Structure-Based Design of a Bromodomain and Extraterminal Domain (BET) Inhibitor Selective for the N-Terminal Bromodomains That Retains an Anti-inflammatory and Antiproliferative Phenotype

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Structure-Based Design of a Bromodomain and Extraterminal

Domain (BET) Inhibitor Selective for the N‑Terminal Bromodomains

That Retains an Anti-inﬂammatory and Antiproliferative Phenotype

Christopher R. Wellaway,* Paul Bamborough, Sharon G. Bernard, Chun-wa Chung, Peter D. Craggs,

Leanne Cutler, Emmanuel H. Demont, John P. Evans, Laurie Gordon, Bhumika Karamshi,

Antonia J. Lewis, Matthew J. Lindon, Darren J. Mitchell, Inmaculada Rioja, Peter E. Soden,

́

Simon Taylor, Robert J. Watson, Rob Willis, James M. Woolven, Beata S. Wyspianska, William J. Kerr,

and Rab K. Prinjha

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ABSTRACT: The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2,

BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind

all eight bromodomains of the family with similar aﬃnity, resulting in a diverse range of biological eﬀects. To enable further

understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or

sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up

to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided

by structural information, a speciﬁc amino acid diﬀerence between BD1 and BD2 domains was targeted for selective interaction with

chemical functionality appended to the previously developed I-BET151 scaﬀold. Data presented herein demonstrate that selective

inhibition of BD1 domains is suﬃcient to drive anti-inﬂammatory and antiproliferative eﬀects.

pan-inhibition. Despite the promising disease-modifying eﬀects

of pan-BET inhibitors, greater insight into how individual

bromodomains contribute to particular biological responses

can be realized with domain-selective inhibitors, and there have

been recent advances in this area.

The emergence of domain-selective BET inhibitors (Figure

1)⁴⁻¹¹such as RVX-208/Apabetalone (1) have provided

further biological understanding. This clinical candidate

originated from a HepG2 Apo-A1 upregulation assay and

INTRODUCTION

■

Bromodomains are a diverse family of protein interaction

modules that bind acetylated lysine (KAc) post-translation

modiﬁcations (PTMs) on histone tails and function as

regulators of transcriptional control.¹The human genome

encodes at least 46 bromodomain-containing proteins (BCPs)

encompassing 61 unique bromodomains, including the

bromodomain and extraterminal domain (BET) family

(BRD2, BRD3, BRD4, and BRDT).²The bromodomains

within the BET family have been targeted with small

molecules, which have been used to probe the function of

the BET proteins in a variety of disease settings including

oncology, inﬂammation, infectious disease, and metabolic

disorders.³Each BET family member contains tandem

bromodomain modules, and most BET inhibitors published

to date bind all eight bromodomains with similar aﬃnity, i.e.,

Received: April 6, 2020

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Figure 1. Structures of domain-selective BET inhibitors.

was subsequently found to inhibit the BET bromodomains

with preferential binding to the C-terminal bromodomains

(BD2s) over the N-terminal bromodomains (BD1s).^4,5The

degree of selectivity was dependent on the method of

measurement, but based on dissociation constants from

isothermal titration calorimetry (ITC), Picaud et al. reported

BD2 selectivities of 23-fold and 8-fold for BRD2 and BRD4,

respectively.⁵Independent ITC measurements by McLure and

co-workers also determined preferential binding to BD2

domains but with an increased magnitude (82-fold for BRD2

and 29-fold for BRD4).⁶Gene-expression studies with RVX-

208 showed that selective inhibition of BD2 only modestly

aﬀected BET-mediated transcription and suggested a dominant

role of BD1 in transcriptional control.⁵

While optimizing the selectivity of a diazobenzene CREBBP

inhibitor toward BRD4 bromodomains, Zhang et al. discovered

MS436 (2, Figure 1), a molecule possessing approximately 10-

fold selectivity for BRD4 BD1 over BRD4 BD2 but with

negligible domain selectivity in BRD3.⁷Cellular studies with

MS436 demonstrated inhibition of the BRD4 function in gene

transcription through blockage of both NF-κB-directed nitric

oxide production and interleukin (IL)-6 cytokine expression in

LPS-stimulated macrophages.

Another report described the structure-based optimization

of a CBP hit compound into Olinone (3, Figure 1), an

inhibitor selective for BET BD1 domains.⁸At the concen-

trations tested, either no or weak (>300 μM) binding of

Olinone to the BD2 domains of BRD2, 3, and 4 was detected

in FA and ITC assays, respectively. However, Olinone

possessed only modest aﬃnities (K_i= 17.2−39.5 μM by FA,

and K_d= 3.3−8.6 μM by ITC) for BD1 domains. Studies with

Olinone demonstrated its ability to promote oligodendrocyte

progenitor diﬀerentiation, whereas a pan-BET inhibitor

suppressed this response.

A publication from Law et al. described the discovery of a

series of BET BD2-selective tetrahydroquinoxalines exempli-

ﬁed by GSK340 (4, Figure 1).⁹The potency of GSK340 for

BD2 domains ranged 316−50 nM in mutant FRET assays with

8−50-fold selectivities over BD1 domains. Potencies and

selectivities were conﬁrmed by surface plasmon resonance

(SPR) and BROMOscan¹²assays. A number of BD2-selective

tetrahydroquinoxalines inhibited the release of the proin-

ﬂammatory cytokine MCP-1 in LPS-stimulated human

peripheral blood mononuclear cells (PBMCs), an eﬀect also

observed for GSK340 in human whole blood. Collectively,

these data indicated selective inhibition of BD2 domains was

suﬃcient to drive immuno-inﬂammatory eﬀects.

Divakaran et al. recently reported the development of the

dual kinase-BET inhibitor (5, Figure 1).¹⁰Several 1,4,5-

trisubstituted imidazoles displayed preferential binding to the

BD1 domains of BET, and IC₅₀values for 5 at BRD4 BD1 and

BRD4 BD2 were determined as 1.8 μM and >100 μM,

respectively; the K_dfor p38α was 0.47 nM. A reduction of the

oncogene c-Myc in MM.1S cells, a hallmark of BET inhibition,

was observed upon treatment with 5, while anti-inﬂammatory

eﬀects in A549 cells, including reduced TNFα-induced

transcription and inhibition of IL-8 cytokine production,

were observed. A subsequent publication from these

researchers demonstrated eﬀective ablation of p38α activity

from triazole-based BET inhibitors, while increasing BRD4

BD1 activity; however, the domain selectivity diminished.¹³

The BD2-selective BET inhibitor ABBV-744 (6, Figure 1)

was very recently reported.^11,14This inhibitor possessed high

potency for the BD2 domains and greater than 290-fold

selectivity over the BD1 domains of BRD2, 3, and 4 in FRET

assays.¹¹The high potency and selectivity for BRD4 BD2 over

BRD4 BD1 were recapitulated in SPR and NanoBRET assays.

Testing in 59 cancer cell lines revealed that the antiproliferative

eﬀects of ABBV-744 were narrowed compared to a pan-BET

inhibitor. In vivo assessment of ABBV-744 demonstrated

eﬃcacy in a prostate cancer xenograft model and reduced side

eﬀects, highlighting the therapeutic potential for selective

inhibition of BET BD2 domains.

Studies with these small molecules have provided initial

insight into the function of the BD1 and BD2 domains.

However, the modest domain selectivities or potencies

displayed by most of these inhibitors limit their usefulness

for fully investigating domain-selective inhibition. For example,

contributory eﬀects on the biological response relating to the

engagement of lower aﬃnity bromodomains cannot be ruled

out for modestly selective inhibitors. Additionally, the high

concentrations required for weak inhibitors to fully engage the

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Figure 2. (A) Bromodomain phylogenetic tree (adapted from Chung et al.²⁰) with an enlarged section showing the BET BD1 and BD2

bromodomains. (B) Domain architecture of the BET proteins. (C) Percentage sequence identity between the eight BET bromodomains (>70%

colored green and ≤40% in red). (D) Sequence alignment of BET bromodomains. (E) BET family BC loop residues colored by amino acid type.

Figure 3. (A) Complex of imidazoquinolinone I-BET151 (7) in BRD4 BD1 (carbon = green, PDB 3zyu²²) superimposed on the BRD4 BD2

protein (carbon = orange, PDB 2yem²¹). (B) Distribution of Asp/His chi1 torsion angles of BRD2 and BRD4 BD1 and BD2 domain X-ray

structures in the Protein Data Bank.

intended bromodomain(s) risk oﬀ-target eﬀects. While other

methods such as protein deletion,¹⁵⁻¹⁷site-directed muta-

genesis,¹⁸and a “bump-and-hole” approach¹⁹have contributed

to our understanding, the development of inhibitors

demonstrating signiﬁcant domain selectivity and potency is

crucial to elucidate the speciﬁc roles of BD1 and BD2 domains

in native protein environments.

Accordingly, we herein report the structure-based design and

development of a probe molecule exhibiting both high aﬃnity

and selectivity for BD1 domains of the BET family. Studies

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with this molecule revealed anti-inﬂammatory and antiprolifer-

ative activities comparable to pan-BET inhibitors.

prospective derivatives to the BD1 and BD2 domains of BET,

biochemical assay development was carried out.

Previous studies with a pan-BET inhibitor revealed apparent

diﬀerences in aﬃnity for isolated BRD2 bromodomains (BD1

and BD2) compared to the tandem BRD2 bromodomain

constructs, which we attribute to diﬀerential stability of the

single-domain proteins.²¹Therefore, tandem bromodomain

protein constructs with site-directed mutations impairing

binding to the KAc pocket of either bromodomain were

developed. This enabled compound IC₅₀determination for

each domain upon displacement of a bromodomain non-

selective ﬂuorescent ligand (Supporting Information, Section

S2). As controls for this approach, BET tandem constructs

were produced with neither or both bromodomains mutated.

BET proteins containing alanine mutants of the conserved

tyrosine residue, whose side chain hydroxyl anchors one of the

key conserved water molecules essential for ligand recognition

in the KAc pocket (for example, Tyr97 in BRD4 BD1), were

expressed in E. coli and puriﬁed utilizing a 6-His tag at the N-

terminus. Subsequently, a ﬂuorescence polarization approach

conﬁrmed that the ﬂuorescent ligand had similar binding

aﬃnities for each of the single bromodomain mutants as well as

the nonmutated proteins but had a signiﬁcant decrease in

aﬃnity for the double mutant constructs (Supporting

Information, Figure S2iii). These results demonstrate that, in

the presence of one mutated bromodomain, the tandem

protein is still able to bind the ﬂuorescent ligand, through the

nonmutated bromodomain, with a similar aﬃnity to the wild-

type construct. However, if both bromodomains are mutated,

then the aﬃnity for the ﬂuorescent ligand is signiﬁcantly

diminished, demonstrating that neither of the bromodomains

can bind in a similar manner to the nonmutated, wild-type

protein. Collectively, these results conﬁrm the validity of this

approach for measuring domain selectivity across the BET

family, and the assays were further developed into a TR-FRET

format for use in this research.

RESULTS AND DISCUSSION

■

The four BET isoforms each contain two bromodomains (BD1

and BD2), all highly conserved with respect to the rest of the

bromodomain phylogenetic tree (Figure 2A,B). The BD1 and

BD2 domains form two distinct sub-branches of the tree, with

higher conservation (68−87% identity) between bromodo-

mains across isoforms (e.g., BRD2 BD1 versus BRD4 BD1,

Figure 2C) and lower conservation (36−45% identity)

between the two bromodomains within each isoform (e.g.,

BRD2 BD1 and BRD2 BD2).²¹One key position close to the

KAc binding site diﬀering between BD1 and BD2 domains is

found in the BC loop (Figure 2D,E). The BD1 domains of all

isoforms possess an aspartic acid at one position (BRD2

Asp160, BRD3 Asp120, BRD4 Asp144, and BRDT Asp113),

whereas the BD2 domains contain a histidine (BRD2 His433,

BRD3 His395, BRD4 His437, and BRDT His356). Two

crystal structures of the BRD4 BD1 and BD2 domains (PDB

codes 3zyu²²and 2yem²¹) are representative of published

high-resolution X-ray structures and show the structural

consequences of this substitution (Figure 3A). Although the

backbones of Asp144 and His437 align closely, their side

chains adopt diﬀerent rotameric states, an observation

conﬁrmed in most BRD2 and BRD4 X-ray structures published

to date (Figure 3B). In BRD4 BD1, Asp144 preferentially

adopts the gauche(+) chi1 rotamer and points away from the

KAc binding site (“out”). In contrast, the BRD4 BD2 His437

lies trans and points toward it (“in”).

A possible explanation for this diﬀerent preference lies in the

hydrogen-bonding interaction between Asp144 and the

backbone NH of Lys141 in BRD4 BD1. Lys141 is conserved

as lysine in all BET BD1 domains, whereas, in all BD2

domains, it is replaced by proline (Pro434 in BRD4). Not only

does Pro434 lack the hydrogen-bonding ability of a backbone

NH, it might also clash with His437 in its gauche(+)

conformation, so in BRD4 BD2, His437 points toward the

KAc site. In doing so, it makes a direct hydrogen bond with the

backbone NH of Val439, the capping residue of the C-helix. In

BRD4 BD1, there is no internal hydrogen bond to Ile146 (the

equivalent residue of BD2 Val439). Instead, the C-helix of

BRD4 BD1 is capped by a water molecule. An equivalent water

molecule has previously been identiﬁed as highly conserved in

numerous fragment complexes of BRD2 BD1.²⁰Using this

insight, we postulated that the selectivity for BD1 could be

obtained by exploiting the steric and electrostatic changes

located around the aspartic acid residue of BD1 and the

histidine of BD2 (the “Asp/His switch”) and set out to test this

hypothesis.

In these assays, I-BET151 exhibited a modest inherent

domain selectivity for BD1 over BD2 (13-fold for BRD4)

(Table 1), which we attribute to a through-water interaction of

Table 1. Activity and Selectivity Proﬁle of I-BET151 in BET

Mutant TR-FRET Assays

a

pIC₅₀

b

BD1

BD2

selectivity (fold)

BRD2

BRD3

BRD4

BRDT

7.3

7.5

7.6

6.9

6.7

7.1

6.5

5.8

4.0

2.5

13

a

b

Expressed as the mean from at least ﬁve test occasions. Calculated

as 10^{(BD1 pIC}

.

₅₀−BD2 pIC₅₀

)

We and others have identiﬁed multiple chemotypes that

bind unselectively to BET bromodomains, the binding modes

of many of which have been solved crystallographically.³Using

this information, we evaluated all available templates to

identify compounds appropriate for chemical modiﬁcation to

interact with the aspartic acid residues of the BD1 domains.

Included in this analysis were quinoline isoxazoles and

inspection of the complex of the key exemplar from this

series; I-BET151 (7, Figure 3A),^23,24in BRD4 BD1,²²

indicated the imidazoquinolinone 8-position as a suitable

vector to target the BRD4 domain-speciﬁc residues Asp144

and His437. To measure the binding of I-BET151 and

the quinoline nitrogen to the side chain of Gln85 in BRD4

BD1, a residue replaced by Lys378 in BRD4 BD2 (Figure 3A).

However, to ensure clear separation of binding from BD2

domains for assessment in phenotypic assays, a focused

program of chemistry was carried out to increase the domain

selectivity of I-BET151 toward a target of >100-fold.

An enantiomerically enriched batch of I-BET151 (ee = 88%

in favor of the R-isomer) available within our laboratories was

used for SAR exploration at the imidazoquinolinone 8-

position. Demethylation of the 8-OCH₃group and reaction

of quinolinol 8 with heteroalkyl halides typically resulted in a

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mixture of mono 8-O (9), mono 3-N (10), and bis 8-O, 3-N

(11) alkylation products (Scheme 1). Rather than optimize the

crystal structure of I-BET151 bound to BRD4 BD1 showed

that the 3-NH vector was directed toward the solvent, and a

methyl substituent at this position was not expected to greatly

aﬀect activity or selectivity. This was subsequently conﬁrmed

upon screening the N-methylated compound 12 in the BRD4

mutant TR-FRET assays (Table 2), and thus, compound 12

served as baseline for SAR comparison. This approach enabled

selective demethylation of the 8-OCH₃group using BBr₃to

give quinolol 13, followed by O-alkylation to access target

compounds 14−39 (Scheme 2). These target compounds

included a functionality that would potentially interact with the

aspartic acid residues (or surrounding water molecules) of the

BD1 domains and/or clash with the His residues of the BD2

domains with the goal of increasing BD1 selectivity. Three

analogues containing carboxylic acids (25, 42, 44) with the

potential to selectively bind the histidine of BD2 were included

in this initial investigation, and those lacking a linking oxygen

atom were accessed from the triﬂate 40 via palladium-catalyzed

cross-coupling and subsequent ester hydrolysis steps (Scheme

3).

Scheme 1. Alkylation of Both the Imidazoquinolinone 3-

a

and 8-Positions

Upon screening these compounds in the BRD4 mutant TR-

FRET assays, it was evident that most substituents led to a

reduction in both BRD4 BD1 and BD2 activity relative to

methoxy 12 (Table 2). However, for basic substituents, BD1

potency was less strongly aﬀected than BD2, leading to

increased BD1 selectivity compared to parent compound 12.

Ethylamine 15 displayed 25-fold greater activity at BRD4 BD1

over BRD4 BD2, while the selectivity for tertiary amine

analogue 16 was >25-fold. The propylamine exhibited

marginally greater BRD4 BD1 activity than ethylamine 15

but maintained the 25-fold level of selectivity. Replacing the

amine of 18 with an alcohol (19), and acetylation to 14,

reduced selectivity. The substitution of the amine with a single

methyl group (22) did not aﬀect activity or selectivity, but two

a

Reagents and conditions: (a) BBr₃(1 M in DCM), −78 °C, 45 min,

then rt, 16 h, then reﬂux, 22 h, 56%. (b) Typical conditions:

heteroalkyl bromide, K₂CO₃, DMF, 100 °C.

reaction conditions for selective mono O-alkylation, we

pursued a synthetic strategy to cap the imidazoquinolinone

3-NH with a methyl group (Scheme 2). Examination of the

a

Scheme 2. Synthesis of O-Substituted Analogues at the Imidazoquinolinone 8-Position

a

Reagents and conditions: (a) NaH, CH₃I, NMP, rt, 5 h, 88%; (b) BBr₃, DCE, 90 °C, 3.5 h, 84%; (c) RX, K₂CO₃, DMF, 45−100 °C, 2−16 h, 24−

86%; (d) 4 M HCl in 1,4-dioxane or TFA, DCM, rt, 1.5−16 h, 12−100%; (e) NaH, CH₃I, NMP, rt, 22 h, 22%; (f) Ac₂O, pyridine, DCM, rt, 2 h,

40%.

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increased domain selectivity to 40-fold, but a greater increase

in BRD4 BD1 activity was observed for the trans-isomer. The

same selectivity was observed for (R)-3-pyrrolidine 31, which

displayed greater BRD4 BD1 activity than the S-isomer 33.

Homologation of these 3-pyrrolidines to the 3-methylene

pyrrolidines 35 and 37 revealed encouraging BD1-selectivity

proﬁles, but less discrimination between each stereoisomer was

observed. Increasing the ring size to 4-methylene piperidine 39

also resulted in signiﬁcant BD1 selectivity. Incorporation of

carboxylic acid groups (25, 42, and 44) led to the diminution

of activity at both domains.

