Application of Sequential Palladium Catalysis for the Discovery of Janus Kinase Inhibitors in the Benzo[ c]pyrrolo[2,3- h][1,6]naphthyridin-5-one (BPN) Series

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Application of Sequential Palladium Catalysis for the Discovery of

Janus Kinase Inhibitors in the

Benzo[c]pyrrolo[2,3‑h][1,6]naphthyridin-5-one (BPN) Series

Mohamed S. A. Elsayed,^†,§Jeﬀery J. Nielsen,^†,§Sungtae Park,^‡,§Jeongho Park,^‡,§Qingyang Liu,^‡,∥

Chang H. Kim,^‡,§,∥Yves Pommier,^⊥Keli Agama,^⊥Philip S. Low,^#,§and Mark Cushman*^,†,§

^†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette,

Indiana 47907, United States

^‡Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907,

United States

^§The Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States

^∥Department of Pathology and Mary H. Weiser Food Allergy Center, University of Michigan Medical School, Ann Arbor, Michigan

48109, United States

^⊥Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer

Institute, Bethesda, Maryland 20892, United States

^#Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States

S

* Supporting Information

ABSTRACT: The present account describes the discovery and development of a new benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-5-one (BPN) JAK inhibitory chemotype that has produced selective JAK inhibitors. Sequential palladium

chemistry was optimized for the rapid access to a focused library of derivatives to explore the structure−activity relationships of

the new scaﬀold. Several compounds from the series displayed potencies in the low nanomolar range against the four members

of the JAK family with various selectivity proﬁles. Compound 20a, with an azetidine amide side chain, showed the best

selectivity for JAK1 kinase vs JAK2, JAK3, and TYK2, with low nanomolar potency (IC₅₀= 3.4 nM). On the other hand, BPNs

17b and 18 had good general activity against the JAK family with excellent kinome selectivity proﬁles. Many of the new BPNs

inhibited JAK3-mediated STAT-5 phosphorylation, the production of inﬂammatory cytokines, and the proliferation of primary

T cells. Moreover, BPN 17b showed very similar in vivo results to tofacitinib in a rheumatoid arthritis animal model.

As shown in Figure 1A, cytokine binding triggers receptor

dimerization, which leads to JAK tyrosine kinase activation in

which the two JAKs phosphorylate each other on a tyrosine

residue in the activation loop. JAK-mediated STAT phosphor-

ylation subsequently induces dimerization of the STAT signal

transduction proteins and their translocation to the nucleus,

which initiates gene transcription.⁵The JAK family has four

members: JAK1, JAK2, JAK3, and TYK2.⁶The JAKs are

activated in various patterns by diﬀerent cytokines (Figure 1B).

JAK1 and JAK3 are activated by the members of the γ

INTRODUCTION

■

The human immune response is a very complex process

involved in defense against external pathogens and foreign

tissues.¹Abnormalities of the immune system often lead to

disease states such as cancer and autoimmune disorders.²In

autoimmune diseases, the immune system attacks tissues

within the same organism. It is estimated that 24 million

people are aﬀected by autoimmune diseases in the United

States.³Cytokines regulate the immune responses by

mediating gene activation and repression.⁴The JAK-STAT

pathway plays a central role in the signal transduction cascades

of many cytokines (Figure 1).⁵

Received: March 31, 2018

A

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Figure 1. (A) The JAK-STAT signaling pathway; (B) various biological responses mediated through the JAK pathway.⁷

common (γ_c) subfamily, namely interleukins IL-2, IL-4, IL-7,

IL-9, IL-15, and IL-21, which cannot activate JAK2 or TYK2.

On the other hand, another subfamily of cytokines that

includes IL-6, IL-11, and IL-17 binds to unique receptors that

share a common glycoprotein 130 (gp130) subunit that is

required for downstream JAK signaling. Several other cytokines

induce JAK1 activation, and JAK2 and TYK2 are also

consistently activated. Erythropoietin (EPO) receptors are

another subfamily of homodimeric receptors that also includes

the receptors for prolactin, thrombopoietin, and growth

hormone. The EPO pathway activates JAK2 exclusively and

is essential for erythropoiesis.

Extensive eﬀorts have been expended over the past decade

to identify novel small-molecule JAK inhibitors with various

subtype selectivity proﬁles to address unmet medical needs

presented by transplant rejection, rheumatoid arthritis, other

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Figure 2. Examples of JAK inhibitors (A) and topoisomerase I poisons (B).

autoimmune diseases, and cancer.⁷The structures of four JAK

inhibitors currently approved or in clinical trials are displayed

in Figure 2A.

The currently approved drugs are mixed inhibitors of all four

JAK kinases with varying potencies against each one.⁸

Tofacitinib is approved by the FDA for the treatment of

rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis,

while ruxolitinib is approved for the treatment of myeloﬁbrosis

and polycythemia vera.⁹These two JAK inhibitors suﬀer from

many side eﬀects, arguing that more selective medications

should be developed to overcome them.^9,10

Palladium catalysis has oﬀered new pathways for the

preparation of pharmaceuticals.¹¹Tandem catalysis is a process

in which the catalyst is used to catalyze more than one reaction

for the construction of complex molecules from simple

building blocks.¹²In the present work, autotandem palladium

catalysis was applied for the rapid construction of pharmaco-

logically active JAK inhibitors. This approach has made the

synthesis of these compounds easier, and it has allowed rapid

diversiﬁcation of newly designed scaﬀolds to obtain a wide

range of derivatives with various activities and selectivities.¹³

Design and Discovery. Potentiation of the eﬀect of

topoisomerase I (Top1) inhibition by checkpoint kinase

(Chk1) inhibitors has been previously demonstrated for the

combination of various Chk1 inhibitors and camptothecin, and

it has also been shown for Chk1 siRNA.¹⁴A selective Chk1

inhibitor also displayed dose-dependent potentiation of the

antitumor eﬀect of irinotecan in MDA-MB-435 breast cancer

xenografts.¹⁵Several other studies showed the potentiating

eﬀect of Chk1 inhibitors on DNA-acting anticancer agents,

including Top1 inhibitors.^16,17For example, Aris et al. reported

the potentiation of the cellular eﬀect of LMP400 by the cell

checkpoint and Chk1-Chk2 inhibitor AZD7762.¹⁸This

encouraged us to attempt the design of potential dual Top1/

Chk1 kinase inhibitors.

Figure 3. Design rationale for the BPN 1 scaﬀold. The structural

similarity between the two compounds was used in the design. The

indenoisoquinoline scaﬀold was modiﬁed by adding an additional

hinge-binding element (hydrogen bond donor) to the hydrogen bond

acceptor, which was changed from a carbonyl oxygen to the pyridine

nitrogen. At the same time, the basic aminopropyl side chain was

retained.

can be oriented in the same direction, and a basic aminopropyl

side chain. Hybridization of the two structures and addition of

the missing hydrogen bond donor required for hinge binding

of kinase inhibitors resulted in the structure of compound 1 as

shown in Figure 3.

BPN 1 was prepared (Scheme 1) and tested against both

Chk1 kinase and Top1 and also for cytotoxic activity. It had

mild Chk inhibitory activity and no Top1 inhibitory activity.

However, BPN 7b had selectivity against blood cancer cell

lines in the cytotoxicity assay (Supporting Information). BPN

1 was therefore tested against a focused library of related

tyrosine kinases that were selected on the basis of a similarity

search using the ChEMBL database.²⁰

Analogues of our Top1 inhibitors LMP400, LMP776, and

LMP744 (Figure 2B) that have been in phase I clinical trials

were designed in an eﬀort to make compounds with dual Top1

and Chk kinase inhibitory activity (Figure 3). The design was

guided by the structural similarities between the reported

pyrazoloquinoline class of potent Chk1 inhibitors¹⁹repre-

sented in Figure 3 and the indenoisoquinolines. Both

structures have a ﬂat scaﬀold, a hydrogen bond acceptor that

Kinase proﬁling of BPN 1 against nine kinases suggested by

the similarity search revealed that it has very good aﬃnity and

some selectivity against JAK kinases (Table 1). Binding assays

of 1 showed high aﬃnities with a K_d= 20 nM against JAK3 and

a K_d= 19 nM against JAK2 kinases. This scaﬀold is a new JAK

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incorporated in the new scaﬀold (BPNs 17e−h).^25,26

A

Table 1. Enzyme Aﬃnity Data for BPN 1 against Nine

Kinases

tofacitinib-like side chain was also incorporated to compare

the resulting product to the clinically used compound (BPN

18, Scheme 5). Finally, the chirality of amino acids was

harnessed to make the chiral BPNs 19−21 with amide side

chains to target the two diﬀerent sides of the binding pocket.²⁷

Chemistry. Initially, the synthetic pathway illustrated in

Scheme 1 was investigated for the synthesis of BPN 1 and two

more analogues 7a and 7b with unsubstituted or with a nitro-

substituted ring D. Compound 2 was protected with MOMCl

using NaH in DMF according to literature methodology.²⁸

Buchwald chemistry was used to add the amine side chain to

aﬀord compound 4. The appropriate 2-bromobenzoic acid

derivatives are commercially available and were used for the

synthesis of compounds 5a−c. Each acid was heated at reﬂux

in SOCl₂to obtain the acid chlorides, which were reacted with

compound 4 in DCM and TEA. The direct intramolecular

arylation reaction was used to obtain compounds 6a−c. The

yield of this reaction was improved by replacing cesium

carbonate with potassium carbonate. Finally, compounds 1 and

7a,b were obtained by removing the MOM group by heating in

concentrated HCl in THF.

a

kinase

%Ctrl @ 10 μM

CDK2

Chk1

Chk2

EGFR

JAK1

JAK2

100

15

5.2

83

5.8

0

JAK3

0

MET

VEGFR-2

19

7.5

a

Values were obtained by comparing the results of the test

compounds to a negative control that was assigned a value of 100%

(nonbinding). Lower numbers indicate stronger binding.

inhibitory chemotype that encouraged the further exploration

of it to see if new JAK inhibitors could be obtained with

optimized activity. The design may be considered as an

extension of the polycyclic JAK inhibitors that have been

reported in the literature.²¹⁻²⁴These BPNs were conceived as

conformationally constrained relatives of tofacitinib that would

direct the placement of the side chain in the binding pocket.

The molecular model in Figure 4 shows the predicted

binding mode of BPN 1 in the ATP binding site of JAK3

overlapped with tofacitinib. On the basis of the molecular

modeling and comparison with other JAK inhibitors, a series of

modiﬁcations were made to BPN 1 to explore the SAR. The

azaindole fragment in the new scaﬀold keeps the essential

hinge binding element by forming two hydrogen bonds with

Glu903 and Leu905. The side chain in BPN 1 is basic in

nature, which often causes problems of nonselective kinome

and hERG binding. Initially, the basic side chain was replaced

with simple side chains like the esters as in BPNs 13a−c

(Scheme 2). These side chains were originally designed to test

whether the binding site can enclose esters and larger groups of

this chemical nature. Afterward, BPNs such as 17a−d with

hydrophobic and alicyclic groups similar to the reported

inhibitors were also considered (BPNs 17a−d, Scheme 3).

Cyclic amines have been reported to be present in some

selective JAK inhibitors, and therefore similar side chains were

The synthetic methodology used in Scheme 1 was not very

useful for the synthesis of the second batch of compounds with

altered side chains because increasing the size of the amine

substituents hindered amide formation with the acid chloride.

Scheme 2 illustrates an alternate synthetic pathway used for the

synthesis of BPNs 13a−c. First, the protected azaindole

compound 8²⁹was converted to the amine 9 using Buchwald

chemistry with benzophenone imine and palladium acetate.

The amine 9 was reacted with the required 2-iodoacyl chloride

derivative to provide the common intermediate 10, which was

alkylated with various reactants using sodium hydride in DMF

to aﬀord 11a−c. An intramolecular ring coupling reaction

yielded intermediates 12a−c, which were easily deprotected by

TFA to give the target compounds 13a−c.

The two schemes used for the synthesis of the target BPNs

were highly convergent, but they were not successful in making

compounds with bulkier side chains as, for example, a

cyclohexyl group. Also, the application of the second scheme

was limited to reactants that have an α-carbonyl relative to the

halide atom that facilitates the S_N2 reaction. To widen the

Figure 4. Binding mode of compound 1 (orange) aligned with tofacitinib (green) in the active site of JAK3 (PDB 3LXK). A surface representation

to the right illustrates the binding mode of the side chain of compound 1 and tofacitinib. The green area represents the methyl pocket occupied by

the tofacitinib side chain.

D

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a

Scheme 1

a

(a) MOMCl, NaH, DMF, 0− 21 °C, 3 h; (b) (H₃C)₂NCH₂CH₂CH₂NH₂, Pd₂(dba)₃, xantphos, Cs₂CO₃, dioxane, 110 °C, 16 h; (c) ArCOCl,

TEA, CH₂Cl₂, 0−60 °C, 5 h; (d) Pd(OAc)₂, K₂CO₃, PCy₃·HBF₄, DMA, 130 °C, 18 h; (e) concd HCl, THF, 60 °C, 20 h.

a

Scheme 2

a

(a) (i) Pd(OAc)₂, benzophenone imine, Cs₂CO₃, xantphos, dioxane, 110 °C, (ii) NH₂OH·HCl, NaOAc, MeOH, 21 °C; (b) ArCOCl, TEA,

CH₂Cl₂, 23 °C, 16 h; (c) R-Br, NaH, DMF, 21 °C, 18 h; (d) Pd(OAc)₂, PCy₃·HBF₄, K₂CO₃, DMA, 130 °C, 18 h; (e) (i) TFA, CH₂Cl₂, (ii)

ethylenediamine, NaOH, MeOH.

range of side chains and facilitate scaﬀold decoration, a new

tandem Pd reaction was engineered that can achieve the

Buchwald amidation and the intramolecular arylation in just

one step.

The reaction portrayed in Table 2 is a modiﬁcation of the

Catellani reaction in which the bifunctionalization of aryl

halides is facilitated by norbornene. The original conditions

reported by Catellani and co-workers failed to provide the

required compound in a good yield.³⁰The aryl iodide in the

present case is diﬀerent from o-iodotoluene in the original

paper. In addition to optimizing the yield, the versatility of the

reaction needed to be augmented in order to facilitate the

synthesis of a broad range of JAK inhibitors. The conditions of

the Catellani reaction were therefore optimized as reported in

Table 2.

The use of DMA as the solvent and PPh₃as the ligand

(entry 4) was not optimal for the reaction as the yield was only

4%. After further investigation of the solvent eﬀect, toluene was

found to be the best one with 56% yield (entry 9). The use of

potassium carbonate as the base proved to be ineﬀective, as

shown in entries 1−4. After some trials, cesium carbonate was

established to be the best base for the reaction with 68% yield

(entry 9). TFP [tri(2-furyl)phosphine] provided optimal yields

relative to the other ligands that were investigated. Finally,

palladium acetate was replaced with palladium triﬂuoroacetate

and this enhanced the yield. Palladium triﬂuoroacetate has

E

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Table 2. Optimization of the Catellani Reaction

a

entry*

catalyst

ligand

base

solvent

additive

temp (C°)/time (h)

yield (%)

b

1

2

3

4

5

6

7

8

Pd(OAc)₂

Pd₂(dba)₃

Pd(TFA)₂

TFP

K₂CO₃

CH₃CN

DMF

CH₃CN

DMA

CH₃CN

dioxane

THF

85/18

105/18

100/18

130/18

85/18

<2

0

20

4

TFP

PPh₃

TFP

xantphos

PCy₃·HB₄

S-Phos

TFP

norbornene

K₂CO₃

Cs₂CO₃

t-BuOK

Cs₂CO₃

t-BuOK

Cs₂CO₃

Na₂CO₃

Cs₂CO₃

24

21

34

56

68

52

42

32

70

75

78

95/18

70/18

toluene

100/18

100/8

9

10

11

12

13

14

15

16

tol/CH₃CN

toluene

100/20

a

b

All the numbers are the isolated yields. Tri(2-furyl)phosphine.

a

Scheme 3

a

(a) Pd(TFA)₂, Cs₂CO₃, norbornene, TFP, toluene, 110 °C; (b) (i) TFA, (ii) H₂N(CH₂)₂NH₂or (i) TFA, (ii) NaOH.

F

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Scheme 4

a

been reported to be more eﬃcient than palladium acetate in

nonpolar solvents like toluene.^31,32

Scheme 5

Scheme 3 illustrates the new reaction and the versatility of

side chain installation that it provides. In this reaction, the

amides 14a−l were reacted with the iodo compound 15 to

form both of the new C−N and C−C bonds and generate

compounds 16a−l in a single step instead of the four steps

employed in Scheme 1. The amides 14a−l can be easily

obtained from the commercially available o-bromo acids, and

compound 15 can be prepared from commercially available 4-

iodo-7-azaindole.³³A mechanism for the reaction is proposed

in Scheme 4.³⁰The versatility of this reaction has been recently

shown in the construction of related scaﬀolds.³⁴

After optimization of the reaction conditions, the reaction

was employed for the synthesis of the target BPNs 17a−m.

