A series of novel aryl-methanone derivatives as inhibitors of FMS-like tyrosine kinase 3 (FLT3) in FLT3-ITD-positive acute myeloid leukemia

European Journal of Medicinal Chemistry 193 (2020) 1 12232

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

A series of novel aryl-methanone derivatives as inhibitors of FMS-like

tyrosine kinase 3 (FLT3) in FLT3-ITD-positive acute myeloid leukemia

Andreas Sellmer ^a, Bernadette Pilsl ^a, Mandy Beyer ^b, Herwig Pongratz ^a, Lukas Wirth ^a,

,

Sigurd Elz ^a, Stefan Dove ^a¹, Sven Julian Henninger ^b, Karsten Spiekermann ^c,

c

d

e

€

Harald Polzer , Susan Klaeger , Bernhard Kuster , Frank D. Bohmer ,

Heinz-Herbert Fiebig , Oliver H. Kramer ¹, Siavosh Mahboobi ^a

f

b,

, *, 1

€

^aInstitute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040, Regensburg, Germany

^bDepartment of Toxicology, University Medical Center, Mainz, Germany

^cDepartment of Medicine III, University Hospital, LMU Munich, Germany

d

€

Technische Universitat München, Wissenschaftszentrum Weihenstephan für Ernahrung, Landnutzung und Umwelt, Germany

e

€

Universitatsklinikum Jena - Bachstrasse 18 - D-07743 Jena, Germany

^f4HF Biotec GmbH, Am Flughafen 14, 79108, Freiburg, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

Mutants of the FLT3 receptor tyrosine kinase (RTK) with duplications in the juxtamembrane domain

(FLT3-ITD) act as drivers of acute myeloid leukemia (AML). Potent tyrosine kinase inhibitors (TKi) of

FLT3-ITD entered clinical trials and showed a promising, but transient success due to the occurrence of

secondary drug-resistant AML clones. A further caveat of drugs targeting FLT3-ITD is the co-targeting of

other RTKs which are required for normal hematopoiesis. This is observed quite frequently. Therefore,

novel drugs are necessary to treat AML effectively and safely. Recently bis(1H-indol-2-yl)methanones

were found to inhibit FLT3 and PDGFR kinases. In order to optimize these agents we synthesized novel

derivatives of these methanones with various substituents. Methanone 16 and its carbamate derivative

17b inhibit FLT3-ITD at least as potently as the TKi AC220 (quizartinib). Models indicate corresponding

interactions of 16 and quizartinib with FLT3. The activity of 16 is accompanied by a high selectivity for

FLT3-ITD.

Received 4 November 2019

Received in revised form

9 March 2020

Accepted 10 March 2020

Available online 13 March 2020

Keywords:

FLT3

FLT3-ITD

FLT3 D835Y

Tyrosine kinase inhibitor

Acute myeloid leukemia

1. Introduction

hematopoiesis, and frequently they are dysregulated in AML [4].

Therefore, RTKs are targets for tailored intervention strategies

Acute myeloid leukemia (AML) is a heterogeneous disorder of

hematopoietic progenitor cells, which lose their ability of natural

differentiation and to respond to normal regulators of proliferation.

Subsequently without treatment this leads to fatal infections,

bleeding, or typical organ inﬁltration within one year after diag-

nosis [1]. Usual treatments are intensive chemotherapy and allo-

geneic hematopoietic stem cell transplantation [2]. However,

60e80% of relapses are eminently frequent [3] especially for elder

patients. Thus, new effective, and precise therapies are urgently

required.

against AML. FMS-like tyrosine kinase 3 (FLT3), macrophage colony

stimulating factor receptor (CSF1R, c-FMS), stem cell factor receptor

(SCFR, KIT), and platelet-derived growth factor receptor (PDGFR

a/

b) belong to this protein family [5]. Each of these receptors consists

of ﬁve immunoglublinelike domains in the extracellular domain, a

transmembrane domain (TMD), a juxtamembrane domain (JM) and

an intracellular kinase domain which is split by a kinase insert

[6e8].

FLT3 is expressed on hematopoietic stem- and progenitor cells

and plays an indispensable role in stem cell development and cell

differentiation [9]. FLT3 is activated by the binding of its ligand.

After ligand-induced dimerization and autophosphorylation of the

receptor, the transduction pathway of the signal is initiated [10].

Mutations in FLT3 are found in about one third of all patients with

AML, and aberrations are associated with poor prognosis [9,1 1,12].

Class III receptor tyrosine kinases (RTKs) are required for normal

* Corresponding author.

E-mail address: Siavosh.mahboobi@chemie.uni-regensburg.de (S. Mahboobi).

Senior authors who contributed equally.

1

https://doi.org/10.1016/j.ejmech.2020.1 12232

2

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

These lesions cause an aberrant activation of FLT3 and its down-

stream signaling pathway [13,14]. Of note is that recent evidence

shows that mutant FLT3 is an oncogenic driver in AML [15].

Three different activating FLT3 mutations are known. 1) Acti-

vating FLT3 internal tandem duplications (FLT3-ITD) are located

within the JM domain of FLT3. They were ﬁrst observed in 1996 by

Nakao et al. [1 1] and are seen in 15e35% of the patients suffering

from AML [1 1,13,16e18]. 2) Point mutations in the activation loop of

the kinase domain (FLT3-TKD) occur in 6e8% of patients with AML

[12,13,19,20]. 3) Point mutations in the JM are detectable in 2% of

AML patients [13,20]. The most common of these mutations, FLT3-

ITD, induces the loss of auto-inhibitory functions. Thus, the kinase

becomes active constitutively. This leads to altered intracellular

signaling [21] and autonomous cell growth [13].

Due to the frequency of FLT3 mutations in AML, the inhibition of

FLT3 and its downstream signaling pathways have triggered sig-

niﬁcant attention to the discovery of new anti-cancer drugs [22].

Under current development most of the potential therapeutics are

direct inhibitors of the FLT3 receptor. These small molecule in-

hibitors are investigated in monotherapy or in combination with

chemotherapy [22e25]. Inhibitors of FLT3 are classiﬁed to the ﬁrst

and the second generation (Fig. 1). The ﬁrst generation of small

molecule inhibitors include midostaurin (1, PKC412)²⁴, approved by

the US FDA in April 2017 for treatment of newly diagnosed FLT3-

mutant AML [26], lestaurtinib (2, CEP-701) [24,27,28], semaxanib

(3, SU 5416) [24,29], and sunitinib (4,SU 11248) [24]. To overcome

therapy acquired resistance also covalent irreversible binding in-

hibitors were published recently [30,31].

receptor tyrosine kinase PDGFR and FLT3 by bis(1H-indol-2-yl)

methanones 14a-c (Fig. 2) [49,50]. These compounds are biologi-

cally active at FLT3 and PDGFR, 14c particularly at FLT3. At least in

part the excellent inhibition of FLT3-ITD by AC220 (quizartinib) (7)

is based on the bisarylurea motive substituted by a tert-butylisox-

azole [51]. Therefore, we generated derivatives of the bis-(1H-

indol-2-yl) methanone skeleton by fusion with this substructure

(Fig. 2).

Here we report on some of our novel compounds which are

potent and selective FLT3 inhibitors. Proliferation assays revealed

AML cell growth inhibition at IC₅₀-values in the low nanomolar

range. Furthermore, one of our new compounds, 16, inhibits FLT3-

ITD as well as FLT3 overexpressing cell lines. Moreover, 16 and

especially its carbamate 17b with an enhanced water solubility of

230 mg/L vs < 100 mg/L for 16, shows excellent selectivity for FLT3

and FLT3-ITD. Moreover, compounds 16 and 17b show excellent

activity against FLT3-ITD and the clinically relevant FLT3 D835Y

mutant in the enzymatic test system, as well as reduced loss off

afﬁnity comparing 16 to quizartinib (7) towards FLT3-ITD-F691 L⁵²

,

an important acquired drug-resistance gatekeeper mutation.

2. Results and discussion

2.1. Chemistry

Based on the promising results from our previous studies, we

designed and synthesized a new series of compounds exploring a

combination of our bisindolylmethanone lead structure [14,49]

with key elements of established tyrosine kinase inhibitors, and

evaluated their pharmacological properties. The synthetic route to

obtain the desired target compounds is outlined in Schemes 1e5

and in the following: 18a-d (Scheme 1) were lithiated using t-

BuLi and the aryl-lithium intermediates reacted with the respective

carbonyl chloride 19a, which was prepared in analogy to Mahboobi

et al. [49] By removal of the sulfonyl protecting group [49] 20a-

d (Scheme 1) were obtained. Acidic cleavage of the Boc-group with

TFA yielded 21a-d. On principle by the same reaction sequence 21e-

f were obtained by coupling of 18b, 18e and 18f with 19b and 19c,

respectively, followed by removals of the sulfonyl protecting group

[49] and the tert-butoxycarbonyl-group. From 21e the hydroxy-

analogue 23e was obtained by hydrogenolytic cleavage of the

benzyloxy-group [49] (way A). Moreover the sequence of steps c)

and d) shown in Scheme 2 can be exchanged. By this alternative

route, way B. e.g., 23i was obtained (see Scheme 6).

Generally the ﬁrst generation of small molecule inhibitors are

multi-kinase inhibitors, and their efﬁcacies are limited, not durable,

and at the expense of undesired side effects [2,25].

Therefore, a second generation of TKi against FLT3-ITD has been

developed in order to yield more selective and high potent agents

for treating AML [32,33]. Prominent representatives of second

generation are tandutinib (5, MLN518) [25,34], KW-2449 (6)

[25,35] and quizartinib (7) [24,25,36] (Fig. 1). Nevertheless, sec-

ondary FLT3 mutations may substantially confer to in vitro resis-

tance of inhibitors, as shown for quizartinib (7) [15].

Further examples are crenolanib (8) [37e39], G-749 (9) [40],

TTT-3002 (10) [41], as well as dual kinase inhibitors (e.g., 11,

CCT137690 against FLT3 and Aurora kinase) [42]. Crenolanib (8)

was originally developed as PDGFR inhibitor [43]. Crenolanib (8)

shows activity against FLT3-ITD in vitro and in vivo. In combination

with sorafenib (12) crenolanib (8) promisingly decreased leukemic

burden and extended the survival of leukemic mice [37]. Ponatinib

(13) is an orally active multi-tyrosine kinase inhibitor speciﬁcally

targeting the BCR-ABL gene mutation, T315I in chronic myeloid

leukemia [44]. Moreover, ponatinib (13) exhibits activity against

AC220-resistant FLT3-ITD/F691 gatekeeper mutations, but is highly

ineffective against FLT3-ITD activation loop mutations, particularly

at the D835 residue [45]. Following the midostaurin approval, in

November 2018, a second-generation FLT3 inhibitor, gilteritinib,

was approved by the US FDA for use as a single agent for adults with

relapsed or refractory FLT3-mutated AML [46]. Beside others,

further FLT3 inhibitors being active towards therapy associated

mutants were described recently. Baska et al. for example found

(phenylethenyl)quinazolines as selective inhibitors of the ITD and

D835Y mutant FLT3 kinases [47]. Yuan et al. prepared and opti-

mized pyrrolo[2,3-d]pyrimidine derivatives for cytotoxic activities

against FLT3-ITD mutant cancer cells, exhibiting nanomolar FLT3

inhibitory activities and subnanomolar inhibitory activities against

MV4-11 and Molm-13 cells. In part they also showed excellent

inhibitory activities in FLT3-ITD-D835V and FLT3-ITD-F691L cells

which were resistant to quizartinib (7) [48].

Amino compounds 21a-d and 23e-i were treated with 5-(tert-

butyl)-3-isocyanatoisoxazole (24a) to obtain 15a-i (Scheme 3) [36].

As shown by the synthesis of 15f (Scheme 4), the sequence of the

steps can be modiﬁed. The benzyl-group can also be removed

ﬁnally from the respective indole.

To obtain the desired aryl-methanone derivative 16, 5-

benzyloxyindole 25 was protected by benzenesulfonyl chloride as

described [14] in order to get 26. Acetylation led to 27, which was

brominated in alpha-position to the carbonyl-group using trime-

thylphenylammonium tribromide to give 28 (Scheme 5). Reaction

of 28 with 2-hydroxy-5-nitrobenzaldehyde (29) resulted in the

formation of (5-(benzyloxy)-1-(phenylsulfonyl)-1H-indol-2-yl)(5-

nitrobenzofuran-2-yl)methanone as an intermediate. By alkaline

removal of the phenylsulfonyl-protecting group 30 was obtained.

By use of ammonium formate and Pd/C the nitro- and Bn-groups

were reduced. Finally, reaction of 31 with 5-(tert-butyl)-3-

isocyanatoisoxazole (24a) in THF led to the target compound 16.

The desired ureas 32b and 32c were formed by coupling the

isocyanate intermediates 24b and 24c with the 5-amino-5⁰-hy-

droxy aryl methanone 23e (Scheme 6). The isocyanates 24b and 24c

were prepared from commercially available arylamines by reaction

We have previously reported on the inhibition of class III

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

3

Fig. 1. FLT3 inhibitors investigated for treatment of AML (1e13).

with trichloromethyl chloroformate in THF [54].

2.2. Biological activity of the novel TKi against human AML cells

It turned out that the bisindolylmethanone skeleton with the

tert-butyl-isoxazol-urea platform in position 5, adopted from

compound 7, was essential for potent biological activity. In position

5⁰of the indole ring the novel lead structure 16 bears a hydroxyl

group (Fig. 2).

In order to modify the phenolic OH group of 16 to the carbamate

17a, 16 was reacted with 1,4⁰-bipiperidine-1⁰-carbonyl chloride in

CH₂Cl₂/pyridine. The respective hydrochloride 17b with improved

water solubility was obtained by treatment of 17a with HCl in 2-

propanol (Scheme 7).

The pharmacological properties of the novel compounds against

FLT3-ITD-positive human MV4-11 cells were evaluated. Based on

the data obtained from the proliferation assay in the human MV4-

11 AML cell line, the IC₅₀values were determined (Table 1).

In a ﬁrst run we performed a fusion of the bis(1H-indol-2-yl)

methanone skeleton of 14b with the bisarylurea-motive taken from

AC220 (quizartinib, 7) and investigated the antiproliferative activity

of the resulting compounds, fused at different positions of the A-

ring system (Fig. 2). It turned out, that an exchange of the phenolic

hydroxy group in position 5 by the bisarylurea-motive in position 5

4

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

Fig. 2. Concept of fusion of the bisarylurea-motive taken from AC220 (quizartinib, 7) with the bisindolylmethanone-skeleton 14 for generation of novel FLT3 inhibitors. To enhance

solubility for in vivo applications, a modiﬁcation of the lead compound was performed, resulting in 17b. Similar modiﬁcations, following the prodrug concept [53], were also

reported in literature.

Scheme 1. Synthesis of the amino compounds 21a-d. (a) 2.2 eq. t-BuLi, ꢀ78 ^ꢁC, THF; (b) NaOH (10%), MeOH, THF (1:1:1), reﬂux: (c) TFA, rt.

or 6 led to the potent compounds 15b and 15c (Table 1). This ﬁnding

was applied to prepare compounds based on the substitution

pattern of 14c (Fig. 2) by modifying one hydroxy-group by the

bisarylurea-motive. The most potent compounds resulting have a

bisaroylmethanone-skeleton substituted with the tert-butyl-iso-

xazol-urea platform in position 5 of one of the selected aroyl-

systems, either indole or benzofurane.

The results obtained in the proliferation assay indicate that the

fusion of the basic 5-hydroxy-substituted bisaroylmethanone-

structure with the 1-(5-(tert-butyl)isoxazol-3-yl)urea residue is

highly active against FLT3-ITD-positive AML cells.

In order to check, whether the antiproliferative activity

observed is due to an inhibition of FLT3, the inhibitory activity of all

compounds exhibiting IC₅₀values below 50 nM in the proliferation

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

5

Scheme 2. Synthesis of the amino compounds 23e, h-i. (a) 2.2 eq. t-BuLi, ꢀ78 ^ꢁC, THF; (b) NaOH (10%), MeOH, THF (1:1:1), reﬂux; (c) TFA, rt; (d) Pd/C (10%), 4.0 eq. NH₄HCOO,

MeOH, THF (1:1), 80 ^ꢁC.

Scheme 3. Synthesis of aryl-methanone derivatives 15a-i. (a) 1M solution of 5-(tert-butyl)-3- isocyanatoisoxazole (24a) in THF, pyridine.

assay on the enzymatic activity of FLT3-ITD was investigated in a

kinase assay using human FLT3-ITD. The therapy-associated FLT3

D835Y mutant was also investigated.

For the FLT3 D835Y mutant, larger differences among the com-

pounds were observed, with several compounds being less active

than against FLT3-ITD.