Table 2. Activity and Selectivity of I-BET151 Derivatives in

BRD4 Mutant TR-FRET Assays

Overall, these data demonstrated that O-linked basic

substituents appended to the imidazoquinolinone 8-position

resulted in enhanced selectivity over BRD4 BD2. However,

incorporation of these substituents generally decreased BRD4

BD1 potency with respect to 12 and did not meet our desired

activity (pIC₅₀∼ 7) for comparison to pan-BET inhibitors in

phenotypic assays. Therefore, we examined other quinoline

isoxazoles available within our collection in the mutant TR-

FRET assays to identify a more potent analogue of I-BET151

suitable for derivatization with an appropriate basic sub-

stituent, the (S)-3-methylene pyrrolidine contained in 37 was

selected as a preferred group for this exercise.

We previously described a strategy to cyclize 4-amino-3-

carboxamide quinoline isoxazoles to imidazoquinolinone

isoxazoles (Figure 4), which conferred several beneﬁts

including increased potency.²³In a similar approach previously

unpublished by our laboratories, an imidazoquinoline scaﬀold

was established (Figure 4), which typically conferred even

higher activity and is exempliﬁed by 8-methoxy imidazoquino-

line 45 (Table 3, see Supporting Information, Section S3ii, for

synthesis and characterization).

The BD1 potency of imidazoquinoline 45 approached the

tight-binding limit of the BRD2, 3, and 4 TR-FRET assays, and

the selectivity over BET BD2 domains was, therefore, likely to

be underestimated. However, the BD1 selectivity at BRDT

(13-fold) was the same as that of I-BET151, and selectivities at

the other BET isoforms were also expected to be similar to I-

BET151. Importantly, 8-methoxy imidazoquinoline 45 ex-

hibited suﬃciently high activity at BET BD1 domains so that

the appendage of the preferred basic substituent at the 8-

position was expected to deliver our potency and selectivity

requirements. In this regard, imidazoquinoline 62 was targeted

for synthesis and was achieved using the route shown in

Scheme 4.

First, the protected phenol moiety was introduced by

nucleophilic substitution of 2-bromo-1-ﬂuoro-4-nitrobenzene

46 with benzyl alcohol to furnish the benzylether 47. A

Suzuki−Miyaura reaction of this material with (3,5-dimethy-

lisoxazol-4-yl)boronic acid formed the cross-coupling product

48 and was followed by nitro reduction to give the aniline 49.

Conjugate substitution with diethyl-2-(ethoxymethylene)-

malonate followed by cyclization in reﬂuxing diphenylether

gave 3-ethyl[quinolin-4(1H)-one]carboxylate 51. Saponiﬁca-

tion and subsequent decarboxylation gave quinolin-4(1H)-one

53, which was then nitrated at the 3-position. After

chlorination, a nucleophilic substitution reaction installed the

(R)-α-methylbenzylamine 4-substituent and reduction of the

3-position nitro unit aﬀorded amine 57. Acylation with 2-

methoxyacetyl chloride, followed by cyclization in hot

propionic acid, constructed the 1H-imidazo[4,5-c]quinoline

ring system in an eﬃcient manner, and upon hydrogenation

over Pd/C, imidazoquinolinol 60 was obtained. The ﬁnal

a

b

Expressed as the mean from at least two test occasions. Calculated

c

as 10^{(BD1 pIC}

⁾. A pIC₅₀value of <4.3 was determined on one

₅₀−BD2 pIC₅₀

test occasion out of ﬁve and was excluded from the reported mean

value. A pIC₅₀value of <4.3 was determined on two test occasions

out of six and was excluded from the reported mean value. Tested as

a mixture of diastereoisomers. A pIC₅₀value of <4.3 was determined

on two test occasions out of four and was excluded from the reported

mean value.

d

e

f

methyls (23) did increase the selectivity to >50-fold. Building

on the encouraging initial results with basic groups connected

by ﬂexible alkyl chains, several rigidiﬁed analogues were then

prepared (Scheme 2). Constraining the propylamine 18 into

the cis- and trans-cyclobutylamines (27 and 29, respectively)

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a

Scheme 3. Synthesis of Carboxylic Acids 42 and 44

a

Reagents and conditions: (a) Tf₂O, pyridine, DCM, 0 °C−rt, 4 h, 90%; (b) CO (g), Pd(OAc)₂(7 mol %), 1,3-bis(diphenylphosphino)propane (7

t

mol %), NEt₃, EtOH, DMF, 70 °C, 3 h, 100%; (c) 2 M NaOH (aq), EtOH, rt, 15 h, then 50 °C, 3 h, 64%; (d) BrZnCH₂CO₂Bu (0.5 M in THF),

Pd(PPh₃)₄(10 mol %), NMP, microwave, 90 °C, 20 min, 17%; (e) TFA, DCM, rt, 36 h, 35%.

Figure 4. Cyclization approach used by Mirguet et al. in the genesis of imidazoquinolinones²³and the related imidazoquinoline scaﬀold.

the stereochemical integrity of the α-methylbenzyl group

remained intact throughout the preparative sequence.

Table 3. Activity and Selectivity Proﬁle of Imidazoquinoline

45 in BET Mutant TR-FRET Assays

Upon testing, greater BRD4 BD1 activity was found for

imidazoquinoline 62 (pIC₅₀= 7.4) compared to imidazoqui-

nolinone 37, validating the switch to an alternative quinoline

isoxazole scaﬀold (Figure 5 and Table 4). We were also

pleased to ﬁnd greater domain selectivity in BRD4 (>150-fold)

was achieved with this modiﬁcation and highlighted the

sensitivity of SAR toward minor scaﬀold changes. The strong

preference for BD1 binding was replicated in the rest of the

BET family, albeit at reduced levels compared to BRD4 (Table

4). With activities at BD1 domains comparable to established

pan-BET inhibitors, and accompanied by suitably large

selectivities over BD2 domains, 62 was investigated in further

experiments.

We next sought to rationalize the selectivity of 62 using X-

ray crystallography, and a high-resolution structure was

obtained in BRD4 BD1 (Figure 6A). The 3,5-dimethylisox-

azole moiety forms characteristic hydrogen-bonding inter-

actions to the conserved Asn140 and the water network at the

base of the binding site, while the aryl ring of the α-

methylbenzyl substituent occupies the WPF shelf and the

quinoline nitrogen retains the through-water interaction to

Gln85. These features are similar to I-BET151 and other

previously reported compounds from this series.²²⁻²⁴As

intended, the 3-methylene pyrrolidine substituent projects

a

pIC₅₀

b

BD1

BD2

selectivity (fold)

BRD2

BRD3

BRD4

BRDT

7.9

8.0

7.6

7.4

7.8

7.4

6.5

3.2

1.6

4.0

13

b

a

Expressed as the mean from at least ﬁve test occasions. Calculated

₅₀−BD2 pIC₅₀

)

as 10^{(BD1 pIC}

.

compound synthesis was accomplished through O-alkylation

with (S)-tert-butyl 3-{[(methylsulfonyl)oxy]methyl}-

pyrrolidine-1-carboxylate, followed by NBoc deprotection,

giving target compound 62. Chiral HPLC analysis of this

material (Supporting Information, Figure S3iv) conﬁrmed that

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a

Scheme 4. Synthesis of Imidazoquinoline 62

a

Reagents and conditions: (a) NaH, BnOH, DMA, 0 °C, 1 h, 73%; (b) (3,5-dimethylisoxazol-4-yl)boronic acid, PEPPSI-IPr (2.5 mol %), Cs₂CO₃,

DME, H₂O, 90 °C, 5 h, 86%; (c) Fe, NH₄Cl, EtOH, H₂O, rt, 15 h, 98%; (d) diethyl 2-(ethoxymethylene)malonate, 130 °C, 1 h, 74%; (e) Ph₂O,

260 °C, 20 min, 90%; (f) 2 M NaOH (aq), EtOH, 95 °C, 3 h, 99%; (g) Ph₂O, 260 °C, 20 min, 89%; (h) HNO₃, EtCO₂H, 100 °C, 1.5 h, 57%; (i)

POCl₃, 100 °C, 1 h, 96%; (j) (R)-α-methylbenzylamine, ⁱPr₂NEt, NMP, rt, 2.5 h, 99%; (k) Fe, AcOH, rt, 1 h, 83%; (l) 2-methoxyacetyl chloride,

pyridine, DCM, rt, 3.5 h, 98%; (m) EtCO₂H, 140 °C, 2 h, 90%; (n) 5% Pd/C, H₂, EtOH, rt, 35 h, 62%; (o) (S)-tert-butyl 3-

{[(methylsulfonyl)oxy]methyl}pyrrolidine-1-carboxylate, K₂CO₃, DMF, 100 °C, 3 h, 57%; (p) 4 M HCl in 1,4-dioxane, 1,4-dioxane, rt, 1 h, 96%.

Table 4. Activity and Selectivity Proﬁles of

Imidazoquinoline 62 in BET Mutant TR-FRET Assays

a

pIC₅₀

b

BD1

BD2

selectivity (fold)

BRD2

BRD3

BRD4

BRDT

7.1

7.4

6.8

5.5

6.0

5.2

4.8

40

25

158

100

a

b

Expressed as the mean from at least eight test occasions. Calculated

as 10^{(BD1 pIC}

.

₅₀−BD2 pIC₅₀

)

Figure 5. Concentration−response curves of 62 in BRD4 mutant TR-

FRET assays highlighting a greater preference for BD1 binding

(crosses) over BD2 (circles). Data points represent the mean from 16

test occasions.

water molecule that also hydrogen bonds to the side-chain

oxygen of Asn140 (Figure 6A,B). Unexpectedly, a second

aspartic acid, Asp145, rotates toward the inhibitor, and its acid

group forms a second interaction connected to the pyrrolidine

nitrogen by another two bridging water molecules. This second

toward Asp144. Rather than making a direct salt-bridge with

Asp144, the pyrrolidine amine interacts with Asp144 via a

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Figure 6. (A) Crystal structure of 62 in BRD4 BD1 (ligand carbon = green, protein carbon = white, PDB 6swn²⁵) overlaid on I-BET151 in BRD2

BD1 (ligand and protein carbon = orange, PDB 4alg²⁴). (B) Magniﬁed view of pyrrolidine water-bridged interactions to BRD4 BD1 Asp144,

Asp145, and Asn140. (C) Crystal structure of 62 in BRD2 BD2 (ligand carbon = cyan, protein carbon = blue, PDB 6swo²⁵) superimposed on the

structure of 62 in BRD4 BD1 (ligand carbon = green, protein carbon = white). (D) Magniﬁed view of pyrrolidine water-bridged interactions with

BRD2 BD2.

H-bonding chain has less ideal geometry and higher B-factors

than the ﬁrst (Supporting Information, Figure S5). The

Asp145 movement displaces the C-helix capping water

hydrogen bonded to Ile146 NH (Wc, Figure 6A), replacing

this interaction with one of the Asp145 side-chain carboxylate

oxygens.

Pro430 creates a BD2 site that cannot accommodate the water

network that coordinates the pyrrolidine nitrogen in the BD1

structure. In BD2, His433 is seen in both chi1 rotamers, a

minor gauche(+) and a major trans (pointing “out” and “in”

with respect to the KAc site, Figure 6D). In its major “in”

conformation, the side chain of His433 overlaps with the

volume occupied by BRD4 BD1 Asp145 and its associated

water chain. As a result, the analogous BRD4 BD2 residue

Asp434 cannot rotate inward toward the pyrrolidine. Even in

its minor “out” conformation, steric constraints from the side

chain of Pro430 push His433 further into the space containing

pyrrolidine-binding water molecules in BD1. Consequently, in

BD2, the pyrrolidine nitrogen of imidazoquinoline 62 is forced

1.8 Å away from its BD1 position and cannot hydrogen-bond

via water to any negatively ionized side chains. The selectivity

of imidazoquinoline 62 can, therefore, be rationalized by the

“Asp-His switch”, even though the pyrrolidine does not directly

interact with these side chains.

We assume that the water-mediated pyrrolidine interactions

of imidazoquinoline 62 would be replicated by imidazoquino-

linone 37, which is 10-fold less potent against BRD4 BD1 than

12 (Table 2). Given all polar interactions, this may seem

unexpected, but the pyrrolidine group is probably well solvated

when unbound, and the reduced BD1 potency suggests that

the interactions it forms in the BD1 complex only partly

compensate for unfavorable eﬀects of extending the methoxy

substituent. We hypothesize that the water seen in this region

of the complex with methoxy substituted compound 7 is quite

stable and resists deformation. However, the increased

selectivity of imidazoquinoline 62 arises not because its

pyrrolidine makes favorable interactions with BD1, but because

its interactions with BD2 are more unfavorable. To gain further

insights into this, a crystal structure of imidazoquinoline 62 in

BRD2 BD2 was obtained (Figure 6C,D). The overall binding

mode is similar to that in BRD4 BD1 (Figure 6C) except for

the pyrrolidine and its surroundings. The dual substitution of

BRD4 BD1 Asp144 and Lys141 for BRD2 BD2 His433 and

The role of the “Asp/His switch” in domain selectivity was

discussed by Divakaran et al. in the context of their dual

kinase/BD1 bromodomain inhibitor 5.¹⁰They observed no

binding to a BRD4 BD2 His437-Asp mutant, suggesting that

selectivity for BRD4 BD1 over BRD4 BD2 was not wholly due

to the “Asp/His switch”. An alternative suggestion from these

researchers was the presence of proline at BRD4 BD2 position

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434 also inﬂuences selectivity by restricting protein ﬂexibility.

While inhibitor 5 is structurally diﬀerent to imidazoquinoline

62 and binds deeper in the KAc site, displacing conserved

water, the selectivity of the two molecules may arise from

similar mechanisms.

Further conﬁrmation of the domain selectivity of imidazo-

quinoline 62 was obtained by SPR (Figure 7 and Figure S6ii).

leukemia (AML)³⁸⁻⁴³prompted us to compare the anti-

proliferative eﬀects of 62, alongside GW841819X and I-

BET151, in MV-4−11 cells, an AML cell line harboring an

MLL-AF4 rearrangement. Upon treatment of these cells over a

three-day period, 62 was found to potently suppress cell

growth with a pIC₅₀value of 7.0 (Figure 8E). This value was in

close accordance with those obtained for GW841819X and I-

BET151 (pIC₅₀= 6.7 and 7.0, respectively), suggesting that

eﬃcacy in MV-4−11 cells is mediated mainly through the BD1

domains.

Overall, these results showed that the anti-inﬂammatory and

antiproliferative phenotype characteristic of pan-BET inhib-

ition was retained upon selective inhibition of BD1 domains.

The potential for 62 to be used as a probe molecule in vivo

was explored next. Following intraperitoneal (ip) adminis-

tration to the male CD1 mouse at 10 mg/kg, compound 62

was well tolerated and demonstrated good exposure (Table 5

and Figure 9A). Visual inspection of the concentration−time

proﬁle (Figure 9A) indicated biphasic elimination with a

moderate (predominant) elimination half-life of approximately

1 h and a longer terminal phase (Table 5). As only unbound

drug is available for eﬃcacy, the unbound fraction in mouse

blood was determined by rapid equilibrium dialysis and found

to be 0.08. To provide a suitability assessment for the

exploration of in vivo activity, the in vivo response (termed

target engagement) in blood over a time course was estimated.

Using a sigmoid Emax model, the blood concentration−time

proﬁle obtained following ip administration was integrated

with measures of in vitro potency (from either BRD4 BD1 and

BD2 mutant TR-FRET assays or the human whole blood IL-6

inhibition assay corrected for free fraction and assuming

equivalent potency across species). The plot shown in Figure

9B illustrates that an ip dose of 10 mg/kg is estimated to

provide over 85% BD1 target engagement at Cmax and

maintain over 45% for approximately 2 h post administration,

while maintaining BD2 levels no greater than 4%, conﬁrming

the properties of 62 allow the selective probing of BD1.

Alternative estimations of in vivo target engagement using a

marker of whole blood activity conﬁrm that an ip dose of 10

mg/kg oﬀers potential for in vivo eﬃcacy (Figure 9C). Studies

with 62 have demonstrated increased survival in a mouse

MLL-AF9 AML model compared to a BD2-selective

inhibitor.²⁵Taken together, these data illustrate that 62 has

suitable disposition, potency, and selectivity properties

appropriate to explore BD1 pharmacology in vivo.

Figure 7. SPR concentration−response curves for 62 highlighting a

greater preference for BRD4 BD1 (crosses) over BRD4 BD2 (circles).

The compound was tested in duplicate, and the individual curves

overlay closely.

Titrations of 62 over immobilized BRD4 BD1 and BRD4 BD2

resulted in pK_D’s of 8.0 and 5.9, respectively, and domain

selectivity >100-fold. Additional proﬁling was conducted using

a qPCR-based assay at DiscoveRx Corp. (Supporting

Information, Table S7i). Here, imidazoquinoline 62 displayed

selective binding within the BET family and over 100-fold

selectivity for BRD4 BD1 over BRD4 BD2. These data also

conﬁrmed the speciﬁcity of 62 for the BET family with

negligible binding detected at 24 other bromodomains.

Having established a clear preference for binding at the BD1

domains of BET in multiple assay formats using recombinant

proteins, 62 was assessed for its ability to act as a chemical

probe in cells. HEK293 cells expressing a nanoluciferase-BRD4

fusion protein containing unmutated bromodomains were

incubated with 62, and inhibition of a bromosporine tracer was

determined with a pIC₅₀value of 7.3. These data conﬁrmed the

ability of 62 to engage the bromodomain modules of BRD4 in

cells and that this compound is, therefore, suitable for use as a

probe molecule for assessment in phenotypic assays.

The anti-inﬂammatory properties of BET inhibitors were

ﬁrst discovered upon studies with I-BET762 in mouse

macrophages where the displacement of BRD2, 3, and 4

from gene promoters suppressed LPS-induced pro-inﬂamma-

tory cytokine and chemokine gene expression.²⁶With anti-

inﬂammatory eﬀects becoming a hallmark of BET inhib-

ition,^7,24,27−37we examined the ability of 62 to block pro-

inﬂammatory cytokine production alongside the pan-BET

inhibitor GW841819X²¹(see Table S4v, conﬁrming un-

selective binding in mutant TR-FRET assays) and the BD1-

biased inhibitor I-BET151 (Figure 8A−D). In both LPS-

stimulated human PBMCs and human whole blood, 62

suppressed IL-6 and monocyte chemotactic protein-1 (MCP-

1) production with comparable activities to GW841819X and

I-BET151 in both cellular environments.

CONCLUSIONS

■

In this report, we have described the development of a small

molecule that retains the wider bromodomain selectivity of I-

BET151 but also exhibits signiﬁcant selectivity for BD1 over

BD2 domains within the BET family of tandem bromodomain-

containing proteins. Structural knowledge was used to identify

aspartic acid residues present only in BD1 domains for

selective targeting with chemical functionality appended to the

8-position of I-BET151. Pendant rigidiﬁed amines enhanced

selectivity for BD1 domains but also decreased activity.

Therefore, a strategy to append the favored 3-methylene

pyrrolidine to a more potent analogue of I-BET151 was

executed and resulted in imidazoquinoline 62 featuring up to

150-fold BD1 selectivity, as well as BD1-activity comparable to

the biased inhibitor I-BET151 and the pan-inhibitor

GW841819X. An X-ray crystal structure of 62 in BRD4 BD1

revealed through-water interactions between the pendant

The profound antileukemic eﬀects of I-BET151 in mixed-

lineage leukemic (MLL) fusion cell lines have, in part, been

attributed to the down-regulation of MLL target genes through

the displacement of BRD3/4 and members of the super

elongation complex (SEC) and polymerase-associated factor

complex (PAFc) from chromatin.²²Further reports demon-

strating the eﬃcacy of BET inhibitors in acute myeloid

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Figure 8. Inhibitory eﬀect of GW841819X, I-BET151, and 62 on IL-6 and MCP-1 production in LPS-stimulated human PBMCs (A and B) and

whole blood (C and D). (E) Growth inhibition of GW841819X, I-BET151, and 62 in MV-4−11 cells.

