Selective targeting of the αC and DFG-out pocket in p38 MAPK

European Journal of Medicinal Chemistry 208 (2020) 1 12721

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

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

Research paper

Selective targeting of the aC and DFG-out pocket in p38 MAPK

a

,

b

a,

Sandra Rohm , Martin Schroder ^b, Jessica E. Dwyer ^c, Caroline S. Widdowson ^c,

€

Apirat Chaikuad ^a^b, Benedict-Tilman Berger ^{a b}, Andreas C. Joerger ^a

,

, b

Andreas Kramer ^b, Jule Harbig ^d, Daniel Dauch ^{d f}, Mark Kudolo ^g, Stefan Laufer ^g,

a,

, e,

€

Mark C. Bagley ^c, Stefan Knapp ^a

, b, *

^aJohann Wolfgang Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438, Frankfurt Am Main, Germany

^bBuchmann Institute for Molecular Life Sciences and Structural Genomics Consortium (SGC), Max-von-Laue-Str. 15, 60438, Frankfurt Am Main, Germany

^cDepartment of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, BN1 9QJ, UK

^dDepartment of Medical Oncology and Pneumology, University Hospital Tuebingen, 72076, Tuebingen, Germany

^eGerman Cancer Research Consortium (DKTK), German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany

^fIFIT Cluster of Excellence EXC 2180 “Image Guided and Functionally Instructed Tumour Therapies”, University of Tuebingen, Tuebingen, Germany

^gDepartment of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of

central importance in inﬂammatory and autoimmune diseases. Although studied extensively, little is

known about how conformation-speciﬁc inhibitors alter signaling outcomes. Here, we have explored the

highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against

known off-targets showed that these fragments maintained the selectivity issues of their parent com-

pound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced in-

Received 15 June 2020

Received in revised form

31 July 2020

Accepted 1 August 2020

Available online 20 August 2020

hibitor selectivity. Further efforts focused therefore on the exploration of the aC-out pocket of p38 MAPK,

Keywords:

yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a

recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-

pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with

good cellular activity targeting also the p38 back pocket.

Selective type-II p38 MAPK inhibitor

Allosteric BIRB fragments

Folded P-loop

Differential scanning ﬂuorimetry (DSF)

NanoBRET^TMassay

1. Introduction

MAPKs p38a and its closely related isoform p38b are highly dy-

namic protein kinases, which have been targeted by type-II in-

hibitors such as BIRB-796, which has high potency for both

isoforms but lacks kinome-wide selectivity. A large fraction of hu-

man kinase domains can adopt a type-II, DFG-out conformation

[5,6], but targeting this structural feature alone is unlikely to yield

selective inhibitors (Fig. 1A) [7e9]. Allosteric fragments inspired by

the BIRB-796 back-pocket binding motif have been studied,

exploring the DFG-out lower selectivity site as well as the allosteric

higher selectivity site tolyl pocket [10e14]. An attractive feature of

the BIRB-796 back-pocket binding moieties is that they have been

associated with very slow off-rate binding kinetics [7,15,16]. How-

ever, whether selectivity of these fragments against BIRB-796 off-

targets can be improved remains to be shown.

The mitogen-activating protein kinases (MAPKs) p38a and p38b

are major mediators of environmental stress signals and they have

been associated with inﬂammatory diseases and cancer. Because of

its strong association with disease development, p38

promising drug target [1e3]. However, to date no p38

a

emerged as a

inhibitor

a

has been approved for treatment.

Many p38 inhibitors have been developed, but review of the

recent literature revealed that early inhibitors that lack speciﬁcity

are most frequently used in functional studies. We have therefore

developed highly selective and potent type-I and type-I½ inhibitors

as well as very recently also a type-II inhibitor (SR-318) [4]. The

An interesting approach has been developed by Decipera et al.,

who reported on allosteric urea-based fragments that explore the

p38 switch pocket region [1 7], by engaging with the less-conserved

residues Arg67, Arg149 and the non-conserved Arg70 residue

* Corresponding author. Johann Wolfgang Goethe University, Institute of Phar-

maceutical Chemistry, Max-von-Laue-Str. 9, 60438, Frankfurt Am Main, Germany.

E-mail address: knapp@pharmchem.uni-frankfurt.de (S. Knapp).

https://doi.org/10.1016/j.ejmech.2020.1 12721

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S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

Fig. 1. Plasticity of p38 allosteric pockets adjacent to the ATP site and comparison with the MAPK ERK2. A) Type-II p38 inhibitor BIRB-796 (PDB: 1KV2) targeting the inactive DFG-

out conformation and the allosteric upper tolyl pocket. B) Type-III p38 inhibitor DP802 (PDB: 3NNW) interacting with the switch-pocket residues. C) Type-II p38 inhibitor VPC-

00628 (PDB: 5LAR) inducing a P-loop folded conformation. D) Type-IIB inhibitor SCH772984 (PDB: 4QTA) targeting the allosteric P-loop/

of ERK2.

aC pocket in a P-loop folded conformation

located in the p38

a

C helix and the HRD¼loop region. This study led

hinge-binding motif. Although it is well known that some kinases,

such as ABL, ACK1, AURORA, cMET, FGFR1, MAP4K4 and p38, can

adopt a folded (inactive) P-loop conformation [22], the enlarged

to the development of type-III inhibitors such as DP802 (Fig. 1B)

[1 7e20]. However, most of these inhibitors showed weak potency

and poor pharmacological properties.

allosteric pocket (P-loop/aC pocket), created by the movement of

Unusual binding modes, targeting non-conserved structural el-

ements in kinases, have been demonstrated to favor selectivity and

potency of corresponding inhibitors. A recently published type-II

the P-loop, has been targeted in the MAPK family only by the ERK1/

2 inhibitor SCH772984. (PDB: 4QTA, Fig. 1D). Targeting this P-loop/

a

C pocket was associated with high inhibitor selectivity and slow

p38

folded P-loop conformation in p38

a type-II cyclohexyl DFG-out pocket binding moiety with a novel

a

MAPK inhibitor VPC-00628 [21] that stabilizes a uniquely

dissociation (off-) rates [23]. Interestingly, although SCH772984 did

not inhibit p38 upstream kinases, this inhibitor abrogated phos-

phorylation by MEK1/2 on the ERK activating residues Thr202/

a

(PDB: 5LAR, Fig. 1C) combined

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European Journal of Medicinal Chemistry 208 (2020) 112721

Tyr204, as well as on Thr185/Thr187. Therefore, SCH772984

inhibited ERK activation by MEK, as well as phosphorylation of ERK

substrates. Thus, a synergistic inhibitory effect on Raf-MEK-ERK

signaling was observed due to the large structural changes in the

N-lobe, normally serving as binding sites for MEKs [24e26]. A

distinct conformation-selective inhibitory effect on p38 and the

MAPK signaling cascade was described by Hari et al.in 2014, using

canonical type-I and type-II inhibitors [27]. Interestingly, this study

also demonstrated different effects on kinase dephosphorylation by

the inactivating phosphatase with dual speciﬁcity, DUSP10. In

addition, Suvillan et al. reported that inhibitors targeting DFG-out

pocket selectivity sites, such as BIRB-796, concurrently inhibited

p38 activation and phosphorylation by MEK6, with K_Dvalues in the

low nanomolar range, without directly affecting MEK6 activity [16].

These reports make a compelling case that conformation-selective

kinase inhibitors may have very different effects on cellular

signaling and as a consequence on phenotypic responses.

further puriﬁcation to obtain 61 in a yield of 46%.

Acetanilide analogues 63e65 were synthesized from 1-(3-(tert-

butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-3-(4-chlorophenyl)urea

ꢀ

(49) using an Fe-catalyzed Bechamp reduction under mild condi-

tions to obtain compound 62 (Scheme 3). Amide functionalities

were then introduced either by activating the carboxylic acid with

HATU, DIPEA in DMF or by a Schotten-Baumann reaction with the

corresponding acid chloride or sulfonyl chloride under basic con-

ditions using aniline 62.

A convergent synthetic approach was used for synthesizing

VPC-00628 derivatives to explore the allosteric back pocket. In the

ﬁrst part of our convergent synthetic route, (E)-ethyl 2-cyano-3-

ethoxyacrylate (66) was reacted with phenylhydrazine to obtain

5-aminopyrazole 67, which was further treated with an aqueous

sodium hydroxide solution to hydrolyze the ester [4]. The acid

function of compound 68 was then activated with EDC-HCl/HOBt

and reacted with methyl 4-(aminomethyl)benzoate hydrochloride

to give the corresponding amide 69. The latter was then hydrolyzed

to the benzoic acid 70 [4]. In the second part, compounds 72e99,

102 and 105 were synthesized by an amide coupling reaction using

the N-protected amino acid 71 as starting material. The subsequent

deprotection of the amine functionality under basic conditions led

to compounds 106e135. Using this method, different amide N-

functionalities were introduced, including linear, branched,

aliphatic, aromatic and heteroaromatic substituents. In the last

step, the amines 106e135 were each coupled with acid 70 using

HATU to obtain the ﬁnal products 136e166 (Scheme 4).

Here, we focused on the exploration of the highly dynamic

allosteric back pocket of p38

a/

b. In order to study the effect of

diverse decorations targeting the

aC-out and DFG-out pocket on

ligand selectivity, we analyzed selectivity of allosteric pyrazole urea

fragments on known BIRB-796 off-targets using differential scan-

ning ﬂuorimetry (DSF). These data showed that off-target activity

followed closely p38a/b activity, suggesting that BIRB-796 off-tar-

gets remain a liability of its pyrazole urea back pocket moiety.

However, signiﬁcant selectivity improvement was achieved after

fusing the allosteric portion of BIRB-796 with the hinge-binding

motif of SR-318, a p38a/b chemical probe that we recently pub-

lished that lacks extensive back-pocket interactions apart from the

DFG-out binding cyclohexyl moiety. Using SR-318, we explored

back pocket modiﬁcations, yielding 137, a potent and selective

3. Results and discussion

Kinase conformations are highly dynamic, allowing precise

modulation of signaling cascades by catalytic and non-catalytic

mechanisms. Allosteric pockets have been discovered coinciden-

tally by trapping less conserved conformational states with small

molecules, thereby providing insights into the conformational

space available for ligand binding. Here we used p38 MAPK as a

well-established model system for which a large diversity of allo-

steric and ATP-competitive inhibitors have been developed to study

allosteric back-pocket interaction, including the DFG-out and the P-

p38

a/b inhibitor representing an alternative type-II chemical probe

targeting p38a/b.

2. Chemistry

The synthesis of the BIRB-796 fragment library compounds

18e57 was performed in a two-step reaction sequence (Scheme 1).

For the preparation of the pyrazole core heterocycle, b-ketonitrile

was reacted with a corresponding hydrazine or hydrazine hydro-

chloride derivative under acidic conditions in ethanol. To maximize

the efﬁciency of the approach, three different microwave-assisted

and non-assisted methods were established and successfully

applied. The resulting aminopyrazole compounds 1e17 were sub-

sequently reacted with aryl isocyanates in methylene chloride to

obtain the ﬁnal urea compounds 18e57 in low to high yields.

For the synthesis of 1-(4-nitrophenyl)-3-(triﬂuoromethyl)-1H-

loop/aC pocket. Allosteric urea-based fragments derived from type-

II inhibitor BIRB-796 were synthesized, and their selectivity proﬁle

against known BIRB-796 off-targets was evaluated. Some of these

allosteric fragments have been studied before; however, their

selectivity proﬁles against BIRB-796 off-targets have not been

determined [7,10,12e14].

Using DSF, we monitored the activity of a set of 47 kinases,

including a selection of BIRB-796 off-targets described by Karaman

and Davis et al. [9,28] (Supplemental Table 1). In our ﬁrst synthe-

sized and investigated library, we focused on the modiﬁcation of

the active-site directed arylic urea moiety, with invariant allosteric

pyrazole decoration. Keeping each 3,5-di-ﬂuoro-, 3,5-dieCF₃e and

4-chloro-benzyl decoration constant, we then varied the allosteric

tolyl-pocket and BIRB-796 DFG-out tert-butyl pocket interactions

pyrazol-5-amine (61),

a one-pot synthesis with ethyl 2,2,2-

triﬂuoroacetate (58) as starting material was used, which was

reacted with acetonitrile and potassium tert-butoxide in tetrahy-

drofuran (Scheme 2). The 4,4,4-triﬂuoro-3-oxobutanenitrile (59)

produced in situ was then heated with (4-nitrophenyl)hydrazine

hydrochloride (60) in hydrochloric acid and ethanol without

Scheme 1. Two-step route for the preparation of urea derivatives 18e57. Reagents and conditions: (a) hydrazine, toluene/AcOH (5:1), 120 ^ꢀC (100 W), MW; (b) hydrazine hy-

drochloride, HCl (cat.), EtOH, 130 ^ꢀC (200 W), MW; (c) hydrazine hydrochloride, HCl (cat.), EtOH, reﬂux, 43-95%; (d) isocyanate, CH₂Cl₂, rt, 8-89%; R¹: tert-butyl, phenyl, cyclopropyl,

CF₃; R²¼ phenyl, 4-tolyl, tert-butyl, methyl, 4-X-phenyl (X ¼ NO₂, Br, F, CF₃, OCF₃, CN, SO₂CH₃); R³¼ 2-naphthyl, 1-naphthyl, 2-tolyl, m-xylol, 3-methoxyphenyl, O-tosyl 3-F-2-tolyl,

3,4-diF-phenyl, 3,5-diCF₃-phenyl, 3,5-diF-phenyl, 4-Y-phenyl (Y ¼ Me, OEt, F, Cl, O-benzyl).

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European Journal of Medicinal Chemistry 208 (2020) 112721

activity for EPHA2, emphasizing the importance of an active-site

directed moiety for potent inhibition of this receptor tyrosine ki-

nase. A second set of tolyl-pocket modiﬁed allosteric compounds,

49e57 and 61e63, was screened against the kinase panel used

before in DSF assay (Table 2).

This set of inhibitors showed that the tolyl-pocket decoration

was important for inhibitor potency on p38 and also most off-

targets. It seems that polar donor and acceptor groups present in

53, 61 and 64 are most potent for p38. For targeting the DFG-out

pocket of p38, the bulky tert-butyl decoration was preferred, as

thermal stabilization was signiﬁcantly smaller for compounds

containing CF₃and negligible for compounds with a cyclopropyl

moiety. However, as observed in the ﬁrst fragment series, the most

potent p38 fragments retained activity against BIRB-796 off-targets

such as BRAF, SLK and STK10. Interestingly, some fragments

showed selective binding to some off-targets, most notably to SLK

and STK10 by compound 63. Thus, the development of selective

inhibitors based on these fragments might be possible.

Scheme 2. One-pot synthesis of compound 61. Reagents and conditions: (a) Aceto-

nitrile, KOtBu, THF, rt; (b) HCl, EtOH, reﬂux, 46%.

(compounds 18e48). During this screen, consistently highest ac-

tivity was found for the kinases p38

STK10 and EPHA2 (Table 1).

a, p38b, BRAF, AURKB, SLK,

This limited screen showed that compounds potently interact-

ing with p38 largely retained activity on kinase targets known to

a/b

bind to BIRB-796. Examples of such inhibitors are 19, 22, 27, 28 and

31. Most compounds harboring a 3,5-di CF₃decoration, 23 and

32e39, lost most binding activity for p38, but, interestingly, some

retained activity for AURKB, suggesting that these fragments could

be developed into chemical probes for this target. From studying

3,5-diﬂuoro decorated compounds 40e48, hydrophobic DFG-out

pocket and tolyl-pocket interactions seem to be important for

p38 activity. However, those compounds also had activity on

AURKB, SLK and BRAF. Interestingly, allosteric fragments lost

Recently, we established a series of type-II p38 inhibitors with

good p38 selectivity [4]. Some of those inhibitors also showed

selectivity for p38

a over the closely related isoform p38b, in

particular when fusing the pyrazole-urea back-pocket decoration of

BIRB-796 with the hinge-binding motif of our initial type-II hit

VPC-00628 [21]. This compound (MCP-081, compound 103 in

€

Rohm et al., 2019) had an about 30-fold selectivity for p38a over

Scheme 3. Synthesis of amino-functionalized compounds 63e65. Reagents and conditions: (a) Fe, NH₄Cl, EtOH/H₂O (4:1), 70 ^ꢀC, 75%; (b) HATU, DIPEA, DMF, rt; (c) TEA, THF, 0 ^ꢀ

/ rt, 29-84%; R¹¼ cyclopropanecarboxamide, iso-butylamide, methanesulfonamide.

C

Scheme 4. Convergent synthetic route for the back-pocket optimization, ﬁnal compounds 136e166. Reagents and conditions: (a) Phenylhydrazine, EtOH, reﬂux, 85%; (b) MeOH/

THF, NaOH aq., reﬂux, 98%; (c) methyl 4-(aminomethyl)benzoate hydrochloride, HOBt, EDC-HCl, DIPEA, DMF, rt, 90%; (d) LiOH, THF/H₂O, rt, 97%; (e) Fmoc-homocyclohexyl-L-

alanine, R¹R²NH, HATU, DIPEA, DMF, rt, 48-96%; (f) piperidine, DMF, rt, quant.; (g) HATU, DIPEA, DMF, rt, 18-96%; R¹¼ C (aliphatic, aromatic) and R²¼ C (aliphatic) or H.

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European Journal of Medicinal Chemistry 208 (2020) 112721

Table 1

Initial BIRB-796 derived urea compound library investigated with DSF assays for off-target inhibition.

DT_maverage derived from two replicates at a compound concentration of

10 mM.

p38

b

(IC₅₀(p38

a

/p38

b

) ¼ 0.055/1.60

m

M) [4]. By screening this

081, we superimposed this structure with the BMS-5c/p38a com-

type-II inhibitor against our DSF kinase panel, a relatively clean

selectivity proﬁle was observed, with off-target activities for

AURKB and BRAF only. In order to understand the remarkable a-

isoform selectivity of MCP-081 and its allosteric back-pocket in-

teractions, we determined the crystal structure of MCP-081 in

plex (PDB: 4KIN, Fig. 2B) [29]. Good isoform selectivity for BMS-5c

has been reported and rationalized based on a backbone ﬂip be-

tween the methionine (Met109) and leucine residue (Leu108) in

the hinge region [29] not observed in the MCP-081 structure. We

previously speculated that a sequence variation in the back pocket

complex with p38

with the BIRB-796/p38

p38 complex (PDB: 5LAR, not shown) revealed similar back-

a

(PDB: 6Y6V). Superposition of this structure

between the p38

selectivity [4]. In position 78 (p38

methionine, while p38 has a leucine. However, the similar binding

a

/p38

b

isoforms could contribute to isoform

a

(PDB: 1KV2, Fig. 2A) and the VPC-00628/

a

numbering), the -isoform has a

a

b

pocket interactions, while contacts to the hinge region had

differing orientations (Fig. 2A). These structural differences may

therefore explain the reduced potency of MCP-081 against p38

MAPK that we previously observed. To gain further insights into the

mode described for BIRB-796, which shows no isoform selectivity,

makes this difference less likely as a potential explanation for the

isoform selectivity of MCP-081. Additional dissimilarities (e.g. dif-

ferences in the water network) may also contribute to the observed

selectivity.

structural reasons for the remarkable a-isoform selectivity of MCP-

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Since structural variations of the BIRB-796 pyrazole-urea motif

did not improve selectivity without losing p38 activity, we were

interested in whether the VPC-00628 DFG-out back-pocket binding

stabilization, in particular for short hydrophobic aliphatic chains,

than with the parent compound VPC-00628 (Table 3). The increase

in potency was also seen in the cellular assays performed. Overall,

137 showed the highest potency, with an IC₅₀value of 14.0 nM in

moiety could be optimized by further expansion into the aC-out

pocket. Targeting this pocket resulted in high target selectivity for

the ERK1/2 inhibitor SCH772984 [23]. In order to explore this

pocket, we expanded this lead structure at the terminal primary

amide with a diverse set of aliphatic and aromatic building blocks.

The obtained compounds 136e166, were ﬁrst tested for p38 ac-

tivity using DSF assays. Inhibition of cellular enzyme activity was

then assessed using NanoBRET™ assays in HEK293T [30] cells

(Table 3, Table 4). At ﬁrst, linear and branched aliphatic residues

were introduced, resulting in comparable or slightly better protein

cells and a thermal stabilization, D

T_m, of 16.4 0.2 ^ꢀC in DSF assays,

and was thus more potent than the lead structure VPC-00628

(

D

T_m¼ 12.9

1.0 ^ꢀC, IC₅₀¼ 38.0 nM). For larger linear and

branched decorations, lower

DT_mvalues and, as expected, lower

potencies were observed. We therefore assumed that the increased

lipophilicity and ﬂexibility of longer-chain ligands adversely

affected the interaction within the more polar allosteric pocket in

p38

a. Interestingly, when one of the methyl groups in the 3-pentyl

moiety (143) was replaced by a hydroxyl group (144), an increase of

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European Journal of Medicinal Chemistry 208 (2020) 112721

2.3 ^ꢀC in

D

T_mwas detected. The hydroxyl group presumably sta-

compounds formed hinge interactions with the backbone of

Met109 and showed a conserved mode of binding. However, for

residues targeting the tolyl pocket, the binding mode differed

bilized the ligand inside the binding pocket by interaction with the

polar environment of this binding pocket.

The most interesting compounds of this series were co-

slightly. The tolyl group of BIRB-796 bound near the aC helix,

crystallized with p38

a

, and their binding mode was compared

introduced linear and branched residues pointed towards the

solvent-exposed region (Fig. 3A). We therefore assumed that the

latter observation may explain the difference in potency towards

BIRB-796. For cyclic systems that were introduced at this position,

with BIRB-796 (Fig. 3). The back-pocket modiﬁed VPC-00628 de-

rivatives induced a folded conformation of the P-loop not observed

in the p38 BIRB-796 complex. All compounds showed a type-II

inhibitor binding mode, extending into the DFG-out pocket via

their cyclohexyl decoration. As expected, all crystallized

interactions closer to the

aC helix were observed (Fig. 3B).

The P-loop/ C pocket of p38 MAPK harbors an aromatic

a

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European Journal of Medicinal Chemistry 208 (2020) 112721

Table 2

Follow-up BIRB-796 derived urea compound library investigated with DSF assays for off-target inhibition.

DT_maverage derived from two replicates at a compound concen-

tration of 10 mM.

histidine residue, His64, at a distance of about 14 Å to the terminal

amide of the lead structure, and the guanidinium groups of Arg70

and Arg67 are within a distance of 9 Å and 11 Å, respectively. We

speculated that the introduction of an aromatic group at the ter-

the DT_mvalues were overall signiﬁcantly lower, although following

the same radius-dependent trend of halogen substitutions. The loss

in afﬁnity for the target protein might be due to the increased steric

demand and the limited plasticity of the binding pocket. To increase

the ﬂexibility of the aforementioned benzylic structures, either a

methylene or ethylene bridge between the back-pocket terminal

amide and a substituted aromatic or heteroaromatic moiety was

introduced (156e158). However, a signiﬁcantly smaller tempera-

ture shift was observed for chlorinated compounds 157 and 158.

Interestingly, for polar picoline compounds (159e161) improved

minal position of our lead structure might form favored

p-p

stacking and cation- stacking interactions with these residues.

p

Based on these considerations, various mono- and di-substituted

halogenated phenyl residues were introduced to extend the lead

structure towards the allosteric back pocket. Subsequently, all

compounds were examined again using DSF assays and cellular

potencies were determined by NanoBRET™ assays (Table 4).

Thermal protein stabilization of para-substituted halogen com-

pounds (150e152) decreased from ﬂuorine to chlorine to bromine.

The ﬂuorine-substituted compound 150 seemed therefore most

activity was observed in

cellular IC₅₀values showing low nanomolar potency. The para-

substituted picoline derivative (159, T_m14.7

0.5 ^ꢀC,

DT_massays, which was conﬁrmed by

D

¼

IC₅₀¼ 21.7 nM) seemed to be slightly more potent than the meta-

optimal within this series, with a

D

T_mvalue of 10.1 0.5 ^ꢀC and an

substituted (160,

substituted (161,

Less potent binding was detected for the smaller, more electron-

D

T_m¼ 13.9 0.4 ^ꢀC, IC₅₀¼ 32.9 nM) and ortho-

IC₅₀value of 22.1 nM, which was comparable to the lead structure

VPC-00628. For 3,5-dihalogen substituted compounds (153e155),

T_m¼ 12.6 0.3 ^ꢀC, IC₅₀¼ 25.2 nM) compounds.

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European Journal of Medicinal Chemistry 208 (2020) 112721

rich, ﬁve-membered thiophene compound 162.

with different aromatic and heteroaromatic structures. In com-

An overlay of the crystal structures of the p38

a

complexes with

parison with the piperazine derivative 148 (

D

T_m¼ 12.6 0.3 ^ꢀC),

chlorine-substituted 150 and 158 with the structure of the BIRB-

796 complex illustrated the high plasticity of the allosteric pocket

region (Fig. 4A). Targeting unique residues such as the non-

substitution of the free NH-hydrogen atom by an aromatic residue

led to a sharp decrease in thermal shift values for all compounds

that were synthesized (163e166). Interestingly, a

6.0

DT_mvalue of

conserved Arg70 in p38

The more rigid compound 150 bound in closer proximity to the

helix, and the complex of p38 with 158 showed that the terminal

a MAPK is therefore a challenging task.

0.2 ^ꢀC was measured for the benzoxazole derivative 166,

aC

whereas replacement of the oxygen atom by a sulfur atom led to an

a

overall loss of stabilization (

All piperazine-aryl derivatives showed cellular activity on p38a,

D

T_mof 2.4 ^ꢀC; 165,

D

T_m¼ 3.6 0.1 ^ꢀC).

decoration extended towards the solvent-exposed region. Inter-

estingly, the picoline nitrogen atom of the potent inhibitor 159 sits

close to a structural water molecule (4.0 Å) that forms a hydrogen

bond with the guanidinium group of Arg70, which is unique to

with IC₅₀values in the low micromolar range.

The most potent compounds 137 and 159 were chosen for a

kinome-wide selectivity screen against a panel of 468 kinases and

kinase mutants. As expected from the binding mode observed in

the crystal structure, 159 showed a very clean selectivity proﬁle.

Main off-targets were DDR1 and EPHA2 (Supplemental Table 2).

Our most potent compound 137 also showed favorable selectivity,

with only seven detected off-targets: DDR1/2, ZAK, ABL1, STK11,

MAP3K4 and EPHA1, using a cut-off value of ꢁ35% residual activity

p38a (Fig. 4B), and may form a water-mediated interaction with

this arginine in solution. In addition, a structural water molecule in

the immediate vicinity bridges the back-pocket carbonyl oxygen of

the switch-pocket residue Arg67 with the amide oxygen of 159.

Targeting of the folded P-loop conformation of ERK2 with

SCH772984 was possible because this inhibitor harbors

piperazine-phenyl-pyrimidine back-pocket decoration adopting a

sharp bend conformation. Because the P-loop/ C pocket might also

a

at 1 mM compound concentration (Fig. 5A). In order to conﬁrm the

a

selectivity screen data, K_Dvalues were determined in dose response

for all targets, showing an inhibition >90% using the KinomeScan

assay format (Table 5). Interestingly, 137 showed excellent potency

be targetable in p38, we synthesized compounds with extensions

resembling the SCH772984 back-pocket decoration. To ensure that

the inhibitors were ﬂexible enough to adopt a similarly kinked

conformation, the piperazine moiety was conserved and decorated

for p38

a

/

b

(K_D¼ 6.3/20 nM) with narrow selectivity for the

detected off-targets DDR1/2 (K_D¼ 31/40 nM), ZAK (K_D¼ 120 nM)

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European Journal of Medicinal Chemistry 208 (2020) 112721

Fig. 2. Comparison of the binding modes of MCP-081 (PDB: 6Y6V), BMS-5c (PDB: 4KIN) and BIRB-796 (PDB: 1KV2) in complex with p38

fragment with the VPC-00628 hinge binding motif in MCP-081 reveals a different binding mode in the active-site region of p38 . The P-loop in the MCP-081 complex is disor-

dered in the crystal structure due to its high ﬂexibility. B) Superposition of MCP-081 with the -isoform selective BMS-5c compound. Although both compounds shown exhibited an

-isoform selectivity, ﬂip of the Leu108 backbone was induced by BMS-5c only, thereby questioning a key role of this backbone-ﬂip induction in -isoform selectivity.

a. A) Superposition of the BIRB-796

a

Table 3

Activity of p38 back-pocket optimized aliphatic compounds 136e149 investigated by DSF and cellular enzyme activity IC₅₀values determined by NanoBRET™ assay.

a

Comp.

Structure/R

D

T_m[^ꢀC]

IC₅₀^b[nM]

Comp.

Structure/R

D

T_m[^ꢀC]

IC₅₀^b[nM]

SD p38

a

p38

a

SD p38

a

p38a

BIRB-796

19.8

0.2

7.7

142

14.0

0.4

20.0

VPC-00628 (lead)

136

12.9

1.0

38.0

21.9

143

144

10.4

0.4

37.6

38.2

15.0

0.2^c

12.7

0.1

137

138

16.4

0.2

14.0

17.7

145

146

8.6

0.5

93.7

47.0

13.7

0.5

9.8

0.2

139

11.3

0.5

33.7

147

13.2

0.3

44.8

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European Journal of Medicinal Chemistry 208 (2020) 112721

Table 3 (continued)

a

Comp.

Structure/R

D

T_m[^ꢀC]

IC₅₀^b[nM]

p38

Comp.

Structure/R

D

T_m[^ꢀC]

IC₅₀^b[nM]

p38a

SD p38

a

SD p38

a

140

141

7.4

0.7

9520

148

12.6

0.3

81.3

4.6

0.6

2070

149

5.6

0.3

n.d.

a

D

T_maverage of four measurements (n ¼ 4).

b

c

IC₅₀values were derived from duplicates (n ¼ 2).

n ¼ 6.

Table 4

Activity of p38 back-pocket optimized aromatic compounds 150e166 measured by DSF (DT_m) and cellular NanoBRET™ assays.

a

Comp.

Structure/R

D

T_m[^ꢀC]

IC₅₀^b[nM] p38

a

Comp.

Structure/R

D

T_m[^ꢀC]

IC₅₀^b[nM] p38

a

SD p38

a

SD p38a

150

10.1

0.5

22.1

159

160

14.7

0.5

21.7

151

7.5

1.1

107

13.9

0.4

32.9

152

153

6.8

0.1

94.4

41.1

161

162

12.6

0.3

25.2

81.3

7.3

0.2

6.1

2.1

154

155

156

157

1.9

0.5

1990

2830

543

163

164

165

166

4.8

0.2

1770

2390

1040

462

2.3

0.3

3.1

0.1

7.8

1.2

3.6

0.1

3.5

1.8

287

6.0

0.2

158

5.1

0.3

144

a

D

T_maverage of four measurements, except for 156 where n ¼ 6.

b

IC₅₀values were derived from duplicates (n ¼ 2).

and ABL1 (K_D¼ 130 nM).