Compounds 16a−l were deprotected using TFA and ethyl-

enediamine (Scheme 3). The target compounds 18, 19a,b,

20a,b, and 21 were synthesized from the corresponding amines

or acids using HATU in DMF (Scheme 5)

Biological Results and Discussion. The JAK inhibitory

activities of all of the target compounds are listed in Table 3.

Generally, the BPNs had very good to excellent activity against

the four enzymes with various selectivity proﬁles. Ring D

substituents are important, as evidenced by the unsubstituted

BPN 7a, which showed higher potency against the four

members of the JAK family when compared to BPN 7b but

lower than that of 1 with a dimethoxy substitution pattern. The

dimethoxylated ring D was kept for the rest of the BPNs in the

series because the docking calculations supported its

importance in the solvent-exposed area. On the other hand,

the side chain modiﬁcations alter both the potency and

selectivity. The elongated basic side chain of BPNs 1 and 7a,b

was not optimal for the JAK inhibitory activity when compared

a

(a) For 18: cyanoacetic acid, HATU, DIPEA, DMF, 21 °C, 18 h. For

the rest: the appropriate amines, HATU, DIPEA, DMF, 21 °C, 18 h.

to the side chains found in 13a−c. Additionally, BPNs 17a−d

with alicyclic side chains were among the most potent in this

series with single-digit nanomolar potency against the diﬀerent

members of the JAK family. On the other hand, BPNs 17e−h

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Table 3. Janus Kinase Inhibitory Activities of the BPNs

compd

JAK1 IC₅₀(nM)

JAK2 IC₅₀(nM)

JAK3 IC₅₀(nM)

TYK2 IC₅₀(nM)

tofacitinib

1

7a

1.42 0.07

95.62 5.18

423.8 4.1

3334 30

1.09 0.17

72.97 2.35

229.25 8.45

489.65 2.15

14.12 0.945

17.56 0.08

10.55 0.41

1.07 0.05

3.57 0.02

0.89 0.09

1.82 0.01

37.07 1.55

14.70 0.12

37.91 2.60

8.91 0.29

63.04 1.69

220.8 16.3

3.12 0.05

1171 128

>1000

0.24 0.02

129.75 0.45

285.9 8.5

1111.5 53.5

9.94 0.091

6.65 0.05

27.5 0.75

2.92 0.14

1.65 0.1

1.09 0.03

1.01 0.026

124.1 7.1

18.31 0.07

143.7 1.3

23.45 0.11

28.94 1.55

95.56 1.74

2.55 0.02

818.9 20.5

26.58 0.58

114.4 0.6

87.03 0.4

75.03 1.57

10.38 0.85

427.45 11.45

708.65 34.35

4766 207

>1000

7b

13a

13b

13c

17a

17b

17c

17d

17e

17f

17g

17h

17i

17j

18

103.15 1.25

9.89 1.18

1.91 0.09

4.23 0.34

125.1 3.6

6.59 0.48

17.86 0.97

37.54 1.08

11.48 0.61

10.61 0.22

5.28 0.19

58.26 1.92

304.45 38.05

1.20 0.22

616.3 20.9

138.1 4.2

3.37 0.13

43.78 3.1

30.17 1.73

18.42 1.05

17.4 0.88

1.93 0.07

39.43 3.95

2.24 0.30

8.38 0.44

166.85 36.35

32.39 3.61

176.3 2.3

45.10 4.8

256.5 16.5

1502 124

4.35 0.38

5730 80

19a

19b

20a

20b

21

1163.5 3.5

73.66 2.4

336.3 12

273.4 2.6

29.6 1.2

140.2

7

109.95 6.1

with cyclic amine side chains had good to moderate activity

with no characteristic selectivity proﬁles. Compound 18, with

an N-(cyanoacetyl)-3-piperidinyl side chain as found in the

clinically used drug tofacitinib, also showed potent activity

against the four enzymes with IC₅₀values in the low

nanomolar range and low selectivity.

good as the cyanoazetidine ring of 20a in enhancing the

selectivity of BPN 13c. Compound 20a is a very good

candidate for further development, with good potency and

signiﬁcant selectivity for JAK1 kinase.

To test the performance in a cellular context, six of the more

potent compounds representing the whole series were tested

for their ability to inhibit the phosphorylation of STAT-5. This

assay is selective for JAK3-mediated phosphorylation of STAT-

5 in SZ4 cells when activated by IL-2. Compound 18 showed

the best inhibition of STAT-5 phosphorylation, with an IC₅₀

value of 1.55 μM relative to tofacitinib. Compound 17a also

showed good activity with 3.72 μM IC₅₀value. On the other

hand, BPNs 17b and 17e had very low activity. Table 4 lists

the STAT-5 phosphorylation assay results.

Furthermore, the activities of BPNs 17a and 18 were further

conﬁrmed by Western blot analyses of STAT-5 phosphor-

ylation. Figure 5 illustrates Western blot-based determination

of the IC₅₀values of STAT-5 phosphorylation for BPNs 17a

and 18. Both compounds showed very good activity in this

cell-based assay, with IC₅₀values of 65.9 and 15.3 nM,

respectively, giving further evidence of the JAK3 inhibitory

activity. The curves for IC₅₀determinations are provided in the

Supporting Information.

The JAKs, particularly JAK3 and TYK2, are highly expressed

by T cells and mediate cytokine receptor signaling.³⁵A T cell

proliferation assay was performed in order to access the

inhibitory activities of the compounds in a functional context

(Table 4 and Figure 6). The prototype JAK inhibitor

tofacitinib was eﬀective in suppressing T cell proliferation.

Although not particularly active in the enzyme assays, BPN

17e, with a methylpiperidine side chain, showed the best

activity in the cellular assay with an IC₅₀value = 0.29 μM,

while BPNs 17b and 17d, with hydrophobic side chains, had

very good activity that was similar to the reference compound

tofacitinib, with IC₅₀values = 0.63 and 0.76 μM, respectively.

A kinome scanning assay for BPN 17b showed some oﬀ-target

Compound 13a had very interesting selectivity for JAK2 and

JAK3 over JAK1 and TYK2, while 13b was not as selective,

with good activity against all four enzymes. On the other hand,

BPN 13c displayed more interesting selectivity for JAK1, with

an IC₅₀value of 1.9 nM and 5-fold selectivity against JAK2 and

14-fold selectivity for JAK3. However, being an ester, BPN 13c

is expected to suﬀer from poor metabolic stability, and it is also

racemic in nature. Compounds 19a and 19b were prepared to

replace the ester of 17i with amides in order to increase the

stability toward metabolic hydrolysis. Unfortunately, BPN 19a

lost potency and selectivity relative to the ester 17i, while BPN

19b was less potent than 17i vs JAK1, JAK2, and TYK2, but it

kept very good potency and selectivity vs JAK3. Comparing the

activities of the two enantiomers 17i and 17j indicates that the

R conﬁguration present in 17i is much better than the S

conﬁguration.

The isopropyl group present in 19a and 19b was changed to

an ethyl in 20a and 20b in order to explore eﬀects on potency

and selectivity. Compound 20a had very good activity and

selectivity vs JAK1 kinase with an IC₅₀= 3.37 nM, 10-fold

selectivity vs JAK2, and 30-fold selectivity vs JAK3. Although

the replacement of the ester group in 13c with this amide side

chain decreased the activity moderately, it enhanced the

selectivity vs JAK1 compared to JAK2, JAK3, and TYK2.

Changing the absolute conﬁguration of the side chain of 20b

did not inﬂuence the activity very much, as shown by

comparing the activities of 20b and 21 against the four

enzymes. Although 21 was consistently more active than 20b,

the conﬁgurational eﬀect is opposite that observed with 17i

and 17j. The triﬂuoroethyl amide side chain of 20b was not as

H

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Table 4. Activities of Selected BPNs for Inhibition of

Primary T Cell Proliferation and STAT-5 Phosphorylation

in SZ4 Cells

IC₅₀(μM) T cell

proliferation

IC₅₀(μM) (JAK3/STAT-5)

a

compd

IL2/SZ4 cells

tofacitinib

7a

0.63 0.26

1.39 0.57

4.45 2.18

3.97 2.14

1.82 0.9

1.21

NT

3.72

76.9

18.34

28.86

61.57

NT

1.55

NT

b

13a

13b

13c

17a

17b

17c

17d

17e

17f

17g

17h

17i

17j

18

0.79 0.26

0.63 0.18

3.24 2.40

0.76 0.21

0.29 0.08

1.45 0.53

2.55 1.48

1.15 0.45

1.12 0.42

1.67 0.44

0.68 0.22

5.79 2.22

5.07 2.03

3.66 2.18

4.66 1.85

19b

20a

20b

21

a

The numbers listed are the result of two determinations. Please see

b

Supporting Information for the data. Not tested.

Figure 5. Western blot analysis of phospho-STAT-5 and anti-α-

tubulin for BPNs 17a and 18.

activity, and in addition, cytotoxicity of this compound was

observed in the NCI-60 cell lines (Supporting Information),

which might explain the higher activity of this compound in

the cell proliferation assay.

Figure 6. Eﬀects of BPNs on proliferation of primary murine T cells.

Naive CD4 T cells were isolated from mouse spleens and cultured

+

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with the indicated BPNs in the presence of T cell activators

(antibodies to CD3 and CD28) and IL-2 for 3 days. T cell

proliferation was assessed by XTT staining and measured at 475 nm

with a microplate reader (SpectraMax i3X, Molecular Devices). The

assay was repeated three times (n = 3 for each dose and inhibitor),

and mean SEM ranges are shown.

The assay results shown in Figure 6 also provide information

on the cytotoxicity of the compounds to T cells. Among the

compounds, 17a, 17b, 17d, 17d, and 18 displayed relatively

high cell killing activity on proliferating T cells at 2 μM or

lower. Moreover, eight diﬀerent representative compounds

were assayed for the general cytotoxicity in the NCI-60 cell

line panel. Generally speaking, the BPNs were not very

cytotoxic, although BPNs 1, 7b, 17d, and 17i produced

negative growth percentages in a few of the cell lines when

tested at a concentration of 10 μM. The results are listed in

Table 1s in Supporting Information. Twelve BPNs were tested

for their Top1 inhibitory activity, and none of them displayed

signiﬁcant activity. Figures 1s, 2s, and 3s in the Supporting

Information show the DNA fragmentation patterns produced

by the 12 compounds.

moderate inhibitory activity on T cell proliferation when

compared to tofacitinib, with IC₅₀values higher than 3 μM.

The absence of a good correlation between the cellular and

enzyme inhibition data may be due to the diﬀerential

expression of various JAK subtypes in T cells. In addition,

the physicochemical properties of the BPNs and diﬀerences in

cell permeability may also play a major role in the cellular

activities as will be discussed shortly.³⁶

Cytokines such as IFNγ and IL-17 play key roles in

mediating the inﬂammatory activities of T cells. These two

cytokines are produced by distinct T cell subsets that are

antagonistic to each other.³⁷The eﬀects of selected JAK

inhibitors on T cell expression of IFNγ and IL-17 were

On the other hand, BPNs 7a and 17f−j displayed good

activity when compared to tofacitinib (Table 4 and Figure 6).

In addition, BPNs 13a, 13b, 17c, 19b, 20a, 20b, and 21 had

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Figure 7. Eﬀects of compounds on IFNγ expression by primary murine T cells. Naive CD4 T cells were isolated from mouse spleens and cultured

with the indicated compounds in the presence of T cell activators (antibodies to CD3 and CD28) and IL-2 for 5 days. (A) T cell expression of

IFNγ was assessed by intracellular staining of activated T cells with an antibody to IFNγ. The cells were analyzed with a Canto II ﬂow cytometer

(Becton Dickinson). (B) % IFNγ expression by T cells in the presence of test compounds compared to control. Note that data for 17b and 19a are

presented with diﬀerent scales on the y-axes of the bar graphs than the other compounds. The assay was repeated three times (n = 3 for each dose

and inhibitor), and mean SEM are shown in part B. *Signiﬁcant diﬀerences from controls (no inhibitor) by Student t test (paired one-tailed).

examined. Tofacitinib was eﬀective in suppressing IFNγ

production (Figure 7). The BPNs 17a, 17c, 17d, 18, and

20a were also similarly eﬀective in suppressing IFNγ

production. In general, there is an inverse relationship between

IFNγ and IL-17 production because the IFNγ produced by T

cells eﬀectively suppresses IL-17 production. There is much

greater T cell production of IL-17 in the presence of tofacitinib

(Figure 8). Similarly, IL-17 production was increased in the

presence of 17a−e, 18, and 20a, but it was not increased

signiﬁcantly by 19a as expected from the data reported in

Figure 7. These results indicate that in general the compounds

have potent modulatory activity on the production of T cell

eﬀector cytokines.

The results from the assays for T cell proliferation and

cytokine production are generally in line with those expected

from the JAK inhibition assay. However, the most potent

compounds in the isolated enzyme assay were not necessarily

the most potent ones in suppressing T cells. This may be

because the compounds need to have additional characteristics

such as membrane permeability and metabolic stability to be

eﬀective as inhibitors within cells. Also, the diﬀerences among

the compounds on target or cytokine signaling selectivity can

account for such diﬀerences among the assays. For example,

IL-2 and IL-23 drive T cell proliferation in the assays that were

used. IL-2 uses JAK1 and JAK3, whereas IL-23 uses JAK2 and

TYK2. If a compound inhibits JAK2 but not JAK1 or JAK3, it

would not eﬀectively inhibit IL-2-driven T cell proliferation.

Thus, the results from the biochemical inhibition assays would

not exactly match the results from cell-based assays or in vivo

experiments.

Kinome Selectivity. For any new kinase inhibitor scaﬀold,

it is always important to show selectivity against the target of

interest to avoid possible side eﬀects and toxicity.^38,39To

investigate the kinome selectivity of the new BPN scaﬀold,

three potent BPNs were tested for their abilities to inhibit

various members of the human kinome (468 kinases)

(KINOMEscan Assay Platform by DiscoverX). Compounds

17b, 18, and 20a were assayed for their ability to displace an

immobilized ATP binding site ligand from 403 normal kinases

and 65 mutant kinases (Figure 9). These compounds

demonstrated excellent selectivity proﬁles. Table 5 shows the

selectivity scores and number of hits for the three compounds

tested. The selectivity score or S-score is a quantitative

measure of compound selectivity. It is calculated by dividing

the number of enzyme hits to by the total number of distinct

kinases tested, excluding mutant variants. This value can be

calculated using %Ctrl as a potency threshold and provides a

quantitative method of describing compound selectivity to

facilitate comparison of diﬀerent compounds.

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Figure 8. Eﬀects of compounds on IL-17 expression by primary murine T cells. Naive CD4 T cells were isolated from mouse spleens and cultured

with the indicated compounds in the presence of T cell activators (antibodies to CD3 and CD28) and the cytokines listed in the method section for

5 days. (A) T cell expression of IL-17 was assessed by intracellular staining of activated T cells with an antibody to IL-17. The cells were analyzed

with a Canto II ﬂow cytometer. (B) Percent IL-17 expression by CD4 T cells in the presence of test compounds compared to control. Note the

diﬀering scales on the y-axes of the bar graphs. The assay was repeated three times (n = 3 for each dose and inhibitor), and mean SEM are shown

in part B. *Signiﬁcant diﬀerences from controls (no inhibitor) by Student t test (paired one-tailed).

Figure 9. Technique used for determination of kinome selectivity.

BPN 20a had outstanding selectivity for the four Janus

kinases (Supporting Information, Figure 4s), and of those, it

was most active vs JAK-1 (Table 3). BPNs 17b and 18 aﬀected

some additional kinases in the kinome in a potent manner.

Table 6 lists the K_dvalues in nM for some of the additional

kinases aﬀected by 17b and 18. Both of them bound to BIKE

kinase in the low nanomolar range. Compound 17b also

showed good binding activity to FLT3(N841I), SNARK, and

TrkA⁴⁰in the low nanomolar range, and it had moderate

binding to Ark5. Compound 18 demonstrated potent

inhibition of YSK4 kinase. A complete list of kinases tested

and the kinome maps are available in Table 2s and Figure 4s in

the Supporting Information.

ADME Properties. To evaluate the preliminary ADME

properties of this new BPN class of JAK inhibitors, ﬁve

diﬀerent compounds were tested for solubility, cell perme-

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transport. On the other hand, three of the BPNs (17e, 18,

and 20a) had excellent metabolic stabilities against human liver

microsomal enzymes with t_1/2values over 60 min.