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carried out on either a Biotage SP4 automated ﬂash chromatography

system using SNAP silica cartridges or a Combiﬂash Companion

automated ﬂash chromatography system using Redisep silica

cartridges. Mass-directed autopreparative HPLC (MDAP) puriﬁca-

tion was conducted on a Waters HPLC using (Method A) Sunﬁre

C18 (150 mm × 30 mm, 5 μm packing diameter), eluting with an

appropriate gradient of 0.1% formic acid in water (mobile phase A)

and 0.1% formic acid in CH₃CN (mobile phase B) at an ambient

temperature and a ﬂow rate of 40 mL/min, (Method B) an Xbridge

C18 column (150 mm × 30 mm, 5 μm packing diameter), eluting

with an appropriate gradient of 10 mM ammonium bicarbonate in

water adjusted to pH 10 with ammonia solution (mobile phase A)

and CH₃CN (mobile phase B) at an ambient temperature and a ﬂow

rate of 40 mL/min, or (Method C) Sunﬁre C18 (150 mm × 30 mm, 5

μm packing diameter), eluting with an appropriate gradient of 0.1%

TFA in water (mobile phase A) and 0.1% TFA in CH₃CN (mobile

phase B) at an ambient temperature and a ﬂow rate of 40 mL/min.

The UV detection was an averaged signal from a wavelength of 210−

350 nm, and mass spectra were recorded on a Waters micromass ZQ

mass spectrometer operating in alternate-scan positive and negative

electrospray ionization modes for Methods A and B and in positive

electrospray ionization mode for Method C. Melting points were

measured using a Stuart SMP40 automatic melting point apparatus.

IR spectra were obtained on a PerkinElmer Spectrum 1 machine.

Optical rotations were measured using a Jasco P-1030 polarimeter. ¹H

NMR spectra were recorded on a Bruker AV-400 spectrometer at 400

MHz or a Bruker AV-600 spectrometer at 600 MHz. ¹³C NMR

spectra were recorded on a Bruker AV-400 spectrometer at 100 MHz

or a Bruker AV-600 spectrometer at 150 MHz. NMR spectra were

acquired at 293 K unless otherwise stated. Signal multiplicities were

assigned using the following abbreviations: s (singlet), br s (broad

singlet), t (triplet), q (quartet), quin (quintet), m (multiplet), dd

(doublet of doublets), dt (doublet of triplets), dq (doublet of

Table 5. Pharmacokinetic Parameters of 62 Following ip

Administration (10 mg/kg) to the Male CD1 Mouse

parameter

mean (n = 3) SD

C_max(μM)

T_max(h)

predominant (0−7 h) T_1/2(h)

terminal (7−24 h) T_1/2(h)

AUC_∞(μM h)

2.40 0.46

0.25−0.50

0.9 0.1

8.2 0.7

3.34 0.31

amine and the BD1-conserved residues Asp144 and Asp145. In

the BD2 domains, the substitutions of histidine for Asp144 and

proline for Lys141 prevent 62 from forming these interactions

and are the likely origin of the observed domain selectivity.

Studies with 62 in human PBMCs and whole blood, and in

MV-4−11 cells, demonstrated that selective inhibition of BD1

retains the anti-inﬂammatory and antiproliferative phenotype

characteristic of pan-BET inhibition. The pharmacokinetic

proﬁle of 62 makes this compound suitable for use to probe

BD1 selective pharmacology in vivo. Biological investigations of

62 (also referred to as GSK778/iBET-BD1) alongside BD2-

selective molecules (GSK046/iBET-BD2 and GSK620)^44,45

have very recently been reported²⁵and further contribute to

other recent progress in this area.^11,46,47

EXPERIMENTAL SECTION

■

General Experimental. All commercial chemicals and solvents

used were reagent grade and used without further puriﬁcation. SCX-2

and aminopropyl cartridges by ISOLUTE were used for catch and

release and scavenging SPE protocols. Column chromatography was

Figure 9. (A) Blood concentration−time proﬁle of compound 62 following ip administration (10 mg/kg) to the male CD1 mouse. Total

concentrations are depicted with closed squares and unbound concentrations with closed triangles. Data are mean n = 3. (B and C) Predicted in

vivo target engagement-time proﬁles for compound 62 integrating the mouse ip PK data and the in vitro IC₅₀values determined in (B) BRD4 BD1

(closed diamonds) and BRD4 BD2 (open circles) mutant TR-FRET assays and (C) LPS-stimulated IL-6 inhibition in human whole blood (open

triangles).

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quartets), td (triplet of doublets), and tt (triplet of triplets). The

purity of all biologically tested compounds was ≥95% as determined

by LC−MS UV traces except for compound 25 (94% purity). The

speciﬁc LC−MS UV method used for purity determination is noted

for each compound and was conducted on a Waters Acquity UPLC

equipped with a BEH C18 column (50 mm × 2.1 mm, 1.7 μm

packing diameter) using a gradient elution carried out at a ﬂow rate of

1 mL/min at 40 °C using either Method A or B. Method A: 0.1%

formic acid in water (mobile phase A) and 0.1% formic acid in

CH₃CN (mobile phase B) 0.0−1.5 min 3−100% mobile phase B,

1.5−1.9 min 100% mobile phase B, 1.9−2.0 min 100−3% mobile

phase B. Method B: 10 mM ammonium bicarbonate in water adjusted

to pH 10 with ammonia solution (mobile phase A) and CH₃CN

(mobile phase B) 0.0−1.5 min 1−97% mobile phase B, 1.5−1.9 min

97% mobile phase B, 1.9−2.0 min 97−1% mobile phase B. The UV

detection was a summed signal from wavelengths of 210−350 nm,

and mass spectra were recorded on a Waters Micromass ZQ mass

spectrometer operating in alternate-scan positive and negative

electrospray ionization modes. High-resolution mass spectra were

acquired on a Micromass Q-Tof Ultima hybrid quadrupole TOF mass

spectrometer.

complete, the reaction was stirred at 90 °C under nitrogen for 3.5 h.

The reaction was cooled in an ice bath and carefully quenched with

10% EtOH in DCM (75 mL) and then MeOH (75 mL). After stirring

for 20 min, the solvent was removed in vacuo. The solid-foam mixture

was suspended in EtOAc (200 mL) and sonicated for 10 min. To the

mixture was added sat. Na₂CO₃(aq) (200 mL) and the biphasic

mixture stirred vigorously for 10 min. The organic layer was separated

and left to stand for 5 min during which time a precipitate formed.

The solid was isolated by vacuum ﬁltration and the ﬁltrate dried

(MgSO₄). The solvent was evaporated in vacuo and the solid

triturated with ether (50 mL). This solid was combined with the

previous solid precipitate to give the title compound as an oﬀ-white

solid (2.96 g, 7.12 mmol, 84% yield): mp 287 °C (decomp); [α]_D²¹

1

−25 (c 0.5, CHCl₃/MeOH [4:1]); H NMR (400 MHz, DMSO-d₆,

393 K) δ 8.70 (s, 1H), 8.54 (d, J = 4.8 Hz, 1H), 7.79 (s, 1H), 7.76−

7.70 (m, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.37 (s, 1H), 7.26 (dd, J = 7.4,

4.8 Hz, 1H), 6.27 (q, J = 7.2 Hz, 1H), 3.54 (s, 3H), 2.31 (s, 3H), 2.14

(s, 3H), 2.12 (d, J = 7.2 Hz, 3H) OH signal not resolved; ¹³C NMR

(150 MHz, DMSO-d₆) δ 165.8, 159.1, 158.7, 153.3 (2 C), 149.0,

139.8, 137.0, 132.9, 130.9, 126.9, 123.3, 122.4, 120.7, 120.2, 115.9,

112.6, 103.6, 53.8, 27.4, 17.5, 11.4, 10.4; 76% ee determined by

HPLC analysis on a Chiralpak ID column (250 × 4.6 mm, 5 μm),

elution with 50% EtOH in heptane, ﬂow rate 1 mL/min, UV

detection at 215 nm; LC−MS (Method A) [M + H]⁺= 416, t_R0.62

min; HRMS [M + H]⁺calcd for C₂₃H₂₂N₅O₃416.1723, found

416.1711.

Chemistry Methods and Characterization for Compounds

8−62. 7-(3,5-Dimethyl-4-isoxazolyl)-8-hydroxy-1-[(1R)-1-(2-

pyridinyl)ethyl]-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (8).

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-1-[(1R)-1-(2-pyridinyl)-

ethyl]-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (I-BET151, 1 g,

2.41 mmol, ee unknown) in DCM (25 mL) under nitrogen at −78 °C

was treated with 1 M BBr₃in DCM (12 mL, 12.00 mmol) added over

30 min. The reaction was stirred at −78 °C for 45 min, then allowed

to warm to rt, and stirred for 16 h. The reaction was heated to reﬂux

for 4 h, and then additional 1 M BBr₃in DCM (6 mL, 6 mmol) was

added. After an additional 2 h, an additional portion of 1 M BBr₃in

DCM (6 mL, 6 mmol) was added, and reﬂux continued for 16 h. The

reaction was cooled in an ice bath, and EtOH (2.5 mL) in DCM (12.5

mL) was added slowly. After stirring for 20 min, the reaction mixture

was concentrated to give a brown solid. The crude material was

puriﬁed on a 100 g silica cartridge eluting with 1−10% 2 M ammonia/

MeOH in DCM. The appropriate fractions were concentrated in

vacuo to give the title compound as a cream solid (0.57 g, 1.35 mmol,

tert-Butyl [2-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-

[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}-

oxy)ethyl]carbamate (14). 7-(3,5-Dimethylisoxazol-4-yl)-8-hydroxy-

3-methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-

2(3H)-one 13 (100 mg, 0.241 mmol), potassium carbonate (43 mg,

0.310 mmol), and tert-butyl (2-bromoethyl)carbamate (54 mg, 0.241

mmol) were combined in DMF (5 mL) and heated at 100 °C

overnight. The reaction mixture was cooled to rt and concentrated in

vacuo. The crude compound was puriﬁed on a 25 g silica cartridge

eluting in with 1% MeOH in DCM for 2 column volumes, 1−5%

MeOH in DCM for 10 column volumes, and then 5% MeOH in

DCM for 5 column volumes. The appropriate fractions were

concentrated in vacuo to give an oil, which was then puriﬁed by

MDAP (Method A). The appropriate fractions were concentrated in

vacuo. The residue was dissolved in MeOH and applied to a 2 g SCX-

2 cartridge, which was then washed with MeOH (10 mL), followed by

2 M ammonia in MeOH solution (10 mL). The basic wash was

concentrated to give the title compound as an orange oil (59 mg,

0.095 mmol, 40% yield): ¹H NMR (400 MHz, CDCl₃) δ 8.70 (d, J =

4.5 Hz, 1H), 8.68 (s, 1H), 7.84 (s, 1H), 7.67−7.61 (m, 1H), 7.38 (br

s, 1H), 7.28−7.24 (m, 1H), 7.01 (br s, 1H), 6.49−6.41 (m, 1H),

4.84−4.75 (m, 1H), 3.94−3.86 (m, 1H), 3.71 (s, 3H), 3.54−3.50 (m,

1H), 3.48−3.41 (m, 2H), 2.33 (s, 3H), 2.17 (s, 3H), 2.14 (d, J = 7.3

Hz, 3H), 1.48 (s, 9H); % ee undetermined; LC−MS (Method A) [M

+ H]⁺= 559, t_R0.86 min.

8-(2-Aminoethoxy)-7-(3,5-dimethylisoxazol-4-yl)-3-methyl-1-[1-

(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one Hydro-

chloride (15). A solution of tert-butyl [2-({7-(3,5-dimethylisoxazol-

4-yl)-3-methyl-2-oxo-1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)ethyl]carbamate 14 (59 mg, 0.106

mmol) in DCM (4 mL) was treated with TFA (0.5 mL, 6.49 mmol)

and the reaction stirred at rt for 90 min. The solvent was removed in

vacuo and the residue dissolved in MeOH and applied to a 2 g SCX-2

cartridge. The cartridge was washed with MeOH (10 mL) followed by

2 M ammonia in MeOH solution (10 mL). The basic wash was

concentrated to give the title compound as an orange oil (39 mg). 1

M HCl in diethyl ether (0.047 mmol, 47 μL) was added to 19 mg of

the free base and the solvent removed under a stream of nitrogen to

give the title compound as a light orange solid (17 mg, 0.033 mmol,

31% yield): ¹H NMR (400 MHz, DMSO-d₆) δ 9.12 (s, 1H), 8.68 (d,

J = 5.0 Hz, 1H), 8.16 (br s, 3H), 8.07 (s, 1H), 7.90−7.84 (m, 1H),

7.59 (d, J = 6.8 Hz, 1H), 7.38 (dd, J = 7.3, 5.0 Hz, 1H), 7.03 (br s,

1H), 6.39 (q, J = 7.3 Hz, 1H), 4.22−4.14 (m, 1H), 3.64 (s, 3H),

3.24−3.19 (m, 2H), 2.31 (s, 3H), 2.11 (s, 3H), 2.08 (d, J = 7.3 Hz,

1

56% yield): H NMR (400 MHz, DMSO-d₆) δ 11.53 (br s, 1H),

10.03 (br s, 1H), 8.54 (s, 1H), 7.79−7.74 (m, 2H), 7.38 (d, J = 8.1

Hz, 1H), 7.30 (m, 1H), 7.18 (br s, 1H), 6.18 (q, J = 7.2 Hz, 1H), 2.29

(s, 3H), 2.12 (s, 3H), 2.08 (d, J = 7.2 Hz, 3H); % ee undetermined;

LC−MS (Method A) [M + H]⁺= 402, t_R0.57 min.

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-3-methyl-1-[(R)-1-(pyr-

idin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one (12). A solu-

tion of 7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-1-[(R)-1-(pyridin-2-

yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one (I-BET151 7, 4.0 g,

9.63 mmol, ee = 88%) in anhydrous NMP (30 mL) under nitrogen at

rt was treated with NaH (60% dispersion in mineral oil) (0.50 g, 12.5

mmol) and the mixture stirred for 20 min. Methyl iodide (0.66 mL,

10.6 mmol) was added and the mixture stirred for 5 h. The reaction

mixture was diluted with brine (50 mL) and extracted with EtOAc

(50 mL). The organic layer was washed with water (4 × 50 mL) and

then passed through a hydrophobic frit. The solvent was evaporated in

vacuo to give the title compound as a cream foam (3.63 g, 8.45 mmol,

1

88% yield): H NMR (400 MHz, DMSO-d₆, 353 K) δ 8.82 (s, 1H),

8.61 (d, J = 4.8 Hz, 1H), 7.81 (s, 1H), 7.80−7.74 (m, 1H), 7.44 (d, J

= 8.1 Hz, 1H), 7.33 (dd, J = 7.3, 4.8 Hz, 1H), 6.94 (s, 1H), 6.33 (q, J

= 7.2 Hz, 1H), 3.62 (s, 3H), 3.55 (s, 3H), 2.26 (s, 3H), 2.07 (d, J =

7.2 Hz, 3H), 2.06 (s, 3H); 76% ee determined by HPLC analysis on a

Chiralpak AD column (250 × 4.6 mm, 10 μm), elution with 40%

EtOH in heptane, ﬂow rate 1 mL/min, UV detection at 215 nm; LC−

MS (Method A) [M + H]⁺= 430, t_R0.62 min.

7-(3,5-Dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-1-(pyri-

din-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one (13). To 7-

(3,5-dimethylisoxazol-4-yl)-8-methoxy-3-methyl-1-[(R)-1-(pyridin-2-

yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 12 (3.63 g, 8.45

mmol) in DCE (75 mL) was added BBr₃(8.0 mL, 85 mmol)

dropwise. A thick precipitate formed, and after the addition was

M

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3H), one OCH₂proton obscured by water; % ee undetermined; LC−

MS (Method A) [M + H]⁺= 459, t_R0.50 min.

(br s, 3H), 7.90−7.82 (m, 1H), 7.60 (d, J = 6.8 Hz, 1H), 7.39 (dd, J =

7.3, 4.8 Hz, 1H), 7.07 (s, 1H), 6.41 (q, J = 7.3 Hz, 1H), 4.03−3.94

(m, 1H), 3.64 (s, 3H), 2.85−2.72 (m, 2H), 2.32 (s, 3H), 2.11 (s, 3H),

2.09 (d, J = 7.3 Hz, 3H), 2.05−1.96 (m, 2H), one OCH₂proton

obscured by water; % ee undetermined; LC−MS (Method A) [M +

H]⁺= 473, t_R0.52 min.

8-[2-(Dimethylamino)ethoxy]-7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-

2(3H)-one Hydrochloride (16). 7-(3,5-Dimethylisoxazol-4-yl)-8-hy-

droxy-3-methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]-

quinolin-2(3H)-one (50 mg, 0.12 mmol), potassium carbonate (43

mg, 0.31 mmol), and 2-bromo-N,N-dimethylamine hydrochloride (24

mg, 0.129 mmol) were combined in DMF (5 mL) under nitrogen and

heated to 100 °C for 16 h. The reaction was cooled and evaporated in

vacuo to give a black solid, which was puriﬁed by MDAP (Method A).

The appropriate fractions were evaporated under a stream of nitrogen

and applied to a 2 g SCX cartridge in the minimum amount of

MeOH. The cartridge was washed with MeOH (10 mL), followed by

2 M ammonia in MeOH solution (10 mL). The basic wash was

concentrated in vacuo to give an orange solid, which was taken up in

the minimum amount of DCM and 1 N HCl in diethyl ether (0.039

mL, 0.039 mmol) added. The solvent was removed under a stream of

nitrogen to give the title compound as a salmon pink solid (17 mg,

0.029 mmol, 24% yield): ¹H NMR (400 MHz, DMSO-d₆) δ 10.40 (br

s, 1H), 9.06 (s, 1H), 8.68 (d, J = 4.5 Hz, 1H), 8.01 (s, 1H), 7.89−7.81

(m, 1H), 7.56 (d, J = 7.3 Hz, 1H), 7.39 (dd, J = 7.3, 4.5 Hz, 1H), 7.12

(br s, 1H), 6.38 (q, J = 7.1 Hz, 1H), 4.37−4.28 (m, 1H), 3.65−3.62

(m, 3H), 2.72−2.66 (m, 6H), 2.30 (s, 3H), 2.11 (s, 3H), 2.08 (d, J =

7.1 Hz, 3H), CH₂protons obscured by water; % ee undetermined;

LC−MS (Method A) [M + H]⁺= 487, t_R0.43 min.

tert-Butyl [3-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-

[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}-

oxy)propyl]carbamate (17). A mixture of 7-(3,5-dimethylisoxazol-4-

yl)-8-hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo-

[4,5-c]quinolin-2(3H)-one 13 (145 mg, 0.349 mmol) and potassium

carbonate (60 mg, 0.349 mmol) in DMF (5 mL) was treated with

tert-butyl (3-bromopropyl)carbamate (102 mg, 0.428 mmol). The

mixture was stirred under nitrogen at 100 °C for 2.5 h and then

allowed to cool to rt. EtOAc (10 mL) was added and washed with

10% LiCl (aq) (10 mL). The aqueous layer was extracted with EtOAc

(10 mL), and the organic layers were combined and washed with 10%

LiCl (aq) (10 mL). The aqueous layer was extracted with EtOAc (10

mL), and the organic layers were combined and passed through a

hydrophobic frit. The solvent was removed by rotary evaporation.

