Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors

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Design and Synthesis of Highly Selective Brain Penetrant p38α

Mitogen-Activated Protein Kinase Inhibitors

Niklas M. Torm ä hlen,^⊥Mariella Martorelli,^⊥Annette Kuhn, Florian Maier, Jamil Guezguez,

Michael Burnet, Wolfgang Albrecht, Stefan A. Laufer, and Pierre Koch *

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ABSTRACT: Stress-induced p38α mitogen-activated protein

(MAP) kinase activation modulates cytokine overproduction and

is associated with neuroinﬂammation and neurodegeneration. As a

potential therapeutic approach, novel Skepinone-based p38α MAP

kinase inhibitors were optimized to cross the blood−brain barrier

via either amino acid transporters or hydrophobic diﬀusion. To

enhance absorption from the oral route, we used methyl ester

prodrugs of the active carboxy analogs. Of these, 3-(8-((2,4-

diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]-

annulene-3-carboxamido)propanoic acid (43; p38α, IC₅₀= 5.5 nM) and 4-(8-((2,4-diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoic acid (44; p38α, IC₅₀= 12 nM) had brain-to-plasma ratios of 1.4 and 4.4,

respectively. Compound 70, 3-(8-((2-aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)-

propanoic acid (p38α, IC₅₀= 1.0 nM), the Skepinone-N counterpart of 43, was most present in the mouse brain (brain-to-

plasma ratio of 4.7; 0.4 mg/kg p.o., 2 h, 580 nmol/kg). Compounds 43, 44, and 70 were p38α-MAP-kinase-selective, metabolically

stable, hERG nonbinding, and able to modulate IL-6 and TNF-α production in cell-based assays.

INTRODUCTION

■

The p38α mitogen-activated protein (MAP) kinase catalyzes

the transfer of the γ-phosphate of its natural cosubstrate ATP

to the hydroxyl group of serine and threonine side chains of its

substrates, thereby activating cell signaling cascades, in

particular, during inﬂammation. This central role made it an

early choice as a drug target for chronic inﬂammatory diseases

as target-based screening evolved in the mid-1990s.¹p38α

MAP kinase is also expressed in glia and neurons, making it

potentially relevant for central nervous system (CNS)

disorders and associated neuroinﬂammatory responses.²⁻⁸

Increased activity of p38α MAP kinase was found in post-

mortem brain tissue from Alzheimer’s disease (AD)

Figure 1. Structures of p38α MAP kinase inhibitors Neﬂamapimod

patients.^9,10In microglia, p38α MAP kinase stimulates the

(1a, VX-745),¹⁵MW150 (1b),¹⁶Skepinone-L (2a), and Skepinone-N

(2b).

release of the pro-inﬂammatory cytokines interleukin (IL)-1β

and tumor necrosis factor α (TNF-α) in response to stressors

including amyloid-β42¹¹as well as during tau localization and

yielded signiﬁcant improvements in cognition using a dose of

neuronal plasticity.¹²

40 mg of Neﬂamapimod three times daily versus b.i.d. (twice a

Given this association with CNS inﬂammation, three phase

day) or placebo (EIP Pharma, Inc., 13th Clinical Trials in

II trials on the selective p38α MAP kinase inhibitor

Neﬂamapimod (1a, VX-745)¹³(Figure 1) for the treatment

of AD were completed (NCT02423200, NCT02423122, and

Special Issue: New Horizons in Drug Discovery -

Understanding and Advancing Kinase Inhibitors

NCT03402659), and another one on brain inﬂammation in

AD (NCT03435861) was launched in 2018.¹⁴In 2019, an

Received: October 9, 2020

additional phase II study began on the cognitive eﬀects of

Neﬂamapimod in early-stage Huntington’s disease

(NCT03980938). According to the sponsors, these trials

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A

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Alzheimer’s Disease (CTAD) meeting, AscenD-LB study).

These data suggest that whereas there is potential to improve

AD signs with compounds from this class, those optimized for

peripheral diseases like rheumatoid arthritis (RA) require

frequent dosing to exert central eﬀects. When applied at 1.5

mg/kg in rats, Neﬂamapimod reached 33 ng/mL (76 nM) in

plasma, which corresponds to 129 nmol/g in brain. This was

the dose reported to improve results in the Morris water maze

model.¹⁵At 4.5 mg/kg, the substance was able to inﬂuence

hippocampal IL-1β, and exposure was reported to be 92 ng/

mL in plasma corresponding to 359 nmol/kg in brain. These

data suggest that depending on the parameter used, the

eﬀective dose corresponds to peak brain levels in the range of

13−36 times the IC₅₀value for p38α MAP kinase.

Using the same rationale, the brain penetrant pyridinylpyr-

idazine-based p38α MAP kinase inhibitor MW150 (1b) was

investigated in diverse animal models of neurologic disorders

including AD and has now progressed to clinical trials.¹⁶⁻²⁰

When applied at 6 mg/kg, reported plasma concentrations are

in the range of 390 to 650 ng/mL,²⁰corresponding to brain

concentrations of 1030 to 1693 nmol/kg. The eﬀective dose

recorded for APP/PS1 mice was 2.5 mg/kg, suggesting that at

eﬀective doses, the peak levels in brain would likely be two to

three times the IC₅₀value for p38α MAP kinase.

dibenzosuberone core targeting the HR II was replaced by

amide moieties to increase interaction possibilities with the

enzyme. Lipophilic and basic moieties, (amino) acids, biogenic

amines, amino alcohols, and glucosamine were introduced as

amide moieties with the potential for active transport into the

brain as amino acid or carbohydrate mimics. In many analogs,

these mimics gave rise to terminal carboxy groups. Given that

carboxy species may be poorly resorbed in the gut, we also

formed ester prodrugs of these compounds to compare with

the free acids in both enzyme inhibition and oral

pharmacokinetic studies .

RESULTS AND DISCUSSION

■

Synthesis. The novel dibenzosuberone derivatives 6−51

(Skepinone-L series) and 54−72 (Skepinone-N series) were

synthesized by a linear approach starting from the reported

methyl 8-chloro-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]-

annulene-3-carboxylate³⁰(3). The amide moiety targeting

HR II of p38α MAP kinase was introduced in the last steps of

the synthetic sequences (Schemes 1 and 4). The synthesis of

Scheme 1. Synthesis of Dibenzosuberones 6−40

a

(Skepinone-L Series)

In 2012, we reported the development of dibenzosuberone

derivatives Skepinone-L (2a) and Skepinone-N (2b) as potent

selective p38α MAP kinase inhibitors (Figure 1).^21,22The

prototypic compound Skepinone-L has been used as a probe to

further investigate the role of p38α MAP kinase in various

disorders.²³⁻²⁷X-ray analysis of this inhibitor complexed with

the target enzyme suggests that it acts as a type I kinase

inhibitor, inducing a glycine ﬂip at the hinge region and

binding hydrophobic region (HR) I and HR II (Figure 2).²¹

Skepinone-L has served as a starting point for the design of

type I 1/2 p38α MAP kinase inhibitors with improved binding

kinetics.^28,29

a

Reagents and conditions: (i) 2,4-diﬂuoroaniline, Cs₂CO₃, XPhos,

Pd(OAc)₂, 1,4-dioxane/t-BuOH (5:1, v/v), 110 °C. (ii) KOH,

MeOH, reﬂux temperature. (iii) CDI, DMF, 50 °C. For the detailed

structures of compounds 6−51, see Tables 1 and 2 and Table S1, SI.

dibenzosuberones 6−51 is depicted in Schemes 1−3 and

Schemes S1−S6 (SI). In the ﬁrst step, the amino function at

position C-8 of the dibenzosuberone core was introduced by

the coupling of 3 to 2,4-diﬂuoroaniline under Buchwald−

Hartwig conditions (Scheme 1). Basic hydrolysis of 4 resulted

in dibenzosuberone 5 bearing a free carboxylic acid function in

position C-3. Activation of the carboxylic acid with 1,1′-

carbonyldiimidazole (CDI) and conversion with the respective

amines resulted in compounds 6−40. The detailed syntheses

of dibenzosuberones 45−51 are depicted in Schemes S1−S6

(Supporting Information, SI).

The Boc group present in dibenzosuberone 23 was cleaved

under acidic conditions to yield the corresponding amino

derivative 41 (Scheme 2).

a

Scheme 2. Synthesis of 41

Figure 2. Binding mode of Skepinone-L (2a) within the ATP binding

site of p38α MAP kinase (PDB code: 3QUE).

The aim of the present study was to identify selective and

metabolically stable Skepinone-based inhibitors of p38α MAP

kinase, which also have the ability to cross the blood−brain

barrier (BBB) in vivo in mouse models. To this end, we

employed amino acid transporters and passive diﬀusion

mechanisms. The ether function at position C-7 of the

a

Reagents and conditions: (i) 2,2,2-triﬂuoroacetic acid (TFA),

dichloromethane (DCM), room temperature (rt).

B

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a

Carboxylic acids 42−44 were obtained by treatment of the

corresponding esters 31, 33, and 34 with methanolic

potassium hydroxide solution at reﬂux temperature (Scheme

3).

Scheme 6. Synthesis of 69

a

Scheme 3. Synthesis of 42−44

a

Reagents and conditions: (i) KOH, MeOH, reﬂux temperature.

a

Scheme 7. Synthesis of 70

a

Reagents and conditions: (i) KOH, MeOH, reﬂux temperature.

Dibenzosuberones 54−72 having an o-phenylenediamine

moiety at position C-8 were prepared using an analogous

synthetic strategy, as in the case of compounds 6−51

(Schemes 4−7 and Scheme S7 (SI)). Buchwald−Hartwig

a

Reagents and conditions: (i) KOH, MeOH, reﬂux temperature.

Dibenzosuberone 76 bearing an N-methyl carboxamide

moiety at position C-3 was synthesized using a slightly

diﬀerent synthetic strategy (Scheme 8). The aniline moiety

Scheme 4. Synthesis of Dibenzosuberones 54−67

a

(Skepinone-N Series)

Scheme 8. Synthesis of Dibenzosuberone 76 (Skepinone-N

a

Series)

a

Reagents and conditions: (i) o-phenylenediamine, Cs₂CO₃, XPhos-

Pd second generation, 1,4-dioxane/t-BuOH (5:1, v/v), 60 °C. (ii)

KOH, MeOH, reﬂux temperature. (iii) CDI, DMF, 50 °C. For the

detailed structures of compounds 54−67 and 71−72, see Tables 3

and 4 and Table S1, SI.

a

Reagents and conditions: (i) 2-nitroaniline, Cs₂CO₃, XPhos,

Pd(OAc)₂, 1,4-dioxane/t-BuOH (5:1, v/v), 110 °C. (ii) KOH,

MeOH, reﬂux temperature. (iii) Methylamine (2 N THF), CDI,

DMF, 50 °C. (iv) SnCl₂, EtOH, reﬂux temperature.

coupling of ester 3 with o-phenylenediamine yielded diaryl-

amine 52, which was subsequently transferred into carboxylic

acid 53 (Scheme 4). The amide moiety at position C-3 of the

dibenzosuberone core was installed using CDI as a coupling

reagent. The modiﬁcation of dibenzosuberone 67 into

compounds 71 and 72 is shown in Scheme S7 (SI).

Dibenzosuberone 68 was obtained in excellent yield by

treatment of Boc-protected derivative 58 with TFA at ambient

temperature (Scheme 5).

targeting HR I was installed via the Buchwald−Hartwig

coupling reaction of aryl chloride 3 with 2-nitroaniline. After

the subsequent hydrolysis of ester 73, the methyl amide moiety

was introduced by treatment of carboxylic acid 74 with

methylamine in the presence of CDI as a coupling reagent.

Finally, compound 76 was obtained by reduction of the nitro

group present in compound 75 with tin(II) chloride in

ethanol.

Biological Evaluation. Kinase Activity Assay. The

dibenzosuberones 6−37, 41−51, 54−66, 68−72, and 76

were evaluated for their ability to inhibit p38α MAP kinase.³¹

The nature of the amide was essential for the inhibitory

activity. Secondary amide 7 (IC₅₀= 21 nM) showed an almost

20 times lower IC₅₀value compared with tertiary amide 8

(Table 1). Similarly, the tertiary amides 6 (IC₅₀= 95 nM) and

24 (IC₅₀= 120 nM) were less potent than the corresponding

secondary N-methylamide (IC₅₀= 3 nM).³⁰For hydrophobic

moieties, the inhibitory activity decreased with chain lengths

over two methylene groups: Compounds 7, 16, and 25 had

lower IC₅₀values than compounds 14, 21, and 22. The

introduction of a ﬂuorine atom at the para position of the

benzene ring of inhibitor 7 resulted in an equipotent

compound 9. The introduction of a hydroxyl group in this

position (compound 10) or the bioisosteric replacement of the

benzene ring by a thiophene ring (compound 12) resulted in a

a

Scheme 5. Synthesis of 68

a

Reagents and conditions: (i) TFA, DCM, rt.

Dicarboxylic acid 69 and carboxylic acid 70 were

synthesized from the corresponding diester 64 and ester 65,

respectively, using similar conditions as described for

compounds 42−44 of the Skepinone-L series (Schemes 6

and 7).

C

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Table 1. Biological Activities of 3-Substituted 2-((2,4-

Diﬂuorophenyl)amino)-5-dibenzosuberones 6−30 and 41

(Skepinone-L Series) (n = 3)

Table 2. Biological Activities of 3-Substituted 2-((2,4-

Diﬂuorophenyl)amino)-5-dibenzosuberones 31−37 and

42−51 (Ester Prodrugs and Corresponding Acids of

Skepinone-L Series) (n = 3)

As observed in the case of Skepinone-L (2a) and Skepinone-

N (2b) (Figure 1), most dibenzosuberone derivatives of our

study bearing an o-phenylenediamine substituent at position

C-8 (Skepinone-N series) were less active than their

corresponding Skepinone-L counterparts (compare 63 vs 18,

65 vs 33, 57 vs 11, 64 vs 36, 69 vs 46, 68 vs 41, 66 vs 37, 71 vs

48, 72 vs 51, Tables 1−4). In the case of inhibitors 55, 58, and

70, the Skepinone-N analog was, however, equal to or more

potent than the corresponding Skepinone-L derivatives 7, 23,

and 43, respectively.

In the series presented in Table 3, the secondary amides 58,

63, and 76 having an N-Boc-protected piperazinoethyl, a 2-

aminoethyl, or a methyl moiety at the carboxamide group,

respectively, have an equal or a better inhibitory activity than

primary amide 62. Hydroxyethyl-substituted dibenzosuberone

59 as well as diols 60 and 61 have similar inhibitory activities

in the low double-digit nanomolar range. Cleavage of the Boc-

group present in compound 58 resulted in a nine-fold loss of

inhibitory activity (compound 68).

Esters 64 and 71 are approximately 1.5 times more active

than their corresponding carboxylic acids 69 and 72,

respectively (Table 4). In the case of ester 65, however, the

corresponding acid 70 displays a 30 times lower IC₅₀value.

Dibenzosuberone 70 represents the most potent p38α MAP

kinase inhibitor of our study.

Metabolism. Inhibitors 11, 13, 15, 19, 26, 31−33, 41−44,

46, 49, 50, 54, 56, 59, 66, 70, and 76 were further evaluated

for their metabolic stability in human liver microsomes

(HLMs) (Tables 5 and 6, Figures S1−S21, SI).

The ester prodrugs were converted into the free carboxylic

acids. Simple esters such as 31 and 33 were cleaved within a

three times lower IC₅₀value. Compound 15 bearing a

spirocyclic morpholine isostere was also a potent inhibitor

(IC₅₀= 5.9 nM). Compound 17 having a branched moiety was

intermediate in potencythe racemic mixture 17 (IC₅₀= 22

nM) was more potent than the (R)-enantiomer (IC₅₀89

nM),³⁰suggesting that the (S)-enantiomer is worthy of

puriﬁcation. Compounds 18 and 20 (IC₅₀= 2.5 and 3.5 nM,

respectively) bearing an 2-aminoethyl and a 2,2,2-triﬂuoroethyl

moiety at the dibenzosuberone-C3-carboxamide function,

respectively, were likewise potent.

The hydroxamic acid derivative 26 (IC₅₀= 5.2 nM) and

compounds having a (poly-)alcohol (27 (IC₅₀= 3.0 nM), 29

(IC₅₀= 10 nM), 30 (IC₅₀= 1.7 nM)) or a glucosamine moiety

(28 (IC₅₀= 5.2 nM)) were also potent p38α MAP kinase

inhibitors (Table 1).

The introduction of amino acid derivatives (31−37 and 42−

51) provided consistently potent inhibitors (Table 2).