As shown by the data of Table 2, all compounds inhibited FLT3-

ITD in the low micromolar until nanomolar range. For 15i, 32b and

16 in particular, the data on antiproliferative activity and FLT3-ITD

inhibition correlate very well.

Within this group of agents and taking both sets into account, 16

revealed to be the most potent derivative. When given once at only

1 nM it inhibited the proliferation of MV4-11 cells by nearly 80%. In

the FLT3-ITD assay 50% inhibition was achieved at 2.3 nM (Table 2).

6

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

Scheme 4. Modiﬁed reaction sequence for the synthesis of 15f by reaction of the isocyanate 24a with 21f. At ﬁrst the urea derivative is formed, followed by hydrogenolytic cleavage

to 15f. (a) 0.1 M Solution of 5-(tert-butyl)-3- isocyanatoisoxazole (24a) in THF, THF, kat. pyridine, rt; (b) Pd/C (10%), 4.0 eq. NH₄HCOO, MeOH, THF (1:1), 80 ^ꢁC.

Scheme 5. (a) NaH, PhSO₂Cl, THF; (b) 1.1 eq. n-BuLi, ꢀ78 ^ꢁC, THF, 11.0 eq. Ac₂O; (c) 1.05 eq. trimethylphenylammonium tribromide, THF; (d) 1.0 eq. K₂CO₃, 2-butanone, 80 ^ꢁC; (e)

NaOH (10%), MeOH, THF (1:1:1), reﬂux; (f) Pd/C (10%), 4.0 eq. NH₄HCOO, MeOH, THF (1:1), 80 ^ꢁC; (g) 1M 5-(tert-butyl)-3-isocyanatoisoxazole 24a in THF, pyridine.

Moreover 16 and its carbamate 17b are also active towards the

D835Y point mutant, whereas for AC220 (quizartinib, 7) a signiﬁ-

cant decrease in activity can be observed. Therefore, compound 16

was analyzed in further detail. The carbamate (17b), prepared in

order to enhance the solubility, was included into these

investigations.

2.3. Western-blotting

To prove whether FLT3 kinase inhibition could be recapitulated

in vitro a western blotting assay was performed. We could see that

16 was able to dephosphorylate FLT3 strongly, after 3 h of incuba-

tion, using doses of 20 nM and 40 nM (Fig. 3).

To investigate the binding afﬁnity towards FLT3-ITD-F691L, an

important acquired drug-resistance gatekeeper mutation [52], K_d

values of compound 16 in comparison to AC220, were determined

(KINOMEscan Proﬁling Service, Euroﬁns DiscoverX Corporation). In

result, 16 (K_d¼ 115 15 nM) remains an enhanced afﬁnity towards

FLT3-ITD-F691L in comparison to AC220 (Kd ¼ 340 60 nM).

2.4. Annexin V/PI staining

To analyze the potency of apoptosis induction of compound 16,

MV4-11 cells were analyzed with AnnexinV/PI staining using ﬂow

cytometry. After 48 h using 20 nM and 40 nM of compound 16,

respectively, we could detect 25% of apoptotic MV4-11 cells. With

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

7

nitrogen of C694 (Fig. 5B). The sulfur atom of AC220 (quizartinib, 7)

may only form a single weak H-bond with this backbone nitrogen

[51], but in the second crystal structure (PDB 4RT) the imidazole

moiety is involved in water mediated, bidentate H-bonds with

backbone atoms of E692 and C694,⁵⁵explaining the similar potency

of both inhibitors (Table 1). In case of 15h, the nearly equipotent

structural isomer of 16 with exchanged benzofuran and indole

moieties, both benzofuran and methanone oxygens may form

hydrogen bonds with the backbone nitrogen of C694.

For the benzofuran moiety of 16, an antiperiplanar (ap, trans)

conformation was assumed which, on the one hand, avoids repul-

sion of the oxygen atoms. On the other hand, this conformation

enables interactions of the lipophilic benzofuran edge with a hy-

drophobic patch consisting of V675, F691 (gatekeeper), L818 and

F830 (DFG motif). The phenyl ring of the benzofuran moiety over-

laps with the central phenyl linker in quizartinib and contacts like

that F691 from one and F830 from the other side. Replacing the

benzofuran in 16 or the indole in 15e by a pyrrolopyrimidine

moiety, 15f eliminates interactions with F830 and reduces potency

by at least two orders of magnitude. However, this large effect may

be additionally due to conformational changes in the activation

loop behind the DFG motif, induced by the looser ﬁt of 15f between

F691 and F830.

Scheme 6. Synthesis of aryl-methanone derivatives 32b-c. (a) 23e, THF, 50 ^ꢁC.

both doses the fraction of cells in late stages of apoptosis and ne-

crosis increased up to 40% (Fig. 4).

The ureido and (tert-butyl)isoxazole moieties of 16 and AC220

(quizartinib, 7) also overlap. The carbonyl and an ureido nitrogen

form hydrogen bonds with the backbone NH of D829 (DFG motif)

and a carboxyl oxygen of E661, respectively. The side chain

conformation of E661 is stabilized by a salt bridge with K644 pre-

sent in other tyrosine kinases, too. M665 is directed towards the

isoxazole plane. The tert-butyl substituent ﬁts well into a hydro-

phobic speciﬁcity pocket consisting of M664, I674, L802 and I827

[51]. Obviously, these hydrophobic interactions of the tert-butyl

group contribute signiﬁcantly to the high inhibitory potency of 16

and AC220 (quizartinib, 7) since 15e is about two orders of

magnitude more potent than its methyl analogue 32c (Table 1).

The 5-hydroxyindole moiety of 16 is stacked between amino

acids of the hinge region, namely Y693 and the backbone of Y696

and G697. The 5-OH group points out of the ATP site. Larger polar 5-

substituents like the 1,4⁰-bipiperidine-1⁰-carbamate group in

2.5. Binding modes

Crystal structures of the FLT3 kinase domain in complex with

quizartinib 7 (PDB 4XUF [51], PDB 4RT7 [55]) may serve as tem-

plates to investigate the binding mode of 16 and its derivatives.

Based on previous models of FLT3-bisindolylmethanone in-

teractions [49] and on the ﬁt of the (tert-butyl)isoxazole moiety of

AC220 (quizartinib, 7) into a hydrophobic speciﬁcity pocket present

in both crystal structures,16 was docked into the ATP binding site of

FLT3 (PDB 4XUF). Fig. 5 shows the ﬁnal model. As illustrated in

Fig. 5A, the binding modes of AC220 (quizartinib, 7) and 16 closely

overlap. Both inhibitors align with the hinge region (F691 e Y696)

in the ATP binding pocket. The synperiplanar (sp, cis) conformation

of the indole moiety of 16 with respect to the methanone oxygen

enables a bidentate hydrogen bond with the backbone oxygen and

Scheme 7. Synthesis of carbamate derivative 17a and its hydrochloride 17b. (a) 1,4⁰-Bipiperidine-1⁰-carbonyl chloride, pyridine; b) HCl (ⁱProp), CH₂Cl₂, MeOH.

8

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

Table 1

Antiproliferative activity of bis(1H-indol-2-yl)methanones and derivatives determined by the trypan blue dye exclusion method with a Vi-CELL™ Cell Viability Analyzer. MV4-

11 cells were treated for 72 h with different concentrations.of inhibitor (N ¼ 3), ranging from 100 to 5 nM, respectively, from 100 to 0.5 nM for more active compounds. IC₅₀

values were calculated with Graphpad Prism. Inﬂuence on the enzymatic activity of FLT3 mutants.

No

Position

IC₅₀[nM]

15a

15b

15c

15d

4

5

6

7

>100

13.5 3.75

23.5 3.50

>100

Compound

X

Z

A

Y

R

IC₅₀[nM]

15e

15f

15h

15i

16

32b

32c

CH

N

CH

N

CH

NH

O

NH

O

BIZ

BTD

MIZ

1.7 0.41

>100

CH

0.78 0.65

29.8 0.59

0.64 0.86

14.4 0.66

>100

O

NH

Midostaurin (1)

21

Table 2

Inhibition of FLT3-ITD by 15be17b in the enzymatic assay in comparison to midostaurin 1 and AC220 (quizartinib, 7) as reference compounds. Compounds were tested by

Reaction Biology in a 10-dose IC₅₀duplicate mode with a 3-fold serial dilution starting at 10 or 1 M. Reactions were carried out at 10 M ATP.

m

No.

15b

133

15c

85

15h

11

15i

34

32b

16

17b

PKC412 (1)

AC220 (quizartinib, 7)

4.2 0.6

FLT3-ITD IC₅₀[nM]

FLT3 D835Y IC₅₀[nM]

2

5

1

2

12

28

1

5

2.3 0.2

4.84 0.4

3.9 0.1

3.03 0.1

0.73 0.01

0.15 0.02

3 530 320

2 055 165

240 53

2 075 515

137

7

(quizartinib, 7) bind to both FLT3 and FLT3-ITD with similar afﬁnity.

In case of the wild type, the inhibitors probably displace the jux-

tamembrane region from the inner binding pocket.

Quizartinib (7) is not very potent at the D835Y mutant (Table 2).

D835 belongs to the activation loop and does not participate in

inhibitor binding, but is adjacent to a hydrophobic patch in a model

of active FLT3 [51]. Hydrophobic D835 mutations may interact with

this patch, stabilizing the activation loop in an extended confor-

mation unfavorable for binding of AC220 (quizartinib, 7) and other

type II TKis. Thus, the high potency of compounds 16, 17b and

32 b at the D835Y mutant (Table 2) is surprising because of their

structural similarity with AC220 (quizartinib, 7) and may even be

due to the stabilization of a speciﬁc, active-like conformation.

Fig. 3. MV4-11 cells were treated with either 20 nM or 40 nM of 16 for 3 h. Western

blot analyses of whole-cell lysates were performed to detect p-FLT3, and Tubulin

served as loading control (n ¼ 4).

compound 17a adopt an outward position within the aqueous

environment like the morpholinoethoxy group of AC220 (qui-

zartinib, 7) and are therefore without signiﬁcant effect on potency

(Table 2).

16 and AC220 (quizartinib, 7) are type II TKis that bind to and

stabilize the inactive DFG-out conformation of FLT3. The data in

Table 2 show that both compounds are potent inhibitors of acti-

vating FLT3-ITD mutations. ITDs release the autoinhibitory in-

teractions of the juxtamembrane segment folded onto the kinase

domain in the inactive wild type [56]. The crystal structure of FLT3

in complex with AC220 (quizartinib, 7) indicates that binding of

this ligand is not compatible with the autoinhibitory juxtamem-

brane segment conformation [51]. Therefore, 16 and AC220

2.6. Determination of the kinase inhibitor proﬁle by proteomics

assay

In target based drug discovery of small molecules, protein ki-

nases are among the most frequently targeted proteins. Almost a

quarter of all kinases are involved in oncogenesis. Therefore, the

development of novel antitumor agents that selectively occupy the

highly conserved ATP binding pocket is a goal in cancer research. To

check the selectivity of our novel compounds, we used a chemical

proteomics assay for kinase inhibitor proﬁling (kinobeads), a

helpful tool to elucidate the target spectrum of a compound and to

anticipate possible side effects [57]. Brieﬂy, immobilized, broad-

spectrum small molecule kinase inhibitors enable puriﬁcation of

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

9

Fig. 4. MV4-11 cells were treated with 20 nM or 40 nM of 16 for 48 h. For analysis of apoptosis, Annexin V/PI staining was performed and measured by ﬂow cytometry, n ¼ 9 SD;

two-way ANOVA; Bonferroni’s multiple comparisons test; ****p ꢂ 0.0001.

endogenous kinases from tissues or cells. When performed as a

competition binding assay and measured by quantitative MS,

kinobeads allow the label free measurement of the physical inter-

action of a compound of interest with thousands of proteins in

parallel.

2.8. In vitro assessment of AC220 (quizartinib, 7), midostaurin 1,

and 17b in 27 selected hematological, human cancer cell lines

Compound 16 and its carbamate 17b, respectively, show strong

activity across the hematological cell lines tested. With a geometric

The selectivity of 16 was proﬁled in a cell mix consisting of a 1:

1: 1: 1 mixture of K562 (CML), Colo205 (human colon adenocar-

cinoma), MV4-11 (AML) and SKNBE2 (human neuroblastoma) ly-

sates. By use of a cell mix, the repertoire of kinases that can be

enriched is much larger than that of a single cell line.

From 249 identiﬁed kinases in addition to FLT3 only RET and

ZAK/MLTK are signiﬁcantly affected by 16 (Table 3). RET is

responsible for the differentiation of neurons in the autonomic

nervous system mainly in embryonic development and has no

function in the adult organism [58]. ZAK, on the other hand, be-

longs to the family of MAP kinase (mitogen-activated protein ki-

absolute mean IC₅₀value of 0.131

potent compound followed by 16 (0.337

AC220 (quizartinib, 7) (0.846 M).

All ﬁve highly sensitive cell lines EOL-1, MOLM-13, MV4-11,

MONO-MAC-1, MONO-MAC-6 exhibit FLT3 overexpression.

MONO-MAC-1, and MONO-MAC-6 have an activating point muta-

tion of FLT3 (V592A) in the juxtamembrane domain [64].

16 and its carbamate 17b achieved IC₅₀values in the sub

nanomolar till low nanomolar range in these cell lines and were

extremely speciﬁc. For the remaining AMLs, the compounds were

m

M Midostaurin 1 was the most

m

M), 17b (0.502 M) and

m

completely ineffective up to the highest dose of 30

mM. The IC₅₀for

nase pathway) and is part of the TGFb/JNK pathway [59], playing a

17b on the sensitive cell lines was 0.09e11 nM. In contrast it

role in the regulation of the cell cycle [60].

showed IC₅₀values between 1 and 5

indicating 100 to 1000-fold selectivity.

mM in the other cell lines,

Most interestingly, no signiﬁcant co-targeting of other RTKs

required for normal hematopoiesis was observed. The further 237

kinases studied in this experiment showed no reduced binding.

2.9. In vivo tolerability of 17b

To investigate possible toxicity effects in vivo, we started a

preliminary tolerability study using NMRI nu/nu mice. The mice

were given 75 mg/kg/day of 17b orally for 3 days. Daily observation

of the treated mice (n ¼ 6) showed no lethality and no impairment

of general condition till euthanasia on day 4. Moreover, a body

weight gain of 3% during the therapy from day 0e3 was observed.

In summary, no signs of toxicity were observed. Comparative data

were obtained for 17b and AC220 in the pharmacologic compari-

son, extended experiments which will be published in a forth-

coming paper by some of the authors.

2.7. Selectivity of 16 and 17b in cell viability assays

Additional experiments to assess the speciﬁcity of 16 were

performed. Other human blood cell lines that overexpress FLT3 or

carry FLT3-ITD as well as further hematological tumor cell lines

were treated with 16 and analyzed by using the CellTiter-Blue® Cell

Viability Assay. The cell line panel comprises 12 cell lines derived

from acute myeloid leukemia (AML) and 7 cell lines derived from

multiple myeloma (MM).

16 showed activity across the 19 hematological cell lines tested

with a geometric absolute mean IC₅₀value of 0.34

mM and a sig-

3. Conclusion

niﬁcant selectivity for the three highly sensitive cell lines EOL-1,

MOLM-13 and MV4-11. At least for MV4-11 and MOLM-13 an ITD

(internal tandem duplicate) of FLT3 was described [61] causing

strong sensitivity towards inhibitors of FLT3. These ﬁndings match

well the results presented in Table 1. The EOL-1 cell line represents

an AML cell line overexpressing wild-type FLT3 [62] and bearing a

PDGFRA mutation in addition [63], sensitizing it towards 7.

Regarding cell lines derived from multiple myeloma, the sensitivity

was clearly lower.

In enzymatic assays and in cell lines the new FLT3 inhibitor 16

and its carbamate derivative 17b show high efﬁciency, compa-

rable to AC220 (quizartinib, 7) in FLT3 activating mutations,

namely FLT3-ITD and activating point mutations. This activity is

accompanied by a high speciﬁcity for FLT3. No signiﬁcant co-

targeting of other RTKs required for normal hematopoiesis can

be observed.

Moreover, 16 and its carbamate 17b exhibit selectivity of 100

10

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

Fig. 5. Probable binding mode of 16 at FLT3. General atom colors, red, O, blue, N, yellow, S. (A) Overview of the binding site with 16 (C atoms grey), quizartinib (C atoms red orange)

and some important amino acids. Speciﬁc regions are highlighted on the ribbon/tube model of the backbone: blue, hinge region, yellow, nucleotide binding loop, orange, catalytic

loop, green, activation loop. Ball and stick model, side chains of M664, L802 and I827 as main components of the hydrophobic speciﬁty pocket. (B) Detailed view of the binding site

of 16 with all amino acids lying within a sphere of 3 Å around the ligand. C and polar H atoms blue, hinge region, orange, catalytic loop, green, activation loop, cyan, other amino

acids of the binding site, grey, 16. Red dashed lines, hydrogen bonds. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of

this article.)