The residue was dissolved in MeOH (1.0 mL) and applied to a

MeOH-preconditioned 5 g SCX-2 cartridge. The cartridge was

washed with MeOH (30 mL) and 2 M ammonia in MeOH solution

(30 mL). The basic wash was evaporated in vacuo and the residue

puriﬁed by MDAP (Method B) to give the title compound as a brown

gum (172 mg, 0.300 mmol, 86% yield): ¹H NMR (400 MHz, DMSO-

d₆, 393 K) δ 8.76 (s, 1H), 8.58 (d, J = 4.8 Hz, 1H), 7.77 (s, 1H),

7.77−7.72 (m, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.29 (dd, J = 7.3, 4.8

Hz, 1H), 7.01 (s, 1H), 6.29 (q, J = 7.3 Hz, 1H), 6.22 (br s, 1H), 3.83

(dt, J = 9.9, 6.3 Hz, 1H), 3.58 (s, 3H), 3.56 (dt, J = 9.9, 6.3 Hz, 1H),

2.99 (t, J = 6.3 Hz, 1H), 2.98 (t, J = 6.3 Hz, 1H), 2.25 (s, 3H), 2.05 (s,

3H), 2.05 (d, J = 7.3 Hz, 3H), 1.77 (quin, J = 6.3 Hz, 2H), 1.38 (s,

9H); % ee undetermined; LC−MS (Method B) [M + H]⁺= 573, t_R

1.07 min.

7-(3,5-Dimethylisoxazol-4-yl)-8-(3-hydroxypropoxy)-3-methyl-1-

[1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one (19). A

solution of 7-(3,5-dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-

1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (270

mg, 0.65 mmol) in anhydrous DMF (4.5 mL) was dispensed evenly

between 6× vessels each containing potassium carbonate (18 mg, 0.13

mmol). To one of these vessels was added 3-bromo-1-propanol (11

μL, 0.13 mmol) and the mixture stirred under nitrogen at 100 °C for

16 h. The reaction mixture was evaporated under a stream of nitrogen

and the crude material puriﬁed by MDAP (Method B) to give the title

compound as a light brown foam (23 mg, 0.05 mmol, 45% yield): ¹H

NMR (400 MHz, CDCl₃) δ 8.72−8.65 (m, 2H), 7.84 (s, 1H), 7.65 (t,

J = 7.3 Hz, 1H), 7.45−7.31 (m, 1H), 7.27−7.23 (m, 1H), 7.14−6.91

(m, 1H), 6.52−6.41 (m, 1H), 4.09−3.96 (m, 1H), 3.81−3.70 (m,

6H), 2.32 (s, 3H), 2.19−2.15 (m, 6H), 2.03−1.94 (m, 2H), OH

signal not resolved; % ee undetermined; LC−MS (Method A) [M +

H]⁺= 474, t_R0.64 min.

N-[3-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-(pyri-

din-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}oxy)-

propyl]acetamide Hydrochloride (20). A solution of 8-(3-amino-

propoxy)-7-(3,5-dimethylisoxazol-4-yl)-3-methyl-1-[(R)-1-(pyridin-2-

yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 18 (23 mg, 0.048

mmol) in DCM (2 mL) and pyridine (0.25 mL) was treated with

acetic anhydride (4.5 μL, 0.048 mmol) and the mixture stirred at rt

for 2 h. The reaction was concentrated in vacuo and the residue

puriﬁed by MDAP (Method A). The residue was dissolved in MeOH

and applied to a 2 g SCX-2 cartridge. The cartridge was washed with

MeOH, followed by 2 M ammonia in MeOH solution. The basic

wash was concentrated, dissolved in DCM, and treated with 1 M HCl

in diethyl ether (30 μL, 0.030 mmol). The solvent was removed under

a stream of nitrogen to give the title compound as an oﬀ-white solid

(11 mg, 0.019 mmol, 40% yield): ¹H NMR (400 MHz, DMSO-d₆) δ

9.30 (s, 1H), 8.57 (d, J = 4.8 Hz, 1H), 8.13 (s, 1H), 7.92−7.84 (m,

1H), 7.66−7.63 (m, 1H), 7.40−7.37 (m, 2H), 6.73 (br s, 1H), 6.43

(q, J = 7.3 Hz, 1H), 3.87 (m, 1H), 3.66 (s, 3H), 3.07 (m, 2H), 2.32

(s, 3H), 2.12 (s, 3H), 2.07 (d, J = 7.3 Hz, 3H), 1.82−1.75 (m, 5 H),

one OCH₂proton obscured by water; % ee undetermined; LC−MS

(Method A) [M + H]⁺= 515, t_R0.64 min.

tert-Butyl [3-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-

[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}-

oxy)propyl](methyl)carbamate (21). A solution of tert-butyl [3-({7-

(3,5-dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-

2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}oxy)propyl]carbamate

17 (153 mg, 0.267 mmol) in anhydrous NMP (1 mL) under nitrogen

was treated with NaH (60% dispersion in mineral oil) (15 mg, 0.374

mmol) and the mixture stirred at rt for 20 min. Methyl iodide (0.018

mL, 0.294 mmol) was added, and the mixture was left to stir for 17 h.

The mixture was treated with NaH (60% dispersion in mineral oil)

(15 mg) and stirred for 20 min before methyl iodide (0.009 mL, 0.147

mmol) was added. After 4.5 h of stirring, water (8 mL) was added.

The mixture was extracted with EtOAc (2 × 10 mL), and the extracts

were combined and passed through a hydrophobic frit. The solvent

was removed by rotary evaporation and the residue puriﬁed by MDAP

(Method B). The appropriate fractions were combined, and the

solvent was removed by rotary evaporation to give the title compound

8-(3-Aminopropoxy)-7-(3,5-dimethylisoxazol-4-yl)-3-methyl-1-

[1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one Hydro-

chloride (18). A solution of tert-butyl [3-({7-(3,5-dimethylisoxazol-4-

yl)-3-methyl-2-oxo-1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)propyl]carbamate 17 (52.4 mg,

0.092 mmol) in DCM (4 mL) was treated with TFA (1 mL, 12.98

mmol) and the reaction stirred at rt for 90 min. The solvent was

removed in vacuo, and the residue was applied to a 2 g SCX-2

cartridge in the minimum amount of MeOH. The cartridge was

washed with MeOH (10 mL) and then 2 M ammonia in MeOH (10

mL). The basic wash was concentrated in vacuo to give the free base

as a brown oil (41 mg). 1 M HCl in diethyl ether (45 μL, 0.045

mmol) was added to 21 mg of the free base and the solvent removed

under a stream of nitrogen to give the title compound as a light brown

solid (14 mg, 0.025 mmol, 28% yield): ¹H NMR (400 MHz, DMSO-

d₆, 393 K) δ 9.20 (s, 1H), 8.61 (d, J = 4.8 Hz, 1H), 8.14 (s, 1H), 7.97

1

as a yellow solid (34 mg, 0.058 mmol, 22% yield): H NMR (400

MHz, DMSO-d₆, 393 K) δ 8.77 (s, 1H), 8.58 (d, J = 4.8 Hz, 1H),

7.78 (s, 1H), 7.77−7.70 (m, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.29 (dd, J

= 7.3, 4.8 Hz, 1H), 7.02 (s, 1H), 6.29 (q, J = 7.3 Hz, 1H), 3.82 (dt, J =

9.9, 6.1 Hz, 1H), 3.58 (s, 3H), 3.55 (dt, J = 9.9, 6.1 Hz, 1H), 3.17 (t, J

= 6.9 Hz, 1H), 3.17 (t, J = 6.9 Hz, 1H), 2.70 (s, 3H), 2.25 (s, 3H),

2.06 (s, 3H), 2.05 (d, J = 7.3 Hz, 3H), 1.88−1.79 (m, 2H), 1.34 (s,

9H); % ee undetermined; LC−MS (Method B) [M + H]⁺= 587, t_R

1.13 min.

N

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Article

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-8-[3-(methylamino)-

propoxy]-1-[1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-

2(3H)-one (22). A solution of tert-butyl [3-({7-(3,5-dimethylisoxazol-

4-yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)propyl](methyl)carbamate 21 (23

mg, 0.039 mmol) in 1,4-dioxane (0.5 mL) was treated with 4 M

HCl in 1,4-dioxane solution (0.5 mL, 0.039 mmol) and the mixture

stirred at rt for 2 h in a stoppered vessel. The reaction mixture was

evaporated under a stream of nitrogen, and the residue was dissolved

in MeOH (0.5 mL). The solution was applied to a MeOH-

preconditioned 2 g SCX-2 cartridge. The cartridge was washed with

MeOH (16 mL), followed by 2 M ammonia in MeOH solution (16

mL). The basic wash was evaporated under in vacuo to give a light

brown oil, which solidiﬁed upon standing (19 mg, 0.039 mmol, 100%

evaporated in vacuo. The solid was dissolved in MeOH (0.5 mL) and

applied to a 0.5 g MeOH-preconditioned SCX-2 cartridge. The

cartridge was washed with MeOH (3 mL), followed by 2 M ammonia

in MeOH solution (3 mL). The basic wash was evaporated under a

stream of nitrogen to give the title compound as an oﬀ-white solid (15

mg, 0.032 mmol, 58% yield): ¹H NMR (400 MHz, DMSO-d₆, 393 K)

8.79 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 7.83 (s, 1H), 7.80−7.73 (m,

1H), 7.46 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 7.3, 4.8 Hz, 1H), 7.15 (s,

1H), 6.31 (q, J = 7.2 Hz, 1H), 4.44−4.34 (m, 2H), 3.60 (s, 3H), 2.31

(s, 3H), 2.14 (s, 3H), 2.07 (d, J = 7.2 Hz, 3H), CO₂H signal not

resolved; % ee undetermined; LC−MS (Method A) [M + H⁺] = 474,

t_R0.61 min, 94% purity by LC−MS UV.

tert-Butyl [cis-3-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-

1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-

yl}oxy)cyclobutyl]carbamate (26). A mixture of trans-3-[(tert-

butoxycarbonyl)amino]cyclobutyl methanesulfonate (281 mg, 1.06

mmol), potassium carbonate (160 mg, 1.16 mmol), and 7-(3,5-

dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)-

ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (400 mg, 0.96

mmol) was suspended in anhydrous DMF (4 mL) and stirred

under nitrogen at 100 °C for 6 h. The reaction mixture was allowed to

cool to rt and diluted with brine (20 mL). The mixture was extracted

with DCM (2 × 20 mL), and the organic layers were combined and

passed through a hydrophobic frit. The solvent was removed in vacuo

to give a brown oil. The oil was dissolved in MeOH (5 mL) and

applied to a MeOH-preconditioned 50 g SCX-2 cartridge. The

cartridge was washed with MeOH (200 mL), followed by 2 M

ammonia in MeOH solution (200 mL). The basic fraction was

evaporated in vacuo to give a brown gum. The crude material was

puriﬁed by MDAP (Method A). The appropriate fractions were

combined, and the solvent was removed by rotary evaporation to give

the title compound as a white solid (233 mg, 0.40 mmol, 41% yield):

¹H NMR (400 MHz, DMSO-d₆, 393 K) δ 8.77 (s, 1H), 8.63 (d, J =

1

yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ 8.76 (s, 1H), 8.59

(d, J = 4.8 Hz, 1H), 7.78 (s, 1H), 7.77−7.71 (m, 1H), 7.41 (d, J = 8.1

Hz, 1H), 7.29 (dd, J = 7.3, 4.8 Hz, 1H), 7.02 (s, 1H), 6.29 (q, J = 7.3

Hz, 1H), 3.86 (dt, J = 9.7, 6.4 Hz, 1H), 3.58 (s, 3H), 3.63−3.57 (m,

1H), 2.52 (t, J = 6.7 Hz, 2H), 2.29 (s, 3H), 2.24 (s, 3H), 2.05 (s, 3H),

2.05 (d, J = 7.1 Hz, 3H), 1.74 (tt, J = 6.7, 6.4 Hz, 2H), NH signal not

resolved; % ee undetermined; LC−MS (Method B) [M + H]⁺= 487,

t_R0.77 min.

8-[3-(Dimethylamino)propoxy]-7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-

2(3H)-one (23). A mixture of 7-(3,5-dimethylisoxazol-4-yl)-8-

hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]-

quinolin-2(3H)-one 13 (45 mg, 0.108 mmol), potassium carbonate

(36 mg, 0.260 mmol), and 3-bromo-N,N-dimethylpropan-1-amine

hydrobromide (32 mg, 0.130 mmol) was suspended in anhydrous

DMF (0.8 mL) and stirred under nitrogen at 100 °C for 2 h. The

reaction mixture was allowed to cool to rt, diluted with MeOH (0.2

mL), and ﬁltered. The solution was puriﬁed by MDAP (Method B).

The appropriate fractions were combined, and the solvent was

removed by rotary evaporation to give the title compound as an oﬀ-

4.8 Hz, 1H), 7.80 (s, 1H), 7.79−7.72 (m, 1H), 7.42 (d, J = 7.8 Hz,

1H), 7.33 (dd, J = 7.8, 4.8 Hz, 1H), 6.89 (s, 1H), 6.64 (d, J = 6.3 Hz,

1H), 6.27 (q, J = 7.2 Hz, 1H), 3.92−3.83 (m, 1H), 3.70−3.60 (m,

1H), 3.58 (s, 3H), 2.82−2.72 (m, 1H), 2.54−2.44 (m, 1H), 2.27 (s,

3H), 2.09 (s, 3H), 2.06 (d, J = 7.2 Hz, 3H), 1.94−1.84 (m, 2H), 1.40

(s, 9H); % ee undetermined; LC−MS (Method A) [M + H]⁺= 585,

t_R0.96 min.

1

white solid (34 mg, 0.068 mmol, 63% yield): H NMR (400 MHz,

DMSO-d₆, 393 K) δ 8.78 (s, 1H), 8.61 (d, J = 4.8 Hz, 1H), 7.81 (s,

1H), 7.79−7.73 (m, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.31 (dd, J = 7.3,

4.8 Hz, 1H), 7.09 (s, 1H), 6.33 (q, J = 7.3 Hz, 1H), 3.87 (dt, J = 9.9,

6.1 Hz, 1H), 3.67−3.59 (m, 4H), 2.30−2.24 (m, 5 H), 2.16 (s, 6H),

2.11−2.07 (m, 6H), 1.80−1.71 (m, 2H); % ee undetermined; LC−

MS (Method A) [M + H]⁺= 501, t_R0.55 min.

8-(cis-3-Aminocyclobutoxy)-7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-1-[1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-

one (27). A solution of tert-butyl [cis-3-({7-(3,5-dimethylisoxazol-4-

yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo-

[4,5-c]quinolin-8-yl}oxy)cyclobutyl]carbamate 26 (229 mg, 0.39

mmol) in DCM (6 mL) was treated with TFA (2 mL, 26.0 mmol).

The mixture was allowed to stand in a stoppered vessel at rt for 2 h.

The reaction mixture was evaporated under a stream of nitrogen and

the residue dissolved in MeOH (2 mL). The solution was applied to a

MeOH-preconditioned 5 g SCX-2 cartridge. The cartridge was

washed with MeOH (30 mL), followed by 2 M ammonia in MeOH

solution (30 mL). The basic wash was evaporated under a stream of

nitrogen to give the title compound as an oﬀ-white solid (178 mg,

0.37 mmol, 94% yield): mp 158−160 °C; ¹H NMR (400 MHz,

DMSO-d₆, 393 K) δ 8.77 (s, 1H), 8.62 (d, J = 4.8 Hz, 1H), 7.80 (s,

1H), 7.78−7.71 (m, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.32 (dd, J = 7.8,

4.8 Hz, 1H), 6.87 (s, 1H), 6.27 (q, J = 7.3 Hz, 1H), 3.83−3.75 (m,

1H), 3.58 (s, 3H), 3.09−3.00 (m, 1H), 2.80−2.71 (m, 1H), 2.52−

2.44 (m, 1H), 2.27 (s, 3H), 2.09 (s, 3H), 2.06 (d, J = 7.3 Hz, 3H),

1.65−1.54 (m, 2H), NH₂signal not resolved; ¹³C NMR (150 MHz,

DMSO-d₆) δ 166.0, 159.1, 158.9, 153.7, 152.4, 149.3, 140.1, 137.5,

133.1, 131.6, 127.1, 124.0, 122.8, 120.8, 120.6, 115.2, 112.1, 101.6,

64.7, 52.7, 41.2, 40.4, 39.7, 27.7, 17.5, 11.5, 10.5; % ee undetermined;

LC−MS (Method A) [M + H]⁺= 485, t_R0.52 min; HRMS [M + H]⁺

calcd for C₂₇H₂₉N₆O₃485.2301, found 485.2292.

tert-Butyl 2-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-

(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}-

oxy)acetate (24). A mixture of 7-(3,5-dimethylisoxazol-4-yl)-8-

hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]-

quinolin-2(3H)-one 13 (60 mg, 0.14 mmol) and potassium carbonate

(24 mg, 0.17 mmol) was suspended in anhydrous DMF (1 mL) and

treated with tert-butyl bromoacetate (0.026 mL, 0.17 mmol). The

mixture was stirred under nitrogen at 100 °C for 5 h. The reaction

mixture was allowed to cool to rt and partially evaporated under a

stream of nitrogen (∼0.9 mL remaining). The mixture was ﬁltered

and puriﬁed by MDAP (Method A). The appropriate fractions were

combined, and the solvent was removed by rotary evaporation. The

solid was dissolved in MeOH (0.5 mL) and applied to a MeOH-

preconditioned 1 g SCX-2 cartridge. The cartridge was washed with

MeOH (3 mL), followed by 2 M ammonia in MeOH solution (3

mL). The basic wash was evaporated under a stream of nitrogen to

give the title compound as a light brown solid (37 mg, 0.07 mmol,

1

48% yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ 8.80 (s, 1H),

8.62 (d, J = 4.8 Hz, 1H), 7.85 (s, 1H), 7.80−7.74 (m, 1H), 7.46 (d, J

= 8.1 Hz, 1H), 7.33 (dd, J = 7.3, 4.8 Hz, 1H), 7.15 (s, 1H), 6.29 (q, J

= 7.2 Hz, 1H), 4.43−4.31 (m, 2H), 3.59 (s, 3H), 2.30 (s, 3H), 2.13

(s, 3H), 2.06 (d, J = 7.2 Hz, 3H), 1.47 (s, 9H); % ee undetermined;

LC−MS (Method A) [M + H]⁺= 530, t_R0.96 min.

