with

the analogous BRD4 residue Leu94 (Figure 5C). The vector

projecting from the C2 hydrogen points toward the side chain

carbonyl oxygen of Asn140, which is only 3.5 Å away (Figure

5A). Even a substituent as small as a methyl group would clash

with this carbonyl oxygen, explaining the (2S) stereochemical

requirement for activity.

Figure 2. Oral activity of 47 in a murine acute LPS challenge model:

PAI-1 mRNA relative to vehicle (mean SEM, n = 8). (*** p value

<0.0001 following a t test comparison to the vehicle-treated group).

The unsaturated ring of the THQ is sandwiched by

hydrophobic residues (mainly Pro82 and Leu92) in a narrow

lipophilic region at the base of the ZA channel (Figure 5C).

The 6-substituent phenyl ring extends further through this

channel toward solvent, passing close by Trp81 in an edge-to-

face arrangement. This location explains the gross SAR seen at

the 6-position in the BET binding assays. The presence of the

6-aryl ring in compounds such as 16 increases BET potency

compared to analogues like 12 because of lipophilic interactions

with the channel, particularly with Trp81. The crystal structure

also shows that the meta and especially the para positions of the

6-aryl ring point away from the protein surface, explaining the

wide range of tolerated substitutions including acidic and basic

groups in compounds 17−25. The carboxylate of 47 makes no

direct interaction with the protein but does lie in the rough

vicinity of the side chain of Lys91, although because of its

ﬂexibility this probably does not contribute much to the

binding free energy. The ortho position of the 6-aryl ring oﬀers

greater opportunities for interaction with the protein. One

ortho position lies close to the BRD4 WPF motif (Trp81−

Pro82−Phe83), with limited room for growth (Figure 5B). The

opposite ortho position has more accessible space, but the SAR

shows that substitution is detrimental to binding (compound

44). This can be understood by considering the torsion angle

between the THQ core and the 6-phenyl ring, which is about

30°. Ortho groups would increase this torsion angle and the

width of the ligand within the ZA channel, leading to closer

steric contacts against Trp81 and Leu92.

Figure 3. Intravenous activity of 47 in a murine septic shock model

(mean of two independent experiments with n = 8 per group in each

study).

mode of fragment 48 in BRD2 (Figure 5A). This includes the

key directional interactions between the THQ scaﬀold and the

acetyl-lysine site of BRD4, namely the hydrogen bond between

the N1-acetyl group and the side chain NH₂of the conserved

asparagine Asn140. The N1-acetyl group forms a second,

shorter hydrogen bond to a water molecule, which itself

hydrogen bonds to the side chain hydroxyl of Tyr97. Our

analysis of fragment complexes with BRD2 suggested that the

water interaction may be the stronger of the two. This water

molecule is one of a chain of four conserved within

bromodomain crystal structures published to date. Lying at

the bottom of the acetyl-lysine pocket, these form a highly

satisﬁed H-bonding network connecting Tyr97 to the backbone

carbonyls of Pro82 and Gln85. The H-bonding interactions of

The THQ 4-position anilino linker adopts near optimal

torsional angles for this group and directs its substituent

chlorophenyl ring toward the hydrophobic “WPF shelf”

(named because of its proximity to the conserved bromodo-

main WPF motif deﬁned above). This lipophilic site has been

shown to be important in other classes of BET inhibitor,^30,31,33

I

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Figure 4. (A) Tetrahydroquinoline fragment 48. (B) Conservation of the BET family and CREBBP bromodomain acetyl-lysine sites. Colors

represent residue properties: green = hydrophobic, red = acidic, blue = basic, orange = polar, purple = proline, white = small. * denotes residue is in

contact (≤4.5 Å) with compound 47 in BRD4 N-terminal bromodomain crystal structure (PDB 3uvw).³⁹

and the poor activity of 26 and 27 which lack this substituent

supports its importance in the THQ series. One edge of the

chlorophenyl ring makes direct hydrophobic contacts with the

WPF shelf (the 2-position with Ile146, and the 3-position with

Trp81 and Pro82) but the opposite face points toward solvent

(Figure 5C). There is therefore plenty of space for ortho or

meta substituents on this ring, explaining the rather ﬂat

substituent eﬀects on activity at these positions. The 4-chloro

group lies near Trp81 and contacts the hydrophobic side chain

of Met149, although this is near the outside of the shelf and

exposed to solvent, so this might make relatively small

contributions to activity. Because all of the interactions made

by the 4-anilino chlorophenyl ring are hydrophobic, they are

not very directional, and modiﬁcations to the ring may be

accommodated by relatively small sliding movements over the

WPF shelf surface, as found previously in a dimethyl isoxazole

series.³¹

Comparison of Binding Mode to Selected Literature

BET Inhibitors. Figure 6A shows the BRD4 N-terminal

bromodomain binding mode of THQ 47 superimposed on that

of compound 1.⁴These diﬀerent chemotypes overlay very

closely, with the N1 acetyl group of 47 fulﬁlling a similar role to

the triazole ring of 1 in its interaction with Asn140 and the

conserved water network. The polycyclic cores of both

inhibitors also overlay very closely, as do the chlorophenyl

rings of each, which approach the WPF shelf from the same

direction and make similar interactions (Figure 6A). Com-

pound 3 binds in a very diﬀerent manner, both in chemical

structure and also in the directionality of the approach of its

aryl ring to the WPF shelf (Figure 6B).²⁵

N-terminal bromodomain of BRD4. This is as expected, given

the conservation of residues involved in direct interactions with

the inhibitor (Figure 4B). The only diﬀerences are relatively

small movements of the 4-anilino chlorophenyl group. Because

of the nonpolar, weakly directional nature of the interactions

between the chlorophenyl and the WPF shelf, these small

movements do not translate into signiﬁcant diﬀerences in

potency between the BET isoforms (Table 5).

An X-ray structure of the N-terminal bromodomain of BRD2

was also solved in complex with 28. The majority of the

structure is well resolved in chains A and B, although the ligand

is not visible in chain C. Compound 28 adopts a very similar

binding mode to that of 47 (Figure 7C), but the electron

density for the methylpiperidine is weak (Supporting

Information Figure S3) and in neither chain does this group

make apparent strong interactions. This is consistent with the

small eﬀect of this substituent on binding aﬃnity (e.g., 28

relative to 19).

CONCLUSION

■

The discovery and optimization of the tetrahydroquinoline

series which led to 47 was accomplished without the beneﬁt of

any knowledge of the molecular target. Its molecular

mechanism is now well understood. This series joins a growing

list of diﬀerent chemotypes which produce potent biological

eﬀects by binding to the BET family of bromodomains. We

have recently shown that 47 is a potent suppressor of MYCN

expression and survival in neuroblastoma cell lines and

xenograft growth.³⁷Here, we have described its discovery and

potent anti-inﬂammatory eﬀects. The THQ series oﬀers

chemical tractability and an attractive range of properties

suitable for optimization and progression to preclinical

development. Further publications will describe more of the

Binding Mode in other BET Bromodomains. X-ray

structures of 47 were also solved in complex with the N-

terminal and C-terminal bromodomains of BRD2 (Figure

7A,B). Both binding modes are very similar to that seen in the

J

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Figure 5. Crystal structure of 47 (cyan carbons) in the BRD4 N-terminal bromodomain (blue carbons). The conserved H-bonding network between

inhibitor, protein, and conserved waters is marked in yellow. (A) Comparison with X-ray structure of fragment 48 in BRD2 N-terminal domain

(orange). Black dotted line indicates the 3.5 Å distance between the C2 carbon of 47 and the side chain oxygen of Asn140. (B) Comparison to

acetyl-lysine (purple carbons) taken from X-ray structure of histone H4(1-12)K5AcK8Ac in BRD4 (PDB 3uvw).³⁹(C) Key hydrogen-bonding

interactions of 47 with BRD4.

Figure 6. (A) THQ 47 (cyan carbons) bound to BRD4 N-terminal bromodomain, with BRD4 molecular surface generated with modeled hydrogen

atoms, aligned to 1 (yellow, PDB 3p5o). (B) Binding mode of 47 in BRD4 N-terminal bromodomain compared to that of 3 (yellow, PDB 3zyu).

K

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reactions were followed by TLC analysis (TLC plates GF254, Merck)

or liquid chromatography mass spectrometry (LCMS) using a Waters

ZQ instrument. NMR spectra were recorded on a Bruker NanoBay

400 MHz or a Bruker AV++ 600 MHz spectrometer and are

1

referenced as follows. H NMR (400 or 600 MHz), internal standard

TMS at δ = 0.00. ¹³C NMR (100.6 or 150.9 MHz), internal standard

CDCl₃at δ = 77.23 or DMSO-d₆at δ = 39.70. Column

chromatography was performed on prepacked silica gel columns

(30−90 mesh, IST) using a Biotage SP4. Mass spectra were recorded

on Waters ZQ (ESI-MS) and Q-Tof 2 (HRMS) spectrometers. Mass

directed auto prep was performed on a Waters 2767 with a MicroMass

ZQ mass spectrometer using Supelco LCABZ++ column. Reactions

performed under microwave irradiation utilized either a Biotage

Initiator or CEM Discover Microwave.

Abbreviations for multiplicities observed in NMR spectra: s; singlet;

br s, broad singlet; d, doublet; t, triplet; q, quadruplet; p, pentuplet;

spt, septuplet; m, multiplet. The purity of all compounds was

determined by LCMS and ¹H NMR and was always >95%. For

detailed LCMS/MDAP methodology see Supplementary Methods,

Supporting Information.

General Procedure for the Formamide Deprotection

(Method A). To a suspension of formamide (1 equiv) in MeOH

(0.3 mol/L) was added 6 N HCl (3 equiv). The resulting mixture was

stirred at reﬂux for 3 h, and the medium was made basic by the

addition of 2N NaOH. The MeOH was evaporated under reduced

pressure and the organic material extracted with EtOAc. The organic

layer was washed with brine, dried over Na₂SO₄, and concentrated to

dryness to aﬀord the desired primary amine.

General Procedure for the Chan−Lam Coupling (Method B).

A mixture of the amine (1 equiv), phenyl boronic acid (3 equiv),

anhydrous copper acetate (1.5 equiv), and triethylamine (3 equiv) in

dry DCM (0.3 mol/L) was stirred at room temperature for 48−72 h.

Progression of the reaction was monitored by TLC, and if necessary an

additional equivalent of phenyl boronic acid and triethylamine was

added and the mixture allowed to stir at room temperature for a

further 12 h. This operation was repeated until the amount of the

expected product exceeded the remaining proportion of the starting

intermediate. The resulting mixture was poured into water, and the

organic phase was extracted with DCM, dried over Na₂SO₄, ﬁltered,

and evaporated to dryness. The residue was puriﬁed by recrystalliza-

tion or chromatography on silica gel.

General Procedure for the Suzuki Coupling (Method C). To a

solution of bromo derivative (100 mg, 0.28 mmol) in 1,2-

dimethoxyethane (0.1 mol/L) was added boronic acid or the

corresponding boronate ester (1.5 equiv), a 2N solution sodium

carbonate in water (5 equiv), and Pd(PPh₃)₄(0.05 equiv), and the

mixture was stirred under reﬂux for 20 h. The solvent was then

removed under reduced pressure, and EtOAc was added to the

residue. The organic phase was washed with brine, dried over Na₂SO₄,

ﬁltered, and evaporated to dryness. The residue was puriﬁed by

recrystallization or chromatography on silica gel.

General Procedure for Ester Hydrolysis (Method D). A

solution of ester (1 equiv) in EtOH (0.1 mol/L) and 1N NaOH (2

equiv) was heated to 60 °C. After 1 h, the mixture was evaporated to

dryness and the residue taken up in water. Acetic acid was added until

PH 4−5, and the aqueous layer was extracted with DCM (2×). The

combined organic layers were washed with brine and dried over

Na₂SO₄. After concentration under reduced pressure, the title

compound was obtained as a solid.

Figure 7. Comparison between X-ray structures of 47 in complex with

BRD4 N-terminal bromodomain (cyan/blue) and (A) BRD2 N-

terminal bromodomain (pink/purple), with BRD4 residue numbering,

and (B) BRD2 C-terminal bromodomain (yellow/orange), with BRD2

residue numbering. (C) Comparison of X-ray structures of BRD2 N-

terminal bromodomain in complex with 47 (pink/purple) and with 28

(chain A, green), with BRD2 N-terminal numbering.

General Procedure for Reductive Amination (Method E). To

a solution of aldehyde (1 equiv) and piperidine (1.2 equiv) in solution

in dichloroethane (0.1 mol/L) were added at room temperature

triacetoxysodiumborohydride (1.3 equiv) and acetic acid (2.2 equiv).