Table 5. Kinome Selectivity Scores of BPNs 17b, 17d, and

18

no. of

screening

Molecular Modeling. The structures of the inhibitors

were docked in the ATP binding sites of the four subtypes of

the JAK family. Compounds 17b, 17d, and 18 were the most

active against the JAK3 enzyme, with IC₅₀values of 1.65, 1.01,

and 2.55 nM, respectively, while BPNs 13c, 18, and 20a were

the most active against JAK1 with IC₅₀values of 1.91, 1.20, and

3.37 nM, respectively. Figure 10 illustrates a comparison

between the binding modes of compound 17d in the ATP

binding sites of JAK1 and JAK3. As expected, the

pyrrolopyridine part in all of the compounds made the

essential hinge binding interactions with Leu-905 and Glu-903

in JAK3 and with Leu-959 and Glu-957 in JAK1 involved in

the formation of hydrogen bonds.

selectivity enzyme

nonmutant concentration selectivity

a

compd score type

hits

kinases

(nM)

score

b

17b

18

20a

S(35)

S(10)

S(1)

S(35)

S(10)

S(1)

S(35)

S(10)

S(1)

24

6

0

40

9

5

4

0

403

100

0.06

c

0.015

d

0

0.099

0.022

0.012

0.01

0

a

The numbers reﬂect the abilities of the test compounds to inhibit the

binding of the kinases to an immobilized ATP binding site ligand.

Lower numbers indicate higher selectivities of the test compounds for

There is only a minor diﬀerence between the structures of

the JAK1 and JAK3 ATP binding sites in the activation loop

(green) (glycine vs alanine). This can partially explain the

diﬀerence in the activity between compound 17d against JAK1

(IC₅₀= 17.86 nM) and JAK3 (IC₅₀= 1.01 nM).

b

the enzymes. (Number of nonmutant kinases with %Ctrl <35)/

(number of nonmutant kinases tested) where %Ctrl is the percentage

of enzymes remaining bound to an immobilized ATP binding site

ligand in the presence of 100 nM concentration of the test compound.

c

(Number of nonmutant kinases with %Ctrl <10)/(number of

d

On the other hand, BPN 18 had excellent inhibitory activity

against both enzymes with very good cellular potency and

kinome selectivity. The hypothetical binding modes of BPN 18

with JAK1 and JAK3 are illustrated in Figure 11. Compound

18 was calculated to have a hydrophobic interaction with the

activation loop (colored green) in both enzymes, but the

hydrophobic part of the piperidine side chain is not large

enough to cause a signiﬁcant diﬀerence in activity between the

two enzymes (Table 3). The cyanoacetate side chain is

directed toward the phosphate binding loop in both enzymes

(colored red in Figure 11), which is a relatively hydrophilic

region that plays a role in the potency of this compound.

Finally, compound 20a was not very active in the T cell

proliferation assay. This can be explained by its good selectivity

against JAK1, which is moderately expressed in the cells that

were used in the assay, in addition to poor cellular

permeability. A clear explanation for the JAK1 selectivity of

20a could not be drawn from the molecular modeling, but it

can be hypothesized that the nonbonded interaction of the

ethyl side chain with the activation loop in JAK1 and JAK3

may contribute to the diﬀerence in activity. The presence of

the larger Ala966 moiety in JAK3 relative to the Gly1020

residue of JAK1 can limit the free movement of the ethyl group

in the hydrophobic pocket of the activation loop while the

smaller glycine residue can accommodate the ethyl side chain

of 20a as shown in Figure 12.

nonmutant kinases tested). (Number of nonmutant kinases with %

Ctrl <1)/(number of nonmutant kinases tested).

Table 6. K_d(nM) Values for Kinases Identiﬁed as Hits

During Kinome Scanning

kinase

ARK5

BIKE

FLT3(D835V)

FLT3(N841I)

SNARK

17b

18

a

42

7.5

NT

15

3.5

2.3

NT

0.22

>100

NT

1.2

TRKA

YSK4

a

NT: Not tested.

ability, and liver microsomal metabolic stability. Table 7 lists

the results for BPNs 17a, 17b, 17e, 18, and 20a. Most of them

had low aqueous solubilities (PBS buﬀer, pH 7.4) with the

exception of compound 17e, which has a methylpiperidine side

chain that is well-known to enhance the solubility. In contrast,

with the exception of 17e, the compounds displayed enhanced

solubilities in gastric and intestinal ﬂuids relative to the PBS

buﬀered media. The Caco-2 cellular permeability proﬁles of

these particular BPNs are relatively unfavorable, with BPNs

17e and 18 being potential substrates for active eﬄux

Table 7. Preliminary ADME Properties of Selected Compounds

aqueous

aqueous solubility

(simulated intestinal

ﬂuid) (μM)

aqueous solubility

(simulated gastric

ﬂuid) (μM)

A−B permeability (Caco-2,

B−A permeability (Caco-2,

human liver

microsomes

(t_1/2) (min)

solubility (PBS,

pH 6.5/7.4) permeability

−6

compd

17a

17b

17e

18

pH 7.4) (μM)

(10 cm/s)

a

c

NS

12.5

8.8

200

NS

192.9

NT

NS

60.7

198.1

187.3

24.2

NT

NP

0.1

43.4

9.6

14

NT

2

36.7

17

7

1.7

195.8

NS

2.9

0.4

0.3

0.1

NT

0.3

>60

NT

>60

20a

13.6

2

tamoxifen

ranitidine

propranolol

b

NT

33.5

a

b

c

NS: Not soluble. NT: Not tested. NP: Not permeable.

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Figure 10. Hypothetical binding modes of compound 17d in JAK1 (left) (PDB 4I5C) and JAK3 (right) (PDB 3LXK).

Figure 11. Hypothetical binding mode of compound 18 in JAK1 (left) (PDB 4I5C) and JAK3 (right) (PDB 3LXK).

Figure 12. Hypothetical binding mode of 20a in JAK1 l (left) (PDB 4I5C) and JAK3 (right) (PDB 3LXK).

Determination of Anti-Inﬂammatory Activity in a

Mouse Model. To access anti-inﬂammatory activity in vivo,

compound 17b, tofacitinib, or vehicle (in 10% DMSO and

10% Cremophor EL) were each administered to groups of

seven mice two months after collagen-induced inﬂammation,

and the activity was measured on each paw using a standard

0−4 rheumatoid arthritis scale. This approach is more realistic

than the usual collagen-induced rheumatoid arthritis studies

because it measures the ability of the drugs to treat existing

inﬂammation rather than prevent it. The total scores, which

reﬂect the degree of inﬂammation and ankylosis, are plotted as

a function of time (Figure 13). The t test reveals that both

tofacitinib and 17b cause a signiﬁcantly diﬀerent response

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concd HCl (2 mL) was added to the solution. The mixture was

heated at 80 °C for 18 h and then cooled to room temperature. The

THF was evaporated under reduced pressure, and the residue was

treated with saturated aq NaHCO₃solution until the eﬀervescence

ceased. The mixture was extracted with CHCl₃(3 × 15 mL), and the

combined organic layer was washed with water (2 × 10 mL) and

brine (1 × 15 mL), dried with sodium sulfate, and evaporated to give

a light-brown residue. The residue was puriﬁed by silica gel column

chromatography using CHCl₃−MeOH (8.5:1.5) to give the ﬁnal

1

compound 1 as a light-brown foam (26 mg, 59%). H NMR (300

MHz, DMSO-d₆) δ 12.11 (s, 1 H), 9.40 (s, 1 H), 8.04 (s, 1 H), 7.73

(s, 1 H), 7.55 (s, 1 H), 6.93 (s, 1 H), 4.69−4.62 (m, 2 H), 4.05 (s, 3

H), 3.91 (s, 3 H), 2.63−2.48 (m, 2 H), 2.26 (s, 6 H), 2.12−1.94 (m, 2

H). MALDIMS m/z 381 (MH)⁺. HRESIMS calcd for C₂₁H₂₅N₄O₃

(MH)⁺381.1927, found 381.1920. C18 HPLC purity: 100%

(MeOH−H₂O, 85:15).

4-Bromo-1-(methoxymethyl)-1H-pyrrolo[2,3-b]pyridine (3).

This compound was prepared according to a published literature

Figure 13. Mean rheumatoid arthritis score. Mouse paw collagen-

induced inﬂammation assay (male DBA-1 n = 7) of compound 17b.

Daily doses of tofacitinib or 17b were administered subcutaneously in

10% DMSO and 10% Cremophor EL to seven mice. Paw

inﬂammation for each paw was expressed on a 0−4 mean rheumatoid

arthritis scale. The t test revealed that both tofacitinib and 17b caused

a signiﬁcantly diﬀerent response compared to the vehicle control and

that there is not a statistically signiﬁcant diﬀerence between the two

drugs. The calculated P values were 0.0481 for week 2, 0.0443 for

week 3, 0.0442 for week 4, and 0.0473 for week 5.

procedure²⁸and isolated as yellow oil (1.16 g, 96%). H NMR (300

1

MHz, CDCl₃) δ 8.15 (d, J = 5.2 Hz, 1 H), 7.40 (d, J = 3.6 Hz, 1 H),

7.31 (d, J = 5.2 Hz, 1 H), 6.59 (d, J = 3.6 Hz, 1 H), 5.64 (s, 2 H), 3.30

(s, 3 H).

N¹-(1-(Methoxymethyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-

N³,N³-dimethylpropane-1,3-diamine (4). Compound 3 (1 equiv,

0.5 g, 2.01 mmol), N¹,N¹-dimethylpropane-1,3-diamine (0.275 g, 2.7

mol, 1.3 equiv), Pd₂(dba)₃(95 mg, 5 mol %), xantphos (120 mg, 10

mol %), and cesium carbonate (1.35 g, 4.15 mmol, 2 equiv) were

mixed in a 45 mL pressure tube, and anhydrous dioxane (15 mL) was

added to the mixture. Argon was bubbled through the reaction

mixture for 2 min, and then the pressure tube was capped and placed

directly in a preheated oil bath at 110 °C. The mixture was heated for

16 h and then allowed to cool to room temperature. CHCl₃(20 mL)

was added to the mixture and then it was ﬁltered through a bed of

Celite and the residue left on the ﬁlter pad was washed with additional

CHCl₃(20 mL). The ﬁltrate was evaporated to aﬀord a dark residue

that was puriﬁed by silica gel column chromatography using a

CHCl₃−MeOH mixture (9:1) as the eluent to provide the amine 4 as

compared to the vehicle control and that there is not a

statistically signiﬁcant diﬀerence between the two drugs.

CONCLUSION

■

A novel tetracyclic BPN series of JAK inhibitors was

discovered by the hybridization of Chk1 inhibitors and Top1

poisons. The new series showed interesting enzymatic activity

proﬁles against the four members of the JAK family with

potencies in the low nanomolar range and variable selectivities.

Cellular kinase and T-cell proliferation assays revealed

promising activity of the new scaﬀold compared to the

clinically used drug tofacitinib. Preliminary testing of the

ADME properties of a limited number of compounds in this

new class indicated that several of them suﬀer from low

aqueous solubility and poor cellular permeability that might be

improved by ﬁne-tuning the side chain. In vivo studies in a

mouse rheumatoid arthritis model revealed BPN 17a has anti-

inﬂammatory activity that is comparable to that of the clinically

used drug tofacitinib. Molecular modeling was used to show

the hypothetical JAK1 and JAK3 binding modes of some of the

more potent compounds in this class.

1

a light-brown oil (0.4 g, 74%). H NMR (300 MHz, CDCl₃) δ 8.06

(d, J = 5.5 Hz, 1 H), 7.08−7.02 (m, 1 H), 6.50 (d, J = 3.6 Hz, 1 H),

6.25 (s, 1 H), 6.19 (d, J = 5.6 Hz, 1 H), 5.57 (s, 2 H), 3.51−3.45 (m,

2 H), 3.29 (s, 3 H), 2.73 (t, J = 6.3 Hz, 2 H), 2.48 (s, 6 H), 2.07−1.87

(m, 2 H). HRESIMS m/z calcd for C₁₄H₂₂N₄O (MH)⁺263.1866,

found 263.1869.

General Procedure for the Preparation of the Amides 5a−c.

The appropriate benzoyl chloride derivative was prepared by heating

the necessary 2-bromobenzoic acid derivative (0.9 mmol, 1 equiv)

and thionyl chloride (5 mL) at reﬂux for 2 h, followed by removal of

excess thionyl chloride under vacuum. The acid chloride (1 equiv, 0.9

mmol) in anhydrous DCM (5 mL) was added dropwise to an ice-cold

solution of the amine 4 (0.25 g, 1 equiv, 0.9 mmol) and TEA (10

equiv) in anhydrous DCM (20 mL). The mixture was heated at reﬂux

for 5 h. After cooling to room temperature, the mixture was washed

with saturated NaHCO₃solution (1 × 20 mL), water (2 × 20 mL),

and brine (1 × 20 mL) and dried with anhydrous sodium sulfate. The

residue obtained after the evaporation of DCM was subjected to silica

gel column chromatography using CHCl₃−MeOH as eluent to yield

the desired amides 5a−c.

N-(3-(Dimethylamino)propyl)-2-bromo-N-(1-(methoxymethyl)-

1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide (5a). This compound was

isolated as a brown gum (0.27 g, 57%). ¹H NMR (300 MHz, CDCl₃)

δ 8.16−8.08 (m, 1 H), 7.72−7.66 (m, 1 H), 7.38 (d, J = 3.6 Hz, 1 H),

6.91−6.85 (m, 4 H), 6.68−6.61 (m, 1 H), 5.59 (s, 2 H), 4.15−4.09

(m, 2 H), 3.27 (s, 3 H), 2.54−2.48 (m, 2 H), 2.26 (s, 6 H), 1.94−1.88

(m, 2 H). HRESIMS m/z calcd for C₂₁H₂₅BrN₄O₂(MH)⁺445.1234/

447.1216, found 445.1231/447.1215.

EXPERIMENTAL SECTION

■

General. Melting points were determined with a Mel-Temp

apparatus using capillary tubes and are uncorrected. The proton

nuclear magnetic resonance spectra (¹H NMR) were recorded using

Bruker ARX300 300 MHz or Bruker DRX500 500 MHz

spectrometers. IR spectra were obtained with a PerkinElmer 1600

series FTIR spectrometer. For biologically tested compounds, the

HPLC major peak accounted for ≥95% of the combined total peak

area when monitored by a UV detector at 254 nm. HPLC analyses

were performed on a Waters 1525 binary HPLC pump/Waters 2487

dual λ absorbance detector system. Analytical thin-layer chromatog-

raphy was conducted on Baker-ﬂex silica gel IB2-F plates, and

compounds were visualized with UV light at 254 nm. Silica gel ﬂash

chromatography was performed using 230−400 mesh silica gel.

4-(3-(Dimethylamino)propyl)-7,8-dimethoxy-1,4-dihydro-

5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one (1). Com-

pound 6b (0.05 g, 0.13 mmol) was dissolved in THF (10 mL), and

2-Bromo-N-(3-(dimethylamino)propyl)-4,5-dimethoxy-N-(1-(me-

thoxymethyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide (5b). This

compound was isolated as a dark-brown gum (0.26 g, 54%). ¹H NMR

(300 MHz, CDCl₃) δ 8.16 (s, 1 H), 7.38 (d, J = 3.6 Hz, 1 H), 6.91 (s,

1 H), 6.82 (s, 1 H), 6.62 (d, J = 3.6 Hz, 1 H), 6.47 (s, 1 H), 5.59 (s, 2

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H), 4.11−4.04 (m, 2 H), 3.75 (s, 3 H), 3.40 (s, 3 H), 3.26 (s, 3 H),

2.64−2.59 (m, 2 H), 2.31 (s, 6 H), 1.97−1.89 (m, 2 H). HRESIMS

m/z calcd for C₂₃H₂₉BrN₄O₄(MH)⁺505.1444/507.1427, found

505.1442/507.1423.

(dd, J = 9.0, 2.6 Hz, 1 H), 7.64 (s, 1 H), 7.01 (s, 1 H), 4.69−4.62 (m,

2 H), 2.55−2.50 (m, 2 H), 2.24 (s, 6 H), 2.05−1.96 (m, 3 H). ¹³C

NMR (126 MHz, DMSO) δ 160.94, 150.55, 145.81, 142.17, 139.52,

139.02, 126.94, 125.93, 124.06, 123.93, 123.42, 106.36, 106.02,

102.34, 56.60, 45.50, 43.52, 26.06. MALDIMS m/z 366 (MH)⁺.

HRESIMS calcd for C₁₉H₂₀N₅O₃(MH)⁺366.1566, found 366.1560.

C18 HPLC purity: 100% (MeOH−H₂O, 85:15).

2-Bromo-N-(3-(dimethylamino)propyl)-N-(1-(methoxymethyl)-

1H-pyrrolo[2,3-b]pyridin-4-yl)-5-nitrobenzamide (5c). This com-

1

pound was isolated as a dark-brown gum (0.35 g, 74%). H NMR

(300 MHz, CDCl₃) δ 8.15 (d, J = 5.0 Hz, 1 H), 7.86 (s, 1 H), 7.80

(dd, J = 8.7, 2.6 Hz, 1 H), 7.56 (d, J = 8.7 Hz, 1 H), 7.43 (d, J = 3.6

Hz, 1 H), 6.96 (d, J = 5.1 Hz, 1 H), 6.64 (d, J = 3.7 Hz, 1 H), 5.57 (s,

2 H), 4.12 (s, 2 H), 3.22 (s, 3 H), 2.54 (s, 2 H), 2.30 (s, 6 H), 2.02−

1.86 (m, 2 H). HRESIMS m/z calcd for C₂₁H₂₄BrN₅O₄(MH)⁺

490.1084/492.1067, found 490.1085/492.1070.

