Scholarly article on 4-(8-methoxy-1-((S)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole 1422554-35-5 from Journal of Medicinal Chemistry p. 12200

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Discovery of a Novel Bromodomain and Extra Terminal Domain

(BET) Protein Inhibitor, I‑BET282E, Suitable for Clinical Progression

Published as part of the Journal of Medicinal Chemistry special issue “Epigenetics 2022”.

Katherine L. Jones,* Dominic M. Beaumont, Sharon G. Bernard, Rino A. Bit, Simon P. Campbell,

Chun-wa Chung, Leanne Cutler, Emmanuel H. Demont, Kate Dennis, Laurie Gordon, James R. Gray,

Michael V. Haase, Antonia J. Lewis, Scott McCleary, Darren J. Mitchell, Susanne M. Moore, Nigel Parr,

Olivia J. Robb, Nicholas Smithers, Peter E. Soden, Colin J. Suckling, Simon Taylor, Ann L. Walker,

Robert J. Watson, and Rab K. Prinjha

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ABSTRACT: The functions of the bromodomain and extra terminal (BET) family of proteins have been implicated in a wide range

of diseases, particularly in the oncology and immuno-inﬂammatory areas, and several inhibitors are under investigation in the clinic.

To mitigate the risk of attrition of these compounds due to structurally related toxicity ﬁndings, additional molecules from distinct

chemical series were required. Here we describe the structure- and property-based optimization of the in vivo tool molecule I-

BET151 toward I-BET282E, a molecule with properties suitable for progression into clinical studies.

into several families, one of which is the “bromodomain (B)

and extra−terminal (ET)”, or BET, proteins. In humans, there

are four BET proteins (BRD2, BRD3, BRD4, and BRDT)

which exhibit similar gene arrangements, domain organiza-

tions, and some functional properties; each of these proteins

contains two bromodomains (BD1 and BD2) and one ET

domain.⁶The four BET proteins have all been shown to bind

acetylated lysines, although their preference for which lysine

mark, and on which histone, diﬀers. There is over 75%

homology between each of the N-terminals (BD1) and each of

the C-terminals (BD2) across the BET family proteins,

although only approximately 44% homology between the N-

terminal and C-terminal bromodomain within the same

INTRODUCTION

■

As a result of a wide range of disease-relevant biology, there

has been signiﬁcant interest in the inhibition of functions of

the bromodomain and extra terminal (BET) family of proteins

by small molecule inhibitors of their bromodomains.

Bromodomains are ∼110 amino acid structural domains

within proteins that were ﬁrst reported in 1992.^1,2Structural

and site-directed mutagenesis studies of the bromodomain of

P300/CBP-associated factor (PCAF) provided evidence that

the bromodomain interacts speciﬁcally with acetylated lysine

residues, an epigenetic histone modiﬁcation that is important

for transcription.³The acetylation of lysine residues on histone

tails has a direct eﬀect on the structure of chromatin. This is

due to the neutralization of the charge present on lysine amino

acid side chains at physiological pH. This results in activated

states of chromatin, as the DNA is more accessible to

transcription factors.⁴

Received: May 11, 2021

Published: August 13, 2021

There are 46 known human bromodomain-containing

proteins, which contain a total of 61 bromodomain sequence

domains.⁵Proteins containing bromodomains can be classiﬁed

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Figure 1. Selected BET bromodomain inhibitors currently being progressed in clinical trials.

Figure 2. I-BET151 bound to the N-terminal bromodomain of BRD4 with key residues labeled (PDB: 3ZYU, cyan). (a) I-BET151 shown in stick

format overlaid with acetylated lysine residue of peptide from PDB: 2DVQ (green). (b) Solvent-accessible protein surface of BRD4 with WPF shelf

highlighted. (PDB: 3ZYU) overlaid with acetylated lysine residue of peptide from PDB: 2DVQ.

protein.⁷However, in all cases, the amino acid residues critical

for binding acetylated lysine residues are conserved.

(+)-JQ1 was shown to disrupt chromatin complexes

responsible for the expression of key inﬂammatory genes in

activated macrophages. Additionally, in an in vivo model of

LPS-induced endotoxic shock, administration of I-BET762

both 1 h prior and 1.5 h after stimulation prevented or delayed

the death of the mice.

Outside the oncology and immuno-inﬂammatory areas, it

has been shown that BRD4 has a role in virus-induced

pathogenesis and is a potential target for drug development

with a view to controlling HPV-induced human diseases,

including genital warts, skin tumors, and cervical cancers.^16,17

In addition, whole-body disruption of BRD2 in mice led to

severe obesity while avoiding the development of Type 2

diabetes and enhancing glucose tolerance. This suggests that

the BET family of proteins may also be relevant in metabolic

diseases.¹⁸

Published research has provided a rationale for targeting

BET proteins in cancer. A cell-penetrant small molecule

inhibitor of the BET family of bromodomains, (+)-JQ1, was

shown to have antitumor eﬃcacy in xenograft models of

nuclear protein in testes (NUT) midline carcinoma (NMC).⁸

NMC is characterized by a recurrent t(15;19) chromosomal

translocation and the expression of a fusion protein containing

NUT and BRD3/4. Subsequent to the initial research on

(+)-JQ1, it has been shown that BET inhibitors may have

therapeutic potential in a wider range of cancers. BET

inhibitors have been shown to down regulate key genes in

cell-proliferation such as c-Myc, and have shown eﬃcacy in in

vivo models of multiple myeloma, prostate cancer, neuro-

blastoma, and leukemia.⁹⁻¹⁴

As well as their potential utility as anticancer agents, strong

evidence has also been provided for targeting BET

bromodomains in inﬂammation. Work within our own

laboratories showed that a potent, selective inhibitor of the

BET family of bromodomains, I-BET762 (1, Figure 1), has

anti-inﬂammatory potential.¹⁵I-BET762, which has a similar

structure, as well as in vitro potency and selectivity proﬁle, to

A number of BET bromodomain inhibitors have already

been reported as having progressed to clinical trials (Figure

1).^19,20Those with disclosed chemical structures included I-

BET762 (1) from within our laboratories,²¹the structurally

related Oncoethix inhibitor OTX015 (2),²²and the structur-

ally distinct Abbvie inhibitor ABBV-075 (3)²³being

progressed for oncology indications, and the structurally

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Figure 3. Medicinal chemistry design strategy for optimization of I-BET151.

distinct pan-BD2 biased Resverlogix inhibitor RVX-208

(4),^24,25being assessed in a range of cardiovascular diseases.

Recent literature has begun to elucidate the phenotype of

inhibitors of only the ﬁrst or second bromodomains of the

BET proteins.²⁶However, given the wide range of profound

Table 1. Comparison of the Developability Proﬁles of I-

BET151 and I-BET762^21,28

I-BET762

I-BET151

(1)

(5)

clogP

PBMC pIC₅₀

HWB pIC₅₀

2.4

6.5

6.2

2.3

6.7

5.9

eﬀects across biological pathways relevant to many diﬀerent

therapeutic areas, the potential of pan-BET bromodomain

inhibition remains worthy of serious investigation. To enhance

the probability of success in providing novel medicines to

patients, multiple clinically enabled molecules of diverse

chemotypes are of signiﬁcant value.²⁷This helps to minimize

the risk of oﬀ-target toxicity associated with any clinical

candidate (since the selectivity proﬁle of any molecule cannot

be fully assessed via proteomics) or any developability issue

associated with the compound itself (such as a reactive

metabolite, lack of a stable crystalline form, limited solubility,

or a risk of genotoxicity). In this manuscript, we describe the

identiﬁcation of I-BET282E, a structurally distinct pan-BET

bromodomain inhibitor with properties suitable for pro-

gression into clinical studies.

a

FaSSIF solubility (μg/mL)

CYP inhibition IC₅₀(μM)

122

>50

<0.86

66

9.9

9.7

<0.86

2C9

3A4 (DEF)

rat

b

CL_i(hepatocytes, mL/min/g

liver)

dog

<1.7

9.8

human

<0.86

a

b

LPS-stimulated PMBC/HWB assay, measuring IL-6. DEF refers to

the probe substrate used to determine the CYP3A4 inhibition proﬁle.

clinical development. A potential strategy to achieve this would

be through lowering the aromatic ring count, with no increase

in lipophilicity (as measured by clogP).^35,36Compound 5

shows low turnover in rat hepatocytes which translates into

good exposure following oral administration in vivo.²⁸

Turnover of compound 5 in human hepatocytes is below the

lowest level of quantiﬁcation of the assay, which is

encouraging. This is in sharp contrast to data in the dog

which indicated the likelihood that high hepatic clearance will

be seen in vivo. This will have to be discharged as it will be

essential to use this species in preclinical safety studies to

enable comparison with the data generated for I-BET762. It is

also of note that the modest CYP activity of I-BET151 (5) will

require improvement to minimize the risk of drug−drug

interactions in clinical settings.

RESULTS AND DISCUSSION

■

In parallel with our clinical investigations with I-BET762,

chemotypes with alternative structural features acting as the

acetyl lysine mimetic were developed. Key among these were

compounds of the dimethylisoxazole family, exempliﬁed by I-

BET151 (5, Figure 3).²⁸Here, the dimethylisoxazole moiety

acted as the acetyl lysine mimetic, with the pyridine bound

within the WPF shelf and the quinoline ring system in the ZA

channel. The dimethylisoxazole motif is widely established

within small molecule bromodomain inhibitors, most exten-

sively in the BET²⁹⁻³³and CBP³⁴proteins.

Compound Design and Biological Activities. Key

parameters of the proﬁle of I-BET151 (5, Figure 3) and I-

BET762 (1, Figure 1) are compared in Table 1. As previously

reported,²⁸I-BET151 is a molecule with encouraging cellular

potency, as measured in peripheral blood mononuclear cells

(PBMCs), but its activity in the human whole blood assay can

be improved upon. This could be achieved either by increasing

biochemical potency or reducing the drop-oﬀ from the

biochemical to cellular assays. Solubility in the biologically

relevant fasted state simulated intestinal ﬂuid (FaSSIF) assay is

limited, and values much greater than 100 μg/mL from

crystalline material would be targeted to mitigate attrition in

As the protein target was not known at the time the

medicinal chemistry to discover I-BET151 was performed, this

compound was optimized from the 7-isoxazoquinoline series

without access to X-ray crystallographic data. With access to

the X-ray crystal structure of I-BET151 bound to the N-

terminal bromodomain of BRD4 (Figure 2), we devised a

medicinal chemistry strategy for further optimization.

Inspection of the X-ray data, and of previous SAR, identiﬁed

the areas of binding which contributed to potency. The

dimethylisoxazole of I-BET151 bound into the acetylated

lysine binding site of the bromodomain via a bridging water

molecule to the hydroxyl of Tyr97.^29,30This structural water

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Table 2. R¹-Subsituent SAR on the I-BET151 Core

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Table 2. continued

a

b

c

Expressed as a mean of at least two experimental replicates. LLE is calculated as BRD4-BD1 pIC₅₀− clogP. Compounds are a 1:1 mixture of

enantiomers.

Table 3. R²-Subsituent SAR on the Des-Methyl I-BET151 Core

a

b

Expressed as a mean of at least two experimental replicates. LLE is calculated as BRD4-BD1 pIC₅₀− clogP.

was part of a network of water molecules in the acetylated

lysine binding site. As this was a critical interaction for potency,

the dimethylisoxazole was not modiﬁed. The pyridine group is

bound into the lipophilic “WPF shelf” region of the protein

which consists of the side chains of Trp81 (W), Pro82 (P), and

Phe83 (F). To access the WPF shelf, the vector from the

imidazoquinoline core was critical, and therefore we did not

make any alterations to the triaryl core structure. However,

although this was identiﬁed as a lipophilic region, the X-ray

structure did not suggest any reason why the substituent

interacting with the WPF shelf needed to be aromatic. This

was therefore an area open to optimization, for example, by

replacement of the pyridine with cyclic and acyclic alkyl, ether,

and amine substituents. The crystallographic data also

identiﬁed the imidazoquinolinone 2-position as a vector

where further substitution would be tolerated; therefore, this

could be investigated as a handle for modulating the

physicochemical properties of the molecule. Indeed, as recently

published work carried out concurrently within our labo-

ratories shows, substitution with a small ether group in this

position increased potency.³⁷The medicinal chemistry strategy

was to optimize both substituents independently, followed by

the combination of favored groups into compounds which

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Table 4. Optimized Molecules Combining Favored R¹- and R²-Substituents

a

b

c

d

Expressed as a means of at least two experimental replicates. LLE is calculated as BRD4-BD1 pIC₅₀− clogP. Not tested. Compounds are a 1:1

e

mixture of enantiomers. Compound 25 from Table 3 included to show HWB data.

would be assessed as potential preclinical candidate molecules

(Figure 3).

The substituents designed for the imidazoquinolone R¹-

position were chosen to reduce the aromatic ring count by

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replacement of the pyridine (Table 2). Because of the

lipophilicity of the WPF shelf, several small alkyl (compounds

6−9) and ether (compounds 10−15) substituents were

designed. The biochemical potency of compounds at the ﬁrst

bromodomain of BRD4 was measured using a TR-FRET assay,

where the second bromodomain of BRD4 was mutated

(Y390A) to prevent compound binding. Lipophilic ligand

eﬃciencies (LLEs)³⁸of the alkyl compounds 6−9 were

signiﬁcantly lower than that of I-BET151; all LLEs were

between 2.6 and 2.9, indicating that potency was driven by

lipophilic interactions. Compound 10, the ether analogue of

compound 8, had reduced potency; however, there was a

signiﬁcant increase in LLE from 2.6 to 4.0. This higher LLE

was consistent across all ethers, with the 4-tetrahydropyranyl

analogues 13 and 14 standing out in terms of higher potency.

Also synthesized were a small number of amines (compounds

16−18) as the pyridine of I-BET151 suggested a basic center

could be tolerated in the WPF shelf region of the protein.

However, these compounds all showed a signiﬁcant reduction

in potency over I-BET151, and basic substituents were not

investigated further. The key advantage of non-aryl R¹-

substituents was seen in the LPS-stimulated PBMC assay,

where in almost all examples there was a lower drop-oﬀ

between the biochemical and cellular assays. This was

particularly striking for compounds 13 and 14. Overall, the

optimization of the R¹-substituent resulted in the identiﬁcation

of several non-aryl pyridine replacements, with the ether-

containing substituents giving a particularly advantageous

balance between good cellular potency and low lipophilicity.

The opportunity for substitution in the imidazoquinoline R²-

position, as hypothesized using the X-ray crystal structure, was

conﬁrmed by exploring a small set of alkyl substituents (20−

23) on the simpliﬁed des-methyl analogue 19 of I-BET151

(Table 3). These conveyed a signiﬁcant increase in potency

over the imidazolone 19, and the isopropyl 23 showed that

branching was tolerated. In compounds 20−22, LLE was

either maintained or increased versus compound 19. On the

basis of these examples, heteroalkyl groups such as amines 24−

25 and ethers 26−27 were introduced. Both the amines

showed increased biochemical and cellular potency over the

baseline compound 19, and as for the R¹-substituent, the

ethers maintained the cellular potency of compound 19 while

reducing lipophilicity.

reﬂecting its observed higher potency in initial SAR studies

(Table 2), as was the compound containing an aryl R¹-

substituent (37).

Of the optimized compounds, three were identiﬁed as the

most likely to deliver the desired preclinical proﬁle. These

included bis-ether 32, the dimethylamino substituted 35, and

the pyridyl analogues 37. The pyridyl analogue 25 (Table 3)

also showed suitable potency in the HWB assay and was

further proﬁled. These preferred compounds were selected

based on their biochemical and cellular potency; additionally,

compounds 34 and 36 were deprioritized due to challenges

associated with their synthesis, which precluded a rapid and

cost-eﬀective large-scale preparation. The CYP2C9 and 3A4

inhibition proﬁles of the lead compounds were generated

(Table 5). Compounds 35 and 37 were discounted for further

Table 5. CYP2C9 and 3A4 Proﬁles of Lead Compounds in

Comparison with I-BET151

CYP inhibition IC₅₀(μM)

a

compound

2C9

3A4(VG)

5

9.9

9.7

32

35

25

37

>50

1.3

31.6

7.9

25.1

10

15.8

7.9

a

VG refers to the probe substrate used to determine the CYP3A4

inhibition proﬁle.

progression due to the lower but signiﬁcant level of CYP

inhibition. Ultimately, compound 32 was selected over

compound 25 due to its lower aromatic ring count and the

associated lower risk of toxicity.³⁵

Compound Synthesis. The synthesis of compounds was

built upon key quinoline-isoxazole intermediates whose

synthesis have been previously described.²³These intermedi-

ates were then diversiﬁed to provide access to the compounds

described within this manuscript.

Modiﬁcations of the R¹-position of the imidazoquinolinone

(Table 2), compounds 6−18, were installed in two steps

(Scheme 1) from the 4-chloroquinoline intermediate 39.²³

Compound 39 underwent nucleophilic aromatic substitution

(S_NAr) with the appropriate aliphatic amines giving

intermediates 40−52. These were converted to ﬁnal products

6−18 via a Hoﬀman-type rearrangement using hypervalent

iodine and potassium hydroxide.⁴⁰This was followed, in the

case of compound 16, by deprotection of the piperidine.

The majority of modiﬁcations of the R²-position of the

imidazoquinoline (Table 3), compounds 20−23, and 26−27,

were installed from the quinoline diamine 53²³via amide

formation and acid-mediated cyclization (Scheme 2). The

remaining compounds were prepared directly from the

quinoline diamine 53 (Scheme 3). Compound 19 was

prepared using a Hoﬀman-type rearrangement, compound 24

using cyanic bromide, and compound 25 using 1-isothiocya-

nato-2-methoxyethane followed by EDC.

Using the results generated for the individual R¹- and R²-

substituent changes (Tables 2 and 3), a set of analogues were

synthesized combining the most promising substituents (Table

4). In addition, two additional amine R²-substituents were

added (dimethylamino 35 and morpholino 36) to maintain the

amine functionality of 24 and 25 while reducing hydrogen

bond donor count to give an increased chance of good passive

permeability.³⁹In silico proﬁling prior to synthesis was used to

prioritize targets with low predicted lipophilicity.

While having reasonable biochemical potency, compounds

with the small methoxymethyl R²-substituents (28−29) had

poor cellular potency. This poor cellular potency was also seen

for analogue 30. Compound 31 had a promising biochemical

and cellular potency and showed minimal potency drop-oﬀ

into the LPS-stimulated human whole blood (HWB) assay.

This compound was therefore separated into the single

enantiomers 32 and 33 using chiral HPLC. Of these, the R-

enantiomer 32 was preferred due to its higher biochemical

potency. Compounds with the 4-tetrahydropyranyl R¹-

substituent (34−36) were among the most potent analogues,

To combine the preferred groups at the R¹- and R²-positions

of the imidazoquinoline, the key 3-nitro,4-chloro intermediate

63 was prepared (Scheme 4). The acid of compound 60²³was

decarboxylated by addition to reﬂuxing diphenyl ether to give

compound 61. Compound 61 was nitrated to give compound

62, which was chlorinated using phosphorus oxychloride, to

give compound 63.

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Scheme 1. Access to Compounds with Diverse R¹

Functionality

Scheme 3. Access to Compounds with Heteroatom Linked

a

R²Functionality

a

Reagents and conditions: (a) R¹NH₂, DIPEA, NMP, 100−120 °C,

14−100%; (b) PhI(OAc)₂, KOH, MeOH, 0 °C, 6−89%, followed by

TFA, DCM, r.t., 83% for compound 16.

a

Reagents and conditions: (a) (i) pyridin-2-ylmethanamine, CH₃CN,

reﬂux; (ii) PhI(O(CO)CF₃)₂, CH₃CN, 50 °C, 54% over two steps;

(b) 24: BrCN, EtOH, 60 °C, 32%, 25: (i) MeO(CH₂)₂NCS, EtOH,

60 °C, (ii) EDC, THF 60 °C, 2%.

Scheme 2. Access to Compounds with C-Linked R²

Functionality

a

Scheme 4. Access to Key Intermediate 62

a

Reagents and conditions: (a) Ph₂O, reﬂux, 88%; (b) HNO₃,

propionic acid, 100 °C, 72%; (c) POCl₃, 120 °C, 75%.

a

Reagents and conditions: (a) (R²CO)₂O/R²COCl, pyridine, DCM,

cyclization as a more rapid alternative to the two-step route

used to prepare earlier compounds. Compound 36 was

synthesized from the imidazolone 13 via formation of the

heteroaryl chloride, and compound 35 was formed using a

commercially available starting material to insert the

dimethylamino substituent. Primary aryl amine 37 was

accessed using a thiocyanate to form the thiourea, followed

by cyclization mediated by EDC.

Preclinical Developability Proﬁle. Compound 32, here-

on named I-BET282, showed an excellent preclinical

developability proﬁle. The compound was tested against an

in-house developability panel of 38 unrelated proteins

representing key safety considerations, and only a weak

inhibition of the hERG potassium ion channel (pIC₅₀4.4−

r.t., 59−72%, or R²COOH, HATU, DIPEA, or NEt₃, DCM, r.t., 61−

82%; (b) AcOH reﬂux, 26−79%.

The chloro-nitro intermediate 63 was converted to the ﬁnal

compounds 28−29, 31, and 34, via nucleophilic aromatic

substitution, reduction of the nitro group, amide formation,

and acid-mediated cyclization (Scheme 5). Compound 31 was

separated into its constituent enantiomers 32 and 33 by chiral

HPLC.

Using intermediates prepared as above (Scheme 5), the

remaining compounds were synthesized via a range of bespoke

conditions to access the desired substituents (Scheme 6).

Compound 30 was prepared using a one-step reductive

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Scheme 5. General Route to Compounds with Diverse R¹

investigated using an ex vivo rabbit wedge model; the data

supported further progression with a 40-fold margin to

concentrations in the range expected in humans for

cardiovascular ﬁndings in this assay system. X-ray crystallo-

graphic data in the ﬁrst bromodomain of BRD4 showed that

the compound had a similar binding mode to I-BET151

(Figure 4a,b), as well as GSK788 (Figure 4c,d) which was

developed concurrently within our laboratories.³⁷As with I-

BET151, the dimethylisoxazole bound to the acetyl lysine

binding site of the bromodomain, with a direct hydrogen bond

to Asn140 and a water-mediated hydrogen bond to Tyr97. The

methoxymethyl of the branched imidazoquinoline N-substitu-

ent bound into the WPF shelf region, with the α-methyl

interacting with the lipophilic surface of Leu92. The THP

substituent exited through the ZA channel formed by Trp81

and Leu92, showing excellent shape complementarity with the

protein surface. In contrast to the BD1 selective GSK788,³⁷I-

BET282 has no equivalent to the pyrrolidine that interacts

with D145 of the “Asp-His” BD1/BD2 selectivity switch

(Figure 4d). Consequently, D145 in the I-BET282 structure

adopts a conformation swung away from the ligand, giving rise

to a more extended WPF shelf, and as a result we expected I-

BET282 to maintain equivalent potency against BD1 and BD2

domains.