NanoBRET™ assays (Table 5). Interestingly, 137 inhibited p38

p38b in cellulo with low nanomolar potencies, whereas only

micromolar potencies were determined for the off-targets DDR1,

DDR2, ZAK, MAP3K4, FGFR3(G297C) and FLT3(D835Y). No activity

(IC₅₀>20 mM) was detected for all other identiﬁed off-targets of 137

a

and

We were interested whether the detected off-targets of 137

were also inhibited in the cellular system. Therefore, all off-targets

that were detected in the kinome-wide selectivity screen (ꢁ35%

activity of control) as well as some mutants were tested using

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European Journal of Medicinal Chemistry 208 (2020) 112721

Fig. 3. Binding mode of back-pocket modiﬁed lead structures with aliphatic linear, branched and cyclic structures in complex with p38

and branched compounds 137 (lime, PDB: 6YK7), 143 (yellow, PDB: 6Y4U) and 146 (violet, PDB: 6Y4V) with BIRB-796 (pink, PDB: 1KV2). B) Overlay of cyclic compounds 146 (lime,

PDB: 6Y4W), 148 (yellow, PDB: 6YJC) and BIRB-796 (pink, PDB: 1KV2). Hydrogen-bond interactions are highlighted in green. The extended P-loop conformation of p38 in the BIRB-

796 complex is highlighted in light pink. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

a. A) Overlay of the binding modes of linear

a

Fig. 4. Binding mode of back-pocket modiﬁed VPC-00628 derivatives with aromatic and heteroaromatic structures in complex with p38

aromatic compounds 150 (yellow, PDB: 6ZWP) and 158 (violet, PDB: 6Y4X) with BIRB-796 (pink, PDB: 1KV2). The extended P-loop conformation of p38

a

. A) Overlay of the binding modes of

in the BIRB-796 complex is

a

highlighted in light pink. B) Binding mode of picoline derivative 159 (green, PDB: 6ZWR). Selected structural water molecules close to the picoline moiety of 159 are depicted as red

spheres. Hydrogen bonds are highlighted with green broken lines. The distance between the picoline ring nitrogen and an Arg70-bound structural water molecule is highlighted in

red. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

Fig. 5. Selectivity-creating binding mode. A) Selectivity proﬁling of 137 against 468 kinases at a compound concentration of 1

control without addition of inhibitor are depicted as red dots with radii matching their potency (see insert). B) Co-crystal structure of 137 in complex with p38

pocket is highlighted. A 2Fo-Fc electron density map for the ligand 137 is shown at a contour level of 1.5

m

M. Kinase targets with residual activity of ꢁ35% of

. The P-loop/

s (PDB: 6YK7). (For interpretation of the references to color in this ﬁgure

a

aC

legend, the reader is referred to the Web version of this article.)

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European Journal of Medicinal Chemistry 208 (2020) 112721

Table 5

Selectivity proﬁling and target activity of 137.

kinome targets

137 % of control at 1

m

M

137 DiscoverX

K_DM]

137 NanoBRET™

[

m

IC₅₀SEM^a

[mM]

FLT3(K663Q)

FLT3(D835Y)

DDR1

0

n.d.

>20

0.1

1.5

3.4

8.8

9.4

11.0

18.0

23.0

28.0

0.031

0.0063

0.02

0.13

0.12

0.04

n.d.

n.d

4.19 0.84^b

p38

a

b

0.014 0.01^b

0.017 0.001^b

ABL1-nonphosphorylated

ZAK

DDR2

MAP3K4

EPHA1

STK11

>20

1.7

4.0 0.72^b

5.80

>20

10.0

>20

FGFR3(G697C)

ABL1(Q252H)-nonphosphorylated

a

IC₅₀values listed are the mean of two experiments (n ¼ 2), unless stated otherwise.

n ꢂ 3

b

Fig. 6. Western blot analysis of the effects of inhibitor 137 on p38 autophosphorylation

and Ser82-phosphorylation of Hsp27. HCT-15 cells were treated for 2 h with different

concentrations of 137 or vehicle (DMSO) and then stimulated with anisomycin (10 mg/

mL) for 30 min. Vinculin was used as loading control.

in cellular NanoBRET™ assays, suggesting that off-target inhibition

in cells is not an issue for this compound (Supplemental Fig. 1). The

differences in activity may be attributed to different K_mvalues for

ATP and discrepancies with inhibiting full-length enzymes used in

BRET assays compared with catalytic domains used in the Kino-

meScan and differences in the activation states of the screened

kinases.

After demonstrating the excellent selectivity of 137 in cells

(Fig. 5B), the inhibitor was evaluated further by probing its efﬁ-

ciency on endogenous p38 MAPKs. Human colon adenocarcinoma

(HCT-15) cells were used to study the impact on the endogenous

p38 substrate Hsp27, whose phosphorylation is increased in cancer

cells such as colorectal cancer and associated with resistance to 5-

ﬂuorouracil treatment [50]. After stimulation with the antibiotic

anisomycin, analysis by Western blotting revealed that compound

137 showed dose-dependent inhibition of activating phosphory-

lation of p38 in HCT-15 cells and phosphorylation of its down-

stream substrate Hsp27 (Fig. 6). However, while effects on p38

activation showed the expected dose response, there was no sig-

niﬁcant reduction of Hsp27 phosphorylation upon inhibitor treat-

ment, probably due to phosphorylation of this protein by several

stress-activated kinases.

Fig. 7. Study on the metabolic stability of selected compounds. A) Metabolic degra-

dation and B) metabolite formation of the lead compound VPC-00628, 137, 150 and 159

over 240 min in human liver microsomes (HLM). Experiments were performed in

triplicate. The values represent the mean with standard deviation.

SB203580 [51e53], which inhibits the p38 active conformation,

assuming a canonical type-I binding mode. At the highest inhibitor

concentration of 10

complete inhibition of TNF-

0.48 0.05 M was calculated. In contrast, the type-I inhibitor with

an IC₅₀value of 1.36 0.34 M and a total inhibition of 83% showed

mM used in this study, 137 led to an almost

a

release (92%), and an IC₅₀value of

m

a signiﬁcantly lower effect on the release of this pro-inﬂammatory

marker.

As p38 MAPK is related to inﬂammatory diseases and regulates

the expression of pro-inﬂammatory cytokines, we were further

The metabolic stability of compounds 137, 150, 159 and the lead

structure VPC-00628 was tested in human liver microsomes (HLM).

The degradation and metabolite formation of the compounds was

followed over 240 min, and each sample was analyzed after 0, 10,

20, 30, 60, 120, 180 and 240 min using LC-MS, ESI(þ). The

interested in the efﬁciency of 137 modulating release of TNF-

Interestingly, the type-II inhibitor 137 showed a signiﬁcantly

stronger inhibitory effect on the TNF- release than the compound

a.

a

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European Journal of Medicinal Chemistry 208 (2020) 112721

concentration of the protein was standardized at 1 mg/mL for all

measurements (Fig. 7).

recorded using a Varian VNMRS instrument operating at 400, 500

or 600 MHz for ¹H NMR and 100 or 126 MHz for ¹³C NMR spec-

All compounds showed good stability in human liver micro-

somes. After 240 min, 99.5 6.3% of compound 159, 83.5 8.5% of

150 and 64.0 2.2% of 137 remained in the media. Compounds 150

and 137 increased the metabolic stability compared with the lead

structure VPC-00628 (66.9 2.1% remaining), whereas 159 showed

signiﬁcant stability, with almost no metabolite formation during

the 240 min time window that had been investigated.

troscopy. The chemical shifts (d) are reported in ppm and are cali-

brated against the residual proton peak of the deuterated solvent. J

values were recorded in Hz, and multiplicities were expressed us-

ing the usual conventions. ESI mass spectra (for compounds 1e9)

were obtained using a Bruker Daltonics Apex III, with ESI source

Apollo ESI, using MeOH as the spray solvent. For EI mass spectra a

Fissons VG Autospec instrument was used at 70 eV and were

captured by Dr. Alla K. Abdul-Sada of the University of Sussex Mass

Spectrometry Centre. Mass spectrometry (ESI) for 10e17, 49e65,

67e70, 72e166 were measured on a VG Platform II spectrometer

from Fisons. A number of high resolution mass spectra for 18e48

were obtained courtesy of the EPSRC Mass Spectrometry Service at

Swansea University, UK using the ionization methods speciﬁed.

High resolution mass spectrometry for 49e57 and 72e166

(FTMS þ p MALDI-HRMS) was performed using a MALDI LTQ XL

Orbitrap spectrometer from Thermo Scientiﬁc. For the ﬁnal com-

pounds the purity was determined >95%, if not otherwise stated,

using HPLC (LC-20A Prominence) Shimadzu. Separations were

4. Conclusion

Here, we have developed a series of small molecules for tar-

geting the allosteric aC- and DFG-out pockets in p38a MAPK. We

ﬁrst synthesized pyrazole urea scaffolds derived from BIRB-796 and

tested the activity of those fragments against a comprehensive set

of 47 diverse kinases using DSF. The results for this set of com-

pounds were somewhat disappointing, though, as either off-targets

described for BIRB-796 were retained or the activity for p38a/b was

signiﬁcantly reduced. We then revisited our recently published

compound MCP-081 that combines the allosteric part of BIRB-796

with the hinge-binding motif of VPC-00628. MCP-081 displayed a

clean selectivity proﬁle in our kinase selectivity panel. Intriguingly,

performed on

a C18 column (Luna 10m C18 (2) 100 Å;

250 ꢄ 4.6 mm) from Phenomenex using the following gradient

proﬁle: 0e2 min 95% B, 2e14 min 95% B, 14e21 min 10% B, 21 min

95% B. As solvent A) acetonitrile Ultra MS-Grade was used and as

solvent B) MS-Grade H₂O with 0.1% formic acid at a ﬂow rate of

1 mL/min. The detection was carried out with a LCMS-2020 mass

spectrometric detector from Shimadzu by a wavelength of 254 and

280 nm respectively.

a signiﬁcant selectivity over the p38b isoform was also observed. As

the potency of MCP-081 was reduced compared with VPC-00628

and our recent chemical probe compound SR-318, the allosteric

tert-butyl pyrazole moiety seemed suboptimal. We synthesized a

set of derivatives for targeting the aC-out pocket region, guided by a

signiﬁcant number of crystal structures. Through extension of the

terminal amide towards this allosteric site, we developed the

potent and selective compound 137, which showed excellent

General procedure A for microwave-assisted synthesis of 5-

aminopyrazoles using hydrazine as free base. A stirred solution

of the hydrazine (1 eq) and b-ketonitrile (1 eq) in PhMe/AcOH (5:1,

cellular activity on p38 MAPK in NanoBRET™ assays (IC₅₀[p38

a

/

1e2 mL) was irradiated in a pressure-rated sealed tube at 120 ^ꢀC for

40 min using a CEM Discover microwave synthesizer by moder-

ating the initial power (100 W). After cooling in a ﬂow of com-

pressed air, the solvent was evaporated in vacuo, and the crude

product was puriﬁed by ﬂash column chromatography on SiO₂.

General procedure B for microwave-assisted synthesis of 5-

aminopyrazoles using hydrazine hydrochloride. A stirred solu-

b

] ¼ 14.0/16.8 nM). In addition, 137 induced an anti-inﬂammatory

response by blocking TNF-a release in whole blood and displayed

a high metabolic stability. Taken together, 137 represents a valuable

chemical probe for studying the structural plasticity of p38 MAPK

and the resulting diverse phenotypic effects targeting speciﬁc

conformational states of kinases.

tion of the hydrazine hydrochloride (1 eq), b-ketonitrile (1 eq) and a

5. Experimental section

drop of HCl (conc.) in EtOH (10e20 mL) was irradiated in a

pressure-rated sealed tube (35 mL) at 130 ^ꢀC for 20e30 min using a

CEM Discover microwave synthesizer by moderating the initial

power (200 W). After cooling in a ﬂow of compressed air, the

mixture was basiﬁed to pH 12 by the addition of an aqueous NaOH

solution (10%) and extracted with EtOAc (3 ꢄ 20 mL). The organic

extracts were combined, washed with brine (30 mL), dried over

MgSO₄and the solvent was evaporated in vacuo.

Chemistry. All reagents and water-free solvents were purchased

from commercial suppliers and were used, if not otherwise stated,

without further puriﬁcation or solvents were dried by standard

procedures. Light petroleum refers to the fraction with bp

40e60 ^ꢀC. Microwave-assisted reactions were carried out using a

CEM Discover™ with a CEM Explorer at the given temperature

using the instrument’s in-built IR temperature measuring device,

by modulating the irradiation power (initial power given in pa-

rentheses). Column chromatography on silica was carried out on a

Biotage Isolera Prime ﬂash puriﬁcation system for compounds 1e3

and 5e9. For all other compounds described, column chromatog-

raphy was performed on silica 60, 0.04e0.63 mm from Machery-

Nagel GmbH & Co.KG. Fully characterized compounds were chro-

matographically homogeneous. Analytical thin layer chromatog-

raphy was carried out using aluminium-backed plates coated with

Merck TLC Silicagel 60 F254. Plates were visualized under UV light

General procedure C for synthesis of 5-aminopyrazoles under

conductive heating. A stirred solution of the hydrazine hydro-

chloride (1 eq), b-ketonitrile (1 eq) and a drop of HCl (conc.) in EtOH

(10 mL) was heated at reﬂux for 18 h. After cooling to rtrt, the

mixture was basiﬁed to pH 12 by the addition of an aqueous NaOH

solution (10%) (compound 6) or a sat. NaHCO₃solution (compounds

10e17) and extracted with EtOAc (3 ꢄ 30 mL). The organic extracts

were combined, washed with brine (40 mL), dried over Na₂SO₄and

the solvent was evaporated in vacuo. Puriﬁcation by ﬂash column

chromatography on SiO₂or recrystallization from a hexane/EtOAc

mixture gave the desired product.

(at

l 254 and/or 360 nm), and/or with ninhydrin and potassium

permanganate solutions. Melting points were determined for 1e9

and 18e48 using a Stanford Research Systems Optimelt and are

uncorrected. IR spectra were recorded in the range from 4000 to

600 cm^ꢃ1using a Perkin Trans FT-IR spectrometer. For 10e17,

49e65, 67e70, 72e166, ¹H NMR and ¹³C NMR spectra were recor-

ded either on a Bruker Avance 400, 500 or a Bruker DRX 600 using

TMS as internal standard. For 1e9 and 18e48, NMR spectra were

General procedure D for synthesis of N-pyrazolyl ureas. Ac-

cording to a modiﬁed procedure [31], the isocyanate (1e1.7 eq) was

added to a stirred solution of the 5-aminopyrazole (1 eq) in CH₂Cl₂

(1e3 mL). The mixture was stirred at rt for the given time and then

solvent was evaporated in vacuo to give the crude product.

General procedure E for amide coupling reaction using HATU

for the preparation of intermediate compounds (72e99, 102,

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European Journal of Medicinal Chemistry 208 (2020) 112721

105). The carboxylic acid (S)-2-((((9H-ﬂuoren-9-yl)methoxy)

carbonyl)amino)-4-cyclohexylbutanoic acid (71, 1 eq) and HATU

(1.2 eq) were dissolved in DMF (8 mL) and DIPEA (1.2 eq) was

added. The solution was stirred at rtrt for 1.5 h and the corre-

sponding amine (1.2 eq) was added, either pure when liquid or

diluted in DMF (4 mL) when solid. The mixture was stirred for

20 h at rt. CH₂Cl₂or EtOAc (50 mL) was added and the organic phase

was washed 4 times with water (50 mL) and dried over MgSO₄. The

solvent was evaporated under reduced pressure and the crude

product was puriﬁed by column chromatography on silica (eluent:

cyclohexane/EtOAc, CH₂Cl₂/MeOH or EtOAc/MeOH) if not stated

otherwise.

General procedure for Fmoc-cleavage and the preparation of

the amines (106e135). The amines (106e135) used in the amide

coupling reaction for general procedure F were freshly prepared

directly before coupling to the acid. The corresponding Fmoc pro-

tected amine (72e99,102,105,1.2 eq) was dissolved in DMF (12 mL)

and piperidine (20%) was added. The mixture was stirred for 1 h at

rt, and CH₂Cl₂(30 mL) or EtOAc (30 mL) was added. The organic

phase was washed 5 times with water (30 mL) and dried over

MgSO₄. The solvent was evaporated under reduced pressure, and

the crude product was used without further puriﬁcation.

5-Amino-1,3-di-tert-butyl-1H-pyrazole (6). Prepared according

to general procedure C using tert-butyl hydrazine hydrochloride

(0.66 g, 5.30 mmol) and 4,4-dimethyl-3-oxopentanenitrile (0.50 g,

4.00 mmol) in EtOH (10 mL). Puriﬁcation by ﬂash column chroma-

tography on SiO₂, gradient eluting with hexane to hexane/EtOAc

(17:3), gave 0.47 g (2.41 mmol, 60%) of 6 as a brown solid, mp

68.7e70.4 ^ꢀC (lit [33]. mp 67e69 ^ꢀC); IR: (neat)

1629, 1544, 1359, 1232; ¹H NMR (500 MHz, DMSO‑d₆):

1H), 4.60 (br s, 2H), 1.49 (s, 9H), 1.14 (s, 9H) ppm; ¹³C NMR (126 MHz,

n

/cm^ꢃ13355, 2962,

¼ 5.24 (s,

d

DMSO‑d₆):

d

¼ 155.9 (C), 146.1 (C), 88.2 (CH), 56.9 (C), 31.5 (C), 30.4

(Me), 29.0 (Me) ppm; MS (EI pos.): m/z (%) ¼ 195.2 (35) ([M]^ꢅþ, calcd.

195.2), 124.2 (100) ([M_fr.]^þ, calcd. 124.2); HRMS (ESI pos.): m/

z ¼ 196.1800 [MþH]^þ, calcd. for [C₁₁H₂₂N₃]^þ¼ 196.1808.

5-Amino-3-tert-butyl-1-(4-bromophenyl)-1H-pyrazole (7).

Prepared according to general procedure

B

using 4-

bromophenylhydrazine hydrochloride (0.35 g, 1.60 mmol) and

4,4-dimethyl-3-oxopentanenitrile (0.20 g, 1.60 mmol) in EtOH

(8 mL) under irradiation for 25 min. Puriﬁcation by ﬂash column

chromatography on SiO₂, gradient eluting with hexane to hexane/

EtOAc (3:1), gave 0.27 g (0.92 mmol, 58%) of 7 as a brown solid, mp

133.7e136.4 ^ꢀC; IR: (neat) /cm^ꢃ13427, 2957, 1635, 1557, 1500,

n

1244, 1065; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 7.62 (d, ³J ¼ 9 Hz,

General procedure F for amide coupling reaction using HATU

for the preparation of ﬁnal compounds (134e164). The carbox-

2H), 7.56 (d, ³J ¼ 9 Hz, 2H), 5.40 ( s, 1H), 5.25 (br s, 2H), 1.21 (s, 9H)

ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 161.2 (C), 147.2 (C), 138.9

ylic

acid

4-((5-amino-1-phenyl-1H-pyrazole-4-carboxamido)

(C), 131.7 (CH), 124.0 (CH), 117.6 (C), 87.4 (CH), 31.7 (C), 30.0 (Me)

ppm; MS (EI pos.): m/z (%) ¼ 293.1 (48) ([M]^ꢅþ, calcd. 293.2), 278.6

(100) ([M_fr.]^þ, calcd. 278.6); HRMS (ESI pos.): m/z ¼ 294.0601

[MþH]^þ, calcd. for [C₁₃H₁₇BrN₃]^þ¼ 294.0600.

methyl)benzoic acid (70, 1 eq) and HATU (1.2 eq) were dissolved in

DMF (8 mL) and DIPEA (1.5 eq) was added. The solution was stirred

at rt for 1.5 h, and a solution of the corresponding amine (106e135,

1.2 eq) in DMF (8 mL) with DIPEA (1.5 eq) was added. The mixture

was stirred for 20 h at rt. CH₂Cl₂or EtOAc (50 mL) was added, and

the organic phase was washed 4 times with water (50 mL) and

dried over MgSO₄. The solvent was evaporated under reduced

pressure, and the crude product was puriﬁed by column chroma-

tography on silica (eluent: cyclohexane/EtOAc, CH₂Cl₂/MeOH or

EtOAc/MeOH) if not stated otherwise.

5-Amino-1-methyl-3-phenyl-1H-pyrazole (8), 5-amino-1,3-

diphenyl-1H-pyrazole (9). Prepared as previously published by

Bagley et al. [31].

3-(tert-Butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-amine

(10).

Prepared as previously published by Zhu et al. [34].

3-(tert-Butyl)-1-(4-(triﬂuoromethyl)phenyl)-1H-pyrazol-5-

amine (11). Prepared as previously published by Li et al. [35].

3-(tert-Butyl)-1-(4-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-

amine (12). Prepared as previously published by King-Underwood

et al. [36].

4-(5-Amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile (13).

Prepared as previously published by Springer et al. [37].

3-(tert-Butyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazol-5-

amine (14). Prepared according to general procedure C using (4-

5-Amino-3-tert-butyl-1-phenyl-1H-pyrazole (1), 5-amino-3-

tert-butyl-1-(p-tolyl)-1H-pyrazole (2), 5-amino-3-tert-butyl-1-

methyl-1H-pyrazole (3). Prepared as previously published by

Bagley et al. [31].

5-Amino-3-tert-butyl-1-(4-ﬂuorophenyl)-1H-pyrazole

(4).

Prepared according to general procedure using 4-

B

ﬂuorophenylhydrazine hydrochloride (0.26 g, 1.60 mmol) and 4,4-

dimethyl-3-oxopentanenitrile (0.20 g, 1.60 mmol) in EtOH (9 mL)

under irradiation for 30 min. Puriﬁcation by trituration with light

petroleum gave 0.32 g (1.37 mmol, 86%) of 4 as red solid, mp

(methylsulfonyl)phenyl)hydrazine

hydrochloride

(1.00

g,

4.49 mmol), 4,4-dimethyl-3-oxopentanenitrile (738 mg,

4.49 mmol) and HCl (conc., 2 mL) in EtOH (30 mL), which was

heated at reﬂux for 20 h. The crude product was recrystallized from

a hexane/EtOAc mixture (1:1) to yield 907 mg (3.09 mmol, 69%) of

14 as a yellowish solid. TLC: R_F¼ 0.75 (SiO₂, hexane/EtOAc 1:3); ¹H

101.0e105.2 ^ꢀC; IR: (neat)

NMR (500 MHz, CDCl₃):

n

/cm^ꢃ13319, 2969, 1633, 1510, 1216; ¹H

d

¼ 7.55 (dd, ³J_HH¼ 9 Hz, ³J_HF¼ 5 Hz, 2H),

3

7.14 (dd, J_HH¼ 9 Hz, J_HF¼ 9 Hz, 2H), 5.53 (s, 1H), 3.65 (br s, 2H),

1.32 (s, 9H) ppm; ¹³C NMR (126 MHz, CDCl₃):

d

¼ 162.4 (C), 161.4 (d,

NMR (400 MHz, DMSO‑d₆):

d

¼ 7.98 (d, ³J ¼ 8.9 Hz, 2H), 7.90 (d,

¹J_CF¼ 246 Hz, C), 144.7 (C), 135.2 (C), 125.9 (d, ³J_CF¼ 9 Hz, CH), 116.1

(d, ²J_CF¼ 23 Hz, CH), 87.9 (CH), 32.2 (C), 30.1 (Me) ppm; HRMS (ESI

pos.): m/z ¼ 234.1399 [MþH]^þ, calcd. for [C₁₃H₁₇FN₃]^þ¼ 234.1401.

³J ¼ 8.9 Hz, 2H), 5.52e5.43 (m, 3H), 1.23 (s, 9H) ppm; ¹³C NMR

(101 MHz, DMSO‑d₆):

d

¼ 162.2,148.0,143.7,136.6,128.1,121.6, 88.2,

43.7, 31.9, 30.0 ppm; MS (ESI pos.): m/z (%) ¼ 294.2 (100) ([MþH]^þ,

5-Amino-3-tert-butyl-1-(4-nitrophenyl)-1H-pyrazole

(5).

calcd. 294.1); 238.1 (25) ([M-SO₂CH₃þNa]^þ, calcd. 185.1).

Prepared according to general procedure

A

using 4-

3-Cyclopropyl-1-(4-(triﬂuoromethoxy)phenyl)-1H-pyrazol-

5-amine (15). Prepared according to general procedure C using (4-

(triﬂuoromethoxy)phenyl)hydrazine hydrochloride (1.00 g,

4.37 mmol), 3-cyclopropyl-3-oxopropanenitrile (643 mg,

4.37 mmol) and HCl (conc., 2 mL) in EtOH (abs., 20 mL), which was

heated at reﬂux for 20 h. The crude product was recrystallized from

a cyclohexane/EtOAc mixture (1:1) to yield 956 mg (3.37 mmol,

77%) of 15 as a brownish solid. TLC: R_F¼ 0.76 (SiO₂, cyclohexane/

nitrophenylhydrazine (0.25 g, 1.60 mmol) and 4,4-dimethyl-3-

oxopentanenitrile (0.20 g, 1.60 mmol) in PhMe/AcOH (5:1, 1 mL).

Puriﬁcation by ﬂash column chromatography on SiO₂, gradient

eluting with CH₂Cl₂/hexane to CH₂Cl₂, gave 0.21 g (0.81 mmol, 51%)

of 5 as a colorless solid, mp 162.1e164.8 ^ꢀC (lit [32]. mp 172 ^ꢀC); IR:

(neat)

NMR (500 MHz, CDCl₃):

n

/cm^ꢃ13395, 2961, 1644, 1592, 1504, 1331, 1241, 1108; ¹H

d

¼ 8.29 (d, ³J ¼ 9 Hz, 2H), 7.90 (d, ³J ¼ 9 Hz,

2H), 5.62 (s, 1H), 3.82 (br s, 2H), 1.31 (s, 9H) ppm; ¹³C NMR

EtOAc 1:1); ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 7.69 (d, ³J ¼ 9.0 Hz,

(126 MHz, CDCl₃):

d

¼ 164.0 (C), 145.4 (C), 145.0 (C), 144.8 (C), 124.9

2H), 7.43 (d, ³J ¼ 9.0 Hz, 2H), 5.35 (br s, 2H), 5.20 (s, 1H), 1.79e1.72

(CH), 122.1 (CH), 90.6 (CH), 32.4 (C), 30.0 (Me) ppm; HRMS (ESI

(m, 1H), 0.85e0.78 (m, 2H), 0.64e0.58 (m, 2H) ppm; ¹³C NMR

pos.): m/z ¼ 261.1348 [MþH]^þ, calcd. for [C₁₃H₁₇N₄O₂]^þ¼ 261.1346.

(126 MHz, DMSO‑d₆):

d

¼ 154.9, 147.7, 145.5 (2x), 138.6, 123.7, 121.8,

15

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European Journal of Medicinal Chemistry 208 (2020) 112721

120.2 (¹J_CF¼ 256.1 Hz), 87.0, 9.4, 7.5 ppm; MS (ESI pos.): m/z

(%) ¼ 284.0 (100) ([MþH]^þ, calcd. 284.3).

in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with

light petroleum/EtOAc (1:1) gave 0.30 g (0.80 mmol, 86%) of 21 as a

4-(5-Amino-3-cyclopropyl-1H-pyrazol-1-yl)benzonitrile (16).

colorless solid, mp 215.7e219.3 ^ꢀC; IR: (neat) /cm^ꢃ13291, 2966,

n

Prepared according to general procedure

C

using 4-

1656, 1543, 1510, 1445, 1245, 1050, 1033; ¹H NMR (500 MHz,

hydrazinylbenzonitrile hydrochloride (1.00 g, 5.90 mmol), 3-

cyclopropyl-3-oxopropanenitrile (643 mg, 5.90 mmol) and HCl

(conc., 2 mL) in EtOH (20 mL) which was heated at reﬂux for 20 h.

The crude product was recrystallized from a cyclohexane/EtOAc

mixture (1:1) to yield 939 mg (4.19 mmol, 71%) of 16 as a maroon

solid. TLC: R_F¼ 0.73 (SiO₂, cyclohexane/EtOAc 1:1); ¹H NMR

DMSO‑d₆):

d

¼ 8.78 (s, 1H, NH), 8.27 (s, 1H, NH), 7.56e7.50 (m, 4H),

7.41e7.38 (m, 1H), 7.28 (d, ³J ¼ 9 Hz, 2H), 6.83 (d, ³J ¼ 9 Hz, 2H), 6.35

(s, 1H), 3.96 (t, ³J ¼ 7 Hz, 2H), 1.34e1.24 (m, 12H) ppm; ¹³C NMR

(126 MHz, DMSO‑d₆):

d

¼ 160.6 (C), 153.8 (C), 151.7 (C), 138.6 (C),

137.3 (C), 132.2 (C), 129.1 (CH), 127.0 (CH), 124.1 (CH), 119.9 (CH),

114.5 (CH), 95.3 (CH), 63.0 (CH₂), 31.9 (C), 30.1 (Me), 14.6 (Me) ppm;

(500 MHz, DMSO‑d₆):

d

¼ 7.93 (d, ³J ¼ 8.7 Hz, 2H), 7.83 (d,

HRMS (EI-TOF pos.): m/z

¼

379.2130 [MþH]^þ, calcd. for

³J ¼ 8.7 Hz, 2H), 5.97 (br s, 2H), 5.33 (s, 1H), 1.85e1.78 (m, 1H),

[C₂₂H₂₇N₄O₂]^þ¼ 379.2134.

0.92e0.87 (m, 2H), 0.73e0.67 (m, 2H) ppm; ¹³C NMR (126 MHz,

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(4-

DMSO‑d₆):

d

¼ 156.1, 149.0, 141.9, 133.5, 122.5, 118.7, 107.9, 88.0, 9.0,

ﬂuorophenyl)urea (22). Prepared according to general procedure

D using 4-ﬂuorophenyl isocyanate (0.15 mL, 1.30 mmol) and 5-

amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g, 0.93 mmol)

in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with

light petroleum/EtOAc (5:1) gave 0.28 g (0.80 mmol, 86%) of 22 as a

8.0 ppm; MS (ESI pos.): m/z (%) ¼ 225.1 (100) ([MþH]^þ, calcd.

225.1); 272.1 (14) ([M þ EtOHeH]^þ, calcd. 271.2).

3-Cyclopropyl-1-(4-nitrophenyl)-1H-pyrazol-5-amine (17).

Prepared according to general procedure C using (4-nitrophenyl)

hydrazine hydrochloride (1.00 g, 5.27 mmol), 3-cyclopropyl-3-

oxopropanenitrile (643 mg, 5.27 mmol) and HCl (conc., 2 mL) in

EtOH (20 mL) which was heated at reﬂux for 20 h. The crude

product was recrystallized from a cyclohexane/EtOAc mixture (1:1)

to yield 631 mg (2.58 mmol, 49%) of 17, as an orange solid. TLC:

R_F¼ 0.68 (SiO₂, cyclohexane/EtOAc 1:1); ¹H NMR (600 MHz,

colorless solid, mp 194.0e201.0 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13294,

2954, 1661, 1544, 1509, 1251, 1033; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.01 (s, 1H, NH), 8.36 (s, 1H, NH), 7.53e7.48(m, 4H), 7.41e7.34

(m, 3H), 7.10 (dd, ³J_HH¼ 9 Hz, ³J_HF¼ 9 Hz, 2H), 6.36 (s, 1H), 1.28 (s,

9H); ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 157.4 (d,

¹J_CF¼ 238 Hz, C), 151.7 (C), 138.5 (C), 137.0 (CH), 135.6 (d, ⁴J_CF¼ 2 Hz,

DMSO‑d₆):

d

¼ 8.29 (d, ³J ¼ 9.3 Hz, 2H), 7.92 (d, ³J ¼ 9.3 Hz, 2H), 5.60

C), 129.1 (CH), 127.1 (CH), 124.1 (CH), 119.9 (d, ³J_CF¼ 8 Hz, CH), 115.2

2

(br s, 2H), 5.28 (s, 1H), 1.82e1.76 (m, 1H), 0.88e0.82 (m, 2H),

(d, J_CF¼ 22 Hz, CH), 95.6 (CH), 31.9 (C), 30.1 (Me); HRMS (EI-TOF

0.83e0.68 (m, 2H) ppm; ¹³C NMR (150 MHz, DMSO‑d₆):

d

¼ 156.6,

pos.): m/z ¼ 353.1772 [MþH]^þ, calcd. for [C₂₀H₂₂FN₄O]^þ¼ 353.1778.

148.7, 144.9, 143.6, 124.7, 121.1, 88.3, 9.3, 7.6 ppm; MS (ESI pos.): m/z

(%) ¼ 245.04 (100) ([MþH]^þ, calcd. 245.1).

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-(3-tert-butyl-1-

phenyl-1H-pyrazol-5-yl)urea (23). Prepared according to general

procedure D using 3,5-bis(triﬂuoromethyl)phenyl isocyanate

(0.18 mL, 1.00 mmol) and 5-amino-3-tert-butyl-1-phenyl-1H-pyr-

azole (1, 0.20 g, 0.93 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Pu-

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(naphth-1-yl)

urea (18). Prepared as previously published by Regan et al. [38].