1-((2-(Trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]-

pyridin-4-amine (9). SEM-protected 4-bromo-7-azaindole 8 (2 g,

8.3 mmol) was added to a mixture of xantphos (0.48 g, 0.83 mmol),

Pd(OAc)₂(0.093 g, 0.41 mmol), benzophenone imine (1.8 g, 9.93

mmol), and Cs₂CO₃(5.4 g, 16.59 mmol) in anhydrous dioxane (50

mL). The reaction mixture was added to a 150 mL pressure vessel,

degassed with Ar for 5 min, and heated at 110 °C for 18 h. The

reaction mixture was cooled to room temperature mixed with

chloroform (50 mL) and ﬁltered through a bed of Celite. The ﬁlterate

was evaporated, and the residue was dissolved in MeOH (100 mL).

Sodium acetate (1.5 g, 19 mmol) and hydroxylamine hydrochloride

(0.95 g, 13.7 mmol) were added to the solution, and the mixture was

stirred at room temperature for 10 h. MeOH was evaporated, and the

residue was partitioned between NaOH solution (1 N, 100 mL) and

DCM (100 mL). The organic layer was washed with water (2 × 50

mL) and brine (1 × 20 mL) and dried. Column chromatography of

the residue obtained after evaporation of the solvent (SiO₂/

CHCl₃:MeOH 9:1) gave 9 as a yellow solid (1.3 g, 81%): mp

General Procedure for the Direct Intramolecular Arylation

Reaction to Aﬀord 6a−c. The benzamides 5a−c (1 equiv, 0.5

mmol) were dissolved in DMA (5 mL), and Pd(OAc)₂(10 mol %),

Cs₂CO₃(2 equiv), Ag₂CO₃(2 equiv), and PCy₃·HBF₄(20 mol %)

were added. The reaction mixture was heated at 130 °C under Ar

atmosphere for 18 h. The reaction mixture was cooled to room

temperature, and EtOAc (10 mL) was then added. The mixture was

ﬁltered through a bed of Celite, and the residue left on the ﬁlter pad

was washed with EtOAc (about 20 mL). The ﬁltrate was transferred

to a separatory funnel and washed with water several times (5 × 15

mL) to remove the DMA and brine (1 × 15 mL). The organic layer

was dried and evaporated, and the residue was puriﬁed with column

chromatography using CHCl₃−MeOH (9:1) as the eluent to give the

tetracyclic compounds 6a−c.

1

135−137 °C. H NMR (300 MHz, CDCl₃) δ 8.04 (d, J = 5.4 Hz, 1

H), 7.17 (d, J = 3.7 Hz, 1 H), 6.42 (d, J = 3.7 Hz, 1 H), 6.35 (d, J =

5.4 Hz, 1 H), 5.64 (s, 2 H), 4.42 (s, 2 H), 3.59−3.45 (m, 2 H), 0.98−

0.85 (m, 2 H), −0.06 (s, 9 H).

4-(3-(Dimethylamino)propyl)-1-(methoxymethyl)-1,4-dihydro-

5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one (6a). This com-

1

pound was isolated as a brown semisolid (0.06 g, 32%). H NMR

2-Iodo-4,5-dimethoxy-N-(1-((2-(trimethylsilyl)ethoxy)-

methyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide (10). 2-Iodo-

4,5-dimethoxybenzoyl chloride was prepared by heating a mixture of

the acid (0.3 mmol, 1 equiv) and thionyl chloride (7 mL) at reﬂux for

2 h, followed by the removal of excess thionyl chloride under vacuum.

A solution of the acid chloride (0.342 g, 0.3 mmol) in anhydrous

DCM (5 mL) was added dropwise to an ice-cold solution of the

amine 9 (0.4 g, 0.3 mmol) and TEA (10 equiv) in anhydrous DCM

(20 mL). The mixture was heated at reﬂux for 5 h. After cooling to

room temperature, the mixture was washed with saturated NaHCO₃

solution (1 × 20 mL), water (2 × 20 mL), and brine (1 × 20 mL) and

dried with anhydrous sodium sulfate. The residue obtained after the

evaporation of DCM was subjected to silica gel column chromatog-

raphy using CHCl₃−EtOAc (8:2) as eluent to yield the desired amide

10 as a creamy white solid (0.43 g, 58%): mp 165−167 °C. ¹H NMR

(300 MHz, CDCl₃) δ 8.37 (d, J = 5.6 Hz, 1 H), 8.20−8.02 (m, 2 H),

7.36 (d, J = 3.8 Hz, 1 H), 7.32 (s, 1 H), 6.66 (s, 1 H), 5.75 (s, 2 H),

3.95 (s, 3 H), 3.93 (s, 3 H), 3.63−2.57 (m, 2 H), 1.04−0.78 (m, 2 H),

−0.05 (s, 9 H). HRESIMS m/z calcd for C₂₂H₂₈IN₃O₄Si (MH)⁺

554.0967, found 554.0968.

General Procedures for the Synthesis of Compounds 11a−

c. A suspension of sodium hydride (0.72 mmol) in anhydrous DMF

(5 mL) was cooled in an ice bath. A solution of 10 (0.2 g, 0.36 mmol)

in DMF (2 mL) was added dropwise to the sodium hydride

suspension over 5 min. The mixture was stirred at 21 °C for 1 h, and

then the appropriate alkyl bromide (0.72 mmol) was added all at

once. The reaction mixture was stirred at room temperature for 18 h

and then quenched with ice-cold water (25 mL). The mixture was

extracted with EtOAc (3 × 15 mL), and the combined organic layers

were washed with water (2 × 15 mL) and brine (1 × 20 mL) and

dried. Puriﬁcation of the residue obtained after evaporating the

EtOAc layer by silica gel column chromatography (CHCl₃−EtOAc,

8:2) yielded 11a−c.

(300 MHz, CDCl₃) δ 9.31 (s, 1 H), 8.53 (d, J = 8.1 Hz, 1 H), 8.39 (d,

J = 8.2 Hz, 1 H), 7.80 (t, J = 7.7 Hz, 1 H), 7.57 (t, J = 7.6 Hz, 1 H),

7.42 (d, J = 3.8 Hz, 1 H), 7.02 (d, J = 3.8 Hz, 1 H), 5.73 (s, 2 H),

4.82−4.73 (m, 2 H), 3.36 (s, 3 H), 2.73 (t, J = 7.2 Hz, 2 H), 2.42 (s, 6

H), 2.29−2.21 (m, 4 H). HRESIMS m/z calcd for C₂₁H₂₄N₄O₂

(MH)⁺365.1972, found 365.1970.

4-(3-(Dimethylamino)propyl)-7,8-dimethoxy-1-(methoxymeth-

yl)-1,4-dihydro-5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one

(6b). This compound was isolated as a light-brown oil (0.06 g, 28%).

¹H NMR (300 MHz, CDCl₃) δ 9.19 (s, 1 H), 7.89 (s, 1 H), 7.71 (s, 1

H), 7.41 (d, J = 3.8 Hz, 1 H), 6.99 (d, J = 3.8 Hz, 1 H), 5.73 (s, 2 H),

4.78 (s, 2 H), 4.10 (s, 3 H), 4.04 (s, 3 H), 3.38−3.31 (m, 2 H), 2.81−

2.75 (m, 2 H), 2.45 (s, 6 H), 2.30−2.24 (m, 2 H). HRESIMS m/z

calcd for C₂₃H₂₈BrN₄O₄(MH)⁺425.2183, found 425.2187.

4-(3-(Dimethylamino)propyl)-1-(methoxymethyl)-7-nitro-1,4-di-

hydro-5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one (6c). This

compound was isolated as a yellow solid (0.03 g, 14%): mp 125−127

°C. ¹H NMR (300 MHz, CDCl₃) δ 9.17 (d, J = 2.5 Hz, 1 H), 8.24 (d,

J = 5.6 Hz, 1 H), 8.19 (dd, J = 8.8, 2.5 Hz, 1 H), 7.76 (s, 1 H), 7.71

(d, J = 8.8 Hz, 1 H), 6.80 (d, J = 5.8 Hz, 1 H), 5.66 (s, 2 H), 4.21−

4.15 (m, 2 H), 3.37 (s, 3 H), 2.61 (s, 2 H), 2.41 (s, 6 H), 2.11−2.05

(m, 2 H). HRESIMS m/z calcd for C₂₁H₂₃BrN₅O₄(MH)⁺410.1823,

found 410.1820.

4-(3-(Dimethylamino)propyl)-1,4-dihydro-5H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-5-one (7a). This compound was

prepared from 6a following the same procedure as for 1 and isolated

as a dark-brown solid (0.026 g, 58%): mp 185−187 °C. IR (thin ﬁlm)

2950, 1648, 1578, 1418, 1338, 1314, 1036 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 12.16 (s, 1 H), 9.38 (s, 1 H), 8.68 (d, J = 8.1 Hz, 1 H),

8.36 (d, J = 8.0 Hz, 1 H), 7.85 (t, J = 7.6 Hz, 1 H), 7.66−7.47 (m, 2

H), 6.99−6.92 (m, 1 H), 4.74−4.59 (m, 2 H), 2.66 (s, 2 H), 2.33 (s, 6

H), 2.08−1.99 (m, 2 H). MALDIMS m/z 321 (MH)⁺. HRESIMS

calcd for C₁₉H₂₁N₄O (MH)⁺321.1716, found 321.1709. C18 HPLC

purity: 99.65% (MeOH−H₂O, 85:15).

tert-Butyl N-(2-Iodo-4,5-dimethoxybenzoyl)-N-(1-((2-

(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-

glycinate (11a). Compound 11a was obtained as a creamy white

solid (0.22 g, 91%): mp 113−115 °C. ¹H NMR (300 MHz, CDCl₃) δ

8.17 (s, 1 H), 7.42 (d, J = 3.6 Hz, 1 H), 7.11 (s, 1 H), 7.05−6.99 (m,

1 H), 6.78 (s, 1 H), 6.67 (s, 1 H), 5.76 (s, 2 H), 4.54 (s, 1 H), 3.80 (s,

2 H), 3.61−3.52 (m, 2 H), 3.47 (s, 2 H), 1.50 (s, 9 H), 0.96−0.87 (m,

4-(3-(Dimethylamino)propyl)-7-nitro-1,4-dihydro-5H-

benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one (7b). This com-

pound was prepared from 6c following the same procedures as for 1

and isolated as a yellow powder (0.032 g, 71%): mp 205−207 °C. IR

(thin ﬁlm) 2864, 1667, 1611, 1572, 1519, 1491, 1433, 1403, 1328,

1135, 1077 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 12.35 (s, 1 H),

9.44 (s, 1 H), 9.04 (d, J = 2.6 Hz, 1 H), 8.93 (d, J = 9.2 Hz, 1 H), 8.54

O

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Article

2 H), −0.06 (s, 9 H). HRESIMS m/z calcd for C₂₈H₃₈IN₃O₆Si

(MH)⁺668.1647, found 668.1644.

mL). The combined organic layer was washed with water and brine

(15 mL each), dried, and evaporated to yield 13a as a white solid (20

mg, 26%): mp 212−214 °C. IR (thin ﬁlm) 2926, 1744, 1647, 1612,

1583, 1462, 1427, 1389, 1244, 1327, 1244, 1225, 1210, 1156, 1107,

1029 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 12.19−12.08 (s, 1 H),

9.42 (s, 1 H), 8.32 (s, 1 H), 8.12−8.04 (m, 1 H), 7.72 (s, 1 H), 7.58−

7.46 (m, 1 H), 6.67−6.58 (m, 1 H), 5.38 (s, 2 H), 4.06 (s, 3 H), 3.91

(s, 3 H), 1.44 (s, 9 H). MALDIMS m/z 410 (MH)⁺. HRESIMS calcd

for C₂₂H₂₃N₃O₅(MH)⁺410.1711, found 410.1709. C18 HPLC

purity: 96.64% (MeOH−H₂O, 85:15).

Methyl N-(2-Iodo-4,5-dimethoxybenzoyl)-N-(1-((2-

(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-

glycinate (11b). This compound was isolated as a yellow oil (0.2 g,

88%). ¹H NMR (300 MHz, CDCl₃) δ 8.17 (d, J = 5.4 Hz, 1 H), 7.41

(d, J = 3.6 Hz, 1 H), 7.09 (s, 1 H), 7.04 (d, J = 5.4 Hz, 1 H), 6.74 (d, J

= 3.6 Hz, 1 H), 6.62 (s, 1 H), 5.70 (s, 2 H), 4.67 (s, 2 H), 3.80 (s, 3

H), 3.78 (s, 3 H), 3.58−3.47 (m, 2 H), 3.44 (s, 3 H), 0.97−0.82 (m, 2

H), −0.08 (s, 9 H). HRESIMS m/z calcd for C₂₅H₃₂IN₃O₄Si (MH)⁺

626.1177, found 626.1176.

Methyl 2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)acetate (13b). This com-

pound was prepared form 12b using the procedure described for 13a

and was isolated as a white solid (25 mg, 45%): mp 212−214 °C. IR

(thin ﬁlm) 2964, 1752, 1658, 1612, 1575, 1513, 1458, 1427, 1387,

1370, 1351, 1302, 1267, 1242, 1177, 1167, 1143, 1105, 1077, 1003

Ethyl 2-(2-Iodo-4,5-dimethoxy-N-(1-((2-(trimethylsilyl)ethoxy)-

methyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzamido)butanoate

1

(11c). This compound was isolated as a yellow oil (0.2 g, 83%). H

NMR (300 MHz, CDCl₃) δ 8.16 (d, J = 5.0 Hz, 1 H), 7.38 (d, J = 3.7

Hz, 1 H), 7.10−7.02 (m, 2 H), 6.92 (d, J = 3.7 Hz, 1 H), 6.53 (s, 1

H), 5.66 (s, 2 H), 4.67−4.56 (m, 1 H), 4.32 (d, J = 7.3 Hz, 3 H), 3.75

(s, 3 H), 3.51−3.45 (m, 2 H), 3.36 (s, 3 H), 1.62−1.53 (m, 5 H), 1.36

(t, J = 7.1 Hz, 3 H), 1.01 (t, J = 7.3 Hz, 3 H), 0.93−0.84 (m, 3 H),

−0.09 (s, 9 H). HRESIMS m/z calcd for C₂₈H₃₈IN₃O₆Si (MH)⁺

668.1647, found 668.1651.

1

cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ 12.16 (s, 1 H), 9.41 (s, 1

H), 8.06 (s, 1 H), 7.70 (s, 1 H), 7.51 (s, 1 H), 6.59 (s, 1 H), 5.48 (s, 2

H), 4.05 (s, 3 H), 3.90 (d, J = 3.8 Hz, 3 H), 3.71 (d, J = 5.8 Hz, 3 H).

MALDIMS m/z 368 (MH)⁺. HRESIMS calcd for C₁₉H₁₇N₃O₅

(MH)⁺368.1241, found 368.1243. C18 HPLC purity: 98%

(MeOH−H₂O, 85:15).

tert-Butyl 2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-4-yl)acetate (12a). Pd(OAc)₂(9 mg, 0.037 mmol),

K₂CO₃(0.15 g, 1.12 mmol), Ag₂CO₃(0.2 g, 0.74 mmol), and PCy₃·

HBF₄(28 mg, 0.075 mmol) were added to a solution of benzamide

11a (0.25 g, 0.37 mmol) in DMA (5 mL). The reaction mixture was

heated at 130 °C under Ar atmosphere for 18 h. The reaction mixture

was cooled to room temperature, and EtOAc (10 mL) was then

added. The mixture was ﬁltered through a bed of Celite, and the

residue was washed thoroughly with EtOAc (20 mL). The ﬁltrate was

transferred to a separatory funnel and washed with water (5 × 15 mL)

and brine (15 mL) to remove DMA. The organic layer was dried and

evaporated, and the residue was puriﬁed by silica gel column

chromatography using CHCl₃−MeOH (9:1) as the eluent to give the

tetracyclic compound 12a (0.15 g, 74%) as a yellow solid: mp 156−

158 °C. ¹H NMR (300 MHz, CDCl₃) δ 9.18 (s, 1 H), 7.93 (s, 1 H),

7.71 (s, 1 H), 7.40 (d, J = 3.9 Hz, 1 H), 6.81 (d, J = 3.5 Hz, 1 H), 5.79

(s, 2 H), 5.45 (s, 2 H), 4.12 (s, 3 H), 4.04 (s, 3 H), 3.65−3.47 (m, 3

H), 1.53−1.41 (m, 9 H), 1.03−0.78 (m, 2 H), −0.05 (m, 9 H).

HRESIMS m/z calcd for C₂₈H₂₇N₃O₆Si (MH)⁺540.2524, found

540.2522.