Assuming that interaction with transporters for central nervous

system (CNS) uptake required the physiological (S)-

conﬁguration, all amino acid derivatives were assayed in this

form. The ester prodrugs were, in most cases, slightly more

inhibitory than their corresponding carboxylic acids (compare

33 vs 43, 34 vs 44, 35 vs 45, 36 vs 46, 37 vs 47, 48 vs 51);

however, in the case of ester 31 and carboxylic acid 42, the

reverse was apparent. Ester 33 (IC₅₀= 1.2 nM) was the most

potent inhibitor of this series.

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Table 3. Biological Activities of 3-Substituted 2-((2-

Aminophenyl)amino)-5-dibenzosuberones 54−63, 68, and

76 (Skepinone-N Series) (n = 3)

Table 5. Metabolic Stability of Selected Dibenzosuberone

Inhibitors after 120 min of Incubation with HLMs (%

Parent Remaining)

Table 4. Biological Activities of 3-Substituted 2-((2-

Aminophenyl)amino)-5-dibenzosuberones 64−66 and 69−

72 (Ester Prodrugs and Corresponding Acids of Skepinone-

N Series) (n = 3)

few minutes. Their resulting acids 42 and 43, respectively, were

metabolically stable. Carboxylic acids 44 and 70 also displayed

excellent metabolic stability. Sterically hindered esters with

terminal amino acids such as 49 and 50 were cleaved more

slowly. Compound 32, whose additional methyl group may

shield the ester function from enzymatic hydrolysis, was

degraded by <10% within 2 h. Compound 66 bearing a

methionine methyl ester substituent formed the sulfoxide in

addition to the carboxylic acid.

In both series, the aromatic moiety used to ﬁll HR I and the

dibenzosuberone core remained stable, whereas the moiety

targeting HR II was labile.

Dibenzosuberone analog 13 bearing a tryptamine at the C-3

amide function underwent a slow oxidative biotransformation

(Table 6). After an incubation time of 120 min, 71% of 13 was

present. In contrast, compound 11 having a histamine moiety

in the same position was stable to HLMs (5% degradation after

120 min).

none-L series) had a half life of 113 min and was more stable

than 56, but more varied metabolites were formed during the

incubation.

The aliphatic dimethylethylamino moiety present in

compound 54 was transformed by HLMs to the N-

demethylated and a hydroxylated metabolite. Compound 19

was stable (94% remaining). Hydroxamic acid 26 and N-

methylamide 76 were rapidly metabolized in HLMs (in vitro

half life of 22 and 12 min, respectively) to the corresponding

acids: 5 (m/z 380.3 [M + H⁺]) and 53 (m/z 359.3 [M + H⁺]),

respectively.

Pharmacokinetic and CNS Penetration Study in RjOrl:S-

wiss CD-1 Mice. To evaluate brain penetration and plasma

stability, compounds 15, 16, 28, 30, 31, 33−36, 41, 48, 49, 54,

56, and 57, were tested in adult male RjOrl:Swiss CD-1 mice.

The mice were treated with a single intravenous (i.v.) injection

(10 mg/kg), and plasma samples were collected 10, 30, and

120 min after the treatment. After the last sample was

Morpholino derivative 56 (Skepinone-N series) had an in

vitro half life in HLMs of <1 h. After an incubation time of 2 h,

<38% of 56 remained. The supposedly metabolically more

stable 2-oxa-6-azaspiro[3.3]heptan-6-yl derivative 15 (Skepi-

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Table 6. Metabolic Stability of Selected Dibenzosuberone

Inhibitors after 120 min of Incubation with HLMs (%

Parent Remaining)

Table 7. Brain-to-Plasma Ratio of i.v. Administered

Prodrugs (Esters 31, 33−36, 48, and 49) and Their

Corresponding Acids (42−46, 51, and S2) after 2 h

prodrug

31

33

34

35

36

48

49

a

brain-to-plasma

NA

active

b

metabolite

42

43

1.25

44

4.38

45

0.31

46

NA

51

0.01

S2

brain-to-plasma 0.13

0.003

a

b

NA, not applicable. For the structure of S2, see the SI.

As observed in the in vitro metabolism study of 49 (Figure

S14, SI), the esters of α-amino acids (compounds 48 and 49)

were not fully converted into their corresponding free α-amino

acids 51 and S2 after 2 h. Only traces of both acids were

detected in the brain (Table S6, SI).

Concentrations of 68 and 49 ng/g of compounds 15 and 16

(Table S3, SI), respectively, were found in the brain after 2 h;

this corresponds, in the case of 16, to a brain-to-plasma ratio of

0.51 (Table 8). The spirocyclic moiety present in 15 as well as

the lipophilic moiety of 16 are unlikely to be recognized by

speciﬁc transporters. Therefore, these inhibitors are expected

to cross the BBB only via passive diﬀusion.

Dibenzosuberones 28, 30, and 41 showed a fast degradation

or elimination (Tables S3 and S4, SI). Inhibitor 28 bearing a

glucosamine residue may be transported by the glucose

transporter. However, only 5.9 ng/g of 28 was detected after

2 h, which correlates to a low brain-to-plasma ratio of 0.09.

Brain-to-plasma ratios of 0.17 and 0.24 were observed for

compounds 30 and 41, respectively (Table 8).

Within the Skepinone-N series, only low amounts of

morpholino-containing inhibitor 56 were detected in the

brain. Compound 54, having a tertiary amine in the amide

chain, showed a four times higher concentration in the brain

accompanied by a higher brain-to-plasma ratio.

Dibenzosuberone 57 bearing the biogenic histamine

substituent was eliminated relatively quickly and is not able

to cross the BBB (Table S4, SI).

In Vitro and in Vivo Study in Balb/c and C57BL/6 Mice.

Eﬀects in disease models can be inﬂuenced by partition to

speciﬁc cell types and degrees of inhibition in these cells (i.e.,

due to acid trapping in myeloid cells vs lymphoid cells). To

assess eﬀects in diﬀerent cells, selected compounds (18, 25, 27,

29, 33, 34, 43, 44, 50, 65, 70, 76) were further evaluated in in

vitro and in vivo studies.

obtained, the mice were sacriﬁced for sampling of the brains.

Blood and brain samples were analyzed via liquid chromatog-

raphy mass spectrometry/mass spectrometry (LC-MS/MS)

after preparation. (See the SI.)

First, the selected inhibitors were characterized in

splenocytes isolated from female Balb/c mice. In total, three

diﬀerent assays were done in mouse splenocytes stimulated

with 500 ng/mL lipopolysaccharide (LPS), and the release of

pro-inﬂammatory cytokines IL-6 and TNF-α was quantiﬁed.

The metabolic activity in these cells was measured via (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

reduction to formazan to control for cytotoxic eﬀects.

There was a trend for the tested compounds to increase the

metabolic activity of splenocytes at a concentration of 100 nM

(66−176% relative to LPS-stimulated splenocytes) (Figure

S23, SI). At concentrations of 1 and 10 nM, there was no eﬀect

on the metabolic activity of splenocytes. No cytotoxicity was

observed, whereas the anti-inﬂammatory activity of all

investigated compounds was dose-dependent, with reductions

in both IL-6 and TNF-α (Figure S24, SI). Inhibitor 70

increased IL-6 at concentrations of 1 and 10 nM (138 and

118%, respectively), then decreased it to 29% at 100 nM

Consistent with the HLM study, esters 31 and 33

underwent a very fast metabolism and are no longer detectable

10 min after i.v. administration (Table S5, SI). Their active

metabolites (corresponding acids 42 and 43, respectively)

were detected after 2 h at a concentration of 8.2 and 102.9 ng/

g, which corresponds to a brain-to-plasma ratio of 0.13 and

1.25, respectively (Table 7 and Table S5, SI). A similar level of

the carboxy product 44 was detected in the brain (98.2 ng/g),

which corresponds to a brain-to-plasma ratio of 4.4. Because

passive transport of the charged compounds 43 and 44 seems

unlikely under physiological conditions, these inhibitors may

have crossed the BBB via transporter systems.

Compared with 31 and 34, esters 35 and 36 were

hydrolyzed more slowly to their corresponding acids 45 and

46. Only low levels of acid 45 (15.0 ng/g) were detected in the

brain (Table S5, SI); however, glutamic acid derivative 46 did

not penetrate into the brain (Table 7 and Table S6, SI).

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Table 8. Brain-to-Plasma Ratio of i.v. Administered Inhibitors 15, 16, 28, 30, 41, 54, 56, and 57 after 2 h

inhibitor

15

16

28

30

41

54

56

57

brain-to-plasma

0.16

0.51

0.09

0.17

0.24

0.59

0.08

0.00

relative to PBS-treated LPS-stimulated splenocytes. Com-

pound 25 caused moderate inhibition of LPS-stimulated IL-6

release (51% at 100 nM). Compound 50 caused the highest

reductions in IL-6 (Figure S24a, SI). Compounds 18, 25, 27,

29, 43, 50, 70, and 76 were potent inhibitors of the LPS-

stimulated TNF-α release (Figure S24b, SI). Compounds 18,

27 and 76 were most potent, whereas compounds 33, 34, 44,

and 65 were only moderate to poor inhibitors.

Heparinized whole blood was prepared from C57BL/6 mice

(MWB) stimulated with 500 ng/mL LPS. As for mouse

splenocytes, all tested compounds reduced the release of IL-6

and TNF-α dose-dependently (Figure S25, SI). Compounds

27, 29, and 50 caused the highest reductions in IL-6 release

(Figure S25a, SI), whereas compounds 33, 34, 44, and 65 were

moderate (85, 64, 71, and 81%, respectively). Dibenzosuber-

ones 18, 25, 27, 29, 43, 50, 70, and 76 were potent inhibitors

of LPS-stimulated TNF-α from MWB (range: 5−23% at 1 nM

in comparison with LPS-stimulated MWB). Compounds 27,

43, and 76 (Figure S25b, SI) were most potent in this assay.

Compounds 33, 34, 44, and 65 had lower activity in this assay

(∼50% inhibition at 10 nM). Consistent with mouse

splenocytes, compound 33 was the least active of the series

in MWB.

(Table S21, SI) and the highest levels in all organs after 4 h

(heart ∼120 nM, liver ∼330 nM, kidney ∼380 nM, and brain

∼540 nM). When used in the context of tissue, “nM” is

shorthand for nmol/kg tissue fresh weight and is used in this

way to allow direct comparison to values for inhibition of the

enzyme.

Human Whole Blood Assay. The human whole blood

(HWB) assay is a means to evaluate the eﬀectiveness of p38α

MAP kinase inhibitors in modulating pro-inﬂammatory

cytokine secretion in a primary cell system. In this ex vivo

assay, the eﬃcacy is estimated with in vivo relevant parameters

like plasma protein binding and cellular permeability. Thus the

IC₅₀values are typically higher than those for the direct

enzyme inhibition assay. Ester 34 is a potent inhibitor of the

LPS-stimulated TNF-α release displaying an IC₅₀value of 64

nM (Table S23, SI). Its active metabolite, the corresponding

acid 44, shows a two-fold decrease in inhibitory activity. The

release of LPS-stimulated TNF-α from HWB was inhibited by

compound 15 at concentrations in the double-digit nanomolar

range.

CYP and hERG Assay. The p38α MAP kinase inhibitors 15,

33, 34, 43, 44, and 70 were tested for the inhibition of hERG

and relevant CYP isoforms (Table 9). At a test concentration

Cellular compound uptake using human buﬀy coat (HBC)

was also evaluated. Cells (1 × 10⁶HBC cells/mL) were treated

with compounds at a ﬁnal concentration of 100 μM, and cell

culture supernatants were collected at diﬀerent time points and

analyzed for compound concentration (Tables S24 and S25,

SI).

The eﬀects of compounds on cytokine release were also

assessed in LPS-stimulated HBC cells in the same format as

mouse splenocytes and whole blood. All compounds had dose-

dependent eﬀects, with 50 being most active (Figure S26, SI)

and 33 being the least active, suggesting broadly similar data in

the human and murine systems.

Because most transgenic animal models of neurodegener-

ative disorders are on a C57BL/6 background, this strain was

used for further pharmacokinetic studies including penetration

of the BBB of a second set of inhibitors (18, 25, 27, 29, 50, 70,

and 76) to augment data from CD1 mice. Blood samples were

taken at 5, 15, 30, 60, 120, and 240 min. Animals were

terminated for brain sampling at 120 and 240 min post

substance application. The substance was quantiﬁed in

peripheral plasma, heart-derived plasma, liver, kidney, brain

right hemisphere, and brain left hemisphere by LC-MS/MS.

Compounds were administered (0.4 mg/kg) in mixtures to

reduce the number of animals used. (See the SI for details.)

The rationale for the lower dose was to model the likely

concentrations following oral application and thus to simulate

the likely conditions for transport systems in long-term

neurodegenerative models in C57BL/6.

The compounds from the Skepinone-L series (18, 25, 27,

29, 47, 50) showed no or only low brain uptake after i.v. or

p.o. application (Tables S9−S13 and S16−S20, SI). Com-

pound 76 was BBB-penetrant after i.v. and p.o. application

(∼20 nM in brain at 2 h), exceeding the IC₅₀value for free

p38α MAP kinase (Tables S15 and S22, SI). Compound 70

(∼590 nM in brain, 2 h p.o.) had a brain-to-plasma ratio of 4.7

Table 9. In vitro CYP and hERG Inhibition Data of

Inhibitors 15, 33, 43, 34, 44, and 70

% inhibition @ 10 μM

Cpd hERG CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4

15

33

43

34

44

70

76.6

59.9

11.8

49.7

14.4

22.5

0

88.7

99.4

68.4

98.5

66.0

13.1

93.6

100

60.4

100

63.2

21.8

18.3

73.7

3.0

36.6

2.0

83.6

68.0

34.9

54.8

38.1

4.1

27.3

11.6

33.6

16.2

28.1

11.4

a

n = 2.

of 10 μM, both esters 33 and 34 display a 59.9 and 49.7%

inhibition of hERG and inhibit the tested CYP isoforms 2C9

and 2C19 by more than 98%. At the same concentration, their

active metabolites 43 and 44 that are formed within a few

minutes show no interaction with hERG and a reduced CYP

inhibition. No aﬃnity toward CYP2D6 (a signiﬁcant CNS

CYP) was observed in the case of 43 and 44. Compound 70,

the Skepinone-N counterpart of 44, had no or a limited eﬀect

on CYP and hERG activity. In contrast, compound 15 had

both a higher inhibition of hERG (76.6%) and an elevated

CYP inhibition proﬁle for isoforms CYP2C9, CYP2C19,

CYP2D6, and CYP3A4 compared with carboxylic acids 43,

44, and 70. No inhibition of CYP1A2 was observed for 15.

Kinome Selectivity. Compounds 31, 42−44, and 57 were

tested at 1 and 5 μM against a panel of 320 kinases to evaluate

their selectivity (Tables S26 and S27, SI).

Ester 31 is a highly selective p38α MAP kinase inhibitor. At

both concentrations, only the closely related kinases p38β

MAP kinase, JNK2, and JNK3³²are inhibited by more than

60% (Table S26, SI), resulting in an S₆₀selectivity score of

0.013. At 1 μM, the active metabolite of 31, carboxylic acid 42,

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Figure 3. Selectivity proﬁle of carboxylic acids 43 (left) and 70 (right). Tested kinases are highlighted with circles and colored according to their

inhibition at 1 μM inhibitor concentration. This ﬁgure was generated with the help of the KinMap.³³

had similar selectivity, interacting only with p38β MAP kinase,

JNK3, and ACV-R2B (Table S26, SI). At a higher

concentration (5 μM), CAMK2G, EGF-R, EIF2AK2, JNK2,

and MYLK were additionally inhibited by more than 60% by

42. Inhibitor 43, having an additional methylene unit between

the carboxylic group and the amide function compared with

42, was similar to 42 (Table S26, SI). At 1 μM, the main target

kinase p38α MAP kinase and the MAP kinase family members

p38β MAP kinase, JNK2, and JNK3 as well as the tyrosine

kinase-like kinase ACV-R2B were inhibited by 43 by more

than 60% (Figure 3). At 5 μM (900 times its IC₅₀), 43

inhibited the tyrosine kinases EGF-R, VEGF-R2, and MYLC as

well as the serine/threonine kinase JNK1. Carboxylic acid 44

interacted with p38β MAP kinase, JNK3, and TLK2) at 1 μM

(Table S27, SI) but inhibited 14 kinases by more than 60% at

5 μM with an S₆₀selectivity score of 0.044. Compound 57 was

similar to 31 with an S₆₀selectivity score of 0.013 at both

concentrations (Table S27, SI).