4. Experimental section

Table 3

4.1. Materials and methods

Kinase

EC₅₀[nM]

Kinase

EC₅₀[nM]

RET

FLT3

MAP4K4

ZAK/MLTK

277

443

5737

1069

DDR2

2494

1768

3857

3255

4.1.1. Cell lines and primaries

CDK16

NTRK1

EPHA7

MV4-11 are human acute myeloid leukemia cells that carry a

FLT3-ITD mutation (American Type Culture Collection, ATCC

Accession No: CRL-9591).

Cells are cultured in Roswell Park Memorial Institute (RPMI)

1640 medium plus 10% fetal bovine serum and 100 U/mL penicillin

and 100 mg/mL streptomycin.

until 1000-fold for sensitive versus resistant cell lines. No signs of

toxicity were observed in a preliminary in vivo study.

Our data suggest that 16 or its carbamate derivative 17b,

respectively, might become a novel and well-tolerated TKi against

FLT3-ITD-positive AML.

4.1.2. Substances

The following commercially available substances and com-

pounds were used: AC220 (quizartinib, 7), Crenolanib, Midostaurin

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

11

(PKC412, 1) and Staurosporine (all substances provided by

Selleckchem).

compounds were applied at 10 concentrations in half-log in-

crements in duplicate in the range from 0.001 M to 30 M (AC220

(quizartinib, 7), 16) or from 0.00003 M to 1 M (Midostaurin).

Highly sensitive cell lines were tested at lower concentrations (up

to 0.1 M for all compounds). After four days of treatment of cells,

20 l/well CellTiter-Blue® reagent was added. Following incubation

m

4.1.3. Proliferation assay

m

Proliferation assays were performed as described before, uti-

lizing the Vi-Cell XR (Beckman Coulter, Munich, Germany) as

described earlier [65].

m

with CellTiter-Blue® reagent for up to 4 h, ﬂuorescence (FU) was

measured by using the Enspire Multimode Plate Reader (excitation

Proliferation/viability across a panel of hematological cell lines

(Tables 4 and 5) was assessed by using the CellTiter-Blue® Cell

Viability Assay (#8081, Promega, Mannheim, Germany). Brieﬂy,

cells were harvested from exponential phase cultures, counted and

plated in 96-well ﬂat-bottom microtiter plates at a cell density of

20,000e60,000 cells/well depending on the cell line’s growth rate.

After a 24 h recovery period to allow the cells to resume expo-

l

¼ 570 nm, emission

l

¼ 600 nm). For calculations, the mean

values of duplicate/sextuplicate (untreated control) data were used.

Cell lines were either kindly provided by the NCI (Bethesda; MD), or

were purchased from ATCC (Rockville, MD) or DSMZ (Braunsch-

weig, Germany). Authenticity of cell lines was proven at the DSMZ

by STR (short tandem repeat) analysis,

a PCR based DNA-

ﬁngerprinting methodology.

nential growth, 10 ml of culture medium (six control wells/plate) or

of culture medium with test compounds were added. The

Table 4

IC₅₀values of 16 in a panel of 19 hematological cell lines derived from patients with acute myeloid leukemia (LEXFAM) and multiple myeloma (MMXF). Individual IC₅₀values

were related to the geometric mean IC₅₀value illustrating more sensitive cell lines by green and more resistant cell lines by red color.

12

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

Table 5

IC₅₀values of Midostaurin (1), 17b and AC220 (quizartinib, 7) in a panel of 27 hematological cell lines derived from patients with acute myeloid leukemia (LEXFAM) and

multiple myeloma (MMXF). Individual IC₅₀values were related to the respective geometric mean IC₅₀value illustrating more sensitive cell lines by green and more resistant

cell lines by red color.

4.1.4. Western blot and antibodies

AnnexinV-FITC (Miltenyi) for 30 min. PI (Sigma) was added and

samples were measured immediately by the ﬂow cytometer BD

FACS canto.

Western blot was performed according to lit [66]. Brieﬂy, cells

were lysed in NET-N buffer, followed by soniﬁcation and centrifu-

gation, to remove cellular debris. Afterwards followed protein

determination. To denaturate proteins, sixfold sample buffer was

added to every lysat. Proteins were transferred to nitrocellulose

membrane and detected by ﬂuorescence coupled antibodies using

LI-COR Odyssey® Infrared Imaging System.

The following antibodies were used: FLT3 (SC-480; Santa Cruz

Biotechnology; Heidelberg, Germany), p-FLT3 (Y591) (3461, Cell

Signaling; Frankfurt, Germany) and Tubulin (ab176560; Abcam;

Berlin, Germany).

4.1.6. Statistics

Statistical analyses were performed with GraphPad Prism 6.

Signiﬁcance was determined with indicated statistical tests. As-

terisks are distributed by p-values (*p ꢂ 0.05; **p ꢂ 0.01;

***p ꢂ 0.001; and ****p ꢂ 0.0001).

4.2. Kinobead assay and liquid chromatography e tandem mass

spectrometry

4.1.5. Flow cytometry

Analysis was performed as described in [67]. In brief, MV4-

11 cells were harvested on ice, washed with PBS and incubated with

Kinobead assays were performed as described [57]. For details

see supporting information.

In vivo tolerability of 17b was investigated by charles rivers

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

13

laboratories Germany, Projekt No. P768A.

4.4.4. 5-(Benzyloxy)benzofuran-2-carbonyl chloride (19c)

K_dmeasurements for 16 and quizartinib (7) were performed

using the KINOMEscan assay platform (DiscoverX, San Diego,

United States). Assays were run in duplicate using an 11-point 3-

Preparation analogues to lit [68].

4.5. Preparation of aryl-methanone derivatives - synthesis of 20a-i.

General procedure 1

fold dilutional series starting at 10 mM inhibitor concentration.

4.3. Chemical procedures

A solution of the respective tert-butyl 1-(phenylsulfonyl)-1H-

indol-ylcarbamate (18a-b) (10.0 mmol) in THF (100 mL) was cooled

to ꢀ78 ^ꢁC under N₂and 2.1 eq. ^tBuli (21.0 mmol 1.6 M in pentane)

were added dropwise whilst stirring. The mixture was allowed to

warm up to ꢀ20 ^ꢁC within 3e4 h, cooled to ꢀ78 ^ꢁC again and

a ꢀ78 ^ꢁC cold solution of the respective 1-(phenylsulfonyl)-1H-

indole-2-carbonyl chloride (10.0 mmol) in THF (50.0 mL) was

added at once. The mixture was stirred for 30 min, poured into

water whilst stirring, extracted with ethyl acetate (3 x 50 mL), and

the solvents were removed under reduced pressure. The resulting

crude product was dissolved in THF (100 mL), MeOH (100 mL) and

aqueous NaOH (10%, 100 mL) were added, and the mixture was

reﬂuxed for 2 h. The organic solvent was removed under reduced

pressure, the precipitating product removed by ﬁltration or

extraction with ethyl acetate, puriﬁed by cc (CH₂Cl₂: ethyl acetate

6:1; dry load method) and crystallized from CH₂Cl₂by dissolving

and removing most of the solvent under reduced pressure.

4.3.1. General informationGeneral information

Solvents and reagents were of analytical grade, purchased from

commercial suppliers and used without further puriﬁcation. Thin

layer chromatography was performed on Merck silica gel 60 F₂₅₄

TLC aluminum plates. Geduran 60 (0.063e0.200 mm, Merck) was

used for column chromatography. ¹H NMR and ¹³C NMR spectra

were measured with a Bruker Avance 300 instrument (7.05 T, ¹H:

300 MHz, ¹³C: 75 MHz) and a Bruker Avance 400 instrument (9.4 T,

¹H: 400 MHz, ¹³C: 101 MHz) (Bruker, Karlsruhe, Germany), using

the respective solvent peak as an internal standard.

IR spectra (KBr) were reported with a Bruker Tensor 27 spec-

trometer. Melting points were determined with a Büchi B-545 in-

strument and are uncorrected. Low-resolution mass spectrometry

(MS) was performed with a Finnigen Thermo Quest TSQ 7000 in-

strument (Thermo Finnigan, San Jose, USA) with electrospray

ionization (ESI) coupled to an Agilent 1100 Series HPLC (Agilent

Technologies, Santa Clara, USA) or a Finnigan MAT SSQ 710 A with

electron ionization (EI). High-resolution mass spectrometry

(HRMS) was performed on an Agilent 6540 UHD Accurate-Mass Q-

TOF LC/MS system with electrospray ionization. HPLC was per-

formed using a waters 2545 Binary Gradient Module equipped with

a Waters Prep inject, a Waters 2489 UV/VIS Detector at 220 nm,

4.6. tert-Butyl (2-(4-methyl-1H-indole-2-carbonyl)-1H-indol-4-yl)

carbamate (20a)

Preparation from tert-butyl 1-(phenylsulfonyl)-1H-indol-4-

ylcarbamate (18a) and 4-methyl-1-(phenylsulfonyl)-1H-indole-2-

carbonyl chloride (19a) as described above. Yield: 3.70 g,

using

a

Phenomenex Kinetex 5U XB-C18 100A. Eluent A:

9.50 mmol (34%). Mp: 216.3 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3459, 3320,

H₂O þ CH₃CN (5%) þ TFA (0.1%), Eluent B: CH₃CN. Gradient: Eluent

A 55%/Eluent B 45% to Eluent A 20%, Eluent B 80% within 30 min at a

ﬂow of 18 mL/min.

1690. ¹H NMR (DMSO‑d₆): d ¼ 11.94 (s, 2H), 9.36 (s, 1H), 8.03 (s, 1H),

7.67e7.47 (m, 2H), 7.33 (d, J ¼ 8.3 Hz, 1H), 7.19 (m, 3H), 6.92 (d,

J ¼ 7.0 Hz, 1H), 2.62 (s, 3H), 1.54 (s, 9H). EI-MS (70eV) m/z (%): 390.2

(45) [MHþ]. Anal. (C₂₂H₂₃N₃O₃): Calcd. C 70.93, H 5.95, N 10.79.

Found: C 70.84, H 6.14, N 10.82. Cc: CH₂Cl₂:EtOAc 6:1.

All reactions were carried out under nitrogen. Elemental ana-

lyzes were performed by the Analytical Laboratory of the University

of Regensburg. Chemical names were created using ChemDraw

professional 15.0.0.107. All test compounds were conﬁrmed to be of

ꢃ95% purity.

4.7. tert-Butyl (2-(4-methyl-1H-indole-2-carbonyl)-1H-indol-5-yl)

carbamate (20b)

For experimental and analytical details of further prepared in-

termediates see supporting information.

Preparation from 4-methyl-1-(phenylsulfonyl)-1H-indole-2-

carbonyl chloride (19a) and tert-butyl 1H-indol-5-ylcarbamate

(18b) as described above. Yield: 0.40 g, 1.0 mmol (33%). Mp:

4.4. Preparation of carbonyl chlorides 19a-c

279.5 ^ꢁC. IR (KBr):

NMR (DMSO‑d₆):

1H), 7.62 (s, 2H), 7.38 (m, 2H), 7.32 (d, J ¼ 8.3 Hz, 1H), 7.19 (t,

J ¼ 7.6 Hz, 1H), 6.90 (d, J ¼ 6.9 Hz, 1H), 1.50 (s, 9H). EI-MS (70eV) m/z

(%): 390 (100) [M^þ$]. Anal. (C₂₃H₂₃N₃O₃): Calcd. C 70.93, H 5.95, N

10.79. Found: C 70.75, H 6.15, N 10.52.

n

(cm^ꢀ1) ¼ 3467, 3358, 3284, 1696, 1580, 1528. ¹H

¼ 11.90 (s, 1H), 11.80 (s, 1H), 9.24 (s, 1H), 7.91 (s,

4.4.1. 4-Methyl-1-(phenylsulfonyl)-1H-indole-2-carboxylic acid

Preparation from 4-methyl-1-(phenylsulfonyl)-1H-indole, ana-

logues to lit [49]. Yield: 20.5 g, 65.1 mmol (88%). Mp:

d

155.2e156.1 ^ꢁC. IR (KBr):

(DMSO‑d₆):

n

(cm^ꢀ1) ¼ 2918, 1702, 1548, 1169. ¹H NMR

d

(ppm) ¼ 2.44 (s, 3H), 7.11 (d, 1H, J ¼ 7.3 Hz), 7.35 (dd,

1H, J ¼ 7.5 Hz, 8.4 Hz), 7.40 (d, 1H, J ¼ 0.6 Hz), 7.61 (m, 2H), 7.71 (m,

1H), 7.83 (d,1H, J ¼ 8.5 Hz), 8.02 (m, 2H),13.58 (s,1H). þp ESI-MS m/

z (%): 316 (20) [MþH]^þ, 357 (100) [M þ NH^þ₄]^þ. -p ESI-MS m/z (%):

314 (5) [M ꢀ H]^-, 629 (100) [2M ꢀ H]^-, 360 (35) [M þ HCOO^ꢀ]^-. Anal.

(C₁₆H₁₃NO₄S): Calcd. C 60.94, H 4.16, N 4.44, S 10.17. Found: C 60.83,

H 4.25, N 4.24, S 10.13.

4.8. tert-Butyl (2-(4-methyl-1H-indole-2-carbonyl)-1H-indol-6-yl)

carbamate (20c)

Preparation from and 4-methyl-1-(phenylsulfonyl)-1H-indole-

2-carbonyl chloride (19a) and tert-butyl 1H-indol-6-ylcarbamate

(18c) as described above. Yield: 0.44 g, 1.1 mmol (37%). ¹H NMR

4.4.2. 4-Methyl-1-(phenylsulfonyl)-1H-indole-2-carbonyl chloride

(19a)

Preparation from 4-methyl-1-(phenylsulfonyl)-1H-indole-2-

carboxylic acid analogues to lit [49]. IR (KBr): (cm^ꢀ1) ¼ 3106,

1763. Mp: 116.0e118.0 ^ꢁC.

(DMSO‑d₆):

d

¼ 11.92 (s, 1H), 11.85 (s, 1H), 9.30 (s, 1H), 7.91 (s, 1H),

7.67 (t, J ¼ 9.7 Hz,1H), 7.58 (d, J ¼ 1.5 Hz,1H), 7.38 (m, 2H), 7.23e7.12

(m, 1H), 7.00 (dd, J ¼ 8.7, 1.8 Hz, 1H), 6.90 (d, J ¼ 7.0 Hz, 1H), 2.59 (s,

3H), 1.59 (s, 9H). Cc: CH₂Cl₂: EtOAc 6:1.

4.9. tert-Butyl (2-(4-methyl-1H-indole-2-carbonyl)-1H-indol-7-yl)

carbamate (20d)

4.4.3. 5-(Benzyloxy)-1-(phenylsulfonyl)-1H-indole-2-carbonyl

chloride (19b)

Preparation analogues to lit [49].

Preparation from and 4-methyl-1-(phenylsulfonyl)-1H-indole-

14

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

2-carbonyl chloride (19a) and tert-butyl 1H-indol-7-ylcarbamate

(18d) as described above. Yield: 0.22 g, 0.56 mmol (6%). Mp:

4.14. Deprotection of the tert-butyl carbamate group e synthesis of

21a-g. General Procedure 2

85.4 ^ꢁC. IR (KBr):

(DMSO‑d₆):

n

(cm^ꢀ1) ¼ 3331, 2926, 1693, 1582, 1512. ¹H NMR

d

¼ 11.94 (s, 2H), 9.46 (s,1H), 7.87 (d, J ¼ 7.8 Hz,1H), 7.72

The tert-butyl carbamate derivative was dissolved in TFA and

stirred at rt. After 15 min the reaction mixture was added to ice

water and alkalized with conz. NH₃(pH ¼ 9). The precipitating

product was ﬁltered off, washed with diluted NH₃and dried.

(d, J ¼ 2.0 Hz,1H), 7.67 (d, J ¼ 1.4 Hz,1H), 7.44 (d, J ¼ 8.0 Hz,1H), 7.33

(d, J ¼ 8.1 Hz, 1H), 7.24e7.16 (m, 1H), 7.08 (d, J ¼ 7.9 Hz, 1H), 6.90 (d,

J ¼ 6.9 Hz, 1H), 2.59 (s, 3H), 1.58 (s, 9H). ESI-MS (70eV) m/z (%):

390.2 (100) [MHþ].