After stirring 2 h, the mixture was poured into a saturated aqueous

solution of NaHCO₃. The organic layer was extracted with DCM and

washed with brine, dried over Na₂SO₄, ﬁltered, and evaporated to

dryness. The residue was puriﬁed by recrystallization or chromatog-

raphy on silica gel.

medicinal chemistry and biology around this template targeting

a range of therapeutic areas.

EXPERIMENTAL SECTION

■

General Experimental Methods (Chemistry). All solvents were

purchased from Sigma-Aldrich (Hy-Dry anhydrous solvents) and

commercially available reagents were used as received. Melting points

were recorded on Buchi B-545 apparatus and are uncorrected. All

cis-1-(2-Methyl-4-(p-tolylamino)-3,4-dihydroquinolin-1(2H)-

yl)ethanone (10) and trans-1-(2-Methyl-4-(p-tolylamino)-3,4-

L

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dihydroquinolin-1(2H)-yl)ethanone (11). To a solution of 1-

acetyl-2-methyl-2,3-dihydroquinolin-4(1H)-one (51) (1 g, 4.9 mmol),

4-methylaniline (0.63 g, 5.9 mmol) and TEA (3.0 mL, 21.2 mmol) in

THF (50 mL) at 0 °C was added TiCl₄(1 M in CH₂Cl₂, 7.4 mL, 7.4

mmol). The reaction mixture was stirred for 2 h at room temperature,

and TiCl₄(1 M in CH₂Cl₂, 4.9 mL, 4.90 mmol) was added again. After

3 more hours, sodium borohydride (1.49 g, 39.36 mmol) was added

portion by portion at room temperature. The reaction mixture was

stirred for 18 h then poured into a 2 M aqueous solution of NaHCO₃.

The organic layer was extracted with CH₂Cl₂and washed with brine,

dried over Na₂SO₄, ﬁltered, and evaporated to dryness to aﬀord a

mixture of two isomers cis/trans. The residue was puriﬁed by

chromatography on silica gel (5% MeOH in CH₂Cl₂) to provide the

two isomers. Compound 10: isolated as an orange oil (65 mg, 4.5%).

¹H NMR (300 MHz, CDCl₃) δ ppm 7.30 (m, 1H), 7.21 (m, 1H), 7.10

(m, 2H), 6.90 (m, 1H), 6.55 (m, 1H), 4.82 (m, 1H), 4.51 (m, 1H),

2.50 (m, 1H), 2.19 (m, 1H), 2.16 (s, 3H), 2.09 (s, 3H), 1.69 (m, 2H),

1.13 (m, 1 H), 1.10 (d, J = 6.6 Hz, 3H). Compound 11: isolated as an

MHz, DMSO-d₆) δ 8.00 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H),

7.63 (d, J = 2.2 Hz, 1H), 7.54 (d, J = 1.5 Hz, 1H), 7.46 (d, J = 8.1 Hz,

1H), 7.12 (m, 2H), 6.74 (d, J = 7.6 Hz, 2H), 6.59 (t, J = 7.8 Hz, 1H),

6.05 (d, J = 8.1 Hz, 1H), 4.67−4.79 (m, 1H), 4.26−4.36 (m, 1H), 3.86

(s, 3H), 2.58−2.71 (m, 1H), 2.08 (s, 3H), 1.18−1.31 (m, 1H), 1.10 (d,

J = 6.4 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-2-methyl-4-(phenylamino)-1,2,3,4-tet-

rahydroquinolin-6-yl)benzoic Acid (19). Method D was used to

hydrolyze methyl 4-((2S*,4R*)-1-acetyl-2-methyl-4-(phenylamino)-

1,2,3,4-tetrahydroquinolin-6-yl)benzoate (18); mp 246 °C. LCMS

1

(method formate): retention time 0.70 min, [M − H]⁻= 399.38. H

NMR (400 MHz, DMSO-d₆) δ 12.20−13.44 (br s, 1H), 7.97 (d, J =

8.3 Hz, 2H), 7.59−7.67 (m, 3H), 7.52 (d, J = 1.5 Hz, 1H), 7.45 (d, J =

8.3 Hz, 1H), 7.11 (dd, J = 7.4, 8.4 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H),

6.59 (t, J = 7.4 Hz, 1H), 6.05 (d, J = 8.1 Hz, 1H), 4.73 (m, 1H), 4.31

(m, 1H), 2.63 (m, 1H), 2.15 (s, 3H), 1.24 (m, 1H), 1.10 (d, J = 6.4

Hz, 3H).

1 - ( ( 2 S * , 4 R * ) - 2 - M e t h y l - 4 - ( p h e n y l a m i n o ) - 6 - ( 4 -

(triﬂuoromethyl)phenyl)-3,4-dihydroquinolin-1(2H)-yl)-

ethanone (20). Method C was used starting from 1-((2S*,4R*)-6-

bromo-2-methyl-4-(phenylamino)-3,4-dihydroquinolin-1(2H)-yl)-

ethanone (56) and (4-(triﬂuoromethyl)phenyl)boronic acid; mp 85

°C. LCMS (method formate): retention time 1.36 min, [M + H]⁺=

425.18. ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (m, 4H), 7.63 (dd, J =

2.1, 8.2 Hz, 1H), 7.51 (d, J = 1.2 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H),

7.11 (dd, J = 7.8, 7.8 Hz, 2H), 6.73 (d, J = 7.8 Hz, 2H), 6.58 (t, J = 7.3

Hz, 1H), 6.06 (d, J = 7.8 Hz, 1H), 4.72 (m, 1H), 4.31 (m, 1H), 2.64

(m, 1H), 2.16 (s, 3H), 1.25 (m, 1H), 1.11 (d, J = 6.4 Hz, 3H).

1-((2S*,4R*)-6-(4-Methoxyphenyl)-2-methyl-4-(phenylami-

no)-3,4-dihydroquinolin-1(2H)-yl)ethanone (21). Method C was

used starting from 1-((2S*,4R*)-6-bromo-2-methyl-4-(phenylamino)-

3,4-dihydroquinolin-1(2H)-yl)ethanone (56) and (4-methoxyphenyl)-

boronic acid; mp 86 °C (becomes gummy). LCMS (method formate):

1

orange oil (130 mg, 9%). H NMR (300 MHz, CDCl₃) δ ppm 7.25

(m, 2H), 7.11 (m, 2H), 6.92 (m, 2H), 6.48 (m, 2H), 4.82 (m, 1H),

4.14 (m, 1H), 3.63 (m, 1H), 2.56 (m, 1H), 2.18 (s, 3H), 2.11 (s, 3H),

1.19 (m, 1H), 1.07 (d, J = 6.4 Hz, 3H). LCMS (method formate, 5

min): retention time 2.07 min, [M + H]⁺= 294.0. HRMS (M + H)⁺

294.18.

1-((2S*,4R*)-2-Methyl-4-(phenylamino)-3,4-dihydroquino-

lin-1(2H)-yl)ethanone (12). To a solution of 1-acetyl-2-methyl-2,3-

dihydroquinolin-4(1H)-one (51) (4.06 g, 20 mmol) and aniline (3.65

mL, 40 mmol) in CH₂Cl₂(300 mL) at 0 °C was added TiCl₄(1 M in

CH₂Cl₂, 30 mL, 30 mmol) over 30 min. The reaction mixture was

allowed to warm to room temperature and stirred for 16 h. NaBH₄

(6.1 g, 160 mmol) was then added portion by portion at room

temperature. The resulting mixture was stirred for 4 h and then was

treated with a 10% w/w Na₂CO₃aqueous solution. The layers were

separated, and the organic phase was extracted with CH₂Cl_2.The

combined organic phases were dried over Na₂SO₄, ﬁltered, and

evaporated to dryness to aﬀord a mixture of two isomers cis/trans. The

residue was puriﬁed by chromatography on silica gel (5% MeOH in

CH₂Cl₂) to give 12 (3.2 g, 57%) as an orange oil. LCMS (method

formate): retention time 1.09 min, [M + H]⁺= 281.24. ¹H NMR (400

MHz, DMSO-d₆) δ 7.22−7.33 (m, 2H), 7.16 (m, 2H), 7.09 (m, 2H),

6.65 (d, J = 7.6 Hz, 2H), 6.56 (t, J = 7.3 Hz, 1H), 6.02 (d, J = 7.8 Hz,

1H), 4.71 (m, 1H), 4.18 (m, 1H), 2.59 (m, 1H), 2.08 (s, 3H), 1.19 (m,

1H), 1.4 (d, J = 6.4 Hz, 3H).

1-((2S*,4R*)-2-Methyl-6-phenyl-4-(phenylamino)-3,4-dihy-

droquinolin-1(2H)-yl)ethanone (16). Method C was used starting

from 1-((2S*,4R*)-6-bromo-2-methyl-4-(phenylamino)-3,4-dihydro-

quinolin-1(2H)-yl)ethanone (56) and phenylboronic acid; mp 186

°C. LCMS (method formate): retention time 1.28 min, [M + H]⁺=

357.28. ¹H NMR (400 MHz, DMSO-d₆) δ 7.48−7.58 (m, 3H), 7.38−

7.47 (m, 4H), 7.28−7.35 (m, 1H), 7.10 (m, 2H), 6.73 (d, J = 7.8 Hz,

2H), 6.58 (t, J = 7.3 Hz, 1H), 6.06 (d, J = 7.8 Hz, 1H), 4.73 (m, 1H),

4.28 (m, 1H), 2.63 (m, 1H), 2.10 (s, 3H), 1.24 (m, 1H), 1.10 (d, J =

6.4 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-2-methyl-4-(phenylamino)-1,2,3,4-tet-

rahydroquinolin-6-yl)benzonitrile (17). Method C was used

starting from 1-((2S*,4R*)-6-bromo-2-methyl-4-(phenylamino)-3,4-

dihydroquinolin-1(2H)-yl)ethanone (56) and (4-cyanophenyl)-

boronic acid; mp 104 °C. LCMS (method formate): retention time

1.19 min, [M + H]⁺= 382.26. ¹H NMR (400 MHz, DMSO-d₆) δ 7.89

(d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.61−7.69 (m, 1H),

7.44−7.55 (m, 2H), 7.11 (d, J = 7.7, 7.8 Hz, 2H), 6.73 (d, J = 7.8 Hz,

2H), 6.55 (t, J = 7.8 Hz, 1H), 6.05 (d, J = 7.8 Hz, 1H), 4.70 (m, 1H),

4.31 (m, 1H), 2.63 (m, 1H), 2.15 (s, 3H), 1.24 (m, 1H), 1.10 (d, J =

6.4 Hz, 3H).

Methyl 4-((2S*,4R*)-1-Acetyl-2-methyl-4-(phenylamino)-

1,2,3,4-tetrahydroquinolin-6-yl)benzoate (18). Method C was

used starting from 1-((2S*,4R*)-6-bromo-2-methyl-4-(phenylamino)-

3,4-dihydroquinolin-1(2H)-yl)ethanone (56) and (4-

(methoxycarbonyl)phenyl)boronic acid; mp 90 °C. LCMS (method

formate): retention time 1.24 min, [M + H]⁺= 415.32. ¹H NMR (400

1

retention time 1.24 min, [M + H]⁺= 387.18. H NMR (400 MHz,

DMSO-d₆) δ 7.42−7.52 (m, 3H), 7.33−7.41 (m, 2H), 7.10 (d, J = 7.7,

7.7 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 7.8 Hz, 2H), 6.58 (t,

J = 7.2 Hz, 1H), 6.05 (d, J = 8.1 Hz, 1H), 4.72 (m, 1H), 4.26 (m, 1H),

3.76 (s, 3H), 2.61 (m, 1H), 2.13 (s, 3H), 1.25 (m, 1H), 1.09 (d, J = 6.4

Hz, 3H).

1-((2S*,4R*)-6-(4-(Hydroxymethyl)phenyl)-2-methyl-4-(phe-

nylamino)-3,4-dihydroquinolin-1(2H)-yl)ethanone (22). Method

C was used starting from 1-((2S*,4R*)-6-bromo-2-methyl-4-(phenyl-

amino)-3,4-dihydroquinolin-1(2H)-yl)ethanone (56) and (4-

(hydroxymethyl)phenyl)boronic acid; mp 102 °C. LCMS (method

formate): retention time 1.02 min, [M + H]⁺= 387.3. ¹H NMR (400

MHz, DMSO-d₆) δ 7.54 (dd, J = 2.0, 8.3 Hz, 1H), 7.43−7.5 (m, 3H),

7.32−7.43 (m, 3H), 7.10 (dd, J = 7.8, 7.8 Hz, 2H), 6.73 (d, J = 7.8 Hz,

2H), 6.58 (t, J = 7.3 Hz, 1H), 6.05 (d, J = 8.1 Hz, 1H), 5.17 (t, J = 5.8

Hz, 1H), 4.72 (m, 1H), 4.50 (d, J = 5.8 Hz, 2H), 4.28 (m, 1H), 2.62

(m, 1H), 2.14 (s, 3H), 1.25 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H).