Ethyl 2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)butanoate (13c). This

compound was prepared form compound 12c using the procedure

described for 13a and was isolated as a white solid (37 mg, 60%): mp

235−237 °C. IR (thin ﬁlm) 2979, 1725, 1640, 1610, 1572, 1514,

1460, 1420, 1386, 1368, 1339, 1318, 1269, 1244, 1211, 1141, 1110,

1090, 1033, 1014 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 12.18 (s, 1

H), 9.41 (s, 1 H), 8.04 (s, 1 H), 7.64 (s, 1 H), 7.53 (s, 1 H), 6.95 (s, 1

H), 5.73 (d, J = 7.6 Hz, 1 H), 4.06 (d, J = 4.7 Hz, 1 H), 4.04 (s, 3 H),

3.88 (s, 3 H), 2.37−2.31 (m, 2 H), 1.05 (t, J = 7.1 Hz, 3 H), 0.79 (t, J

= 7.5 Hz, 3 H). MALDIMS m/z 410 (MH)⁺. HRESIMS calcd for

C₂₂H₂₃N₃O₅(MH)⁺410.1711, found 410.1711. C18 HPLC purity:

100% (MeOH−H₂O, 85:15).

Preparation of the Amides 14a−l. 2-Bromo-3,4-dimethoxy-

benzoic acid (0.3 g, 1.14 mmol) was added to thionyl chloride (4 mL)

and a catalytic amount of DMF. The reaction mixture was heated at

reﬂux for 3 h, and then the thionyl chloride was evaporated to aﬀord

the corresponding acid chloride. The appropriate amine (1.2 mmol)

was dissolved in DCM (5 mL) in an ice bath and mixed with TEA (5

mmol) in a vial, and the acid chloride was dissolved in DCM (2 mL)

and added dropwise to the amine solution. The reaction mixture was

stirred overnight at room temperature, and then the reaction was

quenched with a saturated solution of sodium carbonate. The organic

layer was then separated, washed with water, dried, and evaporated to

give a residue. The residue was puriﬁed with silica gel column

chromatography using either CHCl₃−MeOH or EtOAc−hexanes

mixtures to give the amides 14a−l as white solids.

2-Bromo-N-cyclohexyl-4,5-dimethoxybenzamide (14a). This

compound was isolated as a white solid (0.35 g, 89%): mp 185−

187 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.21 (s, 1 H), 6.98 (s, 1 H),

6.17−6.10 (m, 1 H), 3.99 (s, 1 H), 3.89 (s, 6 H), 2.09−1.99 (m, 2 H),

1.80−1.70 (m, 2 H), 1.50−1.17 (m, 6 H). HRESIMS m/z calcd for

C₁₅H₂₀BrNO₃(MH)⁺342.0699/344.0681, found 342.0701/

344.0683.

2-Bromo-N-(cyclohexylmethyl)-4,5-dimethoxybenzamide (14b).

This compound was isolated as a white solid (0.38 g, 93%): mp 163−

165 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.25 (s, 1 H), 6.99 (s, 1 H),

6.33 (s, 1 H), 3.90 (s, 3 H), 3.31 (s, 3 H), 3.31 (t, J = 6.3 Hz, 2 H),

1.89−1.65 (m, 6 H), 1.33−1.12 (m, 4 H), 1.13−0.94 (m, 2 H).

HRESIMS m/z calcd for C₁₆H₂₂BrNO₃(MH)⁺356.0856/358.0837,

found 356.0855/358.0833.

Methyl 2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-4-yl)acetate (12b). Using the same procedure used

for 12a, compound 12b was prepared from 11b and was isolated as a

1

yellow solid (0.1 g, 62%): mp 136−138 °C. H NMR (300 MHz,

CDCl₃) δ 9.18 (s, 1 H), 7.91 (s, 1 H), 7.70 (s, 1 H), 7.40 (d, J = 3.7

Hz, 1 H), 6.76 (d, J = 3.8 Hz, 1 H), 5.81 (s, 2 H), 5.56 (s, 2 H), 4.12

(s, 3 H), 4.04 (s, 3 H), 3.80 (s, 3 H), 3.69−3.54 (m, 2 H), 1.00−0.84

(m, 2 H), −0.04 (s, 9 H). HRESIMS m/z calcd for C₂₅H₃₁N₃O₆Si

(MH)⁺498.2055, found 498.2047.

Ethyl 2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-4-yl)butanoate (12c). This compound was prepared

from 11c by the same procedure used for 12a and isolated as a yellow

solid (0.09 g, 44%): mp 152−154 °C. ¹H NMR (300 MHz, CDCl₃) δ

9.19 (s, 1 H), 7.85 (s, 1 H), 7.70 (s, 1 H), 7.42 (d, J = 3.6 Hz, 1 H),

6.83 (s, 1 H), 5.78 (s, 2 H), 4.11 (s, 3 H), 4.01 (s, 3 H), 3.67−3.54

(m, 3 H), 3.48 (q, J = 7.0 Hz, 3 H), 2.54−2.47 (m, 2 H), 0.94−0.87

(m, 2 H), −0.04 (s, 9 H). HRESIMS m/z calcd for C₂₈H₃₇IN₃O₆Si

(MH)⁺540.2524, found 540.2523.

tert-Butyl 2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo-

[c]pyrrolo[2,3-h][1,6]naphthyridin-4-yl)acetate (13a). Com-

pound 12a (0.1 g, 0.18 mmol) was dissolved in DCM (5 mL), and

TFA (5 mL) was added to the solution dropwise. The mixture was

stirred at room temperature for 2 h, and then the solvent was

evaporated. The residue obtained was dissolved in MeOH (5 mL)

and NaOH solution (2N, 2 mL). Ethylenediamine (1 mL) was added,

and the mixture was stirred overnight at room temperature. The

mixture was acidiﬁed with TFA and extracted with CHCl₃(3 × 10

2-Bromo-N-cycloheptyl-4,5-dimethoxybenzamide (14c). This

compound was isolated as a white solid (0.36 g, 88%): mp 157−

158 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.20 (s, 1 H), 6.98 (s, 1 H),

6.20 (d, J = 7.8 Hz, 1 H), 4.23−4.12 (m, 1 H), 3.89 (s, 6 H), 2.12−

2.01 (m, 2 H), 1.76−1.50 (m, 13 H). HRESIMS m/z calcd for

C₁₆H₂₂BrNO₃(MH)⁺356.0856/358.0837, found 356.0855/

358.0837.

P

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2-Bromo-N-cyclooctyl-4,5-dimethoxybenzamide (14d). This

compound was isolated as a white solid (0.32 g, 75%): mp 145−

147 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.21 (s, 1 H), 6.99 (s, 1 H),

6.23 (d, J = 8.0 Hz, 1 H), 4.27−4.14 (m, 1 H), 3.89 (s, 6 H), 2.03−

1.90 (m, 2 H), 1.75−1.50 (m, 15 H). HRESIMS m/z calcd for

C₁₇H₂₄BrNO₃(MH)⁺370.1012/372.0994, found 370.1012/

372.0997.

2-Bromo-4,5-dimethoxy-N-(1-methylpiperidin-4-yl)benzamide

(14e). This compound was isolated as a white solid (0.35 g, 85%): mp

201−204 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.19 (s, 1 H), 6.99 (s, 1

H), 6.17 (s, 1 H), 4.06−3.96 (m, 1 H), 3.89 (s, 6 H), 2.90−2.79 (m, 2

H), 2.33 (s, 3 H), 2.27−2.18 (m, 2 H), 2.14−2.04 (m, 2 H), 1.74−

1.60 (m, 2 H). HRESIMS m/z calcd for C₁₅H₂₁BrN₂O₃(MH)⁺

357.0808/359.0789, found 357.0809/359.0791.

equiv). The atmosphere of the vial was evacuated and reﬁlled with

argon once and then the solvent (4 mL) was added via syringe and

the vial was evacuated and reﬁlled with argon twice. The reaction

mixture was heated at the stated temperatures and time in Table 2

and was then cooled to room temperature. The reaction mixture was

ﬁltered through a pad of Celite and the solvent was evaporated and

the residue obtained was loaded on a silica gel column and the

mixture was puriﬁed using EtOAc-hexane mixture to give compounds

16a−l.

4-Cyclohexyl-7,8-dimethoxy-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,4-dihydro-5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

5-one (16a). Compound 16a was isolated as a yellowish powder (0.1

g, 78%): mp 198−200 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.18 (s, 1

H), 7.93 (s, 1 H), 7.71−7.67 (m, 1 H), 7.45 (d, J = 3.7 Hz, 1 H), 6.83

(d, J = 3.8 Hz, 1 H), 5.82 (s, 2 H), 5.02−4.95 (m, 1 H), 4.16 (s, 3 H),

4.09 (s, 3 H), 3.72−3.59 (m, 2 H), 3.08−2.91 (m, 2 H), 2.12−2.01

(m, 4 H), 1.86 (s, 1 H), 1.57−1.49 (m, 3 H), 1.05−0.95 (m, 2 H),

0.02 (s, 9 H). HRESIMS m/z calcd for C₂₈H₃₇N₃O₄Si (MH)⁺

508.2626, found 508.2629.

4-(Cyclohexylmethyl)-7,8-dimethoxy-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1,4-dihydro-5H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-5-one (16b). This compound was isolated as a yellow

solid (0.11 g, 81%): mp 122−123 °C. ¹H NMR (300 MHz, CDCl₃) δ

9.18 (s, 1 H), 7.93 (s, 1 H), 7.70 (s, 1 H), 7.42 (d, J = 3.7 Hz, 1 H),

6.85 (d, J = 3.8 Hz, 1 H), 5.80 (s, 2 H), 4.11 (s, 3 H), 4.05 (s, 3 H),

3.68−3.52 (m, 2 H), 1.73−1.61 (m, 9 H), 1.27−1.18 (m, 3 H), 1.01−

0.85 (m, 2 H), −0.04 (s, 9 H). HRESIMS m/z calcd for

C₂₉H₃₉N₃O₄Si (MH)⁺522.2782, found 522.2780.

4-Cycloheptyl-7,8-dimethoxy-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,4-dihydro-5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

5-one (16c). This compound was isolated as a yellowish-white solid

(70 mg, 50%): mp 184−186 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.20

(s, 1 H), 7.94 (s, 1 H), 7.73 (s, 1 H), 7.46 (d, J = 3.7 Hz, 1 H), 6.94

(d, J = 3.7 Hz, 1 H), 5.81 (d, J = 8.7 Hz, 2 H), 5.24 (d, J = 10.4 Hz, 1

H), 4.16 (s, 3 H), 4.09 (s, 3 H), 3.71−3.55 (m, 3 H), 2.99−2.85 (m, 2

H), 2.18−2.08 (m, 2 H), 2.01 (s, 2 H), 1.80 (d, J = 4.3 Hz, 4 H),

1.71−1.63 (m, 7 H), 1.06−0.96 (m, 3 H), 0.02 (s, 9 H). HRESIMS

m/z calcd for C₂₉H₃₉N₃O₄Si (MH)⁺522.2782, found 522.2782.

4-Cyclooctyl-7,8-dimethoxy-1-((2-(trimethylsilyl)ethoxy)methyl)-

1,4-dihydro-5H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one

(16d). This compound was isolated as a creamy white powder (84

mg, 58%): mp 166−168 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.20 (s,

1 H), 7.93 (s, 1 H), 7.72 (d, J = 10.5 Hz, 1 H), 7.47 (d, J = 3.7 Hz, 1

H), 6.96 (d, J = 3.8 Hz, 1 H), 5.81 (d, J = 9.8 Hz, 2 H), 5.28−5.22

(m, 1 H), 4.16 (s, 3 H), 4.09 (s, 3 H), 3.71−3.59 (m, 2 H), 3.00−2.89

(m, 2 H), 2.13−2.07 (m, 2 H), 2.05−1.96 (m, 3 H), 1.89−1.78 (m, 2

H), 1.74−1.63 (m, 7 H), 1.05−0.98 (m, 2 H), 0.02 (s, 9 H).

HRESIMS m/z calcd for C₃₀H₄₁N₃O₄Si (MH)⁺536.2939, found

536.2939.

tert-Butyl 4-(2-Bromo-4,5-dimethoxybenzamido)piperidine-1-

carboxylate (14f). This compound was isolated as a white solid

1

(0.4 g, 79%): mp 210−215 °C. H NMR (300 MHz, CDCl₃) δ 7.19

(s, 1 H), 6.97 (s, 1 H), 6.20 (d, J = 7.9 Hz, 1 H), 4.18−3.98 (m, 3 H),

3.89 (s, 6 H), 3.03−2.91 (m, 2 H), 2.06−1.97 (m, 2 H), 1.46 (s, 9 H).

HRESIMS m/z calcd for C₁₉H₂₇BrN₂O₃(MNa)⁺465.0995/

467.0977, found 465.0995/467.0977.

tert-Butyl (R)-3-(2-Bromo-4,5-dimethoxybenzamido)pyrrolidine-

1-carboxylate (14g). This compound was isolated as a white solid

(0.38 g, 77%): mp 241−243 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.23

(s, 1 H), 6.99 (s, 1 H), 6.39 (s, 1 H), 4.68−4.61 (m, 1 H), 3.90 (s, 6

H), 3.68 (dd, J = 11.5, 5.9 Hz, 1 H), 3.53−3.45 (m, 2 H), 3.41−3.37

(m, 1 H), 2.28−2.17 (m, 1 H), 2.08−1.95 (m, 1 H), 1.47 (s, 9 H).

HRESIMS m/z calcd for C₁₈H₂₅BrN₂O₅(MNa)⁺451.0839/

453.0821, found 451.0836/453.0819.

tert-Butyl (R)-3-(2-Bromo-4,5-dimethoxybenzamido)piperidine-

1-carboxylate (14h). This compound was isolated as a white solid

(0.41 g, 80%): mp 235−236 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.27

(s, 1 H), 6.99 (s, 1 H), 6.51 (d, J = 7.3 Hz, 1 H), 4.24−4.15 (s, 1 H),

3.90 (s, 6 H), 3.63−3.51 (m, 3 H), 3.35−3.20 (m, 1 H), 1.93−1.71

(m, 3 H), 1.44 (s, 9 H). HRESIMS m/z calcd for C₁₉H₂₇BrN₂O₅

(MNa)⁺465.0995/467.0977, found 465.0991/467.0979.

Methyl (2-Bromo-4,5-dimethoxybenzoyl)-^D-valinate (14i). This

compound was isolated as a white solid (0.38 g, 88%): mp 178−180

°C. ¹H NMR (300 MHz, CDCl₃) δ 7.02 (s, 1 H), 6.86 (d, J = 8.4 Hz,

1 H), 4.78 (dd, J = 8.6, 4.7 Hz, 1 H), 3.91 (s, 3 H), 3.89 (s, 3 H), 3.78

(s, 3 H), 2.37−2.28 (m, 1 H), 1.06 (d, J = 6.9 Hz, 3 H), 1.02 (d, J =

6.9 Hz, 3 H). HRESIMS m/z calcd for C₁₅H₂₀BrNO₅(MNa)⁺

396.0417/398.0398, found 396.0412/398.0392.

Methyl (2-Bromo-4,5-dimethoxybenzoyl)-^L-valinate (14j). This

compound was isolated as a white solid (0.37 g, 87%): mp 181−183

°C. ¹H NMR (300 MHz, CDCl₃) δ 7.02 (s, 1 H), 6.86 (d, J = 8.6 Hz,

1 H), 4.78 (dd, J = 8.6, 4.7 Hz, 1 H), 3.91 (s, 3 H), 3.89 (s, 3 H), 3.78

(s, 3 H), 2.38−2.24 (m, 1 H), 1.06 (d, J = 6.9 Hz, 3 H), 1.02 (d, J =

6.9 Hz, 3 H). HRESIMS m/z calcd for C₁₅H₂₀BrNO₅(MNa)⁺

396.0417/398.0398, found 396.0421/398.0403.

Methyl (R)-2-(2-Bromo-4,5-dimethoxybenzamido)butanoate

(14k). This compound was isolated as a white solid (0.34 g, 82%):

mp 165−168 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.26 (s, 1 H), 7.02

(s, 1 H), 6.91 (d, J = 7.8 Hz, 1 H), 4.79 (dd, J = 13.0, 6.5 Hz, 1 H),

3.91 (s, 3 H), 3.89 (s, 3 H), 3.80 (s, 3 H), 2.13−1.97 (m, 1 H), 1.92−

1.85 (m, 1 H), 1.02 (t, J = 7.5 Hz, 3 H). HRESIMS m/z calcd for

C₁₄H₁₈BrNO₅(MNa)⁺382.0260/384.0241, found 382.0258/

384.0237.

Methyl (S)-2-(2-Bromo-4,5-dimethoxybenzamido)butanoate

(14l). This compound was isolated as a white solid (0.37 g, 89%):

mp 185−187 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.25 (s, 1 H), 7.01

(s, 1 H), 6.92 (d, J = 7.1 Hz, 1 H), 4.82−4.75 (m, 1 H), 3.91 (s, 3 H),

3.89 (s, 3 H), 3.79 (s, 3 H), 2.12−1.97 (m, 1 H), 1.91−1.85 (m, 1 H),

1.01 (t, J = 7.5 Hz, 3 H). HRESIMS m/z calcd for C₁₄H₁₈BrNO₅

(MNa)⁺382.0260/384.0241, found 382.0263/384.0246.