4.15. (4-Amino-1H-indol-2-yl)(4-methyl-1H-indol-2-yl)methanone

(21a)

4.10. tert-Butyl (2-(5-(benzyloxy)-1H-indole-2-carbonyl)-1H-

indol-5-yl)carbamate (20e)

Preparation from 20a as described above. Yield: 0.15 g,

0.52 mmol (33%). ¹H NMR (DMSO‑d₆):

d

¼ 11.86 (s, 1H), 11.56 (s, 1H),

Preparation from tert-butyl 1-(phenylsulfonyl)-1H-indol-5-

ylcarbamate (18b) (3.72 g, 10.0 mmol) and 5-(benzyloxy)-1-(phe-

nylsulfonyl)-1H-indole-2-carbonyl chloride (19b) (10.0 mmol;

7.89 (s, 1H), 7.57 (s, 1H), 7.32 (d, J ¼ 8.5 Hz, 1H), 7.23e7.13 (m, 1H),

7.01e6.87 (m, 2H), 6.61 (dd, J ¼ 13.9, 8.0 Hz, 1H), 6.16 (d, J ¼ 7.5 Hz,

1H), 5.74 (s, 2H), 2.61 (s, 3H). EI-MS (70eV) m/z (%): 290 (100)

[Mþ$]. Anal. (C₁₈H₁₅N₃O x 1/₄H₂O): Calcd. C 73.58, H 5.32, N 14.30.

Found: C 73.82, H 5.26, N 14.14. Cc: CH₂Cl₂:EtOAc 10:1.

4.25 g) Yield: 1.65 g, 3.4 mmol (73%). Mp: 215.7e217.9 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3451, 3278, 2978,1665, 1527. ¹H NMR (DMSO‑d₆):

d

¼ 11.87 (s, 1H), 9.25 (s, 1H), 7.87 (s, 1H), 7.47 (dd, J ¼ 11.9, 9.0 Hz,

4H), 7.45e7.39 (m, 4H), 7.40e7.29 (m, 2H), 7.26 (d, J ¼ 2.2 Hz, 1H),

7.05 (dd, J ¼ 8.9, 2.3 Hz, 1H), 5.14 (s, 2H), 1.49 (s, 9H). ESI-MS (70eV)

m/z (%): 482.0 (100) [MH^þ]. Anal. (C₂₉H₂₇N₃O₄): Calcd. C 72.33, H

5.65, N 8.73. Found: C 72.22, H 5.83, N 8.75. Cc: CH₂Cl₂: EtOAc 5:1.

4.16. (5-Amino-1H-indol-2-yl)(4-methyl-1H-indol-2-yl)methanone

(21b)

Preparation from 20b as described above. Yield: 0.4 g, 1.4 mmol

(92%). Mp: 226.9e227.7 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3428, 3266, 1594,

1579, 1530. ¹H NMR (DMSO‑d₆):

d

¼ 11.85 (s, 1H), 11.50 (s, 1H), 7.37

4.11. tert-Butyl (2-(5-(benzyloxy)-1H-indole-2-carbonyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl) carbamate (20f)

(s, 1H), 7.55 (s, 1H), 7.31 (d, J ¼ 8.3 Hz, 1H), 7.23 (d, J ¼ 8.7 Hz, 1H),

7.18 (m, 1H), 6.89 (d, J ¼ 6.9 Hz, 1H), 6.81 (s, 1H), 6.75 (dd, J ¼ 1.4 Hz,

J ¼ 8.8 Hz, 1H), 2.58 (s, 3H). EI-MS (70eV) m/z (%): 290 (100) [Mþ$].

Anal. (C₁₈H₁₅N₃O x 1/₃H₂O): Calcd. C 73.20, H 5.35, N 14.23. Found:

C 73.16, H 5.22, N 14.29.

Preparation from 5-(benzyloxy)-1-(phenylsulfonyl)-1H-indole-

2-carbonyl chloride (19b) and tert-butyl (1-(phenylsulfonyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)carbamate (18e) as described above.

Yield: 3.4 g, 7.05 mmol (66%). Mp: 205e209 ^ꢁC. IR (KBr):

n

4.17. (6-Amino-1H-indol-2-yl)(4-methyl-1H-indol-2-yl)methanone

(21c)

(cm^ꢀ1) ¼ 3299, 1694, 1525. ¹H NMR (DMSO‑d₆):

d

¼ 12.34 (s, 1H),

11.86 (s,1H), 9.47 (s, 1H), 8.44 (d, J ¼ 2.2 Hz, 1H), 8.30 (s, 1H), 7.50 (d,

J ¼ 1.4 Hz, 1H), 7.48 (d, J ¼ 2.1 Hz, 2H), 7.42 (m, 4H), 7.37e7.31 (m,

1H), 7.27 (d, J ¼ 2.3 Hz, 1H), 7.07 (dd, J ¼ 8.9, 2.4 Hz, 1H), 1.50 (d,

J ¼ 9.1 Hz, 9H). ESI-MS (70eV) m/z (%): 483.2 (100) [MH^þ]. Anal.

(C₂₈H₂₆N₄O₄x ¼EtOAc): Calcd. C 69.07, H 5.55, N 11.11. Found: C

68.96, H 5.40, N 11.43.

Preparation from 30c as described above. Yield: 0.6 g, 2.1 mmol

(90%). The crude product was directly converted by the next step

without puriﬁcation.

4.18. (7-Amino-1H-indol-2-yl)(4-methyl-1H-indol-2-yl)methanone

(21d)

4.12. tert-Butyl (2-(5-(benzyloxy)benzofuran-2-carbonyl)-1H-

indol-5-yl)carbamate (20h)

Preparation from 20d as described above. Yield: 0.5 g, 1.7 mmol

(87%). The crude product was directly converted by the next step

without puriﬁcation.

Preparation from 5-(benzyloxy)benzofuran-2-carbonyl chloride

(19c) and tert-butyl (1-(phenylsulfonyl)-1H-indol-5-yl)carbamate

(18b) as described above. Yield: 2.48 g, 5.14 mmol (26%). Mp:

4.19. (5-Amino-1H-indol-2-yl)(5-(benzyloxy)-1H-indol-2-yl)

methanone (21e)

222.0 ^ꢁC. IR (KBr):

(DMSO‑d₆):

n

(cm^ꢀ1) ¼ 3419, 3301, 1712, 1606. ¹H NMR

d

¼ 11.93 (s, 1H), 9.27 (s, 1H), 7.92 (t, J ¼ 1.9 Hz, 2H),

Preparation from 20e as described above. Yield: 1.30 g,

7.81e7.70 (m, 2H), 7.56e7.46 (m, 2H), 7.47e7.30 (m, 6H), 7.27 (dd,

J ¼ 9.1, 2.6 Hz, 1H), 5.19 (s, 2H), 1.49 (s, 9H). ESI-MS (70eV) m/z (%):

483.2 (80) [MH^þ]. Anal. (C₂₉H₂₆N₂O₅):Calcd. C 72.18, H 5.43, N 5.81.

Found: C 72.17, H 5.63, N 5.65. SC: CH₂Cl₂: EtOAc 20:1.

3.43 mmol (62%). Mp:187.2e191.2 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3375,

3025,1526. ¹H NMR (DMSO‑d₆):

d

¼ 11.78 (s, 1H), 11.51 (s, 1H), 7.47

(t, J ¼ 9.7 Hz, 2H), 7.44e7.29 (m, 5H), 7.24 (dt, J ¼ 16.0, 4.8 Hz, 3H),

7.09e6.97 (m, 1H), 6.88e6.65 (m, 2H), 5.13 (s, 2H), 4.80 (s, 1H). ESI-

MS (70eV) m/z (%): 382.0 (100) [MH^þ]. Anal. (C₂₄H₁₉N₃O₂x ¼H₂O):

Calcd. C 74.69, H 5.09, N 10.89. Found: C 74.66, H 5.37, N 10.79.

4.13. tert-Butyl (2-(5-(benzyloxy)benzofuran-2-carbonyl)-1H-

pyrrolo[2,3-c]pyridin-5-yl) carbamate (20i)

4.20. (5-Amino-1H-pyrrolo[2,3-b]pyridin-2-yl)(5-(benzyloxy)-1H-

indol-2-yl)methanone (21f)

Preparation from 5-(benzyloxy)benzofuran-2-carbonyl chloride

(19c) and tert-butyl (1-(phenylsulfonyl)-1H-pyrrolo[2,3-c]pyridin-

5-yl)carbamate (18f) as described above. Yield: 1.2 g, 2.5 mmol

Preparation from 20f as described above. Yield: 0.70 g,

1.83 mmol (35%). Mp: 290.0 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3410, 1654,

(21%). ¹H NMR (Acetone-d₆):

d

¼ 8.71 (s, 1H), 8.38 (s, 1H), 8.26 (s,

1595. ¹H NMR (DMSO‑d₆):

d

¼ 11.96 (s, 1H), 11.81 (s, 1H), 7.99 (d,

1H), 7.92 (d, J ¼ 0.8 Hz, 1H), 7.85 (s, 1H), 7.73 (d, J ¼ 9.1 Hz, 1H), 7.54

(d, J ¼ 7.3 Hz, 2H), 7.48e7.39 (m, 3H), 7.38e7.30 (m, 2H), 5.23 (s,

2H), 1.54 (s, 9H). ESI-MS (70eV) m/z (%): 484 (100) [MHþ].

J ¼ 2.5 Hz,1H), 7.49 (d, J ¼ 6.9 Hz, 2H), 7.43 (s,1H), 7.39 (d, J ¼ 7.3 Hz,

3H), 7.34 (dd, J ¼ 6,3, 3.8 Hz, 1H), 7.23 (d, J ¼ 2.5 Hz, 1H), 7.05 (dd,

J ¼ 9.0, 2.4 Hz,1H), 5.13 (s, 2H), 5.04 (bs, 2H). ESI-MS (70eV) m/z (%):

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

15

383.2 (100) [MH^þ]. Anal. (C₂₃H₁₈N₄O₂x 2/₃H₂O): Calcd. C 70.04, H

4.94, N 14.20. Found: C 70.71, H 4.91, N 14.64.

1603, 1534. ¹H NMR (DMSO‑d₆):

d

¼ 11.61 (s, 1H), 11.47 (s, 1H), 8.97

(s, 1H), 7.35 (t, J ¼ 4.0 Hz, 1H), 7.29 (dd, J ¼ 11.9, 4.9 Hz, 2H), 7.21 (d,

J ¼ 8.7 Hz, 1H), 7.02 (t, J ¼ 7.7 Hz, 1H), 6.83 (dt, J ¼ 7.8, 3.9 Hz, 1H),

6.80e6.69 (m, 2H), 4.72 (bs, 2H). CI-MS (70eV) m/z (%): 292.0 (100)

[MH^þ]. Anal. (C₁₇H₁₃N₃O₂x 1/₆H₂O): Calcd. C 69.38, H 4.50, N 14.42.

Found: C 69.32, H 4.61, N 14.26.

4.21. Removal of the benzyloxy protection group e synthesis of 22e,

22h and 23e, 23h-i - general procedure 3

The appropriate benzyloxy-derivative was dissolved in

THF:MeOH (1:1), ammonium formate (4 eq) was added and the

mixture was heated to reﬂux. When this temp. was reached, a

catalytic amount of Pd on charcoal (10% Pd containing 50% H₂O)

was added and the reaction was monitored by TLC. After comple-

tion of the reaction, the mixture was ﬁltered off over a small pad of

sodium sulfate to remove the catalyst and an excess of ammonium

formate. The pad was washed with a small amount of EtOH twice.

To the clear yellow solution obtained water (100 mL) was added

and most of the organic solvent removed under reduced pressure

until the product precipitates as yellow crystals. The product was

removed by ﬁltration, washed with water, dried and crystallized

from CH₂Cl₂by dissolution in the necessary amount of CH₂Cl₂and

removing most of the solvent until sufﬁcient crystallization

appears.

4.26. (5-Amino-1H-indol-2-yl)(5-hydroxybenzofuran-2-yl)

methanone (23h)

Preparation from 22h as described above. Yield: 0.3 g,1.03 mmol

(88%). ¹H NMR (DMSO‑d₆):

J ¼ 0.7 Hz,1H), 7.58 (d, J ¼ 1.5 Hz,1H), 7.47 (d, J ¼ 9.5 Hz,1H), 7.31 (d,

J ¼ 8.9 Hz, 1H), 7.01 (d, J ¼ 2.2 Hz, 1H), 6.91e6.84 (m, 2H), 5.12 (s,

2H). ESI-MS (70eV) m/z (%): 293.1 (100) [MH^þ].

d

¼ 11.74 (s, 1H), 9.04 (s, 1H), 7.75 (d,

4.27. (5-Amino-1H-pyrrolo[2,3-c]pyridin-2-yl)(5-

hydroxybenzofuran-2-yl)methanone (23i)

Preparation from 22i as described above. Yield: 0.15 g, 0.5 mmol

(50%). ¹H NMR (DMSO‑d₆):

1H), 8.06 (s, 1H), 7.68e7.60 (m, 2H), 7.21e7.13 (m, 2H), 7.10 (dd,

J ¼ 8.9, 2.5 Hz, 1H). Recrystallization from THF. ESI-MS (70eV) m/z

(%): 294.1 (100) [MH^þ].

d

¼ 12.56 (s, 1H), 9.67 (s, 1H), 8.48 (s,

4.22. tert-Butyl-(2-(5-hydroxy-1H-indol-2-carbonyl)-1H-indol-5-

yl)carbamate (22e)

Preparation from 20e as described above. Yield: 7.43 g,

(19.0 mmol, 77%) yellow solid by crystallization from EtOAc. Mp:

225.5e225.7 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3529, 3345, 2982, 1585, 1534.

4.28. Preparation of the corresponding 5-(tert-butyl)-3-

isocyanatoisoxazole derivatives 15a-i e general procedure 4

¹H NMR (300 MHz, DMSO‑d₆):

d

¼ 11.78 (d, J ¼ 1.7 Hz, 1H, Indole-

NH), 11.66 (d, J ¼ 1.7 Hz, 1H, OH), 9.23 (s, 1H, Indole-NH), 8.98 (s, 1H,

NH), 7.86 (s, 1H, ArH), 7.48 (d, J ¼ 2.1 Hz, 1H, ArH), 7.42e7.27 (m, 4H,

ArH), 7.00 (d, J ¼ 2.3 Hz, 1H, ArH), 6.85 (dd, J ¼ 8.8, 2.3 Hz, 1H, ArH),

1.49 (s, 9H, (CH₃)₃). ESI-MS (120 eV) m/z (%): 392.0 (80) [MH^þ].

Anal. (C₂₂H₂₁N₃O₄): Calcd. C 67.51, H 5.41, N 10.74. Found. C 67.19, H

5.34, N 10.74.

Step A. 5-tert-butyl-3-isoxazolyl isocyanate (24a) was prepared

according to Lit [69]. as follows: To a solution of phosgene (1.5 mL,

2.0 M in toluene, 3.0 mmol) in anhydrous CH₂Cl₂(10 mL) was added

3-amino-5-tert-butylisoxazole (0.10 g, 0.71 mmol) followed by

pyridine (0.5 mL). The mixture was allowed to warm to room temp.

and stirred overnight (ca. 16 h), then the mixture was concentrated

in vacuo. The residue was dissolved in anh. THF (7 mL) and stirred

for 10 min. The precipitate was removed and the isocyanate-

containing ﬁltrate (approximately 0.1 M in THF) was used as a

stock solution.

Step B. The respective arylamine (0.5 mmol) was dissolved in a

septum sealed tube under N₂in the isocyanate-containing stock

solution (10.0 mL, 1.0 mmol) and pyridine (1.0 mL) was added. The

mixture was stirred at 20 ^ꢁC overnight, poured into water (20.0 mL)

and the yellow precipitate removed by ﬁltration. Column chroma-

tograpy (SiO₂, CH₂Cl₂/EtOAc; 2:1), respectively in the given solvent,

and crystallization by removal of most of the solvent under reduced

pressure afforded the desired product as yellow crystals.

4.23. tert-Butyl (2-(5-hydroxybenzofuran-2-carbonyl)-1H-indol-5-

yl)carbamate (22h)

Preparation from 20h as described above. Yield: 0.7 g,1.83 mmol

(35%). Mp: 217.3e220.2 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3311, 2978,1552. ¹H

NMR (DMSO‑d₆):

d

¼ 11.91 (s, 1H), 9.54 (s, 1H), 9.27 (bs, 1H), 7.90

(bs, 1H), 7.87 (d, J ¼ 0.7 Hz, 1H), 7.72 (d, J ¼ 2.0 Hz, 1H), 7.63 (d,

J ¼ 9.0 Hz, 1H), 7.38 (s, 2H), 7.12 (d, J ¼ 2.4 Hz, 1H), 7.03 (dd, J ¼ 8.9,

2.5 Hz, 1H), 1.50 (s, 9H). ESI-MS (70eV) m/z (%): 393.2 (100) [MH^þ].

Anal. (C₂₂H₂₀N₂O₅x1/₃H₂O): Calcd. C 66.32, H 5.23, N 7.03. Found: C

66.45, H 5.24, N 7.03.