Inhibitor 70 was tested at concentrations of 1 and 5 μM

against a panel of 65 kinases consisting of all kinases that were

inhibited in the previous screening by more than 40% by

compounds 31, 42−44, and 57 and all members of the p38

MAP kinase family (Figure 3 and Table S28). Besides the

target kinase p38α MAP kinase, only the closely related p38β

MAP kinase was inhibited by more than 60% by 70 at test

concentrations of 1 and 5 μM.

and 34 were rapidly converted into their corresponding

carboxylic acids 43 and 44, respectively. The active metabolites

43 (p38α, IC₅₀= 6 nM) and 44 (p38α, IC₅₀= 12 nM) crossed

the BBB (concentration in brain after 2 h, ∼100 ng/g, ∼220

nM) and displayed promising brain-to-plasma ratios of 1.4 and

4.4, respectively. Skepinone-L-derived inhibitors 43 and 44

were selective and metabolically stable and had low hERG

aﬃnity. Compound 70 (p38α, IC₅₀= 1 nM), the Skepinone-N

counterpart of 43, had the highest concentrations in the brain

2 h after p.o. application (580 nM) (brain-to-plasma ratio of

4.7).

Given its very good general attributes (selectivity, CYP, and

hERG interaction), this very high partition to the CNS makes

compound 70 a promising candidate for the study of the role

of p38α MAP kinase in the development of CNS pathology in

murine models of CNS degeneration. The combination of a

high aﬃnity for p38α MAP kinase and very high central

exposure has not been obtained in either Neﬂamapimod

(p38α, IC₅₀= 10 nM) or MW150 (p38α, IC₅₀= 230 nM).

Reported brain exposure for Neﬂamapimod in the rat was

approximately 130 to 360 nmol/kg at eﬀective doses (1.5 to

4.5 mg/kg), corresponding to a 13- to 36-fold IC₅₀value for

p38α MAP kinase. The data for MW150 were not directly

reported (pharmacokinetics at 5 or 6 mg/kg in rat) but, as

calculated in the Introduction, vary between two and three

times the IC₅₀value for p38α MAP kinase at an eﬀective dose

of 2.5 mg/kg in the APP/PS1 mouse.

CONCLUSIONS

■

62 novel Skepinone-based p38α MAP kinase inhibitors with

IC₅₀values down to the low single-digit nanomolar range were

characterized. Various substituents (lipophilic and basic

moieties, (amino) acid derivatives, biogenic amines, amino

alcohols, and carbohydrates) at position C-3 of the

dibenzosuberone core remained inhibitory. Ester prodrugs 33

Compound 70 (1 nM) reached 580 times the IC₅₀value for

p38α MAP kinase at 0.4 mg/kg in the C57BL/6 mouse brain,

suggesting that it and its near analogs have adequate potency

and physical properties to eﬀectively modulate p38α MAP

kinase activity in the brain.

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1

solid. H NMR (200 MHz, DMSO-d₆) 13.02 (br. s, 1H), 8.60 (s,

1H), 8.43 (d, J = 1.6 Hz, 1H), 7.95−8.03 (m, 2H), 7.30−7.49 (m,

3H), 7.04−7.16 (m, 1H), 6.75 (d, J = 7.5 Hz, 1H), 6.62 (s, 1H),

3.03−3.18 (m, 4H). Analytical data are in agreement with the

literature.³⁰

EXPERIMENTAL PART

■

General. All reagents and solvents were commercially available

and were used without further puriﬁcation. ¹H and ¹³C NMR spectra

were obtained with a Bruker Avance 200 or Bruker Avance 400

apparatus. The spectra were obtained in the indicated solvent and

calibrated against the residual proton peak of the deuterated solvent.

Chemical shifts (δ) are reported in parts per million. Mass spectra

were performed on an Advion Expression S electrospray ionization

mass spectrometer (ESI-MS) with an Advion Plate Express (TLC

interface) apparatus. TLC analysis was performed on ﬂuorescent silica

gel 60 F254 plates (Merck) and visualized under UV illumination at

254 and 366 nm. Column chromatography was performed using an

Interchim PuriFlash 430 automated ﬂash chromatography system.

The purity of all compounds was, unless otherwise stated, >95% and

was determined via reverse-phase high-performance liquid chroma-

tography on a Hewlett-Packard HP 1090 series II LC equipped with a

UV diode array detector (DAD, detection at 230 and 254 nm). The

chromatographic separation was performed on a Phenomenex Luna

5u C8 column (150 mm × 4.6 mm, 5 μm) at a 35 °C oven

temperature. The injection volume was 5 μL, and the gradient of the

used method was as follows (ﬂow, 1.5 mL/min), with 0.01 M

KH₂PO₄, pH 2.3 (solvent A) and methanol (solvent B): from 40 to

85% B in 8 min, 85% B for 5 min, from 85 to 40% B in 1 min, 40% B

for 2 min, stop time 16 min.

8-((2,4-Diﬂuorophenyl)amino)-N,N-dimethyl-5-oxo-10,11-

dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (6).

Compound 6 was prepared according to General Procedure B using

5 (0.050 g, 0.13 mmol) and dimethylamine hydrochloride (0.012 g,

0.14 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

1:0 to 9:1). Yield: 0.041 g (77%), yellow solid. MS (ESI_neg) (m/z):

404.8 [M − H]⁻. IR (ATR) [cm⁻¹] 3247, 2921, 2852, 1621, 1589,

1

1500, 1392, 1358, 1257, 1212, 1138, 965, 866, 601. H NMR (400

MHz, DMSO-d₆) δ 8.61 (s, 1H), 8.00−7.97 (m, 1H), 7.86 (s, 1H),

7.51−7.50 (m, 1H), 7.48−7.34 (m, 3H), 7.13−7.09 (m, 1H), 6.75 (d,

J = 8.7 Hz, 1H), 6.63 (s, 1H), 3.12−3.04 (m, 4H), 2.98−2.93 (m,

6H).

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-phenethyl-10,11-di-

hydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (7). Com-

pound 7 was prepared according to General Procedure B using 5

(0.075 g, 0.20 mmol) and 2-phenylethylamine (0.026 g, 0.22 mmol).

Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1).

Yield: 0.043 g (45%), yellow solid. MS (ESI_neg) (m/z): 481.9 [M −

H]⁻. IR (ATR) [cm⁻¹] 3283, 2923, 1632, 1338, 1314, 1258, 1141,

965, 851, 599. ¹H NMR (400 MHz, DMSO-d₆) δ 8.69−8.64 (m, 1H),

8.58 (s, 1H), 8.33 (s, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.90 (d, J = 7.3

Hz, 1H), 7.41−7.36 (m, 2H), 7.29−7.18 (m, 6H), 7.15−7.06 (m,

2H), 6.76 (d, J = 8.4 Hz, 1H), 6.63 (s, 1H), 3.51−3.48 (m, 2H),

3.14−3.02 (m, 4H), 2.89−2.85 (m, 2H).

General Procedures. General Procedure A. The carboxylic ester

was dissolved in methanol, and KOH (2.5 equiv) was added. The

reaction mixture was heated to reﬂux temperature until completion of

the reaction. After cooling to rt, the solvent was removed under

reduced pressure, and the residue was taken up in ethyl acetate. The

organic phase was washed with a diluted HCl solution and dried over

Na₂SO₄, and the solvent was evaporated under reduced pressure,

yielding a solid product. If the purity of the product was <95%, then

the product was puriﬁed by ﬂash chromatography (SiO₂, DCM/

MeOH/formic acid 94:4:2).

General Procedure B. The carboxylic acid 5, 53, or 75 was

dissolved in 5 mL of DMF under an argon atmosphere with gentle

heating (50 °C). CDI (2 equiv) was added, and the reaction mixture

was stirred for 2 h. The corresponding amine or alcohol was added to

the reaction mixture, and it was stirred for 1 h at 50 °C. Then, the

reaction was stopped by adding H₂O (10 mL) and extracted with

either ethyl acetate or DCM (three times). The combined organic

phases were dried over Na₂SO₄, ﬁltered, and evaporated under

reduced pressure.

General Procedure C. The Boc-protected compound was dissolved

in dry DCM (5 mL), and TFA (1.5 mL) was added. The reaction

mixture was stirred at rt until completion; then, a saturated aq.

NaHCO₃solution was carefully added. The reaction was extracted

with DCM (three times). The organic extracts were collected, and the

solvent was removed in vacuo. The product was puriﬁed by ﬂash

chromatography.

Methyl 8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carboxylate (4). 2,4-Diﬂuor-

oaniline (0.23 g, 1.79 mmol) was dissolved in 12 mL of 1,4-dioxane/t-

BuOH (5:1, v/v) under an argon atmosphere. XPhos (0.025 equiv),

Cs₂CO₃(1.5 equiv), and Pd(OAc)₂(0.05 equiv) followed by aryl

chloride 3 (0.50 g, 1.79 mmol) were added, and the reaction mixture

was heated for 1 h to 110 °C. The reaction was cooled to rt and

ﬁltered. The residue was washed several times with DCM, MeOH,

and EtOAc. The combined organic extracts were evaporated, and the

brown-black residue was puriﬁed by ﬂash chromatography (SiO₂,

petroleum ether/ethyl acetate 4:1) to yield 4 as a yellow solid (0.52 g,

89%).¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (d, J = 1.8 Hz, 1H),

8.16 (d, J = 8.7 Hz, 1H), 8.06 (dd, J = 1.9 Hz, 8.0 Hz, 1H), 7.41−7.25

(m, 2H), 6.81−6.99 (m, 3H), 6.66 (d, J = 2.4 Hz, 1H), 6.00 (s, 1H),

3.92 (s, 3H), 3.23−3.06 (m, 4H). Analytical data are in agreement

with the literature.³⁰

8-((2,4-Diﬂuorophenyl)amino)-N-methyl-5-oxo-N-pheneth-

yl-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(8). Compound 8 was prepared according to General Procedure B

using 5 (0.040 g, 0.11 mmol) and N-methyl-2-phenylethylamine

(0.029 g, 0.21 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.029 g (55%), yellow solid. MS

(ESI_neg) (m/z): 495.1 [M − H]⁻. IR (ATR) [cm⁻¹] 1602, 1579,

1

1500, 1397, 1354, 1258, 1214, 1139, 964, 842, 698. H NMR (400

MHz, DMSO-d₆) δ 8.60 (s, 1H), 8.00−7.97 (m, 1H), 7.43−7.36 (m,

3H), 7.27 (d, J = 22.0 Hz, 3H), 7.17−7.07 (m, 4H), 6.98−6.96 (m,

1H), 6.77−6.75 (m, 1H), 6.63 (s, 1H), 3.68−3.64 (m, 2H), 3.09−

3.02 (m, 7H), 2.91−2.88 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆)

δ 190.6, 170.5, 159.0 (dd, J = 11.2, 241.9 Hz), 156.2 (dd, J = 12.5,

246.7 Hz), 149.8, 145.7, 143.3, 139.9, 139.1, 135.3, 133.9, 129.2,

129.1, 128.8, 128.7, 127.7, 126.7, 126.6, 125.3 (dd, J = 3.3, 13.3 Hz),

114.0, 112.8, 112.3 (dd, J = 3.4, 21.8 Hz), 105.2 (dd, J = 26.0, 27.0

Hz), 52.7, 51.8, 48.9, 35.9, 34.3.

8-((2,4-Diﬂuorophenyl)amino)-N-(4-ﬂuorophenethyl)-5-

oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (9). Compound 9 was prepared according to General

Procedure B using 5 (0.075 g, 0.20 mmol) and 2-(4-ﬂuorophenyl)-

ethylamine (0.138 g, 0.99 mmol). Puriﬁcation: ﬂash chromatography

(SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.037 g (39%), yellow solid.

MS (ESI_neg) (m/z): 499.8 [M − H]⁻. IR (ATR) [cm⁻¹] 3280, 2923,

1

2853, 1602, 1580, 1257, 1139, 1094, 964, 846, 783, 518. H NMR

(400 MHz, DMSO-d₆) δ 8.69−8.66 (m, 1H), 8.60 (s, 1H), 8.31 (s,

1H), 8.00 7.97 (m, 1H), 7.89 (d, J = 7.3 Hz, 1H), 7.43−7.30 (m, 4H),

7.13−7.00 (m, 4H), 6.76 (d, J = 8.3 Hz, 1H), 6.62 (s, 1H), 3.53−3.48

(m, 2H), 3.13−3.03 (m, 4H), 2.89−2.86 (m, 2H).

8-((2,4-Diﬂuorophenyl)amino)-N-(4-hydroxyphenethyl)-5-

oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (10). Compound 10 was prepared according to General

Procedure B using 5 (0.050 g, 0.13 mmol) and tyramine

hydrochloride (0.069 g, 0.40 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.018 g (28%), yellow

solid. MS (ESI_neg) (m/z): 497.5 [M − H]⁻. IR (ATR) [cm⁻¹] 3292,

1

2922, 2852, 1601, 1505, 1360, 1260, 1097, 967, 843, 540. H NMR

(400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.66−8.62 (m, 1H), 8.60 (s,

1H), 8.32 (s, 1H), 8.01−7.98 (m, 1H), 7.90−7.88 (m, 1H), 7.44−

7.33 (m, 3H), 7.12−7.10 (m, 1H), 7.02 (d, J = 7.7 Hz, 2H), 6.76 (d, J

= 8.4 Hz, 1H), 6.69−6.66 (m, 2H), 6.62 (s, 1H), 3.53−3.48 (m, 2H,

overlain by water peak), 3.12−3.03 (m, 4H), 2.74−2.70 (m, 2H).

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxylic Acid (5). Compound 5

was prepared according to General Procedure A starting from

carboxylic ester 4 (1.00 g, 2.54 mmol). Yield: 0.96 g (99%), yellow

I

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N-(2-(1H-Imidazol-4-yl)ethyl)-8-((2,4-diﬂuorophenyl)-

amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-

carboxamide (11). Compound 11 was prepared according to

General Procedure B using 5 (0.050 g, 0.13 mmol) and histamine

dihydrochloride (0.049 g, 0.26 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.018 g (29%), yellow

solid. MS (ESI_neg) (m/z): 471.5 [M − H]⁻. IR (ATR) [cm⁻¹] 2921,

158.8 (dd, J = 11.5, 244.8 Hz), 155.3 (dd, J = 12.8, 248.3 Hz), 148.1,

145.3, 145.1, 138.9, 134.2, 133.0, 131.4, 129.4, 129.3, 128.6, 124.8

(dd, 3.9, 11.9 Hz), 123.9 (dd, J = 2.5, 9.3 Hz), 114.6, 113.3, 111.4

(dd, J = 3.7, 22.1 Hz), 104.8 (dd, J = 23.6, 26.2 Hz), 81.2 (2× C),

63.7 (2× C), 57.9, 39.2, 37.7, 35.8, 34.7.

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-(1-phenylcyclo-

propyl)-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carbox-

amide (16). Compound 16 was prepared according to General

Procedure B using 5 (0.030 g, 0.08 mmol) and 1-phenyl-1-

cyclopropylamine (0.017 g, 0.10 mmol). Puriﬁcation: ﬂash

chromatography (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.017 g

(44%), yellow solid. MS (ESI_neg) (m/z): 493.0 [M − H]⁻. IR (ATR)

[cm⁻¹] 3268, 2922, 2850, 1632, 1603, 1581, 1505, 1353, 1257, 1139,

964, 844, 598. ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 8.11 (d, J

= 8.6 Hz, 1H), 7.98 (d, J = 7.4 Hz, 1H), 7.36−7.22 (m, 6H + solvent

peak), 7.16−7.14 (m, 2H), 6.93−6.87 (m, 2H), 6.79 (d, J = 8.5 Hz,

1H), 6.63 (s, 1H), 6.00 (s, 1H), 3.15−3.13 (m, 2H), 3.07−3.04 (m,

2H), 1.37−1.30 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 191.4,

166.6, 158.8 (dd, J = 10.1, 244.5 Hz), 155.4 (dd, J = 12.2, 246.3 Hz),

148.2, 145.4, 145.3, 142.3, 138.8, 134.2, 132.9, 131.6, 129.6, 129.2,

128.4 (2× C), 128.3, 126.4, 125.7 (2× C), 124.8 (dd, J = 3.8, 11.8

Hz), 124.0 (dd, J = 2.3 Hz, 9.4 Hz), 114.6, 113.3, 111.4 (dd, J = 3.9,

22.1 Hz), 104.8 (dd, J = 23.5, 26.1 Hz), 35.8, 35.4, 34.7, 17.8 (2× C).

N-(1-Cyclohexylethyl)-8-((2,4-diﬂuorophenyl)amino)-5-oxo-

10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(17). Compound 17 was prepared according to General Procedure B

using 5 (0.030 g, 0.08 mmol) and 1-cyclohexylethylamine (0.012 g,

0.08 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

1:0 to 9:1). Yield: 0.012 g (31%), yellow solid. MS (ESI_neg) (m/z):

487.1 [M − H]⁻. IR (ATR) [cm⁻¹] 3268, 2922, 2850, 2361, 1633,

1

2851, 1603, 1579, 1505, 1354, 1258, 1094, 964, 801, 621. H NMR

(400 MHz, DMSO-d₆) δ 8.69−8.66 (m, 1H), 8.60 (s, 1H), 8.32 (s,

1H), 8.00−7.98 (m, 1H), 7.91−7.89 (m, 1H), 7.57 (s, 5H), 7.43−

7.34 (m, 4H), 7.13−7.09 (m, 1H), 6.83 (s, 1H), 6.76 (d, J = 8.7 Hz,

1H), 6.62 (s, 1H), 3.49−3.46 (m, 2H, overlain by water peak), 3.12−

3.03 (m, 4H), 2.78−2.74 (m, 2H).