7,8-Dimethoxy-4-(1-methylpiperidin-4-yl)-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1,4-dihydro-5H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-5-one (16e). This compound was isolated as a yellow

oil (85 mg, 60%). ¹H NMR (500 MHz, CDCl₃) δ 9.18 (s, 1 H), 7.92

(s, 1 H), 7.72 (s, 1 H), 7.47 (d, J = 3.5 Hz, 1 H), 6.80 (s, 1 H), 5.82

(s, 2 H), 5.02 (s, 1 H), 4.16 (s, 3 H), 4.10 (s, 3 H), 3.69−3.61 (m, 2

H), 3.46−3.39 (m, 2 H), 3.24 (s, 2 H), 2.53 (s, 2 H), 2.02−1.96 (m, 2

H), 1.06−0.94 (m, 2 H), 0.02 (s, 9 H). HRESIMS m/z calcd for

C₂₈H₃₈N₄O₄Si (MH)⁺523.2735, found 523.2737.

tert-Butyl 4-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

4-yl)piperidine-1-carboxylate (16f). This compound was isolated as a

yellowish white solid (95 mg, 58%): mp 189−192 °C. ¹H NMR (500

MHz, CDCl₃) δ 9.19 (s, 1 H), 7.90 (s, 1 H), 7.72 (s, 1 H), 7.48 (d, J

= 3.7 Hz, 1 H), 6.78 (d, J = 3.7 Hz, 1 H), 5.84 (s, 2 H), 5.15−5.11

(m, 1 H), 4.51−4.42 (m, 2 H), 4.16 (s, 3 H), 4.10 (s, 3 H), 3.71−3.62

(m, 2 H), 3.26−3.16 (m, 2 H), 2.98 (s, 2 H), 1.95 (s, 2 H), 1.57 (s, 9

H), 1.05−0.95 (m, 2 H), 0.02 (s, 9 H). HRESIMS m/z calcd for

C₃₂H₄₄N₄O₆Si (MH)⁺609.3102, found 609.3102.

General Procedure for the Synthesis of Compounds 16a−l.

A screw-capped vial with a Teﬂon cap was charged with SEM-

protected 4-iodo-7-azaindole (15) (0.26 mmol, 1 equiv), amides

14a−l (0.26 mmol, 1.05 equiv), Pd(OTFA)₂(10 mol %), TFP (20

mol %), norborene (0.26 mmol, 1.05 equiv), and cesium carbonate (3

tert-Butyl (R)-3-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-4-yl)pyrrolidine-1-carboxylate (16g). This compound

Q

DOI: 10.1021/acs.jmedchem.8b00510

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

was isolated as a creamy white solid (95 mg, 60%): mp 156−158 °C.

¹H NMR (500 MHz, CDCl₃) δ 9.20 (s, 1 H), 7.91 (s, 1 H), 7.73 (s, 1

H), 7.50 (s, 1 H), 6.88 (s, 1 H), 5.84 (s, 2 H), 4.17 (s, 3 H), 4.10 (s, 3

H), 4.04−4.01 (m, 1 H), 3.93−3.89 (m, 1 H), 3.73−3.69 (m, 1 H),

3.67−3.62 (m, 2 H), 3.09−3.02 (m, 1 H), 2.46−2.42 (m, 1 H), 1.56

(s, 9 H), 1.05−0.96 (m, 2 H), 0.02 (s, 9 H). HRESIMS m/z calcd for

C₃₁H₄₂N₄O₆Si (MH)⁺595.2946, found 595.2949.

4-Cyclohexyl-7,8-dimethoxy-1,4-dihydro-5H-benzo[c]pyrrolo-

[2,3-h][1,6]naphthyridin-5-one (17a). This compound was isolated

as a white solid (28 mg, 74%): mp 233−235 °C. IR (thin ﬁlm) 3120,

3002, 2922, 2847, 1654, 1608, 1576, 1509, 1462, 1450, 1417, 1400,

1378, 1357, 1300, 1224, 1207, 1180, 1124, 1040 cm⁻¹. ¹H NMR (500

MHz, DMSO-d₆) δ 12.08 (s, 1 H), 9.30 (s, 1 H), 7.95 (s, 1 H), 7.64

(s, 1 H), 7.54−7.45 (m, 1 H), 6.58 (s, 1 H), 4.85 (s, 1 H), 3.99 (s, 3

H), 3.85 (s, 3 H), 2.85−2.71 (m, 2 H), 1.89−1.81 (m, 4 H), 1.75−

1.67 (m, 1 H), 1.48−1.38 (m, 2 H), 1.27−1.19 (m, 2 H). MALDIMS

m/z 378 (MH)⁺. HRESIMS calcd for C₂₂H₂₃N₃O₃(MH)⁺378.1812,

found 378.1814. C18 HPLC purity: 100% (MeOH−H₂O, 85:15).

4-(Cyclohexylmethyl)-7,8-dimethoxy-1,4-dihydro-5H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-5-one (17b). This compound was

isolated as a white solid (32 mg, 82%): mp 198−199 °C. IR (thin

ﬁlm) 3314, 2922, 2849, 1654, 1634, 1573, 1511, 1463, 1450, 1422,

1385, 1358, 1325, 1259, 1206, 1178, 1124, 1051, 1009 cm⁻¹. ¹H

NMR (300 MHz, DMSO-d₆) δ 12.09 (s, 1 H), 9.38 (s, 1 H), 8.31 (s,

1 H), 8.02 (s, 1 H), 7.72 (s, 1 H), 7.54 (s, 1 H), 6.76 (s, 1 H), 4.61 (s,

2 H), 4.03 (s, 3 H), 3.89 (s, 3 H), 1.84−1.78 (m, 1 H), 1.68−1.59 (m,

5 H), 1.25−0.94 (m, 6 H). MALDIMS m/z 392 (MH)⁺. HRESIMS

calcd for C₂₃H₂₅N₃O₃(MH)⁺392.1969, found 392.1968. C18 HPLC

purity: 97.15% (MeOH−H₂O, 85:15).

4-Cycloheptyl-7,8-dimethoxy-1,4-dihydro-5H-benzo[c]pyrrolo-

[2,3-h][1,6]naphthyridin-5-one (17c). This compound was isolated

as a white solid (35 mg, 89%): mp 242−244 °C. IR (thin ﬁlm) 3120,

2926, 2849, 1651, 1610, 1576, 1508, 1462, 1449, 1397, 1356, 1310,

1258, 1240, 1208, 1175, 1120, 1069 cm⁻¹. ¹H NMR (500 MHz,

DMSO-d₆) δ 12.15 (s, 1 H), 9.38 (s, 1 H), 8.02 (s, 1 H), 7.71 (s, 1

H), 7.57 (dd, J = 10.7, 7.4 Hz, 1 H), 6.75 (s, 1 H), 5.17 (s, 1 H), 4.05

(s, 3 H), 3.92 (s, 3 H), 2.85−2.63 (m, 2 H), 2.04−1.92 (m, 4 H),

1.71−1.54 (m, 6 H), 1.25 (s, 3 H). ¹³C NMR (126 MHz, DMSO) δ

161.75, 153.94, 149.54, 149.27, 141.05, 138.16, 128.85, 124.90,

118.46, 108.07, 107.86, 106.16, 103.20, 100.94, 61.68, 56.51, 55.79,

32.49, 28.21, 26.20. MALDIMS m/z 392 (MH)⁺. HRESIMS calcd for

C₂₃H₂₅N₃O₃(MH)⁺392.1969, found 392.1969. C18 HPLC purity:

96.89% (MeOH−H₂O, 85:15).

tert-Butyl (R)-3-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]-

naphthyridin-4-yl)piperidine-1-carboxylate (16h). This compound

1

was isolated as a yellowish solid (82 mg, 52%): mp 148−152 °C. H

NMR (300 MHz, CDCl₃) δ 9.13 (s, 1 H), 7.82 (s, 1 H), 7.64 (s, 1

H), 7.40 (d, J = 3.7 Hz, 1 H), 7.00−6.92 (m, 1 H), 5.81 (s, 2 H),

4.98−4.92 (m, 1 H), 4.53−4.41 (m, 1 H), 4.28−4.22 (m, 1 H), 4.11

(s, 3 H), 4.03 (s, 3 H), 3.66−3.56 (m, 2 H), 3.08−3.03 (m, 1 H),

2.92−2.79 (m, 2 H), 2.07−2.02 (m, 1 H), 1.89 (s, 2 H), 1.65 (s, 9 H),

1.26 (t, J = 7.2 Hz, 2 H), 0.99−0.89 (m, 1 H), −0.04 (s, 9 H).

HRESIMS m/z calcd for C₃₂H₄₄N₄O₆Si (MH)⁺609.3102, found

609.3104.

Methyl (R)-2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

4-yl)-3-methylbutanoate (16i). This compound was isolated as a

1

yellow powder (0.1 g, 76%): mp 212−214 °C. H NMR (500 MHz,

CDCl₃) δ 9.27 (s, 1 H), 7.91 (s, 1 H), 7.78 (s, 1 H), 7.49 (d, J = 3.8

Hz, 1 H), 6.95 (d, J = 3.8 Hz, 1 H), 5.83 (t, J = 7.1 Hz, 2 H), 5.51 (d,

J = 9.3 Hz, 1 H), 4.18 (s, 3 H), 4.09 (s, 3 H), 3.76 (s, 3 H), 3.71−3.62

(m, 2 H), 3.29−3.22 (m, 1 H), 1.49 (d, J = 6.6 Hz, 3 H), 1.35−1.26

(m, 1 H), 1.04−0.93 (m, 2 H), 0.78 (d, J = 7.1 Hz, 3 H), 0.02 (s, 9

H). HRESIMS m/z calcd for C₂₈H₃₇N₃O₆Si (MH)⁺540.2524, found

540.2524.

Methyl (S)-2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

4-yl)-3-methylbutanoate (16j). This compound was isolated as a

yellowish solid (91 mg, 63%): mp 208−210 °C. ¹H NMR (500 MHz,

CDCl₃) δ 9.27 (s, 1 H), 7.92 (s, 1 H), 7.78 (s, 1 H), 7.48 (d, J = 3.7

Hz, 1 H), 6.94 (d, J = 3.8 Hz, 1 H), 5.82 (t, J = 6.7 Hz, 2 H), 5.51 (d,

J = 9.3 Hz, 1 H), 4.17 (s, 3 H), 4.08 (s, 3 H), 3.76 (d, J = 6.7 Hz, 3

H), 3.72−3.64 (m, 2 H), 3.29−3.21 (m, 1 H), 2.17 (s, 1 H), 1.49 (d, J

= 6.6 Hz, 3 H), 1.03−0.95 (m, 2 H), 0.78 (d, J = 7.1 Hz, 3 H), 0.02

(s, 9 H). HRESIMS m/z calcd for C₂₈H₃₇N₃O₆Si (MH)⁺540.2524,

found 540.2524.

Methyl (R)-2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

4-yl)butanoate (16k). This compound was isolated as a yellowish

powder (0.1 g, 68%): mp 189−193 °C. ¹H NMR (500 MHz, CDCl₃)

δ 9.19 (s, 1 H), 7.85 (s, 1 H), 7.71 (s, 1 H), 7.42 (d, J = 3.6 Hz, 1 H),

6.81 (d, J = 3.6 Hz, 1 H), 5.80−5.72 (m, 2 H), 5.65 (t, J = 7.2 Hz, 1

H), 4.13 (s, 3 H), 4.04 (s, 3 H), 3.74 (s, 3 H), 3.62−3.55 (m, 2 H),

2.66−2.58 (m, 1 H), 2.42−2.37 (m, 1 H), 1.00 (t, J = 7.5 Hz, 3 H),

0.95−0.89 (m, 2 H), −0.05 (s, 9 H). HRESIMS m/z calcd for

C₂₇H₃₅N₃O₆Si (MH)⁺526.2368, found 526.2370.

Methyl (S)-2-(7,8-Dimethoxy-5-oxo-1-((2-(trimethylsilyl)ethoxy)-

methyl)-1,5-dihydro-4H-benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-

4-yl)butanoate (16l). This compound was isolated as a yellowish

solid (0.072 g, 55%): mp 188−192 °C. ¹H NMR (500 MHz, CDCl₃)

δ 9.19 (s, 1 H), 7.85 (s, 1 H), 7.71 (s, 1 H), 7.42 (d, J = 3.6 Hz, 1 H),

6.81 (d, J = 3.6 Hz, 1 H), 5.80−5.72 (m, 2 H), 5.65 (t, J = 7.2 Hz, 1

H), 4.13−4.08 (m, 4 H), 4.10−3.98 (m, 3 H), 3.74 (s, 3 H), 3.62−

3.55 (m, 2 H), 2.65−2.59 (m, 1 H), 2.43−2.37 (m, 1 H), 1.00 (t, J =

7.5 Hz, 3 H), 0.95−0.89 (m, 2 H), −0.05 (s, 9 H). HRESIMS m/z

calcd for C₂₇H₃₅N₃O₆Si (MH)⁺526.2368, found 526.2370.

General Procedure for the Deprotection of Compounds

17a−j. Compounds 16a−j (0.1 mmol) were dissolved in DCM (2

mL), and TFA (2 mL) was added to the solution dropwise. The

mixture was stirred at room temperature for 2 h, and then the solvent

was evaporated. The residue was dissolved in MeOH (5 mL), and

ethylenediamine (1 mL) was added and the mixture was stirred

overnight at 50 °C. The mixture was acidiﬁed with TFA and extracted

with CHCl₃(3 × 10 mL). The combined organic layer was washed

with water and brine (15 mL each), dried, and evaporated to yield

17a−j as white solids.

4-Cyclooctyl-7,8-dimethoxy-1,4-dihydro-5H-benzo[c]pyrrolo-

[2,3-h][1,6]naphthyridin-5-one (17d). This compound was isolated

as a white solid (32 mg, 79%): mp 235−238 °C. IR (thin ﬁlm) 2918,

1644, 1607, 1571, 1510, 1463, 1416, 1378, 1354, 1334, 1263, 1241,

1

1207, 1180, 1164, 1062 cm⁻¹. H NMR (500 MHz, DMSO-d₆) δ

12.16 (s, 1 H), 9.38 (s, 1 H), 8.02 (s, 1 H), 7.71 (s, 1 H), 7.60 (s, 1

H), 6.75 (s, 1 H), 5.18 (s, 1 H), 4.05 (s, 3 H), 3.92 (s, 3 H), 2.82−

2.75 (m, 2 H), 1.98−1.83 (m, 4 H), 1.81−1.71 (m, 2 H), 1.64−1.50

(m, 5 H), 1.29−1.21 (m, 2 H). MALDIMS m/z 406 (MH)⁺.

HRESIMS calcd for C₂₄H₂₇N₃O₃(MH)⁺406.2125, found 406.2127.

C18 HPLC purity: 96.27% (MeOH−H₂O, 85:15).

7,8-Dimethoxy-4-(1-methylpiperidin-4-yl)-1,4-dihydro-5H-

benzo[c]pyrrolo[2,3-h][1,6]naphthyridin-5-one (17e). This com-

pound was isolated as a white solid (25 mg, 63%): mp 215−217

°C. IR (thin ﬁlm) 2936, 1633, 1607, 1569, 1512, 1463, 1418, 1353,

1334, 1309, 1258, 1231, 1185, 1143, 1125, 1037, 1011 cm⁻¹. ¹H

NMR (500 MHz, DMSO-d₆) δ 12.14 (s, 1 H), 9.33 (s, 1 H), 7.97 (s,

1 H), 7.64 (s, 1 H), 7.53 (d, J = 3.4 Hz, 1 H), 6.65 (s, 1 H), 5.04−

4.98 (m, 1 H), 4.00 (s, 3 H), 3.86 (s, 3 H), 2.98−2.82 (m, 8 H),

2.57−2.54 (m, 1 H), 2.02−1.80 (m, 2 H). MALDIMS m/z 393

(MH)⁺. HRESIMS calcd for C₂₂H₂₄N₄O₃(MH)⁺393.1921, found

393.1926. C18 HPLC purity: 99.09% (MeOH−H₂O, 85:15).

7,8-Dimethoxy-4-(piperidin-4-yl)-1,4-dihydro-5H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-5-one (17f). This compound was

isolated as a white solid (18 mg, 48%): mp 258−259 °C. IR (thin

ﬁlm) 2925, 1635, 1610, 1572, 1515, 1313, 1261, 1210, 1038 cm⁻¹. ¹H

NMR (500 MHz, DMSO-d₆) δ 12.07 (s, 1 H), 9.31 (s, 1 H), 7.95 (s,

1 H), 7.64 (s, 1 H), 7.51 (s, 1 H), 6.61 (s, 1 H), 4.98−4.89 (m, 1 H),

3.99 (s, 3 H), 3.86 (s, 3 H), 3.21−3.08 (m, 3 H), 2.89 (d, J = 8.6 Hz,

2 H), 2.69−2.58 (m, 4 H), 1.79−1.69 (m, 2 H). MALDIMS m/z 379

(MH)⁺. HRESIMS calcd for C₂₁H₂₂N₄O₃(MH)⁺379.1764, found

379.1762. C18 HPLC purity: 99.27% (MeOH−H₂O, 85:15).

(R)-7,8-Dimethoxy-4-(pyrrolidin-3-yl)-1,4-dihydro-5H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-5-one (17g). This compound was

R

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isolated as a white solid (10 mg, 27%): mp 274−276 °C. IR (thin

ﬁlm) 2920, 1939, 1608, 1570, 1511, 1455, 1419, 1398, 1379, 1336,

1310, 1262, 1244, 1208, 1161, 1127, 1026 cm⁻¹. ¹H NMR (500 MHz,

DMSO-d₆) δ 12.16 (s, 1 H), 9.39 (s, 1 H), 8.04 (s, 1 H), 7.72 (s, 1

H), 7.58 (s, 1 H), 6.96 (s, 1 H), 5.72 (s, 1 H), 4.06 (s, 3 H), 3.92 (s, 3

H), 3.31−3.21 (m, 2 H), 3.05−2.91 (m, 1 H), 2.38−2.28 (m, 2 H),

0.95−0.84 (m, 1 H). MALDIMS m/z 365 (MH)⁺. HRESIMS calcd

for C₂₀H₂₀N₄O₃(MH)⁺365.1608, found 365.1610. C18 HPLC

purity: 100% (MeOH−H₂O, 85:15).