I-BET282 was conﬁrmed to be a pan-inhibitor of all eight

BET bromodomains, and selectivity over other representative

bromodomain-containing proteins was also established (see

Supporting Information). As the closest oﬀ-target, and as a

result of the potential for associated toxicity,⁴¹the selectivity

over the bromodomain of CBP was established deﬁnitively by

biophysical techniques. Thermal shift data showed binding to

the bromodomain of CBP (ΔT_m= 3.4 °C cf. ΔT_m= 9.9 °C for

BRD4), which was conﬁrmed using isothermal titration

calorimetry (ITC). I-BET282 bound to the bromodomain of

CBP with an aﬃnity constant determined to be 1.73 0.17

μM by ITC, a 36-fold window to BRD4 as measured by the

same technique (47.5 25 nM).

a

and R²Functionality

a

Reagents and conditions: (a) R¹NH₂, DIPEA, NMP, 100 °C, 19−

85% or R¹NH₂, 1,4-dioxane, r.t., 73−92%; (b) H₂, 10% Pd/C EtOAc,

EtOH 20 °C 93−96% or SnCl₂, EtOH, 40 °C 36−96%; (c) (i)

R²COCl, pyridine, DCM r.t. or R²COOH, HATU, DIPEA, DMF, r.t.;

(ii) AcOH 120 °C; or pTSA, toluene, reﬂux; or NH₃, (NH₄)₂HCO₃,

acetonitrile, r.t. 15−74%.

Scheme 6. Access to Compounds with Diverse R¹and R²

Functionality Which Were Not Prepared via the General

a

Method

I-BET282 showed a low potential to inhibit CYP proteins in

vitro, with no evidence of time-dependent inhibition of 2D6 or

3A4 (Table 6). In vitro hepatocyte clearance was low across

mouse, rat, and human; encouragingly, a signiﬁcant increase in

metabolic stability in dog hepatocytes for I-BET282 (2.4 mL/

min/g liver) compared with I-BET151 (9.8 mL/min/g liver)

indicated the pharmacokinetics of I-BET282 in the dog may be

appropriate for use as a preclinical safety species. I-BET282

had high in vitro passive permeability (346 nm/s in MDCK

cells) and a moderate free fraction in blood consistent across

all species investigated (f_ub0.1−0.2).

The pharmacokinetic proﬁle of the compound was assessed

in three preclinical species: mouse, rat, and dog (Table 7).

Total blood clearance was low in the mouse and rat (<25%

hepatic blood ﬂow, HBF) and higher in the dog (68% HBF).

The rate of total clearance in vivo was consistent with the

pattern of clearance determined in hepatocytes in vitro

providing conﬁdence that I-BET282 had the potential for

low/moderate clearance in humans. Renal clearance deter-

mined in rats was negligible at <1% of total clearance, so the

major route of elimination was assumed to be metabolism.

Volume of distribution was moderate across all species

indicating distribution to the tissues. Elimination half-life was

considered moderate across all preclinical species indicating

the potential for once or twice daily administration in humans.

Oral bioavailability of the free base in a suspension formulation

a

Reagents and conditions: (a) Na₂S₂O₄, EtOH, H₂O, R²COH, 100

°C, 27%; (b) (i) POCl₃, PCl₅, 120 °C, (ii) morpholine, NMP, 120

°C, 2%; (c) 35: N-(dichloromethylene)-N-methylmethanaminium

chloride, MeCN, 120 °C, 77%; 37: (i) 1-isothiocyanato-2-methoxy-

ethane, EtOH, 60 °C, (ii) EDC, THF, 60 °C, 19%.

5.1 in a variety of assay formats) was identiﬁed as a cause for

further investigation. The weak hERG inhibition was further

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Figure 4. I-BET282 bound to the N-terminal bromodomain of BRD4 with key residues labeled (PDB: 7O18, magenta). (a) I-BET282 shown in

stick format. (b) Solvent-accessible protein surface of BRD4 with the WPF shelf highlighted. (c, d) Show overlay with GSK788 (PDB: 6SWN,

green).

Table 6. CYP2C9 and 3A4 Proﬁles of Lead Compound I-

BET282

Table 7. Pharmacokinetic Proﬁle of I-BET282 in Male CD1

Mice, Male Wistar Han Rats, and Male Beagle Dogs

a

assay

test system

mouse

<0.86

rat

dog

2.4

human

<0.86

parameters

mouse

1.4, 3

rat

1, 3

20

22

dog

1, 3

27

68

b

c

metabolic stability hepatocytes

(mL/min/g

liver)

<0.86

doses i.v. , p.o. (mg/kg)

Cl_b(mL/min/kg)

% LBF

23

19

8

6

a

free fraction (f_ub

)

blood

singly expressed

P450 enzymes

0.10

0.16

0.24

0.17

V_ss(L/kg)

t_1/2(iv, h)

F, po %

1.9 0.1

1.2 0.5

51 15

2.2 0.5

1.4 0.3

3.1 0.1

1.8 0.2

31 12

CYP inhibition

CYP1A2 ER: > 50

CYP2C9 FCA: > 50

proﬁle IC₅₀

bc

,

(μM)

74

9

CYP2C19 BMC: > 50

CYP2D6 MMC: > 50

CYP3A4 VR: > 50

CYP3A4 VG: > 20

a

b

Values are mean, n = 3 SD. i.v. dose was 1 h infusion in DMSO

and (10%, w/v) Kleptose HPB in saline (2%:98% (v/v)). p.o. dose

vehicle: 1% (w/v) methylcellulose (400 cps) (aq).

c

time-dependent

singly expressed

P450 enzymes

CYP2D6 MMC - no inhibition of

CYP2D6

c

During the preparation of large-scale batches of the free base

I-BET282 (compound 32), powder X-ray diﬀraction studies

(see Supporting Information) revealed multiple crystalline

forms. This was undesirable as the presence of multiple

crystalline forms can have signiﬁcant impact during late-stage

drug development as well as on marketed drugs.⁴²Addition-

ally, the new stable “Form 2” material had a lower measured

fasted state solubility of 130 μg/mL, reduced from 480 μg/mL

for the “Form 1” material.⁴³The low solubility of “Form 2”, as

well as its multiple crystalline forms, led to concerns over the

long-term development of the free base. A salt screen was

therefore performed to improve the physical properties of the

material which identiﬁed the mesylate salt compound 75,

hereon named I-BET282E. Closed samples of the mesylate I-

BET282E were stable under three-month accelerated storage

conditions. As salt formation impacts the kinetic, rather than

thermodynamic, solubility of a compound,⁴⁴the mesylate salt

I-BET282E showed a signiﬁcantly improved dissolution rate

P450 inhibition

CYP3A4

c

DEF - no inhibition of CYP3A4,

but an increase in IC₅₀

c

7BQ - could not be determined

a

b

Mean value determined at 100 ng/mL and 1000 ng/mL. Expressed

c

as an average of at least two experimental replicates. ER, FCA, BMC,

MMC, VR, VG, DEF, and 7BQ refer to the probe substrates used to

determine the CYP inhibition proﬁles.

ranged between 31% in dogs to 74% in rats. This was broadly

consistent with the extent of ﬁrst-pass extraction in the

respective species though with some evidence of absorption

limitations, particularly in the mouse. Overall, the DMPK

proﬁle was suitable for further development oﬀering the

potential for an orally available, low-moderate clearance

molecule in humans.

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than the free base of I-BET282, resulting in a 20-fold solubility

increase at 30 min (1.8 mg/mL for I-BET282E, 92 μg/mL for

I-BET282 Form 2). Additionally, only one crystalline form was

observed during polymorph screening of the mesylate I-

BET282E. The mesylate salt was therefore selected as the salt

form of choice for ongoing preclinical development activities.

Prior to progression into these studies, the oral exposure of

I-BET282E administered as the mesylate salt was compared

with that following administration as the free base (Table 8).

In the rat, C_maxand AUC were comparable, whereas in the dog

an approximate 3-fold increase in both C_maxand AUC was

observed. These data conﬁrmed that the mesylate salt provided

at least equivalent exposure to the free base in preclinical

species.

Further to its excellent pharmacokinetic proﬁle, I-BET282E

also showed in vivo eﬃcacy in a rat model of collagen-induced

arthritis (Figure 5). Brieﬂy, the arthritis was established in 4−5

week old female Wistar rats using intradermal injection of

bovine collagen and boosted 7 days later with a combination of

collagen, muramyl dipeptide (MDP), and incomplete Freund’s

adjuvant. Compounds were dosed in a peri-onset schedule

from day 12 when the rats showed the ﬁrst signs of arthritis

until day 21 when the arthritis was fully established. Dosing

with I-BET282E showed a dose-dependent inhibition of

clinical score with signiﬁcant inhibition observed at 3 and 1

mg/kg. Similarly, analysis of microcomputed tomography

(CT) scans demonstrated a dose-dependent inhibition of

bone resorption and remodeling at the same doses. Dosing of

I-BET282E at 3 mg/kg resulted in a more pronounced eﬃcacy

than I-BET151 at 3 mg/kg, which was similar to I-BET282E

dosed at 1 mg/kg. Both BET inhibitors were well tolerated

over the period of the in vivo study, as demonstrated by a

general improvement in the maintenance of body weight by

the highest dosed groups compared to the vehicle control

group. We believe the improved pharmacology of I-BET282E

over I-BET151 is driven by potency and bioavailability as the

Table 8. Pharmacokinetic Proﬁle of I-BET282 and I-

BET282E in Male Wistar Han Rats and Male Beagle Dogs

a

on Oral Dosing

rat

dog

parameter

I-BET282

I-BET282E

I-BET282

I-BET282E

dose (mg/kg)

1

5

AUC_0‑t

(ng·h/mL)

467 213

348 89

367 122

885 150

C_max(ng/mL)

T_max(h)

125 34

1

101 19

1

177 51

0.75

578 182

0.375

a

All doses were administered as an oral suspension prepared in 1%

hydroxypropylmethyl cellulose (w/v) in water. Key: Values are mean

SD, n = 3 for rat, and n = 4 for dog AUC_0‑t= Area under the plasma

concentration−time curve from 0 h to last quantiﬁable time point.

C_max= maximum observed plasma concentration. T_max= time at

which C_maxis observed and is reported as median.

Figure 5. Eﬀects of I-BET282E in the rat collagen-induced arthritis (CIA) model. Arthritis was induced in female Lewis rats by intradermal

injection of bovine type II collagen on Day 0 followed by an intradermal booster injection of bovine type II collagen and MDP on day 7. Rats were

administered either vehicle, I-BET151 (3 mg/kg) or I-BET282E (0.3, 1, or 3 mg/kg) p.o. once daily from day 12 (at onset of symptoms) until day

28, i.e., for 16 days. (A) The eﬀects of I-BET282E on clinical scores of the hind limbs was visually assessed daily from day 6−28. (B) The eﬀects of

I-BET282E on hind paw volumes was measured via plethysmography on days 12−26, data expressed as AUC. Colors were assigned to doses as in

panel A. (C) The eﬀects of I-BET282E on the number of aﬀected bones and overall severity score were determined by micro computed

tomography (CT). Colors were assigned to doses as in panel A. (D) Eﬀects of I-BET282E on new bone growth illustrated by CT images. Data are

expressed as mean SD, n = 7−8/group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group.

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packing diameter). The UV detection was a summed signal from

wavelength of 210 to 350 nm. MS conditions: The mass spectrometry

was conducted on a Waters ZQ mass spectrometer, with an ionization

mode of alternate-scan positive and negative electrospray. The scan

range was 100−1000 AMU, the scan time was 0.50 s, and the

interscan delay was 0.20 s. Method using ammonium bicarbonate

modiﬁer: “MDAP (high pH)”. The method was as for MDAP (formic

acid), except the solvents employed were A = 10 mM ammonium

bicarbonate in water, adjusted to pH 10 with ammonia solution; B =

acetonitrile. The purity of compounds tested in in vitro and in vivo

assays was of greater than 95% using interpretation of a combination

preference for BD1 compared to BD2 is similar for both

compounds (∼2.5 fold and ∼5 fold respectively).

CONCLUSION

■

Late-stage optimization of I-BET151, with the aim of

increasing cellular potency and improving the pharmacokinetic

proﬁle, identiﬁed I-BET282E as a molecule with the potential

for progression into in vivo studies. This compound showed an

excellent selectivity proﬁle, a pharmacokinetic proﬁle oﬀering

the potential for an orally available, low-moderate clearance

molecule in humans, as well as in vivo eﬃcacy in a model of

collagen-induced arthritis. The pharmacokinetic proﬁle in dogs

was also suitable for this species to be used in preclinical safety

studies. Formation of the mesylate salt improved the

polymorph and solubility proﬁle and delivered a molecule

suitable for progression into preclinical development, and

ultimately I-BET282E was progressed into a clinical dose

escalation study in subjects with advanced or recurrent solid

tumors.⁴⁵During the course of this study, a number of

emerging reports highlighted that despite the high level of

structural diversity of BET inhibitors progressed in clinical

trials, dose limiting safety signals showed strong commonalities

(in particular thrombocytopenia, fatigue, and GI toxicity

(mainly diarrhea)), suggesting pharmacology-driven signals in

the majority of cases⁴⁶even if an unexpected and atypical

safety proﬁle has also been reported.⁴⁷On the basis of these

data and the emerging safety proﬁle of I-BET762 (Figure 1,

compound 1) from Phase I trials, the progression of a back-up

molecule was considered unnecessary leading to the

termination of I-BET282E in 2017.

1

of LCMS and H NMR data, unless stated otherwise. LCMS were

conducted either with a formic acid modiﬁer “LCMS (formic acid)”,

triﬂuoroacetic acid modiﬁer “LCMS (TFA)”, or ammonium

bicarbonate modiﬁer “LCMS (high pH)”. Method using formic acid

modiﬁer: “LCMS (formic acid)”. LC conditions: The UPLC analysis

was conducted on an Acquity UPLC BEH C₁₈column (50 mm × 2.1

mm, i.d. 1.7 μm packing diameter) at 40 °C. The solvents employed

were A = 0.1% v/v solution of formic acid in water; B = 0.1% v/v

solution of formic acid in acetonitrile. The gradient (A:B) employed

was from 97:3 to 3:97 over 2 min. The UV detection was a summed

signal from wavelength of 210 to 350 nm. MS conditions: The mass

spectrometry was conducted on a Waters ZQ mass spectrometer, with

an ionization mode of alternate-scan positive and negative electro-

spray. The scan range was 100−1000 AMU, the scan time was 0.27 s,

and the interscan delay was 0.10 s. Method using triﬂuoroacetic acid

modiﬁer: “LCMS (TFA)”. The method was as for LCMS (formic

acid), except the solvents employed were A = 0.1% v/v solution of

triﬂuoroacetic acid in water; B = 0.1% v/v solution of triﬂuoroacetic

acid in acetonitrile. Method using ammonium bicarbonate modiﬁer:

“LCMS (high pH)”. The method was as for LCMS (formic acid),

except the solvents employed were A = ammonium hydrogen

carbonate in water adjusted to pH 10 with ammonia solution; B =

acetonitrile. High-resolution mass spectrometry chromatography and

analysis conditions: An Agilent 1100 liquid chromatograph equipped

with a model G1367A autosampler, a model G1312A binary pump,

and a HP1100 model G1315B diode array detector was used. The

method used was generic for all experiments. All separations were

achieved using a C₁₈reversed phase column (100 × 2.1 mm, 3 μm

particle size) or equivalent. Gradient elution was carried out with the

mobile phases as (A) water containing 0.1% (v/v) TFA and (B)

acetonitrile containing 0.1% (v/v) TFA. The conditions for the

gradient elution were initially 0% B, increasing linearly to 95% B over

8 min, remaining at 95% B for 0.5 min, then decreasing linearly to 0%

B over 0.1 min followed by an equilibration period of 1.49 min prior

to the next injection. The ﬂow rate was 1 mL/min, split to source, and

the temperature controlled at 40 °C with an injection volume of

between 2 to 5 μL. Mass spectrometry conditions: Positive ion mass

spectra were acquired using a Thermo LTQ-Orbitrap FT mass

spectrometer, equipped with an ESI interface, over a mass range of

100−1100 Da, with a scan time of 1 s. The elemental composition

was calculated using Xcalibur software and processed using

RemoteAnalyzer (Spectral Works Ltd.) for the [M + H]⁺and the

EXPERIMENTAL SECTION

The human biological samples were sourced ethically, and their

research use was in accord with the terms of the informed consents

under an IRB/EC approved protocol.

■

Chemistry Methods and Characterization for Compounds.

All commercial chemicals and solvents used were of reagent grade and

used without further puriﬁcation. Where compounds were hydro-

genated using an H-cube, a Thales H-cube continuous−ﬂow

hydrogenation reactor was used, where hydrogen is generated in

situ. Thales disposable catalyst cartridges were used, either the

CatCart 30 (30 mm cartridge, containing 140 mg of 10% Pd/C

catalyst) or the CatCart 70 (70 mm cartridge, containing 350 mg of

10% Pd/C catalyst). Reactions heated under microwave conditions

were heated in a Biotage Initiator microwave. Unless otherwise stated,

the initial absorption was set as “high”, and 15 s of prestirring was

applied before heating commenced. Hydrophobic frit cartridges by

ISOLUTE, which contain a frit selectively permeable to organic

solutions, were used for separation of organic phases from aqueous

phases under gravity. ISOLUTE aminopropyl and SCX cartridges

were used for scavenging SPE protocols. Column chromatography

was carried out either using manual or automated ﬂash chromatog-

raphy systems using silica SPE cartridges. The names of the

compounds were obtained using ChemDraw. Mass-directed autopre-

parative high-performance liquid chromatography (MDAP) were

conducted either with a formic acid modiﬁer “MDAP (formic acid)”

or an ammonium bicarbonate modiﬁer “MDAP (high pH)”. Method

using formic acid modiﬁer: “MDAP (formic acid)”. LC conditions:

The HPLC analysis was conducted on either a Sunﬁre C₁₈column

(100 mm × 19 mm, i.d. 5 μm packing diameter) or a Sunﬁre C₁₈

column (150 mm × 30 mm, i.d. 5 μm packing diameter) at ambient

temperature. The solvents employed were A = 0.1% v/v solution of

formic acid in water; B = 0.1% v/v solution of formic acid in

acetonitrile. The puriﬁcation was run as a gradient (A:B) over either

15 or 25 min, with a ﬂow rate of 20 mL/min (100 mm × 19 mm, i.d.

5 μm packing diameter) or 40 mL/min (150 mm × 30 mm, i.d. 5 μm

1

mass error quoted as ppm. Unless otherwise speciﬁed, H and ¹³C

NMR spectra were recorded at 400 and 101 MHz respectively, and

chemical shifts are given in ppm (δ) relative to tetramethylsilane

(TMS) as an internal standard. Chemical shifts are given to the

nearest 0.01 ppm (¹H NMR) or 0.1 ppm (¹³C NMR), and coupling

constants are given to the nearest 0.1 Hz. NMR spectra were recorded

at room temperature unless otherwise stated. Infrared (IR) spectra

were recorded using a PerkinElmer Spectrum One FT-IR

spectrometer, and key well-deﬁned peaks were recorded in cm⁻¹.

Optical rotation measurements were recorded using a Jasco P-1030

polarimeter. The concentration was recorded in g/mL, path length in

mm, and temperature in °C.

7-(3,5-Dimethyl-4-isoxazolyl)-1-(1-methylethyl)-8-(methyloxy)-

1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (6). 7-(3,5-Dimethyl-

4-isoxazolyl)-4-[(1-methylethyl)amino]-6-(methyloxy)-3-quinoline-

carboxamide 40 (188 mg, 0.53 mmol) was suspended in methanol

(10 mL), and potassium hydroxide (44.6 mg, 0.796 mmol) was

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added. The mixture was stirred for 20 min, then cooled in an ice bath,

and iodobenzene diacetate (222 mg, 0.69 mmol) added. The reaction

mixture was stirred at 0 °C for 2 h. The reaction mixture was

concentrated in vacuo. The residue was taken up in DCM and puriﬁed

by column chromatography (silica), eluted using 1−10% of MeOH in

DCM. The appropriate fractions were combined and concentrated in

vacuo to give an orange oil (113 mg). The oil was taken up in

dichloromethane (∼1 mL), and 1 M hydrochloric acid (0.31 mL, 0.31

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

nitrogen to give the title compound (117 mg, 0.29 mmol, 54% yield)

as a brown solid.

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-(1-methoxypropan-

2-yl)-1H-imidazo[4,5-c]quinolin-2(3H)-one (10). 7-(3,5-Dimethyli-

soxazol-4-yl)-6-methoxy-4-((1-methoxypropan-2-yl)amino)quinoline-

3-carboxamide 44 (116 mg, 0.30 mmol) and potassium hydroxide (25

mg, 0.45 mmol) were taken up in methanol (5 mL) and stirred at

room temperature for 20 min. The reaction mixture was cooled in an

ice bath, and iodobenzene diacetate (126 mg, 0.39 mmol) was added.

The mixture was stirred at 0 °C for 2 h. The reaction mixture was

concentrated in vacuo and puriﬁed by MDAP (formic acid). The

appropriate fractions were blown down under a stream of nitrogen,

taken up in methanol, and puriﬁed by SCX, and eluted with methanol

and then 2 M NH₃in MeOH. The appropriate fractions were

concentrated in vacuo to give the title compound (44 mg, 0.10 mmol,

34% yield) as a yellow residue.

LCMS (formic acid): rt = 0.69 min, MH⁺= 353.

¹H NMR (CD₃OD, 400 MHz) δ 1.85 (d, 6H, J = 6.8 Hz) 2.23 (s,

3H) 2.40 (s, 3H) 4.11 (s, 3H) 5.38−5.48 (m, 1H) 7.81 (s, 1H) 7.98

(s, 1H) 8.74 (s, 1H).

LCMS (formic acid): rt = 0.68 min, MH⁺= 383,

¹H NMR (CD₃OD, 400 MHz) δ 1.71−1.74 (d, 3H, J = 6.3 Hz),

2.19 (s, 3H), 2.36 (s, 3H), 3.37 (s, 3H), 3.80−3.84 (m, 1H), 4.01 (s,

3H), 4.26−4.31 (m, 1H), 5.29−5.35 (m, 1H), 7.76 (s, 1H), 7.85 (s,

1H), 8.55 (s, 1H).