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-(2-(thiophen-2-yl)-

ethyl)-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (12). Compound 12 was prepared according to General

Procedure B using 5 (0.030 g, 0.08 mmol) and 2-(thiophen-2-

)ethylamine (0.015 g, 0.12 mmol). Puriﬁcation: ﬂash chromatography

(SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.021 g (53%), yellow solid.

MS (ESI_neg) (m/z): 486.9 [M − H]⁻. IR (ATR) [cm⁻¹] 1602, 1580,

1

1504, 1354, 1258, 1139, 964, 845, 696, 594. H NMR (400 MHz,

DMSO-d₆) δ 8.77−8.74 (m, 1H), 8.60 (s, 1H), 8.34 (d, J = 2.0 Hz,

1H), 8.00 (d, J = 8.8 Hz, 1H), 7.91 (dd, J = 7.8, 2.0 Hz, 1H), 7.42−

7.32 (m, 4H), 7.13−7.09 (m, 1H), 6.96−6.91 (m, 2H), 6.77−6.74

(m, 1H), 6.62 (d, J = 1.4 Hz, 1H), 3.53−3.48 (m, 2H), 3.13−3.05 (m,

6H).

N-(2-(1H-Indol-3-yl)ethyl)-8-((2,4-diﬂuorophenyl)amino)-5-

oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (13). Compound 13 was prepared according to General

Procedure B using 5 (0.050 g, 0.13 mmol) and tryptamine

hydrochloride (0.106 g, 0.66 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.053 g (81%), yellow

solid. MS (ESI_neg) (m/z): 520.7 [M − H]⁻. IR (ATR) [cm⁻¹] 2916,

1

1603, 1581, 1505, 1257, 1139, 1094, 964, 598. H NMR (400 MHz,

CDCl₃) δ 8.24 (s, 1H), 8.11 (d, J = 8.2 Hz, 1H), 7.94 (d, J = 6.6 Hz,

1H), 7.32−7.24 (m, 2H), 6.92−6.79 (m, 3H), 6.62 (s, 1H), 6.13 (d, J

= 8.1 Hz, 1H), 6.07 (s, 1H), 4.07−3.99 (m, 1H), 3.14−3.12 (m, 2H),

3.06−3.04 (m, 2H), 1.77−1.62 (m, 3H), 1.21−1.10 (m, 8H), 1.05−

0.96 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.5, 165.9, 160.0

(dd, J = 10.9, 244.7 Hz), 155.4 (dd, J = 12.3, 246.5 Hz), 148.2, 145.4,

144.9, 138.9, 134.2, 133.5, 131.5, 129.4, 129.1, 128.0, 124.9 (dd, J =

3.9, 11.5 Hz), 124.0 (dd, J = 2.3 Hz, 9.4 Hz), 114.6, 113.2, 111.5 (dd,

J = 3.7, 22.1 Hz), 104.8 (dd, J = 23.7, 26.0 Hz), 50.0, 43.3, 38.6, 35.9,

34.7, 29.3, 29.2, 26.4, 26.2, 18.0.

N-(2-Aminoethyl)-8-((2,4-diﬂuorophenyl)amino)-5-oxo-

10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(18). Compound 18 was prepared according to General Procedure B

using 5 (0.040 g, 0.11 mmol) and ethylene-1,2-diamine (0.015 g, 0.26

mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0

to 9:1). Yield: 0.027 g (61%), yellow solid. MS (ESI_neg) (m/z): 420.0

[M − H]⁻. IR (ATR) [cm⁻¹] 1602, 1577, 1504, 1354, 1258, 1214,

1114, 1094, 964, 844, 516. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s,

1H), 8.56−8.53 (m, 1H), 8.34−8.33 (m, 1H), 8.00 (d, J = 8.8 Hz,

1H), 7.92 (dd, J = 1.5, 7.8 Hz, 1H), 7.45−7.34 (m, 3H), 7.13−7.09

(m, 1H), 6.76 (d, J = 8.5 Hz, 1H), 6.63 (s, 1H), 3.38−3.16 (m, 2H +

2H, overlain by water), 3.12−3.03 (m, 4H), 2.69 (t, J = 6.2 Hz, 2H).

¹³C NMR (100 MHz, DMSO-d₆) δ 190.5, 165.7, 149.3, 145.2, 144.5,

1

2850, 1633, 1567, 1486, 1259, 1092, 964, 741, 461. H NMR (400

MHz, DMSO-d₆) δ 10.81 (s, 1H), 8.75−8.72 (m, 1H), 8.60 (s, 1H),

8.35 (s, 1H), 8.01−7.91 (m, 2H), 7.58 (d, J = 7.7 Hz, 1H), 7.46−7.31

(m, 4H), 7.17 (s, 1H), 7.13−7.03 (m, 2H), 7.00−6.96 (m, 1H), 6.76

(d, J = 8.5 Hz, 1H), 6.63 (s, 1H), 3.57−3.52 (m, 2H), 3.14−3.03 (m,

4H), 2.97−2.93 (m, 2H).

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-(3-phenylpropyl)-

10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(14). Compound 14 was prepared according to General Procedure B

using 5 (0.075 g, 0.20 mmol) and 3-phenylpropylamine (0.134 g, 0.99

mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0

to 9:1). Yield: 0.023 g (23%), yellow solid. MS (ESI_neg) (m/z): 495.9

[M − H]⁻. IR (ATR) [cm⁻¹] 3315, 1581, 1603, 1527, 1504, 1262,

1141, 964, 854, 700, 631. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (s,

2H), 8.34 (s, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H),

7.45−7.35 (m, 3H), 7.30−7.21 (m, 4H), 7.19−7.15 (m, 1H), 7.13−

7.09 (m, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.62 (s, 1H), 3.30−3.26 (m,

2H), 3.13−3.03 (m, 4H), 2.65−2.60 (m, 2H), 1.83 (quin, J = 7.1 Hz,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.5, 165.5, 149.3, 145.2,

144.5, 141.7, 138.9, 133.3, 133.0, 130.4, 129.1, 128.8, 128.3 (2× C),

128.2 (2× C), 127.2, 126.2 (dd, J = 2.5, 9.8 Hz), 125.7, 124.8 (dd, J =

2.9, 12.2 Hz), 113.5, 112.3, 111.8 (dd, J = 3.0, 21.8 Hz), 104.9 (dd, J

= 24.0, 26.2 Hz), 38.9, 35.4, 33.7, 32.6, 30.8, (C2′-F and C4′-F not

visible).

N-(2-(2-Oxa-6-azaspiro[3.3]heptan-6-yl)ethyl)-8-((2,4-

diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d]-

[7]annulene-3-carboxamide (15). Compound 15 was prepared

according to General Procedure B using 5 (0.060 g, 0.16 mmol) and

2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)ethylamine (0.034 g, 0.24

mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0

to 9:1). Yield: 0.063 g (79%), yellow solid. MS (ESI_neg) (m/z): 502.1

[M − H]⁻. IR (ATR) [cm⁻¹] 3291, 2924, 2854, 1634, 1603, 1580,

1505, 1355, 1259, 1093, 963, 761, 520. ¹H NMR (400 MHz, CDCl₃)

δ 8.31 (d, J = 1.3 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 7.97 (dd, J = 7.8,

1.5 Hz, 1H), 7.39−7.32 (m, 1H), 7.29 (d, J = 7.9 Hz, 1H), 6.96−6.83

(m, 3H), 6.77−6.74 (m, 1H), 6.66−6.65 (m, 1H), 5.96 (s, 1H), 4.74

(s, 4H), 3.43−3.39 (m, 6H), 3.19−3.16 (m, 2H), 3.11−3.09 (m, 2H),

2.62 (t, J = 5.6 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 191.5, 166.7,

138.8, 133.3, 132.9, 130.4, 129.2, 128.8, 127.2, 126.2 (dd, J = 2.6, 9.2

Hz), 124.8 (dd, J = 2.6, 11.5 Hz), 113.5, 112.3, 111.8 (dd, J = 3.7,

21.9 Hz), 104.9 (dd, J = 24.0, 26.2 Hz), 42.8, 41.1, 35.4, 33.8, (C2′-F

and C4′-F not visible).

N-(3-Amino-3-oxopropyl)-8-((2,4-diﬂuorophenyl)amino)-5-

oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (19). Compound 19 was prepared according to General

Procedure B using 5 (0.040 g, 0.11 mmol) and 3-aminopropanamide

(0.035 g, 0.28 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.042 g (82%), yellow solid.

C₂₅H₂₁F₂N₃O₃(M = 449.16 g/mol). MS (ESI_neg) (m/z): 448.2 [M −

H]⁻. IR (ATR) [cm⁻¹] 1654, 1602, 1544, 1504, 1361, 1258, 1113,

968, 847, 569, 470. ¹H NMR (400 MHz, DMSO-d₆) δ 8.64−8.60 (m,

2H), 8.32 (s, 2H), 7.99 (d, J = 8.8 Hz, 1H), 7.90 (dd, J = 1.4, 7.7 Hz,

1H), 7.45−7.35 (m, 4H), 7.13−7.09 (t, J = 8.2 Hz, 1H), 6.84 (s, 1H),

6.76 (d, J = 8.7 Hz, 1H), 6.62 (s, 1H), 3.50−3.45 (m, 2H, overlain by

water), 3.13−3.10 (m, 2H), 3.05−3.03 (m, 2H), 2.36 (t, J = 7.1 Hz,

J

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.5, 172.6, 165.5, 149.3,

145.2, 144.6, 138.8, 133.3, 132.8, 130.3, 129.1, 128.9, 127.2, 126.2

(dd, J = 2.9, 10.0 Hz), 124.8 (dd, J = 2.5, 11.8 Hz), 113.5, 112.3,

111.8 (dd, J = 3.6, 22.1 Hz), 104.9 (dd, J = 24.1, 26.8 Hz), 36.0, 35.4,

34.9, 33.8 (C2′-F and C4′-F not visible).

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-(2,2,2-triﬂuoroeth-

yl)-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (20). Compound 20 was prepared according to General

Procedure B using 5 (0.075 g, 0.20 mmol) and 2,2,2-triﬂuoroethyl-

amine (0.120 g, 0.99 mmol). Puriﬁcation: ﬂash chromatography

(SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.057 g (63%), yellow solid.

MS (ESI_neg) (m/z): 459.8 [M − H]⁻. IR (ATR) [cm⁻¹] 1668, 1604,

1539, 1355, 1243, 1151, 964, 833, 729. ¹H NMR (400 MHz, DMSO-

d₆) δ 9.22−9.20 (m, 1H), 8.60 (s, 1H), 8.39 (s, 1H), 8.01−7.96 (m,

2H), 7.46−7.34 (m, 3H), 7.12−7.08 (m, 1H), 6.78 (s, 1H), 6.63 (s,

1H), 4.14−4.04 (m, 2H), 3.15−3.12 (m, 2H), 3.06−3.04 (m, 2H).

¹³C NMR (100 MHz, DMSO-d₆) δ 190.6, 166.3, 159.8 (dd, J = 11.1,

2-((2,4-Diﬂuorophenyl)amino)-7-(piperidine-1-carbonyl)-

10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one (24). Com-

pound 24 was prepared according to General Procedure B using 5

(0.011 g, 0.03 mmol) and piperidine (0.018 g, 0.07 mmol).

Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to

9:1). Yield: 0.007 g (40%), yellow solid. MS (ESI_neg) (m/z): 445.1

[M − H]⁻. IR (ATR) [cm⁻¹] 2922, 2852, 2359, 2341, 1602, 1579,

1506, 1437, 1353, 1255, 1093, 799. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.60 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.82 (s, 1H), 7.47−7.35 (m,

4H), 7.12−7.09 (m, 1H), 6.75 (d, J = 8.5 Hz, 1H), 6.62 (s, 1H),

3.73−3.55 (m, 4H), 3.11−3.06 (m, 4H), 1.60−1.45 (m, 6H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 190.1, 168.3, 149.3, 145.3, 142.9,

138.6, 134.6, 133.5, 130.1, 129.0, 128.5, 127.1, 126.2 (J = 2.6, 9.8 Hz),

124.8 (dd, J = 3.3, 12.0 Hz), 113.5, 112.2, 111.8 (dd, J = 3.8, 21.7

Hz), 104.9 (dd, J = 24.2, 27.4 Hz), 48.0 (2× C), 35.4, 33.2, 25.9 (2×

C), 24.0, (C2′-F and C4′-F not visible).

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-phenyl-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carboxamide (25). Com-

pound 25 was prepared according to General Procedure B using 5

(0.043 g, 0.11 mmol) and aniline (0.026 g, 0.28 mmol). Puriﬁcation:

ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.050 g

243.6 Hz), 154.5 (dd, J = 12.6, 248.6 Hz), 149.4, 145.4, 145.3, 139.0,

133.4, 131.5, 130.6, 129.4, 129.1, 127.1, 126.3 (dd) 124.9 (dd), 123.4,

113.5, 112.3, 111.8 (dd, J = 3.6, 22.1 Hz), 104.9 (dd J = 24.4, 26.3

Hz), 40.2 (q, J = 33.7 Hz), 35.3, 33.8.

1

(97%), yellow solid. MS (ESI_neg) (m/z): 453.0 [M − H]⁻. H NMR

8-((2,4-Diﬂuorophenyl)amino)-N-octyl-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carboxamide (21). Com-

pound 21 was prepared according to General Procedure B using 5

(0.075 g, 0.20 mmol) and octylamine (0.240 g, 1.98 mmol).

Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to

9:1). Yield: 0.024 g (25%), yellow solid. MS (ESI_neg) (m/z): 489.9

[M − H]⁻. IR (ATR) [cm⁻¹] 3274, 2923, 2853, 1603, 1505, 1257,

1139, 1094, 964, 845, 597. ¹H NMR (400 MHz, DMSO-d₆) δ 8.58−

8.54 (m, 2H), 8.33 (s, 1H), 8.01−7.90 (m, 2H), 7.42−7. 32 (m, 3H),

7.11−7.07 (m, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.63 (s, 1H), 3.27−3.21

(m, 2H), 3.12−3.02 (m, 4H), 1.50 (br. s, 2H), 1.25−1.23 (s, 10H),

0.83 (br. s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.4, 165.4,

158.4 (dd, J = 11.3, 240.9 Hz), 155.7 (dd, J = 12.6, 246.6 Hz), 149.2,

145.2, 144.4, 138.9, 133.3, 130.3, 133.0, 129.1, 128.8, 127.2, 126.2

(dd, J = 2.7, 9.6 Hz), 124.8 (dd, J = 3.2, 12.1 Hz), 113.5, 112.3, 111.8

(dd, J = 3.4, 21.7 Hz), 105.0 (dd, J = 24.0, 25.9 Hz), 39.2, 35.4, 33.8,

31.1, 29.0, 28.7, 28.6, 26.4, 22.0, 13.8.

8-((2,4-Diﬂuorophenyl)amino)-N-hexadecyl-5-oxo-10,11-di-

hydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (22). Com-

pound 22 was prepared according to General Procedure B using 5

(0.075 g, 0.20 mmol) and hexadecylamine (0.955 g, 3.95 mmol).

Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1).