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(o-tolyl)urea

(19). Prepared according to general procedure D using o-tolyl iso-

cyanate (0.12 mL, 1.60 mmol) and 5-amino-3-tert-butyl-1-phenyl-

1H-pyrazole (1, 0.20 g, 0.93 mmol) in CH₂Cl₂(2 mL) at rt for 20 min.

Puriﬁcation by trituration with light petroleum/EtOAc (1:1) gave

riﬁcation by trituration with light petroleum gave 0.39

(0.83 mmol, 89%) of 23 as a colorless solid, mp 175.7e177.8 ^ꢀC

(MeOH); IR: (neat)

/cm^ꢃ13287, 2967, 1662, 1549, 1502, 1276, 1129,

1033; ¹H NMR (500 MHz, DMSO‑d₆):

g

n

d

¼ 9.70 (s, 1H, NH), 8.67 (s,

0.17

166.6e168.0 ^ꢀC; IR: (neat)

1033; ¹H NMR (500 MHz, DMSO‑d₆):

g

(0.49 mmol, 53%) of 19 as

a

colorless solid, mp

1H, NH), 8.07 (s, 2H), 7.64 (s, 1H), 7.56e7.49 (m, 4H), 7.40 (t,

n

/cm^ꢃ13291, 2967, 1643, 1551, 1502,

³J ¼ 6 Hz, 1H), 6.40 (s, 1H), 1.29 (s, 9H) ppm; ¹³C NMR (126 MHz,

d

¼ 8.71 (s,1H, NH), 8.19 (s, 1H,

DMSO‑d₆):

d

¼ 160.8 (C), 151.9 (C), 141.5 (C), 138.5 (C), 136.3 (C),

NH), 7.54e7.50 (m, 4H), 7.40 (m, 1H), 7.14e7.11 (m, 2H), 6.96 (t,

³J ¼ 7 Hz, 1H), 6.36 (s, 1H), 2.17 (s, 3H), 1.28 (s, 9H) ppm; ¹³C NMR

130.7 (q, ²J_CF¼ 37 Hz, CH), 129.2 (CH), 127.2 (CH), 124.0 (CH), 124.0

(q, ¹J_CF¼ 272 Hz, C), 117.9 (CH), 114.6 (CH), 97.1 (CH), 32.0 (C), 30.1

(Me) ppm; MS (EI pos.): m/z (%) ¼ 471.2 (100) ([M]^ꢅþ, calcd. 471.2);

(126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 152.0 (C), 138.7 (C), 137.2 (C),

136.9 (C), 130.1 (CH), 129.2 (CH), 128.3 (C), 127.0 (CH), 126.0 (CH),

124.0 (CH), 123.2 (CH), 121.8 (CH), 95.8 (CH), 32.0 (C), 30.1 (Me), 17.8

(Me) ppm; HRMS (EI-TOF pos.): m/z ¼ 349.2032 [MþH]^þ, calcd. for

[C₂₁H₂₅N₄O]^þ¼ 349.2028.

HRMS (EI-TOF pos.): m/z

[C₂₂H₂₁F₆N₄O]^þ¼ 471.1620.

¼

471.1617 [MþH]^þ, calcd. for

N-(4-Benzyloxyphenyl)-N⁰-(3-tert-butyl-1-phenyl-1H-pyr-

azol-5-yl)urea (24). Prepared according to general procedure D

using 4-benzyloxyphenyl isocyanate (0.21 g, 0.94 mmol) and 5-

amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g, 0.92 mmol)

in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with

light petroleum/EtOAc (5:1) gave 0.33 g (0.75 mmol, 82%) of 24 as a

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(naphth-2-yl)

urea (20). Prepared according to general procedure D using 2-

naphthyl isocyanate (0.16 g, 0.93 mmol) and 5-amino-3-tert-

butyl-1-phenyl-1H-pyrazole (1, 0.20 g, 0.92 mmol) in CH₂Cl₂(2 mL)

at rt for 20 min. Puriﬁcation by trituration with light petroleum/

EtOAc (1:1) gave 0.29 g (0.75 mmol, 81%) of 20 as a colorless solid,

colorless solid, mp 185.6e186.6 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13297,

2957, 1656, 1542, 1509, 1242, 1214, 1005; ¹H NMR (500 MHz,

mp 200.7e206.3 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13285, 2958, 1654,

DMSO‑d₆):

d

¼ 8.80 (s, 1H, NH), 8.28 (s, 1H, NH), 7.54e7.49 (m, 4H),

1550, 1500, 1033; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.22 (s, 1H,

7.44e7.35 (m, 5H), 7.31e7.26 (m, 3H), 6.92 (d, ³J ¼ 9 Hz, 2H), 5.04 (s,

NH), 8.48 (s, 1H, NH), 8.09 (s, 1H), 7.86e7.73 (m, 3H), 7.56e7.51 (m,

4H), 7.46e7.41 (m, 3H), 7.38e7.32 (m, 1H), 6.43 (s, 1H), 1.30 (s, 9H)

2H), 1.27 (s, 9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

153.6 (C), 151.7 (C), 138.6 (C), 137.3 (C), 137.2 (C), 132.6 (C), 129.1

(CH), 128.3 (CH), 127.6 (CH), 127.5 (CH), 127.1 (CH), 124.2 (CH), 119.8

(CH), 115.0 (CH), 95.3 (CH), 69.4 (CH₂), 31.9 (C), 30.1 (Me) ppm;

d

¼ 160.7 (C),

ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 151.6 (C), 138.5

(C), 137.1 (C), 136.9 (C), 133.6 (C), 129.2 (CH), 129.1 (C), 128.3 (CH),

127.3 (CH), 127.1 (CH), 126.9 (CH), 126.3 (CH), 124.2 (CH), 119.4 (CH),

113.5 (CH), 95.3 (CH), 31.9 (C), 30.1 (Me) ppm; HRMS (EI-TOF pos.):

m/z ¼ 385.2021 [MþH]^þ, calcd. for [C₂₄H₂₅N₄O]^þ¼ 385.2028.

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(4-

ethoxyphenyl)urea (21). Prepared according to general procedure

D using 4-ethoxyphenyl isocyanate (0.15 mL, 0.93 mmol) and 5-

amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g, 0.93 mmol)

HRMS (EI-TOF pos.): m/z

¼

441.2288 [MþH]^þ, calcd. for

[C₂₇H₂₉N₄O₂]^þ¼ 441.2291.

N-(3,5-Dimethylphenyl)-N⁰-(3-tert-butyl-1-phenyl-1H-pyr-

azol-5-yl)urea (25). Prepared according to general procedure D

using 3,5-dimethylphenyl isocyanate (0.13 mL, 0.93 mmol) and 5-

amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.18 g, 0.84 mmol)

in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with

16

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European Journal of Medicinal Chemistry 208 (2020) 112721

light petroleum/EtOAc (1:1) gave 0.27 g (0.75 mmol, 89%) of 25 as a

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(3-

colorless solid, mp 196.7e199.5 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13001,

methoxyphenyl)urea (30). Prepared according to general procedure

D using 3-methoxyphenyl isocyanate (0.13 mL, 1.00 mmol) and 5-

amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g, 0.93 mmol) in

CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with light

petroleum/EtOAc (2:1) gave 0.22 g (0.60 mmol, 65%) of 30 as a

2957, 1657, 1543, 1502, 1372, 1214, 1005; ¹H NMR (500 MHz,

DMSO‑d₆):

¼ 8.84 (s, 1H, NH), 8.34 (s, 1H, NH), 7.52e7.46 (m, 4H),

7.41 (m, 1H), 7.03 (s, 2H), 6.61 (s, 1H), 6.38 (s, 1H), 2.21 (s, 6H), 1.28

(s, 9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 151.4 (C),

139.1 (C), 138.5 (C), 137.7 (C), 137.2 (C), 129.2 (CH), 127.1 (CH), 124.2

(CH), 123.6 (CH), 115.8 (CH), 95.2 (CH), 31.9 (C), 30.1 (Me), 21.0 (Me)

ppm; HRMS (EI-TOF pos.): m/z ¼ 363.2193 [MþH]^þ, calcd. for

[C₂₂H₂₇N₄O]^þ¼ 363.2185.

colorless solid, mp 177.1e179.2 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13285,

¼ 9.00

2958, 1661, 1599,1549, 1045; ¹H NMR (500 MHz, DMSO‑d₆):

d

(1H, s, NH), 8.35 (s, 1H, NH), 7.56e7.49 (m, 4H), 7.41e7.39 (m, 1H),

7.14e7.05 (m, 2H), 6.88 (d, ³J ¼ 8 Hz,1H), 6.55 (dd, ³J ¼ 8 Hz,1H), 6.38

(s, 1H), 3.71 (s, 3H), 1.28 (s, 9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

¼ 160.7 (C), 159.6 (C), 151.4 (C), 140.5 (C), 138.5 (C), 137.0 (C), 129.4

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(5-ﬂuoro-2-

methylphenyl)urea (26). Prepared according to general procedure

D using 5-ﬂuoro-2-methylphenyl isocyanate (0.12 mL, 0.93 mmol)

and 5-amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g,

0.91 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trit-

uration with light petroleum/EtOAc (5:1) gave 0.22 g (0.60 mmol,

66%) of 26 as a colorless solid, mp 164.9e166.4 ^ꢀC (MeOH); IR:

d

(CH), 129.2 (CH), 127.1 (CH), 124.2 (CH), 110.3 (CH), 107.6 (CH), 103.8

(CH), 95.4 (CH), 54.8 (Me), 31.9 (C), 30.1 (Me) ppm; HRMS (EI-TOF

pos.): m/z ¼ 365.1980 [MþH]^þ, calcd. for [C₂₁H₂₅N₄O₂]^þ¼ 365.1978.

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(p-tolyl)urea

(31). Prepared according to general procedure D using p-tolyl iso-

cyanate (0.13 mL, 1.00 mmol) and 5-amino-3-tert-butyl-1-phenyl-

1H-pyrazole (1, 0.20 g, 0.92 mmol) in CH₂Cl₂(2 mL) at rt for 20 min.

Puriﬁcation by trituration with light petroleum/EtOAc (1:1) fol-

lowed by recrystallization gave 0.20 g (0.57 mmol, 62%) of 31 as a

(neat) n

/cm^ꢃ13284, 2958, 1654, 1546, 1503, 1236, 1004; ¹H NMR

(500 MHz, DMSO‑d₆):

d

¼ 8.91 (s, 1H, NH), 8.31 (s, 1H, NH), 7.73 (dd,

³J_HF¼ 12 Hz, ⁴J_HH¼ 2 Hz, 1H), 7.54e7.48 (m, 4H), 7.42e7.39 (m, 1H),

3

7.17 (dd, J_HH¼ 8 Hz, J_HF¼ 8 Hz, 1H), 6.76 (ddd, J_HH¼ 8 Hz,

4

³J_HF¼ 8 Hz, J_HH¼ 2 Hz, 1H), 6.38 (s, 1H), 2.15 (s, 3H), 1.28 (s, 9H)

colorless solid, mp 197.8e199.2 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13267,

ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 161.0 (C), 160.5 (d,

2955, 1659, 1538, 1362, 1140; ¹H NMR (500 MHz, DMSO‑d₆):

¹J_CF¼ 240 Hz, C), 151.9 (C), 138.7 (C), 138.5 (d, ³J_CF¼ 11 Hz, C), 137.0

d

¼ 8.88 (s, 1H, NH), 8.32 (s, 1H, NH), 7.56e7.50 (m, 4H), 7.41e7.39

(CH), 131.2 (d, ³J_CF¼ 10 Hz, CH), 129.4 (CH), 127.3 (CH), 124.2 (CH),

(m, 1H), 7.28 (d, ³J ¼ 8 Hz, 2H), 7.06 (d, ³J ¼ 8 Hz, 2H), 6.37 (s, 1H),

4

2

122.9 (d, J_CF¼ 3 Hz, C), 109.0 (d, J_CF¼ 21 Hz, CH), 107.3 (d,

²J_CF¼ 26 Hz, CH), 96.1 (CH), 32.1 (C), 30.2 (Me), 17.2 (Me) ppm;

2.23 (s, 3H), 1.28 (s, 9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 151.5 (C), 138.5 (C), 137.2 (C), 136.7 (C), 130.8 (C), 129.2

HRMS (EI-TOF pos.): m/z

[C₂₁H₂₄FN₄O]^þ¼ 367.1934.

¼

367.1937 [MþH]^þ, calcd. for

(CH), 129.1 (CH), 127.1 (CH), 124.2 (CH), 118.2 (CH), 95.2 (CH), 31.9

(C), 30.1 (Me), 20.1 (Me) ppm; HRMS (EI-TOF pos.): m/z ¼ 349.2041

[MþH]^þ, calcd. for [C₂₁H₂₅N₄O]^þ¼ 349.2028.

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-(3,4-

diﬂuorophenyl)urea (27). Prepared according to general proced-

ure D using 3,4-diﬂuorophenyl isocyanate (0.11 mL, 0.94 mmol) and

5-amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g, 0.92 mmol)

in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with

light petroleum/EtOAc (5:1) gave 0.23 g (0.62 mmol, 67%) of 27 as a

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-[3-tert-butyl-1-(p-

tolyl)-1H-pyrazol-5-yl]urea (32). Prepared according to general

procedure

D using 3,3-bis(triﬂuoromethyl)phenyl isocyanate

(0.19 g, 0.76 mmol) and 5-amino-3-tert-butyl-1-(p-tolyl)-1H-pyr-

azole (2, 0.21 g, 0.90 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Pu-

riﬁcation by trituration with light petroleum followed by

recrystallization gave 0.16 g (0.33 mmol, 43%) of 32 as colorless

colorless solid, mp 154.1e156.4 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13332,

n

2957, 1660, 1571, 1513, 1226, 1136; ¹H NMR (500 MHz, DMSO‑d₆):

3

d

¼ 9.20 (s, 1H, NH), 8.44 (s, 1H, NH), 7.61 (ddd, J_HF¼ 13 Hz,

crystals, mp 155.3e158.3 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13322, 2959,

n

⁴J_HF¼ 7 Hz, ⁴J_HH¼ 2 Hz, 1H), 7.52e7.46 (m, 4H), 7.41e7.39 (m, 1H),

7.31 (q, ³J ¼ 10 Hz, 1H), 7.07 (d, ³J_HH¼ 9 Hz, 1H), 6.37 (s, 1H), 1.28 (s,

1660, 1561, 1383, 1271, 1172, 1139; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.70 (s, 1H, NH), 8.60 ( s, 1H, NH), 8.06 (s, 2H), 7.64 (s, 1H), 7.40

9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 151.6 (C),

(d, ³J ¼ 8 Hz, 2H), 7.32 (d, ³J ¼ 8 Hz, 2H), 6.38 (s, 1H), 1.28 (s, 9H)

149.0 (dd, ¹J_CF¼ 243 Hz, J_CF¼ 13 Hz, C),144.5 (dd, ¹J_CF¼ 240 Hz,

ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 160.5 (C), 151.8 (C), 141.5

2

3

2

²J_CF¼ 12 Hz, C), 138.5 (C), 136.7 (C), 136.4 (dd, J_CF¼ 9 Hz,

(C), 136.7 (C), 136.2 (C), 136.0 (C), 130.7 (q, J_CF¼ 33 Hz, C), 129.6

(CH), 124.1 (CH), 123.4 (q, ¹J_CF¼ 273 Hz, C), 117.9 (q, ³J_CF¼ 3 Hz, CH),

114.6 (CH), 96.5 (CH), 32.0 (C), 30.1 (Me) ppm; MS (EI pos.): m/z

(%) ¼ 484.2 (100) ([M]^ꢅþ, calcd. 484.2); HRMS (ESI pos.): m/

z ¼ 485.1749 [MþH]^þ, calcd. for [C₂₃H₂₃F₆N₄O]^þ¼ 485.1771.

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-[3-tert-butyl-1-

⁴J_CF¼ 2 Hz, C),129.2 (CH),127.1 (CH),124.1 (CH),117.3 (d, ³J_CF¼ 6 Hz,

⁴J_CF¼ 3 Hz, CH), 114.3 (dd, ²J_CF¼ 21 Hz, CH), 107.1 (d, ²J_CF¼ 22 Hz,

CH), 96.0 (CH), 31.9 (C), 30.1 (Me) ppm; HRMS (EI-TOF pos.): m/

z ¼ 371.1681 [MþH]^þ, calcd. for [C₂₀H₂₁F₂N₄O]^þ¼ 371.1683.

N-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-N⁰-phenylurea

(28). Prepared as previously published by Bagley et al.[31].

N-[(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)carbamoyl]-4-

methylbenzenesulfonamide (29). Prepared according to general

procedure D using p-tolylsulfonyl isocyanate (0.15 mL, 1.10 mmol)

and 5-amino-3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.20 g,

0.91 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trit-

uration with light petroleum/EtOAc (1:1) gave 0.28 g (0.68 mmol,

methyl-1H-pyrazol-5-yl]urea (33). Prepared according to general

procedure

D using 3,3-bis(triﬂuoromethyl)phenyl isocyanate

(0.15 mL, 0.87 mmol) and 5-amino-3-tert-butyl-1-methyl-1H-pyr-

azole (3, 0.13 g, 0.84 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Pu-

riﬁcation by trituration with light petroleum followed by

recrystallization gave 0.10 g (0.25 mmol, 30%) of 33 as colorless

crystals, mp 155.7e159.7 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13307, 2968,

n

75%) of 29 as a colorless solid, mp 166.3e170.6 ^ꢀC; IR: (neat)

n

/cm^ꢃ1

1706, 1573, 1386, 1276, 1166, 1130; ¹H NMR (500 MHz, DMSO‑d₆):

¼ 9.61 (s, 1H, NH), 8.74 (s, 1H, NH), 8.14 (s, 2H), 7.64 (s, 1H), 6.07 (s,

3290, 2957, 1657, 1541, 1502, 1212, 989; ¹H NMR (500 MHz,

d

DMSO‑d₆):

d

¼ 11.09 (br s, 1H, NH), 8.55 (s, 1H, NH), 7.77 (d,

1H), 3.61 (s, 3H), 1.22 (s, 9H) ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

³J ¼ 8 Hz, 2H), 7.47e7.35 (m, 7H), 6.26 (s, 1H), 2.40 (s, 3H), 1.23 (s,

d

¼ 158.6 (C), 152.1 (C), 141.7 (C), 136.2 (C), 130.7 (q, ²J_CF¼ 33 Hz, C),

9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 160.7 (C), 148.8 (C),

123.3 (q, J_CF¼ 274 Hz, C), 118.0 (CH), 114.5 (CH), 95.0 (CH), 34.9

(Me), 31.8 (C), 30.3 (Me) ppm; HRMS (ESI pos.): m/z ¼ 409.1454

[MþH]^þ, calcd. for [C₁₇H₁₉F₆N₄O]^þ¼ 409.1458.

1

143.9 (C), 138.1 (C), 136.7 (C), 135.3 (C), 129.4 (CH), 129.1 (CH), 127.3

(CH), 127.3 (CH), 124.0 (CH), 96.8 (CH), 32.0 (C), 30.0 (Me), 21.0 (Me)

ppm; HRMS (EI-TOF pos.): m/z ¼ 413.1655 [MþH]^þ, calcd. for

[C₂₁H₂₅N₄O₃S]^þ¼ 413.1647.

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-[3-tert-butyl-1-(4-

ﬂuorophenyl)-1H-pyrazol-5-yl]urea (34). Prepared according to

17

€

S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

general procedure D using 3,3-bis(triﬂuoromethyl)phenyl isocyanate

(0.15 mL, 0.87 mmol) and 5-amino-3-tert-butyl-1-(4-ﬂuorophenyl)-

1H-pyrazole (4, 0.19 g, 0.82 mmol) in CH₂Cl₂(2 mL) at rt for 20 min.

(ESI

pos.):

m/z

¼

549.0715

[MþH]^þ,

calcd.

for

[C₂₂H₂₀BrF₆N₄O]^þ¼ 549.0719.

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-(1-methyl-3-phenyl-

1H-pyrazol-5-yl)urea (38). Prepared according to general proced-

ure D using 3,3-bis(triﬂuoromethyl)phenyl isocyanate (0.18 g,

0.71 mmol) and 5-amino-1-methyl-3-phenyl-1H-pyrazole (8,

0.15 g, 0.85 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by

trituration with light petroleum gave 0.22 g (0.51 mmol, 72%) of 38

Puriﬁcation by trituration with light petroleum gave 0.30

(0.61 mmol, 74%) of 34 as a yellow solid, mp 177.6e184.3 ^ꢀC; IR:

(neat)

/cm^ꢃ13350, 2962, 1664, 1569, 1510, 1383, 1271, 1140; ¹H NMR

(500 MHz, DMSO‑d₆):

g

n

d

¼ 9.70 (s, 1H, NH), 8.66 (s, 1H, NH), 8.07 (s,

3 4

2H), 7.63 (s, 1H), 7.57 ( dd, J_HH¼ 9 Hz, J_HF¼ 5 Hz, 2H), 7.35 (dd,

³J_HH¼ 9 Hz, ³J_HF¼ 9 Hz, 2H), 6.39 (s, 1H), 1.29 (s, 9H) ppm; ¹³C NMR

as a colorless solid, mp 195.3e198.9 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ1

(100 MHz, DMSO‑d₆):

d

¼ 160.9 (d, ¹J_CF¼ 245 Hz, C), 160.8 (C), 151.9

3378, 2966, 1674, 1561, 1472, 1276, 1125, 1054; ¹H NMR (500 MHz,

(C), 141.5 (C), 136.4 (C), 135.0 (d, ⁴J_CF¼ 3 Hz, C), 130.7 (q, ²J_CF¼ 32 Hz,

DMSO‑d₆):

d

¼ 9.66 (s, 1H, NH), 8.96 (s, 1H, NH), 8.18 (s, 2H), 7.77 (d,

3

1

C), 126.3 (d, J_CF¼ 9 Hz, CH), 123.2 (q, J_CF¼ 273 Hz, C), 117.9 (q,

³J_CF¼ 3 Hz, CH), 115.9 (d, ²J_CF¼ 23 Hz, CH), 114.6 (CH), 97.2 (CH), 32.0

(C), 30.1 (Me) ppm; MS (EI pos.): m/z (%) ¼ 488.1 (100) ([M]^ꢅþ, calcd.

488.1); HRMS (ESI pos.): m/z ¼ 489.1499 [MþH]^þ, calcd. for

[C₂₂H₂₀F₇N₄O]^þ¼ 489.1520.

³J ¼ 8 Hz, 2H), 7.67 (s, 1H), 7.39 (t, ³J ¼ 8 Hz, 2H), 7.29 (t, ³J ¼ 8 Hz,

1H), 6.67 (s, 1H), 3.74 (s, 3H) ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 152.1 (C), 148.1 (C), 141.6 (C), 137.6 (C), 133.4 (C), 130.7 (q,

²J_CF¼ 33 Hz, C), 128.5 (CH), 127.3 (CH), 124.7 (CH), 123.0 (q,

¹J_CF¼ 273 Hz, C), 118.2 (CH), 114.7 (CH), 96.2 (CH), 35.4 (Me) ppm;

MS (EI pos.): m/z (%) ¼ 428.1 (49) ([M]^ꢅþ, calcd. 428.1), 173.6 (100)

([M_fr.]^þ, calcd. 173.6); HRMS (ESI pos.): m/z ¼ 429.1146 [MþH]^þ,

calcd. for [C₁₉H₁₅F₆N₄O]^þ¼ 429.1144.

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-[3-tert-butyl-1-(4-

nitrophenyl)-1H-pyrazol-5-yl]urea (35). Prepared according to

general procedure D using 3,3-bis(triﬂuoromethyl)phenyl isocyanate

(0.12 g, 0.52 mmol) and 5-amino-3-tert-butyl-1-(4-nitrophenyl)-1H-

pyrazole (5, 0.15 g, 0.58 mmol) in CH₂Cl₂(2 mL) at rt for 20 min.

Puriﬁcation by trituration with light petroleum followed by recrys-

tallization gave 0.10 g (0.19 mmol, 37%) of 35 as orange crystals, mp

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-(1,3-diphenyl-1H-pyr-

azol-5-yl)urea (39). Prepared according to general procedure D

using 3,3-bis(triﬂuoromethyl)phenyl isocyanate (0.19 g, 0.73 mmol)

and 5-amino-1,3-diphenyl-1H-pyrazole (9, 0.20 g, 0.86 mmol) in

CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with light

petroleum followed by recrystallization gave 0.23 g (0.47 mmol,

64%) of 39 as colorless crystals, mp 205.7e206.9 ^ꢀC (MeOH); IR:

215.1e217.8 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13350, 2968, 1671, 1572,

n

1504, 1340, 1272, 1175, 1135; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.79

(s, 1H, NH), 8.92 (s, 1H, NH), 8.35 (d, ³J ¼ 9 Hz, 2H), 8.09 (s, 2H), 7.88 (

d, ³J ¼ 9 Hz, 2H), 7.64 (s, 1H), 6.48 (s, 1H), 1.31 (s, 9H) ppm; ¹³C NMR

(neat)

n

/cm^ꢃ13306, 2967, 1653, 1590, 1574, 1474, 1274, 1126, 1055;

(100 MHz, DMSO‑d₆):

d

¼ 162.4 (C), 152.2 (C), 145.0 (C), 143.9 (C),

¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.72 (s, 1H, NH), 8.86 (s, 1H, NH),

2

141.5 (C), 137.1 (C), 130.6 (q, J_CF¼ 32 Hz, C), 124.7 (CH), 123.6 (q,

8.10 (s, 2H), 7.88 (d, ³J ¼ 7 Hz, 2H), 7.65e7.57 (m, 3H), 7.58 (t,

³J ¼ 8 Hz, 2H), 7.49e7.41 (m, 3H), 7.35 (t, ³J ¼ 7 Hz, 1H), 6.99 (s, 1H)

¹J_CF¼ 274 Hz, C), 123.0 (CH), 118.1 (q, J_CF¼ 3 Hz, CH), 114.7 (CH),

3

100.3 (CH), 32.2 (C), 29.8 (Me) ppm; MS (EI pos.): m/z (%) ¼ 515.1

(100) ([M]^ꢅþ, calcd. 515.1); HRMS (ESI pos.): m/z ¼ 516.1442 [MþH]^þ,

calcd. for [C₂₂H₂₀F₆N₅O₃]^þ¼ 516.1465.

ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 151.9 (C), 150.1 (C), 141.4

2

(C), 138.3 (C), 137.6 (C), 132.9 (C), 130.7 (q, J_CF¼ 32 Hz, C), 129.3

(CH), 128.6 (CH), 127.9 (CH), 127.8 (CH), 125.1 (CH), 124.3 (CH), 123.7

(q, ¹J_CF¼ 273 Hz, C), 118.1 (CH), 114.8 (CH), 98.0 (CH) ppm; MS (EI

pos.): m/z (%) ¼ 490.1 (45) ([M]^ꢅþ, calcd. 490.1), 235.3 (100) ([M_fr.]^þ,

calcd. 235.3); HRMS (ESI pos.): m/z ¼ 491.1288 [MþH]^þ, calcd. for

[C₂₄H₁₇F₆N₄O]^þ¼ 491.1301.

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-(1,3-di-tert-butyl-1H-

pyrazol-5-yl)urea (36). Prepared according to general procedure D

using 3,3-bis(triﬂuoromethyl)phenyl isocyanate (0.11 g, 0.43 mmol)

and 5-amino-1,3-di-tert-butyl-1H-pyrazole (6, 95 mg, 0.49 mmol)

in CH₂Cl₂(2 mL) at rt for 30 min. Puriﬁcation by trituration with

light petroleum gave 0.14 g (0.31 mmol, 72%) of 36 as a colorless

N-(3,5-Diﬂuorophenyl)-N⁰-(3-tert-butyl-1-phenyl-1H-pyr-

azol-5-yl)urea (40). Prepared according to general procedure D

using diﬂuorophenyl isocyanate (0.12 g, 0.74 mmol) and 5-amino-

3-tert-butyl-1-phenyl-1H-pyrazole (1, 0.16 g, 0.74 mmol) in CH₂Cl₂

(2 mL) at rt for 20 min. Puriﬁcation by trituration with light pe-

troleum gave 0.17 g (0.46 mmol, 62%) of 40 as a colorless solid, mp

solid, mp 202.2e203.3 ^ꢀC (MeOH); IR: (neat)

1655, 1576, 1384, 1272, 1184, 1133; ¹H NMR (500 MHz, DMSO‑d₆):

¼ 9.64 (s, 1H, NH), 8.20 (s, 1H, NH), 8.15 (s, 2H), 7.62 (s, 1H), 6.05 (s,

1H), 1.54e1.51 (m, 9H), 1.22 (s, 9H) ppm; ¹³C NMR (100 MHz,

DMSO‑d₆):

n

/cm^ꢃ13310, 2969,

d

¼ 156.6 (C), 153.0 (C), 141.9 (C), 134.5 (C), 130.6 (q,

171.0e173.0 ^ꢀC (MeOH); IR: (neat)

n

/cm^ꢃ13322, 2956, 1670, 1554,

²J_CF¼ 32 Hz, C), 123.2 (q, ¹J_CF¼ 273 Hz, C), 117.9 (CH), 114.4 (CH),

100.7 (CH), 58.8 (C), 31.8 (C), 30.3 (Me), 29.6 (Me) ppm; MS (EI

pos.): m/z (%) ¼ 450.1 (30) ([M]^ꢅþ, calcd. 450.1), 139.2 (100) [M_fr.]^þ,

calcd. 139.2); HRMS (ESI pos.): m/z ¼ 451.1923 [MþH]^þ, calcd. for

[C₂₀H₂₅F₆N₄O]^þ¼ 451.1927.

1500,1231,1117; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.38 (s, 1H, NH),

8.54 (s, 1H, NH), 7.53e7.48 (m, 4H), 7.44e7.36 (m, 1H), 7.14 (d,

³J_HF¼ 9 Hz, 2H), 6.78 (t, J_HF¼ 9 Hz, 1H), 6.38 (s, 1H), 1.29 (s, 9H)

3

ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 162.5 (dd, ¹J_CF¼ 243 Hz,

³J_CF¼ 15 Hz, C), 160.7 (C), 151.5 (C), 142.1 (t, ³J_CF¼ 14 Hz, C), 138.5

N-[3,5-Bis(triﬂuoromethyl)phenyl]-N⁰-[3-tert-butyl-1-(4-

bromophenyl)-1H-pyrazol-5-yl]urea (37). Prepared according to

general procedure D using 3,3-bis(triﬂuoromethyl)phenyl isocya-

nate (0.13 g, 0.50 mmol) and 5-amino-3-tert-butyl-1-(4-

bromophenyl)-1H-pyrazole (7, 0.16 g, 0.54 mmol) in CH₂Cl₂

(1 mL) at rt for 20 min. Puriﬁcation by trituration with light pe-

troleum gave 0.16 g (0.29 mmol, 58%) of 37 as a colorless solid, mp

(C), 136.5 (C), 129.2 (CH), 127.2 (CH), 124.1 (CH), 100.9 (dd,

²J_CF¼ 21 Hz, J_CF¼ 8 Hz, CH), 97.0 (t, J_CF¼ 27 Hz, CH), 96.3 (CH),

4

2

32.0 (C), 30.1 (Me) ppm; MS (EI pos.): m/z (%) ¼ 370.1 (47) ([M]^ꢅþ

,

calcd. 370.1), 139.2 (100) ([M_fr.]^þ, calcd. 139.2); HRMS (EI-TOF pos.):

m/z ¼ 371.1673 [MþH]^þ, calcd. for [C₂₀H₂₁F₂N₄O]^þ¼ 371.1678.