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-1-(2-methylpropyl)-

1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (7). 7-(3,5-Dimethyl-

4-isoxazolyl)-6-(methyloxy)-4-[(2-methylpropyl)amino]-3-quinoline-

carboxamide 41 (762 mg, 1.03 mmol) was taken up in methanol (20

mL). Potassium hydroxide (87 mg, 1.55 mmol) was added, and the

mixture stirred for 20 min. The mixture was cooled in an ice bath, and

iodobenzene diacetate (433 mg, 1.34 mmol) was added. The mixture

was stirred at 0 °C for 2 h. The reaction mixture was concentrated in

vacuo. The residue was loaded in DCM and puriﬁed by column

chromatography (silica), eluted using 1−10% MeOH in DCM. The

appropriate fractions were concentrated in vacuo. Dichloromethane

(∼0.5 mL) and 1 M hydrochloric acid (0.46 mL, 0.46 mmol) were

added, and the solvent removed under a stream of nitrogen to give the

title compound (117 mg, 0.26 mmol, 25% yield) as a brown solid.

LCMS (formic acid): rt = 0.76 min, MH⁺= 367.

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-((tetrahydrofuran-3-

yl)methyl)-1H-imidazo[4,5-c]quinoline-2(3H)-one (11). To a sus-

pension of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-(((tetrahydro-

furan-3-yl)methyl)amino)quinoline-3-carboxamide 39 (667 mg, 1.68

mmol) in methanol (5 mL) was added potassium hydroxide (142 mg,

2.52 mmol), and the reaction mixture was stirred for 20 min at room

temperature. The reaction mixture was cooled in an ice/water bath,

iodobenzene diacetate (704 mg, 2.19 mmol) added, and the reaction

mixture was stirred at 0 °C for a further 2 h. The reaction mixture was

blown down under a stream of nitrogen. The sample was loaded in

methanol/dichloromethane (and the column dried in a vacuum oven)

and puriﬁed by column chromatography (silica), eluted using 0−10%

MeOH in DCM. The appropriate fractions were combined and dried

under a stream of nitrogen to give the title compound (558 mg, 1.42

mmol, 84% yield) as a pale brown foam.

¹H NMR (CDCl₃, 400 MHz) δ 1.15 (br s, 6H) 2.25 (br s, 3H)

2.41 (br s, 4H) 4.02 (br s, 3H) 4.24 (s, 2H) 7.50 (br s, 1H) 8.17 (br s,

1H) 9.09 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-(pentan-2-yl)-1H-

imidazo[4,5-c]quinolin-2(3H)-one (8). 7-(3,5-Dimethylisoxazol-4-

yl)-6-methoxy-4-(pentan-2-ylamino)quinoline-3-carboxamide 42

(483 mg, 1.26 mmol) was taken up in methanol (20 mL). Potassium

hydroxide (106 mg, 1.89 mmol) was added, and the mixture was

stirred for 20 min. The mixture was cooled in an ice bath, and

iodobenzene diacetate (529 mg, 1.64 mmol) was added. The reaction

was stirred at 0 °C for 2 h. The reaction mixture was concentrated in

vacuo. The residue was loaded in the minimum amount of DCM and

puriﬁed by column chromatography (silica), eluted using 1−10%

MeOH in DCM. The appropriate fractions were combined and

concentrated in vacuo to give the title compound (352 mg, 0.88

mmol, 70% yield) as an orange oil.

LCMS (formic acid): rt = 0.65 min, MH⁺= 395.

¹H NMR (DMSO-d₆, 393 K, 400 Hz): δ 1.80−1.90 (m, 1H),

2.01−2.11 (m, 1H), 2.14 (s, 3H), 2.33 (s, 3H), 2.85−2.98 (m, 1H),

3.66−3.77 (m, 3H), 3.87−3.93 (m, 1H), 3.99 (s, 3H), 4.29−4.40 (m,

2H), 7.57 (s, 1H), 7.84 (s, 1H), 8.58 (s, 1H), 10.90−11.31 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-((tetrahydro-2H-

pyran-3-yl)methyl)-1H-imidazo[4,5-c]quinoline-2(3H)-one (12). To

a suspension of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-(((tetra-

hydro-2H-pyran-3-yl)methyl)amino)quinoline-3-carboxamide 46

(610 mg, 1.49 mmol) in methanol (5 mL) was added potassium

hydroxide (125 mg, 2.23 mmol), and the reaction mixture was stirred

at room temperature for 20 min. The reaction mixture was cooled in

an ice/water bath, iodobenzene diacetate (622 mg, 1.93 mmol)

added, and the reaction mixture was stirred at 0 °C for 2 h. The

reaction mixture was blown down under a stream of nitrogen. The

sample was loaded in methanol/dichloromethane (and the column

dried in a vacuum oven) and puriﬁed by column chromatography

(silica, 100 g), eluted using 0−100% EtOAc in cyclohexane followed

by 0−10% MeOH in DCM. The appropriate fractions were combined

and dried under a stream of nitrogen to give the title compound (407

mg, 1.00 mmol, 67% yield) as a pale brown foam.

LCMS (formic acid): rt = 0.83 min, MH⁺= 381.

¹H NMR (CDCl₃,400 MHz) δ 0.98 (t, 2H, J = 7.3 Hz) 1.35−1.56

(m, 1H) 1.82 (d, 1H, J = 6.8 Hz) 2.00−2.11 (m, 1H) 2.13 (s, 1H)

2.24 (s, 2H) 2.38 (s, 2H) 3.98 (s, 1H) 7.56 (br s, 1H) 7.97 (s, 1H)

8.79 (s, 1H).

1-(Cyclopropylmethyl)-7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-

1H-imidazo[4,5-c]quinoline-2(3H)-one (9). To a suspension of 4-

((cyclopropylmethyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-methox-

yquinoline-3-carboxamide 43 (365 mg, 1.00 mmol) in MeOH (4 mL)

was added potassium hydroxide (84 mg, 1.49 mmol), and the reaction

mixture stirred at room temperature for 20 min. The reaction mixture

was cooled to 0 °C (ice/water bath), iodobenzene diacetate (417 mg,

1.23 mmol) was added, and the reaction mixture was stirred at 0 °C

for a further 2 h. The reaction mixture was blown down under a

stream of nitrogen. A portion (100 mg) of the crude material was

dissolved in DMSO (1 mL) and puriﬁed by MDAP (high pH). The

solvent was blown down a stream of nitrogen to give the title

compound (22 mg, 0.06 mmol, 6% yield). The remaining crude

material (902 mg) was retained without puriﬁcation.

LCMS (formic acid): rt = 0.69 min, MH⁺= 409.

¹H NMR (d₆-DMSO, 393 K, 400 MHz): δ 1.43−1.53 (m, 2H),

1.65−1.75 (m, 1H), 1.79−1.87 (m, 1H), 2.12 (s, 3H), 2.14−2.26 (m,

1H), 2.31 (s, 3H), 3.37−3.48 (m, 2H), 3.64−3.79 (m, 2H), 3.96 (s,

3H), 4.23 (m, 2H), 7.50 (s, 1H), 7.82 (s, 1H), 8.55 (s, 1H), 10.91−

11.24 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-((tetrahydro-2H-

pyran-4-yl)methyl)-1H-imidazo[4,5-c]quinoline-2(3H)-one (13). To

a suspension of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-(((tetra-

hydro-2H-pyran-4-yl)methyl)amino)quinoline-3-carboxamide 47 (1.7

g, 4.14 mmol) in MeOH (15 mL) was added potassium hydroxide

(0.35 g, 6.21 mmol), and the reaction mixture was stirred at room

temperature for 20 min. The reaction mixture was cooled in an ice/

water bath, iodobenzene diacetate (1.73 g, 5.38 mmol) was added,

and the reaction mixture was stirred at 0 °C for 2 h. The reaction

LCMS (formic acid): rt = 0.73 min, MH⁺= 365.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 0.46−0.51 (m, 2H),

0.55−0.61 (m, 2H), 1.36−1.46 (m, 1H), 2.14 (s, 3H), 2.33 (s, 3H),

3.99 (s, 3H), 4.29 (d, J = 6.3 Hz, 2H), 7.67 (s, 1H), 7.84 (s, 1H), 8.58

(s, 1H), 10.87−10.99 (br s, 1H).

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mixture was blown down under a stream of nitrogen. The residue was

loaded as a suspension in MeOH and DCM (and the column dried in

a vacuum oven) and puriﬁed by SPE (silica, 100 g) using 0−20%

MeOH in DCM. The appropriate fractions were combined and dried

under a stream of nitrogen to give the title compound (1.50 g, 3.67

mmol, 89% yield) as a pale brown foam.

containing fractions were evaporated in vacuo to give a pale yellow

glass. The glass was triturated with a mixture of EtOAc (5 mL) and

cyclohexane (5 mL), and then the solvent evaporated in vacuo to give

a beige solid (247 mg). TFA (2 mL, 26.0 mmol) was added to a

solution of this solid (200 mg) in DCM (10 mL), and the mixture was

stirred for 2 h and then evaporated in vacuo. The residue was

dissolved in methanol (10 mL) and puriﬁed by SCX, washed with

methanol (50 mL), and then eluted with 2 M methanolic ammonia

(30 mL). The eluant was evaporated in vacuo to give the title

compound (133 mg, 0.34 mmol, 42% yield) as gray solid.

LCMS (formic acid): rt = 0.66 min, MH⁺= 409.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.34−1.48 (m, 2H), 1.60 (d, J

= 11.1 Hz, 2H), 2.14 (s, 3H), 2.17−2.25 (m, 1H), 2.33 (s, 3H),

3.22−3.28 (m, 2H), 3.84 (dd, J = 11.4, 2.0 Hz, 2H), 3.97 (s, 3H),

4.23 (d, J = 7.3 Hz, 2H), 7.46 (s, 1H), 7.87 (s, 1H), 8.57 (s, 1H),

11.45−11.58 (br s, 1H).

LCMS (formic acid): rt = 0.42 min, MH⁺= 394.

¹H NMR (CDCl₃, 400 MHz) δ 2.08−2.22 (m, 2H), 2.25 (s, 3H),

2.40 (s, 3H), 2.92−3.24 (m, 4H), 3.50−3.69 (m, 2H), 4.02 (s, 3H),

4.96−5.08 (m, 1H), 7.45−7.77 (br s, 1H) 7.97 (s, 1H), 8.76 (s, 1H).

The two NH were not observed.

¹³C NMR (d₆-DMSO, 300 K): δ 10.4, 11.4, 30.2, 36.7, 46.9, 55.6,

66.4, 99.0, 112.2, 115.6, 120.6, 122.0, 128.3, 132.1, 133.0, 139.7,

154.3, 155.0, 159.1, 166.1.

IR (neat): 1694 (urea), 1597 (aryl), 1228 (aryl ether), 841 (aryl

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-1-[(1-methyl-4-

piperidinyl)methyl]-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one

(17). 7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-{[(1-methyl-4-

piperidinyl)methyl]amino}-3-quinolinecarboxamide 51 (250 mg,

0.59 mmol), potassium hydroxide (50 mg, 0.89 mmol), and

iodobenzene diacetate (247 mg, 0.77 mmol) were dissolved in

methanol at 0 °C, and the mixture was stirred for 3 h, then evaporated

in vacuo. The residue was dissolved in DCM (3 mL) and loaded onto

a 25g silica column and then eluted with 0−10% 2 M methanolic

ammonia/DCM. Product-containing fractions were evaporated in

vacuo to give an amber solid. The product was suspended in hot

EtOAc (3 mL) and then cooled to room temperature and ﬁltered, and

the solid was washed with EtOAc (3 mL) to give the title compound

(181 mg, 0.43 mmol, 73% yield) as a beige solid.

C−H) cm⁻¹.

M.p.: 233−238 °C.

HRMS: C₂₂H₂₅N₄O₄MH⁺requires 409.1870, found MH⁺

409.1870.

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-(1-(tetrahydro-2H-

pyran-4-yl)ethyl)-1H-imidazo[4,5-c]quinoline-2(3H)-one (14). To a

suspension of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((1-(tetra-

hydro-2H-pyran-4-yl)ethyl)amino)quinoline-3-carboxamide 48 (169

mg, 0.40 mmol) in MeOH (2 mL) was added potassium hydroxide

(34 mg, 0.60 mmol), and the reaction mixture was stirred at room

temperature for 20 min. The reaction mixture was cooled to 0 °C

(ice/water bath), iodobenzene diacetate (167 mg, 0.52 mmol) was

added, and the reaction mixture was stirred at 0 °C for a further 2 h.

The reaction mixture was blown down under a stream of nitrogen and

MDAP (formic acid and high pH). The solvent was dried under a

stream of nitrogen to give the title compound (41 mg, 0.10 mmol,

24% yield).

LCMS (formic acid): rt = 0.80 min, MH⁺= 422.

¹H NMR (CDCl₃, 400 MHz) δ 1.62−1.85 (m, 4H), 1.93−2.15 (m,

3H), 2.25 (s, 3H), 2.30 (s, 3H), 2.40 (s, 3H), 2.92−3.00 (m, 2H),

3.98 (s, 3H), 4.30−4.36 (m, 2H), 7.41 (s, 1H), 7.95 (s, 1H), 8.76 (s,

1H), 10.23−10.44 (br s, 1H).

LCMS (formic acid): rt = 0.71 min, MH⁺= 423.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.20−1.31 (m, 1H),

1.31−1.38 (m, 1H), 1.39−1.52 (m, 1H), 1.66 (d, J = 7.1 Hz, 3H),

1.85−1.94 (m, 1H), 2.12 (s, 3H), 2.30 (s, 3H), 2.55−2.68 (m, 1H),

3.21 (dt, J = 11.6, 2.5 Hz, 1H), 3.37 (dt, J = 11.4, 2.8 Hz, 1H), 3.72−

3.79 (m, 1H), 3.92−3.96 (m, 4H), 4.66−4.76 (m, 1H), 7.53 (s, 1H),

7.82 (s, 1H), 8.11−8.14 (br s, 1H) 8.54 (s, 1H).

1-(2-(Dimethylamino)ethyl)-7-(3,5-dimethylisoxazol-4-yl)-8-me-

thoxy-1H-imidazo[4,5-c]quinolin-2(3H)-one (18). 4-((2-

(Dimethylamino)ethyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-me-

thoxyquinoline-3-carboxamide 52 (501 mg, 1.31 mmol) and

potassium hydroxide (110 mg, 1.96 mmol) were taken up in

methanol (20 mL) and stirred at room temperature for 20 min. The

reaction mixture was cooled in an ice bath and iodobenzene diacetate

(547 mg, 1.70 mmol) was added, and the mixture was stirred at 0 °C

for 3 h. The reaction mixture was concentrated in vacuo. The residue

was taken up in dichloromethane and puriﬁed by silica column

chromatography, eluted with 1−10% MeOH in DCM. The

appropriate fractions were combined and concentrated in vacuo to

give the title compound (234 mg, 0.58 mmol, 45% yield) as a yellow

oil.

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-1-(tetrahydro-2H-

pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (15). 7-

(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-(tetrahydro-2H-pyran-

4-ylamino)-3-quinolinecarboxamide 39 (0.34 g, 0.86 mmol) and

potassium hydroxide (0.072 g, 1.29 mmol) were dissolved in

methanol (10 mL), the mixture was cooled in an ice bath, and then

iodobenzene diacetate (0.36 g, 1.12 mmol) was added, and the

mixture was stirred for 3 h. The reaction mixture was evaporated in

vacuo. The residue was dissolved in DCM and puriﬁed by silica

column and then eluted with 0−50% 2 M methanolic ammonia/

DCM. The product-containing fractions were evaporated in vacuo to

give a brown gum. The product was triturated with EtOAc (3 mL) to

give a pale yellow solid, which was collected by ﬁltration and washed

with ether (5 mL) to give the title compound (0.23 g, 0.58 mmol,

68% yield) as pale yellow powder.

LCMS (formic acid): rt = 0.46 min, MH⁺= 382.

¹H NMR (CDCl₃, 400 MHz) δ 2.25 (s, 3H) 2.40 (s, 3H) 2.42 (s,

6H) 2.88 (t, 2H, J = 7.5 Hz) 4.01 (s, 3H) 4.54 (t, 2H, J = 7.5 Hz)

7.61 (s, 1H) 7.94 (s, 1H) 8.73 (s, 1H) 10.09 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-(pyridin-2-ylmethyl)-

1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (19). A ﬂask was

charged with 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquino-

line-3-carboxamide 39 (6.0 g, 18 mmol) and pyridin-2-ylmethan-

amine (4.23 g, 39.1 mmol) then was ﬁlled with CH₃CN (100 mL),

and the resulting mixture was stirred at reﬂux for 4 h and then was

cooled to room temperature and concentrated in vacuo. The residue

was dissolved in water, and the aqueous phase was extracted with

DCM. The combined organics were dried over Na₂SO₄and

concentrated in vacuo to give 7-(3,5-dimethylisoxazol-4-yl)-6-

methoxy-4-((pyridin-2-ylmethyl)amino)quinoline-3-carboxamide

(5.84 g, 14.5 mmol) which was used in the next step without further

puriﬁcation. This crude material was suspended in CH₃CN and then

was treated at room temperature with phenyl-λ³-iodanediyl bis(2,2,2-

triﬂuoroacetate) (19.4 g, 45.0 mmol), and the resulting mixture was

stirred at 50 °C for 12 h and then was cooled to room temperature

and concentrated in vacuo. The residue was dissolved in water, and

the aqueous phase was extracted with DCM. The combined organics

LCMS (formic acid): rt = 0.64 min, MH⁺= 395.

¹H NMR (CDCl₃, 400 MHz) δ 2.02−2.09 (m, 2H), 2.26 (s, 3H),

2.40 (s, 3H), 2.95−3.05 (m, 2H), 3.66−3.72 (m, 2H), 4.03 (s, 3H),

4.28−4.36 (m, 2H), 5.12−5.27 (br s, 1H), 7.17−7.30 (br s, 1H), 7.97

(s, 1H), 8.76 (s, 1H), 10.10−10.19 (br s, 1H).

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-1-(4-piperidinyl)-

1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one (16). 1,1-Dimethy-

lethyl 4-{[3-(aminocarbonyl)-7-(3,5-dimethyl-4-isoxazolyl)-6-(meth-

yloxy)-4-quinolinyl]amino}-1-piperidinecarboxylate 50 (0.50 g, 1.01

mmol) was dissolved in methanol (5 mL), and then potassium

hydroxide (0.085 g) was added, the mixture was cooled to 0 °C, and

iodobenzene diacetate (0.422 g, 1.31 mmol) was added. The mixture

was stirred for 4 h and then evaporated in vacuo. The residue was

dissolved in DCM (5 mL), loaded onto a 25 g silica column, and

eluted with 0−10% 2 M methanolic ammonia/DCM. Product-

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were dried over Na₂SO₄and concentrated in vacuo. Puriﬁcation of the

residue by ﬂash chromatography on silica gel (eluent: 95/5 EtOAc/

MeOH) gave after trituration with CH₃CN/Et₂O the title compound

(3.90 g, 54% over two steps) as a beige powder.

mg, 0.09 mmol) and cyanic bromide (9.9 mg, 0.09 mmol) were

heated in ethanol (5 mL) for 5 h at 60 °C and then evaporated in

vacuo to give a dark brown solid. This was puriﬁed by MDAP (high

pH) to give the title compound (12 mg, 0.03 mmol, 32% yield) as a

pale yellow solid.

LCMS (formic acid): rt = 0.71 min, MH⁺= 402.

LCMS (formic acid): rt = 0.61 min, MH⁺= 401.

¹H NMR (DMSO-d₆, 400 MHz) δ 2.09 (s, 3H), 2.30 (s, 3H), 3.74

(s, 3H) 5.75 (s, 2H), 7.33−7.37 (m, 1H), 7.49−7.56 (m, 2H), 7.82−

7.88 (m, 1H), 7.94 (s, 1H), 8.48−8.53 (m, 1H), 8.88 (s, 1H), 12.33

(br s, 1H).

¹H NMR (CD₃OD, 400 MHz) δ 2.24 (s, 3H), 2.42 (s, 3H), 3.83

(s, 3H), 6.00 (s, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.46−7.50 (m, 2H),

7.89−7.94 (m, 2H), 8.72−8.76 (m, 1H), 8.89 (s 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-N-(2-methoxyethyl)-1-

(pyridine-2-ylmethyl)-1H-imidazo[4,5-c]quinoline-2-amine (25).

To a solution of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyr-

idine-2-ylmethyl)quinoline-3,4-diamine 25 (875 mg, 2.33 mmol) in

EtOH (50 mL) was added 1-isothiocyanato-2-methoxyethane (0.51

mL, 4.66 mmol), and the reaction mixture was heated at 60 °C for 6

h. The solvent was evaporated under reduced pressure. EDC (894 mg,

4.66 mmol) and THF (50 mL) were added, and the reaction mixture

was stirred at 60 °C for 1 h, followed by stirring at room temperature

overnight. The solvent was removed under reduced pressure. The

sample was loaded in MeOH/DCM (and the column dried in a

vacuum oven) and puriﬁed by SPE (silica, 100 g) using a gradient of

0−10% (2 M ammonia in MeOH) in DCM. The appropriate

fractions were combined and evaporated under reduced pressure to

give a brown gum (310 mg). A portion (40 mg) of this gum was

dissolved in DMSO (1 mL) and puriﬁed by MDAP (high pH). The

solvent was blown down a stream of nitrogen to give the title

compound (23 mg, 0.05 mmol, 2% yield) as a white solid.

LCMS (formic acid): rt = 0.68 min, MH⁺= 459.

7-(3,5-Dimethyl-4-isoxazolyl)-2-methyl-8-(methyloxy)-1-(2-pyri-

dinylmethyl)-1H-imidazo[4,5-c]quinoline (20). A solution of N-(7-

(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((pyridin-2-ylmethyl)-

amino)quinolin-3-yl)acetamide 54 (80 mg, 0.19 mmol) in glacial

acetic acid (1 mL) was reﬂuxed for 2 h. The reaction mixture was

cooled, and the solvent was evaporated. The residue was chromato-

graphed using 2−10% methanol in dichloromethane to give the title

compound (37 mg, 0.09 mmol, 48% yield) as a colorless solid.

LCMS (formic acid): rt = 0.65 min, MH⁺= 400.

¹H NMR (DMSO-d₆, 400 MHz): δ 2.07 (s, 3H), 2.28 (s, 3H), 2.76

(s, 3H), 3.74 (s, 3H), 6.08 (s, 2H), 7.30−7.38 (m, 2H), 7.48 (s, 1H),

7.82 (dt, 1H, J = 10.0 Hz, 4.0 Hz), 7.91 (s, 1H), 8.53 (d, 1H, J = 10.0

Hz), 9.03 (s, 1H).