Yield: 0.055 g (46%), yellow solid. MS (ESI_neg) (m/z): 602.1 [M −

H]⁻. IR (ATR) [cm⁻¹] 3345, 2919, 2847, 1631, 1604, 1495, 1358,

1265, 963, 858, 624. ¹H NMR (400 MHz, CDCl₃) δ 8.30 (br. s, 1H),

8.16 (d, J = 8.8 Hz, 1H), 8.01−7.97 (m, 1H), 7.40−7.30 (m, 2H),

6.98−6.85 (m, 3H), 6.69−6.68 (m, 1H), 6.30 (br. s, 1H), 5.81 (br. s,

1H), 3.48−3.43 (m, 2H), 3.21−3.18 (m, 2H), 3.13−3.11 (m, 2H),

1.65−1.58 (m, 2H), 1.37−1.26 (m, 26H), 0.88 (d, J = 6.9 Hz, 3H).

tert-Butyl 4-(2-(8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-

dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)ethyl)-

piperazine-1-carboxylate (23). Compound 23 was prepared

according to General Procedure B using 5 (0.125 g, 0.33 mmol)

and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (0.378 g, 1.65

mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0

to 9:1). Yield: 0.094 g (49%), yellow solid. MS (ESI_neg) (m/z): 598.8

[M − H]⁻. IR (ATR) [cm⁻¹] 1634, 1581, 1505, 1354, 1259, 1165,

1002, 964, 845, 517. ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (s, 1H),

8.54 (s, 1H), 8.32 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H), 7.90 (d, J = 7.7

Hz, 1H), 7.49−7.31 (m, 3H), 7.11 (t, J = 7.8 Hz, 1H), 6.76 (d, J = 8.7

Hz, 1H), 6.62 (s, 1H), 3.42−3.40 (m, 2H), 3.31−3.28 (s, 4H), 3.13−

3.10 (m, 2H), 3.05−3.03 (s, 2H), 2.50−2.48 (m, 2H), 2.40−2.37 (m,

4H), 1.39 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.4, 165.5,

153.8, 149.3, 145.2, 144.5, 138.9, 133.3, 132.8, 130.3, 129.1, 128.9,

127.2, 126.2 (J = 3.3, 9.6 Hz), 124.8 (dd, J = 3.8, 12.2 Hz), 113.5,

112.3, 111.8 (dd, J = 3.8, 22.0 Hz), 104.9 (dd, J = 24.3, 26.3 Hz),

78.7, 56.8, 52.4 (2× C), 36.7 (2× C), 35.4, 33.8, 28.0 (3× C), (C2′-F

and C4′-F not visible).

(400 MHz, DMSO-d₆) δ 10.35 (s, 1H), 8.62 (s, 1H), 8.44 (d, J = 1.6

Hz, 1H), 8.05−8.00 (m, 2H), 7.77 (d, J = 7.8 Hz, 2H), 7.49−7.33 (m,

5H), 7.14−7.09 (m, 2H), 6.77 (dd, J = 1.3, 8.8 Hz, 1H), 6.64 (s, 1H),

3.17−3.15 (m, 2H), 3.08−3.06 (m, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 190.4, 164.8, 149.4, 145.3, 145.1, 139.1, 138.9, 133.4,

133.2, 130.8, 129.6, 129.0, 128.6 (2× C), 127.1, 126.3 (dd, J = 2.6,

10.0 Hz), 124.8 (dd, J = 3.3, 12.4 Hz), 123.7, 120.4 (2× C), 113.5,

112.3, 111.9 (dd, J = 3.7, 21.8 Hz), 105.0 (dd, J = 24.5, 26.3 Hz),

35.4, 33.8, (C2′-F and C4′-F not visible).

8-((2,4-Diﬂuorophenyl)amino)-N-hydroxy-5-oxo-10,11-di-

hydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (26). Com-

pound 26 was prepared according to General Procedure B using 5

(0.040 g, 0.11 mmol) and hydroxylamine hydrochloride (0.009 g,

0.13 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

1:0 to 9:1). Yield: 0.034 g (77%), yellow solid. MS (ESI_neg) (m/z):

392.5 [M − H]⁻. IR (ATR) [cm⁻¹] 2920, 2851, 1602, 1505, 1354,

1

1258, 1093, 1028, 839, 596, 451. H NMR (400 MHz, DMSO-d₆) δ

11.30 (s, 1H), 9.07 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 7.99 (d, J = 8.7

Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.45−7.35 (m, 3H), 7.13−7.09 (m,

1H), 6.76 (d, J = 8.4 Hz, 1H), 6.62 (s, 1H), 3.12−3.02 (m, 4H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 190.7, 163.6, 160.4, 158.5 (dd, J =

11.0, 244.2 Hz), 155.7 (dd, J = 11.7, 248.0 Hz), 149.8, 145.2, 144.6,

138.9, 133.4, 131.4, 129.9, 129.0, 127.1, 126.1, 124.7, 113.5, 112.3,

111.8 (dd, J = 3.3, 21.9 Hz), 104.9 (dd, J = 22.1, 26.5 Hz), 35.3, 33.8.

8-((2,4-Diﬂuorophenyl)amino)-N-(1,3-dihydroxy-2-

(hydroxymethyl)propan-2-yl)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxamide (27). Compound 27

was prepared according to General Procedure B using 5 (0.080 g, 0.21

mmol) and tris(hydroxymethyl)aminomethane hydrochloride (0.083

g, 0.53 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/

MeOH 1:0 to 9:1). Yield: 0.023 g (23%), yellow solid. MS (ESI_neg

)

(m/z): 480.9 [M − H]⁻. IR (ATR) [cm⁻¹] 3300, 1602, 1580, 1505,

1403, 1354, 1258, 1214, 1042, 846, 595. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.60 (s, 1H), 8.27 (d, J = 1.5 Hz, 1H), 8.00 (d, J = 8.8

Hz, 1H), 7.88 (dd, J = 7.8, 1.6 Hz, 1H), 7.45−7.35 (m, 4H), 7.13−

7.08 (m, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.62 (s, 1H), 4.77 (t, J = 5.7

Hz, 3H), 3.70 (d, J = 5.7 Hz, 6H), 3.13−3.11 (m, 2H), 3.05−3.03 (m,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.4, 166.6, 158.5 (dd, J =

11.5, 241.8 Hz), 155.7 (dd, J = 12.2, 246.4 Hz), 149.3, 145.3, 144.6,

138.8, 133.5, 133.4, 130.6, 129.2, 128.7, 127.1, 126.2 (dd, J = 3.2, 9.6

Hz), 124.8 (dd, J = 3.1, 12.2 Hz), 113.5, 112.3, 111.8 (dd, J = 3.3,

21.9 Hz), 104.9 (dd, J = 24.3, 26.3 Hz), 62.7, 60.5, 35.4, 33.8.

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-((2R,3R,4R,5S,6R)-

2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-

yl)-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (28). Compound 28 was prepared according to General

Procedure B using 5 (0.060 g, 0.16 mmol) and ^D-glucosamine

hydrochloride (0.038 g, 0.17 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.048 g (56%), yellow

K

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

solid. MS (ESI_neg) (m/z): 538.7 [M − H]⁻. IR (ATR) [cm⁻¹] 3300,

DCM/MeOH 1:0 to 9:1). Yield: 0.023 g (38%), yellow solid. MS

(ESI_neg) (m/z): 463.6 [M − H]⁻. IR (ATR) [cm⁻¹] 3304, 2921,

2852, 1738, 1634, 1603, 1505, 1454, 1354, 1172, 1212, 1140, 845. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.94 (d, J = 6.7 Hz, 1H), 8.61 (s, 1H),

8.39 (s, 1H), 8.02 − −8.00 (m, 1H), 7.97−7.94 (m, 1H), 7.43−7.35

(m, 3H), 7.13−7.09 (m, 1H), 6.77 (d, J = 8.7 Hz, 1H), 6.63 (s, 1H),

4.53−4.46 (m, 1H), 3.65 (s, 3H), 3.14−3.12 (m, 2H), 3.06−3.04 (m,

2H), 1.41 (d, J = 7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

190.4, 173.1, 165.6, 149.3, 145.2, 144.9, 139.0, 133.3, 132.0, 130.6,

129.4, 128.9, 127.1, 126.2 (dd, J = 3.4, 9.4 Hz), 124.9 (dd, J = 3.2,

12.1 Hz), 113.5, 112.3, 111.8 (dd, J = 3.7, 22.1 Hz), 104.9 (dd, J =

24.7, 26.5 Hz), 51.8, 48.3, 35.4, 33.8, 16.7, (C2′-F and C4′-F not

visible).

1

1633, 1603, 1505, 1355, 1259, 1094, 1027, 965, 845, 516. H NMR

(400 MHz, DMSO-d₆) δ 8.59 (s,1H), 8.37−8.36 (m, 1H), 8.32 (d, J

= 8.8 Hz, 0.3H), 8.20 (d, J = 7.1 Hz, 0.7H), 8.01−7.93 (m, 2H),

7.45−7.34 (m, 3H), 7.13−7.08 (m, 1H), 6.76 (d, J = 8.8 Hz, 1H),

6.63 (s, 1H), 6.58 (d, J = 6.2 Hz, 0.3H), 6.48 (d, J = 4.2 Hz, 0.7H),

5.10−5.08 (s, 0.7H), 4.99−4.97 (m, 1H), 4.93 (d, J = 5.1 Hz, 0.3H),

4.72 (d, J = 4.6 Hz, 0.7H), 4.64−4.56 (m, 0.6H), 4.50−4.47 (m,

0.7H), 3.81−3.63 (m, 3H), 3.54−3.48 (m, 1H), 3.22−3.17 (m, 1H),

3.12−3.10 (m, 2H), 3.05−3.03 (m, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 190.6, 165.9, 158.5 (dd, J = 11.3, 241.9 Hz), 155.7 (dd, J

= 12.6, 246.8 Hz), 149.3, 145.2, 144.5, 138.9, 133.3, 132.8, 130.7,

129.4, 128.7, 127.2, 126.2 (dd, J = 3.0, 9.7 Hz), 124.9 (dd, J = 3.6,

12.1 Hz), 113.6, 112.3, 111.8 (dd, J = 3.3, 21.9 Hz), 104.9 (dd, J =

24.1, 26.2 Hz), 90.5, 72.1, 71.1, 70.0, 61.2, 55.5, 35.5, 33.8.

8-((2,4-Diﬂuorophenyl)amino)-N-(2-(2-hydroxyethoxy)-

ethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-

carboxamide (29). Compound 29 was prepared according to

General Procedure B using 5 (0.040 g, 0.11 mmol) and 2-(2-

aminoethoxy)ethanol (0.027 g, 0.26 mmol). Puriﬁcation: ﬂash

chromatography (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.017 g

(34%), yellow solid. MS (ESI_neg) (m/z): 464.9 [M − H]⁻. IR (ATR)

[cm⁻¹] 2950, 1651, 1601, 1578, 1506, 1348, 1261, 1211, 1133, 1095,

966, 844, 784. ¹H NMR (400 MHz, DMSO-d₆) δ 8.64−8.60 (m, 2H),

8.34 (d, J = 1.5 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.92 (dd, J = 7.9,

1.6 Hz, 1H), 7.45−7.34 (m, 3H), 7.13−7.09 (m, 1H), 6.76 (d, J = 1.3,

8.9 Hz, 1H), 6.63 (s, 1H), 4.63 (t, J = 5.3 Hz, 1H), 3.55−3.52 (m,

2H), 3.50−3.48 (m, 2H), 3.45−3.41 (m, 4H), 3.12−3.10 (m, 2H),

3.05−3.03 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.4, 165.7,

158.5 (dd, J = 11.3, 241.8 Hz), 156.7 (dd, J = 12.6, 245.4 Hz), 149.3,

145.3, 144.6, 138.9, 133.3, 132.7, 130.4, 129.2, 128.9, 127.1, 126.2

(dd, J = 3.0, 9.8 Hz), 124.8 (dd, J = 3.5, 12.2 Hz), 113.5, 112.3, 111.8

(dd, J = 3.4, 21.9 Hz), 104.9 (dd, J = 24.5, 26.6 Hz), 72.1, 68.8, 60.2,

38.2, 35.4, 33.8.

Methyl 3-(8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoate

(33). Compound 33 was prepared according to General Procedure B

using 5 (0.060 g, 0.16 mmol) and methyl 3-aminopropanoate

hydrochloride (0.024 g, 0.17 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.075 g (95%), yellow

solid. MS (ESI_neg) (m/z): 462.8 [M − H]⁻. IR (ATR) [cm⁻¹] 1717,

1

1602, 1581, 1525, 1434, 1244, 1116, 915, 842, 718, 508. H NMR

(400 MHz, DMSO-d₆) δ 8.69−8.66 (s, 1H), 8.60 (s, 1H), 8.31 (s,

1H), 8.01−7.98 (m, 1H), 7.91−7.89 (m, 1H), 7.45−7.35 (m, 3H),

7.13−7.08 (m, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.62 (s, 1H), 3.61−3.60

(m, 3H), 3.51−3.46 (m, 2H), 3.12−3.10 (m, 2H), 3.05−3.03 (m,

2H), 2.62−2.55 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.4,

171.7, 165.6, 149.3, 145.2, 144.7, 138.8, 133.3, 132.6, 130.4, 129.1,

128.9, 127.1, 126.2 (dd, J = 3.0, 9.5 Hz), 124.8 (dd, J = 3.7, 12.0 Hz),

113.5, 112.3, 111.8 (dd, J = 3.6, 22.1 Hz), 104.9 (dd, J = 24.2, 26.7

Hz), 51.3, 35.5, 35.4, 33.8, 33.5, (C2′-F and C4′-F not visible).

Methyl 4-(8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoate

(34). Compound 34 was prepared according to General Procedure B

using 5 (0.040 g, 0.11 mmol) and methyl 4-aminobutanoate

hydrochloride (0.027 g, 0.17 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.070 g (86%), yellow

solid. MS (ESI_neg) (m/z): 476.8 [M − H]⁻. IR (ATR) [cm⁻¹] 3290,

8-((2,4-Diﬂuorophenyl)amino)-N-(2-((2-hydroxyethyl)-

amino)ethyl)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]-

annulene-3-carboxamide (30). Compound 30 was prepared

according to General Procedure B using 5 (0.040 g, 0.11 mmol)

and 2-((2-aminoethyl)amino)ethanol (0.027 g, 0.26 mmol). Puriﬁca-

tion: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1). Yield:

0.025 g (51%), yellow solid. MS (ESI_neg) (m/z): 463.9 [M − H]⁻. IR

(ATR) [cm⁻¹] 1601, 1557, 1524, 1439, 1354, 1257, 1140, 848, 754,

687. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 8.57 (t, J = 5.3

Hz, 1H), 8.34 (d, J = 1.3 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.92 (dd,

J = 7.8, 1.7 Hz, 1H), 7.45−7.34 (m, 3H), 7.13−7.08 (m, 1H), 6.76

(dd, J = 8.8, 1.4 Hz, 1H), 6.63 (s, 1H), 4.55 (br. s, 1H), 3.46 (t, J =

5.7 Hz, 2H), 3.39−3.35 (m, 2H, overlain by water), 3.12−3.10 (m,

2H), 3.05−3.03 (m, 2H), 2.73 (t, J = 6.4 Hz, 2H), 2.64 (t, J = 5.7 Hz,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.5, 165.6, 158.5 (dd, J =

11.1, 241.9 Hz), 155.7 (dd, J = 12.5, 246.5 Hz), 149.3, 145.2, 144.5,

138.9, 133.3, 132.9, 130.4, 129.2, 128.8, 127.2, 126.2 (dd, J = 2.9, 9.6

Hz) 124.8 (dd, J = 3.6, 12.1 Hz), 113.5, 112.3, 111.8 (dd, J = 3.4, 22.1

Hz), 104.9 (dd, J = 24.4, 26.5 Hz), 60.1, 51.2, 48.3, 35.4, 33.8, 30.6.

Methyl (8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carbonyl)glycinate (31).

Compound 31 was prepared according to General Procedure B

using 5 (0.060 g, 0.16 mmol) and glycine methyl ester hydrochloride

(0.024 g, 0.19 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.041 g (58%), yellow solid. MS

(ESI_neg) (m/z): 448.8 [M − H]⁻. IR (ATR) [cm⁻¹] 3383, 3278,

1

1728, 1601, 1505, 1437, 1357, 1261, 1097, 967, 850, 528. H NMR

(400 MHz, CDCl₃) δ 8.31 (d, J = 1.2 Hz, 1H), 8.10 (d, J = 8.7 Hz,

1H), 7.94−7.92 (m, 1H), 7.36−7.31 (m, 1H), 7.27−7.25 (m, 1H +

solvent peak), 6.93−6.76 (m, 4H), 6.64 (s, 1H), 6.11 (br. s, 1H), 3.66

(s, 3H), 3.51−3.46 (m, 2H), 3.15−3.13 (m, 2H), 3.07−3.05 (m, 2H),

2.42 (t, J = 7.1 Hz, 2H), 1.95 (quin, J = 6.9 Hz, 2H). ¹³C NMR (100

MHz, CDCl₃) δ 191.3, 173.9, 166.8, 158.8 (dd, J = 10.7, 244.5 Hz),

155.4 (dd, J = 11.5, 246.2 Hz), 148.1, 145.2, 145.1, 138.9, 134.2,

133.0, 131.2, 129.4, 129.2, 128.5, 124.9 (dd, J = 3.1, 11.4 Hz), 124.0

(dd, J = 2.3, 9.4 Hz), 114.6, 113.3, 111.d (dd, J = 3.7, 22.0 Hz), 104.8

(dd, J = 23.7, 26.2 Hz), 51.7, 39.6, 35.8, 34.7, 31.6, 24.6.