2-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[(R)-1-(pyri-

din-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}oxy)-

acetic Acid (25). A solution of tert-butyl 2-({7-(3,5-dimethylisoxazol-

4-yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)acetate 24 (29 mg, 0.06 mmol) in

DCM (0.75 mL) was treated with TFA (0.5 mL, 6.49 mmol) and left

to stand in a stoppered vessel for 2 h. The reaction mixture was

tert-Butyl [trans-3-({7-(3,5-Dimethyl-4-isoxazolyl)-3-methyl-2-

oxo-1-[1-(pyridine-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]-

quinolin-8-yl}oxy)cyclobutyl]carbamate (28). A mixture of cis-3-

[(tert-butoxycarbonyl)amino]cyclobutyl methanesulfonate (63 mg,

0.24 mmol), potassium carbonate (36 mg, 0.26 mmol), and 7-(3,5-

O

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dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)-

ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (90 mg, 0.22 mmol)

was suspended in anhydrous DMF (1 mL) and stirred under nitrogen

at rt overnight. The temperature was raised to 100 °C and the

reaction left to stir for 6 h. The reaction mixture was allowed to cool

to rt and ﬁltered. The ﬁltrate was puriﬁed by MDAP (Method B). The

appropriate fractions were combined, and the solvent was removed by

rotary evaporation to give the title compound as a light brown gum

(84 mg, 0.14 mmol, 66% yield): ¹H NMR (400 MHz, DMSO-d₆, 393

K) δ 8.77 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H), 7.81 (s, 1H), 7.78−7.70

(m, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.8, 4.8 Hz, 1H), 6.80

(s, 1H), 6.69 (d, J = 5.6 Hz, 1H), 6.26 (q, J = 7.3 Hz, 1H), 4.36−4.27

(m, 1H), 4.06−3.95 (m, 1H), 3.57 (s, 3H), 2.51−2.42 (m, 1H), 2.27

(s, 3H), 2.32−2.08 (m, 3H), 2.09 (s, 3H), 2.05 (d, J = 7.3 Hz, 3H),

1.41 (s, 9H); % ee undetermined; LC−MS (Method A) [M + H]⁺=

585, t_R0.93 min.

8-(trans-3-Aminocyclobutoxy)-7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-1-[1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-

one (29). A solution of tert-butyl [trans-3-({7-(3,5-dimethylisoxazol-

4-yl)-3-methyl-2-oxo-1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)cyclobutyl]carbamate 28 (75 mg,

0.13 mmol) in DCM (1.5 mL) was treated with TFA (0.5 mL,

6.49 mmol). The mixture was allowed to stand in a stoppered vessel

at rt for 2 h. The reaction mixture was evaporated under a stream of

nitrogen and the crude material puriﬁed by MDAP (Method B). The

appropriate fractions were combined, and the solvent was removed by

rotary evaporation to give the title compound as an oﬀ-white solid (48

mg, 0.10 mmol, 77% yield): ¹H NMR (400 MHz, DMSO-d₆, 393 K)

δ 8.76 (s, 1 H) 8.68 (d, J = 4.8 Hz, 1H), 7.80 (s, 1H), 7.78−7.71 (m,

1H), 7.40 (d, J = 7.8 Hz, 1H), 7.31 (dd, J = 7.8, 4.8 Hz, 1H), 6.81 (s,

1H), 6.27 (q, J = 7.3 Hz, 1H), 4.38−4.31 (m, 1H), 3.58 (s, 3H),

3.56−3.49 (m, 1H), 2.27 (s, 3H), 2.18−2.13 (m, 2H), 2.08 (s, 3H),

2.06 (d, J = 7.3 Hz, 3H), 2.12−2.03 (m, 1H), 1.99−1.91 (m, 1H),

1.73 (br s, 2H); % ee undetermined; LC−MS (Method A) [M + H]⁺

= 485, t_R0.52 min.

heptane with a ﬂow rate of 40 mL/min was used at rt. The UV

detection was performed at 215 nm. The appropriate fractions were

combined and evaporated in vacuo to give the title compound (49 mg,

1

0.08 mmol, 34% yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ

8.79 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 7.81 (s, 1H), 7.78−7.72 (m,

1H), 7.44 (d, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.3, 4.8 Hz, 1H), 7.09 (s,

1H), 6.29 (q, J = 7.3 Hz, 1H), 4.55−4.50 (m, 1H), 3.58 (s, 3H),

3.38−3.29 (m, 2H), 3.19−3.11 (m, 2H), 2.22 (s, 3H), 2.17−2.08 (m,

1H), 2.06 (d, J = 7.3 Hz, 3H), 2.02 (s, 3H), 1.98−1.89 (m, 1H), 1.39

(s, 9H), only a single diasteroisomer was observed; LC−MS (Method

B) [M + H]⁺= 585, t_R1.11 min.

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-1-[(R)-1-(pyridin-2-yl)-

ethyl]-8-[(R)-pyrrolidin-3-yloxy]-1H-imidazo[4,5-c]quinolin-2(3H)-

one (31). A solution of (3R)-tert-butyl 3-({7-(3,5-dimethylisoxazol-4-

yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo-

[4,5-c]quinolin-8-yl}oxy)pyrrolidine-1-carboxylate 30 (45 mg, 0.08

mmol) in 1,4-dioxane (0.5 mL) was treated with 4 M HCl in 1,4-

dioxane solution (0.5 mL, 16.46 mmol). The mixture was left to stir at

rt for 2 h. The reaction mixture was evaporated under a stream of

nitrogen and the solid dissolved in MeOH. The solution was applied

to a MeOH-preconditioned 2 g SCX-2 cartridge, which was then

washed with MeOH (12 mL), followed by 2 M ammonia in MeOH

solution (12 mL). The basic wash was evaporated by rotary

evaporation to give the title compound as a light brown solid (31

mg, 0.06 mmol, 83% yield): ¹H NMR (400 MHz, DMSO-d₆, 393 K)

δ 8.77 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 7.80 (s, 1H), 7.78−7.72 (m,

1H), 7.41 (d, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.3, 4.8 Hz, 1H), 7.04 (s,

1H), 6.29 (q, J = 7.3 Hz, 1H), 4.42−4.35 (m, 1H), 3.58 (s, 3H),

2.98−2.92 (m, 2H), 2.68−2.62 (m, 1H), 2.24 (s, 3H), 2.07 (d, J = 7.3

Hz, 3H), 2.05 (s, 3H), 2.05−1.99 (m, 1H), 1.72−1.64 (m, 1H), one

pyrrolidine proton signal obscured by water, NH signal not resolved,

only a single diasteroisomer was observed; LC−MS (Method B) [M

+ H]⁺= 485, t_R0.75 min.

(R)-tert-Butyl 3-[(Methylsulfonyl)oxy]pyrrolidine-1-carboxylate.

A stirred solution of (R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate

(481 mg, 2.57 mmol) and triethylamine (0.931 mL, 6.68 mmol) in

DCM (6 mL) at 0 °C was treated with methanesulfonyl chloride

(0.250 mL, 3.20 mmol). The mixture was stirred under nitrogen for

10 min and allowed to warm to rt over 1 h. The reaction was diluted

with DCM (10 mL) and washed with water (10 mL). The aqueous

layer was extracted with DCM (10 mL), and the organic layers were

combined and passed through a hydrophobic frit. The solvent was

removed by rotary evaporation and the crude material puriﬁed on a

25 g silica cartridge using a gradient of 0−15% MeOH in DCM over

10 column volumes. The appropriate fractions were combined, and

the solvent was removed by rotary evaporation to give the title

(S)-tert-Butyl 3-[(Methylsulfonyl)oxy]pyrrolidine-1-carboxylate.

A stirred solution of (S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate

(500 mg, 2.67 mmol) and triethylamine (0.931 mL, 6.68 mmol) in

DCM (6 mL) at 0 °C was treated with methanesulfonyl chloride

(0.250 mL, 3.20 mmol). The mixture was stirred under nitrogen for

10 min and allowed to warm to rt over 2 h. The reaction was diluted

with DCM (10 mL) and washed with brine (15 mL). The aqueous

layer was extracted with DCM (10 mL), and the organic layers were

combined and passed through a hydrophobic frit. The solvent was

removed by rotary evaporation and the crude material puriﬁed on a

25 g silica cartridge using a gradient of 0−15% MeOH in DCM over

10 column volumes. The appropriate fractions were combined, and

the solvent was removed by rotary evaporation to give the title

1

compound as a light yellow oil (651 mg, 2.45 mmol, 96% yield): H

NMR (400 MHz, CDCl₃) δ 5.28−5.23 (m, 1H), 3.75−3.41 (m, 4H),

3.04 (s, 3H), 2.37−2.08 (m, 2H), 1.45 (s, 9H).

1

compound as a light yellow oil (691 mg, 2.60 mmol, 98% yield): H

NMR (400 MHz, CDCl₃) δ 5.28−5.23 (m, 1H), 3.75−3.41 (m, 4H),

3.04 (s, 3H), 2.37−2.08 (m, 2H), 1.45 (s, 9H).

(3S)-tert-Butyl 3-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-

1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-

yl}oxy)pyrrolidine-1-carboxylate (32). To a vessel containing (R)-

tert-butyl 3-[(methylsulfonyl)oxy]pyrrolidine-1-carboxylate (82 mg,

0.31 mmol) were added potassium carbonate (43 mg, 0.31 mmol)

and 7-(3,5-dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-1-(pyr-

idin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (100 mg,

0.24 mmol). DMF (2 mL) was added and the mixture stirred under

nitrogen and at 100 °C for 5 h. EtOAc (10 mL) was added to the

reaction mixture, which was then washed with brine (10 mL). The

aqueous layer was extracted with EtOAc, and the organic layers were

combined and passed through a hydrophobic frit. The solvent was

removed by rotary evaporation and the crude product dissolved in

DCM (4 mL) and puriﬁed on a 25 g silica cartridge using a gradient

of 0−15% MeOH in DCM over 10 column volumes. The appropriate

fractions were combined, and the solvent was removed by rotary

evaporation. The crude material was puriﬁed by MDAP (Method A).

The appropriate fractions were combined, and the solvent was

removed by rotary evaporation to give the title compound as an oﬀ-

(3R)-tert-Butyl 3-({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-

1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]-

quinolin-8-yl}oxy)pyrrolidine-1-carboxylate (30). To a vessel

containing (S)-tert-butyl 3-[(methylsulfonyl)oxy]pyrrolidine-1-car-

boxylate (89 mg, 0.34 mmol) were added potassium carbonate (47

mg, 0.34 mmol) and 7-(3,5-dimethylisoxazol-4-yl)-8-hydroxy-3-

methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-

2(3H)-one 13 (103 mg, 0.25 mmol). DMF (2 mL) was added and

the mixture stirred under nitrogen at 100 °C for 4 h. EtOAc (10 mL)

was added to the reaction mixture, which was then washed with brine

(10 mL). The aqueous layer was extracted with EtOAc (10 mL), and

the organic layers were combined and passed through a hydrophobic

frit. The solvent was removed by rotary evaporation and the crude

material puriﬁed on a 25 g silica cartridge using a gradient of 0−15%

MeOH in DCM over 10 column volumes. The appropriate fractions

were combined, and the solvent was removed by rotary evaporation.

The sample was dissolved in EtOH (3 mL) and heptane (3 mL).

Fifteen injections were made onto a Chiralpak IA column (250 mm ×

30 mm, 5 μm packing diameter). An isocratic system of 15% EtOH in

1

white solid (99 mg, 0.17 mmol, 70% yield): H NMR (400 MHz,

DMSO-d₆, 393 K) δ 8.80 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 7.82 (s,

P

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1H), 7.79−7.73 (m, 1H), 7.45 (d, J = 7.3 Hz, 1H), 7.31 (dd, J = 7.3,

4.8 Hz, 1H), 7.06 (s, 1H), 6.30 (q, J = 7.3 Hz, 1H), 4.47 (m, 1H),

3.59 (s, 3H), 3.58−3.52 (m, 1H), 3.39−3.30 (m, 2H), 3.19−3.11 (m,

1H), 2.22 (s, 3H), 2.06 (d, J = 7.3 Hz, 3H), 2.02 (s, 3H), 2.01−1.94

(m, 1H), 1.82−1.73 (m, 1H), 1.42 (s, 1.3 H), 1.39 (s, 7.7 H), a 85:15

mixture of diastereoisomers present; LC−MS (Method A) [M + H]⁺

= 585, t_R0.96 min.

mmol) and 7-(3,5-dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-

1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (70

mg, 0.17 mmol) was suspended in anhydrous DMF (0.8 mL) and

stirred under nitrogen at 45 °C for 16 h. The reaction temperature

was raised to 100 °C and left to stir for 3.5 h. The reaction mixture

was allowed to cool to rt and diluted with MeOH (0.2 mL). The

mixture was ﬁltered and puriﬁed by MDAP (Method A). The

appropriate fractions were combined, and the solvent was removed by

rotary evaporation to give the title compound as a dark brown gum

(79 mg, 0.13 mmol, 78% yield): ¹H NMR (400 MHz, DMSO-d₆, 393

K) δ 8.81 (s, 1H), 8.59 (d, J = 4.8 Hz, 1H), 7.82 (s, 1H), 7.80−7.73

(m, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.32 (dd, J = 7.3, 4.8 Hz, 1H), 7.03

(s, 0.15 H), 7.01 (s, 0.85 H), 6.32 (q, J = 7.1 Hz, 1H), 3.81 (dd, J =

9.6, 6.1 Hz, 0.15 H), 3.76 (dd, J = 9.5, 7.2 Hz, 0.85 H), 3.62 (s, 3H),

3.51−3.42 (m, 1H), 3.42−3.35 (m, 1H), 3.35−3.22 (m, 2H), 3.03−

2.98 (m, 1H), 2.55−2.47 (m, 1H), 2.26 (s, 3H), 2.07 (s, 3H), 2.07 (d,

J = 7.1 Hz, 3H), 1.99−1.88 (m, 1H), 1.64−1.53 (m, 1H), 1.45 (s,

9H), a 85:15 mixture of diastereoisomers present; LC−MS (Method

A) [M + H]⁺= 599, t_R0.98 min.

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-1-[1-(pyridin-2-yl)ethyl]-

8-[(S)-pyrrolidin-3-yloxy]-1H-imidazo[4,5-c]quinolin-2(3H)-one

(33). A solution of (3S)-tert-butyl 3-({7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-

c]quinolin-8-yl}oxy)pyrrolidine-1-carboxylate 32 (103 mg, 0.18

mmol) in anhydrous 1,4-dioxane (0.5 mL) was treated with 4 M

HCl in 1,4-dioxane solution (0.5 mL, 16.46 mmol) and the mixture

allowed to stand in a stoppered vessel for 2 h. The reaction mixture

was evaporated under a stream of nitrogen and the solid dissolved in

MeOH. The solution was applied to a MeOH-preconditioned 2 g

SCX-2 cartridge. The cartridge was washed with MeOH (20 mL),

followed by 2 M ammonia in MeOH solution (20 mL). The basic

wash was evaporated by rotary evaporation. Starting material still

remained so the residue was dissolved in anhydrous 1,4-dioxane (0.5

mL) and 4 M HCl in 1,4-dioxane solution (0.5 mL, 16.46 mmol), and

the mixture was left to stir overnight in a stoppered vessel. The

reaction mixture was evaporated under a stream of nitrogen, and the

solid was dissolved in the minimum volume of MeOH. The solution

was applied to a MeOH-preconditioned 2 g SCX-2 cartridge, which

was then washed with MeOH (20 mL), followed by 2 M ammonia in

MeOH solution (20 mL). The basic wash was evaporated by rotary

evaporation and the crude material puriﬁed by MDAP conducted on

an Xbridge BEH Shield RP18 column (150 mm × 19 mm, 5 μm

packing diameter) at a 20 mL/min ﬂow rate. Gradient elution was

carried out at an ambient temperature, with the mobile phases as (A)

water containing 0.1% (v/v) formic acid and (B) acetonitrile

containing 0.1% (v/v) formic acid. The UV detection was a summed

signal from wavelengths of 210−400 nm. The appropriate fractions

were combined and dried under a stream of nitrogen, and the crude

material was puriﬁed on a 10 g silica cartridge using a gradient of 0−

20% 2 M ammonia/MeOH in DCM over 15 column volumes. The

appropriate fractions were combined and evaporated in vacuo to give

the title compound as a colorless oil (10 mg, 0.02 mmol, 12% yield):

¹H NMR (400 MHz, DMSO-d₆, 393 K) δ 8.77 (s, 1H), 8.60 (d, J =

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-1-[1-(pyridin-2-yl)ethyl]-

8-[(R)-pyrrolidin-3-ylmethoxy]-1H-imidazo[4,5-c]quinolin-2(3H)-

one (35). A solution of (3R)-tert-butyl 3-[({7-(3,5-dimethylisoxazol-

4-yl)-3-methyl-2-oxo-1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)methyl]pyrrolidine-1-carboxylate 34

(73 mg, 0.12 mmol) was dissolved in DCM (1.5 mL) and treated with

TFA (0.5 mL, 6.49 mmol). The mixture was allowed to stand in a

stoppered vessel at rt for 2 h. The reaction mixture was evaporated in

vacuo and the crude material puriﬁed by MDAP (Method B). The

appropriate fractions were combined, and the solvent was removed by

rotary evaporation to give the title compound as a white solid (44 mg,

1

0.09 mmol, 72% yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ

8.77 (s, 1H), 8.58 (d, J = 4.8 Hz, 1H), 7.78 (s, 1H), 7.77−7.72 (m,

1H), 7.42 (d, J = 7.3 Hz, 1H), 7.30 (dd, J = 7.3, 4.8 Hz, 1H), 6.99 (s,

1H), 6.29 (q, J = 7.3 Hz, 1H), 3.74 (dd, J = 9.8, 3.2 Hz, 0.15 H), 3.69

(dd, J = 9.6, 6.3 Hz, 0.85 H), 3.59 (s, 3H), 3.46−3.37 (m, 1H), 2.84−

2.78 (m, 2H), 2.53−2.49 (m, 1H), 2.38−2.27 (m, 1H), 2.24 (s, 3H),

2.04 (s, 3H), 2.05 (d, J = 7.3 Hz, 3H), 1.84−1.74 (m, 1H), 1.40−1.28

(m, 1H), one pyrrolidine CH₂N proton signal obscured by water, NH

signal not resolved, a 85:15 mixture of diastereoisomers present; LC−

MS (Method A) [M + H]⁺= 499, t_R0.53 min.

(S)-tert-Butyl 3-{[(Methylsulfonyl)oxy]methyl}pyrrolidine-1-car-

boxylate. To a solution of (S)-tert-butyl 3-(hydroxymethyl)-

pyrrolidine-1-carboxylate (174 mg, 0.87 mmol) in DCM (3 mL)

was added triethylamine (0.150 mL, 1.08 mmol). This was cooled to

0 °C under a nitrogen atmosphere, and methanesulfonyl chloride

(0.086 mL, 1.10 mmol) added dropwise. The mixture was stirred at 0

°C for 10 min, allowed to warm to rt, and then stirred for 2 h. Water

(10 mL) was added to the reaction mixture, which was then extracted

with DCM (2 × 10 mL). The combined organic layers were passed

through a hydrophobic frit, and the solvent was evaporated in vacuo.

The crude material was puriﬁed on a 25 g silica cartridge using a

gradient of 0−10% MeOH in DCM over 10 column volumes. The

appropriate fractions were combined, and the solvent was removed by

rotary evaporation to give the title compound as a colorless oil (219

4.8 Hz, 1H), 7.79 (s, 1H), 7.77−7.71 (m, 1H), 7.41 (d, J = 7.3 Hz,

1H), 7.31 (dd, J = 7.3, 4.8 Hz, 1H), 7.01 (s, 1H), 6.29 (q, J = 7.3 Hz,

1H), 4.37 (m, 1H), 3.58 (s, 3H), 3.21−3.15 (m, 0.15 H), 3.13−3.07

(m, 0.85 H), 2.80−2.62 (m, 3H), 2.24 (s, 2.6 H), 2.19 (s, 0.4 H),

2.10−2.02 (m, 5.1 H), 2.01 (s, 0.9 H), 1.82−1.71 (m, 1H), 1.55−1.47

(m, 1H), a 85:15 mixture of diastereoisomers present; LC−MS

(Method A) [M + H]⁺= 485, t_R0.52 min.