1-((2S*,4R*)-2-Methyl-6-(4-(morpholinomethyl)phenyl)-4-

(phenylamino)-3,4-dihydroquinolin-1(2H)-yl)ethanone (23). N-

((2S*,4R*)-1-Acetyl-6-(4-formylphenyl)-2-methyl-1,2,3,4-tetrahydro-

quinolin-4-yl)formamide (58a) was reacted with morpholine using

method E. After formamide deprotection using method A, coupling of

phenylboronic acid was carried out using method B; mp 80 °C. LCMS

1

(method formate): retention time 1.18 min, [M + H]⁺= 456.37. H

NMR (400 MHz, DMSO-d₆) δ 7.30−7.58 (m, 7H), 7.10 (d, J = 7.6

Hz, 2H), 6.72 (d, J = 7.6 Hz, 2H), 6.58 (t, J = 6.8 Hz, 1H), 6.05 (d, J =

7.6 Hz, 1H), 4.72 (m, 1H), 4.28 (m, 1H), 3.56 (m, 4H), 3.32 (br s,

2H), 2.62 (m, 1H), 2.35 (m, 4H), 2.14 (s, 3H), 1.25 (m, 1H), 1.10 (d,

J = 5.6 Hz, 3H).

1-((2S*,4R*)-2-Methyl-4-(phenylamino)-6-(3-(piperidin-1-

ylmethyl)phenyl)-3,4-dihydroquinolin-1(2H)-yl)ethanone (24).

Suzuki coupling between N-((2S*,4R*)-1-acetyl-6-bromo-2-methyl-

1,2,3,4-tetrahydroquinolin-4-yl)formamide (54) and (3-

formylphenyl)boronic acid was carried out using method C. The

resulting aldehyde was coupled with piperidine using method E. After

formamide deprotection using method A, phenylboronic acid was

coupled using method B. LCMS (method formate): retention time

M

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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1

1.41 min, [M + H]⁺= 454.31. H NMR (400 MHz, DMSO-d₆) δ

yl)benzoate (32). Method B was used to couple methyl 4-

((2S*,4R*)-1-acetyl-4-amino-2-methyl-1,2,3,4-tetrahydroquinolin-6-

yl)benzoate (57a) and 4-(methylsulfonyl)phenylboronic acid. LCMS

7.49−7.69 (m, 2H), 7.31−7.48 (m, 4H), 7.22 (d, J = 7.3 Hz, 1H), 7.10

(dd, J = 7.8, 7.8 Hz, 2H), 6.72 (d, J = 7.8 Hz 2H), 6.58 (t, J = 7.2 Hz,

1H), 6.07 (d, J = 8.1 Hz, 1H), 4.73 (m, 1H), 4.28 (m, 1H), 3.43 (s,

2H), 2.62 (m, 1H), 2.23−2.39 (m, 4H), 2.14 (s, 3H) 1.16−1.56 (m,

7H), 1.10 (d, J = 6.1 Hz, 3H).

1

(method formate): retention time 1.04 min, [M − H]⁻= 491.43. H

NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 8.3 Hz, 2H), 7.70 (d, J =

8.3 Hz, 2H), 7.66 (dd, J = 2.0, 8.3 Hz, 1H), 7.62 (d, J = 9.0 Hz, 2H),

7.50 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 1.2 Hz, 1H), 7.06 (d, J = 8.1 Hz,

1H), 6.89 (d, J = 8.8 Hz, 2H), 4.73 (m, 1H), 4.51 (m, 1H), 3.86 (s,

3H), 3.32 (s, 3H), 2.64 (m, 1H), 2.16 (s, 3H), 1.29 (m, 1H), 1.12 (d, J

= 6.4 Hz, 3H).

1-((2S*,4R*)-2-Methyl-4-(phenylamino)-6-(4-(piperidin-1-

ylmethyl)phenyl)-3,4-dihydroquinolin-1(2H)-yl)ethanone Hy-

drochloride (25). Method B was used starting from 1-((2S*,4R*)-

4-amino-2-methyl-6-(4-(piperidin-1-ylmethyl)phenyl)-3,4-dihydroqui-

nolin-1(2H)-yl)ethanone (27) and phenylboronic acid; mp 100 °C.

4-((2S*,4R*)-1-Acetyl-2-methyl-4-(p-tolylamino)-1,2,3,4-tet-

rahydroquinolin-6-yl)benzoic Acid (33). Method B was used to

couple methyl 4-((2S*,4R*)-1-acetyl-4-amino-2-methyl-1,2,3,4-tetra-

hydroquinolin-6-yl)benzoate (57a) and 4-tolylboronic acid. This was

followed by hydrolysis using method D. LCMS (method formate):

LCMS (method formate): retention time 1.40 min, [M + H]⁺

=

454.41. ¹H NMR (400 MHz, DMSO-d₆) δ 9.51 (br s, 1H), 7.38−7.68

(m, 7H), 7.11 (dd, J = 7.8, 7.8 Hz, 2H), 6.72 (d, J = 7.8 Hz, 2H), 6.58

(t, J = 7.2 Hz, 1H), 6.07 (d, J = 7.8 Hz, 1H), 4.72 (m, 1H), 4.28 (m,

3H), 2.88 (br s, 2H), 2.64 (m, 1H), 2.15 (s, 3H), 1.50−1.91 (m, 6H),

1.15−1.45 (m, 3H), 1.11 (d, J = 6.4 Hz, 3H).

1

retention time 0.73 min, [M + H]⁺= 415.36. H NMR (400 MHz,

DMSO-d₆) δ 11.90−13.70 (br s, 1H),7.99 (d, J = 8.3 Hz, 2H), 7.66 (d,

J = 8.3 Hz, 2H), 7.62 (dd, J = 1.8, 8.2 Hz, 1H), 7.53 (br s, 1H), 7.45

(d, J = 8.3 Hz, 1H), 6.92 (d, J = 8.3 Hz, 2H), 6.65 (d, J = 8.3 Hz, 2H),

5.84 (d, J = 8.1 Hz, 1H), 4.71 (m, 1H), 4.25 (m, 1H), 2.62 (m, 1H),

2.16 (s, 3H), 2.08 (s, 3H), 1.21 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-4-((4-chlorophenyl)amino)-2-methyl-

1,2,3,4-tetrahydroquinolin-6-yl)benzoic Acid (34). Method B

was used to couple methyl 4-((2S*,4R*)-1-acetyl-4-amino-2-methyl-

1,2,3,4-tetrahydroquinolin-6-yl)benzoate (57a) and (4-chlorophenyl)-

boronic acid. This was followed by hydrolysis using method D; mp 84

°C. LCMS (method formate): retention time 1.08 min, [M − H]⁻=

N-((2S*,4R*)-1-Acetyl-2-methyl-6-(4-(piperidin-1-ylmethyl)-

phenyl)-1,2,3,4-tetrahydroquinolin-4-yl)formamide (26). Meth-

od E was used starting from N-((2S*,4R*)-1-acetyl-6-(4-formylphen-

yl)-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)formamide (58a) and

piperidine. LCMS (method formate): retention time 1.03 min, [M +

H]⁺= 406.24. ¹H NMR (400 MHz, DMSO-d₆) δ 8.57 (d, J = 8.3 Hz,

1H), 8.31 (s, 1H), 7.56−7.63 (m, 3H), 7.35−7.46 (m, 4H), 4.79 (m,

1H), 4.69 (m, 1H), 3.46 (s, 2H), 2.44−2.54 (m, 3H), 2.34 (m, 2H),

2.09 (s, 3H), 1.45−1.56 (m, 4H), 1.40 (m, 2H), 1.28 (m, 1H), 1.08 (d,

J = 6.4 Hz, 3H).

1

1-((2S*,4R*)-4-Amino-2-methyl-6-(4-(piperidin-1-ylmethyl)-

phenyl)-3,4-dihydroquinolin-1(2H)-yl)ethanone (27). Forma-

mide deprotection was carried out upon N-((2S*,4R*)-1-acetyl-2-

methyl-6-(4-(piperidin-1-ylmethyl)phenyl)-1,2,3,4-tetrahydroquinolin-

4-yl)formamide (26) using method A. LCMS (method formate):

433.15. H NMR (400 MHz, DMSO-d₆) δ 12.76−13.15 (br s, 1H),

7.99 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 7.63 (m, 1H), 7.49

(m, 2H), 7.12 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 6.30 (d, J

= 7.8 Hz, 1H), 4.71 (m, 1H), 4.30 (m, 1H), 2.62 (m, 1H), 2.15 (s,

3H), 1.23 (m, 1H), 1.10 (d, J = 6.1 Hz, 3H).

retention time 1.06 min, [M + H]⁺= 378.32. H NMR (400 MHz,

1

Methyl 4-((2S*,4R*)-1-Acetyl-4-((4-ﬂuorophenyl)amino)-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (35). Method

B was used to couple methyl 4-((2S*,4R*)-1-acetyl-4-amino-2-methyl-

1,2,3,4-tetrahydroquinolin-6-yl)benzoate (57a) and (4-ﬂuorophenyl)-

boronic acid. LCMS (method formate): retention time 1.19 min, [M +

DMSO-d₆) δ 7.78 (br s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.52 (dd, J =

1.7, 8.1 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1 Hz, 1H),

4.62 (m, 1H), 3.59 (dd, J = 4.1, 11.9 Hz, 1H), 3.35 (m, 2H), 2.45 (m,

1H), 2.34 (m, 4H), 2.05 (s, 3H), 1.51 (m, 4H), 1.40 (m, 2H), 1.09 (d,

J = 6.1 Hz, 3H), 1.05 (m, 1H). NH₂not observed.

1

H]⁺= 433.23. H NMR (300 MHz, CDCl₃) δ 8.09 (d, J = 8.16 Hz,

1-((2S,4R)-2-Methyl-4-(phenylamino)-6-(4-(piperidin-1-

ylmethyl)phenyl)-3,4-dihydroquinolin-1(2H)-yl)ethanone (28)

and 1-((2R,4S)-2-Methyl-4-(phenylamino)-6-(4-(piperidin-1-

ylmethyl)phenyl)-3,4-dihydroquinolin-1(2H)-yl)ethanone (29).

Compound 25 was dissolved in EtOH and separated in its two

enantiomers using column CHIRALPAK AD 250 mm × 4.6 mm 10

uM, eluting with hexane/EtOH 40/60 for 15 min at a concentration of

1 mg/mL to give 28 (retention time 4.7 min) and 29 (retention time

7.7 min).

2H), 7.55−7.63 (m, 5H), 7.26 (m, 1H), 6.94 (m, 2H), 6.65 (m, 2H),

4.93 (m, 1H), 4.21 (m, 1H), 3.94 (s, 3H), 2.70 (m, 1H), 2.26 (s, 3H),

1.28−1.43 (m, 1H), 1.22 (d, J = 6.3 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-4-((3-ﬂuorophenyl)amino)-2-methyl-

1,2,3,4-tetrahydroquinolin-6-yl)benzonitrile (36). Method E was

used starting from 1-((2S*,4R*)-4-amino-6-bromo-2-methyl-3,4-dihy-

droquinolin-1(2H)-yl)ethanone (55) and 4-(cyanophenyl)boronic

acid, then method B was used with (3-ﬂuorophenyl)boronic acid;

mp 124 °C. LCMS (method formate): retention time 1.20 min, [M +

H]⁺= 400.30. ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (d, J = 8.3 Hz,

2H), 7.75 (d, J = 8.3 Hz, 2H), 7.66 (dd, J = 2.0, 8.3 Hz, 1H), 7.46−

7.51 (m, 2H), 7.11 (dd, J = 7.6, 7.6 Hz, 1H), 6.51−6.61 (m, 2H), 6.40

(d, J = 8.1 Hz, 1H), 6.34 (ddd, J = 2.0, 7.6, 7.6 Hz, 1H), 4.70 (m, 1H),

4.32 (m, 1H), 2.62 (m, 1H), 2.15 (s, 3H), 1.23 (m, 1H), 1.10 (d, J =

6.4 Hz, 3H).

Methyl 4-((2S*,4R*)-1-Acetyl-4-((4-(tert-butyl)phenyl)-

amino)-2-methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate

(30). Method B was used to couple methyl 4-((2S*,4R*)-1-acetyl-4-

amino-2-methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (57a) and 4-

(tert-butyl)phenylboronic acid; mp 144 °C. LCMS (method formate):

1

retention time 1.43 min, [M + H]⁺= 471.39. H NMR (400 MHz,

DMSO-d₆) δ 8.00 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 7.63

(d, J = 8.1 Hz, 1H), 7.57 (br s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.13 (d, J