Methyl (8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carbonyl)-^L-phenylalani-

nate (35). Compound 35 was prepared according to General

Procedure B using 5 (0.080 g, 0.21 mmol) and ^L-phenylalanine

methyl ester hydrochloride (0.068 g, 0.32 mmol). Puriﬁcation: ﬂash

chromatography (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.103 g

(90%), yellow solid. MS (ESI_neg) (m/z): 538.9 [M − H]⁻. IR (ATR)

[cm⁻¹] 2157, 2006, 1738, 1651, 1603, 1581, 1258, 1434, 1177, 1118,

1

1140, 699. H NMR (400 MHz, DMSO-d₆) δ 9.00 (d, J = 7.5 Hz,

1H), 8.61 (s, 1H), 8.33 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.88−7.86

(m, 1H), 7.45−7.34 (m, 3H), 7.30−7.17 (m, 5H), 7.13−7.09 (m,

1H), 6.76 (d, J = 8.7 Hz, 1H), 6.62 (s, 1H), 4.71−4.66 (m, 1H), 3.64

(s, 3H), 3.20−3.10 (m, 4H), 3.05−3.03 (m, 2H). ¹³C NMR (100

MHz, DMSO-d₆) δ 190.3, 172.1, 165.7, 158.5 (dd, J = 11.6, 243.5

Hz), 155.7 (dd, J = 12.6, 248.5 Hz), 149.3, 145.8, 145.0, 139.0, 137.6,

133.3, 131.9, 130.5, 129.3, 129.0 (2× C), 128.9, 128.2 (2× C), 127.1,

126.4, 126.2 (dd, J = 3.0, 9.6 Hz), 124.8 (dd, J = 3.1, 11.9 Hz), 113.5,

112.3, 111.8 (dd, J = 3.4, 22.1 Hz), 104.9 (dd, J = 24.4, 26.5 Hz),

54.2, 51.9, 36.2, 35.3, 34.7.

1

1758, 1650, 1606, 1575, 1258, 1195, 962, 760, 512. H NMR (400

MHz, CDCl₃) δ 8.32−8.30 (s, 1H), 8.05−8.03 (d, J = 7.5 Hz, 1H),

7.87−7.85 (m, 1H), 7.34−7.28 (m, 2H), 6.87−6.84 (m, 2H), 6.76−

6.74 (m, 1H), 6.57 (s, 1H), 4.11 (s, 2H), 3.70 (s, 3H), 3.20 (s, 1H),

3.10−3.04 (m, 4H).

Methyl (8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carbonyl)-^L-alaninate (32).

Compound 32 was prepared according to General Procedure B using

5 (0.050 g, 0.13 mmol) and ^L-alanine methyl ester hydrochloride

(0.092 g, 0.66 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

Dimethyl (8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carbonyl)-^L-glutamate

(36). Compound 36 was prepared according to General Procedure B

using 5 (0.080 g, 0.21 mmol) and dimethyl ^L-glutamate hydrochloride

L

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

(0.049 g, 0.23 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.110 g (97%), yellow solid. MS

(ESI_neg) (m/z): 534.7 [M − H]⁻. IR (ATR) [cm⁻¹] 1732, 1633,

3292, 1715, 1601, 1519, 1397, 1284, 1260, 1211, 1138, 964, 841. H

NMR (400 MHz, MeOD-d₄) δ 8.43 (d, J = 1.6 Hz, 1H), 8.07 (d, J =

8.8 Hz, 1H), 7.92 (dd, J = 7.8, 1.4 Hz, 1H), 7.42−7.36 (m, 2H),

7.10−7.04 (m, 1H), 7.00−6.95 (m, 1H), 6.80−6.77 (m, 1H), 6.65 (s,

1H), 4.10 (s, 2H), 3.18−3.16 (m, 2H), 3.10−3.08 (m, 2H). ¹³C NMR

(100 MHz, MeOD-d₄) δ 193.1, 173.2, 169.7, 160.7 (dd, J = 10.6,

243.6 Hz), 157.7 (dd, J = 12.0, 246.7 Hz), 151.4, 147.2, 147.1, 140.8,

134.9, 133.7, 131.8, 130.7, 130.3, 129.0, 127.1 (dd, J = 2.8, 9.4 Hz),

126.5 (dd, J = 3.6, 12.1 Hz), 115.0, 113.7, 112.4 (dd, J = 3.8, 22.2

Hz), 105.6 (dd, J = 24.2, 26.4 Hz), 42.4, 37.0, 35.6.

1

1603, 1505, 1435, 1354, 1258, 1211, 1094, 964, 846. H NMR (400

MHz, DMSO-d₆) δ 8.90 (d, J = 7.4 Hz, 1H), 8.61 (s, 1H), 8.37 (d, J =

1.8 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.96−7.93 (m, 1H), 7.47−7.36

(m, 3H), 7.13−7.09 (m, 1H), 6.76 (dd, J = 8.8, 1.6 Hz, 1H), 6.63 (d,

J = 1.6 Hz, 1H), 4.50−4.45 (m, 1H), 3.64 (s, 3H), 3.58 (s, 3H),

3.14−3.12 (m, 2H), 3.06−3.04 (m, 2H), 2.45 (t, J = 7.5 Hz, 2H),

2.15−2.08 (m, 1H), 2.06−1.97 (m, 1H). ¹³C NMR (100 MHz,

DMSO-d₆) 190.5, 172.7, 172.2, 166.0, 149.4, 145.3, 145.0, 139.0,

133.4, 131.2, 130.7, 129.4, 128.9, 127.1, 126.2 (dd, J = 3.0, 9.7 Hz),

113.6, 112.3, 111.8 (dd, J = 3.7, 22.0 Hz), 105.0 (dd, J = 24.6, 26.9

Hz), 52.0, 52.0, 51.3, 35.4, 33.8, 30.0, 25.7, (C¹′, C²′-F, and C⁴′-F not

visible).

Methyl (8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carbonyl)-^L-methioninate

(37). Compound 37 was prepared according to General Procedure B

using 5 (0.080 g, 0.21 mmol) and ^L-methionine methyl ester

hydrochloride (0.105 g, 0.53 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.043 g (39%), yellow

solid. MS (ESI_neg) (m/z): 523.1 [M − H]⁻. IR (ATR) [cm⁻¹] 2921,

2852, 1737, 1635, 1602, 1505, 1435, 1354, 1257, 1213, 1094, 845. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.91 (d, J = 7.4 Hz, 1H), 8.60 (s, 1H),

8.38 (s, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.44−

7.35 (m, 3H), 7.13−7.08 (m, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.63 (s,

1H), 4.62−4.57 (m, 1H), 3.65 (s, 3H), 3.14−3.12 (m, 2H), 3.06−

3.04 (m, 2H), 2.63−2.53 (m, 2H), 2.08−2.03 (m, 5H). ¹³C NMR

(100 MHz, DMSO-d₆) δ 190.9, 172.9, 166.5, 149.9, 145.8, 145.5,

139.5, 133.8, 132.5, 131.2, 129.9, 129.4, 127.6, 126.7 (dd, J = 2.9, 9.7

Hz), 125.3 (dd, J = 3.2, 11.2 Hz), 114.0, 112.8, 112.3 (dd, J = 3.3,

22.0 Hz), 105.4 (dd, J = 23.8, 26.4 Hz), 52.4, 52.2, 35.9, 34.3, 30.6,

30.4, 15.0, (C2′-F and C4′-F not visible).

3-(8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxamido)propanoic Acid (43).

Compound 43 was prepared according to General Procedure A

starting from carboxylic ester 33 (0.043 g, 0.09 mmol). Yield: 0.029 g

(68%), yellow solid. MS (ESI_neg) (m/z): 448.8 [M − H]⁻. IR (ATR)

[cm⁻¹] 1644, 1601, 1549, 1519, 1188, 1260, 968, 839, 804, 731, 604.

¹H NMR (400 MHz, DMSO-d₆) δ 12.24 (br. s, 1H), 8.67−8.64 (m,

1H), 8.60 (s, 1H), 8.32 (d, J = 1.7 Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H),

7.90 (dd, J = 7.9, 1.8 Hz, 1H), 7.54−7.34 (m, 3H), 7.13−7.08 (m,

1H), 6.76 (dd, J = 8.8, 1.4 Hz, 1H), 6.62 (s, 1H), 3.48−3.43 (m, 2H),

3.12−3.10 (m, 2H), 3.05−3.03 (m, 2H), 2.55−2.51 (m, 2H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 190.7, 173.1, 165.8, 158.7 (dd, J =

11.5, 241.8 Hz), 155.9 (dd. J = 12.2, 246.5 Hz), 149.6, 145.5, 144.9,

139.1, 133.6, 132.9, 130.6, 129.4, 129.1, 127.4, 126.5 (dd, J = 3.2, 9.7

Hz), 125.0 (dd, J = 3.4, 12.2 Hz), 113.8, 112.5, 112.1 (dd, J = 3.6,

21.9 Hz), 105.1 (dd, J = 23.9, 26.4 Hz), 35.8, 35.6, 34.0, 33.9.

4-(8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxamido)butanoic Acid (44).

Compound 44 was prepared according to General Procedure A

starting from carboxylic ester 34 (0.026 g, 0.05 mmol). Yield: 0.010 g

(41%), yellow solid. MS (ESI_neg) (m/z): 462.9 [M − H]⁻. IR (ATR)

[cm⁻¹] 3281, 1733, 1645, 1601, 1519, 1264, 1096, 969, 850, 571. ¹H

NMR (400 MHz, CDCl₃) δ 8.45 (s, 1H), 8.16 (d, J = 8.7 Hz, 1H),

8.05−8.03 (m, 1H), 7.39−7.29 (m, 3H), 6.96−6.88 (m, 2H), 6.83 (d,

J = 8.3 Hz, 1H), 6.65 (s, 1H), 5.97 (br. s, 1H), 3.56−3.51 (m, 2H),

3.17−3.15 (m, 2H), 3.10−3.08 (m, 2H), 2.50 (t, J = 6.7 Hz, 2H),

2.05−1.97 (m, 2H), (COOH not visible). ¹³C NMR (100 MHz,

CDCl₃) δ 192.1, 176.2, 166.9, 148.4, 145.8, 145.3, 138.5, 134.6, 132.8,

132.0, 129.5, 128.8, 128.7, 124.1 (dd, J = 2.2, 9.4 Hz), 114.5, 113.3,

111.5 (dd, J = 3.5, 22.3 Hz), 104.8 (dd, J = 23.8, 26.2 Hz), 39.6, 35.9,

34.7, 31.4, 24.6, (C¹′, C²′-F, and C⁴′-F not visible).

Methyl N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N6-(8-

((2,4-diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carbonyl)-^L-lysinate (38). Com-

pound 38 was prepared according to General Procedure B using 5

(0.10 g, 0.26 mmol) and N-α-Fmoc-^L-lysine methyl ester (S1) (0.221

g, 0.53 mmol). Product was used in the next step without further

puriﬁcation.

(S)-2-((tert-Butoxycarbonyl)amino)-3-(8-((2, 4-

diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d]-

[7]annulene-3-carboxamido)propanoic Acid (39). Compound

39 was prepared according to General Procedure B using 5 (0.066 g,

0.17 mmol) and (S)-3-amino-2-((tert-butoxycarbonyl)amino)-

propanoic acid (0.053 g, 0.26 mmol). Product was used in the next

step without further puriﬁcation.

(S)-2-((tert-Butoxycarbonyl)amino)-4-(8-((2, 4-

diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d]-

[7]annulene-3-carboxamido)butanoic Acid (40). Compound 40

was prepared according to General Procedure B using 5 (0.066 g, 0.17

mmol) and (S)-4-amino-2-((tert-butoxycarbonyl)amino)butanoic

acid (0.057 g, 0.26 mmol). Product was used in the next step

without further puriﬁcation.

8-((2,4-Diﬂuorophenyl)amino)-5-oxo-N-(2-(piperazin-1-yl)-

ethyl)-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (41). Compound 41 was prepared according to General

Procedure C starting from 23 (0.113 g, 0.19 mmol). Puriﬁcation: ﬂash

chromatography (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.089 g

(95%), yellow solid. MS (ESI_pos) (m/z): 491.9 [M + H]⁺. IR (ATR)

[cm⁻¹] 1632, 1603, 1577, 1584, 1354, 1258, 1139, 964, 844, 729. ¹H

NMR (400 MHz, DMSO-d₆) δ 8.60 (s, 1H), 8.52−8.49 (m, 1H),

8.31 (s, 1H), 7.99 (t, J = 8.7 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.45−

7.35 (m, 3H), 7.13−7.08 (m, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.62 (s,

1H), 3.16 (s, 1H), 3.12−3.10 (m, 2H), 3.05−3.03 (m, 2H), 2.82−

2.76 (m, 2H), 2.45−2.40 (m, 10H).

Methyl 8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carboxylate (52). In a three-

necked round-bottomed ﬂask, o-phenylenediamine (0.194 g, 1.79

mmol), XPhos-Pd second generation (0.03 equiv), and aryl chloride 3

were dissolved in 12 mL of 1,4-dioxane/t-BuOH (5:1, v/v) by gentle

heating. Then, Cs₂CO₃(1.5 equiv) was added, and the reaction

mixture was heated to 60 °C for 4 h. After cooling to rt, the reaction

mixture was ﬁltered, and the residue was washed several times with

DCM, MeOH, and ethyl acetate. The combined organic extracts were

evaporated under reduced pressure. The dark-brown residue was

puriﬁed by ﬂash chromatography (SiO₂, petroleum ether/ethyl

acetate 3:1) to yield 52 as a yellow solid (0.53 g, 95%). MS (ESI_neg

)

(m/z): 370.9 [M − H]⁻. IR (ATR) [cm⁻¹] 1714, 1602, 1577, 1498,

1

1353, 1242, 1116, 837, 743, 626. H NMR (400 MHz, DMSO-d₆) δ

8.45 (d, J = 1.6 Hz, 1H), 8.06 (s, 1H), 8.00−7.96 (m, 2H), 7.43 (d, J

= 7.9 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.97−6.93 (m, 1H), 6.79 (d,

J = 7.9 Hz, 1H), 6.64 (dd, J = 8.9, 1.5 Hz, 1H), 6.61−6.57 (m, 1H),

6.47 (d, J = 1.3 Hz, 1H), 4.86 (s, 2H), 3.86 (s, 3H), 3.12−3.10 (m,

2H), 3.00−2.97 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 189.1,

165.7, 151.1, 146.9, 145.3, 143.7, 139.3, 133.7, 131.8, 131.3, 129.4,

127.9, 126.2, 125.9, 125.5, 124.7, 116.5, 115.5, 112.7, 111.9, 52.1,

35.4, 34.1.

8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxylic Acid (53). Compound 53

was prepared according to General Procedure A starting from 52

(0.26 g, 0.70 mmol). Yield: 0.24 g (96%). MS (ESI_neg) (m/z): 357.1

[M − H]⁻. IR (ATR) [cm⁻¹] 1682, 1579, 1505, 1454, 1353, 1239,

1108, 831, 745, 622. ¹H NMR (400 MHz, DMSO-d₆) δ 13.04 (br. s,

1H), 9.70 (s, 1H), 8.44 (s, 1H), 8.26 (s, 1H), 8.03−7.98 (m, 3H),

(8-((2,4-Diﬂuorophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carbonyl)glycine (42). Compound

42 was prepared according to General Procedure A starting from

carboxylic ester 31 (0.020 g, 0.04 mmol). Yield: 0.012 g (62%),

yellow solid. MS (ESI_neg) (m/z): 434.7 [M − H]⁻. IR (ATR) [cm⁻¹]

M

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

7.44 (s, 1H), 7.36−7.30 (m, 1H), 7.18 (s, 1H), 7.03−6.93 (m, 1H),

6.79−6.47 (m, 3H), 3.14−3.12 (m, 2H), 3.04−3.01 (m, 2H).

8-((2-Aminophenyl)amino)-N-(2-(dimethylamino)ethyl)-5-

oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (54). Compound 54 was prepared according to General

Procedure B using 53 (0.025 g, 0.07 mmol) and 3-aminopropanamide

(0.012 g, 0.14 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.018 g (60%), yellow solid. MS

(ESI_pos) (m/z): 429.1 [M + H]⁺. IR (ATR) [cm⁻¹] 2921, 2852, 1633,

= 8.7, 2.1 Hz, 1H), 6.45 (d, J = 1.8 Hz, 1H), 5.66 (s, 1H), 3.65 (s,

2H), 3.55 (s, 4H), 3.18−3.14 (m, 2H), 3.11−3.08 (m, 2H), 2.75 (br.

s, 2H), 2.60 (br. s, 4H), 1.48 (s, 9H).