N-(3,5-Diﬂuorophenyl)-N⁰-[3-tert-butyl-1-(p-tolyl)-1H-pyr-

azol-5-yl]urea (41). Prepared according to general procedure D

using diﬂuorophenyl isocyanate (0.11 g, 0.68 mmol) and 5-amino-

3-tert-butyl-1-(p-tolyl)-1H-pyrazole (2, 0.16 g, 0.70 mmol) in

CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with light

petroleum gave 0.17 g (0.44 mmol, 65%) of 41 as a colorless solid,

196.5e198.2 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13286, 2962, 1664, 1564,

n

1496, 1272, 1179, 1137; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.70 (s,

1H, NH), 8.71 (s, 1H, NH), 8.07 (s, 2H), 7.70 (d, ³J ¼ 8 Hz, 2H), 7.65 (s,

1H), 7.51 (d, ³J ¼ 8 Hz, 2H), 6.40 (s, 1H), 1.29 (s, 9H) ppm; ¹³C NMR

(100 MHz, DMSO‑d₆):

d

¼ 161.7 (C), 152.4 (C), 142.0 (C), 138.3 (C),

mp 188.9e190.8 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13375, 2963, 1720,

n

136.9 (C), 132.6 (CH), 131.2 (q, ²J_CF¼ 33 Hz, C), 126.2 (CH), 124.2 (q,

¹J_CF¼ 273 Hz, C), 120.3 (C), 118.5 (q, ³J_CF¼ 3 Hz, CH), 115.1 (CH), 98.4

(CH), 32.5 (C), 30.5 (Me) ppm; MS (EI pos.): m/z (%) ¼ 550.2 (37)

([MþH]^ꢅþ, calcd. 550.2), 306.4 (100) ([M_fr.]^þ, calcd. 306.4); HRMS

1619, 1542, 1478, 1196, 1114; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.37

(s, 1H, NH), 8.47 (s, 1H, NH), 7.38 (d, ³J ¼ 8 Hz, 2H), 7.32 (d, ³J ¼ 8 Hz,

3

2H), 7.13 (d, J_HF¼ 9 Hz, 2H), 6.79 (t, J_HF¼ 9 Hz, 1H), 6.35 (s, 1H),

2.37 (s, 3H), 1.27 (s, 9H) ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

18

€

S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

1

3

d

¼ 162.5 (dd, J_CF¼ 243 Hz, J_CF¼ 16 Hz, C), 160.5 (C), 151.4 (C),

for 20 min. Puriﬁcation by trituration with light petroleum followed

by recrystallization gave 0.16 g (0.46 mmol, 68%) of 45 as colorless

3

142.1 (t, J_CF¼ 14 Hz, C), 136.8 (C), 136.5 (C), 136.0 (C), 129.6 (CH),

2

4

124.2 (CH), 100.9 (dd, J_CF¼ 29 Hz, J_CF¼ 8 Hz, CH), 97.0 (t,

²J_CF¼ 26 Hz, CH), 95.7 (CH), 32.0 (C), 30.2 (Me), 20.5 (Me) ppm; MS

(EI pos.): m/z (%) ¼ 384.2 (52) ([M]^ꢅþ, calcd. 384.2), 214.2 (100)

([M_fr.]^þ, calcd. 214.2); HRMS (ESI pos.): m/z ¼ 385.1829 [MþH]^þ,

calcd. for [C₂₁H₂₃F₂N₄O]^þ¼ 385.1834.

crystals, mp 212.9e214.5 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13366, 2957,

n

1663, 1612, 1566, 1478, 1207, 1116; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.31 (s, 1H, NH), 8.01 (s, 1H, NH), 7.18 (d, ³J_HF¼ 8 Hz, 2H), 6.77 (t,

³J_HF¼ 9 Hz, 1H), 6.02 (s, 1H), 1.53 (s, 9H), 1.21 (s, 9H) ppm; ¹³C NMR

(100 MHz, DMSO‑d₆):

d

¼ 162.5 (dd, ¹J_CF¼ 243 Hz, ³J_CF¼ 15 Hz, C),

3

N-(3,5-Diﬂuorophenyl)-N⁰-(3-tert-butyl-1-methyl-1H-pyr-

azol-5-yl)urea (42). Prepared according to general procedure D

using diﬂuorophenyl isocyanate (0.14 g, 0.93 mmol) and 5-amino-

3-tert-butyl-1-methyl-1H-pyrazole (3, 0.16 g, 1.00 mmol) in CH₂Cl₂

(2 mL) at rt for 20 min. Puriﬁcation by trituration with light pe-

troleum followed by recrystallization gave 0.22 g (0.71 mmol, 76%)

156.5 (C), 152.6 (C), 142.5 (t, J_CF¼ 15 Hz, C), 134.6 (C), 100.9 (dd,

²J_CF¼ 30 Hz, ⁴J_CF¼ 9 Hz, CH), 100.4 (CH), 96.7 (t, ²J_CF¼ 27 Hz, CH),

58.7 (C), 31.8 (C), 30.3 (Me), 29.5 (Me) ppm; MS (EI pos.): m/z

(%) ¼ 350.2 (39) ([M]^ꢅþ, calcd. 350.2), 139.2 (100) ([M_fr.]^þ, calcd.

139.2); HRMS (ESI pos.): m/z ¼ 351.1987 [MþH]^þ, calcd. for

[C₁₈H₂₅F₂N₄O]^þ¼ 351.1991.

of 42 as colorless crystals, mp 189.0e191.1 ^ꢀC (MeOH); IR: (neat)

cm^ꢃ13322, 2964, 1711, 1610, 1558, 1478, 1206, 1112; ¹H NMR

n

/

N-(3,5-Diﬂuorophenyl)-N⁰-[3-tert-butyl-1-(4-bromophenyl)-

1H-pyrazol-5-yl]urea (46). Prepared according to general proced-

ure D using diﬂuorophenyl isocyanate (0.17 g, 1.00 mmol) and 5-

amino-3-tert-butyl-1-(4-bromophenyl)-1H-pyrazole (7, 0.21 g,

0.71 mmol) in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trit-

uration with light petroleum followed by recrystallization gave

0.17 mg (0.38 mmol, 54%) of 46 as colorless crystals, mp

(500 MHz, DMSO‑d₆):

d

¼ 9.27 (s, 1H, NH), 8.61 (s, 1H, NH), 7.19 (d,

³J_HF¼ 8 Hz, 2H), 6.80 (t, ³J_HF¼ 9 Hz, 2H), 6.04 (s, 1H), 3.59 (s, 3H),

1.21 (s, 9H) ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 162.5 (dd,

¹J_CF¼ 243 Hz, J_CF¼ 15 Hz, C), 158.5 (C), 151.7 (C), 142.2 (t,

3

³J_CF¼ 14 Hz, C), 136.4 (C), 101.0 (dd, J_CF¼ 29 Hz, J_CF¼ 8 Hz, CH),

2

4

2

96.9 (t, J_CF¼ 26 Hz, CH), 94.4 (CH), 34.9 (Me), 31.8 (C), 30.3 (Me)

158.0e159.4 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ13320, 2964, 1668, 1612,

n

ppm; MS (EI pos.): m/z (%) ¼ 308.1 (56) ([M]^ꢅþ, calcd. 308.1), 138.6

(100) ([M_fr.]^þ, calcd. 138.6); HRMS (ESI pos.): m/z ¼ 309.1517

[MþH]^þ, calcd. for [C₁₅H₁₉F₂N₄O]^þ¼ 309.1521.

1552, 1478, 1228, 1116, 670; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.37

3

(s, 1H, NH), 8.58 (s, 1H, NH), 7.71 (d, J_HH¼ 9 Hz, 2H), 7.50 (d,

³J_HH¼ 9 Hz, 2H), 7.14 (d, ³J_HF¼ 8 Hz, 2H), 6.78 (t, J_HF¼ 9 Hz, 1H),

3

N-(3,5-Diﬂuorophenyl)-N⁰-[3-tert-butyl-1-(4-ﬂuorophenyl)-

1H-pyrazol-5-yl]urea (43). Prepared according to general procedure

D using diﬂuorophenyl isocyanate (80 mg, 0.52 mmol) and 5-amino-

3-tert-butyl-1-(4-ﬂuorophenyl)-1H-pyrazole (4, 0.13 g, 0.55 mmol)

in CH₂Cl₂(2 mL) at rt for 20 min. Puriﬁcation by trituration with light

petroleum followed by recrystallization gave 80 mg (0.21 mmol,

40%) 43 as colorless crystals, mp 165.3e169.3 ^ꢀC (MeOH); IR: (neat)

6.38 (s, 1H), 1.28 (s, 9H) ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 163.8 (dd, ¹J_CF¼ 243 Hz, ³J_CF¼ 16 Hz, C),161.2 (C),151.6 (C),142.0

(t, ³J_CF¼ 14 Hz, C), 137.8 (C), 136.7 (C), 132.1 (CH), 125.9 (CH), 119.8

(C), 101.0 (dd, ²J_CF¼ 21 Hz, ⁴J_CF¼ 9 Hz, CH), 97.0 (2CH), 32.0 (C), 30.0

(Me) ppm; MS (EI pos.): m/z (%) ¼ 449.1 (79) ([MþH]^ꢅþ, calcd.

449.1), 279.4 (100) ([M_fr.]^þ, calcd. 279.4); HRMS (ESI pos.): m/

z ¼ 449.0782 [MþH]^þ, calcd. for [C₂₀H₂₀BrF₂N₄O]^þ¼ 449.0783.

N-(3,5-Diﬂuorophenyl)-N⁰-(1-methyl-3-phenyl-1H-pyrazol-5-

yl)urea (47). Prepared according to general procedure D using

diﬂuorophenyl isocyanate (0.10 g, 0.66 mmol) and 5-amino-1-

methyl-3-phenyl-1H-pyrazole (8, 0.13 g, 0.73 mmol) in CH₂Cl₂

(3 mL) at rt for 20 min. Puriﬁcation by trituration with light petro-

leum followed by recrystallization gave 0.18 g (0.55 mmol, 83%) of 47

n

/cm^ꢃ12966, 1728, 1612, 1553, 1226, 1113; ¹H NMR (500 MHz,

DMSO‑d₆):

d

¼ 9.35 (s,1H, NH), 8.52 (s,1H, NH), 7.55 (dd, ³J_HH¼ 8 Hz,

⁴J_HF¼ 5 Hz, 2H), 7.36 (dd, J_HH¼ 8 Hz, J_HF¼ 8 Hz, 2H), 7.13 (d,

3

³J_HF¼ 8 Hz, 2H), 6.79 (t, J_HF¼ 9 Hz, 1H), 6.36 (s, 1H), 1.28 (s, 9H)

3

ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 161.2 (dd, ¹J_CF¼ 243 Hz,

³J_CF¼ 16 Hz, C), 160.9 (d, ¹J_CF¼ 245 Hz, C),160.8 (C),151.5 (C),142.0 (t,

³J_CF¼ 14 Hz, C),136.7 (C),134.9 (d, ⁴J_CF¼ 3 Hz, C),126.5 (d, ³J_CF¼ 8 Hz,

CH), 116.0 (d, ²J_CF¼ 23 Hz, CH), 100.9 (dd, ²J_CF¼ 21 Hz, ⁴J_CF¼ 9 Hz,

CH), 97.0 (t, ²J_CF¼ 26 Hz, CH), 96.4 (CH), 32.0 (C), 30.1 (Me) ppm; MS

(EI pos.): m/z (%) ¼ 389.1 (72) ([MþH]^ꢅþ, calcd. 389.1), 218.9 (100)

([M_fr.]^þ, calcd. 218.9); HRMS (ESI pos.): m/z ¼ 389.1582 [MþH]^þ,

calcd. for [C₂₀H₂₀F₃N₄O]^þ¼ 389.1584.

as colorless crystals, mp 205.6e206.7 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ1

n

3284, 2973, 1711, 1609, 1558, 1479, 1235, 1114; ¹H NMR (500 MHz,

DMSO‑d₆):

d

¼ 9.34 (s, 1H, NH), 8.82 (s, 1H, NH), 7.76 (d, ³J_HH¼ 8 Hz,

3

2H), 7.39 (t, J_HH¼ 8 Hz, 2H), 7.28 (t, J_HH¼ 8 Hz, 1H), 7.22 (d,

³J_HF¼ 9 Hz, 2H), 6.81 (t, J_HF¼ 9 Hz, 1H), 6.64 (s, 1H), 3.73 (s, 3H)

3

ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 163.0 (dd, ¹J_CF¼ 243 Hz,

N-(3,5-Diﬂuorophenyl)-N⁰-[3-tert-butyl-1-(4-nitrophenyl)-

1H-pyrazol-5-yl]urea (44). Prepared according to general proced-

ure D using diﬂuorophenyl isocyanate (150 mg, 0.58 mmol) and 5-

amino-3-tert-butyl-1-(4-nitrophenyl)-1H-pyrazole (5, 82 mg,

0.52 mmol) in CH₂Cl₂(2 mL) at rt for 18 h. Puriﬁcation by trituration

with light petroleum followed by recrystallization gave 54 mg

(0.13 mmol, 25%) of 44 as yellow crystals, mp 178.6e182.0 ^ꢀC

³J_CF¼ 15 Hz, C), 151.7 (C), 148.1 (C), 142.1 (t, ³J_CF¼ 14 Hz, C), 137.8 (C),

133.5 (C), 128.5 (CH), 127.3 (CH), 124.7 (CH), 101.1 (dd, ²J_CF¼ 21 Hz,

⁴J_CF¼ 8 Hz, CH), 96.8 (t, ²J_CF¼ 26 Hz, CH), 95.5 (CH), 35.4 (Me) ppm;

MS (EI pos.): m/z (%) ¼ 328.0 (44) ([M]^ꢅþ, calcd. 328.0), 173.5 (100)

([M_fr.]^þ, calcd. 173.5); HRMS (ESI pos.): m/z ¼ 329.1204 [MþH]^þ,

calcd. for [C₁₇H₁₅F₂N₄O]^þ¼ 329.1208.

N-(3,5-Diﬂuorophenyl)-N⁰-(1,3-diphenyl-1H-pyrazol-5-yl)

urea (48). Prepared according to general procedure D using

diﬂuorophenyl isocyanate (0.12 g, 0.75 mmol) and 5-amino-1,3-

diphenyl-1H-pyrazole (9, 0.20 g, 0.85 mmol) in CH₂Cl₂(2 mL) at

rt for 20 min. Puriﬁcation by trituration with light petroleum fol-

lowed by recrystallization gave 0.21 g (0.54 mmol, 72%) of 48 as

(MeOH); IR: (neat) n

/cm^ꢃ13298, 2963, 1762, 1610, 1565, 1503, 1334,

1234, 1116; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.45 (s, 1H, NH), 8.78

(s,1H, NH), 8.36 (d, ³J_HH¼ 9 Hz, 2H), 7.87 (d, ³J_HH¼ 9 Hz, 2H), 7.14 (d,

³J_HF¼ 8 Hz, 2H), 6.79 (t, J_HF¼ 9 Hz, 1H), 6.45 (s, 1H), 1.30 (s, 9H)

3

ppm; ¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 162.5 (dd, ¹J_CF¼ 243 Hz,

³J_CF¼ 16 Hz, C), 162.4 (C), 151.8 (C), 145.0 (C), 143.9 (C), 142.0 (t,

³J_CF¼ 14 Hz, C), 137.4 (C), 124.8 (CH), 123.1 (CH), 101.1 (dd,

colorless crystals, mp 195.4e200.4 ^ꢀC (MeOH); IR: (neat) /cm^ꢃ1

n

3324, 2945, 1726, 1615, 1544, 1480, 1194, 1106; ¹H NMR (500 MHz,

²J_CF¼ 21 Hz, J_CF¼ 9 Hz, CH), 99.5 (CH), 97.4 (d, ²J_CF¼ 27 Hz, CH),

DMSO‑d₆):

d

¼ 9.42 (s, 1H, NH), 8.71 (s, 1H, NH), 7.86 (d, ³J_HH¼ 8 Hz,

4

32.2 (C), 29.9 (Me) ppm; MS (EI pos.): m/z (%) ¼ 415.2 (30) ([M]^ꢅþ

,

2H), 7.60e7.55 (m, 4H), 7.46e7.42 (m, 3H), 7.35 (t, ³J_HH¼ 8 Hz, 1H),

calcd. 415.2), 271.3 (100) ([M_fr.]^þ, calcd. 271.3); HRMS (ESI pos.): m/

z ¼ 416.1534 [MþH]^þ, calcd. for [C₂₀H₂₀F₂N₅O₃]^þ¼ 416.1529.

N-(3,5-Diﬂuorophenyl)-N⁰-(1,3-di-tert-butyl-1H-pyrazol-5-

yl)urea (45). Prepared according to general procedure D using

diﬂuorophenyl isocyanate (0.11 g, 0.68 mmol) and 5-amino-1,3-di-

tert-butyl-1H-pyrazole (6, 0.14 g, 0.72 mmol) in CH₂Cl₂(2 mL) at rt

7.16 (d, ³J_HF¼ 9 Hz, 2H), 6.95 (s, 1H), 6.81 (t, ³J_HF¼ 9 Hz, 1H) ppm;

¹³C NMR (100 MHz, DMSO‑d₆):

d

¼ 163.0 (dd, J_CF¼ 243 Hz,

1

³J_CF¼ 17 Hz, C), 152.0 (C), 150.6 (C), 142.5 (t, ³J_CF¼ 14 Hz, C), 138.7

(C), 138.4 (C), 133.4 (C), 129.8 (CH), 129.1 (CH), 128.4 (CH), 128.3

(CH), 125.6 (CH), 124.9 (CH), 101.5 (dd, ²J_CF¼ 21 Hz, ⁴J_CF¼ 8 Hz, CH),

97.6 (2CH) ppm; MS (EI pos.): m/z (%) ¼ 390.0 (47) ([M]^ꢅþ, calcd.

19

€

S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

390.1), 235.2 (100) ([M_fr.]^þ, calcd. 235.2); HRMS (ESI pos.): m/

z ¼ 391.1365 [MþH]^þ, calcd. for [C₂₂H₁₇F₂N₄O]^þ¼ 391.1365.

1-(3-(tert-Butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-3-(4-

chlorophenyl)urea (49). Prepared according to general procedure

D, 3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-amine (10, 2.00 g,

7.68 mmol) and 1-chloro-4-isocyanatobenzene (1.07 g, 6.99 mmol)

were dissolved in CH₂Cl₂(50 mL) and the mixture was stirred at rt

for 20 h. The resultant solid was ﬁltered and washed with hexane

(30 mL). Subsequently, the ﬁltrate was concentrated under reduced

pressure to enforce crystallizing of additional product, which was

then ﬁltered and washed with hexane to yield 1.83 g (4.41 mmol,

81%) of 49 as a yellow solid. TLC: R_F¼ 0.89 (SiO₂, hexane/EtOAc

590 mg, 2.46 mmol) and 1-chloro-4-isocyanatobenzene (514 mg,

3.35 mmol) in CH₂Cl₂(30 mL) were stirred at rt for 20 h. The

resultant solid was ﬁltered and washed with hexane (15 mL).

Subsequently, the ﬁltrate was concentrated under reduced pressure

to crystallize additional product, which was ﬁltered and washed

with hexane. The combined crude product was recrystallized from

hexane/EtOAc (1:1) to yield 539 mg (1.37 mmol, 56%) of 52 as a

yellow solid. TLC: R_F¼ 0.89 (SiO₂, cyclohexane/EtOAc 1:3); HPLC:

t_R¼ 16.5 min; ¹H NMR (600 MHz, DMSO‑d₆):

¼ 9.18 (br s,1H), 8.60

d

(br s, 1H), 7.98 (d, ³J ¼ 8.6 Hz, 2H), 7.80 (d, ³J ¼ 8.6 Hz, 2H), 7.44 (d,

³J ¼ 8.8 Hz, 2H), 7.31 (d, ³J ¼ 8.8 Hz, 2H), 6.42 (s, 1H), 1.29 (s, 9H)

ppm; ¹³C NMR (150 MHz, DMSO‑d₆):

d

¼ 162.1, 151.9, 142.4, 138.3,

1:3); HPLC: t_R¼ 16.83 min; ¹H NMR (500 MHz, DMSO‑d₆):

d

¼ 9.20

137.5, 133.5, 128.8, 125.6, 123.4, 119.8, 118.5, 108.8, 98.5, 32.1,

29.9 ppm; MS (ESI pos.): m/z (%) ¼ 394.2 (100) ([MþH]^þ, calcd.

349.2); 267.2 (32) ([M_fr.IeH]^þ, calcd. 268.1); 241.2 (29) ([M_fr.IIþH]^þ,

calcd. 241.2); HRMS (FTMS þ p MALDI): m/z ¼ 394.1409 [MþH]^þ,

calcd. for [C₂₁H₂₁ClN₅O]^þ¼ 394.1429.

(s, 1H), 8.67 (s, 1H), 8.36 (d, ³J ¼ 9.2 Hz, 2H), 7.88 (d, ³J ¼ 9.2 Hz, 2H),

7.44 (d, ³J ¼ 8.9 Hz, 2H), 7.30 (d, ³J ¼ 8.9 Hz, 2H) 6.45 (s, 1H), 1.30 (s,

9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 162.5, 151.9, 145.0,

144.0, 138.3, 137.8, 128.7, 125.8, 124.9, 123.2, 119.9, 99.9, 32.2,

29.9 ppm; MS (ESI pos.): m/z (%) ¼ 414.2 (100) ([MþH]^þ, calcd.

414.2); HRMS (FTMS þ p MALDI): m/z ¼ 414.1345 [MþH]^þ, calcd. for

[C₂₀H₂₁ClN₅O₃]^þ¼ 414.1327.

1-(3-(tert-Butyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazol-5-

yl)-3-(4-chlorophenyl)urea (53). Prepared according to general

procedure D, 3-(tert-butyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyr-

azol-5-amine (14, 700 mg, 2.39 mmol) and 1-chloro-4-

isocyanatobenzene (333 mg, 2.17 mmol) in CH₂Cl₂(15 mL) were

stirred at rt for 20 h. The resultant solid was ﬁltered and washed with

hexane (15 mL). Subsequently, the ﬁltrate was concentrated under

reduced pressure to crystallize additional product, which was

ﬁltered and washed with hexane. The combined crude product was

recrystallized from hexane/EtOAc (1:1) to yield 761 mg (1.70 mmol,

78%) of 53 as a yellowish solid. TLC: R_F¼ 0.72 (SiO₂, cyclohexane/

1-(3-(tert-Butyl)-1-(4-(triﬂuoromethyl)phenyl)-1H-pyrazol-

5-yl)-3-(4-chlorophenyl)urea (50). Prepared according to general

procedure D, 3-(tert-butyl)-1-(4-(triﬂuoromethyl)phenyl)-1H-pyr-

azol-5-amine (11, 460 mg, 1.62 mmol) and 1-chloro-4-

isocyanatobenzene (340 mg, 2.21 mmol) in CH₂Cl₂(10 mL) were

stirred at rt for 20 h. The resultant solid was ﬁltered and washed

with hexane (15 mL). Subsequently, the ﬁltrate was concentrated

under reduced pressure to crystallize additional product, which

was ﬁltered and washed with hexane. The combined crude product

was recrystallized from hexane/EtOAc (1:1) to yield 226 mg

(0.52 mmol, 32%) of 50 as a colorless solid. TLC: R_F¼ 0.91 (SiO₂,

cyclohexane/EtOAc 1:3); HPLC: t_R¼ 17.5 min; ¹H NMR (500 MHz,

EtOAc 1:3); HPLC: t_R¼ 15.8 min; ¹H NMR (600 MHz, DMSO‑d₆):

3

d

¼ 9.17 (br s, 1H), 8.63 (br s, 1H), 8.06 (d, J ¼ 8.7 Hz, 2H), 7.84 (d,

³J ¼ 8.7 Hz, 2H), 7.45 (d, ³J ¼ 8.9 Hz, 2H), 7.31 (d, ³J ¼ 8.9 Hz, 2H), 6.43

(s, 1H), 3.27 (s, 3H), 1.29 (s, 9H) ppm; ¹³C NMR (150 MHz, DMSO‑d₆):

DMSO‑d₆):

d

¼ 9.16 (br s,1H), 8.59 (br s,1H), 7.89 (d, ³J ¼ 8.3 Hz, 2H),

d

¼ 162.0, 151.8, 142.6, 138.5, 138.3, 137.5, 128.6, 128.3, 125.7, 123.6,

7.81 (d, ³J ¼ 8.3 Hz, 2H), 7.45 (d, ³J ¼ 8.5 Hz, 2H), 7.31 (d, ³J ¼ 8.3 Hz,

119.8, 97.8, 43.5, 32.1, 30.0 ppm; MS (ESI pos.): m/z (%) ¼ 447.2 (100)

2H), 6.43 (br s, 1H), 1.29 (s, 9H); ¹³C NMR (126 MHz, DMSO‑d₆):

([MþH]^þ, calcd. 447.1); 294.2 (28) ([M_fr.IþH]^þ, calcd. 294.1); 320.2

d

¼ 161.8, 151.8, 142.0, 138.4, 137.5, 128.7, 127.0 (²J_CF¼ 32.2 Hz),

(29) ([M_fr.II]^þ, calcd. 320.1); HRMS (FTMS

þ

p MALDI): m/

126.4, 125.8, 124.6 (¹J_CF¼ 274.9 Hz), 123.9, 119.9, 97.5, 30.0 ppm; MS

(ESI pos.): m/z (%) ¼ 437.2 (100) ([MþH]^þ, calcd. 437.1); 310.2 (31)

([M_fr.I]^þ, calcd. 310.1); 284.2 (14) ([M_fr.IIþH]^þ, calcd. 284.1); HRMS

z ¼ 469.1054 [MþNa]^þ, calcd. for [C₂₁H₂₃ClN₄NaO₃S]^þ¼ 469.1072.

1-(4-Chlorophenyl)-3-(3-cyclopropyl-1-(4-(tri-

ﬂuoromethoxy)phenyl)-1H-pyrazol-5-yl)urea (54). Prepared ac-

cording to general procedure D using 3-cyclopropyl-1-(4-

(FTMS

þ

p

MALDI): m/z

¼

437.1330 [MþH]^þ, calcd. for

[C₂₁H₂₁ClF₃N₄O]^þ¼ 437.1351.

(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-amine (15, 500 mg,

1.77 mmol) and 1-chloro-4-isocyanatobenzene (407 mg,

2.65 mmol) in CH₂Cl₂(20 mL) which were stirred at rt for 20 h. The

crude product was recrystallized from hexane/EtOAc (1:1) to yield

605 mg (1.39 mmol, 78%) of 54 as a colorless solid. TLC: R_F¼ 0.94

(SiO₂, cyclohexane/EtOAc 1:3); HPLC: t_R¼ 16.4 min; ¹H NMR

1-(3-(tert-Butyl)-1-(4-(triﬂuoromethoxy)phenyl)-1H-pyr-

azol-5-yl)-3-(4-chlorophenyl)urea (51). Prepared according to

general procedure D, 3-(tert-butyl)-1-(4-(triﬂuoromethoxy)

phenyl)-1H-pyrazol-5-amine (12, 505 mg, 1.69 mmol) and 1-

chloro-4-isocyanatobenzene (236 mg, 1.53 mmol) in CH₂Cl₂

(10 mL) were stirred at rt for 20 h. The resultant solid was ﬁltered

and washed with hexane (15 mL). Subsequently, the ﬁltrate was

concentrated under reduced pressure to crystallize additional

product, which was ﬁltered and washed with hexane. The com-

bined crude product was recrystallized from hexane/EtOAc (1:1) to

yield 480 mg (1.06 mmol, 69%) of 51 as a colorless solid; TLC:

R_F¼ 0.92 (SiO₂, cyclohexane/EtOAc 1:3); HPLC: t_R¼ 17.6 min; ¹H

(500 MHz, DMSO‑d₆):

d

¼ 9.09 (br s, 1H), 8.53 (br s, 1H), 7.65 (d,

3

³J ¼ 9.0 Hz, 2H), 7.51 (d, ³J ¼ 9.0 Hz, 2H), 7.43 (d, J ¼ 8.9 Hz, 2H),

3

7.30 (d, J ¼ 8.9 Hz, 2H), 6.19 (s, 1H), 1.93e1.86 (m, 1H), 0.92e0.87

(m, 2H), 0.73e0.68 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 154.8, 151.7, 146.8, 138.3, 137.5 (2x), 128.6, 125.7, 122.0, 120.1

(¹J_CF¼ 256.9 Hz), 119.8, 96.4, 9.4, 7.8 ppm; MS (ESI pos.): m/z

(%) ¼ 436.9 (100) ([MþH]^þ, calcd. 437.1); HRMS (FTMS þ p MALDI):

m/z ¼ 437.0978 [MþH]^þ, calcd. for [C₂₀H₁₇ClF₃N₄O₂]^þ¼ 437.0987.

1-(4-Chlorophenyl)-3-(1-(4-cyanophenyl)-3-cyclopropyl-1H-

pyrazol-5-yl)urea (55). Prepared according to general procedure D

using 4-(5-amino-3-cyclopropyl-1H-pyrazol-1-yl)benzonitrile (16,

500 mg, 2.23 mmol) and 1-chloro-4-isocyanatobenzene (514 mg,

3.35 mmol), in CH₂Cl₂(20 mL) which were stirred at rt for 20 h. The

crude product was recrystallized from hexane/EtOAc (1:1) to yield

110 mg (0.29 mmol, 13%) of 55 as a colorless solid. TLC: R_F¼ 0.86

(SiO₂, cyclohexane/EtOAc 1:3); HPLC: t_R¼ 15.3 min; ¹H NMR

NMR (600 MHz, DMSO‑d₆):

d

¼ 9.12 (br s, 1H), 8.51 (br s, 1H), 7.67

(d, ³J ¼ 8.9 Hz, 2H), 7.52 (d, ³J ¼ 8.9 Hz, 2H), 7.44 (d, ³J ¼ 8.8 Hz, 2H),

7.31 (d, ³J ¼ 8.8 Hz, 2H), 6.39 (s, 1H), 1.28 (s, 9H) ppm; ¹³C NMR

(150 MHz, DMSO‑d₆):

d

¼ 161.3, 151.7, 146.8, 138.3, 137.7, 137.2,

128.6, 125.7 (2x), 121.9, 120.1 (¹J_CF¼ 259 Hz), 119.8, 96.5, 32.0,

30.1 ppm; MS (ESI pos.): m/z (%) ¼ 453.2 (100) ([MþH]^þ, calcd.

453.1); 326.2 (37) ([M_fr.I]^þ, calcd. 326.1); 300.2 (11) ([MþH]^þ, calcd.

300.1); HRMS (FTMS þ p MALDI): m/z ¼ 453.1308 [MþH]^þ, calcd.

for [C₂₁H₂₁ClF₃N₄O₂]^þ¼ 453.1300.

1-(3-(tert-Butyl)-1-(4-cyanophenyl)-1H-pyrazol-5-yl)-3-(4-

chlorophenyl)urea (52). Prepared according to general procedure

D, 4-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile (13,

(600 MHz, DMSO‑d₆):

d

¼ 9.14 (s, 1H), 8.62 (s, 1H), 7.97 (d,

³J ¼ 8.4 Hz, 2H), 7.77 (d, ³J ¼ 8.4 Hz, 2H), 7.43 (d, J ¼ 8.6 Hz, 2H),

3

7.30 (d, J ¼ 8.6 Hz, 2H), 6.21 (s, 1H), 1.96e1.87 (m, 1H), 0.96e0.88

20

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S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

(m, 2H), 0.77e0.68 (m, 2H) ppm; ¹³C NMR (150 MHz, DMSO‑d₆):

1-(1-(4-Aminophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)-3-(4-

chlorophenyl)urea (62). 1-(3-(tert-Butyl)-1-(4-nitrophenyl)-1H-

pyrazol-5-yl)-3-(4-chlorophenyl)urea (49, 1.14 g, 2.76 mmol) was

dissolved in an EtOH/H₂O mixture (4:1, 3 mL). Subsequently, Fe

powder (1.54 g, 27.55 mmol) was sprinkled on, NH₄Cl (1.47 g,

27.55 mmol) was added and the reaction was heated for 1 h at 70 ^ꢀC.

The mixture was ﬁltered over Celite® and washed with EtOAc. The

crude product was puriﬁed by column chromatography on silica

(cyclohexane/EtOAc: 1:1 /1:3) to yield 782 mg (2.03 mmol, 74%) of

62 as a colorless solid. TLC: R_F¼ 0.42 (SiO₂, cyclohexane/EtOAc 1:3);

d

¼ 155.7, 151.9, 142.4, 138.3, 137.7, 133.4, 128.8, 125.6, 123.4, 119.9,

118.4, 108.8, 98.3, 9.3, 7.8 ppm; MS (ESI pos.): m/z (%) ¼ 377.9 (100)

([MþH]^þ, calcd. 378.1); 225.1 (63) ([M_fr.þ2H]^þ, calcd. 225.0); HRMS

(FTMS

þ

p

MALDI): m/z

¼

378.1100 [MþH]^þ, calcd. for

[C₂₀H₁₇ClN₅O]^þ¼ 378.1116.