(R)-tert-Butyl 3-{[(Methylsulfonyl)oxy]methyl}pyrrolidine-1-car-

boxylate. To a solution of (R)-tert-butyl 3-(hydroxymethyl)-

pyrrolidine-1-carboxylate (162 mg, 0.81 mmol) in DCM (3 mL)

was added triethylamine (0.14 mL, 1.01 mmol). This was cooled to 0

°C under a nitrogen atmosphere, and methanesulfonyl chloride (0.08

mL, 1.03 mmol) was added dropwise. The mixture was stirred at 0 °C

for 10 min, allowed to warm to rt, and then stirred for 2 h. Water (10

mL) was added to the reaction mixture, which was then extracted

with DCM (2 × 10 mL). The combined organic layers were passed

through a hydrophobic frit, and the solvent was evaporated in vacuo.

The crude material was puriﬁed on a 25 g silica cartridge using a

gradient of 0−10% MeOH in DCM over 10 column volumes. The

appropriate fractions were combined, and the solvent was removed by

rotary evaporation to give the title compound as a colorless oil (204

1

mg, 0.78 mmol, 91% yield): H NMR (400 MHz, CDCl₃) δ 4.28−

4.11 (m, 2H), 3.62−3.09 (m, 4H), 3.03 (s, 3H), 2.70−2.56 (m, 1H),

2.11−1.99 (m, 1H), 1.73 (br s, 1H), 1.46 (s, 9H).

(3S)-tert-Butyl 3-[({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-

oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]-

quinolin-8-yl}oxy)methyl]pyrrolidine-1-carboxylate (36). A mixture

of (S)-tert-butyl 3-{[(methylsulfonyl)oxy]methyl}pyrrolidine-1-car-

boxylate (37 mg, 0.13 mmol), potassium carbonate (20 mg, 0.14

mmol) and 7-(3,5-dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-

1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (50

mg, 0.12 mmol) was suspended in anhydrous DMF (0.5 mL) and

stirred under nitrogen at 100 °C for 4 h. The reaction mixture was

allowed to cool to rt and diluted with MeOH (0.5 mL). The mixture

was ﬁltered and was puriﬁed by MDAP (Method B). The appropriate

fractions were combined, and the solvent was removed by rotary

evaporation to give the title compound as a colorless gum (49 mg,

1

mg, 0.73 mmol, 91% yield): H NMR (400 MHz, CDCl₃) δ 4.28−

4.11 (m, 2H), 3.62−3.09 (m, 4H), 3.03 (s, 3H), 2.70−2.56 (m, 1H),

2.11−1.99 (m, 1H), 1.73 (br s, 1H), 1.46 (s, 9H).

(3R)-tert-Butyl 3-[({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-

oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]-

quinolin-8-yl}oxy)methyl]pyrrolidine-1-carboxylate (34). A mixture

of (R)-tert-butyl 3-{[(methylsulfonyl)oxy]methyl}pyrrolidine-1-car-

boxylate (52 mg, 0.19 mmol), potassium carbonate (28 mg, 0.20

Q

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1

0.08 mmol, 68% yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ

in 1,4-dioxane (0.7 mL). To the solution was added 4 M HCl in 1,4-

dioxane solution (0.5 mL, 0.152 mmol) and the mixture left to stir at

rt for 1 h. The reaction was concentrated under a stream of nitrogen

and the residue puriﬁed by MDAP (Method B). The appropriate

fractions were combined, and the solvent was removed by rotary

evaporation to give the title compound as a green solid (53 mg, 0.103

mmol, 68% yield): ¹H NMR (400 MHz, DMSO-d₆, 393 K) δ 8.77 (s,

1H), 8.58 (d, J = 4.8 Hz, 1H), 7.77 (s, 1H), 7.77−7.71 (m, 1H), 7.41

(d, J = 7.8 Hz, 1H), 7.31 (dd, J = 7.8, 4.8 Hz, 1H), 6.97 (s, 1H), 6.29

(q, J = 7.3 Hz, 1H), 3.64−3.60 (m, 1H), 3.59 (s, 3H), 3.28 (dd, J =

9.4, 6.8 Hz, 1H), 2.96−2.89 (m, 2H), 2.46−2.41 (m, 1H), 2.23 (s,

3H), 2.05 (d, J = 7.3 Hz, 3H), 2.03 (s, 3H), 1.77−1.65 (m, 1H),

1.57−1.47 (m, 2H), 1.13−0.99 (m, 2H), one piperidine proton signal

obscured by DMSO, NH signal not resolved; % ee undetermined;

LC−MS (Method B) [M + H]⁺= 513, t_R0.78 min.

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)-

ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl Triﬂuorometha-

nesulfonate (40). A solution of 7-(3,5-dimethylisoxazol-4-yl)-8-

hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]-

quinolin-2(3H)-one 13 (4.42g, 10.64 mmol) in DCM (20 mL) and

pyridine (2.58 mL) under nitrogen was cooled in an ice bath and

Tf₂O (2.16 mL, 12.77 mmol) added dropwise. The mixture was

stirred for 2 h, allowing to warm to rt. Further, Tf₂O (0.05 mL) was

added dropwise and the mixture stirred for 2 h. The reaction mixture

was washed with water (2 × 20 mL), dried, and evaporated. The

residue was puriﬁed by column chromatography using a gradient of

0−10% 2 M ammonia in MeOH/DCM) to give the title compound

as a beige foam (5.25 g, 9.59 mmol, 90% yield): ¹H NMR (400 MHz,

CDCl₃) δ 8.85 (s, 1H), 8.64 (d, J = 4.8 Hz, 1H), 8.03 (s, 1H), 7.85

(br s, 1H), 7.69 (td, J = 7.8, 1.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H),

7.30−7.23 (m, 1H), 6.42 (q, J = 7.3 Hz, 1H), 3.72 (s, 3H), 2.33 (s,

3H), 2.19 (s, 3H), 2.10 (d, J = 7.3 Hz, 3H); % ee undetermined; LC−

MS (Method A) [M + H]⁺= 548, t_R1.08 min.

8.77 (s, 1H), 8.57 (d, J = 4.8 Hz, 1H), 7.79 (s, 1H), 7.76−7.70 (m,

1H), 7.42 (d, J = 7.8 Hz, 1H), 7.29 (dd, J = 7.3, 4.8 Hz, 1H), 7.02 (s,

0.85 H), 7.00 (s, 0.15 H), 6.29 (q, J = 7.1 Hz, 1H), 3.79 (dd, J = 9.6,

6.3 Hz, 0.85 H), 3.73 (dd, J = 9.6, 7.0 Hz, 0.15 H), 3.59 (s, 3H),

3.45−3.39 (m, 1H), 3.37−3.28 (m, 2H), 3.26−3.19 (m, 1H), 2.94

(dd, J = 10.9, 6.8 Hz, 1H), 2.52−2.44 (m, 1H), 2.23 (s, 3H), 2.05 (d,

J = 7.1 Hz, 3H), 2.04 (s, 3H), 1.98−1.87 (m, 1H), 1.62−1.52 (m,

1H), 1.41 (s, 9H), a 85:15 mixture of diastereoisomers present; LC−

MS (Method A) [M + H]⁺= 599, t_R0.94 min.

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-1-[1-(pyridin-2-yl)ethyl]-

8-[(S)-pyrrolidin-3-ylmethoxy]-1H-imidazo[4,5-c]quinolin-2(3H)-

one (37). A solution of (3S)-tert-butyl 3-[({7-(3,5-dimethylisoxazol-4-

yl)-3-methyl-2-oxo-1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-

imidazo[4,5-c]quinolin-8-yl}oxy)methyl]pyrrolidine-1-carboxylate 36

(39 mg, 0.07 mmol) was dissolved in DCM (0.6 mL) and treated with

TFA (0.3 mL, 3.89 mmol). The mixture was allowed to stand in a

stoppered vessel at rt for 2 h. The reaction mixture was evaporated

under a stream of nitrogen and the gum dissolved in MeOH (0.5

mL). The solution was applied to a MeOH-preconditioned 1 g SCX-2

cartridge, which was then washed with MeOH (6 mL), followed by 2

M ammonia in MeOH solution (6 mL). The basic wash was

evaporated under a stream of nitrogen to give the title compound as a

1

white solid (28 mg, 0.06 mmol, 86% yield): H NMR (400 MHz,

DMSO-d₆, 393 K) δ 8.77 (s, 1H), 8.58 (d, J = 4.8 Hz, 1H), 7.78 (s,

1H), 7.77−7.71 (m, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.3,

4.8 Hz, 1H), 6.98 (s, 1H), 6.29 (q, J = 7.1 Hz, 1H), 3.74−3.65 (m,

1H), 3.59 (s, 3H), 3.40−3.34 (m, 1H), 2.79−2.70 (m, 4H), 2.42 (dd,

J = 10.6, 6.3 Hz, 1H), 2.34−2.25 (m, 1H), 2.24 (s, 3H), 2.04 (s, 3H),

2.05 (d, J = 7.1 Hz, 3H), 1.82−1.71 (m, 1H), 1.36−1.26 (m, 1H), one

pyrrolidine proton signal obscured by water, NH signal not resolved,

an estimated 85:15 mixture of diastereoisomers present; LC−MS

(Method A) [M + H]⁺= 499, t_R0.53 min.

Ethyl 7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-(pyri-

din-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinoline-8-carboxy-

late (41). A 100 mL 3-necked ﬂask was charged with palladium

diacetate (15 mg, 0.07 mmol), 1,3-bis(diphenylphosphino)propane

(27 mg, 0.07 mmol), 7-(3,5-dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-

[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl

triﬂuoromethanesulfonate 40 (500 mg, 0.91 mmol), anhydrous DMF

(10 mL), EtOH (8 mL, 137 mmol), and triethylamine (0.28 mL, 2.01

mmol). The ﬂask was ﬁtted with a reﬂux condenser and the apparatus

purged with CO gas. A balloon of CO gas was ﬁtted to the top of the

condenser and the mixture stirred at 70 °C for 3 h. The reaction

mixture was evaporated in vacuo and the resulting gum dissolved in

EtOAc (10 mL). The organic layer was washed with 10% LiCl (aq)

(10 mL) and then water (2 × 10 mL). The organic layer was passed

through a hydrophobic frit and the solvent evaporated in vacuo. The

crude material was puriﬁed on a 100 g silica cartridge using a gradient

of 0−15% MeOH in DCM over 12 column volumes. The appropriate

fractions were combined, and the solvent was evaporated in vacuo to

give the title compound as a light yellow foam (430 mg, 0.91 mmol,

100% yield): mp 90−92 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.12

(s, 1H), 8.54 (d, J = 4.8 Hz, 1H), 8.15 (br s, 1H), 7.91 (s, 1H), 7.78

(dd, J = 7.8, 7.3 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.31 (dd, J = 7.3,

4.8 Hz, 1H), 6.33 (q, J = 7.3 Hz, 1H), 4.19−4.05 (m, 2H), 3.30 (s,

3H), 2.20 (s, 3H), 2.05 (d, J = 7.3 Hz, 3H), 1.96 (s, 3H), 1.18 (t, J =

7.1 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.6, 163.3, 158.2,

158.5, 153.6, 149.2, 145.0, 137.3, 135.8, 133.7, 133.5, 129.0, 126.5,

126.3, 124.2, 122.6, 120.9, 115.2, 113.4, 60.9, 54.9, 27.8, 17.1, 13.8,

10.9, 10.0; % ee undetermined; LC−MS (Method A) [M + H]⁺=

472, t_R0.87 min; HRMS [M + H]⁺calcd for C₂₆H₂₆N₅O₄472.1979,

found 472.1972.

tert-Butyl 4-{[(Methylsulfonyl)oxy]methyl}piperidine-1-carboxy-

late. A stirred solution of tert-butyl 4-(hydroxymethyl)piperidine-1-

carboxylate (310 mg, 1.440 mmol) and triethylamine (0.26 mL, 1.865

mmol) in DCM (3 mL) at 0 °C was treated with methanesulfonyl

chloride (0.13 mL, 1.668 mmol). The mixture was stirred for 1 h

under nitrogen, and then sat. NaHCO₃(aq) (10 mL) was added. The

aqueous layer was separated and extracted with DCM (10 mL). The

organic layers were combined and washed with sat. NaHCO₃(aq)

(10 mL). The organic layer was separated and passed through a

hydrophobic frit, and the solvent was evaporated under a vacuum to

give the title compound as a yellow oil (386 mg, 1.316 mmol, 91%

1

yield): H NMR (400 MHz, CDCl₃) δ 4.23−4.10 (m, 2H), 4.10−

4.04 (m, 2H), 3.01 (s, 3H), 2.78−2.65 (m, 2H), 1.98−1.86 (m, 1H),

1.79−1.69 (m, 2H), 1.46 (br s, 9H), 1.31−1.15 (m, 2H).

tert-Butyl 4-[({7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-

[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}-

oxy)methyl]piperidine-1-carboxylate (38). A mixture of 7-(3,5-

dimethylisoxazol-4-yl)-8-hydroxy-3-methyl-1-[(R)-1-(pyridin-2-yl)-

ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-one 13 (63 mg, 0.152

mmol), tert-butyl 4-{[(methylsulfonyl)oxy]methyl}piperidine-1-car-

boxylate (59 mg, 0.201 mmol), and potassium carbonate (34 mg,

0.246 mmol) in DMF (1.5 mL) was stirred under nitrogen at 100 °C

and for 2.5 h. The reaction mixture was allowed to cool to rt, diluted

with EtOAc (8 mL), and washed with brine (8 mL). The aqueous

layer was separated and extracted with EtOAc (8 mL); the organic

layers were combined and passed through a hydrophobic frit. The

solvent was removed by rotary evaporation and the residue dissolved

in MeOH (1 mL). The solution was applied to a MeOH-

preconditioned 2 g SCX-2 cartridge. The cartridge was washed with

MeOH (10 mL), followed by 2 M ammonia in MeOH solution (10

mL). The basic wash was evaporated in vacuo and the material used

directly in the next step.

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-(pyridin-2-yl)-

ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinoline-8-carboxylic Acid

(42). A solution of ethyl 7-(3,5-dimethylisoxazol-4-yl)-3-methyl-2-

oxo-1-[(R)-1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]-

quinoline-8-carboxylate 41 (300 mg, 0.64 mmol) in EtOH (2 mL)

was treated with 2 M NaOH (aq) (0.33 mL, 0.66 mmol) and the

reaction mixture stirred at rt for 15 h. Further 2 M NaOH (aq) (0.150

7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-8-(piperidin-4-ylme-

thoxy)-1-[1-(pyridin-2-yl)ethyl]-1H-imidazo[4,5-c]quinolin-2(3H)-

one (39). Crude tert-Butyl 4-[({7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-

c]quinolin-8-yl}oxy)methyl]piperidine-1-carboxylate 38 was dissolved

R

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mL) was added and the mixture stirred at 50 °C for 3 h. The reaction

mixture was evaporated in vacuo and the crude material puriﬁed by

MDAP (Method A). The appropriate fractions were combined, and

the solvent removed by rotary evaporation to give the title compound

(50 mol % formic acid) as an oﬀ-white solid (191 mg, 0.41 mmol,

1H), 5.39 (s, 2H); LC−MS (Method A) no mass ion detected, t_R

1.30 min.

4-[2-(Benzyloxy)-5-nitrophenyl]-3,5-dimethylisoxazole (48). Ni-

trogen was bubbled through a mixture of 1-(benzyloxy)-2-bromo-4-

nitrobenzene 47 (10.73 g, 34.8 mmol), cesium carbonate (23 g, 70.6

mmol), DME (100 mL), and water (40 mL) for 30 min. (3,5-

Dimethylisoxazol-4-yl)boronic acid (9.75 g, 69.2 mmol) and PEPPSI-

IPr catalyst (0.6 g, 0.883 mmol) were added, and the mixture was

stirred at 90 °C for 5 h under nitrogen. The reaction was allowed to

cool to rt, and then EtOAc (150 mL) and water (75 mL) were added.

The layers were separated, and the organic layer was washed with 10%

Na₂SO₃(aq) (75 mL), followed by brine (75 mL). The organic layer

was dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The solid was triturated with diethyl ether (50 mL), ﬁltered,

and washed with cyclohexane (100 mL). The solid was dried in a

vacuum oven to give the title compound as a light brown solid (9.72

g, 30.0 mmol, 86% yield): ¹H NMR (400 MHz, CDCl₃) δ 8.26 (dd, J

= 9.1, 2.8 Hz, 1H), 8.07 (d, J = 2.8 Hz, 1H), 7.41−7.31 (m, 3H),

7.30−7.25 (m, 2H), 7.13 (d, J = 9.1 Hz, 1H), 5.19 (s, 2H), 2.30 (s,

3H), 2.16 (s, 3H); LC−MS (Method A) [M + H]⁺= 325, t_R1.19

min.

4-(Benzyloxy)-3-(3,5-dimethylisoxazol-4-yl)aniline (49). A mix-

ture of 4-[2-(benzyloxy)-5-nitrophenyl]-3,5-dimethylisoxazole 48 (9.2

g, 28.4 mmol), iron powder (6.0 g, 107 mmol), and ammonium

chloride (12 g, 224 mmol) was suspended in EtOH (400 mL) and

water (100 mL) and stirred at rt for 15 h. The reaction mixture was

ﬁltered through a pad of Celite and the solid washed with EtOH. The

ﬁltrate was evaporated under a vacuum and the residue partitioned

between EtOAc (150 mL) and water (150 mL). The organic layer

was separated, washed with further with water (100 mL), and dried

over MgSO₄. The solvent was removed under a vacuum to give the

title compound as a dark brown oil (8.2 g, 27.9 mmol, 98% yield): ¹H

NMR (400 MHz, CDCl₃) δ 7.37−7.25 (m, 3H), 7.25−7.20 (m, 2H),

6.91 (d, J = 8.6 Hz, 1H), 6.69 (dd, J = 8.6, 2.9 Hz, 1H), 6.51 (d, J =

2.9 Hz, 1H), 4.92 (s, 2H), 3.53 (br s, 2H), 2.29 (s, 3H), 2.18 (s, 3H);

LC−MS (Method A) [M + H]⁺= 295, t_R0.75 min, 85% purity by

LC−MS UV (10% starting material remains).

Diethyl 2-({[4-(Benzyloxy)-3-(3,5-dimethylisoxazol-4-yl)phenyl]-

amino}methylene)malonate (50). A mixture of 4-(benzyloxy)-3-

(3,5-dimethylisoxazol-4-yl)aniline 49 (8.2 g, 27.9 mmol) and diethyl

2-(ethoxymethylene)malonate (5.6 mL, 28.0 mmol) was stirred at

130 °C for 1 h. The brown oil was concentrated under reduced

pressure. The residue was loaded in DCM (20 mL) and puriﬁed on a

330 g silica cartridge using a gradient of 0−100% EtOAc in DCM

over 10 column volumes. The appropriate fractions were combined,

and the solvent was removed by rotary evaporation. The resulting

solid was triturated with cyclohexane, ﬁltered, and dried in a vacuum

oven to give the title compound as a light brown solid (9.6 g, 20.67

mmol, 74% yield): ¹H NMR (400 MHz, CDCl₃) δ 11.02 (d, J = 13.5

Hz, 1H), 8.44 (d, J = 13.5 Hz, 1H), 7.40−7.24 (m, 5H), 7.15 (dd, J =

8.8, 2.9 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 2.9 Hz, 1H),

5.06 (s, 2H), 4.32 (q, J = 7.1 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 2.31

(s, 3H), 2.18 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H), 1.34 (t, J = 7.1 Hz,

3H); LC−MS (Method A) [M + H]⁺= 465, t_R1.30 min.