7-(3,5-Dimethyl-4-isoxazolyl)-2-ethyl-8-(methyloxy)-1-(2-pyridi-

nylmethyl)-1H-imidazo[4,5-c]quinoline (21). Glacial acetic acid (2

mL) was added to N-{7-(3,5-dimethyl-4-isoxazolyl)-6-(methyloxy)-4-

[(2-pyridinylmethyl)amino]-3-quinolinyl}propanamide 55 (100 mg,

0.18 mmol), and the mixture was reﬂuxed for 2 h. The solvent was

evaporated, and the residue was chromatographed using 2−10%

methanol in dichloromethane, followed by trituration with diethyl

ether to give the title compound (40 mg, 0.10 mmol, 42% yield) as an

oﬀ-white solid.

¹H NMR (DMSO-d₆, 393 K): δ 2.08 (s, 3H), 2.27 (s, 3H), 3.35 (s,

3H), 3.63−3.71 (m, 4H), 3.73 (s, 3H), 5.83 (s, 2H), 6.77−6.88 (br

m, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.27−7.35 (m, 1H), 7.45 (s, 1H),

7.72−7.83 (m, 2H), 8.58 (d, J = 4.3 Hz, 1H), 8.81 (s, 1H).

¹³C NMR (DMSO-d₆, 303 K): δ 10.3, 11.3, 42.5, 55.4, 58.0, 70.5,

98.8, 112.5, 116.8, 118.5, 120.8, 123.0, 131.7, 132.5, 131.7, 132.5,

136.7, 138.8, 139.5, 149.5, 154.3, 155.5, 156.5, 159.1, 165.7.

IR (neat): 3233 (imine NH), 1611 (aryl), 1572 (aryl), 1221 (aryl

ether), 777 (aryl C−H) cm⁻¹.

LCMS (formic acid): rt = 0.70 min, MH⁺= 414.

¹H NMR (CDCl₃, 400 MHz): δ 1.56 (t, 3H, J = 12.0 Hz), 2.15 (s,

3H), 2.32 (s, 3H), 3.10 (q, 2H, J = 12.0 Hz), 3.63 (s, 3H), 5.93 (s,

2H), 6.78 (d, 1H, J = 10.0 Hz), 7.23−7.34 (m, 2H), 7.64 (dt, 1H, J =

10.0 Hz, 4.0 Hz), 7.99 (s, 1H), 8.70 (d, 1H, J = 4.0 Hz), 9.23 (s, 1H).

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-2-propyl-1-(2-pyri-

dinylmethyl)-1H-imidazo[4,5-c]quinoline (22). Glacial acetic acid (2

mL) was added to N-{7-(3,5-dimethyl-4-isoxazolyl)-6-(methyloxy)-4-

[(2-pyridinylmethyl)amino]-3-quinolinyl}butanamide 56 (45 mg, 0.1

mmol), and the mixture was reﬂuxed for 2 h. The reaction mixture

was cooled to room temperature, and the solvent was evaporated. The

residue was chromatographed in 2−5% methanol in dichloromethane

and then triturated with hexane/diethyl ether to give the title

compound (23 mg, 0.054 mmol, 53% yield) as an oﬀ-white solid.

LCMS (formic acid): rt = 0.75 min, MH⁺= 428.

M.p.: 245−252 °C.

HRMS: (C₂₅H₂₇N₆O₃) requires MH⁺459.2145, found MH⁺

459.2151.

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

methyl]-1-(2-pyridinylmethyl)-1H-imidazo[4,5-c]quinoline (26).

Glacial acetic acid (2 mL) was added to N-{7-(3,5-dimethyl-4-

isoxazolyl)-6-(methyloxy)-4-[(2-pyridinylmethyl)amino]-3-quinolin-

yl}-2-(methyloxy)acetamide 58 (70 mg, 0.16 mmol). The mixture

was reﬂuxed for 2 h. The reaction mixture was cooled, and the solvent

was evaporated. The residue was chromatographed using 2−10%

methanol in dichloromethane to give the title compound (53 mg, 0.12

mmol, 79% yield) as an oﬀ-white solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.04 (t, 3H, J = 12.0 Hz),

1.82−1.93 (m, 2H), 2.07 (s, 3H), 2.27 (s, 3H), 3.06 (q, 2H, J = 12.0

Hz), 3.70 (s, 3H), 6.08 (s, 1H), 7.27 (d, 1H, J = 12.0 Hz), 7.30−7.34

(m, 2H), 7.45 (s, 1H), 7.80 (dt, 1H, J = 10.0 Hz, 4.0 Hz), 7.90 (s,

1H), 8.50−8.54 (m, 1H), 9.06 (s, 1H).

LCMS (formic acid): rt = 0.71 min, MH⁺= 430.

¹H NMR (DMSO-d₆, 400 MHz): δ 2.14 (s, 3H), 2.31 (s, 3H), 3.46

(s, 3H), 3.62 (s, 3H,), 4.93 (s, 2H), 6.05 (s, 2H), 6.86 (d, 1H, J = 12.0

Hz) 7.23−7.28 (m, 1H), 7.35 (s, 1H), 7.62 (dt, 1H, J = 12.0 Hz, 4.0

Hz), 7.99 (s, 1H), 8.66 (d, 1H, J = 4.0 Hz), 9.24 (s, 1H).

4-(8-Methoxy-1-(pyridin-2-ylmethyl)-2-(tetrahydro-2H-pyran-4-

yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole (27). To

7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyridin-2-ylmethyl)-

quinoline-3,4-diamine 53 (2.0 g, 5.33 mmol) in DCM was added

HATU (3.65 g, 9.6 mmol), NEt₃(excess), and tetrahydro-2H-pyran-

4-carboxylic acid (693 mg, 9.6 mmol), and the reaction mixture was

stirred at room temperature overnight. The reaction mixture was

partitioned between saturated aqueous sodium bicarbonate solution

and dichloromethane. The organic layer was dried using Na₂SO₄,

ﬁltered, and evaporated to dryness to give the intermediate 59 (2.5 g).

Acetic acid (30 mL) was added, and the reaction mixture was heated

at 90 °C for 6 h. The reaction mixture was evaporated to dryness,

resuspended in saturated aqueous sodium bicarbonate solution, and

extracted with dichloromethane. The organic layer was dried using

Na₂SO₄, ﬁltered, and evaporated to dryness. The material was puriﬁed

using silica column chromatography, eluted with 5% methanol in

7-(3,5-Dimethyl-4-isoxazolyl)-2-(1-methylethyl)-8-(methyloxy)-

1-(2-pyridinylmethyl)-1H-imidazo[4,5-c]quinoline (23). Glacial ace-

tic acid (2 mL) was added to N-{7-(3,5-dimethyl-4-isoxazolyl)-6-

(methyloxy)-4-[(2-pyridinylmethyl)amino]-3-quinolinyl}-2-methyl-

propanamide 57 (50 mg, 0.11 mmol), and the mixture reﬂuxed for 2

h. The reaction mixture was cooled to room temperature, and the

solvent was evaporated. The residue was chromatographed using 2−

5% methanol in dichloromethane, and the product was triturated with

hexane/diethyl ether to give the title compound (25 mg, 0.06 mmol,

52% yield) as an oﬀ-white solid.

LCMS (formic acid): rt = 0.74 min, MH⁺= 428.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.39 (d, 6H, J = 12.0 Hz), 2.07

(s, 3H), 2.27 (s, 3H), 3.52−3.60 (m, 1H), 3.66 (s, 3H), 6.12 (s, 2H),

7.22 (d, 1H, J = 12.0 Hz), 7.29−7.34 (m, 1H), 7.40 (s, 1H), 7.80 (dt,

1H, J = 10.0 Hz, 4.0 Hz), 7.90 (s, 1H,), 8.51−8.54 (m, 1H), 9.07 (s,

1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-(pyridin-2-ylmethyl)-

1H-imidazo[4,5-c]quinolin-2-amine (24). 7-(3,5-Dimethylisoxazol-4-

yl)-6-methoxy-N⁴-(pyridin-2-ylmethyl)quinoline-3,4-diamine 53 (35

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dichloromethane. Recrystallization in ethanol gave the title compound

(1.88 g, 4.0 mmol, 75% yield) as a beige solid.

blown down under a stream of nitrogen, and the residue was dissolved

in DMSO (3 mL) and puriﬁed by MDAP (high pH). The solvent was

blown down under a stream of nitrogen to give the title compound

(32 mg, 0.074 mmol, 27% yield) as a colorless gum.

LCMS (formic acid): rt = 0.64 min, MH⁺= 470.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.75−2.10 (m, 7H), 2.27 (s,

3H), 3.29−3.39 (m, 1H), 3.47−3.62 (m, 2H), 3.67 (s, 3H), 3.90−

4.01 (m, 2H), 6.17 (s, 2H), 7.21−7.27 (m, 1H), 7.28−7.36 (m, 1H),

7.39 (s, 1H), 7.76−7.83 (m, 1H), 7.91 (s, 1H), 8.49−8.54 (m, 1H),

9.10 (s, 1H).

LCMS (formic acid): rt = 0.78 min, MH⁺= 433.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 0.48−0.55 (m, 2H)

0.64−0.68 (m, 2H), 1.38−1.52 (m, 1H), 1.89−1.92 (m, 2H), 2.01−

2.10 (m, 2H), 2.15 (s, 3H), 2.34 (s, 3 H), 3.30−3.43 (m, 1H), 3.56−

3.68 (m, 2H), 3.99−4.09 (m, 5H), 4.71 (d, J = 6.0 Hz, 2H), 7.89 (s,

1H), 7.97 (s, 1H), 9.04 (s, 1H).

4-(8-Methoxy-2-(methoxymethyl)-1-(1-methoxypropan-2-yl)-

1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxazole (28). A sol-

ution of 2-methoxyacetic acid (43 μL, 0.56 mmol), HATU (229 mg,

0.60 mmol), and DIPEA (243 μL, 1.39 mmol) in DMF (2 mL) was

stirred at room temperature for 15 min. 7-(3,5-Dimethylisoxazol-4-

yl)-6-methoxy-N⁴-(1-methoxypropan-2-yl)quinoline-3,4-diamine 70

(165 mg, 0.463 mmol) was added, and the reaction mixture was

stirred at room temperature over the weekend. A solution of 2-

methoxyacetic acid (22 μL, 0.28 mmol), HATU (115 mg, 0.30

mmol), and DIPEA (121 μL, 0.69 mmol) in DMF (1 mL) was stirred

at room temperature for 15 min. This was added to the reaction

mixture, which was then stirred at room temperature for 6 h. The

reaction mixture was blown down under a stream of nitrogen. The

sample was loaded in DCM and puriﬁed by SPE (aminopropyl, 2 g),

eluted using 10% MeOH in DCM. The appropriate fractions were

combined and blown down under a stream of nitrogen to give a

brown gum. The gum was puriﬁed by MDAP (high pH). The solvent

was blown down under a stream of nitrogen to give a colorless gum.

The gum was dissolved in acetic acid (1.5 mL, 0.34 mmol), and the

reaction mixture was heated under microwave conditions at 120 °C

for 1 h. The reaction mixture was blown down under a stream of

nitrogen. The resulting gum was puriﬁed by MDAP (high pH). The

solvent was blown down under a stream of nitrogen to give the title

compound 15 (40 mg, 0.10 mmol, 21% yield) colorless glass.

LCMS (formic acid): rt = 0.75 min, MH⁺= 491.

4-(8-Methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-

pyran-4-yl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxa-

zole (31). A solution of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-

(1-methoxypropan-2-yl)quinoline-3,4-diamine 70 (9.1 g, 25.5 mmol)

in DCM (300 mL) was treated with pyridine (30 mL) and tetrahydro-

2H-pyran-4-carbonyl chloride (5.0 mL, 41.1 mmol). The solution was

stirred under nitrogen at room temperature for 3.5 h and then left

standing overnight. The reaction mixture was concentrated under

reduced pressure, and the residue partitioned between DCM and

water. The organic phase was washed with water (×2), and the

combined aqueous layers were extracted with DCM. The combined

organic layers were washed with brine, dried using a hydrophobic frit,

and evaporated under reduced pressure. The combined aqueous

layers, including the brine wash, were basiﬁed with solid sodium

bicarbonate, and the aqueous layer was twice extracted with DCM.

The organic fractions were dried using a hydrophobic frit, combined

with the previously extracted material, and evaporated to dryness

under reduced pressure to give a brown foam. This foam was further

dried in vacuo to give 13.5 g of material. The material was triturated

with diethyl ether, the suspension was chilled (ice/water bath), and

the solid was isolated by ﬁltration. The solid was washed with a little

diethyl ether and air-dried to give a beige solid (11.95 g). This solid

and p-toluenesulfonic acid (1.2 g, 25.5 mmol) in toluene (250 mL)

was heated at reﬂux, under nitrogen, using a Dean−Stark apparatus

for 3 days. Further p-toluenesulfonic acid (0.2 g, 4.3 mmol) was

added, and heating was continued overnight. The reaction mixture

had boiled dry. Water (750 mL) was added, followed by saturated

aqueous sodium bicarbonate solution until the pH reached 8. The

aqueous layer was extracted with EtOAc (3 × 750 mL). The organic

layers were combined, dried using a hydrophobic frit, and evaporated

under reduced pressure to give a light brown solid, ∼11 g. The solid

was triturated in ether (∼200 mL) and placed brieﬂy in an ultrasonic

bath, and the majority of the solid appeared to be a ﬁne powder. The

ﬁne powder suspended in the ether was decanted, and the solid was

isolated by ﬁltration and dried in a vacuum oven to give 6.3 g of

material. This material was triturated in ether (∼100 mL), placed

brieﬂy in an ultrasonic bath, and stood overnight at room

temperature. The solid was isolated by ﬁltration and dried in a

vacuum oven to give the title compound (4.9 g). The clumped solid

left over from the decanting was triturated in ether (100 mL), placed

in an ultrasonic bath for 15 min, and stood at room temperature for 3

days. The solid was isolated by ﬁltration and dried in a vacuum oven

to give the title compound (3.6 g). The total amount of material

obtained was 8.5 g, 74% yield.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.81 (d, J = 7.1 Hz,

3H), 2.16 (s, 3H), 2.35 (s, 3H), 3.31 (s, 2H), 3.43 (s, 2H), 3.94−4.05

(m, 4H), 4.79−4.92 (m, 2H), 5.42−5.56 (m, 1H), 7.83 (s, 1H), 8.00

(s, 1H), 9.06 (s, 1H).

4-(8-Methoxy-2-(methoxymethyl)-1-((tetrahydro-2H-pyran-4-

yl)methyl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxazole

(29). A solution of 2-methoxyacetic acid (15 μL, 0.19 mmol), HATU

(81 mg, 0.21 mmol), and DIPEA (84 μL, 0.48 mmol) in DMF (2

mL) was stirred at room temperature for 15 min. 7-(3,5-

Dimethylisoxazol-4-yl)-6-methoxy-N⁴-((tetrahydro-2H-pyran-4-yl)-

methyl)quinoline-3,4-diamine 71 (37 mg, 0.097 mmol) was added,

and the reaction mixture was stirred at room temperature for 3 h. The

reaction mixture was blown down under a stream of nitrogen. The

sample was loaded in DCM and puriﬁed by SPE (aminopropyl, 2 g),

eluted using 10% MeOH in DCM. The appropriate fractions were

combined and blown down a stream of nitrogen to give a brown gum.

The gum was puriﬁed by MDAP (high pH). The solvent was blown

down a stream of nitrogen to give a colorless gum (22 mg). To this

was added acetonitrile (1.5 mL) and 10 mM aqueous ammonium

bicarbonate solution, adjusted to pH 10 using ammonia (1.5 mL),

and the reaction mixture stood at room temperature for 4 days. The

reaction mixture was blown down under a stream of nitrogen to give

the title compound (22 mg, 0.050 mmol, 52% yield) as a colorless

glass.

LCMS (formic acid): rt = 0.76 min, MH+ = 451.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.80 (d, J = 7.3 Hz,

3H), 1.83−1.96 (m, 2 H), 2.01−2.10 (m, 2H), 2.13 (s, 3H), 2.32 (s,

3H), 3.25 (s, 3H), 3.38−3.48 (m, 1H), 3.54−3.63 (m, 2H), 3.95−

4.05 (m, 6H), 4.08−4.14 (m, 1H) 5.32−5.52 (m, 1H), 7.71 (s, 1H),

7.96 (s, 1H), 9.01 (s, 1H).

LCMS (formic acid): rt = 0.70 min, MH⁺= 437.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.51−1.64 (m, 4H),

2.16 (s, 3H), 2.35−2.43 (m, 4H), 3.26−3.32 (m, 2H), 3.45 (s, 3H),

3.85−3.90 (m, 2H), 4.02 (s, 3H), 4.69 (d, J = 7.6 Hz, 2H), 4.84 (s,

2H), 7.73 (s, 1H), 8.00 (s, 1H), 9.06 (s, 1H).

4-(8-Methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-

pyran-4-yl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxa-

zole (32). The racemic 4-(1-(1-methoxypropan-2-yl)-2-(tetrahydro-

2H-pyran-4-yl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxa-

zole 31 (85 mg, 0.19 mmol) was separated by chiral HPLC. The

HPLC analysis was carried out on a Chiralpak AD-H column

“ADH10029-01” (250 × 30 mm, i.d. 5 μm packing diameter). The

puriﬁcation was run using 15% EtOH in heptane over 35 min, with a

ﬂow rate of 45 mL/min. The UV detection was at a wavelength of 300

nm. The second eluting enantiomer was collected, between 28 and 31

4-(1-(Cyclopropylmethyl)-8-methoxy-2-(tetrahydro-2H-pyran-4-

yl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxazole (30).

To a suspension of N-(cyclopropylmethyl)-7-(3,5-dimethylisoxazol-

4-yl)-6-methoxy-3-nitroquinolin-4-amine 67 (100 mg, 0.27 mmol)

and sodium hydrosulﬁte (142 mg, 0.81 mmol) in EtOH (0.8 mL) and

water (0.4 mL) was added tetrahydro-2H-pyran-4-carbaldehyde (62

mg, 0.54 mmol), and the reaction mixture was heated in under

microwave conditions at 100 °C for 1 h. The reaction mixture was

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min. Combined fraction solutions were evaporated to dryness under

reduced pressure, the residue was transferred to a vial using EtOH,

and the solvent was dried under a stream of nitrogen to leave the title

compound (39 mg, 0.087 mmol, 46% yield).

3.19 (m, 1H), 3.39−3.53 (m, 2H) 3.58−3.76 (m, 2H), 3.94−4.13 (m,

7H), 4.69−5.12 (m, 1H), 7.75 (s, 1H), 7.99 (s, 1H), 9.04 (s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-N,N-dimethyl-1-((tetra-

hydro-2H-pyran-4-yl)methyl)-1H-imidazo[4,5-c]quinoline-2-amine

(35). 7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N⁴-((tetrahydro-2H-

pyran-4-yl)methyl)quinoline-3,4-diamine 71 (50 mg, 0.13 mmol)

and N-(dichloromethylene)-N-methylmethanaminium chloride (43

mg, 0.26 mmol) were combined in dry acetonitrile (0.5 mL) and

heated under microwave conditions at 120 °C for 10 min. The

reaction mixture was blown down under a stream of nitrogen, and the

residue was dissolved in DMSO (1 mL) and puriﬁed by MDAP (high

pH). The solvent was blown down under a stream of nitrogen to give

the title compound (44 mg, 0.10 mmol, 77% yield) as a yellow gum.

LCMS (formic acid): rt = 0.70 min, MH⁺= 436.

LCMS (formic acid): rt = 0.73 min, MH⁺= 451.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.80 (d, J = 7.3 Hz,

3H), 1.83−1.98 (m, 2H), 2.00−2.10 (m, 2H), 2.13 (s, 3H), 2.32 (s,

3H), 3.26 (s, 3H), 3.39−3.49 (m, 1H), 3.54−3.62 (m, 2H), 3.95−

4.05 (m, 6H), 4.08−4.12 (m, 1H) 5.35−5.47 (br.s., 1H), 7.72 (s,

1H), 7.95 (s, 1H), 9.00 (s, 1H).

Chiral HPLC (conditions as for preparative HPLC, except column

diameter of 4.6 mm): 97% ee.

¹³C NMR (DMSO-d₆, 303 K): δ 10.3, 11.3, 17.6, 31.3, 31.7, 33.2,

51.3, 55.6, 58.5, 66.6, 74.0, 101.2, 102.6, 112.1, 117.4, 119.4, 132.1,

133.1, 137.5, 139.5, 142.7, 154.7, 159.1, 166.0.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.31−1.37 (m, 4H),

2.15 (s, 3H), 2.17−2.25 (m, 1H), 2.34 (s, 3H), 2.99 (s, 6H), 3.13−

3.26 (m, 2H), 3.74−3.84 (m, 2H), 3.99 (s, 3H), 4.51 (d, J = 7.3 Hz,

2H), 7.60 (s, 1H) 7.91 (s, 1H) 8.90 (s, 1H)

IR: 2963 (saturated CH₂), 2940 (saturated CH₂), 2852 (ether

COCH₃), 1618 (aryl), 1583 (aryl), 1221 (aryl ether), 839 (aryl C−

H) cm⁻¹.

¹³C NMR (DMSO-d₆, 300 K): δ 10.4, 11.4, 29.6, 35.0, 42.7, 50.8,

55.8, 66.2, 99.4, 112.4, 117.9, 119.6, 131.9, 132.9, 135.8, 138.9, 141.0,

155.0, 159.2, 160.6, 165.9.

M.p.: 190−192 °C.

HRMS: (C₂₅H₃₁N₄O₄) MH⁺requires 451.2340, found MH⁺

451.2331.

[α_D]^23.1°^C(c 1.0, MeOH): + 2.9°.

IR (neat): 2944 (NCH₃), 2911 (NCH₃), 2853 (NCH₃), 1574

(aryl), 1531 (aryl), 1216 (aryl ether), 845 (aryl C−H) cm⁻¹.

M.p.: 205−207 °C.

4-(8-Methoxy-1-((S)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-

pyran-4-yl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethylisoxa-

zole (33). The racemic 4-(1-(1-methoxypropan-2-yl)-2-(tetrahydro-

2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxa-

zole 31 (85 mg, 0.189 mmol) was puriﬁed by chiral HPLC. The

HPLC analysis was carried out on a Chiralpak AD-H column

“ADH10029-01” (250 × 30 mm, i.d. 5 μm packing diameter). The

puriﬁcation was run using 15% EtOH in heptane over 35 min, with a

ﬂow rate of 45 mL/min. The UV detection was at a wavelength of 300

nm. The ﬁrst eluting enantiomer was collected, between 24 and 26.5

min. Combined fraction solutions were evaporated to dryness under

reduced pressure, the residue was transferred to a vial using EtOH,

and the solvent was dried under a stream of nitrogen to leave the title

compound (38 mg, 0.084 mmol, 44% yield).

HRMS: (C₂₄H₃₀N₅O₃) MH⁺requires 436.2343, found MH⁺

436.2335.