1-(4-Chlorophenyl)-3-(3-cyclopropyl-1-(4-nitrophenyl)-1H-

pyrazol-5-yl)urea (56). Prepared according to general procedure D

using 3-cyclopropyl-1-(4-nitrophenyl)-1H-pyrazol-5-amine (17,

500 mg, 2.05 mmol) and 1-chloro-4-isocyanatobenzene (314 mg,

2.05 mmol) in CH₂Cl₂(20 mL) which were stirred at rt for 20 h. The

crude product was recrystallized from hexane/EtOAc (1:1) to yield

262 mg (0.66 mmol, 32%) of 56 as a colorless solid. TLC: R_F¼ 0.88

(SiO₂, cyclohexane/EtOAc 98:2); HPLC: t_R¼ 15.65 min; ¹H NMR

HPLC: t_R¼ 15.23 min; ¹H NMR (500 MHz, DMSO‑d₆):

¼ 9.19 (s, 1H),

d

8.20 (s, 1H), 7.42 (d, ³J ¼ 8.9 Hz, 2H), 7.30 (d, ³J ¼ 8.9 Hz, 2H), 7.07 (d,

³J ¼ 8.7 Hz, 2H), 6.66 (d, ³J ¼ 8.7 Hz, 2H), 6.30 (s, 1H), 5.40 (br s, 2H),

1.25 (s, 9H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 159.6, 151.1,

(500 MHz, DMSO‑d₆):

d

¼ 9.16 (s, 1H), 8.69 (s, 1H), 8.35 (d,

148.7, 138.5, 137.1, 128.7, 126.6, 126.5, 125.5, 119.5, 113.7, 92.8, 32.0,

30.3 ppm; MS (ESI pos.): m/z (%) ¼ 384.2 (100) ([MþH]^þ, calcd.

384.2); 257.2 (49) ([M_fr.I]^þ, calcd. 257.3); 231.1 (24) ([M_fr.IIþH]^þ, calcd.

231.2); HRMS (FTMS þ p MALDI): m/z ¼ 384.1570 [MþH]^þ, calcd. for

[C₂₀H₂₃ClN₅O₃]^þ¼ 384.1586.

³J ¼ 8.9 Hz, 2H), 7.87 (d, J ¼ 8.9 Hz, 2H), 7.43 (d, J ¼ 8.8 Hz, 2H),

7.30 (d, ³J ¼ 8.8 Hz, 2H), 5.75 (s, 1H), 1.98e1.88 (m, 1H), 0.98e0.86

(m, 2H), 0.77e0.68 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

3

d

¼ 156.2, 151.9, 145.0, 143.8, 138.3, 138.0, 128.6, 125.8, 124.8, 123.1,

119.9, 98.8, 39.5, 9.4, 7.9 ppm; MS (ESI pos.): m/z (%) ¼ 397.94 (100)

([MþH]^þ, calcd. 398.10); HRMS (FTMS þ p MALDI): m/z ¼ 398.1015

[MþH]^þ, calcd. for [C₁₉H₁₇ClN₅O₃]^þ¼ 398.1014.

N-(4-(3-(tert-Butyl)-5-(3-(4-chlorophenyl)ureido)-1H-pyr-

azol-1-yl)phenyl)cyclopropanecarboxamide

(63).

Cyclo-

propanecarboxylic acid (28 mg, 0.33 mmol) and HATU (149 mg,

0.39 mmol) were dissolved in DMF (dry, 8 mL) and DIPEA (51 mg,

0.39 mmol) was added. The mixture was stirred at rt for 1.5 h and a

solution of 1-(1-(4-aminophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)-

3-(4-chlorophenyl)urea (62, 150 mg, 0.39 mmol) in DMF (dry, 2 mL)

was added. The mixture was then stirred for additional 20 h at rt.

Water (20 mL) was added, the precipitated solid was ﬁltered,

washed with water (200 mL) and dried in a vacuum oven to yield

89 mg (0.20 mmol, 50%) of 63 as a colorless solid. TLC: R_F¼ 0.64

(SiO₂, cyclohexane/EtOAc 1:3); HPLC: t_R¼ 15.92 min; ¹H NMR

1-(4-Chlorophenyl)-3-(1-(4-nitrophenyl)-3-(tri-

ﬂuoromethyl)-1H-pyrazol-5-yl)urea (57). Prepared according to

general procedure D using 1-(4-nitrophenyl)-3-(triﬂuoromethyl)-

1H-pyrazol-5-amine (61, 1.00 g, 3.67 mmol) and 1-chloro-4-

isocyanatobenzene (769 mg, 5.01 mmol) in CH₂Cl₂(35 mL) which

were stirred at rt for 20 h. The crude product was recrystallized

from cyclohexane/EtOAc (1:1) to yield 122 mg (0.29 mmol, 8%) of

57 as a brownish solid. TLC: R_F¼ 0.91 (SiO₂, cyclohexane/EtOAc

1:3); HPLC: t_R¼ 16.32 min; ¹H NMR (500 MHz, DMSO‑d₆):

¼ 9.26

d

(s, 1H), 8.98 (s, 1H), 8.44 (d, ³J ¼ 8.9 Hz, 2H), 7.95 (d, ³J ¼ 8.9 Hz, 2H),

7.43 (d, ³J ¼ 8.7 Hz, 2H), 7.32 (d, ³J ¼ 8.7 Hz, 2H), 6.95 (s, 1H) ppm;

(300 MHz, DMSO‑d₆):

d

¼ 10.37 (br s, 1H), 9.13 (br s, 1H), 8.35 (br s,

1H), 7.73 (d, ³J ¼ 8.9 Hz, 2H), 7.40e7.38 (m, 4H), 7.30 (d, ³J ¼ 8.9 Hz,

¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 149.7, 145.6, 143.4, 142.6

2H), 6.35 (s, 1H), 1.86e1.73 (m, 1H), 1.27 (s, 9H), 0.90e0.74 (m, 4H)

(²J_CF¼ 36.9 Hz), 126.4, 124.9, 123.7, 121.4 (¹J_CF¼ 269.2 Hz), 112.4,

87.8 ppm; MS (ESI pos.): m/z (%) ¼ 425.8 (17) ([MþH]^þ, calcd.

426.1); 357.1 (12) ([M_fr.II]^þ, calcd. 357.1); 256.9 (33) ([M_fr.I]^þ, calcd.

256.0); HRMS (FTMS þ p MALDI): m/z ¼ 426.0570 [MþH]^þ, calcd.

for [C₁₇H₁₂ClF₃N₅O₃]^þ¼ 426.0575.

ppm; ¹³C NMR (75 MHz, DMSO‑d₆):

d

¼ 171.8, 160.5, 151.4, 138.6,

138.4, 137.0, 133.2, 128.6, 125.6, 125.1, 119.6, 119.4, 94.8, 32.0, 30.2,

14.6, 7.3 ppm; MS (ESI pos.): m/z (%) ¼ 452.18 (100) ([MþH]^þ, calcd.

452.19); MS (ESI neg.): m/z (%) ¼ 450.26 (100) ([MꢃH]^-, calcd.

450.17); HRMS (FTMS þ p MALDI): m/z ¼ 452.1836 [MþH]^þ, calcd.

for [C₂₄H₂₇ClN₅O₂]^þ¼ 452.1848.

1-(4-Nitrophenyl)-3-(triﬂuoromethyl)-1H-pyrazol-5-amine

(61). Potassium tert-butoxide (3.25 g, 28.96 mmol) was dissolved in

THF (dry, 30 mL) and acetonitrile (0.39 mg, 9.60 mmol). Ethyl 2,2,2-

triﬂuoroacetate (58, 5.44 g, 38.29 mmol) was added dropwise and

the mixture was stirred overnight at rt. The mixture was then

acidiﬁed with hydrochloric acid (conc., 3 mL) and water (10 mL)

was added. The solvent was removed under reduced pressure and

the residue was extracted with EtOAc (3 ꢄ 20 mL). The organic

extracts were combined, dried over MgSO₄and the solvent was

removed under reduced pressure to give 4,4,4-triﬂuoro-3-

oxobutanenitrile (59), which was used without further puriﬁca-

tion. The crude compound was dissolved in EtOH (abs., 20 mL), and

(4-nitrophenyl)hydrazine hydrochloride (60, 1.81 g, 9.57 mmol)

was added. The mixture was heated for 8 h at 70 ^ꢀC and afterwards

basiﬁed to pH~12 with aqueous NaOH solution (3 N, 10 mL). The

mixture was extracted with EtOAc (4 ꢄ 50 mL), and the organic

extracts were combined and dried over MgSO₄. After removing the

solvent under reduced pressure, the crude product was puriﬁed by

N-(4-(3-(tert-Butyl)-5-(3-(4-chlorophenyl)ureido)-1H-pyr-

azol-1-yl)phenyl)isobutyramide (64). 1-(1-(4-Aminophenyl)-3-

(tert-butyl)-1H-pyrazol-5-yl)-3-(4-chlorophenyl)urea (62, 300 mg,

0.78 mmol) was dissolved in THF (dry, 10 mL) and TEA (158 mg,

1.56 mmol) was added. The mixture was cooled to 0 ^ꢀC and isobutyryl

chloride (166 mg,1.56 mmol) was added dropwise. After warming to

rt, the mixturewasstirred for 20h at rt. Subsequently, the solvent was

removed under reduced pressure and the residue was dissolved in

EtOAc. Thecrudeproduct waspuriﬁedbycolumn chromatographyon

silica (cyclohexane/EtOAc: 1:10 / 1:1 / 1:3 / 1:20 / EtOAc) to

yield 298 mg(0.66 mmol, 84%)of 64 as a colorless solid. TLC:R_F¼ 0.75

(SiO₂, cyclohexane/EtOAc 1:3); HPLC: t_R¼ 16.12 min; ¹H NMR

(500MHz,DMSO‑d₆):

d

¼ 10.03(brs,1H), 9.14 (brs,1H), 8.33 (brs,1H),

7.75 (d, ³J ¼ 8.9 Hz, 2H), 7.45e7.39 (m, 4H), 7.30 (d, ³J ¼ 8.9 Hz, 2H),

6.35 (s, 1H), 2.68e2.57 (m, 1H), 1.27 (s, 9H), 1.11 (d, ³J ¼ 6.7 Hz, 6H)

ppm; ¹³C NMR (126 MHz, DMSO‑d₆):

d

¼ 175.4, 160.5, 151.4, 138.8,

138.4, 137.1, 133.2, 128.7, 125.6, 125.1, 119.6, 119.5, 94.7, 35.0, 32.0,

30.72,19.5 ppm; MS (ESI pos.): m/z (%) ¼ 454.2 (100) ([MþH]^þ, calcd.

454.2); 327.2 (28) ([M_fr.I]^þ, calcd. 327.2); HRMS (FTMS þ p MALDI): m/

z ¼ 545.2008 [MþH]^þ, calcd. for [C₂₄H₂₉ClN₅O₂]^þ¼ 454.2004.

N-(4-(3-(tert-Butyl)-5-(3-(4-chlorophenyl)ureido)-1H-pyr-

column

chromatography

on

silica

(cyclohexane/EtOAc

4:1 / 1:1 / 1:20 / EtOAc) to yield 1.19 g (4.37 mmol, 46%) of 61

as an orange solid. TLC: R_F¼ 0.63 (SiO₂, cyclohexane/EtOAc 1:1); ¹H

NMR (500 MHz, DMSO‑d₆):

d

¼ 8.38 (d, ³J ¼ 9.1 Hz, 2H), 7.94 (d,

³J ¼ 9.1 Hz, 2H), 6.10 (s, 2H), 5.88 (s, 1H) ppm; ¹³C NMR (126 MHz,

azol-1-yl)phenyl)methanesulfonamide

(65).

1-(1-(4-

DMSO‑d₆):

d

¼ 149.7, 145.6, 143.4, 142.6 (²J_CF¼ 36.9 Hz), 126.4,

Aminophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)-3-(4-chlorophenyl)

urea (62, 200 mg, 0.52 mmol) was dissolved in THF (dry, 8 mL) and

TEA (53 mg, 0.52 mmol) was added. The mixture was cooled to 0 ^ꢀC

124.9, 123.7, 121.4 (¹J_CF¼ 269.2 Hz), 112.4, 87.8 ppm; MS (ESI pos.):

m/z (%) ¼ 273.0 (100) ([MþH]^þ, calcd. 273.1).

21

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S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

and methanesulfonyl chloride (179 mg, 1.56 mmol) was added

dropwise. After warming to rt, the mixture was stirred for 20 h at rt.

Subsequently, the solvent was removed under reduced pressure,

and the residue was dissolved in EtOAc. The organic phase was

washed 4-times with water and dried over MgSO₄. The crude

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc: 1:10 / 1:20) to yield 70 mg (0.15 mmol, 29%) of 65

as a colorless solid. TLC: R_F¼ 0.70 (SiO₂, cyclohexane/EtOAc 1:3);

HPLC: t_R¼ 15.44 min; ¹H NMR (400 MHz, DMSO‑d₆, 300 K):

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 400 mg,

0.98 mmol) and ethanamine (596 mL, 2 M in THF, 1.18 mmol). The

crude product was puriﬁed by column chromatography on silica

(cyclohexane/EtOAc, 1:1) to afford 224 mg (0.52 mmol, 52%) of 73

as a colorless solid. TLC: R_F¼ 0.68 (SiO₂, cyclohexane/EtOAc, 1:1);

¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.58

(d, ³J ¼ 7.5 Hz, 2H), 7.40 (t, ³J ¼ 7.5 Hz, 2H), 7.31 (t, ³J ¼ 7.5 Hz, 2H),

5.89 (s, 1H), 5.36 (d, ³J ¼ 7.7 Hz, 1H), 4.46e4.35 (m, 2H), 4.22 (t,

³J ¼ 6.9 Hz, 1H), 4.08e3.99 (m, 1H), 3.36e3.22 (m, 2H), 1.90e1.78

(m, 1H), 1.74e1.56 (m, 6H), 1.34e1.05 (m, 9H), 0.94e0.79 (m, 2H)

d

¼ 9.98 (br s, 1H), 9.12 (br s, 1H), 8.40 (br s, 1H), 7.52e7.40 (m, 4H)

7.38e7.30 (m, 4H), 6.36 (s, 1H), 3.05 (s, 3H), 1.27 (s, 9H) ppm; ¹³C

NMR (101 MHz, DMSO‑d₆, 300 K):

d

¼ 160.6, 151.4, 138.4, 137.6,

ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 171.6, 154.3, 143.9,

137.1,134.2,128.7, 125.7, 125.6,120.0,119.7, 95.0, 32.0, 30.2 ppm; MS

141.4, 127.9, 127.2, 125.2, 120.1, 67.1, 55.5, 47.3, 37.6, 37.5, 34.6, 33.4,

33.3, 33.2, 30.2, 26.7, 26.4,14.9 ppm; MS (ESI pos.): m/z (%) ¼ 435.26

(100) ([MþH]^þ, calcd. 435.27), 213.29 (52) ([M-FmocþH]^þ, calcd.

213.20), 179.29 (28) ([M_fr.þH]^þ, calcd. 179.29).

(ESI pos.): m/z (%) ¼ 461.97 (100) ([MþH]^þ, calcd. 462.12); HRMS

(FTMS

þ

p

MALDI): m/z

¼

462.1348 [MþH]^þ, calcd. for

[C₂₁H₂₅ClN₅O₃S]^þ¼ 462.1361.

Ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (67).

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-oxo-1-(propy-

lamino)butan-2-yl)carbamate (74). Compound 74 was prepared

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 400 mg,

€

Prepared as previously published by Rohm et al. [4].

5-Amino-1-phenyl-1H-pyrazole-4-carboxylic acid (68). Pre-

€

pared as previously published by Rohm et al. [4].

Methyl 4-((5-amino-1-phenyl-1H-pyrazole-4-carboxamido)

0.98 mmol) and propan-1-amine (97 mL, 1.18 mmol). The crude

€

methyl)benzoate (69). Prepared as previously published by Rohm

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc, 1:1) to afford 248 mg (0.55 mmol, 56%) of 74. TLC:

R_F¼ 0.15 (SiO₂, cyclohexane/EtOAC, 1:1); ¹H NMR (500 MHz, CDCl₃,

et al.[4]5-Amino-1-phenyl-1H-pyrazole-4-carboxylic acid (68,

2.00 g, 9.84 mmol), HOBt (1.56 g, 11.81 mmol) and EDC-HCl (2.26 g,

11.81 mmol) were dissolved in DMF (dry, 20 mL) and DIPEA (1.91 g,

14.77 mmol) was added. The mixture was stirred for 30 min at rt,

and a solution of methyl 4-(aminomethyl)benzoate hydrochloride

(1.95 g, 11.81 mmol) in DMF/DMSO (dry, 20 mL, 1:1) and DIPEA

(1.91 g, 14.77 mmol) was added. The reaction mixture was stirred

for 20 h at rt. CH₂Cl₂(100 mL) was added, and the mixture was

washed with H₂O (5 ꢄ 50 mL). The organic phase was dried over

MgSO₄, and the solvent was removed under reduced pressure. The

crude product was puriﬁed by column chromatography on silica

(CH₂Cl₂/MeOH, 98:2) to afford 3.10 g (8.85 mmol, 90%) of 69 as a

colorless solid. TLC: R_F¼ 0.33 (SiO₂, CH₂Cl₂/MeOH 10:1); ¹H NMR

300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.57 (d, ³J ¼ 7.5 Hz, 2H), 7.39 (t,

³J ¼ 7.4 Hz, 2H), 7.30 (t, ³J ¼ 7.4 Hz, 2H), 6.13 (s, 1H), 5.48 (d,

³J ¼ 7.9 Hz, 1H), 4.48e4.28 (m, 2H), 4.20 (t, ³J ¼ 7.0 Hz, 1H),

4.12e4.02 (m, 1H), 3.30e3.11 (m, 2H), 1.91e1.76 (m, 1H), 1.73e1.58

(m, 6H), 1.56e1.43 (m, 2H), 1.36e1.06 (m, 6H), 0.94e0.79 (m, 5H)

ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 171.8, 156.3, 143.9,

141.4, 127.9, 127.2, 125.2, 120.1, 67.1, 55.5, 47.2, 41.3, 37.6, 33.4, 33.3,

33.1, 30.4, 26.7, 26.4, 22.9, 11.4 ppm; MS (ESI pos.): m/z (%) ¼ 897.62

(84) ([2 M þ H]^þ, calcd. 897.54), 449.24 (92) ([MþH]^þ, calcd.

449.27), 227.18 (100) ([M-FmocþH]^þ, calcd. 227.20).

(S)-(9H-Fluoren-9-yl)methyl (1-(butylamino)-4-cyclohexyl-

1-oxobutan-2-yl)carbamate (75). Compound 75 was prepared

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 400 mg,

(400 MHz, DMSO‑d₆, 300 K):

d

¼ 8.54 (t, ³J ¼ 6.1 Hz,1H), 7.99 (s, 1H),

7.94 (d, ³J ¼ 8.2 Hz, 2H), 7.60e7.49 (m, 4H), 7.45 (d, ³J ¼ 8.2 Hz, 2H),

7.42e7.36 (m, 1H), 6.38 (s, 2H), 4.50 (d, ³J ¼ 6.1 Hz, 2H), 3.85 (s, 3H)

ppm; ¹³C NMR (101 MHz, DMSO‑d₆, 300 K):

d

¼ 166.1, 164.2, 149.2,

0.98 mmol) and butan-1-amine (120 mL, 1.18 mmol). The crude

145.9, 138.4, 138.2, 129.3, 129.2, 128.0, 127.3, 127.1, 123.1, 97.3, 52.0,

41.3 ppm; MS (ESI pos.): m/z (%) ¼ 351.13 (100) ([MþH]^þ, calcd.

351.15).

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc, 4:1 / 1:1) to afford 301 mg (0.65 mmol, 66%) of 75

as a colorless solid. TLC: R_F¼ 0.78 (SiO₂, cyclohexane/EtOAc, 1:1);

4-((5-Amino-1-phenyl-1H-pyrazole-4-carboxamido)methyl)

¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.57

benzoic acid (70). Prepared as previously published by Rohm

(d, ³J ¼ 7.5 Hz, 2H), 7.40 (t, ³J ¼ 7.3 Hz, 2H), 7.30 (t, ³J ¼ 7.3 Hz, 2H),

6.03 (s, 1H), 5.48e5.37 (m, 1H), 4.45e4.32 (m, 2H), 4.21 (t,

³J ¼ 7.0 Hz, 1H), 4.11e4.00 (m, 1H), 3.33e3.14 (m, 2H), 1.91e1.78 (m,

1H), 1.74e1.56 (m, 6H), 1.52e1.39 (m, 2H), 1.37e1.05 (m, 8H),

0.93e0.79 (m, 5H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

€

et al.[4].

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-(methylamino)

-1-oxobutan-2-yl)carbamate (72). Compound 72 was prepared

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 400 mg,

d

¼ 171.7, 156.3,143.9, 141.4, 127.9, 127.2, 125.2, 120.1, 67.1, 55.5, 47.3,

0.98 mmol) and methanamine (596

m

L, 2 M in THF, 1.18 mmol). The

39.4, 37.6, 33.4, 33.3, 33.1, 31.7, 30.4, 26.7, 26.4, 20.1, 13.8 ppm; MS

(ESI pos.): m/z (%) ¼ 925.64 (12) ([2 M þ H]^þ, calcd. 925.59), 463.21

(100) ([MþH]^þ, calcd. 463.30), 241.23 (25) ([M-FmocþH]^þ, calcd.

241.23), 179.21 (11) ([M_fr.þH]^þ, calcd. 179.08).

crude product was puriﬁed by column chromatography on silica

(cyclohexane/EtOAc, 1:1) to afford 334 mg (0.80 mmol, 81%) of 72

as a colorless solid. TLC: R_F¼ 0.40 (SiO₂, cyclohexane/EtOAc, 1:1);

¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H),

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-(hexylamino)-

1-oxobutan-2-yl)carbamate (76). Compound 76 was prepared

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 500 mg,

7.62e7.54 (m, 2H), 7.40 (t, ³J ¼ 7.5 Hz, 2H), 7.30 (t, ³J ¼ 7.5 Hz, 2H),

6.11 (s, 1H), 5.45 (d, ³J ¼ 8.0 Hz, 1H), 4.46e4.33 (m, 2H), 4.26e4.17

(m, 1H), 4.11e4.01 (m,1H), 2.81 (s, 3H), 1.89e1.79 (m, 1H),1.72e1.56

(m, 6H), 1.30e1.05 (m, 6H, H-1), 0.93e0.78 (m, 2H) ppm; ¹³C NMR

1.23 mmol) and hexan-1-amine (190

mL, 1.47 mmol). The crude

(126 MHz, CDCl₃, 300 K):

d

¼ 172.6, 156.4, 143.9, 143.8, 141.4, 127.9,

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc, 4:1 /1:1) to afford 396 mg (0.81 mmol, 66%) of 76.

TLC: R_F¼ 0.87 (SiO₂, cyclohexane/EtOAc, 1:1); ¹H NMR (500 MHz,

127.2 (2x), 125.2, 120.1, 67.1, 55.5, 47.3, 38.8, 37.6, 33.4, 33.3, 33.2,

30.2, 26.7, 26.4 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 421.35 (100)

([MþH]^þ, calcd. 421.24), 199.24 (66) ([M-FmocþH]^þ, calcd. 199.17),

179.21 (29) ([M_fr.þH]^þ, calcd. 179.08).

CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.57 (d, ³J ¼ 7.5 Hz), 7.40

(t, ³J ¼ 7.4 Hz, 2H), 7.30 (t, ³J ¼ 7.4 Hz, 2H), 6.05 (s, 1H), 5.45 (d,

³J ¼ 7.5 Hz, 1H), 4.46e4.31 (m, 2H), 4.20 (t, ³J ¼ 6.9 Hz, 1H),

4.13e4.02 (m, 1H), 3.33e3.13 (m, 2H), 1.92e1.76 (m, 1H), 1.73e1.57

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-(ethylamino)-

1-oxobutan-2-yl)carbamate (73). Compound 73 was prepared

22

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European Journal of Medicinal Chemistry 208 (2020) 112721

(m, 6H), 1.53e1.40 (m, 2H), 1.35e1.05 (m, 12H), 0.94e0.79 (m, 5H)

(9H-Fluoren-9-yl)methyl

((S)-4-cyclohexyl-1-(((SR)-1-

ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 171.7, 156.3, 143.9,

hydroxybutan-2-yl)amino)-1-oxobutan-2-yl)carbamate

(80).

Compound 80 was prepared according to general procedure E using

(S)-2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

141.4, 127.9, 127.2, 125.2, 120.1, 67.1, 55.5, 47.3, 39.7, 37.6, 33.4, 33.3,

33.1, 31.5, 30.4, 29.6, 26.7 (2x), 26.4, 22.7, 14.1 ppm; MS (ESI pos.):

m/z (%) ¼ 513.30 (18) ([MþNa]^þ, calcd. 513.31), 491.33 (100)

([MþH]^þ, calcd. 491.33), 269.33 (27) ([M-FmocþH]^þ, calcd. 269.25),

179.30 (20) ([M_fr.þH]^þ, calcd. 179.29).

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and (rac)-2-

aminobutan-1-ol (133 mg, 1.47 mmol). The crude product was

triturated with cyclohexane/CH₂Cl₂(1:1) to afford 345 mg

(0.72 mmol, 59%) of 80. TLC: R_F¼ 0.30 (SiO₂, cyclohexane/EtOAc,

1:1); ¹H NMR (400 MHz, DMSO‑d₆, 300 K): (mixture of di-

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-(decylamino)-

1-oxobutan-2-yl)carbamate (77). Compound 77 was prepared

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 500 mg,

astereomers)

d

¼ 7.95e7.84 (m, 3H), 7.77e7.69 (m, 2H), 7.95e7.84

(m, 3H), 7.36e7.28 (m, 2H), 4.32e4.17 (m, 3H), 4.03e3.91 (m, 1H),

3.90e3.76 (m, 1H), 3.66e3.51 (m, 2H), 1.72e1.49 (m, 8H), 1.48e1.37

(m, 1H), 1.27e1.04 (m, 6H), 0.90e0.77 (m, 5H) ppm; ¹³C NMR

1.23 mmol) and decan-1-amine (290

mL, 1.47 mmol). The crude

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc, 4:1 / 1:1) to afford 565 mg (1.03 mmol, 84%) of 77.

TLC: R_F¼ 0.86 (SiO₂, cyclohexane/EtOAc, 1:1); ¹H NMR (500 MHz,

(101 MHz, DMSO‑d₆, 300 K): (mixture of diastereomers)

d

¼ 172.0,

155.8, 143.9, 143.8, 140.7, 127.6, 127.0, 125.3, 120.1, 65.5, 55.0, 54.9,

51.4, 51.2, 47.2 (2x), 36.8, 36.7, 32.9 (2x), 32.7, 29.7, 29.6, 26.1, 25.7,

24.3, 24.1, 10.2 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 501.26 (100)

([MþNa]^þ, calcd. 501.27), 479.29 (95) ([MþH]^þ, calcd. 479.29).

CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.57 (d, ³J ¼ 7.5 Hz, 2H),

7.40 (t, ³J ¼ 7.4 Hz, 2H), 7.30 (t, ³J ¼ 7.4 Hz, 2H), 6.00 (s, 1H), 5.42 (d,

³J ¼ 7.5 Hz, 1H), 4.44e4.33 (m, 2H), 4.21 (t, ³J ¼ 6.9 Hz, 1H),

4.10e4.01 (m, 1H), 3.32e3.14 (m, 2H), 1.90e1.96 (m, 1H), 1.73e1.58

(m, 6H), 1.51e1.40 (m, 2H), 1.33e1.07 (m, 20H), 0.93e0.80 (m, 5H)

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-(iso-

pentylamino)-1-oxobutan-2-yl)carbamate (81). Compound 81

was prepared according to general procedure E using (S)-2-((((9H-

ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid

ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 171.7, 156.3, 143.9,

141.4, 127.9, 127.2, 125.2, 120.1, 67.1, 55.5, 47.3, 39.7, 37.6, 33.4, 33.3,

33.1, 32.0, 30.4, 29.7 (2x), 29.6, 29.4 (2x), 27.0, 26.7, 26.4, 22.8,

14.3 ppm; MS (ESI pos.): m/z (%) ¼ 569.32 (35) ([MþNa]^þ, calcd.

569.37), 547.34 (100) ([MþH]^þ, calcd. 547.39), 325.26 (11) ([M-

FmocþH]^þ, calcd. 325.33).

(71, 500 mg, 1.23 mmol) and 3-methylbutan-1-amine (171

mL,

1.47 mmol). The crude product was puriﬁed by column chroma-

tography on silica (cyclohexane/EtOAc, 4:1 / 2:1) to afford 498 mg

(1.05 mmol, 85%) of 81. TLC: R_F¼ 0.30 (SiO₂, cyclohexane/EtOAc,

(9H-Fluoren-9-yl)methyl

((S)-1-((SR)-sec-butylamino)-4-

1:1); ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.6 Hz, 2H),

cyclohexyl-1-oxobutan-2-yl)carbamate (78). Compound 78 was

prepared according to general procedure E using (S)-2-((((9H-ﬂu-

7.57 (d, ³J ¼ 7.6 Hz, 2H), 7.40 (t, ³J ¼ 7.6 Hz, 2H), 7.30 (t, ³J ¼ 7.6 Hz,

2H), 6.03 (s, 1H), 5.44 (d, ³J ¼ 8.0 Hz, 1H), 4.45e4.31 (m, 2H), 4.20 (t,

³J ¼ 6.8 Hz, 1H), 4.11e4.02 (m, 1H), 3.34e3.16 (m, 2H), 1.91e1.77 (m,

1H), 1.73e1.52 (m, 7H), 1.42e1.31 (m, 2H), 1.28e1.06 (m, 6H),

0.94e0.79 (m, 8H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

oren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic

(71, 500 mg, 1.23 mmol) and (rac)-butan-2-amine (160

acid

L,

m

1.47 mmol). The crude product was puriﬁed by column chroma-

tography on silica (cyclohexane/EtOAc, 4:1 /1:1) to afford 363 mg

(0.79 mmol, 64%) of 78 as a colorless solid. TLC: R_F¼ 0.76 (SiO₂,

d

¼ 171.7,156.3,143.9,141.4,127.9,127.2,125.2,120.2, 67.1, 55.5, 47.2,

38.5, 38.0, 37.6, 33.4, 33.3, 33.1, 30.4, 26.7, 26.4, 25.9, 22.5 (2x) ppm;

MS (ESI pos.): m/z (%) ¼ 499.21 (37) ([MþNa]^þ, calcd. 499.29),

477.26 (100) ([MþH]^þ, calcd. 477.31), 186.20 (97)([M_fr.]^þ, calcd.

186.10).

cyclohexane/EtOAc,1:1); ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76

(d, ³J ¼ 7.5 Hz, 2H), 7.58 (d, ³J ¼ 7.5 Hz, 2H), 7.40 (t, ³J ¼ 7.4 Hz, 2H),

7.30 (t, ³J ¼ 7.4 Hz, 2H), 5.86e5.74 (m, 1H), 5.52e5.41 (m, 1H),

4.43e4.32 (m, 2H), 4.21 (t, ³J ¼ 7.1 Hz, 1H), 4.12e4.00 (m, 1H),

3.95e3.84 (m, 1H), 1.90e1.76 (m, 1H), 1.74e1.57 (m, 6H), 1.50e1.38

(m, 2H), 1.30e1.04 (m, 9H), 0.94e0.79 (m, 5H) ppm; ¹³C NMR

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-(cyclo-

hexylamino)-1-oxobutan-2-yl)carbamate (82). Compound 82

was prepared according to general procedure E using (S)-2-((((9H-

ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid

(126 MHz, CDCl₃, 300 K):

d

¼ 171.7, 156.3, 143.9, 141.4, 127.9, 127.2,

125.2, 120.1, 67.1, 55.5, 47.2, 37.6, 33.4, 33.3, 33.1, 30.6, 30.4, 29.7,

26.7, 26.4, 20.6, 20.5, 10.5 ppm; MS (ESI pos.): m/z (%) ¼ 925.68 (12)

([2 M þ H]^þ, calcd. 925.59), 463.25 (100) ([MþH]^þ, calcd. 463.30),

241.23 (25) ([M-FmocþH]^þ, calcd. 241.23), 179.15 (9) ([M_fr.þH]^þ,

calcd. 179.08).