= 8.3 Hz, 2H), 6.68 (d, J = 8.3 Hz, 2H), 5.84 (d, J = 8.1 Hz, 1H), 4.71

(m, 1H), 4.28 (m, 1H), 3.86 (s, 3H), 2.62 (m, 1H), 2.15 (s, 3H), 1.29

(m, 1H), 1.22 (s, 9H), 1.10 (d, J = 6.1 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-4-((3-methoxyphenyl)amino)-2-meth-

yl-1,2,3,4-tetrahydroquinolin-6-yl)benzonitrile (37). Method E

was used starting from 1-((2S*,4R*)-4-amino-6-bromo-2-methyl-3,4-

dihydroquinolin-1(2H)-yl)ethanone (55) and 4-(cyanophenyl)-

boronic acid, then method B was used with (3-methoxyphenyl)-

boronic acid; mp 104 °C. LCMS (method formate): retention time

1.17 min, [M + H]⁺= 412.26. ¹H NMR (400 MHz, DMSO-d₆) δ 7.90

(d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 7.65 (dd, J = 2.0, 8.1 Hz,

1H), 7.53 (d, J = 1.2 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.01 (t, J = 8.1

Hz, 1H), 6.28−6.36 (m, 2H), 6.19 (dd, J = 2.0, 8.1 Hz, 1H), 6.06 (d, J

= 8.3 Hz, 1H), 4.70 (m, 1H), 4.32 (m, 1H), 3.31 (s, 3H), 2.62 (m,

1H), 2.13 (s, 3H), 1.24 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-4-((3-methoxyphenyl)amino)-2-meth-

yl-1,2,3,4-tetrahydroquinolin-6-yl)benzoic Acid (38). Method B

was used starting from methyl 4-((2S*,4R*)-1-acetyl-4-amino-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (57a) and (3-

methoxyphenyl)boronic acid. This was followed by hydrolysis using

4-((2S*,4R*)-1-Acetyl-2-methyl-4-((4-(methylsulfonyl)-

phenyl)amino)-1,2,3,4-tetrahydroquinolin-6-yl)benzoic Acid

(31). Method D was used to hydrolyze methyl 4-((2S*,4R*)-1-

acetyl-2-methyl-4-((4-(methylsulfonyl)phenyl)amino)-1,2,3,4-tetrahy-

droquinolin-6-yl)benzoate (32). LCMS (method formate): retention

1

time 0.62 min, [M − H]⁻= 477.39. H NMR (400 MHz, DMSO-d₆)

δ 12.00−13.65 (br s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.59−7.69 (m,

5H), 7.49 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 1.2 Hz, 1H), 7.06 (d, J = 8.1

Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 4.74 (m, 1H), 4.53 (m, 1H), 3.01

(m, 3H), 2.64 (m, 1H), 2.16 (s, 3H), 1.32 (m, 1H), 1.12 (d, J = 6.1

Hz, 3H).

Methyl 4-((2S*, 4R *)-1-Acetyl-2-methyl-4-((4-

(methylsulfonyl)phenyl)amino)-1,2,3,4-tetrahydroquinolin-6-

N

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

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method D; mp 148 °C. LCMS (method formate): retention time 0.69

min, [M + H]⁺= 431.31. ¹H NMR (400 MHz, DMSO-d₆) δ 7.99 (d, J

= 8.1 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.54

(br s, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.01 (dd, J = 8.1, 8.1 Hz, 1H),

6.29−6.36 (m, 2H), 6.19 (d, J = 8.1 Hz, 1H), 6.07 (d, J = 8.1 Hz, 1H),

4.71 (m, 1H), 4.31 (m, 1H), 3.67 (s, 3H), 2.62 (m, 1H), 2.15 (s, 3H),

1.22 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H). CO₂H not observed.

DMSO-d₆) δ 7.96 (br s, 1H), 7.93 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 2.0

Hz, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.46 (m, 2H), 7.35 (br s, 1H), 7.13

(d, J = 7.8 Hz, 2H), 6.75 (d, J = 7.8 Hz, 2H), 6.28 (d, J = 8.1 Hz, 1H),

4.72 (m, 1H), 4.31 (m, 1H), 2.62 (m, 1H), 2.15 (s, 3H), 1.23 (m, 1H),

1.10 (d, J = 6.4 Hz, 3H).

1-((2S*,4R*)-4-((4-Chlorophenyl)amino)-2-methyl-6-(o-

tolyl)-3,4-dihydroquinolin-1(2H)-yl)ethanone (44). Method C

was used starting from 1-((2S*,4R*)-6-bromo-4-((4-chlorophenyl)-

amino)-2-methyl-3,4-dihydroquinolin-1(2H)-yl)ethanone (56a) and

o-tolylboronic acid. LCMS (method formate): retention time 1.39

4-((2S*,4R*)-1-Acetyl-4-((2-methoxyphenyl)amino)-2-meth-

yl-1,2,3,4-tetrahydroquinolin-6-yl)benzoic Acid (39). Method B

was used starting from methyl 4-((2S*,4R*)-1-acetyl-4-amino-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (57a) and (2-

methoxyphenyl)boronic acid. This was followed by hydrolysis using

method D. LCMS (method formate): retention time 0.72 min, [M +

1

min, [M + H]⁺= 405.32. H NMR (300 MHz, CDCl₃) δ 7.08−7.34

(m, 11H), 6.59 (d, J = 8.2 Hz, 1H), 4.94 (m, 1H), 4.23 (m, 1H), 2.70

(m, 1H), 2.28 (s, 3H), 2.19 (s, 3H), 1.30 (m, 1H), 1.23 (d, J = 6.3 Hz,

3H).

1

H]⁺= 431.34. H NMR (400 MHz, DMSO-d₆) δ 12.05−13.82 (br s,

(R)-1-(6-(4-Methoxyphenyl)-4-(phenylamino)-3,4-dihydro-

quinolin-1(2H)-yl)ethanone (45). To a solution of 1-acetyl-6-(4-

methoxyphenyl)-2,3-dihydroquinolin-4(1H)-one (62) (280 mg, 0.95

mmol), aniline (0.25 mL, 2.85 mmol), and NEt₃(0.67 mL, 4.75

mmol) in anhydrous CH₂Cl₂(10 mL) at 0 °C was added dropwise a

1.0 M solution of titanium tetrachloride in CH₂Cl₂(0.95 mL, 0.95

mmol). After stirring 18 h at room temperature, the reaction mixture

was poured into water and extracted with CH₂Cl₂. The organic phase

was dried over Na₂SO₄, ﬁltered, and evaporated to dryness. The

residue was taken up in iPr₂O, and the ﬁltrate was evaporated to

dryness and directly dissolved in AcOH. NaB(OAc)₃H was added

portionwise, then the reaction mixture was stirred for 18 h and basiﬁed

with concentrated NaOH aqueous solution. The aqueous solution was

extracted with CH₂Cl₂and washed with brine, dried over Na₂SO₄,

ﬁltered, and evaporated to dryness. The residue was puriﬁed by ﬂash

chromatography on silica gel (CH₂Cl₂), and the resulting solid was

recrystallized in CH₃CN to deliver the title compound (65 mg, 15%)

as a white powder. LCMS (method formate): retention time 1.22 min,

[M + H]⁺= 373.27. ¹H NMR (400 MHz, DMSO-d₆) δ 7.42−7.56 (m,

5H), 7.11 (dd, J = 7.9, 7.9 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.74 (d, J

= 7.8 Hz, 2H), 6.57 (t, J = 7.3 Hz, 1H), 6.07 (d, J = 8.3 Hz, 1H), 4.56

(m, 1H), 4.02 (m, 1H), 3.58 (m, 1H), 3.32 (s, 3H), 2.24 (s, 3H), 2.22

(m, 1H), 1.82 (m, 1H).

4-((2S,4R)-1-Acetyl-2-methyl-4-(p-tolylamino)-1,2,3,4-tetra-

hydroquinolin-6-yl)benzoic Acid (46). Compound 46 can be

obtained in a similar manner to compound 33 from the single

enantiomer of compound 57a, which can be obtained from the

racemate as follows: A solution of methyl 4-(1-acetyl-4-amino-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (57a) (103 g, 0.305

mol) in EtOH (500 mL) was treated with ^L-(+)-lactic acid (20% w/w

solution in water, 260 mL, 0.610 mmol), and the resulting mixture was

reﬂuxed for 30 min then cooled to room temperature and

concentrated in vacuo. The residue was suspended in hexane (160

mL) and triturated at reﬂux for 10 min. The mixture was cooled to

room temperature, the solid ﬁltered oﬀ, and the trituration in hexane

repeated once. The solid obtained was suspended in EtOAc (1.3 L)

and triturated at reﬂux for 30 min to give a white precipitate. The

mixture was then cooled to room temperature and left still for 16 h.

The solid was ﬁltered oﬀ and rinsed with EtOAc. The ﬁltrate was

treated with a saturated Na₂CO₃aqueous solution, and the resulting

mixture was stirred for 30 min. The aqueous phase was extracted with

AcOEt (1.5 L) and CH₂Cl₂(1 L), and the combined organics were

dried over Na₂SO₄and concentrated in vacuo to give 50.3 g of amine.

This residue was dissolved in THF (500 mL) and treated with ^L(−)-

dibenzoyltartaric acid (26.7 g, 74.6 mmol), and the resulting mixture

was reﬂuxed for 20 min then was cooled to room temperature and left

still for 72 h then at 0 °C for 30 min. The precipitate obtained was

ﬁltered oﬀ and rinsed with THF (100 mL). This precipitate was

reﬂuxed in EtOH (1.1 L), and the mixture was left at room

temperature for 16 h then at 0 °C for 30 min. The solid was ﬁltered oﬀ

and dried under vacuum. It was then basiﬁed with a saturated Na₂CO₃

aqueous solution. The aqueous phase was extracted with CH₂Cl₂(3 L

overall), and the organic phase was dried over Na₂SO₄and

concentrated in vacuo to give methyl 4-((2S,4R)-1-acetyl-4-amino-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (28.6 g, 28%, ee

>99.5%) as a white solid.

1H), 7.97 (d, J = 8.3 Hz, 2H), 7.58−7.65 (m, 3H), 7.45 (d, J = 8.1 Hz,

1H), 7.41 (br s, 1H), 6.90 (d, J = 7.8 Hz, 1H), 6.75 (t, J = 7.8 Hz, 1H),

6.63 (m, 2H), 5.10 (d, J = 8.1 Hz, 1H), 4.72 (m, 1H), 4.31 (m, 1H),

3.85 (s, 3H), 2.61 (m, 1H), 2.15 (s, 3H), 1.43 (m, 1H), 1.10 (d, J = 6.4

Hz, 3H).

1-((2S*,4R*)-4-((4-Chlorophenyl)amino)-2-methyl-6-(4-(pi-

peridin-1-ylmethyl)phenyl)-3,4-dihydroquinolin-1(2H)-yl)-

ethanone (40). Method B was used to couple 1-((2S*,4R*)-4-amino-

2-methyl-6-(4-(piperidin-1-ylmethyl)phenyl)-3,4-dihydroquinolin-

1(2H)-yl)ethanone (27) and (4-chlorophenyl)boronic acid; mp 128

°C. LCMS (method formate): retention time 1.49 min, [M + H]⁺=

1

490.31. H NMR (400 MHz, DMSO-d₆) δ 7.28−7.60 (m, 7H), 7.12

(d, J = 8.6 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H), 6.28 (d, J = 8.1 Hz, 1H),

4.72 (m, 1H), 4.28 (m, 1H), 3.45 (br s, 2H), 2.61 (m, 1H), 2.35 (m,

4H), 2.14 (s, 3H), 1.50 (m, 4H), 1.40 (m, 2H), 1.24 (m, 1H), 1.09 (d,

J = 6.1 Hz, 3H).

3-(1-Acetyl-4-((4-chlorophenyl)amino)-2-methyl-1,2,3,4-tet-

rahydroquinolin-6-yl)benzoic Acid (41). Hydrolysis of 42 was

carried out using method D; mp 152 °C. LCMS (method formate):

1

retention time 0.79 min, [M − H]⁻= 433.35. H NMR (400 MHz,

DMSO-d₆) δ 12.74−13.20 (br s, 1H), 8.10 (s, 1H), 7.90 (dd, J = 1.2,

7.8 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.53−7.64 (m, 2H), 7.41−7.50

(m, 2H), 7.12 (d, J = 9.0 Hz, 2H), 6.74 (d, J = 9.0 Hz, 2H), 6.31 (d, J

= 8.1 Hz, 1H), 4.72 (m, 1H), 4.30 (m, 1H), 2.62 (m, 1H), 2.15 (s,

3H), 1.24 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H).