7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-2-(4-morpholinyl)-

1-(tetrahydro-2H-pyran-4-ylmethyl)-1H-imidazo[4,5-c]quinoline

(36). 7-(3,5-Dimethyl-4-isoxazolyl)-8-(methyloxy)-1-(tetrahydro-2H-

pyran-4-ylmethyl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one 13

(220 mg, 0.54 mmol) was dissolved in POCl₃(2 mL, 21.5 mmol),

PCl₅(0.2 g, 0.96 mmol) was added, and the mixture was heated at

120 °C for 24 h. The reaction mixture was evaporated in vacuo to give

a beige solid. The solid was dissolved in NMP (0.5 mL), and

morpholine (0.3 mL, 3.44 mmol) was added, and then the solution

was heated in the microwave at 150 °C for 1 h. The solution was

loaded onto a 20 g SCX cartridge and washed with methanol (30 mL)

and then eluted with 2 M methanolic ammonia (30 mL), and the

eluant waa evaporated in vacuo to give a beige gum. This was

dissolved in DCM (3 mL) , loaded onto a 25 g silica column and then

eluted with 0−10% 2 M methanolic ammonia/DCM. Product-

containing fractions were evaporated in vacuo to give the title

compound (4.7 mg, 9.84 μmol, 2% yield) as a beige solid.

LCMS (formic acid): rt = 0.70 min, MH⁺= 478.

LCMS (formic acid): rt = 0.73 min, MH⁺= 451.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.80 (d, J = 7.3 Hz,

3H), 1.82−1.98 (m, 2 H), 2.00−2.10 (m, 2H), 2.13 (s, 3H), 2.32 (s,

3H), 3.26 (s, 3H), 3.54−3.63 (m, 2H), 3.96−4.05 (m, 7H), 5.34−

5.49 (br m, 1H), 7.72 (s, 1H), 7.95 (s, 1H), 9.00 (s, 1H).

Chiral HPLC (conditions as for preparative HPLC, except column

diameter of 4.6 mm): >99% ee.

4-(8-Methoxy-2-(tetrahydro-2H-pyran-4-yl)-1-(1-(tetrahydro-2H-

pyran-4-yl)ethyl)-1H-imidazo[4,5-c]quinoline-7-yl)-3,5-dimethyli-

soxazole (34). To a suspension of tetrahydro-2H-pyran-4-carboxylic

acid (51 mg, 0.39 mmol) and HATU (168 mg, 0.44 mmol) in DMF

(2 mL) was added DIPEA (0.136 mL, 0.779 mmol), and the resulting

solution was stirred at room temperature for 15 min. 7-(3,5-

Dimethylisoxazol-4-yl)-6-methoxy-N⁴-(1-(tetrahydro-2H-pyran-4-yl)-

ethyl)quinoline-3,4-diamine 72 (103 mg, 0.26 mmol) was added, and

the reaction mixture was stirred at room temperature overnight. The

reaction mixture was blown down under a stream of nitrogen, and the

residue was loaded in DCM and puriﬁed by SPE (aminopropyl, 5 g),

eluted using 10% MeOH in DCM. The appropriate fractions were

combined and dried under a stream of nitrogen to give a brown gum.

The gum was dissolved in DMSO (2 mL) and puriﬁed by MDAP

(formic acid). The solvent was dried under a stream of nitrogen to

give a light brown solid (45 mg). To the solid was added glacial acetic

acid (1 mL, 0.09 mmol), and the reaction mixture was heated under

microwave conditions at 120 °C for a total of 29 h. The reaction

mixture was blown down under a stream of nitrogen. The resulting

gum was dissolved in DMSO (1 mL) and puriﬁed by MDAP (high

pH). The solvent was blown down under a stream of nitrogen to give

the title compound (18.7 mg, 0.038 mmol, 15% yield) as a light pink

solid.

¹H NMR (CDCl₃, 400 MHz) δ 1.33−1.40 (m, 2H), 1.48−1.61 (m,

2H), 2.26 (s, 3H), 2.41 (s, 3H), 3.22−3.37 (m, 6H), 3.95−4.00 (m,

10H), 4.38−4.42 (m, 2H), 7.39 (s, 1H), 8.03 (s, 1H), 9.16 (s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-N-(2-methoxyethyl)-1-

((R)-1-(pyridine-2-yl)ethyl)-1H-imidazo[4,5-c]quinoline-2-amine

(37). To a solution of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-

((R)-1-(pyridine-2-yl)ethyl)quinoline-3,4-diamine 74 (538 mg, 1.38

mmol) in EtOH (40 mL) was added 1-isothiocyanato-2-methoxy-

ethane (300 μL, 2.76 mmol), and the reaction mixture was heated at

60 °C for 5.5 h. Further, 1-isothiocyanato-2-methoxyethane (150 μL,

1.38 mmol) was added, and the reaction mixture was heated at 60 °C

overnight. The solvent was removed under reduced pressure, the

residue was dissolved in THF (40 mL), EDC (530 mg, 2.76 mmol)

was added, and the mixture was heated at 60 °C for 4 h. The solvent

was removed under reduced pressure, and the sample was loaded in

MeOH/DCM (and the column dried in a vacuum oven) and puriﬁed

by SPE (silica, 100 g) using a gradient of 0−10% (2 M ammonia in

MeOH) in DCM. The appropriate fractions were combined and

evaporated under reduced pressure to give crude product (∼600 mg).

The crude product was dissolved in DMSO (5 mL) and puriﬁed by

MDAP (high pH). The samples were blown down under a stream of

nitrogen to give the title compound (122 mg, 0.26 mmol, 19% yield)

as a white solid.

LCMS (formic acid): rt = 0.75 min, MH⁺= 491.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 0.83−0.96 (m, 1H),

1.19−1.34 (m, 1H), 1.55−1.70 (m, 1H), 1.80−1.97 (m, 6H), 2.01−

2.11 (m, 2H), 2.17 (s, 3H), 2.35 (s, 3H), 2.55−2.70 (m, 1H), 3.06−

LCMS (formic acid): rt = 0.73 min, MH⁺= 473.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 2.06−2.11 (m, 6H),

2.27 (s, 3H), 3.35 (s, 3H), 3.59 (s, 3H), 3.62−3.73 (m, 4H), 6.32 (q,

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J = 7.1 Hz, 1H), 6.77−6.85 (br m, 1H), 6.96 (s, 1H), 7.30 (d, J = 7.8

Hz, 1H), 7.33−7.39 (m, 1H), 7.75−7.81 (m, 2H), 8.68 (d, J = 4.0 Hz,

1H), 8.82 (s, 1H).

¹H NMR (CDCl₃, 400 MHz) δ 0.86 (t, 3H, J = 7.2 Hz) 1.27−1.49

(m, 5H) 1.52−1.76 (m, 2H) 2.16 (s, 3H) 2.30 (s, 3H) 3.88 (s, 3H)

4.07−4.26 (m, 1H) 6.49−7.02 (m, 1H) 7.47 (s, 1H) 7.67 (s, 1H)

8.71 (d, 1H, J = 9.3 Hz) 8.77 (s, 1H).

¹³C NMR (DMSO-d₆, 303 K): δ 10.3, 11.3, 17.7, 42.8, 55.3, 58.1

(br, 2 C), 70.5, 112.3, 113.9, 116.7, 118.3, 121.1, 121.2, 122.9, 132.7,

137.8, 138.5, 139.9, 149.5, 149.6, 154.0, 155.7, 158.9, 159.1, 165.8

IR (neat): 3239 (imine NH), 1591 (aryl), 1556 (aryl), 1219 (aryl

ether), 772 (aryl C−H) cm⁻¹.

Batch 2: the title compound (178 mg, 0.42 mmol, 28% yield) as a

yellow solid.

LCMS (formic acid): rt = 0.73 min, MH⁺= 383,

¹H NMR (CDCl₃, 400 MHz) δ 0.89−0.94 (t, 3H, J = 6.3 Hz), 1.41

(d, 3H, J = 6.3 Hz), 1.42−1.55 (m, 2H), 1.61−1.79 (m, 2H), 2.22 (s,

3H), 2.36 (s, 3H), 3.95 (s, 3H), 4.17−4.24 (m, 1H), 6.5−7.5 (br s,

2H), 7.53 (s, 1H), 7.72 (s, 1H), 8.83−8.88 (m, 2 H).

M.p.: 109−111 °C.

HRMS: (C₂₆H₂₉N₆O₃) MH⁺requires 473.2296, found MH⁺

473.2284.

[α_D]^23.1°^C(c 1.0, MeOH): + 118.7°.

4-((Cyclopropylmethyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-

methoxyquinoline-3-carboxamide (43). To a suspension of 4-

chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinoline-3-carboxa-

mide 39 (500 mg, 1.51 mmol) in dry NMP (2.5 mL) were added

DIPEA (0.658 mL, 3.77 mmol) and cyclopropylmethanamine (0.163

mL, 1.88 mmol). The reaction mixture was heated under nitrogen at

100 °C for 3.5 h. Further cyclopropylmethanamine (0.163 mL, 1.88

mmol) was added, and the reaction mixture was heated under

nitrogen at 100 °C overnight. The sample was loaded directly onto

and puriﬁed by SCX. The column was washed with 10% MeOH in

DCM and eluted using 2 M ammonia in MeOH. The appropriate

fractions were combined and evaporated under reduced pressure to

give a brown gum. The compound was triturated in EtOAc; the solid

was removed and dried in a vacuum oven overnight to give the title

compound (371 mg, 1.01 mmol, 67% yield) as a yellow solid.

LCMS (formic acid): rt = 0.70 min, MH⁺= 367.

7-(3,5-Dimethyl-4-isoxazolyl)-4-[(1-methylethyl)amino]-6-

(methyloxy)-3-quinolinecarboxamide, (40). 4-Chloro-7-(3,5-di-

methyl-4-isoxazolyl)-6-(methyloxy)-3-quinolinecarboxamide 39 (500

mg, 1.51 mmol) was taken up in NMP (1.5 mL). DIPEA (0.66 mL,

3.77 mmol) was added, followed by 2-propanamine (0.16 mL, 1.88

mmol). The mixture was stirred at 120 °C for 2 h. Another equivalent

of 2-propanamine was added, and the reaction mixture was left to

stand for three nights and then reheated to 120 °C for a further 1 h.

The brown solution was loaded on to a 20 g SCX cartridge. The

cartridge was eluted with methanol and then 2 M NH₃in methanol.

The appropriate fractions were concentrated in vacuo. The crude

product was loaded in the minimum amount of DCM and puriﬁed by

column chromatography (silica), eluted using 1−10% of NH₃/MeOH

in DCM. The appropriate fractions were combined and concentrated

in vacuo to give the title compound (224 mg, 0.57 mmol, 38% yield)

as a brown solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 0.27−0.34 (m, 2H), 0.48−0.56

(m, 2H), 1.12−1.23 (m, 1H), 2.13 (s, 3H) 2.32 (s, 3H), 3.50−3.56

(m, 2H) 3.91 (s, 3H), 7.33−7.44 (br s, 1H), 7.64−7.67 (m, 2H),

7.97−8.07 (br s, 1H), 8.60 (s, 1H) 8.75 (t, J = 5.3 Hz, 1 H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-((1-methoxypropan-

2-yl)amino)quinoline-3-carboxamide (44). 4-Chloro-7-(3,5-dime-

thylisoxazol-4-yl)-6-methoxyquinoline-3-carboxamide 39 (100 mg,

0.30 mmol), 1-methoxypropan-2-amine (0.04 mL, 0.38 mmol), and

DIPEA (0.13 mL, 0.75 mmol) were taken up in N-methyl-2-

pyrrolidone (0.5 mL). The mixture was stirred at 120 °C for 4 h.

Further 1-methoxypropan-2-amine was added (0.016 mL), and the

mixture continued to be stirred at 120 °C for another 4 h. The

reaction mixture was cooled and puriﬁed by SCX, eluted with

methanol and then 2 M NH₃in MeOH. The appropriate fractions

were combined and concentrated in vacuo to give the crude title

compound (116 mg) as a brown solid which was used directly in the

next reaction without further puriﬁcation.

LCMS (formic acid): rt = 0.66 min, MH⁺= 355.

¹H NMR (CDCl₃, 400 MHz) δ 1.42 (d, 6H, J = 6.3 Hz) 2.24 (s,

3H) 2.39 (s, 3H) 3.93 (s, 3H) 4.26−4.40 (m, 1H) 5.79−6.07 (m,

2H) 7.51 (s, 1H) 7.74 (s, 1H) 8.69 (s, 2H).

7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-[(2-

methylpropyl)amino]-3-quinolinecarboxamide (41). 4-Chloro-7-

(3,5-dimethyl-4-isoxazolyl)-6-(methyloxy)-3-quinolinecarboxamide

39 (500 mg, 1.51 mmol) was taken up in NMP (1.5 mL). DIPEA

(0.66 mL, 3.77 mmol) was added, followed by isobutylamine (0.19

mL, 1.88 mmol). The reaction mixture was stirred at 120 °C for 2 h.

The brown solution was puriﬁed by SCX, eluted with methanol and

then 2 M NH₃in MeOH. The basic fractions were concentrated in

vacuo. The methanol fraction was repuriﬁed by SCX, eluted with

methanol and then 2 M NH₃in MeOH. The appropriate fractions

were concentrated in vacuo. NMP was found to be in the product.

This mixture was repuriﬁed by SCX, eluted with methanol and 2 M

NH₃in MeOH. The appropriate fractions were concentrated in vacuo.

The NH₃in MeOH fractions from all the SCX puriﬁcations was

combined. The crude product was loaded in the minimum amount of

DCM and puriﬁed by column chromatography (silica), eluted using

1−10% NH₃in MeOH. The appropriate fractions were combined and

concentrated in vacuo to give the title compound (879 mg, 1.19

mmol, 79% yield) as an orange solution. Minimum purity was 50%

due to residual NMP.

LCMS (formic acid): rt = 0.69 min, MH⁺= 385.

¹H NMR (CDCl₃, 400 MHz) δ 1.36 (d, 3H, J = 6.6 Hz) 2.23−2.27

(m, 5H) 2.40 (s, 4H) 3.44 (s, 3H) 3.55 (d, 2H, J = 5.6 Hz) 3.95 (s,

3H) 4.25−4.38 (m, 1H) 7.76 (s, 1H) 7.77 (s, 1H) 8.34 (d, 1H, J =

1.0 Hz) 8.73 (s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-(((tetrahydrofuran-

3-yl)methyl)amino)quinoline-3-carboxamide (45). To a suspension

of 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinoline-3-car-

boxamide 39 (1 g, 3.01 mmol) in dry N-methyl-2-pyrrolidinone

(2.5 mL) was added DIPEA (1.32 mL, 7.54 mmol) and

(tetrahydrofuran-3-yl)methanamine (0.381 g, 3.77 mmol). The

reaction mixture was heated under nitrogen at 100 °C for 2 h and

then stood at room temperature overnight. The solution was added

directly onto, and puriﬁed by, SCX. The column was washed with

10% MeOH in DCM and eluted with 50% (2 M ammonia in MeOH)

in DCM. The appropriate fractions were combined and evaporated

under reduced pressure to give a brown gum. The gum was triturated

in EtOAc (∼15 mL); the solid was removed by ﬁltration and dried in

a vacuum oven for 2 h to give the title compound (671 mg, 1.69

mmol, 56% yield) as a pale brown solid.

LCMS (formic acid): rt = 0.73 min, MH⁺= 369.

¹H NMR (CDCl₃, 400 MHz) δ 1.07 (d, 6H, J = 6.8 Hz) 2.21 (s,

3H) 2.38 (s, 3H) 3.62 (dd, 1H, J = 6.4, 5.4 Hz) 3.89 (s, 3H) 6.16 (bs,

2H) 7.57 (s, 1H) 7.69 (s, 1H) 8.68 (s, 1H) 9.36 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-(pentan-2-ylamino)-

quinoline-3-carboxamide (42). 4-Chloro-7-(3,5-dimethylisoxazol-4-

yl)-6-methoxyquinoline-3-carboxamide 39 (500 mg, 1.51 mmol) was

taken up in NMP (1.5 mL), pentan-2-amine (0.22 mL, 1.88 mmol)

and DIPEA (0.66 mL, 3.77 mmol) were added, and the mixture was

stirred at 120 °C for 3 h. The reaction mixture was cooled and

puriﬁed by SCX, eluted with methanol and then 2 M NH₃in MeOH.

The appropriate fractions were concentrated in vacuo and loaded in

the minimum amount of DCM and puriﬁed by column chromatog-

raphy (silica), eluted using 1−10% NH₃in MeOH. The fractions were

combined and concentrated in vacuo in two batches. Batch 1: the title

compound (316 mg, 0.78 mmol, 52% yield) as an orange solid.

LCMS (formic acid): rt = 0.73 min, MH⁺= 383.

LCMS (formic acid): rt = 0.61 min, MH⁺= 397.

¹H NMR (DMSO-d₆): δ 1.55−1.65 (m, 1H), 1.96−2.05 (m, 1H),

2.13 (s, 3H), 2.32 (s, 3H), 2.55−2.64 (m, 1H), 3.47 (dd, J = 9.0, 6.0

Hz, 1 H) 3.56−3.66 (m, 3H) 3.71−3.80 (m, 2H), 3.92 (s, 3H), 7.42−

7.51 (br s, 1H), 7.66 (s, 1H), 7.67 (s, 1H), 8.00−8.08 (br s, 1H), 8.45

(br t, 1H), 8.57 (s, 1H).

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7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-(((tetrahydro-2H-

pyran-3-yl)methyl)amino)quinoline-3-carboxamide (46). To a

suspension of 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyqui-

noline-3-carboxamide 39 (1 g, 3.01 mmol) in dry N-methyl-2-

pyrrolidinone (2.5 mL) was added DIPEA (1.32 mL, 7.54 mmol) and

(tetrahydro-2H-pyran-3-yl)methanamine (0.434 g, 3.77 mmol). The

reaction mixture was heated under nitrogen at 100 °C for 2 h and

then stood at room temperature overnight. The solution was added

directly onto, and puriﬁed by, SCX. The column was washed with

10% MeOH in DCM and eluted with 50% (2 M ammonia in MeOH)

in DCM. The appropriate fractions were combined and evaporated

under reduced pressure to give a brown gum. The gum was triturated

in EtOAc (∼15 mL); the solid was removed by ﬁltration and dried in

a vacuum oven overnight to give the title compound (614 mg, 1.50

mmol, 50% yield) as a pale brown solid.

mL), and the eluant was dried in vacuo. The residue was dissolved in

DCM (10 mL) and loaded onto a 50 g silica column and then eluted

with 0−50% 2 M methanolic ammonia/DCM to give the title

compound (0.34 g, 0.86 mmol, 57% yield) as a beige crystalline solid.

LCMS (formic acid): rt = 0.59 min, MH⁺= 397.

¹H NMR (CDCl₃, 400 MHz) δ 1.78−1.88 (m, 2H), 2.07−2.15 (m,

2H), 2.18 (s, 3H), 2.23 (s, 3H), 3.48−3.54 (m, 2H), 3.92 (s, 3H),

4.02−4.07 (m, 2H), 4.11−4.18 (m, 1H), 6.0−6.5 (br s, 2H), 7.43 (s,

1H), 7.76 (s, 1H), 8.73 (s, 1H), 8.70−8.79 (m, 1H).

tert-Butyl 4-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-me-

thoxyquinolin-4-yl)amino)piperidine-1-carboxylate (50). 4-Chloro-

7-(3,5-dimethyl-4-isoxazolyl)-6-(methyloxy)-3-quinolinecarboxamide

39 (0.5 g, 1.51 mmol) and 1,1-dimethylethyl 4-amino-1-piperidine-

carboxylate (0.302 g, 1.51 mmol) were combined in NMP (3 mL)

and DIPEA (0.7 mL), and the mixture was heated at 120 °C for 3 h,

then cooled and added to water (30 mL). The mixture was extracted

with EtOAc (30 mL), and the solvent was washed with water (2 × 30

mL), dried, and evaporated to give a brown gum. The gum was

dissolved in DCM (5 mL) and loaded onto a 50 g silica column and

then eluted with 0−50% 2 M methanolic ammonia/DCM. Product-

containing fractions were evaporated in vacuo to give the title

compound (0.52 g, 1.05 mmol, 70% yield).

LCMS (formic acid): rt = 0.66 min, MH⁺= 411.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.23−1.35 (m, 1H), 1.42−1.53

(m, 1H), 1.54−1.63 (m, 1H), 1.81−1.97 (m, 2H), 2.13 (s, 3H), 2.33

(s, 3H), 3.18 (dd, J = 11.1, 9.1 Hz, 1H), 3.49 (t, J = 6.2 Hz, 1H),

3.67−3.75 (m, 1H), 3.79−3.86 (m, 1H), 3.93 (s, 3H), 7.44−7.51 (br

s, 1H), 7.67 (s, 2H), 8.02−8.09 (br s, 1H), 8.36 (br t, 1H), 8.55 (s,

1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-(((tetrahydro-2H-

pyran-4-yl)methyl)amino)quinoline-3-carboxamide (47). To a

suspension of 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyqui-

noline-3-carboxamide 39 (2 g, 6.03 mmol) in N-methyl-2-

pyrrolidinone (5 mL) was added DIPEA (2.63 mL, 15.07 mmol)

and (tetrahydro-2H-pyran-4-yl)methanamine (0.868 g, 7.54 mmol),

and the reaction mixture was heated at 120 °C for 2 h. The reaction

mixture was loaded directly onto and puriﬁed by SCX washed with

20% MeOH in DCM and eluted using 2 M ammonia in MeOH. The

appropriate fractions were combined and evaporated under reduced

pressure. The resulting gum was triturated using EtOAc to give the

title compound (1.76 g, 4.29 mmol, 71% yield) as a pale brown solid.

LCMS (formic acid): rt = 0.64 min, MH⁺= 411.

LCMS (formic acid): rt = 0.82 min, MH⁺= 496,

1H NMR (CDCl₃, 400 MHz) δ 1.49 (s, 9H), 1.65−1.80 (m, 2H),

2.05−2.13 (m, 2H), 2.25 (s, 3H), 2.39 (s, 3H), 2.95−3.07 (m, 2H),

3.92 (s, 3H), 3.93−4.15 (m, 4H), 5.75−6.15 (br s, 2H), 7.44 (s, 1H),

7.78 (s, 1H), 8.71 (s, 1H), 8.74−8.80 (m, 1H).