4.24. tert-Butyl (2-(5-hydroxybenzofuran-2-carbonyl)-1H-pyrrolo

[2,3-c]pyridin-5-yl)carbamate (22i)

4.29. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(4-methyl-1H-indole-2-

carbonyl)-1H-indol-4-yl)urea (15a)

Preparation from 20i as described above. Yield: 0.40 g,

1.02 mmol (45%). ¹H NMR (DMSO‑d₆):

d

¼ 12.29 (s, 1H), 9.59 (s, 1H),

9.50 (s, 1H), 8.61 (s, 1H), 8.06 (s, 1H), 7.98 (d, J ¼ 7.6 Hz, 1H), 7.73 (d,

J ¼ 7.9 Hz, 1H), 7.63 (d, J ¼ 8.9 Hz, 1H), 7.14 (d, J ¼ 2.4 Hz, 1H), 7.06

(dd, J ¼ 8.9, 2.5 Hz, 1H), 1.49 (s, 9H). ESI-MS (70eV) m/z (%): 394

(100) [MH^þ]. Recrystallization from heptane. Anal. (C₂₁H₁₉N₃O₅x

1/₂Heptan): Calcd. C 66.35, H 6.14, N 9.47. Found: C 66.23, H 6.04, N

9.11.

Preparation from 21a as described above. Yield: 0.07 g,

0.15 mmol (33%). Mp: 265.9 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3471, 3230,

2966, 1739. ¹H NMR (DMSO‑d₆):

d

¼ 12.11 (d, J ¼ 2.4 Hz, 1H), 12.03

(d, J ¼ 2.4 Hz, 1H), 9.78 (s, 1H), 9.07 (s, 1H), 7.77 (d, J ¼ 7.6 Hz, 1H),

7.68 (d, J ¼ 1.9 Hz, 1H), 7.57 (d, J ¼ 2.2 Hz, 1H), 7.37e7.16 (m, 4H),

6.93 (d, J ¼ 6.9 Hz,1H), 6.56 (s,1H), 2.63 (s, 3H),1.32 (s, 9H). ¹³C NMR

(75 MHz, DMSO‑d₆):

d

¼ 180.81, 176.79, 158.85, 151.55, 138.75,

4.25. (5-Amino-1H-indol-2-yl)(5-hydroxy-1H-indol-2-yl)

methanone (23e)

138.09, 134.56, 134.25, 133.36, 132.21, 127.90, 126.74, 126.03, 120.76,

119.33, 110.73, 108.26, 108.19, 107.57, 106.57, 92.78, 32.97, 28.81,

19.05. ESI-MS (70eV) m/z (%): 456 (100) [MHþ]. Anal.

(C₂₆H₂₅N₅O₃): Calcd. C 68.56, H 5.53, N 15.37. Found: C 68.13, H 5.61,

N 15.56. Cc: CH₂Cl₂: EtOAc 10:1.

Preparation from 22e as described above. Yield: 0.82 g,

2.8 mmol (74%). Mp: 289.1e290.4 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3416,

16

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

4.30. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(4-methyl-1H-indole-2-

carbonyl)-1H-indol-5-yl)urea (15b)

0.64 mmol (97%). Mp: 245.0 ^ꢁC IR (KBr):

1634,1652,1606. ¹H NMR (DMSO‑d₆):

n

(cm^ꢀ1) ¼ 3419, 3301,

d

¼ 13.98 (s,1H),12.40 (s,1H),

11.71 (s, 1H), 10.23 (s, 1H), 9.01 (s, 1H), 8.49 (s, 1H), 8.31 (d,

J ¼ 2.1 Hz,1H), 7.51 (d, J ¼ 2.0 Hz,1H), 7.40 (d, J ¼ 1.8 Hz,1H), 7.32 (d,

J ¼ 5.4 Hz, 1H), 7.00 (t, J ¼ 2.6 Hz, 1H), 6.87 (dd, J ¼ 8.8, 2.3 Hz, 1H),

1.08 (s, 9H). ESI-MS (70eV) m/z (%): 461(100) [MH^þ]. Anal.

(C₂₄H₂₂N₆O₄xH₂O þ MeOH): Calcd. C 59.05, H 5.55, N 16.53. Found:

C 59.46, H 5.42, N 16.35. Cc: EtOAc:MeOH 20:1.

Preparation from 21b as described above. Yield: 0.05 g,

0.12 mmol (58%). ¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 11.92 (s, 1H),

11.88 (s, 1H), 9.46 (s, 1H), 8.76 (s, 1H), 7.96 (s, 1H), 7.63 (d, J ¼ 2.5 Hz,

2H), 7.45 (d, J ¼ 8.8 Hz,1H) 7.37e7.27 (m, 2H), 7.20 (t, J ¼ 7.6 Hz,1H),

6.90 (d, J ¼ 7.0 Hz,1H), 6.52 (s,1H), 2.59 (s, 3H),1.30 (s, 9H). ¹³C NMR

(DMSO‑d₆,101 MHz):

d

¼ 180.1, 176.5,158.6,151.7,137.6,135.1,134.4,

134.0, 132.0, 131.9, 127.6, 127.4, 125.5, 120.2, 119.5, 112.9, 111.5, 110.2,

109.3, 108.3, 92.4, 32.5, 28.4, 18.5. EI-MS (70eV) m/z (%): 456 (100)

[Mþ$]. Anal. (C₂₆H₂₇N₅O₄x 1H₂O): Calcd. C 65.95, H 5.75, N 14.79.

Found: C 65.99, H 5.72, N 14.96.

4.35. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(5-hydroxybenzofuran-

2-carbonyl)-1H-indol-5-yl)urea (15h)

Preparation from 23h as described above. Yield: 0.31g,

0.68 mmol (32%). Mp: 285.1e285.6 ^ꢁC IR (KBr):

n

(cm^ꢀ1) ¼ 3297,

4.31. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(4-methyl-1H-indole-2-

carbonyl)-1H-indol-6-yl)urea (15c)

2970, 1630. ¹H NMR (DMSO‑d₆):

d

¼ 11.98 (s, 1H), 9.54 (s, 1H), 9.47

(s, 1H), 8.77 (s, 1H), 7.95 (d, J ¼ 1.7 Hz, 1H), 7.88 (d, J ¼ 0.7 Hz, 1H),

7.74 (d, J ¼ 1.4 Hz,1H), 7.63 (d, J ¼ 9.0 Hz,1H), 7.44 (d, J ¼ 8.8 Hz,1H),

7.32 (dd, J ¼ 8.9, 2.0 Hz, 1H), 7.13 (d, J ¼ 2.4 Hz, 1H), 7.04 (dd, J ¼ 9.0,

2.5 Hz, 1H), 6.52 (s, 1H), 1.30 (d, J ¼ 4.5 Hz, 9H). ¹³C NMR (101 MHz,

Preparation from 21c as described above. Yield: 0.07 g,

0.15 mmol (20%). Mp: 281.4 ^ꢁC. IR (KBr):

y

(cm-1) ¼ 3283, 2969,

1728, 1595, 1564, 1552. ¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 11.89 (d,

DMSO‑d₆):

d

¼ 180.7 (C_q), 173.8 (C_q), 159.2 (C_q), 154.7 (C_q), 153.2

J ¼ 1.5 Hz, 1H), 11.85 (s, 1H), 9.48 (s, 1H), 8.98 (s, 1H), 7.92 (s, 1H),

7.69 (d, J ¼ 8.6 Hz, 1H), 7.60 (dd, J ¼ 18.1, 1.1 Hz, 2H), 7.33 (d,

J ¼ 8.3 Hz, 1H), 7.22e7.15 (m, 1H), 7.01 (dd, J ¼ 8.7, 1.6 Hz, 1H), 6.89

(d, J ¼ 7.0 Hz, 1H), 6.55 (s, 1H), 2.59 (s, 3H), 1.31 (s, 9H). ¹³C NMR

(C_q), 152.2 (C_q), 150.2 (C_q), 135.4 (C_q), 134.8 (C_q), 132.7 (C_q), 128.3

(C_q), 127.9 (C_q), 120.8 (þ, CH), 118.5 (þ, CH), 114.8 (þ, CH), 113.5 (þ,

CH),113.2 (þ, CH), 112.1 (þ, CH), 111.3 (þ, CH), 107.0 (þ, CH), 92.9 (þ,

CH, Isoxazol-CH), 28.8 (þ,(CH₃)₃). ESI-MS (70eV) m/z (%):459.2

(100) [MH^þ]. Anal. (C₂₅H₂₂N₄O₅): Calcd. C 65.49, H 4.84, N 12.22.

Found: C 65.02, H 4.95, N 11.81. Cc: EtOAc: CH₂Cl₂, 2:1.

(DMSO‑d₆, 101 MHz):

136.8, 134.4, 134.1, 131.9, 127.6, 125.2, 123.2, 123.2,120.1, 113.9, 110.2,

110.1, 107.7, 100.9, 92.5, 32.5, 28.4, 18.5. Cc: CH₂Cl₂: EtOAc 6:1.

d

¼ 180.2, 176.1, 158.4, 151.3, 138.5, 137.5,

4.36. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(5-hydroxybenzofuran-

2-carbonyl)-1H-pyrrolo[2,3-c]pyridin-5-yl)urea (15i)

4.32. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(4-methyl-1H-indole-2-

carbonyl)-1H-indol-7-yl)urea (15d)

Preparation from (23i) as described above. Cc: EtOAc: MeOH

Preparation from 21d as described above. Yield: 0.06 g,

10:1.Yield: 0.31g, 0.68 mmol (32%). Mp. 97.0e99.8 ^ꢁC. IR (KBr):

y

0.14 mmol (43%). Mp: 235.9 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3321, 2968,

(cm^ꢀ1) ¼ 2923,1606, 1510. ¹H NMR (400 MHz, DMSO- d₆):

¼ 12.35

d

1690, 1601, 1582, 1534. ¹H NMR (DMSO‑d₆):

d

¼ 11.94 (s, 1H), 11.61

(s, 1H), 9.57 (s, 1H), 9.31 (s, 1H), 8.66 (s, 1H), 8.06 (s, 1H), 7.99 (d,

J ¼ 0.7 Hz,1H), 7.74 (d, J ¼ 1.3 Hz, 1H), 7.64 (d, J ¼ 9.0 Hz, 1H), 7.15 (d,

J ¼ 2.4 Hz, 1H), 7.07 (dd, J ¼ 8.9, 2.5 Hz, 1H), 6.56 (s, 1H), 1.32 (s, 9H).

ESI-MS (120eV) m/z (%): 460.2 (100) [MH^þ]. HR-MS (120eV) m/z:

Calcd. 459.1537. Found 459.1579 (C₂₄H₂₁N₅O₅) [MH^þ].

(s, 1H), 9.58 (d, J ¼ 11.0 Hz, 1H), 9.02 (s, 1H), 7.73 (t, J ¼ 8.0 Hz, 1H),

7.68 (d, J ¼ 1.4 Hz, 1H), 7.55 (dt, J ¼ 10.5, 6.3 Hz, 2H), 7.33 (d,

J ¼ 8.3 Hz, 1H), 7.25e7.16 (m, 1H), 7.09 (dd, J ¼ 13.1, 5.3 Hz, 1H), 6.91

(d, J ¼ 6.9 Hz, 1H), 6.55 (s, 1H), 2.65e2.54 (m, 3H), 1.31 (d, J ¼ 3.2 Hz,

9H). ESI-MS (70eV) m/z (%): 465.1 (100) [MHþ]. Anal. (C₂₆H₂₅N₅O₃x

[22/₃H₂O): Calcd. C 66.80, H 5.68, N 14.98. Found: C 66.46, H 5.67, N

15.02. Cc: CH₂Cl₂:EtOAc 10:1.

4.37. 1-(2-(5-(Benzyloxy)-1H-indole-2-carbonyl)-1H-pyrrolo[2,3-

b]pyridin-5-yl)-3-(5-(tert-butyl)isoxazol-3-yl)urea (23f)

4.33. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(5-hydroxy-1H-indole-2-

carbonyl)-1H-indol-5-yl)urea (15e)

Preparation from 21f as described in general procedure 4. Yield

0.35 g (0.64 mmol, 24% brown solid) after cc (SiO₂, EtOAc: MeOH

20:1). Mp. 249.7e252.1 ^ꢁC (Decomposition). IR (KBr):

n

Preparation from 23e as described above. Yield: 0.80 g,

(cm^ꢀ1) ¼ 3260, 2967, 1653. ¹H NMR (300 MHz, DMSO‑d₆):

¼ 12.45

d

1.72 mmol (24%). Mp: 268.7 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3367, 2968,

(s, 1H, NH), 11.90 (s 1H, NH), 9.65 (s, 1H, NH), 8.92 (s, 1H, ArH), 8.43

(t, J ¼ 2.8 Hz, 1H, ArH), 8.38e8.31 (m, 1H, ArH), 7.52e7.45 (m, 4H,

ArH), 7.43 (s, 1H, ArH), 7.40 (d, J ¼ 1.5 Hz, 1H, ArH), 7.36e7.32 (m,

1H, ArH), 7.28 (d, J ¼ 2.3 Hz, 1H, ArH), 7.13e7.03 (m, 1H, ArH), 6.53

(s, 1H, Isoxazol-CH), 5.14 (s, 2H, OeCH₂-Ph), 1.30 (s, 9H, (CH₃)₃). ESI-

MS (120 eV) m/z (%): 549.2 (100) [MH^þ] (MW ¼ 548.22 g/mol).

Anal. (C₃₁H₂₈N₆O₄x 0.20 CH₂Cl₂): Calcd. C 66.26, H 5.06, N 14.86.

Found. C 66.42, H 5.08, N 14.88.

1627. ¹H NMR (DMSO‑d₆):

d

¼ 11.86 (d, J ¼ 1.2 Hz, 1H), 11.69 (d,

J ¼ 1.3 Hz, 1H), 9.46 (s, 1H), 9.00 (s, 1H), 8.75 (s, 1H), 7.91 (d,

J ¼ 1.5 Hz, 1H), 7.52e7.38 (m, 3H), 7.40e7.23 (m, 2H), 7.02 (d,

J ¼ 2.1 Hz, 1H), 6.86 (dd, J ¼ 8.8, 2.3 Hz, 1H), 6.52 (s, 1H), 1.30 (s, 9H).

¹³C NMR (101 MHz, DMSO‑d₆):

d

¼ 180.0 (C_q,C]O, Bisindol). 158.6

(C_q),176.4 (C_q),158.6 (C_q),151.8 (C_q),151.5 (C_q),135.2 (C_q),134.8 (C_q),

134.3 (C_q), 132.2 (C_q), 128.0 (C_q), 127.4 (C_q), 119.3 (þ, CH), 117.1 (þ,

CH), 113.2 (þ, CH), 112.8 (þ, CH), 111.2 (þ, CH), 109.0 (þ, CH), 108.6

(þ, CH), 104.9 (þ, CH), 92.5 (þ, CH, Isoxazol), 32.5 (C_q), 28.4

(þ,(CH₃)₃). ESI-MS (70eV) m/z (%): 458.0 (100) [MH^þ]. Anal.

(C₂₅H₂₃N₅O₄): Calcd. C 65.63, H 5.07, N 15.31. Found: C 65.13, H 4.95,

N 15.24. Cc: EtOAc: CH₂Cl₂10:1.

4.38. Reaction sequenze for synthesis of 16

4.38.1. 1-(5-Benzyloxy-1-phenylsulfonyl-1H-indol-2-yl)ethan-1-

one (27)

27 Was prepared from 5-(benzyloxy)-1-(phenylsulfonyl)-1H-

indole (26) [70] according to lit [71]. Yield: 10.2 g (28 mmol, 89%)

colorless solid after crystallization from EtOH. Mp.155.6e159.1 ^ꢁC.