(71, 500 mg, 1.23 mmol) and cyclohexanamine (170 mL, 1.47 mmol).

The crude product was puriﬁed by column chromatography on

silica (cyclohexane/EtOAc, 4:1 /1:1) to afford 513 mg (1.05 mmol,

86%) of 82 as a colorless solid. TLC: R_F¼ 0.29 (SiO₂, cyclohexane/

EtOAc, 4:1); ¹H NMR (500 MHz, CDCl₃, 300 K): (mixture of di-

3

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-oxo-1-(pentan-

3-ylamino)butan-2-yl)carbamate (79). Compound 79 was pre-

pared according to general procedure E using (S)-2-((((9H-ﬂuoren-

9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71,

astereomers)

d

¼ 7.76 (d, J ¼ 7.5 Hz, 2H), 7.58 (d, ³J ¼ 7.5 Hz, 2H),

7.40 (t, ³J ¼ 7.5 Hz, 2H), 7.30 (t, ³J ¼ 7.5 Hz, 2H), 5.90 (d, ³J ¼ 7.2 Hz,

1H), 5.47 (d, ³J ¼ 7.8 Hz, 1H), 4.43e4.31 (m, 2H), 4.21 (t, ³J ¼ 7.1 Hz,

1H), 4.10e4.01 (m, 1H), 3.80e3.70 (m, 1H), 1.93e1.77 (m, 3H),

1.74e1.54 (m, 10H), 1.40e1.28 (m, 2H), 1.27e1.04 (m, 9H), 0.95e0.78

(m, 2H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K): (mixture of di-

500 mg, 1.23 mmol) and pentan-3-amine (170 mL, 1.47 mmol). The

crude product was puriﬁed by column chromatography on silica

(cyclohexane/EtOAc, 4:1 /1:1) to afford 458 mg (0.96 mmol, 78%)

of 79. TLC: R_F¼ 0.82 (SiO₂, cyclohexane/EtOAc, 1:1); ¹H NMR

astereomers)

d

¼ 170.7, 156.3, 143.9 (2x), 141.4, 127.9, 127.2, 125.2

(2x), 120.1 (2x), 67.1, 55.5, 48.4, 47.2, 37.6, 33.4, 33.3, 33.2, 33.0, 30.6,

26.7, 26.4, 25.6, 24.9 ppm; MS (ESI pos.): m/z (%) ¼ 511.24 (100)

([MþNa]^þ, calcd. 511.65).

(500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.58 (d,

³J ¼ 7.5 Hz, 2H), 7.40 (t, ³J ¼ 7.5 Hz, 2H), 7.30 (t, ³J ¼ 7.5 Hz, 2H), 5.75

(d, ³J ¼ 8.5 Hz, 1H), 5.49 (d, ³J ¼ 7.9 Hz, 1H), 4.42e4.32 (m, 2H), 4.21

(t, ³J ¼ 7.0 Hz, 1H), 4.14e4.06 (m, 1H), 3.81e3.71 (m, 1H), 1.91e1.78

(m, 1H), 1.76e1.60 (m, 6H),1.59e1.46 (m, 2H), 1.40e1.30 (m, 2H),

1.29e1.07 (m, 6H), 0.94e0.78 (m, 8H) ppm; ¹³C NMR (126 MHz,

(S)-(9H-Fluoren-9-yl)methyl (4-cyclohexyl-1-morpholino-1-

oxobutan-2-yl)carbamate (83). Compound 83 was prepared ac-

cording to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 500 mg,

1.23 mmol) and morpholine (128 mg, 1.47 mmol). The crude

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc 4:1 /1:1 /1:3) to afford 613 mg (1.07 mmol, 87%)

of 83 as a colorless solid. TLC: R_F¼ 0.55 (SiO₂, cyclohexane/EtOAc,

CDCl₃, 300 K):

d

¼ 171.5, 156.3, 143.9, 141.4, 127.9, 127.2, 125.2, 120.1,

67.1, 55.5, 52.3, 47.2, 37.6, 33.4, 33.3, 33.1, 30.5, 26.7, 26.4, 10.4 ppm;

MS (ESI pos.): m/z (%) ¼ 499.21 (9) ([MþNa]^þ, calcd. 499.29), 477.25

(100) ([MþH]^þ, calcd. 477.31), 255.28 (69) ([M-FmocþH]^þ, calcd.

255.23).

1:1); ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H),

23

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European Journal of Medicinal Chemistry 208 (2020) 112721

7.65e7.53 (m, 2H), 7.40 (t, ³J ¼ 7.4 Hz, 2H), 7.31 (t, ³J ¼ 7.5 Hz, 2H),

5.70 (d, ³J ¼ 8.5 Hz, 1H), 4.65e4.56 (m, 1H), 4.42e4.31 (m, 2H), 4.22

(t, ³J ¼ 7.1 Hz, 1H), 3.75e3.45 (m, 8H), 1.78e1.51 (m, 7H), 1.34e1.07

(m, 6H), 0.95e0.81 (m, 2H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-((3,5-

diﬂuorophenyl)amino)-1-oxobutan-2-yl)carbamate (87). Com-

pound 87 was prepared according to general procedure E using (S)-

2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

d

¼ 170.7, 156.1, 144.1, 143.9, 141.4, 127.8, 127.2, 125.3, 120.1, 67.1,

cyclohexylbutanoic acid (71, 400 mg, 0.98 mmol) and 3,5-

diﬂuoroaniline (152 mg, 1.18 mmol). The crude product was puri-

ﬁed by column chromatography on silica (cyclohexane/EtOAc

4:1 / 1:1 / 1:3 / 1:20) and further recrystallized from acetone

to afford 410 mg (0.79 mmol, 81%) of 87 as a colorless solid. TLC:

R_F¼ 0.37 (SiO₂, cyclohexane/EtOAc, 4:1); ¹H NMR (600 MHz,

67.0, 66.8, 50.8, 47.3, 46.2, 42.6, 37.7, 33.5, 33.3, 32.6, 30.8, 26.7, 26.4

(2x) ppm; MS (ESI pos.): m/z (%) ¼ 499.26 (100) ([MþNa]^þ, calcd.

499.26), 477.31 (65) ([MþH]^þ, calcd. 477.28), 517.36 (23) ([MþK]^þ,

calcd. 515.28).

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-((4-ﬂuoro

phenyl)amino)-1-oxobutan-2-yl)carbamate (84). Compound 84

was prepared according to general procedure E using (S)-2-((((9H-

ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid

DMSO‑d₆, 300 K):

d

¼ 10.41 (s, 1H), 7.89 (d, ³J ¼ 7.4 Hz, 2H),

7.76e7.69 (m, 3H), 7.44e7.39 (m, 2H), 7.37e7.29 (m, 4H), 6.96e6.86

(m, 1H), 4.33e4.17 (m, 3H), 4.10e4.03 (m, 1H), 1.75e1.54 (m, 7H),

1.35e1.25 (m, 1H), 1.24e1.05 (m, 5H), 0.91e0.80 (m, 2H) ppm; ¹³C

(71, 400 mg, 0.98 mmol) and 4-ﬂuoroaniline (112 mL, 1.18 mmol).

The crude product was recrystallized from acetone to afford 438 mg

(0.88 mmol, 89%) of 84 as a colorless solid. TLC: R_F¼ 0.26 (SiO₂,

cyclohexane/EtOAc, 4:1); ¹H NMR (600 MHz, DMSO‑d₆, 300 K):

NMR (150 MHz, DMSO‑d₆, 300 K):

d

¼

172.5, 171.9, 162.4

(¹J_CF¼ 243.5 Hz), 162.3 (¹J_CF¼ 243.5 Hz), 156.1, 143.8, 143.7, 141.5,

141.4, 140.7, 127.6, 127.0, 125.2, 120.0, 102.0 (²J_CF¼ 28.9 Hz), 98.5

(²J_CF¼ 28.9 Hz), 65.6, 55.8, 46.6, 36.8, 33.1, 32.8, 32.6, 29.0, 26.1, 25.7

(2x) ppm; MS (ESI pos.): m/z (%) ¼ 541.21 (16) ([MþNa]^þ, calcd.

541.23), 519.18 (22) ([MþH]^þ, calcd. 519.25), 297.20 (11) ([M-

FmocþH]^þ, calcd. 297.18), 179.30 (100) ([M_fr.þH]^þ, calcd. 179.09).

d

¼ 10.06 (s, 1H), 7.89 (d, ³J ¼ 7.5 Hz, 2H), 7.74 (t, ³J ¼ 7.8 Hz, 2H),

7.66e7.60 (m, 3H), 7.41 (td, ³J ¼ 7.5, 3.3 Hz, 2H), 7.32 (td, ³J ¼ 7.5,

3.2 Hz, 2H), 7.14 (t, ³J ¼ 8.9 Hz, 2H), 4.32e4.18 (m, 3H), 4.12e4.02

(m, 1H), 1.75e1.55 (m, 7H), 1.33e1.26 (m, 1H), 1.25e1.07 (m, 5H),

0.90e0.79 (m, 2H) ppm; ¹³C NMR (150 MHz, DMSO‑d₆, 300 K):

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-((3,5-

d

¼ 171.0, 158.0 (¹J_CF¼ 241.9 Hz), 156.0, 143.8, 143.7, 140.7, 135.3,

dichlorophenyl)amino)-1-oxobutan-2-yl)carbamate (88). Com-

pound 88 was prepared according to general procedure E using (S)-

2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

127.6, 127.0, 125.3, 120.9, 120.0 (³J_CF¼ 8.1 Hz), 115.2 (²J_CF¼ 22.3 Hz),

65.6, 55.6, 46.6, 36.8, 33.1, 32.8, 32.6, 29.3, 26.1, 25.7 (2x) ppm; MS

(ESI pos.): m/z (%) ¼ 523.23 (52) ([MþNa]^þ, calcd. 523.24), 501.27

(47) ([MþH]^þ, calcd. 501.26), 279.25 (25) ([M-FmocþH]^þ, calcd.

279.25), 179.28 (100) ([M_fr.þH]^þ, calcd. 179.29).

cyclohexylbutanoic acid (71, 400 mg, 0.98 mmol) and 3,5-

dichloroaniline (191 mg, 1.18 mmol). The crude product was puri-

ﬁed by column chromatography on silica (cyclohexane/EtOAc

4:1 / 1:1 / 1:3) and further recrystallized from MeOH to afford

285 mg (0.55 mmol, 56%) of 88 as a colorless solid. TLC: R_F¼ 0.45

(SiO₂, cyclohexane/EtOAc, 4:1); ¹H NMR (500 MHz, DMSO‑d₆,

(S)-(9H-Fluoren-9-yl)methyl (1-((4-chlorophenyl)amino)-4-

cyclohexyl-1-oxobutan-2-yl)carbamate (85). Compound 85 was

prepared according to general procedure E using (S)-2-((((9H-ﬂu-

oren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic

acid

300 K):

d

¼ 10.38 (s, 1H), 7.89 (d, ³J ¼ 7.6 Hz, 2H), 7.76e7.71 (m, 3H),

(71, 400 mg, 0.98 mmol) and 4-chloroaniline (150 mg, 1.18 mmol).

The crude product was puriﬁed by column chromatography on

silica (cyclohexane/EtOAc, 4:1 / 1:1 / 1:3) to afford 439 mg

(0.85 mmol, 86%) of 85 as a colorless solid. TLC: R_F¼ 0.87 (SiO₂,

cyclohexane/EtOAc, 1:1); ¹H NMR (250 MHz, DMSO‑d₆, 300 K):

7.69 (d, ⁴J ¼ 1.8 Hz, 2H), 7.41 (t, ³J ¼ 7.5 Hz, 2H), 7.32 (t, ³J ¼ 7.5 Hz,

2H), 7.28 (t, ⁴J ¼ 1.7 Hz, 1H), 4.33e4.12 (m, 3H), 4.08e4.01 (m, 1H),

1.76e1.55 (m, 7H), 1.34e1.24 (m, 1H), 1.23e1.05 (m, 5H), 0.90e0.78

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 172.0,156.1,

143.8 (2x), 141.2, 140.7, 134.1, 127.6, 127.0, 125.3 (2x), 122.5, 120.1

(2x), 117.3, 65.6, 55.9, 46.7, 36.8, 33.2, 32.9, 32.6, 29.0, 26.1, 25.8 (2x)

ppm; MS (ESI pos.): m/z (%) ¼ 573.17 (12) ([MþNa]^þ, calcd. 573.13),

551.20 (18) ([MþH]^þ, calcd. 551.19), 179.17 (100) ([M_fr.þH]^þ, calcd.

179.09).

d

¼ 10.14 (s, 1H), 7.89 (d, ³J ¼ 7.4 Hz, 2H), 7.74e7.59 (m, 5H),

7.46e7.26 (m, 6H), 4.32e4.17 (m, 3H), 4.15e4.03 (m, 1H), 1.78e1.52

(m, 7H), 1.35e1.04 (m, 6H), 0.96e0.75 (m, 2H) ppm; ¹³C NMR

(126 MHz, CDCl₃, 300 K):

d

¼ 170.2, 156.8, 143.9, 141.4, 136.2, 129.6,

129.1,128.0,127.3, 125.1,121.4, 120.2, 67.4, 56.2, 47.2, 37.6, 33.4, 33.3,

29.8, 29.6, 27.1, 26.6, 26.4 ppm; MS (ESI pos.): m/z (%) ¼ 539.23 (58)

([MþNa]^þ, calcd. 539.21), 517.26 (100) ([MþH]^þ, calcd. 517.23),

295.21 (23) ([M-FmocþH]^þ, calcd. 295.16), 179.29 (95) ([M_fr.þH]^þ,

calcd. 179.09).

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-((3,5-

dibromophenyl)amino)-1-oxobutan-2-yl)carbamate (89). Com-

pound 89 was prepared according to general procedure E using (S)-

2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

cyclohexylbutanoic acid (71, 400 mg, 0.98 mmol) and 3,5-

dibromoaniline (296 mg, 1.18 mmol). The crude product was puri-

ﬁed by column chromatography on silica (cyclohexane/EtOAc

4:1 / 1:1) to afford 404 mg (0.63 mmol, 64%) of 89 as a colorless

solid. TLC: R_F¼ 0.43 (SiO₂, cyclohexane/EtOAc, 4:1); ¹H NMR

(S)-(9H-Fluoren-9-yl)methyl (1-((4-bromophenyl)amino)-4-

cyclohexyl-1-oxobutan-2-yl)carbamate (86). Compound 86 was

prepared according to general procedure E using (S)-2-((((9H-ﬂu-

oren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic

acid

(71, 400 mg, 0.98 mmol) and 4-bromoaniline (203 mg, 1.18 mmol).

The crude product was puriﬁed by column chromatography on

silica (cyclohexane/EtOAc 4:1 / 1:1 / 1:3 / 1:20) and further

recrystallized from acetone to afford 487 mg (0.87 mmol, 88%) of 86

as a colorless solid. TLC: R_F¼ 0.36 (SiO₂, cyclohexane/EtOAc, 4:1);

(600 MHz, DMSO‑d₆, 300 K):

d

¼ 10.31 (s, 1H), 7.91e7.85 (m, 4H),

7.76e7.68 (m, 3H), 7.50e7.48 (m, 1H), 7.43e7.39 (m, 2H), 7.32 (t,

³J ¼ 7.4 Hz, 2H), 4.34e4.19 (m, 3H), 4.07e4.00 (m,1H),1.75e1.56 (m,

7H), 1.33e1.25 (m, 1H), 1.23e1.06 (m, 5H), 0.90e0.80 (m, 2H) ppm;

¹³C NMR (150 MHz, DMSO‑d₆, 300 K):

d

¼ 171.9, 156.1, 143.8, 143.7,

¹H NMR (600 MHz, DMSO‑d₆, 300 K):

d

¼ 10.15 (s, 1H), 7.89 (d,

141.5, 141.4, 140.7, 127.8, 127.6, 127.0, 125.2, 122.3, 120.5, 120.0, 65.6,

55.9, 46.6, 36.8, 33.1, 32.8, 32.6, 28.9, 26.1, 25.7 (2x) ppm; MS (ESI

neg.): m/z (%) ¼ 675.20 (8) ([MþCl]^-, calcd. 675.53), 417.23 (100)

([M-FmocþH]^-, calcd. 417.00).

³J ¼ 7.6 Hz, 2H), 7.74 (t, ³J ¼ 7.9 Hz, 2H), 7.66 (d, ³J ¼ 7.9 Hz, 1H), 7.58

(d, ³J ¼ 8.7 Hz, 2H), 7.48 (d, ³J ¼ 8.7 Hz, 2H), 7.44e7.38 (m, 2H),

7.34e7.29 (m, 2H), 4.32e4.20 (m, 3H), 4.12e4.06 (m, 1H), 1.76e1.56

(m, 7H), 1.33e1.25 (m, 1H), 1.24e1.06 (m, 5H), 0.91e0.80 (m, 2H)

(S)-(9H-Fluoren-9-yl)methyl (1-(benzylamino)-4-cyclohexyl-

1-oxobutan-2-yl)carbamate (90). Compound 90 was prepared

according to general procedure E using (S)-2-((((9H-ﬂuoren-9-yl)

methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid (71, 400 mg,

0.98 mmol) and phenylmethanamine (126 mg, 1.18 mmol). The

crude product was puriﬁed by column chromatography on silica

ppm; ¹³C NMR (150 MHz, DMSO‑d₆, 300 K):

d

¼ 170.3, 155.0, 142.8,

142.7, 139.7, 137.3, 130.5, 126.6, 126.0, 124.3, 120.1, 119.0, 113.8, 64.6,

54.7, 45.6, 35.8, 32.1, 31.8, 31.6, 28.2, 25.1, 24.7 (2x) ppm; MS (ESI

pos.): m/z (%) ¼ 563.17 (16) ([MþH]^þ, calcd. 563.18), 561.17 (15)

([MþH]^þ, calcd. 561.18), 179.29 (100) ([M_fr.þH]^þ, calcd. 179.09).

24

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S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

(cyclohexane/EtOAc, 1:4 / EtOH) to afford 373 mg (0.75 mmol,

76%) of 90 as a colorless solid. TLC: R_F¼ 0.83 (SiO₂, cyclohexane/

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-oxo-1-((pyr-

idin-2-ylmethyl)amino)butan-2-yl)carbamate (94). Compound

94 was prepared according to general procedure E using (S)-2-

((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

EtOAc, 1:3); ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.41 (t,

³J ¼ 5.9 Hz, 1H), 7.89 (d, ³J ¼ 7.6 Hz, 2H), 7.77e7.70 (m, 2H), 7.50 (d,

³J ¼ 8.2 Hz,1H), 7.42 (t, ³J ¼ 7.4 Hz, 2H), 7.35e7.18 (m, 7H), 4.37e4.16

(m, 5H), 4.02e3.94 (m, 1H), 1.73e1.50 (m, 7H), 1.28e1.05 (m, 6H),

0.90e0.75 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and pyridin-2-

ylmethanamine (159 mg, 1.47 mmol). The crude product was trit-

urated with cyclohexane/CH₂Cl₂(1:1) to afford 495 mg (1.00 mmol,

81%) of 94 as a colorless solid. TLC: R_F¼ 0.31 (SiO₂, cyclohexane/

d

¼ 172.0, 156.0, 143.9, 143.8, 140.7, 139.5, 128.2, 127.6, 127.1, 127.0,

126.7, 125.3, 120.1, 65.6, 55.0, 46.7, 42.0, 36.7, 33.0, 32.0, 32.7, 29.4,

26.2, 25.8 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 519.23 (100)

([MþNa]^þ, calcd. 519.26), 520.24 (34) ([MþNa]^þ, calcd. 520.28).

(S)-(9H-Fluoren-9-yl)methyl (1-((4-chlorobenzyl)amino)-4-

cyclohexyl-1-oxobutan-2-yl)carbamate (91). Compound 91 was

prepared according to general procedure E using (S)-2-((((9H-ﬂu-

EtOAc, 1:3); ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.50 (t,

³J ¼ 5.8 Hz, 1H), 8.48e8.45 (m, 1H), 7.89 (d, ³J ¼ 7.6 Hz, 2H),

7.77e7.68 (m, 3H), 7.55 (d, ³J ¼ 8.1 Hz, 1H), 7.41 (t, ³J ¼ 7.5 Hz, 2H),

7.35e7.20 (m, 4H), 4.37 (d, ³J ¼ 5.6 Hz, 2H), 4.34e4.18 (m, 3H),

4.04e3.96 (m, 1H), 1.76e1.50 (m, 7H), 1.28e1.05 (m, 6H), 0.90e0.77

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 172.3,

oren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic

acid

158.5, 156.0, 148.7, 143.9, 143.8, 140.7, 136.6, 127.6, 127.0, 125.3,

122.1, 120.8, 120.1, 65.6, 55.0, 46.7, 44.1, 36.8, 33.1, 32.9, 32.7, 29.3,

26.2, 25.8 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 498.29 (100) ([MþH]^þ,

calcd. 498.28).

(71, 400 mg, 0.98 mmol) and (4-chlorophenyl)methanamine

(167 mg, 1.18 mmol). The crude product was puriﬁed by column

chromatography on silica (cyclohexane/EtOAc, 8:1 /1:1 / EtOAc)

to afford 350 mg (0.66 mmol, 67%) of 91 as a colorless solid. TLC:

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-oxo-1-((thio-

R_F¼ 0.51 (SiO₂, cyclohexane/EtOAc, 8:1); ¹H NMR (500 MHz,

phen-2-ylmethyl)amino)butan-2-yl)carbamate (95). Compound

95 was prepared according to general procedure E using (S)-2-

((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

3

DMSO‑d₆, 300 K):

d

¼ 8.45 (t, ³J ¼ 5.7 Hz, 1H), 7.89 (d, J ¼ 7.7 Hz,

2H), 7.77e7.70 (m, 2H), 7.50 (d, ³J ¼ 8.0 Hz, 1H), 7.41 (t, ³J ¼ 7.4 Hz,

2H), 7.38e7.22 (m, 6H), 4.35e4.13 (m, 5H), 3.99e3.92 (m, 1H),

1.72e1.47 (m, 7H), 1.25e1.02 (m, 6H), 0.89e0.75 (m, 2H) ppm; ¹³C

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and thiophen-2-

ylmethanamine (113 mg, 1.47 mmol). The crude product was trit-

urated with cyclohexane/CH₂Cl₂(1:1) to afford 468 mg (0.93 mmol,

76%) of 95. TLC: R_F¼ 0.86 (SiO₂, cyclohexane/EtOAc, 1:1); ¹H NMR

NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 172.1, 156.0, 143.9, 143.8,

140.7, 138.6, 131.3, 129.0, 128.1, 127.6, 127.1, 127.0, 125.3, 120.1, 65.6,

55.0, 46.7, 41.4, 36.7, 33.0, 32.9, 32.6, 29.4, 26.2, 25.8 (2x) ppm; MS

(ESI pos.): m/z (%) ¼ 553.21 (33) ([MþNa]^þ, calcd. 553.22), 531.25

(100) ([MþH]^þ, calcd. 531.24), 309.17 (35) ([M-Fmoc þ H]^þ, calcd.

309.18), 179.29 (69) ([M_fr.þH]^þ, calcd. 179.29).

(500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.51 (t, ³J ¼ 5.7 Hz, 1H), 7.89 (d,

³J ¼ 7.6 Hz, 2H), 7.77e7.70 (m, 2H), 7.49 (d, ³J ¼ 8.3 Hz, 1H), 7.42 (t,

³J ¼ 7.4 Hz, 2H), 7.38e7.35 (m, 1H), 7.32 (t, ³J ¼ 7.4 Hz, 2H),

6.98e6.90 (m, 2H), 4.59e4.34 (m, 2H), 4.33e4.15 (m, 3H),

4.00e3.90 (m, 1H), 1.71e1.47 (m, 7H), 1.27e1.04 (m, 6H), 0.89e0.76

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-oxo-1-((pyr-

idin-4-ylmethyl)amino)butan-2-yl)carbamate (92). Compound

92 was prepared according to general procedure E using (S)-2-

((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 171.8,155.9,

143.9, 143.8, 142.5, 140.7, 127.6, 127.0, 126.6, 125.4, 125.2, 125.0,

120.1, 65.6, 54.8, 46.7, 37.2, 36.7, 33.0, 32.9, 32.6, 29.4, 26.2, 25.8 (2x)

ppm; MS (ESI pos.): m/z (%) ¼ 525.22 (100) ([MþNa]^þ, calcd.

525.22).

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and pyridin-4-

ylmethanamine (159 mg, 1.47 mmol). The crude product was trit-

urated with cyclohexane/CH₂Cl₂(1:1) to afford 501 mg (1.01 mmol,

82%) of 92 as a colorless solid. TLC: R_F¼ 0.27 (SiO₂, cyclohexane/

(S)-(9H-Fluoren-9-yl)methyl (1-((4-chlorophenethyl)amino)-

4-cyclohexyl-1-oxobutan-2-yl)carbamate (96). Compound 96

was prepared according to general procedure E using (S)-2-((((9H-

ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-cyclohexylbutanoic acid

(71, 500 mg, 1.23 mmol) and 2-(4-chlorophenyl)ethanamine

(229 mg, 1.47 mmol). The crude product was puriﬁed by column

chromatography on silica (CH₂Cl₂/MeOH, 10:1) to afford 418 mg

(0.77 mmol, 62%) of 96 as a colorless solid. TLC: R_F¼ 0.89 (SiO₂,

EtOAc, 1:20); ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.54 (t,

³J ¼ 5.9 Hz, 1H), 8.46 (d, ³J ¼ 5.7 Hz, 2H), 7.89 (d, ³J ¼ 7.6 Hz, 2H),

7.77e7.71 (m, 2H), 7.57 (d, ³J ¼ 8.3 Hz, 1H), 7.41 (t, ³J ¼ 7.5 Hz, 2H),

7.31 (t, ³J ¼ 7.4 Hz, 2H), 7.23 (d, ³J ¼ 5.7 Hz, 2H), 4.36e4.18 (m, 5H),

4.02e3.94 (m, 1H), 1.75e1.51 (m, 7H), 1.27e1.05 (m, 6), 0.90e0.76

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 172.4,156.1,

149.4, 148.5, 143.9, 143.8, 140.7 (2x), 127.6, 127.0, 125.3, 122.1, 120.1,

65.6, 55.0, 46.7, 41.1, 36.7, 33.1, 32.9, 32.7, 29.2, 26.4, 26.2, 25.8 (2x)

ppm; MS (ESI pos.): m/z (%) ¼ 498.29 (100) ([MþH]^þ, calcd. 498.28).

cyclohexane/EtOAc,1:1); ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76

(d, ³J ¼ 7.5 Hz, 2H), 7.56 (d, ³J ¼ 7.5 Hz, 2H), 7.40 (t, ³J ¼ 7.5 Hz, 2H),

7.30 (t, ³J ¼ 7.5 Hz, 2H), 7.22 (d, ³J ¼ 8.4 Hz, 2H), 7.07 (d, ³J ¼ 8.3 Hz,

2H), 5.95 (s, 1H), 5.24 (d, ³J ¼ 7.8 Hz, 1H), 4.47e4.27 (m, 2H), 4.22 (t,

³J ¼ 6.9 Hz, 1H), 4.08e3.99 (m, 1H), 3.60e3.30 (m, 2H), 2.83e2.64

(m, 2H), 1.91e1.46 (m, 7H), 1.35e1.02 (m, 6H), 0.95e0.73 (m, 2H);

(S)-(9H-Fluoren-9-yl)methyl

(4-cyclohexyl-1-oxo-1-((pyr-

idin-3-ylmethyl)amino)butan-2-yl)carbamate (93). Compound

93 was prepared according to general procedure E using (S)-2-

((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 171.9, 156.3, 143.9, 141.4,

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and pyridin-3-

ylmethanamine (159 mg, 1.47 mmol). The crude product was trit-

urated with cyclohexane/CH₂Cl₂(1:1) to afford 384 mg (0.77 mmol,

63%) of 93 as a colorless solid. TLC: R_F¼ 0.36 (SiO₂, cyclohexane/

137.2, 132.5, 130.2, 128.8, 127.9, 127.2, 125.1, 120.1, 67.2, 55.5, 47.3,

40.5, 37.6, 35.1, 33.4, 33.3, 33.1, 30.2, 26.7, 26.4 ppm; MS (ESI pos.):

m/z (%) ¼ 511.18 (100) (M-Clþ2H]^þ, calcd. 511.30).

(S)-tert-Butyl

4-(2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)

EtOAc, 1:20); ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.50 (t,

amino)-4-cyclohexylbutanoyl)piperazine-1-carboxylate

Compound 97 was prepared according to general procedure E using

(S)-2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

(97).

³J ¼ 5.7 Hz, 1H), 8.47 (s, 1H), 8.46e8.40 (m, 1H), 7.89 (d, ³J ¼ 7.6 Hz,

2H), 7.77e7.70 (m, 2H), 7.63 (d, ³J ¼ 7.9 Hz, 1H), 7.53 (d, ³J ¼ 8.0 Hz,

1H), 7.41 (t, ³J ¼ 7.5 Hz, 2H), 7.36e7.26 (m, 3H), 4.37e4.14 (m, 5H),

3.99e3.90 (m, 1H), 1.72e1.47 (m, 7H), 1.25e1.04 (m, 6H), 0.88e0.74

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and a solution of

tert-butyl piperazine-1-carboxylate (274 mg, 1.47 mmol) in DMF

(4 mL) with DIPEA (80 mg, 0.61 mmol). The crude product was

puriﬁed by column chromatography on silica (cyclohexane/EtOAC

4:1 / 1:1 / 1:3) to afford 613 mg (1.07 mmol, 87%) of 97. TLC:

R_F¼ 0.73 (SiO₂, cyclohexane/EtOAc, 1:1); ¹H NMR (500 MHz, CDCl₃,

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 172.2,

156.0, 148.7, 148.0, 143.9, 143.8, 140.7 (2x), 135.0, 134.9, 127.6, 127.0,

125.4, 125.3, 123.4, 120.1, 65.6, 55.0, 46.7, 36.7, 33.0, 32.9, 32.6, 29.3,

26.3, 26.2, 25.8, 25.7 ppm; MS (ESI pos.): m/z (%) ¼ 498.30 (100)

([MþH]^þ, calcd. 498.28).

300 K):

d

¼ 7.76 (d, ³J ¼ 7.5 Hz, 2H), 7.64e7.57 (m, 2H), 7.40 (t,

25

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European Journal of Medicinal Chemistry 208 (2020) 112721

³J ¼ 7.5 Hz, 2H), 7.31 (t, ³J ¼ 7.5 Hz, 2H), 5.69 (d, ³J ¼ 8.5 Hz, 1H),

4.68e4.55 (m, 1H), 4.41e4.31 (m, 2H), 4.22 (t, ³J ¼ 7.2 Hz, 1H),

3.71e3.23 (m, 8H), 1.79e1.53 (m, 7H), 1.48 (s, 9H), 1.31e1.04 (m,

6H), 0.90e0.80 (m, 2H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

([M-BocþEtOH]^þ, calcd. 264.12), 220.08 (61) ([M-BocþH]^þ, calcd.

220.09).