(R)-7,8-Dimethoxy-4-(piperidin-3-yl)-1,4-dihydro-5H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-5-one (17h). This compound was

isolated as a white solid (18 mg, 48%): mp 286−287 °C. IR (thin

ﬁlm) 3130, 2919, 1649, 1610, 1577, 1509, 1463, 1407, 1378, 1334,

1310, 1242, 1209, 1175, 1127, 1022 cm⁻¹. ¹H NMR (500 MHz,

DMSO-d₆) δ 12.13 (s, 1 H), 9.37 (s, 1 H), 8.02 (s, 1 H), 7.69 (s, 1

H), 7.57 (s, 1 H), 6.86 (s, 1 H), 5.03−4.97 (m, 1 H), 4.06 (s, 3 H),

3.92 (s, 3 H), 3.88−2.84 (m, 1 H), 3.14−3.08 (m, 1 H), 3.02−2.96

(m, 2 H), 1.93−1.85 (m, 1 H), 1.84−1.78 (m, 1 H), 1.68−1.60 (m, 1

H). MALDIMS m/z 379 (MH)⁺. HRESIMS calcd for C₂₁H₂₂N₄O₃

(MH)⁺379.1764, found 379.1765. C18 HPLC purity: 99.29%

(MeOH−H₂O, 85:15).

Methyl (R)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-3-methylbutanoate (17i).

This compound was isolated as a white solid (12 mg, 29%): mp

256−258 °C. IR (thin ﬁlm) 2922, 1726, 1608, 1572, 1514, 1461,

1420, 1386, 1334, 1268, 1184, 1137, 1021 cm⁻¹. ¹H NMR (500 MHz,

DMSO-d₆) δ 12.29 (s, 1 H), 9.47 (s, 1 H), 8.09 (s, 1 H), 7.69 (s, 1

H), 7.64−7.53 (m, 1 H), 6.91 (s, 1 H), 5.54 (d, J = 9.3 Hz, 1 H), 4.08

(s, 3 H), 3.93 (s, 3 H), 3.57 (s, 3 H), 3.10−2.90 (m, 1 H), 1.37 (d, J =

6.6 Hz, 3 H), 0.61 (d, J = 7.1 Hz, 3 H). MALDIMS m/z 410 (MH)⁺.

HRESIMS calcd for C₂₂H₂₃N₃O₅(MH)⁺410.1711, found 410.1712.

C18 HPLC purity: 100% (MeOH−H₂O, 85:15).

Methyl (S)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-3-methylbutanoate (17j).

This compound was isolated as a white solid (26 mg, 68%): mp

274−276 °C. IR (thin ﬁlm) 2956, 1741, 1652, 1608, 1570, 1513,

1461, 1420, 1384, 1365, 1334, 1314, 1264, 1246, 1210, 1145, 1119,

1071, 1028 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆) δ 12.21 (s, 1 H),

9.40 (s, 1 H), 8.03 (s, 1 H), 7.63 (s, 1 H), 7.57−7.47 (m, 1 H), 6.85

(s, 1 H), 5.48 (d, J = 9.2 Hz, 1 H), 4.02 (s, 3 H), 3.87 (s, 3 H), 3.51

(s, 3 H), 3.01−2.91 (m, 1 H), 1.31 (d, J = 6.6 Hz, 3 H), 0.55 (d, J =

7.1 Hz, 3 H). MALDIMS m/z 410 (MH)⁺. HRESIMS calcd for

C₂₂H₂₃N₃O₅(MH)⁺410.1711, found 410.1710. C18 HPLC purity:

98.30% (MeOH−H₂O, 85:15).

(R)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)butanoic Acid (17k).

Compound 16k (0.14 g, 0.226 mmol) was dissolved in DCM (2

mL), and TFA (2 mL) was added to the solution dropwise. The

mixture was stirred at room temperature for 2 h, and then the solvent

was evaporated. The residue was dissolved in MeOH (5 mL) and

THF (5 mL) and ethylenediamine (1 mL) and NaOH (2 mL, 1 M aq

solution) were added and the mixture was stirred overnight at 60 °C.

The volatile solvents were removed, and the solution was acidiﬁed

with HCl to pH 3. The mixture was extracted with EtOAc (3 × 10

mL). The combined organic layer was washed with water and brine

(15 mL each), dried, and evaporated to yield 17k as a white solid (80

mg, 81%): mp 212−214 °C. IR (thin ﬁlm) 3262, 2921, 1722, 1657,

1608, 1574, 1463, 1422, 1387, 1338, 1314, 1264, 1246, 1211, 1137,

1082 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆) δ 12.15 (s, 1 H), 9.38 (s,

1 H), 8.02 (s, 1 H), 7.63 (s, 1 H), 7.50 (s, 1 H), 6.90 (s, 1 H), 5.64 (s,

1 H), 4.02 (s, 3 H), 3.86 (s, 3 H), 2.30−2.23 (m, 2 H), 0.78 (t, J = 7.5

Hz, 3 H). MALDIMS m/z 382 (MH)⁺. HRESIMS calcd for

C₂₀H₁₉N₃O₅(MH)⁺382.1397, found 382.1397.

H), 6.98 (s, 1 H), 5.70 (s, 1 H), 4.08 (s, 3 H), 3.93 (s, 3 H), 2.48−

2.37 (m, 2 H), 2.32 (d, J = 6.6 Hz, 1 H), 0.85 (t, J = 7.5 Hz, 3 H).

MALDIMS m/z 382 (MH)⁺. HRESIMS calcd for C₂₀H₁₉N₃O₅

(MH)⁺382.1397, found 382.1399.

(R)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-3-methylbutanoic Acid

(17m). Compound 16i (0.1 mmol) was dissolved in DCM (2 mL),

and TFA (2 mL) was added to the solution dropwise. The mixture

was stirred at room temperature for 2 h, and then the solvent was

evaporated. The residue was dissolved in MeOH (5 mL) and THF (5

mL) and ethylenediamine (1 mL) and NaOH (2 mL, 1 M soln) were

added and the mixture was stirred overnight at 60 °C. The volatile

solvents were removed, and the solution was acidiﬁed with HCl to pH

3. The mixture was extracted with EtOAc (3 × 10 mL). The

combined organic layer was washed with water and brine (15 mL

each), dried, and evaporated to yield 17m as a yellow solid (0.11 g,

96%): mp 198−200 °C. IR (thin ﬁlm) 3513, 2968, 1720, 1665, 1606,

1514, 1459, 1442, 1388, 1329, 1264, 1183, 1136, 1017 cm⁻¹. ¹H

NMR (500 MHz, DMSO-d₆) δ 12.22 (s, 1 H), 9.40 (s, 1 H), 8.02 (s,

1 H), 7.68 (m, 1 H), 7.60−7.40 (m, 1 H), 6.82 (s, 1 H), 5.35 (d, J =

9.3 Hz, 1 H), 4.02 (s, 3 H), 3.87 (s, 3 H), 3.12−2.94 (m, 1 H), 1.32

(d, J = 8.6 Hz, 3 H), 0.56 (d, J = 7.1 Hz, 3 H). MALDIMS m/z 396

(MH)⁺. HRESIMS calcd for C₂₁H₂₁N₃O₅(MH)⁺396.1554, found

396.1553.

(R)-3-(3-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)piperidin-1-yl)-3-oxopro-

panenitrile (18). Compound 17h (30 mg, 0.08 mol) was added to a

solution of HATU (30 mg, 0.08 mmol), DIPEA (30 mg 0.24 mmol),

and cyanoacetic acid (5 mg) in DMF (4 mL). The mixture was stirred

at room temperature for 24 h and then quenched with saturated

ammonium chloride (15 mL). The solution was extracted with EtOAc

(3 × 15 mL). The combined organic layer was washed with saturated

sodium bicarbonate (15 mL), water, and brine and dried using

anhydrous sodium sulfate. The residue obtained after evaporation of

the organic layer was puriﬁed using column chromatography

(CH₂Cl₂−MeOH 9.8:0.2) to give compound 18 as white solid (26

mg, 73%): mp 185−187 °C. IR (thin ﬁlm) 2928, 2262, 1645, 1608,

1573, 1512, 1446, 1417, 1378, 1333, 1308, 1261, 1208, 1129, 1063

1

cm⁻¹. H NMR (500 MHz, DMSO-d₆) δ 12.11 (s, 1 H), 9.33 (s, 1

H), 7.98 (s, 1 H), 7.64 (d, J = 4.1 Hz, 1 H), 7.51 (d, J = 12.7 Hz, 1

H), 6.59 (m, 1 H), 4.82 (s, 1 H), 4.68−4.61 (m, 1 H), 4.48−4.42 (m,

1 H), 4.24−4.18 (m, 1 H), 4.13−4.08 (m, 1 H), 3.99 (s, 3 H), 3.86 (s,

3 H), 3.76−3.70 (m, 2 H), 3.11−3.02 (m, 1 H), 2.93−2.84 (m, 2 H),

2.50 (s, 2 H), 1.88−1.84 (m, 1 H), 1.72−1.65 (m, 1 H). MALDIMS

m/z 446 (MH)⁺. HRESIMS calcd for C₂₄H₂₃N₅O₄(MH)⁺446.1823,

found 446.1822. C18 HPLC purity: 95.85% (MeOH−H₂O, 85:15).

(R)-1-(2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-3-methylbutanoyl)-

azetidine-3-carbonitrile (19a). Following the same procedure used

for compound 18 and starting with 17m, compound 19a was isolated

as a white solid (48 mg, 86%): mp 236−238 °C. IR (thin ﬁlm) 3248,

2965, 1649, 1608, 1570, 1514, 1413, 1367, 1332, 1264, 1210, 1138,

1115, 1022 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆) δ 12.25 (s, 1 H),

9.42 (s, 1 H), 8.03 (s, 1 H), 7.66 (s, 1 H), 7.58 (s, 1 H), 7.05−6.88

(m, 1 H), 5.48 (s, 1 H), 4.02 (s, 3 H), 4.00−3.95 (m, 1 H), 3.88 (s, 3

H), 3.83−3.79 (m, 1 H), 3.45−3.41 (m, 1 H), 3.07−3.01 (m, 1 H),

1.26 (d, J = 6.6 Hz, 3 H), 0.50 (d, J = 7.2 Hz, 3 H). ¹³C NMR (126

MHz, DMSO-d₆) δ 160.95, 154.52, 149.78, 149.44, 141.44, 138.14,

129.34, 126.42, 124.97, 116.50, 108.29, 107.52, 105.77, 103.52,

101.91, 56.63, 55.84, 26.69, 21.76, 18.10. MALDIMS m/z 460

(MH)⁺. HRESIMS calcd for C₂₅H₂₅N₅O₄(MH)⁺460.1979, found

460.1981. C18 HPLC purity: 100% (MeOH−H₂O, 85:15).

(R)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-3-methyl-N-(2,2,2-

triﬂuoroethyl)butanamide (19b). Following the same procedure

used for compound 18 and starting with 17m, compound 19b was

isolated as a white solid (45 mg, 74%): mp 247−248 °C. IR (thin

ﬁlm) 3123, 2970, 2878, 1677, 1627, 1608, 1548, 1514, 1465, 1418,

1397, 1387, 1362, 1334, 1309, 1282, 1233, 1213, 1159, 1120, 1063,

1034 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆) δ 12.17 (s, 1 H), 9.40 (s,

1 H), 8.63 (t, J = 6.3 Hz, 1 H), 8.03 (s, 1 H), 7.64 (s, 1 H), 7.53 (s, 1

(S)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)butanoic Acid (17l). Fol-

lowing the same procedure used for 17k, compound 17l was isolated

as a white solid (84 mg, 85%): mp 217−218 °C. IR (thin ﬁlm) 3544,

2937, 1704, 1676, 1601, 1515, 1459, 1425, 1390, 1330, 1310, 1264,

1229, 1211, 1181, 1147, 1028 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆)

δ 12.24 (s, 1 H), 9.45 (s, 1 H), 8.08 (s, 1 H), 7.69 (s, 1 H), 7.58 (s, 1

S

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H), 6.82 (s, 1 H), 5.25 (d, J = 9.5 Hz, 1 H), 4.02 (s, 3 H), 3.99−3.95

(m, 1 H), 3.86 (s, 3 H), 3.73−3.68 (m, 1 H), 3.04−2.96 (m, 1 H),

1.19 (d, J = 6.5 Hz, 3 H), 0.38 (d, J = 6.8 Hz, 3 H). MALDIMS m/z

477 (MH)⁺. HRESIMS calcd for C₂₃H₂₃F₃N₄O₄(MH)⁺477.1744,

found 477.1744. C18 HPLC purity: 97.59% (MeOH−H₂O, 85:15).

(R)-1-(2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)butanoyl)azetidine-3-

carbonitrile (20a). Compound 17k (30 mg, 0.08 mmol) was added

to a solution of HATU (30 mg, 0.08 mmol) and DIPEA (30 mg 0.24

mmol) in DMF (4 mL). After stirring for 5 min, azetidine-3-

carbonitrile hydrochloride (13 mg, 0.09 mmol) was added to the

mixture. The mixture was stirred at room temperature for 24 h, and

then the reaction was quenched with saturated ammonium chloride

(15 mL). The solution was extracted with EtOAc (3 × 15 mL). The

combined organic layer was washed with saturated sodium

bicarbonate (15 mL), water, and brine and dried using anhydrous

sodium sulfate. The residue obtained after evaporation of the organic

layer was puriﬁed using silica gel column chromatography

(CH₂Cl₂:MeOH 9.8:0.2) to yield compound 20a as a white solid

(28 mg, 60%): mp 257−260 °C. IR (thin ﬁlm) 3313, 2957, 1649,

1609, 1572, 1514, 1464, 1421, 1385, 1310, 1262, 1209, 1152, 1040

cofactors were added to the substrate solution. Puriﬁed JAK kinases

were added into the substrate solution and gently mixed. A solution of

the compounds in DMSO was then added into the kinase reaction

mixture by Acoustic technology (Echo550; nanoliter range), and the

mixture was incubated for 20 min at room temperature. ³³P-ATP

(speciﬁc activity 10 Ci/mmol) was then added into the reaction

mixture to initiate the reaction. The kinase reaction was incubated for

2 h at room temperature, and then the mixtures were spotted onto

P81 ion exchange paper. Finally, the kinase activity was detected by

the ﬁlter-binding method.

STAT-5 Phosphorylation Inhibition Assay. The experiments

performed by Reaction Biology Corporation. SZ4 cells from growing

stock were seeded in 96-well plates at 1 × 10⁵cells/well in 100 μL of

complete media and then incubated overnight at 37 °C, 5% CO₂. The

cells were treated with test compounds (starting at 30 μM, 8 doses

with 3-fold dilution) for 1 h. The cells were then stimulated with hIL-

2 at a ﬁnal concentration of 100 ng/mL for 15 min. The cells were

spun down, and the culture medium was aspirated. The cells were

lysed with 50 μL of 1× cell lysis buﬀer. The cell lysates (25 μL) were

used for MSD assay immediately or stored at −80 °C. MSD assay was

performed according to the kit manual (https://www.mesoscale.

c o m / ~ / m e d i a / ﬁ l e s / p r o d u c t % 2 0 i n s e r t s /

phospho%20tyr694%20total%20stat5a%20b%20wcl.pdf). IC₅₀values

were calculated using GraphPad Prism 4 based on a sigmoidal dose−

response equation. Western blot assay experimental details are

provided in the Supporting Information.