4.34. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(5-hydroxy-1H-indole-2-

carbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)urea (15f)

IR (KBr):

n

(cm^ꢀ1) ¼ 1672, 1530. ¹H NMR (300 MHz, DMSO‑d₆):

Preparation from 23f according to general procedure 3 by

removal of the benzyloxy protection group. Yield: 0.29 g,

d

¼ 7.97 (d, J ¼ 9.2 Hz, 1H, ArH), 7.91 (s, 1H, ArH), 7.89 (t, J ¼ 1.7 Hz,

1H, ArH), 7.74e7.66 (m, 1H, ArH), 7.59 (dd, J ¼ 10.4, 4.8 Hz, 2H, ArH),

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

17

7.51 (s, 1H, ArH), 7.49e7.43 (m, 2H, ArH), 7.43e7.30 (m, 3H, ArH),

7.27 (d, J ¼ 2.5 Hz, 1H, ArH), 7.20 (dd, J ¼ 9.1, 2.6 Hz, 1H, ArH), 5.13 (s,

Pd (0.10 g, 10% on charcoal) were added. After consumption of the

starting material, the mixture was ﬁltered over a pad of celite, the

solution poured into water (300 mL) and the organic solvents

removed until the product precipitates. Yield 0.67 g (2.29 mmol,

2H, OeCH₂-Ph), 2.59 (s, 3H, CH₃). ¹H NMR (300 MHz, CDCl₃):

d 8.07

(d, J ¼ 9.2 Hz, 1H, ArH), 7.89 (dd, J ¼ 5.3, 3.4 Hz, 2H, ArH), 7.57e7.50

(m, 1H, ArH), 7.40 (m, 7H, ArH), 7.16 (dd, J ¼ 9.2, 2.5 Hz, 1H, ArH),

7.03 (d, J ¼ 2.5 Hz, 2H, ArH), 5.07 (s, 2H, OeCH₂-Ph), 2.62 (s, 3H,

95% orange solid). Mp. 228.4e231.2 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3453,

1612, 1518. ¹H NMR (300 MHz, DMSO‑d₆):

d

¼ 11.74 (s, 1H, Indole-

CH₃). ¹³C NMR (75 MHz, CDCl₃):

d

191.70 (s), 156.14 (s), 140.68 (s),

NH), 9.04 (s, 1H, OH), 7.75 (d, J ¼ 0.7 Hz, 1H, ArH), 7.58 (d,

J ¼ 1.5 Hz, 1H, ArH), 7.47 (m, 1H, ArH), 7.31 (m, 1H, ArH), 7.01 (d,

J ¼ 2.2 Hz, 1H, ArH), 6.88 (dd, J ¼ 4.8, 2.2 Hz, 1H, ArH), 6.86 (d,

J ¼ 2.5 Hz, 2H, ArH), 5.12 (s, 2H, NH₂). ESI-MS (120 eV) m/z (%):

137.94 (s), 136.66 (s), 133.78 (s), 133.72 (s), 129.44 (s), 128.83 (s),

128.67 (s), 128.14 (s), 127.51 (s), 127.25 (s), 117.96 (s), 117.83 (s),

116.90 (s), 105.70 (s), 70.51 (s), 29.72 (s). ESI-MS (120 eV) m/z (%):

406.1 (100) [MH^þ]. Anal. (C₂₃H₁₉NO₄S): Calcd. C 68.13, H 4.72, N

3.45, S 7.91. Found. C 67.98, H 4.76, N 3.37, S 7.71.

293.1 (100) [MH^þ]. ¹³C NMR (75 MHz, DMSO‑d₆)

d 173.03 (s), 152.17

(s), 151.45 (s), 148.62 (s), 145.51 (s), 133.91 (s), 132.90 (s), 127.91 (s),

127.57 (s), 117.69 (s), 117.55 (s), 113.77 (s), 113.25 (s), 112.08 (s),

109.73 (s), 104.80 (s), 104.07 (s).HR-MS (120 eV) m/z: Calcd.

293.0921. Found. 293.0924 (C₁₇H₁₃N₂O₃) [MH^þ]. Anal. (C₁₇H₁₇N₂O₃

x 0.4 DMSO): Calcd. C 66.08, H 4.49, N 8.66. Found. C 66.04, H 4.73,

N 8.14.

4.38.2. 1-(5-Benzyloxy-1-phenylsulfonyl-1H-indol-2-yl)-2-

bromoethan-1-one (28)

To a solution of 27 (11.18 g, 27.57 mmol) in THF (280 mL) was

added

trimethylphenylammonium

tribromide

(10.87

g,

28.95 mmol) portionwise (over 30 min). After consumption of the

starting material (TLC control) water was added. The mixture was

extracted with ethyl acetate (2x 200 mL) and the washed with brine

(200 mL). The organic layer was dried (Na₂SO₄) and the solvent

removed under reduced pressure. Yield 7.92 g (16.35 mmol, 59%)

after cc (SiO₂; light petrol, CH₂Cl₂2:1). Mp. 139.9e141.4 ^ꢁC. IR (KBr):

4.38.5. 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(2-(5-hydroxy-1H-indole-

2-carbonyl)benzofuran-5-yl)urea (16)

Step A. 5-tert-butyl-3-isoxazolyl isocyanate (24a) was prepared

according to Lit [69]. as follows: To a solution of phosgene (1.5 mL,

2.0 M in toluene, 3.0 mmol) in anhydrous CH₂Cl₂(10 mL) was added

3-amino-5-tert-butylisoxazole (0.10 g, 0.71 mmol) followed by

pyridine (0.5 mL). The mixture was allowed to warm to room temp.

and stirred overnight (ca. 16 h), then the mixture was concentrated

in vacuo. The residue was dissolved in dry THF (7 mL) and stirred for

10 min. The precipitate was removed and the isocyanate-

containing ﬁltrate (approximately 0.1 M in THF) was used as a

stock solution.

Step B. (5-Aminobenzofuran-2-yl)(5-hydroxy-1H-indol-2-yl)

methanone (26) (0.1 g, 0.34 mmol) was dissolved in a septum

sealed tube under nitrogen atmosphere in the isocyanate-

containing stock solution (6.8 mL, 0.64 mmol) and pyridine

(0.64 mL) was added. The mixture was stirred at r.t. overnight,

poured into water (20.0 mL) and the yellow precipitate removed by

ﬁltration. Column chromatograpy (SiO₂, CH₂Cl₂/MeOH; 20:1) and

crystallization from EtOAc afforded the desired product as yellow

crystals. Yield 0.077 g (0.17 mmol, 50%. Mp. 171.2e173.7 ^ꢁC

n

(cm^ꢀ1) ¼ 3454, 1672, 1530. ¹H NMR (300 MHz, DMSO‑d₆):

¼ 8.01

d

(ddd, J ¼ 14.1, 9.0, 3.2 Hz, 3H, ArH), 7.79e7.70 (m, 2H, ArH), 7.63 (td,

J ¼ 7.6, 4.2 Hz, 2H, ArH), 7.48 (t, J ¼ 7.0 Hz, 3H, ArH), 7.44e7.32 (m,

4H, ArH), 5.27 (s, 2H, OeCH₂-Ph), 5.02 (s, 2H, CH₂). ¹H NMR

(300 MHz, CDCl₃):

d

8.02 (d, J ¼ 9.1 Hz, 1H, ArH), 7.82e7.73 (m, 2H,

ArH), 7.57e7.48 (m, 1H, ArH), 7.45e7.30 (m, 7H, ArH), 7.16 (dd,

J ¼ 9.2, 2.6 Hz, 1H, ArH), 7.10 (d, J ¼ 0.7 Hz, 1H, ArH), 7.01 (d,

J ¼ 2.4 Hz, 1H, ArH), 5.05 (s, 2H, OeCH₂-Ph), 4.53 (s, 2H, CH₂). ¹³

C

NMR (75 MHz, CDCl₃)

d 186.86 (s), 156.43 (s), 137.23 (s), 136.40 (s),

136.28 (s), 134.08 (s), 133.25 (s), 129.78 (s), 128.88 (s), 128.60 (s),

128.11 (s), 127.42 (s), 127.22 (s), 119.78 (s), 118.36 (s), 116.86 (s),

105.73 (s), 70.42 (s), 33.82 (s). ESI-MS (120 eV) m/z (%): 484.02

⁷⁹BrC₂₃H₁₈NO₄S (91) [MH^þ]. 486.02 ⁸¹BrC₂₃H₁₈NO₄S (90) [MH^þ]

(MW ¼ 483.01 g/mol). Anal. (C₂₃H₁₈BrNO₄S): Calcd. C 57.03, H 3.75,

N 2.89, S 6.62. Found. C 57.04, H 3.76, N 2.62, S 6.51.

4.38.3. (5-Benzyloxy-1H-indol-2-yl)(5-nitrobenzofuran-2-yl)

methanone (30)

(decomposition). IR (KBr):

(300 MHz, DMSO‑d₆):

n

(cm^ꢀ1) ¼ 3454, 1636, 1577. ¹H NMR

d

¼ 11.81 (s, 1H, Indole-NH), 9.57 (s, 1H, NH),

A mixture of 2-hydroxy-5-nitrobenzaldehyde (29) (3.67 g,

7.58 mmol), K₂CO₃(1.05 g, 7.59 mmol) and 1-(5-benzyloxy-1-

phenylsulfonyl-1H-indol-2-yl)-2-bromethan-1-one (28) (1.27 g,

7.59 mmol) in 2-butanone (170 mL) was heated to reﬂux for 2 h.

The mixture was ﬁltered and the solvent removed under reduced

pressure. The crude solid obtained was dissolved in a mixture of in

THF/MeOH/NaOH_aqu. (10%) 1:1:1 (v/v/v) (150 mL) and heated to

reﬂux for 2 h. Water (100 mL) and EtOAc (30 mL) were added, the

precipitating product was removed by ﬁltration, washed with wa-

ter and dried. Yield 2.51 g (6.09 mmol, 80% over 2 steps of a yellow

9.07 (s, 1H, NH), 8.98 (s, 1H, OH), 8.07 (d, J ¼ 2.1 Hz, 1H, ArH), 7.96 (s,

1H, ArH), 7.76 (d, J ¼ 8.9 Hz, 1H, ArH), 7.64 (s, 1H, ArH), 7.50 (dd,

J ¼ 9.0, 2.2 Hz, 1H, ArH), 7.33 (d, J ¼ 8.8 Hz, 1H, ArH), 7.03 (s, 1H,

ArH), 6.90 (dd, J ¼ 8.8, 2.3 Hz, 1H, ArH), 6.53 (s, 1H, Isoxazole-CH),

1.31 (s, 9H, (CH₃)₃). ¹³C NMR (101 MHz, DMSO‑d₆):

d

¼ 180.6 (C_q).

180.2 (C_q), 173.1 (C_q), 158.4 (C_q), 157.9 (C_q), 153.0 (C_q), 151.6 (C_q),

135.3 (C_q), 133.9 (C_q), 133.2 (C_q), 128.0 (C_q), 127.2 (C_q), 120.7 (þ, CH),

118.1 (þ, CH), 114.3 (þ, CH), 113.4 (þ, CH), 112.5 (þ, CH), 112.0 (þ,

CH), 110.3 (þ, CH), 105.0 (þ, CH), 92.5 (þ, CH, Isoxazole-CH), 28.4

(þ,(CH₃)₃). ESI-MS (120 eV) m/z (%): 459.2 (100) [MH^þ]

(MW ¼ 458.16 g/mol). Anal. (C₂₅H₂₂N₄O₅x 0.33 EtOAc): Calcd. C

64.88, H 5.03, N 11.49. Found. C 64.67, H 5.13, N 11.21.

solid). Mp. 278.2e281.1 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3292, 2862, 1610,

¼ 12.08 (s, 1H, Indole NeH),

1557. ¹H NMR (300 MHz, DMSO‑d₆):

d

8.83 (d, J ¼ 2.4 Hz,1H, ArH), 8.44 (dd, J ¼ 9.1, 2.5 Hz,1H, ArH), 8.17 (s,

1H, ArH), 8.10 (d, J ¼ 9.1 Hz,1H, ArH), 7.76 (s,1H, ArH), 7.61e7.22 (m,

7H, ArH), 7.12 (dd, J ¼ 9.0, 2.4 Hz, 1H, Ar), 5.15 (s, 2H, OeCH₂-Ph).

ESI-MS (120 eV) m/z (%):413.1 (100) [MH^þ] (MW ¼ 412.11 g/mol).

Anal. (C₂₄H₁₆N₂O₅): Calcd. C 69.90, H 3.91, N 6.79. Found. C 69.72, H

3.95, N 6.74.

4.39. Preparation of 32b and 32c

Compounds 32b and 32c were prepared by the reaction of iso-

cyanates 24b and 24c with (5-amino-1H-indol-2-yl)(5-hydroxy-

1H-indol-2-yl)methanone (23e) as described above for 15a-i. The

used isocyanates 24b and 24d were obtained from the respective

arylamines by reaction with trichloromethyl chloroformate in THF

analogous to Lit [54]. Coloumn chromatograpy (SiO₂, CH₂Cl₂/EtOAc;

2:1), respectively in the given solvent, and crystallization by

removal of most of the solvent under reduced pressure afforded the

desired product as yellow crystals.

4.38.4. (5-Aminobenzofuran-2-yl)(5-hydroxy-1H-indol-2-yl)

methanone (31)

(5-Benzyloxy-1H-indol-2-yl)(5-nitrobenzofuran-2-yl)meth-

anone (30) (1.00 g, 2.42 mmol) in THF:MeOH (120 mL) (2:1) was

heated to reﬂux and ammonium formate (0.61 g, 9.68 mmol) and

18

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

4.39.1. 1-(5-tert-Butyl-1,3,4-thiadiazol-2-yl)-3-(2-(5-hydroxy-1H-

indole-2-carbonyl)-1H-indol-5-yl)urea (32b)

(101 MHz, DMSO‑d₆):

d

¼ 180.1, 173.3, 158.4, 153.4, 152.7, 151.8,

151.3, 145.2, 135.7, 135.6, 134.6, 127.2, 127.1, 121.5, 120.7, 114.8, 114.6,

113.1, 112.5, 111.6, 111.1, 92.5, 62.2, 48.9, 42.9, 32.5, 28.4, 25.4, 22.5,

21.6. ESI-MS (70 eV) m/z (%): 653.3 (100) [MH^þ]. RP-HPLC (220 nm):

99.0%, tR ¼ 5.6 min.

Yield: 0.9 g, 1.9 mmol (8%). Mp: 235.0 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3299, 2961, 1699, 1527. ¹H NMR (DMSO‑d₆):

d

¼ 11.90 (s,

1H), 11.69 (s, 1H), 10.21 (s, 1H), 9.06 (s, 1H), 8.99 (s, 1H), 7.95 (s, 1H),

7.62e7.27 (m, 5H), 7.01 (d, J ¼ 2.1 Hz, 1H), 6.86 (d, 1 H), 1.46 (s, 9H).

13C NMR (101 MHz, DMSO‑d₆):

d

¼ 176.8 (Cq, C]O, Bisindol), 152.0

4.40. Determination of the water solubility of 17b by UV-

measurements

(Cq), 135.8 (Cq), 135.2 (Cq), 135.0 (Cq), 133.2 (Cq), 128.5 (Cq), 127.8

(Cq), 119.2 (þ, CH), 116.9 (þ, CH), 113.0 (þ, CH), 112.6 (þ, CH), 108.8

(þ, CH), 108.4 (þ, CH), 104.6 (þ, CH), 35.9 (Cq), 30.9 (þ,(CH₃)₃). ESI-

MS (70eV) m/z (%): 475.0 (100) [MH^þ]. Anal. (C₂₄H₂₂N₆O₃S): Calcd.

C 60.75, H 4.67, N 17.71, S 6.76. Found: C 60.57, H 4.68, N 17.53, S

6.36.

To determine the water solubility of 17b a calibration curve was

created by a 5-point measurement of an aqueous solution of 17b

(fourfold determination) at the long wave absorption maximum of

368.2 nm in a range for E ¼ 0.050e0.742.

A saturated solution of 17b was prepared in the following by

subjecting 10 mg of 17b in water (10.00 mL) to ultrasound for 3 h.

Insoluble excess of 17b was allowed to set down on the ground and

the clear solution ﬁltered. The solution was diluted 1:20, resulting

in an extinction of 0.259 at 368.2 nm, which corresponds to a sol-

ubility of 0.23 mg/mL.

4.39.2. 1-(2-(5-Hydroxy-1H-indol-2-carbonyl)-1H-indol-5-yl)-3-

(5-methylisoxazol-3-yl) urea (32c)

Yield: 1.45g, 3.50 mmol (46%). Mp: 280.6e281.5 ^ꢁC. IR (KBr):

n

(cm^ꢀ1) ¼ 3343, 1526. ¹H NMR (DMSO‑d₆):

d

¼ 11.85 (d, J ¼ 1.5 Hz,

1H), 11.69 (t, J ¼ 7.2 Hz, 1H), 9.40 (s, 1H), 8.99 (s, 1H), 8.76 (s, 1H),

7.91 (d, J ¼ 1.8 Hz,1H), 7.52 (d, J ¼ 1.5 Hz,1H), 7.44 (d, J ¼ 8.8 Hz, 2H),

7.35e7.24 (m, 2H), 7.01 (d, J ¼ 2.2 Hz, 1H), 6.93e6.81 (m, 1H), 6.55

(d, J ¼ 0.8 Hz, 1H), 2.37 (d, J ¼ 0.6 Hz, 3H). ¹³C NMR (101 MHz,

In contrast, 1 mg of 16 was not completely soluble in 10 mL of

water.