2-(Piperazin-1-yl)benzo[d]thiazole (101). tert-Butyl 4-(benzo

[d]thiazol-2-yl)piperazine-1-carboxylate (100, 365 mg, 1.14 mmol)

was dissolved in CH₂Cl₂(15 mL), cooled to 0 ^ꢀC and TFA (2 mL) was

added. The mixture was stirred for 3 h at rt and the solvent was

removed under reduced pressure. The crude product was dissolved

in MeOH and K₂CO₃(3.6 g, 25.96 mmol) was added. The mixture

was stirred for 5 min at 5 ^ꢀC, ﬁltered and the solvent was removed

under reduced pressure. Water was added and the aqueous phase

was extracted 3-times with EtOAc (50 mL). The organic extracts

were combined, dried over MgSO₄and the solvent was evaporated

under reduced pressure to afford 246 mg (1.12 mmol, quant.) of 101.

TLC: R_F¼ 0.51 (SiO₂, cyclohexane/EtOAc, 20:1); ¹H NMR (500 MHz,

d

¼ 170.8, 156.1, 154.6, 144.0, 143.9, 141.4, 127.8, 127.2, 125.3, 120.1,

80.6, 67.1, 51.0, 47.3, 45.6, 42.1, 37.6, 33.5, 33.2, 32.7, 30.9, 28.5, 26.7,

26.4 ppm; MS (ESI pos.): m/z (%) ¼ 520.28 (100) ([M-Bocþ2Na]^þ,

calcd. 520.26), 598.32 (89) ([MþNa]^þ, calcd. 598.33), 576.35 (21)

([MþH]^þ, calcd. 576.35).

(S)-(9H-Fluoren-9-yl)methyl

(1-(4-(4-chlorophenyl)piper-

azin-1-yl)-4-cyclohexyl-1-oxobutan-2-yl)carbamate (98). Com-

pound 98 was prepared according to general procedure E using (S)-

2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and 1-(4-

chlorophenyl)piperazine (290 mg, 1.47 mmol). The crude product

was triturated with cyclohexane/CH₂Cl₂(1:1) to afford 651 mg

(1.11 mmol, 91%) of 98 as a colorless solid. TLC: R_F¼ 0.68 (SiO₂,

cyclohexane/EtOAc, 1:1); ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

3

DMSO‑d₆, 300 K):

d

¼ 7.74 (d, J ¼ 7.9 Hz, 1H), 7.44 (d, J ¼ 8.1 Hz,

1H), 7.26 (dt, ³J ¼ 7.9, 1.3 Hz, 1H), 7.05 (dt, ³J ¼ 7.7, 1.1 Hz, 1H),

3.51e3.43 (m, 4H), 2.86e2.74 (m, 4H) ppm; ¹³C NMR (126 MHz,

DMSO‑d₆, 300 K):

d

¼ 168.4, 152.2, 130.2, 125.9, 121.1, 121.0, 118.5,

3

d

¼ 7.88 (d, J ¼ 7.5 Hz, 2H), 7.72 (dd, J ¼ 7.6, 3.5 Hz, 2H), 7.63 (d,

49.3, 45.0 ppm; MS (ESI pos.): m/z (%) ¼ 220.10 (100) ([MþH]^þ,

³J ¼ 8.3 Hz, 1H), 7.40 (dt, ³J ¼ 7.6, 3.1 Hz, 2H), 7.30 (t, ³J ¼ 7.4 Hz, 2H),

7.24 (d, ³J ¼ 9.0 Hz, 2H), 6.94 (d, ³J ¼ 9.0 Hz, 2H), 4.46e4.36 (m, 1H),

4.32e4.17 (m, 3H), 3.72e3.44 (m, 4H), 3.17e2.99 (m, 4H), 1.71e1.48

(m, 7H), 1.30e1.02 (m, 6H), 0.91e0.75 (m, 2H) ppm; ¹³C NMR

calcd. 220.09.

(S)-(9H-Fluoren-9-yl)methyl

(1-(4-(benzo[d]thiazol-2-yl)

piperazin-1-yl)-4-cyclohexyl-1-oxobutan-2-yl)carbamate (102).

Compound 102 was prepared according to general procedure E

(126 MHz, DMSO‑d₆, 300 K):

d

¼ 170.1, 155.9, 149.5, 143.8, 140.7,

using

(S)-2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

128.7, 127.6, 127.0, 125.3, 122.7, 120.1, 117.3, 65.6, 50.7, 48.6, 48.1,

46.7, 44.6, 41.2, 37.0, 32.9, 32.7, 29.0, 26.4, 26.2, 25.8 (2x) ppm; MS

(ESI pos.): m/z (%) ¼ 586.20 (9) (MþH]^þ, calcd. 586.29), 364.23

(100) ([M-Fmocþ2H]^þ, calcd. 364.22), 295.22 (93) ([M_fr.]^þ, calcd.

295.07).

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and a solution

of 2-(piperazin-1-yl)benzo[d]thiazole (101, 260 mg, 1.19 mmol, 0.9

eq) in DMF/THF (7 mL, 6:1). The crude product was puriﬁed by

column chromatography on silica (CH₂Cl₂/MeOH, 10:1) to afford

466 mg (0.77 mmol, 62%) of 102. TLC: R_F¼ 0.96 (SiO₂, CH₂Cl₂/

(S)-(9H-Fluoren-9-yl)methyl

(1-(4-(4-bromophenyl)piper-

MeOH, 10:1); ¹H NMR (400 MHz, DMSO‑d₆, 300 K):

d

¼ 7.91e7.85

azin-1-yl)-4-cyclohexyl-1-oxobutan-2-yl)carbamate (99). Com-

pound 99 was prepared according to general procedure E using (S)-

2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

(m, 2H), 7.79 (d, ³J ¼ 7.5 Hz, 1H), 7.73 (dd, ³J ¼ 7.7, 3.5 Hz, 2H), 7.64

(d, ³J ¼ 8.2 Hz, 1H), 7.48 (d, ³J ¼ 7.6 Hz, 1H), 7.44e7.40 (m, 2H),

7.35e7.26 (m, 3H), 7.13e7.06 (m, 1H), 4.47e4.38 (m, 1H), 4.32e4.18

(m, 3H), 3.74e3.46 (m, 8H), 1.71e1.51 (m, 7H), 1.27e1.04 (m, 6H),

0.92e0.77 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and 1-(4-

bromophenyl)piperazine (355 mg, 1.47 mmol). The crude product

was triturated with cyclohexane/CH₂Cl₂(1:1) to afford 610 mg

(0.97 mmol, 79%) of 99. TLC: R_F¼ 0.79 (SiO₂, cyclohexane/EtOAc,

d

¼ 173.3, 168.1, 168.0, 152.3, 143.9, 130.4, 127.3 (2x), 126.0, 124.6,

123.9, 121.4 (2x), 121.2, 120.0, 118.7, 67.8, 50.4, 47.1, 40.9, 37.1, 33.0,

32.9, 32.7, 32.6, 32.1, 26.2, 25.8 (2x) ppm; MS (ESI pos.): m/z

(%) ¼ 387.24 (100) ([M-FmocþH]^þ, calcd. 387.23).

1:1); ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 7.88 (d, ³J ¼ 7.6 Hz,

2H), 7.72 (dd, ³J ¼ 7.6, 3.4 Hz, 2H), 7.63 (d, ³J ¼ 8.3 Hz, 1H), 7.40 (dt,

³J ¼ 7.4, 3.0 Hz, 2H), 7.35 (d, ³J ¼ 8.9 Hz, 2H), 7.30 (t, ³J ¼ 7.4 Hz, 2H),

6.89 (d, ³J ¼ 9.1 Hz, 2H), 4.45e4.37 (m, 1H), 4.31e4.17 (m, 3H),

3.69e3.45 (m, 4H), 3.17e2.99 (m, 4H), 1.69e1.48 (m, 7H), 1.28e1.03

(m, 6H), 0.91e0.74 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆,

tert-Butyl 4-(benzo[d]oxazol-2-yl)piperazine-1-carboxylate

(103). 2-Chlorobenzo[d]oxazole (153 mg, 1.00 mmol) and tert-

butyl piperazine-1-carboxylate (373 mg, 2.00 mmol) were sus-

pended in H₂O (2 mL) and stirred for 3 d at rt. The precipitate was

dissolved in EtOAc, the aqueous phase was extracted 3 times with

EtOAc (50 mL) and the organic extracts were combined and dried

over MgSO₄. The solvent was removed under reduced pressure to

afford 303 mg (0.99 mmol, quant.) of 103. TLC: R_F¼ 0.67 (SiO₂,

300 K):

d

¼ 170.1, 155.9, 149.5, 143.8, 140.7, 131.5, 127.6, 127.0, 125.3,

120.1, 117.7, 110.5, 65.6, 50.7, 48.4, 47.9, 46.7, 44.6, 41.2, 37.0, 32.9,

32.7, 29.0, 26.2, 25.8 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 632.27 (22)

([MþH]^þ, calcd. 632.23), 408.20 (41) ([M-Fmocþ2H]^þ,calcd.

408.17), 339.11 (100) ([M_fr.]^þ, calcd. 339.02).

cyclohexane/EtOAc,1:1); ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.36

tert-Butyl 4-(benzo[d]thiazol-2-yl)piperazine-1-carboxylate

(100). 2-Chlorobenzo[d]thiazole (170 mg, 1.00 mmol) and tert-

butyl piperazine-1-carboxylate (373 mg, 2.00 mmol) were sus-

pended in water (2 mL) and stirred for 5 d at rt. The precipitate was

dissolved in EtOAc, the aqueous phase was extracted 3-times with

EtOAc (50 mL) and dried over MgSO₄. The solvent was evaporated

under reduced pressure and the crude product was puriﬁed by

column chromatography on silica (cylcohexane/EtOAc 20:1 /

EtOAc) to afford 282 mg (0.88 mmol, 88%) of 100 as a colorless solid.

TLC: R_F¼ 0.81 (SiO₂, cyclohexane/EtOAc, 1:1); ¹H NMR (500 MHz,

(d, ³J ¼ 7.9 Hz, 1H), 7.25 (d, ³J ¼ 7.8 Hz, 1H), 7.17 (t, ³J ¼ 7.7 Hz, 1H),

7.03 (t, ³J ¼ 7.7 Hz, 1H), 3.71e3.36 (m, 4H), 3.60e3.51 (m, 4H), 1.48

(s, 9H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 162.1, 154.7,

148.9, 143.0, 124.2, 121.1, 116.6, 108.9, 80.5, 45.5, 43.3, 28.5 ppm; MS

(ESI pos.): m/z (%) ¼ 304.25 (14) ([MþH]^þ, calcd. 304.17), 248.20

(100) ([M_fr.þ2H]^þ, calcd. 248.11), 204.12 (49) ([M-Bocþ2H]^þ, calcd.

204.12).

2-(Piperazin-1-yl)benzo[d]oxazole (104). tert-Butyl 4-(benzo

[d]oxazol-2-yl)piperazine-1-carboxylate (103, 500 mg, 1.65 mmol)

was dissolved in CH₂Cl₂(15 mL), cooled to 0 ^ꢀC and TFA (2 mL) was

added. The mixture was stirred for 3 h at rt and the solvent was

removed under reduced pressure. The crude product was dissolved

in MeOH, K₂CO₃(3.6 g, 25.96 mmol) was added and the mixture

was stirred for 5 min at 5 ^ꢀC. The mixture was ﬁltered and the

solvent was removed under reduced pressure. Water was added

and the aqueous phase was extracted 3-times with EtOAc (50 mL).

CDCl₃, 300 K):

d

¼ 7.61 (d, ³J ¼ 7.9 Hz, 1H), 7.56 (d, ³J ¼ 8.0 Hz, 1H),

7.30 (dt, ³J ¼ 7.7, 1.3 Hz, 1H), 7.09 (dt, ³J ¼ 7.7, 1.2 Hz, 1H), 3.64e3.54

(m, 8H), 1.49 (s, 9H) ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 168.8, 154.7, 152.7, 130.8, 126.2, 121.8, 120.9, 119.4, 80.6, 48.3,

43.4, 28.5 ppm; MS (ESI pos.): m/z (%) ¼ 342.18 (65) ([MþNa]^þ,

calcd. 342.13), 320.24 (36) ([MþH]^þ, calcd. 320.15), 264.19 (100)

26

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European Journal of Medicinal Chemistry 208 (2020) 112721

The organic extracts were combined, dried over MgSO₄and the

solvent was evaporated under reduced pressure to afford 298 mg

(1.47 mmol, 89%) of 104. TLC: R_F¼ 0.17 (SiO₂, CH₂Cl₂/MeOH, 10:1);

(FTMS

þ

p

MALDI): m/z

¼

531.3063 [MþH]^þ, calcd. for

[C₃₀H₃₉N₆O₃]^þ¼ 531.3078.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-oxo-1-(propylamino)

butan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

¹H NMR (250 MHz, CDCl₃, 300 K):

d

¼ 7.36 (d, ³J ¼ 7.5 Hz, 1H), 7.26

(d, ³J ¼ 7.9 Hz, 1H), 7.17 (dt, ³J ¼ 7.7, 1.1 Hz, 1H), 7.04 (dt, ³J ¼ 7.7,

1.1 Hz, 1H), 3.89e3.65 (m, 4H), 3.17e2.96 (m, 4H) ppm; MS (ESI

pos.): m/z (%) ¼ 204.27 (100) ([MþH]^þ, calcd. 204.11).

carboxamide (138). Compound 138 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 100 mg, 0.30 mmol) and

(S)-2-amino-4-cyclohexyl-N-propylbutanamide (108, 81 mg,

0.36 mmol). The crude product was puriﬁed by column chroma-

tography on silica (CH₂Cl₂/MeOH 20:1) to afford 121 mg

(0.22 mmol, 75%) of 138. TLC: R_F¼ 0.17 (SiO₂, CH₂Cl₂/MeOH 20:1);

HPLC: 14.9 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

(S)-(9H-Fluoren-9-yl)methyl

(1-(4-(benzo[d]oxazol-2-yl)

piperazin-1-yl)-4-cyclohexyl-1-oxobutan-2-yl)carbamate (105).

Compound 105 was prepared according to general procedure E

using

(S)-2-((((9H-ﬂuoren-9-yl)methoxy)carbonyl)amino)-4-

cyclohexylbutanoic acid (71, 500 mg, 1.23 mmol) and 2-(piper-

azin-1-yl)benzo[d]oxazole (104, 298 mg, 1.47 mmol). The crude

product was puriﬁed by column chromatography on silica (CH₂Cl₂/

MeOH, 10:1) to afford 571 mg (0.96 mmol, 79%) of 105. TLC:

R_F¼ 0.94 (SiO₂, CH₂Cl₂/MeOH, 10:1); ¹H NMR (400 MHz, DMSO‑d₆,

d

¼ 8.55e8.47 (m, 1H), 8.28 (d, ³J ¼ 8.1 Hz, 1H), 7.98 (s, 1H), 7.92 (t,

³J ¼ 5.7 Hz, 1H), 7.85 (d, ³J ¼ 8.3 Hz, 2H), 7.59e7.49 (m, 4H),

7.41e7.31 (m, 3H), 6.38 (br s, 2H), 4.49e4.43 (m, 2H), 4.39e4.32 (m,

1H), 3.10e2.93 (m, 2H), 1.81e1.71 (m, 1H), 1.70e1.55 (m, 6H), 1.40

3

300 K):

d

¼ 7.91e7.84 (m, 2H), 7.79e7.69 (m, 2H), 7.63 (d,

(sext, J ¼ 7.3 Hz, 2H), 1.28e1.04 (m, 6H), 0.90e0.77 (m, 5H) ppm;

³J ¼ 8.2 Hz, 1H), 7.45e7.36 (m, 3H), 7.35e7.27 (m, 3H), 7.17 (t,

³J ¼ 7.5 Hz, 1H), 7.04 (t, ³J ¼ 7.5 Hz, 1H), 4.48e4.39 (m, 1H),

4.33e4.18 (m, 3H), 3.76e3.40 (m, 8H), 1.71e1.50 (m, 7H), 1.27e1.04

(m, 6H), 0.92e0.77 (m, 2H) ppm; ¹³C NMR (101 MHz, DMSO‑d₆,

¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 171.7, 168.8, 166.0, 164.1,

149.2,143.6,141.4, 138.4,138.2,134.9,132.7, 129.4,127.6,127.1,127.0,

126.8, 126.7, 123.1, 97.4, 53.6, 41.3, 36.8, 33.3, 32.9, 32.7, 29.4, 26.2,

25.8 (2x), 24.1, 22.3, 11.4 ppm; MS (ESI pos.): m/z (%) ¼ 545.32 (100)

([MþH]^þ, calcd. 545.33), 486.27 (30) ([M_fr.]^þ, calcd. 486.25); HRMS

300 K):

d

¼ 170.5, 161.6, 155.9, 148.3, 143.8 (2x), 142.8, 140.7, 127.6,

127.0, 125.3, 124.0, 120.7, 116.0, 109.0, 65.6, 50.8, 46.7, 37.0, 32.9,

32.7, 28.9, 26.1, 25.8, 25.7 ppm; MS (ESI pos.): m/z (%) ¼ 371.09 (100)

([M-Fmocþ2H]^þ, calcd. 371.25).

(FTMS

þ

p

MALDI): m/z

¼

545.3222 [MþH]^þ, calcd. for

[C₃₁H₄₁N₆O₃]^þ¼ 545.3240.

(S)-5-Amino-N-(4-((1-(butylamino)-4-cyclohexyl-1-

(S)-5-Amino-N-(4-((4-cyclohexyl-1-(methylamino)-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (139). Compound 139 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 100 mg, 0.30 mmol) and

(S)-2-amino-4-cyclohexyl-N-butylbutanamide (109, 120 mg,

0.50 mmol). The crude product was puriﬁed by column chroma-

tography on silica (CH₂Cl₂/MeOH 20:1) to afford 140 mg

(0.25 mmol, 85%) of 139. TLC: R_F¼ 0.30 (SiO₂, CH₂Cl₂/MeOH 20:1);

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (136). Compound 136 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 100 mg, 0.29 mmol) and

(S)-2-amino-4-cyclohexyl-N-methylbutanamide (106, 71 mg,

0.36 mmol). The crude product was puriﬁed by column chroma-

tography on silica (CH₂Cl₂/MeOH 20:1 / 10:1 / EtOH) to afford

90 mg (0.17 mmol, 59%) of 136. TLC: R_F¼ 0.38 (SiO₂, EtOAc/MeOH

10:1); HPLC: 13.9 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

HPLC: 15.5 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.51 (t,

³J ¼ 6.1 Hz, 1H), 8.27 (d, ³J ¼ 8.0 Hz, 1H), 7.98 (s, 1H), 7.91 (t,

³J ¼ 5.1 Hz, 1H), 7.85 (d, ³J ¼ 8.1 Hz, 2H), 7.61e7.48 (m, 4H),

7.42e7.35 (m, 3H), 6.38 (br s, 2H), 4.47 (d, ³J ¼ 6.0 Hz, 2H),

4.39e4.31 (m, 1H), 3.14e2.97 (m, 2H), 1.80e1.71 (m, 1H), 1.70e1.55

(m, 6H), 1.42e1.33 (m, 2H), 1.31e1.04 (m, 8H), 0.91e0.77 (m, 5H)

d

¼ 8.51 (t, ³J ¼ 6.0 Hz, 1H), 8.33 (d, ³J ¼ 8.1 Hz, 1H), 7.96 (s, 1H),

7.89e7.84 (m, 3H), 7.60e7.49 (m, 4H), 7.42e7.33 (m, 3H), 6.38 (s,

2H), 4.47 (d, ³J ¼ 6.0 Hz, 2H), 4.38e4.30 (m, 1H), 2.58 (d, ³J ¼ 4.6 Hz,

3H), 1.81e1.72 (m, 1H), 1.71e1.54 (m, 6H), 1.28e1.03 (m, 6H),

0.90e0.76 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 171.6, 166.0, 164.1,

d

¼ 172.3, 166.1, 164.1, 149.2, 143.6, 138.4, 138.2, 132.6, 129.4, 127.6,

149.2, 143.6, 138.4, 138.2, 132.7, 129.4, 127.6, 126.8, 123.1, 97.4, 53.6,

41.3, 38.1, 36.8, 33.3, 32.9, 32.7, 31.2, 29.4, 26.2, 25.8 (2x), 19.5,

13.7 ppm; MS (ESI neg.): m/z (%) ¼ 557.47 (100) ([MꢃH]^-, calcd.

557.31), 603.44 (41) ([MþEtOHeH]^-, calcd. 603.36); HRMS

127.1, 126.7, 123.1, 97.4, 53.6, 41.3, 36.8, 33.4, 32.9, 32.7, 29.2, 26.2,

25.8 (2x), 25.6 ppm; MS (ESI pos.): m/z (%) ¼ 517.20 (100) ([MþH]^þ,

calcd. 517.29), 486.17 (87) ([M_fr.]^þ, calcd. 486.25), 319.09

(56)([M_fr.II]^þ, calcd. 319.25); HRMS (FTMS

þ

p

MALDI): m/

(FTMS

þ

p

MALDI): m/z

¼

581.3211 [MþNa]^þ, calcd. for

z ¼ 539.2742 [MþNa]^þ, calcd. for [C₂₉H₃₆N₆NaO₃]^þ¼ 539.2747.

[C₃₂H₄₂N₆NaO₃]^þ¼ 581.3216.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-(ethylamino)-1-

(S)-5-Amino-N-(4-((4-cyclohexyl-1-(hexylamino)-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (137). Compound 137 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 100 mg, 0.30 mmol) and

(S)-2-amino-4-cyclohexyl-N-ethylbutanamide (107, 76 mg,

0.36 mmol). The crude product was puriﬁed by column chroma-

tography on silica (CH₂Cl₂/MeOH 20:1 / 10:1 / EtOH) to afford

82 mg (0.16 mmol, 52%) of 137. TLC: R_F¼ 0.25 (SiO₂, CH₂Cl₂/MeOH

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (140). Compound 140 was prepared according to

general procedure B using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 150 mg, 0.45 mmol) and

(S)-2-amino-4-cyclohexyl-N-hexylbutanamide (110, 144 mg,

0.54 mmol). The crude product was puriﬁed by column chroma-

tography on silica (cyclohexane/EtOAc 1:1 / 1:6 / EtOAc) to

afford 206 mg (0.35 mmol, 79%) of 104. TLC: R_F¼ 0.33 (SiO₂,

cyclohexane/EtOAc 1:6); HPLC: 16.7 min; ¹H NMR (500 MHz,

20:1); HPLC: 14.4 min; ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.75

(s, 1H), 7.68 (d, ³J ¼ 8.2 Hz, 2H), 7.57e7.47 (m, 4H), 7.42e7.36 (m,

1H), 7.29 (d, ³J ¼ 8.2 Hz, 2H), 7.08 (d, ³J ¼ 8.0 Hz, 1H), 6.67e6.55 (m,

2H), 5.55 (br s, 2H), 4.63e4.51 (m, 3H), 3.36e3.17 (m, 2H),1.98e1.88

(m, 1H), 1.81e1.57 (m, 6H), 1.33e1.05 (m, 9H), 0.92e0.78 (m, 2H)

DMSO‑d₆, 300 K):

d

¼ 8.52 (t, J ¼ 6.1 Hz, 1H), 8.28 (d, J ¼ 8.1 Hz,

3

1H), 7.99 (s, 1H), 7.92 (t, ³J ¼ 5.6 Hz, 1H), 7.86 (d, ³J ¼ 8.3 Hz, 2H),

7.60e7.49 (m, 4H), 7.41e7.35 (m, 3H), 6.39 (br s, 2H), 4.47 (d,

³J ¼ 5.9 Hz, 2H), 4.39e4.31 (m, 1H), 3.16e3.06 (m, 1H), 3.04e2.94

(m, 1H), 1.81e1.55 (m, 7H), 1.43e1.32 (m, 2H), 1.31e1.04 (m, 12H),

0.91e0.77 (m, 5H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

ppm; ¹³C NMR (126 MHz, CDCl₃, 300 K):

d

¼ 171.8, 167.2, 164.8,

148.9, 142.9, 137.8, 137.7, 132.9, 129.9, 128.2, 127.6, 123.9, 97.9, 54.1,

42.6, 37.7, 34.6, 33.4, 33.3, 30.3 (2x), 26.7, 26.4, 14.8 ppm; MS (ESI

pos.): m/z (%) ¼ 531.23 (100) ([MþH]^þ, calcd. 531.31); HRMS

d

¼ 171.6, 166.0, 164.1, 149.2, 143.6, 138.4, 138.2, 132.7, 129.4, 127.6,

127.1, 126.8, 123.1, 97.4, 53.6, 41.3, 38.4, 36.8, 33.3, 32.9, 32.7, 31.0,

27

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European Journal of Medicinal Chemistry 208 (2020) 112721

29.4, 29.0, 26.2, 26.0, 25.8, 22.1, 13.9 ppm; MS (ESI pos.): m/z

(%) ¼ 587.38 (48) ([MþH]^þ, calcd. 587.37), 486.27 (13) ([M_fr.I]^þ,

calcd. 486.25), 190.35 (100) ([M_fr.IIþNa]^þ, calcd. 190.13); HRMS

ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 171.7, 167.1, 164.8,

149.0, 142.9, 137.8, 137.7, 133.0, 129.9, 128.2, 127.6 (2x), 123.9, 98.0,

54.3, 52.5, 42.6, 37.7, 33.4, 33.3, 33.2, 30.4, 27.5 (2x), 26.7, 26.4, 10.5,

10.4 ppm; MS (ESI pos.): m/z (%) ¼ 573.27 (100) ([MþH]^þ, calcd.

573.36), 486.20 (61) ([M_fr.]^þ, calcd. 486.25); HRMS (FTMS þ p

(FTMS

þ

p

MALDI): m/z

¼

609.3523 [MþNa]^þ, calcd. for

[C₃₄H₄₆N₆NaO₃]^þ¼ 609.3529.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-(decylamino)-1-

MALDI):

m/z

¼

573.3540

[MþH]^þ,

calcd.

for

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (141). Compound 141 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 200 mg, 0.60 mmol) and

(S)-2-amino-4-cyclohexyl-N-decylbutanamide (111, 239 mg,

0.74 mmol). The crude product was puriﬁed by column chroma-

tography on silica (cyclohexane/EtOAc 1:6 / EtOAc) to afford

217 mg (0.34 mmol, 57%) of 141. TLC: R_F¼ 0.41 (SiO₂, cyclohexane/

EtOAc 1:6); HPLC: 21.0 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

[C₃₃H₄₅N₆O₃]^þ¼ 573.3553.

5-Amino-N-(4-(((S)-4-cyclohexyl-1-(((SR)-1-hydroxybutan-2-

yl)amino)-1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyr-

azole-4-carboxamide (144). Compound 144 was prepared according

to general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 200 mg, 0.60 mmol) and (S)-

2-amino-4-cyclohexyl-N-((SR)-1-hydroxybutan-2-yl)butanamide

(114, 189 mg, 0.71 mmol). The crude product was puriﬁed by column

chromatography on silica (cyclohexane/EtOAc 1:20) to afford 166 mg

(0.29 mmol, 49%) of 144. TLC: R_F¼ 0.76 (SiO₂, EtOAc/MeOH 10:1);

HPLC: 13.78,14.1 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K): (mixture

d

¼ 8.51 (t, ³J ¼ 5.9 Hz, 1H), 8.27 (d, ³J ¼ 8.0 Hz, 1H), 7.98 (s, 1H), 7.91

(t, ³J ¼ 5.6 Hz, 1H), 7.85 (d, ³J ¼ 8.3 Hz, 2H), 7.59e7.50 (m, 4H),

7.41e7.35 (m, 3H), 6.38 (br s, 2H), 4.47 (d, ³J ¼ 5.9 Hz, 2H), 4.39e4.31

(m, 1H), 3.16e3.06 (m, 1H), 3.03e2.94 (m, 1H), 1.80e1.71 (m, 1H),

1.70e1.55 (m, 6H), 1.44e1.33 (m, 2H), 1.32e1.03 (m, 20H),

0.90e0.77 (m, 5H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

ofdiastereomers)

d

¼ 8.52(t, ³J ¼ 6.1 Hz,1H), 8.30(d, ³J ¼ 8.1 Hz, 0.7H),

8.25 (d, ³J ¼ 8.1 Hz, 0.3H), 7.99 (s,1H), 7.85 (d, ³J ¼ 8.3 Hz, 2H), 7.68 (d,

³J ¼ 8.5 Hz, 0.3H), 7.60e7.49 (m, 4.7H), 7.42e7.33 (m, 3H), 6.38 (br s,

2H), 4.65e4.58 (m, 1H), 4.47 (d, ³J ¼ 5.9 Hz, 2H), 4.45e4.34 (m, 1H),

3.66e3.56 (m, 1H), 3.40e3.30 (m, 1H), 3.29e3.20 (m, 1H), 1.81e1.53

(m, 7H), 1.36e1.04 (m, 7H), 0.90e0.77 (m, 5H) ppm; ¹³C NMR

d

¼ 171.6, 166.0, 164.2, 149.2, 143.6, 138.4, 138.2, 132.7, 129.4, 127.6,

127.1, 126.8, 123.1, 97.4, 53.7, 41.3, 36.9, 33.3, 32.9, 32.8, 31.3, 29.4,

29.1, 29.0 (2x), 28.8, 26.3, 26.2, 25.8, 22.1, 14.0 ppm; MS (ESI pos.):

m/z (%) ¼ 643.46 (96) ([MþH]^þ, calcd. 643.44), 486.33 (25) ([M_fr.I]^þ,

calcd. 486.25), 190.35 (100) ([M_fr.IIþNa]^þ, calcd. 190.13); HRMS

(126 MHz, DMSO‑d₆, 300 K): (mixture of diastereomers)

d

¼ 171.7,

166.0 (2x), 164.2, 149.2, 143.9, 138.4, 138.2, 132.7, 129.4, 127.5 (2x),

127.1,126.8,123.1, 97.4, 63.0 (2x), 53.8, 53.7, 52.2, (2x), 41.4, 36.9, 36.8,

33.3, 33.2, 33.0, 32.7, 29.7, 29.4, 26.2, 25.8(2x), 23.7,10.4 (2x) ppm; MS

(ESI pos.): m/z (%) ¼ 575.17 (100) ([MþH]^þ, calcd. 575.34), 486.08 (33)

([M_fr.I]^þ, calcd. 486.25), 319.04 (12) ([M_fr.II]^þ, calcd. 319.12); HRMS

(FTMS

þ

p

MALDI): m/z

¼

665.4169 [MþNa]^þ, calcd. for

[C₃₈H₅₄N₆NaO₃]^þ¼ 466.4155.