7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-{[(1-methyl-4-

piperidinyl)methyl]amino}-3-quinolinecarboxamide (51). 4-

Chloro-7-(3,5-dimethyl-4-isoxazolyl)-6-(methyloxy)-3-quinolinecar-

boxamide 39 (0.37 g, 1.12 mmol) and (1-methylpiperidin-4-

yl)methanamine (0.200 g, 1.56 mmol) were combined in NMP (3

mL) and DIPEA (0.7 mL), and the mixture was heated at 120 °C for

3 h and then cooled and dissolved in methanol (20 mL). The brown

solution was loaded onto a 50 g SCX cartridge and washed with

methanol (100 mL), then eluted with 2 M methanolic ammonia, and

the eluant evaporated in vacuo to give the title compound (253 mg,

0.60 mmol, 54% yield) as a brown gum.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.20−1.30 (m, 2H), 1.60−1.72

(m, 2H), 1.84−1.96 (m, 1H), 2.13 (s, 3H), 2.32 (s, 3H), 3.22−3.33

(m, 2H), 3.53 (d, J = 6.1 Hz, 2H), 3.86 (dd, J = 11.2, 3.2 Hz, 2H),

3.92 (s, 3H), 7.39−7.51 (m, 1H), 7.66−7.67 (m, 2H), 7.98−8.09 (br

s, 1H), 8.58 (s, 1H), 8.61 (t, J = 5.3 Hz, 1H).

LCMS (formic acid): rt = 0.43 min, MH⁺= 424.

¹H NMR (CDCl₃, 400 MHz) δ 1.85−1.99 (m, 4H), 2.24 (s, 3H),

2.26 (s, 3H), 2.88−2.97 (m, 4H), 3.92 (s, 3H), 5.75−5.95 (br s, 2H),

7.58 (s, 1H), 7.74 (s, 1H), 8.65 (s, 1H), 9.31−9.37 (m, 1H). Sample

contains unreacted amine starting material that obscures some of the

signals.

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-((1-(tetrahydro-2H-

pyran-4-yl)ethyl)amino)quinoline-3-carboxamide (48). 4-Chloro-7-

(3,5-dimethylisoxazol-4-yl)-6-methoxyquinoline-3-carboxamide 39 (1

g, 3.01 mmol) was suspended in N-methyl-2-pyrrolidinone (5 mL), 1-

(tetrahydro-2H-pyran-4-yl)ethanamine (0.487 g, 3.77 mmol) and

DIPEA (1.32 mL, 7.54 mmol) added, and the reaction mixture was

heated at 100 °C for 3.5 h. The sample was loaded directly, and

puriﬁed by SCX, washed with 50% MeOH in DCM, and eluted using

2 M ammonia in MeOH. The appropriate fractions were combined

and evaporated under reduced pressure to give a brown gum. The

gum was loaded in MeOH/DCM (and the column dried in a vacuum

oven) and puriﬁed by SPE (100 g, silica) using a gradient of 0−100%

EtOAc in cyclohexane followed by 0−10% (2 M ammonia in MeOH)

in DCM. The appropriate fractions were combined and evaporated

under reduced pressure to give the title compound (173 mg, 0.41

mmol, 14% yield) as a brown gum.

4-((2-(Dimethylamino)ethyl)amino)-7-(3,5-dimethylisoxazol-4-

yl)-6-methoxyquinoline-3-carboxamide (52). 4-Chloro-7-(3,5-dime-

thylisoxazol-4-yl)-6-methoxyquinoline-3-carboxamide 39 (500 mg,

1.51 mmol), N¹,N¹-dimethylethane-1,2-diamine (0.206 mL, 1.88

mmol) and DIPEA (0.66 mL, 3.77 mmol) were taken up in N-methyl-

2-pyrrolidone (1.5 mL). The mixture was stirred at 120 °C for 3 h.

The reaction mixture was loaded onto a 70 g SCX cartridge. The

cartridge was eluted with methanol and then 2 M NH₃in MeOH. The

appropriate fractions were concentrated in vacuo and then loaded

onto a 100 g silica cartridge in the minimum amount of DCM. The

column was eluted with 1−10% NH₃in MeOH. The appropriate

fractions were combined and concentrated in vacuo to give the title

compound (501 mg, 1.24 mmol, 82% yield) as a yellow solid.

LCMS (formic acid): rt = 0.41 min, MH⁺= 384.

LCMS (formic acid): rt = 0.68 min, MH⁺= 425, 77% pure by UV.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.23−1.29 (m, 5H) 1.27 (d, J =

6.6 Hz, 3H) 1.52−1.62 (m, 1H) 1.62−1.74 (m, 2H) 2.14 (s, 3H) 2.34

(s, 3H) 3.22−3.30 (m, 3H) 3.82−3.90 (m, 2H) 3.93 (s, 3H) 7.54 (s,

1H) 7.70 (s, 1H) 8.38 (d, J = 9.9 Hz, 1H) 8.65 (s, 1H). Compound

>70% pure.

¹H NMR (CDCl₃, 400 MHz) δ 2.24 (s, 3H), 2.36 (s, 6H), 2.39 (s,

3H), 2.65 (t, J = 5.9, 2H), 3.79 (q, J = 6.0 Hz, 2H), 3.94 (s. 3H),

5.80−6.50 (br s, 2H), 7.54 (s, 1H), 7.75 (s, 1H), 8.30−8.39 (br t,

1H), 8.71 (s, 1H).

7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-(tetrahydro-2H-

pyran-4-ylamino)-3-quinolinecarboxamide (49). 4-Chloro-7-(3,5-

dimethyl-4-isoxazolyl)-6-(methyloxy)-3-quinolinecarboxamide 39

(0.5 g, 1.51 mmol) and 4-aminotetrahydropyran hydrochloride

(0.270 g, 1.96 mmol) were heated in a mixture of DIPEA (0.66

mL, 3.77 mmol) and N-methyl-2-pyrrolidone (3 mL) for 3 h at 120

°C. The brown solution was cooled and puriﬁed by SCX, washed with

methanol (200 mL), then eluted with 2 M methanolic ammonia (100

N-{7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-[(2-

pyridinylmethyl)amino]-3-quinolinyl}acetamide (54). Acetic anhy-

dride (27 mg, 25 μL, 0.26 mmol) was added to a solution of 7-(3,5-

dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyridin-2-ylmethyl)quinoline-

3,4-diamine 53 (100 mg, 0.26 mmol) in dichloromethane (5 mL) and

pyridine (0.5 mL). The reaction mixture was stirred at room

temperature for 2 h. The solvent was evaporated, and the residue was

chromatographed using 5−10% methanol in dichloromethane to give

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the title compound (80 mg, 0.19 mmol, 72% yield) as a colorless solid

after trituration with diethyl ether.

¹H NMR (CDCl₃, 400 MHz) δ 2.21 (s, 3H), 2.35 (s, 3H), 3.61 (s,

3H), 3.96 (s, 3H), 4.23 (s, 2H), 4.79 (d, J = 6.1 Hz, 2H), 5.38 (t, J =

6.1 Hz, 1H), 7.19−7.21 (m, 1H), 7.23−7.25 (m, 1H), 7.47 (s, 1H),

7.66−7.70 (m, 1H), 7.79 (s, 1H), 8.65−8.68 (m, 1H), 8.89 (s, 1H),

9.86 (s 1H).

LCMS (formic acid): rt = 0.63 min, MH⁺= 418.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.86 (s, 3H), 2.13 (s, 3H), 2.32

(s, 3H), 3.89 (s, 3H), 4.79 (d, J = 6.0 Hz, 2H), 7.01 (t, J = 6.0 Hz,

1H), 7.26−7.29 (m, 1H), 7.34 (d, J = 6.8 Hz, 1H), 7.67 (s, 1H), 7.72

(s, 1H), 7.73−7.77 (m, 1H), 8.16 (s, 1H), 8.54−8.57 (m, 1H), 9.59

(s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxyquinolin-4-ol (61). To

reﬂuxing diphenyl ether (120 mL) was added 7-(3,5-dimethylisox-

azol-4-yl)-4-hydroxy-6-methoxyquinoline-3-carboxylic acid 60 (6.1 g,

19.3 mmol) in small portions over 8 min. Vigorous bubbling was seen

on addition. Safety note: because of the high temperature and volume

of gas produced, extreme caution should be taken to add in small

portions and to use a wide-necked vessel with capacity for the gas to

escape. The reaction mixture was heated at reﬂux for 16 min and then

allowed to cool to room temperature over 2 h. Diethyl ether (100

mL) was added, and the mixture stood at room temperature for 2 h.

The solid was removed by ﬁltration, washed on the ﬁlter with diethyl

ether (2 × 50 mL), and dried in a vacuum oven overnight to give the

title compound (4.6 g, 17.0 mmol, 88% yield) as a pale brown

powder.

N-{7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-[(2-

pyridinylmethyl)amino]-3-quinolinyl}propanamide (55). Propionyl

chloride (28 mg, 0.30 mmol) was added to a solution of 7-(3,5-

dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyridin-2-ylmethyl)quinoline-

3,4-diamine 54 (100 mg, 0.26 mmol) in dichloromethane (5 mL) and

pyridine (0.5 mL). The reaction mixture was stirred at room

temperature for 2 h. The solvent was evaporated, and the residue was

chromatographed using 5−10% methanol in dichloromethane] to give

the title compound (68 mg, 0.16 mmol, 59% yield) as a colorless solid

after trituration with diethyl ether.

LCMS (formic acid): rt = 0.63 min, MH⁺= 432.

¹H NMR (DMSO-d₆, 400 MHz): δ 0.97 (t, 3H, J = 12.0 Hz), 2.13

(s, 3H), 2.16 (q, 2H, J = 12.0 Hz), 2.33 (s, 3H), 3.91 (s, 3H), 4.36 (br

s, 2H), 4.84−4.89 (m, 1H), 7.26−7.35 (m, 2H), 7.71 (s, 1H,), 7.73−

7.79 (m, 1H), 7.81 (s, 1H), 8.25 (s, 1H), 8.52−8.55 (m, 1H), 9.58 (s,

1H).

LCMS (formic acid): rt = 0.62 min, MH⁺= 271.

¹H NMR (DMSO-d₆, 400 MHz): δ 2.12 (s, 3H), 2.32 (s, 3H), 3.31

(s, 3H), 6.03 (d, J = 7.3 Hz, 1H), 7.43 (s, 1H), 7.62 (s, 1H), 7.86−

7.91 (m, 1H), 11.66−11.76 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-3-nitroquinolin-4-ol

(62). To a stirred solution of 7-(3,5-dimethylisoxazol-4-yl)-6-

methoxyquinolin-4-ol 61 (8.7 g, 32.2 mmol) in propionic acid (88

mL) was added, dropwise, fuming nitric acid (3.16 mL, 70.8 mmol).

Safety note: nitration reactions have potential for a runaway exotherm.

Maintain close temperature monitoring and control via slow addition

of the nitric acid. After addition was complete, the reaction mixture

was stirred at 100 °C for 1 h. The reaction mixture was allowed to

cool to room temperature. The solid was removed by ﬁltration,

washed on the ﬁlter with diethyl ether (100 mL), and dried in a

vacuum oven to give the title compound (7.3 g, 23.2 mmol, 72%

yield) as a mustard yellow solid.

N-{7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-[(2-

pyridinylmethyl)amino]-3-quinolinyl}butanamide (56). n-Butanoyl

chloride (20 mg, 0.19 mmol) was added to a stirred solution of 7-

(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyridin-2-ylmethyl)-

quinoline-3,4-diamine 53 (70 mg, 0.19 mmol) in dichloromethane (3

mL) and pyridine (0.3 mL). The reaction mixture was stirred at room

temperature for 2 h. The solvent was evaporated, and the residue was

twice re-evaporated from toluene. The residue was chromatographed

using 2−5% methanol in dichloromethane, followed by trituration

with diethyl ether, to give the title compound (52 mg, 0.12 mmol,

63% yield) as a pale yellow solid.

LCMS (formic acid): rt = 0.70 min, MH⁺= 316.

LCMS (formic acid): rt = 0.67 min, MH⁺= 446.

¹H NMR (DMSO-d₆, 400 MHz) δ 2.13 (s, 3H), 2.33 (s, 3H), 3.92

(s, 3H), 7.61 (s, 1H), 7.78 (s, 1H), 9.17 (br s, 1H), 12.8−13.01 (br s,

1H).

¹H NMR (DMSO-d₆, 400 MHz): δ 0.88 (t, 3H, J = 12.0 Hz),

1.47−1.55 (m, 2H), 2.10−2.15 (m, 5H), 2.33 (s, 3H), 3.90 (s, 3H),

4.80−4.87 (m, 2H), 7.20−7.30 (m, 2H), 7.69 (s, 1H), 7.73−7.80 (m,

3H), 8.23 (s, 1H), 8.53−8.57 (m, 1H), 9.58 (s, 1H).

¹³C NMR (DMSO-d₆, 303 K): δ 10.3, 11.4, 55.9, 105.6, 111.7,

122.3, 125.2, 129.2, 130.4, 132.4, 141.3, 155.1, 158.8, 166.6, 166.8.

IR: 1613 (aryl), 1536 (aryl), 1536 (NO₂), 1361 (NO₂), 1221 (aryl

ether), 786 (aryl C−H) cm⁻¹.

N-{7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-[(2-

pyridinylmethyl)amino]-3-quinolinyl}-2-methylpropanamide (57).

Isobutyryl chloride (20 mg, 0.19 mmol) was added to a stirred

solution of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyridin-2-

ylmethyl)quinoline-3,4-diamine 53 (70 mg, 0.19 mmol) in dichloro-

methane (3 mL) and pyridine (0.3 mL). The reaction mixture was

stirred at room temperature for 2 h. The solvent was evaporated, and

the residue was twice re-evaporated from toluene. The residue was

chromatographed using 2−5% methanol in dichloromethane and then

triturated with diethyl ether to give the title compound (58 mg, 0.13

mmol, 70% yield) as a pale yellow solid.

M.p.: 257−267 (decomposition).

HRMS: (C₁₅H₁₅N₃O₅) MH⁺requires 316.0933, found MH⁺

316.0937.

4-(4-Chloro-6-methoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxa-

zole (63). A solution of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-3-

nitroquinolin-4-ol 62 (3.5 g, 11.1 mmol) in phosphorus oxychloride

(35 mL, 375 mmol) was heated at 120 °C for 2 h. The reaction

mixture was allowed to cool to room temperature and evaporated

under reduced pressure. Twice, toluene (50 mL) was added, and the

mixture was evaporated under reduced pressure. Safety note: Ensure

very cautious quenching of the evaporated excess phosphorus

oxychloride. The residual brown liquid was dissolved in DCM (250

mL), and the organic layer was washed with saturated aqueous

sodium bicarbonate solution (3 × 150 mL), until the aqueous layer

removed was neutral. The organic layer was dried using a

hydrophobic frit and evaporated under reduced pressure. The sample

was loaded in DCM and puriﬁed by SPE (silica, 100 g) using 0−50%

EtOAc in cyclohexane. The appropriate fractions were combined and

evaporated under reduced pressure to give the title compound (2.76

g, 8.27 mmol, 75% yield) as a yellow solid.

LCMS (formic acid): rt = 0.68 min, MH⁺= 446.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.04 (d, 6H, J = 12.0 Hz), 2.13

(s, 3H), 2.33 (s, 3H), 3.90 (s, 3H), 4.80−4.85 (m, 2H), 6.98−7.05

(m, 1H), 7.25−7.29 (m, 1H), 7.38 (d, 1H, J = 12.0 Hz), 7.68 (s, 1H),

7.70−7.78 (m, 2H), 8.19 (s, 1H), 8.52−8.56 (m, 1H), 9.51 (s, 1H).

Methine proton of the isopropyl group was obscured by DMSO.

N-{7-(3,5-Dimethyl-4-isoxazolyl)-6-(methyloxy)-4-[(2-

pyridinylmethyl)amino]-3-quinolinyl}-2-(methyloxy)acetamide

(58). A mixture of 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-

(pyridin-2-ylmethyl)quinoline-3,4-diamine 53 (100 mg, 0.27 mmol),

DIPEA (42 mg, 57 μL, 0.33 mmol), 2-methoxyacetic acid (25 mg,

0.28 mmol), and HATU (122 mg, 0.32 mmol) in DMF (2 mL) was

stirred at room temperature overnight. Saturated NaHCO₃(5 mL)

was added, and the mixture was extracted with ethyl acetate (3 × 5

mL). The combined extracts were washed with water and brine. The

organic phase was dried and evaporated. The residue was chromato-

graphed using 2−5% methanol in dichloromethane to give the title

compound (73 mg, 0.16 mmol, 61% yield) as a light brown oil.

LCMS (formic acid): rt = 0.61 min, MH⁺= 448.

LCMS (formic acid): rt = 1.13 min, MH⁺= 334.

¹H NMR (DMSO-d₆, 400 MHz): δ 2.16 (s, 3H), 2.36 (s, 3H), 4.05

(s, 3H), 7.74 (s, 1H), 8.15 (s, 1H), 9.27 (s, 1H).

¹³C NMR (DMSO-d₆, 303 K): δ 10.3, 11.4, 55.9, 105.6, 111.7,

122.3, 125.2, 129.2, 130.4, 132.4, 141.3, 155.1, 158.8, 166.6, 166.8.

IR (neat): 1612 (aryl), 1589 (aryl), 1535 (NO₂), 1361 (NO₂),

1223 (aryl ether), 784 (aryl C−H) cm⁻¹.

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M.p.: 279−>300 °C (decomposition).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-(pyridine-2-

ylmethyl)quinoline-4-amine (68). To a suspension of 4-(4-chloro-6-

methoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxazole 63 (3 g, 8.99

mmol) in 1,4-dioxane (20 mL) was added pyridine-2-ylmethanamine

(1.40 mL, 13.5 mmol), and the reaction mixture stirred at room

temperature for 5 h. Further pyridine-2-ylmethanamine (0.7 mL, 6.74

mmol) was added and the reaction mixture stirred at room

temperature overnight. The solvent was removed under reduced

pressure and the residue loaded in MeOH/DCM (and the column

dried in a vacuum oven) and puriﬁed by SPE (silica, 2 × 100 g) using

a gradient of 50−100% EtOAc in cyclohexane. The appropriate

fractions were combined and evaporated under reduced pressure to

give the title compound (2.67 g, 6.59 mmol, 73% yield) as a yellow

solid.

HRMS: (C₁₅H₁₄N₃O₅- consistent with hydroxyl replacement of

chloro) MH⁺requires 316.0928, found MH⁺: 316.0926.

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N-(1-methoxypropan-

2-yl)-3-nitroquinolin-4-amine (64). To a solution of 4-(4-chloro-6-

methoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxazole 63 (20 g, 59.9

mmol) in 1,4-dioxane (200 mL) was added 1-methoxypropan-2-

amine (32 mL, 300 mmol) and the reaction mixture heated at 70 °C

for 1.5 h. The solvent was removed under reduced pressure and the

resulting solid partitioned between EtOAc (3 × 750 mL) and water

(750 mL). The organic layers were combined, dried using a

hydrophobic frit and evaporated under reduced pressure to give the

title compound (25.8 g, 98% yield).

LCMS (formic acid): rt = 0.97 min, MH⁺= 387.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.41 (d, J = 6.6 Hz, 3H), 2.15

(s, 3H), 2.34 (s, 3H), 3.29 (s, 3H), 3.55−3.58 (m, 2H), 3.97 (s, 3H),

4.34−4.44 (m, 1H), 7.79 (s, 1H), 7.83 (s, 1H), 8.63 (d, J = 9.1 Hz,

1H), 9.04 (s, 1H).

LCMS (formic acid): rt = 0.87 min, MH⁺= 406.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 2.14 (s, 3H), 2.34 (s,

3H), 3.90 (s, 3H), 5.17 (d, J = 4.8 Hz, 2H), 7.29−7.39 (m, 1H), 7.51

(d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.80−7.84 (m, 1H), 7.96 (s, 1H),

8.60 (d, J = 4.5 Hz, 1H), 9.02 (s, 1H), 9.47−9.66 (br s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-((R)-1-(pyri-

dine-2-yl)ethyl)quinoline-4-amine (69). To a suspension of 4-(4-

chloro-6-methoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxazole 63 (1

g, 3.00 mmol) in 1,4-dioxane (6.5 mL) was added (R)-1-(pyridine-

2-yl)ethanamine (0.55 g, 4.5 mmol), and the reaction mixture stirred

at room temperature for 5 h. Further (R)-1-(pyridine-2-yl)-

ethanamine (0.26 g, 2.3 mmol) was added and the reaction mixture

stirred at room temperature overnight. The solvent was removed

under reduced pressure and the residue loaded in MeOH/DCM (and

the column dried in a vacuum oven) and puriﬁed by SPE (silica, 2 ×

100 g) using a gradient of 50−100% EtOAc in cyclohexane. The

appropriate fractions were combined and evaporated under reduced

pressure to give the title compound (1.15 g, 2.74 mmol, 92% yield) as

an orange solid.

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-((tetrahydro-

2H-pyran-4-yl)methyl)quinoline-4-amine (65). To a suspension of

4-(4-chloro-6-methoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxazole 63

(2 g, 5.99 mmol) in N-methyl-2-pyrrolidone (5 mL) was added

DIPEA (1.05 mL, 5.99 mmol) and (tetrahydro-2H-pyran-4-yl)-

methanamine (1.04 g, 8.99 mmol) and the reaction mixture heated at

100 °C overnight. The resulting solid was triturated in EtOAc to give

the title compound (2.1 g, 5.09 mmol, 85% yield) as a yellow solid.

LCMS (formic acid): rt = 0.87 min, MH⁺= 413.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.18−1.29 (m, 2H), 1.56−1.65

(m, 2H), 1.91−2.04 (m, 1H), 2.14 (s, 3H), 2.33 (s, 3H), 3.23−3.28

(m, 2H), 3.29 (s, 3H), 3.47 (t, J = 6.0 Hz, 2H), 3.85 (dd, J = 11.2, 3.2

Hz, 2H), 3.97 (s, 3H), 7.79 (s, 1H) 7.92 (s, 1H) 8.67−8.72 (m, 1H)

8.93 (s, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-(1-(tetrahy-

dro-2H-pyran-4-yl)ethyl)quinoline-4-amine (66). 4-(4-Chloro-6-me-

thoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxazole 63 (520 mg, 1.56

mmol) was dissolved in N-methyl-2-pyrrolidinone (5 mL), DIPEA

(0.27 mL, 1.56 mmol) and 1-(tetrahydro-2H-pyran-4-yl)ethanamine

(302 mg, 2.34 mmol) added, and the reaction mixture heated at 100

°C for 3.5 h. The sample was loaded directly, and puriﬁed by, SPE

(SCX, 50 g). The column was washed with 50% MeOH in DCM and

eluted using 2 M ammonia in MeOH. The appropriate fractions were

combined and evaporated under reduced pressure to give a brown

gum. The gum was loaded in MeOH/DCM (and the column dried in

a vacuum oven) and puriﬁed by SPE (silica, 25 g) using 0−20% (2 M

ammonia in MeOH) in DCM. The appropriate fractions were

combined and dried under a stream of nitrogen to give the title

compound (124 mg, 0.29 mmol, 19% yield) as a brown gum.

LCMS (formic acid): rt = 0.97 min, MH⁺= 427.