Methyl 3-((2S*,4R*)-1-Acetyl-4-((4-chlorophenyl)amino)-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (42). Method

B was used starting from 1-((2S*,4R*)-4-amino-6-bromo-2-methyl-

3,4-dihydroquinolin-1(2H)-yl)ethanone (55) and (4-chlorophenyl)-

boronic acid. Suzuki coupling with (3-(methoxycarbonyl)phenyl)-

boronic acid was then eﬀected using method C; mp 98 °C. LCMS

1

(method formate): retention time 1.31 min, [M − H]⁻= 447.34. H

NMR (400 MHz, DMSO-d₆) δ 8.07 (s, 1H), 7.92 (dd, J = 1.2, 7.8 Hz,

1H), 7.80 (d, J = 7.8 Hz, 1H), 7.52−7.66 (m, 2H), 7.45 (d, J = 9.0 Hz,

2H), 7.13 (d, J = 9.0 Hz, 2H), 6.74 (d, J = 9.1 Hz, 2H), 6.32 (d, J = 8.1

Hz, 1H), 4.71 (m, 1H), 4.30 (m, 1H), 3.87 (s, 3H), 2.62 (m, 1H), 2.15

(s, 3H), 1.25 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H).

4-((2S*,4R*)-1-Acetyl-4-((4-chlorophenyl)amino)-2-methyl-

1,2,3,4-tetrahydroquinolin-6-yl)benzamide (43). A solution of 4-

((2S*,4R*)-1-acetyl-4-((4-chlorophenyl)amino)-2-methyl-1,2,3,4-tet-

rahydroquinolin-6-yl)benzoic acid (34) (250 mg, 0.575 mmol) in

DMF (5 mL) at room temperature was treated with EDCI (220 mg,

1.15 mmol), HOBT (116 mg, 0.862 mmol), and NEt₃(0.16 mL, 1.15

mmol). The resulting mixture was stirred at this temperature for 15

min then treated with a 25% w/w NH₄OH aqueous solution (0.06 mL,

1.15 mmol). After continued stirring at this temperature for 16 h, more

EDCI (50 mg, 0.26 mmol) and 25% w/w NH₄OH aqueous solution

(0.06 mL, 1.15 mmol) were added and the mixture was stirred for

another 2 h, then was concentrated in vacuo. The residue was

dissolved in EtOAc, and the organic phase was washed successively

with a 0.5N NaOH aqueous solution, a 0.5 N HCl aqueous solution,

water and brine, then dried over Na₂SO₄and concentrated in vacuo.

Puriﬁcation of the residue by ﬂash chromatography on silica gel (5%

MeOH in CH₂Cl₂) and recrystallization in EtOH gave 43 (82 mg,

33%) as white crystals; mp 235 °C. LCMS (method formate):

1

retention time 1.00 min, [M + H]⁺= 434.27. H NMR (400 MHz,

O

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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4-((2S,4R)-1-Acetyl-4-((4-chlorophenyl)amino)-2-methyl-

1,2,3,4-tetrahydroquinolin-6-yl)benzoic Acid (47). A solution of

ethyl 4-{(2S,4R)-1-acetyl-4-[(4-chlorophenyl)amino]-2-methyl-

1,2,3,4-tetrahydro-6-quinolinyl}benzoate (69) (28.0 g, 60.5 mmol)

in EtOH (380 mL) at room temperature was treated with a 2 M

NaOH aqueous solution (190 mL, 60.5 mmol). The resulting mixture

was stirred at this temperature for 3 h. Most of EtOH was removed in

vacuo. The resulting orange emulsion was diluted with water, and the

aqueous phase was extracted twice with CH₂Cl₂. The combined

organic phases were washed with water, and the combined aqueous

phases were acidiﬁed with a 2 N HCl aqueous solution. The aqueous

phase was then extracted twice with EtOAc. The combined organic

phases were dried over MgSO₄and concentrated in vacuo. The

residual orange foam was suspended in EtOAc, and the resulting

mixture was stirred at room temperature for 15 min. The solid formed

was ﬁltered oﬀ, rinsed with EtOAc, and dried under vacuum at 50 °C

toluene (300 mL). Acetaldehyde (64.7 mL, 1.17 mol) in solution in

toluene (200 mL) was added dropwise. The reaction mixture became

progressively homogeneous and then gave a precipitate. The resulting

mixture was stirred 12 h under nitrogen atmosphere then ﬁltered. The

precipitate was recrystallized in toluene to aﬀord intermediate 52 as a

white solid (304 g, 82%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (d, J

= 8.6 Hz, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.51 (m, 2H), 7.43 (m, 1H),

7.16 (d, J = 8.2 Hz, 2H), 6.63 (d, J = 8.2 Hz, 2H), 1.86 (d, J = 6.8 Hz,

3H). NH not observed.

N-((2S*,4R*)-6-Bromo-2-methyl-1,2,3,4-tetrahydroquinolin-

4-yl)formamide (53). A 3 L, four-neck ﬂask under nitrogen

atmosphere was charged with N-vinyl formamide (66.2 g, 0.946

mol) and dry THF (400 mL). BF₃·Et₂O (239 mL, 1.9 mol) was added

dropwise at −5 °C to the milky mixture. After 15 min, 52 (150 g,

0.473 mol) in solution in THF (1 L) was added at −5 °C. After 2 h,

the mixture was slowly and carefully poured in a NaHCO₃saturated

aqueous solution (5 L). EtOAc (2 L) was added, and the mixture was

transferred to a separatory funnel. The organic layer was separated and

was washed with water (1 × 200 mL) and brine (1 × 200 mL) and

dried over Na₂SO₄. The mixture was ﬁltered and the solid washed with

EtOAc (1 × 50 mL). The ﬁltrate was concentrated progressively until

a precipitate appeared and the mixture cooled in an ice bath for 2 h.

The precipitate was ﬁltered through a Buchner funnel and washed with

iPr₂O (2 × 100 mL) to deliver intermediate 53 (71 g, 56%) as a solid.

1

to give 47 (20.2 g, 77%) as a cream solid. H NMR, ¹³C NMR, and

chiral analysis of the product showed no evidence of epimerization, ee

>99%. LCMS (method formate): retention time 1.08 min, [M − H]⁻

1

= 433.15. H NMR (600 MHz, DMSO-d₆) δ ppm 12.92 (br s, 1H),

8.00−7.97 (m, 2H), 7.68−7.65 (m, 2H), 7.63 (dd, J = 8.2, 2.1 Hz,

1H), 7.48 (d, J = 1.5 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.19−7.09 (m,

2H), 6.70−6.78 (m, 2H), 6.26 (d, J = 8.1 Hz, 1H), 4.66−4.77 (m,

1H), 4.30 (ddd, J = 11.8, 7.9, 4.1 Hz, 1H), 2.62 (ddd, J = 12.5, 8.4, 4.2

Hz, 1H), 2.15 (s, 3H), 1.27−1.20 (m, 1H), 1.10 (d, J = 6.2 Hz, 3H).

¹³C NMR (150.9 MHz, DMSO-d₆) δ ppm 168.6 (s, 1C), 167.0 (s,

1C), 147.1 (s, 1C), 143.9 (s, 1C), 137.9 (s, 1C), 136.7 (s, 1C), 135.8

(s, 1C), 130.0 (s, 2C), 129.5 (s, 1C),128.7 (s, 2C), 126.6 (s, 1C),

126.5 (s, 2C), 125.3 (s, 1C), 121.8 (s, 1C), 119.5 (s, 1C), 114.1 (s,

2C), 48.8 (s, 1C), 47.2 (s, 1C), 39.9 (s, 1C), 22.9 (s, 1C), 21.3 (s, 1C).

See Supporting Information Figure S4 for NMR spectra. [α]D =

+40.8° (T = 21.4 °C, 10 mm cell, c = 10 mg/mL, MeOH).

LCMS (method formate): retention time 0.79 min, [M + H]⁺

=

269.05, 272.18 (1 Br). ¹H NMR (400 MHz, DMSO-d₆) δ 8.37 (d, J =

8.8 Hz, 1H), 8.21 (s, 1H), 7.05 (dd, J = 2.1, 8.4 Hz, 1H), 6.97 (s, 1H),

6.44 (d, J = 8.8 Hz, 1H), 5.93 (s, 1H), 5.10 (m, 1H), 3.41 (m, 1H),

1.95 (m, 1H), 1.40 (m, 1H), 1.14 (d, J = 6.4 Hz, 3H).

N-((2S*,4R*)-1-Acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydro-

quinolin-4-yl)formamide (54). Acetyl chloride (21 mL, 0.29 mol)

was added dropwise at 0 °C to a solution of 53 (71 g, 0.26 mol) in a

mixture of CH₂Cl₂(1 L) and pyridine (350 mL). After stirring 2 h at 0

°C, the mixture was poured into a mixture of crushed ice (2 kg) and

concentrated HCl (450 mL). The product was extracted with CH₂Cl₂

(1 L), washed with brine, and dried over Na₂SO₄. Concentration

under vacuum aﬀorded intermediate 54 (82 g, 100%) as an oﬀ-white

solid. LCMS (method formate): retention time 0.73 min, [M + H]⁺=

311.08, 313.04 (1 Br). ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (d, J =

8.3 Hz, 1H), 8.27 (s, 1H), 7.49 (dd, J = 1.8, 8.4 Hz, 1H), 7.29−7.34

(m, 2H), 4.58−4.77 (m, 2H), 2.45 (m, 1H), 2.08 (s, 3H), 1.25 (m,

1H), 1.05 (m, 3H).

3-(Phenylamino)butanoic Acid (49). A solution of aniline (93.3

g, 1 mol) and crotonic acid (66 g, 0.77 mol) in toluene (600 mL) was

heated to reﬂux. After 24 h, the reaction mixture was evaporated to

dryness and the residue was puriﬁed by ﬂash chromatography on silica

1

gel to aﬀord intermediate 49 as a brown oil (77.5 g, 43%). H NMR

(300 MHz, CDCl₃) δ ppm 7.23 (t, J = 7.6 Hz, 2H), 6.81 (t, J = 7.6 Hz,

1H), 6.73 (d, J = 7.6 Hz, 2H), 3.95 (m, 1H), 2.70 (m, 1H), 2.55 (m,

1H), 1.32 (d, J = 6.6 Hz, 3H).

2-Methyl-2,3-dihydroquinolin-4(1H)-one (50). A mixture of 3-

(phenylamino)butanoic acid (49) (30 g, 0.168 mol) and PPA (300

mL) was heated to 110 °C. After 5 h, the reaction mixture was poured

into ice and a saturated aqueous solution of NaHCO₃was added until

pH 7. The aqueous layer was extracted with EtOAc (3 × 1 L). The

combined organic layers were washed with brine and dried over

Na₂SO₄and concentrated under reduced pressure. The residue was

1-((2S*,4R*)-4-Amino-6-bromo-2-methyl-3,4-dihydroquino-

lin-1(2H)-yl)ethanone Hydrochloride (55). Method A was used

starting from N-((2S*,4R*)-1-acetyl-6-bromo-2-methyl-1,2,3,4-tetra-

hydroquinolin-4-yl)formamide (54). Yield: 91% (oil). LCMS (method

1

formate): retention time 0.76 min, [M + H]⁺= 283.285 (1 Br). H

puriﬁed by ﬂash chromatography on silica gel (8 g, 29%). H NMR

NMR (400 MHz, DMSO-d₆) δ 9.03 (br s, 3H), 7.61 (d, J = 1.5 Hz,

1H), 7.58 (dd, J = 1.5, 8.3 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 4.56−

4.72 (m, 1H), 4.13−4.30 (m, 1H), 2.74 (m, 1H), 2.04 (s, 3H), 1.25−

1.41 (m, 1H), 1.06 (d, J = 6.36 Hz, 3H).

(300 MHz, CDCl₃) δ ppm 7.74 (dd, J = 1.5 and 7.9 Hz, 1H), 7.25 (m,

1H), 6.66 (m, 1H), 6.57 (d, J = 7.9 Hz, 1H), 3.70 (m, 1H), 2.55 (dd, J

= 3.8 and 16.1 Hz, 1H), 2.44 (dd, J = 13.0 and 16.1 Hz, 1H), 1.27 (d, J

= 6.4 Hz, 3H).

1-((2S*,4R*)-6-Bromo-2-methyl-4-(phenylamino)-3,4-dihy-

droquinolin-1(2H)-yl)ethanone (56). Method B was used starting

from 1-((2S*,4R*)-4-amino-6-bromo-2-methyl-3,4-dihydroquinolin-

1(2H)-yl)ethanone hydrochloride (55) and phenylboronic acid.