5-Amino-N-(4-(((S)-1-((SR)-sec-butylamino)-4-cyclohexyl-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (142). Compound 142 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 150 mg, 0.446 mmol) and

(FTMS

þ

p

MALDI): m/z

¼

597.3173 [MþNa]^þ, calcd. for

[C₃₂H₄₂N₆NaO₄]^þ¼ 597.3165.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-(isopentylamino)-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (145). Compound 145 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 200 mg, 0.60 mmol) and

(S)-2-amino-4-cyclohexyl-N-isopentylbutanamide (115, 182 mg,

0.71 mmol). The crude product was puriﬁed by column chroma-

tography on silica (cyclohexane/EtOAc 1:1 / 1:6 / EtOAc) to

afford 327 mg (0.57 mmol, 96%) of 145. TLC: R_F¼ 0.41 (SiO₂,

cyclohexane/EtOAc 1:6); HPLC: 16.0 min; ¹H NMR (500 MHz,

(S)-2-amino-N-((SR)-sec-butyl)-4-cyclohexylbutanamide

(112,

81 mg, 0.36 mmol). The crude product was puriﬁed by column

chromatography on silica (CH₂Cl₂/MeOH 20:1) to afford 155 mg

(0.28 mmol, 78%) of 142. TLC: R_F¼ 0.19 (SiO₂, CH₂Cl₂/MeOH 20:1);

HPLC: 15.5 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.51 (t,

³J ¼ 5.9 Hz, 1H), 8.22 (dd, ³J ¼ 8.0, 12.3 Hz, 1H), 7.98 (s, 1H), 7.85 (d,

³J ¼ 8.0 Hz, 2H), 7.72 (dd, ³J ¼ 8.2, 20.7 Hz, 1H), 7.59e7.49 (m, 4H),

7.41e7.34 (m, 3H), 6.38 (br s, 2H), 4.47 (d, ³J ¼ 5.8 Hz, 2H),

4.40e4.32 (m, 1H), 3.67 (sext, ³J ¼ 6.7 Hz, 1H), 1.78e1.55 (m, 7H),

1.43e1.32 (m, 2H), 1.28e1.08 (m, 6H), 1.01 (dd, ³J ¼ 6.7, 10.3 Hz, 3H),

0.88e0.77 (m, 5H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

3

DMSO‑d₆, 300 K):

d

¼ 8.52 (t, J ¼ 6.1 Hz, 1H), 8.27 (d, J ¼ 8.0 Hz,

1H), 7.99 (s, 1H), 7.90 (t, ³J ¼ 5.6 Hz, 1H), 7.85 (d, ³J ¼ 8.3 Hz, 2H),

7.59e7.49 (m, 4H), 7.41e7.35 (m, 3H), 6.38 (br s, 2H), 4.47 (d,

³J ¼ 6.0 Hz, 2H), 4.38e4.31 (m, 1H), 3.16e2.99 (m, 2H), 1.80e1.71

(m, 1H), 1.70e1.52 (m, 6H), 1.33e1.03 (m, 9H), 0.90e0.79 (m, 8H)

d

¼ 171.7, 165.9 (2x), 164.2, 149.2, 143.6, 138.4, 138.2, 132.7, 129.4,

127.5, 127.1, 126.8, 123.1, 97.4, 53.6 (2x), 45.7 (2x), 41.3, 36.8, 33.3,

33.0, 32.7, 29.4, 28.8 (2x), 26.2, 25.8 (2x), 20.3 (2x), 10.5, 10.4 ppm;

MS (ESI pos.): m/z (%) ¼ 581.39 (100) ([MþNa]^þ, calcd. 581.32);

HRMS (FTMS þ p MALDI): m/z ¼ 581.3209 [MþNa]^þ, calcd. for

[C₃₂H₄₂N₆NaO₃]^þ¼ 581.3216.

ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 171.5, 166.0, 164.1,

149.2, 143.6, 138.4, 138.2, 132.7, 129.4, 127.6, 127.1, 126.8, 123.1, 97.4,

53.6, 41.3, 38.1, 36.8, 36.7, 33.3, 32.9, 32.7, 29.3, 26.2, 25.8, 25.1,

22.4 ppm; MS (ESI pos.): m/z (%) ¼ 573.38 (100) ([MþH]^þ, calcd.

573.35), 486.30 (26) ([M_fr.I]^þ, calcd. 486.25), 319.22 (22) ([M_fr.II]^þ,

calcd. 319.12); HRMS (FTMS þ p MALDI): m/z ¼ 573.3530 [MþH]^þ,

calcd. for [C₃₃H₄₅N₆O₃]^þ¼ 573.3548.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-oxo-1-(pentan-3-

ylamino)butan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-

4-carboxamide (143). Compound 143 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 150 mg, 0.45 mmol) and

(S)-5-Amino-N-(4-((4-cyclohexyl-1-(cyclohexylamino)-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (146). Compound 146 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 200 mg, 0.60 mmol) and

(S)-2-amino-4-cyclohexyl-N-(pentan-3-yl)butanamide

(113,

136 mg, 0.54 mmol). The crude product was puriﬁed by column

chromatography on silica (CH₂Cl₂/MeOH 20:1) to afford 148 mg

(0.26 mmol, 58%) of 143. TLC: R_F¼ 0.20 (SiO₂, CH₂Cl₂/MeOH 20:1);

(S)-2-amino-N,4-dicyclohexylbutanamide

(116,

159

mg,

HPLC: 15.9 min; ¹H NMR (500 MHz, CDCl₃, 300 K):

d

¼ 7.76 (s, 1H),

0.71 mmol). The crude product was puriﬁed by column chroma-

tography on silica (cyclohexane/EtOAc 2:3 / 1:6 / EtOAc) to

afford 138 mg (0.24 mmol, 40%) of 146. TLC: R_F¼ 0.38 (SiO₂,

7.67 (d, ³J ¼ 8.2 Hz, 2H), 7.56e7.46 (m, 4H), 7.38 (t, ³J ¼ 7.2 Hz, 1H),

7.29 (d, ³J ¼ 8.2 Hz, 2H), 7.15 (d, ³J ¼ 7.9 Hz, 1H), 6.66 (t, ³J ¼ 5.9 Hz,

1H), 6.33 (d, ³J ¼ 9.0 Hz, 1H), 5.52 (s, 2H), 4.65e4.51 (m, 3H),

3.78e3.68 (m, 1H), 2.00e1.88 (m, 1H), 1.82e1.72 (m, 1H), 1.71e1.59

(m, 5H), 1.58e1.44 (m, 2H), 1.43e1.05 (m, 8H), 0.93e0.78 (m, 8H)

cyclohexane/EtOAc 1:6); HPLC: 16.3 min; ¹H NMR (500 MHz,

3

DMSO‑d₆, 300 K):

d

¼ 8.52 (t, J ¼ 6.0 Hz, 1H), 8.24 (d, ³J ¼ 8.2 Hz,

1H), 7.99 (s, 1H), 7.89e7.78 (m, 3H), 7.60e7.49 (m, 4H), 7.42e7.30

28

€

S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

(m, 3H), 6.39 (br s, 2H), 4.48 (d, ³J ¼ 6.0 Hz, 2H), 4.42e4.33 (m, 1H),

3.61e3.47 (m, 1H), 1.78e1.48 (m, 12H), 1.33e1.03 (m, 11H),

0.90e0.76 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

DMSO‑d₆, 300 K):

d

¼ 8.58e8.47 (m, 2H), 7.98 (s, 1H), 7.85 (d,

³J ¼ 8.2 Hz, 2H), 7.59e7.49 (m, 4H), 7.41e7.32 (m, 3H), 6.38 (br s,

2H), 4.87e4.79 (m, 1H), 4.47 (d, ³J ¼ 5.9 Hz, 2H), 3.64e3.18 (m, 8H),

1.77e1.54 (m, 7H), 1.40 (s, 9H), 1.28e1.05 (m, 6H), 0.91e0.78 (m,

d

¼ 170.8, 165.9, 164.1, 149.2, 143.6, 138.4, 138.2, 132.7, 129.4, 127.5,

127.1, 126.8, 123.1, 97.4, 59.8, 53.5, 47.5, 41.3, 36.8, 33.3, 32.9, 32.7,

32.4, 32.2, 29.6, 29.2, 26.2, 25.8, 25.2, 24.5 ppm; MS (ESI pos.): m/z

(%) ¼ 585.25 (100) ([MþH]^þ, calcd. 585.36), 486.16 (80) ([M_fr.]^þ,

calcd. 486.25); HRMS (FTMS þ p MALDI): m/z 607.3360 [MþNa]^þ,

calcd. for [C₃₄H₄₄N₆NaO₃]^þ¼ 607.3376.

2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 170.2, 165.9,

164.1, 159.3, 149.2, 143.7, 138.4, 138.2, 132.4, 129.4, 127.6, 127.1,

126.8, 123.1, 97.4, 79.2, 59.8, 49.5, 44.7, 41.4, 41.3, 38.2, 37.0, 33.1,

32.9, 32.7, 28.8, 26.2, 25.8 ppm; MS (ESI pos.): m/z (%) ¼ 672.42 (24)

([MꢃH]^þ, calcd. 672.42), 486.26 (11) ([M_fr.I]^þ, calcd. 486.26), 190.36

(100) ([M_fr.IIþNa]^þ, calcd. 6190.13); HRMS (FTMS þ p MALDI): m/

z ¼ 694.3688 [MþNa]^þ, calcd. for [C₃₇H₄₉N₇NaO₅]^þ¼ 694.3693.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-((4-ﬂuorophenyl)

(S)-5-Amino-N-(4-((4-cyclohexyl-1-morpholino-1-oxobutan-

2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-carboxamide

(147). Compound 147 was prepared according to general procedure

F

using

4-((5-amino-1-phenyl-1H-pyrazole-4-carboxamido)

amino)-1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyr-

azole-4-carboxamide (150). Compound 150 was prepared ac-

cording to general procedure F using 4-((5-amino-1-phenyl-1H-

pyrazole-4-carboxamido)methyl)benzoic acid (70, 200 mg,

0.60 mmol) and (S)-2-amino-4-cyclohexyl-N-(4-ﬂuorophenyl)

butanamide (118, 270 mg, 0.71 mmol). The crude product was

puriﬁed by column chromatography on silica (cyclohexane/EtOAc

1:1 /1:3 / 1:20) and further recrystallized from acetone to afford

149 mg (0.25 mmol, 42%) of 150. TLC: R_F¼ 0.73 (SiO₂, cyclohexane/

EtOAc 1:3); HPLC: 16.1 min; ¹H NMR (600 MHz, DMSO‑d₆, 300 K):

methyl)benzoic acid (70, 200 mg, 0.60 mmol) and (S)-2-amino-4-

cyclohexyl-1-morpholinobutan-1-one (117, 182 mg, 0.71 mmol).

The crude product was puriﬁed by column chromatography on

silica (cyclohexane/EtOAc 1:3 / 1:10) to afford 90 mg (0.16 mmol,

26%) of 147. TLC: R_F¼ 0.27 (cyclohexane/EtOAc 1:10); HPLC:

14.6 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.54 (d,

³J ¼ 8.1 Hz, 1H), 8.52 (t, ³J ¼ 6.1 Hz, 1H), 7.99 (s, 1H), 7.86 (d,

³J ¼ 8.3 Hz, 2H), 7.59e7.55 (m, 2H), 7.52 (t, ³J ¼ 7.9 Hz, 2H) 7.41e7.35

(m, 3H), 6.38 (br s, 2H), 4.86e4.78 (m, 1H), 4.47 (d, ³J ¼ 5.9 Hz, 2H),

3.61e3.39 (m, 8H), 1.77e1.55 (m, 7H), 1.27e1.05 (m, 6H), 0.90e0.78

d

¼ 10.15 (s, 1H), 8.57e8.44 (m, 2H), 7.99 (s, 1H), 7.89 (d, ³J ¼ 7.5 Hz,

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 170.1, 165.8,

2H), 7.68e7.48 (m, 6H), 7.43e7.35 (m, 3H), 7.14 (t, ³J ¼ 8.1 Hz, 2H),

6.37 (br s, 2H), 4.56e4.43 (m, 3H), 1.88e1.75 (m, 2H), 1.73e1.56 (m,

5H), 1.39e1.30 (m, 1H), 1.28e1.05 (m, 5H), 0.92e0.81 (m, 2H) ppm;

164.2, 149.2, 143.7, 138.4, 138.2, 132.4, 129.4, 127.6, 127.1, 126.8,

123.1, 97.4, 66.2, 49.3, 45.6, 42.0, 41.3, 37.0, 33.1, 32.9, 32.7, 28.8,

26.2, 25.8 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 573.33 (51) ([MþH]^þ,

calcd. 573.32), 486.28 (23) ([M_fr.I]^þ, calcd. 486.25), 190.35 (100)

¹³C NMR (150 MHz, DMSO‑d₆, 300 K):

d

¼ 170.9, 166.3, 164.1, 157.9

(¹J_CF¼ 240.8 Hz), 149.2, 143.6, 138.3, 138.2, 135.3, 132.4, 129.3, 127.6,

127.0, 126.7, 123.0, 121.0 (³J_CF¼ 7.9 Hz), 115.2 (²J_CF¼ 22.3 Hz), 97.4,

54.5, 41.3, 36.8, 33.3, 32.8, 32.6, 29.1, 26.1, 25.8 (2x) ppm; MS (ESI

pos.): m/z (%) ¼ 619.20 (13) ([MþNa]^þ, calcd. 619.28), 597.20 (100)

([MþH]^þ, calcd. 597.31), 486.19 (99) ([M_fr.I]^þ, calcd. 486.25), 458.21

(16) ([M_fr.II]^þ, calcd. 458.26), 319.04 (28) ([M_fr.III]^þ, calcd. 319.12);

HRMS (FTMS þ p MALDI): m/z ¼ 619.2816 [MþNa]^þ, calcd. for

[C₃₄H₃₇FN₆NaO₃]^þ¼ 619.2809.

([M_fr.IIþNa]^þ, calcd. 190.13); HRMS(FTMS

þ

p MALDI): m/

z ¼ 595.2997 [MþNa]^þ, calcd. for [C₃₂H₄₀N₆NaO₄]^þ¼ 595.3009.

(S)-5-Amino-N-(4-((4-cyclohexyl-1-oxo-1-(piperazin-1-yl)

butan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (148). (S)-tert-Butyl-4-(2-(4-((5-amino-1-phenyl-

1H-pyrazole-4-carboxamido)methyl)benzamido)-4-cyclo-hex-

ylbutanoyl)piperazine-1-carboxylate (162, 80 mg, 0.120 mmol, 1

eq) was dissolved in CH₂Cl₂(10 mL) and cooled to 0 ^ꢀC. TFA (2 mL)

was added dropwise and the mixture was stirred for 1.5 h at rt. The

solvent was removed under reduced pressure and the crude

product was dissolved in MeOH. The solution was cooled to 5 ^ꢀC

and neutralized with K₂CO₃. After 10 min stirring, the mixture was

ﬁltered, and the solvent was removed under reduced pressure. The

crude product was puriﬁed by column chromatography on silica

(cyclohexane/EtOAc 1:9 / EtOAc / EtOHþ1% TEA) to afford 35 mg

(0.06 mmol, 51%) of 148. TLC: R_F¼ 0.04 (EtOAc/MeOH 10:1); HPLC:

5-Amino-N-(4-((1-((4-chlorophenyl)amino)-4-cyclohexyl-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (151). Compound 151 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 185 mg, 0.55 mmol) and

(S)-2-amino-N-(4-chlorophenyl)-4-cyclohexyl-butanamide (119,

195 mg, 0.66 mmol). The crude product was puriﬁed by column

chromatography on silica (cyclohexane/EtOAc 1:1 / 1:3 / 1:20)

to afford 91 mg (0.148 mmol, 27%) of 151. TLC: R_F¼ 0.40 (SiO₂,

cyclohexane/EtOAc 1:3); HPLC: 16.8 min; ¹H NMR (500 MHz,

10.1 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

d

¼ 8.56 (t,

³J ¼ 8.2 Hz, 1H), 8.50 (t, ³J ¼ 8.2 Hz 2H), 8.00 (s, 1H), 7.84 (d,

³J ¼ 8.2 Hz, 2H), 7.60e7.49 (m, 4H), 7.41e7.34 (m, 3H), 6.38 (br s,

2H), 4.88e4.76 (m, 1H), 4.46 (d, ³J ¼ 5.9 Hz, 2H), 3.64e3.45 (m, 4H),

2.47e2.29 (m, 4H), 1.76e1.54 (m, 7H), 1.29e1.04 (m, 7H), 0.90e0.77

DMSO‑d₆, 300 K):

d

¼ 10.26 (s, 1H), 8.60e8.48 (m, 2H), 7.99 (s, 1H),

7.88 (d, ³J ¼ 8.0 Hz, 2H), 7.65 (d, ³J ¼ 8.6 Hz, 2H), 7.60e7.49 (m, 4H),

7.44e7.31 (m, 5H), 6.38 (br s, 2H), 4.56e4.42 (m, 3H),1.88e1.75 (m,

2H), 1.73e1.55 (m, 5H), 1.40e1.29 (m, 1H), 1.28e1.04 (m, 5H),

0.92e0.79 (m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

(m, 2H) ppm; ¹³C NMR (126 MHz, DMSO‑d₆, 300 K):

d

¼ 169.8,

165.8, 164.2, 149.2, 143.7, 138.4, 138.2, 132.4, 129.4, 127.6, 127.1,

126.8, 123.1, 97.4, 51.5, 50.9, 45.1, 41.5, 41.3, 37.0, 33.1, 32.9, 32.7,

29.0, 26.2, 25.8 ppm; MS (ESI pos.): m/z (%) ¼ 572.43 (100)

([MþH]^þ, calcd. 572.34); HRMS (FTMS þ p MALDI): m/z ¼ 572.3328

[MþH]^þ, calcd. for [C₃₂H₄₂N₇O₃]^þ¼ 572.3344.

d

¼ 172.3, 166.4, 164.2, 149.2, 143.7, 138.4, 138.2, 138.0, 132.4, 129.4,

128.6,127.7, 127.1,126.8,123.1, 120.8, 97.4, 54.7, 41.3, 36.9, 33.4, 32.9,

32.7, 29.1, 26.1, 25.8 (2x) ppm; MS (ESI pos.): m/z (%) ¼ 613.21 (76)

([MþH]^þ, calcd. 613.27), 486.20 (100) ([M_fr.]^þ, calcd. 486.26); HRMS

(FTMS

þ

p

MALDI): m/z

¼

613.2684 [MþH]^þ, calcd. for

(S)-tert-Butyl-4-(2-(4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzamido)-4-cyclohexylbutanoyl)piper-

azine-1-carboxylate (149). Compound 149 was prepared accord-

[C₃₄H₃₈ClN₆O₃]^þ¼ 613.2694.

(S)-5-Amino-N-(4-((1-((4-bromophenyl)amino)-4-

cyclohexyl-1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-

pyrazole-4-carboxamide (152). Compound 152 was prepared ac-

cording to general procedure F using 4-((5-amino-1-phenyl-1H-

pyrazole-4-carboxamido)methyl)benzoic acid (70, 200 mg,

0.60 mmol) and (S)-2-amino-N-(4-bromophenyl)-4-cyclohexyl-

butanamide (120, 204 mg, 0.60 mmol). The crude product was

puriﬁed by column chromatography on silica (cyclohexane/EtOAc

1:1 /1:3 / 1:20) and further recrystallized from acetone to afford

ing to general procedure

F using 4-((5-amino-1-phenyl-1H-

pyrazole-4-carboxamido)methyl)benzoic acid (70, 200 mg,

0.60 mmol) and (S)-tert-butyl 4-(2-amino-4-cyclohexylbutanoyl)

piperazine-1-carboxylate (131, 252 mg, 0.71 mmol). The crude

product was puriﬁed by column chromatography on silica (cyclo-

hexane/EtOAc) to afford 138 mg (0.21 mmol, 35%) of 149. TLC:

R_F¼ 0.58 (EtOAc/MeOH 10:1); HPLC: 16.4 min; ¹H NMR (500 MHz,

29

€

S. Rohm et al.

European Journal of Medicinal Chemistry 208 (2020) 112721

69 mg (0.10 mmol, 18%) of 152. TLC: R_F¼ 0.17 (SiO₂, cyclohexane/

EtOAc 1:1); HPLC: 17.0 min; ¹H NMR (600 MHz, DMSO‑d₆, 300 K):

d

2H), 7.60 (d, ³J ¼ 9.0 Hz, 2H), 7.57 (d, ³J ¼ 7.8 Hz, 2H), 7.52 (t,

³J ¼ 8.0 Hz, 2H), 7.48 (d, ³J ¼ 9.0 Hz, 2H), 7.41e7.37 (m, 3H), 6.37 (br

s, 2H), 4.54e4.46 (m, 3H), 1.87e1.75 (m, 2H), 1.72e1.55 (m, 5H),

1.37e1.29 (m, 1H), 1.28e1.06 (m, 5H), 0.92e0.81 (m, 2H) ppm; ¹³C

HRMS (FTMS þ p MALDI): m/z ¼ 669.2099 [MþNa]^þ, calcd. for

[C₃₄H₃₆ClN₆NaO₃]^þ¼ 669.2124.

¼ 10.23 (s, 1H), 8.55e8.48 (m, 2H), 7.98 (s, 1H), 7.88 (d, ³J ¼ 8.2 Hz,

(S)-5-Amino-N-(4-((4-cyclohexyl-1-((3,5-dibromophenyl)

amino)-1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyr-

azole-4-carboxamide (155). Compound 155 was prepared accord-

ing to general procedure

F using 4-((5-amino-1-phenyl-1H-

pyrazole-4-carboxamido)methyl)benzoic acid (70, 200 mg,

0.60 mmol) and (S)-2-amino-4-cyclohexyl-N-(3,5-dibromophenyl)

butanamide (123, 251 mg, 0.60 mmol). The crude product was pu-

riﬁed by column chromatography on silica (cyclohexane/EtOAc

1:1 / 1:3 / 1:20) and further recrystallized from acetone to afford

79 mg (0.11 mmol, 18%) of 155. TLC: R_F¼ 0.67 (SiO₂, cyclohexane/

EtOAc 1:3); HPLC: 18.8 min; ¹H NMR (600 MHz, DMSO‑d₆, 300 K):

NMR (75 MHz, DMSO‑d₆, 300 K):

d

¼ 171.3,166.4,164.2,149.2,143.7,

138.4, 138.2, 132.4, 131.5, 129.4, 127.6, 127.1, 126.8, 123.1, 121.2, 114.8,

97.4, 54.7, 41.3, 36.9, 33.4, 32.9, 32.7, 29.1, 26.1, 25.8 (2x) ppm; MS

(ESI pos.): m/z (%) ¼ 681.14 (15) ([MþNa]^þ, calcd. 681.20), 659.14

(41) ([MþH]^þ, calcd. 659.22), 486.19 (100) ([M_fr.I]^þ, calcd. 486.25),

392.00 (60) ([M_fr.II]^þ, calcd. 392.24), 324.21 (57) ([M_fr.III]^þ, calcd.

324.10); HRMS (FTMS þ p MALDI): m/z ¼ 679.2019 [MþNa]^þ, calcd.

for [C₃₄H₃₇BrN₆NaO₃]^þ¼ 679.2008.

d

¼ 10.39 (s, 1H), 8.57 (d, ³J ¼ 7.6 Hz, 1H), 8.51 (t, ³J ¼ 5.6 Hz, 1H), 7.99

(s, 1H), 7.92e7.84 (m, 4H), 7.60e7.48 (m, 5H), 7.42e7.35 (m, 3H), 6.37

(br s, 2H), 4.52e4.42 (m, 3H), 1.88e1.75 (m, 2H), 1.72e1.55 (m, 5H),

1.38e1.30 (m, 1H), 1.27e1.06 (m, 5H), 0.91e0.81 (m, 2H) ppm; ¹³C

(S)-5-Amino-N-(4-((4-cyclohexyl-1-((3,5-diﬂuorophenyl)

amino)-1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyr-

azole-4-carboxamide (153). Compound 153 was prepared ac-

cording to general procedure F using 4-((5-amino-1-phenyl-1H-

pyrazole-4-carboxamido)methyl)benzoic acid (70, 200 mg,

0.60 mmol) and (S)-2-amino-4-cyclohexyl-N-(3,5-diﬂuorophenyl)

butanamide (121, 178 mg, 0.60 mmol). The crude product was pu-

riﬁed by column chromatography on silica (cyclohexane/EtOAc

1:1 /1:3 / 1:20) and further recrystallized from acetone to afford

202 mg (0.33 mmol, 55%) of 153. TLC: R_F¼ 0.19 (SiO₂, cyclohexane/

EtOAc 1:1); HPLC: 16.7 min; ¹H NMR (600 MHz, DMSO‑d₆, 300 K):

NMR (75 MHz, DMSO‑d₆, 300 K):

d

¼ 171.9, 166.5, 164.1, 149.2, 143.8,

141.6, 138.4, 138.2, 132.3, 129.3, 127.8, 127.6, 127.0, 126.8, 123.1, 122.3,

120.5, 97.4, 54.9, 41.3, 36.9, 33.3, 32.8, 32.7, 28.8, 26.1, 25.8 (2x) ppm;

MS (ESI pos.): m/z (%) ¼ 737.14 (84) ([MþH]^þ, calcd. 737.13), 486.21

(100) ([M_fr.I]^þ, calcd. 486.25); HRMS (FTMS þ p MALDI): m/

z ¼ 737.1271 [MþH]^þ, calcd. for [C₃₄H₃₇Br₂N₆O₃]^þ¼ 737.1273.

(S)-5-Amino-N-(4-((1-(benzylamino)-4-cyclohexyl-1-

oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (156). Compound 156 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 150 mg, 0.45 mmol) and

(S)-2-amino-N-benzyl-4-cyclohexylbutanamide (124, 147 mg,

0.54 mmol). The crude product was triturated with cyclohexane/

EtOAc (1:1) to afford 202 mg (0.34 mmol, 76%) of 156. TLC: R_F¼ 0.67

(SiO₂, EtOAc/MeOH 10:1); HPLC: 15.6 min; ¹H NMR (500 MHz,

3

d

¼ 10.50 (s, 1H), 8.58 (d, J ¼ 7.4 Hz, 1H), 8.48 (t, J ¼ 5.9 Hz, 1H),

8.00 (s, 1H), 7.90 (d, ³J ¼ 8.2 Hz, 2H), 7.58 (d, ³J ¼ 7.9 Hz, 2H), 7.53 (t,

³J ¼ 8.0 Hz,2H), 7.43e7.34 (m, 5H), 6.91 (tt, ³J ¼ 9.2, 2.0 Hz, 1H), 6.38

(br s, 2H), 4.53e4.46 (m, 3H), 1.88e1.77 (m, 2H), 1.74e1.57 (m, 5H),

1.40e1.32 (m, 1H), 1.29e1.07 (m, 5H), 0.92e0.82 (m, 2H) ppm; ¹³C

NMR (75 MHz, DMSO‑d₆, 300 K):

d

¼ 171.9, 166.5, 164.1, 162.5

(¹J_CF¼ 243.3 Hz), 162.3 (¹J_CF¼ 243.3 Hz), 149.2, 143.8, 141.7 (2x),

141.3, 138.4, 138.2, 132.3, 129.4, 127.6, 127.1, 126.8, 123.1, 102.1

(²J_CF¼ 19.3 Hz), 102.0 (²J_C-F¼ 19.3 Hz), 98.7, 98.4, 98.0, 97.4, 54.8,

41.3, 36.9, 33.4, 32.9, 32.7, 28.9, 26.1, 25.8 (2x) ppm; MS (ESI pos.):

m/z (%) ¼ 615.26 (100) ([MꢃH]^þ, calcd. 615.28), 486.11 (69) ([M_fr.]^þ,

calcd. 486.25); HRMS (FTMS þ p MALDI): m/z ¼ 615.2887 [MþH]^þ,

calcd. for [C₃₄H₃₇F₂N₆O₃]^þ¼ 615.2895, 637.2710 [MþNa]^þ, calcd.

for [C₃₄H₃₆F₂N₆NaO₃]^þ¼ 637.2709.

DMSO‑d₆, 300 K):

d

¼ 8.70 (t, J ¼ 6.1 Hz, 1H); 8.62 (t, J ¼ 6.0 Hz,

3

1H), 8.49 (d, ³J ¼ 8.1 Hz, 1H), 8.07 (s, 1H), 7.88 (d, J ¼ 8.3 Hz, 2H),

7.59e7.49 (m, 4H), 7.41e7.35 (m, 3H), 7.31e7.35 (m, 5H), 6.37 (br s,

2H), 4.49e4.38 (m, 3H), 4.33e4.22 (m, 2H), 1.86e1.53 (m, 7H)

1.33e1.03 (m, 6H), 0.88e0.75 (m, 2H) ppm; ¹³C NMR (126 MHz,

3

DMSO‑d₆, 300 K):

d

¼ 172.1, 166.1, 164.2, 149.2, 143.7, 139.6, 138.6,

132.6, 129.4, 128.2, 127.6, 127.1, 126.8, 126.7, 123.1, 97.5, 53.9, 42.0,

41.3, 36.8, 33.3, 32.9, 29.2, 26.2 (2x) ppm; MS (MALDI pos.): m/z

(%) ¼ 614.8 (100) ([MþNa]^þ, calcd. 615.31), 592.86 (55) ([M]^þ, calcd.

592.32); HRMS (FTMS þ p MALDI): m/z ¼ 631.2784 [MþK]^þ, calcd.

for [C₃₅H₄₀KN₆O₃]^þ¼ 631.2799.

5-Amino-N-(4-((4-cyclohexyl-1-((3,5-dichlorophenyl)amino)-

1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (154). Compound 154 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 140 mg, 0.42 mmol) and

(S)-2-amino-4-cyclohexyl-N-(3,5-dichlorophenyl)butanamide (122,

165 mg, 0.50 mmol). The crude product was puriﬁed by column

chromatography on silica (cyclohexane/EtOAc 1:1 / 1:3 / 1:20 /

EtOAc) and further recrystallized from acetone to afford 97 mg

(0.15 mmol, 32%) of 154. TLC: R_F¼ 0.48 (SiO₂, cyclohexane/EtOAc

1:3); HPLC: 18.27 min; ¹H NMR (500 MHz, DMSO‑d₆, 300 K):

(S)-5-Amino-N-(4-((1-((4-chlorobenzyl)amino)-4-cyclohexyl-

1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-

carboxamide (157). Compound 157 was prepared according to

general procedure F using 4-((5-amino-1-phenyl-1H-pyrazole-4-

carboxamido)methyl)benzoic acid (70, 200 mg, 0.60 mmol) and

(S)-2-amino-N-(4-chlorobenzyl)-4-cyclohexylbutanamide

(125,

221 mg, 0.74 mmol). The crude product was puriﬁed by column

chromatography on silica (cyclohexane/EtOAc 1:1 / 1:4 / 1:7 /

EtOAc) to afford 203 mg (0.32 mmol, 54%) of 157. TLC: R_F¼ 0.66

(SiO₂, EtOAc/MeOH 10:1); HPLC: 16.1 min; ¹H NMR (500 MHz,

d

¼ 10.49 (s, 1H), 8.60 (d, ³J ¼ 7.3 Hz, 1H), 8.53 (t, ³J ¼ 6.1 Hz, 1H), 7.99

(s,1H), 7.88 (d, ³J ¼ 8.3 Hz, 2H), 7.71 (d, ⁴J ¼ 1.9 Hz, 2H), 7.58e7.49 (m,

4H), 7.42e7.36 (m, 3H), 7.28 (t, ⁴J ¼ 1.9 Hz, 1H), 6.39 (br s, 2H),

4.51e4.23 (m, 3H), 1.88e1.75 (m, 2H), 1.73e1.55 (m, 5H), 1.39e1.30

(m, 1H), 1.28e1.05 (m, 5H), 0.91e0.80 (m, 2H) ppm; ¹³C NMR

DMSO‑d₆, 300 K):

d

¼ 8.57e8.44 (m, 2H), 8.39 (d, ³J ¼ 7.9 Hz, 1H),

8.00 (s,1H), 7.88 (d, ³J ¼ 8.3 Hz, 2H), 7.61e7.49 (m, 4H), 7.42e7.33 (m,

5H), 7.28 (d, ³J ¼ 8.4 Hz, 2H) 6.39 (br s, 2H), 4.48 (d, ³J ¼ 6.0 Hz, 2H),

4.44e4.37 (m, 1H), 4.27 (d, ³J ¼ 6.3 Hz, 2H) 1.86e1.53 (m, 7H),

1.28e1.04 (m, 6H), 0.90e0.73 (m, 2H) ppm; ¹³C NMR (126 MHz,

(126 MHz, DMSO‑d₆, 300 K):

d

¼ 172.0, 166.6, 164.2, 149.2, 143.8,

141.3, 138.4, 138.2, 134.1, 132.3, 129.4, 127.7, 127.1, 126.8, 123.1, 122.5,

117.3, 97.4, 54.9, 41.3, 36.9, 33.4, 32.9, 32.7, 28.8, 26.1, 25.8 (2x) ppm;

MS (ESI pos.): m/z (%) ¼ 647.13 (4) ([MþH]^þ, calcd. 647.23), 354.99

(19) ([M_fr.I]^þ, calcd. 355.10), 329.02 (100) ([M_fr.II]^þ, calcd. 329.10);

DMSO‑d₆, 300 K):

138.2, 132.6, 131.2, 129.4, 129.0, 128.1, 127.6, 127.1, 126.8, 123.1, 97.4,

d

¼ 172.1, 166.2, 164.1, 149.2, 143.6, 138.6, 138.4,

53.6, 41.4, 36.7, 33.3, 32.9, 32.7, 29.1, 26.2, 25.8 ppm; MS (ESI pos.): m/

30

Article Doi

Selective targeting of the αC and DFG-out pocket in p38 MAPK

DOI: 10.1016/j.ejmech.2020.112721

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