LCMS (formic acid): rt = 0.98 min, MH⁺= 420.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.74 (d, J = 6.6 Hz,

3H), 2.13 (s, 3H), 2.32 (s, 3H), 3.86 (s, 3H), 5.51−5.58 (m, 1H),

7.29−7.33 (m, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.78 (s,

1H), 7.79−7.82 (m, 1H), 8.58 (d, J = 4.8 Hz, 1H), 9.05 (s, 1H), 9.24

(d, J = 7.6 Hz, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N4-(1-methoxypro-

pan-2-yl)quinoline-3,4-diamine (70). 7-(3,5-Dimethylisoxazol-4-yl)-

6-methoxy-N-(1-methoxypropan-2-yl)-3-nitroquinolin-4-amine 64

(25.8 g, 66.8 mmol) was dissolved in a mixture of EtOAc (1000

mL) and dimethyl sulfoxide (50 mL) and the solution was

hydrogenated using an H-cube (settings: 20 °C, 1 bar, 1 mL/min

ﬂow rate) using 10% Pd/C CatCart 70 cartridges containing the

catalyst. The catalyst cartridge was changed whenever it became

blocked. The reaction mixture was evaporated under reduced pressure

and partitioned between EtOAc (750 mL) and water (3 × 750 mL).

The aqueous layers were combined and extracted with EtOAc (750

mL). Both organic layers were combined, dried using a hydrophobic

frit, and evaporated under reduced pressure. The residue was loaded

in DCM and puriﬁed by SPE (silica, 8 × 100 g) using a gradient of 0−

4% (2 M ammonia in MeOH) in DCM. The appropriate fractions

were combined and evaporated under reduced pressure to give

batches of product (13.6 g) and recovered starting material (6.5 g).

The recovered starting material was dissolved in EtOAc (250 mL)

and the reaction was hydrogenated using an H-cube (settings: 20 °C,

1 bar, 1 mL/min ﬂow rate) using a 10% Pd/C CatCart 70 as the

catalyst. The reaction mixture was evaporated under reduced pressure

and the residue was loaded in DCM and puriﬁed by SPE (silica, 2 ×

100 g) using a gradient of 0−4% (2 M ammonia in MeOH) in DCM.

The appropriate fractions were combined and evaporated under

reduced pressure to give a second batch of product. Both batches of

product were combined to give the title compound (15.6 g, 43.8

mmol, 66% yield) as a sticky dark brown gum.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.46 (d, J = 6.3 Hz, 3H) 1.53−

1.61 (m, 2H) 2.16 (s, 3H) 2.16−2.22 (m, 3H) 2.35 (s, 3H) 3.19−

3.29 (m, 1H) 3.81−3.90 (m, 4H) 3.99 (s, 3H) 7.75 (s, 1H) 7.81 (s,

1H) 8.45−8.53 (m, 1H) 8.99 (s, 1H) Compound 60% pure.

N-(Cyclopropylmethyl)-7-(3,5-dimethylisoxazol-4-yl)-6-me-

thoxy-3-nitroquinolin-4-amine (67). 4-(4-Chloro-6-methoxy-3-ni-

troquinolin-7-yl)-3,5-dimethylisoxazole 63 (1 g, 3.00 mmol) was

dissolved in N-methyl-2-pyrrolidinone (4 mL), DIPEA (0.523 mL,

3.00 mmol) and cyclopropylmethanamine (0.378 mL, 4.49 mmol)

added, and the reaction mixture heated at 100 °C for 3.5 h. The

sample was loaded directly, and puriﬁed by, SPE (SCX, 20 g), washed

with 50% MeOH in DCM and eluted using (2 M ammonia in

MeOH). The appropriate fractions were combined and evaporated

under reduced pressure to give a brownish yellow solid. The solid was

triturated in EtOAc, ﬁltered, and dried in a vacuum oven to leave the

title compound (259 mg, 0.70 mmol, 23% yield) as a yellow solid.

LCMS (formic acid): rt = 0.97 min, MH⁺= 369.

¹H NMR (DMS-d₆400 MHz): δ 0.33−0.39 (m, 2H), 0.53−0.61

(m, 2H), 1.14−1.26 (m, 1H), 2.14 (s, 3H), 2.33 (s, 3H), 3.58 (dd, J =

6.2, 4.9 Hz, 2H), 3.97 (s, 3H), 7.78 (s, 1H), 7.88 (s, 1H), 8.97 (s,

1H), 9.00−9.04 (m, 1H).

LCMS (formic acid): rt = 0.73 min, MH⁺= 357.

¹H NMR (DMSO-d₆, 393 K): δ 1.20 (d, J = 6.6 Hz, 2 H), 2.11 (s, 3

H), 2.30 (s, 3 H), 3.32 (s, 3 H), 3.35−3.43 (m, 2 H) 3.52−3.65 (m, 1

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H), 3.89 (s, 3 H), 4.31 (d, J = 10.1 Hz, 1 H), 4.96 (s, 1 H), 7.45 (s, 1

H), 7.55 (s, 1 H), 8.32 (s, 1 H).

4.80−4.97 (m, 2H), 7.18−7.27 (m, 1H), 7.38 (s, 1H), 7.45 (d, J = 7.8

Hz, 1H), 7.53 (s, 1H), 7.67−7.73 (m, 1H), 8.33 (s, 1H), 8.56 (d, J =

4.8 Hz, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N⁴-((tetrahydro-2H-

pyran-4-yl)methyl)quinoline-3,4-diamine (71). To a suspension of

7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-((tetrahydro-2H-

pyran-4-yl)methyl)quinoline-4-amine 65 (1.1 g, 2.67 mmol) in EtOH

(20 mL) was added tin(II) chloride (1.77 g, 9.33 mmol) and the

reaction mixture stirred at 40 °C for 1.5 h. The reaction mixture was

basiﬁed to pH 12 using 2 M aqueous sodium hydroxide solution.

Water (50 mL) was added and the aqueous suspension extracted with

DCM (3 × 50 mL). The organic layers were combined, dried using a

hydrophobic frit and blown down under a stream of nitrogen. The

sample was loaded in DCM and puriﬁed by SPE (silica, 100 g) using a

gradient of 0−10% (2 M ammonia in MeOH) in DCM. The

appropriate fractions were combined and blown down under a stream

of nitrogen to give the title compound (984 mg, 2.57 mmol, 96%

yield) as a brown gum.

4-(8-Methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-

pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole,

methanesulfonic acid salt (75). 4-(8-Methoxy-1-((R)-1-methoxy-

propan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]-

quinolin-7-yl)-3,5-dimethylisoxazole 32 (1.02 g, 2.26 mmol) was

dissolved in the minimum amount of hot MeOH, and methane-

sulfonic acid, 70% weight in water, (0.21 mL, 2.26 mmol) added. The

solution was blown down under a stream of nitrogen to leave a white

gum. The gum was triturated in diethyl ether and then dried in a

vacuum oven to give the title compound (1.18 g, 2.16 mmol, 96%

yield).

LCMS (formic acid): rt = 0.73 min, MH⁺= 451.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.83 (d, J = 7.3 Hz,

3H), 1.86−2.01 (m, 2H), 2.03−2.13 (m, 2H), 2.16 (s, 3H), 2.35 (s,

3H), 2.40 (s, 3H), 3.26 (s, 3H), 3.50−3.65 (m, 3H), 3.98−4.08 (m,

6H), 4.11−4.16 (m, 1H), 5.48−5.63 (m, 1H), 7.87 (s, 1H), 8.12 (s,

1H), 9.36 (s, 1H).

LCMS (formic acid): rt = 0.69 min, MH⁺= 383.

¹H NMR (DMSO-d₆, 400 MHz): δ 1.20−1.31 (m, 2H), 1.72−1.80

(m, 3H), 2.10 (s, 3H), 2.29 (s, 3H), 3.05 (t, J = 6.6 Hz, 2H), 3.22−

3.28 (m, 2H), 3.82−3.93 (m, 5H), 4.74 (t, J = 7.1 Hz, 1H), 5.07 (br s,

2H), 7.39 (s, 1H), 7.55 (s, 1H), 8.27 (s, 1H).

IR (neat): 2849 (ether COCH₃), 1610 (aryl), 1389 (OSO₂), 1223

(aryl ether), 1163 (OSO₂) 841 (aryl C−H) cm⁻¹.

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N⁴-(1-(tetrahydro-2H-

pyran-4-yl)ethyl)quinoline-3,4-diamine (72). 7-(3,5-Dimethylisoxa-

zol-4-yl)-6-methoxy-3-nitro-N-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-

quinoline-4-amine 66 (120 mg, 0.28 mmol) was dissolved in EtOAc

(4.5 mL) and EtOH (1.5 mL) and the reaction mixture was

hydrogenated using an H-cube (20 °C, 1 bar, 1 mL/min ﬂow rate)

with a 10% Pd/C CatCart 30. After the ﬁrst pass through the H-cube

the reaction mixture was recirculated for 60 min. The reaction mixture

was blown down under a stream of nitrogen to give the title

compound (104 mg, 0.26 mmol, 93% yield) as a brown gum.

LCMS (formic acid): rt = 0.74 min, MH⁺= 397, 69% pure.

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N⁴-(pyridine-2-

ylmethyl)quinoline-3,4-diamine (73). To a suspension of 7-(3,5-

dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-(pyridine-2-ylmethyl)-

quinoline-4-amine 68 (2.7 g, 6.6 mmol) in EtOH (50 mL) was added

tin(II) chloride (4.4 g, 23.0 mmol) and the reaction mixture stirred at

40 °C for 2.5 h. The reaction mixture was basiﬁed to pH 12 using 2 M

aqueous sodium hydroxide solution. Water (100 mL) was added and

the aqueous suspension extracted with DCM (3 × 100 mL). The

organic layers were combined, dried using a hydrophobic frit and

blown down a stream of nitrogen. The sample was loaded in DCM

and puriﬁed by SPE (silica, 100 g) using a gradient of 0−10% (2 M

ammonia in MeOH) in DCM. The appropriate fractions were

combined and blown down a stream of nitrogen to give the title

compound (879 mg, 2.34 mmol, 36% yield) as a dark brown gum.

LCMS (formic acid): rt = 0.63 min, MH⁺= 376.

M.p.: 256−259 °C.

HRMS: (C₂₅H₃₁N₄O₄) MH⁺requires 451.2340, found MH⁺

451.2338.

[α_D]^23.1°^C(c 1.0, MeOH): +22.4°.

BRD4 Protein Expression and Puriﬁcation. Recombinant

human bromodomains BRD4 (1−477) (Y390A) (BD2 mutation to

monitor binding to BD1) were expressed in Escherichia coli cells using

a pET15b vector with a 6-His tag at the N-terminal. The His-tagged

bromodomain pellet was resuspended in 50 mM 4-(2-hydroxyethyl)-

piperazine-1-ethanesulfonic acid (HEPES) (pH 7.5), 300 mM NaCl,

10 mM imidazole, and 1 μL/mL protease inhibitor cocktail and

extracted from the E. coli cells using sonication. Puriﬁcation was

carried out using a nickel sepharose high-performance column, and

the proteins were washed and then eluted with a linear gradient of 0−

500 mM imidazole with buﬀer 50 mM HEPES (pH 7.5), 150 mM

NaCl, and 500 mM imidazole, over 20 column volumes. Final

puriﬁcation was completed by using a Superdex 200 prep grade size

exclusion column. Puriﬁed protein was stored at −80 °C in 20 mM

HEPES (pH 7.5) and 100 mM NaCl. The protein identity was

conﬁrmed by peptide mass ﬁngerprinting, and the predicted

molecular weight was conﬁrmed by mass spectrometry.

BRD4 TR-FRET Assay. All assay components were diluted in a

buﬀer composition of 50 mM HEPES (pH 7.4), 150 mM NaCl, 5%

glycerol, 1 mM DTT, and 1 mM 3-[(3-cholamidopropyl)-

dimethylammonio]-1-propanesulfonate. The ﬁnal concentration of

bromodomain protein was 10 nM. An Alexa Fluor 647 derivative of I-

BET762 (1) was used as the competing ligand and was used at a

concentration equal to the K_d. These components were premixed, and

5 μL of this reaction mixture was added to wells containing 50 nL of

various concentrations of the test compound or DMSO vehicle (0.5%

DMSO ﬁnal) in 384-well black low volume microtiter plates and

incubated in the dark for 30 min at rt. The bromodomain protein/

ﬂuorescent ligand interaction was detected using TR-FRET following

5 μL addition of a 1.5 nM europium chelate-labeled anti-6His

antibody in assay buﬀer. Time-resolved ﬂuorescence (TRF) was then

detected on a TRF laser equipped with a PerkinElmer EnVision

multimode plate reader (excitation = 337 nm; emission 1 = 615 nm;

emission 2 = 665 nm; dual wavelength bias dichroic = 400, 630 nm).

The TR-FRET ratio was calculated using the following equation: ratio

= ((acceptor ﬂuorescence at 665 nm)/(donor ﬂuorescence at 615

nm)) × 1000. TR-FRET ratio data were normalized to a mean of 16

replicates per microtiter plate of both 10 μM I-BET151 (5) and 1%

DMSO controls, and IC₅₀values were 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.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 2.10 (s, 3H), 2.29 (s,

3H), 3.82 (s, 3H), 4.52 (d, J = 3.8 Hz, 2H), 4.82−5.01 (br s, 2H)

5.12−5.25 (br s, 1H), 7.24−7.27 (m, 1H), 7.47 (s, 1H), 7.49 (d, J =

7.8 Hz, 1H), 7.55 (s, 1H), 7.72−7.76 (m, 1H), 8.33 (s, 1H), 8.57 (d, J

= 4.3 Hz, 1H).

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N⁴-((R)-1-(pyridine-2-

yl)ethyl)quinoline-3,4-diamine (74). To a suspension of 7-(3,5-

dimethylisoxazol-4-yl)-6-methoxy-3-nitro-N-((R)-1-(pyridine-2-yl)-

ethyl)quinoline-4-amine 69 (1.1 g, 2.50 mmol) in EtOH (20 mL) was

added tin(II) chloride (1.66 g, 8.76 mmol) and the reaction mixture

stirred at 40 °C for 2.5 h. The reaction mixture was basiﬁed to pH 12

using 2 M aqueous sodium hydroxide solution. Water (100 mL) was

added and the aqueous suspension extracted with DCM (3 × 100

mL). The organic layers were combined, dried using a hydrophobic

frit and blown down a stream of nitrogen. The sample was loaded in

DCM and puriﬁed by SPE (silica, 100 g) using a gradient of 0−10%

(2 M ammonia in MeOH) in DCM. The appropriate fractions were

combined and blown down a stream of nitrogen to give the title

compound (543 mg, 1.39 mmol, 56% yield) as a dark brown gum.

LCMS (formic acid): rt = 0.70 min, MH⁺= 390.

¹H NMR (DMSO-d₆, 393 K, 400 MHz): δ 1.58 (d, J = 6.8 Hz,

3H), 2.09 (s, 3H), 2.28 (s, 3H), 3.83 (s, 3H), 4.66−4.80 (m, 1H),

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LPS-Stimulated PBMC Assay. For cell preparation, the plating

medium (500 mL of DMEM, 50 mL of heat-inactivated Australian

fetal bovine serum, 5 mL penicillin/streptomycin, 5 mL Glutamax)

was prewarmed in a 37 °C water bath for at least 20 min prior to

thawing frozen peripheral blood mononuclear cells (PBMCs). The

compound plates were prepared before commencing cell prepara-

tiontwo replicates of each master plate are required to account for

PBMC donor variability. Suﬃcient vials of PBMCs from two diﬀerent

donors were selected from the −140 °C freezer and placed in the

water bath until the cells were just defrosted. In the primary cell

biosafety cabinet, the PBMCs were transferred to 50 mL centrifuge

tubes, with each donor being processed separately. Warm medium

was added dropwise to each tube of cells, and they were then

centrifuged at 1600 rpm for 5 min at ambient temperature. The

supernatant was discarded, and the PBMCs were resuspended in a

suitable volume of warm media. A total of 300 μL of cell suspension

was removed for counting on the Cedex cell counter (Innovatis). The

viable cell count and percentage viability for each donor were entered

into a spreadsheet which calculated the correct volumes of cell

suspension, warm media, and lipopolysaccharide required to make up

suﬃcient plating suspension for the compound plates. A total of 50 μL

of cell suspension was added to each well of the compound plates

using a Multidrop Combi (Thermo). The seeded plates were then

lidded and placed in the humidiﬁed primary cell incubator for 18−24

h at 37 °C, 5% CO₂.

The activity of compounds in the IL-6 assay were determined by

using Mesoscale Discovery (MSD) technology to measure electro-

chemiluminescence. Ten microliters of cell supernatant was trans-

ferred to a low-proﬁle 384-well MSD plate, precoated with human IL-

6 capture antibody using the Biomek FX. The plates were then sealed

and placed on a shaker at 600 rpm at room temperature for 2 h. A 1%

solution of MSD blocker A was prepared by dissolving solid blocker A

in PBS, and this was then used to dilute the antihuman IL-6 antibody

labeled with MSD SULFO-TAG reagent to a working solution of 1

μg/mL (a 1 in 50 dilution). Ten microliters of the working antibody

solution was then added to each well of the MSD plates using a

Multidrop Combi. The plates were then resealed and returned to the

shaker for another 2 h at 600 rpm at room temperature. The plates

were then washed three times with PBS, ensuring there was no liquid

remaining in the plate after washing. A total of 35 μL of 2× MSD

Read Buﬀer T (stock 4× MSD Read Buﬀer T was diluted 50:50 with

deionized water) was then added to each well using a Multidrop

Combi, and the plates were then read on the MSD Sector Imager

6000. The resulting data were used for data analysis.

months: 40 °C, 75% relative humidity, open; 50 °C, ambient

humidity, closed; 30 °C, 65% relative humidity, closed. PXRD

diﬀractograms of the closed samples were concordant with the input

material. Additional peaks were visible in the PXRD diﬀractograms of

the open samples showing that some degradation of the material was

observed.

Cocrystallization and Structure Determination. One micro-

liter of 200 mM of I-BET282 32 was dissolved in DMSO and added

to 100 μL of BRD4 (44-168). This solution was incubated for 3 h on

ice before crystallization was performed using four diverse screens

(PACT, PEGI, PEGII, INDEX). 150 nL + 150 nL drops of the

protein complex and well solution were used for crystallization on

Innoadyne SD2 96-well plates. The crystallization plates were stored

at 20 °C. Data were collected from a single crystal grown in 200 mM

CaCl₂25% PEG4K, 100 mM trisHCl pH8.5 on a house RIGAKU

FRE+ SUPERBRIGHT/A200 system. The crystal had been brieﬂy

transferred into well solution with the addition of 20% ethylene glycol

as a cryoprotectant prior to ﬂash freezing in liquid nitrogen. The

diﬀraction images were processed and scaled using D*Trek to give a

1.7 Å data set. The P2₁2₁2₁orthorhombic cell (α = β = γ = 90°, a =

42.55 Å, b = 46.29 Å, c = 61.41 Å) has one molecule in the ASU. After

rigid body reﬁnement using a previously determined in-house

structure, model building was performed using Coot⁴⁸and reﬁned

using refmac.⁴⁹There was clear diﬀerence density for the ligand at the

acetyl lysine binding site, allowing I-BET282 32 to be unambiguously

placed. The statistics for the data collection and reﬁned coordinates

are given in Table S1. The ﬁnal reﬁned model (Rfac/Rfree = 18.1%/

21.5%) showed excellent density for the ligand and an unambiguous

binding mode for this molecule. The chirality of this compound was

also unambiguously determined.

In Vitro DMPK Studies. The human biological samples were

sourced ethically, and their research use was in accordance with the

terms of the informed consents under an IRB/EC approved protocol.

Metabolic Stability in Hepatocytes. Cryopreserved hepatocytes

from mouse, rat, dog, and human were obtained from Invitrogen UK

and stored in liquid nitrogen prior to use. A 5 mM stock solution of I-

BET282 32 was prepared and diluted to 100 μM with DMSO. An

aliquot was taken and further diluted to 1 μM with Williams Medium

E (WME) and prewarmed at 37 °C. The cryopreserved hepatocytes

for each species were thawed in the manner recommended by the

supplier. Cell concentration and viability were ascertained using the

Trypan blue exclusion method. The cell suspension was then diluted

to 1.4 million cells per mL with prewarmed (37 °C) WME. A total of

300 μL of cell suspension was added to each well of a 12-well plate to

which 300 μL of 1 μm I-BET282 32 was also added to give a ﬁnal

incubation concentration of 0.5 μM I-BET282 32 and 0.7 million

cells/mL. The incubations were maintained at 37 °C for the duration

of the experiment. At set intervals (0, 10, 20, 30, 45, 60, 75, 90, 105,

and 120 min), 25 μL of cell suspension was transferred to a 96-well

block containing 100 μL of stopping solution containing an internal

standard. No compound controls, replacing I-BET282 32 with 300 μL

of WME, were performed, with samples being taken at 120 min only.

No hepatocyte controls, replacing 300 μL of cell suspension with 300

μL of WME, were also performed, with samples being taken at 0 and

120 min only. Incubations were repeated with 0.05 μM I-BET282 32

using mouse, rat, dog, and human hepatocytes. Samples were analyzed

by LCMS/MS. The peak area of I-BET282 32 was quantiﬁed at each

time point, and the ratio of this relative to internal standard peak area

over a time course was plotted. The intrinsic clearance (CLi) was

determined from the ﬁrst-order elimination rate constant by nonlinear

regression using Graﬁt v 5.0.8 (Erithacus Software Ltd.).

LPS-Stimulated HWB Assay. Compounds diluted to 4.2 mM in

100% DMSO were serially diluted (1:3) in DMSO and 1 μL stamped

into 96-well ﬂat-bottomed tissue culture plates. A total of 130 μL of

human whole blood (containing 10 IU/mL sodium heparin) was

added to the compound wells, the plates were incubated (37 °C, 5%

CO₂) for 30 min, and 10 μL of 2.8 μg/mL LPS (diluted in 1% BSA)

was added. The plates were incubated (37 °C, 5% CO₂) for 24 h. A

total of 140 μL of PBS was added, and the plate was shaken for 1 min.

The plates were centrifuged at either 2000 or 2500 rpm for 10 min,

and 100 μL of supernatant was removed. Twenty-ﬁve microliters of

supernatant (diluted 1 in 40 in MSD diluent) was added to IL-6 MSD

assay plates (MSD MA6000 96-well plate), and the plates were

shaken for 2 h. Twenty-ﬁve microliters of Sulfo-tag anti-IL-6 antibody

(1 mg/mL dilution in MSD antibody diluent) was added, and the

plates were shaken for 2 h. A total of 150 μL of MSD read buﬀer T

(diluted 1:1 in distilled water) was added, and the plates were read on

an MSD 6000 plate reader.