1-Acetyl-2-methyl-2,3-dihydroquinolin-4(1H)-one (51). To a

solution of 2-methyl-2,3-dihydroquinolin-4(1H)-one (50) (6.44 g, 40

mmol) in anhydrous CH₂Cl₂(150 mL) at 0 °C was added acetyl

chloride (3.45 g, 45 mmol) followed by DMAP (0.48 g, 4 mmol). The

mixture was stirred for 18 h at room temperature, and then 1N NaOH

was added until pH 7−8. The organic layer was separated, dried over

Na₂SO₄, and concentrated under reduced pressure. The residue was

taken up in pentane, ﬁltered, then washed with iPr₂O to deliver

intermediate 51 as an oﬀ-white powder (6.95 g, 86%). LCMS (method

LCMS (method formate): retention time 1.20 min, [M + H]⁺

=

359.36 (1 Br). ¹H NMR (400 MHz, DMSO-d₆) δ 7.45 (dd, J = 2.0, 8.4

Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.11 (dd, J

= 7.3, 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 6.59 (t, J = 7.3 Hz, 1H),

6.06 (d, J = 7.8 Hz, 1H), 4.67 (m, 1H), 4.22 (m, 1H), 2.58 (m, 1H),

2.08 (s, 3H), 1.19 (m, 1H), 1.06 (d, J = 6.4 Hz, 3H).

1

formate): retention time 2.12 min. [M + H]⁺= 204.0. H NMR (300

1-((2S*,4R*)-6-Bromo-4-((4-chlorophenyl)amino)-2-methyl-

3,4-dihydroquinolin-1(2H)-yl)ethanone (56a). Method B was

used starting from 1-((2S*,4R*)-4-amino-6-bromo-2-methyl-3,4-dihy-

droquinolin-1(2H)-yl)ethanone (55) and (4-chlorophenyl)boronic

acid. LCMS (method formate): retention time 1.28 min, [M + H]⁺

= 393.14, 395.01 (1 Br). ¹H NMR (400 MHz, DMSO-d₆) δ 7.45 (dd, J

= 1.7, 8.3 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 1.7 Hz, 1H),

7.12 (d, J = 8.9 Hz, 2H), 6.69 (d, J = 8.9 Hz, 2H), 6.28 (d, J = 7.8 Hz,

MHz, CDCl₃) δ ppm 8.01 (dd, J = 1.7 and 7.7 Hz, 1H), 7.57 (m, 1H),

7.39 (br s, 1H), 7.28 (m, 1H), 5.35 (br s, 1H), 3.05 (dd, J = 5.8 and

17.9 Hz, 1H), 2.58 (dd, J = 1.7 and 17.9 Hz, 1H), 2.35 (s, 3H), 1.21

(d, J = 7.0 Hz, 3H).

N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)ethyl)-4-bromoaniline

(52). To a suspension of benzotriazole (139 g, 1.16 mol) in toluene (2

L) in a 3 L, four-neck ﬂask under nitrogen atmosphere was added at

room temperature a solution of 4-bromoaniline (200 g, 1.16 mol) in

P

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 4.67 (m, 1H), 4.23 (m, 1H), 2.58 (m, 1H), 2.07 (s, 3H), 1.17 (m,

1H), 1.06 (d, J = 6.4 Hz, 3H).

(E)-Isopropyl But-2-enoylcarbamate (63). Isopropyl carbamate

(30 g, 291 mmol) was charged to a 3 L Lara vessel, and dry THF (150

mL) added. (2E)-2-Butenoyl chloride (31.2 mL, 326 mmol) was

added under nitrogen and the jacket cooled to −30 °C. When the

solution temperature reached −17 °C, lithium tert-butoxide (655 mL,

655 mmol, 1 M in THF) was added by peristaltic pump over 2 h,

keeping the reaction temperature between −10 °C and −18 °C. Once

the addition was complete, the mixture was stirred for 30 min and

brought to 0 °C. Et₂O (450 mL) and HCl (375 mL, 1 M aqueous

solution) were added and the mixture brought to 20 °C with vigorous

stirring. The stirring was stopped and the layers allowed to separate

and the aqueous layer to run oﬀ. Brine (375 mL) was added and the

mixture stirred vigorously. The stirring was stopped, and the layers

were allowed to separate and the aqueous layer to run oﬀ. The organic

layer was dried (MgSO₄), ﬁltered, and evaporated to a brown oil (60

g). The mixture was loaded onto a 40+M Biotage silica column and

eluted with CH₂Cl₂/EtOAc (1:1 → 0:1, 10CV). The product

containing fractions were evaporated to dryness and loaded on to a

1500 g Redisep Isco silica column and eluted with cyclohexane/EtOAc

(0 → 40%, 17CV). The clean, product containing fractions were

evaporated to an oﬀ-white solid (15.4 g, 29%). LCMS (method TFA):

Methyl 4-((2S*,4R*)-1-Acetyl-4-amino-2-methyl-1,2,3,4-tet-

rahydroquinolin-6-yl)benzoate (57a). Method B was used to

couple (4-(methoxycarbonyl)phenyl)boronic acid to N-((2S*,4R*)-1-

acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)formamide

(54) before formamide deprotection using method A; mp 250 °C.

LCMS (method formate): retention time 0.89 min, [M + H]⁺

=

1

339.25. H NMR (400 MHz, DMSO-d₆) δ 8.06 (d, J = 8.1 Hz 2H),

7.86−7.90 (m, 3H), 7.62 (dd, J = 2.0, 8.3 Hz, 1H), 7.38 (d, J = 8.3 Hz,

1H), 4.62 (m, 1H), 3.88 (s, 3H), 3.61 (dd, J = 4.2, 12.0 Hz, 1H), 2.44

(m, 1H), 2.08 (s, 3H), 1.05 (d, J = 6.4 Hz, 3H), 1.02 (m, 1H). NH₂

not observed.

N-((2S*,4R*)-1-Acetyl-6-(4-formylphenyl)-2-methyl-1,2,3,4-

tetrahydroquinolin-4-yl)formamide (58a). Method C was used

starting from N-((2S*,4R*)-1-acetyl-6-bromo-2-methyl-1,2,3,4-tetra-

hydroquinolin-4-yl)formamide (54) and (4-formylphenyl)boronic

acid; mp 86 °C. LCMS (method formate): retention time 0.78 min,

[M + H]⁺= 337.24. ¹H NMR (400 MHz, DMSO-d₆) δ 10.06 (s, 1H),

8.75 (d, J = 8.6 Hz, 1H), 8.31 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.93

(d, J = 8.1, 2H), 7.71 (dd, J = 1.5, 8.2 Hz, 1H), 7.58 (d, J = 1.5 Hz,

1H), 7.49 (d, J = 8.2 Hz, 1H), 4.82 (m, 1H), 4.69 (m, 1H), 2.51 (m,

1H), 2.12 (s, 3H), 1.30 (m, 1H), 1.05−1.11 (d, J = 6.4 Hz, 3H).

N-((2S*,4R*)-1-Acetyl-2-methyl-6-(4-(piperidin-1-ylmethyl)-

phenyl)-1,2,3,4-tetrahydroquinolin-4-yl)formamide (59). Meth-

od E was used starting from N-((2S*,4R*)-1-acetyl-6-(4-formylphen-

yl)-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)formamide (58a) and

piperidine. LCMS (method formate): retention time 1.03 min, [M +

H]⁺= 406.24. ¹H NMR (400 MHz, DMSO-d₆) δ 8.57 (d, J = 8.3 Hz,

1H), 8.31 (s, 1H), 7.56−7.63 (m, 3H), 7.35−7.46 (m, 4H), 4.79 (m,

1H), 4.69 (m, 1H), 3.46 (s, 2H), 2.44−2.54 (m, 3H), 2.34 (m, 2H),

2.09 (s, 3H), 1.45−1.56 (m, 4H), 1.40 (m, 2H), 1.28 (m, 1H), 1.08 (d,

J = 6.4 Hz, 3H).

1

retention time 0.68 min, [M + H]⁺= 172.0. H NMR (400 MHz,

CDCl₃) δ ppm 1.30 (d, J = 6.3 Hz, 6H), 1.94 (dd, J = 6.9, 1.6 Hz, 3H),

4.94−5.06 (m, 1H), 6.87 (dd, J = 15.4, 1.5 Hz, 1H), 7.14 (dq, J = 15.4,

6.9 Hz, 1H), 7.35 (br s, 1H).

(S)-Isopropyl (3-((4-Bromophenyl)amino)butanoyl)-

carbamate (64). (E)-Isopropyl but-2-enoylcarbamate (63) (9.38 g,

54.8 mmol) was stirred in toluene (281 mL) under nitrogen and ((R)-

BINAP)ditriﬂatebis(acetonitrile)palladium(II) (3.35 g, 3.01 mmol)

added. The catalyst formed a gummy ball, and the solution turned to

an opaque yellow mixture and was stirred for 20 min. 4-Bromoaniline

(14.14 g, 82 mmol) was added, the solution turned a clear light brown,

and the gummy catalyst dissolved further. The mixture was stirred for

16 h. Similarly, a second batch of 1-methylethyl (2E)-2-butenoylcar-

bamate (8.51 g, 49.7 mmol) was stirred in toluene (255 mL) under

nitrogen and ((R)-BINAP)ditriﬂatebis(acetonitrile)palladium(II)

(3.04 g, 2.73 mmol) added. The catalyst formed a gummy ball, and

the solution turned to an opaque yellow mixture and was stirred for 20

min. 4-Bromoaniline (12.83 g, 74.6 mmol) was added, the solution

turned a clear light brown, and the gummy catalyst dissolved further.

The mixture was stirred for 16 h. The two reaction mixtures were

combined and loaded on to a 1.5 kg Isco silica Redisep column. The

column was eluted with CH₂Cl₂/MeOH (0% → 0.5%, 19CV). The

clean product containing fractions were evaporated to a pale-brown

oil. The mixture was dried in a vacuum oven overnight at 40 °C to give

a white solid (24.2 g, 67% overall). LCMS (method TFA) retention

time = 0.91 min, [M + H]⁺= 342.9.. Chiral HPLC: 92% ee (method:

∼ 1 mg sample dissolved in EtOH/heptane (1 mL), 20 μL injected on

4.6 mm i.d. × 25 cm Chiralpak AD column (lot no. AD00CE-

FE126)). Column eluted with 15% EtOH/heptane, f = 1.0 mL/min.

6-Bromo-2,3-dihydroquinolin-4(1H)-one (60). To a solution of

2,3-dihydroquinolin-4(1H)-one (1.85 g, 12.6 mmol) in CH₂Cl₂(15

mL) at 0 °C was added N-bromosuccinimide (2.28 g, 12.8 mmol), and

the mixture was stirred for 1.5 h. CH₂Cl₂was added to the reaction

mixture, then the organic layer was separated and washed with 1N

NaOH aqueous solution and brine and dried over Na₂SO₄. The

residue was puriﬁed by ﬂash chromatography on silica gel (0−1%

MeOH in CH₂Cl₂) to aﬀord 6-bromo-2,3-dihydroquinolin-4(1H)-one

(60) (2.43 g, 86%) as a yellow solid. LCMS (method formate, 5 min):

1

retention time 2.52 min, [M + H]⁺= 225.23. H NMR (300 MHz,

CDCl₃) δ ppm 7.87 (d, J = 2.5 Hz, 1H), 7.27 (dd, J = 2.3 and 8.7 Hz,

1H), 6.53 (d, J = 8.7 Hz, 1H), 3.51 (t, J = 6.8 Hz, 2H), 2.62 (t, J = 6.8

Hz, 2H).

1-Acetyl-6-bromo-2,3-dihydroquinolin-4(1H)-one (61). A

mixture of 6-bromo-2,3-dihydroquinolin-4(1H)-one (60) (1.10 g, 4.9

mmol) and acetic anhydride (11 mL) was stirred of 2 h at 80 °C and

15 h at 40 °C then concentrated under reduced pressure. The residue

was recrystallized in acetonitrile to deliver 61 (0.91 g, 69%) as pale-

yellow solid. LCMS (method formate, 5 min): retention time 2.44

min, [M + H]⁺= 268.00. ¹H NMR (300 MHz, DMSO) δ ppm 7.90 (s,

1H), 7.75 (m, 2H), 4.12 (t, J = 6.1 Hz, 2H), 2.81 (t, J = 6.1 Hz, 2H),

2.30 (s, 3H).

1

UV detector wavelength = 215 nm). H NMR (400 MHz, CDCl₃) δ

ppm 1.26−1.35 (m, 9H), 2.90 (dd, J = 16.0, 6.2 Hz, 1H), 3.10 (dd, J =

15.9, 5.8 Hz, 1H), 3.86−3.94 (m, 1H), 3.94−4.06 (m, 1H), 4.92−5.03

(m, 1H), 6.51 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.44 (m,

1H).