8-((2-Aminophenyl)amino)-N-(2-hydroxyethyl)-5-oxo-

10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(59). Compound 59 was prepared according to General Procedure B

using 53 (0.050 g, 0.14 mmol) and 2-aminoethanol (0.026 g, 0.42

mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0

to 9:1). Yield: 0.007 g (13%), yellow solid. MS (ESI_neg) (m/z): 400.1

[M − H]⁻. IR (ATR) [cm⁻¹] 3305, 2923, 1603, 1557, 1498, 1354,

1266, 1216, 1113, 1065, 746, 451. ¹H NMR (400 MHz, DMSO-d₆) δ

8.54−8.51 (m, 1H), 8.33 (s, 1H), 8.01−7.97 (m, 2H), 7.92−7.98 (m,

1H), 7.38 (d, J = 7.9 Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 6.97−6.93

(m, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.65−6.62 (m, 1H), 6.60−6.57 (m,

1H), 6.47 (s, 1H), 4.84 (s, 2H), 4.75 (br. s, 1H), 3.53−3.48 (m, 2H),

3.35−3.30 (m, 2H), 3.10−3.08 (m, 2H), 3.00−2.98 (m, 2H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 190.5, 166.2, 151.4, 145.8, 145.0,

144.1, 139.6, 134.0, 133.3, 130.7, 129.7, 129.2, 126.6, 126.4, 126.2,

125.3, 117.0, 116.0, 113.3, 112.3, 60.2, 42.7, 36.2, 34.3.

8-((2-Aminophenyl)amino)-N-(2,3-dihydroxypropyl)-5-oxo-

10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(60). Compound 60 was prepared according to General Procedure B

using 53 (0.040 g, 0.11 mmol) and 3-aminopropane-1,2-diol (0.011 g,

0.12 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

1:0 to 9:1). Yield: 0.010 g (21%), yellow solid. MS (ESI_neg) (m/z):

429.7 [M − H]⁻. IR (ATR) [cm⁻¹] 3306, 2920, 2851, 1603, 1574,

1557, 1455, 1354, 1263, 1111, 1036, 745. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.51−8.48 (m, 1H), 8.34−8.33 (m, 1H), 8.01−7.97 (m,

2H), 7.91 (dd, J = 7.8, 2.0 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.02

(dd, J = 7.8, 1.2 Hz, 1H), 6.97−6.93 (m, 1H), 6.78 (dd, J = 8.1, 1.5

Hz, 1H), 6.64 (dd, J = 8.9, 2.3 Hz, 1H), 6.60−6.56 (m, 1H), 6.47 (d, J

= 2.4 Hz, 1H), 4.86 (s, 2H), 4.82−4.81 (m, 1H), 4.57 (t, J = 5.7 Hz,

1H), 3.67−3.60 (m, 1H), 3.41−3.34 (m, 3H), 3.22−3.16 (m, 1H),

3.11−3.07 (m, 2H), 3.02−2.98 (m, 2H).

8-((2-Aminophenyl)amino)-N-(1,3-dihydroxypropan-2-yl)-

5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (61). Compound 61 was prepared according to General

Procedure B using 53 (0.040 g, 0.11 mmol) and 2-aminopropane-1,3-

diol (0.011 g, 0.12 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.011 g (22%), yellow solid. MS

(ESI_neg) (m/z): 430.3 [M − H]⁻. IR (ATR) [cm⁻¹] 3307, 2918,

2850, 1603, 1574, 1557, 1456, 1353, 1266, 1036, 746, 565. ¹H NMR

(400 MHz, DMSO-d₆) δ 8.34 (d, J = 1.7 Hz, 1H), 8.08 (d, J = 8.1 Hz,

1H), 8.01−7.97 (m, 2H), 7.92 (dd, J = 7.8, 2.0 Hz, 1H), 7.39 (d, J =

7.8 Hz, 1H), 7.03−7.00 (m, 1H), 6.97−6.93 (m, 1H), 6.78 (dd, J =

7.9, 1.3 Hz, 1H), 6.64 (dd, J = 8.9, 2.3 Hz, 1H), 6.60−6.56 (m, 1H),

6.47 (d, J = 2.0 Hz, 1H), 4.86 (s, 2H), 4.68−4.64 (m, 2H), 4.00−3.94

(m, 1H), 3.52−3.49 (m, 4H), 3.11−3.07 (m, 2H), 3.02−2.98 (m,

2H).

1

1603, 1574, 1455, 1353, 1262, 1112, 852, 744, 543. H NMR (400

MHz, CDCl₃) δ 8.37 (s, 1H), 8.13 (d, J = 8.7 Hz, 1H), 7.96 (d, J =

7.6 Hz, 1H), 7.26−7.24 (m, 1H), 7.15−7.08 (m, 2H), 6.84−6.76 (m,

2H), 6.65 (dd, J = 8.7, 2.0 Hz, 1H), 6.43 (d, J = 1.9 Hz, 1H), 5.71 (s,

1H), 3.62−3.58 (m, 2H), 3.14−3.12 (m, 2H), 3.06−3.04 (m, 2H),

2.67−2.65 (m, 2H), 2.37 (s, 6H), (NH, NH, NH₂not visible). ¹³C

NMR (100 MHz, CDCl₃) δ 191.3, 166.9, 150.0, 145.5, 145.0, 142.8,

139.3, 134.3, 132.9, 131.0, 129.1, 128.9, 128.1, 127.3, 126.9, 125.8,

119.1, 116.4, 113.5, 112.7, 57.9, 44.9 (2× C), 37.0, 35.9, 34.7.

8-((2-Aminophenyl)amino)-5-oxo-N-phenethyl-10,11-dihy-

dro-5H-dibenzo[a,d][7]annulene-3-carboxamide (55). Com-

pound 55 was prepared according to General Procedure B using 53

(0.043 g, 0.12 mmol) and 2-phenylethylamine (0.013 g, 0.03 mmol).

Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1).

Yield: 0.013 g (24%), yellow solid. MS (ESI_neg) (m/z): 460.1 [M −

H]⁻. IR (ATR) [cm⁻¹] 3313, 2923, 2854, 1358, 2341, 1633, 1575,

1

1354, 1261, 1112, 745, 698. H NMR (400 MHz, CDCl₃) δ 8.28 (s,

1H), 8.06 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 7.4 Hz, 1H), 7.23 (d, J =

7.6 Hz, 1H), 7.17−7.04 (m, 5H), 6.95−6.82 (m, 3H), 6.64 (d, J = 8.2

Hz, 1H), 6.44 (s, 1H), 6.08 (br. s, 1H), 3.72−3.69 (m, 2H), 3.09−

3.07 (m, 2H), 3.00−2.98 (m, 2H), 2.65 (t, J = 5.7 Hz, 2H), (NH,

NH, NH₂not visible).

8-((2-Aminopheinyl)amino)-N-(2-morpholinoethyl)-5-oxo-

10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide

(56). Compound 56 was prepared according to General Procedure B

using 53 (0.032 g, 0.09 mmol) and 2-morpholinoethan-1-amine

(0.040 g, 0.31 mmol). Puriﬁcation: ﬂash chromatography (SiO₂,

DCM/MeOH 1:0 to 9:1). Yield: 0.029 g (70%), yellow solid. MS

(ESI_pos) (m/z): 471.2 [M + H]⁺. IR (ATR) [cm⁻¹] 3304, 2921, 2852,

1

1633, 1602, 1574, 1498, 1353, 1264, 1112, 859, 745, 537. H NMR

(400 MHz, DMSO-d₆) δ 8.55−8.52 (m, 1H), 8.33 (s, 1H), 8.04−7.99

(m, 2H), 7.90 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.06 (d, J

= 7.6 Hz, 1H), 6.98−6.94 (m, 1H), 6.80−6.78 (m, 1H), 6.66−6.58

(m, 2H), 6.48 (br. s, 1H), 4.88 (br. s, 2H), 3.57 (br. s, 4H), 3.40−

3.37 (m, 2H), 3.11−3.09 (m, 2H), 3.01−2.99 (m, 2H), 2.46 (d, J =

6.9 Hz, 2H), 2.41 (br. s, 4H).

N-(2-(1H-Imidazol-5-yl)ethyl)-8-((2-aminophenyl)amino)-5-

oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (57). Compound 57 was prepared according to General

Procedure B using 53 (0.050 g, 0.14 mmol) and histamine

dihydrochloride (0.039 g, 0.21 mmol). Puriﬁcation: ﬂash chromatog-

raphy (SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.028 g (46%), yellow

solid. MS (ESI_neg) (m/z): 450.1 [M − H]⁻. IR (ATR) [cm⁻¹] 2920,

8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxamide (62). Compound 62

was prepared according to General Procedure B using 53 (0.050 g,

0.14 mmol) and aq. ammonia (0.019 g, 0.28 mmol). Puriﬁcation:

ﬂash chromatography (SiO₂, DCM/MeOH [7 N NH₃] 1:0 to 9:1).

Yield: 0.031 g (62%), yellow solid. MS (ESI_neg) (m/z): 356.0 [M −

H]⁻. IR (ATR) [cm⁻¹] 3305, 2922, 2853, 1652, 1602, 1567, 1513,

1

2851, 1602, 1557, 1495, 1353, 1263, 1112, 829, 746, 619. H NMR

(400 MHz, DMSO-d₆) δ 11.81 (s, 1H), 8.68 (t, J = 5.4 Hz, 1H), 8.32

(d, J = 1.6 Hz, 1H), 8.04 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.89 (dd, J

= 7.9, 1.7 Hz, 1H), 7.53 (s, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.02 (d, J =

7.6 Hz, 1H), 6.97−6.93 (m, 1H), 6.85−6.77 (m, H), 6.64 (dd, J = 8.9,

2.1 Hz, 1H), 6.60−6.57 (m, 1H), 6.47 (d, J = 1.9 Hz, 1H), 4.86 (s,

2H), 3.49−3.46 (m, 2H, overlain by water), 3.10−3.08 (m, 2H),

3.00−2.98 (m, 2H), 2.77−2.74 (m, 2H).

1

1353, 1270, 1108, 830, 145, 524. H NMR (400 MHz, DMSO-d₆) δ

8.34 (d, J = 1.4 Hz, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.98 (d, J = 8.9

Hz, 1H), 7.92 (dd, J = 7.9, 1.4 Hz, 1H), 7.38−7.36 (m, 2H), 7.02 (d, J

= 7.6 Hz, 1H), 6.99−6.93 (m, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.63

(dd, J = 8.9, 2.0 Hz, 1H), 6.61−6.56 (m, 1H), 6.47 (d, J = 1.7 Hz,

1H), 4.84 (s, 2H), 3.10−3.08 (m, 2H), 3.01−2.98 (m, 2H). ¹³C NMR

(100 MHz, DMSO-d₆) δ 190.5, 167.9, 151.4, 145.8, 145.1, 144.1,

139.6, 134.0, 133.0, 130.9, 130.1, 129.2, 126.6, 126.4, 126.2, 125.3,

117.0, 116.0, 113.3, 112.3, 36.2, 34.3.

N-(2-Aminoethyl)-8-((2-aminophenyl)amino)-5-oxo-10,11-

dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamide (63).

Compound 63 was prepared according to General Procedure B

using 53 (0.050 g, 0.14 mmol) and ethylene-1,2-diamine (0.025 g,

0.42 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

[7 N NH₃] 1:0 to 9:1). Yield: 0.012 g (22%), yellow solid. MS

tert-Butyl 4-(2-(8-((2-Aminophenyl)amino)-5-oxo-10,11-di-

hydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)ethyl)-

piperazine-1-carboxylate (58). Compound 58 was prepared

according to General Procedure B using 53 (0.110 g, 0.31 mmol)

and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (0.077 g, 0.34

mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0

to 9:1). Yield: 0.081 g (46%), yellow solid. MS (ESI_neg) (m/z): 568.1

[M − H]⁻. IR (ATR) [cm⁻¹] 3289, 2923, 1660, 1603, 1573, 1417,

1

1246, 1164, 1113, 860, 786. H NMR (400 MHz, CDCl₃) δ 8.37 (s,

1H), 8.17 (d, J = 8.7 Hz, 1H), 7.98 (dd, J = 1.5, 7.8 Hz, 1H), 7.31 (d,

J = 7.0 Hz, 1H), 7.17−7.11 (m, 3H), 6.86−6.79 (m, 3H), 6.68 (dd, J

N

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

(ESI_neg) (m/z): 399.1 [M − H]⁻. IR (ATR) [cm⁻¹] 3290, 2920,

NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 8.12 (d, J = 8.8 Hz, 1H),

7.92 (d, J = 7.8 Hz, 1H), 7.29 (s, 1H), 7.16−7.06 (m, 2H), 6.84−6.75

(m, 2H), 6.64 (dd, J = 1.6, 8.6 Hz, 1H), 6.42 (d, J = 5.7 Hz, 1H),

3.55−3.51 (m, 2H), 3.16−3.06 (m, 8H), 2.68−2.64 (m, 6H), (NH,

NH, NH, NH₂not visible).

(8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carbonyl)-^L-glutamic Acid (69).

Compound 69 was prepared according to General Procedure A

starting from carboxylic ester 64 (0.066 g, 0.13 mmol). Yield: 0.052 g

(84%), yellow solid. MS (ESI_neg) (m/z): 486.1 [M − H]⁻. IR (ATR)

[cm⁻¹] 2922, 2852, 1714, 1603, 1519, 1451, 1394, 1266, 1101, 743.

¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (d, J = 7.7 Hz, 1H), 8.38 (d, J

= 1.8 Hz, 1H), 8.03 (s, 1H), 8.00−7.97 (m, 2H), 7.94 (dd, J = 7.9, 1.9

Hz, 1H), 7.73−7.71 (m, 1H), 7.57−7.53 (m, 1H), 7.43−7.40 (m,

2H), 7.04 (dd, J = 1.1, 7.7 Hz, 1H), 6.99−6.94 (m, 1H), 6.81 (dd, J =

7.9, 1.1 Hz, 1H), 6.66−6.59 (m, 2H), 6.48 (d, J = 2.0 Hz, 1H), 4.44−

4.38 (m, 1H), 3.12−3.09 (m, 2H), 3.01−2.99 (m, 2H), 2.35 (t, J =

7.4 Hz, 2H), 2.14−2.05 (m, 1H), 2.00−1.92 (m, 1H). ¹³C NMR (100

MHz, DMSO-d₆) δ 190.0, 173.8, 173.3, 166.0, 150.9, 145.3, 144.8,

143.0, 139.2, 133.5, 132.2, 130.5, 129.4, 128.7, 126.1, 125.9, 125.7,

124.6, 116.9, 115.9, 112.9, 111.9, 52.0, 35.7, 33.8, 30.4, 25.8.

1

2851, 1602, 1564, 1495, 1352, 1262, 1112, 831, 745, 598. H NMR

(400 MHz, DMSO-d₆) 8.53 (s, 1H), 8.36−8.31(m, 1H), 8.07 (s, 1H),

8.00−7.96 (m, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 8.1 Hz,

1H), 7.02 (d, J = 7.6 Hz, 1H), 6.97−6.95 (m, 1H), 6.78 (d, J = 7.9

Hz, 1H), 6.64 (dd, J = 1.8, 8.9 Hz, 1H), 6.60−6.57 (m, 1H), 6.47 (s,

1H), 4.87 (s, 2H), 3.50 (s, 2H), 3.27−3.23 (m, 2H), 3.10−3.08 (m,

2H), 3.00−2.97 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.5,

166.2, 151.4, 145.8, 144.9, 144.1, 139.6, 134.0, 133.4, 130.7, 129.7,

129.1, 126.6, 126.3, 126.2, 125.3, 116.9, 116.0, 113.3, 112.3, 79.6,

70.3, 36.2, 34.3.

Dimethyl (8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carbonyl)-^L-glutamate (64).

Compound 64 was prepared according to General Procedure B

using 53 (0.070 g, 0.20 mmol) and dimethyl ^L-glutamate (0.045 g,

0.21 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

1:0 to 9:1). Yield: 0.066 g (65%), yellow solid. MS (ESI_neg) (m/z):

514.1 [M − H]⁻. IR (ATR) [cm⁻¹] 2921, 2852, 1732, 1606, 1580,

1

1505, 1447, 1356, 1308, 1208, 1105, 848, 744. H NMR (400 MHz,

DMSO-d₆) δ 8.89 (d, J = 7.4 Hz, 1H), 8.36 (d, J = 1.8 Hz, 1H), 8.02

(s, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.92 (dd, J = 7.9, 1.9 Hz, 1H), 7.41

(d, J = 8.0 Hz, 1H), 7.03−7.00 (m, 1H), 6.97−6.93 (m, 1H), 6.78

(dd, J = 8.0, 1.1 Hz, 1H), 6.64 (dd, J = 8.9, 2.2 Hz, 1H), 6.61−6.56

(m, 1H), 6.47 (d, J = 2.0 Hz, 1H), 4.84 (s, 2H), 4.50−4.44 (m, 1H),

3.64 (s, 3H), 3.58 (s, 3H), 3.12−3.09 (m, 2H), 3.00−2.98 (m, 2H),

2.44 (t, J = 7.5 Hz, 2H), 2.16−1.96 (m, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 189.9, 172.7, 172.1, 166.1, 151.0, 145.3, 145.0, 143.6,

139.2, 133.5, 131.8, 130.5, 129.4, 128.7, 126.1, 125.9, 125.6, 124.7,

116.5, 115.5, 112.8, 111.8, 52.0, 51.9, 51.3, 35.7, 33.8, 29.9, 25.6.