FaSSIF Solubility. FaSSIF GI medium (pH 6.5) was added to a

sample of compound in order to produce a known concentration of

suspension. The samples were left on a roller mixer for 30 min or 4

hm at which point a 200 μL aliquot was removed. The samples were

ﬁltered and centrifuged (Durapore PVDF 0.45 μm ﬁlters, 10 000

rpm), and then 100 μL of the supernatant was diluted in 1:1 MeCN/

H₂O. Samples were analyzed by gradient HPLC

Fraction Unbound in Blood. Control male Balb/C mouse, male

Wistar Han rat, and male Beagle dog blood was obtained on the day

of experimentation from in-house GSK stock animals. Control human

blood from a single nonmedicated donor was obtained from a GSK

blood donation unit on the day of the experiment (gender unknown).

A nominal 10 mg/mL DMSO stock solution of I-BET282 32 was

diluted to 4 mg/mL and further diluted to 200 μg/mL and 20 μg/mL

in 50:50 acetonitrile/water, and a 5 μL aliquot of each was spiked into

995 μL of whole blood from each species to achieve nominal

Three-Month Accelerated Storage. One milligram and 100 mg

of I-BET282E were stored under the following conditions for three

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concentrations of 1000 ng/mL and 100 ng/mL respectively. The

fraction unbound of I-BET282 32 at nominal concentrations of 1000

ng/mL and 100 ng/mL in the blood was determined using a rapid

equilibrium dialysis technology (RED) plate. Spiked blood was

incubated at 37 °C on a roller for 10 min, prior to being dialyzed

against phosphate buﬀered saline solution by incubating the dialysis

units at 37 °C for 4 h on an orbital shaker operating at 100 rpm.

Following incubation, aliquots of blood and buﬀer were matrix

matched and extracted using acetonitrile containing an analytical

internal standard. The samples were shaken for 20 min on a vortex

mixer and centrifuged for 30 min at 1600g, prior to analysis by LC−

MS/MS. The unbound fraction was determined using the peak area

ratios between analyte and internal standard in buﬀer and in blood

(mean of n = 6 replicates).

Potential for Inhibition of P450 Enzymes CYP1A2, CYP2C9,

CYP2C19, CYP2D6, and CYP3A4. Bactosomes expressing CYP450

enzymes were obtained from Cypex Ltd. and were stored at

approximately −80 °C prior to experimental use. The inhibition of

CYP450-mediated NADPH-dependent mono-oxygenase metabolism

of proﬂuorescent probe substrates by test compounds was assessed in

a ﬂuorometric assay, with the following recombinant human

cytochrome P450s coexpressed in E. coli with human NADPH

reductase: CYP1A2 with the substrate ethoxyresoruﬁn (ER);

CYP2C9 with the substrate 7-methoxy-4-triﬂuoromethylcoumarin-3-

acetic acid (FCA); CYP2C19 with the substrate 3-butyryl-7-

methoxycoumarin (BMC); CYP2D6 with the substrate 4-methyl-

aminomethyl-7-methoxycoumarin (MMC); and CYP3A4 with the

substrates VividGreen and VividRed. For the ﬂuorometric IC₅₀

determination assay, DMSO stock solutions of test compounds

were incubated for 15−60 min at room temperature (22 0.5 °C)

over 11 successive 3-fold dilutions of 50 μM, with bactosomes

expressing recombinant human CYP1A2, 2C9, 2C19, 2D6, or 3A4

(Cypex Ltd., Dundee; 0.1 mg protein/mL) in the presence of a

proﬂuorescent substrate for the speciﬁc CYP isoform under test. At

the end of the incubation, the ﬂuorescent intensity of each sample was

read on a plate reader (EnVision or ViewLux, PerkinElmer), and

pIC₅₀values were determined by automated curve-ﬁtting using

ActivityBase (IDBS).

Time-Dependent Inhibition of P450 Enzymes CYP2D6 and

CYP3A4. Bactosomes expressing CYP3A4 or CYP2D6 were obtained

from Cypex Ltd. and were stored at approximately −80 °C prior to

experimental use. A 5 mM stock solution of I-BET282 32 was

prepared in methanol and serially diluted to give 1.65, 0.5, 0.165, 0.05,

0.0165, and 0.005 mM solutions, and aliquots of each concentration

were incubated with probe substrate (either diethoxyﬂuorescein,

DEF; 7-benzyloxyquinoline, 7-BQ; or 4-methylaminomethyl-7-

methoxycoumarin, MMC) and CYP3A4 or CYP2D6 bactosomes in

50 mM phosphate buﬀer, pH7.4, for 10 min at 37 °C. Reactions were

initiated by the addition of a cofactor (an NADPH-regenerating

system), and the ﬂuorescent products of metabolism were measured

at 1 min intervals over a 30 min time course in a Cytoﬂuor series 4000

ﬂuorescent plate reader. Final incubation concentrations were 0.1−

100 μM I-BET282 32, 1 μM DEF or 25 μM, 7-BQ or 10 μM of MMC

and 0.1 mg/mL bactosomal protein. Control incubations, containing

methanol only, and incubations with the positive control CYP3A4

inhibitor, troleandomycin or the CYP2D6 inhibitor, MDMA at a

concentration range 0.01−10 μM were also performed. The rates of

ﬂuorescent metabolite production in each well, over each successive 5

min interval, were expressed as a percentage of the uninhibited

control rates (in wells containing methanol only), and plotted against

I-BET282 32 concentration to determine the IC50. Graphical analysis

of data was performed using GraFit v 5.0 (Erithacus Software Ltd.).

In Vivo DMPK Studies. All animal studies were ethically reviewed

and carried out in accordance with The Animals (Scientiﬁc

Procedures) Act 1986 and the GSK Policy on the Care, Welfare

and Treatment of Laboratory Animals. For all in vivo studies, the

known contaminants in the diet or water at concentrations that could

interfere with the outcome of the studies.

Mouse Surgical Preparation for IV Infusion Study. Male CD1

mice (supplied by Charles River UK Ltd.) were surgically prepared at

GSK with cannulae implanted into the jugular vein (for drug

administration) while under isoﬂurane anesthesia. The mice received

ampicillin and carprofen (both administered subcutaneously) as a

preoperative antibiotic and analgesic, respectively. The mice were

allowed to recover for at least 4 days prior to dosing and had free

access to food and water throughout.

Mouse IV n = 3 PK Study. Three surgically prepared male CD1

mice received a 1 h intravenous (iv) infusion of I-BET282 32 as a

discrete dose formulated in DMSO and 10% (w/v) Kleptose HPB in

0.9% (w/v) saline (2%:98% (v/v)) at a concentration of 0.2 mg/mL,

administered at a constant rate of 5 mL/kg/h to achieve a target dose

of 1 mg/kg/h. Serial blood samples (20 μL) were collected via the tail

vein up to 24 h after the start of the iv infusion and an additional

terminal blood sample via cardiac puncture at 26 h after the start of

the iv infusion. Diluted blood samples were analyzed for parent

compound using a speciﬁc LC−MS/MS assay (LLQ = 5 ng/mL). At

the end of the study the mice were euthanized by a Schedule 1

method.

̈

Mouse PO n = 3 PK Study. Three naive male CD1 mice (supplied

by Charles River UK Ltd.) with no surgical preparation each received

an oral gavage administration of I-BET282 32 as a discrete dose

suspended in 1% (w/v) methylcellulose 400 aq. at a concentration of

0.6 mg/mL to achieve a target dose of 3 mg/kg. Serial blood samples

(20 μL) were collected via direct venipuncture of the tail vein up to 8

h after oral dosing and an additional terminal blood sample via cardiac

puncture at 12 h after oral dosing. Diluted blood samples were

analyzed for parent compound using a speciﬁc LC−MS/MS assay

(LLQ = 5 ng/mL). At the end of the study the mice were euthanized

by a Schedule 1 method.

Rat Surgical Preparation for IV Infusion Study. Male Wistar Han

rats (supplied by Charles River UK Ltd.) were surgically prepared at

GSK with cannulae implanted into the vena cava via the femoral vein

(for drug administration) and jugular vein (for blood sampling) while

under isoﬂurane anesthesia. The rats received ampicillin (100 mg/kg)

and carprofen (7.5 mg/kg), both administered subcutaneously, as a

preoperative antibiotic and analgesic, respectively. The rats were

allowed to recover for at least 6 days prior to dosing and had free

access to food and water throughout.

Rat IV PO n = 3 Crossover PK Study. This study was conducted as

a crossover design over two dosing occasions with at least 3 days

between dose administrations, in three surgically prepared male

Wistar Han Rats. On the ﬁrst dosing occasion, each rat received a 1 h

intravenous (iv) infusion of I-BET282 32 as a discrete dose

formulated in DMSO and 10% (w/v) Kleptose HPB in 0.9% (w/v)

saline (2%:98% (v/v)) at a concentration of 0.2 mg/mL, administered

at a constant rate of 5 mL/kg/h to achieve a target dose of 1 mg/kg/

h. On the second dosing occasion, the same three rats received an oral

gavage administration of I-BET282 32 as a discrete dose suspended in

1% (w/v) methylcellulose 400 aq. at a concentration of 0.6 mg/mL to

achieve a target dose of 3 mg/kg. Serial blood samples (60 μL) were

collected predose and up to 26 h after the start of the iv infusion and

after oral dosing. Diluted blood samples were analyzed using a speciﬁc

LC−MS/MS assay (LLQ = 10 ng/mL). At the end of the study the

rats were euthanized by a Schedule 1 method.

̈

Rat PO n = 6 PK Study. Six naive male Wistar Han rats (supplied

by Charles River UK Ltd.) with no surgical preparation received an

oral gavage administration of I-BET282 32 (n = 3) or I-BET282E 75

(n = 3) as a discrete dose suspended in 1% hydroxypropylmethyl

cellulose (w/v), at a concentration of 0.1 mg/mL to achieve a target

dose of 1 mg/kg. Serial blood samples (200 μL) were collected up to

24 h after oral dosing and were centrifuged to obtain plasma. Plasma

samples were analyzed for parent compound using a speciﬁc HPLC−

MS/MS assay (LLQ = 0.5 ng/mL). At the end of the study the rats

were euthanized by a Schedule 1 method.

temperature and humidity were nominally maintained at 21 °C

2

°C and 55% 10%, respectively. The diet for rodents was 5LF2

Eurodent Diet 14% (PMI Labdiet, Richmond, IN) and for dogs was

Harlan Teklad 2021C (HarlanTeklad, Madison, WI). There were no

Dog IV PO n = 3 Crossover PK Study. Three healthy, laboratory-

bred, male Beagle dogs (supplied by Harlan Laboratories, U.K.) were

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used. The dogs were fasted overnight prior to each dose

administration and fed approximately 4 h after the start of dosing

and had free access to water throughout the study. This study was

conducted as a crossover design, with 7 days between dose

administrations. On the ﬁrst dosing occasion, each dog received a 1

h intravenous (iv) infusion of I-BET282 32 as a discrete dose via

cannulation of the saphenous vein. I-BET282 32 was formulated in

DMSO and 10% (w/v) Kleptose HPB in 0.9% (w/v) saline (2%:98%

(v/v)) at a concentration of 0.2 mg/mL, and administered at a rate of

5 mL/kg/h to achieve a target dose of 1 mg/kg/h. On the second

dosing occasion, the same dogs received an oral gavage administration

of I-BET282 32 as a discrete dose suspended in 1% (w/v)

methylcellulose 400 aq. at a concentration of 1.5 mg/mL to achieve

a target dose of 3 mg/kg. A temporary cannula was inserted into the

cephalic vein from which serial blood samples (200 μL) were

collected predose and up to 2 h after the start of dosing. After

collection of the 2 h sample, the cannula was removed and serial

blood samples (200 μL) were taken via direct venipuncture of the

jugular vein up to 28 h after the start of dosing. Diluted blood samples

were analyzed for parent drug concentration using a speciﬁc LC−MS/

MS assay (LLQ = 1 ng/mL). At the end of the study, the dogs were

returned to the colony.

Dog PO n = 4 Crossover PK Study. Four healthy, laboratory-bred,

male Beagle dogs (supplied by Harlan Laboratories, U.K.) were used.

The dogs were fasted overnight prior to each dose administration and

fed approximately 4 h after the start of dosing and had free access to

water throughout the study. This study was conducted as a crossover

design, with 7 days between dose administrations. On the ﬁrst dosing

occasion, each dog received an oral gavage administration of I-

BET282 32 as a discrete dose suspended in 1% hydroxypropylmethyl

cellulose (w/v) at a concentration of 0.5 mg/mL to achieve a target

dose of 5 mg/kg. On the second dosing occasion, the same dogs

received an oral gavage administration of I-BET282E 75 as a discrete

dose suspended in 1% hydroxypropylmethyl cellulose (w/v) at a

concentration of 0.5 mg/mL to achieve a target dose of 5 mg/kg.

Serial blood samples (500 μL) were collected via direct venipuncture

of the jugular vein predose and up to 24 h after the start of dosing and

centrifuged to obtain plasma. Plasma samples were analyzed for

parent drug concentration using a speciﬁc HPLC−MS/MS assay

(LLQ = 0.5 ng/mL). At the end of the study, the dogs were returned

to the colony.

Sample Analysis. Diluted blood samples (1:1 with water) were

extracted using protein precipitation with acetonitrile containing an

analytical internal standard. An aliquot of the supernatant was

analyzed by reverse phase LC−MS/MS using a heat assisted

electrospray interface in positive ion mode. Samples were assayed

against calibration standards prepared in control blood. Plasma

samples were analyzed for I-BET282 32 concentrations using a

method based on extraction of plasma by addition of acetonitrile,

followed by HPLC-MS/MS analysis.

PK Data Analysis from PK Studies. PK parameters were obtained

from the blood or plasma concentration−time proﬁles using

noncompartmental analysis with WinNonlin Professional 6 (Phar-

sight, Mountain View, CA).

Rat Collagen-Induced Arthritis (CIA). Female Lewis rats (5−6

weeks age) were obtained from Charles River Laboratories UK,

housed in groups of 4 according to treatment (n = 8 per group) and

acclimatized for 14 days to the laboratory environment. Animals

received food and water ad libitum. On day 0, arthritis was induced by

intradermal injection at the base of the tail of 100 μL containing 200

μg bovine type II collagen (MD Bioproducts, Switzerland) and

Incomplete Freund’s Adjuvant (IFA; Sigma-Aldrich, UK) emulsion.

On day 7 a booster injection containing 200 μg collagen, 3 μg MDP

and IFA was given. All rats were dosed orally (p.o.) with 1%

methylcellulose (vehicle) once daily from day 0 until day 11. Vehicle

or I-BET282E 75 (0.3, 1, or 3 mg/kg) were administered p.o. once

daily from day 12 (at onset of symptoms) until day 28, i.e. for 16 days.

A further group of rats were orally dosed daily with I-BET151 5 (3

mg/kg) over the same dosing period. Disease onset is typically 5 days

following boost (D12), and is deﬁned as the ﬁrst day that swelling or

erythema of the paws is observed. CIA was assessed visually as a

clinical score of hind limbs on days 12−28 and by measurement of

paw volume on days 12−26. Body weights were measured daily for

the entire duration of the study. Arthritis was assessed blinded by

clinical score of hind limbs. Clinical score was recorded on a 0−4

scale per paw according to the following criteria: 0 = no signs of

arthritis; 1 = slight edema and erythema on the foot or ankle; 2 =

slight edema and erythema to the entire paw; 3 = moderate edema

and erythema to the entire paw; 4 = severe edema and ankylosis (the

maximum clinical score attainable per animal being 8). Paw swelling

was measured using a plethysmometer (Linton Instrumentation, UK)

on day 6, 12, 13, 14, 15, 18, 19, and 21 and recorded as the mean paw

volume of both hind paws. On day 21, blood was collected via cardiac

puncture under terminal anesthesia. Blood was assessed for antirat

collagen IgG1 antibodies by ELISA (Chondrex; AMS Biotechnology,

UK).

Rats were culled by cervical dislocation and both hind limbs were

removed, skinned and ﬁxed in 4% paraformaldehyde, prior to

obtaining Micro Computed Tomography (CT) scans processing for

histological analysis. After a minimum of 24 h of ﬁxation, the hind

limbs were removed from ﬁxative and analyzed (Skyscan 1176

microCT scanner, Bruker). Projection images were acquired through

180° with source voltages and current of 50 kV and 500 mA

respectively. Individual 3D reconstructions were generated with

individual voxel dimensions of 35 × 35 × 35 μm. A subjective scoring

technique examining new bone growth, erosion/uneven bone surface

and apparent reduction in joint space was applied to determine the

degree of arthritic changes seen in the ankle joints. All scoring was

blinded to the treatment group. The number of aﬀected bones per

hind limb (maximum of 14 per hind limb: 5 metatarsals, 5 main

tarsals, calcaneus, talus, tibia and ﬁbula), and an overall severity

between 0 and 4 (none, minimal, mild, moderate, marked) were

determined. The severity for each hind limb was calculated as the

total number of aﬀected bones multiplied by the severity with a

maximum possible score per animal of 112 (sum of both hind limbs).

Following 7 days ﬁxation the hind paws and knees were decalciﬁed

in 10% EDTA for 14 days at 4 °C, then embedded in paraﬃn. Serial

paraﬃn sections (4 μm) were stained with hematoxylin and eosin

(H&E). All sections were evaluated histologically by two independent

observers, and the gradation of arthritis was scored as described.

H&E-stained hind limb sections were examined by light microscopy.

Each section was assigned a qualitative score for the severity of

arthritic changes; 0 = normal joint architecture, 1 = inﬂammation

virtually absent, minimal synovial thickening, no bone remodeling or

ﬁbrosis, 2 = minimal mononuclear cell inﬂammation, some bone

remodeling with rare new bone formation and ﬁbrosis, synovial

thickening present, 3 = moderate inﬂammatory changes and ﬁbrosis

with bone remodeling; lesion usually focal, 4 = marked remodeling of

the bones, with degeneration/necrosis and new bone formation,

chronic active inﬂammation and ﬁbrosis, extensive lesions, involving

all the joints/bones and 5 = severe remodeling of the bones, with

degeneration/necrosis and new bone formation, chronic active

inﬂammation, ﬁbrosis and cyst formation, extensive lesions, involving

all the joints/bones. Statistical analysis was performed using

GraphPad Prism v5.0.4, with an ANOVA test followed by a Dunnett’s

multiple comparisons test.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00855.

■

sı

Crystallography data collection and coordinates, BET

bromodomain FRET potency data for I-BET282 (32),

ITC of I-BET282 (32) with BRD4 and CBP, T_m

selectivity determination, Powder X-ray diﬀraction

1

(PXRD), HPLC traces of key compounds, H NMR,

¹³C NMR, and chiral HPLC traces for I-BET282 (32)

(PDF)

12224

https://doi.org/10.1021/acs.jmedchem.1c00855

J. Med. Chem. 2021, 64, 12200−12227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Rab K. Prinjha − GlaxoSmithKline, Medicines Research

Molecular formula strings (CSV)

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Accession Codes

7O18 (32); authors will release the atomic coordinates and

experimental data upon article publication.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00855

Notes

AUTHOR INFORMATION

The authors declare no competing ﬁnancial interest.

■

Corresponding Author

ACKNOWLEDGMENTS

Katherine L. Jones − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.;

orcid.org/0000-0003-4853-8283;

■

We thank Frédéric Donche, Amanda Emmons, Hannah

Fleetwood, and Alex Phillipou for supporting laboratory

contributions, and Inma Rioja for her assistance with

manuscript preparation.

Email: katherine.jones@crl.com

Authors

Dominic M. Beaumont − GlaxoSmithKline, Medicines

Research Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Sharon G. Bernard − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Rino A. Bit − GlaxoSmithKline, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, U.K.

Simon P. Campbell − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Chun-wa Chung − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.;

orcid.org/0000-0002-2480-3110

Leanne Cutler − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Emmanuel H. Demont − GlaxoSmithKline, Medicines

Research Centre, Stevenage, Hertfordshire SG1 2NY, U.K.;

orcid.org/0000-0001-7307-3129

ABBREVIATIONS USED

■

ATAD2, ATPase family AAA domain-containing protein 2;

AUC, area under the curve; BAZ2A, bromodomain adjacent to

zinc ﬁnger domain protein 2A; BD1, ﬁrst (N-terminal)

bromodomain; BD2, second (C-terminal) bromodomain;

BET, bromodomain and extraterminal; CBP, CREB-binding

protein; CIA, collagen-induced arthritis; CYP, cytochrome

P450; DIPEA, diisopropylethylamine; DMPK, drug metabo-

lism and pharmacokinetic; EDC, N¹-((ethylimino)methylene)-

N³,N³-dimethylpropane-1,3-diamine; FaSSIF, fasted state

simulated intestinal ﬂuid; HATU, 1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]-pyridinium 3-oxide hexa-

ﬂuorophosphate; HEPES, 4-(2-hydroxyethyl) piperazine-1-

̀

ethanesulfonic acid; hERG, human ether-a-go-go-related

gene; HPV, human papillomavirus; HPLC, high-performance

liquid chromatography; HWB, human whole blood; IL-6,

interleukin 6; ITC, isothermal titration calorimetry; LLE,

lipophilic ligand eﬃciency; LPS, lipopolysaccharide; MDAP,

mass-directed autopreparative high-performance liquid chro-

matography; MDCK, Madin-Darby canine kidney; NMC,

nuclear protein in testis midline carcinoma; PBMC, peripheral

blood mononuclear cell; PCAF, P300/CBP-associated factor;

SAR, structure activity relationship; SP140, nuclear body

protein SP140; TRF, time-resolved ﬂuorescence; PXRD,

powder X-ray diﬀraction

Kate Dennis − GlaxoSmithKline, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, U.K.

Laurie Gordon − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

James R. Gray − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Michael V. Haase − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Antonia J. Lewis − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Scott McCleary − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Darren J. Mitchell − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Susanne M. Moore − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Nigel Parr − GlaxoSmithKline, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, U.K.

Olivia J. Robb − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Nicholas Smithers − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Peter E. Soden − GlaxoSmithKline, Medicines Research

Centre, Stevenage, Hertfordshire SG1 2NY, U.K.

Colin J. Suckling − Department of Pure & Applied Chemistry,

University of Strathclyde, Glasgow G1 1XL, U.K.

Simon Taylor − GlaxoSmithKline, Medicines Research Centre,

Stevenage, Hertfordshire SG1 2NY, U.K.

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Article Doi

Discovery of a Novel Bromodomain and Extra Terminal Domain (BET) Protein Inhibitor, I-BET282E, Suitable for Clinical Progression

DOI: 10.1021/acs.jmedchem.1c00855

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Article abstract of DOI:10.1021/acs.jmedchem.1c00855

Full text of DOI:10.1021/acs.jmedchem.1c00855

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