1

cm⁻¹. H NMR (500 MHz, DMSO-d₆) δ 12.26 (s, 1 H), 9.47 (s, 1

H), 8.09 (s, 1 H), 7.72 (s, 1 H), 7.62 (s, 1 H), 7.06 (s, 1 H), 5.77 (s, 1

H), 4.08 (s, 3 H), 4.03−3.97 (m, 1 H), 3.94 (s, 3 H), 3.91−3.86 (m, 1

H), 3.54 (s, 1 H), 2.47−2.36 (m, 2 H), 2.21 (d, J = 7.6 Hz, 1 H), 0.81

(t, J = 7.6 Hz, 3 H). MALDIMS m/z 446 (MH)⁺. HRESIMS calcd for

C₂₄H₂₃N₅O₄(MH)⁺446.1823, found 446.1828. C18 HPLC purity:

96.72% (MeOH−H₂O, 85:15).

Primary T Cell Inhibition Assay. CD4⁺T cells were isolated

from mouse spleen and cultured in vitro with antibodies to CD3 (5

μg/mL) and CD28 (2 μg/mL) in the presence of IL-2 (100 U/mL)

for nonpolarizing conditions as described before.⁴¹Inhibitors were

added to culture at the indicated concentrations. After culture, the

cells were stained with XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-

sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt)

and analyzed at 475 nm by a microplate reader (SpectraMax i3X,

Molecular Devices). The cells were cultured for 5 days and stained

with antibodies to mouse CD4 (RM4-5). The cells were activated

with phorbol myristate acetate (PMA, 50 ng/mL, Sigma-Aldrich),

ionomycin (1 μM, Sigma-Aldrich), and monensin (2 mM, Sigma-

Aldrich) for 4 h followed by ﬁxation with 4% PFA in PBS and

permeabilization in 1% saponins in PBS. The cells were stained with

antibodies to mouse IFNγ (XGM1.2) or mouse IL-17 (TC11-

18H10.1) for ﬂow cytometry (Canto II, Becton Dickinson). For IL-17

production, the T cells were cultured with anti-IL2, anti-IFNγ, anti-

IL-4, IL-23, TNF-α, and IL-1β as previously described.⁴¹

Kinome Scanning Assay. Selected compounds were tested for

kinome selectivity at Euroﬁns DiscoverX Corporation, Fremont,

California, USA, by applying their KINOMEscan technology.

Diﬀerent kinases were labeled with a unique DNA tag for qPCR. A

known kinase ligand was immobilized on a solid support and

incubated with the labeled kinase in the presence of either DMSO or

a speciﬁed concentration of the test compounds. After equilibration,

the solid support was washed to remove unbound kinase, and then the

kinase captured on solid support was quantiﬁed by qPCR. Finally, the

captured kinase levels in the test compound and DMSO control

samples were compared.

(R)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-N-(2,2,2-triﬂuoroethyl)-

butanamide (20b). Compound 17k (30 mg, 0.08 mmol) was added

to a solution of HATU (30 mg, 0.08 mmol) and DIPEA (30 mg 0.24

mmol) in DMF (4 mL). After stirring for 5 min, triﬂuoroethylamine

hydrochloride (11 mg, 0.09 mmol) was added to the mixture. The

mixture was stirred at room temperature for 24 h and then quenched

with saturated ammonium chloride (15 mL). The solution was

extracted with EtOAc (3 × 15 mL). The combined organic layer was

washed with saturated sodium bicarbonate (15 mL), water, and brine

and dried using anhydrous sodium sulfate. The residue obtained after

evaporation of the organic layer was puriﬁed using silica gel column

chromatography (CH₂Cl₂:MeOH 9.8:0.2) to provide compound 20b

as a white solid (20 mg, 55%): mp 267−268 °C. IR (thin ﬁlm) 3508,

2979, 2872, 2249, 1649, 1610, 1572, 1517, 1499, 1416, 1386, 1315,

1247, 1210, 1186, 1166, 1112, 1041 cm⁻¹. ¹H NMR (500 MHz,

DMSO-d₆) δ 12.10 (s, 1 H), 9.37 (s, 1 H), 8.50 (t, J = 6.4 Hz, 1 H),

8.02 (s, 1 H), 7.62 (s, 1 H), 7.49 (s, 1 H), 6.64 (s, 1 H), 5.56 (t, J =

7.5 Hz, 1 H), 4.01 (s, 3 H), 3.86 (s, 3 H), 3.68−3.61 (m, 2 H), 2.65

(s, 1 H), 2.37−2.29 (m, 2 H), 0.52 (t, J = 7.4 Hz, 3 H). MALDIMS

m/z (rel intensity) 463 (MH)⁺. HRESIMS calcd for C₂₂H₂₁F₃N₄O₄

(MH)⁺463.1588, found 463.1602. C18 HPLC purity: 98.86%

(MeOH−H₂O, 85:15).

(S)-2-(7,8-Dimethoxy-5-oxo-1,5-dihydro-4H-benzo[c]-

pyrrolo[2,3-h][1,6]naphthyridin-4-yl)-N-(2,2,2-triﬂuoroethyl)-

butanamide (21). Following the same procedure used for 20b but

using the acid 17l, compound 21 was isolated as a white solid (24 mg,

60%): mp 235−237 °C. IR (thin ﬁlm) 3318, 2959, 1699, 1649, 1610,

1572, 1513, 1465, 1421, 1365, 1337, 1310, 1260, 1248, 1208, 1151,

1136 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆) δ 12.16 (s, 1 H), 9.43 (s,

1 H), 8.56 (t, J = 6.3 Hz, 1 H), 8.08 (s, 1 H), 7.68 (s, 1 H), 7.58−7.52

(m, 1 H), 6.70 (s, 1 H), 5.62 (t, J = 7.6 Hz, 1 H), 4.08 (s, 3 H), 4.07−

3.99 (m, 1 H), 3.92 (s, 3 H), 3.71 (s, 2 H), 2.39 (t, J = 7.5 Hz, 3 H),

0.59 (t, J = 7.4 Hz, 3 H). MALDIMS m/z 463 (MH)⁺. HRESIMS

calcd for C₂₂H₂₁F₃N₄O₄(MH)⁺463.1588, found 463.1590. C18

HPLC purity: 100% (MeOH−H₂O, 85:15).

Pharmacokinetic Proﬁling. The experiments were performed at

Euroﬁns Scientiﬁc Inc., Lancaster, PA, USA.

Caco-2 Permeability. Caco-2 cells were grown in tissue culture

ﬂasks, trypsinized, and suspended in medium, and known

concentrations of cell suspensions were seeded onto wells of a

Millipore 96-well Caco-2 plate. The cells were allowed to grow and

diﬀerentiate for 3 weeks, feeding at two-day intervals. For apical to

basolateral (A → B) permeability, the test article was added to the

apical (A) side and the amount of permeation on the basolateral (B)

side was determined; for basolateral to apical (B → A) permeability,

the test article was added to the B side and the amount of permeation

on the A side was determined. To test tight junctions and monolayer

integrity, the A-side buﬀer contained 100 μM Lucifer yellow dye in

transport buﬀer (1.98 g/L glucose in 10 mM HEPES, 1× Hank’s

Balanced Salt Solution) with pH 6.5, while the B-side buﬀer was

Enzyme Inhibition Assay. The enzyme assay was performed by

Reaction Biology Corporation according to the following protocol:

The base reaction buﬀer consisted of 20 mM Hepes (pH 7.5), 10 mM

MgCl₂, 1 mM EDTA, 0.02% Brij-35, 0.02 mg/mL BSA, 0.1 mM

Na₃VO₄, 2 mM DTT, and 1% DMSO. The indicated substrate was

added to freshly prepared base reaction buﬀer, and then the required

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

transport buﬀer with pH 7.4. Caco-2 cells were incubated with these

buﬀers for 2 h, and the receiver-side buﬀer was removed for analysis

by LC/MS/MS. To verify the tight junctions and integrity of Caco-2

cell monolayers, aliquots of the cell buﬀers were analyzed by

ﬂuorescence (Lucifer yellow transport ≤2%). Any deviations from

(Chondrix) containing 4 mg/mL of Mycoplasma tuberculosis. Mice

were maintained under anesthesia during the inculcation via

inhalation of isoﬂurane. Each mouse received a 100 μL injection of

the emulsion transdermally in the tail. Three weeks later, the mice

received a booster inculcation the same way but with an emulsion

made from equal parts of Incomplete Freund’s Adjuvant (Chondrix)

and type II bovine collagen (Chondrix). After 8 weeks, all mice

displayed symptoms of inﬂammation. They were sorted into one of

the three treatment groups so that the mean RA score for each of the

treatment groups was the same, and seven mice were assigned to each

treatment group. Sorting in this manner controlled for the variability

in the response to a CIA induction. The treatment groups consisted of

17b, tofacitinib, and vehicle (10% DMSO and 10% Cremophor EL).

Daily doses of 5 mg/kg/day were administered subcutaneously in

10% DMSO and 10% Cremophor EL for 5 weeks. Every week

inﬂammation was assessed by measuring paw volume with a digital

caliper and scoring all four paws with the 0−4 rheumatoid arthritis

score. The paws were scored by a trained technician who was blinded

to the treatment groups and who referred to a visual key for scoring

every time (Figure 7s, Supporting Information). Paw volume was

calculated as the product of the length, width, and height of the paws.

Measurements of height and width were taken at the same location on

the paw every time. Mouse weight was monitored throughout the

treatment period to watch for any toxicities, and no large ﬂuctuations

in weight were seen as a result of any of the treatments (Figure 8s,

Supporting Information). After 5 weeks of treatment, the mice were

euthanized, and the paws were collected for micro CT analysis. The

Student’s t test revealed that both tofacitinib and 17b cause a

signiﬁcantly diﬀerent response compared to the vehicle control and

that there is not a statistically signiﬁcant diﬀerence between the two

drugs. Statistical signiﬁcance was determined using the Holm−Sidak

method, with α = 0.05.

control values are reported. Data are expressed as permeability (P_app

)

= (dQ/dt)/(C₀A) where dQ/dt is the rate of permeation, C₀is the

initial concentration of test agent, and A is the area of the monolayer.

In bidirectional permeability studies, the eﬄux ratio (R_e) is also

calculated: R_e= (P_appB → A)/(P_appA → B); R^e> 2 indicates a

potential substrate for P-glycoprotein or other active eﬄux trans-

porter(s).

Metabolic Stability Analysis. The compounds were incubated in

duplicate with pooled human liver microsomes at 37 °C. The reaction

mixtures contained microsomal protein in 100 mM potassium

phosphate buﬀer (pH 7.4), 2 mM NADPH, and 3 mM MgCl₂. A

control was run for each test article omitting NADPH to detect

NADPH-free degradation. At predetermined time points, an aliquot

was removed from each experimental and control reaction and mixed

with an equal volume of ice-cold methanol containing propranolol as

the internal standard to stop the reaction and precipitate proteins.

Stopped reactions were kept on ice for at least 10 min followed by an

addition of an equal volume of water. The samples were centrifuged

to remove precipitated protein, and the supernatants were analyzed by

LC-MS/MS to quantify parent remaining. Data was calculated as %

parent remaining by assuming zero-minute time point peak area ratio

(analyte/IS) as 100% and dividing remaining time point peak area

ratios by the zero minute time point peak area ratio. Data were

subjected to ﬁt a ﬁrst-order decay model to calculate slope and

thereby half-life.

PBS Solubility Screen. Serial dilutions of the tested compounds,

reserpine, tamoxifen, and verapamil were prepared in phosphate

buﬀered saline (PBS) at 100× the ﬁnal concentration. The solutions

were diluted 100-fold into PBS in a 96-well plate and mixed. The

absorbance of the PBS-containing plate was measured prior to

addition of the test agents to determine the background absorbance.

After 2 h, the presence of precipitate was detected by turbidity

(absorbance at 540 nm). An absorbance value of greater than (mean

+ 3× standard deviation of the blank), after subtracting the

preexperiment background, is indicative of turbidity. The solubility

limit is reported as the highest experimental concentration with no

evidence of turbidity.

Molecular Modeling. The X-ray crystal structures of JAK kinases

were obtained from the Protein Data Bank (JAK1 PDB 4I5C; JAK2

PDB 3LXK). Each protein structure was cleaned, inspected for errors

and missing residues, hydrogens were added, and the water molecules

were deleted using Accelry’s Discovery studio 2.5 software. Structures

of the inhibitors were constructed using ChemBioDraw Ultra 13 and

saved in SDF ﬁle format and were corrected and optimized using

Accelrys Discovery studio 2.5 software. GOLD 4.1 was used for

docking with the default parameters except that the iterations were

increased to 300000 and the early termination option was disabled.

The centroids of the binding sites were deﬁned by the ligands in the

cocrystal structures. The top 10 docking poses per ligand were

inspected visually following the docking runs. Energy minimizations

were performed for selected ligand poses. The CHARMM force ﬁeld

was utilized within the Accelrys Discovery Studio 2.5 for energy

minimization.

Collagen-Induced Inﬂammation Model. All procedures

performed on live mice were approved by the Purdue Animal Use

and Care Committee and conformed with NIH guidelines. Eight-

week-old, male DBA-1 mice were obtained from Jackson Laboratories.

The mice were kept in housing facilities that maintained a constant

temperature of 22 °C and a constant humidity of 50%. The mice were

maintained on a 12-h light/dark cycle. Collagen-induced arthritis was

induced in the mice after a one-week acclimation period in our

housing facility. To induce the inﬂammation, equal volumes of the

following compounds were mixed on ice with a homogenizer until a

stable emulsion formed: 2 mg/mL of bovine type II collagen in 0.05

M acetic acid (Chondrix) and Complete Freund’s Adjuvant

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b00510.

Table for the % growth of the NCI-60 cell lines, gels

monitoring Top1-mediated DNA cleavage, table of

kinome selectivity data, kinome maps, EC₅₀determi-

nation curves and experimental procedure for the JAK3-

mediated STAT-5 phosphorylation assay, mouse paw

collagen-induced inﬂammation assay (RA paw volume

and percent weight change results) (PDF)

SMILES molecular formula strings (CSV)

Figure 9 JAK1 molecular model (PDB)

Figure 9 JAK3 molecular model (PDB)

Figure 10 JAK1 molecular model (PDB)

Figure 10 JAK3 molecular model (PDB)

Figure 11 JAK1 molecular model (PDB)

Figure 11 JAK3 molecular model (PDB)

AUTHOR INFORMATION

Corresponding Author

*Phone: (765)494-1465. Fax: (765)494-6970. E-mail:

cushman@purdue.edu.

■

ORCID

Mohamed S. A. Elsayed: 0000-0002-1353-7259

Philip S. Low: 0000-0001-9042-5528

Mark Cushman: 0000-0002-0152-5891

Notes

The authors declare no competing ﬁnancial interest.

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

isomerase I Poisons in Vitro and in Vivo. Clin. Cancer Res. 2007, 13,

591−602.

ACKNOWLEDGMENTS

■

We gratefully acknowledge support from the Purdue University

Center for Cancer Research, and NIH grants P30 CA023168,

NIH 5R01AI080769, and NIH R01AI121302. Mohamed S. A.

Elsayed was partially supported by the McKeehan Graduate

Scholarship in Pharmacy Award. This investigation was also

supported by the NCI Center for Cancer Research (Z01

BC006161).

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ABBREVIATIONS USED

■

DCM, dichloromethane; DMA, N,N-dimethylacetamide;

DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;

EtOAc, ethyl acetate; HATU, (1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-

ﬂuorophosphate); MOM, methoxymethyl; SEM, 2-

(trimethylsilyl)ethoxymethyl; TEA, triethylamine; TFA, tri-

ﬂuoroacetic acid; THF, tetrahydrofuran

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G.; Dyke, H. J.; Eigenbrot, C.; Ghilardi, N.; Gibbons, P.; Harrison, T.

K.; Hewitt, P. R.; Liimatta, M.; Hurley, C. A.; Johnson, A.; Johnson,

T.; Kenny, J. R.; Bir Kohli, P.; Maxey, R. J.; Mendonca, R.; Mortara,

K.; Murray, J.; Narukulla, R.; Shia, S.; Steffek, M.; Ubhayakar, S.;

Ultsch, M.; van Abbema, A.; Ward, S. I.; Waszkowycz, B.; Zak, M.

Identification of Imidazo-Pyrrolopyridines as Novel and Potent JAK1

Inhibitors. J. Med. Chem. 2012, 55, 5901−5921.

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(25) Kim, M. K.; Shin, H.; Park, K.-s.; Kim, H.; Park, J.; Kim, K.;

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Blair, W. S.; Chang, C.; Deshmukh, G.; DeVoss, J.; Dragovich, P. S.;

Eigenbrot, C.; Ghilardi, N.; Gibbons, P.; Gradl, S.; Hamman, C.;

Hanan, E. J.; Harstad, E.; Hewitt, P. R.; Hurley, C. A.; Jin, T.;

Johnson, A.; Johnson, T.; Kenny, J. R.; Koehler, M. F. T.; Bir Kohli,

P.; Kulagowski, J. J.; Labadie, S.; Liao, J.; Liimatta, M.; Lin, Z.;

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DOI: 10.1021/acs.jmedchem.8b00510

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Article abstract of DOI:10.1021/acs.jmedchem.8b00510

Full text of DOI:10.1021/acs.jmedchem.8b00510

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