Methyl 3-(8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoate (65).

Compound 65 was prepared according to General Procedure B using

53 (0.050 g, 0.14 mmol) and methyl 3-aminopropanoate (0.021 g,

0.15 mmol). Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH

1:0 to 9:1). Yield: 0.061 g (99%), yellow solid. MS (ESI_pos) (m/z):

3-(8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxamido)propanoic Acid (70).

Compound 70 was prepared according to General Procedure A

starting from carboxylic ester 65 (0.050 g, 0.12 mmol). Yield: 0.042 g

1

(85%), yellow solid. MS (ESI_pos) (m/z): 428.4 [M − H]⁻. H NMR

(400 MHz, DMSO-d₆) 12.20 (br. s, 1H), 8.66−8.64 (m, 1H), 8.32 (s,

1H), 8.02−7.98 (m, 2H), 7.90 (d, J = 7.3 Hz, 1H), 7.39 (d, J = 7.8

Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 6.97−6.93 (m, 1H), 6.79 (d, J =

7.8 Hz, 1H), 6.66−6.57 (m, 2H), 6.48 (br. s, 1H), 4.93 (br. s, 2H),

3.51−3.45 (m, 2H), 3.11−3.09 (m, 2H), 3.00−2.98 (m, 2H), 2.54−

2.50 (m, 2H + solvent peak).

Methyl 8-((2-nitrophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxylate (73). 2-Nitroaniline

(0.247 g, 1.79 mmol) was dissolved in 12 mL of 1,4-dioxane/t-

BuOH (5:1, v/v) under an argon atmosphere. XPhos (0.025 equiv),

Cs₂CO₃(1.5 equiv), and Pd(OAc)₂(0.05 equiv) followed by aryl

chloride 3 (0.50 g, 1.79 mmol) were added, and the reaction mixture

was heated for 1 h to 110 °C. The reaction was cooled to rt and

ﬁltered. The residue was washed several times with DCM, MeOH,

and EtOAc. The combined organic extracts were evaporated, and the

brown-black residue was puriﬁed by ﬂash chromatography (SiO₂,

petroleum ether/ethyl acetate 3:1) to yield 73 as a yellow solid (0.48

g, 80%). MS (ESI_neg) (m/z): 401.1 [M − H]⁻. IR (ATR) [cm⁻¹]

1722, 1601, 1563, 1506, 1435, 1348, 1240, 1156, 838, 761, 640, 539.

¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (d, J = 1.7 Hz, 1H), 8.10 (dd,

J = 0.9, 8.3 Hz, 1H), 8.04−8.01 (m, 2H), 7.64−7.60 (m, 1H), 7.58−

7.56 (m, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.20 (dd, J = 8.7, 2.1 Hz, 1H),

7.14−7.09 (m, 2H), 3.87 (s, 3H), 3.21−3.19 (m, 2H), 3.14−3.12 (m,

2H), (NH not visible). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.8,

165.6, 147.1, 145.2, 144.8, 138.6, 137.7, 137.5, 135.3, 132.9, 132.2,

131.1, 130.6, 129.8, 128.1, 126.1, 121.2, 120.5, 118.9, 116.9, 52.0,

34.5, 33.9.

1

442.1 [M − H]⁻. H NMR (400 MHz, DMSO-d₆) 8.69−8. 67 (m,

1H), 8.32 (s, 1H), 8.03−7.98 (m, 2H), 7.90 (d, J = 7.8 Hz, 1H), 7.40

(d, J = 7.8 Hz, 1H), 7.03 (d, J = 7.8 Hz, 1H), 6.98−6.94 (m, 1H),

6.80 (d, J = 7.8 Hz, 1H), 6.66−6.58 (m, 2H), 6.48 (br. s, 1H), 4.88

(br. s, 2H), 3.61 (s, 3H), 3.53−3.47 (m, 2H), 3.11−3.08 (m, 2H),

3.03−2.98 (m, 2H), 2.61 (t, J = 6.8 Hz, 2H).

Methyl (8-((2-Aminophenyl)amino)-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carbonyl)-^L-methioninate (66).

Compound 66 was prepared according to General Procedure B using

53 (0.070 g, 0.20 mmol) and ^L-methionine methyl ester hydro-

chloride (0.043 g, 0.21 mmol). Puriﬁcation: ﬂash chromatography

(SiO₂, DCM/MeOH 1:0 to 9:1). Yield: 0.012 g (12%), yellow solid.

MS (ESI_neg) (m/z): 502.1 [M − H]⁻. IR (ATR) [cm⁻¹] 2920, 2852,

1737, 1604, 1575, 1504, 1444, 1353, 1256, 1209, 742. ¹H NMR (400

MHz, CDCl₃) δ 8.34 (s, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.90 (d, J =

7.8 Hz, 1H), 7.45−7.21 (m, 3H + solvent peak), 7.16−7.05 (m, 3H),

7.01−6.97 (m 1H), 6.66−6.64 (m, 1H), 6.48 (s, 1H), 4.95−4.90 (m,

2H), 3.80−3.73 (m, 4H), 3.05−3.03 (m, 2H), 2.95−2.93 (m, 2H),

2.59 (t, J = 7.4 Hz, 2H), 2.32−2.22 (m, 1H), 2.18−2.00 (m, 4H). ¹³C

NMR (100 MHzCDCl₃) δ 191.3, 172.6, 166.5, 149.2, 145.5, 145.4,

143.6, 139.1, 138.0, 134.1, 132.2, 132.0, 131.7, 130.1, 129.3, 128.9,

128.1, 126.3, 126.1, 114.1, 113.0, 52.6, 52.1, 35.7, 34.6, 30.2, 29.7,

15.4.

Methyl N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N6-(8-((2-

aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d]-

[7]annulene-3-carbonyl)-^L-lysinate (67). Compound 67 was

prepared according to General Procedure B using 53 (0.100 g, 0.28

mmol) and N-α-Fmoc-^L-lysine methyl ester (S1) (0.234 g, 0.56

mmol). Product was used in the next step without further puriﬁcation.

8-((2-Aminophenyl)amino)-5-oxo-N-(2-(piperazin-1-yl)-

ethyl)-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxa-

mide (68). Compound 68 was prepared according to General

Procedure C starting from 58 (0.010 g, 0.02 mmol). Puriﬁcation: ﬂash

chromatography (SiO₂, DCM/MeOH [7 N NH₃] 1:0 to 9:1). Yield:

0.006 g (67%), yellow solid. MS (ESI_pos) (m/z): 470.2 [M + H]⁺. ¹H

8-((2-Nitrophenyl)amino)-5-oxo-10,11-dihydro-5H-

dibenzo[a,d][7]annulene-3-carboxylic Acid (74). Compound 74

was prepared according to General Procedure A starting from

carboxylic ester 73 (0.15 g, 2.37 mmol). Yield: 0.137 g (95%), orange

solid. MS (ESI_neg) (m/z): 387.0 [M − H]⁻. IR (ATR) [cm⁻¹] 1690,

1

1598, 1571, 1495, 1345, 1253, 1115, 882, 741, 507. H NMR (400

MHz, DMSO-d₆) δ 9.31 (s, 1H), 8.44 (d, J = 1.8 Hz, 1H), 8.10 (d, J =

8.4, 1.3 Hz, 1H), 8.04−8.00 (m, 2H), 7.65−7.61 (m, 1 H), 7.57−7.49

(m, 2H), 7.17 (dd, J = 2.1, 8.7 Hz, 1H), 7.14−7.10 (m, 2H), 3.12−

3.10 (m, 2H), 3.21−3.18 (m, 2H), (COOH not visible). ¹³C NMR

(100 MHz, DMSO-d₆) δ 190.9, 165.7, 147.2, 145.6, 144.8, 138.7,

137.8, 137.8, 135.5, 133.0, 132.4, 131.2, 130 6, 130.0, 128.2, 126.2,

121.5, 120.9, 119.0, 117.0, 34.6, 34.0.

N-Methyl-8-((2-nitrophenyl)amino)-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carboxamide (75). Compound

75 was prepared according to General Procedure B using 74 (0.050

g, 0.13 mmol) and methylamine (2 N THF) (0.010 g, 0.32 mmol).

O

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Puriﬁcation: ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1).

Yield: 0.045 g (87%), yellow solid. M (ESI_neg) (m/z): 400.3 [M −

H]⁻. ¹H NMR (400 MHz, DMSO-d₆) δ 9.32 (s, 1H), 8.58−8.54 (m,

1H), 8.35 (s, 1H), 8.11 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H),

7.94 (d, J = 7.4 Hz, 1H), 7.65−7.61 (m, 1H), 7.57−7.55 (m, 1H),

7.44 (d, J = 7.9 Hz, 1H), 7.22−7.19 (m, 1H), 7.14−7.10 (m, 2H),

3.19−3.16 (m, 2H), 3.14−3.12 (m, 2H), 2.79 (d, J = 4.2 Hz, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 191.4, 165.8, 145.2, 144.7, 144.5,

138.4, 137.8, 137.6, 135.3, 132.9, 132.7, 130.9, 130.5, 129.2, 128.9,

126.1, 121.2, 120.6, 119.1, 117.0, 34.7, 33.7, 26.2.

AUTHOR INFORMATION

Corresponding Author

■

Pierre Koch − Department of Pharmaceutical and Medicinal

Chemistry, Institute of Pharmaceutical Sciences, Eberhard

̈ ̈

Karls Universität Tubingen, 72076 Tubingen, Germany;

Department of Pharmaceutical/Medicinal Chemistry II,

Institute of Pharmacy, University of Regensburg, 93053

Regensburg, Germany; orcid.org/0000-0003-4620-4650;

Phone: +49 941 943 4827; Email: pierre.koch@uni-

tuebingen.de, pierre.koch@ur.de

8-((2-Aminophenyl)amino)-N-methyl-5-oxo-10,11-dihydro-

5H-dibenzo[a,d][7]annulene-3-carboxamide (76). The nitro

compound 75 (0.030 g, 0.07 mmol) was dissolved in EtOH (40

mL) with heating (50 °C). SnCl₂(3 equiv) was added in one portion,

and the reaction was heated to reﬂux temperature. After completion of

the reaction, the reaction was cooled to rt, and NaHCO₃was carefully

added and stirred for another 15 min. Then, EtOH was removed

under reduced pressure, and the residue was extracted three times

with EtOAc. The combined organic phases were dried over Na₂SO₄,

ﬁltered, and evaporated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography (SiO₂, DCM/MeOH 1:0 to 9:1) to

yield 76 (0.020 mg, 69%) as a yellow solid. MS (ESI_neg) (m/z): 370.1

[M − H]⁻. IR (ATR) [cm⁻¹] 1633, 1603, 1574, 1455, 1403, 1353,

Authors

Niklas M. Tormählen − Department of Pharmaceutical and

Medicinal Chemistry, Institute of Pharmaceutical Sciences,

Eberhard Karls Universität Tu

Germany

Mariella Martorelli − Synovo GmbH, 72076 Tu

Germany

̈

bingen, 72076 Tu

̈

bingen,

̈

bingen,

Annette Kuhn − Department of Pharmaceutical and

Medicinal Chemistry, Institute of Pharmaceutical Sciences,

Eberhard Karls Universität Tu

Germany

Florian Maier − Synovo GmbH, 72076 Tu

Jamil Guezguez − Synovo GmbH, 72076 Tu

̈

bingen, 72076 Tu

̈

bingen,

1

1262, 1111, 853, 745. H NMR (400 MHz, DMSO-d₆) δ 8.54−8.53

̈

bingen, Germany

(m, 1H), 8.33 (s, 1H), 8.02 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.92−

7.84 (m, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.95

(t, J = 7.4 Hz, 1H), 6.78 (d, J = 7.7 Hz, 1H), 6.64 (dd, J = 8.8, 1.6 Hz,

1H), 6.61−6.57 (m, 1H), 6.48−6.47 (m, 1H), 4.86 (s, 2H), 3.11−

3.07 (m, 2H), 3.02−2.99 (m, 2H), 2.78 (d, J = 4.3 Hz, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 189.9, 166.0, 150.9, 145.3, 144.4,

143.7, 139.1, 133.5, 132.8, 130.0, 129.1, 128.7, 126.1, 125.8, 125.7,

124.8, 116.4, 115.5, 112.8, 111.8, 35.7, 33.8, 26.2.

̈

Michael Burnet − Synovo GmbH, 72076 Tu

̈

Wolfgang Albrecht − Teva-ratiopharm, 89079 Ulm,

Germany

Stefan A. Laufer − Department of Pharmaceutical and

Medicinal Chemistry, Institute of Pharmaceutical Sciences,

̈ ̈

Eberhard Karls Universität Tubingen, 72076 Tubingen,

Germany; orcid.org/0000-0001-6952-1486

p38α MAP Kinase Activity Assay. The inhibitory activity of the

ﬁnal compounds was tested using a protocol reported by Goettert et

al.³¹In detail, 96-well plates were coated with 50 μL/well (10 μg/mL)

in Tris-buﬀered saline (TBS) of ATF-2 and stored overnight at 4 °C.

In the next morning, each plate was washed with water (three times),

and the remaining binding sites were blocked with blocking buﬀer

(0.05% Tween 20, 0.025% bovine serum albumin (BSA), and 0.02%

NaN₃in TBS) for 30 min at rt and washed again with water (three

times). A 10 mM stock solution of the test compound in DMSO was

further diluted in a kinase buﬀer (12 ng/50 μL activated p38α MAPK,

50 mM Tris of pH 7.5, 10 mM MgCl₂, 10 mM β-glycerophosphate,

100 μg/mL BSA, 1 mM dithiothreitol, 0.1 mM Na₃VO₄, and 100 μM

ATP). 50 μL of each dilution was pipetted into the corresponding

wells and incubated for 1 h at 37 °C. After washing with water (three

times), blocking for 15 min, and washing with water (three times), a

diluted monoclonal antiphospho-ATF-2 (Thyr69/71)-peroxidase-

conjugated antibody (1:5000) in blocking buﬀer (50 μL) adjusted

to a pH of 6.5 was added to each well. After incubation for 1 h at 37

°C, 3,3′,5,5′-tetramethylbenzidine (50 μL) was added to all wells, and

the peroxide-labeled conjugates developed a deﬁnitive blue color that

was measured photometrically at 650 nm.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01773

Author Contributions

^⊥N.M.T. and M.M. contributed equally.

Notes

The authors declare the following competing ﬁnancial

interest(s): M.M., F.M., J.G., and M.B. are employees of

Synovo GmbH, a pharmaceutical company that has an interest

in the development of this class of compounds.

ACKNOWLEDGMENTS

■

We thank Jens Strobach and Katharina Bauer for their

assistance in the p38α MAP kinase ELISA activity assay. Dr.

Michael Forster and Dr. Stanislav Andreev are gratefully

acknowledged for synthetic and analytical support. We thank

Mark Kudolo for his support with metabolism data of

compounds 44 and 70. This study was supported by the

Federal Ministry of Education and Research (BMBF) within

the BioPharma-Neuroallianz consortium (Neuro-T8B project).

We also thank the pharmacology staﬀ of Synovo GmbH,

Luciano De Oliveira, Thilo Weinstein, Christina Frech,

Natascha Cloos, and Santiago Cruces, for their assistance.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01773.

ABBREVIATIONS USED

Detailed procedures for the preparation of compounds

45−51, 70−72, and S1, CNS penetration studies, in

vitro metabolic stability study, CYP and hERG

inhibition, the selectivity screening as well as selected

HPLC traces and NMR spectra (PDF)

■

AD, Alzheimer’s disease; BBB, blood−brain barrier; CDI, 1,1′-

carbonyldiimidazole; CNS, central nervous system; HBC,

human buﬀy coat; HLM, human liver microsome; HR,

hydrophobic region; HWB, human whole blood; IL,

interleukin; i.v., intravenous; LC-MS, liquid chromatography

mass spectrometry; LPS, lipopolysaccharide; MWB, mouse

SMILES strings of tested compounds (CSV)

P

https://doi.org/10.1021/acs.jmedchem.0c01773

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors

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