1-Acetyl-6-(4-methoxyphenyl)-2,3-dihydroquinolin-4(1H)-

one (62). To a solution of 1-acetyl-6-bromo-2,3-dihydroquinolin-

4(1H)-one (61) (0.91 g, 3.4 mmol) in 1,2-dimethoxyethane (25 mL)

was added (4-methoxyphenyl)boronic acid (0.67 g, 4.4 mmol), a 2.5

M solution of cesium carbonate in water (3.4 mL, 3.5 mmol) and

Pd(PPh₃)₄(0.11 g, 0.035 mmol), and the mixture was stirred under

reﬂux for 3.3 h. The solvent was then removed under reduced

pressure, and CH₂Cl₂was added to the residue. The organic phase was

washed with water and brine, dried over Na₂SO₄, ﬁltered, and

evaporated to dryness. The residue was puriﬁed by ﬂash chromatog-

raphy on silica gel (2% MeOH in CH₂Cl₂) to deliver the title

compound (0.67 g, 67%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃)

δ ppm 8.15 (s, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.7 Hz, 3H),

6.92 (d, J = 8.7 Hz, 2H), 4.20 (t, J = 6.1 Hz, 2H), 3.80 (s, 3H), 2.78 (t,

J = 61 Hz, 2H), 2.33 (s, 3H).

Isopropyl ((2S,4R)-6-Bromo-2-methyl-1,2,3,4-tetrahydroqui-

nolin-4-yl)carbamate (65). (S)-Isopropyl (3-((4-bromophenyl)-

amino)butanoyl)carbamate (64) (17.9 g, 52.2 mmol) was taken up

in EtOH (150 mL) and cooled to below −10 °C (internal

temperature) in a CO₂/acetone bath. Sodium borohydride (1.381 g,

36.5 mmol) was added, followed by magnesium chloride hexahydrate

(11.35 g, 55.8 mmol) in water (25 mL), keeping the temperature

below −5 °C. The mixture was allowed to stir at <0 °C for 1 h then

warmed to room temperature and stirred for 1 h. The resulting thick

suspension was poured into a mixture of citric acid (25.05 g, 130

mmol), HCl (205 mL, 205 mmol, 1 M aqueous solution), and CH₂Cl₂

(205 mL). The biphasic mixture was stirred at room temperature for 1

h. The layers were separated and the organic layer dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo to yield the product as a single cis

isomer, a light-brown solid (14.1 g, 78%). LCMS (method formic):

Q

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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retention time = 1.13 min, [M + H]⁺= 327.2 (99% purity). ¹H NMR

(400 MHz, DMSO-d₆) δ ppm 1.14 (d, J = 6.3 Hz, 3H), 1.20 (d, J = 6.3

Hz, 3H), 1.23 (d, J = 6.3 Hz, 3H), 1.46 (q, J = 11.9 Hz, 1H), 1.88−

1.97 (m, 1H) 3.40−3.51 (m, 1H), 4.69−4.79 (m, 1H), 4.78−4.88 (m,

1H), 6.48 (d, J = 8.6 Hz, 1H), 6.98 (s, 1H), 7.07 (dd, J = 8.5, 1.9 Hz,

1H), 7.42 (d, J = 9.1 Hz, 1H).

batch of the title compound (1.7 g, 2.7%) as a very pale-gray solid,

which was used in the next step without further puriﬁcation. LCMS

1

(method formic) retention time = 1.16 min, [M + H]⁺= 439.16. H

NMR (400 MHz, DMSO-d₆) δ 8.06 (d, J = 8.3 Hz, 2H), 7.80 (d, J =

8.3 Hz, 2H), 7.71 (d, J = 8.8 Hz, 1H), 7.65 (dd, J = 1.8, 8.8 Hz, 1H),

7.46 (m, 2H), 4.85 (spt, J = 6.9 Hz, 1H), 4.66 (m, 1H), 4.45 (m, 1H),

4.34 (q, J = 7.1 Hz, 2H), 2,47 (m, 1H), 2.10 (s, 3H), 1.35 (t, J = 7.1

Hz, 3H), 1.25 (t, J = 6.9 Hz, 6H), 1.19 (m, 1H), 1.06 (d, J = 6.3 Hz,

3H).

Isopropyl ((2S,4R)-1-Acetyl-6-bromo-2-methyl-1,2,3,4-tetra-

hydroquinolin-4-yl)carbamate (66). Isopropyl ((2S,4R)-6-bromo-

2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)carbamate (65) (14.1 g, 43.1

mmol) was taken up in CH₂Cl₂(400 mL) under nitrogen at room

temperature. Pyridine (10.46 mL, 129 mmol) followed by acetyl

chloride (4.60 mL, 64.6 mmol) were added and the reaction stirred for

16 h. The reaction mixture was partitioned between EtOAc (2000 mL)

and a saturated NaHCO₃aqueous solution (800 mL). The organic

layer was extracted and washed with water (1500 mL) and brine (1500

mL) and then dried over Na₂SO₄, ﬁltered, and concentrated in vacuo

to yield a purple solid. The crude product was taken up in the

minimum of dichloromethane and applied to a Companion XL

column (330 g) and eluted with 12% EtOAc in cyclohexane for 1CV,

then 12−63% EtOAc over 12 CV, then held at 63% for 4CV. The

appropriate fractions were collected to aﬀord the title product as an

oﬀ-white solid (12.37 g, 78%). LCMS (method formic): retention time

1.03 min, [M + H]⁺= 369.1. ¹H NMR (600 MHz, DMSO-d₆) δ ppm

7.61 (br d, J = 8.6 Hz, 1H), 7.47 (dd, J = 8.4, 2.4 Hz, 1H), 7.29 (d, J =

8.4 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 4.83 (spt, J = 6.2 Hz, 1H),

4.66−4.58 (m, 1H), 4.40−4.33 (m, 1H), 2.44 (ddd, J = 12.7, 8.5, 4.5

Hz, 1H), 2.05 (s, 3H), 1.24 (dd, J = 16.3, 6.1 Hz, 6H), 1.21−1.16 (m,

2H), 1.02 (d, J = 6.4 Hz, 3H). ¹³C NMR (150.9 MHz, DMSO-d₆) δ

ppm 168.4 (s, 1C), 155.7 (s, 1C), 139.2 (s, 1C), 135.4 (br s, 1C),

129.6 (s, 1C),128.1 (br s, 1C), 125.5 (s, 1C), 117.9 (s, 1C), 67.3 (s,

1C), 46.9 (br s, 1C), 46.6 (s, 1C), 39.6 (s, 1C), 22.5 (s, 1C), 22.0 (s,

2C), 21.2 (s, 1C), See Supporting Information Figure S4 for NMR

spectra.

Separation of enantiomers: Chiralpak AD column, 20 μm particle

size, internal diameter 75 mm, bed length 258 mm. Eluent 80:20

heptane:ethanol. Flow: 400 mL/min. Temperature: ambient. Injection

volume: ∼44 mL containing 4 g of compound 66 in 35:65 v/v

heptane:ethanol. Run time: 4.5 min. Pure fractions containing the

required ﬁrst eluting enantiomer from all the injections were

concentrated to dryness on a rotary evaporator to give product as a

solid. This was slurried in heptane, stirred for 1 h at 20−25 °C, ﬁltered,

washed on the ﬁlter with heptane, and dried under vacuum at 40 °C to

give the product as a solid (89% recovery). HPLC analysis showed that

the product contained <0.5% of the second eluting enantiomer. [α]D

= +281.1° (T = 20.7 °C, 10 mm cell, c = 0.508 g/100 mL, EtOH).

Ethyl 4-((2S,4R)-1-Acetyl-4-((isopropoxycarbonyl)amino)-2-

methyl-1,2,3,4-tetrahydroquinolin-6-yl)benzoate (67). Isoprop-

yl ((2S,4R)-1-acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydroquinolin-4-

yl)carbamate (66) (53.47 g, 145 mmol), {4-[(ethyloxy)carbonyl]-

phenyl}boronic acid (29.51 g, 152 mmol), and tetrakis-

(triphenylphosphine)palladium (2.51 g, 2.17 mmol) were mixed in

DME (500 mL) and the mixture treated with a 2N Na₂CO₃aqueous

solution (290 mL, 580 mmol). The mixture was degassed and then

heated at 105 °C under nitrogen for 16 h. The mixture was then

cooled to room temperature and was partitioned between EtOAc and

water. The layers were separated, and the aqueous phase was extracted

twice with EtOAc. The combined organics were washed with water

and then brine, dried using a hydrophobic frit, and concentrated in

vacuo. The residual gray−brown solid was dissolved in EtOAc and

ﬁltered through a plug of silica. The silica was washed with EtOAc, and

the combined ﬁltrate and washings were concentrated in vacuo to give

a cream solid. This material was triturated with Et₂O, ﬁltered, and the

solid washed with a small volume of Et₂O. The solid was partially air-

dried and then dried under vacuum at 40−50 °C to give the title

compound (56.2 g, 89%) as a white solid, which was used in the next

step without further puriﬁcation. The ﬁltrate and washings were

concentrated in vacuo. The gummy brown residue obtained was

triturated with Et₂O and the solvent removed in vacuo. The trituration

with Et₂O was repeated. The solid obtained was collected by ﬁltration,

washed with Et₂O, and dried under vacuum at 40 °C to give another

Ethyl 4-((2S,4R)-1-Acetyl-4-amino-2-methyl-1,2,3,4-tetrahy-

droquinolin-6-yl)benzoate (68). Ethyl 4-[(2S,4R)-1-acetyl-2-meth-

yl-4-({[(1-methylethyl)oxy]carbonyl}amino)-1,2,3,4-tetrahydro-6-

quinolinyl]benzoate (67) (25.33g, 57.8 mmol) was added to a ice-cold

suspension of AlCl₃(29.24 g, 219 mmol) in CH₂Cl₂(450 mL), and

the resulting solution was stirred at 0 °C for 20 min. A solution of

NEt₃(96 mL, 693 mmol) and MeOH (50 mL) was then added over

approximately 2 min (initially dropwise and then faster after ﬁrst third

of the solution was used). The mixture was stirred for 30 min in the ice

bath, then was diluted with EtOAc, ﬁltered through Celite, and the

residue washed with EtOAc. The residue was partitioned between

EtOAc and a saturated sodium NaHCO₃aqueous solution. The

biphasic mixture was ﬁltered through Celite, and the residue was

washed with EtOAc. The residue was further washed with acetone and

the acetone washings combined with the previous biphasic mixture.

Most of the organics were concentrated in vacuo, and the aqueous

residue was partitioned between EtOAc and water. The layers were

separated, and the aqueous phase was extracted with EtOAc. The

combined organic phases were washed with brine, dried using a

hydrophobic frit, and concentrated in vacuo to give the title compound

(14.7 g, 72%) as a cream solid which was used in the next step without

further puriﬁcation. The solid residue was rewashed with MeOH, and

most of the MeOH was concentrated in vacuo. The residue was

partitioned between EtOAc and water, and the layers were separated.

The aqueous phase was extracted with EtOAc, and the combined

organic phases were dried using a hydrophobic frit and concentrated in

vacuo to give the title compound (5.3 g, 26%) as a cream solid, which

was used in the next step without further puriﬁcation. LCMS (method

formic) retention time 0.73 min, [M + H − NH₂]⁺= 337.3. ¹H NMR

(400 MHz, DMSO-d₆) δ 8.06 (d, J = 8.3 Hz, 2H), 7.87−7.91 (m, 3H),

7.63 (dd, J = 2.0, 8.3 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 4.62 (m, 1H),

4.36 (q, J = 7.0 Hz, 2H), 3.62 (m, 1H), 2.46 (m, 1H), 2.08 (s, 3H),

1.36 (t, J = 7.0 Hz, 3H), 1.19 (m, 1H), 1.06 (d, J = 6.3 Hz, 3H). NH₂

not observed.

Ethyl 4-{(2S,4R)-1-Acetyl-4-[(4-chlorophenyl)amino]-2-meth-

yl-1,2,3,4-tetrahydro-6-quinolinyl}benzoate (69). Ethyl 4-

[(2S,4R)-1-acetyl-4-amino-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl]-

benzoate (68) (24.6 g, 69.8 mmol), 1-bromo-4-chlorobenzene (26.7 g,

140 mmol), bis(dibenzylideneacetone)palladium (3.20 g, 5.56 mmol),

and [2′-(dicyclohexylphosphanyl)-2-biphenylyl]dimethylamine (Dave-

phos, 2.19 g, 5.56 mmol) were mixed in toluene (450 mL), and the

mixture was treated with sodium t-butoxide (7.38 g, 77 mmol). The

resulting mixture was degassed and stirred at 70 °C under nitrogen for

24 h, then was cooled to room temperature and ﬁltered through

Celite. The residue was washed with toluene and then with Et₂O. The

combined ﬁltrate and washings were washed twice with water then

twice with a 2 N HCl aqueous solution. The orange oil which

precipitated was collected with the aqueous phase. The acid extracts

were extracted with EtOAc, and the combined organics were washed

with water and brine. This water and brine were combined with the

acidic washes in order to recover the starting material (vide infra). The

organic solution was further dried over MgSO₄and concentrated in

vacuo to give a dark-orange oil. This experiment was repeated, and the

two residues hence obtained were combined for further puriﬁcation.