DMSO‑d₆):

d

¼ 176.4 (C_q, C]O, Bisindol), 169.1 (C_q), 158.9 (C_q), 151.6

4.41. Molecular modeling

(C_q),151.5 (C_q),135.2 (C_q),134.8 (C_q),131.9 (C_q),128.0 (C_q),127.4 (C_q),

119.5 (þ, CH), 117.1 (þ, CH), 113.2 (þ, CH), 112.8 (þ, CH), 111.5 (þ,

CH), 109.0 (þ, CH), 108.6 (þ, CH), 104.8 (þ, CH), 95.5 (þ, CH,

Isoxazol-CH), 12.1 (þ, CH₃). ESI-MS (70eV) m/z (%): 416.0 (100)

[MH^þ]. Anal. (C₂₂H₁₇N₅O₄x ½EtOAc): Calcd. C 62.45, H 4.35, N

15.50. Found: C 62.05, H 4.55, N 15.95.

Docking studies were performed with the molecular modeling

package SYBYLx 2.0 (Certara, L.P., St. Louis, MO, USA) using the

crystal structure of FLT3 in complex with quizartinib 7 (PDB ID

4XUF, [51]).16 was manually docked based on the binding modes of

bisindolylmethanones [49] and quizartinib 7. A model of FLT3 in

complex with 16 was prepared as follows: Hydrogens were added

and charges were assigned (proteins and water molecules e

AMBER_FF99, ligands e Gasteiger-Hueckel). The complex was

reﬁned in two steps: ﬁrst, minimization with ﬁxed ligand using the

AMBER_FF99 force ﬁeld [72]; second, subset minimization of 16

and the surrounding (distance up to 6 Å) protein residues (Tripos

force ﬁeld [73]). In both minimization steps, the Powell conjugate

gradient method with termination at a root-mean-square force of

0.05 kcal/mol ꢄ Å^ꢀ1was applied.

4.39.3. 2-(5-(3-(5-(tert-Butyl)isoxazol-3-yl)ureido)benzofuran-2-

carbonyl)-1H-indol-5-yl [1,4⁰-bipiperidine]-1⁰-carboxylate (17a)

100 mg 16 (0.22 mmol) were dissolved in a mixture of CH₂Cl₂

(5 ml) and pyridine (5 ml). After addition of 86 mg 1,4⁰-bipiper-

idine-1⁰-carbonyl chloride (0.37 mmol) in 0.5 ml CH₂Cl₂the solu-

tion was stirred at r.t. for 16 h. The mixture was diluted with 40 ml

of CH₂Cl₂, washed with water and dried with Na₂SO₄. The solvent

was removed under reduced pressure. The residue was suspended

in ether and the beige crystals were ﬁltered off, washed with ether

and dried. Yield: 140 mg, 0.21 mmol (95%). Mp: 261.3e262.9 ^ꢁC. IR

Associated content

(KBr):

n

(cm^ꢀ1) ¼ 3279, 2931, 1711. ¹H NMR (DMSO‑d₆)

d 12.15 (d,

J ¼ 2.3 Hz, 1H), 9.58 (s, 1H), 9.01 (s, 1H), 8.09 (d, J ¼ 2.2 Hz, 1H), 8.01

(s,1H), 7.81e7.74 (m, 2H), 7.54e7.45 (m, 3H), 7.10 (dd, J ¼ 8.9, 2.3 Hz,

1H), 6.53 (s, 1H), 4.30e3.99 (m, 2H), 3.09e2.78 (m, 2H), 2.49e2.41

(m, 4H), 1.86e1.70 (m, 2H), 1.56e1.33 (m, 9H), 1.31 (s, 9H). ¹³C NMR

Supporting Information. Additional Schemes illustrating the

synthesis of intermediates as building blocks, NMR spectra and

performance of kinobead assay and liquid chromatography e tan-

dem mass spectrometry are supported. This material is available

free of charge via the Internet at http://pubs.acs.org.

(151 MHz, DMSO‑d₆):

d

¼ 180.1, 173.3, 158.4, 153.4, 152.7, 151.7,

151.3, 145.4, 135.7, 135.4, 134.6, 127.2, 127.1, 121.7, 120.9, 114.8, 114.6,

113.1, 112.5, 112.0, 111.1, 92.5, 61.4, 49.7, 43.5, 32.5, 28.4, 27.7, 26.0,

24.5. ESI-MS (70 eV) m/z (%): 653.3 (100) [MH^þ]. Anal. (C₃₆H₄₀N₆O₆

x H₂O) Calcd. C 64.46; H 6.31; N 12.53; Found: C 64.41; H 6.10; N

12.38.

Notes

The authors declare the following competing ﬁnancial interest:

Mahboobi, Siavosh; Sellmer, Andreas; Pongratz, Herwig; Pilsl,

€

Bernardette; Kramer, Oliver; Beyer, Mandy are also the inventors of

4.39.4. 2-(5-(3-(5-(tert-Butyl)isoxazol-3-yl)ureido)benzofuran-2-

carbonyl)-1H-indol-5-yl [1,4⁰-bipiperidine]-1⁰-carboxylate

hydrochloride (17b)

this entity: PCT Int. Appl. (2019), WO 2019034538 A1 20190221.

Funding

70 mg 17a (0.11 mmol) were dissolved in a mixture of CH₂Cl₂

(10 ml) and methanol (10 ml). After addition of 0.5 ml 6N HCl in 2-

propanol the mixture was stirred for 2 h at r.t. The solvent was

removed in vacuum and the residue was dried. Yield: 72 mg,

We thank the Deutsche Forschungsgemeinschaft (DFG) for

ﬁnancial support. Grant No. MA 2183/3-1, KR 2291/8-1.

0.10 mmol (90%). Mp: 173 ^ꢁC (decomp.). IR (KBr):

n

(cm^ꢀ1) ¼ 3370,

Contributors

3280, 2961, 1700. ¹H NMR (DMSO‑d₆)

d

12.17 (d, J ¼ 2.3 Hz, 1H),

10.27 (s, 1H), 9.73 (s, 1H), 9.47 (s, 1H), 8.10 (d, J ¼ 2.2 Hz, 1H), 8.02 (s,

1H), 7.87e7.69 (m, 2H), 7.58e7.43 (m, 3H), 7.12 (dd, J ¼ 8.8, 2.3 Hz,

1H), 6.54 (s, 1H), 4.44e4.07 (m, 2H), 3.51e3.33 (m, 4H), 3.10e2.83

(m, 4H), 2.24e2.06 (m, 2H), 1.91e1.64 (m, 7H), 1.31 (s, 9H). ¹³C NMR

The manuscript was written by contributions of all authors. All

authors have given approval to the ﬁnal version of the manuscript.

Andreas Sellmer, Bernadette Pilsl, Herwig Pongratz, Lukas

Wirth, and Siavosh Mahboobi were responsible for drug design and

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

19

[13] S. Kayser, M.J. Levis, FLT3 tyrosine kinase inhibitors in acute myeloid leuke-

mia: clinical implications and limitations, Leuk. Lymphoma 55 (2014)

243e255.

[14] S. Mahboobi, S. Teller, H. Pongratz, H. Hufsky, A. Sellmer, A. Botzki, A. Uecker,

T. Beckers, S. Baasner, C. Schaechtele, F. Ueberall, M.U. Kassack, S. Dove, F.-

D. Boehmer, Bis(1H-2-indolyl)methanones as a novel class of inhibitors of the

platelet-derived growth factor receptor kinase, J. Med. Chem. 45 (2002)

1002e1018.

synthesis.

Sigurd Elz was responsible for revision of the manuscript,

careful re-reading and helpful discussions of chemical and biolog-

ical data.

€

Mandy Beyer, Sven Julian Henninger, Oliver H. Kramer and Frank

€

D. Bohmer were responsible for western blotting, annexin staining,

[15] C.C. Smith, Q. Wang, C.-S. Chin, S. Salerno, L.E. Damon, M.J. Levis, A.E. Perl,

K.J. Travers, S. Wang, J.P. Hunt, P.P. Zarrinkar, E.E. Schadt, A. Kasarskis,

J. Kuriyan, N.P. Shah, Validation of ITD mutations in FLT3 as a therapeutic

target in human acute myeloid leukaemia, Nature 485 (2012) 260e263.

[16] S. Schenone, C. Brullo, M. Botta, Small molecules ATP-competitive inhibitors of

FLT3: a chemical overview, Curr. Med. Chem. 15 (2008) 3113e3132.

[17] S. Yokota, H. Kiyoi, M. Nakao, T. Iwai, S. Misawa, T. Okuda, Y. Sonoda, T. Abe,

K. Kahsima, Y. Matsuo, T. Naoe, Internal tandem duplication of the FLT3 gene

is preferentially seen in acute myeloid leukemia and myelodysplastic syn-

drome among various hematological malignancies. A study on a large series of

patients and cell lines, Leukemia 11 (1997) 1605e1609.

interpretation of biochemical experiments and review of the bio-

logical part.

Karsten Spiekermann and Harald Polzer were responsible for

proliferation assays in the human MV4-11 AML cell line.

Susan Klaeger and Bernhard Kuster were responsible for

determination and interpretation of the kinase inhibitor proﬁle by

proteomics assay.

Heinz-Herbert Fiebig was responsible for determination and

interpretation of the selectivity in cell viability assays.

Stefan Dove performed the modeling calculations.

[18] S. Frohling, R.F. Schlenk, J. Breitruck, A. Benner, S. Kreitmeier, K. Tobis,

H. Dohner, K. Dohner, Prognostic signi ﬁ cance of activating FLT3 mutations in

younger adults (16 to 60 years) with acute myeloid leukemia and normal

cytogenetics:

4372e4380.

a study of the AML study group Ulm, Blood 100 (2002)

Declaration of competing interests

[19] F.M. Abu-Duhier, A.C. Goodeve, G.A. Wilson, R.S. Care, I.R. Peake, J.T. Reilly,

Identi ﬁ cation of novel FLT-3 Asp835 mutations in adult acute myeloid

leukaemia, Br. J. Haematol. 113 (2001) 983e988.

[20] C. Reindl, K. Bagrintseva, S. Vempati, S. Schnittger, J.W. Ellwart, K. Wenig, K.-

P. Hopfner, W. Hiddemann, K. Spiekermann, Point mutations in the juxta-

membrane domain of FLT3 de ﬁ ne a new class of activating mutations in AML,

Blood 107 (2006) 3700e3707.

[21] D. Schmidt-Arras, S.-A. Bohmer, S. Koch, J.P. Muller, L. Blei, H. Cornils, R. Bauer,

S. Korasikha, C. Thiede, F.-D. Bohmer, Anchoring of FLT3 in the endoplasmic

reticulum alters signaling quality, Blood 113 (2009) 3568e3576.

[22] H. Konig, C.D. Santos, Signal transduction in acute myeloid leukemia - im-

plications for novel therapeutic concepts, Curr. Cancer Drug Targets 15 (2015)

803e821.

The authors declare the following ﬁnancial interests/personal

relationships which may be considered as potential competing

interests: Mahboobi, Siavosh; Sellmer, Andreas; Pongratz, Herwig;

€

Pilsl, Bernardette; Kramer, Oliver; Beyer, Mandy are also the in-

ventors of this entity: PCT Int. Appl. (2019), WO 2019034538 A1

20190221.

Acknowledgments

[23] M.R. Grunwald, M.J. Levis, FLT3 tyrosine kinase inhibition as a paradigm for

targeted drug development in acute myeloid leukemia, Semin. Hematol. 52

(2015) 193e199.

We thank Prof. Dr. Dr. Wolfgang Wiegrebe for his discussion.

[24] T. Kindler, D.B. Lipka, T. Fischer, FLT3 as a therapeutic target in AML: still

challenging after all these years, Blood 116 (2010) 5089e5102.

[25] A. Fathi, M. Levis, FLT3 inhibitors: a story of the old and the new, Curr. Opin.

Hematol. 18 (2011) 71e76.

[26] M.R. Luskin, D.J. DeAngelo, Midostaurin/PKC412 for the treatment of newly

diagnosed FLT3 mutation-positive acute myeloid leukemia, Expet Rev. Hem-

atol. 10 (2017) 1033e1045.

[27] B.D. Smith, M. Levis, M. Beran, F. Giles, H. Kantarjian, K. Berg, K.M. Murphy,

T. Dauses, J. Allebach, D. Small, Single-agent CEP-701, a novel FLT3 inhibitor,

shows biologic and clinical activity in patients with relapsed or refractory

acute myeloid leukemia, Blood 103 (2004) 3669e3676.

[28] S. Knapper, A.K. Burnett, T. Littlewood, W.J. Kell, S. Agrawal, R. Chopra,

R. Clark, M.J. Levis, D. Small, A phase 2 trial of the FLT3 inhibitor lestaurtinib

(CEP701) as ﬁ rst-line treatment for older patients with acute myeloid leu-

kemia not considered ﬁ t for intensive chemotherapy, Blood 108 (2006)

3262e3270.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.112232.

References

[1] U. Creutzig, d.H.-E.M. M. van, B. Gibson, M.N. Dworzak, S. Adachi, B.E. de,

J. Harbott, H. Hasle, D. Johnston, A. Kinoshita, T. Lehrnbecher, G. Leverger,

E. Mejstrikova, S. Meshinchi, A. Pession, S.C. Raimondi, L. Sung, J. Stary,

C.M. Zwaan, G.J.L. Kaspers, D. Reinhardt, Diagnosis and management of acute

myeloid leukemia in children and adolescents: recommendations from an

international expert panel, Blood 120 (2012) 3187e3205.

[2] A.Y.H. Leung, C.H. Man, Y.L. Kwong, FLT3 inhibition: a moving and evolving

target in acute myeloid leukaemia, Leukemia 27 (2013) 260e268.

[3] M.S. Tallman, D.G. Gilliland, J.M. Rowe, Drug therapy for acute myeloid leu-

kemia, Blood 106 (2005) 1154e1163.

[4] R. Berenstein, Class III receptor tyrosine kinases in acute leukemia - biological

functions and modern laboratory analysis, Biomark. Insights 10 (2015) 1e14.

[5] J. Xu, E.H.Q. Ong, J. Hill, A. Chen, C.L.L. Chai, Design, synthesis and biological

evaluation of FLT3 covalent inhibitors with a resorcylic acid core, Bioorg. Med.

Chem. 22 (2014) 6625e6637.

[6] D. Small, M. Levenstein, E. Kim, C. Carow, S. Amin, P. Rockwell, L. Witte,

C. Burrow, M.Z. Ratajczak, et al., STK-1, the human homolog of Flk-2/Flt-3, is

selectively expressed in CD34 þ human bone marrow cells and is involved in

the proliferation of early progenitor/stem cells, Proc. Natl. Acad. Sci. U. S. A 91

(1994) 459e463.

[29] F.J. Giles, A.T. Stopeck, L.R. Silverman, J.E. Lancet, M.A. Cooper, A.L. Hannah,

J.M. Cherrington, A.-M. O ’Farrell, H.A. Yuen, S.G. Louie, W. Hong, J.E. Cortes,

S. Verstovsek, M. Albitar, S.M. O ’Brien, H.M. Kantarjian, J.E. Karp, SU5416, a

small molecule tyrosine kinase receptor inhibitor, has biologic activity in

patients with refractory acute myeloid leukemia or myelodysplastic syn-

dromes, Blood 102 (2003) 795e801.

[30] D. Bensinger, D. Stubba, A. Cremer, V. Kohl, T. Wassmer, J. Stuckert,

V. Engemann, K. Stegmaier, K. Schmitz, B. Schmidt, Virtual screening identi ﬁ es

irreversible FMS-like tyrosine kinase

3 inhibitors with activity toward

resistance-conferring mutations, J. Med. Chem. 62 (2019) 2428e2446.

[31] T. Yamaura, T. Nakatani, K. Uda, H. Ogura, W. Shin, N. Kurokawa, K. Saito,

N. Fujikawa, T. Date, M. Takasaki, D. Terada, A. Hirai, A. Akashi, F. Chen,

Y. Adachi, Y. Ishikawa, F. Hayakawa, S. Hagiwara, T. Naoe, H. Kiyoi, A novel

irreversible FLT3 inhibitor, FF-10101, shows excellent ef ﬁ cacy against AML

cells with FLT3 mutations, Blood 131 (2018) 426e438.

[7] S.D. Lyman, Biology of ﬂ t3 ligand and receptor, Int. J. Hematol. 62 (1995)

63e73.

[32] C. Ustun, D.L. DeRemer, A.P. Jillella, K.N. Bhalla, Investigational drugs targeting

FLT3 for leukemia, Expet Opin. Invest. Drugs 18 (2009) 1445e1456.

[33] W.-H. Lin, J.T.A. Hsu, S.-Y. Hsieh, C.-T. Chen, J.-S. Song, S.-C. Yen, T. Hsu, C.-

T. Lu, C.-H. Chen, L.-H. Chou, Y.-N. Yang, C.-H. Chiu, C.-P. Chen, Y.-J. Tseng, K.-

J. Yen, C.-F. Yeh, Y.-S. Chao, T.-K. Yeh, W.-T. Jiaang, Discovery of 3-phenyl-1H-

5-pyrazolylamine derivatives containing a urea pharmacophore as potent and

ef ﬁ cacious inhibitors of FMS-like tyrosine kinase-3 (FLT3), Bioorg. Med. Chem.