1

64% yield): H NMR (400 MHz, DMSO-d₆, 353 K) δ 9.04 (s, 1H),

8.52 (d, J = 4.8 Hz, 1H), 8.30 (s, 1H), 8.14 (s, 0.5 H, HCO₂H), 7.85

(s, 1H), 7.74 (dd, J = 7.8, 7.3 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.28

(dd, J = 7.3, 4.8 Hz, 1H), 6.33 (q, J = 7.3 Hz, 1H), 3.63 (s, 3H), 2.21

(s, 3H), 2.07 (d, J = 7.3 Hz, 3H), 1.99 (s, 3H); % ee undetermined;

LC−MS (Method A) [M + H]⁺= 444, t_R0.61 min.

tert-Butyl 2-{7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-

(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}-

acetate (43). A mixture of 7-(3,5-dimethylisoxazol-4-yl)-3-methyl-2-

oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-

8-yl triﬂuoromethanesulfonate 40 (250 mg, 0.457 mmol) and

Pd(PPh₃)₄(53 mg, 0.046 mmol) in a sealed vessel under nitrogen

was diluted with anhydrous NMP (0.5 mL) and [2-(tert-butoxy)-2-

oxoethyl]zinc(II) chloride (0.5 M solution in THF) (2.5 mL, 1.250

mmol). The mixture was heated in a Biotage I60 microwave at 90 °C

for 20 min. The reaction mixture was diluted with sat. NH₄Cl (aq) (2

mL) and extracted with EtOAc (3 × 2 mL). The organic extracts were

combined and passed through a hydrophobic frit. The solvent was

removed in vacuo, and the residue was loaded in DCM (2 mL) and

puriﬁed on a 100 g silica cartridge using a gradient of 0−100%

acetone in cyclohexane over 12 column volumes. The appropriate

fractions were combined, the solvent was removed by rotary

evaporation, and the solid was puriﬁed by MDAP (Method B). The

appropriate fractions were combined, and the solvent removed by

rotary evaporation to give the title compound as an oﬀ-white solid (39

mg, 0.076 mmol, 17% yield): ¹H NMR (400 MHz, DMSO-d₆, 393 K)

δ 8.92 (s, 1H), 8.56 (d, J = 4.8 Hz, 1H), 7.81 (s, 1H), 7.78 (s, 1H),

7.77−7.72 (m, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.27 (dd, J = 7.8, 4.8

Hz, 1H), 6.35 (q, J = 7.2 Hz, 1H), 3.60 (s, 3H), 3.36 (br s, 2H), 2.20

(s, 3H), 2.08 (d, J = 7.2 Hz, 3H), 2.01 (s, 3H), 1.32 (s, 9H); % ee

undetermined; LC−MS (Method A) [M + H]⁺= 514, t_R0.91 min.

2-{7-(3,5-Dimethylisoxazol-4-yl)-3-methyl-2-oxo-1-[1-(pyridin-2-

yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-c]quinolin-8-yl}acetic acid

(44). A solution of tert-butyl 2-{7-(3,5-dimethylisoxazol-4-yl)-3-

methyl-2-oxo-1-[1-(pyridin-2-yl)ethyl]-2,3-dihydro-1H-imidazo[4,5-

c]quinolin-8-yl}acetate 43 (35 mg, 0.068 mmol) in DCM (0.5 mL)

was treated with TFA (0.5 mL, 6.49 mmol) and the mixture left to

stand at rt in a stoppered vessel for 36 h. The reaction mixture was

evaporated under a stream of nitrogen and was puriﬁed by MDAP

(Method C). The appropriate fractions were combined, and the

solvent was evaporated under a stream of nitrogen. The solid was

dissolved in MeOH (0.5 mL) and applied to a 0.5 g MeOH-

preconditioned SCX-2 cartridge. The cartridge was washed with

MeOH (3 mL), followed by 2 M ammonia in MeOH solution (3

mL). The basic wash was evaporated under a stream of nitrogen to

give the title compound as an oﬀ white solid (11 mg, 0.024 mmol,

35% yield): ¹H NMR (400 MHz, DMSO-d₆, 393 K) δ 8.94−8.88 (m,

1H), 8.56 (d, J = 4.8 Hz, 1H), 7.81−7.77 (m, 2H), 7.77−7.71 (m,

1H), 7.44 (d, J = 8.1 Hz, 1H), 7.27 (dd, J = 7.3, 4.8 Hz, 1H), 6.35 (q,

J = 7.2 Hz, 1H), 3.60 (s, 3H), 3.42−3.30 (m, 2H), 2.19 (s, 3H), 2.08

(d, J = 7.2 Hz, 3H), 2.00 (s, 3H); % ee undetermined; LC−MS

(Method A) [M + H]⁺= 458, t_R0.59 min.

Ethyl 6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-4-oxo-1,4-di-

hydroquinoline-3-carboxylate (51). Diphenyl ether (75 mL) was

heated with stirring to 260 °C (internal). Solid diethyl 2-({[4-

(benzyloxy)-3-(3,5-dimethylisoxazol-4-yl)phenyl]amino}methylene)-

malonate 50 (9.6 g, 20.67 mmol) was added in portions, followed by

diphenyl ether (10 mL). The mixture was stirred for 20 min. The

mixture was allowed to cool to rt and diluted with DCM (20 mL).

The solution was loaded onto a 330 g silica cartridge and puriﬁed

using a gradient of 0−20% MeOH in DCM over 10 column volumes.

The appropriate fractions were combined, and the solvent was

removed by rotary evaporation to give the title compound as a light

1-(Benzyloxy)-2-bromo-4-nitrobenzene (47). To a slurry of NaH

(60% dispersion in mineral oil) (2.8 g, 70.0 mmol) and DMA (60

mL) was added benzyl alcohol (4.75 mL, 45.7 mmol) dropwise with

stirring under nitrogen. The mixture was stirred for 2 h at rt and then

cooled to 0 °C. 2-Bromo-1-ﬂuoro-4-nitrobenzene 46 (11 g, 50.0

mmol) was added to the cooled solution and the mixture stirred for 1

h. The reaction mixture was poured onto sat. NH₄Cl (aq) (80 mL),

stirred for 10 min, ﬁltered, and washed with water. The solid was

triturated with diethyl ether (25 mL) and ﬁltered to give the title

1

brown solid (7.8 g, 18.64 mmol, 90% yield): H NMR (400 MHz,

1

compound as a tan solid (11.25 g, 36.5 mmol, 73% yield): H NMR

DMSO-d₆) δ 12.27 (br s, 1H), 8.55 (s, 1H), 7.82 (s, 1H), 7.53 (s,

1H), 7.40−7.29 (m, 5H), 5.24 (s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 2.30

(s, 3H), 2.10 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); LC−MS (Method A)

(400 MHz, DMSO-d₆) δ 8.45 (d, J = 2.8 Hz, 1H), 8.28 (dd, J = 9.1,

2.8 Hz, 1H), 7.53−7.47 (m, 2H), 7.47−7.40 (m, 3H), 7.40−7.34 (m,

S

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i

[M + H]⁺= 419, t_R0.93 min, 85% purity by LC−MS UV (9%

decarboxylated material present).

mmol) in anhydrous NMP (25 mL) was treated with Pr₂NEt (4.26

mL, 24.40 mmol), followed by (R)-1-phenylethanamine (1.71 mL,

13.42 mmol). The solution was stirred under nitrogen at rt for 2.5 h.

The reaction mixture was diluted with water (50 mL) and extracted

with EtOAc (75 mL). The organic layer was washed with 10% LiCl

(aq) (2 × 50 mL), followed by water (50 mL) and passed through a

hydrophobic frit. The solvent was removed in vacuo and dried in a

vacuum oven to give the title compound as a yellow foam (5.96 g,

12.05 mmol, 99% yield): ¹H NMR (400 MHz, CDCl₃) δ 9.54 (d, J =

7.8 Hz, 1H), 9.27 (s, 1H), 7.70 (s, 1H), 7.53−7.46 (m, 2H), 7.44−

7.37 (m, 3H), 7.36−7.29 (m, 1H), 7.28−7.21 (m, 3H), 7.00−6.95

(m, 2H), 5.32 (dq, J = 7.8, 6.7 Hz, 1H), 4.46 (d, J = 11.8 Hz, 1H),

4.07 (d, J = 11.8 Hz, 1H), 2.20 (s, 3H), 2.07 (s, 3H), 1.69 (d, J = 6.7

Hz, 3H); LC−MS (Method A) [M + H]⁺= 495, t_R1.31 min.

6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-N⁴-[(R)-1-

phenylethyl]quinoline-3,4-diamine (57). Iron powder (5.36 g, 96

mmol) was added to a solution of 6-(benzyloxy)-7-(3,5-dimethylisox-

azol-4-yl)-3-nitro-N-[(R)-1-phenylethyl]quinolin-4-amine 56 (5.93 g,

11.99 mmol) in acetic acid (50 mL). The mixture was stirred in an

open vessel at rt for 1 h. EtOAc (50 mL) was added to the ﬂask and

the mixture stirred for 1 h. The mixture was ﬁltered through a Celite

cartridge, which was washed further with EtOAc (100 mL). The

ﬁltrate was washed with 0.5 M NaOH (aq) (2 × 100 mL) and water

(100 mL). The organic layer was separated and passed through a

hydrophobic frit, and the solvent was removed by rotary evaporation.

The resulting foam was dissolved in MeOH (10 mL) and applied to a

MeOH-preconditioned 50 g aminopropyl cartridge. The cartridge was

washed with MeOH (160 mL) and the solvent evaporated under a

vacuum to give the title compound as a light brown foam (4.62 g, 9.94

mmol, 83% yield): ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.73

(s, 1H), 7.41−7.25 (m, 10 H), 6.99 (s, 1H), 5.02−4.93 (m, 2H), 4.52

(q, J = 6.7 Hz, 1H), 3.69 (br s, 2H), 3.56 (br s, 1H), 2.34 (s, 3H),

2.21 (s, 3H), 1.61 (d, J = 6.7 Hz, 3H); LC−MS (Method A) [M +

H]⁺= 465, t_R0.99 min.

6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-4-oxo-1,4-dihydro-

quinoline-3-carboxylic Acid (52). To ethyl 6-(benzyloxy)-7-(3,5-

dimethylisoxazol-4-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate 51

(7.8 g, 18.64 mmol) were added EtOH (50 mL) and 2 M NaOH

(aq) (30 mL, 60.0 mmol), and the mixture was stirred at 95 °C for 3

h. The reaction was allowed to cool to rt, and the volatiles were

evaporated under a vacuum. The remaining mixture was acidiﬁed to

pH = 1 with 12.5% HCl (aq) (∼30 mL) and the resulting precipitate

isolated by vacuum ﬁltration. The solid was washed with water (200

mL) and diethyl ether (50 mL) and then dried in a vacuum oven

overnight to give the title compound as an oﬀ-white solid (7.2 g,

18.44 mmol, 99% yield): ¹H NMR (400 MHz, DMSO-d₆) δ 15.64 (br

s, 1H), 8.85 (s, 1H), 7.90 (s, 1H), 7.76 (s, 1H), 7.43−7.30 (m, 5 H),

5.31 (s, 2H), 2.31 (s, 3H), 2.12 (s, 3H); LC−MS (Method A) [M +

H]⁺= 391, t_R1.00 min, 82% purity by LC−MS UV (10%

decarboxylated material present).

6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)quinolin-4(1H)-one

(53). Diphenyl ether (70 mL) was heated with stirring to 260 °C

(internal). Solid 6-(benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-4-oxo-

1,4-dihydroquinoline-3-carboxylic acid 52 (7.2 g, 18.44 mmol) was

added in one portion, followed by diphenyl ether (10 mL). The

mixture was stirred for 20 min. The reaction was allowed to cool to rt

and the solution applied to a 330 g silica cartridge with DCM (20

mL). The mixture was puriﬁed using a gradient of 0−20% MeOH in

DCM over 10 column volumes. The appropriate fractions were

combined, and the solvent was removed by rotary evaporation to give

the title compound as a brown solid (5.7 g, 16.46 mmol, 89% yield):

¹H NMR (400 MHz, DMSO-d₆) δ 11.72 (br s, 1H), 7.92−7.86 (m,

1H), 7.75 (s, 1H), 7.44 (s, 1H), 7.41−7.29 (m, 5 H), 6.03 (d, J = 7.2

Hz, 1H), 5.21 (s, 2H), 2.29 (s, 3H), 2.11 (s, 3H); LC−MS (Method

A) [M + H]⁺= 347, t_R0.83 min.

6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-3-nitroquinolin-

4(1H)-one (54). To a 500 mL round-bottomed ﬂask containing 6-

(benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)quinolin-4(1H)-one 53 (5.7

g, 16.46 mmol) was added propionic acid (75 mL, 1002 mmol),

followed by 70% nitric acid (1.6 mL, 35.8 mmol). A suspension

formed, which was stirred at 100 °C for 1.5 h. The reaction mixture

was allowed to cool to rt, ﬁltered under a vacuum, and washed with

cyclohexane (100 mL). The solid was dried in a vacuum oven to give

N-[6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-4-{[(R)-1-

phenylethyl]amino}quinolin-3-yl]-2-methoxyacetamide (58). A

solution of 6-(benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-N⁴-[(R)-1-

phenylethyl]quinoline-3,4-diamine 57 (1.89 g, 4.07 mmol) and

pyridine (0.50 mL, 6.18 mmol) in DCM (20 mL) was cooled to 0

°C and 2-methoxyacetyl chloride (0.41 mL, 4.48 mmol) added. The

mixture was stirred under nitrogen at 0 °C for 10 min and allowed to

warm to rt over 3.5 h. The reaction mixture was washed sequentially

with 0.5 M HCl (aq) (20 mL) and water (2 × 20 mL). The organic

layer was separated and passed through a hydrophobic frit and the

solvent removed by rotary evaporation to give the title compound as a

1

the title compound as a tan solid (3.7 g, 9.45 mmol, 57% yield): H

NMR (400 MHz, DMSO-d₆) δ 12.97 (br s, 1H), 9.18 (s, 1H), 7.91

(s, 1H), 7.63 (s, 1H), 7.42−7.29 (m, 5 H), 5.29 (s, 2H), 2.30 (s, 3H),

2.11 (s, 3H); LC−MS (Method A) [M + H]⁺= 392, t_R0.94 min.

4-[6-(Benzyloxy)-4-chloro-3-nitroquinolin-7-yl]-3,5-dimethyli-

soxazole (55). To 6-(benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-3-

nitroquinolin-4(1H)-one 54 (3.53 g, 9.02 mmol) was added POCl₃

(20 mL, 215 mmol) and the suspension stirred at 100 °C under

nitrogen for 1 h. The reaction mixture was allowed to cool to rt and

evaporated under a vacuum. Toluene (20 mL) was added and the

mixture evaporated. The residue was dissolved in DCM (25 mL), and

sat. NaHCO₃(aq) (25 mL) was added. The mixture was stirred

vigorously for 30 min and the organic layer separated. The aqueous

layer was extracted with DCM (25 mL), and the organics were

combined and passed through a hydrophobic frit. The solvent was

removed under a vacuum, and the resulting solid was triturated with

cyclohexane/diethyl ether (8:2) (40 mL). The solid was dried in

vacuo to give the title compound as a light brown solid (3.54 g, 8.64

mmol, 96% yield): mp 230−232 °C; IR (solid) ν (cm⁻¹) 3130, 1619,

1556, 1526, 1420, 1381, 1331, 1227, 1023, 739; ¹H NMR (400 MHz,

CDCl₃) δ 9.16 (s, 1H), 8.01 (s, 1H), 7.82 (s, 1H), 7.47−7.33 (m, 5

H), 5.32 (s, 2H), 2.37 (s, 3H), 2.23 (s, 3H); ¹³C NMR (100 MHz,

CDCl₃) δ 167.1, 159.3, 157.3, 144.9, 142.6, 141.7, 135.0, 134.2, 133.0,

129.2, 128.9 (2 C), 128.7, 127.4 (2 C), 126.7, 111.7, 104.7, 71.3, 12.0,

10.9; LC−MS (Method A) [M + H]⁺= 410, 412, t_R1.32 min; HRMS

[M + H]⁺calcd for C₂₁H₁₇³⁵ClN₃O₄410.0902, found 410.0897.

6-(Benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-3-nitro-N-[(R)-1-

phenylethyl]quinolin-4-amine (56). A solution of 4-[6-(benzyloxy)-

4-chloro-3-nitroquinolin-7-yl]-3,5-dimethylisoxazole 55 (5 g, 12.20

1

light brown foam (2.14 g, 3.99 mmol, 98% yield): H NMR (400

MHz, CDCl₃) δ 8.80 (s, 1H), 8.25 (s, 1H), 7.82 (s, 1H), 7.40−7.29

(m, 8 H), 7.26−7.21 (m, 3H), 4.92−4.85 (m, 2H), 4.62 (dq, J = 8.6,

6.8 Hz, 1H), 4.27 (d, J = 8.6 Hz, 1H), 4.10 (s, 2H), 3.53 (s, 3H), 2.34

(s, 3H), 2.21 (s, 3H), 1.61 (d, J = 6.8 Hz, 3H); LC−MS (Method A)

[M + H]⁺= 537, t_R0.91 min.

4-{8-(Benzyloxy)-2-(methoxymethyl)-1-[(R)-1-phenylethyl]-1H-

imidazo[4,5-c]quinolin-7-yl}-3,5-dimethylisoxazole (59). A solution

of N-[6-(benzyloxy)-7-(3,5-dimethylisoxazol-4-yl)-4-{[(R)-1-

phenylethyl]amino}quinolin-3-yl]-2-methoxyacetamide 58 (2.14 g,

3.99 mmol) in propionic acid (6.0 mL, 80 mmol) was stirred at 140

°C under nitrogen for 2 h. The reaction mixture was allowed to cool

to rt and the solvent removed in vacuo. The residue was dissolved in

DCM (25 mL) and washed with 1 M NaOH (aq) (25 mL). The

aqueous layer was extracted with DCM (25 mL), and the organic

layers were combined and passed through a hydrophobic frit. The

solvent was removed by rotary evaporation, and the foam was

dissolved in DCM (5 mL) and puriﬁed on a 100 g silica cartridge

using a gradient of 0−15% MeOH in DCM over 12 column volumes.