Puriﬁcation of these combined residues by ﬂash chromatography on

silica gel (750 g column, 5−80% AcOEt in hexane) gave the title

compound (16.5 g, 26% mean yield) as a pale-yellow foam. The

impure fractions containing product were combined and concentrated

in vacuo. Puriﬁcation of this residue by ﬂash chromatography on silica

gel (330 g column, 5−80% AcOEt in hexane) gave the title compound

(7.3 g, 11% mean yield) as a pale-brown foam. The combined acidic

R

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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phases (vide supra) were basiﬁed using solid Na₂CO₃and then were

extracted three times with EtOAc. The combined organics were dried

over MgSO₄and combined with the same organic phases from the

parallel experiment, then were concentrated in vacuo to give 69 (28.0

g, 57% average yield) as an orange foam which partially crystallized

upon standing overnight. This material could be used in a repeat of

this coupling without further puriﬁcation. LCMS (method formic)

parameters: stoichiometry of 1.93, K_Dof 4.4 0.9 nM, ΔH −15.9

0.08 kcal/mol, ΔS −15.2 cal/mol/deg.

ITC titration of CREBBP was carried out using a Microcal

AutoITC200 in 50 mM Hepes, pH 7.4, 150 mM NaCl, at 25 °C. First,

44 μM of CREBBP bromodomain was placed in the cell, and 500 μM

ligand was titrated into this to achieve a ﬁnal ligand:protein excess of

∼2.2:1. The data was then ﬁtted within Origin (Microcal version) to

give the following parameters: stoichiometry of 1.1, K_Dof 6.3 0.5

μM, ΔH −4.35 0.08 kcal/mol, ΔS 9.2 cal/mol/deg.

1

retention time 1.35 min, [M + H − NHPhCl]⁺= 337.23. H NMR

(400 MHz, DMSO-d₆) δ 8.01 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 8.6 Hz,

2H), 7.64 (dd, J = 2.0, 8.1 Hz, 1H), 7.47 (m, 2H), 7.12 (d, J = 8.8 Hz,

2H), 6.74 (d, J = 8.8 Hz, 2H), 6.30 (d, J = 8.1 Hz, 1H), 4.71 (m, 1H),

4.32 (q, J = 7.1 Hz, 2H), 4.28 (m, 1H), 2.62 (m, 1H), 2.15 (s, 3H),

1.33 (t, J = 7.1 Hz, 3H), 1.23 (m, 1H), 1.10 (d, J = 6.1 Hz, 3H).

FaSSIF Solubility. See Supporting Information, Supplementary

Methods, for details of this high-throughput method to determine the

solubility of compounds in fasted-state simulated intestinal ﬂuid by

precipitation.

ApoA1 Luciferase Assay. HepG2 cells were stably transfected

with a plasmid encoding ﬁreﬂy luciferase under the control of the

human ApoA1 promoter region (−1162, +232) and the 3′-UTR of the

human ApoA-1 gene (+1037, +1091). ApoA1 potency was deﬁned as

the concentration of compound, resulting in a 70% increase in

luciferase luminescence (ApoA1 Luc EC₁₇₀). See Supporting

Information, Supplementary Methods. High correlation has been

shown between ApoA1-luciferase reporter activity and ApoA1 protein

secretion in HepG2 cells.²⁴

Recombinant Protein Binding Assays. The tandem bromodo-

main constructs of BRD2, BRD3, and BRD4 were screened in two

formats. The FA (ﬂuorescence anisotropy) assay measures displace-

ment of a labeled benzodiazepine analogue of 1, as previously

described.⁴The TR-FRET (time-resolved ﬂuorescence resonance

energy transfer) assay measures displacement of a tetra-acetylated

biotinylated peptide. See Supporting Information, Supplementary

Methods, for full details.

IL-6 PBMC and Whole Blood Assays. Human whole blood and

peripheral blood mononuclear cells (PBMCs) puriﬁed from whole

blood were incubated with LPS and varying concentrations of test

compounds for 18−24 h. Samples were prepared, and inhibition of IL-

6 was measured as described in Supporting Information, Supple-

mentary Methods.

X-ray Crystallography. General methods for the crystallography

of the N-terminal bromodomain of Brd2 have been previously

described.²⁹The complexes of 28 and 47 were determined using

Escherichia coli expressed His-tagged Brd2 (67−200). Speciﬁc details

for each complex determined are given in Supporting Information,

Supplementary Methods. For the C-terminal bromodomain of Brd2, E.

coli expressed His-tagged Brd2-BD2 (338−473) was puriﬁed to

homogeneity using standard protocols in a similar manner to the N-

terminal bromodomain of Brd4 using His aﬃnity capture, Tev

protease cleavage, and gel ﬁltration.⁷See Supporting Information,

Supplementary Methods, for full details. Ligand OMIT maps and

reﬁnement statistics are shown in Supporting Information Figure S3

and Table S5. Methods for the determination of the complex of 47

with the Brd4 N-terminal bromodomain and 48 with the Brd2 N-

terminal bromodomain have previously been described.^29,37

Cytochrome P450 Inhibition and Time-Dependent Inhib-

ition Studies. All cytochrome P450 inhibition studies were

performed as described in Supporting Information, Supplementary

Methods, at GlaxoSmithKline Medicines Research Centre (Stevenage,

UK).

Hepatocyte Clearance. Intrinsic clearance studies were per-

formed as described in Supporting Information, Supplementary

Methods, at GlaxoSmithKline Medicines Research Centre (Ware,

UK).

Permeability Assay. Artiﬁcial membrane permeability was

measured as described in Supporting Information, Supplementary

Methods.

Blood Protein Binding. All measurements of blood protein

binding were performed as described in Supporting Information,

Supplementary Methods, at GlaxoSmithKline Medicines Research

Centre (Stevenage, UK).

Animal Studies. All animal studies were ethically reviewed and

carried out in accordance with the GSK Policy on the Care, Welfare,

and Treatment of Animals and European Directive 86/609. The rat

PK study was performed at GSK Stevenage, UK, the dog PK study at

GSK Ware, UK, the mouse PK studies at GSK Les Ulis, France, and

the primate PK study at CIT, France. For all studies, the temperature

and humidity were nominally maintained at 21 °C 2 °C and 55%

10%, respectively. There were no known contaminants in the diet or

water at concentrations that could interfere with the outcome of the

studies. See Supporting Information, Supplementary Methods.

Pharmacokinetics Studies. Diluted blood samples (1:1 with

water) or plasma were extracted using protein precipitation with

acetonitrile containing an analytical internal standard. An aliquot of the

supernatant was analyzed by reverse phase LC-MS/MS using a heat

assisted electrospray interface in positive ion mode. Samples were

assayed against calibration standards prepared in control blood. PK

parameters were obtained from the blood or plasma concentration−

time proﬁles using noncompartmental analysis with WinNonlin

Professional 4.1a (Pharsight, Mountain View, CA).

Thermal Shift Assay (T_m). Thermal shifts of bromodomains were

analyzed on a FluoDiaT70 instrument. The temperature of the protein

sample was ramped from 26/30 to 74 °C in the presence or absence of

100 μM of the ligand. Denaturation was visualized using 1:1000

dilution of a ﬂuorescent Sypro orange dye in a buﬀer of PBS, 10%

glycerol. The temperature was ramped at a rate of 1 °C/min and

ﬂuorescence readings taken every 1 °C. Bromodomains tested

included ATAD2, BAZ2B, BRD2 (tandem bromodomains), BRD4

(N- and C-terminal and tandem bromodomains), CREBBP, PCAF,

SMARCA2, SP140, and TAF1 (tandem bromodomains). Results

reported are the mean of at least two experiments.

Surface Plasmon Resonance (BIAcore) Analysis of Binding

to BRD2, 3, and 4. BIAcore data of His6-tagged BRD2 (1−473),

BRD3 (1−434), and BRD4 (1−477) was acquired and analyzed on a

T100 BIAcore instrument at 25 °C. In all cases, a CM5 chip with ∼5−

12kU amine coupled protein immobilized on the dextran surface was

used with a running buﬀer of 30 mM Hepes pH 7, 150 mM NaCl, 1

mM EGTA, NaN3. Compounds were titrated as a tripling dilution

starting from 10 μM. Sensorgrams and binding curves were analyzed

with BIAevaluation (GE Healthcare) using a 1:1 binding model. The

In Vivo Murine Eﬃcacy Models. The eﬀects of single-dose

administration of compound 47 on LPS-induced PAI-1 RNA

expression, and in a mouse model of septic shock, were determined

as described in Supporting Information, Supplementary Methods.

equilibrium K_Dwas calculated using response = concentration × R_max

/

(concentration + K_D) + oﬀset. Example dose response ﬁts are given in

Supporting Information, Figure S2.

Isothermal Titration Calorimetry.³⁷ITC titration of BRD4 was

carried out using a Microcal VPITC in 50 mM Hepes, pH 7.5, 150

mM NaCl, at 25 °C. First, 9 μM His-tagged BRD4 (1−477) tandem

bromodomain was placed in the cell and 200 μM ligand was titrated

into this to achieve a ﬁnal ligand:protein excess of ∼4:1. The data was

then ﬁtted within Origin (Microcal version) to give the following

ASSOCIATED CONTENT

■

S

* Supporting Information

Methods, bromodomain selectivity proﬁle of 47, BET binding

curves of 47 by SPR, X-ray ligand density maps, NMR spectra,

tables showing mean, standard deviation and number of test

S

dx.doi.org/10.1021/jm5010539 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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reﬁnement statistics. This material is available free of charge via

the Internet at http://pubs.acs.org.

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Accession Codes

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Data Bank. The codes are 4uyf (BRD2 N-terminal

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main with 47), and 4uyh (BRD2 N-terminal bromodomain

with 28).

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AUTHOR INFORMATION

Corresponding Authors

*For P.B.: phone, +44 (0)20 8047 5000; fax, +44 1438 764486;

E-mail, paul.a.bamborough@gsk.com.

■

*For R.K.P. (requests for material, research collaboration, etc.):

phone, +44 1438 764303; E-mail: rabinder.prinjha@gsk.com.

Present Addresses

^¶For R.G.: Galapagos SASU, 102 Avenue Gaston Roussel,

93230 Romainville, France.

⁺For C.S.: 71 Avenue Franklin Roosevelt, 94550 Chevilly

Larue, France.

^▲For O.M.: Institut de Recherche Servier, 11 rue des

Moulineaux, 92150 Suresnes, France.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the DiscoveRx Corporation for BROMOscan^SM

selectivity screening. We thank Richard Upton (GlaxoSmithK-

line, Stevenage) for NMR support, Catherine Cartwright and

James Clarke (GlaxoSmithKline, Ware) for in vitro preclinical

developability proﬁle information, and Nigel Parr and Iain

Uings (GlaxoSmithKline, Stevenage) for leadership and helpful

guidance given to the BET bromodomain program.

(15) Miyoshi, S.; Ooike, S.; Iwata, K.; Hikawa, H.; Sugahara, K.

Antitumor agent. WO2008-JP73864, 2008.

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access chemical and clinical probes to support drug discovery. Nature

Chem. Biol. 2009, 5, 436−440.

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ABBREVIATIONS USED

■

AcK, acetyl-lysine; ApoA1, apolipoprotein A-1; BD, bromodo-

main; BET, bromodomain and extra-terminal; BRD2/3/4,

bromodomain-containing protein 2/3/4; BZD, benzodiaze-

pine; CREBBP, cAMP response element-binding binding

protein; FA, ﬂuorescence anisotropy; IL6, interleukin-6; LPS,

lipopolysaccharide; NUT, nuclear protein in testis; THQ,

tetrahydroquinoline; TR-FRET, time-resolved ﬂuorescence

resonance energy transfer

(18) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.;

Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.;

Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.;

West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La, T. N.;

French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E.

Selective inhibition of BET bromodomains. Nature 2010, 468, 1067−

1073.

(19) Mertz, J. A.; Conery, A. R.; Bryant, B. M.; Sandy, P.;

Balasubramanian, S.; Mele, D. A.; Bergeron, L.; Sims, R. J., III.

Targeting MYC dependence in cancer by inhibiting BET bromodo-

mains. Proc. Natl. Acad. Sci. U. S. A 2011, 108, 16669−16674.

(20) Dawson, M. A.; Prinjha, R. K.; Dittmann, A.; Giotopoulos, G.;

Bantscheff, M.; Chan, W. I.; Robson, S. C.; Chung, C. W.; Hopf, C.;

Savitski, M. M.; Huthmacher, C.; Gudgin, E.; Lugo, D.; Beinke, S.;

Chapman, T. D.; Roberts, E. J.; Soden, P. E.; Auger, K. R.; Mirguet,

O.; Doehner, K.; Delwel, R.; Burnett, A. K.; Jeffrey, P.; Drewes, G.;

Lee, K.; Huntly, B. J.; Kouzarides, T. Inhibition of BET recruitment to

chromatin as an effective treatment for MLL-fusion leukaemia. Nature

2011, 478, 529−533.

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The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor

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