21 (2013) 2856e2867.

[34] M.M. Schittenhelm, K.M. Kampa, K.W.H. Yee, M.C. Heinrich, The FLT3 inhibitor

tandutinib (formerly MLN518) has sequence-independent synergistic effects

with cytarabine and daunorubicin, Cell Cycle 8 (2009) 2621e2630.

[35] K.W. Pratz, J. Cortes, G.J. Roboz, N. Rao, O. Arowojolu, A. Stine, Y. Shiotsu,

A. Shudo, S. Akinaga, D. Small, J.E. Karp, M. Levis, A pharmacodynamic study of

the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response,

[8] S. Meshinchi, F.R. Appelbaum, Structural and functional alterations of FLT3 in

acute myeloid leukemia, Clin. Canc. Res. 15 (2009) 4263e4269.

[9] D.G. Gilliland, J.D. Grif ﬁ n, The roles of FLT3 in hematopoiesis and leukemia,

Blood 100 (2002) 1532e1542.

[10] C.-H. Heldin, Dimerization of cell surface receptors in signal transduction, Cell

80 (1995) 213e223.

[11] M. Nakao, S. Yokota, T. Iwai, H. Kaneko, S. Horiike, K. Kashima, Y. Sonoda,

T. Fujimoto, S. Misawa, Internal tandem duplication of the ﬂ t3 gene found in

acute myeloid leukemia, Leukemia 10 (1996) 1911e1918.

[12] Y. Yamamoto, H. Kiyoi, Y. Nakano, R. Suzuki, Y. Kodera, S. Miyawaki, N. Asou,

K. Kuriyama, F. Yagasaki, C. Shimazaki, H. Akiyama, K. Saito, M. Nishimura,

T. Motoji, K. Shinagawa, A. Takeshita, H. Saito, R. Ueda, R. Ohno, T. Naoe,

Activating mutation of D835 within the activation loop of FLT3 in human

hematologic malignancies, Blood 97 (2001) 2434e2439.

20

A. Sellmer et al. / European Journal of Medicinal Chemistry 193 (2020) 112232

Blood 113 (2009) 3938e3946.

[54] S. Bhagwat, Q. Chao, R.M. Grotzfeld, H.K. Patel, K.G. Sprankle, Preparation of

Imidazolothiazole Compounds for Treatment of Proliferative Diseases or

Autoimmune Diseases, WO2007109120A2, 2007.

[36] Q. Chao, K.G. Sprankle, R.M. Grotzfeld, A.G. Lai, T.A. Carter, A.M. Velasco,

R.N. Gunawardane, M.D. Cramer, M.F. Gardner, J. James, P.P. Zarrinkar,

H.K. Patel, S.S. Bhagwat, Identi ﬁ cation of N-(5-tert-Butyl-isoxazol-3-yl)-N ’-{4-

[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}

urea dihydrochloride (AC220), a uniquely potent, selective, and ef ﬁ cacious

FMS-like tyrosine kinase-3 (FLT3) inhibitor, J. Med. Chem. 52 (2009)

7808e7816.

[37] E.I. Zimmerman, D.C. Turner, J. Buaboonnam, S. Hu, S. Orwick, M.S. Roberts,

L.J. Janke, A. Ramachandran, C.F. Stewart, H. Inaba, S.D. Baker, Crenolanib is

active against models of drug-resistant FLT3-ITD-positive acute myeloid leu-

kemia, Blood 122 (2013) 3607e3615.

[55] C.C. Smith, C. Zhang, K.C. Lin, E.A. Lasater, Y. Zhang, E. Massi, L.E. Damon,

M. Pendleton, A. Bashir, R. Sebra, A. Perl, A. Kasarskis, R. Shellooe, G. Tsang,

H. Carias, B. Powell, E.A. Burton, B. Matusow, J. Zhang, W. Spevak, P.N. Ibrahim,

M.H. Le, H.H. Hsu, G. Habets, B.L. West, G. Bollag, N.P. Shah, Characterizing and

overriding the structural mechanism of the quizartinib-resistant FLT3 "gate-

keeper" F691L mutation with PLX3397, Canc. Discov. 5 (2015) 668e679.

[56] J. Grif ﬁ th, J. Black, C. Faerman, L. Swenson, M. Wynn, F. Lu, J. Lippke, K. Saxena,

The structural basis for autoinhibition of FLT3 by the juxtamembrane domain,

Mol. Cell. 13 (2004) 169e178.

[38] A. Galanis, H. Ma, T. Rajkhowa, A. Ramachandran, D. Small, J. Cortes, M. Levis,

Crenolanib is a potent inhibitor of FLT3 with activity against resistance-

conferring point mutants, Blood 123 (2014) 94e100.

[39] C.C. Smith, E.A. Lasater, K.C. Lin, Q. Wang, M.Q. McCreery, W.K. Stewart,

L.E. Damon, A.E. Perl, G.R. Jeschke, M. Sugita, M. Carroll, S.C. Kogan, J. Kuriyan,

N.P. Shah, Crenolanib is a selective type I pan-FLT3 inhibitor, Proc. Natl. Acad.

Sci. U. S. A. 111 (2014) 5319e5324.

[57] G. Medard, F. Pachl, B. Ruprecht, S. Klaeger, S. Heinzlmeir, D. Helm, H. Qiao,

X. Ku, M. Wilhelm, T. Kuehne, Z. Wu, A. Dittmann, C. Hopf, K. Kramer,

B. Kuster, Optimized chemical proteomics assay for kinase inhibitor pro ﬁ ling,

J. Proteome Res. 14 (2015) 1574e1586.

[58] Y. Honma, M. Kawano, S. Kohsaka, M. Ogawa, Axonal projections of mecha-

noreceptive dorsal root ganglion neurons depend on Ret, Development 137

(2010) 2319e2328.

[40] H.K. Lee, H.W. Kim, I.Y. Lee, J. Lee, J. Lee, D.S. Jung, S.Y. Lee, S.H. Park, H. Hwang,

J.-S. Choi, J.-H. Kim, S.W. Kim, J.K. Kim, J. Cools, J.S. Koh, H.-J.G. Song, 749, a

novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of

acute myeloid leukemia, Blood 123 (2014) 2209e2219.

[41] H. Ma, B. Nguyen, L. Li, S. Greenblatt, A. Williams, M. Zhao, M. Levis, M. Rudek,

A. Duf ﬁ eld, D. Small, TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with

activity against FLT3-associated leukemias in vitro and in vivo, Blood 123

(2014) 1525e1534.

[59] H. Vin, G. Ching, S.S. Ojeda, C.H. Adelmann, V. Chitsazzadeh, D.W. Dwyer,

H. Ma, K. Ehrenreiter, M. Baccarini, R. Ruggieri, J.L. Curry, A.M. Ciurea,

M. Duvic, N.L. Busaidy, N.M. Tannir, K.Y. Tsai, Sorafenib suppresses JNK-

dependent apoptosis through inhibition of ZAK, Mol. Canc. Therapeut. 13

(2014) 221e229.

[60] J.-J. Yang, A novel zinc ﬁ nger protein, ZZaPK, interacts with ZAK and stimu-

lates the ZAK-expressing cells re-entering the cell cycle, Biochem. Biophys.

Res. Commun. 301 (2003) 71e77.

[42] A.S. Moore, A. Faisal, d.C.D. Gonzalez, V. Bavetsias, C. Sun, B. Atrash, M. Valenti,

H.B.A. de, S. Avery, D. Mair, F. Mirabella, J. Swansbury, A.D.J. Pearson,

P. Workman, J. Blagg, F.I. Raynaud, S.A. Eccles, S. Linardopoulos, Selective FLT3

inhibition of FLT3-ITD þ acute myeloid leukaemia resulting in secondary

D835Y mutation: a model for emerging clinical resistance patterns, Leukemia

26 (2012) 1462e1470.

[43] N.L. Lewis, L.D. Lewis, J.P. Eder, N.J. Reddy, F. Guo, K.J. Pierce, A.J. Olszanski,

R.B. Cohen, Phase I study of the safety, tolerability, and pharmacokinetics of

oral CP-868,596, a highly speci ﬁ c platelet-derived growth factor receptor

tyrosine kinase inhibitor in patients with advanced cancers, J. Clin. Oncol. 27

(2009) 5262e5269.

[44] F.H. Tan, T.L. Putoczki, S.S. Stylli, R.B. Luwor, Ponatinib: a novel multi-tyrosine

kinase inhibitor against human malignancies, OncoTargets Ther. 12 (2019)

635e645.

[45] C.C. Smith, E.A. Lasater, X. Zhu, K.C. Lin, W.K. Stewart, L.E. Damon, S. Salerno,

N.P. Shah, Activity of ponatinib against clinically-relevant AC220-resistant

kinase domain mutants of FLT3-ITD, Blood 121 (2013) 3165e3171.

[46] N.J. Short, H. Kantarjian, F. Ravandi, N. Daver, Emerging treatment paradigms

with FLT3 inhibitors in acute myeloid leukemia, Ther. Adv. Hematol. 10

(2019), 2040620719827310.

[61] H. Quentmeier, J. Reinhardt, M. Zaborski, H.G. Drexler, FLT3 mutations in acute

myeloid leukemia cell lines, Leukemia 17 (2003) 120e124.

[62] K. Reiter, H. Polzer, C. Krupka, A. Maiser, B. Vick, M. Rothenberg-Thurley,

K.H. Metzeler, D. Doerfel, H.R. Salih, G. Jung, E. Noessner, I. Jeremias,

W. Hiddemann, H. Leonhardt, K. Spiekermann, M. Subklewe, P.A. Greif,

Tyrosine kinase inhibition increases the cell surface localization of FLT3-ITD

and enhances FLT3-directed immunotherapy of acute myeloid leukemia,

Leukemia 32 (2018) 313e322.

[63] K.M. Kampa-Schittenhelm, M.C. Heinrich, F. Akmut, H. Dohner, K. Dohner,

M.M. Schittenhelm, Quizartinib (AC220) is a potent second generation class III

tyrosine kinase inhibitor that displays a distinct inhibition pro ﬁ le against

mutant-FLT3, -PDGFRA and -KIT isoforms, Mol. Canc. 12 (2013) 1476e4598.

[64] K. Spiekermann, R.J. Dirschinger, R. Schwab, K. Bagrintseva, F. Faber, C. Buske,

S. Schnittger, L.M. Kelly, D.G. Gilliland, W. Hiddemann, The protein tyrosine

kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and

apoptosis in AML-derived cell lines expressing a constitutively activated FLT3,

Blood 101 (2003) 1494e1504.

[65] H. Polzer, H. Janke, D. Schmid, W. Hiddemann, K. Spiekermann, Casitas B-

lineage lymphoma mutants activate AKT to induce transformation in coop-

eration with class III receptor tyrosine kinases, Exp. Hematol. 41 (2013)

271e280.e4.

[47] F. Baska, A. Sipos, Z. Or ﬁ , Z. Nemes, J. Dobos, C. Szantai-Kis, E. Szabo, G. Szenasi,

L. Dezsi, P. Hamar, M.T. Cserepes, J. Tovari, R. Garamvolgyi, M. Kreko, L. Or ﬁ ,

Discovery and development of extreme selective inhibitors of the ITD and

D835Y mutant FLT3 kinases, Eur. J. Med. Chem. 184 (2019), 111710.

[48] X. Yuan, Y. Chen, W. Zhang, J. He, L. Lei, M. Tang, J. Liu, M. Li, C. Dou, T. Yang,

L. Yang, S. Yang, Y. Wei, A. Peng, T. Niu, M. Xiang, H. Ye, L. Chen, Identi ﬁ cation

of pyrrolo[2,3-d]pyrimidine-based derivatives as potent and orally effective

Fms-like tyrosine receptor kinase 3 (FLT3) inhibitors for treating acute mye-

logenous leukemia, J. Med. Chem. 62 (2019) 4158e4173.

€

[66] M. Beyer, N. Kiweler, S. Mahboobi, O.H. Kramer, How to distinguish between

the activity of HDAC1-3 and HDAC6 with western blot, in: Methods Mol Biol,

1510, 2017, pp. 355e364. New York, USA.

[67] A. Sellmer, H. Stangl, M. Beyer, E. Gruenstein, M. Leonhardt, H. Pongratz,

E. Eichhorn, S. Elz, B. Striegl, Z. Jenei-Lanzl, S. Dove, R.H. Straub, O.H. Kraemer,

S. Mahboobi, Marbostat-100 de ﬁ nes a new class of potent and selective

antiin ﬂ ammatory and antirheumatic histone deacetylase 6 inhibitors, J. Med.

Chem. 61 (2018) 3454e3477.

[68] S. Mahboobi, A. Sellmer, H. Hoecher, C. Garhammer, H. Pongratz, T. Maier,

T. Ciossek, T. Beckers, 2-Aroylindoles and 2-aroylbenzofurans with N-

hydroxyacrylamide substructures as a novel series of rationally designed

histone deacetylase inhibitors, J. Med. Chem. 50 (2007) 4405e4418.

[69] J. Dumas, U. Khire, T.B. Lowinger, H. Paulsen, B. Riedl, W.J. Scott, R.A. Smith,

J. Wood, H. Hatoum-Mokdad, W. Lee, A. Redman, J. Johnson, R. Sibley, Prep-

aration of Substituted Heterocyclic Ureas as P38 Kinase Inhibitors, 2012.

US20120046290A1.

[70] S. Mahboobi, A. Uecker, C. Cenac, A. Sellmer, E. Eichhorn, S. Elz, F.-D. Boehmer,

S. Dove, Inhibition of FLT3 and PDGFR tyrosine kinase activity by bis(benzo[b]

furan-2-yl)methanones, Bioorg. Med. Chem. 15 (2007) 2187e2197.

[71] J. Jiang, G.W. Gribble, A direct lithiation route to 2-acyl-1-(phenylsulfonyl)

indoles, Synth. Commun. 32 (2002) 2035e2040.

[72] W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz Jr., D.M. Ferguson,

D.C. Spellmeyer, T. Fox, J.W. Caldwell, P.A. Kollman, A second generation force

ﬁ eld for the simulation of proteins, nucleic acids, and organic molecules, J. Am.

Chem. Soc. 117 (1995) 5179e5197.

€

[49] S. Mahboobi, A. Ücker, A. Sellmer, C. Cenac, H. Hocher, H. Pongratz,

E. Eichhorn, H. Hufsky, A. Trümpler, M. Sicker, F. Heidel, T. Fischer, C. Stocking,

€

S. Elz, F.-D. Bohmer, S. Dove, Novel bis(1H-indol-2-yl)methanones as potent

inhibitors of FLT3 and platelet-derived growth factor receptor tyrosine kinase,

J. Med. Chem. 49 (2006) 3101e3115.

€

[50] S. Teller, D. Kramer, S.A. Bohmer, K.F. Tse, D. Small, S. Mahboobi, C. Wallrapp,

€

T. Beckers, K. Kratz-Albers, J. Schwable, H. Serve, F.D. Bohmer, Bis(1H-2-

indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase

Flt3, Leukemia 16 (2002) 1528e1534.

[51] J.A. Zorn, Q. Wang, E. Fujimura, T. Barros, J. Kuriyan, Crystal structure of the

FLT3 kinase domain bound to the inhibitor quizartinib (AC220), PloS One 10

(2015) e0121177/1ee0121177/15.

[52] C. Albers, H. Leischner, M. Verbeek, C. Yu, A.L. Illert, C. Peschel, N. von Bubnoff,

J. Duyster, The secondary FLT3-ITD F691L mutation induces resistance to

AC220 in FLT3-ITD þ AML but retains in vitro sensitivity to PKC412 and

Sunitinib, Leukemia 27 (2013) 1416e1418.

[53] D.H. Jornada, G.F.d.S. Fernandes, D.E. Chiba, T.R.F. de Melo, J.L. dos Santos,

M.C. Chung, The prodrug approach: a successful tool for improving drug

solubility, Molecules 21 (2016) 42/1e42/31.

[73] M. Clark, R.D. Cramer III, N. Van Opdenbosch, Validation of the general pur-

pose Tripos 5.2 force ﬁ eld, J. Comput. Chem. 10 (1989) 982e1012.

Article Doi

A series of novel aryl-methanone derivatives as inhibitors of FMS-like tyrosine kinase 3 (FLT3) in FLT3-ITD-positive acute myeloid leukemia

DOI: 10.1016/j.ejmech.2020.112232

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2020.112232

Full text of DOI:10.1016/j.ejmech.2020.112232

Products guided by the article

R&D Labs maybe for 51508-05-5

Relevant to this article

Hot Product