The appropriate fractions were combined, and the solvent was

removed by rotary evaporation to give the title compound as a light

1

brown foam (1.87 g, 3.61 mmol, 90% yield): H NMR (400 MHz,

DMSO-d₆, 393 K) δ 9.15 (s, 1H), 7.96 (s, 1H), 7.46−7.30 (m, 8 H),

7.24−7.20 (m, 2H), 7.09 (s, 1H), 6.47 (q, J = 7.1 Hz, 1H), 4.88−4.85

(m, 2H), 4.80 (d, J = 11.9 Hz, 1H), 4.41 (d, J = 11.9 Hz, 1H), 3.40 (s,

T

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3H), 2.24 (s, 3H), 2.07−2.03 (m, 6 H); LC−MS (Method A) [M +

H]⁺= 519, t_R1.08 min.

ammonia in MeOH solution (30 mL). The basic wash was evaporated

in vacuo to give the title compound as an oﬀ-white solid (150 mg,

0.293 mmol, 96% yield): mp 130−132 °C; [α]²_D¹−27 (c 0.5, CHCl₃);

IR (solid) ν (cm⁻¹) 2929, 1618, 1495, 1444, 1393, 1367, 1215, 1095;

¹H NMR (400 MHz, DMSO-d₆, 393 K) δ 9.13 (s, 1H), 7.92 (s, 1H),

7.44−7.37 (m, 2H), 7.37−7.29 (m, 3H), 6.95 (s, 1H), 6.48 (q, J = 7.0

Hz, 1H), 4.91 (d, J = 13.0 Hz, 1H), 4.87 (d, J = 13.0 Hz, 1H), 3.65

(dd, J = 9.3, 6.3 Hz, 1H), 3.41 (s, 3H), 3.19−3.12 (m, 1H), 2.92−

2.81 (m, 3H), 2.44 (dd, J = 10.6, 6.0 Hz, 1H), 2.34−2.22 (m, 4H),

2.11 (d, J = 7.0 Hz, 3H), 2.07 (s, 3H), 1.85−1.74 (m, 1H), 1.40−1.29

(m, 1H), NH not resolved; ¹³C NMR (100 MHz, DMSO-d₆, 393 K)

δ 165.2, 158.3, 153.9, 151.8, 142.7, 139.7, 139.6, 137.1, 132.5, 132.4,

128.6 (2C), 127.0, 125.1 (2C), 120.2, 117.4, 111.8, 104.1, 71.3, 66.8,

57.5, 54.1, 49.4, 45.6, 38.1, 28.5, 18.7, 10.6, 9.5; >99% de determined

by HPLC analysis on a Chiralpak AD column (250 × 4.6 mm, 10

μm), elution with 20% EtOH in heptane containing 0.1% isopropyl-

amine, ﬂow rate 1 mL/min, UV detection at 235 nm; LC−MS

(Method A) [M + H]⁺= 512, t_R0.59 min; HRMS [M + H]⁺calcd for

C₃₀H₃₄N₅O₃512.2656, found 512.2650.

Mutant TR-FRET Assays. Compounds were screened against N-

terminal 6His-tagged single mutant tandem bromodomain proteins in

a dose−response format using an Alexa Fluor 647 derivative of I-

BET762 (63, Supporting Information, Method S2ii). Compounds

were titrated from 10 mM in 100% DMSO and 100 nL transferred to

a low volume black 384-well microtiter plate using a Labcyte Echo

555. A Thermo Scientiﬁc Multidrop Micro was used to dispense 5 μL

of 10 nsM protein in 50 mM HEPES, 50 mM NaCl, and 1 mM

CHAPS, pH 7.4, in the presence of 50 nM compound 63 (∼K_d

concentration for the interaction between all BET single mutant

tandem bromodomain proteins and 63, except for BRDT Y66A,

which used 200 nM). After equilibrating for 1 h in the dark at rt, the

bromodomain protein/ﬂuorescent ligand interaction was detected

using TR-FRET, following a 5 μL addition of 1.5 nM europium

chelate labeled anti-6His antibody (PerkinElmer, W1024, AD0111) in

assay buﬀer. Time resolved ﬂuorescence (TRF) was then detected on

a TRF laser equipped PerkinElmer Envision multimode plate reader

(excitation = 337 nm; emission 1 = 615 nm; emission 2 = 665 nm;

dual wavelength bias dichroic = 400 nm, 630 nm). The TR-FRET

ratio was calculated using the following equation: ratio = ((acceptor

ﬂuorescence at 665 nm)/(donor ﬂuorescence at 615 nm)) × 1000.

The TR-FRET ratio data was normalized to a mean of 16 replicates

per microtiter plate of both 10 μM I-BET151 and 1% DMSO controls

and IC₅₀values determined for each of the compounds tested by

ﬁtting the ﬂuorescence ratio data to a four parameter model: y = a +

((b−a)/(1 + (10^x/10^c)^d), where a is the minimum, b is the Hill slope,

c is the IC₅₀, and d is the maximum. All compounds were screened

against BRD4 (1−477) Y97A and Y390A with selected compounds

also screened against BRD2 (1−473) Y113A and Y386A, BRD3 (1−

435) Y73A and Y348A, and BRDT (1−397) Y66A and Y309A.

Mouse Pharmacokinetic (PK) Studies. Experimental Protocol. All

animal studies were ethically reviewed and carried out in accordance

with Animals (Scientiﬁc Procedures) Act 1986 and the GlaxoSmithK-

line (GSK) Policy on the Care, Welfare, and Treatment of Laboratory

Animals. Mice were individually housed in a plastic solid-bottom cage

and had free access to food (5LF2 EURodent Diet 14% supplied by

PMI Labdiet, Richmond, Indiana, USA) and water. There were no

known contaminants in the diet or water at concentrations that could

interfere with the outcome of this study. Temperature and humidity

7-(3,5-Dimethylisoxazol-4-yl)-2-(methoxymethyl)-1-[(R)-1-phe-

nylethyl]-1H-imidazo[4,5-c]quinolin-8-ol (60). A solution of 4-{8-

(benzyloxy)-2-(methoxymethyl)-1-[(R)-1-phenylethyl]-1H-imidazo-

[4,5-c]quinolin-7-yl}-3,5-dimethylisoxazole 59 (1.81 g, 3.49 mmol) in

EtOH (20 mL) was added to 5% palladium on carbon (180 mg, 1.692

mmol) and stirred at rt under atmospheric hydrogen for 15 h. The

reaction mixture was ﬁltered through Celite and the cake washed with

EtOH (50 mL). The ﬁltrate was evaporated under a vacuum and the

residue dissolved in EtOH (40 mL). The solution was added to 5%

palladium on carbon (360 mg, 3.38 mmol) and stirred at rt under

atmospheric hydrogen for 20 h. The reaction mixture was ﬁltered

through Celite and the cake washed with EtOH (50 mL). The ﬁltrate

was evaporated under a vacuum and the residue dissolved in DCM (8

mL). The solution was loaded onto a 220 g silica cartridge and

puriﬁed using a gradient of 0−15% MeOH in DCM over 10 column

volumes. The appropriate fractions were combined, and the solvent

was removed by rotary evaporation to give an oﬀ-white solid, which

was puriﬁed by MDAP (Method B). The appropriate fractions were

combined, and the solvent was removed by rotary evaporation to give

the title compound as a light-yellow solid (923 mg, 2.154 mmol, 62%

1

yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ 9.50 (br s, 1H),

9.04 (s, 1H), 7.89 (s, 1H), 7.45−7.23 (m, 6H), 6.51 (q, J = 7.1 Hz,

1H), 4.69 (d, J = 12.9 Hz, 1H), 4.62 (d, J = 12.9 Hz, 1H), 3.33 (s,

3H), 2.31 (s, 3H), 2.17 (d, J = 7.1 Hz, 3H), 2.14 (s, 3H); LC−MS

(Method A) [M + H]⁺= 429, t_R0.72 min.

Fractions containing a minor peak were combined, and the solvent

was removed by rotary evaporation to give byproduct (R)-1-{2-

(methoxymethyl)-8-methyl-1-(1-phenylethyl)-1H-furo[2,3-g]imidazo-

[4,5-c]quinolin-7-yl}ethanone 63 as a light-yellow foam (129 mg,

1

0.312 mmol, 9% yield): H NMR (400 MHz, DMSO-d₆, 393 K) δ

9.24 (s, 1H), 8.71 (s, 1H), 7.70 (s, 1H), 7.44−7.36 (m, 2H), 7.36−

7.25 (m, 3H), 6.57 (q, J = 7.1 Hz, 1H), 4.82 (s, 2H), 3.38 (s, 3H),

2.79 (s, 3H), 2.67 (s, 3H), 2.13 (d, J = 7.1 Hz, 3H); LC−MS

(Method A) [M + H]⁺= 414, t_R0.81 min.

(3S)-tert-Butyl 3-[({7-(3,5-Dimethylisoxazol-4-yl)-2-(methoxy-

methyl)-1-[(R)-1-phenylethyl]-1H-imidazo[4,5-c]quinolin-8-yl}oxy)-

methyl]pyrrolidine-1-carboxylate (61). A mixture of 7-(3,5-dime-

thylisoxazol-4-yl)-2-(methoxymethyl)-1-[(R)-1-phenylethyl]-1H-

imidazo[4,5-c]quinolin-8-ol 60 (233 mg, 0.544 mmol), potassium

carbonate (107 mg, 0.778 mmol), and (S)-tert-butyl 3-

{[(methylsulfonyl)oxy]methyl}pyrrolidine-1-carboxylate (190 mg,

0.680 mmol) in anhydrous DMF (1.2 mL) was stirred at 100 °C

under nitrogen for 3 h. The reaction mixture was allowed to cool to rt,

diluted with 10% LiCl (aq), (10 mL) and extracted with EtOAc (3 ×

10 mL). The organic layers were combined and passed through a

hydrophobic frit. The solvent was removed by rotary evaporation and

the residue puriﬁed by MDAP (Method B). The appropriate fractions

were combined and evaporated in vacuo to give the title compound as

1

an oﬀ-white solid (191 mg, 0.312 mmol, 57% yield): H NMR (400

MHz, DMSO-d₆, 373 K) δ 9.15 (s, 1H), 7.93 (s, 1H), 7.44−7.38 (m,

2H), 7.37−7.31 (m, 3H), 6.92 (s, 1H), 6.49 (q, J = 7.0 Hz, 1H), 4.93

(d, J = 13.0 Hz, 1H), 4.88 (d, J = 13.0 Hz, 1H), 3.71 (dd, J = 9.4, 6.4

Hz, 1H), 3.41 (s, 3H), 3.39−3.25 (m, 3H), 3.13−3.07 (m, 1H),

2.93−2.91 (m, 1H), 2.49−2.41 (m, 1H), 2.27 (s, 3H), 2.11 (d, J = 7.0

Hz, 3H), 2.08 (s, 3H), 2.00−1.89 (m, 1H), 1.65−1.54 (m, 1H), 1.46

(s, 9H); LC−MS (Method A) [M + H]⁺= 612, t_R1.08 min; HRMS

[M + H]⁺calcd for C₃₅H₄₂N₅O₅612.3181, found 612.3170.

4-{2-(Methoxymethyl)-1-[(R)-1-phenylethyl]-8-[(S)-pyrrolidin-3-

ylmethoxy]-1H-imidazo[4,5-c]quinolin-7-yl}-3,5-dimethylisoxazole

(62). A solution of (3S)-tert-Butyl 3-[({7-(3,5-dimethylisoxazol-4-yl)-

2-(methoxymethyl)-1-[(R)-1-phenylethyl]-1H-imidazo[4,5-c]-

quinolin-8-yl}oxy)methyl]pyrrolidine-1-carboxylate 61 (186 mg,

0.304 mmol) in anhydrous 1,4-dioxane (2 mL) was treated with 4

M HCl in 1,4-dioxane (2 mL, 8.00 mmol) and the mixture allowed to

stand in a stoppered vessel for 1 h. The reaction mixture was

evaporated in vacuo and the solid dissolved in MeOH (2 mL). The

solution was applied to a MeOH-preconditioned 5 g SCX-2 cartridge,

which was then washed with MeOH (30 mL), followed by 2 M

were nominally maintained at 21 °C

2 °C and 55%

10%,

respectively. Three male CD1 mice (30−33 g, supplied by Charles

River U.K. Ltd.) each received an intraperitoneal administration of 62

formulated in 10% Kleptose in saline at a concentration of 2 mg/mL

and administered at 5 mL/kg to achieve a target dose of 10 mg/kg.

The pH of the dose formulation was adjusted to 5 using 2 M HCl

(aq). Following dose administration, serial blood samples (ca. 20 μL)

were collected up to 12 h after the start of dosing, via direct puncture

of the tail vein, a terminal sample was taken at 24 h post dose via

cardiac puncture under terminal isoﬂurane anesthesia. Blood samples

were collected into blank tubes, diluted with an equal volume of

U

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Article

puriﬁed water, and stored at −20 °C prior to analysis by LC−MS/

MS.

developability and cross screening panel data for

compound 62 (PDF)

Blood Sample Analysis. Diluted blood samples were extracted

using protein precipitation with 250 μL of acetonitrile containing an

analytical internal standard. An aliquot of the supernatant was

analyzed by reverse-phase LC−MS/MS in positive ion mode. Samples

were assayed against calibration standards prepared in the control

blood.

Accession Codes

The atomic coordinates of 62 bound to BRD4 BD1 and BRD2

BD2 have been deposited with the Protein Data Bank under

the accession codes 6swn and 6swo, respectively.²⁵

PK Data Analysis. PK proﬁles were obtained from the blood

concentration−time proﬁles using noncompartmental analysis with

WinNonlin Phoenix 6.3 (Certara, Princetown, NJ).

AUTHOR INFORMATION

Corresponding Author

■

Fraction Unbound in Blood. Control blood from CD1 mice was

obtained on the day of experimentation from in house GSK stock

animals. Control human blood was obtained on the day of

experimentation from a single nonmedicated consenting donor from

an in-house GSK blood donation unit. The human biological samples

were sourced ethically, and their research use was in accordance with

the terms of the informed consent. The fraction unbound was

determined using rapid equilibrium dialysis technology (RED plate

[Linden Bioscience, Woburn, MA]) at concentrations of 200 ng/mL

and 1000 ng/mL. Blood was dialyzed against phosphate buﬀered

saline solution incubating the dialysis units at 37 °C for 4 h. Following

incubation, aliquots of blood and buﬀer were matrix matched prior to

analysis by LC−MS/MS. The unbound fraction was determined using

the peak area ratios in buﬀer and in blood.

Prediction of the In Vivo Response (Target Engagement) for

Compound 62 in the Male CD1 Mouse. Mean (n = 3 mice) blood

concentration−time proﬁles adjusted for the free fraction in mouse

blood (0.08) were transformed using an inhibitory sigmoid I_maxmodel

incorporating in vitro eﬃcacy data to simulate response-time (target

engagement) proﬁles for compound 62 following ip administration at

10 mg/kg.

Christopher R. Wellaway − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom;

orcid.org/0000-0003-0077-3452;

Email: christopher.x.wellaway@gsk.com

Authors

Paul Bamborough − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom;

orcid.org/0000-0001-9479-2894

Sharon G. Bernard − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

Chun-wa Chung − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom; orcid.org/0000-

0002-2480-3110

Peter D. Craggs − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Leanne Cutler − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Emmanuel H. Demont − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom;

orcid.org/0000-0001-7307-3129

The inhibitory sigmoid I_maxmodel was deﬁned as

I_max× C^γ

response =

IC₅^γ₀+ C^γ

John P. Evans − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

where I_maxis the maximum induced drug eﬀect, IC₅₀is the unbound

drug concentration at 50% of maximal eﬀect, C is the blood

concentration, and γ is the sigmoidicity factor or hill slope.

The IC₅₀and hill slope were determined from in vitro studies

investigating the inhibition of IL-6 production in LPS-stimulated

human whole blood or from BRD4 BD1 and BD2 mutant TR-FRET

assays. The IC₅₀determined in human whole blood was adjusted for

the free fraction (0.17), and it was assumed that potency in human

blood reﬂects potency in mouse blood. The TR-FRET assays were

assumed to be protein free, and no adjustment for free fraction was

made.

Laurie Gordon − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Bhumika Karamshi − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

Antonia J. Lewis − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Matthew J. Lindon − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

Darren J. Mitchell − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

Inmaculada Rioja − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

Peter E. Soden − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Simon Taylor − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Robert J. Watson − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Rob Willis − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

James M. Woolven − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00566.

■

sı

Molecular formula strings and biological data for ﬁnal

compounds (CSV)

Protein production, determination of binding constants

of ﬂuorescent ligand 63 in wild type and mutant tandem

BET proteins, NMR spectra, synthesis and character-

ization of compound 45, LC−MS traces of compound

62, diastereomeric purity of 62 by chiral HPLC, BET

and BRD4 mutant TR-FRET data tables for tested

compounds, magniﬁed view of the crystal structure of 62

in BRD4 BD1 displaying distances, angles and B-factors

for interactions of the pyrrolidine group, SPR exper-

imental and sensorgrams, bromodomain binding assay

protocol, data and concentration−response curves,

nanoBRET method and concentration−response curves

for compound 62, protocols for phenotypic assays,

́

Beata S. Wyspianska − GSK, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, United Kingdom

William J. Kerr − Department of Pure and Applied Chemistry,

WestCHEM, University of Strathclyde, Glasgow G1 1XL,

United Kingdom; orcid.org/0000-0002-1332-785X

Rab K. Prinjha − GSK, Medicines Research Centre, Stevenage,

Hertfordshire SG1 2NY, United Kingdom

Complete contact information is available at:

V

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Suto, R. K.; Wong, N. C. W.; Wagner, G. S.; Hansen, H. C.; Young, P.

R. RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain

antagonist. PLoS One 2013, 8, No. e83190.

https://pubs.acs.org/10.1021/acs.jmedchem.0c00566

Author Contributions

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K.; Joshua, J.; Zeng, L.; Mujtaba, S.; Ohlmeyer, M.; Zhou, M.-M.

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BET bromodomains. J. Med. Chem. 2013, 56, 9251−9264.

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chemical modulation of gene transcription favors oligodendrocyte

lineage progression. Chem. Biol. 2014, 21, 841−854.

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Demont, E. H.; Gordon, L. J.; Lindon, M.; Prinjha, R. K.; Watson, A.

J. B.; Hirst, D. J. Discovery of tetrahydroquinoxalines as

bromodomain and extra-terminal domain (BET) inhibitors with

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Wang, L.; Sehgal, V.; Lin, X.; Huang, X.; Lu, X.; Uziel, T.; Hessler, P.;

Lam, L. T.; Bellin, R. J.; Mehta, G.; Fidanze, S.; Pratt, J. K.; Liu, D.;

Hasvold, L. A.; Sun, C.; Panchal, S. C.; Nicolette, J. J.; Fossey, S. L.;

Park, C. H.; Longenecker, K.; Bigelow, L.; Torrent, M.; Rosenberg, S.

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The manuscript was written with contributions from all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

́

David Hemming, Abigail Lucas, and Frederic Donche are

warmly acknowledged for their laboratory assistance. We are

indebted to Steve Richards and Sean Lynn for their NMR

assistance, Eric Hortense for conducting chiral HPLC analysis,

Richard Gregory for generating the fraction unbound in blood

data, Davina Angell for assistance in producing ﬁgures, and

David House for proof-reading.

ABBREVIATIONS USED

■

AML, acute myeloid leukemia; ApoA1, apolipoprotein A1;

BCP, bromodomain-containing protein; BET, bromodomain

and extraterminal domain; BRD2/3/4, bromodomain-contain-

ing protein 2/3/4; CREBBP, cyclic adenosine monophosphate

response element binding protein binding protein; IL-6,

interleukin 6; ITC, isothermal titration calorimetry; KAc,

acetyl-lysine; MCP-1, monocyte chemotactic protein-1;

MDAP, mass-directed autopreparative HPLC; MLL, mixed-

lineage leukemia; NMC, NUT midline carcinoma; NUT,

nuclear protein in testis; PAFc, polymerase-associated factor

complex; PEPPSI, pyridine enhanced precatalyst preparation

stabilization and initiation; PBMC, peripheral blood mono-

nuclear cell; Pol II, RNA polymerase II; P-TEFb, positive

transcription elongation factor b; PTM, post-translational

modiﬁcation; qPCR, quantitative real-time polymerase chain

reaction; SEC, superelongation complex; SPR, surface plasmon

resonance; TNFα, tumor necrosis factor α; TR-FRET, time-

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T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.;

Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.-M.;

Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel,

H. K.; Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase

interaction map for clinical kinase inhibitors. Nat. Biotechnol. 2005,

23, 329−336.

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Structure-Based Design of a Bromodomain and Extraterminal Domain (BET) Inhibitor Selective for the N-Terminal Bromodomains That Retains an Anti-inflammatory and Antiproliferative Phenotype

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